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Biomedical subjects

D FitzGerald

Publications and source records attributed to D FitzGerald.

At least 19 recordsLinked to original sources
PubMed2000

The quality of care for Medicare patients with peptic ulcer disease.

OBJECTIVE: The aim of this study was to examine quality of care for hospitalized Medicare beneficiaries with peptic ulcer disease. METHODS: Collaborating with five Peer Review Organizations, we used 1995 Medicare claim files to select samples of inpatients with a principal diagnosis of peptic ulcer disease. Quality of care indicators developed by content experts included percentages for ulcer patients tested for Helicobacter pylori (H. pylori); biopsied patients who received tissue tests; H. pylori-positive patients who received appropriate therapy; and ulcer patients screened for preadmission nonsteroidal anti-inflammatory drug (NSAID) use and counseled about risks. RESULTS: Of 2,644 patients eligible for medical record review, 56% were tested for H. pylori, and 73% of those testing positive were treated appropriately; 84% of patients with endoscopic biopsies received a tissue test for H. pylori; 74% of patients were screened for preadmission NSAID use, 24% had documented counseling of NSAID use, and only 2% had documented counseling on the ulcer risk of NSAID use. Statistically significant regional variation occurred in four of six quality indicators. Outpatient records were reviewed for 529 patients to document prior outpatient H. pylori in this population; only 2% (n = 12) were tested for H. pylori in the year before admission. CONCLUSIONS: Opportunities exist to improve quality of care by testing for and treating H. pylori in hospitalized Medicare beneficiaries with peptic ulcer disease and to improve screening for NSAIDs and counseling on ulcer risks.

Aged↗
PubMed2000

New approaches to antigen delivery.

An improved understanding of immunologic events associated with immunization, the identification of promising new antigens, and an increased capacity to generate these antigens through chemical and biotechnology methods have led to many new vaccine opportunities. Inappropriate antigen exposure, however, can result in unwanted outcomes, such as incomplete protection, allergic reactions, autoimmunity, infection, or even tolerization. Thus, proper antigen delivery is critical for achieving the desired outcome. A number of vaccination approaches have now been described with varied degrees of success. The relative success of these approaches can be correlated with antigen delivery to specific presentation cells and stimulation of the immune system at sites where protective immunity is most appropriate. In addition, a greater understanding of mechanisms involving cells and effector molecules in the events of immunity may allow for improved possibilities for initiating, augmenting, and maintaining the response to a delivered antigen. This review provides insights into the various strategies currently being explored to optimize antigen delivery and the immune response to that antigen.

Animals↗
PubMed1999

Efficacy of treatment modalities in refractive amblyopia.

BACKGROUND: The pediatric clinic of the State College of Optometry/University Optometric Center develops a yearly quality management plan to monitor patient care. One of the areas retrospectively reviewed for all outcomes is refractive amblyopia. METHODS: A retrospective review of records was performed on patients diagnosed with refractive amblyopia. With the use of a prescribed protocol, each patient's progress was tracked for a period of 6 months. Major emphasis was placed on outcome as related to treatment modality. Treatment alternatives were optical correction alone, optical correction in conjunction with patching, and optical correction and patching with vision therapy. RESULTS: Improvement criteria included a 2-line increase in visual acuity on the Snellen chart and an increase of 20 seconds of arc of stereopsis, as measured by the Wirt circles. The groups that patched with correction and those that received vision therapy had similar visual acuity improvement's; however, the latter group had a significantly greater improvement in stereopsis. Both groups performed significantly better in both categories when compared to the group receiving optical correction alone. CONCLUSIONS: Though patching alone may be sufficient for improvement of visual acuity, binocular performance is significantly better when vision therapy is included in the treatment regimen.

Adolescent↗
PubMed1998

Functional expression of the chicken low density lipoprotein receptor-related protein in a mutant chinese hamster ovary cell line restores toxicity of Pseudomonas exotoxin A and degradation of alpha2-macroglobulin.

