Transcranial direct current stimulation for the outpatient treatment of poor-responder depressed patients.

Transcranial direct current stimulation (tDCS) is a selective, painless, brain stimulation technique that allows the electric stimulation of specific cortical regions. TDCS has been recently used as investigational intervention for major depression and treatment resistant depression (TRD) with encouraging results. The present study was aimed to investigate the efficacy and tolerability of tDCS in major depressives with poor response to pharmacological treatment. Twenty-three depressed patients, with a diagnosis of major depressive disorder or bipolar disorder, were treated with augmentative tDCS for 5 days, two sessions per day in a blind-rater trial. The course of depressive symptoms was analyzed using repeated measures ANOVA for HAM-D and MADRS total scores. A qualitative analysis on the basis of the HAM-D response was performed as well. Both analyses were conducted at three time-points: T0 (baseline), T1 (endpoint tDCS) and T2 (end of the first week of follow-up). All patients completed the trial without relevant side-effects. A significant reduction of HAM-D and MADRS total scores was observed during the study (P<0.0001). Treatment response (endpoint HAM-D reduction ≥50%) was obtained by four patients (17.4%) at T1 and by seven patients (30.4%) at T2 and remission (endpoint HAM-D<8) by three patients (13.0%) at T1 and by four subjects (17.4%) at T2. Present findings support the efficacy and good tolerability of tDCS in the acute treatment of patients with TRD with clinical benefit being progressive and extended to the first week of follow-up. Further sham-controlled trials with longer follow-up are needed to confirm present results.

Treatment of woodchuck hepatitis virus infection in vivo with 2', -3'-dideoxycytidine (ddC) and 2',-3'-dideoxycytidine monophosphate coupled to lactosaminated human serum albumin (L-HSA ddCMP).

Dideoxycytidine (ddC) is a nucleoside analogue active against human immunodeficiency virus and with in vitro activity against human hepatitis B virus. We investigated the ability of ddC to inhibit one of the Hepadnaviridae, the woodchuck hepatitis virus and compared the results with the effect obtained by a conjugate of lactosaminated human serum albumin 2',-3'-dideoxycytidine monophosphate (L-HSA ddCMP). This compound specifically enters the hepatocyte via the asialoglycoprotein receptor. We treated five chronic woodchuck hepatitis virus carriers with intravenous injections of 0.5 mg/kg body weight of ddC for 5 consecutive days, and under the same protocol five woodchucks with 10.4 mg/ kg L-HSA ddCMP, a dose equivalent to 0.25 mg/kg of free ddC. A reduction of serum woodchuck hepatitis virus DNA (5-125 fold) was observed during therapy in three out of five animals receiving ddC and in two of the five animals treated with L-HSA ddCMP. In responding woodchucks, virus DNA levels rebounded immediately after stopping therapy. No signs of toxicity were observed during or after the course of therapy. These preliminary results of short-term treatment indicate that ddC has anti-viral activity against woodchuck hepatitis virus. When the dose was reduced by 50%, L-HSA ddCMP showed anti-viral activity to an even lesser degree.

The effects of using recombinant vaccinia viruses expressing either large or small HDAg to protect woodchuck hepadnavirus carriers from HDV superinfection.

Live rVVs expressing either p24 delta or p27 delta were produced and used to immunize woodchuck hepadnavirus carriers. Upon challenge with infectious HDV, circulating HDV RNA levels appeared to be similar in both controls and vaccinees. Although extended follow-up studies of these animals is necessary before making firm conclusions, including an analysis of circulating HDAg levels, these preliminary results provide no evidence for a protective immunity conferred by the rVVs. In contrast, we have shown in other studies that repeated immunization of woodchucks with purified, recombinant p24 delta subunit does confer significant protection against HDV challenge in some of the vaccinees (A. Ponzetto, et al., this volume). The underlying immunological mechanisms responsible for the different outcome of these varied vaccination regimens remain to be elucidated.

Chronic infection in woodchucks infected by a cloned hepatitis delta virus.

