Method for early diagnosis of proliferative diabetic retinopathy

The present invention provides an ex vivo method for diagnosis, especially early diagnosis, of proliferative diabetic retinopathy. Furthermore, it provides a kit for performing said method.

Background of the Invention

Diabetic retinopathy (DRP) is worldwide the leading cause of visual loss in diabetic patients. It can be one of the earliest events observed even in a very young diabetic population. DRP is a disease of the small blood vessels of the retina of the eye. When retinopathy first starts, the tiny blood vessels in the retina become swollen, and they leak a little fluid into the center of the retina. The person's sight may be blurred. This condition is called background retinopathy or non-proliferative DRP. About 80 percent of people with background retinopathy never have serious vision problems, and the disease never goes beyond this first stage. However, if retinopathy progresses, the harm to sight can be more serious. Many new, tiny blood vessels grow out and across the eye (neovascularization). The vessels may break and bleed into the clear gel that fills the center of the eye, blocking vision. Scar tissue may also form near the retina, pulling it away from the back of the eye. This stage is called proliferative retinopathy, and it can lead to impaired vision and even blindness.

A micro vascular disorder is implicated in the pathomechanisms of DRP (Katsumori, K. et al., Diabetes Res. Clin. Pract. 29(3): 195-201 (1995); Akasaka, T. et al., J. Am. Coll. Cardiol. 30(4) :935-41 (1997); Oku, H. et al., Invest. Ophthalmol. Vis. Sci. 42(8) : 1915-20 (2001)). Abnormalities in the microcirculation observed in diabetes mellitus (DM) patients correlate significantly with the severity of hyperglycemia. However, both the occurrence and the severity of diabetic micro vascular disease vary among individual patients and do not necessarily correlate with the duration of DM (Cheung, AT. et al., Endocrin. Pract. 7(5) : 358-363 (2001)). Moreover, even in cases of well-balanced glycemia (e.g. no periodic high- low glucose fluctuations in blood), there are three main subgroups among diabetic patients, namely groups with (1) no visible secondary complications, (2) mild complications and (3) severe complications. Therefore, a predisposition to the development of secondary complications which is independent from hyperglycemia and DM duration has been proposed for DM patients (Bodansky, H.J. et al.,

Diabetes 31(l) :70-4 (1982); Strippoli, G. F. et al., J. Nephrol. 16(4):487-99 (2003)).

The deposition of advanced glycation end products is enhanced in DM and has been linked to the micro vascular disease underlying DRP (Wautier, J. L., and Guillausseau, P. J., Vase. Med. 3(2): 131-7 (1998)). Glycated proteins have receptors on mononuclear blood cells (MBCs) (one kind of leukocytes) and generate reactive oxygen species altering gene expression and modifying cellular targets, such as endothelial cells (Schmidt, A.M. et al., Arterioscler. Thromb. 14(10) : 1521-8 (1994)). Modified endothelial cells, further, play the key role in the pathology of both diabetic cardiovasculopathy and nephropathy (Petty, R. G. et al., Diabetes Res. 17(3) : 115-23 (1991)). This cause-and-effect chain is the reason why the development of proliferative retinopathy is usually the most early and best recognizable pathology among diverse diabetic complications, whilst it is only extremely rarely observed in non-diabetic population. Furthermore, once occurred, the proliferative diabetic retinopathy is usually followed by later diabetic complications such as cardiomyopathy, nephropathy and others. Therefore, the appearance of proliferative retinopathy can be considered as an early indicator for a predisposition for development of secondary complications in diabetic patients. Thus, one of the objectives of present invention is to present a connection between this predisposition and the molecular interplay leading to the occurrence of proliferative retinopathy and further diabetic complications.

Diabetic retinopathy has an underlying inflammatory component, manifesting itself in leukocyte recruitment and up-regulation of genes responsive to inflammatory processes (van Hecke, M.V. et al., Diabetologia 48(7) : 1300-6 (2005)). Adhesion of leukocytes to the retinal vasculature is one of the earliest events in experimental DM that results in breakdown of the blood-retinal barrier, endothelial cell damage, and capillary non-perfusion (Ogura, Y., Jpn. J. Ophthalmol. 44(3) : 322-3 (2000); Boeri, D. et al., Diabetes 50(6) : 1432-9 (2001); Hofman, P. et al., Arch. Ophthalmol. 119(6) :861-6 (2001)). By blocking adhesion molecules on the vascular endothelium, the leukocyte-endothelial interactions and concomitantly a retinal atrophy were effectively inhibited in animal models (Ogura, Y., Jpn. J. Ophthalmol. 44(3) : 322-3 (2000)). This indicates a functional relationship between leukocytes recruitment and development of retinal atrophy. DRP is currently detected mainly by microscopic, particularly by angiographic methods. Said methods provide no possibility to predict any development of DRP or

of any other secondary complications in diabetes. Instead, they just allow assertion of the status quo.

Moreover, Golubnitschaja, O. et al., Amino Acids (Vienna) 28(2) : 239-237 (2005);

Uemura, S. et al, Circulation Res. 88(12): 1291-8 (2001); Yeghiazayan, K. et al., Amino Acids (Vienna) 25(2) : 150-1 (2003); and Kapalla, M. et al., Amino Acids

(Vienna) 28(2) : 221-227 (2005) disclose the determination of the levels of NF- kappaB or XIAP and/or of the activity of MMP-2 and/or MMP9 in diabetes mellitus patients and propose that such determination is suitable to predict whether a patient is likely to develop diabetic retinopathy. However the reliability of a prediction based on the determination of one or two factors as in said documents was found to be insufficient.

Thus, there is a need for a method, which allows a meaningful prediction of the risk to develop proliferative DRP and a prediction on the probability of DRP progression.

Such risk evaluation is essential for improvement of life quality of the patients, of the blindness emergency response and of prediction and prevention of further secondary diabetic complications besides DRP.

Summary of the invention

The present invention is based on the finding that activated leukocytes and matrix metalloproteinases play a central role in the pathophysiology of DRP. It presents a functional link between specific alterations in gene expression of leukocytes, the increased activity of tissue-remodeling matrix metalloproteinases in serum, and the proliferative retinopathy in DM.

Differential gene expression in leukocytes was analyzed in order to identify molecular markers specifically expressed during progression and possibly even during development of the disease. The identified genes, which are involved in retinal angiogenesis also represent potential targets for medicaments, which suppress retinal neovascularization in DM.

