Pro-inflammatory cytokines derived from West Nile virus (WNV)-infected SK-N-SH cells mediate neuroinflammatory markers and neuronal death

<p>Abstract</p> <p>Background</p> <p>WNV-associated encephalitis (WNVE) is characterized by increased production of pro-inflammatory mediators, glial cells activation and eventual loss of neurons. WNV infection of neurons is rapidly progressive and destructive whereas infection of non-neuronal brain cells is limited. However, the role of neurons and pathological consequences of pro-inflammatory cytokines released as a result of WNV infection is unclear. Therefore, the objective of this study was to examine the role of key cytokines secreted by WNV-infected neurons in mediating neuroinflammatory markers and neuronal death.</p> <p>Methods</p> <p>A transformed human neuroblastoma cell line, SK-N-SH, was infected with WNV at multiplicity of infection (MOI)-1 and -5, and WNV replication kinetics and expression profile of key pro-inflammatory cytokines were analyzed by plaque assay, qRT-PCR, and ELISA. Cell death was measured in SK-N-SH cell line in the presence and absence of neutralizing antibodies against key pro-inflammatory cytokines using cell viability assay, TUNEL and flow cytometry. Further, naïve primary astrocytes were treated with UV-inactivated supernatant from mock- and WNV-infected SK-N-SH cell line and the activation of astrocytes was measured using flow cytometry and ELISA.</p> <p>Results</p> <p>WNV-infected SK-N-SH cells induced the expression of IL-1β, -6, -8, and TNF-α in a dose- and time-dependent manner, which coincided with increase in virus-induced cell death. Treatment of cells with anti-IL-1β or -TNF-α resulted in significant reduction of the neurotoxic effects of WNV. Furthermore treatment of naïve astrocytes with UV-inactivated supernatant from WNV-infected SK-N-SH cell line increased expression of glial fibrillary acidic protein and key inflammatory cytokines.</p> <p>Conclusion</p> <p>Our results for the first time suggest that neurons are one of the potential sources of pro-inflammatory cytokines in WNV-infected brain and these neuron-derived cytokines contribute to WNV-induced neurotoxicity. Moreover, cytokines released from neurons also mediate the activation of astrocytes. Our data define specific role(s) of WNV-induced pro-inflammatory cytokines and provide a framework for the development of anti-inflammatory drugs as much-needed therapeutic interventions to limit symptoms associated with WNVE.</p

Comparison of real-time PCR and hemagglutination assay for quantitation of human polyomavirus JC

Human polyomavirus JC (JCV), the etiological agent of the disease progressive multifocal leukoencephalopathy (PML) affects immunocompromised patients particularly patients with AIDS. In vitro studies of JCV infection are hampered by the lack of sensitive JCV quantitation tests. Although the hemagglutination (HA) assay has been routinely employed for in vitro quantitation of JCV, its sensitivity is severely limited. We have employed a real-time PCR assay which compares favorably with the HA assay for the in vitro quantitation of JCV. JCV(Mad1), propagated in primary human fetal glial (PHFG) cells in two independent laboratories, was purified and quantitated by the HA assay. Both batches of purified JCV(Mad1) were then serially diluted in Dulbecco's Modified Eagle's Medium to obtain HA titers ranging from 64 to 0.001 HA units (HAU) per 100 μL of virus suspension. DNA was extracted from 100 μL of virus suspension and eluted in 50 μL of buffer, and DNA amplification and quantitation were performed in the Bio-Rad iCycler iQ Multicolor Real-Time PCR Detection System using T-antigen as the target gene. Real-time PCR for quantitation of JCV was sensitive and consistently detected 1.8 × 10(1 )copies of JCV DNA, and as low as 0.001 HAU equivalent of JCV. Moreover, there was a strong linear correlation between the HA assay and the DNA copy number of JCV(Mad1). The intra-run and inter-run coefficients of variation for the JCV standard curve were 0.06% to 4.8% and 2.6% to 5.2%, respectively. Based on these data, real-time PCR can replace the less-sensitive HA assay for the reliable detection, quantitation and monitoring of in vitro JCV replication

