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.

Genetic manipulation of an Ixodes scapularis cell line.

Although genetic manipulation is one of the hallmarks of model organisms, its applicability to non-model species has remained difficult due to our limited understanding of their fundamental biology. For instance, manipulation of a cell line originated from the black-legged tick Ixodes scapularis, an arthropod that serves as a vector for several human pathogens, has yet to be established. Here, we demonstrate the successful genetic modification of the commonly used tick ISE6 line through ectopic expression and clustered regularly interspaced palindromic repeats [(CRISPR)/CRISPR-associated protein 9 (Cas9)] genome editing. We performed ectopic expression using nucleofection and attained CRISPR-Cas9 editing via homology-dependent recombination. Targeting the E3 ubiquitin ligase x-linked inhibitor of apoptosis (xiap) and its substrate p47 led to an alteration in molecular signaling within the immune deficiency network and increased infection of the rickettsial agent Anaplasma phagocytophilum in I. scapularis ISE6 cells. Collectively, our findings complement techniques for the genetic engineering of I. scapularis ticks, which currently limit efficient and scalable molecular genetic screens in vivo.IMPORTANCEGenetic engineering in arachnids has lagged compared to insects, largely because of substantial differences in their biology. This study unveils the implementation of ectopic expression and CRISPR-Cas9 gene editing in a tick cell line. We introduced fluorescently tagged proteins in ISE6 cells and edited its genome via homology-dependent recombination. We ablated the expression of xiap and p47, two signaling molecules present in the immune deficiency (IMD) pathway of Ixodes scapularis. Impairment of the tick IMD pathway, an analogous network of the tumor necrosis factor receptor in mammals, led to enhanced infection of the rickettsial agent Anaplasma phagocytophilum. Altogether, our findings provide a critical technical resource to the scientific community to enable a deeper understanding of biological circuits in the black-legged tick I. scapularis.

Impaired vision in children prenatally exposed to methadone: an observational cohort study.

BACKGROUND/OBJECTIVES: To examine prevalence of failed visual assessment at 8-10 years in children born to methadone-maintained opioid dependent (MMOD) mothers and relate this to known in utero substance exposure. SUBJECTS/METHODS: Follow up of observational cohort study of methadone-exposed and comparison children matched for birthweight, gestation and postcode of residence at birth. Participants were 144 children (98 exposed, 46 comparison). Prenatal drug exposure was previously established via comprehensive maternal and neonatal toxicology. Children were invited to attend for visual assessment and casenotes were reviewed. Presence of acuity poorer than 0.2 logMAR, strabismus, nystagmus and/or impaired stereovision constituted a 'fail'. Fail rates were compared between methadone-exposed and comparison children after adjusting for known confounding variables. RESULTS: 33 children attended in person: data were also derived from casenote review for all children. After controlling for maternal reported tobacco use, methadone-exposed children were more likely to have a visual 'fail' outcome, adjusted odds ratio 2.6, 95% CI 1.1-6.2; adjusted relative risk 1.8 (95% CI 1.1-3.4). Visual 'fail' outcome rates did not differ between methadone-exposed children who had (n = 47) or had not (n = 51) received pharmacological treatment for neonatal abstinence/opioid withdrawal syndrome (NAS/NOWS); fail rate 62% vs 53% (95% CI of difference-11-27%). CONCLUSIONS: Children born to MMOD mothers are almost twice as likely as unexposed peers to have significant visual abnormalities at primary school age. Prenatal methadone exposure should be considered in the differential diagnosis of nystagmus. Findings support visual assessment prior to school entry for children with any history of prenatal opioid exposure. TRIAL REGISTRATION: The study was prospectively registered on ClinicalTrials.gov (NCT03603301), https://clinicaltrials.gov/ct2/show/NCT03603301 .

Tick extracellular vesicles impair epidermal homeostasis through immune-epithelial networks during hematophagy.

Hematophagous ectoparasites, such as ticks, rely on impaired wound healing for skin attachment and blood feeding. Wound healing has been extensively studied through the lens of inflammatory disorders and cancer, but limited attention has been given to arthropod-borne diseases. Here, we used orthogonal approaches combining single-cell RNA sequencing (scRNAseq), flow cytometry, murine genetics, and intravital microscopy to demonstrate how tick extracellular vesicles (EVs) disrupt networks involved in tissue repair. Impairment of EVs through silencing of the SNARE protein vamp33 negatively impacted ectoparasite feeding and survival in three medically relevant tick species, including Ixodes scapularis. Furthermore, I. scapularis EVs affected epidermal γδ T cell frequencies and co-receptor expression, which are essential for keratinocyte function. ScRNAseq analysis of the skin epidermis in wildtype animals exposed to vamp33-deficient ticks revealed a unique cluster of keratinocytes with an overrepresentation of pathways connected to wound healing. This biological circuit was further implicated in arthropod fitness when tick EVs inhibited epithelial proliferation through the disruption of phosphoinositide 3-kinase activity and keratinocyte growth factor levels. Collectively, we uncovered a tick-targeted impairment of tissue repair via the resident γδ T cell-keratinocyte axis, which contributes to ectoparasite feeding.

