Using a Deep Learning Method and Data from Two-Dimensional (2D) Marker-Less Video-Based Images for Walking Speed Classification.

Human body measurement data related to walking can characterize functional movement and thereby become an important tool for health assessment. Single-camera-captured two-dimensional (2D) image sequences of marker-less walking individuals might be a simple approach for estimating human body measurement data which could be used in walking speed-related health assessment. Conventional body measurement data of 2D images are dependent on body-worn garments (used as segmental markers) and are susceptible to changes in the distance between the participant and camera in indoor and outdoor settings. In this study, we propose five ratio-based body measurement data that can be extracted from 2D images and can be used to classify three walking speeds (i.e., slow, normal, and fast) using a deep learning-based bidirectional long short-term memory classification model. The results showed that average classification accuracies of 88.08% and 79.18% could be achieved in indoor and outdoor environments, respectively. Additionally, the proposed ratio-based body measurement data are independent of body-worn garments and not susceptible to changes in the distance between the walking individual and camera. As a simple but efficient technique, the proposed walking speed classification has great potential to be employed in clinics and aged care homes.

Denosumab Regulates Gut Microbiota Composition and Cytokines in Dinitrobenzene Sulfonic Acid (DNBS)-Experimental Colitis.

The pro-inflammatory mediator receptor activator of nuclear factor-kappa B ligand (RANKL) plays a significant role in the development of rheumatoid arthritis; however, its role in inflammatory bowel disease is unknown. Genome-wide association meta-analysis for Crohn's disease (CD) identified a variant near the TNFSF11 gene that encodes RANKL and CD risk allele increased expression of RANKL in specific cell lines. This study aims to elucidate if the RANKL inhibitor denosumab can reduce the severity of experimental colitis and modify the gut microbiota composition using murine dinitrobenzenesulfonic acid (DNBS)-experimental model of colitis mimicking CD. In colitic conditions, denosumab treatment significantly decreased the pro-inflammatory cytokines IL-6, IL-1ß, and TNF-α within the colonic mucosa. Moreover, colitis was accompanied by disruption of gut microbiota, and preventative treatment with denosumab modulated this disruption. Denosumab treatment also modified the alpha- and beta diversity of colonic mucosa and fecal microbiota. These results provide a rationale for considering denosumab as a future potential therapy in CD; however, more detailed experimental and clinical studies are warranted.

Reactivation of Intestinal Inflammation Is Suppressed by Catestatin in a Murine Model of Colitis via M1 Macrophages and Not the Gut Microbiota.

While there is growing awareness of a relationship between chromogranin-A (CHGA) and susceptibility to inflammatory conditions, the role of human catestatin [(hCTS); CHGA352-67] in the natural history of established inflammatory bowel disease is not known. Recently, using two different experimental models, we demonstrated that hCTS-treated mice develop less severe acute colitis. We have also shown the implication of the macrophages in this effect. The aims of this study were to determine (1) whether hCTS treatment could attenuate the reactivation of inflammation in adult mice with previously established chronic colitis; (2) whether this effect is mediated through macrophages or the gut microbiota. Quiescent colitis was induced in 7-8-week-old C57BL6 mice using four cycles (2-4%) of dextran sulfate sodium. hCTS (1.5 mg/kg/day) treatment or vehicle started 2 days before the last induction of colitis and continuing for 7 days. At sacrifice, macro- and microscopic scores were determined. Colonic pro-inflammatory cytokines [interleukin (IL)-6, IL-1ß, and TNF- α], anti-inflammatory cytokines (IL-10, TGF- ß), classically activated (M1) (iNOS, Mcp1), and alternatively activated (M2) (Ym1, Arg1) macrophages markers were studied using ELISA and/or RT-qPCR. In vitro, peritoneal macrophages isolated from naïve mice and treated with hCTS (10-5 M, 12 h) were exposed to either lipopolysaccharide (100 ng/ml, 12 h) to polarize M1 macrophages or to IL-4/IL-13 (20 ng/ml) to polarize M2 macrophages. M1/M2 macrophage markers along with cytokine gene expression were determined using RT-qPCR. Feces and mucosa-associated microbiota (MAM) samples were collected, and the V4 region of 16 s rRNA was sequenced. Micro- and macroscopic scores, colonic IL-6, IL-1ß, TNF- α, and M1 macrophages markers were significantly decreased in the hCTS-treated group. Treatment did not have any effect on colonic IL-10, TGF-ß, and M2 markers nor modified the bacterial richness, diversity, or the major phyla in colitic fecal and MAM samples. In vitro, pro-inflammatory cytokines levels, as well as their gene expression, were significantly reduced in hCTS-treated M1 macrophages. hCTS treatment did not affect M2 macrophage markers. These findings suggest that hCTS treatment attenuates the severity of inflammatory relapse through the modulation of the M1 macrophages and the release of pro-inflammatory cytokines.

