Biomedical subjects

J T Evans

Publications and source records attributed to J T Evans.

At least 19 recordsLinked to original sources

PubMed2002

Purification of native alpha-enolase from Streptococcus pneumoniae that binds plasminogen and is immunogenic.

Many pathogenic bacteria express plasminogen receptors on their surface, which may play a role in the dissemination of organisms by binding plasminogen that, when converted to plasmin, can digest extracellular matrix proteins. A 45-kDa protein was purified from Streptococcus pneumoniae and confirmed as an alpha-enolase by its ability to catalyse the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate and by N-terminal sequencing. The activity of alpha-enolase was found in the cytoplasm and in whole cells. Activity was also demonstrated in cell wall fractions, which confirmed that alpha-enolase is a cytoplasmic antigen also expressed on the surface of S. pneumoniae. The plasminogen-binding activity of alpha-enolase was examined by Western blot, which showed that purified alpha-enolase was able to bind human plasminogen. Immunoblots of the purified 45-kDa alpha-enolase with 22 sera from patients with pneumococcal disease showed binding in 15 cases, indicating that pneumococcal enolase is immunogenic.

Amino Acid Sequence↗

PubMed2001

Pre-clinical evaluation of an in vitro selection protocol for the enrichment of transduced CD34+ cell-derived human dendritic cells.

The efficient genetic modification of CD34+ cell-derived dendritic cells (DC) will provide a significant advancement towards the development of immunotherapy protocols for cancer, autoimmune disorders and infectious diseases. Recent reports have described the transduction of CD34+ cells via retrovirus- and lentivirus-based gene transfer vectors and subsequent differentiation into functional DC. Since there is significant apprehension regarding the clinical uses of HIV-based vectors, in this report, we compare a murine leukemia virus (MLV)- and a human immunodeficiency virus (HIV)-based bicistronic vector for gene transfer into human CD34+ cells and subsequent differentiation into mature DC. Each vector expressed both EGFP and the dominant selectable marker DHFR(L22Y) allowing for the enrichment of marked cells in the presence of the antifolate drug trimetrexate (TMTX). Both MLV-based and HIV-based vectors efficiently transduced cytokine mobilized human peripheral blood CD34+ cells. However, in vitro expansion and differentiation in the presence of GM-CSF, TNF-alpha, Flt-3L, SCF and IL-4 resulted in a reduction in the percentage of DC expressing the transgene. Selection with TMTX during differentiation increased the percentage of marked DC, resulting in up to 79% (MLV vector) and up to 94% (lentivirus-vector) transduced cells expressing EGFP without loss of DC phenotype. Thus, MLV-based vectors and in vitro selection of transduced human DC show great promise for immunotherapy protocols.

Adult↗

PubMed2000

Differentiation and expansion of lentivirus vector-marked dendritic cells derived from human CD34(+) cells.

The in vitro genetic manipulation of dendritic cells (DCs) for the expression of foreign proteins or peptides will assist in the development of immunotherapeutic approaches to treat cancer, immunological disorders, and/or infectious diseases. Reports have shown the expansion and differentiation of CD34(+) progenitor cells into mature DCs. In this article we describe the differentiation and expansion of lentivirus vector-marked DCs from umbilical cord blood, bone marrow, and cytokine-mobilized peripheral blood CD34(+) cells in the presence of GM-CSF, TNF-alpha, SCF, Flt-3, and IL-4. Lentivirus-marked DCs expressed high levels of enhanced green fluorescent protein and the characteristic DC surface markers CD1a, CD83, HLA-DR, and CD80. Transduced DCs activated allogeneic CD3(+) T cells as efficiently as control (nontransduced) DCs in mixed lymphocyte reactions. These results demonstrate the potential utility of lentivirus-transduced DCs in future immunotherapy protocols.

Antigens, CD↗

PubMed2000

Lentivirus vector mobilization and spread by human immunodeficiency virus.

