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Biomedical subjects

C A Mullen

Publications and source records attributed to C A Mullen.

At least 37 records · Page 2Linked to original sources
PubMed1999

Tacrolimus for prevention of graft-versus-host disease after mismatched unrelated donor cord blood transplantation.

Ten children with hematologic malignancies or a storage disease underwent transplantation using cord blood cells from an unrelated donor mismatched for 1 (n = 7) or 2 (n = 3) HLA antigens. The median total nucleated cell dose was 4.0 (range, 2.2-7.1) x 10(7)/kg. GVHD prophylaxis consisted of tacrolimus dose-adjusted to maintain a whole blood level of 5-15 ng/ml with or without methotrexate 5 mg/m2 i.v. on days 1, 3, 6 and 11. Corticosteroids were not administered prophylactically. Median follow-up is 12 months (range, 5-28 months). One patient had autologous recovery and subsequently relapsed 153 days post transplant. For the remainder of the patients, the median time to an ANC >0.5 x 10(9)/l was 21 days (range, 19-38 days), and the median time to platelets >20 x 10(9)/l was 39 days (range, 21-97 days). The actuarial risk of grade 2 GVHD was 77% (95% CI, 49-100%), and no patient had grades 3-4 GVHD. Two patients developed chronic GVHD. The survival rate is 90% (95% CI, 81-100%). The combination of tacrolimus and minidose methotrexate is active for the prevention of severe but not moderate acute GVHD after mismatched unrelated donor cord blood transplantation.

Antimetabolites, Antineoplastic↗
PubMed1998

Ganciclovir chemoablation of herpes thymidine kinase suicide gene-modified tumors produces tumor necrosis and induces systemic immune responses.

The goal of this work was to identify potential host immune responses to thymidine kinase (TK) suicide gene-modified tumors undergoing chemoablation induced by the prodrug ganciclovir (GCV). The aims were to measure the efficacy and specificity of immunity induced against unmodified tumor, to identify qualitative or quantitative changes in the host response to TK+ tumors undergoing chemoablation that may contribute to the induction of antitumor immunity, and to compare critically the induction of immunity by chemoablation of TK-modified tumors with that of other methods of immunization in this tumor model and in response to other well-defined model antigens. Animals treated with TK+ tumors and GCV developed specific resistance to rechallenge with unmodified tumor. GCV induced significant tumor necrosis, which was associated with a pronounced host cell infiltrate composed of polymorphonuclear cells, both CD4+ and CD8+ T lymphocytes, and increased intratumoral IL-12. Cyclophosphamide-treated mice exhibited no such host response despite the induction of tumor necrosis. CTL responses to defined antigens in TK+ cells were greater in animals treated with prodrug than were those in animals not treated with prodrug but harboring live TK+ cells. Similar degrees of immunity were produced by immunization with irradiated cells.

Animals↗
PubMed1998

A pH-dependent stabilization of an active site loop observed from low and high pH crystal structures of mutant monomeric glycinamide ribonucleotide transformylase at 1.8 to 1.9 A.

A mutation in the dimer interface of Escherichia coli glycinamide ribonucleotide transformylase (GarTfase) disrupts the observed pH-dependent association of the wild-type enzyme, but has no observable effect on the enzyme activity. Here, we assess whether a pH effect on the enzyme's conformation is sufficient by itself to explain the pH-dependence of the GarTfase reaction. A pH-dependent conformational change is observed between two high-resolution crystal structures of the Glu70Ala mutant GarTfase at pH 3.5 (1.8 A) and 7.5 (1.9 A). Residues 110 to 131 in GarTfase undergo a transformation from a disordered loop at pH 3.5, where the enzyme is inactive, to an ordered loop-helix structure at pH 7.5, where the enzyme is active. The ordering of this flexible loop-helix has a direct effect on catalytic residues in the active site, binding of the folate cofactor and shielding of the active site from solvent. A main-chain carbonyl oxygen atom from Tyr115 in the ordered loop forms a hydrogen bond with His108, and thereby provides electronic and structural stabilization of this key active site residue. Kinetic data indicate that the pKa of His108 is in fact raised to 9. 2. The loop movement can be correlated with elevation of the His pKa, but with further stabilization, probably from Asp144, after the binding of folate cofactor. Leu118, also in the loop, becomes positioned near the p-amino benzoic acid binding site, providing additional hydrophobic interactions with the cofactor 10-formyl tetrahydrofolate. Thus, the pH-dependence of the enzyme activity appears to arise from local active site rearrangements and not from differences due to monomer-dimer association.

