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ID PMID Title PublicationDate abstract
8612351 Genetically programmed development of salivary gland abnormalities in the NOD (nonobese di 1996 Apr NOD (nonobese diabetic) mice develop chronic lymphocytic infiltrates of the salivary glands (sialoadenitis) that correlate with a temporal decline in saliva production. To differentiate autoimmune and nonautoimmune components in this decline, we evaluated glandular function in NOD-scid mice. Although saliva volumes and protein concentrations appeared normal, amylase and EGF activities declined 50 and 20%, respectively, in NOD-scid mice between 10 and 25 weeks of age. Salivary protein profiles on SDS-polyacrylamide gels showed a profound decline in two prominent proteins of 32 and 20 kDa, and the emergence of a new 27-kDa protein. All three proteins exhibited amino acid sequence homology to parotid secretory protein (PSP) and reacted with PSP-specific antibody, suggesting an age-dependent alteration in PSP. In addition, there was an induced expression of proline-rich protein in the salivary glands and saliva of NOD and NOD-scid mice that was not detectable in mouse strains lacking autoimmune disease. Submandibular gland histology revealed selective loss of acinar tissue despite an absence of sialoadenitis. These changes in salivary protein composition and histology in the absence of detectable lymphocytic infiltration suggest that glandular defects in the NOD genetic background may contribute to the triggering of the autoimmune response in the salivary glands.
7673733 SLE-like autoantibodies and Sjögren's syndrome-like lymphoproliferation in TGF-beta knock 1995 Sep 15 Mice bearing the TGF-beta 1 null mutation (-/-) develop lymphoid infiltrates in the heart, lungs, salivary glands, and other organs similar to those seen in the pseudolymphoma of Sjögren's Syndrome. We studied sera from -/- mice and found elevated Ab levels to dsDNA, ssDNA, and Sm ribonucleoprotein. No Abs to SSA/Ro or SSB/La and no IgM rheumatoid factor were found. Serum autoantibodies were predominately IgG and were specific as shown by ELISA inhibition studies. Antinuclear Ab patterns on Western blots varied from one mouse to the next, indicating a random process responsible for the diversity. Wild-type and heterozygote mice had no autoantibodies. Ig glomerular deposits were found in -/- mice, indicating that these autoantibodies may be pathogenic. Treatment of -/- mice with dexamethasone or TGF-beta 1 failed to suppress autoantibody production. These mice represent an overlap combining the autoimmune serology of SLE with the tissue infiltrates of SS. Our results support the concept that TGF-beta 1 is an important naturally occurring immunosuppressive cytokine whose absence can lead to a systemic autoimmune disease.
7564269 The cell proliferation-associated protein Ki-67 is a target of autoantibodies in the serum 1995 Sep BACKGROUND: Ab in the serum of patients with autoimmune diseases have been used to identify, characterize, and purify many autoantigens. EXPERIMENTAL DESIGN: Serum from a patient (Ge) with Sjögren's syndrome was used to identify cDNA clones encoding novel autoantigens. This patient's serum was chosen for study because it contained antinuclear Ab that were different from those frequently detected in patients with autoimmune diseases. RESULTS: Ge serum identified a cDNA clone encoding part of protein Ki-67, a cell proliferation-associated protein. The Ki-67 protein (pKi-67) was not previously known to be a target of autoantibodies. To investigate the association between Ab directed against pKi-67 and autoimmune diseases, sera from autoimmune mice were tested for reactivity with a recombinant fragment of pKi-67. Ab were detected in serum from MRL/MpJ(-)+/+ and MRL/MpJ-lpr/lpr mice but not in serum from other autoimmune mice or control animals. CONCLUSIONS: Protein Ki-67 joins the proliferating cell nuclear Ag (PCNA) as an example of a cell proliferation-associated protein that is a target of autoantibodies. The presence of anti-pKi-67 Ab in MRL mice, but not other autoimmune mice, suggests that anti-pKi-67 Ab may be specific markers for the systemic lupus erythematosus-like illness that develops in these animals. Further characterization of the immune response directed against this Ag may provide clues to the etiology and pathogenesis of autoimmune disease in these animals.
