Expression of 5-hydroxytryptamine 7 receptor in intestinal mucosa correlates with the degree of intestinal inflammation in Crohn's disease.

BACKGROUND: Crohn's disease (CD), an inflammatory bowel disease (IBD), is a complex and heterogeneous disease characterized by nonspecific transmural inflammation of the gastrointestinal tract. CD has a variety of potential causes with no effective treatment available yet. Current clinical laboratory findings from patients do not provide direct indication of the status of mucosal inflammation in the intestine. Recently, it has been found that intestinal inflammation is generally associated with increased levels of 5-hydroxytryptamine (5-HT), which acts as an important gastrointestinal signaling molecule in intestinal homeostasis by stimulating specific receptors. Most previous researches were carried out in vitro or with animal models, and there was a lack of authentic clinical research. In this study, clinical specimens from patients with Crohn's disease were used to investigate the expression of 5-hydroxytryptamine 7 receptor (5-HT7R) in the induction and development of chronic non-specific inflammatory bowel disease. METHODS: Patients with CD admitted to the Department of Gastroenterology in the First Affiliated Hospital of Anhui Medical University between June 2014 and January 2018 were recruited, among which 28 were in active disease and 32 were in remission. In addition, 20 patients who had no obvious abnormality by colonoscopy in the hospital during the same time period were recruited into the control group. Data of clinical disease activity (CDAI), CD endoscopic score (SES-CD) and magnetic resonance score (MaRIA) were collected from those two groups of patients. The expression and distribution of 5-HT7R were investigated and their correlations with clinical CDAI, MaRIA, and endoscopic SES-CD scores were analyzed. RESULTS: Our study demonstrated that 5-HT7R is expressed in intestinal neurons and CD11C-positive cells in human colon. In CD11c/CD86 double-positive cells in the bowel, 5-HT7R expression was significantly increased in the inflammatory area in the bowel of CD patients, and it was closely related to disease severity, MaRIA, and SES-CD scores. CONCLUSION: The expression of 5-HT7R was significantly correlated with the degree of gut inflammation in CD patients and could be a potential biomarker for disease activity and the therapeutic efficacy in patients with Crohn's Disease.

Decreased local immune response and retained HPV gene expression during chemoradiotherapy are associated with treatment resistance and death from cervical cancer.

More than one-third of patients with locally advanced cervical cancer do not respond to chemoradiation therapy (CRT). We aimed to characterize the transcriptional landscape of paired human cervical tumors before and during CRT in order to gain insight into the evolution of treatment response and to elucidate mechanisms of treatment resistance. We prospectively collected cervical tumor biopsies from 115 patients both before and 3 weeks into CRT. RNA-sequencing, Gene Set Enrichment Analysis and HPV gene expression were performed on 20 paired samples that had adequate neoplastic tissue mid-treatment. Tumors from patients with no evidence of disease (NED) at last follow-up had enrichment in pathways related to the immune response both pretreatment and mid-treatment, while tumors from patients dead of disease (DOD) demonstrated enrichment in biosynthetic and mitotic pathways but not in immune-related pathways. Patients DOD had decreased expression of T-cell and cytolytic genes and increased expression of PD-L2 mid-treatment compared to patients NED. Histological and immunohistochemical analysis revealed a decrease in tumor-associated lymphocytes (TAL) during CRT in all patients but tumors from patients DOD had a significantly more pronounced decrease in TALs and CD8+ cells mid-treatment, which was validated in a larger mid-treatment cohort. Finally, patients DOD retained more HPV E6/E7 gene expression during CRT and this was associated with increased expression of genes driving mitosis, which was corroborated in vitro. Our results suggest that decreased local immune response and retained HPV gene expression may be acting together to promote treatment resistance during CRT in patients with cervical cancer.

WDHD1 facilitates G1 checkpoint abrogation in HPV E7 expressing cells by modulating GCN5.

