Immunohistochemical classification of primary and recurrent macular corneal dystrophy in Germany: subclassification of immunophenotype I A using a novel keratan sulfate antibody.

Macular corneal dystrophy (MCD) is an autosomal recessive disease characterized by abnormal deposition of glycosaminoglycans in corneal stroma, keratocytes, Descemet's membrane and corneal endothelium. According to the presence and distribution of sulfated keratan sulfate (KS)-epitopes in serum and cornea (using mAb 5-D-4), MCD can be classified into three immunophenotypes: type I, I A and II. The purpose of this study is to evaluate the immunophenotype of primary and recurrent MCD and to analyze the reactions of a novel KS-antibody in MCD corneas, which recognizes an epitope localized in the binding region of KS-chains to the core protein (mAb 3D12/H7). Indirect immunohistochemistry for KS (mAbs 3D12/H7 and 5-D-4) was performed on 44 corneas of 37 patients with MCD including two recurrences. Immunogold labeling was used to localize KS ultrastructurally within keratocytes. The serum concentration of KS (cKS) was determined in a serum antigen-inhibition assay. Immunohistochemically, no reaction was observed using mAb 5-D-4 in 18 corneas of 16 patients (43% of 37 patients; immunophenotype I). Positive reactions within single keratocytes but not in the stroma, were seen in 22 corneas of 17 patients (46% of 37 patients; immunophenotype I A) and positive reactions in keratocytes and extracellular stroma were found in four corneas of four patients (11% of 37 patients: immunophenotype II). For analysis of cKS a total of seven samples was available. Whereas in the samples of the five patients with immunophenotypes I and I A cKS was below the limit of detection, in the two sera from patients with immunophenotype II, cKS was normal (cKS = 1243 and 1380 nmol l(-1)). The two recurrences demonstrated immunophenotype II. Using mAb 3D12/H7, MCD immunophenotype I A can be further subclassified in type I A 1 (lacking reaction with mAb 3D12/H7 in keratocytes; 77%) and type I A 2 (positive reaction with mAb 3D12/H7 within keratocytes; 23%). MCD immunophenotype I A can not only be found in Saudi Arabia, but is as common as immunophenotype I in German patients. The only recurrences of MCD necessitating regrafting occurred in two patients with immunophenotype II possibly suggesting a higher risk for recurrence in this immunophenotype. The mAb 3D12/H7 allows a further subclassification of immunophenotype I A into type I A1 and 2. This points to a broader spectrum of MCD immunophenotypes and indirectly to a broader corneal proteoglycan pathology in MCD.

A large keratan sulfate proteoglycan present in human cervical mucous appears to be involved in the reorganization of the cervical extracellular matrix at term.

OBJECTIVE: To elucidate the function of keratan sulfate proteoglycan (KS-PG) in the human uterine cervix, we analyzed its distribution with respect to physiologic conditions. METHODS: Immunohistochemistry was used to localize KS bearing proteoglycans (mAb 5D4) and decorin (mAb 6B6) in the lower uterine segment. Proteins present in cervical mucous were labeled with biotin, glycosaminoglycan chains were digested enzymatically, and the samples were analyzed by Western blot. RESULTS: Decorin was detected throughout the extracellular matrix, in tissues from menstruating nonpregnant women, in early pregnancy, from women who had cesarean at term, at postpartum hysterectomy, and from postmenopausal women. In menstruating nonpregnant women, in early pregnancy (first trimester), and in postmenopausal women, KS-PG was detectable only in epithelial, mucous-producing cells. Interestingly, in samples obtained either at the time of cesarean at term (lower uterine segment) or after postpartal hysterectomy, KS-PG was detectable throughout the extracellular matrix, indicating that the expression of KS-PG is associated with reorganization of the tissue. Biochemical analysis of the KS present in mucous revealed a core protein in the range of 220 kDa, suggesting an identity with the large KS-PG described previously. CONCLUSION: At parturition, a large KS-PG, which is virtually exclusively present in the cervical mucous of either early or nonpregnant women, was detected in the extracellular matrix. This finding indicates that cervical ripening is accompanied not only by quantitative but also by qualitative changes in the composition of the extracellular matrix.

Expression of glypican-4 in haematopoietic-progenitor and bone-marrow-stromal cells.

