A different perspective on sofosbuvir-ledipasvir treatment of patients with HCV genotype 1b cirrhosis: The ital-c network study.

The effectiveness of a 12-week course of sofosbuvir-ledipasvir in treatment-experienced HCV genotype 1b-infected patients with cirrhosis is still under debate. Our primary endpoint was to compare the sustained virological response at post-treatment week 12 (SVR12) of sofosbuvir-ledipasvir in combination with ribavirin for 12 weeks, and sofosbuvir-ledipasvir alone for 24 weeks. This was a prospective observational study that enrolled 424 (195 naive, 229 experienced; 164 treated for 12 weeks with Ribavirin and 260 with sofosbuvir-ledipasvir alone for 24 weeks) consecutive HCV genotype 1b-infected patients with cirrhosis. The SVR12 rates were 93.9% and 99.2% in patients treated for 12 and 24 weeks, respectively (P = .002). The baseline characteristics of patients treated for 12 weeks were significantly different from those treated for 24 weeks as regards their younger age (P = .002), prevalence of Child-Pugh class A (P = .002), lower MELD scores (P = .001) and smaller number of nonresponders (P = .04). The shorter treatment was significantly associated with a lower SVR12 in univariate and multivariate analyses (P = .007 and P = .008, respectively). The SVR rate was unaffected by age, gender, BMI, Child-Pugh class, MELD score or previous antiviral treatment. Patients receiving ribavirin experienced more episodes of ascites and headache but less recurrence of hepatocellular carcinoma (HCC), and were prescribed more diuretics and cardiopulmonary drugs. No patient discontinued treatment. The therapeutic regimen of sofosbuvir-ledipasvir plus ribavirin administered for 12 weeks was less effective than sofosbuvir-ledipasvir alone given for 24 weeks. At odds with European guidelines, the recommended 12-week treatment with sofosbuvir-ledipasvir alone might be suboptimal for this setting of patients.

The impact of proteomics in the understanding of the molecular basis of Paclitaxel-resistance in ovarian tumors.

The current therapy for ovarian cancer has advanced from alkylating agents, to a combination of carboplatinum and paclitaxel offering increased survival. Although most patients respond to this first-line therapy, initially, the majority of these patients relapse within 2 years. The mechanisms responsible for acquired drug resistance in ovarian cancer have been elucidated only in part. They include i) enhanced drug export, ii) activation/inhibition of intracellular signalling pathways, iii) molecular alterations in tubulin isotype composition. A better understanding of these mechanisms is needed, in order to develop new approaches, aimed at overcoming resistance to anticancer agents, and to reveal the complexity of causes, which contribute to drug resistance. In this review we offer an updated overview of proteomic studies on the molecular mechanisms of paclitaxel resistance. These proteomic studies also identify potential targets for modulating drug resistance, that could be predictive of response to chemotherapy in ovarian carcinomas.

Helicobacter pylori infection and autoimmune thyroid disease in young patients: the disadvantage of carrying the human leukocyte antigen-DRB1*0301 allele.

CONTEXT AND OBJECTIVE: Pathogenesis of autoimmune thyroid disease (ATD) is multifactorial. Helicobacter pylori (Hp) infection has been proposed to be involved in nongastrointestinal conditions and reported more frequently in ATD adult patients. We evaluated the prevalence of Hp antibodies in young ATD patients and investigated the possibility that a susceptible immunogenetic profile could influence the development of ATD in subjects with Hp infection. SUBJECTS AND METHODS: We retrospectively studied 90 children with ATD (median age 11.2 yr), 70 age- and sex-matched healthy subjects as controls, and 65 patients with Turner syndrome (median age 18.8 yr). Antibodies to Hp were determined at diagnosis in ATD patients and, in Turner patients, at the last control in cases without ATD and before the appearance of thyroid autoantibodies in the others. Serological and molecular human leukocyte antigen (HLA) typing for classes I and II polymorphisms was performed. RESULTS: Prevalence of positive Hp serology resulted significantly higher in ATD patients than controls (P = 0.032). No association was found between individual HLA alleles and Hp serology. HLA-A1, B8, and DRB1*0301 were found significantly associated with ATD. A significant interaction between HLA-DRB1*0301 and Hp infection was present in ATD patients and not controls (P = 0.007), suggesting that the copresence of these two factors might favor ATD development. A similar phenomenon was observed in Turner syndrome patients (P = 0.02; cumulative Mantel test, P = 0.0001). CONCLUSIONS: Another target of Hp-elicited immune inflammatory response might be the thyroid gland in subjects with a peculiar immunogenetic profile so that ATD may be a consequence. Our findings suggest the opportunity of eradicating Hp infection in children with ATD and/or susceptible HLA alleles.

