Enrichment for murine keratinocyte stem cells based on cell surface phenotype

The identification and physical isolation of epithelial stem cells is critical to our understanding of their growth regulation during homeostasis, wound healing, and carcinogenesis. These stem cells remain poorly characterized because of the absence of specific molecular markers that permit us to distinguish them from their progeny, the transit amplifying (TA) cells, which have a more restricted proliferative potential. Cell kinetic analyses have permitted the identification of murine keratinocyte stem cells (KSCs) as slowly cycling cells that retain [(3)H]thymidine ([(3)H]Tdr) label, termed label-retaining cells (LRCs), whereas TA cells are visualized as rapidly cycling cells after a single pulse of [(3)H]Tdr, termed pulse-labeled cells (PLCs). Here, we report on the successful separation of KSCs from TA cells through the combined use of in vivo cell kinetic analysis and fluorescence-activated cell sorting. Specifically, we demonstrate that murine dorsal keratinocytes characterized by their high levels of alpha(6) integrin and low to undetectable expression of the transferrin receptor (CD71) termed alpha(6)(bri)CD71(dim) cells, are enriched for epithelial stem cells because they represent a minor ( approximately 8%) and quiescent subpopulation of small blast-like cells, with a high nuclear:cytoplasmic ratio, containing approximately 70% of label-retaining cells, the latter being a well documented characteristic of stem cells. Conversely, TA cells could be enriched in a phenotypically distinct subpopulation termed alpha(6)(bri)CD71(bri), representing the majority ( approximately 60%) of basal keratinocytes that are actively cycling, and importantly contain approximately 70% of [(3)H]Tdr pulse-labeled cells. Importantly, immunostaining of dorsal skin revealed the presence of CD71(dim) cells in the hair follicle bulge region, a well documented location for KSCs.     

Splenectomy in a case of splenic vein thrombosis unmasks essential thrombocythemia

We report a patient with splenic vein thrombosis (SVT) in whom splenectomy resulted in the unmasking of essential thrombocythemia (ET). He had portal hypertension with haematemesis, resulting in anaemia requiring repeated blood transfusions. Investigations revealed SVT. Following splenectomy, he suffered a transient ischaemic attack episode, associated with persistent thrombocytosis (> 2000 x 10(9)/l). Other myeloproliferative disorders were excluded and a diagnosis of ET was established. He responded to hydroxyurea but, due to financial constraints, he discontinued treatment and subsequently relapsed. The association of ET with SVT is rare and the diagnosis of ET was missed initially as the platelet count was normal prior to splenectomy.     

Neural Network Analysis of Anal Sphincter Repair

PURPOSE: Prediction of success after anterior sphincter repair for incontinence is difficult. Standard multivariate analysis techniques have only 75 to 80 percent accuracy. Artificial intelligence, including artificial neural networks, has been used in the analysis of complex clinical data and has proved to be successful in predicting the outcome of other surgical procedures. Using a neural network algorithm, we have assessed the probability of success after anterior sphincter repair. METHODS: Prospective anorectal physiology data of 72 patients undergoing anterior sphincter repair was collected between 1995 and 1999. Complete data sets of 75 percent of the series were used to train an artificial neural network; the remaining 25 percent were used for data validation. The output was continence grading, ranging from 0 to 4 (worse to continent). RESULTS: The outcome at 3, 6, and 12 months postoperatively was obtained and assessed. The best correlation between actual data value and artificial neural network value was found at 12 months (r = 0.931; P = 0.0001). Clear correlations also were found at three months (r = 0.898; P = 0.0001) and six months (r = 0.742; P = 0.002). Results of applying a net to details excluding pudendal nerve latency were poor. CONCLUSIONS: Artificial neural networks are more accurate (93 percent correlation) than standard statistics (75 percent) when applied to the prediction of outcome after anterior sphincter repair. This assessment also confirms the usefulness of pudendal latency in the prediction of anterior sphincter repair outcome. The results obtained highlight the obvious usefulness of artificial neural networks, which could now be used in a prospective evaluation for application of the technique.     

