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Supplementary Figure 1 from Differential Regulation of Hypoxia-Induced CXCR4 Triggering during B-Cell Development and Lymphomagenesis

Supplementary Figure 2 from Differential Regulation of Hypoxia-Induced CXCR4 Triggering during B-Cell Development and Lymphomagenesis

The chemokine receptor CXCR4 plays a central role in organ-specific homing and tumor spreading and is induced by hypoxia. B lymphocytes are exposed to low oxygen tensions during their development, but the influence of hypoxia on their physiology is poorly understood. Here, we show that hypoxia is associated with up-regulation of CXCR4 expression in human normal and malignant B cells, through both transcriptional and posttranslational mechanisms. However, a dichotomic functional response to CXCR4 triggering was observed: both peripheral B cells and lymphomas arising from mature B cells displayed increased responses to CXCR4 triggering under hypoxia, whereas germinal center (GC) B cells as well as GC-derived lymphomas showed CXCR4 receptor desensitization. This phenomenon was associated with differential modulation of key signal-transducing molecules, including mitogen-activated protein kinase phosphatase-1 and regulator of G protein signaling molecule-1. The unresponsiveness of GC-derived lymphomatous B cells to CXCR4 triggering under hypoxia may have implications for the development and pathogenesis of GC-derived lymphoid tumors. [Cancer Res 2007;67(18):8605–14]

Supplementary Figure 4 from Differential Regulation of Hypoxia-Induced CXCR4 Triggering during B-Cell Development and Lymphomagenesis

Supplementary Methods and Materials, Figure Legends 1-4 from Differential Regulation of Hypoxia-Induced CXCR4 Triggering during B-Cell Development and Lymphomagenesis

Supplementary Table 1 from Differential Regulation of Hypoxia-Induced CXCR4 Triggering during B-Cell Development and Lymphomagenesis

Supplementary Figure 3 from Differential Regulation of Hypoxia-Induced CXCR4 Triggering during B-Cell Development and Lymphomagenesis

International audience; Even though an estimated 10-20 million people worldwide are infected with the oncogenic retrovirus, human T-lymphotropic virus type 1 (HTLV-1), its epidemiology is poorly understood, and little effort has been made to reduce its prevalence. In response to this situation, the Global Virus Network launched a taskforce in 2014 to develop new methods of prevention and treatment of HTLV-1 infection and promote basic research. HTLV-1 is the etiological agent of two life-threatening diseases, adult T-cell leukemia and HTLV-associated myelopathy/tropical spastic paraparesis, for which no effective therapy is currently available. Although the modes of transmission of HTLV-1 resemble those of the more familiar HIV-1, routine diagnostic methods are generally unavailable to support the prevention of new infections. In the present article, the Taskforce proposes a series of actions to expand epidemiological studies; increase research on mechanisms of HTLV-1 persistence, replication and pathogenesis; discover effective treatments; and develop prophylactic and therapeutic vaccines.

RNA interference (RNAi) has been suggested to represent a promising therapeutic approach in different disease settings. Primary effusion lymphoma (PEL) is a plasmablastic lymphoma consistently expressing B lymphocyte-induced maturation protein 1 (Blimp-1), a pivotal transcriptional regulator during

Loss of menin, a tumor suppressor coded by the MEN1 gene, is a key factor in the pathogenesis of multiple endocrine neoplasia type I and in a percentage of sporadic endocrine tumors of the pancreas and parathyroid glands. This study investigated expression of the menin protein in the normal exocrine pancreas and in pancreatic ductal adenocarcinoma (PDAC), the most common pancreatic tumor. Immunofluorescence (IF) analyses showed that menin is expressed at high levels in normal acinar and duct cells. Examination of 24 clinical samples of PDAC revealed a pronounced decrease in menin expression in all tumors examined. To identify alterations underlying this defect, we searched for disruption and epigenetic silencing of the MEN1 gene. Analysis of nine laser-microdissected tumors revealed loss of heterozygosity of intragenic (one tumor) or adjacent (three tumors) MEN1 microsatellite markers. Methylation of CpG sites in the MEN1 promoter was documented in five of 24 tumors. IF analyses also revealed low to undetectable menin expression in the PDAC cell lines MiaPaCa-2 and Panc-1. Ectopic expression of menin in these cells resulted in a marked alteration of the cell cycle, with an increase in the G1/S+G2 ratio. These findings represent the first evidence that the MEN1 gene is a target of mutation and methylation in PDAC and that menin influences the cell cycle profile of duct cells.

