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Biomedical subjects

R F James

Publications and source records attributed to R F James.

At least 127 records · Page 7Linked to original sources
PubMed1989

Monoclonal antibodies to human secretory "pregnancy-associated endometrial alpha 1-globulin," an insulin-like growth factor binding protein: characterization and use in radioimmunoassay, Western blots, and immunohistochemistry.

Monoclonal antibodies were raised against pregnancy-associated endometrial alpha 1-globulin (alpha 1-PEG), a 32 KD insulin-like growth factor binding protein (IGF-BP), which represents a major secretory product of the human decidualized endometrium during pregnancy. This class of IGF-BP has been implicated in the modulation of action, inhibitory and stimulatory, of insulin-like growth factors. Immunization with the protein purified from pregnancy endometrium resulted after myeloma fusion in the isolation of six hybridoma clones and the antibodies produced were characterized. The Ka of the antibodies ranged between 4.75 x 10(9) M-1 and 0.7 x 10(8) M-1. In Western blots all monoclonal antibodies reacted with purified protein of molecular weight 32 KD and specifically detected this IGF-BP species in culture medium and cytosolic extracts of pregnancy endometrium and amniotic fluid. The monoclonal antibodies appear to define three epitope-bearing regions as evidenced by their reactivity to polypeptide fragments of the protein. After synthesis and secretion by tissue explants in vitro the protein is susceptible to cleavage into fragments possessing different monoclonal antibody-defined reactivity. Employing immunohistochemical techniques the protein was principally localized to decidual cells in tissue sections of pregnancy endometrium and solely to these cells after enzymic digestion of the tissue. The implications of these results are discussed with respect to potential role of IGF-BP in the action of IGF upon the IGF-1 receptor-bearing populations, including lymphocytes and trophoblast cells, D in the decidua.

Antibodies, Monoclonal↗
PubMed1989

Subarachnoid haemorrhage in Plasmodium falciparum malaria.

A 41 year old man with cerebral malaria was found to have numerous bilateral retinal haemorrhages and very high parasitaemia. Despite intensive treatment his condition deteriorated and he died. Autopsy showed subarachnoid haemorrhage, which has not been previously described in cerebral malaria.

Adult↗
PubMed1989

Human 'pregnancy-associated endometrial alpha 1-globulin', an insulin-like growth factor-binding protein: immunohistological localization in the decidua and placenta during pregnancy employing monoclonal antibodies.

We have previously shown that pregnancy-associated endometrial alpha 1-globulin, a small molecular weight insulin-like growth factor-binding protein (IGF-BP), is quantitatively the major secretory protein product of the decidualized endometrium during human pregnancy. In the present study, employing monoclonal antibodies raised against this protein in an immunohistological technique, the cellular localization of the protein has been examined in the decidua and placenta during pregnancy. During the first trimester the protein was principally associated with the decidual cell in the decidualized decidua compacta region of the endometrium with both cytoplasmic and extracellular matrix-associated staining patterns being detected. No extensive staining was observed in the placenta. At term the protein was localized in similar cells in the placental bed and endometrium associated with the amniochorion but not in the placenta. These studies suggest that the decidual cell represents the major source of IGF-BP during pregnancy and have relevance to the origin of amniotic fluid IGF-BP and the paracrine role of the decidual cell in the control of trophoblast growth.

Antibodies, Monoclonal↗
PubMed1989

Successful reversal of diabetes in nude rats by transplantation of isolated adult human islets of Langerhans.

A method is described in which the viability of isolated adult human islets of Langerhans can be assessed in vivo. The Rowett nude rat, made diabetic with streptozocin (STZ), has been used as the islet recipient in these studies. Although these animals are athymic and are able to accept xenogeneic grafts for prolonged periods, they are very susceptible to dehydration and infection once made diabetic. Therefore, a considerably shortened diabetes induction period was used. The basis of the study was to prepare pure adult human pancreatic islets that were cultured for 48 h. Nude rats were given 80 mg/kg i.v. STZ during islet isolation and were transplanted with 800-1000 islets under the renal capsule at 48 h. To monitor islet function, animals were bled regularly for random blood glucose measurements and were given a glucose tolerance test at day 20. The kidney containing the graft was removed on day 21 to allow histological assessment of the graft and to confirm that glucose control was due to the transplanted islets and was not secondary to reversion of the animal's own islets. Seven rats were transplanted, and five were deemed to have received viable human islets. Two rats that received islets from the same donor did not reverse their diabetes and were found by histology to have vacuolated islet structures with scant insulin-staining tissue under the kidney capsule. This method allows a definitive judgment of the ability of isolated adult human islets to reverse diabetes.

Adult↗
PubMed1989

A microfluorometric viability assay for isolated human and rat islets of Langerhans.

