The impact of e-health system implementation on UNWRA health services: an observational study.

BACKGROUND: The United Nations Relief and Works Agency for Palestine Refugees in the Near East (UNRWA) provides primary-health-care services to more than 5 million Palestinian refugees in five operational fields (Jordan, Syria, Lebanon, West Bank, and Gaza) through 144 health centres. UNRWA developed its electronic health records (e-Health) system to improve monitoring and facilitation of health services provided to Palestinian refugees. By the end of 2017, the system had been deployed in 129 health centres, included the health files of 3 million patients, and managed more than 8 million visits per year. We assessed whether preventive-health-care services had improved following implementation of the system. METHODS: This observational study used three key performance indicators to assess preventive-health-care services reported in UNRWA's annual reports in 2012-17: the percentage of targeted people aged 40 years and older screened for diabetes; the percentage of pregnant women with a livebirth who attended at least four antenatal visits; and the prevalence of growth problems (underweight, stunting, wasting, and overweight or obesity) in children younger than 5 years. Simple descriptive analysis was conducted with Microsoft Excel 2010. Ethical approval was obtained from the UNRWA Headquarters Department of Health. FINDINGS: Screening for diabetes significantly increased from 13% in 2012 to 21% in 2017 (p<0·0001) since the e-Health system started sending alerts to clerks at health centres. The percentage of pregnant women with a livebirth who attended at least four antenatal visits, increased from 87% in 2012 to 92% in 2017, when the e-Health System allowed health-care providers to log all pregnant women who missed their appointments to enable follow-up. Additionally, an electronic maternal and child health mobile application, sends regular reminders to mothers about appointments for themselves and their children. The percentages of underweight, stunting, wasting, and overweight or obesity among children younger than 5 years increased respectively from 3%, 4%, 2%, and 2% in 2014 to 5%, 7%, 4%, and 5% in 2017, but this was due to the increased detection of growth problems through the e-Health system. INTERPRETATION: The e-Health system improved detection and monitoring by UNRWA health-care providers and access to health services for Palestinian refugees in all three indicator categories. This study has several limitations. It is an observational study based on assessing health records of children rather than a prospective study over time. The focus of the study was to assess the impact of the eHealth system on health-care delivery and not to assess the changes in health-care delivery itself. The electronic health records that were reviewed are for the Palestine refugees who are registered at UNRWA health clinics and not for other refugees or residents of the five countries. Strengths of the study are that it is based on electronic health records which contain data recorded by the treating staff; the e-health system used by UNRWA clinics staff is centralised and the data are aggregated agency wide; and the growth monitoring indicators used by UNRWA were derived from the WHO Multi-center Growth Reference Study to assess the growth of children from birth up to age 5 years. FUNDING: None.

HIV integration and the establishment of latency in CCL19-treated resting CD4(+) T cells require activation of NF-κB.

BACKGROUND: Eradication of HIV cannot be achieved with combination antiretroviral therapy (cART) because of the persistence of long-lived latently infected resting memory CD4(+) T cells. We previously reported that HIV latency could be established in resting CD4(+) T cells in the presence of the chemokine CCL19. To define how CCL19 facilitated the establishment of latent HIV infection, the role of chemokine receptor signalling was explored. RESULTS: In resting CD4(+) T cells, CCL19 induced phosphorylation of RAC-alpha serine/threonine-protein kinase (Akt), nuclear factor kappa B (NF-κB), extracellular-signal-regulated kinase (ERK) and p38. Inhibition of the phosphoinositol-3-kinase (PI3K) and Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/ERK signalling pathways inhibited HIV integration, without significant reduction in HIV nuclear entry (measured by Alu-LTR and 2-LTR circle qPCR respectively). Inhibiting activation of MEK1/ERK1/2, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1) and NF-κB, but not p38, also inhibited HIV integration. We also show that HIV integrases interact with Pin1 in CCL19-treated CD4(+) T cells and inhibition of JNK markedly reduced this interaction, suggesting that CCL19 treatment provided sufficient signals to protect HIV integrase from degradation via the proteasome pathway. Infection of CCL19-treated resting CD4(+) T cells with mutant strains of HIV, lacking NF-κB binding sites in the HIV long terminal repeat (LTR) compared to infection with wild type virus, led to a significant reduction in integration by up to 40-fold (range 1-115.4, p = 0.03). This was in contrast to only a modest reduction of 5-fold (range 1.7-11, p > 0.05) in fully activated CD4(+) T cells infected with the same mutants. Finally, we demonstrated significant differences in integration sites following HIV infection of unactivated, CCL19-treated, and fully activated CD4(+) T cells. CONCLUSIONS: HIV integration in CCL19-treated resting CD4(+) T cells depends on NF-κB signalling and increases the stability of HIV integrase, which allow subsequent integration and establishment of latency. These findings have implications for strategies needed to prevent the establishment, and potentially reverse, latent infection.

