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Biomedical subjects

D S Kumararatne

Publications and source records attributed to D S Kumararatne.

At least 37 records · Page 2Linked to original sources
PubMed1998

Endomysial antibody detection using human umbilical cord tissue as substrate: reactivity of cells in Wharton's jelly.

The measurement of immunoglobulin A (IgA) endomysial antibodies is now established as an important diagnostic test in gluten-sensitive disease. Monkey oesophagus is the commonly used tissue substrate, but it has been proposed that human umbilical cord tissue may be a suitable alternative for antibody detection. In this study, we report a modified method of examining endomysial antibody reactivity with cord tissue. This involves examination of antibody reactivity with cells in Wharton's jelly, and with umbilical cord blood vessel. A total of 370 patients being investigated for coeliac disease were studied and this diagnosis was established in 42. Endomysial antibodies were found in all 42 using umbilical cord tissue, and the test results were confirmed with monkey oesophagus substrate. In three of the remaining 328 non-coeliacs, a false-positive endomysial antibody test was noted and small intestine histology was normal in these patients. All positive sera were found to react with cells in Wharton's jelly, and reticular staining of blood vessels was also present. Examination for immunofluorescence in both Wharton's jelly and blood vessel components of cord tissue greatly simplified test interpretation.

Adolescent↗
PubMed1998

BPL plasma products: new source, enhanced products.

Bio Products Laboratory (BPL), the main fractionator of plasma-derived medicines, are undergoing a change from UK plasma to plasma from the USA. BPL aim to maintain continuity of supply and a seamless changeover, despite meticulous cleaning of its production plant and months of auditing by both the organization and regulatory authorities.

Blood Banks↗
PubMed1997

ATP-induced killing of mycobacteria by human macrophages is mediated by purinergic P2Z(P2X7) receptors.

The death of BCG-infected human macrophages induced in vitro by ligation of surface CD95 (Fas), CD69, or complement-mediated lysis was shown not to result in the death of intracellular mycobacteria, whereas exposure to extracellular ATP initiated both macrophage death and killed the intracellular bacteria. ATP acted via P2Z receptors because these effects were mimicked by benzoylbenzoic ATP (a known agonist of P2Z receptors) and blocked by oxidized ATP, DIDS, suramin, amiloride, and KN62 (known inhibitors of P2Z-mediated responses). ATP-mediated bacterial killing was independent of reactive nitrogen and oxygen intermediates and of actinomycin D or cycloheximide inhibition. ATP-induced macrophage cell death, BCG killing, and lucifer yellow dye incorporation were minimal in 2 out of 19 healthy donors. The results suggest possible genetic heterogeneity of this mechanism of mycobacterial killing associated with P2Z-mediated pore formation.

Adenosine Triphosphate↗
PubMed1997

Sjögren's syndrome in women presenting with chronic dyspareunia.

OBJECTIVE: To identify cases of Sjögren's syndrome among women with chronic dyspareunia who did not already have a diagnosed rheumatological disorder. DESIGN: Prospective recruitment over 12 months. SETTING: Tertiary referral service for the assessment of vulval disease. PARTICIPANTS: Women with chronic dyspareunia who had musculoskeletal symptoms, Raynaud's phenomenon or symptoms of ocular or oral dryness. METHODS: The women underwent a Schirmer tear test and a comprehensive auto-antibody screen including latex fixation test for rheumatoid factor, antinuclear, anti-Ro, anti-La and anti-salivary duct antibodies. A labial salivary gland biopsy and vaginal biopsy were taken for routine histological analysis. MAIN OUTCOME MEASURES: Cases of definite and probable Sjögren's syndrome were identified using the European criteria. RESULTS: Eleven women were assessed for features of Sjögren's syndrome. Four had definite primary Sjögren's syndrome, two had probable primary Sjögren's syndrome and one had probable secondary Sjögren's syndrome. Among these seven women the median duration of vaginal symptoms was seven years (range 0.25-20), of ocular symptoms was one year (range 0.25-2) and of oral symptoms was 1.5 years (range 0-6). In all but one woman dyspareunia presented before ocular or oral symptoms, often by many years. CONCLUSIONS: Although well-recognised as a feature of established Sjögren's syndrome, this study emphasises that chronic dyspareunia can be a presenting feature in these women, antedating the emergence of ocular or oral symptoms by many years. Symptoms of ocular or oral dryness, Raynaud's phenomenon or musculoskeletal symptoms should be sought in women with chronic dyspareunia to identify those who merit further investigation.

