A human stomach cell type transcriptome atlas.

BACKGROUND: The identification of cell type-specific genes and their modification under different conditions is central to our understanding of human health and disease. The stomach, a hollow organ in the upper gastrointestinal tract, provides an acidic environment that contributes to microbial defence and facilitates the activity of secreted digestive enzymes to process food and nutrients into chyme. In contrast to other sections of the gastrointestinal tract, detailed descriptions of cell type gene enrichment profiles in the stomach are absent from the major single-cell sequencing-based atlases. RESULTS: Here, we use an integrative correlation analysis method to predict human stomach cell type transcriptome signatures using unfractionated stomach RNAseq data from 359 individuals. We profile parietal, chief, gastric mucous, gastric enteroendocrine, mitotic, endothelial, fibroblast, macrophage, neutrophil, T-cell, and plasma cells, identifying over 1600 cell type-enriched genes. CONCLUSIONS: We uncover the cell type expression profile of several non-coding genes strongly associated with the progression of gastric cancer and, using a sex-based subset analysis, uncover a panel of male-only chief cell-enriched genes. This study provides a roadmap to further understand human stomach biology.

No association between circulating concentrations of vitamin D and risk of lung cancer: an analysis in 20 prospective studies in the Lung Cancer Cohort Consortium (LC3).

Background: There is observational evidence suggesting that high vitamin D concentrations may protect against lung cancer. To investigate this hypothesis in detail, we measured circulating vitamin D concentrations in prediagnostic blood from 20 cohorts participating in the Lung Cancer Cohort Consortium (LC3). Patients and methods: The study included 5313 lung cancer cases and 5313 controls. Blood samples for the cases were collected, on average, 5 years before lung cancer diagnosis. Controls were individually matched to the cases by cohort, sex, age, race/ethnicity, date of blood collection, and smoking status in five categories. Liquid chromatography coupled with tandem mass spectrometry was used to separately analyze 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] and their concentrations were combined to give an overall measure of 25(OH)D. We used conditional logistic regression to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for 25(OH)D as both continuous and categorical variables. Results: Overall, no apparent association between 25(OH)D and risk of lung cancer was observed (multivariable adjusted OR for a doubling in concentration: 0.98, 95% CI: 0.91, 1.06). Similarly, we found no clear evidence of interaction by cohort, sex, age, smoking status, or histology. Conclusion: This study did not support an association between vitamin D concentrations and lung cancer risk.

Human cytomegalovirus replication induces endothelial cell interleukin-11.

Endothelial cells (EC) are critical sites of human cytomegalovirus (hCMV) infection in vivo. Infection can induce the production of various EC cytokines, such as interleukin (IL-)6, which can have autocrine and/or paracrine effector functions. Here, we report that hCMV induces the production of EC IL-11, a relatively understudied member of the IL-6-type cytokine family. We detail temporal EC IL-11 translation and protein secretion dynamics in response to hCMV infection, and reveal distinct differences compared to EC IL-6. Viral replication had markedly opposing effects on the regulation of these closely related cytokines, representing a major driving force behind IL-11 production, whilst concurrently suppressing IL-6 expression. This is the first report of any biological agent that stimulates EC IL-11 production.

Human cytomegalovirus induces a biphasic inflammatory response in primary endothelial cells.

Contradictory studies report either pro- or anti-inflammatory endothelial cell (EC) responses to human cytomegalovirus (hCMV) infection, hindering the validation of a potential link between this virus and associated inflammatory pathologies. Clarifying this issue, we report that hCMV induces a biphasic response. Early after inoculation, hCMV promoted lymphocyte and, to a lesser extent, neutrophil capture under in vivo relevant shear stresses. In contrast, later stages of infection rendered EC refractory to basal, or cytokine-induced, leukocyte recruitment.

Circulating microRNAs predict biochemical recurrence in prostate cancer patients.

