TET2 binds the androgen receptor and loss is associated with prostate cancer.

Genetic alterations associated with prostate cancer (PCa) may be identified by sequencing metastatic tumour genomes to identify molecular markers at this lethal stage of disease. Previously, we characterized somatic alterations in metastatic tumours in the methylcytosine dioxygenase ten-eleven translocation 2 (TET2), which is altered in 5-15% of myeloid, kidney, colon and PCas. Genome-wide association studies previously identified non-coding risk variants associated with PCa and melanoma. We perform fine-mapping of PCa risk across TET2 using genotypes from the PEGASUS case-control cohort and identify six new risk variants in introns 1 and 2. Oligonucleotides containing two risk variants are bound by the transcription factor octamer-binding protein 1 (Oct1/POU2F1) and TET2 and Oct1 expression are positively correlated in prostate tumours. TET2 is expressed in normal prostate tissue and reduced in a subset of tumours from the Cancer Genome Atlas (TCGA). Small interfering RNA-mediated TET2 knockdown (KD) increases LNCaP cell proliferation, migration and wound healing, verifying loss drives a cancer phenotype. Endogenous TET2 bound the androgen receptor (AR) and AR-coactivator proteins in LNCaP cell extracts, and TET2 KD increases prostate-specific antigen (KLK3/PSA) expression. Published data reveal TET2 binding sites and hydroxymethylcytosine proximal to KLK3. A gene co-expression network identified using TCGA prostate tumour RNA-sequencing identifies co-regulated cancer genes associated with 2-oxoglutarate (2-OG) and succinate metabolism, including TET2, lysine demethylase (KDM) KDM6A, BRCA1-associated BAP1, and citric acid cycle enzymes IDH1/2, SDHA/B, and FH. The co-expression signature is conserved across 31 TCGA cancers suggesting a putative role for TET2 as an energy sensor (of 2-OG) that modifies aspects of androgen-AR signalling. Decreased TET2 mRNA expression in TCGA PCa tumours is strongly associated with reduced patient survival, indicating reduced expression in tumours may be an informative biomarker of disease progression and perhaps metastatic disease.

Partial characterization and tissue distribution of the feline mu opiate receptor.

Heroin abuse is a common route of acquiring HIV-1 infection. However, the effects of opiates on lentivirus disease progression are not well understood. Feline immunodeficiency virus is recognized as a good animal model for HIV-1, but characterization of the opiate receptor system in cats is lacking. Here we report the partial sequencing of the feline mu opiate receptor (MOR) and demonstrate a homology of 92 and 93% to the published human MOR sequences. Additionally, MOR transcripts were detected in the feline brain and tonsil but not in the spleen. Also, specific receptor ligand interactions were observed using microphysiometry.

Methamphetamine and HIV-1: potential interactions and the use of the FIV/cat model.

The interaction of methamphetamine with human immunodeficiency virus (HIV), the aetiologic agent of Acquired Immune Deficiency Syndrome (AIDS), has not been thoroughly investigated. However, increasingly, a larger proportion of HIV infected individuals acquire the virus through methamphetamine use or are exposed to this drug during their disease course. In certain populations, there is a convergence of methamphetamine use and HIV-1 infection; yet our understanding of the potential effects that simultaneous exposure to these two agents have on disease progression is extremely limited. Studying the interactions between methamphetamine and lentivirus in people is difficult. To thoroughly understand methamphetamine's effects on lentivirus disease progression, an animal model that is both clinically relevant and easily manipulated is essential. In this report, we identified potential problems with methamphetamine abuse in individuals with a concurrent HIV-1 infection, described the Feline Immunodeficiency Virus (FIV)/cat model for HIV-1, and reported our early findings using this modelling system to study the interaction of methamphetamine and lentivirus infections.

Borna disease virus persistence causes inhibition of glutamate uptake by feline primary cortical astrocytes.