Adult↗
PubMed1996

Candidate antigens for improved serodiagnosis of tuberculosis.

OBJECTIVE: To explore the possibility that an analysis of antibody specificity to separated components of mycobacteria in a group of tuberculous patients may reveal a combination of target antigens whose antibodies could form the basis of a useful serodiagnostic test. DESIGN: Immunoblots of 1-dimensional (SDS-PAGE) and 2-dimensional (isoelectric focusing/SDS-PAGE) separation of antigenic extracts of Mycobacterium tuberculosis H37Rv (MTSE) and M. bovis bacille Calmette-Guérin (BCG) (MBSE) with 52 tuberculous and 59 BCG-vaccinated control human sera were analyzed for band and spot reactivity patterns that are indicative of infection with M. tuberculosis. RESULTS: Reactivity to antigens banding in the 10-18 kDa, 37-43 kDa and 70-90 kDa regions allowed a good discrimination between patients and normal subjects. Patients' sera reacting with antigens in the 22-30 and 70-88 kDa regions differentiated responses to MTSE and MBSE. In 2-D immunoblotting, patients' sera only reacted with antigens separating at approximately pI 6.5/26-28 kDa, pI 4.8/38 kDa and pI 6.5/70-79 kDa position and the responses were specific for M. tuberculosis (MTSE). CONCLUSION: These results provide evidence that a combination of these M. tuberculosis antigens may be a useful basis for developing a diagnostic antibody test. Additionally, they may help to define antigens, and host antibody responses that are specific to one but not the other of the two closely related species.

Antibodies, Bacterial↗
PubMed1996

T-cell response to mycobacterial proteins: a comparative study of tuberculous and control immunoblots of Mycobacterium tuberculosis and M. bovis BCG.

OBJECTIVE: To evaluate and compare the lymphoproliferative response of human peripheral blood mononuclear cells (PBMC) to fractionated soluble extracts of Mycobacterium tuberculosis H37Rv (MTSE) and M. bovis bacille Calmette-Guérin (BCG) (MBSE), and thereby determine responses that correlate to infection, and to contrast antibody and T-cell responses. DESIGN: Membrane blots of SDS-PAGE fractionated M. tuberculosis H37Rv and M. bovis BCG were employed for antibody immunoblotting and T-cell proliferative responses using sera and PBMC from seven tuberculous and seven BCG vaccinated control subjects. RESULTS: The profiles of responses contrasted rather interestingly, with antibody and T-cells responding more to higher and lower molecular weight fractions respectively. T-cells responding to antigens in the 59-88 kDa region discriminated between tuberculous and BCG vaccinated controls (P < 0.05) even though the differences were more toward the 70-75 kDa fractions within the region in question. Responses to smaller molecular weight fractions of both MTSE and MBSE were high in direct contrast to antibody responses. Additionally, responses to MBSE in these regions were generally higher than for MTSE in vaccinated controls. The reverse was the case with tuberculous subjects where responses to MTSE were generally higher, though not sufficiently significant in enough of the tuberculous subjects to be considered discriminatory. CONCLUSION: T-cell proliferative responses to mycobacterial antigens in the 59-88 kDa region, and particularly antigens in the 70-75 kDa region, can be an indication of infection with M. tuberculosis, as well as the basis for discriminating between active disease and vaccination with BCG.

Antigens, Bacterial↗
PubMed1996

Simple method for pretreatment of tissue sections for the detection of apoptosis by in situ end-labelling and in situ nick translation.

