Immunogenicity of a fractional or full third dose of AZD1222 vaccine or BNT162b2 messenger RNA vaccine after two doses of CoronaVac vaccines against the Delta and Omicron variants.

OBJECTIVES: The study aimed to compare the immunogenicity and safety of fractional (half) third doses of heterologous COVID-19 vaccines (AZD1222 or BNT162b2) to full doses after the two-dose CoronaVac and when boosting after three different extended intervals. METHODS: At 60-<90, 90-<120, or 120-180 days intervals after the two-dose CoronaVac, participants were randomized to full-dose or half-dose AZD1222 or BNT162b2, followed up at day 28, 60, and 90. Vaccination-induced immune responses to Ancestral, Delta, and Omicron BA.1 strains were evaluated by antispike, pseudovirus, and microneutralization and T cell assays. Descriptive statistics and noninferiority cut-offs were reported as geometric mean concentration or titer and concentration or titer ratios comparing baseline to day 28 and day 90 and different intervals. RESULTS: No safety concerns were detected. All assays and intervals showed noninferior immunogenicity between full doses and half doses. However, full-dose vaccines and/or longer 120-180-day intervals substantially improved the immunogenicity (measured by antispike or measured by pseudotyped virus neutralizing titers 50; P <0.001). Seroconversion rates were over 90% against the SARS-CoV-2 strains by all assays. Immunogenicity waned more quickly with half doses than full doses but remained high against the Ancestral or Delta strains. Against Omicron, the day 28 immunogenicity increased with longer intervals than shorter intervals for full-dose vaccines. CONCLUSION: Immune responses after day 28 when boosting at longer intervals after the two-dose CoronaVac was optimal. Half doses met the noninferiority criteria compared with the full dose by all the immune assays assessed.

Cell-type-specific transcriptional profiles of the dimorphic pathogen Penicillium marneffei reflect distinct reproductive, morphological, and environmental demands.

Penicillium marneffei is an opportunistic human pathogen endemic to Southeast Asia. At 25° P. marneffei grows in a filamentous hyphal form and can undergo asexual development (conidiation) to produce spores (conidia), the infectious agent. At 37° P. marneffei grows in the pathogenic yeast cell form that replicates by fission. Switching between these growth forms, known as dimorphic switching, is dependent on temperature. To understand the process of dimorphic switching and the physiological capacity of the different cell types, two microarray-based profiling experiments covering approximately 42% of the genome were performed. The first experiment compared cells from the hyphal, yeast, and conidiation phases to identify "phase or cell-state-specific" gene expression. The second experiment examined gene expression during the dimorphic switch from one morphological state to another. The data identified a variety of differentially expressed genes that have been organized into metabolic clusters based on predicted function and expression patterns. In particular, C-14 sterol reductase-encoding gene ergM of the ergosterol biosynthesis pathway showed high-level expression throughout yeast morphogenesis compared to hyphal. Deletion of ergM resulted in severe growth defects with increased sensitivity to azole-type antifungal agents but not amphotericin B. The data defined gene classes based on spatio-temporal expression such as those expressed early in the dimorphic switch but not in the terminal cell types and those expressed late. Such classifications have been helpful in linking a given gene of interest to its expression pattern throughout the P. marneffei dimorphic life cycle and its likely role in pathogenicity.

Interleukin-25 (IL-25) promotes efficient protective immunity against Trichinella spiralis infection by enhancing the antigen-specific IL-9 response.

Mammalian hosts often develop distinct immune response against the diverse parasitic helminths that have evolved for immune evasion. Interleukin-25 (IL-25), an IL-17 cytokine family member, plays a key role in initiating the protective immunity against several parasitic helminths; however, the involvement and underlying mechanisms by which IL-25 mediates immune response against Trichinella spiralis infection have not been investigated. Here we showed that IL-25 functions in promoting protective immunity against T. spiralis infection. Mice treated with IL-25 exhibited a lower worm burden and fewer muscle larvae in the later stage of T. spiralis infection. In contrast, mice treated with neutralizing antibody against IL-25 failed to expel T. spiralis effectively. During T. spiralis infection, intestinal IL-25 expression was rapidly elevated before the onset of IL-4 and IL-9 induction. While antigen-specific Th2 and Th9 immune responses were both developed during T. spiralis infection, an antigen-specific Th9 response appeared to be transiently induced in the early stage of infection. Mice into which antigen-specific T cells deficient in IL-9 were transferred were less effective in worm clearance than those given wild-type T cells. The strength of the antigen-specific Th9 immune response against T. spiralis could be enhanced or attenuated after treatment with IL-25 or neutralizing antibody against IL-25, respectively, correlating positively with the levels of intestinal mastocytosis and the expression of IL-9-regulated genes, including mast cell- and Paneth cell-specific genes. Thus, our study demonstrates that intestinal IL-25 promotes protective immunity against T. spiralis infection by inducing antigen-specific Th9 immune response.