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.

Dancing partners at the ball: Rational selection of next generation anti-CD20 antibodies for combination therapy of chronic lymphocytic leukemia in the novel agents era.

The anti-CD20 antibodies represent a major advancement in the therapeutic options available for chronic lymphocytic leukemia. The addition of rituximab, ofatumumab and obinutuzumab to various chemotherapy regimens has led to considerable improvements in both response and survival. Ocaratuzumab, veltuzumab and ublituximab are currently being explored within the trial setting. We review the current status of these antibodies, and discuss how their mechanisms of action may impact on the choice of combinations with novel small molecule agents.

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.

Apolipoprotein B100 danger-associated signal 1 (ApoBDS-1) triggers platelet activation and boosts platelet-leukocyte proinflammatory responses.

Low-density lipoproteins (LDL), occurring in vivo in both their native and oxidative form, modulate platelet function and thereby contribute to atherothrombosis. We recently identified and demonstrated that 'ApoB100 danger-associated signal 1' (ApoBDS-1), a native peptide derived from Apolipoprotein B-100 (ApoB100) of LDL, induces inflammatory responses in innate immune cells. Platelets are critically involved in the development as well as in the lethal consequences of atherothrombotic diseases, but whether ApoBDS-1 has also an impact on platelet function is unknown. In this study we examined the effect of ApoBDS-1 on human platelet function and platelet-leukocyte interactions in vitro. Stimulation with ApoBDS-1 induced platelet activation, degranulation, adhesion and release of proinflammatory cytokines. ApoBDS-1-stimulated platelets triggered innate immune responses by augmenting leukocyte activation, adhesion and transmigration to/through activated HUVEC monolayers, under flow conditions. These platelet-activating effects were sequence-specific, and stimulation of platelets with ApoBDS-1 activated intracellular signalling pathways, including Ca2+, PI3K/Akt, PLC, and p38- and ERK-MAPK. Moreover, our data indicates that ApoBDS-1-induced platelet activation is partially dependent of positive feedback from ADP on P2Y1 and P2Y12, and TxA2. In conclusion, we demonstrate that ApoBDS-1 is an effective platelet agonist, boosting platelet-leukocyte's proinflammatory responses, and potentially contributing to the multifaceted inflammatory-promoting effects of LDL in the pathogenesis of atherothrombosis.

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.

A randomised, double-blind, placebo-controlled, crossover study to assess the efficacy and safety of three dosing schedules of agalsidase alfa enzyme replacement therapy for Fabry disease.

Anecdotal reports suggest that the currently approved dosing interval of agalsidase alfa (0.2 mg/kg/2 weeks) for Fabry disease treatment is too long. This randomised, double-blind, placebo-controlled, crossover study investigated three altered dosing intervals. 18 Fabry patients received three agalsidase alfa dosing schedules, each for four weeks (A: 0.2 mg/kg∗2 weeks, B: 0.1 mg/kg/week, C: 0.2 mg/kg/week). Health state, pain levels, sweat volume and latency and plasma and urinary globotriaosylceramide levels were recorded throughout the study. No significant differences were found among the schedules for the primary efficacy outcome of self-assessed health state, or for pain scores. A trend toward increased sweat volume on QSART testing, and reduced urine globotriaosylceramide concentration were seen with treatment schedule C. Agalsidase alfa was safe and well tolerated with all schedules. In conclusion, the primary analyses did not find weekly infusions of agalsidase alfa to be statistically better than the approved dosing schedule however the data indicates that further studies with more patients over a longer period are required to more accurately determine the optimum dose and schedule.

Targeting cell cycle and hormone receptor pathways in cancer.