Binding Sites↗
PubMed1998

A single mutation disrupts the pH-dependent dimerization of glycinamide ribonucleotide transformylase.

Monomeric GART reversibly associates into a dimeric form as a function of decreasing solution pH. The transition is consistent with a three-proton transfer reaction with an apparent pKa near 7. We now report that a single mutation, which replaces a glutamic acid at position 70 in the dimer interface with alanine (E70A), disrupts the pH-dependent dimerization of GART based on dynamic light scattering and gel filtration studies. A comparison of data obtained from UV-absorbance difference spectroscopy for both the wild-type and mutant forms of GART indicates that a tyrosine residue(s) undergoes a change in solvent exposure over the pH range 6.55 to 8.19. A conformational change in tertiary structure that accompanies dimerization accounts for 60% of the observed optical difference, while the remaining 40% can be attributed to a pH-dependent process unrelated to dimerization. In addition, fluorescence studies of the mutant protein indicate that a pH-dependent change in tryptophan fluorescence exhibited by the wild-type protein is unrelated to quaternary structural changes and is likely a result of simple fluorescence quenching by nearby protonated histidine side-chains. Taken together, our results indicate that a single amino acid change at the dimer interface is sufficient to interrupt the highly specific, pH-dependent assembly reaction of GART, although pH-dependent conformational changes present in the wild-type protein also occur in E70A GART. This work is a first application of structure-based site-directed mutagenesis to the analysis of this pH-dependent assembly reaction.

Amino Acid Substitution↗
PubMed1998

Peripheral expansion of pre-existing mature T cells is an important means of CD4+ T-cell regeneration HIV-infected adults.

The CD4+ T-cell pool in HIV-infected patients is in a constant state of flux as CD4+ T cells are infected and destroyed by HIV and new cells take their place. To study T-cell survival, we adoptively transferred peripheral blood lymphocytes transduced with the neomycin phosphotransferase gene between syngeneic twin pairs discordant for HIV infection. A stable fraction of marked CD4+ T cells persisted in the circulation for four to eighteen weeks after transfer in all patients. After this time there was a precipitous decline in marked cells in three of the patients. At approximately six months, marked cells were in lymphoid tissues in proportions comparable to those found in peripheral blood. In two patients, the proportion of total signal for the transgene (found by PCR analysis) in the CD4/CD45RA+ T-cell population relative to the CD4/CD45RO+ population increased in the weeks after cell infusion. These findings indicate that genetically-marked CD4+ T cells persist in vivo for weeks to months and that the CD4+ T-cell pool in adults is maintained mostly by the division of mature T cells rather than by differentiation of prethymic stem cells. Thus, after elements of the T-cell repertoire are lost through HIV infection, they may be difficult to replace.

Adult↗
PubMed1998

Allogeneic peripheral blood stem cell transplantation for active hemophagocytic lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a lethal disorder of immune dysregulation. Successful allogeneic BMT is the only therapy that has produced long-term disease-free survival. However, for patients with active HLH the outcome of BMT remains poor when compared with those transplanted in remission. We describe a patient with active, recurrent HLH treated with high-dose chemotherapy and related allogeneic peripheral blood stem cell (PBSC) transplantation. Hematologic recovery was rapid and the post-transplant course was uncomplicated. The patient is alive and well 2 1/2 years after transplant. Allogeneic PBSC transplantation may deserve further evaluation as an alternative source of stem cells for transplantation in patients with active HLH.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1998

Review: analogies between trophoblastic and malignant cells.

