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Biomedical subjects

K A Fitzgerald

Publications and source records attributed to K A Fitzgerald.

At least 19 recordsLinked to original sources
PubMed2004

Helicobacter pylori activates the early growth response 1 protein in gastric epithelial cells.

The early growth response 1 (Egr-1) transcription factor is rapidly induced by various stimuli and is implicated in the regulation of cell growth, differentiation, and gene expression. The aim of this study was to examine the effect of Helicobacter pylori on the expression of Egr-1 and Egr-1-regulated genes in gastric epithelial AGS cells. Egr-1 expression was assayed by immunoblotting and electrophoretic mobility shift assays using H. pylori-stimulated AGS cells. Transient transfection experiments with promoter-reporter constructs of CD44, ICAM-1, and CD95L were used for expression studies. H. pylori induced the expression of Egr-1 in gastric epithelial cell lines in a dose-dependent manner, with the rapid kinetics that are typical of this class of transcription factors. Immunohistochemical studies of biopsies revealed that Egr-1 expression is more abundant in H. pylori-positive patients than in uninfected individuals. Reporter-promoter transfection studies indicated that Egr-1 binding is required for the H. pylori-induced transcriptional promoter activity of the CD44, ICAM-1, and CD95L (APO-1/Fas) constructs. The blocking of egr-1 with an antisense sequence prevented H. pylori-induced Egr-1 and CD44 protein expression. The MEK1/2 signaling cascade participates in H. pylori-mediated Egr-1 expression, but the p38 pathway does not. The data indicate that H. pylori induces Egr-1 expression in AGS cells in vitro and that the Egr-1 protein is readily detectable in biopsies from H. pylori-positive subjects. These observations suggest that H. pylori-associated Egr-1 expression may play a role, in part, in H. pylori-induced pathology.

Cell Line, Tumor↗
PubMed2001

Mal (MyD88-adapter-like) is required for Toll-like receptor-4 signal transduction.

The recognition of microbial pathogens by the innate immune system involves Toll-like receptors (TLRs), which recognize pathogen-associated molecular patterns. Different TLRs recognize different pathogen-associated molecular patterns, with TLR-4 mediating the response to lipopolysaccharide from Gram-negative bacteria. All TLRs have a Toll/IL-1 receptor (TIR) domain, which is responsible for signal transduction. MyD88 is one such protein that contains a TIR domain. It acts as an adapter, being involved in TLR-2, TLR-4 and TLR-9 signalling; however, our understanding of how TLR-4 signals is incomplete. Here we describe a protein, Mal (MyD88-adapter-like), which joins MyD88 as a cytoplasmic TIR-domain-containing protein in the human genome. Mal activates NF-kappaB, Jun amino-terminal kinase and extracellular signal-regulated kinase-1 and -2. Mal can form homodimers and can also form heterodimers with MyD88. Activation of NF-kappaB by Mal requires IRAK-2, but not IRAK, whereas MyD88 requires both IRAKs. Mal associates with IRAK-2 by means of its TIR domain. A dominant negative form of Mal inhibits NF-kappaB, which is activated by TLR-4 or lipopolysaccharide, but it does not inhibit NF-kappaB activation by IL-1RI or IL-18R. Mal associates with TLR-4. Mal is therefore an adapter in TLR-4 signal transduction.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Factors associated with antibiotic resistance in coliform organisms from community urinary tract infection in Wales.

Routine susceptibility data for urinary coliform isolates from community practice were analysed in comparison with dispensed antibiotic prescriptions for all conditions and social deprivation data for Bro Taf and North Wales Health Authorities for financial years 1996--1998. Prescribing rates and resistance rates varied widely between practices. Among isolates from practices with high usage of an antibiotic, rates of resistance to that antibiotic tended to be high, and usage correlated significantly with resistance between practice population units. Cross-correlations were found between usage of one antibiotic and resistance to another, particularly for trimethoprim and ampicillin. Usage, particularly of trimethoprim, was associated with multi-resistance to up to four antibiotics. Resistance was more frequent in isolates from males, children and the elderly. Ampicillin resistance correlated with social deprivation. Analyses including or excluding potential repeat isolates yielded closely similar results. Indices reflecting sampling behaviour (laboratory coliform positivity rates, positivity per 1000 registered patients, specimens submitted per 1000 registered patients) varied widely between surgeries, suggesting lack of consensus on urine sampling policies. These indices showed only weak correlations with usage or resistance. Associations between resistance and usage were compared for isolates from two patient subsets that were likely to differ in their proportions of non-Escherichia coli isolates: female patients aged 16--55 years; and males, children and patients aged >55 years. The latter showed higher base levels of resistance, but the associations of resistance with usage were statistically indistinguishable for the two populations. The results suggest that usage of antibiotics in a practice population may affect the rate of urinary infection caused by resistant coliform organisms in that population.

