Potential serological use of a recombinant protein that is a replica of a Mycobacterium tuberculosis protein found in the urine of infected mice

The recent availability of numerous well-characterized Mycobacterium tuberculosis recombinant proteins has revived interest in the serological diagnosis of tuberculosis. Several promising results have been reported, particularly when more than one antigen is used in the test. However, thus far these antigens have not been used in routine diagnostic tests because they lack sufficient sensitivity. In addition, with the exception of one antigen, most recombinant M. tuberculosis proteins do not identify the majority of tuberculosis patients coinfected with human immunodeficiency virus (HIV). Here, we report a newer M. tuberculosis protein that is a promising candidate for increasing the sensitivity of the serological tests, in particular for patients coinfected with HIV. The protein was found in the urine of mice during the early stages of infection with M. tuberculosis (10 to 14 days), thus suggesting that the antigen is abundantly released during the in vivo growth of the mycobacterium. Reverse genetics was used to produce the recombinant protein, which we named U1 (for urine protein 1). Using a conventional enzyme-linked immunosorbent assay (ELISA), antibody to U1 could be detected in 60% of patients with pulmonary tuberculosis with no signs of coinfection with HIV (n = 83). Conversely, anti-U1 antibody was detected in 87% of the sera from tuberculosis patients coinfected with HIV (n = 47). Out of 12 HIV-infected nontuberculosis patients' sera, 9 did not react with U1 and three sera gave borderline ELISA signals (signal/cutoff of < or =1.75). These results suggest that the high efficiency of U1 in identifying tuberculosis patients coinfected with HIV may be related to abundant release of this protein during the initial phase of the HIV coinfection. The immediate availability of the antigen at a time point in which the patient's immune system is still competent would lead to a secondary immune response to U1 that persists for months in the patient's serum.     

Precipitation of nanohydroxyapatite on PLLA/PBLG/Collagen nanofibrous structures for the differentiation of adipose derived stem cells to osteogenic lineage

Tissue engineering and nanotechnology have enabled engineering of nanostructured materials to meet the current challenges in bone treatment owing to rising occurrence of bone diseases, accidental damages and defects. Poly(l-lactic acid)/Poly-benzyl-l-glutamate/Collagen (PLLA/PBLG/Col) scaffolds were fabricated by electrospinning and nanohydroxyapatite (n-HA) was deposited by calcium-phosphate dipping method for bone tissue engineering (BTE). The abundance and accessibility of adipose derived stem cells (ADSC) may prove to be novel cell therapeutics for bone repair and regeneration. ADSCs were cultured on these scaffolds and were induced to undergo osteogenic differentiation in the presence of PBLG/n-HA for BTE. The cell-biomaterial interactions were analyzed using cell proliferation, SEM and CMFDA dye extraction techniques. Osteogenic differentiation of ADSC was confirmed using alkaline phosphatase activity (ALP), mineralization (ARS) and dual immunofluorescent staining using both ADSC marker protein and Osteocalcin, which is a bone specific protein. The utmost significance of this study is the bioactive PBLG/n-HA biomolecule introduced on the polymeric nanofibers to regulate and improve specific biological functions like adhesion, proliferation and differentiation of ADSC into osteogenic lineage. This was evident from the immunostaining and CMFDA images of ADSCs showing cuboidal morphology, characteristic of osteogenic lineage. The observed results proved that the PLLA/PBLG/Col/n-HA scaffolds promoted greater osteogenic differentiation of ADSC as evident from the enzyme activity and mineralization profiles for bone tissue engineering.     

