Biomedical subjects

T R Kozel

Publications and source records attributed to T R Kozel.

At least 19 recordsLinked to original sources

PubMed1999

B7 costimulatory ligand regulates development of the T-cell response to Cryptococcus neoformans.

The contribution of B7 molecules to the induction and maintenance of the T-cell response to the human pathogenic fungus Cryptococcus neoformans was investigated. T-cell activation by C. neoformans was regulated by B7 molecules. This costimulatory signal was necessary for initiation and maintenance of the T-cell response, through early and late requirements for B7-CD28 interaction. Blocking B7-2 inhibited the normal T-cell proliferative response. This inhibition was due, in part, to a reduced capability of T cells to produce interleukin-2 (IL-2). In contrast, the same T-cell population produced more interferon-gamma. Suppression of the normal lymphoproliferation and IL-2 secretion responses to encapsulated C. neoformans by antibodies to B7 was largely reversed by addition of the monoclonal antibody 2H1, that is reactive with the major capsular polysaccharide, glucuronoxylomannan. Overall, our data indicate that B7 molecules play a critical role in T-cell activation by C. neoformans and suggest that appropriate manipulation could drive T helper type 1 cell development.

Antibodies, Monoclonal↗

PubMed1999

The capsule of Cryptococcus neoformans reduces T-lymphocyte proliferation by reducing phagocytosis, which can be restored with anticapsular antibody.

Cell-mediated immunity is critical for the host defense to Cryptococcus neoformans, as demonstrated by numerous animal studies and the prevalence of the infection in AIDS patients. Previous studies have established that the polysaccharide capsule contributes to the virulence of C. neoformans by suppressing T-lymphocyte proliferation, which reflects the clonal expansion of T lymphocytes that is a hallmark of cell-mediated immunity. The present studies were performed to identify the major mechanism by which polysaccharide impairs lymphocyte proliferation, since capsular polysaccharide has the potential to affect the development of T-lymphocyte responses by stimulating production of interleukin-10 (IL-10), inhibiting phagocytosis, and inducing shedding of cell surface receptors. We demonstrate that polysaccharide inhibits lymphocyte proliferation predominantly by blocking uptake of C. neoformans, which is crucial for subsequent lymphocyte proliferation. In addition, we show that polysaccharide did not suppress lymphocyte proliferation via an IL-10-dependent mechanism, nor did it affect critical surface receptor interactions on the T cell or antigen-presenting cell. Having established that polysaccharide impairs phagocytosis, we performed studies to determine whether opsonization with human serum or with anticapsular antibody could reverse this effect. Impaired uptake and lymphocyte proliferation that were induced by polysaccharide can be enhanced through opsonization with monoclonal antibodies or human serum, suggesting that antipolysaccharide antibodies might enhance the host defense by restoring uptake of the organism and subsequent presentation to T lymphocytes. These studies support the therapeutic potential of stimulating cell-mediated immunity to C. neoformans with anticapsular antibody.

Adult↗

PubMed1998

Monoclonal antibodies reveal additional epitopes of serotype D Cryptococcus neoformans capsular glucuronoxylomannan that elicit protective antibodies.

Epitope specificity and isotype influence mAb efficacy against Cryptococcus neoformans; however, the relative contribution of each attribute is poorly understood. To date, only mAbs that recognize two epitopes of capsular glucuronoxylomannan (GXM), defined by the IgG1 mAbs 2H1 and E1, consistently mediate protection against C. neoformans. The role of epitope specificity was further examined using six additional IgG1 mAbs and serotype D C. neoformans ATCC 24067. mAbs 3C2, 439, and 471 recognize the 2H1 epitope, whereas mAbs 339, 1255, and 302 recognize two separate epitopes. mAbs 3C2, 439, and 471 competed for GXM with the IgA mAb 18G9, a 2H1 mAb family member, whereas mAbs 302, 339, and 1255 did not. Each mAb bound GXM similarly, as determined by agglutination, direct Ag binding, Ag inhibition, and indirect capsular immunofluorescence assays. mAb apparent affinity constants for GXM ranged from 5 to 26 x 10(7) M(-1) with mAb 1255 > 3C2 > 339 > 439 > 471 > 302. Each mAb significantly prolonged survival (p < 0.05); the average survival times of control and mice passively immunized with mAbs 3C2, 302, 339, 439, 471, and 1255 were 10.8, 36.6, 33, 25.5, 24.9, 17, and 22.6 days, respectively. Although each mAb enhanced J774.16 cell fungicidal activity, differences were observed in the ability of each mAb to facilitate attachment and ingestion of cryptococci. These results indicate 1) two additional epitope specificities associated with mAb efficacy, 2) differences in opsonic and protective efficacy for IgG1 anti-GXM mAbs, 3) an association between affinity and protective efficacy, and 4) additional support for association between an annular indirect capsular immunofluorescence pattern and mAb efficacy.

