Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in HTLV-I-associated myelopathy

Matrix metalloproteinases (MMPs) have been reported to be involved in inflammatory disorders of the central nervous system (CNS). However, little is known about the role of MMPs in the pathogenesis of HTLV-I-associated myelopathy (HAM)/Tropical spastic paraparesis (TSP). To address this issue, we examined the tissue expression and localization of MMPs and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs) in the spinal cord lesions of HAM/TSP using immunohistochemistry. In addition, the blood and cerebrospinal fluid (CSF) levels of MMPs and TIMPs of the patients with HAM/TSP were determined using sandwich enzyme immunoassays (SIA) and gelatin zymography. Immunohistochemical studies revealed that collagen IV and decorin immunoreactivity on the basement membrane of CNS parenchymal vessels was partially disrupted where inflammatory mononuclear cells infiltrated in active-chronic lesions of HAM/TSP. In these lesions, MMP-2 (gelatinase A) was immunostained mainly on the surface of foamy macrophages and lymphocytes, whereas MMP-9 (gelatinase B) expression was positive in the intravascular and perivascular mononuclear cells but not on foamy macrophages. In contrast, inactive chronic lesions of the spinal cords of the HAM/TSP contained fewer MMP-2-positive or MMP-9-positive mononuclear cells than active-chronic lesions. Many parenchymal vessels had thickened vascular walls which showed increased immunoreactivity to decorin. SIA revealed that production levels of MMP-2 and MMP-9 in both blood and CSF were higher in the patients with HAM/TSP than those in non-inflammatory other neurological disease controls (ONDs). Using zymography, proMMP-9 was detected more frequently in the CSF of patients with HAM/TSP than those in ONDs. Taken together, our data indicate that MMP-2 and MMP-9 may play an important role in the blood-brain barrier breakdown and tissue remodeling in the CNS of HAM/TSP.     

The regulation of neovascularization of matrix metalloproteinases and their inhibitors

This study sought to produce a cognitive profile of herpes simplex encephalitis (HSE) survivors from a large group of definitively diagnosed, acyclovir-treated participants. Results from 22 adults who underwent a battery of neuropsychological tests indicated anterograde memory dysfunction to be the most severe and common deficit (although the variation was great), with less severe and less frequent impairments in the areas of retrograde memory, executive functions, and language functioning. Overall, neuropsychological outcome was unimpaired in six participants, mildly impaired in thirteen, moderately impaired in one, severely impaired in two. Older participants and those with a lower level of consciousness before the start of treatment produced poorer scores on certain aspects of cognitive outcome (p < 0.05). A significantly better cognitive outcome was found in participants for whom there was a short delay (fewer than 5 days) between symptom onset and acyclovir treatment compared with those participants for whom there was a longer delay. The two children in the study had disparate results on most tests, the exception being those assessing memory functioning on which both children had scores at population norms. On a naming task designed to explore category-specific knowledge deficits, the adults as a group made more errors on pictures of living things than nonliving things (matched pair-wise for word frequency and visual familiarity), although this difference disappeared on a smaller subset of pictures also matched for visual complexity.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in reactive and neoplastic lymphoid cells

We have studied the expression of gelatinase A, gelatinase B, interstitial collagenase, tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in reactive lymphoid cells, as well as in a series of cell lines derived from neoplasms of B- and T-cell lineage. Using both Northern blot analysis and zymography, gelatinase B activity was detected by zymography in two Burkitt cell lines and in a tonsillar cell suspension, while gelatinase A and interstitial collagenase activities were not detected by either method. TIMP-1 expression was demonstrated by Northern blot analysis in the multipotential neoplastic K-562 cell line, the high grade Burkitt's B-cell lymphoma lines, isolated tonsillar B cells and at low levels in peripheral blood T cells, but was not expressed in any of the neoplastic T-cell lines or isolated peripheral blood B cells. In contrast, TIMP-2 expression was restricted to tissues containing cells of T-cell lineage with high levels being observed in the neoplastic T-cell lines and lower levels in normal peripheral blood T cells and hyperplastic tonsil. Expression of TIMP-1 and TIMP-2 was confirmed at the protein level by reverse zymography and immunofluorescence assays using antihuman TIMP polyclonal antibodies. Expression of gelatinase B by the high grade B-cell Burkitt's lymphoma cell lines is consistent with previous findings in large cell immunoblastic lymphomas and indicates that this enzyme may play an important role in high grade non-Hodgkin's lymphomas. TIMP expression correlated with cell lineage in that TIMP-1 was primarily observed in B cells and TIMP-2 was restricted to T cells.     

