Diversity of Culturable Actinobacteria Producing Protease Inhibitors Isolated from the Intertidal Zones of Maharashtra, India.

Phylogenetic diversity of culturable actinobacteria isolated from the intertidal regions of west coast of Maharashtra, India was studied using 16S rRNA gene sequencing. Total of 140 actinobacterial isolates were obtained, which belonged to 14 genera, 10 families and 65 putative species with Streptomyces being the most dominant (63%) genus followed by Nocardiopsis and Micromonospora. Isolates were screened for production of extracellular protease inhibitors (PI) against three pure proteases viz. chymotrypsin, trypsin, subtilisin and a crude extracellular protease from Pseudomonas aeruginosa. Eighty percent of the isolates showed PI activity against at least one of the four proteases, majority of these belonged to genus Streptomyces. Actinobacterial diversity from two sites Ade (17° 52' N, 73° 04' E) and Harnai (17° 48' N, 73° 05' E) with varying anthropological pressure showed that more putative species diversity was obtained from site with lower human intervention i.e. Ade (Shannon's H 3.45) than from Harnai (Shannon's H 2.83), a site with more human intervention. However, in Ade, percentage of isolates not showing PI activity against any of the proteases was close to 21% and that in Harnai was close to 9%. In other words, percentage of PI producers was lower at a site with lesser human intervention.

Identification of Cell Surface Straight Chain Poly-N-Acetyl-Lactosamine Bearing Protein Ligands for VH4-34-Encoded Natural IgM Antibodies.

B cell binding and cytotoxicity by human VH4-34-encoded Abs of the IgM isotype has been well documented. A VH4-34-IgM has recently shown a favorable early response in a phase 1 trial for treatment of B cell acute lymphoblastic leukemia. Although its B cell ligand has been identified as straight chain poly-N-acetyl-lactosamine (SC-PNAL), the carrier of the sugar moiety has not been identified. Using nanoelectrospray ionization mass spectrometry, we identify the metabolic activation related protein complex of CD147-CD98 as a major carrier of poly-N-acetyl-lactosamine (SC-PNAL) on human pre-B cell line Nalm-6. Previous studies have suggested CD45 as the SC-PNAL carrier for VH4-34-encoded IgG Abs. Because Nalm-6 is CD45 negative, human peripheral blood B lymphocytes and human B cell line, Reh, with high CD45 expression, were examined for SC-PNAL carrier proteins. Western blot analysis shows that the CD147-98 complex is indeed immunoprecipitated by VH4-34-encoded IgMs from human peripheral blood B lymphocytes and human B cell lines, Reh, OCI-Ly8, and Nalm-6. However, CD45 is immunoprecipitated only from peripheral B lymphocytes, but not from Reh despite the high expression of CD45. These results suggest that human B cells retain SC-PNAL on the CD147-98 complex, but modulate the sugar moiety on CD45. Because the carbohydrate moiety may act as a selecting Ag for VH4-34 autoantibody repertoire, its differential expression on proteins may provide a clue to the intricate atypical regulation of the VH4-34 gene.

IgG Subclasses and Isotypes of VH4-34 Encoded Antibodies.

VH4-34 gene encoded autoantibodies are elevated in systemic lupus erythematosus (SLE) and in other diseases associated with B-cell hyperproliferation/dysfunction. One of the autoantigens recognized by VH4-34-encoded antibodies are branched/linear poly N-acetyl lactosamine chains. Since the anti-carbohydrate response in humans is dominated by the IgG2 subclass, here we tested whether VH4-34 encoded IgG showed similar subclass segregation. Serum samples from SLE, infectious mononucleosis, nasopharyngeal carcinoma and hepatitis-C were analyzed. Levels of VH4-34-encoded IgM and IgA isotypes were also tested. VH4-34-IgM and IgA were elevated in all four clinical conditions. VH4-34-IgG was detected in the IgG1 and IgG3 subclass but not in the IgG2 and IgG4 subclass. Interestingly, VH4-34-IgG3 was also detected in serum samples of normal healthy adults. These observations are discussed in context of the VH4-34 gene regulation. VH4-34 repertoire development is of interest since it is the only human VH gene profoundly overrepresented in the naïve repertoire but counter-selected for antibody secretion. VH4-34 B-cell could thus become a unique tool to inspect germinal center independent/dependent pathways of subclass and isotype-specific antibody secretion.