The chemokine receptor CXCR4 plays a central role in organ-specific homing and tumor spreading and is induced by hypoxia. B lymphocytes are exposed to low oxygen tensions during their development, but the influence of hypoxia on their physiology is poorly understood. Here, we show that hypoxia is associated with up-regulation of CXCR4 expression in human normal and malignant B cells, through both transcriptional and posttranslational mechanisms. However, a dichotomic functional response to CXCR4 triggering was observed: both peripheral B cells and lymphomas arising from mature B cells displayed increased responses to CXCR4 triggering under hypoxia, whereas germinal center (GC) B cells as well as GC-derived lymphomas showed CXCR4 receptor desensitization. This phenomenon was associated with differential modulation of key signal-transducing molecules, including mitogen-activated protein kinase phosphatase-1 and regulator of G protein signaling molecule-1. The unresponsiveness of GC-derived lymphomatous B cells to CXCR4 triggering under hypoxia may have implications for the development and pathogenesis of GC-derived lymphoid tumors. [Cancer Res 2007;67(18):8605–14]

Experiments have been performed with the aim of elucidating the nature and the extent of the in vivo interactions between murine leukemia viruses (MuLVs) and murine sarcoma virus (MSV). BALB/c and CBA mice, injected neonatally with Graffi or passage A Gross viruses (MuLV-Gi, MuLV-G), have been inoculated as young adults with murine sarcoma virus, Moloney strain (MSV-M). A higher percentage of nonregressing sarcomas appeared in these animals, sometimes accompanied simultaneously by leukemia. The immune reactivity of mice receiving MuLV-Gi at birth was found to be significantly depressed when evaluated by the hemolytic palque-forming cell (PFC) technique. However, in mice infected with MuLV-Gi and MSV-M the number of PFC ranged within the control values or slightly increased. The potentiation of MSV-M oncogenicity following infection with MuLV was studied in a more natural situation. Adult AKR mice, known to release endogenous MuLV continuously, were injected with MSV-M. The incidence of induced sarcomas was similar to that observed in control BALB/c mice inoculated with MSV-M. Moreover, tumors developed with a very long latent period. On the other hand, the great majority of tumors showed no regression and ultimately killed the host. Additional experiments, making use of immunologic manipulation of the host and Fl hybrids, suggest that the relative resistance to MSV-M oncogenesis in AKR mice is influenced by genetic and immunologic factors. MSV recovered from MSV-M-induced tumors in AKR and C58 mice was typed by highly specific mouse antisera. The results clearly showed that formation of a new MSV pseudotype occurred in vivo, the endogenous Gross virus acting as helper.

Defects in the regulation of programmed cell death play a fundamental role in the development of neoplasia and neurological disorders, both of which are linked to the human T-cell leukemia/lymphoma virus type 1 (HTLV-1) infection. We previously showed that the HTLV-1 Tax protein protects from apoptosis induced by serum starvation by preventing cytochrome c release and Bax relocation to mitochondria, two early events in the mitochondrial apoptotic pathway. As a natural extension of these findings, and to better define the action of Tax, in the present study, we investigated the outcome of Tax and two mutants which are inactive in CREB/ATF (M47) or NF-κB (M22) pathways, in the control of apoptosis induced by the proapoptotic Bax protein. We found that activation of CREB, rather than NF-κB, is a key phenomenon in preventing apoptosis. Furthermore, the importance of CREB activation is strengthened by experiments with CREB mutants, treatment with forskolin, and in situ analysis of P-CREB status in cells transfected with Tax or its nonprotecting M47 mutant. Considered together, these results underscore a primary role of CREB in preventing apoptosis triggered by Bax, and suggest that Tax might act by affecting the phosphorylation state of CREB.

