MRC OX8 Research Articles

Reduction of graft-versus-host reactivity after small bowel transplantation: ex vivo treatment of intestinal allografts with an anti-T cell immunotoxin

A specific T lymphocyte immunotoxin was used to pre-treat small bowel grafts in an attempt to prevent graft-versus-host (GVH) reactivity and GVH disease in a rat transplant model. The immunotoxin used was a conjugate of the anti-CD5 MoAb MRC OX-19 with ricin A chain. The grafts were perfused ex vivo with a standard solution of immunotoxin followed by incubation at 4 degrees C for 1 h before transplantation. In a semi-allogeneic strain combination (parent to F1 hybrid offspring) graft treatment with immunotoxin led to a prolongation of recipient survival compared with groups receiving similar transplants without immunotoxin treatment. An additive effect on survival was observed when the host was treated with cyclosporin. The effect of immunotoxin was greater than that of mesenteric lymphadenectomy in increasing host survival. The effect of graft treatment with the immunotoxin on cellular migration from graft to host lymphoid tissues was assessed in fully allogeneic transplantation (PVG to DA). Host lymphoid tissues were subjected to immunohistochemical analysis using a MoAb specific for donor class I MHC antigens. Graft treatment with the immunotoxin led to a significant decrease in the number of graft cells found in host lymphoid tissues 7 days after transplantation. However, this effect was less marked than that achieved by graft mesenteric lymphadenectomy. With our current protocol graft treatment with a specific T cell immunotoxin can significantly reduce but not abolish GVH reactivity in rat small bowel transplantation.

Immunization with a Nontoxic/Nonfibrillar Amyloid-β Homologous Peptide Reduces Alzheimer's Disease-Associated Pathology in Transgenic Mice

Immunization with a Nontoxic/Nonfibrillar Amyloid-β Homologous Peptide Reduces Alzheimer's Disease-Associated Pathology in Transgenic Mice

Effect of CD4(+) and CD8(+) cell depletion on wound healing.

Depression of the immune system can result in poor or delayed wound healing. Thymectomized rats were depleted of CD4(+) and CD8(+) lymphocytes by intraperitoneal injection of Medical Research Council Oxford (MRC OX)38 antibodies and MRC OX8. Significant depletion was demonstrated throughout the wound healing process by immunofluorescence studies of peripheral blood. Following depletion the rats underwent laparotomy incisions which were allowed to heal for 10 weeks. Differences in healing were demonstrated by analysing the wounds biomechanically by tensiometry to obtain values of ultimate strength, resilience, toughness, maximum extension and elastic constant. Wounds of animals depleted of CD4+ lymphocytes showed a significant decrease in ultimate strength, resilience and toughness. Wounds of animals depleted of CD8(+) lymphocytes showed a significant increase in ultimate strength, resilience and toughness. Wounds healed in the absence of T lymphocytes. However, the subsets have an opposing regulatory role, with CD4(+) lymphocytes upregulating and CD8(+) lymphocytes downregulating wound healing. Presented to the Surgical Research Society in Nottingham, UK, 11 July 1997 and published in abstract form as Br J Surg 1997; 84: 1618

Maturation of rat dendritic cells during intrahepatic translocation evaluated using monoclonal antibodies and electron microscopy.

Specific populations of hepatic sinusoidal cells were stained with monoclonal antibodies that recognize monocytes/macrophages (ED1), tissue macrophages (Kupffer cells) (ED2), MHC class II (Ia) antigen (MRC OX6), and dendritic cells/gamma,delta T-cells (MRC OX62) and analyzed by light and electron microscopy. The majority of ED1(+) and/or ED2(+) cells were localized to the hepatic parenchyma, whereas OX6(+) and/or OX62(+) cells were more densely distributed within Glisson's sheath than in the hepatic parenchyma. Double-immunoperoxidase staining of normal liver for ED1, ED2, and OX6 identified dendritic cells (DC) of two different phenotypes, ED1(+)ED2(-)OX6(+) and ED1(-)ED2(-)OX6(+). DC can be classified into three different types based on ultrastructural characteristics. The first type (type I) is characterized by one or more long cytoplasmic processes and a well-developed lysosomal system. The second type (type II) has an inconspicuous lysosomal system, abundant hyaloplasm, and characteristic short cytoplasmic processes. The third type (type I-II) has cytologic features intermediate between those of type I and type II DC. At the electron-microscopic level, these three cell types are found in the sinusoidal lumen, whereas the majority of type II DC are located in the space of Disse and Glisson's sheath. Furthermore, some OX6-labeled elongated DC appeared to traverse the lumen of sinusoids through endothelial pores to enter the space of Disse. One hour after intravenous injection of latex particles (0.81 micrometer in diameter), numerous latex-laden dendritic cells (ED1(+)OX6(+), type I and type I-II) were detected in the lumen of hepatic sinusoids, but not in the space of Disse or Glisson's sheath. These findings suggest that normal rat liver contains resident dendritic cells which downregulate phagocytic activity and mature into potent accessory cells during migration from the portal vein toward the central vein. These DC then traverse the sinusoidal lumen to the hepatic lymph system via the space of Disse.

