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At least 1,045 records · Page 58Linked to original sources
PubMed2005

In vivo depletion of CD4+CD25+ regulatory T cells enhances the antigen-specific primary and memory CTL response elicited by mature mRNA-electroporated dendritic cells.

We previously described mRNA electroporation as an efficient gene delivery method to introduce tumor-antigens (Ag) into murine immature dendritic cells (DC). Here, we further optimize the protocol and evaluate the capacity of mRNA-electroporated DC as a vaccine for immunotherapy. First, the early DC maturation kinetics and the effect of different lipopolysaccharide incubation periods on the phenotypic maturation profile of DC are determined. Next, we show that either immature or mature DC are equally well electroporated and express and present the transgene at a comparable level after electroporation. We point out that the mRNA electroporation results in a negative effect on the interleukin (IL)-12p70, IL-6, and tumor necrosis factor-alpha secretion after maturation. Nevertheless, mRNA-electroporated DC induce an effective cytotoxic T lymphocyte (CTL) response in vivo. Mature electroporated DC are significantly more potent in eliciting an Ag-specific CD8+ CTL response compared to their immature electroporated counterparts. In addition, a significant improvement in CTL response is obtained both in the primary and in the memory effector phases when CD4+CD25+ regulatory T cells (Treg) are depleted in vivo prior to immunization. These findings are further substantiated in tumor protection experiments and hold convincing evidence for the merit of Treg cell depletion prior to immunization with mRNA-electroporated DC.

Animals↗
PubMed1991

Clinical experience with technetium 99m teboroxime.

Technetium 99m teboroxime is a boronic acid adduct of technetium dioxime complex, a neutral lipophilic technetium-containing myocardial perfusion imaging agent with high myocardial extraction, rapid myocardial washout, and entero-hepatic excretion. Early "redistribution" occurs, and, based on preliminary animal data, appears to be due to differential washout alone since there is no reuptake of teboroxime into the heart; however, for practical purposes two injections of tracer, at stress and at rest, are used clinically. Due to the high photon flux of technetium, diagnostic images can be acquired in the narrow time window between the end of blood clearance and peak hepatic uptake. To begin an acquisition within 2 minutes of tracer injection, the patient must be moved rapidly from the treadmill to a camera that is set and ready to count. A complete set of planar images can be completed in less than 5 minutes. Optimal single photon emission computed tomography (SPECT) imaging requires a continuously counting single detector camera or a three-headed camera to complete acquisition in less than 10 minutes. The image quality is comparable to thallium 201 and the imaging time is a fraction of the time required to complete SPECT thallium scans. Both left ventricular function (ejection fraction) and planar imaging can be performed in less than 4 minutes, without moving the patient from the treadmill, using the Sim-400 (Scinticor, Milwaukee, WI), a portable high count-rate, multicrystal scintillation camera. Because teboroxime maintains high myocardial extraction at high myocardial flow, it may be uniquely suited to be used in combination with pharmacological stress, which does not require patient movement between stress and imaging. Work is in progress to further refine and optimize imaging protocols using teboroxime.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1998

The cost-effectiveness of treating women with a cervical vaginal smear diagnosis of atypical squamous cells of undetermined significance.

OBJECTIVE: The purpose was to determine the optimal treatment protocol for women with a cervical vaginal diagnosis of atypical squamous cells of undetermined significance. STUDY DESIGN: By means of decision analysis, 8 strategies were compared in terms of cost, life expectancy, cost-effectiveness, and the number of cancers and complications from treatment. Data were obtained from the medical literature and the University of Iowa Hospitals and Clinics. RESULTS: Compared with more aggressive strategies, such as those that use immediate colposcopy, strategies featuring repeated smears were less expensive and carried fewer complications but had lower life expectancies per patient and more cancers. The strategy of repeating a smear annually had a lower cost per patient than did the other strategies, ranging from $112 to $989, and had a similar discounted life expectancy to that of the strategy with the longest discounted life expectancy. CONCLUSIONS: In most clinical scenarios strategies that used repeated smears were the most cost-effective.

Adult↗
PubMed1996

The impact of quadruple immunosuppression with OKT3 on kidney transplantation in black recipients.