Expression of matrix metalloproteinases and their inhibitors in aneurysms and normal aorta

BACKGROUND: Abdominal aortic aneurysms (AAAs) are characterized by degradation of collagen and elastin resulting from increases in matrix metalloproteinase (MMP) activity. Previous authors have identified isolated increases in expression of specific MMPs in AAAs, but none have compared relative levels of expression of particular MMPs to one another or to those of their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). This study proposes to quantify relative mRNA levels for interstitial collagenase (MMP-1), 72 kd type IV collagenase (MMP-2), 92 kd type IV collagenase (MMP-9), TIMP-1, and TIMP-2 in normal aorta (NA) and AAA to provide insight as to the relative importance of each in aneurysm formation. METHODS: Competitive polymerase chain reactions (PCRs) with gene-specific external standards and cDNA derived from AAAs (n = 8; mean age, 67.4 years) and NA (n = 5; mean age, 40.6 years) were used to quantify mRNA levels. Results were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels, determined by means of competitive PCR, and compared by means of Mann-Whitney statistics. RESULTS: Significant increases in MMP mRNA expression in AAA over NA were observed for MMP-1 (3.64 versus 0.3, p = 0.007), MMP-9 (78.03 versus 3.35, p = 0.003), TIMP-1 (835.32 versus 477.2, p = 0.027), and TIMP-2 (18.09 versus 4.14, p = 0.003). The ratio of MMP to TIMP mRNA levels was higher in AAA than NA (0.135 versus 0.045, p = 0.018). CONCLUSIONS: Increases in expression of MMP-1, MMP-9, and MMP/TIMP ratios may result in increased proteolysis and matrix degradation, which characterize AAAs. MMP-9 appears to be the predominant metalloproteinase expressed in AAA, because its mRNA levels were more than 20 times and 2 times higher than those of MMP-1 and MMP-2, respectively. TIMP-1 mRNA levels were in molar excess to those of any of the metalloproteinases studied.     

5,5-trans lactone-containing inhibitors of serine proteases: identification of a novel, acylating thrombin inhibitor

Synthesis of a variety of 5,5-trans fused lactones, related to compounds found in extracts of Lantana camara, has provided a series of novel acylating inhibitors of human thrombin, trypsin, chymotrypsin and human leucocyte elastase. The most effective thrombin inhibitor is 7 with an IC50 of 130 nM and a Kobs/[1] of 4,000 M-1 s-1.     

Expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in the mouse uterus during the peri-implantation period

In this study the SEB-activated LAK cytotoxicity was identified and characterized in human peripheral blood lymphocytes (PBMC). After 3 days of SEB stimulation, the PBMC acquired a cytotoxicity against traditional LAK targets, K-562 and Daudi, beside that human glomerular endothelial cells (HGEC) were effectively lysed. The magnetic separation of SEB-stimulated CD5+ T cells revealed that the dominant LAK cytotoxicity remained in the CD5- lymphocyte fraction. The major part of the SEB-generated cytotoxicity of CD5- cells could be blocked with specific antibodies to IL-2 and IFN-gamma. The IFN-gamma pretreatment of HGEC reduced the target sensitivity, but because of the upregulation of MHC class II on HGEC surface, these cells were able to present SEB to CD5+ cells. These results suggested that in bacterial superantigen-mediated infection, the non-T (NK cells-derived) LAK cells might have a primary pathogenic role, and the adverse effect of IFN-gamma, that was massively secreted from superantigen-stimulated cells, requires greater consideration.     