Class II MHC-expressing cells in the rat adrenal gland defined by monoclonal antibodies.

The detailed distribution and heterogeneity of various immunocompetent cells were characterized in the normal adrenal gland of the rat, with special emphasis on major histocompatibility complex (MHC) class II-expressing cells and macrophages. All adrenals contained at least two different populations of cells reactive with the dendritic cell or the macrophage antibodies. These cells were clearly distinguished from adrenal parenchymal cells by their morphology and location. The majority of dendritic cells were immunoreactive for the MHC class II (Ia) antigen (MRC OX6) and/or the dendritic cell antibodies (MRC OX62), and negative for the macrophage antibodies (ED1, ED2, and/or MRC OX42), whereas the main population of macrophages was immunonegative for the former antibodies and positive for the latter. The OX62-positive cells and the OX42-labeled cells occurred exclusively throughout the medulla. The cellular density of dendritic cells in the adrenal cortex was significantly higher than that of macrophages. Double-immunoperoxidase staining for ED1 and OX6 revealed that positively stained cells could be classified into the following categories: ED1+OX6+, ED1+OX6-, and ED1-OX6+. More then 40% of OX6+ cells were immunoreactive for ED1 in the zona glomerulosa, while approximately 15%, 20%, and 30% of OX6+ cells were positive for ED1 in the zona fasciculata, zona reticularis and medulla, respectively. ED1+ED2- cells were more frequently detected in the zona glomerulosa than in other adrenal zones. Only a few ED1-ED2+ cells were located in the zona glomerulosa, whereas a large number of them were found in the zona fasciculata. In the zona reticularis and medulla, ED1+ED2+, ED1+ED2-, and ED1-ED2+ cells were detected in the ratio 2:1:3. Our rsults suggest that dendritic cells and macrophages mature during their migration within the adrenal gland. These immunocompetent cells may contribute to a paracrine regulation of adrenal function under physiological conditions.

Effects of castration on the lymphocytes of the thymus, spleen and lymph nodes

It is well known that the thymus plays an important role in the development and maintenance of a competent immune system. The thymus atrophies with age, a process that is accelerated after puberty when there is elevation of serum sex steroid levels. We have used a panel of commercial monoclonal antibodies against various T and B cell surface markers to investigate the post-castration histological alterations in the thymus, spleen and lymph nodes of male Sprague-Dawley rats. Castration of 5-week-old male rats produced a significant increase in thymic weight ( P < 0.05) compared to age-matched intact animals. The major observations from the immunohistochemical studies were post-castration elevations in staining for total T cells (MRC OX 19 and W 3/13), CD8 cells (MRC OX 8), B cells (MRC OX 12 and MARK-1) and cells bearing activation markers such as IL-2 receptor (MRC OX 39), transferrin receptor (MRC OX 26) and major histocompatibility class II antigen (MRC OX 6). These data suggest that following castration there is an increase in the ability of lymphocytes to respond to activation. As a result, there are elevated numbers of immature thymocytes within the thymus that undergo differentiation/maturation and consequently produce an increase in peripheral T and B cells.

Concordant lung xenograft rejection in a hamster-to-rat model.

