Optimal analysis of composite cytokine responses during alloreactivity

The one-way mixed lymphocyte reaction (MLR) is a useful model of the graft-vs.-host (GvH) response that occurs following bone-marrow transplantation (BMT). Previous studies of the MLR have shown high levels of type-1 cytokine production, such as IL-1, IL-6, IFN-γ and TNF-, but low or undetectable levels of type-2 cytokines, such as IL-4 and IL-10. Here, through establishing optimal conditions for the examination of levels and kinetics of a more definitive panel of type-1/type-2 cytokines (IL-4, IL-5, IL-10 and IL-13, IFN-γ, TNF- and the soluble IL-4 receptor) we show that, contrary to previously published data, the human alloresponse is truly heterogeneous, resulting in abundant type-2 as well as type-1 cytokine secretion. The kinetics of cytokine levels in the MLR show surprising complexity, suggesting a well-defined regulation as the alloresponse develops over time. Furthermore, each MLR responder:stimulator combination tested produces a composite cytokine profile that is intrinsic to that particular pairing. These combination-specific cytokine responses are reproducible when tested on multiple occasions over time. These data reveal a potential clinical application for the cytokine MLR in selecting donors for BMT with the least inflammatory cytokine profile. Additional analysis of this system reveals that the bulk of cytokine measured is both allospecific and T-cell-derived, with comparatively low levels produced through an autologous mechanism. Interestingly, although most of the cytokine detected is produced by CD45RO+ ‘mature/activated’ T cells, CD45RA+ ‘naive’ T cells are responsible for transient early production of IL-4. This novel finding suggests that naive T cells themselves could regulate type-1/type-2 developmental fate through an autocrine IL-4 mechanism.     

The interface between tapasin and MHC class I: identification of amino acid residues in both proteins that influence their interaction

Prior to the binding of antigenic peptide, a complex of chaperone proteins associates with the Major Histocompatibility Complex (MHC) class I heavy chain/β₂m heterodimer. Although each dornain of the MHC class I heavy chain contains amino acid resid uses that influence chaperone binding, there are several pieces of evidence that point to an interaction between the MHC clas 1α2/α3 domains and tapasin. In egard to the site on tapasin involved in the tapasin/MHC interface, we have found that a particular region of tapasin (containing amino acid residues 334–342) is necessary for the binding of tapasin to the MHC class I heavy chain. Our results also indicate that amino acids in this region of tapasin also affect the proportion of MHC class I open forms expressed at the cell surface and MHC class I egress from the endoplasmic reticulurn. Based on these results and those obtained by other laboratories, a model for MHC class I/tapasin interaction is proposed.     

The response of NG2-expressing oligodendrocyte progenitors to demyelination in MOG-EAE and MS

Remyelination of primary demyelinated lesions is a common feature of experimental models of multiple sclerosis (MS) and is also suggested to be the normal response to demyelination during the early stages of MS itself. Many lines of evidence have shown that remyelination is preceded by the division of endogenous oligodendrocyte precursor cells (OPCs) in the lesion and its borders. It is suggested that this rapid response of OPCs to repopulate the lesion site and their subsequent differentiation into new oligodendrocytes is the key to the rapid remyelination. Antibodies to the NG2 chondroitin sulphate proteoglycan have proved exceedingly useful in following and quantitating the response of endogenous OPCs to demyelination. Here we review the literature on the response of NG2-expressing OPCs to demyelination and provide some new evidence on their response to the chronic inflammatory demyelinating environment seen in recombinant myelin oligodendrocyte glycoprotein (MOG) induced experimental allergic encephalomyelitis (EAE) in the DA rat. NG2-expressing OPCs responded to the inflammatory demyelination in this model by becoming reactive and increasing in number in a very focal manner. Evidence of NG2⁺OPCs in lesioned areas beginning to express the oligodendrocyte marker CNP was also seen. The response of OPCs appeared to occur following successive relapses but did not always lead to remyelination, with areas of chronic demyelination observed in the spinal cord. The presence of OPCs in the adult human CNS is clearly of vital importance for repair in multiple sclerosis (MS). As in rat tissue, the antibody labels an evenly distributed cell population present in both white and grey matter, distinct from HLA-DR⁺microglia. NG2⁺cells are sparsely distributed in the centre of chronic MS lesions. These cells apparently survive demyelination and exhibit a multi-processed or bipolar morphology in the very hypocellular environment of the lesion.     

