Dopamine enhancement of NMDA currents in dissociated medium-sized striatal neurons: role of D1 receptors and DARPP-32

Dopamine (DA), via activation of D1 receptors, enhances N-methyl-D-aspartate (NMDA)-evoked responses in striatal neurons. The present investigation examined further the properties of this enhancement and the potential mechanisms by which this enhancement might be effected. Dissociated medium-sized striatal neurons were obtained from intact rats and mice or mutant mice lacking the DA and cyclic adenosine 3',5' monophosphate (cAMP)-regulated phosphoprotein of M(R) 32,000 (DARPP-32). NMDA (10-1,000 microM) induced inward currents in all neurons. In acutely dissociated neurons from intact rats or mice, activation of D1 receptors with the selective agonist, SKF 81297, produced a dose-dependent enhancement of NMDA currents. This enhancement was reduced by the selective D1 receptor antagonist SKF 83566. Quinpirole, a D2 receptor agonist alone, produced small reductions of NMDA currents. However, it consistently and significantly reduced the enhancement of NMDA currents by D1 agonists. In dissociated striatal neurons, in conditions that minimized the contributions of voltage-gated Ca(2+) conductances, the D1-induced potentiation was not altered by blockade of L-type voltage-gated Ca(2+) conductances in contrast to results in slices. The DARPP-32 signaling pathway has an important role in D1 modulation of NMDA currents. In mice lacking DARPP-32, the enhancement was significantly reduced. Furthermore, okadaic acid, a protein phosphatase 1 (PP-1) inhibitor, increased D1-induced potentiation, suggesting that constitutively active PP-1 attenuates D1-induced potentiation. Finally, activation of D1 receptors produced differential effects on NMDA and gamma aminobutyric acid (GABA)-induced currents in the same cells, enhancing NMDA currents and inhibiting GABA currents. Thus simultaneous activation of D1, NMDA, and GABA receptors could predispose medium-sized spiny neurons toward excitation. Taken together, the present findings indicate that the unique potentiation of NMDA receptor function by activation of the D1 receptor signaling cascade can be controlled by multiple mechanisms and has major influences on neuronal function.     

Human TLR-7-, -8-, and -9-mediated induction of IFN-alpha/beta and -lambda Is IRAK-4 dependent and redundant for protective immunity to viruses

Five TLRs are thought to play an important role in antiviral immunity, sensing viral products and inducing IFN-alpha/beta and -lambda. Surprisingly, patients with a defect of IRAK-4, a critical kinase downstream from TLRs, are resistant to common viruses. We show here that IFN-alpha/beta and -lambda induction via TLR-7, TLR-8, and TLR-9 was abolished in IRAK-4-deficient blood cells. In contrast, IFN-alpha/beta and -lambda were induced normally by TLR-3 and TLR-4 agonists. Moreover, IFN-beta and -lambda were normally induced by TLR-3 agonists and viruses in IRAK-4-deficient fibroblasts. We further show that IFN-alpha/beta and -lambda production in response to 9 of 11 viruses tested was normal or weakly affected in IRAK-4-deficient blood cells. Thus, IRAK-4-deficient patients may control viral infections by TLR-3- and TLR-4-dependent and/or TLR-independent production of IFNs. The TLR-7-, TLR-8-, and TLR-9-dependent induction of IFN-alpha/beta and -lambda is strictly IRAK-4 dependent and paradoxically redundant for protective immunity to most viruses in humans.     

Pulmonary and mediastinal "sarcoidosis" following surgical resection of cancer

Previous reports indicate that enlarged hilar and mediastinal lymph nodes caused by sarcoid-like reactions may develop after curative resection of cancer, and their presence does not necessarily denote neoplastic recurrence. Reports further suggest that coexisting pulmonary infiltrates in this setting may be related to sarcoidosis. In this study, we describe two patients who had resected lung and gastric cancer and who later developed pulmonary interstitial infiltrate, concurrent with progressive mediastinal lymphadenopathy initially thought to be caused by intrathoracic dissemination of their cancer. These changes were shown by open lung biopsy to be a benign, granulomatous reaction interpreted as sarcoidosis. Thus, it is important to recognize this clinical pattern when pulmonary infiltrates develop after complete treatment of cancer in an otherwise relapse-free patient and to encourage lung or lymph node biopsy in these particular settings in order to confirm a sarcoid-like reaction, thereby avoiding unnecessary chemotherapy for presumed tumor recurrence.     

Quantitation of hepatitis B viral DNA by solution hybridization: Comparison with DNA polymerase and hepatitis B e antigen during antiviral therapy

Serological markers of hepatitis B virus (HBV) replication were assessed in a randomized, controlled trial of prednisone withdrawal followed by α -interferon in the treatment of chronic hepatitis B. HBV DNA levels in more than 700 serial serum samples from 41 patients were determined by a sensitive and quantitative solution hybridization assay. Results were compared with HBV DNA polymerase (DNAp) activity and hepatitis B e antigen (HBeAg) in 21 untreated controls and 20 treated patients. Among treated patients, the mean pretherapy HBV DNA values were higher in nonresponders than in responders. During prednisone treatment, DNA levels increased an average of 2.1-fold in responders and 1.4-fold in nonresponders. During the 2-week rest interval between prednisone and interferon, DNA values fell an average of 57% in responders. In contrast, the mean DNA values in nonresponders did not change during the same interval. This early distinction between responders and nonresponders was not apparent from DNAp or HBeAg results. During interferon treatment, HBV DNA became undetectable in responders and remained negative during a 1-year follow-up. DNA in nonresponders declined to 14% of baseline during interferon treatment but increased to pretherapy levels after treatment. DNAp values generally paralleled HBV DNA values, but DNAp activity showed more variability and lower sensitivity than did the hybridization assay results. HBeAg values varied independently of HBV DNA and DNAp with a much delayed decline in responders. These results indicate that HBV DNA, when measured quantitatively by a sensitive solution hybridization assay, is an early predictor of the effects of antiviral agents on replication.     