BACKGROUND: Circulating microRNAs (miRNAs) are emerging as promising biomarkers for prostate cancer. Here, we investigated the potential of these molecules to assist in prognosis and treatment decision-making. METHODS: MicroRNAs in the serum of patients who had experienced rapid biochemical recurrence (BCR) (n=8) or no recurrence (n=8) following radical prostatectomy (RP) were profiled using high-throughput qRT-PCR. Recurrence-associated miRNAs were subsequently quantitated by qRT-PCR in a validation cohort comprised of 70 patients with Gleason 7 cancers treated by RP, 31 of whom had undergone disease progression following surgery. The expression of recurrence-associated miRNAs was also examined in tumour tissue cohorts. RESULTS: Three miRNAs - miR-141, miR-146b-3p and miR-194 - were elevated in patients who subsequently experienced BCR in the screening study. MiR-146b-3p and miR-194 were also associated with disease progression in the validation cohort, as determined by log-rank tests and Cox proportional hazards regression. Multivariate analysis revealed that miR-146b-3p possessed prognostic information beyond standard clinicopathological parameters. Analysis of tissue cohorts revealed that miR-194 was robustly expressed in the prostate, elevated in metastases, and its expression in primary tumours was associated with a poor prognosis. CONCLUSION: Our study suggests that circulating miRNAs, measured at the time of RP, could be combined with current prognostic tools to predict future disease progression in men with intermediate risk prostate cancers.

Kaposi's sarcoma-associated herpesvirus inhibits expression and function of endothelial cell major histocompatibility complex class II via suppressor of cytokine signaling 3.

Endothelial cells (EC) can present antigen to either CD8(+) T lymphocytes through constitutively expressed major histocompatibility complex class I (MHC-I) or CD4(+) T lymphocytes through gamma interferon (IFN-γ)-induced MHC-II. Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), an EC neoplasm characterized by dysregulated angiogenesis and a substantial inflammatory infiltrate. KSHV is understood to have evolved strategies to inhibit MHC-I expression on EC and MHC-II expression on primary effusion lymphoma cells, but its effects on EC MHC-II expression are unknown. Here, we report that the KSHV infection of human primary EC inhibits IFN-γ-induced expression of the MHC-II molecule HLA-DR at the transcriptional level. The effect is functionally significant, since recognition by an HLA-DR-restricted CD4(+) T-cell clone in response to cognate antigen presented by KSHV-infected EC was attenuated. Inhibition of HLA-DR expression was also achieved by exposing EC to supernatant from KSHV-inoculated EC before IFN-γ treatment, revealing a role for soluble mediators. IFN-γ-induced phosphorylation of STAT-1 and transcription of CIITA were suppressed in KSHV-inoculated EC via a mechanism involving SOCS3 (suppressor of cytokine signaling 3). Thus, KSHV infection resulted in transcriptional upregulation of SOCS3, and treatment with RNA interference against SOCS3 relieved virus-induced inhibition of IFN-γ-induced STAT-1 phosphorylation. Since cell surface MHC-II molecules present peptide antigens to CD4(+) T lymphocytes that can function either as direct cytolytic effectors or to initiate and regulate adaptive immune responses, inhibition of this antigen-presenting pathway would provide a survival advantage to the virus.

Establishing Clinically Meaningful Change on Outcome Assessments Frequently Used in Trials of Mild Cognitive Impairment Due to Alzheimer's Disease.

BACKGROUND: Consensus is lacking on what constitutes a meaningful score change for individual patients on clinical outcome assessments (COAs) that are commonly used in clinical trials of Alzheimer's disease. Such thresholds are one important approach to help contextualize trial results and demonstrate meaningful treatment benefit. OBJECTIVES: To estimate meaningful within-patient change thresholds for the Clinical Dementia Rating Scale - Sum of Boxes (CDR-SB), Alzheimer's Disease Assessment Scale - Cognitive Subscale (ADAS-Cog), and the Mini-Mental State Examination (MMSE) among participants with mild cognitive impairment (MCI). DESIGN: Retrospective anchor- and distribution-based analyses of data from the ADC-008 (NCT00000173) study were used to estimate thresholds for meaningful within-patient change on the target measures. SETTING: Analyses were conducted using data from ADC-008 a Phase III, multicenter, randomized, double-blind, placebo-controlled, parallel-group study among participants with the amnestic subtype of MCI, which was conducted by the Alzheimer's Disease Cooperative Study (ADCS) between March 1999 and January 2004 in the United States and Canada. PARTICIPANTS: Analyses were based on 769 eligible participants who completed the baseline assessment from 69 ADCS sites in the United States and Canada. MEASUREMENTS: The target outcome measures for this analysis included the CDR-SB, the ADAS-Cog, and the MMSE. The anchor measures for this analysis included the Global Deterioration Scale and the MCI-Clinical Global Impression of Change. RESULTS: Focusing on the 12-month time point, within-patient increases of 1-2.5 points in the CDR-SB and increases of 2-5 points on the 11-item ADAS-Cog and 13-item ADAS-Cog, on average, reflect minimal-to-moderate levels of deterioration, respectively. CONCLUSIONS: These thresholds may be useful to aid the interpretation of Alzheimer's disease clinical trial data by illustrating meaningful within-patient progression over the course of a clinical trial via supplementary progressor analyses, which may in turn be informative for treatment decisions. Estimates generated via these methods are specifically intended to evaluate within-patient change and are not intended to assess the magnitude and meaningfulness of differences between group-level changes over time.

