TECHNICAL FIELD OF THE INVENTION This invention relates to medical devices suitable for irradiating pancreatic beta- cells and methods for such irradiation.

BACKGROUND Beta-cells are an important group of cells located in the human pancreas. The key functions of beta-cells are insulin production, storage and release and thereby the control of blood glucose concentration. T1 D, also known as insulin dependent diabetes, is believed to be caused by an autoimmune response where the immune system destroys beta-cells [1 ]. In addition, beta-cell death is implicated in Type 2 diabetes mellitus [2]. Promoting the proliferation of residual or newly-generated beta- cells has the potential to improve patient health in some forms of diabetes, and thus this is pursued and supported by many parties. These parties include industry (e.g. beta-cell proliferation is a beneficial effect of treatment with glucagon-like peptide 1 (GLP1 ) [3]), funding agencies and many academic researchers.

Very few methods are currently available to control beta-cell proliferation in patients. Chemical factors promoting the growth of beta-cells, such as peptide growth factors including hepatocyte growth factor (HGF) or GLP1 , are efficacious in animal models of diabetes in vitro and in vivo [4, 5]. However, many of these factors and their cognate cell receptors have widespread tissue distribution, and systemic factor administration may result in non-specific effects, such as undesired proliferation of non-beta-cells and an increased risk of cancer [6-9]. In addition, many factors exhibit limited half-life in the circulation and thus are difficult to apply to patients. Finally, factor delivery using gene therapy is sustained and cannot be withdrawn at later stages or after treatment.

Phototherapy, the illumination of tissues affected by or causative for disease, has been described for controlling cellular functions. Phototherapy methods either employ photosensitizers (chemical molecules that respond to light and result in a modification of cellular functions [10]) or optogenetic methods and constructs (e.g., engineering microbial opsins into mammalian cells or tissue [1 1 ]). Reinbothe et al. and Kushibiki et al. [12, 13] describe optogenetic control of insulin secretion in beta -cells using transgenic cells expressing the light-sensitive cation channel Channelrhodopsin-2 (ChR2), an algal protein. Blue light stimulation of the beta-cells triggered increases in intracellular Ca ⁺⁺ and enhanced insulin secretion. There was no effect of light stimulation on Ca ⁺⁺ in non-ChR2 expressing control beta- cells, and glucose-induced insulin secretion of control beta-cells was unaffected by blue light illumination.

US2012/0197358A1 discloses techniques for managing diabetes and prediabetes. A light generating device is positioned close to a body area rich in adipose tissue, such as the abdominal area, thigh, buttocks, and upper arm of a patient, and a low energy light beam (wavelength (λ) ranging from 400 to 1300 nm, power (P) ranging from 1 to 12 mW) is used. Such light therapy is described to increase adiponectin synthesis by the adipose tissue and modulate the immune system.

There is a need to develop effective diabetes treatment optionally used as complementary physical treatment in addition to administration of pharmaceuticals.

SUMMARY

It is the object of the invention to manage diabetes in a patient by ex vivo and in vivo phototherapy treatment.

The object is solved by the subject matter as claimed and further described herein.

According to the invention, there is provided a biocompatible device for invasive phototherapy comprising a light emitting phototherapy source (LEPTS) incorporating an outlet or connected with an outlet, suitable for emitting light to a target, wherein the outlet and optionally the LEPTS or the device is suitable to be located implanted in a patient suffering from diabetes into an area within or in close proximity to the pancreas, to deliver adequate light intensity onto pancreatic beta-cells and/or the pancreas, and the LEPTS is suitable to irradiate the target pancreatic beta-cells with an effective energy dose, using at least one wavelength of visible light to activate pancreatic opsin proteins. According to a specific aspect, such device is used with the exception of such uses that comprise or encompass an invasive step representing a substantial physical intervention on the body of a human or an animal, which requires professional medical expertise to be carried out and which entail a substantial health risk even when carried out with the required professional care and expertise.

According to a further specific aspect, the device is applied to a patient by a physician and/or provided for the use by a patient without specific professional medical expertise.

In specific cases, the LEPTS is connected with an outlet suitable for emitting light to a target which forms a distant part of the LEPTS. Such system provides for an outlet which is e.g., suitable to illuminate the target cells, and connected to an LEPTS that is situated in a convenient location, such as within the fascia of the pectoral or axiallary region, the fascia of the abdominal and pleural cavities, subcutaneous tissue, etc.

According to a preferred embodiment, the target cells are not of adipose tissue.

According to a specific embodiment, the LEPTS is extracorporeal and the outlet comprises optical fibers to conduct light to the remote, internal treatment site.

