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Background: Liver disease remains a significant contributor to morbidity and mortality in HIV-infected individuals, even during successful treatment with combination antiretroviral therapy (cART). In non-human primates, SIV infection is associated with gut microbiome dysbiosis as well as bacterial translocation into the colonic lamina propria and liver via the portal vein. Here the liver microbiome was evaluated in rhesus macaques to discern the influence of SIV infection alone (SIV+) and during cART administration (SIV+cART) on liver bacterial dysbiosis and neutrophil infiltration.Results: Dysbiosis in liver bacterial composition was observed, encompassing changes in a number of genera, during SIV infection in the absence and presence of cART. The most striking finding was an increase in the level of Mycobacterium, which while barely detectable in the uninfected macaques, was the most abundant genus observed in the livers of a majority SIV+ and SIV+cART macaques. Multi-gene sequencing analyses identified a species of environmental mycobacteria similar to the opportunistic pathogen M. smegmatis. The effect of M. smegmatis on host gene expression in primary hepatocytes was evaluated in vitro utilizing PILAM, a glycolipid cell wall component found in atypical Mycobacteria. PILAM induced an upregulation of inflammatory responses, including an increase in the chemokines associated with neutrophil chemotaxis (CXCL1, CXCL5, and CXCL6). Assessment of the macaque livers by microscopy determined that neutrophil levels were reduced in SIV+cART macaques, suggesting that the SIV infection and/or cART treatment influence the liver-associated neutrophil response. Conclusions: A number of liver bacteria genera were altered following SIV infection even in the context of cART, possibly as a consequence of reduced neutrophil recruitment. Mycobacteria became a major component of the SIV infected macaque liver microbiome, raising the possibility that bacteria of this genus might contribute to liver disease in HIV infected patients.

There is an ongoing need for clinically relevant models of perinatal infection and hypoxia-ischemia (HI) in which to test therapeutic interventions for infants with the neurological sequela of prematurity. Ferrets are ideal candidates for modeling the preterm human brain, as they are born lissencephalic and develop gyrencephalic brains postnatally. At birth, ferret brain development is similar to a 13 week human fetus, with postnatal-day (P) 17 kits considered to be equivalent to an infant at 32-36 weeks' gestation. We describe an injury model in the P17 ferret, where lipopolysaccharide administration is followed by bilateral cerebral ischemia, hypoxia, and hyperoxia. This simulates the complex interaction of prolonged inflammation, ischemia, hypoxia, and oxidative stress experienced in a number of neonates who develop brain injury. Injured animals display a range of gross injury severity, with morphological changes in the brain including narrowing of multiple cortical gyri and associated sulci. Injured animals also show slowed reflex development, slower and more variable speed of locomotion in an automated catwalk, and decreased exploration in an open field. This model provides a platform in which to test putative therapies for infants with neonatal encephalopathy associated with inflammation and HI, study mechanisms of injury that affect cortical development, and investigate pathways that provide resilience in unaffected animals.

Benzofulvenes and their derivatives are useful molecular entities having applications as biologically active molecules, polymer precursors, and optoelectronic devices. We have developed a simple and mild synthetic method for the formulation of a variety of these interesting compounds. Using carbonyl coupling techniques combined with microwave heating, a wide variety of functionalized benzofulvenes can be accessed rapidly in good yield. Furthermore, we have obtained five crystal structures further expanding a limited number of benzofulvene structures available.

ATP-sensitive potassium (KATP) channels are found in the nervous system and are downstream targets of opioid receptors. KATP channel activity can effect morphine efficacy and may beneficial for relieving chronic pain in the peripheral and central nervous system. Unfortunately, the KATP channels exists as a heterooctomers, and the exact subtypes responsible for the contribution to chronic pain and opioid signaling in either dorsal root ganglia (DRG) or the spinal cord are yet unknown. Chronic opioid exposure (15 mg/kg morphine, s.c., twice daily) over 5 days produces significant downregulation of Kir6.2 and SUR1 in the spinal cord and DRG of mice. In vitro studies also conclude potassium flux after KATP channel agonist stimulation is decreased in neuroblastoma cells treated with morphine for several days. Mice lacking the KATP channel SUR1 subunit have reduced opioid efficacy in mechanical paw withdrawal behavioral responses compared to wild-type and heterozygous littermates (5 and 15 mg/kg, s.c., morphine). Using either short hairpin RNA (shRNA) or SUR1 cre-lox strategies, downregulation of SUR1 subtype KATP channels in the spinal cord and DRG of mice potentiated the development of morphine tolerance and withdrawal. Opioid tolerance was attenuated with intraplantar injection of SUR1 agonists, such as diazoxide and NN-414 (100 μM, 10 μL) compared to vehicle treated animals. These studies are an important first step in determining the role of KATP channel subunits in antinociception, opioid signaling, and the development of opioid tolerance, and shed light on the potential translational ability of KATP channel targeting pharmaceuticals and their possible future clinical utilization. These data suggest that increasing neuronal KATP channel activity in the peripheral nervous system may be a viable option to alleviate opioid tolerance and withdrawal.

