Rapid oxidative release of fungal mannan for detection by immunoassay.

Detection of fungal cells in infected tissue by procedures such as potassium hydroxide (KOH) microscopy and histopathology are well-established methods in medical mycology. However, microscopy requires skilled personnel, specialized equipment, and may take considerable time to a result. An alternative approach is immunoassay for detection of fungal mannans in tissue as a biomarker for the presence of fungal cells. However, mannan is a component of the fungal cell wall, and detection of mannan would require a facile means for mannan extraction prior to detection by immunoassay. In this study, we evaluated a broad spectrum of extraction reagents using Trichophyton rubrum mycelia and Saccharomyces cerevisiae Mnn2 blastoconidia as model fungi. Oxidative release by treatment with dilute bleach proved to be a novel and highly effective procedure. Complete extraction occurred in as little as 2-4 min. Detergents, chaotropes, and acid were ineffective. Strong base released mannan but was less efficient than oxidative release and required the use of highly corrosive reagents. Oxidative release of cell wall mannans from fungal mycelia and blastoconidia may be an effective first step in immunodetection of fungi in tissues from infected humans, animals, or plants that could be done at or near the diagnostic point of need.

Mannans are components of the fungal cell wall that play a role in disease production and are potential biomarkers for the diagnosis of infection. Oxidative release of mannans from intact cell walls is a novel method for mannan extraction that is rapid, uses relatively mild reagents, and yields soluble mannans that are readily detected by immunoassay.

Point-of-Care Testing for Infectious Diseases: Past, Present, and Future.

Point-of-care (POC) diagnostics provide rapid actionable information for patient care at the time and site of an encounter with the health care system. The usual platform has been the lateral flow immunoassay. Recently, emerging molecular diagnostics have met requirements for speed, low cost, and ease of use for POC applications. A major driver for POC development is the ability to diagnose infectious diseases at sites with a limited infrastructure. The potential use in both wealthy and resource-limited settings has fueled an intense effort to build on existing technologies and to generate new technologies for the diagnosis of a broad spectrum of infectious diseases.

Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence.

Upon ingestion by macrophages, Cryptococcus neoformans can survive and replicate intracellularly unless the macrophages become classically activated. The mechanism enabling intracellular replication is not fully understood; neither are the mechanisms that allow classical activation to counteract replication. C. neoformans-induced lysosome damage was observed in infected murine bone marrow-derived macrophages, increased with time, and required yeast viability. To demonstrate lysosome damage in the infected host, we developed a novel flow cytometric method for measuring lysosome damage. Increased lysosome damage was found in C. neoformans-containing lung cells compared with C. neoformans-free cells. Among C. neoformans-containing myeloid cells, recently recruited cells displayed lower damage than resident cells, consistent with the protective role of recruited macrophages. The magnitude of lysosome damage correlated with increased C. neoformans replication. Experimental induction of lysosome damage increased C. neoformans replication. Activation of macrophages with IFN-γ abolished macrophage lysosome damage and enabled increased killing of C. neoformans. We conclude that induction of lysosome damage is an important C. neoformans survival strategy and that classical activation of host macrophages counters replication by preventing damage. Thus, therapeutic strategies that decrease lysosomal damage, or increase resistance to such damage, could be valuable in treating cryptococcal infections.

Influence of IgG Subclass on Human Antimannan Antibody-Mediated Resistance to Hematogenously Disseminated Candidiasis in Mice.

