Effects of injury severity on regional and temporal mRNA expression levels of calpains and caspases after traumatic brain injury in rats.

Despite a preponderance of studies demonstrating gene expression and/or enzymatic activation of calpain and caspase proteases after traumatic brain injury (TBI), no studies have examined the effects of injury magnitude on expression levels of these cell death effectors after TBI. Determination of the degree to which injury severity affects specific expression profiles is critical to understanding the relevant pathways contributing to post-trauma pathology and for developing targeted therapeutics. This investigation tested the hypothesis that different injury magnitudes (1.0, 1.2, and 1.6 mm) cause alterations in the regional and temporal patterns of mRNA expression of calpain-related (calpain-1 and -2, calpastatin) and caspase-related (caspases -3, -8, -9, BID) gene products after cortical impact in rats. Quantitative RT-PCR was used to compare effects of injury severity on mRNA levels in ipsilateral (injured) cortex and hippocampus, 6 h to 5 days post-injury. TBI caused increases in mRNA expression of all proteins examined, with the highest expression detected in the cortex. Generally, injury magnitude and levels of gene expression were positively correlated. High levels of gene induction were observed with BID, caspase-3, and -8, while caspase-9 mRNA had the lowest level of induction. Interestingly, although calpains are activated within minutes of TBI, calpain mRNA expression was highest 72 h to 5 days post-TBI. This study is the first analysis of the regional and temporal expression of calpains and caspases after TBI. These data provide insight into the inter-relationship of these two protease families and on the distinct but overlapping cascades of cell death after TBI.

In situ XAS characterization of the electrochemical insertion of copper in polypyrrole films.

We report the electrochemical insertion of copper in polypyrrole films followed by XAS spectroscopy. Depending on the applied potential the copper remains as an ionic form like Cu(+n)-(OSO3-)n. For more reductive potentials metal particles are synthesized. Small particles with an estimated average diameter of 20 A were obtained.

Open transarterial pulmonary valvulotomy for pulmonic stenosis utilizing hypothermia (preliminary report of two cases with notes on clinical features)

Two cases of pulmonic valvular stenosis were performed under hypothermia utilizing a media sternotomy. The stenosed valves were incised in one and finger-fractured in the other under direct vision through an anterior pulmonary arteriotomy. Complete circulatory occlusion during the procedure averaged one to two minutes. Recovery was rapid in both and clinical improvement was evident from the second week up. There were no serious complications except for a transient hypotension in the younger patientThe clinical features, the technical aspects, as well as the merits and pitfalls of the procedures are discussed. (Summary)

Region-specific central nervous system expression and axotomy-induced regulation in sympathetic neurons of a VIP-beta-galactosidase fusion gene in transgenic mice.

To assess the activity of cis-acting elements that direct human vasoactive intestinal peptide (VIP) expression in vivo, two independent transgenic mouse lines were created using a transgene comprised of 1.9 kb of 5'-flanking sequence of the human VIP gene joined to the Escherichia coli beta-galactosidase reporter gene. Transgene expression in brain was assessed using beta-galactosidase histochemistry and compared to the distribution of endogenous VIP expression. Transgene expression was observed in most central and peripheral nervous system sites in which endogenous VIP is expressed. We investigated whether the VIP-beta-galactosidase transgene was regulated in sympathetic neurons in experimental paradigms in which VIP regulation is dependent on the release of leukemia inhibitory factor (LIF). After dissociation in vitro and postganglionic axotomy in vivo there were parallel increases in endogenous VIP and transgene expression in superior cervical ganglia. These results indicate that the 1.9 kb region of 5'-flanking sequence of the human VIP gene includes genomic elements important for cell-specific expression and LIF-dependent regulation in neurons.

Regulation of vasoactive intestinal polypeptide and galanin mRNA stabilities.

The stabilities of vasoactive intestinal polypeptide (VIP) and galanin mRNAs were examined in a human neuroblastoma cell line (NBFL) treated with agents that alter second-messenger pathways. VIP and galanin mRNA stabilities were estimated by the decay of steady-state levels of transcripts following transcriptional arrest with actinomycin D or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). In the presence of actinomycin D, phorbol ester treatment stabilized VIP mRNA while treatment with adenylate cyclase activators, calcium ionophore, or CNTF did not. In the presence of DRB, VIP mRNA was not stabilized in phorbol ester-treated cells but instead was stabilized in cells treated with adenylate cyclase activators. With either transcriptional inhibitor, stability of galanin mRNA was not significantly altered. The difference in the behavior of VIP mRNA in the presence of actinomycin D and DRB may result from their different mechanisms of action-actinomycin D intercalates into nucleic acids while DRB is a kinase inhibitor. Using an assay for RNA stability that did not require transcriptional inhibitors, an in vitro transcribed VIP RNA fragment was relatively stable in extracts from phorbol ester-treated cells. Although treatment with phorbol ester alone resulted in stabilization of VIP mRNA, treatment with a combination of phorbol ester and adenylate cyclase activator, calcium ionophore, or CNTF did not-implying a complex interaction of these second-messenger pathways in the regulation of RNA stability.

