In vitro correlate of tranplantation immunity: spleen cell migration inhibition in the lizard, Calotes Versicolor.

Sensitization to skin allografts in the lizard, Calotes versicolor, was assessed using the in vitro capillary migration inhibition (MI) assay. In the presence of the respective donor antigen, an appreciable degree of MI of sensitized spleen cells was observed as early as 4 days after grafting. A maximum level was attained on day 7 and this response was maintained as long as one month after grafting with only slight fluctuations in the level of MI. When the clinical manifestations of graft rejection culminated on day 35, the degree of MI was still at the maximum level. MI of allograft-sensitized spleen cells is an antigen specific event, and the same level of inhibition is observed whether the specific antigen is provided in the form of intact spleen cells or spleen extract.     

Effect of trypsin-chymotrypsin (Chymoral Forte D.S.) preparation on the modulation of cytokine levels in burn patients

The present study was carried out in burn patients administered with Trypsin-Chymotrypsin (Chymoral Forte D.S.) preparation to observe if the acute-phase protein levels in the serum are modulated through the synthesis of IL-1beta and IL-6 and if the severity of the inflammatory phase could be regulated. The effects of Trypsin-Chymotrypsin preparation on the modulation of cytokine levels particularly, IL-6 and IL-1beta were analyzed in serum samples of 15 burn patients and compared with untreated controls. Significant differences in cytokine levels (P<0.05) were observed in untreated burn patients and Trypsin-Chymotrypsin preparation treated patients. There were significant variations in serum IL-6 and IL-1beta on the day of admission and post burn day 10 in treated as well as untreated burn patients. Patients with a higher percentage of total body surface area and sepsis showed a significant increase in IL-1beta and IL-6 in the serum. An increase in serum levels of both cytokines was observed on post burn day 1 and a significant decrease was observed in Trypsin-Chymotrypsin preparation treated patients on days 7 and 10. The possible role of the inflammatory cytokines in the pathophysiology of burns is discussed.     

Tufted angioma in a case of neurofibromatosis type I

Tufted angiomas are rare benign tumours with a characteristic histopathological appearance. Their occurrence in neurofibromatosis type 1, a genodermatosis in which vascular malformations are not uncommon, has hitherto not been reported. Some of the characteristic features of tufted angiomas are discussed.     

Interferon gamma modulates trauma-induced muscle wasting and immune dysfunction

OBJECTIVE: To test the effect of burn injury in mice congenitally deficient in interferon gamma (IFN-gamma) and as well as in wild-type animals treated with IFN-gamma neutralizing antibody. SUMMARY BACKGROUND DATA: The mechanisms underlying muscle wasting following burn trauma are incompletely characterized, although the hypercatabolic state is a consequence of increased proteasomal degradation. Concurrently, burn injury results in an immunocompromised state, and subsequent infections are the leading cause of morbidity and mortality in these patients. IFN-gamma, best conceptualized as a macrophage activating protein, modulates a variety of biologic pathways potentially relevant to muscle wasting and immune dysfunction. METHODS: Mice received either a 20% total body surface area burn or a control sham treatment. At days 1, 2, and 7 following treatment, skeletal muscle, peripheral blood, and spleen were harvested from both groups. Protein synthesis and degradation rates were measured. Lymphocyte subpopulation expression of major histocompatibility complex I (MHC I) molecules was assessed by flow cytometry, and proliferation capacity was measured using mixed lymphocyte reaction. RESULTS: IFN-gamma is critically involved in burn-induced weight loss; moreover, absence of IFN-gamma virtually abolished skeletal muscle hypercatabolism following burn injury. Lymphocyte proliferation and MHC I expression in the setting of burn trauma are also normalized in the absence of IFN-gamma. Both antigen presentation and proliferation functions are independently affected. CONCLUSIONS: IFN-gamma plays a fundamental role in mediating the hypercatabolic state of multiple cell types following burn trauma.     

