The ion channel TRPV1 gain-of-function reprograms the immune microenvironment to facilitate colorectal tumorigenesis.

Transient receptor potential vanilloid 1 (TRPV1) is a Ca2+-permeable ion channel that acts as cellular sensor and is implicated in the tumor microenvironment cross talk. However, the functional role of TRPV1 in colorectal cancer (CRC) is still controversial. By using a TRPV1 gain-of-function model, we previously reported that hyperfunctional TRPV1 exacerbated experimental colitis by modulating mucosal immunity. Here, we found that TRPV1 gain-of-function significantly promoted tumor initiation and progression in colitis-associated cancer, as evidenced by the increase in the number and size of tumor. Systemic TRPV1 hyperactivation fostered a tumor permissive microenvironment through altering macrophage activation status and shifting the Th1/Th2 balance towards Th2 phenotype. Mechanistically, TRPV1 gain-of-function directly potentiated M1 cytokine production in macrophage and enhanced Th2 immune response by promoting Calcineurin/nuclear factor of activated T cells (NFATc2) signaling activation. In patients with CRC, TRPV1 expression was increased in tumor immune infiltrating cells. TRPV1 level was associated with CRC progression and could impact clinical outcome. Our study reveals an important role for TRPV1 in regulating the immune microenvironment during colorectal tumorigenesis. TRPV1 might be a potential target for CRC immunotherapy.

Genotype‒Phenotype Correlation of TRPV3-Related Olmsted Syndrome.

We have previously shown that gain-of-function variations in transient receptor potential vanilloid-3 (TRPV3) underlay Olmsted syndrome, a rare hyperkeratotic skin channelopathy. In this study, we attempt to establish a genotype‒phenotype correlation in Olmsted syndrome, which has been unclear owing to the rarity and heterogeneity of the condition. We identified five previously unreported TRPV3 variations (R416Q, R416W, L655P, W692S, and L694P) and three recurrent variations (G568D, G568V, and L673F) in nine unrelated patients. Seven variants were expressed in human embryonic kidney 293 cells, and channel behavior was characterized electrophysiologically, with results compared with the clinical severity. These variant TRPV3 channels, in either homomeric or heteromeric form, exhibited differentially elevated basal open probability, increased voltage sensitivity, and cytotoxicity. Functional changes were particularly pronounced in variants corresponding to severer Olmsted syndrome (e.g., L673F and W692S) but not in mild Olmsted syndrome variants (e.g., R416Q). Interestingly, the extent of functional rescue by wild-type TRPV3 in vitro was also consistent with the clinical severity of the variants. These findings, in combination with all reported cases, indicate a preliminary genotype‒phenotype correlation, that is, variations in the S4‒S5 linker and transient receptor potential domain of TRPV3 significantly enhance channel function, causing severe phenotype, whereas other variations appear to exert milder effects on channel function and disease phenotype.

Gain of Function of Ion Channel TRPV1 Exacerbates Experimental Colitis by Promoting Dendritic Cell Activation.

Dysregulated mucosal immunity plays an essential role in the pathophysiology of inflammatory bowel disease (IBD). Transient receptor potential vanilloid 1 (TRPV1) is a Ca2+-permeable ion channel that is implicated in modulating immune responses. However, its role in the pathogenesis of intestinal inflammation remains elusive. Here, we found that TRPV1 gain of function significantly increased the susceptibility of mice to experimental colitis, and that was associated with excessive recruitment of dendritic cells and enhanced Th17 immune responses in the lamina propria of colon. TRPV1 gain of function promoted dendritic cell activation and cytokine production upon inflammatory stimuli, and consequently enhanced dendritic cell-mediated Th17 cell differentiation. Further mechanistic studies showed that TRPV1 gain of function in dendritic cells enhanced activation of calcineurin/nuclear factor of activated T cells (NFATc2) signaling induced by inflammatory stimuli. Moreover, in patients with IBD, TRPV1 expression was increased in lamina propria cells of inflamed colon compared with healthy controls. Our findings identify an important role for TRPV1 in modulating dendritic cell activation and sustaining Th17 responses to inflammatory stimuli, which suggest that TRPV1 might be a potential therapeutic target in controlling mucosal immunity and IBD.

Ginsenoside Rg3 Sensitizes Colorectal Cancer to Radiotherapy through Downregulation of Proliferative and Angiogenic Biomarkers.

