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Biomedical subjects

F Graham

Publications and source records attributed to F Graham.

At least 19 recordsLinked to original sources
PubMed2004

Implications of the increasing female participation in the general practice workforce in Ireland.

Almost one in three Irish general practitioners (GPs) are now women, a ratio that has doubled since the early 1990s. The increase in numbers of women entering general practice training looks likely to continue and will have a formative influence on the future of general practice. This study investigates the implications for Irish general practice of the increasing feminization of the workforce. Questionnaires were sent to all (200) Irish vocationally trained female General Practitioners qualifying between 1995 and 2001. Sixty eight percent of respondents were currently working in full-ltime general practice. A majority wish to remain in general practice (88%) and potentially up to 90% intend to work part-time in the future. The ideal future work practices of this cohort are part-time partnerships, and over 50% are not in a position to work out of hours. To facilitate the career and working intentions of female vocationally trained GPs and to retain their services, there needs to be increased flexibility of hours of work, increased part-time partnerships and an Irish retainer scheme that will accommodate female GPs who cannot work full-time because of family commitments.

Family Practice↗
PubMed2000

Evaluation of a combination rapid immunoassay for detection of Giardia and Cryptosporidium antigens.

A combination cassette format nonenzymatic rapid immunoassay for detection of Giardia and Cryptosporidium antigens was evaluated by using 556 patient stool specimens from three clinical laboratories. This assay (Genzyme Diagnostics Contrast Giardia/Cryptosporidium), which can be used with fresh or formalin-fixed specimens, had unadjusted sensitivities and specificities of 96.1 and 98.5% for Giardia and 100 and 98.7% for Cryptosporidium, respectively, in this study.

Animals↗
PubMed1999

A cluster of transfusion-associated babesiosis cases traced to a single asymptomatic donor.

CONTEXT: The risk of acquiring babesiosis by blood transfusion is largely unknown since in areas where it is endemic it is often an asymptomatic infection. OBJECTIVE: To investigate and treat a cluster of blood transfusion-associated babesiosis cases. DESIGN: Case series and epidemiologic investigation. SETTING: Urban inner-city hospital. PATIENTS: Six persons who received Babesia microti-infected blood components from a donor. MAIN OUTCOME MEASURE: Diagnosis and successful therapy of babesiosis following transfusion. RESULTS: Six individuals (1 adult, 1 child, and 4 neonates) were exposed to products from a single blood donation by an asymptomatic Babesia-infected donor. Three of the 6 exposed patients became parasitemic. Polymerase chain reaction testing, animal inoculation studies, and indirect immunofluorescent antibody testing were used to confirm the presence of Babesia microti in the donor's blood and to establish the presence of infection in 3 of the 6 recipients. The 3 infected recipients and 1 additional recipient were treated without incident. CONCLUSION: Physicians should consider babesiosis in the differential diagnosis of a febrile hemolytic disorder after blood transfusion. Prompt diagnosis is important since babesiosis is responsive to antibiotic therapy and, untreated, can be a fatal disease in certain risk groups.

Aged↗
PubMed1999

Use of helper-dependent adenoviral vectors of alternative serotypes permits repeat vector administration.

We have developed a new helper adenovirus (Ad) based on serotype 2, Ad2LC8cCARP, for use in the Cre/loxP system (Parks et al. Proc Natl Acad Sci USA, 1996; 93: 13565-13570) to generate Ad vectors deleted of all protein coding sequences (helper-dependent Ad vectors (hdAd)). A comparison of Ad2LC8cCARP and our original helper virus (based on serotype 5, Ad5LC8cluc) showed that the two helper viruses amplified hdAd with a similar efficiency, and resulted in a similar yield and purity after large-scale preparation of vector. In vitro, the resulting hdAd2 had a similar transduction efficiency and expression kinetics of transgene (beta-gal) as the hdAd5. An important feature of the helper-dependent system is that all virion components, except the virion DNA, derive from the helper virus. Consequently, vectors produced with help from Ad2LC8cCARP were not neutralized by antibodies against Ad5, and vectors produced with Ad5 helper were resistant to neutralizing antibodies against Ad2. Analysis of transgene expression in mouse liver after intravenous injection of the Ad2-based hdAd showed that the vector could efficiently transduce the liver, and produce high levels of a foreign transgene, similar to those expressed by the hdAd generated with the Ad5 helper virus. Mice immunized with hdAd2 produced Ad2-neutralizing antibodies, which did not cross-react with hdAd5. To determine if successful repeat Ad vector administration could be achieved by sequential use of alternative Ad serotypes, we injected mice with hdAd2 (hSEAP) followed 3 months later by a lacZ-expressing hdAd of either the same or different serotype. Repeated administration of hdAd2 resulted in a 30- to 100-fold reduction in transgene expression compared with naive animals. In contrast, no decrease in transgene expression was observed when the second vector was of a different serotype. These results demonstrate that effective vector readministration can be achieved by the sequential use of hdAds based on alternative serotypes.