Phase I trial of a novel human monoclonal antibody mAb216 in patients with relapsed or refractory B-cell acute lymphoblastic leukemia.

BACKGROUND: This phase I trial was conducted to determine the safety and pharmacokinetics of monoclonal antibody 216, a human monoclonal Immunoglobulin M antibody targeting a linear B-cell lactosamine antigen, administered alone and in combination with vincristine in patients with relapsed or refractory B-cell acute lymphoblastic leukemia, and to preliminarily assess tumor targeting and efficacy. DESIGN AND METHODS: Three cohorts of patients received escalating doses of monoclonal antibody 216 administered as an intravenous infusion. In the case of poor response to the first dose of monoclonal antibody 216 alone, defined as less than 75% reduction in peripheral blood blast count, a second dose of the antibody with vincristine was given between days 4 and 7. Responses were assessed weekly until day 35. Serum concentration of monoclonal antibody 216 was measured before and after infusion. Monoclonal antibody 216 targeting was determined with an anti-idiotypic antibody to monoclonal antibody 216 and preliminary efficacy was analyzed by changes in peripheral blood blasts. RESULTS: Thirteen patients were enrolled. One episode of grade 3 epistaxis was the only dose-limiting toxicity observed. All patients showed a poor response to the first monoclonal antibody 216 infusion with a decrease in peripheral blasts from 6-65% in 9 patients. In 8 patients, addition of vincristine to monoclonal antibody 216 resulted in an average reduction of the peripheral blasts of 81%. One patient without peripheral blasts achieved a hypoplastic marrow without evidence of leukemia after one infusion of monoclonal antibody 216 and monoclonal antibody 216/vincristine each. Monoclonal antibody 216 was detected on peripheral blasts in all patients. CONCLUSIONS: Treatment with monoclonal antibody 216 in combination with vincristine is feasible and well tolerated in patients with relapsed or refractory B-cell acute lymphoblastic leukemia. Binding of monoclonal antibody 216 to leukemic blasts was efficient, and favorable early responses were observed.

Taxol-oligoarginine conjugates overcome drug resistance in-vitro in human ovarian carcinoma.

OBJECTIVE: Multidrug resistance is the major cause of failure of many chemotherapeutic agents. While resistance can arise from several factors, it is often dominated by drug efflux mediated by P-glycoprotein (P-gp), a membrane-bound polysubstrate export pump expressed at high levels in resistant cells. While co-administration of pump inhibitors and a drug could suppress efflux, this two-drug strategy has not yet advanced to therapy. We recently demonstrated that the reversible attachment of a guanidinium-rich molecular transporter, polyarginine, to a drug provides a conjugate that overcomes efflux-based resistance in cells and animals. This study is to determine whether this strategy for overcoming resistance is effective against human disease. METHODS: Tumor samples from ovarian cancer patients, both malignant ascites cells and dissociated solid tumor cells, were exposed to Taxol-oligoarginine conjugates designed to release free drug only after cell entry. Cell viability was determined via propidium-iodide uptake by flow cytometry. To analyze bystander effect, toxicity of the drug conjugates was also tested on peripheral blood leucocytes. RESULTS: Human ovarian carcinoma specimens resistant to Taxol in vitro demonstrated increased sensitivity to killing by all Taxol-transporter conjugates tested. These studies also show that the drug conjugates were not significantly more toxic to normal human peripheral blood leukocytes than Taxol. CONCLUSIONS: These studies with human tumor indicate that oligoarginine conjugates of known drugs can be used to overcome the efflux-based resistance to the drug, providing a strategy that could improve the treatment outcomes of patients with efflux-based drug-resistance.

Study of personal-indoor-ambient fine particulate matters among school communities in mixed urban-industrial environment in India.

