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After having used virtual reality with patients, the hospital of Valenciennes (59) made the bet that its use, associated with a hypnotic speech combining cardiac coherence, selected images and special filming techniques, would be likely to cause a significant reduction of stress and a release of emotions in health professionals. A use which would allow, during the same session, to re-energize them so that they can resume their activity.

This article discusses skill proficiency of providers related to emergency cricothyroidotomies. Various techniques to improve procedural skills were studied. Accurate identification of the cricothyroid membrane via palpation remained consistently inadequate. High-fidelity simulation including the use of human cadavers may be the preferred method of skill training for crisis management. The authors emphasize that additional research is needed regarding a method for rapid cricothyroid membrane identification as well as needle cricothyroidotomy versus surgical airway on cadavers. More consistent training will enable emergency care providers to perform this rare but lifesaving skill.

Total ankle arthroplasty (TAA) is a common surgical approach for patients with end-stage ankle osteoarthritis (OA). However, very little is known about the path of the center of pressure (COP) of the foot, and thus important aspects of load transfer, muscle mechanical advantage, and balance, in patients before or after surgery. The objective of this study was to trace the pathway of the COP under the foot in patients with symptomatic ankle OA, comparing asymmetry between affected and unaffected limbs. From force plate data, proximo-distal and medio-lateral position of the COP beneath the foot were calculated and compared for the affected and unaffected foot in patients with unilateral ankle OA (N=93) before and after TAA. Gender and age at surgery had little or no effect in this study. Patients with ankle OA had minimal COP position asymmetry before surgery, and this asymmetry was reduced following surgery. Prior to surgery, patients had a slower walking speed and a shorter path of the COP which began relatively distal to the heel and ended relatively proximal to the hallux. TAA increased the proximo-distal distance the COP traveled under both the unaffected and affected foot, a pattern that was maintained for over two years post-surgery. TAA allows patients with ankle OA to maintain a longer COP path than they had before surgery on both sides that is closer to that reported for unaffected individuals, extending effectively from the heel to the hallux, potentially improving pedal mechanics. This article is protected by copyright. All rights reserved.

Autophagy is dependent on appropriate lipid supply for autophagosome formation. The regulation of lipid acquisition and the autophagy network response to lipid-limiting conditions are mostly elusive. Here, we show that the knockout of the RAB GTPase RAB18 interferes with lipid droplet catabolism, causing an impaired fatty acid release. The resulting reduced lipid-droplet-derived lipid availability influences autophagy and provokes adaptive modifications of the autophagy network. These adjustments include increased expression and phosphorylation of ATG2B as well as augmented formation of the ATG12-ATG5 conjugate. Moreover, ATG9A shows an enhanced phosphorylation at amino acid residues tyrosine 8 and serine 14, resulting in an increased ATG9A trafficking. Via pharmacological inhibition of Y8 phosphorylation, we demonstrate that this ATG9A modification is important to maintain basal autophagy under RAB18 knockout conditions. However, while the network adaptations are sufficient to maintain basal autophagic activity, they are incapable of ensuring autophagy induction upon starvation, which is characterized by an enhanced lipid demand. Thus, here, we define the molecular role of RAB18 in connecting lipid droplets and autophagy, emphasize the significance of lipid droplets as lipid sources for the degradative pathway, and uncover a remarkable autophagy network plasticity, including phosphorylation-dependent ATG9A activation, to compensate reduced lipid availability in order to rescue basal autophagic activity.

Previous studies of level running suggest runners adjust foot strike to control leg stiffness. This study aimed to determine how runners adjusted mechanical stiffness and foot strike prior to, during, and after a drop in surface height. Ten healthy subjects (5 male, 5 female; 24.32 ± 5.0 years) were video recorded as they ran on an outdoor path with a single drop in surface height (12.5 cm). Foot strike was recorded, while subject velocity, duty factor (DF), normalized maximum ground reaction force (GRF

