Novel method to quantify phenotypic markers of HIV-associated neurocognitive disorder in a murine SCID model.

Despite combined antiretroviral therapy (cART), HIV infection in the CNS persists with reported increases in activation of macrophages (MΦ), microglia, and surrounding astrocytes/neurons, conferring HIV-induced inflammation. Chronic inflammation results in HIV-associated neurocognitive disorders (HAND) with reported occurrence of up to half of individuals with HIV infection. The existing HAND mouse model used by laboratories including ours, and the effect of novel agents on its pathology present with labor-intensive and time-consuming limitations since brain sections and immunohistochemistry assays have to be performed and analyzed. A novel flow cytometry-based system to objectively quantify phenotypic effects of HIV using a SCID mouse HAND model was developed which demonstrated that the HIV-infected mice had significant increases in astrogliosis, loss of neuronal dendritic marker, activation of murine microglia, and human macrophage explants compared to uninfected control mice. HIV p24 could also be quantified in the brains of the infected mice. Correlation of these impairments with HIV-induced brain inflammation and previous behavioral abnormalities studies in mice suggests that this model can be used as a fast and relevant throughput methodology to quantify preclinical testing of novel treatments for HAND.

A mouse model of HIV-associated neurocognitive disorders: a brain-behavior approach to discover disease mechanisms and novel treatments.

HIV-associated neurocognitive disorders (HAND) remain highly prevalent despite combined antiretroviral therapy (cART). Although the most common forms of HAND are mild and identified through neuropsychological testing, there is evidence that with aging these mild forms become more prevalent and may advance to the most severe form of HAND, HIV-associated dementia. Therefore, novel therapies must be developed that can be used adjunctively with cART to prevent deterioration or restore normal cognitive function. In order to develop innovative treatments, animal models are used for preclinical testing. Ideally, a HAND animal model should portray similar mild cognitive deficits that are found in humans. A mouse model of HAND is discussed, which demonstrates mild behavioral deficits and has been used to investigate cART and novel treatments for HAND. This model also shows correlations between abnormal mouse behavior due to HIV in the brain and pathological parameters such as gliosis and neuronal abnormalities. A recent advancement utilizes the object recognition test to monitor mouse behavior before and after treatment. It is postulated that this model is well suited for preclinical testing of novel therapies and provides correlations of mild cognitive impairment with pathological markers that can give further insight into the pathophysiology of HAND.

Peroxisome proliferator-activated receptor-γ agonists attenuate biofilm formation by Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a significant contributor to recalcitrant multidrug-resistant infections, especially in immunocompromised and hospitalized patients. The pathogenic profile of P. aeruginosa is related to its ability to secrete a variety of virulence factors and to promote biofilm formation. Quorum sensing (QS) is a mechanism wherein P. aeruginosa secretes small diffusible molecules, specifically acyl homo serine lactones, such as N-(3-oxo-dodecanoyl)-l-homoserine lactone (3O-C12-HSL), that promote biofilm formation and virulence via interbacterial communication. Strategies that strengthen the host's ability to inhibit bacterial virulence would enhance host defenses and improve the treatment of resistant infections. We have recently shown that peroxisome proliferator-activated receptor γ (PPARγ) agonists are potent immunostimulators that play a pivotal role in host response to virulent P. aeruginosa Here, we show that QS genes in P. aeruginosa (strain PAO1) and 3O-C12-HSL attenuate PPARγ expression in bronchial epithelial cells. PAO1 and 3O-C12-HSL induce barrier derangements in bronchial epithelial cells by lowering the expression of junctional proteins, such as zonula occludens-1, occludin, and claudin-4. Expression of these proteins was restored in cells that were treated with pioglitazone, a PPARγ agonist, before infection with PAO1 and 3O-C12-HSL. Barrier function and bacterial permeation studies that have been performed in primary human epithelial cells showed that PPARγ agonists are able to restore barrier integrity and function that are disrupted by PAO1 and 3O-C12-HSL. Mechanistically, we show that these effects are dependent on the induction of paraoxonase-2, a QS hydrolyzing enzyme, that mitigates the effects of QS molecules. Importantly, our data show that pioglitazone, a PPARγ agonist, significantly inhibits biofilm formation on epithelial cells by a mechanism that is mediated via paraoxonase-2. These findings elucidate a novel role for PPARγ in host defense against P. aeruginosa Strategies that activate PPARγ can provide a therapeutic complement for treatment of resistant P. aeruginosa infections.-Bedi, B., Maurice, N. M., Ciavatta, V. T., Lynn, K. S., Yuan, Z., Molina, S. A., Joo, M., Tyor, W. R., Goldberg, J. B., Koval, M., Hart, C. M., Sadikot, R. T. Peroxisome proliferator-activated receptor-γ agonists attenuate biofilm formation by Pseudomonas aeruginosa.

