LED-Fluorescence Microscopy for Diagnosis of Pulmonary Tuberculosis under Programmatic Conditions in India

Background

Light-emitting diode fluorescence microscopy (LED-FM) has been shown to be more sensitive than conventional bright field microscopy using Ziehl-Neelsen (ZN) stain in detecting sputum smear positive tuberculosis in controlled laboratory conditions. In 2012, Auramine O staining based LED-FM replaced conventional ZN microscopy in 200 designated microscopy centres (DMC) of medical colleges operating in collaboration with India’s Revised National Tuberculosis Control Programme. We aimed to assess the impact of introduction of LED-FM services on sputum smear positive case detection under program conditions.

Methods

This was a before and after comparison study. In 15 randomly selected medical college DMCs, all presumptive TB patients who underwent sputum smear examination in the years 2011 (before LED-FM) and 2012 (after LED-FM) were compared. An additional 15 comparable DMCs that implemented conventional ZN sputum smear microscopy were also selected for comparison between 2011 and 2012.

Results

The proportion of presumptive TB patients (PTP)found sputum smear positive increased by 30%- from 13.6% (3432/25159) in 2011 to 17.8% (4706/26426) in 2012 (P value <0.01) in the sites that implemented LED-FM microscopy, whereas in DMCs where the ZN staining procedure is followed the proportion of sputum smear positive had remained unchanged (13.0%versus 12.6%;P value0.31).

Conclusion

Use of LED-FM significantly increased the proportion of smear positive cases among presumptive TB patients under routine program conditions in high workload laboratories. The study provides operational evidence needed to scale-up the use of LED-FM in similar settings in India and beyond.     

Suppression of islet allogeneic immune response by indoleamine 2,3 dioxygenase-expressing fibroblasts

Success of transplantation of pancreatic islets which is a promising way for restoring efficient insulin regulation in type 1 diabetes depends on lifelong use of immunosuppressive drugs. To eliminate the use of systemic immunosuppressive drugs for islet transplantation, we examined the potential use of a local immunosuppressive factor, indoleamine 2,3-dioxygenase (IDO). Thus, the aim of this study was to determine whether local expression of IDO in bystander syngeneic fibroblasts could prevent islet allogeneic immune response in vitro. C57BL/6 (B6) mouse fibroblasts were induced to express IDO by either IFN-gamma treatment or transduction with an adenoviral vector and were co-cultured with B6 mouse lymphocytes and BALB/c mouse pancreatic islets in the presence or absence of an IDO inhibitor. Proliferation of lymphocytes were then assessed using [(3)H]-thymidine incorporation assay. IDO-expression by co-cultured syngeneic fibroblasts resulted in a five-fold decrease in lymphocyte proliferation rate upon stimulation of lymphocytes by allogeneic mouse pancreatic islets (21.9% +/- 5.3 and 22.1% +/- 4.9 in the preparations with IFN-gamma treated and genetically modified IDO-expressing fibroblasts, respectively vs. 100% in control groups, P < 0.01). Allogeneic response was restored when IDO inhibitor was added to the culture indicating that suppression was due to IDO. In conclusion, this study shows that local expression of IDO by syngeneic bystander fibroblasts can suppress in vitro proliferation of lymphocytes in response to stimulation with allogeneic pancreatic islets. This local immunosuppressive function of IDO may be employed for development of a novel alternative strategy for preventing allogeneic islet graft rejection.     

Enzymatic Synthesis of 2′,5′-Dideoxv Purine Nucleosides and Related Compounds

Abstract

A wide range of 2′,5′-dideoxy-nucleosides, including 6- substituted purine, pyrazolo[3,4-d]pyrimidine and 1-deazapurine derivatives, has been enzymatically prepared using purine nucleoside phosphorylase. Specificity towards cleavage by bacterial versus mammalian purine nucleoside phosphorylase was evaluated.     

Immunogenicity of Salmonella enterica serovar Enteritidis virulence protein,InvH, and cross-reactivity of its antisera with Salmonella strains

