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V M Sanders

Publications and source records attributed to V M Sanders.

At least 55 records · Page 3Linked to original sources
PubMed1986

Isolation of antigen-binding virgin and memory B cells.

One major obstacle in studying the activation of antigen-specific B cells is the small number of B cells reactive with a particular antigenic epitope. In this report, we describe a method by which large numbers of highly purified antigen-binding cells can be obtained. We have shown that by varying the haptenation level of the erythrocytes used for rosetting, we can purify antigen-binding B cells which have different affinities for the antigenic epitope. Thus, memory cells (which have receptors of higher affinity) can be prepared and these cells are essentially free of contaminating virgin cells. The effects of varying the haptenation levels on the red cells used for purifying the B cells can, in turn, be related to the precursor frequency of secreting cells following their activation with T cells and antigen.

Animals↗
PubMed1986

In vitro activation of murine antigen-specific memory B cells by a T-dependent antigen.

A procedure that generates an enriched population (60 to 85%) of memory B cells specific for TNP (TNP-MABC) was employed. The activation requirements of TNP-MABC for the T-dependent antigen TNP-KLH and carrier-primed helper T (Th) cells were compared to those for TNP-binding cells from nonimmune mice (TNP-ABC). Proliferation and differentiation of TNP-MABC in response to cognate recognition of antigen requires less antigen and fewer carrier-primed Th cells than the activation of TNP-ABC. Furthermore, responses of the TNP-MABC were of a greater magnitude. Non-cognate activation induces a low level of proliferation of both TNP-ABC and TNP-MABC, but induces differentiation of TNP-MABC only. Percoll density fractionation of spleen cells prior to enrichment for TNP-MABC suggests that the small, dense cell population responds to cognate, but not to non-cognate activation. FACS separation of TNP-MABC by surface Ig isotype reveals that approximately 80% of the secondary IgG response is derived from cells expressing sIgG. Such cells constitute less than 10% of the total number of TNP-MABC. Limiting dilution studies with sorted TNP-MABC indicate that sIgG+ TNP-MABC are enriched for precursors that give rise to a large clone size. The in vitro results indicate the existence of three putative pathways for antigen-specific memory B cell activation by a T-dependent antigen: 1) sIgG+ cells differentiating into IgG-secreting cells; 2) sIgM+ cells differentiating into IgG-secreting cells; and 3) sIgM+ cells differentiating into IgM-secreting cells.

Animals↗
PubMed1986

Interleukin 4 mediates autocrine growth of helper T cells after antigenic stimulation.

The role of interleukin 4 (IL-4) (previously called B-cell stimulatory factor 1) in the response of T helper (TH) cells to antigen presented by antigen-specific B cells or splenic adherent cells was investigated. Antigenic stimulation of either a keyhole-limpet-hemocyanin-specific TH-cell line or two keyhole-limpet-hemocyanin-specific T-cell clones resulted in the secretion of IL-4 but not interleukin 2 (IL-2). The secretion of IL-4 was first detected in the culture supernatant 6-8 hr after antigenic stimulation. Induction of IL-4 secretion was antigen specific and major histocompatibility complex restricted. Antigenic stimulation also resulted in increased responsiveness of the TH cells to exogenously added or endogenously produced IL-4. The antigen-induced proliferation of the TH cells could be inhibited by an anti-IL-4 antibody but not by an anti-IL-2-receptor antibody. These results suggest that IL-4 mediates the proliferation of some TH cells by an antigen-induced autocrine mechanism. Taken together with past results, these data indicate that, during T-cell-B-cell interactions involving some soluble protein antigens, IL-4 and not IL-2 is the critical lymphokine for activating resting B cells and inducing proliferation of the TH cells.

Animals↗
PubMed1985

Immunotoxicological investigations in the mouse: general approach and methods.