Donor-recipient incompatibility at CD31-codon 563 is a major risk factor for acute graft-versus-host disease after allogeneic bone marrow transplantation from a human leucocyte antigen-matched donor.

Disparities at minor histocompatibility antigens (mHA) are thought to be responsible for acute graft-versus-host disease (aGVHD) in patients receiving bone marrow transplantation (BMT) from a human leucocyte antigen (HLA)-matched donor. Although some mHA have been identified in humans, their role in aGVHD has not. Patients (n = 150) receiving a BMT from an HLA-matched donor were investigated for a correlation between aGVHD and donor/recipient incompatibility for seven polymorphisms previously proposed for mHA (HA-1, H-Y, CD31-codon 125, CD31-codon 563, HPA-1, HPA-3 and HPA-5). Only mismatch at CD31-codon 563 predicted grade II-IV aGVHD. The risk derived from CD31-codon 563 mismatch was the same as that derived from the use of bone marrow from an unrelated donor. We suggest that donor/recipient compatibility at CD31-codon 563 should be added to HLA-typing for donor selection and/or adjustment of aGVHD prophylaxis.

N-acetylcysteine inhibits in vivo nitric oxide production by inducible nitric oxide synthase.

This in vivo study evaluates the effect of N-acetylcysteine (NAC) administration on nitric oxide (NO) production by the inducible form of nitric oxide synthase (iNOS). NO production was induced in the rat by the ip administration of 2 mg/100 g lipopolysaccharide (LPS). This treatment caused: (1) a decrease in body temperature within 90 min, followed by a slow return to normal levels; (2) an increase in plasma levels of urea, nitrite/nitrate, and citrulline; (3) the appearance in blood of nitrosyl-hemoglobin (NO-Hb) and in liver of dinitrosyl-iron-dithiolate complexes (DNIC); and (4) increased expression of iNOS mRNA in peripheral blood mononuclear cells (PBMC). Rat treatment with 15 mg/100 g NAC ip, 30 min before LPS, resulted in a significant decrease in blood NO-Hb levels, plasma nitrite/nitrate and citrulline concentrations, and liver DNIC complexes. PBMC also showed a decreased expression of iNOS mRNA. NAC pretreatment did not modify the increased levels of plasma urea or the hypothermic effect induced by the endotoxin. The administration of NAC following LPS intoxication (15 min prior to sacrifice) did not affect NO-Hb levels. These results demonstrate that NAC administration can modulate the massive NO production induced by LPS. This can be attributed mostly to the inhibitory effect of NAC on one of the events leading to iNOS protein expression. This hypothesis is also supported by the lack of effect of late NAC administration.

1-Benzopyran-4-one antioxidants as aldose reductase inhibitors.

Starting from the inhibitory activity of the flavonoid Quercetin, a series of 4H-1-benzopyran-4-one derivatives was synthesized and tested for inhibition of aldose reductase, an enzyme involved in the appearance of diabetic complications. Some of the compounds obtained display inhibitory activity similar to that of Sorbinil but are more selective than Quercetin and Sorbinil with respect to the closely related enzyme, aldehyde reductase, and also possess antioxidant activity. Remarkably, these compounds possess higher pKa values than carboxylic acids, a characteristic which could make the pharmacokinetics of these compounds very interesting. Molecular modeling investigations on the structures of inhibitors bound at the active site of aldose reductase were performed in order to suggest how these new inhibitors might bind to the enzyme and also to interpret structure-activity relationships.