The rapid advancement of lentivirus-based gene transfer systems and their demonstrated utility in a variety of in vitro and in vivo settings has heightened the need for assays to evaluate the safety of these vectors prior to human clinical trials. Two major concerns relating to the use of lentivirus-based vectors in a clinical setting are the presence of contaminating replication-competent retroviruses in vector preparations and the efficiency of vector mobilization and spread by wild-type helper virus (rescue). This article describes an in vitro system to study the rescue of lentivirus-based vectors by wild-type HIV. We show that lentivirus-based vectors can be readily rescued from T cell lines and to a lesser extent from primary human lymphocytes by wildtype HIV, resulting in the spread of mobilized vector particles to previously untransduced cells. Furthermore, we show that vector mobilization can be prevented by antiretroviral drugs such as AZT. In contrast to recently published reports by Bukovsky et al. and An et al., the lentivirus vectors used in these studies had little or no effect on the replication and spread of HIV in transduced cells [Bukovsky et al. (1999). J. Virol. 73, 7087-7092; An et al. (1999). J. Virol. 73, 7671-7677]. Whereas vector spread is a significant concern for most gene therapy applications, in the context of gene therapy for HIV infection it may have beneficial effects.

Cells, Cultured↗

PubMed2000

Thymocyte differentiation from lentivirus-marked CD34(+) cells in infant and adult human thymus.

Changes in thymic function and immune system homeostasis associated with HIV infection or chemotherapy have significant effects on the ability of patients to maintain a complete T cell receptor repertoire. Therefore, the development of in vitro systems to evaluate thymic function in children and adults may aid in the understanding of thymopoiesis and the development of new therapies to improve thymic output. Here we use a lentivirus-based gene transfer system to mark CD34(+) cells with EGFP and follow their differentiation into CD4(+) and CD8(+) single positive thymocytes in human thymic organ cultures. Lentivirus-marked cells entered the thymus and were detected in both the cortex and medulla. Pretreatment of the thymus with 2-deoxyguanosine depleted resident thymocytes and significantly increased the percentage of EGFP(+) thymocytes. High frequency gene transfer into CD34(+) cells and maintained expression throughout differentiation allows for the in vitro assessment of thymic function. In thymuses ranging in age from fetal to adult we observed EGFP(+) thymocytes at all stages of development suggesting that thymuses of all ages are capable of accepting new T cell progenitors and contributing to the maintenance of T cell homeostasis.

Adult↗

PubMed2000

In vitro selection of lentivirus vector-transduced human CD34+ cells.

Human CD34(+) cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous hematopoietic disorders. However, the efficient introduction of exogenous genes into this small, quiescent population of cells continues to present a significant challenge. To circumvent the need for high initial transduction efficiency of human hematopoietic cells, we investigated a dominant selection strategy using a variant of the DHFR gene (DHFR(L22Y)). For this purpose, we constructed a lentivirus-based bicistronic vector expressing EGFP and DHFR(L22Y). Here we demonstrate efficient in vitro selection and enrichment of lentivirus vector-transduced human CD34(+) hematopoietic cells from fetal liver, umbilical cord blood, bone marrow, and peripheral blood after cytokine mobilization. Growth of transduced human CD34(+) cells in semisolid culture under selective pressure resulted in enrichment of transduced progenitor cells to 99.5% (n = 14). Selection for DHFR(L22Y)(+) cells after expansion of transduced progenitors in liquid culture resulted in a 7- to 13-fold increase in the percentage of marked cells. Thus we have shown that transduced human hematopoietic cells may be effectively enriched in vitro by dominant selection, suggesting that development of such strategies holds promise for future in vivo application.

Antigens, CD34↗

PubMed2000

Role of platelet-derived growth factor in allograft vasculopathy.