Tumor growth is currently viewed as a phenomenon associated with neovascularization and sustained production of angiogenic factors, but whether a transient angiogenic switch may trigger tumor growth remains unclear. Here, we report that leukemia cells (MOLT-3) were poorly angiogenic and remained dormant when injected s.c. into immunodeficient mice. However, progressive growth of lymphoid tumors was invariably recorded when irradiated angiogenic cells from Kaposi’s sarcoma (KS-IMM) were locally coinjected with MOLT-3 cells or administered later. The persistence of KS-IMM cells in vivo was tracked by flow cytometry and real-time PCR analysis, and it was limited to a few days, during which angiogenesis was induced and preceded tumor growth. The engraftment of other types of poorly tumorigenic cancer cells was also greatly improved by irradiated KS-IMM cells. Moreover, short-term treatment with angiogenic factors, including basic FGF or VEGF, either given as recombinant factors or delivered by retroviral vectors, also accelerated tumor growth. These findings may emphasize that tumor angiogenesis is a process requiring a higher amount of angiogenic factors for its induction than maintenance.

Patterns of endemicity of human herpesvirus 8 (HHV8) are still undefined in some European populations, such as those from Western Balkan countries. Serum samples from 605 human immunodeficiency virus-seronegative subjects (299 Albanians and 306 Kosovars) were tested for the presence of HHV8 antibodies to a capsid-related open reading frame (ORF65)-encoded protein and a latency-associated nuclear antigen (LANA) to determine HHV8 seroprevalence in populations from Albania and from the Kosovo region of former Yugoslavia. Levels of co- circulation with hepatitis A (HAV) and hepatitis B (HBV) viruses were also determined. HHV8 antibodies to at least one of the two antigens were detected in 28.8% of Albanians and 18% of Kosovars. The seroprevalence of HHV8 was found to be 25.0 and 16.8% in Albanian and Kosovar children (

Ovarian cancer represents a suitable disease for gene therapy because of the containment of neoplastic cells in the peritoneal cavity even at advanced tumor stages. The aim of this study was to investigate whether intraperitoneal administration of a lentiviral vector encoding murine interferon-alpha (LV-IFN) could have therapeutic activity in a transplantable ovarian cancer model. Multiple injections of low amounts of LV-IFN into severe combined immunodeficiency (SCID) mice bearing IGROV-1 or OC316 ovarian cancer cells elicited remarkable antitumor activity, leading to prolongation of survival in the majority of animals. A definitive cure was obtained in animals bearing PD-OVA#1 tumors, generated by injecting tumor cells isolated from the ascitic fluid of a patient into SCID mice. Interferon-alpha levels were detected in the peritoneal fluids but not in the serum of treated mice, indicating that production of the cytokine is mainly local, by both tumor and normal cells of the host. Antitumor effects were associated with a remarkable decrease in the formation of hemorrhagic ascites, an increase in ischemic tumor necrosis, and a reduction in microvessel density. In conclusion, our findings show that intracavitary IFN-alpha gene therapy, using a lentiviral vector, provides strong antitumor effects in murine models of ovarian cancer and reinforces the evidence that angiogenesis inhibition is a promising strategy for the treatment of localized tumors.

Establishment of latently infected cell lines from primary effusion lymphomas (PEL) presently is the most efficient system for the propagation of clinical strains of human herpesvirus 8 (HHV-8) in culture. Here we describe a new approach to culture productively replicating HHV-8 from patient samples. A BJAB-derived B-cell line, BBF, was found to retain HHV-8 longer, to support the latent and lytic replication programs, and to produce transmissible virus. Supernatants from n -butyrate-treated peripheral blood mononuclear cells of 24 HHV-8-seropositive renal transplant recipients were used to infect BBF cells, and replicating virus was detected in cultures from 11 patients. Moreover, BBF cells infected with saliva strains showed a highly productive profile regardless of the initial viral load, which confirms that infectious HHV-8 can be present in saliva and also suggests that saliva strains may exhibit a high tropism for B lymphocytes. In conclusion, we established an in vitro system that efficiently detects HHV-8 in samples with low viral loads and that produces infectious progeny. BBF cells can be used to propagate HHV-8 from different biological samples as well as to clarify important issues related to virus-cell interactions in a context distinct from endothelial and PEL-derived cell lines.