'Free' iron, as detected by electron paramagnetic resonance spectroscopy, increases unequally in different tissues during dietary iron overload in the rat.

'Free' iron concentration, as determined by electron paramagnetic resonance (EPR) spectroscopy, and lipid peroxidation (LPO), as determined by thiobarbituric acid test, were assessed in the lung, heart, liver, spleen, brain and kidney of rats subjected to experimental iron overload. Two tests, Desferal- and NO-available iron, were used to measure 'free' iron and gave comparable results. The most pronounced accumulation of 'free' iron was observed in liver, kidney and spleen. Differences between control and iron loaded animals increased during the initial 90 days of treatment. Between 90 and 180 days 'free' iron concentration reached a steady state level, or even decreased, as in the case of liver. Lipid peroxidation level, measured in the organs of both treated and matched controls, did not give any significant difference during the initial 90 days of treatment. A significant augmentation was observed in liver, kidney, spleen and heart at 180 days. The results of the present research show that, under conditions of moderate siderosis, the occurrence of LPO is partially related to the level of 'free' iron.

Acute promyelocytic leukemia toluidine blue subtype.

In the hypergranular group of acute promyelocytic leukemia (APL) a rare subvariant with basophilic granules, metachromatic for toluidine blue, is recognizable. To evaluate the incidence as well as the biological and clinical significance of this subtype, we studied 53 consecutive untreated patients with APL with morphological, cytochemical, immunological and cytogenetic methods. In 10 cases (19% of the total) granules stained metachromatically in percentages of promyelocytes ranging from 16 to 60. In these cases peroxidase positivity was weaker than in the classic hypergranular and microgranular M3 and activities of esterases were usually present; at the ultrastructural level granules contained particulate material. Immunophenotypic and cytogenetic characteristics seemed not to differ from those of other M3 cases. Coagulopathy was usually life-threatening, notwithstanding the low white cell count, and the median survival was short. Hyperhistaminemia-related symptoms were not observed. Cytochemical, immunologic and cytogenetic findings are useful to differentiate this form from M2 with basophilic differentiation and from mast cell leukemia.

Free radical production during metabolism of organic hydroperoxides by normal human keratinocytes.

Evidence of a relationship between tumor production induced by various organic (hydro)peroxides and free radical formation has been shown in cultured murine keratinocytes and human skin-tumor cell line. In the present study the bioactivation of cumene hydroperoxide, t-butyl-hydroperoxide, and benzoyl peroxide via one-electron oxidation or reduction was compared in freshly isolated and in cultured normal human keratinocytes. The formation of methyl free radicals during the metabolism of cumene and t-butyl-hydroperoxide was shown by the electron spin resonance-spin trapping technique. Radical formation increased under hypoxic conditions. An intracellular activation site was demonstrated by the use of two spin-trapping agents, the hydrophilic, membrane-impermeable, 3,5-dibromo-4-nitrosobenzenesulfonic acid and the lipophilic, membrane-permeable alpha-(4-pyridyl-1-oxide)-N-t-butylnitrone. At 30 min incubation and 25 mM concentration, hydroperoxides exhibited cytotoxicity, as indicated by trypan blue exclusion and lactate dehydrogenase release assay; free radicals were concurrently trapped. Hydroperoxides at a lower concentration (1 mM) did not significantly affect cell viability. However, free radical production was still detected using a membrane-permeable spin trap. The incubation of keratinocytes with benzoyl peroxide did not show any peroxide-dependent radical adduct. No significant differences in bioactivation capability were demonstrated between freshly isolated and cultured human keratinocytes. The results indicate that cultured human keratinocytes can be used as a model system for the study of the metabolic activation to free radical intermediates of toxic and carcinogenic compounds in the epidermis.

