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T R Kozel

Publications and source records attributed to T R Kozel.

At least 55 records · Page 3Linked to original sources
PubMed1995

Adherence to and damage of endothelial cells by Cryptococcus neoformans in vitro: role of the capsule.

Escape from the intravascular compartment is likely a critical step in the development of hematogenously disseminated cryptococcal infections, such as meningitis. The capsule of Cryptococcus neoformans is considered to be a virulence factor because of its antiphagocytic properties. To further investigate the role of the capsule in escape from the intravascular compartment, we used isogenic strain pairs, an acapsular mutant, and an encapsulated clinical isolate to determine the effects of the capsule of C. neoformans on adherence to, phagocytosis by, and damage of endothelial cells in vitro. Acapsular C. neoformans adhered significantly more to endothelial cells and caused greater endothelial cell injury than did encapsulated organisms. Coating of an acapsular strain with cryptococcal glucuronoxylomannan decreased both adherence to and damage of endothelial cells by 61.7% +/- 9.1% and 76.6% +/- 10.2%, respectively. Transmission electron microscopy demonstrated internalization of acapsular, but not encapsulated, organisms by endothelial cells. Internalization of an acapsular strain occurred through endothelial cell phagocytosis and was inhibited by cytochalasin D. Phagocytosis required a heat-labile serum factor, probably complement. These results suggest that acapsular or poorly encapsulated C. neoformans may be the form(s) that escapes from the vasculature during initiation of hematogenously disseminated disease.

Cell Adhesion↗
PubMed1995

Downregulation by cryptococcal polysaccharide of tumor necrosis factor alpha and interleukin-1 beta secretion from human monocytes.

The regulation by Cryptococcus neoformans encapsulation of interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) production by human monocytes was investigated. By using encapsulated and acapsular C. neoformans, we demonstrated that both strains induce cytokine production, although the acapsular strain was a better stimulator than the thinly encapsulated strain. The cytokine levels produced by cells stimulated by the two strains were lower and followed a different kinetic than those stimulated by lipopolysaccharide (LPS). Purified capsular polysaccharide inhibits TNF-alpha secretion induced by LPS or acapsular C. neoformans. In contrast, no regulator effect on IL-1 beta was observed when LPS was used. The secretory response of these cytokines follows different pathways of macrophage activation; in fact, complete inhibition of TNF-alpha does not affect IL-1 beta production and vice versa. These data indicate that purified capsular polysaccharide of C. neoformans could contribute to the in vivo progress of cryptococcosis by suppressing cytokine production of macrophages and suggest that a therapeutic approach to address the suppressive effect of cryptococal polysaccharide could be devised.

Adult↗
PubMed1994

Specificity of the thioester-containing reactive site of human C3 and its significance to complement activation.

The specificity of the thioester-containing site in three plasma proteins is regulated by elements of their protein structures other than the thioester bond itself. Human C4A and alpha 2-macroglobulin preferentially form amide linkages while human C3 primarily forms ester linkages with hydroxyl groups. We have examined the thioester in C3 and found evidence of strong preferences for certain carbohydrates, indications of selectivity for specific positions on those carbohydrates and a preference for terminal sugars in polysaccharides. A testable set of rules are derived from these findings which predict preferred attachment sites on polysaccharides. A computer model of the effect of different reactivities on activation of the alternative pathway of complement suggested that organisms might greatly alter their susceptibility to complement with small changes in carbohydrate structure. While a random selection of 20 biological particles showed no correlation between activation and C3b attachment efficiency, subsets of related organisms differing primarily in their surface polysaccharide exhibited stronger correlations. The strongest correlation occurred in a series of the yeasts (Cryptococcus neoformans) possessing capsular polysaccharides with one, two, three or four branching xylose sugars per repeating unit. These organisms exhibited capture efficiencies for metastable C3b from 12% (one-xylose strain) to 41% (four-xylose strain).

