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HIV-1–associated nephropathy (HIVAN) is a severe complication of HIV-1 infection. To gain insight into the pathogenesis of kidney disease in the setting of HIV, a transgenic (Tg) mouse model [CD4C/HIV-negative regulator factor (Nef)] was used in which HIV-1 nefexpression is under control of regulatory sequences (CD4C) of the human CD4gene, thus allowing expression in target cells of the virus. These Tg mice develop a collapsing focal segmental glomerulosclerosis associated with microcystic dilatation, similar to human HIVAN. To identify kidney cells permissive to the CD4C promoter, CD4C reporter Tg lines were used. They showed preferential expression in glomeruli, mainly in mesangial cells. Breeding CD4C/HIV Tg mice on 10 different mouse backgrounds showed that HIVAN was modulated by host genetic factors. Studies of gene-deficient Tg mice revealed that the presence of B and T cells and that of several genes was dispensable for the development of HIVAN: those involved in apoptosis (Trp53, Tnfsf10, Tnf, Tnfrsf1b, and Bax), in immune cell recruitment (Ccl3, Ccl2, Ccr2, Ccr5, and Cx3cr1), in nitric oxide (NO) formation (Nos3and Nos2), or in cell signaling (Fyn, Lck, and Hck/Fgr). However, deletion of Srcpartially and that of Hck/Lynlargely abrogated its development. These data suggest that Nef expression in mesangial cells through hematopoietic cell kinase (Hck)/Lck/Yes novel tyrosine kinase (Lyn) represents important cellular and molecular events for the development of HIVAN in these Tg mice.

Macrophages are motile leukocytes, targeted by HIV-1, thought to play a critical role in host dissemination of the virus. However, whether infection impacts their migration capacity remains unknown. We show that 2-dimensional migration and the 3-dimensional (3D) amoeboid migration mode of HIV-1–infected human monocyte-derived macrophages were inhibited, whereas the 3D mesenchymal migration was enhanced. The viral protein Nef was necessary and sufficient for all HIV-1–mediated effects on migration. In Nef transgenic mice, tissue infiltration of macrophages was increased in a tumor model and in several tissues at steady state, suggesting a dominant role for mesenchymal migration in vivo. The mesenchymal motility involves matrix proteolysis and podosomes, cell structures constitutive of monocyte-derived cells. Focusing on the mechanisms used by HIV-1 Nef to control the mesenchymal migration, we show that the stability, size, and proteolytic function of podosomes are increased via the phagocyte-specific kinase Hck and Wiskott-Aldrich syndrome protein (WASP), 2 major regulators of podosomes. In conclusion, HIV-1 reprograms macrophage migration, which likely explains macrophage accumulation in several patient tissues, which is a key step for virus spreading and pathogenesis. Moreover, Nef points out podosomes and the Hck/WASP signaling pathway as good candidates to control tissue infiltration of macrophages, a detrimental phenomenon in several diseases.

Macrophages are motile leukocytes, targeted by HIV-1, thought to play a critical role in host dissemination of the virus. However, whether infection impacts their migration capacity remains unknown. We show that 2-dimensional migration and the 3-dimensional (3D) amoeboid migration mode of HIV-1–infected human monocyte-derived macrophages were inhibited, whereas the 3D mesenchymal migration was enhanced. The viral protein Nef was necessary and sufficient for all HIV-1–mediated effects on migration. In Nef transgenic mice, tissue infiltration of macrophages was increased in a tumor model and in several tissues at steady state, suggesting a dominant role for mesenchymal migration in vivo. The mesenchymal motility involves matrix proteolysis and podosomes, cell structures constitutive of monocyte-derived cells. Focusing on the mechanisms used by HIV-1 Nef to control the mesenchymal migration, we show that the stability, size, and proteolytic function of podosomes are increased via the phagocyte-specific kinase Hck and Wiskott-Aldrich syndrome protein (WASP), 2 major regulators of podosomes. In conclusion, HIV-1 reprograms macrophage migration, which likely explains macrophage accumulation in several patient tissues, which is a key step for virus spreading and pathogenesis. Moreover, Nef points out podosomes and the Hck/WASP signaling pathway as good candidates to control tissue infiltration of macrophages, a detrimental phenomenon in several diseases.

ABSTRACTCryptococcus neoformansvar. grubiiis the most frequent cause of AIDS-associated cryptococcosis worldwide, while Cryptococcus gattiiusually infects immunocompetent people. To understand the mechanisms which cause differential susceptibility to these cryptococcal species in HIV infection, we established and characterized a model of cryptococcosis in CD4C/HIVMutAtransgenic (Tg) mice expressing gene products of HIV-1 and developing an AIDS-like disease. Tg mice infected intranasally with C. neoformansvar. grubiistrain H99 or C23 consistently displayed reduced survival compared to non-Tg mice at three graded inocula, while shortened survival of Tg mice infected with C. gattiistrain R265 or R272 was restricted to a single high inoculum. HIV-1 transgene expression selectively augmented systemic dissemination to the liver and spleen for strains H99 and C23 but not strains R265 and R272. Histopathologic examination of lungs of Tg mice revealed large numbers of widely scattered H99 cells, with a minimal inflammatory cell response, while in the non-Tg mice H99 was almost completely embedded within extensive mixed inflammatory cell infiltrates. In contrast to H99, R265 was dispersed throughout the lung parenchyma and failed to induce a strong inflammatory response in both Tg and non-Tg mice. HIV-1 transgene expression reduced pulmonary production of CCL2 and CCL5 after infection with H99 or R265, and production of these two chemokines was lower after infection with R265. These results indicate that an altered immune response in these Tg mice markedly enhances C. neoformansbut not C. gattiiinfection. This model therefore provides a powerful new tool to further investigate the immunopathogenesis of cryptococcosis.

CD4C/HIVNef Tg mice express HIV-1 Nef in relevant target cells through the regulatory sequences of the human CD4 gene. These mice develop a severe disease showing many characteristics of human AIDS. A summary of the results obtained with these Tg mice is presented.

