Angelo Messina

IL-4 Protects Tumor Cells from Anti-CD95 and Chemotherapeutic Agents via Up-Regulation of Antiapoptotic Proteins

We recently proposed that Th1 and Th2 cytokines exert opposite effects on the pathogenesis and clinical outcome of organ-specific autoimmunity by altering the expression of genes involved in target cell survival. Because a Th2 response against tumors is associated with poor prognosis, we investigated the ability of IL-4 to protect tumor cells from death receptor- and chemotherapy-induced apoptosis. We found that IL-4 treatment significantly reduced CD95 (Fas/APO-1)- and chemotherapeutic drug-induced apoptosis in prostate, breast, and bladder tumor cell lines. Analysis of antiapoptotic protein expression revealed that IL-4 stimulation resulted in up-regulation of cellular (c) FLIP/FLAME-1 and Bcl-xL. Exogenous expression of cFLIP/FLAME-1 inhibited apoptosis induced by CD95 and to a lesser extent by chemotherapy, while tumor cells transduced with Bcl-xL were substantially protected both from CD95 and chemotherapeutic drug stimulation. Moreover, consistent IL-4 production and high expression of both cFLIP/FLAME-1 and Bcl-xL were observed in primary prostate, breast, and bladder cancer in vivo. Finally, primary breast cancer cells acquired sensitivity to apoptosis in vitro only in the absence of IL-4. Thus, IL-4 protects tumor cells from CD95- and chemotherapy-induced apoptosis through the up-regulation of antiapoptotic proteins such as cFLIP/FLAME-1 and Bcl-xL. These findings may provide useful information for the development of therapeutic strategies aimed at restoring the functionality of apoptotic pathways in tumor cells.

V gamma 9V delta 2 T lymphocytes efficiently recognize and kill zoledronate-sensitized, imatinib-sensitive, and imatinib-resistant chronic myelogenous leukemia cells.

Imatinib mesylate (imatinib), a competitive inhibitor of the BCR-ABL tyrosine kinase, is highly effective against chronic myelogenous leukemia (CML) cells. However, because 20–30% of patients affected by CML display either primary or secondary resistance to imatinib, intentional activation of Vγ9Vδ2 T cells by phosphoantigens or by agents that cause their accumulation within cells, such as zoledronate, may represent a promising strategy for the design of a novel and highly innovative immunotherapy capable to overcome imatinib resistance. In this study, we show that Vγ9Vδ2 T lymphocytes recognize, trogocytose, and efficiently kill imatinib-sensitive and -resistant CML cell lines pretreated with zoledronate. Vγ9Vδ2 T cell cytotoxicity was largely dependent on the granule exocytosis- and partly on TRAIL-mediated pathways, was TCR-mediated, and required isoprenoid biosynthesis by zoledronate-treated CML cells. Importantly, Vγ9Vδ2 T cells from patients with CML can be induced by zoledronate to develop antitumor activity against autologous and allogeneic zoledronate-treated leukemia cells, both in vitro and when transferred into immunodeficient mice in vivo. We conclude that intentional activation of Vγ9Vδ2 T cells by zoledronate may substantially increase their antileukemia activities and represent a novel strategy for CML immunotherapy.

Altered Expression of c-IAP1, Survivin, and Smac Contributes to Chemotherapy Resistance in Thyroid Cancer Cells

Resistance to chemotherapy predicts an unfavorable outcome for patients with radioiodine-insensitive thyroid cancer. To investigate the mechanisms underlying this resistance, we evaluated the expression of four different inhibitor of apoptosis proteins, and their antagonist, Smac, in thyroid cancer cells that survived 48 hours of exposure to cisplatin, doxorubicin, or taxol. We found high levels of c-IAP1 after cisplatin treatment and increased expression of survivin following exposure to doxorubicin. Cells that endured treatment with taxol showed reduced expression of Smac and released minimal amounts of this protein from the mitochondria. Down-regulation of c-IAP1 and survivin increased the cytotoxicity of cisplatin and doxorubicin, whereas overexpression of Smac improved the efficacy of taxol. Finally, thyroid cancer cells permanently resistant to doxorubicin or cisplatin showed increased expression of c-IAP1 and survivin, respectively. However, silencing of these proteins by RNA interference restored sensitivity to doxorubicin and cisplatin. Thus, in thyroid cancer cells, early resistance to chemotherapeutic agents requires high levels of c-IAP1 and survivin and low levels of Smac. Furthermore, increased expression of c-IAP1 and survivin contributes to the acquisition of permanent resistance to cytotoxic compounds.

