Roberto Caporale

Natural fluorescence of white blood cells: spectroscopic and imaging study

Autofluorescence has been proved to be an intrinsic parameter of biological substrates that may aid in both the characterization of the physiological state and the discrimination of pathological from normal conditions of cells, tissues and organs. In this work, the fluorescence properties of human white blood cells have been studied in suspension and on single cells at microscopy. The results indicate that suspensions of agranulocytes and granulocytes differ in the amplitude of the fluorescence signal on excitation at wavelengths in the range 250-370 nm. The differences are particularly enhanced when excitation is performed in the 250-265 nm range. Microspectrofluorometric analysis, performed on single cells, allows several leukocyte families to be characterized. Lymphocytes, monocytes, neutrophils and eosinophils can be distinguished according to the intensity and spectral shape of the autofluorescence emission in the visible range from 440 to 580 nm. Both the nature and extent of the differences change when the excitation wavelength is moved from 366 to 436 nm. Differences in the intrinsic metabolic engagement, rather than in the cell dimensions, seem to be responsible for the differences observed between the leukocyte populations. The results identify interesting perspectives for autofluorescence as a discriminating parameter in the differential counting of human white blood cells.

Attenuated-dose idarubicin in acute myeloid leukaemia of the elderly: pharmacokinetic study and clinical results.

AML in the elderly is characterized by intrinsic biological features implying an enhanced chemoresistance. Intensive chemotherapy should be the treatment of choice, but the standard doses could induce unacceptable rates of aplastic deaths. We evaluated the efficacy of an induction protocol with attenuated‐dose idarubicin (IDA) 8 mg/m2 for 3 d plus cytarabine and etoposide in 26 AML patients aged >60. 18 patients (69%) achieved CR, five (19%) were non‐responders and three (12%) died during induction. To compare the pharmacokinetics of IDA between elderly and young patients, we assayed daily the serum level of the drug and of its metabolite (idarubicinol, IDAol) in a group of eight elderly patients who received a dose of 8 mg/m2 (group A) and in a group of nine younger AML patients treated with 12 mg/m2 (group B). The apparent terminal half‐life of IDAol was significantly longer in the elderly than in the younger patients (mean half‐life 59–7h versus 41–4h, P< 0–05). The values of the area under the serum concentration curve of IDAol indicated that the two patient groups received a very similar exposure to the drug despite the different doses.

Selective ex vivo expansion of cytomegalovirus-specific CD4+ and CD8+ T lymphocytes using dendritic cells pulsed with a human leucocyte antigen A*0201-restricted peptide

Adoptive transfer of ex vivo‐generated cytomegalovirus (CMV)‐specific T lymphocytes may be effective in preventing CMV disease in allogeneic haematopoietic stem cell transplantation (HSCT) recipients. We developed a procedure for expansion of CMV‐specific T lymphocytes based on the antigen‐presenting function of donor dendritic cells (DCs), pulsed with a human leucocyte antigen A*0201‐restricted pp65 nonamer peptide. CMV‐specific T lymphocytes were identified following induction of interferon γ (IFN‐γ) secretion prompted by peptide exposure. Both CD8+ and CD4+ CMV‐specific T lymphocytes were selectively produced in these cultures and showed CMV‐restricted cytotoxicity. The simultaneous and selective expansion of CD4+ and CD8+ CMV‐specific lymphocytes might be instrumental for more efficient in vivo function of infused CMV‐specific lymphocytes.