The Nef protein of HIV-1 is important for AIDS pathogenesis, but it is not targeted by current antiviral strategies. Here, we describe a single-domain antibody (sdAb) that binds to HIV-1 Nef with a high affinity (Kd = 2 × 10−9M) and inhibited critical biologic activities of Nef both in vitro and in vivo. First, it interfered with the CD4 down-regulation activity of a broad panel of nef alleles through inhibition of the Nef effects on CD4 internalization from the cell surface. Second, it was able to interfere with the association of Nef with the cellular p21-activated kinase 2 as well as with the resulting inhibitory effect of Nef on actin remodeling. Third, it counteracted the Nef-dependent enhancement of virion infectivity and inhibited the positive effect of Nef on virus replication in peripheral blood mononuclear cells. Fourth, anti-Nef sdAb rescued Nef-mediated thymic CD4+ T-cell maturation defects and peripheral CD4+ T-cell activation in the CD4C/HIV-1Nef transgenic mouse model. Because all these Nef functions have been implicated in Nef effects on pathogenesis, this anti-Nef sdAb may represent an efficient tool to elucidate the molecular functions of Nef in the virus life cycle and could now help to develop new strategies for the control of AIDS.

ABSTRACTThe critical impairments of innate and adaptive immunity that cause susceptibility to mucosal candidiasis in human immunodeficiency virus (HIV) infection have not been fully determined. We therefore conducted an analysis of macrophage-mediated responses to Candida albicansin transgenic (Tg) mice expressing Nef, Env, and Rev of HIV type 1 (HIV-1) in CD4+T cells, dendritic cells, and macrophages and developing an AIDS-like disease (CD4C/HIVMutATg mice). Macrophages were successfully recruited to the oral and gastric mucosae of these Tg mice in response to chronic carriage of C. albicansand displayed polarization toward an alternatively activated phenotype. Functionally, peritoneal macrophages from uninfected Tg mice exhibited increased phagocytosis of C. albicansand enhanced production of interleukin 6 and monocyte chemoattractant protein 1, demonstrating that the HIV-1 transgene independently activates selected macrophage functions. Production of H2O2by macrophages from Tg mice primed with gamma interferon and treated with phorbol 12-myristate 13-acetate or C. albicanswas moderately reduced, but expression of the HIV-1 transgene did not alter production of nitric oxide or reduce killing of C. albicans. A knockout of the inducible nitric oxide synthase (NOS2) gene in these Tg mice did not augment oral or gastrointestinal burdens during chronic carriage of C. albicansor cause systemic dissemination, likely due to a redundancy provided by partially preserved production of H2O2and oxygen-independent candidacidal mechanisms. Thus, the macrophage response to C. albicansis largely preserved in these Tg mice, and no functional macrophage defect appears to primarily determine the susceptibility to mucosal candidiasis.

The critical impairments of innate and adaptive immunity that cause susceptibility to mucosal candidiasis in human immunodeficiency virus (HIV) infection have not been fully determined. We therefore conducted an analysis of macrophage-mediated responses to Candida albicans in transgenic (Tg) mice expressing Nef, Env, and Rev of HIV type 1 (HIV-1) in CD4+T cells, dendritic cells, and macrophages and developing an AIDS-like disease (CD4C/HIVMutATg mice). Macrophages were successfully recruited to the oral and gastric mucosae of these Tg mice in response to chronic carriage of C. albicans and displayed polarization toward an alternatively activated phenotype. Functionally, peritoneal macrophages from uninfected Tg mice exhibited increased phagocytosis of C. albicans and enhanced production of interleukin 6 and monocyte chemoattractant protein 1, demonstrating that the HIV-1 transgene independently activates selected macrophage functions. Production of H2O2by macrophages from Tg mice primed with gamma interferon and treated with phorbol 12-myristate 13-acetate or C. albicans was moderately reduced, but expression of the HIV-1 transgene did not alter production of nitric oxide or reduce killing of C. albicans. A knockout of the inducible nitric oxide synthase (NOS2) gene in these Tg mice did not augment oral or gastrointestinal burdens during chronic carriage of C. albicans or cause systemic dissemination, likely due to a redundancy provided by partially preserved production of H2O2and oxygen-independent candidacidal mechanisms. Thus, the macrophage response to C. albicans is largely preserved in these Tg mice, and no functional macrophage defect appears to primarily determine the susceptibility to mucosal candidiasis.

The North is of great importance to the future of our planet. Climate change and the accelerating development of northern resources are putting unprecedented pressure on the land and the people living on it. If done in a sustainable way, this devel¬opment will contribute to the quality of life of northern communities and, indirectly, to the well-being of all Arctic circumpolar countries. A high- quality, accurate and up-to-date geospatial data infrastructure is increasingly being considered as an essential component upon which can be built a more complete knowledge-based infrastructure to support exploration, land management, impact-assessment studies, security issues and, down the road, long-term sustainable development. The core of this paper will be around the Canadian Northern Geospatial Infrastructure and how it is supporting northern development. Available information and ongoing developments to better use new technologies like Earth-observation data, to upgrade and complement this infrastructure through time, will be presented.

We previously reported that truncation of Notch1(N1) by provirus insertion leads to overexpression of both the intracellular (N1IC) and the extracellular (N1EC) domains. We produced transgenic (Tg) mice expressing N1ECin T cells and in cells of the myeloid lineage under the regulation of the CD4 gene. These CD4C/N1ECTg mice developed vascular disease, predominantly in the liver: superficial distorted vessels, cavernae, lower branching of parenchymal vessels, capillarized sinusoids, and aberrant smooth muscle/endothelial cell topography. The disease developed in lethally irradiated normal mice transplanted with Tg bone marrow or fetal liver cells as well as in Rag−/−Tg mice. In nude mice transplanted with fetal liver cells from (ROSA26 × CD4C/N1EC) F1Tg mice, abnormal vessels were of recipient origin. Transplantation of Tg peritoneal macrophages into normal recipients also induced abnormal vessels. These Tg macrophages showed impaired functions, and their conditioned medium inhibited the proliferation of liver sinusoid endothelial cells in vitro. The Egr-1gene and some of its targets (Jag1, FIII, FXIII-A, MCP-1, and MCP-5), previously implicated in hemangioma or vascular malformations, were overexpressed in Tg macrophages. These results show that myeloid cells can be reprogrammed by N1ECto induce vascular malformations through a paracrine pathway.