Update on disease-modifying therapies for multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune, demyelinating disease of the central nervous system (CNS). It predominantly affects young women and is one of the most common causes of disability in young adults. MS is characterized by formation of white matter lesions in the CNS as a result of inflammation, demyelination, and axonal loss. Treatment has been a focus of neurological research for over 60 years. A number of disease-modifying therapies (DMTs) have become available making MS a treatable disease. These compounds target the inflammatory response in MS. They work by decreasing the chances of relapse, decreasing the chances of new lesion formation seen on MRI of the CNS and slowing the accumulation of disability. The first drugs for MS to be available were interferon-ß and glatiramer acetate. These work by modulating the inflammatory response via different mechanisms that are briefly discussed. Newer agents have since become available and have significantly changed the dynamics of MS treatment. These include fingolimod, dimethyl fumarate and teriflunomide, which are oral agents. Other second-line and third-line Food and Drug Administration (FDA) approved medications include natalizumab and alemtuzumab. Natalizumab is considered one of the most potent treatments for relapse prevention. However, the high risk of progressive multifocal leukoencephalopathy (PML), which is caused by JC virus infection in the brain, tempers the more widespread use of this agent; nevertheless, JC virus antibody tests have helped to stratify the risk of PML. Alemtuzumab, which also has a considerable side effect profile, is likewise highly efficacious. Ocrelizumab, a monoclonal antibody to CD20 on B cells, is a highly effective agent for MS that is likely to be approved soon by the FDA. MS is a major contributor to healthcare costs and it is critical that healthcare providers be aware of the availability and benefits of DMTs. It is imperative that prompt and adequate treatment be established on diagnosis. Changes in therapy should be considered when there is evidence of disease activity as well as accumulation of disability or safety or tolerability concerns.

The Janus kinase inhibitor ruxolitinib reduces HIV replication in human macrophages and ameliorates HIV encephalitis in a murine model.

A hallmark of persistent HIV-1 infection in the central nervous system is increased activation of mononuclear phagocytes and surrounding astrogliosis, conferring persistent HIV-induced inflammation. This inflammation is believed to result in neuronal dysfunction and the clinical manifestations of HIV-associated neurocognitive disorders (HAND). The Jak/STAT pathway is activated in macrophages/myeloid cells upon HIV-1 infection, modulating many pro-inflammatory pathways that result in HAND, thereby representing an attractive cellular target. Thus, the impact of ruxolitinib, a Janus Kinase (Jak) 1/2 inhibitor that is FDA approved for myelofibrosis and polycythemia vera, was assessed for its potential to inhibit HIV-1 replication in macrophages and HIV-induced activation in monocytes/macrophages in culture. In addition, a murine model of HIV encephalitis (HIVE) was used to assess the impact of ruxolitinib on histopathological features of HIVE, brain viral load, as well as its ability to penetrate the blood-brain-barrier (BBB). Ruxolitinib was found to inhibit HIV-1 replication in macrophages, HIV-induced activation of monocytes (CD14/CD16) and macrophages (HLA-DR, CCR5, and CD163) without apparent toxicity. In vivo, systemically administered ruxolitinib was detected in the brain during HIVE in SCID mice and markedly inhibited astrogliosis. Together, these data indicate that ruxolitinib reduces HIV-induced activation and infiltration of monocytes/macrophages in vitro, reduces the replication of HIV in vitro, penetrates the BBB when systemically administered in mice and reduces astrogliosis in the brains of mice with HIVE. These data suggest that ruxolitinib will be useful as a novel therapeutic to treat humans with HAND.

Morphine exposure during HIV encephalitis in SCID mice.

HIV encephalitis (HIVE) is often complicated by opiate abuse. Based on human pathological, animal and in vitro studies, opiates are thought to exacerbate HIVE. To test this hypothesis we exposed 10 week old SCID mice with HIVE to morphine and examined histopathological parameters. Mice inoculated intracerebrally with either HIV-infected or uninfected (control mice) human macrophages were immediately implanted subcutaneously with pellets containing saline, morphine or morphine plus naltrexone. They were sacrificed after 10 days. Immunostaining for astrocytes (GFAP), mouse mononuclear phagocytes (CD45) and neuronal dendrites (MAP2) was analyzed by densitometry. HIVE mice exposed to either saline, morphine or morphine plus naltrexone also had brain sections counted for HIV+ human macrophages. Typical HIVE pathology was present, consistent with previously published studies. Surprisingly, there were no effects on astrogliosis, microgliosis and MAP2 decreases in the HIVE, morphine treated group. There was also no effect of morphine exposure on numbers of p24+ human macrophages. These results emphasize the complexities of modeling opiate effects in HIVE and the potential significance of opiate abuse on HIVE in humans.