The invention further provides for a method of treating a patient suffering from diabetes by invasive or otherwise in vivo phototherapy, comprising activating target beta-cells in the pancreas (herein also referred to as pancreatic beta -cells) of said patient by irradiation using a device comprising a LEPTS incorporating an outlet or connected with an outlet, which is suitable for emitting light to the target beta-cells, comprising the steps:

a) inserting the outlet and optionally the LEPTS or the device into an area within or in close proximity to the pancreas, thereby delivering adequate light intensity onto the target beta-cells and/or the pancreas, and

b) irradiating the target beta-cells with an effective energy dose, using at least one wavelength of visible light thereby activating pancreatic opsin proteins.

Specifically, the target beta-cells are repeatedly irradiated according to step b). Specifically, the outlet is positioned within the patient's body and is connected to the LEPTS that is positioned outside the patient's body.

Specifically, the outlet is part of the LEPTS which is implanted into the patient's body. Specifically, the method is combined with any anti-diabetic treatment, e.g., by administering an active substance or pharmaceutical preparation for treating diabetes.

According to a certain aspect, the invention provides for a method of treating a patient suffering from diabetes by administering an effective amount of a preparation of human pancreatic beta-cells that have been activated by ex vivo irradiation as further described herein, in particular wherein said irradiation has been performed in a manner to promote the pancreatic beta-cells's cell growth, proliferation and/or insulin

production.

Specifically, the pancreatic beta -cells are autologous to said patient.

Specifically, the target pancreatic beta-cells are not modified transgenically, but of mammalian origin, such as isolated from a subject e.g., human, dog, pig. Such pancreatic cells particularly do not comprise microbial gene products or transgenes.

Specifically, the device allows an energy dose which effectively activates opsin proteins in the pancreas and in beta -cells, and specifically in close proximity to the pancreas.

According to a specific aspect, the LEPTS is suitable for irradiating the pancreatic beta-cells and/or the pancreas with visible light. Specifically, one or more wavelengths ranging from 400 to 650 nm, specifically a wavelength of at least 425 nm, or at least 440, or at least 450 nm, and further specifically a wavelength of up to 600 nm, or up to 550 nm may be used. One or more specific wavelengths optionally be selected at 470 nm (+/- 20 nm, or +/- 10 nm); or at 535 nm (+/- 20 nm, or +/- 10 nm); or at both wavelengths, 470 nm (+/- 20 nm, or +/- 10 nm) and 535 nm (+/- 20 nm, or +/- 10 nm), in particular by both wavelengths consecutively.

There are several types of light sources suitable for the purpose of the phototherapy and irradiation as described herein. These include e.g., a laser, superluminous diode, laser diode, or a light-emitting diode (LED), such as laser diodes (including also semiconductor laser diodes) and superluminous diodes which provide for highly directional light that is limited in its frequency range. In particular, laser diodes produce a beam of light or radiation that is essentially monochromatic, is sharply collimated and is coherent. Laser diodes are available with continuous wave emission capability and as devices that are pulsed.

Specifically, the LEPTS is suitable for irradiating the pancreatic beta-cells and/or the pancreas with an effective energy dose. Specifically, the energy dose is ranging from 1 LiW/cm ² to 10 mW/cm ² , e.g., at least 1 pW/cm ² , or at least 10 pW/cm ² , or at least 100 pW/cm ² , or at least 1 mW/cm ² , or at least 2, 3, 4, 5, 6, 7, 8, or 9 mW/cm ² . The target beta-cells are irradiated with a suitable energy dose, such that the effective energy dose is directly reaching the target cells.

Specifically, the energy dose is effective for phototherapy so to effectively treat the patient's disorder, in particular by stimulation of cell proliferation with light having the selected optical parameters.

Specifically, the patient is suffering from a disorder of diabetes (diabetes type 1 or diabetes type 2), and specifically hyperglycemia, hypoinsulinemia or insulin resistance.

Specifically, the device is a medical device which can optionally include one or more of a sensing circuit, a controller circuit, a memory circuit, a communication circuit, a power source such as a battery, a battery status circuit, an activity sensor, configured to sense a physical activity signal of a patient, and a physiologic sensor configured to sense a physiologic signal of the patient.

Specifically, the LEPTS provides for manipulating and controlling the outlet and/or the administration of the effective energy dose to the target, and comprises at least one or more controllers for controlling phototherapy. Optionally control of phototherapy is provided via manual, mechanical, electrical, optical, laser, magnetic, hydraulic, pneumatic, motorized, ultrasonic, acoustic, wired and/or wireless technology, or any combination thereof.

The device and phototherapy system as described herein may be associated with an auxiliary device such as a catheter, endoscope, trocar, or the like.