Agglutination Tests↗

PubMed1998

Cryptococcus neoformans and Candida albicans regulate CD4 expression on human monocytes.

This study examined the capability of Candida albicans and Cryptococcus neoformans to modulate CD4 expression on human monocytes. C. albicans and an acapsular strain of C. neoformans induced higher levels of CD4 expression than encapsulated strains. Purified glucuronoxylomannan did not regulate CD4 expression on monocytes, but down-regulation of CD4 expression compared with stimulation by acapsular C. neoformans alone was observed when glucuronoxylomannan was used in combination with acapsular C. neoformans. The ability of opsonic factors to facilitate fungal-mediated CD4 overexpression suggests that binding or internalization (or both) of the yeast cells is a critical event. Protein synthesis was required, excluding redistribution of the intracellular pool of CD4 receptors to the cellular surface as the sole possible mechanism. Results demonstrate a new effect of fungi on professional phagocytic cells and raise the possibility that modulation of CD4 could influence gp120-mediated human immunodeficiency virus entry.

CD4 Antigens↗

PubMed1998

Characterization of a murine monoclonal antibody to Cryptococcus neoformans polysaccharide that is a candidate for human therapeutic studies.

The murine monoclonal antibody (MAb) 18B7 [immunoglobulin G1(kappa)] is in preclinical development for treatment of Cryptococcus neoformans infections. In anticipation of its use in humans, we defined the serological and biological properties of MAb 18B7 in detail. Structural comparison to the related protective MAb 2H1 revealed conservation of the antigen binding site despite several amino acid differences. MAb 18B7 was shown by immunofluorescence and agglutination studies to bind to all four serotypes of C. neoformans, opsonize C. neoformans serotypes A and D, enhance human and mouse effector cell antifungal activity, and activate the complement pathway leading to deposition of complement component 3 (C3) on the cryptococcal capsule. Administration of MAb 18B7 to mice led to rapid clearance of serum cryptococcal antigen and deposition in the liver and spleen. Immunohistochemical studies revealed that MAb 18B7 bound to capsular glucuronoxylomannan in infected mouse tissues. No reactivity of MAb 18B7 with normal human, rat, or mouse tissues was detected. The results show that both the variable and constant regions of MAb 18B7 are biologically functional and support the use of this MAb in human therapeutic trials.

Amino Acid Sequence↗

PubMed1998

Mannan-specific immunoglobulin G antibodies in normal human serum accelerate binding of C3 to Candida albicans via the alternative complement pathway.

Candida albicans activates the classical and alternative complement pathways, leading to deposition of opsonic complement fragments on the cell surface. Our previous studies found that antimannan immunoglobulin G (IgG) in normal human serum (NHS) allows C. albicans to initiate the classical pathway. The purpose of this study was to determine whether antimannan IgG also plays a role in initiation of the alternative pathway. Pooled NHS was rendered free of classical pathway activity by chelation of serum Ca2+ with EGTA alone or in combination with immunoaffinity removal of antimannan antibodies. Kinetic analysis revealed a 6-min lag in detection of C3 binding to C. albicans incubated in EGTA-chelated NHS, compared to a 12-min lag in NHS that was both EGTA chelated and mannan absorbed. The 12-min lag was shortened to 6 min by addition of affinity-purified antimannan IgG. The accelerating effect of antimannan IgG on alternative pathway initiation was dose dependent and was reproduced in a complement binding reaction consisting of six purified proteins of the alternative pathway. Both Fab and F(ab')2 fragments of antimannan IgG facilitated alternative pathway initiation in a manner similar to that observed with intact antibody. Immunofluorescence analysis showed that addition of antimannan IgG to EGTA-chelated and mannan-absorbed serum promoted an early deposition of C3 molecules on the yeast cells but had little or no effect on distribution of the cellular sites for C3 activation. Thus, antimannan IgG antibodies play an important regulatory role in interactions between the host complement system and C. albicans.