Effect of an inhibitor of matrix metalloproteinases on spontaneous osteoarthritis in guinea pigs

A 67-year-old patient with large granular lymphocyte (LGL) leukemia is described. At fluorescence-activated cell sorting (FACS) analysis of the peripheral blood, the lymphocytes were positive for CD3, CD4, CD5, CD29, CD45RA, CD57, and TCR alpha/beta and negative for CD7, CD8, CD16, CD56, CD19, CD22, and TCR gamma/delta. Bone marrow histology and immunohistochemistry did not reveal any lymphocyte infiltration. Cytogenetic examination of peripheral blood cultures showed a clone with the karyotype 46,XY,t(3;5)(p26;q13). Molecular analysis revealed rearrangement of the gamma-T-cell-receptor chain. The region 3p25-3p26 which harbors the von Hippel-Lindau tumor suppressor gene and the RAF1 oncogene has been rearranged in a few cases of T-cell leukemia. The translocation in this case has not yet been described and may reflect an alternative mechanism in the pathogenesis of these disorders.     

Immunohistochemical study of matrix metalloproteinases and their tissue inhibitors in pulmonary Langerhans' cell granulomatosis

OBJECTIVE: To evaluate the role of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in the pathogenesis of the lesions of pulmonary Langerhans' cell granulomatosis. DESIGN: Immunohistochemical and confocal microscopic studies were made of lung biopsy specimens from five patients with pulmonary Langerhans' cell granulomatosis. RESULTS: The reactivity of Langerhans' cells was moderate to intense for MMP-2, weaker for MMP-9, and faint for TIMP-1 and TIMP-2. Type IV collagen colocalized with MMP-2 in areas of damage to epithelial basement membranes, a finding that emphasizes the potential importance of this enzyme in the pathogenesis of the destructive lesions of pulmonary Langerhans' cell granulomatosis. In the more advanced fibrotic lesions, TIMP-2 colocalized with basement membranes and with fibrillar collagen, suggesting that it contributes to the permanence of the fibrosis. CONCLUSION: These results indicate an important role for MMPs and TIMPs in pulmonary Langerhans' cell granulomatosis.     

Structure-based design and synthesis of a series of hydroxamic acids with a quaternary-hydroxy group in P1 as inhibitors of matrix metalloproteinases

In the ovarian follicular fluid (FF) of Holstein cows, calcium (Ca) and magnesium (Mg) levels and their roles on thrombin generation were examined and compared with the blood samples. Total Ca levels in FF increased while the total Mg levels decreased with follicular development from preantral to preovulatory stage of follicles. These changes resulted in Ca values being significantly (p < 0.05) higher in FF from the most developed follicles and the Mg values being significantly higher (p < 0.05) in the least developed follicles. To determine whether the high level of Mg might function to regulate thrombin generation in FF as occurs in plasma, the influence of Mg supplementation of FF from various types of follicles was examined. In FF from small size follicles, Mg accelerated the prothrombin time, an estimation of the overall rate of thrombin production, although a similar effects was not observed in FF from medium and large size follicles. The addition of Mg to FF from all sizes of follicles resulted an inhibition in factor X activation. Since activation of factor X is a precursor step for thrombin formation it is concluded that Mg can function as a slow accelerator of thrombin generation in FF from follicles at the antral stage of development. It is likely to have a more important role in regulating the rate of thrombin generation as the follicle develops.     

Discovery of a novel tetrahydroacridine acetylcholinesterase inhibitor through an indexed combinatorial library

BACKGROUND: Methods for the rapid and efficient preparation of drug candidates through combinatorial chemistry are of increasing interest. We have previously reported an indexed combinatorial library method that allows both the preparation and testing of compounds in solution. We set out to apply this method to develop more effective analogs of the known, marketed drug tacrine, an acetylcholinesterase inhibitor. RESULTS: A one-step condensation of cyclohexanones with cyanoanilines to generate tetrahydroacridine pools was developed. The resulting library of (formally) 72 tetrahydroacridines was screened against acetylcholinesterase, and a compound 10-fold more potent than tacrine, 7-nitrotacrine, was discovered. Its increased potency could be readily explained by examining the known structure of the complex of acetylcholinesterase with tetrahydroacridine. CONCLUSIONS: In this work, we have provided a relatively rare example of carbon-carbon bond formation in a pool synthesis and have discovered a potentially useful acetylcholinesterase inhibitor.     

