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Biomedical subjects

J W Murphy

Publications and source records attributed to J W Murphy.

At least 91 records · Page 5Linked to original sources
PubMed1986

Correlation of natural killer cell activity and clearance of Cryptococcus neoformans from mice after adoptive transfer of splenic nylon wool-nonadherent cells.

Previous reports demonstrate that natural killer (NK) cells inhibit the growth of Cryptococcus neoformans in vitro, but conclusive evidence supporting the effectiveness of NK cells in host resistance to cryptococci is not available. The objective of these studies was to assess the ability of NK cells to clear C. neoformans from the lungs, livers, and spleens of infected mice. CBA/J mice were depleted of NK cells, as well as other natural effector cells, by an intraperitoneal injection of cyclophosphamide (Cy), 240 mg/kg of body weight. One day later, 7.5 X 10(7) nylon wool-nonadherent (NWN) spleen cells, either untreated or treated with anti-asialo GM1 and complement to remove NK cells, were adoptively transferred to Cy-pretreated mice. On day 2 after Cy treatment, the mice were injected intravenously with 2 X 10(4) cryptococci. At 4 and 6 days after Cy treatment, tissues were assayed for NK reactivity, using a 4-h 51Cr-release assay, and for in vivo clearance of cryptococci as reflected by mean log10 CFU per organ. We observed that Cy treatment depleted NK activity against YAC-1 targets and reduced in vivo clearance of C. neoformans from the tissues of infected mice. Additionally, Cy treatment depleted the total lung and spleen cellularity and the total number of peripheral blood lymphocytes when compared with those in normal untreated control mice. Also, spleen weights were significantly decreased in comparison with those of untreated animals 4 days after Cy treatment. Adoptive transfer of untreated NWN spleen cells into Cy-depressed mice restored the NK cell activity which correlated with enhanced clearance of cryptococci from lungs, livers, and spleens. In contrast, treatment of NWN spleen cells with anti-asialo GM1 and complement before adoptive transfer abrogated the ability of these cells to restore NK activity or reduce the numbers of cryptococci present in tissues of infected mice. Taken together, these data indicate that NK cells are the cells effective in diminishing the numbers of cryptococci in tissues of infected mice. Consequently, NK cells may play a role in first-line host resistance against C. neoformans.

Animals↗
PubMed1986

Antibody-dependent natural killer cell-mediated growth inhibition of Cryptococcus neoformans.

Previous data from this laboratory indicate that normal murine nylon wool nonadherent splenic cells with characteristics of natural killer (NK) cells effectively inhibit in vitro growth of Cryptococcus neoformans, a yeastlike pathogen. Since NK cells have been shown to be involved in antibody-dependent, cell-mediated cytotoxicity against immunoglobulin G (IgG)-coated tumor cells and xenogenic erythrocytes, we were interested in assessing the effects of the IgG fraction of rabbit anticryptococcal serum on NK cell-mediated inhibition of C. neoformans growth. Early in the study it became apparent that the conventional method of determining the numbers of CFU that was used previously for assessment of viable cryptococci at the end of the growth inhibition assay was not reliable for these studies, owing to minor clumping of the organisms in the presence of anticryptococcal antibody. Therefore, the BACTEC radiometric system was evaluated and determined to be a reliable replacement for the CFU count method. Using the BACTEC methodology, we showed that the anticryptococcal antibody significantly augmented the in vitro ability of NK cells to inhibit the growth of C. neoformans compared with normal rabbit serum or tissue culture medium. Furthermore, the antibody alone did not have an adverse effect on the organism, confirming that reduced growth indices obtained from test wells containing antibody, NK cells, and cryptococci were due to the effects of the NK cells. Maximum anticryptococcal activity of the NK cells was observed in the presence of 16 micrograms of IgG per ml; however, significant augmentation of anticryptococcal activity was seen with antibody concentrations as low as 3 micrograms/ml. Using different populations of murine splenic cells which had varying degrees of NK cell activity, we were able to show that NK cell activities, as determined by 51Cr release from YAC-1 targets, directly correlated with antibody-dependent, cell-mediated growth inhibition against cryptococci, suggesting that NK cells were effector cells in the antibody-dependent assays. Furthermore, in every case, the antibody-dependent activity of NK cells against C. neoformans was higher than the spontaneous activity of NK cells against the organism, emphasizing that NK cell activity against cryptococci can be augmented by specific antibody. When NK cell numbers were enriched by Percoll fractionation of nylon wool nonadherent splenic cells, antibody-dependent and spontaneous growth inhibitory activities of the effector cells were concomitantly augmented, confirming that NK cells were the effector cells in antibody-dependent growth inhibition of cryptococci.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

Immunoadsorption of Cryptococcus-specific suppressor T-cell factors.

