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G P Sandilands

Publications and source records attributed to G P Sandilands.

At least 19 recordsLinked to original sources
PubMed1999

B-cell antigens within normal and activated human T cells.

In this study we compared cell surface staining for human peripheral blood lymphocyte (PBL) CD antigens by flow cytometry, with staining obtained following permeabilization of PBL using the Cytoperm method (Serotec). Six CD antigens (CD20, CD21, CD22, CD32, CD35 and major histocompatibility complex class II antigen) normally found on the surface of B cells, were also found to be expressed within T cells. We also showed, by immunoelectron microscopy, that these inappropriately expressed ('occult') CD antigens are located within cytoplasmic vesicles or within the rough endoplasmic reticulum. Following in vitro activation of T cells a distinct increase in expression of all of these cytoplasmic antigens was observed but staining at the cell surface was, by comparison, weak. We therefore propose that up-regulation of various B-cell CD antigens occurs within the cytoplasm of T cells following activation and that these antigens may be synthesized and released into the fluid-phase as soluble immunoregulatory molecules.

Antigens, CD↗
PubMed1997

Differential expression of CD32 isoforms following alloactivation of human T cells.

Receptors for the Fc region of immunoglobulin G (IgG) (Fc gamma Rs) exist in three main forms: membrane bound, soluble and cytoplasmic. The function of cytoplasmic Fc gamma Rs is poorly understood. We have previously demonstrated cytoplasmic Fc gamma RII (cCD32) within most normal human peripheral blood lymphocytes (PBL), including T cells. In this study we have investigated the hypothesis that following lymphocyte activation, up-regulation of cCD32 occurs, resulting in increased expression at the cell surface. Normal PBL were activated in vitro using a two-way mixed lymphocyte reaction (MLR) and expression of CD32 monitored by flow cytometry and by immunoperoxidase staining using specific monoclonal antibodies and aggregated mouse IgG subclasses. Furthermore, we designed oligonucleotide probes specific for the three main isoforms of CD32 and looked for changes in mRNA expression throughout the MLR using an in situ hybridization technique. Increased surface expression of CD32 was found on both activated human T and B lymphocytes, but this was found only in the early stages of the MLR, on days 3 and 4, and was virtually absent by day 7. An inverse relationship between cell surface expression of CD32 and mRNA for the IIb isoforms was noted with strong mRNA expression for IIb isoforms occurring in the later stages of the MLR (days 6-7) when interleukin-2R (IL-2R)-positive T cells were predominant. A soluble IgG binding factor (soluble CD32?) was also detected in the MLR culture supernatant. These observations provide support for the hypothesis that synthesis of IIb isoforms of CD32 occurs following alloantigen activation of human T lymphocytes.

Flow Cytometry↗
PubMed1997

Demonstration of cytoplasmic CD32 (Fc gamma RII) within human lymphocytes following microwave treatment.

We have recently described a cytoplasmic from of CD32 (Fc gamma RII) within the vast majority of normal human peripheral blood lymphocytes (PBL) including T cells. The function of cytoplasmic CD32 is not known. These flow cytometric studies were conducted using single cell suspensions of PBL that had been pre-fixed and permeabilized using methanol/triton-X-100. In this study we have attempted to visualize cytoplasmic CD32 by immunocytochemistry using normal PBL processed in various ways and have also looked for CD32 within tissue lymphocytes. Weak cytoplasmic CD32 staining was observed in paraffin sections of normal lymphocytes but only when sections were microwave treated. The intensity of staining for CD32 did however, appear to be much stronger within infiltrating lymphocytes found in autoimmune diseases or in rejecting allografts: an observation that suggests that up-regulation of cytoplasmic CD32 may occur when T cells become activated in vivo. Microwave treatment of PBL suspensions was shown to disrupt the outer cell membrane, thus effectively permeabilizing the cell and allowing for the detection of cytoplasmic components, like CD32, by flow cytometry. Microwave treatment may, therefore, afford an alternative method for cell permeabilization and may prove to be a useful method for the study of cytoplasmic molecules in cell suspensions and in paraffin-embedded tissues.

Antibodies, Monoclonal↗
PubMed1995

Occult expression of CD32 (Fc gamma RII) in normal human peripheral blood mononuclear cells.

Three main classes of Fc gamma receptor (Fc gamma R) have been described on the surface of normal human peripheral blood peripheral blood mononuclear cells. These receptors are thought to play an important role in many immune mechanisms. Following interaction with ligand, i.e. IgG in the form of an immune complex, receptor cross-linking occurs and some isoforms of Fc gamma R become internalized and will recycle back to the cell surface. This mechanism may be important in signal transduction pathways. In this study we have shown that a particular type of Fc gamma R (CD32), which is normally expressed on the surface of B cells, can be detected by flow-cytometry within the cytoplasm of up to 90% of normal human peripheral blood lymphocytes. The function of this 'occult' CD32 is not known but may represent an internal receptor pool that is up-regulated following cell activation.

