Probes for INS

Zika virus (ZIKV) is a teratogenic mosquito-borne flavivirus which can be sexually transmitted from man to woman. High viral loads and prolonged viral shedding in semen suggest that ZIKV replicates within the human male genital tract, but its target organs are unknown. Using ex vivo infection of organotypic cultures, we demonstrated here that ZIKV replicates in human testicular tissue and infects a broad range of cell types, including germ cells, which we also identified as infected in the semen from ZIKV-infected donors. ZIKV had no major deleterious effect on the morphology and hormonal production of the human testis explants. Infection induced a broad antiviral response but no interferon up-regulation and minimal pro-inflammatory response in testis explants, with no cytopathic effect. Finally, we studied ZIKV infection in mouse testis, and compared it to human infection. This study provides key insights into how ZIKV may persist in semen and alter semen parameters, as well as a valuable tool for testing antiviral agents.

The lack of representative nasopharyngeal carcinoma (NPC) models has seriously hampered research on EBV carcinogenesis and preclinical studies in NPC. Here we report the successful growth of five NPC patient-derived xenografts (PDXs) from fifty-eight attempts of transplantation of NPC specimens into NOD/SCID mice. The take rates for primary and recurrent NPC are 4.9% and 17.6%, respectively. Successful establishment of a new EBV-positive NPC cell line, NPC43, is achieved directly from patient NPC tissues by including Rho-associated coiled-coil containing kinases inhibitor (Y-27632) in culture medium. Spontaneous lytic reactivation of EBV can be observed in NPC43 upon withdrawal of Y-27632. Whole-exome sequencing (WES) reveals a close similarity in mutational profiles of these NPC PDXs with their corresponding patient NPC. Whole-genome sequencing (WGS) further delineates the genomic landscape and sequences of EBV genomes in these newly established NPC models, which supports their potential use in future studies of NPC.

Abstract

Macrocyclic lactone-resistant Parascaris have been reported throughout the world. In part, the drug resistant phenotype is hypothesized to be associated with ATP-binding cassette transporters known as P-glycoproteins. In many systems, P-glycoproteins efflux drugs out of cells thereby precluding drug binding to target receptors. Parascaris may evade macrocyclic lactone-mediated death by effluxing drugs away from target receptors in the nervous system. Alternatively, P-glycoprotein expression in the gut or body wall could prevent penetration of drugs into the body of the parasite altogether. In the present study, we evaluate expression of Peq-pgp-11 and Peq-pgp-16 using a novel multiple nucleic acid hybridization method. This method allowed for visualization of individual mRNA transcripts within fixed tissue sections of Parascaris adults. Our investigation revealed expression of Peq-pgp-11 and Peq-pgp-16 in the intestine, body wall, nerves, lateral cords, and reproductive tissues of male and female parasites. These results suggest that Pgp could efflux drugs locally at the level of parasite neuronal tissue as well as at sites of entry for drugs such as the hypodermis and intestine. The multiple nucleic acid hybridization method could be useful for providing tissue context for gene expression in a variety of nematode parasites.

Human endogenous retroviruses (HERVs) play pivotal roles in the development of breast cancer. However, the detailed mechanisms of noncoding HERVs remain elusive. Here, our genome-wide transcriptome analysis of HERVs revealed that a primate long noncoding RNA, which we dubbed TROJAN, was highly expressed in human triple-negative breast cancer (TNBC). TROJAN promoted TNBC proliferation and invasion and indicated poor patient outcomes. We further confirmed that TROJAN could bind to ZMYND8, a metastasis-repressing factor, and increase its degradation through the ubiquitin-proteasome pathway by repelling ZNF592. TROJAN also epigenetically up-regulated metastasis-related genes in multiple cell lines. Correlations between TROJAN and ZMYND8 were subsequently confirmed in clinical samples. Furthermore, our study verified that antisense oligonucleotide therapy targeting TROJAN substantially suppressed TNBC progression in vivo. In conclusion, the long noncoding RNA TROJAN promotes TNBC progression and serves as a potential therapeutic target.