OBJECTIVE: To test the hypothesis that platelet-derived growth factor (PDGF) accelerates the formation of allograft vascular disease. SUMMARY BACKGROUND DATA: Allograft vasculopathy, characterized by myointimal hyperplasia of the coronary arteries in the transplanted heart, is the most common cause of late graft failure and death in heart transplant recipients. The cause of the process is unclear, and no treatment exists. PDGF has been implicated in alterations in vascular endothelial biology and in vascular restenosis, but the role of PDGF in allograft vasculopathy has not been explored. METHODS: An orthotopic heart transplant model was established in the rat mismatched at one class II locus using the PVGR8 and PVGR23 strains. No immunosuppressive regimen was used. Six treatment groups (PDGF-A, PDGF-A antibody, and PDGF-A receptor antibody) using 10 rats per group were examined. An untreated group of 10 rats manifesting chronic rejection as well as the native hearts were used as controls. PDGF-A at 1 ng/dL (10 rats) or 10 ng/dL (10 rats) was administered intraperitoneally to each transplant group. Similar groups were treated with PDGF-A antibody and PDGF-A receptor antibody. The animals were killed after 50 days; transplanted and native hearts were removed and coronary arteries were examined morphometrically. Smooth muscle proliferation was confirmed by immunohistochemistry. Statistical analysis was performed using multivariate analysis of variance. RESULTS: Coronary myointimal hyperplasia was seen in the chronic rejection group. The PDGF-A groups showed significant myointimal hyperplasia. Administration of PDGF-A antibody did not attenuate the process. Administration of PDGF-A receptor antibody at 1 ng/dL resulted in reduction of the hyperplasia, and 10 ng/dL significantly attenuated the process. CONCLUSIONS: This study establishes a cause-and-effect relation between PDGF-A and coronary myointimal hyperplasia in the rat transplant model. Blockade of the PDGF-A receptor clearly attenuates the process, indicating a potential mode of therapy to be explored.

Animals↗

PubMed2000

Anal duct carcinoma: case report and review of the literature.

This report details the clinical course of two patients with true anal duct carcinoma. The incidence of this malignancy is low. The tissues of origination are the glands of the anal duct. The features that differentiate this tumor from the usual rectal carcinoma are prominent ductal structures, abundant mucin production with organized mucinous pools, and infiltration into the perirectal soft tissue. The clinical management of anal duct carcinoma remains a surgical challenge. The extent of surgical resection must be radical because of the infiltrative nature of the tumor. This report describes treatment of two patients with anal duct carcinoma. The first patient was a black woman with no previous history of rectal disease. Her operative procedure was an abdominoperineal resection with posterior vaginectomy. Nine months after initial surgery a local recurrence was resected. The second patient was a white man with a previous history of hemorrhoidectomy and anal fissure. He underwent an abdominoperineal resection but had positive dermal skin margins on permanent sections despite wide perirectal soft tissue resection. A secondary resection with confirmed clear margins of the skin was performed 2 weeks postoperatively. One management aspect of anal duct carcinoma that needs emphasis is the need for wide local excision of the perirectal soft tissues.

Adenocarcinoma, Mucinous↗

PubMed1999

Human cord blood CD34+CD38- cell transduction via lentivirus-based gene transfer vectors.

The efficient transfer and sustained expression of a transgene in human hematopoietic cells with in vivo repopulating potential would provide a significant advancement in the development of protocols for the treatment of hematopoietic diseases. Recent advances in the ability to purify and culture hematopoietic cells with the CD34+CD38- phenotype and with in vivo repopulating potential from human umbilical cord blood provide a direct means of testing the ability of transfer vectors to transduce these cells. Here we demonstrate the efficient transduction and expression of enhanced green fluorescent protein (EGFP) in human umbilical cord-derived CD34+CD38- cells, without prestimulation, using a lentivirus-based gene transfer system. Transduced CD34+CD38- cells cultured in serum-free medium supplemented with SCF, Flt-3, IL-3, and IL-6 maintained their surface phenotype for 5 days and expressed readily detectable levels of the transgene. The average transduction efficiency of the CD34+CD38- cells was 59 +/- 7% as determined by flow cytometry. Erythroid and myeloid colonies derived from transduced CD34+CD38- cells were EGFP positive at a high frequency (66 +/- 9%). In contrast, a murine leukemia virus-based vector transduced the CD34+CD38- cells at a low frequency (<4%). These results demonstrate the utility of lentiviral-based gene transfer vectors in the transduction of primitive human hematopoietic CD34+CD38- cells.

Animals↗

PubMed1999

Efficient transduction of human lymphocytes and CD34+ cells via human immunodeficiency virus-based gene transfer vectors.