Aims-To overcome the problems associated with proteolytic pretreatment of tissue sections for the detection of apoptosis.Methods-Formalin fixed, paraffin wax embedded tissue sections of reactive lymph nodes and biopsy specimens of Burkitt lymphoma were pretreated by pressure cooking for the detection of apoptosis using the in situ end-labelling and in situ nick translation methods.Results-The results achieved with the in situ end-labelling and nick translations methods were compared with those obtained using a novel anti-apoptosis specific protein (ASP) antibody. The staining patterns generated using the three methods were similar and consistent, although the ASP antibody seemed to be more sensitive and detected higher numbers of apoptotic cells within sections.Conclusions-Pressure cooking is advocated as an alternative method to proteolytic enzyme digestion for pretreating paraffin wax sections. It is reliable, inexpensive, reduces the need to optimise pretreatment variables for different tissues, and permits double immunostaining of sections.

Journal Article↗
PubMed1995

Lysis of human macrophages by cytolytic CD4+ T cells fails to affect survival of intracellular Mycobacterium bovis-bacille Calmette-Guérin (BCG).

Human CD4+, mycobacteria-specific, cytolytic T cell clones were used to lyse BCG-infected macrophages, and the effect on the subsequent growth and viability of the organisms was examined. The survival of released bacteria following cell lysis was assessed by both 3H-uridine labelling and colony-forming unit (CFU) estimation. The results indicate that even when effective antigen-specific or lectin-mediated cytolysis of the infected macrophages was achieved, there was no evidence for a direct mycobactericidal effect on the intracellular bacteria. This remained the case even if the period of co-culture of T cells and macrophages was extended up to 48 h. Pretreatment of the macrophages with interferon-gamma (IFN-gamma) was not able to act together with T cell-mediated lysis to produce inhibition of mycobacterial growth.

CD4-Positive T-Lymphocytes↗
PubMed1995

CD4+ cytolytic T cells can destroy autologous and MHC-matched macrophages but fail to kill intracellular Mycobacterium bovis-BCG.

Mycobacterium bovis-BCG infected macrophages were exposed in vitro to PPD-stimulated T lymphocytes from tuberculin responsive donors or to a panel of mycobacterial-antigen specific CD4+ T cell clones. Both polyclonal and clonal T cells caused considerable antigen-specific lysis of autologous or MHC class II matched macrophages. However, lysis of infected macrophages did not significantly affect the number of viable mycobacteria which were released into the culture media from lysed macrophages. In tuberculosis, CD4+ cytolytic T cells may be primarily involved in tissue destruction and lack a significant role in acquired cellular immunity.

CD4-Positive T-Lymphocytes↗
PubMed1994

Interleukin 1 beta, hand and foot bone mineral content and the development of joint erosions in rheumatoid arthritis.

OBJECTIVE: To assess the relationship between plasma levels of the cytokine interleukin-1 beta (IL-1 beta) and the progression of rheumatoid arthritis (RA). METHODS: Two subgroups of patients, one with persistently raised ESR (>/= 50 mm/hour, n = 16, group A) and one with persistently low ESR (</= 28 mm/hour), n = 18, group I) were chosen to represent stable extremes of inflammatory activity from a prospective study of 106 patients with active RA studied over one year in a single centre. The change from baseline in hand, foot and calcaneal bone mineral content measured by single photon absorptiometry and radiographic score of joint damage was measured over 12 months, together with plasma IL-1 beta and erythrocyte sedimentation rate. RESULTS: Significant progression of joint damage occurred in both subgroups over one year (p < 0.0001, paired t test) though progression was significantly less in the subgroup with low ESR (p < 0.05, ANOVA). Hand and foot bone mineral content decreased by almost 10% in the subgroup with raised ESR (p < 0.005, paired t test). Stepwise linear regression analysis revealed significant independent relationships between radiographic progression over one year and plasma IL-1 beta and ESR (multiple R 0.674, F = 11.64, p < 0.0002). No such relationships were observed for changes in bone mineral content parameters. CONCLUSIONS: Plasma IL-1 beta levels correlate weakly with progression of joint damage though not with loss of peripheral bone density in RA. A significant reduction in peripheral bone mineral content occurs over one year in patients with active RA with persistently raised ESR.