7600243 Proteolytic enzymes as indicators of periodontal health in gingival crevicular fluid of pa 1995 Feb The present study characterizes the periodontal status of patients with Sjögren's syndrome (SS) and measures collagenase, elastase and gelatinase in gingival crevicular fluid (GCF) from these patients compared with adult individuals with periodontitis and healthy controls. The periodontal status was assessed by the Gingival Bleeding Index (GBI), the Visible Plaque Index (VPI), and pocket depth. Activity measurements were performed for collagenase with SDS-PAGE/laser densitometry, for elastase spectrophotometrically using a synthetic N-succinyl-Ala-Ala-Val p-nitroanilide peptide substrate, and for gelatinase with zymography. Seven of the 8 patients with SS and xerostomia showed a periodontium comparable to that seen in healthy controls. The GCF collagenase and elastase were significantly lower in patients with SS and in healthy controls than in patients with adult periodontitis. It was noteworthy that the one SS patient with periodontitis had high GCF collagenase and elastase activity. In all study groups multiple forms of gelatinases were present, indicating that they represent constitutively expressed proteinases involved in normal tissue remodeling processes. Our findings suggest that periodontal pockets/GCF form a micromilieu not affected by involvement of glandular tissue and, therefore, patients with SS show, clinically and biochemically, a periodontal status comparable to that seen in healthy controls.
7821973 MRL/lpr mice produce anti-Ro 52,000 MW antibodies: detection, analysis of specificity and 1994 Sep MRL/lpr mice are studied as one of the animal models of the human autoimmune disease Sjögren's syndrome. The mice develop inflammatory exocrinopathy resembling that of patients with Sjögren's syndrome. To investigate if MRL/lpr mice produce the anti-Ro/SS-A and anti-La/SS-B autoantibodies common to Sjögren's syndrome patients, mouse sera were tested in ELISA and Western blot with recombinant Ro 60,000 MW, Ro 52,000 MW and La antigen. Thirty per cent of mice aged 4 months and 5% of mice aged 2 months produced antibodies to human Ro 52,000 MW. Antibodies to Ro 60,000 MW and La were found in a low percentage of the older mice but not at all in the younger mice. Immunohistological staining of mouse organ sections demonstrated anti-Ro 52,000 MW-producing cells in spleen, lymph nodes and salivary glands of seropositive animals. These findings provide further evidence for the usefulness of the MRL/lpr mouse as a model for Sjögren's syndrome.
7511655 Autoantibody responses to the "native" 52-kDa SS-A/Ro protein in neonatal lupus syndromes, 1994 Apr 1 Abs to the 52-kDa SS-A/Ro protein are found in high prevalence in patients with Sjogren's syndrome (SS) and mothers whose children have the neonatal lupus syndrome (NLS). This study further defines the specificity of this response. By ELISA, 97% of 59 mothers of offspring with NLS had Abs to the 52-kDa recombinant protein compared with 80% in 132 non-NLS sera with anti-SS-A/Ro Abs (p < 0.004). Antigenic regions on the 52-kDa protein were evaluated by immunoprecipitation of [35S]-radiolabeled in vitro translation products. Ninety-five percent of 99 sera that contained anti-52-kDa Abs by ELISA reacted with a large fragment spanning amino acids (aa) 1-291. Two antigenic regions were identified, aa169-291 containing the leucine zipper that was recognized by 83% of the anti-52-kDa sera tested and aa1-78 containing the zinc finger domains that was recognized by only half the sera. No sera immunoprecipitated this N-terminal fragment exclusively. Recognition of one or both regions was not unique to any clinical subset of patients; however, a greater number of sera from patients with SS contained both specificities, whereas asymptomatic mothers whose children had NLS comprised the only clinical group in which the majority recognized the central region of the molecule. Reactivity with both epitopes was demonstrated significantly more often in sera with high titers of Abs to the 60-kDa rSS-A/Ro protein by ELISA in association with the anti-52-kDa response compared with anti-52-kDa responses associated with low titers of anti-60-kDa Abs (p < 0.04). Eighty-one percent of 16 sera that recognized the N-terminal epitope were from patients with the combination of HLA-DRB1*0301, DQA1*0501, and DQB1*0201 alleles, compared with 30% of 10 that recognized only the central epitope (p < 0.02). In summary, this study demonstrates that there are at least two antigenic determinants on the 52-kDa SS-A/Ro protein, one "immunodominant" and the other recognized by a more "restricted" subset of anti-52-kDa SS-A/Ro Abs.