Specifically, the device comprises signal communication means for repeatedly transmitting light to the target pancreatic beta -cells with the same or a different energy dose according to a predefined dose regimen. The typical treatment period may be from 0.5 minutes to 20 minutes or 30 minutes, specifically at least 1 minute and up to 15 minutes. In certain embodiments treatment is shorter, e.g. 1 millisecond or longer, such as at least 1 millisecond or at least 10 milliseconds, up to 0.5 minutes. Treatment may also be continuous for a longer period, e.g. comprising illumination for 0.5 hours or longer, such as at least 0.5 hours, or at least 1 hour, up to 2, 3, 4, or 5 hours.

The dose regimen may include regular intervals of treatment or follow a treatment schedule on demand within a predefined range, depending on the patient's physical condition or diet. Specifically, the activation of pancreatic beta-cells is reversible, thus, repeated treatment is indicated at least once in regular intervals.

Because of activating endogeneous pancreatic beta-cells (or those which originate from allogenic or heterologous sources and have previously been implanted and considered as endogenous), the likelihood of overdosing is low. Thus, specific dose/treatment schedules comprise a lower limit in regular intervals, and further repeated treatment on demand, to improve the patient's conditions.

According to specific dose/ treatment schedule, the patient is treated at least 1 x a day, or at least 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 1 1 x, or 12x a day, e.g. in regular or irregular intervals.

Specifically, the LEPTS is suitable to be positioned outside the patient's body.

According to a specific aspect, the LEPTS is situated outside the patient's body carried by the patient, and the outlet is directly attached to the body, or implanted.

Specifically, the outlet is positioned into an area within the pancreas.

Specifically, the outlet is positioned into an area in close proximity to the pancreas, which is non-adipotic tissue. At least a portion of the device or LEPTS outlet is physically shaped suitable to be situated at a predetermined position. Positioning and operations can be facilitated via a channeling device such as a guiding catheter and/or via a remote visualization device as is known and accepted in the art for example emplyong a scope or an endoscope.

According to a certain aspect, the outlet is situated in the inner pancreas organ, or in close proximity (e.g., within a distance of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cm) to the outer organ, such as in the abdominal cavity behind the stomach. According to a specific embodiment, the outlet is shaped to physically contact the pancreas.

According to a further embodiment, the LEPTS is suitable to be implanted into the patient's body. According to a further embodiment, the device is an implantable device.

Specific embodiments have a manual control carried by the patient outside the body e.g., as a remote control or directly connected to implanted LEPTS and/or implanted outlet of the LEPTS. Such implanted parts can comprise the implantable light source and/or the light outlet, suitable for intracorporeal phototherapy.

The method of insertion, such as implantation, may optionally include endoscopy with or without ultrasound, stereotactic methods laparoscopy, such as implantation with a laparoscope into abdominal organs, the pancreatic wall and body cavities.

Optionally, at least those parts of the device that are implanted into a subject are biocompatible. Thus, the device may optionally be biocompatible as a whole, or at least the implantable parts, such as the LEPTS and/or its outlet, may be biocompatible.

The biocompatible device specifically comprises those parts that are in direct contact with the patient's body or tissue which at least partially (or completely) consist of or are at least partially (or completely) coated with a biocompatible inert material, in particular a non-degradable or non-absorbable material that is designed and engineered to provide for the necessary stability and flexibility of specific device parts. Such biocompatible inert material can be based on a metal, alloy, polymer, biologic scaffolding, or a combination comprising at least one of the foregoing . For example, the biocompatible material is a biocompatible polymer. The use of a biocompatible material helps to prevent inflammation, toxic or allergic reactions and ensures that the implant system causes no complications after implantation. Exemplary biocompatible materials are conventional linear or crossl inked block copolymers or other thermoplastic polymers e.g., (crosslinked) polyurethane and block copolymer of polyethylene terephthalate and polyethyleneoxide.

According to a further aspect, there is provided a pharmaceutical composition comprising an active substance for treating diabetes in a patient, for use in combination with the device as described herein.

The invention particularly further refers to a combination treatment, wherein a subject is treated both, by standard treatment with a pharmaceutical composition, in combination with the irradiation treatment as described herein.

Specific active substances as used for combination purposes can be any of an antidiabetic agent, such as preferably any of insulin, sulfonylureas, incretins, other secretagogues, glitazones, metformin, GLP-1 agonists, DPP4 inhibitors, glucosidease inhibitors, amylin analogs, or SGLT2 inhibitors. As an active substance, retinal may be used.

