Davide Gibellini

Quantitative detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells by SYBR green real-time PCR technique

BACKGROUND The persistence of proviral human immunodeficiency virus type 1 (HIV-1) DNA reservoir represents one of the major drawbacks to the total eradication of HIV-1. The quantitative determination of proviral HIV-1 DNA load offers significant therapeutic information, especially when the HIV-1 RNA levels drop below the detectable limits during the highly active retroviral therapy (HAART) treatment. Moreover, the detection of HIV-1 proviral DNA is an important diagnostic marker in the evaluation of HIV-1 infection of newborns of HIV-1 seropositive women. OBJECTIVE We evaluated a real-time PCR based on LightCycler technology revealed through SYBR green fluorochrome (SYBR green real-time PCR) to quantify the HIV-1 proviral DNA load in peripheral blood mononuclear cells (PBMC) of HIV-1 seropositive patients. STUDY DESIGN Firstly, we assessed the SYBR green real-time quantitative PCR for HIV-1 proviral DNA load detection determining the specificity and sensitivity of the assay using the LightCycler system. Secondly, we tested the performance of our SYBR green real-time PCR on 50 HIV-1 seropositive patients under HAART and 20 blood donors. RESULTS/CONCLUSIONS The results of this study showed that our SYBR green real-time PCR is able to detect five copies of the HIV-1 genome. Moreover, our method revealed HIV-1 proviral DNA in all the 50 HIV-1 seropositive patients ( 627 +/- 1068 HIV-1 proviral DNA copies per 10(6) PBMC, with a range of 30-6300 copies), whereas no positive signal was observed in any PBMC blood donors. Our SYBR green real-time PCR represents a sensitive and useful approach that could be applied both in HIV-1 proviral DNA reservoir determination and in HAART monitoring, particularly when the HIV-1 plasmatic RNA is undetectable.

Antibodies against full-length Tat protein and some low-molecular-weight Tat-peptides correlate with low or undetectable viral load in HIV-1 seropositive patients

BACKGROUND The efficacy of a specific humoral response to transactivating Tat protein was studied in a group of HIV-1 seropositive drug addicts, who had previously received a similar course of anti-retroviral treatment with two reverse transcriptase inhibitors. OBJECTIVES The aim of the study was to evaluate the meaning of an immune response to Tat protein in HIV-1 seropositive patients with different levels of HIV-1 RNA viremia. STUDY DESIGN The study analyzed the presence of anti-Tat antibody reacting either with full-length Tat or with individual overlapping Tat-peptides (Tat(6-14), Tat(11-24), Tat(36-50), Tat(46-60), Tat(56-70) and Tat(65-80)), in a group of HIV-1 seropositive subjects with different peripheral blood viral loads. Plasma samples were examined by immunoenzymatic assay for the presence of anti-Tat IgG antibody and for the quantification of peripheral blood (plasma) viral load by branched DNA assay. RESULTS The large majority of HIV-1 patients showed detectable levels of serum IgG to full-length-Tat, and the anti-Tat antibody level presented an inverse correlation with viral load magnitude. The analysis of antibody levels against individual overlapping Tat-peptides clearly showed that an undetectable viral load was significantly associated with the presence of a high antibody concentration against Tat(6-14), Tat(36-50) and Tat(46-60) (P=0.002, P=0.027 and P<0.001, respectively). CONCLUSION In HIV-1-infected patients, a strong humoral immune response against HIV-1 Tat protein is inversely correlated to peripheral blood viral load and, in particular, a high level of antibody against Tat peptides containing amino acid residues 6-14 (Tat(6-14)), 36-50 (Tat(36-50)) and 46-60 (Tat(46-60)) is associated with an undetectable plasma viral load. These findings may help to tailor anti-HIV-1 Tat-containing vaccines.

HIV-1 triggers apoptosis in primary osteoblasts and HOBIT cells through TNFα activation

Several HIV‐1 infected patients show bone loss and osteopenia/osteoporosis during the course of disease. The mechanisms underlying this degenerative process are largely unsettled and it has not been determined yet whether bone dysfunction is linked to HIV‐1‐mediated direct and/or indirect effects on osteoblasts/osteoclasts cross‐talk regulation. This study investigated the effects of HIV‐1IIIb and HIV‐1ADA strains on osteoblasts using the osteoblast‐derived cell line (HOBIT) and primary human osteoblasts as cellular models. The challenge of these cell cultures by both HIV‐1 strains triggered a significant apoptosis activation unrelated to viral infection, since proviral HIV‐1 DNA and supernatant HIV‐1 RNA were not detected by real time PCR or b‐DNA assays respectively. Under the experimental conditions, even heat‐inactivated HIV‐1 or cross‐linked recombinant gp120 treatment of HOBIT and osteoblasts induced programmed cell death, suggesting that apoptosis is regulated by the interaction between HIV‐1 gp120 and cell membrane. The analysis of cell culture supernatants showed a significant up‐regulation of TNFα, a pleiotropic protein considered an apoptosis inducer in the osteoblast model. In fact, pretreatment of HOBIT and osteoblast cell cultures with anti‐TNFα polyclonal antibody tackled effectively HIV‐1 related induction of cell apoptosis. As a whole, these results indicate that HIV‐1 may impair bone mass structure homeostasis by TNFα regulated osteoblast apoptosis. J. Med. Virol. 80:1507–1514, 2008.

