Formononetin Protects LPS-Induced Mastitis Through Suppressing Inflammation and Enhancing Blood-Milk Barrier Integrity via AhR-Induced Src Inactivation.

Formononetin (FOR), a natural flavonoid derived from Radix Astragali, has been reported to have anti-inflammatory and anti-oxidative effects. However, its protective mechanism against mastitis is still unknown. Nuclear factor kappa-B (NF-κB) signaling pathway plays an important role in inflammation, especially mastitis. Aryl hydrocarbon receptor (AhR) is involved in inflammatory regulation and defense against diseases. We investigated the protective effect of FOR on LPS-induced mastitis in mice and the effect of Ahr and NF-κB signaling pathways on the development of mastitis. In this study, mastitis model was induced by LPS injection through the nipple duct. Protective effect of FOR on LPS-induced mastitis was assessed by FOR pretreatment. The protective mechanism of FOR against mastitis was further investigated using LPS stimulation on mouse mammary epithelial cells EpH4-Ev. The results showed that LPS-induced mammary histological injury was inhibited by FOR. FOR significantly inhibited LPS-induced MPO activity. FOR administration enhanced the integrity of blood-milk barrier. In vitro and in vivo experiments showed that FOR inhibited LPS-induced NF-κB signaling pathway activation and the production of inflammatory factors TNF-α and IL-1ß. Moreover, FOR increased the expression of tight junction protein and enhanced blood-milk barrier integrity. LPS activated AhR and Src expression. But FOR induced significant increase in AhR inhibited Src phosphorylation to exert anti-inflammatory effects. In addition, AhR antagonist CH223191 reversed the inhibition of FOR on Src expression. And the inhibition of FOR on NF-κB activation and inflammatory cytokine production were reversed by AhR antagonist CH223191. In conclusion, FOR had protective effects against LPS-induced mastitis via suppressing inflammation and enhancing blood-milk barrier integrity via AhR-induced Src inactivation.

Changes in the Intestinal Histomorphometry, the Expression of Intestinal Tight Junction Proteins, and the Bone Structure and Liver of Pre-Laying Hens Following Oral Administration of Fumonisins for 21 Days.

Fumonisins (FB) are metabolites found in cereal grains (including maize), crop products, and pelleted feed. There is a dearth of information concerning the effects of FB intoxication on the intestinal histomorphometry, the expression of intestinal tight junction proteins, and the bone structure and liver in pre-laying hens. The current experiment was carried out on hens from the 11th to the 14th week of age. The hens were orally administered an extract containing fumonisin B1 (FB1) and fumonisin B2 (FB2) at doses of 0.0 mg/kg b.w. (body weight), 1.0 mg/kg b.w., 4.0 mg/kg b.w., and 10.9 mg/kg b.w. for 21 days. Following FB intoxication, the epithelial integrity of the duodenum and jejunum was disrupted, and dose-dependent degenerative changes were observed in liver. An increased content of immature collagen was observed in the bone tissue of FB-intoxicated birds, indicating intensified bone turnover. A similar effect was observed with regards to the articular cartilage, where enhanced fibrillogenesis was observed mainly in the group of birds that received the FB extract at a dose of 10.9 mg/kg b.w. In conclusion, FB intoxication resulted in negative structural changes in the bone tissue of the hens, which could result in worsened bone mechanics and an increase in the risk of bone fractures. Fumonisin administration, even at a dose of 1.0 mg/kg b.w., can lead to degradation of the intestinal barrier and predispose hens to intestinal disturbances later in life.

Effect of Heat-killed Lactobacillus casei DKGF7 on a Rat Model of Irritable Bowel Syndrome.

