Multivariate analysis of adaptive response to ferulic acid and p-coumaric acid after physiological stresses in Cronobacter sakazakii.

AIM: The present study demonstrated the antimicrobial activity of ferulic acid and p-coumaric acid against unstressed and stressed (cold stressed, starved and desiccated) Cronobacter sakazakii in laboratory media (37°C) and reconstituted powdered infant formulation (PIF) with mild heat treatment (50°C). METHODS AND RESULTS: Five phenolics, namely, quercetin, rutin, caffeic acid, ferulic acid and p-coumaric acid, were tested for antimicrobial activities against five strains of C. sakazakii either unstressed or stressed. Strain specific higher resistance to ferulic acid and p-coumaric acid was observed after stress adaptation in laboratory media. The effect of cross protection was validated using reconstituted PIF as delivery vehicle of selected compounds. Both p-coumaric acid and ferulic acid showed inhibition of C. sakazakii in a dose and time dependent manner as revealed by their viable cell counts. Principal component analysis revealed that the desiccated cells were more sensitive to phenolics in reconstituted PIF. CONCLUSIONS: Only ferulic acid and p-coumaric acid showed marked antibacterial activity with minimum inhibitory concentration in the range of 2·5-5 mg ml-1 for unstressed C. sakazakii cells in tryptone soy broth. The maximum inhibition was achieved with 20 mg ml-1 of both the tested polyphenols in reconstituted PIF. Cold stress and starvation stress did not impart any protection nor increased the susceptibility of C. sakazakii, whereas desiccation resulted in increased susceptibility to phenolic compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained in this study helps in understanding the effect of environmental stresses during processing on susceptibility of C. sakazakii to natural antimicrobial agents. Future transcriptomic studies and functional genetic studies are warranted to understand the strain specific stress responses for the development of better control methods possibly by using these natural antagonists.

Whole blood interleukin-2 release test to detect and characterize rare circulating gluten-specific T cell responses in coeliac disease.

Whole blood cytokine release assays (CRA) assessing cellular immunity to gluten could simplify the diagnosis and monitoring of coeliac disease (CD). We aimed to determine the effectiveness of electrochemiluminescence CRA to detect responses to immunodominant gliadin peptides. HLA-DQ2·5+ CD adults (cohort 1, n = 6; cohort 2, n = 12) and unaffected controls (cohort 3, n = 9) were enrolled. Cohort 1 had 3-day gluten challenge (GC). Blood was collected at baseline, and for cohort 1 also at 3 h, 6 h and 6 days after commencing 3-day GC. Gliadin peptide-stimulated proliferation, interferon (IFN)-γ enzyme-linked immunospot (ELISPOT) and 14- and 3-plex electrochemiluminescence CRA were performed. Poisson distribution analysis was used to estimate responding cell frequencies. In cohort 1, interleukin (IL)-2 dominated the gliadin peptide-stimulated cytokine release profile in whole blood. GC caused systemic IL-2 release acutely and increased gliadin peptide-stimulated IFN-γ ELISPOT and whole blood CRA responses. Whole blood CRA after GC was dominated by IL-2, but also included IFN-γ, C-X-C motif chemokine ligand 10/IFN-γ-induced protein 10 (CXCL10/IP-10), CXCL9/monokine induced by IFN-γ (MIG), IL-10, chemokine (C-C motif) ligand 3/macrophage inflammatory protein 1-alpha (CCL3/MIP-1α), TNF-α and IL-8/CXCL8. In cohorts 2 and 3, gliadin peptide-stimulated whole blood IL-2 release was 100% specific and 92% sensitive for CD patients on a gluten-free diet; the estimated frequency of cells in CD blood secreting IL-2 to α-gliadin peptide was 0·5 to 11 per ml. Whole blood IL-2 release successfully mapped human leucocyte antigen (HLA)-DQ2·5-restricted epitopes in an α-gliadin peptide library using CD blood before and after GC. Whole blood IL-2 release assay using electrochemiluminescence is a sensitive test for rare gliadin-specific T cells in CD, and could aid in monitoring and diagnosis. Larger studies and validation with tetramer-based assays are warranted.

Serum cytokines elevated during gluten-mediated cytokine release in coeliac disease.