Kaposi's sarcoma-associated herpesvirus infection of endothelial cells inhibits neutrophil recruitment through an interleukin-6-dependent mechanism: a new paradigm for viral immune evasion.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), an endothelial cell (EC) neoplasm characterized by dysregulated angiogenesis and inflammation. KSHV infection of EC causes production of proinflammatory mediators, regarded as possible initiators of the substantial mononuclear leukocyte recruitment seen in KS. Conversely, KSHV immune evasion strategies exist, such as degradation of EC leukocyte adhesion receptors by viral proteins. Here, we report the effects of KSHV infection of primary EC on recruitment of flowing leukocytes. Infection did not initiate adhesion of any leukocyte subset per se. However, on cytokine-stimulated EC, KSHV specifically inhibited neutrophil, but not PBL or monocyte, transmigration, an observation consistent with the inflammatory cell profile found in KS lesions in vivo. This inhibition could be recapitulated on uninfected EC using supernatant from infected cultures. These supernatants contained elevated levels of human interleukin 6 (hIL-6), and both the KSHV- and the supernatant-induced inhibitions of neutrophil transmigration were abrogated in the presence of a hIL-6 neutralizing antibody. Furthermore, preconditioning of EC with hIL-6 mimicked the effect of KSHV. Using RNA interference (RNAi), we show that upregulation of suppressor of cytokine signaling 3 (SOCS3) was necessary for this effect of hIL-6. These studies reveal a novel paracrine mode of KSHV immune evasion, resulting in reduced recruitment of neutrophils, a cell type whose antiviral and antitumor roles are becoming increasingly appreciated. Moreover, the findings have implications for our understanding of the contribution of hIL-6 to the pathogenesis of other inflammatory disorders and tumors in which this cytokine is abundant.

Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor-2 inhibits type 1 interferon signalling by targeting interferon-stimulated gene factor-3.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes four viral interferon regulatory factors (vIRF-1-4). We investigated the mechanism and consequences of vIRF-2-mediated inhibition of interferon-response element signalling following type I interferon (IFN) induction. Western blot and electrophoretic mobility-shift assays identified the interferon-stimulated gene factor-3 (ISGF-3) components STAT1 and IRF-9 as the proximal targets of vIRF-2 activity. The biological significance of vIRF-2 inhibition of ISGF-3 was demonstrated by vIRF-2-mediated rescue of the replication of the IFN-sensitive virus encephalomyocarditis virus. This study provides both a mechanism and evidence for KSHV vIRF-2-mediated suppression of the consequences of type 1 IFN-induced signalling.

A population-based study of how children are exposed to saliva in KwaZulu-Natal Province, South Africa: implications for the spread of saliva-borne pathogens to children.

