Structure-based molecular characterization of a putative aspartic proteinase from Bacteroides fragilis.

Bacteroides fragilis resides in mammals and human intestines and secrete series of proteins and molecules outside that cause various diseases such as colon cancer and chronic colitis in the host. B. fragilis has been shown to produce numerous proteins to the infected cell surface which are involved in host colonization, microbial interactions, and pathogenicity. Among secreted proteins, a B. fragilis toxin (BFT) is a metalloprotease and disintegrates the epithelial cell layer and causes colon cancers. Except the BFT, information of secreted proteases from B. fragilis is limited and no structure is available. Aspartic proteinase cleaves a peptide bond using two aspartate residues in a catalytic site in acidic conditions, pH ranges from 3 to 6. Aspartic proteinase have been characterized mostly from eukaryotes and retroviruses but rare from bacteria including B. fragilis. A putative aspartic proteinase is identified from the B. fragilis genome and prepared recombinantly as a Bacteroides aspartic proteinase (BAPtase). The crystal structure of BAPtase was determined at 2.6 Å. Structure-based comparative and endopeptidase analyses demonstrated that BAPtase presents a two-domain structure and is a functional aspartic proteinase in unusually weak basic pHs, which would propose to be a critical in bacterial pathogenesis and in host immunity. Our observations on the distinct structural and catalytic properties of BAPtase would benefit the future development of B. fragilis-specific drugs or preventatives.

Expression of Caspases in the Pig Endometrium Throughout the Estrous Cycle and at the Maternal-Conceptus Interface During Pregnancy and Regulation by Steroid Hormones and Cytokines.

Caspases, a family of cysteine protease enzymes, are a critical component of apoptotic cell death, but they are also involved in cellular differentiation. The expression of caspases during apoptotic processes in reproductive tissues has been shown in some species; however, the expression and regulation of caspases in the endometrium and placental tissues of pigs has not been fully understood. Therefore, we determined the expression of caspases CASP3, CASP6, CASP7, CASP8, CASP9, and CASP10 in the endometrium throughout the estrous cycle and pregnancy. During the estrous cycle, the expression of all caspases and during pregnancy, the expression of CASP3, CASP6, and CASP7 in the endometrium changed in a stage-specific manner. Conceptus and chorioallantoic tissues also expressed caspases during pregnancy. CASP3, cleaved-CASP3, and CASP7 proteins were localized to endometrial cells, with increased levels in luminal and glandular epithelial cells during early pregnancy, whereas apoptotic cells in the endometrium were limited to some scattered stromal cells with increased numbers on Day 15 of pregnancy. In endometrial explant cultures, the expression of some caspases was affected by steroid hormones (estradiol-17ß and/or progesterone), and the cytokines interleukin-1ß and interferon-γ induced the expression of CASP3 and CASP7, respectively. These results indicate that caspases are dynamically expressed in the endometrium throughout the estrous cycle and at the maternal-conceptus interface during pregnancy in response to steroid hormones and conceptus signals. Thus, caspase action could be important in regulating endometrial and placental function and epithelial cell function during the implantation period in pigs.

Seroprevalence of neutralizing antibodies against human adenovirus type 55 in the South Korean military, 2018-2019.

We conducted a seroprevalence study of a large ongoing outbreak of human adenovirus type 55 (HAdV-55) among the military in South Korea. Serum samples were collected between 2018 and 2019 from military-exposed (military group) and non-exposed (non-military group) populations. The plaque reduction neutralization test (PRNT) was used to assess neutralization activity against HAdV-55. A total of 100 sera was collected from the non-military group, of which 18.8% showed HAdV-55 neutralizing antibody activity. Ninety-six sera were tested from the military group, which had significantly higher prevalence of neutralizing antibodies (56.0%, P <0.001). A significantly higher proportion of the military group had PRNT titers ≥1:1,000 than the non-military group (85.7% vs. 50.0%, P = 0.004). Among the military group, 48.9% of active-duty soldiers had PRNT titers ≥1:5,000, while none of the discharged civilians did (P = 0.007). In conclusion, Koreans were exposed to HAdV-55 in their communities, but the exposure risk was higher among people in military service.