A microfluorometric viability assay for isolated human and rat islets of Langerhans has been developed using the fluorochromes fluorescein diacetate and propidium iodide. Fluoroscein diacetate causes live cells to fluoresce green under blue light excitation (490 nm); propidium iodide causes dead cells to fluoresce red under green light excitation (545 nm). The fluorescence intensity from the live and dead cells within a single islet was selectively measured by photometry using 520 nm and 610 nm barrier filters with blue and green light excitation respectively. All measurements were corrected for background fluorescence. It was necessary to incubate single islets with the fluorochrome mixture for 105 min in order to achieve maximum fluorescence intensity. It was found that when 50 microliters of a fluorochrome mixture containing 0.67 mumol/l fluorescein diacetate and 4.0 mumol/l propidium iodide was incubated with a single islet and the fluorescence from live (blue light excitation) and dead (green light excitation) cells measured, then the proportion of dead cells within the islet was equal to the (propidium iodide fluorescence)--(0.04 x fluorescein fluorescence) divided by the sum of the (fluorescein fluorescence) and (propidium iodide fluorescence--(0.04 x fluorescein fluorescence]. The proportion of dead cells within single human or rat islets measured by microfluorometry was found to correlate highly significantly (r = 0.99, p less than 0.001) with the proportion of dead cells measured by dissociating the same islet into a single cell suspension and counting the actual proportion of dead cells. This assay therefore provides a rapid, accurate and objective measurement of the proportion of dead cells within isolated human and rat islets.

Animals↗
PubMed1988

Do CD4-positive cytotoxic T cells damage islet beta cells in type 1 diabetes?

The mechanism by which islet beta cells are destroyed in type 1 diabetes is still unknown. Because in diabetes the majority of T cells activated in vivo express CD4 and the islet beta cells selectively express the HLA class II antigens needed for recognition by CD4-positive T cells, the possibility that selective damage to islet beta cells may be caused by CD4-positive cytotoxic cells was investigated. Activated T cells were cloned from a newly diagnosed diabetic patient, and many CD4 cytotoxic clones were detected. The clone with the highest cytolytic capacity lysed HLA class II compatible islet cells which had been induced by interferon gamma and tumour necrosis factor to express class II antigens. The specificity of the lysis was demonstrated by use of histoincompatible islets, other histocompatible target cells, and blocking by anti-class-II monoclonal antibodies. The results show that a CD4-positive T cell clone can lyse HLA class II matched islet cells; this process may be important in the pathogenesis of type 1 diabetes.

Adult↗
PubMed1988

In vivo assessment of isolated pancreatic islet viability using the streptozotocin-induced diabetic nude rat.

In order to establish a model for the in vivo assessment of islet function we have used the Rowett nude rat with transplantation of allogeneic and xenogeneic (mouse) islets into the renal subcapsular space following a minimal period of diabetic induction. Thirty-one nude rats were given streptozotocin and 30 became diabetic with blood glucose levels of greater than 20 mmol/l at 48 h. Rat and mouse islets were prepared by intraductal collagenase and bovine serum albumin density gradient isolation. Eight rats received transplants of freshly prepared allogeneic islets and 8 rats received transplants of 48 h cultured allogeneic islets. Seven rats received transplants of 48 h cultured mouse islets. Diabetes was reversed in all animals and all remained normoglycaemic for 21 days. Graft removal by nephrectomy resulted in hyperglycaemia in 22 out of 23 animals. Histological examination of the grafts showed a band of endocrine tissue beneath the renal capsule which stained strongly positive for insulin and there was no evidence of lymphocytic infiltration/rejection. One rat remained normoglycaemic after graft removal, which may represent recovery of the animal's own islets from the streptozotocin-induced diabetes. Control rats remained diabetic until death. In conclusion, the athymic nude rat can be used for the assessment of allogeneic and xenogeneic islet function when a short (48 h) period of streptozotocin-induced diabetes is used. This model offers a potential method for assessing in vivo function of isolated human islets.

Animals↗
PubMed1988

Dietary fat in relation to fatty acid composition of red cells and adipose tissue in colorectal cancer.

Fatty acids were determined in erthrocytes in 49 patients with colorectal cancer and compared with age and sex-matched controls. Marginally increased levels of stearic acid (P = 0.057) and oleic acid (P = 0.064) and decreased arachidonic acid (P = 0.043) occurred in cancer patients. There was no difference in the stearic to oleic acid ratio between the two groups. Dietary intake, assessed by dietary recall and adipose tissue analysis was also not different. In control subjects the polyunsaturated:saturated (P:S) fatty acid ratio correlated between diet and adipose tissue (P less than 0.01, at least). In contrast cancer patients showed different correlations; in particular dietary and erythrocyte P:S fatty acid ratios correlated (P less than 0.01). These findings may indicate disturbed fat metabolism in cancer patients. The erythrocyte stearic to oleic acid ratio is of no diagnostic value.

