CD95-Mediated Calcium Signaling Promotes T Helper 17 Trafficking to Inflamed Organs in Lupus-Prone Mice.

CD95 ligand (CD95L) is expressed by immune cells and triggers apoptotic death. Metalloprotease-cleaved CD95L (cl-CD95L) is released into the bloodstream but does not trigger apoptotic signaling. Hence, the pathophysiological role of cl-CD95L remains unclear. We observed that skin-derived endothelial cells from systemic lupus erythematosus (SLE) patients expressed CD95L and that after cleavage, cl-CD95L promoted T helper 17 (Th17) lymphocyte transmigration across the endothelial barrier at the expense of T regulatory cells. T cell migration relied on a direct interaction between the CD95 domain called calcium-inducing domain (CID) and the Src homology 3 domain of phospholipase Cγ1. Th17 cells stimulated with cl-CD95L produced sphingosine-1-phosphate (S1P), which promoted endothelial transmigration by activating the S1P receptor 3. We generated a cell-penetrating CID peptide that prevented Th17 cell transmigration and alleviated clinical symptoms in lupus mice. Therefore, neutralizing the CD95 non-apoptotic signaling pathway could be an attractive therapeutic approach for SLE treatment.

Effects of in ovo injection of bacterial peptides and CpG-ODN on Salmonella enterica serovar Heidelberg infection in specific pathogen-free (SPF) chicks.

RESEARCH HIGHLIGHTS: Peptides + CpG-ODN reduced SH in caeca at the first week post-infection.Administered formulations did not reduce SH-faecal excretion.Levels of intestinal IgA were similar between all groups.CpG-ODN improved some parameters associated with chick intestinal health.

Whole-genome sequencing and gene sharing network analysis powered by machine learning identifies antibiotic resistance sharing between animals, humans and environment in livestock farming.

Anthropogenic environments such as those created by intensive farming of livestock, have been proposed to provide ideal selection pressure for the emergence of antimicrobial-resistant Escherichia coli bacteria and antimicrobial resistance genes (ARGs) and spread to humans. Here, we performed a longitudinal study in a large-scale commercial poultry farm in China, collecting E. coli isolates from both farm and slaughterhouse; targeting animals, carcasses, workers and their households and environment. By using whole-genome phylogenetic analysis and network analysis based on single nucleotide polymorphisms (SNPs), we found highly interrelated non-pathogenic and pathogenic E. coli strains with phylogenetic intermixing, and a high prevalence of shared multidrug resistance profiles amongst livestock, human and environment. Through an original data processing pipeline which combines omics, machine learning, gene sharing network and mobile genetic elements analysis, we investigated the resistance to 26 different antimicrobials and identified 361 genes associated to antimicrobial resistance (AMR) phenotypes; 58 of these were known AMR-associated genes and 35 were associated to multidrug resistance. We uncovered an extensive network of genes, correlated to AMR phenotypes, shared among livestock, humans, farm and slaughterhouse environments. We also found several human, livestock and environmental isolates sharing closely related mobile genetic elements carrying ARGs across host species and environments. In a scenario where no consensus exists on how antibiotic use in the livestock may affect antibiotic resistance in the human population, our findings provide novel insights into the broader epidemiology of antimicrobial resistance in livestock farming. Moreover, our original data analysis method has the potential to uncover AMR transmission pathways when applied to the study of other pathogens active in other anthropogenic environments characterised by complex interconnections between host species.

The Extraperitoneal Approach to Left-Sided Colorectal Resections: A Human Cadaveric Study.