Furthermore, the activity of MMP-9 and MMP-2 was analyzed, since an activation of these genes is known to play a key role in tissue remodeling and neovascularization and is supposed to be involved in vascular pathomechanisms conditioned by hyperglycemia (Uemura, S. et al., Circ. Res. 88(12) : 1291-8 (2001); Mene, P. et al.,

Nephrol. Dial. Transplant. 16(5) :913-22 (2001)). Thus, the present invention provides

(1) a method for evaluation of the risk of a diabetes mellitus (DM) patient to develop diabetic retinopathy (DRP), for early diagnosis of diabetic retinopathy, for determination of the progression risk of an existing diabetic retinopathy, and for determination of the risk to develop secondary complications to DM, which comprises

(a) determining ex vivo in a body fluid sample from the patient at least one, preferably at least three, more preferably at least five parameter(s) selected from the group consisting of (i) the expression level of one or more proteins of the group consisting of NF-kappaB, XIAP, TIMP-I, TIMP-2 and Ly-GDI, (ii) the activity of the matrix metalloproteinases MMP-2 and/or MMP-9, and (iii) the ratio MMP-2 activity:TIMP-2 expression level (MMP-2:TIMP-2) and/or MMP-9 activity:TIMP-l expression level (MMP-9:TIMP-1);

(b) comparing the parameter(s) determined in step (a) with the corresponding mean value(s) of a non-diabetic control group or with the corresponding earlier value(s) of the same patient ("reference value") and identifying of increased or decreased parameter level(s); and

(c) identifying one or more risk factors based on said increased or decreased parameter level(s);

(2) a preferred embodiment of (1) above, wherein in step (c) said risk factors are selected from the group consisting of (A) increased Ly-GDI expression, increased

MMP-2 activity, increased ratio MMP-2 :TIMP-2, increased MMP-9 activity, increased ratio MMP-9:TIMP-1, and (B) decreased TIMP-I expression, decreased NF-kappaB expression, decreased XIAP expression, and at most 2 times increased, preferably decreased TIMP-2 expression, in comparison to the reference value; (3) a preferred embodiment of (1) and (2) above, wherein step (a) comprises determining at least the expression level of TIMP-I or TIMP-2, and preferably comprises further determining the activity of the matrix metalloproteinases MMP-2 and/or MMP-9 and/or the ratio MMP-2 activity:TIMP-2 expression level (MMP- 2:TIMP-2) and/or MMP-9 activity:TIMP-l expression level (MMP-9:TIMP-1); (4) a kit for performing the method of (1), (2) or (3) above comprising reagents and/or devices for

(i) detecting the expression of the genes listed in (1) above, and/or (ii) determining the activity of MMP-2 and/or MMP-9; and (5) a method for treating diabetic retinopathy (DRP) which comprises evaluating of the risk of a diabetes mellitus (DM) patient to develop DRP, early diagnosing of

DRP, determining the progression risk of an existing DRP, or determining the risk to develop secondary complications to DM in a patient as defined in (1) to (3) above and treating the DRP of the patient.

A patient showing at least one, preferably more than one of the risk factors listed under (2) has an increased risk to develop DRP or that an existing DRP will progress to later stages, especially to later proliferative stages, or that secondary complications will develop.

Short Description of the Figures Fig. 1 : Expression Array ("Atlas Array") image of cDNA from pooled mRNA of 13 diabetic patients (subject information see example 1, Pilot Study; cDNA synthesis see example 3). The use of cDNA from pooled mRNA allows the detection of general differences between patient and control groups and equalizes individual differences between the subjects. The DNA array comprised double-spotted probes for altogether 588 different cardiovascular diseases specific genes. See Example 4. The differentially expressed genes are listed in Tab. 1.

Fig. 2: 2D-PAGE of total protein from circulating leukocytes of diabetic female and male patients and controls (see example 5A). Arrow: position of Ly-GDI protein spot. Ly-GDI expression is enhanced in diabetic men and women (lower part) in comparison to samples of non-diabetic patients.

Sequence Listing - Free Text

SEQ ID NO Description

1 DNA encoding human beta actin

2 human beta actin

3 DNA encoding human XIAP

4 human XIAP

5 DNA encoding human NF-kappaB

6 human NF-kappaB

7 DNA encoding human TIMP-2

8 human TIMP-2

9 DNA encoding human TIMP-I

10 human TIMP-I

11 DNA encoding human MMP-2

12 human MMP-2

13 DNA encoding human MMP-9

14 human MMP-9

15 DNA encoding human Ly-GDI

16 human Ly-GDI

17 to 26 primers (see example 6)

Definitions

In the context of present invention, the following abbreviations, terms and definitions are used:

Abbreviations: DRP, diabetic retinopathy; DM, Diabetes mellitus; XIAP, X-linked inhibitor of apoptosis protein, or expression level of said protein; MMP-9, matrix metalloproteinase 9, or the activity level thereof; MMP-2, matrix metallo-proteinase 2, or activity level thereof; TIMP-I, tissue inhibitor of metallo-proteinase 1, or expression level of said inhibitor; TIMP-2 tissue inhibitor of metalloproteinase 2, or expression level of said inhibitor; NF-kappaB, NF-kappaB or expression level of NF- kappaB; Ly-GDI, lymphoid-specific guanosine diphosphate (GDP) dissociation inhibitor, or expression level of said inhibitor.

The term "reference value" pertains to the mean value of a respective parameter determined in a control group of non-diabetic subjects or to the corresponding value of the same patient, which was determined by the same detection method before DRP set in. The control group of non-diabetic subjects preferably consists of at least 5 subjects, more preferably 10 subjects. Alternatively, reference values for non-diabetic subjects can be taken from literature.

"Relative units" (r.u.) is a terminus technicus used for gene expression levels (both transcription and translation) as well as zymography results in molecular biology. The term "enhanced" pertains to expression or activity levels which are higher than that in the control group (i.e. the reference value), preferably at least two times the mean value of the control group ("slightly increased"). It also encompasses "high" expression and activity levels, namely levels, which are increased several times ("highly increased"), i.e. more than two times, preferably more than three times compared to the mean values of the control group. "DRP" is diabetic retinopathy. "Proliferative" DRP in the context of present invention means a DRP which has passed the background DRP stadium and is characterized by a diffuse image instead of clearly formed micro-vessels at angiographic detection. A "highly proliferative DRP" is additionally characterized by a diffuse image not only in the area of micro-vessels, but even in the area of macro-vessels. The system of "DRP classes" used in present invention is based on the following classification : class I (proliferation "-", "(+)"), class II (proliferation "+", "+(+)"), class III (proliferation "++", "+ ++", "+ + ++"). Therein, "+" and "-" correlate to the following symptoms: "-", "(+)": angiography images similar to those of non-diabetic controls without retinopathy;

"+ ", "+(+)": no microcirculation detectable in angiography images; "++": diffuse image typical for neovascularization of micro-vessels with increasing severity grade;

"+ ++", "+ + ++": diffuse image typical for neovascularization of macro-vessels with increasing severity grade.

Thus, "proliferative DRP" corresponds to class II, "highly proliferative DRP" corresponds to class III, and "no active proliferative DRP" corresponds to class I. "Risk" means a patient's chance to develop a disease, especially proliferative DRP, or secondary complications, especially secondary complications typical for DM. A "risk factor" in the context of present application is a condition, which increases the probability that a patient develops a disease, especially proliferative DRP. Risk factors do not cause the disease, and not everyone who has a risk factor will get the disease. However, risk factors help a medical practitioner to focus risk reduction efforts on the people who are most "at risk" for a disease, especially for proliferative DRP. Particularly, the summarized risk factors presented in Tab. 3 provide the most valuable information for a calculation of the probability that a patient will develop proliferative DRP. Thus, a useful risk factor to prognose development of proliferative DRP is characterized by a correlation of its alteration in comparison to a healthy control group ("disease specific alteration") with disease severity. The same applies mutatis mutandis to ,,risk factors" for DRP progression and for development of further secondary complications.