In vitro antifungal activities of longan (Dimocarpus longan Lour.) seed extract.

Longan, Dimocarpus longan Lour., contains polyphenolic compounds which exhibit several pharmacological properties. This study aims to evaluate antifungal activities of longan fruit extract in comparison to its active compounds. The results showed that longan seed exhibited antifungal activity against the opportunistic yeasts (Candida species and Cryptococcus neoformans). In contrast, longan pulp and whole fruit did not demonstrate any inhibitory effects. Ellagic acid showed the most potent antifungal activity followed by corilagin and gallic acid, respectively. Ellagic acid inhibited Candida parapsilosis and C. neoformans more effectively than Candida krusei and also some Candida albicans clinical strains. Baidam cultivar possessed higher antifungal activity (MIC=500-4000 µg/ml) as it contained higher contents of ellagic acid and gallic acid than Edor (MIC=1000-8000 µg/ml). For antibacterial activity, only corilagin and gallic acid possessed weak to moderate inhibitory effects against Staphylococcus aureus and Streptococcus mutans, respectively. Longan seed was then applied in the oral care products. Longan effervescent granule (5% extract) significantly reduced adhesion of C. albicans to acrylic strips. Mouthwash containing 0.5% extract exhibited good antifungal activity compared to a commercial product. These findings indicated that longan seed extract and its polyphenolic compounds can be used as an antifungal agent in oral care products for the treatment of opportunistic yeast infection.

Effects of paraoxon on neuronal and lymphocytic cholinergic systems.

The cholinergic system in lymphocytes is hypothesized to be a key target for neurotoxic organophosphates (OPs). The present study determined the comparative effects of paraoxon, the active metabolite of OP-parathion, which is detected in the human neuroblastoma line, SH-SY5Y, and leukemic T-lymphocytes, MOLT-3, in vitro. Paraoxon induced cytotoxic effects in a dose- and time-dependent manner in both cells. Further, the paraoxon-induced modulatory effects were comparable despite different cell types, including over-expression of N-terminus acetylcholinesterase (N-AChE) protein, a marker of apoptosis, down-regulations of mRNA encoding M1, M2, and M3 muscarinic acetylcholine receptors (mAChRs), and induction in expression of c-Fos gene, an indication of certain mAChR subtype(s) activation. Furthermore, the non-selective cholinergic antagonist atropine partially attenuated the paraoxon-induced N-AChE and c-Fos activations in both types of cells. These results provide initial and additional information that OPs may similarly induce neuro- and immuno-toxic effects through mAChRs activation, and they underline the potential of using lymphocytes for assessing OPs-induced neurotoxicity.

Atorvastatin affects TLR4 clustering via lipid raft modulation.

Statins, HMG-CoA reductase inhibitors, are used widely in the treatment of hypercholesterolemia. Apart from lowering lipid levels, statins have been shown to have anti-inflammatory effects. Previously we showed that atorvastatin inhibits NF-kappaB activation, dose and time dependently, in LPS-TLR4 signaling pathway. In this study, we investigated the anti-inflammatory mechanism of atorvastatin via Toll-like receptor 4 (TLR4) in murine pro-B cell lines transfected with TLR4. Co-treatment of LPS-stimulated cells with both atorvastatin and mevalonate rescued NF-kappaB activation and TLR4 blockade demonstrated that atorvastatin does not exert its inhibitory effect via TLR4 receptor-ligand binding mechanism. Further investigation into the anti-inflammatory mechanism has shown that atorvastatin causes an impairment of TLR4 recruitment into the lipid raft thereby affecting anti-inflammatory responses. In contrast, mevalonate repaired lipid raft function leading to TLR4 clustering in the lipid raft. Together, these data suggest that atorvastatin exerts its anti-inflammatory effect via lipid raft modification. This novel finding offers another insight into the pleiotropic effects of atorvastatin and may be applicable to other pattern recognition receptors that utilize membrane lipid raft as a platform for signal transduction.

