The potential impact of gut microbiota on your health:Current status and future challenges.

Our health and probably also our behaviors and mood depend not only on what we eat or what we do (lifestyle behaviors), but also on what we host. It is well established for decades that all vertebrates including humans are colonized by a wide array of bacteria, fungi, eukaryotic parasites and viruses, and that, at steady state (homeostasis), this community of microbes establishes a friendly mutual relationship with the host. The term microbiota was originally meant to represent an ecological community of commensals and potentially pathogenic microbes that live within our bodies, but it is now used interchangeably with the term microbiome which was initially meant to represent a collective genome of the microbiota. Although the number of microbes that live in or on our body was previously estimated to outnumber that of their hosts by 10 to 1, the latest estimate put the ratio to be closer to 1:1. On the other hand, their collective genomes (microbiome) outnumber those of the host by 100-200 times. It is not surprising therefore that these microbes not only provide the host with a variety of metabolic impact, but can also modulate tissue integrity and immune defense, all of which lead to a healthy ecosystem (symbiosis) that is unfavorable for colonization and invasion of pathogens. Microbiota is well known for its role in development and education of immune system. However, its link with diseases is less known and it is only recently that there is a surge of interest in the potential impact of microbiota on human health and disease. The diversity and composition of microbiota (healthy microbiota profile) are dynamics, depending not only on the host physical status, genotype and immune phenotype, but also on the environmental factors like diet, antibiotic usage and lifestyle behaviors. These environmental factors may adversely alter gut ecosystem (dysbiosis) that is frequently associated with increased susceptibility to infections as well as to non-communicable diseases like obesity, metabolic syndromes (e.g., diabetes and cardiovascular diseases), allergy and other inflammatory diseases. Emerging evidence from more recent studies also demonstrate the existence of a bidirectional communication route linking gut and microbiota with brain, thus suggesting that these microbes may play a role in neurological disorders as well as in host perception, behavior and emotional response. However, whether the observed alteration of the microbiota profile in these diverse conditions is the cause or the consequence of the disease remains to be established. These observations imply that it may be possible to design new strategies for the management of diseases by manipulating gut microbiota. The common practice now available is the use of probiotics to rehabilitate gut ecosystem. The microbiota-based therapeutics like fecal transplantation for the treatment of recurrent antibiotic-resistant Clostridium difficile infection is now under clinical trial and reported to be highly successful. In the next decade, we will probably see even more exciting approaches, for example, using advanced microbiota engineering technologies to create "intelligent" or "smart" bacteria for use in diagnosis, prevention, prediction and treatment of inflammatory diseases and possibly of some gastrointestinal cancers. The microbiota-based therapeutics together with personalized medicine may be the most accurate and optimal strategy for the future treatment of some difficult-to-manage diseases. However, many challenges remain to be solved before the translational potential of this new knowledge can be implemented clinically. In this review, I highlight some important recent developments and advances that contribute to our understanding in the role of microbiota in human health and disease and on how to best manipulate the microbiome to promote greater human health.

The pleiotropic role of vitamin A in regulating mucosal immunity.