This study was conducted to investigate the functional and morphological aspects of orthotopic lung xenograft rejection in a concordant hamster-to-rat donor-recipient species combination. By postoperative day (POD) 3, allotransplanted grafts demonstrated good aeration, but infiltrates were seen in all the xenotransplanted lungs. Antihamster lymphocytotoxic antibody titers increased to 5.2 +/- 1.1 by POD 3 (P < 0.05 vs POD 1) and reached 7.0 +/- 0.8 by POD 5 (P < 0.05 vs PODs 1 and 3). The CD4+/CD8+ ratio in peripheral blood lymphocytes increased significantly on POD 3 (P < 0.05 vs untransplanted), but decreased by POD 5 (P < 0.05). Histologically, on POD 3 the xenotransplanted grafts were characterized by perivascular cellular infiltrates and edema. Cytologically, the cells consisted of small round lymphocytes, monocytes, and occasional neutrophils. Immunohistochemical analysis showed heavy IgM and C3 deposits in the vascular endothelium, without any IgG deposits. Allotransplanted grafts showed moderate W 3/25 + (28.3 +/- 17.3%) and MRC OX8 + (38.7 +/- 0.7%) cellular infiltrations on POD 3, but ED1 + (8.0 +/- 3.7%) cells were rare. Conversely, in the xenotransplanted grafts, ED1 + (34.2 +/- 16.4%) cells were more prevalent than MRC OX8 + (18.1 +/- 16.5%) cells on POD 3, at P < 0.01 and P < 0.05 vs allograft, respectively. These results indicate that both antidonor antibodies and macrophages/monocytes play an important role in the concordant lung xenograft rejection.

Soybean agglutinin binds a 160-kDa rat macrophage membrane glycoprotein and enhances cell differentiation and activation

Mature macrophages (Mφ) differ from other rat leukocytes by their ability to bind soybean agglutinin (SBA). In this study we identify the SBA-binding structure on rat bone marrow-derived Mφ (BMDMφ). Precipitation of iodinated membrane proteins from rat bone marrow cells (BMC) and BMDMφ with SBA revealed a major glycoprotein of Mr 160 kDa on BMDMφ but not on BMC. In addition minor bands migrating at 70 and 26 kDa were seen. Stimulation of BMDMφ with 100 nM SBA induced a decrease in surface density of Thy1.1 (MRC OX7) and His54 and an increase in the expression of MRC OX6 (RT1.B/I-A), MRC OX17 (RT1.D/I-E), MRC OX41 (gp 110 120 ), MRC OX42 ( CD11b c ), Mac1 ( CD11b CR3 ) and Mac2 (galectin-3/IgE binding protein) antigen. Expression of other Mφ differentiation antigens recognized by mAb MRC OX43 (Mφ, endothelial cells) and ED9 ( Mφ CD14 like) were not significantly altered. BMDMφ derived from cultures with Mφ colony-stimulating factor (M-CSF) and SBA showed increased oxidative burst and phagocytic activity compared to cells cultured with M-CSF alone. Our data suggest that binding of a 160-kDa membrane glycoprotein on Mφ by N-acetylgalactosamine-specific lectins stimulates Mφ differentiation and activation.

Prolongation of lung xenograft survival in rats with a short course of deoxyspergualin and cyclosporin A

Abstract The efficacy of 15-deoxy-spergualin (DSG), cyclosporin A (CyA), and splenectomy - alone or in combination - in prolonging the survival of concordant lung xeno-transplants was studied in the hamster-to-rat model. In the untreated group, rejection occurred within 3 days, with an elevation of lympho-cytotoxic antibody titers. The rejected lung revealed that ED1+ cells were more prevalent than MRC OX8+ cells in the perivascular infiltrates. In the DSG group, the antibody response was suppressed and median survival increased to 7.5 days. The rejected lungs demonstrated a highly significant depression in ED1+ cellular infiltration and a moderate MRC OX8+ cellular infiltration. When maintenance CyA was combined with a short course of DSG, survival dramatically increased to beyond 100 days. There were no deposits of IgM, IgG, or C3 or of any cell infiltrate in the grafts of two animals sacrificed 107 and 119 days post-transplantation. We conclude that initial treatment with DSG combined with continuous CyA can suppress acute rejection in the hamster-to-rat lung xenograft model, resulting in long-term graft survival.

Prolongation of lung xenograft survival in rats with a short course of deoxyspergualin and cyclosporin A.

