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Biomedical subjects

J Farrant

Publications and source records attributed to J Farrant.

At least 73 records · Page 4Linked to original sources
PubMed1984

The role of B cell differentiation factors and specific T cell help in the pathogenesis of primary hypogammaglobulinemia.

We have examined the function of T and B cells from patients with late onset primary acquired hypogammaglobulinemia (PHG). T cells from these patients give effective help to normal B cells for antigen-dependent antibody synthesis. PHG mononuclear cells also synthesize normal quantities of B cell differentiation factors, which enhance IgG, IgM and antigen-dependent antibody synthesis by normal lymphocytes. While patient T cells appear to behave appropriately, the responsiveness of patient B cells is abnormal. Although they respond to differentiation factors with increased synthesis of IgM, overall levels are 10-50-fold lower than normal B cells, and they produce little or no IgG. This pattern of response is not altered if normal T cells are the source of help. The poor response of the B cell appears to represent immaturity rather than an inherent defect, as IgG-secreting clones can be obtained after Epstein-Barr virus transformation of lymphocytes from certain patients, and some of these clones respond to differentiation factors with increased IgG production. The lack of any functional defect in the T population, and the apparent immaturity rather than abnormality of the B cells, may implicate accessory cells in the pathogenesis of the disease.

Agammaglobulinemia↗
PubMed1984

Non-specific factor enhancement of human in vitro antigen-dependent antibody synthesis: role of B cell activation and T cell help.

Lectin-free supernatants obtained from PWM-stimulated lymphocytes, enable B cells to proliferate and secrete immunoglobulin. Both functions are augmented by the addition of irradiated T cells. In the presence of antigen, these supernatants also enhance specific anti-tetanus toxoid antibody production. The components of the supernatant responsible for these activities have a molecular weight between 30,000 and 60,000, and have the characteristics of non-specific factors: they are genetically unrestricted, and do not bind to either antigen or anti-DR affinity columns. There is no evidence that the partial T dependency of these factors is an indication that their target is a T cell. Instead, T cells appear necessary to move the B cell into a state of activation in which it becomes responsive to the factor. Alternative activation signals such as Staph. A. Cowan can substitute for T cell help in the proliferative response, but not for immunoglobulin or antibody synthesis. The implications of these results for the approaches used to detect and classify B cell growth factors are discussed.

Antibody Formation↗
PubMed1983

The interaction of specific T-cell help and non-specific B-cell growth factors in the production of anti-tetanus antibody by human B cells grown in serum-free microcultures.

Apart from a brief period after in-vivo immunization, only a minority of human donors provide peripheral lymphocytes that synthesize specific antibody on stimulation with tetanus toxoid in vitro. A 20 microliters hanging drop microculture technique using serum-free medium has been adapted to analyse the conditions under which B cells mature into antibody-secreting cells. Multiple permutations of antigen dose, cell concentration and T:B cell ratios have been examined. The results indicate that in-vitro failure of antigen response by the majority of donors is not due simply to an inappropriate choice of culture conditions. The addition to antigen-stimulated cultures of a lectin-free conditioned medium derived from pokeweed mitogen-stimulated peripheral lymphocytes, enables B cells from the majority of donors to produce high titres of specific antibody, in a T-dependent manner, for up to 24 months after immunization. The observed failure of prolonged antigen responsiveness in vitro thus appears to represent a failure to expand a population of antigen-specific B cells, rather than indicating an absence of such clones.

Antibodies, Bacterial↗
PubMed1981

Simple hanging drop (20 microliter) double antibody radioimmunoassay of human IgM, IgG, IgA, IgD and IgE.

A simple hanging drop radioimmunoassay has been developed for different classes of human immunoglobulin from serum and culture supernatant samples. The immune precipitate was formed in inverted Terasaki plates (20 microliter per well) using 4 aliquots (5 microliter) as follows: (A) a standard or unknown sample; (B) rabbit antisera to the appropriate immunoglobulin class; (C) 125I-labelled human immunoglobulin of the appropriate class; (D) a developing goat-anti rabbit serum. The immune precipitate in the hanging drop was harvested and washed without centrifugation with a simple Terasaki plate harvester. The method gives a great saving both in time and in sample and reagent volumes in comparison with an established double antibody assay in tubes (4 X 100 microliter aliquots).

Animals↗
PubMed1981

Relative ability to provide help: an explanation for Con A-induced suppression.

Concanavalin A (Con A) induced suppression of human peripheral lymphocytes has been studied in vitro at different cell concentrations using a 20 microliters inverted Terasaki plate culture system in which the uptake of 3H-thymidine was measured. Conventional assessment of suppression relates the response of cells to Con A in the presence of Con A-pretreated autologous cells, with the response of cells preincubated without Con A (control cells). The level of suppression so calculated was affected both by serum-related high background counts and by autologous stimulation of responding cells by mitomycin-treated Con A-pretreated cells. The high background was largely removed by the use of serum-free Iscove's medium. The data involving different cell concentrations and periods of cultivation indicate that the assessment of suppression is inadequate. Both Con A-pretreated and control cells are shown to 'help' the response of autologous responder cells to added soluble Con A. When the help by Con A-treated cells is less marked than that by control cells, apparent suppression is seen since the control cell provide the conventional baseline from which suppression is judged. After longer periods of cultivation the Con A-pretreated cells increased responses more than did the control cells leading to apparent enhancement.

Cells, Cultured↗
PubMed1980

Conditions for measuring DNA synthesis in PHA stimulated human lymphocytes in 20 microliters hanging drops with various cell concentrations and periods of culture.