HIV/AIDS in Pakistan: the battle begins

Pakistan, the second most populous Muslim nation in the world, has started to finally experience and confront the HIV/AIDS epidemic. The country had been relatively safe from any indigenous HIV cases for around two decades, with most of the infections being attributable to deported HIV positive migrants from the Gulf States. However, the virus finally seems to have found a home-base, as evidenced by the recent HIV outbreaks among the injection drug user community. Extremely high-risk behavior has also been documented among Hijras (sex workers) and long-distance truck drivers. The weak government response coupled with the extremely distressing social demographics of this South-Asian republic also helps to compound the problem. The time is ripe now to prepare in advance, to take the appropriate measures to curtail further spread of the disease. If this opportunity is not utilized right now, little if at all could be done later

Momordica charantia (bitter melon) attenuates high-fat diet-associated oxidative stress and neuroinflammation

<p>Abstract</p> <p>Background</p> <p>The rising epidemic of obesity is associated with cognitive decline and is considered as one of the major risk factors for neurodegenerative diseases. Neuroinflammation is a critical component in the progression of several neurological and neurodegenerative diseases. Increased metabolic flux to the brain during overnutrition and obesity can orchestrate stress response, blood-brain barrier (BBB) disruption, recruitment of inflammatory immune cells from peripheral blood and microglial cells activation leading to neuroinflammation. The lack of an effective treatment for obesity-associated brain dysfunction may have far-reaching public health ramifications, urgently necessitating the identification of appropriate preventive and therapeutic strategies. The objective of our study was to investigate the neuroprotective effects of <it>Momordica charantia </it>(bitter melon) on high-fat diet (HFD)-associated BBB disruption, stress and neuroinflammatory cytokines.</p> <p>Methods</p> <p>C57BL/6 female mice were fed HFD with and without bitter melon (BM) for 16 weeks. BBB disruption was analyzed using Evans blue dye. Phosphate-buffered saline (PBS) perfused brains were analyzed for neuroinflammatory markers such as interleukin-22 (IL-22), IL-17R, IL-16, NF-κB1, and glial cells activation markers such as Iba1, CD11b, GFAP and S100β. Additionally, antioxidant enzymes, ER-stress proteins, and stress-resistant transcription factors, sirtuin 1 (Sirt1) and forkhead box class O transcription factor (FoxO) were analyzed using microarray, quantitative real-time RT-PCR, western immunoblotting and enzymatic assays. Systemic inflammation was analyzed using cytokine antibody array.</p> <p>Results</p> <p>BM ameliorated HFD-associated changes in BBB permeability as evident by reduced leakage of Evans blue dye. HFD-induced glial cells activation and expression of neuroinflammatory markers such as NF-κB1, IL-16, IL-22 as well as IL-17R were normalized in the brains of mice supplemented with BM. Similarly, HFD-induced brain oxidative stress was significantly reduced by BM supplementation with a concomitant reduction in FoxO, normalization of Sirt1 protein expression and up-regulation of Sirt3 mRNA expression. Furthermore, plasma antioxidant enzymes and pro-inflammatory cytokines were also normalized in mice fed HFD with BM as compared to HFD-fed mice.</p> <p>Conclusions</p> <p>Functional foods such as BM offer a unique therapeutic strategy to improve obesity-associated peripheral inflammation and neuroinflammation.</p

Deletion of Pregnancy Zone Protein and Murinoglobulin-1 Restricts the Pathogenesis of West Nile Virus Infection in Mice

