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Context: Therapeutic modality control variables are thought to be thermal neutral, a term sometimes used interchangeably with room temperature. We question this common assumption. Objective: To determine thermal neutrality of common therapeutic modality control variables. Design: We performed 5 laboratory experiments, including (1) water temperature over 3 weeks in 3 different containers (glass, plastic, and polystyrene); (2) water temperature and volume of 4 beakers (2 insulated, 2 uninsulated) over 4 weeks, with 1 beaker of each type covered by polyethylene; and skin interface temperature of (3) a dry, nonheated hydrocollator pack held against the chest, (4) kitty litter applied to the knee, and (5) room-temperature ultrasound gel to the forearm. Setting: Therapeutic modalities laboratory. Patients or Other Participants: College student volunteers were subjects in experiments 3, 4, and 5. Main Outcome Measure(s): We measured temperature and volume change. Data were evaluated using descriptive and interferential statistics. Results: Water temperature plateaued significantly below room temperature. Temperatures significantly increased in all but the open, insulated container. Open containers plateaued at approximately 2°C below room temperature and lost significant amounts of water; closed containers plateaued at room temperature with negligible water loss. In experiments 3 through 5, skin temperatures rose significantly during hydrocollator pack, kitty litter, and ultrasound gel application. Conclusions: Room-temperature water baths, dry hydrocollator packs, kitty litter, and ultrasound gel were not thermally neutral. Room temperature should not be used synonymously with thermal neutral. Care must be taken to ensure that control variables truly are controlled. [ABSTRACT FROM AUTHOR]

Context: The amount of retained ankle flexibility gains and the effects of diathermy on those gains are unclear. Objective: To determine the retention of flexibility 3 weeks after an 18-day stretching regime and the effect of pulsed, shortwave diathermy on that retention. Design: We used a 2x4 factorial with repeated measures on day (1, 19, 24, and 39). The other independent variable was treatment (stretch only versus diathermy and stretch). The dependent variable was ankle-dorsiflexion angular displacement as measured on a digital inclinometer. Setting: Therapeutic Modality Research Laboratory. Patients or Other Participants: 23 healthy college-aged volunteers (8 males, 15 females; age = 22.7 +/- 2.1 years, height = 171.1 +/- 8.8 cm, mass = 70.4 +/- 13.5 kg). Interventions: All subjects performed 3 weeks (not including weekends) of low-load, prolonged, long-duration stretching. One group performed stretching only; the other group also received diathermy. Main Outcome Measure(s): After an 18-day stretching regime and 7-day retention study, subjects returned 14 days later for the 3-week retention measure. The angle of inclination from the posterior Achilles tendon to the sole of the shoe near the heel was measured on each treatment and test day. Results: Regardless of group (F(1,21) = 0.74, P = 0.40), the flexibility gained between days 1 (99.7 +/- 4.0 degrees), 19 (102.9 +/- 5.8 degrees ), and 24 (105.0 +/- 6.2 degrees) were maintained at day 39 (104.8+/- 7.2 degrees) (P < .05). Conclusions: Flexibility gains in normal ankles with 3 weeks of training were retained for at least 3 weeks after training ceased. The application of pulsed, shortwave diathermy during stretching did not appear to influence the chronic retention of flexibility gains in normal subjects. [ABSTRACT FROM AUTHOR]

Context: Neoprene sleeves are alleged to increase heat in skeletal muscle, but no published research supports this belief. Objectives: To quantify anterior thigh skin and intramuscular temperature changes in varsity athletes wearing a neoprene thigh sleeve. Design: A 2 x 2 x 3 x 5 factorial design with replicated measures on 3 variables. Independent variables included sex (male or female), neoprene sleeve (yes or no), exercise intensity (control, 50% intensity bicycle, 70% intensity bicycle), and exercise phase (5 minutes pre-exercise, 15 minutes of exercise, 0-10 minutes postexercise, 11-20 minutes postexercise, 21- 30 minutes postexercise). Setting: Research laboratory. Patients or Other Participants: 12 male (23 +/- 0.95 years) and 12 female (20.59 +/- 1.44 years) National Collegiate Athletic Association Division I collegiate athletes actively engaged in offseason strength and conditioning programs. Intervention(s): We sampled skin and muscle temperatures every 10 seconds for 50 minutes with a telethermometer interfaced to a personal computer. Each 60-minute session included 15 minutes pre-exercise (temperatures recorded during the last 5 minutes), 15 minutes of exercise (control, 50% intensity, and 70% intensity), and 30 minutes postexercise. Skin temperature was measured on the anterior aspect of the thigh using surface thermocouples. Intramuscular temperature was measured in the vastus lateralis muscle at a depth of 2 cm below the subcutaneous fat using implantable thermocouples. Main Outcome Measure(s): Skin and intramuscular temperature. Results: Skin temperature was greater when subjects were wearing neoprene sleeves (1.4 degrees C during control, 3.1 degrees C during 70% exercise). Wearing a thigh sleeve had no effect on intramuscular temperature before or during exercise, but postexercise temperatures averaged 0.5 degrees C higher. Exercise intensity showed 1.3 degrees C to 2.0 degrees C increases in temperature with the sleeve. Females had higher intramuscular temperatures than males when wearing sleeves (1.4 degrees C during control, 1 degrees C during 50% exercise, and 0.8 degrees C during 70% exercise). Males had higher skin temperatures than females (0.8 degrees C during 50% exercise, 1 degrees C during 70% exercise). Conclusions: Neoprene sleeves are effective in maintaining intramuscular temperature after 15 minutes of exercise and in increasing skin temperature during and after exercise. These results may be attributable to the insulating effects of the neoprene sleeve. [ABSTRACT FROM AUTHOR]

Context: Data from electrothermometers are used to determine therapeutic modality usage, but the value of experimental results is only as good as the data collected.Objective: To determine the reliability and validity of 3 electrothermometers from 2 manufacturers.Design: A 3 x 4 x 17 factorial with repeated measures on 2 factors. Independent variables were trial (1, 2, 3), thermometer (mercury thermometer, Iso-Thermex calibrated from -50 degreesC to 50 degreesC, Iso-Thermex calibrated from -20 degreesC to 80 degreesC, and Data-logger), and time (17).Setting: Human Performance Research Center.Intervention(s): Eighteen thermocouples were inserted through the wall of a foamed polystyrene cooler, and 6 were connected to each of the 3 electrothermometers. The cooler was positioned on a stir plate and filled with room-temperature water (18.4 degreesC). A mercury thermometer was immersed into the water bath. Measurements of the water bath were taken every 10 seconds for three 3-minute trials.Main Outcome Measure(s): The temperature variability of 3 electrothermometers was taken from a calibrated mercury thermometer.Results: The Iso-Thermex electrothermometers did not differ statistically from each other in uncertainty (validity error +/- reliability error = 0.06 degreesC +/- 0.03 degreesC +/- 0.03 degreesC +/- 0.02 degreesC, P < .05), but both differed from the Datalogger (0.64 degreesC +/- 0.20 degreesC, P < .05). The Datalogger temperature was consistently higher than the mercury thermometer temperature.Conclusions: The Iso-Thermex electrothermometers were more stable than the Datalogger, and values were within the published uncertainty (+/-0.1 degreesC) when used with PT-6 thermocouples. The Datalogger we used had an uncertainty of measurement greater than that indicated in the user's manual (approximately +/-0.52 degreesC). Uncertainty of +/-0.84 degreesC can significantly influence the interpretation of results when intramuscular temperature changes are usually less than 5 degreesC. [ABSTRACT FROM AUTHOR]