Two woodchucks (Marmota monax) intrahepatically inoculated with hepatitis delta virus (HDV) complementary DNA clones pSVL-D3 and pSVL-Ag showed virological and pathological signs of acute and chronic HDV infection. HDV-RNA and hepatitis delta antigen (HDAg) were detected in serum by slot-blot hybridization and by western blot five weeks after inoculation. Liver biopsy specimens collected at 8th week post inoculum were positive for HDV-RNA. Anti-HDV antibodies were detected at the 11th and 9th weeks, respectively. Histological finding of hepatocarcinoma and persistence of circulating HDV-RNA and anti-HDV were observed up to the 10th month. Both woodchucks produced "small" and "large" HDAg antigen, although the inoculated cloned DNA bears the coding capability solely for the small antigen. A transient decrease of woodchuck hepatitis virus DNA (WHV-DNA) level was observed during the peak of HDV infection. Successive inoculation of acute-phase serum in three woodchucks resulted in a successful infection in one of the animals.

Hepatitis C virus-related chronic liver disease with autoantibodies to liver-kidney microsomes (LKM). Clinical characterization from idiopathic LKM-positive disorders.

This study was carried out on 33 patients who were sero-positive for liver-kidney microsomal antibodies (LKM) in order to examine clinical features and the presence of underlying hepatitis C virus infection. Twenty-four sera were positive for antibodies against HCV (anti-HCV) as detected by enzyme immunoassay and confirmed by recombinant immunoblot assay. These patients had chronic liver disease and the majority of those treated with interferon responded favourably. Three of the nine anti-HCV-negative patients had idiopathic chronic hepatitis and two responded favourably to steroids. Two patients were diagnosed as having toxic hepatitis and the other four had various extrahepatic disorders without evidence of liver involvement. The immunoblotting analysis showed reactivity with a 50 kDa microsomal protein which presumably corresponded to cytochrome P-450 db1 both in anti-HCV-positive and -negative sera. In addition a few anti-HCV-positive sera also reacted with a 35 kDa microsomal antigen. Autoimmune markers different from LKM were absent in both groups. The high prevalence of antibodies to the hepatitis C virus among LKM-positive sera confirms that this infection plays a role in forms of chronic hepatitis that had previously been labelled autoimmune. In patients with LKM the presence of anti-HCV may help to forecast a therapeutic response to interferon, while its absence may forecast response to steroid therapy.

Chronic active hepatitis B. Interferon-activated natural killer-like cells against a hepatoma cell line transfected with the hepatitis B virus nucleic acid.

In a rapid 51Cr release assay, peripheral blood mononuclear cells from 12 healthy donors did not lyse the hepatitis B virus deoxyribonucleic-acid-transfected human hepatoma cell line 2.2.15, but under the same experimental conditions they did lyse K562 cells. Peripheral blood mononuclear cells from 10 out of 16 patients with chronic active hepatitis B exhibited cytotoxic activity against 2.2.15 cells in the presence of a relatively reduced natural killer cell activity to the K562 cell target. Enhancement of the cytotoxic activity to 2.2.15 cells was statistically significant in the group of patients being treated with leukocyte alpha-interferon. The activity was not influenced by the degree of human leukocyte antigen type matching between effector and target, and was enhanced by depletion of T-cells and by in vitro interferon treatment. These results therefore support the concept of a natural killer-like cell activated by clinical administration of interferon in chronic active hepatitis B patients. This cell effector was lytic for the virus B negative HEP-G2 cells also. However, T-cells purified from a few patients failed to lyse the HEP-G2 while lysing the 2.2.15 target, thus indicating that a preferential recognition of the virus-infected target may be exerted by certain T-lymphocyte subsets. The use of the human leukocyte antigen type defined, highly differentiated, hepatitis B virus releasing 2.2.15 cell line as target for fresh lymphocytes in this cytolytic assay did not disclose cytolytic T-cells in an obvious way. Further manipulation of this system perhaps using T-cell clones may be the next step to exploit the investigative possibilities offered by the availability of the 2.2.15 cell target.

Vinculin, talin, and integrins are localized at specific adhesion sites of malignant B lymphocytes.