Heparan sulphate proteoglycans and the extracellular matrix of bone-marrow-stromal cells are important components of the microenvironment of haematopoietic tissues and are involved in the interaction of haematopoietic stem and stromal cells. Previous studies have emphasized the role of heparan sulphate proteoglycan synthesis by bone-marrow-stromal cells. In the present study we describe the expression of glypican-4 (GPC-4), belonging to the glypican family, in bone-marrow-stromal cells and haematopoietic-progenitor cells of human and murine origin. Expression of GPC-4 was shown on the mRNA-level by reverse transcription-PCR and Northern blot analysis. Amplification products were cloned and sequenced, to confirm these results. To analyze the expression of GPC-4 on the protein level, polyclonal antibodies against selected peptides were raised in rabbits. Western blot analysis showed expression of GPC-4 as a heparan sulphate proteoglycan in the human haematopoietic-progenitor cell line TF-1 and normal human bone marrow. These results were confirmed by FACS analysis of TF-1 cells. Furthermore, GPC-4-positive progenitor cells and stromal cells were enriched from normal human bone marrow by magnetic-cell sorting and analysed by confocal laser-scanning microscopy.

Induction of alpha1-antitrypsin synthesis in human articular chondrocytes by interleukin-6-type cytokines: evidence for a local acute-phase response in the joint.

OBJECTIVE: We have previously shown that human articular chondrocytes synthesize large amounts of interleukin-6 (IL-6) upon stimulation with proinflammatory cytokines and that they express the IL-6 receptor. The present study was undertaken to analyze whether different IL-6-type cytokines can induce synthesis of the acute-phase protein alpha1-antitrypsin in human articular chondrocytes. METHODS: Chondrocytes from human articular cartilage, cultured in agarose, were stimulated with IL-6-type cytokines. Total RNA was isolated and analyzed by Northern blotting. Levels of alpha1-antitrypsin protein were determined by enzyme immunoassay. RESULTS: Stimulation of chondrocytes with oncostatin M (OSM) and IL-6 led to a 5-10-fold increase in alpha1-antitrypsin synthesis. This increase was dose and time dependent. Furthermore, OSM and IL-6 induced IL-6 synthesis in chondrocytes, resulting in an autocrine amplification loop. CONCLUSION: Our data strongly suggest the existence of a local acute-phase response in the joint. Synthesis of the acute-phase protein alpha1-antitrypsin, a major inhibitor of serine proteinases, may be an important protective mechanism of articular chondrocytes to prevent cartilage damage in inflammatory joint diseases.

Heparan sulfate proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells.

Heparan sulfate (HS) proteoglycans of bone marrow (BM) stromal cells and their extracellular matrix are important components of the microenvironment of hematopoietic tissues and are involved in the interaction of hematopoietic stem and stromal cells. Although previous studies have emphasized the role of HS proteoglycan synthesis by BM stromal cells, we have recently shown that the human hematopoietic progenitor cell line TF-1 also expressed an HS proteoglycan. Immunochemical, reverse transcriptase-polymerase chain reaction (RT-PCR), and Northern blot analysis of this HS proteoglycan showed that it was not related to the syndecan family of HS proteoglycans or to glypican. To answer the question of whether the expression of HS proteoglycans is associated with the differentiation state of hematopoietic progenitor cells, we have analyzed the proteoglycan synthesis of several murine and human hematopoietic progenitor cell lines. Proteoglycans were isolated from metabolically labeled cells and purified by several chromatographic steps. Isolation and characterization of proteoglycans from the cell lines HEL and ELM-D, which like TF-1 cells have an immature erythroid phenotype, showed that these cells synthesize the same HS proteoglycan, previously detected in TF-1 cells, as a major proteoglycan. In contrast, cell lines of the myeloid lineage, like the myeloblastic/promyelocytic cell lines B1 and B2, do not express HS proteoglycans. Taken together, our data strongly suggest that expression of this HS proteoglycan in hematopoietic progenitor cell lines is associated with the erythroid lineage. To prove this association we have analyzed the proteoglycan expression in the nonleukemic multipotent stem cell line FDCP-Mix-A4 after induction of erythroid or granulocytic differentiation. Our data show that HS proteoglycan expression is induced during early erythroid differentiation of multipotent hematopoietic stem cells. In contrast, during granulocytic differentiation, no expression of HS proteoglycans was observed.

Interleukin-1beta and interleukin-8 concentrations in the lower uterine segment during parturition at term.

OBJECTIVE: To assess the roles of interleukin-1beta, interleukin-8, and fibroblasts in the lower uterine segment during parturition. METHODS: Lower uterine segment biopsy specimens were obtained from 36 women undergoing cesarean delivery at various stages of cervical dilation (less than 2 cm, n = 8; 2 to less than 4 cm, n = 9; 4-6 cm, n = 10; more than 6 cm, n = 9). The concentrations of interleukin-1beta and interleukin-8 in protein extracts prepared from the tissue samples were measured by enzyme immunoassays. The effect of incubation with interleukin-1beta (30 U/mL) on interleukin-8 secretion by lower uterine segment fibroblasts in vitro also was determined. RESULTS: The median interleukin-1beta concentration in the specimens increased from 1.3 pg/mg of total protein at less than 2 cm of dilation to 22.2 pg/mg of total protein at 4-6 cm of dilation (P < .05). No further increase was detectable after 6 cm of dilation. The interleukin-8 concentration increased from 17.2 pg/mg of total protein at less than 2 cm of dilation to 2080.7 pg/mg of total protein at 4-6 cm of dilation (P < .05), thus paralleling the increase in interleukin-1beta concentration. Interleukin-1beta induced a significant increase in interleukin-8 secretion by fibroblasts in vitro, from 0.8 ng/10(6) cells to 35.6 ng/10(6) cells. CONCLUSION: The increase in interleukin-8 concentration in the lower uterine segment during parturition may be induced by interleukin-1beta and fibroblasts may be one of the sources of this interleukin-8.

Proteoglycan synthesis in haematopoietic cells: isolation and characterization of heparan sulphate proteoglycans expressed by the bone-marrow stromal cell line MS-5.

Proteoglycans of bone-marrow stromal cells and their extracellular matrix are important components of the haematopoietic microenvironment. Recently, several studies have indicated that they are involved in the interaction of haematopoietic stem and stromal cells. However, a detailed characterization of the heparan sulphate proteoglycans synthesized by bone-marrow stromal cells is still lacking. Here we report on the isolation and characterization of proteoglycans from the haematopoietic stromal cell line MS-5, that efficiently supports the growth and differentiation of human and murine haematopoietic progenitor cells. Biochemical characterization of purified proteoglycans revealed that the haematopoietic stromal cell line MS-5 synthesizes, in addition to chondroitin sulphate proteoglycans, several different heparan sulphate proteoglycans. Immunochemical analysis, using specific antibodies against the different members of the syndecan family, glypican, betaglycan and perlecan, showed that MS-5 cells synthesize all these different heparan sulphate proteoglycans. These data were further supported by reverse-transcriptase PCR and confirmed by sequence and Northern blot analysis. The relative abundance of the different heparan sulphate proteoglycans was estimated on the protein and mRNA levels.

Development of an enzyme immunoassay specific for a core protein epitope of a novel small basement membrane associated heparan sulphate proteoglycan from human kidney.

Heparan sulphate proteoglycans are major components of the glomerular basement membrane and play a key role in their molecular organization and function. Moreover, their presence is essential for the maintenance of the selective permeability of the glomerular basement membrane. Recently, we have isolated and characterized a novel, small basement membrane associated heparan sulphate proteoglycan from human aorta and kidney. Using specific monoclonal antibodies we have shown that the novel heparan sulphate proteoglycan is predominantly located in the glomerular basement membrane, to a lesser extent in the basement membrane of tubuli, and also in the mesangium. Turnover or, in the course of kidney diseases, degradation of heparan sulphate proteoglycan from glomerular basement membranes may lead to urinary excretion of heparan sulphate proteoglycan. Therefore, changes in the structure and function of glomerular basement membranes may be directly detected by measuring the excretion of a component of this basement menbrane, e. g. heparan sulphate proteoglycan into urine. Here we describe the establishment of an enzyme immunoassay for the sensitive detection of the novel, small heparan sulphate proteoglycan in urine. In this assay the specific monoclonal antibody 1F10/B8, which recognizes a core protein epitope, was used to detect the polyanionic heparan sulphate proteoglycan bound to the surface of a cationic charge modified microtitre plate. This assay allows the sensitive and specific detection of the small heparan sulphate proteoglycan, which is released from the glomerular basement membrane into urine during normal turnover and also in the course of kidney diseases.

