Direct measurement of stool consistency by texture analyzer and calculation of reference value in Belgian general population.

Stool consistency is evaluated mainly in reference to indirect indicators such as water content or the appearance of stool forms using Bristol Stool Form Scale (BSFS). Methods of measurement are limited. We thus aimed to develop a simple protocol for direct measurement of stool consistency using the TA.XTExpress Texture Analyser (Stable Micro Systems Ltd.). We developed a protocol which enables mechanical quantification of the gram-force against a cylindrical probe (ø 6 mm) pushed into the stool surface at 2.0 mm/s to 5 mm depth. The consistency of 252 stools collected from 40 healthy Belgians was evaluated by the direct method and by the indirect indicators (water content and BSFS) for comparison. The log-transformed stool consistency values measured by the texture analyzer had a negative linear correlation with the stool water contents (rrm = - 0.781) with homoscedastic variance, suggesting the appropriateness of the new protocol. They showed a similar correlation with the BSFS, but with a large variance in the consistency values of normal stool forms. This correlation was much smaller for BSFS scored by subjects (rrm = - 0.587) than by experts (rrm = - 0.789), collectively indicating BSFS as a rough indicator of stool consistency susceptible to subjective bias despite its effectiveness in clinical use. The optimized direct method using the texture analyzer enables the accurate quantification of stool consistency, which facilitates understanding of the intestinal environment and function and thus may enhance the value of the stool as a predictor of human health.

Short communication: Probiotic induction of interleukin-10 and interleukin-12 production by macrophages is modulated by co-stimulation with microbial components.

Probiotic lactobacilli stimulate macrophages and dendritic cells to secrete cytokines and thereby regulate the immune responses of the host. The balance of the IL-10 and IL-12 production induced by a probiotic is crucial for determining the direction of the immune response. In the present study, we examined the ability of microbial components to modify IL-10 and IL-12 production induced by a popular probiotic strain, Lactobacillus casei strain Shirota (LcS), which itself predominantly induces IL-12 production. Microbial ligands for toll-like receptor (TLR)3 and TLR5 further enhanced the IL-12 induction by LcS, whereas ligands for TLR2, TLR4, TLR7, and TLR9 converted the cytokine production pattern from IL-12 predominant to IL-10 predominant. These results indicate that the probiotic induction of IL-10 and IL-12 production can be flexibly modified by co-stimulation with microbial components. This could explain the variety of immunomodulatory functions (immunoactivation or anti-inflammation) exerted by this probiotic strain.

Microbiota of the Small Intestine Is Selectively Engulfed by Phagocytes of the Lamina Propria and Peyer's Patches.

Phagocytes such as dendritic cells and macrophages, which are distributed in the small intestinal mucosa, play a crucial role in maintaining mucosal homeostasis by sampling the luminal gut microbiota. However, there is limited information regarding microbial uptake in a steady state. We investigated the composition of murine gut microbiota that is engulfed by phagocytes of specific subsets in the small intestinal lamina propria (SILP) and Peyer's patches (PP). Analysis of bacterial 16S rRNA gene amplicon sequences revealed that: 1) all the phagocyte subsets in the SILP primarily engulfed Lactobacillus (the most abundant microbe in the small intestine), whereas CD11bhi and CD11bhiCD11chi cell subsets in PP mostly engulfed segmented filamentous bacteria (indigenous bacteria in rodents that are reported to adhere to intestinal epithelial cells); and 2) among the Lactobacillus species engulfed by the SILP cell subsets, L. murinus was engulfed more frequently than L. taiwanensis, although both these Lactobacillus species were abundant in the small intestine under physiological conditions. These results suggest that small intestinal microbiota is selectively engulfed by phagocytes that localize in the adjacent intestinal mucosa in a steady state. These observations may provide insight into the crucial role of phagocytes in immune surveillance of the small intestinal mucosa.

Enhanced differentiation of intraepithelial lymphocytes in the intestine of polymeric immunoglobulin receptor-deficient mice.

To clarify the effect of secretory IgA (sIgA) deficiency on gut homeostasis, we examined intraepithelial lymphocytes (IELs) in the small intestine (SI) of polymeric immunoglobulin receptor-deficient (pIgR(-/-) ) mice. The pIgR(-/-) mice exhibited the accumulation of CD8αß(+) T-cell receptor (TCR)-αß(+) IELs (CD8αß(+) αß-IELs) after weaning, but no increase of CD8αß(+) γδ-IELs was detected in pIgR(-/-) TCR-ß(-/-) mice compared with pIgR(+/+) TCR-ß(-/-) mice. When 5-bromo-2'-deoxyuridine (BrdU) was given for 14 days, the proportion of BrdU-labelled cells in SI-IELs was not different between pIgR(+/+) mice and pIgR(-/-) mice. However, the proportion of BrdU-labelled CD8αß(+) -IELs became higher in pIgR(-/-) mice than pIgR(+/+) mice 10 days after discontinuing BrdU-labelling. Intravenously transferred splenic T cells migrated into the intraepithelial compartments of pIgR(+/+) TCR-ß(-/-) mice and pIgR(-/-) TCR-ß(-/-) mice to a similar extent. In contrast, in the case of injection of immature bone marrow cells, CD8αß(+) αß-IELs increased much more in the SI of pIgR(-/-) TCR-ß(-/-) mice than pIgR(+/+) TCR-ß(-/-) mice 8 weeks after the transfer. αß-IELs from pIgR(-/-) mice could produce more interferon-γ and interleukin-17 than those of pIgR(+/+) mice, and intestinal permeability tended to increase in the SI of pIgR(-/-) mice with aging. Taken together, these results indicate that activated CD8αß(+) αß-IELs preferentially accumulate in pIgR(-/-) mice through the enhanced differentiation of immature haematopoietic precursor cells, which may subsequently result in the disruption of epithelial integrity.

