Expression and localization of the deubiquitinating enzyme mUBPy in wobbler mouse testis during spermiogenesis.

Mouse ubiquitin-specific processing protease (mUBPy) is a deubiquitinating enzyme highly expressed in both brain and testis. In testis, it interacts with the DnaJ protein, MSJ-1; both mUBPy and MSJ-1 are located on the cytoplasmic surface of the developing acrosome and in the centrosomal region during spemiogenesis. Present data show the first appearance in testis of mUbpy mRNA and protein at 10 days post-partum (d.p.p.). In addition, to investigate on a possible role of mUBPy in sperm formation, we took advantage of mutant wr/wr (wobbler) mice characterized by male infertility, which is likely due to the lack of a real, functional acrosome. RT-PCR and Northern blot analyses show that mUbpy is up-regulated in adult wobbler testis. Furthermore, in wild-type testis mUBPy protein is primarily detected by Western blot in the soluble (cytosolic/nuclear) fraction during the first round of spermatogenesis and in the adult. By contrast, mUBPy is primarily detected in membranous/insoluble protein fraction when wobbler phenotype is clearly shown (30 d.p.p.) and in adult wobbler testis. By immunohistochemistry, whereas in wild-type animals mUBPy marks the profile of the acrosomic vesicle in differentiating spermatids, in wobbler mice only a detergent pre-treatment procedure allows to detect mUBPy immunoreactivity, which results in diffuse spotted granules inside the cytoplasm and around the nuclear shape. In conclusion, in wobbler testis expression of mUbpy is up-regulated, while a differential sorting of the protein characterizes wobbler spermatids where acrosome formation is impaired.

Structure of msj-1 gene: a comparative analysis.

Msj-1 gene encodes a DnaJ protein highly expressed in spermatids and spermatozoa of both rodents and amphibians. We isolated and characterized the msj-1 gene in mice. A bioinformatic approach was then used to predict the putative promoter region, chromosomal localization, and its presence in the human genome. The analysis of msj-1 genomic sequence revealed that msj-1 is an intronless gene. Interestingly, two regions (A and B, separated by 10,682 bp) on human chromosome 2 having respectively 78% and 77% nucleotide identity with the murine msj-1 coding region were identified. This suggests the existence of an msj-1-like gene also in humans.

Impaired insulin-mediated amino acid plasma disappearance in non-alcoholic fatty liver disease: a feature of insulin resistance.

BACKGROUND AND AIM: Insulin resistance is a main feature, and possibly a pathogenic factor, of non-alcoholic fatty liver disease. It is usually measured on glucose metabolism; the effects on amino acid regulation have never been assessed. In particular, no data are available on insulin-dependent branched-chain amino acid metabolism, which is under insulin control. MATERIALS AND METHODS: We measured amino acid disappearance from plasma during an euglycemic glucose clamp in 39 biopsy-proven non-alcoholic fatty liver disease patients and in ten control subjects. A primed-constant infusion of insulin (constant rate, 40 mU/m2 per min for 2 h) was used to raise plasma insulin to approximately 100 mU/l. Euglycemia was maintained by a variable glucose infusion, a measure of tissue insulin sensitivity. Plasma amino acids were assayed during the clamp after ninhidrin derivatization. RESULTS: Fasting plasma amino acids were similar in the two groups. Steady-state insulin levels were significantly higher in non-alcoholic fatty liver disease patients, whereas tissue sensitivity to insulin was reduced by 50%. The plasma disappearance of branched-chain amino acids, as well as the disappearance of the sum of glutamine and glutamate and that of serine were significantly reduced in non-alcoholic fatty liver disease. Differences were maintained after adjustment for steady-state insulin, and correlated with reduced tissue sensitivity to glucose. CONCLUSION: Insulin resistance in non-alcoholic fatty liver disease patients also affects amino acid metabolism, especially for amino acids involved in peripheral muscle nitrogen exchange. The metabolic effects of altered protein/amino acid metabolism must be considered.