In the murine cryptococcal suppressor cell circuit, two different T-cell suppressor factors, TsF1 and TsF2, have been identified which specifically suppress the delayed-type hypersensitivity (DTH) response to cryptococcal culture filtrate antigen (CneF). TsF1 is produced by a first-order T suppressor (Ts1) cell population and suppresses the afferent limb of the DTH response, whereas TsF2 is produced by a second-order T suppressor (Ts2) cell population and suppresses the efferent limb of the cryptococcal DTH response. The objective of this study was to ascertain whether TsF1 or TsF2 could bind to cryptococcal antigen. To assess this, adsorption of TsF1 and TsF2 was performed with heat-killed Cryptococcus neoformans cells and by solid-phase immunoadsorption (SPIA) on columns containing cryptococcal antigens, i.e., CneF covalently bound to Sepharose 4B. The suppressive effect of TsF1 was removed by adsorption with intact heat-killed cryptococci and by SPIA on CneF-Sepharose 4B. The binding of cryptococcal TsF1 to the cryptococcal SPIA column was shown to be specific since Sepharose 4B columns either coupled with Saccharomyces cerevisiae mannan or blocked with glycine did not adsorb the suppressor activity. In contrast, the suppressive component of TsF2 did not bind to heat-killed cryptococci, CneF-Sepharose 4B, S. cerevisiae mannan-Sepharose 4B, or glycine-Sepharose 4B columns. These results, together with the finding that cryptococcal antigen, anticryptococcal antibody, and C1q-binding immune complexes were not demonstrated in either TsF1 or TsF2, establish that TsF1 and TsF2 can be differentiated on the basis of their affinity for cryptococcal antigen.

Animals↗
PubMed1985

Effects of first-order Cryptococcus-specific T-suppressor cells on induction of cells responsible for delayed-type hypersensitivity.

Cell-mediated immunity is an important aspect of host resistance against Cryptococcus neoformans. Using a CBA/J murine model, we demonstrated that injection of cryptococcal antigen (CneF) at dosages sufficient to stimulate the antigenemia observed in cryptococcosis patients induces specific T-cell-mediated suppression of the cryptococcal delayed-type hypersensitivity response. The purpose of this study was to establish whether Lyt 1+, first-order T-suppressor (Ts1) cells block the induction of T cells responsible for delayed-type hypersensitivity (TDH cells) or whether they function by inducing Lyt 2+, efferent suppressor (Ts2) cells. In one set of experiments, suppression was observed when Ts1 cells were adoptively transferred to recipient animals the day before, the day of, or the day after immunization; however, when Ts1 cells were transferred after TDH cells were present, no suppression occurred. In other experiments, putative TDH cells from lymph nodes (LN) or spleens were adoptively transferred from mice after immunization or after a suppressive dose of CneF or adoptive transfer of Ts1 cells and immunization. Delayed-type hypersensitivity could not be transferred with LN or spleen cells from mice receiving the suppressive dose of CneF or the Ts1 cells, even when the LN or spleen cells were treated with anti-Lyt 2.1 antibody and complement to remove any Ts2 cells. Delayed-type hypersensitivity was readily transferred with LN or spleen cells from immunized mice whether the cells were or were not treated with anti-Lyt 2 and complement. Furthermore, the cells in the tolerized LN cell pools responsible for suppression of TDH cell induction were Lyt 1+ 2-, I-J+ cells, which is the phenotype of the Ts1 cells. Taken together, these data indicate that Ts1 cells inhibit the induction of TDH cells. This finding, coupled with the previous demonstration that Ts1 cells or a Ts1 cell-derived soluble factor (TsF1) induces Ts2 cells, establishes that the cryptococcal Ts1 cells are bifunctional in the suppressive pathway.