Adolescent↗
PubMed2000

Topoisomerase II is required for mitoxantrone to signal nuclear factor kappa B activation in HL60 cells.

Topoisomerase II is a target for a number of chemotherapeutic agents used in the treatment of cancer. Its essential physiological role in modifying the topology of DNA involves the generation of transient double-strand breaks. Anti-cancer drugs, such as mitoxantrone, that target this enzyme interrupt its catalytic cycle and give rise to persistent double strand breaks, which may be lethal to a cell. We investigated the role of such lesions in signaling the activation of the transcription factor nuclear factor kappaB (NFkappaB) by this drug. Mitoxantrone activated NFkappaB and stimulated IkappaBalpha degradation in the promyelocytic leukemia cell line HL60 but not in the variant cells, HL60/MX2 cells, which lack the beta isoform of topoisomerase II and express a truncated alpha isoform that results in an altered subcellular distribution. Treatment of sensitive HL60 cells with mitoxantrone led to a depletion of both isoforms, suggesting the stabilization of transient DNA-topoisomerase II complexes. This depletion was absent in the variant cells, HL60/MX2. Activation of caspase 3 by mitoxantrone was also impaired in the HL60/MX2 cells. NFkappaB activation in response to tumor necrosis factor and bleomycin, the latter causing topoisomerase II-independent DNA damage, was intact in both cell lines. An inhibitor rather than a poison of topoisomerase II, Imperial Cancer Research Fund 187 (ICRF 187) the mechanism of which does not involve the generation of double strand breaks, did not activate NFkappaB, nor did it induce apoptosis in parental HL60 cells. However, ICRF 187 protected against IkappaB degradation in parental HL60 cells in response to mitoxantrone. This protection was also shown with another topoisomerase II inhibitor, merbarone, which is structurally and functionally distinct from ICRF 187. Their effects were specific, as neither protected against tumor necrosis factor-stimulated IkappaB degradation. The poisoning of topoiso- merase II with resultant DNA damage is therefore a critical signal for NFkappaB activation.

Antigens, Neoplasm↗
PubMed2000

Ras, protein kinase C zeta, and I kappa B kinases 1 and 2 are downstream effectors of CD44 during the activation of NF-kappa B by hyaluronic acid fragments in T-24 carcinoma cells.

We have investigated the ability of hyaluronic acid (HA) fragments to activate the transcription factor NF-kappa B. HA fragments activated NF-kappa B in the cell lines T-24, HeLa, MCF7, and J774. Further studies in T-24 cells demonstrated that HA fragments also induced I kappa B alpha phosphorylation and degradation, kappa B-linked reporter gene expression, and ICAM-1 promoter activity in an NF-kappa B-dependent manner. The effect of HA was size dependent as neither disaccharide nor native HA were active. CD44, the principal cellular receptor for HA, was critical for the response because the anti-CD44 Ab IM7.8.1 blocked the effect on NF-kappa B. HA fragments activated the I kappa B kinase complex, and the effect on a kappa B-linked reporter gene was blocked in T-24 cells expressing dominant negative I kappa B kinases 1 or 2. Activation of protein kinase C (PKC) was required because calphostin C inhibited NF-kappa B activation and I kappa B alpha phosphorylation. In particular, PKC zeta was required because transfection of cells with dominant negative PKC zeta blocked the effect of HA fragments on kappa B-linked gene expression and HA fragments increased PKC zeta activity. Furthermore, damnacanthal and manumycin A, two mechanistically distinct inhibitors of Ras, blocked NF-kappa B activation. Transfection of T-24 cells with dominant negative Ras (RasN17) blocked HA fragment-induced kappa B-linked reporter gene expression, and HA fragments activated Ras activity within 5 min. Taken together, these studies establish a novel signal transduction cascade emanating from CD44 to Ras, PKC zeta, and I kappa B kinase 1 and 2.

