HIV-1 inhibits haematopoiesis via microRNA secreted by virus-infected CD4+ T cells.

INTRODUCTION: HIV-1-infected patients develop haematological disorders such as cytopenias. One possible explanation is the inhibition of haematopoiesis at the level of differentiation of CD34+ haematopoietic progenitor stem cells. Based on our previous studies, we hypothesised that there may be viral encoded, or host cellular factors which participate in the process of inhibition of haematopoiesis. MATERIALS AND METHODS: Virus-depleted media from infected CD4+ T cells was prepared by filtration and added to CD34+ cell differentiation semisolid medium. We have also used the virus-depleted media to isolate host/viral factors including miRNA. Isolated miRNAs were screened for their haematopoietic inhibitory function using the miRNA mining approach. RESULTS: Addition of virus-depleted media caused a 40% inhibition of differentiation of CD34+ cells into myeloid and erythroid colony formation. Real-time RT-PCR showed miR-15a and miR-24 from both pIndie-C1 and pNL4.3 HIV-1-infected cells showed a significant differential expression when compared to control media. CONCLUSION: In this study, we have identified two miRNAs, miR-15a and miR-24 secreted from purified HIV-1-infected CD4+ T cells that inhibited CD34+ haematopoietic progenitor stem cell differentiation into myeloid and erythroid colonies in vitro.

Expression of domain III of the envelope protein from GP-78: a Japanese encephalitis virus.

Acute encephalitis caused by the Japanese encephalitis virus (JEV) represents a growing epidemic and is a cause for concern in Southeast Asia. JEV is transmitted to humans through the bite of the Culicine mosquito species. The virus genome comprising of an RNA strand also encodes the envelope protein (E) which surrounds the virus. The E protein aids in fusion of virus with the cellular membrane of the host cell with the help of three structurally distinct domains (DI, DII, DIII) that are connected by flexible hinge regions. Of these domains, DIII (JEV-DIII) has been reported to interact with the cellular membrane, aid viral entry and viral replication. Hence JEV-DIII has the potential to be an antigen that can provide immune protection to a JEV infection. In this study, we describe the cloning and expression of DIII of GP-78, a virulent strain of JEV prevalent in India. Our data clearly shows that JEV-DIII expressed from pVAC1 in HEK293T cells is membrane targeted. To our knowledge, this is the first demonstration of a recombinant construct that may block JEV entry into the cells and/or evoke specific antibodies against JEV. Future studies will reveal if our construct will elicit significant immune responses which will alleviate or ameliorate the pro-inflammatory responses induced by JEV.

Bacillus Calmette-Guérin Confers Neuroprotection in a Murine Model of Japanese Encephalitis.

OBJECTIVE: Japanese encephalitis (JE) is a debilitating disease caused by infection with the JE virus (JEV; family: Flaviviridae), which leaves neurological sequelae in survivors but more often leads to mortality. Neurodegeneration caused by inflammation is the primary pathology behind the clinical manifestation of encephalitis caused by JEV. Bacillus Calmette-Guérin (BCG) has been used in immunoprophylaxis for tuberculosis and in the adjuvant therapy of many malignancies, and has exhibited neuroprotective activities in experimental models of Parkinson and Alzheimer disease. This study aimed at assessing the neuroprotective role of BCG in a murine model of JE. METHODS: Suckling mice were inoculated with 106 CFU of BCG and at 18 days postinoculation were challenged with 100 LD50 of JEV. PBS-inoculated mice were used as controls. Mice were sacrificed on days 2, 4, 6, and 8. Brain tissue was homogenized for RNA extraction. One-step real-time RT-PCR was performed to assess the relative gene expressions of TNF-α, IL-6, and iNOS. RESULTS: The BCG-inoculated (BCG+JEV) group exhibited a significant delay in the presentation of neuropathological symptoms, longer survival, and a downregulation in the expression of TNF-α, IL-6, and iNOS on days 2, 4, and 6 post-JEV challenge compared to the JEV group. CONCLUSION: These findings indicate that the administration of BCG offers neuroprotection in the murine model of JE. BCG should therefore be further investigated as an adjuvant in the management of JE. BCG is an accepted vaccine for tuberculosis in many countries that are endemic for JEV. This approach may have a significant impact on the public health burden in these countries.

Early cognitive impairment after sedation for colonoscopy: the effect of adding midazolam and/or fentanyl to propofol.

