Biomedical subjects
Publications and source records attributed to M J Browning.
As part of the Fourth Asia-Oceania Histocompatibility (4AOH) Workshop, the authors have demonstrated a method of DNA-based tissue typing of the HLA-A locus using ARMS-designed primers in a panel of specific PCR reactions. The study was carried out blind under Workshop conditions and the results confirm the method as an accurate means of determining HLA-A locus tissue types.
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A system devised for tissue typing the HLA-A locus by PCR from genomic DNA has been used to investigate abnormalities of HLA expression in a panel of 30 colorectal tumor cell lines, by comparing the HLA-A locus genotype with surface expression of HLA. Three cell lines showed complete lack of HLA expression associated with failure to express beta 2-microglobulin. In two other cell lines, loss of expression of HLA-A2 was observed, in spite of the presence of the gene in genomic DNA. Eleven cell lines gave a single HLA-A locus specificity on PCR typing. In one of these cell lines we have demonstrated the loss of an HLA-A locus gene in the tumor cell by comparison with DNA from a lymphoblastoid B-cell line derived from the same patient. These data indicate that at least three independent mechanisms were involved in the loss of HLA expression on the colorectal tumor cell lines.
A system devised for tissue typing the human leukocyte antigen-A (HLA-A) locus from genomic DNA by the polymerase chain reaction (PCR) has been used to investigate abnormalities of HLA expression in a panel of 30 cell lines derived from colorectal adenocarcinomas, by comparison of the HLA-A locus genotype with surface expression of HLA. Eleven cell lines gave single HLA-A locus specificity on PCR typing, suggesting that loss of HLA alleles is a common abnormality. In one of these cell lines the loss of an HLA-A locus allele was confirmed by comparison with DNA from a lymphoblastoid B cell line derived from the same patient. In three cell lines, loss of expression of an HLA-A locus determinant was observed in spite of the presence of the relevant allele in genomic DNA. Three cell lines showed absent HLA expression associated with failure to express beta 2-microglobulin. These data indicate that at least three independent mechanisms were involved in the loss of HLA expression on the colorectal tumor cell lines.
We describe sequence-specific primer (SSP) combinations for use in a one-step polymerase chain reaction (PCR) typing system to determine HLA-A locus subtypes of A9 (A23, A24), A10 (A25, A26, A43), A28 (A*6801, A*6802, A*6901) and A19 (A*2901, A*2902, A*3001, A*3002, A31, A32, A33) from genomic DNA. SSP's were designed on the basis of the amplification refractory mutation system (ARMS) in which a mismatch at the 3' residue inhibits non-specific amplification. The SSP combinations described extend our low-resolution typing system, to provide a high-definition typing of the HLA-A locus.
A region of feline immunodeficiency virus (FIV)/Glasgow-8 external envelope glycoprotein (env) incorporating the third and fourth variable regions (V3/V4) was cloned, inserted into the pGEX vector and expressed in Escherichia coli to yield milligram quantities of the recombinant polypeptide as a fusion protein with glutathione S-transferase. The fusion protein V3/V4GST was used in lymphocyte proliferation assays, where it consistently caused peripheral blood lymphocytes from naive cats to proliferate in a dose-dependent manner. Other FIV fusion proteins produced under identical conditions (V5GST and p24GST) and glutathione S-transferase alone did not cause proliferation in this system. The monoclonal antibody vpg15, which has been shown to block infection of susceptible cells in vitro, did not decrease the response to V3/V4GST. Human peripheral blood lymphocytes did not proliferate in response to V3/V4GST.
Evidence for a direct involvement of cell-mediated immune mechanisms in the control of human tumours remains sketchy. The past year, however, has seen advances in our understanding of the relationship between the immune system and tumour cells, and has given rise to new prospects for immunological treatment and the prevention of cancers.
By immunizing inbred mice with purified replication-competent, defective virus particles, or an admixture of the two, differential effects on the cellular immune system have been uncovered. Defective virus, exemplified by the vesicular stomatitis virus (VSV) defective interfering particle (DI 0.33), induced in BALB/c mice low levels of proliferating, IL-2 secreting, and cytolytic Ag-specific T lymphocytes. This was not caused by a dominant suppressor cell response, or by a failure to stimulate lymphokine-secreting cells, but appeared to reflect a reduced efficiency of priming as compared with standard virus. Mice primed with a mixture of wt and DI virus showed reduced proliferation compared with mice primed with wt virus. When histocompatible target cells were sensitized by pure DI particles, they were neither recognized nor lysed by CD8+ CTL. Cells co-infected with wt and DI particles were not as readily lysed by CD8+ CTL as cells infected by VSV alone. The extent of this reduction was dependent on the concentration of DI particles. This suggests that DI particles may have prevented the proper presentation of endogenously synthesized Ag for recognition by CD8+ CTL. Metabolic labeling studies indicated that the presence of DI particles suppressed the synthesis of viral proteins in dually infected cells. However, CD4+ T lymphocyte clones recognized and efficiently lysed histocompatible Ia+ cells infected with DI particles alone or co-infected with replication-competent and defective virus.
