Background: Most lactic acid bacteria are non-motile but some of them are flagellated and exhibit motility. So far, motile lactobacilli have rarely been studied, and characteristics of their flagellins are poorly understood. In this study, a highly motile strain of Lactobacillus agilis was recruited for transcriptional analysis and characterization of its flagellins. Results: Unlike another motile lactic acid bacteria of intestinal isolate, Lactobacillus ruminis, flagellar filaments of the L. agilis strain probably consist of two homologous but distinct flagellins. Glycosylation of the flagellar filaments and their resistance to heat, acid and SDS were also observed. The immunological activity of the flagellins was evaluated through the stimulation of Caco-2 cells. The results show that TLR5-stimulating activity of the protein is attenuated, likely due to an incomplete TLR5-recognition site.

Sixty healthy Japanese women with a defaecation frequency of 2-4 times/week participated in this randomised, double-blind crossover trial. Participants received 2 g/day lactulose for 2 weeks and placebo in a random order, separated by a washout period of 3 weeks. Eight participants were excluded who did not satisfy the conditions, and therefore data from 52 were analysed. The primary outcome was defaecation frequency and the secondary outcomes were the number of defaecation days, faecal consistency, faecal volume, and the number and percentage of Bifidobacterium in faeces. The defaecation frequency (times/week) was significantly higher during lactulose (4.28±0.23) than placebo (3.83±0.23) treatment (delta (Δ) 0.45 [95% confidence interval (CI) 0.10-0.80], P=0.013). The defaecation days (days/week) was significantly higher during lactulose (3.77±0.17) than placebo (3.47±0.17) treatment (Δ0.30 [95% CI 0.04-0.56], P=0.024). Faecal consistency using the Bristol Stool Scale (/defaecation) was significantly higher during lactulose (3.84±0.10) than placebo (3.68±0.10) treatment (Δ0.16 [95% CI 0.00-0.31], P=0.044). Faecal volume (/week) was significantly higher during lactulose (21.73±3.07) than placebo (17.65±3.07) treatment (Δ4.08 [95% CI 0.57-7.60], P=0.024). The number of Bifidobacterium in faeces (log colony forming units/g faeces) was significantly higher during lactulose (9.53±0.06) than placebo (9.16±0.06) treatment (Δ0.37 [95% CI 0.23-0.49], P<0.0001). The percentage of Bifidobacterium in faeces was also significantly higher during lactulose (25.3±1.4) than placebo (18.2±1.4) treatment (Δ7.1 [95% CI 2.9-11.4], P=0.0014). Finally, straining at defaecation (/defaecation) during lactulose (3.62±0.24) treatment was significantly lower than during placebo (3.97±0.24) treatment (Δ0.35 [95% CI -0.69 – -0.02], P=0.037). No significant difference was observed between lactulose and placebo with regard to flatulence. Severe adverse effects did not occur. Thus, oral ingestion of 2 g/day lactulose had a prebiotic effect, increasing the number and percentage of bifidobacteria in faeces, softening the faeces, and increasing defaecation frequency, but without increasing flatulence.

Vaccine delivery systems using lactic acid bacteria are under development, but their efficiency is insufficient. Autologous cytokines, such as interleukin-1␤ (IL-1␤), are potential adjuvants for mucosal vaccines and can be provided by recombinant lactic acid bacteria. The aim of this study was the construction and evaluation of recombinant Lactobacillus casei producing IL-1␤ as an adjuvant delivery agent. The recombinant strain was constructed using an expression/secretion vector plasmid, including a mature IL-1␤ gene from mouse. The biological activity of the cytokine was confirmed by IL-8 production from Caco-2 cells. In response to the recombinant L. casei secreting IL-1␤, expression of IL-6 was detected in vivo using a ligated-intestinal-loop assay. The release of IL-6 from Peyer's patch cells was also detected in vitro. Intragastric immunization with heat-killed Salmonella enterica serovar Enteritidis (SE) in combination with IL-1␤-secreting lactobacilli resulted in relatively high SE-specific antibody production. In this study, it was demonstrated that recombinant L. casei secreting bioactive murine IL-1␤ provided adjuvant effects for intragastric immunization.

Clinical outcomes in colorectal cancer (CRC) have been correlated with T cell infiltrates, but the specific populations of T cells, their functions, and how they influence clinical outcomes remains unclear. To comprehensively investigate the diverse phenotype and function of T cells in CRC, we profiled 37,931 single T cells from tumors and adjacent normal colon of 16 treatment-naïve CRC patients with respect to transcriptome, TCR sequence, and 23 cell surface markers. Our single-cell analysis identified phenotypically and functionally distinguishable effector CD4 + and CD8 + T celltypes within human tumors. We employed single-cell gene signatures from these T cell subsets to query the TCGA database to assess the prognostic significance of these subsets. Among CD8 + Tcell infiltrates, we found two distinct cytotoxic T cell types differentiated into clonally-expanded exhausted T cells. GZMK + KLRG1 + cytotoxic T cells with a less dysfunctional phenotype were enriched in CRC patients with good outcomes. Strikingly, GNLY + CD103 + cytotoxic T cells, including intraepithelial lymphocytes (IELs) with a more dysfunctional phenotype, were not associated with good clinical outcomes, despite high co-expression of CD39 and CD103, markers which denote tumor-reactivity. Together, this suggests that tumor-reactive cytotoxic T cells are effectively targeted to the tumor, yet their presence alone does not contribute to anti-tumor activity due to their impaired function, as reflected in clinical outcomes. Among CD4 + T cell-infiltrates, we found two distinct regulatory T cells (Treg) subtypes associated with opposite clinical outcomes. While total Tregs, predominantly Helios + cells, were associated with good outcomes, Helios -CD38 + peripherally-induced Treg cells (pTregs) were strongly associated with bad outcomes independent of stage. CD38 + pTregs, which shared gene signatures with Th17 cells, possessed a highly suppressive phenotype, suggesting they are the elusive Treg population that inhibits anti-tumor immunity in CRC. These findings highlight the potential utility of these 4 subpopulations in predicting clinical outcomes independent of stage. Furthermore, these observations support the potential for novel CRC therapies directed at CD38 + pTregs or CD8 + CD103 + T cells to augment existing T cell-targeted immunotherapies.

To investigate the prebiotic effect of lactulose at low dosages, we assessed changes in defaecation frequency following ingestion of 1, 2, or 3 g/day of lactulose for 2 weeks. Each test was carried out after a 2-week washout period. This was an open-label, before-after trial that enrolled 26 healthy Japanese women. The defaecation frequency, number of defaecation days, and number of faecal bifidobacteria increased significantly compared with before ingestion of 1, 2, and 3 g/day of lactulose. These results suggest that even 1 g/day of lactulose could have a prebiotic effect.