Adenocarcinoma↗
PubMed1988

Immunohistological localization of the human endometrial secretory protein pregnancy-associated endometrial alpha 1-globulin, an insulin-like growth factor-binding protein, during the menstrual cycle.

Pregnancy-associated endometrial alpha 1-globulin (alpha 1-PEG), a 29,000 mol wt insulin-like growth factor-binding protein, is the major secretory protein of the human endometrium from the latter half of the first trimester to the end of pregnancy. It is also produced and secreted by nonpregnant endometrial tissue, especially during the late secretory phase of the menstrual cycle. We studied the localization of this protein in the endometrium during different phases of the cycle using immunohistochemistry with monoclonal antibodies. Immunostaining was first observed in the midsecretory phase and was most consistently detected during the late secretory phase, during which it was principally associated with stromal cell populations. These cells were localized initially surrounding spiral arteries and subsequently also in the subluminal epithelial region of the endometrium. These results suggest that alpha 1-PEG synthesis is principally associated with the process of stromal cell differentiation. i.e. predecidualization. However, the presence of alpha 1-PEG was not directly correlated with the presence of mature predecidual cells, suggesting that its synthesis is not an inherent feature of this cell.

Adult↗
PubMed1988

Glycosylated haemoglobin: an index of glucose control in pancreatic islet transplanted rats.

In this study we have measured glycosylated haemoglobin in the blood of 22 rats made diabetic with streptozotocin. This significantly increased from a normal level of 1.6 +/- 0.2% to reach a maximum, plateau level, of 6.6 +/- 0.3% at 45 days. At this time, 16 of these animals were given intra-portal transplants of purified isologous pancreatic islets. By varying the number of islets transplanted, animals were produced with normal blood glucose levels on random sampling, but with a spectrum of glucose control as measured by the glucose tolerance test. The glycosylated haemoglobin level was also measured at 30, 60 and 120 days post transplantation. This was significantly reduced (in some cases back to normal) and at 60 and 120 days there was a significant correlation (p less than 0.001) between glycosylated haemoglobin and the glucose clearance (K value). These results suggest that the measurement of glycosylated haemoglobin can be used as a simple and accurate index of glucose metabolism in islet transplanted rats.

Animals↗
PubMed1988

Inhibition of expression of cell-mediated immunity by a cell surface-binding antibody directed against T-cell helper factors.

In this study, the effects of a monoclonal antibody, AF3.44.4, which is directed against antigen-specific, T-cell-derived helper factors, were investigated in two models of T-cell-macrophage co-operation. In vitro, cultures of nylon-wool-enriched peritoneal cells from mice immune to Listeria monocytogenes (T cells), macrophages from normal mice and Listeria antigen produced an activity mitogenic for mouse thymocytes (TMF). When AF3.44.4 was added to such cultures, the production of this activity was inhibited in a dose-dependent fashion. Preincubation of T cells with AF3.44.4, followed by its removal, was not sufficient for the inhibitory effect to be manifested. In an in vivo system, when spleen cells from Listeria-immunized mice were transferred to unprimed mice, the latter were protected against subsequent challenge with Listeria. Treatment of spleen cells with AF3.44.4 prior to transfer did not affect their ability to confer resistance upon recipients. It is postulated that AF3.44.4 acts on a population of T cells that is involved in macrophage-T-cell co-operation in vitro and that this population may be different from that involved in macrophage T-cell co-operation in vivo.

Animals↗
PubMed1987

Bovine serum albumin density gradient isolation of rat pancreatic islets.

The use of a bovine serum albumin (BSA) density gradient for isolation of rat pancreatic Islets of Langerhans after collagenase digestion has been compared with the standard Ficoll separation technique. The criteria studied were islet yield (insulin extraction of the islet interfaces and pellet), purity of preparation (amylase content of the islet preparation), insulin release characteristics, and the result of isologous transplantation in diabetic rats. The islet interface of the BSA gradient contained 62.2% of the total insulin, whereas the corresponding interface of the Ficoll gradient contained only 36.6% (P less than 0.001). The amylase content of the Ficoll-separated islet preparation was 6133 U/L, as opposed to 1230 U/L with BSA (P less than 0.001). BSA-isolated islets gave similar insulin release characteristics to non-density-gradient-isolated islets, whereas Ficoll-separated islets showed suboptimal insulin release. Single-donor-single-recipient transplantation was successfully performed with BSA-isolated islets whereas multiple donors were required with Ficoll-separated islets. Thus significantly improved results were found with the bovine serum albumin density gradient separation in all criteria and consequently the use of this gradient represents an advance in islet isolation techniques.

Animals↗