Liver disease is a leading contributor to morbidity and mortality during HIV infection, despite the use of combination antiretroviral therapy (cART). The precise mechanisms of liver disease during HIV infection are poorly understood partially due to the difficulty in obtaining human liver samples as well as the presence of confounding factors (e.g. hepatitis co-infection, alcohol use). Utilizing the simian immunodeficiency virus (SIV) macaque model, a controlled study was conducted to evaluate the factors associated with liver inflammation and the impact of cART. We observed an increase in hepatic macrophages during untreated SIV infection that was associated with a number of inflammatory and fibrosis mediators (TNFα, CCL3, TGFβ). Moreover, an upregulation in the macrophage chemoattractant factor CCL2 was detected in the livers of SIV-infected macaques that coincided with an increase in the number of activated CD16+ monocyte/macrophages and T cells expressing the cognate receptor CCR2. Expression of Mac387 on monocyte/macrophages further indicated that these cells recently migrated to the liver. The hepatic macrophage and T cell levels strongly correlated with liver SIV DNA levels, and were not associated with the levels of 16S bacterial DNA. Utilizing in situ hybridization, SIV-infected cells were found primarily within portal triads, and were identified as T cells. Microarray analysis identified a strong antiviral transcriptomic signature in the liver during SIV infection. In contrast, macaques treated with cART exhibited lower levels of liver macrophages and had a substantial, but not complete, reduction in their inflammatory profile. In addition, residual SIV DNA and bacteria 16S DNA were detected in the livers during cART, implicating the liver as a site on-going immune activation during antiretroviral therapy. These findings provide mechanistic insights regarding how SIV infection promotes liver inflammation through macrophage recruitment, with implications for in HIV-infected individuals., Author summary Liver disease is common in HIV-infected individuals and is one of the leading causes of death in this population. Currently, the factors that contribute to liver disease during HIV infection are not known, as human studies are difficult to conduct. Therefore, pathogenic SIV infection of macaques is a valuable model system for understanding immune changes that occur in tissues during infection, including the liver. Using liver samples from uninfected and SIV-infected macaques, we observed an increase in liver macrophage infiltration that was likely mediated by the macrophage-attracting chemokine, CCL2. Importantly, liver macrophage number strongly correlated with liver SIV levels and the expression of liver inflammatory and fibrosis mediators during SIV infection. Further, we identified an upregulation of immune and inflammatory pathways in the liver of SIV-infected macaques, including a strong antiviral response. Treatment with antiretroviral drugs decreased macrophage infiltration and reduced liver inflammation, consistent with reduced SIV levels. In summary, these findings provide mechanistic insights into how SIV infection promotes liver inflammation through macrophage infiltration, which can predispose this population to liver complications during infection.

The ATP-sensitive K+ channel (KATP) is involved in hypersensitivity during chronic pain and is presumed to be a downstream target of mu opioid receptors. Multiple subtypes of KATP channels exist in the peripheral and central nervous system and their activity may be inversely correlated to chronic pain phenotypes in rodents. In this study, we investigated the different KATP channel subunits that could be involved in neuropathic pain in mice. In chronic pain models utilizing spinal nerve ligation, SUR1 and Kir6.2 subunits were found to be significantly downregulated in dorsal root ganglia and the spinal cord. Local or intrathecal administration of SUR1-KATP channel subtype agonists resulted in analgesia after spinal nerve ligation but not SUR2 agonists. In ex-vivo nerve recordings, administration of the SUR1 agonist diazoxide to peripheral nerve terminals decreased mechanically evoked potentials. Genetic knockdown of SUR1 through an associated adenoviral strategy resulted in mechanical hyperalgesia but not thermal hyperalgesia compared to control mice. Behavioral data from neuropathic mice indicate that local reductions in SUR1-subtype KATP channel activity can exacerbate neuropathic pain symptoms. Since neuropathic pain is of major clinical relevance, potassium channels present a target for analgesic therapies, especially since they are expressed in nociceptors and could play an essential role in regulating the excitability of neurons involved in pain-transmission.