Alcohols↗
PubMed1994

Protein-polysaccharide interactions. A monoclonal antibody specific for the capsular polysaccharide of Cryptococcus neoformans.

Monoclonal antibodies that react with the capsular polysaccharide, termed glucuronoxylomannan (GXM), of Cryptococcus neoformans have potential roles in the diagnosis, monitoring of disease progress, and immunotherapy of cryptococcal GXM of all four serotypes. A molecular model of the Fab fragment of monoclonal antibody 439 was constructed from the amino acid sequence and a template antibody molecule, Fab 4-4-20. A tryptophan is present on the surface between light chain CDR3 and heavy chain CDR3 in the putative binding site. This tryptophan residue proved to be a reporter group, and a fluorescence study of Fab 439 was performed to analyze the interaction between cryptococcal GXM and Fab 439. Binding of the polysaccharide enhanced the intrinsic fluorescence and caused a blue shift in the emission maximum, indicating that the environment of a tryptophan changes from a polar to less polar environment. This is consistent with the loss of water from the binding site caused by the binding of antigen. This interpretation was confirmed by acrylamide quenching, which showed that 1 less tryptophan was exposed to solvent in the Fab-GXM complex than in free Fab. Fluorescence titration was used to determine binding and dissociation constants (KD). The apparent KD values for serotypes A-C were approximately the same; the KD for serotype D GXM was 5-11-fold greater. De-O-acetylation of serotype A GXM produced a 31-fold increase in the KD, indicating that the O-acetyl groups are important, but not essential, for binding. Carboxyl groups appear to be essential for strong binding because the KD for carboxyl-reduced GXM was so large that it could not be determined.

Amino Acid Sequence↗
PubMed1994

Occurrences, specificities, and functions of ubiquitous antibodies in human serum that are reactive with the Cryptococcus neoformans cell wall.

Previous studies found that normal human serum (NHS) contains an immunoglobulin G (IgG) antibody that mediates initiation of the classical complement pathway by nonencapsulated Cryptococcus neoformans. The present study used an enzyme-linked immunosorbent assay with whole nonencapsulated yeast cells as solid-phase antigens to demonstrate the presence of high levels of IgG antibody in each of 11 sera from normal adult donors. The IgG antibodies were of the IgG2 subclass. The antibody activity was blocked completely by treatment of serum with isolated yeast glucan. Treatment of serum with mannan or chitin had no effect on antibody levels. Antibody activity was adsorbed completely by treatment of serum with zymosan particles. Adsorption with intact cells of Saccharomyces cerevisiae or Candida albicans had no effect, suggesting that the glucan on S. cerevisiae or C. albicans is not surface exposed. Assessment of the opsonic requirements for phagocytosis of nonencapsulated cryptococci by monocyte-derived human macrophages (MO-M phi) showed that high levels of phagocytosis occurred when yeast cells were opsonized with NHS. Removal of anti-glucan antibody by adsorption with whole nonencapsulated cryptococci did not diminish opsonic activity. Heat-inactivated serum or anti-glucan antibody affinity purified from NHS lacked opsonic activity. Taken together, these results indicate that phagocytosis of nonencapsulated cryptococci by monocyte-derived human macrophages has an obligatory requirement for opsonic ligands of the complement system, with no contribution by the anti-glucan IgG that is found in NHS.

Antibodies, Fungal↗
PubMed1994

Occurrences, immunoglobulin classes, and biological activities of antibodies in normal human serum that are reactive with Cryptococcus neoformans glucuronoxylomannan.

