Cytotoxic T cell responses to haptenated cells III. Isolation and specificity analysis of continuously growing clones

Various procedures were used to derive continuously growing cytotoxic T lymphocyte (CTL) clones from a primary culture containing responder cells from immunized mice and 3-(p-sulfophenyldiazo)-4-hydroxylphenyl acetic acid (SP)-or fluorescein isothiocyanate (FL)-coupled stimulator cells. It seems likely that CTL have to undergo some change, possibly genetic, to be able to grow continuously in T cell growth factor conditioned medium in the absence of any stimulator or filler cells. The most convenient and reliable procedure to generate CTL clones with different specificities was to establish from several aliquots of a primary culture cell populations continuously growing in medium conditioned with T cell growth factor(s). Clones with different specificities segregated in the different populations. SP-and FL-specific CTL clones restricted to H-2K^k, and H-2D^d and two FL-specific CTL clones with no apparent H-2 restriction are described.     

General practitioners and psychiatrists: comparison of attitudes to depression using the depression attitude questionnaire.

BACKGROUND. Variation in the management of depression may be linked to doctors' attitudes to depression. AIM. A study was undertaken comparing the attitudes to depression between general practitioners and psychiatrists. METHOD. A sample of 74 general practitioners and 65 psychiatrists in Wales was surveyed by postal questionnaire. Attitudes were assessed by the depression attitude questionnaire and patient management was assessed by a questionnaire on prescribing practice. RESULTS. General practitioners differed significantly from psychiatrists in attitudes, particularly in areas covering professional ease in dealing with patients with depression and identification of depression. Those general practitioners who reported use of low antidepressant doses were significantly more likely than general practitioners prescribing standard doses to believe in psychotherapeutic treatments. Users of short-term continuation therapy expressed a lack of therapeutic optimism and comfort in dealing with depressed patients. CONCLUSION. General practitioners and psychiatrists differ significantly in their attitudes to depression. The attitudes which vary among general practitioners reflect practice. The depression attitude questionnaire may prove useful in indicating how educational initiatives to improve primary care detection and management should be directed.     

Immunoglobulin heavy chain switch region restriction fragment length polymorphisms are associated with renal disease.

We describe here, to our knowledge for the first time, associations between polymorphisms at the genomic DNA level in the immunoglobulin gene region and renal diseases which lead to chronic renal failure. Recent studies have shown that protein polymorphisms, present in immunoglobulin (Ig) heavy chains (Gm allotypes) are associated with certain forms of renal disease and with end stage renal failure per se. To investigate this association at the DNA level we have used probes which recognize Ig heavy chain genes and this report describes results obtained with one of these, the S mu switch region probe. With the restriction endonuclease Sst 1 (or the isoschizomer; Sac I) a number of restriction fragment length polymorphisms (RFLP) can be obtained which are recognized by this probe and there is a highly significant association between certain of these and renal disease. This is the first report of Ig switch region polymorphisms being associated with disease, yet our results suggest that S mu RFLP are more closely linked to renal disease than Ig protein polymorphisms.     

Coping strategies,hostility, and depressive symptoms: A path model

Previous studies of coping, hostility, and depressive symptoms have highlighted the significant relations between all possible pairs of these 3 variables. To more completely explore the nature of depressive symptoms, we link them all together in this study by testing a coping→hostility→depressive symptoms path model. One hundred forty participants completed psychological questionnaires measuring coping strategies, hostility, and depressive symptoms. While controlling age and social class as covariates, SPSS stepwise regression analyses were used to examine relations among these 3 constructs. Results suggest that coping has a direct relation with depressive symptoms as well as an indirect relation mediated by hostility. Passive coping may lead to increased hostility, resulting in depressive symptoms. Active coping may have the opposite effect. These findings suggest that the inclusion of measures of both coping strategies and hostility yields a more thorough understanding of concomitants of depressive symptoms. From a clinical perspective, knowing what coping strategies a person uses and how much anger they experience and express may be useful in guiding the management of depressive symptoms.     

Pseudomonas aeruginosa pyocyanin and 1-hydroxyphenazine inhibit fungal growth

AIM: To examine strains of Pseudomonas aeruginosa for specific antifungal factors. METHODS: Two clinical strains of P aeruginosa with strong in vitro inhibition (by cross streak assay) of Candida albicans and Aspergillus fumigatus were examined. Both strains were isolated from sputum--one from a patient with cystic fibrosis and one from a patient with bronchiectasis. Bacterial extracts were fractionated by high performance liquid chromatography and examined by ultraviolet absorbance and mass spectroscopy. Antifungal activity against C albicans and A fumigatus was determined in a well plate assay. RESULTS: Pyocyanin was the major antifungal agent of P aeruginosa; 1-hydroxy-phenazine also possessed activity. Pyocyanin MICs for C albicans and A fumigatus were > 64 micrograms/ml. These phenazines were active against nine other yeast species pathogenic for man. Preliminary experiments also suggested possible inhibition of yeast mycelial transformation in C albicans by pyocyanin. CONCLUSIONS: There may be a role for pyocyanin and 1-hydroxyphenazine in the prevention of pulmonary candidiasis in patients colonised by P aeruginosa.     

Inactivation of the peroxisomal ABCD2 transporter in the mouse leads to late-onset ataxia involving mitochondria, Golgi and endoplasmic reticulum damage

ATP-binding cassette (ABC) transporters facilitate unidirectional translocation of chemically diverse substances, ranging from peptides to lipids, across cell or organelle membranes. In peroxisomes, a subfamily of four ABC transporters (ABCD1 to ABCD4) has been related to fatty acid transport, because patients with mutations in ABCD1 (ALD gene) suffer from X-linked adrenoleukodystrophy (X-ALD), a disease characterized by an accumulation of very-long-chain fatty acids (VLCFAs). Inactivation in the mouse of the abcd1 gene leads to a late-onset neurodegenerative condition, comparable to the late-onset form of X-ALD [Pujol, A., Hindelang, C., Callizot, N., Bartsch, U., Schachner, M. and Mandel, J.L. (2002) Late onset neurological phenotype of the X-ALD gene inactivation in mice: a mouse model for adrenomyeloneuropathy. Hum. Mol. Genet., 11, 499-505.]. In the present work, we have generated and characterized a mouse deficient for abcd2, the closest paralog to abcd1. The main pathological feature in abcd2-/- mice is a late-onset cerebellar and sensory ataxia, with loss of cerebellar Purkinje cells and dorsal root ganglia cell degeneration, correlating with accumulation of VLCFAs in the latter cellular population. Axonal degeneration was present in dorsal and ventral columns in spinal cord. We have identified mitochondrial, Golgi and endoplasmic reticulum damage as the underlying pathological mechanism, thus providing evidence of a disturbed organelle cross-talk, which may be at the origin of the pathological cascade.     

Bacteremia due to a novel Microbacterium species in a patient with leukemia and description of Microbacterium paraoxydans sp. nov

A yellow-pigmented coryneform rod was isolated from the blood of a child with acute lymphoblastic leukemia who was perfused with a central venous catheter. The culture bottles were positive twice, at a 2-month interval. The isolate was identified as a Microbacterium sp. and studied along with five other similar strains. Phenotypic, chemotaxonomic, and genetic characteristics indicated that they are closely related to Microbacterium oxydans but that they belong to a distinct species, for which the name Microbacterium paraoxydans sp. nov. is proposed. The type strain of M. paraoxydans is CF36(T) = DSM 15019(T). The G+C content of its DNA is 69.9 mol%.     

Molecular Analysis of SV-40-CAL,a New Slow Growing SV-40 Strain from the Kidney of a Caged New World Monkey with Fatal Renal Disease

A decline of the Callimico goeldii population in American zoos is presently occurring due to glomerulonephritis of unknown etiology. We hypothesized that this emerging idiopathic fatal renal disease (IFRD) was caused by a virus. We therefore attempted to isolate virus from the kidneys three C. goeldi in Illinois that had IFRD. Along with other viruses, Simian virus 40 (SV-40) strain CAL was isolated. SV-40-CAL is currently the slowest-growing natural isolate of SV-40 in CV-1 cells. Inefficient SV-40-CAL growth in CV-1 cells stems from two features: a suboptimal protoarchetypal regulatory region, and a Large tumor antigen gene sequence like that of SV-40 strain T302, previously considered the slow-growing natural isolate of SV-40. To our knowledge, this is the first documented isolation of SV-40 from a New World monkey outside of a laboratory setting. Though SV-40 is renaltropic, the role of SV-40-CAL in IFRD is uncertain. Transmission of SV-40 to C. goeldii through anthropogenic activity is suspected.     

Reevaluation of a genetic model for the development of exostosis in hereditary multiple exostosis

EXT1 and EXT2 are genes that have been shown to cause hereditary multiple exostosis (HME), a syndrome marked by the formation of bony growths juxtaposed to the growth plate. These genes are members of a growing family of proteins with glycosyltransferase activity required for the synthesis of heparan sulfate chains. This protein activity is predicted to play a role in the expression of proteoglycans on the cell surface and in the extracellular matrix. We and others have previously suggested that a two-hit mutational model applies to the development of an exostosis where a germline mutation coupled with a somatic mutation results in the loss of EXT1 or EXT2 function and subsequent tumor formation. We report the direct sequencing and loss of heterozygosity (LOH) analysis of 12 exostoses from 10 HME families, 4 solitary exostoses, and their corresponding constitutional DNA. Of the 16 exostoses screened, we find only one solitary case in which two somatic mutations, a deletion and an LOH, are present. This provides limited support for the two-hit hypothesis involving the EXT1 and EXT2 genes for the development of an exostosis. Alternative models are developed based on the functional significance of EXT proteins in heparan sulfate biosynthesis.     

A new selective differential medium for isolation ofStenotrophomonas maltophilia

A new selective differential medium for the isolation ofStenotrophomonas (formerlyXanthomonas) maltophilia was developed. The medium, VIA agar, contained vancomycin, imipenem, and amphotericin B as selective agents and incorporated a mannitol/bromothymol blue indicator system. Compared withXanthomonas maltophilia Selective Medium (XMSM), VIA agar was less inhibitory toStenotrophomonas maltophilia and was more selective than XMSM in preventing the growth of unwanted bacteria from contaminated specimens. Although vancomycin-resistant strains ofEnterococcus faecium may grow on VIA agar, these can be easily distinguished fromStenotrophomonas maltophilia because of mannitol fermentation.