Maternal Metabolic Biomarkers are Associated with Obesity and Excess Gestational Weight Gain.

OBJECTIVE: The purpose of this study was to evaluate the independent contribution of maternal obesity and gestational weight gain (GWG) in excess of the Institute of Medicine's guidelines on levels of maternal serum inflammatory and metabolic measures. STUDY DESIGN: Banked maternal serum samples from 120 subjects with documented prepregnancy or first trimester body mass index (BMI) were utilized for analyte analyses. Validated, BMI-specific formulas were utilized to categorize GWG as either insufficient, at goal or excess based on the Institute of Medicine guidelines with gestational age adjustments. Serum was analyzed for known inflammatory or metabolic pathway intermediates using the Luminex xMap system with the MILLIPLEX Human Metabolic Hormone Magnetic Bead Panel. Measured analytes included interleukin-6, monocyte chemoattractant protein-1, and tumor necrosis factor-α and metabolic markers amylin, c-peptide, ghrelin, gastric inhibitory polypeptide, glucagon-like peptide-1, glucagon, insulin, leptin, pancreatic polypeptide, and peptide YY. Kruskal-Wallis ANOVA and Pearson's correlation coefficients were calculated for each marker. RESULTS: C-peptide, insulin, and leptin all varied significantly with both obesity and GWG while glucagon-like peptide-1 varied by BMI but not GWG. These analytes covaried with other metabolic analytes, but not with inflammatory analytes. CONCLUSION: Maternal metabolic biomarkers at delivery vary significantly with both obesity and GWG. Taken together, these findings suggest that GWG (with and without comorbid obesity) is an important mediator of measurable metabolites in pregnancy but is not necessarily accompanied by inflammatory measures in serum. These findings are consistent with GWG being an independent risk factor for metabolic disturbances during pregnancy.

Lactobacillus rhamnosus L34 and Lactobacillus casei L39 suppress Clostridium difficile-induced IL-8 production by colonic epithelial cells.

BACKGROUND: Clostridium difficile is the main cause of hospital-acquired diarrhea and colitis known as C. difficile-associated disease (CDAD).With increased severity and failure of treatment in CDAD, new approaches for prevention and treatment, such as the use of probiotics, are needed. Since the pathogenesis of CDAD involves an inflammatory response with a massive influx of neutrophils recruited by interleukin (IL)-8, this study aimed to investigate the probiotic effects of Lactobacillus spp. on the suppression of IL-8 production in response to C. difficile infection. RESULTS: We screened Lactobacillus conditioned media from 34 infant fecal isolates for the ability to suppress C. difficile-induced IL-8 production from HT-29 cells. Factors produced by two vancomycin-resistant lactobacilli, L. rhamnosus L34 (LR-L34) and L.casei L39 (LC-L39), suppressed the secretion and transcription of IL-8 without inhibiting C. difficile viability or toxin production. Conditioned media from LR-L34 suppressed the activation of phospho-NF-κB with no effect on phospho-c-Jun. However, LC-L39 conditioned media suppressed the activation of both phospho-NF-κB and phospho-c-Jun. Conditioned media from LR-L34 and LC-L39 also decreased the production of C. difficile-induced GM-CSF in HT-29 cells. Immunomodulatory factors present in the conditioned media of both LR-L34 and LC-L39 are heat-stable up to 100°C and > 100 kDa in size. CONCLUSIONS: Our results suggest that L. rhamnosus L34 and L. casei L39 each produce factors capable of modulating inflammation stimulated by C. difficile. These vancomycin-resistant Lactobacillus strains are potential probiotics for treating or preventing CDAD.

Anti-inflammatory properties of gastric-derived Lactobacillus plantarum XB7 in the context of Helicobacter pylori infection.