Adenoviridae↗
PubMed1999

Adenovirus vector expressing mouse oncostatin M induces acute-phase proteins and TIMP-1 expression in vivo in mice.

Mouse oncostatin M (MuOSM) regulates the production of acute-phase proteins by hepatocytes as well as tissue inhibitor of metalloproteinases-1 (TIMP-1) production by fibroblasts in vitro. We have generated an adenovirus (Ad) encoding MuOSM and tested the effects of administration of recombinant AdMuOSM to mice in vivo. On intramuscular injection, AdMuOSM (5 X 10(7) plaque-forming units, pfu) induced an increase in serum levels of interleukin-6 (IL-6) as well as the acute-phase proteins serum amyloid A (SAP) and alpha1-acid glycoprotein (AGP) at day 1. SAP and AGP concentrations were elevated to greater levels at day 3 and decreased to near control levels at day 7. Intratracheal treatment with AdMuOSM induced TIMP-1 mRNA levels (as assessed by Northern blots) that corresponded to the presence of transgene MuOSM mRNA levels. TIMP-1 was elevated at day 1 and day 3 and less consistently at day 7 after administration. Intraperitoneal treatment with AdMuOSM also resulted in elevation of TIMP-1 mRNA in lung tissue. These results show that AdMuOSM can induce both local and systemic effects and demonstrate in vivo effects of OSM that are consistent with in vitro studies on acute-phase protein and TIMP-1 expression.

Acute-Phase Proteins↗
PubMed1997

Dendritic cells transduced with an adenoviral vector encoding a model tumor-associated antigen for tumor vaccination.

Evaluation of the potential role of dendritic cells (DCs) as adjuvants for tumor vaccination has focused primarily on techniques that load DCs with peptide tumor antigens. Our aim has been to optimize the induction of antitumor immunity by enhancing the ability of DCs to present tumor-associated antigens endogenously to the afferent lymphatic system in the appropriate major histocompatibility complex (MHC)-restricted context. We have used replication-defective adenovirus vectors (Ads) to transduce DCs with various genes, including tumor antigen genes. We found that 90% of murine bone marrow derived-DCs could be infected with an Ad vector expressing the beta-galactosidase gene and still retain their physiologic and phenotypic characteristics. Furthermore, we demonstrated that transgene expression was detectable in the spleen for at least 3 days following intravenous injection of Ad-transduced DCs. Using a polyoma middle T (PymT) transgenic murine mammary carcinoma model, we have shown that a single injection of 10(5)-4 x 10(6) DCs transduced with an Ad vector expressing PymT provided complete and specific protection against tumor cell challenge in 100% of vaccinated animals. Immunization against the PymT tumor by injection with the PymT expressing Ad vector alone resulted in varying degrees of effectiveness, was highly dependent upon the route of administration, and led to significant hepatic toxicity that was not seen in mice immunized with DC transduced with the Ad vector. Our results suggest that: (i) DCs can be very efficiently modified by ex vivo Ad transduction to express tumor-specific antigens, (ii) such modified DCs appear nontoxic and stimulate a potent antitumor response.

Adenoviridae↗
PubMed1997

Transgene-induced production of IL-4 alters the development and collagen expression of T helper cell 1-type pulmonary granulomas.