The present study is focused on the relationship of school community personal exposure of respirable particulate matter (RPM) with its indoor and ambient outdoor component. A representative longitudinal study design has been adopted. School community is divided into two categories: (1) subjects of science discipline and (2) subjects of nonscience discipline. On the basis of local meteorological parameters and school building constructions, three schools have been identified for the study. Selected subjects have been monitored for 24-h personal RPM exposure along with school indoor and respective outdoor RPM. Paired samples of ambient PM(10) and RPM were collected to establish the correlation between them. Regressions between school indoor and ambient outdoor RPM have shown strong positive relationships in case of two schools having moderate ventilation. The highest RPM to PM(10) ratio (0.62) has occurred at school X located near the steel plant downwind. Almost all indoor and ambient outdoor RPM levels exceed Indian National Ambient Air Quality Standards (NAAQS; 60 microg m( - 3)). Highest levels of indoor and ambient outdoor RPM have occurred in school X located near the steel plant. Subjects belonging to the school located near the steel plant (school X) have shown five to six times higher values of RPM compared to NAAQS standards.

Microbiome and imputed metagenome study of crude and refined petroleum-oil-contaminated soils: Potential for hydrocarbon degradation and plant-growth promotion.

Microbial community structure of crude petroleum oil (CP)- and refined petroleum oil (RP)-contaminated soil was investigated. The taxonomical and functional diversity of such soils can be a great source of information about microbial community and genes involved in petroleum hydrocarbon (PHC) degradation. In this study, microbial diversity of soils contaminated by RP from urban biome of Pune, India, and CP from agricultural biome of Gujarat, India, were assessed by 16S rRNA amplicon sequencing on Illumina MiSeq platform. Association between the soil microbial community and the physicochemical parameters were investigated for their potential role. In RP- and CP-contaminated soils, the microbiome analysis showed Proteobacteria as most dominant phylum followed by Actinobacteria. Interestingly, Firmicutes were most prevailing in a CP-contaminated sample while they were least prevailing in RP-contaminated soils. Soil moisture content, total organic carbon and organic nitrogen content influenced the taxa diversity in these soils. Species richness was more in RP as compared to CP soils. Further prediction of metagenome using PICRUSt revealed that the RP and CP soils contain microbial communities with excellent metabolic potential for PHC degradation. Microbial community contributing to genes essential for soil health improvement and plant growth promotion was also gauged. Our analysis showed promising results for future bioaugmentation assisted phytoremediation (BAP) strategies for treating such soils.

Rapid Estimate of Adult Literacy in Medicine and Dentistry-20 and oral health status among adolescents, India: A cross-sectional study.

INTRODUCTION: Health literacy is an important issue in public health. Individuals with low health literacy skills often have poorer health knowledge and health status than those with higher literacy level. Research documented on the assessment of oral health literacy in health settings and its association with oral health outcomes for adolescents was scarce. AIMS AND OBJECTIVES: The aim of this study is to assess oral health literacy about oral health status among adolescents attending pre-university colleges in Mysore, India. MATERIALS AND METHODS: A cross-sectional study was conducted over a period of 2 months among 401 adolescents attending pre-university colleges. Rapid Estimate of Adult Literacy in Medicine and Dentistry-20 (REALMD-20) and the WHO oral health assessment pro forma for adults (2013) were used. Data were analyzed using SPSS version 22 and tests employed were Chi-square test, ANOVA, and multiple linear regression. RESULTS: Mean REALMD-20 score was 10.31 ± 5.7. The study participants belonging to science course (12.69 ± 5.0) and private pre-university colleges (11.76 ± 5.8) had significantly higher REALMD-20 scores. Mean decayed, missing, and filled teeth among the study participants was (0.42 ± 0.9). Type of college, course, dental history, and number of dental visits was significantly associated with oral health literacy while oral health parameters were not significantly associated. CONCLUSION: Oral health literacy was not significantly associated with oral health status. However, long-term studies are recommended to validate the results of the present study.

Ovarian carcinoma glyco-antigen targeted by human IgM antibody.