Antibodies, Fungal↗

PubMed1998

Contrasting roles of mannan-specific monoclonal immunoglobulin M antibodies in the activation of classical and alternative pathways by Candida albicans.

Polyclonal antimannan immunoglobulin G (IgG) activates the classical complement pathway and accelerates initiation of the alternative pathway by Canidida albicans. This dual role was assessed for two antimannan IgM monoclonal antibodies (MAbs). MAb B6.1 is specific for an epitope on the acid-labile portion of C. albicans phosphomannan; MAb B6 is specific for an epitope on the acid-stable region. Both MAbs were potent activators of the classical pathway but poor facilitators of alternative pathway initiation.

Antibodies, Monoclonal↗

PubMed1998

Encapsulation of Cryptococcus neoformans with glucuronoxylomannan inhibits the antigen-presenting capacity of monocytes.

This report examines the effect of the major capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), on the antigen-presenting capability of human monocytes treated with acapsular cells of C. neoformans. We found that pretreatment of acapsular cryptococci with GXM downregulates, in a dose-dependent manner, the antigen-presenting capacity of monocytes, leading to reduced proliferative T-lymphocyte responses. Similar levels of suppression occurred when monocytes were exposed to encapsulated cryptococci or acapsular cryptococci that were pretreated with GXM. The magnitude of the T-cell response correlated with the ability of monocytes to ingest the yeast. Supernatant fluids from cocultures of monocytes and T cells cultured with encapsulated cryptococci contained higher levels of interleukin-10 (IL-10) than supernatant fluids of cells with acapsular cryptococci. Addition of anti-IL-10 monoclonal antibodies to the incubation medium of monocytes and T cells cultured with encapsulated cryptococci restored proliferative T-cell responses to levels observed during culture with acapsular cryptococci. Finally, treatment of monocytes with encapsulated cryptococci or GXM-treated acapsular cryptococci suppressed expression of class II major histocompatibility complex (MHC) molecules in a manner consistent with previous reports of IL-10-mediated suppression of class II MHC molecules and suppression of proliferative T-cell responses. These results suggest a link between GXM encapsulation, increased IL-10 synthesis by monocytes, decreased expression of class II MHC molecules on monocytes, and reduced proliferative T-cell responses.

Antigen Presentation↗

PubMed1998

Characterization of anticapsular monoclonal antibodies that regulate activation of the complement system by the Cryptococcus neoformans capsule.

Incubation of the encapsulated yeast Cryptococcus neoformans in human serum leads to alternative pathway-mediated deposition of C3 fragments in the capsule. We examined the ability of monoclonal antibodies (MAbs) specific for different epitopes of the major capsular polysaccharide to alter the kinetics for classical and alternative pathway-mediated deposition of C3 onto a serotype A strain. We studied MAbs reactive with capsular serotypes A, B, C, and D (MAb group II); serotypes A, B, and D (MAb group III); and serotypes A and D (MAb group IV). The MAb groupings are based on antibody variable region usage which determines the antibody molecular structure. When both the classical and alternative pathways were operative, group II MAbs induced early classical pathway-mediated binding of C3 but reduced the overall rate of C3 accumulation and the amount of bound C3. Group III MAbs closely mimicked the effects of group II MAbs but exhibited reduced support of early classical pathway-facilitated accumulation of C3. Depending on the antibody isotype, group IV MAbs slightly or markedly enhanced early binding of C3 but had no effect on either the rate of C3 accumulation or the amount of bound C3. When the classical pathway was blocked, group II and III MAbs markedly suppressed C3 binding that normally would have occurred via the alternative pathway. In contrast, MAbs of group IV had no effect on alternative pathway-mediated C3 binding. These results indicate that anticapsular antibodies with different epitope specificities may have distinct regulatory effects on activation and binding of C3.

Adult↗

PubMed1998

Bivalency is required for anticapsular monoclonal antibodies to optimally suppress activation of the alternative complement pathway by the Cryptococcus neoformans capsule.