The adverse effects of chemicals on the lymphoreticular system have generated considerable toxicological interest. In this series of papers, the effects of selected environmentally relevant compounds are reported. This first paper describes the methods and general approach used in judging a chemical's potential risk to the immune system. Risk evaluation was approached utilizing acute, 14- and 90-day studies. Both sexes of the CD-1 random-bred mouse were employed. The immune system was evaluated against a background of more standard toxicological parameters, which included fluid consumption, body and organ weights, hematology, serum and liver chemistries, hepatic microsomal enzyme activities and blood coagulation. Bone marrow status was evaluated by assessing DNA synthesis. Humoral immunity was evaluated by determining the number of IgM spleen antibody-forming cells (AFC) to sheep erythrocytes (sRBC), the serum antibody level to sRBC, and spleen lymphocyte response to the B cell mitogen, lipopolysaccharide (LPS). The status of cell-mediated immunity was assessed by quantitating the delayed type hypersensitivity (DTH) response to sRBC, proliferation of the popliteal lymph node, and the spleen cell response to the T lymphocyte mitogen, Concanavalin A (Con A). Macrophage function was evaluated by measurement of the vascular clearance rate and distribution of radiolabeled sRBC in the liver, spleen, lungs, and thymus, and recruitability, adherence, chemotaxis, and phagocytic activity of peritoneal exudate cells (PEC). Historical control data from six 14- and 90-day studies conducted over a one year period are given. The data resulting from these types of studies can provide a basis for the initial evaluation of a chemical's adverse effect on the immune system.

Animals↗
PubMed1985

Toxicology of 1,1,2-trichloroethane in the mouse.

1,1,2-Trichloroethane (TCE) was administered to male and female CD-1 mice to evaluate its effect on standard toxicological parameters. Following determination of the acute LD50 (378 mg/kg in males and 491 mg/kg in females), and a 14-day range-finding study, a 90-day drinking water study was performed in which the doses consumed were 4.4, 46, and 305 mg/kg for males and 3.9, 44, and 384 mg/kg for females. The liver was a target of TCE toxicity in both sexes as demonstrated by dose-dependent alterations in hepatic microsomal enzyme activities and serum enzyme levels. The erythroid element of the female mice was also affected, as indicated by significantly decreased hematocrit and hemoglobin levels.

Animals↗
PubMed1985

Humoral and cell-mediated immune status of mice exposed to 1,1,2-trichloroethane.

The purpose of this study was to assess the immunological effects of 1,1,2-trichloroethane (TCE) on random-bred CD-1 mice following 14 and 90 days of oral exposure. A toxicological evaluation conducted at the same time revealed the target organs to be the liver of both sexes and the erythroid elements of the females. The 14-day immunological range-finding study in males exposed to doses 1/10 and 1/100 the LD50 (38 and 3.8 mg/kg) revealed no alterations in either humoral or cell-mediated immune status. Following 90 days of exposure in the drinking water (4.4., 46, and 305 mg/kg for males and 3.9, 44, and 384 mg/kg for females), a more detailed series of immunological parameters was assessed. Cell-mediated immunity was unaltered in both sexes, while humoral immune status was depressed in both sexes, particularly when determined by hemagglutination titers. Macrophage function was depressed only in the males as indicated by the ability of thioglycolate-recruited peritoneal exudate cells (PEC) to phagocytize sheep erythrocytes (sRBC).

Animals↗
PubMed1985

Toxicology of trans-1,2-dichloroethylene in the mouse.

Trans-1,2-dichloroethylene (DCE) was administered to male and female CD-1 mice in order to evaluate its effects on standard toxicological parameters. Following an acute LD50 determination (2122 mg/kg in males and 2391 mg/kg in females) and a 14-day range-finding study, a 90-day drinking water study was performed using levels of DCE calculated to deliver approximately 1/100, 1/10, and 1/5 the LD50. Various toxicological assessments were made, including body and organ weights, hematology, serum chemistries, and hepatic microsomal activities. Few alterations were observed in either sex following 90 days of exposure. The most noteworthy changes occurred in the males exposed to the highest level of DCE, where there was a significant decrease in glutathione levels, and in the females exposed to all three DCE levels, where there was a significant decrease in aniline hydroxylase activity. These data served as background for the immunotoxicological evaluation presented in the following manuscript.