Metabolic bone disease in HIV infection.

HIV mainly replicates in CD4þ T lymphocytes andmonocyte/macrophages causing severe immunologicalimpairment. In addition to the immune system, HIVinfection affects tissues and organs such as kidney, liver,the central nervous system, heart and bone showing acomplex pathogenesis [1].The advent and widespread use of highly activeantiretroviral therapy (HAART) in the last two decadeshasled toa markedimprovementinthe treatmentofHIVdisease even though viral infection cannot be eradicatedbecause HAART does not completely eliminate the viralreservoirs [2]. HAART has dramatically changed thecourseofHIVinfection froma fatalinfectiontoachronicand relatively manageable disease. The increased lifeexpectancyofHIVpatientsandtheeffectsofHAARThavechanged the management of HIV infection. Nowadaysmedical treatment is no longer focused solely on HIVinfection, opportunistic diseases and monitoring immunederangement, but also includes the control of metabolic,cardiovascular, liver, bone and kidney complications. Inparticular, bone alterations have been observed in thecourse of HIV disease representing a pivotal clinicalprobleminthemanagementofHIVpatientsespeciallyforapossible development of bone fractures [3]. The majorbonelesionsdetectableinHIVpatientsarerelatedtobonedemineralization (osteopenia/osteoporosis and osteoma-lacia) and osteonecrosis ([4] for a review).This report will discuss the pathogenesis, diagnosis andtreatment of major bone complications represented bybone demineralization diseases during HIV infection andHAART treatment.

HIV-1 Tat-mediated Inhibition of the Tyrosine Hydroxylase Gene Expression in Dopaminergic Neuronal Cells

Treatment of dopaminergic rat PC12 cells with human immunodeficiency virus, type 1 (HIV-1) Tat protein ortat cDNA inhibited the expression of tyrosine hydroxylase (TH), the rate-limiting enzyme for the dopamine biosynthetic pathway, as well as the production and release of dopamine into the culture medium. Moreover, the Tat addition to PC12 cells up-regulated the expression of the inducible cAMP early repressor (ICER), a specific member of the cAMP-responsive element modulator transcription factor family, in a cAMP-dependent manner. In turn, ICER overexpression abrogated the transcription activity of the TH promoter in PC12 cells, strongly suggesting ICER involvement in Tat-mediated inhibition of TH gene expression. In vivoinjection of synthetic HIV-1 Tat protein into the striatum of healthy rats induced a subclinical Parkinsons-like disease that became manifested only when the animals were treated with amphetamine. As early as one week postinjection, the histochemical examination of the rat substantia nigra showed a reduced staining of neurons expressing TH followed by a loss of TH+ neurons at later time points. As Tat protein can be locally released into the central nervous system by HIV-1-infected microglial cells, our findings may contribute to the explanation of the pathogenesis of the motorial abnormalities often reported in HIV-1 seropositive individuals.

Recombinant human immunodeficiency virus type-1 (HIV-1) Tat protein sequentially up-regulates IL-6 and TFG-β1 mRNA expression and protein synthesis in peripheral blood monocytes

Summary. In this study we evaluated the effect of human immunodeficiency virus type 1 (HIV‐1) recombinant Tat protein on mRNA expression and protein synthesis of two inflammatory cytokines ‐ interleukin‐6 (IL‐6) and transforming growth factor‐β1 (TGF‐β1) ‐ by peripheral blood (PB) monocytes. Whereas maximal levels of IL‐6 protein were recovered in PB monocyte culture supernatants after 24–48 h from the addition of 1 μg/ml of recombinant Tat, TGF‐β1 showed a slower and progressive increase, reaching maximal levels only after 72–96h of culture. Consistently, the analysis of the steady‐state levels of mRNA showed a sharp increse of IL‐6 mRNA expression after 24h of culture, with a slow decline thereafter. On the other hand, TGF‐β1 mRNA expression showed a slow increase only after 72–96h of culture. Moreover, IL‐6 appeared involved in the up‐regulation of TGF‐β1, because the addition of a neutralizing anti‐IL‐6 antibody to Tat‐treated PB monocyte cultures significantly reduced the amounts of TGF‐β1 recovered in the culture supernatants after 96 h.

