[0001] This application claims priority from U.S. Provisional Application No.

62/533,589, filed July 17, 2017, having the same title, and having the same inventors, and which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates generally to gene expression modulation, and more particularly to polytherapy which modulates cathelicidin gene expression for the treatment of medical conditions such as Alzheimer's disease.

BACKGROUND OF THE DISCLOSURE

Initial Discovery of Mammalian Cathelicidins, and Human Cathelicidin LL-37.

[0003] In 1991, Agerberth et al. first discovered the cathelicidin peptide and reported its antibacterial activity. That first literature report appeared in December 1991 and described the porcine cathelicidin PR-39. [REF: Amino acid sequence of PR-39. Isolation from pig intestine of a new member of the family of proline-argi nine-rich antibacterial peptides. Agerberth B, Lee JY, Bergman T, Carlquist M, Soman HG, Mutt V, Jornvali H. Eur J Biochem. 1991 Dec 18;202(3):849-54. Pubmed ID PMID: 1765098] The first studies of the mechanisms of antibacterial action of PR-39 were reported in 1993. [REF. Mechanisms of action on Escherichia coli of cecropi PI and PR-39, two antibacterial peptides from pig intestine. Bom an HG, Agerberth B, Bom an A, Infect Immun. 1993 Jul;61 (7):2978-84. Pubmed ID PMID: 8514403] [0004] In 1995, the first report of the human peptide analogue of PR-39 was reported by the same group. It was originally called FALL-39, but the name was changed later to LL-37. [REF: FALL-39, a putative human peptide antibiotic, is cysieine-free and expressed in bone marrow and testis. Agerberth B, Gunne I I, Odeberg J, Kogner P, Boman HG. Gudmundsson GH. Proc Nail Acad Sci U S A. 1995 Jan 3;92(1): 195-9. Pubrned PMID. 7529412.] Also in 1995, the rabbit cathelicidin protein was reported by Larrick et al, and was shown to bind to and detoxify bacterial lipopolysaccharide (LPS), which is a dangerous proinflammatory molecule in infections. [REF: Human CAP] 8; a novel antimicrobial 1 i popoly saccharide-binding protein. Larrick JW, Hiraia M, Baiirit RF, Lee J, Zhong J, Wright. SC. Infect Immun. 1995 Apr;63(4): 129I-7. Pubrned ID PMID 7890387 ^" | .

[0005] The human cathelicidin gene was initially called FALL39 by its Swedish discoverers, and later renamed CAMP. Human cathelicidin protein initially was shown to be expressed and processed to its final, active peptide form, LL-37, in human

granulocytes (such as neutrophils), which are mature white blood cells that contain, in their cytoplasm, granules of antipathogenic peptides, such as LL-37 and defensins. [REF: The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes. Gudmundsson GH, Agerberth B, Odeberg J, Bergman T, Oisson B, Salcedo R. Eur J Biochem. 1996 Jun l;238(2):325-32. Pubrned ID PMID: 8681941 ]

[0006] It is to be noted that granulocytes are sometimes also called

Polymorphonuclear Leukocytes (variously abbreviated PMN, PML, or PMNL), and include many immune cell types in the human system, including for example neutrophils, macrophages, B cells, and T cells. [REF: The human antimicrobial and chemotactic peptides LL-37 and alpha-defensins are expressed by specific lymphocyte and monocyte populations. Agerberth B, Charo J, Werr J, Oisson B, Idali F, Lindbom L, Kiessling R, Jornval! II Wigzel! H, Gudmundsson GH. Blood. 2000 Nov 1 ;96ί9):308ό~93. Pubrned ID PMID: 1 1049988] The effects of LL-37 on a number of different human cell types, including human peripheral blood mononuclear cells, CD14+ monocytes, myeloid and plasmocytoid dendritic cells and T- and B-lymphocytes, was studied by flow cytometry and DNA microarrays, and was shown to be both significant and complex, with all cell types responding to treatment with LL-37 peptide. [REF: Mo! Biosyst. 2009

May;5(5):483-96. Systems biology evaluation of immune responses induced by human host defence peptide LL-37 in mononuclear cells. Mookherjee NI , Fia.mil 1 P, Gardy J, Blimkie D, Faisafi R. Chikatamarla A, Arenillas DJ, Doria S, oiimann TR, Hancock RE. Pubmed PMID 19381363] In other words, white blood cells make and release LL-37 as an antimicrobial peptide. Once released, however, the LL-37 peptide is also immunomodulatory of white blood cell phenotype.

The Human LL-37 Gene CAMP and LL-37 Mechanism of Antibacterial Action.

[0007] In 1997 it was reported that the expression of the human gene encoding LL-37 is induced in keratinocytes during inflammatory disorders, highlighting the complexity of LL-37's in vivo immunomodulatory functions. [REF: The expression of the gene coding fbr tiie antibacterial peptide LL-37 is induced in human keratinocytes during

inflammatory disorders. Frohm M, Agerberth B, Ahangari G, Stahle-Backdahl M, Li den S, Wigzell H, Gudmundsson GPL J Bio! Chem. 1997 Jun 13 ,272(24): ^' ] 5258-63. Pubmed PMID: 9182550] In 1998, further studies of the antibacterial activity and mechanism of LL-37 were published, as interest in this peptide as a broad-spectrum endogenous antibiotic peptide grew. [REF: Conformation-dependent antibacterial activity of the naturally occurring human peptide LL-37. Johansson J, Gudmundsson GH, Rottenberg ME, Berndt KD, Agerberth B. J Biol Chem, 1998 Feb 6;273(6):3718-24. Pubmed ID PMID: 9452503] and [REF: Structure and activity of cathelicidin antibacterial proteins. Wang Y, Agerberth B, Johansson J. J Protein Chem. 1998 Aug; 17(6):522-3. Pubmed ID PMID: 9723727] Initial reports focused on bacterial membrane binding and disruption as the predominant mechanism of bacterial killing, but a 2017 report showed that LL-37 peptides rapidly cross bacterial cell membranes and have profound intracellular effects, aggregating cytosolic bacterial biomolecules irreversibly. [REF: intracellular bioraass flocculation as a key mechanism of rapid bacterial killing by caiiomc, amphipathic antimicrobial peptides and peptoids. Chongsiriwatana NP, Lin JS, apoor R, Wetzler M, Ilea JAC, Didwariia MK, Contag (Ί I Barron All. Sci Rep. 2017 Dec i ;7(I): 16718. dor 10.1038/s41598-017-16180-0. Pubmed ID PMID: 29196622] Injury to the skin has been shown to cause the expression and release of cathelicidin, which then can prevent infection by group A Streptococcus, a common skin pathogen. [REF: Cutaneous injury induces the release of cathelicidin anti -microbial peptides active against group A

Streptococcus. Dorschner RA, Pestonjamasp V , Tamakuwa!a S, Ohtake T, udi sill J, Nizet V, Agerberth B, Gudmundsson GPL Gallo RE. J Invest Dermatol. 2001

Jul; ] J 7( l):9i-7. Pubmed ID PMID: 11442754]

LL-37 is a Critical Element of Both the Formation and Clearance of Extracellular Traps

[0008] Another important aspect of the human body's response to invasion by pathogens is the formation of Neutrophil Extracellular Traps (NETs). [REF. Beneficial suicide: why neutrophils die to make NETs. Brinkmann V, Zychlmsky A. Nat Rev Microbiol . 2007 Aug;5(8):577-82. Pubmed ID PMID 17632569] Neutrophils, which are the most abundant class of white blood cells, upon stimulation (e.g., by the presence of bacteria) not only engulf pathogens, but can also produce extracellular structures called NETs. These structures, which are mostly DNA-based, directly capture and kill microorganisms, functioning like antibiotic spider webs. Other types of white blood cells with more specialized functions, such as eosinophils, are also able to make extracellular traps, which immobilize and subsequently kill pathogens. [REF. Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense. Yousefi S, Gold JA, Andina N, Lee J J, Kelly AM, Kozlowski E, Schmid I, Straumann A, Reichenbach J, G!eich Gi . Simon HU. Nat Med. 2008 Sep; 14i9):949-53. Pubmed ID PMID 18690244] [0009] Mast cells are also able to make extracellular traps, and presumably other cell types as well. [REF: Phagocytosis-independent antimicrobial activity of mast cells by means of extracellular trap formation, von Kockritz-Bikkwede M, Goldmann O, Timlin P, Heinemann K, Norrby-Teglund A, Rohde M, Medina E. Blood. 2008 Mar

15; l l l(6):3070-80. Pubmed ID PMID 18182576] NETs have been shown to be formed primarily from human DNA molecules, which are physically crosslinked by two classes of cationic proteins: histones (which function to spool DNA within the nucleus) and the cathelicidin LL-37. Thus, LL-37 plays an important role in stabilizing extracellular traps, which are an essential part of the human innate immune response to infection. [REF: The antimicrobial peptide LL-37 facilitates the formation of neutrophil extracellular traps. Neumann A, Berends ET, Ner!ich A, Molhoek EM, (Jal!o RE, Meerioo T, Nizet V, Nairn BY, von Kockritz-Blickwede M. Biochern 1. 2014 Nov 15;464(1):3-1 1 . Pubrned PMID: 25.181554] and [REF: Novel role of the antimicrobial peptide LL-37 in the protection of neutrophil extracellular traps against degradation by bacterial nucleases. Neumann A, Vol!ger L, Berends ET, Molhoek EM, Stape!s DA, Midon M, Friaes A, Pingoud A, Rooijakkers SH, Gallo RL, Morgelin M, Nizet V, Nairn HY, von Kockritz-Blickwede M. J Innate Xrnmun. 201 ;6(6):860-8. Pubmed PMID: 25012862]

[0010] Some pathogens that escape killing by NETs, do so by releasing Ml peptides (known as virulence factors) that specifically bind to and inactivate LL-37. [REF: M l protein allows Group A streptococcal survival in phagocyte extracellular traps through cathelicidin inhibition. Lauth XI , von oekritz-Bli ckwede M, cNamara CW,

Myskowski S, Zinkernagel AS, Beall B, Ghosh P, Gallo RL, Nizet V. J Innate Immun. 2009;1(3):202-14. Pubmed ID PMID 20375578] Impaired clearance and degradation of extracellular traps, which reflects a dysfunction of phagocytosis and macroautophagy, occurs in several types of chronic autoimmune diseases, including dermatomyositis and polymyositis. [REF: Enhanced formation and impaired degradation of neutrophil extracellular traps in dermatomyositis and polymyositis: a potential contributor to interstitial lung disease complications. Zhang S, Shu X, Tian X, Chen F„ Lu X, Wang G. Clin Exp Immunol. 2014 Jul;177(l): 134-41. Pubmed PMID: 24611519] and [KEF:

Neutrophil extracellular traps and their role in the development of chronic inflammation and autoimmunity. Pinegin B, Vorobjeva N, Pinegin V. Autoimmun Rev. 2015

Jul ; 14(7):633-40. Review. Pubmed PMID: 25797532]

[0011] In Systemic Lupus Erythematosus, uncleared ETs appear to be a critical autoimmune irritant, which are involved in the chronic disease inflammatory process. [REF: Neutrophils activate plasmaeytoid dendritic cells by releasing seif-DNA -peptide complexes in systemic lupus erythematosus. Lande R, Ganguly D, Facchinetti V, Frasca L, Conrad C, Gregorio J, Meiler S, Chami!os G, Sebasigari R, Riccieri V, Bassett R, Amuro H, Fukuhara S, Ito T, Liu YJ, Gilliet M. Sci Trans! Med. 201 1 Mar

9;3(73):73ral9. Pubmed ID PMID 21389263] and [REF: Netting neutrophils are major inducers of type I IF production in pediatric systemic lupus erythematosus. Garcia- Ror o GS1, Caielli S, Vega B, Connolly J, Allantaz F, Xu Z, Punaro M, Baisch J, Guiducci C. C off man RL, Barrat FJ, Banchereau J, Pascual V. Sci Transl Med. 201 1 Mar 9;3(73):73ra20. Pubmed ID PMID 21389264] This also appears to be the case in the autoimmune skin condition psoriasis, for which it was found that complexes between self-DNA (from the person's own damaged skin cells) and LL-37 induce autoimmune inflammation in the skin. [REF: Plasmaeytoid dendritic cells sense self-DNA. coupled with antimicrobial peptide. Lande R, Gregorio J, Facchinetti V, Chatter] ee B, Wang YH, Homey B, Cao W, Wang VI i Su B, Nestle FO, Zal T, Me!lman L Schroder JM, Liu YJ, Gilliet M, Nature. 2007 Oct 4;449(7162):564-9. Pubmed ID PMID 17873860]

[0012] As will be discussed in more detail below, LL-37 is also a critical player in the process of macroautophagy, i.e., the clearance of extracellular debris and inactivated or killed pathogens by macrophages, which is the body's way of clearing away extracellular traps, once their purpose has been served. [REF: Vitamin D3 induces autophagy in human monocytes / macrophages via cathelicidin. Yuk JM, Shin DM, Lee HM, Yang CS, Jin I IS Ki !< K. Lee ZW, Lee Si !. Kim JM, Jo I K Cell Host Microbe. 2009 Sep 17;6(3):231-43. Pubmed ID PMID: 19748465] and [REF: Antimicrobial peptide LL-37 promotes bacterial phagocytosis by human macrophages. Wan M, van der Does AM, Tang X, Lindbom L, Agerberth B, Haeggstrom JZ. J Leukoc Biol. 2014 Jun:95(6);971- 81. Pubmed ID PMID: 24550523] Thus, as discussed above, LL-37 plays at least three distinct roles in the control of pathogens by extracellular traps: (1) LL-37 directly crosslinks and stabilizes NETs; (2) LL-37 directly kills pathogens trapped in NETs; and (3) LL-37 stimulates autophagic clearance of NETs by macrophages. Thus, the expression of LL-37 is important for many aspects of human innate immune response to pathogens and to clearance of damaged host cell debris and NETs as well.

LL-37 is Antifungal.

[0013] In addition to being antibacterial, the LL-37 peptide is antifungal. A fungal species that is often responsible for acute or chronic infections in human patients, particularly in the human oral cavity and urogenital tract, is Candida Albicans . For instance, patients who have been treated with radiation or chemotherapy for cancer, or AIDs patients who are immunosuppressed, often suffer from painful fungal infections of the mouth. The LL-37 peptide has been shown to be antifungal, by preventing Candida Albicans ' adhesion to tissue and cell surfaces, via binding to the fungal carbohydrate mannan. [REF: Human antimicrobial peptide LL-37 inhibits adhesion of Candida albicans by interacting with yeast cell-wall carbohydrates. Tsai P L Yang CY, Chang HI, Lan CY. PLoS One. 201 1 Mar 14;6(3):el7755. Pubmed ID PMID 21448240] Moreover, the cathelicidin peptide LL-37 also directly inhibits fungal growth and kills Candida Albicans cells rapidly by inducing cell membrane permeabilization, causing vacuolar expansion. [REF: Fungicidal mechanisms of cathelicidins LL-37 and CATH-2 revealed by live-cell imaging. Ordonez SR, A arullah IH, Wubbolts RW, Veldhuizen EJ, Haagsraan HP. Antimicrob Agents Chemother. 2014;58( ^' 4):2240-8. Pubmed ID PMID 24492359] Mechanistic studies reveal that LL-37 is most effective at killing cutaneous, rather than subcutaneous fungal pathogens, emphasizing LL-37's essential barrier function, and the importance for human immunity to maintain a steady, normal concentration of LL-37 in the skin, in order to prevent infections. [REF: Anti-fungal activity of cathelicidins and their potential role in Candida albicans skin infection. Lopez- Garcia Bl, Lee PH, Yamasaki , Galio RL. J Invest Dermatol. 2005 Jul, 125(1): 108-15.

Pubmed ID PMID 15982310]

LL-37 is Antiviral.

[0014] The human cathelicidin peptide LL-37 is also a broad-spectrum antiviral host defense peptide. For instance, LL-37 has strong activity against the Herpesvirus HSV-1 and Adenovirus Adl9; this was demonstrated in the context of the epithelium of the eye, or ocular surface [REF: Human cathelicidin (LL-37), a multifunctional peptide, is expressed by ocular surface epithelia and has potent antibacterial and antiviral activity. Gordon YJ1, Huang LC. Roma owski EG. Yates Κ Λ. Proske RJ, McDermoti AM. Curr Eye Res. 2005 May,30(5):385-94. Pubmed ID PMID 16020269]

[0015] A painful skin condition known as atopic dermatitis eczema herpeticum (ADEH) can result from chronic infection and dissemination of the Herpesvirus HSV-2 in damaged skin of patients suffering from atopic dermatitis. It was shown that lower- than-normal endogenous levels of cathelicidin LL-37 predispose these patients to ADEH; and further, that LL-37 is able to inactivate the Herpesvirus HSV-2 directly. [REF:

Cathelicidin deficiency predisposes to eczema herpeticum. Howell MD, Wolienberg A, Gailo RL, laig M, Streih JE, Wong C, Pavicic T, Boguniewicz M, Leung DY. J Allergy Clin Immunol. 2006 Apr; 1 1 7(4):836-41. Pubmed ID PMID 16630942]

[0016] The essential role of antimicrobial peptides, including cathelicidin, as defense against skin infections by Vaccinia virus were also demonstrated in murine studies using transgenic mice deficient in cathelicidin peptide. [REF: Skin mast cells protect mice against vaccinia virus by triggering mast ceil receptor S1PR2 and releasing antimicrobial peptides. Wang Zl, Lai Y, Bernard JJ, Macleod DT, Cogen A ! .. Moss B, Di Nardo A. J Immunol . 2012 Jan l;188(!):345-57. Pubmed ID PMID 22140255] A virus that can cause severe respiratory infections, especially in infants and children but also in adults, is the Respiratory Syncytial Virus (RSV). It has been shown in both mice and humans that cathelicidins have important, direct anti-RSV protective functions in vivo. [KEF:

Cathelicidins Have Direct Antiviral Activity against Respiratory Syncytial Virus In Vitro and Protective Function in Vivo in Mice and Humans. Carrie SM, Gwyer Find! ay E,

McFariane Al Fiich PM, Bottcher B, Colegrave N, Paras A, Jozwik A, Chiu C,

Schwarze J, Davidson DJ. J Imraunol. 2016 Mar 1 5; 196(6).2699-710 Pubmed ID PMID 26873992]

[0017] Another widespread viral infection in the world's population, especially in Asia, is the Hepatitis C Virus (HCV), which affects liver tissue and can predispose those who are infected to liver cancer. The human cathelicidin LL-37 is active against HCV, and attenuates HCV infection. It has been shown that Vitamin D3 supplementation can improve the efficacy of Interferon Gamma therapy, a long-term (1-year) treatment for HCV infection of the liver; this was stated to be the result of better LL-37 expression induced by Vitamin D3 [REF: Antimicrobial peptide LL-37 attenuates infection of hepatitis C virus. Matsumura T, Sugiyama N, Mura ama A, Yamada N, Shiina M, Asa.be S, Wakita T, Imawari M5, Kaio T. Hepatol Res. 2016 Aug;46(9):924-32. Pubmed ID PMID 26606891].

[0018] Additionally, the human cathelicidin peptide LL-37 has been demonstrated to play an important role in host defense against influenza A viruses (IAV), exerting both direct antiviral effects, by disrupting IAV viral membranes, and also modulating host inflammatory responses to infection. [REF: The human cathelicidin LL-37 inhibits influenza A viruses through a mechanism distinct from that of surfactant protein D or defensins. Tripathi SI, Tecle T, Verma A. Crouch E, White M, Hartshorn KL. J Gen Virol. 2013 Jan;94(Pt l ).4()-9. Pubmed PMID 23052388] and [REF: LL-37 modulates human neutrophil responses to influenza. A virus. Tripathi S, Verma A, Kim EJ, White MR, Hartshorn KL. J Leukoc Biol. 2014 Nov;96(5):931-8. Pubmed PMID: 25082153]. LL-37 is able to directly inhibit the replication of the HIV-1 virus, and thus is important in preventing the progression of HIV infection to AIDS. [REF: The antimicrobial peptide LL-37 inhibits HIV-1 replication, Bergman P, Waiter-Jallow L, Broliden , Agerberth B, Soderlund J. Curr HIV Res. 2007 Jul;5(4):410-5. ΡΜΪΪ): 17627504].

[0019] In examining the progress of latent HIV-1 infection and looking at CD4 levels in T cells (essential for health maintenance of HIV-infected individuals), and looking at a group that had been treated with antiretroviral drugs (ARTs) vs. individuals who had not, researchers found that LL-37 levels were positively correlated with serum Vitamin D3 levels in controls, but not in HIV-infected groups. LL-37 levels were also positively correlated with current CD4 and DCD4 (current-nadir) in a previously ART-treated group. After adjusting for age, race, sex, and HIV infection duration, the association between LL-37 and CD4 levels remained significant. Thus, LL-37 levels were found to be significantly correlated with immune system strength in HIV-infected persons. [KEF: LL-37 concentrations and the relationship to vitamin D, immune status, and inflammation in HIV-infected children and young adults. Tangpricha V„ Judd SE, Ziegler TR, Hao L, Alvarez J A, Fitzpatrick AM, McComsey GA. Eckard AR. AIDS Res Hum Retroviruses. 2014 Jul;30(7):670-6. PMID: 2479823 1 ]

[0020] Remarkably, the known natural resistance of certain individuals to HIV infection appears to be related to their higher-than-usual endogenous, natural levels of expression of LL-37. [REF: Antiviral molecules correlate with vitamin D pathway genes and are associated with natural resistance to HIV-l infection. Aguilar-Jimenez W, Zapata W, Ruge!es T. Microbes Infect. 2016 Jul-Aug; l 8(7-8):510-6. Puhmed ID PMID 27083474] Thus, HIV-infected individuals would likely benefit from maintaining the highest possible levels of the cathelicidin LL-37 in their bloodstream.