Borna disease virus (BDV), a nonsegmented, negative-stranded (NNS) RNA virus, causes central nervous system (CNS) disease in a broad range of vertebrate species, including felines. Both viral and host factors contribute to very diverse clinical and pathological manifestations associated with BDV infection. BDV persistence in the CNS can cause neurobehavioral and neurodevelopmental abnormalities in the absence of encephalitis. These BDV-induced CNS disturbances are associated with altered cytokine and neurotrophin expression, as well as cell damage that is very restricted to specific brain regions and neuronal subpopulations. BDV also targets astrocytes, resulting in the development of prominent astrocytosis. Astrocytes play essential roles in maintaining CNS homeostasis, and disruption of their normal activities can contribute to altered brain function. Therefore, we have examined the effect of BDV infection on the astrocyte's physiology. We present here evidence that BDV can establish a nonlytic chronic infection in primary cortical feline astrocytes that is associated with a severe impairment in the astrocytes' ability to uptake glutamate. In contrast, the astrocytes' ability to uptake glucose, as well as their protein synthesis, viability, and rate of proliferation, was not affected by BDV infection. These findings suggest that, in vivo, BDV could also affect an important astrocyte function required to prevent neuronal excitotoxicity. This, in turn, might contribute to the neuropathogenesis of BDV.

Effects of multiple acute morphine exposures on feline immunodeficiency virus disease progression.

Drug abuse is a common method of human immunodeficiency virus type 1 transmission, but the role of opiates on lentivirus disease progression is not well understood. The feline immunodeficiency virus (FIV)/cat system was used to model the weekend opiate abuser: the nondependent, nonaddicted, and nontolerant person. Sixteen cats were placed into 4 groups: FIV only, morphine only, morphine/FIV, and controls. Multiple acute morphine exposure did not increase the severity of early lentivirus infection. On the contrary, it delayed or moderated the FIV-induced disease progression. Although the animals were exposed to only 1 injection of morphine per day for 2 consecutive days per week, the morphine-treated FIV-infected animals had a delayed onset of the FIV-induced lymphadenopathy, did not develop or had a significant delay in the FIV-induced effects on brain stem auditory evoked potentials, and demonstrated a trend toward decreased virus load.

Replication rate of feline immunodeficiency virus in astrocytes is envelope dependent: implications for glutamate uptake.

Feline immunodeficiency virus (FIV) induces neurological abnormalities in domestic cats. Previously, we demonstrated that two disparate strains of FIV (FIV-34TF10 and FIV-PPR) varied greatly in the ability to replicate in feline cortical astrocytes. To investigate the impact of the env region on the replication efficiency of these strains, we constructed two env chimera viruses, FIV-34TF10-PPRenv and FIV-PPR-34TF10env, to infect feline cortical astrocytes in vitro. Although all of these viruses infected cortical astrocytes, the efficiency of replication depended on strain, and the env region played an essential role. The viruses containing the env of 34TF10, FIV-34TF10, and FIV-PPR-34TF10env had the greatest replication rate, whereas the viruses containing the env of PPR replicated at a lower level. Other viral regions had modulatory effects on the replication rate, with the FIV-PPR genome providing a slight replication advantage over the FIV-34TF10 genome. We also monitored the effects of these viruses on an important astrocyte function, glutamate uptake; all viruses significantly decreased this activity, but only the viruses containing the env of PPR significantly impaired glutamate uptake without altering the culture viability. These results may be particularly relevant in the context of lentivirus-induced central nervous system disease in which a selective breakdown of astroglial function may contribute to neurodegeneration.

Neurotoxic effects of feline immunodeficiency virus, FIV-PPR.

FIV is a lentivirus of domestic cats that causes neurologic disorders which are remarkably similar to those found in HIV-1 infected people. Using feline neuron cultures, we investigated the potential of both FIV virus and FIV-Env protein to cause neuronal damage through the excitotoxicity mechanism. The neuron swelling and lactate dehydrogenase (LDH) release assays were used as measures of cellular damage. The effects of FIV Env protein on glutamate receptor mediated increases in intracellular calcium were also examined. We found that FIV virus and FIV-Env protein significantly increased LDH release from the neuron cultures. Additionally, an increase in neuron size was detected in the cultures exposed to the virus, while swelling did not occur with exposure to either saline, denatured virus, or FIV-Env by itself. However, when both 20 microM glutamate and the FIV-PPR Env protein were added to the culture, a significant increase in neuron cell size was observed. The NMDA calcium signals were similar in general form between the control and FIV-PPR Env exposed cultures. However, the FIV - PPR Env protein treated cultures resulted in significant enhancement of the NMDA induced calcium signal. Our results indicate that FIV Env protein (either within the virion or baculovirus expressed) induced neurotoxicity as measured by neuron swelling and LDH release assays and that exposure of feline neurons to FIV Env protein alters the handling of intracellular calcium. These findings help to validate the FIV/cat system as a potential animal model for evaluating therapeutic approaches that target the excitotoxicity mechanisms of lentivirus induced CNS disease.