The efficacy of 15-deoxyspergualin (DSG), cyclosporin A (CyA), and splenectomy--alone or in combination--in prolonging the survival of concordant lung xenotransplants was studied in the hamster-to-rat model. In the untreated group, rejection occurred within 3 days, with an elevation of lymphocytotoxic antibody titers. The rejected lung revealed that ED1 + cells were more prevalent than MRC OX8 + cells in the perivascular infiltrates. In the DSG group, the antibody response was suppressed and median survival increased to 7.5 days. The rejected lungs demonstrated a highly significant depression in ED1 + cellular infiltration and a moderate MRC OX8 + cellular infiltration. When maintenance CyA was combined with a short course of DSG, survival dramatically increased to beyond 100 days. There were no deposits of IgM, IgG, or C3 or of any cell infiltrate in the grafts of two animals sacrificed 107 and 119 days post-transplantation. We conclude that initial treatment with DSG combined with continuous CyA can suppress acute rejection in the hamster-to-rat lung xenograft model, resulting in long-term graft survival.

Expression of class II MHC molecules in the rat pineal gland during development and effects of treatment with carbon tetrachloride.

Cells expressing major histocompatibility complex (MHC) class II (Ia) antigen have been examined during the development of rat pineals and in the pineal gland of adult rats treated with carbon tetrachloride. Cells positive for MHC class II are first detected in the pineal gland of the 7-day-old rat. These positive cells increase in number gradually during development, MHC class II immunoreactivity reaching adult levels at 4 weeks after birth. The MHC class II antigen is intensely labeled on the cell surface, and labeled cells are distributed throughout the organ, several positive cells being gathered into groups. The positive cells are small (7-12 microm in diameter), irregular in shape, and frequently exhibit one or more processes. At the electron-microscopic level, the cytoplasm of positive cells contains few organelles, variously sized empty vacuoles, and a few electron-dense lysosome-like structures. Pinealocytes with synaptic ribbons have been found adjacent to immunoreactive cells. Double-immunoperoxidase staining for MRC OX6, MRC OX42, and ED1 results in OX6(-)/ED1(+)/OX42(+), OX6(-)/ED1(-)/OX42(+), and OX6(+)/ED1(-)/OX42(- )cells. These findings suggest that OX6-positive cells in the pineal can be considered as peripheral dendritic cells. The number of cells expressing MHC class II (Ia) antigen significantly increases in the pineal gland of rats after treatment with carbon tetrachloride (P<0.005). Our results indicate that at least some of the OX6-positive cells migrate into the gland from the circulation under these conditions.

698 Biochemical analysis of AD brains for amyloid B protein with amino-terminal pyroglutamate

Mature macrophages (Mφ) differ from other rat leukocytes by their ability to bind soybean agglutinin (SBA). In this study we identify the SBA-binding structure on rat bone marrow-derived Mφ (BMDMφ). Precipitation of iodinated membrane proteins from rat bone marrow cells (BMC) and BMDMφ with SBA revealed a major glycoprotein of Mr 160 kDa on BMDMφ but not on BMC. In addition minor bands migrating at 70 and 26 kDa were seen. Stimulation of BMDMφ with 100 nM SBA induced a decrease in surface density of Thy1.1 (MRC OX7) and His54 and an increase in the expression of MRC OX6 (RT1.B/I-A), MRC OX17 (RT1.D/I-E), MRC OX41 (gp 110120), MRC OX42 (CD11bc), Mac1 (CD11bCR3) and Mac2 (galectin-3/IgE binding protein) antigen. Expression of other Mφ differentiation antigens recognized by mAb MRC OX43 (Mφ, endothelial cells) and ED9 (MφCD14 like) were not significantly altered. BMDMφ derived from cultures with Mφ colony-stimulating factor (M-CSF) and SBA showed increased oxidative burst and phagocytic activity compared to cells cultured with M-CSF alone. Our data suggest that binding of a 160-kDa membrane glycoprotein on Mφ by N-acetylgalactosamine-specific lectins stimulates Mφ differentiation and activation.