Objective: To review the 44-year history of the Journal of Athletic Training. Description: We examined the 179 issues of the Journal and selected 9 years (every 5 years beginning in 1957) for a page-by-page analysis of content. We interviewed 8 former Editors and Editors-in-Chief and drew upon our own experiences during a combined 56 years of association with the Journal. The Journal has undergone significant changes in both structure and quality; its growth mirrors the growth of the athletic training profession. During its infancy, the Journal relied greatly on reprints from other journals. Now, most authors are certified athletic trainers, sharing the results of their own scholarship. Conclusions: The Journal, like the athletic training profession, is alive and strong. It has become an independent leader in the dissemination of athletic health care information. [ABSTRACT FROM AUTHOR]

Objective: To outline the essential elements of scholarship, identify its role in the development of the athletic training profession, and encourage athletic trainers to increase scholarly activities. Background: In the process of writing 2 manuscripts for the Journal of Athletic Training about how to write journal manuscripts, we felt something needed to be said concerning why one should write such manuscripts and how individual manuscripts should tie together to advance knowledge. This led us to search the scientific literature for information concerning scholarship and its attainment. Finding no comprehensive discussion on the topic, we then began to investigate components of scholarship and activities that lead to its attainment. Description: Scholarship, knowledge, truth, and theory are defined. The attributes or characteristics of a scholar delineated and discussed include seeking to establish truth and develop new knowledge, developing and refining theory, being focused in one's work, being honest about one's work, communicating ideas and stirring thinking, being open minded, and recognizing the difference between scholarship and pseudoscholarship. It is important to promote scholarship among undergraduate and graduate students; numerous suggestions are outlined. Application: Scholarship is essential to our survival as a profession. The guidelines presented here will help individuals examine and improve their scholarship. [ABSTRACT FROM AUTHOR]

Objective: Most of the research on cold applications has been performed on nonexercising supine subjects during a single cold pack application. Most athletic injuries occur during exercise, which increases skin temperature. Exercise before ice application will also increase ankle skin temperature during the rewarming phase. The purpose of this study was to examine the effects of activity on subsequent ice pack applications and rewarming using standard immediate care procedures. Design and Setting: Three experimental conditions (20-, 30-, and 40-minute ice pack applications) were applied to 12 subjects in a repeated measures design. Subjects rode a bicycle ergometer for 15 minutes before ice application to the ankle and opposite thigh, and were active (walking with crutches, simulated showering and dressing) for 20 minutes following application. Subjects rested with the limb elevated for an additional 40 minutes. Ice packs were then reapplied for the appropriate time (20, 30, or 40 minutes) followed by 60 minutes of rest with the limb elevated. Subjects: Twelve (8 males, 4 females) college-aged volunteers. Only subjects with good-to-high fitness levels were accepted for this study. Measurements: Ankle skin, thigh skin, and atmospheric temperatures were measured every minute using an Isothermex (Columbus Instruments, Columbus, OH). Results: Thigh temperature changes during the first ice application were greater during the 30- and 40-minute conditions than the 20-minute condition. Ankle and thigh temperature changes during the first ice application and rewarming, and for the entire trial were greater during the 40-minute condition than the 20- or 30-minute conditions. Throughout the first ice application and rewarming, and the entire trial, thigh temperature changes were greater during the 30-minute condition than the 20-minute condition. Conclusions: During immediate care procedures following injury, ice packs should be reapplied immediately following showering, changing clothes, and returning home. [ABSTRACT FROM AUTHOR]

Objective: To share with potential authors tips for constructing a scholarly manuscript and for organizing information in various types of scholarly manuscripts: experimental reports, literature reviews, case reports, and clinical techniques. Description: The goal of writing a scientific/technical/medical article is to communicate new information that hopefully has clinical relevance and will improve health care. This information must be organized and presented clearly and logically. We present 66 tips for organizing a scholarly manuscript. We tell not only what goes where in the manuscript but also how to construct each of the elements so as to logically communicate the author's message. The tips are numbered to facilitate referencing. Conclusion: By becoming familiar with these tips, potential authors can avoid making mistakes that may hinder publication of their manuscripts. [ABSTRACT FROM AUTHOR]

Objective: To share with potential authors tips for communicating their ideas more clearly in a scholarly manuscript. Description: Communicating scientific, technical, or medical information so that readers can understand its meaning requires logical organization and proper use of language. These 30 tips review basic English grammar and suggest ways authors can clearly and concisely present their material. We admonish authors to avoid common errors such as writing in the passive voice, overusing abbreviations, and emphasizing unimportant facts. Conclusion: Attention to matters of writing style enhances clear communication, which must be the prime objective of scientific writing. [ABSTRACT FROM AUTHOR]

OBJECTIVE: To measure tissue-temperature rise in the lateral aspect of the ankle during 10-minute ultrasound treatments with ultrasound gel (gel), a gel pad with a thin layer of ultrasound gel on the top (gel/pad), and a gel pad with a thin layer of ultrasound gel on the top and the bottom coated with ultrasound gel (gel/pad/gel). DESIGN AND SETTING: We used a 1 x 3 repeated-measures, crossover design. The dependent variables were tissue-temperature change and time to peak temperature. The independent variable was the type of ultrasound coupling medium. Treatment orders were randomly assigned, and all possible orders were assigned 3 times. A thermocouple was inserted through a 32-mm catheter at a depth of 1 cm into the target-tissue space, the posterior lateral aspect of the left ankle, halfway between the lateral malleolus and the Achilles tendon. Each treatment consisted of ultrasound delivered topically at 1 W/cm(2), 3 MHz, in the continuous mode for 10 minutes. SUBJECTS: Eighteen healthy, college-aged student volunteers (13 women, 5 men), with no history of ankle injury in the previous 6 months. MEASUREMENTS: Intratissue temperature, measured every 30 seconds for 10 minutes. RESULTS: Intratissue temperature increases during the 10-minute treatments were significantly greater for the ultrasound gel (7.72 degrees C +/- 0.52 degrees C) and the gel/pad/gel (6.68 degrees C +/- 0.52 degrees C) than for the gel/pad (4.98 degrees C +/- 0.52 degrees C). CONCLUSIONS: When ultrasound is applied over bony prominences, a gel pad should be covered with ultrasound gel on both sides to ensure optimal heating. [ABSTRACT FROM AUTHOR]