1391725 Absence of H(+)-ATPase in cortical collecting tubules of a patient with Sjogren's syndrome 1992 Aug Distal urinary acidification abnormalities may arise from transepithelial voltage defects, permeability defects, or proton-secretory defects, but tests to determine the cellular mechanisms underlying secretory abnormalities have not previously been reported. A patient with Sjogren's syndrome and distal renal tubular acidosis due to a secretory defect is described, whose kidney biopsy was examined by fluorescent immunocytochemistry with an antibody to the M(r) 31,000 subunit of the mammalian kidney vacuolar H(+)-ATPase and was compared with normal human kidney. Staining with the anti-H(+)-ATPase antibody in normal human kidney was detected in the brush border microvilli and subvillar invaginations of the proximal tubule and in intercalated cells in the collecting duct. A biopsy sample from the patient was devoid of any anti-H+-ATPase staining in the intercalated cells. Staining was also absent from the proximal tubule brush border microvilli but was present in the subvillar invaginations. Although autoantibodies to normal human kidney membrane proteins were detected in the serum by immunoblot analysis, no immunocytochemical evidence for anti-intercalated cell autoantibodies was observed in the patient's serum. This report demonstrates that the basis for the proton secretory defect in some patients with distal renal tubular acidosis is likely the absence of H(+)-ATPase in the intercalated cells. It also illustrates the potential diagnostic utility of anti-H(+)-ATPase antibodies in the classification of distal renal tubular acidoses.
9025991 Immunohistochemical findings of arterial fibrinoid necrosis in major and lingual minor sal 1996 Dec Arterial fibrinoid lesions in major salivary glands and lingual minor salivary glands from four autopsied patients with primary Sjögren's syndrome were studied histologically and immunohistochemically. On a morphological basis, the preceding arterial fibrinoid necrosis was regarded as medial damage, particularly of smooth muscle cells. The medial smooth muscle cells underwent vacuolated degeneration and disappeared, and resulted in full-blown fibrinoid arteritis. By means of the immunoperoxidase method the distribution of the immunoglobulins, fibrin, complement (C3), transferrin, ferritin, vimentin and lysozyme was studied. The normal arterial wall reacted with the lambda light chain of immunoglobulin, transferrin and vimentin Vacuolated degeneration of medial smooth muscle cells, regarded as the initial change in cases of vascular fibrinoid lesion, was positive for IgG, C3 and vimentin. We suggest that IgG antibody is a useful marker to detect the initial phase of arterial fibrinoid necrosis. In the foci of fibrinoid necrosis, fibrin, C3 and vimentin were detected. Among these three antibodies, only fibrin was negative in the normal arterial wall and vacuolated degenerates of medial smooth muscle cells. Mononuclear cells surrounding areas of fibrinoid necrosis stained strongly with antisera to immunoglobulins, transferrin, ferritin and vimentin, and negatively with fibrin, C3 and lysozyme antibodies.
7828370 V gamma 9-V delta 2+ gamma delta T cells from a patient with Felty syndrome that exhibit a 1995 Feb Expanded populations of T lymphocytes bearing gamma delta T cell receptors have been detected in several patients with Felty syndrome. The goal of this study was to functionally characterize these lymphocytes in a newly described patient with this disease. For this, fluorescence-activated cell sorter analysis of T cell surface antigens, proliferation, and tumor necrosis factor alpha enzyme-linked immunosorbent assays, as well as quantitative assays of immunoglobulins secreted by pokeweed mitogen-driven B cells were performed. The gamma delta cells, that expressed a CD3+CD4-V gamma 9-V delta 2+C delta + phenotype, and constituted 60% of the peripheral blood T cells, did not proliferate after triggering with anti-CD3, but did secrete tumor necrosis factor alpha and the addition of these cells to pokeweed mitogen-stimulated B cells from the patient decreased their secretion of immunoglobulin M while augmenting IgG secretion. These data suggest that the expanded anergic V gamma 9-V delta 2+ gamma delta cells can play an immunoregulatory role in the patient.