Serum obtained from normal human subjects contains antibodies reactive in an enzyme-linked immunosorbent assay with the glucuronoxylomannan (GXM) of Cryptococcus neoformans. The frequency of occurrence of class-specific antibodies among normal subjects was 28% for immunoglobulin G (IgG), 98% for IgM, and 3% for IgA. Anti-GXM antibodies with kappa light chains occurred in 98% of normal subjects, while the occurrence of lambda light chains was 28%. Each of five subjects with high levels of anti-GXM IgG antibodies had readily detectable antibodies of the IgG2 isotype; two of the five subjects had readily detectable IgG1 antibody. An examination of sera from human immunodeficiency virus-infected patients showed that human immunodeficiency virus infection was accompanied by a significant decrease in the occurrence of IgM antibodies and anti-GXM antibodies with kappa light chains; these decreases occurred early in infection when CD4 counts were still > or = 500 cells per microliter. A slight but not statistically significant decrease in the occurrence of anti-GXM IgG antibodies was seen only in patients with CD4 levels of < 200 cells per microliter. Sera from normal subjects with high levels of anti-GXM IgG antibodies were examined to identify any contribution of the antibodies to complement activation or to opsonization of the yeast cells. An analysis of the kinetics for activation and binding of C3 to the yeast cell showed no pattern of quantitative or qualitative differences between sera with high or low levels of anti-GXM IgG antibodies. Phagocytosis studies showed that the naturally occurring IgG antibodies did not contribute to opsonization of the yeast cells.

Antibodies, Fungal↗
PubMed1994

Molecular and idiotypic analysis of antibodies to Cryptococcus neoformans glucuronoxylomannan.

Antibodies to the Cryptococcus neoformans capsular glucuronoxylomannan (GXM) form the basis of two potential therapeutic intervention strategies, i.e., conjugate vaccines and passive antibody therapy. To better understand the molecular basis of the antibody response, the heavy- and light-chain immunoglobulin variable region (VH and VL, respectively) sequences of seven monoclonal antibodies (MAbs) to GXM were determined. Rabbit anti-idiotypic serum was made to the previously characterized murine MAb 2H1 and used to study MAb 2H1 idiotype expression in other GXM-binding MAbs and immune sera. MAb E1 originated from a C3H/HeJ mouse immunized with C. neoformans serotype A polysaccharide. MAbs 471, 1255, 339, 3C2, 386, and 302 originated from BALB/c mice immunized with polysaccharide of serotypes A, A, B, C, D, and D, respectively, conjugated to sheep erythrocytes. In the E1, VH uses V11 from the T15 gene family and JH3 and has a D segment of three amino acids, and the VL uses a VKSer-like gene family element and JK5. In MAbs 471 and 3C2, the VH uses VH7183-like gene family elements and JH2 and has D segments of seven amino acids, and the VL uses VK5.1 and JK1. In MAbs 1255 and 339, the VH uses VH10-like gene elements and JH4 and has six codon D segments, and the VL uses a VK21-like gene element and JK5. In MAbs 302 and 386, respectively, the VH uses VHGAM-like gene elements and JH2 and JH3 and has six and four codon D segments, and VL uses VK4/5-like gene elements and JK1.VH usage, MAb 2H1 idiotype expression, and fine specificity mapping define a minimum of three GXM epitopes which elicit protective antibodies. The results confirm that the antibody response is highly restricted, suggest a close relationship between molecular structure and serological properties, and provide insight into protein structural motifs important for GXM binding.

Animals↗
PubMed1993

Accelerated decay of C3b to iC3b when C3b is bound to the Cryptococcus neoformans capsule.

Incubation of encapsulated and nonencapsulated Cryptococcus neoformans in normal human serum (NHS) leads to activation and binding of potentially opsonic fragments of complement component C3 to the yeast cells. Analysis of the molecular forms of C3 after incubation of encapsulated cryptococci in NHS showed that the percentage of bound C3 occurring as iC3b approached 100% after 8 min. The percentage of bound C3 occurring as iC3b on nonencapsulated cryptococci never exceeded 70%, even after 60 min of incubation in NHS. Conversion of C3b to iC3b was assessed further by incubating C3b-coated cryptococci for various times with a mixture of complement factors H and I at 40% of their respective physiological concentrations. Most, if not all, of the C3b on encapsulated cryptococci was converted to iC3b at a single fast rate. Conversion of C3b to iC3b on nonencapsulated cryptococci did not follow a single rate constant and appeared to have a fast and a slow component. Studies of the requirements for factors H and I in cleavage of C3b to iC3b showed steep dose-response curves for both factors in the case of encapsulated cryptococci and shallow curves with C3b bound to nonencapsulated cryptococci. Taken together, our results indicate that C3b molecules bound to encapsulated cryptococci have a uniformly high susceptibility to conversion to iC3b by factors H and I. In contrast, a significant portion of the C3b bound to nonencapsulated cryptococci is very resistant to conversion to iC3b by factors H and I.