Cytokines have been extensively studied in coeliac disease, but cytokine release related to exposure to gluten and associated symptoms has only recently been described. Prominent, early elevations in serum interleukin (IL)-2 after gluten support a central role for T cell activation in the clinical reactions to gluten in coeliac disease. The aim of this study was to establish a quantitative hierarchy of serum cytokines and their relation to symptoms in patients with coeliac disease during gluten-mediated cytokine release reactions. Sera were analyzed from coeliac disease patients on a gluten free-diet (n = 25) and from a parallel cohort of healthy volunteers (n = 25) who underwent an unmasked gluten challenge. Sera were collected at baseline and 2, 4 and 6 h after consuming 10 g vital wheat gluten flour; 187 cytokines were assessed. Confirmatory analyses were performed by high-sensitivity electrochemiluminescence immunoassay. Cytokine elevations were correlated with symptoms. Cytokine release following gluten challenge in coeliac disease patients included significant elevations of IL-2, chemokine (C-C motif) ligand 20 (CCL20), IL-6, chemokine (C-X-C motif) ligand (CXCL)9, CXCL8, interferon (IFN)-γ, IL-10, IL-22, IL-17A, tumour necrosis factor (TNF)-α, CCL2 and amphiregulin. IL-2 and IL-17A were earliest to rise. Peak levels of cytokines were generally at 4 h. IL-2 increased most (median 57-fold), then CCL20 (median 10-fold). Cytokine changes were strongly correlated with one another, and the most severely symptomatic patients had the highest elevations. Early elevations of IL-2, IL-17A, IL-22 and IFN-γ after gluten in patients with coeliac disease implicates rapidly activated T cells as their probable source. Cytokine release after gluten could aid in monitoring experimental treatments and support diagnosis.

A method for early detection of antibiotic resistance in positive blood cultures: experience from an oncology centre in eastern India.

PURPOSE: For antibiotic susceptibility results, conventional culture and sensitivity methods takes 48 hours after a blood culture is flagged positive by automated systems. Early initiation of targeted antibiotic therapy is essential for effective management of sepsis to reduce morbidity, mortality, cost of treatment and prevent antibiotic resistance. Objective of this study was to evaluate Direct Sensitivity Test (DST) as a potential tool to get reliable antibiotic susceptibility results 24 hours earlier. MATERIALS AND METHODS: Blood cultures flagged positive between May 2011 to December 2012 by BacT/ALERT were Gram stained. All uni-microbial gram-negative blood cultures were simultaneously cultured and processed for DST from broth using disk diffusion method using British Society of Antimicrobial Chemotherapy (BSAC) guidelines. DST results available next day were compared with conventional antibiotic susceptibility test (AST) performed by Vitek-2 on isolated colonies. Results of DST (test method) and AST (reference method) were compared for agreements or errors. RESULTS: Of the 840 antibiotic gram-negative organism combinations tested, Categorical and essential agreements were 83.7% and 96.2% respectively. Minor, major and very major errors were 12.5%, 3.33% and 0.47%, respectively. CONCLUSIONS: DST using disk diffusion from positive blood culture broths helps to initiate early targeted antibiotic therapy. There is high concordance between DST and AST.

Helminth infection reactivates latent γ-herpesvirus via cytokine competition at a viral promoter.

Mammals are coinfected by multiple pathogens that interact through unknown mechanisms. We found that helminth infection, characterized by the induction of the cytokine interleukin-4 (IL-4) and the activation of the transcription factor Stat6, reactivated murine γ-herpesvirus infection in vivo. IL-4 promoted viral replication and blocked the antiviral effects of interferon-γ (IFNγ) by inducing Stat6 binding to the promoter for an important viral transcriptional transactivator. IL-4 also reactivated human Kaposi's sarcoma-associated herpesvirus from latency in cultured cells. Exogenous IL-4 plus blockade of IFNγ reactivated latent murine γ-herpesvirus infection in vivo, suggesting a "two-signal" model for viral reactivation. Thus, chronic herpesvirus infection, a component of the mammalian virome, is regulated by the counterpoised actions of multiple cytokines on viral promoters that have evolved to sense host immune status.

Effect of supplementing coconut or krabok oil, rich in medium-chain fatty acids on ruminal fermentation, protozoa and archaeal population of bulls.

