Guideline for collection, analysis and presentation of safety data in clinical trials of vaccines in pregnant women.

Vaccination during pregnancy is increasingly being used as an effective approach for protecting both young infants and their mothers from serious infections. Drawing conclusions from published studies in this area can be difficult because of the inability to compare vaccine trial results across different studies and settings due to the heterogeneity in the definitions of terms used to assess the safety of vaccines in pregnancy and the data collected in such studies. The guidelines proposed in this document have been developed to harmonize safety data collection in all phases of clinical trials of vaccines in pregnant women and apply to data from the mother, fetus and infant. Guidelines on the prioritization of the data to be collected is also provided to allow applicability in various geographic, cultural and resource settings, including high, middle and low-income countries

Lactobacilli regulate Staphylococcus aureus 161:2-induced pro-inflammatory T-cell responses in vitro.

There seems to be a correlation between early gut microbiota composition and postnatal immune development. Alteration in the microbial composition early in life has been associated with immune mediated diseases, such as autoimmunity and allergy. We have previously observed associations between the presence of lactobacilli and Staphylococcus (S.) aureus in the early-life gut microbiota, cytokine responses and allergy development in children. Consistent with the objective to understand how bacteria modulate the cytokine response of intestinal epithelial cell (IEC) lines and immune cells, we exposed IEC lines (HT29, SW480) to UV-killed bacteria and/or culture supernatants (-sn) from seven Lactobacillus strains and three S. aureus strains, while peripheral blood mononuclear cells (PBMC) and cord blood mononuclear cells (CBMC) from healthy donors were stimulated by bacteria-sn or with bacteria conditioned IEC-sn. Although the overall IEC response to bacterial exposure was characterized by limited sets of cytokine and chemokine production, S. aureus 161:2-sn induced an inflammatory response in the IEC, characterized by CXCL1/GROα and CXCL8/IL-8 production, partly in a MyD88-dependent manner. UV-killed bacteria did not induce a response in the IEC line, and a combination of both UV-killed bacteria and the bacteria-sn had no additive effect to that of the supernatant alone. In PBMC, most of the Lactobacillus-sn and S. aureus-sn strains were able to induce a wide array of cytokines, but only S. aureus-sn induced the T-cell associated cytokines IL-2, IL-17 and IFN-γ, independently of IEC-produced factors, and induced up regulation of CTLA-4 expression and IL-10 production by T-regulatory cells. Notably, S. aureus-sn-induced T-cell production of IFN- γ and IL-17 was down regulated by the simultaneous presence of any of the different Lactobacillus strains, while the IEC CXCL8/IL-8 response was unaltered. Thus these studies present a possible role for lactobacilli in induction of immune cell regulation, although the mechanisms need to be further elucidated

Cytokine production by intestinal epithelial cells and immune cells simultaneously co-cultured with <i>S. aureus</i> 161:2 and each <i>Lactobacillus</i> strain supernatant.

<p>(A) CXCL8/IL-8 level in IEC-sn after co-cultured with <i>S. aureus</i> 161:2-sn together with each <i>Lactobacillus</i> strains-sn measured by ELISA. (B) IL-17 and (C) IFN-γ levels in PBMC-sn after PBMC co-cultured with <i>S. aureus</i> 161:2-sn together with each <i>Lactobacillus</i> strains-sn measured by ELISA. The graphs represent mean + SEM value of independent experiments using PBMC from 3 healthy donors. (D) Shows IFN-γ production by CBMC simultaneously co-cultured with <i>S. aureus</i> 161:2-sn and either <i>L. reuteri</i> DSM 17938-sn or <i>L. rhamnosus</i> GG culture–sn. One representative CB donor out of four. nd signifies below detection. (E) IL-2 and (F) TNF-α levels in PBMC-sn after PBMC co-cultured with <i>S. aureus</i> 161:2-sn together with each <i>Lactobacillus</i> strains-sn measured by ELISA. One representative experiment is shown. ***<i>P</i> < 0·001, **<i>P</i> < 0·01 and *<i>P</i> < 0·05. Background generated by bacterial medium is subtracted.</p

Cytokine production by PBMC stimulated with bacteria conditioned IEC-sn

<p>(A) IL-6, (B) IL-17, (C) IFN-γ, (D) IL-2 and (E) TNF-α levels measured by ELISA in supernatant from PBMC stimulated with bacteria conditioned IEC-sn for 24 hours. (A-C) The graphs represent mean + SEM value of independent experiments using PBMC from 3 healthy donors. ***<i>P</i> < 0·001, **<i>P</i> < 0·01 and *<i>P</i> < 0·05. (D-E) One representative experiment is shown. Background generated by bacterial medium is subtracted.</p

<i>Lactobacillus</i> strains differentially produce histamine.

<p>ELISA analysis of histamine level in the bacteria supernatants. Histamine production by <i>L. reuteri</i> ATCC PTA 6475 was used as a positive control. One representative experiment out of two is shown.</p

Cytokine production by IEC stimulated with bacteria supernatant.

<p>HT29 cells were cultured in 48 well plates for 24 hours with the bacteria-sn. (A) Shows semi quantitative analysis of human cytokine array in IEC-sn by measuring the integrated pixel density (Pi) (area x mean gray value). (B) Shows the level of CXCL8/IL-8 in IEC-sn following ELISA. Data are shown as means + SEM of 3-4 experiments respectively. (C) Shows the effect of MyD88-silencing on IEC CXCL8/IL-8 production normalized with the control culture medium response. One representative experiment out of three is shown. ***<i>P</i> < 0·001, **<i>P</i> < 0·01 and *<i>P</i> < 0·05. Background generated by bacterial medium is subtracted.</p

<i>S. aureus</i>-sn induces IL-17 and IFN-γ producing T-helper cells and up-regulation of CTLA-4 and IL-10 production by T-regulatory cells after <i>in</i><i>vitro</i> stimulation of PBMC.

<p>Flow cytometry analysis of (A) IL-17⁺ and IFN-γ⁺ CD4⁺ T-cells and (B) CTLA-4⁺ and IL-10⁺ T-regulatory cells following stimulation with <i>S. aureus</i> 161:2-sn, <i>L. reuteri</i> DSM 17938-sn or a combination of both. Numbers refer to percentages of positive cells. One representative experiment out of five, using healthy adult donors. (C) Shows IL-10 production by PBMC stimulated with <i>S. aureus</i> 161:2-sn, <i>L. reuteri</i> DSM 17938-sn or a combination of both. The graphs represent mean + SEM value of independent experiments using PBMC from 3 healthy donors.</p