PROBLEM: The question of how trophoblastic and malignant cells evade immunologic recognition and rejection by their host was studied. METHOD OF STUDY: A literature review was conducted. RESULTS: Trophoblastic and malignant cells share a number of similarities. These include a lack of major histocompatibility complex antigen expression, resistance to lysis by natural killer cells, T-helper cell-2 (TH2)-biased response, prostaglandin E production, and response to transforming growth factor beta. In addition, the analogies between trophoblastic and malignant cells extend into immunotherapy in which anti-idiotype therapy has a viable role in the prevention of pregnancy loss and the treatment of cancer. CONCLUSIONS: Trophoblastic and malignant cells use a number of similar mechanisms to resist rejection by their host. By using similar strategies these cells are able to successfully co-exist in an immunologically hostile environment.

Female↗
PubMed1997

Autologous lymphoblastoid cell lines stably transfected with Plasmodium falciparum circumsporozoite protein as targets in cytotoxic T-lymphocyte assays.

To produce cell lines that can be used as a continuous source of antigen presenting cells for stimulating T-cell lines and clones and as targets in cytotoxic T-lymphocyte (CTL) assays, we used a retroviral vector with a simian virus (SV40) early promotor to transfer a Plasmodium falciparum circumporozoite (PfCSP) gene into human EBV transformed B-lymphoblastoid cell lines (B-LCL). We herein report successful, stable transfection and cell surface expression of this gene, as confirmed by PCR, Western blot analysis and immunoelectron microscopy. One of three successfully transfected autologous cell lines expressed PfCSP on the cell surface and was lysed by CD8+ T-cell dependent CTL from a donor volunteer who had been immunized with irradiated P. falciparum sporozoites. Such cell lines should provide excellent tools for characterizing human CD8+ T-cell responses against Plasmodium sp. proteins.

Animals↗
PubMed1997

Mechanism of 'bystander effect' killing in the herpes simplex thymidine kinase gene therapy model of cancer treatment.

'Bystander' killing of adjacent wild-type tumor cells was seen when tumors transduced with the herpes thymidine kinase gene were treated with the antiviral agent ganciclovir (GCV). Some tumors were 'bystander-sensitive' while others were 'bystander-resistant'. Mixtures of different 'sensitive' tumor lines showed cross-transfer of bystander killing, while in mixtures of 'resistant' with 'sensitive' tumors, the resistant phenotype was predominant. Using 3H-GCV with 'sensitive' mixtures, phosphorylated 3H-GCV was found in both herpes thymidine kinase transduced and unmodified cells, while 'resistant' cell combinations showed little or no transfer of phosphorylated GCV between cells. The capacity of intracellularly produced nucleotide toxin to spread from cell to cell within a tumor mass effectively amplifies the apparent efficiency of gene transfer in the tumor and makes feasible the use of this system for therapy of localized cancer.

Adenocarcinoma↗
PubMed1996

Glycinamide ribonucleotide transformylase undergoes pH-dependent dimerization.

Glycinamide ribonucleotide transformylase (GART) exhibits closely packed dimers in all crystal forms (pH 6.75), but was demonstrated to be monomeric in solution under conditions of optimal catalytic efficiency (pH 7.5). We undertook a study of the pH-dependent behavior of GART in solution to determine whether side-chain ionization is responsible for the observed difference in association state. In the pH range 6.8 to 7.5, dimeric GART reversibly dissociates into a monomeric form as demonstrated by dynamic light scattering. The data give a best fit to a cooperative three-proton transfer mechanism: [formula: see text] A comparison of normalized data obtained from difference UV-absorption spectroscopy with the dynamic light scattering data indicates that two or more tyrosine residues per monomer undergo a local conformational change concomitant with dimerization. Fluorescence studies show that the environment of one or both of the tryptophan residues distal to the dimer interface are also perturbed by dimerization. Fitting of the normalized titration curves yields an apparent pKa = 7.16(+/-0.02) and a subnanomolar KD for the transition. Examination of the dimer interface in the crystal structure indicates that there are two histidine residues, H54 and H73, that are likely responsible for the pH-dependent dimerization. There are also two tyrosine residues, Y67 and Y78, which are adjacent to the interface and which may be exposed during dimerization. Our study indicates that under physiological pH conditions, GART exists as a mixture of monomer and dimer in solution. Taken together, the fact that the monomer-dimer transition displays a sharp pH dependence, and the fact that the enzyme activity is maximal under conditions where it is fully monomeric, suggest that enzyme activity may be modulated by subtle pH changes in the cell.

