Additional file 1: of Antibiotic therapy for skin and soft tissue infections: a protocol for a systematic review and network meta-analysis

Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) Checklist. (DOCX 32 kb

Additional file 2: of Antibiotic therapy for skin and soft tissue infections: a protocol for a systematic review and network meta-analysis

Medline Search Strategy. (DOC 37 kb

Additional file 1: of Using tocolysis in pregnant women with symptomatic placenta praevia does not significantly improve prenatal, perinatal, neonatal and maternal outcomes: a systematic review and meta-analysis

Appendices 1–5 (DOCX 222 kb

Influenza Transmission in the Mother-Infant Dyad Leads to Severe Disease, Mammary Gland Infection, and Pathogenesis by Regulating Host Responses

<div><p>Seasonal influenza viruses are typically restricted to the human upper respiratory tract whereas influenza viruses with greater pathogenic potential often also target extra-pulmonary organs. Infants, pregnant women, and breastfeeding mothers are highly susceptible to severe respiratory disease following influenza virus infection but the mechanisms of disease severity in the mother-infant dyad are poorly understood. Here we investigated 2009 H1N1 influenza virus infection and transmission in breastfeeding mothers and infants utilizing our developed infant-mother ferret influenza model. Infants acquired severe disease and mortality following infection. Transmission of the virus from infants to mother ferrets led to infection in the lungs and mother mortality. Live virus was also found in mammary gland tissue and expressed milk of the mothers which eventually led to milk cessation. Histopathology showed destruction of acini glandular architecture with the absence of milk. The virus was localized in mammary epithelial cells of positive glands. To understand the molecular mechanisms of mammary gland infection, we performed global transcript analysis which showed downregulation of milk production genes such as Prolactin and increased breast involution pathways indicated by a STAT5 to STAT3 signaling shift. Genes associated with cancer development were also significantly increased including JUN, FOS and M2 macrophage markers. Immune responses within the mammary gland were characterized by decreased lymphocyte-associated genes CD3e, IL2Ra, CD4 with IL1β upregulation. Direct inoculation of H1N1 into the mammary gland led to infant respiratory infection and infant mortality suggesting the influenza virus was able to replicate in mammary tissue and transmission is possible through breastfeeding. In vitro infection studies with human breast cells showed susceptibility to H1N1 virus infection. Together, we have shown that the host-pathogen interactions of influenza virus infection in the mother-infant dyad initiate immunological and oncogenic signaling cascades within the mammary gland. These findings suggest the mammary gland may have a greater role in infection and immunity than previously thought.</p></div

Virus replication and pathology in mammary glands of mothers nursing 2009 H1N1-infected infants.

<p>Infant ferrets were intranasally inoculated with A/Cal and housed with mother ferrets for a 7 Day time course where mammary glands, milk, blood, and feces were collected as shown in the schematic (<b>A</b>). Live virus was quantified by MDCK titration of homogenized mammary tissue (<b>B</b>), nipples (<b>C</b>), and expressed milk (<b>D</b>) from nursing-mothers of intranasal inoculated infant ferrets. qRT-PCR was performed on expressed milk for 2009 H1N1 viral RNA (vRNA) (<b>E</b>). Viral presence was also determined by qRT-PCR in infant feces (post-direct-inoculation) (<b>F</b>) and infant, mother, and adult ferret blood (3/4 days post-inoculation) (<b>G</b>). ND = Not Detected. Samples were collected and analyzed from at least 3 independent litter inoculated/infected infants and 3 mothers per time point. Results show the mean or are representative of 3 litters per time point. Mother ferrets have variable numbers of active mammary glands per pregnancy/postpartum.</p

STAT5 protein is decreased and STAT3 protein is nuclear localized in H1N1+ Mammary Glands.

<p>STAT5 (right panels) and STAT3 (left panels) protein expression was visualized in Control and H1N1+MG on Day 7 post-infant-inoculation by IHC. IHC analysis of paraffin-embedded mammary sections from nursing-mothers of infected infants stained with STAT5 or STAT3 antibodies were visualized using an Aperio ScanScope XT, Leica Biosystems, Nußloch, Germany for high resolution scans. The inset picture shows a magnification of the scanned image to show cellular detail. Scale bars indicate 100 μm or 10 μm. The images show a representative of mammary glands collected from infant intranasal virus or mock inoculations (three inoculations each).</p

Human “normal” and adenocarcinoma mammary epithelial cells are susceptible and permissive to 2009 H1N1 infection <i>in vitro</i>.

