Natural killer cells and Cryptococcus neoformans.
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Animals↗
Biomedical subjects
J W Murphy
Publications and source records attributed to J W Murphy.
Cytokine profiles associated with induction of the anticryptococcal cell-mediated immune response.
Previous studies with a murine model have shown that immunization with cryptococcal culture filtrate antigen (CneF) emulsified in complete Freund adjuvant (CFA) induces two populations of anticryptococcal reactive CD4+ T cells. One population (TDH cells) transfers anticryptococcal delayed-type hypersensitivity (DTH), and the other population (Tamp cells) amplifies the anticryptococcal DTH response of given to recipient mice at the time of immunization of the recipient. Treatment of mice with cyclosporin A (CsA) ablates the induction of Tamp cells but not TDH cells. The present study focused on assessing the cytokines produced by spleen cells taken from CsA-treated and control (solvent-treated) mice at days 1, 2, 4, and 6 after immunization. Supernatants from the spleen cells cultured in vitro for 24 or 48 h in medium alone or with CneF, concanavalin A, or phorbol 12-myristate 13-acetate plus calcium ionophore were assessed for the presence of interleukin-2 (IL-2), gamma interferon (IFN-gamma), IL-4, IL-5, and tumor necrosis factor. Spleen cells from CneF-CFA-treated mice produced IL-2 and IFN-gamma, but not IL-4 or IL-5, constitutively and in response to CneF, indicating that CneF-CFA induces a Th1 response. Tumor necrosis factor was not produced. Anticryptococcal TDH cells developed in spleens in which there were low levels of IFN-gamma and IL-2 (CsA-treated, immunized mice), whereas anticryptococcal Tamp cells along with TDH cells matured in spleens in which production of IFN-gamma and IL-2 was high (solvent-treated, immunized mice). The data also suggest that IL-2 and IFN-gamma produced by Tamp cells early after adoptive transfer are influential in the development of the amplified anticryptococcal DTH response that has been observed in Tamp cell-recipient mice.
Animals↗
Mobility of human neutrophils in response to Cryptococcus neoformans cells, culture filtrate antigen, and individual components of the antigen.
The mobility of human neutrophils (PMN) in response to encapsulated or nonencapsulated Cryptococcus neoformans cells or cryptococcal culture filtrate (CneF) and its components was studied by using a 48-well modified Boyden chamber. Encapsulated C. neoformans (isolate 184A) cells and CneF-184A stimulated directed migration of human PMN in the absence of serum (direct chemotactic activity) and activated a heat-labile component(s) in fresh human serum to become a chemoattractant(s) for human PMN (indirect chemotactic activity). At a 1:8 dilution (0.25 mg of carbohydrate per ml), CneF-184A displayed chemokinetic activity when assessed with a checkerboard assay. Nonencapsulated C. neoformans isolate 602 cells did not have direct chemotactic activity but did have indirect chemotactic activity. The capsule of C. neoformans is composed predominantly of glucuronoxylomannan (GXM). Purified GXM displayed both direct and indirect chemotactic activity. CneF-184A contains, in addition to GXM, a concanavalin A-binding mannoprotein (MP), whereas CneF-602 contains no GXM but does contain MP. CneF-184A showed direct chemotactic activity and CneF-602 did not. Both CneF-184A and CneF-602 displayed indirect chemotactic activity for human PMN. In addition, purified MP from CneF-184A, like CneF-602, showed only indirect chemotactic activity. These results indicate that GXM contributes to the direct chemotactic activity of PMN observed with the whole encapsulated yeast cells and the unfractionated CneF derived from the encapsulated cells. Both MP and GXM from encapsulated C. neoformans cells mediate indirect chemotactic activity on human PMN.
Antigens, Fungal↗
Characterization of cellular infiltrates and cytokine production during the expression phase of the anticryptococcal delayed-type hypersensitivity response.
