In Vitro Roles of Burkholderia Intracellular Motility A (BimA) in Infection of Human Neuroblastoma Cell Line.

The bacterial pathogen Burkholderia pseudomallei causes human melioidosis, which can infect the brain, leading to encephalitis and brain abscesses. Infection of the nervous system is a rare condition but is associated with an increased risk of mortality. Burkholderia intracellular motility A (BimA) was reported to play an important role in the invasion and infection of the central nervous system in a mouse model. Thus, to gain insight of the cellular mechanisms underlying the pathogenesis of neurological melioidosis, we explored the human neuronal proteomics to identify the host factors that are up- and downregulated during Burkholderia infection. When infected the SH-SY5Y cells with B. pseudomallei K96243 wild-type (WT), 194 host proteins showed a fold change of >2 compared with uninfected cells. Moreover, 123 proteins showed a fold change of >2 when infected with a knockout bimA mutant (ΔbimA) mutant compared with WT. The differentially expressed proteins were mainly associated with metabolic pathways and pathways linked to human diseases. Importantly, we observed the downregulation of proteins in the apoptosis and cytotoxicity pathway, and in vitro investigation with the ΔbimA mutant revealed the association of BimA with the induction of these pathways. Additionally, we disclosed that BimA was not required for invasion into the neuron cell line but was necessary for effective intracellular replication and multinucleated giant cell (MNGC) formation. These findings show the extraordinary capacity of B. pseudomallei in subverting and interfering with host cellular systems to establish infection and extend our understanding of B. pseudomallei BimA involvement in the pathogenesis of neurological melioidosis. IMPORTANCE Neurological melioidosis, caused by Burkholderia pseudomallei, can result in severe neurological damage and enhance the mortality rate of melioidosis patients. We investigate the involvement of the virulent factor BimA, which mediates actin-based motility, in the intracellular infection of neuroblastoma SH-SY5Y cells. Using proteomics-based analysis, we provide a list of host factors exploited by B. pseudomallei. The expression level of selected downregulated proteins in neuron cells infected with the ΔbimA mutant was determined by quantitative reverse transcription-PCR and was consistent with our proteomic data. The role of BimA in the apoptosis and cytotoxicity of SH-SY5Y cells infected by B. pseudomallei was uncovered in this study. Additionally, our research demonstrates that BimA is required for successful intracellular survival and cell fusion upon infection of neuron cells. Our findings have significant implications for understanding the pathogenesis of B. pseudomallei infections and developing novel therapeutic strategies to combat this deadly disease.

Early Effects of Rhodomyrtone on Membrane Integrity in Methicillin-Resistant Staphylococcus aureus.

Strong evidence of high potency of rhodomyrtone as a promising antibacterial agent against pathogenic gram-positive bacteria has been clearly demonstrated in our previous work. The aim of this study was to provide insight into early action of rhodomyrtone, an acylphloroglucinol, on membrane damage in multidrug-resistant methicillin-resistant Staphylococcus aureus (MRSA). Early effects of rhodomyrtone on the bacterial membrane integrity were detected in a time-course study. Flow cytometry revealed a reduction in green fluorescent emission and increase in uptake of propidium iodide in rhodomyrtone-treated bacterial cells in a concentration- and time-dependent manner. Disruption of cytoplasmic membrane was further monitored by measuring cellular adenosine triphosphate (ATP) and potassium ion (K+). Leakage of both ATP and K+ and significant decrease in intracellular ATP in MRSA were observed following treatment. Pronounced changes in the bacterial ultrastructure and morphology were confirmed by transmission electron microscopy and scanning electron microscopy. Bacterial cell disruption, holes in cell surface, and bulge formations were noted in rhodomyrtone-treated cells. In this study, we provided relevant data to clarify that rhodomyrtone is a bacterial cell membrane-damaging agent. A possible early effect of this novel compound involves bacterial membrane disruption.

Allergenicity of native and recombinant major allergen groups 1 and 2 of Dermatophagoides mites in mite sensitive Thai patients.