We have studied conditions for measuring the uptake of [3H]thymidine ([3H]Tdr) by human lymphocytes in inverted microcultures, varying cell concentrations and periods in culture. Analysis of variance of the log values for [3H]Tdr uptake may be used to separate effects of variables and their interactions. A pulse time of 2 h, a total thymidine concentration of about 1 microgram/ml and a specific activity of [3H]Tdr of 2 Ci/mmole were optimal. Variables such as cell concentration, period of culture, type of serum and dose of PHA were shown to interact, suggesting that these variables should be examined together especially when comparing different samples of lymphocytes. Conditions for doing this simply in small volumes are now available for culturing, thymidine pulsing, harvesting and analysis of data.

Cell Division↗
PubMed1979

HLA-D typing of human lymphocytes using frozen and thawed spermatozoa.

A method is described for the preservation of haploid populations of human spermatozoa in liquid nitrogen for their subsequent use as stimulators in HLA-D (SL) lymphocyte typing. The typing results obtained using thawed spermatozoa correlated well with those obtained when using fresh sperm and also with other cellular typing methods.

Freezing↗
PubMed1979

Curve -shift analysis of cryopreserved killer T cell function.

The conditions for cryopreservation and reconstitution after thawing of cytotoxic effector cells for cell mediated lympholysis (CML) tests are studied. Percentage recovery of functional activity is analysed not by the commonly used method of comparisons at a single concentration of effector cells but by the movement of the curve of functional activity on the axis of cell concentration in culture. By this method the position of the ascending slope of the dose-response curve, at several different effector to target cell ratios, is compared between fresh and frozen-thawed cells. Cooling rates giving optimal recovery were found to vary between experiments. Using simple techniques, optimal cooling rates were found to range from 0.3 to 1.0 degrees C/min, when using dimethyl sulphoxide (10% v/v). Dead cell debris, but not intact dead cells (which take up eosin), were shown to inhibit the lytic ability of functionally active frozen-thawed effector cells. This could be removed by centrifuging the thawed cells over Ficoll-Hypaque. In the recovered population the proportion of cells which excluded the vital dye, eosin, was greater than the proportion of functioning effector cells. This suggested that the cells which excluded eosin contained both functional and nonfunctional, partially damaged effector cells. Thus dye exclusion methods generally over-estimated the functional activity of thawed effector cells. When cells to be used as targets were preserved prior to treatment with phytohaemagglutinin (PHA) no abnormalities were detected in their behaviour to fresh cells.

Aged↗
PubMed1979

Help and suppression by lymphoid cells as a function of cellular concentration.

The phytohemagglutinin-stimulated uptake of [3H]thymidine in mixtures of human lymphocytes from the same source was shown to depend on the cell concentration in vitro as well as on the period of cultivation. "Helper" and "suppressor" effects were obtained by varying the concentration of cells and the periods of cultivation. The possibility that helper and suppressor subpopulations were responsible was avoided by mixing lymphoid cell line cells with others of the same monoclonal origin. Even under these conditions, both the direction and the extent of activity depended on the same two variables. This weakens the case for postulating the existence of distinct subpopulations of lymphocytes with helper or suppressor properties. This case was based on the use of damaging treatments believed to separate cell populations which were then found to differ in their helper and suppressor properties. We propose instead that the effect of such treatments is mediated through changes in the concentrations of interacting cells. Our data make it clear that the function of lymphoid cells ascertained in one set of conditions need not apply within a different cellular environment.

Cell Count↗
PubMed1979

Survival of frozen mouse embryos after rapid thawing from -196 degrees C.

The effect of the rate of rewarming on the survival of 8-cell mouse embryos and blastocysts was examined. The samples were slowly cooled (0.3--0.6 degrees C/min) in 1.5 M-DMSO to temperatures between -10 and -80 degrees C before direct transfer to liquid nitrogen (-196 degrees C). Embryos survived rapid thawing (275--500 degrees C/min) only when slow cooling was terminated at relatively high subzero temperatures (-10 to -50 degrees C). The highest levels of survival in vitro of rapidly thawed 8-cell embryos were obtained after transfer to -196 degrees C from -35 and -40 degrees C (72 to 88%) and of rapidly thawed blastocysts after transfer from -25 to -50 degrees C (69 to 74%). By contrast, for embryos to survive slow thawing (8 to 20 degrees C/min) slow cooling to lower subzero temperatures (-60 degrees C and below) was required before transfer to -196 degrees C. The results indicate that embryos transferred to -196 degrees C from high subzero temperatures contain sufficient intracellular ice to damage them during slow warming but to permit survival after rapid warming. Survival of embryos after rapid dilution of DMSO at room temperature was similar to that after slow (stepwise) dilution at 0 degrees C. There was no difference between the viability of rapidly and slowly thawed embryos after transfer to pseudopregnant foster mothers. It is concluded that the behaviour of mammalian embryos subjected to the stresses of freezing and thawing is similar to that of other mammalian cells. A simpler and quicker method for the preservation of mouse embryos is described.

Animals↗
PubMed1977

Water transport and cell survival in cryobiological procedures.

Living cells may be cooled to 77 K (liquid nitrogen) either to destroy them selectively or to store them for long periods. Water transport across the cell membranes during freezing and thawing is a primary factor determining whether the cells survive. These water movements are controlled by phase changes both intracellular and extracellular and by other factors such as the nature of any cryoprotective agent present, and the rates of cooling and thawing. The relation between cooling procedure, water transport and cell survival is discussed. In particular, the crucial rôle of dilution shock is emphasized: this is the damage to cells induced during the dilution that occurs both as ice melts during rewarming and when any cryoprotective additives are removed after thawing. Apart from the usefulness of understanding these processes for maximizing preservation or controlling selective destruction, the diverse responses of cells to different combinations of water transport and temperature changes appear likely to provide basic information on the properties of cell membranes.

Animals↗