Cryptococcosis, an increasingly important opportunistic infection caused by the encapsulated yeast-like organism Cryptococcus neoformans, is limited by an anticryptococcal cell-mediated immune (CMI) response. Gaining a thorough understanding of the complex anticryptococcal CMI response is essential for developing means of controlling infections with C. neoformans. The murine cryptococcosis model utilizing footpad swelling to cryptococcal antigen (delayed-type hypersensitivity [DTH]) has proven to be a valuable tool for studying the induction and regulation of the anticryptococcal CMI response, but this technique has limitations with regard to evaluating the role of the final effector cells recruited by an ongoing CMI response. The purpose of this study was to assess the types of cells and cytokines induced into the site of cryptococcal antigen deposition in C. neoformans-infected and -immunized mice compared with those for control mice. We used a gelatin sponge implant model to examine the cells and cytokines present at the site of an anticryptococcal DTH response. Sponges implanted in infected mice and injected with cryptococcal culture filtrate antigen (CneF) 24 h before assessment had significantly increased numbers of infiltrating leukocytes compared with saline-injected sponges in the same animals. Exaggerated influxes of neutrophils and mononuclear cells were the major contributors to the increase in total numbers of cells in the DTH-reactive sponges. The numbers of CD4+ and LFA-1+ cells were found to be significantly increased in the CneF-injected sponges of infected and immunized mice over the numbers in control sponges. The numbers of large granular lymphocytes were also increased in DTH-reactive sponges compared with control sponges. Gamma interferon, interleukin 2 (IL-2), and IL-5 are clearly relevant cytokines in the anticryptococcal CMI response, since they were produced in greater amounts in the CneF-injected sponges from C. neoformans-infected and -immunized mice than in control sponges. IL-4 was not associated with the expression of DTH to cryptococcal antigen. The gelatin sponge model is an excellent tool for studying cells and cytokines involved in specific CMI responses.
Animals↗
Direct interactions of human lymphocytes with the yeast-like organism, Cryptococcus neoformans.
Lymphocytes, especially CD4+ T cells, are essential for clearance of the yeast-like organism Cryptococcus neoformans from the infected host. The mechanism(s) by which the lymphocytes facilitate elimination of cryptococci has not been elucidated. It is generally thought, however, that lymphocytes reactive with C. neoformans indirectly function by production of lymphokines to enhance clearance of the organism by natural effector cells such as macrophages. In the present study, we assessed the ability of freshly isolated human lymphocytes to interact directly with C. neoformans and to limit the growth of the organism in vitro. We found that large granular lymphocytes (LGL) as well as T cells bound to cryptococcal cells when the lymphocytes were mixed with the cryptococcal cells at a 2:1 ratio. The physical binding interactions of the two lymphocyte populations were different. LGL attached to the cryptococcal cells by many microvilli; T lymphocytes associated with the yeast through broad areas of membrane attached to the cryptococcal cell surface. The two types of lymphocyte interactions did not result in phagocytosis but resulted in direct inhibition of cryptococcal growth, making these lymphocyte interactions with cryptococci distinctly different from interactions of monocytes with cryptococci. With the human natural killer (NK) cell line, NK 3.3, we confirmed that NK cells that were present in the LGL population were capable of limiting the growth of C. neoformans. Through immunoelectron microscopy, human CD3+ lymphocytes were seen attached to cryptococcal cells and by mass cytolysis, human CD3+ lymphocytes were shown to be responsible for inhibition of C. neoformans growth. The direct inhibitory interactions of NK cells and T lymphocytes with cryptococcal cells may be important means of host defense against this ubiquitous organism that frequently causes life-threatening disease in AIDS patients.
CD3 Complex↗
Postmodernism and clinical practice: a critical analysis of the disease model.
The disease model attempts to explain a variety of phenomena ranging from mental illness to criminal behavior. This approach is grounded in the Western philosophical tradition of dualism. The limitations of the disease model are analyzed through a postmodern perspective. Socially sensitive strategies for clinical intervention are offered.
Adaptation, Psychological↗
Directional coronary atherectomy in acute myocardial infarction.
To date, application of directional coronary atherectomy (DCA) in acute myocardial infarction (AMI) has had limited reports. In eleven patients with AMI, DCA was applied. In three of these patients, DCA was used as a stand-alone procedure without use of thrombolytic agents. In each case a guidewire was placed across the stenosis, and in eight patients balloon angioplasty was utilized as a predilating modality prior to DCA. The thrombolytic agent urokinase was utilized in five of these eight patients, either before, during, or after angioplasty and/or DCA. DCA success (defined as ability to cross the lesion, reduction of less than or equal to 20% in stenosis and thrombolysis--when a thrombus is present) was achieved in 10 of 11 patients. One patient had persistent abrupt reclosure of an LAD lesion, accompanied by hemodynamic compromise, necessitating intra-aortic balloon pump insertion and subsequent emergent coronary artery bypass graft surgery. Final angiograms revealed residual stenoses less than or equal to 20%, and adequate thrombolysis. Significant cardiac events were limited to one emergent CABG, Q wave MI in four patients, and non-Q wave MI in two patients. Clinically all eleven patients improved, survived the AMI/CABG, and were discharged. This clinical experience demonstrates the feasibility and safety of DCA application in selected patients who experience acute myocardial infarction.