Acute promyelocytic leukemia: morphological and clinical features.

BACKGROUND AND METHODS: Acute promyelocytic leukemia (APL) is not a morphologically homogeneous entity: to verify whether there is any relationship between this heterogeneity and other biological and clinical aspects, we studied 43 cases of APL with morphological, cytochemical, cytogenetic and immunological methods. RESULTS: Three morphological categories were present: a classic hypergranular type (30 cases), a microgranular type (6 cases) and a form with basophilic granules (M3b) that stained metachromatically with toluidine blue (7 cases). In all these groups there were cases with cytochemical features of both myeloid and monocytic type (alpha-naphthyl-acetate esterase positive). No immunological and cytogenetic differences were observed; the morphological variant with basophilic granules was more frequent in females; age distribution was not related to the morphological subtype; organomegaly was extremely rare in M3b. A low white blood cell count was constant in M3b, whereas no differences were observed in hemoglobin and platelet values. Severity of bleeding was worst in the group with toluidine blue metachromasia; this and the microgranular type had poor prognosis. CONCLUSIONS: Our study confirms the importance of identifying different cytologic categories in APL. In particular we focused our attention on a new variant with basophilic granules.

Transient aplasia preceding adult acute lymphoblastic leukemia.

A 62-year-old woman developed common acute lymphoblastic leukemia (ALL) after spontaneous recovery from transient marrow aplasia. Although the mechanisms underlying bone marrow suppression in acute leukemia are obscure, it is important to know that transient aplasia may be observed as a prodromal feature in ALL in adult patients as well as in children.

[A case of thyroid ectopia: its original diagnostic procedure]. / Un caso di ectopia tiroidea: originale iter diagnostico.

A case of thyroid ectopy in a 8-years-old girl is here described. The diagnostic course of this case is quite particular. In fact the first sign of the disease was a morphological evidence of thyroid sketch at the basis of the tongue. There has never been evidence of thyroid bad functioning.

Immunocytochemical detection of factor XIII A--subunit in acute leukemia.

Factor XIII (FXIII) is a plasma pro-transglutaminase consisting of A and B subunits in a tetrameric structure. A cellular form of FXIII consisting exclusively of A subunits exists in platelets and monocytes: monocyte FXIII may be involved in connective tissue organization. To evaluate the expression and diagnostic significance of FXIII A subunit (FXIIIA) in acute leukemia, we performed an immunocytochemical study (PAP technique) with rabbit antiserum against FXIIIA on leukemic blasts of 48 cases. FXIIIA was detected only in myelomonocytic (M4), monocytic (M5) and megakaryocytic (M7) cases: in M4 and M5 samples the amount of blast cytoplasmic FXIIIA was closely correlated with the expression of monocyte-specific antigenic and cytochemical markers. Our data show immunocytochemical detection of FXIIIA to be useful for acute leukemia characterization.

In vitro and in vivo evidence for the formation of methyl radical from procarbazine: a spin-trapping study.

Electron spin resonance (ESR) analysis combined with the use of 4-pyridyl-1-oxide-t-butyl nitrone (4-POBN) and dibromonitroso benzenesulfonic acid (DBNBS) as spin-trapping agents was used to characterize free radical generation during the metabolism of the anticancer agent procarbazine [N-isopropyl-a-(2-methylhydrazino)-p-toluamide hydrochloride]. The formation of free radical species, identified as methyl radicals, was observed during oxidation of procarbazine in rat liver microsomes and isolated hepatocytes in vitro, as well as in several organs following administration of the drug in vivo. A cytochrome P450-mediated reaction, involving P450IA and IIB isoenzymes, was responsible for the activation process. The metabolic pathway leading to free radical formation was characterized using various procarbazine metabolites and revealed strict analogies with previously published data on methane production from procarbazine. These results supported the identification of the trapped species as methyl free radical and suggested that C-oxidation of azoprocarbazine is the main source of radical intermediates derived from this anticancer drug.