BACKGROUND AND OBJECTIVE: Natural allergenic extracts using for diagnosis and immunotherapy may have batch-to-batch variations and contaminations with unrefined allergens or non-allergenic components. Thus, recombinant allergen is believed to overcome these shortcomings. In this study, native and recombinant allergens of group 1 and 2 of Dermatophagoides mites were produced and their allergenicities were compared. METHODS: Native allergens were prepared by MAb affinity chromatography. All recombinant allergens were produced in E. coli expression system. IgE reactivities of these allergens were determined by IgE-ELISA. RESULTS: The native and recombinant Der p 1, Der p 2, Der f 1, Der f 2 had molecular weights of approximately 25, 15, 25 and 15 kDa, respectively. IgE reactivities of nDer p 1, nDer f 1, rDer p 1 and rDer f 1 were 96.67%, 90%, 43.33% and 46.67%, respectively. Allergenicities of nDer p 2, nDer f 2, rDer p 2 and rDer f 2 were 86.67%, 96.43%, 76.67% and 89.29%, respectively. The findings indicated that recombinant group-1 products were minor allergens which revealed no correlation with their native forms. In contrast, recombinant group-2 allergens were major allergens and showed a significant correlation to their native allergens. CONCLUSION: We successfully produced native and recombinant group-1 and group-2 allergens. According to their allergenicities, recombinant Der p 2 and rDer f 2 have potential to replace native allergen in diagnostic and therapeutic extracts. Moreover, they can employ as a standard reagent to measure the amount of group 2 allergen in the environment by sandwich-ELISA and utilise this as an immunogen for MAb production.

Quantification of Der f 1 in houses of patients allergic to house dust mite, Dermatophagoides farinae, using a locally produced detection reagents.

BACKGROUND: House dust mite (HDM) allergen quantification in house dust samples before and after the allergen elimination is one means of convincing the target population about the health benefits of allergen removal from their environment. OBJECTIVE: To produce local reagents for quantification of Der f 1 (major allergen of Dermatophagoides farinae) in dust samples from houses of HDM allergic Thai patients. METHODS: Recombinant Der f 1 was used for immunization of a BALB/c mouse for hybridoma production. Polyclonal antibody (PAb) to whole body extract of D. farinae was prepared from an immunized rabbit. A sandwich ELISA (MAb-allergen-PAb) was used, in comparison with the commercialized reagents (Indoor Biotechnology, UK), to quantify Der f 1 in dust samples. RESULTS: Two hybridoma clones, Dfl-1 and Dfl-2, were established. Their secreted MAbs (MAbDfl-1 and MAbDfl-2, respectively) bound to the homologous antigen as well as native Der f 1 and a crude extract of D. farinae. Epitopes of MAbDfI-1 and MAbDfl-2 were located at amino acid residues 206NSQHYGISNYCQ217 and 283DYW---NSWD-WGDSG298 of Der f 1. MAbDf-1 had higher affinity to Der f 1 than the MAbDfl-2. A sandwich ELISA (MAbDfl-1-allergen-PAb) and commercialized reagents (MAbl-allergen-MAb2 sandwich ELISA) were used in comparison for quantification of Der f 1 in 42 dust samples collected from bedrooms and living rooms of 21 houses of the HDM allergic patients. All of the 42 dust samples measured by both ELISAs had the Der f 1 levels higher than 2 mg per gram of fine dust which is the HDM allergy sensitizing level. In addition, Der f 1 levels in 41 samples (except 1 sample from a living room) measured by the MAbDfI-1-PAb and MAbl-MAb2 sandwich ELISAs were higher than 10 mg per g of dust which is the morbidity level of HDM allergen. The local sandwich ELISA showed a high coefficient correlation (r = 0.91) in measuring known amounts of recombinant and native Der f 1. The results indicate that the reagents produced in the present study can be used for measuring the environmental levels of HDM Der f 1. The assay can also be used for standardization of the HDM extract for monitoring patient's allergenic status or for immunotherapeutic purpose.

Human monoclonal ScFv that inhibits cellular entry and metalloprotease activity of tetanus neurotoxin.