Expression of MHC Class II Antigens on Rat Bone Marrow Cells and Macrophages, and Their Modulation during Culture with Murine GM-CSF or M-CSF

Flow cytometric analysis employing MRC OX 6 and MRC OX 17 monoclonal antibodies recognizing determinants on RT1.B or RT1.D molecules, equivalent to murine I-A and I-E, respectively, was used to detect rat MHC class II antigen (Ag) expression. Approximately 5 % of freshly isolated rat bone marrow cells (BMC) expressed RT1.B and over 30 % displayed RT1.D molecules. The RT1.D + cells were W3/13 +, OX 7 +, OX 19 − and OX 22 −. After one week culture of BMC with murine recombinant granulocyte/ macrophage colony-stimulating factor (GM-CSF), regardless of concentrations, 90 to 95 % of the cells were scored as bone marrow-derived macrophages (BMDMΦ), and over 30 % expressed both RT1.B and RT1.D Ag. GM-CSF increases the percentage of BMDMΦ bearing MHC class II Ag in a concentration-dependent manner. This effect seems to be specific because antibodies to interferon-γ, tumor necrosis factor-α or interleukin-4 did not reduce the number of cells expressing RT1.B and RT1.D Ag. Furthermore, GM-CSF was able to trigger expression of class II molecules on rat peritoneal macrophages (MΦ) and BMDMΦ resulted from cultures of BMC with mouse MΦ-CSF (M-CSF), and the RT1.B and RT1.D inducing effect of GM-CSF was opposed by M-CSF, and by anti-GM-CSF antibodies. The induction of MHC class II Ag synthesis by GM-CSF on rat BMDMΦ was confirmed at the mRNA level by Northern blot analysis employing cDNA probes encoding the RT1.B α.

Antigen-antibody binding kinetics for biosensors: the fractal dimension and the binding rate coefficient.

The diffusion-limited binding kinetics of antigen (or antibody or substrate) in solution to antibody (or antigen or enzyme) immobilized on a biosensor surface is analyzed within a fractal framework. The change in the fractal dimension, Df, is in the same direction as that in the forward binding rate coefficient, k1. An increase or decrease in the fractal dimension, Df leads to an increase or decrease in the forward binding rate coefficient, k1, respectively, irrespective of whether the antigen or the antibody is immobilized on the biosensor surface. For example, an increase in the concentration of IgG (MRC OX-19) (antigen) in solution from 1 to 10 micrograms/mL bound to polyclonal sheep anti-mouse IgG (antibody) immobilized on immunomagnetic beads leads to decreases in Df from 1.93 to 0.517 and in k1 from 2.72 to 0.064, respectively. The different examples analyzed and presented together indicate one means by which the forward binding rate coefficient, k1, may be controlled, that is, by changing the fractal dimension or the disorder on the surface. The versatility of the analysis is indicated by applying it to different examples for both types of systems, wherein either the antigen or antibody is immobilized on the surface. The analysis should assist in improving the stability, sensitivity, and response time of biosensors.

Migration pathways of CD4 T cell subsets in vivo: the CD45RC- subset enters the thymus via alpha 4 integrin-VCAM-1 interaction.

The present investigation examines the localization and migration of purified T cell subsets in comparison with B cells, CD8 T cells and CD4+ CD8- single-positive thymocytes. CD4 T cell subsets in the rat are defined by mAb MRC OX22 (anti-CD45RC), which distinguishes resting CD4 T cells (CD45RC+) from those (CD45RC-) which have encountered antigen in the recent past--subpopulations often referred to as 'naive' and 'memory'. Purified, 51Cr-labelled CD45RC+ CD4 T cells broadly reflected the migration pattern of CD8 T cells and B cells. Early localization to the spleen was followed by a redistribution to mesenteric lymph nodes (MLN) and cervical lymph nodes (CLN), B cells migrating at a slightly slower tempo. There was almost no localization of these subpopulations to the small or large intestine [Peyer's patches (PP) excluded]. In contrast, CD45RC- CD4 T cells (indistinguishable in size from the CD45RC+ subset) localized in large numbers to the intestine; they were present here at the earliest time point (0.5 h), persisted for at least 48 h but did not accumulate, indicating a rapid exit. Numerically, localization of CD45RC- CD4 T cells in the MLN could be accounted for entirely by afferent drainage from the intestine. Unexpectedly, CD45RC- CD4 T cells (but not other subsets) localized and accumulated in the thymus. In vivo treatment with mAb HP2/1 against the integrin alpha 4 subunit inhibited almost entirely CD45RC- CD4 T cell migration into the PP (98.1%), intestine (87.1%), MLN (89.1%) and thymus (93.5%); migration into the CLN was only reduced by half. To distinguish between recognition of MAdCAM-1 and VCAM-1 by alpha 4-containing integrins, recipients were treated with mAb 5F10 against rat VCAM-1. Except for the thymus and a small reduction in CLN, localization of CD45RC- CD4 T cells was unaffected; entry to the thymus was almost completely blocked (92.3%) by anti-VCAM-1. The results indicated (i) that CD45RC- CD4 T cells alone showed enhanced localization to the gut and PP, probably via alpha 4 beta 7-MAdCAM-1 interaction; (ii) that many CD45RC- cells entered non-mucosal LN independently of alpha 4 integrin or VCAM-1; and (iii) that entry of mature recirculating CD45RC- CD4 T cells into the thymus across thymic endothelium was apparently regulated by alpha 4 integrin-VCAM-1 interaction.

