Anti-Inflammatory Properties of Plasma from Children with Short Bowel Syndrome

Sepsis, resulting from a dysregulated host immune response to invading pathogens, is the leading cause of mortality in critically ill patients worldwide. Immunomodulatory treatment for sepsis is currently lacking. Children with short bowel syndrome (SBS) may present with less severe symptoms during gram-negative bacteremia. We, therefore, tested the hypothesis that plasma from children with SBS could confer protection against Escherichia coli sepsis. We showed that SBS plasma at 5% and 10% concentrations significantly (p < 0.05) inhibited the production of both TNF-α and IL-6 induced by either E. coli- or LPS-stimulated host cells when compared to plasma from healthy controls. Furthermore, mice treated intravenously with select plasma samples from SBS or healthy subjects had reduced proinflammatory cytokine levels in plasma and a significant survival advantage after E. coli infection. However, SBS plasma was not more protective than the plasma of healthy subjects, suggesting that children with SBS have other immunomodulatory mechanisms, in addition to neutralizing antibodies, to alleviate their symptoms during gram-negative sepsis

Synergistic effect of high-mobility group box-1 and lipopolysaccharide on cytokine induction in bovine peripheral blood mononuclear cells

High-mobility group box 1 (HMGB1) is one of the potent endogenous adjuvants released by necrotic and activated innate immune cells. HMGB1 modulates innate and adaptive immune responses in humans and mice by mediating immune cells crosstalk. However, the immuno-modulatory effects of HMGB1 in the bovine immune system are not clearly known. In this study, the effect of bovine HMGB1 alone or in combination with LPS on the expression kinetics of cytokines upon in vitro stimulation of bovine peripheral blood mononuclear cells (PBMCs) was investigated by quantitative PCR assay. The biological activity of bovine HMGB1 expressed in this prokaryotic expression system was confirmed by its ability to induce nitric oxide secretion in RAW 264.7 cells. The present results indicate that HMGB1 induces a more delayed TNF-α response than does LPS in stimulated PBMCs. However, IFN-γ, IFN-β and IL-12 mRNA transcription peaked at 6 hr post stimulation after both treatments. Further, HMGB1 and LPS heterocomplex up-regulated TNF-α, IFN-γ and IL-12 mRNA expression significantly than did individual TLR4 agonists. The heterocomplex also enhanced the expression of TLR4 on bovine PBMCs. In conclusion, the data indicate that HMGB1 and LPS act synergistically and enhance proinflammatory cytokines, thereby eliciting Th1 responses in bovine PBMCs. These results suggest that HMGB1 can act as an adjuvant in modulating the bovine immune system and thus lays a foundation for using HMGB1 as an adjuvant in various bovine vaccine preparations

Morphological and phenotypic characterization of immature MoDCs.

<p>(A) Light microscopy (20X) of freshly isolated monocyte. (B) Light microscopy (20X) of 4^th day culture of presumably immature MoDCs showing clusters of veiled cells with pseudopodia. (C) Freshly isolated monocytes and 5 day culture of MoDCs were analysed for phenotypic changes by evaluating mRNA expression levels of various TLRs and costimulatory genes by qRT-PCR assay. Results are expressed as fold change (log10) in mRNA transcription of monocytes and MoDCs. Data presented are mean ± standard deviation of values obtained from three independent experiments involving two cattle of Hallikar breed. *<i>p</i>< 0.05.**<i>p</i>< 0.01.</p

Characterization of <i>E</i>. <i>coli</i> ghosts by electron microscopy.

<p>(A) Field emission scanning electron micrograph (FE-SEM) of protein E lysed <i>E</i>. <i>coli</i> ghosts showing transmembrane tunnels, indicated by arrow heads. (B) FESEM of intact <i>E</i>. <i>coli</i> before lysis. (C) Transmission electron micrograph (TEM) of <i>E</i>. <i>coli</i> ghosts with a loss of cytoplasmic contents but intact cellular morphology. (D) TEM of intact <i>E</i>. <i>coli</i> prior to gene <i>E</i> induction.</p

Kinetics of cytokine mRNA transcription from MoDCs treated with either BGs or LPS.

<p>RNA was extracted and gene transcription was quantified by qRT-PCR at 0, 6 and 12 h. Results are expressed as fold induction (log10) of cytokine mRNA transcription by BGs or LPS stimulated cells compared to the media treated (Med) cells. GAPDH was used as an internal control and mRNA levels at 0 h was used as calibrator. Histograms represent mean cytokine levels and bars represent standard deviation of three independent experiments. *p < 0.05, **p<0.01, ns = non-significant.</p