Objective: To compare the effects of 3 treatments on ankle dorsiflexion range of motion: prolonged long-duration stretching, pulsed shortwave diathermy followed by stretching, and pulsed shortwave diathermy, stretching, and ice combined.Design and Setting: A 2 x 5 x 15 repeated-measures (on 2 factors) design guided this study. Range-of-motion change in triceps surae flexibility was the dependent variable. The 3 independent variables were treatment group, pretest and posttest measurements, and day. Treatment group had 4 levels: control, stretching (10 minutes of stretching via the weight and pulley), diathermy and stretching (20 minutes of diathermy and 10 minutes of stretching), and diathermy, stretching, and ice (20 minutes of diathermy, 10 minutes of stretching applied after 15 minutes of diathermy, and 5 minutes of ice applied during the last 5 minutes of stretching). Each subject received 14 treatments throughout 3 weeks, with a follow-up measurement taken 6 days after the last treatment.Subjects: Forty-four healthy college-student volunteers not involved in any flexibility program.Measurements: We measured ankle dorsiflexion using a digital inclinometer before and after treatment.Results: After 14 days of treatment, the range-of-motion increase was greater after heat and stretching than after stretching alone. After 6 additional days of rest, the heat and stretching range-of-motion increase was greater than that for stretching alone.Conclusion: Pulsed shortwave diathermy application before prolonged long-duration static stretching was more effective than stretching alone in increasing flexibility throughout 3 weeks. After 14 treatments, prolonged long-duration stretching combined with pulsed shortwave diathermy followed by ice application caused greater immediate and net range-of-motion increases than prolonged long-duration stretching alone. [ABSTRACT FROM AUTHOR]

Objective: To compare the effects of low-load, short-duration stretching with or without high-intensity, pulsed short-wave diathermy on hamstring flexibility.Design and Setting: We used a single-blind, repeated-measures design (pretest and posttest for all treatments) that included a placebo. The 3 independent variables were treatment mode, pretest and posttest measurements, and day. Treatment mode had 3 levels: diathermy and stretching, stretching alone, and control. The dependent variable was range of motion. Subjects were randomly assigned to the diathermy and stretching, stretching-only, or control group. Subjects were treated and tested each day (at approximately the same time) for 5 days, with a follow-up test administered 72 hours later. Hamstring flexibility was tested using a sit-and-reach box before and after each treatment. Diathermy and stretching subjects received a 15-minute diathermy treatment on the right hamstring at a setting of 7000 pulses per second, with an average pulse width of 95 microsec. Stretching-only subjects received a 15-minute sham diathermy treatment. Both diathermy and stretching and stretching-only subjects then performed three 30-second stretches (short duration) before being retested. Control subjects lay prone for 15 minutes before being retested.Subjects: Thirty-seven healthy college students (11 men, 26 women, age = 20.46 +/- 1.74 years) volunteered.Measurements: Hamstring flexibility was measured using a sit-and-reach box before and after each treatment.Results: The average increases in hamstring flexibility over the 5 treatment days for the diathermy and stretching, stretching-only, and control groups were 6.06 cm (19.6%), 5.27 cm (19.7%), and 3.38 cm (10.4%), respectively. Three days later (after no treatment), the values for the diathermy and stretching, stretching-only, and control groups were 8.27 cm (26.7%), 6.83 cm (25.3%), and 4.15 cm (14.2%), respectively. No significant differences in hamstring flexibility were noted among the groups.Conclusions: Diathermy and short-duration stretching were no more effective than short-duration stretching alone at increasing hamstring flexibility. The effects of diathermy with longer stretching times need to be researched. [ABSTRACT FROM AUTHOR]

Objective: To compare tissue temperature rise and decay after 20-minute diathermy and ultrasound treatments. Design and Setting: We inserted 3 26-gauge thermistor microprobes into the medial aspect of the anesthetized triceps surae muscle at a depth of 3 cm and spaced 5 cm apart. Eight subjects received the diathermy treatment first, followed by the ultrasound treatment. This sequence was reversed for the remaining 8 subjects. The diathermy was applied at a frequency of 27.12 MHz at the following settings: 800 bursts per second, 400-microsecond burst duration, 850-microsecond interburst interval, peak root mean square amplitude of 150 W per burst, and an average root mean square output of 48 W per burst. The ultrasound was delivered at a frequency of 1 MHz and an intensity of 1.5 W/cm2 in the continuous mode for 20 minutes over an area of 40 times the effective radiating area. The study was performed in a ventilated research laboratory. Subjects: Sixteen (11 men, 5 women) healthy subjects (mean age = 23.56 +/- 4.73 years) volunteered to participate in this study. Measurements: We recorded baseline, final, and decay temperatures for each of the 3 sites. Results: The average temperature increases over baseline temperature after pulsed short-wave diathermy were 3.02 degrees C 1.02 degrees C in site 1, 4.58 degrees C +/- 0.87 degrees C in site 2, and 3.28 degrees C +/- 1.64 degrees C in site 3. The average temperature increases over baseline temperature after ultrasound were only 0.17 degrees C +/- 0.40 degrees C, 0.09 degrees C +/- 0.56 degrees C, and -0.43 degrees C +/- 0.41 degrees C in sites 1, 2, and 3, respectively. The temperature dropped only 1 degree C in 7.65 +/- 4.96 minutes after pulsed short-wave diathermy. Conclusions: We conclude that pulsed short-wave diathermy was more effective than 1-MHz ultrasound in heating a large muscle mass and resulted in the muscles' retaining heat longer. [ABSTRACT FROM AUTHOR]