The development of gene transfer systems for the efficient transduction of human primary cells including lymphocytes and CD34+ cells is a significant step in the advancement of gene therapy and cell marking protocols. Efficient gene transfer systems also represent useful tools for basic research. Here we show that human primary lymphocytes and CD34+ cells can be efficiently transduced using a VSV-G pseudotyped HIV-1-based gene transfer system. The enhanced green fluorescent protein (EGFP) was chosen as the marker transgene, because it can be easily visualized and quantitated using fluorescence microscopy and flow cytometry, thus eliminating the need for selection or PCR to score transduction. Vectors produced with this system did not generate replication-competent retroviruses (RCRs) and efficiently transduced human cell lines (40-90%), PBMCs (60%), mobilized CD34+ cells (39%), and CD34+ cells from umbilical cord blood (60%) as measured by flow cytometry. Cells treated with AZT prior to infection did not express EGFP, ruling out passive protein or plasmid DNA transfer. This was further confirmed in methylcellulose cultures, where expression in myeloid and erythroid colonies was maintained for at least 3 weeks. In addition, this HIV-based vector was able to efficiently transduce freshly isolated, not-prestimulated CD34+ cells (70% EGFP positive) in serum-free medium. Under these same conditions, a Moloney murine leukemia virus-based vector failed to transduce not-prestimulated CD34+ cells. These characteristics make this gene transfer system an excellent choice for both basic science and possible gene therapy applications.

Antigens, CD34↗

PubMed1999

Sequence and analysis of the genome of a baculovirus pathogenic for Lymantria dispar.

The genome of the Lymantria dispar multinucleocapsid nucleopolyhedrovirus (LdMNPV) was sequenced and analyzed. It is composed of 161,046 bases with a G + C content of 57.5% and contains 163 putative open reading frames (ORFs) of >/=150 nucleotides. Homologs were found to 95 of the 155 genes predicted for the Autographa californica MNPV (AcMNPV) genome. More than 9% of the LdMNPV genome was occupied by 16 repeated genes related to AcMNPV ORF2. Readily identifiable homologs of several genes that have been reported to play important roles in the AcMNPV life cycle are not present; these include ie-2, a transcriptional transactivator, and gp64, a major envelope glycoprotein of the nonoccluded form of the virus. A number of genes lacking in AcMNPV but present in other baculoviruses were identified; these include two viral enhancing factor homologs, a second copy of a conotoxin-like gene, and a dutpase homolog. Although a single gene predicted to encode a large subunit of ribonucleotide reductase was found, two different copies of the small subunit gene were present. In addition, homologs of genes not previously reported for baculoviruses were identified, including a predicted protein with homology to DNA ligases and another that has motifs most closely related to a yeast mitochondrial helicase. Thirteen homologous regions (hrs) containing 54 repeated sequences that include 30-bp imperfect palindromes were identified. The imperfect palindromes are related to those from other baculoviruses.

Amino Acid Sequence↗

PubMed1999

Characterization of the interaction between the baculovirus ssDNA-binding protein (LEF-3) and putative helicase (P143).

LEF-3 and P143 are two of six proteins encoded by the Autographa californica multinucleocapsid nucleopolyhedrovirus genome which are required for DNA replication in transient replication assays. LEF-3 has the properties of an ssDNA-binding protein and P143 exhibits amino acid sequence homology to helicases. In this report, the interaction of LEF-3 and P143 was studied by yeast two-hybrid and immunoaffinity analyses. Using the yeast two-hybrid system, the interaction domain of LEF-3 (385 aa) was mapped to amino acids between positions 1 and 165. Deletion analysis of P143 failed to reveal an interaction domain, suggesting that there were either multiple interaction domains or that the deletions disrupted the secondary structures required for the interaction between LEF-3 and P143.

Base Sequence↗

PubMed1997

The sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus genome.

The nucleotide sequence of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was completed and analyzed. It is composed of 131,990 bases with a G + C content of 55% and contains 152 putative genes of 150 nucleotides or greater. Major differences in gene content and arrangement between OpMNPV and the Autographa californica MNPV were found. These include the presence in OpMNPV of three complete iap gene homologs, two conotoxin gene homologs, two protein tyrosine phosphatase homologs, and genes encoding homologs of dUTPase and the large and small subunits of ribonucleotide reductase. Seven major intergenic repeated regions were identified. Five of these are homologous regions that are related to similar regions from other baculoviruses.

Amino Acid Sequence↗

PubMed1997

Assessment of surgical operations for ductal carcinoma in situ of the breast.

The choice of surgical procedure for the treatment of ductal carcinoma in situ (DCIS) remains clinically based. A meta-analysis was used to synthesize the results of 24 published clinical studies. Partial breast tissue excision appears to be as efficacious as mastectomy for the treatment of DCIS of the breast.