Stability of Reference Genes for Messenger RNA Quantification by Real-Time PCR in Mouse Dextran Sodium Sulfate Experimental Colitis.

BACKGROUND: Many animal models have been developed to characterize the complexity of colonic inflammation. In dextran sodium sulfate (DSS) experimental colitis in mice the choice of reference genes is critical for accurate quantification of target genes using quantitative real time PCR (RT-qPCR). No studies have addressed the performance of reference genes in mice DSS-experimental colitis. This study aimed to determine the stability of reference genes expression (RGE) in DSS-experimental murine colitis. METHODS: Colitis was induced in male C57BL/6 mice using DSS5% for 5 days, control group received water. RNA was extracted from inflamed and non-inflamed colon. Using RT-qPCR, comparative analysis of 13 RGE was performed according to predefined criteria and relative colonic TNF-α and IL-1ß gene expression was determined by calculating the difference in the threshold cycle. RESULTS: Colitis significantly altered the stability of mucosal RGE. Commonly used glyceraldehyde-3-phosphate dehydrogenase (Gapdh), ß-actin (Actb), or ß2-microglobulin (ß2m) showed the highest variability within the inflamed and control groups. Conversely, TATA-box-binding protein (Tbp) and eukaryotic translation elongation factor 2 (Eef2) were not affected by inflammation and were the most stable genes. Normalization of colonic TNF-α and IL-1ß mRNA levels was dependent on the reference gene used. Depending on the genes used to normalize the data, statistical significance varied from significant when TBP / Eef2 were used to non-significant when Gapdh, Actb or ß2m were used. CONCLUSIONS: This study highlights the appropriate choice of RGE to ensure adequate normalization of RT-qPCR data when using this model. Suboptimal RGE may explain controversial results from published studies. We recommend using Tbp and Eef2 instead of Gapdh, Actb or ß2m as reference genes.

Human Catestatin Alters Gut Microbiota Composition in Mice.

The mammalian intestinal tract is heavily colonized with a dense, complex, and diversified microbial populations. In healthy individuals, an array of epithelial antimicrobial agents is secreted in the gut to aid intestinal homeostasis. Enterochromaffin cells (EC) in the intestinal epithelium are a major source of chromogranin A (CgA), which is a pro-hormone and can be cleaved into many bioactive peptides that include catestatin (CST). This study was carried out to evaluate the possible impact of CST on gut microbiota in vivo using a mouse model. The CST (Human CgA352-372) or normal saline was intrarectally administered in C57BL/6 male mice for 6 days and then sacrificed. Feces and colonic mucosa tissue samples were collected, DNA was extracted, the V4 region of bacterial 16S rRNA gene was amplified and subjected to MiSeq Illumina sequencing. The α-diversity was calculated using Chao 1 and ß-diversity was determined using QIIME. Differences at the genus level were determined using partial least square discriminant analysis (PLS-DA). Phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) was used to predict functional capacity of bacterial community. CST treatment did not modify bacterial richness in fecal and colonic mucosa-associated microbiota; however, treatment significantly modified bacterial community composition between the groups. Also, CST-treated mice had a significantly lower relative abundance of Firmicutes and higher abundance of Bacteroidetes, observed only in fecal samples. However, at lower phylogenetic levels, PLS-DA analysis revealed that some bacterial taxa were significantly associated with the CST-treated mice in both fecal and colonic mucosa samples. In addition, differences in predicted microbial functional pathways in both fecal and colonic mucosa samples were detected. The results support the hypothesis that CST treatment modulates gut microbiota composition under non-pathophysiological conditions, however, the result of this study needs to be further validated in a larger experiment. The data may open new avenues for the development of a potential new line of antimicrobial peptides and their use as therapeutic agents to treat several inflammatory conditions of the gastrointestinal tract, such as inflammatory bowel disease (IBD), inflammatory bowel syndrome (IBS), or other health conditions.