Association between heparan sulfate proteoglycan excretion and proteinuria after renal transplantation.

The aim of the study presented here was to investigate whether, in patients showing immediate graft function after renal transplantation, cold-ischemia and reperfusion lead to damage of the glomerular basement membrane and consequently to a loss of heparan sulfate proteoglycans. Loss of these heparan sulfate proteoglycans is a major cause of proteinuria. Time-dependent changes in urinary excretion rates of heparan sulfate proteoglycans but also of total protein, albumin, low- and high-molecular-weight proteins were analyzed quantitatively and by polyacrylamid-gel-electrophoresis in eight patients. Immediately after renal transplantation, severe proteinuria with an excretion rate of up to 251 +/- 108 mg/min was apparent and rapidly declined within 24 h to 4.11 +/- 2.80 mg/min. The gel-electrophoretic pattern showed a nonselective glomerular and tubular proteinuria. The excretion rate of heparan sulfate proteoglycan was increased in this initial reperfusion phase (up to 7 h), most probably because of ischemia- and reperfusion-induced damage of the glomerular basement membrane. The initial nonselective glomerular proteinuria disappeared within 48 h, changing to a mild selective glomerular proteinuria. In this second phase (7 to 48 h), lower levels of heparan sulfate proteoglycan excretion were observed (0.54 +/- 0.54 microgram/min versus 1.66 +/- 1.93 micrograms/min, P < 0.05). However, during the repair process of the glomerular basement membrane, heparan sulfate proteoglycan is synthesized de novo, leading to an increasing heparan sulfate proteoglycan content of the glomerular basement membrane. This second phase is paralleled by the change from a nonselective to a selective glomerular proteinuria. In the third phase, when the heparan sulfate proteoglycan content of the glomerular basement membrane normalizes, glomerular proteinuria was abolished in most of the patients.

Evidence for the presence of a large keratan sulphate proteoglycan in the human uterine cervix.

Profound changes occur in the uterine cervix during pregnancy. In particular, the extracellular matrix of the connective tissue is remodelled extensively. To elucidate the mechanisms involved in this process, we have analysed the proteoglycan pattern in the human cervix from pregnant and non-pregnant women. Proteoglycans of the cervix tissue specimen were extracted with 4 M guanidine hydrochloride and precipitated with 80% ethanol. Purification of proteoglycans was performed by several chromatographic steps. Characterization of proteoglycans was done by SDS/PAGE before and after digestion with glycosaminoglycan-specific enzymes. Proteoglycans were detected by combined Alcian Blue/silver staining or, after blotting of biotin-labelled proteoglycans on to poly(vinylidene difluoride) membrane, with peroxidase-conjugated avidin or by the use of keratan sulphate- or decorin-specific monoclonal antibodies. In contrast with previous reports, where only chondroitin/dermatan sulphate proteoglycans have been found in the uterine cervix, we have shown in the present study the existence of a large keratan sulphate proteoglycan with an M(r) > 220,000 in cervix samples from non-pregnant and pregnant women. This proteoglycan showed a strong reaction with the keratan sulphate-specific monoclonal antibody 5D4 and could be degraded by keratanases. The size of the core protein of this keratan sulphate proteoglycan was estimated to be about M(r) 220,000.

Oncostatin M differentially regulates tissue inhibitors of metalloproteinases TIMP-1 and TIMP-3 gene expression in human synovial lining cells.

Tissue inhibitor of metalloproteinases (TIMP) 1, 2 and 3 are related proteins that can form complexes with all known matrix metalloproteinases (MMPs). They inhibit the action of MMPs on extracellular matrix components. The balance of MMPs and TIMPs is important for tissue remodeling and its disturbance is believed to play a crucial role in pathophysiological processes such as tumor metastasis, destruction of cartilage and fibrosis. Cytokines and growth factors were found to regulate TIMPs and MMPs in a complex manner. In order to better understand the role of TIMPs in inflammatory joint diseases we have studied in vitro the regulation of TIMP-1 and TIMP-3 by inflammatory cytokines in cultured human synovial lining cells. We found that transforming growth factor beta 1 as well as interleukin-1 beta induce gene expression of both TIMP-1 and TIMP-3. In contrast, oncostatin M, an interleukin-6-type cytokine produced by activated T-lymphocytes and monocytes, had a differential effect on TIMP mRNA levels. After oncostatin M treatment, TIMP-1 expression was up-regulated but basal, as well as interleukin-1 beta-induced, TIMP-3 expression was inhibited. Interleukin-6 itself had no effect on synovial lining cells but a complex of interleukin-6 and the soluble interleukin-6 receptor induced activation of signal transducer and activator of transcription (STAT) factors in these cells and regulated TIMP-1 and TIMP-3 expression in a similar fashion as oncostatin M. Since TIMP-3 is matrix-associated whereas TIMP-1 is found in many body fluids, the role of oncostatin M during inflammatory processes might be to promote ECM degradation in the local environment but to prevent it systemically.