[0021] Many review articles have been written about the family of cathelicidin proteins, which discuss and consider their many different functions and physiological effects. [REF: Cathelicidins - a fam ly of multifunctional antimicrobial peptides. Bais R, Wilson JM. Cell Mol Life Sci. 2003 Apr;60(4):7 1-20. Puhmed ED PMID 12785718], Signaling its high significance to human health, a number of reviews have focused solely on the human cathelicidin protein, LL-37, in particular. [REF: Pulmonary defense and the human cathelicidm hCAP-18/LL~37. Fahy RJL Wewers MD. Immunol Res.

2005:31(2):75-89. Pubmed ID P ID 15778507] and REF; LL-37, the only human member of the cathe!icidin family of antimicrobial peptides. Diirr UH1 , Sudheendra US, Ramamoorthy A Biochim Biophys Acta. 2006 Sep; ] 758(9): 1408-25 Pubmed ID PMID 16716248] and [REF: The chemistry and biology of LL-37. Burton MF, Steel PG. Nat Prod Rep. 2009 Dec;26(.l 2): 1572-84. Review. Pubmed ID PMID: 19936387] and [REF: Catheiicidin LL-37: a muititask antimicrobial peptide. Bucki R, Leszczynska K, amiot A, Sokoiowski W. Arch Immunol Ther Exp (Warsz). 2010 Feb;58(l): 15-25. Pubmed ID PMID 20049649] and [REF: A comprehensive summary of LL-37, the factotum human catheiicidin peptide. Vandamme Dl , Landuyt B, Luyten W„ Schoofs L. Cell Immunol. 2012 Nov;280(1 ):22-35. Pubmed ID PMID 23246832] and [REF: Unique features of human catheiicidin LL-37. Bandurska K, Berdowska A, Barczynska-Felusiak R, rupa P. Biofactors. 2015 Sep-Oct;41 (5):289-300. Pubmed ID PMID] These numerous review articles have been written and published because the LL-37 peptide is both important and unique in the human proteome and immune system. At this time, LL-37 is continually being discovered to play additional, complex roles in human health and disease. This makes the proper expression and regulation of the human gene that encodes LL-37, CAMP, essential to human health.

LL-37 Is An Essential Element of Cellular Autophagy, An Important Physiological Process

[0022] Besides being directly antibacterial, the peptide LL-37 is an essential participant in innate immune process known as autophagy, which includes the engulfment of pathogens such as bacteria or viruses by white blood cells such as macrophages, neutrophils, and natural killer (NK) cells. It was shown in 2009 that autophagy in human monocytes and macrophages is enhanced by the presence of Vitamin D3, specifically because Vitamin D3 enhances the expression of the catheiicidin LL-37, which was identified as being directly involved in autophagy [REF: Vitamin D3 induces autophagy in human monocytes / macrophages via cathelicidin. Yuk JM, Shin DM, Lee B ^' M, Yang CS, Tin HS, Kim KK, Lee ZW, Lee Sit Kim JM, Jo EK. ( oi l Host Microbe. 2009 Sep 17;6(3):231-43. Pubmed ID PM1D: 19748465] and [REF: Antimicrobial peptide LL-37 promotes bacterial phagocytosis by human macrophages. Wan M, van der Does AM, Tang X, Lindbom L, Agerberth B, Haeggstrom JZ. J Leukoc Bioi. 2014 Jun;95(6):971 -81 Pubmed ID PMID: 24550523].

[0023] This stimulation of autophagy was shown specifically in the case of mycobacteria, which cause tuberculosis. [REF: Phenylbutyrate induces LL-. 7-dependent autophagy and intracellular killing of Mycobacterium tuberculosis in human

macrophages. Rekha RS, Rao Muwa SS, Wan M, Raqib R, Bergman P, Brighenti S, Gudmundsson GH, Agerberth B. Autophagy. 20 I5; 11(9): 1688-99. Pubmed ID PM1D: 26218841 ] Thus, the clearance of infectious pathogens by white blood cells is enhanced by the upregulation of the cathelicidin LL-37. Additionally, other diseases that would benefit from enhancement of autophagic processes might be treated by upregulation of cathelicidin.

[0024] As an example, the natural process of autophagy appears to be dysfunctional in neurodegenerative diseases. [REF: Autophagy gone awry in neurodegenerative diseases. Wong E, Cuervo AM. at eurosci. 2010 Ju! ; 13(7):805~l l . Pubmed ID PM1I) 20581817] Examination of brain tissue from patients who died from Alzheimer's Disease showed the extensive involvement of disorders of autophagy in this chronic

neurodegenerative condition. [REF: Extensive involvement of autophagy in Alzheimer disease: an immuno-el eetron microscopy study. Nixon RA, Wegiel J, Kumar A, Yu WH, Peterhoff C, Cataldo A, Cuervo AM. J europathol Exp Neurol. 2005 Feb;64(2): l 13-22.] Another example is in Chronic Granulomatous Disease (CGD); a deficit in autophagy appears to play a key role in the pathogenesis of granulomatous colitis in CGD [REF: Deficient autophagy unravels the ROS paradox in chronic granulomatous disease, van de Veerdonk F, Dinarel!o CA. Autophagy. 2014 Jun; 10(6): 1 141-2. Pubmed ID PMID 24879159] As another example, the clearance of chronic Herpesvirus infections is dependent upon macro-autophagic clearance processes; and there is evidence that Herpesviruses often may evade clearance by modulating autophagy, as a way to escape elimination by innate and adaptive immune responses. [REF: Autophagy in herpesvirus immune control and immune escape. Taylor GS, Mautner J, Munz C. Herpesviridae.

201 1 Jan 5;2(1):2. Pubmed ID PMID 21429245],

[0025] Specifically in the case of viral infections of neuronal tissue, autophagy is a critical mechanism of neuronal self-defense against viruses, since neuronal cells are long- lived in the body, according to their function in memory for instance, and cannot undergo apoptosis without harming neural functions. [REF: Autophagy and viral neurovirulence.

Orvedahl A, Levi e B. Cell Microbiol, 2008 Sep; 10(9): 1747-56. Pubmed ID PMID 1 8503639] Thus, the treatment of chronic viral infections by pathogens such as

Herpesvirus should be enhanced by LL-37 upregulation.

[0026] Remarkably, the process of autophagy has been shown to be essential for lifespan extension in model studies in animals, where various compounds were studied for that purpose. [REF: Autophagy mediates pharmacological lifespan extension by spermidine and resveratrol . orseili EL Gal kizzi L, Kepp O, Criollo A, aiuri MC, Tavemarakis N, Madeo F, Kroemer G. Aging (Albany NY). 2009 Dec 23;l(12):961-70. Pubmed ID PMID 201.57579]. In general, the process of autophagy is critical for healthy tissue homeostasis, especially in the case of maintaining neuronal tissue health, which can include both brain tissue and the peripheral neurons important for the senses of sight, smell, taste, and touch. [REF: Autophagy for tissue homeostasis and neuroprotection. Marino G, Madeo F, Kroemer G. Curr Opin Cell Biol. 2011 A.pr;23(2): .198-206. Pubmed ID PMID 21030235], This presents the possibility that enhancement of autophagic processes, by increasing LL-37 expression above a baseline or uninduced level, would have the potential of enhancing a person's longevity and increasing the ability of peripheral neurons to provide a keen sense of sight, smell, taste, and touch into old age. LL-37 Increases Mitochondrial Biogenesis and Maintains Mitochondrial

Homeostasis.

[0027] Relevant to the topic of longevity, LL-37 expression has been shown to protect the health of mitochondria, the energy production organelles of cells. In a HaCaT cell culture system, the presence of LL-37 was demonstrated to dramatically reduce the mRNA levels and protein secretion of inflammatory cytokines including IL-6, IL-8, IL- la and tumor necrosis factor-a (TNF-a), which are induced by lipopolysaccharides (LPS). The anti-inflammatory effects of LL-37 resulted from its demonstrated ability to increase mitochondrial biogenesis and to maintain mitochondrial homeostasis. [REF: 1.1.-· 37 attenuates inflammatory impairment via mTOR signaling-dependent mitochondrial protection. Sun W, Zheng Y, Lu Z, Wang E, Feng Z, Wang J, Xiao S, Liu F, Liu J. int J Biochem Cell Biol. 2014 Sep;54:26-35. Pubmed ID PMID: 24984264]

LL-37 Affects Stem Cells and Stimulates Wound Healing.

[0028] LL-37 is a pleiotropic peptide, i.e., it has a wide range of different biological functions and physiological effects ("pleiotropy" occurs when one gene influences two or more seemingly unrelated phenotypic traits). As early as 2004, it was understood that within the extracellular matrix (i.e., the intracellular space of human tissue), LL-37 is chemotactic (i.e., that LL-37 can induce cell migration) for neutrophils, monocytes, mast cells, and T cells. Additionally, LL-37 has been shown to induce the degranulation of mast cells; to alter transcriptional responses in macrophages; and finally, to stimulate wound vascularization and the re-epithelialization of healing skin [REF- Cathelicidins, multifunctional peptides of the innate immunity. Zanetti M J Leukoc Biol. 2004

Jan;75(l):39-48. Pubmed ID PMID 12960280]

[0029] In an angiogenesis model, experiments showed that LL-37 can sensitize stem cells to become migratory at lower chemokine concentrations, enhancing

neovascularization by embryonic stem cells. [REF: NF kappaB activation in embryonic endothelial progenitor cells enhances neovascularization via PSGL-1 mediated recruitment: novel role for LL-37. Pfosser A, El-Aouni C, Pfisterer I, Dietz M, Giobisch F, Stacbel G, Trenkwalder T, Pinkenburg O, Horstkotte J, Hi kel R, Sperandio , Haizopoulos A , Boekstegers P, Bals R, Kupatt C. Stem Cells, 2010 Feb;28(2):376~85. Pubmed ID PMID: 20014279] In a chronic ischemic hind-limb animal model, delivery of the CAMP gene encoding using an adenovirus vector was shown to result in enhanced wound neovascularization, when LL-37 was expressed. [R EF: Recombinant adeno-associated virus-based gene transfer of catheiicidin induces therapeutic neovascularization preferentially via potent collateral growth. Pinkenburg O, Pfosser A, Hinkei R, Bottcher M, Dinges C, Lebherz C, Sultana S, Enssie J, El-Aouni C, Buning H, Boekstegers P, Bals R, Kupatt C. Hum Gene Then 2009 Feb;2G(2): 159-67. Pubmed ID PMID: 20377367].

[0030] Another study showed that LL-37 enhances angiogenesis by stimulating

PGE2-EP3 signaling in endothelial cells. [Catheiicidin LL-37 induces angiogenesis via PGE2-EP3 signaling in endothelial cells, in vivo inhibition by aspirin. Salvado MD, Di Gennaro A, Lindbora L, Agerberth B, Haeggstrom JZ. Arteiioscler Thromb Vase Biol. 2013 Aug;33(8): 1965-72. Pubmed ID PMID: 23766266] Additionally, it was shown that CAMP gene expression to produce the LL-37 peptide is essential in cellular

differentiation of cord blood-derived CD34+ cells into early endothelial progenitor cells, which in turn are essential for the healing and regeneration of blood vessels. [REF:

Successful in vitro expansion and differentiation of cord blood derived CD34+ cells into early endothelial progenitor cells reveals highly differential gene expression. Ahrens I , Domeij lii Topcic D, Havrv I, Merivirta RM, Agrotis A, Leifcner E, Jowett .IB. Bode C, Lappas M, Peter K. PLoS One. 201 l;6(8):e23210. Pubmed ID PMID 21858032] Thus, enhanced expression of LL-37, up to appropriate and normal levels, in a person suffering from wounds or who has experienced physical trauma to their tissues, may enhance the rate and improve the quality of wound healing and neovascularization. LL-37 Is Important in Lung Health.

[0031] LL-37 is produced not only by white blood cells and granulocytes, but also by the skin and all epithelial cells types, for instance, epithelial cells that line the internal surfaces of the lungs. LL-37 is detected in bronchoalveolar lavage fluid. In epithelial cells, LL-37 is produced in response to inflammation. In the lung, the antibacterial peptide LL-37 is produced by alveolar macrophages, bronchial epithelial cells, and bronchial glands, suggesting that it has key defensive roles in airway mucosa. [REF: Antibacterial components in bronchoalveolar lavage fluid from healthy individuals and sarcoidosis patients. Agerberth B, Grunewald J, CastaSos-Velez E, Oisson B, Jornvall H, Wigzell H, Eklund A, Gudmundsson GH. Am J Respir Crit Care Med. 1999

;160( l):283-90. Pubmed ID PMID: 10390413] Thus, the enhanced expression of LL- 37 in patients who suffer from lung injury, insults to lung tissue including aspiration or inhalation of toxins, or lung infections involving inhalation of pathogens, could improve health outcomes for these patients.

[0032] Another dangerous bacterial lung pathogen is Pseudomonas Aeruginosa. LL- 37 has been shown to play important roles in lung defense against this pathogen, by increasing lung epithelial cell stiffness so that bacteria cannot cross through lung tissue. [REF: Caihe!icidin LL-37 increases lung epithelial cell stiffness, decreases transepitheliai permeability, and prevents epithelial invasion by Pseudomonas aeruginosa. Byfield FJ, owa!ski M, Cruz K, Leszezynska , Naraiot A, Savage PB, Buck! R. Janmey PA. J Immunol. 201 1 Dec 15: 187(12):6402-9. Pubmed ID PMID 22095714] Here, it should be noted that enhancing tissue stiffness is another important aspect of LL-37' s host defense. Similar protective effects are seen in blood vessel endothelial cells, so that LL-37 levels directly affect the permeability of endothelial cell barriers to pathogen invasion. [REF: Catheiicidin LL-37 peptide regulates endothelial ceil stiffness and endothelial barrier permeability. Byfield FJ, Wen Q, Leszezynska , Kulakowska A, Namiot Z, Janmey PA, Bucki R. Am J Physiol Cell Physiol. 201 1 Jan;300(l ):C105-12. Pubmed ID PMID:

20943960], LL-37 Works with Vitamin D3 To Form and Heal Bone.

[0033] LL-37 is also important in the nucleation of new bone, and may be important for the regeneration of bone tissue, as important for the prevention of osteoporosis. Bone cell types include osteoblasts, osteoclasts, and osteocytes, each of which originate from different precursor cells, and are stimulated by various growth factors to differentiate into new bone. The formation of new bone tissue is necessary, for instance, in the healing of bone fractures. Sites of bone injury will naturally recruit a white blood cell type called a monocyte. It was shown that LL-37 (which is produced from its precursor protein, hCAP- 18) can cause monocytes to differentiate into large, adherent cells, which form nascent bone-like structures both in vitro and in vivo. [REF: Generation of novel bone forming cells (monoosteophils) from the cathelici din-derived peptide LL-37 treated monocytes. Zhang Z l , Shive!y JE. PLoS One. 2010 Nov 15,5(1 1 ):el3985. Pubmed ID PMID

21085494] Thus, patients who have bone fractures that need to heal, or patients who suffer from osteoporosis, would benefit from enhanced expression of the CAMP gene that encodes LL-37.

LL-37 Can Protect Against Sexually Transmitted Diseases.

[0034] The precursor protein to LL-37, hCAP-18, has been identified as being present in human seminal fluid, and appears to play an important role in natural host defense during sexual activity. [REF: Isolation of human cationic antimicrobial protein- 18 from seminal plasma and its association with prostasomes. Andersson El . Soxensen OE, Frohm B, Borregaard N, Egesien A, Malm J. Hum Reprod. 2002 Oct; 17(10):2529- 34 Pubmed ID PMID 12351523]. People with higher endogenous levels of LL-37 might be less likely to become infected by sexually transmitted diseases. HIV has already been discussed above, but another sexually transmitted pathogen is Chlamydia trachomatis, an obligate intracellular bacterial pathogen. The human cathelicidin LL-37 has demonstrated activity against Chlamydia trachomatis, a pathogen that typically infects the lower genital tract first, but then if untreated, can also cause damage to ocular surfaces and even cause blindness. Additionally, LL-37 was shown to be significantly induced in the genital tracts of women who were diagnosed positive for C. trachomatis, indicating its importance in natural host defense. [REF: Chlamydial plasmid-encoded virulence factor Pgp3 interacts with human cathelicidin peptide LL-37 to modulate immune response. Hou S. Sun X, Dong X, Lin H, Tang I., Xue M, Zhong G Microbes Infect. 2018 Jun 26. pi; . S I 286- 4579(18)30142-4. Pubmed ID PMID 29959096] Thus, persons who are sexually active may benefit from maintaining normal to high levels of CAMP gene expression, to increase natural resistance to C. trachomatis infection.

[0035] Additionally, for females, sexual activity can often result in chronic urinary tract infections. This can also occur when urinary catheterization is necessary, for medical reasons such as paralysis. Studies have been done demonstrating LL-37' s natural ability to prevent infections, for example in the urinary tract epithelium and the urinary bladder. [REF: The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection. Chromek M, Siamova Z, Bergman P, Kovacs L, Podracka L, Ehren L Hokfelt T, Gudraundsson GH, Gallo RL, Agerbe/th B, Brauner A. Nat Med. 2006 Jim; 12(6): 636-41. Pubmed ID PMID 16751768] and [REF: Vitamin D induction of the human antimicrobial Peptide cathelicidin in the urinary bladder. Hettting O, Holm A, Luthje P, Brauner FI Dyrdak R, Jonasson AF, Wiklund P, Chromek M, Brauner A. PL.oS One. 2010 Dec 14;5(12):el5580. Pubmed ID PMID 21 179490] Maintaining healthy levels of LL-37 expression may reduce the incidence of these infections.

[0036] Newborn infants, at birth, are covered in a waxy substance called vernix caseosa, which has potent antimicrobial properties by virtue of its expression of antimicrobial peptides including LL-37. [REF: The newborn infant is protected by an innate antimicrobial barrier: peptide antibiotics are present in the skin and vernix caseosa. Marchim G, Lindow S, Brismar H, Stabi B, Berggren V, Ulfgren AK, Lonne-Rahm S, Agerberth B, Gudmundsson GET Br J Dermatol. 2002 Dec; 147(6): 1127-34. Pubmed ID PMID: 12452861] Thus, LL-37 is an important human host defense peptide, from the earliest stages of life.

LL-37 Protects Against Stomach Ulcers and Gastric Cancer.

[0037] The strain of bacteria that can cause infection of the human stomach lining, Helicobacter Pylori or H. Pylori, is also susceptible to killing by the cathelicidin peptide LL-37 ^' . It was shown that LL-37, either alone or in synergy with another antimicrobial peptide, human beta-defensin 1, was bactericidal for several H. pylori strains. [REF: Expression of LL-37 by human gastric epithelial cells as a potential host, defense mechanism against Helicobacter pylori. Hase , Murakami M, iimura M, Cole SP, Horibe Y, Ohtake T, Obonyo M, Gai!o RL, Eckmann L, agnoff MF. Gastroenterol og . 2003 Dec; 125(6): 1613-25, Pubmed ID PMID 14724813] Chronic H. Pylori infections of the stomach lining can cause ulcers, and also increase susceptibility to stomach cancer. Hence, patients who suffer from H. Pylori infection of the stomach lining would likely benefit from an upregulation of the CAMP gene expression of LL-37. Indeed, there is direct evidence that expression suppresses the growth of gastric cancer cells.

[REF: The host defense peptide LL-37 activates the tumor-suppressing bone

morphogenetic protein signaling via inhibition of proteasome in gastric cancer cells. Wu WK, Sung J J, To KF, Yu L, Li HT, Li ZI Chu KM, Yu J, Cho CH. J Cell Physiol. 2010 Apr;223(l ): 178-86. Pubmed II) ΡΜΠ) 20054823]

Complex Associations of LL-37 with Atherosclerosis and Cardiovascular Disease.