Animals↗
PubMed2000

The role of the interleukin-1/Toll-like receptor superfamily in inflammation and host defence.

The IL-1 receptor/Toll-like receptor superfamily comprises a diverse family of cell surface receptors defined by a characteristic conserved sequence in their cytosolic regions, termed the Toll/IL-1 receptor domain, which function in inflammation and host defence against microbial pathogens. Members include receptors for the proinflammatory cytokines IL-1 and IL-18 and Toll-like receptors 2 and 4, which are involved in host responses to Gram-positive and Gram-negative bacteria, respectively. Signalling pathways activated by these receptors are conserved and the superfamily represents a pan-genomic system involved in the host response to infection and injury.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Characterization of CD44 induction by IL-1: a critical role for Egr-1.

The adhesion molecule CD44 is a multifunctional, ubiquitously expressed glycoprotein that participates in the process of leukocyte recruitment to sites of inflammation and to their migration through lymphatic tissues. In this study, we have investigated the effect of the proinflammatory cytokine IL-1alpha on CD44 gene expression in the human immortalized endothelial cell line ECV304. Immunoblotting of cell extracts showed constitutive expression of a 85-kDa protein corresponding to the standard form of CD44, which was potently up-regulated following IL-1alpha treatment. Furthermore, IL-1alpha induced expression of v3- and v6-containing isoforms of CD44, which migrated at 110 and 140-180 kDa, respectively. The effect of IL-1alpha on CD44 standard, v3- and v6-containing isoforms was dose and time dependent and was inhibited in the presence of IL-1 receptor antagonist. To elucidate the molecular mechanisms regulating CD44 expression in response to IL-1alpha, we investigated the effect of IL-1alpha on CD44 mRNA expression. Reverse-transcriptase PCR and Northern analysis demonstrated an increase in CD44 mRNA expression indicating a transcriptional mechanism of control by IL-1alpha. Furthermore, IL-1alpha increased expression of a reporter gene under the control of the CD44 promoter (up to -1.75 kb). The effect of IL-1alpha was critically dependent on the site spanning -151 to -701 of the promoter. This effect required the presence of an Egr-1 motif at position -301 within the CD44 promoter since mutation of this site abolished responsiveness. IL-1alpha also induced Egr-1 expression in these cells. These studies therefore identify Egr-1 as a critical transcription factor involved in CD44 induction by IL-1alpha.

Cell Line, Transformed↗
PubMed1992

Mechanism of action of chlorhexidine diacetate and phenoxyethanol singly and in combination against gram-negative bacteria.

Chlorhexidine diacetate and the aromatic alcohol, phenoxyethanol in combination had an enhanced bacteriostatic action against Escherichia coli and Pseudomonas aeruginosa strains. Investigations of potassium (K+) ion leakage by means of a potassium electrode and a radioactive method, employing 86Rb, indicated that the combination accelerated the rate of leakage from the cell. Leakage of pentose was also found to be enhanced in the presence of the combination compared with either drug alone.

Anti-Infective Agents, Local↗
PubMed1992

Bacterial uptake of 14C-chlorhexidine diacetate and 14C-benzyl alcohol and the influence of phenoxyethanol and azolectin: studies with gram-negative bacteria.

The uptake of 14C-chlorhexidine (14C-CHA) by Pseudomonas aeruginosa and smooth, rough and deep rough strains of Escherichia coli was very rapid with maximum uptake occurring within 20 s. Despite the rapid binding, the lethal action of CHA, although concentration-dependent, is comparatively slow and occurs in minutes rather than seconds. This indicates that the initial rapid binding is followed by a second slower action, responsible for the lethal effects of CHA. The lethal action could be accelerated, particularly at modest concentrations of CHA, by the simultaneous presence of phenoxyethanol (POE) or benzyl alcohol (BZA), although the magnitude of the effect was small. Both alcohols had little effect on the binding of 14C-CHA, which does not explain the enhanced bactericidal action of CHA. Uptake of 14C-benzyl alcohol (14C-BZA) by the same strains showed very different patterns with slower and time-related binding. CHA had a marked effect on BZA absorption but no direct link was established between binding patterns and cell death. The CHA neutraliser, azolectin, removed bound CHA (in the presence or absence of POE) very efficiently even at contact times of only 20 s.

