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Biomedical subjects

M Whitmore

Publications and source records attributed to M Whitmore.

9 recordsLinked to original sources
PubMed2001

High-level production and purification of P30P2MSP1(19), an important vaccine antigen for malaria, expressed in the methylotropic yeast Pichia pastoris.

P30P2MSP1(19) is a recombinant subunit vaccine derived from merozoite surface protein 1 (MSP1) of Plasmodium falciparum, the causative agent of malaria. P30P2MSP1(19) consists of two universal T-cell epitopes fused to the most C-terminal 19-kDa portion of MSP1, and this protein has previously shown promising potential as a vaccine for malaria. However, previous attempts at producing this molecule in Saccharomyces cerevisiae resulted in the production of a truncated form of the molecule missing most of the universal T-cell epitopes. Here, we report the production of full-length P30P2MSP1(19) in Pichia pastoris. As salt precipitation is a common problem during P. pastoris high-density fermentation, we utilized an alternative low-salt, fully defined medium that did not reduce growth rates or biomass yields to avoid precipitation. A total of 500 mg/L of secreted purified protein was produced in high cell density fermentation and the protein was purified in one step utilizing nickel-chelate chromatography. P30P2MSP1(19) produced in Pichia was reactive with monoclonal antibodies that recognize only conformational epitopes on correctly folded MSP1. Rabbits immunized with this molecule generated higher and more uniform antibody titers than rabbits immunized with the protein produced in Saccharomyces. P30P2MSP1(19) produced in Pichia may prove to be a more efficacious vaccine than that produced in Saccharomyces and Pichia would provide a system for the cost-effective production of such a vaccine.

Amino Acid Sequence↗
PubMed2000

Habitability research priorities for the International Space Station and beyond.

Advanced technology and the desire to explore space have resulted in increasingly longer manned space missions. Long Duration Space Flights (LDSF) have provided a considerable amount of scientific research on the ability of humans to adapt and function in microgravity environments. In addition, studies conducted in analogous environments, such as winter-over expeditions in Antarctica, have complemented the scientific understanding of human performance in LDSF. These findings indicate long duration missions may take a toll on the individual, both physiologically and psychologically, with potential impacts on performance. Significant factors in any manned LDSF are habitability, workload and performance. They are interrelated and influence one another, and therefore necessitate an integrated research approach. An integral part of this approach will be identifying and developing tools not only for assessment of habitability, workload, and performance, but also for prediction of these factors as well. In addition, these tools will be used to identify and provide countermeasures to minimize decrements and maximize mission success. The purpose of this paper is to identify research goals and methods for the International Space Station (ISS) in order to identify critical factors and level of impact on habitability, workload, and performance, and to develop and validate countermeasures. Overall, this approach will provide the groundwork for creating an optimal environment in which to live and work onboard ISS as well as preparing for longer planetary missions.

Antarctic Regions↗
PubMed1999

LPD lipopolyplex initiates a potent cytokine response and inhibits tumor growth.

Our laboratory has recently developed a lipopolyplex consisting of DOTAP:cholesterol liposomes, protamine sulfate, and plasmid DNA (LPD) that provides improved systemic gene delivery compared with lipoplex following tail vein injection in mice. Because endothelial cells are the primary cells transfected in the lung, it was hypothesized that LPD might be an effective vector for gene therapy of pulmonary metastases. This hypothesis was examined by testing the efficacy of cytokine (IL-12) and tumor suppressor (p53) strategies for treatment of an experimental model of pulmonary metastasis in C57Bl/6 mice. Surprisingly, all LPD complexes including those containing an 'empty' plasmid provided a potent (>50% inhibition) and dose-dependent antitumor effect, compared with dextrose-treated controls. In addition, i.v. injections of LPD containing 'empty' plasmid also inhibited tumor growth in a subcutaneous model of C3 fibrosarcomma. The antitumor effect correlated well with a strong and rapid proinflammatory cytokine (TNF-alpha, IL-12 and IFN-gamma) response. Naked plasmid DNA did not elicit a cytokine response and the response required assembly of DNA into a lipoplex or the LPD lipopolyplex. Except for the heart, elevated levels of cytokine were observed in all organs (lung, liver, kidney and spleen) where LPD is known to have gene transfer activity. Methylation of immune-stimulatory CpG motifs in the plasmid component of LPD inhibited the proinflammatory cytokine response as well as the antitumor effect of LPD in both tumor systems. This suggests that i. v. administration of LPD elicits a systemic proinflammatory cytokine response that mediates the antitumor activity of the lipopolyplex. In addition, the antitumor activity was not observed in SCID mice suggesting a possible role for B or T lymphocytes in the antitumor response initiated by LPD. This represents the first demonstration that an intravenously administered cationic liposome-based nonviral vector can promote a systemic, Th1-like innate immune response. The immune adjuvant properties of LPD might prove to be suitable for delivering tumor-specific antigens in the context of DNA vaccination.