Quantitative analysis of the expression of a VIP transgene.

We have analyzed the expression of a transgene bearing 2 kilobases of the 5' flanking region of the human vasoactive intestinal polypeptide (VIP) gene coupled to beta-galactosidase. Expression was assayed by beta-galactosidase histochemistry and by mRNA quantitation using polymerase chain reaction (PCR)-mediated amplification; we compared beta-galactosidase activity against both transgene and endogenous VIP mRNA levels. We found that the human 5' flanking sequence in this construct is able to direct tissue-specific expression of beta-galactosidase similar to the pattern for endogenous VIP. However, the transgene is also expressed in smooth muscle and Schwann cells, where VIP mRNA is rare. In various tissues where the transgene and endogenous gene are both active, the ratio between their message levels differs dramatically--transgene mRNA is more abundant where VIP is relatively scarce, but is much less abundant than the endogenous message at sites where VIP mRNA is most concentrated. These results suggest that sequence elements that may restrict VIP transcription or cause tissue-specific VIP mRNA accumulation are missing from the transgene. In the testis there is a high level of transgene message but no significant beta-galactosidase activity; this discrepancy is caused by transcription from a cryptic promoter within the beta-galactosidase sequence.

Early signaling defects in human T cells anergized by T cell presentation of autoantigen.

Major histocompatibility complex class II-positive human T cell clones are nontraditional antigen-presenting cells (APCs) that are able to simultaneously present and respond to peptide or degraded antigen, but are unable to process intact protein. Although T cell presentation of peptide antigen resulted in a primary proliferative response, T cells that had been previously stimulated by T cells presenting antigen were completely unresponsive to antigen but not to interleukin 2 (IL-2). In contrast, peptide antigen presented by B cells or DR2+ L cell transfectants resulted in T cell activation and responsiveness to restimulation. The anergy induced by T cell presentation of peptide could not be prevented by the addition of either autologous or allogeneic B cells or B7+ DR2+ L cell transfectants, suggesting that the induction of anergy could occur in the presence of costimulation. T cell anergy was induced within 24 h of T cell presentation of antigen and was long lasting. Anergized T cells expressed normal levels of T cell receptor/CD3 but were defective in their ability to release [Ca2+]i to both alpha CD3 and APCs. Moreover, anergized T cells did not proliferate to alpha CD2 monoclonal antibodies or alpha CD3 plus phorbol myristate acetate (PMA), nor did they synthesize IL-2, IL-4, or interferon gamma mRNA in response to either peptide or peptide plus PMA. In contrast, ionomycin plus PMA induced both normal proliferative responses and synthesis of cytokine mRNA, suggesting that the signaling defect in anergized cells occurs before protein kinase C activation and [Ca2+]i release.

Characterization of the enantiomers of cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1- pyrrolidinyl)cyclohexylamine (BD737 and BD738): novel compounds with high affinity, selectivity and biological efficacy at sigma receptors.

A novel class of compounds with very high affinity and selectivity for sigma receptors has been discovered. BD614 [(+/-)-cis-N-[2-(3,4-dichlorophenyl)ethyl]-N-methyl-2-(1- pyrrolidinyl)cyclohexylamine] and its optically pure 1S,2R-(-)-[BD737] and 1R,2S-(+)-[BD738]enantiomers bound to sigma receptors of guinea pig brain with Ki = 2.0 +/- 0.4, 1.3 +/- 0.3 and 6 +/- 3 nM, respectively. These compounds exhibited little or no affinity for dopamine-D2, kappa opiate or phencyclidine receptors and displayed high biological efficacy in assays of sigma receptor function, ability to produce alterations in motor behavior and inhibition of the muscarinic cholinergic phosphoinositide response. Microinjection of BD614 into the rat red nucleus or substantia nigra produced a dose-dependent alteration in head position and contralateral circling, respectively. BD614, BD737 and BD738 inhibited stimulation of inositol phosphate production by carbachol or oxotremorine-M in a dose-dependent manner. Thus, N-substituted cis-2-(1-pyrrolidinyl)cyclohexylamines may prove useful in studies of sigma receptor structure and function.

Decreased interferon response by lymphocytes from children with chronic hepatitis.