Risk Factors for Classical Kaposi Sarcoma in a Population-based Case-control Study in Sicily

Background: Classical Kaposi sarcoma is a rare complication of Kaposi sarcoma-associated herpes virus (KSHV) infection. We conducted a population-based, frequency-matched case-control study in Sicily to further investigate the reported inverse relationship between smoking and classical Kaposi sarcoma and to identify other factors associated with altered risk. Methods: All incident, histologically confirmed classical Kaposi sarcoma cases in Sicily were eligible. A two-stage cluster sample design was applied to select population controls. KSHV seropositivity was determined using four antibody assays (K8.1 and orf73 enzyme immunoassays and two immunofluorenscence assays). Using SAS-callable SUDAAN, we compared the characteristics of classical Kaposi sarcoma cases and KSHV-seropositive controls. Odds ratios (OR) and 95% confidence intervals (CI) are presented. Results: In total, 142 classical Kaposi sarcoma cases and 123 KSHV-seropositive controls were recruited. Current cigarette smoking was associated with reduced risk of classical Kaposi sarcoma amongst males (OR, 0.20; 95% CI, 0.06-0.67). Edema was associated with classical Kaposi sarcoma, but only when it presented on the lower extremities (OR, 3.65; 95% CI, 1.62-8.23). Irrespective of presentation site, diabetes and oral corticosteroid medications were associated with increased risk (OR, 4.73; 95% CI, 2.02-11.1 and OR, 2.34; 95% CI, 1.23-4.45, respectively). Never smoking, diabetes, and oral corticosteroid medication use were all independently associated with classical Kaposi sarcoma risk. Discussion: We confirmed previous reports that cigarette smoking was associated with a reduced risk of classical Kaposi sarcoma, and we found that risk was lowest among current smokers. We also found that classical Kaposi sarcoma risk was strongly and independently associated with oral corticosteroid use and diabetes. Corroboration of these observations and investigation of possible underlying mechanisms are warranted. (Cancer Epidemiol Biomarkers Prev 2008;17(12):3435–43)

Virologic, hematologic, and immunologic risk factors for classic Kaposi sarcoma

Classic Kaposi sarcoma (CKS) is an inflammatory‐mediated neoplasm that develops in the presence of KS‐associated herpesvirus (KSHV) and immune perturbation. In the current study, the authors compared CKS cases with age‐matched and sex‐matched KSHV‐seropositive controls without human immunodeficiency virus‐1 infection and markers of viral control, blood counts, CD4‐positive and CD8‐positive lymphocytes, and serum β‐2‐microglobulin and neopterin levels.

Associations of Classic Kaposi Sarcoma with Common Variants in Genes that Modulate Host Immunity

Classic Kaposi sarcoma (CKS) is an inflammatory-mediated neoplasm primarily caused by Kaposi sarcoma–associated herpesvirus (KSHV). Kaposi sarcoma lesions are characterized, in part, by the presence of proinflammatory cytokines and growth factors thought to regulate KSHV replication and CKS pathogenesis. Using genomic DNA extracted from 133 CKS cases and 172 KSHV-latent nuclear antigen-positive, population-based controls in Italy without HIV infection, we examined the risk of CKS associated with 28 common genetic variants in 14 immune-modulating genes. Haplotypes were estimated for IL1A, IL1B, IL4, IL8, IL8RB, IL10, IL12A, IL13, and TNF. Compared with controls, CKS risk was decreased with 1235T/−1010G–containing diplotypes of IL8RB (odds ratio, 0.49; 95% confidence interval, 0.30-0.78; P = 0.003), whereas risk was increased with diplotypes of IL13 containing the promoter region variant 98A (rs20541, alias +130; odds ratio, 1.88; 95% confidence interval, 1.15-3.08; P = 0.01) when considered in multivariate analysis. Risk estimates did not substantially vary by age, sex, incident disease, or disease burden. Our data provide preliminary evidence for variants in immune-modulating genes that could influence the risk of CKS. Among KSHV-seropositive Italians, CKS risk was associated with diplotypes of IL8RB and IL13, supporting laboratory evidence of immune-mediated pathogenesis. (Cancer Epidemiol Biomarkers Prev 2006;15(5):926–34)

The Italian study of the Mitroflow postoperative results (ISTHMUS): a 20-year, multicenter evaluation of Mitroflow pericardial bioprosthesis