INTRODUCTION: Technical challenges during laparoscopic and robotic anterior resection include identification of key retroperitoneal structures and obtaining clear views of the inferior mesenteric artery (IMA) pedicle and total mesorectal excision (TME) plane. Steep head-down position improves surgical exposure but is associated with cerebral oedema, high intrapulmonary pressures, and rare neurological complications. In this article we describe the key steps of an anterior resection performed via the extra-peritoneal (XP) space in the supine position. METHODS: The technique of same-side lateral-to-medial XP dissection has been developed and refined in serial cadaveric workshops. A standard periumbilical port is inserted for initial laparoscopic exploration. Dissection is then performed in the left XP space via a 5 cm skin incision (later used as the extraction site) to allow for insertion of four (latterly three) working ports. The colon is mobilized along its lateral attachments, reflecting retroperitoneal structures down and away. The IMA pedicle is taken proximally, next to the duodenum. If required, TME dissection can be continued in the same plane. A short intraperitoneal phase is then required to complete the procedure. RESULTS: Eight cadavers were studied (seven males; median 78 y). Four operations were performed laparoscopically and four robotically. Excellent views of the key retroperitoneal structures were achieved early in the procedure. Anatomical identification was performed sequentially for left-sided structures-psoas tendon, gonadal vessel, ureter, common iliac artery, IMA, and duodenum before ligation of the IMA pedicle. High ligation of IMA on the aorta and splenic flexure mobilization were performed in all eight procedures. CONCLUSIONS: This novel study shows it is feasible to perform the key steps of an anterior resection using the XP space in the supine position. This will reduce the need for steep head-down positioning which may have meaningful clinical benefits. Prospective clinical studies are required to validate the technique within a patient population.

Salmonella Heidelberg side-step gene loss of respiratory requirements in chicken infection model.

Among the important recent observations involving anaerobic respiration was that an electron acceptor produced as a result of an inflammatory response to Salmonella Typhimurium generates a growth advantage over the competing microbiota in the lumen. In this regard, anaerobically, salmonellae can oxidize thiosulphate (S2O32-) converting it into tetrathionate (S4O62-), the process by which it is encoded by ttr gene cluster (ttrSRttrBCA). Another important pathway under aerobic or anaerobic conditions is the 1,2-propanediol-utilization mediated by the pdu gene cluster that promotes Salmonella expansion during colitis. Therefore, we sought to compare in this study, whether Salmonella Heidelberg strains lacking the ttrA, ttrApduA, and ttrACBSR genes experience a disadvantage during cecal colonization in broiler chicks. In contrast to expectations, we found that the gene loss in S. Heidelberg potentially confers an increase in fitness in the chicken infection model. These data argue that S. Heidelberg may trigger an alternative pathway involving the use of an alternative electron acceptor, conferring a growth advantage for S. Heidelberg in chicks.

Heat stress impairs egg production in commercial laying hens infected by fowl typhoid.

Salmonella Gallinarum (SG) is an avian-restricted pathogen that causes fowl typhoid in poultry. Although it has been reported frequently over many decades in poultry flocks worldwide, the microorganism is more commonly associated with poultry in developing countries, particularly those with high ambient temperatures, where the acute form of the disease results in considerable economic losses. A more detailed investigation of environmental factors that affect the course of disease may assist in identifying effective prevention and control measures. Heat stress is known to impair the immunological response to a variety of pathogens and clearly may be an important contributory factor in the prevalence of disease in countries with warm or hot climates. Thus, the objective of the present study was to evaluate the effects of heat stress on chickens infected with SG. For this, light and semi-heavy commercial laying hens were distributed randomly within four groups as follows: infected and non-infected groups in rooms held at ambient temperature, and infected and non-infected groups under heat stress. Clinical signs, egg production, and mortality were recorded daily. Bacteriological counts in liver and spleen samples were estimated at 2, 5, 7, and 14 days post-infection. The results showed that both SG infection and heat stress had similar effects on egg production and a synergistic effect of the two stressors was observed. The data show an interaction between disease and heat stress which could point towards environmental and biosecurity approaches to resolving the possible 30% fall in production observed in such countries.

Diagnosis of sub-clinical coccidiosis in fast growing broiler chickens by MicroRNA profiling.

Coccidiosis in broiler chickens, caused by infection with Eimeria spp. remains one of the most economically important production diseases. Development of a genetic biomarker panel of sub-clinical infection would be an important biological tool for the management of broiler flocks. We analysed expression of MicroRNAs (miRNAs) to determine the potential for these in diagnosing coccidiosis in broiler flocks. miRNA expression, in the ilea of Ross 308 broilers, was compared between chickens naturally clinically or sub-clinically infected with Eimeria maxima and Eimeria acervulina using NextSeq 500 sequencing. 50 miRNAs with greatest coefficient of variance were determined and principal component analysis showed that these miRNAs clustered within the clinical and sub-clinical groups much more closely than uninfected controls. Following false detection rate analysis and quantitative PCR we validated 3 miRNAs; Gallus gallus (gga)-miR-122-5p, gga-miR-205b and gga-miR-144-3p, which may be used to diagnose sub-clinical coccidiosis.