Candida albicans is a yeast-like pathogen and can cause life-threatening systemic candidiasis. Its cell surface is enriched with mannan that is resistant to complement activation. Previously, we developed the recombinant human IgG1 antimannan antibody M1g1. M1g1 was found to promote complement activation and phagocytosis and protect mice from systemic candidiasis. Here, we evaluate the influence of IgG subclass on antimannan antibody-mediated protection. Three IgG subclass variants of M1g1 were constructed: M1g2, M1g3, and M1g4. The IgG subclass identity for each variant was confirmed with DNA sequence and subclass-specific antibodies. These variants contain identical M1 Fabs and exhibited similar binding affinities for C. albicans yeast and purified mannan. Yeast cells and hyphae recovered from the kidney of antibody-treated mice with systemic candidiasis showed uniform binding of each variant, indicating constitutive expression of the M1 epitope and antibody opsonization in the kidney. All variants promoted deposition of both murine and human C3 onto the yeast cell surface, with M1g4 showing delayed activation, as determined by flow cytometry and immunofluorescence microscopy. M1g4-mediated complement activation was found to be associated with its M1 Fab that activates the alternative pathway in an Fc-independent manner. Treatment with each subclass variant extended the survival of mice with systemic candidiasis (P < 0.001). However, treatment with M1g1, M1g3, or M1g4, but not with M1g2, also reduced the kidney fungal burden (P < 0.001). Thus, the role of human antimannan antibody in host resistance to systemic candidiasis is influenced by its IgG subclass.

IgG subclass and heavy chain domains contribute to binding and protection by mAbs to the poly γ-D-glutamic acid capsular antigen of Bacillus anthracis.

Bacterial capsules are common targets for antibody-mediated immunity. The capsule of Bacillus anthracis is unusual among capsules because it is composed of a polymer of poly-γ-d-glutamic acid (γdPGA). We previously generated murine IgG3 monoclonal antibodies (mAbs) to γdPGA that were protective in a murine model of pulmonary anthrax. IgG3 antibodies are characteristic of the murine response to polysaccharide antigens. The goal of the present study was to produce subclass switch variants of the γdPGA mAbs (IgG3 → IgG1 → IgG2b → IgG2a) and assess the contribution of subclass to antibody affinity and protection. Subclass switch antibodies had identical variable regions but differed in their heavy chains. The results showed that a switch from the protective IgG3 to IgG1, IgG2b or IgG2a was accompanied by i) a loss of protective activity ii) a change in mAb binding to the capsular matrix, and iii) a loss of affinity. These results identify a role for the heavy chain constant region in mAb binding. Hybrid mAbs were constructed in which the CH1, CH2 or CH3 heavy chain constant domains from a non-protective, low binding IgG2b mAb were swapped into the protective IgG3 mAb. The IgG3 mAb that contained the CH1 domain from IgG2b showed no loss of affinity or protection. In contrast, swapping the CH2 or CH3 domains from IgG2b into IgG3 produced a reduction in affinity and a loss of protection. These studies identify a role for the constant region of IgG heavy chains in affinity and protection against an encapsulated bacterial pathogen.

Antigenic Relatedness between Mannans from Coccidioides immitis and Coccidioides posadasii Spherules and Mycelia.

Immunoassays for cell wall mannans that are excreted into serum and urine have been used as an aid in the diagnosis of many disseminated fungal infections, including coccidioidomycosis. Antigen-detection immunoassays are critically dependent on the detection of an analyte, such as mannan, by antibodies that are specific to the analyte. The goal of this study was to evaluate the extent of cross-reactivity of polyclonal antibodies raised against Coccidioides spp. Analysis of antigenic relatedness between mannans from C. posadasii and C. immitis spherules and mycelia showed complete relatedness when evaluated by the method of Archetti and Horsfall, which was originally used to study the antigenic relationships between Influenzae virus isolates. In a further effort to validate the suitability of the antigenic relatedness calculation methodology for polysaccharide antigens, we also applied the method of Archetti and Horsfall to published results that had previously identified the major capsular serotypes of Cryptococcus species. The results of this analysis showed that Archetti and Horsfall's antigenic relatedness calculation correctly identified the major cryptococcal serotypes. Together, these results suggest that the method is applicable to polysaccharide antigens, and that immunoassays that detect Coccidioides mannans are likely to have good reactivity across Coccidioides species (inclusivity) due to the species' high level of antigenic relatedness.

A defect in ATP-citrate lyase links acetyl-CoA production, virulence factor elaboration and virulence in Cryptococcus neoformans.

