HE4 suppresses the expression of osteopontin in mononuclear cells and compromises their cytotoxicity against ovarian cancer cells.

Ovarian cancers are known to evade immunosurveillance and to orchestrate a suppressive immune microenvironment. Here we examine the role of human epididymis protein 4 (HE4), an ovarian cancer biomarker, in immune evasion. Through modified subtractive hybridization analyses we have characterized the gene targets of HE4 in human peripheral blood mononuclear cells (PBMCs), and established a preliminary mechanism for HE4-mediated immune failure in ovarian tumours. Upon exposure of purified PMBCs to HE4, osteopontin (OPN) and dual-specificity phosphatase 6 (DUSP6) emerged as the most suppressed and up-regulated genes, respectively. SKOV3 and OVCAR8, human ovarian carcinoma cell lines, exhibited enhanced proliferation in conditioned media from HE4-exposed PBMCs, an effect that was attenuated by the addition of recombinant OPN or OPN-inducible cytokines [interleukin (IL)-12 and interferon (IFN)-Ɣ]. Additionally, upon co-culture with PBMCs, HE4-silenced SKOV3 cells were found to be more susceptible to cytotoxic cell death. The relationship between HE4 and OPN was reinforced further through the analysis of serous ovarian cancer patient samples. In these biopsy specimens, the number of OPN+ T cells correlated positively with progression free survival (PFS) and inversely with serum HE4 level. Taken together, these findings show that HE4 enhances ovarian cancer tumorigenesis by compromising OPN-mediated T cell activation.

Review: Collagen markers in early arthritic diseases.

In arthritic diseases e.g. osteoarthritis (OA) and rheumatoid arthritis (RA), the stability of the collagen type II (CII) fibers, a major component of articular cartilage, is compromised with extensive proteolytic breakdown leading to cartilage erosion and joint deterioration. A clinical need for molecular markers that give instantaneous measure of rate of joint deterioration has developed, as other measurements e.g. arthroscopy, and joint space narrowing are insensitive to small changes in disease status over short periods of time. Owing to its exclusive presence in cartilaginous tissues, markers of CII synthesis and degradation have been extensively studied. Assays that measure these markers in biological fluids e.g. synovial fluid (SF), serum, and urine have been developed and applied to detect early disease onset, monitor disease progression, and response to anti-arthritic drugs. CII synthesis markers include the procollagen type II C-propeptide (PIICP) and the procollagen type IIA N-propeptide (PIIANP). CII degradation markers include CII C-telopeptide (CII-X), CII neoepitope (TIINE), helix II, C2C, CNBr 9.7, Coll 2-1, and Coll 2-1 NO(2). Most of these markers differentiate between early stages of OA, RA and reference controls. The best correlations with structural changes occur when measurements are made in SF while serum measurement frequently did not correlate with structural changes. Although the selection of an optimal marker or a set of markers is still problematic, few markers are of considerable utility in early detection and monitoring of arthritic diseases. The current challenge is to improve the discriminatory power of these markers so they can be used to guide therapeutic decisions.

Association of articular cartilage degradation and loss of boundary-lubricating ability of synovial fluid following injury and inflammatory arthritis.