Immunomodulation by vasoactive intestinal peptide is associated with increased survival and growth of Salmonella Typhimurium in mice.

Studies have shown that administration of vasoactive intestinal peptide (VIP) in mice rescues them from lethal endotoxaemia and that this is correlated with decreased concentration of inflammatory cytokines. VIP has, therefore, been proposed as a novel anti-inflammatory which could be used in the treatment of Gram negative sepsis. However, the effect of VIP has not been reported in mice infected with viable Gram negative bacteria. Here, we show that Salmonella enterica serovar Typhimurium 4/74 significantly increased expression of mRNA of a type 1 receptor (VPAC1) for anti-inflammatory vasoactive intestinal peptide (VIP) in murine ileum and mesenteric lymph nodes at day 6 post-infection (d6 pi) and in the spleen at d3 pi. When VIP (5 nmol/ml) was administered to S. Typhimurium-infected mice, there was a significant increase in the number of S. Typhimurium cultured from murine faeces and ileum at d3 and 6 pi and in MLN and spleen at d3 dpi, compared to faeces and tissues examined from mice infected with S. Typhimurium (without VIP administration). Administration of VIP to S. Typhimurium-infected mice also altered the splenic architecture, resulting in a lack of discernable periarterial lymphoid sheaths or marginal zones at d6 pi but liver histology appeared similar on both d3 and d6 pi. The effects of VIP administration were correlated with a significant decrease in expression of inflammatory cytokine mRNA, associated with systemic inflammatory response syndrome (SIRS) of bacteraemia and acute sepsis. We conclude that VIP inhibits expression of diagnostic/prognostic cytokine biomarkers of sepsis in S. Typhimurium-infected mice. However, this occurred with a concomitant increase in Salmonella growth in tissues and increased bacterial shedding in faeces. Thus, VIP may have potential as an adjunctive therapy to antibiotics in sepsis.

Viruses of protozoan parasites and viral therapy: Is the time now right?

Infections caused by protozoan parasites burden the world with huge costs in terms of human and animal health. Most parasitic diseases caused by protozoans are neglected, particularly those associated with poverty and tropical countries, but the paucity of drug treatments and vaccines combined with increasing problems of drug resistance are becoming major concerns for their control and eradication. In this climate, the discovery/repurposing of new drugs and increasing effort in vaccine development should be supplemented with an exploration of new alternative/synergic treatment strategies. Viruses, either native or engineered, have been employed successfully as highly effective and selective therapeutic approaches to treat cancer (oncolytic viruses) and antibiotic-resistant bacterial diseases (phage therapy). Increasing evidence is accumulating that many protozoan, but also helminth, parasites harbour a range of different classes of viruses that are mostly absent from humans. Although some of these viruses appear to have no effect on their parasite hosts, others either have a clear direct negative impact on the parasite or may, in fact, contribute to the virulence of parasites for humans. This review will focus mainly on the viruses identified in protozoan parasites that are of medical importance. Inspired and informed by the experience gained from the application of oncolytic virus- and phage-therapy, rationally-driven strategies to employ these viruses successfully against parasitic diseases will be presented and discussed in the light of the current knowledge of the virus biology and the complex interplay between the viruses, the parasite hosts and the human host. We also highlight knowledge gaps that should be addressed to advance the potential of virotherapy against parasitic diseases.

Reduction of Salmonella contamination on the surface of chicken skin using bacteriophage.