Antibodies, Monoclonal↗
PubMed1993

Detection of lymphocyte Fc gamma receptor-blocking factors by the EA rosette inhibition assay. Refinement of the conventional method and development of a novel flow-cytometric assay.

Serum factors which interact with human peripheral blood lymphocyte Fc gamma receptors (Fc gamma Rs) may be detected in vitro by the EA rosette inhibition assay (EARIA). This assay has been used to detect circulating immune complexes and certain alloantibodies directed against cell surface antigens situated in close proximity to Fc gamma Rs. Three main types of FcR-blocking factor have been demonstrated by the EARIA in human serum following exposure to alloantigens. A strong correlation was observed between the presence of one of these FcR-blocking factors (FcBF1) and human renal allograft survival. This factor was previously shown to bind preferentially to CD32+ B cells and to inhibit antibody synthesis. In this study we have shown that detection of FcBF1 by the EARIA depends on the type of erythrocyte and on the amount of antibody used to sensitise the erythrocytes. Furthermore, we have developed a flow-cytometric version of the EARIA which is rapid, reproducible and, most importantly, objective. Inter-laboratory comparisons using this standardised EARIA should now be possible.

Erythrocytes↗
PubMed1992

Immunohistochemical and functional studies of glycoprotein 60 (gp60) in platelets.

We showed by immunofluorescence, immunoelectron microscopy and Western blot analysis that the plasma glycoprotein (gp60), an Fc gamma binding protein which inhibits complement-mediated prevention of immune precipitation, is present in platelets. The gp60 content of platelets in normal individuals and patients with rheumatoid arthritis was similar (mean 0.028 and 0.024 fg/platelet respectively). Immunoelectron microscopic studies showed that gp60 was present in the cytoplasm and the surface connecting structures but not in the alpha granules, dense granules or lysosomes. Using this technique gp60 was also found on platelet membranes, an observation which was confirmed by immunofluorescence. Activation of platelets with thrombin, calcium ionophore, and immune complexes (IC) resulted in the release of the contents of the alpha granules (beta-thromboglobulin), dense granules (5-hydroxytryptamine) and lysosomes (beta-glucuronidase) but did not induce gp60 secretion. The inability of Fab anti-gp60 to inhibit IC-mediated platelet aggregation and of F(ab')2 anti-gp60 to produce platelet aggregation suggested that IC-mediated platelet aggregation did not occur as a result of the interaction of IC with platelet gp60. However, as the preincubation of IC with purified gp60 produced dose-dependent inhibition of the ability of IC to aggregate platelets it is possible that fluid-phase plasma gp60 modulates the interaction of IC with platelets.

Blood Platelets↗
PubMed1990

Isolation and characterization of a high molecular weight lymphocyte Fc gamma receptor-blocking factor associated with renal allograft survival.

We have confirmed our previous observation that improved human renal allograft survival is associated with the presence in pre-transplant serum of a high molecular weight lymphocyte Fc gamma receptor-blocking factor. Serum fractionation studies suggest that this factor is a complex protein consisting of IgG together with an IgG-binding protein which has an apparent molecular weight of approximately 60 kD.

Antigens, Differentiation↗
PubMed1990

Immunohistochemical localization of a plasma protein (glycoprotein 60) which inhibits complement-mediated prevention of immune precipitation.

Using various immunocytochemical techniques it has been shown that glycoprotein 60 (gp60), an IgG Fc-binding protein recently isolated from normal human plasma, is found localized in human hepatocytes, platelets and a subpopulation of normal peripheral blood lymphocytes (PBL). This protein, which appears to be identical to a 60,000 MW IgG-binding protein (60-IBF) previously isolated from normal human PBL culture supernatants, also appears to be distinct from the three well-defined leucocyte Fc gamma-receptors: Fc gamma RI, Fc gamma RII and Fc gamma RIII.

Antigens, CD↗
PubMed1990

FcR blocking activity in serum of actively enhanced rat renal allograft recipients due to IgG anti-class II MHC alloantibody.

In some rat strain combinations, pre-operative donor-specific blood transfusion produces long-term renal allograft survival, although the underlying mechanisms are unclear. This study has examined whether Fc receptor (FcR)-blocking activity could be detected in the serum of unmodified PVG strain recipients bearing a rejecting renal allograft and in recipients bearing an actively enhanced graft following pre-operative blood transfusion. Serum harvested on Day 5 from actively enhanced PVG recipients of DA rat renal allografts was shown to specifically inhibit erythrocyte-antibody (EA) rosette formation with donor strain, but not third-party, splenocytes, while the levels of EA rosette inhibition (EAI) in Day 5 serum from rejecting rats remained markedly lower. This FcR-blocking activity was present in enhanced serum fractions, prepared by discontinuous density gradient centrifugation, which corresponded to the 7 S peak. Purified IgG prepared from enhanced serum was also found to inhibit EA rosette formation with donor splenocytes, and absorption of the IgG preparations with donor strain erythrocytes failed to abrogate EA rosette inhibition. Further experiments, in which absorbed IgG from enhanced animals was tested for FcR blocking activity against splenocytes of defined major histocompatability complex (MHC) subregion specificities, established that FcR-blocking activity was mediated by IgG alloantibodies directed against donor MHC class II antigens. Whether the presence of such antibodies early after transplantation contributes to the beneficial effect of blood transfusion on graft survival remains to be determined.