Transcriptomic analysis of interferon-γ-regulated genes in endometrial explants and their possible role in regulating maternal endometrial immunity during the implantation period in pigs, a true epitheliochorial placentation species.

The implantation process requires precisely controlled interactions between the maternal uterine endometrium and the implanting conceptus. Conceptus-derived secretions affect endometrial cells to facilitate the adhesion and attachment of trophoblasts, and endometrial secretions support the growth and development of the conceptus. In pigs, the conceptus secretes a large amount of type II interferon, interferon-γ (IFNG), during the implantation period. However, the role of IFNG in the implantation process has not been fully understood in pigs. Thus, to determine the role of IFNG in the endometrium during early pregnancy in pigs, we treated endometrial explant tissues with increasing doses of IFNG and analyzed the transcriptome regulated by IFNG using an RNA-sequencing analysis. Data analyses identified 276 differentially regulated genes, their Gene Ontology terms, and 94 signature genes in a Gene Set Enrichment Analysis. Furthermore, we analyzed the expression of IFNG-regulated genes, including CIITA, KYNU, IDO1, WARS, and MHC class II molecules, in the endometrium throughout pregnancy and found that levels of those genes in the endometrium were highest on Day 15 of pregnancy, corresponding to the time of peak IFNG secretion by porcine conceptuses. In addition, immunohistochemical analyses revealed that CIITA, KYNU, and IDO proteins were expressed in a cell type- and pregnancy status-specific manner in the endometrium. These results show that genes overrepresented in endometrial tissues in response to IFNG were mainly related to immune responses, suggesting that conceptus-derived IFNG could play critical roles in regulating the maternal immune response for the establishment of pregnancy in pigs.

Cytokine Production in Cholangiocarcinoma Cells in Response to Clonorchis sinensis Excretory-Secretory Products and Their Putative Protein Components.

Clonorchis sinensis is a carcinogenic human liver fluke that promotes hepatic inflammatory environments via direct contact or through their excretory-secretory products (ESPs), subsequently leading to cholangitis, periductal fibrosis, liver cirrhosis, and even cholangiocarcinoma (CCA). This study was conducted to examine the host inflammatory responses to C. sinensis ESPs and their putative protein components selected from C. sinensis expressed sequenced tag (EST) pool databases, including TGF-ß receptor interacting protein 1(CsTRIP1), legumain (CsLeg), and growth factor binding protein 2 (CsGrb2). Treatment of CCA cells (HuCCT1) with the ESPs or bacterial recombinant C. sinensis proteins differentially promoted the secretion of proinflammatory cytokines (IL-1ß, IL-6, and TNF-α) as well as anti-inflammatory cytokines (IL-10, TGF-ß1, and TGF-ß2) in a time-dependent manner. In particular, recombinant C. sinensis protein treatment resulted in increase (at maximum) of ~7-fold in TGF-ß1, ~30-fold in TGF-ß2, and ~3-fold in TNF-α compared with the increase produced by ESPs, indicating that CsTrip1, CsLeg, and CsGrb2 function as strong inducers for secretion of these cytokines in host cells. These results suggest that C. sinensis ESPs contribute to the immunopathological response in host cells, leading to clonorchiasis-associated hepatobiliary abnormalities of greater severity.

Clonorchis sinensis excretory-secretory products increase malignant characteristics of cholangiocarcinoma cells in three-dimensional co-culture with biliary ductal plates.

