CD38 regulation in activated astrocytes: implications for neuroinflammation and HIV-1 brain infection.

Reactive astrogliosis is a key pathological aspect of neuroinflammatory disorders including human immunodeficiency virus type 1 (HIV-1)-associated neurological disease. On the basis of previous data that showedastrocytes activated with interleukin (IL)-1beta induce neuronal injury, we analyzed global gene changes in IL-1beta-activated human astrocytes by gene microarray. Among the up-regulated genes, CD38, a 45-kDa type II single chain transmembrane glycoprotein, was a top candidate, with a 17.24-fold change that was validated by real-time polymerase chain reaction. Key functions of CD38 include enzymatic activities and involvement in adhesion and cell signaling. Importantly, CD38(+)CD8(+) T-cell expression is a clinical correlate for progression of HIV-1 infection and biological marker for immune activation. Thus, CD38 expression in HIV-1 and/or IL-1beta-stimulated human astrocytes and human brain tissues was analyzed. IL-1beta and HIV-1 activation of astrocytes enhanced CD38 mRNA levels. Both CD38 immunoreactivity and adenosine 5'-diphosphate (ADP)-ribosyl cyclase activity were up-regulated in IL-1beta-activated astrocytes. CD38 knockdown using specific siRNAs significantly reduced astrocyte proinflammatory cytokine and chemokine production. However, CD38 mRNA levels were unchanged in IL-1beta knockdown conditions, suggesting that IL-1beta autocrine loop is not implicated in this process. Quantitative immunohistochemical analysis of HIV-seropositive without encephalitis and HIV-1 encephalitis brain tissues showed significant up-regulation of CD38, which colocalized with glial fibrillary acidic protein-positive cells in areas of inflammation. These results suggest an important role of CD38 in the regulation of astrocyte dysfunction during the neuroinflammatory processes involved in neurodegenerative/neuroinflammatory disorders such as HIV-1 encephalitis.

Evaluation of the efficacy of a canine influenza virus (H3N8) vaccine in dogs following experimental challenge.

Canine influenza virus (CIV) subtype H3N8 is an emerging pathogen with sustained horizontal transmission in the dog population in the United States. This study evaluated the efficacy of an inactivated CIV vaccine in 6- to 8-week-old beagle pups challenged with virulent CIV. One group of CIV-seronegative pups was vaccinated with two doses of a CIV vaccine 3 weeks apart; a second group of pups received adjuvanted placebo as a control. Blood samples were collected at various times to determine antibody titers. All pups were challenged with a virulent CIV isolate 13 days after the second vaccination and monitored for clinical signs of respiratory disease, virus shedding, and lung consolidation. Vaccinated pups developed hemagglutination inhibition antibody titers after vaccination. The severity of clinical signs (P < .001) and the magnitude and duration of virus shedding (P < .0001) were significantly lower in vaccinated pups compared with control pups. These results demonstrate that the CIV vaccine used in this study provides protection against virulent CIV challenge in dogs.

Efficacy of the canine influenza virus H3N8 vaccine to decrease severity of clinical disease after cochallenge with canine influenza virus and Streptococcus equi subsp. zooepidemicus.

Since first emerging in the North American canine population in 2004, canine influenza virus (CIV) subtype H3N8 has shown horizontal transmission among dogs, with a high level of adaptation to this species. The severity of disease is variable, and coinfection by other respiratory pathogens is an important factor in the degree of morbidity and mortality. The first influenza vaccine for dogs, an inactivated vaccine containing CIV subtype H3N8, was conditionally approved by the U.S. Department of Agriculture (USDA) for licensure in May 2009 and fully licensed in June 2010. This study evaluates the efficacy of this vaccine to reduce the severity of illness in dogs cochallenged with virulent CIV and Streptococcus equi subsp. zooepidemicus.

Transmission of canine influenza virus (H3N8) among susceptible dogs.