The mechanisms by which intraperitoneal injection of peripheral blood mono-nuclear cells (PBMCs) from Epstein-Barr virus (EBV)–seropositive donors into severe combined immunodeficient (SCID) mice gives rise to lymphomas (hu/SCID tumors) are far from clear. This study addressed whether chemokine receptors and their ligands could be implicated in this experimental model. CXCR4 was found to be highly expressed in hu/SCID tumors; surface expression of CXCR4 was prevalently limited to a tumor cell subset poorly expressing CD23, whereas the CXCR4 ligand, CXCL12, was predominantly expressed by the tumor subpopulation expressing CD23. In vitro inhibition of this autocrine/paracrine CXCL12/CXCR4 axis significantly inhibited lymphoma proliferation and survival. Furthermore, CXCL12 was expressed in cells recovered from the mouse peritoneal cavity early after PBMC transfer as well as by EBV-transformed B cells but not by resting or activated B lymphocytes; also, lymphoma development was associated with a dramatic increase in the levels of murine CXCL12 present in the peritoneal cavity. Finally, antagonizing the CXCL12/CXCR4 axis in vivo strongly counteracted lymphoma development. These studies demonstrate that CXCL12 expression may be associated with EBV infection and suggest that the CXCR4/CXCL12 axis may participate in the EBV-associated lymphomagenesis process in immunodeficient hosts.

BACKGROUND: Up to 20 million persons are infected with the human retroviruses human T-lymphotropic virus (HTLV)-I and HTLV-II globally. Most data on the seroprevalence of HTLV-I and HTLV-II in Europe are from studies of low-risk blood donors or high-risk injection drug users (IDUs). Little is known about the general population. METHODS: A prospective anonymous study of HTLV-I and HTLV-II seroprevalence among 234,078 pregnant women in Belgium, France, Germany, Italy, Portugal, Spain, and the United Kingdom was conducted. Maternal antibody status was determined by standard methods using sera obtained for routine antenatal infection screens or eluted from infant heel prick dried blood spots obtained for routine neonatal metabolic screens. RESULTS: Anti-HTLV-I/II antibodies were detected and confirmed in 96 pregnant women (4.4 per 10,000, 95% confidence interval [CI]: 3.5-5.2). Of these, 73 were anti-HTLV-I, 17 were anti-HTLV-II, and 6 were specifically anti-HTLV but untyped. The seroprevalence ranged from 0.7 per 10,000 in Germany to 11.5 per 10,000 in France. CONCLUSIONS: Pregnant women better reflect the general population than blood donors or IDUs. The seroprevalence of HTLV-I and HTLV-II in Western Europe is 6-fold higher among pregnant women (4.4 per 10,000) than among blood donors (0.07 per 10,000). These data provide a robust baseline against which changes in HTLV-I and HTLV-II seroprevalence in Europe can be measured.

Patients infected with human immunodeficiency virus type 1 (HIV-1) develop a spectrum of B cell lymphoproliferative disorders ranging from polyclonal B cell activation to B cell lymphomas. While a direct role of Epstein-Barr virus (EBV) is well recognized for most of these lesions, recent findings have suggested that transactivator HIV-1 Tat protein might be involved in the pathogenesis of B cell lymphomas. Tat-expressing EBV-positive B cells were generated by transduction with a retroviral Tat-encoding vector. B(Tat+) cells expressed lower levels of anti-apoptotic protein Bcl-2 than parental and control B(Tat-) cells, generated by transduction with an empty retroviral vector, and were more prone to apoptosis upon serum withdrawal, as assessed by analysis of annexin V-stained cells and cleavage of poly-ADP-ribose-polymerase by caspase 3. Nevertheless, in serum starvation, B(Tat-) cells mainly exhibited the Rb hypo-phosphorylated form, underwent cell cycle arrest, and grew in single cell suspension, while B(Tat+) cells displayed the Rb hyper-phoshorylated form, progressed throughout the cell cycle, and retained the ability to grow in small clumps. Finding that B(Tat+) cells maintained proliferative capacity upon serum withdrawal suggests that cells expressing Tat have growth advantages among the EBV-driven cell proliferations and may originate B cell clones with more oncogenic potential.