Non-viable bacteria, referred to as "paraprobiotics," have attracted attention as potentially safer alternatives to probiotics. The aim of this study was to investigate the efficacy of heat-killed Lactobacillus casei DKGF7 on the symptomatic improvement of irritable bowel syndrome (IBS) in a rat disease model and to elucidate the underlying mechanisms that contribute to the beneficial effects of heat-killed probiotics. Seven male Wistar rats were induced with IBS by restraint stress and administered heat-killed L. casei DKGF7 for four weeks and then compared with seven rats in the control group. Stool consistency measured four weeks after initial treatment was the primary outcome measure. To investigate the mechanism of action of the heat-killed bacteria on IBS, we measured serum corticosterone levels, inflammatory cytokines in colon tissue, and expression of tight junction proteins (TJPs) in the epithelium. The treatment group showed significantly better stool consistency scores than the control group at week 4, as well as at every measured time point (all p values < 0.05). The treatment group showed lower serum corticosterone levels, lower colonic inflammatory cytokine levels, and higher expression of TJPs compared with the control group. Paraprobiotics such as heat-killed L. casei DKGF7 can improve stool consistency in a rat IBS model, which may indicate a potential therapeutic strategy for IBS treatment.

Anti-Obesity Effect of Dictyophora indusiata Mushroom Polysaccharide (DIP) in High Fat Diet-Induced Obesity via Regulating Inflammatory Cascades and Intestinal Microbiome.

Obesity is a multifactorial metabolic disorder characterized by low-grade chronic inflammation, hyper-permeability of the gut epithelium, and perturbation of the intestinal microbiome. Despite the numerous therapeutic efficacies of Dictyophora indusiata mushroom, its biological activity in alleviating obesity through regulation of the gut microbiota and inflammatory cascades remain obscure. Henceforth, we determined the modulatory impact of D. indusiata polysaccharide (DIP) in the high-fat diet (HFD)-induced obesity mice model. The experimental subjects (BALB/C mice) were supplemented with chow diet (Control group), high-fat diet (HFD group), or HFD along with DIP at a low dose [HFD + DIP(L)] and high dose [HFD + DIP(H)]. Obesity-related parameters, including body weight gain, epididymal adipocyte size, fat accumulation, adipogenic markers, lipogenic markers, inflammatory associated markers, intestinal integrity, and intestinal microbiome, were elucidated. Our findings demonstrated that the oral administration of DIP at low dose partially and at high dose significantly reversed HFD-induced obesity parameters. Furthermore, the body weight, fat accumulation, adipocyte size, adipogenic and liver associated markers, glucose levels, inflammatory cytokines, and endotoxin (Lipopolysaccharide, LPS) levels were reduced considerably. Moreover, the study revealed that DIP treatment reversed the dynamic alterations of the gut microbiome community by decreasing the Firmicutes to Bacteroidetes ratio. These findings led us to infer the therapeutic potential of DIP in alleviating HFD-induced obesity via regulating inflammatory cascades, modulating intestinal integrity and intestinal microbiome community.

Effect of a Synbiotic Containing Lactobacillus paracasei and Opuntia humifusa on a Murine Model of Irritable Bowel Syndrome.

The administration of a combination of probiotics and prebiotics is expected to be a promising strategy for improving irritable bowel syndrome (IBS) symptoms. This study aimed to investigate the efficacy of a synbiotic containing Lactobacillus paracasei and Opuntia humifusa extract for symptomatic improvement of IBS in a murine model and to evaluate the mechanism underlying the beneficial effects of this synbiotic. A total of 20 male Wistar rats aged 8 weeks with IBS induced by restraint stress were assigned into four groups and administered L. paracasei as a probiotic and O. humifusa extract as a prebiotic for 4 weeks. The primary outcome was stool consistency at week 4. To evaluate the mechanism underlying the beneficial effects of the synbiotic, fecal microbial analysis was conducted, and the serum corticosterone levels, tumor necrosis factor-α (TNF-α) levels in the colon tissue, and expression of tight junction proteins were investigated. All three treatment groups showed significantly lower scores for stool consistency than the control group at week 4 (all p < 0.001). When compared with the control group, the synbiotic groups showed a significantly greater abundance of L. paracasei in fecal microbial analysis, lower serum corticosterone levels, lower TNF-α levels in the colon tissue, and higher expression of tight junction proteins. This novel synbiotic containing L. paracasei and O. humifusa extract can improve the stool consistency in a murine model of IBS. It may be a promising treatment option for IBS, and human studies are warranted.

Brain tight junction protein expression in sepsis in an autopsy series.