Effects of feline immunodeficiency virus on astrocyte glutamate uptake: implications for lentivirus-induced central nervous system diseases.

Feline immunodeficiency virus (FIV) is a lentivirus of domestic cats that causes a spectrum of diseases remarkably similar to AIDS in HIV-infected humans. As part of this spectrum, both HIV-1 and FIV induce neurologic disorders. Because astrocytes are essential in maintaining the homeostasis of the central nervous system, we analyzed FIV for the ability to infect feline astrocytes. Through immunocytochemistry and reverse transcriptase activity, it was demonstrated that two molecular clones of FIV (FIV-34TF10 and FIV-PPR) produce a chronic low level productive infection of feline astrocyte cultures. To investigate the consequences of this infection, selected astrocyte functions were examined. Infection with FIV-34TF10 significantly decreased the ability of astrocytes to scavenge extracellular glutamate (with a peak inhibition of 74%). The effects of the infection did not appear to be a result of toxicity but rather were more selective in nature because the glucose uptake function of the infected astrocyte cultures was not altered. Our data demonstrate that FIV productively infected, at a low level, feline astrocyte cultures, and as a consequence of this infection, an important astroglial function was altered. These findings suggest that a chronic low grade infection of astrocytes may impair the ability of these cells to maintain homeostasis of the central nervous system that, in turn, may contribute to a neurodegenerative disease process that is often associated with lentivirus infections.

Primary in vitro sensitization of human T-helper lymphocytes by peptides derived from the V3 loop of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein.

To generate CD4+ T-helper cell lines, lymphocytes from HIV-seronegative subjects were primed in vitro with peptides derived from the V3 loop of HIV-1 gp120. Antigen-specific reactivity was inhibited by an anti-DR monoclonal antibody, indicating HLA-class II dependency, but peptides could be recognized in different HLA-class II contexts. Three sites on V3LAI and two on V3MN were identified as targets of the respective V3LAI- and V3MN-specific lines. Recognition of V3 peptides was isolate-specific. The lines did not react against whole gp160, which suggests that V3 may be differently presented when used as such rather than as part of the entire glycoprotein. Similar results were obtained in chimpanzees immunized in vivo against V3LAI.

Immunogenicity of the human immunodeficiency virus (HIV) recombinant nef gene product. Mapping of T-cell and B-cell epitopes in immunized chimpanzees.

The nonstructural nef gene product of human immunodeficiency virus (HIV), p27, is a regulatory "early phase" protein produced by HIV-infected cells. As a possible negative regulator of transcription, it has been suggested that p27 may be involved in the control of HIV proviral latency. Immune reactivity to p27 may result in early destruction of HIV-replicating cells before viral assembly or of latently infected cells. It appeared, thus, of interest to investigate the immunogenicity of the molecule in chimpanzees immunized against HIV antigens. Two of the six chimpanzees that were injected with soluble recombinant p27 in association with other HIV proteins, displayed significant and sustained T-helper lymphocyte proliferative responses to p27 and to the other antigens. Using a set of synthetic peptides spanning the entire p27 sequence, two T-cell epitopes could be located: one within the last 20 amino-acids of the C terminus of the molecule, the other around the region of residues 118-122. Sera from the same animals also reacted to p27 in a radioimmunoassay as well as to some of the peptides in enzyme-linked immunosorbent assay. Sequential B-cell epitopes could thus be determined as being located in the regions of amino acids: 17-35, 52-66, and 185-205. The results obtained with peptides spanning the region between amino acid residues 65 and 172 indicate that at least two additional B-cell epitopes were present in the region comprised between amino acid 65 and 146. Interestingly, the extreme C terminus of the molecule encompasses both immunodominant T- and B-cell epitopes. Taken together, these observations should prove useful for the rational design of a HIV vaccine.