BACKGROUND: Genomic instability is a hallmark of cancer. The G1 checkpoint allows cells to repair damaged DNA that may lead to genomic instability. The high-risk human papillomavirus (HPV) E7 gene can abrogate the G1 checkpoint, yet the mechanism is still not fully understood. Our recent study showed that WDHD1 (WD repeat and high mobility group [HMG]-box DNA-binding protein 1) plays a role in regulating G1 checkpoint of E7 expressing cells. In this study, we explored the mechanism by which WDHD1 regulates G1 checkpoint in HPV E7 expressing cells. METHODS: NIKS and RPE1 derived cell lines were used. Real-time PCR, Rescue experiment, FACS and BrdU labeling experiments were performed to examine role of GCN5 in G1 checkpoint abrogation in HPV-16 E7 expressing cells. RESULTS: In this study, we observed that WDHD1 facilitates G1 checkpoint abrogation by modulating GCN5 in HPV E7 expressing cells. Notably, depletion of WDHD1 caused G1 arrest while overexpression of GCN5 rescued the inhibitory effects of WDHD1 knockdown on G1/S progression. Furthermore, siWDHD1 significantly decreased cell cycle proliferation and DNA synthesis that was correlated with Akt phosphorylation (p-Akt), which was reversed by GCN5 overexpression in HPV E7 expressing cells. CONCLUSIONS: In summary, our data identified a WDHD1/GCN5/Akt pathway leading to the abrogation of G1 checkpoint in the presence of damaged DNA, which may cause genomic instability and eventually HPV induced tumorigenesis.

High-Speed Integrated Endoscopic Photoacoustic and Ultrasound Imaging System.

Endoscopic integrated photoacoustic and ultrasound imaging has the potential for early detection of the cancer in the gastrointestinal tract. Currently, slow imaging speed is one of the limitations for clinical translation. Here, we developed a high speed integrated endoscopic PA and US imaging system, which is able to perform PA and US imaging simultaneously up to 50 frames per second. Using this system, the architectural morphology and vasculature of the rectum wall were visualized from a Sprague Dawley rat in-vivo.

Computational analysis of six optical coherence tomography systems for vocal fold imaging: A comparison study.

OBJECTIVES: There have been many advancements in laryngeal imaging using optical coherence tomography (OCT), with varying system design and probes for use in research, office, and operating room settings. We evaluated the performance of six distinct OCT systems in imaging porcine vocal folds (cords) using computational image processing and segmentation. METHODS: Porcine vocal folds were scanned using six OCT systems. Imaging system and probe performance were quantitatively assessed for signal penetration, layer differentiation, and epithelium (EP) measurement. Fitted exponential decay curves with corresponding α constant and intensity thresholding segmentation were utilized to quantify the aforementioned parameters. RESULTS: The smallest average α constant and deepest signal penetration was of the SS-OCT 1700 nm 90 kHz microscope system (α = -1.74), followed by the SS-OCT 1310 nm 200 kHz VCSEL microscope system (α = -1.99), and SS-OCT 1310 nm 50 kHz rigid forward viewing endoscope system (α = -2.23). The EP was not readily visualized for three out of six systems, but was detected using automated segmentation. Average EP thickness (mean ± SD) was calculated as 55.79 ± 31.86 µm which agrees favorably with previous literature. CONCLUSION: Comparisons of OCT systems are challenging, as they encompass different probe design, optical path, and lasers, depending on application. Practical evaluation of different systems using computer based quantitative image processing and segmentation revealed basic, constructive information, such as EP measurements. To further validate the comparisons of system performance with clinical usability, in vivo human laryngeal imaging will be conducted. Further development of automated image processing and segmentation can be useful in rapid analysis of information. Lasers Surg. Med. 51:412-422, 2019. © 2019 Wiley Periodicals, Inc.

The lysine acetyltransferase GCN5 contributes to human papillomavirus oncoprotein E7-induced cell proliferation via up-regulating E2F1.

