TNF-α induced endothelial MAdCAM-1 expression is regulated by exogenous, not endogenous nitric oxide

BACKGROUND: MAdCAM-1 is an adhesion molecule expressed in Peyer's patches and lymphoid tissues which is mobilized by cytokines like TNF-α and is a major determinant of lymphocyte trafficking to the gut in human inflammatory bowel disease (IBD). It has been suggested that both reactive oxygen and nitrogen metabolites participate in regulating adhesion molecule expression in response to TNF-α. METHODS: To examine how exogenous and endogenous sources of NO modulate MAdCAM-1 induction by TNF-α, we pre-treated mouse lymphatic endothelial cells with either long or short acting NO donors prior to TNF-α-stimulation, and measured MAdCAM-1 induction at 24 h. RESULTS AND DISCUSSION: DETA-NO, a long-acting NO donor, and SperNO, a rapid releasing NO donor both inhibited TNF-α-stimulated MAdCAM-1 expression in a concentration dependent manner. Both NO donors also reduced a4b7-dependent lymphocyte endothelial adhesion. Inhibition of endogenous NO production by either L-NAME, a non-selective NOS inhibitor, or by 1400 w, a selective iNOS inhibitor failed to induce, or potentiate TNF-α regulated MAdCAM-1 expression. CONCLUSIONS: Exogenous NO donors may be beneficial in the treatment of IBD, while endogenous nitric oxide synthases may be less effective in controlling adhesion molecule expression in response to cytokines

Gastrointestinal-Sparing Effects of Novel NSAIDs in Rats with Compromised Mucosal Defence

Nonsteroidal anti-inflammatory drugs are among the most commonly used prescription and over-the-counter medications, but they often produce significant gastrointestinal ulceration and bleeding, particularly in elderly patients and patients with certain co-morbidities. Novel anti-inflammatory drugs are seldom tested in animal models that mimic the high risk human users, leading to an underestimate of the true toxicity of the drugs. In the present study we examined the effects of two novel NSAIDs and two commonly used NSAIDs in models in which mucosal defence was expected to be impaired. Naproxen, celecoxib, ATB-346 (a hydrogen sulfide- and naproxen-releasing compound) and NCX 429 (a nitric oxide- and naproxen-releasing compound) were evaluated in healthy, arthritic, obese, and hypertensive rats and in rats of advanced age (19 months) and rats co-administered low-dose aspirin and/or omeprazole. In all models except hypertension, greater gastric and/or intestinal damage was observed when naproxen was administered in these models than in healthy rats. Celecoxib-induced damage was significantly increased when co-administered with low-dose aspirin and/or omeprazole. In contrast, ATB-346 and NCX 429, when tested at doses that were as effective as naproxen and celecoxib in reducing inflammation and inhibiting cyclooxygenase activity, did not produce significant gastric or intestinal damage in any of the models. These results demonstrate that animal models of human co-morbidities display the same increased susceptibility to NSAID-induced gastrointestinal damage as observed in humans. Moreover, two novel NSAIDs that release mediators of mucosal defence (hydrogen sulfide and nitric oxide) do not induce significant gastrointestinal damage in these models of impaired mucosal defence

Gene deletion of P-Selectin and ICAM-1 does not inhibit neutrophil infiltration into peritoneal cavity following cecal ligation-puncture