BACKGROUND: Black recipients of kidney transplants have been shown to have lower graft survival than other racial groups. There is ongoing controversy about the optimal immunosuppressive protocol for blacks after kidney transplantation. PATIENTS AND METHODS: Five hundred-eighty-two recipients of kidney transplants performed between 1985 and 1994 were divided into three groups: (1) nonblacks who received cyclosporine and prednisone (N = 292); (2) blacks who received "quadruple" cyclosporine based immunosuppression with OKT3 induction (N = 98); and (3) blacks who received cyclosporine and prednisone only (N = 192). Patient and graft survival and incidence of acute rejection episodes were compared among the groups. RESULTS: Blacks had lower graft survival when compared with nonblacks. However, in the subgroup of black recipients who received quadruple immunosuppression, graft survival at 1 year was higher and the incidence of acute rejection episodes was significantly decreased compared with blacks without induction. Graft survival and the incidence of acute rejection in blacks on quadruple therapy was comparable with nonblacks. CONCLUSIONS: Our data support the principle that quadruple immunosuppression should be used routinely for black recipients of kidney transplants.

Adult↗
PubMed1995

Mechanical and fatigue properties of wrist flexor muscles during repetitive contractions after cervical spinal cord injury.

OBJECTIVES: Force generation and fatigue properties of wrist flexor muscles were examined in subjects with chronic (> 1 year) cervical spinal cord injury (SCI, n = 16), and also in a control group of able-bodied (AB, n = 9) subjects. DESIGN: Using surface electrodes, wrist flexor muscles were stimulated with 126 trains of 26 stimuli at a frequency of 40Hz. The offset of each train was followed by a 1.5-second pause, for a total fatigue-test time of approximately 4.2 minutes. Isometric wrist flexion force was measured with a strain gauge. SETTING: This study was conducted at a research and rehabilitation center for spinal cord injury. MAIN OUTCOME MEASURES: Force profiles were analyzed for the maximum (peak) amplitude, the rise time, and the time constant of relaxation. RESULTS: At the outset, the average peak isometric measured in the SCI group was approximately one half that of the AB subjects. Although the relative decline in force with repeated stimulation was comparable between groups, the slowing of relaxation rate was much more pronounced in the SCI group. CONCLUSIONS: These findings are consistent with alterations in the metabolic profiles of wrist flexor muscles in the SCI group, probably reflected their altered activation pattern. When designing stimulation protocols for optimizing force and fatigue resistance in muscle left partially-paralyzed after spinal cord injury, particular care must be taken to allow adequate time for complete muscle relaxation, to avoid overdriving of the muscle and a loss of functional capacity.

Adult↗
PubMed2000

Magnetic resonance spectroscopy in schizophrenia: methodological issues and findings--part I.

Our knowledge of the biological basis of schizophrenia has significantly increased with the contribution of in vivo proton and phosphorus magnetic resonance spectroscopy (MRS), a noninvasive tool that can assess the biochemistry from a localized region in the human body. Studies thus far suggest altered membrane phospholipid metabolism at the early stage of illness and reduced N-acetylaspartate, a measure of neuronal volume/viability in chronic schizophrenia. Inconsistencies remain in the literature, in part due to the complexities in the MRS methodology. These complexities of in vivo spectroscopy make it important to understand the issues surrounding the design of spectroscopy protocols to best address hypotheses of interest. This review addresses these issues, including 1) understanding biochemistry and the physiologic significance of metabolites; 2) the influence of acquisition parameters combined with spin-spin and spin-lattice relaxation effects on the MRS signal; 3) the composition of spectral peaks and the degree of overlapping peaks, including the broader underlying peaks; 4) factors affecting the signal-to-noise ratio; 5) the various types of localization schemes; and 6) the objectives to produce accurate and reproducible quantification results. The ability to fully exploit the potentials of in vivo spectroscopy should lead to a protocol best optimized to address the hypotheses of interest.

Animals↗
PubMed2001

Quantitative study of electroporation-mediated molecular uptake and cell viability.

Electroporation's use for laboratory transfection and clinical chemotherapy is limited by an incomplete understanding of the effects of electroporation parameters on molecular uptake and cell viability. To address this need, uptake of calcein and viability of DU 145 prostate cancer cells were quantified using flow cytometry for more than 200 different combinations of experimental conditions. The experimental parameters included field strength (0.1-3.3 kV/cm), pulse length (0.05-20 ms), number of pulses (1-10), calcein concentration (10-100 microM), and cell concentration (0.6-23% by volume). These data indicate that neither electrical charge nor energy was a good predictor of electroporation's effects. Instead, both uptake and viability showed a complex dependence on field strength, pulse length, and number of pulses. The effect of cell concentration was explained quantitatively by electric field perturbations caused by neighboring cells. Uptake was shown to vary linearly with external calcein concentration. This large quantitative data set may be used to optimize electroporation protocols, test theoretical models, and guide mechanistic interpretations.

