Inhibition of aquaporin-1 prevents myocardial remodeling by blocking the transmembrane transport of hydrogen peroxide.

Pathological remodeling of the myocardium has long been known to involve oxidant signaling, but strategies using systemic antioxidants have generally failed to prevent it. We sought to identify key regulators of oxidant-mediated cardiac hypertrophy amenable to targeted pharmacological therapy. Specific isoforms of the aquaporin water channels have been implicated in oxidant sensing, but their role in heart muscle is unknown. RNA sequencing from human cardiac myocytes revealed that the archetypal AQP1 is a major isoform. AQP1 expression correlates with the severity of hypertrophic remodeling in patients with aortic stenosis. The AQP1 channel was detected at the plasma membrane of human and mouse cardiac myocytes from hypertrophic hearts, where it colocalized with NADPH oxidase-2 and caveolin-3. We show that hydrogen peroxide (H2O2), produced extracellularly, is necessary for the hypertrophic response of isolated cardiac myocytes and that AQP1 facilitates the transmembrane transport of H2O2 through its water pore, resulting in activation of oxidant-sensitive kinases in cardiac myocytes. Structural analysis of the amino acid residues lining the water pore of AQP1 supports its permeation by H2O2 Deletion of Aqp1 or selective blockade of the AQP1 intrasubunit pore inhibited H2O2 transport in mouse and human cells and rescued the myocyte hypertrophy in human induced pluripotent stem cell-derived engineered heart muscle. Treatment of mice with a clinically approved AQP1 inhibitor, Bacopaside, attenuated cardiac hypertrophy. We conclude that cardiac hypertrophy is mediated by the transmembrane transport of H2O2 by the water channel AQP1 and that inhibitors of AQP1 represent new possibilities for treating hypertrophic cardiomyopathies.

Sequential LASER ART and CRISPR Treatments Eliminate HIV-1 in a Subset of Infected Humanized Mice.

Elimination of HIV-1 requires clearance and removal of integrated proviral DNA from infected cells and tissues. Here, sequential long-acting slow-effective release antiviral therapy (LASER ART) and CRISPR-Cas9 demonstrate viral clearance in latent infectious reservoirs in HIV-1 infected humanized mice. HIV-1 subgenomic DNA fragments, spanning the long terminal repeats and the Gag gene, are excised in vivo, resulting in elimination of integrated proviral DNA; virus is not detected in blood, lymphoid tissue, bone marrow and brain by nested and digital-droplet PCR as well as RNAscope tests. No CRISPR-Cas9 mediated off-target effects are detected. Adoptive transfer of human immunocytes from dual treated, virus-free animals to uninfected humanized mice fails to produce infectious progeny virus. In contrast, HIV-1 is readily detected following sole LASER ART or CRISPR-Cas9 treatment. These data provide proof-of-concept that permanent viral elimination is possible.

Removal of HIV DNA by CRISPR from Patient Blood Engrafts in Humanized Mice.

We used NOD/SCID mice, also known as NRG, to assess the ability of lentivirus-mediated intravenous delivery of CRISPR in editing the HIV-1 genome from the circulating PBMC engrafts, some of which homed within several animal solid tissues. Lentivirus-mediated delivery of a multiplex of guide RNAs accompanied by Cas9 endonuclease led to the excision of the targeted region of the viral genome positioned within the HIV-1 LTR from the in-vitro-infected human peripheral blood mononuclear cells (PBMCs) embedded in the spleens of NRG mice. Similarly, the treatment of NRG mice harboring PBMC engrafts derived from HIV-1-positive patients with the therapeutic lentivirus eliminated the presence of the viral DNA fragment in the blood, as well as in the spleen, lung, and liver, of the engrafted animals. Sanger sequence analysis of the viral DNA after treatment with the lentiviral vectors expressing Cas9 and gRNAs verified the editing and removal of the proviral DNA fragment from the viral genome at the predicted sites. This proof-of-concept study, for the first time, demonstrates successful excision of the HIV-1 proviral DNA from patient immune cell engrafts in humanized mice upon treatment with lentivirus-expressing CRISPR and causes a decline in the level of replication-competent virus.

SV40 Infection of Mesenchymal Stromal Cells From Wharton's Jelly Drives the Production of Inflammatory and Tumoral Mediators.

