Detection of Bacillus anthracis in animal tissues using InBios active anthrax detect rapid test lateral flow immunoassay.

The Active Anthrax Detect (AAD) Rapid Test lateral flow immunoassay is a point-of-care assay that was under investigational use for detecting Bacillus anthracis capsular polypeptide (polyglutamic acid) in human blood, serum and plasma. Small sample volumes, rapid results and no refrigeration required allow for easy use in either the field or laboratory. Although the test was developed for use in suspect cases of human inhalation anthrax, its features also make it a potentially powerful tool for testing suspect animal cases. We tested animal tissue samples that were confirmed or ruled out for B. anthracis. The AAD Rapid Tests were also deployed in the field, testing animal carcasses during an anthrax outbreak in hippopotami (Hippopotamus amphibius) and Cape buffalo (Syncerus caffer) in Namibia. Evaluation of all samples showed a specificity of 82% and sensitivity of 98%. However, when the assay was used on specimens from only fresh carcasses (dead for <24 h), the specificity increased to 96%. The AAD Rapid Test is a rapid and simple screening assay, but confirmatory testing needs to be done, especially when the age of the sample (days animal has been deceased) is unknown. SIGNIFICANCE AND IMPACT OF THE STUDY: In countries where anthrax is endemic, many human outbreaks are often caused by epizootics. Earlier detection of infected animals may allow for identification of exposed people, early implementation of prevention and control methods, and ultimately lessen the number of people and animals affected. Detection of Bacillus anthracis in animal tissues using a simple, rapid and field-deployable method would allow for faster outbreak response. We evaluated a simple sample collection and processing method for use with the Active Anthrax Detect Rapid Test lateral flow immunoassay to screen dead animals for anthrax.

A purified capsular polysaccharide markedly inhibits inflammatory response during endotoxic shock.

Capsular material of the opportunistic fungus Cryptococcus neoformans is composed mainly of a polysaccharide named glucuronoxylomannan (GXM). In this study, the effects of GXM were analyzed in an in vivo experimental system of lipopolysaccharide (LPS)-induced shock. Endotoxic shock was induced in mice by a single intraperitoneal injection of LPS from Escherichia coli. GXM treatment reduced the mortality of mice at early stages. Mice treated with LPS alone showed markedly increased plasma levels of tumor necrosis factor alpha (TNF-α), interleukin-1ß (IL-1ß), and IL-6, whereas mice that were also treated with GXM showed significantly lower plasma levels of these cytokines. This effect was related to a marked suppression of Akt and IκBα activation. Importantly, the inhibitory effect of GXM on proinflammatory cytokine secretion was reproduced by treatment with wortmannin, an inhibitor of the Akt transcription pathway. Our results indicate that GXM has a beneficial effect on endotoxic shock, resulting in a significant increase in the rate of survival by dampening the hyperinflammatory response.

A critical role for FcgammaRIIB in up-regulation of Fas ligand induced by a microbial polysaccharide.

The microbial capsular polysaccharide glucuronoxylomannan (GXM) from the opportunistic fungus Cryptoccocus neoformans is able to alter the innate and adaptive immune response through multi-faceted mechanisms of immunosuppression. The ability of GXM to dampen the immune response involves the induction of T cell apoptosis, which is dependent on GXM-induced up-regulation of Fas ligand (FasL) on antigen-presenting cells. In this study we elucidate the mechanism exploited by GXM to induce up-regulation of FasL. We demonstrate that (i) the activation of FasL is dependent on GXM interaction with FcgammaRIIB (FcγRIIB); (ii) GXM induces activation of c-Jun NH(2) -terminal kinase (JNK) and p38 signal transduction pathways via FcγRIIB; (iii) this leads to downstream activation of c-Jun; (iv) JNK and p38 are simultaneously, but independently, activated; (v) FasL up-regulation occurs via JNK and p38 activation; and (vi) apoptosis occurs via FcγRIIB engagement with consequent JNK and p38 activation. Our results highlight a fast track to FasL up-regulation via FcγRIIB, and assign to this receptor a novel anti-inflammatory role that also accounts for induced peripheral tolerance. These results contribute to our understanding of the mechanism of immunosuppression that accompanies cryptococcosis.

Virulence factors of Cryptococcus neoformans.