Clonorchis sinensis is a carcinogenic human liver fluke, prolonged infection which provokes chronic inflammation, epithelial hyperplasia, periductal fibrosis, and even cholangiocarcinoma (CCA). These effects are driven by direct physical damage caused by the worms, as well as chemical irritation from their excretory-secretory products (ESPs) in the bile duct and surrounding liver tissues. We investigated the C. sinensis ESP-mediated malignant features of CCA cells (HuCCT1) in a three-dimensional microfluidic culture model that mimics an in vitro tumor microenvironment. This system consisted of a type I collagen extracellular matrix, applied ESPs, GFP-labeled HuCCT1 cells and quiescent biliary ductal plates formed by normal cholangiocytes (H69 cells). HuCCT1 cells were attracted by a gradient of ESPs in a concentration-dependent manner and migrated in the direction of the ESPs. Meanwhile, single cell invasion by HuCCT1 cells increased independently of the direction of the ESP gradient. ESP treatment resulted in elevated secretion of interleukin-6 (IL-6) and transforming growth factor-beta1 (TGF-ß1) by H69 cells and a cadherin switch (decrease in E-cadherin/increase in N-cadherin expression) in HuCCT1 cells, indicating an increase in epithelial-mesenchymal transition-like changes by HuCCT1 cells. Our findings suggest that C. sinensis ESPs promote the progression of CCA in a tumor microenvironment via the interaction between normal cholangiocytes and CCA cells. These observations broaden our understanding of the progression of CCA caused by liver fluke infection and suggest a new approach for the development of chemotherapeutic for this infectious cancer.

Investigation of bovine tuberculosis outbreaks by using a trace-back system and molecular typing in Korean Hanwoo beef cattle.

Bovine tuberculosis is a chronic contagious disease responsible for major agricultural economic losses. Abattoir monitoring and trace-back systems are an appropriate method to control bovine tuberculosis, particularly in beef cattle. In the present study, a trace-back system was applied to bovine tuberculosis cases in Korean native Hanwoo beef cattle. Bovine tuberculosis was detected in three index beef cattle during abattoir monitoring in Jeonbuk Province, Korea, and the original herds were traced back from each index cow. All cattle in each original herd were subjected to tuberculin skin test. The positive rates in the tuberculin skin test were 64.6% (62 of 96), 4.8% (2 of 42), and 8.1% (3 of 37) at farms A, B, and C, respectively. On post-mortem examination of 56 tuberculin-positive cattle, 62% had granulomatous lesions, and Mycobacterium bovis was cultured from 40 (71.4%) of the cattle. Molecular typing by spoligotyping and the mycobacterial interspersed repetitive unit-variable-number tandem repeat assay revealed the genotype of the M. bovis strains from the index cattle were same as the M. bovis genotype in each original herd. The results suggest that tracing back from index cattle to the original herd is an effective method to control bovine tuberculosis in beef cattle.

Discovery of Parasite Eggs in Archeological Residence during the 15th Century in Seoul, Korea.

During civil engineering construction near Sejong-ro, Jongro-ku, Seoul, cultural sites were found that are thought to have been built in the 15th century. This area was home to many different people as well as the leaders of the Yi dynasty. To gain further insight into the life styles of the inhabitants of the old capital, soil samples were collected from various areas such as toilets, water foundations, and drainage ways. Parasite eggs were examined by microscopy after 5 g soil samples were rehydrated in 0.5% trisodium phosphate solution. A total of 662 parasite eggs from 7 species were found. Species with the highest number of eggs found were Ascaris lumbricoides (n=483), followed by Trichuris trichiura (138), Trichuris vulpis (21), Fasciola hepatica (8), Clonorchis sinensis (6), Paragonimus westermani (4), and Metagonimus yokogawai (2). These findings indirectly indicate the food habits of the people in Yi dynasty.

Mycobacterium bovis infection in a wild sow (Sus scrofa): first case in Korea.

Mycobacterium (M.) bovis causes tuberculosis and has a broad host range, including humans, livestock, and wild animals. M. bovis infection of wild boar has been reported in several European countries. We report here the first case of M. bovis infection in a domesticated wild sow in Korea. Granulomatous and necrotizing lesions with small numbers of acid-fast bacilli were observed in nodules of the lung of wild sow. Furthermore, the M. bovis isolate from the wild sow had spoligotype SB0140 and a novel MIRU-VNTR allelic profile, which is not found in cattle and deer in Korea.

