Characterization of pain-related behaviors in a rat model of acute-to-chronic low back pain: single vs. multi-level disc injury.

Introduction: Low back pain is the most common type of chronic pain. We examined pain-related behaviors across 18 weeks in rats that received injury to one or two lumbar intervertebral discs (IVD) to determine if multi-level disc injuries enhance/prolong pain. Methods: Twenty-three Sprague-Dawley adult female rats were used: 8 received disc puncture (DP) of one lumbar IVD (L5/6, DP-1); 8 received DP of two lumbar IVDs (L4/5 & L5/6, DP-2); 8 underwent sham surgery. Results: DP-2 rats showed local (low back) sensitivity to pressure at 6- and 12-weeks post-injury, and remote sensitivity to pressure (upper thighs) at 12- and 18-weeks and touch (hind paws) at 6, 12 and 18-weeks. DP-1 rats showed local and remote pressure sensitivity at 12-weeks only (and no tactile sensitivity), relative to Sham DP rats. Both DP groups showed reduced distance traveled during gait testing over multiple weeks, compared to pre-injury; only DP-2 rats showed reduced distance relative to Sham DP rats at 12-weeks. DP-2 rats displayed reduced positive interactions with a novel adult female rat at 3-weeks and hesitation and freezing during gait assays from 6-weeks onwards. At study end (18-weeks), radiological and histological analyses revealed reduced disc height and degeneration of punctured IVDs. Serum BDNF and TNFα levels were higher at 18-weeks in DP-2 rats, relative to Sham DP rats, and levels correlated positively with remote sensitivity in hind paws (tactile) and thighs (pressure). Discussion: Thus, multi-level disc injuries resulted in earlier, prolonged and greater discomfort locally and remotely, than single-level disc injury. BDNF and TNFα may have contributing roles.

A longitudinal characterization of sex-specific somatosensory and spatial memory deficits in HIV Tg26 heterozygous mice.

The pathogenesis of human immunodeficiency virus associated neurological disorders is still not well understood, yet is known to result in neurological declines despite combination anti-retroviral therapy. HIV-1 transgenic (Tg26) mice contain integrated non-infectious HIV-1 proviral DNA. We sought to assess the integrity of neurocognitive function and sensory systems in HIV-1 Tg26 mice using a longitudinal design, in both sexes, to examine both age- and sex-related disease progression. General neurological reflexive testing showed only acclimation to repeated testing by all groups. Yet, at 2.5 months of age, female Tg26 +/- mice showed hyposensitivity to noxious hot temperatures, compared to wild types (both sexes) and male Tg26 +/- mice, that worsened by 10 months of age. Female Tg26 +/- mice had short-term spatial memory losses in novel object location memory testing at 2.5 and 7 months, compared to female wild types; changes not observed in male counterparts. Female Tg26 +/- mice showed mild learning deficits and short- and long-term spatial memory deficits in olfactory and visually cued Barnes Maze testing at 3 months of age, yet greater learning and memory deficits by 8 months. In contrast, male Tg26 +/- mice displayed no learning deficits and fewer spatial memory deficits (mainly heading errors in nontarget holes). Thus, greater sex-specific temperature hyposensitivity and spatial memory declines were observed in female HIV Tg26 +/- mice, than in male Tg26 +/- mice, or their wild type littermates, that increased with aging. Additionally, tibial bones were examined using ex vivo micro-CT after tissue collection at 11 months. Sex-dependent increases in bone volume and trabecular number were seen in males, matching their greater weights at this age. These results indicate that HIV-1 Tg26 mice is a promising model in which to study neuropathic mechanisms underlying peripheral pathology as well as cognitive deficits seen with HIV.

Suppression of Zika Virus Infection in the Brain by the Antiretroviral Drug Rilpivirine.

Zika virus (ZIKV) infection is associated with microcephaly in neonates and Guillain-Barré syndrome in adults. ZIKV produces a class of nonstructural (NS) regulatory proteins that play a critical role in viral transcription and replication, including NS5, which possesses RNA-dependent RNA polymerase (RdRp) activity. Here we demonstrate that rilpivirine (RPV), a non-nucleoside reverse transcriptase inhibitor (NNRTI) used in the treatment of HIV-1 infection, inhibits the enzymatic activity of NS5 and suppresses ZIKV infection and replication in primary human astrocytes. Similarly, other members of the NNRTI family, including etravirine and efavirenz, showed inhibitory effects on viral infection of brain cells. Site-directed mutagenesis identified 14 amino acid residues within the NS5 RdRp domain (AA265-903), which are important for the RPV interaction and the inhibition of NS5 polymerase activity. Administration of RPV to ZIKV-infected interferon-alpha/beta receptor (IFN-A/R) knockout mice improved the clinical outcome and prevented ZIKV-induced mortality. Histopathological examination of the brains from infected animals revealed that RPV reduced ZIKV RNA levels in the hippocampus, frontal cortex, thalamus, and cerebellum. Repurposing of NNRTIs, such as RPV, for the inhibition of ZIKV replication offers a possible therapeutic strategy for the prevention and treatment of ZIKV-associated disease.