Acyltransferases↗
PubMed1996

Molecular analysis of T lymphocyte-directed gene therapy for adenosine deaminase deficiency: long-term expression in vivo of genes introduced with a retroviral vector.

Peripheral blood lymphocytes from a patient with adenosine deaminase (ADA) deficiency were transduced in vitro with a replication-defective retroviral vector containing a human ADA-cDNA. Eighteen months after the last of a series of infusions of autologous retroviral vector-treated cells, vector sequences were detectable in DNA isolated from peripheral blood mononuclear cells (PBMCs), with an average copy number approaching one per cell. Increased ADA enzyme activity reaching approximately one-quarter normal levels was found in this population of cells. Other evidence of long-term retroviral vector expression in vivo included neomycin phosphotransferase (NPT) activity and demonstration of persistent vector mRNA by reverse transcriptase polymerase chain reaction (RT-PCR). No evidence of spontaneous reversion of either mutant endogenous ADA allele was found.

Adenosine Deaminase↗
PubMed1996

Treatment of microscopic pulmonary metastases with recombinant autologous tumor vaccine expressing interleukin 6 and Escherichia coli cytosine deaminase suicide genes.

Poorly immunogenic tumor cells genetically transduced to simultaneously express the cytokine interleukin 6 (IL-6) and the bacterial metabolic suicide gene cytosine deaminase (205-IL6-CD) become highly immunogenic. They are rejected by normal mice without 5-fluorocytosine prodrug treatment. Mice with preexisting wild-type pulmonary micrometastases exhibit prolonged survival and an increased rate of cure when treated with live 205-IL6-CD cells as a therapeutic vaccine. Treatment with these autologous tumor cells producing both the cytokine and the bacterial protein was more effective than treatment with exogenous IL-6 and/or irradiated wild-type tumor cells. Irradiation of the 205-IL6-CD cells significantly reduced their therapeutic efficacy. Therapeutic vaccination with 205-IL6-CD was more effective in animals with wild-type 205 tumor than in animals bearing an unrelated syngeneic tumor. Vaccine efficacy was significantly reduced in animals pretreated with high-dose cyclophosphamide. The results indicate that genetically engineered autologous tumor vaccines may be capable of inducing significant antitumor immunity in hosts of preexisting micrometastatic disease.

Adenocarcinoma↗
PubMed1996

Strategies for success: preparing for the certification examination.

Preparing for one of the certification examinations in perianesthesia nursing can be as challenging as taking them. For many, a certification examination will be the first test taken since graduation from a nursing education program. Identifying and practicing successful testing behaviors will assist the candidate in organizing an approach to learning and reviewing for examination. Developing a realistic time frame for a study strategy, along with objectives and completed goals, is imperative. Test anxiety often is a major obstacle to effective test performance. Strategies to deal with anxiety can be learned and used to successfully prepare for and take the examination. A confident approach to the certification examination is the candidate's greatest asset.

Certification↗
PubMed1996

Radiosensitization by 5-fluorocytosine of human colorectal carcinoma cells in culture transduced with cytosine deaminase gene.