Central muscarinic cholinergic activation alters interaction between splenic dendritic cell and CD4+CD25- T cells in experimental colitis.

BACKGROUND: The cholinergic anti-inflammatory pathway (CAP) is based on vagus nerve (VN) activity that regulates macrophage and dendritic cell responses in the spleen through alpha-7 nicotinic acetylcholine receptor (a7nAChR) signaling. Inflammatory bowel disease (IBD) patients present dysautonomia with decreased vagus nerve activity, dendritic cell and T cell over-activation. The aim of this study was to investigate whether central activation of the CAP alters the function of dendritic cells (DCs) and sequential CD4+/CD25-T cell activation in the context of experimental colitis. METHODS: The dinitrobenzene sulfonic acid model of experimental colitis in C57BL/6 mice was used. Central, intracerebroventricular infusion of the M1 muscarinic acetylcholine receptor agonist McN-A-343 was used to activate CAP and vagus nerve and/or splenic nerve transection were performed. In addition, the role of α7nAChR signaling and the NF-kB pathway was studied. Serum amyloid protein (SAP)-A, colonic tissue cytokines, IL-12p70 and IL-23 in isolated splenic DCs, and cytokines levels in DC-CD4+CD25-T cell co-culture were determined. RESULTS: McN-A-343 treatment reduced colonic inflammation associated with decreased pro-inflammatory Th1/Th17 colonic and splenic cytokine secretion. Splenic DCs cytokine release was modulated through α7nAChR and the NF-kB signaling pathways. Cholinergic activation resulted in decreased CD4+CD25-T cell priming. The anti-inflammatory efficacy of central cholinergic activation was abolished in mice with vagotomy or splenic neurectomy. CONCLUSIONS: Suppression of splenic immune cell activation and altered interaction between DCs and T cells are important aspects of the beneficial effect of brain activation of the CAP in experimental colitis. These findings may lead to improved therapeutic strategies in the treatment of IBD.

Catestatin decreases macrophage function in two mouse models of experimental colitis.

Mucosal inflammation in patients with inflammatory bowel disease (IBD) is characterized by an alteration of prohormone chromogranin A (CgA) production. The recent demonstration of an implication of CgA in collagenous colitis and immune regulation provides a potential link between CgA-derived peptides (catestatin, CTS) and gut inflammation. Colitis was induced by administration of dextran sulfate sodium or 2, 4 dinitrobenzenesulfonic acid to C57BL/6 mice. Treatment with human (h)CTS or its proximal or distal part was started one day before colitis induction and colonic inflammatory markers were determined. Pro-inflammatory cytokines were evaluated in peritoneal isolated and bone marrow derived macrophages (BMDMs); p-STAT3 level was studied. Serum levels of CgA and CTS were assessed in experimental colitis and in a separate study in IBD patients and healthy controls. We show that sera from IBD patients and that in experimental colitis conditions the colonic level of mouse (m)CgA and mCTS are significantly increased. Moreover, in vivo treatment with human (h)CTS reduces the disease onset and suppresses exacerbated inflammatory responses in preclinical settings of colitis associated with an increase of p-STAT3. In vitro, hCTS treatment decreases proinflammatory cytokine release by peritoneal macrophages and BMDMs and increases p-STAT3 levels. These results support the hypothesis that CTS is increased during colitis and that hCTS modulates intestinal inflammation via the macrophage population and through a STAT-3 dependent pathway in a murine model of colitis. Identification of the molecular mechanism underlying the protective role of this peptide may lead to a novel therapeutic option in IBD.