Comparison of the performance of the NucliSENS EasyQ HPV E6/E7 mRNA assay and HPV DNA chip for testing squamous cell lesions of the uterine cervix.

This study aims to evaluate the clinical performance of the NucliSENS EasyQ assay and compare it with HPV DNA genotyping for the detection of high-grade squamous intraepithelial lesions (HSIL) and cancer in a Korean population. In 188 total thin prep samples, the remaining fluid after cytology slide preparation was tested with Goodgene HPV DNA chips and the NucliSENS EasyQ HPV E6/E7 messenger RNA (mRNA) assay. The sensitivity and specificity of each test were calculated with HSIL and squamous cell carcinoma (SCC) as the disease endpoint. Out of the 188 samples, 139 (74%) were positive for DNA of 14 HPV types, while 57 (30%) cases were positive for E6/E7 mRNA. The DNA test was positive in cytology cases of SCC, HSIL, and atypical squamous cell. The mRNA test yielded results of 75%, 74%, 60%, 56%, and 29% positivity in abnormal cytology cases of SCC, HSIL, atypical squamous cells - cannot exclude HSIL, atypical squamous cells of undetermined significance, and low-grade squamous intraepithelial lesion, respectively. In normal cytology cases, the positivity rates were 9% and 53% for the mRNA and DNA tests, respectively. For detection of HSIL and SCC, the sensitivity of the mRNA test was 74.36% and that of the DNA test was 100%, while the specificities of the tests were 85% and 40.83%, respectively. These findings suggest that the HPV E6/E7 mRNA assay can overcome the shortcoming of low specificity of DNA assays for clinical detection of high-grade cervical lesions and malignancies.

Differentiation of antigen-specific T cells with limited functional capacity during Mycobacterium tuberculosis infection.

Despite the generation of Mycobacterium tuberculosis-specific T cell immune responses during the course of infection, only 5 to 10% of exposed individuals develop active disease, while others develop a latent infection. This phenomenon suggests defective M. tuberculosis-specific immunity, which necessitates more careful characterization of M. tuberculosis-specific T cell responses. Here, we longitudinally analyzed the phenotypes and functions of M. tuberculosis-specific T cells. In contrast to the functional exhaustion of T cells observed after chronic infection, M. tuberculosis-specific CD8(+) T cells differentiated into either effector (CD127(lo) CD62L(lo)) or effector memory (CD127(hi) CD62L(lo)) cells, but not central memory cells (CD127(hi) CD62L(hi)), with low programmed death 1 (PD-1) expression, even in the presence of high levels of bacteria. Additionally, M. tuberculosis-specific CD8(+) and CD4(+) T cells produced substantial levels of tumor necrosis factor alpha (TNF-α) and gamma interferon (IFN-γ), but not interleukin 2 (IL-2), upon in vitro restimulation. Among M. tuberculosis-specific CD8(+) T cells, CD127(hi) effector memory cells displayed slower ongoing turnover but greater survival potential. In addition, these cells produced more IFN-γ and TNF-α and displayed lytic activity upon antigen stimulation. However, the effector function of M. tuberculosis-specific CD8(+) CD127(hi) effector memory T cells was inferior to that of canonical CD8(+) CD127(hi) memory T cells generated after acute lymphocytic choriomeningitis virus infection. Collectively, our data demonstrate that M. tuberculosis-specific T cells can differentiate into memory T cells during the course of M. tuberculosis infection independent of the bacterial burden but with limited functionality. These results provide a framework for further understanding the mechanisms of M. tuberculosis infection that can be used to develop more effective vaccines.

REBA HPV-ID® for efficient genotyping of human papillomavirus in clinical samples from Korean patients.