Memory deficits, gait ataxia and neuronal loss in the hippocampus and cerebellum in mice that are heterozygous for Pur-alpha.

Pur-alpha is a highly conserved sequence-specific DNA and RNA binding protein with established roles in DNA replication, RNA translation, cell cycle regulation, and maintenance of neuronal differentiation. Prior studies have shown that mice lacking Pur-alpha (-/-) display decreased neurogenesis and impaired neuronal differentiation. We sought to examine for the first time, the behavioral phenotype and brain histopathology of mice that are heterozygous (+/-) for Pur-alpha. Standardized behavioral phenotyping revealed a decreased escape response to touch, limb and abdominal hypotonia, and gait abnormalities in heterozygous Pur-alpha (+/-) mice, compared to wild-type (+/+) littermates. Footprint pattern analyses showed wider-based steps, increased missteps and more outwardly rotated hindpaws in heterozygous Pur-alpha (+/-) mice, suggestive of cerebellar pathology. The Barnes maze and novel object location testing revealed significant memory deficits in heterozygous Pur-alpha mice, suggestive of hippocampal pathology. Quantitative immunohistochemical assays of the vermal region of the cerebellum and CA1-3 regions of the hippocampus revealed reduced numbers of neurons in general, as well as reduced numbers of Pur-alpha+-immunopositive neurons and dendrites in heterozygous Pur-alpha mice, compared to wild-type littermates. Past studies have implicated mutations in Pur-alpha in several diseases of brain development and neurodegeneration. When combined with these new findings, the Pur-alpha heterozygous knockout mice may provide an animal model to study mechanisms of and treatments for Pur-alpha-related cognitive deficiencies and neuropathology.

Combined proteasome and Bcl-2 inhibition stimulates apoptosis and inhibits growth in EBV-transformed lymphocytes: a potential therapeutic approach to EBV-associated lymphoproliferative diseases.

OBJECTIVES: Epstein-Barr virus (EBV) transforms B-cells into immortalized lymphoblastoid cells (LCLs) by triggering signaling pathways that lead to activation of multiple transcription factors and anti-apoptotic proteins, including NF-kappaB and Bcl-2, respectively. Since proteasome inhibition suppresses NF-kappaB activity, we sought to determine whether the proteasome inhibitor, bortezomib, alone or in combination with Bcl-2 inhibition, has potential as a therapeutic strategy in EBV-driven B-cell neoplasms. METHODS: We evaluated the effects of bortezomib in LCLs in vitro, in the presence and absence of the small molecular inhibitor of Bcl-2, HA14-1, on proliferation, apoptosis, caspase activation, and expression of Bcl-2 family members, and in vivo in the severe combined immunodeficiency (SCID) model of EBV+ lymphoproliferative disease. RESULTS: Bortezomib inhibited proliferation, stimulated apoptosis, and activated caspases-3 and -9 in a dose-dependent manner in LCLs. In vivo, bortezomib completely abrogated development of EBV+ lymphoproliferative disease in LCL-bearing SCID mice. When HA14-1 was added to bortezomib in vitro, we observed a synergistic anti-proliferative effect and enhancement of apoptosis and caspase activation, including activation of caspase-8, in LCLs. These events were associated with modulation of expression of Bcl-2 family members towards a pro-apoptotic profile with translocation of cytochrome C from mitochondria to cytoplasm. CONCLUSIONS: These studies demonstrated that bortezomib mediates anti-tumor effects in EBV-associated lymphoproliferations both in vitro and in vivo, and that its anti-proliferative and apoptotic effects are synergistically enhanced in the presence of a Bcl-2 inhibitor. These findings support further investigation of bortezomib in EBV+ lymphoproliferative diseases, and suggest that bortezomib in combination with Bcl-2 antagonists represents a potential therapeutic strategy for EBV-driven B-cell neoplasms.

Systemic Bcl-2 antisense oligodeoxynucleotide in combination with cisplatin cures EBV+ nasopharyngeal carcinoma xenografts in SCID mice.

