Clinical validation of the "in silico" prediction of immunogenicity of a human recombinant therapeutic protein.

Antibodies elicited by protein therapeutics can cause serious side effects in humans. We studied immunogenicity of a recombinant fusion protein (FPX) consisting of two identical, biologically active, peptides attached to human Fc fragment. EpiMatrix, an in silico epitope-mapping tool, predicted promiscuous T-cell epitope(s) within the 14-amino-acid carboxy-terminal region of the peptide portion of FPX. On administration of FPX in 76 healthy human subjects, 37% developed antibodies after a single injection. A memory T-cell response against the above carboxy-terminus of the peptide was observed in antibody-positive but not in antibody-negative subjects. Promiscuity of the predicted T-cell epitope(s) was confirmed by representation of all common HLA alleles in antibody-positive subjects. As predicted by EpiMatrix, HLA haplotype DRB1*0701/1501 was associated with the highest T-cell and antibody response. In conclusion, in silico prediction can be successfully used to identify Class II restricted T-cell epitopes within therapeutic proteins and predict immunogenicity thereof in humans.

Application of disposable plastic microfluidic device arrays with customized chemistries to multiplexed biochemical assays.

Plastic microfluidic array platforms and synergistic multiplexed assay chemistries are under development for a variety of applications, including assays of gene expression, proteomics, genotyping, DNA sequencing and fragment analysis, sample preparation and high-throughput pharmaceutical discovery. The low production costs of plastic substrates makes possible economical single-use device arrays, eliminating cleaning and sample-to-sample carryover contamination. Hundreds of microchannels and reservoirs are readily included on a single microtitre-plate-size substrate, enabling the manufacture of highly parallel fluidic array systems to increase throughput and speed.

Effects of a 10-minute back rub on cardiovascular responses in healthy subjects.

This study determined the cardiovascular responses to a 10-minute back rub. Twelve healthy, college-age males and females (mean age = 22 years) volunteered to participate as subjects. Using an ABA design, the subjects tested for 10 minutes (Control #1) on a padded plinth lying on one side. During the Treatment period, the back rub was administered, which was followed by Control #2. Oxygen consumption (VO2) was determined via the Medical Graphics CPX/D metabolic analyzer, which also estimated cardiac output (Q) using the CO2 rebreathing (equilibrium) method. A repeated measures ANOVA was performed to statistically compare the cardiovascular responses across the three periods. The back rub, when compared to Control #1, had no significant effect on VO2, but the central and peripheral components of VO2 were changed. Cardiac output was decreased as a result of the decreased stroke volume (SV), as a function of the increased peripheral vascular resistance (PVR). We also found an increase in the extraction of oxygen (a-vO2 diff) in the peripheral tissues. These results indicate that the VO2 response during the back rub was achieved by reciprocal central (SV,Q) and peripheral (a-vO2 diff) adjustments. Following the back rub, (i.e., Control #2 vs. Treatment), the decrease in VO2, VCO2, Ve, and a-vO2 diff appears to indicate that it was effective in inducing relaxation. Since HR, SV, and Q were unchanged, the VO2 response was a result of the decreased a-vO2 diff. Hence, the findings suggest certain positive implications for the health care industry.

The "bends" and neurogenic bladder dysfunction.

Decompression sickness (the "bends") is a well-known risk of scuba diving. The pathophysiology and treatment is well documented. In the urologic data, no reference to the development of a neurogenic bladder as a result of an episode of the bends was found. We present the evaluation and management of a previously asymptomatic man who developed detrusor hyperreflexia after an episode of decompression sickness. Urologists in coastal communities should be aware of the potential risk of the development of neurogenic bladder.

Recent advances in the management of the neurogenic bladder.

Proper evaluation of the neurogenic bladder remains the cornerstone for accurate management of the neurologically impaired patient. Due to the inherent progressive nature of many neurologic disorders causing bladder dysfunction and lack of targeted medical therapy, much work has been done and needs to be done to advance the management of this often-difficult patient population. This article reviews the latest advances in managing the neurogenic bladder. For ease of review, the neurogenic bladder can be divided into 2 basic categories: first, bladders that fail to empty successfully and, second, those that fail to store urine adequately. Therapy should be based on these categories because poor therapeutic results are seen when a standard treatment is prescribed for the wrong bladder condition. Given the success of other specialties (physical medicine and rehabilitation, orthopedics, and neurology) at improving and prolonging the lives of the neurologically impaired patient, the urologist has an increasing responsibility to evaluate and treat the neurogenic bladder effectively over a life span that is approaching that of the normal population.