Myeloid dendritic cells induce HIV-1 latency in non-proliferating CD4+ T cells.

Latently infected resting CD4(+) T cells are a major barrier to HIV cure. Understanding how latency is established, maintained and reversed is critical to identifying novel strategies to eliminate latently infected cells. We demonstrate here that co-culture of resting CD4(+) T cells and syngeneic myeloid dendritic cells (mDC) can dramatically increase the frequency of HIV DNA integration and latent HIV infection in non-proliferating memory, but not naïve, CD4(+) T cells. Latency was eliminated when cell-to-cell contact was prevented in the mDC-T cell co-cultures and reduced when clustering was minimised in the mDC-T cell co-cultures. Supernatants from infected mDC-T cell co-cultures did not facilitate the establishment of latency, consistent with cell-cell contact and not a soluble factor being critical for mediating latent infection of resting CD4(+) T cells. Gene expression in non-proliferating CD4(+) T cells, enriched for latent infection, showed significant changes in the expression of genes involved in cellular activation and interferon regulated pathways, including the down-regulation of genes controlling both NF-κB and cell cycle. We conclude that mDC play a key role in the establishment of HIV latency in resting memory CD4(+) T cells, which is predominantly mediated through signalling during DC-T cell contact.

An in-depth comparison of latent HIV-1 reactivation in multiple cell model systems and resting CD4+ T cells from aviremic patients.

The possibility of HIV-1 eradication has been limited by the existence of latently infected cellular reservoirs. Studies to examine control of HIV latency and potential reactivation have been hindered by the small numbers of latently infected cells found in vivo. Major conceptual leaps have been facilitated by the use of latently infected T cell lines and primary cells. However, notable differences exist among cell model systems. Furthermore, screening efforts in specific cell models have identified drug candidates for "anti-latency" therapy, which often fail to reactivate HIV uniformly across different models. Therefore, the activity of a given drug candidate, demonstrated in a particular cellular model, cannot reliably predict its activity in other cell model systems or in infected patient cells, tested ex vivo. This situation represents a critical knowledge gap that adversely affects our ability to identify promising treatment compounds and hinders the advancement of drug testing into relevant animal models and clinical trials. To begin to understand the biological characteristics that are inherent to each HIV-1 latency model, we compared the response properties of five primary T cell models, four J-Lat cell models and those obtained with a viral outgrowth assay using patient-derived infected cells. A panel of thirteen stimuli that are known to reactivate HIV by defined mechanisms of action was selected and tested in parallel in all models. Our results indicate that no single in vitro cell model alone is able to capture accurately the ex vivo response characteristics of latently infected T cells from patients. Most cell models demonstrated that sensitivity to HIV reactivation was skewed toward or against specific drug classes. Protein kinase C agonists and PHA reactivated latent HIV uniformly across models, although drugs in most other classes did not.

Establishment of HIV-1 latency in resting CD4+ T cells depends on chemokine-induced changes in the actin cytoskeleton.

Eradication of HIV-1 with highly active antiretroviral therapy (HAART) is not possible due to the persistence of long-lived, latently infected resting memory CD4(+) T cells. We now show that HIV-1 latency can be established in resting CD4(+) T cells infected with HIV-1 after exposure to ligands for CCR7 (CCL19), CXCR3 (CXCL9 and CXCL10), and CCR6 (CCL20) but not in unactivated CD4(+) T cells. The mechanism did not involve cell activation or significant changes in gene expression, but was associated with rapid dephosphorylation of cofilin and changes in filamentous actin. Incubation with chemokine before infection led to efficient HIV-1 nuclear localization and integration and this was inhibited by the actin stabilizer jasplakinolide. We propose a unique pathway for establishment of latency by direct HIV-1 infection of resting CD4(+) T cells during normal chemokine-directed recirculation of CD4(+) T cells between blood and tissue.