Diagnostic and prognostic value of flow cytometric immunophenotyping in malignant hematological diseases.

Flow cytometry is a diagnostic cell analysis technique with ever increasing applications in modern hematological practice. To date immunophenotyping of clonal hematological diseases represents one of the primary clinical applications of flow cytometry. Immunophenotyping of abnormal cells is now considered a fundamental tool to establish the cell lineage assignment and to obtain a more precise identification of the various cell subtypes. A number of observations have emerged showing strong association between specific immunophenotypes and genetic recurrent abnormalities underlying the malignant transformation, with prognostic value.

Absolute TCD4+ counting by a minimalist dual-platform flow cytometric method.

UNLABELLED: The aim of this work was to compare the performance of an absolute TCD4+ counting method based on total WBC gating versus the standard lymphocyte (Ly) gating method, in order to develop a flow cytometric (FCM) minimalist strategy for TCD4+ enumeration. METHOD: 132 routine peripheral blood samples, mainly from HIV infected patients, were labelled with CD3-FITC/CD4-PE/CD45-PECy5 and analyzed by two gating methods: a) standard method based on Ly immunological gating (CD45++SSClow), followed by the determination of CD3+CD4+ percentage and absolute number (# calculation using Ly # from hematological analyser (HA); b) total WBC immunological gate on biparametric scatter CD45/CD4, followed by CD4++SSClow percentage determination and absolute number calculation using WBC absolute number from hematological counter without using the WBC differential. Moreover on 63 samples Ly # based on Ly % from FCM and WBC counting from HA was compared with Ly # from HA. RESULTS: The TCD4+/microL ranged from 3 to 3277 and the statistical analysis results showed: a) linear regression: r2 = 0.9847; b) Bland & Altman analysis: difference mean = -56.22; agreement range = +95.68 / -208.12; c) the mean of result difference/mean value*100 between two methods was -9.06%; d) comparison between regression line and the boundaries for acceptable residual values based on regressed confidence limits found by A. Kunkl et al showed regression line within boundaries near the upper limits. The Ly/microL count ranged from 635 to 8752. The statistical analysis results showed: a) linear regression: r2 = 0.9764; b) Bland & Altman analysis: difference mean = -362.93; agreement range = +134.51 / -860.37; c) the mean of result difference/mean value*100 between two methods was -16.12%. CONCLUSIONS: Our results suggest a fair agreement between the two gating methods, but the one based on total WBC gate gives TCD4+/microL counts systematically higher than the standard method. This finding can be attributed to a systematic lower estimation of Ly% by HA.

Monoclonal antibody panels for acute leukemia and myelodysplastic syndrome diagnosis. Results of a co-operative quality control group.

The need for standardization criteria and result reproducibility in immunophenotyping hematological diseases has increased along with their clinical importance. Our group "Policentric Study Group on Immunological Markers", is composed of 40 laboratories. Its aim, over recent years, has been to find a standardized way of immunophenotypic analysis applicable to various hematological diseases. The objective of this study is to contribute to the debate concerning standardization of monoclonal antibody panels and immunophenotypic analysis procedures in acute leukemia (AL) and myelodysplastic syndrome (MDS), with the following targets: to improve interlaboratory reproducibility of the immunophenotyping data, and interpretative results; to study, with improved feasibility, correlation between immunophenotype and clinical or biological findings on a large number of AL and MDS cases; to verify the utility of the proposed monoclonal antibody panels for proper AL and MDS classification, and to detect minimal residual disease. In the field of AL and MDS our experience is based on about 1800 and 700 cases respectively analyzed over the last five years. Starting from these experiences and data of the literature we have elaborated the proposed panels of monoclonal antibodies and the methods of analysis. We have suggested a standardized immunophenotypic approach to study AL and MDS. In particular our work has focused on the gating strategy. This aims at drawing a gate of analysis having high purity and recovery, and on the choice of monoclonal antibody combinations for multiparametric analysis, particularly the normal antigen expression on each step of lineage differentiation or their clinically relevant aberrant expressions. A standardized criteria has become a necessary starting point in any kind of analytical process. In the field of acute leukemias and myelodysplastic syndromes the work of this polycentric group has focused on the pre-analytical and analytical steps to be taken in cytometric evaluation of hematological malignancies. The results obtained may contribute to reaching intra and inter-laboratory reproducibility.