The active substance can as well be an immunomodulatory drug, including vaccine-based approaches using beta-cell autoantigens, anti-CD3 antibodies, anti- CD20 antibodies, anti-CTLA4 antibodies, nicotinamide, rapamycin, cyclosporine A, azatiopirine, anti-thymocyte globulin (ATG), or prednisolone. The pharmaceutical composition of an active substance may be formulated for oral, parenteral, systemic, mucosal, topic, rectal, sublingual, buccal or implant use. Such preparation typically comprises a pharmaceutically acceptable carrier appropriate for a desired route of administration, preferably wherein the pharmaceutical preparation is a tablet, dermal or transdermal formulation, ointment, gel, cream, lotion, patch, solution, injectable, ophtalmic solution, disperse system, emulsion, microencapsulated drug system, osmotic pump, subdermal implant, granule, microsphere, modified release system, targeted release system, granules, or pill. Pharmaceutically acceptable carriers generally include any and all suitable solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, sustained release agents, and the like that are physiologically compatible with an active agent or related composition. Further examples of pharmaceutically acceptable carriers include sterile water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations of any thereof.

Examplary formulations as used for parenteral administration include subcutaneous, intramuscular or intravenous injection as, for example, a sterile solution or suspension. Formulations for topical application include a number of forms such as creams or ointments, patches, pastes and gels. Specific pharmaceutically acceptable carriers and pharmaceutical compositions are known in the art and described in, e.g. REMINGTON'S PHARMACEUTICAL SCIENCES.

According to a further aspect, there is provided a method of ex vivo treatment of a population of human pancreatic beta -cells to promote cell growth, proliferation and/or insulin production, comprising

a) providing a preparation of human pancreatic beta-cells;

b) treating the cells by exposure to light with an effective energy dose, using at least one wavelength of visible light thereby activating pancreatic opsin proteins in the treated cells; and optionally further

c) determining the extent of cell growth, proliferation and/or insulin production in the treated cells.

The population of human pancreatic beta-cells may be obtained from a human donor, in particular a healthy (or dead) subject or else from a patient suffering from diabetes. Specific populations are obtained from cell culture, wherein the beta -eel Is are cultivated e.g., to obtain a well-characterized standard population. Specific culture techniques employ handling and/or cultivating cells in a suited growth medium supplemented with additives to promote survival and/or growth of beta -cells and at a suited temperature under sterile conditions.

A specific aspect relates to the cultivation of human pancreatic beta -cells in a cell culture, wherein the cells are exposed to light treatment as described herein, thereby obtaining a cell culture of pancreatic beta-cells with improved proliferation and insulin production.

The light treatment specifically is employed such that the opsin proteins located close to or within the pancreas and/or pancreatic beta -cells (i.e. the pancreatic opsin proteins) are activated. Specifically, the energy dose is effective for phototherapy so to effectively stimulate pancreatic beta-cells for an improved proliferation and insulin production by stimulation with light having the selected optical parameters.

Specifically, in the course of the ex vivo treatment, the beta-cells are treated in a medium which is suited for maintenance of the cells. The treatment period mainly depends on the type of light and power of energy, to obtain the effective energy dose required for the opsin activation.

Specifically, the selection of the wavelength and the effective energy dose directly reaching the target beta-cells is as described above. Preferably, the light emitting source is placed in close proximity to the beta-cells but without physical contact, and the opsin activation and/or beta -eel I proliferation controlled. As a result, beta-cells are stimulated, such as by excitatory stimulation resulting in a high proliferation rate over at least two generations as measured by assessing number, metabolic activity or composition of cells.

Specifically, the treated pancreatic beta-cells have preserved and/or protected beta-cell function and are characterized e.g., by the following expression of insulin and response to glucose with insulin secretion. Specifically, the expression of insulin is at least 10% of the control beta-cells, where control cells are defined as untreated cells, and preferably at least 25%, 50% or 75%, up to 100% of the control cells, or more than 100%. Further, the glucose concentration that triggers insulin secretion in treated cells is less than 5-fold the concentration that triggers insulin sectration in control cells, where control cells are defined as untreated cells, preferably less than 4, 3 or 2-fold or equal than of the effective concentration at the provided cells.

Specifically, the opsin proteins are one or more of

a) human short-wave-sensitive opsin 1 characterized by the amino acid sequence SEQ ID 1 ; b) human medium-wave-sensitive opsin 1 characterized by the amino acid sequence SEQ ID 2;

c) human long-wave-sensitive opsin 1 characterized by the amino acid sequence SEQ ID 3;

d) human rhodopsin characterized by the amino acid sequence SEQ ID 4;

e) human opsin-3 characterized by the amino acid sequence SEQ ID 5;

f) human melanopsin (opsin-4) characterized by the amino acid sequence SEQ ID 6; or

g) human opsin-5 characterized by the amino acid sequence SEQ ID 7.

Specifically, the treatment is such that at least one, two, three, four, five, six, or all of the opsins are activated. In some instances, it may be sufficient to activate less than 7, 6, 5, 4, 3, 2, or only one of the opsins, e.g. at least 1 , 2, 3, or all of the opsins identified by SEQ ID 1 to 5.

