Michele La Placa

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.

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.

Impaired survival of bone marrow GPIIb/IIIa+ megakaryocytic cells as an additional pathogenetic mechanism of HIV-1-related thrombocytopenia

Glycoproteic (GP) IIb/IIIa+ megakaryocytic cells were purified from the bone marrow (BM) of 15 HIV‐1 seropositive thrombocytopenic patients, eight HIV‐1 seronegative patients affected by immune thrombocytopenic purpura (ITP) and 14 HIV‐1 seronegative normal donors. The presence of apoptosis was evaluated in freshly isolated GPIIb/IIIa+ cells by flow cytometry after propidium iodide staining and electron microscopy. GPIIb/IIIa+ cells from HIV‐1 seropositive thrombocytopenic patients showed a significant ( P < 0.001) increase of apoptosis with respect to both HIV‐1 seronegative ITP patients and normal donors. Moreover, the degree of apoptosis in bone marrow GPIIb/IIIa+ cells purified from HIV‐1 seropositive thrombocytopenic patients was inversely ( P < 0.01) related to the count of circulating platelets, whereas it did not show any significant correlation with the absolute number of circulating CD4 T cells, the CD4/CD8 ratio or the presence of proviral gag DNA sequences. Therefore neither an advanced stage of HIV‐1 disease nor a direct infection with HIV‐1 seems to play a primary role in the impaired survival of BM GPIIb/IIIa+ megakaryocytic cells. These findings strengthen the notion that, besides the immune targeting of peripheral platelets, an impairment of the bone marrow megakaryocyte compartment may also contribute to the pathogenesis of HIV‐1 related thrombocytopenia.

Impaired telomerase activity in uninfected haematopoietic progenitors in HIV-1-infected patients

Objective: To evaluate the role of the selective forces exerted by the host on the HIV‐1 structures involved in viral entry. Design and methods: The V3 region of the env gene was analysed in cell‐free HIV‐1 RNA from 17 infected subjects: 11 long‐term non‐progressors (LTNP) and six symptomless, typical progressor patients. To evaluate the potential biological significance of one of the rare variants detected in the LTNP, it was reproduced by recombinant PCR into a HIV‐1 molecular clone. Results: The intrapatient divergence of the V3‐loop sequences averaged 8.62% in LTNP and 5.29% in progressors, although LTNP displayed lower divergence from the clade B consensus than progressors (16.65 and 19.76%, respectively). The analysis of non‐synonymous and synonymous substitutions indicated that selective pressure was exerted in this region in both LTNP and progressors. Individual peculiarities (unique and rare V3‐loop variants) emerged, however, in most sequences from LTNP, and variants bearing mutations in a domain crucial for the V3‐loop structure were more prevalent in LTNP (P = 0.0012). The pNL4–3‐derived mutant reproducing a V3‐loop variant detected in a LTNP was efficiently expressed upon transfection, but the mutant virus was nearly completely unable to infect CD4+ cell lines, activated primary peripheral blood lymphocytes, or monocyte‐derived macrophages, suggesting that a defect impaired the entry phase of the replication cycle. Conclusions: The results indicate that host factors impose selective constraints on the evolution of the HIV‐1 structures involved in viral entry. In LTNP, these factors are likely to force the virus into attenuated variants.Background: Haematopoietic progenitor cells (HPC) of HIV‐1‐infected patients are severely compromised in their replication and clonogenic capacities, and show an enhanced propensity to apoptosis, despite the lack of productive or latent HIV‐1 infection. Objective: To investigate telomerase enzyme levels in CD34+ HPC isolated from HIV‐1‐infected patients, because the absence of telomerase activity has been found to be correlated with a diminished replication potential. Methods: Telomerase levels were measured by a PCR‐based telomeric repeat amplification protocol. CD34+ HPC isolated from the peripheral blood of 11 HIV‐1‐infected patients were compared with CD34+ HPC isolated from peripheral blood (nine subjects) or bone marrow (six subjects) from 15 healthy donors. Telomerase levels were also studied in normal HPC after exposure to either gp120 or transforming growth factor (TGF)‐β1. Results: CD34+ HPC isolated from either peripheral blood or bone marrow from healthy donors expressed a high level of telomerase activity. On the contrary, CD34+ HPC isolated from HIV‐1‐seropositive patients did not express any detectable telomerase activity in nine patients, and a clearly reduced enzymatic activity in two patients. Furthermore, telomerase activity in normal CD34+ HPC exposed to recombinant gp120 was significantly reduced, and to a higher extent than in CD34+ HPC exposed to recombinant TGF‐β1. Conclusions: This is the first study to demonstrate severely impaired telomerase activity in uninfected CD34+ HPC isolated from HIV‐1‐infected patients. The mechanism underlying this impairment probably involves the interaction of HIV‐1 envelope glycoprotein gp120 with the cell membrane. These results may add to our understanding of the pathogenesis of the lesion of the HPC compartment.

