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Biomedical subjects

R F James

Publications and source records attributed to R F James.

At least 109 records · Page 6Linked to original sources
PubMed1991

Cytotoxic effect of IFN-gamma plus TNF-alpha on human islet cells.

We have previously reported that the combination of IFN-gamma plus TNF-alpha is able to induce the de novo expression of HLA class II on human beta cells. In the present study, we have investigated the effect of these cytokines, alone or in combination, on the function and viability of human islet cells in vitro. Three hour insulin release was markedly reduced in human islet monolayer cultures after 4 days' exposure to 1000 U/ml of the combination TNF-alpha plus IFN-gamma (36.7 +/- 7.7, % of the control +/- SEM) or to TNF-alpha alone (49.5 +/- 7% of the control) while IFN-gamma had little effect. On direct inspection cell damage was clearly detected only in the cultures treated with TNF-alpha plus IFN-gamma in which staining by indirect immunofluorescence (IFL) for insulin revealed that the number of beta cells was also significantly reduced, thus suggesting a real cytotoxic effect of this cytokine combination. This effect was not beta cell specific since glucagon release and the number of alpha cells were also reduced in the cultures exposed to IFN-gamma plus TNF-alpha. 51Cr release experiments supported the cytoxicity of these cytokines to normal islet cells. There was a time course relationship between class II induction (2 days) and the cytotoxic effect of IFN-gamma plus TNF-alpha (4 days) on the same islet cells. In conclusion, these results indicate that the combination of IFN-gamma and TNF-alpha exerts a cytotoxic effect on human islet cells in vitro.

Cell Survival↗
PubMed1991

Transfection with SV40 gene of human pancreatic endocrine cells.

At present, only islet cell lines of animal origin have been successfully generated (e.g. RIN, HIT). A fully differentiated human beta cell line would be advantageous for diabetes research. We now report the generation of a human endocrine pancreatic cell line obtained by transfection using a plasmid containing the early region of SV40 viral DNA. Viral integration and transcription was assessed by Southern and Northern blotting. This cell line has been growing continuously for more than 2 years and maintains several of the characteristics of the parental cells from which they were generated. The presence of Neuron Specific Enolase, Protein Gene Product 9.5, cytokeratin, microvilli, cytoplasmic electrodense granules and the secretion of insulin, glucagon and somatostatin supports the neuroendocrine origin of this cell line. However, hormone production progressively decreased and finally stopped at passage 8. Flow cytometric analysis showed that HLA expression in this cell line is readily induced by IFN-gamma and modulated by TNF-alpha. The establishment of this human endocrine cell line indicates the feasibility of immortalizing human islets by transfection with viral oncogenes. To obtain a fully differentiated cell line it may be necessary to use other DNA constructs which immortalize the cells without fully transforming their phenotype.

Antigens, Viral, Tumor↗
PubMed1991

Renal subcapsular islet transplantation in the rat: a comparison of three techniques.

The purpose of this study was to investigate whether the results of renal subcapsular islet transplantation in the rat could be improved by placing the islets in a blood or plasma clot. Islets were isolated and transplanted into diabetic recipients using a standardized technique and islet function post transplantation measured by the ability to restore normoglycemia and by intravenous glucose tolerance testing. It was found that 750 islets in minimal essential medium reversed diabetes (6/6 recipients) and produced a normal IVGTT whereas the success rate if the islets were placed in a blood or plasma clot was only 0/6 and 1/6 recipients respectively (p less than 0.008). We conclude that any possible benefit from trophic factors within blood or plasma is outweighed by the fact that islets in a blood or plasma clot are distanced from the blood supply on the kidney surface. This finding has important implications for human islet transplantation.

Animals↗
PubMed1991

The effect of class II gene transfection on the tumourigenicity of the H-2K-negative mouse leukaemia cell line K36.16.

There is much evidence to suggest that potentially immunogenic tumour cells can escape cytolytic immune destruction by loss of class I antigen expression. Many tumours are allele-specific class I negative and, in murine systems, reconstitution of class I expression by gene transfection leads to an increase in tumour immunogenicity. In many systems where mice have rejected class I transfected tumour cells they are also immune to a subsequent challenge with the untransfected parent tumour. In this study we have examined the effect of stable class II antigen expression (induced by gene transfection) on a class I loss mutant (H-2Kk negative) murine cell line, K36.16. We show that H-2Ek expression is more effective at increasing tumour immunogenicity than the reconstitution of H-2Kk expression in these cells. This suggests that the induction of class II antigen expression on tumour cells may provide an effective way of enhancing tumour-specific immune responses in vivo.