[0038] In 1998, it was discovered that the human LL-37 peptide binds specifically to the High Density Lipoprotein (HDL)-associated protein Apolipoprotein A-l (ApoAl); and specifically, that this binding occurs in the context of lipid- and cholesterol -bearing HDL particles, i.e., LL-37 does not bind to ApoAl in the absence of lipids and cholesterol [REF ^' : Apolipoprotein Α-Ϊ binds and inhi its the human

antibacterial/cytotoxic peptide LL-37. Wang Y, Agerberth B, Lothgren A, Almstedt A, Johansson J. J Biol Chem. 1998 Dec 1 1 ;273(50):331 15-8. Pubmed ID PMID: 9837875] and [REF: The antimicrobial peptide LL-37 binds to the human plasma protein apoiipoprotein A-I. Wang Y„ Johansson J, Agerberth B, Jdrnvall H, Griffiths WJ. Rapid Cornmun Mass Spectrom. 2004; 18(5):588-9. Pubmed ID PMID- 14978805] and [REF: Apoptosis of airway epithelial cells: human serum sensitive induction by the cathelicidin LL-37. Lau YE, Bowdish DM, Cosseau C, Hancock RE, Davidson DJ. Am J Respir Ceil Mol Biol. 2006 Apr;34(4):399-409. Pubmed ID PMID 16340000],

[0039] It is significant that LL-37, in the human blood circulation, is found bound to HDL particles, not free in the plasma, because LL-37 peptide, at concentrations above 13 pg/mL, can be cytotoxic to endothelial cells lining blood vessels. However, the normal concentration of LL-37 in human blood (virtually all of which is bound to HDL particles) is 1.15 μ /Γηί, 11 -fold lower than the cytotoxic concentration. [REF: Apoiipoprotein Α-Ϊ attenuates LL-37-induced endothelial cell cytotoxicity. Svensson D, Lagersiedt: JO, Niisson BO, Del Giudice R. Biochem Biophys Res Commun. 2017 Nov 4;493(l):71 -76. Pubmed ID PMID. 28919413] While its mechanism of involvement is unclear, and LL- 37 expression in the cardiovascular system has not been shown to be harmful in human patients, there does appear to be a correlation between cardiovascular disease risk, and CAMP gene expression levels (as measured by mRNA) in blood. [REF ^' : Associat on of human cathelicidin (hC AP- 18/LL-37) gene expression with cardiovascular disease risk factors. Benachour H, Zaiou M, Samara A, Herheth B, Pfisier M, Lambert D, Siest G, Visvikis-Siest S, Nuir Metab Cardiovasc Dis, 2009 Dec; 19{ Ί0):720-8. Pubmed ID PMID

19346112]

[0040] Additionally, it has been shown that LL-37 peptide is found in atherosclerotic lesions and plaques. At very high (quite unusually high) concentrations of 30 μg/mL, it can be cytotoxic to endothelial cells. [REF: Human antimicrobial peptide LL-37 is present in atherosclerotic plaques and induces death of vascular smooth muscle cells: a laboratory study. Ciornei CD, Tapper H, Bja/tell A, Sternby H, Bodeisson M. BMC Cardiovasc Disord. 2006 Dec 20;6:49, Pubmed ID PMID 17181861] Another, related study also identified the presence of LL-37 in human atherosclerotic lesions, at 6-fold higher concentration than the normal concentration in the blood, and concluded instead that LL-37 acts as a protective immune modulator within atherosclerotic plaques. [REF: Involvement of the antimicrobial peptide LL-37 in human atherosclerosis. Edfe!dt K, Agerberth B, Rottenberg ME, Gudmundsson GH, Wang XB, Mandal K, Xu Q, Yan ZQ. Arterioscler Thromb Vase Biol. 2006 Jul;26(7);1551-7. Pubraed ID PMID 16645154]

[0041] It has further been shown that LL-37 peptide is actively produced within atherosclerotic lesions, being expressed mainly by macrophages within the plaque, but also by the endothelial cells of the blood vessel lining. [REF . Involvement of the antimicrobial peptide LL-37 in human atherosclerosis. Edfeldt K, Agerberth B,

Rottenberg ME, Gudmundsson GH, Wang XB, Mandal K, Xu Q, Yan ZQ. Arterioscler Thromb Vase Biol. 2006 Jul;26(7): l 551-7. Epub 2006 Apr 27. Pubmed ID PMID 16645154] On the other hand, studies of the effects of neutrophil-derived LL-37 in the context of injured arteries showed benefit, finding that LL-37 promoted healthy re- endothelialization (healing of blood vessels), preventing the formation of neointimal hyperplasia (aberrant, scab-like tissues within blood vessels). [REF: Set Trans! Med. 2011 Oct 5;3(103): 103ra98. Neutrophil-derived cathelicidin protects from neointimal hyperplasia. Soehnlein O, Wantha S, Simsekyilmaz S. Doring Y, Megens RT, Mause SF. Drechsler M, Smeets R, Weinandy S, Schreiber F, Gries T, Jockenhoevel S, Moiler M, Vijayan S, van Zandvoort MA, Agerberth B, Pham CT, Gallo RL, Hackeng TM, Li elm EA, Zernecke A, lee D, Weber C. Pubmed ID PMID 21974936]

[0042] Taken all together, these data indicate that highly elevated levels of LL-37 expression in the cardiovascular system (greater than 10X normal concentrations) can have cytotoxic effects on the human circulatory system. However, it is also worth noting that, while the benefits of cathelicidin expression are clear in the context of preventing numerous types of infections of bacterial, fungal, or viral origin, it is not the case that highly elevated concentrations will be beneficial in every circumstance. Hence, monitoring of cathelicidin expression levels within the blood, to ensure that it is less than 6X the normal concentration, would be beneficial.

The Role of LL-37 in Cancer Is Complex.

[0043] The cathelicidin peptide has also been studied in the context of cancer.

Initially, it was shown that, if the mouse gene for cathelicidin {camp) was knocked out, creating a camp{-l-) mouse, that transgenic mouse was much less able to fight cancer in an orthotopic tumor cell injection model, specifically because the mouse' s Natural Killer (NK) cells did not express the cathelicidin peptide as an anticancer molecule. [REF: The host defense peptide cathelicidin is required for NK cell-media ted suppression of tumor growth. Buchau AS, Morizane S, Trowbridge J, Schauber J, Koto! P, Bui JIX Gal io RL. J Immunol. 2010 Jan 1 : 1 84(1 ): 369~78. Pubmed ID PMID 19949065] It has also been shown, in the context of B cell lymphoma (a blood cell cancer), that Vitamin D3-induced expression of cathelicidin results in beneficial cytotoxic activity of Ml inflammatory macrophages against high-grade B cell lymphoma. [REF: Vitamin D-dependent induction of cathelicidin in human macrophages results in cytotoxicity against high-grade B cel l lymphoma. Bruns H, Buttner M, Fabri M, Mougiakakos D, Bittenbring JT, Hoffmann MH, Beier F, Pasemann S, Jitschin R, Fiofmann AD, Neumann F, Daniel€, Maurberger A, Kempkes B, Amann K, Mackensen A, Gerbitz A. Sci Transl Med. 2015 Apr

8;7(282):282ra47. Pubmed ID PMID 25855493] The mechanism by which the cathelicidin peptide can exert anti-cancers on Jurkat T-cell lymphoma also has been studied, and LL-37 was shown to induce cancer cell death in a calpain- and apoptosis- inducing factor (AIF) dependent manner involving Bax activity. [REF: The human host defense peptide LL-37 induces apoptosis in a calpain- and apoptosis-inducing factor- dependent manner involving Bax activity. Moi Cancer Res. 2009 May;7(5):689-702. Mader J S. Mookheqee N, Hancock RE, Bieackley RC, Pubmed ID PMID 19435812]

[0044] Another study found that LL-37 can suppress cancer growth in gastric adenocarcinoma (stomach cancer) cells, by activating a tumor-suppressing gene BMP (bone morphogenic protein) signaling pathway. [REF: The host defense peptide LL-37 activates the tumor-suppressing bone morphogenetic protein signaling via inhibition of proteasome in gastric cancer cells. J Cell Physiol. 2010 Apr;223(l): 178-86. Wu W , Sung jj, To KF, Yu L, Li ί Π . Li XT Chu KM, Yu J, Cho€1:1] However, whereas LL-37 shows beneficial anticancer activities for lymphomas, in some tissue-based cancers involving solid tumors, the results of studies are mixed, and seem to indicate that LL-37 expression is upregulated in the tumor microenvironment and may promote metastasis, acting as a growth factor for epithelial cell-type cancers (which makes sense, given its natural involvement in wound healing and angiogenesis). [REF: Antimicrobial protein hCAP18/LL-37 is highly expressed in breast, cancer and is a putative growth factor for epithelial cells. Ffeilborn ID, Nilsson MF, Jimenez CI, Sandstedt B, Borregaard N, Tham E, S0rensen OE, Weber G, Stable . Int J Cancer. 2005 May l ;l 14(5}:713--9. Pubmed ID PMID 15609314]

[0045] What is unclear, is whether this is a consequence of the tumor, a byproduct of the tumor, or a pathogenic element of solid tumors. In the case of ovarian cancer, LL-37 is overexpressed and appears to play a negative role, increasing ovarian cancer cell proliferation and invasion. [REF: Ovarian cancers overexpress the antimicrobial protein hCAP-18 and its derivative LL-37 increases ovarian cancer cell proliferation and invasion. Coffelt SB, Waterman RS, Florez L, Honer zu Bentrup K, Zwezdaryk KJ, Torachuck SL. LaMarca HL, Danka ES, Morris CA, Scandurro AB. Int J Cancer. 2008 Mar 1 ; 122(5): 1030-9. Pubmed ID PMID 17960624] A negative role for LL-37 also has been found in the case of malignant melanoma. [REF: The antimicrobial peptide human cationic antimicrobial protein- 18/catheiicidin LL-37 as a putative growth factor for malignant melanoma. Kim JIT Kim HI, Choi IM, Lee KB, Kim TY, Cho BK, Jung JY, Chung KY, Clio D, Park 1 U . Br J Dermatol . 2010 Nov; 163i ^" 5):959-67. Pubmed ID PM1T ⁾ 20977442]

[0046] Based on this literature, speaking broadly, it is not yet clear whether LL-37 expression plays a positive, anticancer role or negative, pro-metastasis role in cancer. It appears to be beneficial to upregulate LL-37 expression in the context of blood cancers like B cell lymphoma and Jurkat T cell lymphoma. On the other hand, in cases of breast cancer, malignant melanoma, and ovarian cancer, increasing LL-37 concentrations may not be beneficial.

SUMMARY OF THE DISCLOSURE

[0047] In one aspect, a method is provided for upregulating cathelicidin gene expression in the brain of a subject. The method comprises forming a pharmaceutically acceptable composition which includes a mixture of materials selected from the group consisting of Vitamin D3, phenylbutyrate, bexarotene, DHA, curcumin, resveratrol, and pharmaceutically acceptable salts thereof; and administering the pharmaceutically acceptable composition to the subject.

[0048] In another aspect, a method is provided for treating a human subject for Alzheimer's disease. The method comprises diagnosing the human subject as suffering from Alzheimer's disease; and applying to the subject a pharmaceutically acceptable composition which upregulates cathelicidin gene expression in the brain of the subject.

[0049] In a further aspect, a method is provided for treating β-amyloid aggregation and accumulation in a subject. The method comprises detecting the presence of β- amyloid aggregate accumulation in tissues of the subject; and administering to the subject a pharmaceutically acceptable composition which upregulates cathelicidin gene expression in the tissues of the subject.

[0050] In yet another aspect, a method is provided for treating a subject. The method comprises monitoring levels of the cathelicidin peptide LL-37 and β-amyloid in tissues of a subject; and, when the condition L/B< k is detected, where L is the level of LL-37 detected, B is the level of β-amyloid detected, and & is a predetermined threshold value, upregulating cathelicidin gene expression in the tissues of the subject.

[0051] In still another aspect, a method is provided for modulating in vivo β-amyloid (Αβ) fibril formation. The method comprises monitoring the level of β-amyloid in tissues of a subject; and administering to the subject a pharmaceutically active composition which modulates in vivo fibril formation in said tissues by inducing the expression of a physiologically effective binding partner for β-amyloid.

[0052] In another aspect, a method is provided for modulating in vivo fibril formation. The method comprises co-incubating β-amyloid (Αβ) with a physiologically effective binding partner for β-amyloid, thereby obtaining co-incubated peptides; creating a pharmaceutical composition from the co-incubated polypeptides; and administering the pharmaceutical composition to a subject.

[0053] In a further aspect, a method for modulating microglia-mediated

neuroinflammation is provided. The method comprises monitoring the level of a cytokine in microglia tissues of a subject, wherein the cytokine is selected from the group consisting of TNFa and IL-6; and administering to the subject a pharmaceutically active composition which reduces the level of the cytokine in the microglia tissues of the subject, wherein the pharmaceutically active composition induces the expression of a physiologically effective binding partner for β-amyloid in the microglia tissues of the subject.

[0054] In another aspect, a method is provided for inhibiting in vivo β-amyloid (Αβ) fibril formation in the tissues of a subject in which an equilibrium exists between smaller and larger MW species of β-amyloid (Αβ). The method comprises administering to the subject a pharmaceutical composition which shifts the equilibrium toward the smaller species of Αβ.

[0055] In yet another aspect, a method is provided for modulating in vivo β-amyloid (Αβ) fibril formation. The method comprises monitoring the level of β-amyloid in tissues of a subject; determining that the monitored level of β-amyloid has exceeded a predetermined threshold; and reducing the level of β-amyloid in the tissues below the threshold amount by inducing LL-37 production in the tissues through the stimulation of glial cells with a stimulant selected from the group consisting of

lipopolysaccharide/interferon-gamma (LPS/IFNY) and IFNy. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] FIGs. 1-3 are a series of CE electropherograms. FIG. 1 is for Αβ ₄ ο (t = 0 days), FIG. 2 is for Αβ ₄ 2 (t = 0 days), and FIG. 3 is for Αβ ₄₂ (t = 24 days). In the CE traces, the designation · refers to low MW-oligomers, and the designation * refers to high

MW-oligomers.

[0057] FIG2. 4-9 depict the results of binding studies performed by SPRi. The SPRi- chip was functionalized with Αβ ₄ ο (t = 0 days) (FIGs. 4 and 7); Αβ ₄₂ (t = 0 days) (FIGs. 5 and 8), and Αβ ₄₂ (t = 24 days) (FIGs. 6 and 9). All the Αβ peptides were immobilized in replicate (n=8) on the same SPRi chip at the same concentration (20 μΜ). SPRi reference-corrected responses related to LL-37 (10 μΜ) (black) and ovalbumin (10 μΜ) (gray) (negative control) flowed on the SPRi-chip functionalized with different Αβ forms (FIGs. 4-6). The three SPRi sensograms show the injection of running buffer (baseline) (1), the injection of the analyte (association phase) (2) and the subsequent injection of buffer (dissociation phase) (3). Calibration curve of LL-37 flowed onto different Αβ forms immobilized on the SPRi-chip Αβ ₄₀ (t=0 days, FIG. 7), Αβ ₄₂ (t=0 days, FIG. 8), Αβ ₄₂ (t=24 days, FIG. 9). The equilibrium binding constants (KA and KD) values were calculated using a nonlinear curve fit of the SPRi response at equilibrium (see FIG. 9).

[0058] FIGs. 10-14 are Transmission Electron Microscopy (TEM) images for 50 μΜ Αβ ₄₂ at t=0 (FIG. 10); 100 μΜ LL-37 at t=10 days (FIG. 11); equimolar mixtures of 50 μΜ Αβ ₄₂ and LL-37 at t=0 (FIG. 12); t=3 days (FIG. 13); and t=9 days (FIG. 14). Scale bar: 200 nm, magnification 60,000x.

[0059] FIGs. 15-16 are circular dichroism spectra of peptide solutions, recorded at t = 0 min (FIG. 15) and t = 24 hours (FIG. 16). Blue line: 50 μΜ Αβ ₄₂ ; red line: 50 μΜ LL-37; green line: 1 : 1 mixture. [0060] FIG. 17 is a listing of amino acid sequences for human cathelicidin peptide LL-37 (so named because it comprises 37 amino acids) and the human amyloid-β peptide, Αβ ₄₂ .

[0061] FIGs. 18-20 are graphs depicting the effects of treatment with Αβ, LL-37, or their mixture on the viability changes of SH-SY5Y cells induced by microglial-mediated toxicity in 72 h (FIG. 18) and levels of cytokines, TNFa (FIG. 19) and IL-6 (FIG. 20) in microglial toxic supernatant. Values are mean±SEM, n=4. One-way ANOVA was carried out to test significance. Multiple group comparisons were followed by a post-hoc Bonferroni test where necessary. * P<0.01 for Αβ-treated cells and LL-37-exposed cells compared with control (CON) group and ** P<0.01 for Aβ-LL-37-treated groups compared with Αβ-treated cells and LL-37-exposed cells. Note that Αβ and LL-37 inhibit the microglial activation of each other.

[0062] FIG. 21 is a series of plasmonic curves acquired before the injection of LL-37 peptide. The shift between these curves is related to the amount of molecules

immobilized on the SPRi chip surface. In particular, the plasmonic curves related to both Αβ ₄ ο (t = 0 days) and Αβ ₄₂ (t = 24 days) show a significant shift from that related to Αβ ₄ ο solubilized in PBS (t = 0) and from that of the reference peptide (negative) and to the chip (no molecules adsorbed).

[0063] FIG. 22 is a CCD image of the chip surface. The size of spots measured directly by this type of images was around 190 μπι.

[0064] FIGs. 23-25 are reference-corrected responses related to seven dilutions of LL-37 injected on the SPRi-chip functionalized with (FIG. 23) Αβ40) solubilized in PBS; (FIG. 24) Αβ42 1 = 0 days and (FIG. 25) Αβ42 1 = 24 days, used to calculate the equilibrium binding constants (KD) in FIG. 4. All the responses reached the equilibrium (plateau) before the end of the injection (500 s)

[0065] FIG. 26 is a CCD differential image of the SPRi-biochip during the late association phase (500 s) of LL-37 injected at concentration 25 μΜ. "Ref ' is the reference peptide used as negative control on the chip surface. [0066] FIGs. 27-29 depict the results of an equilibrium-binding analysis of the control scrambled LL-37 peptide, performed by surface plasmon resonance imaging (SPRi) using the same procedures and conditions used to study LL-37 (compare with FIG. 16). The SPRi-chip was functionalized with Αβ40 (t = 0 days) (FIG. 27); Αβ42 (t = 0 days) (FIG. 28) and Αβ42 (t = 24 days) (FIG. 29). The equilibrium binding constants and the fitting curves could not be calculated, due to the irregular and negative dose- response trend.

[0067] FIG. 30 is a graph depicting SH-SY5Y, Microglia and SH-SY5Y/Microglia viability changes induced by Αβ peptide and LL-37 peptide (30 μΜ each). Reductions in numbers of live cells are indicated by the MTT assay. Values are mean±SEM, n=4. Oneway ANOVA was carried out to test significance. Multiple group comparisons were followed by a post-hoc Bonferroni test where necessary. * P<0.01 for cells in 72 h compared with the ones in 0 h and ** P<0.01 for SH-SY5Y plus microglia without Αβ or LL-37 peptides group compared with SH-SY5Y cells or microglial cells in the same incubation time. Note that the cell viability of SH-SY5Y and microglia co-culture is nearly equal to the sum of two cell culture group in 72 h, and that treatment with either Αβ or LL-37 alone did not change viability of either cell type (alone) over the course of 72 h.

[0068] FIG. 31 is a graph depicting the results of induction of the camp (cathelicidin) gene in the brain of wild type c57bl/6j mice treated for 2 weeks with oral polytherapy. Mice also had access to a running wheel.

[0069] FIGs. 32-34 are a series of graphs depicting the effects of treatment on 5XFAD model mice vs. untreated 5XFAD mice and wild type C57BL/6J mice, and vs. treated C57BL/6J mice. FIG. 32 depicts the results for contextual memory total (5 min), FIG. 33 depicts the results for contextual memory total excluding the first minute, and FIG. 34 depicts contextual memory at each minute of the trial. In Contextual Fear Conditioning testing (72 hours), which tests memory and recall, treated 5XFAD mice show an improved memory of contextual fear, as compared to untreated 5XFAD mice. Treated 5XFAD mice show memory comparable to wild type mice.

[0070] FIGs. 35-36 depict the chemical structures of orally delivered cathelicidin gene inducers.

DETAILED DESCRIPTION

[0071] It will be appreciated from the literature review and discussion presented in the Background Section that the protein product of the CAMP gene, the innate immune effector LL-37, has been studied extensively in the context of infectious disease. LL-37 has also been studied, to a lesser degree, in the context of wound healing, angiogenesis and stem cells, and cancer. However, it has now been discovered that a direct relation exists between LL-37 and Alzheimer's Disease. See [REF: Evidence that the Human Innate Immune Peptide LL-37 may be a Binding Partner of Amyioid-β and Inhibitor of Fibril Assembly. De Lorenzi. E, Chiaii M, Colombo R, Cretich M, Sola L, Vanna R, Gagm P, Bisceg!Ia F, Morasso C, Lin JS, Lee M, McGeer PL, Barron AE. J Alzheimers Dis. 2017;59(4): 1213-1226. Pubmed ID PMID 28731438], which is incorporated by reference in its entirety. This surprising discovery has not been appreciated in the relevant art, and led to the further discovery by the present inventors that Alzheimer's Disease may be treated by inducing expression of the CAMP gene encoding the LL-37 peptide. This discovery is a key technical foundation of the methodologies disclosed herein.

Cathelicidin LL-37 in the Context of Alzheimer's Disease.

