Passive experimental autoimmune encephalomyelitis in C57BL/6 with MOG: evidence of involvement of B cells

Experimental autoimmune encephalomyelitis (EAE) is the most relevant animal model to study demyelinating diseases such as multiple sclerosis. EAE can be induced by active (active EAE) or passive (at-EAE) transfer of activated T cells in several species and strains of rodents. However, histological features of at-EAE model in C57BL/6 are poorly described. The aim of this study was to characterize the neuroinflammatory and neurodegenerative responses of at-EAE in C57BL/6 mice by histological techniques and compare them with that observed in the active EAE model. To develop the at-EAE, splenocytes from active EAE female mice were harvested and cultured in presence of MOG 35-55 and IL-12, and then injected intraperitoneally in recipient female C57BL6/J mice. In both models, the development of EAE was similar except for starting before the onset of symptoms and presenting a higher EAE cumulative score in the at-EAE model. Spinal cord histological examination revealed an increased glial activation as well as more extensive demyelinating areas in the at-EAE than in the active EAE model. Although inflammatory infiltrates composed by macrophages and T lymphocytes were found in the spinal cord and brain of both models, B lymphocytes were significantly increased in the at-EAE model. The co-localization of these B cells with IgG and their predominant distribution in areas of demyelination would suggest that IgG-secreting B cells are involved in the neurodegenerative processes associated with at-EAE

Physician career satisfaction within specialties

<p>Abstract</p> <p>Background</p> <p>Specialty-specific data on career satisfaction may be useful for understanding physician workforce trends and for counseling medical students about career options.</p> <p>Methods</p> <p>We analyzed cross-sectional data from 6,590 physicians (response rate, 53%) in Round 4 (2004-2005) of the Community Tracking Study Physician Survey. The dependent variable ranged from +1 to -1 and measured satisfaction and dissatisfaction with career. Forty-two specialties were analyzed with survey-adjusted linear regressions</p> <p>Results</p> <p>After adjusting for physician, practice, and community characteristics, the following specialties had significantly higher satisfaction levels than family medicine: pediatric emergency medicine (regression coefficient = 0.349); geriatric medicine (0.323); other pediatric subspecialties (0.270); neonatal/prenatal medicine (0.266); internal medicine and pediatrics (combined practice) (0.250); pediatrics (0.250); dermatology (0.249);and child and adolescent psychiatry (0.203). The following specialties had significantly lower satisfaction levels than family medicine: neurological surgery (-0.707); pulmonary critical care medicine (-0.273); nephrology (-0.206); and obstetrics and gynecology (-0.188). We also found satisfaction was significantly and positively related to income and employment in a medical school but negatively associated with more than 50 work-hours per-week, being a full-owner of the practice, greater reliance on managed care revenue, and uncontrollable lifestyle. We observed no statistically significant gender differences and no differences between African-Americans and whites.</p> <p>Conclusion</p> <p>Career satisfaction varied across specialties. A number of stakeholders will likely be interested in these findings including physicians in specialties that rank high and low and students contemplating specialty. Our findings regarding "less satisfied" specialties should elicit concern from residency directors and policy makers since they appear to be in critical areas of medicine.</p

Rituximab therapy reduces organ-specific T cell responses and ameliorates experimental autoimmune encephalomyelitis.

Recent clinical trials have established B cell depletion by the anti-CD20 chimeric antibody Rituximab as a beneficial therapy for patients with relapsing-remitting multiple sclerosis (MS). The impact of Rituximab on T cell responses remains largely unexplored. In the experimental autoimmune encephalomyelitis (EAE) model of MS in mice that express human CD20, Rituximab administration rapidly depleted peripheral B cells and strongly reduced EAE severity. B cell depletion was also associated with diminished Delayed Type Hypersensitivity (DTH) and a reduction in T cell proliferation and IL-17 production during recall immune response experiments. While Rituximab is not considered a broad immunosuppressant, our results indicate a role for B cells as a therapeutic cellular target in regulating encephalitogenic T cell responses in specific tissues

Rituximab administration alters MOG-specific recall responses.

<p><b>Panels A–E</b>. WTLM and hCD20Tg mice were treated with Rituximab daily for 3 days (Day -3,-2,-1) followed by immunization with MOG_1–125 on Day 0. On day 20 post-immunization, bulk draining lymph node cells (LNC) were isolated and recall response determined. <b>Panels A and B</b>. Identification of MOG-reactive T cells by tetramer staining. Bulk LNC were cultured for 3 days in the presence of MOG_1–125 prior to labeling with antibodies to CD3, CD4 and I-Ab tetramers to either human (A) CLIP_103–117 or (B) MOG_38–48. Numbers above boxes indicate percentages of T cells in the tetramer positive gate. <b>Panel C</b>. Secondary T cell proliferative responses were determined by CFSE dilution assay. LNC were labeled with CFSE and placed in culture with 20 µg/ml MOG_1–125 and proliferation determined by flow cytometry on day 6 of culture. Results shown are gated on CD4+ events. Numbers indicate the percentage of total cells that diluted CFSE from WTLM and hCD20Tg mice. <b>Panels D and E</b>. 48-hour supernatants from the Panel C experiments were examined for the presence of IL-17 (D) or IFNγ (E) by ELISA. Asterices indicate a significant decrease in IL-17 production (p<0.05). Results are representative of at least 2 independent experiments.</p

B and T cell frequency in therapeutic model of Rituximab administration.