BACKGROUND: Neuroinflammation often develops in sepsis along with increasing permeability of the blood-brain barrier (BBB), which leads to septic encephalopathy. The barrier is formed by tight junction structures between the cerebral endothelial cells. We investigated the expression of tight junction proteins related to endothelial permeability in brain autopsy specimens in critically ill patients deceased with sepsis and analyzed the relationship of BBB damage with measures of systemic inflammation and systemic organ dysfunction. METHODS: The case series included all (385) adult patients deceased due to sepsis in the years 2007-2015 with available brain specimens taken at autopsy. Specimens were categorized according to anatomical location (cerebrum, cerebellum). The immunohistochemical stainings were performed for occludin, ZO-1, and claudin. Patients were categorized as having BBB damage if there was no expression of occludin in the endothelium of cerebral microvessels. RESULTS: Brain tissue samples were available in 47 autopsies, of which 38% (18/47) had no expression of occludin in the endothelium of cerebral microvessels, 34% (16/47) developed multiple organ failure before death, and 74.5% (35/47) had septic shock. The deceased with BBB damage had higher maximum SOFA scores (16 vs. 14, p = 0.04) and more often had procalcitonin levels above 10 µg/L (56% vs. 28%, p = 0.045) during their ICU stay. BBB damage in the cerebellum was more common in cases with C-reactive protein (CRP) above 100 mg/L as compared with CRP less than 100 (69% vs. 25%, p = 0.025). CONCLUSIONS: In fatal sepsis, damaged BBB defined as a loss of cerebral endothelial expression of occludin is related with severe organ dysfunction and systemic inflammation.

Therapeutic effect of Lactobacillus rhamnosus SHA113 on intestinal infection by multi-drug-resistant Staphylococcus aureus and its underlying mechanisms.

Staphylococcus aureus, especially multi-drug-resistant (MDR) pathogenic S. aureus, poses a severe threat to food safety and human health. Probiotics offer promising potential for the control of MDR pathogens because of their safe and biofunctional properties. This study shows that Lactobacillus rhamnosus SHA113, a strain isolated from the milk of healthy women, could efficiently inhibit MDR S. aureus both in vitro and in vivo. In vitro, L. rhamnosus efficiently inhibited and even killed drug resistant and drug sensitive S. aureus strains. In vivo experiments showed that SHA113 could efficiently decrease the number of S. aureus cells, inhibit the expression of inflammatory factors TNF-α and IL-6, and restore the level of white cells and neutrophils in the blood. SHA113 could also efficiently repair damage of the intestinal barrier and other functions impaired by S. aureus infection. This was indicated by a change of intestinal villi length and structure, and an up-regulated expression of tight junction proteins ZO-1 and occludin. SHA113 also restored the structural damage of immune organs, such as the enlargement of the spleen and the increased level of inflammatory cytokines caused by S. aureus infection. More importantly, L. rhamnosus SHA113 showed more effective inhibitory and therapeutic effects on MDR S. aureus strain ZBQ006 than on drug sensitive S. aureus strain 29213. These results illustrated that L. rhamnosus SHA113 has great potential for the treatment of MDR S. aureus contamination as food control and for therapeutic treatment.

Idiopathic pulmonary fibrosis is associated with tight junction protein alterations.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterized by an abnormal healing response to injury of the alveolar epithelium. Tight junctions provide a physical barrier at the apical intercellular space between epithelial cells and regulate paracellular flux. While tight junction alterations are known to contribute to barrier dysfunction in a number of disease states, the role of tight junction proteins in IPF is poorly defined. To determine a potential role for tight junction protein alterations in IPF, we performed immunohistochemical staining for tight junction proteins ZO-1, occludin, claudin-2, claudin-3, claudin-4, claudin-5, and claudin-18. Staining intensity and localization were compared between IPF and control lung tissues. IPF was associated with type II pneumocyte hyperplasia and altered tight junction protein expression. While there was no difference in the expression of ZO-1, claudin-3, or claudin-5, between IPF and normal control, there was an overall increase in claudin-2 expression in bronchiolar and alveolar epithelium and a decrease in claudin-4 expression in type II pneumocytes. There was also increased occludin and decreased claudin-18 expression in pneumocytes overlying fibroblastic foci. These findings suggest that epithelial barrier alterations may be important to the pathogenesis of IPF.

Tandem mass tag (TMT) proteomic analysis of fetal lungs revealed differential expression of tight junction proteins in a rat model of congenital diaphragmatic hernia.