According to a specific aspect, the invention further provides for a preparation of human pancreatic beta-cells with preserved and/or protected beta-cell function, obtainable by a method as described herein. Such preparation can be provided as isolated cell culture and optionally be provided in the context of an implantable material (such as tissue) or device. Specifically, the cultivated cells may be provided as research tool, e.g., for analytical, diagnostic or medical purposes.

Specifically, the treated pancreatic beta-cells are further used e.g., for medical purposes freshly prepared, such as within 24 hours upon irradiating.

Specifically, the preparation treated pancreatic beta-cells may be provided as a medical device, a medicinal product, or a pharmaceutic product.

Specifically, the preparation is provided for use in a method of treating a patient suffering from diabetes. Specific preparations may be heterologous (e.g., allogenic), or autologous to a patient, preferably wherein the pancreatic beta-cells are autologous to said patient.

The patient is treated e.g., by implanting the beta-cells, and by further combination treatment, such as an additional immunosuppressive regimen.

Optionally, the preparation comprises the treated beta -cells as such, or those which are obtained from a further cell culture to increase the amount of implantable material. According to a specific aspect, the invention further provides for the use of a LEPTS for ex vivo treatment of isolated pancreatic beta-cells to control cell growth , proliferation and/or insulin production, by exposing the cells to light with an effective energy dose, using at least one wavelength of visible light to activate pancreatic opsin proteins.

FIGURES

Figure 1 . Light stimulation (arrow from left to right) supports the proliferation of beta-cells (increased transition from left to right) and protects beta-cells against attack (reduced transition from right to left).

Figure 2. INS-1 E cells were stimulated with blue light (wavelength (λ) = 470 nm, intensity (I) = 1 uW/cm ² ) and cell proliferation was measured using a colorimetric assay (tetrazolium dye reduction; increasing cell number and/or metabolic activity is indicated by increasing absorbance difference measured for 570 and 690 nm). Increased proliferation upon light stimulation and retinal addition is observed. After depletion retinal, only cells treated with retinal but not untreated or retinoic acid treated cells respond to light (λ = 470 nm, I = 1 LiW/cm ² ) with an increase in proliferation.

Figure 3. INS-1 E cells were stimulated with blue light (λ = 470 nm, I = 250 iiW/cm ² ) and cell proliferation was measured using a DNA synthesis assay. The modified thymidine analogue EdU is incorporated into newly synthesized DNA and fluorescently labeled. Increased proliferation upon light stimulation is observed.

Figure 4. Phosphorylation of ERK1 /2 and Akt is observed in INS-1 E cells upon light stimulation (λ = 470 nm, I = 300 iiW/cm ² )

Figure 5. Sequences of human opsin proteins (SEQ ID 1 -7).

SEQ ID 1 : >Short-wave-sensitive opsin 1 (predominant color of light absorbed: blue, max = 420 nm, _range = 200 - 510 nm)

SEQ ID 2: >Medium-wave-sensitive opsin 1 (predominant color of light absorbed: green, X _max = 534 nm, X _rang e - 400 - 620 nm)

SEQ ID 3: >Long-wave-sensitive opsin 1 (predominant color of light absorbed: red, X _max = 564 nm, _range = 400 - 660 nm)

SEQ ID 4: > Rhodopsin (predominant color of light absorbed: green, X _max = 498 nm, range = 400 - 580 nm) SEQ ID 5: >Opsin-3 (predominant color of light absorbed: green (non-visual), ληΐ3χ = 500 nm, _range = 400 - 580 nm)

SEQ ID 6: >Melanopsin (predominant color of light absorbed: green (non- visual), max = 500 nm, X _range = 400 - 580 nm)

SEQ ID 7: >Opsin-5 (predominant color of light absorbed: nearUV/blue (non- visual), X _max = 375 nm, _rang e = 300 - 450 nm)

Indications:

m _ax : Wavelength at which light is maximally absorbed by the opsin

r _ange : Wavelength range in which light is absorbed by the opsin

Figure 6: LEPTS (1 ) suitable for emitting light targeted to the pancreas. Extracorporeal LEPTS is connected to an outlet that is placed close to the target pancreas and emitting light to the target pancreas (left); Intracorporeal LEPTS is placed into the body close to the target pancreas and included in a housing or device incorporating both, the LEPTS and the outlet (right).

Figure 7: Schematic construction of a LEPTS (1 ) incorporating an outlet (3) capable of emitting light to a target, comprises the following parts: connector (2) connecting the light source (4) and the outlet (3); light source (4); controller (5); power source (6); circuit (7) (e.g. memory circuit or communication circuit); activity/physiological/physical sensor (8); auxiliary device (9) (e.g. catheter, endoscope, or trocar).

Figure 8: Human pancreatic islets were stimulated with blue and green light (λ = 470 nm; I = 64 pW/cm ² ; λ = 535 nm; I = 64 pW/cm ² ) for 96 hours and cell proliferation was measured using a DNA synthesis assay. The modified thymidine analogue EdU is incorporated into newly synthesized DNA and fluorescently labeled. Increased proliferation upon light stimulation is observed in two independent experiments.

