Cristina Ponti

NK Cells and Cancer

In this review, we overview the main features and functions of NK cells, focusing on their role in cell-mediated immune response to tumor cells. In parallel, we discuss the information available in the field of NK cell receptors and offer a wide general overview of functional aspects of cell targeting and killing, focusing on the recent acknowledgments on the efficacy of NK cells after cytokine and mAb administration in cancer therapy. Since efficacy of NK cell-based immunotherapy has been proven in KIR-mismatch regimens or in TRAIL-dependent apoptosis, the ability to manipulate the balance of activating and inhibitory receptors on NK cells and of their cognate ligands, as well as the sensitivity of tumor cells to apoptosis, opens new perspectives for NK cell-based immunotherapy.

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.

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.

HIV-1 Tat protein concomitantly down-regulates apical caspase-10 and up-regulates c-FLIP in lymphoid T cells: a potential molecular mechanism to escape TRAIL cytotoxicity.

In this study, we showed the existence of a positive correlation between the amount of human immunodeficiency virus‐type 1 (HIV‐1) RNA in HIV‐1 seropositive subjects and the plasma levels of TRAIL. Since it has been previously demonstrated that HIV‐1 Tat protein up‐regulates the expression of TRAIL in monocytic cells whereas tat‐expressing lymphoid cells are more resistant to TRAIL cytotoxicity, we next investigated the effect of Tat on the expression/activity of both apical caspase‐8 and ‐10, which play a key role in mediating the initial phases of apoptosis by TRAIL, and c‐FLIP. Jurkat lymphoblastoid human T cell lines stably transfected with a plasmid expressing wild‐type (HIV‐1) tat gene showed normal levels of caspase‐8 but significantly decreased levels of caspase‐10 at both mRNA and protein levels with respect to Jurkat transfected with the control plasmid or with a mutated (cys22) non‐functional tat cDNA. A significant decrease of caspase‐10 expression/activity was also observed in transient transfection experiments with plasmid carrying tat cDNA. Moreover, c‐FLIPL and c‐FLIPS isoforms were up‐regulated in tat‐expressing cells at both mRNA and protein level in comparison with control cells. Taken together, these results provide a molecular basis to explain the resistance of tat‐expressing Jurkat cells to apoptosis induced by TRAIL and, possibly, to other death‐inducing ligands.

Lignan Derivatives from Krameria lappacea Roots Inhibit Acute Inflammation in Vivo and Pro-inflammatory Mediators in Vitro

The roots of Krameria lappacea are used traditionally against oropharyngeal inflammation. So far, the astringent and antimicrobial properties of its proanthocyanidin constituents are considered to account for the anti-inflammatory effect. The aim of the present study was to characterize pharmacologically a lipophilic extract of K. lappacea roots and several isolated lignan derivatives (1–11) in terms of their putative anti-inflammatory activity. The dichloromethane extract (ID50 77 μg/cm2) as well compounds 1–11 (ID50 0.31–0.60 μmol/cm2) exhibited topical antiedematous properties comparable to those of indomethacin (ID50 0.29 μmol/cm2) in a mouse ear in vivo model. Two of the most potent compounds, 2-(2-hydroxy-4-methoxyphenyl)-5-(3-hydroxypropyl)benzofuran (5) and (+)-conocarpan (7), were studied regarding their time-dependent edema development and leukocyte infiltration up to 48 h after croton oil-induced dermatitis induction, and they showed activity profiles similar to that of hydrocortisone. In vitro studies of the isolated lignan derivatives demonstrated the inhibition of NF-κB, cyclooxygenase-1 and -2, 5-lipoxygenase, and microsomal prostaglandin E2 synthase-1 as well as antioxidant properties, as mechanisms possibly contributing to the observed in vivo effects. The present findings not only support the ethnopharmacological use of K. lappacea roots but also reveal that the isolated lignan derivatives contribute strongly to the anti-inflammatory activity of this herbal drug.