Tat‐expressing Jurkat cells show an increased resistance to different apoptotic stimuli, including acute human immunodeficiency virus‐type 1 (HIV ‐1) infection

Human CD4+ T lymphoblastoid Jurkat cells were stably transfected with two different plasmid vectors containing the cDNA of human immunodeficiency virus-type 1 (HIV-1) tat gene under the control of either the promoter of simian virus 40 (pRPneo/tat) or the long terminal repeat region of SL3 murine leukaemia virus (pRPneo/SL3/tat). Both pRPneo/tat and pRPneo/SL3/tat Jurkat cell lines showed a constant and high production of bioactive Tat in transient co-transfection assays with an HIV-1 long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) reporter plasmid. Tat-positive and mock-transfected Jurkat cells were cultured with various cytotoxic agents, which have been associated to the progressive loss of CD4 T-lymphocytes characteristic of HIV-1 disease. In the presence of recombinant tumour necrosis factor-alpha (TNF-alpha), anti-fas antibody, Leu3a anti-CD4 antibody, the percentage of apoptosis, evaluated in a 24-72 h short-term assay, was lower (P < 0.05) in tat-positive Jurkat cells than in mock-transfected controls. The low susceptibility to the cytotoxic activity of TNF-alpha and anti-fas antibody of tat-transfected cells was confirmed by counting viable cells up to 15 d of culture. Also, recombinant Tat protein was able to prevent the increase of apoptosis induced in mock-transfected Jurkat by TNF-alpha. Of note, tat-expressing cells showed a better survival with respect to mock-transfected control cells even when acutely infected with high doses (500,000 cpm of reverse transcriptase) of HIV-1 (strain IIIB) or treated with heat-inactivated HIV-1. These data demonstrate that the expression of the regulatory HIV-1 Tat protein is able to rescue Jurkat lymphoblastoid cells from apoptosis induced by a variety of cytotoxic agents. Since Tat protein expression is restricted to the initial phases of an active HIV-1 replication, the anti-apoptotic effect of Tat could have the physiological significance of selectively protecting HIV-1 producing cells from death, at least for the time necessary to allow virus production and spreading.

Uninfected haematopoietic progenitor (CD34+) cells purified from the bone marrow of AIDS patients are committed to apoptotic cell death in culture

ObjectiveTo determine the mechanism underlying the poor growth in vitro of haematopoietic progenitor cells isolated from HIV-1-infected patients. MethodApoptotic death in liquid culture of bone-marrow CD34+ cells obtained from 11 HIV-1-seropositive patients and 18 HIV-1-seronegative donors was quantitatively monitored by a flow cytometry procedure. ResultsNo significant differences in the percentage of apoptotic cells were noted between the two groups immediately after purification. When CD34 + cells were placed in liquid cultures supplemented with 2 ng/ml interleukin-3, the number of apoptotic cells progressively and significantly (P < 0.05) increased in all HIV-1-seropositive patients, while it remained constant in HIV-1-seronegative individuals. Although all HIV-1-seropositive patients showed signs of active viral replication in the bone-marrow micro-environment, progenitor CD34 + cells did not show the presence of active and/or latent HIV-1 infection. ConclusionOur data demonstrate that CD34 + cells isolated from AIDS patients with active HIV-1 replication in bone-marrow accessory cells are committed to apoptotic death without being directly affected by productive infection.

HIV-1 and HCV detection in dried blood spots by SYBR Green multiplex real-time RT-PCR

Dried blood spot (DBS) is a reliable method of blood collection used for the diagnosis of several human diseases. DBS is particularly useful for diagnosing children and for the screening of high-risk populations especially in countries where health facilities are not readily accessible. This report describes a qualitative SYBR Green-based real-time multiplex RT-PCR for the simultaneous detection of hepatitis C virus (HCV) and human immunodeficiency virus type 1 (HIV-1) genomes in DBS. Specific viral amplicons were identified in the same sample by their distinctive melting temperatures. The analysis of scalar concentrations of the reference samples indicated that this multiplex procedure detects at least 2500 copies/ml of HCV and 400 copies/ml of HIV-1. HIV-1 and HCV viral loads in 20 patients infected with HIV-1 and/or HCV and in 5 healthy blood donors were also tested, confirming the sensitivity and specificity of the assay. This method may represent a reliable alternative for the detection of HIV-1/HCV co-infection, in rapid and relatively inexpensive screening programmes.

Molecular profiling of blastic plasmacytoid dendritic cell neoplasm reveals a unique pattern and suggests selective sensitivity to NF-kB pathway inhibition

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare disease of controversial origin recently recognized as a neoplasm deriving from plasmacytoid dendritic cells (pDCs). Nevertheless, it remains an orphan tumor with obscure biology and dismal prognosis. To better understand the pathobiology of BPDCN and discover new targets for effective therapies, the gene expression profile (GEP) of 25 BPDCN samples was analyzed and compared with that of pDCs, their postulated normal counterpart. Validation was performed by immunohistochemistry (IHC), whereas functional experiments were carried out ex vivo. For the first time at the molecular level, we definitely recognized the cellular derivation of BPDCN that proved to originate from the myeloid lineage and in particular, from resting pDCs. Furthermore, thanks to an integrated bioinformatic approach we discovered aberrant activation of the NF-kB pathway and suggested it as a novel therapeutic target. We tested the efficacy of anti-NF-kB-treatment on the BPDCN cell line CAL-1, and successfully demonstrated by GEP and IHC the molecular shutoff of the NF-kB pathway. In conclusion, we identified a molecular signature representative of the transcriptional abnormalities of BPDCN and developed a cellular model proposing a novel therapeutic approach in the setting of this otherwise incurable disease.