The seroprevalence of human herpesvirus 8 (HHV-8) in a group of HIV-1-infected pregnant women and in mother–child pairs from Southeastern Italy (Apulia) was determined. Blood was collected from 49 HIV-1-infected women during pregnancy or at delivery as well as from their children. Samples were analysed for the presence of antibodies to the latency-associated nuclear antigen and a structural antigen encoded by open reading frame 65. The presence of antibodies to hepatitis C virus (HCV) was also determined. Nineteen women (38.7%) were found to be positive for HHV-8 antibodies to at least one of the two antigens, and 21 (42.9%) for HCV antibodies. HHV-8 antibodies were more common in injecting drug users (56.3%) than in women infected through heterosexual intercourse (30.3%). HCV antibodies were significantly more prevalent in HHV-8-seropositive (66.7%) than HHV- 8-seronegative (29%) women. Thirteen children born to HIV-1/HHV-8 co-infected women were HHV-8-seroreactive, with a variable pattern of reactivity to the analysed antigens. Follow-up of children showed a prolonged persistence of antibodies, in two cases for more than 12 months. This study has provided serological evidence for a high rate of HHV-8 infection in HIV-1-infected women in the Apulia region, and has identified a possible association between HHV-8 infection, past use of injection drugs and HCV infection. Parenteral transmission may, therefore, be a mode of virus spread. J. Med. Virol. 72:656–660, 2004. © 2004 Wiley-Liss, Inc.

Deregulation of the apoptotic process can lead to pathophysiological changes that result in either degenerative diseases or cancer. Although the transactivator Tax has been established as an essential effector of human T-lymphotropic virus type-1 (HTLV-1)-mediated diseases, which include both a neurodegenerative pathology and leukemia/lymphoma, its exact role(s) in the pathogenesis remains to be clarified. Because the apoptotic potential of Tax is still being debated, we addressed this question by testing the susceptibility of Tax(-) and Tax(+) cells to apoptosis under conditions of growth factor withdrawal and Bax overexpression. Results showed that Tax(+) cells are protected from apoptosis triggered by depletion of growth factors. This protective effect seems to be the result of a block in the apoptotic program regulated by mitochondria. Furthermore, in an attempt to elucidate which transcriptional pathway activated by Tax is important in the observed Tax-induced resistance, we found that CREB/ATF activity plays a relevant role in protecting cells from apoptosis induced by Bax overexpression. All together, these data might suggest that the ability of Tax to inhibit certain apoptotic stimuli could be important in its role as a viral transforming protein.

Mutations in BRCA1 and BRCA2 show different expressivity with respect to cancer risk, and allelic heterogeneity may be present in both genes. We collected 179 pedigrees with identified germline mutation (104 BRCA1 and 75 BRCA2), ascertained in six collaborating centers of the Italian Consortium for Hereditary Breast and Ovarian Cancer. Significant heterogeneity was detected for several variables, and a logistic regression model including age of diagnosis in the proband, presence of ovarian cancer in the family, presence of prostate or pancreatic cancer in the family, and presence of male breast cancer in the family proved to be effective in predicting the presence of a mutation in a gene rather than the other. Excess of familial aggregation of both breast and ovarian cancer was observed in both genes. Proportion of ovarian cancer was increased in the 5' portion of BRCA1, and presence of prostate or pancreatic cancer in a family was correlated with presence of ovarian cancer in BRCA2.