A number of complex mechanisms regulate the size and cellularity of an Ag-dependent granulomatous reaction and their accompanying cytokine production profiles. In the present study, Th1 (purified protein derivative (PPD)) and Th2 (schistosome egg Ag)-type granulomas were established to examine the role of IL-4 in lesion development and procollagen type III expression. PPD-sensitized mice were transfected via the airway with an adenovirus construct containing an IL-4 cDNA cassette, and the lungs were embolized with sized Ag-coated Sepharose beads. Granuloma development in response to the PPD bead challenge in control mice primarily consisted of mononuclear cells. In contrast, the overexpression of IL-4 in the IL-4 adenovirus-transfected animals demonstrated a significant increase in cellularity, size, procollagen type III expression, and accumulation of eosinophils. Analysis of mRNA and protein within the lung demonstrated significant expression of IL-4 in only the IL-4 adenovirus-transfected animals. The granuloma lesion size was significantly increased in the IL-4 adenovirus-transfected animals on days 2 and 5, reaching an approximate 50% increase compared with the control groups. Furthermore, procollagen type III mRNA expression was increased in the IL-4 adenovirus-transfected, PPD bead-embolized lungs. In contrast, when IL-4 was neutralized during Th2-type schistosome egg Ag bead granulomas, a decrease in procollagen type III was observed. These data indicate that the progression of certain granulomas may be regulated by the production of IL-4, thus altering the cellularity, size, and matrix composition of the inflammatory response.

Animals↗
PubMed1997

Intramarrow cytokine gene transfer by adenoviral vectors in dogs.

Daily systemic administration of hematopoietic growth factors can be associated with dose-limiting systemic side effects. To overcome this, we have investigated hematopoietic cytokine gene transfer to the marrow cavity of dogs by direct intramarrow injection of adenoviral vectors. In marrow culture, replication-deficient (E1-deleted) adenoviral vectors were able to transduce marrow stromal cells, demonstrating 30-fold greater expression than from other marrow cell types. High-level (ng/ml) cytokine production from transduced stromal cells persisted for 14 days in culture. Because adenovectors could efficiently transduce marrow stromal cells in culture, we investigated if stromal cells could also be transduced in vivo following direct intramarrow vector injection. Adenovectors with genes for interleukin 6 (IL-6) and Lac Z (beta-galactosidase) were injected directly into the marrow cavity of dogs resulting in protein expression localized to within the treated marrow. To evaluate this approach further in dogs, we constructed a vector expressing biologically active canine granulocyte-macrophage colony stimulating factor (GM-CSF). 293 cells infected with ADGM-CSF demonstrated prevalent GM-CSF mRNA by Northern blot and 135 +/- 30 ng/ml of protein as measured by enzyme-linked immunosorbent assay (ELISA). In vitro bioactivity of protein expressed was confirmed by canine GM colony-forming assay (CFU-GM). In vivo high-level protein production was noted in supernatants of marrow aspirates 72 hr following direct intramarrow administration of ADGM-CSF (baseline mean +/- SEM, 27 +/- 22 ng/ml, 72-hr sample 921 +/- 461 ng/ml). A localized myeloid expansion of marrow and significant peripheral leukocytosis (neutrophilia) were noted in all ADGM-CSF-treated dogs. Peripheral blood changes lasted for up to 3 weeks in dogs following single intramarrow injection. Thus, adenoviral cytokine expression from the marrow of a single large bone (ilium) led to compartmentalized expression of growth factor and an increase of hematopoiesis sufficient to cause peripheral blood changes in a large animal model.

Adenoviridae↗
PubMed1996

Adenovirus-vector-mediated cytokine gene transfer to lung tissue.

In summary, we have been able to demonstrate that adenovirus vectors are valuable tools in examining the roles played by individual cytokines in lung responses and inflammation. These viral vectors have marked trophism for the epithelial cells of the lung and are highly efficient in transferring the genes into these lining cells. This results in significant expression of cytokines both within the lumen and the parenchyma of the lung. As a result of an individual cytokine being overexpressed, there are cytokine-specific changes seen-IL-6 resulting in lymphocytosis, MIP2 resulting in neutrophil accumulation, RANTES resulting in monocyte accumulation, TGF beta resulting in monocytosis but no fibrosis, and GM-CSF most surprisingly resulting in tissue eosinophilia, granuloma formation, and subsequently the onset of fibrosis. These vectors have helped pinpoint the role of a number of the cytokines in inducing chronic inflammatory changes to the lung and imply that a single cytokine may not be the only trigger resulting in chronic changes within the lung parenchyma.

Adenoviridae↗
PubMed1996

Transfer of granulocyte-macrophage colony-stimulating factor gene to rat lung induces eosinophilia, monocytosis, and fibrotic reactions.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine whose expression is increased in numerous respiratory diseases, particularly in asthma. However, the role of GM-CSF in the pathogenesis of these conditions in vivo remains unclear. Here, we report the functional activities of GM-CSF highly expressed in rat lung after intrapulmonary transfer of the gene coding for murine GM-CSF by using an adenoviral vector. This high, transient expression of GM-CSF led to the sustained but self-limiting accumulation of eosinophils and macrophages associated with tissue injury in the lung followed by varying degrees of irreversible fibrotic reactions observed in later stages. These results suggest that GM-CSF plays a previously unrealized role in the development of respiratory conditions characterized by eosinophilia, granuloma and/or fibrosis and provide the rationale for targeting this molecule in these diseases.