Cardiac responses to pressor challenges in congenital central hypoventilation syndrome

Summary Question of the Study   Congenital central hypoventilation syndrome (CCHS) subjects exhibit diminished respiratory-related heart rate variation in addition to defining characteristics of CO₂ insensitivity and reduced ventilatory drive during sleep. Loss of cardiovascular and breathing coupling may diminish blood pressure influences on breathing; such influences may be determined by evaluating cardiorespiratory responses to different pressor challenges.
Patients and Methods   Ten children with CCHS and 10 age- and gender-matched controls were subjected to a forehead cold pressor challenge and to Valsalva maneuvers. Heart and respiratory rates and variability during 30-s baseline and 120-s challenge periods were assessed with scatterplot displays and by analysis of variance procedures.
Results   Cold pressor challenges enhanced breathing efforts and increased respiratory-related heart rate variation in controls but not in CCHS patients, while lower frequency heart rate variability increased in both controls and CCHS subjects. Heart rate variation resulting from voluntary expiratory efforts was present but slightly reduced in CCHS. Respiratory and cardiac rate trends differed in control and CCHS cases.
Conclusions   More-rapidly changing heart rate variation from spontaneous or reflexively-induced sources is diminished in CCHS but remains intact from voluntary expiratory ­efforts, as does slower variation. Loss of reflexive influences on breathing from blood pressure changes may attenuate a source of respiratory drive.     

Decellularized ureter for tissue-engineered small-caliber vascular graft

Previous attempts to create small-caliber vascular prostheses have been limited. The aim of this study was to generate tissue-engineered small-diameter vascular grafts using decellularized ureters (DUs). Canine ureters were decellularized using one of four different chemical agents [Triton-X 100 (Tx), deoxycholate (DCA), trypsin, or sodium dodecyl sulfate (SDS)] and the histology, residual DNA contents, and immunogenicity of the resulting DUs were compared. The mechanical properties of the DUs were evaluated in terms of water permeability, burst strength, tensile strength, and compliance. Cultured canine endothelial cells (ECs) and myofibroblasts were seeded onto DUs and evaluated histologically. Canine carotid arteries were replaced with the EC-seeded DUs (n = 4). As controls, nonseeded DUs (n = 5) and PTFE prostheses (n = 4) were also used to replace carotid arteries. The degree of decellularization and the maintenance of the matrix were best in the Tx-treated DUs. Tx-treated and DCA-treated DUs had lower remnant DNA contents and immunogenicity than the others. The burst strength of the DUs was more than 500 mmHg and the maximum tensile strength of the DUs was not different to that of native ureters. DU compliance was similar to that of native carotid artery. The cell seeding test resulted in monolayered ECs and multilayered alpha-smooth muscle actin-positive cells on the DUs. The animal implantation model showed that the EC-seeded DUs were patent for at least 6 months after the operation, whereas the nonseeded DUs and PTFE grafts become occluded within a week. These results suggest that tissue-engineered DUs may be a potential alternative conduit for bypass surgery.     