Candida albicans causes oropharyngeal candidiasis (OPC) but rarely disseminates to deep organs in human immunodeficiency virus (HIV) infection. Here, we used a model of OPC in CD4C/HIVMut transgenic (Tg) mice to investigate the role of polymorphonuclear leukocytes (PMNs) and CD8+ T cells in limiting candidiasis to the mucosa. Numbers of circulating PMNs and their oxidative burst were both augmented in CD4C/HIVMutA Tg mice expressing rev, env, and nef of HIV type 1 (HIV-1), while phagocytosis and killing of C. albicans were largely unimpaired compared to those in non-Tg mice. Depletion of PMNs in these Tg mice did not alter oral or gastrointestinal burdens of C. albicans or cause systemic dissemination. However, oral burdens of C. albicans were increased in CD4C/HIVMutG Tg mice expressing only the nef gene of HIV-1 and bred on a CD8 gene-deficient background (CD8–/–), compared to control or heterozygous CD8+/– CD4C/HIVMutG Tg mice. Thus, CD8+ T cells contribute to the host defense against oral candidiasis in vivo, specifically in the context of nef expression in a subset of immune cells.

ABSTRACTCandida albicanscauses oropharyngeal candidiasis (OPC) but rarely disseminates to deep organs in human immunodeficiency virus (HIV) infection. Here, we used a model of OPC in CD4C/HIVMuttransgenic (Tg) mice to investigate the role of polymorphonuclear leukocytes (PMNs) and CD8+T cells in limiting candidiasis to the mucosa. Numbers of circulating PMNs and their oxidative burst were both augmented in CD4C/HIVMutATg mice expressing rev, env, and nefof HIV type 1 (HIV-1), while phagocytosis and killing of C. albicanswere largely unimpaired compared to those in non-Tg mice. Depletion of PMNs in these Tg mice did not alter oral or gastrointestinal burdens of C. albicansor cause systemic dissemination. However, oral burdens of C. albicanswere increased in CD4C/HIVMutGTg mice expressing only the nefgene of HIV-1 and bred on a CD8 gene-deficient background (CD8−/−), compared to control or heterozygous CD8+/−CD4C/HIVMutGTg mice. Thus, CD8+T cells contribute to the host defense against oral candidiasis in vivo, specifically in the context of nefexpression in a subset of immune cells.

The mouse mammary tumor virus (MMTV) provirus was found to target the Notch1gene, producing insertional mutations in mammary tumors of MMTV/neu transgenic (Tg) mice. In these mammary tumors, the Notch1gene is truncated upstream of the transmembrane domain, and the resulting Notch1 in-tracellular domain (Notch1intra), deleted of most extracellular sequences, is overexpressed. Although Notch1intratransforms mammary epithelial cells in vitro, its role in mammary gland tumor formation in vivowas not studied. Therefore, we generated MMTV/Notch1intraTg mice that overexpress murine Notch1intrain the mammary glands. We observed that MMTV/Notch1intraTg females were unable to feed their pups because of impaired ductal and lobulo-alveolar mammary gland development. This was associated with decreased proliferation of ductal and alveolar epithelial cells during rapid expansion at puberty and in early pregnancy, as well as decreased production of β-casein. Notch1intrarepressed expression of the β-casein gene promoter, as assessed in vitrowith a β-casein/luciferase reporter construct. The MMTV/Notch1intraTg females developed mammary gland tumors, confirming the oncogenic po-tential of Notch1intrain vivo. Furthermore, MMTV/Notch3intraTg mice exhibited a very similar pheno-type. Thus, these Tg mice represent novel models for studying the role of Notch1 or Notch3 in the development and transformation of the mammary gland.

SUMMARYOropharyngeal and esophageal candidiases remain significant causes of morbidity in human immunodeficiency virus (HIV)-infected patients, despite the dramatic ability of antiretroviral therapy to reconstitute immunity. Notable advances have been achieved in understanding, at the molecular level, the relationships between the progression of HIV infection, the acquisition, maintenance, and clonality of oral candidal populations, and the emergence of antifungal resistance. However, the critical immunological defects which are responsible for the onset and maintenance of mucosal candidiasis in patients with HIV infection have not been elucidated. The devastating impact of HIV infection on mucosal Langerhans' cell and CD4+cell populations is most probably central to the pathogenesis of mucosal candidiasis in HIV-infected patients. However, these defects may be partly compensated by preserved host defense mechanisms (calprotectin, keratinocytes, CD8+T cells, and phagocytes) which, individually or together, may limit Candida albicans proliferation to the superficial mucosa. The availability of CD4C/HIV transgenic mice expressing HIV-1 in immune cells has provided the opportunity to devise a novel model of mucosal candidiasis that closely mimics the clinical and pathological features of candidal infection in human HIV infection. These transgenic mice allow, for the first time, a precise cause-and-effect analysis of the immunopathogenesis of mucosal candidiasis in HIV infection under controlled conditions in a small laboratory animal.