Cryptococcosis is a serious fungal disease in patients with AIDS or other defects in T-cell-mediated host defenses. Cryptococcus neoformans produces several virulence factors, most notably the polysaccharide capsule and phenol oxidase. Molecular studies of cryptococcal virulence factors have contributed to our understanding of the pathobiology of this yeast, and will enable the identification of new targets for antifungal therapy.

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.

Benzoquinone activation of Cryptococcus neoformans capsular polysaccharide for construction of an immunoaffinity column.

p-benzoquinone was used as a two-step coupling reagent for preparation of an immunoaffinity absorbent in which the capsular polysaccharide of Cryptococcus neoformans was linked to an agarose gel. Cryptococcal polysaccharide is a difficult subject for chemical modification because it contains immunogenic O-acetyl groups which are sensitive to alkaline hydrolysis. The polysaccharide was activated by treatment with benzoquinone. The 'activated' polysaccharide was reactive with amino groups on AH-Sepharose. A pH of 8-9 was optimal for activation of the polysaccharide. Once activated, the polysaccharide was reactive with a model substrate, L-alanine-4-nitroanilide, over a pH range of 6-10. Since the O-acetyl groups are hydrolyzed at pH above 8.0, an activation pH of 8.0 and a coupling pH of 7.5 were used to prepare the conjugated gel. The polysaccharide immunoaffinity column was used successfully for isolation of rabbit antibodies to cryptococcal polysaccharide.

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.

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.

Dissociation of a hydrophobic surface from phagocytosis of encapsulated and non-encapsulated cryptococcus neoformans.

Cryptococcus neoformans is surrounded by a capsular polysaccharide that inhibits phagocytosis of the yeast by macrophages. This capsular polysaccharide also confers several physicochemical properties to the cell surface, including a negative surface charge and a hydrophilic surface. The present study was designed to determine whether a hydrophobic surface was necessary or sufficient for phagocytosis of C. neoformans cells. The hydrophobic nature of the cell surface was measured by hydrophobic interaction chromatography on octyl-Sepharose. Liability to phagocytosis was determined by use of mouse peritoneal macrophages. The surface properties of C. neoformans cells were modified by (i) preincubation of cryptococcal cells with nonimmune serum or immune anticapsular serum, (ii) chemical modification of the carboxyl and O-acetyl groups in the capsular polysaccharide, and (iii) use of various serotypes of C. neoformans with different degrees of O-acetyl and xylosyl substitution. The results showed that it was possible to experimentally vary the surface hydrophobic-hydrophilic characteristics of the cell surface; however, the antiphagocytic character of the capsule remained unchanged. The results further suggest that a hydrophobic surface was neither necessary nor sufficient for phagocytosis of C. neoformans cells by macrophages.

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.

Non-encapsulated variant of Cryptococcus neoformans. II. Surface receptors for cryptococcal polysaccharide and their role in inhibition of phagocytosis by polysaccharide.

The binding of cryptococcal polysaccharide to a non-encapsulated strain of Cryptococcus neoformans was studied. Binding of purified polysaccharide to the yeast was determined by inhibition of phagocytosis and by indirect immunofluorescence techniques. The ability of cryptococcal polysaccharide to prevent phagocytosis of the non-encapsulated strain appears to be directly related to adherence of polysaccharide to the yeast via specific receptors on the cell surface. Addition of varying doses of cryptococcal polysaccharide to non-encapsulated yeast cells inhibited phagocytosis only at polysaccharide concentrations at which the polysaccharide could be demonstrated on the yeast surface by immunofluorescence. Macrophages treated with cryptococcal polysaccharide had no detectable amounts of cryptococcal polysaccharide adherent to their surface, and they had a normal ability to phagocytize the yeast. Kinetic studies showed that inhibition of phagocytosis is directly related to the presence of cryptococcal polysaccharide at the yeast surface rather than to some indirect effect by the polysaccharide on serum components necessary for phagocytosis. Purified polysaccharide from C. neoformans serotypes A, B, C, and D bound to the yeast, but type III pneumococcal polysaccharide did not inhibit phagocytosis of the nonencapsulated yeast. Cryptococcal polysaccharide did not bind to cells of Candida albicans, C. pseudotropicalis, Torulopsis sp., Rhodotorula sp., or Saccharomyces cerevisiae.

