Luigi Chieco-Bianchi

Identification of Genes Selectively Regulated by IFNs in Endothelial Cells

IFNs are highly pleiotropic cytokines also endowed with marked antiangiogenic activity. In this study, the mRNA expression profiles of endothelial cells (EC) exposed in vitro to IFN-α, IFN-β, or IFN-γ were determined. We found that in HUVEC as well as in other EC types 175 genes were up-regulated (>2-fold increase) by IFNs, including genes involved in the host response to RNA viruses, inflammation, and apoptosis. Interestingly, 41 genes showed a >5-fold higher induction by IFN-α in EC compared with human fibroblasts; among them, the gene encoding the angiostatic chemokine CXCL11 was selectively induced by IFN-α in EC along with other genes associated with angiogenesis regulation, including CXCL10, TRAIL, and guanylate-binding protein 1. These transcriptional changes were confirmed and extended by quantitative PCR analysis and ELISA; whereas IFN-α and IFN-β exerted virtually identical effects on transcriptome modulation, a differential gene regulation by type I and type II IFN emerged, especially as far as quantitative aspects were concerned. In vivo, IFN-α-producing tumors overexpressed murine CXCL10 and CXCL11, guanylate-binding protein 1, and TRAIL, with evidence of CXCL11 production by tumor-associated EC. Overall, these findings improve our understanding of the antiangiogenic effects of IFNs by showing that these cytokines trigger an antiangiogenic transcriptional program in EC. Moreover, we suggest that quantitative differences in the magnitude of the transcriptional activation of IFN-responsive genes could form the basis for cell-specific transcriptional signatures.

Antigen detection, virus culture, polymerase chain reaction, and in vitro antibody production in the diagnosis of vertically transmitted HIV-1 infection

Polymerase chain reaction (PCR), virus culture (V), antigen detection (Ag), and in vitro antibody production (IVAP) assays may be useful for the early detection of vertically transmitted HIV-1 infection in infants under 18 months of age, when a diagnosis cannot be based on seropositivity because of maternal antibody persistence. To assess the reliability of these procedures and to correlate diagnostic results with infection status, 101 children born to HIV-1-seropositive mothers were evaluated by all these techniques within the first 6 months of life. The children were then followed up to the age of at least 18 months, when diagnosis was made on the basis of AIDS or AIDS-related complex (ARC) onset or persistence of HIV-1 seropositivity. Out of 27 children classified as infected according to the above criteria, 25 (92.5%) were repeatedly positive in IVAP test, 22 (81.5%) in the first PCR analysis, and only 19 (70.3%) in the initial V assay. On further testing, a total of 24 children (88.9%) were found positive in PCR assay, and 23 (85.2%) in V test. All these assays were found to be more sensitive than antigen detection for HIV-1 infection diagnosis, but the antigenaemia was shown to be a useful prognostic marker of disease onset. We also found that both Ag and IVAP assays could give false-positive results in the first 2 months of life, which severely limits their diagnostic value during this period of time. False-positive results in PCR assay could occur at any time of the tested period and were unrelated to the childs age.(ABSTRACT TRUNCATED AT 250 WORDS)

Dynamics of viral replication in infants with vertically acquired human immunodeficiency virus type 1 infection.

About one-third of vertically HIV-1 infected infants develop AIDS within the first months of life; the remainder show slower disease progression. We investigated the relationship between the pattern of HIV-1 replication early in life and disease outcome in eleven infected infants sequentially studied from birth. Viral load in cells and plasma was measured by highly sensitive competitive PCR-based methods. Although all infants showed an increase in the indices of viral replication within their first weeks of life, three distinct patterns emerged: (a) a rapid increase in plasma viral RNA and cell-associated proviral DNA during the first 4-6 wk, reaching high steady state levels (> 1,000 HIV-1 copies/10(5) PBMC and > 1,000,000 RNA copies/ml plasma) within 2-3 mo of age; (b) a similar initial rapid increase in viral load, followed by a 2.5-50-fold decline in viral levels; (c) a significantly lower (> 10-fold) viral increase during the first 4-6 wk of age. All infants displaying the first pattern developed early AIDS, while infants with slower clinical progression exhibited the second or third pattern. These findings demonstrate that the pattern of viral replication and clearance in the first 2-3 mo of life is strictly correlated with, and predictive of disease evolution in vertically infected infants.

