Intermediate filament expression in human vascular smooth muscle and in arteriosclerotic plaques

Summary Different regions of human aorta and of other human arteries obtained at autopsy were analyzed with regard to their topography and to the different stages of arteriosclerosis. Material was studied by immunocytochemical techniques with antibodies specific for either desmin (D) or for vimentin (V), the two types of intermediate filament proteins present in vascular smooth muscle cells. In normal arteries endothelial cells as well as the adjacent intimal cells were D–V+. In the media D+V+ as well as D–V+ cells were present, with the relative numbers of each cell type dependent on the particular blood vessel. When cells in arteriosclerotic plaques at different stages of development were examined an occasional plaque showed cells of the D+V+ type. In the majority of plaques however the cells were V– D+. In plaques where severe ulceration and necrotic material was present D–V+ cells were found at the border of the lesion: foam cells when they could be identified appeared to be D–V+.     

Airway eosinophilia and expression of interleukin-5 protein in asthma and in exacerbations of chronic bronchitis

Background An increased nutnber of eosinophils in the bronchial mucosa has been demonstrated both in asthma and in exacerbations of chronic bronchitis. Oiyective To investigate whether the airway eosinophilia present in asthma and in chronic bronchitis during exacerbations is associated with interleukin (IL)-5 protein expression in the bronchial mucosa. Methods We obtained bronchial biopsies in 18 subjects with asthma (four intrinsic, seven extrinsic and seven occupational) and in II subjects with chronic bronchitis examined during an exacerbation. The findings were compared wilh those of bronchial biopsies from 10 subjects with chronic bronchitis examined under baseline conditions and from seven normal subjects, taken as controls. By immunohistochemistry, we assessed the expression of IL-5 protein and the number of eosinophils (EG2), mast cells ftryptase), and T-lymphocytes (CD3) in the submucosa. Results As compared with controls, the number of eosinophils was increased to a similar degree in both asthma (P < 0.001) and in exacerbations of ehronic bronchitis (P < 0.001). whereas the number of I L-5 immunopositive cells was increased significantly only in asthma (P < 0.01). No diflerences were observed in the number of tnast cells and T-lymphocytes between the four groups of subjects examined. Conciusions This study shows that the degree of airway eosinophilia is similar in asthma and in exacerbations of ehronic bronchitis, but only in asthma is it associated with an increased expression of I L-5 protein in the bronchial tnucosa.     

Virulence and attenuation of murine cytomegalovirus

Murine cytomegalovirus (MCMV) was rapidly and regularly attenuated by passage through mouse embryo cell culture. This attenuation was manifested by alteration of lethality for suckling mice and by a striking loss of capacity to multiply in liver and spleen of weanling mice. The attenuation was selective in that the passaged virus multiplied vigorously in other organs and established high titer infections in submaxillary glands and pancreas comparable to those seen with wild virus. Furthermore, attenuated virus no longer induced transient suppression of antibody and interferon responsiveness which was a regular feature of wild MCMV infection. Wild and attenuated MCMV shared the property of being poor immunogens. They induced anti-CMV complement-fixing or neutralizing antibody very slowly with barely detectable levels present at the end of the first 2 weeks of infection. The close antigenic relationship between wild and attenuated agents was demonstrated by nearly identical neutralization by a rabbit antiserum induced with wild MCMV. Furthermore, survivors of neonatal infection with attenuated virus were fully protected against subsequent challenge with potentially lethal doses of wild MCMV. Virulence could be rapidly restored by back passage in mice.     

Immunohistochemical localization of neurofilaments and neuron-specific enolase in 29 cases of neuroblastoma.

Twenty-nine neuroblastomas have been examined with the use of rabbit antibodies specific for each of the three neurofilament polypeptides, with a monoclonal antibody specific for the NF-L polypeptide, and with a rabbit antibody specific for neuron-specific enolase. When frozen material was used, all neuroblastomas were positive with the neurofilaments antibodies. When alcohol-fixed paraffin-embedded material was used, neurofilament staining was weaker and the fixation procedure appeared to destroy the epitopes recognized by the NF-L antibodies preferentially. Although all neuroblastomas were positive for neurone-specific enolase, so were two rhabdomyosarcomas, suggesting that NSE is not an appropriate marker to distinguish the different small blue cell tumors of children.     