OBJECTIVE: Congenital diaphragmatic hernia (CDH) is a common and often lethal birth defect characterized by congenital lung malformation, which severely affects neonate prognosis and mortality. This study aimed to investigate differences in protein expression in order to elucidate the mechanism of CDH-associated pulmonary hypoplasia during the early stage of lung development using tandem mass tag (TMT) quantitative proteomics. METHODS: Nitrofen was administered orally to establish a rat CDH model, and pathological changes were evaluated through hematoxylin-eosin (H&E), PCNA, and Ki67 staining at the pseudoglandular stage. Fetal lungs were then collected, pooled before TMT labeling, and subjected to mass spectrometry. Immunohistochemistry (IHC), Western blotting, and Q-PCR were used to further validate the candidate proteins. RESULTS: A total of 79 differentially expressed proteins (DEPs) were identified when CDH and control lungs were compared, and further bioinformatics analysis showed that these proteins play important roles in tight-junctions, phospholipase D signaling, and the HIF-1 signaling pathway. Three differentially expressed proteins, Cldn3, Magi1, and Myh9 are involved in the tight-junction pathway (P < 0.05), and their differential expressions were confirmed by IHC, Western blotting, and Q-PCR. CONCLUSION: These findings indicate that alterations of tight-junction protein expression may play an important role in the pathogenesis of abnormal lung development in CDH. Further studies are warranted to verify the mechanism by which these tight-junction proteins influence the pathogenesis of CDH-associated pulmonary hypoplasia.

Impact of chronic immobilization stress on parameters of colonic homeostasis in BALB/c mice.

The intestinal epithelium is a monolayer of cells arranged side­by­side and connected by tight junction (TJ) proteins expressed at the apical extreme of the paracellular membrane. This layer prevents stress­induced inflammatory responses, thus helping to maintain gut barrier function and gut homeostasis. The aim of the present study was to evaluate the effects of chronic immobilization stress on the colonic expression of various parameters of homeostasis. A total of two groups of female BALB/c mice (n=6) were included: A stressed group (short­term immobilization for 2 h/day for 4 consecutive days) and an unstressed (control) group. Colon samples were obtained to detect neutrophils and goblet cells by optical microscopy, TJ protein expression (occludin, and claudin ­2, ­4, ­7, ­12 and ­15) by western blotting, mRNA levels of TJ genes and proinflammatory cytokines [tumor necrosis factor (TNF)­α, interleukin (IL)­1ß, ­6 and ­8] by reverse transcription­quantitative PCR, fecal lactoferrin by ELISA and the number of colony­forming units of aerobic bacteria. Compared with goblet cells in control mice, goblet cells were enlarged and reduced in number in stressed mice, whereas neutrophil cellularity was unaltered. Stressed mice exhibited reduced mRNA expression for all evaluated TJ mRNAs, with the exception of claudin­7, which was upregulated. Protein levels of occludin and all claudins (with the exception of claudin­12) were decreased in stressed mice. Fecal lactoferrin, proinflammatory cytokine mRNA levels and aerobic bacterial counts were all increased in the stressed group. These results indicated that immobilization stress induced proinflammatory and potential remodeling effects in the colon by decreasing TJ protein expression. The present study may be a useful reference for therapies aiming to regulate the effects of stress on intestinal inflammatory dysfunction.

F5-Peptide and mTORC1/rpS6 Effectively Enhance BTB Transport Function in the Testis-Lesson From the Adjudin Model.