OBJECTIVE: To study the relationship between the boundary-lubricating ability of synovial fluid (SF) and articular cartilage damage in a rabbit knee injury model, to correlate collagen markers of such damage with SF boundary-lubricating ability and elastase activity, and to examine the lubricating ability of SF, together with collagen markers of articular cartilage damage, under the inflammatory conditions of knee joint synovitis (KJS) and rheumatoid arthritis (RA). METHODS: SF was aspirated weekly from the affected knee joints of 10 adult rabbits following transection of the anterior and posterior cruciate ligaments. The boundary-lubricating ability of SF was determined in vitro using a previously described friction apparatus. Lubricin concentrations and type II collagen (CII) peptides were quantified by sandwich enzyme-linked immunosorbent assays (ELISAs). Levels of the C-terminal neoepitope 9A4 (derived from collagenase degradation of CI, CII, and CIII) and of epitope 5-D-4 of keratan sulfate (a marker of proteoglycan depletion) were quantified by inhibition ELISAs. Elastase activity was measured spectrophotometrically. The sensitivity of purified human lubricin to digestion by neutrophil elastase (NE) was examined by Western blotting. RESULTS: The lubricating ability of SF from injured rabbit knees was significantly decreased at weeks 2 and 3 compared with week 1 after injury. Lubricin concentrations were significantly higher at week 1 than at weeks 2 and 3. CII peptide concentrations increased significantly at weeks 2 and 3 compared with week 1, while 9A4 neoepitope concentrations increased significantly at week 3 compared with weeks 1 and 2. There were no significant differences in epitope 5-D-4 concentrations among the 3 weeks. Elastase activity in SF increased significantly at weeks 2 and 3 compared with week 1. Elastase activity correlated significantly with diminishing lubrication at weeks 1, 2, and 3. SF from patients with KJS or RA exhibited deficient lubrication and elevated levels of CII peptides compared with SF from normal controls. NE was shown to completely degrade purified human lubricin in vitro. CONCLUSION: Loss of boundary-lubricating ability of SF after injury is associated with damage to the articular cartilage matrix. This can be attributed to inflammatory processes resulting from the injury, particularly in the early phases. This association also exists in patients with acute knee injuries or progressive chronic inflammatory arthritis.

Detection of collagen type II and proteoglycans in the synovial fluids of patients diagnosed with non-infectious knee joint synovitis indicates early damage to the articular cartilage matrix.

OBJECTIVE: We have sought to determine if markers of proteoglycans and collagen type II (CII) degradation can be detected at an early stage following acute knee injury in the synovial fluid (SF) from a group of patients diagnosed with non-infectious knee joint synovitis (KJS). CII, proteoglycans and elastase activity in the SF from patients with KJS were compared to SF from patients with two chronic arthritis conditions: osteoarthritis (OA) and rheumatoid arthritis (RA) as well as normal SF controls. METHODS: CII peptides were measured by sandwich ELISA using two monoclonal antibodies: 8:6:D8, a CII-specific antibody, and 14:7:D8 which binds to an amino acid sequence on CII as well as collagens type I, III and V. Epitope 9A4, a neo-epitope resulting from collagenase digestion of CI, CII, and CIII was measured by inhibition ELISA. Proteoglycans measurement included total sulfated glycosaminoglycans (sGAG) by dye-binding assay and 5-D-4 epitope, a keratan sulfate epitope, by inhibition ELISA. Elastase activity was measured colorimetircally using N-succinyl trialanine p-nitroanilide (SANA) substrate. RESULTS: The quantified CII peptide concentrations by sandwich and inhibition ELISA were significantly higher in SF from patients with KJS (P<0.05) compared to SF from patients with OA, RA and normal aspirates. 5-D-4 and sGAG concentrations were significantly lower (P<0.05) in SF from patients with KJS compared to SF from patients with OA and RA. Elastase activity in SF from patients with KJS and RA were significantly higher (P<0.05) than SF from patients with OA. A significant correlation exists between elastase activity and 9A4 epitope concentration in SF from patients with KJS. CONCLUSION: The elevated CII peptides concentrations in KJS SF compared to normal and OA aspirates indicate early signs of cartilage network damage. The low proteoglycans concentrations in SF from patients with KJS may indicate that injury is limited to the superficial zone of cartilage in the patient population studied. The high elastase activity in SF from patients with KJS and RA are linked to the high CII peptides concentration. The elastase activity in the SF from patients with KJS is due to the action of neutrophil elastase (NE) and collagenases, where both contribute to the destruction of the articular cartilage.

Articular collagen degradation in the Hulth-Telhag model of osteoarthritis.