Probiotic upregulation of peripheral IL-17 responses does not exacerbate neurological symptoms in experimental autoimmune encephalomyelitis mouse models.

CONTEXT: It is of great importance to evaluate the safety of probiotics in dysregulated immune conditions, as probiotics can possibly modulate immune functions in the host. OBJECTIVE: We tried to confirm the safety of using Lactobacillus casei strain Shirota (LcS) to help prevent autoimmunity in the central nervous system. METHODS: We used two chronic experimental autoimmune encephalomyelitis (EAE) models, a relapse and remission type EAE model in SJL/J mice and a durable type model in C57BL/6 mice. LcS was administered from 1 week before antigen sensitization until the end of the experiments, and neurological symptoms and histopathological changes of the spinal cord were observed. Immunological parameters were also examined in the SJL/J mouse model. RESULTS: LcS administration did not exacerbate neurological symptoms or histopathological changes of the spinal cord in either model but instead tended to improve neurological symptoms in the SJL/J mouse EAE model. LcS administration transiently upregulated IL-17 production by antigen-stimulated lymphocytes of draining lymph nodes 7 days after sensitization. Enhanced production of IL-10 and an increase in the percentage of CD4(+)CD25(+) T regulatory cells were also observed at the same sites. Strong expression of IL-17 mRNA was detected in the spinal cord of mice that displayed severe neurological symptoms on day 12, but this expression was not enhanced by LcS administration. CONCLUSION: These results demonstrate that LcS does not exacerbate, but instead may improve EAE depending on the immunization conditions, and that IL-17 responses at peripheral sites may not always result in a worsening of autoimmune diseases.

Duox maturation factors form cell surface complexes with Duox affecting the specificity of reactive oxygen species generation.

Dual oxidases (Duox1 and Duox2) are plasma membrane-targeted hydrogen peroxide generators that support extracellular hemoperoxidases. Duox activator 2 (Duoxa2), initially described as an endoplasmic reticulum resident protein, functions as a maturation factor needed to deliver active Duox2 to the cell surface. However, less is known about the Duox1/Duoxa1 homologues. We identified four alternatively spliced Duoxa1 variants and explored their roles in Duox subcellular targeting and reconstitution. Duox1 and Duox2 are functionally rescued by Duoxa2 or the Duoxa1 variants that contain the third coding exon. All active maturation factors are cotransported to the cell surface when coexpressed with either Duox1 or Duox2, consistent with detection of endogenous Duoxa1 on apical plasma membranes of the airway epithelium. In contrast, the Duoxa proteins are retained in the endoplasmic reticulum when expressed without Duox. Duox1/Duoxa1alpha and Duox2/Duoxa2 pairs produce the highest levels of hydrogen peroxide, as they undergo Golgi-based carbohydrate modifications and form stable cell surface complexes. Cross-functioning pairs that do not form stable complexes produce less hydrogen peroxide and leak superoxide. These findings suggest Duox activators not only promote Duox maturation, but they function as part of the hydrogen peroxide-generating enzyme.

Subcellular localization and function of alternatively spliced Noxo1 isoforms.

Nox organizer 1 (Noxo1), a p47(phox) homolog, is produced as four isoforms with unique N-terminal PX domains derived by alternative mRNA splicing. We compared the subcellular distribution of these isoforms or their isolated PX domains produced as GFP fusion proteins, as well as their ability to support Nox1 activity in several transfected models. Noxo1alpha, beta, gamma, and delta show different subcellular localization patterns, determined by their PX domains. In HEK293 cells, Noxo1beta exhibits prominent plasma membrane binding, Noxo1gamma shows plasma membrane and nuclear associations, and Noxo1alpha and delta localize primarily on intracellular vesicles or cytoplasmic aggregates, but not the plasma membrane. Nox1 activity correlates with Noxo1 plasma membrane binding in HEK293 cells, since Noxo1beta supports the highest activity and Noxo1gamma and Noxo1alpha support moderate or low activities, respectively. In COS-7 cells, where Noxo1alpha localizes on the plasma membrane, the activities supported by the three isoforms (alpha, beta, and gamma) do not differ significantly. The PX domains of beta and gamma bind the same phospholipids, including phosphatidic acid. These results indicate that the variant PX domains are unique determinants of Noxo1 localization and Nox1 function. Finally, the overexpressed Noxo1 isoforms do not affect p22(phox) localization, although Nox1 is needed to transport p22(phox) to the plasma membrane.

A regulated adaptor function of p40phox: distinct p67phox membrane targeting by p40phox and by p47phox.

In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47(phox), p67(phox), p40(phox), and Rac) translocate and associate with the membrane-spanning flavocytochrome b(558), leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40(phox) that regulate its translocation to membranes upon stimulation. GFP-p40(phox) translocates to early endosomes, whereas GFP-p47(phox) translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67(phox) does not translocate to membranes when expressed alone, but it is dependent on p40(phox) and p47(phox) for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40(phox) or GFP-p47(phox) to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67(phox) translocation to either membrane is abolished by mutations that disrupt its interaction with p40(phox) or p47(phox). Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40(phox) in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40(phox) and p47(phox) function as diverse p67(phox) "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms.