Biomedical subjects

L J Warnock

Publications and source records attributed to L J Warnock.

8 recordsLinked to original sources

PubMed1999

TNF is essential for the cell-mediated protective immunity induced by the radiation-attenuated schistosome vaccine.

C57BL/6 mice exposed to the radiation-attenuated schistosome vaccine exhibit high levels of protective immunity. The cell-mediated pulmonary effector mechanism involves IFN-gamma-producing CD4+ T cells in a focal response around challenge larvae. IFN-gamma can promote production of TNF and can synergize with this cytokine in its actions on responder cells. We have examined whether TNF plays a role in lung phase immunity to schistosomes using mice with a disrupted gene for TNFRI (TNFRI-/-). The most dramatic finding was that the schistosome vaccine elicited no protection whatsoever in these mice. However, this could not be attributed to a lack of responder cells, because more lymphocytes were lavaged from the airways of TNFRI-/- than wild-type mice. Furthermore, CD4+ T cells were equally represented in airway populations from the two groups and produced IFN-gamma upon Ag stimulation in vitro. In contrast, pulmonary macrophage function was defective in TNFRI-/- mice, as indicated by a failure to up-regulate inducible NO synthase mRNA. Histopathological analysis revealed that focal infiltrates were of similar size and cell composition in the two groups but that more parasites were free of foci in the TNFRI-/- mice. These animals had a greatly impaired IgG response to schistosomes, which may explain their lack of residual protection due to Ab in a situation where cell-mediated immunity is disabled. We suggest that the absence of protective immunity could result from a retarded build-up of leukocytes around migrating lung worms and/or a deficit in accessory cell function within a focus, both of which would permit parasite escape.

Animals↗

PubMed1997

The 3' untranslated region of IL-1beta regulates protein production.

IL-1beta, a pro-inflammatory cytokine, has sequences in its 3' untranslated region (UTR) that may play a role in the post-transcriptional regulation of IL-1beta production. To test this hypothesis, a series of chimeric reporter genes were developed consisting of a chloramphenicol acetyltransferase (CAT) gene the native 3'-UTR of which was replaced by the full-length IL-1beta 3'-UTR or various 3'-UTR deletion mutants. Expression of these constructs under the SV40 late promoter in THP-1 cells showed that the full-length 3'-UTR repressed constitutive CAT activity to 28% of control CAT activity. Further analysis of the 3'-UTR localized the repressor signal to an adenosine-thymidine (AdT)-rich region. Upon exposure to LPS, the full-length IL-1beta 3'-UTR mediated almost a fivefold increase in CAT activity. The LPS response was not simply loss of AdT-mediated repression; when this sequence was tested alone, it did not respond to LPS. The LPS response effect was localized to the terminal 177 base pairs of the IL-1beta 3'-UTR. The increase in CAT activity following LPS stimulation was associated with an increased CAT.IL-1beta 3'-UTR mRNA half-life, indicating at least one effect of the 3'-UTR on a post-transcriptional process. These studies demonstrate that the IL-1beta 3'-UTR is involved in the regulation of IL-1beta protein production, and a LPS response element may be in the IL-1beta 3'-UTR.

Cell Line↗

PubMed1996

Betamethasone modulation of sphingomyelin hydrolysis up-regulates CTP:cholinephosphate cytidylyltransferase activity in adult rat lung.

Glucocorticoids appear to play an integral role in stimulating surfactant synthesis by activating the rate-regulatory enzyme for phosphatidylcholine synthesis, CTP:cholinephosphate cytidylyltransferase (CT). The activity of liver CT, in vitro, has been shown to be inhibited by the sphingomyelin hydrolysis product, sphingosine. In order to investigate the mechanisms by which glucocorticoids alter CT activity, in vivo, we administered betamethasone (1 mg/kg intraperitoneally) sequentially to adult male rats for 5 days. Betamethasone increased CT activity 2-fold relative to control in whole lung. The hormone also increased membrane-bound activity, but did not affect cytosolic enzyme activity. Betamethasone modestly increased CT mRNA as determined by the reverse-transcription PCR and Southern analysis of PCR products, but did not alter the levels of immunoreactive enzyme in lung membranes as demonstrated by Western blotting. The hormone did, however, produce a nearly 3-fold increase in membrane-associated sphingomyelin, and co-ordinately a substantial decrease in the levels of sphingosine in lung membranes. Sphingosine, but not sphinganine, was a competitive, reversible inhibitor of lung CT with respect to the enzyme activator, phosphatidylglycerol. Betamethasone decreased the activities of the sphingomyelin hydrolases: acid sphingomyelinase by 33% and of alkaline ceramidase by 21%. The hormone also inhibited the generation of sphingosine from lysosphingomyelin in lung membranes. There was no significant effect of the hormone on serine palmitoyltransferase activity, the first committed enzyme for sphingolipid biosynthesis. Further, administration of L-cycloserine, an inhibitor of sphingosine formation, was shown to stimulate CT activity by 74% and increase disaturated phosphatidylcholine in alveolar lavage by 52% relative to control. These observations suggest that glucocorticoids up-regulate surfactant synthesis at the level of a key regulatory enzyme by significantly altering the availability of inhibitory metabolites resulting from sphingomyelin hydrolysis.

