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Biomedical subjects

K D Keeler

Publications and source records attributed to K D Keeler.

15 recordsLinked to original sources
PubMed1988

Sexing of sperm by flow cytometry.

Economics dictate that livestock producers will be under increasing pressure to optimise output. A technique for sex pre-selection could help by reducing the number of females required to produce a given number of progeny of the desired sex; the technique would be particularly useful to the dairy industry. Live mammalian sperm, stained with a vital dye and analysed by flow cytometry, show a bimodal fluorescence distribution. Such bimodality may represent two overlapping subpopulations of X- and Y-chromosome bearing sperm. To test this hypothesis, sperm from the two subpopulations were separated using the sorting capacity of a flow cytometer and were used for the insemination of suitably prepared females. The sex of the resulting progeny was determined either by anatomical criteria or by identification of the sex chromosomes by karyotyping. Insufficient data are available so far to provide statistically significant evidence in support of the hypothesis, but a preliminary sequential analysis indicates a progressive tendency towards significance.

Animals↗
PubMed1984

Activation and proliferation signals in mouse B cells. I. A comparison of the capacity of anti-Ig antibodies or phorbol myristic acetate to activate B cells from CBA/N or normal mice into G1.

B lymphocytes from the CBA/N mouse do not synthesize DNA when cultured with anti-Ig antibodies. However, these cells like normal B cells, do manifest increased Ia antigen expression and RNA synthesis (i.e. enter G1) when stimulated by anti-Ig, even at doses which are nonmitogenic for normal B cells. Pretreatment of both normal and CBA/N B cells with anti-Ig also primes them to give an enhanced proliferative response to lipopolysaccharide (LPS). The tumor promoter phorbol myristic acetate (PMA) also enhances RNA synthesis and Ia antigen expression in B cells from both normal and CBA/N mice. However, PMA only primes CBA/N B cells to respond to LPS: pretreatment of normal B cells with PMA causes a modest suppression of LPS-induced, and a marked suppression of anti-Ig induced, DNA synthesis. These results therefore confirm and extend earlier data showing that there are distinct activating (G0 to G1) vs. proliferative (G1 to S) signals discernible in B cells. They also suggest that the activation mechanism of CBA/N B cells is subtly different from that of any known subpopulation of normal B cells.

Animals↗
PubMed1984

Flip-flop in the Lyt 2 phenotype of T cells from radiation chimaeras between Thy 1 congenic donor and recipient mice.

We present further evidence that T helper cells change their surface phenotype from Lyt 2- to Lyt 2+, after secondary adoptive transfer. Chimaeras were established with Lyt 2- spleen cells from normal, or carrier-primed A-Thy 1a donors and irradiated, A-Thy 1b recipients. Despite efficient depletion of Lyt 2+ cells from the initial donor, in the fluorescence-activated cell sorter, the majority of chimaeric T cells expressed Lyt 2 and were resistant to monoclonal Thy 1.2 alloantibody and complement treatment. In functional studies, the T helper activity of chimaeras, reconstituted with carrier-primed spleen cells, was present in both Lyt 2+ and Lyt 2- subsets.

Animals↗
PubMed1984

Activation and proliferation signals in mouse B cells. III. Intact (IGG) anti-immunoglobulin antibodies activate B cells but inhibit induction of DNA synthesis.

Intact (IgG) rabbit anti-immunoglobulin antibodies are generally not mitogenic for mouse B cells but, on the contrary, inhibit proliferation induced by either F(ab')2 anti-Ig or by lipopolysaccharide. We show here, however, that IgG anti-Ig activates mouse B cells, since it causes B cells to depolarize, to enlarge and to express increased levels of Ia antigens. In the continuing presence of IgG anti-Ig, B cells do not synthesise DNA. However, if cells cultured with IgG antibody are then washed, they start to proliferate earlier in response to F(ab')2 anti-Ig, i.e. they have become primed. We therefore conclude that IgG anti-Ig is an example of a 'step-one activator' for mouse B cells, which drives resting B cells out of Go, but actively prevents the cells from progressing into S. The latter effect appears to result from cross-linking of surface Ig and Fc receptors on B cells, although the mechanism is, as yet, unknown.

Animals↗
PubMed1984

A flow microfluorimetric analysis of the binding of immunoglobulins to Fc gamma receptors on brush borders of the neonatal mouse jejunal epithelium.

In order to determine whether the neonatal mouse small intestine has the ability to be selective in the binding of homologous immunoglobulin isotypes, mouse immunoglobulins were tested for their ability to bind to brush borders isolated from jejunal enterocytes of neonatal and adult mice. The binding of immunoglobulins to brush borders was detected by fluorescence-activated cell sorter (FACS) analysis. The results showed that brush borders from 12-day-old mice bound mouse immunoglobulins selectively, binding IgG2a, IgG2b, IgG3 and IgE but not IgG1, IgM or IgA. Similarly, at 16 days, only IgG2a, IgG2b, IgG3 and IgE were bound significantly. The ability of jejunal brush borders to bind gamma-globulins declined with age and their selectivity was lost such that in mice of 18 days and over not one of the immunoglobulins tested was bound. It was concluded that neonatal mouse jejunal Fc gamma receptors bind IgG2a and IgG2b almost exclusively. The sudden drop in binding between 16 and 18 days coincides with the previously reported age at which immunoglobulin transmission across the gut in the neonatal mouse ceases (closure).

Aging↗
PubMed1983

Flow microfluorometric analysis of living spermatozoa stained with Hoechst 33342.

