Th2 cells and GATA-3 in asthma: new insights into the regulation of airway inflammation.
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Asthma↗
Biomedical subjects
A Ray
Publications and source records attributed to A Ray.
NHMRC Clinical Trials Centre. Advancing research.
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Australia↗
HSV-1 migration in latently infected and naive rabbits after penetrating keratoplasty.
PURPOSE: To investigate the migration of herpes simplex virus type 1 (HSV-1) between latently infected and naive corneal tissues and trigeminal ganglion (TG) in rabbits after penetrating keratoplasty (PKP) and transcorneal epinephrine iontophoresis. METHODS: Two mutants, genetically constructed from HSV-1 strain 17syn+, were used to inoculate rabbit corneas: 17deltaPst, a latency associated transcript (LAT) negative, low-reactivating virus and 17Pr, a high-reactivating, LAT-positive rescuant of 17deltaPst. Latently infected rabbits were given corneal allografts from naive rabbits, and naive rabbits received grafts from latently infected rabbits. Ninety days after PKP, groups of the transplanted rabbits were induced to reactivate by transcorneal epinephrine iontophoresis, but others were not induced. Viral shedding was monitored by tear film cultures. Rabbits were killed 5 days after iontophoresis. Transplanted grafts, recipient corneal rims, and corresponding TG were obtained. Nucleic acids were extracted and amplified for detection of HSV-1 DNA and viral gene transcription. RESULTS: In naive rabbits receiving grafts transplanted from rabbits latently infected with 17Pr (LAT+), 3 of 6 corneal rims contained HSV DNA after induction. In contrast, none of the 5 corneal rims from naive rabbits receiving grafts from rabbits latent with 17deltaPst (LAT-) contained viral DNA. Viral DNA and gene transcripts were detected in 2 of 6 TG from naive rabbits that received grafts from 17Pr (LAT+) latently infected rabbits. In recipient corneal rims and TG of latently infected rabbits receiving grafts from naive rabbits, viral DNA concentration was significantly greater with induced reactivation, compared with the results in noninduced rabbits. The amount of viral DNA in naive grafts transplanted into 17Pr (LAT+) latently infected rabbits was significantly higher with induction than without induction (P = 0.018). More viral DNA and viral gene transcripts were found in tissues from rabbits latently infected with 17Pr (LAT+) than in rabbits latently infected with 17deltaPst (LAT-). CONCLUSIONS: Corneas from latently infected rabbits contain HSV-1 DNA that can replicate after induced reactivation. Viral migration can occur in both anterograde and retrograde directions between the transplanted graft and the recipient corneal rim and TG. The LAT negative HSV-1 construct 17deltaPst has a significantly reduced ability to replicate and migrate.
Animals↗
Immunohistochemical co-expression of c-erbb-2/Neu oncoprotein, altered tumour suppressor (p53) protein, EGF-R and EMA in histological subtypes of infiltrating duct carcinoma of the breast.
Carcinoma of the breast has an unpredictable biological behaviour. Several oncogenes have been implicated in the progression of breast cancer. Immunohistochemical staining of c-erbB-2 (Neu) oncoprotein and mutant p53 protein on 45 cases of infiltrating duct carcinoma (IDC) of the breast revealed 33% membrane positivity of c-erbB-2 oncoprotein, 46% nuclear positivity of mutated p53 protein, 33% and 84% membrane positivity of EGF-R and EMA respectively. Staining profile of c-erb-B2 oncoprotein in various histological subtypes of IDC of the breast indicated a high positivity rate in comedo followed by NOS and cibriform subtype. Similarly, high incidence of immunopositivity of mutated p53 protein was observed in comedo and cibriform subtypes while papillary carcinoma were found exclusively positive for mutated p53 protein. Interestingly, tubular subtype of IDC was not positive for c-erbB-2 oncoprotein as well as p53 mutant protein. Further, comedo and cibriform subtypes of IDC revealed 'high grade' histological features of tumour of the breast with high mitotic count, presence of marked pleomorphism and multinucleation thus, reflecting a positive relationship with overexpression of c-erbB-2 (Neu) oncoprotein as well as mutant p53 protein. The results on immunoexpression of c-erbB-2 oncoprotein and mutated p53 protein in various histological subtypes of IDC of the breast demonstrated c-erbB-2 status as an important predictor and also indicated that oncogene product may be involved in growth factor response pathway.
Breast Neoplasms↗
Glutathione level and its relation to radiation therapy in patients with cancer of uterine cervix.