The microanatomy of the dot-shaped, close-contact sites called podosomes and the mechanism of their formation have been investigated in vitro in the malignant lymphocytes of B chronic lymphocytic leukemia (B-CLL). In this paper the authors demonstrate that in B-CLL podosomes: (1) vinculin, talin, and beta 2 integrin (CD18) rings surround an F-actin core; (2) the beta 1 integrin is localized within the F-actin core; (3) the beta 3 integrin is not present. This distribution and organization of adhesion-related molecules appears to be unique to B-CLL lymphocytes, since it has not been observed in normal B cells. B-CLL adhesion and podosome formation are inhibited by the synthetic peptide GRGDSP that contains the Arg-Gly-Asp (RGD) sequence.

Vanadate-treated baby hamster kidney fibroblasts show cytoskeleton and adhesion patterns similar to their Rous sarcoma virus-transformed counterparts.

Rous sarcoma virus-transformed baby hamster kidney fibroblasts (RSV/B4-BHK) adhere to a fibronectin-coated substratum by means of dot-like adhesion sites called podosomes in view of their shape and function as cellular feet (Tarone et al.: Exp Cell Res 159:141, 1985). Podosomes concentrate tyrosine-phosphorylated proteins, including pp60v-src, and appear in many cells transformed by oncogenes coding for tyrosine kinases. In this paper we used orthovanadate, an inhibitor of phosphotyrosine phosphatases, in order to increase the cellular concentration of phosphotyrosine and to study whether this treatment induced the cytoskeleton remodeling leading to the formation of podosomes. Indeed, orthovanadate (10-100 microM) induced in a time- and dose-dependent manner the redistribution of F-actin and the formation of podosomes in BHK cells. Cytoskeleton remodeling occurred along with a marked increase of tyrosine phosphorylated proteins. The vanadate effect on the cytoskeletal phenotype was enhanced by the simultaneous treatment of cells with a phorbol ester. Under the latter conditions almost all BHK cells showed podosomes. The vanadate effect was reversible insofar as podosomes and tyrosine-phosphorylated proteins disappeared. Then, vanadate treatment of normal cells induced the cascade of events leading to the cytoskeletal changes typical of transformation and suggested that the transformed cytoskeletal phenotype may be primarily induced by the tyrosine phosphorylation of unknown target(s) operated by endogenous kinases.

Human endothelial cells are target for platelet-activating factor. I. Platelet-activating factor induces changes in cytoskeleton structures.

Platelet-activating factor (PAF), but not its deacetylated and biologically inactive metabolite lyso-PAF, in a dose dependent-manner (0.1 to 10 nM), induces human endothelial cells (EC) in culture to change their shape. EC retract and tend to lose reciprocal contact, whereas the distribution of stress fibers is changed and the cells tend to assume a migratory phenotype. The normal localization of vinculin at streaks corresponding to adhesion plaques and located at stress fiber endings is mostly lost and replaced by diffuse distribution of this protein related to the cell-substratum adhesion complex. The effects of PAF are appreciable after 10 min, become maximal after 30 min, and are fully reversible. The disappearance of F-actin in stimulated EC was analyzed by measuring the fluorescence of the cells after staining with fluorosceinated phalloidin, PAF, but not lyso-PAF and the enantiomer of PAF ([S] form), decreases in a time- and concentration-dependent fashion the fluorescence of the cells stained with fluoresceinated phalloidin. EC grown on fibronectin-coated polycarbonate filters restrict the diffusion of 125I-albumin; PAF promotes 125I-albumin diffusion with a concentration dependency similar to that for the PAF-induced cytoskeleton changes. Four different PAF-receptor antagonists belonging to four different series, CV-3988 (PAF-related framework), BN52021 (a natural terpenoid), BN53013 (a natural lignan) and 48740 RP (a synthetic antagonist) prevent the alteration induced by PAF. These findings, coupled to the lack of effect of the enantiomer of PAF ([S] form), support the existence of a specific mechanism of PAF-mediated activation of EC, most probably mediated by a putative receptor. These results explain part of the PAF mechanism of action in inducing the increase of vascular permeability in vivo.

Plasma and semen androgens in normospermic and dysspermic subjects.

The seminal and plasmatic concentrations have been examined of testosterone, 5 alpha-dihydrotestosterone, and delta 4-androstenedione in normospermic and dysspermic subjects. The results obtained did not provide evidence of significant differences of androgen levels in fertile, hypofertile and sterile men.