Increased dietary fat consumption is associated with colon cancer development. The exact mechanism by which fat induces colon cancer is not clear, however, increased bile acid excretion in response to high-fat diet may promote colon carcinogenesis. The farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily, and bile acids are endogenous ligands of FXR. FXR is highly expressed in the intestine and liver where FXR is essential for maintaining bile acid homeostasis. The role of FXR in intestine cancer development is not known. The current study evaluated the effects of FXR deficiency in mice on intestinal cell proliferation and cancer development. The results showed that FXR deficiency resulted in increased colon cell proliferation, which was accompanied by an up-

Chemokines play a key role in the regulation of central nervous system disease. CXCL10 over-expression has been observed in several neurodegenerative diseases, including multiple sclerosis, Alzheimer's disease and HIV-associated dementia. More recent studies by others and us have shown that CXCL10 elicits apoptosis in fetal neurons. The mechanism of CXCL10-mediated neurotoxicity, however, remains unclear. In this study, we provide evidence for the direct role of Ca(2+) dysregulation in CXCL10-mediated apoptosis. We demonstrate that treatment of fetal neuronal cultures with exogenous CXCL10 produced elevations in intracellular Ca(2+) and that this effect was modulated via the binding of CXCL10 to its cognate receptor, CXCR3. We further explored the association of intracellular Ca(2+) elevations with the caspases that are involved in CXC10-induced neuronal apoptosis. Our data showed that increased Ca(2+), which is available for uptake by the mitochondria, is associated with membrane permeabilization and cytochrome c release from this compartment. The released cytochrome c then activates the initiator active caspase-9. This initiator caspase sequentially activates the effector caspase-3, ultimately leading to apoptosis. This study identifies the temporal signaling cascade involved in CXCL10-mediated neuronal apoptosis and provides putative targets for pharmaceutical intervention of neurological disorders associated with CXCL10 up-regulation.

Inflammatory mediators play a crucial role in the pathophysiology of several neurodegenerative diseases including acquired immune deficiency syndrome dementia complex. In the present study we identified a link between CXCL10 overexpression in the brain and human immunodeficiency virus dementia and demonstrated the presence of the chemokine CXCL10 and its receptor, CXCR3, in the neurons in the brains of macaques with simian human immunodeficiency virus encephalitis. Using human fetal brain cultures, we showed that treatment of these cells with either SHIV89.6P or viral gp120 resulted in induction of CXCL10 in neurons. Cultured neurons treated with the chemokine developed increased membrane permeability followed by apoptosis via activation of caspase-3. We confirmed the relevance of these findings in sections of human and macaque brains with encephalopathy demonstrating that neurons expressing CXCL10 also expressed caspase-3.

Previous studies have shown that the transmembrane (TM) domain of the subtype B Vpu enhances virion release from cells and some studies have shown that this domain may form an oligomeric structure with properties of an ion channel. To date, no studies have been performed to assess the role of this domain in virus pathogenesis in a macaque model of disease. Using a pathogenic molecular clone of simian human immunodeficiency virus (SHIVKU-1bMC33), we have generated a novel virus in which the transmembrane domain of the Vpu protein was scrambled but maintained hydrophobic in nature (SHIVTM), which presumably would disrupt any ion channel TM properties of this protein. Vectors expressing the Vpu as a fusion protein with the enhanced green fluorescent protein (VpuTMEGFP) indicate that it was transported to the same intracellular compartment as the unmodified Vpu protein but did not down-regulate cell surface expression of CD4. To assess the pathogenicity of SHIVTM, three pig-tailed macaques were inoculated with the SHIVTM and monitored for 6-8 months for CD4+ T cell levels, viral loads and the stability of the sequence of the vpu gene. Our results indicated that unlike the parental SHIVKU-1bMC33, inoculation of macaques with SHIVTM did not cause a severe CD4+ T cell loss over the course of their infections. Sequence analysis of the vpu gene analyzed from sequential PBMC samples derived from macaques revealed that the scrambled TM was stable during the course of infection. At necropsy, examination of tissues revealed low viral loads and none of the pathology commonly observed in lymphoid and non-lymphoid tissues following inoculation with the pathogenic parental SHIVKU-1bMC33 virus. Thus, these results show for the first time that the TM domain of Vpu contributes to the pathogenicity of SHIVKU-1bMC33 in pig-tailed macaques.