Animals↗
PubMed1990

The cryobiology of rat and human dendritic cells: preservation and destruction of membrane integrity by freezing.

Dendritic cells (DCs) are now regarded as specialized leucocytes with distinctive morphological and functional characteristics as accessory or stimulator cells for many lymphocyte responses. While knowledge of the response of other leucocytes (e.g., lymphocytes, macrophages, and granulocytes) to freezing and thawing has been established for some years, an understanding of the cryobiological properties of DCs has not, hitherto, been determined specifically. Such information is important both for establishing procedures for the long-term storage of these cells for use in immunological procedures and for defining freezing conditions that might selectively kill DCs in attempts to modulate the immunogenicity of transplantable tissues during cryopreservation. Preparations of rat and human spleen cells enriched for DCs were frozen to -60 degrees C at one of six cooling rates (0.3, 1.5, 10, 20, 70, or 150 degrees C/min) using a procedure that was established for pancreatic islets with 2 M dimethyl sulfoxide (Me2SO) as the cryoprotectant. Following storage at -196 degrees C the survival of thawed cells was assessed by evaluating both the numbers of cells recovered after the complete process and the membrane integrity of the recovered cells using a supravital fluorescent probe assay. Survival profiles for DCs showed a dependence upon cooling rate similar to other lymphoid cells but DCs were more sensitive to freezing injury than either lymphocytes or macrophages: Optimum survival (75% recovery of numbers and 57% membrane integrity) of rat DCs was achieved by slow cooling (0.3 degrees C/min). Optimal recovery of human DCs was significantly higher (83% recovery of numbers and 72% membrane integrity) after cooling at either 0.3 or 1.5 degrees C/min. The viable yield of DCs from both species declined abruptly as cooling rate was increased, with less than 10% survival after cooling at 20 degrees C/min and negligible survival after cooling at 70 degrees C/min or greater. Analysis of variance of the survival data showed that the response of DCs to freezing and thawing was significantly different (P less than 0.005) from that of either lymphocytes or macrophages, thus providing additional evidence that DCs are distinct from other leucocytes, especially macrophages. This study defines conditions that either will provide effective cryopreservation of DCs for immunological purposes or are most likely to bring about their inactivation in cryobiological approaches to modulating tissue immunogenicity.

Animals↗
PubMed1990

A simple method for the release of islets by controlled collagenase digestion of the human pancreas.

A simple technique for the controlled collagenase digestion of the human pancreas is described. The pancreas is distended with collagenase, and a biopsy taken and divided into 5 pieces that are placed in Universals containing minimal essential medium and dithizone at 39 degrees C. The pancreas itself is incubated in MEM at 39 degrees C. Starting at 5 min and at intervals thereafter, a Universal is removed from the water bath, shaken for 30 sec, and the contents examined by microscopy. As soon as free cleaved islets are seen, the pancreas is placed into one compartment of a kidney-bowl divided in half by a 1-mm mesh. The pancreas is gently teased apart and fluid digest in the empty half of the bowl aspirated and passed through a 500-micron mesh into ice-cold MEM containing 20% newborn calf serum. This process is repeated until the digestion process has ceased. Using this technique on 20 consecutive pancreata, median wt. (range) 53.9 (45.2-72.9) g, we have counted 131,672 (43,516-400,000) islets in the digest, equivalent to 2394 (715-8000) islets/g pancreas. The volume of islet tissue in the digest was 299 (26-1341) mm3 equivalent to 5.81 (0.36-26.81) mm3/g pancreas. In conclusion, we have found this simple technique to be an effective method for the controlled collagenase digestion of the human pancreas.

Adolescent↗
PubMed1990

Differential effects of beta-adrenergic agonists on insulin secretion from pancreatic islets isolated from rat and man.