A "secondary disease" is a disease that follows and results from an earlier disease. In the context of present invention, said earlier disease is DM. Said secondary diseases are especially DRP and other cardiovascular complications observed in DM patients.

A "secondary complication" is a secondary disease, an accident, or a negative reaction occurring during the course of an illness and usually aggravating the illness. Particular secondary complications connected to DRP and/or DM are disturbed microcirculation, pathologic remodelling of the vascular system, and possibly even silent myocardial infarcts.

"Expression level" of a protein means its level of expression. It may be determined on the mRNA or protein level. Preferably, the expression level is determined by quantification of mRNA, including quantification by detection with fluorescent reporter molecules and Real-time-PCR, especially PCR using intercalation dye/fluorescent reporter molecule detection or molecular beacons based PCR.

Detailed Description of the Invention

The present invention provides an ex vivo method for diagnosis and prognosis of proliferative DRP. Said method is based on gene expression of a set of molecular markers and on the activity of matrix metalloproteinases.

In one preferred aspect of embodiments (1), (2) and (3) of the present invention, the patient shows more than one, preferably at least four, more preferably at least six of the risk factors. Such patient has an increased risk to develop DRP or that an existing DRP will progress to later stages, especially to later proliferative stages, or that secondary complications will develop.

In the method of embodiments (1), (2) and (3), preferably a risk factor which is shown by the patient is an increased ratio MMP-9:TIMP-1. This is especially important if only a few of the parameters listed under embodiment (1) can be measured. MMP-9:TIMP-1 is the most reliant indicator for an increased risk of a DM patient to develop DRP or to progress in DRP.

Furthermore preferred is the use of whole blood or a blood component as the body fluid in the method of embodiments (1), (2) and (3). More preferably, a blood component is used which is either circulating leukocytes, blood plasma or blood serum, or a combination thereof. Most preferred is the use of blood serum or plasma for determination of MMP activity, and of circulating leukocytes for determination of the expression levels listed under (i) in embodiment (1) above. The determination steps of the method according to the present invention are preferably performed by DNA or RNA detection, more preferably by mRNA detection, for determination of gene expression levels and by zymography or ELISA for determination of MMP activity.

One preferred aspect of the present invention is a method for evaluation of the risk of DRP progression in all DRP stages. A second preferred aspect of the present invention is a method for evaluation of the risk of DRP development in diabetic patients with no retinopathy detected. Both of said methods comprise the determination of one or more, preferably of at least three, most preferably of all, of the parameters of the group consisting of MMP-9 activity, NF-kappaB expression level, TIMP-I expression level, Ly-GDI expression level, XIAP expression level and the calculation of the ratio of MMP-9:TIMP-1. In a preferred aspect of said methods, one or more of (i) a ratio MMP-9:TIMP-1 which is at least 1.5 times higher than the reference value;

(ii) a MMP-9 activity level which is at least 1.3 times higher than the reference value;

(iii) a NF-kappaB expression level which is at least 1.5 times lower than the reference value; (iv) a Ly-GDI expression level which is at least 1.3 times higher than the reference value; and

(v) a XIAP expression level which is at least 1.5 times lower than the reference value indicate a risk of DRP development or progression from nonproliferative to proliferative DRP.

In an even more preferred aspect, said methods comprise the determination of one or more, preferably of at least three, most preferably of all, of the parameters of the group consisting of MMP-9 activity, NF-kappaB expression level, TIMP-I expression level and the calculation of the ratio of MMP-9:TIMP-1. In cases where the XIAP and/or Ly-GDI expression level is determined in addition to one or more of said latter parameters,

(i) a Ly-GDI expression level which is higher than the reference value; and

(ii) a XIAP expression level which is at least 1.5 times lower than the reference value is an additional indicator for the risk of DRP progression, especially for the risk of

DRP progression from nonproliferative to proliferative DRP.

A third preferred aspect of the present invention is a method for evaluation of the risk of DRP progression specifically in late proliferative DRP stages, especially from proliferative (class II) to highly proliferative (class III) DRP. Said method comprises the determination of one or more, preferably of at least three, most preferably of all, of the parameters of the group consisting of MMP-9 and MMP-2 activity, TIMP-I expression level, TIMP-2 expression level, NF-kappaB expression level and the calculation of the ratio MMP-2:TIMP-2 and MMP-9:TIMP-1. In a preferred aspect of said method, one or more of (i) a TIMP-2 level which is equal or less than two times the reference value, preferably is lower than the reference value;

(ii) a TIMP-I level which is at least 2 times lower than the reference value;

(iii) a MMP-2 activity level which is at least 1.5 times higher than the reference value; (iv) a ratio MMP-2:TIMP-2 which is at least 1.5 times higher than the reference

value;

(v) a MMP-9 activity level which is at least 1.5 times higher than the reference value;

(vi) a ratio MMP-9:TIMP-1 which is at least 2.5 times higher than the reference value; and

(vii) a NFkappaB expression level which is at least 3 times lower than the reference value indicate a risk of DRP progression from proliferative DRP to highly proliferative DRP.

In an even more preferred aspect, said method comprises the determination of one or more, preferably of at least three, most preferably of all, of the parameters of the group consisting of MMP-2 activity, TIMP-I expression level, TIMP-2 expression level and the calculation of the ratio MMP-2:TIMP-2 and MMP-9:TIMP-1. In cases where the NF-kappaB expression level and/or MMP-9 activity level is determined in addition to one or more of said latter parameters, (i) a NFkappaB expression level which is at least 3 times lower than the reference value; and/or

(ii) a MMP-9 activity level which is at least 1.5 times higher than the reference value is an additional indicator for the risk of DRP progression from proliferative DRP to highly proliferative DRP.

The kit of embodiment (4) preferably contains reagents and/or devices for

(i) detecting the expression of the proteins listed in embodiment (1) above by

"Real-Time"-PCR, especially by primer or molecular beacons based PCR, and/or

(ii) determining the activity of MMP-2 and/or MMP-9 by means to measure the gelatinase activity of MMP-2 and MMP-9, e.g. by zymography, or by ELISA.

The treatment of the DRP in a patient according to embodiment (5) of the invention is effected by a balanced (i.e. antioxidant-rich) diet or by administration of pharmaceuticals capable of modulating (balancing) the increased or decreased levels/activity of the parameters (a). Suitable modulating agents include blocking agents (inhibitors) for MMP-2 and MMP-9, blocking agents (inhibitors) for the growth hormone NF-kappaB; and compound enhancing the activity of TIMP-I or of

TIMP-2.

A comparative investigation of gene expression in circulating leukocytes isolated from DM-patients with versus without proliferative retinopathy as well as versus non-diabetic controls was performed (Examples 2 to 6). In addition, the activity of

two matrix metalloproteinases (MMP-2 and MMP-9) in the serum of patients was measured (Example 7), since an activation of these enzymes is known to play a key role in tissue remodeling and neovascularization (Uemura, S. et al., Circ. Res. 22;88(12) : 1291-8 (2001); Mene, P. et al., Nephrol. Dial. Transplant. 16(5) : 913-22 (2001)).