Breast Neoplasms↗

PubMed1997

Characterization of the interaction between the baculovirus replication factors LEF-1 and LEF-2.

The Autographa californica multinucleocapsid nuclear polyhedrosis virus has six genes required and three genes stimulatory for transient DNA replication. We demonstrate that the products of two of these genes, LEF-1 and LEF-2, interact in both two-hybrid assays using Saccharomyces cerevisiae and glutathione S-transferase fusion affinity assays. Using yeast-two-hybrid assays, we mapped the interaction domain of LEF-2 to amino acids between positions 20 and 60. Extensive deletion analyses of LEF-1 failed to reveal a delimited interaction domain, suggesting that there may be essential secondary structural elements that are inactivated by these deletions. All clones expressing LEF-1 and LEF-2 that were unable to interact also failed to support significant levels of transient DNA replication, suggesting that this interaction is required for DNA replication. Sequence analysis of LEF-1 revealed a primase-like motif, WVVDAD. When this motif was mutated to WVVQAD, LEF-1 no longer supported transient DNA replication.

Animals↗

PubMed1997

The baculovirus single-stranded DNA binding protein, LEF-3, forms a homotrimer in solution.

LEF-3 is one of six proteins from Autographa californica multinucleocapsid polyhedrosis virus required for transient DNA replication and has the properties of a single-stranded DNA binding protein. In this report we demonstrate that LEF-3 interacts with itself in both yeast two-hybrid assays and glutathione S-transferase fusion affinity assays. LEF-3 deletion clones which were unable to interact with full-length LEF-3 also failed to support transient DNA replication, suggesting that this interaction is required for the proper function of LEF-3. LEF-3 was purified to homogeneity and characterized by analytical ultracentrifugation and native polyacrylamide gel electrophoresis. These studies revealed that LEF-3 was present as a 132-kDa complex, indicating that its native conformation is that of a homotrimer. This result was confirmed by cross-linking with glutaraldehyde followed by matrix-assisted laser desorption/ionization mass spectrometry.

DNA-Binding Proteins↗

PubMed1996

The relationship between nonenforcement of state pornography laws and rates of sex crime arrest.

The nonoperation of antipornography statutes in four states (Maine, North Carolina, Pennsylvania, and Washington) for varying periods between 1973 and 1986 provided an opportunity to examine the impact of such statutes and pornography availability on sex crimes because nonenforcement is associated with an increase in the availability of sexually explicit materials. Arrests for property offenses and for rape, prostitution, and other sex offenses during the period before the suspension of the laws, when compared with the period during suspension, reflected no significant changes. Findings are consistent with other foreign and American studies that have failed to find a link between exposure to sexually explicit media materials and rates of reports of rape and other sex offenses.

Crime↗

PubMed1996

A clinical trial of wound closure by constant tension approximation.

BACKGROUND: Wounds that heal slowly may pass through a subacute phase and proceed to reach a chronic situation. The latter may heal slowly, if at all. These wounds often heal by secondary intention with a single layer of epithelium that has a tendency to break down. Methods that expedite healing by full-thickness skin usually involve operative procedures. A nonoperative method of achieving coverage of tardy wounds with full-thickness skin, partial or complete depending on the wound, would be valuable. PATIENTS AND METHODS: A device has been designed that approximates the wound margins by applying constant low-grade tension over a period of days or weeks. One or more devices have been applied to the wounds of 25 patients to date. RESULTS: Healing has been obtained in those patients (20) where the devices were used optimally. In 5 patients, optimal use was not possible, mostly for socioeconomic reasons; the latter included noncompliance, insurance problems, economic difficulties, and personal considerations. In these 5 cases, healing was obtained in the 3 that were available for follow-up, full-thickness skin coverage being estimated by grid photography to be in the 80% to 90% range. One patient was lost to follow-up after the wound was 95% covered by full-thickness skin. One patient whose wound reduced in size declined further treatment after 12 days of treatment. CONCLUSIONS: It appears that when constant-tension, low-grade force is applied to subacute and chronic wounds, healing is accelerated and, depending on the wound, a considerable number of ulcers can be closed by full-thickness skin.

Adolescent↗