Figure 9. Phosphorylation of ERK1/2 and Akt is observed in human pancreatic islets upon 5 minutes blue and green light illumination (λ = 470 nm; I = 64 pW/cm ² ; λ = 535 nm; I = 64 pW/cm ² ). DETAILED DESCRIPTION OF THE INVENTION

Therefore, a method for controlling the growth of beta-cells is specifically provided that is based on light treatment. It has surprisingly turned out that beta -cells express several light-sensitive proteins that are members of the opsin protein superfamily. Members of the opsins are well-known for their role in vision (four of the seven human opsins are used as the primary photoreceptors for dim light and three color vision) but also play a role outside of visual light detection [14-16]. Also, cellular signaling cascades exist that should allow opsins to activate proliferative signals via, e.g. G-protein coupled receptor kinases [17], arrestins [18], tyrosine-protein kinase Src, receptor tyrosine kinases and phosphoinositide 3-kinase (PI3K) [19, 20].

Thus, the proliferation of beta-cells (such as the experimental INS-1 E cell line model [21 ]) can be controlled e.g., by blue light, which is a color of light that should activate many opsins (in particular, both blue and green-light absorbing opsins). Light effectively augmented the proliferation of beta-cells (Fig. 2, 3 and 8) and augmentation was comparable to that achieved e.g. with the potent chemical factor HGF in this cell model in a previous study [22].

While some experiments may investigate an increase in proliferation in light- vs. dark-treated cells, these differences may potentially be reduced by normal cell culture facility handling light. Because all opsins utilize retinal as the light-sensing co-factor, cells were prepared in absence of retinal (dextran-coated charcoal-stripped fetal bovine serum) and in the presence of retinal (10 μΜ). Proliferation was observed to be light-dependent for the retinal treated but not for the untreated cells, and that the increase in proliferation is larger than observed previously (Fig. 2). To exclude that added retinal increases cell signaling alone via a retinoic acid intermediate, the same experiments were performed with retinoic acid and no changes in cell proliferation were observed (Fig. 2).

It further can be demonstrated that cellular signaling pathways, particularly the Mitogen-activated protein kinase/Extracellular signal-regulated kinase (MAPK/ERK) and Phosphoinositide 3-kinase/Protein kinase B (PI3K/AKT) pathways, be activated (Fig. 4 and 9), and beta-cell function be protected against attack (e.g., toxins or stress factors, such as by the toxin streptozotocin, cytokines or H2O2.

The present phototherapy or treatment of beta-cells for controlling cell proliferation has the following advantages: - the treatment can be local by applying/focusing the light to/on the tissue of interest. This is not possible with chemicals as these diffuse in tissues and act in the circulation;

- because of this local application, growth will only be augmented in specific cells and not in other unrelated cells. This reduces the risk of uncontrolled cell proliferation and cancer;

- only the application of light but neither of chemicals (i.e. drugs) nor genes (gene therapy) is required, though a combination of treatments may be indicated;

- light control can be started and stopped quickly and with varying repetition rates, as light can be switched on and off quickly and precisely from the outside;

- in contrast to replacement of beta-cells using outside sources of cells [23], which involves a surgical transplant procedure, internal regeneration of beta-cells is more direct and does not require donor tissue the supply of which is limited.

The foregoing description will be more fully understood with reference to the following example. Such example is, however, merely representative of methods of practicing one or more embodiments of the present invention and should not be read as limiting the scope of invention.

EXAMPLE

Example 1 : Light activation of opsins endoqenously expressed in a pancreatic beta-cell line and their effects on proliferation and signaling pathways.

Autoimmune destruction of beta-cells in the pancreas leads to the development of Type I diabetes (TID). Replacement of insulin-producing tissue by transplantation of allogenic islets together with immune suppression has corrected hyperglycemia in a number of TID patients. However, a major limitation of this approach is the limited number of donor tissue and the non-proliferative status of beta-cells ex vivo. Current evidence indicates that the activation of pro-proliferative signaling pathways like MAPK/ERK and PI3K/AKT pathways play a crucial role in beta-cell survival and growth [4, 5, 24]. In former studies in cells other than beta-cells, it has been shown, that the activation of opsins by light leads to the activation of these pro-proliferative signaling pathways (PI3K/AKT, MAPK/ERK) [20, 25, 26]. In the present method, light stimulation supports the proliferation of beta-cells and may also protect cells against attack (Fig. 1 ). This method is based on the activation of endogenous expressed opsins in beta- cells. INS-1 E cells were used as a model cell line [21 ]. This cell line is able to secrete insulin in response to elevated glucose concentration and their concentration dependence curve is similar to that of rat pancreatic islets. INS-1 E cells are an isolated clone from INS-1 cells which are derived from a rat insulinoma induced by x-ray irradiation [27].

