Inhibition of ADAMTS-7 and ADAMTS-12 degradation of cartilage oligomeric matrix protein by alpha-2-macroglobulin.

OBJECTIVE: As we previously reported, ADAMTS-7 and ADAMTS-12, two members of ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) family, degrade cartilage oligomeric matrix protein (COMP) in vitro and are significantly induced in the cartilage and synovium of arthritic patients [Liu CJ, Kong W, Ilalov K, Yu S, Xu K, Prazak L, et al. ADAMTS-7: a metalloproteinase that directly binds to and degrades cartilage oligomeric matrix protein. FASEB J 2006;20(7):988-90; Liu CJ, Kong W, Xu K, Luan Y, Ilalov K, Sehgal B, et al. ADAMTS-12 associates with and degrades cartilage oligomeric matrix protein. J Biol Chem 2006;281(23):15800-8]. The purpose of this study was to determine (1) whether cleavage activity of ADAMTS-7 and ADAMTS-12 of COMP are associated with COMP degradation in osteoarthritis (OA); (2) whether alpha-2-macroglobulin (a(2)M) is a novel substrate for ADAMTS-7 and ADAMTS-12; and (3) whether a(2)M inhibits ADAMTS-7 or ADAMTS-12 cleavage of COMP. METHODS: An in vitro digestion assay was used to examine the degradation of COMP by ADAMTS-7 and ADAMTS-12 in the cartilage of OA patients; in cartilage explants incubated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1-beta (IL-1beta) with or without blocking antibodies; and in human chondrocytes treated with specific small interfering RNA (siRNA) to knockdown ADAMTS-7 or/and ADAMTS-12. Digestion of a(2)M by ADAMTS-7 and ADAMTS-12 in vitro and the inhibition of ADAMTS-7 or ADAMTS-12-mediated digestion of COMP by a(2)M were also analyzed. RESULTS: The molecular mass of the COMP fragments produced by either ADAMTS-7 or ADAMTS-12 were similar to those observed in OA patients. Specific blocking antibodies against ADAMTS-7 and ADAMTS-12 dramatically inhibited TNF-alpha- or IL-1beta-induced COMP degradation in the cultured cartilage explants. The suppression of ADAMTS-7 or ADAMTS-12 expression by siRNA silencing in the human chondrocytes also prevented TNF-alpha- or IL-1beta-induced COMP degradation. Both ADAMTS-7 and ADAMTS-12 were able to cleave a(2)M, giving rise to 180- and 105-kDa cleavage products, respectively. Furthermore, a(2)M inhibited both ADAMTS-7- and ADAMTS-12-mediated COMP degradation in a concentration (or dose)-dependent manner. CONCLUSION: Our observations demonstrate the importance of COMP degradation by ADAMTS-7 and ADAMTS-12 in vivo. Furthermore, a(2)M is a novel substrate for ADAMTS-7 and ADAMTS-12. More significantly, a(2)M represents the first endogenous inhibitor of ADAMTS-7 and ADAMTS-12.

A sensitive single reverse passive haemagglutination test for detecting both HBsAg and anti-HBs.

A trial of a modified reverse passive haemagglutination test for HBsAg using a 0.1% cell suspension instead of the recommended 1% showed an approximately eight-fold increase in detection sensitivity. The test can be performed within 30 minutes and lends itself to mass screening techniques. Confirmation tests can be done using the 0.1% method. In addition, the same serological plates and cells used for HBsAg screening can then be used to screen for high-titre anti-HBs. This makes the overall screening for both HBsAg and high-titre anti-HBs donors cheap and convenient.

A sensitive immunoradiometric assay for the detection of hepatitis B surface antigen.

A solid-phase immunoradiometric assay for hepatitis B surface antigen is described which has been in use since 1971. Initially it was used for reference laboratory work, but from 1974 it has also been used for screening blood and blood products. Methods for the production of reagents and their use in blood transfusion and reference work are outlined.

Donors whose blood reacts falsely positive in transfusion microbiology screening assays need not be lost to transfusion.

Donations that repeatedly react in transfusion microbiology screening assays are usually discarded; with appropriate confirmatory testing on the index and on follow-up samples, the great majority of these can be shown to be falsely positive. Under carefully controlled conditions, with secure information transfer, these donations, although still reactive in the primary screening assays, can be made available for clinical use after testing and obtaining negative results with alternative assays from a list of assays evaluated as suitable for the release of blood donations. We will describe a generic algorithm that can be applied to all markers.

The 2', 3'-cyclic AMP 3'-phosphohydrolase in normal and psoriatic epidermis.

The activity of the enzyme 2', 3'-cyclic AMP 3'-phosphohydrolase is significantly greater in the 'involved' psoriatic skin than in the 'uninvolved' psoriatic skin or in skin samples taken from persons having clinically normal skin. Although the physiological function of this enzyme is not established, it is possible that, besides being associated with myelin, it may also play some role in cell proliferation and maturation, probably at the membrane level.