The interaction of Cryptococcus neoformans with phagocytic cells of the innate immune system is a key step in disseminated disease leading to meningoencephalitis in immunocompromised individuals. Transcriptional profiling of cryptococcal cells harvested from cell culture medium or from macrophages found differential expression of metabolic and other functions during fungal adaptation to the intracellular environment. We focused on the ACL1 gene for ATP-citrate lyase, which converts citrate to acetyl-CoA, because this gene showed elevated transcript levels in macrophages and because of the importance of acetyl-CoA as a central metabolite. Mutants lacking ACL1 showed delayed growth on medium containing glucose, reduced cellular levels of acetyl-CoA, defective production of virulence factors, increased susceptibility to the antifungal drug fluconazole and decreased survival within macrophages. Importantly, acl1 mutants were unable to cause disease in a murine inhalation model, a phenotype that was more extreme than other mutants with defects in acetyl-CoA production (e.g. an acetyl-CoA synthetase mutant). Loss of virulence is likely due to perturbation of critical physiological interconnections between virulence factor expression and metabolism in C. neoformans. Phylogenetic analysis and structural modelling of cryptococcal Acl1 identified three indels unique to fungal protein sequences; these differences may provide opportunities for the development of pathogen-specific inhibitors.

Rapid Capsular Antigen Immunoassay for Diagnosis of Inhalational Anthrax: Preclinical Studies and Evaluation in a Nonhuman Primate Model.

Inhalational anthrax is a fatal infectious disease. Rapid and effective treatment is critically dependent on early and accurate diagnosis. Blood culture followed by identification and confirmation may take days to provide clinically relevant information. In contrast, immunoassay for a shed antigen, the capsular polypeptide gamma-d-polyglutamate (γDPGA), can provide rapid results at the point of care. In this study, a lateral flow immunoassay for γDPGA was evaluated in a robust nonhuman primate model of inhalational anthrax. The results showed that the time to a positive result with the rapid test using either serum or blood as a clinical specimen was similar to the time after infection when a blood culture became positive. In vitro testing showed that the test was equally sensitive with cultures of the three major clades of Bacillus anthracis. Cultures from other Bacillus spp. that are known to produce γDPGA also produced positive results. The test was negative with human sera from 200 normal subjects and 45 subjects with culture-confirmed nonanthrax bacterial or fungal sepsis. Taken together, the results showed that immunoassay for γDPGA is an effective surrogate for blood culture in a relevant cynomolgus monkey model of inhalational anthrax. The test would be a valuable aid in early diagnosis of anthrax, which is critical for rapid intervention and a positive outcome. Use of the test could facilitate triage of patients with signs and symptoms of anthrax in a mass-exposure incident and in low-resource settings where laboratory resources are not readily available. IMPORTANCE Patient outcome in anthrax is critically dependent on early diagnosis followed by effective treatment. We describe a rapid lateral flow immunoassay that detects capsular antigen of Bacillus anthracis that is shed into blood during infection. The test was evaluated in a robust cynomolgus monkey model of inhalational anthrax. Rapid detection of capsular antigen is an effective surrogate for the time-consuming and laboratory-intensive diagnosis by blood culture, direct fluorescent antibody staining, or other molecular testing. The test can be performed at the point of patient contact, is rapid and inexpensive, and can be used by individuals with minimal training.

Evaluation of a novel point-of-care cryptococcal antigen test on serum, plasma, and urine from patients with HIV-associated cryptococcal meningitis.

BACKGROUND: Many deaths from cryptococcal meningitis (CM) may be preventable through early diagnosis and treatment. An inexpensive point-of-care (POC) assay for use with urine or a drop of blood would facilitate early diagnosis of cryptococcal infection in resource-limited settings. We compared cryptococcal antigen (CRAG) concentrations in plasma, serum, and urine from patients with CM, using an antigen-capture assay for glucuronoxylomannan (GXM) and a novel POC dipstick test. METHODS: GXM concentrations were determined in paired serum, plasma, and urine from 62 patients with active or recent CM, using a quantitative sandwich enzyme-linked immunosorbent assay (ELISA). A dipstick lateral-flow assay developed using the same monoclonal antibodies for the sandwich ELISA was tested in parallel. Correlation coefficients were calculated using Spearman rank test. RESULTS: All patients had detectable GXM in serum, plasma, and urine using the quantitative ELISA. Comparison of paired serum and plasma showed identical results. There were strong correlations between GXM levels in serum/urine (r(s) = 0.86; P < .001) and plasma/urine (r(s) = 0.85; P < .001). Levels of GXM were 22-fold lower in urine than in serum/plasma. The dipstick test was positive in serum, plasma, and urine in 61 of 62 patients. Dipstick titers correlated strongly with ELISA. Correlations between the methods were 0.93 (P < .001) for serum, 0.94 (P < .001) for plasma, and 0.94 (P < .001) for urine. CONCLUSIONS: This novel dipstick test has the potential to markedly improve early diagnosis of CM in many settings, enabling testing of urine in patients presenting to health care facilities in which lumbar puncture, or even blood sampling, is not feasible.