The effect of vitamin A on mucosal immunity has never been subjected to extensive studies until recently. We started to work in this area in the early 1970s when we observed that children with protein-calorie malnutrition (PCM) often had defective mucosal immunity, judging from the incidence of respiratory tract infections and diarrhea. We reported that these children had depressed secretory IgA (sIgA) levels in their nasal wash fluids. The IgA level in specimens collected from those superimposed with some degrees of vitamin A deficiency state appeared to be more severely affected. In order to better understand the underlying mechanism associated with this condition, we started to study more detail the deficiency state using experimental vitamin A-deficient rats. From a series of experiments using this animal model, we proposed that vitamin A was needed for transport and/or secretion of sIgA across the mucosa. This conclusion was based on the observation that the secretory component of sIgA synthesized by the epithelial cells of these vitamin A deficient animals was adversely affected as compared to the control animals. From that time onward, much progress has been made by several other groups showing that other mechanisms could also influence the integrity and immune function of the mucosa. For instance, recent studies demonstrated that retinoic acid which is a biologically active form of vitamin A has an essential role in mucosal homeostasis, controlling tolerance and immunity in these non-lymphoid tissues. Such a conclusion was made possible by the availability of sophisticated new molecular biology and genetic engineering techniques together with advances in the field of immunoregulation, e.g., the discovery of dendritic cells (DCs) and T helper cell subsets in 1980s, and the role of Toll-like receptors (TLRs) together with other innate immune regulators in controlling adaptive immune response in the early 1990s. These advances provided considerable new insights into the pleiotropic roles of vitamin A including educating mucosal DCs, differentiation of lymphocyte lineages and imprinting them with mucosal-homing properties as well as in regulating tolerance and immunity. The identification of a novel lymphocyte subpopulation, innate lymphoid cells (ILCs), at the beginning of this century has provided us with an additional insight into a new role of vitamin A in regulating homeostasis at the mucosal surface through influencing ILCs. Another new player that regulates intestinal homeostasis and mucosal immune response is microbiota whose composition is known to vary with vitamin A status. So it appears now that the role of vitamin A on mucosal immunity is far beyond regulating the adaptive Th1-Th2 cell response, but is highly pleiotropic and more complicating, e.g., polarizing the phenotype of mucosal DCs and macrophages, directing gut-homing migration of T and B cells, inducing differentiation of effector T cells and Treg subpopulation, balancing mucosal ILCs subpopulation and influencing the composition of microbiota. In this review, I will attempt to bring together these important advances to provide a comprehensive and contemporary perspective on the role of vitamin A in regulating mucosal immunity.

Evolutionary insights into the origin of innate and adaptive immune systems: different shades of grey.

To struggle for survival, all living organisms, from protists to humans, must defend themselves from attack by predators. From the time when life began around 3,500 million years ago, all living cells have evolved mechanisms and strategies to optimally defend themselves, while the invaders also need to survive by evading these immune defenses. The end results would be healthy co-evolution of both parties. Classically, immune host defense is divided into two main categories, namely, innate and adaptive systems. It is well documented that while vertebrates possess both systems, invertebrates and prokaryotes like bacteria and archaea depend almost exclusively on the innate immune functions. Although the adaptive immune system like antibodies and cellular immunity or their equivalents are believed to have evolved at the time when the vertebrates first appeared about 550 million years ago, more recent information from molecular and genomic studies suggest that different forms of adaptive immune system may also be present in the invertebrates as well. These forms of "adaptive" immune system exhibit, for instance, limited degrees of memory, diversity and similarities of their immune receptors with the immunoglobulin domains of the conventional adaptive immune system of vertebrates. Organized lymphoid tissues have been identified in all vertebrates. Very recent molecular and genetic data further suggest that a special type of adaptive system functioning like RNAi of vertebrates is also present in the very ancient form of life like the bacteria and archaea. In this review, I provide some insights, based on recent information gathering from evolutionary data of innate and adaptive immune receptors of invertebrate and vertebrate animals that should convince the readers that our current view on the innate and adaptive immunity may need to be modified. The distinction between the two systems should not be thought of in terms of a "black and white" phenomenon anymore, as recent molecular and genomic information points to the fact that a line of distinction is not as sharp as it was once thought to be, but it is blurred by different shades of grey.

Differentiation in pyroptosis induction by Burkholderia pseudomallei and Burkholderia thailandensis in primary human monocytes, a possible cause of sepsis in acute melioidosis patients.