Biological Transport, Active↗
PubMed1997

Gene transfer technologies for the production of enzyme and protein reference materials.

To maintain the success of recommended methods and to allow comparison among various methods of enzyme analysis, enzyme reference materials are required, having catalytic properties as close as possible to those of the corresponding human enzymes. Though human sources are preferable, ethical reasons require the extraction and purification from animal tissues. By providing theoretically unlimited amounts of material, gene transfer technologies and mass culture can overcome the need of human or mammalian tissues. We have used these technologies to produce human gamma-glutamyltransferase (GGT) and pancreatic lipase (PL) in various types of host cells. Different strategies were tested, especially for GGT, depending on the inherent properties and requirements of the human enzyme. Expression and purification protocols were optimized, yielding good amounts of recombinant enzymes which share many physico-chemical and catalytic features with their natural counterparts. Kinetic constants and catalytic behavior were very similar, demonstrating the usefulness of these products as reference materials. We assume recombinant DNA technologies could be successfully applied to most enzymes or proteins assayed in clinical chemistry laboratories.

Animals↗
PubMed2003

Pseudomonas stutzeri soluble nitrate reductase alphabeta-subunit is a soluble enzyme with a similar electronic structure at the active site as the inner membrane-bound alphabetagamma holoenzyme.

A two-subunit (alphabeta) form of dissimilatory nitrate reductase from Pseudomonas stutzeri strain ZoBell was separated from the membrane-residing gamma-subunit by a heat solubilization step. Here we present an optimized purification protocol leading to a soluble alphabeta form with high specific activity (70 U/mg). The soluble form has the stoichiometry alpha(1)beta(1) consisting of the 130 kDa alpha-subunit and the 58 kDa beta-subunit. We did not observe any proteolytic cleavage in the course of the heat solubilization. The enzyme is competively inhibited by azide, but not by chlorate. It exhibits a K(M) value of 3.2 mM for nitrate. We compare the enzymatic and electron paramagnetic resonance (EPR) spectroscopic properties of the alphabeta form with the alphabetagamma holoenzyme which resides in the membrane and can be prepared by detergent extraction. The nearly identical EPR spectra for the Mo(V) signal of both enzyme preparations show that the active site is unaffected by the heat step. The factors influencing the binding of the alpha- and beta-subunit to the gamma-subunit are discussed.

Azides↗
PubMed2000

Basal antral follicle number and mean ovarian diameter predict cycle cancellation and ovarian responsiveness in assisted reproductive technology cycles.

OBJECTIVE: To determine the predictive value and define threshold levels for basal antral follicle number and mean ovarian diameter in patients undergoing ART cycles. DESIGN: Retrospective. SETTING: Tertiary care center. PATIENTS: Two hundred seventy-eight patients who had ovarian measurements performed on cycle day 3 before beginning treatment with gonadotropins. INTERVENTION: Pretreatment ovarian ultrasound measurements. MAIN OUTCOME MEASURE: Number of oocytes retrieved, hormone levels, and cycle outcomes. RESULTS: A direct linear correlation was observed between mean ovarian diameter and basal follicle number. Both measures demonstrated a positive linear correlation with recovered oocytes, basal E(2), and peak E(2). Both demonstrated a negative linear correlation with ampules of gonadotropins administered, days of stimulation, patient age, cycle day 3 FSH, and FSH:LH ratio. An antral follicle count of </=10 or a mean ovarian diameter of <20 mm was associated with an increased risk of cycle cancellation. CONCLUSIONS: Ovarian diameter and basal antral follicle number identify patients who may respond poorly to ART stimulation. These ovarian measures correlate well with ART screening and stimulation parameters. This knowledge allows physicians to evaluate and counsel patients immediately before an ART stimulation and to optimize stimulation protocols.

Female↗
PubMed2003

The London nail bombings: the St. Thomas' Hospital experience.