[0072] Alzheimer's Disease is a terminal diagnosis suffered by over 42 million patients globally, with no effective therapy or treatment. The handful of drugs currently prescribed to Alzheimer's patients at best ameliorate symptoms ('disease-modifying' as opposed to 'disease-curative' treatments). Most 'symptomatic therapies' currently available are neurotransmitter-focused, and fall into two classes: cholinesterase inhibitors, and the MDA (glutamatergic) receptor agonist memantine. There are no FDA-approved drugs that address the underlying causes of Alzheimer's disease, which remain poorly understood despite 110 years of research. [REF: Alzheimer's Disease: Lessons Learned from Amyloidocentr c Clinical Trials. Soejitno A, Tjan A, Purwata TE. CNS Drugs. 201 5 Jun;29(6):487-502. PMID: 26187557] Alzheimer's disease risk is known to correlate with the risks of several other chronic diseases, in particular metabolic syndrome / type 2 diabetes and cardiovascular disease. However, these connections are understudied, given the complexity of studying such associations. In practice, pharmaceutical drug development is typically undertaken for only one chronic disease target at a time, although some FDA-approved drugs are repurposed for other conditions.

[0073] As of 2018, it has been 14 years since a new drug for Alzheimer's disease was approved by FDA. The majority of the more than 400+ failed clinical trials pursued strategies to reduce the distinct, observable pathophysiological protein signatures of Alzheimer's disease. These protein signatures include Αβ amyloid plaque accumulation in the brain, which is primarily extracellular (outside of neurons), and (much less commonly) phosphorylated tau protein aggregates, which form 'neurofibrillary tangles' within neuronal cells. Clinical trials aimed at reducing the burden of Αβ and tau aggregate protein deposits have so far failed to improve patient cognition and function, as was required by the FDA for drug approval (until recently, when FDA relaxed these criteria somewhat) [REF: Prevention of sporadic Alzheimer's disease: lessons learned from clinical trials and future directions. Andrieu S. Coley N, Lovestone S, Aisen PS, Vellas B Lancet Neurol . 201 5 Sep;14(9):926-944. PMID: 26213339].

[0074] Within the aging human brain, Alzheimer's disease (AD) involves the assembly of β-amyloid (Αβ) peptides from soluble monomers into oligomers, fibrils and plaques [REF: Alzheimer mechanisms and therapeutic strategies. Huang Y, Mucke L. Cell . 2012 Mar 16; 148(6): 1204-22. Review, Pubmed ID PMID: 22424230]. Studies of the spatiotemporal interplay between diffusible Αβ oligomers and fibrillar deposits, as well as intracellular tau tangles, have been aimed at investigating drivers of neuronal dysfunction, which are still not well understood [REF: Alzheimer's and Parkinson's diseases: The prion concept in relation to assembled Ap, tau, arid a-symtclein. Goedert M. Science, 2015 Aug 7;349(6248): 1255555. Pubmed PMID 26250687] and [REF:

Alzheimer's Disease: Lessons Learned from Amyloidoce tric Clinical. Trials. Soejit.no A, Tjan A, Purwata TE. CNS Drugs. 2015 Jun;29(6):487-502. doi : 10. ^' i 007/s40263-015- 0257-8. Pubmed ID PMID 26187557] The root physiological causes of sporadic (i.e., spontaneously arising) Alzheimer's Disease— i.e., instances of disease in patients for whom there is no known genetic predisposition, which is the most common type of AD— remain unspecified, preventing the development of effective therapies to prevent, halt, or reverse the disease [REF: Prevention of sporadic Alzheimer's disease: lessons learned from clinical trials and future directions. Andneu S, Coley N, Lovestone S, Aisen PS, Vellas B. Lancet Neurol. 201 5 Sep; 14(9):926-944. Pubmed ID PMID 26213339].

[0075] Identification of physiologically relevant binding partners of Αβ that modulate fibril formation in vivo may yield new insights into causes of AD and help identify early instigators of Αβ accumulation and neurotoxic effects. A number of natural proteins that can bind to, or interact with, Αβ have been identified by various assays [REF: Molecules that target beta-amyloid. Stains CI, Mondai K, Ghosh I. ChemMedChem. 2007

Dec;2( 12).T 674-92. Review. Pubmed ID PMID: 1 7952881 ]. However, their roles in sporadic AD are often challenging to assess.

[0076] Literature reports exist which might arguably be interpreted to suggest, in an indirect, "connect-the-dots" fashion, that the biophysical activities and signaling functions of Αβ peptides and LL-37 (the only cathelicidin-derived innate immune system peptide found in humans) are related in vivo. For instance, the vitamin D receptor (VDR) and retinoid X receptor (RXR) are both connected with AD, as well as with expression levels of LL-37. Vitamin D3 treatment was shown to reduce cerebral amyloid-β accumulation and to improve cognition in a mouse model of AD, although the

mechanism of action was not stated [REF: 1 a,25~Di hydrox -vitamin 1 ⁾ 3 reduces cerebral amyloid-β accumulation and improves cognition in mouse models of Alzheimer's disease. Durk MR, Han K, Chow EC, Ahrens R, Henderson JT, Fraser PE, Pang KS. J Neurosci. 2014 May 21;34(21):709ΐ -101. doi : l 0.1523/jNEUROSCI.2711-i 3.20i 4. Pubmed ID PMID 248493 5], while RXR activation reduced neuronal loss and improved cognition in an aggressive mouse model of AD (but again, with no mention of cathelicidin involvement) [REF: Neuronaily directed effects of RXR activation in a mouse model of Alzheimer's disease. Mariani MM, Malm T, Lamb R, Jay TR, eilson L, Casali B, Medarametla L, Landreth GE. Sci Rep. 201 7 Feb 16:7:42270. Pubmed ID PMID

282.05585]. The above AD papers do not mention that expression levels of the human CAMP gene that encodes hCAP-18 (the protein precursor for LL-37) are upregulated by activation of VDR. [REF: The chemistry and biology of LL-37, Burton MF, Steel PG. Nat Prod Rep. 2009 Dee;26(12): 1572-84. Review. PMID: 19936387] and [REF: Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitamin D receptor and is strongly up-regulated in myeloid cells by 1 ,25-dihydroxy vitamin D3. Gombart AF, Borregaard N, Koeffler HP. FASEB J. 2005 Jul; 19(9): 1067-77. Pubmed ID PMID 15985530] These references also do not mention that RXR activation is an obligate part of CAMP gene expression, although this has been shown. [REF: Label-free quantitative mass spectrometry reveals novel pathways involved in LL-37 expression. Cederlund A, Ny!en F, M raglia E, Bergman P, Gudraundsson GH, Agerberth B. J innate Immun. 2014;6(3):365-76. Pubmed ID PMID 24246949] and [REF; Cell Tissue Res. 2016 Nov;366(2):353-362. Vitamin D-induced up-regulation of human keratinocyte cathelicidin anti-mi crobial peptide expression involves retinoid X receptor a. Svensson D, Nebel D, Voss U, Ekblad E, Nilsson BO. Pubmed ID PMID 27357S04]

[0077] Another indirect connection between Alzheimer's Disease mechanism and cathelicidin LL-37 is the hypothesis that sporadic AD is essentially 'Type 3 Diabetes' occurring in brain tissue [REF: Type 3 diabetes is sporadic Alzheimer's disease mini- review, de la Monte SM. Eur Neuropsychopharmacol. 2014 Dec;24( 12): 1954-60.

Pubmed ID PMID 25088942] and [REF: Alzheimer's disease is type 3 d abetes-evidence reviewed, de la Monte SM, Wands JR. J Diabetes Sci Technof . 2008 Nov;2(6): 1101-13. Pubrned ID PMID 19885299]. In a 2015 study, it was shown that intraperitoneal administration of the murine cathelicidin peptide, CRAMP (which, like LL-37 for humans, is unique in the mouse proteome) protects Non-Obese Diabetic (NOD) mice against the development of autoimmune diabetes. [REF: Pancreatic β-Cells Limit Autoimmune Diabetes via an Immunoreguiatory Antimicrobial Peptide Expressed under the Influence of the Gut Microbiota. Sun J, Furio L, Mecheii R, van der Does AM, Lundeberg E, Saveanu L. Chen Y, van Endert P, Agerberth B, Diana J. Immunity. 2015 Aug 18;43(2):304-17. Pubrned ID PMID 26253786] This treatment was motivated by the authors' discovery that the genetic defect of NOD mice that creates susceptibility to autoimmune diabetes is a deficit in their ability to express cathelicidin. This defect is more pronounced in female NOD mice, who have a higher incidence of disease as compared with males. In this animal model, immunomodulatory effects of the

cathelicidin peptide, including effects on the phenotypes of white blood cells including macrophages, dendritic cells, T and B cells, reduced risks of inflammatory disease [REF: Pancreatic β-Cells Limit Autoimmune Diabetes via. an Immunoreguiatory Antimicrobial Peptide Expressed under the influence of the Gut Microbiota. Sun J, Furio L, Mecheri R, van der Does AM, Lundeberg E, Saveanu L, Chen Y, van Endert P, Agerberth B, Diana J. Immunity. 201.5 Aug 18;43<2):304-17. Pubrned ID PMID 26253786]. What this shows is that, in vivo, the cathelicidin peptide is strongly immunomodulatory of white blood cell phenotypes. Thus, macrophages, dendritic cells, T cells, and B cells, were all switched from a proinflammatory, diabetogenic phenotype by the injection of cathelicidin peptide, to a noninflammatory, healing phenotype that prevented the development of autoimmune diabetes.

[0078] To date, most approaches to AD have relied on the supposition that pathological overexpression or hindered degradation of Αβ lays the primary foundation for disease. [REF: Alzheimer mechanisms and therapeutic strategies. Huang Y, Mucke L.

Cell. 2012 Mar 16;] 48(6): 1204-22. Review. Pubrned ID PMID: 22424230]. Recently, it has been shown that the chronic under-expression of the cathelicidin LL-37, which normally opposes Αβ fibril formation, likely plays a key role in the pathological accumulation of Αβ, as will be discussed below [ EF: Evidence that the Human Innate Immune Peptide LL-37 may be a Binding Partner of Amyloid-β and Inhibitor of Fibril Assembly. De Lorenzi E, Chiari M, Colombo K, Cretich M, Sola L, Vanna R, Cagni P, Bisceglia F, orasso C. Lin IS, Lee M, McGeer PL, Barron AE. j Alzheimers Dis.

2017;59(4}.1213-1226. Pubmed 03 PMID 28731438]. It is, of course, difficult for researchers to identify a systemic element that should be present, or perhaps should be better regulated, but is not. This may explain why the central and important role of LL- 37 under-expression in Alzheimer's Disease was not discovered prior to 2017.

[0079] Recently, a multifaceted approach was taken to confirm and characterize, in vitro, the interactions between Αβ and LL-37, and inhibitory effects of LL-37 on Αβ oligomer/fibril formation. It was demonstrated that LL-37 and Αβ42 (residues 1-42 of the Αβ peptide), which are both individually toxic and proinflammatory to neuroblastoma cell line SH-SY5 Y via the stimulation of microglial production of inflammatory cytokines, lose most of their cytotoxicity to neurons if the two peptides are co-incubated prior to being added to the cell culture media.

[0080] The interactions between LL-37 and Αβ peptides were investigated by SPR imaging (SPRi), a recent evolution of traditional SPR, which couples the label-free monitoring of molecular interactions by scanning angle SPR measurements with simultaneous CCD-based imaging of the whole surface for signal detection [REF ^' .

Surface piasmon resonance imaging for affinity-based biosensors. Scarano S, Mascini M, Turner AP, Mi urmi M. Biosens Rioelectro . 2010 Jan 15,25(5):957~66. Pubmed ID PMID 19765967]. The multi-array SPRi configuration improves the overall accuracy of the study by allowing simultaneous detection of signals originating from both positive and negative control ligands immobilized on the same chip. In addition, the real-time imaging of the entire SPR-biochip allows for verification of the quality and the optical properties of different ligands after their immobilization on the chip surface. To evaluate the aggregation state and the presence of soluble oligomers in the Αβ samples used for SPRi, capillary electrophoresis (CE) analysis was carried out. The inhibitory effect on fibril formation was demonstrated through transmission electron microscopy (TEM) by investigating fibril formation in quasi-physiological conditions. Conformational analyses of Αβ42 peptide in solution, in the absence and presence of LL-37, were carried out by circular dichroism (CD) spectroscopy.

[0081] In this work [REF: Evidence that: the Human innate Immune Peptide LL-37 may be a Binding Partner of Amyioid-β and Inhibitor of Fibril Assembly. De Lorenzi E, Chiari M, Colombo R, Cretich M, Sola L, Vanna R, Gagni P, Bisceglia F, Morasso C, Lin JS. Lee M, McGeer PL, Barron AE. J Aizheimers Dis. 2017;59(4): 1213-1226.

Pubrned ID PMID 28731438], it was demonstrated that Αβ and LL-37 bind to each other specifically, and that LL-37 inhibits the adoption by Αβ of ordered β-type secondary structures. Additionally, a variety of different published findings were discussed that indicate a physiological interplay between (and potential co-regulation of) these two peptides as an aspect of human innate immunity that may affect the initiation and progression of Alzheimer's Disease-related pathology.

Materials and Methodologies

[0082] The methodologies disclosed herein may be further appreciated with respect to the following non-limiting examples. The following materials and methodologies were utilized in the examples provided herein.

Sourcing Peptides and Buffer Chemicals; Materials and Methods for Sample Preparation

[0083] Αβ40, Αβ42 free base peptides and 1% H40H can be purchased from AnaSpec (Fremont CA, USA). LL-37 was from Innovagen (Lund, Sweden). An unrelated peptide to serve as a binding study control (sequence SYSVQDQYQALLQQHAQYK) was kindly gifted by Dr. Alessandro Gori from the Peptide Synthesis Lab, ICRM, CNR, Milan. "Scrambled sequence" LL-37 peptide was from AnaSpec, and has the same amino acid composition as natural LL-37, but with the following sequence:

GLKLRFEF SKIKGEFLKTPEVRFRDIKLKD RIS VQR. 1, 1,1,3,3,3 -Hexafluoro-2- propanol (FIFIP), acetonitrile, sodium carbonate, sodium hydroxide as well as NaH2P04 and Na2FIP04 were from Sigma-Aldrich (Stenheim, Germany). Buffer solutions were prepared daily using Millipore Direct-Q™-deionized water, filtered with 0.45 μπι Millipore membrane filters (Bedford, MA, USA) and degassed by sonication.

Samples and Methods Used for SPRi and CE Experiments

[0084] In SPRi and CE experiments, 1 mg/mL stock solutions of Αβ40 and unrelated peptide or scrambled-sequence LL-37 in PBS were diluted to the desired concentration. Αβ42 was prepared as described by Bartolini et al. [REF: Kinetic char cter zat on of amyioid-heta 1-42 aggregation with a multimethodoiogical approach. Bartolini M, Naldi M, Fiori I, Yal!e F, Biscarini F, Nico!au DV, Andrisano V. Ana) Bioehem. 2011 Jul 15;414(2):215-25. Pu med ID PMID 21435333]. Briefly, 0.5 mg of lyophilized Αβ42 powder was dissolved in HFIP (149 μΜ) and kept overnight at room temperature. After 12 hours, the solution was aliquoted and the HFIP was left to evaporate overnight at room temperature. The Αβ42 film was redissolved in CH3CN/300 μΜ Na2CO3/250 mM NaOH (48.3 :48.3 :3.4, v/v/v). The obtained 500 μΜ Αβ42 solution was diluted to the desired concentration with 20 mM phosphate buffer, pH 7.4 (SPRi: 20 μΜ; CE: 100 μΜ).

Methods for TEM and CE Analysis

[0085] For TEM and CD analysis, the samples were prepared as described in Brogi et al. [REF: Disease-modifying anti-Alzheimer's drugs: inhibitors of human cholinesterases interfering with β-arnyloid aggregation. Brogi. S, Butini S, Maramai S, Colombo R, Verga L, Lanni€, De Lorenzi E, Lamponi S, Andreassi M, Bartolini , Andrisano V,

Novellino E, Campiani G, Brindisi M, Gemma S, CNS Neurosci Ther. 2,014

Jul;20(7):624-32. Pubmed ID PMID 24935788], and analyzed at 50 and/or 100 μΜ concentrations. Briefly, to 1 mg lyophilized Αβ42 powder (AnaSpec) 80 L of 1% H4OH solution was added. After 1 minute sonication, the solution was diluted with 20 mM phosphate buffer, pH 7.4, to the desired concentration, and divided into aliquots, which were freeze-dried and stored at -80°C. Each lyophilized peptide aliquot was resuspended in 20 mM phosphate buffer, pH 7.4 immediately before use. Samples were sonicated 3 min in an ultrasonic bath, to break down pre-formed aggregates and increase the

peptide's effective concentration [REF: Sonication of proteins causes formation of aggregates that resemble amyloid. Stathopulos PB, Scholz GA, Hwang YM, Rumfeldt JA, Lepock JR, Meiering EM. Protein Sci. 2004 Nov; 13(1 l):3Q 17-27. Pubraed ID PMID 15459333]. For samples incubated with LL-37, lyophilized peptide was re-suspended in appropriately diluted LL-37 solutions, so as to keep the peptides at the desired molar concentrations and obtain proper AP42/LL-37 molar ratios. Samples were analyzed either immediately after solubilization, or after different lengths of incubation time, as noted in FIG. legends.

Methods for Surface Plasmon Resonance imaging (SPRi) Experiments with LL-37 and Αβ

[0086] Bare gold SPRi-biochips (HORIBA Jobin Yvon SAS, France) were treated for 10 minutes with an Oxygen Plasma Generator (Harrick Plasma Cleaner), then coated using a solution of co-poly(DMA-NAS-MAPS) (MCP-2, Lucidant Polymers, Sunnyvale, CA, USA) via surface immersion for 30 min (MCP-2 copolymer was dissolved at 1% w/v in aqueous 20% ammonium-sulfate). Coated SPRi-biochips were then washed with distilled water and dried at 80 °C for 15 minutes. Residual reactive groups were then reacted with 50 mM ethanolamine / 0.1M Tris pH 9.0 for 1 hour, washed with water, and dried with nitrogen.

[0087] The SPRi-biochip surface coated with the copolymer was functionalized by spotting the following 20 μΜ solutions: 1) Αβ40 (t = 0 days); 2) Αβ42 (t = 0 days); 3) Αβ42 (t = 24 days, i.e. spotted after 24 days of incubation at room temperature) and 4) an unrelated peptide used as reference. These peptides were spotted in replicate (8 spots, around 190 μιη diameter each) using a piezoelectric spotter (SciFLEXARAYER S5; Scienion) at 20 °C and 50% humidity. After spotting, the SPRi-biochip was incubated overnight in a sealed chamber saturated with sodium chloride at room temperature.

[0088] The binding analysis was performed using a Horiba XelPleX SPRi imaging instrumentation (HORIBA Jobin Yvon SAS, France) after an overnight instrument equilibration with HBS-T (10 mM HEPES, 150 mM NaCl, 0,05% Tween) used also as running buffer. Thereafter increasing concentrations of LL-37 peptide solution (1.0, 2.5, 5.0, 10, 25, 50, 100 μΜ in phosphate buffer 20 mM, pH 7.4) were injected onto the chip at 50 μΕ/ιηίη for 10 minutes, at 25 °C, in order to reach a binding equilibrium for all the responses. Ovalbumin (Sigma-Aldrich, A5503) was used as the negative control sample. After each injection, the chip surface was regenerated by injecting 50 mM glycine at pH 2.0, 50 μΕ/ιηίη for 4 minutes, thus obtaining a complete regeneration of the chip surface without significant loss of binding capacity. The results were preprocessed using EzSuite (HORIBA Jobin Yvon SAS, France), then equilibrium-binding constants (KD and KA (1/ KD) values were calculated using Scrubbergen2 (licensed by HORIBA Jobin Yvon SAS). Additional binding analysis experiments were performed using the same procedures and conditions, but by injecting a control scrambled-sequence LL-37 peptide.

Methods for Capillary Electrophoresis (CE) Analysis of Αβ Peptides

[0089] All CE experiments were carried out by slightly modifying the method reported by Brogi et al. [REF: Disease-modifying anti-Alzheimer's drugs: inhibitors of human cholinesterases interfering with β-amyioid aggregation. Brogi S, Butini S, Maramai S. Colombo R, Verga L, Lanni C, De Lorenzi E, Lamponi S, Andreassi M, Bartolim M, Andrisano V, NovelHno E, Campia G, Brindisi M, Gemma S. CNS Neurosci Ther. 2014 Jui;20(7):624-32. Pubraed ID PMID 24935788]. Briefly, an Agilent Technologies 3D CE system with built-in diode-array detector (Waldbronn, Germany) was used and data were collected using Chemstation A.10.02 software. The fused-silica capillary (50 μιτι id, 360 μιτι od, total length 33 cm, effective length 24.5 cm) was from Polymicro Technologies (Phoenix, Arizona, USA). A new capillary was flushed at 1 bar with 1 M NaOH and water for 60 min each and background electrolyte (BGE, 80 mM sodium phosphate buffer, pH 7.4) for 90 min. The between-run rinsing cycle consisted of 50 mM SDS (1.5 min), water (1.5 min) and BGE (2 min). Sample injection (100 μΜ) was carried out by applying a pressure of 30 mbar for 3 s. Separations were carried out at 25°C and +12 kV (current 75-80 μΑ). The acquisition wavelength was 200 nm.