Cardiovascular responses to a hot tub bath.

This study was conducted to determine the cardiovascular effects of 15 minutes of hot tub immersion at 39 degrees C. Five college-age subjects (4 males and 1 female) volunteered to participate in this study. Assessments were made while sitting first in a chair for 5 minutes and then in the hot tub for 15 minutes. Oxygen consumption (VO2) and cardiac output (Q) measurements were made using a Medical Graphics CPX/D metabolic analyzer. Cardiac output was determined at minute 15 using the indirect CO2 rebreathing procedure. The data were analyzed using the analysis of variance with repeated measures, which indicated that at minute 15, heart rate (HR) and Q were increased, which increased VO2. The increase in Q was due to the heart rate (HR) response and the decrease in systemic vascular resistance (SVR). Mean arterial pressure (MAP) and systolic blood pressure (SBP) were decreased while double product (DP) was increased. There were no changes in stroke volume (SV) or arteriovenous oxygen difference (a-vO2 diff). These findings indicate that the HR and Q responses are necessary to the increase in metabolism (VO2). Hot tube use within these time and temperature constraints should reduce concern over hot tub safety in college-age subjects.

Molecular cloning and functional characterization of a novel CC chemokine, stimulated T cell chemotactic protein (STCP-1) that specifically acts on activated T lymphocytes.

A novel human chemokine STCP-1 (stimulated T cell chemotactic protein) was isolated from an activated macrophage cDNA library. The chemokine has four cysteines positioned in a manner that identifies STCP-1 as a member of the CC chemokine family. The amino acid sequence shows 34% identity with RANTES. The gene consists of 3 exons and 2 introns with the position of intron/exon boundaries similar to that of RANTES. The gene is expressed as a 3.4-kilobase transcript on lymph node, thymus, and Appendix. STCP-1 induces Ca2+ mobilization in a small percentage of primary activated T lymphocytes, but on repeated stimulation the percentage of T lymphocytes that respond to STCP-1 increases. The chemokine STCP-1 does not induce Ca2+ mobilization in monocytes, dendritic cells, neutrophils, eosinophils, lipopolysaccharide-activated B lymphocytes, and freshly isolated resting T lymphocytes. Similarly, STCP-1, while acting as a mild chemoattractant for primary activated T lymphocytes, is a potent chemoattractant for chronically activated T lymphocytes but has no chemoattractant activity for monocytes, neutrophils, eosinophils, and resting T lymphocytes. As STCP-1 acts specifically on activated T lymphocytes, it may play a role in the trafficking of activated/effector T lymphocytes to inflammatory sites and other aspects of activated T lymphocyte physiology.

Pegylated megakaryocyte growth and development factor abrogates the lethal thrombocytopenia associated with carboplatin and irradiation in mice.

Megakaryocyte growth and development factor (MGDF) is a potent inducer of megakaryopoiesis in vitro and thrombopoiesis in vivo. The effects of MGDF appear to be lineage-selective, making this cytokine an ideal candidate for use in alleviating clinically relevant thrombocytopenias. This report describes a murine model of life-threatening thrombocytopenia that results from the combination treatment of carboplatin and sublethal irradiation. Mortality of this regimen is 94% and is associated with widespread internal bleeding. The daily administration of pegylated recombinant human MGDF (PEG-rMGDF) significantly reduced mortality (to < 15%) and ameliorated the depth and duration of thrombocytopenia. The severity of leucopenia and anemia was also reduced, although it was not clear whether these effects were direct. Platelets generated in response to PEG-rMGDF were morphologically indistinguishable from normal platelets. PEG-rMGDF administered in combination with murine granulocyte colony-stimulating factor completely prevented mortality and further reduced leukopenia and thrombocytopenia. These data support the concept that PEG-rMGDF may be useful to treat iatrogenic thrombocytopenias.

The effect of massage on oxygen consumption at rest.

This study determined the effect of massage on oxygen consumption at rest. Ten healthy, adult males (mean age = 28 years) volunteered to serve as subjects. During the Control Session, each subject was placed in the supine position on a massage table to remain motionless for 30 minutes. During the Treatment Session, each subject received a 30-minute sports massage of the lower extremities. Oxygen consumption was determined via the Beckman Metabolic Measurement Cart, which was upgraded to estimate cardiac output using the CO2 rebreathing (equilibrium) method. Paired t-tests were used for all tests of statistical significance. There was no significant difference in the subjects' oxygen consumption with the massage. Also, there were no significant differences in heart rate, stroke volume, cardiac output, and arteriovenous oxygen difference during the massage. These findings indicate (1) that massaging the lower extremities results in neither an increase nor a decrease in the subjects' expenditure of energy at rest and (2) that the energy cost of metabolism at rest is determined by the same central and/or peripheral adjustments.