The Mesenchymal Stromal Cells from umbilical cord Wharton's jelly (WJSCs) are a source of cells with high potentiality for the treatment of human immunological disorders. Footprints of the oncogenic viruses Simian Virus 40 (SV40) and JC Virus (JCPyV) have been recently detected in human WJSCs specimens. The aim of this study is to evaluate if WJSCs can be efficiently infected by these Polyomaviruses and if they can potentially exert tumoral activity. Cell culture experiments indicated that WJSCs could sustain both SV40 and JCPyV infections. A transient and lytic replication was observed for JCPyV, while SV40 persistently infected WJSCs over a long period of time, releasing a viral progeny at low titer without evident cytopathic effect (CPE). Considering the association between SV40 and human tumors and the reported ability of the oncogenic viruses to drive the host innate immune response to cell transformation, the expression profile of a large panel of immune mediators was evaluated in supernatants by the Bioplex platform. RANTES, IL-3, MIG, and IL-12p40, involved in chronic inflammation, cells differentiation, and transformation, were constantly measured at high concentration comparing to control. These findings represent a new aspect of SV40 biological activity in the humans, highlighting its interaction with specific host cellular pathways. In view of these results, it seems to be increasingly urgent to consider Polyomaviruses in the management of WJSCs for their safely use as promising therapeutic source. J. Cell. Physiol. 232: 3060-3066, 2017. © 2016 Wiley Periodicals, Inc.

Negative Feedback Regulation of HIV-1 by Gene Editing Strategy.

The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells.

Interaction Between Human Polyomavirus BK and Hypoxia Inducible Factor-1 alpha.

BK polyomavirus (BKV) has a worldwide seroprevalence of approximately 90%. After primary infection, BKV establishes a life-long latency within the urogenital tract. The severe immunological impairment occurring in renal transplant recipients leads to BKV reactivation, which may result in polyomavirus associated nephropathy (PVAN). While the transplanted kidney is transiently unperfused, Hypoxia Inducible Factors (HIFs) mediate the cellular response to hypoxia. The α-subunit of HIF isoform 1 (HIF-1α) may interact with several viruses, but until now, there has been no information regarding the interaction between BKV and HIF-1α. The aim of this study is to investigate the possible interaction between HIF-1α and BKV and its potential effect on the pathogenesis of PVAN. Screening of 17 kidney tissue samples revealed that HIF-1α expression was 13.6-fold higher in PVAN tissues compared to control tissues. A luminometric assay in co-transfected African green monkey kidney cells (VERO) demonstrated BKV promoter activation ranging from two to sixfold (P < 0.05) when HIF-1α was over-expressed. A Chromatin ImmunoPrecipitation (ChIP) assay showed structural binding between the BKV promoter and HIF-1α. The amount of BKV DNA increased by threefold in VERO infected cells that were exposed to simulated hypoxia, compared to the cells not subjected to hypoxia. Both ex vivo and in vitro interactions between HIF-1α and BKV were observed, suggesting that HIF-1α, stabilized during transplantation, may be able to bind the BKV promoter and enhance BKV replication. Thus, hypoxia should be considered a risk factor for the development of PVAN in kidney transplant recipients.

Human polyomavirus 6 DNA in the cerebrospinal fluid of an HIV-positive patient with leukoencephalopathy.

BACKGROUND: Leukoencephalopathies in HAART-treated, HIV-positive patients include progressive multifocal leukoencephalopathy (PML), a result of lytic infection oligodendrocytes by JC polyomavirus (JCV), and another form characterized by the absence of JCV genome in cerebrospinal fluid (CSF). OBJECTIVES: To test the potential viral etiology of JCV-negative leukoencephalopathy. STUDY DESIGN: CSF was collected from 43 HIV-positive patients with MRI suggestive of leukoencephalopathies. DNA was isolated and real-time PCR assays for neurotropic viruses (Herpes Simplex Viruses 1/2, Varicella Zoster Virus, Epstein Barr Virus, Human Cytomegalovirus, Human Herpesvirus 6, JCV and HIV) were conducted. CSF from 14 non-reactive cases were subjected to random nucleic acid amplification, deep sequencing, and in silico search for viral sequences. RESULTS: JCV genome was detected in the CSF of 19/43 PML patients, HIV genome in the CSF of 5 PML patients including 2 JCV negative patients, and no viruses were detected in 22 patients. Human Polyomavirus 6 (HPyV6) DNA was detected by deep sequencing in one JCV-negative leukoencephalopathy CSF sample. CONCLUSIONS: HPyV6 DNA was detected in CSF of a case of demyelinating disease. HPyV6 has not been previously reported in CSF or associated with any disease. Demonstrating a causative role will require further studies.