OBJECTIVES: In sub-Saharan Africa, many viral infections, including Epstein-Barr virus, cytomegalovirus, Kaposi's sarcoma-associated herpesvirus and hepatitis B are acquired in childhood. While saliva is an important transmission conduit for these viruses, little is known about how saliva is passed to African children. We endeavoured to identify the range and determinants of acts by which African children are exposed to saliva. METHODS: To identify the range of acts by which African children are exposed to saliva, we conducted focus groups, semi-structured interviews and participant observations in an urban and a rural community in South Africa. To measure the prevalence and determinants of the identified acts, we administered a questionnaire to a population-based sample of caregivers. RESULTS: We identified 12 caregiving practices that expose a child's oral-respiratory mucosa, cutaneous surfaces or anal-rectal mucosa to saliva. Several acts were heretofore not described in the contemporary literature (e.g., caregiver inserting finger lubricated with saliva into child's rectum to relieve constipation). Among 896 participants in the population-based survey, many of the acts were commonly practised by all respondent types (mothers, fathers, grandmothers and siblings). The most common were premastication of food, sharing sweets and premastication of medicinal plants that are spit onto a child's body. CONCLUSIONS: African children are exposed to saliva through a variety of acts, practised by a variety of caregivers, with no single predominant practice. This diversity poses challenges for epidemiologic work seeking to identify specific saliva-passing practices that transmit viruses. Most acts could be replaced by other actions and are theoretically preventable.

Prospective study of dietary patterns and colorectal cancer among Singapore Chinese.

An influence of Western diet and lifestyle factors observed among Singapore Chinese may contribute to the population's marked rise in colorectal cancer incidence over the past two decades. Thus far, however, there is little evidence for individual nutrients and foods as major contributing factors in this population. We evaluated whether patterns of food intake were associated with colorectal cancer in a population-based cohort of 61,321 Singapore Chinese that was established in 1993-98. Two dietary patterns, meat-dim sum and vegetable-fruit-soy, were previously identified by principal components analysis using baseline dietary data from a validated 165-item food frequency questionnaire. As of 31 December 2005, 961 incident colorectal cancer cases were diagnosed. Proportional hazards regression was used to calculate adjusted hazard ratios. Using nearly 10 years of follow-up data, we observed no association with either the meat-dim sum or vegetable-fruit-soy pattern for colorectal cancer. In conclusion, neither individual nutrients or foods nor dietary patterns appear to explain the rise in colorectal cancer among Singapore Chinese population.

A ZEB1-miR-375-YAP1 pathway regulates epithelial plasticity in prostate cancer.

MicroRNA-375 (miR-375) is frequently elevated in prostate tumors and cell-free fractions of patient blood, but its role in genesis and progression of prostate cancer is poorly understood. In this study, we demonstrated that miR-375 is inversely correlated with epithelial-mesenchymal transition signatures (EMT) in clinical samples and can drive mesenchymal-epithelial transition (MET) in model systems. Indeed, miR-375 potently inhibited invasion and migration of multiple prostate cancer lines. The transcription factor YAP1 was found to be a direct target of miR-375 in prostate cancer. Knockdown of YAP1 phenocopied miR-375 overexpression, and overexpression of YAP1 rescued anti-invasive effects mediated by miR-375. Furthermore, transcription of the miR-375 gene was shown to be directly repressed by the EMT transcription factor, ZEB1. Analysis of multiple patient cohorts provided evidence for this ZEB1-miR-375-YAP1 regulatory circuit in clinical samples. Despite its anti-invasive and anti-EMT capacities, plasma miR-375 was found to be correlated with circulating tumor cells in men with metastatic disease. Collectively, this study provides new insight into the function of miR-375 in prostate cancer, and more broadly identifies a novel pathway controlling epithelial plasticity and tumor cell invasion in this disease.

The histone deacetylase inhibitor, CBHA, inhibits growth of human neuroblastoma xenografts in vivo, alone and synergistically with all-trans retinoic acid.

Histone deacetylase inhibitors (HDACIs) inhibit the growth of a variety of transformed cells in culture. We demonstrated previously that the hybrid-polar HDACI m-carboxycinnamic acid bis-hydroxamide (CBHA) induces apoptosis of human neuroblastoma in vitro and is effective in lower doses when combined with retinoids. The current study investigates the effect of CBHA on the growth of human neuroblastoma in vivo, both alone and in combination with all-trans retinoic acid (atRA), using a severe combined immunodeficiency-mouse xenograft model. CBHA (50, 100, and 200 mg/kg/day) inhibited growth of SMS-KCN-69n tumor xenografts in a dose-dependent fashion, with 200 mg/kg CBHA resulting in a complete suppression of tumor growth. The efficacy of 50 and 100 mg/kg CBHA was enhanced by the addition of 2.5 mg/kg atRA. This dose of atRA was ineffective when administered alone. Treatment was accompanied by mild weight loss in all groups except the lowest dose of CBHA. Our results suggest HDACIs alone or combined with retinoids may have therapeutic utility for neuroblastoma.