BACKGROUND: Helicobacter pylori colonization of the gastric epithelium induces interleukin-8 (IL-8) production and inflammation leading to host cell damage. We searched for gastric-derived Lactobacillus with the ability to suppress H. pylori-induced inflammation. MATERIALS AND METHODS: Conditioned media from gastric-derived Lactobacillus spp. were tested for the ability to suppress H. pylori-induced IL-8 production in AGS gastric epithelial cells. IL-8 protein and mRNA levels were measured by ELISA and qPCR, respectively. The changes on host cell signaling pathway were analyzed by Western blotting and the anti-inflammatory effect was tested in a Sprague-Dawley rat model. RESULTS: Conditioned media from L. salivarius B101, L. rhamnosus B103, and L. plantarum XB7 suppressed IL-8 production and IL-8 mRNA expression in H. pylori-induced AGS cells without inhibiting H. pylori growth. Conditioned media from LS-B101, LR-B103, and LP-XB7 suppressed the activation of NF-κB in AGS cells, while strain LP-XB7 also suppressed c-Jun activation. The anti-inflammatory effect of LP-XB7 was further assessed in vivo using a H. pylori-infected Sprague-Dawley rat model. Strain LP-XB7 contributed to a delay in the detection and colonization of H. pylori in rat stomachs, attenuated gastric inflammation, and ameliorated gastric histopathology. Additionally, the administration of LP-XB7 correlated with the suppression of TNF-α and CINC-1 in sera, and suppression of CINC-1 in the gastric mucosa of H. pylori-infected rats. CONCLUSIONS: These results suggest that L. plantarum XB7 produces secreted factors capable of modulating inflammation during H. pylori infection, and this probiotic Lactobacillus strain shows promise as an adjunctive therapy for treating H. pylori-associated disease.

Probiotics stimulate enterocyte migration and microbial diversity in the neonatal mouse intestine.

Beneficial microbes and probiotics show promise for the treatment of pediatric gastrointestinal diseases. However, basic mechanisms of probiosis are not well understood, and most investigations have been performed in germ-free or microbiome-depleted animals. We sought to functionally characterize probiotic-host interactions in the context of normal early development. Outbred CD1 neonatal mice were orally gavaged with one of two strains of human-derived Lactobacillus reuteri or an equal volume of vehicle. Transcriptome analysis was performed on enterocyte RNA isolated by laser-capture microdissection. Enterocyte migration and proliferation were assessed by labeling cells with 5-bromo-2'-deoxyuridine, and fecal microbial community composition was determined by 16S metagenomic sequencing. Probiotic ingestion altered gene expression in multiple canonical pathways involving cell motility. L. reuteri strain DSM 17938 dramatically increased enterocyte migration (3-fold), proliferation (34%), and crypt height (29%) compared to vehicle-treated mice, whereas strain ATCC PTA 6475 increased cell migration (2-fold) without affecting crypt proliferative activity. In addition, both probiotic strains increased the phylogenetic diversity and evenness between taxa of the fecal microbiome 24 h after a single probiotic gavage. These experiments identify two targets of probiosis in early development, the intestinal epithelium and the gut microbiome, and suggest novel mechanisms for probiotic strain-specific effects.

Host response to probiotics determined by nutritional status of rotavirus-infected neonatal mice.

OBJECTIVES: Beneficial microbes and probiotics are promising agents for the prevention and treatment of enteric and diarrheal diseases in children; however, little is known about their in vivo mechanisms of action. We used a neonatal mouse model of rotavirus diarrhea to gain insight into how probiotics ameliorate acute gastroenteritis. METHODS: Rotavirus-infected mice were treated with 1 of 2 strains of human-derived Lactobacillus reuteri. We assessed intestinal microbiome composition with 16S metagenomic sequencing, enterocyte migration and proliferation with 5-bromo-2'-deoxyuridine, and antibody and cytokine concentrations with multiplex analyses of intestinal explant cultures. RESULTS: Probiotics reduced diarrhea duration, improved intestinal histopathology, and enhanced intestinal microbiome richness and phylogenetic diversity. The magnitude of reduction of diarrhea by probiotics was strain specific and influenced by nutritional status. L reuteri DSM 17938 reduced diarrhea duration by 0, 1, and 2 days in underweight, normal weight, and overweight pups, respectively. The magnitude of reduction of diarrhea duration correlated with increased enterocyte proliferation and migration. Strain ATCC PTA 6475 reduced diarrhea duration by 1 day in all of the mice without increasing enterocyte proliferation. Both probiotic strains decreased concentrations of proinflammatory cytokines, including macrophage inflammatory protein-1α and interleukin-1ß, in all of the animals, and increased rotavirus-specific antibodies in all but the underweight animals. Body weight also influenced the host response to rotavirus, in terms of diarrhea duration, enterocyte turnover, and antibody production. CONCLUSIONS: These data suggest that probiotic enhancement of enterocyte proliferation, villus repopulation, and virus-specific antibodies may contribute to diarrhea resolution, and that nutritional status influences the host response to both beneficial microbes and pathogens.

The Human Nose Organoid Respiratory Virus Model: an Ex Vivo Human Challenge Model To Study Respiratory Syncytial Virus (RSV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Pathogenesis and Evaluate Therapeutics.