The low density lipoprotein receptor-related protein (LRP) is responsible for the clearance of several physiological ligands including a complex of proteinase and alpha2-macroglobulin (alpha2M) and for the entrance of Pseudomonas exotoxin A (PEA) into cells. We have prepared expression plasmids for the full-length chicken LRP (designated LRP100) and two intermediates encoding 25 and 67% of the receptor (designated LRP25 and LRP67, respectively) using overlapping cDNA fragments. LRP25 and LRP67 encode the N-terminal 22 and 64%, respectively, of LRP100 plus the transmembrane and intracellular domains. Transient transfection of these plasmids into COS-7 cells yielded recombinant proteins of expected molecular mass and immunoreactivity. However, LRP100 was incompletely processed into alpha- (515-kDa) and beta- (85-kDa) chains and was poorly transported from the endoplasmic reticulum to the Golgi compartment. Stable transformants of LRP100, LRP67, and LRP25 were generated in a mutant Chinese hamster ovary cell line that lacked expression of endogenous LRP and was resistant to PEA. All forms of recombinant LRP proteins were transported from the endoplasmic reticulum to the Golgi apparatus in Chinese hamster ovary cells as shown by their sensitivity to endoglycosidase H and resistance to neuraminidase. Cell surface iodination and subcellular fractionation studies indicated that all three LRP variants were expressed on the plasma membrane. Furthermore, expression of the three LRP variants restored, to various degrees, sensitivity to PEA and the ability to degrade methylamine-activated alpha2M (alpha2M*). These data suggest that deletion of large internal portions of LRP, including the processing site, does not prevent transport of LRP to the plasma membrane, nor does it abolish the interaction of LRP with alpha2M* or PEA. This LRP expression system may allow for the characterization of domains within LRP responsible for its multifunctionality.

ADP Ribose Transferases↗
PubMed1997

Furin-mediated cleavage of Pseudomonas exotoxin-derived chimeric toxins.

Pseudomonas exotoxin (PE) requires proteolytic cleavage to generate a 37-kDa C-terminal fragment that translocates to the cytosol and ADP-ribosylates elongation factor 2. Cleavage within cells is mediated by furin, occurs between arginine 279 and glycine 280, and requires an arginine at both P1 and P4 residues. To study the proteolytic processing of PE-derived chimeric toxins, TGFalpha-PE38 (transforming growth factor fused to the domains II and III of PE) and a mutant form, TGFalpha-PE38gly279, were each produced in Escherichia coli. When assessed on various epidermal growth factor (EGF) receptor-positive cell lines, TGFalpha-PE38 was 100-500-fold more toxic than TGFalpha-PE38gly279. In contrast to PE, where cleavage by furin is only evident at pH 5.5, furin cleaved TGFalpha-PE38 over a broad pH range, while TGFalpha-PE38gly279 was resistant to cleavage. TGFalpha-PE38 was poorly toxic for furin-deficient LoVo cells, unless it was first pretreated in vitro with furin. Furin treatment produced a nicked protein that was 30-fold more toxic than its unnicked counterpart. Using the single chain immunotoxin HB21scFv-PE40 as a substrate, furin-mediated processing of an antibody-based immunotoxin was also evaluated. HB21scFv-PE40, which targets cells expressing the transferrin receptor, was cleaved in a similar fashion to that of TGFalpha-PE38 and nicked HB21scFv-PE40 exhibited increased toxicity for LoVo cells. In short-term experiments, the rate of reduction in protein synthesis by furin-nicked immunotoxins was increased compared with unnicked protein, indicating that cleavage by furin can be a rate-limiting step. We conclude that furin-mediated cleavage of PE-derived immunotoxins is important for their cytotoxic activity.

Animals↗
PubMed1996

Recombinant immunotoxins.

Pseudomonas exotoxin has been genetically modified so that it targets cancer cells. This was accomplished by deleting its cell binding domain and replacing it with Fv fragments of antibodies that react with breast, colon, and other cancers. Several recombinant immunotoxins are now in clinical trials.

ADP Ribose Transferases↗
PubMed1996

Informed consent in emergency medicine: ethics under fire.