Complement C3b↗
PubMed1993

Effects of strain variation, serotype, and structural modification on kinetics for activation and binding of C3 to Cryptococcus neoformans.

Incubation of encapsulated cells of Cryptococcus neoformans in normal human serum leads to activation of the alternative complement pathway and deposition of opsonic fragments of C3 into the capsule. We determined whether the variation in capsular structure that occurs among the four major cryptococcal serotypes was reflected in the kinetics for activation and binding of C3. We also examined the effects on activation kinetics of de-O-acetylation or periodate oxidation of the capsule. Binding kinetics were characterized in terms of the time required to deposit 5% of the maximal amount of C3 on the yeast (t5%), the first-order rate constant for amplification of C3 deposition (k'), and the maximum amount of C3 that could be deposited in the capsule (C3max). Our results showed that variations in the capsular structure that characterized each serotype had no significant influence on C3max but that the rate of C3 deposition depended significantly on the serotype. C3 accumulated at a higher rate on cells of serotypes A and D than on cells of serotypes B and C. There was a significant correlation between capsular volume and C3max, although the relationship was not linear. Periodate treatment of encapsulated cryptococci of all four serotypes led to decapsulation. Periodate-oxidized encapsulated cells displayed kinetics for activation and binding of C3 that were identical to kinetics observed with nonencapsulated cryptococci. Finally, de-O-acetylation led to a significant but relatively minor increase in C3max.

Acetylation↗
PubMed1993

Activation of the complement system by the capsule of Cryptococcus neoformans.

In vitro studies indicate that encapsulated cryptococci are among the most powerful particulate activators of the complement system reported to date. The capsule itself is the site at which activation occurs. Activation occurs solely via the alternative complement pathway. Initiation occurs at apparently random focal sites in the capsule that expand through alternative pathway amplification to fill the capsule with C3 fragments. The C3 fragments are rapidly converted to iC3b, suggesting that phagocyte receptors for iC3b will be important in phagocytosis of the yeast. There is abundant evidence that a similar form of activation occurs in vivo during a cryptococcal infection. Cryptococcemia in humans and experimental animals is accompanied by a depletion of serum complement levels. Studies with complement deficient guinea pigs and mice indicate that the complement system plays an essential role in resistance to cryptococcosis. It is likely that the complement system contributes to host resistance by opsonization of the yeast to facilitate attachment and ingestion by phagocytic cells as well as by releasing chemotactic fragments of the complement cascade which contribute to the inflammatory response. The absence or unavailability of a functional complement system in the central nervous system may account in part for the predilection of the yeast for the brain.

Cell Wall↗
PubMed1993

Opsonization and phagocytosis of Cryptococcus neoformans.

