HIV-1 cellular and tissue replication patterns in infected humanized mice.

Humanized mice have emerged as a testing platform for HIV-1 pathobiology by reflecting natural human disease processes. Their use to study HIV-1 biology, virology, immunology, pathogenesis and therapeutic development has served as a robust alternative to more-well developed animal models for HIV/AIDS. A critical component in reflecting such human pathobiology rests in defining the tissue and cellular sites for HIV-1 infection. To this end, we examined the tissue sites for viral infection in bone marrow, blood, spleens, liver, gut, brain, kidney and lungs of human CD34+ hematopoietic stem cell engrafted virus-infected NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. Cells were analyzed by flow cytometry and sorted from species mixtures defined as CD34+ lineage negative progenitor cells, CD14+CD16+ monocyte-macrophages and central, stem cell and effector memory T cells. The cell distribution and viral life cycle were found dependent on the tissue compartment and time of infection. Cell subsets contained HIV-1 total and integrated DNA as well as multi-spliced and unspliced RNA in divergent proportions. The data support the idea that humanized mice can provide a means to examine the multifaceted sites of HIV-1 replication including, but not limited to progenitor cells and monocyte-macrophages previously possible only in macaques and human

HIV-1 cellular and tissue replication patterns in infected humanized mice.

Humanized mice have emerged as a testing platform for HIV-1 pathobiology by reflecting natural human disease processes. Their use to study HIV-1 biology, virology, immunology, pathogenesis and therapeutic development has served as a robust alternative to more-well developed animal models for HIV/AIDS. A critical component in reflecting such human pathobiology rests in defining the tissue and cellular sites for HIV-1 infection. To this end, we examined the tissue sites for viral infection in bone marrow, blood, spleens, liver, gut, brain, kidney and lungs of human CD34+ hematopoietic stem cell engrafted virus-infected NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ mice. Cells were analyzed by flow cytometry and sorted from species mixtures defined as CD34+ lineage negative progenitor cells, CD14+CD16+ monocyte-macrophages and central, stem cell and effector memory T cells. The cell distribution and viral life cycle were found dependent on the tissue compartment and time of infection. Cell subsets contained HIV-1 total and integrated DNA as well as multi-spliced and unspliced RNA in divergent proportions. The data support the idea that humanized mice can provide a means to examine the multifaceted sites of HIV-1 replication including, but not limited to progenitor cells and monocyte-macrophages previously possible only in macaques and human

A mature macrophage is a principal HIV-1 cellular reservoir in humanized mice after treatment with long acting antiretroviral therapy.

BACKGROUND: Despite improved clinical outcomes seen following antiretroviral therapy (ART), resting CD4+ T cells continue to harbor latent human immunodeficiency virus type one (HIV-1). However, such cells are not likely the solitary viral reservoir and as such defining where and how others harbor virus is imperative for eradication measures. To such ends, we used HIV-1ADA-infected NOD.Cg-Prkdc (scid) Il2rg (tm1Wjl) /SzJ mice reconstituted with a human immune system to explore two long-acting ART regimens investigating their abilities to affect viral cell infection and latency. At 6 weeks of infection animals were divided into four groups. One received long-acting (LA) cabotegravir (CAB) and rilpivirine (RVP) (2ART), a second received LA CAB, lamivudine, abacavir and RVP (4ART), a third were left untreated and a fourth served as an uninfected control. After 4 weeks of LA ART treatment, blood, spleen and bone marrow (BM) cells were collected then phenotypically characterized. CD4+ T cell subsets, macrophages and hematopoietic progenitor cells were analyzed for HIV-1 nucleic acids by droplet digital PCR. RESULTS: Plasma viral loads were reduced by two log10 or to undetectable levels in the 2 and 4ART regimens, respectively. Numbers and distributions of CD4+ memory and regulatory T cells, macrophages and hematopoietic progenitor cells were significantly altered by HIV-1 infection and by both ART regimens. ART reduced viral DNA and RNA in all cell and tissue compartments. While memory cells were the dominant T cell reservoir, integrated HIV-1 DNA was also detected in the BM and spleen macrophages in both regimen-treated mice. CONCLUSION: Despite vigorous ART regimens, HIV-1 DNA and RNA were easily detected in mature macrophages supporting their potential role as an infectious viral reservoir

Increased liver stiffness promotes hepatitis B progression by impairing innate immunity in CCl4-induced fibrotic HBV\u3csup\u3e+\u3c/sup\u3e transgenic mice