General control nondepressible 5 (GCN5), the first identified transcription-related lysine acetyltransferase (KAT), is an important catalytic component of a transcriptional regulatory SAGA (Spt-Ada-GCN5-Acetyltransferase) and ATAC (ADA2A-containing) complex. While GCN5 has been implicated in cancer development, its role in cervical cancer is not known. The human papillomavirus (HPV) oncoprotein E7 abrogates the G1 cell cycle checkpoint and induces genomic instability, which plays a central role in cervical carcinogenesis. In this study, we observed that GCN5 was up-regulated in HPV E7-expressing cells, knockdown of GCN5 inhibited cell cycle progression and DNA synthesis in HPV E7-expressing cells. Notably, GCN5 knockdown reduced the steady-state levels of transcription factor E2F1. Depletion of E2F1 caused G1 arrest while overexpression of E2F1 rescued the inhibitory effects of GCN5 knockdown on G1/S progression in HPV E7-expressing cells. Results from chromatin immunoprecipitation (ChIP) assays demonstrated that GCN5 bound to the E2F1 promoter and increased the extent of histone acetylation within these regions. GCN5 also acetylated c-Myc and increased its ability to bind to the E2F1 promoter. Knockdown of c-Myc reduced the steady-state levels of E2F1 and caused G1 arrest. These results revealed a novel mechanism of E7 function whereby elevated GCN5 acetylates histones and c-Myc to regulate E2F1 expression and cell cycle progression.

CIP2A facilitates the G1/S cell cycle transition via B-Myb in human papillomavirus 16 oncoprotein E6-expressing cells.

Infection with high-risk human papillomaviruses (HR-HPVs, including HPV-16, HPV-18, HPV-31) plays a central aetiologic role in the development of cervical carcinoma. The transforming properties of HR-HPVs mainly reside in viral oncoproteins E6 and E7. E6 protein degrades the tumour suppressor p53 and abrogates cell cycle checkpoints. Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an oncoprotein that is involved in the carcinogenesis of many human malignancies. Our previous data showed that CIP2A was overexpressed in cervical cancer. However, the regulation of CIP2A by HPV-16E6 remains to be elucidated. In this study, we demonstrated that HPV-16E6 significantly up-regulated CIP2A mRNA and protein expression in a p53-degradation-dependent manner. Knockdown of CIP2A by siRNA inhibited viability and DNA synthesis and caused G1 cell cycle arrest of 16E6-expressing cells. Knockdown of CIP2A resulted in a significant reduction in the expression of cyclin-dependent kinase 1 (Cdk1) and Cdk2. Although CIP2A has been reported to stabilize c-Myc by inhibiting PP2A-mediated dephosphorylation of c-Myc, we have presented evidence that the regulation of Cdk1 and Cdk2 by CIP2A is dependent on transcription factor B-Myb rather than c-Myc. Taken together, our study reveals the role of CIP2A in abrogating the G1 checkpoint in HPV-16E6-expressing cells and helps in understanding the molecular basis of HPV-induced oncogenesis.

Role of WDHD1 in Human Papillomavirus-Mediated Oncogenesis Identified by Transcriptional Profiling of E7-Expressing Cells.

UNLABELLED: The E7 oncoprotein of the high-risk human papillomavirus (HPV) plays a major role in HPV-induced carcinogenesis. E7 abrogates the G1 cell cycle checkpoint and induces genomic instability, but the mechanism is not fully understood. In this study, we performed RNA sequencing (RNA-seq) to characterize the transcriptional profile of keratinocytes expressing HPV 16 (HPV-16) E7. At the transcriptome level, 236 genes were differentially expressed between E7 and vector control cells. A subset of the differentially expressed genes, most of them novel to E7-expressing cells, was further confirmed by real-time PCR. Of interest, the activities of multiple transcription factors were altered in E7-expressing cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were investigated. The upregulated genes were enriched in cell cycle and DNA replication, as well as in the DNA metabolic process, transcription, DNA damage, DNA repair, and nucleotide metabolism. Specifically, we focused our studies on the gene encoding WDHD1 (WD repeat and high mobility group [HMG]-box DNA-binding protein), one of the genes that was upregulated in E7-expressing cells. WDHD1 is a component of the replisome that regulates DNA replication. Recent studies suggest that WDHD1 may also function as a DNA replication initiation factor as well as a G1 checkpoint regulator. We found that in E7-expressing cells, the steady-state level of WDHD1 protein was increased along with the half-life. Moreover, downregulation of WDHD1 reduced E7-induced G1 checkpoint abrogation and rereplication, demonstrating a novel function for WDHD1. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications. IMPORTANCE: The high-risk HPV types induce cervical cancer and encode an E7 oncoprotein that plays a major role in HPV-induced carcinogenesis. However, the mechanism by which E7 induces carcinogenesis is not fully understood; specific anti-HPV agents are not available. In this study, we performed RNA-seq to characterize transcriptional profiling of keratinocytes expressing HPV-16 E7 and identified more than 200 genes that were differentially expressed between E7 and vector control cells. Through bioinformatics analysis, pathways altered in E7-expressing cells were identified. Significantly, the WDHD1 gene, one of the genes that is upregulated in E7-expressing cells, was found to play an important role in E7-induced G1 checkpoint abrogation and rereplication. These studies shed light on mechanisms by which HPV induces genomic instability and have therapeutic implications.