EVIDENCE OF ALLOREACTIVE T SUPPRESSOR CELLS IN THE MAINTENANCE PHASE OF SPONTANEOUS TOLERANCE AFTER ORTHOTOPIC LIVER TRANSPLANTATION IN THE RAT

In the DA-->PVG rat strain combination liver allografts are spontaneously tolerated and induce a state of systemic tolerance towards donor antigens. Thirty long-term (> 100 days)-surviving liver recipients in this strain combination were used to study humoral and cellular mechanisms of tolerance in the maintenance phase after liver transplantation. In the popliteal lymph node assay splenocytes of liver grafted or native PVG rats displayed a similar proliferative response in (DAxPVG)F1 hybrid rats, indicating an unaffected capacity of these cells to mount graft-versus-host reactions. Using adoptive transfer assays T-splenocytes (1x10(7) cells, MRC Ox 19+, MRC Ox12-, CD 5) of long-term-surviving liver recipients were able to suppress the rejection of DA hearts (donor-specific), of DA and AO kidney grafts in irradiated (300 cGy) PVG recipients. Serum of long-term-surviving liver recipients was able to suppress the rejection of semiallogeneic (DAxPVG)F1 heart grafts after double injection of 1 ml serum, while fully allogeneic DA heart as well as skin and pancreaticoduodenal grafts were resistant to passive enhancement. The results confirm the development of spontaneous tolerance after liver grafting in the DA-->PVG strain combination, and the demonstration of T cells with suppressive capacity in adoptive transfer experiments indicates a role for T suppressor cells in the maintenance phase of spontaneous tolerance after liver grafting.

Evidence that recipient CD8+ T cell depletion does not alter development of chronic vascular rejection in a rat heart allograft model.

Progressive chronic vascular rejection is a central feature of indefinitely surviving WF.1L LEW/Gut (RT1(1)) heart grafts transplanted to LEW (RT1(1)) recipients in unmodified donor-recipient combinations. At 70 days posttransplantation, large vessels of the grafts are characterized by the presence of vasculitis, vasculitis with associated variable myointimal thickening, and occlusive myointimal thickening with minimal or absent concomitant vasculitis. To assess the potential role of CD8+ T cells as critical effectors of chronic vascular rejection in this model, LEW recipients of WF.1L heart grafts were effectively depleted of CD8+ T cells as a result of prior thymectomy and anti-CD8 (MRC OX8) monoclonal antibody administration prior to transplantation. WF.1L heart grafts transplanted to LEW recipients that had undergone prior sham thymectomy and MRC OX8 administration, or thymectomy and administration of antibody-free culture supernatant, provided appropriate controls. At 70 days posttransplantation, large vessels of WF.1L heart grafts in all 3 transplantation groups showed similar morphologic features, which were comparable to those observed in heart grafts of long-surviving unmodified donor-recipient pairs. This study has shown that profound selective depression of recipient CD8+ T cells does not alter the characteristic features of chronic vascular rejection in this rat cardiac model, and provides evidence that CD8+ T cells play no critical role in the initiation or development of progressive vascular damage in this setting.

MRC OX19 recognizes the rat CD5 surface glycoprotein, but does not provide evidence for a population of CD5bright B cells.

