MCMV Dissemination from Latently-Infected Allografts Following Transplantation into Pre-Tolerized Recipients.

Transplantation tolerance is achieved when recipients are unresponsive to donor alloantigen yet mobilize against third-party antigens, including virus. After transplantation, cytomegalovirus (CMV) reactivation in latently-infected transplants reduces allograft viability. To determine if pre-tolerized recipients are resistant to viral dissemination in this setting, we transfused chemically-fixed donor splenocytes (1-ethyl-3- (3'-dimethyl-aminopropyl)-carbo-diimide (ECDI)-treated splenocytes (ECDIsp)) to induce donor antigen tolerance without immunosuppression. In parallel, we implanted donor islet cells to validate operational tolerance. These pre-tolerized recipients were implanted with murine CMV (MCMV) latently-infected donor kidneys (a validated model of CMV latency) to monitor graft inflammation and viral dissemination. Our results indicate that tolerance to donor islets was sustained in recipients after implantation of donor kidneys. In addition, kidney allografts implanted after ECDIsp and islet implantation exhibited low levels of fibrosis and tubulitis. In contrast, kidney cellular and innate immune infiltrates trended higher in the CMV group and exhibited increased markers of CD8+ T cell activation. Tolerance induction was unable to prevent increases in MCMV-specific CD8+ T cells or dissemination of viral IE-1 DNA. Our data suggest that latently-infected allografts are inherently more susceptible to inflammation that is associated with viral dissemination in pre-tolerized recipients. Thus, CMV latently-infected allografts require enhanced strategies to protect allograft integrity and viral spread.

A novel murine model of differentiation-mediated cytomegalovirus reactivation from latently infected bone marrow haematopoietic cells.

CD34+ myeloid lineage progenitor cells are an important reservoir of latent human cytomegalovirus (HCMV), and differentiation to macrophages or dendritic cells (DCs) is known to cause reactivation of latent virus. Due to its species-specificity, murine models have been used to study mouse CMV (MCMV) latency and reactivation in vivo. While previous studies have shown that MCMV genomic DNA can be detected in the bone marrow (BM) of latently infected mice, the identity of these cells has not been defined. Therefore, we sought to identify and enrich for cellular sites of MCMV latency in the BM haematopoietic system, and to explore the potential for establishing an in vitro model for reactivation of latent MCMV. We studied the kinetics and cellular characteristics of acute infection and establishment of latency in the BM of mice. We found that while MCMV can infect a broad range of haematopoietic BM cells (BMCs), latent virus is only detectable in haematopoietic stem cells (HSCs), myeloid progenitor cells, monocytes and DC-enriched cell subsets. Using three separate approaches, MCMV reactivation was detected in association with differentiation into DC-enriched BMCs cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) followed by lipopolysaccharide (LPS) treatment. In summary, we have defined the kinetics and cellular profile of MCMV infection followed by the natural establishment of latency in vivo in the mouse BM haematopoietic system, including the haematopoietic phenotypes of cells that are permissive to acute infection, establish and harbour detectable latent virus, and can be stimulated to reactivate following DC enrichment and differentiation, followed by treatment with LPS.

A clinically relevant murine model unmasks a "two-hit" mechanism for reactivation and dissemination of cytomegalovirus after kidney transplant.

Reactivation of latent cytomegalovirus remains an important complication after transplant. Although immunosuppression (IS) has been implicated as a primary cause, we have previously shown that the implantation response of a kidney allograft can lead to early transcriptional activation of latent murine cytomegalovirus (MCMV) genes in an immune-competent host and to MCMV reactivation and dissemination to other organs in a genetically immune-deficient recipient. We now describe a model that allows us to separately analyze the impact of the implantation effect vs that of a clinically relevant IS regimen. Treatment with IS of latently infected mice alone does not induce viral reactivation, but transplant of latently infected allogeneic kidneys combined with IS facilitates MCMV reactivation in the graft and dissemination to other organs. The IS regimen effectively dampens allo-immune inflammatory pathways and depletes recipient anti-MCMV but does not affect ischemia-reperfusion injury pathways. MCMV reactivation similar to that seen in allogeneic transplants combined with also occurs after syngeneic transplants. Thus, our data strongly suggest that while ischemia-reperfusion injury of the implanted graft is sufficient and necessary to initiate transcriptional reactivation of latent MCMV ("first hit"), IS is permissive to the first hit and facilitates dissemination to other organs ("second hit").

STAT-5 Regulates Transcription of the Topoisomerase IIß-Binding Protein 1 (TopBP1) Gene To Activate the ATR Pathway and Promote Human Papillomavirus Replication.