Role of Cdc6 in re-replication in cells expressing human papillomavirus E7 oncogene.

The E7 oncoprotein of high-risk human papillomavirus (HPV) types induces DNA re-replication that contributes to carcinogenesis; however, the mechanism is not fully understood. To better understand the mechanism by which E7 induces re-replication, we investigated the expression and function of cell division cycle 6 (Cdc6) in E7-expressing cells. Cdc6 is a DNA replication initiation factor and exhibits oncogenic activities when overexpressed. We found that in E7-expressing cells, the steady-state level of Cdc6 protein was upregulated and its half-life was increased. Cdc6 was localized to the nucleus and associated with chromatin, especially upon DNA damage. Importantly, downregulation of Cdc6 reduced E7-induced re-replication. Interestingly, the level of Cdc6 phosphorylation at serine 54 (S54P) was increased in E7-expressing cells. S54P was associated with an increase in the total amount of Cdc6 and chromatin-bound Cdc6. DNA damage-enhanced upregulation and chromatin binding of Cdc6 appeared to be due to downregulation of cyclin-dependent kinase 1 (Cdk1) as Cdk1 knockdown increased Cdc6 levels. Furthermore, Cdk1 knockdown or inhibition led to re-replication. These findings shed light on the mechanism by which HPV induces genomic instability and may help identify potential targets for drug development.

Role of Cdk1 in the p53-independent abrogation of the postmitotic checkpoint by human papillomavirus E6.

UNLABELLED: Specific types of human papillomavirus (HPV) are strongly associated with the development of cervical carcinoma. The HPV E6 oncoprotein from HPV degrades p53 and abrogates cell cycle checkpoints. Nonetheless, functional p53 has been observed in cervical cancer. We have previously identified a p53-independent function of E6 in attenuating the postmitotic G1-like checkpoint that can lead to polyploidy, an early event during cervical carcinogenesis that predisposes cells to aneuploidy. How E6 promotes cell cycle progression in the presence of p53 and its target, p21, remains a mystery. In this study, we examined the expression of cell cycle-related genes in cells expressing wild-type E6 and the mutant that is defective in p53 degradation but competent in abrogating the postmitotic checkpoint. Our results demonstrated an increase in the steady-state levels of G1- and G2-related cyclins/Cdks in E6-expressing keratinocytes. Interestingly, only Cdk1 remained active in E6 mutant-expressing cells while bypassing the postmitotic checkpoint. Furthermore, the downregulation of Cdk1 impaired the ability of both wild-type and mutant E6 to induce polyploidy. Our study thus demonstrated an important role for Cdk1, which binds p21 with lower affinity than Cdk2, in abrogating the postmitotic checkpoint in E6-expressing cells. We further show that E2F1 is important for E6 to upregulate Cdk1. Moreover, reduced nuclear p21 localization was observed in the E6 mutant-expressing cells. These findings shed light on the mechanisms by which HPV induces genomic instability and hold promise for the identification of drug targets. IMPORTANCE: HPV infection is strongly associated with the development of cervical carcinoma. HPV encodes an E6 oncoprotein that degrades the tumor suppressor p53 and abrogates cell cycle checkpoints. Nonetheless, functional p53 has been observed in cervical cancer. We have recently demonstrated a p53-independent abrogation of the postmitotic checkpoint by HPV E6 that induces polyploidy. However, the mechanism is not known. In this study, we provide evidence that Cdk1 plays an important role in this process. Previously, Cdk2 was thought to be essential for the G1/S transition, while Cdk1 only compensated its function in the absence of Cdk2. Our studies have demonstrated a novel role of Cdk1 at the postmitotic G1-like checkpoint in the presence of Cdk2. These findings shed light on the mechanisms by which HPV induces genomic instability and hold promise for the identification of drug targets.