The selective beta 2-adrenergic agonist clenbuterol was ineffective as a stimulus for insulin secretion when isolated rat pancreatic islets were incubated with glucose at concentrations between 4 and 20 mM. Inclusion of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine led to potentiation of glucose-induced insulin secretion, but did not facilitate stimulation by clenbuterol. Furthermore, maintenance of isolated rat islets for up to 3 days in tissue culture also failed to result in the appearance of a secretory response to beta-agonists. By contrast, clenbuterol induced a dose-dependent increase in insulin release from isolated human islets incubated with 20 mM glucose. Clenbuterol did not increase the basal rate of insulin secretion (4 mM glucose) in human islets. Under perifusion conditions, the secretory response of human islets to clenbuterol was rapid, of similar magnitude to that seen under static incubation conditions and could be sustained for at least 30 min. The increase in insulin secretion induced by clenbuterol was inhibited by propranolol, indicating that the response was mediated by activation of beta-receptors. In support of this, a similar enhancement of glucose-induced insulin secretion was elicited by a different beta 2-agonist, salbutamol, in human islets. The results indicate that the B cells of isolated rat islets are unresponsive to beta-agonists, whereas those of human islets are equipped with functional beta-receptors which can directly influence the rate of insulin secretion.

Adrenergic beta-Agonists↗
PubMed1990

Human 'pregnancy-associated endometrial alpha 1-globulin', a 32 kDa insulin-like growth factor-binding protein: immunohistological distribution and localization in the adult and fetus.

We have previously shown that pregnancy-associated alpha 1-globulin, a small molecular weight (32 kDa) insulin-like growth factor-binding protein (IGF-BP), is quantitatively the major secretory protein product of the decidualized endometrium during human pregnancy and is localized principally in the decidual cell. In the present study, employing monoclonal antibodies in immunohistological techniques, the distribution and localization of IGF-BP has been examined in normal and pathological tissues of the adult and first trimester fetus. In the adult, most intense reactivity was associated with endometrial stroma and their derived decidual cells in the uterus or in ectopic sites in patients with endometriosis. During the menstrual cycle, the appearance of IGF-BP in endometriotic tissue was linked with its appearance in uterine endometrial tissue. The only other adult cells where significant staining was detected was in the luteal cells of the corpus luteum. Production of the protein was not a feature of carcinomas. In the fetus, the protein was localized in lymphoid-myeloid progenitor cells and hepatocytes of the liver and at lower levels in testicular Leydig cells and adenocortical cells. These observations suggest highly specific tissue expression of this protein and support a specialized role for this protein in progenitor cells of the lymphomyeloid system, in certain steroid hormone-producing cells and in the decidual cell in pregnancy.

Endometrium↗
PubMed1990

A fluorometric viability assay for single human and rat islets.

A microfluorometric viability assay for isolated human and rat islets of Langerhans has been developed using the fluorochromes fluorescein diacetate and propidium iodide. Fluorescein 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 (454 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. 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↗
PubMed1990

Epidermal growth factor and transforming growth factor-alpha suppress HLA class II induction in human thyroid epithelial cells.

Inappropriate expression of HLA class II by human thyroid epithelial cells (thyrocytes) occurs in autoimmune thyroid diseases where it may contribute to the pathogenesis. Several substances have been found to induce or up-regulate thyrocyte HLA class II expression in vitro. The present investigations show that the induction of HLA class II in human thyrocytes cultured with interferon (IFN)-gamma can be partially suppressed by exposure of the thyrocytes to epidermal growth factor (EGF): this occurs when the thyrocytes are treated with the two reagents simultaneously and also when the exposure to EGF is before or after that with IFN-gamma. Concentrations of EGF at least as low as 0.1 ng/ml show this inhibitory effect, which can be over-ridden by very high concentrations of IFN-gamma. Thyrocyte HLA class II expression stimulated by thyroid-stimulating hormone (TSH) (in the presence or absence of IFN-gamma) is also suppressed by EGF. Transforming growth factor-alpha (TGF alpha), which is structurally related to EGF and interacts with the same cell surface receptor, has a similar inhibitory activity on the induction of thyrocyte HLA class II expression. The existence of substances which can down-regulate, as well as those which can up-regulate, thyrocyte HLA class II expression raises the possibility that the occurrence of such expression in vivo may be determined by the balance between factors with opposing modulatory effects.

Cells, Cultured↗
PubMed1990

A study of the effect of capsule composition on the viability of cultured alginate/poly-l-lysine--encapsulated rat islets.

