Biomedical subjects

T R Kozel

Publications and source records attributed to T R Kozel.

At least 19 recordsLinked to original sources

PubMed2001

Capsular reactions of Cryptococcus neoformans with polyspecific and oligospecific polyclonal anticapsular antibodies.

Monoclonal antibodies to the capsular polysaccharide of Cryptococcus neoformans produce distinct capsular reactions and have biological activities that are determined by serotype specificity. In the present study, polyclonal rabbit anticapsular antibodies were cross-absorbed to produce serotype specificities similar to those of monoclonal antibodies. The results showed that polyclonal and monoclonal antibodies with similar serotype specificities have similar capsular reactions and biological activities.

Animals↗

PubMed2000

Interleukin-12 counterbalances the deleterious effect of human immunodeficiency virus type 1 envelope glycoprotein gp120 on the immune response to Cryptococcus neoformans.

The mechanism involved in the envelope glycoprotein gp120-induced Th2 response to Cryptococcus neoformans was investigated. Peripheral blood mononuclear cells (PBMC) from healthy donors were treated with human immunodeficiency virus gp120 and an encapsulated or acapsular strain of C. neoformans in the presence or absence of glucuronoxylomannan, the major capsular polysaccharide. gp120 inhibited early and late production of interleukin (IL)-12 by PBMC. This reduction paralleled IL-10 induction and inhibited translocation of CD40 to the surface of monocytes. Flow cytometric analysis revealed that gp120 down-regulated the expression of IL-12 receptor beta2 subunit on T cells responding to C. neoformans. Because the IL-12/IL-12 receptor beta2 subunit pathway is critical for the Th1 differentiation process, underexpression demonstrates that gp120 contributes to Th2 bias. Exogenous IL-12 added simultaneously with gp120 up-regulated interferon-gamma secretion and limited IL-4 production. These results suggest that gp120 limits the Th1 response to C. neoformans and that exogenous IL-12 could offset this effect.

CD40 Antigens↗

PubMed2000

Biological correlates of capsular (quellung) reactions of Cryptococcus neoformans.

The capsular swelling or quellung reaction was reported almost 100 years ago and described the effect of Abs on the appearance of microbial capsules. Despite widespread use to assess Ab binding to capsules, relatively little is known as to the mechanism of this effect or its biological consequences. The fungus Cryptococcus neoformans is an attractive system to study capsule reactions because it has a large polysaccharide capsule that is readily visible by light microscopy. When viewed by differential interference contrast microscopy, binding of mAb to C. neoformans cells produced two distinct capsular reactions that depended on the Ab epitope specificity and the yeast serotype. In the first pattern, termed "rim," the capsule appears transparent with a highly refractive outer edge. In the second pattern, termed "puffy," the capsule appears opaque and lacks a highly refractive outer rim. mAbs that bind with a rim pattern suppress the overall rate of C3 deposition on the yeast via the classical and alternative complement pathways. In contrast, mAbs that bind with a puffy pattern do not affect C3 deposition. Protective and nonprotective IgM mAbs produce rim and puffy patterns, respectively. These results indicate that: 1) capsule reactions are a consequence of Ab-induced changes in capsular refractive index; 2) the type of capsule reaction depends on the Ab specificity; and 3) Ab-induced changes in refractive index correlate with biological activities important for host defense against C. neoformans. Our results provide the first evidence associating distinct capsule reaction patterns with Ab biological activity.

Antibodies, Monoclonal↗

PubMed2000

T lymphocyte and monocyte interaction by CD40/CD40 ligand facilitates a lymphoproliferative response and killing of Cryptococcus neoformans in vitro.