Gallic acid induces apoptosis in human cervical epithelial cells containing human papillomavirus type 16 episomes.

The high-risk human papillomaviruses (HPV) that infect the anogenital tract are strongly associated with the development of cervical carcinoma, which is the second most common cancer in women worldwide. Therapeutic drugs specifically targeting HPV are not available. Polyphenolic compounds have gained considerable attention because of their cytotoxic effects against a variety of cancers and certain viruses. In this study, we examined the effects of several polyphenols on cellular proliferation and death of the human cervical cancer cells and human cervical epithelial cells containing stable HPV type 16 episomes (HPVep). Our results show that three polyphenols inhibited proliferation of HeLa cells dose-dependently. Furthermore, one of the examined polyphenols, gallic acid (GA), also inhibited the proliferation of HPVep cells and exhibited significant specificity towards HPV-positive cells. The anti-proliferative effect of GA on HPVep and HeLa cells was associated with apoptosis and upregulation of p53. These results suggest that GA can be a potential candidate for the development of anti-HPV agents.

Human papillomavirus E7 induces rereplication in response to DNA damage.

Human papillomavirus (HPV) infection is necessary but not sufficient for cervical carcinogenesis. Genomic instability caused by HPV allows cells to acquire additional mutations required for malignant transformation. Genomic instability in the form of polyploidy has been demonstrated to play an important role in cervical carcinogenesis. We have recently found that HPV-16 E7 oncogene induces polyploidy in response to DNA damage; however, the mechanism is not known. Here we present evidence demonstrating that HPV-16 E7-expressing cells have an intact G(2) checkpoint. Upon DNA damage, HPV-16 E7-expressing cells arrest at the G(2) checkpoint and then undergo rereplication, a process of successive rounds of host DNA replication without entering mitosis. Interestingly, the DNA replication initiation factor Cdt1, whose uncontrolled expression induces rereplication in human cancer cells, is upregulated in E7-expressing cells. Moreover, downregulation of Cdt1 impairs the ability of E7 to induce rereplication. These results demonstrate an important role for Cdt1 in HPV E7-induced rereplication and shed light on mechanisms by which HPV induces genomic instability.

Infected peripheral blood mononuclear cells transmit latent varicella zoster virus infection to the guinea pig enteric nervous system.

Latent wild-type (WT) and vaccine (vOka) varicella zoster virus (VZV) are found in the human enteric nervous system (ENS). VZV also infects guinea pig enteric neurons in vitro, establishes latency and can be reactivated. We therefore determined whether lymphocytes infected in vitro with VZV secrete infectious virions and can transfer infection in vivo to the ENS of recipient guinea pigs. T lymphocytes (CD3-immunoreactive) were preferentially infected following co-culture of guinea pig or human peripheral blood mononuclear cells with VZV-infected HELF. VZV proliferated in the infected T cells and expressed immediate early and late VZV genes. Electron microscopy confirmed that VZV-infected T cells produced encapsulated virions. Extracellular virus, however, was pleomorphic, suggesting degradation occurred prior to release, which was confirmed by the failure of VZV-infected T cells to secrete infectious virions. Intravenous injection of WT- or vOka-infected PBMCs, nevertheless, transmitted VZV to recipient animals (guinea pig > human lymphocytes). Two days post-inoculation, lung and liver, but not gut, contained DNA and transcripts encoding ORFs 4, 40, 66 and 67. Twenty-eight days after infection, gut contained DNA and transcripts encoding ORFs 4 and 66 but neither DNA nor transcripts could any longer be found in lung or liver. In situ hybridization revealed VZV DNA in enteric neurons, which also expressed ORF63p (but not ORF68p) immunoreactivity. Observations suggest that VZV infects T cells, which can transfer VZV to and establish latency in enteric neurons in vivo. Guinea pigs may be useful for studies of VZV pathogenesis in the ENS.

Expression and transcriptional profiling of the LKB1 tumor suppressor in cervical cancer cells.