Melioidosis caused by Burkholderia pseudomallei is an infectious disease with a high mortality rate. In acute melioidosis, sepsis is a major cause of death among patients. Once the bacterium enters the bloodstream, immune system dysregulation ensues, leading to cytokine storms. In contrast to B. pseudomallei, a closely related but non-virulent strain B. thailandensis has rarely been reported to cause cytokine storms or death in patients. However, the mechanisms in which the virulent B. pseudomallei causes sepsis are not fully elucidated. It is well-documented that monocytes play an essential role in cytokine production in the bloodstream. The present study, therefore, determined whether there is a difference in the innate immune response to B. pseudomallei and B. thailandensis during infection of primary human monocytes and THP-1 monocytic cells by investigating pyroptosis, an inflammatory death pathway known to play a pivotal role in sepsis. Our results showed that although both bacterial species exhibited a similar ability to invade human monocytes, only B. pseudomallei can significantly increase the release of cytosolic enzyme lactate dehydrogenase (LDH) as well as the increases in caspase-1 and gasdermin D activations in both cell types. The results were consistent with the significant increase in IL-1ß and IL-18 production, key cytokines involved in pyroptosis. Interestingly, there was no significant difference in other cytokine secretion, such as IL-1RA, IL-10, IL-12p70, IL-15, IL-8, and IL-23 in cells infected by both bacterial species. Furthermore, we also demonstrated that ROS production played a crucial role in controlling pyroptosis activation during B. pseudomallei infection in primary human monocytes. These findings suggested that pyroptosis induced by B. pseudomallei in the human monocytes may contribute to the pathogenesis of sepsis in acute melioidosis patients.

Insight into the mechanisms regulating immune homeostasis in health and disease.

Innate and adaptive immune systems consist of cells and molecules that work together in concert to fight against microbial infection and maintain homeostasis. Hosts encounter microbes / exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) all the time and they must have proper mechanisms to counteract the danger such that appropriate responses (e.g., degree of inflammation and types of mediators induced) can be mounted in different scenarios. Increasing numbers of endogenous danger signals of host origin are being identified including, for example, uric acid and cholesterol crystals, high mobility group box1 (HMGB1) protein, oxidized LDL, vesicans, heat shock proteins (HSPs) and self DNA. Many of these endogenous ligands have been shown to be associated with inflammation-related diseases like atherosclerosis, gout and type 2 diabetes. Several DAMPs appear to have the ability to interact with more than one receptor. We are now beginning to understand how the immune system can distinguish infection from endogenous ligands elaborated following cellular insults and tissue damage. Appropriate responses to maintain the homeostatic state in health and disease depend largely on the recognition and response to these stimuli by germline encoded pattern-recognition receptors (PRRs) present on both immune and non-immune cells. These receptors are, for example, Toll-like receptors (TLRs), C-type lectin receptors (CLRs) and cytosolic receptors (e.g., RLRs, NLRs and some intracellular DNA sensors). Atypical PRR "danger" receptors, like the receptor for advanced glycation end products (RAGE) and their ligands have been identified. A proper response to maintain homeostasis relies on specific negative regulators and regulatory pathways to dampen its response to tissue injury while maintaining the capacity to eliminate infection and induce proper tissue repair. Moreover, some PRRs (e.g., TLR2,TLR4 and NLRP3) and atypical PRRs can recognize both PAMPs and DAMPs, either as single entities or after forming complexes (e.g., immune complexes, or DNA- HMGB1 and DNA-LL37 complexes), so there must be a mechanism to selectively depress or alleviate the inflammatory response to DAMPs, while leaving that of PAMPs intact. Excessive inflammatory responses can induce considerable tissue damage and can be highly detrimental to the host. For example, CD24 reacting with HMGB1 and HSPs has been implicated to function as negative regulator for RAGE. In this review, I will briefly overview the information on various host and microbial components and bring together the information to synthesize a model to explain how homeostasis can be maintained in states of health and disease. Understanding the molecular mechanisms by which the immune system functions under different scenarios will provide us with ways and means to design appropriate approaches, for example, to prevent or treat autoimmune and inflammatory diseases or the ability to design new drugs or formulate safe chemicals for vaccine adjuvants.

Antiviral immune responses in H5N1-infected human lung tissue and possible mechanisms underlying the hyperproduction of interferon-inducible protein IP-10.