Many ethical issues in emergency medicine involve the question of informed consent. In this article, the ethical basis for informed consent, the essential elements of a morally valid informed consent, and the inadequacy of the law as a moral guide for informed consent are discussed. The ways in which the nature of emergency medicine affects the application of moral principles are examined, and specific guidelines for assessing a patient's decision-making capacity regarding informed consent are provided.

Decision Making↗
PubMed1994

Pseudomonas exotoxin A mutants. Replacement of surface-exposed residues in domain III with cysteine residues that can be modified with polyethylene glycol in a site-specific manner.

Pseudomonas exotoxin A (PE) is composed of three structural and functional domains. Domain Ia is responsible for cell recognition, domain II for translocation of PE across the cell membrane, and domain III for ADP-ribosylation of elongation factor 2. To investigate the role of the amino acids exposed on the surface of domain III, we replaced 15 of these, generating 29 different mutants at positions 412, 416, 418, 490, 513, 516, 522, 551, 576, 590, 599, 604, 606, 607 and 608. All but one mutant retained substantial ADP-ribosylation and cytotoxic activities. Modification of proteins with monomethoxy-polyethylene glycol (mPEG) prolongs their circulation in the blood stream and reduces their immunogenicity. Unlike PEGylated enzymes acting on small molecule substrates, PEGylated toxins may lose those functions that are based on macromolecular interactions. Therefore, we selectively PEGylated mutant PEs at positions 490, 513, 516, 522, 604, and 606. Most PEs modified by a 5-kDa mPEG via a disulfide or a thioether bond retained high cytotoxic activity. However, when a 20-kDa mPEG was used there was a decrease in cytotoxic activity with the disulfide-bonded molecules being more active. Positions 522 and 604 are good sites for PEGylation, but 490 is not. We also found that PEGylation of PE 522C prolonged its in vivo circulation time in mice.

ADP Ribose Transferases↗
PubMed1994

TGF alpha-PE40 inhibits non-small cell lung cancer growth.

The ability of a chimeric toxin containing transforming growth factor alpha (TGF alpha) and truncated Pseudomonas exotoxin A to inhibit NSCLC growth was investigated. TGF alpha-PE40 inhibited binding of 125I-EGF to NSCLC cell lines with an IC50 value of 0.5-3 micrograms/ml. Similarly, other forms of the fusion protein, TGF alpha-PE38 and TGF alpha-PE40Asp553, which have active TGF alpha binding domains, inhibited specific 125I-EGF binding to NSCLC cells with IC50 values of 0.1-2 and 0.05-05 microgram/ml respectively. TGF alpha-PE40 inhibited 35S-methionine uptake by NSCLC cells with an ED50 value of 1-30 ng/ml. TGF alpha-PE38, which has one of the two disulfide pairs of PE40, inhibited amino acid uptake with ED50 values of 3-50 ng/ml whereas TGF alpha-PE40Asp553, which lacks ADP ribosylation activity, had an ED50 > 100 ng/ml. TGF alpha-PE40 inhibited colony formation of NSCLC cells with an LD50 value of 0.008-0.1 ng/ml. Similarly, TGF alpha-PE38 inhibited NSCLC colony formation with LD50 values of 0.002-0.1 ng/ml whereas TGF alpha-PE40Asp553 had an LD50 > 10 ng/ml. Also, TGF alpha-PE40 and TGF alpha-PE38 inhibited NSCLC xenograft formation in nude mice whereas TGF alpha-PE40Asp553 was inactive. These data suggest that TGF alpha-PE40 and TGF alpha-PE38 may be useful agents to inactivate NSCLC cells.

Animals↗
PubMed1993

The N-terminal region of the 37-kDa translocated fragment of Pseudomonas exotoxin A aborts translocation by promoting its own export after microsomal membrane insertion.

The 37-kDa C-terminal fragment of Pseudomonas exotoxin A (PE; termed PE37 and composed of aa 280-613 of PE) translocates to the cell cytosol to cause cell death. PE37 requires a C-terminal endoplasmic reticulum retention sequence to be cytotoxic, indicating that the toxin may translocate to the cytosol from the endoplasmic reticulum. We show here that the N-terminal region of nascent PE37 can be inserted into the membrane of canine pancreatic microsomes by the preprocecropin signal sequence but then is exported or released from microsomes. The 34 N-terminal amino acids of the toxin fragment are sufficient to arrest translocation and prevent the microsomal accumulation of nascent chains that otherwise are sequestered into microsomes. These data support a role for the N-terminal region of PE37 in the translocation of the toxin from the endoplasmic reticulum to the cytosol in mammalian cells.