Blood clearance of dextran magnetite particles determined by a noninvasive in vivo ESR method.

Dextran magnetite (DM) is a potential MR contrast agent with superparamagnetic properties. Its fast clearance from the blood and selective uptake by tissue macrophages provide advantages for imaging tumors in the liver and spleen. DM consists of a suspension of solid particles with a wide distribution of sizes. In this study we have used ESR spectroscopy to determine the blood clearance of DM injected iv in mice. The spectra are obtained on living animals by inserting the tail of a mice into the waveguide cavity of the ESR spectrometer and recording the ESR spectrum continuously. This procedure allows the direct measurement of the plasma clearance of DM from individual animals, without blood sampling. We applied this method to study the clearance of suspensions of DM particles with different average sizes.

Selective uptake of 2-thiouracil into melanin-producing systems depends on chemical binding to enzymically generated dopaquinone.

2-Thiouracil (TU), an antithyroid drug, is receiving growing interest as a specific tumor marker for malignant melanoma, owing to its capability of being selectively accumulated into active melanin-producing tissues. However, up until now, the molecular mechanism of TU uptake by growing melanin has remained largely unknown. In an attempt to fill this gap, we have investigated the effect of TU on the tyrosinase catalyzed oxidation of tyrosine. At a concentration of 0.5 mM, TU was found to totally inhibit melanin formation by tyrosinase catalyzed oxidation of 0.25 mM tyrosine in phosphate buffer at pH 6.8. Polarographical monitoring of oxygen consumption under conditions of complete suppression of melanogenesis revealed a significant tyrosinase activity, with TU acting as a modest non-competitive inhibitor of the enzyme (Ki = 0.6 mM). HPLC and TLC analysis of the tyrosine-tyrosinase reaction in the presence of excess TU showed that the substrate is progressively consumed and a major hitherto unknown product (lambda max = 284 nm), positive to ninhydrin and ferric chloride, is concomitantly formed. This was isolated by repeated gel filtration chromatography of the reaction mixture on Sephadex G-10 and was formulated as the TU-dopa adduct 3,4-dihydroxy-6-(4'-hydroxypyrimidinyl-2'-thio)phenylalanine by spectral analysis. These results suggest that selective TU incorporation in pigmented melanomas and other melanin-producing systems is due to the covalent binding to dopaquinone, produced by tyrosinase catalyzed oxidation of tyrosine.

Metabolism of nitroxide spin labels in subcellular fractions of rat liver. II. Reduction in the cytosol.

As part of an ongoing study of the role of subcellular fractions on the metabolism of nitroxides, we studied the metabolism of a set of five nitroxides in cytosol derived from rat hepatocytes. The nitroxides were chosen to provide information on the effects of the type of charge and the ring on which the nitroxyl group is located. The rates of reduction were fastest for a six-membered positively charged nitroxide ('CAT-1') and slowest for an anionic five-membered ring nitroxide ('PCA'). Changing levels of glutathione, sulphydryl groups in general, NADPH or NADH had little or no effect on the rates of reduction, while the addition of ascorbate oxidase essentially abolished reduction of the nitroxides. The products of reduction by the cytosol were the corresponding hydroxylamines. The overall rates of reduction of neutral or anionic nitroxides were much slower than those observed with intact cells. We conclude that the primary source of metabolism of nitroxides by cytosol is reduction by ascorbate and that under most conditions reduction of nitroxides in the cytosol is not a major factor in the metabolism of nitroxides by cells.

Bone marrow uptake of liposome-entrapped spin label after liver blockade with empty liposomes.

Using an ESR spectrometer, we studied the time course of the uptake of the liposome-entrapped spin label 2,2,6,6-tetramethylpiperidine-N-oxyl-4-trimethylammonium in liver, spleen, and bone marrow following reticuloendothelial liver blockade. Our results show that suppression of the phagocytic activity of the liver increases the delivery of liposomes to the spleen and bone marrow without substantially altering uptake by the liver.