Since a decreased interferon response has been linked with certain chronic viral infections, a preliminary study was undertaken to determine whether an altered interferon response may be involved in the pathogenesis of chronic hepatitis, a complication of type B hepatitis. The production of interferon by lymphocytes from patients with chronic hepatitis was compared with the production by lymphocytes from normal subjects. The amount of interferon produced by paramyxovirus-stimulated lymphocytes from 16 patients was substantially smaller than that produced by lymphocytes from 12 healthy donors. The results indicate that decreased production of interferon may be responsible for the chronicity of the disease. However, further studies are necessary to establish causality.

LY 171555-induced hyperdefensiveness in the mouse does not implicate benzodiazepine receptors.

In naive mice the selective D2 agonist LY171555 dose-dependently (0.5-5 mg/kg) induces defensive responses toward non-aggressive conspecifics. In order to investigate possible anxiogenic properties of the D2 agonist, its behavioural effects were compared with those produced by the benzodiazepine receptor inverse agonist methyl-beta-carboline-3-carboxylate(beta-CCM) in the elevated plus maze and in social interactions with non-aggressive opponents. When tested in the elevated plus maze, mice injected with LY 171555 (0.005-1 mg/kg) showed no decrease either of the number of entries or of the time spent in the open arms. At 5 mg/kg an actual increase of these two measures was observed. By contrast, beta-CCM (1-3 mg/kg) dose-dependently decreased both the number of entries and the time spent in the open arms without altering locomotion. The effects of beta-CCM were antagonized by the benzodiazepine receptor antagonist RO 15-1788 (3 mg/kg) showing a selective involvement of benzodiazepine receptors in their modulation. On the other hand, beta-CCM, (1-3 mg/kg) did not produce significant effects on defensive behaviour of mice interacting with non-aggressive opponents and the defensive responses of mice treated with 1 mg/kg LY 171555 were not prevented by 5 mg/kg chlordiazepoxide. These results show that DA D2-mediated hyperdefensiveness and anxiety modulated by benzodiazepine receptors are unrelated phenomena and suggest that this behavioural response may represent a model of those forms of fear-related reaction that do not respond to benzodiazepine treatment.

trans-dominant inhibition of herpes simplex virus transcriptional regulatory protein ICP4 by heterodimer formation.

Herpes simplex virus encodes a 175-kilodalton immediate-early transactivating protein referred to as ICP4. A mutant ICP4 molecule expressed from a stable transformed cell line lacks the sequences required for transactivation yet retains the ability to specifically associate with DNA and to form homodimers. Expression of the mutant ICP4 peptide from this cell line, designated X25, resulted in the inhibition of herpes simplex virus growth. Wild-type ICP4 homodimers were depleted in X25-infected cells by the formation of heterodimers containing the wild-type ICP4 molecule and the mutant peptide. While the ICP4 heterodimer retained DNA-binding activity, immunological studies suggest that the wild-type subunit of the heterodimer is conformationally altered in a region that serves as the antigenic epitope. Physical studies that determined the composition of the heterodimer and its native size and approximate shape support this observation. The structural change is in a region of ICP4 genetically implicated as important for transactivation and may result in an alteration in an interaction between ICP4 and a target protein essential to promote transcriptional activation. Sequestering wild-type monomers of a viral regulatory protein into heterodimers which are less proficient in transactivation may explain the dominant inhibitory activity of the X25 cells, resulting in attenuation of viral growth.

Metabolites of haloperidol display preferential activity at sigma receptors compared to dopamine D-2 receptors.

Haloperidol bound with equal affinity to sigma and dopamine D-2 receptors (KI = 2.8 nM). Compared to haloperidol, its carbonyl-reduced metabolite bound to sigma receptors with nearly equal affinity. However, reduced haloperidol bound to dopamine receptors with 85-fold lower affinity compared to haloperidol (KI = 239 nM). The chlorophenyl-hydroxy-piperidine metabolite of haloperidol lacked affinity for dopamine receptors, but bound with moderate affinity to sigma receptors (KI = 326 nM). The carboxylic acid metabolite lacked affinity for either receptor. Like haloperidol, (+)-pentazocine, and 1,3-di-o-tolylguanidine, reduced haloperidol potently inhibited the phosphoinositide response to muscarinic agonists in rat brain synaptoneurosomes, an assay which monitors sigma agonist activity. This metabolite also produced a dystonic alteration of head position in rats when microinjected into the red nucleus. However, unlike observations with haloperidol and other sigma ligands, this effect was associated with pathological changes in the red nucleus. Therefore, it cannot be attributed to sigma receptor interactions with certainty. These findings suggest that administration of haloperidol results initially in effects mediated through both dopamine and sigma receptors, but as metabolism proceeds the sigma actions would be expected to decline at a significantly slower rate than the dopaminergic actions.

Infection with the delta agent in children.