7588294 Cytokine-stimulated expression of inducible nitric oxide synthase by mouse, rat, and human 1995 Dec Recent evidence suggests that the production of nitric oxide (NO) may have important roles in the regulation of osteoblast and osteoclast metabolism. The present study was performed to investigate the effects of interleukin-1 beta (IL-1 beta), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) on the expression of inducible NO-synthase (iNOS) and to measure high-output production of NO by primary rat osteoblasts and osteoblastic cell lines ROS 17/2.8, MC3T3-E1 and MG-63. In addition, we have investigated if NO may mediate some of the effects of these cytokines on osteoblast metabolism. Northern blots and immunocytochemistry revealed time-dependent iNOS messenger RNA and protein expression in primary rat osteoblasts in response to cytokine treatment. Reverse transcription polymerase chain reaction amplified an 807-base pair (bp) product from ROS 17/2.8 cells, which had a size and restriction enzyme-cut pattern identical to that predicted for authentic rat iNOS. Nitrite accumulation in culture medium was induced by IFN-gamma in a time- and dose-dependent manner and inhibited by cotreatment with inhibitors of NOS activity and by dexamethasone. IL-1 beta, TNF-alpha, and bacterial lipopolysaccharide were found to have weak stimulatory effects on nitrite production on their own. However, IL-1 beta and TNF-alpha showed strong synergy with IFN-gamma, but, surprisingly, lipopolysaccharide was found to exert potent inhibitory effects on IFN-gamma-induced nitrite synthesis. Basal production of nitrite and induction of its synthesis was similarly observed with primary rat osteoblasts as well as ROS 17/2.8, MC3T3-E1, and MG-63 cell lines. Cytokine-induced NO production significantly reduced osteoblast activity, as was evidenced by inhibition of DNA synthesis, cell proliferation, alkaline phosphatase activity, and osteocalcin production. The results provide evidence for a basal expression of iNOS activity and show that the iNOS messenger RNA, protein, and enzyme activity are all induced by cytokines across the species. The data further suggest that osteoblast-derived NO may have an important role in mediation of localized bone destruction associated with inflammatory bone diseases such as rheumatoid arthritis.
7876550 Overexpression of vascular permeability factor (VPF/VEGF) and its endothelial cell recepto 1995 Mar 15 Delayed hypersensitivity (DH) is a T cell-mediated form of immune response characterized by a predominantly perivascular, mononuclear cell infiltrate. The venules in DH reactions are hyperpermeable to plasma proteins, leading to extravasation of plasma fibrinogen and its extravascular clotting to form a fibrin gel that promotes induration and angiogenesis. The mechanisms responsible for microvascular hyperpermeability in DH are unknown. Recently, a cytokine named vascular permeability factor (VPF, also known as vascular endothelial growth factor or VEGF) has been implicated in the chronic vascular hyperpermeability and angiogenesis of solid and ascites tumors, healing wounds, rheumatoid arthritis, and psoriasis. These findings suggested that VPF/VEGF might also have a role in the pathogenesis of DH. Two model systems were studied: allergic contact dermatitis to poison ivy in human volunteers and classical tuberculin hypersensitivity in rats. In both, in situ hybridization revealed that the mRNAs encoding VPF/VEGF were strikingly overexpressed in keratinocytes of the epidermis; scattered mononuclear cells infiltrating the dermis also overexpressed VPF/VEGF mRNA, to a greater extent in rat tuberculin than in human contact reactions. In contact reactions, mRNAs for two VPF/VEGF vascular endothelial cell receptors, flt-1 and KDR, were also strikingly overexpressed. Abundant fibrin deposition in both models confirmed that dermal microvessels were indeed hyperpermeable to plasma fibrinogen. These results implicate VPF/VEGF as a potentially important mediator in the pathogenesis of cell-mediated immunity and provide further evidence that products of epithelial cells may regulate the inflammatory response.