<p>Cells from each organ were pooled from at least three mice per group and counted. Cell counts were then normalized by dividing the total counts by the numbers of mice in each group and then multiplied by the percentage of each cell type as identified by flow cytometry. Total leukocytes were identified by CD45+ events within FSC and SSC gates. B cells were identified using gates for CD19 and B220. T cells were identified by expression of CD3 and either CD4 or CD8. Numbers in parenthesis indicate the percent change in cell counts in the hCD20Tg mice as compared to WTLM controls.</p

B and T cell dynamics following Rituximab treatment.

<p><b>Panels A and E</b>. Expression of human CD20 by hCD20Tg B cells and hCD20Tg T cells. Splenocytes from WTLM and hCD20Tg mice were stained with antibodies to human CD20, CD4 and CD19 and flow cytometry performed. Results shown are gated on CD19+CD4− events to identify B cells or gated on CD19−CD4+ events to identify T cells. <b>Panels B/C/D/F/G</b>. WTLM and hCD20Tg mice received three daily injections of Rituximab (100 µg) beginning on day 0. At 144 hours after Rituximab treatment was initiated, tissues were harvested for flow cytometry analysis. <b>Panel B</b>. Peripheral B cells are rapidly depleted following Rituximab treatment. <b>Panel C</b>. Splenic B cells are depleted following Rituximab treatment. <b>Panel D</b>. B cells in the LN (Axilary, Brachial and Inguinal) are depleted following Rituximab treatment. <b>Panel E</b>. CD4 T cells do not express human CD20. <b>Panel F</b>. Splenic CD4 T cells are reduced following Rituximab treatment. <b>Panel G</b>. CD4 T cells in the LN (Axilary, Brachial and Inguinal) are reduced following Rituximab treatment. <b>Panel H</b>. Rituximab administration does not prevent priming of inflammatory T-effector cells. WTLM and hCD20Tg mice were treated with Rituximab daily for 3 days (Day -3,-2,-1), followed by immunization with MOG_1–125 on Day 0. On day 10 post-immunization, DTH responses were elicited by subcutaneous injection of MOG_1–125 (10 µg) in the ear. The net ear swelling responses were determined at 24 hours. Results shown indicate the mean ear swelling in mmX10E-3 (background subtracted) +/− SEM. Significant differences were detected by unpaired t-test (*, p<0.05; **, p<0.01). These results are representative of at least two independent experiments.</p

Disruption of EAE pathogenesis by B cell depletion.

<p><b>Panel A</b>. EAE severity is similar in WTLM and hCD20Tg mice. EAE onset and severity were monitored using a 5-point scale on WTLM and hCD20Tg mice immunized with MOG_1–125. These results are representative of at least two independent experiments. <b>Panels B–D</b>. Rituximab administration prevents the induction of EAE. WTLM or hCD20Tg mice were either left untreated or were injected with 100 µg Rituximab daily for three days (Day -3,-2,-1). On Day 0, EAE was induced by immunization with MOG_1–125. <b>Panel B</b>. Disease course of WTLM and hCD20Tg mice, EAE onset and severity was monitored using a 5-point scale. Shown are the mean clinical score +/− SEM. <b>Panel C</b>. B cell depletion results in reduced levels of anti-MOG IgG in the serum. Serum was harvested on day 21 post-immunization and MOG-specific IgG levels were determined by ELISA. Results shown are the mean IgG concentration +/− SEM. Asterix indicates significant decrease as compared to Rituximab-treated WTLM mice. <b>Panel D</b>. Rituximab administration results in rapid depletion of B cells in the peripheral blood. Blood was taken from WTLM or hCD20Tg mice 3 days following the final dose of Rituximab (day 2 post-immunization). B cells were identified by flow cytometry using gates to identify lymphocytes and CD19 expressing cells. Results shown are the mean percentages of CD19+ B cells +/−SEM (*, p<0.01). <b>Panels E/F</b>. Treatment with Rituximab reduces EAE severity. EAE was initiated in WTLM and hCD20Tg mice on Day 0. Upon the appearance of clinical signs of EAE, Rituximab (100 µg) was administered daily for three treatments. <b>Panel E</b>. Disease course of WTLM and hCD20Tg mice, EAE onset and severity was monitored using a 5-point scale. Shown are the mean clinical score +/− SEM. <b>Panel F</b>. B cell depletion in peripheral blood on day 20. Significant differences were determined using an unpaired t-test (*, p<0.05; **, p<0.01). These results are representative of at least two independent experiments with Rituximab and two experiments using the 1F5 anti-human CD20 mAb (data not shown).</p