Epstein-Barr virus (EBV)-positive B-cell lymphoproliferative disease develops in severe combined immunodeficient (SCID) mice inoculated with peripheral blood mononuclear cells (PBMC) from EBV(+) individuals (SCID/hu mice). In this study, we investigated the contribution of EBV reactivation and de novo infection of B lymphocytes to tumor outgrowth in SCID/hu mice. Evaluation of BZLF-1, an early EBV activation transcript, in cells recovered from the mouse peritoneal cavity within 16 days following PBMC transfer did not reveal EBV reactivation, while BZLF-1 expression was only detected in tumor masses or in vitro established lymphoblastoid cell lines. To confirm these data by a different strategy, we coinjected PBMC from seropositive donors with purified B cells from seronegative donors of different sex. Fluorescence in situ hydridization analysis of the resulting tumor masses disclosed that the overwhelming majority of lymphoma cells originated from the seropositive donor, implying that no substantial in vivo production and transmission of virus had occurred. Further, treatment of SCID/hu mice with ganciclovir did not prevent lymphoma development. Our results suggest that in the SCID/hu mouse, early EBV replication and secondary infection of bystander B cells does not occur, and that the direct outgrowth of the transformed B lymphocytes present within the PBMC inoculum is the predominant mechanism, which leads to lymphoma generation in this experimental model.

Human T-cell leukemia virus type 1 encodes a number of “accessory” proteins of unclear function; one of these proteins, p13II, is targeted to mitochondria and disrupts mitochondrial morphology. The present study was undertaken to unravel the function of p13II through (i) determination of its submitochondrial localization and sequences required to alter mitochondrial morphology and (ii) an assessment of the biophysical and biological properties of synthetic peptides spanning residues 9–41 (p139–41), which include the amphipathic mitochondrial-targeting sequence of the protein. p139–41 folded into an α helix in micellar environments. Fractionation and immunogold labeling indicated that full-length p13II accumulates in the inner mitochondrial membrane. p139–41 induced energy-dependent swelling of isolated mitochondria by increasing inner membrane permeability to small cations (Na+, K+) and released Ca2+ from Ca2+-preloaded mitochondria. These effects as well as the ability of full-length p13II to alter mitochondrial morphology in cells required the presence of four arginines, forming the charged face of the targeting signal. The mitochondrial effects of p139–41 were insensitive to cyclosporin A, suggesting that full-length p13II might alter mitochondrial permeability through a permeability transition pore-independent mechanism, thus distinguishing it from the mitochondrial proteins Vpr and X of human immunodeficiency virus type 1 and hepatitis B virus, respectively.

Background The spontaneous in-vitro antibody synthesis observed in unstimulated lymphocyte cultures from HIV-infected patients closely reflects the in-vivo activation of the B cell compartment; however, the mechanisms underlying this phenomenon are far from clear. Methods We compared the ability of peripheral blood mononuclear cells (PBMC) and lymph-node cells (LNC) from 10 HIV-infected patients to produce in vitro HIV-specific and total Ig spontaneously, and we correlated these parameters with tumour necrosis factor alpha (TNF-alpha) expression by CD4 T cells, viral dissemination in the organism, and the extent of HIV spread into lymph-node germinal centres, measured by in-situ hybridization (ISH). Results In-vitro spontaneous synthesis of both HIV-specific and total antibody was significantly higher in PBMC than in LNC; the two variables showed a good correlation in LNC, but not in PBMC. In both compartments, no correlation was found between B cell activation and the percentage of CD4 T cells expressing TNF-alpha, which was increased compared with seronegative donors. Furthermore, no correlation was found between in-vitro spontaneous antibody synthesis and the number of T cells containing proviral HIV in PBMC and LNC, or the plasma levels of HIV RNA. On the contrary, a good correlation was found between HIV-specific B cell activation and the extent of viral spread into lymph-node germinal centres, evaluated by ISH. Conclusion These data suggest that the adhesion of HIV virions to the follicular dendritic cell network in lymph-node germinal centres may primarily contribute to sustaining the steady B cell activation observed in HIV-infected patients.