Objective: Although ankle braces supposedly protect the ankle by providing mechanical support of the joint and enhancing proprioceptive input, their proprioceptive effects are unclear. Measuring the center of pressure during posture provides a reasonably well-controlled evaluation of proprioceptive input at the ankle. We, therefore, compared the changes in the center of pressure resulting from wearing ankle braces and wearing no brace (control). Design and Setting: Center-of-pressure variables were measured during a one-legged modified Romberg test with six variations. The six test conditions systematically altered the three sensory modalities that control posture: visual input, vestibular input, and proprioceptive input. Subjects performed three 16-second trials of each Romberg variation for each brace condition. Subjects: Twenty-four male volunteers (age = 18 to 26 yr) with no history of ankle injuries in the past 5 years and no difficulty with balance. Measurements: Center of pressure, transmitted through the bottom of the foot, was monitored during each trial and transformed into total distance traveled, anterior-posterior (AP) position, and medial-lateral (ML) position. Results: Average AP and ML center-of-pressure positions were increased only during brace wearing when all sensory modalities were functioning normally (control condition). Total center-of-pressure distance was the same for all three brace conditions. Conclusions: Our results do not support or refute the concept that bracing enhances proprioception. The fact that subjects relocated their center of pressure only during the control condition is perplexing. If braces were to enhance proprioception, one would expect to see lower average ML and average AP center-of-pressure values when comparing the braced with the unbraced conditions. Alternatively, the relocated position may represent a more stable position resulting from enhanced proprioception. [ABSTRACT FROM AUTHOR]

Objective: To compare the effects of tape, with and without prewrap, on dynamic ankle inversion before and after exercise. Design and Setting: Doubly multivariate analyses of variance were used to compare the taping and exercise conditions. Subjects were randomly assigned to a fixed treatment order as determined by a balanced latin square. The independent variables were tape application (no tape, tape with prewrap, tape to skin) and exercise (before and after). The dependent variables were average inversion velocity, total inversion, maximum inversion velocity, and time to maximum inversion. Subjects: Thirty college-age male and female students (17 males, 13 females; mean age = 24.9 +/- 4.3 years, range, 19 to 39 years) were tested. Subjects were excluded from the study if they exhibited a painful gait or painful range of motion or had a past history of ankle surgery or an ankle sprain within the past 4 weeks. Measurements: We collected data using electronic goniometers while subjects balanced on the right leg on an inversion platform tilted about the medial-lateral axis to produce 15 degrees of plantar flexion. Sudden ankle inversion was induced by pulling the inversion platform support, allowing the platform support base to rotate 37 degrees. Ten satisfactory trials were recorded on the inversion platform before and after a prescribed exercise bout. We calculated total inversion, time to maximum inversion, average inversion velocity, and maximum inversion velocity after sudden inversion. Results: We found no significant differences between taping to the skin and taping over prewrap for any of the variables measured. There were significant differences between both taping conditions and no-tape postexercise for average inversion velocity, maximum inversion, maximum inversion velocity, and time to maximum inversion. The total inversion mean for no-tape postexercise was 38.8 degrees +/- 6.3 degrees, whereas the means for tape and skin and for tape and prewrap were 28.3 degrees +/- 4.6 degrees and 29.1 degrees +/- 4.7 degrees, respectively. After exercise, inversion increased by 1.0 degrees +/- 2.8 degrees for the no-tape condition, whereas the tape-to-skin and tape-over-prewrap inversion increased by 2.1 degrees +/- 3.2 degrees and 1.7 degrees +/- 2.2 degrees, respectively. Conclusions: There was no difference in the amount of inversion restriction when taping with prewrap was compared with taping to the skin. Tape and tape with prewrap significantly reduced the average inversion velocity, maximum inversion, maximum inversion velocity, and the time to maximum inversion. Both taping conditions offered residual restriction after exercise. [ABSTRACT FROM AUTHOR]

Objective: Therapeutic ultrasound is an effective deep heating modality commonly applied alone or after cooling or heating of the treatment area. The purpose of this study was to examine the tissue temperature rise in the human triceps surae muscle group after ultrasound with prior heating via a silicate gel hot pack. Design and Setting: This study was designed as a 2 x 2 x 3 factorial with repeated measures on two factors (depth and time). Independent variables were temperature of pack (hot and room temperature), depth of measurement (1 cm and 3 cm), and time (beginning, after pack application, and after ultrasound). The dependent variable was tissue temperature. Subjects were assigned to one of two treatment groups: ultrasound preceded by a 15-minute hot pack treatment or ultrasound preceded by a 15-minute application with a silicate gel pack at room temperature. Measurements were taken while subjects were treated in a university training room. Subjects: Twenty-one uninjured male and female college student volunteers were randomly assigned to one of the two pack groups. Measurements: The hot packs were stored in 75 degrees C water. A 1-MHz ultrasound treatment was administered for 10 minutes at an intensity of 1.5 W/cm2 . Tissue temperature was measured every 30 seconds using 23-gauge hypodermic microprobes interfaced with a telethermometer and inserted 1 and 3 cm below the surface of anesthetized triceps surae muscle. Results: At both tissue depths, there was a 0.8 degrees C greater increase in tissue temperature with hot packs and ultrasound. At 1 cm, ultrasound increased temperature 3.5 degrees C after a 0.5 degrees C rise during the room temperature-pack application, but only 0.6 degrees C after a 3.8 degrees C increase during hot-pack application. At 3 cm, ultrasound increased temperature 3.85 degrees C following a slight (-0.26 degrees C) decrease during the room temperature-pack application and 3.68 degrees C after a 0.74 degrees C increase during hot-pack application. Conclusions: Vigorous increases in deep muscle temperature (>/= 4 degrees C) can be reached with 2 to 3 minutes less total sonation time when preheated with a hot pack. Thus, ultrasound and hot packs have an additive effect on intramuscular temperature, but the characteristics of the additive effect are different, primarily because there appears to be a tissue temperature plateau. [ABSTRACT FROM AUTHOR]