Adult↗
PubMed1994

Two enzyme linked immunosorbent assays for detecting antibodies against meningococcal capsular polysaccharides A and C.

AIMS: To evaluate two of the recent methods of coating microtitre plates in the enzyme linked immunosorbent assay (ELISA) for detecting human antibodies against meningococcal capsular polysaccharides A and C with a view to validating a specific meningococcal antibody assay for routine clinical use. METHODS: Two four-layer ELISA protocols were standardised: one method utilised meningococcal polysaccharides conjugated to poly-L-lysine polypeptide for coating the microtitre plates; another used polysaccharides mixed with methylated human serum albumin (mHSA). Titration curves were plotted for the ELISAs and the squared Pearson correlation coefficient (R2) was used to determine the degree of accuracy of fit of the curves. Specificity tests were performed by inhibition and adsorption studies. RESULTS: Both methods gave good titration curves with a high R2 of > 0.98, indicating a high degree of accuracy in forming the curves. The titration end point after vaccination, obtained by the mHSA method, was 20 times higher, however, than that obtained by the poly-L-lysine method. Specificity tests showed that in the ELISA using polysaccharide/poly-L-lysine, antibody activity of a pre-vaccination serum sample was inhibited by 37%, and of post-vaccination serum by 50% with 1000-fold excess antigen. Antibody activity (post-vaccination) was reduced by 51% and 59%, respectively, by adsorption with antigen-coated Sepharose beads or adsorption with suspensions of killed meningococci. In contrast, antibody activity of a pre-vaccination serum was inhibited by 60% and a post-vaccination serum by 90% in ELISA employing polysaccharides mixed with mHSA. Reproducibility was better with the use of methylated human serum albumin than with poly-L-lysine; the former showed intrabatch and interbatch coefficients of variation of 4% and 2%, respectively, compared with 43% (intrabatch) and 16% (interbatch) obtained with the poly-L-lysine. CONCLUSION: It is concluded that the antibody assay using meningococcal polysaccharides groups A and C mixed with mHSA is much better than that using polysaccharides coupled with poly-L-lysine.

Antibodies, Bacterial↗
PubMed1993

Human CD4+ cytolytic T cells kill antigen-pulsed target T cells by induction of apoptosis.

Stimulation of human CD4+ T cell clones with appropriate specific peptides or with lectins in the absence of APC induces a substantial degree of cell death. We have investigated the mechanisms of induction of this cell death and show that it occurs by apoptosis, identified by morphology and the characteristic pattern of DNA degradation. We also investigated whether this T cell death was a result of a suicide process activated in the T cell after "inappropriate" recognition of Ag on the surface of another T cell clone (and in the absence of other accessory signals), or was due to a conventional lethal hit delivered by one cytolytic T cell to another "target" T cell. Our results strongly suggest that peptide-induced cell death of human CD4+ CTL is due to active killing of Ag-presenting target T cells by effector T cells of the same clone. The cell death that is induced in the target T cell occurs via apoptosis that requires de novo RNA transcription and translation in the effector T cell.

Apoptosis↗
PubMed1993

The acquisition of anti-pneumococcal capsular polysaccharide Haemophilus influenzae type b and tetanus toxoid antibodies, with age, in the UK.