This study was conducted to evaluate the overall performance of a reverse blot hybridization-based assay, REBA HPV-ID® (Molecules and Diagnostics, Wonju, Korea) for genotyping human papillomaviruses (HPV). HPV Genotyping on 356 specimens examined cytologically was performed using the REBA HPV-ID®, and its results were compared with those obtained using the MyHPV DNA Chip® (Mygene, Seoul, Korea), DNA chip-based HPV genotyping assay. The results from this study showed that the positivity rate of the REBA HPV-ID® for abnormal cytological samples was higher (80.9%) than that of the MyHPV DNA chip (69.8%). In addition, the REBA HPV-ID® positivity rate with normal cytological samples was higher (64.4%) than that obtained using DNA chips (34.4%). Subsequently, sequence analysis was performed with specimens that generated conflicting test results. Sequence analysis confirmed that the specimens which were positive by REBA HPV-ID® did indeed contain HPV sequences. The results of this study suggest that the REBA HPV-ID® is a sensitive test for genotyping HPV of clinical specimens.

Neutrophils Promote Mycobacterial Trehalose Dimycolate-Induced Lung Inflammation via the Mincle Pathway.

Trehalose 6,6'-dimycolate (TDM), a cord factor of Mycobacterium tuberculosis (Mtb), is an important regulator of immune responses during Mtb infections. Macrophages recognize TDM through the Mincle receptor and initiate TDM-induced inflammatory responses, leading to lung granuloma formation. Although various immune cells are recruited to lung granulomas, the roles of other immune cells, especially during the initial process of TDM-induced inflammation, are not clear. In this study, Mincle signaling on neutrophils played an important role in TDM-induced lung inflammation by promoting adhesion and innate immune responses. Neutrophils were recruited during the early stage of lung inflammation following TDM-induced granuloma formation. Mincle expression on neutrophils was required for infiltration of TDM-challenged sites in a granuloma model induced by TDM-coated-beads. TDM-induced Mincle signaling on neutrophils increased cell adherence by enhancing F-actin polymerization and CD11b/CD18 surface expression. The TDM-induced effects were dependent on Src, Syk, and MAPK/ERK kinases (MEK). Moreover, coactivation of the Mincle and TLR2 pathways by TDM and Pam3CSK4 treatment synergistically induced CD11b/CD18 surface expression, reactive oxygen species, and TNFα production by neutrophils. These synergistically-enhanced immune responses correlated with the degree of Mincle expression on neutrophil surfaces. The physiological relevance of the Mincle-mediated anti-TDM immune response was confirmed by defective immune responses in Mincle⁻/⁻ mice upon aerosol infections with Mtb. Mincle-mutant mice had higher inflammation levels and mycobacterial loads than WT mice. Neutrophil depletion with anti-Ly6G antibody caused a reduction in IL-6 and monocyte chemotactic protein-1 expression upon TDM treatment, and reduced levels of immune cell recruitment during the initial stage of infection. These findings suggest a new role of Mincle signaling on neutrophils during anti-mycobacterial responses.

Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling.

The "enhanced intracellular survival" (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.

Effect of glasswort (Salicornia herbacea L.) on microbial community variations in the vinegar-making process and vinegar characteristics.

Three types of nuruk were made from rice, wheat, and a rice-glasswort (6:4) mixture. Nuruk, makgeolli, and vinegar were manufactured with rice nuruk (RN), wheat nuruk (WN), and rice-glasswort nuruk (RGN). The saccharifying activity and ethanol productivity of nuruk, polyphenol content in makgeolli, and acetic acid and polyphenol content in the vinegar were increased as the result of the addition of glasswort. The variable region of 18S- or 16S-rDNA amplified with genomic DNA extracted directly from nuruk, makgeolli- and vinegar-making cultures was analyzed via temperature gradient gel electrophoresis (TGGE). The sequence of the 18S-rDNA variable region extracted from the TGGE gel for nuruk was more than 95% homologous with Aspergillus sp and that for the makgeolli-making culture was more than 95% homologous with Saccharomyces sp and Saccharomycodes sp. The sequence of the 16S-rDNA variable region extracted from TGGE gel for the vinegar-making culture was more than 95% homologous, primarily with the Acetobacter sp. The eukaryotic and prokaryotic diversity in the nuruk, makgeolli-making, and vinegar-making cultures was not significantly altered by the addition of glasswort. Prokaryotic diversity was higher than eukaryotic diversity in the nuruk, but eukaryotic diversity was higher than prokaryotic diversity in the makgeolli-making culture, on the basis of the TGGE patterns. No 18S-rDNA was amplified from the DNA extracted from the vinegar-making culture. In conclusion, the glasswort may be not simply an activator for the growth of microorganisms during the fermentation of nuruk, makgeolli, or vinegar, but also a nutritional supplement that improves the quality of vinegar.