During spermatogenesis, the blood-testis barrier (BTB) undergoes cyclic remodeling that is crucial to support the transport of preleptotene spermatocytes across the immunological barrier at stage VIII to IX of the epithelial cycle. Studies have shown that this timely remodeling of the BTB is supported by several endogenously produced barrier modifiers across the seminiferous epithelium, which include the F5-peptide and the ribosomal protein S6 [rpS6; a downstream signaling molecule of the mammalian target of rapamycin complex 1 (mTORC1)] signaling protein. Herein, F5-peptide and a quadruple phosphomimetic (and constitutively active) mutant of rpS6 [i.e., phosphorylated (p-)rpS6-MT] that are capable of inducing reversible immunological barrier remodeling, by making the barrier "leaky" transiently, were used for their overexpression in the testis to induce BTB opening. We sought to examine whether this facilitated the crossing of the nonhormonal male contraceptive adjudin at the BTB when administered by oral gavage, thereby effectively improving its BTB transport to induce germ cell adhesion and aspermatogenesis. Indeed, it was shown that combined overexpression of F5-peptide and p-rpS6-MT and a low dose of adjudin, which by itself had no noticeable effects on spermatogenesis, was capable of perturbing the organization of actin- and microtubule (MT)-based cytoskeletons through changes in the spatial expression of actin- and MT-binding/regulatory proteins to the corresponding cytoskeleton. These findings thus illustrate the possibility of delivering drugs to any target organ behind a blood-tissue barrier by modifying the tight junction permeability barrier using endogenously produced barrier modifiers based on findings from this adjudin animal model.

Brain Infection by Hepatitis E Virus Probably via Damage of the Blood-Brain Barrier Due to Alterations of Tight Junction Proteins.

Extrahepatic injury, particularly neurologic dysfunctions such as Guillain-Barré syndrome, neurologic amyotrophy, and encephalitis/meningoencephalitis/myositis were associated with HEV infection, which was supported by both clinical and laboratory studies. Thus, it is crucial to figure out how the virus invades into the central nervous system (CNS). In this study, CNS lesions were determined in rabbits and Mongolian gerbils inoculated with genotype 4 HEV. Junctional proteins were detected in HEV infected primary human brain microvascular cells (HBMVCs). Viral encephalitis associated perivascular cuffs of lymphocytes and microglial nodules were observed in HEV infected rabbits. Both positive- and negative-strand of HEV RNA was detected in brain and spinal cord in rabbits intraperitoneally infected with HEV at 28 dpi (days postinoculation), but not in rabbits gavaged with HEV. HEV ORF2 protein was further examined in both brain and spinal cord sections of infected rabbits, with positive signals located mainly in neural cells and perivascular areas. Ultrastructural study showed thickened and reduplicated basement membranes of capillary endothelium in HEV RNA positive brain tissues. In vitro study showed loss of tight junction proteins including Claudin5, Occludin, and ZO-1 (zonula occludens-1) in HBMVCs inoculated with HEV for 48 h. These findings indicated that disruption of the blood-brain barrier (BBB) might be potential mechanisms of HEV invasion into the CNS. It provides new insights to further study HEV associated neurologic disorders and will be helpful for seeking potential therapeutics for HEV infection in the future.

Low-Luminance Blue Light-Enhanced Phototoxicity in A2E-Laden RPE Cell Cultures and Rats.

N-retinylidene-N-retinylethanolamine (A2E) and other bisretinoids are components of lipofuscin and accumulate in retinal pigment epithelial (RPE) cells-these adducts are recognized in the pathogenesis of retinal degeneration. Further, blue light-emitting diode (LED) light (BLL)-induced retinal toxicity plays an important role in retinal degeneration. Here, we demonstrate that low-luminance BLL enhances phototoxicity in A2E-laden RPE cells and rats. RPE cells were subjected to synthetic A2E, and the effects of BLL on activation of apoptotic biomarkers were examined by measuring the levels of cleaved caspase-3. BLL modulates the protein expression of zonula-occludens 1 (ZO-1) and paracellular permeability in A2E-laden RPE cells. Early inflammatory and angiogenic genes were also screened after short-term BLL exposure. In this study, we developed a rat model for A2E treatment with or without BLL exposure for 21 days. BLL exposure caused fundus damage, decreased total retinal thickness, and caused neuron transduction injury in the retina, which were consistent with the in vitro data. We suggest that the synergistic effects of BLL and A2E accumulation in the retina increase the risk of retinal degeneration. These outcomes help elucidate the associations between BLL/A2E and angiogenic/apoptotic mechanisms, as well as furthering therapeutic strategies.

Modulation of intestinal epithelial permeability and mucin mRNA (MUC2, MUC5AC, and MUC5B) expression and protein secretion in Caco-2/HT29-MTX co-cultures exposed to aflatoxin M1, ochratoxin A, and zearalenone individually or collectively.

