COS-7-based model: methodological approach to study John Cunningham virus replication cycle.

John Cunningham virus (JCV) is a human neurotropic polyomavirus whose replication in the Central Nervous System (SNC) induces the fatal demyelinating disease, progressive multifocal leukoencephalopathy (PML). JCV propagation and PML investigation have been severely hampered by the lack of an animal model and cell culture systems to propagate JCV have been very limited in their availability and robustness. We previously confirmed that JCV CY strain efficiently replicated in COS-7 cells as demonstrated by the progressive increase of viral load by quantitative PCR (Q-PCR) during the time of transfection and that archetypal regulatory structure was maintained, although two characteristic point mutations were detected during the viral cycle. This short report is an important extension of our previous efforts in defining our reliable model culture system able to support a productive JCV infection.Supernatants collected from transfected cells have been used to infect freshly seeded COS-7 cell line. An infectious viral progeny was obtained as confirmed by Western blot and immunofluorescence assay. During infection, the archetype regulatory region was conserved.Importantly, in this study we developed an improved culture system to obtain a large scale production of JC virus in order to study the genetic features, the biology and the pathogenic mechanisms of JC virus that induce PML.

Effect of cigarette smoke extract on the polymorphonuclear leukocytes chemiluminescence: influence of a filter containing glutathione.

Cigarette smoking is known to be a risk factor for several chronic and neoplastic diseases. Many compounds formed by cigarette burning, ranging from particulate materials to water solutes and gaseous extracts, are considered to be noxious agents, and many biochemical and molecular mechanisms have been proposed for the toxic effects of cigarette smoke. The oral cavity and the upper respiratory tract represent the first contact areas for smoke compounds; even a single cigarette can produce marked effects on some components of the oral cavity, either chemical compounds, such as glutathione and enzymes, or cellular elements, such as polymorphonuclear leukocytes. Several studies suggest a protective role of glutathione against the noxious effects of tobacco smoke; the sulphydril groups of glutathione, in fact, could react with some smoke products, such as unsaturated aldehydes, leading to the formation of harmless intermediate compounds and simultaneously preventing the inactivation of metabolically essential molecules, such as some enzymes. In this paper we analyse the effect of a filter containing glutathione on the respiratory burst of polymorphonuclear leukocytes exposed to aqueous extract of cigarette smoke, measuring their chemiluminescence activity. The results of this paper indicate that the GSH-containing filter has a likely protective effect against the inhibition of cigarette smoke extract on polymorphonuclear leukocyte activity.

Return to normal values of lipid pattern after effective chemotherapy in acute lymphoblastic leukemia.

In the present work we investigated HDL-C and its subfractions HDL2 and HDL3 as well as total cholesterol (TC), triglycerides (TG), LDL-C, VLDL-C, apolipoproteins A1 (ApoA1) and B (ApoB) and lipoprotein (a) (Lp(a) in 25 patients with newly diagnosed acute lymphocytic leukemia (ALL) before and after induction treatment. The mean basal plasma levels of TC, HDL-C and its subfractions, LDL-C, and ApoA1 were significantly lower than the mean values observed in normal subjects, whereas TG and VLDL-C were significantly higher. The patients (n = 22) who achieved complete remission after chemotherapy showed a significant increase of TC, HDL-C, HDL2, HDL3, ApoA1 and a significant decrease of TG and VLDL-C. These data suggest that ALL patients are characterized by a lipid metabolic derangement mainly of HDL and TG-rich lipoproteins, that is reversed by effective treatment of disease.

Prognostic relevance of lipoprotein cholesterol levels in acute lymphocytic and nonlymphocytic leukemia.

We studied serum lipid and lipoprotein changes before and after induction treatment in 25 acute nonlymphocytic leukemia (ANLL) and in 18 acute lymphocytic leukemia (ALL) patients in order to investigate their relationship with disease activity and their prognostic relevance. ANLL at diagnosis is associated with significantly low levels of all lipid parameters, the same applies to ALL patients apart from plasma triglycerides and very-low-density-lipoprotein cholesterol (VLDL-C) which are significantly higher than in the normal population. In ANLL responders, after effective chemotherapy, a significant increase of total cholesterol, low-density-lipoprotein cholesterol (LDL-C) and apolipoprotein B levels, without changes of high-density-lipoprotein cholesterol (HDL-C) values, is observed. A further decrease of total cholesterol and LDL-C was found in nonresponders and in ANLL responders treated with granulocyte-macrophage colony-stimulating factor (GM-CSF), known for its cholesterol-lowering action; in fact after the completion of GM-CSF therapy, these parameters returned progressively toward normal values. In ALL responders an increase of total cholesterol, HDL-C and apolipoprotein A1 with a simultaneous decrease of triglycerides and VLDL-C is evident; no variation was found in the nonresponder group. These results suggest a close correlation between serum lipids and acute leukemia: total cholesterol and LDL-C in ANLL, and HDL-C and VLDL-C in ALL may be considered reliable markers of complete remission and may be useful in the follow-up of leukemic patients.

Choice of time for urine collection for detecting early kidney abnormalities in hypertensives.

