Journal: Science translational medicine

Article Title: The systemic anti-microbiota IgG repertoire can identify gut bacteria that translocate across gut barrier surfaces

doi: 10.1126/scitranslmed.abl3927

Figure Lengend Snippet: (A) Shown is the IgA-seq workflow. Fecal bacteria from mice and humans were incubated with phycoerythrin (PE)–conjugated anti-IgA antibodies, after which anti-PE microbeads facilitated magnetic separation of IgA-bound bacteria from IgA-unbound bacteria. These two fractions were subjected to 16S rRNA sequencing, and IgA scores for each taxon [i.e., amplicon sequence variant (ASV)] were calculated as the log ratio of taxon relative abundance in the IgA-bound fraction over that in the unbound fraction. (B) Shown is the IgG-seq workflow. Fecal bacteria were incubated with autologous sera and stained with PE anti-IgG antibodies, fractionated, and sequenced as for IgA-seq. ( C ) Many gut bacterial taxa in stool samples exhibited differential binding by mucosal IgA and serum IgG from healthy individuals ( n = 12). Taxa with 10-fold higher IgA scores than IgG scores are depicted in purple, whereas taxa with 10-fold higher IgG scores are in green. ( D and E ) IgA scores correlated more strongly with baseline fecal relative abundances than IgG scores. (D) Shown are absolute values of all Spearman rho values for correlations comparing IgA or IgG scores to fecal relative abundances among taxa whose IgA and IgG scores differed by 10-fold [green and purple dots in (C)]. (E) Spearman P values for the same comparisons as in (D). Paired ratio t tests were performed for (D) and on log-transformed values of (E).

Article Snippet: Taxon IgG scores were converted to z scores (centered and scaled across all participants on a per-translocator basis) and averaged within each participant category (healthy, IBD translocation-positive, and IBD translocation-negative) as inputs into the linear mixed effects model. IBD translocation-positive and IBD translocation-negative participants together consisted of 18 participants with CD and 11 participants with UD ( n = 29 total), whereas healthy, non-IBD participants that did not undergo mesenteric sampling were n = 9.

Techniques: Bacteria, Incubation, Sequencing, Amplification, Variant Assay, Staining, Binding Assay, Transformation Assay

Journal: Science translational medicine

Article Title: The systemic anti-microbiota IgG repertoire can identify gut bacteria that translocate across gut barrier surfaces

doi: 10.1126/scitranslmed.abl3927

Figure Lengend Snippet: ( A ) Shown is the experimental plan. Livers and spleens of acute T. gondii –infected mice were processed and subjected to bacterial culture at day 3 after infection. Bacterial colonies were identified using full-length 16 S rRNA sequencing. IgA-seq and IgG-seq were performed at day 28 after infection ( n = 17 mice for IgA-seq and n = 15 mice for IgG-seq, across three independent experiments). ( B ) Shown are translocating bacterial species that were cultured from mouse livers or spleens in one or more independent experiments. Translocation index refers to the number of independent experiments ( n = 4 to 7 T. gondii –infected mice per experiment) in which translocating bacterial species was cultured. Mean fold differences in IgA and IgG scores between T. gondii –infected and uninfected mice were calculated for each taxon, and Mann-Whitney U tests were performed to calculate P values.

Article Snippet: Taxon IgG scores were converted to z scores (centered and scaled across all participants on a per-translocator basis) and averaged within each participant category (healthy, IBD translocation-positive, and IBD translocation-negative) as inputs into the linear mixed effects model. IBD translocation-positive and IBD translocation-negative participants together consisted of 18 participants with CD and 11 participants with UD ( n = 29 total), whereas healthy, non-IBD participants that did not undergo mesenteric sampling were n = 9.

Techniques: Bacteria, Translocation Assay, Infection, Sequencing, Cell Culture, MANN-WHITNEY

Journal: Science translational medicine

Article Title: The systemic anti-microbiota IgG repertoire can identify gut bacteria that translocate across gut barrier surfaces

doi: 10.1126/scitranslmed.abl3927

Figure Lengend Snippet: ( A ) Shown is the SFC-IgG-seq workflow. In the absence of stool samples from study participants, a surrogate fecal community (SFC) comprising a combination of healthy donor feces from a separate cohort was prepared [see fig. S3 ( A and B )]. Fecal bacteria and sera were co-incubated, and the IgG-unbound fraction was sequenced along with triplicate pre-enrichment aliquots of the SFC. IgG scores were calculated, comparing taxon abundances to the pre-enrichment community (see fig. S3C ). ( B ) To identify translocating bacteria in a cohort of patients with IBD undergoing medically indicated bowel resection surgery, mesenteric adipose tissue from resected small and large intestinal samples was subjected to bacterial culture. Full-length 16 S rRNA sequencing was then performed on bacterial colonies to identify translocating bacteria for each individual. ( C ) SFC-IgG-seq was performed using serum samples from n = 29 patients with IBD and n = 9 healthy individuals. For each taxon, mean scaled IgG scores were compared across healthy individuals, patients with IBD with no detected translocation for that taxon, and patients with IBD for whom that taxon was cultured from the mesenteric adipose tissue (“translocation positive”). These groups were treated as ordinal variables in that order, and linear regression on IgG scores was performed, grouping all taxa ( P = 0.00015). IgG scores for translocating bacterial taxa (translocator) were elevated in translocation-positive patients with IBD compared to translocation-negative patients with IBD ( P = 0.0268, paired two-sided Student’s t test). * P < 0.05.

Article Snippet: Taxon IgG scores were converted to z scores (centered and scaled across all participants on a per-translocator basis) and averaged within each participant category (healthy, IBD translocation-positive, and IBD translocation-negative) as inputs into the linear mixed effects model. IBD translocation-positive and IBD translocation-negative participants together consisted of 18 participants with CD and 11 participants with UD ( n = 29 total), whereas healthy, non-IBD participants that did not undergo mesenteric sampling were n = 9.

Techniques: Bacteria, Incubation, Sequencing, Translocation Assay, Cell Culture