Wasting and renal diseases are frequent complications of HIV (human immunodeficiency virus) infection and are associated with accelerated disease progression and increased mortality. Transgenic mice expressing HIV1 under control of the CD4 promoter develop an AIDS-like disease and were used in the present work to study HIV1-induced wasting and kidney pathology. In this study, we reported that disease evolution paralleled increases in serum urea and creatinine levels, indicating an early and progressive deterioration of kidney function; meanwhile the wasting syndrome characterized by up-regulation of the ubiquitine-proteasome pathway and increased level of serum 3-methyl-histidine levels occurred at later stages just prior to death. Further examination of kidney and muscle pathologies revealed a progressive accumulation of CD45+cells, first affecting the kidneys. In addition, the onset of disease is accompanied by elevated levels of circulating “regulated on activation, normal and secreted T cell expressed and secreted” (RANTES). These results prompted us to assess the effects of AS602868, a specific small molecule inhibitor of IkappaB kinase 2 (IKK2) on disease progression. Inhibition of the NF-κB pathway indeed resulted in increased lifespan, kidney and lean body mass preservation. These beneficial results were associated with a reduction of CD45+cells infiltrating the kidneys, amelioration of the renal architecture, and reduced level of circulating RANTES. Together our data provide evidence that IKK2 inhibitors have therapeutic relevance in the treatment of HIV1-associated disorders.

While studying the unique Nramp1 (Slc11a1)–independent susceptibility to Mycobacterium bovis (BCG) infection of BXH-2 mice, we noted that these mice develop important splenomegaly and enlargement of lymph nodes. Segregation analyses in several F2 crosses showed that splenomegaly segregates as a single recessive trait caused by a novel mutation in BXH-2, independent of the infection. Histologic and fluorescence-activated cell sorter (FACS) analyses indicated that splenomegaly is associated with a large increase in Mac1+/GR1+ (macrophage antigen-1+/granulocyte differentiation antigen 1+) granulocyte precursors in spleen, lymph nodes, and bone marrow, resembling a myeloproliferative syndrome. This is concomitant to extramedullary erythropoiesis in the spleen, as measured by proportion of Ter119+ erythroid cells. The locus controlling this myeloproliferative syndrome and splenomegaly was designated Myls and maps to an 18 centimorgan (cM) region of chromosome 8, which also contains an integrated copy of an N-ecotropic murine leukemia virus (MuLV) provirus (Emv2). The relationship between Myls, expansion of Mac1+/GR1+ cells, and Emv2 was investigated. Homozygosity at Myls is necessary but not sufficient for B-ecotropic virus replication in splenocytes, the extent of which appears to be under separate genetic control. Our results suggest a model in which Myls-dependent myeloproliferation in BXH-2 acts as a predisposing factor for the subsequent development of virally induced myeloid leukemia characteristic of this strain.

While studying the unique Nramp1(Slc11a1)–independent susceptibility to Mycobacterium bovis(BCG) infection of BXH-2 mice, we noted that these mice develop important splenomegaly and enlargement of lymph nodes. Segregation analyses in several F2 crosses showed that splenomegaly segregates as a single recessive trait caused by a novel mutation in BXH-2, independent of the infection. Histologic and fluorescence-activated cell sorter (FACS) analyses indicated that splenomegaly is associated with a large increase in Mac1+/GR1+(macrophage antigen-1+/granulocyte differentiation antigen 1+) granulocyte precursors in spleen, lymph nodes, and bone marrow, resembling a myeloproliferative syndrome. This is concomitant to extramedullary erythropoiesis in the spleen, as measured by proportion of Ter119+erythroid cells. The locus controlling this myeloproliferative syndrome and splenomegaly was designated Mylsand maps to an 18 centimorgan (cM) region of chromosome 8, which also contains an integrated copy of an N-ecotropic murine leukemia virus (MuLV) provirus (Emv2). The relationship between Myls, expansion of Mac1+/GR1+cells, and Emv2was investigated. Homozygosity at Mylsis necessary but not sufficient for B-ecotropic virus replication in splenocytes, the extent of which appears to be under separate genetic control. Our results suggest a model in which Myls-dependent myeloproliferation in BXH-2 acts as a predisposing factor for the subsequent development of virally induced myeloid leukemia characteristic of this strain.

ABSTRACTThe Ahi-1locus was initially identified as a common helper provirus integration site in Abelson pre-B-cell lymphomas and shown to be closely linked to the c-mybproto-oncogene. Since no significant alteration of c-mybexpression was found in Abelson murine leukemia virus-induced pre-B-lymphomas harboring a provirus inserted within the Ahi-1locus, this suggested that it harbors another gene whose dysregulation is involved in tumor formation. Here we report the identification of a novel gene (Ahi-1) targeted by these provirus insertional mutations and the cloning of its cDNA. The Ahi-1 proviral insertions were found at the 3' end of the gene, in an inverse transcriptional orientation, with most of them located around and downstream of the last exon, whereas another insertion was within intron 22. In addition, another previously identified provirus insertion site, Mis-2, was found to map within the 16th intron of the Ahi-1gene. The Ahi-1cDNA encodes a 1,047-amino-acid protein. The predicted Ahi-1 protein is a modular protein that contains one SH3 motif and seven WD40 repeats. The Ahi-1gene is conserved in mammals and encodes two major RNA species of 5 and 4.2 kb and several other shorter splicing variants. The Ahi-1gene is expressed in mouse embryos and in several organs of the mouse and rat, notably at high levels in the brain and testes. In tumor cells harboring insertional mutations in Ahi-1, truncated Ahi-1/viral fused transcripts were identified, including some splicing variants with deletion of the SH3 domain. Therefore, Ahi-1is a novel gene targeted by provirus insertion and encoding a protein that exhibits several features of a signaling molecule. Thus, Ahi-1may play an important role in signal transduction in normal cells and may be involved in tumor development, possibly in cooperation with other oncogenes (such as v-abland c-myc) or with a tumor suppressor gene (Nf1), since Ahi-1 insertion sites were identified in tumors harboring v-abldefective retroviruses or a c-myctransgene or in tumors exhibiting deletion of Nf1.