To clone the rat CD5 gene we first produced two rat CD5 probes. The probes were obtained by polymerase chain reaction (PCR) on rat genomic DNA using primers designed on conserved regions between mouse and human CD5. The screening of a rat cDNA library at high stringency using these probes resulted in a 1.5-kb positive clone. The DNA sequence of this clone confirmed its CD5 nature, but the clone appeared to lack part of the 5' and part of the 3' end. These missing 5' and 3' ends were obtained by PCR on rat thymus RNA. By ligating these PCR products to the original 1.5-kb CDM8 clone, a full-length rat CD5 gene was constructed. The full-length clone showed high identity with mouse and human CD5; however, at the 5' site of the gene a region of 36 nucleotides is present which is not seen in either mouse or human CD5. We have evidence that this sequence is a normal constituent of the rat CD5 gene: first, it is in frame with the rest of the CD5 coding sequence; second, it does not contain a stop codon; and third, it is also present in the CD5 gene of other rat strains. We transfected the full-length CD5 construct in COS cells and demonstrated that indeed the CD5 protein is recognized by MRC OX19. Although we showed that CD5 mRNA is present in rat B cells, extensive flow cytometry analysis using MRC OX19 as antibody failed to detect B cells expressing significant levels of CD5 on their cell surface compared to other B cells in any tissue or cell suspension tested from a variety of rat strains. This is in contrast with the mouse where a distinct population of B cells (B-1a cells) can be found expressing more CD5 than the other B cells. Either B-1 cells are not present in rats or CD5 is not the right phenotypic marker for rat B-1 cells. It still remains to be investigated whether a population of B cells with functions similar to those of murine B-1 cells is present in rats.

New immunocytochemical evidence for a neuronal/oligodendroglial origin for corpora amylacea

Studies employing a bank of antisera applied to sections of LR White embedded AD and normal ageing brain tissue, may throw new light on the derivation of CA. Conspicuous levels of immunoreactivity were found in the CA of both tissues with markers for oligodendrocytic proteins such as antisera against myelin basic proteolipid protein, galactocerebroside and myelin/oligodendrocyte glycoprotein. CA were unreactive with MRC OX-42, a marker for microglia and macrophages. In a previous publication we demonstrated that the much more abundant CA in the brains of Alzheimer's disease (AD) sufferers, although slightly more varied in their immunoreactivity than those found in normally ageing controls, were universally immunoreactive with anti-tau, a neuronally derived protein and often also contained amyloid. The cores of CA were not immunoreactive with anti-GFAP, suggesting a lack of involvement with astrocytes. Our results now show that in addition to amyloid and neuronal proteins, a significant proportion of the content of CA is derived from oligodendrocytes and/or myelin. The substantial Fe peak previously reported following X-ray microanalysis of CA was probably due to ferritin. However, immunostaining with antisera to ferritin showed that high ferritin immunoreactivity was common to both micro- and macroglia as well as CA. More significantly, the immunoreactivity of CA with anti-ubiquitin suggests that degeneration of neuronal/oligodendrocytic elements may precede CA formation.

Antigenic determinants encoded by alternatively spliced exons of CD45 are determined by the polypeptide but influenced by glycosylation.

Antibodies recognising the products of alternatively spliced exons near the N-terminus of the leukocyte common antigen, CD45, have been widely used to distinguish populations of lymphocytes with different functional properties. These alternatively spliced regions contain a high content of serine and threonine residues (average 35%) and are heavily O-glycosylated. Despite evidence that the O-glycosylation contributes significantly to the antigenic character of this region of CD45, work with leukosialin and mucin glycoproteins leads to the prediction that the majority of epitopes in the N-terminal exons should be linear protein determinants. In this study the exons of CD45 were expressed in Escherichia coli as non-glycosylated proteins fused to glutathione S-transferase (GST). Fourteen out of 17 mAbs specific for human CD45R reacted with a fusion protein containing exons 4, 5 and 6 (ABC) of human CD45, and four out of six mAbs specific for rat CD45R reacted with an equivalent rat protein. mAbs recognising the product of rat exon B are reported for the first time. Kinetic analysis of MRC OX22 antibody binding to spleen CD45 and to GST fusion proteins showed that the carbohydrate affected the kinetics of binding of antibodies to the protein backbone. In conclusion, heterogeneity in the glycosylation of heavily O-glycosylated cell surface proteins can affect interactions of these proteins both directly through the carbohydrate and indirectly through effects on the protein backbone.