Animals↗
PubMed1999

Effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors.

Cationic lipid-mediated intravenous gene delivery shows promise in treating pulmonary diseases including lung tumor metastases, pulmonary hypertension, and acute respiratory distress syndrome. Nevertheless, clinical applications of cationic lipidic vectors via intravenous administration are limited by their transient gene expression. In addition, repeated dosing is not effective at frequent intervals. In an effort to elucidate the mechanism of gene inactivation, we report in this study that cationic lipid-protamine-DNA (LPD) complexes, but not each component alone, can induce a high level of cytokine production, including interferon-gamma and tumor necrosis factor-alpha. Furthermore, we demonstrate that LPD administration triggers apoptosis in the lung, a phenomenon that may be mediated in part by the two cytokines. Treatment of mice with antibodies against the two cytokines prolongs the duration of gene expression and also improves lung transfection on a second administration of LPD. Although the mechanism underlying LPD-induced cytokine production is unclear, methylation of the DNA significantly decreased the level of both interferon-gamma and tumor necrosis factor-alpha, suggesting that unmethylated CpG sequences in plasmid DNA play an important role. These data suggest that decreasing the CpG-mediated immune response while not affecting gene expression may be a useful therapeutic strategy to improve cationic lipid-mediated intravenous gene delivery to the lung.

Animals↗
PubMed1998

Habitability and performance issues for long duration space flights.

Advancing technology, coupled with the desire to explore space has resulted in increasingly longer manned space missions. Although the Long Duration Space Flights (LDSF) have provided a considerable amount of scientific research on human ability to function in extreme environments, findings indicate long duration missions take a toll on the individual, both physiologically and psychologically. These physiological and psychological issues manifest themselves in performance decrements; and could lead to serious errors endangering the mission, spacecraft and crew. The purpose of this paper is threefold: 1) to document existing knowledge of the effects of LDSF on performance, habitability, and workload, 2) to identify and assess potential tools designed to address these decrements, and 3) to propose an implementation plan to address these habitability, performance and workload issues.

Adaptation, Psychological↗
PubMed1995

Metallothionein protects against the cytotoxic and DNA-damaging effects of nitric oxide.

In inflammatory states, nitric oxide (.NO) may be synthesized from precursor L-arginine via inducible .NO synthase (iNOS) in large amounts for prolonged periods of time. When .NO acts as an effector molecule under these conditions, it may be toxic to cells by inhibition of iron-containing enzymes or initiation of DNA single-strand breaks. In contrast to molecular targets of .NO, considerably less is known regarding mechanisms by which cells become resistant to .NO. Metallothionein (MT), the major protein thiol induced in cells exposed to cytokines and bacterial products, is capable of forming iron-dinitrosyl thiolates in vitro. Therefore, we tested the hypothesis that overexpression of MT reduces the sensitivity of NIH 3T3 cells to the .NO donor, S-nitrosoacetylpenicillamine (SNAP), and to .NO released from cells (NIH 3T3-DFG-iNOS) after infection with a retroviral vector expressing human iNOS gene. There was a 4-fold increase in MT in cells transfected with the mouse MT-1 gene (NIH 3T3/MT) compared to cells transfected with the promoter-free inverted gene (NIH 3T3/TM). NIH 3T3/MT cells were more resistant than NIH 3T3/TM cells to the cytotoxic effects of SNAP (0.1-1.0 mM) or .NO released from NIH 3T3-DFG-iNOS cells. A brief (1 h) exposure to 10 mM SNAP caused DNA single-strand breaks that were 9-fold greater in NIH 3T3/TM compared to NIH 3T3/MT cells. Electron paramagnetic resonance spectroscopy of NIH 3T3 cells revealed a greater peak at g = 2.04 (e.g., iron-dinitrosyl complex) in NIH 3T3/MT than NIH 3T3/TM cells. These data are consistent with a role for cytoplasmic MT in interacting with .NO and reducing .NO-induced cyto- and nuclear toxicity.

3T3 Cells↗
PubMed1990

Everted glove-pouch technique: a simple method for the hygienic disposal of contaminated guide-wires, catheters and tubes.

In order to reduce the risk of nosocomial infection during endoscopic procedures, a simple, safe and economical technique for disposal of contaminated guide-wires, tubes and catheters is described. A disposable glove, everted over a coiled wire or other endoscopic accessory, creates a pouch which then contains the contaminated object to be discarded. This everted glove-pouch technique prevents dispersal of secretions into the endoscopic environment and provides a convenient means for disposal of these otherwise awkward endoscopic accessories.

Cross Infection↗