MATRIX METALLOPROTEINASE-9 AND INFLAMMATION IN DIFFERENT TYPES OF MULTIPLE SCLEROSIS

39 for patients with inflammatory breast cancer: SBCCSG-04 study. Journal of Clinical Oncology, 24 (18S), 10783. [18] Veyret, C., Levy, C., Chollet, P., Merrouche, Y., Roche, H., Kerbrat, P. et. al (2006). Inflammatory breast cancer outcome with epirubicin-based induction and maintenance chemotherapy. Cancer, 107 (11), 2535–2544. doi: 10.1002/cncr.22227 [19] Yang, W. T., Le-Petross, H. T., Macapinlac, H., Carkaci, S., Gonzalez-Angulo, A. M., Dawood, S. et. al. (2007). Inflammatory breast cancer: PET/CT, MRI, mammography, and sonography findings. Breast Cancer Research and Treatment, 109 (3), 417–426. doi: 10.1007/s10549-007-9671-z [20] Rousseau, C., Devillers, A., Sagan, C., Ferrer, L., Bridji, B., Campion, L. et. al (2006). Monitoring of Early Response to Neoadjuvant Chemotherapy in Stage II and III Breast Cancer by [18F]Fluorodeoxyglucose Positron Emission Tomography. Journal of Clinical Oncology, 24 (34), 5366–5372. doi: 10.1200/jco.2006.05.7406


Introduction
Data received in the recent years using morphological, immunological and neuroimaging research methods have greatly changed the traditional notion of multiple sclerosis (MS) as a remittent central nervous system (CNS) disease causing destruction of myelin of brain and spinal cord conductors only. It turned out that the pathological process continues even in the phase of clinical remission; axons are damaged from the initial stage of disease; besides the CNS white matter, grey matter of the cortex and subcortex is also damaged. However, a number of issues still remain unsolved. Different clinical courses of MS, heterogeneity of its clinical implications, different effect of immunomodulatory therapy for the same clinical forms implies various pathogenetic mechanisms of the CNS damage in this disease [1][2][3][4][5]. The scientific and practical objective in researching MS lies in studying mechanisms of neurodegenerative process development, assessment of interrelation between inflammatory, immunopathological and degenerative processes. There have not yet been established any informative immunobiochemical markers allowing the monitoring of pathological process activity at different MS courses using process activity estimates and prognosis; mechanisms that trigger exacerbations and remission are not clear either. Applicability of immunological and biochemical markers for the estimation of immunocorrecting and anti-inflammatory therapy efficacy is disputable [6][7][8]. Many of the proposed methods are complicated, laborious and costly. In this respect, it deems relevant to look for informative and widely available markers suitable for pathological process monitoring and prediction.
Search for new, more efficient MS treatment methods is closely related to the profound research of pathogenesis links of the disease, many aspects of which still unclear. In particular, the role of matrix metalloproteinase (MMP) which is normally a physiological mediator of tissues remodeling in MS pathogenesis has not been sufficiently studied. Most of the investigations are based on experiments on animals and MMP in vitro studies [9][10][11][12]. One of the main pathogenetic links in MS such as blood-brain barrier (BBB) disruption and as a result migration of plasma proteins to the brain parenchyma is MMP. MMPs form a series of enzymes whose principal activity is remodeling of the intercellular matrix. MMP-9 or gelatinase В is the most complicated metalloproteinase in terms of its domain structure and activity regulation. MMP-9 activity is strictly regulated at the different levels: regulation of genetic transcription by cytokines and various cellular interactions; regulation of proenzyme activation by the enzyme cascade including serine proteases and other MMPs; regulation by specific tissue inhibitors of MMPs (TIMPs) or non-specific inhibitors. MMP's main function is degradation of extracellular matrix components.
Matrix metalloproteinases are involved in multiple processes represented in Table 1.
Scholars attach great importance to MMPs when studying inflammations. It is known that most cells involved in immune reactions and inflammation (T-lymphocytes, macrophages, eosinophils and neutrophils) produce certain MMPs [13][14][15][16]. MMP production depends on different inflammatory mediators. MMP synthesis in macrophages for example is induced by contact with collagen and is further intensified by T-lymphocyte membrane determinants. Through matrix damage MMPs prepare the vascular wall to the adhesion of immune cells and facilitate migration of cells, proteins, antibodies, complement, etc. through the basal membrane to tissues [17][18][19]. According to the latest publications MMPs are very important for CNS development and differentiation and may be produced by neurons [20,21]. Besides through ECM remodeling it can influence different functions of the nerve tissue and growth factor concentration as well as impact synapse formation and stabilization and further interneuronic and neuroglia interaction. There are studies dedicated to MMP's role in metastatic dissemination of CNS tumors, stroke, neurodegenerative and demyelinization diseases. Some studies deal with MMPs' activity and intensity in infectious diseases of the CNS. MMP's role in bacterial meningitis is paid special attention to. The fundamental pathogenetic mechanism causing secondary damage at demyelinization diseases is BBB disruption. There are a lot of inflammation mediators that are known to play a certain role in BBB disruption. These are above all radicals of oxygen, nitrogen oxide, metabolites of arachidonic acid (E2 prostaglandines) and cytokines such as TNF-α, interleukin (IL)-1 β, IL-6 and IL-10 [22,23]. However mechanisms of BBB disruption have not yet received comprehensive examination.
Thus on the one hand MMPs are physiological mediators important for CNS growth, development and functioning and on the other hand they are proteolytic enzymes that actively interact with cellular and humoral factors of the immune system (epidermal growth factor, growth factor of thrombocyte origin, IL-1, IL-4, etc.) and trigger pathological processes.
This research aims at improvement of pathological process stages diagnostics at multiple sclerosis and further therapy optimization depending on the activity of inflammatory process.