Metallo-beta-lactamase VIM-2 in clinical isolates of Pseudomonas aeruginosa from Portugal

Resistance to carbapenems is emerging, and it is a great problem to therapeutics. Three isolates of Pseudomonas aeruginosa from a Portuguese hospital identified in urine and sputum, in 1995, presented a high-level resistance to imipenem (> 32 mg/L). Afterward, one isolate of P. aeruginosa recovered from urine of an ambulatory patient in 1998 showed high resistance to imipenem and meropenem. The resistance to carbapenems in these strains was associated with the production of a class B beta-lactamase, as was demonstrated by imipenem hydrolysis and inhibition by EDTA. Using primers described for bla(IMP) and bla(VIM), the amplification of the latter was observed in all isolates and a VIM-2 metallo-enzyme was identified. The pulsed-field gel electrophoresis (PFGE) patterns of these isolates were indistinguishable, suggesting dissemination to the community of this VIM-2 producer.     

Gender-related cytokine patterns in sera of schistosomiasis patients with Symmers' fibrosis

Cytokine levels were compared between schistosomiasis patients affected by intense fibrosis defined by ultrasound examination and graded from F-0 to F-3. The concentrations of interleukin-1beta (IL-1beta), IL-4, IL-5, IL-10, IL-13, gamma interferon, and tumor necrosis factor alpha (TNF-alpha) were determined by enzyme-linked immunosorbent assay of serum samples. Levels of IL-4, IL-5, and TNF-alpha in the sera of F-3 patients were significantly higher than those found in F-0 individuals, while levels of IL-13 were lower. Levels of IL-4, IL-5, and TNF-alpha in serum were significantly higher in F-3 males than in F-0 males or F-3 females. Conversely, levels of IL-13 were significantly lower in F-3 females than in F-0 females and males.     

Genome analysis of dengue type-1 virus isolated between 1990 and 2001 in Brazil reveals a remarkable conservation of the structural proteins but amino acid differences in the non-structural proteins

We have investigated the genetic diversity of dengue type-1 (DEN-1) virus in Brazil. The full nucleotide sequences of three DEN-1 virus isolated from DEN fever (DF) and DEN hemorrhagic fever patients in northeastern Brazil in 1997 (BR/97) and one from a DF patient in the south of Brazil in 2001 (BR/01) were compared to that of the reference strain BR/90 obtained in the city of Rio de Janeiro in 1990. Sequence analysis showed that the structural proteins were remarkably conserved between all isolates. A total of 27 amino acid changes occurred throughout the non-structural proteins. Among them, nine amino acid substitutions were specific of BR/97 and BR/01 isolates, indicating that in situ evolution of these strains had occurred. Within the BR/97 and BR/01 samples, some amino acid substitutions have been previously identified in DEN-1 virus strains sequenced so far, suggesting that recombination events might have occurred.     

Liposomes as immunological adjuvants in eliciting antibodies specific to the synthetic polypeptide poly(LTyr,LGlu)-poly(DLAla)–poly(LLys) with high frequency of site-associated idiotypic determinants

The antibody response to the synthetic polypeptide, poly(LTyr, LGlu)-poly(DLAla)–poly(LLys), [(T, G)-A–L], injected entrapped in liposomes which served as adjuvant, has been analyzed. The liposomes used were composed of phosphatidylcholine, cholesterol, dicetylphosphate and DL α-tocopherol (molar ratios as 4:3:0.1:0.5) and therefore, were negatively charged. Since the (T, G)-A–L is also negatively charged, no free complexes were formed. The (T, G)-A–L was found to be entrapped inside the enclosed volume of the liposomes, and no (T, G)-A–L antigenic determinants could be detected on the liposomal membranes. Injection of high-responder C3H.SW (H-2^b) mice with (T, G)-A–L-bearing liposomes demonstrated that the i.p. and the i.v. routes of immunization were efficient in eliciting (T, G)-A–L-specific antibodies, whereas the i.d. injection led to poor antibody responses. The latter route of immunization is the most effective when (T, G)-A–L is injected in complete Freund's adjuvant (CFA). When low doses (0.1 and 1 μg) of (T, G)-A–L were used for immunization, the liposomes were better adjuvants than CFA. The effectiveness of the liposomes as immunological adjuvants was also shown in their ability to induce high-potential, primed memory cells. The pattern of low (H-2^{k, a}) and high (H-2^b) responsiveness to (T, G)-A–L was retained following immunization with (T, G)-A–L entrapped in liposomes, as tested in two pairs of congenic strains. (T, G)-A–L-specific antibodies induced by injection with 1μ antigen entrapped in liposomes bear the (T, G)-A–L site-related idiotypic markers of C3H.SW (Igh-1^a) mice in a significantly higher frequency than the homologous idiotypes, namely the antibodies elicited in this strain against (T, G)-A–L in CFA. Thus, liposomes may serve as adjuvants for the production of relatively restricted (T, G)-A–L-specific antibodies of high qualitiy.