ADP Ribose Transferases↗
PubMed1993

[Gene cloning, expression of fusion protein TGF alpha-PE 40 and its inhibition activity on cancer cell growth].

Recombinant plasmid pYX382 and pYX3825 were constructed by fusing the cDNA encoding transforming growth factor type alpha (TGF alpha) to Pseudomonas exotoxin gene (PE) in which the cell recognition domain was deleted. The chimeric proteins produced by host E. Coli cells BL21 transformed by plasmid pYX382 and pYX3825 are termed TGF alpha-PE 40 which reacts with antibody against TGF alpha or antibody against PE in immunoblotting to show a 46 kd protein band reflecting the fusion of 56 kD TGF alpha peptide and 40 kD truncated Pseudomonas exotoxin molecule. An additional signal sequence OmpA was inserted into upstream region of TGF alpha cDNA in plasmid pYX3825 resulting in the partly secreting of expression product into medium and periplasm of the cells. TGF alpha-PE 40 was purified from medium by MONO Q ion exchange column and TSK 250 gel filtration column attached to Pharmacia EPLC system. The TGF alpha-PE 40 molecules showed a very strong activities inhibiting the protein synthesis and killing the cancer cells overexpressing EGF receptor on the cell surface.

Cloning, Molecular↗
PubMed1993

Ethical considerations.

The doctor's oath to serve the good of the patient requires the recognition that the patient's good consists of more than simply his or her biomedical health status. This article proposes a scheme for identifying the primary decision maker in clinical settings, particularly when the patient's own decision-making capacity is in question. A few of the many ethical controversies encountered in the practice of trauma medicine are examined and discussed.

Advance Directives↗
PubMed1992

Alanine scanning mutagenesis identifies surface amino acids on domain II of Pseudomonas exotoxin required for cytotoxicity, proper folding, and secretion into periplasm.

Pseudomonas exotoxin A (PE) is a single polypeptide chain that contains 613 amino acids and is arranged into three major structural domains. Domain Ia is responsible for cell recognition, domain II for translocation of PE across the membrane, and domain III for ADP-ribosylation of elongation factor 2. Recombinant PE can be produced in Escherichia coli and is efficiently secreted into the periplasm when an OmpA signal sequence is present. To investigate the role of the amino acids located on the surface of domain II in the action of the toxin against mammalian cells, we substituted alanine for each of the 27 surface amino acids present in domain II. Surprisingly, all 27 mutant proteins had some alteration in cytotoxicity when tested on human A431 or MCF7 cells or mouse L929 cells. Native PE has a compact structure and therefore is relatively protease resistant and very little ADP-ribosylation activity is detected in the absence of the denaturing agents like urea and dithiothreitol. Several of the mutations resulted in altered protease sensitivity of the toxin. Seven of the mutant molecules exhibited ADP-ribosylation activity without urea and dithiothreitol, indicating they are partially unfolded. Out of these seven mutants, six had increased cytotoxic activity on at least one of the target cell lines and the other retained its native cytotoxic potency.

ADP Ribose Transferases↗
PubMed1992

A recombinant form of Pseudomonas exotoxin directed at the epidermal growth factor receptor that is cytotoxic without requiring proteolytic processing.