The asymmetric synthesis and in vitro characterization of succinyl mercaptoalcohol and mercaptoketone inhibitors of matrix metalloproteinases

A series of succinyl based mercaptoketones and diastereomeric mercaptoalcohols were prepared and evaluated in vitro as inhibitors of the matrix metalloproteinases collagenase-1 (MMP-1), stromelysin (MMP-3), and gelatinase-B (MMP-9).     

Structural characterizations of nonpeptidic thiadiazole inhibitors of matrix metalloproteinases reveal the basis for stromelysin selectivity

The binding of two 5-substituted-1,3,4-thiadiazole-2-thione inhibitors to the matrix metalloproteinase stromelysin (MMP-3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1-P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond interactions with enzyme structural elements that are conserved across all enzymes in the matrix metalloproteinase class. Strong hydrophobic interactions between the inhibitors and the side chain of tyrosine-155 appear to be responsible for the very high selectivity of these inhibitors for stromelysin. In these enzyme/inhibitor complexes, the S1' enzyme subsite is unoccupied. A conformational rearrangement of the catalytic domain occurs that reveals an inherent flexibility of the substrate binding region leading to speculation about a possible mechanism for modulation of stromelysin activity and selectivity.     

Matrix metalloproteinases and tissue inhibitors of metalloproteinases in loose artificial hip joints

The role of extracellular matrix metalloproteinase enzymes and the tissue inhibitors of metalloproteinase in the periprostetic connective tissue matrix of loose artificial hip joints is reviewed. In the periprosthetic granulomatous interface connective tissues between bone and implants and inner cellular regenerating pseudocapsular tissues, matrix metalloproteinase 1, matrix metalloproteinase 2, matrix metalloproteinase 3, matrix metalloproteinase-9, and membrane type 1 matrix metalloproteinase enzymes can be shown in the light of immunohistochemistry, enzyme activity analysis, and messenger ribonucleic acid levels. Tissue inhibitors of metalloproteinase 1 and tissue inhibitors of metalloproteinase 2 also are found in the corresponding tissues. Analysis of matrix metalloproteinase and tissue inhibitors of metalloproteinase interaction shows imbalance between the enzymes and the endogenous inhibitors in favor of matrix metalloproteinase. This induces pathologic connective tissue remodeling in the interface and pseudocapsule. The data suggest that matrix metalloproteinase and tissue inhibitors of metalloproteinase system participate in the extracellular matrix degradation and tissue remodeling in artificial hip joints, and may contribute to the periprosthetic weakening, implant loosening, and osteolysis around implants. More evidence for their active involvement is sought by intervention studies with type specific matrix metalloproteinase inhibitors.     

Effect of adenosine analogues on the expression of matrix metalloproteinases and their inhibitors from human dermal fibroblasts

The effect of the cytostatic and antiviral adenosine analogues 3-deazaadenosine (c3Ado) and 3-deaza-(+/-)-aristeromycin (c3Ari) on human skin fibroblasts was studied. Variables examined were cell morphology, viability, DNA fragmentation, expression of matrix metalloproteinases (MMPs) and matrix metalloproteinase inhibitors (TIMPs). None of these variables were changed when cells were exposed to c3Ari concentrations ranging from 10(-5) to 10(-3) M or 10(-5) M c3Ado. However, large changes in cell morphology, viability and expression of MMPs and MMP inhibitors occurred when fibroblasts were treated with 10(-4) or 10(-3) M c3Ado. Cells rounded up, shrank in volume, some detached and viability was lost without any detectable fragmentation of DNA. These changes in morphology and viability were associated with a differentiated expression of MMPs and MMP inhibitors. A large increase in collagenase activity occurred, and depending on the concentration of the adenosine analogue and the length of treatment, this change in activity could be shown to be due to one or a combination of the following factors: an increased synthesis of the collagenase protein, a decreased production of TIMP-1 or an increased activity of the collagenase superactivator, stromelysin. In contrast to this, treatment with c3Ado resulted in a decreased gelatinase activity, which in part could be attributed to an increased production of an inhibitor that seemed to affect gelatinase but not collagenase. The cellular changes induced by c3Ado seemed to reflect some of the alteration in the metabolic machinery that appears during a drug-induced or programmed/controlled death of a dermal cell. The different effects exerted by these two adenosine analogues on dermal fibroblasts can at least in part explain why c3Ado have previously been shown to be more toxic than c3Ari in animal models.     