OBJECTIVE: This study employed immunohistochemistry to investigate the pattern of type II collagen (CII) degradation in an acute injury model of osteoarthritis. It was hypothesized, based on previous studies of primary osteoarthritis (OA), that the worst areas of CII degradation would be located in the superficial and upper middle zones of the articular cartilage, with staining extending into the deep zone as the OA became more severe. DESIGN: In this model of secondary OA, rabbits were made osteoarthritic by transecting the anterior and posterior cruciate ligaments and removing the meniscus. At various times post surgery, articular cartilage was examined for CII degradation using monoclonal antibody 18:6:D6. This antibody reacts to an epitope that is exposed when CII is degraded as the result of protease cleavage. Proteoglycans (PG) were localized using Safranin-O/Fast Green. Staining intensities were quantitated using image analysis software. RESULTS: In the joints with surgically induced OA, degradation of CII was seen as early as 3 weeks with the majority of the degradation localized in zones I and III. At 14 weeks the destruction of CII was more pronounced, but there was a surprising lack of degradation in zone II. There were also several other unexpected findings. The sham-operated joints, for example, were intended to serve as controls yet CII degradation was observed in rabbits killed 3 weeks after surgery. It was also expected that greater CII degradation would occur in cartilage from medial condyles, but after 14 weeks there was no significant difference between medial and lateral condyles. Finally, the loss of staining for PG correlated with the degradation of CII except in zone III where limited PG loss was observed. CONCLUSION: Differences were observed between the pattern of articular cartilage destruction in this model of secondary OA and that of primary OA. Further investigation of the mechanical and biochemical processes underlying the development of both types of OA needs to be conducted.

Chondrogenic differentiation of cultured human mesenchymal stem cells from marrow.

In the adult human, mesenchymal stem cells (MSCs) resident in bone marrow retain the capacity to proliferate and differentiate along multiple connective tissue lineages, including cartilage. In this study, culture-expanded human MSCs (hMSCs) of 60 human donors were induced to express the morphology and gene products of chondrocytes. Chondrogenesis was induced by culturing hMSCs in micromass pellets in the presence of a defined medium that included 100 nM dexamethasone and 10 ng/ml transforming growth factor-beta(3) (TGF-beta(3)). Within 14 days, cells secreted an extracellular matrix incorporating type II collagen, aggrecan, and anionic proteoglycans. hMSCs could be further differentiated to the hypertrophic state by the addition of 50 nM thyroxine, the withdrawal of TGF-beta(3), and the reduction of dexamethasone concentration to 1 nM. Increased understanding of the induction of chondrogenic differentiation should lead to further progress in defining the mechanisms responsible for the generation of cartilaginous tissues, their maintenance, and their regeneration.

Immunological detection of type II collagen degradation: use in the evaluation of anti-arthritic therapies.

Propagated Swaram rat chondrosarcoma cells, rabbit chrondrocytes (from articular cartilage of knee, shoulder and hip joints), and bovine nasal cartilage explant cultures were studied. Type II collagen (CII) and its peptide fragments were quantitated in cell medium and cell layer separately, using two previously developed assays; one assay employed a monoclonal antibody, C4F6, that reacts specifically with triple helical CII and the other assay used an antibody, EIE5, that reacts specifically with a peptide of CII. A time-dependent increase in the content of CII and CII-derived peptides was observed in both rat and rabbit cultures. In both culture systems the majority of the native type II collagen is found associated with the cell layer (97% in rat cultures and 73% in rabbit cultures), while the major part of the CII peptides is found in the media (73% in rat cultures, 88% in the rabbit cultures). The concentration of peptides in the media reaches approximately 2 micrograms mL-1 in both chondrocyte monolayer cultures after 4 days. The CII peptide assay employing E1E5 was well suited to quantitate articular cartilage collagen degradation in explant culture. thus it can be used to evaluate potential therapeutic agents that can modify or inhibit cartilage degradation. The assay has the added potential that it could be used in-vivo to evaluate the effectiveness of potential metalloproteinase inhibitors in animal models of osteoarthritis or in clinical trials.

Effects of certain antiarthritic agents on the synthesis of type II collagen and glycosaminoglycans in rat chondrosarcoma cultures.