Osteoarthritis (OA) is the most common form of joint disease in middle-aged and older people. Previous studies have shown that over expression of matrix-degrading proteinases and proinflammatory cytokines is associated with osteoarthritic cartilage degradation. However, it remains unclear which transcription factors regulate the expression of these cartilage-degrading molecules in articular chondrocytes. This study demonstrated that mice lacking Nfat1, a member of the nuclear factor of activated T-cells (NFAT) transcription factors, exhibited normal skeletal development but displayed loss of type-II collagen (collagen-2) and aggrecan with overexpression of specific matrix-degrading proteinases and proinflammatory cytokines in young adult articular cartilage of weight-bearing joints. These initial changes are followed by articular chondrocyte proliferation/clustering, progressive articular surface destruction, periarticular chondro-osteophyte formation, and exposure of thickened subchondral bone, all of which resemble human OA. Forced expression of Nfat1 delivered with lentiviral vectors in cultured 3-month-old primary Nfat1 knockout (Nfat1−/−) articular chondrocytes partially or completely rescued the abnormal catabolic and anabolic activities of Nfat1−/− articular chondrocytes. These new findings revealed a previously unrecognized critical role of Nfat1 in maintaining the physiological function of differentiated adult articular chondrocytes through regulating the expression of specific matrix-degrading proteinases and proinflammatory cytokines. Nfat1 deficiency causes OA due to an imbalance between catabolic and anabolic activities of adult articular chondrocytes, leading to articular cartilage degradation and failed repair activities in and around articular cartilage. These results may provide new insights into the aetiology, pathogenesis and potential therapeutic strategies for osteoarthritis.

Although Francisella tularensis subsp. tularensis is known to cause extensive tissue necrosis, the pathogenesis of tissue injury has not been elucidated. To characterize cell death in tularemia, C57BL/6 mice were challenged by the intranasal route with type A F. tularensis, and the pathological changes in infected tissues were characterized over the next 4 days. At 3 days postinfection, well-organized inflammatory infiltrates developed in the spleen and liver following the spread of infection from the lungs. By the next day, extensive cell death, characterized by the presence of pyknotic cells containing double-strand DNA breaks, was apparent throughout these inflammatory foci. Cell death was not mediated by activated caspase-1, as has been reported for cells infected with other Francisella subspecies. Mouse macrophages and dendritic cells that had been stimulated with type A F. tularensis did not release interleukin-18 in vitro, a response that requires the activation of procaspase-1. Dying cells within type A F. tularensis-infected tissues expressed activated caspase-3 but very little activated caspase-1. When caspase-1-deficient mice were challenged with type A F. tularensis, pathological changes, including extensive cell death, were similar to those seen in infected wild-type mice. In contrast, type A F. tularensis-infected caspase-3-deficient mice showed much less death among their F4/80 ؉ spleen cells than did infected wild-type mice, and they retained the ability to express tumor necrosis factor alpha and inducible NO synthase. These findings suggest that type A F. tularensis induces caspase-3-dependent macrophage apoptosis, resulting in the loss of potentially important innate immune responses to the pathogen.