Aflatoxin M1 (AFM1), ochratoxin A (OTA), and zearalenone (ZEA) are mycotoxins commonly found in milk. Mycotoxin contamination has caused food safety concerns worldwide since most of the toxic effects in humans are serious. The combined toxic effects of these mycotoxins on intestinal epithelial cells have not been reported. Herein, we investigated the combined effects of AFM1, OTA, and ZEA on intestinal integrity and define the underlying mechanisms(s) of their effects in Caco-2/HT29-MTX co-cultures. Our results showed that the mixtures of AFM1 + OTA, AFM1 + ZEA, and AFM1 + ZEA + OTA significantly increased epithelial permeability. Immunofluorescence analysis and transmission electron microscopy revealed that mycotoxins altered TJ proteins morphology and disrupted their structures. Also, the present study showed that mixtures of mycotoxins significantly modulated MUC5AC and MUC5B mRNA levels and protein secretion. This study demonstrated that the effects of mixtures of mycotoxins on intestinal barrier function were more significant than AFM1 alone. More importantly, the damage of intestinal integrity caused by mycotoxins was correlated to the change of the TJ proteins location and the decrease of mucin secretion. Mixtures of AFM1, OTA, and ZEA in food might pose a health risk to consumers, particularly in children, and toxin risks should be considered.

Licochalcone A Protects the Blood-Milk Barrier Integrity and Relieves the Inflammatory Response in LPS-Induced Mastitis.

Background/Aims: Mastitis is an acute clinical inflammatory response. The occurrence and development of mastitis seriously disturb women's physical and mental health. Licochalcone A, a phenolic compound in Glycyrrhiza uralensis, has anti-inflammatory properties. Here, we examined the effect of licochalcone A on blood-milk barrier and inflammatory response in LPS-induced mice mastitis. Methods:In vivo, we firstly established mice models of mastitis by canal injection of LPS to mammary gland, and then detected the effect of licochalcone A on pathological indexes, inflammatory responses and blood-milk barrier in this model. In vivo, Mouse mammary epithelial cells (mMECs) were treated with licochalcone A prior to the incubation of LPS, and then the inflammatory responses, tight junction which is the basic structure of blood-milk barrier were analyzed. Last, we elucidated the anti-inflammatory mechanism by examining the activation of mitogen-activated protein kinase (MAPK) and AKT/NF-κB signaling pathways in vivo and in vitro. Result: The in vivo results showed that licochalcone A significantly decreased the histopathological impairment and the inflammatory responses, and improved integrity of blood-milk barrier. The in vitro results demonstrated that licochalcone A inhibited LPS-induced inflammatory responses and increase the protein levels of ZO-1, occludin, and claudin3 in mMECs. The in vivo and in vitro mechanistic study found that the anti-inflammatory effect of licochalcone A in LPS-induced mice mastitis was mediated by MAPK and AKT/NF-κB signaling pathways. Conclusions and Implications: Our experiments collectively indicate that licochalcone A protected against LPS-induced mice mastitis via improving the blood-milk barrier integrity and inhibits the inflammatory response by MAPK and AKT/NF-κB signaling pathways.

Distinct molecular signature of phospholamban p.Arg14del arrhythmogenic cardiomyopathy.

Phospholamban (PLN) p.Arg14del cardiomyopathy is characterized by a distinct arrhythmogenic biventricular phenotype that can be predominantly left ventricular, right ventricular, or both. Our aim was to further elucidate distinct features of this cardiomyopathy with respect to the distribution of desmosomal proteins observed by immunofluorescence (IF) in comparison to desmosomal arrhythmogenic cardiomyopathy and co-existent genetic variants. We studied eight explanted heart specimens from PLN p.Arg14del mutation carriers. Macro- and microscopic examination revealed biventricular presence of fibrofatty replacement and interstitial fibrosis. Five out of 8 (63%) patients met consensus criteria for both arrhythmogenic right ventricular cardiomyopathy (ARVC) and dilated cardiomyopathy (DCM). In four cases, targeted next-generation sequencing revealed one additional pathogenic variant and six variants of unknown significance. IF showed diminished junction plakoglobin signal intensity at the intercalated disks in 4 (67%) out of 6 cases fulfilling ARVC criteria but normal intensity in both cases fulfilling only DCM criteria. Notably, the four cases with diminished junction plakoglobin were also those where an additional gene variant was detected. IF for two proteins recently investigated in desmosomal arrhythmogenic cardiomyopathy (ACM), synapse-associated protein 97 and glycogen synthase kinase-3 beta, showed a distinct distributional pattern in comparison to desmosomal ACM. In 7 (88%) out of 8 cases we observed both a strong synapse-associated protein 97 signal at the sarcomeres and no glycogen synthase kinase-3 beta translocation to the intercalated discs. Phospholamban p.Arg14del cardiomyopathy is characterized by a distinct molecular signature compared to desmosomal ACM, specifically a different desmosomal protein distribution. This study substantiates the idea that additional genetic variants play a role in the phenotypical heterogeneity.