We previously reported that a severe acquired immune deficiency syndrome-like disease develops in transgenic (Tg) mice expressing the human immunodeficiency virus-1 in its natural target cells: immature and mature CD4+T cells and cells of the macrophage/dendritic lineage. Here, we show that these mice also develop cardiac disease, characterized most prominently by a focal myocytolysis, occasionally by myocarditis and by deposition of endogenous immunoglobulin on cardiomyocytes. Microfil perfusion demonstrated widespread coronary arteriospasm and echocardiographic analysis revealed depressed cardiac function in Tg mice. A higher (but still modest) level of cardiomyocyte apoptosis was detected in Tg as compared to non-Tg hearts. Tg expression was detected in some of the infiltrating mononuclear cells, but not in cardiomyocytes or in cells of the heart vessels, suggesting a human immunodeficiency virus-1-induced disease process mediated by cells of the immune system. The similarity of the heart disease observed in these Tg mice to that observed in acquired immune deficiency syndrome patients suggests a common pathogenesis.

ABSTRACTIn order to study the functions of simian immunodeficiency virus (SIV) Nef in vivo in a small-animal model, we constructed transgenic (Tg) mice expressing the SIVmac239 nefgene in the natural target cells of the virus under the control of the human CD4 gene promoter (CD4C). These CD4C/SHIV-nefSIVTg mice develop a severe AIDS-like disease, with manifestations including premature death, failure to thrive or weight loss, wasting, thymic atrophy, an especially low number of peripheral CD8+T cells as well as a low number of peripheral CD4+T cells, diarrhea, splenomegaly, and kidney (interstitial nephritis, segmental glomerulosclerosis), lung (lymphocytic interstitial pneumonitis), and heart disease. In addition, these Tg mice fail to mount a class-switched antibody response after immunization with ovalbumin, they produce anti-DNA autoantibodies, and some of them develop Pneumocystis cariniilung infections. All these results suggest a generalized Nef-induced immunodeficiency. The low numbers of peripheral CD8+and CD4+T cells are likely to reflect a thymic defect and may be similar to the DiGeorge-like “thymic defect” immunophenotype described for a subgroup of human immunodeficiency virus type 1-infected children. Therefore, it appears that SIV Nef alone expressed in mice, in appropriate cell types and at sufficient levels, can elicit many of the phenotypes of simian and human AIDS. These Tg mice should be instrumental in studying the pathogenesis of SIV Nef-induced phenotypes.

To identify the regulatory elements controlling expression of the human CD4 (hCD4) gene in different cell types of the immune system, deletion and chimeric (human/murine) reporter genes were constructed and tested in transgenic (Tg) mice. Regulatory elements required for the proper hCD4 expression in the immature double-positive thymic T cells were identified in the enhancer and in the 3′ end of intron 1. Expression of hCD4 in macrophages is controlled by at least 2 sets of regulatory elements: one present in front of exon 1 and the second at the 5′ end of intron 1. The hCD4 elements required for expression on both myeloid and lymphoid CD8α+ dendritic cells (DCs) from lymph node and thymus were found to be different from those required for macrophage expression. The results indicate that expression of hCD4 in T cells, macrophages, and DCs is controlled by distinct regulatory elements.

ABSTRACTThe human immunodeficiency virus type 1 (HIV-1) Nef protein is an important determinant of AIDS pathogenesis. We have previously reported that HIV-1 Nef is responsible for the induction of a severe AIDS-like disease in CD4C/HIV transgenic (Tg) mice. To understand the molecular mechanisms of this Nef-induced disease, we generated Tg mice expressing a mutated Nef protein in which the SH3 ligand-binding domain (P72XXP75XXP78) was mutated to A72XXA75XXQ78. This mutation completely abolished the pathogenic potential of Nef, although a partial downregulation of the CD4 cell surface expression was still observed in these Tg mice. We also studied whether Hck, one of the effectors previously found to bind to this PXXP motif of Nef, was involved in disease development. Breeding of Tg mice expressing wild-type Nef on an hck-/-(knockout) background did not abolish any of the pathological phenotypes. However, the latency of disease development was prolonged. These data indicate that an intact PXXP domain is essential for inducing an AIDS-like disease in CD4C/HIV Tg mice and suggest that interaction of a cellular effector(s) with this domain is required for the induction of this multiorgan disease. Our findings indicate that Hck is an important, but not an essential, effector of Nef and suggest that another factor(s), yet to be identified, may be more critical for disease development.

To identify the regulatory elements controlling expression of the human CD4(hCD4) gene in different cell types of the immune system, deletion and chimeric (human/murine) reporter genes were constructed and tested in transgenic (Tg) mice. Regulatory elements required for the proper hCD4 expression in the immature double-positive thymic T cells were identified in the enhancer and in the 3′ end of intron 1. Expression of hCD4 in macrophages is controlled by at least 2 sets of regulatory elements: one present in front of exon 1 and the second at the 5′ end of intron 1. The hCD4 elements required for expression on both myeloid and lymphoid CD8α+dendritic cells (DCs) from lymph node and thymus were found to be different from those required for macrophage expression. The results indicate that expression of hCD4 in T cells, macrophages, and DCs is controlled by distinct regulatory elements.

Morphologic revertants of FBJ murine sarcoma virus (v-fos)-transformed rat-1 fibroblasts were isolated using a novel selection procedure based on prolonged retention of rhodamine 123 within mitochondria of v-fos-transformed versus normal fibroblasts. Two classes of revertants were isolated: class I revertants have sustained mutations in cellular genes, and a class II revertant has a nonfunctional v-fosprovirus. Somatic-cell hybridization studies suggested that the revertant phenotype was recessive to the transformed phenotype. Class I revertants were also resistant to retransformation by v-gag-fos-fox, v-Ha-ras, v-abl, and v-mos, but could be retransformed by the trkoncogene and polyoma virus middle T antigen. These results suggest that the class I revertants sustained mutations in one or more cellular genes essential for transformation by some, but not all, oncogenes. Our data suggest the existence of common biochemical pathways for transformation.