A novel keratan sulphate domain preferentially expressed on the large aggregating proteoglycan from human articular cartilage is recognized by the monoclonal antibody 3D12/H7.

Monoclonal antibodies (mAbs) were prepared against aggrecan which has been isolated from human articular cartilage and purified by several chromatographic steps. One of these mAbs, the aggrecan-specific mAb 3D12/H7, was selected for further characterization. The data presented indicate that this mAb recognizes a novel domain of keratan sulphate chains from aggrecan: (1) immunochemical staining of aggrecan is abolished by treatment with keratanase/keratanase II, but not with keratanase or chondroitin sulphate lyase AC/ABC; (2) after chemical deglycosylation of aggrecan no staining of the core-protein was observed; (3) different immunochemical reactivity was observed against keratan sulphates from articular cartilage, intervertebral disc and cornea for the mAbs 3D12/H7 and 5D4. For further characterization of the epitope, reduced and 3H-labelled keratan sulphate chains were prepared. In an IEF-gel-shift assay it was shown that the 3H-labelled oligosaccharides obtained after keratanase digestion of reduced and 3H-labelled keratan sulphate chains were recognized by the mAb 3D12/H7. Thus it can be concluded that the mAb 3D12/H7 recognizes an epitope in the linkage region present in, at least some, keratan sulphate chains of the large aggregating proteoglycan from human articular cartilage. Moreover, this domain seems to be expressed preferentially on those keratan sulphate chains which occur in the chondroitin sulphate-rich region of aggrecan, since the antibody does not recognize the keratan sulphate-rich region obtained after combined chondroitinase AC/ABC and trypsin digestion of aggrecan.

Proteoglycan synthesis in human and murine haematopoietic progenitor cell lines: isolation and characterization of a heparan sulphate proteoglycan as a major proteoglycan from the human haematopoietic cell line TF-1.

Proteoglycans of bone-marrow stromal cells and their extracellular matrix are important components of the microenvironment of haematopoietic tissues. Proteoglycans might also be involved in the interaction of haematopoietic stem and stromal cells. Recently, several studies have been reported on the proteoglycan synthesis of stromal cells, but little is known about the proteoglycan synthesis of haematopoietic stem or progenitor cells. Here we report on the isolation and characterization of proteoglycans from two haematopoietic progenitor cell lines, the murine FDCP-Mix A4 and the human TF-1 cell line. Proteoglycans were isolated from metabolically labelled cells and purified by several chromatographic steps, including anion-exchange and size-exclusion chromatography. Biochemical characterization was performed by electrophoresis or gel-filtration chromatography before and after digestion with glycosaminoglycan-specific enzymes or HNO2 treatment. Whereas FDCP-Mix A4 cells synthesize a homogeneous chondroitin 4-sulphate proteoglycan, isolation and characterization of proteoglycans from the human cell line TF-1 revealed, that TF-1 cells synthesize, in addition to a chondroitin sulphate proteoglycan, a heparan sulphate proteoglycan as major proteoglycan. For this heparan sulphate proteoglycan a core protein size of approx. 59 kDa was determined. Immunochemical analysis of this heparan sulphate proteoglycan revealed that it is not related to the syndecan family nor to glypican.

Limited heterogeneity of rearranged T cell receptor V alpha and V beta transcripts in synovial fluid T cells in early stages of rheumatoid arthritis.

