Differences in inflammatory pain in nNOS-, iNOS- and eNOS-deficient mice.

To assess the relative importance of the isoforms of nitric oxide synthase (NOS) in inflammatory pain, we directly compared pain behaviour and paw thickness after intraplantar injection of complete Freund's adjuvant (CFA) in wild-type (WT) mice and in mice lacking either inducible (iNOS), endothelial (eNOS) or neuronal NOS (nNOS). In mice deficient for nNOS, thermal hyperalgesia was reduced by approximately 50% compared to wild type mice at 4 and 8h after CFA injection, and mechanical hypersensitivity was absent. The only change in pain behaviour in iNOS and eNOS deficient mice compared to WT mice was a more rapid recovery from thermal hyperalgesia. A compensatory up-regulation of nNOS in dorsal root ganglia (DRG) and spinal cords of iNOS and eNOS knockout mice was excluded using RT-PCR. However, an increase of iNOS gene expression was found in spinal cords of eNOS and nNOS deficient mice. To study the downstream effects of nNOS deficiency on DRG neurones, we assessed their immunoreactivity for calcitonin gene-related peptide (CGRP) and cytokines. We found a significant reduction in the CFA induced increase in CGRP immunoreactive neurones as well as in CGRP gene expression in nNOS deficient mice, whereas the percentage of cells immunopositive for tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) was unchanged. These results support the proposed role of nNOS in sensitization of DRG neurones, and might indicate that CGRP is involved in this process.     

Dopamine receptor subtypes modulate olfactory bulb gamma-aminobutyric acid type A receptors

The gamma-aminobutyric acid type A (GABAA) receptor is the predominant Cl- channel protein mediating inhibition in the olfactory bulb and elsewhere in the mammalian brain. The olfactory bulb is rich in neurons containing both GABA and dopamine. Dopamine D1 and D2 receptors are also highly expressed in this brain region with a distinct and complementary distribution pattern. This distribution suggests that dopamine may control the GABAergic inhibitory processing of odor signals, possibly via different signal-transduction mechanisms. We have observed that GABAA receptors in the rat olfactory bulb are differentially modulated by dopamine in a cell-specific manner. Dopamine reduced the currents through GABA-gated Cl- channels in the interneurons, presumably granule cells. This action was mediated via D1 receptors and involved phosphorylation of GABAA receptors by protein kinase A. Enhancement of GABA responses via activation of D2 dopamine receptors and phosphorylation of GABAA receptors by protein kinase C was observed in mitral/tufted cells. Decreasing or increasing the binding affinity for GABA appears to underlie the modulatory effects of dopamine via distinct receptor subtypes. This dual action of dopamine on inhibitory GABAA receptor function in the rat olfactory bulb could be instrumental in odor detection and discrimination, olfactory learning, and ultimately odotopic memory formation.     

The snare-assisted technique for transcatheter coil occlusion of moderate to large patent ductus arteriosus: immediate and intermediate results.

OBJECTIVES: The purpose of this study was to evaluate the feasibility, safety and efficacy of using a snare-assisted technique to coil occlude the moderate to large size patent ductus arteriosus (PDA). BACKGROUND: Transcatheter occlusion of small PDAs using Gianturco coils is safe and effective. However, in larger size PDAs and/or those with short PDA length, the procedure still carries risks of coil embolization, incomplete occlusion and failure to implant the coil. METHODS: From January 1994 to June 1997, the records of 104 consecutive snare-assisted coil occlusions of moderate to large PDAs (minimum diameter >2.0 mm) were reviewed. Immediate and intermediate outcomes including complete and partial occlusion, failure to implant and complications were analyzed with respect to ductal type and size. RESULTS: Patient age ranged from 0.1 to 70.1 years (median 3.3 years). Minimum PDA diameter ranged from 2.1 to 6.8 mm (mean 3.0 +/- 0.9 mm). Angiographic types were A-62, B-13, C-6, D-14 and E-9. Using the snare-assisted technique, coil placement was successful in 104/104 patients (100%), irrespective of size or angiographic type. Immediate complete closure was observed in 73/104 (70.2%) and was related to smaller PDA size, but not to angiographic type. Complete closure was documented in 102/104 (98.1%) at 2- to 16-month follow-up. Successful closure was unrelated to PDA size or type. Coil embolization to the pulmonary artery occurred in 3/104 (2.9%) patients and was not related to PDA size or type. The need for multiple coils was found in 28/104 patients (26.9%), and was related to larger PDA size, but not to angiographic type. CONCLUSIONS: The snare-assisted delivery technique allows successful occlusion of moderate to large PDAs up to 6.8 mm, irrespective of angiographic type. This technique permits improved control and accuracy of coil placement, and facilitates delivery of multiple coils.     

Targeting brain angiotensin and corticotrophin-releasing hormone systems interaction for the treatment of mood and alcohol use disorders

The brain renin–angiotensin system (RAS) participates importantly in the regulation of endocrine, autonomic, and behavioral response to stress. Recent data indicate that central action of AT₁ receptor antagonists can reduce anxiety symptoms in experimental animals. Furthermore, central inhibition of RAS activity decreases ethanol intake in an animal model of alcoholism. Pathological anxiety responses and the development of substance dependence are both critically mediated through corticotrophin-releasing hormone (CRH) systems, and the RAS is positioned to interact both with hypothalamic as well as extrahypothalamic CRH systems. The thesis of this paper is that the RAS is part of the neurochemical dysregulation underlying negative affective states, anxiety disorders, and ethanol dependence and that medications targeting the RAS should be considered to augment the treatment of these disorders.     