Recently, use of the suicide gene, cytosine deaminase (CD), has shown a selective antitumor activity of 5-fluorocytosine (5-FC) on human colorectal carcinoma cells grown in vitro and in vivo. We hypothesized that the radiosensitivity of human colorectal carcinoma cells transduced with a retroviral vector encoding the bacterial CD gene would be selectively enhanced by the nontoxic prodrug 5-FC. The radiobiological rationale of using suicide gene therapy is based on the fact that a toxic metabolite of 5-FC, 5-fluorouracil, is a well-known radiation enhancer for the treatment of gastrointestinal and other tumors. 5-FC was found to enhance selectively the radiation cytotoxicity of human colorectal carcinoma cells expressing the CD gene. Colorectal carcinoma cells transduced with the CD gene (WiDr-CD) were highly sensitive to radiation compared with parental cells (WiDr) when exposed to 20 microgram/ml 5-FC for 72 h prior to irradiation. The sensitization enhancement ratio was 2.38. This magnitude of radiation enhancement is comparable to that obtained with 5-fluorouracil. These results suggest that the addition of radiation would substantially improve the therapeutic potential of CD gene therapy for the treatment of locally advanced colorectal carcinomas.

Cell Survival↗
PubMed1995

T lymphocyte-directed gene therapy for ADA- SCID: initial trial results after 4 years.

In 1990, a clinical trial was started using retroviral-mediated transfer of the adenosine deaminase (ADA) gene into the T cells of two children with severe combined immunodeficiency (ADA- SCID). The number of blood T cells normalized as did many cellular and humoral immune responses. Gene treatment ended after 2 years, but integrated vector and ADA gene expression in T cells persisted. Although many components remain to be perfected, it is concluded here that gene therapy can be a safe and effective addition to treatment for some patients with this severe immunodeficiency disease.

Adenosine Deaminase↗
PubMed1995

5-Fluorocytosine-induced eradication of murine adenocarcinomas engineered to express the cytosine deaminase suicide gene requires host immune competence and leaves an efficient memory.

The nonmammalian cytosine deaminase (CD) enzyme converts the nontoxic prodrug 5-fluorocytosine (5-FC) to the toxic metabolite 5-fluorouracil. Parental cells of a mammary adenocarcinoma (TSA-pc) of BALB/c mice were transfected with the CD gene (TSA-CD), and the ability of 5-FC to hamper their growth was evaluated. A quantity amounting to 0.5 mg of 5-FC/0.3 ml of medium inhibits the proliferation of TSA-CD cells, but not that of TSA-pc, nor that of TSA-pc transfected with neomycin-resistance gene only (TSA-neo). In BALB/c mice, 800 mg 5-FC/kg of body weight injected daily i.p. for 30 days causes total regression of incipient (1-day-old), and established (3- and 7-day-old) TSA-CD tumors, and of 3-day-old experimental lung metastases, but does not impair TSA-pc nor TSA-neo cell growth. Because in CD8+ T lymphocyte- and granulocyte-depleted mice 5-FC no longer impairs TSA-CD growth, immune mechanisms appear to play an important role in this regression. Following, regression, all mice are resistant to subsequent s.c. or i.v. lethal challenges with TSA-pc. The induction of this immune memory is dependent on CD4+ lymphocytes, whereas its effector phase depends on both CD4+ and CD8+ lymphocytes. The memory elicited in tumor-bearing mice by the 5-FC-dependent regression of TSA-CD tumors cures a significant number of mice with 4-day-old TSA-pc metastases, but does not impair the growth of 4-day-old solid s.c. tumors. The reliability of this regression and the subsequent establishment of an efficient immune memory against poorly immunogenic TSA-pc offer the prospect that CD-transduced tumor cells and 5-FC can be used as components of a live antitumor vaccine.

Adenocarcinoma↗
PubMed1995

Retrovirus mediated gene transfer as therapy for adenosine deaminase (ADA) deficiency.

ADA deficiency manifests as a severe combined immunodeficiency with profound T-lymphocytopenia. Affected individuals have variable defects of both T- and B-lymphocyte function and greatly increased morbidity and mortality caused by frequent viral and bacterial infection. In 1990 a clinical protocol for the treatment of this disease using retrovirus mediated transfer of the ADA gene into peripheral lymphocytes was begun and in 1993 an amendment permitting gene transfer to CD34+ stem cells isolated from peripheral blood or from umbilical cord blood was approved. Five patients have been treated on this protocol and have been analyzed for the persistence of cells containing the transferred gene and for immunologic reconstitution.

Adenosine Deaminase↗