BALB/c-H-2dm2 mice (H-2KdI-AdI-EdDd), a congenic strain of BALB/c mice, have a deletion of the class I MHC Ag, H-2Ld. This gene encodes the exclusive class I MHC-restricting gene product for vesicular stomatitis virus-specific cytolytic T lymphocytes. When dm2 mice were immunized with infectious vesicular stomatitis virus, a specific CTL response was generated. These CTL lysed VSV-infected targets that expressed Iad gene products, but not VSV-infected Iad- targets. The CTL were used initially as long term cytolytic lines; 13 CTL clones were derived by limit dilution. All of the clones expressed the phenotype CD3+, CD4+, CD8-; some clones expressed TCR that are members of the V beta 8 family, others did not. The clones were restricted by class II MHC Ag, both I-Ad and I-Ed serving as restricting elements for individual clones of the panel. All of the clones derived from dm2 mice were specific for the immunizing serotype, Indiana, of VSV and did not lyse syngeneic cells infected with VSV of the New Jersey serotype. Studies using defective interfering virus particles, UV light-inactivated virus, and purified micelles of the viral glycoprotein indicated that infectious virus was not required for sensitization of target cells for immune recognition by the class II MHC-restricted CTL clones. Additional studies using recombinant vaccinia virus vectors to sensitize targets confirmed the specificity of the clones for the viral glycoprotein. These studies also demonstrated a cryptic population of class II-restricted CTL in BALB/c lines specific for VSV G. Naturally occurring variant viruses and mutant viruses, selected for escape from neutralization by mAb, were used in an effort to map the determinant(s) recognized; on the basis of patterns of target cell lysis, three groups of epitopes recognized by the clones were defined. Therefore, in the absence of the class I MHC Ag required for a CTL response to VSV, dm2 mice generated CTL with the CD4+ phenotype that recognized different epitopes on the viral glycoprotein, and lysed cells in a class II-MHC restricted, Ag-specific manner.
A six-month-old British female, living in Glasgow was admitted in June 1986 with a four-day history of fever and lower limb weakness following immunisation with oral polio and triple (DTP) vaccines. Examination revealed paralysis of all limbs, facial muscles and right diaphragm, scoliosis, opsoclonus and ocular flutter. Poliovirus types 1, 2 and 3, isolated from her stool specimens were all vaccine-like strains. Her serial serum IgA levels were persistently low and salivary IgA was undetectable. This appears to be the first fully authenticated case of poliovaccine damage in Scotland. It is unclear whether the selective IgA deficiency contributed to her vulnerability. It is essential to investigate elaborately and process viral isolates in every suspected case of acute poliomyelitis so as to determine the dimension and ramifications of poliovaccine damage in the UK population which is known to be rather apprehensive about vaccine dangers.
We have investigated the genetic basis of oral tolerance to OVA in a number of inbred mouse strains. Our results emphasise the efficiency of the oral route for inducing tolerance and provide evidence for both MHC and non-MHC linked control of oral tolerance.
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The pharmacokinetics of a single intravenous dose of cefotetan were studied in 17 volunteer patients with end-stage renal failure, requiring intermittent haemodialysis in 12 cases or undergoing continuous ambulatory peritoneal dialysis in 5 cases. Between haemodialysis the mean plasma elimination half life was 20.4 h (S.E.M. +/- 2.1). This decreased to 7.5 h (S.E.M. +/- 0.6) during haemodialysis. In patients treated by continuous ambulatory peritoneal dialysis the mean plasma elimination half life was 15.5 h (S.E.M. +/- 1.9). Small amounts of cefotetan (5-9% of the administered dose) were recovered in the peritoneal dialysates removed over the 24 h following the dose.
Intraperitoneal absorption of human insulin (crb) and porcine insulin was examined in 9 diabetic patients with renal failure, and on Continuous Ambulatory Peritoneal Dialysis (CAPD). Serial blood sampling was performed to determine the characteristics of insulin absorption and glycaemic control. Four patients received successive equal doses of human and porcine insulins, 2 received different doses and 3 received human insulin only. Glycaemic control was similar after both insulins. Mean insulin levels were consistently higher after human insulin with significant differences at 90, 300 and 360 min. It is concluded that, if required, patients may be transferred from i.p. purified porcine to i.p. human insulin without a change in insulin dose.
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The aetiology, clinical course and affected organs were studied in 124 patients with acquired systemic amyloidosis and seven patients with organ-limited amyloid deposits. Seventy-five patients had reactive systemic AA amyloidosis, which was associated with rheumatic disease in 55 and with chronic infection in 13 cases. Forty-nine patients had systemic AL amyloidosis. Thirteen of these cases were associated with myelomatosis and 11 with non-malignant immunocyte dyscrasias. In 25 patients with systemic AL disease no immunocyte dyscrasia was identified. Renal involvement dominated the clinical course of both forms of systemic amyloidosis, and renal failure was the most common cause of death. Gastrointestinal disturbance and hepatosplenomegaly were found in both AA and AL disease, although differences were noted in the distribution of amyloid protein within rectal biopsies. Amyloid cardiomyopathy, neuropathy and macroglossia were present in patients with AL amyloidosis only. These clinical patterns were reflected by tissue distribution at necropsy in 67 patients.
Three patients with end-stage renal failure complicating systemic amyloidosis have been treated with continuous ambulatory peritoneal dialysis for periods of 10, 14 and 18 months respectively. In each case satisfactory control of uraemia and fluid balance has been achieved.