Lipoperoxide plasma levels in patients with liver cirrhosis.

BACKGROUND/AIMS: Oxygen free radicals might play a role in the pathogenesis of tissue damage in many pathological conditions, including liver diseases where antioxidant tissue systems are reduced. The leading mechanism of free radical toxicity is the peroxidation of membrane phospholipids. Lipoperoxide hydrolysis produces aldehydes -the most represented being malondialdehyde-which reacts with thiobarbituric acid and whose concentration is considered a marker of lipid peroxidation. MATERIALS AND METHODS: We developed a fast and cheap HPLC method for measuring malondialdehyde concentration in plasma using a 3 mm C18 Baker column (4.6 x 50 mm). Thiobarbituric acid-reacting substances were eluted isocratically with a mobile phase containing methanol/KH2PO4 (35/65), and detected fluorometrically. The whole analysis lasts 2.5 minutes. The fasting levels of thiobarbituric acid-reactive substances were measured in 30 non-smoking blood donors and in 45 patients with liver cirrhosis. RESULTS: In control subjects they were on average 0.84 [SD 0.41] mumol/L and were increased to 1.59 [SD 1.23] mumol/L in patients with cirrhosis, where they inversely correlated with hepatocellular function. CONCLUSIONS: The method compares favorably with previous techniques in terms of cost and analytical time. It can be used for serial measurement of plasma lipoperoxide concentrations in response to oxidative stress or following drug administration. Our preliminary data confirms the presence of an oxidative stress in cirrhotic patients. Normal lipoperoxide levels in subjects with very advanced disease may be due to polyunsaturated fatty acid deficiency and/or enzyme defects.

Ticlopidine administration to patients does not inhibit the ability of their monocytes to form rosettes and to phagocytize erythrocytes in vitro.

Inhibition of monocyte function is known to have important clinical consequences and to be present in a variety of diseases: Accordingly we evaluated the effect of Ticlopidine, a powerful and widely employed inhibitor of platelet aggregation, on the capacity of peripheral blood monocytes to ingest IgG-coated erythrocytes. Our results show that in vivo administration of this drug has no effect on monocyte phagocytosis, when tested with an in vitro assay. This finding suggests that Ticlopidine can be safely used in patients with depressed monocyte function and in those in whom an impairment of monocyte phagocytosis could lead to a deterioration of the clinical picture.

Thymomodulin increases the depressed production of superoxide anion by alveolar macrophages in patients with chronic bronchitis.

Thymomodulin is an immunomodulating agent which is derived from calf thymus by partial acid lysis. It promotes T-cell maturation, enhances antibody synthesis and improves the phagocytic response of neutrophils. Clinical trials have revealed the effectiveness of this thymic derivative in the prevention of recurrent respiratory infections (RRI) in children and in adults; 11 patients (8 males and 3 females; age range 18-76 years) with chronic bronchitis dominated by recurrent respiratory infections were studied. They were treated orally for 6 months during the winter season with 120 mg/day of thymomodulin. All the subjects were asked to keep a diary recording the intensity of their symptoms, the number of working days lost (days of illness) and the use of antibiotic and/or mucolytic drugs. At the beginning and at the end of the trial each patient was subjected to a control with a flexible fibreoptic bronchoscope with bronchoalveolar lavage to evaluate the phagocytic response of alveolar macrophages. At the end of therapy a significant improvement of the clinical status, evaluated by the above-mentioned parameters, of the bronchial mucosa aspect and an increase in alveolar macrophage superoxide production was noticed (from 0.1 +/- 0.09 and 0.8 +/- 0.5 nmol to 1.6 +/- 0.8 and 4.1 +/- 2.2 nmol with PMA or zymosan particles respectively; p less than 0.001). During thymomodulin treatment no side-effects were recorded.