Animals↗
PubMed1985

Humoral and cell-mediated immune status of mice exposed to trans-1,2-dichloroethylene.

This study assessed possible adverse immunological effects of trans-1,2-dichloroethylene (DCE) on random-bred CD-1 mice following 14 and 90 days of exposure. A 14-day range-finding study was performed on male mice by gavage at doses 1/10 and 1/100 the LD50 (210 and 21 mg/kg). No alterations in either humoral or cell-mediated immunity were observed following this exposure. A 90-day study was conducted in which DCE was administered in the drinking water of male and female mice. The levels of DCE in the drinking water were calculated to deliver levels equivalent to, and higher than, those delivered for 14 days (17, 175, and 387 mg/kg for males and 23, 224, and 452 mg/kg for females). No changes were observed in the cell-mediated immune status of either sex or in the humoral immune status of females. However, a marked suppression in humoral immune status was observed in male mice exposed to all three levels of DCE, as indicated by a decreased ability of spleen cells to produce antibody against sheep erythrocytes (sRBC). Macrophage function was depressed only in females, as indicated by the decreased ability of thioglycollate-recruited peritoneal exudate cells (PEC) to phagocytize sRBC.

Animals↗
PubMed1985

Preparation and analysis of antigen-specific memory B cells.

A procedure has been developed for the enrichment of TNP-binding memory B cells (TNP-MABC) from spleens of immunized mice. More than 75% of the cells expressed surface IgM (sIgM) and IgD (sIgD) and about 9% expressed surface IgG (sIgG). The TNP-MABC consisted of small resting lymphocytes with high affinity antigen-binding receptors. These cells expressed increased densities of Ia antigens and decreased densities of sIgD. Adoptive transfer of the cells into irradiated, carrier-primed syngeneic recipients resulted in their differentiation into IgG anti-TNP antibody-secreting cells. TNP-MABC secreted high affinity IgG anti-TNP antibodies when cultured in vitro with carrier-primed T cells and antigen. Limiting dilution analysis revealed that TNP-MABC contained a relatively low frequency of precursors for IgG-secreting cells that had an exceptionally large clone size. These results show that a highly enriched population of antigen-specific memory B cells can now be prepared and used to analyze their activation requirements.

Animals↗
PubMed1985

Role of alpha adrenoceptor activation in modulating the murine primary antibody response in vitro.

The present study shows that positive and negative modulation of the immunoglobulin M antibody response in mouse spleen cells immunized with sheep erythrocytes can be achieved by selective activation of alpha adrenoceptor subtypes. Alpha-1 adrenoceptor activation by methoxamine produced a number of spleen cells secreting immunoglobulin M antibody which was enhanced 63% above control on day 4 after immunization and which returned to control levels on days 5 (peak day of control antibody response), 6 and 7. This response mimicked the previously reported response produced by norepinephrine in the presence of propranolol, but not by norepinephrine alone. Alpha-2 adrenoceptor activation by clonidine produced no change when compared to control on days 4, 6 or 7, but produced a 50% suppression on day 5. Activation of both adrenoceptor subtypes by phenylephrine produced a control response on day 4, a depressed response on day 5 and an elevated response on days 6 and 7 by 50 and 64% above control, respectively. All drug responses were concentration-dependent and the methoxamine and clonidine responses were antagonized by phentolamine. These results suggest that antibody responses can be modulated by alpha-1 adrenoceptor activation to produce an enhanced response 1 day sooner than the peak control response and by alpha-2 adrenoceptor activation to produce a depressed response at the time of the peak control response.

Animals↗
PubMed1984

Beta adrenoceptor mediation of the enhancing effect of norepinephrine on the murine primary antibody response in vitro.