Defective aggregation in cirrhosis is independent of in vivo platelet activation

BACKGROUND/AIMS Platelet function abnormalities contribute to the hemostatic defect in patients with cirrhosis. In this study we evaluated the occurrence of in vivo platelet activation as a possible mechanism of defective platelet aggregation in patients with cirrhosis. METHODS Nine patients with severe (Child B-C) cirrhosis and defective platelet aggregation were studied in comparison with age- and sex-matched healthy controls. The presence of activated platelets in the bloodstream was evaluated by fluorescence-activated flow cytometry using antibodies directed against activation-dependent platelet proteins and by measuring plasma levels of beta-thromboglobulin and platelet factor 4. Urinary levels of 11-dehydro-TXB2 and of 2,3-dinor-TXB2 were assayed by radioimmunoassay following chromatographic separation. RESULTS In unstimulated platelets, the expression of both GMP 140 and GP 53 was not significantly different in patients with cirrhosis and in controls. After stimulation with ADP and epinephrine, expression of activation-dependent antigens was lower in platelets from patients (GMP 140: 0.64 +/- 0.09 vs 0.73 +/- 0.04, p = 0.02; GP 53: 0.41 +/- 0.13 vs 0.54 +/- 0.14). Plasma levels of beta-thromboglobulin and platelet factor 4, as indexes of in vivo platelet activation, were also comparable in the two groups of subjects. Urinary levels of 11-dehydro-TXB2 and of 2,3-dinor-TXB2, the major systemic metabolites of TXA2, were significantly higher in patients with cirrhosis (1807 +/- 518 vs 341 +/- 121 ng/pg creatinine and 693 +/- 512 vs 205 (93 ng/pg creatinine, respectively, p < 0.001). However, increased excretion of TXB2 metabolites was also observed in three patients with chronic autoimmune thrombocytopenia. CONCLUSIONS These data indicate that circulating platelets are not activated in cirrhosis, and that defective aggregation is most likely dependent on the alteration of the transmembrane signaling pathways. The increased urinary excretion of systemic TXA2 metabolites may be related to increased intrasplenic platelet destruction.

The frequency of granulocytes with spontaneous somatic mutations: a wide distribution in a normal human population.

Germ-line mutation rate has been regarded classically as a fundamental biological parameter, as it affects the prevalence of genetic disorders and the rate of evolution. Somatic mutation rate is also an important biological parameter, as it may influence the development and/or the course of acquired diseases, particularly of cancer. Estimates of this parameter have been previously obtained in few instances from dermal fibroblasts and lymphoblastoid cells. However, the methodology required has been laborious and did not lend itself to the analysis of large numbers of samples. We have previously shown that the X-linked gene PIG-A, since its product is required for glycosyl-phosphatidylinositol-anchored proteins to become surface bound, is a good sentinel gene for studying somatic mutations. We now show that by this approach we can accurately measure the proportion of PIG-A mutant peripheral blood granulocytes, which we call mutant frequency, ƒ. We found that the results are reproducible, with a variation coefficient (CV) of 45%. Repeat samples from 32 subjects also had a CV of 44%, indicating that ƒ is a relatively stable individual characteristic. From a study of 142 normal subjects we found that log ƒ is a normally distributed variable; ƒ variability spans a 80-fold range, from less than 1×10−6 to 37.5×10−6, with a median of 4.9×10−6. Unlike other techniques commonly employed in population studies, such as comet assay, this method can detect any kind of mutation, including point mutation, as long as it causes functional inactivation of PIG-A gene. Since the test is rapid and requires only a small sample of peripheral blood, this methodology will lend itself to investigating genetic factors that underlie the variation in the somatic mutation rate, as well as environmental factors that may affect it. It will be also possible to test whether ƒ is a determinant of the risk of cancer.

Induction of CD83+CD14+ Nondendritic Antigen-Presenting Cells by Exposure of Monocytes to IFN-α

