Acute phase response in two consecutive experimentally induced E. coli intramammary infections in dairy cows

<p>Abstract</p> <p>Background</p> <p>Acute phase proteins haptoglobin (Hp), serum amyloid A (SAA) and lipopolysaccharide binding protein (LBP) have suggested to be suitable inflammatory markers for bovine mastitis. The aim of the study was to investigate acute phase markers along with clinical parameters in two consecutive intramammary challenges with <it>Escherichia coli </it>and to evaluate the possible carry-over effect when same animals are used in an experimental model.</p> <p>Methods</p> <p>Mastitis was induced with a dose of 1500 cfu of <it>E. coli </it>in one quarter of six cows and inoculation repeated in another quarter after an interval of 14 days. Concentrations of acute phase proteins haptoglobin (Hp), serum amyloid A (SAA) and lipopolysaccharide binding protein (LBP) were determined in serum and milk.</p> <p>Results</p> <p>In both challenges all cows became infected and developed clinical mastitis within 12 hours of inoculation. Clinical disease and acute phase response was generally milder in the second challenge. Concentrations of SAA in milk started to increase 12 hours after inoculation and peaked at 60 hours after the first challenge and at 44 hours after the second challenge. Concentrations of SAA in serum increased more slowly and peaked at the same times as in milk; concentrations in serum were about one third of those in milk. Hp started to increase in milk similarly and peaked at 36–44 hours. In serum, the concentration of Hp peaked at 60–68 hours and was twice as high as in milk. LBP concentrations in milk and serum started to increase after 12 hours and peaked at 36 hours, being higher in milk. The concentrations of acute phase proteins in serum and milk in the <it>E. coli </it>infection model were much higher than those recorded in experiments using Gram-positive pathogens, indicating the severe inflammation induced by <it>E. coli</it>.</p> <p>Conclusion</p> <p>Acute phase proteins would be useful parameters as mastitis indicators and to assess the severity of mastitis. If repeated experimental intramammary induction of the same animals with <it>E. coli </it>is used in cross-over studies, the interval between challenges should be longer than 2 weeks, due to the carry-over effect from the first infection.</p

Treatment of mastitis during lactation

Treatment of mastitis should be based on bacteriological diagnosis and take national and international guidelines on prudent use of antimicrobials into account. In acute mastitis, where bacteriological diagnosis is not available, treatment should be initiated based on herd data and personal experience. Rapid bacteriological diagnosis would facilitate the proper selection of the antimicrobial. Treating subclinical mastitis with antimicrobials during lactation is seldom economical, because of high treatment costs and generally poor efficacy. All mastitis treatment should be evidence-based, i.e., the efficacy of each product and treatment length should be demonstrated by scientific studies. Use of on-farm written protocols for mastitis treatment promotes a judicious use of antimicrobials and reduces the use of antimicrobials

Effect of Fructooligosaccharide Metabolism on Chicken Colonization by an Extra-Intestinal Pathogenic Escherichia coli Strain

Extra-intestinal pathogenic Escherichia coli (ExPEC) strains cause many diseases in humans and animals. While remaining asymptomatic, they can colonize the intestine for subsequent extra-intestinal infection and dissemination in the environment. We have previously identified the fos locus, a gene cluster within a pathogenicity island of the avian ExPEC strain BEN2908, involved in the metabolism of short-chain fructooligosaccharides (scFOS). It is assumed that these sugars are metabolized by the probiotic bacteria of the microbiota present in the intestine, leading to a decrease in the pathogenic bacterial population. However, we have previously shown that scFOS metabolism helps BEN2908 to colonize the intestine, its reservoir. As the fos locus is located on a pathogenicity island, one aim of this study was to investigate a possible role of this locus in the virulence of the strain for chicken. We thus analysed fos gene expression in extracts of target organs of avian colibacillosis and performed a virulence assay in chickens. Moreover, in order to understand the involvement of the fos locus in intestinal colonization, we monitored the expression of fos genes and their implication in the growth ability of the strain in intestinal extracts of chicken. We also performed intestinal colonization assays in axenic and Specific Pathogen-Free (SPF) chickens. We demonstrated that the fos locus is not involved in the virulence of BEN2908 for chickens and is strongly involved in axenic chicken cecal colonization both in vitro and in vivo. However, even if the presence of a microbiota does not inhibit the growth advantage of BEN2908 in ceca in vitro, overall, growth of the strain is not favoured in the ceca of SPF chickens. These findings indicate that scFOS metabolism by an ExPEC strain can contribute to its fitness in ceca but this benefit is fully dependent on the bacteria present in the microbiota

Developing in vitro expanded CD45RA+ regulatory T cells as an adoptive cell therapy for Crohn's disease.