Analysis of Foxp3, CD25, and CD127 expressed on regulatory T cells in Thai subjects.

Foxp3+ natural regulatory T cells (nTreg) play a distinct role in maintaining self tolerance at the periphery. CD25hi and CD127lo were proposed for the identification and purification of nTreg but they have not been confirmed in non-Caucasian populations. This study examined the sensitivity and purity of Foxp3 nTreg identified by CD25 and CD127 in the peripheral blood of Thai subjects (13 males, 15 females with age range of 20-42 years old). The proportions of nTreg/CD4+ as identified by the different markers were as follows: Foxp3+, 18.3 +/- 6.4%; CD25hi 6.4 +/- 3.2%; and CD127lo, 54.3 +/- 14.2%. Sensitivity tests showed the following results: CD25hi, 23.1%; CD127lo, 40.6%; CD25hiCD127lo, 7.4%. Purity tests concluded as follows: CD25hi, 63.6%; CD25int, 24.9%; CD2510, 8.7%, CD127lo, 26.5%; CD127hi, 14.9%, and CD25hiCD127lo, 52.0%. In conclusion, the proportions of nTreg in Thai subjects are similar to Caucasian populations. CD25hi is superior to CD127lo for separating Foxp3+ nTreg. Combining CD25hi and CD127lo does not improve the nTreg purity.

Atorvastatin attenuates TLR4-mediated NF-kappaB activation in a MyD88-dependent pathway.

Antigen presenting cells such as dendritic cells and macrophages have recently been detected in atherosclerotic plaques. Toll-like receptors expressed on the surface of these cells, have been implicated in ongoing inflammatory responses in the plaques. In this study, we investigated the anti-inflammatory effect of atorvastatin, a lipid lowering drug, via Toll-like receptor 4 (TLR4) in vitro, employing murine pro-B cell lines transfected with hTLR4/MD2 and MyD88/hTLR4/MD2 systems. The results showed that atorvastatin at 10 microM significantly attenuated NF-kappaB activation within 24 hours while at lower doses of 0.1 and 1 microM, treatment time had to be prolonged up to 48 hours for a significant inhibition to occur. The inhibition of NF-kappaB was also observed in a cell line cotransfected with MyD88 and TLR4 suggesting that the attenuation of NF-kappaB by atorvastatin occurred in a MyD88 dependent fashion.

Differential gene expression profiles of lung epithelial cells exposed to Burkholderia pseudomallei and Burkholderia thailandensis during the initial phase of infection.

Burkholderia pseudomallei is the causative agent of melioidosis, and its infection usually affects patients' lungs. The organism is a facultative intracellular Gram-negative bacillus commonly found in soil and water in endemic tropical regions. Another closely related Burkholderia species found in soil and water is B. thailandensis. This bacterium is a non-pathogenic environmental saprophyte. B. pseudomallei is considerably more efficient than B. thailandensis in host cell invasion and adherence. A previous study by our group demonstrated that after successfully invading cells, there was no difference in the ability to survive and to replicate between both Burkholderia species in cultured A549 human lung epithelial cells. In this study, Human Affymetrix GeneChips were used to identify the difference in gene expression profiles of A549 cells after a 2-h exposure to B. pseudomallei and B. thailandensis. A total of 280 of 22,283 genes were expressed at higher levels in the B. pseudomallei-infected cells than in the B. thailandensis-infected cells, while 280 genes were expressed at lower levels in the B. pseudomallei-infected cells. Approximately 9% of these genes were involved in immune response and apoptosis. Those genes were further selected for gene expression analysis using reverse transcription PCR and/or real-time RT-PCR. The results of RT-PCR and real-time RT-PCR are in accordance with data from the microarray data in that bcl2 gene expression in the B. pseudomallei-infected cells was 2-fold higher than the level in the B. thailandensis-infected cells even though no apoptosis was seen in the infected cells. The levels of E-selectin, ICAM-1, IL-11, IRF-1, IL-6, IL-1beta and LIF genes expression in the B. pseudomallei-infected cells were 1.5-5 times lower than in the B. thailandensis-infected cells. However, both species stimulated the same level of IL-8 production from the tested epithelial cell line, and no difference in the ratio of adherent polymorphonuclear cells (PMNs) to infected A549 cells of both species was observed. Taken together, our results suggest that B. pseudomallei manipulates host response in favor of its survival in the host cell, which may explain the more virulent characteristics of B. pseudomallei when compared with B. thailandensis.