Effectiveness of integrating case studies in online and face-to-face instruction of pathophysiology: a comparative study.

Due to growing demand from students and facilitated by innovations in educational technology, institutions of higher learning are increasingly offering online courses. Subjects in the hard sciences, such as pathophysiology, have traditionally been taught in the face-to-face format, but growing demand for preclinical science courses has compelled educators to incorporate online components into their classes to promote comprehension. Learning tools such as case studies are being integrated into such courses to aid in student interaction, engagement, and critical thinking skills. Careful assessment of pedagogical techniques is essential; hence, this study aimed to evaluate and compare student perceptions of the use of case studies in face-to-face and fully online pathophysiology classes. A series of case studies was incorporated into the curriculum of a pathophysiology class for both class modes (online and face to face). At the end of the semester, students filled out a survey assessing the effectiveness of the case studies. Both groups offered positive responses about the incorporation of case studies in the curriculum of the pathophysiology class. This study supports the argument that with proper use of innovative teaching tools, such as case studies, online pathophysiology classes can foster a sense of community and interaction that is typically only seen with face-to-face classes, based on student responses. Students also indicated that regardless of class teaching modality, use of case studies facilitates student learning and comprehension as well as prepares them for their future careers in health fields.

Expression and reactivation of HIV in a chemokine induced model of HIV latency in primary resting CD4+ T cells.

BACKGROUND: We recently described that HIV latent infection can be established in vitro following incubation of resting CD4+ T-cells with chemokines that bind to CCR7. The main aim of this study was to fully define the post-integration blocks to virus replication in this model of CCL19-induced HIV latency. RESULTS: High levels of integrated HIV DNA but low production of reverse transcriptase (RT) was found in CCL19-treated CD4+ T-cells infected with either wild type (WT) NL4.3 or single round envelope deleted NL4.3 pseudotyped virus (NL4.3- Δenv). Supernatants from CCL19-treated cells infected with either WT NL4.3 or NL4.3- Δenv did not induce luciferase expression in TZM-bl cells, and there was no expression of intracellular p24. Following infection of CCL19-treated CD4+ T-cells with NL4.3 with enhanced green fluorescent protein (EGFP) inserted into the nef open reading frame (NL4.3- Δnef-EGFP), there was no EGFP expression detected. These data are consistent with non-productive latent infection of CCL19-treated infected CD4+ T-cells. Treatment of cells with phytohemagluttinin (PHA)/IL-2 or CCL19, prior to infection with WT NL4.3, resulted in a mean fold change in unspliced (US) RNA at day 4 compared to day 0 of 21.2 and 1.1 respectively (p = 0.01; n = 5), and the mean expression of multiply spliced (MS) RNA was 56,000, and 5,000 copies/million cells respectively (p = 0.01; n = 5). In CCL19-treated infected CD4+ T-cells, MS-RNA was detected in the nucleus and not in the cytoplasm; in contrast to PHA/IL-2 activated infected cells where MS RNA was detected in both. Virus could be recovered from CCL19-treated infected CD4+ T-cells following mitogen stimulation (with PHA and phorbyl myristate acetate (PMA)) as well as TNFα, IL-7, prostratin and vorinostat. CONCLUSIONS: In this model of CCL19-induced HIV latency, we demonstrate HIV integration without spontaneous production of infectious virus, detection of MS RNA in the nucleus only, and the induction of virus production with multiple activating stimuli. These data are consistent with ex vivo findings from latently infected CD4+ T-cells from patients on combination antiretroviral therapy, and therefore provide further support of this model as an excellent in vitro model of HIV latency.

Coinfection of hepatic cell lines with human immunodeficiency virus and hepatitis B virus leads to an increase in intracellular hepatitis B surface antigen.