NK-active cytokines IL-2, IL-12, and IL-15 selectively modulate specific protein kinase C (PKC) isoforms in primary human NK cells

Natural killer (NK) cell function is largely modulated by growth factors and cytokines. In particular, interleukin (IL)‐2, IL‐12, and IL‐15 have major effects on the proliferative and cytotoxic activities of NK cells against tumor and virus‐infected cells. It is thought that the members of the protein kinase C (PKC) family of serine/threonine kinases play an important role in mediating the pleiotropic effects of cytokines on their target cells. We have investigated the downstream effects generated in purified human NK cells by IL‐2, IL‐12, and IL‐15 on PKCα and PKCϵ—a canonical and a novel isoform of PKC, respectively. By means of Western blotting, PKC activity assays, and immunofluorescence performed on highly purified preparations of primary human NK cells, we demonstrate that: 1) the three cytokines have similar effects on PKCα and PKCϵ activities; 2) whereas PKCϵ activity is induced by cytokine stimulation, PKCα activity is inhibited; and 3) both the induction of PKCϵ and the inhibition of PKCα functional activity are relatively early events in NK cells, while longer cytokine stimulations do not generate significant variations in enzyme activity, suggesting that the activation of both the canonical and novel isoforms of PKC are events required in the early phases of cytokine‐induced NK cell stimulation. Anat Rec 266:87–92, 2002.

HIV-1 Tat protein enhances RANKL/M-CSF-mediated osteoclast differentiation.

Impaired osteoblast/osteoclast cross-talk and bone structure homeostasis resulting in osteopenia/osteoporosis are often observed in HIV seropositive patients but the causal mechanisms remain unsettled. This study analyzed the biological effects of Tat on peripheral blood monocyte-derived osteoclast differentiation. Tat enhances osteoclast differentiation and activity induced by RANKL plus M-CSF treatment increasing both the mRNA expression of specific osteoclast differentiation markers, such as cathepsin K and calcitonin receptor, and TRAP expression and activity. These Tat-related biological effects may be related, at least in part, to the induction of c-fos expression and AP-1 activity. c-fos up-regulation was triggered by Tat when cell cultures were co-treated with RANKL/M-CSF and an analysis of c-fos promoter with c-fos deletion mutant constructs disclosed specific c-fos promoter domains targeted by Tat. Together, these results show that Tat may be considered a viral factor positively modulating the osteoclastogenesis and then bone resorption activity suggesting a pathogenetic role of this viral protein in the HIV-related osteopenia/osteoporosis.

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.

Role of CREB transcription factor in c‐fos activation in natural killer cells

In natural killer (NK) cells, interleukin‐2 (IL‐2) differentially regulates the expression of several transcription factors, including JunB and c‐fos. The cAMP response element binding protein, CREB, is a key transcriptional regulator of a large number of genes containing the octanucleotide CRE consensus sequence in their upstream regulatory regions. We studied here thefunctional role of CREB in the IL‐2‐mediated transcriptional regulation of c‐fos in human NK cells. Our results show that IL‐2 activates CREB in human NK cells and that CREB activation hasa prominent regulatory role on the IL‐2‐induced expression of functional c‐fos and AP‐1 in NK cells. We identify two domains of the c‐fos promoter, containing three CRE sites, which are critical for the transcriptional activity induced by IL‐2. The first domain is located within the first 220 nucleotides of the c‐fos promoter, while the second encompasses the nucleotides − 440 and − 220. Our results show that CREB has a relevant role in the cytokine‐mediated activation of NK cells, and are particularly remarkable in the light of the several genes that are positively regulated by c‐fos and AP‐1, such as IFN‐γ, IL‐2 and GM‐CSF genes.

Effects of human immunodeficiency virus on the erythrocyte and megakaryocyte lineages

Anaemia and thrombocytopenia are haematological disorders that can be detected in many human immunodeficiency virus (HIV)-positive patients during the development of HIV infection. The progressive decline of erythrocytes and platelets plays an important role both in HIV disease progression and in the clinical and therapeutic management of HIV-positive patients. HIV-dependent impairment of the megakaryocyte and erythrocyte lineages is multifactorial and particularly affects survival, proliferation and differentiation of bone marrow (BM) CD34+ haematopoietic progenitor cells, the activity of BM stromal cells and the regulation of cytokine networks. In this review, we analyse the major HIV-related mechanisms that are involved in the genesis and development of the anaemia and thrombocytopenia observed in HIV positive patients.