Background: Hepatitis B virus (HBV) infection develops as an acute or chronic liver disease, which progresses from steatosis, hepatitis, and fibrosis to end-stage liver diseases such as cirrhosis and hepatocellular carcinoma (HCC). An increased stromal stiffness accompanies fibrosis in chronic liver diseases and is considered a strong predictor for disease progression. The goal of this study was to establish the mechanisms by which enhanced liver stiffness regulates HBV infectivity in the fibrotic liver tissue. Methods: For in vitro studies, HBV-transfected HepG2.2.15 cells were cultured on polydimethylsiloxane gels coated by polyelectrolyte multilayer films of 2 kPa (soft) or 24 kPa (stiff) rigidity mimicking the stiffness of the healthy or fibrotic liver. For in vivo studies, hepatic fibrosis was induced in C57Bl/6 parental and HBV+ transgenic (HBVTg) mice by injecting CCl4 twice a week for 6 weeks. Results: We found higher levels of HBV markers in stiff gel-attached hepatocytes accompanied by up-regulated OPN content in cell supernatants as well as suppression of anti-viral interferon-stimulated genes (ISGs). This indicates that pre-requisite “fibrotic” stiffness increases osteopontin (OPN) content and releases and suppresses anti-viral innate immunity, causing a subsequent rise in HBV markers expression in hepatocytes. In vitro results were corroborated by data from HBVTg mice administered CCl4 (HBVTg CCl4). These mice showed higher HBV RNA, DNA, HBV core antigen (HBcAg), and HBV surface antigen (HBsAg) levels after liver fibrosis induction as judged by a rise in Col1a1, SMA, MMPs, and TIMPs mRNAs and by increased liver stiffness. Importantly, CCl4-induced the pro-fibrotic activation of liver cells, and liver stiffness was higher in HBVTg mice compared with control mice. Elevation of HBV markers and OPN levels corresponded to decreased ISG activation in HBVTg CCl4 mice vs HBVTg control mice. Conclusion: Based on our data, we conclude that liver stiffness enhances OPN levels to limit anti-viral ISG activation in hepatocytes and promote an increase in HBV infectivity, thereby contributing to end-stage liver disease progression

Pharmacodynamics of folic acid receptor targeted antiretroviral nanotherapy in HIV-1-infected humanized mice.

Long-acting nanoformulated antiretroviral therapy (nanoART) can sustain plasma drug levels and improve its biodistribution. Cell targeted-nanoART can achieve this and bring drug efficiently to viral reservoirs. However, whether such improvements affect antiretroviral responses remains unknown. To these ends, we tested folic acid (FA)-linked poloxamer407-coated ritonavir-boosted atazanavir (FA-nanoATV/r) nanoparticles for their ability to affect chronic HIV-1 infection in humanized mice. Following three, 100mg/kg FA-nanoATV/r intramuscular injections administered every other week to infected animals, viral RNA was at or below the detection limit, cell-associated HIV-1p24 reduced and CD4+ T cell counts protected. The dosing regimen improved treatment outcomes more than two fold from untargeted nanoATV/r. We posit that these nanoformulations have potential for translation to human use

Alcohol-Induced Lysosomal Damage and Suppression of Lysosome Biogenesis Contribute to Hepatotoxicity in HIV-Exposed Liver Cells

Although the causes of hepatotoxicity among alcohol-abusing HIV patients are multifactorial, alcohol remains the least explored “second hit” for HIV-related hepatotoxicity. Here, we investigated whether metabolically derived acetaldehyde impairs lysosomes to enhance HIV-induced hepatotoxicity. We exposed Cytochrome P450 2E1 (CYP2E1)-expressing Huh 7.5 (also known as RLW) cells to an acetaldehyde-generating system (AGS) for 24 h. We then infected (or not) the cells with HIV-1ADA then exposed them again to AGS for another 48 h. Lysosome damage was assessed by galectin 3/LAMP1 co-localization and cathepsin leakage. Expression of lysosome biogenesis–transcription factor, TFEB, was measured by its protein levels and by in situ immunofluorescence. Exposure of cells to both AGS + HIV caused the greatest amount of lysosome leakage and its impaired lysosomal biogenesis, leading to intrinsic apoptosis. Furthermore, the movement of TFEB from cytosol to the nucleus via microtubules was impaired by AGS exposure. The latter impairment appeared to occur by acetylation of α-tubulin. Moreover, ZKSCAN3, a repressor of lysosome gene activation by TFEB, was amplified by AGS. Both these changes contributed to AGS-elicited disruption of lysosome biogenesis. Our findings indicate that metabolically generated acetaldehyde damages lysosomes and likely prevents their repair and restoration, thereby exacerbating HIV-induced hepatotoxicity

Loss of neuronal integrity during progressive HIV-1 infection of humanized mice.