Epithelial Ovarian Cancer (EOC) cells expression of a novel carbohydrate antigen was defined using a human VH4-34 encoded IgM monoclonal antibody (mAb216). MAb216 binds to a poly N-acetyllactosamine epitope expressed on B cells and kills normal and malignant B cells in vitro and in vivo. EOC patient ascites and EOC cell lines were used to study the anti tumor effect of mAb216. Various assays were used to characterize the epitope and demonstrate antibody-mediated binding and cytotoxicity in EOC. Drug and antibody combination effects were determined by calculating the combination index values using the Chou and Talalay method. MAb216 displays direct antibody mediated cytotoxicity on a population of human EOC tumor and ascites samples and EOC cell lines, which express high amounts of poly N-acetyllactosamine epitope, carried by CD147/CD98. Eighty four percent of patient samples, including platin resistant, had a tumor population that bound the monoclonal antibody. The binding pattern of mAb216 and mechanism of cytotoxicity was similar to that seen on normal and malignant B cells with unique general membrane disruption and "pore" formation. In vitro incubation with mAb216 and cisplatin enhanced killing of OVCAR3 cell line. In EOC cell lines percent cytotoxicity correlated with percent expression of epitope. Although in vitro data shows specific EOC cytotoxicity, for possible treatment of EOC MAb216 would need to be evaluated in a clinical trial with or without chemotherapy.

B cell lymphoproliferative disorders and VH4-34 gene encoded antibodies.

The VH4-34 represents an unusual Ig heavy chain variable region gene given that it is conserved and overexpressed despite its autoreactivity. Besides RBC 'I/i' recognition, a subset of VH4-34 encoded Igs bind and kill human B-lymphocytes via interaction with a cytoskeletally-associated ligand similar in structure to the cord RBC 'i' antigen. In vivo, secretion of VH4-34 gene encoded antibodies is minimal in healthy individuals. The turn on signal occurs in few clinical conditions such as, systemic lupus erythematosus, AIDS and infectious mononucleosis. Here we show that secretion of VH4-34 Abs is also switched on in hepatitis C and nasopharyngeal carcinoma; but not in diseases such as HPV-associated cervical carcinoma, multiple sclerosis and sarcoidosis. All syndromes with increased VH4-34 Igs appear to be associated with B cell hyperproliferation and B cell lymphotropic viruses, particularly EBV. The significance of the tightly controlled secretion of an autoreactive, conserved Ig gene is discussed.

Historical perspective on the usage of perfumes and scented Articles in ancient Indian literatures.

In India perfumes and scented articles were in use from pre Vedic and Vedic periods for religious practices, social customs, and domestic rituals and later gradually became part and parcel of human life. Perfumes were also used in cosmetics and beauty aids. Medicinal values of many perfumes were well known to ancient Indians and were used in both rituals and to treat diseases. Medicated fumigation (dhupan) was an advanced method for medicinal purposes. Medicated oils, collyriums, powders were prepared from perfumes used externally in many diseases. Perfumes were also anointed in various body parts (Anulepan). Chewing betel leaves along with fragrant material like nutmeg, mace, etc. (Tambulam) was used with a view to rendering mouth clean and fragrant. usage of scented oils to massage body(Abhyanga) which keeps the skin smooth, healthy and invigorating; Udvartanam, massaging various body parts; Udgarshanam, scrubbing; Utsadanam rubbing with scented powders etc., were some of the health protective and disease eliminating procedures. Scented drugs and perfumes enhance the quality, activity and pleasantness of these processes.

Effects of human monoclonal antibody 216 on B-progenitor acute lymphoblastic leukemia in vitro.