Regression of AIDS-related Kaposi's sarcoma following antiretroviral therapy with protease inhibitors: biological correlates of clinical outcome

The clinical response of AIDS-related Kaposis sarcoma (KS) to highly active antiretroviral therapy (HAART), a combination of human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase inhibitors, was studied in 11 patients, all but one with progressive KS. CD4+ cell counts, plasma HIV-1 RNA levels, and antibody titres to lytic ORF65 and latency-associated human herpes virus type 8 (HHV-8) proteins were determined in sequential samples. Six complete and three partial clinical responses were achieved in a median time of 6 and 3 months, respectively, and confirmed after a median time of 16 months on HAART. 2 patients showed disease progression. A consistent decrease in HIV-1 RNA levels, paralleled by an increase in CD4+ cell counts, was observed in all patients who showed complete or partial clinical response; HIV-1 RNA levels remained persistently high in the two patients who progressed, despite a change in HAART. HHV-8 antibody titres were generally higher in patients with mucosal/visceral involvement compared with patients with limited disease; a decrease in ORF65 antibody titre was significantly associated with a clinical response. These results indicate that HAART is effective for AIDS-related KS; the clinical response correlates with a decrease in plasma HIV-1 RNA levels, an increase in CD4+ lymphocytes, and a decrease in antibodies to ORF65 HHV-8 protein.

Viral phenotype and host-cell susceptibility to HIV-1 infection as risk factors for mother-to-child HIV-1 transmission

Objective: To investigate the role of maternal HIV‐1 isolate phenotype and a childs cell susceptibility/resistance to viral infection in mother‐to‐child HIV‐1 transmission. Patients and methods: Forty‐nine women were studied at the time of delivery. Primary isolates, obtained by culturing patient peripheral blood mononuclear cells (PBMC) with PBMC from healthy donors, were characterized for tropism and syncytium‐inducing capability in monocyte‐derived macrophages (MDM), peripheral blood lymphocytes (PBL), and in the MT‐2 and MOLT‐3 T‐cell lines. Results: Seven women transmitted HIV‐1 to their children. Primary isolates were obtained from six and 28 transmitting and non‐transmitting mothers, respectively. All primary isolates from transmitting mothers and their infants but only 50% of those from non‐transmitting mothers replicated in MDM, regardless of their replication capacity in T‐cell lines. PBL and MDM cells from six uninfected children were exposed to the corresponding maternal isolates. Polymerase chain reaction analysis of HIV‐1 DNA in cells and p24 antigen assay in culture supernatants disclosed that two PBL and five MDM cultures were resistant to viral infection; two other PBL cultures, although HIV‐1‐infected, were negative for p24 production. Depletion of CD8+ cells only partially restored productive infection in CD4+ cell cultures. Moreover, all six PBL but only one MDM cultures were productively infected by an isolate obtained from a transmitting mother, thus suggesting that MDM resistance to HIV‐1 infection is not viral isolate‐restricted. Conclusions: Our findings strongly suggest that mother‐to‐child HIV‐1 transmission is influenced by both monocyte‐macrophage tropism of the maternal isolate and susceptibility of the childs target cells, in particular monocyte‐macrophages, to HIV‐1 infection. AIDS 1995, 9:427‐434

Mitochondrial targeting of the p13II protein coded by the x-II ORF of human T-cell leukemia/lymphotropic virus type I (HTLV-I).

The X region of the HTLV-I genome contains four major open reading frames (ORFs), two of which, termed x-I and x-II, are of still undefined biological significance. By indirect immunofluorescence and dual labeling with marker proteins, we demonstrate that p13II, an 87-amino acid protein coded by the x-II ORF, is selectively targeted to mitochondria. Mutational analysis revealed that mitochondrial targeting of p13II is directed by an atypical 10-amino acid signal sequence that is not cleaved upon import and is able to target the Green Fluorescent Protein to mitochondria. Expression of p13II results in specific alterations of mitochondrial morphology and distribution from a typical string-like, dispersed network to round-shaped clusters, suggesting that p13II might interfere with processes relying on an intact mitochondrial architecture. Functional studies of mitochondria with the cationic fluorochrome tetramethylrhodamine revealed that a subpopulation of the cells with p13II-positive mitochondria show a disruption in the mitochondrial inner membrane potential (Δψ), an early event observed in cells committed to apoptosis. Taken together, these results suggest novel virus-cell interactions that might be important in HTLV-I replication and/or pathogenicity.