Graft-versus-host disease (GVHD) is a primary and often lethal complication of allogenic hematopoietic stem cell transplantation (HSCT). Prophylactic regimens for GVHD are given as standard pretransplantation therapy; however, up to 50% of these patients develop acute GVHD (aGVHD) and require additional immunosuppressive intervention. Using a mouse GVHD model, we previously showed that injecting mice with exopolysaccharide (EPS) from Bacillus subtilis prior to GVHD induction significantly increased 80-day survival after transplantation of complete allogeneic major histocompatibility complex-mismatched cells. To ask whether EPS might also inhibit GVHD in humans, we used humanized NSG-HLA-A2 mice and induced GVHD by i.v. injection of A2 neg human peripheral blood mononuclear cells (PBMCs). Because we could not inject human donors with EPS, we transferred EPS-pretreated dendritic cells (DCs) to inhibit aGVHD. We derived these DCs from CD34 + human cord blood cells, treated them with EPS, and then injected them together with PBMCs into the NSG-HLA-A2 mice. We found that all mice that received untreated DCs were dead by day 35, whereas 25% of mice receiving EPS-treated DCs (EPS-DCs) survived. This DC cell therapy could be readily translatable to humans, because we can generate large numbers of human EPS-DCs and use them as an "off the shelf" treatment for patients undergoing HSCT., (Copyright © 2023 The American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Introduction: Many well-known risk factors for breast cancer are associated with dysbiosis (an aberrant microbiome). However, how bacterial products modulate cancer are poorly understood. In this study, we investigated the effect of an exopolysaccharide (EPS) produced by the commensal bacterium Bacillus subtilis on breast cancer phenotypes. Although B. subtilis is commonly included in probiotic preparations and its EPS protects against inflammatory diseases, it was virtually unknown whether B. subtilis -derived EPS affects cancer., Methods: This work investigated effects of EPS on phenotypes of breast cancer cells as a cancer model. The phenotypes included proliferation, mammosphere formation, cell migration, and tumor growth in two immune compromised mouse models. RNA sequencing was performed on RNA from four breast cancer cells treated with PBS or EPS. IKKβ or STAT1 signaling was assessed using pharmacologic or RNAi-mediated knock down approaches., Results: Short-term treatment with EPS inhibited proliferation of certain breast cancer cells (T47D, MDA-MB-468, HCC1428, MDA-MB-453) while having little effect on others (MCF-7, MDA-MB-231, BT549, ZR-75-30). EPS induced G1/G0 cell cycle arrest of T47D cells while increasing apoptosis of MDA-MB-468 cells. EPS also enhanced aggressive phenotypes in T47D cells including cell migration and cancer stem cell survival. Long-term treatment with EPS (months) led to resistance in vitro and promoted tumor growth in immunocompromised mice. RNA-sequence analysis showed that EPS increased expression of pro-inflammatory pathways including STAT1 and NF-κB. IKKβ and/or STAT1 signaling was necessary for EPS to modulate phenotypes of EPS sensitive breast cancer cells., Discussion: These results demonstrate a multifaceted role for an EPS molecule secreted by the probiotic bacterium B. subtilis on breast cancer cell phenotypes. These results warrant future studies in immune competent mice and different cancer models to fully understand potential benefits and/or side effects of long-term use of probiotics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Nguyen, Ma, Wyatt, Knight and Osipo.)

Regulating B cell death is essential for generating antibodies and maintaining immune tolerance. B cells can die by apoptosis, and we report that human tonsil B cells, but not peripheral blood B cells also die by NETosis. This cell death is density-dependent, characterized by the loss of cell and nuclear membrane integrity, release of reactive oxygen species, and chromatin decondensation. Tonsil B cells secrete high levels of TNF, and inhibiting TNF prevented chromatin decondensation. By in situ fluorescence microscopy, B cell NETosis, as identified by the hyper citrullination of Histone-3, was localized to the light zone (LZ) of germinal centers in normal tonsil and overlapped with the B cell marker CD19/IgM. We propose a model in which stimulation of B cells in the LZ induces NETosis, driven in part by TNF. We also provide evidence that NETosis of tonsil B cells may be inhibited by an unidentified factor in tonsil. The results describe a previously unidentified form of B cell death and suggest a new mechanism to maintain B cell homeostasis during immune responses., (© 2023. The Author(s).)

The fetal and neonatal immune systems are uniquely poised to generate tolerance to self, maternal and environmental antigens encountered in the womb and shortly after birth. However, the tolerogenic nature of fetal and neonatal immunity can be detrimental in the context of pathogens, leading to overwhelming bacterial infections or chronic viral infections. A variety of mechanisms contribute to fetal and neonatal tolerance, including a propensity to generate Foxp3 + regulatory T cells (Treg cells). However, the mechanism(s) of fetal Foxp3 + T-cell differentiation, the specific antigen-presenting cells required and factors that inhibit Treg generation after the neonatal period are poorly understood. Here, we demonstrate that a subset of CD14 + monocytes expressing the scavenger molecule, CD36, can generate CD4 + and CD8 + T cells that coexpress Foxp3 and T-bet from both umbilical cord blood. These Foxp3 + T-bet + T cells potently suppress T-cell proliferation and ameliorate xenogeneic graft-versus-host disease. CD14 +  CD36 + monocytes provide known Treg-inducing signals: membrane-bound transforming growth factor-beta and retinoic acid. Unexpectedly, adult peripheral blood monocytes are also capable of inducing Foxp3 + T cells from both cord blood and adult peripheral naïve T cells. The induction of Foxp3 + T cells in umbilical cord blood by monocytes was inhibited by the lymphoid fraction of adult peripheral blood cells. These studies highlight a novel immunoregulatory role of monocytes and suggest that antigen presentation by CD36 hi monocytes may contribute to the peripheral development of Foxp3 + T-bet + T cells with regulatory functions in both neonates and adults., (© 2021 John Wiley & Sons Ltd.)

Fatty liver is the earliest and most common response of the liver to consumption of excessive alcohol. Steatosis can predispose the fatty liver to develop progressive liver damage. Chief among the many mechanisms involved in development of hepatic steatosis is dysregulation of insulin-mediated adipose tissue metabolism. Particularly, it is the enhanced adipose lipolysis-derived free fatty acids and their delivery to the liver that ultimately results in hepatic steatosis. The adipose-liver axis is modulated by hormones, particularly insulin and adiponectin. In recent studies, we demonstrated that an alcohol-induced increase in serum ghrelin levels impairs insulin secretion from pancreatic β-cells. The consequent reduction in circulating insulin levels promotes adipose lipolysis and mobilization of fatty acids to the liver to ultimately contribute to hepatic steatosis. Because many tissues, including adipose tissue, express ghrelin receptor we hypothesized that ghrelin may directly affect energy metabolism in adipocytes. We have exciting new preliminary data which shows that treatment of premature 3T3-L1 adipocytes with ghrelin impairs adipocyte differentiation and inhibits lipid accumulation in the tissue designed to store energy in the form of fat. We further observed that ghrelin treatment of differentiated adipocytes significantly inhibited secretion of adiponectin, a hepatoprotective hormone that reduces lipid synthesis and promotes lipid oxidation. These results were corroborated by our observations of a significant increase in serum adiponectin levels in ethanol-fed rats treated with a ghrelin receptor antagonist verses the un-treated ethanol-fed rats. Interestingly, in adipocytes, ghrelin also increases secretion of interleukin-6 (IL-6) and CCL2 (chemokine [C-C motif] ligand 2), cytokines which promote hepatic inflammation and progression of liver disease. To summarize, the alcohol-induced increase in serum ghrelin levels dysregulates adipose-liver interaction and promotes hepatic steatosis by increasing the free fatty acid released from adipose for hepatic uptake, and by altering adiponectin and cytokine secretion. Taken together, our data indicates that targeting the activity of ghrelin may be a powerful treatment strategy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Staphylococcus aureus is a Gram-positive opportunistic pathogen that causes a variety of diseases. Bloodstream infection is the most severe, with mortality rates reaching 20 to 50%. Exopolysaccharide (EPS) from the probiotic Bacillus subtilis reduces bacterial burden and inflammation during S. aureus bloodstream infection in mice. Protection is due, in part, to hybrid macrophages that restrict S. aureus growth through reactive oxygen species and to limiting superantigen-induced T cell activation and interferon gamma (IFN-γ) production during infection. A decrease in IFN-γ production was observed within 24 h after infection, and here, we investigated how EPS abrogates its production. We discovered that S. aureus uses a rapid, superantigen-independent mechanism to induce host IFN-γ and that this is mediated by interleukin-12 (IL-12) activation of NK cells. Furthermore, we found that EPS limits IFN-γ production by modulating host immunity in a Toll-like receptor 4 (TLR4)-dependent manner, a signaling pathway that is required for EPS-mediated protection from S. aureus infection in vivo We conclude that EPS protects hosts from acute bloodstream S. aureus infection not only by inducing macrophages that restrict S. aureus growth and inhibit superantigen-activated T cells but also by limiting NK cell production of IFN-γ after S. aureus infection in a TLR4-dependent manner., (Copyright © 2020 American Society for Microbiology.)