Liver-related mortality is increased in the setting of HIV-hepatitis B virus (HBV) coinfection. However, interactions between HIV and HBV to explain this observation have not been described. We hypothesized that HIV infection of hepatocytes directly affects the life cycle of HBV. We infected human hepatic cell lines expressing HBV (Hep3B and AD38 cells) or not expressing HBV (Huh7, HepG2, and AD43 cells) with laboratory strains of HIV (NL4-3 and AD8), as well as a vesicular stomatitis virus (VSV)-pseudotyped HIV expressing enhanced green fluorescent protein (EGFP). Following HIV infection with NL4-3 or AD8 in hepatic cell lines, we observed a significant increase in HIV reverse transcriptase activity which was infectious. Despite no detection of surface CD4, CCR5, and CXCR4 by flow cytometry, AD8 infection of AD38 cells was inhibited by maraviroc and NL4-3 was inhibited by AMD3100, demonstrating that HIV enters AD38 hepatic cell lines via CCR5 or CXCR4. High-level infection of AD38 cells (50%) was achieved using VSV-pseudotyped HIV. Coinfection of the AD38 cell line with HIV did not alter the HBV DNA amount or species as determined by Southern blotting or nucleic acid signal amplification. However, coinfection with HIV was associated with a significant increase in intracellular HBsAg when measured by Western blotting, quantitative HBsAg, and fluorescence microscopy. We conclude that HIV infection of HBV-infected hepatic cell lines significantly increased intracellular HBsAg but not HBV DNA synthesis and that increased intrahepatic HBsAg secondary to direct infection by HIV may contribute to accelerated liver disease in HIV-HBV-coinfected individuals.

The antibody response to Plasmodium falciparum Merozoite Surface Protein 4: comparative assessment of specificity and growth inhibitory antibody activity to infection-acquired and immunization-induced epitopes.

BACKGROUND: Malaria remains a global public health challenge. It is widely believed that an effective vaccine against malaria will need to incorporate multiple antigens from the various stages of the parasite's complex life cycle. Plasmodium falciparum Merozoite Surface Protein 4 (MSP4) is a vaccine candidate that has been selected for development for inclusion in an asexual stage subunit vaccine against malaria. METHODS: Nine monoclonal antibodies (Mabs) were produced against Escherichia coli-expressed recombinant MSP4 protein and characterized. These Mabs were used to develop an MSP4-specific competition ELISA to test the binding specificity of antibodies present in sera from naturally P. falciparum-infected individuals from a malaria endemic region of Vietnam. The Mabs were also tested for their capacity to induce P. falciparum growth inhibition in vitro and compared against polyclonal rabbit serum raised against recombinant MSP4. RESULTS: All Mabs reacted with native parasite protein and collectively recognized at least six epitopes. Four of these Mabs recognize reduction-sensitive epitopes within the epidermal growth factor-like domain found near the C-terminus of MSP4. These sera were shown to contain antibodies capable of inhibiting the binding of the six Mabs indicating infection-acquired responses to the six different epitopes of MSP4. All of the six epitopes were readily recognized by human immune sera. Competition ELISA titres varied from 20 to 640, reflecting heterogeneity in the intensity of the humoral response against the protein among different individuals. The IgG responses during acute and convalescent phases of infection were higher to epitopes in the central region than to other parts of MSP4. Immunization with full length MSP4 in Freund's adjuvant induced rabbit polyclonal antisera able to inhibit parasite growth in vitro in a manner proportionate to the antibody titre. By contrast, polyclonal antisera raised to individual recombinant fragments rMSP4A, rMSP4B, rMSP4C and rMSP4D gave negligible inhibition. Similarly, murine Mabs alone or in combination did not inhibit parasite growth. CONCLUSIONS: The panel of MSP4-specific Mabs produced were found to recognize six distinct epitopes that are also targeted by human antibodies during natural malaria infection. Antibodies directed to more than three epitope regions spread across MSP4 are likely to be required for P. falciparum growth inhibition in vitro.

Preparing CLS professionals to be consumers and producers of research.

Research proficiency is part of the curriculum in all NAACLS accredited CLS programs. Learning the basic research tools enables students to understand and interpret published research as informed consumers of research. This paper describes an improved and innovative approach to prepare future CLS professionals to be both analytical consumers and active producers of pertinent research.