Encapsulated cells of Cryptococcus neoformans are potent activators of the alternative complement pathway. Previous studies found that monoclonal antibodies (MAbs) specific for the major capsular polysaccharide, termed glucuronoxylomannan (GXM), can markedly suppress the ability of the capsule to accumulate C3 from normal human serum via the alternative pathway. The present study examined the abilities of F(ab)2 and Fab fragments of three MAbs (MAbs 439, 3C2, and 471) to mediate the suppressive effect. The results showed that F(ab)2 fragments of all three MAbs suppressed activation and binding of C3 via the alternative pathway in a manner similar to that of intact antibodies. In contrast, Fab fragments of MAb 439 and MAb 3C2 showed no suppressive activity, and Fab fragments of MAb 471 were markedly reduced in suppressive activity. Indeed, there was an earlier accumulation of C3 on encapsulated cryptococci in the presence of the Fab fragments. Study of subclass switch families of MAb 439 and MAb 471 found that MAbs of an immunoglobulin G (IgG) subclass with increased flexibility in the hinge region (IgG2b) had less suppressive activity than MAbs of IgG subclasses with less flexibility (IgG1 or IgG2a). Taken together, these results indicate that cross-linking of the capsular matrix is an essential component in suppression of the alternative complement pathway by anti-GXM MAbs.

Adult↗

PubMed1998

Involvement of C3a and C5a in interleukin-8 secretion by human polymorphonuclear cells in response to capsular material of Cryptococcus neoformans.

In a previous paper we demonstrated that human polymorphonuclear cells (PMN) in the presence of normal human serum (NHS) secrete proinflammatory cytokines in response to Cryptococcus neoformans or its major capsular component, glucuronoxylomannan (GXM). The hypothesis that activation of the complement system could be responsible for the observed phenomenon is supported by the fact that encapsulated and acapsular C. neoformans isolates are activators of the complement system and, in particular, large encapsulated isolates are powerful activators. In the present study we demonstrate that (i) interleukin-8 (IL-8) release in response to acapsular or encapsulated strains of C. neoformans is significantly reduced in the presence of heat-inactivated serum rather than NHS and is completely abrogated in the absence of human serum; (ii) GXM-induced IL-8 release is strictly dependent on the presence of NHS, is inhibited by specific antibodies to either C3a and C5 complement components, and is completely abrogated by the combined use of these antibodies; (iii) the addition of purified C3a and C5a directly stimulates IL-8 release by PMN; and (iv) monoclonal antibody to GXM in combination with GXM or encapsulated C. neoformans potentiates IL-8 release by PMN. These data shed light on the mechanism involved in GXM-induced IL-8 secretion by PMN, provide an additional potential role for complement in the control of C. neoformans infections, and suggest a complex interplay between the complement system, humoral immunity, and cytokine regulation.

Cells, Cultured↗

PubMed1997

Reactivity patterns and epitope specificities of anti-Cryptococcus neoformans monoclonal antibodies by enzyme-linked immunosorbent assay and dot enzyme assay.

Cryptococcus neoformans glucuronoxylomannans (GXM) are capsular polysaccharides important for virulence in cryptococcosis. This study used dot enzyme assays (DEA) and enzyme-linked immunosorbent assays (ELISA) to determine the reactivity patterns of 21 murine monoclonal antibodies (MAbs) with structurally defined GXMs from five serotypes. The MAbs were categorized into eight groups on the basis of DEA and five groups on the basis of ELISA. MAbs 302, 339, and 439 were studied extensively for their binding to various native and chemically modified GXMs. Quantitative variation in the inhibitory effects of GXMs on the binding of MAbs 302, 339, and 439 were observed by competitive ELISA. O-Deacetylation of serotype A, B, and D GXM resulted in the complete loss of their inhibitory properties. Carboxyl group reduction of GXMs from serotypes A and D resulted in a significant decrease of inhibitory activity for MAb. Xylomannans and methyl glycosides exhibited no detectable inhibitory activity on MAb binding to GXM. The results indicate (i) the existence of five to eight MAb-defined distinct epitopes in C. neoformans GXM that can elicit antibody responses, (ii) MAb detection of antigenic variation within GXMs assigned to a particular serotype, (iii) good correspondence between the patterns of MAb reactivities and polyclonal rabbit factor sera, (iv) good agreement between MAb molecular structure and serotype reactivity, and (v) a dependence of the serotype reactivity profile for a given MAb on the technique used to measure binding.