Adult↗
Cryptococcal immunity and immunostimulation.
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Animals↗
Immunomodulation in the mycoses.
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Animals↗
A method for hyperthermic treatment of mouse skin.
The Sencar mouse skin system is a recognized model for tumour initiation, promotion and progression. The current interest in the effect of hyperthermia on this multi-stage tumorigenesis model prompted the need for a technique to accurately heat a section of dorsal skin of a large number of mice for 30 min per heat treatment. In the technique described, experimental groups of 25 female Sencar mice were treated at 7-8 weeks of age under general methoxyflurane anaesthesia. Treatment consisted of the application of initiating and/or promoting agents with or without hyperthermia. For hyperthermic skin treatments, each group of mice was placed onto a platform in a water bath so that the dorsal skin of the mice was in contact with 44 degrees C temperature controlled water.
Anesthesia, General↗
Effects of Cryptococcus neoformans-specific suppressor T cells on the amplified anticryptococcal delayed-type hypersensitivity response.
Cell-mediated immunity is an important host resistance mechanism against Cryptococcus neoformans, the etiological agent of cryptococcosis. Previous studies from our laboratory have shown that the anticryptococcal cell-mediated immune response as measured by delayed-type hypersensitivity (DTH) is down-regulated by a cascade of antigen-specific T suppressor (Ts) cells. Recently, we have identified a population of CD4 T cells that up-regulate the anticryptococcal DTH response (Tamp cells). The Tamp cells are found in the spleens of donor mice at 6 days after immunization with cryptococcal antigen, and they amplify the anticryptococcal DTH response when transferred to syngeneic recipients at the time of immunization of the recipients. In this study, we determined the effects of C. neoformans-specific Ts cells on the induction of the Tamp cells in the Tamp cell-donor mice and on the induction and expression of the amplified anticryptococcal DTH response in the Tamp cell-recipient mice. When cryptococcal-specific Ts1 cells were given at the time of immunization of the Tamp cell-donor mice, induction of Tamp cells was inhibited. In contrast, when Ts1 cells were given at the time of adoptive transfer of Tamp cells, the recipients displayed amplified DTH responses, indicating that Ts1 cells do not affect the Tamp cells' function once the Tamp cells have been produced. C. neoformans-specific Ts2 cells given at the time of either immunization or footpad challenge of the Tamp cell-recipient mice did not alter, to any measurable extent, the amplified DTH response. These results indicate that in addition to amplifying the anticryptococcal DTH response, Tamp cells may protect the anticryptococcal TDH cells from suppression by C. neoformans-specific Ts cells, much like contrasuppressor cells do in other systems. However, further characterization of the Tamp cells revealed that they are not adherent to Viscia villosa lectin, indicating that the anticryptococcal Tamp cells do not have this characteristic in common with contrasuppressor cells of other antigen systems.
Animals↗
Binding interactions of murine natural killer cells with the fungal target Cryptococcus neoformans.
Murine natural killer (NK) cells have been shown to inhibit the growth of the yeastlike organism Cryptococcus neoformans both in vivo and in vitro. An essential first step in NK cell-mediated damage of cryptococcal cells is the binding of the NK cell to the cryptococcal cell. The studies presented here focused on the binding event. Electron photomicrographs and three-dimensional reconstructions of NK cell-C. neoformans conjugates show that NK cells bind to cryptococci through many microvilli. This is in contrast to the broad membrane-membrane interactions which form the binding site of NK cell-YAC-1 tumor cell conjugates. NK cell binding to cryptococci is much slower than NK cell binding to YAC-1 targets. Maximal conjugate formation with cryptococcal targets is reached after 2 h, whereas maximal conjugate formation with YAC-1 targets is obtained after 20 min. Once maximum NK cell-C, neoformans conjugate formation is obtained, another 4 h is required before damage to the cryptococcal cells can be detected with the CFU assay. These data indicate that the binding and action of NK cells on C. neoformans cells requires considerably more time than is necessary for similar events to occur in the NK cell-tumor cell model. NK cell membrane integrity is necessary for NK cells to bind to tumor targets, since some disruption of membrane integrity with 0.1 M dimethyl sulfoxide reduces conjugate formation and tumor cell lysis. In contrast, 0.1 M dimethyl sulfoxide did not diminish NK cell binding to cryptococcal targets; however, it significantly reduced cryptococcal growth inhibition. Although we have observed several differences in NK cell binding to the cryptococcal target compared with NK cell binding to tumor cell targets, there are some similarities in binding interactions of NK cells with the two different targets. Disulfide bonding appears to play a role in the binding of NK cells to both targets, since 5 mM 2-mercaptoethanol, a reagent that reduces disulfide bonds, prevented NK cells from binding to the tumor targets as well as the cryptococcal targets. Actin filaments, components of the cytoskeletal network, must be intact for NK cells to bind to YAC-1 cells or cryptococci. Taken together, our data confirm that binding of NK cells to the cryptococcal target is prerequisite to the stages that result in damage to the cryptococcal cell and that there are similarities and differences in NK cell-binding interactions with structurally different target cells.