A microbial polysaccharide reduces the severity of rheumatoid arthritis by influencing Th17 differentiation and proinflammatory cytokines production.

Rheumatoid arthritis (RA) is a chronic and debilitating autoimmune disease characterized by chronic joint inflammation with subsequent cartilage and bone destruction. RA is emerging as a model of IL-17-driven autoimmune inflammatory disease. IL-17 is a marker for Th17 cells, with its master regulator being the retinoic acid receptor-related orphan receptor (RORgammat) regulated by STAT3 signaling. Glucuronoxylomannan (GXM), a polysaccharide representing the main component of the capsular material of the opportunistic yeast Cryptococcus neoformans, exhibits potent immunosuppressive properties both in vitro and in vivo. The present study investigates the effects of GXM treatment on the progression of collagen-induced arthritis. GXM suppressed clinical signs of collagen-induced arthritis and blocked joint erosion progression. This effect was mediated by down-regulation of key cytokines involved in the pathogenesis of RA such as TNF-alpha and IL-1beta, and up-regulation of the inhibitory cytokine IL-10. Moreover, a reduction of IL-6 and TGF-beta, which inhibit Th17 differentiation with consequent decreased IL-17 production at the local and systemic level, was observed. The effect of GXM on Th17 differentiation mirrored the reduction in STAT3 activation and inhibition of RORgammat synthesis. Consequently, this work highlights the beneficial properties of an efficacious compound that could eventually be destined to the clinic.

Influence of mannan and glucan on complement activation and C3 binding by Candida albicans.

The complement system is important for host resistance to hematogenously disseminated candidiasis. However, modulation of complement activation by cell wall components of Candida albicans has not been characterized. Although intact yeast display mannan on the surface, glucan, typically located in the interior, becomes exposed during C. albicans infection. We show here the distinct effects of mannan and glucan on complement activation and opsonophagocytosis. Previous studies showed that intact cells are resistant to initiation of complement activation through the alternative pathway, and antimannan antibody reverses this resistance via an Fc-independent mechanism. The present study shows that this mannan-dependent resistance can be overcome by periodate-borohydride conversion of mannose polysaccharides to polyalcohols; cells treated with periodate-borohydride initiate the alternative pathway without the need for antibody. These observations identify an inhibitory role for intact mannan in complement activation. Next, removal of the surface-displayed mannan by acid treatment of periodate-borohydride cells exposes glucan. Glucan-displaying cells or purified beta-glucan initiate the alternative pathway when incubated with the purified proteins of the alternative pathway alone, suggesting that C. albicans glucan is a natural activator of the alternative pathway. Finally, ingestion of mannan-displaying cells by human neutrophils requires anti-mannan antibody, whereas ingestion of glucan-displaying cells requires complement. These results demonstrate a contrasting requirement of natural antibody and complement for opsonophagocytosis of C. albicans cells displaying mannan or glucan. Thus, differential surface expression of mannan and glucan may influence recognition of C. albicans by the complement system.

Phenotypic heterogeneity in expression of epitopes in the Cryptococcus neoformans capsule.