BACKGROUND: Neutrophil infiltration is one of the critical cellular components of an inflammatory response during peritonitis. The adhesion molecules, P-selectin and intercellular adhesion molecule (ICAM)-1, mediate neutrophil-endothelial cell interactions and the subsequent neutrophil transendothelial migration during the inflammatory response. Despite very strong preclinical data, recent clinical trials failed to show a protective effect of anti-adhesion therapy, suggesting that the length of injury might be a critical factor in neutrophil infiltration. Therefore, the objective of this study was to determine the role of P-selectin and ICAM-1 in neutrophil infiltration into the peritoneal cavity during early and late phases of peritonitis. METHODS: Peritonitis was induced in both male wild-type and P-selectin/ICAM-1 double deficient (P/I null) mice by cecal ligation-puncture (CLP). Peripheral blood and peritoneal lavage were collected at 6 and 24 hours after CLP. The total leukocyte and neutrophil contents were determined, and neutrophils were identified with the aid of in situ immunohistochemical staining. Comparisons between groups were made by applying ANOVA and student t-test analysis. RESULTS: CLP induced a severe inflammatory response associated with a significant leukopenia in both wild-type and P/I null mice. Additionally, CLP caused a significant neutrophil infiltration into the peritoneal cavity that was detected in both groups of mice. However, neutrophil infiltration in the P/I null mice at 6 hours of CLP was significantly lower than the corresponding wild-type mice, which reached a similar magnitude at 24 hours of CLP. In contrast, in peritonitis induced by intraperitoneal inoculation of 2% glycogen, no significant difference in neutrophil infiltration was observed between the P/I null and wild-type mice at 6 hours of peritonitis. CONCLUSIONS: The data suggest that alternative adhesion pathway(s) independent of P-selectin and ICAM-1 can participate in neutrophil migration during peritonitis and that the mode of stimuli and duration of the injury modulate the neutrophil infiltration

The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity

Although the murine immune response to Venezuelan equine encephalitis virus (VEEV) is well-characterized, little is known about the human antibody response to VEEV. In this study we used phage display technology to isolate a panel of 11 VEEV-specfic Fabs from two human donors. Seven E2-specific and four E1-specific Fabs were identified and mapped to five E2 epitopes and three E1 epitopes. Two neutralizing Fabs were isolated, E2-specific F5 and E1-specific L1A7, although the neutralizing capacity of L1A7 was 300-fold lower than F5. F5 Fab was expressed as a complete IgG1 molecule, F5 native (n) IgG. Neutralization-escape VEEV variants for F5 nIgG were isolated and their structural genes were sequenced to determine the theoretical binding site of F5. Based on this sequence analysis as well as the ability of F5 to neutralize four neutralization-escape variants of anti-VEEV murine monoclonal antibodies (mapped to E2 amino acids 182–207), a unique neutralization domain on E2 was identified and mapped to E2 amino acids 115–119

Transfection of IL-10 expression vectors into endothelial cultures attenuates α4β7-dependent lymphocyte adhesion mediated by MAdCAM-1

BACKGROUND: Enhanced expression of MAdCAM-1 (mucosal addressin cell adhesion molecule-1) is associated with the onset and progression of inflammatory bowel disease. The clinical significance of elevated MAdCAM-1 expression is supported by studies showing that immunoneutralization of MAdCAM-1, or its ligands reduce inflammation and mucosal damage in models of colitis. Interleukin-10 (IL-10) is an endogenous anti-inflammatory and immunomodulatory cytokine that has been shown to prevent inflammation and injury in several animal studies, however clinical IL-10 treatment remains insufficient because of difficulties in the route of IL-10 administration and its biological half-life. Here, we examined the ability of introducing an IL-10 expression vector into endothelial cultures to reduce responses to a proinflammatory cytokine, TNF-α METHODS: A human IL-10 expression vector was transfected into high endothelial venular ('HEV') cells (SVEC4-10); we then examined TNF-α induced lymphocyte adhesion to lymphatic endothelial cells and TNF-α induced expression of MAdCAM-1 and compared these responses to control monolayers. RESULTS: Transfection of the IL-10 vector into endothelial cultures significantly reduced TNF-α induced, MAdCAM-1 dependent lymphocyte adhesion (compared to non-transfected cells). IL-10 transfected endothelial cells expressed less than half (46 ± 6.6%) of the MAdCAM-1 induced by TNF-α (set as 100%) in non-transfected (control) cells. CONCLUSION: Our results suggest that gene therapy of the gut microvasculature with IL-10 vectors may be useful in the clinical treatment of IBD