Assessment of the knowledge, attitudes, and practices (KAP) among UNRWA* health staff in Jordan concerning mental health programme pre-implementation: a cross-sectional study.

BACKGROUND: Mental health is a major public health priority, especially among refugees. The United Nations Relief and Works Agency for Palestine Refugees (UNRWA) started to integrate mental health and psychosocial support (MHPSS) into its primary healthcare services in Jordan in late 2017. In this study, we aimed to assess of the knowledge, attitudes, and practices (KAP) among UNRWA health staff (HS) in Jordan concerning mental health programme pre-implementation, and their perceived barriers about this MHPSS programme. METHODS: A cross-sectional study was conducted among doctors, dentists, nurses, and midwives who work at 16 of the 25 UNRWA health centres in Jordan. The assessment was made using a validated self-administered questionnaire. Data analysis was performed using SPSS (version 22). RESULTS: Of the participants, 73% (161 of 220) believed that their knowledge of MHPSS programmes was insufficient, with no significant difference (p = 0·116) between different categories of staff. Furthermore, 88% (194 of 220) said that they needed more training, 67% (147 of 220) reported that the number of mental health cases is increasing, and 50% (110 of 220) that dealing with these cases is difficult. Reflecting on the past 12 months, 31% of staff (69 out of 220) reported meeting between one and ten children, and 45% (100 out of 220) reported meeting between one and ten adults suspected of having mental illnesses. The most suspected condition was depression (84%; 150 of 220), followed by epilepsy (64%; 140 of 220). The main perceived barriers to implementation included the limited availability of MHPSS policies (87%; 192 of 220), MH professionals (86%; 190 of 220), resources (86%; 189 out of 220), and lack of privacy (14%; 31 out of 220). CONCLUSIONS: Most health staff had positive attitudes towards MHPSS programme implementation but felt they lacked the required knowledge. There is a need for training and clear technical guidelines. Perceived barriers to MHPSS programme implementation were consistent with the previous studies and need to be tackled with a structured plan of action.

Anaemia prevalence in children newly registered at UNRWA schools: a cross-sectional study.

OBJECTIVE: Children entering first grade at the United Nations Relief and Works Agency for Palestine Refugees in the Near East (UNRWA) schools in West Bank, Gaza, Lebanon, Jordan and Syria complete a comprehensive medical examination at UNRWA health centres (HCs) as a requirement for their acceptance. Our study aimed to assess anaemia prevalence and undernutrition indicators among new entrant school children during their preschool medical examination. SETTINGS: In 2017, we conducted a cross-sectional study in 59 UNRWA HCs, targeting children entering first grade at UNRWA schools in four of UNRWA's countries of operation (known as fields), namely Gaza, West Bank, Syria and Lebanon. PARTICIPANTS: 2419 completed the study. Boys and girls living inside or outside Palestine refugee camps were included. Verbal consent was obtained from their parents. PRIMARY AND SECONDARY OUTCOME MEASURES: Sociodemographic and anthropometric data on each child were collected. Underweight (weight-for-age z-score <-2 SD), stunting (height-for-age z-score <-2 SD), thinness (body mass index-for-age z-score <-2 SD) and obesity (body mass index-for-age z-score >+2 SD) were examined according to WHO growth indicators (5-10 years). RESULTS: 2419 students (1278 girls and 1141 boys) aged 6.1±0.4 years were examined. The prevalence of anaemia (haemoglobin (Hb) <11.5 g/L) was 25.0% (Gaza: 29.3%; West Bank: 22.0%; Syria: 30.0%; Lebanon: 18.3%). The mean Hb level was 12.0±0.9 g/L. The overall prevalence of stunting, thinness and underweight was 3.2%, 3.5% and 5.6%, respectively, with the highest levels found in Syria (4.3%, 6.3% and 10.1%, respectively). The highest prevalence of overweight was in Lebanon (8.6%), and the lowest was in Gaza (2.6%). Significant differences were found among fields with regard to undernutrition indicators (p=0.001). Also, children with anaemia had significantly higher prevalence of being underweight (5.2%) in comparison with those without anaemia (p=0.001). CONCLUSIONS: The prevalence of anaemia among the surveyed children increased to 25.0%, compared with the previous study conducted by UNRWA in 2005 (19.5%). Thus, it is recommended that Hb testing be included in the medical examination of new entrant school children attending UNRWA schools.