Functions of the unique N-terminal region of glycoprotein E in the pathogenesis of varicella-zoster virus infection

Varicella-zoster virus (VZV) is an alphaherpesvirus that infects skin, lymphocytes, and sensory ganglia. VZV glycoprotein E (gE) has a unique N-terminal region (aa1-188), which is required for replication and includes domains involved in secondary envelopment, efficient cell-cell spread, and skin infection in vivo. The nonconserved N-terminal region also mediates binding to the insulin-degrading enzyme (IDE), which is proposed to be a VZV receptor. Using viral mutagenesis to make the recombinant rOka-ΔP27-G90, we showed that amino acids in this region are required for gE/IDE binding in infected cells; this deletion reduced cell-cell spread in vitro and skin infection in vivo. However, a gE point mutation, linker insertions, and partial deletions in the aa27-90 region, and deletion of a large portion of the unique N-terminal region, aa52-187, had similar or more severe effects on VZV replication in vitro and in vivo without disrupting the gE/IDE interaction. VZV replication in T cells in vivo was not impaired by deletion of gE aa27-90, suggesting that these gE residues are not essential for VZV T cell tropism. However, the rOka-ΔY51-P187 mutant failed to replicate in T cell xenografts as well as skin in vivo. VZV tropism for T cells and skin, which is necessary for its life cycle in the human host, requires this nonconserved region of the N-terminal region of VZV gE.     

Mutations in the factor IX gene (F9) during the past 150 years have relative rates similar to ancient mutations

Pollutants and dietary mutagens have been associated with somatic mutation and cancer, but the extent of their influence on germline mutation is not clear. Since deleterious germline mutations can be transmitted for thousands of years, any influence on germline mutation from the vast increase in man‐made chemicals of the past 150 years would be an important public health issue. Observed disease causing mutations in the X‐linked factor IX gene (F9) of hemophilia B patients originated predominantly in the past 150 years, since the half‐life of these mutations in human populations had been about two generations before effective treatment became available about a generation ago. Recent changes in germline mutational processes may be detected by comparison of the observed hemophilia B causing mutation pattern in F9 with the pattern of neutral polymorphisms which occurred over a much longer period of time. By scanning a total of 1.5 megabases of deep intronic regions of F9 in the genomic DNA from 84 individuals, 42 neutral polymorphisms were found in 23 haplotypes that differed by at least 11 mutations from the ancestral primate haplotype. By sequencing F9 in seven non‐human primates, 39 of these polymorphisms were characterized as ancient mutations relative to a unanimous ancestral primate allele. This ancient mutation pattern was compared to the recent pattern of hemophilia B causing mutations. Remarkably, no significant difference was found (P=0.5), suggesting that the vast increase in man‐made chemicals during the past 150 years has not had a major impact on the pattern of human germline mutation. This result is consistent with the hypothesis that endogenous processes dominate germline mutation. Hum Mutat 19:49–57, 2002. © 2001 Wiley‐Liss, Inc.     

Transdifferentiation of endothelial and renal tubular epithelial cells into myofibroblast-like cells under in vitro conditions: a morphological analysis

Renal fibrosis is a hallmark of progressive kidney disease and is characterized by an accumulation of extracellular-matrix-synthesizing cells in the glomerulus and tubulointerstitium. This population of myofibroblast-like cells (MFLC) is heterogeneous. It has been experimentally shown, for example, that tubular epithelial cells could change their phenotype into MFLC under certain circumstances, a process called epithelial-mesenchymal transdifferentiation. However, MFLC may also originate from other sources. Therefore, we examined whether endothelial cells (EDC) are able to transdifferentiate into MFLC in vitro. We compared potential differences between syngeneic tubular epithelial cells (EPC) and EDC during transdifferentiation into MFLC using bovine and porcine EDC isolated from pulmonary arteries, and glomerular capillaries. Renal tubular EPC were prepared from bovine renal cortical tissue by collagenase digestion and isolation from homogeneous cell monolayers. Bovine renal tubular EPC stained positive for cytokeratin. Furthermore, tubular EPC selectively incorporated labeled bovine serum albumin, a typical property of differentiated renal tubular cells. EDC were characterized by the absence of epithelial markers (e.g. cytokeratin), but stained positive for vWF. The transdifferentiation of EDC into MFLC occurs sequentially in two steps: First, by a rapid reversible transformation in postconfluent or clonal cultures without the need of cytokine stimulation and second, by a prolonged secondary step in the presence of the transformation-accelerating cytokines and the absence of adherently growing EDC. Thus, EDC that are able to sprout can also irreversibly transdifferentiate into MFLC. On the other hand, prolonged incubation of EPC in the presence of cytokines such as transforming growth factor-beta1 and tumor necrosis factor-alpha leads only to a very small number of MFLC without the ability to further proliferate. Our in vitro data suggest that EDC can more easily transdifferentiate into MFLC than syngeneic renal tubular EPC.