Chronic colitis-induced visceral pain is associated with increased anxiety during quiescent phase.

Inflammatory bowel diseases (IBD) are characterized by repetition of flares and remission periods leading to chronic postinflammatory sequelae. Among postinflammatory sequelae, one-third of patients with IBD are suffering from functional symptoms or psychological comorbidities that persist during remission. The aim of our study was to assess functional and behavioral sequelae of chronic colitis in rats with quiescent intestinal inflammation. Chronic colitis was induced by a weekly intrarectal injection of increasing concentrations of trinitrobenzene sulfonic acid (TNBS) for 3 wk (15-45 mg of TNBS) in 30 rats, whereas the control rats (n = 24) received the vehicle. At 50 days post-TNBS, visceral sensitivity was assessed by visceromotor response to colorectal distension, and transient receptor potential vanilloid type 1 (TRPV1) expression was also quantified in the colon and dorsal root ganglia. Barrier function and inflammatory response were assessed by studying intestinal permeability, tight junction protein, myeloperoxidase activity, histological score, and cytokine production (IL-6, IL-10, and TNF-α). Anxiety behavioral tests were performed from 50 to 64 days after the last TNBS injection. Chronic TNBS induced 1) a visceral hypersensitivity (P = 0.03), 2) an increased colon weight-to-length ratio (P = 0.01), 3) higher inflammatory and fibrosis scores (P = 0.0390 and P = 0.0016, respectively), and 4) a higher colonic IL-6 and IL-10 production (P = 0.008 and P = 0.005, respectively) compared with control rats. Intestinal permeability, colonic production of TNF-α, myeloperoxidase activity, and TRPV1 expression did not differ among groups. Chronic TNBS increased anxiety-related behavior in the open-field test and in the acoustic stress test. In conclusion, chronic colitis induced functional sequelae such as visceral hypersensitivity and increased anxiety with a low-grade intestinal inflammation. Development of a representative animal model will allow defining novel therapeutic approaches to achieve a better management of IBD-related sequelae. NEW & NOTEWORTHY Patients with inflammatory bowel diseases have impaired quality of life. Therapeutic progress to control mucosal inflammation provides us an opportunity to develop novel approaches to understand mechanisms behind postinflammatory sequelae. We used a chronic colitis model to study long-term sequelae on visceral pain, gut barrier function, and psychological impact. Chronic colitis induced functional symptoms and increased anxiety in the remission period. It might define novel therapeutic approaches to achieve a better inflammatory bowel disease-related sequelae management.

Immunoreactivity patterns of tight junction proteins are useful for differential diagnosis of human salivary gland tumors.

The expression pattern of tight junction proteins (TJPs) varies among organs and tumor types. In this study, we examined the immunoreactivity of claudin (CLDN)-1, -4, and -7, and JAM-A in salivary gland tumors (SGTs) by histological types and cell types to estimate their usefulness as differential diagnostic markers. Immunoreactivity of CLDN1 was higher in ductal epithelium cells of SGTs than in non-tumor tissues. Conversely, immunoreactivity of CLDN1 was significantly decreased in basal/myoepithelium cells of SGTs compared with that in non-tumor tissues. There was no significant difference between the immunoreactivity of CLDN1 in benign tumors and that in malignant tumors. Immunoreactivity of CLDN4, CLDN7, and JAM-A in ductal epithelium cells was higher in many SGTs than in non-tumor tissues. There was a difference depending on the histological type of SGT in immunoreactivity of CLDN4, CLDN7, and JAM-A in basaloid/myoepithelial cells. It was possible to classify SGTs by a hierarchical clustering using immunoreactivity of TJPs. The results suggest that an immunohistochemical marker panel including these TJPs may be useful for differential diagnosis of SGTs and that CLDN1 is associated with tumorigenesis of SGTs.