Glutathione functions as an important antioxidant in the destruction of hydrogen peroxide and lipid peroxides by providing substrate for the glutathione peroxidase and also promotes the ascorbic acid. Glutathione plays a vital role in detoxification of xenobiotics, carcinogens, free radicals and maintenance of immune functions. The study was aimed to determine plasma glutathione as well as erythrocyte glutathione and glutathione peroxidase in patients with invasive cervical carcinoma (n = 30) before initiation and after completion of radiotherapy and subsequently, at the time of first three monthly follow-up visit. The levels of plasma glutathione, erythrocyte glutathione and glutathione peroxidase activity were found to be lower in all cervical cancer patients as compared to age matched normal control women. The study indicates a change in antioxidant status in relation with the glutathione system among patients with invasive carcinoma of the uterine cervix. This study also demonstrates the effect of radiation therapy on this antioxidant system.
Adult↗
Relation between chubby cheeks and visceral fat.
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Abdomen↗
SH3 binding domains in the dopamine D4 receptor.
The dopamine D4 receptor is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit adenylyl cyclase. The dopamine D4 receptor as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the D4 receptor are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the D4 receptor can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2-SH3 adapter proteins Grb2 and Nck. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the dopamine D4 receptor resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to adenylyl cyclase and MAPK, although dopamine modulated receptor-G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the D4 receptor contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.
Adaptor Proteins, Signal Transducing↗
Essential role of nuclear factor kappaB in the induction of eosinophilia in allergic airway inflammation.
The molecular mechanisms that contribute to an eosinophil-rich airway inflammation in asthma are unclear. A predominantly T helper 2 (Th2)-type cell response has been documented in allergic asthma. Here we show that mice deficient in the p50 subunit of nuclear factor (NF)- kappaB are incapable of mounting eosinophilic airway inflammation compared with wild-type mice. This deficiency was not due to a block in T cell priming or proliferation in the p50(-/-) mice, nor was it due to a defect in the expression of the cell adhesion molecules VCAM-1 and ICAM-1 that are required for the extravasation of eosinophils into the airways. The major defects in the p50(-/-) mice were the lack of production of the Th2 cytokine interleukin 5 and the chemokine eotaxin, which are crucial for proliferation and for differentiation and recruitment, respectively, of eosinophils into the asthmatic airway. Additionally, the p50(-/-) mice were deficient in the production of the chemokines macrophage inflammatory protein (MIP)-1alpha and MIP-1beta that have been implicated in T cell recruitment to sites of inflammation. These results demonstrate a crucial role for NF-kappaB in vivo in the expression of important molecules that have been implicated in the pathogenesis of asthma.
Animals↗
Differential responsiveness of the IL-5 and IL-4 genes to transcription factor GATA-3.
The cytokines IL-4 and IL-5 are often coordinately produced by Th2 cells as in asthma. However, it is unclear whether similar molecular mechanisms underlie transcription of the two genes. We have previously shown that the transcription factor GATA-3 is expressed in Th2 but not Th1 cells and is crucial for activation of the IL-5 promoter by different stimuli. In a different study, GATA-3 was shown to be sufficient for the expression of IL-4 and other Th2 cytokine genes. Here, we show that ectopic expression of GATA-3 is sufficient to drive IL-5 but not IL-4 gene expression. Also, in Th2 cells, antisense GATA-3 RNA inhibits IL-5 but not IL-4 promoter activation. The induction of IL-5 gene expression by GATA-3 involves high affinity binding of GATA-3 to an inverted GATA repeat in the IL-5 promoter.
Animals↗
Alteration in the brain content of substance P (1-7) during withdrawal in morphine-dependent rats.
Previous studies have shown that substance P (SP) may modulate the abstinence reaction to opioid withdrawal. Its N-terminal fragment SP1-7 may inhibit the intensity of the withdrawal reactions in morphine dependent mice. This study was designed to determine whether the endogenous concentrations of the SP1-7 fragment in the brain are affected during naloxone-precipitated withdrawal in the male rat. The amounts of the peptide was assessed by a specific radioimmunoassay in extracts of discrete brain regions (including the cerebral cortex, hippocampus, hypothalamus, nucleus accumbens, striatum, substantia nigra, ventral tegmental area and the spinal cord) during morphine tolerance and withdrawal. The results indicated that the concentrations of SP1-7 were significantly elevated in the ventral tegmental area both in morphine tolerant rats and during naloxone-precipitated withdrawal. During morphine withdrawal significant increases in the peptide concentration were also observed in the hypothalamus and the spinal cord. It was concluded that the enhanced content of SP1-7 may also indicate the involvement of the SP system during opioid withdrawal in the rat. The enhanced production of SP1-7 may reflect an increased release and/or metabolism of SP, which, in turn, counteracts the withdrawal.
Animals↗
Immunohistochemical expression of c-erbB-2 oncoprotein and EGF-R in pre- and postmenopausal breast cancer.