There is an unmet need for preclinical models to understand the pathogenesis of human respiratory viruses and predict responsiveness to immunotherapies. Airway organoids can serve as an ex vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a noninvasive technique to generate human nose organoids (HNOs) as an alternative to biopsy-derived organoids. We made air-liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hypersecretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation), while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration-dependent manner. Thus, the HNO-ALI model can serve as an alternative to lung organoids to study respiratory viruses and test therapeutics. IMPORTANCE Preclinical models that recapitulate aspects of human airway disease are essential for the advancement of novel therapeutics and vaccines. Here, we report a versatile airway organoid model, the human nose organoid (HNO), that recapitulates the complex interactions between the host and virus. HNOs are obtained using noninvasive procedures and show divergent responses to SARS-CoV-2 and RSV infection. SARS-CoV-2 induces severe damage to cilia and the epithelium, no interferon-λ response, and minimal mucus secretion. In striking contrast, RSV induces hypersecretion of mucus and a profound interferon-λ response with ciliary damage. We also demonstrated the usefulness of our ex vivo HNO model of RSV infection to test the efficacy of palivizumab, an FDA-approved monoclonal antibody to prevent severe RSV disease in high-risk infants. Our study reports a breakthrough in both the development of a novel nose organoid model and in our understanding of the host cellular response to RSV and SARS-CoV-2 infection.

The human nose organoid respiratory virus model: an ex-vivo human challenge model to study RSV and SARS-CoV-2 pathogenesis and evaluate therapeutics.

There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.

From prediction to function using evolutionary genomics: human-specific ecotypes of Lactobacillus reuteri have diverse probiotic functions.

The vertebrate gut symbiont Lactobacillus reuteri has diversified into separate clades reflecting host origin. Strains show evidence of host adaptation, but how host-microbe coevolution influences microbial-derived effects on hosts is poorly understood. Emphasizing human-derived strains of L. reuteri, we combined comparative genomic analyses with functional assays to examine variations in host interaction among genetically distinct ecotypes. Within clade II or VI, the genomes of human-derived L. reuteri strains are highly conserved in gene content and at the nucleotide level. Nevertheless, they share only 70-90% of total gene content, indicating differences in functional capacity. Human-associated lineages are distinguished by genes related to bacteriophages, vitamin biosynthesis, antimicrobial production, and immunomodulation. Differential production of reuterin, histamine, and folate by 23 clade II and VI strains was demonstrated. These strains also differed with respect to their ability to modulate human cytokine production (tumor necrosis factor, monocyte chemoattractant protein-1, interleukin [IL]-1ß, IL-5, IL-7, IL-12, and IL-13) by myeloid cells. Microarray analysis of representative clade II and clade VI strains revealed global regulation of genes within the reuterin, vitamin B12, folate, and arginine catabolism gene clusters by the AraC family transcriptional regulator, PocR. Thus, human-derived L. reuteri clade II and VI strains are genetically distinct and their differences affect their functional repertoires and probiotic features. These findings highlight the biological impact of microbe:host coevolution and illustrate the functional significance of subspecies differences in the human microbiome. Consideration of host origin and functional differences at the subspecies level may have major impacts on probiotic strain selection and considerations of microbial ecology in mammalian species.

Rapid prenatal diagnosis using uncultured amniocytes and oligonucleotide array CGH.

OBJECTIVE: Oligonucleotide-based array comparative genomic hybridization (array CGH) is an established method for detecting chromosomal abnormalities. Here, we explored the feasibility of using DNA extracted from uncultured amniocytes in amniotic fluid for array CGH on an oligonucleotide array platform. METHODS: Fifteen fetuses from 14 ongoing pregnancies were studied by array CGH on targeted oligonucleotide arrays with DNA isolated from direct amniotic fluid using a modified DNA extraction protocol. RESULTS: High-quality array CGH results were obtained for 13 samples with suboptimal but interpretable results in only 2 samples due to limited DNA amounts. Array CGH using whole genome amplification (WGA) of DNA for the two cases with limited DNA was successful, and results were consistent with those from unamplified DNA. For another five samples, the results of array CGH with amplified DNA matched those with unamplified DNA. Chromosome analysis was performed for 14 cases and was consistent with array CGH results. CONCLUSION: This study demonstrates the feasibility of prenatal genetic diagnosis using oligonucleotide array CGH analysis for direct analysis of amniocytes without culturing cells. The use of oligonucleotide arrays increases the sensitivity and accuracy of detection over previous bacterial artificial chromosome (BAC)-based arrays. Furthermore, the direct analysis allows for rapid array CGH results and shorter reporting time.