Cryptococcus neoformans is unique among the pathogenic fungi because the yeast is surrounded by an antiphagocytic polysaccharide capsule. Host resistance to cryptococcosis is dependent on natural defense mechanisms that cope with this essential fungal virulence factor. Recent studies in several laboratories indicate that, under appropriate circumstances, the antiphagocytic activity of the capsule can be overcome. A reversal of the antiphagocytic properties of the capsule requires (i) activation of the alternative complement pathway by encapsulated cryptococci, leading to deposition of the opsonic ligand iC3b at the capsular surface, (ii) presence of essential cytokines which up-regulate the efficiency of complement-dependent phagocytosis, and (iii) availability of phagocytic cells whose complement receptors are capable of appropriate up-regulation. Deficiencies in one or more components of the opsonization-phagocyte-cytokine triad may account, in part, for several features of the pathogenesis of cryptococcosis as well as the susceptibility of some immunocompromised patients to cryptococcosis. It may be possible to overcome some deficiencies by passive immunization with anticapsular IgG which takes advantage of phagocyte Fc receptors that are constitutively competent for phagocytosis or by therapeutic use of cytokines that up-regulate complement-dependent phagocytosis.

AIDS-Related Opportunistic Infections↗
PubMed1992

Contribution of antibody in normal human serum to early deposition of C3 onto encapsulated and nonencapsulated Cryptococcus neoformans.

Encapsulated and nonencapsulated cryptococci differ in their activation of the complement system. Incubation of nonencapsulated cryptococci in normal human serum (NHS) initiates both the classical and alternative pathways. This activation is characterized by an immediate, synchronous activation and binding of C3 to the yeast cells. Encapsulated cryptococci activate only the alternative pathway. This activation is characterized by a delayed (4 to 5 min), asynchronous activation and binding of C3. We examined the properties of antibodies in NHS that mediate immediate, synchronous binding of C3 to nonencapsulated cryptococci and zymosan. Adsorption of NHS with nonencapsulated cryptococci or zymosan produced a 4- to 6-min delay in the kinetics for activation and binding of C3 from the adsorbed serum to each respective yeast cell. This delay was similar to the delay observed when nonencapsulated cryptococci or zymosan was incubated in NHS in which the classical pathway was blocked by chelation of Ca2+. Proteins bound to serum-treated nonencapsulated cryptococci or zymosan were eluted and found to be predominantly immunoglobulin G (IgG), with lesser amounts of IgM. The eluted IgG could restore to adsorbed serum the rapid early kinetics for activation and binding of C3 characteristic of classical pathway initiation. Cross-adsorption showed that there was considerable cross-reactivity between the antibodies which restored rapid, early activation kinetics to NHS adsorbed with zymosan or nonencapsulated cryptococci. Encapsulated cryptococci were unable to adsorb the antibodies from NHS that mediated the rapid, early activation and binding of C3 to zymosan and nonencapsulated cryptococci. The latter results show that occlusion of antigenic sites at the cryptococcal cell wall is a newly recognized property that can be added to the repertoire of biological activities of the cryptococcal capsule.

Adsorption↗
PubMed1992

Kinetic analysis of the amplification phase for activation and binding of C3 to encapsulated and nonencapsulated Cryptococcus neoformans.

Encapsulated and nonencapsulated cryptococci exhibit quantitative and qualitative differences in their activation of the complement system. We examined the kinetics for the rapid amplification phase in which C3 was activated and bound to encapsulated cryptococci, nonencapsulated cryptococci, and zymosan particles. Yeast cells were incubated in normal human serum containing 125I-labeled C3, and bound C3 fragments were measured after 1 to 64 min of incubation. A kinetic analysis showed that the apparent first-order rate constant (k') for binding of C3 to nonencapsulated cryptococci did not differ significantly from k' for binding of C3 to zymosan particles (P greater than 0.05). However, the rate constant for binding of C3 to encapsulated cryptococci was significantly (P less than 0.001) greater than k' for binding of C3 to nonencapsulated cryptococci and zymosan particles. A plot of C3 molecules bound to encapsulated cryptococci versus time cubed was nearly linear, suggesting that accumulation of C3 in the cryptococcal capsule follows the kinetics predicted by an expanding sphere. In contrast, the plot of C3 molecules bound to nonencapsulated cryptococci or zymosan particles against time was nearly linear, but those plots against time squared or time cubed were not. This result indicates that the rate-limiting step for the addition of C3 fragments to these latter yeast cells follows the kinetics of neither the perimeter of an expanding circle nor the surface of an expanding sphere. Taken together, the results indicate that the high rate of accumulation of C3 in the cryptococcal capsule is consistent with the expected geometry of an expanding sphere of bound C3 within the three-dimensional matrix of the capsule.