OBJECTIVE: The identification of activated T cells in synovial fluid and synovium, and the association of rheumatoid arthritis (RA) with specific HLA-DR restriction elements, strongly suggest that these T cells play a critical role in the etiology and pathogenesis of RA. Analysis of the T cell receptor (TCR) repertoire in the early stages of RA might be an approach to identify those T cells involved in the initiation and/or perpetuation of the disease. METHODS: TCR V alpha and V beta transcripts of synovial T cells, sampled at the early stages of RA, were amplified by reverse transcriptase-polymerase chain reaction. HLA-DR subtyping was determined by serologic analysis and dot-blot hybridization of polymerase chain reaction amplification products using digoxigenin-labeled, sequence-specific oligonucleotide probes. RESULTS: Our findings showed a limited heterogeneity of V alpha and V beta TCRs in synovial fluid T cells, and a preferential usage of TCR V alpha 17 in early RA. In contrast, in the later stages of RA, a more polyclonal TCR V alpha and V beta gene usage was observed. CONCLUSION: Our results support the view that induction of RA is driven by an oligoclonal immune response to an unknown antigen. These findings also suggest a pathogenetic role for V alpha 17 T cells in the early stages of RA.

CD66a (BGP), an adhesion molecule of the carcinoembryonic antigen family, is expressed in epithelium, endothelium, and myeloid cells in a wide range of normal human tissues.

CD66a, also called biliary glycoprotein (BGP), is a member of the carcinoembryonic antigen (CEA) family and of the immunoglobulin superfamily. CD66a is the human homologue of Cell-CAM, a well-defined cell adhesion molecule of the rat. In the present study a monoclonal antibody specific for CD66a was used to locate CD66a in human tissues. CD66a is expressed in epithelia, in certain endothelia, and in cells of the myeloid lineage. Hepatocytes were stained along the bile canaliculi. A characteristic apical membranous staining was observed in enterocytes, superficial absorptive cells of the colon, in the epithelia of esophageal and Brunner's glands, bile ducts and gallbladder, pancreatic ducts, proximal tubules of the kidney, prostate, endometrium, and mammary ducts. Selective staining of endothelia was present in glomeruli and vasa recta of the kidney, small placental vessels, adrenal sinusoids, endometrium, the prostate. Among the cells of the myeloid lineage, granulocytes and myelocytes were positive. The expression of CD66a by human cells and tissues is well comparable with the expression reported for Cell-CAM, the rat counterpart of CD66a. The wide tissue distribution of CD66a indicates that CD66a is a prominent human adhesion molecule.

Transforming growth factor beta 1, a major stimulator of hyaluronan synthesis in human synovial lining cells.

OBJECTIVE: To investigate the role of cytokines and growth factors in the regulation of hyaluronan synthesis in human synovial lining cells. METHODS: Synovial lining cells were obtained from human knee joints, isolated by the explant method, and characterized by immunocytochemistry using monoclonal antibodies against monocyte/macrophage markers as well as antibodies against hyaluronan synthase. After stimulation by cytokines and growth factors, hyaluronan was measured by radiometric assay. The molecular weight distribution of the hyaluronan synthesized was determined by high-performance gel-permeation liquid chromatography. To test the effect of oxygen-derived free radicals, the concentration and molecular weight distribution of hyaluronan were determined in the presence and absence of catalase and superoxide dismutase. RESULTS: Hyaluronan synthesis was stimulated in synovial lining cells by transforming growth factor beta 1 (TGF beta 1), interleukin-1 beta (IL-1 beta), and to a lesser extent by tumor necrosis factor alpha (TNF alpha). Analysis of the molecular weight distribution of hyaluronan after stimulation of synovial lining cells with TGF beta 1, IL-1 beta, and TNF alpha indicated that hyaluronan is synthesized in a high molecular weight form and might be degraded in the course of inflammatory processes by oxygen-derived free radicals. CONCLUSION: Our findings suggest that TGF beta 1 is a major stimulator of hyaluronan synthesis in human synovial lining cells and might be involved in the pathogenic mechanisms of joint swelling in inflammatory and degenerative joint diseases.

Decreased glomerular basement membrane heparan sulfate proteoglycan in essential hypertension.

