In-vitro antiviral action of Eupatorium perfoliatum against dengue virus infection: Modulation of mTOR signaling and autophagy.

ETHNOPHARMACOLOGICAL RELEVANCE: Dengue virus (DENV) is a re-emerging mosquito-borne flavivirus that has recently engendered large epidemics around the world. Consequently antivirals with effective anti-DENV therapeutic activity are urgently required. In the 18th century, Europeans, as well as native inhabitants of North America, were known to adapt the medicinal property of the common perennial plant Eupatorium perfoliatum L. to treat fever and infections. Previous studies have shown that Eupatorium perfoliatum L. possesses anti-inflammatory, anti-oxidative, anti-plasmodial, anti-bacterial and antiviral activities. However, to the best of our knowledge, no anti-DENV activity of E. perfoliatum L. has been investigated at the molecular level so far. AIM OF STUDY: Here, for the first time we have attempted to study the action of E. perfoliatum extract and its few bioactive components i.e., quercetin, caffeic acid and eupafolin against wild primary clinical isolate of DENV-2 infection in an in vitro model. MATERIALS AND METHODS: The presence of the bioactive components in the E. perfoliatum extract, were analyzed by HPLC- DAD. Then, CC50 as well as IC50 values of the extract and its bioactive components were measured against DENV in HepG2 cell line. After that, the antiviral activity was studied by Time of addition assay using qRT-PCR. Further, the downstream signalling action of E. perfoliatum extract, was studied by Human phosphorylation MAPK antibody array, followed by immunofluorescence microscopy. Moreover, a molecular docking analysis was done to study the binding affinity of bioactive components of E. perfoliatum extract with TIM-1 transmembrane receptor protein, which is known for viral internalization. RESULT: We found that E. perfoliatum extract has marked antiviral activity during pre-treatment against DENV infection in HepG2 cell line. The extract also significantly reduced the DENV induced autophagy in HepG2 cell line as detected by LC3 II localization. The presence of different bioactive compounds in E. perfoliatum extract were confirmed by HPLC-DAD. In the bioactive components, in parallel to earlier studies, quercetin showed the most significant preventive action against DENV infection. Further, in molecular docking analysis also, quercetin showed the strongest binding affinity towards DENV membrane receptor TIM-1 protein. CONCLUSION: Our findings suggests that E. perfoliatum extract has significant potential to be an anti-DENV therapeutic agent. Moreover, among the bioactive components, quercetin may have a prophylaxis role in executing the antiviral activity of E. perfoliatum extract against DENV infection.

Correlates of treatment outcomes and drug resistance among pulmonary tuberculosis patients attending tertiary care hospitals of Kolkata, India.

BACKGROUND: Worldwide highest number of new pulmonary tuberculosis (PTB) cases, was reported from India in 2012. Adverse treatment outcomes and emergence of drug resistance further complicated the prevailing scenario owing to increased duration, cost and toxicity associated with the treatment of drug-resistant cases. Hence to reinforce India's fight against TB, identification of the correlates of adverse treatment outcomes and drug resistance, seemed critical. METHODS: To estimate the associations between diagnostic findings, patient types (based on treatment outcomes), drug resistance and socio-demographic characteristics of PTB patients, a cross-sectional study was conducted in two tertiary-care hospitals in Kolkata between April 2010 and March 2013. Altogether, 350 consenting Mycobacterium tuberculosis sputum-culture positive PTB patients were interviewed about their socio-demographic background, evaluated regarding their X-ray findings (minimal/moderately advanced/far advanced/cavities), sputum-smear positivity, and treatment history/outcomes (new/defaulter/relapse/treatment-failure cases). Multiple-allele-specific polymerase chain reaction (MAS-PCR) was conducted to diagnose drug resistance. RESULTS: Among all participants, 31.43% were newly diagnosed, while 44%, 15.43% and 9.14% patients fell into the categories of relapsed, defaulters and treatment-failures, respectively. 12.29% were multi-drug-resistant (MDR: resistant to at least isoniazid and rifampicin), 57.71% had non-MDR two-drug resistance and 12% had single-drug resistance. Subjects with higher BMI had lower odds of being a relapse/defaulter/treatment failure case while females were more likely to be defaulters and older age-groups had more relapse. Elderly, females, unmarried, those with low BMI and higher grade of sputum-smear positivity were more likely to have advanced X-ray features. Higher grade of sputum-smear positivity and advanced chest X-ray findings were associated with relapse/treatment-failures. Elderly, unmarried, relapse/defaulter/treatment-failure cases had higher odds and those with higher BMI and moderately/far advanced X-ray findings had lower odds of having MDR/non-MDR two-drug resistant PTB. CONCLUSION: Targeted intervention and appropriate counseling are needed urgently to prevent adverse treatment outcomes and development of drug resistance among PTB patients in Kolkata.