Effects of fluid flow on elution of hydrophilic modifier from dialysis membrane surfaces

When uremic blood flows through dialyzers during hemodialysis, dialysis membrane surfaces are exposed to shear stress and internal filtration, which may affect the surface characteristics of the dialysis membranes. In the present study, we evaluated changes in the characteristics of membrane surfaces caused by shear stress and internal filtration using blood substitutes: water purified by reverse osmosis and 6.7 wt% dextran70 solution. We focused on the levels of a hydrophilic modifier, polyvinylpyrrolidone (PVP), on the membrane surface measured by attenuated total reflectance Fourier transform infrared spectroscopy. Experiments involving 4 h dialysis, 0-144 h shear-stress loading, and 4 h dead-end filtration were performed using polyester-polymer alloy (PEPA) and polysulfone (PS) membranes. After the dialysis experiments with accompanying internal filtration, average PVP retention on the PEPA membrane surface was 93.7% in all areas, whereas that on the PS membrane surface was 98.9% in all areas. After the shear-stress loading experiments, PVP retention on the PEPA membrane surface decreased as shear-stress loading time and the magnitude of shear stress increased. However, with the PS membrane, PVP retention scarcely changed. After the dead-end filtration experiments, PVP retention decreased in all areas for both PEPA and PS membranes, but PVP retention on the PEPA membrane surface was lower than that on the PS membrane surface. PVP on the PEPA membrane surface was eluted by both shear stress and internal filtration, while that on the PS membrane surface was eluted only by internal filtration.     

R497K polymorphism in epidermal growth factor receptor gene is associated with the risk of acute coronary syndrome

Background  

Previous studies suggested that genetic polymorphisms in the epidermal growth factor receptor (EGFR) gene had been implicated in the susceptibility to some tumors and inflammatory diseases. EGFR has been recently implicated in vascular pathophysiological processes associated with excessive remodeling and atherosclerosis. Acute coronary syndrome (ACS) is a clinical manifestation of preceding atherosclerosis. Our purpose was to investigate the association of the EGFR polymorphism with the risk of ACS. In this context, we analyzed the HER-1 R497K and EGFR intron 1 (CA)_n repeat polymorphisms in 191 patients with ACS and 210 age- and sex-matched controls in a Chinese population, using a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) strategy and direct sequencing.     

Development of a novel pulsatile bioreactor for tissue culture

The construction of tissue-engineered parts such as heart valves and arteries requires more than just the seeding of cells onto a biocompatible/biodegradable polymeric scaffold. It is essential that the functionality and mechanical integrity of the cell-seeded scaffold be investigated in vitro prior to in vivo implantation. The correct hemodynamic conditioning would lead to the development of tissues with enhanced mechanical strength and cell viability. Therefore, a bioreactor that can simulate physiological conditions would play an important role in the preparation of tissue-engineered constructs. In this article, we present and discuss the design concepts and criteria, as well as the development, of a multifunctional bioreactor for tissue culture in vitro. The system developed is compact and easily housed in an incubator to maintain sterility of the construct. Moreover, the proposed bioreactor, in addition to mimicking in vivo conditions, is highly flexible, allowing different types of constructs to be exposed to various physiological flow conditions. Initial verification of the hemodynamic parameters using Laser doppler anemometry indicated that the bioreactor performed well and produced the correct physiological conditions.     

Variable selection under multiple imputation using the bootstrap in a prognostic study

Background  

Missing data is a challenging problem in many prognostic studies. Multiple imputation (MI) accounts for imputation uncertainty that allows for adequate statistical testing. We developed and tested a methodology combining MI with bootstrapping techniques for studying prognostic variable selection.     

Competency-based evaluation tools for integrative medicine training in family medicine residency: a pilot study

Background  

As more integrative medicine educational content is integrated into conventional family medicine teaching, the need for effective evaluation strategies grows. Through the Integrative Family Medicine program, a six site pilot program of a four year residency training model combining integrative medicine and family medicine training, we have developed and tested a set of competency-based evaluation tools to assess residents' skills in integrative medicine history-taking and treatment planning. This paper presents the results from the implementation of direct observation and treatment plan evaluation tools, as well as the results of two Objective Structured Clinical Examinations (OSCEs) developed for the program.