Antibody levels specific for capsular polysaccharides of Streptococcus pneumoniae and Haemophilus influenzae type b (Hib) and to tetanus toxoid (TT), were measured in serum samples of 750 age-stratified subjects from the UK. The study subjects comprised healthy adult volunteers and hospitalized children undergoing elective surgery, excluding those with a history of infection or under investigation for immunological or haematological disorders. These antibody levels were calibrated by comparison with serum pool obtained from healthy adult volunteers, who were immunized with Hib polyribose-phosphate vaccine (Merieux). The data are intended to provide reference ranges to assist in the interpretation of specific antibody measurements in the clinical setting. Maternal IgG pneumococcal capsular polysaccharide (PCP) specific antibody levels, geometric-mean titre (GMT) 1/22, were lost by 6 months of age (GMT of 1/9). They remained low until 3-5 years (GMT of 1/20), and consisted principally of IgG1. Thereafter, IgG anti-PCP antibody titres increased steadily to adult levels (GMT of 1/275), of which 80% was IgG2. Anti-PCP antibody titres of the IgM isotype rose steadily from a GMT 1/21 (0-6 months) to 1/420 (3-5 years), a level which was maintained until adulthood. Anti-Hib antibody concentrations, determined by RABA, again demonstrated the decline in maternal antibody, from 0.18 micrograms/ml in the 0-6 month age cohort, to 0.09 microgram/ml between 6 and 12 months. Geometric-mean antibody concentrations remained below 0.2 micrograms/ml until 3-5 years, then increased with age, attaining the mean adult level of 1.02 micrograms/ml. Anti-TT antibody concentrations were measured in the same sera, by ELISA. Two peaks in anti-TT antibody levels were seen in children of 0.059 IU/ml and 0.166 IU/ml corresponding to the schedule of routine childhood immunization in the first year and at 5 years of age.

Adolescent↗
PubMed1992

Mucosal cell-mediated immunity to mycobacterial, enterobacterial and other microbial antigens in inflammatory bowel disease.

Culture studies have suggested that Mycobacterium paratuberculosis may play a role in the aetiology of Crohn's disease. However, evidence of sensitization to mycobacterial antigens amongst patients with Crohn's disease has not yet been adequately demonstrated. Previous studies of cell-mediated immunity (CMI) in Crohn's disease were restricted to responses of peripheral blood mononuclear cells (PBMC) to mycobacterial antigens. In this study we have investigated the proliferative responses of both PBMC and mesenteric lymph node mononuclear cells (MLNMC) to a range of mycobacterial and non-mycobacterial antigens. There was no evidence of specific sensitization in the responses of MLNMC and PBMC from patients with inflammatory bowel disease (IBD) to the mycobacterial antigens. However, anergy to M. paratuberculosis could not be excluded. IBD MLNMC responses to most antigens were generally greater than those of PBMC, which were often undetectable. When compared with controls, there was evidence of increased CMI to a range of non-mycobacterial antigens, especially Yersinia enterocolitica, amongst both MLNMC and PBMC from patients with Crohn's disease and ulcerative colitis (UC). These results do not provide support to the proposed role of mycobacteria in the pathogenesis of Crohn's disease, but indicate that further investigation may determine a role for bacterial-specific T cell-mediated responses in the pathogenesis of IBD.

Antigens, Bacterial↗
PubMed1992

An association between homozygous C3 deficiency and low levels of anti-pneumococcal capsular polysaccharide antibodies.

Inherited deficiencies of complement components are associated with an increased risk of infection by encapsulated, high grade bacterial pathogens such as Streptococcus pneumoniae, Haemophilus influenzae type b and Neisseria meningitidis. Hence, the levels of antibodies to bacterial capsular polysaccharide antigens were measured using ELISA in 65 patients with inherited deficiencies covering the classical, alternative and terminal components of the complement cascade. Three of the four C3-deficient individuals studied were found to be almost totally deficient in specific anti-pneumococcal capsular polysaccharide (PCP) antibodies. These individuals had a history of recurrent pneumococcal sepsis. While single individuals with C1r, C2 and C1Inh deficiency were found to have low anti-PCP antibody levels, no other group of complement deficiency had significantly reduced anti-PCP antibody levels compared with 100 controls. Antibody levels to the other two polysaccharides were not significantly lower in the patient groups. These findings suggest that C3 may be able to provide a stimulatory signal to promote the production of anti-PCP antibodies.

Adolescent↗