Engagement of Penicillium marneffei conidia with multiple pattern recognition receptors on human monocytes.

P. marneffei is a thermal dimorphic fungus which causes penicilliosis, an opportunistic infection in immunocompromised patients in South and Southeast Asia. Little is known about the innate immune response to P. marneffei infection. Therefore, the initial response of macrophages to P. marneffei conidia was evaluated by us. Adhesion between monocytes from healthy humans and fungal conidia was examined and found to be specifically inhibited by MAbs against PRR, such as MR, (TLR)1, TLR2, TLR4, TLR6, CD14, CD11a, CD11b, and CD18. To study the consequences of these interactions, cytokines were also examined by ELISA. Binding of P. marneffei conidia to monocytes was significantly inhibited, in a dose-dependent manner, by MAbs against MR, TLR1, TLR2, TLR4, TLR6, CD14, CD11b and CD18. When monocytes were co-cultured with the conidia, there was an increase in the amount of surface CD40 and CD86 expression, together with TNF-alpha and IL-1beta production, compared to unstimulated controls. In assays containing anti-TLR4 or anti-CD14 antibody, reduction in the amount of TNF-alpha released by monocytes stimulated with P. marneffei conidia was detected. In addition, it was found that production of TNF-alpha and IL-1beta from adherent peripheral blood monocytes was partially impaired when heat-inactivated autologous serum, in place of untreated autologous serum, was added to the assay. These results demonstrate that various PRR on human monocytes participate in the initial recognition of P. marneffei conidia, and the engagement of PRR could partly initiate proinflammatory cytokine production.

Serum antibodies and cytokines in C4-deficient mice and their responses to exercise.

Psychological stress is believed to be one of the predisposing factors for systemic lupus erythematosus (SLE), whereas physical stress such as exercise has never been reported to be related. We measured the circulating levels of antibodies (IgM, IgG, anti-dsDNA IgG), Th1 (IFN-gamma), Th2 (IL-4, IL-6), and of pro-inflammatory (TNF-alpha, IL-1beta) and anti-inflammatory (TGF-beta) cytokines of C4(-l-) female mice at rest, after acute exercise and after exercise training, using an antibody-capture ELISA. Prior to the exercise, the C4(-l-) mice had higher levels of IgG and anti-dsDNA IgG but lower levels of IFN-gamma, IL-1beta, IL-6 and IL-4 than wild-type C57BL/6 (B6) mice. A single bout of exercise to exhaustion increased serum IgG, TNF-alpha, IL-1beta and TGF-beta in the B6 mice but only TGF-beta in the C4(-l-) mice was increased. We conclude that exhaustive or moderate exercise has no effect on the levels of serum antibodies and cytokines and is thus unlikely to promote the onset of SLE.

Differential gene expression profiles of human monocyte-derived antigen presenting cells in response to Penicillium marneffei: roles of DC-SIGN (CD209) in fungal cell uptake.

