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Biomedical subjects

L O'Reilly

Publications and source records attributed to L O'Reilly.

At least 19 recordsLinked to original sources
PubMed2005

Subversion of the Bcl-2 life/death switch in cancer development and therapy.

The Bcl-2 protein family, which largely determines commitment to apoptosis, has central roles in tumorigenesis and chemoresistance. Its three factions of interacting proteins include the BH3-only proteins (e.g., Bim, Puma, Bad, Noxa), which transduce diverse cytotoxic signals to the mammalian pro-survival proteins (Bcl-2, Bcl-x(L), Bcl-w, Mcl-1, A-1), whereas Bax and Bak, when freed from pro-survival constraint, provoke the mitochondrial permeabilization that triggers apoptosis. We have discovered unexpected specificity in their interactions. Only Bim and Puma, which mediate multiple cytotoxic signals, engage all the pro-survival proteins. Noxa and Bad instead bind subsets and cooperate in killing, indicating that apoptosis requires neutralization of different pro-survival subsets. Furthermore, Mcl-1 and Bcl-x(L), but not Bcl-2, directly sequester Bak in healthy cells, and Bak is freed only when BH3-only proteins neutralize both its guards. BH3-only proteins such as Bim are tumor suppressors and mediate many of the cytotoxic signals from anticancer agents. Hence, compounds mimicking them may prove valuable for therapy. Indeed, the recently described ABT-737 is a promising "BH3 mimetic" of Bad. We find that, like Bad, ABT-737 kills cells efficiently only if Mcl-1 is absent or down-regulated. Thus, manipulation of apoptosis by targeting the Bcl-2 family has exciting potential for cancer treatment.

Animals↗
PubMed2003

Real-time application of automated ribotyping and DNA macrorestriction analysis in the setting of a listeriosis outbreak.

A cluster of three cases of listeriosis cases occurred against a background of endemic listeriosis in Western Australia. Human and environmental isolates of Listeria monocytogenes obtained during the outbreak investigation were rapidly subtyped by automated ribotyping using an EcoRI protocol and a RiboPrinter. DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE) was used to confirm the relatedness of isolates. Serogroup 1/2 predominated among the food samples and the four clinical isolates from the outbreak cluster were also of this serogroup. All isolates from chicken material were serogroup 1/2 and indistinguishable by ribotype pattern. PFGE subdivided strains of this ribotype into four subtypes. The preliminary analysis had an immediate impact on hypothesis generation, environmental health investigations, environmental specimen collection and initial control measures. Sufficient typing data to guide environmental health and disease control initiatives was generated in less than one week by combining automated ribotyping with PCR-based detection of L. monocytogenes in suspect foodstuffs and an L. monocytogenes DNA probe. There were no further cases of bacteriologically confirmed listeriosis in Western Australia for six months after completion of the investigation.

Automation↗
PubMed2002

Tissue distribution of Diablo/Smac revealed by monoclonal antibodies.

Diablo/Smac is a mammalian pro-apoptotic protein that can antagonize the inhibitor of apoptosis proteins (IAPs). We have produced monoclonal antibodies specific for Diablo and have used these to examine its tissue distribution and subcellular localization in healthy and apoptotic cells. Diablo could be detected in a wide range of mouse tissues including liver, kidney, lung, intestine, pancreas and testes by Western blot analysis. Immunohistochemical analysis found Diablo to be most abundant in the germinal cells of the testes, the parenchymal cells of the liver and the tubule cells of the kidney. In support of previous subcellular localization analysis, Diablo was present within the mitochondria of healthy cells, but released into the cytosol following the induction of apoptosis by UV.

3T3 Cells↗
PubMed2001

The LIM domain gene LMO4 inhibits differentiation of mammary epithelial cells in vitro and is overexpressed in breast cancer.