UNLABELLED: The life cycle of high-risk human papillomaviruses (HPVs) is dependent upon epithelial differentiation. Following infection of basal cells, HPV genomes are stably maintained at low copy numbers, and productive replication or amplification is restricted to highly differentiated suprabasal cells. In high-risk HPV infections, the ATM pathway is constitutively activated in the absence of external DNA-damaging agents and is required for productive viral replication. The ataxia telangiectasia (ATM) pathway repairs double-strand breaks in DNA, while the ataxia telangiectasia and Rad3-related (ATR) pathway targets single-strand breaks. Our studies show that the ATR pathway, like the ATM pathway, is activated in HPV-positive cells and that inhibitors of ATR or CHK1 phosphorylation block both amplification and late viral gene expression in differentiated cells while moderately reducing stable copy numbers in undifferentiated cells. TopBP1 is a critical upstream activator of the ATR pathway and is expressed at elevated levels in HPV-positive cells. This increased expression of TopBP1 is necessary for ATR/CHK1 activation in HPV-positive cells, and knockdown blocks amplification. Furthermore, TopBP1 activation is shown to be regulated at the level of transcription initiation by the innate immune regulator STAT-5, which is activated by HPV proteins. STAT-5 has also been shown to be a regulator of the ATM response, demonstrating that these two pathways are coordinately regulated in HPV-positive cells. These findings identify a novel link between the innate immune response and activation of the ATR DNA damage response in regulating the life cycle of high-risk HPVs. IMPORTANCE: High-risk human papillomaviruses (HPVs) are the causative agents of cervical and other anogenital cancers, as well as many oral cancers. HPVs infect epithelial cells and restrict productive viral replication or amplification and virion production to differentiated cells. Our studies demonstrate that HPVs activate the ATR single-strand DNA repair pathway and this activation is necessary for HPV genome amplification. The innate immune regulator STAT-5 is shown to regulate transcription of the ATR binding factor TopBP1, and this is critical for the induction of the ATR pathway. Our study identifies important links between innate immune signaling, the ATR DNA damage pathway, and productive HPV replication that may lead to the characterization of new targets for the development of therapeutics to treat HPV-induced infections.

The HIV protease inhibitor indinavir down-regulates the expression of the pro-angiogenic MT1-MMP by human endothelial cells.

In addition to contrast human immunodeficiency virus (HIV) replication, the HIV protease inhibitors (HIV-PI) have reduced tumour incidence or clinical progression in infected patients. In this regard, we have previously shown that, independently of its anti-viral activity, the HIV-PI indinavir (IDV) directly blocks matrix metalloproteinase (MMP)-2 proteolytic activation, thus efficiently inhibiting tumour angiogenesis in vitro, in animal models, and in humans. Herein we investigated the molecular mechanism for IDV anti-angiogenic effect. We found that treatment of human primary endothelial cells with therapeutic IDV concentrations decreases the expression of membrane type (MT)1-MMP, which is the major activator of MMP-2. This occurs for both the constitutive expression of MT1-MMP and that up-regulated by angiogenic factors. In either cases, reduction of MT1-MMP levels by IDV is preceded by the inhibition of the binding of the specificity protein (Sp)1 transcription factor to the promoter region of the MT1-MMP gene in endothelial cell nuclei. As MT1-MMP is key for tumour angiogenesis, these results support the use of IDV or its derivatives in anti-cancer therapy. This is recommended by the low toxicity of the drug, and the large body of data on its pharmacokinetic.

Ritonavir or saquinavir impairs the invasion of cervical intraepithelial neoplasia cells via a reduction of MMP expression and activity.

OBJECTIVE AND DESIGN: Treatment of human immunodeficiency virus (HIV)-infected women with the highly active antiretroviral therapy (HAART) has reduced the onset of uterine cervical intraepithelial neoplasia (CIN), and halted its progression to cervical carcinoma. We and others demonstrated that the HIV protease inhibitors (HIV-PIs) used in HAART can exert direct antitumour activities also in HIV-free preclinical or clinical models. As uterine cervical carcinoma is a leading cause of death in women independently of HIV infection, herein we assessed the impact of therapeutic concentrations of HIV-PIs including indinavir (IDV), saquinavir (SQV) or ritonavir (RTV) on cells obtained from CIN or cervical carcinoma lesions of HIV-negative women. METHODS: HIV-PI effects were evaluated by cell invasion, growth or toxicity assays, and by RNA, protein or zymogram analyses. RESULTS: Both SQV and RTV inhibited CIN cell invasion, and this was paralleled by a reduced expression and proteolytic activity of the matrix metalloproteinase (MMP)-2 and 9 in treated cells. SQV and RTV also reduced CIN cell growth rate, but did not affect the invasion or growth of cells derived from highly progressed cervical carcinoma. CONCLUSION: As MMP-2 and MMP-9 have a key role in CIN evolution into cervical carcinoma, these results support the use of SQV or RTV for the block of CIN clinical progression in either HIV-infected or uninfected patients.

