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A G Pockley

Publications and source records attributed to A G Pockley.

At least 55 records · Page 3Linked to original sources
PubMed1995

Flow cytometric analysis of peripheral blood lymphocyte subset light scatter characteristics as a means of monitoring the development of rat small bowel allograft rejection.

This investigation used flow cytometry to monitor peripheral blood lymphocyte morphology after rat small bowel transplantation. Preliminary studies demonstrated that in vitro activated peripheral blood lymphocytes exhibited increased cell size and granularity as measured by flow cytometric analysis of forward (FSc) and side (SSc) light scatter characteristics. The formation of distinct 'activated' light scatter regions by such lymphoblastoid transformation occurred concomitantly with up-regulated p55IL-2R expression. Heterotopic small bowel transplantation was performed between PVG donor and DA recipient rats without immunosuppression. Animals receiving isografts served as controls. Peripheral blood lymphocyte subsets were identified using appropriate MoAbs, and the light scatter characteristics of each cell subset were determined by backgating strategies. Increased proportions of activated alpha/beta T cell receptor (TCR)-positive cells could be detected in allografted animals as early as day 2 post-transplantation. B cells showed peak activation by day 4, at which time the proportion of activated cells was over two-fold greater than that seen in untransplanted animals--few activated B cells were detected in isografted animals. Resting natural killer (NK) cell light scatter regions only partially overlap with those of resting T and B lymphocytes, but in allografted animals almost the entire NK population fell outside the resting lymphocyte gate by day 2 post-transplantation, an activation state which was maintained until day 4. These findings associate peripheral blood cell subset lymphoblastoid transformation with developing small bowel allograft rejection. Importantly, changes were detected early and prior to the onset of overt rejection. These data suggest that analysis of peripheral blood lymphocyte light scatter properties may provide an insight into in vivo immune status after small bowel transplantation.

Animals↗
PubMed1995

An enzyme immunoassay for rat soluble MHC class I molecules (RT1a) and the release of soluble class I from mitogenically stimulated mononuclear cells.

Soluble MHC class I antigens can be detected in the serum of humans and various animals and appear in the circulation shortly after liver transplantation. The precise role of these antigens is currently uncertain, but soluble MHC class I may be involved in immunomodulation. We have developed an enzyme linked immunosorbent assay for soluble rat MHC class I (RT1a) molecules and monitored the kinetics of antigen release following in vitro stimulation of splenic mononuclear cells. A 4 day DA splenocyte Con A supernatant provided a source of soluble class I antigens and was arbitrarily assigned a concentration of 1000 units/ml. Ninety six well plates were coated with a rat RT1a-specific mAb (MN4-91-6) and soluble class I binding was detected using a biotinylated mAb reactive with a monomorphic region of the rat MHC class I molecule (OX18) followed by a streptavidin-alkaline phosphatase conjugate and substrate. The intra- and interassay variations were typically less than 5% and 10% respectively, to give a working range for the assay of between 62.5 and 1000 units/ml. Mitogenic stimulation led to a progressive increase in soluble class I levels in culture supernatants. This assay will be valuable in differentiating recipient and graft responses following experimental organ transplantation.

Animals↗
PubMed1994

Activation antigen expression on peripheral blood neutrophils following rat small bowel transplantation. NKR-P1 is a novel antigen preferentially expressed during allograft rejection.

This study used flow cytometric analyses to monitor activation antigen expression (MHC class II; interleukin-2 receptor, p55IL-2R and 3.2.3/NKR-P1 antigen) on peripheral blood neutrophils following rat small bowel transplantation. The rat 3.2.3 antigen is a member of the NKR-P1 family of natural killer (NK) cell-associated molecules, which are expressed at high levels on NK cells and lymphokine-activated killer cells, and low levels on at least one T cell subset. Peripheral blood neutrophils in normal animals express very low or undetectable levels of NKR-P1. Detectable levels of NKR-P1 were induced as early as day 1 following small bowel transplantation in all allografted animals, whereas expression was only rarely detected in isografted animals. In addition, NKR-P1 density was significantly higher in allografted animals and was maintained as rejection developed. MHC class II and p55IL-2R expression was also induced following transplantation. The mechanisms of induction and functional relevance of NKR-P1 expression on neutrophils remain to be defined. However, the concomitant increased expression of MHC class II and p55IL-2R suggest NKR-P1 to be a neutrophil activation marker and implicate a potential role for NKR-P1+ neutrophils in small bowel allograft rejection. This hypothesis is further supported by the loss of detectable peripheral blood neutrophils only with developing rejection. Flow cytometric analysis of neutrophil activation antigen expression may be useful for monitoring human small bowel transplant recipients.

Animals↗
PubMed1994

Effect of small bowel transplantation, denervation and ischaemia on rat intestinal microflora.