LMO4 belongs to a family of LIM-only transcriptional regulators, the first two members of which are oncoproteins in acute T cell leukemia. We have explored a role for LMO4, initially described as a human breast tumor autoantigen, in developing mammary epithelium and breast oncogenesis. Lmo4 was expressed predominantly in the lobuloalveoli of the mammary gland during pregnancy. Consistent with a role in proliferation, forced expression of this gene inhibited differentiation of mammary epithelial cells. Overexpression of LMO4 mRNA was observed in 5 of 10 human breast cancer cell lines. Moreover, in situ hybridization analysis of 177 primary invasive breast carcinomas revealed overexpression of LMO4 in 56% of specimens. Immunohistochemistry confirmed overexpression in a high percentage (62%) of tumors. These studies imply a role for LMO4 in maintaining proliferation of mammary epithelium and suggest that deregulation of this gene may contribute to breast tumorigenesis.

Adaptor Proteins, Signal Transducing↗
PubMed2001

Medium-chain acyl-CoA dehydrogenase (MCAD) mutations identified by MS/MS-based prospective screening of newborns differ from those observed in patients with clinical symptoms: identification and characterization of a new, prevalent mutation that results in mild MCAD deficiency.

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently diagnosed mitochondrial beta-oxidation defect, and it is potentially fatal. Eighty percent of patients are homozygous for a common mutation, 985A-->G, and a further 18% have this mutation in only one disease allele. In addition, a large number of rare disease-causing mutations have been identified and characterized. There is no clear genotype-phenotype correlation. High 985A-->G carrier frequencies in populations of European descent and the usual avoidance of recurrent disease episodes by patients diagnosed with MCAD deficiency who comply with a simple dietary treatment suggest that MCAD deficiency is a candidate in prospective screening of newborns. Therefore, several such screening programs employing analysis of acylcarnitines in blood spots by tandem mass spectrometry (MS/MS) are currently used worldwide. No validation of this method by mutation analysis has yet been reported. We investigated for MCAD mutations in newborns from US populations who had been identified by prospective MS/MS-based screening of 930,078 blood spots. An MCAD-deficiency frequency of 1/15,001 was observed. Our mutation analysis shows that the MS/MS-based method is excellent for detection of MCAD deficiency but that the frequency of the 985A-->G mutant allele in newborns with a positive acylcarnitine profile is much lower than that observed in clinically affected patients. Our identification of a new mutation, 199T-->C, which has never been observed in patients with clinically manifested disease but was present in a large proportion of the acylcarnitine-positive samples, may explain this skewed ratio. Overexpression experiments showed that this is a mild folding mutation that exhibits decreased levels of enzyme activity only under stringent conditions. A carrier frequency of 1/500 in the general population makes the 199T-->C mutation one of the three most prevalent mutations in the enzymes of fatty-acid oxidation.

Acyl-CoA Dehydrogenase↗
PubMed2000

Bcl-w is an important determinant of damage-induced apoptosis in epithelia of small and large intestine.

The potential role of the bcl-2 relative bcl-w as a physiological regulator of apoptosis in intestinal epithelia has been investigated. Immunoblots for bcl-w with new monoclonal antibodies revealed that it was expressed in the small intestine and colon, among other murine tissues, as well as in six human tumour cell lines of epithelial origin, including two colon carcinoma lines. To assess whether bcl-w regulates either spontaneous or damage-induced apoptosis in the small intestine or colon, apoptosis in intestinal crypts of bcl-w -/- and wild-type mice was quantified microscopically on a cell positional basis. Spontaneous apoptosis within crypt epithelia was not significantly increased by loss of bcl-w, in either the small intestine or midcolon. However, after treatment with the cytotoxic drug 5-fluorouracil or with gamma-radiation, the bcl-w-null animals exhibited substantially more apoptosis than their wild-type counterparts in both tissues. The greatest enhancement of apoptosis attributable to the absence of bcl-w (up to sixfold) occurred in the small intestine. Hence, bcl-w is an important determinant of damage-induced apoptosis in intestinal epithelia, and unlike bcl-2, which regulates only colonic apoptosis, plays a major role in small intestinal epithelium.

Animals↗
PubMed2000

Second-site changes affect viability of amphotropic/ecotropic chimeric enveloped murine leukemia viruses.