OBJECTIVE A multicentre experience with the Mitroflow pericardial bioprosthesis has been evaluated longitudinally over a 20-year period. METHODS From 1988 through 2008, 1591 patients (mean age, 75.3±6.8 years, and 60.1% female) from 12 centres had a Mitroflow in the aortic position. Concomitant coronary artery bypass was performed in 41.9% (n=666) of patients, urgency/emergency surgery in 9.5% (n=152) and replacement of degenerated prosthesis in 2.3% (n=36). Follow-up (7.447 patient-years) was 99.2% complete. Median follow-up was 61.9 months (interquartile range (IQR) 30.8-90.9 months). The study was carried out following American Association for Thoracic Surgery/Society for Thoracic Surgeons/European Association for Cardio-Thoracic Surgery (AATS/STS/EACTS) Guidelines for reporting valve morbidity and mortality. RESULTS The early (30-day) mortality was 6.5% (n=104). Actuarial survival rates at 10, 15 and 18 years were 53%, 34% and 27%, respectively (2.2 patient/year). Re-operation was required in 96 patients (5.9%), of whom 59 patients (3.7%) for structural valve degeneration. Actuarial freedom from prosthetic valve degeneration at 18 years was 65.5% (78% in patients>70 years) with a linearised rate of 1.4 patient/year (0.8 patient/year in patients>70 years). At 18 years, freedom from embolism was 82% (0.9 patient/year), freedom from valve endocarditis was 89% (0.6 patient/year) and freedom from bleeding episodes was 95% (0.2 patient/year), respectively. CONCLUSIONS This independent multicentre study indicates that the Mitroflow pericardial bioprosthesis provides favourable long-term postoperative results with a low rate of valve-related events and need of re-intervention, particularly in patients older than 70 years.

Polyamine involvement in functional activation of human macrophages

Polyamines naturally occur in all living beings and play an important role in the regulation of cell proliferation, differentiation, and functional stimulation of terminally differentiated cells. Our studies, using specific inhibitors of polyamine biosynthesis such as α‐difluoro‐ methylornithine and methylglyoxal‐bis[guanylhydrazone] to prevent polyamine accumulation, have indicated that polyamines are associated with functional activation of human macrophages. Both inhibitors diminished the respiratory burst activity of macrophages induced by lipopolysaccharide and interferon gamma. The methyl‐ glyoxal‐bis[guanylhydrazone] inhibitory effect was concentration‐dependent and could be reversed by spermine, which is the final product of polyamine biosynthesis.

Cell death in human acute myelogenous leukemic cells induced by pyrrolidinedithiocarbamate

Pyrrolidinedithiocarbamate (PDTC) is a metal chelating compound, which exerts both pro-apoptotic effect and pro-oxidant activity on many cells. Our objective was to investigate whether PDTC was able to interfere with apoptotic process in leukemic and normal bone marrow CD34+ cells. Since hematopoietic growth factors stimulate growth and differentiation and prevent apoptosis, we therefore studied the effect of growth factors pretreatment, such as interleukin-3 and granulocyte-macrophage colony stimulating factor, in human myeloid CD34+ cells to evaluate whether they protect the cells from the apoptotic action of PDTC.We revealed that PDTC exerted an apoptotic effect in leukemic CD34+ cells. This effect was dependent on the ability of this compound to generate the oxidation of cellular glutathion to its disulphide and consequently to induce an intracellular oxidative stress. Hematopoietic growth factors did not protect cells from apoptosis induced by previous treatment with PDTC. The ability of PDTC to induce apoptosis was restricted to acute myelogenous leukaemia CD34+ cells, since normal CD34+ cells were insensitive to the pro-oxidant effect of PDTC.These findings imply that normal cells are equipped with mechanisms by which they respond differently to PDTC effects with respect to leukemic cells.

Host Immunogenetics and Control of Human Herpesvirus–8 Infection

BACKGROUND Kaposi sarcoma (KS) is primarily caused by human herpesvirus (HHV)-8 infection, and the risk is increased with high HHV-8 lytic or latent antibody titers or the detection of HHV-8 DNA in peripheral blood mononuclear cells (PBMCs). Host genes important for control of HHV-8 infection are not well characterized. METHODS In 172 HHV-8 latent nuclear antigen (LANA)-seropositive adults in Italy without KS, we examined correlations of common variants in host immune genes with the detection of HHV-8 DNA in PBMCs and with high lytic and latent antibody titers. Twenty-eight single-nucleotide polymorphisms in 14 genes were analyzed. We detected HHV-8 DNA in PBMCs with real-time amplification of the K6 gene, anti-K8.1 (lytic) titers with enzyme-linked immunosorbent assay, and anti-LANA (latent) titers with immunofluorescence. RESULTS Detection of HHV-8 DNA in PBMCs was not significantly related to any variant examined. In contrast, a 3-locus haplotype of IL4, which contains the -1098G allele (rs2243248), was overrepresented among subjects with high lytic titers (odds ratio [OR], 2.8 [95% confidence interval {CI}, 1.1-6.7]), compared with those with low titers, as was the functional promoter variant of IL6, C-236C (rs1800795) (OR, 3.7 [95% CI, 1.1-12.8]). Compared with subjects with low HHV-8 latent antibody titers, analysis of inferred haplotypes for IL12A revealed an overrepresentation of -798T/277A in subjects with high HHV-8 latent antibody titers (OR, 2.4 [95% CI, 1.1-5.2]). CONCLUSIONS Our observations are the first to provide preliminary evidence suggesting that common variants in key host immune genes could influence the control of HHV-8 infection.