DNA microarray technology was used to determine the gene expression profile of human monocyte-derived macrophages (hMDMs) after stimulation by Penicillium marneffei yeast. The expression levels of 175 macrophage genes were found to be altered by a minimum of two-fold in magnitude following 4 hours of P. marneffei exposure. Among those, 41 genes were upregulated in activated hMDMs while 134 genes were downregulated. Real-time PCR and RT-PCR were performed to further examine gene expression associated with the inflammatory response. Increased levels of TNF-alpha and IL-1 beta gene expression in both hMDMs and human monocyte-derived dendritic cells (hMoDCs) were observed after stimulation by P. marneffei yeast. Furthermore, the genes encoding T-bet, IL-6 and ICAM-1 were also upregulated in hMDMs. Functional analysis of the adhesion of P. marneffei to dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN, CD209) was performed in hMoDCs since the microarray data revealed an increased expression of DC-SIGN in activated hMDMs. We found that DC-SIGN-Fc bound preferentially to P. marneffei yeast rather than to conidia. Moreover, an anti-DC-SIGN monoclonal antibody inhibited the binding of P. marneffei yeast to hMoDCs, but did not inhibit endocytosis of P. marneffei yeast. The mannose receptor, on the other hand, was important in both adhesion and phagocytosis. These results suggest that P. marneffei may exploit DC-SIGN as a receptor to facilitate the systemic spread of infection. Taken together, our study demonstrates the usefulness of microarray technology in generating valuable expression data to permit conventional immunologic investigations of host-fungal interactions.

Molecular typing of Vibrio cholerae O1 isolates from Thailand by pulsed-field gel electrophoresis.

The aim of the present study was to genotypically characterize Vibrio cholerae strains isolated from cholera patients in various provinces of Thailand. Two hundred and forty V. cholerae O1 strains, isolated from patients with cholera during two outbreaks, i.e. March 1999-April 2000 and December 2001-February 2002, in Thailand, were genotypically characterized by NotI digestion and pulsed-field gel electrophoresis (PFGE). In total, 17 PFGE banding patterns were found and grouped into four Dice-coefficient clusters (PF-I to PF-IV). The patterns of V. cholerae O1, El Tor reference strains from Australia, Peru, Romania, and the United States were different from the patterns of reference isolates from Asian countries, such as Bangladesh, India, and Thailand, indicating a close genetic relationship or clonal origin of the isolates in the same geographical region. The Asian reference strains, regardless of their biotypes and serogroups (classical O1, El Tor O1, O139, or O151), showed a genetic resemblance, but had different patterns from the strains collected during the two outbreaks in Thailand. Of 200 Ogawa strains collected during the first outbreak in Thailand, two patterns (clones)--PF-I and PF-II--predominated, while other isolates caused sporadic cases and were grouped together as pattern PF-III. PF-II also predominated during the second outbreak, but none of the 40 isolates (39 Inaba and 1 Ogawa) of the second outbreak had the pattern PF-I; a minority showed a new pattern--PF-IV, and others caused single cases, but were not groupable. In summary, this study documented the sustained appearance of the pathogenic V. cholerae O1 clone PF-II, the disappearance of clones PF-I and PF-III, and the emergence of new pathogenic clones during the two outbreaks of cholera. Data of the study on molecular characteristics of indigenous V. cholerae clinical isolates have public-health implications, not only for epidemic tracing of existing strains but also for the recognition of strains with new genotypes that may emerge in the future.

Involvement of the lymphocytic muscarinic acetylcholine receptor in methylmercury-induced c-Fos expression and apoptosis in human leukemic T cells.

Methylmercury (MeHg) is an environmental toxicant that is known to induce lymphocyte apoptosis; however, little is known about the molecular mechanism involved. Data showed that MOLT-3 cells were more sensitive to MeHg-induced cytotoxic effects than Jurkat clone E6-1 cells, suggesting that the lymphocytic muscarinic cholinergic system may be involved since the expressions of five subtypes (M1-M5) of muscarinic acetylcholine receptor (mAChR) in MOLT-3 cells are higher than in Jurkat cells. The role of mAChR-linked pathways in MeHg-induced apoptosis in human leukemic T cells was examined in this study. Treatment of the MOLT-3 cells with 1 microM MeHg produced induction of c-Fos expression, apoptotic cell death, and downregulation of mAChR. MeHg-induced c-Fos expression was significantly reduced by pretreatment with atropine (a nonselective mAChR antagonist), or 4-DAMP (a selective M1/M3 mAChR antagonist), whereas pirenzipine (a selective M1 mAChR antagonist) or himbazine (a selective M2/M4 mAChR antagonist) did not reduce this induction, suggesting that MeHg-induced c-Fos expression through the activation of the mAChR, at least M3 subtype, is involved. Pretreatment with 4-DAMP or SB 203580 (a specific p38 inhibitor) resulted in decreases in the level of phosphorylated p38, c-Fos expression, and apoptotic cell death induced by MeHg. Taken together, these data suggest that the mAChR-p38-dependent pathway participates in the increase of c-Fos expression, which is involved in MeHg-induced lymphocyte apoptosis. In addition, a noncytotoxic concentration of MeHg (0.1 microM) inhibited PHA/PMA-stimulated interleukin (IL)-2 production, and this inhibition was reversed by pretreatment with atropine or 4-DAMP. Overall, this study provides initial evidence that MeHg may alter the immune system by targeting the lymphocytic mAChR.