BACKGROUND: Radiation therapy is an important mode of colorectal cancer treatment. However, most people die of local recurrence after tumors become resistant to radiotherapy, and little progress has been made in treating radiotherapy-resistant colorectal cancer. Hence, novel agents that are nontoxic and can sensitize colorectal cancer to radiotherapy are urgently needed. Ginsenoside Rg3, a saponin extracted from ginseng, shows cytotoxicity against a variety of cancer cells through suppression of pathways linked to oncogenesis, including cell survival, proliferation, invasion, and angiogenesis. In this article, we investigated whether Rg3 can sensitize colorectal cancer to radiation in vivo. METHODS AND MATERIALS: We established CT-26 xenografts in BALB/c mice and treated them with vehicle, Rg3, radiation, and combined Rg3 + radiation. Mouse quality of life, survival, tumor volumes, and inhibitive rates were estimated. NF-κB activation was ascertained using electrophoretic mobility shift assay and immunohistochemistry. We also tested for markers of proliferation, angiogenesis, and invasion using immunohistochemistry and Western blot analysis. RESULTS: Rg3 significantly enhanced the efficacy of fractionated radiotherapy by improving the quality of life of mice. Moreover, tumors from mice xenografted with CT-26 cells and treated with combined Rg3 + radiotherapy showed significantly lower tumor volumes (P < 0.01 versus controls; P < 0.05 versus radiation alone), NF-κB activation, and expression of NF-κB-regulated gene products (cyclin D1, survivin, cyclooxygenase-2 (COX-2), and vascular endothelial growth factor (VEGF)) compared with controls. The combination treatment was also effective in suppressing angiogenesis, as indicated by lower CD31+ microvessel density compared with controls (P < 0.05). CONCLUSION: Our results suggest that Rg3 enhances the antitumor effects of radiotherapy for colorectal cancer by suppressing NF-κB and NF-κB-regulated gene products, leading to inhibition of tumors and prolongation of the lifespan of CT-26 xenograft BALB/c mice.

TRPV1 gain-of-function mutation impairs pain and itch sensations in mice.

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel, which can detect various noxious stimuli that cause pain, inflammation, hyperalgesia, and itch. TRPV1 knock-out mice show deficiency in nociception, but the in vivo effects of persistent activation of TRPV1 are not completely understood. Here, we generated TRPV1 knock-in mice with a G564S mutation. In the heterologous expression system, an electrophysiological study showed that the G564S mutation in mouse TRPV1 caused increased basal current and a leftward shift of voltage dependence. Intriguingly, using behavioral analysis, we found that knock-in mice showed a thermosensory defect, impaired inflammatory thermal pain, and capsaicin sensitivity. We also demonstrated an attenuated behavioral response to the pruritic agent histamine in the knock-in mice. Indeed, calcium imaging together with electrophysiology showed that the overactive mutant had decreased capsaicin sensitivity. Western blot analysis revealed that the G564S mutant reduced TRPV1 phosphorylation and cell membrane trafficking. Together, we have generated a mouse model with a gain-of-function mutation in Trpv1 gene and demonstrated that the pain and histamine-dependent itch sensations in these mice are impaired due to a decreased phosphorylation level and reduced membrane localization of TRPV1.

Clinical and molecular epidemiological study of xeroderma pigmentosum in China: A case series of 19 patients.

Xeroderma pigmentosum (XP) is a rare genetic disorder which is divided into eight complementation groups: XP-A to XP-G and XP-V. Some XP patients demonstrate severe cutaneous and neurological manifestations, management of which requires timely diagnosis and intervention. We performed clinical evaluation and genetic analysis on 19 patients, the largest cohort of XP to date in China. Twenty-three mutations from six groups were identified, 16 of which were novel. All patients developed marked freckle-like pigmentation on sun-exposed sites while patients with XP-A, XP-D, XP-F and XP-G showed acute sunburn reactions. Only XP-A patients displayed progressive neurological degeneration. A relatively larger proportion of XP-A and XP-C were found in Chinese XP patients. One XP case and two carriers were prenatally determined. This study extended the mutation spectrum of XP in China and may aid in the diagnosis and treatment of Chinese XP patients.

Exome sequencing reveals novel BCS1L mutations in siblings with hearing loss and hypotrichosis.

As a powerful tool to identify the molecular pathogenesis of Mendelian disorders, exome sequencing was used to identify the genetic basis of two siblings with hearing loss and hypotrichosis and clarify the diagnosis. No pathogenic mutations in GJB2, GJB3 and GJB6 genes were found in the siblings. By analysis of exome of the proband, we identified a novel missense (p.R306C) mutation and a nonsense (p.R186*) mutation in the BCS1L gene. Mutations were confirmed by Sanger sequencing. The siblings were compound heterozygotes, and the inheritance mode of autosomal recessive was postulated. BCS1L is the causative gene of Björnstad syndrome, which is characterized by sensorineural hearing loss and pili torti. The longitudinal gutters along the hair shaft were found by scanning electron microscopy in our patient. Therefore the diagnosis of Björnstad syndrome was eventually made for the patients. Our study extends the phenotypic spectrum of Björnstad syndrome and highlights the clinical applicability of exome sequencing as a diagnostic tool for atypical Mendelian disorders.