Metaplastic ossification in the cartilage of the bronchus of a patient with chronic multi-drug resistant tuberculosis: a case report.

INTRODUCTION: Pulmonary ossification has been rarely observed in pulmonary fibrosis and in some chronic respiratory diseases such as chronic obstructive pulmonary disease. We report here a metaplastic ossification in the bronchial cartilage of a patient with multi-drug resistant tuberculosis. CASE PRESENTATION: We report the case of a 41-year-old Asian man from Korea with chronic multi-drug resistant tuberculosis with a rare focus of bone formation from the cartilage of a bronchus subtending an active cavity. The patient had a large multi-lobed, thick-walled cavitary tuberculosis lesion in his left upper lobe. Severe infiltration of his lymphocytes and epithelioid cells, along with some giant cells and neutrophils, was observed in the patient's bronchial wall. Desquamated bronchial epithelium and acid-fast bacilli were found inside his bronchus. A small focus of bony metaplasia was found in the cartilage of his bronchial wall. Histopathological examination confirmed calcification and showed hematopoietic cells forming in his marrow cavity. CONCLUSIONS: Chronic inflammation in the lungs of our patient, caused by underlying tuberculosis, probably played a role in the development of osseous metaplasia from the associated cartilage of the bronchial wall.

Evaluation of enzyme-linked immunosorbent assay using milk samples as a potential screening test of bovine tuberculosis of dairy cows in Korea.

An enzyme-linked immunosorbent assay (ELISA) using bulk tank milk samples was evaluated as a screening test for bovine tuberculosis (TB), a contagious chronic disease of cattle. An ELISA with MPB70, a major antigen of Mycobacterium bovis was performed using paired sets of milk and sera samples from 33 tuberculin-positive and 43 tuberculin-negative cattle. Anti-MPB70 antibodies were detected in milk samples and there was a significant correlation between seroreactivities of milk and sera samples (R(2)=0.83). Using the tuberculin skin test as the reference test, the sensitivities of ELISA using milk and sera samples were 87.8% and 81.8%, respectively, and the specificities were 97.7% and 100%, respectively. In the screening test using bulk tank milk samples from 931 dairy herds in Whasung, Gyeonggi-do, Korea, the positive rate for anti-MPB70 antibody was 4.5% (42/931) and the tuberculin-positive rate was 2.8% (26/931). Individual milk samples (n=253) were collected from randomly selected 8 problematic and 3 negative herds (positive and negative in the screening test by MPB70 ELISA using bulk tank milk samples, respectively) and tested by MPB70 milk ELISA. In the problematic herds, positive rates were 10.5% (20/190) for anti-MPB70 antibodies in milk ELISA and 2.1% (4/190) in the tuberculin skin test. More than one dairy cows were positive by milk ELISA among the problematic herds, and all tuberculin-positive dairy cows were positive in the milk ELISA. Further, no positive cows were detected in negative herds both by milk ELISA and tuberculin skin test. These results suggest that an ELISA, using bulk tank milk samples, might be a potential efficient screening test for bovine TB of dairy cows.

Expression and evaluation of Chikungunya virus E1 and E2 envelope proteins for serodiagnosis of Chikungunya virus infection.