Chimeras were previously generated between the ecotropic (Moloney-MuLV) and amphotropic (4070A) SU and TM proteins of murine leukemia virus (MuLV). After passage in D17 cells, three chimeras with junctions in the C terminus of SU (AE5, AE6, and AE7), showed improved kinetics of viral spreading, suggesting that they had adapted. Sequencing of the viruses derived from the D17 cell lines revealed second-site changes within the env gene. Changes were detected in the receptor binding domain, the proline-rich region, the C terminus of SU, and the ectodomain of TM. Second-site changes were subcloned into the parental DNA, singly and in combination, and tested for viability. All viruses had maintained their original cloned mutations and junctions. Reconstruction and passage of AE7 or AE6 virus with single point mutations recovered the additional second-site changes identified in the parental population. The AE5 isolate required changes in the VRA, the VRC, the VRB-hinge region, and the C terminus of SU for efficient infection. Passage of virus, including the parental 4070A, in D17 cells resulted in a predominant G100R mutation within the receptor binding domain. Viruses were subjected to titer determination in three cell types, NIH 3T3, canine D17, and 293T. AE6 viruses with changes in the proline-rich region initially adapted for growth on D17 cells could infect all cell types tested. AE6-based chimeras with additional mutations in the C terminus of SU could infect D17 and 293T cells. Infection of NIH 3T3 cells was dependent on the proline-rich mutation. AE7-based chimeras encoding L538Q and G100R were impaired in infecting NIH 3T3 and 293T cells.

3T3 Cells↗
PubMed1998

Improving cancer pain management through a system wide commitment.

Despite the well documented need for better cancer pain management there is little evidence that conventional educational approaches have improved clinical practice. This article discusses strategies to improve cancer pain management, focusing particular attention to institutional approaches that contain quality assurance (QA) and quality improvement (QI) efforts. An innovative pain management project is described that includes an in-depth evaluation of system and provider strengths and weaknesses; a system-wide commitment to cancer pain management; patient/family, nurse, pharmacist, and physician collaborative teams; use of a Washington State law allowing pharmacist prescriptive authority of narcotics and other drugs within a prescribed treatment protocol; and QA and QI efforts to improve and document performance. The authors strongly believe that the development of collaborative programs that integrate the power of the institutional process with clinical care will produce the greatest improvement in patient care not only for cancer pain, but also for other difficult domains of end-of-life.

Journal Article↗
PubMed1997

Causes of death of HIV-infected persons in Ottawa, Ontario, 1984-1995.

BACKGROUND: Acquired immunodeficiency syndrome (AIDS) has become a leading cause of death of young men in the United States. With the introduction of prophylaxes and antiretrovirals for opportunistic infection, there have been significant changes in the clinical history of human immunodeficiency virus (HIV) infection. OBJECTIVE: To determine the cause of death of the patients followed up by our clinic from 1984 to 1995. METHODS: A critical chart review was performed on the records of all patients affiliated with the Ottawa General Hospital HIV/AIDS Clinic, Ottawa, Ontario, who died between 1984 and July 15, 1995. Data regarding the cause of death, last CD4 T-lymphocyte cell count before death, medication use at time of death, and location and year of death were collected. Data were analyzed for 1984 through 1988, 1989 through 1991, and 1992 through 1995, corresponding to the evolution of HIV-related medical care. RESULTS: The median CD4 T-lymphocyte cell count at death had declined. Pneumocystis carinii pneumonia has decreased significantly as cause of death (28.6%-3.8%, P < .001). No other specific attributable terminal illness has decreased in frequency during 11 years. The wasting illnesses, particularly HIV wasting syndrome (3.6%-13.7%, P = .04), and untreatable illnesses have increased in frequency as causes of death. Patients are increasingly likely to die at home (0%-25%, P < .001) and less likely to die in hospital (54%-35%, P < .001). CONCLUSIONS: The HIV-infected persons are dying with more advanced HIV immunosuppression. Advances in P carinii pneumonia prophylaxis and treatment have had a dramatic effect on the cause of death of HIV-infected persons. Improved prophylaxis and treatment for non-P carinii pneumonia opportunistic infections and malignancies and HIV wasting syndrome are required.

AIDS-Related Opportunistic Infections↗
PubMed1996

Characterization of chimeras between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A envelope proteins.

