DMSO Represses Inflammatory Cytokine Production from Human Blood Cells and Reduces Autoimmune Arthritis

Dimethyl sulfoxide (DMSO) is currently used as an alternative treatment for various inflammatory conditions as well as for cancer. Despite its widespread use, there is a paucity of data regarding its safety and efficacy as well as its mechanism of action in human cells. Herein, we demonstrate that DMSO has ex-vivo anti-inflammatory activity using Escherichia coli- (E. coli) and herpes simplex virus-1 (HSV-1)-stimulated whole human blood. Specifically, we found that between 0.5%– 2%, DMSO significantly suppressed the expression of many pro-inflammatory cytokines/chemokines and prostaglandin E₂ (PGE₂). However, a significant reduction in monocyte viability was also observed at 2% DMSO, suggesting a narrow window of efficacy. Anti-inflammatory concentrations of DMSO suppressed E. coli-induced ERK1/2, p38, JNK and Akt phosphorylation, suggesting DMSO acts on these signaling pathways to suppress inflammatory cytokine/chemokine production. Although DMSO induces the differentiation of B16/F10 melanoma cells in vitro, topical administration of DMSO to mice subcutaneously implanted with B16 melanoma cells was ineffective at reducing tumor growth, DMSO was also found to block mouse macrophages from polarizing to either an M1- or an M2-phenotype, which may contribute to its inability to slow tumor growth. Topical administration of DMSO, however, significantly mitigated K/BxN serum-induced arthritis in mice, and this was associated with reduced levels of pro-inflammatory cytokines in the joints and white blood cell levels in the blood. Thus, while we cannot confirm the efficacy of DMSO as an anti-cancer agent, the use of DMSO in arthritis warrants further investigation to ascertain its therapeutic potential.     

The presence of X- and Y-chromosomes in oocytes leads to impairment in the progression of the second meiotic division

The oocytes of B6.Y(TIR) sex-reversed female mice can be fertilized but the resultant embryos die at early cleavage stages. In the present study, we examined chromosome segregation at meiotic divisions in the oocytes of XY female mice, compared to those of XX littermates. The timing and frequency of oocyte maturation in culture were comparable between the oocytes from both types of females. At the first meiotic division, the X- and Y-chromosomes segregated independently and were retained in oocytes at equal frequencies. However, more oocytes retained the correct number of chromosomes than anticipated from random segregation. The oocytes that had reached MII-stage were activated by fertilization or incubation with SrCl(2). As expected, the majority of oocytes from XX females completed the second meiotic division and reached the 2-cell stage in 24 h. By contrast, more than half of oocytes from XY females initially remained at the MII-stage while the rest precociously entered interphase after SrCl(2) activation; very few oocytes were seen at the second anaphase or telophase and they often showed impairment of sister-chromatid separation. Eventually the majority of oocytes entered interphase and formed pronuclei, but very few reached the 2-cell stage. Similar results were obtained after fertilization. We conclude that the XY chromosomal composition in oocyte leads to impairment in the progression of the second meiotic division.     

Tridimensional evaluation and optimization of the orthotic treatment of adolescent idiopathic scoliosis

Adolescent idiopathic scoliosis involves complex tridimensional deformities of the spine, rib cage and pelvis. Moderate curves generally are treated using an orthosis. This paper presents different studies performed over the last fifteen years related to the biomechanical evaluation and optimization of the orthopedic treatment of scoliotic deformities. Patient specific 3D models of the spine, pelvis and rib cage are computed from calibrated radiographs, and are used to calculate 2D and 3D clinical indices. The torso shape is acquired using surface topography. With such internal and external 3D models, the efficacy of the most frequently used orthoses can be analyzed and new treatments can be developed. Pressures generated by a brace on the patient's trunk were measured using a flexible matrix of pressure sensors and displayed over the patient's internal geometry in order to analyze the brace efficacy. Patient specific finite element models have been developed, including the osseo-ligamentous structures as well as the muscles, the neuro-control, trunk growth and its adaptation to the stress. These models were used to analyze the effects of the Boston brace. The electro-myographic activity also was measured to analyze the < active > correction mechanisms. Adjustment techniques and software are used to help the orthotists with real time feedback when the brace is being fabricated and adjusted to the patient. Residual growth potential is also being added to the computer model to simulate the long term effect of a brace. The improvement of the orthotic treatments of scoliotic deformities is very encouraging. The exploitation of such tools is expected to allow reaching optimal treatment personalized to each patient. double dagger.     