BACKGROUND: Enteric infections caused by Salmonella spp. remain a major public health burden worldwide. Chickens are known to be a major reservoir for this zoonotic pathogen. The presence of Salmonella in poultry farms and abattoirs is associated with financial costs of treatment and a serious risk to human health. The use of bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization of chickens could be reduced. In a prior study, phages Eϕ151 and Tϕ7 significantly reduced broiler chicken caecal colonization by S. Enteritidis and S. Typhimurium respectively. METHODS: Salmonella-free Ross broiler chickens were orally infected with S. Enteritidis P125109 or S. Typhimurium 4/74. After 7 days of infection, the animals were euthanased, and 25cm2 sections of skin were collected. The skin samples were sprayed with a phage suspension of either Eϕ151 (S. Enteritidis), Tϕ7 phage suspension (S. Typhimurium) or SM buffer (Control). After incubation, the number of surviving Salmonellas was determined by direct plating and Most Probable Number (MPN). To determine the rate of reduction of Salmonella numbers on the skin surface, a bioluminescent S. Typhimurium DT104 strain was cultured, spread on sections of chicken breast skin, and after spraying with a Tϕ11 phage suspension, skin samples were monitored using photon counting for up to 24 h. RESULTS: The median levels of Salmonella reduction following phage treatment were 1.38 log10 MPN (Enteritidis) and 1.83 log10 MPN (Typhimurium) per skin section. Treatment reductions were significant when compared with Salmonella recovery from control skin sections treated with buffer (p < 0.0001). Additionally, significant reduction in light intensity was observed within 1 min of phage Tϕ11 spraying onto the skin contaminated with a bioluminescent Salmonella recombinant strain, compared with buffer-treated controls (p < 0.01), implying that some lysis of Salmonella was occurring on the skin surface. CONCLUSIONS: The results of this study suggest that phages may be used on the surface of chicken skin as biocontrol agents against Salmonella infected broiler chicken carcasses. The rate of bioluminescence reduction shown by the recombinant Salmonella strain used supported the hypothesis that at least some of the reduction observed was due to lysis occurred on the skin surface.

Reviving Phage Therapy for the Treatment of Cholera.

Cholera remains a major risk in developing countries, particularly after natural or man-made disasters. Vibrio cholerae El Tor is the most important cause of these outbreaks, and is becoming increasingly resistant to antibiotics, so alternative therapies are urgently needed. In this study, a single bacteriophage, Phi_1, was used to control cholera prophylactically and therapeutically in an infant rabbit model. In both cases, phage-treated animals showed no clinical signs of disease, compared with 69% of untreated control animals. Bacterial counts in the intestines of phage-treated animals were reduced by up to 4 log10 colony-forming units/g. There was evidence of phage multiplication only in animals that received a V. cholerae challenge. No phage-resistant bacterial mutants were isolated from the animals, despite extensive searching. This is the first evidence that a single phage could be effective in the treatment of cholera, without detectable levels of resistance. Clinical trials in human patients should be considered.

Immune evasion by Salmonella: exploiting the VPAC1/VIP axis in human monocytes.

Immune evasion is a critical survival mechanism for bacterial colonization of deeper tissues and may lead to life-threatening conditions such as endotoxaemia and sepsis. Understanding these immune evasion pathways would be an important step for the development of novel anti-microbial therapeutics. Here, we report a hitherto unknown mechanism by which Salmonella exploits an anti-inflammatory pathway in human immune cells to obtain survival advantage. We show that Salmonella enterica serovar Typhimurium strain 4/74 significantly (P < 0·05) increased expression of mRNA and surface protein of the type 1 receptor (VPAC1) for anti-inflammatory vasoactive intestinal peptide (VIP) in human monocytes. However, we also show that S. Typhimurium induced retrograde recycling of VPAC1 from early endosomes to Rab11a-containing sorting endosomes, associated with the Golgi apparatus, and anterograde trafficking via Rab3a and calmodulin 1. Expression of Rab3a and calmodulin 1 were significantly increased by S. Typhimurium infection and W-7 (calmodulin antagonist) decreased VPAC1 expression on the cell membrane while CALP-1 (calmodulin agonist) increased VPAC1 expression (P < 0·05). When infected monocytes were co-cultured with VIP, a significantly higher number of S. Typhimurium were recovered from these monocytes, compared with S. Typhimurium recovered from monocytes cultured only in cell media. We conclude that S. Typhimurium infection exploits host VPAC1/VIP to gain survival advantage in human monocytes.

The SseL protein inhibits the intracellular NF-κB pathway to enhance the virulence of Salmonella Pullorum in a chicken model.