Suberoylanilide hydroxamic acid, an inhibitor of histone deacetylase, suppresses the growth of prostate cancer cells in vitro and in vivo.

Suberoylanilide hydroxamic acid (SAHA) is the prototype of a family of hybrid polar compounds that induce growth arrest in transformed cells and show promise for the treatment of cancer. SAHA induces differentiation and/or apoptosis in certain transformed cells in culture and is a potent inhibitor of histone deacetylases. In this study, we examined the effects of SAHA on the growth of human prostate cancer cells in culture and on the growth of the CWR22 human prostate xenograft in nude mice. SAHA suppressed the growth of the LNCaP, PC-3, and TSU-Pr1 cell lines at micromolar concentrations (2.5-7.5 microM). SAHA induced dose-dependent cell death in the LNCaP cells. In mice with transplanted CWR222 human prostate tumors, SAHA (25, 50, and 100 mg/kg/day) caused significant suppression of tumor growth compared with mice receiving vehicle alone; treatment with 50 mg/kg/day resulted in a 97% reduction in the mean final tumor volume compared with controls. At this dose, there was no detectable toxicity as evaluated by weight gain and necropsy examination. Increased accumulation of acetylated core histones was detected in the CWR22 tumors within 6 h of SAHA administration. SAHA induced prostate-specific antigen mRNA expression in CWR22 prostate cancer cells, resulting in higher levels of serum prostate-specific antigen than predicted from tumor volume alone. The results suggest that hydroxamic acid-based hybrid polar compounds inhibit prostate cancer cell growth and may be useful, relatively nontoxic agents for the treatment of prostate carcinoma.

Expanding the relationship context for couple-based HIV prevention: Elucidating women's perspectives on non-traditional sexual partnerships.

INTRODUCTION: HIV prevention interventions targeting couples are efficacious, cost-effective and a key strategy for preventing HIV transmission. Awareness of the full spectrum of relationship types and underlying complexities, as well as available support mechanisms in a given context, are critical to the design of effective couple-based interventions. OBJECTIVE: This paper is based on a sub-analysis of a qualitative research study investigating HIV disclosure dynamics amongst pregnant women living with HIV in Durban, South Africa. The sub-analysis explored the nature of participants' social and relationship contexts and consequences of these dynamics on women's feelings of trust towards partners and perceptions of partner commitment. METHODS: Between June and August 2008, we conducted in-depth interviews with 62 pregnant women living with HIV and accessing Prevention of Mother-to-Child Transmission (PMTCT) services in Durban, South Africa. Transcripts were coded for emergent themes and categories using a grounded theoretical approach. RESULTS: The median age of participants was 26 years (interquartile range: 22-29 years). Three major themes with accompanying sub themes were identified: 1) relationship types (sub themes included unmarried status, minimal cohabitation with partners, presence of concurrent relationships), 2) relationship quality/functioning (sub themes included low trust and expectation of partner commitment, relationship turbulence, and lack of communication/ability to negotiate protective behaviours), and 3) factors underlying the relationship functioning (sub themes included dynamics of concurrent relationships coinciding with concurrent pregnancies, gender roles and unequal relationship power, intimate partner violence or threat thereof, and lack of social support). CONCLUSIONS: Our research findings indicate a lack of many of the dyadic relationship elements underlying couple-counselling frameworks for successful risk reduction coordination. Understanding sexual behaviour and the accompanying relationship dynamics within different types of partnerships is crucial for the optimal design of couple-based HIV prevention interventions.

Inhibition of transformed cell growth and induction of cellular differentiation by pyroxamide, an inhibitor of histone deacetylase.