OBJECTIVES: To characterize the biological activities of LKB1, examine LKB1 protein expression and identify LKB1-regulated genes that may serve as therapeutic targets in cervical cancer. METHODS: Proliferation of cervical cancer HeLa cells expressing LKB1 was examined. LKB1 expression in normal cervical tissues and cervical cancers was assessed by immunohistochemistry. Gene expression profiles of cervical cancer HeLa cells stably expressing LKB1 were analyzed by microarray. Differentially expressed genes were analyzed using Gene Ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) PATHWAY database. Quantitative RT-PCR was used to validate the microarray data. The expression of lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) was confirmed by western blotting. RESULTS: Expression of LKB1 inhibited HeLa cell proliferation, activated AMPK and was lost in more than 50% of cervical carcinomas. More than 200 genes were differentially expressed between HeLa cells with and without LKB1. Bioinformatics analysis with GO annotation indicated that LKB1 plays a role in receiving diverse stimuli and converting them into molecular signals. KEGG PATHWAY analysis showed that 8 pathways were significantly regulated. These include arginine and proline metabolism and inositol phosphate metabolism. The differential expression of 7 randomly selected genes was confirmed by quantitative RT-PCR. Furthermore, the steady-state level of INPP4B protein was up-regulated in LKB1-overexpressing cells. CONCLUSIONS: This study establishes LKB1 as an important tumor suppressor in cervical cancer and sheds light on a novel signaling pathway regulated by LKB1.

Slit/Robo-mediated chemorepulsion of vagal sensory axons in the fetal gut.

BACKGROUND: The vagus nerve descends from the brain to the gut during fetal life to reach specific targets in the bowel wall. Vagal sensory axons have been shown to respond to the axon guidance molecule netrin and to its receptor, deleted in colorectal cancer (DCC). As there are regions of the gut wall into which vagal axons do and do not extend, it is likely that a combination of attractive and repellent cues are involved in how vagal axons reach specific targets. We tested the hypothesis that Slit/Robo chemorepulsion can contribute to the restriction of vagal sensory axons to specific targets in the gut wall. RESULTS: Transcripts encoding Robo1 and Robo2 were expressed in the nodose ganglia throughout development and mRNA encoding the Robo ligands Slit1, Slit2, and Slit3 were all found in the fetal and adult bowel. Slit2 protein was located in the outer gut mesenchyme in regions that partially overlap with the secretion of netrin-1. Neurites extending from explanted nodose ganglia were repelled by Slit2. CONCLUSIONS: These observations suggest that vagal sensory axons are responsive to Slit proteins and are thus repelled by Slits secreted in the gut wall and prevented from reaching inappropriate targets.

A novel synthetic nucleic acid mixture for quantification of microbes by mNGS.

Metagenomic next-generation sequencing (mNGS) provides considerable advantages in identifying emerging and re-emerging, difficult-to-detect and co-infected pathogens; however, the clinical application of mNGS remains limited primarily due to the lack of quantitative capabilities. This study introduces a novel approach, KingCreate-Quantification (KCQ) system, for quantitative analysis of microbes in clinical specimens by mNGS, which co-sequence the target DNA extracted from the specimens along with a set of synthetic dsDNA molecules used as Internal-Standard (IS). The assay facilitates the conversion of microbial reads into their copy numbers based on IS reads utilizing a mathematical model proposed in this study. The performance of KCQ was systemically evaluated using commercial mock microbes with varying IS input amounts, different proportions of human genomic DNA, and at varying amounts of sequence analysis data. Subsequently, KCQ was applied in microbial quantitation in 36 clinical specimens including blood, bronchoalveolar lavage fluid, cerebrospinal fluid and oropharyngeal swabs. A total of 477 microbe genetic fragments were screened using the bioinformatic system. Of these 83 fragments were quantitatively compared with digital droplet PCR (ddPCR), revealing a correlation coefficient of 0.97 between the quantitative results of KCQ and ddPCR. Our study demonstrated that KCQ presents a practical approach for the quantitative analysis of microbes by mNGS in clinical samples.

Respiratory syncytial virus induces leukotriene C4 synthase expression in bronchial epithelial cells.