Objective Patients infected with HIV-1 are at high risk of developing Epstein–Barr virus (EBV)-associated lymphoproliferative disorders. This study evaluated the impact of highly active antiretroviral therapy (HAART) on EBV infection. Methods To measure EBV content in peripheral blood lymphocytes (PBL) and in plasma, we set up a quantitative analysis using the real-time PCR. EBV latent membrane protein 1 (LMP1) expression was determined by reverse transcriptase-PCR. Results EBV levels were determined in 33 HIV-1- and EBV-coinfected patients at the start of HAART, and during therapy. At baseline, EBV content in PBL samples ranged from 8 to 14 532 copies/μg DNA. EBV levels transiently increased in nine out of 17 patients in whom HIV-1 plasmaviraemia declined to undetectable levels (virological response) and CD4 cell counts increased (immunological response), while they remained fairly stable or decreased in the other eight virological and immunological responders, and in seven patients who showed a virological response only. Of interest, a significant increase in EBV load was observed in five out of nine patients who showed an increase in CD4 cell counts but lack of HIV-1 suppression during HAART. This EBV increase was accompanied by the detection of both LMP1 transcripts in PBL and EBV DNA in plasma, and was paralleled by an increase in immunoglobulin levels, a marker of B-cell stimulation. Conclusions These findings suggest that peripheral immune reconstitution during HAART without a reduction in HIV-1 replication may increase B-cell stimulation and the number of EBV-infected B cells.

Intraperitoneal transfer of peripheral blood mononuclear cells (PBMC) from human EBV+ donors into severe combined immunodeficiency (SCID) mice is a suitable model for studying some aspects of lymphomagenesis and immune activation. Neopterin is a soluble immune marker which was found to be a useful indicator for immune activation processes in humans, e.g. to monitor immunological complications in allograft recipients or to predict prognosis in HIV-infected individuals. In contrast, this pteridine compound is normally synthesized in murine organism in only very low amounts. The measurement of neopterin concentrations in serum and urine should be feasible in SCID mice reconstituted with human PBMC. In this study, we examined the usability of this experimental model for monitoring human T cell activation by neopterin measurements. The production of neopterin by SCID mice after injection of freshly isolated human PBMC, purified B or T cells and cultured Epstein-Barr virus (EBV)+ lymphoblastoid cells (LCL) was determined. It was found that neopterin can be detected early after injecting SCID mice with PBMC, whereas injection of purified human T or B cells did not result in neopterin production. Highest neopterin levels were detected in mice treated with LCL cells when developing lymphoma. We discuss the possible sources of neopterin along this process and its usefulness in this model.

The effect of human immunodeficiency virus type 1 (HIV-1) on telomerase activity in peripheral blood lymphocytes (PBL) was examined. Telomerase is an enzyme that is involved in mechanisms that control cell life span and replicative potential. HIV-1 reduced telomerase activity in in vitro-infected PBL and impaired enzyme activation upon cell stimulation. Telomerase activity was significantly lower in PBL from 23 HIV-1-infected patients than in PBL from healthy donors and significantly increased during highly active antiretroviral therapy (HAART) in 10 patients who had both a virological and an immunological response and in 5 and 8 patients with a virological or an immunological response, respectively. Further analyses of fractionated cells revealed that telomerase activity increased mainly in CD4(+) lymphocytes. Overall, these findings demonstrate that HIV-1 infection down-modulates telomerase activity and suggest that both the HIV-1 decline and immunorestoration in response to HAART contribute to increased telomerase activity in CD4(+) lymphocytes.

A serological survey for HTLV infection identified an AIDS patient with HTLV-I/HTLV-II dual seroreactivity. Two further sequential blood samples were collected (samples A and B) for PCR, restriction fragment length polymorphism (RFLP), and sequence analyses of HTLV-I and HTLV-II strains. PCR analyses confirmed dual infection in both samples. Restriction digests of the env region amplified from sample A showed the presence of an HTLV-IIa subtype; the HTLV-II provirus was found to be defective in the pol and env regions in the second sample from this patient. RFLP analysis of the HTLV-II LTR region of both samples confirmed this finding and identified an a5/bzl restriction type. Nucleotide sequence analyses revealed full homology in the HTLV-I env and LTR regions and in the HTLV-II LTR region between the two samples. These findings document the first case of an HTLV-I/HTLV-II coinfection that was fully confirmed and characterized by means of molecular analyses.