Serological evidence of infection with the hepatitis B virus associated delta agent (delta) was found in 34 of 270 Italian children with HBsAg-positive liver disease. In different histological forms of chronic HBsAg hepatitis the prevalence of delta infection increased in parallel with the activity of the disease and was maximal in children with cirrhosis. During two to seven years of follow up the hepatitis deteriorated in 38% of the 34 patients with delta infection and ameliorated only in 9%. By contrast the disease usually ran a mild course in the 236 delta-negative carriers of HBsAg, with remission in 55% of these children and deterioration in only 7%. The outcome of chronic hepatitis associated with delta infection was not influenced by treatment with steroids and azathioprine. Chronic delta infection in children is usually accompanied by serious liver disease, that has a tendency to progress and is unresponsive to conventional immunosuppressive treatment.

A long-term study of hepatitis B virus-related antigens and immunological parameters in children with chronic hepatitis.

Forty-two children with HBsAg positive chronic hepatitis who received immunosuppressive therapy underwent multiple liver biopsies over periods ranging from one to five years. The presence of HBsAg, HBcAg, deltaAg, antinuclear antibodies and immune complexes was studied using immunofluorescent techniques. The data were correlated with the clinical status of the patients. The presence of HBsAg, HBcAg and deltaAg remained unchanged throughout the study in most positive patients. All 30 HBcAg positive cases were still positive at the end of the study. One of the negative cases became positive. Eight of the ten delta antigen positive cases showed no change. Intrahepatic immunoglobulins, immune complexes and antinuclear antibodies showed a decrease in positivity in a significant number of patients. Fifteen of the 36 patients positive for intrahepatic immunoglobulins became negative during the study, as did 12 of the 29 immune complex positive subjects and 22 of the 33 antinuclear antibody-positive cases. The persistence of HBV antigens seems to be independent of the clinical course. On the other hand, the persistence of positive immunological indices corresponds to a less favourable outcome.

Immunosuppressive treatment of chronic HBsAg-positive hepatitis in childhood.

The results of immunosuppressive treatment (a corticosteroid-azathioprine combination) in 104 children suffering from chronic HBsAg-positive hepatitis are reviewed. A recovery and/or improvement of the majority of the cases treated was observed after two to seven years of therapy, in contradiction to the opinion of other authors who treated adult patients. A biochemical and/or bioptic deterioration was observed in some cases in which treatment was interrupted too soon; this was followed by an improvement when treatment was re-initiated. No serious side-effects were observed with the combination treatment.

[Differential diagnostic problems in acute and chronic hepatitis and hepato-cholangiographies of various etiologies in childhood]. / Problemi diagnostico-differenziali tra epatiti acute o croniche ed epato-colangiopatie di diversa eziologia in età pediatrica.

The necessity is emphasized to perform always a careful anamnesis and a clinical inspection of patients before achieving laboratory examinations (avoiding to demand to a "check up" the diagnosis). This is particularly important in those diseases, like viral hepatitis, which become even more frequent, so that such diagnosis may be supposed basing on unreliable laboratory findings. The possibility is mentioned that different hepato-colangiopathies (Wilson's disease, intolerance to fructose, hepatic congenital fibrosis, hepatic ductal hypoplasia, granulomatous hepatitis, bacterial, micotic and protozoarian cholangitis, liver sufference in onchologic diseases) may simulate viral hepatitis.

[Relation between viral antigens in hepatic tissue detected by immunofluorescence and serological markers of HBV infection in patients with chronic hepatitis]. / Rapporto tra gli antigeni virali presenti nel tessuto epatico rilevati in immunofluorescenza e i markers sierologici di infezione da HBV in soggetti affetti da epatite cronica.

In 21 subjects HBsAg positive affected by different forms of chronic hepatitis and in one carrier we compared the presence in the liver of HBsAg, HBcAg and delta studied by means of I.F. and the presence of serological markers of hepatitis B virus infection. By the I.F. test, HBsAg was present in all cases, HBcAg in 15 cases (71%), delta in 8 cases (38%). These percentages are almost the same as those of other researches in chronic hepatitis in immunodepressive treatment. About the serological markers, HBsAg and HBcAb were present in 100% of cases, while, regarding the e-anti e system, HBeAg was present in 12 cases (57%) and HBeAb in 7 (33%). In the work effected there is no relation between delta and HBeAg because in 50% of the positive delta subjects there was HBeAb. On the contrary HBcAg and HBeAg are strictly correlated. Therefore the antigens core and delta are not interdependent. We wanted to verify the existence of relation between HBcAg and HBeAg and came to the conclusion that this relation exists in 17 out of 21 subjects (81%). In our researches is clear the long persistence of the hepatic antigens without any connection with the time of treatment and the course of the various chronic hepatitis. The change in the serum from HBeAg to HBeAb occurred in many improved cases but not in all of them. We presume that the long immunodepressive treatment (about 4 years) has influenced our results.