Fibroblast growth factor-2 transiently activates the p53 oncosuppressor protein in human primary vascular smooth muscle cells: implications for atherogenesis.

OBJECTIVE: The development of atherosclerotic lesions associates with the proliferation of vascular smooth muscle cells (VSMC), and their migration from arterial tunica media to the intima. Fibroblast growth factor (FGF)-2 can trigger either phenomena, which are accompanied by the functional impairment of the p53 transcription factor. However, FGF-2 impact on p53 function in VSMC is largely unknown. METHODS AND RESULTS: RT-PCR and Western blot analyses assayed FGF-2 effect on human primary VSMC expression of p53-induced molecules with a role in atherogenesis. Confocal microscopy evaluated whether FGF-2 could affect p53 distribution inside VSMC. Results indicate that VSMC exposure to FGF-2 at amounts stimulating the proliferation and migration of these cells promotes p53 phosphorylation and transient accumulation in VSMC nuclei. This is followed by an increase in the expression of the p53-induced thrombospondin (TSP)-1, a VSMC growth and motility factor, and human double minute 2 (HDM2), an antagonist of p53 transcriptional and growth suppressive activity. At later time points, in agreement with the increase of HDM2 and with the capability of this protein to export nuclear p53 to the cytoplasm, the content of p53 in VSMC nuclei is reduced, and the expression of the p53-targeted TSP-l and HDM2 is diminished. CONCLUSIONS: Since FGF-2, p53, TSP-1, and HDM2 are expressed in human atherosclerotic lesions, the in vitro effects of FGF-2 described herein may be operative in vivo, providing a molecular mechanism for FGF-2 pro-atherogenic activity.

Neuroendocrine phenotype analysis in five patients with isolated hypogonadotropic hypogonadism due to a L102P inactivating mutation of GPR54.

CONTEXT: Loss of function of the G protein-coupled receptor of kisspeptins (GPR54) was recently described as a new cause of isolated hypogonadotropic hypogonadism. In vivo studies performed in several species have confirmed the major role of kisspeptins in neuroendocrine regulation of the gonadotropic axis and therefore sexual maturation. OBJECTIVE: The objective of this study was to specify the exact contribution of kisspeptins and GPR54 to the initiation of puberty in humans. DESIGN: Detailed neuroendocrine descriptions were performed in five patients with isolated hypogonadotropic hypogonadism bearing a new GPR54-inactivating mutation. RESULTS: A homozygous mutation (T305C) leading to a leucine substitution with proline (L102P) was found in the five affected patients. This substitution completely inhibited GPR54 signaling. Phenotypic analysis revealed variable expressivity in the same family, either partial or complete gonadotropic deficiency. LH pulsatility analysis showed peaks with normal frequency but low amplitude. Repeated GnRH tests performed between 12 and 21 yr of age in one affected male revealed progressive changes in pituitary response from an early pubertal to an almost full pubertal pattern. Double GnRH test stimulations performed at a 120-min interval showed reduced dynamic pituitary response in GPR54-mutated patients. CONCLUSION: GPR54 inactivation does not impede neuroendocrine onset of puberty; rather, it delays and slows down pubertal maturation of the gonadotropic axis. The L102P loss of function mutation in GPR54 results in a more quantitative than qualitative defect of gonadotropic axis activation.

New insights in the genetics of isolated hypogonadotropic hypogonadism.

Isolated gonadotropic deficiency or isolated hypogonadotropic hypogonadism is defined as a low sexual hormone secretion by the gonads associated with low LH and FSH plasma levels. Kallmann syndrome is defined as a congenital isolated gonadotropic deficiency associated with anosmia whereas the phenotype of the idiopathic form is limited to the gonadotropic axis. For several years, it has been known that mutations of the KAL-1 gene or loss-of-function mutations of GnRH receptor did not explain all familial cases of isolated gonadotropic deficiency with or without anosmia. Thus the existence of other genes playing a major role in the physiology of the gonadotropic axis was highly suggested. In 2003, fibroblast growth factor receptor 1 (FGFR1) and GPR54 were shown to be two of these genes. FGFR1 loss-of-function mutations were reported in Kallmann syndrome whereas inactivating mutations of GPR54 were described in the idiopathic form of the gonadotropic deficiency. These genetic studies have opened up a new chapter in the physiology and the pharmacology of the gonadotropic axis.