Eastern equine encephalitis in Tennessee: 2002-2008

Human and equine outbreaks caused by eastern equine encephalomyelitis virus (EEEV) typically occur in North America adjacent to coastal wetlands associated with the presence of Culiseta melanura (Coquillet) mosquitoes. Eastern equine encephalomyelitis (EEE) is an emerging disease in Tennessee, as the first records of equine disease began in 2002. In 2006 we trapped and tested mosquitoes for EEEV at hardwood swamps in western Tennessee that were at the epicenter of a multi-equine outbreak in fall of 2005. Additionally, the Tennessee Valley Authority tested mosquito pools collected in Tennessee swamps from 2000 to 2007 for the presence of arboviruses. Two pools of EEEV positive Culex erraticus (Dyer and Knab) mosquitoes were found (one each in 2003 and 2004) in a county adjacent to where the 2005 outbreak occurred. In 2008, another EEE outbreak involving multiple horses occurred in West Tennessee. A brain specimen was collected from a horse during this outbreak and the first isolate of EEEV from Tennessee was obtained. In total, 74,531 mosquitoes collected from 2000 to 2008 were tested via polymerase chain reaction and VecTest for EEEV. The traditional enzootic vector, Cs. melanura, was found in low numbers at all collection sites. Cx. erraticus, however, was consistently found in high numbers and was the only mosquito species in which EEEV was detected. We suggest that EEE transmission may be maintained by Cx. erraticus in a nontraditional cycle. We discuss the importance of a nontraditional cycle from the perspective of EEEV adaptation and emergence.     

Kinetics of humoral and memory B cell response induced by the Plasmodium falciparum 19-kilodalton merozoite surface protein 1 in mice

The 19-kDa carboxyl-terminal fragment of the merozoite surface protein-1 (MSP-1(19)) has been shown to regulate antibody (Ab)-mediated protective immunity to blood-stage malaria infection. But the serological memory to this antigen tends to be short-lived, and little is known of the mechanisms that regulate the formation of B cell memory to MSP-1(19) antigen. We studied the formation of B cell memory response after immunization with the recombinant 19-kDa Plasmodium falciparum merozoite surface protein 1 (PfMSP-1(19)). Immunization with PfMSP-1(19) resulted in delayed increase in germinal center (GC) B cell numbers. This poor GC reaction correlated with short-lived PfMSP-1(19)-specific antibodies in serum and the short life of PfMSP-1(19)-specific plasma cells and memory B cells (MBCs) in spleen and bone marrow. PfMSP-1(19)-specific MBCs were capable of producing antigen (Ag)-specific Ab-secreting cell (ASC) responses that were short-lived following challenge immunization of the immune mice with antigen or transgenic Plasmodium berghei parasite expressing PfMSP-1(19) in place of native P. berghei MSP-1(19) at 8 weeks after the last immunization or following adoptive transfer into naive hosts. However, no protection was achieved in PfMSP-1(19) immune mice or recipient mice with PfMSP-1(19)-specific MBCs following challenge with transgenic P. berghei. Our findings suggest that PfMSP-1(19)-specific IgG production by short-lived plasma cells combined with the poor ability of the PfMSP-1(19)-induced MBCs to maintain the anamnestic IgG responses failed to contribute to protection against infection.     

Provider-initiated HIV testing in rural Haiti: low rate of missed opportunities for diagnosis of HIV in a primary care clinic

As HIV treatment is scaled-up in resource-poor settings, the timely identification of persons with HIV infection remains an important challenge. Most people with HIV are unaware of their status, and those who are often present late in the course of their illness. Free-standing voluntary counseling and testing sites often have poor uptake of testing. We aimed to evaluate a 'provider-initiated' HIV testing strategy in a primary care clinic in rural resource-poor Haiti by reviewing the number of visits made to clinic before an HIV test was performed in those who were ultimately found to have HIV infection. In collaboration with the Haitian Ministry of Health, a non-governmental organization (Partners In Health) scaled up HIV care in central Haiti by reinforcing primary care clinics, instituting provider-initiated HIV testing and by providing HIV treatment in the context of primary medical care, free of charge to patients. Among a cohort of people with HIV infection, we assessed retrospectively for delays in or 'missed opportunities' for diagnosis of HIV by the providers in one clinic. Of the first 117 patients diagnosed with HIV in one clinic, 100 (85%) were diagnosed at the first medical encounter. Median delay in diagnosis for the remaining 17 was only 62 days (IQR 19 – 122; range 1 – 272). There was no statistical difference in CD4 cell count between those with and without a delay. 3787 HIV tests were performed in the period reviewed. Provider-initiated testing was associated with high volume uptake of HIV testing and minimal delay between first medical encounter and diagnosis of HIV infection. In scale up of HIV care, provider-initiated HIV testing at primary care clinics can be a successful strategy to identify patients with HIV infection.     