Tetanus is a deadly disease of warm blooded animals and humans caused by an exotoxin called tetanospasmin or tetanus neurotoxin (TeNT) produced by anaerobic bacterium named Clostridium tetani TeNT is an A-B toxin; each molecule consists of a heavy chain (HC) containing cellular receptor binding domain and a light chain (LC) with zinc metalloprotease activity. TeNT produced in the infected tissue by the bacteria grown under anaerobic condition binds to ganglioside receptors of peripheral nerve, and endocytosed. The A subunit exits from the endosome and undergoes a retrograde transport via the nerve axon to the spinal cord. This highly toxic enzyme specifically cleaves one of the nerve cell SNARE proteins, i.e., synaptobrevin, resulting in inhibition of the release of neurotransmitters (glycine and GABA) from inhibitory interneuron causing spastic paralysis, the characteristic of tetanus. Current treatment mainstay of human tetanus is by passively administering anti-tetanus toxin produced from animals immunized with adjuvanted tetanus toxoid (TT). There are several obstacles in production and use of the animal derived therapeutic antibody especially the allergic reaction and serum sickness induced by the host immune response to the foreign protein. The animal antibody, mainly IgG, blocks nerve cell entry of the TeNT but does not neutralize the TeNT protease activity per se and cannot reverse the tetanus symptoms. In this study, fully human single chain antibody fragments (HuScFv) were produced from a human antibody phage display library. TT was used as antigen in a single round phage bio-panning to select phage clones that display TT bound-HuScFv from the library. HuScFv from 4 selected huscfv-phagemid transformed E. coli clones inhibited binding of the native TeNT to retinoic acid pulsed human neuroblastoma cells when used at the molecular TeNT:HuScFv ratio of 1:100. HuScFv from one of the 4 clones also inhibited the TeNT mediated cleavage of recombinant synaptobrevin. Further investigation is needed for identification of epitope specificity of these HuScFv and HuScFv effector mechanisms towards the TeNT. Cell penetrating version of the HuScFv that inhibited the TeNT zinc metalloprotease activity should be made. The HuScFv produced in this study either singly or in their suitable combination warrant developing further to a real use in humans as a surrogate of the animal antibody for treatment of tetanus.

Giardia intestinalis in Thailand: identification of genotypes.

This study was undertaken to determine the genetic diversities of Giardia intestinalis isolated in Thailand. G. intestinalis cysts were collected from stool samples of 61 subjects residing in Bangkok or in rural communities of Thailand with and without gastrointestinal symptoms. All the cyst samples gave positive tpi amplicons (100% sensitivity), either of the 148- or the 81-bp tpi segments. Cyst assemblage identification of the 148- and 81-bp tpi gene segments by polymerase chain reaction showed that 8% of the cysts were assemblage A, 41% assemblage A and B combined, and 51% assemblage B. The prevalence of assemblage A was significantly lower than that of assemblage B and the mixed types. Restriction fragment length polymorphism (RFLP) of the 384-bp beta-giardin gene segment revealed that 12% and 88% of the assemblage A cysts were AI and AII respectively. RFLP, based on the 432-bp gdh gene segment, showed 45.5% of the assemblage B cysts to be BIII and 54.5% to be BIV. The AI sub-assemblage was less prevalent than the others. All subjects with AI and 50% of the subjects with BIII sub-assemblage cysts were symptomatic; 80% of symptomatic Bangkok residents were adults/elderly while 85% of the rural cases were children.

Growth factor combination for chondrogenic induction from human mesenchymal stem cell.

During the last decade, many strategies for cartilage engineering have been emerging. Stem cell induction is one of the possible approaches for cartilage engineering. The mesenchymal stem cells (MSCs) with their pluripotency and availability have been demonstrated to be an attractive cell source. It needs the stimulation with cell growth factors to make the multipluripotent MSCs differentiate into chondrogenic lineage. We have shown particular patterns of in vitro chondrogenesis induction on human bone marrow MSCs (hBMSCs) by cycling the growth factors. The pellet cultures of hBMSCs were prepared for chondrogenic induction. Growth factors: TGF-beta3, BMP-6, and IGF-1 were used in combination for cell induction. Gene expression, histology, immunohistology, and real-time PCR methods were measured on days 21 after cell induction. As shown by histology and immunohistology, the induced cells have shown the feature of chondrocytes in their morphology and extracellular matrix in both inducing patterns of combination and cycling induction. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, collagen type II and aggrecan. This study has demonstrated that cartilage tissue can be created from bone marrow mesenchymal stem cells. Interestingly, the combined growth factors TGF-beta3 and BMP-6 or TGF-beta3 and IGF-1 were more effective for chondrogenesis induction as shown by the real-time PCR assay. The combination of these growth factors may be the important key for in vitro chondrogenesis induction.