Homozygosity for a 32-base pair deletion (delta32) within the CC-chemokine receptor 5 (CCR5) gene confers resistance to infection by R5-type HIV-1 isolates. To ascertain how CCR5delta32 heterozygosity influences the susceptibility of lymphocytes and macrophages to HIV-1 infection, peripheral blood lymphocytes (PBLs) and monocyte-derived macrophages (MDMs) from three HIV-1-uninfected CCR5delta32 heterozygous infants and three HIV-1-uninfected CCR5 wild-type homozygous infants were exposed to two R5-type primary isolates. HIV-1 infection was monitored by DNA-PCR and p24 antigen determination; CCR5 and CCR5delta32 transcripts were quantified by competitive reverse transcription-PCR. Wild-type homozygous MDMs and PBLs and heterozygous PBLs were infected by both viral isolates, albeit with different efficiencies, but heterozygous MDMs showed restriction to HIV-1 infection. Lower levels of CCR5 mRNA and protein expression were found in heterozygous versus wild-type homozygous MDMs and PBLs. Interestingly, wild-type homozygous MDMs showed higher levels of CCR5 mRNA expression compared with wild-type homozygous PBLs, while heterozygous MDMs had lower levels of CCR5 wild-type mRNA and a higher CCR5delta32/CCR5 mRNA ratio compared with heterozygous PBLs. These findings suggest that CCR5delta32 heterozygosity confers a different degree of protection against HIV-1 in PBLs and MDMs, depending on the ratio of wild-type and mutant CCR5 mRNA in the two cell types, and may delay virus spread in the host by preventing infection of monocytes and macrophages.

Antiangiogenic therapy may represent a promising approach to cancer treatment. Indeed, the efficacy of endogenous angiogenesis inhibitors, including angiostatin, endostatin and TIMPs, has been demonstrated in many types of solid tumors in animal models. In view of the possible problems associated with long-term administration of inhibitors as recombinant proteins, we propose their delivery as nucleic acids through a gene therapy approach. To this end, eukaryotic expression constructs for murine angiostatin and endostatin as well as human TIMP-2 were generated, and characterized in vitro. All constructs carry the relevant cDNAs under the control of the strong HCMV promoter/enhancer, and cleavable leader signals to allow protein secretion. Expression of the angiogenesis inhibitors was detected by in vitro transcription/translation experiments as well as transfection of 293T cells, followed by Western blotting (WB) or radioimmunoprecipitation analysis of both cell lysates and supernatants (SNs). These constructs might be used for in vivo intramuscular delivery of plasmid DNA and as a set of reagents for the development of retroviral as well as adeno-associated viral (AAV) vectors expressing angiogenesis inhibitors.

The CD4/CD8 ratio has long been used for the follow-up and monitor of many infectious diseases. Following the demonstration in 1983 that the CD4/CD8 ratio in the mouse is under genetic control, it was subsequently shown to be controlled by a major locus in man. To define the mode of inheritance of the CD4/CD8 ratio, we addressed the absolute number of CD4 and CD8 cells in a large unselected control sample and in members of 70 nuclear families. Pedigrees of nuclear families were analyzed by complex segregation analysis. Data was adjusted prior to this analysis to remove the effects of relevant covariates. The non-genetic-transmission and the multifactorial model could be easily rejected for both CD4 and CD8 cells. Among the different inheritance models, involving both a major gene and a multifactorial (MFT) component, a major autosomal recessive gene with a residual MFT effect controlling the high number of CD4 and a major autosomal recessive gene with a residual MFT effect controlling the high number of CD8 cells were the significantly best-fitting ones. Our findings have some practical implications. Among all, the knowledge of the CD4+ cell number and the proportion between CD4+ and CD8+ T cells could be a useful parameter in predicting human immunodeficiency virus infection outcome.