A Quick and Easy Approach to Prepare Ground Bone Slides from Undecalcified Skeleton

Ground bone slides are not only necessary for teaching histology of Haversian system to undergraduate and postgraduate students, but also essential for rapid pathological diagnosis in related diseases as well as to be used in polarizing microscopy and studying bone architectures from fossils. This technique is also applicable for hard tissues like teeth. Present histology manual-cum-practical literatures though prefer bone-slide preparation from decalcified skeleton, but Haversian architectures with osteons are vivid in ground bone slides. Here a technique is briefed; which have practiced well to prepare the ground bone slides with very simple, cheap and easily available equipments in a very short time, which can be adopted by anatomists, odontologists and geologists for quick making of compact bone slides without hampering the bone continuity avoiding hazardous and time consuming methods or costly equipments like freezing microtome.     

Integrating genetic and gene expression evidence into genome-wide association analysis of gene sets

Single variant or single gene analyses generally account for only a small proportion of the phenotypic variation in complex traits. Alternatively, gene set or pathway association analyses are playing an increasingly important role in uncovering genetic architectures of complex traits through the identification of systematic genetic interactions. Two dominant paradigms for gene set analyses are association analyses based on SNP genotypes and those based on gene expression profiles. However, gene-disease association can manifest in many ways, such as alterations of gene expression, genotype, and copy number; thus, an integrative approach combining multiple forms of evidence can more accurately and comprehensively capture pathway associations. We have developed a single statistical framework, Gene Set Association Analysis (GSAA), that simultaneously measures genome-wide patterns of genetic variation and gene expression variation to identify sets of genes enriched for differential expression and/or trait-associated genetic markers. Simulation studies illustrate that joint analyses of genomic data increase the power to detect real associations when compared with gene set methods that use only one genomic data type. The analysis of two human diseases, glioblastoma and Crohn's disease, detected abnormalities in previously identified disease-associated pathways, such as pathways related to PI3K signaling, DNA damage response, and the activation of NFKB. In addition, GSAA predicted novel pathway associations, for example, differential genetic and expression characteristics in genes from the ABC transporter family in glioblastoma and from the HLA system in Crohn's disease. These demonstrate that GSAA can help uncover biological pathways underlying human diseases and complex traits.     

Expansion of CTG repeat in myotonin protein kinase gene on Alu(ins)‐Hinf1‐1 background in a myotonic dystrophy patient from India

To determine the founder of Indian myotonic dystrophy mutation, we have studied the expansion of CTG repeats in myotonin protein kinase gene and two intragenic linked loci Alu(ins) / Alu(del) and G/T intron 9 Hinf1 polymorphism in ten unrelated DM patients from eastern India. Out of these ten patients, reconstruction of haplotype was possible for five patients unambiguously. In the other five cases, haplotype for the normal allele was assumed to be the most common haplotype found in normal individuals from Indian populations. Such analysis showed that in nine cases, the expansion of CTG repeats took place on Alu(ins)‐Hinf1‐2 background indicating common founder with other DM mutation published. However, in one case we observed a different haplotype [Alu(ins)‐Hinf1‐1] which could be a new mutation or due to admixture. © 1998 Wiley‐Liss, Inc.     

Chromatin interactions in differentiating keratinocytes reveal novel atopic dermatitis– and psoriasis-associated genes

1. Download : Download high-res image (202KB)
2. Download : Download full-size image