A series of 22 chimeric envelope (env) genes were generated between the ecotropic Moloney murine leukemia virus and the amphotropic 4070A isolate. The chimeric envelopes were expressed within the complete, replication-competent provirus and tested for virus viability by transient expression assays. Eleven of the 22 viruses were viable. Five of these chimeric viruses showed an ecotropic host range, and six exhibited an amphotropic host range and viral interference. The host range determinants map to the first half of the surface (SU) protein. The N-terminal 72 amino acids of 4070A (42 of processed SU) are not required for amphotropic receptor usage. Ecotropic and amphotropic viruses differ in their ability to form large, multinucleated syncytia when cocultured with the rat XC cell line. Ecotropic murine leukemia virus forms large syncytia with XC cells, whereas no syncytia are reported for amphotropic virus. All chimeras which contained the N-terminal half of the ecotropic SU protein, encoding the receptor binding domain, formed the large multinucleated syncytia with XC cells.

3T3 Cells↗
PubMed1995

Aspartate at position 57 of nonobese diabetic I-Ag7 beta-chain diminishes the spontaneous incidence of insulin-dependent diabetes mellitus.

MHC class II genes have been shown to influence the development of the autoimmune disease insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse. In human IDDM it has been suggested that the presence of an aspartate at position 57 of the DQ beta-chain might be important in determining resistance to development of IDDM. The involvement of MHC class II genes in IDDM was investigated through the introduction of MHC encoding transgenes. We show that introduction of a mutated I-Ag7 Ab gene which encodes an aspartate at position 57 reduces the incidence of IDDM but does not prevent insulitis, sialadenitis, or the development of insulin and nuclear autoantibodies.

Amino Acid Sequence↗
PubMed1995

The role of infiltrating macrophages in islet destruction and regrowth in a transgenic model.

The expression of interferon-gamma (IFN-gamma) in pancreatic beta cells leads to a complex pathology that represents the processes of both islet destruction and islet regeneration. Inflammatory cells and the factors elicited from them participate in the development of pathology in this transgenic model. To dissect the role of infiltrating macrophages in these events, the monoclonal directed against the type 3 complement receptor (5C6) was utilized to inhibit the extravasation of macrophages. This was approached by treating transgenic mice with 5C6 for 3 or 4 months, starting from 5-7 days of age. The data presented in this report demonstrate that infiltrating macrophages are important in the observed induction of diabetes in our transgenic model. We also found that infiltrating macrophages did not play a major role in the observed proliferation and islet regeneration, but some interesting subtleties regarding the regulation of this proliferative process emerged.

Animals↗
PubMed1995

Tolerance induction as a therapeutic strategy for the control of autoimmune endocrine disease in mouse models.

This chapter aims to describe ways in which autoimmunity can be prevented or reversed and 'self-tolerance' re-established. To this end we have largely restricted our overview to the two main autoimmune disease models with which we are involved, i.e. IDDM in NOD mice and EAT in H-2k mice although, where appropriate and to demonstrate a particular point, other models are mentioned. The chapter has been divided into sections covering protection afforded by 1) transgenes, 2) autoantigen and 3) by reagents targetting T-cell surface molecules. Where established, the mechanism by which protection or tolerance is achieved is described but where, as in most cases, it is unknown the possibilities are discussed. Investigations using T-cell lines and clones and on islet regeneration which are currently being followed as part of a comprehensive approach to the study of autoimmunity are included as separate sections and their relevance discussed.

Animals↗
PubMed1994

Non-diabetogenic insulitis in NOD<-->B10.GD allophenic mice in spite of permissive class I MHC antigens.

Allophenic mice (embryo aggregation mouse chimeras) enable us to dissect the process of spontaneous autoimmunity under physiological conditions. Our previous experiments showed that the autoimmune process in allophenic mice of the NOD<-->C57B1/6 strain combination does not progress from insulitis to diabetes. One possible explanation for this protection is that H-2 Kd-restricted CD8+ T cells kill only NOD beta cells (Kd,Db) in the chimeric islets, while the B6 beta cells (Kb,Db) are spared from destruction. To test this hypothesis we analysed 22 NOD<-->B10.GD chimeras in which the class I MHC are shared by both parental strains. Therefore all the beta cells in these chimeras express H-2 Kd molecules. Ten allophenic mice were killed at 7 weeks and studied for early pathology. No evidence for intra-islet infiltration was obtained at this age, suggesting that the autoimmune process in NOD<-->B10.GD chimeras is slower than in NOD mice. Twelve chimeras were followed up for 1 year for disease development and all failed to progress to full-blown diabetes, despite the occurrence of intra-insulitis in six out of 12 mice. The lack of disease in NOD<-->B10.GD chimeras demonstrates that class I MHC chimerism does not account for diabetes resistance in NOD-allophenic mice.