Rational design of novel antimicrobials: blocking purine salvage in a parasitic protozoan

All parasitic protozoa obtain purine nucleotides solely by salvaging purine bases and/or nucleosides from their host. This observation suggests that inhibiting purine salvage may be a good way of killing these organisms. To explore this idea, we attempted to block the purine salvage pathway of the parasitic protozoan Tritrichomonas foetus. T. foetus is a good organism to study because its purine salvage depends primarily on a single enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), and could provide a good model for rational drug design through specific enzyme inhibition. Guided by the crystal structure of T. foetus HGXPRTase, we used structure-based drug design to identify several non-purine compounds that inhibited this enzyme without any detectable effect on human HGPRTase. One of these compounds, 4-[N-(3, 4-dichlorophenyl)carbamoyl]phthalic anhydride (referred to as TF1), was selected for further characterization. TF1 was shown to be a competitive inhibitor of T. foetus HGXPRTase with respect to both guanine (in the forward reaction; Ki = 13 microM) and GMP (in the reverse reaction; Ki = 10 microM), but showed no effect on the homologous human enzyme at concentrations of up to 1 mM. TF1 inhibited the in vitro growth of T. foetus with an EC50 of approximately 40 microM. This inhibitory effect was associated with a decrease in the incorporation of exogenous guanine into nucleic acids, and could be reversed by supplementing the growth medium with excess exogenous hypoxanthine or guanine. Thus, rationally targeting an essential enzyme in a parasitic organism has yielded specific enzyme inhibitors capable of suppressing that parasite's growth.     

CombiDOCK: structure-based combinatorial docking and library design

BACKGROUND: Pathogen-negative diarrhea is common in HIV infection and has been associated with clinical symptoms, histopathology, HIV expression, CD4+ lymphocyte depletion, cytokine mRNA expression, and apoptosis of lamina propria mononuclear cells. OBJECTIVES AND METHODS: To examine the short-term (7-day) effects of treatment with combination antiretroviral therapies upon gastrointestinal symptoms and rectal mucosa in 15 HIV-infected subjects. RESULTS: Treatment was associated with significant decreases in the perception of abdominal bloating and cramps. Similar declines in RNA burden and rises in CD4+ lymphocyte counts were found in blood and mucosa. Treatment was also associated with a fall in the number of lamina propria mononuclear cells undergoing apoptosis by in situ labeling, a change that correlated with the change in mucosal viral burden. CONCLUSIONS: Peripheral blood and mucosal compartments are equally responsive to effective antiretroviral therapies. The detection of significant changes within 7 days of starting antiviral therapy implies that intestinal dysfunction may be a direct result of local HIV infection.     

Metallopeptidase inhibitors of tetanus toxin: A combinatorial approach

The bacterial protein tetanus toxin (TeNt), which belongs to the family of zinc endopeptidases, cleaves synaptobrevin, an essential synaptic protein component of the neurotransmitter exocytosis apparatus, at a single peptide bond (Gln76-Phe77). This protease activity is a particularly attractive target for designing potent and selective synthetic inhibitors as a possible drug therapy for tetanus. beta-Aminothiols mimicking Gln76 of synaptobrevin have been previously shown to inhibit the tetanus neurotoxin enzymatic activity in the 35-250 microM range. These compounds have now been modified to interact with S' subsites of the TeNt active site, with the aim of increasing their inhibitory potencies. Combinatorial libraries of pseudotripeptides, containing an ethylene sulfonamide or an m-sulfonamidophenyl moiety as the P1 side chain and natural amino acids in P1' and P2' positions, were synthesized. The best inhibitory activity was observed with Tyr and His as P1' and P2' components, respectively. This led to new inhibitors of TeNt with Ki values in the 3-4 microM range. These molecules are the most potent inhibitors of TeNt described so far.