Animals↗
Responses of murine natural killer cells to binding of the fungal target Cryptococcus neoformans.
Natural killer (NK) cells bind to and inhibit the growth of the fungal target Cryptococcus neoformans. Since C. neoformans is structurally and chemically distinct from the standard tumor cell target used in the model of NK cell-mediated cytotoxicity, this study was designed to investigate the NK cell response after binding to cryptococci. Transmission electron micrographs and three-dimensional reconstructions of NK cell-cryptococci conjugates demonstrated focusing of the NK cell centrioles and Golgi apparatus toward the cryptococcal attachment site. NK cell cytoskeletal changes after cryptococcal binding were confirmed by immunofluorescence studies in which NK cells were allowed to bind to cryptococci in Mg2(+)-containing, Ca2(+)-free medium. One hour after the addition of Ca2+ to the preformed conjugates, the bound NK cells demonstrated a significant increase in the percentage of microtubule organizing centers focused toward the cryptococcal binding site. Colchicine, a drug that inhibits microtubule assembly, did not affect NK cell-cryptococci binding but abrogated NK cell-mediated cryptococcal growth inhibition, indicating that microtubule assembly, an important prerequisite for the secretory process, is not required for NK cell-cryptococci binding but is essential for inhibition of cryptococcal growth. In addition, the Ca2+ channel-blocking reagents, lidocaine and verapamil, did not affect NK cell-cryptococci binding but blocked the NK cell-mediated anticryptococcal activity, suggesting that a Ca2+ flux is essential for inhibition of cryptococcal growth. Considered together, these data indicate that NK cells respond to binding of a target cell that has a capsule and cell wall, in addition to a cell membrane, in a manner similar to that seen following binding to target cells that are surrounded by only a cell membrane; however, the response of the NK cells to the binding of C. neoformans is slower and possibly less efficient than the response after tumor cell binding.
Animals↗
Murine natural killer cells are fungicidal to Cryptococcus neoformans.
Murine natural killer (NK) cells have been shown to bind to and inhibit the growth of Cryptococcus neoformans in vitro and to contribute to clearance of the organism in vivo. However, it is unclear whether NK cells actually kill cryptococci or simply inhibit proliferation of the fungal target. Therefore, the studies presented here were designed to determine whether NK cells are fungicidal to C. neoformans targets. C. neoformans viability was determined on the basis of the metabolic function of two different enzyme systems, as measured by the two vital stains MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] and fluorescein diacetate. Cryptococcal viability, as determined by vital stains, was compared with cryptococcal proliferation, as measured by microcolony formation in agarose at the individual cell level and by CFU counts or extinction dilution analysis in the total cell suspension. Initial comparisons of the vital stains and proliferation assays indicated that these methods effectively distinguished between live and heat-killed cryptococci at the individual cell level and in the total cell suspensions. After cryptococci were incubated with murine NK cells for 18 h, vital stains demonstrated that at the single conjugate level and in the total cell suspension, NK cells kill bound C. neoformans target cells. In addition, the numbers of dead cryptococci in the NK cell-C. neoformans suspensions as determined by the vital stains were comparable to the numbers of cryptococci that were unable to proliferate. Kinetics of NK cell-mediated C. neoformans binding and killing at the single conjugate level and in the total cell suspension were assessed by MTT staining at 2-h intervals after mixing effector and target cells, and the data support the concept that NK cell-C. neoformans binding precedes cryptococcal death. Furthermore, unbound, dead fungal cells were observed in the NK cell-C. neoformans suspensions after 18 h, suggesting that NK cell-C. neoformans interactions may involve both effector cell recycling and killing of unbound cryptococci by soluble cytotoxic factors. In conclusion, the results of these studies firmly establish that NK cells kill C. neoformans.