Immune subversion by Mycobacterium tuberculosis through CCR5 mediated signaling: involvement of IL-10.

Tuberculosis is characterized by severe immunosuppression of the host macrophages, resulting in the loss of the host protective immune responses. During Mycobacterium tuberculosis infection, the pathogen modulates C-C Chemokine Receptor 5 (CCR5) to enhance IL-10 production, indicating the possible involvement of CCR5 in regulation of the host immune response. Here, we found that Mycobacterium infection significantly increased CCR5 expression in macrophages there by facilitating the activation of its downstream signaling. These events culminated in up-regulation of the immunosuppressive cytokine IL-10 production, which was further associated with the down-regulation of macrophage MHC-II expression along with the up-regulation of CCR5 expression via engagement of STAT-3 in a positive feedback loop. Treatment of macrophages with CCR5 specific siRNA abrogated the IL-10 production and restored MHCII expression. While, in vivo CCR5 silencing was also effective for the restoration of host immune responses against tuberculosis. This study demonstrated that CCR5 played a very critical role for the immune subversion mechanism employed by the pathogen.

A molecular approach to identification and profiling of first-line-drug-resistant mycobacteria from sputum of pulmonary tuberculosis patients.

Conventional and molecular techniques were applied to detect and characterize drug resistance of mycobacteria in the sputum samples of clinically confirmed tuberculosis. The sensitivities of mycobacterium detection by ZN staining, culture, multiplex PCR, and restriction fragment length polymorphism (RFLP) were 27.7%, 19.9%, 92.9%, and 95.7%, respectively, but all were 100% specific. The conventional and multiple-allele-specific PCR (MAS-PCR) methods enabled establishment of the drug resistance in 19.3% and 86.9% cases, respectively. We demonstrated that molecular techniques have potential in the accurate diagnosis of tuberculosis.

Oral itraconazole for the treatment of severe seborrhoeic dermatitis.

BACKGROUND: Seborrheic dermatitis (SD) is an inflammatory skin disorder in which colonies of Malassezia furfur have been found in affected areas. AIM: The aim of this study was to evaluate the efficacy of itraconazole in the treatment of severe SD. MATERIALS AND METHODS: Itraconazole was given to 30 patients of SD in a dose of 100 mg twice daily for 1 week followed by 200 mg/day for first 2 days of the following 2 months. The response was noted on day 15, 30, 60, and 90. The clinical response was graded as markedly effective, effective, or ineffective. RESULTS: Clinical improvement (evaluated as markedly effective or effective) was observed in 83.3% cases. CONCLUSION: The anti-inflammatory activity of oral itraconazole suggests that it should be the first-line therapy in severe SD.

Bacteriological study of aerobic isolates from plantar ulcers of paucibacillary leprosy patients.

BACKGROUND: Plantar ulcers commonly occur in leprosy patients, which usually recur and cause morbidity in such cases. AIMS: The aim of the study is to find out the bacteriological profile of these ulcers and to find out the antibiotic susceptibility of the isolates so that appropriate drugs may be chosen for treatment and for prevention of recurrence. MATERIALS AND METHODS: Fifty-six samples from recurrent plantar ulcers of paucibacillary leprosy patients (attending the outpatient department of Calcutta School of Tropical Medicine) were studied for the purpose. Proper sample collection, gram staining, inoculation on culture media, and final identification by biochemical methods were undertaken. Antibiotic susceptibility testing was done for appropriate choice of drugs. RESULTS: Mixed growth of bacteria was seen in 20 (36%) cases while single organism was isolated from the rest. Staphylococcus aureus is the predominant single isolate followed by E. coil, Proteus sp. and Pseudomonas sp. Chloramphenicol and gentamycin are the two drugs that have shown efficacy to the extent of 75 to 100% and 25 to 100% respectively in vitro studies. CONCLUSION: Bacteriological study of plantar ulcers of leprosy patients has revealed Staphylococcus aureus as the main pathogen. Treatment with chloramphenicol and gentamycin holds good prospect as per our study.