Complement Activation↗
PubMed1992

In vivo complement activation and binding of C3 to encapsulated Cryptococcus neoformans.

Tissues from mice infected with Cryptococcus neoformans were examined by immunofluorescence to determine the extent of deposition of complement component C3 on encapsulated cryptococci. The relative percentages of cryptococci in each tissue having readily visible C3 were greatest for liver and lung tissues, with the kidney tissue having the next highest percentage and the spleen having the lowest percentage. Binding of C3 fragments to cryptococci in brain tissue was essentially absent.

Animals↗
PubMed1991

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↗
PubMed1990

Opsonization of Cryptococcus neoformans by a family of isotype-switch variant antibodies specific for the capsular polysaccharide.

A family of immunoglobulin isotype-switch variants was isolated by sib selection from a murine hybridoma which produced an immunoglobulin G subclass 1 (IgG1) antibody specific for the capsular polysaccharide of Cryptococcus neoformans. Antibodies of the IgG1, IgG2a, and IgG2b isotypes had similar serotype specificity patterns in double immunodiffusion assays which used polysaccharides of the four cryptococcal serotypes as antigens. A quantitative difference in the ability of the isotypes to form a precipitate with the polysaccharide was observed in a double immunodiffusion assay and confirmed in a quantitative precipitin assay. The relative precipitating activity of the antibodies was IgG2a greater than IgG1 much greater than IgG2b. Analysis by enzyme-linked immunosorbent assay of the reactivity of the three isotypes with cryptococcal polysaccharide showed identical titers and slopes, suggesting that the variable region of the class-switch antibodies was unaltered. This system allowed us to examine the effect of the Fc portion of the antibody on opsonization of encapsulated cryptococci. Yeast cells were precoated with antibodies of each isotype and incubated with murine macrophages or cultured human monocytes. Antibodies of all three isotypes exhibited a dose-dependent opsonization for phagocytosis by both human and murine phagocytes. The relative opsonic activity of the antibodies was IgG2a greater than IgG1 greater than IgG2b.

Animals↗
PubMed1990

Passive immunization against Cryptococcus neoformans with an isotype-switch family of monoclonal antibodies reactive with cryptococcal polysaccharide.

The in vivo properties of an immunoglobulin isotype-switch family of monoclonal antibodies specific for the polysaccharide capsule of Cryptococcus neoformans were examined in a murine model of cryptococcosis. Subclass-switch variants were isolated by sequential sublining of an immunoglobulin G subclass 1 (IgG1)-secreting cell line. Antibodies of the IgG1, IgG2a, and IgG2b isotypes with identical reactivities with cryptococcal polysaccharide were prepared. The antibodies had the distinct biological properties associated with the heavy chains of each respective isotype. The antibodies were used prophylactically or therapeutically in an attempt to alter the course of cryptococcal infection in mice. Survival of mice and a tissue census of the numbers of viable cryptococci in the lung, spleen, and brain were used as indicators of efficacy. Passive immunization with the IgG2a and IgG2b antibodies effected a reduction in the numbers of cryptococci in lung and spleen. Passive immunization with the IgG1 antibody was markedly less effective. Passive immunization had little or no effect on the numbers of cryptococci in brain tissue, regardless of the immunoglobulin isotype. Despite apparent efficacy with regard to reduction in the numbers of yeast cells in the lung and spleen, the results showed no improvement in survival from murine cryptococcosis. Our results indicate that passive immunization produces a modest effect on the course of murine cryptococcosis in tissues other than brain. However, under the experimental conditions used, such treatment does not have a measurable impact on the ultimate outcome of the infection.

Animals↗