The opportunistic yeast Cryptococcus neoformans is surrounded by a polysaccharide capsule comprised primarily of glucuronoxylomannan (GXM). GXM is a key component of the antigenic character of the capsule. Expression of the epitope that allows for binding of mAbs that require O-acetylation of GXM for mAb recognition was greatly influenced by cell age, growth conditions and serotype. Yeast cells of serotype A grown in vitro under capsule induction conditions showed considerable cell-to-cell variability in binding of two O-acetyl-dependent mAbs, and such mAbs uniformly failed to bind to GXM that covers yeast buds. Expression of the O-acetyl-dependent epitope increased with cell age. In contrast, all serotype A cells harvested from brain tissue bound the same O-acetyl-dependent mAbs. The ability of the cryptococcal capsule to activate the complement cascade and bind C3 occurred uniformly over the surface of all yeast cells, including the bud. Finally, the cell-to-cell variability in binding of O-acetyl-dependent mAbs with strains of serotype A was not found with strains of serotype D; almost all cells of serotype D showed homogeneous binding of O-acetyl-dependent mAbs. These results indicate that variability in expression of antigenic epitopes by GXM should be considered in selection of mAbs used for immunodiagnosis or immunotherapy.

Isolation and characterization of Cryptococcus neoformans spores reveal a critical role for capsule biosynthesis genes in spore biogenesis.

Spores are essential particles for the survival of many organisms, both prokaryotic and eukaryotic. Among the eukaryotes, fungi have developed spores with superior resistance and dispersal properties. For the human fungal pathogens, however, relatively little is known about the role that spores play in dispersal and infection. Here we present the purification and characterization of spores from the environmental fungus Cryptococcus neoformans. For the first time, we purified spores to homogeneity and assessed their morphological, stress resistance, and surface properties. We found that spores are morphologically distinct from yeast cells and are covered with a thick spore coat. Spores are also more resistant to environmental stresses than yeast cells and display a spore-specific configuration of polysaccharides on their surfaces. Surprisingly, we found that the surface of the spore reacts with antibodies to the polysaccharide glucuronoxylomannan, the most abundant component of the polysaccharide capsule required for C. neoformans virulence. We explored the role of capsule polysaccharide in spore development by assessing spore formation in a series of acapsular strains and determined that capsule biosynthesis genes are required for proper sexual development and normal spore formation. Our findings suggest that C. neoformans spores may have an adapted cell surface that facilitates persistence in harsh environments and ultimately allows them to infect mammalian hosts.

Characteristics of Fc-independent human antimannan antibody-mediated alternative pathway initiation of C3 deposition to Candida albicans.

The complement system has an important role in host resistance to systemic candidiasis but regulation of complement activation by Candida albicans remains poorly defined. Previous studies have identified a requirement for naturally occurring antimannan IgG antibody in initiation of C3 opsonization of C. albicans through either the classical or alternative pathway. This study characterized antibody-dependent initiation of the alternative pathway using the recombinant human monoclonal antimannan Fab fragment M1 and its full-length IgG1 antibody M1g1. Kinetic analysis of C3b deposition onto C. albicans with flow cytometry demonstrated the ability of M1g1 to restore the activity of either the classical or alternative pathway to the yeast-absorbed normal human serum, but the Fc-free M1 Fab restored only the activity of the alternative pathway. This Fc-independent, antimannan Fab-mediated C3 deposition through the alternative pathway was also observed in a serum-free assay containing the six alternative pathway proteins and in C1q- or C2-depleted serum but not in factor B-depleted serum. M1- or M1g1-dependent alternative pathway initiation of C3b deposition occurred in an asynchronous manner at discrete sites that expanded to cover the entire cell surface over time as revealed with immunofluorescence microscopy, in contrast to a uniform appearance of initial C3 deposition through the classical pathway. Furthermore, antimannan Fab M1 promoted the assembly of the alternative pathway convertase on the cell surface seen as colocalization of C3 and factor B with immunofluorescence microscopy. Thus, human antimannan antibody has a distinct Fc-independent effector function in regulation of C3 deposition to C. albicans.

Tracheal colonization factor A (TcfA) is a biomarker for rapid and specific detection of Bordetella pertussis.