Bovine spermatozoa were stained with Hoechst 33342. The fluorescence distribution of stained spermatozoa was complex. Non-motile spermatozoa displayed a higher fluorescence than did motile spermatozoa. The fluorescence profile of the motile spermatozoa was bimodal. Sort and reanalysis, and orientation experiments suggested that there are two distinct populations of motile spermatozoa.

Animals↗
PubMed1983

The expression of surface IgD on B cells responsive to thymus-independent and thymus-dependent antigens and its requirement for B-cell triggering.

Cells separated by the fluorescence activated cell sorter on the basis of their surface IgD (sIgD) phenotype have been examined for responsiveness to thymus-dependent and thymus-independent antigens. The ability of monoclonal anti-IgD alloantibodies to inhibit responses in vitro to the various classes of antigen has also been investigated. Evidence is presented indicating that both sIgD positive and sIgD negative cells can respond to all types of antigen tested. However, although the presence of sIgD was necessary for the response of sIgD positive cells to thymus-dependent antigens, the presence of this isotype was not obligatory for the response of the sIgD positive population to thymus independent antigens. The possible role of sIgD as the obligatory purveyor of a B-cell activation signal is discussed in the light of these findings.

Animals↗
PubMed1983

FACS analysis of changes in T and B lymphocyte populations in the blood, spleen and lymph nodes of pregnant mice.

Using the fluorescence-activated cell sorter (FACS), the percentages of T and B lymphocytes in the peripheral blood, spleen and lymph nodes of CBA syngeneic and allogeneic pregnancies were determined. At 19 days of syngeneic pregnancy, the level of T cells in the blood had decreased and that of B cells had increased. In the spleen, a decrease in both T and B cells occurred earlier in pregnancy and had returned to control levels by 19 days. During the last few days of pregnancy, the para-aortic lymph nodes (PALN) draining the pregnant uterus of syngeneically mated mice showed a 1 . 7-fold increase in the level of B cells over that in virgin controls. The T-cell level dropped at the end of pregnancy by approximately 10%. There was no change in T- or B-cell status in the brachial (underarm) nodes taken from the same animals.

Animals↗
PubMed1982

Autofluorescence as an index of ageing in human fibroblasts in culture.

An exponential increase in autofluorescence (AF) during serial passaging of human diploid fibroblasts, strain MRC-5, was observed using a fluorescence-activated cell sorter. Skin fibroblasts from patients with premature-ageing syndromes had high levels of AF, whereas virus-transformed cell lines had negligible amounts of AF.

Adult↗
PubMed1980

Vital DNA staining and cell sorting by flow microfluorometry.

A procedure has been investigated for sorting viable cells according to their DNA content. Cells are stained with the U.V. activated fluorochromes 4'6-diamidino-2-phenylindole (DAPI), Hoechst 33258 or Hoechst 33342, and sorted with a Fluorescence Activated Cell Sorter. Hoechst 33342 is a suitable vital stain for a variety of cell types. Hoechst 33258 and DAPI, however, are quantitative vital stains for CHO cells only. Cloning efficiency is unaffected by the sorting procedure, and these stains are not mutagenic at concentrations suitable for vital staining. Potential applications of this procedure to cell biology are discussed.

Amidines↗
PubMed1979

Restricted heterogeneity of antibody synthesized by T-cell deprived mice.

Mice which had been thymectomized and injected with anti-thymocyte serum to remove long-lived recirculating T cells, initially failed to produce haemagglutinating and haemolysing antibody after injection of sheep erythrocytes. After six fortnightly injections of heterologous erythrocytes, however, haemolysin titres in the T-cell deprived mice were comparable to those in similarly challenged but immunologically intact animals. Isoelectric focusing of these sera indicated that the anti-sheep erythrocyte antibody eventually synthesized by the T-cell deprived mice was less heterogeneous than antibodies found in the sera of control mice.

Animals↗
PubMed1979

IgM rheumatoid factor as a source of non-specificity in murine anti-allotype sera.

Anti-allotype sera have shown a breakdown of the expected specificity especially when used for the development of haemolysis of fragile target erythrocytes. IgM anti-IgG rheumatoid factor has been shown to be the source of non-specificity. Removal or destruction of IgM in an anti-allotype serum restores the expected specificity.

Animals↗
PubMed1976

Immunoglobulin M receptors on memory cells of immunoglobulin G antibody-forming cell clones.

The memory cells of two antibody-forming cell clones had receptors of the IgM class, even though the clones had been producing IgG1 or IgG2a anti-2,4-dinitrophenyl antibodies for 9-15 months previously (on exposure to antigen). Thus a phenotypic switch in heavy chain constant region evidently occurred after re-exposure of these memory cells to antigen. To show that, we first removed the clonal cells' surface immunoglobins by "capping" and "stripping", with class- or subclass-specific antisera. Then, to assay their remaining receptor activity, the cells were incubated with antigen in vitro, washed and transferred (together with carrier primed cells) to irradiated recipients, and their antibody responses to this in vitro boost were assayed by iselectric focusing. Pretreatment with anti-mu serum, as well as with anti-Fab(kappa), prevented the responses of the IgG1 and IgG2a clones to an in vitro boost, while anti-gamma1 and anti-gamma2a antisera had no effect. An antiserum to the putative mouse IgD also had no effect. The anti-mu serum failed to react with the IgG1 and IgG2A clonal serum antibodies in the test tube. Some other contaminating clones were suppressed completely only by the anti-Fab serum. This result strongly suggests that switching in class commitment may occur during the differentiation of memory cells to antibody producers, and may therefore be antigen-dependent. It also implies that some apparently naive cells with surface IgM may, in reality, be B memory cells.

Animals↗