Structure of msj-1 gene in mice and humans: a possible role in the regulation of male reproduction.

Msj-1 gene encodes a DnaJ protein highly expressed in spermatids and spermatozoa of both rodents and amphibians, possibly involved in vesicle fusion and protein quality control by means of interaction with heat shock proteins. We isolated and characterized the entire murine msj-1 gene and searched for putative msj-1-like genes into the human genome. Furthermore, ultrastructural localization of MSJ-1 was analyzed in mouse germ cells by immunogold electron microscopy. The analysis of murine msj-1 genomic sequence reveals that it is an intron less gene. Putative promoter region was predicted within the 600 bp upstream the transcription start site. In mouse, msj-1 maps on chromosome 1, into an intronic region of UDP glucuronosyl-transferase 1 family cluster. At ultrastructural level, MSJ-1 marks the developing acrosomic vesicle and the sperm centriolar region. A blast search against the human genome database revealed two closed regions (Ha and Hb) on human chromosome 2 having high nucleotide identity with murine msj-1 coding region. Similarly to mouse, in human both regions map into an intronic region of UDP glycosyl-transferase 1 family polypeptide A cluster (ugt1a@). A significant ORF encoding a putative DnaJ protein of 145 aa was predicted from Ha. Finally, expression analysis, conducted by RT-PCR in human sperm cells, demonstrated that Ha mRNA is effectively present in humans; by Western blot, a specific MSJ-1 band of approximately 30kDa was detected in human sperm. Taken together, these data suggest that msj-1 gene might be conserved among vertebrates and might exert fundamental functions in reproduction.

Increased frequency of antibodies to ubiquitous viruses in essential mixed cryoglobulinaemia.

Antibodies to ubiquitous Herpes viruses have been studied in 13 patients with type II essential mixed cryoglobulinemia (EMC), and in two different control groups. All the EMC patients had monoclonal IgM kappa in their cryoprecipitates. IgM antibodies to the viral capsid antigen (VCA) of Epstein-Barr virus (EBV) were found in the sera of 11 EMC patients but all the cryoprecipitates were negative. IgG antibodies were also present in the sera of all and in the cryoprecipitates of some patients. In contrast, the number of subjects with antibodies to Cytomegalovirus (CMV) and Hepatitis B Virus (HBV) was not higher than in controls. Possible correlations between EMC and EBV infection are discussed.

Increase in peripheral benzodiazepine receptors on monocytes in fibromyalgia.

OBJECTIVE: The aim of this study is to evaluate the expression of Peripheral Benzodiazepine Receptors (PBRs) on leukocytes in patients affected by primary fibromyalgia and to argue their possible role in pain perception and in modulation of immunologic process. METHODS: The expression of PBRs has been evaluated by flow cytometry on monocytes, on lymphocytes and on granulocytes in twenty patients with primary fibromyalgia, with indirect immunofluorescence methods. RESULTS: Upregulation of leukocyte PBRs expression has been demonstrated in fibromyalgia. A statistically significant difference has been documented only in monocytes. The monocyte PBRs expression was 26.74 +/- 14.84 MIF in fibromyalgia versus 17.45 +/- 8.54 MIF in controls (P < 0.023). Upregulation of PBRs expression, although not statistically significant, was also observed in lymphocytes and granulocytes. CONCLUSIONS: The monocyte PBRs overincrease in fibromyalgia may be due to abnormalities in the regulation of pain or to inflammation. It might perhaps explicate the possible mechanisms of therapeutic response to benzodiazepine in fibromyalgia.

Bacillus subtilis selectively stimulates the synthesis of membrane bound and secreted IgA.