Cartilage destruction is a characteristic feature of osteoarthritis. Treatment with certain nonsteroidal anti-inflammatory drugs could exacerbate cartilage destruction by impairing the synthesis of cartilage matrix proteins, type II collagen and proteoglycan. In order to monitor the changes occurring in cartilage collagen synthesis, we developed a type II collagen specific ELISA. The effects of antiarthritic agents on type II collagen and glycosaminoglycan synthesis were examined in rat chondrosarcoma cultures. Drugs were added to the monolayer cultures and 4 days later the total type II collagen, as determined by the type II collagen ELISA, and glycosaminoglycan content, as measured by dimethyl-methylene blue dye binding assay, was measured. All drugs except tiaprofenic acid decreased type II collagen synthesis by at least 40% at 100 micrograms/ml. Tiaprofenic acid at 1 microgram/ml increased type II collagen content by 54% of the controls. Glycosaminoglycan synthesis was decreased by acetylsalicylic acid, diclofenac and tiaprofenac acid, at 50 micrograms/ml or above. Indomethacin, naproxen and dexamethasone had no effect. Interestingly, tenidap stimulated the glycosaminoglycan synthesis by 32% at 100 micrograms/ml. We show that the combination of chondrosarcoma cultures, type II collagen specific ELISA and dimethylmethylene blue dye binding assay serves as a useful model for screening the effects of agents capable of modulating type II collagen and glycosaminoglycan synthesis.

Production of type II collagen specific monoclonal antibodies.

Immunoassays are used for the specific measurement of type II collagen, a major cartilage protein, which is lost in osteoarthritic joints. Poor immunogenicity and species dependent immune response to type II collagen make it difficult to obtain specific antibodies required for immunoassay development. In addition, type II collagen antibodies exhibit reactivity to structurally dissimilar antigens such as actin, myoglobin, thyroglobulin and ssDNA, complicating the isolation of specific antibodies. It is therefore necessary to characterize the antibody reactivity against both noncollagenous antigens and different collagen types. In this study, immune response to type II collagen was improved by conjugation to carrier proteins, KLH and BSA. Hybridomas were generated by fusions of lymphocytes derived from lymph nodes or spleens with X63-653-Ag8 myeloma cells. Compared to spleens, the utilization of lymph nodes as a source of lymphocytes resulted in a 23% higher number of hybridomas secreting type II collagen antibodies. Hybridomas secreting polyreactive antibodies were identified based on their reactivity to thyroglobulin and eliminated. Extensive testing of the remaining monoclonal antibodies with other structurally dissimilar antigens and various types of collagen for reactivity, allowed us to isolate specific monoclonal antibodies to type II collagen. We emphasize the importance of characterization of the reactivity of type II collagen monoclonal antibodies before employing them for immunoassays.

Extracellular matrix gene expression by human bone marrow stroma and by marrow fibroblasts.

In the marrow space hemopoietic progenitors interact with stromal cells allowing homing, migration and the expression of developmental programs. Some of these interactions are mediated by extracellular matrix molecules (ECM) which are produced by marrow stromal cells. In the murine system the nature of these interactions and its biological significance have been studied in vitro by using indistinctly, either whole stroma (obtained from long-term marrow cultures) or isolated stromal (fibroblast-like) cells. In an attempt to analyze whether in the human system both sources of stromal cells are equivalent in terms of production of ECM components, we measured the expression of genes encoding for several ECM proteins. The results obtained show that whole stroma as well as marrow fibroblasts express genes for collagen types I, III, IV, V and VI as well as for fibronectin and laminin. However, the relative abundance of mRNA transcripts for some of these proteins was higher in marrow fibroblasts as compared to whole stroma. The latter was further supported by an increased collagen synthesis observed in marrow fibroblasts as compared to whole stroma.

Effects of doxazosin on atherosclerosis in cholesterol-fed rabbits.

Doxazosin was administered to rabbits fed diets enriched in cholesterol and peanut oil for 7.5 or 12 weeks, in 2 separate experiments. Doxazosin suppressed the accumulation of cholesterol and formation of atherosclerotic plaques in the aortas of treated rabbits and prevented a diet-induced increase in aortic collagen and wall mass. Doxazosin was more effective in the thoracic and abdominal segments of the aorta than in the aortic arch. Pharmacokinetic analysis indicated that treated rabbits were exposed to concentrations of doxazosin, integrated over 24 h, which were consistent with the therapeutic range of doxazosin measured in patients treated for hypertension. Doxazosin did not alter serum levels of cholesterol or triglycerides, nor were there any consistent effects on glucose, free fatty acid or ketone levels. Hypotheses of the mechanism of action of doxazosin are discussed, including the possible involvement of alpha 1-adrenergic receptors in recruitment of smooth muscle cells by subintimal macrophages and nonadrenergic mechanisms of inhibition of lipid infiltration.