Tissues from 40 cases each of premenopausal and postmenopausal breast cancer were studied immunohistochemically for epidermal growth factor receptor (EGF-R) and c-erbB-2 oncoprotein. In the premenopausal group, immunopositivity for c-erbB-2 was 15% and for EGF-R 22.5%, whereas in the postmenopausal group, 45% of cases were positive for c-erbB-2 and 42.5% for EGF-R. The difference in immunoexpression of c-erbB-2 between the two groups was significant. A significant correlation was observed between the concomitant expression of c-erbB-2 as well as EGF-R and lymph node involvement. Furthermore, an association was found between c-erbB-2 positivity and histological grading of the tumour. It is interesting that the pattern of the investigated parameters indicates the difference in the pathological events of pre- and postmenopausal breast cancer.
Adult↗
Isolation and functional characterization of cDNA of serum amyloid A-activating factor that binds to the serum amyloid A promoter.
Serum amyloid A (SAA), a plasma protein inducible in response to many inflammatory conditions, is associated with the pathogenesis of several diseases including reactive amyloidosis, rheumatoid arthritis, and atherosclerosis. We have previously reported an element of the SAA promoter, designated SAA-activating sequence (SAS), that is involved in the inflammation-induced SAA expression, and a nuclear factor, SAS-binding factor (SAF), that interacts with the SAS element has been identified previously (A. Ray and B. K. Ray, Mol. Cell. Biol. 16:1584-1594, 1996). To evaluate how SAF is involved in SAA promoter activation, we have investigated structural features and functional characteristics of this transcription factor. Our studies indicate that SAF belongs to a family of transcription factors characterized by the presence of multiple zinc finger motifs of the Cys2-His2 type at the carboxyl end. Of the three cloned SAF cDNAs (SAF-1, SAF-5, and SAF-8), SAF-1 isoform showed a high degree of homology to MAZ/ZF87/Pur-1 protein while SAF-5 and SAF-8 isoforms are unique and are related to SAF-1/MAZ/ZF87/Pur-1 at the zinc finger domains but different elsewhere. Although structurally distinct, all members are capable of activating SAS element-mediated expression and display virtually identical sequence specificities. However, varying levels of expression of members of this gene family were observed in different tissues. Functional activity of SAF is regulated by a posttranslational event as SAF DNA-binding and transactivation abilities are increased by a protein phosphatase inhibitor, okadaic acid, and inhibited by a protein kinase inhibitor, H7. Consistent with this observation, increased DNA binding of the cloned SAF and its hyperphosphorylation, in response to okadaic acid treatment of the transfected cells, were observed. Taken together, our results suggest that, in addition to tissue-specific expression, SAFs, a family of zinc finger transcription factors, undergo a modification by a posttranslational event that confers their SAA promoter-binding activity and transactivation potential.
Amino Acid Sequence↗
The C terminus of the major yeast telomere binding protein Rap1p enhances telomere formation.
The telomeres of most organisms consist of short repeated sequences that can be elongated by telomerase, a reverse transcriptase complex that contains its own RNA template for the synthesis of telomere repeats. In Saccharomyces cerevisiae, the RAP1 gene encodes the major telomere binding protein Rap1p. Here we use a quantitative telomere formation assay to demonstrate that Rap1p C termini can enhance telomere formation more than 30-fold when they are located at internal sites. This stimulation is distinct from protection from degradation. Enhancement of formation required the gene for telomerase RNA but not Sir1p, Sir2p, Sir3p, Sir4p, Tel1p, or the Rif1p binding site in the Raplp C terminus. Our data suggest that Rap1p C termini enhance telomere formation by attracting or increasing the activity of telomerase near telomeres. Earlier work suggests that Rap1p molecules at the chromosome terminus inhibit the elongation of long telomeres by blocking the access of telomerase. Our results suggest a model where a balance between internal Rap1p increasing telomerase activity and Rap1p at the termini of long telomeres controlling telomerase access maintains telomeres at a constant length.
Binding Sites↗
Organochlorine pesticide-induced oxidative stress and immune suppression in rats.
Effects of subchronic DDT and lindane exposure were evaluated on lipid peroxidation, antioxidant mechanisms and humoral immune response in rats. Oral administration of DDT, (100 and 200 ppm) and lindane (40 and 80 ppm) dose dependently increased thiobarbituric acid reactive substance (TBARS) levels in serum after 8 wk of treatment. Superoxide dismutase (SOD) activity in red blood cells (RBC) was also dose dependently increased by these compounds. In addition, such DDT or lindane exposure markedly suppressed the humoral immune response as assessed by anti-sheep RBC antibody titres. Simultaneous treatment with ascorbic acid (100 mg/kg) markedly attenuated the effects of DDT and lindane on (a) lipid peroxidation, (b) SOD activity and (c) humoral immune suppression. These results indicate the possible involvement of free radicals in organochlorine-induced immunotoxicity.