HSV-1 establishes latency in both peripheral nerve ganglia and the central nervous system (CNS). The outcome of acute and latent infections in these different anatomic sites appear to be distinct. It is becoming clear that many of the existing culture models using animal primary neurons to investigate HSV-1 infection of the CNS are limited and not ideal, and most do not recapitulate features of CNS neurons. Human induced human pluripotent stem cells (hiPSCs) and neurons derived from them as tools to study aspects of neuropathogenesis are documented but few have focused on modeling infections of the (CNS). Here, we characterize functional two-dimensional (2D) CNS-like neuron cultures and three-dimensional (3D) brain organoids made from hiPSCs to model HSV-1-human-CNS interactions. Our results show that: i) hiPSC-derived CNS neurons are permissive for HSV-1 infection; ii) a quiescent state exhibiting key landmarks of HSV-1 latency described in animal models can be established in hiPSC-derived CNS neurons; iii) the complex laminar structure of the organoids can be efficiently infected with HSV, with virus being transported from the periphery to the central layers of the organoid; iv) the organoids support reactivation of HSV, albeit less efficiently than 2D cultures. Collectively, our results indicate that hiPSC-derived neuronal platforms, especially 3D organoids, offer an extraordinary opportunity for modeling the interaction of HSV-1 with the complex cellular and architectural structure of the human CNS.IMPORTANCE This study employed human induced pluripotent stem cells (hiPSCs) to model acute and latent HSV-1 infections in two-dimensional (2D) and three-dimensional (3D) CNS neuronal cultures. We successfully established acute HSV-1 infections and infections showing features of latency. HSV-1 infection of the 3D organoids was able to spread from the outer surface of the organoid and was transported to the interior lamina providing a model to study HSV-1 trafficking trough complex neuronal tissue structures. HSV-1 could be reactivated in both culture systems tough in contrast to 2D cultures, it appears to be more difficult to reactivate HSV-1 in 3D cultures, potentially paralleling the low efficiency of HSV-1 reactivation in the CNS of animal models. The reactivation events were accompanied by dramatic neuronal morphological changes and cell-cell fusion. Together our results provide substantive evidence of the suitability of hiPSC-based neuronal platforms to model HSV-1 CNS interactions in a human context.

Abstract

BACKGROUND:

Microglia are the principal innate immune defense cells of the centeral nervous system (CNS) and the target of the human immunodeficiency virus type one (HIV-1). A complete understanding of human microglial biology and function requires the cell's presence in a brain microenvironment. Lack of relevant animal models thus far has also precluded studies of HIV-1 infection. Productive viral infection in brain occurs only in human myeloid linage microglia and perivascular macrophages and requires cells present throughout the brain. Once infected, however, microglia become immune competent serving as sources of cellular neurotoxic factors leading to disrupted brain homeostasis and neurodegeneration.

METHODS:

Herein, we created a humanized bone-marrow chimera producing human "microglia like" cells in NOD.Cg-PrkdcscidIl2rgtm1SugTg(CMV-IL34)1/Jic mice. Newborn mice were engrafted intrahepatically with umbilical cord blood derived CD34+ hematopoietic stem progenitor cells (HSPC). After 3 months of stable engraftment, animals were infected with HIV-1ADA, a myeloid-specific tropic viral isolate. Virologic, immune and brain immunohistology were performed on blood, peripheral lymphoid tissues, and brain.

RESULTS:

Human interleukin-34 under the control of the cytomegalovirus promoter inserted in NSG mouse strain drove brain reconstitution of HSPC derived peripheral macrophages into microglial-like cells. These human cells expressed canonical human microglial cell markers that included CD14, CD68, CD163, CD11b, ITGB2, CX3CR1, CSFR1, TREM2 and P2RY12. Prior restriction to HIV-1 infection in the rodent brain rested on an inability to reconstitute human microglia. Thus, the natural emergence of these cells from ingressed peripheral macrophages to the brain could allow, for the first time, the study of a CNS viral reservoir. To this end we monitored HIV-1 infection in a rodent brain. Viral RNA and HIV-1p24 antigens were readily observed in infected brain tissues. Deep RNA sequencing of these infected mice and differential expression analysis revealed human-specific molecular signatures representative of antiviral and neuroinflammatory responses.