Stroma-derived factor 1α induces a selective inhibition of human erythroid development via the functional upregulation of Fas/CD95 ligand

CXC chemokine receptor 4 (CXCR4), the high‐affinity receptor for stroma‐derived factor 1α (SDF‐1α), shows distinct patterns of expression in human CD34+ haematopoietic progenitor cells induced to differentiate in vitro along the granulocytic and erythroid lineages. In serum‐free liquid cultures supplemented with stem cell factor (SCF), interleukin 3 (IL‐3) and granulocyte colony‐stimulating factor, the expression of surface CXCR4 progressively increased in cells differentiating along the granulocytic lineage. The addition in culture of 200 ng/ml of SDF‐1α, a concentration which maximally activated intracellular Ca2+ flux, only modestly affected the expression levels of CD15 and CD11b granulocytic antigens, as well as the total number of viable cells. On the other hand, in liquid cultures supplemented with SCF, IL‐3 and erythropoietin, SDF‐1α induced the downregulation of glycophorin A erythroid antigen, accompanied by a progressive decline in the number of viable erythroblasts. Moreover, in semisolid assays, SDF‐1α significantly reduced the number of plurifocal erythroid colonies (erythroid blast‐forming units; BFU‐E), whereas it did not affect granulocyte–macrophage colony‐forming units (CFU‐GM). We also demonstrated that the inhibitory effect of SDF‐1α on glycophorin A+ erythroid cell development was mediated by the functional upregulation of CD95L in erythroid cultures. These data indicate that SDF‐1α plays a role as a negative regulator of erythropoiesis.

Development of transplantable ascites tumours which continuously produce polyclonal antibodies in pristane primed BALB/c mice immunized with bacterial antigens and complete Freund's adjuvant

Bacterial immunogens (whole cells of Borrelia burgdorferi, elementary bodies of Chlamydia trachomatis and purified proteins of 22 and 24 kDa of Borrelia hermsii) were emulsified with an excess of complete Freunds adjuvant and injected (i.p.) on days 0, 7, 14 and 21, into BALB/c mice treated with pristane on day 6. This procedure induced the development of antibody-producing ascites tumours which could be serially transplanted in pristane-conditioned mice. Ascites tumours continued to yield a consistent amount of specific polyclonal antibody after ten serial transplants. The method described appears to be particularly useful for the production of a large amount of antibody when only small amounts of immunogen are available.

Differentially expressed genes in HIV-1 tat-expressing CD4+ T-cell line

Several studies have indicated that human immunodeficiency virus type-1 (HIV-1) transactivating Tat protein is essential for proviral DNA transcription and virus replication. In addition, it is actively released from acutely HIV-1-infected cells and interacts either with the same virus-infected and virus producing cell, or with bystander uninfected cells, influencing the expression of several genes and related cellular functions. The main goal of this paper was to determine the Tat-related expression of basic cellular genes in a permanently tat transfected CD4+ cell line, to identify the cellular genes influenced by the presence of endogenous-exogenous Tat protein. For this purpose, we analyzed, by a cDNA-membrane-array assay, cellular mRNAs expressed in serum-free cultures of lymphoblastoid CD4(+) Jurkat cells, stably transfected with a plasmid constitutively expressing tat gene, in comparison with Jurkat cells transfected with the backbone plasmid only, and parental Jurkat cells. The expression of mRNAs in permanently tat-transfected Jurkat cells showed significant differences in 24 out of 1176 analyzed genes in comparison with parental or backbone plasmid transfected cells. Most of the genes overexpressed in permanently tat-transfected Jurkat cells, belong to transcription factors, or to receptors, adaptors, and mediators of signal transduction pathways, and to factors involved in response to oxidative stress, suggesting a complex regulation of CD4(+) T-lymphoid cell survival and proliferation by HIV-1 Tat protein.

A hybrido-immunocytochemical assay for the in situ detection of cytomegalovirus DNA using digoxigenin-labeled probes.

A non-radioactive hybrido-immunocytochemical assay for the detection of cytomegalovirus (CMV) DNA in infected cells was developed. Two different DNA fragments belonging to the repeated sequences of CMV genome were used to construct the hybridization probe. The probe was constructed by incorporating deoxyuridine triphosphate labeled with digoxigenin. The in situ hybridized CMV DNA probe was immunocytochemically visualized by anti-digoxigenin. Fab fragments labeled with alkaline phosphatase. This procedure permitted the DNA detection, in the nuclei of infected cells fixed at 48 h after infection, of the Towne CMV reference strain and 21 different laboratory-isolated CMV strains. Our assay demonstrated a high specificity, sensitivity and reproducibility.

Serological response to chlamydial infection in sheep, studied by enzyme‐linked immunosorbent assay and immunoblotting

Two enzyme-linked immunosorbent assays (ELISA) using highly purified elementary bodies (EB) of Chlamydia psittaci A/22 strain (ovine) or 6BC strain (psittacine) were set up for the detection of antichlamydial antibodies in sheep. No significant differences were observed between the two ELISAs, whereas these tests proved to be more sensitive than complement fixation test and showed a good correlation (r = 0.75) with immunofluorescence assay. The periodate treatment of chlamydial antigens modified the results of serological responses studied by ELISA, depending on the sera. The average reduction of ELISA values by periodate was 28%, ranging between 5% and 65%. The immunoblot analysis of sheep sera showed high cross reactivity between the polypeptides of A/22 and 6BC strains. However, some differences were observed. The major outer membrane protein (MOMP) of 6BC strain was recognized at different molecular weight position (40,000 kDa) in comparison with the MOMP of A/22 strain (38,000 kDa). In addition, a clear band of 97,000 kDa was detected by all sheep sera tested with A/22 strain. This band was undetectable in the blots performed with 6BC strain.