Quantitative immunoassays for type II collagen and its cyanogen bromide peptides.

This paper describes the development of quantitative immunoassays utilizing mouse monoclonal antibodies which are monospecific to type II collagen. The monoclonal antibodies were characterized and tested extensively for reactivity against a panel of antigens including actin, myoglobin, thyroglobulin, ssDNA, tetanus toxin and different types of collagens including their CnBr-derived peptides. Four monoclonal antibodies having strict monospecificity to type II collagen were selected. Quantitative immunoassays developed with these antibodies can measure either type II collagen in its native conformation or type II collagen-derived cyanogen bromide peptides. The assay conditions such as coating concentration of antigen, monoclonal antibody concentration, second antibody concentration and incubation times were optimized to obtain maximum possible sensitivity. These quantitative immunoassays can be employed to measure type II collagen or type II collagen-derived peptides in low amounts ranging from 20 to 100 ng/ml. The assays can be applied to chondrocyte cultures without interference from serum components or other collagen types.

STZ-induced diabetes in SHR and renovascular hypertensive rats: dissociation between changes in arterial pressure and vascular collagen synthesis.

Streptozotocin (STZ)-induced diabetes depresses the rate of vascular collagen synthesis in the spontaneously hypertensive rat (SHR), but it also reduces arterial pressure (SAP) in this strain. We investigated this phenomenon further by comparing the SHR with the renovascular hypertensive (RVH) rat, because diabetes does not affect SAP in the latter model of hypertension. Renovascular hypertension was induced by clipping the left renal artery of Wistar-Kyoto (WKY) rats; sham-operated WKY were included as normotensive controls. Collagen synthesis of arterial tissue in vitro was quantified as prolyl hydroxylase activity and the rate of radioactive proline incorporation into collagen. Arterial collagen synthesis of nondiabetic SHR and RVH animals was elevated compared to that of the nonhypertensive WKY controls. STZ-induced diabetes (8 weeks) reduced SAP of SHR, but had no effect on SAP of either RVH or normotensive WKY rats. However, diabetes significantly depressed vascular collagen synthesis of both SHR and RVH rats, and, less consistently, of the WKY. The results strongly suggest that STZ-induced diabetes in SHR impairs arterial collagen synthesis independent of associated changes in arterial pressure.

The generation of hybridomas secreting human monoclonal antibodies reactive with type II collagen.

Human monoclonal antibodies reactive with type II collagen were obtained from patients with rheumatoid arthritis and relapsing polychondritis by fusion of cells with the human-mouse myeloma analogue HMMA2.11 TG/0. Direct fusion of peripheral blood or bone marrow mononuclear cells was unsuccessful in obtaining antibody producing hybridomas although fusion efficiency was high (1 hybridoma per 25,400 mononuclear cells fused). Polyclonal, Epstein Barr Virus transformed B cell lines derived from peripheral blood and bone marrow mononuclear cells after direct transformation, in vitro secondary immunization, and/or CD8 depletion were found to produce antibodies that reacted with type II collagen. Antibody producing EBV transformed B cells were fused with the human-mouse myeloma analogue HMMA2.11 TG/0 and six separate, stable IgM antibody producing hybridomas obtained. These results demonstrate the difficulty in obtaining human monoclonal autoantibodies, particularly of IgG isotypes, using readily accessible sources of cells in patients with chronic autoimmune diseases without expansion and preselection.

Comparison of in vitro, animal, and clinical determinations of iron bioavailability: International Nutritional Anemia Consultative Group Task Force report on iron bioavailability.

Relative bioavailability of two iron fortificants, electrolytic Fe and ferric orthophosphate, was related to that of the reference ferrous sulfate with in vitro and rat model depletion-repletion methods in four laboratories to compare values directly with those obtained in a parallel human study. In vitro testing was performed on Fe compounds with both solubility and dialysis in a simulated in vitro gastrointestinal digestion system. Two depletion-repletion techniques, hemoglobin-regeneration efficiency (HRE) and an official method of the Association of Official Analytical Chemists (AOAC), were examined. AOAC relative biological values (RBV) of electrolytic Fe were 0.66 and 0.78 and of FePO4 were 0.25 and 0.34. HRE values were 0.78 and 0.58 for electrolytic Fe and FePO4, respectively. When compared with FeSO4 in a radiolabeled farina-based meal fed to humans, the RBV of FePO4 was 0.25 and electrolytic Fe 0.75. Results obtained with the AOAC method serve as the most reliable prediction of Fe bioavailability in the human although in vitro dialysis is a promising screening technique.