In silico prediction and immunological validation of common HLA-DRB1-restricted T cell epitopes of Candida albicans secretory aspartyl proteinase 2.

ABSTRACT Sap2 is the most abundant virulence factor expressed during Candida infection, and the principal protein known to induce antibody response during Candida infection in humans. Its role in T-cell activation however, has not yet been determined. Sequence analysis revealed that Sap2 contains two variable regions: Var1 and Var2. Computational predictions by the Hotspot Hunter program identified that Var1 contains three candidate T-cell epitopes, whereas Var2 contains four. Thirty-nine overlapping peptides of Sap2 were then synthesized, and tested for their ability to induce proliferation of PBMC from 12 donors. Peptides P11, P17 and P31 exhibited significantly higher proliferative indices when compared with those of other peptides or controls. P17 and P31 are located in the areas of prediction, while P11 is not. There were other peptides outside the prediction areas that could stimulate PBMC proliferation at low levels. Nevertheless, the proliferative noise caused by such peptides was ruled out by IL-2 ELISpot analysis. Only P17 and P31 were shown to induce clonal proliferation of IFN-gamma producing lymphocytes, suggesting that these two peptides contain T cell epitopes. P11, which stimulated IL-2 producing clones, contains a known B-cell epitope. Interestingly, P17 and P31 elicited both Th1 and Th2 cell responses with significant numbers of IL-13 secreting clones in response to stimulation. Taken together, the computer-based T cell epitope prediction method could identify the immunogenic T cell epitopes of C. albicans Sap2 that promiscuously bind to the HLA-DRB1 supertype.

Promiscuous T cell epitope prediction of Candida albicans secretory aspartyl protienase family of proteins.

Candida albicans is one of the most important opportunistic dimorphic fungi responsible for hospital acquired fungal infection in humans. Candida infection rarely occurs in healthy individuals but it is frequently associated with patients who suffer from acquired immunodeficiency syndromes. To date, there is no effective vaccine against this fungal infection. Herein we demonstrated the use of immunomics to characterize promiscuous T cell epitope of C. albicans virulence factors by utilizing CandiVF, a C. albicans database previously constructed to be equipped with protein sequence analysis tool, three dimensional structure visualization software, sequence variable analysis program and Hotspot Hunter epitope prediction tool. Secretory aspartyl proteinase (Sap) family was chosen as a model to validate the Hotspot Hunter prediction. Analysis of Saps1-10 protein entries from CandiVF database revealed that a consensus T cell epitope was located at the C-terminal region of Saps1-10. The result of the in silico prediction was subsequently validated by conventional immunological methods. By using overlapping peptides span the predicted consensus T cell epitopes of Saps1-10 as stimulators, it was demonstrated that peptides S6 and S7 could stimulate PBMC proliferation in 9 of 12 blood donors. Interestingly, S2, the predicted T cell epitope of Sap2, was able to induce proliferation of all donors' PBMC. ELISpot assay for the detection of gamma-interferon producing clones confirmed that the peptide S2 actually stimulated T cell proliferation. The results suggest that S2 might be a potential candidate for vaccine development against C. albicans infection or to be utilized as an adjuvant to stimulate the pre-existing CD4+ T cell in other vaccine development.

Lipopolysaccharide heterogeneity among Burkholderia pseudomallei from different geographic and clinical origins.