Loss-of-function mutations in CAST cause peeling skin, leukonychia, acral punctate keratoses, cheilitis, and knuckle pads.

Calpastatin is an endogenous specific inhibitor of calpain, a calcium-dependent cysteine protease. Here we show that loss-of-function mutations in calpastatin (CAST) are the genetic causes of an autosomal-recessive condition characterized by generalized peeling skin, leukonychia, acral punctate keratoses, cheilitis, and knuckle pads, which we propose to be given the acronym PLACK syndrome. In affected individuals with PLACK syndrome from three families of different ethnicities, we identified homozygous mutations (c.607dup, c.424A>T, and c.1750delG) in CAST, all of which were predicted to encode truncated proteins (p.Ile203Asnfs∗8, p.Lys142∗, and p.Val584Trpfs∗37). Immunohistochemistry shows that staining of calpastatin is reduced in skin from affected individuals. Transmission electron microscopy revealed widening of intercellular spaces with chromatin condensation and margination in the upper stratum spinosum in lesional skin, suggesting impaired intercellular adhesion as well as keratinocyte apoptosis. A significant increase of apoptotic keratinocytes was also observed in TUNEL assays. In vitro studies utilizing siRNA-mediated CAST knockdown revealed a role for calpastatin in keratinocyte adhesion. In summary, we describe PLACK syndrome, as a clinical entity of defective epidermal adhesion, caused by loss-of-function mutations in CAST.

Molecular profile of drug resistance in tuberculous meningitis from southwest china.

BACKGROUND: Tuberculous meningitis (TBM) is the most severe form of extrapulmonary tuberculosis and causes high mortality and morbidity. Isoniazid resistance is strongly predictive of death in patients with TBM. METHODS: In the present study, using polymerase chain reaction (PCR) and Genotype MTBDRplus line-probe assay, we investigated the drug resistance in patients with TBM living in Southwest China. RESULTS: Our results showed that only one-third of patients with TBM had a positive result for Mycobacterium tuberculosis culture from cerebrospinal fluid (CSF). PCR-based detection of M. tuberculosis DNA in CSF is not only an alternative diagnostic approach for TBM but also can be further used for the detection of drug resistance when combined with the MTBDRplus assay, the results of which were consistent with the classic drug susceptibility test. However, it further provided the molecular profile of the mutations can be conducted much faster than the classic drug susceptibility test can (1 day vs 30-40 days, respectively). In the studied 30 CSF samples from patients with TMB, we found a rate of 64.29% for isoniazid resistance, 39.29% for rifampicin resistance, and 32.14% for multidrug-resistant tuberculosis, which is relatively higher than the reported resistance in pulmonary tuberculosis. However, the molecular profile indicated that the most frequently observed mutations in the rpoB and katG genes are also responsible for drug resistance in TBM. CONCLUSIONS: Our data suggest that the MTBDRplus line-probe assay is capable of detecting drug resistance for the CSF samples that have a PCR-positive result. We recommend PCR-based diagnosis and drug resistance test as routine assays for patients with suspected TBM.

[GenoType MTBDRplus assay for rapid detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis in Sichuan].

OBJECTIVE: To explore the molecular and epidemic characteristics of rifampin (RFP) and isoniazid (INH) resistance of mycobacterium tuberculosis (MTB) in Sichuan. METHODS: GenoType reg; MTBDRplus Assay GTplus was used to examine 68 clinical isolates of MTB and 105 clinical specimens for mutations in rpoB, katG and inhA genes related to RFP and INH resistance. RESULTS: Of the 151 valid tests obtained, 44 (29.14%) and 26 (17.22%) showed drug resistance and multidrug resistance, respectively. Resistance to RFP and INH was found in 21.85% (33/151) and 24.50% (37/151) of the samples, respectively. The most prevalent mutations were rpoB S531L, katG S315T1 and inhA C-15T. The multidrug resistance rate in the sputum specimens was significantly higher than that in the non-respiratory samples (19.35% vs 7.41%). CONCLUSION: Drug-resistant, especially multidrug-resistant tuberculosis is highly prevalent in Sichuan. The multidrug-resistant bacteria most frequently show rpoB S531L combined with katG S315T1 mutations, suggesting the necessity of developing rapid clinical identification methods for drug-resistant MTB to control the spread of the resistant strains.

Application of genotype MTBDRplus in rapid detection of the Mycobacterium tuberculosis complex as well as its resistance to isoniazid and rifampin in a high volume laboratory in Southern China.