HIV latency can be established in proliferating and nonproliferating resting CD4+ T cells in vitro: implications for latency reversal.

OBJECTIVE: To determine whether latency can be established and reversed in both proliferating and nonproliferating CD4+ T cells in the same model in vitro. METHODS: Activated CD4+ T cells were infected with either a nonreplication competent, luciferase reporter virus or wild-type full-length enhanced green fluorescent protein (EGFP) reporter virus and cultured for 12 days. The cells were then sorted by flow cytometry to obtain two distinct T-cell populations that did not express the T-cell activation markers, CD69, CD25 and human leukocyte antigen (HLA)-DR: CD69CD25HLA-DR small cells (nonblasts) that had not proliferated in vitro following mitogen stimulation and CD69CD25HLA-DR large cells (which we here call transitional blasts) that had proliferated. The cells were then reactivated with latency-reversing agents and either luciferase or EGFP quantified. RESULTS: Inducible luciferase expression, consistent with latent infection, was observed in nonblasts and transitional blasts following stimulation with either phorbol-myristate-acetate/phytohemagglutinin (3.8 ±â€Š1 and 2.9 ±â€Š0.5 fold above dimethyl sulfoxide, respectively) or romidepsin (2.1 ±â€Š0.6 and 1.8 ±â€Š0.2 fold above dimethyl sulfoxide, respectively). Constitutive expression of luciferase was higher in transitional blasts compared with nonblasts. Using wild-type full-length EGFP reporter virus, inducible virus was observed in nonblasts but not in transitional blasts. No significant difference was observed in the response to latency-reversing agents in either nonblasts or transitional blasts. CONCLUSION: HIV latency can be established in vitro in resting T cells that have not proliferated (nonblasts) and blasts that have proliferated (transitional blasts). This model could potentially be used to assess new strategies to eliminate latency.

Parent-child communication and preventive practices for child sexual abuse among the general population: A community-based study.

OBJECTIVES: This study was conducted to investigate parent-child communication and preventive practices centred on child sexual abuse among a cohort of Saudi population. METHODS: A cross-sectional descriptive study was carried out at a single primary health care centre (PHC) in Dammam city, KSA. Parent-child communication and preventive practice were measured by a Self-Reporting Questionnaire with binary answers (yes/no). A total of 400 subjects were selected using a stratified random sampling method. RESULTS: This study found that 82.5% (n = 329) of parents talked with their children about the latter's bodily privacy. With regard to supervision, most parents (91.7%) said that their children were under their direct supervision all the time. Other variables wear wearing appropriate clothes (94.7%), supervision (93.5%), and teaching a child about their bodily privacy (93.2%). A majority of the respondents (76.2%) were achieved a good preventive practice score, while 95 (23.8%) were observed to have poor communication practice. Logistic regression analyses for gender (p < 0.020; odds ratio 3.031; 95% CI), marital status (p < 0.026; odds ratio 0.081; 95% CI), and family size of more than one wife (p < 0.041; odds ratio 0.081; 95% CI) were significantly associated with good preventive practice. CONCLUSION: The study showed that parent-child communication practices in KSA seem to be reasonably good. Further parental supervision of children with respect to sexual abuse needs attention. Parents are advised to communicate with their children and to provide them with information and material on the prevention of sexual abuse.

Factors associated with perceptions of child sexual abuse and lack of parental knowledge: a community-based cross-sectional study from the Eastern Province of Saudi Arabia.