The beta adrenoceptor has been identified in this study to be the receptor responsible for the enhanced immunoglobulin M antibody response produced by norepinephrine in mouse spleen cells immunized with sheep erythrocytes in vitro. The magnitude and kinetics of the enhanced antibody response to norepinephrine alone, or to norepinephrine in the presence of phentolamine, were more closely mimicked with a beta-2 adrenoceptor agonist (terbutaline) than with a beta-1 adrenoceptor agonist (dobutamine). Norepinephrine alone, norepinephrine in the presence of phentolamine, or terbutaline exposure produced a number of spleen cells secreting immunoglobulin M antibody that is equal to control on day 4 after immunization and which is enhanced above control on days 5, 6 and 7. Dobutamine causes no change when compared to control on days 4 and 5, but causes a delayed decline in the response on days 6 and 7. All drug responses were concentration-dependent and propranolol antagonized the enhanced response observed in the presence of terbutaline or dobutamine alone. When norepinephrine was added to immunized spleen cell cultures in the presence of propranolol, an alpha adrenoceptor-mediated component was unmasked which produced an enhanced response on day 4 after immunization and returned to control levels on days 5, 6 and 7. These results suggest that antibody responses can be modulated positively by a sympathetic neurotransmitter. This up-modulation by norepinephrine is beta adrenoceptor-mediated at the time of, and after, peak control response and alpha adrenoceptor-mediated 1 day before peak control response.

Animals↗
PubMed1984

Kinetics of the enhancing effect produced by norepinephrine and terbutaline on the murine primary antibody response in vitro.

Experiments were performed to determine the minimal drug exposure time required for norepinephrine and a relatively selective beta-2 adrenoceptor agonist to produce a maximal enhancing effect on the murine primary antibody response in vitro. The antibody response was determined by counting the number of spleen cells producing immunoglobulin M antibody directed against sheep erythrocytes 5 days after exposure of spleen cells to antigen and drugs. Norepinephrine (10(-5) M) or terbutaline (10(-5) M) were added to mouse spleen cell suspensions at the time of immunization with sheep erythrocytes on day 0. Phentolamine (10(-5) M) and/or propranolol (10(-6) M) were added to the spleen cell cultures to block adrenoceptor activation either at the time of agonist exposure (time = 0 hr) or at various times after agonist exposure (time = 15 min-96 hr). The addition of adrenoceptor antagonists at times after agonist exposure allowed for determination of the minimal time required for adrenoceptor activation to produce a maximally enhanced antibody response. Norepinephrine and terbutaline produced a maximally enhanced antibody response, as measured 5 days later, after 5 to 6 hr of agonist exposure before the addition of the antagonists. Addition of the antagonists at times later than 5 to 6 hr after agonist had no effect on the maximal antibody response. When antagonists were added before this time, the magnitude of the enhancing effect attained was time-dependent. These results indicate that early adrenoceptor activation in vitro is responsible for initiating events which culminate in an enhanced primary antibody response measured a number of days after drug exposure.

Animals↗
PubMed1982

In vitro immunotoxicological assays for detection of compounds requiring metabolic activation.

A system for metabolic activation of cyclophosphamide (CP), consisting of a crude microsomal fraction of mouse liver and necessary cofactors (S9 mix), was interfaced with three murine cell culture assays for immunotoxicity. These assays were: the Mishell-Dutton assay for in vitro antibody formation, splenic lymphocyte responsiveness to mitogens and bone marrow cell cultures. There was no effect of CP at doses up to 261 microgram/ml (lmM) on any of the parameters measured unless S9 mix was included. Much greater potency was achieved if the S9 mix was prepared from livers of mice pretreated with phenobarbital. Under these conditions and dose-related inhibition of plaque-forming cells (PFC) in the Mishell-Dutton assay was observed, yielding an ED50 of 6.3 microgram/ml. When splenic lymphocytes were exposed to CP in the presence of induced S9 mix, a dose related inhibition of the response to the B-cell mitogen, lipopolysaccharide (LPS), and to the T-cell mitogen, concanavalin A (Con A), was observed. For the optimum LPS concentration, the ED50 for CP was 8.1 microgram/ml; for the optimum concentration of Con A, the ED50 was 6.7 microgram/ml. DNA synthesis was not inhibited by the doses used. When bone marrow cells were exposed to CP in the presence of induced S9 mix, the stem cell population, enumerated by colonization in semisolid medium, was reduced in a dose-dependent manner, with an ED50 of 5.2 microgram/ml. Again, DNA synthesis was not affected unless higher doses of CP were used.