Retrospective follow-up of recipients and donors of blood donations reactive for anti-HBc or for single HCV antibodies.

A retrospective study was undertaken to investigate the infectivity of blood donations that were HCV RIBA indeterminate or reactive for 'anti-HBc only'. Samples from 6032 blood donations were tested for anti-HBc; 17 of them were positive for anti-HBc but were negative when tested for anti-HBs. None of the recipients of the red cells from these donations, when tested 9 months after transfusion, had evidence of HBV infection. Samples from 3000 blood donations were tested for HCV antibodies using RIBA-3; 53 had single lines. Samples taken 9 months after transfusion from the recipients of the red cells from these donations were tested for evidence of infection with hepatitis C, and all were negative.

Antibody to hepatitis C virus in blood donors found positive for other agents. I. Treponemal infection.

Stored serum samples from 169 blood donors found positive for TPHA were tested for antibodies to hepatitis C virus (anti-HCV) and to hepatitis B core (anti-HBc), as evidence of previous HBV infection, a condition known to be sexually transmissible. Only three donors were positive with a 'first generation' anti-HCV and all three failed to confirm with a recombinant immunoblot assay (RIBA). In contrast, 33 (19.5%) of the TPHA-positive donors were positive for anti-HBc. The results suggest that sexual transmission is not a major factor in the spread of HCV.

Rapid transfer of small volumes of serum into microplates.

A method has been devised to transfer small volumes of serum, as an adjunct to larger volume sampling, into microplates. The Costar Transplate 96 machine is used routinely in our laboratory for transferring serum samples into reaction wells for anti-HIV screening. Using this additional method a mean of 4 microliters of serum was transferred, within acceptable limits of error (coefficient of variation, c.v., 16%).

Contrasting patterns and frequency of antibodies to the surface, core, and e antigens of hepatitis B virus in blood donors and in homosexual patients.

An attempt was made to assess the significance of the relatively frequent low-titre positive reactions in radioimmunoassays for antibody to hepatitis B surface antigen. Serum specimens negative for surface antigen were assayed for antibodies to the surface, core, and e antigens of the Hepatitis B virus. Two populations were studied - one of low hepatitis B incidence (2000 blood donors) and one of high incidence (200 male patients attending a clinic for sexually transmitted diseases). The validity of some singly occurring low-titre anti-HBs and anti-HBc reactions is questioned, and the occurrence of positive reactions unrelated to previous hepatitis B infection inferred.

A comparison of different methods of screening blood donations for HBsAg.

In a retrospective comparison between countermigration immunoelectrophoresis (CIEP) and reverse passive haemagglutination (RPHA) for screening 260,500 blood donations, the latter's 10-fold increase in sensitivity resulted in 36% more HBsAg detections. In a prospective comparison between RPHA and radioimmunoassay (RIA) the latter's 40-fold increase in sensitivity over RPHA resulted in 11% more detections than RPHA in 27,094 new donors. One in 500 new donors was HBsAg-positive by RPHA, compared with 1 in 11,000 established donors who had donated and been tested previously. Acute hepatitis B infections, though uncommon, accounted for a greater proportion of the HBsAg-positive found in "established" rather than new donors. Reported post-transfusion hepatitis cases have declined following the introduction of screening tests in 1971. The feasibility of RIA testing at a transfusion centre supplied simply with the two basic RIA reagents has been demonstrated.

Anti-HIV-1: extended validation of a modified gelatin particle agglutination assay, and its use with an image analyser.

Modification of a commercial gelatin particle agglutination assay for anti-HIV-1 (MAT) has increased sensitivity, reduced reaction time and lowered the cost by 90%. We tested over 10,000 blood donations in parallel with the ELISA currently in use, and found the modified test to be highly sensitive and to enhance cost-effectiveness as it substantially reduces false-positive rates. The patterns of agglutination are clearly reproducible and readable by the naked eye and/or Image Analyzer, which provides objectivity and hard-copy documentation of results.

Reduction of enzyme levels by propranolol after acute myocardial infarction.

The effect of propranolol (0.1 mg/kg intravenously followed by 320 mg given over 27 hour orally) on serum levels of creatine kinase enzyme was studied in a randomized trial involving 95 patients seen within 12 hours of onset of symptoms of uncomplicated myocardial infarction. In 15 patients who were treated with propranolol within 4 hours of onset, and who eventually developed pathological Q waves, peak measured enzyme levels were 27% (P less than 0.0125) lower than in 19 control patients who were also seen within 4 hours of the onset but had no specific treatment. Total calculated enzyme appearance was also lower in the treated patients (reduced 25%, P less than 0.05) as was the calculated rate of the appearance (33%, P less than 0.005). No significant difference was found for treated compared with control patients entering the trial more than 4 hours after the onset of chest pain. This evidence suggests that propranolol may reduce the size of uncomplicated infarctions if it is given intravenously within 4 hours of the onset.

