Giuliana Gobbi

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.

Hydrogen sulfide prevents apoptosis of human PMN via inhibition of p38 and caspase 3.

Hydrogen sulfide, together with carbon monoxide and nitric oxide, is now considered a gasotransmitter able to induce specific cellular responses. As hydrogen sulfide is a component of several natural compounds known to be effective in many inflammatory pathologies, particularly of the respiratory tract, we studied its effects in vitro on the survival and bactericidal activity of purified human neutrophils. We found that (1) HS− ions promote the survival of granulocytes, but not that of lymphocytes or eosinophils, cultured in serum-free medium; (2) the pro-survival effect of HS− is due to inhibition of caspase-3 cleavage and p38 MAP kinase phosphorylation; (3) the bactericidal activity of neutrophils is not impaired by hydrogen sulfide. We conclude that HS− promotes the short-term survival of neutrophils potentially accelerating the resolution of inflammatory processes and preventing the occurrence of new ones.

Abnormal VWF modifies megakaryocytopoiesis: studies of platelets and megakaryocyte cultures from patients with von Willebrand disease type 2B

von Willebrand factor (VWF) is an essential mediator of platelet adhesion to the vessel wall, but little is known about its role in megakaryocytopoiesis. VWF and its platelet receptor, glycoprotein Ibalpha (GPIbalpha), are both expressed during megakaryocyte (MK) maturation. This study was designed to evaluate whether the enhanced VWF-GPIbalpha interactions typical of patients with von Willebrand disease type 2B (VWD2B) modify platelet production. Platelets from 9 patients with VWD2B with 7 different gain-of-function mutations were examined by electron microscopy (EM) and immunofluorescence labeling. For the patients with VWD2B, EM characteristically showed variable numbers of structurally abnormal giant platelets, sometimes in agglutinates. Cultures of MKs from controls performed with or without purified VWF confirmed a positive influence of VWF on platelet production with specific inhibition by an antibody blocking VWF binding to GPIbalpha. VWD2B MK cultures examined by EM showed a disorganized demarcation membrane system and abnormal granule distribution. They produced platelets with structural abnormalities typical of VWD2B. Confocal examination of MK revealed limited extension of pseudopods with few large proplatelets. These results confirm that megakaryocytopoiesis is modified by the enhanced VWF-GPIbalpha interactions. These data obtained for controls and patients with VWD2B suggest a novel regulatory role of VWF-GPIbalpha interactions in platelet production.

Involvement of INK4A gene products in the pathogenesis and development of human osteosarcoma

The INK4A tumor suppressor gene plays a crucial role in the regulation of the G1 cell cycle phase. It encodes two transcripts, p16 and p14 alternate reading frame (ARF), involved in retinoblastoma protein (pRb)‐ and p53‐ cell growth control pathways, respectively.

Presence of telomerase activity in different musculoskeletal tumor histotypes and correlation with aggressiveness.

Telomerase is a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomeres. Telomerase activity was observed and correlated with aggressiveness in different neoplasms such as breast, prostate, blood and brain cancers, among others. To investigate whether telomerase activity is an index of aggressiveness in bone and soft tissue lesions of the extremities, 66 biopsy samples from our tissue bank were studied. These samples included 43 high‐grade sarcomas, 9 aggressive benign tumors and 14 totally benign lesions. The samples were collected from patients homogeneously treated at the Rizzoli Orthopaedic Institute with a follow‐up ranging from 4 to 11 years (median, 7 years). A non‐radioactive polymerase chain reaction‐based enzyme‐linked immunosorbent assay was used for the study. All tumors investigated were positive for telomerase activity. Among benign lesions, only 2 aneurysmal bone cysts showed higher telomerase activity than the cut‐off point, whereas all the other benign lesions had lower activity. Our results indicate that high levels of telomerase activity in bone and soft tissue lesions correlate with more aggressive clinical behavior in patients treated with surgery alone. An interesting inverse correlation between telomerase activity and occurrence of pulmonary metastasis was detected in osteosarcoma patients treated with chemotherapy. A parallel increase of telomerase activity and malignancy was observed in the adipose and cartilagineous tissue lesions. Our data suggest that telomerase activity could be considered a marker of tumor aggressiveness for bone and soft tissue lesions. The results obtained in osteosarcoma samples suggest that low levels of telomerase activity may be predictive of the prognosis and should influence the therapeutic protocol.

Flow cytometry detection of serotonin content and release in resting and activated platelets

Summary. Early detection of platelet activation is important for the diagnosis and follow‐up of several pathological conditions that primarily or secondarily involve platelets in their pathogenesis. The golden standard assay to detect thrombocyte activation is represented by the release of serotonin, classically performed by demanding methodologies, such as high‐performance liquid chromatography, 14C‐labelling and enzyme‐linked immunosorbent assay (ELISA). We developed a non‐radioactive method, based on individual cells, for the detection of serotonin content in activated and resting platelets by flow cytometry. The assay was standardized on cells activated by Ca2+ ionophore or by sera from patients with heparin‐induced thrombocytopenia (HIT). Cells were identified by CD41a surface staining and their serotonin content measured by specific antiserotonin intracytoplasmic staining, while their activation was independently shown by annexin V binding. Cellular degranulation was detected by flow cytometry in all the cases that were also positive by standard ELISA. Moreover, multiparametric flow cytometry analysis revealed that, although virtually all activated cells bind annexin V, serotonin was released only by the platelet subset that downmodulates surface CD41a.