IFN-α is a well-known agent for treatment of viral and malignant diseases. It has several modes of actions, including direct influence on the immune system. We investigated IFN-α effects on PBMC in terms of dendritic cell (DC) differentiation, as PBMC are exposed to high IFN-α levels during treatment of infections and cancers. We show that in vitro IFN-α exposure induced rapid and strong up-regulation of the DC-maturation markers CD80, CD86, and CD83 in bulk PBMC. Consistently, IFN-α induced up-regulation of these molecules on purified monocytes within 24 h. Up-regulation of CD80 and CD83 expression was IFN-α concentration-dependent. In contrast to GM-CSF + IL-4-generated DCs, most of the IFN-α-challenged CD83+ cells coexpressed the monocyte marker CD14. Despite a typical mature DC immunophenotype, IFN-α-treated monocytes conserved phagocytic activity and never acquired a dendritic morphology. In mixed lymphocyte reactions IFN-α-treated monocytes were less potent than GM-CSF + IL-4-generated DCs but significantly more potent than untreated monocytes to induce T cell proliferation in bulk PBMC. However, only GM-CSF + IL-4-generated DCs were able to induce a significant proliferation of naive CD4+ T cells. Notably, autologous memory CD4+ T cells proliferated when exposed to tetanus toxoid-pulsed IFN-α-treated monocytes. At variance with untreated or GM-CSF + IL-4-exposed monocytes, those challenged with IFN-α showed long-lasting STAT-1 phosphorylation. Remarkably, CD83+CD14+ cells were present in varicella skin lesions in close contact with IFN-α-producing cells. The present findings suggest that IFN-α alone promptly generates nondendritic APCs able to stimulate memory immune responses. This may represent an additional mode of action of IFN-α in vivo.

Growth inhibition and differentiation of human breast cancer cells by the PAFR antagonist WEB-2086

WEB-2086 – an antagonist of platelet-activating factor receptor (PAFR) with known anti-inflammatory, antiangiogenic and antileukaemic properties – also proved to inhibit the proliferation in human solid tumour cell lines of different histology, and with much higher efficacy than in normal fibroblasts. A detailed analysis of WEB-2086 anticancer activity was then performed focusing on breast adenocarcinoma MCF-7 and MDA-MB-231 cells. WEB-2086-treated cells, either expressing (MCF-7) or unexpressing (MDA-MB-231) the oestrogen receptor (ER)α, underwent a dose-dependent growth arrest (IC50=0.65±0.09 and 0.41±0.07 mM, respectively) and accumulation in G0–G1 phase. WEB-2086 also induced morphological and functional changes typical of mature mammary phenotype including (i) cell enlargement and massive neutral lipid deposition (best accomplished in MCF-7 cells); (ii) decrease in motility and active cathepsin D levels (mainly observed in highly invasive MDA-MB-231 cells). The expression of ERα was neither increased nor reactivated in treated MCF-7 or MDA-MB-231 cells, respectively. WEB-2086-induced differentiation in breast cancer cells involved the upregulation of PTEN, a key tumour suppressor protein opposing tumorigenesis, and was apparently independent of p53, PAFR, peripheral benzodiazepine receptor and ERα status. Overall, WEB-2086 can be proposed as an effective antiproliferative and differentiative agent with interesting translational opportunities to treat breast cancers in support to conventional chemotherapy.

Replicating neuroblastoma cells in different cell cycle phases display different vulnerability to amyloid toxicity

A key role of mitotic activation in neuronal cell death in early stages of Alzheimer’s disease (AD) has been suggested. Apparently, terminally differentiated neurons are precluded from mitotic division, yet some phenotypic markers of cell cycling are present in AD-vulnerable brain areas. In this paper, we investigated whether dividing human neuroblastoma cells are preferentially vulnerable to amyloid aggregate toxicity in some specific cell cycle stage(s). Our data indicate that Aβ1–40/42 aggregates added to the cell culture media bind to the plasma membrane and are internalized faster in the S than in the G2/M and G1 cells possibly as a result of a lower content in membrane cholesterol in the former. Earlier and sharper increases in reactive oxygen species production triggered a membrane oxidative injury and a significant impairment of antioxidant capacity, eventually culminating with apoptotic activation in S and, to a lesser extent, in G2/M exposed cells. G1 cells appeared more resistant to the amyloid-induced oxidative attack possibly because of their higher antioxidant capacity. The high vulnerability of S cells to aggregate toxicity extends previous data suggesting that neuronal loss in AD could result from mitotic reactivation of terminally differentiated neurons with arrest in the S phase.