Pertussis is a highly contagious disease for which prompt, point-of-care diagnosis remains an unmet clinical need. Results from conventional test modalities (nucleic acid detection, serology, and culture) take hours to days. To overcome this challenge, we identified a new biomarker (tracheal colonization factor A, TcfA) for detection of Bordetella pertussis infection by lateral flow immunoassay (LFIA). We developed a library of 28 epitope-mapped monoclonal antibodies against TcfA and incorporated three antibodies into a LFIA. The LFIA did not cross-react with common bacterial or fungal organisms, but did react with nine distinct B. pertussis strains. The minimal linear epitope sequences targeted by the LFIA were conserved in 98% of 954 B. pertussis isolates collected across 12 countries from 1949-2017. The LFIA's limit of detection was 3.0 × 105 CFU/mL with B. pertussis cells in buffer, 6.2 × 105 CFU/mL with nasopharyngeal washes from a non-human primate model, and 2.3 ng/mL with recombinant TcfA. The LFIA reacted with patient nasopharyngeal swab specimens containing as few as 1.8 × 106 B. pertussis genomes/mL and showed no false-positives. Rapid (< 20 min) LFIA detection of TcfA as a biomarker for B. pertussis infection is feasible and may facilitate early detection of pertussis.

Macrophage uptake, intracellular localization, and degradation of poly-gamma-D-glutamic acid, the capsular antigen of Bacillus anthracis.

Bacillus anthracis is surrounded by a capsular polypeptide composed of poly-gamma-D-glutamic acid (PGA). This antiphagocytic capsule is an essential virulence factor and is shed into body fluids during a murine model of pulmonary anthrax. Our previous studies of a murine model for antigen clearance showed that purified PGA accumulates in the liver and spleen, most notably in splenic macrophages and the Kupffer cells and sinusoidal endothelial cells of the liver. Although the tissue and cellular depots have been identified, there is little known about the uptake and intracellular fate of PGA. As a consequence, we examined the cellular uptake and organelle localization of PGA in the murine macrophage-like cell line J774.2. We found that PGA binds to and is internalized by J774.2 cells and accumulates in CD71 transferrin receptor-positive endosomes. The receptor-mediated endocytosis inhibitors amantadine and phenylarsine oxide inhibited the binding and uptake of PGA in these cells. Cytochalasin D and vinblastine, actin and microtubule inhibitors, respectively, failed to completely inhibit binding and uptake. Finally, we found that PGA is degraded in J774.2 cells starting 4 h after uptake, with continued degradation occurring for at least 24 h. This degradation of PGA may explain the rapid clearance of PGA that is observed in vivo compared to the slow clearance noted with capsular polysaccharides.

Efficacy of opsonic and nonopsonic serotype 3 pneumococcal capsular polysaccharide-specific monoclonal antibodies against intranasal challenge with Streptococcus pneumoniae in mice.

Serotype-specific antibodies to pneumococcal capsular polysaccharide (PPS) are a critical component of vaccine-mediated immunity to Streptococcus pneumoniae. In this study, we investigated the in vitro opsonophagocytic activities of three PPS-specific mouse immunoglobulin G1 monoclonal antibodies (MAbs), 1E2, 5F6, and 7A9, and determined their in vivo efficacies against intranasal challenge with WU2, a serotype 3 pneumococcal strain, in normal and immunodeficient mice. The MAbs had different in vitro activities in a pneumococcal killing assay: 7A9 enhanced killing by mouse neutrophils and J774 cells in the presence of a complement source, whereas 5F6 promoted killing in the absence, but not the presence, of complement, and 1E2 did not promote killing under any conditions. Nonetheless, all three MAbs protected normal and complement component 3-deficient mice from a lethal intranasal challenge with WU2 in passive-immunization experiments in which 10 mug of the MAbs were administered intraperitoneally before intranasal challenge. In contrast, only 1E2 protected Fcgamma receptor IIB knockout (FcgammaRIIB KO) mice and mice that were depleted of neutrophils with the MAb RB6, whereas 7A9 and 5F6 required neutrophils and FcgammaRIIB to mediate protection. Conversely, 7A9 and 5F6 protected FcgammaR KO mice, but 1E2 did not. Hence, the efficacy of 1E2 required an activating FcgammaR(s), whereas 5F6 and 7A9 required the inhibitory FcgammaR (FcgammaRIIB). Taken together, our data demonstrate that both MAbs that do and do not promote pneumococcal killing in vitro can mediate protection in vivo, although their efficacies depend on different host receptors and/or components.