Rise and Fall of an Anti-MUC1 Specific Antibody

So far, human antibodies with good affinity and specificity for MUC1, a transmembrane protein overexpressed on breast cancers and ovarian carcinomas, and thus a promising target for therapy, were very difficult to generate.A human scFv antibody was isolated from an immune library derived from breast cancer patients immunised with MUC1. The anti-MUC1 scFv reacted with tumour cells in more than 80% of 228 tissue sections of mamma carcinoma samples, while showing very low reactivity with a large panel of non-tumour tissues. By mutagenesis and phage display, affinity of scFvs was increased up to 500fold to 5,7×10(-10) M. Half-life in serum was improved from below 1 day to more than 4 weeks and was correlated with the dimerisation tendency of the individual scFvs. The scFv bound to T47D and MCF-7 mammalian cancer cell lines were recloned into the scFv-Fc and IgG format resulting in decrease of affinity of one binder. The IgG variants with the highest affinity were tested in mouse xenograft models using MCF-7 and OVCAR tumour cells. However, the experiments showed no significant decrease in tumour growth or increase in the survival rates. To study the reasons for the failure of the xenograft experiments, ADCC was analysed in vitro using MCF-7 and OVCAR3 target cells, revealing a low ADCC, possibly due to internalisation, as detected for MCF-7 cells.Antibody phage display starting with immune libraries and followed by affinity maturation is a powerful strategy to generate high affinity human antibodies to difficult targets, in this case shown by the creation of a highly specific antibody with subnanomolar affinity to a very small epitope consisting of four amino acids. Despite these "best in class" binding parameters, the therapeutic success of this antibody was prevented by the target biology

The impact of viral mutations on recognition by SARS-CoV-2 specific T cells.

We identify amino acid variants within dominant SARS-CoV-2 T cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T cells assessed by IFN-γ and cytotoxic killing assays. Complete loss of T cell responsiveness was seen due to Q213K in the A∗01:01-restricted CD8+ ORF3a epitope FTSDYYQLY207-215; due to P13L, P13S, and P13T in the B∗27:05-restricted CD8+ nucleocapsid epitope QRNAPRITF9-17; and due to T362I and P365S in the A∗03:01/A∗11:01-restricted CD8+ nucleocapsid epitope KTFPPTEPK361-369. CD8+ T cell lines unable to recognize variant epitopes have diverse T cell receptor repertoires. These data demonstrate the potential for T cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T cell as well as humoral immunity.This work is supported by the UK Medical Research Council (MRC); Chinese Academy of Medical Sciences(CAMS) Innovation Fund for Medical Sciences (CIFMS), China; National Institute for Health Research (NIHR)Oxford Biomedical Research Centre, and UK Researchand Innovation (UKRI)/NIHR through the UK Coro-navirus Immunology Consortium (UK-CIC). Sequencing of SARS-CoV-2 samples and collation of data wasundertaken by the COG-UK CONSORTIUM. COG-UK is supported by funding from the Medical ResearchCouncil (MRC) part of UK Research & Innovation (UKRI),the National Institute of Health Research (NIHR),and Genome Research Limited, operating as the Wellcome Sanger Institute. T.I.d.S. is supported by a Well-come Trust Intermediate Clinical Fellowship (110058/Z/15/Z). L.T. is supported by the Wellcome Trust(grant number 205228/Z/16/Z) and by theUniversity of Liverpool Centre for Excellence in Infectious DiseaseResearch (CEIDR). S.D. is funded by an NIHR GlobalResearch Professorship (NIHR300791). L.T. and S.C.M.are also supported by the U.S. Food and Drug Administration Medical Countermeasures Initiative contract75F40120C00085 and the National Institute for Health Research Health Protection Research Unit (HPRU) inEmerging and Zoonotic Infections (NIHR200907) at University of Liverpool inpartnership with Public HealthEngland (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford.L.T. is based at the University of Liverpool. M.D.P. is funded by the NIHR Sheffield Biomedical ResearchCentre (BRC – IS-BRC-1215-20017). ISARIC4C is supported by the MRC (grant no MC_PC_19059). J.C.K.is a Wellcome Investigator (WT204969/Z/16/Z) and supported by NIHR Oxford Biomedical Research Centreand CIFMS. The views expressed are those of the authors and not necessarily those of the NIHR or MRC