8247037 Lack of immune responsiveness to bovine serum albumin in insulin-dependent diabetes. 1993 Dec 16 BACKGROUND: Epidemiologic studies have implicated the ingestion of cow's milk in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). Moreover, in a recent study, 100 percent of patients with new-onset IDDM had antibodies against bovine serum albumin (BSA), with a majority directed against a 17-amino-acid BSA peptide (ABBOS). Cellular immune mechanisms are thought to be the principal mediators of pancreatic beta-cell destruction in IDDM. METHODS: We measured the responses of peripheral-blood mononuclear cells to BSA and ABBOS or serum IgG anti-BSA antibodies (by particle-concentration fluorescence immunoassay) in 71 patients with IDDM, 55 subjects at various degrees of risk for IDDM, 36 patients with other autoimmune disorders (chronic autoimmune thyroiditis, rheumatoid arthritis, and systemic lupus erythematosus), and 48 normal subjects. RESULTS: The responses of peripheral-blood mononuclear cells to BSA or ABBOS were positive in 2 of 24 patients with new-onset IDDM, 1 of 25 first-degree relatives of patients with IDDM who were negative for islet-cell antibodies, 2 of 30 first-degree relatives of patients with IDDM who were positive for islet-cell antibodies, 1 of 28 patients with established IDDM, and 1 of 29 normal subjects. Similarly, anti-BSA antibodies were not detected significantly more often in patients with new-onset IDDM (3 of 31, 10 percent) than in normal subjects (1 of 37, 3 percent; P = 0.32). However, many patients with autoimmune disease and subjects at increased risk for IDDM had anti-BSA antibodies (frequency, 10 to 31 percent). CONCLUSIONS: Anti-BSA antibodies may reflect a general defect in the process of immunologic tolerance associated with a predisposition to autoimmunity rather than immunity specific to beta cells. The absence of cellular immunity to BSA and ABBOS in IDDM does not support a role for this antigen in the pathogenesis of the disorder.
8885242 Redox-mediated activation of latent transforming growth factor-beta 1. 1996 Sep Transforming growth factor beta 1 (TGF beta) is a multifunctional cytokine that orchestrates response to injury via ubiquitous cell surface receptors. The biological activity of TGF beta is restrained by its secretion as a latent complex (LTGF beta) such that activation determines the extent of TGF beta activity during physiological and pathological events. TGF beta action has been implicated in a variety of reactive oxygen-mediated tissue processes, particularly inflammation, and in pathologies such as reperfusion injury, rheumatoid arthritis, and atherosclerosis. It was recently shown to be rapidly activated after in vivo radiation exposure, which also generates reactive oxygen species (ROS). In the present studies, the potential for redox-mediated LTGF beta activation was investigated using a cell-free system in which ROS were generated in solution by ionizing radiation or metal ion-catalyzed ascorbate reaction. Irradiation (100 Gray) of recombinant human LTGF beta in solution induced 26% activation compared with that elicited by standard thermal activation. Metal-catalyzed ascorbate oxidation elicited extremely efficient recombinant LTGF beta activation that matched or exceeded thermal activation. The efficiency of ascorbate activation depended on ascorbate concentrations and the presence of transition metal ions. We postulate that oxidation of specific amino acids in the latency-conferring peptide leads to a conformation change in the latent complex that allows release of TGF beta. Oxidative activation offers a novel route for the involvement of TGF beta in tissue processes in which ROS are implicated and endows LTGF beta with the ability to act as a sensor of oxidative stress and, by releasing TGF beta, to function as a signal for orchestrating the response of multiple cell types. LTGF beta redox sensitivity is presumably directed toward recovery of homeostasis; however, oxidation may also be a mechanism of LTGF beta activation that can be deleterious during disease mechanisms involving chronic ROS production.