To persist in the host, Salmonella is known to facultatively parasitize cells to escape the immune response. Intracellular Salmonella enterica can replicate using effector proteins translocated across the Salmonella-containing vacuolar membrane via a type III secretion system (T3SS) encoded by Salmonella pathogenicity island-2 (SPI-2). One of these factors, Salmonella secreted factor L (SseL), is a deubiquitinase that contributes to the virulence of Salmonella Typhimurium in mice by inhibiting the cellular NF-κB inflammatory pathway. However, the nature of its effect on the NF-κB pathway is controversial, and little research has been performed in other animal models. In this study, the SseL of Salmonella Pullorum was studied, and chickens were used as an infection model. An sseL gene deletion strain, a complementation strain and a eukaryotic expression plasmid were used to clarify the means by which SseL regulates Salmonella virulence and the cellular inflammatory response. SseL significantly enhanced the virulence of Salmonella Pullorum in chickens and suppressed activation of the cellular NF-κB pathway, thus inhibiting cellular inflammatory cytokine expression.

Confirmation that somatic mutations of beta-2 microglobulin correlate with a lack of recurrence in a subset of stage II mismatch repair deficient colorectal cancers from the QUASAR trial.

AIMS: Beta2-microglobulin (B2M) forms part of the HLA class I complex and plays a role in metastatic biology. B2M mutations occur frequently in mismatch repair-deficient colorectal cancer (dMMR CRC), with limited data suggesting they may protect against recurrence. Our experimental study tested this hypothesis by investigating B2M mutation status and B2M protein expression and recurrence in patients in the stage II QUASAR clinical trial. METHODS AND RESULTS: Sanger sequencing was performed for the three coding exons of B2M on 121 dMMR and a subsample of 108 pMMR tumours; 52 with recurrence and 56 without. B2M protein expression was assessed by immunohistochemistry. Mutation status and protein expression were correlated with recurrence and compared to proficient mismatch repair (pMMR) CRCs. Deleterious B2M mutations were detected in 39 of 121 (32%) dMMR tumours. Five contained missense B2M-variants of unknown significance, so were excluded from further analyses. With median follow-up of 7.4 years, none of the 39 B2M-mutant tumours recurred, compared with 14 of 77 (18%) B2M-wild-type tumours (P = 0.005); six at local and eight at distant sites. Sensitivity and specificity of IHC in detecting B2M mutations was 87 and 71%, respectively. Significantly (P < 0.0001) fewer (three of 104, 2.9%) of the 108 pMMR CRCs demonstrated deleterious B2M mutations. One pMMR tumour, containing a frameshift mutation, later recurred. CONCLUSION: B2M mutations were detected in nearly one-third of dMMR cancers, none of which recurred. B2M mutation status has potential clinical utility as a prognostic biomarker in stage II dMMR CRC. The mechanism of protection against recurrence and whether this protection extends to stage III disease remains unclear.

Differential immune response to Eimeria maxima infection in fast- and slow-growing broiler genotypes.

Very little has been reported comparing resistance to coccidiosis in fast or slow growing broilers, the latter of which are becoming more prevalent in the broiler industry. We examined mRNA expression in the intestines of fast and slow growing broilers following Eimeria infection. We show that by day 13 post-infection (d pi) with 2500 or 7000 oocysts of Eimeria maxima, slower-growing (Ranger Classic) broilers significantly (P < 0.01) upregulated expression of proinflammatory cyclooxygenase genes (LTB4DH, PTSG1 and PTSG2) above that detected in fast growing (Ross 308) broilers. Expression of CD8α mRNA was downregulated in Ross 308 at day 6d pi with either 2500 or 7000 oocysts of E maxima (P < 0.05), compared to uninfected controls, but was not differentially expressed in Ranger Classic. CD4 genes were not differentially expressed in either chicken line infected with either infectious oocyst dose at d6 pi, compared to uninfected controls. However, at d13 pi, CD4 expression was significantly upregulated in both chicken lines infected with either infectious oocyst dose, compared to uninfected controls (P < 0.05) but this was significantly greater in Ranger Classic broilers compared to Ross 308 (P < 0.05). At d13 pi, expression of CD3 chains (required for T lymphocyte activation) was significantly increased in Ranger Classic compared to Ross 308, infected with either oocyst dose (P < 0.05-0.01). Expression of IL-2 and IL-15 mRNA, required for T lymphocyte proliferation was also significantly upregulated, or maintained longer, in Ranger Classic broilers compared to Ross 308. These differences in immune response to E maxima corresponded with a reduction in E maxima genome detected in the intestines of Ranger Classic compared to Ross 308.

Model of Persistent Salmonella Infection: Salmonella enterica Serovar Pullorum Modulates the Immune Response of the Chicken from a Th17-Type Response towards a Th2-Type Response.