Amidohydrolases↗

PubMed1995

Multiple second messenger pathways regulate IL-1 beta-induced expression of PGHS-2 mRNA in normal human skin fibroblasts.

Very little is known about the specific regulation of PGHS-2 mRNA compared with PGHS-1 mRNA. Using normal human fibroblasts, we show that at baseline there is constitutive expression of PGHS-1 mRNA and barely detectable amounts of PGHS-2 mRNA. There was a marked increase in PGHS-2 mRNA transcription following exposure to IL-1 beta. Maximal expression of PGHS-2 mRNA occurred with concentrations of IL-1 beta > or = 1 ng/ml at 3 hours after stimulation. Downregulation of protein kinase C (PKC) activity by pretreating fibroblast cultures with PMA inhibited IL-1-induced PGHS-2 mRNA expression without affecting the constitutive expression of PGHS-1 mRNA. The addition of various PKC inhibitors also blocked the IL-1 beta induction of PGHS-2 mRNA but did not alter PGHS-1 mRNA expression; inhibitors of protein kinase A (PKA) or tyrosine kinase (TK) had only a limited effect on IL-1 beta-induced PGHS-2 mRNA expression. These findings show that IL-1 beta increases PGHS-2 mRNA, at least in part, via activation of PKC. Activation of PKA or TK appears to have a more limited role in this process.

Fibroblasts↗

PubMed1993

Attenuated prostaglandin formation in peroxisomal-deficient human skin fibroblasts.

Peroxisomal-deficient skin fibroblasts from patients with Zellweger's syndrome or infantile Refsum's disease produced fewer prostaglandins than normal skin fibroblasts. Radioimmunoassay indicated a 45-55% decrease in prostaglandin E2 (PGE2) production when Zellweger's fibroblasts were incubated with arachidonic acid. This deficiency was not overcome by pretreatment of the Zellweger's fibroblasts with media containing arachidonic acid, and it was not due to channeling of arachidonic acid into other eicosanoid products. Modifications in the peroxide tone of the Zellweger's fibroblasts by addition of H2O2 or catalase failed to increase PGE2 production. Using Northern analysis, we were unable to detect an mRNA transcript for PGH synthase in unstimulated Zellweger fibroblasts but identified a 4.2-kb mRNA transcript after treatment with phorbol myristate acetate (PMA). Treatment for 6 h with 10 nM PMA raised PGE2 production in normal and Zellweger fibroblasts to equivalent levels. These increases were prevented by addition of H-7, staurosporine, cycloheximide, or actinomycin D. Our findings suggest that the reduced PGE2 production in peroxisomal deficient fibroblasts is due to a decrease in PGH synthase mRNA. The reduction in PGH synthase can be overcome by treatment of the cells with agents which enhance gene expression.

Arachidonic Acid↗

PubMed1991

The application of molecular biology techniques to haemostasis and thrombosis.

One may no longer consider coagulation research without encompassing the wealth of knowledge available from the application of molecular biology techniques. This innovative form of research has not proceeded to the exclusion of more conventional techniques, and moreover has provided support for basic scientific hypotheses. This paper discusses the molecular biology techniques currently used in the study of haemostatic and thrombotic disorders and gives an outline of the evolution of such techniques which have taken place at a staggering pace. The article includes specific examples of how results from molecular studies are directly applied to hereditary haemostatic and thrombotic diseases to support clinical diagnosis and to increase our understanding of the biochemical processes involved in the disorders. The reader is referred to several reviews for detailed lists of genetic defects revealed by molecular biological techniques.

Blood Coagulation Disorders↗

PubMed1991

GENESRCH: a computer program for restriction fragment analysis.

We describe a program GENESRCH which has been written to enable the design of efficient restriction fragment analysis of DNA from patients with hereditary disorders. The program identifies homology of DNA sequences up to 20 base pairs in length, calculates the fragment sizes and indicates the position of enzyme restriction sites within the gene of interest. GENESRCH is written in the BASIC language and will run on any IBM-compatible PC with a parallel printer. A hard disc is recommended for efficient storage and retrieval of data. An example of the use of the program is cited.

Base Sequence↗

PubMed1990

Rapid assessment of haemophilia A carrier state by non-invasive techniques using the polymerase chain reaction.

The polymerase chain reaction (PCR) was used to amplify two polymorphic regions in the factor VIII gene. In vitro synthesis of DNA was achieved using samples obtained from buccal cells, urine, and hair follicles in addition to purified genomic and crude DNA samples prepared from whole blood. Female members of two kindreds affected with haemophilia A were assessed for carrier state using direct restriction fragment length polymorphism analysis of amplified gene products in the Bc1I and XbaI regions. It is concluded that this is a non-invasive, rapid, and inexpensive technique for carrier detection.

Carrier State↗