Timing and Expression Level of Protein Kinase Cε Regulate the Megakaryocytic Differentiation of Human CD34 Cells

Protein kinase C (PKC)‐mediated intracellular signaling participates in several key steps of hematopoietic cell differentiation. The ε isoform of PKC has been associated with erythroid differentiation as well as with the early phases of megakaryocytic (MK) lineage commitment. Here, we worked on the hypothesis that PKCε expression levels might be modulated during MK differentiation, with a specific role in the early as well as in the late phases of thrombopoiesis. We demonstrate that—at variance with the erythroid lineage development—PKCε is completely downmodulated in TPO‐induced CD34 cells from day 6 onward. The forced expression of PKCε in the late phases of MK differentiation delays the phenotypic differentiation of progenitors likely via Bcl‐xL upregulation. Moreover, tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL), known as a negative regulator of early erythroid expansion, is not apoptogenic for thrombopoietin‐induced CD34 cells, but rather accelerates their maturation. However, PKCε levels negatively interfere also with the effects of TRAIL in MK differentiation. PKCε can therefore be considered a signaling intermediate whose expression levels are finely tuned, with a virtually opposite kinetic, in erythroid versus megakaryocytic lineages, to adequately respond to the signaling requirements of the specific hematopoietic lineage.

Exogenous hydrogen sulfide induces functional inhibition and cell death of cytotoxic lymphocytes subsets

The toxic effects of exogenous hydrogen sulfide on peripheral blood lymphocytes have been investigated in detail. Hydrogen sulfide is now considered as a gasotransmitter with specific functional roles in different cell types, like neurons and vascular smooth muscle. Here we show that exogenous hydrogen sulfide induces a caspase‐independent cell death of peripheral blood lymphocytes that depends on their intracellular glutathion levels, with a physiologically relevant subset specificity for CD8+ T cells and NK cells. Although lymphocyte activation does not modify their sensitivity to HS−, after 24 h exposure to hydrogen sulfide surviving lymphocyte subsets show a dramatically decreased proliferation in response to mitogens and a reduced IL‐2 production. Overall, our data demonstrate that HS− reduces the cellular cytotoxic response of peripheral blood lymphocytes as well as their production of IL‐2, therefore de‐activating the major players of local inflammatory responses, adding new basic knowledge to the clinically well known anti‐inflammatory effects of sulfur compounds. J. Cell. Physiol. 213:826–833.

Troglitazione affects survival of human osteosarcoma cells

Activation of PPARγ, a transcription factor member of the family of peroxisome proliferator‐activated receptors, induces apoptosis in several normal and tumor cell lines. In our study, we investigated whether treatment with troglitazone (TRO), a known PPARγ agonist, induced apoptosis in the human osteosarcoma (OS) cell lines G292, MG63, SAOS and U2OS that express PPARγ. In our experiments, TRO never induced apoptosis of OS cells; on the contrary, TRO increased cell number, based on MTT proliferation assay. Remarkably, the TRO‐induced cell number increase depended on a decrease of apoptosis that naturally occurred in the culture and was not due to an increased cell proliferation rate. TRO also prevented staurosporin‐induced apoptosis. The TRO‐mediated survival effect correlated with the activation of Akt, a well‐known mediator of survival stimuli. Our work describes a new function for TRO and indicates that the Akt survival pathway may be a mediator of TRO‐induced increase of survival.

HIV‐1 matrix protein p17 enhances the proliferative activity of natural killer cells and increases their ability to secrete proinflammatory cytokines

Summary. We investigated the effects of human immunodeficiency type‐1 virus (HIV‐1) matrix protein p17 on freshly isolated and purified human natural killer (NK) cells. HIV‐1 p17 increased the cytokines interleukin (IL) 2, IL‐12 and IL‐15, and induced natural killer cell proliferation, but not cytotoxicity. This effect was specific because it was abrogated by anti‐p17 monoclonal antibody. Moreover, HIV‐1 p17 enhanced the cytokine‐induced production of tumour necrosis factor (TNF)‐α and interferon (IFN)‐γ by NK cells. IL‐4 downregulated IFN‐γ and TNF‐α secretion in IL‐2‐ and IL‐15‐treated NK cells. HIV‐1 p17 restored the ability of NK cells to produce both cytokines when added to the cultures simultaneously with IL‐4. The property of p17 to increase the production of TNF‐α and IFN‐γ might be a mechanism used by HIV‐1 to modulate the immune system to support its replication and spreading.