Salmonella enterica infection affects a wide range of animals and humans, and a small number of serovars cause typhoid-like infections, one characteristic of which is persistent infection in convalescents. Avian-specific S. enterica serovar Pullorum produces systemic disease in young chickens, which is followed by a carrier state in convalescent birds, leading to infection of the ovary at sexual maturity and vertical transmission. However, the immunological basis of persistent infection remains unclear. S. enterica serovar Enteritidis is taxonomically closely related but does not show this characteristic. Differences in the immune responses between S Pullorum and S Enteritidis were compared by using Salmonella-infected chicken monocyte-derived macrophages (chMDMs) and CD4+ T lymphocytes that had been cocultured with infected chMDMs or chicken splenocytes in vitro and also in 2-day-old chickens in vivo In comparison with S Enteritidis, S Pullorum-infected chMDMs showed reduced mRNA expression levels of interleukin-12α (IL-12α) and IL-18 and stimulated the proliferation of Th2 lymphocytes, with reduced expression of gamma interferon (IFN-γ) and IL-17 and increased expression levels of IL-4 and IL-13 There was little evidence of clonal anergy or immune suppression induced by S Pullorum in vitro. S Pullorum also increased the levels of expression of IL-4 and decreased the levels of IFN-γ in the spleen and cecal tonsil of infected birds. This suggests that S Pullorum is able to modulate host immunity from a dominant IFN-γ-producing Th17 response toward a Th2 response, which may promote persistent infection in chickens. S Pullorum in chickens is presented as a good model of the typhoid group to study persistent infection.

Differential Immune Phenotypes in Human Monocytes Induced by Non-Host-Adapted Salmonella enterica Serovar Choleraesuis and Host-Adapted S. Typhimurium.

We studied the effects of two Salmonella enterica serovar Typhimurium (host-adapted) strains (14028 and 4/74) and three S Choleraesuis (non-host-adapted) strains (A50, A45, and B195) in human monocytes between 2 and 24 h postinfection (p.i.) to investigate whether differences in immune response may explain the much higher prevalence of sepsis in individuals infected with S Choleraesuis. Both serovars significantly increased the production of cytokines associated with acute sepsis (tumor necrosis factor alpha [TNF-α], interleukin ß [IL-ß], and IL-6), but temporal differences occurred between these serovars and between different S Choleraesuis strains. Generally, all S Choleraesuis strains induced significantly higher production of inflammatory cytokines than S Typhimurium strains (P < 0.01 to 0.05). All S Choleraesuis strains very significantly increased IL-10 production by monocytes at 6 and 24 h p.i. in comparison to S Typhimurium strains (P < 0.01). In addition, ∼80% of monocytes were viable at 24 h p.i. with S Choleraesuis A50, compared to only ∼40% following S Typhimurium infection. Using S Typhimurium 14028 and S Choleraesuis A50 as examples of these two serovars, we also showed differential expression of genes within the Janus tyrosine kinase (JAK) and signal transducer and activator of transcription (STAT) (JAK/STAT) pathway via quantitative PCR (qPCR) microarray analysis. High serum IL-10 concentration and monocyte survival have been reported as markers of the development of human sepsis. We therefore conclude that high production of IL-10 by monocytes may, in part, explain the greater propensity for S Choleraesuis to induce human sepsis and that this may be greater in strains such as A50, which induces both high IL-10 production and monocyte survival.

Dysregulation of JAK/STAT genes by vasoactive intestinal peptide (VIP) in Salmonella-infected monocytes may inhibit its therapeutic potential in human sepsis.