Neuronal damage induced by ongoing human immunodeficiency virus type 1 (HIV-1) infection was investigated in humanized NOD/scid-IL-2Rγ(c)(null) mice transplanted at birth with human CD34-positive hematopoietic stem cells. Mice infected at 5 months of age and followed for up to 15 weeks maintained significant plasma viral loads and showed reduced numbers of CD4(+) T-cells. Prospective serial proton magnetic resonance spectroscopy tests showed selective reductions in cortical N-acetyl aspartate in infected animals. Diffusion tensor imaging revealed structural changes in cortical gray matter. Postmortem immunofluorescence brain tissue examinations for neuronal and glial markers, captured by multispectral imaging microscopy and quantified by morphometric and fluorescence emission, showed regional reduction of neuronal soma and synaptic architectures. This was evidenced by loss of microtubule-associated protein 2, synaptophysin, and neurofilament antigens. This study is the first, to our knowledge, demonstrating lost neuronal integrity after HIV-1 infection in humanized mice. As such, the model permits studies of the relationships between ongoing viral replication and virus-associated neurodegeneration

Immune Activations and Viral Tissue Compartmentalization During Progressive HIV-1 Infection of Humanized Mice

Human immunodeficiency virus type one (HIV-1) tissue compartments are established soon after viral infection. However, the timing in which virus gains a permanent foothold in tissue and the cellular factors that control early viral-immune events are incompletely understood. These are critical events in studies of HIV-1 pathogenesis and in the development of viral reservoirs after antiretroviral therapy. Moreover, factors affecting the permanence of viral-tissue interactions underlie barriers designed to eliminate HIV-1 infection. To this end we investigated the temporal and spatial viral and host factors during HIV-1 seeding of tissue compartments. Two humanized NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ mouse models were employed. In the first, immune deficient mice were reconstituted with human CD34+ cord blood hematopoietic stem cells (HSC) (hu-HSC) and in the second mice were transplanted with adult mature human peripheral lymphocytes (hu-PBL). Both, in measure, reflect relationships between immune activation and viral infection as seen in an infected human host. Following humanization both mice models were infected with HIV-1ADA at 104 50% tissue culture infective doses. Viral nucleic acids and protein and immune cell profiles were assayed in brain, lung, spleen, liver, kidney, lymph nodes, bone marrow, and gut from 3 to 42 days. Peripheral CD4+ T cell loss began at 3 days together with detection of HIV-1 RNA in both mouse models after initiation of HIV-1 infection. HIV-1 was observed in all tested tissues at days 3 and 14 in hu- PBL and HSC mice, respectively. Immune impairment was most prominent in hu-PBL mice. T cell maturation and inflammation factors were linked directly to viral tissue seeding in both mouse models. We conclude that early viral tissue compartmentalization provides a roadmap for investigations into HIV-1 elimination

Cisplatin-loaded core cross-linked micelles: comparative pharmacokinetics, antitumor activity, and toxicity in mice

Polymer micelles with cross-linked ionic cores are shown here to improve the therapeutic performance of the platinum-containing anticancer compound cisplatin. Biodistribution, antitumor efficacy, and toxicity of cisplatin-loaded core cross-linked micelles of poly(ethylene glycol)-b-poly(methacrylic acid) were evaluated in a mouse ovarian cancer xenograft model. Cisplatin-loaded micelles demonstrated prolonged blood circulation, increased tumor accumulation, and reduced renal exposure. Improved antitumor response relative to free drug was seen in a mouse model. Toxicity studies with cisplatin-loaded micelles indicate a significantly improved safety profile and lack of renal abnormalities typical of free cisplatin treatment. Overall, the study supports the fundamental possibility of improving the potential of platinum therapy using polymer micelle-based drug delivery

Human-like NSG Mouse Glycoproteins Sialylation Pattern Changes the Phenotype of Human Lymphocytes and Sensitivity to HIV-1 Infection

BACKGROUND: The use of immunodeficient mice transplanted with human hematopoietic stem cells is an accepted approach to study human-specific infectious diseases such as HIV-1 and to investigate multiple aspects of human immune system development. However, mouse and human are different in sialylation patterns of proteins due to evolutionary mutations of the CMP-N-acetylneuraminic acid hydroxylase (CMAH) gene that prevent formation of N-glycolylneuraminic acid from N-acetylneuraminic acid. How changes in the mouse glycoproteins\u27 chemistry affect phenotype and function of transplanted human hematopoietic stem cells and mature human immune cells in the course of HIV-1 infection are not known. RESULTS: We mutated mouse CMAH in the NOD/scid-IL2Rγ CONCLUSION: NSG-cma