BACKGROUND: The sedative drug combination that produces minimal cognitive impairment and optimal operating conditions during colonoscopy has not been determined. We sought to determine if the use of propofol alone results in less cognitive impairment at discharge than the use of propofol plus midazolam and/or fentanyl in patients presenting for elective outpatient colonoscopy. METHODS: Two hundred adult patients presenting for elective outpatient colonoscopy were randomized to receive propofol alone or propofol plus midazolam, and/or fentanyl for IV sedation. Baseline cognitive function was measured using the computerized CogState test battery (Cogstate, Melbourne, Australia) before sedation. During the procedure, sedative drug doses, depth of sedation (via the bispectral index and observer's assessment of alertness/sedation score), complications, and treatability were recorded. Patients were interviewed about recall immediately after emerging from sedation, and cognitive testing was repeated at hospital discharge. Recovery times, quality of recovery, and satisfaction with care were also recorded. RESULTS: In the propofol plus adjuvants group, 84 patients received fentanyl 50 microg (25-100) (median [range]) and 57 patients received midazolam 2 mg (0.5-10). Patients' cognitive function at discharge was worse than their performance at baseline. However, the changes in cognitive function between discharge and baseline were not significantly different between the two groups. At discharge, 18.5% of patients were cognitively impaired to an extent equivalent to a blood-alcohol concentration of 0.05%. Sedation with propofol plus midazolam and/or fentanyl produced better operating conditions than sedation with propofol alone and was associated with shorter procedure times. Recovery times, recall, dreaming, quality of recovery, and patient satisfaction with care were similar between the groups. Administration of >2 mg of midazolam was a predictor of impaired cognitive function at discharge. CONCLUSIONS: Significant cognitive impairment was common at discharge from elective outpatient colonoscopy. However, the addition of midazolam and/or fentanyl to propofol sedation did not result in more cognitive impairment than the use of propofol alone. Furthermore, the use of adjuvants improved the ease of colonoscopy without increasing the rate of complications or prolonging early recovery times..

Propofol dose and incidence of dreaming during sedation.

BACKGROUND AND OBJECTIVE: Dreaming is commonly reported after propofol-based sedation. We measured the incidence of dreaming and bispectral index (BIS) values in colonoscopy patients sedated with combinations of propofol, midazolam and fentanyl. METHODS: Two hundred patients presenting for elective outpatient colonoscopy were sedated with combinations of propofol, midazolam and fentanyl. BIS was monitored throughout the procedure. Patients were interviewed immediately after they emerged from sedation. The primary end point was a report of dreaming during sedation. RESULTS: Ninety-seven patients were administered propofol alone, 44 were administered propofol and fentanyl, 16 were administered propofol and midazolam and 43 were administered propofol, midazolam and fentanyl. Dreaming was reported by 19% of patients. Dreamers received higher doses of propofol and had lower BIS values during sedation. Age of 50 years or less, preoperative quality of recovery score of less than 14, higher home dream recall, propofol dose of more than 300 mg and time to Observers' Assessment of Alertness/Sedation score equalling 5 of 8 min or less were independent predictors of dreaming. CONCLUSION: Dreaming during sedation is associated with higher propofol dose and lower BIS values.

Characterization of a selective inhibitor of inositol hexakisphosphate kinases: use in defining biological roles and metabolic relationships of inositol pyrophosphates.

Inositol hexakisphosphate kinases (IP6Ks) phosphorylate inositol hexakisphosphate (InsP(6)) to yield 5-diphosphoinositol pentakisphosphate (5-[PP]-InsP(5) or InsP(7)). In this study, we report the characterization of a selective inhibitor, N(2)-(m-(trifluoromethy)lbenzyl) N(6)-(p-nitrobenzyl)purine (TNP), for these enzymes. TNP dose-dependently and selectively inhibited the activity of IP6K in vitro and inhibited InsP(7) and InsP(8) synthesis in vivo without affecting levels of other inositol phosphates. TNP did not inhibit either human or yeast Vip/PPIP5K, a newly described InsP(6)/InsP(7) 1/3-kinase. Overexpression of IP6K1, -2, or -3 in cells rescued TNP inhibition of InsP(7) synthesis. TNP had no effect on the activity of a large number of protein kinases, suggesting that it is selective for IP6Ks. TNP reversibly reduced InsP(7)/InsP(8) levels. TNP in combination with genetic studies was used to implicate the involvement of two pathways for synthesis of InsP(8) in yeast. TNP induced a fragmented vacuole phenotype in yeast, consistent with inhibition of Kcs1, a Saccharomyces cerevisiae IP6K. In addition, it also inhibited insulin release from Min6 cells in a dose-dependent manner further implicating InsP(7) in this process. TNP thus provides a means of selectively and rapidly modulating cellular InsP(7) levels, providing a new and versatile tool to study the biological function and metabolic relationships of inositol pyrophosphates.