Acellular vaccines containing bacterial immunodominant components such as surface proteins may be potent alternatives to live attenuated vaccines in order to reduce salmonellosis risk to human health. invH gene, an important part of needle complex in type three secretion system (TTSS) plays important role in efficient bacterial adherence and entry into epithelial cells. In this work we hypothesize that use of a 15 kDa recombinant InvH as Salmonella enterica serovar Enteritidis surface protein could provoke antibody production in mouse and would help us study feasibility of its potential for diagnosis and/or a recombinant vaccine. The purified InvH provoked significant rise of IgG in mice. Active protection induced by immunization with InvH against variable doses of S. enterica serovar Enteritidis, indicated that the immunized mice were completely protected against challenge with 10⁴ LD₅₀. The immunoreaction of sera from immunized mice with other Salmonella strains or cross reaction with sera of Salmonella strains inoculated mice is indicative of possessing by Salmonella strains of the surface protein, InvH, that can be employed in both prophylactic and diagnostic measures against S. enterica. Bacteria free spleen and ileum of the immunized mice in this study indicate that the invH gene affects bacterial invasion. Efficacy of the virulence protein, InvH, in shuttling into host cells in injectisome of S. enterica serovar Enteritidis and inhibition of this phenomenon by active immunization was shown in this study. In conclusion immunization with InvH protein can develop protection against S. enterica serovar Enteritidis infections. InvH in Salmonella strains can be exploited in protective measures as well as a diagnostic tool in Salmonella infections.     

Promoter methylation analysis of WNT/β-catenin pathway regulators and its association with expression of DNMT1 enzyme in colorectal cancer

Background

Aberrant DNA methylation as the most important reason making epigenetic silencing of genes is a main mechanism of gene inactivation in patients with colorectal cancer. In this study, we decided to identify promoter methylation status of ten genes encoding WNT negative regulators, and measure the expression of DNMT1 enzyme in colorectal cancer samples.

Results

Aberrant methylation of APC gene was statistically significant associated with age over 50 (p = 0.017), DDK3 with male (p < 0.0001), SFRP4, WIF1, and WNT5a with increasing tumor stage (p = 0.004, p = 0.029, and p = 0.004), SFRP4 and WIF1 with tumor differentiation (p = 0.009 and p = 0.031) and SFRP2 and SFRP5 with histological type (p = 0.001 and p = 0.025). The increasing number of methylated genes correlated with the expression levels of the DNMT1 mRNA.

Conclusions

The rate of gene promoter methylation of WNT pathway regulators is high in colorectal cancer cells. Hyper-methylation is associated with increased expression of the DNMT1 enzyme.     

Redescription and remarks on the species Minniza persica (Pseudoscorpiones: Olpiidae) from Iran

Minniza persica, which has been described briefly by Beier in 1951 on the basis of specimens from Hormozgan and Mazandaran provinces of Iran, was recently collected again from Hormozgan and Fars provinces and is described and illustrated here. The subspecies M. persica deminuta Beier is regarded as synonymous with the nominate subspecies.     

Prediction of shape and internal structure of the proximal femur using a modified level set method for structural topology optimisation

A computational framework was developed to simulate the bone remodelling process as a structural topology optimisation problem. The mathematical formulation of the Level Set technique was extended and then implemented into a coronal plane model of the proximal femur to simulate the remodelling of internal structure and external geometry of bone into the optimal state. Results indicated that the proposed approach could reasonably mimic the major geometrical and material features of the natural bone. Simulation of the internal bone remodelling on the typical gross shape of the proximal femur, resulted in a density distribution pattern with good consistency with that of the natural bone. When both external and internal bone remodelling were simulated simultaneously, the initial rectangular design domain with a regularly distributed mass reduced gradually to an optimal state with external shape and internal structure similar to those of the natural proximal femur.     

Contamination level and human health hazard assessment of heavy metals and polycyclic aromatic hydrocarbons (PAHs) in street dust deposited in Mahshahr,southwest of Iran

Mahshahr has a strategic position and is considered as industrial hub of Iran. Selected heavy metals and polycyclic aromatic hydrocarbons (PAHs) contamination and health risk, and the mineralogical composition of street dust from Mahshahr were investigated. Results indicated that geology is the main determinant of the dust mineralogical composition. Calculated enrichment factor (EF) and principal component analysis (PCA) showed that Pb, Hg, Zn, and Cu accumulations are greatly influenced by anthropogenic sources including traffic and industry. High heavy metals content poses great ecological risk in the study area and exposure doses revealed that ingestion is the main exposure route to street dust, especially for children in residential/commercial areas. It was found that the total amount of PAHs (∑PAHs) varies from 161 to 1996 µg/kg, dominated by four-ring PAHs. Diagnostic ratios and PCA showed that both petrogenic and pyrogenic sources of PAHs in Mahshahr street dust and traffic play important roles in this respect. Furthermore, toxic equivalents and incremental lifetime cancer risk of PAHs in street dust indicated a high potential carcinogenic risk for inhabitants mainly via dermal contact and ingestion pathways particularly for outdoor workers in industrial use scenario. Finally, distribution maps of total hazard index of heavy metals and cancer risk of PAHs indicated the most impacted zones for different groups and use scenarios.