Experimental and Statistical Investigation to Evaluate Impact Strength Variability and Reliability of Preplaced Aggregate Concrete Containing Crumped Rubber and Fibres

The proper disposal of used rubber tires has emerged as a primary concern for the environment all over the globe. Millions of tires are thrown away, buried and discarded every year, posing a major environmental concern owing to their slow decomposition. As a result, it is advantageous to use recycled waste rubber aggregates as an additional building resource. Recycling crushed rubber would lead to a long-term solution to the problem of decreasing natural aggregate resources while conserving the environment. This study examines the impact strength variability and reliability of preplaced aggregate concrete containing crumped rubber and fibres. Ten different mixtures were prepared by replacing natural aggregate with crumped rubber (5, 10, 15 and 20%). The crumped rubber was pretreated by the water with sodium hydroxide dilution for 30 min before usage. Hooked-end steel fibres were used at a dosage of 1.5%. The compressive strength, impact strength, impact ductility index and failure pattern were examined and discussed. In addition, a statistical method called Weibull distribution is used to analyze the scattered experimental results. The results showed that when the crumb rubber content was raised, the retained first cracking and failure impact numbers increased. As a result of substituting crumb rubber for 20% of the coarse aggregate in plain and fibrous mixes, the percentage development in first crack and failure was between 33% and 76% and 75% to 129%, respectively.     

Short chain fatty acids exhibit selective estrogen receptor downregulator (SERD) activity in breast cancer

Early stage estrogen receptor α (ERα, ESR1)-positive breast cancer patients can develop more aggressive endocrine-resistant tumors that express constitutively active mutant forms of ERα including ERα-Y537S and ERα-D538G. These patients are treated with selective ER down regulators (SERDs) such as the ERα antagonist fulvestrant. Previous studies show that histone deacetylase (HDAC) inhibitors downregulate ERα and since some dietary derived short chain fatty acids (butyrate, propionate and acetate) exhibit HDAC inhibitory activity we investigated their effects as SERDs in MCF-7 and T47D cells expressing wild-type and mutant ERα-D538G and ERα-Y537S. The SCFAs exhibited SERD-like activity in both cell lines expressing wild-type and mutant ERα. The results for propionate and butyrate correlated with parallel induction of histone acetylation and this was also observed for the HDAC inhibitors Panobinostat, Vorinostat and Entinostat which also downregulated wild-type and mutant ERα and induced histone acetylation. Although acetate induced ERα degradation the mechanisms may be independent of the HDAC inhibitory activity of this compound. These results suggest that high fibre diets that induce formation of SCFAs may have some clinical efficacy for treating ER-positive endocrine resistant breast cancer patients and this is currently being investigated.     

Utility and limitations of exome sequencing in the molecular diagnosis of pediatric inherited platelet disorders

Inherited platelet disorders (IPD) are a heterogeneous group of rare disorders that affect platelet number and function and often predispose to other significant medical complications. In spite of the identification of over 50 IPD disease‐associated genes, a molecular diagnosis is only identified in a minority (10%) of affected patients without a clinically suspected etiology. We studied a cohort of 21 pediatric patients with suspected IPDs by exome sequencing (ES) to: (1) examine the performance of the exome test for IPD genes, (2) determine if this exome‐wide diagnostic test provided a higher diagnostic yield than has been previously reported, (3) to evaluate the frequency of variants of uncertain significance identified, and (4) to identify candidate variants for functional evaluation in patients with an uncertain or negative diagnosis. We established a high priority gene list of 53 genes, evaluated exome capture kit performance, and determined the coverage for these genes and disease‐related variants. We identified likely disease causing variants in 5 of the 21 probands (23.8%) and variants of uncertain significance in 52% of patients studied. In conclusion, ES has the potential to molecularly diagnose causes of IPD, and to identify candidate genes for functional evaluation. Robust exome sequencing also requires that coverage of genes known to be associated with clinical findings of interest need to be carefully examined and supplemented if necessary. Clinicians who undertake ES should understand the limitations of the test and the full significance of results that may be returned.     