Animals↗
PubMed1988

A proposed mechanism of action for ALG and ATG in severe acquired aplastic anaemia.

In this study we have shown that various batches of anti-lymphocyte globulin (ALG), anti-thymocyte globulin (ATG) and human intravenous immunoglobulin (IV Ig) all contain antibodies with the capacity to block lymphocyte receptors for the Fc region of IgG, i.e., Fc gamma receptors. These antibodies may exert an effect on Fc gamma receptor bearing suppressor T cell function in vivo. Since T cells have been implicated in the pathogenesis of acquired severe aplastic anaemia, it is conceivable that administration of Fc gamma receptor blocking antibodies in the form of ALG, ATG or IV Ig preparations may be important in the treatment of the disease. The level of Fc gamma receptor blocking produced by these IgG preparations was however found to vary from batch to batch and one lymphocyte donor to another. In vivo Fc gamma receptor modulation will therefore only occur when the "correct" batch of IgG is used, thus affording a possible explanation for the variable clinical response of patients to this type of therapy.

Anemia, Aplastic↗
PubMed1987

Correlation between high-molecular-weight Fc gamma-receptor-blocking factors in serum and renal allograft survival.

In this study, we have demonstrated the occurrence of IgG class noncytotoxic, lymphocyte Fc gamma-receptor-blocking antibodies in a proportion of sera obtained from transfused uremic patients. The presence of these antibodies was not found to correlate with subsequent renal allograft survival. Serum fractionation studies did however reveal a striking correlation between graft survival and Fc gamma-receptor blocking mediated by a serum factor(s) with a sedimentation coefficient of greater than 19S. The precise nature of this factor remains to be clarified.

Blood Proteins↗
PubMed1987

Spontaneous release of Fc gamma receptors from normal human peripheral blood lymphocytes in vitro.

Certain normal human peripheral blood lymphocyte (PBL) membrane receptors for the Fc region of IgG (i.e. Fc gamma receptors) are known to be released spontaneously when incubated at 37 degrees in serum-free medium into the culture supernatant. However, the mechanisms involved in spontaneous Fc gamma receptor release are not clear. In this study we have shown that Fc gamma receptors appear to be released in two stages. Stage 1 occurs within the first hour of incubation, and is probably mediated by limited proteolysis at the cell surface. Stage 2 occurs between 2 hr and 4 hr, and requires active synthesis of Fc gamma receptors. The kinetics of Fc gamma receptor release from activated PBL was found to be slightly different from normal, but no appreciable increase in Fc gamma receptor 'activity' was found in the culture supernatants when compared with supernatants from normal 'resting' cells. It is doubtful whether these mechanisms operate in vivo since spontaneous release of Fc gamma receptors was completely inhibited in the presence of serum. The serum factors that prevent receptor release were found to be confined to two distinct fractions corresponding to those that contain the major serum protease enzyme inhibitors-alpha 2-macroglobulin and alpha 1-trypsin inhibitor.

Cells, Cultured↗
PubMed1987

Lymphocyte Fc gamma-receptor blocking antibodies in intravenous gammaglobulin preparations.

Various batches of intravenous gammaglobulin (IV-IgG) were found to contain antibodies with the capacity to block human peripheral blood lymphocyte receptors for the Fc region of IgG i.e., Fc gamma-receptors (Fc gamma Rs). The level of Fc gamma R-blocking produced was found to vary considerably from batch to batch and from lymphocyte donor to donor. IgG purified from normal human serum pooled from only 20 donors was also found to produce significant Fc gamma R-blockade. Analysis of the individual contributors to this normal IgG pool indicated that Fc gamma R-blocking antibodies may in fact be auto-antibodies.

Adult↗
PubMed1987

In vivo and in vitro blocking of human lymphocyte Fc gamma-receptors by intravenous gammaglobulin.

Lymphocyte subpopulations were measured in patients with idiopathic thrombocytopenic purpura before and immediately after high dose intravenous gammaglobulin (IV-IgG) therapy. A significant relative and absolute reduction in the Fc gamma-receptor bearing lymphocyte subpopulation was observed in 5 out of 7 patients tested. In vivo modulation of lymphocyte Fc gamma-receptors therefore probably occurs: an effect which may be attributable to the Fc gamma R-blocking anti-lymphocytic antibodies found in IV-IgG preparations. In vitro studies showed that IV-IgG preparations also contain antibodies with the capacity to block Fc gamma-receptors on human monocytes and polymorphs and that these antibodies can inhibit the T cell response to phytohaemagglutinin. These anti-lymphocyte antibodies may be important in the mode of action of high dose IV-IgG therapy.

Aged↗