Medium-chain fatty acids (MCFA), for example, capric acid (C10:0), myristic (C14:0) and lauric (C12:0) acid, have been suggested to decrease rumen archaeal abundance and protozoal numbers. This study aimed to compare the effect of MCFA, either supplied through krabok (KO) or coconut (CO) oil, on rumen fermentation, protozoal counts and archaeal abundance, as well as their diversity and functional organization. KO contains similar amounts of C12:0 as CO (420 and 458 g/kg FA, respectively), but has a higher proportion of C14:0 (464 v. 205 g/kg FA, respectively). Treatments contained 35 g supplemental fat per kg DM: a control diet with tallow (T); a diet with supplemental CO; and a diet with supplemental KO. A 4th treatment consisted of a diet with similar amounts of MCFA (i.e. C10:0+C12:0+C14:0) from CO and KO. To ensure isolipidic diets, extra tallow was supplied in the latter treatment (KO+T). Eight fistulated bulls (two bulls per treatment), fed a total mixed ration predominantly based on cassava chips, rice straw, tomato pomace, rice bran and soybean meal (1.5% of BW), were used. Both KO and CO increased the rumen volatile fatty acids, in particular propionate and decreased acetate proportions. Protozoal numbers were reduced through the supplementation of an MCFA source (CO, KO and KO+T), with the strongest reduction by KO. Quantitative real-time polymerase chain reaction assays based on archaeal primers showed a decrease in abundance of Archaea when supplementing with KO and KO+T compared with T and CO. The denaturing gradient gel electrophoresis profiles of the rumen archaeal population did not result in a grouping of treatments. Richness indices were calculated from the number of DGGE bands, whereas community organization was assessed from the Pareto-Lorenz evenness curves on the basis of DGGE band intensities. KO supplementation (KO and KO+T treatments) increased richness and evenness within the archaeal community. Further research including methane measurements and productive animals should elucidate whether KO could be used as a dietary methane mitigation strategy.

Succinate is an inflammatory signal that induces IL-1ß through HIF-1α.

Macrophages activated by the Gram-negative bacterial product lipopolysaccharide switch their core metabolism from oxidative phosphorylation to glycolysis. Here we show that inhibition of glycolysis with 2-deoxyglucose suppresses lipopolysaccharide-induced interleukin-1ß but not tumour-necrosis factor-α in mouse macrophages. A comprehensive metabolic map of lipopolysaccharide-activated macrophages shows upregulation of glycolytic and downregulation of mitochondrial genes, which correlates directly with the expression profiles of altered metabolites. Lipopolysaccharide strongly increases the levels of the tricarboxylic-acid cycle intermediate succinate. Glutamine-dependent anerplerosis is the principal source of succinate, although the 'GABA (γ-aminobutyric acid) shunt' pathway also has a role. Lipopolysaccharide-induced succinate stabilizes hypoxia-inducible factor-1α, an effect that is inhibited by 2-deoxyglucose, with interleukin-1ß as an important target. Lipopolysaccharide also increases succinylation of several proteins. We therefore identify succinate as a metabolite in innate immune signalling, which enhances interleukin-1ß production during inflammation.

Chondrolipoma of tongue.

Although lipomas can occur in any part of the body, they are found infrequently in the oral cavity. Variants of lipoma have been described according to the type of tissue present; a lipoma with cartilaginous metaplasia is a chondrolipoma which is a rare variant. We describe a chondrolipoma of tongue in a 36 year old lady.

Superficial angiomyxoma of the external ear not associated with Carney's complex: a case report.

Superficial angiomyxomas are rare tumours and multiple tumours occurring in the external ear are invariably associated with Carney's complex. In the present study, a solitary superficial angiomyxoma was found in a 20 year old lady; and there was no evidence of any of the components of Carney's complex at the time of presentation or at the end of 2 years of follow-up, after the surgical excision of the tumour.

Regulation of glycolysis in Lactococcus lactis: an unfinished systems biological case study.

The unexpectedly long, and still unfinished, path towards a reliable mathematical model of glycolysis and its regulation in Lactococcus lactis is described. The model of this comparatively simple pathway was to be deduced from in vivo nuclear magnetic resonance time-series measurements of the key glycolytic metabolites. As to be expected from any nonlinear inverse problem, computational challenges were encountered in the numerical determination of parameter values of the model. Some of these were successfully solved, whereas others are still awaiting improved techniques of analysis. In addition, rethinking of the model formulation became necessary, because some generally accepted assumptions during model design are not necessarily valid for in vivo models. Examples include precursor-product relationships and the homogeneity of cells and their responses. Finally, it turned out to be useful to model only some of the metabolites, while using time courses of ubiquitous compounds such as adenosine triphosphate, inorganic phosphate, nicotinamide adenine dinucleotide (oxidised) and nicotinamide adenine dinucleotide (reduced) as unmodelled input functions. With respect to our specific application, the modelling process has come a long way, but it is not yet completed. Nonetheless, the model analysis has led to interesting insights into the design of the pathway and into the principles that govern its operation. Specifically, the widely observed feedforward activation of pyruvate kinase by fructose 1,6-bisphosphate is shown to provide a crucial mechanism for positioning the starving organism in a holding pattern that allows immediate uptake of glucose, as soon as it becomes available.

Transrectal fine needle aspiration of prostate.