By animal-to-animal passage of simian/human immunodeficiency virus (SHIV) in pig-tailed macaques, we have developed a macaque model of human immunodeficiency virus type 1 (HIV-1) disease in humans. Passaging was begun with a chimeric virus containing the env gene of HIV-1 HXBc2 and the gag and pol genes of simian immunodeficiency virus SIVmac239. SHIV was passaged serially in cohorts of two macaques each, using bone marrow-to-bone marrow transfers at 5, 5, and 16 weeks for passages 2, 3, and 4, respectively. The fifth passage was done by using cell-free virus isolated from cerebrospinal fluid of a passage 4 macaque. The virus became more virulent with each passage. Virus replication was restricted in all three animals in passages 1 and 2 but not in five of the six animals in passages 3, 4, and 5. In these animals, intense virus replication in the lymphoid tissues resulted in almost total elimination of CD4 ؉ T cells within weeks of inoculation, and three of these animals developed AIDS in less than 1 year. The more uniform virus-host interaction initiated by the cell-free virus in the passage 5 animals contrasted with a more variable pattern of disease initiated by infectious bone marrow cells during earlier passages. The virulent cell-free SHIV can now be used to screen the efficacy of vaccines directed against the envelope of HIV-1. Human immunodeficiency virus type 1 (HIV-1) can infect nonhuman primates such as chimpanzees but does not cause disease in these animals (16). This is a major impediment to understanding the mechanisms of pathogenesis of HIV-1 infection and has also retarded efforts to evaluate the efficacy of anti-HIV drugs and vaccines. At present, simian immunodeficiency virus SIVmac infection in macaques is the best-characterized model of HIV-1 disease (3). HIV-2 infection in baboons represents another useful model (1). SIVmac and HIV-2 are closely related viruses, but both have genetic, antigenic, and structural features in their env genes that clearly distinguish them from HIV-1. Chimeric simian/human immunodeficiency viruses (SHIVs) that bear the envelope of HIV-1 and are infectious in macaques potentially offer a solution to this problem. However, SHIVs have so far proven avirulent in macaques (2, 9, 13, 15, 17). Since the bone marrow (BM) of SIVmac-infected macaques has high virus burdens (7) and since specific pathogenic viral genotypes can be selected by serial passage of infectious BM in animals (14), we hypothesized that similar passaging of SHIV-infected BM cells would also result in selection of pathogenic genotypes of this virus. We report here on derivation of a pathogenic SHIV by using this procedure and the initial characterization of the disease process caused by this virus. MATERIALS AND METHODS Passage in macaques. SHIV, obtained as described below, was used to begin passaging in young (7 to 19 months old) rhesus (Macaca mulatta) and pig-tailed (Macaca nemestrina) macaques (Fig. 1). Macaques were housed in American Association for Laboratory Animal Care-accredited facilities an...

This study highlights the possible pathological role of MMP-12 in the context of ischemic stroke. Male rats were subjected to a two-hour middle cerebral artery occlusion (MCAO) procedure. MMP-12 shRNA expressing plasmid formulation was administered to these rats twenty-four hours after reperfusion. The results showed a predominant upregulation of MMP-12 (approximately 47, 58, 143, and 265 folds on days 1, 3, 5, 7 post-ischemia, respectively) in MCAO subjected rats. MMP-12 expression was localized to neurons, oligodendrocytes and microglia, but not astrocytes. Transcriptional inactivation of MMP-12 significantly reduced the infarct size. The percent infarct size was reduced from 62.87 ± 4.13 to 34.67 ± 5.39 after MMP-12 knockdown compared to untreated MCAO subjected rats. Expression of myelin basic protein was increased, and activity of MMP-9 was reduced in ischemic rat brains after MMP-12 knockdown. Furthermore, a significant reduction in the extent of apoptosis was noticed after MMP-12 knockdown. TNFα expression in the ipsilateral regions of MCAO-subjected rats was reduced after MMP-12 knockdown in addition to the reduced protein expression of apoptotic molecules that are downstream to TNFα signaling. Specific knockdown of MMP-12 after focal cerebral ischemia offers neuroprotection that could be mediated via reduced MMP-9 activation and myelin degradation as well as inhibition of apoptosis.