Methods for Transmission Electron Microscopy (TEM) Analysis of Αβ and LL-37 Peptides

[0090] For each TEM sample, a ΙΟ-μΙ. droplet of suspension or solution was applied to a carbon-coated Formvar nickel grid (200 mesh) (Electron Microscopy Sciences, Washington, PA, USA). Each sample was allowed to sediment onto the carbon film for 15 min, then negative staining was performed with 10 μΐ. of 2% w/v uranyl acetate solution (Electron Microscopy Sciences). After carefully draining off excess staining solution using filter paper, the specimen was transferred to a Philips CM12 transmission electron microscope for examination, operating the TEM at 80 kV. Electron micrographs of negatively stained samples were photographed using Kodak film.

Methods for Circular Dichroism Analysis of Αβ and LL-37 Peptides

[0091] CD spectra were collected at 20 °C using a Jasco-810 spectrophotometer and a quartz cuvette with a 0.1 cm path length. All experiments were performed with an Αβ42 concentration of 50 μΜ. Spectra were registered from 190 to 250 nm and run at a scan speed of 20 nm/min, with a time response of 2 sec and data pitch of 0.2 nm. All spectra were baseline-corrected. Molar mean residue ellipticity [Θ] is expressed in degrees cm2 dmol-1, and calculated as [Θ] = Gobs MWR/(10 1 c), where Θ is observed ellipticity in degrees; MWR, peptide mean residue molar weight; 1, the optical path length in cm; and c, peptide concentration in g/mL. Methods of Neuronal Cell Culture for Testing Effects of Αβ and LL-37 Peptides

[0092] Cells from the human neuroblastoma SH-SY5 Y cell line were grown in DMEM/F12 medium containing 10% fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) and 100 IU/mL penicillin and 100 g/mL streptomycin (Invitrogen, Carlsbad, CA) under humidified 5% CO2 and 95% air.

[0093] Human microglial cells were isolated from surgically resected temporal lobe tissue as described in Lee et al. [REF: Depiction of GSH in glial cells induces neurotoxicity: relevance to aging and degenerative neurological diseases. Lee M, Cho T, Janiaramotai N, Wang Y Γ, McGeer E, McGeer PL. FASEB J . 2010 ,24(7):2533~45. Pubrned ID PMID 20228251 ]. Briefly, tissues were rinsed with a PBS solution and chopped into small pieces (< 2 mm ³ ) with a sterile scalpel. They were treated with 10 mL of a 0.25%) trypsin solution at 37°C for 20 min. Subsequently DNase I (from bovine pancreas, Pharmacia Biotech, Baie d'Urfe, PQ, Canada) was added to reach a final concentration of 50 μg/mL. Tissues were incubated for an additional 10 min at 37°C. After centrifugation at 275 g for 10 min, the cell pellet was resuspended in the serum- containing medium and passed through a 100 μπι nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). The cell suspension was centrifuged again (275 g for 10 min) and re-suspended in 10 mL of DMEM/F12 medium with 10% FBS containing gentamicin (50 μg/mL), and plated onto tissue culture plates (Becton Dickinson) in a humidified 5% CO2, 95%) air atmosphere at 37°C for 2 h. This achieved adherence of microglial cells. Cells were allowed to grow by replacing the medium once per week.

[0094] For estimating the purity of microglial cell cultures, aliquots of the cultures were placed on glass slides at 37°C for 48 h. The attached cells were then fixed with 4% paraformaldehyde for 1 h at 4°C, and made permeable with 0.1%> Triton X-100 for 1 h at room temperature. After washing twice with PBS, the culture slides were treated with the monoclonal anti-GFAP antibody (1/4,000, DAKO) for astrocytic staining and polyclonal anti-Iba-1 antibody (1/500, Wako Chemicals, Richmond, VA) for microglial staining for 3 h at room temperature. The slides were then incubated with Alexa Fluor 488- conjugated goat anti -mouse IgG antibody (Invitrogen, 1 :500) and Alexa Fluor 546- conjugated goat anti -rabbit IgG antibody (Invitrogen, 1 :500) in the dark for 3 h at room temperature to yield a positive red fluorescence. To visualize all cells, the slides were washed twice with PBS and counterstained with the nuclear dye DAPI (100 μg/mL, Sigma) to give a blue fluorescent color. Images were acquired using an Olympus BX51 microscope and a digital camera (Olympus DP71). Fluorescent images were co-localized with ImagePro software (Improvision Inc., Waltham, MA). The purity of microglia was more than 99% (1.93 ± 0.54 astrocytes in 500 total cells in microglial culture, n=30).

[0095] To achieve SH-SY5Y differentiation, the undifferentiated cells were treated for 4 days with 5 M retinoic acid (RA) in DMEM/F12 medium containing 5% FBS, 100 IU/mL penicillin, and 100 g/mL streptomycin [REF: Tissue transglutaminase mediates activation of RhoA and MAP kinase pathways during retinoic acid-induced neuronal differentiation of SH-SY5Y cells Singh US, Pan J, Kao YL, Joshi S, Young KL, Baker KM. J Biol Chem. 2003 Jan 3 278(1 ):391-9. Pubmed ID PMID 12401808]. The RA- containing medium was changed every two days. Differentiated SH-SY5Y cells demonstrated neurite extension, indicative of their differentiation [REF: Neurotoxins released from interferon-gamma-stimulated human astrocytes, Lee M, McGeer E, McGeer PL. euroscience. 2013 Jan J 5;229: 164-75. Pubmed ID PMID 23098801].

Methods for Neuronal Cell Culture Treatment with Αβ and LL-37 Peptides

[0096] Human microglial cells (5x 10 ⁴ cells per well) or human neuroblastoma SH- SY5Y cells (5 ^χ 10 ⁵ cells per well) or their mixture were seeded into 24-well plates in 1 mL of DMEM/F12 medium containing 5% FBS. Αβ42 peptide (AnaSpec, Fremont, CA) was dissolved in mixture of 1% NH40H and 0.1% Thioflavin S (Sigma, St. Louis, MO) in water to make 30 mM stock solution. Further dilution was made with water to reach 30 μΜ. Similar methods were used to prepare LL-37 peptide solutions (AnaSpec, Fremont, CA). Cultured cells were exposed to either 30 μΜ Αβ42 or 30 μΜ LL-37; or to a combined 1 : 1 equimolar mixture of the two peptides at 30 μΜ each. After incubation for 72 h cell free medium was collected to measure levels of proinflammatory cytokines such as TNFa and IL-6. For SH-SY5Y cell viability, MTT assays were performed as described by Lee et al. REF: Acidic fibroblast growth factor (FGF) potentiates giial-mediated neurotoxicity by activati g FGFR2 Illb protein . Lee M, Kang Y, Suk K, Schwab€, Yu

S, M:cGeer PL. J Biol Chem. 201 1 Dec 2;286(48):41230-45. Pubmed ID PM D

21990352].

Methods of SH-SY5Y-microglial Cell Viability Assays

[0097] The viability of SH-SY5Y cells following incubation with glial cell supernatants was evaluated by MTT assays as described by Lee et al. [REF: Acidic fibroblast growth factor (FGF) potentiates g!ial-raediaied neurotoxicity by activating FGFR2 Illb protein. Lee M, Kang Y, Suk K, Schwab C, Yu S, McGeer PL. J Biol Chem. 201 1 Dec 2:286(48}:41230~45. Pubmed 03 PMID 21990352]. Briefly, the viability was determined by adding MTT to the cell cultures to reach a final concentration of 1 mg/mL. Following 1 h incubation at 37°C, the dark crystals formed were dissolved by adding a SDS/DMF extraction buffer (300 pL, 20% sodium dodecyl sulfate, 50% N, N- dimethylformamide, pH 4.7). Subsequently plates were incubated overnight at 37°C and optical densities at 570 nm were measured by transferring 100 pL aliquots to 96-well plates and using a plate reader with a corresponding filter. Data are presented as a percentage of the values obtained from cells incubated in fresh medium only.

Methods for the Measurement of TNF and IL-6 release From Cultured Neuronal Cells

[0098] Cytokine levels were measured in cell-free supernatants after 72 h.

Quantitation was performed with ELISA detection kits (Peprotech, NJ) following protocols described by the manufacturer. Methods for Statistical Data Analysis of Results Obtained in Neuronal Cell Culture

Assays

[0099] The significance of differences between data sets was analyzed by one-way ANOVA tests. Multiple group comparisons were followed by a post-hoc Bonferroni test, e.g. cell viability in control condition versus cells treated with LL-37 alone; cell viability in control condition versus cells treated with Αβ42 alone; and viability in control condition versus cells treated with mixed LL-37 + Αβ42. P values are provided in the legends of the figures that show the results.

EXAMPLE 1 - SPRi analysis of LL-37 binding to immobilized Αβ; and CE analysis

[00100] To demonstrate interaction between LL-37 and Αβ42, a Surface Plasmon Resonance imaging (SPRi) biochip was functionalized with copoly(DMA-NAS-MAPS), a polydimethylacrylamide based copolymer, widely used to immobilize biomolecules on microarray slides [REF: Overcoming mass transport limitations to achieve femtomoiar detection limits on silicon protein microarray s. Cretich M, Bagnati M, Damin F, Sola L, Chiari M. Anal Biochem. 201 1 Nov 1 ;41 S(1 ): 164-6. Pubmed ID PMID 21802399]. Three different solutions of 20 μΜ Αβ-peptides, in different states of aggregation, were spotted onto the SPRi-chip surface. The aggregation state and the presence of soluble oligomers were determined by CE analysis [REF: Disease-modifying anti -Alzheimer's drugs:

inhibitors of human cholinesterases interfering with β-arayloid aggregation. Brogi S, Butini S, aramai S, Colombo R, Verga L, Lanni C, De Lorenzi E, Lamponi S,

Andreassi M, Bartolini M, Andrisano V, Novellino E, Campiam G, Brindisi M, Gemma S. CNS Neurosci Ther. 2014 Jul;20(7}:624~32. Pubmed ID PMID 24935788] and [REF: Capillary electrophoresis studies on the aggregation process of beta-amyloid .1 -42 and 1- 40 peptides. Sabe! ia S, Quagiia M, Lanni C, Racchi , Govoni S, Cacdalanza G, Cailigaro A, Bellotti V, De Lorenzi E, Electrophoresis, 2004 Oct;25(18-19):3 I 86-94. Pubmed ID PMID 1 5472964]. Αβ40 (a) dissolved in PBS, Αβ42 (b) prepared as in [16], both solubilized immediately before spotting (t = 0 days), and Αβ42 (c) prepared as sample (b), but stored in the buffer for 24 days (t = 24 days) were spotted in different subarrays. Αβ42 was dissolved following the protocol detailed in the Material and Methods section. The solubilization buffer consisted of i) HFIP, a low-polarity solvent that stabilizes the a-helix and disrupts the interstrand hydrogen bonds of the β-sheets and ii) a mixture of acetonitrile/Na2C03/NaOH. While acetonitrile stabilizes the

unordered/a-helix structure, the basic pH increases the solubility of Αβ peptide. Overall, this procedure disfavors aggregation and provides a time window long enough to appreciate the prevailing formation of low-MW oligomers at early stages (t = 0 days) and the building up of high-MW oligomers at late-stages (t = 24 days) of the in vitro fibrillogenesis process. Indeed, this was confirmed by analyzing in CE the three samples at 100 μΜ concentration using a method slightly modified from Sabella et al. [KEF: Disease-modifying anti-Alzheimer's drugs: inhibitors of human cholinesterases interfering with β-amyioid aggregation. Brogi S, Butini S, Maramai S, Colombo R, Verga L, Lanni C, De Lorenzi E, Lamponi S, Andreassi , Bartolini M, Andrisano V,

Nove!lino E, Campiani G, Biindisi M„ Gemma S. CNS Neurosci Ther. 2014

;20(7):624-32. Pubmed ID PMID 24935788]. FIGs. 1-3 show CE electropherograms of the same Αβ peptide solutions spotted on the SPRi chip; consistent with what has been reported previously in literature [KEF: Disease-modifying anti-Alzheimer's drugs:

inhibitors of human cholinesterases interfering with β-amyloid aggregation. Brogi S, Buiini S, Maramai S, Colombo R, Verga L, Lanni C, De Lorenzi E, Lamponi S,

Andreassi M. Bartolini M, Andrisano V, Noveliino E, Campiani G, Brindisi M, Gemma S. CNS Neurosci Ther. 2014 Jui;20(7):624-32. Pubmed ID PM D 24935788] and [RF.F: Capillar}' electrophoresis studies on the aggregation process of beta-amyloid 1 -42 and 1 - 40 peptides. Sabella S, Quaglia M, Lanni C, Racchi M, Govoni S, Caccialanza G, Cal iigaro A, Bellotti V, De Lorenzi E. Electrophores s. 2004 Oct;25(18-I 9):3186-94. Pubmed ID PMID 15472964] and [REF: CE can identify small molecules that selectively target soluble oligomers of am loid beta protein and display antifibril iogenic activity , Colombo Rl , Carotti A, Catto M, Racchi M, Lanni C, Verga L, Caccialanza G, De Lorenzi E. Electrophoresis. 2009 Apr;30(8): 1418-29, Pubmed ID PMID 19306269], the fast-migrating group of peaks can be attributed to low MW-oligomers whereas the slow- migrating broad band corresponds to high MW-oligomers. In particular, in the Αβ42 sample, the high MW-oligomers build up over time, as evident in FIG. 2 and FIG. 3.

[00101] The interaction of Αβ in different aggregation states with LL-37 was investigated by SPRi. The CCD image of the chip surface, and the plasmonic curves acquired before the injection of LL-37 (FIG. 22), show the three Αβ forms conjugated to the surface. Specifically, the plasmonic curves of the different samples indicate that the sample containing a higher amount of Αβ42 high-MW oligomers (t = 24 days) binds to the surface more, compared to Αβ42 low-MW oligomers (t = 0 days) which, in turn, binds more than Αβ40 low-MW oligomers (t = 0 days). Differences in the immobilized mass could result from the increased molecular weight of the samples in different aggregation states from low to high MW-oligomers [REF: Kinetic characterization of amyloid-beta 1 -42 aggregation with a multimethodological approach. Bartoiini M. aldi M, Fiori J, Valle F, Bi scarini F, Nicolau DV, Andrisano V. Anal Biochem . 201 1 Jul 1 5;414(2):215-25. Pubmed ID PMID 21435333] and [REF; Disease-modifying anti- Alzheimer's drugs: inhibitors of human cholmesterases interfering with β-amyloid aggregation. Brogi S, Butini S, Maramai S, Colombo R, Verga L, Lanni€, De Lorenzi E, Lamport! S, Andreassi M, Bartoiini M, Andrisano V, Noveilino E, Campiani G, Brindisi M, Gemma. S. CNS eurosci i ^' her 2014 Ju! ;20(7):624-32. Pubmed ID PMID 24935788]. The sensograms taken after flowing a solution of LL-37 onto the SPRi chip for 10 minutes, revealed specific and reversible interactions with all the three Αβ forms (FIGs. 2-4). The injection of ovalbumin, a non-correlated protein, did not produce interaction signals. Analogously, the amount of LL-37 captured by non-correlated peptides (negative controls), spotted in parallel on the same SPRi chip, was significantly lower compared to that of positive signals. In a follow-up experiment, seven serial dilutions of LL-37, ranging from 1 to 100 μΜ, were injected to investigate the dependency of the SPR signal on ligand concentration for affinity studies (FIGs. 23-25). An equilibrium analysis (steady-state analysis) was performed by fitting the SPRi response at the equilibrium state for each concentration (FIGs. 7-9). It is noteworthy that the system reached the equilibrium state at each LL-37 concentration and a complete regeneration of the SPRi chip surface was obtained after each injection. As expected, the maximal response value, or maximum binding capacity (Rmax), which is related to the maximum number of LL-37 peptides that can bind the Αβ peptides at a certain time, decreases in the following order: Αβ42 (t = 24 days) » Αβ42 (t = 0 days) > Αβ40 (t = 0 days) (FIGs. 4-9, TABLE 1 below). This is in agreement with the amount of Αβ peptide immobilized on the SPRi chip, which increased with Αβ aggregation. To the contrary, as shown in TABLE 1 below, the affinity of LL-37 for the different Αβ forms shows the opposite trend, i.e. LL- 37 binds to low-MW Αβ oligomers more strongly than to high-MW Αβ oligomers. The binding specificity of LL-37 was verified by performing the same equilibrium analysis with a control scrambled-sequence LL-37. For this experiment, it was difficult to extract adequate calibration curves (see FIGs. 27-29), mostly due to non-specific and irregular interactions. For this reason, the binding affinities constants could not be calculated for experiments using scrambled LL-37.

TABLE 1 Values of the maximal response (Rmax), (related to the absolute number of LL- 37 molecules bound on the Αβ peptides at a certain time) and equilibrium binding constants (KA and KD), calculated using a nonlinear curve fit of the SPRi response at equilibrium.

Peptide R _ma x K^M ¹ ) Κ _Β (μΜ)

Αβ40 (t = 0 days) 0.59 1.20xl0 ⁵ 8.3

Αβ42 (t = 0 days) 1.56 7.51xl0 ⁴ 13.3

Αβ42 (t = = 24 days) 4.43 4.92xl0 ⁴ 20.3 EXAMPLE 2 - Results of Transmission Electron Microscopy Analysis of Αβ and LL-37 peptides

[00102] An inhibitory effect of LL-37 peptide binding on fibril formation was demonstrated by transmission electron microscopy (TEM). For these experiments (see Methods), samples were prepared according to a more aggregating protocol [REF:

Disease-modifying arrti- Alzheimer's drugs: inhibitors of human cholmesterases interfering with β-amyioid aggregation. Brogi S, Butini S, Maramai S, Colombo R, Verga L, Lanni C, De Lorenzi E, Lamponi S, Andreassi M, Bartolini M, Andrisano V,

Novel lino E, Carapiani G, Brindisi M, Gemraa S. CNS Neurosci Ther. 2014

Jul;20(7):624-32. Pubmed ED PMID 24935788], to mimic quasi-physiological conditions and to better verify LL-37 anti-fibrillogenic activity. Αβ42 reproducibly forms a dense network of interpenetrating, μιη-long, straight, unbranched filaments with a diameter of about 10 nm (FIG. 10, n=3), corresponding to known features of classic, mature amyloid fibrils [REF: Molecular mechanisms of amyloidosis. Merlini G, Bellotti V. N Engl j Med. 2003 Aug 7;349(6):583-96. Pubmed ID PMID. 12904524]. This network of long, straight, interconnected fibrils covered the TEM plate uniformly. On the other hand, LL- 37 alone in solution produced globular, amorphous aggregates (FIG. 11, n=3). To assess the influence of LL-37 on Αβ fibril formation, 50 μΜ Αβ42 was co-incubated with an equimolar amount of LL-37 peptide. Mixtures of Αβ42 and LL-37, prepared as described in Methods, were analyzed by TEM, either immediately after solubilization or after 3 or 9 days of incubation. In the equimolar solution of the two peptides at t=0, shortened, less defined, more sparsely represented fibrils (as compared to FIG. 10) were observed, in mixture with clumps of amorphous material (FIG. 12, n=2). These evolved into (as compared to FIG. 10) sparser clumps of shorter and more curved fibrils at t=3 days

(FIG. 13, n=2), and then into globular amorphous aggregates at t=9 days, with no visible fibrils present (FIG. 14, n=2). As reported in literature, molecules with a similar time- dependent antifibrillogenic effect are considered inhibitors of Αβ42 fibril formation

[REF: N-Methyiated peptide inhibitors of beta-amyloid aggregation and toxicity. Optimization of the inhibitor structure, Kokkoni N, Stott , Amijee H, Mason JM, Doig AJ. Biochemistry. 2006 Aug 15;45C32):9906-18. Pubmed i: PMID 16893191 ] and

[REF: Discovery and structure activity relationship of small molecule inhibitors of toxic p-amyloid~42 fibril formation. roth. H, A saloni A, Varisco Y, Jan A, Sreenivasachary N, Rezaei-Ghaieh N, Girie s V, Lohmann S, Lopez-Deber MP, Adolfsson O, Pihlgren M, Paganetti P, Froestl W, Nagel-Steger L, Wiliboid D, Schrader T, Zweckstetter M, Pfeifer A, Lashuel HA, Muhs A. J Biol Chem. 2012 Oct 5;287(41):34786-800. Pubmed ID PMID 22891248], thus from these data it can be deduced that LL-37 inhibits Αβ42 fibril formation.