BACKGROUND: Child sexual abuse (CSA) has serious consequences that can affect the physical, social and mental health of a child. In the last two decades, concern about CSA has increased around the world including Saudi Arabia. OBJECTIVE: Evaluate factors associated with parental perceptions and knowledge of CSA. DESIGN: Cross-sectional survey. SETTINGS: Primary health care clinic. SUBJECTS AND METHODS: Simple random sampling was used to select participants. The main tool for data collection was a self-administered questionnaire. MAIN OUTCOME MEASURES: Factors associated with knowledge and perceptions of CSA. SAMPLE SIZE: 400. RESULTS: Most respondents (69%) had good knowledge of the signs of sexual abuse in children. For perception scores, statistically significant variables were age (P=.004), educational level (P=.005), income (P less than .001), number of wives (P=.004), number of male children (P=.021), and number of female children (P=.027). For knowledge scores, statistically significant variables were income (P=.008), number of wives (P less than .005), number of male children (P=.003) and number of female chil.dren (P less than .003). Logistic regression showed that the older age group was significantly associated with a good perception score (P less than .046). CONCLUSIONS: Risk factors for parental lack of knowledge and poor perception associated with CSA are poverty and low education. Protective factors included the older parent age, size of the family and families with more than one wife. Education should be designed for parents and the community to increase the knowledge and perception of CSA. LIMITATIONS: Single-center study and short study period. CONFLICT OF INTEREST: None.

Insight into HIV-2 latency may disclose strategies for a cure for HIV-1 infection.

HIV-1 and HIV-2 originate from two distinct zoonotic transmissions of simian immunodeficiency viruses from primate to human. Although both share similar modes of transmission and can result in the development of AIDS with similar clinical manifestations, HIV-2 infection is generally milder and less likely to progress to AIDS. HIV is currently incurable due to the presence of HIV provirus integrated into the host DNA of long-lived memory cells of the immune system without active replication. As such, the latent virus is immunologically inert and remains insensitive to the administered antiviral drugs targeting active viral replication steps. Recent evidence suggests that persistent HIV replication may occur in anatomical sanctuaries such as the lymphoid tissue due to low drug penetration. At present, different strategies are being evaluated either to completely eradicate the virus from the patient (sterilising cure) or to allow treatment interruption without viral rebound (functional cure). Because HIV-2 is naturally less pathogenic and displays a more latent phenotype than HIV-1, it may represent a valuable model that provides elementary information to cure HIV-1 infection. Insight into the viral and cellular determinants of HIV-2 replication may therefore pave the way for alternative strategies to eradicate HIV-1 or promote viral remission.

LEDGIN-mediated Inhibition of Integrase-LEDGF/p75 Interaction Reduces Reactivation of Residual Latent HIV.

Persistence of latent, replication-competent Human Immunodeficiency Virus type 1 (HIV-1) provirus is the main impediment towards a cure for HIV/AIDS (Acquired Immune Deficiency Syndrome). Therefore, different therapeutic strategies to eliminate the viral reservoirs are currently being explored. We here propose a novel strategy to reduce the replicating HIV reservoir during primary HIV infection by means of drug-induced retargeting of HIV integration. A novel class of integration inhibitors, referred to as LEDGINs, inhibit the interaction between HIV integrase and the LEDGF/p75 host cofactor, the main determinant of lentiviral integration site selection. We show for the first time that LEDGF/p75 depletion hampers HIV-1 reactivation in cell culture. Next we demonstrate that LEDGINs relocate and retarget HIV integration resulting in a HIV reservoir that is refractory to reactivation by different latency-reversing agents. Taken together, these results support the potential of integrase inhibitors that modulate integration site targeting to reduce the likeliness of viral rebound.

Effect of protein kinase Cgamma on gap junction disassembly in lens epithelial cells and retinal cells in culture.