Activation of the complement system by Cryptococcus neoformans leads to binding of iC3b to the yeast.

The complement system plays a key role in resistance to cryptococcosis. In the present study, we examined several factors that influence the binding of C3 cleavage fragments to Cryptococcus neoformans. Binding of C3 was determined by using normal human serum supplemented with 125I-labeled C3. Incubation of encapsulated cryptococci in 20% serum led to the binding of approximately 3.2 X 10(6) molecules of C3 to each cell. The binding of C3 was markedly inhibited by heating the serum at 56 degrees C for 30 min or by chelation of the serum with EDTA. Chelation of the serum with EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] reduced binding of C3 by 37%. These results indicated that activation of C3 cleavage fragments and their binding to C. neoformans was primarily dependent upon the alternative pathway. Bound C3 could be removed by incubation with 1.0 M hydroxylamine (pH 10) but not by incubation with 3.5 M NaSCN or with phosphate-buffered saline containing 0.1% sodium dodecyl sulfate. These results suggested that C3 fragments were bound to C. neoformans by ester bonds. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of C3 fragments eluted from the yeast showed the presence of protein bands consistent with the presence of iC3b. C3b was not detected on the yeast after incubation with serum for time intervals as short as 2.5 min, indicating a rapid conversion of cell-bound C3b to iC3b. These results indicate that iC3b is the ligand which most likely interacts with the phagocyte C3 receptors involved in the phagocytosis of C. neoformans.

Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide.

Cryptococcus neoformans is surrounded by a capsular polysaccharide. There are at least four known serotypes of the polysaccharide. The objective of this study was to produce monoclonal antibodies (MAbs) that could be used to study the distribution of epitopes among the serotypes of C. neoformans. BALB/c mice were immunized with cryptococcal polysaccharides of serotype A or D that were coupled to sheep erythrocytes. Splenocytes were isolated, and hybridomas secreting MAbs specific for cryptococcal polysaccharides were isolated. Two hybridomas, designated MAbs 439 and 1255, were produced from mice immunized with serotype A polysaccharide. One hybridoma, designated MAb 302, was produced from mice immunized with serotype D polysaccharide. All three antibodies were of the immunoglobulin G1 isotype. MAb 302 showed a specificity for serotypes A and D in Ouchterlony diffusion, agglutination, and opsonophagocytosis assays. MAb 1255 was reactive with polysaccharides and cells of serotypes A, B, and D. MAb 439 was reactive with polysaccharides and cells of serotypes A, B, C, and D. The reactivity of these MAbs closely matched the distribution of epitopes among cryptococcal polysaccharides predicted in previous studies of polyclonal antibodies reactive with cryptococcal polysaccharides. The ability to produce a MAb against an epitope shared by all four serotypes may have value for the detection of cryptococcal antigens in body fluids.

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.

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.

Opsonization of Cryptococcus neoformans by human immunoglobulin G: masking of immunoglobulin G by cryptococcal polysaccharide.

Previous studies have shown that attachment of non-encapsulated cryptococci to macrophages is highly dependent on opsonizing immunoglobulin G (IgG) and that cryptococcal polysaccharide inhibits the attachment phase of phagocytosis. We investigated various mechanisms by which cryptococcal polysaccharide might interfere with the opsonizing action of IgG. Cryptococcal polysaccharide did not appreciably prevent binding of opsonizing IgG to the yeast. Furthermore, cryptococcal polysaccharide acted as a noncompetitive inhibitor with respect to the opsonizing action of IgG. These experiments suggested that cell wall-bound IgG is masked in some manner such that it is unable to participate in Fc-mediated phagocytosis. This appeared to be the case, since cryptococcal polysaccharode inhibited agglutination of IgG-opsonized yeast cells by antiserum to IgG. There was good dose-response correlation between the amount of polysaccharide needed to inhibit phagocytosis of non-encapsulated Cryptococcus neoformans and the amount of polysaccharide needed to prevent agglutination of IgG-opsonized cryptococci by antiserum to IgG. The ability of cryptococcal polysaccharide to prevent agglutination of IgG-opsonized cryptococci by antiserum to IgG was lost if dextran, a substance known to enhance agglutination of several particles, was incorporated into the medium.

Opsonization of Cryptococcus neoformans by human immunoglobulin G: role of immunoglobulin G in phagocytosis by macrophages.