Canine influenza virus (CIV) is an emerging pathogen that causes acute respiratory disease in dogs. As with any communicable disease, dog-to-dog transmission of CIV occurs when infected dogs come in contact with other susceptible dogs. We demonstrate that CIV transmission occurs readily from CIV-infected dogs to susceptible dogs following co-mingling. Four experimentally infected dogs were co-mingled with a group of eight CIV-negative dogs at 1 day post-infection and both groups were observed for CIV-associated respiratory disease. The onset of clinical signs, virus shedding, seroconversion, and appearance of lung lesions were observed earlier in experimentally infected dogs; however, the severity of the clinical signs and lung lesions were very similar in both groups. One hundred percent of the experimentally infected dogs and 75% of the contact-exposed dogs excreted virus in their nasal secretions. Additionally, 100% of experimentally infected dogs and 75% of the contact-exposed dogs exhibited varying degrees of pneumonia. Our study results demonstrate that CIV spreads readily from infected dogs to other susceptible dogs through direct contact.

Potential mechanisms for astrocyte-TIMP-1 downregulation in chronic inflammatory diseases.

The pathogenesis of many neurodegenerative disorders, including human immunodeficiency virus (HIV)-1 associated dementia, is exacerbated by an imbalance between matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of metalloproteinases (TIMPs). In the context of disease, TIMP-1 has emerged as an important multifunctional protein capable of regulating inflammation. We previously reported differential TIMP-1 expression in acute versus chronic activation of astrocytes. This study investigates possible mechanisms underlying TIMP-1 downregulation in chronic neuroinflammation. We used interleukin (IL)-1beta as a model pro-inflammatory stimulus and measured TIMP-1 binding to extracellular matrix, cell death, receptor downregulation, TIMP-1 mRNA stability and transcriptional regulation in activated astrocytes. TIMP-1 remained localized to the cell body or was secreted into the cell supernatant. DNA fragmentation ELISA and MTT assay showed that prolonged IL-1beta activation of astrocytes induced significant astrocyte death. In acute and chronic IL-1beta-activated astrocytes, IL-1 receptor levels were not significantly different. TIMP-1 mRNA stability was measured in astrocytes and U87 astroglioma cells by real-time PCR, and TIMP-1 promoter activation was studied using TIMP-1-luciferase reporter constructs in transfected astrocytes. Our results indicated that TIMP-1 expression is regulated through multiple mechanisms. Transcriptional control and loss of mRNA stabilization are, however, the most likely primary contributors to chronic downregulation of TIMP-1. These data are important for unraveling the mechanisms underlying astrocyte responses during chronic neuroinflammation and have broader implications in other inflammatory diseases that involve MMP/TIMP imbalance.

Induction of cytotoxic T-lymphocytes specific for bovine herpesvirus-1 by DNA immunization.

Cytotoxic T-lymphocytes (CTLs) are critical for the defense against herpesvirus infections, in which cell-to-cell spread occurs earlier than the hematogenous spread. The ability of bovine herpesvirus-1 (BHV-1) to undergo latency, to induce apoptosis of CD4(+) T-lymphocytes, and to down-regulate the expression of major histocompatibility complex (MHC) class I molecules, necessitates the development of immunization strategies that do not involve the live virus. The objective of this study was to evaluate the feasibility of DNA immunization as a means of induction of CTLs against BHV-1. Mice were injected either by intramuscular (IM) or intradermal (ID) route with a Sindbis virus-based plasmid carrying the gene encoding the glycoprotein D (gD) of BHV-1. Splenocytes from the immunized mice were re-stimulated in vitro with gD-transduced syngeneic fibroblasts. The CTLs generated specifically lysed syngeneic targets, either transduced with gD or infected with BHV-1. IM route of inoculation induced a better CTL response when compared to ID route with respect to onset, magnitude and duration of immunity. These results indicate the feasibility of using a plasmid carrying the gene encoding BHV-1 gD as an immunogen to induce CTLs against BHV-1.