The study consisted of three main parts:

A. Pilot study: differential gene expression analysis comparing transcriptional pools isolated from peripheral leukocytes of 13 diabetic patients with those of 11 non-diabetic controls using a DNA array technique (see example 1, Tab. 4 for information on the individuals);

B. verification of the differential expression of genes selected in part A by a quantification of the specific expression rates on the transcriptional level using real-time-PCR and on the level of protein products using both Western blot and zymography; for this aim newly recruited patients (38 subjects) versus non-diabetic pools (22 subjects) were tested (Example 1, Tab. 5, Main

Study);

C. statistical analysis of the differences in terms of correlation between individual gene expression rates and enzymatic activities, respectively, and the severity grade of proliferative retinopathy (active neovascularization of retina, determined by angiography).

The hybridization of labeled cDNAs to a DNA array specific for human cardiovascular diseases (Human Cardiovascular Array; Atlas) with altogether 588 cardiovascular diseases relevant genes (Fig. 1, Example 4) revealed a differential expression of 152 genes in leukocytes of the DM-group (13 subjects) which were either activated or suppressed versus the non-diabetic group (11 subjects) (Tab. 1) in study part A (pilot study).

Tab. 1 : Differentially expressed genes in leukocytes of DM patients in the pilot study; Position: probe position on Atlas Array; Block: Atlas Array blocks

The differential expression of the identified genes was further verified on a newly recruited pool of non-diabetic controls (22 subjects) versus a newly recruited DM patients pool (38 subjects) in study part B (Main Study). In said study part B, expression of single genes was quantified and parallel ophthalmologic examinations were performed. All further experimental data discussed below is from said study part B.

Of the genes listed in Tab. 1, NF-kappa-B (D05m), XIAP (C04k), MMP-9 (F07m), MMP-2 (F03m), TIMP-I (F05n) and TIMP-2 (F06n) were selected for further evaluation as potential molecular markers for DRP due to significant differences in their expression levels in diabetic patients in comparison to non-diabetic controls. In said further evaluation, MMP-2 and MMP-9 were evaluated by enzyme activity assays, the other potential molecular markers by measurement of their expression levels.

Furthermore, Ly-GDI has been identified as potential molecular marker for DRP due to its enhanced expression in diabetic patients (compare example 5, Fig. 2). MMP-2 activity in serum was classified as follows (all values are given in relative units) : negative (-; 0.00-0.34), enhanced ( + ; 0.35-0.79), highly increased ( + + ; 0.80-1.29) and extremely increased ( + + + ; higher than 1.29).

MMP-9 activity in serum was classified as follows (all values are given in relative units) : negative (-; 0.00-0.49), enhanced ( + ; 0.50-0.89), highly increased ( + + ; 0.90-1.44) and extremely increased ( + + + ; higher than 1.44). Enhanced and increased MMP-2 activities were found in the serum of 72 %, 67 %, and 63 % of patients with proliferative, highly proliferative and no active proliferative DRP, respectively (Example 7, Tab. 8). The MMP-2 activities were highly increased in 44 %, 44 %, and 19 % of patients with proliferative, highly proliferative and no active proliferative DRP. 62 % of smokers and 78 % of non- smokers demonstrated at least enhanced MMP-2 activities; exactly one half of these subjects in each group (31 % and 39 % respectively) revealed highly increased MMP-2 serum levels.

Enhanced and increased MMP-9 activities (Example 7, Tab. 8) were found in the serum of 72 % and 81 % of cases with and without proliferative DRP, respectively, and, with only one exception, in all patients of the subgroup with highly proliferative DRP. The MMP-9 activities were highly increased in 56 %, 67 %, and 31 % of patients with proliferative, highly proliferative and no active proliferative DRP. In the subgroups B and C (compare Tab. 3) these numbers were 80 %, 100 %, 40 % and 44 %, 63 %, 14 %, respectively. Almost an equal amount of smokers and non-smokers (85 % and 83 %, respectively) demonstrated at least enhanced MMP-9 activities; 38 % of smokers and 48 % of non-smokers demonstrated highly increased MMP-9 serum levels.

The correlation between DRP and the levels (MMP-2 and MMP-9: activity levels; all other molecular markers: expression levels) of the different potential molecular markers was statistically evaluated (example 8). Analysis of variance was done using ANOVA, pairwise testing was done by the Tukey test. The results are shown in Tab. 2.

Tab. 2: Mean statistical values of risk factors in different DRP classes. Significance for relationship of said risk factors to DRP progression. Pairweise testing with a post hoc multiple comparison procedure (Tukey test) was used, since ANOVA three-subgroup (class I, class II, and class III) comparison indicated significant differences. The level of significance for Tukey test was p=0.1. For easy data orientation a, b, and ab letters are

used to express different mean value levels. Significant differences were found between different levels (letters), such as between a and JD and not between JD and JD in the case of TIMP-2. Additionally, the most significant differences between single sub-groups (classes) are pronounced with grey fields.

Relationship between DRP and

Relationship between DRP and

In the following, "increased/decreased value" means "increased/decreased in comparison to the mean values for the DRP classes listed in Tab. 2".

There are significant differences between the DRP classes for the following parameters: NFkappaB, TIMP-2, MMP-2, ratio MMP-2/TIMP-2, MMP-9 and ratio MMP-9/TIMP-1. Said significant differences are marked by underlined figures in Tab. 2. The increasing potential for DRP progression to the next higher level is marked in Tab. 2 by grey shading. Thus, increased values of Ly-GDI in DRP class I indicate a potential for progression into class II; decreased TIMP-2 values in class II indicate a potential for progression into class III, etc. .

Taken together, statistically significant is the correlation between

(A) the potential DRP progression and increased values of MMP-9 and MMP-9/TIMP-

1 and decreased values of NF-kappaB, in all DRP classes;

(B) the potential DRP progression of class I into class II and increased values of Ly- GDI as well as decreased values of XIAP; and

(C) the potential DRP progression of class II into class III and decreased values of TIMP-I, TIMP-2 values, which are at most 2 times the reference value but preferably decreased, as well as increased values of MMP-2 and MMP-2/TIMP-2. The following parameters are therefore relevant for a prediction of a potential DRP progression between the three DRP classes:

Class I to Class II (altogether 5 parameters) : Ly-GDI, NF-kappaB, XIAP, MMP-9, and MMP-9/TIMP-1; for sure prognosis at least the following 3 parameters should be measured : NF-kappaB, MMP-9, and MMP-9/TIMP-1.

Preferably, the single parameter values are considered as contributing to the risk for DRP progression, if

- Ly-GDI patient values (PV) are higher than the respective reference value: in our study those values PV > 0.85 relative units (later r.u.); - NF-kappaB patient values are at least 1.5 times lower than the respective reference value: in our study those values PV < 0.14 r.u.;

- XIAP patient values are at least 1.5 times lower than the respective reference value: in our study those values PV < 0.013 r.u.;

- MMP-9 patient values are at least 1.3 times higher than the respective reference value: in our study those values PV > 0.9 r.u.; and/or

- MMP-9/TIMP-1 patient values are at least 1.5 times higher than the respective reference value: in our study those values PV > 5.0 r.u.