Animals↗
Early events in initiation of alternative complement pathway activation by the capsule of Cryptococcus neoformans.
The capsule of Cryptococcus neoformans is a powerful activator of the alternative complement pathway. This study examined the manner in which the cryptococcal capsule influences initiation of and early events in complement activation by C. neoformans. These studies examined the effects of the classical and alternative pathways on the kinetics and early sites for deposition of C3 fragments on encapsulated cryptococci, nonencapsulated cryptococci, and zymosan. The results showed that nonencapsulated cryptococci and zymosan are qualitatively and quantitatively similar in the manner in which they initiate complement activation. Both utilize the classical and alternative pathways. Initiation via the classical pathway occurs suddenly and simultaneously at sites distributed over the entire cell surface. Initiation of the alternative pathway by zymosan and nonencapsulated cryptococci is characterized by a lag of 6 to 8 min before appreciable amounts of C3 accumulate on the cells. Alternative pathway initiation by zymosan and nonencapsulated cryptococci occurs at a limited number of focal initiation sites that expand with alternative pathway amplification to cover the cell surface. Presence of the cryptococcal capsule blocks classical pathway initiation, which would normally occur at the cryptococcal cell wall, and produces an initiation that is dependent solely on the alternative pathway. Initiation of the alternative pathway by the cryptococcal capsule is characterized by a lag in C3 accumulation and the appearance of a limited number of focal initiation sites which resemble those observed when the alternative pathway is activated by zymosan and nonencapsulated cryptococci.
Complement Activation↗
Mechanisms of natural resistance to human pathogenic fungi.
Natural host-resistance mechanisms are essential first-line defenses against most mycotic agents; however, these defenses are often not sufficient for complete protection. The host relies on the immune responses to provide the additional antifungal activity necessary for maximum protection. In systemic mycotic diseases, the immune system must be functional to prevent the host from succumbing to the disease. Natural resistance mechanisms act together in a coordinated manner early in the disease process to either kill or prevent the fungal agent from proliferating and gaining entrance to other tissues. The early defensive measures of the natural effector mechanisms are usually sufficiently effective to provide time for the generally more effective immune defenses to develop. Together, the natural defenses and the immune defenses regulate each other through cytokine networks, and generally these systems provide the host with adequate protection against threatening mycotic agents.
Disease Susceptibility↗
A residency-based information system.
The medical literature has grown to an unmanageable size, and we need to develop information systems that provide better accessibility. An internal medicine residency program is an ideal setting for developing a system in which articles are screened for usefulness in clinical decision making and organized to be rapidly accessible and relatively inexpensive to use. In our residency training program, we use clinical problem-solving, journal club article review, and teaching conference preparation to generate appropriate articles from PAPERCHASE (MEDLINE) database searches. With the chief resident supervising the process, articles are then selected and organized in a computerized article file. The software program, Reference Manager (Research Information Systems, Inc., Encinitas, California), stores article citations and supports searches using standard, medical subject heading vocabulary key words. In an 18-month period, residents have collected more than 1800 references appropriate to clinical decision making and have established an article file that is now used daily as a medical information source.
Abstracting and Indexing↗
Cytoplasmic components of natural killer cells limit the growth of Cryptococcus neoformans.
Murine natural killer (NK) cell-mediated inhibition of growth of a yeast-like target cell, Cryptococcus neoformans, was completely abrogated by blocking the effector cell secretory process with monensin. Therefore, further studies were performed to determine the ability of various cytoplasmic fractions of NK cells to mediate inhibition of cryptococcal growth. Percoll-fractionated homogenates of rat LGL tumor cells demonstrated that the granule-containing fractions plus three additional sets of less dense cytoplasmic fractions displayed anti-cryptococcal activity; whereas only the cytoplasmic granule-containing fractions had cytotoxic activity against YAC-1 tumor cell and sheep erythrocyte targets. Maximal cryptococcal growth inhibition induced by LGL granules occurred after a 1 h incubation, required the presence of Ca2+ (1.0 mM) or Mg2+ (0.5 mM or 5.0 mM), and was completely abrogated in the presence of rabbit anti-LGL granule IgG. Cytolysin, the granule component which mediates tumor cell and sheep erythrocyte lysis, effectively limited the growth of cryptococci. Since Percoll gradient fractionation of the LGL homogenates demonstrated three separate peaks of anti-cryptococcal activity other than the granule peak, it is possible that the cytolysin-containing granules are not the only subcellular component of NK cells playing a role in inhibition of C. neoformans growth.
Animals↗