Islets of Langerhans were isolated from WAGola rats and encapsulated in high mannuronic acid sodium alginate droplets. The capsules were completed in the following ways: (a) poly-l-lysine alone, (b) poly-l-lysine plus high guluronic acid alginate, (c) poly-l-lysine plus high mannuronic acid alginate. Islet viability was assessed every 3-4 days over a period of 4 weeks, by a microfluorometric assay using fluorescein diacetate and propidium iodide, and every 7 days by perifusion. The perifusion results were corrected for DNA content to take islet size differences into account. The results from the microfluorometric assay showed a generally high viability of the islets in all groups at each time interval. The perifusion experiments showed that the response time and stimulation indices of the islets was similar in all groups, but the absolute amount of insulin released was slightly lower from encapsulated islets relative to unencapsulated controls. It was concluded that the presence of a capsule, regardless of its composition, did not adversely affect the membrane integrity of the islets, their response time, nor their proportionate increase in insulin release following glucose stimulation. However, the absolute amount of insulin released from encapsulated islets was reduced during perifusion. This reduction was apparent after 1 week in culture, but remained stable thereafter.

Alginates↗
PubMed1990

The effect of varying fibronectin concentration on the attachment of endothelial cells to polytetrafluoroethylene vascular grafts.

Endothelial cell seeding onto untreated polytetrafluoroethylene vascular prostheses is inefficient. In an effort to improve cell attachment, numerous investigators have used fibronectin as a coating material to pretreat the luminal surfaces of these prostheses. The concentrations of fibronectin used have varied enormously, and no one has yet determined the most efficient concentration in terms of cell attachment and cost. Using endothelial cells labeled with indium 111 oxine we have studied the effect of varying fibronectin concentration on the attachment of these cells onto polytetrafluoroethylene vascular grafts. Seeding efficiency was significantly better in all groups of coated grafts, at all times (10, 30, 60, and 120 minutes), compared to uncoated controls (p less than 0.01). Overall, fibronectin at a concentration of 20 micrograms/ml was found to be the most efficient in terms of cell attachment and cost since any further increase in concentration was not accompanied by increased cell attachment. We now routinely use fibronectin at a concentration of 20 micrograms/ml to coat our grafts before endothelial cell seeding.

Blood Vessel Prosthesis↗
PubMed1989

Attachment of indium-111 labelled endothelial cells to pretreated polytetrafluoroethylene vascular grafts.

This study investigated the effect of different surface coatings on endothelial cell attachment to polytetrafluoroethylene (PTFE) vascular grafts. Small segments of PTFE vascular grafts were precoated with one of a number of substances: gelatin, poly-l-lysin, fibronectin, or collagen type I, III or IV. Indium-111 labelled endothelial cells were then seeded on to the grafts and left for either 10, 30, 60 or 120 min. The unattached cells were removed and the degree of cell attachment was calculated. All coatings were significantly better at enhancing endothelial cell attachment at all times compared with controls, and fibronectin was significantly better as a coating material than any other material used. By pretreating with a substance such as fibronectin, the number of endothelial cells attaching to PTFE vascular grafts can be greatly increased, thereby enhancing the cell seeding process.

Blood Vessel Prosthesis↗
PubMed1989

Gamma irradiation of isolated rat islets pretransplantation produces indefinite allograft survival in cyclosporine-treated recipients.

In this study we have examined the use of low-dose gamma-irradiation for the reduction of islet immunogenicity in the strong allogeneic combination of WAG rat islets transplanted into diabetic AUG recipients. First, we determined that gamma-irradiation reduced immunogenicity in vitro by use of a modified MLR with WAG islets as stimulators and AUG splenocytes as responders. We then determined the maximum dose of gamma-irradiation that could be used (250 rads) before islet function was affected. As 250 rads islet pretreatment alone was ineffective in prolonging allograft survival, we combined the pretreatment with a short course (days 0, 1, 2; 30 mg/kg) of cyclosporine. We found that CsA was only effective in significantly prolonging allograft survival when given subcutaneously in olive oil. The CsA treatment alone gave a significantly prolonged survival time for the islet allografts (median, 37 days vs. 6 days for controls), but when combined with the 250 rads islet pretreatment a synergistic effect was seen with 100% becoming long-term survivors (greater than 100 days). The long-term surviving AUG rats from both the CsA alone group and the CsA plus 250 rads pretreated islets group were challenged with WAG dendritic cells (DC). The islets from the 250 rads pretreated group were subsequently rejected (day 6) while the CsA alone group were not affected. The role of low dose gamma-irradiation when combined with CsA treatment of islet graft recipients in inducing specific unresponsiveness will be discussed.

Animals↗