Animals↗
PubMed1996

B7-1 gene transfer into human cancer cells by infection with an adenovirus-B7 (Ad-B7) expression vector.

BACKGROUND: Transfection of the costimulatory molecule B7-1 into some murine tumors can increase antitumor immunity and eradicate tumor growth. The purpose of this work was to construct an adenovirus-B7 (Ad-B7) expression vector and study B7-1 gene transfer into human cancer cells. METHODS: The human B7-1 cDNA was ligated into an expression cassette containing the human cytomegalovirus immediate early gene promoter and then inserted into the E1 region of the Ad5 genome by homologous recombination. The resulting Ad-B7 vector was used to infect established cancer cell lines and freshly resected cancers. Resected tumors were disaggregated into single cell suspensions by mechanical mincing and enzymatic digestion. Surface expression of B7-1 after infection was verified by flow cytometry. RESULTS: Expression kinetics in three cell lines showed that infected cells began to express B7-1 within 24 h. The proportion of B7-1+ cells continued to increase during the next 48 h, after which expression remained relatively constant during the next 5 days (up to 98% B7-1+ cells). Fresh tumor cells from various cancers displayed similar kinetics, but with greater variability in the proportion of cells expressing B7-1 (13% to 95% B7-1+ cells). Cancers which were successfully infected included 3 colorectal adenocarcinomas, 2 leiomyosarcomas, 2 lung squamous cell carcinomas, and 1 renal cell carcinoma. CONCLUSIONS: The Ad-B7 vector is a rapid and efficient means of gene transfer which does not require host cell proliferation. The ultimate objective is to engineer autologous tumors to express B7-1 and vaccinate cancer patients in an adjuvant or palliative setting.

Adenocarcinoma↗
PubMed1996

Tropism of human adenovirus type 5-based vectors in swine and their ability to protect against transmissible gastroenteritis coronavirus.

The infection of epithelia] swine testicle and intestinal porcine epithelial (IPEC-1) cell lines by adenovirus type 5 (Ad5) has been studied in vitro by using an Ad5-luciferase recombinant containing the firefly luciferase gene as a reporter. Porcine cell lines supported Ad5 replication, showing virus titers, kinetics of virus production, and luciferase expression levels similar to those obtained in human 293 cells, which constitutively express the 5'-end 11% of the Ad5 genome. The tropism of Ad5-based vectors in swine and its ability to induce an efficient immune response against heterologous antigens expressed by foreign genes inserted in these vectors has been determined. Ad5 vectors replicate and express heterologous antigens in porcine lungs and mediastinal and mesenteric lymph nodes. Significant levels of heterologous antigen expression were also demonstrated in the small intestine (jejunum and ileum), but Ad5 replication in this organ was very poor, suggesting that Ad vectors undergo an abortive replication in the porcine small intestine. The tissues infected by Ad5 were dependent on the inoculation route. The oronasal route appeared to be best for inoculation of bronchus-associated lymphoid tissue infection, while the intraperitoneal route was best for gut-associated lymphoid tissue infection. Epithelial cells of bronchioles, macrophages, type II pneumocytes, and follicular dendritic cells were identified as targets for Ad5, while epithelial cells of the intestine were not infected by Ad5. Viruses with a deletion from 79.5 to 84.8 map units in the E3 region, with or without heterologous inserted genes, replicated to lower levels in porcine tissues than did wild-type Ad5. It was also shown that an Ad5 recombinant expressing the four antigenic sites (A, B, C, and D) of transmissible gastroenteritis coronavirus (TGEV) spike protein induced in swine immune responses which neutralized TGEV infectivity. In addition, porcine serum from Ad-TGEV-immune animals provide passive protection when mixed with fully virulent TGEV and orally administered to highly susceptible newborn piglets. These results taken together indicate that swine may be a good animal model for human Ad5 lung infection to aid in the evaluation of candidate adenovirus vaccines and that Ad5 may be suitable as a recombinant viral vaccine or for other applications in swine.

Adenoviruses, Human↗
PubMed1996

Adenoviral gene transfer of macrophage inflammatory protein-2 in rat lung.