Heterogeneous patterns were obtained for lipopolysaccharide (LPS) from 1,327 Burkholderia pseudomallei isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, silver staining, and immunoblot analysis. Two LPS serotypes (A and B) possessing different ladder profiles and a rough LPS without ladder appearances were identified. All three LPS types were antigenically distinct by immunoblotting. The predominant type A (97%) produced the lowest amount of biofilm. The two less common types (smooth type B and rough type) were found more in clinical than environmental isolates and more in Australian isolates than Thai isolates. These isolates were more often associated with relapse than with primary infection.

Chondroitin sulfate B and heparin mediate adhesion of Penicillium marneffei conidia to host extracellular matrices.

Penicilliosis is a disseminated infection in immunocompromised individuals caused by the dimorphic fungus, Penicillium marneffei. Very little is known about its route of infection, however, it is thought that initial infection occurs through inhalation of conidia. We investigated the role played by various extracellular matrix glycosaminoglycans (GAGs) in the initial adherence of P. marneffei conidia using a direct adhesion assay. GAGs were further used to block the binding of fungal spores to human lung epithelial cells and highly sulfated GAGs were tested for their inhibitory effects owing to their degree of sulfation. Our results demonstrated high levels of conidial adhesion to chondroitin sulfate B, heparin and highly sulfated chitosan (CP-3). No direct adherence was observed to immobilized chondroitin sulfate (CS) A, CSC, CSD and hyaluronic acid, as well as chitosans with low sulfate content. The results suggested that P. marneffei conidia bind to iduronic acid (IdoA) of the polysaccharide chains. Involvement of negatively charged sulfate groups in adhesion was also indicated. Furthermore, significant inhibition of conidial adherence to A549 cells was observed in the presence of CSB, heparan sulfate (HS), heparin and CP-3. It was further demonstrated that GAGs can affect the adhesion of conidia to fibronectin and laminin, glycoproteins that have previously been implicated as adhesive receptors for fungal conidia. CSB and HS could partially inhibit the adhesion of fungal conidia to laminin and fibronectin implying that conidia can weakly interact with the IdoA GAG-binding domain(s) of these molecules. The data indicated that, in addition to fibronectin and laminin, IdoA-containing GAGs may play an important role in fungal adherence to the surface of human lung epithelium.

Conditional lethal disruption of TATA-binding protein gene in Penicillium marneffei.

Problems can arise in studying the regulation of transcription in fungi if gene disruption is employed to evaluate the role of essential transcription factors. Herein, we have developed a method to characterize the essential genes of Penicillium marneffei. This has been used to examine the significance of P. marneffei TATA-binding protein (TBP) in growth and development. Strains in which the expression of TbpA could be regulated were constructed by placing tbpA under the control of the xylP promoter. The construct was introduced into P. marneffei and the resulting strains were used to produce P. marneffei tbpA deletion strains. Phenotypic examination of growth of the tbpA overexpressing strains revealed that high levels of TbpA expression inhibit fungal growth at conidial germination in both filamentous and yeast forms. Under repressing conditions, the tbpA deletion strains failed to grow at 25 degrees C whilst showing reduced growth at 37 degrees C. The results suggested that TbpA is essential for P. marneffei filamentous growth, but plays a less significant role in growth and development during the yeast phase.

Immunohistological characterization of macrophage migration inhibitory factor expression in Plasmodium falciparum-infected placentas.

Previously, we have shown that macrophage migration inhibitory factor (MIF) was highly elevated in the placental intervillous blood (IVB) of Plasmodium falciparum-infected women. Here, we compared the expression of MIF in placental tissues obtained from P. falciparum-infected and -uninfected women. Immunoperoxidase staining showed a consistent pattern of MIF expression in syncytiotrophoblasts, extravillous trophoblasts, IVB mononuclear cells, and amniotic epithelial cells, irrespective of their malaria infection status. Cytotrophoblast, villous stroma, and Hofbauer cells showed focal staining. Only amniotic epithelial and IVB mononuclear cells from P. falciparum-infected placentas exhibited significantly higher level of MIF expression than uninfected placentas. Stimulation of syncytilized human trophoblast BeWo cells with P. falciparum-infected erythrocytes that were selected to bind these cells resulted in significant increases in MIF secretion, whereas control erythrocytes, lipopolysaccharides, and synthetic beta-hematin had minimal effect. These findings suggest that placental malaria modulates MIF expression in different placental compartments.