At 18:00 h on 17 April 1999 a nail bomb exploded outside a supermarket in Brixton, London. This was the first of a series of three identical nail bombs. The injuries were due to penetrating nails, most were relatively minor, with only three patients requiring general anaesthesia for removal of nails and debridement. One 6-year-old child subsequently underwent onward referral for neurosurgical treatment. At 18:40 h on 30 April 1999, the third bomb exploded (there were no casualties at our hospital from the second bomb, that in Brick Lane on 24 April 1999) in the confined environment of the Admiral Duncan public house in Soho. The injuries were much more severe than those seen from the Brixton bomb. Two persons died on the scene and 81 were injured; 27 were transferred to our hospital. Three primary lower limb amputations were performed. Within the first 24h, four patients required ITU care and two onward referral to the regional burns unit. Injuries such as those seen in these two bombings are common in war situations. In peacetime it is rare to see this spectrum of injury and hence surgeons can be unaware of optimal management protocols. Our approach in these patients, based on thorough initial debridement and delayed closure/split skin grafting is similar to that advocated by the International Committee of the Red Cross based on their war surgery experience. We emphasise debridement without any initial reconstructive procedures. We also discuss some logistic problems of major incidents.

Adolescent↗
PubMed1998

Heat-induced changes in the mechanics of a collagenous tissue: pseudoelastic behavior at 37 degrees C.

The outcome of many clinical heat therapies depends on the post-treatment structural integrity of the tissue. Unfortunately, there are few data on heat-induced changes in the mechanical properties of tissues due to temperature levels commonly achieved in laser, microwave, and radio-frequency-based modalities. Without such information, one cannot design optimal clinical protocols. Hence, we present new findings on the uniaxial stress strain behavior at 37 degrees C of a model collagenous tissue (chordae tendineae) both before and after thermal damage. This damage was induced via a variety of different thermo-mechanical loads: isothermal heatings at temperatures from 65 degrees C to 90 degrees C, durations of heating from 120 to 3600 s, and isotonic loads during heating from 0 to 0.65 MPa. Our data reveal that chordae exhibit pseudoelastic responses both before and after heating, but the extensibility, hysteresis, and compliance all increase with increased thermal damage. Fortunately, these complex heat-induced changes in behavior can be parameterized using a single measure of the prior thermal damage. This will clearly simplify the requisite constitutive formulations.

Biomechanical Phenomena↗
PubMed2003

CD1a and CD1c cell sorting yields a homogeneous population of immature human Langerhans cells.

There is increasing evidence that ex vivo generated Langerhans cells (LCs) cannot fully substitute for their physiological counterparts in normal epidermis when studying the immunobiology of this prototype of a tissue-residing immature dendritic cell (DC). Here, we present CD1-based magnetic-activated cell-sorting (MACS) protocols for the effective isolation of human epidermal LCs. CD1c selection yielded a homogeneous population of pure and viable HLA-DR(+)/CD1a(+) DCs, with the ultrastructural features, surface antigen expression and cytokine profile, characteristic of epidermis-resident immature LCs. The immature state and functional integrity were established by allogeneic mixed lymphocyte reactions showing a weak stimulatory capacity of freshly isolated cells and upregulation upon stimulation. Characterizing the cells in more detail, we could demonstrate for the first time that normal human LCs express CXCR4, CD40 ligand (CD40L), and Fas and Fas ligand (FasL). The observed constitutive transcription of TGF-beta suggests that the viability and immature state of epidermal LCs are maintained not only by the TGF-beta production from the microenvironment, but also in an autocrine or paracrine manner. LPS and IFN-omega stimulated the expression of the inflammatory cytokines TNF-alpha and IL-1beta, and there was secretion of IL-12p70 after CD40 ligation. Remarkably, the CD1-sorted LCs showed no loss of their Birbeck granules and CD1a expression upon culturing and no spontaneous phenotypic and functional maturation into potent antigen-presenting cells (APCs). We conclude that human epidermal LCs obtained by the CD1c cell-sorting protocol are optimal candidates with which to elucidate the properties and capabilities of immature cells and to develop immunotherapeutic vaccines.

Antigens, CD↗
PubMed1997

Diagnosis and management of duodenal injuries in children.