Peripheral blood lymphocyte subpopulations and IgA production in vitro have been studied in 30 elderly subjects. Fifteen patients were treated with Bacillus subtilis (Enterogermina, Midy) and 15 with a placebo preparation. At the end of the treatment, a significant increase was noticed in lymphocytes bearing membrane IgA and Ia+ cells in the group treated with Bacillus subtilis, but not in the controls. Similarly, a significantly enhanced spontaneous production of IgA in vitro was seen in the treated subjects. The relevance of these findings to the understanding of the mode of action of Bacillus subtilis and its usefulness in different immunodeficiency states is discussed.

Comparison of serum and liver hepatitis C virus quasispecies in HCV-related hepatocellular carcinoma.

BACKGROUND/AIMS: The hepatitis C virus (HCV) genome consists of quasispecies populations of heterogeneous variants, especially in the hypervariable region. To assess the profiles of viral quasispecies in HCV-related hepatocellular carcinoma, we studied the viral population patterns in serum and liver tissues of 13 HCV-positive patients with hepatocellular carcinoma developed on cirrhotic and non-cirrhotic livers (5 and 8 cases, respectively). METHODS: HCV genome heterogeneity was analyzed by polymerase chain reaction-mediated single-strand conformation polymorphism analysis, which showed multiple DNA bands representing different hypervariable region sequences. RESULTS: The HCV populations were different between tumorous and nontumorous tissues in 3/5 hepatocellular carcinomas with cirrhosis and in 6/8 without cirrhosis. At least one or more than one common band was detected in both compartments in all but one case. No significant differences in the complexity of HCV quasispecies were found in hepatocellular carcinoma with or without underlying cirrhosis. Comparison of the HCV quasispecies profiles in serum and liver tissues showed a different distribution of HCV variants between these two compartments in 6/7 patients. In four cases, both common and compartmentalized sequences were detected, whereas in two cases, both without cirrhosis, the HCV population in serum was completely different from that found in the liver. CONCLUSIONS: These results suggest that the complexity of HCV populations is influenced by the presence of hepatocellular carcinoma rather than by the severity of the underlying chronic liver disease. The different quasispecies patterns found in serum and liver may reflect different biological properties of circulating and intrahepatic HCV particles or the existence of extrahepatic sites of replication.

Complete eradication of hepatitis C virus after interferon treatment for chronic hepatitis C.

BACKGROUND: Alpha-interferon therapy can lead to a persistent biochemical response, but discordant opinions have been expressed on the definition of sustained response and on the real possibility of complete eradication of hepatitis C virus (HCV). AIMS: To define the clinical, virological and histologic profiles of the patients with sustained response. PATIENTS: Twenty-eight patients with three different biochemical and virological patterns of response to interferon therapy (16 sustained responders, 6 responders with relapse and 6 non responders) were studied for a follow-up period of 36 months. METHODS: HCV-RNA sequences were investigated in serum, peripheral blood mononuclear cells and in liver tissue by means of reverse transcriptase-polymerase chain reaction, targeted to the 5' non coding region. Viral load in serum was quantified by branched-DNA signal amplification. HCV genotypes were evaluated using a line probe assay. RESULTS: All sustained responders showed persistent normal ALT values and loss of serum HCV-RNA during the treatment and in the entire follow-up period. The HCV clearance was also demonstrated in peripheral blood mononuclear cells and in liver tissue. Pre-treatment HCV-RNA quantitation showed that sustained responders had a significantly lower viral load compared to relapsers and non responders (p = 0.005). HCV genotyping showed that patients infected by genotypes 2a, 3a were more likely to achieve a sustained response. Interestingly, a prolonged response was also observed in the only three patients with pre-treatment detectable viral load infected by genotype 3a and in patients with genotype 1b and low viraemia levels. To assess the histologic outcome following HCV eradication, all sustained responders underwent a second liver biopsy in the follow-up period (6-18 months). Periportal necrosis and portal inflammation were significantly improved. CONCLUSIONS: Our results suggest that persistent loss of HCV-RNA in serum, peripheral blood mononuclear cells and liver as well as histologic improvement are consistent with the complete HCV eradication even from intracellular compartments and from potential extra-hepatic sites of viral persistence. Moreover, pre-treatment viral load, HCV infecting genotypes and histologic features may influence the clinical outcome of hepatitis C and the response to interferon therapy.