Endothelin as a therapeutic target in the treatment of cardiovascular disease

Endothelins, a family of peptides derived from the vascular endothelium and smooth muscle cells possess vasoconstrictor and mitogenic properties. By acting predominantly in a paracrine fashion, these peptides activate specific receptors and have protean effects in normal and diseased organ systems. The wide distribution of these receptors in various tissues mediate the multiplicity of physiologic actions attributed to endothelins. Much of our understanding about endothelins has come from the development of an array of receptor-specific and mixed receptor antagonists. Based on the promising results from animal studies, active research and drug development programs are under way to investigate the clinical potential of endothelin antagonism for treatment of cardiovascular disease.     

Acute-phase reactants during murine tuberculosis: unknown dimensions and new frontiers

SETTING: Serum amyloid P-component (SAP) plays important roles in host defense during various infectious diseases; however, nothing is known in tuberculosis (TB). OBJECTIVE: To study the SAP response of Mycobacterium tuberculosis H37Rv- and H37Ra-infected mice, and to determine the effect(s) of purified mouse SAP both on their intra-alveolar macrophage (AM) uptake and intra-AM growth in vitro. DESIGN: The SAP levels of mice intratracheally infected with M. tuberculosis H37Rv and H37Ra were determined by ELISA. Mycobacterial AM uptake and intra-AM growth in vitro were determined using fluorescence microscopy and plating, respectively. RESULTS: M. tuberculosis H37Rv-infected mice showed significantly (p < 0.05) increased SAP levels (352.8+/-36.1 microg/ml) with compared mice infected with M. tuberculosis H37Ra (170+/-18.5 microg/ml). During the acute phase of both these infections, enhanced SAP levels correlated with the lung mycobacterial load. In vitro, purified mouse SAP (1-80 microg/ml) inhibited the AM uptake of both the mycobacteria in a concentration-dependent manners to a similar extent; 20 microg/ml SAP appeared optimal. Mycobacterial uptake inhibition was divalent cation- and pH-dependent, and was unaffected both by heat-inactivated and deglycosylated SAP, separately. Curiously, purified mouse SAP (1-80 microg/ml), in a concentration-dependent manner, inhibited the intra-AM growth of both M. tuberculosis H37Rv and H37Ra in vitro; the effect was 0.8 log10 CFUs greater on the latter. Both the mannose-based simple sugars and rabbit anti-mouse SAP polyclonal antibody, separately, annulled the inhibition of mycobacterial growth in vitro. CONCLUSION: This initial study demonstrates that both the SAP response of M. tuberculosis-infected mice, and the SAP-induced intra-AM mycobacterial growth inhibition in vitro were apparently dependent on mycobacterial virulence.     

GC-FID,Antioxidants Analysis and Antimicrobial,Anti-Inflammatory,Anti-Diabetic Activities of Tisanes Derived from Underutilized Lemon Grass Cymbopogon khasianus (Hack.)

Proceedings of the National Academy of Sciences, India Section B: Biological Sciences - Cymbopogon khasianus (Hack.) is widely distributed and an underutilized plants in tropical and subtropical...     

Status of TIM-1 exon 4 haplotypes and CD4+T cell counts in HIV-1 seroprevalent North Indians

The TIM (T cell/transmembrane, immunoglobulin and mucin) proteins are crucial regulators of Th1/Th2 immune responses and have been implicated in several diseases including HIV-1/AIDS. The TIM1 exon 4 that codes for mucin domain is highly diverse, with sequence variants associated with varying phenotypes. In this study, TIM1 exon 4 was sequenced among 227 HIV-1 seroprevalent and 288 healthy non infected individuals from North Indian population and haplotypes established. A novel but rare haplotype D1^∗ was identified among the healthy and differed from D1 by a synonymous substitution G>T at Thr208Thr. The TIM1 haplotype diversity showed no association with susceptibility to HIV-1 infection. The seroprevalent individuals carrying D3A had relatively higher median CD4+T cell counts (368/μl) than those without (313/μl; p = 0.02). A comparison of CD4+T counts between D3-A individuals on ART or ART naïve did not show any significant difference plausibly due to confounding nature of ART and other factors.     