PURPOSE: To determine the effects of protein kinase Cgamma (PKCgamma) on phosphorylation of Cx43, the gap junction protein of lens epithelial cells, and on cell surface assembly/disassembly of Cx43-gap junction complexes. METHODS: Association and phosphorylation of Cx43 by PKCgamma was determined using co-immunoprecipitation and reaction with phosphoserine antisera. Activation of PKCgamma was with 200 nM phorbol ester for 30 to 60 min. Effects of specific PKC isoforms was determined after overexpression of either PKCalpha or PKCgamma for 24 h in N/N 1003A rabbit lens epithelial cells or in two retinal cell lines, WERI and Y79. Gap junction plaques were counted on the cell surface by immunolabeling of Cx43 using confocal microscopy. RESULTS: Co-immunoprecipitation of Cx43 with PKCgamma was observed only in cells over expressing PKCgamma and in cells activated with phorbol ester. Both overexpression and phorbol ester produced a rapid phosphorylation of Cx43 on serine. Cx43 cell surface gap junction plaques decreased in cells over expressing PKCgamma and in cells treated with phorbol ester. Similar results were observed using the retinal cell lines, WERI and Y79. The effect of PKCgamma overexpression was persistent for 7 days but total cell Cx43 was not decreased. Overexpression of PKCa resulted in an increase in cell surface gap junction plaques. CONCLUSIONS: PKCgamma can be co-immunoprecipitated with Cx43 from lens epithelial cells using phorbol ester activation. PKCgamma phosphorylates Cx43 on serine and this causes disassembly and loss of gap junction Cx43 from the cell surface. Overexpression of PKCgamma confirmed that only this PKC isoform caused the loss of cell surface Cx43. Overexpression of PKCalpha, the other major lens PKC isoform, caused an increase in cell surface Cx43. The presence of PKCgamma and loss of surface Cx43 from two retinal cell lines, WERI and Y79, upon phorbol ester activation further suggests that activation of PKCgamma may be a common mechanism for control of cell surface Cx43.

Antigenic and genetic typing of Whataroa viruses in Australia.

We recently characterized three novel alphaviruses isolated from mosquitoes captured in New South Wales, Australia. Initial cross-neutralization studies revealed antigenic similarity to the Sindbis virus (SINV)-like Whataroa virus (WHAV), heretofore found only in New Zealand. Nucleotide sequence analysis showed that the WHAV-like viruses shared >99% nucleotide sequence similarity with each other, and 96-97% similarity with prototype WHAV. Enzyme-linked immunosorbent assay reactions of a panel of monoclonal antibodies to SINV showed that the novel WHAV-like viruses displayed identical binding patterns and were antigenically distinct from all SINV isolates examined. Although these viruses displayed a similar binding pattern to prototype WHAV, three monoclonal antibodies discriminated them from the New Zealand virus. Our results suggest that these novel alphaviruses are antigenic variants of WHAV and represent the first reported isolations of this virus from outside New Zealand. The monoclonal antibodies used in this study will be useful for typing new SINV and SINV-like isolates.

Entinostat is a histone deacetylase inhibitor selective for class 1 histone deacetylases and activates HIV production from latently infected primary T cells.

OBJECTIVES: To compare the potency, toxicity and mechanism of action of multiple histone deacetylase inhibitors (HDACi) in activating HIV production from latency. DESIGN: In-vitro analysis of HDACi in a primary T-cell model of HIV latency and latently infected cell lines. METHODS: Latently infected chemokine ligand 19 (CCL19)-treated CD4⁺ T cells and the latently infected cell lines ACH2 and J-Lat were treated with a panel of HDACi, including entinostat, vorinostat, panonbinostat and MCT3. Viral production and cell viability were compared. Expression of cellular HDACs was measured by western blot and PCR. Association of HDACs with the HIV long-terminal repeat (LTR) using latently infected CCL19-treated primary CD4⁺ T cells in the presence and absence of specific HDACi was determined by chromatin immunoprecipitation (ChIP). RESULTS: We demonstrated considerable variation in the potency and toxicity of HDACi in latently infected primary CD4⁺ T cells and cell lines. All HDACi tested activated HIV production in latently infected primary T cells with greatest potency demonstrated with entinostat and vorinostat and greatest toxicity with panobinostat. Following the addition of HDACi in vitro, there were no changes in markers of T-cell activation or expression of the HIV coreceptors chemokine (C-X-C motif) receptor 4 (CXCR4) or chemokine (C-C motif) receptor type 5 (CCR5). ChIP analysis of latently infected CCL19-treated primary CD4⁺ T cells showed binding by HDAC1, HDAC2 and HDAC3 to the LTR with removal of HDAC1 and HDAC2 following treatment with the HDACi vorinostat and HDAC1 only following treatment with entinostat. CONCLUSION: The HDACi entinostat, selective for inhibition of class I HDACs, induced virus expression in latently infected primary CD4⁺ T cells making this compound an attractive novel option for future clinical trials.