The effects of denervation and warm ischaemia on quantitative and qualitative changes in small intestinal microflora following rat heterotopic small-bowel isotransplantation were assessed. Animals with Thiry-Vella fistula, but without transplants, acted as controls. Thirty and 40-fold increases in bacterial colony counts were seen in the isografts compared to controls at 2 and 7 days, respectively (P < 0.05). Aerobic faecal organisms predominated at 2 and 7 days, but an overgrowth of Flavobacterium meningosepticum occurred at 28 days in the transplanted and host bowels. The effect of warm ischaemia on intestinal microflora was assessed by the application of a microvascular clamp to the superior mesenteric artery for 90 min. The effect of denervation was assessed following microsurgical division of all nervous tissue around the superior mesenteric artery. After 7 days, lengths of jejunum and ileum were removed and intraluminal microflora assessed. The number of bacterial colonies isolated from the ileum in the warm ischaemia group was six times greater than the number in the control group, whereas no significant changes were seen in the upper bowel. In contrast, denervation led to a slight, but consistent, decrease in colony counts. These findings suggest that the increase in bacterial numbers in an isografted small bowel primarily results from warm ischaemia rather than from mesenteric denervation, and that physical aspects of the procedure may affect the development of sepsis following small-bowel transplantation.

Animals↗
PubMed1993

Effect of red cell lysis protocols on the expression of rat peripheral blood lymphocyte subset and activation antigens.

The effect of ammonium chloride and a commercial red cell lysing agent (Erythrolyse) on cell subset (CD4, CD8, B cell common leucocyte antigen) and activation (p55IL-2R [OX39], MHC class II) antigen expression by freshly isolated and in vitro activated rat peripheral blood lymphocytes was compared. Freshly isolated cells treated with the commercial lysing agent Erythrolyse were 42 +/- 12% p55IL-2R+, whereas p55IL-2R expression was not detected following red cell lysis with ammonium chloride. The expression of cell subset and MHC class II antigens was unaffected by either of the treatment protocols. Density gradient separation of freshly isolated cells had variable effects on antigen expression. In some cases up to a two-fold increase in p55IL-2R expression was observed, whilst in others there was a reduced receptor expression. Similarly, MHC class II antigen expression was either increased or reduced following density gradient separation. IL-2R expression on cells activated in vitro with the mitogen concanavalin A (95 +/- 2% IL-2R+) was unaffected by ammonium chloride lysis. Incubation of freshly isolated lymphocytes with p55IL-2R antibody prior to ammonium chloride treatment did not prevent the reduction in receptor expression. These findings suggest that ammonium chloride lysis of freshly isolated rat peripheral blood prior to IL-2R expression analysis should be avoided.

Ammonium Chloride↗
PubMed1993

Method for diagnosing rejection in small bowel transplantation.

Diagnosis of rejection in small bowel transplantation by the identification of a host-cell infiltrate is hampered by the physiological trafficking of host lymphocytes to the 'gut-associated lymphoid tissue' of the graft. This study compared physiological host-cell infiltration of small bowel grafts with that occurring in rejection and stable immunosuppression. Physiological host-cell infiltration, where the graft does not present an immune stimulus to the host, was assessed by transplanting bowel from DA to (DA x PVG) F1 hybrid rats. The extent of host-cell infiltration was determined by immunohistochemical analysis. In the lamina propria, considerable infiltration by host cells was seen, although it was significantly less than that in rejection or stable immunosuppression. By contrast, host cells were seen in the intraepithelial compartment only in rejection. Host-cell infiltration in the absence of an allogeneic stimulus suggests that histological identification of host cells in the lamina propria is not necessarily indicative of rejection. However, the presence of host cells in the intraepithelial compartment is specific for rejection in small bowel transplantation.

Animals↗
PubMed1993

The effect of rejection and graft-versus-host disease on small intestinal microflora and bacterial translocation after rat small bowel transplantation.

Bacterial translocation and the development of sepsis after small bowel transplantation may be promoted by immunological damage to the intestinal mucosa or by quantitative and qualitative changes in intestinal microflora. This study assessed the effects of rejection, graft-versus-host disease (GVHD) and immunosuppression on intestinal microflora and bacterial translocation after heterotopic rat small bowel transplantation. Isografts, allografts with and without CsA immunosuppression, and the semi-allogeneic parent to the F1 hybrid GVHD model were studied. Intestinal microflora in graft and host loops and bacterial translocation to host organs and the graft mesenteric lymph node were determined. Bacterial colonies were counted and individual colonies identified using API 20E nutrient and fermentation indicator techniques. Colony counts in isografts and allografts were significantly higher than in the native intestine, whereas there was a massive overgrowth in the native intestine in the GVHD group. The species profile for the host and graft loops was similar in animals that had received isografts, allografted animals receiving CsA, and animals undergoing GVHD. However, there was a large increase in Staphylococcus epidermidis in animals with rejection. Bacterial translocation was not detected in isografted animals, but was observed in all other animal groups, with S. epidermidis being the most prevalent organism. These findings demonstrate that rejection and GVHD are associated with shifts in intestinal microflora toward potentially pathogenic organisms and that bacterial translocation into recipient tissues poses a major threat for the development of sepsis.

Animals↗