Flow cytometric determination of mitochondrial membrane potential changes during apoptosis of T lymphocytic and pancreatic beta cell lines: comparison of tetramethylrhodamineethylester (TMRE), chloromethyl-X-rosamine (H2-CMX-Ros) and MitoTracker Red 580 (MTR580)

The mitochondria-specific dyes, TMRE, H2-CMX-Ros and MTR580 were determined for their suitability to measure mitochondrial potential changes of the T cell leukemia cell line Jurkat and insulin-secreting beta cell line NIT-1 during apoptosis. Both freshly harvested Jurkat and NIT-1 cells induced to undergo apoptosis displayed poor retention of the potential-sensitive, intrinsically fluorescent dye, TMRE. Treatment with formaldehyde or paraformaldehyde completely abolished TMRE uptake in both cell types regardless of apoptosis induction. Interestingly, freshly harvested apoptotic Jurkat cells exhibited lower retention of H2-CMX-Ros, indicating marked reduction in the oxidative status of lymphoid cells during apoptosis. This is in contrast to NIT-1 cells which failed to display significant reduction in H2-CMX-Ros retention after anoikis induction. Paraformaldehyde treatment reduced the retention of H2-CMX-Ros in live Jurkat cells but still allowed the discrimination of apoptotic cells which poorly retained H2-CMX-Ros. However, live Jurkat cells lost their ability to retain H2-CMX-Ros after formaldehyde treatment. In contrast, treatment with paraformaldehyde or formaldehyde did not have significant impact on the retention of H2-CMX-Ros in both live and apoptotic NIT-1 cells. The uptake of MTR580 was independent of mitochondrial membrane potential in both T and beta cell lines. However, MTR580 was comparable to H2-CMX-Ros for confocal microscopic analysis of apoptotic Jurkat cells following fixation with formaldehyde and cell permeabilization. These data demonstrate that while TMRE and H2-CMX-Ros are suitable for determining mitochondrial membrane potential changes during apoptosis in lymphoid cells, only TMRE is suitable for such analysis in beta cells. Both H2-CMX-Ros and MTR580 proved to be suitable for confocal imaging of mitochondria.     

Regulation of parasite antigen-driven immune responses by interleukin-10 (IL-10) and IL-12 in lymphatic filariasis.

We investigated the mechanisms by which interleukin-10 (IL-10) regulates antigen-specific hyporesponsiveness in asymptomatic microfilaremic (MF) individuals. Peripheral blood mononuclear cells from MF individuals (n = 11) were stimulated in vitro with Brugia malayi antigen (BMA) or mycobacterial purified protein derivative (PPD) in the presence of neutralizing anti-IL-10 or isotype control monoclonal antibodies. As expected, BMA stimulated little or no gamma interferon (IFN-gamma) secretion in MF individuals, whereas PPD stimulated IFN-gamma in all but one. Neutralization of endogenous BMA-driven IL-10 secretion led to augmentation of IFN-gamma in seven of nine MF individuals (1.5- to 10-fold) and did so in a BMA-specific manner (PPD-driven IFN-gamma was augmented in only two of eight MF individuals and only 1.5- to 2-fold), indicating that IL-10 downregulates type 1 responses in these individuals. Type 2 responses (IL-5 secretion) were unaffected by the IL-10 blockade. To assess whether IL-12 could reverse the type 1 downregulation observed, the effect of recombinant human IL-12 (rhIL-12) on BMA-driven IL-5 and IFN-gamma production was also evaluated. rhIL-12 augmented both BMA- and PPD-driven IFN-gamma production 5- to 10-fold in six of nine MF individuals. These data demonstrate that IL-10 downregulates BMA-driven type 1 responses and that IL-12 can overcome downregulation of Th1 responses associated with MF but does so in a non-antigen-specific manner.