Incomplete rescue of cystic fibrosis transmembrane conductance regulator deficient mice by the human CFTR cDNA

We have used a mouse model to study the ability of human CFTR to correct the defect in mice deficient of the endogenous protein. In this model, expression of the endogenous Cftr gene was disrupted and replaced with a human CFTR cDNA by a gene targeted 'knock-in' event. Animals homozygous for the gene replacement failed to show neither improved intestinal pathology nor survival when compared to mice completely lacking CFTR. RNA analyses showed that the human CFTR sequence was transcribed from the targeted allele in the respiratory and intestinal epithelial cells. Furthermore, in vivo potential difference measurements showed that basal CFTR chloride channel activity was present in the apical membranes of both nasal and rectal epithelial cells in all homozygous knock-in animals examined. Ussing chamber studies showed, however, that the cAMP-mediated chloride channel function was impaired in the intestinal tract among the majority of homozygous knock-in animals. Hence, failure to correct the intestinal pathology associated with loss of endogenous CFTR was related to inefficient functional expression of the human protein in mice. These results emphasize the need to understand the tissue- specific expression and regulation of CFTR function when animal models are used in gene therapy studies.      

Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases

The expansion of trinucleotide repeat sequences is associated with several neurodegenerative diseases. The mechanism of this expansion is unknown but may involve slipped-strand structures where adjacent rather than perfect complementary sequences of a trinucleotide repeat become paired. Here, we have studied the interaction of the human mismatch repair protein MSH2 with slipped-strand structures formed from a triplet repeat sequence in order to address the possible role of MSH2 in trinucleotide expansion. Genomic clones of the myotonic dystrophy locus containing disease-relevant lengths of (CTG)n x (CAG)n triplet repeats were examined. We have constructed two types of slipped-strand structures by annealing complementary strands of DNA containing: (i) equal numbers of trinucleotide repeats (homoduplex slipped structures or S-DNA) or (ii) different numbers of repeats (heteroduplex slipped intermediates or SI-DNA). SI-DNAs having an excess of either CTG or CAG repeats were structurally distinct and could be separated electrophoretically and studied individually. Using a band-shift assay, the MSH2 was shown to bind to both S-DNA and SI-DNA in a structure- specific manner. The affinity of MSH2 increased with the length of the repeat sequence. Furthermore, MSH2 bound preferentially to looped-out CAG repeat sequences, implicating a strand asymmetry in MSH2 recognition. Our results are consistent with the idea that MSH2 may participate in trinucleotide repeat expansion via its role in repair and/or recombination.      

Molecular heterogeneity at the phenylalanine hydroxylase locus in the population of the south-west of England.

The phenylalanine hydroxylase gene locus has been studied in 35 independent phenylketonuric families in the south-west of England using RFLP haplotype patterns and allele specific oligonucleotide probes. Haplotype 3 was the most common pattern on mutant chromosomes and there was strict linkage disequilibrium between this haplotype and the splice mutation in exon 12. The R408W mutation in exon 12 occurred on both haplotypes 1 and 2. The R126Q mutation in exon 7 was found only on a rare haplotype 28 pattern. No gene carried the R158Q mutation. More than 60% of mutant genes did not carry these four mutations which were originally described in other European populations. We suggest that the splice mutation arose as a single event and spread throughout northern Europe by population migration and admixture. In addition, we believe the haplotype/mutation associations seen in our population are a reflection of the mixed ancestry of the inhabitants of the British Isles.     

Desmin is a specific marker for rhabdomyosarcomas of human and rat origin.

Putative human rhabdomyosarcoma (RMS) has been divided into two groups according to desmin content. Twenty-five tumors with histologic features consistent with but not necessarily sufficient to prove a diagnosis of RMS were desmin-positive. More than 95% of the tumor cells were desmin-positive, suggesting a muscle origin and supporting the diagnosis of RMS. Nine tumors for which the preferred first histologic diagnosis was also RMS were desmin-negative. Reexamination of the original histologic slides together with results from intermediate filament typing resulted in a diagnosis other than RMS for all tumors in this second group, and in several instances other tests were used to prove the correctness of the final diagnosis. The results on human material were extended to a rat model system in which RMS was induced by nickel sulfide. Again, all 24 tumors tested were desmin-positive. Vimentin was coexpressed in a varying percentage of tumor cells in RMS of human and rat origin. The results show that desmin is an excellent marker for rhabdomyosarcoma, yielding few if any false-positive or false-negative results in frozen or alcohol-fixed material.     

Short term dynamic psychotherapy goes to Hollywood: the treatment of performance anxiety in cinema

This article uses characters in popular films to demonstrate the theory and application of short term dynamic psychotherapy (STDP) in the treatment of performance anxiety. The reader is taught to identify affect phobias that are hypothesized to underlie performance anxiety. Similar in function to external phobias, affect phobias or internal phobias involve the avoidance of feelings (e.g., fear about feeling anger, shame about showing grief, pain about closeness), which thwarts adaptive responding and generates numerous behavioral problems. STDP treatment focuses on the restructuring of defenses, conflicted affects, and attachments. The resolution of the affect phobia requires systematic desensitization of affective responses (i.e., exposure and desensitization of underlying conflicted feelings). When patients learn to access adaptive forms of feelings, performance anxiety can often be resolved.