The role of immunoglobulin G (IgG) as an opsonin in phagocytosis of Cryptococcus neoformans by macrophages was investigated. Labeling with 125I showed that IgG isolated from normal human serum bound to non-encapsulated C. neoformans. Furthermore, IgG-opsonized cryptococci were agglutinated by anti-serum to IgG heavy chains, indicating that normal human serum contains antibody that will bind to the yeast surface. The IgG isolated from normal serum accounted for all opsonizing activity found in normal human serum, since differences were not noted between the opsonizing activities of whole serum, heat-inactivated serum and purified IgG when these opsonins were compared at equivalent concentrations of IgG. Phagocytosis of IgG-opsonized cryptococci was inhibited by anti-macrophage IgG, a reagent known to block Fc-mediated attachment and ingestion, and by pepsin digestion of opsonizing IgG. Thus, IgG opsonization is an Fc-dependent process. Opsonizing IgG appears to play its major role during the attachment phase of phagocytosis, since antimacrophage IgG blocked attachment of cryptococci to macrophages but could not block ingestion of IgG-opsonized cryptococci that had been allowed to attach to macrophages. Ingestion of opsonized cryptococci was not blocked by 2-deoxy-D-glucose, a reagent known to block Fc-mediated ingestion, thus confirming that IgG has a primary role in attachment and suggesting that ingestion is mediated by a process that is not Fc dependent.

Phagocytosis of Cryptococcus neoformans by normal and thioglycolate-activated macrophages.

Phagocytosis of Cryptococcus neoformans by normal and thioglycolate-activated mouse peritoneal macrophages was studied. Thioglycolate-activated macrophages exhibited a lower percent phagocytosis than did normal macrophages. Differences in phagocytosis could not be attributed to differences in macrophage viability, minor variations in the concentration of adherent macrophages, or a general depression in activated macrophage phagocytosis. Thioglycolate-activated macrophages required heat-labile opsonins for optimal phagocytosis of non-encapsulated cryptococci, whereas nonactivated macrophages did not require heat-labile opsonins for phagocytosis of the yeast. Both types of macrophages exhibited similar sensitivity to the phagocytosis-inhibiting properties of cryptococcal polysaccharide. The results show that depletion of heat-labile opsonins from serum or inactivation of yeast-bound, heat-labile opsonins by polysaccharide cannot account for the phagocytosis-inhibiting properties of cryptococcal polysaccharide.

Chemotaxigenesis and activation of the alternative complement pathway by encapsulated and non-encapsulated Cryptococcus neoformans.

In the presence of serum, whole cells of encapsulated and non-encapsulated Cryptococcus neoformans generated a chemotactic response by neutrophils. Heat inactivation of serum ablated all chemotactic activity. Cryptococcal polysaccharide was not chemotaxigenic. Assays for alternative complement pathway activation such as depletion of alternative complement pathway factor B or electrophoretic conversion of factor B closely paralleled chemotaxis assays. Cells of encapsulated and non-encapsulated C. neoformans activated the alternative complement pathway, whereas cryptococcal polysaccharide was inactive. Failure of the capsular material to activate the alternative pathway was not due to serotype specificity because polysaccharide of several serotypes failed to achieve activation. The results suggest that chemotaxigenesis and alternative complement pathway activation are functions of the yeast cell wall. The results support our proposal that the cryptococcal capsul does not prevent potential opsonins from reaching binding and activation sites at the yeast cell wall or the release of biologically active soluble cleavage products into the surrounding medium; however, cell wall-bound cleavage products remain bound to the cell wall beneath the capsule. Therefore, they are unable to participate as opsonins in phagocytosis.

Nonencapsulated Variant of Cryptococcus neoformans I. Virulence Studies and Characterization of Soluble Polysaccharide.

A weakly virulent nonencapsulated variant of Cryptococcus neoformans is described. The chemical structure and antigenicity of the soluble polysaccharides produced by the variant strain and a typical virulent strain were compared. The soluble polysaccharides produced by both strains were composed of the same constituent monosaccharides; however, the virulent strain produced a polysaccharide having a greater uronic acid content and a larger molecular size than that of the variant strain. Soluble polysaccharides from the two strains are not closely related immunologically. Soluble polysaccharide obtained from the virulent strain did not affect persistence of the variant strain in mice.