To investigate whether light activation of endogenously expressed opsins lead to an increase of INS-1 E cell growth, INS-1 E cells were stimulated 16 hours with blue light (λ = 470 nm; I = 1 pW/cm ² ) and cell proliferation was measured using the 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, Vienna, Austria) assay. INS-1 E cells were seeded at a density of 3 x 10 ⁴ cells in each well of a 96-well plate in complete medium (RPMI 1640 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 pg/ml streptomycin, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 μΜ beta-mercaptoethanol). After 24 hours, cells were starved in reduced serum medium (RPMI 1640 supplemented with 0.1 % fetal bovine serum, 100 units/ml penicillin, 100 pg/ml streptomycin, 10 mM HEPES, 2 mM L- glutamine, 1 mM sodium pyruvate, and 50 μΜ beta-mercaptoethanol) for 16 hours before 6 hour pre-treatment with 10 μΜ 9-cis retinal (Sigma) or 10 μΜ retinoic acid (Sigma) or only vehicle followed by blue light stimulation for 16 hours. MTT assay was performed by incubating the cells with 0.5 mg/ml MTT for 2 hours at 37°C in 5% CO ₂ . Formazan produced in the cells was dissolved with acidic isopropanol containing 0.1 N HCL (Sigma), and absorbance was read at wavelengths of 570 nm and 690 nm with a microplate reader (Synergy H1 , BioTek, Bad Friedrichshall, Germany). Increased proliferation was observed upon light stimulation and retinal addition (Fig. 2). After depletion of cellular and medium retinal, only cells treated with retinal but not untreated or retinoic acid treated cells respond to blue light with an increase in proliferation.

Additionally, a Click-iT ^® EdU Microplate Assay (Life Technologies, Vienna, Austria) was performed to verify light-induced proliferation of INS-1 E cells (Fig. 3). 5 x 10 ³ cells were seeded in each well of 96-well plates in complete medium. After 24 hours cells were starved in reduced serum medium for 16 hours before 6 hour pre- treatment with 1 μΜ 9-cis retinal. After 6 hours of pre-treatment, 10 μΜ EdU was added to each well and cells were stimulated with blue light (λ = 470 nm, I = 250 μνν/cm ² ) for 16 hours. Subsequently, newly synthesized DNA was stained with Click- iT ^® EdU Microplate Assay following the manufacturer's protocol. Fluorescence (λ (Ex/Em)= 565/595 nm) was read with a microplate reader (Synergy H1 , BioTek). INS- I E cells showed an increase in proliferation upon light stimulation compared to unstimulated cells (Fig. 3).

Moreover, an immunoblot was performed to investigate downstream signaling activation in INS-1 E cells upon light stimulation. In particular, the activation of the pro- proliferative MAPK/ERK and PI3K/AKT pathway was analyzed. 1 x 10 ⁶ cells were seeded in each 35 mm dish. After 24 hours, medium was replaced with reduced serum medium. After additional 16 hours, cells were pre-treated for 6 hours with 10 μΜ 9-cis retinal or only vehicle. 6 hours after pre-treatment cells were illuminated for 10 minutes (λ = 470 nm, I = 300 to 1500 W/cm ² ). Cells were harvested in 250 μΙ lysis buffer on ice and lysates were shaken for 30 minutes at 4°C and centrifuged (12,000 rpm, 20 minutes, 4°C). 18 μΙ of lysate per lane were separated by SDS-PAGE and electro- blotted onto PVDF membranes. The blot was incubated with the primary antibody, phospho-p44/42 MAPK (Erk1 /2) (Thr202/Tyr204) (#9101 ; Cell Signaling Technology, Danvers, MA, USA), diluted 1 :1 ,000 in blocking solution (5% BSA in TBST) overnight at 4°C. The secondary antibody, HRP-coupled goat anti-rabbit (#170-6515; BioRad Vienna, Austria) was applied at a dilution of 1 :10,000 in TBST for 1 hour at room temperature. Chemiluminescence was developed with Clarity Western ECL Substrate (Biorad) and signals recorded with Molecular Imager® VersaDoc™MP Substrate (Biorad).

These results demonstrated that MAPK/ERK pathway and PI3K/AKT is activated upon light stimulation of INS-1 E cells (Fig. 4).

Herein a method for inducing beta-cell proliferation by light stimulation was established. In particular, light induced proliferation of INS-1 E cells is induced by endogenous expressed opsins and the resultant activation of pro-proliferating signaling pathways. Example 2: Light activation of opsins endogenously expressed in human pancreatic islets and their effects on proliferation and signaling pathways.