Hepatitis B virus infection in patients attending a genitourinary medicine clinic: risk factors and vaccine coverage.

BACKGROUND: The hepatitis B virus (HBV) immunisation policy in the United Kingdom includes offering vaccines selectively to those at risk by sexual contact. Among genitourinary medicine (GUM) clinic attenders, homosexual men are offered vaccine, but estimates of the vaccine uptake are required to monitor policy and estimate the possible impact on transmission; heterosexuals are not routinely offered vaccine, but this policy might change if the prevalence was found to be high. OBJECTIVE: To determine the prevalence of HBV infection and vaccine uptake among patients attending a GUM clinic. METHODS: HBV seroprevalence determined by unlinked anonymous testing of consecutive blood samples sent for syphilis serology. Demographic and risk factor data and history of HBV immunization extracted from clinic notes before unlinking. Prevalence data were compared with a population of first time blood donors from the same area. SETTING: Open access GUM clinic in central London. RESULTS: Samples were obtained and tested from 441 homosexual and 527 heterosexual men and from 821 women over a 4 month period in 1990. After exclusion of injecting drug users and their sexual partners (n = 30) and HBV carriers attending for follow up (n = 12), the prevalence of antibody to HBV core (anti-HBc) was 38.7% in homosexual men, 5.9% in heterosexual men, and 3.5% in women (50.0%, 6.0%, 3.7% respectively if previous vaccinees were also excluded). The prevalence of HBV surface antigen positivity was 4.2%, 0.60%, and 0.39% respectively after exclusion of vaccinees (chi(2) p < 0.001 for homosexual men versus others). The prevalence of the anti-HBc in first time blood donors was 1.1% (8/749). Among male GUM clinic attenders, the prevalence of anti-HBc was higher in those of non-UK origin or place of birth and/or non-white ethnicity (odds ratios 2.87, 95% CI 1.57-5.24 and 8.06, CI 3.39-19.1, in homosexuals and heterosexuals respectively). In homosexual men anti-HBc prevalence increased with age (OR 1.05, CI 1.02-1.07 for each year) and lifetime number of STDs (OR 6.36, CI 3.77-10.8 for > or = 2 versus < 2) and in clinic reattenders compared with new patients (OR 5.42, 95% CI 3.32-9.16). Among heterosexuals, age was associated with anti-HBc prevalence in women (OR 1.09, CI 1.04-1.12) but not men (OR 0.99, 95% CI 0.95-1.02). There were no other associations in heterosexuals. A history of HBV immunisation in homosexual men was recorded in 13/131 (9.9%) of new patients and 103/305 (33.8%; OR 4.63, CI 2.49-8.60) clinic reattenders. CONCLUSIONS: Although higher than a sample of blood donors, the prevalence of serological markers of HBV infection among heterosexuals was low, providing little support for extending HBV immunisation to all heterosexuals attending GUM clinics as a targeted strategy for control of HBV infection. Homosexual men remain at high risk of infection, but many are now being immunised. Efforts to improve compliance with existing vaccine policies in GUM clinics should be encouraged.

Low incidence of non-A, non-B post-transfusion hepatitis in London confirmed by hepatitis C virus serology.

To see whether the introduction of screening tests for post-transfusion non-A, non-B hepatitis (NANBH) in the UK would be worth while, the incidence of such hepatitis was assessed among patients receiving blood during operations at five hospitals served by the North London Blood Transfusion Centre. 387 patients, who each received blood or blood components from an average of 3 donors were followed up prospectively and blood samples were taken every 2 weeks for 3 months and then each month for a further 3 months. 229 patients also provided a sample at 12 months. All available patient and donor samples were tested for alanine aminotransferase concentrations and for antibody to hepatitis C virus (anti-HCV) by ELISA. Repeatedly anti-HCV positive samples were submitted to supplementary HCV assays. 1 of the 387 patients showed biochemical evidence of acute post-transfusion NANBH after exclusion of non-viral causes. Anti-HCV developed in this patient and the seroconversion was confirmed by recombinant immunoblot assay and polymerase chain reaction. Serum from 1 of the 8 donors whose blood he received was positive for anti-HCV by all three methods. In another patient HCV seroconversion was shown by ELISA but alanine aminotransferase concentrations remained normal throughout follow-up. His samples and those of his 2 donors were negative for HCV by the polymerase chain reaction. A third patient showed rises in alanine aminotransferase compatible with post-transfusion NANBH, but serology and polymerase chain reaction assays for HCV were negative for her samples and those of her donors. Anti-HCV reactivity likely to be false positive (negative by both confirmatory tests and no adverse effects in recipients) was seen in 6 of 1283 donors. This study, despite its being carried out in the part of the UK with the highest frequency of infectious markers in blood donations, has shown a very low incidence of post-transfusion NANBH.