Murine/LPS models of Gram negative sepsis indicate that vasoactive intestinal peptide (VIP) has therapeutic potential. We investigated the unknown effect of VIP on JAK/STAT proteins and genes in human monocytes infected with Salmonella Typhimurium 14028. S. Typhimurium 14028 increased expression of both IL-6 receptor (IL-6R) and interferon gamma receptor 1 (IFNγR1) on monocytes but co-culture of infected monocytes with VIP (10-7 M) only decreased expression of IFNγR1 (P < 0.05). In contrast, S. Typhimurium 14028 infection or co-culture with VIP had no effect on IL-10 receptor expression on the monocyte surface. However, S. Typhimurium 14028 down regulated IFNGR1 gene expression and this was not altered by co-culture with VIP, suggesting that changes in IFNγR1 protein may be due to an effect on cytoplasmic transport. 15 JAK/STAT genes, out of 84 studied, were up-regulated by S. Typhimurium 14028 infection and five were down-regulated. Co-culture with VIP significantly decreased expression of two genes (IFNG and IL-20) and increased expression of three genes (SOCS1, SOCS3 and STAT4) (P < 0.05). S. Typhimurium 14028 also increased expression of PTPN1, which dephosphorylates JAK2 and TYK2. This was unaltered by co-culture with VIP but S. Typhimurium 14028-induced expression of ISG15, associated with susceptibility to Gram negative infection, was further increased by VIP. We conclude that the effect of VIP on JAK/STAT genes may preclude its therapeutic use in human Gram negative sepsis.

Gastric cooling and menthol cause an increase in cardiac parasympathetic efferent activity in healthy adult human volunteers.

NEW FINDINGS: What is the central question of this study? How do gastric stretch and gastric cooling stimuli affect cardiac autonomic control? What is the main finding and its importance? Gastric stretch causes an increase in cardiac sympathetic activity. Stretch combined with cold stimulation result in an elimination of the sympathetic response to stretch and an increase in cardiac parasympathetic activity, in turn resulting in a reduction in heart rate. Gastric cold stimulation causes a shift in sympathovagal balance towards parasympathetic dominance. The cold-induced bradycardia has the potential to decrease cardiac workload, which might be significant in individuals with cardiovascular pathologies. ABSTRACT: Gastric distension increases blood pressure and heart rate in young, healthy humans, but little is known about the effect of gastric stretch combined with cooling. We used a randomized crossover study to assess the cardiovascular responses to drinking 300 ml of ispaghula husk solution at either 6 or 37°C in nine healthy humans (age 24.08 ± 9.36 years) to establish the effect of gastric stretch with and without cooling. The effect of consuming peppermint oil capsules to activate cold thermoreceptors was also investigated. The ECG, respiratory movements and continuous blood pressure were recorded during a 5 min baseline period, followed by a 115 min post-drink period, during which 5 min epochs of data were recorded. Cardiac autonomic activity was assessed using time and frequency domain analyses of respiratory sinus arrhythmia to quantify parasympathetic autonomic activity, and corrected QT (QTc) interval analysis to quantify sympathetic autonomic activity. Gastric stretch only caused a significant reduction in QTc interval lasting up to 15 min, with a concomitant but non-significant increase in heart rate, indicating an increased sympathetic cardiac tone. The additional effect of gastric cold stimulation was significantly to reduce heart rate for up to 15 min, elevate indicators of cardiac parasympathetic tone and eliminate the reduction in QTc interval seen with gastric stretch only. Stimulation of gastric cold thermoreceptors with menthol also caused a significant reduction in heart rate and concomitant increase in the root mean square of successive differences. These findings indicate that gastric cold stimulation causes a shift in the sympathovagal balance of cardiac control towards a more parasympathetic dominant pattern.

An innovative investigative approach to characterize the effects observed in a combined fertility study in male and female rats.

This paper describes the characterization of male- and female-mediated effects in a standard ICH rat fertility and early embryonic development study with a discontinued clinical small molecule. In the standard study, the test item had no effect on the number of treated females becoming pregnant, but litter sizes were reduced at the high dose level. In the treated male rats, increased incidences of abnormal sperm, decreases in average sperm path and straight line velocities, and minimal retention of mature sperm in the seminiferous tubules were observed at all dose-levels tested. These findings were unexpected in view of a lack of histopathological changes in the reproductive organs of either gender in 4-week repeat dose studies in rats and monkeys. A follow-up fertility study was conducted using an innovative flexible study design and a single high-dose level. In the first instance, treated male rats were mated with untreated females, followed by necropsy of a subset of males. The intention was then to re-mate the males after an 8-week wash-out period with either treated or untreated females depending on the outcome of the first mating. On this occasion, litter sizes were not affected, but the testicular effects were reproduced. A second mating with treated females reproduced the reduced litter sizes due to increased pre- and post-implantation loss, demonstrating that the effect on fecundity was female-mediated. The testicular changes in males were shown to be reversible after a 12-week recovery period.