CONCLUSIONS:

This humanized microglia mouse reflected human HIV-1 infection in its known principal reservoir and showed the development of disease-specific innate immune inflammatory and neurotoxic responses mirroring what can occur in an infected human brain.

Solute carrier family 7 member 2 (SLC7A2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in immune responses to pathogens. We assessed the role of SLC7A2 in murine infection with Citrobacter rodentium, an attaching and effacing enteric pathogen that causes colitis. Induction of SLC7A2 was upregulated in colitis tissues, and localized predominantly to colonic epithelial cells. Compared to wild-type mice, Slc7a2-/-mice infected with C. rodentium had improved survival and decreased weight loss, colon weight, and histologic injury; this was associated with decreased colonic macrophages, dendritic cells, granulocytes, and Th1 and Th17 cells. In infected Slc7a2-/-mice, there were decreased levels of the proinflammatory cytokines G-CSF, TNF-α, IL-1α, IL-1β, and the chemokines CXCL1, CCL2, CCL3, CCL4, CXCL2, and CCL5. In bone marrow chimeras, the recipient genotype drove the colitis phenotype, indicative of the importance of epithelial, rather than myeloid SLC7A2. Mice lacking Slc7a2 exhibited reduced adherence of C. rodentium to the colonic epithelium and decreased expression of Talin-1, a focal adhesion protein involved in the attachment of the bacterium. The importance of SLC7A2 and Talin-1 in the intimate attachment of C. rodentium and induction of inflammatory response was confirmed in vitro, using conditionally-immortalized young adult mouse colon (YAMC) cells with shRNA knockdown of Slc7a2 or Tln1. Inhibition of L-Arg uptake with the competitive inhibitor, L-lysine (L-Lys), also prevented attachment of C. rodentium and chemokine expression. L-Lys and siRNA knockdown confirmed the role of L-Arg and SLC7A2 in human Caco-2 cells co-cultured with enteropathogenic Escherichia coli. Overexpression of SLC7A2 in human embryonic kidney cells increased bacterial adherence and chemokine expression. Taken together, our data indicate that C. rodentium enhances its own pathogenicity by inducing the expression of SLC7A2 to favor its attachment to the epithelium and thus create its ecological niche.

Follicular helper CD4 T cells, TFH, residing in B-cell follicles within secondary lymphoid tissues, are readily infected by AIDS viruses and are a major source of persistent virus despite relative control of viral replication. This persistence is due at least in part to a relative exclusion of effective antiviral CD8 T cells from B-cell follicles. To determine whether CD8 T cells could be engineered to enter B-cell follicles, we genetically modified unselected CD8 T cells to express CXCR5, the chemokine receptor implicated in cellular entry into B-cell follicles. Engineered CD8 T cells expressing CXCR5 (CD8hCXCR5) exhibited ligand specific signaling and chemotaxis in vitro. Six infected rhesus macaques were infused with differentially fluorescent dye-labeled autologous CD8hCXCR5 and untransduced CD8 T cells and necropsied 48h later. Flow cytometry of both spleen and lymph node samples revealed higher frequencies of CD8hCXCR5 than untransduced cells, consistent with preferential trafficking to B-cell follicle-containing tissues. Confocal fluorescence microscopy of thin-sectioned lymphoid tissues demonstrated strong preferential localization of CD8hCXCR5 T cells within B-cell follicles with only rare cells in extrafollicular locations. CD8hCXCR5 T cells were present throughout the follicles with some observed near infected TFH In contrast, untransduced CD8 T cells were found in the extrafollicular T-cell zone. Our ability to direct localization of unselected CD8 T cells into B-cell follicles using CXCR5 expression provides a strategy to place highly effective virus-specific CD8 T cells into these AIDS virus sanctuaries and potentially suppress residual viral replication.IMPORTANCE AIDS virus persistence in individuals under effective drug therapy or those who spontaneously control viremia remains an obstacle to definitive treatment. Infected follicular helper CD4 T cells, TFH, present inside B-cell follicles represent a major source of this residual virus. While effective CD8 T-cell responses can control viral replication in conjunction with drug therapy or in rare cases spontaneously, most antiviral CD8 T cells do not enter B-cell follicles and those that do fail to robustly control viral replication in the TFHpopulation. Thus, these sites are a sanctuary and a reservoir for replicating AIDS viruses. Here, we demonstrate that engineering unselected CD8 T cells to express CXCR5, a chemokine receptor on TFH associated with B-cell follicle localization, redirects them into B-cell follicles. These proof of principle results open a pathway for directing engineered antiviral T cells into these viral sanctuaries to help eliminate this source of persistent virus.