Mismatch and base excision repair proficiency in murine embryonic stem cells

Accumulation of mutations in embryonic stem (ES) cells would be detrimental to an embryo derived from these cells, and would adversely affect multiple organ systems and tissue types. ES cells have evolved multiple mechanisms to preserve genomic integrity that extend beyond those found in differentiated cell types. The present study queried whether mismatch repair (MMR) and base-excision repair (BER) may play a role in the maintenance of murine ES cell genomes. The MMR proteins Msh2 and Msh6 are highly elevated in mouse ES cells compared with mouse embryo fibroblasts (MEFs), as are Pms2 and Mlh1, albeit to a lesser extent. Cells transfected with an MMR reporter plasmid showed that MMR repair capacity is low in MEFs, but highly active in wildtype ES cells. As expected, an ES cell line defective in MMR was several-fold less effective in repair level than wildtype ES cells. Like proteins that participate in MMR, the level of proteins involved in BER was elevated in ES cells compared with MEFs. When BER activity was examined biochemically using a uracil-containing oligonucleotide template, repair activity was higher in ES cells compared with MEFs. The data are consistent with the suggestion that ES cells have multiple mechanisms, including highly active MMR and BER that preserve genetic integrity and minimize the accumulation of mutations.     

Beta turn propensity and a model polymer scaling exponent identify intrinsically disordered phase-separating proteins

The complex cellular milieu can spontaneously demix, or phase separate, in a process controlled in part by intrinsically disordered (ID) proteins. A protein''s propensity to phase separate is thought to be driven by a preference for protein–protein over protein–solvent interactions. The hydrodynamic size of monomeric proteins, as quantified by the polymer scaling exponent (v), is driven by a similar balance. We hypothesized that mean v, as predicted by protein sequence, would be smaller for proteins with a strong propensity to phase separate. To test this hypothesis, we analyzed protein databases containing subsets of proteins that are folded, disordered, or disordered and known to spontaneously phase separate. We find that the phase-separating disordered proteins, on average, had lower calculated values of v compared with their non-phase-separating counterparts. Moreover, these proteins had a higher sequence-predicted propensity for β-turns. Using a simple, surface area-based model, we propose a physical mechanism for this difference: transient β-turn structures reduce the desolvation penalty of forming a protein-rich phase and increase exposure of atoms involved in π/sp² valence electron interactions. By this mechanism, β-turns could act as energetically favored nucleation points, which may explain the increased propensity for turns in ID regions (IDRs) utilized biologically for phase separation. Phase-separating IDRs, non-phase-separating IDRs, and folded regions could be distinguished by combining v and β-turn propensity. Finally, we propose a new algorithm, ParSe (partition sequence), for predicting phase-separating protein regions, and which is able to accurately identify folded, disordered, and phase-separating protein regions based on the primary sequence.     

Discovery and optimization of piperidyl benzamide derivatives as a novel class of 11β-HSD1 inhibitors

Discovery and optimization of a piperidyl benzamide series of 11β-HSD1 inhibitors is described. This series was derived from a cyclohexyl benzamide lead structures to address PXR selectivity, high non-specific protein binding, poor solubility, limited in vivo exposure, and in vitro cytotoxicity issues observed with the cyclohexyl benzamide structures. These efforts led to the discovery of piperidyl benzamide 15 which features improved properties over the cyclohexyl benzamide derivatives.     