8594243 Self-reported breast implants and connective-tissue diseases in female health professional 1996 Feb 28 OBJECTIVE: To evaluate the association of breast implants with connective-tissue diseases. DESIGN AND PARTICIPANTS: Retrospective cohort study of 395,543 female health professionals who completed mailed questionnaires for potential participation in the Women's Health Study. A total of 10,830 women reported breast implants and 11,805 reported connective-tissue diseases between 1962 and 1991. Cox proportional hazards regression models were used in analyses. MAIN OUTCOME MEASURE: Self-reported connective-tissue diseases. RESULTS: Compared with women who did not report breast implants, the relative risk (RR) of the combined end point of any connective-tissue disease among those who reported breast implants was 1.24 (95% confidence interval, 1.08 to 1.41, P = .0015). With respect to the individual diseases, the finding for other connective-tissue diseases (including mixed) was statistically significant (P = .017), the findings for rheumatoid arthritis, Sjogren's syndrome, dermatomyositis or polymyositis, or scleroderma were of borderline statistical significance (.05 < P < .10), and the finding for systemic lupus erythematosus was not statistically significant (P = .44). There were no clear trends in RR with increasing duration of breast implants. CONCLUSION: These self-reported data from female health professionals are compatible with prior reports from other cohort studies that exclude a large hazard, but do suggest small increased risks of connective-tissue diseases among women with breast implants. The very large sample size makes chance an unlikely explanation for the results, but bias due to differential overreporting of connective-tissue diseases or selective participation by affected women with breast implants remains a plausible alternative explanation. The major contribution of this and other observational analytic studies has been to exclude large risks of connective-tissue diseases following breast implants.
7887825 Review of immunomodulation by photopheresis: treatment of cutaneous T-cell lymphoma, autoi 1994 Dec Photopheresis is an apheresis-based therapy that is currently available at approximately 70 medical centers worldwide. Recent evidence indicates that extracorporeal photopheresis can significantly prolong life as well as induce a 60-75% response rate among individuals with advanced cutaneous T-cell lymphoma (CTCL). Moreover, a 10-15% cure rate, in response to photopheresis alone, or in combination with interferon-alpha, has been obtained at our institution. These complete responses have been characterized by the complete disappearance of morphologically atypical cells from the skin and blood. Southern blot analysis of peripheral blood specimens has also confirmed the indefinite disappearance of the malignant T-cell clone from the blood of patients with complete responses. Current immunological data obtained from in vitro human studies and from animal models suggest that the basis for the responses of CTCL patients are related to activation of treated macrophages resulting in release of cytokines, including substantial levels of tumor necrosis factor alpha (TNF-alpha), and perhaps, to the induction of anticlonotypic immunity directed against pathogenic clones of T lymphocytes. In addition to the treatment of CTCL, a potential role for photopheresis in the therapy of autoimmune disease has been suggested by recent pilot studies of pemphigus vulgaris, rheumatoid arthritis, and systemic lupus erythematosus. Furthermore, a randomized, single-blinded trial involving 79 patients with early onset, aggressive systemic sclerosis suggested that photopheresis could benefically affect the course of the cutaneous thickening in this form of the disease. Lastly, two independent pilot studies of cardiac transplantation have indicated that photopheresis can reverse acute cardiac allograft rejection and potentially suppress ongoing chronic rejection. Randomized, controlled trials for these new indications for photopheresis therapy are currently in the early stages of implementation.
7984435 Regulation of collagenase gene expression by IL-1 beta requires transcriptional and post-t 1994 Nov 11 Interleukin-1 beta is believed to contribute to the pathophysiology of rheumatoid arthritis by activating collagenase gene expression. We have used a cell culture model of rabbit synovial fibroblasts to examine the molecular mechanisms of IL-1 beta-mediated collagenase gene expression. Stimulation of rabbit synovial fibroblasts with 10 ng/ml recombinant human IL-1 beta resulted in a 20-fold increase in collagenase mRNA by 12 h. Transient transfection studies using collagenase promoter-CAT constructs demonstrated that proximal sequences responded poorly to IL-1 beta, possibly due to insufficient activation of AP-1 by this cytokine. More distal sequences were required for IL-1 beta responsiveness, with a 4700 bp construct showing approximately 5-fold induction above control. To examine post-transcriptional mechanisms, transcript from a human collagenase cDNA was constitutively produced by the simian virus 40 early promoter. IL-1 beta stabilized the constitutively expressed human transcript. Furthermore, mutation of the ATTTA motifs in the 3' untranslated region of the human gene also stabilized the transcript. Finally, the rabbit collagenase 3' untranslated region destabilized a constitutively transcribed chloramphenicol acetyltransferase transcript. These data indicate that in addition to activating transcription, IL-1 beta increases collagenase transcript stability by reversing the destabilizing effects of sequences in the 3' untranslated region.