Kinetics of lethal factor and poly-D-glutamic acid antigenemia during inhalation anthrax in rhesus macaques.

Systemic anthrax manifests as toxemia, rapidly disseminating septicemia, immune collapse, and death. Virulence factors include the anti-phagocytic gamma-linked poly-d-glutamic acid (PGA) capsule and two binary toxins, complexes of protective antigen (PA) with lethal factor (LF) and edema factor. We report the characterization of LF, PA, and PGA levels during the course of inhalation anthrax in five rhesus macaques. We describe bacteremia, blood differentials, and detection of the PA gene (pagA) by PCR analysis of the blood as confirmation of infection. For four of five animals tested, LF exhibited a triphasic kinetic profile. LF levels (mean +/- standard error [SE] between animals) were low at 24 h postchallenge (0.03 +/- 1.82 ng/ml), increased at 48 h to 39.53 +/- 0.12 ng/ml (phase 1), declined at 72 h to 13.31 +/- 0.24 ng/ml (phase 2), and increased at 96 h (82.78 +/- 2.01 ng/ml) and 120 h (185.12 +/- 5.68 ng/ml; phase 3). The fifth animal had an extended phase 2. PGA levels were triphasic; they were nondetectable at 24 h, increased at 48 h (2,037 +/- 2 ng/ml), declined at 72 h (14 +/- 0.2 ng/ml), and then increased at 96 h (3,401 +/- 8 ng/ml) and 120 h (6,004 +/- 187 ng/ml). Bacteremia was also triphasic: positive at 48 h, negative at 72 h, and positive at euthanasia. Blood neutrophils increased from preexposure (34.4% +/- 0.13%) to 48 h (75.6% +/- 0.08%) and declined at 72 h (62.4% +/- 0.05%). The 72-h declines may establish a "go/no go" turning point in infection, after which systemic bacteremia ensues and the host's condition deteriorates. This study emphasizes the value of LF detection as a tool for early diagnosis of inhalation anthrax before the onset of fulminant systemic infection.

Biosensing with nanofluidic diodes.

Recently reported nanofluidic diodes with highly nonlinear current-voltage characteristics offer a unique possibility to construct different biosensors. These sensors are based on local changes of the surface charge on walls of single conical nanopores induced by binding of an analyte. The analyte binding can be detected as a change of the ion-current rectification of single nanopores defined as a ratio of currents for voltages of one polarity, and currents for voltages of the opposite polarity. In this article, we provided both modeling and experimental studies of various biosensing routes based on monitoring changes of the rectification degree in nanofluidic diodes used as a biosensing platform. A prototype of a sensor for the capsular poly gamma-D-glutamic acid (gammaDPGA) from Bacillus anthracis is presented. The nanopore used for the sensing was locally modified with the monoclonal antibody for gammaDPGA. The proof of principle of the rectification degree-based sensing was further shown by preparation of sensors for avidin and streptavidin. Our devices also allowed for determination of the isoelectric point of the minute amounts of proteins immobilized on the surface.

Capsular polysaccharide induction of apoptosis by intrinsic and extrinsic mechanisms.

A purified microbial capsular polysaccharide of Cryptococcus neoformans, glucuronoxylomannan (GXM), induces Fas ligand (FasL) upregulation on macrophages and, as a consequence, apoptosis of lymphocytes. The mechanisms that lead to lymphocyte apoptosis in both in vitro and in vivo systems were investigated by cytofluorimetric analysis and Western blotting experiments. Caspase 8 cleaves caspase 3 in two different pathways: directly as well as indirectly by activation of Bcl-2 interacting domain, which initiates caspase 9 cleavage. Therefore, the caspase 8 and caspase 9 pathways cooperate in an amplification loop for efficient cell death, and noteworthily we provide evidence that they are both activated in one single cell. Furthermore, both activation of GXM-mediated caspase 8 and apoptosis were also found in in vivo systems in an experimental model of murine candidiasis. Collectively, our data show that GXM-induced apoptosis involves, in a single cell, a cross-talk between extrinsic and intrinsic pathways. Such a finding offers opportunities for the therapeutic usage of this polysaccharide in appropriate clinical settings for taming T-cell responses.