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Univerzitet u Zenici
  Doctoral theses defended at the University in Zenica

Date and place of thesis defense:
10.01.2013., Zenica  
prim. doc. dr. sci. med. Mersija Kasumović
prof.dr.sci. Đemo Subašić

Thesis title:

Study of TIMP-1 and MMP-1 gene expression correlation as well as some specific biomarkers in patients with systemic lupus erithematosus

Systemic lupus erythematosus (SLE) is a complex autoimmune disorder with multigenic character. It was found that a large number of genes have altered expression in patients suffering from this disease and that their role is mainly linked to apoptosis process, repair of damaged DNA molecules, adhesion receptors and determination of receptors in immune system. One of genes that is often the object of research is TIMP-SEL 1 gene (Tissue inhibitor of matrix metalloproteinase 1 gene). TIMP-1 gene encoding TIMP-1 protein which is in fact a natural inhibitor of certain matrix metalloproteinases such as MMP-1 and MMP-9.

So the body is balanced relationship between matrix metalloproteinases and their inhibitors on the basis of feedback, which maintains autotolerance and somehow prevent its violation. Any change in this expression, leads to loss of autotolerance and induction of autoimmunity. SEL patients reduces the level of expression of TIMP-1 gene, which has a logical consequence of increased level of MMP-1 and MMP-9 protein enzymes. Altered expression of over 1000 genes was detected in systemic lupus erythematosus, except of  these genes.

Besides the aspects related to matrix metalloproteinases and their natural inhibitors, it was observed that the level of essential components such as complement C3 and C4 as well as the number of T lymphocytes (CD4 and CD8) that are involved in the regulation of peripheral tolerance was redused, and thus prevent synthesis of autoantibodies, as a key initial event in the SEL etiopatology.

Every serious study of molecular basis of systemic lupus erythematosus include genetic aspect, enzymatic aspect and the level of CD4 and CD8 lymphocytes, NK cells and B lymphocytes, all in light of specific biomarkers of disease activity. So, besides SEL diagnostic biomarkers (ANA, anti-Sm, anti-ds-DNA), there are biomarkers of activity such as levels of C3 and C4 components of complement, anti-cardiolipin antibodies and anti-Sm/RNPs antibodies. Concerning SEL patients, there can be a passive and active stage of the disease, so called “overlap syndrome”, where the symptomatology of several systemic autoimmune diseases overlap, antyphospholipid syndrome and other clinical characteristics.

 It should not be ignored the role of cytokines in the induction of SEL since the MMPS and TIMPs genes are regulated by the cytokines IL-1 beta and TGF-B.

Otherwise, the clinical management of SEL patients, monitors the level of CD4 + and CD8 + T lymphocytes in order to monitor disease activity. Some studies have shown an increased level of double-negative T lymphocytes (CD4-CD8-) or DNT (double negative T-cells) in the SEL patients. Flow cytometry monitors the level of DNT in SEL patients in mononuclear cells of peripheral blood, in which, the increased levels of DNT the cells was observed.

It is believed that these cells do not play a role in SEL activities, although it is not very reliable evidence. However, research conducted by Sawada et al. 1993 showed that the level of CD4 + and CD8 + T lymphocytes significantly increased in cases of active systemic lupus and a positive correlation with the erythrocyte sedimentation rate and the level of anti-ds-DNA and IgG was found, while a negative correlation with levels of complement components was observed. All these parameters are important when the intensity of disease activity in SEL patients is monitored.

Thus, flow cytometry defines lymphocyte phenotypes in peripheral blood samples of SEL patients <nd NK cells surface receptors (G2A and G2D) because it can lead to gignificant information relevant etiopatology of sistemic lupus erythematosus.

Based on all the facts stated in this research, the molecular bases of SEL was covered by aspect of the expression of TIMP-1 and MMP1 genes, levels of specific activity biomarkers such as C3, C4, anti-ds-DNA, anti-Sm, anti-Sm/RNPs, ACA-G, ACA-M, Beta-2-G, Beta-2-M and the level of immune system cells that are responsible for maintaining of autotolerance-CD4 and CD8 T lymphocytes.