Heparan sulfate proteoglycans are major components of the glomerular basement membrane and play a key role in the molecular organization and function of the basement membrane. Moreover, their presence is essential for maintenance of the selective permeability of the glomerular basement membrane. Recently, we isolated and characterized a novel small basement membrane-associated heparan sulfate proteoglycan from human aorta and kidney. Partial amino acid sequence data clearly show that this heparan sulfate proteoglycan is distinct from the large basement membrane-associated heparan sulfate proteoglycan (perlecan). Using specific monoclonal antibodies, we have shown that the novel heparan sulfate proteoglycan is located predominantly in the glomerular basement membrane and, to a lesser extent, in the basement membrane of tubuli. Turnover or, in the course of kidney diseases, degradation of heparan sulfate proteoglycan from glomerular basement membranes may lead to urinary excretion of heparan sulfate proteoglycan, which can be measured by a sensitive enzyme immunoassay. The aim of the present study was to analyze whether changes in the structure and function of glomerular basement membranes can be directly detected by measurement of the excretion of a component of this basement membrane, eg, heparan sulfate proteoglycan into urine. The excretion of this small heparan sulfate proteoglycan was compared after physical exercise in normotensive and hypertensive subjects. Normotensive subjects and treated, essential hypertensive patients underwent a standardized workload on a bicycle ergometer. Biochemical characterization of the urinary proteins and heparan sulfate proteoglycan was performed before and 15 and 45 minutes after exercises.(ABSTRACT TRUNCATED AT 250 WORDS)

Development of enzyme immunoassays specific for keratan sulphate- and core-protein-epitopes of the large aggregating proteoglycan from human articular cartilage.

In the course of chronic inflammatory and degenerative joint diseases proteoglycans are degraded by the action of proteases and oxygen radicals. Therefore, proteoglycan fragments, released from cartilage into the peripheral blood, might be useful markers of cartilage degradation. Sensitive enzyme immunoassays are useful for the detection of these proteoglycan fragments in serum. We therefore developed specific monoclonal antibodies against the large aggregating proteoglycan (aggrecan), which has been isolated and purified from human articular cartilage. Two monoclonal antibodies which recognize a novel cartilage-specific epitope on the keratan sulphate chain of aggrecan (mAb 4B3/D10) and an epitope of the core-protein of aggrecan (4G4/A10) were selected for the development of competitive enzyme-immunoassays. These assays allow the sensitive and specific detection of cartilage-derived proteoglycan fragments, not only in synovial fluid but also in serum. They can now be used for the study of inflammatory and degenerative joint diseases.

Transforming growth factor beta 1 regulates tissue inhibitor of metalloproteinases-1 expression in differentiated human articular chondrocytes.

OBJECTIVE: To investigate the role of interleukin-6 (IL-6) and transforming growth factor beta 1 (TGF beta 1) in the regulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) synthesis in human articular chondrocytes. METHODS: Articular cartilage was obtained from human knee joints 24 hours after death. Chondrocytes were isolated by collagenase digestion and embedded in low-gelling-temperature agarose. After stimulation by cytokines, total RNA was isolated and analyzed by Northern blotting. TIMP-1 protein levels were determined using a competitive enzyme-linked immunosorbent assay. RESULTS: Human chondrocytes in agarose culture expressed messenger RNA (mRNA) for the IL-6 receptor (gp80) and its signal-transducing subunit gp130. In contrast to the findings in a previous study, IL-6 did not stimulate TIMP-1 expression in these cells, whereas TGF beta 1 was an important inducer of TIMP-1 mRNA and protein synthesis. CONCLUSION: Our findings suggest that TGF beta 1 has a protective effect on the extracellular matrix of human articular chondrocytes.

PMN elastase in bone and joint infections.

PMN (polymorphonuclear neutrophil) elastase is a proteolytic enzyme which is a biochemical marker for abnormal granulocyte stimulation. In inflammation and sepsis, excessive neutrophil stimulation results in significant amounts of PMN elastase being released into the plasma which indicates the severity of the disease and its prognosis. In 62 patients with osteomyelitis or suppurative arthritis, PMN elastase had a diagnostic sensitivity of 81%, which is comparable to the nonspecific erythrocyte sedimentation rate. Sensitivity of C-reactive protein (CRP) was 71%, fibrinogen 54% and leucocyte count 26%. PMN elastase was also useful in the follow up of patients with bone and joint infections; in the early post-operative period it became normal more quickly than the other findings unless the patients developed complications. Ten days after operation, PMN elastase was normal in 75% of the patients compared to the CRP which became normal in only 25%. Later both results were similar: on discharge from hospital, PMN elastase was normal in 77% and CRP in 71%.