Sarah Shapiro

Regulation of Endothelial Matrix Metalloproteinase-2 by Hypoxia/Reoxygenation

Among the consequences resulting from the exposure of endothelial cells (ECs) to ischemia/reperfusion is angiogenesis, involving degradation of vascular basement membrane and extracellular matrix. Matrix metalloproteinase (MMP)-2, a member of the MMP family, partakes in this process. MMP-2, secreted as a proenzyme, undergoes activation through interaction with membrane type (MT)1-MMP and the endogenous tissue inhibitor of MMPs (TIMP)-2. Although hypoxia and reoxygenation (H/R) are major constituents of ischemia/reperfusion processes, their direct effects on endothelial MMP-2 have been scarcely investigated. This study examined the in vitro effects of H/R on human macrovascular ECs (EAhy 926). The level of MMP-2 mRNA (Northern blot) and protein (zymography, ELISA) and the mRNA of its activator (MT1-MMP) and inhibitor (TIMP-2) were analyzed. Short (6-hour) hypoxia inhibited the mRNA expression of MMP-2, MT1-MMP, and TIMP-2, culminating in reduced latent and active MMP-2 protein. Prolonged (24-hour) hypoxia further suppressed MT1-MMP and TIMP-2 mRNA, whereas it enhanced MMP-2 mRNA and enzyme secretion (after 48-hour hypoxia). Reoxygenation did not influence the inhibited TIMP-2 but upregulated MMP-2 and MT1-MMP mRNA expression, leading to enhanced secretion of active MMP-2 protein. These results demonstrate H/R-mediated modulation of EC MMP-2 at both transcriptional and posttranscriptional levels. Prolonged hypoxia of ECs appears to enhance MMP-2 production and secretion, whereas reoxygenation further increases its level. These H/R-mediated effects on MMPs have the potential of enabling EC migration and possible angiogenesis.

Matrix metalloproteinases and their tissue inhibitors as markers of disease subtype and response to interferon-β therapy in relapsing and secondary-progressive multiple sclerosis patients

Matrix metalloproteinases (MMPs) have recently been implicated in the pathogenesis of multiple sclerosis. Their suggested role includes the disruption of the blood–brain barrier, immune cell transmigration into the central nervous system, and myelin degradation. The present study characterized the mRNA level of a wide spectrum of MMPs and tissue inhibitors of metalloproteinases (TIMPs) expressed by peripheral blood leukocytes from relapsing‐remitting (n = 16) and secondary‐progressive (n = 12) multiple sclerosis patients. The expression of the same MMPs and TIMPs was evaluated also in a prospective 12‐month follow‐up of 6 patients randomly chosen from each of the 2 groups during interferon‐β‐1a treatment. Reverse transcription‐polymerase chain reaction assessment demonstrated elevated levels of MT1‐MMP and MMP‐7 mRNA levels in both groups of patients, and no significant differences in MMP‐9 levels, compared with healthy controls. Divergent expression of MMP‐2 between relapsing‐remitting and secondary‐progressive patients compared with controls was observed. IFN‐β treatment was associated with significant suppression of MMP‐9 and MMP‐7 mRNA in relapsing‐remitting patients, though no significant changes were observed in the secondary‐progressive group. These results contribute to the understanding of the interferon‐β‐mediated immunomodulatory and therapeutic effects in multiple sclerosis patients and also support evidence for distinct immune mechanism(s) underlying relapsing‐remitting‐ versus secondary‐progressive multiple sclerosis. The study also suggests that MMPs may be considered as potential biomarkers for response to treatment as well as targets for immunotherapy in multiple sclerosis.

Three different GB virus C/hepatitis G virus genotypes: Phylogenetic analysis and a genotyping assay based on restriction fragment length polymorphism

The 5′‐untranslated region (5′‐UTR) sequences of 33 GB virus C/hepatitis G virus (GBV‐C/HGV) obtained from different geographic areas were determined through reverse‐transcription polymerase chain reaction and dideoxy chain termination sequencing, the alignment of sequences, the estimation of the number of nucleotide substitution per site, and construction of phylogenetic trees. The 5′‐UTR of GBV‐HGV was found to be heterogeneous, with 70.9–99.5% homology. Three distinct phylogenetic branches were observed consistently in all phylogenetic trees. GBV‐C is the prototype for one, HGV for another, and there is a new branch which consisted of GBV‐C/HGV isolates from Asia. Genotype‐specific restriction sites for the restriction enzymes, ScrFI and BsmFI, were identified, and a simple restriction fragment polymorphism analysis was developed for genotyping. These data provide evidence that GBV‐C/HGV consists of three different genotypes. Our simple genotyping assay will also provide a tool for epidemiological studies of GBV‐C/HGV infection.

Intravenous gamma globulin inhibits the production of matrix metalloproteinase‐9 in macrophages

Degradation of the extracellular matrix (ECM) is essential for progression and metastasis of cancer cells. The ECM‐degrading enzymes, matrix metalloproteinases (MMPs), are produced mainly by intratumor monocytes/macrophages. MMPs, particularly MMP‐9, are reported to be of crucial significance for both growth and tumor invasiveness. Inhibition of the expression of MMP‐9 may prevent tumor development. High‐dose intravenous gamma globulins (IVIG) effectively inhibit metastatic spread of tumors in mice and humans and a variety of mechanisms have been suggested to explain this effect.

Expression of matrix metalloproteinases, sICAM-1 and IL-8 in CSF from children with meningitis.