EXAMPLE 3 - Results of Conformational Analysis by Circular Dichroism (CD) Spectroscopy

[00103] Conformational analyses of Αβ42 peptide in solution, in the absence and presence of LL-37, were carried out by circular dichroism (CD) spectroscopy. As in TEM analysis, spectra were recorded immediately after dissolving peptide according to [REF: Disease-modifying anti- lzheimer's drugs: inhibitors of human cholinesterases interfering with β-amyloid aggregation. Brogi S, Butini S, Maramai S, Colombo R, Verga L, Lanni€, De Lorenzi E, Lamponi S, Andreassi M, Bartolini M, Andrisano V,

Noveliino E, Campiani G, Brindisi M, Gemma S. CNS Ne rosci Ther. 2014·

;20(7):624-32. Pubmed ID PMID 24935788] (FIG. 15) and again after 24 hours (FIG. 16). We found that at t=0, Αβ42 assumed an unordered conformation (FIG. 15) whereas over 24 hours, adoption of β-type conformations was observed (FIG. 16), with a characteristic positive band at 195 nm and negative band at 215 nm. On the other hand, 50 uM LL-37 at t=0, immediately adopted a-helical conformations and maintained this secondary structure over 24 hours (FIGs. 15-16). Remarkably, in the presence of LL-37, the Αβ42 peptide in solution seemed to be prevented, to a large degree, from forming typical β structure (FIGs. 15-16). [00104] While CD is essentially an "averaging" spectroscopic method, the spectra of 1 : 1 mixed Αβ42 and LL-37 peptides do not show ordered β structure of the type that predominates in pure Αβ42 solutions. No time-dependence, within 24 hours, of peptide secondary structure was observed in the mixed solutions, in contrast to the behavior of Αβ42 solutions. As discussed below, this interaction between the peptides makes sense, considering their opposite charges and mutually high and similar proportions of aromatic and aliphatic amino acids (FIG. 17). We can only conclude, definitively, that, in the presence of LL-37 we do not observe the typical CD spectrum of self-associated Αβ42.

EXAMPLE 4 - Effects of Αβ42, LL-37, and AP42 LL-37 complexes on human microglia, SH-SY5Y neuronal cells, and microglia-mediated cytotoxicity to SH- SY5Y neuronal cells

[00105] We studied the effects of Αβ42 alone, LL-37 alone, and Αβ42/ίί-37 complexes on SH-SY5Y neuroblastoma cells, human microglia, and on cytokine release from microglia that impact SH-SY5Y cells. Specifically, we exploited a simplified model that reproduces the cross-talk between the major players involved in neuronal damage by looking at the effect of cell-free supernatant from microglial cell culture, on cultured neuronal cells. Cultured cells were exposed to either Αβ42 alone or LL-37 alone (each at 30 uM concentration). After incubation for 72 h, MTT assays were performed and the cell-free medium was collected to measure levels of proinflammatory cytokines TNFa and IL-6. Note, as shown in the control experiments (FIG. 30), neither Αβ nor LL- 37 peptide was toxic to SH-SY5Y cells directly, nor to human microglia alone, at any concentration.

[00106] However, we find evidence for microglia-mediated neuroinflammation, for both peptides. Human microglia were exposed to either Αβ alone, LL-37 alone, or an equimolar LL-37/ Αβ mixture for 2 days. Then cell-free supernatants from microglia cultures were transferred to differentiated SH-SY5Y cells. SH-SY5Y cell viability was measured after 72 h. We also measured TNFa and IL-6 levels in microglial cell-free supernatants after 2 days' incubation with the peptides. Data are presented in FIGs. 18- 20. Treatment of microglia with Αβ or LL-37 induced release of toxic microglial cytokines TNFa and IL-6 (FIG. 19 and FIG. 20) and resulted in a reduction in SH-SY5Y cell viability (down to 70% after 3 days, FIG. 18). However, when microglia were exposed to an equimolar AP:LL-37 mixture (30 μΜ each), the complexation of these two peptides reduced TNFa and IL-6 release, and attenuated SH-SY5Y cell viability loss by more than 90%, which is a major reduction in the deleterious impacts of either peptide, i.e., their complex is non-toxic to neuronal cells.

Discussion of In Vitro Results of Ap:LL-37 Interaction Studies by TEM, CD, SPRi, and Neuronal Cell Culture

[00107] Among the many inhibitors of Αβ aggregation and neurotoxicity that have been identified previously by various assays, or designed, synthesized, and/or selected. [REF: Molecules that target beta-amy!oid. Stains CI, Mondaf , Ghosh I.

ChemMedChem. 2007 Dec;2(12): 1674-92. Review. Pubmed ID PMID: 17952881] and [REF: A molecular ehaperone breaks the catalytic cycle that generates toxic Αβ oligomers. Cohen SIA, Arosio P, Presto J, Kurudenkandy FR, Biverstal H, Dolfe L, Dunning C, Yang X, Frohra B, Vendruscolo M, Johansson J, Dobson CM, Fisahn A, nowles TPJ, Linse S, Nat Struct Mol Biol. 2015 Mar;22(3):207-213. Pubmed ID PMID 25686087] However, no naturally occurring peptides that match Αβ in size were ever identified. The cathelicidin LL-37 is a pleiotropic, 37-residue innate immune effector that corresponds to residues 134-170 of the human cationic antimicrobial protein [REF: The chemistry and biology of LL-37. Burton MF, Steei PG. Nat Prod Rep. 2009

Dec;26(1.2): 1572-84. Review PMID: 19936387]. It is one of a kind in the human proteome and is expressed in many organs, including the brain [REF: The chemistry and biology of LL-37. Burton MF, Steel PG. Nat Prod Rep. 2009 Dec;26(12): 1572-84.

Review. PMID: 19936387] and [REF: Human antimicrobial peptide LL-37 induces g!ial- mediated neuroinflammation. Lee M, Shi L Barron AE, McGeer E, McGeer PL. Biochern Pharmacol. 2015 Mar 15;94(2): 130-41. Pubmed ID PMID 25686659]. LL-37 fulfills myriad systemic roles as an immune system effector and modulator [ EF: Mo! Bios st. 2009 May,5(5):483~96. Systems biology evaluation of immune responses induced by human, host defence peptide LL-37 in. mononuclear cells. Mookherjee N L, Hamill P, Gardy J, Blimkie D, Falsafi R, Chikatamarla A, Arenil!as DJ, Doria S, ollmarm TR, Hancock RE. Pubmed PMID .19381363] Additionally, LL-37' s immunomodulatory functions and effects on cells, links the endocrine and immune systems. [REF: Cathelicidms link the endocrine and immune systems. Mayer ML, Hancock RE. Cell Host Microbe. 2010 Apr 22;7(4):257-9. Pubmed ID PMID 20413092] Moreover, LL-37 plays a central role in Vitamin D3 -stimulated monocyte/macrophage autophagy. [REF: Vitamin D3 induces autophagy in human monocytes/macrophages via cathelieidin. Yuk JM, Shin DM, Lee HM, Yang CS, Jin HS, Kim KK, Lee ZW, Lee SH, Kirn JM, Jo EK. Ceil Host Microbe 2009 Sep I7,6i3):23 ! -43 Pubmed ID PMID 19748465]

[00108] Thus, these experiments that were described above, have both discovered and quantified a sequence-specific molecular binding interaction between Αβ and LL-37, indicating that these two peptides appear to be natural binding partners. If so, the dynamics of spatiotemporal expression of LL-37 and Αβ are likely to hold major significance to human health. While there are myriad well-studied functions of LL-37 as an antimicrobial and antiviral weapon of immune cells, the physiological functions of Αβ are not yet fully elucidated [REF: Physiological roles for amyloid beta peptides. 1*6318011 HA, Peers C. J Physiol. 2006 Aug 15;575(Pt I).540. Pubmed PMID: 16809372].

Recently published work suggests that Αβ may itself function in vivo, particularly in the human brain, as an antimicrobial and antiviral peptide [REF: The Alzheimer's disease- associated am loid beta-protein is an antimicrobial peptide. Soscia SJ, Kirby JE, Washicosky KJ, Tucker SM, Ingelsson M, Hyman B, Burton MA, Goldstein LE, Duong S, Tanzi RE, Moir RD. PLoS One. 2010 Mar 3;5(3):e9505. Pubmed ID PM ID:

20209079] and [REF: Sci Trans! Med. 2016 May 25;8(340):340ra72. Amyioid-β peptide protects against microbial infection in mouse and worm models of Alzheimer's disease. umar DK, Choi SH, Washicosky KJ, Eimer VV A, Tucker S, Ghofrani J, Lefkowitz A, McColl G, Goldstein LE, Tanzi RE, Moir RE). Pubmed ID PMID 27225182] and {KEF: Bioge ontology. 201.5 Feb;16(l):85-98. β-Arnyloid peptides display protective activity against the human Alzheimer's disease-associated herpes simplex virus- 1. Bourgade K, Garneau H, Giroux G, Le Page AY, Bocti C, Dupuis G, Frost EH, Fulop T Jr. Pubmed ID PMID 25376108] and [REF: Aizlieimer's Disease-Associated β-Amyloid Is Rapidly Seeded by Herpesviridae to Protect against Brain Infection. Elmer WA, Kumar DKV, Shanraugam K , Rodriguez AS, Mitchell T, Washicosky KJ.Gyorgy B, Breaker! eld XO. Tanzi RE, Moir RD. Neuron, 2018 Jul 1 1 ;99(1):56~63].

[00109] The study of Ι -37:Αβ interaction was initially inspired by an observation of a strong, apparent complementarity between the peptides' amino acid (aa) sequences (FIG. 17). They are similar in size and molecular weight (Αβ: 39-42 aa, Αβ42 is 4493 g/mol; and LL-37: 37 aa, 4514 g/mol). Αβ42 has a net negative charge of -3 (discounting the charge of histi dines), while LL-37 has a positive charge of +6, providing for strong electrostatic attraction. Αβ has four aromatic residues (F or Y) with a binary "FF" motif within the KLVFF sequence known to be necessary for Αβ'β assembly. [REF: Molecules that target beta-amyloid. Stains CI, Mondal K, Ghosh I. ChemMedChem . 2007

Dec;2(l 2): 1674-92. Review. Pubmed ID PMID: 17952881] LL-37 has four aromatic residues as well, and comprises the same "FF" motif within residues 4-8 (DFFRK). LL- 37 has nine hydrophobic, aliphatic residues (L, V, I) while Αβ has eleven, again making them biophysically similar. And like Αβ, LL-37 associates into homo-oligomers when alone in solution. [REF: Kinetic characterization of amyloid-beta 1-42 aggregation with a muitimethodological approach. Baitolini M, Naldi M, Fiori J, Val!e F, Biscarini F, Nicolau DV, Andrisano V. A al Biochem. 2011 Jul 15;414(2):215-25. Pubmed ID PMID 21435333] and [REF: Structure and organization of the human antimicrobial peptide LL- 37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Oren Z, Lerman JC, Gudmundsson GH, Agerberth B, Shai Y. Biochem J. 1999 Aug 1:3 1 ( Pt 3): 501-13. Pubmed ID PMID 1041731 1] and [REF: New aspects of the structure and mode of action of the human catheiicidin LL-37 revealed by the intrinsic probe p-cyanophenylaianine. Xhindoli D, Morgera F, Zinth U„ Rizzo R, Pacor S, Tossi A. Biochem J. 2015 Feb I:465(3 ).443-57. Pubmed ID PMID 25378136] When dissolved in aqueous solution, LL-37 peptides may self-assemble, but remain helical in character, unlike Αβ which slowly self-assembles via adoption of β-type secondary structure when alone in solution.

[00110] In Lee et al. in 2015, it was reported that the human catheiicidin precursor protein hCAP18 and its downstream cleavage product, the peptide LL-37, are both expressed in many organs of the human body, with the highest basic levels of LL-37 being expressed in the gastrointestinal tract and the brain. [REF: Human antimicrobial peptide LL-37 induces glial -mediated neuromfl animation. Lee M, Shi XI , Barron AE, McGee E, McCee PL. Biochem Pharmacol . 2015 Mar 15;94(2): 130-41. Pubmed ΪΙ ⁾ PMID 25686659] Prior to that 2015 Lee et al. publication, LL-37's expression and functional role in the human central nerve system (CNS) had not been reported. Thus, LL-37 has been proven to be expressed in brain tissue in the human body, as is the Αβ peptide. However, that 2015 publication purported to show, only, that LL-37 might be pro-inflammatory in human brain tissue, like Αβ itself. That 2015 paper did not mention or suggest any possibility of a binding interaction between LL-37 and Αβ.

Results of the SPRi interaction study of LL-37: Αβ Interaction

[00111] The physical interaction and binding specificity between LL-37 and different Αβ forms was demonstrated by SPRi, taking advantage of the simultaneous monitoring of different interactions on the same chip enabled by the imaging configuration of the technique. CCD images and related plasmonic curves (FIGs. 4-9) correlated to the amount of molecules adsorbed on the surface, showed an increasing immobilization density from low-MW oligomers to high-MW oligomers. [REF: Kinetic characterization of amyloid-beta 1-42 aggregation with a multimethodological approach. Bartolini M, Naldi M, Fiori J, Valle F, Biscarini F, Nicolau DV, Andrisano V. Anal Biochera. 201 1 Jul l 5:414(2):215-25. Pubmed ID PMID 21435333] and [ EF: Multifunctional cholinesterase and amyloid Beta fibrillization modulators. Synthesis and biological investigation. B tini S, Brindisi M, Brogi S, Mararnai S, Guarino E, Panico A, Saxena A, Chauhan V, Colombo R, Verga L, De Lorenzi E, Bartolini M, Andrisano V, Novel iino E, Campiam G, Gemma S. ACS Med Chem Lett. 2013 Oct 6;4(12): 1 178-82. Pubmed ID PMID 24900626]. While SPR measurements revealed higher absolute SPRi signals (Rmax) for Αβ with oligomeric features, the affinity for LL-37 was higher for the low-MW oligomeric forms present at the early stage of aggregation (i.e., Αβ40 (t = 0 days) and Αβ42 (t = 0 days)). [REF: Kinetic characterization of amyloid-beta 1-42 aggregation with a multimethodologieal approach. Bartolini M, Nakli , Fiori J, Valle F, Biscarini F, Nicolau D , Andrisano V. Anal Biochem. 201 1 Jul 15;414(2):215-25. Pubmed ID PMID 21435333] and [REF: Capillary electrophoresis studies on the aggregation process of beta-amyioid 1 -42 and 1-40 peptides. Sabeila S, Quagiia M, Lanni C, Racchi M. Govoni S, Caccialanza G, Calligaro A, Bellotti V, De Lorenzi E. Electrophoresis. 2004

Oci;25(18-19):3186-94. Pubmed ID PMID 15472964]

[00112] Several studies have reported the use of conventional SPR approaches to study Αβ potential interactors. Kai et al. reported the inhibition of Αβ fibril formation by tabersonine and demonstrated the binding between this small molecule and Αβ42 monomers and oligomers immobilized on the SPR chip surface. [REF. Tabersonine inhibits amyloid fibril formation and cytotoxicity of Αβ(1 -42). Kai T, Zhang L, Wang X, Jmg A, Zhao B, Yu X, Zheng J, Zhou F. ACS Chem Neurosci. 2015 Jim 17;6(6).879-88. Pubmed ID PMID 25874995] Equilibrium binding studies yield KD values of 69 and 535 μΜ for Αβ42 monomers and oligomers, respectively. In a similar study, Barr et al.

verified the inhibition of Αβ fibril formation by a 15-amino acid peptide.

[REF: Validation and Characterization of a Novel Peptide That Binds Monomelic and Aggregated β-Arayloid and Inhibits the Formation of Neurotoxic Oligomers. Barr RK. Verdiie G, Wijaya LK, Morici M, Taddei K, Gupta VB, Pedrini S, Jin L, Nieoiazzo JA, Knock E, Fraser PE, Martins RN. J Biol Chem. 2016 Jan 8;291(2):547-59. Pubmed ID PMID 26538562] SPR surfaces functionalized with monomeric, oligomeric and fibrillar Αβ42 revealed a stronger affinity of the polypeptide toward the fibrillar form, although only a KD value of 11 μΜ was measured by injecting Αβ42 onto immobilized peptides and no affinity constant was given. In the SPRi studies described above, kinetic constants in the range between 8 to 20 μΜ were found, similar to what is published for other Αβ fibril inhibitors, as noted above. On the other hand, as mentioned earlier (and differently to what was previously shown) [REF: Tabersonine inhibits amyloid fibril formation and cytotoxicity of Αβ( 1-42). ai T, Zhang L, Wang X, ling A, Zhao B, Yu X, Zheng J,

Zhou F. ACS Chem Neuroscl. 2015 Jun I7,6(6):879~88. Pubmed ID PMID 25874995] and [KEF: Validation and Characterization of a Novel Peptide That Binds Monomeric and

Aggregated β- Amyloid and Inhibits the Formation of Neurotoxic Oligomers. Barr RK, Verdi ie G, Wijaya L , Morici M, Taddei K, Gupta VB, Pedrini S, Jin L, Nicolazzo J A, Knock E, Fraser PE. Martins RN. J Biol Chem. 2016 Jan 8;291 (2}:547~59. Pubmed ID PMID 26538562], LL-37 shows higher affinity for low-MW Αβ oligomers. This strongly supports the ability of LL-37 to inhibit fibril formation by shifting the equilibrium toward smaller species of Αβ, according to the fibrillization mechanisms proposed by Cohen et al. [KEF: Proliferation of amyloid-p42 aggregates occurs through a secondary nue!eation mechanism. Cohen Si, Linse S, Luheshi LM, Hellstrand E, White DA, Rajah L, Otzen DE, Vendruscolo M, Dobson CM, Knowies TP. Proc Natl Acad Sci U S A. 2013 Jun 1 1; 1 10(24):9758-63. Pubmed ID PMID 23703910]

Effects of LL-37 and Αβ on cytotoxicity and inflammatory cytokine release

[00113] The results of the MTT assays showed that Αβ42 and LL-37 both induced microglial-mediated toxicity to SH-SY5Y cells. At the same time, the results of quantitative ELISA assays on IL-6 and TNFa showed that the levels of these

proinflammatory markers released from microglia were markedly elevated compared with that of the untreated control group, when each peptide was added alone. However, the effects of each peptide on cell viability and inflammatory marker production were largely mitigated when the two peptides were co-incubated prior to being added to the culture medium. These data support the hypothesis that Αβ42 interacts with LL-37 as seen by the considerable attenuation of the toxicity of the peptides when they are in complexation.

[00114] Certain aspects of our results are not surprising, as previously we have shown that microglia, upon stimulation by LL-37, can release proinflammatory cytokines that affect neurons. [REF: Human antim crobial peptide LL-37 induces glial-mediated neuroinflamraatioxi . Lee M, Shi XI. Barron AE, McGeer E, McGeer PL. Btocbem Pharmacol. 2015 Mar 15;94(2): 130-41. Pubmed ID PMID 25686659] Similarly, Αβ has been shown to activate microglia to produce inflammatory cytokines [REF: M croglia- mediated neurotoxicity: uncovering the molecular mechanisms. Block MLl, Zecca L, Hong JS. Nat Rev eurosci. 2007 Jan;8(l):57-69. Pubmed ID PMID 17180163]

However, an attenuation of the release of proinflammatory factors (IL-6 and TNFa) upon treatment of microglia with equimolar LL-37 and Αβ peptide mixtures is a novel finding, which strongly supports our finding that these two peptides may be natural binding partners, which perhaps the body seeks to maintain in balance over the longer term (after an acute innate immune response). Unlike either peptide alone, which causes human microglia to release proinflammatory and neurotoxic cytokines, the complex of the two peptides lacks that neurotoxic effect.

Correlation of biophysical activities of Αβ peptides and LL-37

[00115] Prior to this new work, no one has established a correlation between cathelicidin expression levels and Alzheimer's disease. However, in addition to those mentioned above, other literature reports suggest that the biophysical activities and signaling functions of Αβ peptides and LL-37 are related in vivo. For instance, the Formyl-like Peptide Receptor 1 (FPRL1) is activated by both Αβ [REF: Amyloid (beta)42 activates a G-protein~coupled chemoattractant receptor, FPR~Hke~l . Le Y, Gong W, Tiffany HL, Turnanov A, Nedospasov S, Shen W, Dunlop MM, Gao JL, Murphy PM, Oppenheim JJ, Wang JM. J eurosci. 2001 Jan 1 5;21 (2):RC123. Pubmed ID PMID 11160457] and LL-37 [REF: LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1.) as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and T ceils. De Yang] , Chen Q, Schmidt AP, Anderson GM, Wang JM, Wooters J, Oppenheim JJ, Chertov O J Exp Med. 2000 Oct 2; 192(7): 1069-74. Pubmed ID PMID 1 1015447]. This receptor is reportedly involved in inflammatory aspects of AD [REF: Potential role of the formyl peptide receptor-like 1 (FPRL1 ) in inflammatory aspects of Alzheimer's disease. Cut Y, Le Y, Yazawa H, Gong W, Wang JM. J Leukoc Biol. 200:2 Oct;72(4):628-35. Review. Pubmed ΪΪ) PMID. 12377930] via its effects on phagocyte responses [REF: Role of formyl peptide receptor -like 1 (FPRL 1 FPR2) in mononuclear phagocyte responses in Alzheimer disease, iribarren P, Zhou Y, Hu J, Le Y, Wang JM. Immunol Res.