Anti-Infective Agents, Local↗
PubMed1990

Evaluation in vitro of adriamycin immunoconjugates synthesized using an acid-sensitive hydrazone linker.

A novel method for linking Adriamycin (ADM) to monoclonal antibodies is described in which the 13-keto position of the anthracycline is used as the attachment site to the linker arm. A new ADM acylhydrazone derivative, Adriamycin 13-[3-(2-pyridyldithio)propionyl]hydrazone hydrochloride, which contains a pyridyl-protected disulfide, was synthesized and used for conjugation to monoclonal antibodies (MAbs) that were thiolated with N-succinimidyl 3-(pyridyldithiol)propionate or 2-iminothiolane. This resulted in formation of a linker between MAb and drug that contained a disulfide bond. Conjugation conditions were optimized to yield conjugates with high ADM:MAb molar ratios. The final immunoconjugate yields were found to decrease as the ADM:MAb molar ratio of the conjugates increased. Stability studies indicated that ADM was released from the immunoconjugates at mildly acidic pHs ranging from 4.5-6.5. Treatment of immunoconjugates with mild reducing agent dithiothreitol resulted in release of an acylhydrazone derivative of ADM. Flow-cytometric studies showed that the binding activity of various MAbs following conjugation to ADM was preserved at ADM:MAb molar ratios up to 10. Antibody-directed cytotoxicity was demonstrated under several assay conditions using combinations of antigen-positive and antigen-negative cells and binding and nonbinding immunoconjugates. In several experiments, ADM immunoconjugates were more potent than equivalent amounts of unconjugated ADM.

Antibodies, Monoclonal↗
PubMed1990

Inhalation injuries.

Inhalation injuries comprise three distinct clinical entities that may be classified according to the time of onset of symptoms, etiologic agents, and the anatomic location of injury. These entities are carbon monoxide toxicity, upper airway obstruction, and smoke inhalation or chemical injury. Each has a distinct pathophysiology, clinical manifestations, treatment, and prognosis. The emergency management of inhalation injury is frequently based on the health professional's degree of suspicion despite the availability of sophisticated diagnostic tests. Early aggressive treatment, including maintaining a patent airway, administering humidified oxygen and bronchodilators, and providing pulmonary toilet, is necessary to ensure the best possible outcome. Understanding the pathophysiology, clinical manifestations, diagnosis, medical management, and nursing implications of inhalation injuries can improve patient survival.

Airway Obstruction↗
PubMed1989

Uptake of 14C-chlorhexidine diacetate to Escherichia coli and Pseudomonas aeruginosa and its release by azolectin.

Uptake of 14C-labelled chlorhexidine diacetate (14C-CHA) by wild-type and envelope mutant strains of Escherichia coli and Pseudomonas aeruginosa was very rapid. Maximum uptake was observed within a contact time of 20 s with no additional binding on increased contact, and was concentration-dependent. In contrast to this rapid binding of 14C-CHA, bactericidal studies revealed that the lethal activity of low concentrations of unlabelled CHA was slow, although higher concentrations had a rapid effect. Comparison of a wild-type strain with its envelope mutants indicated that there was little difference in 14C-CHA uptake, in minimal inhibitory concentrations or in bactericidal activity. Azolectin was found to be an effective neutralising agent of biguanide action, but in in vitro agar tests and in reducing or removing the amount of 14C-CHA taken up by the cells.

Chlorhexidine↗
PubMed1987

Calcium and phosphate solubility in neonatal parenteral nutrient solutions containing Aminosyn PF.

Factors affecting the solubility of calcium and phosphate in neonatal total parenteral nutrient (TPN) solutions containing a new amino acid formulation were studied. Six TPN solutions containing various concentrations of Aminosyn PF, an amino acid solution for infants and children, were prepared in 10% dextrose injection. Some of the solutions also contained cysteine hydrochloride 40 mg per gram of protein. Various concentrations of calcium gluconate and monobasic and dibasic potassium phosphate were added to 20-mL samples of the TPN solutions. A total of 27 samples of each TPN solution was prepared. Samples were visually inspected after 18 hours at 25 degrees C and again after 30 minutes in a water bath at 37 degrees C. Clear samples at this time were also examined microscopically. Solubility curves were prepared by plotting the concentrations at which either visual or microscopic precipitation occurred. Solubility curves for TPN formulations containing Aminosyn PF revealed a decrease in calcium solubility of 5 to 15 meq/L and a decrease in phosphate solubility of 5 to 15 mmol/L compared with previously published calcium-phosphate solubility curves for another similar amino acid solution, TrophAmine. Calcium and phosphate solubilities were also influenced by temperature and the time after solution preparation. In these TPN formulations containing Aminosyn PF as the amino acid source, the solubilities of calcium and phosphate were substantially less than reported in a previous study of solutions containing TrophAmine.