Mice inoculated with replication-competent stocks of the murine acquired immunodeficiency syndrome (MAIDS) virusare severely immunocompromised and proned to the development of T- and B-cell lymphomas. We have studied the development of T-cell lymphomas in C57BL/6 and RF/J mice inoculated with helper-free stocks of the MAIDS defective virus. We observed the expansion of T cell clones (detected by TCR gene rearrangements and by transplantation) only rarely in diseased C57BL/6 mice and slightly more frequently in RF/J mice. We succeeded in establishing four transplantable T cell tumors and malignant cell lines. The three cell lines from RF/J mice were immature T-cells (Thy-1+, CD3−, CD4+, CD8+, Mac-1+), while the line from the C57BL/6 mouse had the phenotype of mature T-cells (Thy-1+, CD3*, CD4+, CD8−). All lines were virus-producers despite the fact that helper-free stocks of the virus were inoculated. These helper MuLVs most likely originated from endogenous MuLV sequences. Also, the defective viral genome was clearly detectable in one cell line and was rearranged in two other lines. These established cell lines may be useful to determine whether they share some of the characteristics of the anergic T-cells in vivo and to study the role of the MAIDS defective virus in T cell transformation.

ABSTRACTWe have constructed transgenic (Tg) mice expressing the entire human immunodeficiency virus type 1 (HIV-1) coding sequences in cells targeted by HIV-1 infection in humans. These Tg mice developed a severe AIDS-like disease leading to early death (<1 month). They developed muscle wasting, severe atrophy and fibrosis of lymphoid organs, tubulointerstitial nephritis, and lymphoid interstitial pneumonitis. In addition the expression of RANTES was increased in various tissues of these Tg mice relative to that in the normal controls. Disease appearance was correlated with the levels of transgene expression. The numerous pathologies observed in these mice are remarkably similar to those observed in human AIDS and, more specifically, in pediatric AIDS.

The unintegrated closed circular DNA intermediates from B- and N-tropic endogenous BALB/c murine leukemia virus (MuLV) were isolated from acutely infected mouse fibroblasts and cleaved at their unique HindIII sites. The linearized B- and N-tropic MuLV DNAs were then inserted in ? vector Charon 21A and cloned. Seventeen B-tropic and ten N-tropic recombinants were analyzed. The viral insert of these 27 MuLV recombinants could be grouped into 3 classes. The first class included permuted full-length molecules of 8.8 kilobase pairs (kbp) having two long terminal repeats (LTR) (0.55 kbp) mixed with 8.25-kbp molecules. The second class included 8.25-kbp molecules which harbored only one LTR copy. The third class of inserts was made of molecules with deletions of various lengths (0.1 to 3 kbp). These deletions were localized by restriction enzyme analysis. Significantly more deletions were observed in N-tropic than in B-tropic recombinants, although these two genomes isolated from acutely infected cells seemed to differ only by the absence of a 70-base-pair (bp) sequence in the N-tropic LTR. The biological activity of each of the recombinants of the first and second classes was assayed by transfection on NIH/3T3 cells. Two recombinants, one B-tropic (? B-16) and one N-tropic (?N-20) were found to be infectious. Both were able to give rise to replication-competent MuLV which could form large XC plaques and had kept the tropism of its progenitor. Like its progenitor in vivo DNA, ?N-20-cloned DNA lacked a 70-bp sequence in its unique LTR. The emerging N-tropic MuLV after transfection with ?N-20 was used to acutely infect NIH/3T3 cells. The viral DNA intermediates isolated were now found to have two LTR copies. They had also acquired a 70-bp sequence in each LTR. The acquisition or loss of this 70-bp sequence did not seem to affect the replication, the XC plaque formation, or the tropism of the virus.

We have found that levels of unintegrated linear viral DNA were nearly identical in several Fv-1resistant cell lines, whereas levels of closed circular viral DNA are markedly reduced in these resistant cells, to the same extent as virus production (P. Jolicoeur and E. Rassart, J. Virol. 33:183-195, 1980). To determine the fate of linear viral DNA made in resistant cells we performed pulse-chase experiments, labeling viral DNA with 5-bromodeoxyuridine and following it with a thymidine chase. 5-Bromodeoxyuridine-labeled viral DNA (HH) recovered by banding on cesium chloride gradients was sedimented on neutral sucrose density gradients or separated by the agarose gel-DNA transfer procedure and detected by hybridization with complementary DNA. Levels of linear viral DNA made in Fv-1b/b(JLS-V9 and SIM.R) and Fv-1n/n(NIH/3T3 and SIM) cells were found to decrease during the chase period at about the same rate in permissive and nonpermissive conditions, indicating that linear viral DNA is not specifically degraded in Fv-1resistant cells. Levels of the two species of closed circular viral DNA made in Fv-1permissive cells increased relative to the levels of linear DNA during the chase period. This confirmed the precursor-product relationship between linear DNA and the two species of circular DNA. In Fv-1resistant cells, this apparent conversion of linear viral DNA into circular forms was not seen, and no supercoiled viral DNA could be detected. To determine whether the transport of linear viral DNA from the cytoplasm into the nucleus was prevented by the Fv-1gene product, SIM.R cells were fractionated into cytoplasmic and nuclear fractions, and viral DNA was detected in each fraction by the agarose gel-DNA transfer procedure. Levels of linear viral DNA were nearly identical in both cytoplasmic and nuclear fractions of permissive or resistant cells. Circular viral DNA could be detected in the nuclear fraction of permissive cells, but not in that of resistant cells. A pulse-chase experiment was also performed with SIM.R cells. During the thymidine chase period, linear viral DNA was seen to accumulate in nuclei of both permissive and resistant cells, whereas supercoiled viral DNA accumulated only in nuclei of permissive cells. These results indicate that the Fv-1gene product does not interfere with the transport of linear viral DNA into the nucleus. Our data also suggest that the Fv-1restriction does not operate through a degradation process. Therefore, the Fv-1gene product could either block the circularization of linear viral DNA directly or promote the synthesis of a faulty linear viral DNA whose defect (yet undetected) would prevent its circularization.