Vitiligo is impacted by environmental triggers. We studied the contribution of the microbiome in FH mice, in which depigmentation is mediated by tyrosinase-reactive T cells. The mice received oral antibiotics and were monitored for depigmentation. The microbiome was studied in fecal and skin samples using 16S rRNA analysis. The resulting T-cell distributions were evaluated. In untreated mice, pigment loss did not expand to the pelage, whereas mice in the ampicillin group were approximately 1/3 depigmented at 30 weeks. In contrast to models of autoimmunity that are less dependent on IFN-γ, ampicillin but not neomycin treatment correlated with accelerated disease and reduced bacteria in the fecal pellets. Modified cytokine patterns in the tissue and serum suggest a response that transcends the gut. Ampicillin-induced depigmentation was accompanied by gut but not skin dysbiosis, and reduced T cell numbers in both sites. Neomycin induced a redistribution of gut T cells and an accumulation of skin regulatory T cells. This treatment spurred a Bacteroides-dominated population of fecal bacteria. Reduced diversity is prominent particularly after ampicillin treatment, when the gut is dominated by Pseudomonas species. In line with current concepts relating the microbiome and the immune system, we predict that dietary measures might promote skin health and delay vitiligo onset., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Staphylococcus aureus causes severe systemic infection with high mortality rates. We previously identified exopolysaccharide (EPS) from a probiotic, Bacillus subtilis , that induces anti-inflammatory macrophages with an M2 phenotype and protects mice from Citrobacter rodentium -induced colitis. We tested if EPS could protect from systemic infection induced by S. aureus and found that EPS-treated mice had enhanced survival as well as reduced weight loss, systemic inflammation, and bacterial burden. While macrophages from EPS-treated mice display an M2 phenotype, they also restrict growth of internalized S. aureus through reactive oxygen species (ROS), reminiscent of proinflammatory phagocytes. These EPS-induced macrophages also limit T cell activation by S. aureus superantigens, and EPS abrogates systemic induction of gamma interferon after infection. We conclude that B. subtilis EPS is an immunomodulatory agent that induces hybrid macrophages that bolster antibacterial immunity and simultaneously limit inflammation, reducing disease burden and promoting host survival., (Copyright © 2018 American Society for Microbiology.)

In mammals, the most striking IgA system belongs to Lagomorpha. Indeed, 14 IgA subclasses have been identified in European rabbits, 11 of which are expressed. In contrast, most other mammals have only one IgA, or in the case of hominoids, two IgA subclasses. Characteristic features of the mammalian IgA subclasses are the length and amino acid sequence of their hinge regions, which are often rich in Pro, Ser and Thr residues and may also carry Cys residues. Here, we describe a new IgA that was expressed in New Zealand White domestic rabbits of IGHVa1 allotype. This IgA has an extended hinge region containing an intriguing stretch of nine consecutive Ser residues and no Pro or Thr residues, a motif exclusive to this new rabbit IgA. Considering the amino acid properties, this hinge motif may present some advantage over the common IgA hinge by affording novel functional capabilities. We also sequenced for the first time the IgA14 CH2 and CH3 domains and showed that IgA14 and IgA3 are expressed., Competing Interests: The authors have declared that no competing interests exist.

Studies using the European rabbit Oryctolagus cuniculus contributed to elucidating numerous fundamental aspects of antibody structure and diversification mechanisms and continue to be valuable for the development and testing of therapeutic humanized polyclonal and monoclonal antibodies. Additionally, during the last two decades, the use of the European rabbit as an animal model has been increasingly extended to many human diseases. This review documents the continuing wide utility of the rabbit as a reliable disease model for development of therapeutics and vaccines and studies of the cellular and molecular mechanisms underlying many human diseases. Examples include syphilis, tuberculosis, HIV-AIDS, acute hepatic failure and diseases caused by noroviruses, ocular herpes, and papillomaviruses. The use of rabbits for vaccine development studies, which began with Louis Pasteur's rabies vaccine in 1881, continues today with targets that include the potentially blinding HSV-1 virus infection and HIV-AIDS. Additionally, two highly fatal viral diseases, rabbit hemorrhagic disease and myxomatosis, affect the European rabbit and provide unique models to understand co-evolution between a vertebrate host and viral pathogens.

Although IgD first appeared, along with IgM, in the cartilaginous fishes and has been retained throughout subsequent vertebrate evolution, it has been lost in a diverse group of vertebrate species. We previously showed that, unlike vertebrates that express IgD, the rabbit lacks an IgD (Cδ) gene within 13.5 kb downstream of the IgM gene. We report here that, by conducting BLAST searches of rabbit Ig heavy chain genomic DNA with known mammalian IgD exons, we identified the remnant of the rabbit Cδ gene approximately 21 kb downstream of the IgM gene. The remnant Cδ locus lacks the δCH1 and hinge exons, but contains truncated δCH2 and δCH3 exons, as well as largely intact, but non-functional, secretory and transmembrane exons. In addition, we report that the Cδ gene probably became non-functional in leporids at least prior to the divergence of rabbits and hares ~12 million years ago.