Animals↗
PubMed1985

In vitro binding of natural killer cells to Cryptococcus neoformans targets.

Nylon wool-nonadherent splenic cells from 7- to 8-week-old CBA mice were further fractionated on discontinuous Percoll gradients. Enrichment of natural killer (NK) cells in Percoll fractions 1 and 2 was confirmed by morphological examination, by immunofluorescent staining, and by assessing the cytolytic activity of each Percoll cell fraction against YAC-1 targets in the 4-h51Cr release assay. Cells isolated from each Percoll fraction were tested for growth-inhibitory activity against Cryptococcus neoformans, a pathogenic yeastlike organism, by using an in vitro 18-h growth inhibition assay. The results showed that NK cell enrichment was concomitant with enrichment of anti-Cryptococcus activity in Percoll fractions 1 and 2. Cells from NK cell-rich fractions formed conjugates with the mycotic targets similar to the conjugates reported in NK cell-tumor systems. In addition, the percentage of effector cell-Cryptococcus conjugates was directly proportional to the level of the C. neoformans growth-inhibitory activity of the effector cells used. Scanning electron microscopy of the effector cell-Cryptococcus conjugates showed direct contact between the effector cells and the cryptococcal targets. An immunolabeling method combined with scanning electron microscopy was used to demonstrate that the effector cells attached to C. neoformans were asialo GM1 positive and, therefore, had NK cell characteristics.

Animals↗
PubMed1985

Regulation of cell-mediated immunity in cryptococcosis. III. Characterization of second-order T suppressor cells (Ts2).

Frequently, patients with systemic cryptococcosis have depressed or absent in vivo or in vitro cell-mediated immune responses to cryptococcal antigen. In addition, these patients have relatively high levels of circulating cryptococcal antigen, which is generally considered indicative of a poor prognosis. Because acquired cell-mediated immunity is an important host defense mechanism in cryptococcosis, we have been studying the effects of cryptococcal antigen on such responses. Using a murine model, we have demonstrated that cryptococcal antigen given i.v. to CBA/J mice, to simulate antigen levels found in patients, can trigger the production of a series of suppressor cells that specifically inhibit anticryptococcal cell-mediated immune responses. Briefly, cryptococcal antigen induces afferent suppressor and/or suppressor inducer (Ts1) cells in the lymph nodes of mice, and the Ts1 cells, or a soluble factor derived therefrom (TsF1), stimulate the production of second-order or efferent suppressor (Ts2) cells. This report provides data that demonstrate that the Ts2 cells were induced in spleens from cyclophosphamide (Cy)-resistant precursors in response to cryptococcal Ts1 cells or TsF1 and that a proliferative process sensitive to Cy was not required for Ts2 induction. The Ts2 cells were detectable 6 days after Ts1 injection, were specific in suppressing the cryptococcal delayed-type hypersensitivity (DTH) response, were T cells with an Lyt-1-,2+,Ia+ (I-J+) phenotype, were effective only on the efferent limb of the cryptococcal DTH response, and mediated their activity via a soluble factor (TsF2). Thus far, the suppressive pathway induced by cryptococcal antigen has characteristics more like those of the suppressor circuits described for the DTH responses to the haptens azobenzenearsonate and 4-hydroxy-3-nitrophenyl acetyl than those of the modulating circuits induced by other antigens. We postulate that a suppressive circuit similar to the one we have defined in the mouse model is operating to suppress the in vivo and in vitro cell-mediated immune responses in cryptococcosis patients who have significant amounts of circulating cryptococcal antigen.

Animals↗
PubMed1984

Abdominal and pelvic imaging as part of liver-spleen scintigraphy for the detection of mesenteric bleeding in trauma. A case report.

Liver--spleen scintigraphy in patients who have incurred blunt abdominal trauma is accepted as a useful diagnostic tool. Abdominal and pelvic imaging to detect extrahepatic or extrasplenic sites of hemorrhage should be a routine part of liver--spleen imaging in the evaluation of blunt abdominal injury. A patient with mesenteric bleeding and a normal liver and spleen detected by liver--spleen scintigraphy is reported.

Abdomen↗
PubMed1984

In vitro effects of natural killer cells against Paracoccidioides brasiliensis yeast phase.