Cognitive dysfunction in HIV encephalitic SCID mice correlates with levels of Interferon-alpha in the brain.

BACKGROUND: Interferon alpha (IFNalpha) is an antiviral cytokine produced in response to viral infection. IFNalpha also acts as a neuromodulatory molecule in the central nervous system (CNS). Elevated IFNalpha in the CNS causes cognitive deficits. OBJECTIVE: To determine if elevated levels of IFNalpha in an HIV encephalitis mouse model correlate with cognitive deficits. METHODS: C57BL/6J SCID mice were inoculated intracerebrally (i.c.) with HIV infected or uninfected (control) macrophages and cognitively tested in a water escape radial arm maze. After behavioral testing was completed, immunohistochemistry and ELISA were used to examine brain pathology and IFNalpha expression. RESULTS: Mice injected i.c. with HIV infected macrophages exhibited significantly more working memory errors, particularly in trials with the highest memory load. Immunohistochemistry indicated increased mouse IFNalpha staining prevalent on neurons and glial cells in the brains of mice with HIV infected macrophages compared to mice with uninfected control macrophages. In addition, IFNalpha levels in the brain correlated directly with working memory errors for mice with HIV infected macrophages. CONCLUSIONS: These data suggest that the cognitive deficit noted for the C57BL/6J SCID mice with HIV infected macrophages is mediated by the infection induced increase in IFNalpha.

Highly active antiretroviral therapy of cognitive dysfunction and neuronal abnormalities in SCID mice with HIV encephalitis.

Our objective was to determine if highly active antiretroviral therapy (HAART), previously shown to ameliorate several pathological features of HIV encephalitis (HIVE) in a SCID mouse model, would also reduce additional established pathological features of HIV: cognitive dysfunction, TNF-alpha, production, and reduced MAP-2 expression. SCID mice with HIVE and control mice inoculated with uninfected monocytes were administered HAART or saline. The HIV pathological features evaluated included astrogliosis, viral load, neuronal apoptosis, MAP-2 expression, mouse TNF-alpha mRNA production and learning acquisition and retention. HAART reduced the HIV-induced viral load, and the astro- and microgliosis as previously observed; this effect was extended to HIV-induced increases in TNF-alpha mRNA production. In contrast, although HIV produced the cognitive deficits previously observed and also decreased MAP-2 expression in the area surrounding the injected HIV-infected human monocytes, HAART did not attenuate these effects. Interestingly, there was no neuronal apoptosis evident at the time point reflecting the above pathology. The results of this study combined with previous reports indicate that HAART reduces TNF-alpha mRNA, viral load and astrogliosis; however, HAART does not improve HIV-induced cognitive dysfunction or MAP-2 decreases. These results suggest that viral load, astrogliosis, TNF- alpha and apoptosis are not prominent in the pathogenesis of early functional deficits related to decreased MAP-2 expression or cognitive dysfunction in HIVE in SCID mice.

Treatment of spinal cord impact injury in the rat with transforming growth factor-beta.

To investigate the contribution of cytokines, proinflammatory TNF-alpha and inhibitory TGF-beta, to spinal cord injury (SCI) in a rat model, two studies were performed using adult Sprague-Dawley rats which were injured at T9/T10. In the first study, rats were sacrificed at 1, 6, 24, 96 and 168 h after SCI for immunocytochemistry of coronal sections for the presence of mononuclear phagocytes, astrocytes, TNF-alpha and TGF-beta, among other markers. From intervening frozen sections, RNA was extracted for semiquantitative polymerase chain reaction (RT-PCR) analysis of TNF-alpha and TGF-beta. In the second experiment, rats were treated with intravenous TGF-beta 30 min after injury and sacrificed at 6 and 48 h after injury. Spinal cord sections were immunocytochemically stained and RNA extracted for semiquantitative PCR as mentioned above, as well as quantitation of lesion volume. There were increases in mononuclear phagocytes and astrocytes, as early as 1 h after SCI, with steady progression over 168 h after injury. TNF-alpha and TGF-beta was produced locally by mononuclear phagocytes and astrocytes. There was an 18-h delay in peak mRNA production of TGF-beta compared to TNF-alpha. The treatment of SCI rats with TGF-beta reduced lesion volume by 50% at 48 h and this was associated with decreased accumulation of mononuclear phagocytes in and around the injury site. This reduction of mononuclear phagocyte numbers around the site of trauma would reduce their contribution to secondary injury.