BACKGROUND AND AIM: Thymus-derived regulatory T cells (Tregs) mediate dominant peripheral tolerance and treat experimental colitis. Tregs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. Treg cell therapy is also conceptually attractive for Crohn's disease (CD). However, barriers exist to this approach. The stability of Tregs expanded from Crohn's blood is unknown. The potential for adoptively transferred Tregs to express interleukin-17 and exacerbate Crohn's lesions is of concern. Mucosal T cells are resistant to Treg-mediated suppression in active CD. The capacity for expanded Tregs to home to gut and lymphoid tissue is unknown. METHODS: To define the optimum population for Treg cell therapy in CD, CD4(+)CD25(+)CD127(lo)CD45RA(+) and CD4(+)CD25(+)CD127(lo)CD45RA(-) Treg subsets were isolated from patients' blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background. RESULTS: Tregs can be expanded from the blood of patients with CD to potential target dose within 22-24 days. Expanded CD45RA(+) Tregs have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA(-) Tregs. CD45RA(+) Tregs highly express α4β7 integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA(+) Tregs also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA(+) Tregs. These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohn's mucosa. CONCLUSIONS: CD4(+)CD25(+)CD127(lo)CD45RA(+) Tregs may be the most appropriate population from which to expand Tregs for autologous Treg therapy for CD, paving the way for future clinical trials

Correction of defective T-regulatory cells from patients with Crohn's disease by Ex Vivo ligation of retinoic acid receptor alpha

BACKGROUND & AIMS: Crohn's disease (CD) is characterized by an imbalance of effector and regulatory T cells in the intestinal mucosa. The efficacy of anti-adhesion therapies led us to investigate whether impaired trafficking of T-regulatory (Treg) cells contributes to the pathogenesis of CD. We also investigated whether proper function could be restored to Treg cells by ex vivo expansion in the presence of factors that activate their regulatory activities. METHODS: We measured levels of the integrin α4β7 on Treg cells isolated from peripheral blood or lamina propria of patients with CD and healthy individuals (controls). Treg cells were expanded ex vivo and incubated with rapamycin with or without agonists of the retinoic acid receptor alpha (RARA) and their gene expression profiles were analyzed. We also studied the cells in cytokine challenge, suppression, and flow chamber assays and in SCID mice with human intestinal xenografts. RESULTS: We found that Treg cells from patients with CD express lower levels of the integrin α4β7 than Treg cells from control patients. The pathway that regulates expression of integrin subunit alpha is induced by retinoic acid (RA). Treg cells from patients with CD incubated with rapamycin and an agonist of RARA (RAR568) expressed high levels of integrin α4β7, as well as CD62L and FOXP3, compared to cells incubated with rapamycin or rapamycin and ATRA. These Treg cells had increased suppressive activities in assays and migrated under conditions of shear flow; they did not produce inflammatory cytokines and RAR568 had no effect on cell stability or lineage commitment. Fluorescently labelled Treg cells incubated with RAR568 were significantly more likely to traffic to intestinal xenografts than Treg cells expanded in control medium. CONCLUSIONS: Treg cells from patients with CD express lower levels of the integrin α4β7 than Treg cells from control patients. Incubation of patients' ex vivo expanded Treg cells with rapamycin and an RARA agonist induced expression of α4β7 and had suppressive and migratory activities in culture and in intestinal xenografts in mice. These cells might be developed for treatment of CD. clinicaltrials.gov no: NCT03185000

Correction of Defective T-regulatory Cells From Patients With Crohn’s Disease by Ex Vivo Ligation of Retinoic Acid Receptor Alpha

BACKGROUND & AIMS: Crohn's disease (CD) is characterized by an imbalance of effector and regulatory T cells in the intestinal mucosa. The efficacy of anti-adhesion therapies led us to investigate whether impaired trafficking of T-regulatory (Treg) cells contributes to the pathogenesis of CD. We also investigated whether proper function could be restored to Treg cells by ex vivo expansion in the presence of factors that activate their regulatory activities. METHODS: We measured levels of the integrin α4β7 on Treg cells isolated from peripheral blood or lamina propria of patients with CD and healthy individuals (controls). Treg cells were expanded ex vivo and incubated with rapamycin with or without agonists of the retinoic acid receptor alpha (RARA) and their gene expression profiles were analyzed. We also studied the cells in cytokine challenge, suppression, and flow chamber assays and in SCID mice with human intestinal xenografts. RESULTS: We found that Treg cells from patients with CD express lower levels of the integrin α4β7 than Treg cells from control patients. The pathway that regulates expression of integrin subunit alpha is induced by retinoic acid (RA). Treg cells from patients with CD incubated with rapamycin and an agonist of RARA (RAR568) expressed high levels of integrin α4β7, as well as CD62L and FOXP3, compared to cells incubated with rapamycin or rapamycin and ATRA. These Treg cells had increased suppressive activities in assays and migrated under conditions of shear flow; they did not produce inflammatory cytokines and RAR568 had no effect on cell stability or lineage commitment. Fluorescently labelled Treg cells incubated with RAR568 were significantly more likely to traffic to intestinal xenografts than Treg cells expanded in control medium. CONCLUSIONS: Treg cells from patients with CD express lower levels of the integrin α4β7 than Treg cells from control patients. Incubation of patients' ex vivo expanded Treg cells with rapamycin and an RARA agonist induced expression of α4β7 and had suppressive and migratory activities in culture and in intestinal xenografts in mice. These cells might be developed for treatment of CD. clinicaltrials.gov no: NCT03185000