<p>Mammary epithelial cells (MCF-7, MDA-MB-231, MCF-10A cells) inoculated at an MOI of 1 with A/Cal (H1N1) were fixed at 24 hours post-inoculation and stained for filamentous actin (red), DNA (green), and influenza A virus NP protein (blue), and imaged by confocal microscopy (<b>A</b>). Mammary epithelial cells (MCF-7, MDA-MB-231, MCF-10A cells) inoculated at an MOI of 1 with A/Cal (H1N1) were collected at 3, 24, 48, and 72 h post-inoculation for quantification of viral RNA segment 7 by qRT-PCR (<b>B</b>), determination of cell viability (<b>C</b>), and live virus quantification in supernatant (<b>D</b>). Confocal pictures are representative of three independent experiments. White arrows indicate nuclear localization of influenza NP protein. Yellow arrows show virus budding along the plasma membrane. BC, Baseline Control.</p

Influenza virus inoculation into active mammary glands of nursing-mother ferrets leads to respiratory infection, severe disease and mortality in infants.

<p>A schematic depicting the mammary gland inoculation design is shown (<b>A</b>). Temperature (left panel) and weights (right panel) of lactating mother ferrets following 2009 H1N1 (pink lines) or PBS (light grey lines) mammary inoculation (<b>B</b>). Temperature (left panel) and weights (right panel) of infants feeding from lactating mothers with 2009 H1N1 (pink lines) or PBS (light grey lines) inoculated mammary glands (<b>C</b>). Survival of mother and infant ferrets following mammary 2009 H1N1 virus inoculation (<b>D</b>). Live viral load determined from nasal wash in both infant and mothers (<b>E</b>), and live virus shedding from expressed milk (<b>F</b>) following mammary gland 2009 H1N1 inoculation. * Indicates a p-value less than 0.05 determined by ANOVA. Error bars indicate +/- SD. Results are the mean of or represent three independent mammary gland inoculation experiments (3 mammary inoculated mothers, 18 infants) and mock mammary inoculation controls (3 mammary mock inoculated mothers, 21 infants).</p

2009 H1N1 transmission from mothers to infants results in severe lower respiratory tract pathology.

<p>Harvested lungs from control infants and infants of inoculated nursing-mothers were processed for histopathological assessment. Tissue morphology was assessed by hematoxylin & eosin staining. Data was collected from three independent litter inoculations/infections (3 inoculated/infected mothers, 16 infants, and 3 mock inoculated/infected mothers) and results are a representative of the inoculations/infections. pmi = Post-Mother-Inoculation</p

Transmission of H1N1 influenza from infants to mother ferrets causes upper and lower respiratory tract infection with significant pathology.

<p>Inoculated infants (A/Cal H1N1 influenza (10⁵ EID₅₀)) were housed with their nursing-mothers. Nasal washes were collected daily from infants and mothers. Live viral loads were determined by MDCK titration assay in nasal wash (NW) (<b>A</b>) and mother trachea and mother lungs at specific time points (<b>B</b>). Lungs were harvested on Day 3/4 and 7 post-infant-inoculation for hematoxylin & eosin (H&E) histopathological assessment from nursing-mothers of inoculated infants and directly inoculated adult ferrets as control (<b>C</b>). Green arrows denote dense cell accumulation; black arrows denote diffuse immune cell infiltration. Black arrows not included on Day 7 adult tissue due to widespread infiltration. High resolution scans were performed using an Aperio ScanScope XT, Leica Biosystems, Nußloch, Germany. The left and central columns of mother images represent a low and high magnification of each lung scan. The scale bars indicate the relative 100 μm. Results of <b>A</b> are representative of 3 independent litter inoculations where nasal washes were collected from 3 mothers and 9 infants (3 per litter) daily. Results of <b>B</b> and <b>C</b> are from 6 independent litter inoculations of serial tissue collections (3 collected Day 3/4 and 3 collected Day 7). Error bars indicate +/- SD. Mock: mother ferrets nursing mock-inoculated 4-week-old infant ferrets. Results show the mean or are representative of independent litter inoculations/infections.</p