Information on the immune response against H5N1 within the lung is lacking. Here we describe the sustained antiviral immune responses, as indicated by the expression of MxA protein and IFN-alpha mRNA, in autopsy lung tissue from an H5N1-infected patient. H5N1 infection of primary bronchial/tracheal epithelial cells and lung microvascular endothelial cells induced IP-10, and also up-regulated the retinoic acid-inducible gene-I (RIG-I). Down-regulation of RIG-I gene expression decreased IP-10 response. Co-culturing of H5N1-infected pulmonary cells with TNF-alpha led to synergistically enhanced production of IP-10. In the absence of viral infection, TNF-alpha and IFN-alpha also synergistically enhanced IP-10 response. Methylprednisolone showed only a partial inhibitory effect on this chemokine response. Our findings strongly suggest that both the H5N1 virus and the locally produced antiviral cytokines; IFN-alpha and TNF-alpha may have an important role in inducing IP-10 hyperresponse, leading to inflammatory damage in infected lung.

Differential intracellular fate of Burkholderia pseudomallei 844 and Burkholderia thailandensis UE5 in human monocyte-derived dendritic cells and macrophages.

BACKGROUND: Burkholderia pseudomallei (Bp) is a category B biothreat organism that causes a potentially fatal disease in humans and animals, namely melioidosis. Burkholderia thailandensis (Bt) is another naturally occurring species that is very closely related to Bp. However, despite this closely related genotype, Bt is considered avirulent as it does not cause the disease. In the present study, we compared the growth kinetics of B. pseudomallei strain 844 (Bp-844) in human monocyte-derived dendritic cells (MoDCs) and macrophages (Mphis), as well as its ability to stimulate host cell responses with those of B. thailandensis strain UE5 (Bt-UE5). RESULTS: Primary human MoDCs and Mphis were infected with Bp-844 and its intracellular growth kinetics and ability to induce host cell responses were evaluated. The results were compared with those obtained using the Bt-UE5. In human MoDCs, both bacteria were similar in respect to their ability to survive and replicate intracellularly, induce upregulation of costimulatory molecules and cytokines and bias T helper cell differentiation toward a Th1 phenotype. By contrast, the two bacteria exhibited different growth kinetics in human Mphis, where the intracellular growth of Bt-UE5, but not Bp-844, was significantly suppressed. Moreover, the ability of Mphis to kill Bp-844 was markedly enhanced following stimulation with IFN-gamma. CONCLUSION: The data presented showed that while both strains were similar in their ability to survive and replicate in human MoDCs, only Bp-844 could readily replicate in human Mphis. Both bacteria induced similar host cellular responses, particularly with regard to their ability to bias T cell differentiation toward a Th1 phenotype.

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.

Immune responses of selected phagotopes from monoclonal antibodies of Burkholderia pseudomallei.

Random peptide libraries displayed by bacteriophage T7 and M13 were employed to identify mimotopes from 4 monoclonal antibodies (MAbs) specific to Burkholderia pseudomallei. Insert DNA sequences of bound phages selected from four rounds of panning with each MAb revealed peptide sequences corresponding to B. pseudomallei K96243 hypothetical protein BPSL2046, hypothetical protein BpseP_02000035, B. pseudomallei K96243 hypothetical protein BPSS0784, B. pseudomallei 1710b hypothetical protein BURPS1710b_1104, and B. cenocepacia H12424 TonB-dependent siderophore receptor, all located at the outer membrane. The immune responses from all selected phagotopes were significantly higher than that of lipopolysaccharide. The study demonstrates the feasibility of identifying mimotopes through screening of phage-displayed random peptide libraries with B. pseudomallei MAbs.

Reduced interleukin-17 expression of Burkholderia pseudomallei-infected peripheral blood mononuclear cells of diabetic patients.