Class II to Class III (altogether 7 parameters) : TIMP-2, TIMP-I, NF-kappaB, MMP- 2, MMP2-/TIMP-2, MMP-9, and MMP-9/TIMP-1; for sure prognosis at least the following 5 parameters should be measured: TIMP-2, TIMP-I, MMP-2, MMP2-/TIMP- 2, and MMP-9/TIMP-1.

Preferably, the single parameter values are considered as contributing to the the risk for DRP progression, if

- TIMP-2 patient values are not higher than 2 times compared to the respective reference value: in our study those values PV < 0.034 r.u. The lower the TIMP-2 level, the more probable is a Class II to Class III progression. Nevertheless, a slight increase in TIMP-2 up to 2 times in comparison to the control group is not lowering the risk of progression from Class II to Class III;

- TIMP-I patient values are lower than 0.5 times of the respective reference value: in our study those values PV < 0.18 r.u.;

- NF-kappaB patient values are at least 3 times lower than the respective reference value : in our study those values PV < 0.065 r. u . ;

- MMP-2 patient values are at least 1.5 times higher than the respective reference value : in our study those values PV > 0.9 r. u . ; - MMP2-/TIMP-2 patient values are at least 1.5 times higher than the respective reference value : in our study those values PV > 120 r. u . ;

- MMP-9 patient values are at least 1.5 times higher than the respective reference value : in our study those values PV > 1.1 r. u . ; and/or

- MMP-9/TIMP- 1 patient values are at least 2.5 times higher than the respective reference value : in our study those values PV > 8.6 r. u .

In summary, the following 9 parameter values are considered as risk factors in the context of present invention : increased values of Ly-GDI, MMP-2, MMP-2/TIMP-2, MMP-9, MMP-9/TIMP- 1 ; values of TIMP-2, which are not higher than two times the reference value, and which are preferably decreased; decreased values of TIMP- I, NF-kappaB, XIAP.

The Ly-GDI, MMP-2, MMP-2/TIMP-2, MMP-9, MMP-9/TIMP- 1, TIMP-2, TiMP- I, NR- kappaB and XIAP values were determined in DM patients with different duration of DM and different levels of proliferative DRP. The results are summarized in Tab. 3.

Tab. 3 : Summarized information about the parameters and DRP risk factors evaluated in DM patients. The column "summarized points" indicates the grade of DRP risk calculated for individual patients. The final sum of points (maximum 9) is calculated by addition of individual points given for each of the 9 parameters (NFkappaB, XIAP, etc.) : "-" parameter not measured"; "0" contribution of the parameter to the overall risk is minimal and therefore negligible for risk calculation (i.e. no risk factor); "1" contribution of the parameter is considerable and should be included in the overall risk calculation (i.e. risk factor).

The sum of risk factors (0 : risk factor not present; 1 : risk factor present) should be set in relation the amount of parameters that could be measured. For example, for the patients No. 34, No. 56 and No. 11 in Tab. 3 this would give the values 2/5 x 100 = 40%, 3/9 x 100 = 33%, and 6/6 x 100 = 100%, respectively. Resulting values equal or over 67 % (i.e. at least two thirds of the measured parameters are risk factors) indicate a highly increased risk of DRP development or progression; resulting values equal or below 33 % (i.e. one third or less of the measured

parameters are risk factors) indicate a low risk of DRP development or progression. Patients with middle values - above 33 % and below 67 % - should be advised to repeat the test in a few months time in order to evaluate the trend of development. The measurements of the activity in serum showed an increased or even highly increased level of both MMP-2 and MMP-9 enzymatic activity in the majority of DM- patients compared to non-diabetic controls. Particularly high levels were observed for MMP-9 activity, and those were increased in patients with proliferative DRP. With only one exception noted, the MMP-9 activity was increased in all patients with highly proliferative retinopathy. This correlation can be seen well in subgroups B and C with a longer DM-duration of 10-19 and 20-43 years respectively. Highly increased activities of both MMP-2 and MMP-9 were observed in more than twice as many cases with highly proliferative retinopathy compared to patients with no detectable active proliferation. Moreover, and quite surprisingly, not the activity of MMP-2 and MMP-9 in blood serum/plasma alone, but the ratio of activity of said MMPs in blood serum/plasma to the expression of the corresponding tissue inhibitor of MMP (MMP- 2/TIMP-2 and MMP-9/TIMP-1) in circulating leukocytes is an even more important - if not the most important - parameter for the risk evaluation. Furthermore, it is possible to differentiate risk factors between classes I, II and III: while an increasing ratio of MMP-9 to TIMP-I is the risk factor for both class I and class II in the DRP progression to the next higher class, the ratio MMP-2 to TIMP-2 is only important for the prediction of the progression from class II to class III.

The calculated statistic to overall high risk in single diabetic sub-groups is well in agreement with current data published by other authors (Cheung, AT. et al., Endocr. Pract. 7: 358-363 (2001)). Moreover, roughly after the first decade of DM duration, indeed, the risk for secondary complications increases considerably, at least more than twofold. Contrary, in advanced age the potential risk is slightly decreased due to slowing of all metabolic processes generally. Furthermore, present data indicates that the previously suggested prediction of the occurrence or the severity of DRP based on a single HbAIc value alone is impossible (compare Tab. 2). Said prediction was based on the positive correlation between individual HbAIc (long term blood glucose level) values and the status of proliferative retinopathy (Lovestam -Adrian, M. et al., J. Diabetes Complications 15: 287-294 (2001) ; Porta, M. et al., Diabetologia 44: 2203-2209 (2001); Henricsson, M. et al., Diabetes Care 26: 349-354 (2003)).

In case that only a limited number of parameters can and will be used for clinical evaluation of DRP progression, the ratio MMP-9/TIMP-1 should be calculated for all three classes of patients, whereas the ratio MMP-2/TIMP-2 should be calculated for patients of class II only. The present invention is described in more detail by reference to the following examples. It should be understood that these examples are for illustrative purpose only and are not to be construed as limiting the invention.

Examples In the following examples, standard techniques of recombinant DNA technology were used that were described in various publications, e.g. Sambrook et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, or Ausubel et al. (1987), Current Protocols in Molecular Biology 1987-1988, Wiley Interscience. Unless otherwise indicated, all devices, enzymes, reagents and kits were used according to the manufacturers ^' instructions.

Example 1 : Subjects

DM patients as well as non-diabetic controls were recruited for these investigations.

The diagnosis of DM was based on a detailed clinical history and biochemical examination of the recruited persons. All investigations conformed to the principles outlined in the Declaration of Helsinki and were performed with permission of the local Ethics Committee.

Patients that came to the Department of Ophthalmology for evaluation of diabetic retinopathy and that were subsequently scheduled for fluorescein angiography were asked to participate. Informed consent was obtained, and multiple data regarding DM as well as details possibly affecting the patient's general health status were recorded. Before angiography, approximately 20 ml of full blood anti-coagulated with K ₃ EDTA were drawn and delivered immediately for molecular biological examination.