2005:31 (3): Ί 65-76. Review. Pubmed PMID: 15888909]; and a decreased phagocyte clearance of CNS β-amyloid is a hallmark of AD [REF: Decr eased clearance of CNS beta-amyloid in Alzheimer's disease. Ma uenyega G1 , Sigurdson W, Ovod V, Munsell L, Kasten T, Morris JC. Yarasheski E, Bateman RJ. Science. 2010 Dec

24,330(6012): 1774. Pubmed ID PMID 21148344]. As mentioned above, LL-37 itself is involved in monocyte and macrophage autophagy; its expression, stimulated by Vitamin D3 [REF: Human cathelicidin antim crobial peptide {("AMP) gene is a direct target of the vitamin D receptor and is strongly up-reguiated in myeloid cells by 1,25- dihydroxy vitamin D3. Gombart AF, Borregaard N, Koeffler HP. FASF.B J. 2005

Jul; 19(9): 1067-77. Pubmed ID PMID 15985530], engenders enhanced autophagy [REF: Vitamin D3 induces autophagy in human monocytes/macrophages via cathelicidin. Yuk JM1 , Shin DM, Lee MM , Yang CS Jin M S. Kim !< K. Lee ZW, Lee SH, Kim JM, Jo i .K Cell Host Microbe. 2009 Sep 17;6(3):23 i-43. Pubmed ID PMID 19748465]. Thus, a dearth of LL-37 generally would reduce phagocytic activity. Phenylbutyrate and Vitamin D3 have each, individually, been shown to be neuroprotective in Alzheimer's Disease mouse models [REF: la,25-Dihydroxyvitamin D3 reduces cerebral amyloid-β

accumulation and improves cognition in mouse models of Alzheimer's disease. Durk MR, Han K, Chow EC, Ahrens R, Henderson JT, Eraser PE, Pang K.S. J Neurosci. 2014 May 21 ;34(21 ): 7091-7101 . Pubraed ID P ID 24849345] and [REF- Curr Phann Des.

2013; 19(28):5076-84. Phenyibutyrate is a multifaceted drug that exerts neuroprotective effects and reverses the Alzheimer ^' s disease-like phenotype of a commonly used mouse model. Cuadrado-Tej edor M, Ricobaraza AL, Torrijo R, Franco R, Garcia-Osta A.

Pubmed ID PMED 23448463], and these same two compounds are known cathelicidin inducers, although this connection between cathelicidin induction and Alzheimer's Disease has not been noticed prior to this patent [REF: Phenyibutyrate induces antimicrobial peptide expression. Steinmami J, Halldorsson S, Agerbex h B,

Gudmundsson GH. Antimicrob Agents Chemother. 2009 Dec;53(12):5 ^' i 27-33. Pubmed ID PMID 19770273]. Finally, there is mounting evidence that innate immunity plays a larger role in Alzheimer's disease than previously thought, and that AP's normal function may be as an antimicrobial and antiviral peptide [REF: The Alzheimer's disease- associated am loid beta-protein is an antimicrobial peptide. Soscia SJ, Kirby JE,

Washicosky KJ, Tucker SM, Ingeisson M, Hyman B, Burton MA, Goldstein LE, Duong S, Tanzi RE, Moir RD. PLoS One. 2010 Mar 3;5(3):e9505. Pubmed ID PMID:

20209079] and [REF: Sci Trans! Med. 2016 May 25;8(340):340ra72. Amyioid-β peptide protects against microbial infection in mouse and worm models of Alzheimer's disease. Kumar DK, Choi SH, Washicosky KJ, Eimer WA, Tucker S. Ghofrani J, Lefkowitz A, McColl G, Goldstein LE, Tanzi RE, Moir RD. Pubmed ID PMID 27225182] and [REF: Bi ©gerontology. 2015 Feb; 16(1 ):85-98. β-Amyioid peptides display protective activity against the human Alzheimer's disease-associated herpes simple . virus- 1. Bourga.de K, Garaeau H, Ciiroux G, Le Page AY, Bocti C, Dupuis G, Frost EH, Fulop T Jr. Pubmed ID PMID 25376108] and [REF: Alzheimer's Disease-Associated β-Amyioid Is Rapidly Seeded by Herpesviridae to Protect against Brain Infection. Eimer WA, Kumar DKV, Shanmugam KN, Rodriguez AS, Mitchell T, Washicosky KJ,Gyorgy B, Breakefl eid. O. Tanzi RE, Moir RD. Neuron. 2018 Jul l l;99(l):56-63]. Kumar et al. showed that Αβ expression protects against fungal and bacterial infections in mouse, nematode, and cell culture models of AD [REF; Sci Trans! Med. 2016 May 25,8(340):340ra72.

Aray!oid-β peptide protects against microbial infection in mouse and worm models of

Alzheimer's disease. Kumar DK, Choi SH, Washicosky KJ, Eimer WA, Tucker S, Ghofrarii J, Leikowitz A, McColl G, Goldstein LE, Tanzi RE, Moir RD. Pubmed ID PMID 27225182]. And, part of the body's natural response to infection is, of course, to upregulate cathelicidin [REF: The chemistry and biology of LL-37, Burton MF, Steel PG. Nat Prod Rep. 2009 Dec;26(! 2): 1572-84. Review. Pubmed ID PMID: 19936387].

Significance of Studies of LL-37: Αβ Interaction and Binding In Vitro

[00116] The work described above demonstrates that these two peptides, the cathelicidin peptide LL-37 and the Alzheimer's Disease-associated peptide Αβ, which are both ubiquitous in human tissues and expressed in the human brain, bind to and interact with each other in different aggregation states, with affinity constants typical of protein- protein interactions and close to values reported in the literature for the binding of Αβ with optimized synthetic peptides. The addition of LL-37 to Αβ42, in a buffer that mimics quasi -physiological conditions, strongly inhibits fibril formation. The protective effect of LL-37' s presence against microglia-mediated Αβ42 toxicity to SH-SY5Y neuroblastoma cells is an important new finding, revealing that in combination the two peptides are 90% less pro-inflammatory than either peptide alone. Literature reports suggest that numerous biophysical activities and signaling functions of Αβ peptides and LL-37 are related in vivo. The in vitro data presented here were the starting point from which we are further investigating whether factors that affect cathelicidin gene CAMP regulation, which will in turn control the production of LL-37 in vivo, modulate Αβ aggregation and/or microglia-induced neuroinflammation. Correlations Between Infections of Brain Tissue, and Alzheimer's Disease

[00117] As has been mentioned above, it has been hypothesized that the Alzheimer's Disease-associated peptide Αβ may itself be an important host defense peptide. However, it is not yet well understood what causes the expression of the Αβ peptide or its precursor protein, APP. [REF: The Alzheimer's disease-associated amyloid beta-protein is an antimicrobial peptide. Soscia SJ, Kirby JE, Washicosky J, Tucker SM, Inge!sson M, Hyman B, Burton MA, Goldstein LE, Duong S, Tanzi RE, Moir RD. PLoS One. 2010 Mar 3;5(3):e9505. Pubmed ID PMiD: 20209079] and [REF: Sci Trans! Med, 2016 May 25;8(340):340ra72. Ara loid-β peptide protects against microbial infection in mouse and worm models of Alzheimer's disease. Kumar DK, Choi SH, Washicosky KJ, Eimer WA, Tucker S, Ghofrani J, Lefkowitz A, McColi G. Goldstein LE, Tanzi RE, Moir RD.

Pubmed ID PMID 27225182] and [REF: Biogerontoiogy. 201 5 Feb; 16(l):85-98. β- Amyloid peptides display protective activity against the human Alzheimer's disease- associated herpes simplex virus- 1. Bourgade K, Gameau H, Giroux G, Le Page AY, Bocti C, Dupuis G, Frost EH, Fiilop T Jr. Pubmed 03 PMID 25376108] Kumar et al. showed that Αβ expression protects against fungal and bacterial infections in mouse, nematode, and cell culture models of AD [REF: Sci Trans! Med. 2016 May

25;8(340):340ra72. Ara loid-β peptide protects against microbial infection in mouse and worm models of Alzheimer's disease. Kumar DK, Choi SH, Washicosky KJ, Eimer WA, Tucker S, Ghofrani J, Lefkowitz A, McColi G. Goldstein LE, Tanzi RE, Moir RD.

Pubmed ID PMID 27225 ! 82]. It has been shown that infection of brain tissue with Herpesvirus results in rapid expression of Αβ peptide, which then entraps the Herpes viral particles in beta-amyloid, which may mean that viral infections actually stimulate the expression of Αβ. [REF: Alzheimer's Disease- Associated β- Amyloid Is Rapidly Seeded by Herpesviridae to Protect against Brain Infection. Eimer WA, Kumar DKV,

Shanmugam NKN, Rodriguez AS, Mitchell T, Washicosky KJ.Gyorgy B, Breakefieid XO. Tanzi RE, Moir RD. Neuron. 2018 Jul l l ;99i l):56-63. Pubmed ID PMID

30001512]. Other pathogens, besides Herpesvirus, which seem to infect brain tissue and correlate with higher risk of Alzheimer's Disease include the bacterial pathogens

Chlamydia pneumoniae and the family of Spirochete bacteria. [REF: Historic evidence to support a causal relationship between spirochetal infections and Alzheimer's disease. Miklossy J. Front Aging Neurosci . 2015 Apr 16;7:46. Pubmed ID PMID 25932012] and [REF: Chlamydia pneumoniae infection of monocytes in vitro stimulates innate and adaptive immune responses relevant to those in Alzheimer's disease. Lim C, Hammond CJ, Hmgley ST, Balin BJ. J Neuromfi animation. 2014 Dec 24: 1 :21 7, Pubmed ID PMID: 25540075] and [Bacterial Amyloid and DNA are Important Constituents of Senile Plaques: Further Evidence of the Spirochetal and Biofilm Nature of Senile Plaques.

Miklossy J. J Alzheimers Dis. 2016 Jun 13;53(4): 459-73. Pubmed ED 27.314530] A meta-analysis of all studies that have examined the relationship between bacterial infections of brain tissue, and Alzheimer's Disease (AD), found over a ten-fold increased occurrence of AD when there was detectable evidence of spirochetal infection (OR:

10.61; 95% CI: 3.38-33.29), with over a four-fold increased occurrence of AD as a conservative risk estimate (OR: 4.45; 95% CI: 2.33-8.52). The same meta-analysis also found over a five-fold increased occurrence of AD with Chlamydophila pneumoniae infection (OR: 5.66; 95% CI: 1.83-17.51). Therefore, there is a strongly positive association between bacterial infection of the brain and AD. [REF: Bacterial infection and Alzheimer's disease: a meta-analysis. Maheshwari P, Eslick GD. J Alzheimers Dis. 2015;43( ^" 3):957-66. Pubmed ID PMID: 25182736] If indeed, as these studies indicate, Αβ is a host defense peptide just as LL-37 is, then their role as binding partners that can detoxify each other also makes sense. Also, it should be noted that the LL-37 peptide has been shown to be active against both HSV-1 and HSV-2, two different strains of

Herpesvirus against which it has been tested. [REF: Human cathelicidin (LL-37), a.

multifunctional peptide, is expressed by ocular surface epithelia and has potent antibacterial and antiviral activity. Gordon YJ1, Huang LC, Romanowski EG, Yates A, Proske Ri, McDermoti AM . Curr Eye Res. 2005 May;30(5):385-94. Pubmed Π) PMID 16020269] and [REF: Cathelicidin deficiency predisposes to eczema herpeticum. Howell MD, Wollenberg A, Gallo RL, Flaig M, Streib JE, Wong C, Pavicic T, Boguniewicz M, Leung DY. J Allergy Clin Immunol 2006 Apr; 117(4):836-41. Pubmed ID PMID

16630942] Interestingly, the particular bacterial pathogen Chlamydia Pneumoniae , which is associated with AD, appeared in one study to resist the antibacterial activity of LL-37 peptide. [REF: Involvement of the antimicrobial peptide LL-37 in human atherosclerosis. Edfeldt K, Agerberth B, Rottenberg ME, Gudmundsson Gil Wang XI Mandal K, Xu Q, Yan ZQ. Artenoscler Thromh Vase Biol. 2006 Ju!;26(7): 155 ^' i -7. Pubmed ID PMID 16645 154] Finally, a 2018 study of the brains of patients with Alzheimer's Disease found increased prevalence of human herpesvirus 6A (HHV-6A) and human herpesvirus 7 (HHV-7) within the brain tissue of subjects with Alzheimer's Disease, compared with controls. These results were replicated in two additional, independent and geographically dispersed cohorts. [REF: Multi scale Analysis of independent Alzheimer's Cohorts Finds Disruption of Molecular, Genetic, and Clinical Networks by Human Herpesvirus.

Readhead B, Haure-Mirande IV, Funk CC, Richards MA, Shannon P, Haroutunian V, Sano M, Liang WS, Beokmann ND, Pnce ND, Reman EM, Schadi EE, Ehrlich ML, Gandy S, Dudley JT. Neuron. 2018 Jul 1 l;99(l):64-82.e7. Pubmed ID PMID 29937276] Whether this statistically significant, increased burden of Herpesviridae in the brains of Alzheimer's Disease sufferers is causative of neurodegeneration, or correlative with the disease process, is not yet clear; however, this finding is important. If, as put forward by Moir and Tanzi et al., Αβ amyloid oligomers, fibrils and plaques serve to sequester and inactivate Herpesvirus particles, then LL-37-stimulated macroautophagy may serve to clear away these molecular debris from the brain, once the virus has been rendered inactive by immobilization in Αβ plaques. [REF: Vitamin D3 induces autophag in human monocytes / macrophages via cathelicidin. Yuk JM, Shin DM, Lee FIM, Yang CS, Jin HS, Kim KK, Lee ZW, Lee SH, Kim JM, Jo EK. Cel l Host Microbe, 2009 Sep 17;6(3):231-43. Pubmed ID PMID: 19748465] Inducers of Cathelicidin Gene, CAMP, and Obligate Co-Factors for CAMP

Expression

[00118] There are a number of known inducers and essential cofactors for expression of the cathelicidin gene, which is called CAMP in humans, and camp in mice. One important compound for the induction of CAMP gene expression is Vitamin D3. [REP: Human cathelicidin antimicrobial peptide (CAMP) gene is a direct target of the vitaxnin D receptor and is strongly up-regulated in myeloid ceils by 1 ,25-di hydroxy vitamin D3. Gombart API, Borregaard N, Koeffler HP, FASEB J. 2005 Jul; 19(9): 1067-77. Pubmed ID PMID 15985530] and [REF: Vitamin D as an inducer of cathelicidin antimicrobial peptide expression: past, present and future. White JH. J Steroid Biochem Moi Biol. 2010 ;121( l-2):234-8. Pubmed ID PMID 20302931 ] Vitamin D3 is a particularly potent inducer of cathelicidin in human immune cells. [REF: Regulation of the human cathelicidin antimicrobial peptide gene by la,25-dibydroxy vitamin D3 in primaiy immune cells. Lowry MB, Guo C, Borregaard N, Gombart AF. J Steroid Biochem Mol Biol . 2014 Sep;l 43 -.183-91. Pubmed PMID: 24565560] Interestingly, this Vitamin D3 requirement for CAMP expression in human beings and primates, especially, is related to the existence of a Vitamin D Receptor (VDR) binding domain in transcriptional control region of the gene. [REF: Exaptation of an ancient Alu short interspersed element provides a highly conserved vitamin D-mediaied innate immune response in humans and primates. Gombart AF, Saito T, Koeffler HP. BMC Genomics. 2009 Jul 16, 10:321. doi: 10. 1 186/1471-2164-10-321. Pubmed ID PMID 19607716] It has been shown that the regulation of cathelicidin expression by Vitamin D3 differs, in different tissues of the body. [REF: Regulation of the CAMP gene by l,25(OH)2D3 in various tissues. Gombart AF, O'Kelly J, Saito T, Koeffler HP. J Steroid Biochem Mol Biol . 2007 Mar, 103(3- 5):552-7. Pubmed 113 PMID. 17368184] There is also a Retinoid X Receptor (RXR) binding domain in the CAMP transcriptional control region, which makes RXR agonists obligate cofactors for CAMP gene expression, along with Vitamin D3. RXR agonists include, for instance, natural molecules such as Retinoic Acid (Vitamin A), which is a metabolic by-product of the dietary retinoid β-Carotene, and/or Dodecahexanoic Acid (DHA). A potent non-natural RXR agonist is the compound Bexarotene, which is an FDA-approved drug for cancer treatment. [REF: Vitamin D- duced up-regulation of human keratinoeyte cathelicidin antimicrobial peptide expression involves retinoid X receptor a. Svensson D, Nebe! D, Voss U, Ekhiad E, Nilsson BO. Cell Tissue Res. 2016 Nov;366(2):353-362. Jun 30. Pubmed ID PMID 27357804] and [REF: Label-free quantitative mass spectrometry reveals novel pathways involved in LL-37 expression. Cederlund A, Nylen F, Miraglia E, Bergman P, Gudmundsson GH, Agerberth B. J Innate immun. 2014;6(3):365-76. doi: 10.1 159/000355931. Epuh 2013 Nov 16. Pubmed PMID: 24246949]

[00119] The use of the RXR agonist DHA in combination with Bexarotene shows synergy and more efficient activation of Retinoid X Receptors, such that a lower dosage of the FDA-approved drug Bexarotene can be used as an RXR agonist, reducing any potential toxicity to liver or kidneys. [REF: Omega-3 Fatty Acids Augment the Actions of Nuclear Receptor Agonists in a Mouse Model of Alzheimer's Disease. Casali BT, Corona AW, Mariani MM, Karlo JC, Ghosai , Landreth GE, J Neurosci . 2015 Jun 17;.35(24):9173-81. Pubmed ED PMID 26085639]

[00120] In addition to Vitamin D3, certain short-chain fatty acids are able to induce the expression of the CAMP gene, especially Butyrate. [REF: Expression of the cathelicidin LL-37 is modulated by short chain fatty acids in coionocytes: relevance of signalling pathways. Schauber J, Svanholm C, Termen S, Iffland K, Menzel T,

Scheppach W, Melcher R, Agerberth B, Luhrs FL Gudmundsson GH. Gut. 2003

May;52(5):735-41. Pubmed ID PMID: 12692061 ] However, Butyrate is an undesirable therapeutic compound, as it has a noxious smell. As a replacement for Butyrate induction of CAMP gene expression, it was discovered the Phenylbutyrate is highly effective; and does not smell bad. [REF: Phenylbutyrate induces antimicrobial peptide expression. Steinmann J, Haildorsson S, Agerberth B, Gudmundsson GIL A timicrob Agents Chemother. 2009 Dec;53(12):5127-33. doi: 10. 128/AAC, 00818-09. Epub 2009 Sep 21. Pubrned ID PMID: 19770273] The combination of Vitamin D3 and Phenylbutyrate is synergistic in its ability to induce CAMP gene expression, providing enhanced expression of LL-37 and antibacterial activity, relative to what can be achieved with just one of the two compounds. [REF: Vitamin D?and phenylbutyrate promote development of a human dendritic ce!i subset displaying enhanced antimicrobial properties, van der Does AM, enne E, oppelaar E, Agerberth B, Lindbom L. J Leukoc Biol. 2014 jim,95(6):883-9L Pubmed ID PMID: 24550524] In the United States, the compound Phenylbutyrate is an FDA-approved drug for the treatment of Urea-Cycle Disorders. [REF: Profile of sodium phenylbutyrate granules for the treatment of urea-cycle disorders: patient perspectives. Pefia-Qumtana L, Llarena M, Reyes- Suarez D, Al damiz-Echevarria L. Patient Prefer Adherence. 2017 Sep 6;1 1 : 1489-1496. Review. Pubmed ID PMID: 28919721] It has been shown that Phenylbutyrate induction of CAMP gene also occurs via interaction with the Vitamin D receptor, like Vitamin D3 itself. [REF; Phenylbutyrate induces cathelicidin expression via the vitamin D receptor: Linkage to inflammatory and growth factor cytokines pathways. Kulkarni N , Yi Z, Huehnken C, Agerberth B, Gudmundsson GH. Mol Immunol. 2015 Feb;63f2):530-9. Pubmed ID PMID: 25458314] The synergistic induction of the CAMP gene by the combination of Vitamin D3 and Phenylbutyrate has been developed as a novel treatment for bacterial infections, for instance, for the treatment of tuberculosis. A dosage study was done in humans, and the ideal dose for induction of cathelicidin for the purpose of treating lung infection was 5000 IU Vitamin D3 taken daily, plus 500 mg Phenylbutyrate per dose taken twice per day. [REF:

Induction of the human cathelicidin LL-37 as a novel treatment against bacterial infections, van der Does AM, Bergman P. Aaerherth B. Lindbom L. J Leukoc Biol. 2012 Oct;92(4):735-42. Review. Pubmed ID PMID: 2270.1042] and [REF: Oral intake of phenylbutyrate with or without vitamin D3 upregulates the cathelicidin LL-37 in human macrophages: a dose fi nding study for treatment of tuberculosis. Mily A, Rekha RS, Kama! SM, Akhtar E. Sarker P, Rahira Z, Gudmundsson GH, Agerberth R, Raqib R. BMC Pulm Med, 2013 Apr 16;13:23. doi: 10.1186/1471-2466-13-23. Pubmed ID PMID: 23590701] and [REF: Significant Effects of Oral Phenylbutyrate and Vitamin D3