Burkholderia pseudomallei is the causative agent of melioidosis. One of the main risk factors for B. pseudomallei infection in endemic areas is diabetes mellitus. The present study investigated IL-17 mRNA and protein expression by peripheral blood mononuclear cells in response to B. pseudomallei infection in 10 diabetic patients in comparison to 10 healthy blood donors. The IL-17 expression in diabetic patients was significantly lower (p < 0.05) than in the controls. However, IL-23 mRNA expression of the 2 groups was comparable. The present findings suggest that melioidosis affects T cell IL-17 production and that patients with diabetes mellitus have a defective IL-17 production in response to this type of infection.

Melioidosis in Thailand: Present and Future.

A recent modelling study estimated that there are 2800 deaths due to melioidosis in Thailand yearly. The Thailand Melioidosis Network (formed in 2012) has been working closely with the Ministry of Public Health (MoPH) to investigate and reduce the burden of this disease. Based on updated data, the incidence of melioidosis is still high in Northeast Thailand. More than 2000 culture-confirmed cases of melioidosis are diagnosed in general hospitals with microbiology laboratories in this region each year. The mortality rate is around 35%. Melioidosis is endemic throughout Thailand, but it is still not uncommon that microbiological facilities misidentify Burkholderia pseudomallei as a contaminant or another organism. Disease awareness is low, and people in rural areas neither wear boots nor boil water before drinking to protect themselves from acquiring B. pseudomallei. Previously, about 10 melioidosis deaths were formally reported to the National Notifiable Disease Surveillance System (Report 506) each year, thus limiting priority setting by the MoPH. In 2015, the formally reported number of melioidosis deaths rose to 112, solely because Sunpasithiprasong Hospital, Ubon Ratchathani province, reported its own data (n = 107). Melioidosis is truly an important cause of death in Thailand, and currently reported cases (Report 506) and cases diagnosed at research centers reflect the tip of the iceberg. Laboratory training and communication between clinicians and laboratory personnel are required to improve diagnosis and treatment of melioidosis countrywide. Implementation of rapid diagnostic tests, such as a lateral flow antigen detection assay, with high accuracy even in melioidosis-endemic countries such as Thailand, is critically needed. Reporting of all culture-confirmed melioidosis cases from every hospital with a microbiology laboratory, together with final outcome data, is mandated under the Communicable Diseases Act B.E.2558. By enforcing this legislation, the MoPH could raise the priority of this disease, and should consider implementing a campaign to raise awareness and melioidosis prevention countrywide.

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.

Triptolide sensitizes resistant cholangiocarcinoma cells to TRAIL-induced apoptosis.

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) promotes apoptosis by binding to transmembrane receptors. It is known to induce apoptosis in a wide variety of cancer cells, but TRAIL-resistant cancers have also been documented. In this study, the relative resistance of human cholangiocarcinoma (CCA) cell lines against TRAIL-induced apoptosis is reported and the possible potential synergistic effect with triptolide, a diterpene triepoxide extracted from the Chinese herb Tripterygium wilfordii, in killing TRAIL-resistant CCA cells is investigated. MATERIALS AND METHODS: Six human CCA cell lines were treated with various concentrations of TRAIL and the resistant cells were identified and subsequently tested for their sensitivity to a combination of TRAIL and triptolide. The susceptibility and resistance of the cells were based on analysis of cytotoxic and apoptotic induction and expression of anti-apoptotic factors (Mcl-1 and cFLIP). RESULTS: The treatment of TRAIL induced a dose-dependent decrease in cell viability in 4 out of the 6 cell lines. A combination of TRAIL and triptolide enhanced cytotoxicity and apoptosis in these 2 resistant cell lines. The combined treatment enhanced activation of caspase-8 and its downstream signaling processes compared with the treatment with either one alone. CONCLUSION: The results presented show that human CCA cells were heterogeneous with respect to susceptibility to TRAIL-induced apoptosis. The combination of TRAIL and triptolide could enhance susceptibility to TRAIL-induced apoptotic killing in these TRAIL-resistant CCA cells, thus offering an alternative approach for the treatment of TRAIL-resistant cholangiocarcinoma.