Murine AIDS (MAIDS) is a disease that shows many similarities with human AIDS. Several immunological parameters of the disease have been analyzed and genetic studies have mapped a gene (or genes) of resistance in the H‐2 complex and shown that the genetic background of the mouse can significantly modify some features of the disease. The etiologic agent of MAIDS is a defective murine leukemia virus that seems able to induce disease in the absence of virus replication. This defective virus induces proliferation of its target cells and the cell expansion was found to be oligoclonal, thus suggesting that the immunodeficiency observed in these mice is a paraneoplastic syndrome. The excellent response of MAIDS mice to antineoplastic agents is consistent with this notion. This animal model has already been useful in stimulating the emergence of novel questions and the formulation of new hypotheses about human AIDS, namely about the role of defective HIV, the role of HIV replication in the progression of the disease, and the importance to identify the target cells of HIV in vivo. Although MAIDS and AIDS are not identical and are induced by retroviruses of different classes, the availability of such a model in an easily accessible small animal species, whose genetics is very sophisticated, may be instrumental in understanding the pathogenesis of AIDS if some of the cellular and molecular affected pathways are common in both diseases.—Jolicoeur, P. Murine acquired immunodeficiency syndrome (MAIDS): an animal model to study the AIDS pathogenesis. FASEB J.5: 2398–2405;1991.

Certain strains of transgenic mice that express the rat neu oncogene (neuT) in mammary epithelial cells develop breast tumours at an average of 44 weeks of age. In this study, intraperitoneal injection of a monoclonal anti-receptor antibody specific for the rat neuT oncogene product dramatically affected tumour development in these transgenic mice in a dose-dependent manner. A significant proportion (50%) of mice, when injected with anti-receptor antibodies, did not develop tumours even after 90 weeks of age. The phosphotyrosine levels of the membrane fraction of breast tissues in the anti-receptor antibody-treated mice were almost completely abolished when a higher dose of antibodies was used. This study demonstrates, for the first time, that immunologic manipulation of an oncogene product can effectively prevent the development of tumours in a rodent transgenic model.

Vacuolar myelopathy is a common neurological complication in AIDS patients. The pathogenesis of this spinal cord white matter disease remains unclear and it is still debated whether infection of spinal cord with the human immunodeficiency virus type 1 (HIV–1) is causing the disease. We have generated transgenic mice expressing the entire HIV–1 genome under the regulation of an oligodendrocyte–specific promoter. These mice develop spinal cord vacuolar lesions similar to those found in AIDS patients. This animal model provides in vivo evidence linking the expression of HIV–1 proteins in oligodendrocytes to the spinal cord damage found in vacuolar myelopathy.

Mice inoculated with the murine AIDS (MAIDS)-defective virus develop severe B and T cell dysfunctions. The primary event in the development of this disease is the infection and polyclonal expansion of the target cells of this defective virus, which have been reported to belong to the B cell lineage. To further study the central role that these cells play in the development of MAIDS, we attempted to establish MAIDS-defective virus-infected B cell linesin vitro.We succeeded in establishing two cell lines, SD1 and CSTB5, from the enlarged organs of C57BL/6 mice inoculated with helper-free stocks of the MAIDS-defective virus. Both cell lines are not transplantable in syngeneic C57BL/6 mice or in nude or CD8−/−mice and are apparently not malignant. They both belong to the B lineage, as their immunoglobulin (Ig) genes, but not the T cell receptor (TcR) β locus, are rearranged, suggesting that they are relatively mature B cells. However, analysis of cell surface marker expression by FACS revealed a surface phenotype similar to that of pre-B cells (MHC I+, MHC II+, B7.2+, sIgM−, sIgG−, κ−, B220−, CD5−, Thy1.2−, TcR−, CD3−, CD4−, CD8−, Mac-1−, 33D1−). Additionally, the CSTB5 cells express CD40 and the SD1 cells express CD43. Both cell lines contain the MAIDS-defective provirus and express the expected 4.2-kb viral RNA and the corresponding Pr60gagprotein. The CSTB5 cells are nonproducer, while the SD1 cell line produces what appears to be an endogenous MuLV. The phenotype of these cell lines is very similar to what is known about the target B cells of this virusin vivo.These new established cell lines are likely to be useful in elucidating the mechanism(s) by which the MAIDS-defective virus causes its target B cells to proliferate and induce T cell anergy in infected animals.

In normal hemopoietic cells that are dependent on specific growth factors for cell survival, the expression of the basic helix-loop-helix transcription factor SCL/Tal1 correlates with that of c-Kit, the receptor for Steel factor (SF) or stem cell factor. To address the possibility that SCL may function upstream of c-kit, we sought to modulate endogenous SCL function in the CD34+ hemopoietic cell line TF-1, which requires SF, granulocyte/macrophage colony–stimulating factor, or interleukin 3 for survival. Ectopic expression of an antisense SCL cDNA (as-SCL) or a dominant negative SCL (dn-SCL) in these cells impaired SCL DNA binding activity, and prevented the suppression of apoptosis by SF only, indicating that SCL is required for c-Kit–dependent cell survival. Consistent with the lack of response to SF, the level of c-kit mRNA and c-Kit protein was significantly and specifically reduced in as-SCL– or dn-SCL– expressing cells. c-kit mRNA, c-kit promoter activity, and the response to SF were rescued by SCL overexpression in the antisense or dn-SCL transfectants. Furthermore, ectopic c-kit expression in as-SCL transfectants is sufficient to restore cell survival in response to SF. Finally, enforced SCL in the pro–B cell line Ba/F3, which is both SCL and c-kit negative is sufficient to induce c-Kit and SF responsiveness. Together, these results indicate that c-kit, a gene that is essential for the survival of primitive hemopoietic cells, is a downstream target of the transcription factor SCL.

No relevant conflicts of interest to declare.