9049977 Development of recombinant adenoviruses that drive high level expression of the human meta 1996 Dec Remodelling of the extracellular matrix resulting from increased secretion of metalloproteinase enzymes (MMPs) is implicated in many pathological conditions, including rheumatoid arthritis, restenosis following balloon angioplasty, atherosclerosis and cancer cell invasion and metastasis. Clear definition of the normal and pathological function of individual MMPs will benefit from approaches that use gene transfer to produce increases in MMP levels that mimic those observed in pathological conditions. Similarly, gene transfer methods leading to controlled increases in levels of the tissue inhibitor of metalloproteinases (TIMPs) will help to define the function of MMPs both in vitro and in vivo. Gene transfer of TIMPs may also have therapeutic potential in pathological conditions where inhibition of MMP activity may be beneficial. We have used the adenovirus serotype 5 vector system to generate replication-deficient recombinant adenoviruses capable of expressing the MMP-9, TIMP-1 or -2 genes. High level expression is driven by the cytomegalovirus major immediate early promoter (CMV IEP). Efficient and selective over-production of each recombinant protein was shown by immunofluorescence in either rabbit smooth muscle cells (SMC) or human MCF-7 adenocarcinoma cells. High level secretion directly dependent on the multiplicity of infection (MOI) was observed for each functional transgene by gelatin zymography. Using a quantitative ELISA assay, levels of recombinant TIMP-1 were detected when SMC were infected with as low as three plaque forming units (pfu) of virus per cell in vitro. A linear increase in TIMP-1 secretion was observed up to 1000 pfu/cell of virus (0.75 ng/10(4) cells/24 h at 3 pfu/cell to 1243 ng/10(4) cells/24h at 1000 pfu/cell). Similar levels of secretion of MMP-9 and TIMP-2 were observed by Western blot analysis using the same MOI of adenovirus. Thus, recombinant adenoviruses are an efficient and flexible system for high level expression of MMPs and TIMPs and will be useful tools in the study of matrix remodelling in vivo and in vitro.
8921246 Increased matrix metalloproteinases in the aqueous humor of patients and experimental anim 1996 Oct PURPOSE: Matrix metalloproteinases (MMPs) play a major role in connective tissue remodelling, wound healing and embryogenesis. They have also been implicated in pathological tissue degradation in diseases such as rheumatoid arthritis (RA), osteoarthritis (OA), and tumor invasion. The aim of this study was to define the potential role of MMPs in the inflammatory process of uveitis by identifying these proteases in the aqueous humor (AH) of patients with uveitis and in rabbits with endotoxin-induced uveitis (EIU). METHODS: Aqueous humor samples from 6 patients with uveitis and 5 control patients who had undergone elective cataract surgery were examined. The profile of MMPs in the AH of experimentally-induced acute anterior uveitis in rabbits was also assessed. Western blot analysis and SDS-PAGE substrate zymography were used to detect metalloenzymes and their natural inhibitor, tissue inhibitor of metalloproteinase (TIMP-1) in aqueous samples. RESULTS: Aqueous humor from all patients contained interstitial collagenase (MMP-1), stromelysin (MMP-3), gelatinase B (MMP-9) and TIMP-1. Although the amount of MMPs varied considerably, TIMP-1 levels remained unchanged in the aqueous of uveitis patients. Using substrate gel zymography, we were able to reveal several gelatinolytic bands, including one major band at approximately 92-kDa whose activity differed between uveitis and cataract AH. The gelatinase activity found in human AH samples was shown to be inhibited by 10 mM EDTA and activated in vitro by APMA, indicating that these enzymes were indeed of the metalloproteinase class. Aqueous humor samples from the rabbit EIU model revealed a 100-kDa molecular weight species likely to correspond to gelatinase B. This gelatinolytic activity was maximal at 6 hours after the lipopolysaccharide (LPS) injection, declined at 12 and 24 hours post LPS, and was absent at later time points. The induction of gelatinase activity in rabbit AH preceded the increase in cell number during the inflammatory process in the anterior chamber. CONCLUSIONS: Metalloproteinases found in normal human AH may participate in physiological turnover of extracellular matrix in the eye. Elevated levels of MMPs were found in the AH of patients with uveal inflammation and animals with LPS-induced uveitis, where they are likely to be critical to tissue destructive and repair processes. It is likely that pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 alpha (IL-1 alpha), which are known modulators of MMPs, induce their secretion in acute anterior uveitis.