Animals↗
PubMed1982

In vivo assessment of immunotoxicity.

The organs, tissues, and cells of the lymphoreticular system have received considerable attention as targets for chemicals causing adverse effects. A basic toxicological approach is described for assessing the risk of a chemical perturbing the immune system. CD-1 mice were exposed for 14 or 90 days to one of several chlorinated hydrocarbons: 1,2-dichloroethane, 1,2-dichloroethylene or 1,1,2-trichloroethylene. Other mice were exposed to dexamethasone, a known immunosuppressive agent. The immune system is evaluated against a background of the more standard toxicological parameters such as fluid consumption, body and organ weights, hematology, clinical chemistries, and blood coagulation. Reported here are the results for the male mice after 14-day exposure to three chlorinated hydrocarbons and after 90-day exposure to 1,2-dichloroethane and dexamethasone.Acute toxicity studies were performed to provide a basis for doses used in the subchronic studies. The LD(50) values are reported. The status of the humoral immune system was determined by measuring the number of IgM spleen antibody-forming cells to sRBC, the serum antibody level to sRBC, and the lymphocyte response to the B-cell mitogen, LPS. Of the three chlorinated hydrocarbons, only dichloroethane produced a significant (p < 0.05) reduction in antibody-forming cells. The other two chemicals produced trends towards suppression. Mice exposed to dichloroethane in the drinking water for 90 days showed no alteration in AFC, serum antibody titers or response to the B-lymphocyte mitogen, LPS. Subchronic 90-day exposure to dexamethasone produced a dose-dependent inhibition of AFC/spleen but not AFC/10(6) spleen cells when measured on the peak day of response. Response to LPS was not altered, and spleen weight and spleen cell number were reduced as much as 42%. These data suggest that dexamethasone administered in the drinking water is nonspecifically cytotoxic to the spleen cells.Cell-mediated immunity was assessed by measuring the DTH response to sRBC and the response to the T-lymphocyte mitogen, concanavalin A. After 14 days of exposure, trichloroethylene produced a 15 and 60% suppression at 24 and 240 mg/kg, respectively. Dichloroethylene produced a non-dose-dependent inhibition at 4.9 and 49 mg/kg, which was slight, but significant (p < 0.05). Subchronic 90-day exposure to dichloroethane did not alter the DTH response or spleen lymphocyte response to concanavalin A. In contrast, dexamethasone produced a dose-dependent inhibition of the DTH response and a hyperresponsiveness to concanavalin A.Dichloroethane did not alter the functional activity of the reticuloendothelial system, as measured by the vascular clearance rate and tissue uptake of (51)Cr sRBC. In the case of dexamethasone exposure, only the spleen and thymus showed decreased uptake of (51)Cr sRBC, which was directly related to decrease in size. The approaches and results from these types of studies provide a basis for judging a chemical's potential risk to the immune system.

Animals↗
PubMed1982

Toxicology of chloral hydrate in the mouse.

Chloral hydrate has been found in our drinking water supplies at levels up to 5 micrograms/1. The purpose of this study was to evalute the acute and subchronic toxicology of chloral hydrate in the random-bred CD-1 mouse, to provide data for risk assessment. The acute oral LD50 of this compound was 1442 and 1265 mg/kg in male and female mice, respectively. Acute toxicity appeared to be related to depression of the central nervous system. Fourteen-day exposure by gavage in male mice at doses 1/10 and 1/100 the LD50 caused an increase in liver weight and a decrease in spleen weight at the highest dose level. Based on the data derived from 14 days of exposure, a 90-day study was performed. The compound was delivered via the drinking water; levels of the compound delivered per day were equivalent to those dosed in the 14-day study. The target organ in both sexes appeared to be the liver, with the males most affected. Male mice demonstrated a dose-related hepatomegaly accompanied by significant changes in serum chemistries and hepatic microsomal parameters. The females did not demonstrate the hepatomegaly observed in males, but did show alterations in hepatic microsomal parameters. No other significant toxicological changes were observed in either sex following 90 days of exposure.

Administration, Oral↗