Differential effects of angiostatin, endostatin and interferon-α1 gene transfer on in vivo growth of human breast cancer cells

The administration of different angiogenesis inhibitors by gene transfer has been shown to result in inhibition of tumor growth in animal tumor models, but the potency of these genes has been only partially evaluated in comparative studies to date. To identify the most effective anti-angiogenic molecule for delivery by retroviral vectors, we investigated the effects of angiostatin, endostatin and interferon(IFN)-α1 gene transfer in in vivo models of breast cancer induced neovascularization and tumor growth. Moloney leukemia virus-based retroviral vectors for expression of murine angiostatin, endostatin and IFN-α1 were generated, characterized, and used to transduce human breast cancer cell lines (MCF7 and MDA-MB435). Secretion of the recombinant proteins was confirmed by biological and Western blotting assays. Their production did not impair in vitro growth of these breast cancer cells nor their viability, and did not interfere with the expression of angiogenic factors. However, primary endothelial cell proliferation and migration in vitro were inhibited by supernatants of the transduced cells containing angiostatin, endostatin, and IFN-α1. Stable gene transfer of the IFN-α1 cDNA by retroviral vectors in both MCF7 and MDA-MB435 cells resulted in a marked and long-lasting inhibition of tumor growth in nude mice that was associated with reduced vascularization. Endostatin reduced the in vivo growth of MDA-MB435, but not MCF7 cells, despite similar levels of in vivo production, and angiostatin did not impair the in vivo growth of either cell line. These findings indicate heterogeneity in the therapeutic efficacy of angiostatic molecules delivered by viral vectors and suggest that gene therapy with IFN-α1 and endostatin might be useful for treatment of breast cancer.

Spontaneous in vitro production of virus-specific antibody by lymphocytes from HIV-infected subjects

In vitro synthesis of IgG directed against HIV components was detected by ELISA and Western blot assay of lymphocyte culture supernatants. Lymphocytes from HIV-infected individuals spontaneously produced antibody against HIV proteins very early in culture, suggesting in vivo activation of HIV-specific antibody-forming cells. The frequency of circulating B cells spontaneously secreting HIV-specific IgG was very high in some cases, but spontaneous HIV-specific antibody synthesis was not accompanied by polyclonal reactivation of B-cell clones of different specificity. The pattern of specificity of the anti-HIV antibody produced in vitro did not reflect the serum pattern consistently. These findings indicate a new approach potentially useful for the study of the immunobiology of HIV infection. The possible implications of the in vitro production of HIV-specific antibody for the diagnosis, prognosis and clinical management of this infection are also discussed.

Dynamics of Epstein-Barr virus in HIV-1-infected subjects on highly active antiretroviral therapy.

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.

Vertical transmission of HIV-1 : lack of detectable virus in peripheral blood cells of infected children at birth

ObjectiveTo evaluate the time-course of HIV-1 detection in peripheral blood mononuclear cells (PBMC) from newborns at risk of vertically acquired infection. Design and methodForty-six infants born to HIV-1-infected mothers were enrolled at birth and examined virologically and clinically in the perinatal period and every 30 days for the first 3 months of life. Follow-up was conducted at intervals of 2–3 months. HIV-1 detection in PBMC was performed using virus culture and polymerase chain reaction (PCR) assay. ResultsOnly one out of 24 newborns tested within 48 h of delivery and two out of 22 infants tested between 3 and 15 days of age were found to be HIV-1-positive by both PCR and virus culture. Further testing performed between 30 and 60 days of life identified an additional eight HIV-1-positive children. Subsequent viral, immunological and clinical follow-up confirmed PCR and virus culture results obtained in 30–60-day-old children. ConclusionsInfected infants had detectable levels of HIV-1 in their PBMC at 1 month of age. The negative PCR and virus culture findings in PBMC of newborns indicate strongly that HIV-1 cannot be diagnosed at birth in the majority of cases, and suggests that viral transmission could occur during late pregnancy and/or delivery.