Pseudomonas exotoxin A is composed of three structural domains that mediate cell recognition (I), membrane translocation (II), and ADP-ribosylation (III). Within the cell, the toxin is cleaved within domain II to produce a 37-kDa carboxyl-terminal fragment, containing amino acids 280-613, which is translocated to the cytosol and causes cell death. In this study, we constructed a mutant protein (PE37), composed of amino acids 280-613 of Pseudomonas exotoxin A, which does not require proteolysis to translocate. PE37 was targeted specifically to cells with epidermal growth factor receptors by inserting transforming growth factor-alpha (TGF-alpha) after amino acid 607 near the carboxyl terminus of Pseudomonas exotoxin A. PE37/TGF-alpha was very cytotoxic to cells with epidermal growth factor receptors. It was severalfold more cytotoxic than a derivative of full-length Pseudomonas exotoxin A containing TGF-alpha in the same position, probably because the latter requires intracellular proteolytic processing to exhibit its cytotoxicity, and proteolytic processing is not 100% efficient. Deletion of 2, 4, or 7 amino acids from the amino terminus of PE37/TGF-alpha greatly diminished cytotoxic activity, indicating the need for a proper amino-terminal sequence. In addition, a mutant containing an internal deletion of amino acids 314-380 was minimally active, indicating that other regions of domain II are also required for the cytotoxic activity of Pseudomonas exotoxin A.

ADP Ribose Transferases↗
PubMed1992

In vitro and in vivo suppression of interleukin-2-activated killer cell activity by chimeric proteins between interleukin-2 and Pseudomonas exotoxin.

The biological effects of IL-2 are mediated through high (complex of alpha and beta chain) or intermediate (beta chain) affinity IL-2 receptors. Previously, chimeric proteins composed of IL-2 and Pseudomonas exotoxin (IL-2-PE) were shown to be specifically cytotoxic to cells bearing IL-2 receptors. It has also been shown that IL-2-PE chimeric proteins can abrogate T cell-mediated immune response in vitro. In the current study, we have investigated the effects of IL-2-PE on LAK activity both in vivo and in vitro. We administered either IL-2-PE40 (comprised of IL-2 and 40-kDa portion of PE) or IL-2-PE66 (comprised of IL-2 and 66-kDa molecule of PE) to normal C57BL/6 mice for 3 or 8 days and LAK activity was assessed in various organs of mice. We found that IL-2-PE40 generated LAK activity in various compartments of mice and the level of activity was slightly lower than that observed with an equivalent amount of recombinant (r) IL-2 alone. However, IL-2-PE66 failed to generate LAK activity which would have been induced due to an equivalent concentration of rIL-2. IL-2-PE66 also did not induce LAK activity from the splenocytes during in vitro culture while IL-2-PE40 generated good LAK activity. An equivalent amount of IL-2 also generated potent LAK activity. The suppression of LAK activity by IL-2-PE66 was also evident in cells preactivated with IL-2; however, this inhibition was partial. The suppressive activity of IL-2-PE66 was shown to be mediated through IL-2 receptor interactions as excess amounts of rIL-2 were able to abrogate its effect. Both IL-2 toxins were equivalently cytotoxic to IL-2 receptor-bearing HUT 102 cells and both were able to compete from high and intermediate affinity IL-2 receptors. Taken together, our data indicate that IL-2-PE66 is highly cytotoxic to LAK cells while IL-2-PE40 is less cytotoxic. Thus, data from our study and from other published reports indicate that IL-2-PE66 is more potent immunosuppressive agent than IL-2-PE40.

ADP Ribose Transferases↗
PubMed1992

Targeting growth factor receptors with fusion toxins.

Recombinant toxins which bind to growth factor receptors have been prepared and used to kill cells responsible for malignant or autoimmune disease. Our strategy has been to genetically fuse ligands to different forms of Pseudomonas exotoxin which due to mutations or deletions do not bind to normal cells. The resulting recombinant chimeric toxins, in concentrations often less than 1 ng/ml, selectively kill cells expressing the appropriate growth factor receptor. The ligand may be a growth factor, such as transforming growth factor alpha (TGF alpha), interleukin 6 (IL6) or interleukin 2 (IL2), or single chain antigen binding proteins, such as the variable heavy and light regions of the monoclonal antibody anti-Tac. These chimeric toxins kill not only established cell lines but also fresh tumor cells from patients and display anti-tumor activity toward human malignant tumors in nude mice. While clinical trials are beginning with some of these agents, work continues to improve the effectiveness of recombinant chimeric toxins, and to widen the scope of disorders which might be treated by this approach.

ADP Ribose Transferases↗