Recently, data have been reported suggesting natural killer (NK) cells may function in natural resistance against a fungus, Cryptococcus neoformans. The primary objective of this study was to examine the reactivity of murine splenic cells against another fungus, Paracoccidioides brasiliensis. Levels of NK activity in effector cell pools were varied by: (i) removing nylon wool-adherent cells, (ii) fractionating splenic cells on Percoll discontinuous gradients, (iii) using old and young effector cell donor mice, (iv) using donors from different strains, and (v) pretreating donors with NK-augmenting and -depressing agents. The various effector cell pools were simultaneously used in the 4-h 51Cr release assay with YAC-1 targets to determine the NK reactivity and in the in vitro growth inhibition assay against P. brasiliensis yeast phase targets. In each case, the level of NK reactivity correlated with the ability of the effector cells to inhibit the in vitro growth of P. brasiliensis. NK activity and P. brasiliensis growth-inhibiting ability could be augmented by fractionation of splenic cells through nylon wool or Percoll gradients. The effector cells responsible for the NK activity and P. brasiliensis growth inhibition were characterized as being nylon wool nonadherent, being found in the low-density fractions from Percoll discontinuous gradients, and having no detectable Thy-1 antigen or immunoglobulin but having asialo GM1 on their surface. These data support the contention that NK or NK-like cells are responsible for limiting the in vitro growth of P. brasiliensis.

Animals↗
PubMed1983

Cellular immunity in a cutaneous model of cryptococcosis.

The temporal development of cellular immune responses in mice inoculated cutaneously with viable Cryptococcus neoformans 145 was determined in vivo and in vitro by comparing several antigen preparations for their efficacy in the assays selected. Three antigens derived from C. neoformans 145, viz., a culture filtrate preparation (CneF-145), a membrane extract (B-HEX), and soluble cytoplasmic substances (SCS), were compared for their ability to detect delayed hypersensitivity (DH) in vivo in a footpad assay or to stimulate lymphocytes in vitro in a thymidine incorporation assay. DH to B-HEX could be demonstrated as early as 1 week after infection, whereas significant responses to SCS and CneF-145 were not regularly detected until 3 weeks after infection. Substantial reactions were observed to all three antigens up to 12 weeks, although they peaked at 2 to 3 weeks. Reactions to B-HEX and SCS were somewhat better than those to CneF. Differences in the efficacies of the three antigens were not obvious after the sixth week of infection, however. In vitro, lymph node cells from infected animals were stimulated significantly with all three antigens beginning at week 1. As with DH, however, responses to CneF-145 were usually less than those to SCS and B-HEX. In vitro lymphocyte responses waned after approximately 6 weeks, whereas DH responses were clearly positive through 12 weeks. In addition to the studies in infected animals, animals immunized with heat-killed cells of C. neoformans 145 or 184 were tested 6 to 8 days later for DH with CneF-145, CneF-184, or B-HEX derived from C. neoformans 145. The CneF-145 and CneF-184 were equally effective for detecting DH, regardless of the cryptococcal strain used for immunization. Likewise, the B-HEX detected equivalent responses in mice sensitized with each cryptococcal strain. Since all three antigens were soluble and easily extracted and since each elicited significant cellular immune responses in infected animals, further studies involving their specificity and the nature of their reactive components seems warranted as they may help evaluate immune responses in humans infected with this fungus.

Animals↗
PubMed1983

Regulation of cell-mediated immunity in cryptococcosis. II. Characterization of first-order T suppressor cells (Ts1) and induction of second-order suppressor cells.

Cryptococcosis patients frequently have high levels of cryptococcal antigen in their body fluids, and the levels of circulating antigen can generally be used to predict the patient's recovery, with high or rising antigen titers indicating a poor prognosis and low or decreasing levels a good prognosis. In a previous study, we reported on a murine model for studying the effects of cryptococcal antigen on host defense mechanisms. In that work, we demonstrated that an i.v. injection of cryptococcal antigen (CneF) into CBA/J mice, to simulate the antigenemia known to occur in human cryptococcosis, induced a population of T suppressor cells (Ts1) in the lymph nodes (LN). Upon adoptive transfer, the Ts1 cells specifically suppressed the afferent limb of the delayed-type hypersensitivity (DTH) response to cryptococcal antigen. In the present study, we show that the precursors of the Ts1 cells are sensitive to low-dose cyclophosphamide treatment and that the phenotype of the Ts1 cells is Lyt-1+, Ia+ (I-J+). LN cells from CneF-injected mice or a soluble factor derived therefrom can induce in the spleens of recipient mice a second-order suppressor cell population that suppresses the efferent limb of the DTH response. The cells that induce the second-order or efferent suppressor cells have the same phenotype as the cells that appear to suppress the afferent limb of the DTH response. The findings in this study indicate that a complex regulatory mechanism is responsible for the observed suppression of the DTH response in this infectious disease model. Furthermore, the suppressive circuit thus far defined for cryptococcal antigen is similar to the antigen-specific suppressor cell pathway outlined for certain chemically defined haptenic systems.