Antibodies, Monoclonal↗

PubMed1997

Binding of Cryptococcus neoformans to heterologously expressed human complement receptors.

Previously, we demonstrated that monoclonal antibodies (MAb) directed against any of the three defined complement receptors (CR) for the third component of complement (CR1, CR3, and CR4) profoundly inhibited the binding of serum-opsonized Cryptococcus neoformans to monocyte-derived macrophages. These studies suggested either that a synergistic interaction between multiple CR was required for optimal binding of C. neoformans or that the MAb were exerting nonspecific effects (such as receptor coassociation). In the present studies, we took a novel approach to dissecting out the contributions of individual receptors to binding of a microbial pathogen. Chinese hamster ovary (CHO) cells stably transfected with human CR1, CR3, or CR4 were challenged with serum-opsonized C. neoformans. We found that CHO cells transfected with any of the three receptors bound C. neoformans, with the avidity of binding to CR3 being the greatest followed in decreasing order by CR1 and CR4. Following binding of C. neoformans to transfected CHO cells, most organisms remained surface attached only, although for each receptor a significant percentage (18.5 to 27.3%) of C. neoformans was internalized. Both C. neoformans and sheep erythrocytes that were selectively opsonized with the fragments of the third component of complement, C3b and iC3b, were bound preferentially by CHO cells transfected with CR1 and CR3, respectively. These data establish CR1, CR3, and CR4 as receptors independently capable of binding C. neoformans opsonized with fragments of C3. Moreover, our study demonstrates the usefulness of transfected cell lines as a powerful tool for identifying the contribution of individual receptors to the binding of a microbial pathogen.

Animals↗

PubMed1997

Structure and biological activities of acapsular Cryptococcus neoformans 602 complemented with the CAP64 gene.

The extracellular polysaccharide capsule of Cryptococcus neoformans is a well-recognized virulence factor. Strain 602 is an acapsular clinical isolate of unknown serotype which has been widely used in studies of virulence and host-parasite interactions. In previous studies, strain 602 was compared with genetically unrelated strains of various serotypes because the wild-type equivalent of strain 602 was not available. We created an encapsulated strain, TYCC38-602, by transforming strain 602 with the CAP64 gene which was isolated from a serotype D strain. Serological tests and chemical analysis of the major polysaccharide capsule of TYCC38-602 indicated that strain 602 was originally derived from a serotype A strain. Restoration of the ability to produce a capsule enabled strain 602 to cause fatal infection in mice, whereas the acapsular strain 602 remained avirulent. Capsule-restored yeast cells of strain 602 activated the human complement system and bound C3 fragments in a manner that is characteristic of encapsulated cryptococci. In addition, the capsule in TYCC38-602 masked the ability of the organism to induce tumor necrosis factor alpha and subsequent nitric oxide synthase production in primed macrophage-like cells. These results indicate that the lack of capsule in strain 602 is the reason for its inability to cause fatal infection. Moreover, the acapsular phenotype accounts for differences in various biological activities of strain 602 compared to encapsulated strains. The results also indicate that the gene product of CAP64 does not contribute to serotype specificity of capsules in C. neoformans.

Animals↗

PubMed1997

Acute lethal toxicity following passive immunization for treatment of murine cryptococcosis.

Passive immunization with monoclonal antibodies (MAbs) specific for the major capsular polysaccharide of Cryptococcus neoformans alters the course of murine cryptococcosis. During studies of passive immunization for treatment of murine cryptococcosis, we noted the occurrence of an acute, lethal toxicity. Toxicity was characterized by scratching, lethargy, respiratory distress, collapse, and death within 20 to 60 min after injection of antibody. The toxic effect was observed only in mice with a cryptococcal infection and was reduced or absent in the early and late stages of disease. The clinical course and histopathology were consistent with those for shock. There was considerable variation between mouse strains in susceptibility to toxicity. Swiss Webster mice from the Charles River colony were most susceptible, followed by C3H/He, BALB/c, and C57BL/6 mice. DBA/2 mice and Swiss Webster mice from the Simonsen colony were resistant. Acute toxicity was mimicked by injection of preformed complexes of MAb and purified polysaccharide. The toxic effect was also produced by injection of MAbs into mice that were preloaded with polysaccharide. The toxic effect was not blocked by treatment of mice with chloropheniramine or anti-tumor necrosis factor alpha antibodies or by depletion of complement components via pretreatment with cobra venom factor. Toxicity was reduced by treatment of mice with high doses of epinephrine, dexamethasone, or chlorpromazine. Finally, the toxic effect was completely blocked by treatment of mice with the platelet-activating factor antagonist WEB 2170 BS or by pretreatment of mice with the liposome-encapsulated drug dichloromethylene diphosphonate, a procedure which depletes macrophages from the spleen and liver.