Adjunctive Therapy in Pulmonary Tuberculosis: A Randomized Controlled Trial. Miiy A, Rekha RS, Kamal SM, Arifuzzaman AS, Rahim Z, Khan L, Haq MA, Zaman K, Bergman P, Brighenti S, Gudmundsson GH, Agerberth B, Raqib R. PLoS One. 2015 Sep 22; 10(9):e0138340. Pubmed ID PMID: 26394045] and [REF: immune responses in the treatment of drug-sensitive pulmonary tuberculosis with phenylbutyrate and vitami D3 as host directed therapy. Rekha RS, Miiy A, Sultana T, Haq A, Ahmed S, Mostafa Kamal SM, van Schadewijk A, Hiemstra PS, Gudmundsson GH, Agerberth B, Raqib R. BMC Infect Dis. 2018 Jul 4; I8i l ):303. doi: 10.1186/s Ϊ 2879-0 Ϊ 8-3203 - . Pubmed II ⁾ PMID: 29973153] Phenylbutyrate has also been tested in a preclinical animal trial, using rabbits, for the treatment of enteropathogenic E. Co/z ^' -induced diarrhea, with good success. [REF . Treatment with phenylbutyrate in a pre-ciinical trial reduces diarrhea due to

e teropathogeni c Escherichia coli: link to cath.elicid.in induction. Al-Mamun A, Miiy A, Sarker P, Tiash S, Navarro A, Akter M, Taiukder KA, Islam MF, Agerberth B,

Gudmundsson GH, Cravioio A, Raqib R. Microbes Infect. 2013 Nov; 15(13):939-50. Pubmed ID PMID: 24016414]

[00121] Other compounds have also been shown, more recently, to induce the expression of the CAMP gene, but by Vitamin D3 receptor-independent mechanisms that are not fully understood. For instance, the compound Curcumin induces CAMP gene expression by a non- Vitamin D3 receptor dependent mechanism. [REF. Curcumin induces human cathelicidin antimicrobial peptide gene expression through a vitamin D receptor-independent pathway. Guo C, Rosoha E, Lowry MB, Borregaard N, Gombait AF. J Nutr Biochem. 2013 May:24(5):754-9. Pubmed ID PMID: 22841393] Additionally, compounds from the family of stilbenoids, in particular Resveratrol or Pterostilbene, also induce CAMP gene expression by a Vitamin D3 receptor-independent mechanism, which is also synergistic with Vitamin D3. [REF: Synergistic induction of human cathelicidin antimicrobial peptide gene expression by vitamin D and stilbenoids. Guo€, Sinnott B, Niu B, Lowry MB, Fantacone ML, Gornbart AF. Moi Nutr Food Res. 201 Mar;58(3):.528-536. Pubmed ID PMID: 24039193] It has been shown that the manner in which Resveratrol induces cathelicidin expression is by a novel mechanism involving sphingosine-1 -phosphate pathway signaling. [REF: Resveratrol stimulates sphingosine-l- phosphate signaling of cathelicidin production. Park K, Elias PM, Hupe M, Borkowski AW, Gallo RL, Shin KG, Lee YM, Holleran WM, Uchida Y. J Invest Dermatol. 2013 Aug, 133(8); 1942-9. Pubmed 03 PMID: 23856934] and [REF: Sphingosine kinase 1 activation enhances epidermal innate immunity through sphingosine- 1 -phosphate stimulation of cathelicidin production. Jeong SK, Kim YI, Shin KO, Kim BW, Lee SH, Jeon JE, Kim HJ, Lee YM, auro T , Elias PM, IJchida Y, Park K. J Dermatol Sci . 2015 Sep;79(.3):229-34. Pubmed ID PMID: 261 131 14] One lipid molecule called

Genistein was shown in 2015 also to induce cathelicidin expression by a sphingosine- 1- phosphate stimulation mechanism. [REF: The dietary ingredient, genistein, stimulates cathelicidin antimicrobial peptide expression through a novel Si -dependent mechanism. Park K, Kim YI, Shin KO, Seo HS, Krra JY, Mann I, Oda Y, Lee YM, Holleran WM, Elias PM, IJchida Y J utr Biochem. 2014 Jul;25(7);734-40. Pubmed PMID: 24768661] Genistein is a soy (or other plant, such as red clover)-derived isoflavanoid compound that has a multitude of health benefits. [REF; Bioactivity of genistein: A review of in vitro and in vivo studies. Ganai AA, Farooqi H. Bioraed Pharmacother. 2015 Dee;76:30~8, doi : 10.1016/j.biopha.2015.10.026. Epub 2015 Nov 10. Review. Pubmed ID PMID:

26653547] Interestingly, Genistein and Resveratrol have been shown to have synergistic anticancer effects (with no reference to cathelicidin gene expression); this highlights that natural compounds of this type often show synergies. [REF: A new class of hybrid anticancer agents inspired by the synergistic effects of eurcumin and genistein: Design, synthesis, and a ti-proliferative evaluation. Chen QH, Yu K, Zhang X, Chen G, Hoover A, Leon F, Wang R, Subrahman am N, Addo Mekuria E, Harinantenaina Rakotondraibe L. Bioorg Med Chem Lett. 2015 Oct 15;25(20):4553-6. doi: 1.0.1016/j .brncI.2015.08.064. Epub 2015 Aug 24. Pubmed ID PMID: 26341 135] And again, it should be noted that Curcumin's induction of cathelicidin expression is also by a non- Vitamin D receptor dependent mechanism; but perhaps different from that of Genistein, given their synergistic effects in treating cancer. [REF: Curcumin induces human cathelicidin antimicrobial peptide gene expression through a vitamin D receptor-independent pathway. Guo C, R.osoha E, Lowry MB, Borregaard N, Gombart AF. J Nutr Biochem . 2013 May;24(5):754-9. Pubmed D PMiD: 22841393]

[00122] In 2016, a novel family of compounds called aroylated phenylenediamines was developed to achieve the potent induction of the cathelicidin gene, which includes the compound Entinostat, for the treatment of infections such as Cholera. [REF: Potent Inducers of Endogenous Antimicrobial Peptides for Host Directed Therapy of infections. Ottosson H, NyJen F, Sarker P, Miraglia E, Bergman P, Gudmundsson GH, Raqib R, Agerberth B, Stromberg R. Set Rep. 2016 Nov 9;6:36692. Pubmed ID PMID: 27827460] and [REF; Entinostat up-reguiates the CAMP gene encoding LL-37 via activation of STAT3 and HIF-Ι transcription factors. Miraglia E, Ny!en F, Johansson K, Araer E, Cebuia M, Farm and S, Ottosson H. Stromberg R, Gudmundsson GH, Agerberth B, Bergman P. Sci Rep. 2016 Sep 16,6:33274. Pubmed ID PMID: 27633343] and [REF: Treatment with Entinostat Heals Experimental Cholera by Affecting Physical and Chemical Barrier Functions of Intestinal Epithel a, Sarker P, Banik A, Stromberg R, Gudmundsson GH, Raqib R, Agerberth B. Antimicrob Agents Chemother. 201.7 Jun 27;61 (7). pii: e02570-16. Pubmed ID PMID: 28438947]

[00123] The combination of Vitamin D3 and Phenylbutyrate has been shown to be effective for the treatment of infections such as tuberculosis, and the aroylated phenylenediamine Entinostat is able to induce cathelicidin gene expression via activation of STAT3 and HIF-la transcription factor, again to treat cholera. However, until now, no combination of G MP-inducing compounds has ever been proposed for the potential treatment of Alzheimer's Disease. This is because it has only recently been discovered that the LL-37 peptide can bind to and detoxify the Alzheimer's Disease-related peptide Αβ. Thus, until 2017 when this patent was filed, no one would have considered CAMP gene induction as a potential therapy for Alzheimer's Disease. [REF: Evidence that the Human innate Immune Peptide LL-37 may be a Binding Partner of Amyloid-β and Inhibitor of Fibril Assembly. De Lorenzi E, C iari , Colombo R, Cretich M, Sola L, Vanna R, Gagni P, Bisceglia F, Morasso C, Lin JS, Lee M, McGeer PL, Barron AE. J Alzheimers Dis. 2017;59(4): 1213-1226. Pubmed ID PMID 28731438]

[00124] Levels of cathelicidin in the human body can be measured by quantitative PCR of DNA extracted from white blood cells, by fluorescent ELISA assays of protein levels, or by radioimmunoassays, as disclosed in these two papers: [REF: Oral intake of phenylbutyrate with or without vitamin D3 upregulates the cathelicidin LL-37 in human macrophages: a dose finding study for treatment of tuberculosis. Miiy A, Rekha RS, amal SM, Akhtar E, Sarker P, Rahim Z, Gudmundsson GH, Agerberth B, Raqib R. BMC Pu!m Med. 2013 Apr 16; 13 :23. doi : 10.1 186/1471-2466-13-23. Pubmed ID PMID: 23590701 ] and [REF: PLoS One. 2016 May 6; 1 1 (5 }:e0152? 1 1. Circulating Cathelicidin Concentrations in a Cohort of Healthy Children: Influence of Age, Body Composition, Gender and Vitamin D Status. Stukes I ^' M, Shary JR, Wei W. Ebeling MD, Dezsi KB, Shary FS, Forestieri NE, Mollis BW, Wagner CL. Pubmed 03 PMID 27152524]

[00125] We disclose here a novel polytherapy, i.e., mixture of a number of different compounds, for the purposes of inducing the expression of the cathelicidin gene CAMP (see FIGs. 35-36). As all of these C4 P-inducing compounds are hydrophobic or lipophilic, and not soluble in water, we dissolved them in liquid coconut oil, or medium- chain triglyceride oil, composed of capric acid, caprylic acid, and lauric acid. The mixture of compounds that we disclose for the synergistic induction of CAMP gene expression, which can have many human health benefits, including the potential treatment of Alzheimer's Disease and other conditions is as follows: Vitamin D3, Phenylbutyrate, Curcumin, Resveratrol, Bexarotene, and DHA, all dissolved in liquid coconut oil (which comprises capric acid, caprylic acid, and lauric acid) (FIGs. 35-36). The rationale for using this mixture of six different compounds, dissolved in coconut oil, is that all of these have been shown to induce LL-37 expression, with a variety of different mechanisms of induction, and with synergy of induction occurring (by different mechanisms), at least for the pair of Vitamin D3 and Phenylbutyrate, and Vitamin D3 and Resveratrol. The other rationale for combining these six compounds are that all of them are proven to be safe for dosing in humans. Vitamin D3, Curcumin, Resveratrol, and DHA are all GRAS (Generally Recognized As Safe) compounds according to the U.S. FDA, whereas both Phenylbutyrate and Bexarotene are already FDA-approved as therapeutic compounds, which means that safe doses have been established. Given that all of these compounds have different structures and degrees of lipophilicity /

hydrophilicity (FIGs. 35-36), they will each partition differently into different body compartments, and can broaden the mechanism of cathelicidin induction by being used simultaneously.

[00126] We have shown that this particular combination of six compounds, dissolved in liquid coconut oil, and orally dosed in mice every other day, can induce the expression of the cathelicidin gene in the brain. The particular dosage used to show this brain induction of cathelicidin expression, for the first time, is shown in TABLE 2 below.

TABLE 2: Dosages for Induction of Cathelicidin Expression in C57BL/6J Mice

DHA 66.67 1.67

Coconut oil (MCT oil) 100

[00127] Preliminary studies in wild-type (C57BL/6J) mice using the oral polytherapy treatment, dosed as described in the table above, demonstrated significantly increased levels of cathelicidin gene expression in brain tissue compared to untreated controls. (FIG. 12). In this study, groups of 4-5 wild type mice were dosed every other day, for 2 weeks, with the polytherapy formulation shown in the table. Then, after two weeks of dosing, at particular and exact times after the last dose was given, the mice were sacrificed, and their brain tissue was tested by quantitative PCR for the levels of cathelicidin gene expression. This allowed us to see the time-dependent cathelicidin gene expression after dosing, at 30, 60, and 180 minutes after dosing. This polytherapy provided, on average, between 3-fold and 7.5-fold induction of cathelicidin gene expression, within 3 hours. This is the first time that oral induction of cathelidin gene expression in the brain has ever been shown; this is a new and unpublished result.

[00128] We have also tested this oral polytherapy using the 5XFAD mouse model as an inflammatory preclinical model of Alzheimer' s disease with an aggressive amyloid beta accumulation pathology, to model cerebral amyloidosis with intracellular amyloid beta and amyloid beta plaques beginning to appear as early as 2 months' age. [REF: Neuron loss in the 5XFAD mouse model of Alzheimer's disease correlates with intratieuronal Α.β42 accumulation and Caspase-3 activation. Eiraer WA, Vassar R . Mol Neurodegener. 2013 Jan 14;8:2. Pubmed ID PMID: 23316765] and [REF: Genetic mouse models of brain ageing and Alzheimer's disease. Bilkei-Gorzo A. Pharmacol Ther. 2014 May; 142(2):244-57. Review. Pubmed ID PMID: 24362083] and [REF: Molecular Differences and Similarities Between Alzheimer's Di sease and the 5XFAD Transgenic Mouse Model of Amyloidosi s. Maarouf CL, Kokjohn TA, Whiteside CM, Macias MP, alback WM, Sabbagh MN. Beach TG, Vassar R, oher AE. Biochem Insights. 2013 Nov 21;6:1 -10. Pubmed ID PMID: 25210460] Experiments commenced at 5 months age. Mice were dosed with the polytherapy as described in Table 2, every other day, for 70 days total. The 5XFAD transgenic model reliably replicates neuropathology and cognitive symptoms of Alzheimer's disease not present in alternative models. In order to obtain sufficient power in our statistical analysis and taking into account the inherent variability in behavioral testing and stereotaxic injections, we used 14-15 mice in each treatment group as well as in the untreated control groups. We compared four groups: (1) treated and (2) untreated wild type C57BL/6J mice, and (3) treated and (4) untreated 5XFAD mice. Mouse studies were carried out with APLAC and IACUC approval.

[00129] We have completed the dosing portion of the study and are in the process of analyzing the data to look for treatment effects on amyloid plaque loads, inflammation, autophagy and cell senescence. As shown in FIGs. 32-34, the results from behavioral testing are promising (contextual fear conditioning) in which treated 5XFAD mice showed memory comparable to wild type mice, and significantly improved over untreated 5XFAD. This indicates efficacy of this six-component oral polytherapy in treating Alzheimer's Disease, in the well-accepted 5XFAD mouse model of this neurodegenerative disease (unpublished data).

[00130] Interestingly, in 2015 it was shown that neutrophils and their creation of Neutrophil Extracellular Traps (NETs) play a role in the inflammatory brain pathology in the 5XFAD mouse model of Alzheimer's Disease. It was found that activated neutrophils could extravasate and cross the blood brain barrier, and release NETs inside brain tissue. [REF: Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin. Zenaro E, Pietronigro E, Delia Bianca V, Piacentino G, Marongiu L, Budui S, Turano E, Rossi B, Angiari. S, Dusi S, Montresor A, Carlucci T, Nam S, Tosadori G, Caiciano L, Catalucci D, Berton G, Bonetti B, Constantin G. Nat Med. 2015 Aug;2.1 (8): 880-6. Pubmed ID PMID: 26214837] Since, as discussed above, the cathelicidin peptide plays a central role in the clearance of neutrophil NETs, which otherwise can be proinflammatory if left in place, it makes sense that the induction of the camp cathelicidin gene in 5XFAD mice was able to enhance their cognitive function, as shown in FIGs. 32-34, where the treated 5XFAD mice had memory that was equivalent to the wild type mice, when tested in the same fear conditioning experiment.

[00131] In a prior study in which CAMP induction with Vitamin D3 and

Phenylbutyrate to treat tuberculosis, the proper dosing for humans of Vitamin D3 was found to be 5000 IU, and for Phenylbutyrate was 500 mg per dose, twice per day. [REF: Oral intake of phenylbutyrate with or without vitamin D3 upreguiates the cathelicidin LL-37 in human macrophages: a dose finding study for treatment of tuberculos s. Mily A, Reklia RS. Kama! SM, Akhtar E, Sarker P, Rahim Z, Gudmundsson GH, Agerberth B. Raqib R. BMC Pulm Med, 2013 Apr 16; 13 :23. doi: 10.1 186/1471 -2466-13-23. Pubmed ID PMID: 23590701 ] The FDA-recommended and approved initial dosage of the drug Bexarotene is 300 mg/m ² /day. The dosages of the other compounds, Curcumin,

Resveratrol, and DHA, could for example be set at typical levels they are provided as GRAS daily supplements: Curcumin, 500 mg per dose, twice per day; Resveratrol, 250 mg per dose, twice per day; and DHA, 500 mg per dose, twice per day. However, since these G MP-induced compounds are provided in a polytherapy, much lower doses of each or any individual compound may be effective for the intended purpose, in combination with the others.

[00132] It is expected that this synergistic combination of compounds that all help induce cathelicidin expression by causing the expression of the LL-37 peptide in the brain. Without wishing to be bound by theory, this is expected to serve as a useful treatment for Alzheimer' s Disease by (1) binding to and detoxifying the Alzheimer' s Disease-associated peptide Αβ; (2) enhancing the autophagic clearance of Αβ fibrils and plaques in the brain, as well as the macroautophagy of any dead and damaged tissue within the brain; (3) enhancing tissue regeneration and angiogenesis, improving the oxygenation of brain tissue; (4) enhancing mitochondrial biogenesis in brain cells, and supporting mitochondrial homeostasis; and (5) helping to enhance the clearance of any pathogens that may infect brain tissue, including viruses such as Herpevirus, and bacteria such as Chlamydia Pneumoniae and Spirochetes, which are associated with Alzheimer's Disease and dementia. This is a novel approach to inducing the CAMP gene, which may prove beneficial not only for the treatment of Alzheimer's Disease, but for the treatment of other conditions and diseases as well. In the preferred embodiment, a polytherapy of compounds has been intentionally selected, each of which has already been proven to be safe for humans in extensive testing, since this treatment may be most effective if taken regularly.

[00133] The pharmaceutical compositions disclosed herein may utilize one or more active ingredients (and will preferably utilize multiple active ingredients) which may be dissolved, suspended or disposed in various media. Such media may include, for example, various liquid, solid or multistate media such as, for example, emulsions, gels or creams. Such media may include liquid media, which may be hydrophobic or may comprise one or more triglycerides or oils. Such media may include, but is not limited to, vegetable oils, fish oils, animal fats, hydrogenated vegetable oils, partially hydrogenated vegetable oils, synthetic triglycerides, modified triglycerides, fractionated triglycerides, and mixtures thereof. Triglycerides used in these pharmaceutical compositions may include those selected from the group consisting of almond oil; babassu oil; borage oil; blackcurrant seed oil; black seed oil; canola oil; castor oil; coconut oil; corn oil;

cottonseed oil; evening primrose oil; grapeseed oil; groundnut oil; mustard seed oil;

olive oil; palm oil; palm kernel oil; peanut oil; rapeseed oil; safflower oil; sesame oil; shark liver oil; soybean oil; sunflower oil; hydrogenated castor oil; hydrogenated coconut oil; hydrogenated palm oil; hydrogenated soybean oil; hydrogenated

vegetable oil; hydrogenated cottonseed and castor oil; partially hydrogenated soybean oil; soy oil; glyceryl tricaproate; glyceryl tricaprylate; glyceryl tricaprate; glyceryl triundecanoate; glyceryl trilaurate; glyceryl trioleate; glyceryl trilinoleate; glyceryl trilinolenate; glyceryl tricaprylate/caprate; glyceryl tricaprylate/caprate/laurate; glyceryl tricaprylate/caprate/linoleate; glyceryl tricaprylate/caprate/stearate; saturated

polyglycolized glycerides; linoleic glycerides; caprylic/capric glycerides; modified triglycerides; fractionated triglycerides; and mixtures thereof. The use of coconut oil is especially preferred.

[00134] Various fatty acids may be utilized in the pharmaceutical compositions disclosed herein. These include, without limitation, both long and short chain fatty acids. Examples of such fatty acids include, but are not limited to, docosahexaenoic acid, caprylic acid, capric acid, lauric acid, butyric acid, and pharmaceutically acceptable salts thereof.

[00135] The pharmaceutical compositions disclosed herein may be applied in various manners. Thus, for example, these compositions may be applied as oral, transdermal, transmucosal, intravenous or injected treatments, or via cell-based drug delivery systems. Moreover, these compositions may be applied in a single dose, multi-dose or controlled release fashion.

[00136] The pharmaceutical compositions disclosed herein may be manufactured as tablets, liquids, gels, foams, ointments or powders. In some embodiments, these compositions may be applied as microparticles or nanoparticles.

[00137] In some embodiments, the pharmaceutically acceptable compositions disclosed herein preferably include a mixture of at least four more preferably at least five, and most preferably at least six materials (preferably active materials) selected from the group consisting of phenylbutyrate, bexarotene, curcumin, resveratrol, retinol, phenylbutyrate, cholecalciferol, fatty acids, and pharmaceutically acceptable salts thereof. In other embodiments, the pharmaceutically acceptable compositions disclosed herein preferably include a mixture of at least four more preferably at least five, and most preferably at least six materials (preferably active materials) selected from the group consisting of phenylbutyrate, bexarotene, curcumin, resveratrol, retinol, phenylbutyrate, cholecalciferol, docosahexaenoic acid, caprylic acid, capric acid, lauric acid, and pharmaceutically acceptable salts thereof.

[00138] Various counterions may eb utilized in forming pharmaceutically acceptable salts of the materials disclosed herein. One skilled in the art will appreciate that the specific choice of counterion may be dictated by various considerations. However, the use of sodium and hydrochloride salts may be preferred in some applications.

[00139] The above description of the present invention is illustrative, and is not intended to be limiting. It will thus be appreciated that various additions, substitutions and modifications may be made to the above described embodiments without departing from the scope of the present invention. Accordingly, the scope of the present invention should be construed in reference to the appended claims.