Traumatic duodenal perforations in children pose a diagnostic and therapeutic challenge. To identify specific diagnostic criteria and define an optimal therapeutic protocol, we reviewed all duodenal injuries treated at our institution in the past 10 years. There were 14 hematomas and 13 perforations. The diagnosis was confirmed by computed tomography (CT), ultrasound scan (US), upper gastrointestinal contrast studies (UGI), or at laparotomy. The clinical findings and CT findings of the two groups were compared. Children with suspected duodenal hematomas were treated expectantly, and children with duodenal perforations were treated surgically. Twenty-five associated injuries (10 pancreatic) occurred in 19 children. Children with perforations had higher injury severity scores (ISS) (25 v 9), but the two groups could not be differentiated based on presenting signs, symptoms, or laboratory findings. CT findings of retroperitoneal air or contrast were seen in 9 of 9 perforations and in 0 of 10 hematomas. CT findings of intraabdominal or retroperitoneal fluid, mesenteric enhancement, and thickened duodenal wall did not differentiate the two groups. Duodenojejunostomy was performed in one patient, and primary repair was performed in 11 children who had perforation. In five children, duodenostomy tube drainage with feeding jejunostomy or gastrojejunostomy were added. Complications occurred in three of four children in the first 5 years of the study and in two of nine children in the last 5 years. The decreased morbidity rate correlated with reduced time to definitive therapy (28 v 7.8 hours). Duodenal fistulae resulted in three of seven children treated without duodenostomy tube drainage and zero of five treated with drainage. Enteral feeds resumed faster (average, 12 v 27 days) if repair of perforation was combined with feeding jejunostomy or pyloric exclusion and gastrojejunostomy. Children with duodenal hematoma resumed eating an average of 16 days after injury. Only one child required surgery for persistent obstruction. The findings of retroperitoneal air and contrast extravasation on CT accurately distinguish duodenal perforation from hematoma. Conservative management of hematoma is safe and effective. Primary repair of perforation with duodenal drainage results in fewer postoperative complications, and gastrojejunostomy or feeding jejunostomy shorten the time to resumption of feeds.

Child↗
PubMed2002

Pharmacogenetics place in modern medical science and practice.

Pharmacogenetic evidence-based treatment strategies will have major implications for all aspects of the product pipeline, including drug discovery, high throughput target screening protocols, lead optimization, and drug formulation to produce series of medicines for a particular disease which will meet the efficacy needs of the majority of patients. The initial proof of principle experiments involves whole genome screening for DNA variants and determination of specific patterns of variants associated with adverse events of marketed products [SNP Print(sm)]. Pharmacogenetics has the potential of changing the pipeline model of drug discovery, clinical development, and mass customization marketing.

Drug Therapy↗
PubMed2000

Mechanisms of stem-/progenitor-cell mobilization: the anti-VLA-4 paradigm.

Since the introduction of mobilized peripheral blood stem cells for transplantation purposes, many studies have been performed using mobilized cells with different mobilization schemes, primarily to optimize mobilization protocols. Studies aiming at mechanisms of mobilization have been few, but have provided useful insights. However, conclusions about mobilization mechanisms were largely inferential. We have attempted to analyze the mobilization process Involving the VLA-4/VCAM-1 pathway. Our findings are summarized and an attempt is made to put our experience into a general model of mobilization.

Animals↗
PubMed2005

Molecular fragment replacement approach to protein structure determination by chemical shift and dipolar homology database mining.

A novel approach is described for determining backbone structures of proteins that is based on finding fragments in the protein data bank (PDB). For each fragment in the target protein, usually chosen to be 7-10 residues in length, PDB fragments are selected that best fit to experimentally determined one-bond heteronuclear dipolar couplings and that show agreement between chemical shifts predicted for the PDB fragment and experimental values for the target fragment. These fragments are subsequently refined by simulated annealing to improve agreement with the experimental data. If the lowest-energy refined fragments form a unique structural cluster, this structure is accepted and side chains are added on the basis of a conformational database potential. The sequential backbone assembly process extends the chain by translating an accepted fragment onto it. For several small proteins, with extensive sets of dipolar couplings measured in two alignment media, a unique final structure is obtained that agrees well with structures previously solved by conventional methods. With less dipolar input data, large, oriented fragments of each protein are obtained, but their relative positioning requires either a small set of translationally restraining nuclear Overhauser enhancements (NOEs) or a protocol that optimizes burial of hydrophobic groups and pairing of beta-strands.

Computer Simulation↗