Comparative sensitivities of purified preparations of lysyl oxidase and other amine oxidases to active site-directed enzyme inhibitors.

Recent evidence has revealed that lysyl oxidase, plasma amine oxidase and diamine oxidase each contain copper and pyrroloquinoline quinone at their active sites as cofactors essential to their catalytic functions. It thus seems likely that these enzymes will share similar mechanisms of action. Since mechanism-based inhibitors of lysyl oxidase have important chemotherapeutic potential for the control of fibrotic disease, the relative inhibitory potential of such agents toward catalytically similar amine oxidases was assessed in the present study using purified preparations of lysyl oxidase, diamine oxidase, plasma amine oxidase and the flavin-dependent mitochondrial monoamine oxidase A and B. The results indicate that there is sufficient difference between the sensitivities of lysyl oxidase and the other amine oxidases to beta-aminopropionitrile to warrant its consideration as an antifibrotic agent in vivo, while also revealing that aminoguanidine, clorgyline and deprenyl are sufficiently selective for diamine oxidase, monoamine oxidase A and monoamide oxidase B, respectively, to differentiate between lysyl oxidase and these enzymes at appropriate concentrations.

Effects of doxazosin on vascular collagen synthesis, arterial pressure and serum lipids in the spontaneously hypertensive rat.

Hypertension in various experimental models, including spontaneously hypertensive rats (SHR), is associated with elevated rates of vascular collagen synthesis. The sympathetic nervous system is an important factor in the etiology of hypertension in SHR. The primary purpose of this study was to determine the effects of the alpha 1-adrenergic receptor antagonist doxazosin on aortic collagen synthesis and on systolic arterial pressure in SHR. Doxazosin was administered either short-term (20 or 200 mg/kg/day by gavage over 5 days) or long-term (0.02 or 0.20 g/L in the drinking water over 8 weeks). Rates of collagen synthesis were determined by incubating aortic segments with 14C-proline in vitro and then measuring either the formation of 14C-hydroxyproline by means of high-performance liquid chromatography, or the amount of radioactivity liberated by collagenase digestion. Systolic arterial pressure was monitored with the standard tail-cuff technique. Both doses of doxazosin depressed aortic collagen synthesis at 8 weeks of treatment, but neither dose had any effect at 4 weeks. In the short-term study only the higher acute dose of doxazosin significantly reduced aortic collagen synthesis; the lower dose had no effect. In the short-term study doxazosin reduced systolic arterial pressure, with a maximum effect at 1-2 days. Tolerance to the depressor effect developed over the remaining 3-4 days, especially with the higher dose. In the 8-week study, the lower doxazosin dose had no effect on systolic arterial pressure, and the higher dose exerted a biphasic effect, moderately but significantly reducing systolic arterial pressure at 1 and 8 weeks of treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Monoclonal antibodies to human lysyl oxidase.

Hybridoma antibodies against human lysyl oxidase were produced by fusing Sp. 2.0-Ag 14 myeloma cells with spleen cells from mice hyperimmunized with lysyl oxidase isolated from umbilical cords. Hybridomas positive by enzyme-linked immunosorbent assay (ELISA) for human lysyl oxidase were cloned by the dilution method. Eight hybridomas producing antibodies were isolated, three of which also recognized purified bovine aortic lysyl oxidase in an ELISA. One of these antibodies, monoclonal antibody I, was purified and attached to Sepharose CL-4B. The immobilized antibody was effective in binding an enzymatically active 30,000 dalton species. Immunoblot analysis of four of these antibodies showed reactivity against the 30,000 dalton catalytically active enzyme and the 24,000 dalton fragment of lysyl oxidase. These monoclonal antibodies should be useful tools for studying the localization and biosynthesis of lysyl oxidase.