All patients and non-diabetic controls received a full eye exam, which included complex ophthalmologic, particularly angiographic investigations in terms of occurrence and severity grade of proliferative retinopathy.

Pilot Study

Sub-group mRNA pools (DM-females; DM-males; control-females; control-males; compare Tab.4) from isolated peripheral leukocytes were used for the pilot study using the cDNA array whose results are listed in Tab. 1. Individual protein samples

of the subjects were used for 2D-PAGE and subsequent comparative 2D-PAGE imaging.

Tab. 4: information about the subjects in the pilot study. ,,n": Number of subjects

Main Study

Clinical data : Non-diabetic controls were on the average 49.1 years old. From 22 subjects no one demonstrated an proliferative retinopathy. DM-patients were on the average 54.6 (subgroup A), 58.7 (subgroup B), and 59.3 years (subgroup C) old. They formed subgroups with no ("-", "(+)"), middle ("proliferative") ("+","+( + )", and severe ("highly proliferative") C++", "+ ++", "+ + ++") DRP, respectively (Tab. 5). Of 38 patients, 35 subjects suffered from DM type 2, and 3 subjects from DM type 1. 76 % of all patients were insulin dependent. 34% were smokers. DM duration varied considerably from 3 to 43 years. Therefore, 3 subgroups A, B, and C with a DM-duration of 3-9, 10-19, and more than 20 years respectively, were formed. A proliferative retinopathy was found in exactly 50 % of all DM-patients (with a male to female ratio of about 1 : 1, the mean age being 60.7±7.9 years). There were 50, 45, and 56 % of patients with proliferative disease in subgroups A, B, and C respectively. The most prominent proliferations (noted as "++", "+ ++" and "+ + ++"; class III) were observed in 28 % of all DM-patients, and in 33, 9, and 37.5 % of subgroups A, B, and C, respectively. Proliferative disease (class II), highly proliferative disease (class III) and no active proliferative disease (class I) was observed in 58, 25, and 42 % of non-smokers and in 42, 33, and 58 % of smokers, respectively. No correlation between individual HbAIc (an indicator for the long-term blood glucose level) values and the occurrence or a grade of proliferative retinopathy severity was found. The clinical data of all patients and of the members of the control group is summarized in Tab. 5.

Tab. 5: Information about the patients, the control group and about the parameters measured. Patients are grouped according to the DM-duration. HbAIc shows long-term blood glucose levels (normal values are 5.0-6.9); APT indicates any type of anti-proliferative

therapy. VO: venous occlusion; FS: Fuch ^' s spot; M : myopia; R: non-diabetic retinosis; MD: macular degeneration; T: trauma; EG: epiretinal gliosis.

Example 2: Isolation of leukocytes

Blood samples (20 ml) anti-coagulated with heparin were collected from patients and controls. Leukocytes (including monocytes and stem cells) were separated

using Ficoll-Histopaque gradients (Histopaque 1077, Sigma, Switzerland) as described previously (Golubnitschaja-Labudova, O. et al., Curr. Eye Res. 21 :867-876 (2000)). Briefly, blood samples were diluted with an equal volume of physiological buffer solution (PBS, GibcoBRL, Switzerland). Then, 2 ml of histopaque were placed into 10 ml sterile centrifuge tubes and 5 ml of diluted blood samples were carefully layered onto each histopaque gradient. Gradients were centrifuged at 475 g and 20 ⁰ C for 15 min. The leukocyte bands were removed from the interface between the plasma and histopaque layers of each tube and collected into one 50 ml tube. The total volume was brought to 50 ml with cold Dulbecco's Modified Eagle Medium (DMEM, GibcoBRL, Switzerland). The cell suspension was washed three times with DMEM and the total number of cells was determined. Cells were finally suspended in PBS and aliquoted into Eppendorf tubes at 10 ⁷ cells/tube. After centrifugation cell pellets were dried and stored at -70 ⁰ C until either mRNA or protein isolation.

Example 3: Isolation of total RNA, mRNA and first-strand-cDNA synthesis

Isolation of total RNA from aliquoted samples of isolated leukocytes (Example 2) and the concomitant mRNA isolation were performed using RNAzol™ B (WAK- Chemie Medical GmbH, Germany) and Oligotex mRNA Mini Kit (Qiagen, Germany), respectively, according to the protocols supplied by the manufacturers. cDNA synthesis was performed using the "First-Strand cDNA Synthesis Kit" (Amersham Biosciences, UK). For each cDNA synthesis, 1 μ g mRNA was reverse transcribed using an oligo(dT)i ₈ primer in a final volume of 15 μl each, according to the protocol supplied by the manufacturer.

Example 4: Hybridization to DNA Array

A) Preparation of biotin-labeled cDNA probes: 100 ng of cDNA (Example 3) were labeled using a SpotLight™ Random Primer Labeling Kit (BD Biosciences Clontech, USA). Template cDNA together with 5 μl of 10x Random Primer Mix were heated to 97 ⁰ C for 3 min in a final volume of 31 μl and chilled quickly on ice. After adding the reaction mix (5 μl 10x Klenow Reaction Buffer, 5 μl 10x Klenow Labeling Mix, 1 μl Klenow Enzyme and 8 μl ddH ₂ O) the labeling reaction was performed at 37 ⁰ C for 30 min. Then the reaction was stopped by adding 2 μl of 0.5 M EDTA (pH 8.0).

B) Purification of biotin-labeled probe: Unincorporated biotin-labeled nucleotides and small (<0.1 kb) cDNA fragments were removed using the NucleoSpin Extraction columns according to the protocol supplied by the manufacturer (BD Biosciences Clontech, USA). The concentration of the newly synthesized biotin-

labeled probes was determined by UV spectroscopy. Finally, the biotin-labeled probes were stored at -20 ⁰ C until hybridization to an Atlas Array. O Hybridization of biotin-labeled probes to Atlas Array: For the hybridization, an Atlas™ Human Cardiovascular Array (Cat. #7734-1, BD Biosciences Clontech, USA) with altogether 588 cardiovascular diseases relevant genes, as well as a SpotLight™ Chemiluminescent Hybridization & Detection kit (Clontech) were used. Each Atlas Array (membrane) was wetted by placing it in a dish of de-ionized H ₂ O, transferred to the hybridization bottle and pre-hybridized in the hybridization mix at 42 ⁰ C for 3 h before the hybridization. Each hybridization was performed overnight at 42 ⁰ C with individual biotinylated cDNA probes. These probes were denatured in 100 mM NaOH at 68 ⁰ C for 20 min and neutralized with 0.5 M NaH ₂ PO ₄ (pH 7.0) at 68 ⁰ C for 10 min before the hybridization in the Hybridizer (Techne, UK).

D) Stringency Washes: Hybridization solution was discarded and the membranes were washed 4 times in 200 ml of Wash solution 1 (2xSSC and 1 % SDS) for 30 min at 60 ⁰ C with continuous agitation. Wash solution 1 was then replaced with Wash solution 2 (0. IxSSC and 0.5 % SDS), and the membranes were washed two more times for 30 min at 48 ⁰ C.