Animals↗
A comparative evaluation of immunotoxicity of malathion after subchronic exposure in experimental animals.
The effects of sub-chronic doses of malathion exposure on humoral and cell-mediated immune (CMI) responses were studied in male albino mice, rats and rabbits using sheep red blood cells (SRBC), tetanus toxoid and ovalbumin as antigens. The humoral immune response was assessed by estimating serum immunoglobulin (IgM and IgG) concentrations, antibody titre against antigens and splenic-plaque forming cells (PFC). The CMI response was studied by using the leucocyte migration inhibition (LMI) and macrophage migration inhibition (MMI) tests. In general there were (a) attenuation in antigen induced antibody response, (b) suppression of PFC, and (c) marked inhibition of LMI and MMI factors. Sub-chronic malathion exposure induced differential degrees of humoral and CMI suppression in these experimental animals. However, both cellular and humoral immune responses were decreased in a dose-time dependent pattern and a consistent trend was observed. The threshold level of the malathion for inducing immune suppression depends on the animal species, type of antigen used, and the method of immunological assay. In view of the widespread use of malathion a comparative assessment of immune responses using different experimental animals and antigens is an important aspect of its safety evaluation.
Animals↗
The effects of mutation of the anr gene on the aerobic respiratory chain of Pseudomonas aeruginosa.
The anr gene of Pseudomonas aeruginosa encodes a transcriptional regulator of anaerobic gene expression, homologous to the Fnr protein of Escherichia coli. We report here that Anr has a role in regulating the activity of the aerobic respiratory chain of P. aeruginosa. Strains with internal deletions in their anr gene had lowered levels of membrane bound cytochromes whilst the activity of the cytochrome c oxidase, cytochrome co (likely to be a cytochrome cbb3-type oxidase), and the cyanide-insensitive respiratory pathway was markedly higher than in the wild-type strains. These data, and the finding that provision of multiple copies of the anr gene led to severe repression of these respiratory activities, suggest that Anr is a repressor of aerobic respiratory pathways and possibly the terminal oxidases themselves. In contrast, Anr activated cytochrome c peroxidase, a respiratory chain linked enzyme induced under low oxygen conditions.
Aerobiosis↗
Induction of serum amyloid A (SAA) gene by SAA-activating sequence-binding factor (SAF) in monocyte/macrophage cells. Evidence for a functional synergy between SAF and Sp1.
Serum amyloid A (SAA) is a plasma protein that is associated with many inflammatory diseases including amyloidosis, arthritis, and atherosclerosis. SAA level is significantly increased during inflammatory condition, and such abnormal expression of this protein is linked to the pathogenesis of the above-mentioned diseases. A promoter element, designated as SAA-activating sequence (SAS), located between -280 and -226 has been implicated in the induction mechanism and a nuclear factor, SAS-binding factor (SAF), has been shown to bind to this region. In this report, using a cloned SAF gene in transient transfection assay, we provide evidence that SAF potentiates SAA gene expression through SAS element. Furthermore, we show that during lipopolysaccharide-mediated induction of SAF, heteromeric complex with transcription factor Sp1 is formed. Transfection assays using both transcription factor genes have demonstrated that SAF-Sp1 heteromer is a highly potent transactivator of SAA expression.
Amyloid↗
Transcription factor GATA-3 is differentially expressed in murine Th1 and Th2 cells and controls Th2-specific expression of the interleukin-5 gene.
Interleukin-5 (IL-5), which is produced by CD4(+) T helper 2 (Th2) cells, but not by Th1 cells, plays a key role in the development of eosinophilia in asthma. Despite increasing evidence that the outcome of many diseases is determined by the ratio of the two subsets of CD4(+) T helper cells, Th1 and Th2, the molecular basis for Th1- and Th2-specific gene expression remains to be elucidated. We previously established a critical role for the transcription factor GATA-3 in IL-5 promoter activation in EL-4 cells, which express both Th1- and Th2-type cytokines. Our studies reported here demonstrate that GATA-3 is critical for expression of the IL-5 gene in bona fide Th2 cells. Whereas mutations in the GATA-3 site abolished antigen- or cAMP-stimulated IL-5 promoter activation in Th2 cells, ectopic expression of GATA-3 in Th1 cells or in a non-lymphoid, non-IL-5-producing cell line activated the IL-5 promoter. During the differentiation of naive CD4(+) T cells isolated from T cell receptor transgenic mice, GATA-3 gene expression was up-regulated in developing Th2 cells, but was down-regulated in Th1 cells, and antigen- or cAMP-activated Th2 cells (but not Th1 cells) expressed the GATA-3 protein. Thus, GATA-3 may play an important role in the balance between Th1 and Th2 subsets in immune responses. Inhibition of GATA-3 activity has therapeutic potential in the treatment of asthma and other hypereosinophilic diseases.
Animals↗