Animals↗
PubMed1993

The regulation of autoimmunity through CD4+ T cells.

Our experiments imply that it is possible to use monoclonal antibody therapy to reestablish self tolerance to self antigens. This can be achieved by using a short course of an nd anti-CD4 antibody thus avoiding the problem of long term immunosuppression. The mechanism by which such a state of self tolerance is achieved remains to be clarified but possible mechanisms include deletion or anergy of autoreactive T cells or some form of suppression mediated through local cytokine production. As this antibody induced state of tolerance can be reversed in the NOD mouse by cyclophospamide deletion cannot be the method by which autoreactivity is prevented. The mixing experiments which have been described in the thyroiditis experiments strongly suggest that anery is not the mechanism. It therefore remains most likely that tolerance induced following administration of nd anti-CD4 is an active process maintained through the production of an inhibitory cytokine. This ability to reprogram the immune system using monoclonal antibodies makes it not beyond the realms of possibility that individuals suffering from IDDM may become tolerant of their beta cell antigens and thus be able to regenerate their own beta cell mass. If this could indeed occur it might mean that a lifetime of insulin injections and the development of the life threatening complications that may accompany a disease like IDDM may be avoided.

Animals↗
PubMed1992

The use of a non-depleting anti-CD4 monoclonal antibody to re-establish tolerance to beta cells in NOD mice.

The use of immunosuppressive drugs in the management of autoimmunity penalizes a large part of the immune system for the misdemeanors of a small minority of T cells. An ideal form of therapy would be one in which it were possible to render the immune system tolerant of the inciting antigens with minimal effects on other responses. We here show that it is possible to re-establish self tolerance in an animal model of insulin-dependent diabetes mellitus without prior deletion of CD4+ T cells using a short course of therapy with a non-lytic monoclonal antibody to the CD4 adhesion receptor on T cells. This tolerance can be achieved even when diabetogenic cells are already in the pancreas. Primary responses to antigens given after therapy has ceased are normal and secondary responses to antigens seen prior to, but not during, the period of antibody therapy can remain unaffected. This suggests that intervention with selected CD4 antibodies may have significant advantages over and above that provided not only by conventional immunosuppression but also over that provided by a depleting antibody.

Animals↗
PubMed1992

Expression of major histocompatibility complex class I antigens at low levels in the thymus induces T cell tolerance via a non-deletional mechanism.

Transgenic CBA (H-2k haplotype) mice expressing the H-2 Kb major histocompatibility complex (MHC) class I gene under control of transcriptional promoter elements from a milk protein gene display high-level H-2 Kb transcription in lactating mammary glands and low-level transcription in skin and thymus of male and virgin female transgenic mice. However, H-2 Kb antigen could be detected only in lactating mammary gland epithelial cells by immunohistological methods. All transgenic mice are tolerant of H-2 Kb since they fail to reject skin grafts from mice expressing H-2 Kb molecules. Furthermore, anti-H-2 Kb cytotoxic responses could not be generated using responder T cells from transgenic mice but T cells from the same mice proliferated, in the presence of interleukin-2, in response to stimulator cells expressing H-2 Kb. Tolerance to H-2 Kb is induced in the thymus since CBA mice grafted with thymus tissue from transgenic mice fail to reject H-2 Kb disparate skin grafts. However, experiments with double-transgenic mice also expressing a T cell receptor with anti-H-2 Kb specificity reveal that tolerance induction is not brought about by elimination of thymocytes bearing H-2 Kb-reactive receptors. Instead, a non-deletional mechanism which results in down-modulation of both CD8 and T cell receptor expression in peripheral T cells correlates with the induction of tolerance in these mice. These data reveal that extremely low levels of self-antigen expression in the thymus are sufficient to induce tolerance via non-deletional mechanisms.

Animals↗