This study explored the role of CD40 / CD40 ligand (CD40L) in the induction of a lymphoproliferative response and killing of Cryptococcus neoformans in vitro. In our experimental system, monocytes exposed to C. neoformans were used as antigen-presenting cells (APC) and co-cultured with autologous T cells. The results showed that CD40 / CD40L strongly regulated the blastogenic response of T cells to C. neoformans. The fungus up-regulated CD40 expression on APC. An acapsular strain appeared to be a better inducer than an encapsulated strain. Time course experiments showed optimal regulation of CD40 expression at 48 h of incubation. Blocking the interaction of CD40 on APC with CD40L on T cells using mAb to CD40L resulted in a significant inhibition of IFN-gamma production. The anti-cryptococcal activity of monocytes was greatly influenced by the CD40 / CD40L interaction, and a positive correlation was found between nitric oxide secretion and enhanced killing of C. neoformans. Finally, the CD40 / CD40L interaction was critical for induction of optimal secretion of pro-inflammatory cytokines such as TNF-alpha and IL-1beta. These results indicate an important role for CD40 / CD40L interaction in inducing activation of T cells. Such cell-to-cell contact promotes anti-cryptococcal activity as well as secretion of pro-inflammatory cytokines by monocytes.

Antigens, Differentiation, T-Lymphocyte↗

PubMed2000

Antibody interactions with the capsule of Cryptococcus neoformans.

Monoclonal antibodies to the encapsulated fungus Cryptococcus neoformans produce different immunofluorescence (IF) patterns after binding to the polysaccharide capsule. To explore the relationship between the IF pattern and the location of antibody binding, two immunoglobulin M (IgM) monoclonal antibodies (MAbs) (12A1 and 13F1) that differ in protective efficacy and IF pattern and one protective IgG1 MAb (2H1) were studied by IF and electron microscopy (EM). Fixing C. neoformans cells in lung tissue for EM resulted in significantly better preservation of the capsule than fixing yeast cells in suspension. The localization of MAbs 12A1 and 13F1 by immunogold EM differed depending on whether the MAb was bound to cells in cut tissue sections embedded in plastic or to cells in solution. In cut tissue sections, MAbs 12A1 and 13F1 bound throughout the capsule, whereas in solution both MAbs bound near the capsule surface. To investigate whether antibody binding to the C. neoformans capsule affected the binding of other primary or secondary reagents, various combinations of MAbs 12A1, 13F1, and 2H1 were studied by direct and indirect IF. The IF pattern and location of binding for MAbs 12A1, 13F1, and 2H1 varied depending on the presence of other capsule-binding MAbs and the method of detection. The results show that (i) binding of MAbs to the C. neoformans polysaccharide capsule can modify the binding of subsequent primary or secondary antibodies; (ii) the IgM MAbs bind primarily to the outer capsule regions despite the occurrence of their epitopes throughout the capsule; and (iii) MAb 2H1 staining of newly formed buds is reduced, suggesting quantitative or qualitative differences in bud capsule.

Animals↗

PubMed1999

HIV type 1 envelope glycoprotein gp120 induces development of a T helper type 2 response to Cryptococcus neoformans.

OBJECTIVE: To analyse the contribution of HIV type 1 envelope glycoprotein gp120 to regulation of a T-cell response to Cryptococcus neoformans. DESIGN: Monocytes treated with recombinant gp120 and exposed to C. neoformans were used as antigen presenting cells (APC) in coculture with autologous T lymphocytes. METHODS: Costimulatory and major histocompatibility complex class II molecules were evaluated on APC by flow cytometry analysis. T-cell proliferation was determined as 3H thymidine incorporation. Cytokine production was analysed by enzyme-linked immunosorbent assay. RESULTS: gp120 had multiple effects on APC and the T-cell response including: (i) up-regulation of major histocompatibility complex class II antigens on the APC surface resulting from both redistribution of molecules from the intracellular pool and synthesis of new molecules; (ii) up-regulation of B7-2 molecules on the APC surface; (iii) altered T-cell proliferation; and (iv) promotion of interleukin-4 and inhibition of interferon-gamma synthesis and release. CONCLUSIONS: These data indicate that gp120 alters the normal T-cell response to C. neoformans, promoting a T-helper type 2 response. The altered T-cell response produced by gp120 may play an important role in the pathogenesis of cryptococcosis in the patient with AIDS.

Antigens, Fungal↗

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↗