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the diagnosis of Penicillium marneffei infection.

A monoclonal antibody (MAb)-based sandwich enzyme-linked immunosorbent assay was developed for the detection of Penicillium antigen in clinical specimens from patients with Penicillium marneffei infection. The IgM from clone 8C3, reactive with both yeast and mycelial antigens, was immobilized on a microtiter plate. The antigen in serum or urine was captured and detected with biotinylated polyclonal rabbit anti-P. marneffei antibody. The test was sensitive in detecting soluble yeast exoantigen at a concentration as low as 4.88 ng/mL. The reliability of the assay method was evaluated using sera at a dilution of 1:2 from 18 patients with culture-proven penicilliosis, 23 patients with other fungal and bacterial infections, and 125 healthy volunteers. The method exhibited a sensitivity of 72%, a specificity of 100%, a positive predictive value of 100%, a negative predictive value of 97%, and an accuracy of 97%. However, the sensitivity could be increased to 90% when the specimens were used undiluted. The method was also useful for the detection of antigen secreted in the urine of the patients. The results showed that the newly described assay method can be used in the diagnosis of P. marneffei infection with a high degree of reliability.

Short report: a rapid method for the differentiation of Burkholderia pseudomallei and Burkholderia thailandensis.

A rapid method for the identification and differentiation of Burkholderia pseudomallei and Burkholderia thailandensis colonies is described. It consists of simultaneous use of 2 monoclonal antibody-based latex agglutination test systems. The anti-lipopolysaccharide test reacts with both species, whereas the anti-exopolysaccharide reacts only with B. pseudomallei. Compared with classical biochemical tests, the method is highly reproducible and accurate. It is particularly useful for the identification of the organisms in environmental specimens, which may contain both of these Burkholderia species.

Synergistic cytotoxicity and apoptosis induced in human cholangiocarcinoma cell lines by a combined treatment with tumor necrosis factor-alpha (TNF-alpha) and triptolide.

Cholangiocarcinoma is known to be relatively resistant to chemotherapy. One alternative approach is to use a combination of an immunomodulating agent with an anticancer drug. Here we studied the synergistic actions of TNF-alpha and triptolide (a diterpene epoxide prepared from Tripterygium wilfordii), previously shown to have antitumor activity against hamster cholangiocarcinoma (CCA) cells. Three human CCA cell lines (HuCCA-1, HubCCA-1, KKU-100 cell lines) were subjected to a combined treatment of TNF-alpha (0.1-10 ng/ml) and triptolide (5-50 ng/ml) for 24 hours in microculture plates. The combination of TNF-alpha and triptolide had a significantly increased cytotoxic activity over that of triptolide alone (p < 0.05). Under the same conditions, TNF-alpha by itself was not cytotoxic to these cell lines. Similarly, the combined treatment could also accelerate apoptotic cell death in all three human cholangiocarcinoma cell lines. The combined treatment of TNF-alpha at 10 ng/ml and triptolide at 50 ng/ml for 6-10 hours achieved a percentage of apoptotic cells shown by DAPI staining of 18-65%, compared to only 6-20% apoptotic cells for triptolide alone. Analyzing the possible mechanisms of the combined treatment, we found by Western blot that at 6 hours, there was a poly (ADP-ribose) polymerase (PARP) cleavage which was not detectable by the treatment of either TNF-alpha or triptolide alone. The cleavage of PARP was inhibited when the cells were pretreated with the enzyme inhibitor AC-DEVD-CMK, suggesting that apoptosis induced by the combination of TNF-alpha and triptolide involved activation of caspase 3. These results indicate that apoptosis of human cholangiocarcinoma cell lines as induced by a combination of TNF-alpha and triptolide is mediated through caspase 3 activation.

Involvement of GRIM-19 in apoptosis induced in H5N1 virus-infected human macrophages.