E) Probe detection and signal visualization : The membranes were incubated first in 25 ml Blocking Buffer per Atlas Array at room temperature for 1 hour before an incubation with Streptavidin-HRP Conjugate (final dilution of 1 : 300) for 1 hour with constant gentle agitation. Afterwards the membranes were washed 4 times in Ix Wash Buffer (SpotLight™ Chemiluminescent Hybridization & Detection kit, BD Biosciences Clontech) for 10 min with concomitant equilibration in Substrate Equilibration Buffer (kit) for 5 min at room temperature before the incubation in 8 ml Working Solution (the mix of Luminol/Enhancer Solution and Stable Peroxide Solution, supplied in the kit) per membrane for 5 min. After removing excess liquid the Atlas Arrays were processed by autoradiography with exposure times of 1, 2, 5, 10 and 30 min. The exposed spots were scanned and analyzed with Atlaslmage software (BD Biosciences Clontech). A resulting radiograph (exposure time 5 min) is shown in Fig. 1.

Example 5: 2D-Page, identification and guantification of Lv-GDI Ly-GDI was identified as potentially relevant for DRP using comparative 2D-PAGE imaging combined with MALDI-TOF protein sequence analysis followed by Western- blot quantification. 2D-PAGE images showing differential expression of Ly-GDI in

four sub-groups (DM-females, DM-males; control-females, control-males) of the pilot study (Tab. 4) are given in Fig. 2.

In more detail, comparative 2D-PAGE imaging followed by tandem mass spectrometry analysis was performed as describe below. The identified differentially expressed Ly-GDI protein was further quantified in individual samples using "Western-blot" analysis as described below.

A) Two-dimensional PAGE (2D-PAGE) : A 400 μg aliquot of each protein sample isolated from circulating leukocytes (example 2) of either individual patients or controls was utilized for 2D-PAGE analysis performed two times for each sample. First-dimensional separation was performed in immobilized pH gradient (IPG) strips (Bio-Rad) in the range of pH 4-7 following the manufacturer's protocol. 125 μl protein samples containing rehydration buffer (8 M urea, 10 mM DTT, 1 % CHAPS, 0.25 % Bio-Lyte, pH 4-7) was loaded on the IPG-strips and subjected to 14 kVh overnight at 20 ⁰ C in a PROTEAN IEF Cell. Following first-dimensional separation, the extruded IPGstrips were equilibrated in gel equilibration buffer I (50 mM Tris- HCI, 6 M urea, 30 % glycerol, 2 % SDS, 1 % DTT), followed by equilibration in buffer II (50 mM Tris-HCI, 6 M urea, 30 % glycerol, 2 % SDS and 260 mM iodacetamide) for 10 min before loading them onto polyacrylamide gels (12 % SDS-PAGE) for the second-dimensional resolution in Mini-PROTEAN 3 (Bio-Rad). For size determination of the separated proteins, colored protein standards (Cat. Nr. 161-0318, Bio-Rad) were loaded onto the gels. After electrophoresis, the separated proteins were visualized using silver staining which provides a high sensitivity for protein expression analysis. Differential gene expression was analyzed using specific software (Bio-Rad). Differentially expressed protein spots were excised from the gels and analysed by tandem mass spectrometry.

B) Tandem mass spectrometry: Non-separated peptide mixtures from the tryptic, in-gel digest of unknown proteins from the 2D-PAGE were analyzed by nanoelectrospray tandem mass spectrometry. The tryptised peptides from the wells of the microtiter plates were desalted and concentrated on a pulled capillary containing approximately 100 nl of POROS R2 reverse phase material (Perseptive Biosystems, Framingham, MA) and eluted with 1 ml of 60 % methanol in 5 % formic acid directly into the nanoelectrospray needle. Electrospray mass spectra were acquired on a QSTAR Pulsar I quadrupole TOF tandem mass spectrometer (AB/MDS-Sciex, Toronto, Canada) equipped with a nanoelectrospray ion source (MDS Protana, Odense, Denmark). Fragmentation by tandem mass spectrometry

yielded a stretch of sequence together with its location in the peptide (peptide sequence tag). With these sequence tags appropriate databases were searched. O Western -Bl ot analysis: All analyses were performed two times for each sample. Leukocytes were lysed by homogenisation in lysis buffer (9 M urea (Merck, Germany), 1 % DTT (Sigma, USA), 2 % CHAPS (Merck, Germany), 0.8 % Bio-Lyte, pH 3-10 (Bio-Rad, USA), 5 mM Pefabloc ^® (Roche, Switzerland)), followed by a centrifugation step. The protein concentration was quantified by the DC- Protein Assay (Bio-Rad, USA). 40 μg protein of each sample were loaded onto 12 % SDS-polyacrylamide gels and electrophoresed to separate proteins. The proteins were then transferred to nitrocellulose membranes (Hybond ECL, Amersham Biosciences, UK) and afterwards incubated at room temperature in blocking-buffer (58 mM Na ₂ HPO ₄ , 17 mM NaH ₂ PO ₄ , 68 mM NaCI, 5 % nonfat dry milk powder; 0.1 % Tween 20 ^® ) for I h. Primary antibody incubation was performed at room temperature using a 1 : 250 dilution of Ly-GDI C-20 sc-6047 antibodies (goat polyclonal IgG, 200 μg/ml; Santa Cruz, USA) in washing buffer I (58 mM Na ₂ HPO ₄ , 17 mM NaH ₂ PO ₄ , 68 mM NaCI, 1 % non-fat dry milk powder, 0.1 % Tween 20 ^® ) for 1 h. The membranes were then washed four times in the same solution. The horseradish peroxidase-labelled anti-goat secondary antibody was incubated 1 h at room temperature with the membranes in washing buffer I, followed by three washes in washing buffer II (58 mM Na ₂ HPO ₄ , 17 mM NaH ₂ PO ₄ , 68 mM NaCI, 1 % nonfat dry milk powder; 0.3 % Tween 20 ^® ) and three washes in washing buffer I. Then the membranes were reacted with chemiluminescent reagent ECL plus (Detection Kit, Amersham Biosciences, UK) for 1 h and processed for auto-radiography. The individual signals were measured densitometrically using the "Quantity One" imaging system (Bio-Rad, USA). LY-GDI protein expression rates of individual subjects in leukocytes quantified by Western blot are shown in Tab. 6.

Tab. 6: Quantification (in relative units) of Ly-GDI expression in leukocytes. Control Numbers 61 to 67: additional control subjects.