The cyclin/cyclin-dependent kinase (CDK)/retinoblastoma (RB)-axis is a critical modulator of cell cycle entry and is aberrant in many human cancers. New nodes of therapeutic intervention are needed that can delay or combat the onset of malignancies. The antitumor properties and mechanistic functions of PD-0332991 (PD; a potent and selective CDK4/6 inhibitor) were investigated using human prostate cancer (PCa) models and primary tumors. PD significantly impaired the capacity of PCa cells to proliferate by promoting a robust G1-arrest. Accordingly, key regulators of the G1-S cell cycle transition were modulated including G1 cyclins D, E and A. Subsequent investigation demonstrated the ability of PD to function in the presence of existing hormone-based regimens and to cooperate with ionizing radiation to further suppress cellular growth. Importantly, it was determined that PD is a critical mediator of PD action. The anti-proliferative impact of CDK4/6 inhibition was revealed through reduced proliferation and delayed growth using PCa cell xenografts. Finally, first-in-field effects of PD on proliferation were observed in primary human prostatectomy tumor tissue explants. This study shows that selective CDK4/6 inhibition, using PD either as a single-agent or in combination, hinders key proliferative pathways necessary for disease progression and that RB status is a critical prognostic determinant for therapeutic efficacy. Combined, these pre-clinical findings identify selective targeting of CDK4/6 as a bona fide therapeutic target in both early stage and advanced PCa and underscore the benefit of personalized medicine to enhance treatment response.

Rescue of platinum-damaged oocytes from programmed cell death through inactivation of the p53 family signaling network.

Non-proliferating oocytes within avascular regions of the ovary are exquisitely susceptible to chemotherapy. Early menopause and sterility are unintended consequences of chemotherapy, and efforts to understand the oocyte apoptotic pathway may provide new targets for mitigating this outcome. Recently, the c-Abl kinase inhibitor imatinib mesylate (imatinib) has become the focus of research as a fertoprotective drug against cisplatin. However, the mechanism by which imatinib protects oocytes is not fully understood, and reports of the drug's efficacy have been contradictory. Using in vitro culture and subrenal grafting of mouse ovaries, we demonstrated that imatinib inhibits the cisplatin-induced apoptosis of oocytes within primordial follicles. We found that, before apoptosis, cisplatin induces c-Abl and TAp73 expression in the oocyte. Oocytes undergoing apoptosis showed downregulation of TAp63 and upregulation of Bax. While imatinib was unable to block cisplatin-induced DNA damage and damage response, such as the upregulation of p53, imatinib inhibited the cisplatin-induced nuclear accumulation of c-Abl/TAp73 and the subsequent downregulation of TAp63 and upregulation of Bax, thereby abrogating oocyte cell death. Surprisingly, the conditional deletion of Trp63, but not ΔNp63, in oocytes inhibited apoptosis, as well as the accumulation of c-Abl and TAp73 caused by cisplatin. These data suggest that TAp63 is the master regulator of cisplatin-induced oocyte death. The expression kinetics of TAp63, c-Abl and TAp73 suggest that cisplatin activates TAp63-dependent expression of c-Abl and TAp73 and, in turn, the activation of TAp73 by c-Abl-induced BAX expression. Our findings indicate that imatinib protects oocytes from cisplatin-induced cell death by inhibiting c-Abl kinase, which would otherwise activate TAp73-BAX-mediated apoptosis. Thus, imatinib and other c-Abl kinase inhibitors provide an intriguing new way to halt cisplatin-induced oocyte death in early follicles and perhaps conserve the endocrine function of the ovary against chemotherapy.

Increasing productivity, reducing cost and improving quality in elective surgery in New Zealand: the Waitemata District Health Board joint arthroplasty pilot.

BACKGROUND: In 2010, Waitemata District Health Board piloted a new model of care for total hip and knee arthroplasties. The pilot was incentive based and clinically led. The participating surgeons and anaesthetists were responsible for increasing surgical throughput. The pilot aimed to increase productivity, reduce cost and increase quality for patients. AIM: To compare costs and outcomes for elective hip and knee arthroplasties carried out at the pilot site (Waitakere Hospital) compared with the main District Health Board hospital site (North Shore Hospital (NSH)). METHODS: A retrospective matched cohort study of hip and knee replacements discharged between 1 July 2010 and 31 March 2011, comparing costs and outcomes at the pilot site compared with the NSH site. Only non-complex procedures were included, and routinely collected data were used. RESULTS: One hundred and seventy-seven hip replacements (77 NSH, 100 pilot) and 158 knee replacements (88 NSH, 70 pilot) were analysed. Total inpatient event costs were 12% and 17% lower for hip and knee replacements, respectively, at the pilot site compared with NSH. Significant reduction in operation length (39% hip, 36% knee) and length of stay (38% hip, 39% knee) were found in the pilot groups compared with NSH. CONCLUSION: Implementation of an innovative new model in a public hospital setting has produced significant increases in productivity and reduced overall costs. This model could potentially be used in other public healthcare settings for non-complex elective surgery.