B lymphopoiesis is necessary to generate a diverse pool of naïve B cells that are able to respond to a broad spectrum of antigens during immune responses to pathogens and to vaccination. Rabbits have been utilized for many years to generate high affinity monoclonal and polyclonal antibodies. Specific antibodies generated in rabbits have greatly advanced scientific discoveries, but the unique qualities of rabbit B cell development have been underappreciated. Unlike in humans and mice, where B lymphopoiesis declines in mid to late life, B lymphopoiesis in rabbits arrests early in life, between 2 and 4 months of age. This review focuses on the early loss of B cell development in rabbits and the contribution of the bone marrow microenvironment to this process. We also propose directions for future research in this area, and discuss how the rabbit can be used as a model to understand the decline of B lymphopoiesis that occurs in humans late in life. Such studies will be important for developing therapeutics targeted to prevent and/or reverse declining B lymphopoiesis in the elderly, as well as boosting immunity and antibody responses after infection or vaccination., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Clostridium difficile infection (CDI) is a major cause of health care-associated disease. CDI initiates with ingestion of C. difficile spores, germination in the gastrointestinal (GI) tract, and then colonization of the large intestine. The interactions between C. difficile cells and other bacteria and with host mucosa during CDI remain poorly understood. Here, we addressed the hypothesis that, in a mouse model of CDI, C. difficile resides in multicellular communities (biofilms) in association with host mucosa. To do this, we paraffin embedded and then sectioned the GI tracts of infected mice at various days postinfection (p.i.). We then used fluorescent in situ hybridization (FISH) with 16S rRNA probes targeting most bacteria as well as C. difficile specifically. The results revealed that C. difficile is present as a minority member of communities in the outer (loose) mucus layer, in the cecum and colon, starting at day 1 p.i. To generate FISH probes that identify bacteria within mucus-associated communities harboring C. difficile, we characterized bacterial populations in the infected mouse GI tract using 16S rRNA gene sequence analysis of bacterial DNA prepared from intestinal content. This analysis revealed the presence of genera of several families belonging to Bacteroidetes and Firmicutes. These data suggest that formation of multispecies communities associated with the mucus of the cecum and colon is an important early step in GI tract colonization. They raise the possibility that other bacterial species in these communities modulate the ability of C. difficile to successfully colonize and, thereby, cause disease., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Clostridium difficile rivals methicillin-resistant Staphylococcus aureus as the primary hospital-acquired infection. C. difficile infection (CDI) caused by toxins A and/or B can manifest as mild diarrhea to life-threatening pseudomembranous colitis. Although most patients recover fully from CDI, ~20% undergo recurrent disease. Several studies have demonstrated a correlation between anti-toxin antibody (Ab) and decreased recurrence; however, the contributions of the systemic and mucosal Ab responses remain unclear. Our goal was to use the CDI mouse model to characterize the protective immune response to C. difficile. C57BL/6 mice infected with epidemic C. difficile strain BI17 developed protective immunity against CDI and did not develop CDI upon rechallenge; they generated systemic IgG and IgA as well as mucosal IgA Ab to toxin. To determine if protective immunity to C. difficile could be generated in immunodeficient individuals, we infected CD4(-/-) mice and found that they generated both mucosal and serum IgA anti-toxin Abs and were protected from CDI upon rechallenge, with protection dependent on major histocompatibility complex class II (MHCII) expression; no IgG anti-toxin Ab was found. We found that protection was likely due to neutralizing mucosal IgA Ab. In contrast, pIgR(-/-) mice, which lack the receptor to transcytose polymeric Ab across the epithelium, were also protected from CDI, suggesting that although mucosal anti-toxin Ab may contribute to protection, it is not required. We conclude that protection from CDI can occur by several mechanisms and that the mechanism of protection is determined by the state of immunocompetence of the host.

The ability to grow as a biofilm can facilitate survival of bacteria in the environment and promote infection. To better characterize biofilm formation in the pathogen Clostridium difficile, we established a colony biofilm culture method for this organism on a polycarbonate filter, and analyzed the matrix and the cells in biofilms from a variety of clinical isolates over several days of biofilm culture. We found that biofilms readily formed in all strains analyzed, and that spores were abundant within about 6 days. We also found that extracellular DNA (eDNA), polysaccharide and protein was readily detected in the matrix of all strains, including the major toxins A and/or B, in toxigenic strains. All the strains we analyzed formed spores. Apart from strains 630 and VPI10463, which sporulated in the biofilm at relatively low frequencies, the frequencies of biofilm sporulation varied between 46 and 65%, suggesting that variations in sporulation levels among strains is unlikely to be a major factor in variation in the severity of disease. Spores in biofilms also had reduced germination efficiency compared to spores obtained by a conventional sporulation protocol. Transmission electron microscopy revealed that in 3 day-old biofilms, the outermost structure of the spore is a lightly staining coat. However, after 6 days, material that resembles cell debris in the matrix surrounds the spore, and darkly staining granules are closely associated with the spores surface. In 14 day-old biofilms, relatively few spores are surrounded by the apparent cell debris, and the surface-associated granules are present at higher density at the coat surface. Finally, we showed that biofilm cells possess 100-fold greater resistance to the antibiotic metronidazole then do cells cultured in liquid media. Taken together, our data suggest that C. difficile cells and spores in biofilms have specialized properties that may facilitate infection.

Context: In the electrically induced cramp model, the tibial nerve is stimulated at an initial frequency of 4 Hz with increases in 2-Hz increments until the flexor hallucis brevis cramps. The frequency at which cramping occurs (ie, threshold frequency [TF]) can vary considerably. A potential limitation is that multiple subthreshold stimulations before TF might induce fatigue, which is operationally defined as a decrease in maximal voluntary isometric contraction (MVIC) force, thereby biasing TF., Objective: To determine if TF is similar when initially stimulated at 4 Hz or 14 Hz and if MVIC force is different among stimulation frequencies or over time (precramp, 1 minute postcramp, and 5 minutes postcramp)., Design: Crossover study., Setting: Laboratory. Patients or Other Participants: Twenty participants (13 males: age = 20.6 ± 2.9 years, height = 184.4 ± 5.7 cm, mass = 76.3 ± 7.1 kg; 7 females: age = 20.4 ± 3.5 years, height = 166.6 ± 6.0 cm, mass = 62.4 ± 10.0 kg) who were prone to cramps., Intervention(s): Participants performed 20 practice MVICs. After a 5-minute rest, three 2-second MVICs were recorded and averaged for the precramp measurement. Participants were stimulated at either 4 Hz or 14 Hz, and the frequency was increased in 2-Hz increments from each initial frequency until cramp. The MVIC force was reevaluated at 1 minute and 5 minutes postcramp., Main Outcome Measure(s): The TF and MVIC force., Results: Initial stimulation frequency did not affect TF (4 Hz = 16.2 ± 3.8 Hz, 14 Hz = 17.1 ± 5.0 Hz; t(19) = 1.2, P = .24). Two participants had inaccurate TFs when initially stimulated at 14 Hz; they cramped at 10 and 12 Hz in the 4-Hz condition. The MVIC force did not differ between initial frequencies (F(1,19) = 0.9, P = .36) but did differ over time (F(2,38) = 5.1, P = .01). Force was lower at 1 minute postcramp (25.1 ± 10.1 N) than at precramp (28.7 ± 7.8 N; P, .05) but returned to baseline at 5 minutes postcramp (26.7 ± 8.9 N; P > .05)., Conclusions: The preferred initial stimulation frequency might be 4 Hz because it did not alter or overestimate TF. The MVIC force was lower at 1 minute postcramp, suggesting the induced cramp rather than the varying electrical frequencies affected force. A 1- to 5-minute rest should be provided postcramp induction if multiple cramps are induced.