Research materials and methods
135 patients (54 males and 81 females) of different age (from 18 to 67) with multiple sclerosis (diagnosed according to the McDonald criteria, 2010) of different course types and at different activity stages of the pathological process were examined.
Quantitative determination of MMP-9 concentration was performed in the blood serum using an immune-enzyme test kit (Human MMP-9 ELISA, Bio Tech Lab-S).

Results and discussion
The highest MMP-9 rate was observed in patients with relapsing-remittent multiple sclerosis (RRMS) (average rate ММР-9 av =212.37±17.51). The lowest rate was in patients with primary progressive MS (PPMS) (ММР-9 av =135.33±6.87). The medium rate was in patients with secondary progressive (SPMS) (ММР-9 av =252.19±10.36). Obtained factor is F=5.238; its statistical significance is р<0.01. ANOVA variance analysis was used to determine interrelation between the clinical course type and MMP-9 rate; graphic results are provided in Fig. 1.  Fig. 1. Dependence of MMP-9 rate on the clinical course type Besides the metalloproteinase rate was analyzed at different activity stages of the pathological process (exacerbation and remission in RRMS, progression and stabilization in progredient course types). Average MMП-9 av rates for the groups under examination were as follows: in RRMS at the remission stage -122.05±7.82, at the exacerbation stage -378.68±21.54. In progredient course types: at the stage of progression -164.73±12.21, at the stage of stabilization -114.64±8.43. The data obtained can be found in Fig. 2.   Fig. 2. Dependence of MMP-9 rate on the clinical course type The value of factor F is so high that probability of error was almost equal to zero (р<0.0001). It is shown on the graph that the highest MMP-9 rates were observed at the exacerbation and progression stages of the pathological process. At the stages of remission and stabilization MMP-9 rates were within the normal range. Thus it was proved with a high degree of confidence that the average MMP-9 rate depends on the stage of disease.
MMP-9 rate is influenced by all of the factors collectively but not individually. Therefore clinical course types and disease stages as factors influencing MMP-9 rate most of all were analyzed using the multi-factor variance analysis. The results obtained are given in Table 2.
According to Table 2, the highest MMP-9 rates were observed in the RRMS group at the stage of disease exacerbation (381.54±22.19). MMP-9 rate was within the normal range at the stage of remission and reached 122.05±7.82. MMP-9 rates were high at the active stage in SPMS and PPMS: 169.98±15.64 and 147.03±6.78 respectively. The results obtained proved that inflammatory reactions in SPMS are more apparent than in PPMS when neurodegeneration processes are of primary importance. Unexpectedly MMP-9 rates at the stage of stabilization were higher in PPMS (121. 96±10 between the number of patients in the respective groups -there were three times fewer patients in the PPMS group at the stage of stabilization than in the SPMS group at the same stage. The obtained results are represented graphically in Fig. 3.