PURPOSE: Chikungunya virus (CHIKV) causes endemic or epidemic outbreaks of CHIKV fever, which is a mosquitoe- transmitted viral disease in Africa, India, South-East Asia, and recently Southern Europe. Currently, serological diagnostic tests such as hemagglutination inhibition test (HI test), in-house IgM capture enzyme-linked immunosorbent assays (ELISA), and indirect immunofluorescence test were used for diagnosis of chikungunya fever, which are based on whole virus antigens. MATERIALS AND METHODS: CHIKV E1, and E2 envelope proteins for the CHIKV-specific serodiagnostic reagents for chikungunya fever were expressed in baculovirus expression system. The seroreactivity of recombinant CHIKV E1 and E2 envelope proteins were evaluated using sera panels of patients from Laboratoire Marcel Merieux by indirect IgM capture ELISA. RESULTS: The recombinant CHIKV E1 and E2 envelope protein showed sensitivity of 77.5% and 90%, respectively. The specificities of both CHIKV E1 and E2 envelope proteins were 100%. CONCLUSION: The recombinant CHIKV E1 and E2 envelope proteins could be a useful diagnostic reagent for CHIKV infection.

Mutation in the transcriptional regulator PhoP contributes to avirulence of Mycobacterium tuberculosis H37Ra strain.

Attenuated strains of mycobacteria can be exploited to determine genes essential for their pathogenesis and persistence. To this goal, we sequenced the genome of H37Ra, an attenuated variant of Mycobacterium tuberculosis H37Rv strain. Comparison with H37Rv revealed three unique coding region polymorphisms. One polymorphism was located in the DNA-binding domain of the transcriptional regulator PhoP, causing the protein's diminished DNA-binding capacity. Temporal gene expression profiles showed that several genes with reduced expression in H37Ra were also repressed in an H37Rv phoP knockout strain. At later time points, genes of the dormancy regulon, typically expressed in a state of nonreplicating persistence, were upregulated in H37Ra. Complementation of H37Ra with H37Rv phoP partially restored its persistence in a murine macrophage infection model. Our approach demonstrates the feasibility of identifying minute but distinct differences between isogenic strains and illustrates the consequences of single point mutations on the survival stratagem of M. tuberculosis.

Tumor necrosis factor-alpha and interleukin-10 in whole blood is associated with disease progression in pulmonary multidrug-resistant tuberculosis patients.

BACKGROUND: Cytokine production profiles may reflect the clinical pictures of patients with tuberculosis (TB). OBJECTIVE: We examined the relationship between cytokine levels and clinical parameters indicating the state of disease in active pulmonary TB patients. METHODS: We measured interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 levels in whole blood after stimulation with culture filtrate protein of Mycobacterium tuberculosis in 33 multi-drug resistant (MDR)-TB and 51 non-MDR-TB patients. RESULTS: No significant difference was found in IFN-gamma production between non-MDR-TB and MDR-TB patients, but there was a marked reduction in TNF-alpha production in MDR-TB patients accompanied by a moderate increase in IL-10 levels. In contrast, the presence of cavity was associated with a significant increase in IFN-gamma, whereas no difference in TNF-alpha between the cavity and non-cavity group was observed. Those who have TB lesions in the left lung showed lower levels of IFN-gamma and TNF-alpha and higher IL-10 levels than the patients with lesions on the right side. IFN-gamma levels were significantly increased in those with moderate or advanced lesions compared with patients with mild lesions. TNF-alpha and IL-10 levels did not change with disease severity. The number of M. tuberculosis bacilli in sputum was closely associated with TNF-alpha levels. The patient group with high value (+++) of sputum culture acid-fast bacilli produced significantly reduced levels of TNF-alpha compared with medium (++) and low (+) values. CONCLUSION: These findings suggest that IFN-gamma, TNF-alpha or IL-10 production patterns in whole blood are associated with disease progression in active pulmonary TB.