Context: Researchers have observed slower cooling rates in thigh muscle with greater overlying adipose tissue, suggesting that cryotherapy duration should be based on the adipose thickness of the treatment site. Skinfold data do not exist for other common cryotherapy sites, and no one has reported how those skinfolds might vary because of physical activity level or sex., Objective: To determine the variability in skinfold thickness among common cryotherapy sites relative to sex and activity level (National Collegiate Athletic Association Division I athletes, recreationally active college athletes)., Design: Descriptive laboratory study., Setting: Field., Patients or Other Participants: Three hundred eighty-nine college students participated; 196 Division I athletes (157 men, 39 women) were recruited during preseason physicals, and 193 recreationally active college athletes (108 men, 85 women) were recruited from physical education classes., Intervention(s): Three skinfold measurements to within 1 mm were taken at 8 sites (inferior angle of the scapula, middle deltoid, ulnar groove, midforearm, midthigh, medial collateral ligament, midcalf, and anterior talofibular ligament [ATF]) using Lange skinfold calipers., Main Outcome Measure(s): Skinfold thickness in millimeters., Results: We noted interactions among sex, activity level, and skinfold site. Male athletes had smaller skinfold measurements than female athletes at all sites except the ATF, scapula, and ulnar groove (F₇,₂₇₀₂ = 69.85, P < .001). Skinfold measurements were greater for recreationally active athletes than their Division I counterparts at all sites except the ATF, deltoid, and ulnar groove (F₇,₂₇₀₂ = 30.79, P < .001). Thigh skinfold measurements of recreationally active female athletes were the largest, and their ATF skinfolds were the smallest., Conclusions: Skinfold thickness at common cryotherapy treatment sites varied based on level of physical activity and sex. Therefore, clinicians should measure skinfold thickness to determine an appropriate cryotherapy duration.

Commensals limit disease caused by invading pathogens; however, the mechanisms and genes utilized by beneficial microbes to inhibit pathogenesis are poorly understood. The attaching and effacing mouse pathogen Citrobacter rodentium associates intimately with the intestinal epithelium, and infections result in acute colitis. C. rodentium is used to model the human pathogens enterohemorrhagic Escherichia coli and enteropathogenic E. coli. To confirm that Bacillus subtilis, a spore-forming bacterium found in the gut of mammals, could reduce C. rodentium-associated disease, mice received wild-type B. subtilis spores and 24 h later were infected by oral gavage with pathogenic C. rodentium. Disease was assessed by determining the extent of colonic epithelial hyperplasia, goblet cell loss, diarrhea, and pathogen colonization. Mice that received wild-type B. subtilis prior to enteric infection were protected from disease even though C. rodentium colonization was not inhibited. In contrast, espH and hag mutants, defective in exopolysaccharides and flagellum production, respectively, did not protect mice from C. rodentium-associated disease. A motAB mutant also failed to protect mice from disease, suggesting that B. subtilis-mediated protection requires functional flagella. By expanding our current mechanistic knowledge of bacterial protection, we can better utilize beneficial microbes to prevent intestinal disease caused by pathogenic bacteria, ultimately reducing human disease. Our data demonstrate that wild-type B. subtilis reduced disease caused by C. rodentium infection through a mechanism that required espH and functional flagella.

Objective: A human thymic epithelial cell (TEC) line expressing human leukocyte antigen-ABC and human leukocyte antigen-DR was engineered to overexpress murine Delta-like 1 (TEC-Dl1) for the purpose of establishing a human culture system that supports T lymphopoiesis from hematopoietic progenitor cells (HPCs)., Materials and Methods: Cord blood or bone marrow HPCs were co-cultured with either the parental TEC line expressing low levels of the Notch ligands, Delta-like 1 and Delta-like 4, or with TEC-Dl1 to determine if these cell lines support human lymphopoiesis., Results: In co-cultures with cord blood or bone marrow HPCs, TEC-Dl1 cells promote de novo generation of CD7(pos)CD1a(pos) T-lineage committed cells. Most CD7(pos)CD1a(hi) cells are CD4(pos)CD8(pos) double-positive (DP). We found that TEC-Dl1 cells are insufficient to generate mature CD3(hi) CD4(pos) or CD3(hi) CD8(pos) single-positive (SP) T cells from the CD4(pos)CD8(pos) DP T cells; however, we detected CD3(lo) cells within the DP and SP CD4 and CD8 populations. The CD3(lo) SP cells expressed lower levels of interleukin-2Rα and interleukin-7Rα compared to CD3(lo) DP cells. In contrast to the TEC-Dl1 line, the parental TEC-84 line expressing low levels of human Notch ligands permits HPC differentiation to the B-cell lineage., Conclusions: We report for the first time a human TEC line that supports lymphopoiesis from cord blood and bone marrow HPC. The TEC cell lines described herein provide a novel human thymic stroma model to study the contribution of human leukocyte antigen molecules and Notch ligands to T-cell commitment and maturation and could be utilized to promote lymphopoiesis for immune cell therapy., (Copyright © 2011 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

B lymphopoiesis arrests in rabbits by 4 months of age. To identify molecules that contribute to this arrest, cDNA-representational difference analysis on BM stromal cells from young and adult rabbits showed that expression of Postn that encodes for the extracellular matrix protein periostin dramatically reduced with age. Postn-small interfering RNA OP9 cells lost their capacity to support B-cell development from rabbit or murine BM cells, and reexpression of periostin restored this potential, indicating an in vitro requirement for periostin in B lymphopoiesis. In our system, we determined that periostin deficiency leads to increased cell death and decreased proliferation of B-lineage progenitors. Further, RGD peptide inhibition of periostin/α(v)β(3) interaction resulted in a marked decrease in B lymphopoiesis in vitro. Microarray analysis of the Postn-small interfering RNA OP9 cells showed decreased expression of key B-lymphopoietic factors, including IL-7 and CXCL12. In vivo, unidentified molecule(s) probably compensate periostin loss because Postn(-/-) mice had normal numbers of B-cell progenitors in BM. We conclude that the decline in periostin expression in adult rabbit BM does not solely explain the arrest of B lymphopoiesis. However, the interaction of periostin with α(v)β(3) on lymphoid progenitors probably provides both proliferative and survival signals for cells in the B-cell development pathway.