[Aerosol thymopentin in the prevention of relapse of chronic bronchitis]. / Timopentina per via aerosolica nella prevenzione delle riacutizzazioni della bronchite cronica.

In this study the Authors have verified the efficacy of Thymopentin administered via aerosol in the prevention of recurrent catarrhal episodes in the patients affected by COPD during the winter season, compared to the previous winter. In October, 1 phial of Thymopentin was administered via aerosol to 15 patients affected by COPD, daily, for 10 consecutive days; all patients were evaluated at monthly clinical control for 4 months and all patients were invited to keep a diary of daily variations. After 4 months from treatment a net improvement was observed in the clinical parameters studied: sputum (volume and purulence), cough and dyspnoea, but the most interesting datum was the total absence of recurrent episodes of infection, associated to the reduction in quantity of antibiotics, mucolytics and number of days of illness and with noticeable improvement in the quality of life of the patients.

Ambroxol for the prevention of chronic bronchitis exacerbations: long-term multicenter trial. Protective effect of ambroxol against winter semester exacerbations: a double-blind study versus placebo.

In a 6-month, double-blind multicenter trial conducted over the winter, the effects of daily administration of ambroxol retard (75 mg) were compared with those of placebo in preventing exacerbations and improving symptoms and clinical signs in chronic bronchitis patients. The trial was completed by 110 patients in the ambroxol group and by 104 in the placebo group. Initially, there were no significant differences between the groups. By the end of the 2nd month of treatment, 67.2% of the ambroxol group had had no exacerbations compared to 50.4% in the placebo group. At the end of the 6-month trial, 45.5% of the treatment group had had no exacerbations, compared to only 14.4% of the control group. These differences were statistically significant. Patients in the treatment group lost significantly fewer days through illness (442) and had fewer days when they needed antibiotic therapy (371) compared to the placebo group patients (837 and 781). Ambroxol also produced statistically significant symptomatic improvement, measured as difficulty in expectoration, coughing, presence of dyspnea and the auscultatory signs as compared to controls. Since ambroxol was well tolerated and compliance was good, it appears like a drug of choice for pharmacological prophylaxis of chronic bronchitis.

Cytofluorometric methods for assessing absolute numbers of cell subsets in blood. European Working Group on Clinical Cell Analysis.

The enumeration of absolute levels of cells and their subsets in clinical samples is of primary importance in human immunodeficiency virus (HIV)+ individuals (CD4+ T- lymphocyte enumeration), in patients who are candidates for autotransplantation (CD34+ hematopoietic progenitor cells), and in evaluating leukoreduced blood products (residual white blood cells). These measurements share a number of technical options, namely, single- or multiple-color cell staining and logical gating strategies. These can be accomplished using single- or dual-platform counting technologies employing cytometric methods. Dual-platform counting technologies couple the percentage of positive cell subsets obtained by cytometry and the absolute cell count obtained by automated hematology analyzers to derive the absolute value of such subsets. Despite having many conceptual and technical limitations, this approach is traditionally considered as the reference method for absolute cell count enumeration. As a result, the development of single-platform technologies has recently attracted attention with several different technical approaches now being readily available. These single-platform approaches have less sources of variability. A number of reports clearly demonstrate that they provide better coefficients of variation (CVs) in multicenter studies and a lower chance to generate aberrant results. These methods are therefore candidates for the new gold standard for absolute cell assessments. The currently available technical options are discussed in this review together with the results of some cross-comparative studies. Each analytical system has its own specific requirements as far as the dispensing precision steps are concerned. The importance of precision reverse pipetting is emphasized. Issues still under development include the establishment of the critical error ranges, which are different in each test setting, and the applicability of simplified low-cost techniques to be used in countries with limited resources.