Replication-defective adenoviral vectors are capable of localized transfer and expression of incorporated gene product in lung tissue. We have constructed an adenoviral vector that expresses rat macrophage inflammatory protein (MIP)-2, a C-X-C chemokine specifically chemotactic for neutrophils, Supernatants from 293 cells, infected with the adenoviral MIP-2 (ADMIP-2) construct, showed potent chemotactic activity and the ability of the ADMIP-2 vector to transcribe and make functional protein was confirmed. In vivo analysis of bronchoalveolar lavage fluid from rats after intratracheal instillation of ADMIP-2 (10(9) plaque-forming units) showed a 10-fold increase in the absolute number of neutrophils in bronchoalveolar lavage fluid as opposed to rats treated with an equal titer of an E1-disabled control virus expressing firefly luciferase (ADCA-18). Neutrophils constituted 65% of total BAL cells with alveolar macrophages being the other major cell type recovered. Rat MIP-2 protein was increased (nanograms per milliliter) in bronchoalveolar lavage fluid over a period of 7 days in ADMIP-2-treated animals. MIP-2 mRNA was demonstrated by Northern blot analysis in lung tissue, and histological analysis confirmed the presence of massive localized tissue neutrophilia. Evidence of chronic tissue injury and repair (ie, fibrosis) was not detected up to 2 weeks after the neutrophil infiltrate had resolved, subsequent to decreased chemokine presence. Adenoviral gene transfer proved an effective tool for the assessment of lung tissue expression of this chemokine in vivo and is useful in developing rodent models of tissue neutrophilia.

Adenoviridae↗
PubMed1995

Induction of antibodies protecting against transmissible gastroenteritis coronavirus (TGEV) by recombinant adenovirus expressing TGEV spike protein.

Ten recombinant adenoviruses expressing either fragments of 1135, 1587, or 3329 nt or the full-length spike gene of transmissible gastroenteritis coronavirus (TGEV) have been constructed. These recombinants produce S polypeptides with apparent molecular masses of 68, 86, 135, and 200 kDa, respectively. Expression of the recombinant antigen driven by Ad5 promoters was inhibited by the insertion of an exogenous SV-40 promoter. Most of the recombinant antigens remain intracytoplasmic in infected cells. All the recombinant-directed expression products contain functional antigenic sites C and B (Gebauer et al., 1991, Virology 183, 225-238). The recombinant antigen of 135 kDa and that of 200 kDa, which represents the whole spike protein, also contain antigenic sites D and A, which have previously been shown to be the major inducers of TGEV-neutralizing antibodies. Interestingly, here we show that recombinant S protein fragments expressing only sites C and B also induced TGEV-neutralizing antibodies. The chimeric Ad5-TGEV recombinants elicited lactogenic immunity in hamsters, including the production of TGEV-neutralizing antibodies. The antisera induced in swine by the Ad5 recombinants expressing the amino-terminal 26% of the spike protein (containing sites C and B) or the full-length spike protein, when mixed with a lethal dose of virus prior to administration to susceptible piglets, delayed or completely prevented the induction of symptoms of disease, respectively.

Adenoviruses, Human↗
PubMed1995

Adenovirus vectors for cytokine gene expression.

Recombinant Adenovirus type 5 constructs containing IL-6 cDNA can be used to infect cells in vitro and obtain a high level of IL-6 expression and secretion into culture media. Furthermore, Ad5-IL-6 viruses can also be used to infect Balb/c mice or Sprague-Dawley rats and obtain a high level of IL-6 expression that is sustained over a period of 3-5 days. Intratracheal infection was accompanied by dramatic increases in virus-encoded IL-6 mRNA levels in rat lung tissue, raised levels of IL-6 detected in bronchoalveolar lavage fluids and in serum, and IL-6-dependent sequelae such as liver acute phase responses. This occurs in a tissue-specific manner, depending on routes of infection by the virus. Rat lungs showed a prominent expansion (10 fold in numbers) of all classes of lymphocytes, including B cells, T helper cells (CD4+) and CTL (CD8+) at day 7 after infection which resolved significantly by day 12. Thus the associated biological effects of viral vector mediated IL-6 over-expression was also transient in nature. Other tissues can be infected with Ad5 and thus can also be induced to express selected genes in a transient fashion. We are currently examining the potential for Ad recombinant cytokine vectors in therapy for cancer and for bone marrow reconstitution after transplantation. Thus the use of recombinant Ad5 vectors may have a broad application in the study of cytokine function and possibly in future therapy as a transient gene transfer approach.

Adenoviruses, Human↗