PURPOSE: We have synthesized a series of hybrid polar compounds that induce differentiation and/or apoptosis of various transformed cells. These agents are also potent inhibitors of histone deacetylases (HDACs). Pyroxamide (suberoyl-3-aminopyridineamide hydroxamic acid) is a new member of this class of compounds that is currently under development as an anticancer agent. We investigated the activity of pyroxamide as an inducer of differentiation and/or apoptosis in transformed cells. EXPERIMENTAL DESIGN AND RESULTS: Pyroxamide, at micromolar concentrations, induced terminal differentiation in murine erythroleukemia (MEL) cells and caused growth inhibition by cell cycle arrest and/or apoptosis in MEL, prostate carcinoma, bladder carcinoma, and neuroblastoma cells. Administration of pyroxamide (100 or 200 mg/kg/day) to nude mice at doses that caused little evident toxicity significantly suppressed the growth of s.c. CWR22 prostate cancer xenografts. Despite the potent growth-inhibitory effects of pyroxamide in this tumor model, serum prostate-specific antigen levels in control versus pyroxamide-treated mice were not significantly different. Pyroxamide is a potent inhibitor of affinity-purified HDAC1 (ID(50) = 100 nM) and causes the accumulation of acetylated core histones in MEL cells cultured with the agent. Human CWR22 prostate tumor xenografts from mice treated with pyroxamide (100 or 200 mg/kg/day) showed increased levels of histone acetylation and increased expression of the cell cycle regulator p21/WAF1, compared with tumors from vehicle-treated control animals. CONCLUSIONS: The findings suggest that pyroxamide may be a useful agent for the treatment of malignancy and that induction of p21/WAF1 in transformed cells by pyroxamide may contribute to the antitumor effects of this agent.

The activity of caspase-3-like proteases is elevated during the development of colorectal carcinoma.

Activated caspase-3-like proteases promote apoptotic cell death by cleaving cellular substrates. Caspase-3-like activity was measured in colonic carcinomas and in matched normal colonic mucosa from 31 patients and was significantly elevated in 25/ 31 colonic carcinomas and adenomas when compared to normal mucosa (P < 0.0001). Caspase-3-like activity was much higher in normal mucosa and tumours of female subjects than of males (P < 0.0001). No correlation was obtained between caspase-3-like activity and location of the tumour, tumour grade, stage, or patient age. The marked increase in caspase-3-like activity in colorectal carcinomas may reflect an increase in the proportion of cells undergoing spontaneous apoptosis.

Methylation of CpG sites in exon 2 of the bcl-2 gene occurs in colorectal carcinoma.

BACKGROUND: Aberrant bcl-2 expression frequently occurs in colorectal carcinoma. The current study investigated if CpG sites in bcl-2 were methylated in colorectal carcinoma and if methylation correlated with loss of expression of bcl-2 mRNA. METHODS: Methylation was assessed in 23 matched normal mucosae and colonic carcinomas by Southern blotting with methylation-sensitive enzymes. Expression of bcl-2 mRNA was assessed by Northern blotting. RESULTS: A SacII site in exon 2 of the bcl-2 gene was methylated in 5 carcinomas, plus an adjacent HpaII sites in 1 tumour. SacII site in the bcl-2 promoter were not methylated. Elevated levels of bcl-2 mRNA were detected in 3 carcinomas, 5 showed decreased expression and 4 were unchanged. CONCLUSIONS: De novo methylation of CpG sites in exon 2 of the bcl-2 gene occurs during the development of colorectal carcinoma. However, there was no relationship between expression of bc1-2 mRNA and methylation of specific CpG sites.

Lymphedema. Clinical signs, diagnosis, and treatment.

Medical management of lymphedema is warranted in all dogs with suspected congenital lymphedema, before surgical intervention. Although pharmaceutical agents, such as the benzo-pyrones, have not been investigated for clinical use in dogs, such studies appear to be justified. None of the surgical techniques discussed will cure lymphedema. The only technique reported with any frequency in the dog has been excision of affected tissues, and although some successes have been reported with this procedure, others have found it to be of no benefit or severe complications have occurred. Excisional techniques require meticulous attention to prevent infection intraoperatively and postoperatively. Staging the procedure may decrease problems associated with devascularization of remaining tissues. Evaluation of other techniques may be warranted in dogs; however, no technique has proven to be consistently beneficial in human beings with lymphedema.