During 2011 in Clinical Center University of Sarajevo in Institute of Clinical Immunology, 300 serum samples of patients suspected to SEL were analyzed and selected by the clinicians of Rheumatology Clinic. There were 24 patients suspected to SEL. The majority of these patients were hospitalized later, and their serum were analyzed in order to determine these biomarkers and activity levels of expression of TIMP-1 and MMP-1 gene at the level of mRNA molecules, as well as levels of CD4 and CD8 T lymphocytes. These analyzes were carried out by following methods:

 - IFT (Fluorescent antibody technique)
-  ELISA test
-  nephelometry
-  Flow cytometry
-  RT-PCR meStudies have shown the following:

-  High values of ??anti-Sm/RNPs have always been in correlation with the
    values ?? of ACA- G and ACA-M, and that this biomarker is more important
    in the diagnosis, but it is not so reliable when it comes to disease activity.

- Beta-2 G and  Beta-2-M are shown to be bad indicators of SEL disease
   activity because their level was not significantly changed regardless of the
   variation of other clinical and laboratory parameters.

- Levels of C3 and C4 have been regularly diminished in the vast majority of
   patients with active disease, which indicates that it is a good biomarkers of
   SEL activity as well as therapy effectiveness. The level of C3 and C4 should
   be monitored for several weeks and in cases of their persistently low level,
   renal biopsy was regularly done and laboratory analysis of the important
   components of complement.

-  Determined-scale sensitivity and specificity of analyzed SEL activity
    biomarkers have the following values:

Beta-2-G --------------- 13,   0%

Beta-2-M --------------  13,   0%

Anti-Sm/RNPs--------- 29,  10%

ACA-IgG -------------- 50,    0%

ACA-IgM -------------  58,    3%

C4 levels ----------------79,    1%

C3 level ---------------- 87,     5%

- Increased levels of immunoglobulins were in correlation with phenotyping
   parameters, and in most cases the levels of CD3 were increased and levels of
   CD3+/HLA-DR and total HLA-DR expressed on lymphocytes were reduced.

It should be noted that levels of CD4+CD25+ and CD4+CD25- which were analyzed do not have referential value, so that it could not be correlated with other parameters, but jet in the further research it is necessary to evaluate their level with the appropriate number of specifically selected, healthy people, in order to determine referential values ??for these parameters for the Bosnia and Herzegovina population. Each world laboratory establishes its own referential values ??for these parameters, because individual genetic variation takes into account.

           Otherwise, in these studies was established a positive correlation between CD3 (T-ly), and CD3 + / HLA-DR and total HLA-DR.

In OS patients other than antiphospholipid syndrome other clinical complications such as myocardial infarction, pulmonary thromboembolism, renal complications, pancreatic complications and anemia were found.

           Therapy was more effective when it comes to normalization of T lymphocytes in correlation to B lymphocytes.

Patients with predominant symptoms of joints inflammation, does not have so apparent immunophenotype changes as SEL patients with overlap syndrome.

It was obvious that in such cases would be easier to monitor disease activity and efficacy of therapy using parameters such as levels of immunoglobulins, C3, C4, CIC and anticardiolipine antibodies.

It should be emphasized that serum C3 and C4 are specific parameter in the diagnosis and monitoring of lupus nephritis, because their level is usually low in such cases.

         Determined  TIMP1 gene  expression ranged  from 8,742 to 49,253.

         Determined  MMP1 gene  expression ranged  from 40,087 to 46,479.

          MMP1 gene expression was much more stable in comparison to TIMP1 gene at SLE patients. The extreme differencies  in expression of these two genes  probably, have been caused  by individual genetic variation. Only one  serum specimen showed this extreme difference (TIMP1-19,45 versus  MMP1- 46,47).

          Also, only one serum specimen showed undetectable level of TIMP1 gene expresssion and MMP1  expression 44,44. These findings were in corelation with increasing of  CD3+/HLA-DR   level and ACA level as well.

           Lower expression of TIMP-1 gene and increased expression of MMP-1 gene, although there are some discrepancies when it comes to SEL patients with OS and antiphospholipide syndrome.

In the following research it would certainly be interesting to include molecular-biological studies of expression of MMP-9 gene,IL-2,  IL-10 gene, IL-15 gene, IL-17 gene  and TGF-B gene, which would give a closer picture of coordination of metalloproteinases and their inhibitors concerning measurement of specific biomarkers SEL activities. Otherwise some of the SEL hospitalized patients had overlap syndrome and received appropriate symptomatic therapy, and it was overlapping symptomatology of few systemic illnesses such as SEL, RA and Sjögren syndrome. Such patients are extremely good model for selective distinguishing of quantitative values of SEL biomarkers individually for each systemic disease.

Key words :
SLE, biomarkers, ANA-IFA, ELISA, nephelometry, flow cytometry, PCR

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