Animals↗

PubMed1997

Mannan-specific immunoglobulin G antibodies in normal human serum mediate classical pathway initiation of C3 binding to Candida albicans.

Candida albicans activates both the classical and alternative complement pathways. Previous studies found that immunoglobulin G (IgG) in normal human serum (NHS) mediates classical pathway initiation. The goal of this study was to determine the role of candidal mannan-specific human IgG antibodies in complement activation. Mannan was purified from the yeast cells, and naturally occurring antimannan IgG was isolated from pooled NHS or plasma samples by immunoaffinity chromatography. Early activation and binding of C3, characteristics of classical pathway activity, were abolished in yeast- or mannan-absorbed serum but could be restored to absorbed serum with added purified antimannan IgG in a dose-dependent manner. Microscopically, the immunofluorescence pattern of initial C3 binding was diffuse over the entire cell surface for yeast cells incubated in NHS or in mannan-absorbed NHS supplemented with antimannan IgG but was asynchronous and focal for yeast cells incubated in EGTA-treated or mannan-absorbed NHS. The antimannan IgG level in serum samples from 21 donors varied from 17 to 570 microg/ml of serum compared to 220 microg in pooled NHS samples. The rate of initial C3 binding to yeast cells correlated with the level of antimannan IgG in sera from different individuals (r2 = 0.94) and could be accelerated in sera containing lower amounts of antimannan IgG with exogenous antimannan IgG. These observations identify antimannan IgG as the initiator of classical pathway C3 deposition on C. albicans. Given the variability in the levels of antimannan antibodies in sera from different individuals, the presence or absence of these antibodies may be an important determinant of host resistance to disseminated candidiasis.

Antibodies, Fungal↗

PubMed1996

Activation of the complement system by pathogenic fungi.

Fungi have been studied as prototype activators of the complement cascade since the early 1900s. More recently, attention has focused on the role of the complement system in the pathogenesis of fungal infections. The interactions of Cryptococcus neoformans and Candida albicans with the complement system are the most widely characterized; however, all pathogenic fungi examined to date have the ability to initiate the complement cascade. The molecular mechanisms for initiation and regulation of the complement cascade differ from one fungus to another, most likely reflecting differences in the structure of the outer layers of the cell wall. The molecular bases for such differences remain to be identified. Studies of mycoses in experimental animals with induced or congenital deficiencies in the complement system demonstrate that complement is an important innate system for control of fungal infection. Contributions to host resistance include opsonization and generation of inflammatory mediators. Inflammation induced by chemotactic products of the complement system may contribute to the pathogenesis of some fungal infections.

Aspergillosis↗

PubMed1996

Influence of opsonization conditions on C3 deposition and phagocyte binding of large- and small-capsule Cryptococcus neoformans cells.

Previous studies demonstrated that, following opsonization with normal human serum (NHS), phagocytes bind greater numbers of small-capsule Cryptococcus neoformans cells than yeast cells with large capsules. The present study tested the hypothesis that suboptimal deposition of opsonic C3 fragments contributes to this disparity. C neoformans was grown under conditions promoting large or small capsules and was incubated at various concentrations in NHS. At low concentrations of yeast cells (125 cells per microl of NHS), the deposition of C3 fragments per unit of capsule volume and the binding of yeast cells to cultured human monocytes were similar for yeast cells having large and small capsules. However, at higher cell concentrations, large-capsule cells exhibited suboptimal coating with C3 fragments and markedly diminished monocyte binding compared with small-capsule cells. Thus, the inverse correlation between capsule size and phagocyte binding can be overcome by conditions promoting optimal C3 deposition.

Complement C3↗