56 patients with prostatic enlargement were subjected to transrectal fine needle aspiration (FNA) of prostate. The cytologic diagnosis was correlated with histology in 55 cases. Benign prostatic hyperplasia (BPH) was diagnosed in 44 cases. In 18 of these, there was associated chronic prostatitis in three cases acute prostatitis and in two cases granulomatous prostatitis. One case of tuberculous prostatitis and 11 cases of prostatic carcinoma could also be diagnosed on cytologic basis. There was only one false negative and no false positive cytodiagnosis was made.

Fine needle aspiration cytologic diagnosis of the solitary cold thyroid nodule. Comparison with ultrasonography, radionuclide perfusion study and xeroradiography.

Thirty-six cases of solitary and scintigraphically "cold" thyroid nodules were studied by fine needle aspiration (FNA) cytology, ultrasonography, radionuclide perfusion study (RPS) and xeroradiography with the aim of differentiating the neoplastic from the nonneoplastic nodules. Histologic study of the excised specimens provided the definitive diagnosis in all cases. Of the techniques used in this study, FNA cytology and RPS had the highest sensitivities and specificities. Ultrasonography and xeroradiography were of limited use due to their low sensitivity rates.

Low fertility rate in vasovasostomized males and its possible immunologic mechanism.

Contradictory views have been expressed about the role of the various antisperm antibodies which develop after vasoligation. The present study was conducted in 50 normal fertile males, 50 vasectomized subjects and 25 subjects after recanalization of their vas deferens in order to investigate the development of various anti-sperm antibodies after vasectomy, along with their incidence, their persistence after successful relief of vaso-obstruction by vasovasostomy and their role in the causation of infertility in vasoanatomized normospermia males. Sperm agglutinating, immobilizing and haemagglutinating antibodies showed rises in titres with increase during the post-vasectomy period, indicating continuous antigenic stimulus. Age, post-operative complications and blood group did not seem to alter the results. 86% of subjects developed antisperm agglutinins, mostly tail-to-tail type (54.5%), 1-12 years after vasoligation, while only 2% of fertile men had circulating spermagglutinins. A lower incidence of positive sperm in the immobilization test than in the agglutination test suggests either that different antibodies are detected by these two tests or these tests have differing sensitivities. Of the 25 vasovasostomized subject, 13 (52%) cases became normospermic and 4 (16%) oligospermic while 8 (32%) remained azoospermics. Except for 3 oligospermic subjects, all had circulating spermagglutinins. Among the 13 normospermic vasovasostomized persons, a significant correlation was found between the titres of circulating antisperm agglutinins and autoagglutination of spermatozoa in their ejaculates; and also between the sperm immobilization values of their sera and the degree of their sperm motility. Three normospermic recanalized men, having low levels of sperm agglutinins and haemagglutinins with normal seminogram and no sperm immobilizing antibody, successfully impregnated their wives. Another 10 vasovasotomized infertile subjects had sperm agglutinins in significant titre; 5 showed positive sperm immobilization values, a similar number showed autoagglutination of sperm, while a decreased degree of motility of sperms was noted in 6 cases. Thus there was a significant correlation between the titres of anti-sperm antibodies and autoagglutination of spermatozoa, which might be an important cause of male infertility after successful anatomic relief of vasoobstruction. Histological studies of testicular biopsy showed normal spermatogenesis in azoospermic recanalized subjects, although they had high levels of antisperm antibodies. This suggests that these antibodies do not affect normal spermatogenesis, and sperm counts.

PIP: A study was undertaken in light of the contradictory views concerning the role of various antisperm antibodies which develop following vasoligation. 50 normal fertile males, 50 vasectomized males, and 25 males with recanalized vas deferens were observed. Sperm agglutinating, immobilizing, and hemagglutinating antibodies showed rises in titers with increase during the postvasectomy period indicating continuous antigenic stimulus. 86% of subjects developed antisperm agglutinins 1-12 years after vasoligation while only 2% of fertile men had circulating spermagglutinins. Of the 25 vasovasostomized subjects 13 (52%) became normospermic and 4(16%) oligospermic while 32% remained azoospermic. All had circulating spermagglutinins except for 3 with oli gospermia. A significant correlation was found between the titers of ci rculating antisperm agglutinins and autoagglutinin of spermatozoa in their ejaculates and between sperm immobilization values of their sera and the degree of sperm motility among the 13 normospermic vasovasostomized subjects. 3 normospermic recanalized men successfully impregnated their wives. 10 vasovasostomized infertile subjects had sperm agglutinins in significant titers; 5 had positive sperm immobilization values; a similar number showed autoagglutination of sperm and 6 had a decreased degree of sperm motility. Histological studies of testicular biopsy showing normal spermatogenesis in azoospermic recanalized subjects despite high levels of antisperm antibodies suggest that these antibodies fail to affect normal spermatogenesis and sperm counts.