7645853 CD8+ T-cells in psoriatic lesions preferentially use T-cell receptors V beta 3 and/or V be 1995 Jul 7 Restricted T-cell receptor V beta gene use in animal models of autoimmune disease has led to the development of strategies to treat autoimmune disease by targeting the T-cell receptors of the pathogenic T-cells. Restricted T-cell receptor gene use has been noted in human autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. We report here the finding of restricted T-cell receptor gene use in psoriasis vulgaris, as well. Our results show an elevated skin (over PBL) expression of V beta 3 and/or V beta 13.1 messages in the CD8+ T-cells in a majority of patients studied. CDR3 sequence analysis on these two V beta s from the skin demonstrated monoclonality or marked oligoclonality. A second biopsy performed 3.5 to 8 months later in four patients, at the same or different lesions, again revealed an elevated V beta 3 and/or V beta 13.1 expression and clonality. Moreover, in three of the four patients, the same TcR V beta CDR3 rearrangement was found in both biopsies, although there was no V beta CDR3 homology noted between patients. In two patients in which V beta 3 and/or V beta 13.1 was not elevated in the CD8+ T-cell population, an increase in V beta 17 gene use and clonality was found. The persistence of V beta 3- and/or V beta 13.1-bearing CD8+ T-cells in lesions that did not undergo resolution suggests their role as effector cells rather than as regulatory cells. The effector function of these CD8+ T-cells is further supported by the clonality of TcR V beta sequence data, which indicates they are recruited and expanded in situ. The V beta s identified in this study are candidate targets for selective immunotherapeutic intervention in psoriasis.
7522625 CD3+, CD56+ aggressive variant of large granular lymphocyte leukemia. 1994 Oct 1 Clonal expansions of CD3+ large granular lymphocytes (LGL) have been classified as T-LGL leukemia. The majority of patients with T-LGL leukemia have a chronic disease (years) manifested often by severe neutropenia, rheumatoid arthritis, and mild-to-moderate splenomegaly. The characteristic phenotype of the leukemic LGL is CD3+, CD8+, CD16+, CD57+, and CD56-. In this report we describe an aggressive variant of T-LGL leukemia in which leukemic LGL also expressed CD56, as identified by two-color flow-cytometry analysis. In contrast to the chronic nature typical of T-LGL leukemia, these patients presented with a severe systemic illness that was rapidly progressive and resistant to treatment. Atypical clinical features included rapidly increasing spleen size to massive proportions, extensive lymphadenopathy, and the presence of B symptoms (fever, nightsweats, weight loss). Hematologic and pathologic features were also unusual for T-LGL leukemia. These patients had very high LGL counts at diagnosis (range 11,692 to 26,312 microL), which increased rapidly despite treatment. Histopathologic examination of splenic sections showed extensive infiltration of red pulp cords and sinuses by leukemic cells with atrophy of the white pulp. These clinicopathologic features are similar to those described for patients with natural killer cell (NK)-LGL leukemia, whose cells are also CD56+. However, unlike NK-LGL leukemia, we could not show a direct pathogenic role for Epstein-Barr virus (EBV), as Southern-blot analyses using an EBV-joined termini probe were negative in these patients. Our findings suggest that CD3+, CD56+ LGL leukemia is a distinct clinicopathologic entity separate from the usual CD3+, CD56- T-LGL leukemia. The expression on leukemic LGL of CD56, an adhesion molecule, may determine the aggressive biologic nature of this newly described disease.