Abstract 447

Hematopoietic differentiation is controlled to a large extent by a network of transcription factors and chromatin modifiers and disruption of this system can lead to leukemia or lymphoma. One of the transcription factor genes, which is aberrantly expressed in human T-cell lymphoma is Growth Factor Independence 1 (Gfi1). Since over expression of Gfi1 can accelerate experimentally induced T-cell tumors in mice, it is likely that Gfi1 plays a crucial role in establishing or maintaining lymphoid neoplasms. To test this hypothesis we have used, N-ethyl-N-nitrosourea (ENU) to induce T-cell tumors in WT mice (Gfi1+/+), Gfi1-deficient mice (Gfi1−/−) or mice transgenically over expressing Gfi1 under the control of the pan-hematopoietic vav-promoter (vav-Gfi1). As expected, most of Gfi1+/+ mice (25/27) developed T-cell tumors and acute myeloid leukemia within 118 days. Similarly, vav-Gfi1 mice (10/10) developed T-cell lymphoma, but within a shorter latency period (88 days). In contrast, only 3/14 Gfi1−/− mice developed hematopoietic neoplasia with a prolonged median latency period of 126 days. Other approaches using infection of newborn mice with Moloney Murine leukemia virus (MoMuLV) to induce T-cell lymphoma or co expression of an Eμ-myc transgene to induce B-cell lymphoma showed a similar dependency of tumor formation on the presence and expression of Gfi1. Closer analysis of tumors forming in Gfi1−/− mice demonstrated that Gfi1 deficiency correlated with a smaller size of the tumors and a noticeably increased rate of cell death within the tumor samples. This pointed to a potential role of Gfi1 in the regulation of apoptosis. To explore this hypothesis, we exposed both thymocytes and hematopoietic stem cells (Lin-, Sca1+, c-kit+, LSK) to ENU or gamma-irradiation in vitro. We could observe that Gfi1−/− thymocytes and stem cells (LSK cells) have a higher rate of cell death following exposure to these DNA damage inducing agents in vitro than the WT controls. To validate these results, we recapitulated these experiments in vivo. Gfi1−/− mice exhibited severe bone marrow failure and a more pronounced loss of hematopoietic stem cells (LSK) than Gfi1+/+ mice after ENU treatment or gamma irradiation in vivo. To explore this mechanism on the molecular basis we evaluated expression of the different pro and antiapoptotic components in Gfi1+/+ and Gfi1−/− thymocytes after irradiation. Strikingly, Gfi1−/− thymocytes expressed higher levels of the pro-apoptotic proteins such as Bax and Noxa and lower levels of the CDK inhibitor p21WAF than WT thymocytes following induction of DNA damage. Our model would be that Gfi1 represents a new regulator in the cellular response to DNA damage in the hematopoietic system by inhibiting different proapoptotic factors. We propose that Gfi1 is essential for the development of lymphoid and potentially myeloid neoplasms by inhibiting apoptosis. We suggest that Gfi1 could represent a possible new target structure for therapeutic intervention.

Hematopoietic differentiation is controlled to a large extent by a network of transcription factors and chromatin modifiers and disruption of this system can lead to leukemia or lymphoma. One of the transcription factor genes, which is aberrantly expressed in human T-cell lymphoma is Growth Factor Independence 1(Gfi1). Since over expression of Gfi1 can accelerate experimentally induced T-cell tumors in mice, it is likely that Gfi1 plays a crucial role in establishing or maintaining lymphoid neoplasms. To test this hypothesis we have used, N-ethyl-N-nitrosourea (ENU) to induce T-cell tumors in WT mice (Gfi1+/+), Gfi1-deficient mice (Gfi1−/−) or mice transgenically over expressing Gfi1under the control of the pan-hematopoietic vav-promoter (vav-Gfi1). As expected, most of Gfi1+/+mice (25/27) developed T-cell tumors and acute myeloid leukemia within 118 days. Similarly, vav-Gfi1mice (10/10) developed T-cell lymphoma, but within a shorter latency period (88 days). In contrast, only 3/14 Gfi1−/−mice developed hematopoietic neoplasia with a prolonged median latency period of 126 days. Other approaches using infection of newborn mice with Moloney Murine leukemia virus (MoMuLV) to induce T-cell lymphoma or co expression of an Eμ-myc transgene to induce B-cell lymphoma showed a similar dependency of tumor formation on the presence and expression of Gfi1. Closer analysis of tumors forming in Gfi1−/−mice demonstrated that Gfi1deficiency correlated with a smaller size of the tumors and a noticeably increased rate of cell death within the tumor samples. This pointed to a potential role of Gfi1in the regulation of apoptosis. To explore this hypothesis, we exposed both thymocytes and hematopoietic stem cells (Lin-, Sca1+, c-kit+, LSK) to ENU or gamma-irradiation in vitro. We could observe that Gfi1−/−thymocytes and stem cells (LSK cells) have a higher rate of cell death following exposure to these DNA damage inducing agents in vitrothan the WT controls. To validate these results, we recapitulated these experiments in vivo. Gfi1−/−mice exhibited severe bone marrow failure and a more pronounced loss of hematopoietic stem cells (LSK) than Gfi1+/+mice after ENU treatment or gamma irradiation in vivo. To explore this mechanism on the molecular basis we evaluated expression of the different pro and antiapoptotic components in Gfi1+/+and Gfi1−/−thymocytes after irradiation. Strikingly, Gfi1−/−thymocytes expressed higher levels of the pro-apoptotic proteins such as Bax and Noxa and lower levels of the CDK inhibitor p21WAFthan WT thymocytes following induction of DNA damage. Our model would be that Gfi1 represents a new regulator in the cellular response to DNA damage in the hematopoietic system by inhibiting different proapoptotic factors. We propose that Gfi1 is essential for the development of lymphoid and potentially myeloid neoplasms by inhibiting apoptosis. We suggest that Gfi1 could represent a possible new target structure for therapeutic intervention.