West Nile virus (WNV) is an enveloped positive-stranded RNA virus that causes meningitis, encephalitis, and acute flaccid paralysis in humans. There are no therapeutic agents available for use against WNV infection. Alpha-2 macroglobulin (A2M) is a major plasma proteinase inhibitor that also has important role in immune modulation. In mice, pregnancy zone protein (PZP) and murinoglobulin-1 (MUG-1) are two close homologous of human A2M. In this study, we investigated the role of PZP and MUG-1 proteins in the pathogenesis of WNV infection in mice. Adult C57BL/6J wild-type and PZP/MUG-1 double knockout (DKO) mice were inoculated subcutaneously with WNV and mortality, virus burden, and immune responses were analyzed. Infection of wild-type (WT) mice with WNV resulted in significantly high morbidity and mortality. In comparison, no mortality was observed in DKO mice, suggesting that PZP and MUG-1 play a deleterious role in WNV infection. Increased survival in WNV-infected DKO mice was associated with significantly low viral burden in serum, spleen, kidney, and brain compared to WT mice. In addition, significantly reduced levels of type 1 interferon and WNV-specific antibodies were observed in the DKO mice compared to WT mice. We further demonstrated that protein levels of inflammatory cytokines and chemokines in the serum, spleen, and brain were significantly reduced in DKO mice compared to WT mice. Collectively our data demonstrate that lack of PZP and MUG-1 restricts the pathogenesis of WNV infection in mice

Minority Health and Health Disparities Research Training (MHRT) Program at the University of Hawaii

The objective of the Minority Health Research Training (MHRT) program at the University of Hawaii at Manoa (UHM) is to encourage students from underrepresented (including minority) backgrounds to pursue careers in science; and expose students to biomedical, clinical, and behavioral health research and global health issues that relate to health disparities. The program also aims to enable collaborations between colleges/universities and out-of-state research programs. Funded by the National Institute of Minority Health and Health Disparities (NIMHD), National Institutes of Health (NIH), the UHM MHRT program is in its ninth year. The MHRT program is a short-term research training opportunity offered to undergraduate, post-baccalaureate, and pre-doctoral students from under-represented backgrounds. MHRT students are from various academic disciplines at UH and have diverse ethnic backgrounds. To date, the MHRT program has trained eight (8) cohorts of students totaling 85 students. Selected students learn to conduct research during the spring semester and spend 8-9 weeks during the summer at their international training sites under the guidance of their assigned in-country mentor and their UH mentor. In addition to life-changing research and cultural experiences, program benefits include up to10 credits of directed research courses in the spring and summer semesters, and while abroad students are provided with a stipend, travel, and living expenses.NIMHD/NIH-T37MD008636-0

Evidence for a Founder Effect among HIV-infected injection drug users (IDUs) in Pakistan.

Background: We have previously reported a HIV-1 subtype A infection in a community of injection drug users (IDUs) in Karachi, Pakistan. We now show that this infection among the IDUs may have originated from a single source. Methods: Phylogenetic analysis was performed of partial gag sequences, generated using PCR, from 26 HIV-positive IDU samples. Results: Our results showed formation of a tight monophyletic group with an intra-sequence identity of \u3c 98% indicating a founder effect . Our data indicate that the HIV-1 epidemic in this community of IDUs may have been transmitted by an HIV positive overseas contract worker who admitted to having contact with commercial sex workers during stay abroad. Conclusion: Specific measures need to implemented to control transmission of HIV infection in Pakistan through infected migrant workers

Sequence analysis of human T cell lymphotropic virus type I strains from southern India: gene amplification and direct sequencing from whole blood blotted onto filter paper

Human T cell lymphotropic virus type I (HTLV-I) infection in India has been found to be associated with adult T cell leukaemia/lymphoma (ATLL) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP) among life-long residents of southern India. To examine the heterogeneity of HTLV-I strains from southern India and to determine their relationship with the sequence variants of HTLV-I from Melanesia, 1149 nucleotides spanning selected regions of the HTLV-I gag, pol, env and pX genes were amplified and directly sequenced from DNA extracted from whole blood blotted onto filter paper and from peripheral blood mononuclear cells, obtained from one patient with HAM/TSP, two with ATLL and eight asymptomatic carriers from Andhra Pradesh, Kerala and Tamil Nadu. Sequence alignments and comparisons indicated that the 11 HTLV-I strains from southern India were 99.2% to 100% identical among themselves and 98.7% to 100% identical to the Japanese prototype HTLV-I ATK. The majority of base substitutions were transitions and silent. No frameshifts, insertions, deletions or possibly disease-specific base changes were found in the regions sequenced. The observed clustering of the Indian HTLV-I strains with those from Japan, as determined by the maximum parsimony method, suggested a common source of HTLV-I infection with subsequent parallel evolution. Amplification of DNA from blood specimens collected on filter paper may be useful for the study of other blood-borne pathogens