Effects of qigong on cardiorespiratory changes: a preliminary study.

Qigong, a special form of breathing exercise, was investigated to examine its effect on cardiorespiratory changes. Ten volunteers (five males and five females) participated in a 20-minute group instructional session for 10 consecutive days before testing of its treatment effects. The testing protocol followed a C1-T-C2 design, where C1, T, and C2 represented the first, treatment, and second control period, respectively. Each period consisted of a 5-minute interval, and thus each testing session consisted of 15 minutes. The results indicated there were no statistically significant differences (p > 0.05) in heart rate or tidal volume for the three 5-minute periods. There was a significant decrease (p < 0.05) in respiratory exchange ratio between T and C2. A significant increase in ventilatory efficiency for carbon dioxide production was found between C1 and T. Statistically significant differences (p < 0.05) were found in the volume of oxygen consumed and carbon dioxide produced, frequency of breath, expired ventilation, and ventilatory efficiency for oxygen produced between the T and the two control periods. This preliminary study of Qigong demonstrates that the subjects were able to learn the technique in a short period of time. The data also suggest that, with an improvement of nearly 20% in ventilatory efficiency for oxygen uptake and carbon dioxide production, this technique may have useful therapeutic value.

Transurethral intravesical electrotherapy for neurogenic bladder dysfunction in children with myelodysplasia: a prospective, randomized clinical trial.

Myelomeningocele is the most common cause of neurogenic bladder dysfunction in children. Urinary incontinence is socially disabling for many of these children and undetected elevations in detrusor pressure can lead to serious upper tract damage. Sensory receptors in the bladder mucosa and submucosa provide afferent information to the central nervous system regulating the micturition reflex. Since 1959 Katona and several other investigators have used intravesical electrotherapy for diagnosis and treatment of the neuropathic bladder. Our objective was to conduct a randomized, sham controlled and blinded clinical study on the efficacy of transurethral intravesical electrotherapy in treating urinary incontinence in the myelodysplastic child. A total of 36 children was enrolled in the study and 31 completed the entire protocol. Of the patients completing the study 13 were randomly selected to serve as an internal sham control having the electrocatheter placed without activating the stimulator. These patients were subsequently treated with a 3-week course of electrotherapy. The remaining 18 patients completing the study were randomly selected to undergo 2, 3-week courses of intravesical bladder stimulation. Urodynamic studies were performed before and after each treatment series. Detailed daily questionnaires were submitted to each participant covering subjective improvement in urinary continence and any development of bladder sensory awareness. Analysis of the urodynamic data and questionnaires failed to reveal any statistically significant increase in bladder capacity, development of detrusor contractions, improvement in detrusor compliance or the acquisition of bladder sensation allowing timely intermittent catheterization preventing urinary incontinence.

Dissimilarities between purified human interleukin-1 and recombinant human interleukin-2 in the induction of fever, brain prostaglandin, and acute-phase protein synthesis.

The lymphokine interleukin-2 (IL-2) has been shown to enhance natural cell-mediated cytotoxicity, the generation of cytolytic T lymphocytes, and several other aspects of cellular immune function. The gene coding for human IL-2 has been cloned, and recombinant IL-2 will be available for clinical trials in patients with neoplastic, infectious, and immunodeficiency diseases. The present investigation was undertaken to determine if IL-2 was similar to interleukin-1 (IL-1) in its ability to induce fever and the acute-phase response. These studies were based on recent work with recombinant human interferon (IFN)-alpha, which is intrinsically pyrogenic and capable of producing fever by inducing the synthesis of prostaglandin E2 (PGE2). The prospect that IL-2 might exert similar physiologic effects is of critical concern since elevated temperature, PGE2, and acute-phase reactants may profoundly inhibit natural cell-mediated cytotoxicity. Our studies have shown that recombinant human IL-2 is not intrinsically pyrogenic in rabbits at doses as high as 10,000 units/kg when administered by a single intravenous injection. In contrast to IL-1, IL-2 does not stimulate cultured hypothalamus cells to synthesize PGE2, and, furthermore, IL-2 does not elevate serum C-reactive protein levels. These results predict that the administration of IL-2 to patients in doses that stimulate cellular immune function will not induce fever and other toxic side effects frequently seen in individuals receiving IFN.