Distinct Signal Transduction Abnormalities and Erythropoietin Response in Bone Marrow Hematopoietic Cell Subpopulations of Myelodysplastic Syndrome Patients

Purpose: Myelodysplastic syndromes (MDS) are heterogeneous clonal diseases characterized by cytopenias as a result of ineffective hematopoiesis. Little is known about alterations in signal transduction pathways in MDS. Experimental Design: Multiparameter flow cytometry was used to evaluate the proteolytic activation of caspase-3 and the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and STAT5 specifically in defined CD34+, CD45+, or CD71+CD45− bone marrow (BM) cells from 60 MDS cases and normal controls, both at baseline and following stimulation with granulocyte colony-stimulating factor (G-CSF) and erythropoietin. Results: In CD71+CD45− cells from a subpopulation of 36 MDS cases who were predicted to be responsive by clinical parameters (endogenous erythropoietin levels, transfusion dependency, percentage of blasts in the BM), erythropoietin failed to activate ERK1/2 or STAT5 in 23 of 36 cases, but it was effective in 13 of 36 cases, although to a significantly lower degree than in CD71+CD45− cells from healthy donor BM. The erythropoietin response in vivo correlated with in vitro erythropoietin-dependent STAT5 activation in 20 of 22 cases. STAT5 was significantly activated at baseline in MDS cells compared with normal controls, whereas caspase-3 was activated in CD34+ and CD45+ MDS cells, and was activated more often in the RA and RAEB-1 MDS subtypes. G-CSF stimulation activated ERK1/2 and STAT5 equally in MDS and normal CD34+ cells. Conclusions: Abnormalities in the response to growth factors are restricted to erythropoietin stimulation in CD71+CD45− cells and correlate with the clinical response to erythropoietin. Activation of baseline signal transduction for proliferative and apoptotic signals is altered in MDS but with different patterns among the various BM subpopulations. Clin Cancer Res; 18(11); 3079–89. ©2012 AACR.

Differing molecular mechanisms appear to underlie early toxicity of prefibrillar HypF-N aggregates to different cell types.

Considerable attention has been paid to the high cytotoxic potential of small, prefibrillar aggregates of proteins/peptides, either associated or not associated with amyloid diseases. Recently, we reported that different cell types are variously affected by early aggregates of the N‐terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF‐N), a protein not involved in any disease. In this study, we provide detailed information on a chain of events triggered in Hend murine endothelial cells and IMR90 fibroblasts, which have previously been shown to be highly vulnerable or very resistant, respectively, to HypF‐N aggregates. Initially, both cell lines displayed impaired viability upon exposure to HypF‐N toxic aggregates; however, at longer exposure times, IMR90 cells recovered completely, whereas Hend cells did not. In particular, significant initial mitochondrial permeability transition (MPT) pore opening was found in IMR90 cells followed by a sudden repair of membrane integrity with rapid and efficient inhibition of cytochrome c and AIF release, and upregulation of Bcl‐2. The greater resistance of IMR90 fibroblasts may also be due to a higher cholesterol content in the plasma membrane, which disfavours interaction with the aggregates. In contrast, Hend cells, which have less membrane cholesterol, showed delayed MPT opening with prolonged translocation of cytochrome c into the cytosol. Finally, the caspase 9 active fragment was increased significantly in both Hend and IMR90 cells; however, only Hend cells showed caspase 8 and caspase 3 activation with DNA fragmentation. From our data, the different responses of the two cell types to the same aggregates appear to be associated with two key events: (a) aggregate interaction with the plasma membrane, disfavoured by a high level of membrane cholesterol; and (b) alterations in mitochondrial functionality, leading to the release of pro‐apoptotic stimuli, which are counteracted by upregulation of Bcl‐2.