The combined expression of the inflammatory mediators, matrix metalloproteinases (MMPs), soluble form of intracellular adhesion molecule ICAM-1 (sICAM-1) and interleukin (IL)-8, was evaluated in children infected with bacterial or viral meningitis. MMP-2 and IL-8 were detected in all CSF samples and were enhanced in both bacterial and viral infected samples, compared to those from control children. The expression of MMP-9 as well as sICAM-1 was not detected in control CSF while observed in viral infected and further elevated in bacterial infected samples. This pilot study supports a role for MMPs, IL-8 and sICAM in infectious meningitis and suggests further research to determine their possible use as biomarkers for various forms of meningeal infection as well as the use of their specific antagonists as potential therapeutic agents for central nervous system (CNS) inflammatory processes.

Bio-markers of disease activity and response to therapy in multiple sclerosis

The pathogenesis of multiple sclerosis (MS) involves immune-mediated as well as neurodegenerative processes, each comprising both acute and chronic phases. The vast variety of cell types playing a part in the cascade of these processes, including lymphocytes, monocytes, glia cells, neurons, and endothelial cells, are sources for many biological activities and mediators [1–6]. Measurement of these activities (as cell proliferation, migration) and related mediators (as cytokines, proteolytic enzymes), conducted at different biological levels such as mRNA, protein level and activity, in accessible body fluids, to characterization of useful bio-markers for MS, has gained much attention in recent years. Aditionally, MS appears to include distinct clinical subtypes. Their main forms are RRMS, which tends to convert, eventually, to an SP form, and PPMS [7,8]. Immuno-pathological as well as neuro-radiological studies point to possible differences in the basic mechanisms underlying these distinct clinical subtypes [9–12]. Thus, the studies of disease-related indicators are directed towards the identification of markers specific to the distinct MS clinical subtypes and underlying pathogenic mechanisms. This review will focus on some of the bio-markers the mea-

Inorganic lead enhances cytokine-induced elevation of matrix metalloproteinase MMP-9 expression in glial cells.

Inorganic lead (Pb) is a ubiquitous environmental contaminant that produces a variety of deleterious effects in the central nervous system (CNS). Matrix metalloproteinases (MMPs), specifically MMP-9, induced by inflammatory cytokines, are increasingly being implicated in CNS pathology. The present study demonstrates that low concentrations of either pro-inflammatory cytokines (TNF-alpha and IL-1beta) or Pb did not influence the MMP-9 expression in a glial cell line (C6) when added separately. However, combined administration of Pb and cytokines induced a marked synergized elevation of MMP-9 expression in spite of a reduction in the number of glial cells. These results demonstrate a possible new mechanism by which Pb may induce neuropathological processes.

Interferon-β and -γ, but not tumor necrosis factor-α, demonstrate immunoregulatory effects on carcinoma cell lines infected with human papillomavirus

Mechanisms whereby cells infected with human papillomavirus (HPV) escape immune surveillance, ultimately leading to invasive cervical carcinoma, may involve changes in local cytokine production, loss of responsiveness to cytokines, and alterations in the expression of immune‐regulatory molecules such as histocompatibility‐related leukocyte antigen (HLA) Class 1 and 2 and ICAM‐I. This study examined the separate and combined effects of immune‐activating cytokines, interferon (IFN)‐γ, IFN‐β, and tumor necrosis factor (TNF)‐α, on the expression of these molecules.

A role for complement as the major opsonin in the sequestration of erythrocytes from elderly and young donors

Summary. Erythrocytes from elderly donors (> 70 years), but not young donors (18‐35 years), are shown to undergo sequestration in an in vitro erythrophagocytosis assay. Comparable levels of sequestration are observed for high density erythrocytes from young individuals and both low density and high density erythrocytes from elderly individuals. These cells, which are susceptible to phagocytosis with no additional treatment are collectively termed ‘in situ aged’erythrocytes. We present evidence for the involvement of complement in the sequestration of ‘in situ aged’erythrocytes and correlate levels of complement bound to ‘in situ aged’erythrocytes from young and elderly donors with levels of phagocytosis. We also demonstrate that the in vitro sequestration of erythrocytes from elderly donors can be inhibited by β‐galactosyl sugars and arginine‐glycine‐aspartic acid (RGD) but not by mannose nor by Protein‐G, a specific inhibitor of Fc‐γ mediated phagocytosis. These experiments show that IgG is not the major opsonin in the sequestration of red cells from elderly donors. In support of the role of complement rather than IgG as the major opsonin in red cell sequestration, we further demonstrate that C′3 bearing immune complexes block in vitro sequestration of erythrocytes from elderly donors. This competition is not dependent upon the isotype of the immunoglobulin (IgM or IgG) in the complex but rather on the presence of active complement.

Matrix metalloproteinases and their inhibitors in hypoxia/reoxygenation and stroke

Hypoxia, resulting from ischemic events, leads to a variety of pathological processes, depending on the duration and severity of the primary insult. The ensuing reoxygenation, resulting from reperfusion, may have opposing effects; either aggravation of the primary hypoxia-induced injury or stimulation of repair processes in an attempt to minimize the tissue damage. Amongst the cells directly exposed to changes in oxygen tension are endothelial cells (ECs), lining the vascular bed, as well as peripheral blood leukocytes (PBL). The response of these cells to hypoxia involves a complex cascade of events including: increased release of thrombolytic factors, increased permeability of the endothelium, immune activation, release of chemokines, cytokines and growth factors, as well as up-regulation of surface adhesion molecules. Includes. Two additional processes are part of the responses to hypoxia: immune cell transvasation and angiogenesis of ECs, processes which are dependent on the activity of enzymes, members of the matrix metalloproteinases (MMPs) family [1–4].