In order to confirm the effects observed in INS-1 E cells, light activation of endogenously expressed opsins in human pancreatic islets was investigated. Light- dependent increase of cell proliferation as well as activation of pro-proliferative signaling pathways in human pancreatic islets upon light illumination was observed. Human adult pancreatic islets from a healthy donor with a purity of 85% were used.

To investigate whether light-activation of endogenously expressed opsins lead to an increase of proliferation in human pancreatic islets, islets were illuminated 96 hours with blue and green light (λ = 470 nm; I = 64 pW/cm ² ; λ = 535 nm; I = 64 pW/cm ² ) and cell proliferation was assessed using Click-iT ^® Plus EdU Alexa Fluor ^® 488 Imaging Kit (Life Technologies). 500 islet equivalents (IEQ) in total, 250 lEQs per condition were cultured in a 35 mm dish in complete medium (CMRL 1066 supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 pg/ml streptomycin and 2 mM L-glutamine) for 24 hours at 37°C in 5% CO ₂ . After 24 hours, medium was changed to fresh complete medium supplemented with 5 μΜ EdU followed by blue and green light illumination for 96 hours (the control dish was kept in the dark). After 48 hours, 1 ml fresh complete medium was added to each dish. After 96 hours of light illumination cells were dispersed by incubating with trypsin-EDTA (Life Technologies) for 20 minutes at 37°C followed by mechanical dissociation. For determining the percentage of proliferating cells, cytospin slides were prepared with dispersed cells. Cells were fixed with 4% paraformaldehyde for 8 minutes followed by washing three times with blocking buffer (5% BSA in PBST). Subsequently, newly synthesized DNA was stained with Click-iT ^® Plus EdU Alexa Fluor ^® 488 Imaging Kit following the manufacturer's protocol. For visualizing cell nuclei, the slides were also stained with DAPI. After washing with PBST, the slides were mounted with Mowiol ^® 4- 88 (Sigma). Images were taken with an EVOS ^® fluorescence microscope (Thermo Scientific, Vienna, Austria) at 10x magnification. Image analysis was done using ImageJ. Percentage of proliferation in human pancreatic islets was defined by counting total number of cells (DAPI) and proliferating cells (EdU positive).

An increase in proliferating cells upon light illumination was observed compared to dark control (Fig. 8). ln addition, an immunoblot was performed to investigate downstream signaling activation in human pancreatic islets upon light illumination. In particular, the activation of the pro-proliferative MAPK/ERK and PI3K/AKT pathway was analyzed. 600 IEQ were seeded in a 60 mm dish. After 24 hours, lEQs were collect in a 15 ml falcon tube, centrifuged for 5 minutes at 200 rpm and resuspended in A+AB buffer (135 mM NaCI, 5.6 mM KCI, 1 .2 mM MgCI ₂ , 1 .28 mM CaCI ₂ , 10 mM HEPES, 3 mM glucose, 1 % (v/v) Penicillin/Streptomycin, 0.1 mg/ml BSA; pH 7.4). 330 lEQs per condition were transferred into separate microcentrifuge tubes and incubated for 2 hours in the dark. After 2 hours of dark incubation lEQs were illuminated with blue and green light (λ = 470 nm; I = 64 pW/cm ² ; λ = 535 nm; I = 64 pW/cm ² ) for 5 minutes followed by lysing with 30 μΙ ice-cold lysis buffer. Dark control was immediately put one ice and lysed after 2 hours of dark incubation. Cell lysates were shaken for 30 minutes at 4°C and centrifuged (12,000 rpm, 20 minutes, 4°C). 30 μΙ of lysate per lane were separated by SDS-PAGE and electro-blotted onto PVDF membranes. The blot was incubated with the primary antibodies, phospho-p44/42 MAPK (Erk1 /2) (Thr202/Tyr204) (#9101 ; Cell Signaling Technology) and Akt (#9272; Cell Signaling Technology), or phosph-Akt (Ser473)(D9E) (#4060; Cell Signaling Technology) and ERK2 (K-23) (sc-153; Santa Cruz Biotechnology, Heidelberg, Germany), diluted 1 :1 ,000 in blocking solution (5% BSA in TBST) overnight at 4°C. The secondary antibody, HRP-coupled goat anti-rabbit (#170-6515; BioRad) was applied at a dilution of 1 :10,000 in TBST for 1 hour at room temperature. Chemiluminescence was developed with Clarity Western ECL Substrate (Biorad) and signals recorded with Molecular Imager® VersaDoc™MP Substrate (Biorad).

These results demonstrated that MAPK/ERK pathway and PI3K/AKT is activated upon blue and green light illumination of human pancreatic islets (Fig. 9).

These results could confirm the results in INS-1 E cells and showed that light stimulation of human pancreatic islets results in increased proliferation and activation of pro-proliferating signaling pathways. References

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