BACKGROUND AND OBJECTIVE: Respiratory syncytial virus (RSV) results in acute wheezing in infants and is frequently associated with recurrent wheezing. Although RSV-induced wheezing clinically resembles that of asthma, corticosteroids are not equivalently effective in RSV-associated wheezing. The study sought to determine the mechanisms of RSV-induced wheezing by establishing an in vitro model of RSV-infected human bronchial epithelial cells (16-HBEC). METHODS: Leukotriene C4 synthase (LTC4 S) messenger RNA (mRNA) expression in 16-HBEC was detected using fluorescence quantitative polymerase chain reaction, and the relative level of LTC4 S mRNA was expressed as quotient cycle threshold (qCt) based on the threshold cycle number value compared with that of ß-actin. Cysteinyl leukotrienes (CysLT) in culture supernatant were measured by enzyme-linked immunosorbent assay. RSV-infected 16-HBEC was incubated with gradient concentration of budesonide (BUD) to assess its effects on LTC4 S expression and CysLT secretion. RESULTS: RSV infection resulted in increased LTC4 S mRNA expression between 48 and 96 h post-infection. High level of CysLT was detected in the supernatant of RSV-infected 16-HBEC. BUD at concentrations of 10(-10) to 10(-5) mol/L did not significantly alter LTC4 S mRNA expression. CONCLUSIONS: RSV infection upregulated LTC4 S expression in HBEC leading to increased CysLT secretion. Such induction was not attenuated by BUD, suggesting that CysLT might contribute to the pathogenesis of RSV-induced wheezing.

Optical coherence tomography evaluation of vaginal epithelial thickness during CO2 laser treatment: A pilot study.

Genitourinary syndrome of menopause (GSM) negatively affects more than half of postmenopausal women. Energy-based therapy has been explored as a minimally invasive treatment for GSM; however, its mechanism of action and efficacy is controversial. Here, we report on a pilot imaging study conducted on a small group of menopause patients undergoing laser treatment. Intravaginal optical coherence tomography (OCT) endoscope was used to quantitatively monitor the changes in the vaginal epithelial thickness (VET) during fractional-pixel CO2 laser treatment. Eleven patients with natural menopause and one surgically induced menopause patient were recruited in this clinical study. Following the laser treatment, 6 out of 11 natural menopause patient showed increase in both proximal and distal VET, while two natural menopause patient showed increase in VET in only one side of vaginal tract. Furthermore, the patient group that showed increased VET had thinner baseline VET compared to the patients that showed decrease in VET after laser treatment. These results demonstrate the potential utility of intravaginal OCT endoscope in evaluating the vaginal tissue integrity and tailoring vaginal laser treatment on a per-person basis, with the potential to monitor other treatment procedures.

Varicella zoster virus (VZV) infects and establishes latency in enteric neurons.

Case reports have linked varicella-zoster virus (VZV) to gastrointestinal disorders, including severe abdominal pain preceding fatal varicella and acute colonic pseudoobstruction (Ogilvie's syndrome). Because we had previously detected DNA and transcripts encoding latency-associated VZV gene products in the human gut, we sought to determine whether latent VZV is present in the human enteric nervous system (ENS) and, if so, to identify the cells in which it is located and its route to the bowel. Neither DNA, nor transcripts encoding VZV gene products, could be detected in resected gut from any of seven control children (<1 year old) who had not received the varicella vaccine or experienced varicella; however, VZV DNA and transcripts were each found to be present in resected bowel from 6/6 of children with a past history of varicella and in that of 6/7 of children who received the varicella vaccine. Both wild-type (WT) and vaccine-type (vOka) VZV thus establish latent infection in human gut. To determine routes by which VZV might gain access to the bowel, we injected guinea pigs with human or guinea pig lymphocytes expressing green fluorescent protein (GFP) under the control of the VZV ORF66 gene (VZV(OKA66.GFP)). GFP-expressing enteric neurons were found throughout the bowel within 2 days and continued to be present for greater than 6 weeks. DNA encoding VZV gene products also appeared in enteric and dorsal root ganglion (DRG) neurons following intradermal administration of WT-VZV and in enteric neurons after intradermal injection of VZV(OKA66.GFP); moreover, a small number of guinea pig DRG neurons were found to project both to the skin and the intraperitoneal viscera. Viremia, in which lymphocytes carry VZV, or axonal transport from DRG neurons infected through their epidermal projections are thus each potential routes that enable VZV to gain access to the ENS.