The alarmingly worsening epidemics of drug-resistant tuberculosis (TB) call urgent need for a simple method for the rapid detection of drug-resistant TB in clinical settings. In an attempt to establish a rapid procedure for laboratory diagnosis of TB and investigate the local TB epidemiology, molecular line probe assay of the Genotype MTBDRplus was used to identify Mycobacterium tuberculosis complex (MTBC) and detect mutations conferring resistance to two most active first-line drugs against TB: Rifampin and Isoniazid. 96 acid-fast bacillus (AFB) smear- positive sputums and 18 PCR-positive non-sputum specimens have been determined for the MTBC and resistance to Rifampin and Isoniazid. The MTBC detection rates in two sources of specimens were 93.8% (90/96) and 77.8% (14/18) respectively. The overall drug resistance (Rifampin or Isoniazid) occurred in 34.6% (36/104). Resistance to rifampin (RMP) was 28.8% (30/104) and 25% (26/104) was to Isoniazid (INH), in which high level drug resistance accounted for 88.5% (23/26) and low level drug resistance accounted for 7.7% (2/26). Multidrug resistance (MDR), defined as resistant to both RMP and INH, was found in 19.2% (20/104) of clinical samples, which was double that of official statistics. In addition, 63.3% (19/30) RMP-resistant mutations were identified in the region of RopB 530-533 and 57.9% (11/19) were the S531L mutation. 84.6% (22/26) of resistance to INH was mediated by Kat S315T1 mutations which conferred the high-level resistance to INH. The Genotype MTBDRplus line probe assay is a suitable and applicable method for establishing the rapidness in detection of drug-resistant TB in clinical laboratory. It will be a valuable addition to the conventional TB diagnostic approaches.

The -159C/T polymorphism in the CD14 gene and the risk of asthma: a meta-analysis.

The -159C/T polymorphism in the CD14 gene has been implicated in susceptibility to asthma, but a large number of studies have reported inconclusive results. The aim of this study is to investigate the association between the -159C/T polymorphism in the CD14 gene and the risk of asthma by meta-analysis. We searched Pubmed, Embase, CNKI database, Wanfang database, Weipu database, and Chinese Biomedical database, covering all publications (last search been performed on April 20, 2010). Statistical analysis was performed by using the softwares Revman 4.2 and STATA 10.0. A total of 17 case-control studies in 17 articles (4,246 cases and 3,631 controls) were included in this meta-analysis. There was no association between this polymorphism and asthma risk in combined analyses (odds ratio (OR) = 0.86 and 95% confidence interval (95% CI) = 0.72-1.02, P = 0.09 for TC + TT vs. CC). In the subgroup analysis by age, ethnicity, and atopic status, no significant associations of asthma risks were obtained from age groups, ethnic groups, and atopic groups for TC + TT vs. CC comparison. For atopic population, significant decreased atopic asthma risks were found among Asian population (OR = 0.69, 95% CI 0.52-0.92, P = 0.01) and children population (OR = 0.69, 95% CI 0.54-0.89, P = 0.0004) for TC + TT vs. CC comparison. This meta-analysis suggests that CD14 is a candidate gene for atopic asthma susceptibility. The -159C/T polymorphism may be a protective factor for atopic asthma in Asian and children. More studies are needed to validate these associations.

Increasing imipenem resistance and dissemination of the ISAba1-associated blaOXA-23 gene among Acinetobacter baumannii isolates in an intensive care unit.

The antibiotic susceptibility of Acinetobacter calcoaceticus-Acinetobacter baumannii complex strains recovered from the intensive care unit (ICU) of West China Hospital, Sichuan, PR China, from 2006 to 2009 was investigated. The identification of A. baumannii and analysis of carbapenemase-encoding genes and their relationship with ISAba1 were performed by PCR. Furthermore, a DiversiLab repetitive extragenic palindromic sequence-based PCR (rep-PCR) microbial typing system and a multilocus sequence typing (MLST) scheme were applied to assess the genetic relationship of the isolates. The results showed that the antibiotic susceptibility of the A. calcoaceticus-A. baumannii complex isolates changed and imipenem resistance increased rapidly between 2006 and 2009. The blaOXA-51-like and ISAba1-associated blaOXA-23 genes were prevalent in the imipenem-resistant A. baumannii isolates. However, the blaOXA-58-like gene was found in only one isolate and no metallo-ß-lactamase genes were detected. The representative multidrug-resistant A. baumannii isolates were identified as one cluster by rep-PCR fingerprinting and belonged to the clonal complex 92 (CC92) according to MLST. These findings indicate a situation of increasing resistance and wide distribution of class D ß-lactamase genes, especially the acquired ISAba1-associated blaOXA-23 gene, in A. baumannii isolates in the ICU of West China Hospital, probably caused by expansion of the CC92 clone.