Over half of adults are seropositive for JC polyomavirus (JCV), but rare individuals develop progressive multifocal leukoencephalopathy (PML), a demyelinating JCV infection of the central nervous system. Previously, PML was primarily seen in immunosuppressed patients with AIDS or certain cancers, but it has recently emerged as a drug safety issue through its association with diverse immunomodulatory therapies. To better understand the relationship between the JCV life cycle and PML pathology, we studied autopsy brain tissue from a 70-year-old psoriasis patient on the integrin alpha-L inhibitor efalizumab following a ~2 month clinical course of PML. Sequence analysis of lesional brain tissue identified PML-associated viral mutations in regulatory (non-coding control region) DNA, capsid protein VP1, and the regulatory agnoprotein, as well as 9 novel mutations in capsid protein VP2, indicating rampant viral evolution. Nine samples, including three gross PML lesions and normal-appearing adjacent tissues, were characterized by histopathology and subject to quantitative genomic, proteomic, and molecular localization analyses. We observed a striking correlation between the spatial extent of demyelination, axonal destruction, and dispersion of JCV along white matter myelin sheath. Our observations in this case, as well as in a case of PML-like disease in an immunocompromised rhesus macaque, suggest that long-range spread of polyomavirus and axonal destruction in PML might involve extracellular association between virus and the white matter myelin sheath.

Human papillomavirus (HPV) is an independent risk factor for development of oral squamous cell carcinoma (OSCC). This study aimed to investigate the role of HPV infection and the trend in percentage of HPV-associated OSCC over a five-year period in northeastern Thailand. In this case-control study, 91 exfoliated oral cell samples and 80 lesion cell samples from OSCC cases and exfoliated oral cells from 100 age/gender-matched controls were collected. HPV infection was investigated by PCR using GP5+/GP6+ primers followed by HPV genotyping using reverse line blot hybridization. Quantitative RT-PCR was used to evaluate HPV oncogene transcription. Temporal trends of HPVinfection were evaluated in archived formalin-fixed paraffin-embedded (FFPE) OSCC tissues using in situ hybridization. HPV DNA was found in 17.5% (14/80) of lesion samples from OSCC cases and 29.7% (27/91) of exfoliated oral cell samples from the same cases. These values were significantly higher than in exfoliated oral cell samples from controls (13%, 13/100). HPV-16 was the genotype most frequently found in OSCC cases (92.8%, 13/14 infected cases). Interestingly, HPV oncogene mRNA expression was detected and correlated with OSCC cases (P < 0.005). Of 146 archived FFPE OSCC samples, 82 (56.2%) were positive for high-risk HPV DNA and 64 (43.8%) cases were positive for HPV E6/E7 mRNA expression. There was a trend of increasing percentage of HPV-associated OSCC from 2005 to 2010. This was especially so for females with well-differentiated tumors in specific tongue sub-sites. We suggest that HPV infection plays an important role in oral carcinogenesis in northeastern Thailand.