Animals↗
PubMed1982

Soluble factors in tolerance and contact sensitivity to 2,4-dinitrofluorobenzene in mice. IV. Characterization of migration inhibition factor-producing lymphocytes and genetic requirements for activation.

The production of migration inhibition factor (MIF) in vitro by lymph node cells from mice with contact sensitivity to 2,4-dinitrofluorobenzene (DNFB) was investigated. MIF activity of cell-free culture supernatants was measured using a micro, indirect "hanging-drop" assay system. We found that DNFB-sensitized lymph node cells are stimulated to produce MIF by co-culture with DNP-labeled spleen cells or splenic adherent cells. The stimulation was quantitatively antigen-specific, as co-culture with TNP-spleen cells or TNP-splenic adherent cells induced only low levels of MIF activity. Pretreating the immune lymph node cells with different antisera plus complement, before addition of DNP-spleen cells, showed that MIF production is dependent on Ia- T cells. Additional experiments showed that in order for the T cells to be stimulated, homology at the I-A subregion of the major histocompatibility complex between the T cells and DNP-spleen cells is required. Collectively, these results correlate with our previous finding that transfer of contact sensitivity is mediated by Ia- T cells and indicate that both tests, i.e., transfer in vivo and MIF production in vitro, are measuring effector functions of the same T cell subset.

Animals↗
PubMed1982

The use of management by objectives in medical education enrichment programs.

Since numerous medical education enrichment programs have been established throughout the United States, their effectiveness must eventually be evaluated. It is difficult to evaluate the goals of these programs. The programs do have goals, yet most program managers do not have a simple tool for transforming these broad statements into measurable performance standards. No program can be properly prepared for evaluation unless a goal clarification process is initiated. The authors maintain that "management by objectives" is an appropriate process for refining a program's goals and enables the enrichment programs to be fairly evaluated.

Education, Premedical↗
PubMed1982

In vitro interactions of immune lymphocytes and Cryptococcus neoformans.

CBA/J mice immunized subcutaneously with emulsions of heat-killed Cryptococcus neoformans in complete Freund adjuvant displayed delayed-type hypersensitivity to cryptococcal culture filtrate antigen and developed sensitized splenic lymphoid cells which inhibited the growth of C. neoformans in vitro. The in vitro assay of growth inhibition served to investigate further the kinetics of the effect of sensitized lymphoid cells on the pathogen. There was a close correlation between the delayed-type hypersensitivity response in mice and inhibition of growth of C. neoformans by lymphoid cells. Sensitized splenic lymphocytes capable of inhibiting the growth of the cryptococci were detected at day 6 after immunization and reached maximum levels by days 8 through 16. Inhibition of growth was highest with effector-to-target cell ratios of 300:1 or greater. Inhibition of growth of C. neoformans by sensitized lymphoid cells was detectable as early as 4 h after effector and target cells were mixed and increased gradually, reaching a maximum at 24 h, but dropped significantly by 48 h. By supplementing the reaction mixtures with fresh medium or additional sensitized effector cells during incubation, the inhibition of growth of C. neoformans could be maintained through 48 h. C. neoformans-sensitized effector lymphoid populations not only inhibited the growth of the pathogen in vitro but also restricted C. neoformans proliferation in various vital organs upon transfer to naive recipient animals, indicating that the in vitro growth inhibition assay may be a means of assessing the resistance of animals to C. neoformans. The effector cells from sensitized animals were nylon wool-nonadherent Thy-1+ and Ia+ lymphocytes.

Animals↗