Amino Acids↗
PubMed1986

Calcium and phosphate solubility in neonatal parenteral nutrient solutions containing TrophAmine.

Factors affecting solubilities of calcium and phosphate in neonatal total parenteral nutrient (TPN) solutions containing a new amino acid formulation were examined. Twelve TPN solutions containing various concentrations of TrophAmine, an amino acid formulation specific for infants and young children, were prepared in 10% dextrose injection. Some of the solutions also contained cysteine hydrochloride 40 mg/g of protein and either sodium bicarbonate or hydrochloric acid (lipid emulsion buffer) to buffer the solution pH to simulate that produced by simultaneously administering lipid emulsion through the i.v. line. Calcium gluconate and monobasic and dibasic potassium phosphate were added to 20-mL samples of the TPN solutions to achieve calcium concentrations of 10, 20, 30, 40, or 50 meq/L with phosphate concentrations of either 10, 20, 30, or 40 mmol/L; a total of 20 samples of each TPN solution was prepared. Samples were inspected visually for precipitation or crystallization after 18 hours at 25 degrees C and again after 30 minutes in a water bath at 37 degrees C. Clear samples at this time were also examined microscopically for evidence of microcrystallization. Solubility curves were prepared by plotting graphically the concentrations at which either visual or microscopic precipitation occurred. Temperature, amino acid concentration, and the addition of cysteine hydrochloride and lipid emulsion buffer each influenced the solubilities of calcium and phosphate in the TPN solutions. The use of TrophAmine as the amino acid source allowed slightly greater concentrations of phosphate to be solubilized as compared with older amino acid formulations.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1985

Activation of human thymocytes via the 50KD T11 sheep erythrocyte binding protein induces the expression of interleukin 2 receptors on both T3+ and T3- populations.

Recent studies have demonstrated that the 50KD T11 molecule is a surface component of a macrophage-independent alternative pathway of human T cell activation that is unrelated to the T3/Ti antigen-MHC receptor complex. Given the expression of T11 on all human thymocytes, it was of interest to determine whether they could be activated via this pathway. The triggering of T11 by monoclonal antibodies anti-T112 and anti-T113, directed at two unique epitopes on the molecule, induced IL 2 receptor expression on both T3+ and T3- thymocytes but did not induce IL 2 production. Consequently, in contrast to peripheral blood T cells, thymocytes did not proliferate in response to anti-T112 and anti-T113 in the absence of exogenous IL 2. These studies suggest that IL 2 receptor gene activation precedes IL 2 gene activation in T cell development. The ability of the alternative pathway of T cell activation to induce IL 2 receptor expression on T3- thymocytes implies that the T11 molecule may have an important role in early thymocyte ontogeny.

Antibodies, Monoclonal↗
PubMed1984

An alternative pathway of T-cell activation: a functional role for the 50 kd T11 sheep erythrocyte receptor protein.

A series of seven monoclonal antibodies was produced against the T-lineage-specific 50 kd T11 sheep erythrocyte rosette (SRBC) receptor protein in order to define the function of the molecule. Three distinct epitopes were detected: T11(1), the SRBC binding site expressed on all T lymphocytes and thymocytes; T11(2), an epitope unrelated to the SRBC binding site but with a similar distribution; and T11(3), a neo-epitope expressed only upon T-cell activation. Simultaneous triggering of T11(2) and T11(3) epitopes by monoclonal antibodies induces T lymphocytes to proliferate and mediate their functional programs in the absence of antigen and/or antigen-presenting cells. This antigen-independent mode of triggering is distinct from that involving the T3-Ti antigen receptor complex and represents an alternate pathway of T-cell activation. Given that T11 is the earliest T-lineage surface glycoprotein to appear in thymic ontogeny and is thus expressed before T3-Ti, the former may be involved in clonal expansion and/or differentiation during early development.

Animals↗