Growth plate cartilage shows different strain patterns in response to static versus dynamic mechanical modulation

Longitudinal growth of long bones and vertebrae occurs in growth plate cartilage. This process is partly regulated by mechanical forces, which are one of the underlying reasons for progression of growth deformities such as idiopathic adolescent scoliosis and early-onset scoliosis. This concept of mechanical modulation of bone growth is also exploited in the development of fusionless treatments of these deformities. However, the optimal loading condition for the mechanical modulation of growth plate remains to be identified. The objective of this study was to evaluate the effects of in vitro static versus dynamic modulation and of dynamic loading parameters, such as frequency and amplitude, on the mechanical responses and histomorphology of growth plate explants. Growth plate explants from distal ulnae of 4-week-old swines were extracted and randomly distributed among six experimental groups: baseline (\(n=10\)), control (\(n=10\)), static (\(n=10\)) and dynamic (\(n=3\times 10\)). For static and dynamic groups, mechanical modulation was performed in vitro using an Indexed CartiGen bioreactor. A stress relaxation test combined with confocal microscopy and digital image correlation was used to characterize the mechanical responses of each explant in terms of peak stress, equilibrium stress, equilibrium modulus of elasticity and strain pattern. Histomorphometrical measurements were performed on toluidine blue tissue sections using a semi-automatic custom-developed MATLAB toolbox. Results suggest that compared to dynamic modulation, static modulation changes the strain pattern of the tissue and thus is more detrimental for tissue biomechanics, while the histomorphological parameters are not affected by mechanical modulation. Also, under dynamic modulation, changing the frequency or amplitude does not affect the biomechanical response of the tissue. Results of this study will be useful in finding optimal and non-damaging parameters for the mechanical modulation of growth plate in fusionless treatments.     

Spatial and temporal activation of the small GTPases RhoA and Rac1 by the netrin-1 receptor UNC5a during neurite outgrowth

Netrin-1 attracts or repels growing axons during development. The UNC5 receptors mediate the repulsive response, either alone or in complex with DCC receptors. The signaling mechanisms activated by UNC5 are poorly understood. Here, we examined the role of Rho GTPases in UNC5a signaling. We found that UNC5a induced neurite outgrowth in N1E-115 neuroblastoma cells in a netrin-1- and Rac1-dependent manner. UNC5a lacking its cytoplasmic tail also mediated this effect. In fibroblasts, UNC5a was able to activate RhoA and to a lower extent Rac1 and Cdc42 in response to netrin-1. Using Fluorescence Resonance Energy Transfer (FRET) intermolecular probes, we visualized the spatial and temporal activation of Rac1, Cdc42 and RhoA in live N1E-115 cells expressing UNC5a during neurite outgrowth. We found that Rac1 but not Cdc42 was transiently activated at the leading edge of the cell during neurite initiation. However, at later times when well-developed neurites were formed, active RhoA was found in the cell body and at the base of the neuronal leading process in UNC5a-expressing cells. Together, these findings demonstrate that the netrin-1 receptor UNC5a is able to induce neurite outgrowth and to differentially activate RhoA and Rac1 during neurite extension in a spatial and temporal manner.     

Biomechanical influence of disk properties on the load transfer of healthy and degenerated disks using a poroelastic finite element model

Spine degeneration is a pathology that will affect 80% of the population. Since the intervertebral disks play an important role in transmitting loads through the spine, the aim of this study was to evaluate the biomechanical impact of disk properties on the load carried by healthy (Thompson grade I) and degenerated (Thompson grades III and IV) disks. A three-dimensional parametric poroelastic finite element model of the L4/L5 motion segment was developed. Grade I, grade II, and grade IV disks were modeled by altering the biomechanical properties of both the annulus and nucleus. Models were validated using published creep experiments, in which a constant compressive axial stress of 0.35 MPa was applied for 4 h. Pore pressure (PP) and effective stress (S(E)) were analyzed as a function of time following loading application (1 min, 5 min, 45 min, 125 min, and 245 min) and discal region along the midsagittal profile for each disk grade. A design of experiments was further implemented to analyze the influence of six disk parameters (disk height (H), fiber proportion (%F), drained Young's modulus (E(a),E(n)), and initial permeability (k(a),k(n)) of both the annulus and nucleus) on load-sharing for disk grades I and IV. Simulations of grade I, grade III, and grade IV disks agreed well with the available published experimental data. Disk height (H) had a significant influence (p<0.05) on the PP and S(E) during the entire loading history for both healthy and degenerated disk models. Young's modulus of the annulus (E(a)) significantly affected not only S(E) in the annular region for both disk grades in the initial creep response but also S(E) in the nucleus zone for degenerated disks with further creep response. The nucleus and annulus permeabilities had a significant influence on the PP distribution for both disk grades, but this effect occurred at earlier stages of loading for degenerated than for healthy disk models. This is the first study that investigates the biomechanical influence of both geometrical and material disk properties on the load transfer of healthy and degenerated disks. Disk height is a significant parameter for both healthy and degenerated disks during the entire loading. Changes in the annulus stiffness, as well as in the annulus and nucleus permeability, control load-sharing in different ways for healthy and degenerated disks.     