The fatal H5N1 infection has a high mortality rate among infected patients. The pathogenesis of H5N1 viral infection is associated with the ability of the virus to induce apoptotic cell death. However, the molecular mechanism of apoptosis induced by H5N1 remains unclear. In the present study we demonstrate that H5N1 virus is able to up-regulate the expression of gene associated with retinoid and interferon induced mortality-19 (GRIM-19) in human monocyte-derived macrophages (hMDMs). GRIM-19 has been identified as a novel gene with apoptotic effects in virus-infected cells. The percentage of apoptotic cells is significantly decreased in H5N1-infected GRIM-19 depleted hMDMs, which is also associated with a decrease of BH3-interacting domain death agonist cleavage and apoptosis-inducing factor (AIF) release to the cytosol. These results suggested the involvement of GRIM-19 in apoptosis induced by H5N1 virus. Furthermore, neutralizing-IFN-ß Ab is able to suppress GRIM-19 expression in H5N1-infected cells resulting in a decrease in apoptotic cell number, indicating that IFN-ß secreted by H5N1-infected hMDMs regulates GRIM-19 expression leading to apoptosis. Altogether, the results presented here provide additional insight on the regulatory mechanism of H5N1 viral-induced apoptotic cell death in hMDMs.

Apoptosis induced by avian H5N1 virus in human monocyte-derived macrophages involves TRAIL-inducing caspase-10 activation.

Avian influenza virus H5N1 is a potentially fatal disease not only in birds, but also in humans. The virus is able to induce apoptosis in many cell types including macrophages and dendritic cells. In the present study, we demonstrated that TNF-related apoptosis-inducing ligand (TRAIL) is involved in apoptosis-associated mechanisms of apoptosis downstream of the TRAIL receptor in H5N1 virus-infected human monocyte-derived macrophages (MDMs). Activation of caspase-10 was also observed in avian virus H5N1-infected MDMs. In the presence of caspase-10 inhibitor, Z-AEVD-FMK, the activation of Bid and a release of apoptotic-inducing factor (AIF) from mitochondria were markedly reduced, resulting in a significant decrease of apoptotic cells which suggested the involvement of caspase-10 activation in mitochondria leakage. Furthermore, neutralizing Ab against TRAIL significantly reduced caspase-10 activities, which paralleled with a decrease in the number of apoptotic cells. Together, this study demonstrated that apoptosis in avian virus H5N1-infected MDMs was induced by TRAIL-activated caspase-10, resulting in the activation of Bid and the release of AIF from mitochondria.

Cationic liposomes extend the immunostimulatory effect of CpG oligodeoxynucleotide against Burkholderia pseudomallei infection in BALB/c mice.

Melioidosis is a severe disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Previously we showed that pretreatment of mice with CpG oligodeoxynucleotide (CpG ODN) 2 to 10 days prior to B. pseudomallei challenge conferred as high as 90% protection, but this window of protection was rather short. In the present study, we therefore aimed to prolong this protective window and to gain further insight into the mechanisms underlying the protection induced by CpG ODN against B. pseudomallei infection. It was found that the CpG ODN incorporated with cationic liposomes (DOTAP) but not zwitterionic liposomes (DOPC) provided complete protection against bacterial challenge. Although marked elevation of gamma interferon (IFN-γ) was found in the infected animals 2 days postinfection, it was significantly lowered by the DOTAP-plus-CpG ODN pretreatment. When appropriately activated, the phagocytic index and oxidative burst responses of neutrophils appeared not to be elevated. However, macrophages from stimulated mice showed higher levels of nitric oxide production and exhibited higher levels of antimicrobial activities, judging from lower numbers of viable intracellular bacteria. Taken together, our results demonstrate that DOTAP can enhance the protective window period of CpG ODN to at least 30 days and provide 100% protection against B. pseudomallei infection. The protective effect of DOTAP plus CpG ODN could provide an alternative approach to preventing this lethal infection, for which no vaccine is yet available.