BACKGROUND: Human monoclonal antibody (mAb) 216 is a naturally occurring IgM cytotoxic mAb that binds to a glycosylated epitope on the surface of B-lymphocytes. This study investigated if this mAb could bind and kill acute lymphoblastic leukemia (ALL) B-progenitor lymphoblasts in vitro. ALL cell lines were used to determine if combining mAb 216 with chemotherapeutic drugs would enhance killing and cell lines were used to measure cytotoxicity by mAb 216 with human complement. PROCEDURE: Expression of cell surface markers and mAb 216 epitope on fresh and banked ALL bone marrow samples was determined by flow cytometry. Fresh lymphoblasts were incubated for 20 hr with mAb 216 without complement to measure cytotoxicity. Cytotoxicity of ALL cell lines incubated with mAb 216 and vincristine (VCR) or human complement was determined using flow cytometry. RESULTS: Pre-B-ALL cells but not T-ALL cells are bound and killed by mAb 216. The combination of mAb 216 and VCR at sub-therapeutic levels demonstrated enhanced cytotoxicity beyond that observed for either agent alone. Incubation of mAb 216 with human complement increased cytotoxicity of ALL cell lines. CONCLUSION: This increased cytotoxicity with chemotherapy and the functional ability of mAb 216 to use multiple pathways to induce cell death identify mAb 216 as a potentially novel therapeutic tool in the treatment of B-progenitor ALL. Based on the results from this preclinical study, a Phase I clinical trial with mAb 216 for the treatment of patients with relapsed or refractory B-lineage ALL is ongoing.

Validation of the inactivant binary ethylenimine for inactivating rabies virus for veterinary rabies vaccine production.

The rabies vaccine is produced by inactivation of rabies virus propagated on BHK21 cells. In the rabies inactivation process, BEI is added at a final concentration of 1.6 mM to the viral harvest at 37 degrees C, followed by a second dose of BEI at 24 h post-inactivation. Inactivation was confirmed by the mice innocuity test and tissue culture amplification test as per B.P (Vet) 2004. Validation of test procedure is essential as per cGMP requirement. The dose of BEI was validated by using lower and higher concentrations of BEI in inactivation process. The study indicated that BEI at a lower concentration (0.4 mM) was able to inactivate the rabies virus within 30 h and the routine concentration (1.6 mM) of BEI is effective in inactivating rabies virus within 18 h. The amplification test used for confirming the inactivation of the live virus was validated by spiking the sample with different dilutions of pretitrated live rabies virus. The test revealed that the amplification method is sensitive to detect live rabies virus if present in the inactivated sample. The validation of BEI as an inactivant and the amplification test are discussed.

VH4-34 encoded antibody in systemic lupus erythematosus: effect of isotype.

OBJECTIVE: To determine the clinical significance of elevated serum levels of VH4-34 encoded IgM and IgG antibodies with respect to the clinical characteristics of systemic lupus erythematosus (SLE). METHODS: VH4-34 encoded IgM and IgG immunoglobulin was measured in 95 patients with SLE by ELISA using antiidiotype monoclonal antibody (Mab) 9G4. SLE disease activity, severity, and damage were assessed by visual analog scales, Systemic Lupus Activity Measure, Lupus Severity of Disease Index, and Systemic Lupus International Collaborating Clinics/American College of Rheumatology Damage Index. Presence of VH4-34 encoded antibodies on patients' B lymphocytes was analyzed by flow cytometry using Mab 9G4. RESULTS: Fifty-two of 95 patients with SLE had elevated levels of VH4-34 encoded antibodies of IgG isotype; 17 patients with VH4-34 IgG had elevated VH4-34 of the IgM isotype. Forty-three of the 95 patients had normal levels of VH4-34 encoded antibodies. When disease severity was correlated to VH4-34 isotype, patients with circulating VH4-34 IgG but without IgM had significantly more severe disease compared to patients who had VH4-34 of both isotypes. Eighty-six percent of patients with SLE nephritis and 100% of those with central nervous system (CNS) lupus had VH4-34 IgG without IgM. In vivo, VH4-34 encoded antibodies were found to bind autologous B lymphocytes. CONCLUSION: Presence of VH4-34 IgG in the absence of VH4-34 IgM was the finding most strongly associated with severe SLE, nephritis, and CNS lupus, suggesting that isotype switching of VH4-34 encoded antibodies or loss of VH4-34 IgM encoded antibodies may influence the progression of disease in SLE.