Aerobic Fitness and the Brain: Increased N-Acetyl-Aspartate and Choline Concentrations in Endurance-Trained Middle-Aged Adults

Engagement in regular aerobic exercise is associated with cognitive benefits, but information on the mechanisms governing these changes in humans is limited. The goal of the current study was to compare neurometabolite concentrations relating to cellular metabolism, structure, and viability in endurance-trained and sedentary middle-aged adults. Twenty-eight endurance-trained and 27 sedentary adults, aged 40–65 years, underwent general health assessment, cardiorespiratory fitness measurement, neuropsychological testing, and proton magnetic resonance spectroscopy (¹H MRS). ¹H MRS was used to examine N-acetyl-aspartate (NAA), creatine (Cr), myo-inositol (mI), choline (Cho), and glutamate (Glu) concentrations in frontal and occipitoparietal grey matter. Group differences in concentrations of NAA, Cho, mI, and Glu, calculated as ratios over Cr, were explored using ANOVA. There were no significant differences in global cognitive function, memory, and executive function performance between the groups. In comparison to sedentary adults, the endurance-trained group displayed significantly higher NAA/Cr in the frontal grey matter (F(1, 53) = 5.367, p = 0.024) and higher Cho/Cr in the occipitoparietal grey matter (F(1, 53) = 5.138, p = 0.028). Within our middle-aged sample, endurance-trained adults demonstrated higher levels of NAA/Cr in the frontal grey matter and higher Cho/Cr in the occipitoparietal grey matter. Higher levels of NAA may indicate greater neuronal integrity and higher cerebral metabolic efficiency in association with cardiorespiratory fitness, whereas increased Cho may represent increased phospholipid levels secondary to neural plasticity.     

Antineoplastic drug, carboplatin, protects mice against visceral leishmaniasis

In the present study, the leishmanicidal effect of two doses (5 and 10 mg/kg body weight) of the carboplatin was studied in Leishmania donovani-infected BALB/c mice. Mice were infected intracardially with promastigotes of L. donovani, and a month after infection, they were treated intraperitoneally with the two doses of the drug (5 and 10 mg/kg body weight) for five continuous days. Animals were sacrificed on 1 and 15 posttreatment days. Hepatic parasite load was assessed on Geimsa-stained imprints. Immune responses were studied by measuring delayed-type hypersensitivity (DTH) responses, serum IgG isotype levels (IgG1 and IgG2a) and cytokine levels [γ-interferon (IFN-γ), interleukin (IL)-10 and IL-2] in spleen cell cultures by ELISA. To study the drug-induced side effects, various haematological (haemoglobin and total leukocyte count), biochemical (liver and kidney function tests) and histological investigations (kidney, liver and spleen) were carried out. The antileishmanial potential of the drug was revealed by significant reduction in the parasite burden. The infected and treated animals were also found to exhibit increased DTH responses, higher IgG2a levels, lower IgG1 levels and greater cytokine (IFN-γ, IL-10 and IL-2) concentrations pointing towards the generation of mixed Th1/Th2 response. Liver and kidney function tests and histological studies of kidney, liver and spleen of treated mice revealed no side effects. Carboplatin cures mice of visceral leishmaniasis without causing any serious side effects, and the drug was found be more effective at a dose of 10 mg/kg body weight as compared to 5 mg/kg body weight.     

Mechanistic and physiological approaches of fecal microbiota transplantation in the management of NAFLD

Inflammation Research - Non-alcoholic fatty liver disease (NAFLD) is a multifaceted disease allied with various metabolic disorders, obesity and dysbiosis. Gut microbiota plays an influential role...