Example 6: Reverse Transcriptase PCR (RT-PCRI and Real-Time Quantitative PCR (RT-QPCR1

In order to detect the expression of the target genes in leukocytes and to optimize the reaction conditions for Real-Time Quantitative PCR, reverse transcriptase PCR was performed using specific primer sets designed to the house-keeping gene β- actin and the target genes. The primer sets were as follows: β-actin :

Forward : 5 ¹ GATGGTG GG CATG GGTCAG 3 ¹ (SEQ ID NO: 17) Reverse: 5'TGGGGTTCAGGGGGGCCT 3 ¹ (SEQ ID NO: 18)

NF-kappaB:

Forward : 5'CTACAACCCAGGTCTGGAT 3 ¹ (SEQ ID NO: 19)

Reverse: 5 'GTCACTGTGTGTGTTACCAG G 3 ¹ (SEQ ID NO: 20)

XIAP:

Forward : 5 ¹ GAAGATGACTTTTAACAGTTTTGAAG 3 ¹ (SEQ ID NO: 21)

Reverse: 5 ¹ AATTTGGGGATACTTTCCTGTG 3 ¹ (SEQ ID NO: 22) TIMP-I :

Forward : 5'GGCCCCCTTTGAGCCC 3 ¹ (SEQ ID NO: 23)

Reverse: 5 ¹ CTCAGGCTGTTCCAGGGA 3 ¹ (SEQ ID NO: 24)

TIMP-2:

Forward : 5 'TCACCCTCTGTG ACTTCATC 3 ¹ (SEQ ID NO: 25) Reverse: 5'GTCGAGAAACTCCTGCTTG 3 ¹ (SEQ ID NO: 26) cDNA synthesis was performed using a "First-Strand cDNA Synthesis Kit" (Amersham Biosciences, UK). The PCR mixture contained IxPCR buffer (16.6 mM ammonium sulfate, 67 mM Tris, pH 8.8, 6.7 mM MgCI ₂ , 10 mM 2-mercaptoethanol), dNTPs (each at 1.25 mM), primer pairs (100 pM each per reaction), and 10 ng of cDNA-template in a final volume of 50 μl. Reactions were hot-started at 95 ⁰ C for 5 min before adding 1.5 units of Taq Polymerase (Red-Hot ^® , ABgene, UK) at the annealing temperature of 56 ⁰ C followed by polymerization at 72 ⁰ C for 1 min. Amplification was carried out in a Perkin Elmer DNA Thermal Cycler TC480 for 45 cycles (penetration for 45 s at 95 ⁰ C, annealing for 45 sec at 56 ⁰ C, and polymerization at 72 ⁰ C for 30 s), followed by a final 7 min extension at 72 ⁰ C. Negative controls without DNA as well as positive controls with a sequenced template were performed for each set of PCR experiments. PCR products (50 μl) were directly loaded onto 3 % agarose gels (,,Wide Range"- Agarose for analysis of DNA fragments longer than 50 bp, Sigma), stained with ethidium bromide after electrophoresis, directly visualized under UV illumination, and imaged using a specialized imaging system (MWG-Biotech, Germany). The specificity of each PCR amplification was controlled using the site specific restriction analysis of target PCR products. The amplification products underwent an extraction from the agarose gel using a DNA isolation kit (DNACIean™, Hybaid-AGS GmbH, Germany) before digestion. They were digested in a final volume of 50 μl with 20 units of each restriction endonuclease for 4 h, according to conditions specified by the manufacturer (Roche, Switzerland), and imaged after electrophoresis (MWG- Biotech, Germany). Results: RT-PCR of the house keeping gene, β-actin, and all target genes in the cDNA samples demonstrated the specific amplification of the target PCR products with the expected DNA fragment lengths. Further restriction analysis carried out on the isolated PCR products confirmed the expected sequences.

In order to profile precisely the changes in expression of target genes, RT-QPCR was used. SYBR ^® Green I (Molecular Probes, USA) was utilized as the intercalation dye and fluorescent reporter molecule detecting the accumulation of the amplified double-stranded product in the iCycler iQIM Detection System (Bio-Rad, USA) according to the protocol supplied by the manufacturer. 50 ng of the cDNAs

synthesized by above RT-PCR were used for each real-time PCR analysis. The reaction mixtures had the same contents as for RT-PCR with an exception of Red- Hot ^® polymerase (ABgene, UK), which was substituted for Taq DNA polymerase (Roche, Switzerland) in order to avoid color signal disturbances. The same amplification program was used in both qualitative RT-PCR and quantitative realtime PCR analysis. The algorithm of the iCycler iQIM Detection System normalizes the reporter signal (non-intercalated SYBR ^® Green I) to a passive reference and multiplies the standard deviation (SD) of the background signal in the first few cycles by a default factor of 10, to determine a threshold. The cycle at which this baseline level is exceeded is defined as threshold cycle (C _t ). C _t depends on the initial template copy number and is proportional to the log of the starting amount of nucleic acid (Heid, CA. et al., Genome Res. 6:986-994 (1996)). By subtracting the Ct values of the target genes from those of the housekeeping gene (β-actin), the data were normalized. The relative levels were calculated for each sample based on the differences in C _t values. The results of the quantification of the specific target gene transcription rates in leukocytes for NF-kappaB, XIAP, TIMP-I and TIMP-2 are shown in Tab. 7. The mean values and statistical significance are listed in Tab. 2.

Tab. 7: Gene transcription rates (normalized to β-actin)

Example 7: Zvmoqraphv

For determination of gelatinase activity of MMP-2 (gelatinase A) and MMP-9 (gelatinase B) in blood serum "Ready-Gelatin-Gels" (Bio-Rad, USA) were used according to the instructions of the manufacturer. Two micro liters from individual serum samples were electrophoresed under non-reducing conditions using Criterion™ Precast Gel System (Bio-Rad, USA). After electrophoresis, each gel was incubated at room temperature in 2 % Triton X- 100 ^® for 2 x 30 min in order to remove the traces of sodium dodecyl sulphate, and then incubated overnight at 37 ⁰ C in buffer (150 mM NaCI, 50 mM Tris-HCI, pH 7.6, containing 5 mM CaCI ₂ and 0.02 % NaN ₃ ). Afterwards a staining with 0.5 % Coomassie blue G-250 (Sigma, USA) was performed for each gel. The proteolytic activity of each gelatinase was identified as a clear band on a blue background according to the correspondent molecular weight of each gelatinase. Gels were dried between cellophane sheets

with a GelAir™ Drying System (Bio-Rad, USA) and then scanned with a yellow filter using Adobe Photoshop (Adobe System, USA) in grey-scale mode. Densitometric analysis of zymographic lysis zones at 66 and 86 kDa for gelatinases A and B, respectively, was performed using "Quantity One" imaging system (Bio-Rad, USA). The zymography results are show in Tab. 8.

Tab. 8: Results of densitometric quantification (in relative units) of the activity of MMP-2 and MMP-9 in blood serum. Column 3: expression of TIMP-2 in leukocytes; column 4: ratio MMP-2/TIMP-2; column 6: expression of TIMP-I in leukocytes; column 7: ratio MMP- 9/TIMP-l.

Example 8: Statistical evaluation

ANOVA (ANalysis Of VAriables), i.e. a test of the statistical significance of the differences among the mean scores of two or more groups on one or more variables, has been used for statistical evaluations. The Tukey HSD procedure was used to conduct post hoc pairwise comparisons following mean value ratios from ANOVA, since interclass (between class I, class II, and class III of DRP) differences were found to be statistically significant. The JMP statistical software version 5.1. (SAS institute, Cary, NC, USA) is a suitable software for performing the statistical evaluation.