Arsenic induces autophagy in developmental mouse cerebral cortex and hippocampus by inhibiting PI3K/Akt/mTOR signaling pathway: involvement of blood-brain barrier's tight junction proteins.

For the past decade, there has been an increased concern about the health risks from arsenic (As) exposure, because of its neurotoxic effects on the developing brain. The exact mechanism underlying As-induced neurotoxicity during sensitive periods of brain development remains unclear, especially the role of blood-brain barrier's (BBB) tight junction (TJ) proteins during As-induced neurotoxicity. Here, we highlight the involvement of TJ proteins in As-induced autophagy in cerebral cortex and hippocampus during developmental periods [postnatal day (PND) 21, 28, 35 and 42]. Here, the administration of arsenic trioxide (As2O3) at doses of 0.15 mg or 1.5 mg or 15 mg As2O3/L in drinking water from gestational to lactational and continued to the pups till PND42 resulted in a significant decrease in the mRNA expression levels of TJ proteins (Occludin, Claudin, ZO-1 and ZO-2) and Occludin protein expression level. In addition, As exposure significantly decreased PI3K, Akt, mTOR, and p62 with a concomitant increase in Beclin1, LC3I, LC3II, Atg5 and Atg12. Moreover, As exposure also significantly downregulated the protein expression levels of mTOR with a concomitant upregulation of Beclin 1, LC3 and Atg12 in all the developmental age points. However, no significant alterations were observed in low and medium dose-exposed groups of PND42. Histopathological analysis in As-exposed mice revealed decreased number of pyramidal neurons in hippocampus; and neurons with degenerating axons, shrinkage of cells, remarkable vacuolar degeneration in cytoplasm, karyolysis and pyknosis in cerebral cortex. Ultrastructural analysis by transmission electron microscopy revealed the occurrence of autophagosomes and vacuolated axons in the cerebral cortex and hippocampus of the mice exposed to high dose As at PND21 and 42. The severities of changes were found to more persist in the cerebral cortex than in the hippocampus of As-exposed mice. Finally, we conclude that the leaky BBB in cerebral cortex and hippocampus may facilitate the transfer of As and induces autophagy by inhibiting PI3K/Akt/mTOR signaling pathway in an age-dependent manner, i.e., among the four different developmental age points, PND21 animals were found to be more vulnerable to the As-induced neurotoxicity than the other three age points.

Marked Changes in Blood-Brain Barrier Biomarkers After Direct Bypass Surgery for Moyamoya Angiopathy: Preliminary Study.

OBJECTIVE: The blood-brain barrier (BBB) of patients with moyamoya angiopathy (MMA) is unstable, which may contribute to transient neurologic symptoms (TNS) after direct bypass surgery. However, BBB-related proteins have never been investigated. The purpose of this study was to evaluate the perioperative serum levels of biomarkers representing BBB function in MMA patients based on the hypothesis that postoperative hemodynamic change may disrupt the BBB. METHODS: A total of 12 hemispheres in 11 patients with MMA were prospectively examined. Direct revascularization surgery was performed for all cases. The serum levels of tight junction (occludin and claudin 5), adherens junction (vascular endothelial-cadherin) proteins, and matrix metalloproteinase (MMP)-2 and MMP-9 were measured quantitatively 1 day before surgery and on postoperative days 1, 4, and 7. RESULTS: Successful patency of the direct bypass was achieved in all. The serum level of occludin was significantly increased on postoperative day 1, and the levels in 2 cases with TNS were markedly elevated over 10-fold higher than baseline. Furthermore, the postoperative MMP-9 levels were significantly elevated on each day. On the other hand, there was no significant fluctuation in claudin 5, vascular endothelial-cadherin, and MMP-2 level. CONCLUSIONS: Marked changes in biomarkers representing the tight junction of the BBB were observed. These preliminary results suggest that marked hemodynamic change and TNS in some patients are associated with disruption of the BBB after direct bypass surgery for MMA.