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.

The long-term effectiveness of generic adult fixed-dose combination antiretroviral therapy for HIV-infected Ugandan children.

BACKGROUND: Access to pediatric antiretroviral formulations is increasing in resource-limited countries, however adult FDCs are still commonly used by antiretroviral therapy (ART) programs. OBJECTIVE: To describe long-term effectiveness of using adult FDC of d4T+3TC+NVP (Triomune) in children for HIV treatment. METHODS: Clinical, immunologic, and virologic outcomes of HIV-infected ART-naïve children aged six months to 12 years, were evaluated up to 96 weeks post-ART initiation. RESULTS: From March 2004 to June 2006, 104 children were followed with a median age of 5.4 years, median CD4 cell percent and HIV-1 RNA were 11.0% (IQR 6.7-13.9) and 348,846copies/mL (IQR 160,941-681,313) respectively at baseline. Using Kaplan-Meir estimates, 75% of children had undetectable viral loads (<400copies/mL) at 96 weeks of ART. Children with a baseline CD4 cell percent >15% were 3 times more likely to achieve viral load <400copies/mL than those with baseline CD4 cell percent <5% after adjusting for baseline age {aHR = 3.03 (1.10-8.32), p=0.03}; no difference was found among those with CD4 cell percent >5-14.9% and <5%. CONCLUSION: Treatment with generic adult FDC for HIV-infected Ugandan children led to sustained clinical, immunologic and virologic response during 96 weeks of ART. Early initiation of ART is key to achieving virological success.

Attitudinal and demographic determinants of diet quality and implications for policy targeting.

BACKGROUND: Poor diet quality is a major public health concern that has prompted governments to introduce a range of measures to promote healthy eating. For these measures to be effective, they should target segments of the population with messages relevant to their needs, aspirations and circumstances. The present study investigates the extent to which attitudes and constraints influence healthy eating, as well as how these vary by demographic characteristics of the UK population. It further considers how such information may be used in segmented diet and health policy messages. METHODS: A survey of 250 UK adults elicited information on conformity to dietary guidelines, attitudes towards healthy eating, constraints to healthy eating and demographic characteristics. Ordered logit regressions were estimated to determine the importance of attitudes and constraints in determining how closely respondents follow healthy eating guidelines. Further regressions explored the demographic characteristics associated with the attitudinal and constraint variables. RESULTS: People who attach high importance to their own health and appearance eat more healthily than those who do not. Risk-averse people and those able to resist temptation also eat more healthily. Shortage of time is considered an important barrier to healthy eating, although the cost of a healthy diet is not. These variables are associated with a number of demographic characteristics of the population; for example, young adults are more motivated to eat healthily by concerns over their appearance than their health. CONCLUSIONS: The approach employed in the present study could be used to inform future healthy eating campaigns. For example, messages to encourage the young to eat more healthily could focus on the impact of diets on their appearance rather than health.

Differential effects of exogenous androgen and an androgen receptor antagonist in the peri- and postpubertal murine mammary gland.

There is emerging evidence that androgens inhibit proliferation of normal and malignant breast epithelial cells, but the actions of androgens in normal mammary gland morphogenesis are not well understood. In this study, we investigated whether development of the murine mammary gland could be altered by stimulating or suppressing androgen receptor (AR) signaling in vivo. Intact virgin female mice aged 5 wk (midpuberty) or 12 wk (postpuberty) were implanted with slow-release pellets containing either placebo, 5α-dihydrotestosterone (1.5 mg) or the AR antagonist flutamide (60 mg). Treatment with 5α-dihydrotestosterone from midpuberty to 12 wk of age-retarded ductal extension by 40% (P = 0.007), but treatment from 12-21 wk had no significant effect on gland morphology. In contrast, inhibition of AR signaling with flutamide from midpuberty had no effect on the mammary gland, but flutamide treatment from 12-21 wk increased ductal branching (P = 0.004) and proliferation (P = 0.03) of breast epithelial cells. The increased proliferation in flutamide-treated mice was not correlated with serum estradiol levels or estrogen receptor-α (ERα) expression. In control mice, the frequency and intensity of AR immunostaining in mammary epithelial cells was significantly increased in the 12- to 21-wk treatment group compared with the 5- to 12-wk group (P < 0.001). In contrast, no change in ERα occurred, resulting in a marked increase in the AR to ERα ratio from 0.56 (±0.12) to 1.47 (±0.10). Our findings indicate that androgen signaling influences development and structure of the adult mammary gland and that homeostasis between estrogen and androgen signaling in mature glands is critical to constrain the proliferative effects of estradiol.

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.

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.

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.