The aim of this study was to determine the most appropriate urine collection for detecting differences in the excretion rates of albumin, gamma glutamyl transpeptidase (GGT) and N-acetyl-beta-D-glucosaminidase (NAGA) between normotensive subjects and hypertensive patients on treatment. Twenty treated hypertensive patients, mean (SEM, standard error of mean) age; 52.2 (6.2) years and 20 normotensive subjects, mean age 49.2 (4.2) years, were studied in a consecutive sampling design. Urinary excretion rates of albumin, GGT and NAGA were determined in consecutive timed urine samples collected overnight and during 3-5 h the next morning. Mean (SEM) overnight excretion rates for albumin, GGT and NAGA for normotensive subjects were 11.05 (1.18) micrograms/min, 17.00 (2.20) mU/min and 6.55 (0.39) mU/min, respectively, which were significantly lower than those of hypertensive subjects which were 20.77 (2.14) micrograms/min, 21.84 (1.65) mU/min and 10.92 (0.87) mU/min, respectively (P < 0.05). The mean (SEM) percentage increases in urinary albumin, GGT and NAGA in morning urine collections of normotensive subjects of 15.22 (3.88)%, 34.04 (6.45)% and 11.54 (3.63)%, respectively were significantly lower than 107.03 (15.04)%, 121.96 (16.71)% and 72.75 (7.50)% found in hypertensive patients (P < 0.05). These data suggest that were urinary albumin and tubular enzyme excretion to be used as correlates of hypertensive renal damage, ambulatory urine collections may be more sensitive than overnight collections.

Lipids and lipoproteins in acute lymphoblastic leukaemia (ALL).

Current studies suggest a derangement of the cholesterol homeostasis in certain types of leukaemia. The low serum cholesterol levels observed in patients with AML have been related to the tumoral mass and to the disease activity. Only a few studies have taken into consideration a possible change of the serum lipids in ALL. The aim of this study was to evaluate a possible prognostic significance of serum levels of total cholesterol (TC), triglycerides, HDL-C, LDL-C, VLDL-C, apolipoproteins A1 and B and lipoprotein (a), investigated in 10 newly diagnosed ALL patients before and after induction treatment. At diagnosis all these parameters were not significantly different in our patients vs control group except cholesterol and HDL-C levels, which were significantly lower. After the induction treatment, we found a significant increase of HDL-C and Apo A1 values only in those patients that achieved a complete remission. These results support the idea that some serum lipids, such as HDL-C and Apo A1, may have a role as early and reliable markers of the effectiveness of chemotherapy.

T lymphocyte subsets and platelet-associated IgG in idiopathic thrombocytopenic purpura: effect of splenectomy.

The modifications of cell-mediated immunity in idiopathic thrombocytopenic purpura (ITP) were investigated using the technique of Moretta et al. [J. exp. Med. 151: 969-974, 1980] to study T lymphocyte subsets in 35 ITP subjects at various clinical stages. In all these patients, platelet-associated IgG (PAIgG) were measured by a complement lysis inhibition technique. Untreated ITP patients did not show significant modifications of T lymphocyte subsets. Patients with thrombocytopenia not responsive to corticoids or splenectomy and increased PAIgG levels showed significant reduction of Fc-gamma-bearing T lymphocytes (T-gamma). Patients 'cured' by splenectomy (12 out of 17) showed a normal PAIgG level, a marked increase of T-gamma cells and a reduction of T-mu cells; patients 'not cured' by splenectomy showed an increased PAIgG level and a reduction of T-gamma cells. An increase of PAIgG levels and a reduction of T-gamma cells were found in 2 splenectomized patients after relapse. An association between chronic nonresponsive ITP and decrease of T-gamma cells was established.

Transient deficiency of peripheral blood accessory cells in supporting T cell mitogenesis in patients suffering from chronic idiopathic thrombocytopenic purpura after intravenous gammaglobulin treatment.

Mitogenic response of blood lymphocytes to phytohemagglutinin (PHA) and to OKT3 monoclonal antibodies was investigated in 7 patients suffering from chronic idiopathic thrombocytopenic purpura (ITP) before, during and after high-dose intravenous (i.v.) immunogammaglobulin (IgG) infusion. The platelet count rose above the pre-treatment values during infusion therapy in all patients but one. Five out of seven patients presented elevated platelet-associated IgG (PA-IgG) levels at the time of the first infusion; four of these showed an increase in platelet count and a transient reduction or normalization of PA-IgG after IgG infusion. Five out of seven patients showed an impairment of T lymphocyte mitogenic response to PHA and OKT3 before therapy. All patients responded to IgG therapy with a transient deficiency of FcR mediated monocytes (Mo) in supporting T cell mitogenesis induced by both mitogens during and after IgG infusion. This reduced cooperative capability of Mo disappeared at various times after the end of therapy (range 3-12 days). The transient alteration of Mo function, possibly due to a modification in the surface number or in the affinity of Fc-receptors, can explain in part, the increase in platelet count during and after IgSRK infusion.

Platelet activation in patients with benign and malignant ovarian diseases.

Plasma concentration of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) were measured by radioimmunoassay in 45 patients with benign and malignant ovarian diseases. All patients with ovarian carcinoma showed increased beta-TG and PF4 levels. Among benign ovarian diseases the patients with serous cystadenoma more frequently showed signs of platelet activation, whereas those with endometriotic cyst and mucinous cystadenoma generally had normal beta-TG and PF4 values. These results indicate that an increased platelet activation is consistently associated with malignant tumors of the ovary, whereas benign tumors show a different capacity to induce platelet activation.

Coagulation disorders in patients with tumors of the uterus.

Sixty-eight previously untreated female subjects were studied: 26 patients with cervical carcinoma, 22 with endometrial carcinoma, and 20 with benign uterine diseases. These patients, together with 25 healthy female control subjects, underwent several coagulation tests, including beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4) plasma levels. Of all the parameters considered, the variations in beta-TG and PF4 were the most interesting. They were increased in patients with cervical and endometrial carcinoma. The sensitivity of the two tests reached 79% (15/19) for patients with invasive cervical carcinoma and 74% (16/22) for all patients with endometrial carcinoma. Our data demonstrate that among the investigated parameters beta-TG and PF4 are the earliest disorders of the hemostatic system and are more frequently increased in the gynecologic malignancies.