Clostridium difficile-associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity

Background

Recent reports suggest that Clostridium difficile colitis may be evolving into a more severe disease. During the second half of 2002 we noted an increase in the number of patients with severe C. difficile-associated diarrhea (CDAD) in our institution. We describe cases of CDAD at our institution over a 13-year period and investigate changes in illness severity.

Methods

We undertook a retrospective chart review of all cases of CDAD diagnosed at the Centre hospitalier universitaire de Sherbrooke from Jan. 1, 1991, to Dec. 31, 2003. Because the hospital serves a well-defined population of Quebec, we were also able to calculate population-based incidence during this period. We abstracted data on individual patients from patient charts and from hospital and pharmacy computer databases. We defined cases of CDAD as having a positive C. difficile cytotoxicity assay result, or endoscopic or histopathological evidence of pseudomembranous colitis. A case was considered complicated if one or more of the following was observed: megacolon, perforation, colectomy, shock requiring vasopressor therapy, or death within 30 days after diagnosis.

Results

A total of 1721 cases of CDAD were diagnosed during the study period. The incidence increased from 35.6 per 100 000 population in 1991 to 156.3 per 100 000 in 2003; among patients aged 65 years or more, it increased from 102.0 to 866.5 per 100 000. The proportion of cases that were complicated increased from 7.1% (12/169) in 1991–1992 to 18.2% (71/390) in 2003 (p < 0.001), and the proportion of patients who died within 30 days after diagnosis increased from 4.7% (8/169) in 1991– 1992 to 13.8% (54/390) in 2003 (p < 0.001). A high leukocyte count (20.0 х 10⁹/L or greater) and an elevated creatinine level (200 μmol/L or greater) were strongly associated with adverse outcomes: in 2003, 45 (40.9%) of 110 patients with a high leukocyte count or creatinine level, or both, had complicated CDAD and 28 (25.5%) died within 30 days after diagnosis. After adjustment for age and other confounding factors, patients initially given oral vancomycin therapy had a risk of progression to complicated CDAD that was 79% lower than the risk among patients initially treated with metronidazole (adjusted odds ratio 0.2, 95% confidence interval 0.06–0.8, p = 0.02).

Interpretation

An epidemic of CDAD with an increased case-fatality rate has had important consequences on the elderly population of our region. Our observational data suggest that the equivalence of vancomycin and metronidazole in the treatment of CDAD needs to be questioned.Clostridium difficile is an important cause of diarrhea in industrialized countries and the leading cause of infectious diarrhea among patients in hospital.^1,2,3,4,5,6 In the second half of 2002 we noted an increase in the number of patients with fulminant C. difficile colitis in our institution who required an emergency colectomy. Informal discussions with clinicians from other hospitals in southern Quebec suggested that this phenomenon was occurring in other cities as well. To investigate whether this was related merely to an increase in the number of cases of C. difficile-associated diarrhea (CDAD) or to an increase in the proportion of cases that developed severe colitis, we undertook a retrospective study of all cases of CDAD diagnosed at our institution over a 13-year period and investigated changes in illness severity.