Antibiotic Cocktail for Pediatric Acute Severe Colitis and the Microbiome : The PRASCO Randomized Controlled Trial

Background: Alterations in the microbiome have been postulated to drive inflammation in IBD. In this pilot randomized controlled trial, we evaluated the effectiveness of quadruple antibiotic cocktail in addition to intravenous-corticosteroids (IVCSs) in acute severe colitis (ASC). Methods: Hospitalized children with ASC (pediatric ulcerative colitis activity index [PUCAI] >= 65) were randomized into 2 arms: the first received antibiotics in addition to IVCS (amoxicillin, vancomycin, metronidazole, doxycycline/ciprofloxacin [IVCS+AB]), whereas the other received only IVCS for 14 days. The primary outcome was disease activity (PUCAI) at day 5. Microbiome was analyzed using 16S rRNA gene and metagenome. Results: Twenty-eight children were included: 16 in the AB + IVCS arm and 12 in the IVCS arm (mean age 13.9 +/- 4.1 years and 23 [82%] with extensive colitis). The mean day-5 PUCAI was 25 +/- 16.7 vs 40.4 +/- 20.4, respectively (P = 0.037). Only 3 and 2 children, respectively, required colectomy during 1-year follow-up (P = 0.89). Microbiome data at time of admission were analyzed for 25 children, of whom 17 (68%) had a predominant bacterial species (>33% abundance); response was not associated with the specific species, whereas decreased microbiome diversity at admission was associated with day-5 response in the IVCS arm. Conclusion: Patients with ASC have alterations in the microbiome characterized by loss of diversity and presence of predominant bacterial species. Quadruple therapy in addition to IVCS improved disease activity on day 5, but larger studies are needed to determine whether this is associated with improved long-term outcomes.Peer reviewe

Low Interferon Relative-Response to Cytomegalovirus Is Associated with Low Likelihood of Intrauterine Transmission of the Virus.

BACKGROUND:Congenital Cytomegalovirus (CMV) is a very common intrauterine infection which can cause severe mental and hearing impairments. Notably, only 40% of primarily infected women transmit CMV to the fetus. CMV-specific T-cell response has a role in CMV disease but individual immune heterogeneity precludes reliable correlation between measurable T-cells response and intrauterine transmission. STUDY AIM:To establish a correlation between maternal T-cells response and fetal CMV transmission using an individual normalized immune response. METHODS:We analyzed IFN-γ secretion upon whole blood stimulation from primary CMV-infected pregnant women, with either CMV-peptides or PHA-mitogen. RESULTS:We established a new normalization method of individual IFN-γ response to CMV by defining the ratio between specific-CMV response and non-specific mitogen response (defined as IFN-γ relative response, RR), aiming to overcome high person-to-person immune variability. We found a unique subpopulation of women with low IFN-γ RR strongly correlated with absence of transmission. IFN-γ RR lower than 1.8% (threshold determined by ROC analysis) reduces the pre-test probability of transmission from 40% to 8%, revealing an unexpected link between low IFN-γ RR and non-transmission. CONCLUSION:In pregnant women with primary CMV infection, low IFN-γ RR is associated with low risk of transmission

Normalization method of individual IFN-γ specific response.

<p>A. Schematic representation of the <i>in-vitro</i> stimulation assay. Blood samples of 76 pregnant women with primary CMV were stimulated with either CMV peptides or PHA, for 24 hours at 37°C. The amounts of IFN-γ, TNF-α, IL-10 and IL-6 in the supernatant were determined by CBA analysis. B. Comparing normalizations of IFN-γ induced after CMV stimulation to Nil or Mitogen tubes. We compared the ratios between the levels of IFN-γ induced by CMV peptides divided by that of 'nil' (CMV/N) or 'Mitogen' (CMV/M). Blood samples of five patients (P-I to P-V) were taken in two different time points, T1 and T2 (intervals of 5–8 weeks). Delta determined as the ratio at T1 subtracted from the ratio at T2. C. Comparing IFN-γ TB/M to IFN-g CMV/M ratio. Blood samples of 19 women were collected into Nil, CMV and mitogen tubes, and an additional tube containing Mycobacterium tuberculosis (TB) antigens. We compared the IFN-γ induced by CMV peptides to the IFN-γ induced by TB peptides both normalized to the amount of IFN-γ in the Mitogen tube.</p

Low IFN-γ RR in non transmitters does not stem primarily from high IFN-γ level caused by mitogen activation.

<p>Comparison of IFN-γ levels after mitogen activation in transmitters and non transmitters. Women with IFN-γ RR <1.8% are marked with filled triangles. T = transmitters, NT = non transmitters.</p

PD-1 expression in PBMC’s of women with high and low IFN-γ relative response is similar.

<p>The expression of PD-1 in PBMC was performed by TaqMan Real time PCR of PD-1 and GUSB (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147883#sec007" target="_blank">methods</a> section). ∆Ct was calculated (average ∆Ct of PD-1minus the average ∆Ct of GUSB). Relative expression (2^(-∆Ct)±SE) values of women with high and low IFN-γ RR were compared by <i>t</i>-test.</p

Normalization method of individual IFN-γ specific response.

<p>A. Schematic representation of the <i>in-vitro</i> stimulation assay. Blood samples of 76 pregnant women with primary CMV were stimulated with either CMV peptides or PHA, for 24 hours at 37°C. The amounts of IFN-γ, TNF-α, IL-10 and IL-6 in the supernatant were determined by CBA analysis. B. Comparing normalizations of IFN-γ induced after CMV stimulation to Nil or Mitogen tubes. We compared the ratios between the levels of IFN-γ induced by CMV peptides divided by that of 'nil' (CMV/N) or 'Mitogen' (CMV/M). Blood samples of five patients (P-I to P-V) were taken in two different time points, T1 and T2 (intervals of 5–8 weeks). Delta determined as the ratio at T1 subtracted from the ratio at T2. C. Comparing IFN-γ TB/M to IFN-g CMV/M ratio. Blood samples of 19 women were collected into Nil, CMV and mitogen tubes, and an additional tube containing Mycobacterium tuberculosis (TB) antigens. We compared the IFN-γ induced by CMV peptides to the IFN-γ induced by TB peptides both normalized to the amount of IFN-γ in the Mitogen tube.</p

Clinical and laboratory characteristics of CMV-infected pregnant women- transmitters and non-transmitters.

<p>Clinical and laboratory characteristics of CMV-infected pregnant women- transmitters and non-transmitters.</p

Post-test probability of women with low IFN-γ relative response to CMV.

<p>The post-test probability of women with IFN-g RR<1.8% reduced from 40% to 7%-8% in all tested women (n = 75), for women with low IgG avidity (n = 62) and for viremic women (n = 37).</p