Nasopharyngeal carcinoma (NPC) is causally linked to Epstein-Barr virus (EBV), and the EBV oncoprotein, latent membrane protein 1 (LMP-1), is expressed in the majority of NPCs. LMP-1 upregulates antiapoptotic genes, including bcl-2, and Bcl-2 protein is overexpressed in NPC. Given the antiapoptotic and chemoprotective effects of Bcl-2, it represents a rational therapeutic target in NPC. We have investigated the antitumor and chemosensitizing effects of the Bcl-2 antisense oligodeoxynucleotide G3139 (oblimersen, Genasense) in NPC. For these studies, we used the C666-1 line, a stably infected NPC-derived line that co-expresses LMP-1 and Bcl-2. We have shown that G3139 treatment of C666-1 in vitro caused sequence-dependent suppression of Bcl-2 protein, inhibition of cell growth and enhanced sensitivity to cisplatin (CDDP), as measured by increased antiproliferative and apoptotic effects. In vivo, G3139 treatment (25 mg/kg every 3 days x 5 doses) delayed engraftment and significantly inhibited growth of established C666-1 xenografts in SCID mice compared to control oligo-treated animals. However, G3139 alone did not prevent engraftment or cure established tumors in any animals. In contrast, G3139 treatment (25 mg/kg every 3 days x 5 starting on day 7) in combination with CDDP (8 mg/kg on day 14) completely abrogated tumor engraftment in 80% of animals compared to CDDP (0%) or CDDP + control oligo (0%). When treatment was delayed until tumor was established, G3139 in combination with CDDP significantly inhibited tumor growth compared to CDDP or CDDP + control oligo, and cured 69% animals with established tumors. No animals treated with G3139, CDDP or CDDP + control oligo were cured. Tumor burden and response to treatment correlated with EBV DNA load in serum, measured by real-time PCR. Western blots of tumor extracts obtained during oligo treatment showed that Bcl-2 levels were significantly decreased in G3139-treated animals. Our studies have demonstrated that the Bcl-2 antisense oligodeoxynucleotide, G3139, has proapoptotic effects in C666-1, and in combination with CDDP, is curative in C666-1 NPC xenograft tumors in vivo. The sequence-dependency of these effects is consistent with an antisense mechanism. These studies suggest that Bcl-2 may represent a biologically relevant target for the development of novel combinatorial therapies for NPC.

Bcl-2 antisense (G3139, Genasense) enhances the in vitro and in vivo response of Epstein-Barr virus-associated lymphoproliferative disease to rituximab.

Bcl-2 is up-regulated by EBV in immortalized lymphoblastoid B cells and is expressed in the majority of EBV-associated lymphoproliferative diseases, including posttransplant lymphoproliferative disorder (PTLD) and AIDS-related lymphoma (ARL). Given the antiapoptotic and chemoprotective effect of Bcl-2, it represents a logical target for modulation using antisense strategies in PTLD and ARL. We previously examined the antitumor effects of a fully phosphorothioated Bcl-2 antisense oligonucleotide, G3139, in EBV(+) lymphoproliferative disease in vitro and in vivo using the human/severe combined immunodeficient (SCID) chimeric model of PTLD. These studies showed that G3139 treatment decreased Bcl-2 protein levels in association with antiproliferative and proapoptotic effects in lymphoblastoid cell lines (LCLs) in vitro. In vivo, although G3139 treatment completely abrogated EBV(+) lymphoid tumor engraftment in the human/SCID model of PTLD, antisense treatment alone was not curative in animals with established tumors. Because the humanized anti-CD20 antibody, rituximab, has antitumor activity in patients with PTLD and stimulates apoptosis in some lymphoid cell lines, we sought to determine whether Bcl-2 antisense treatment potentiates the antitumor effects of rituximab in EBV-associated lymphoproliferative disease in vitro and in vivo. Proliferation assays by thymidine uptake in LCLs showed that G3139 but not control oligonucleotides augmented the antiproliferative effect of rituximab. Flow cytometric terminal deoxynucleotidyltransferase-mediated nick end labeling assays confirmed that G3139 treatment enhanced the apoptotic response of LCLs to rituximab, and this interaction was oligonucleotide sequence dependent. To test the in vivo efficacy of G3139 and rituximab in the human/SCID model of PTLD, we used a delayed treatment schedule that permitted detection of enhanced antitumor activity of combination therapy. Although G3139 or rituximab treatment significantly prolonged survival compared with untreated controls, 89% of animals in the monotherapy arms died with disseminated tumors. In contrast, 79% of animals in the combined G3139 and rituximab arm remained tumor free for the duration of follow-up (>160 days) with no evidence of tumors at the time of sacrifice, indicating that G3139 in combination with rituximab was curative therapy in the majority of tumor-bearing animals. These studies demonstrated that G3139 potentiates the antitumor response of PTLD to rituximab in vivo and augments the antiproliferative and apoptotic effects of rituximab in vitro in LCLs. This is the first report of G3139 potentiating the antitumor activity of an antibody-based therapy both in vitro and in vivo. Bcl-2 antisense oligonucleotide therapy in combination with rituximab may represent a promising nontoxic and effective targeted therapy for EBV-associated lymphoproliferative diseases such as PTLD and ARL. Furthermore, this approach may have broader applications to other Bcl-2- and CD20-expressing lymphoid malignancies.