OPTIMIZED WORKFLOW FOR SARS-COV-2 WHOLE GENOME SEQUENCING: THREE-WAY ANALYSIS BETWEEN DNA CONCENTRATION, GEL ELECTROPHORESIS IMAGING AND SUCCESSFUL SUBMISSION OF SEQUENCES TO GENBANK

Introduction: Whole Genome sequencing (WGS) is essential for monitoring mutations and detecting the emergence of SARS-CoV-2 variants as the virus makes its way through the population. However, WGS of every specimen is not feasible due to several clinical and technical variables. Further, WGS is a time consuming and expensive technique, which requires highly trained personnel, therefore it is not practical to conduct WGS on every sample. Laboratories must select the ideal samples for WGS based on time post infection, collection media including swabs, sample quality and quantity, primers, and CT value. Objective: The objective of this study was to correlate variables such as DNA concentration and gel electrophoresis based image analysis, prior to conducting viral cDNA library, to identify ideal samples for successful WGS and submission to GenBank. Methods: Nasal swabs, mid-turbinate swabs, and nasopharyngeal swabs were collected from individuals confirmed to be SARS-CoV-2 RT-PCR positive, from clinical laboratories on Oahu. Total RNA from the samples was extracted and the entire SARS-CoV-2 genome was amplified using the ARTIC Network V3 primer pools and RT-PCR. Gel electrophoresis was conducted on purified PCR products to verify band size and quality followed by image analysis using GelAnalyzer. Further, DNA concentration of PCR products were measured using the Quant-iT PicoGreen dsDNA Assay. PCR products were submitted to the Advanced Studies in Genomics, Proteomics and Bioinformatics (ASGPB) facility at UH Manoa and sequenced using the Illumina MiSeq platform. Sequencing reads were mapped to the original Wuhan sequence (MN908947.3), assembled into whole genomes using the iVar workflow, and submitted to GenBank. To determine ideal samples for successful WGS, three-way correlation analysis was conducted using DNA concentration, an arbitrary unit assigned by the GelAnalyzer and sequences submitted to the GenBank using GraphPad Prism. Results: Of the 149 samples submitted for WGS, 99 (66.4%) samples were successfully sequenced and submitted to the GenBank. DNA concentration (ng/µL) of samples successfully submitted to GenBank was significantly higher (median 27.24, IQR 11.73-61.36, ps = 0.91). Conclusion: These data demonstrate that samples successfully submitted to the GenBank, have a minimum band to ladder ratio of 0.167 and/or a DNA concentration of at least 1.096 ng/µL. Beta testing of 100 samples using the techniques mentioned above is ongoing. These data will assist in determining the ideal samples for which WGS and subsequent GenBank submission will be most successful, to conserve precious reagents, personnel resources, and to cut down on sequencing time. Acknowledgments: This research was supported by a COBRE grant (P30GM114737) from the Pacific Center for Emerging Infectious Diseases Research, a grant (P20GM103466) from the INBRE, NIGMS, and a grant (T37MD008636) from the NIMHD, NIH. We thank Dr. Jennifer Saito at the ASGPB Core, UH Manoa for her expertise with WGS, and Dr. Eileen Nakano and Dr. Sandra Chang for their assistance with sample procurement. We also thank the Tropical Medicine Clinical Laboratory, Kaiser Permanente Clinical Laboratory, and National Kidney Foundation of Hawaii for providing de-identified patient samples.COBRE grant (P30GM114737) from the Pacific Center for Emerging Infectious Diseases Research, a grant (P20GM103466) from the INBRE, NIGMS, and a grant (T37MD008636) from the NIMHD, NIH

West Nile Virus Detection in Urine

We report West Nile virus (WNV) RNA in urine collected from a patient with encephalitis 8 days after symptom onset. Viral RNA was detected by reverse transcriptase–polymerase chain reaction (RT-PCR). Sequence and phylogenetic analysis confirmed the PCR product to have ≥99% similarity to the WNV strain NY 2000-crow3356