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is a predominantly fatal common malignancy with inadequate treatment options. Glycogen synthase kinase 3β (GSK-3β) is an emerging target in human malignancies including PDAC. Experimental Design: Pancreatic cancer cell lines and patient-derived xenografts were treated with a novel GSK-3 inhibitor 9-ING-41 alone or in combination with chemotherapy. Activation of the DNA damage response pathway and S-phase arrest induced by gemcitabine were assessed in pancreatic tumor cells with pharmacologic inhibition or siRNA depletion of GSK-3 kinases by immunoblotting, flow cytometry, and immunofluorescence. Results: 9-ING-41 treatment significantly increased pancreatic tumor cell killing when combined with chemotherapy. Inhibition of GSK-3 by 9-ING-41 prevented gemcitabine-induced S-phase arrest suggesting an impact on the ATR-mediated DNA damage response. Both 9-ING-41 and siRNA depletion of GSK-3 kinases impaired the activation of ATR leading to the phosphorylation and activation of Chk1. Mechanistically, depletion or knockdown of GSK-3 kinases resulted in the degradation of the ATR-interacting protein TopBP1, thus limiting the activation of ATR in response to single-strand DNA damage. Conclusions: These data identify a previously unknown role for GSK-3 kinases in the regulation of the TopBP1/ATR/Chk1 DNA damage response pathway. The data also support the inclusion of patients with PDAC in clinical studies of 9-ING-41 alone and in combination with gemcitabine.

Objectives Foot and ankle dysfunction in barefoot/minimally shod populations remains understudied. Although factors affecting musculoskeletal pain in Western populations are well-studied, little is known about how types of work, gender, and body shape influence bone and joint health in non-Western and minimally shod communities. This study examines the effect of human variation on locomotor disability in an agrarian community in Madagascar. Materials and methods Foot measurements were collected along with height, weight, age, and self-report data on daily activity and foot and ankle pain from 41 male and 48 female adults. A short form revised foot function index (FFI-R), that measures functional disability related to foot pain, was calculated. Raw and normalized foot measurements were compared by gender and used in a multiple linear regression model to determine predictors of FFI-R. Results Compared to men, women reported higher FFI-R scores (p = 0.014), spent more time on their feet (p = 0.019), and had higher BMIs (p = 0.0001). For their weight, women had significantly smaller and narrower feet than men. Bimalleolar breadth (p = 0.0005) and foot length (p = 0.0223) standardized by height, time spent on feet (p = 0.0102), ankle circumference standardized by weight (p = 0.0316), and age (p = 0.0090) were significant predictors of FFI-R score. Discussion Our findings suggest that human variation in anatomical and behavioral patterns serve as significant explanations for increased foot and ankle pain in women in this non-Western rural population. Foot and ankle pain were prevalent at similar levels to those in industrialized populations, indicating that research should continue to examine its effect on similar barefoot/minimally shod communities.

Targeting the B-cell receptor and phosphatidylinositol 3-kinase/mTOR signaling pathways has shown meaningful, but incomplete, antitumor activity in lymphoma. Glycogen synthase kinase 3 (GSK3) α and β are 2 homologous and functionally overlapping serine/threonine kinases that phosphorylate multiple protein substrates in several key signaling pathways. To date, no agent targeting GSK3 has been approved for lymphoma therapy. We show that lymphoma cells abundantly express GSK3α and GSK3β compared with normal B and T lymphocytes at the messenger RNA and protein levels. Utilizing a new GSK3 inhibitor 9-ING-41 and by genetic deletion of GSK3α and GSK3β genes using CRISPR/CAS9 knockout, GSK3 was demonstrated to be functionally important to lymphoma cell growth and proliferation. GSK3β binds to centrosomes and microtubules, and lymphoma cells treated with 9-ING-41 become arrested in mitotic prophase, supporting the notion that GSK3β is necessary for the progression of mitosis. By analyzing recently published RNA sequencing data on 234 diffuse large B-cell lymphoma patients, we found that higher expression of GSK3α or GSK3β correlates well with shorter overall survival. These data provide rationale for testing GSK3 inhibitors in lymphoma patient trials.

BACKGROUND total hip arthroplasty (THA) is associated with decreased pain and improved function, including increased walking speed, but it does not always improve overall joint mechanics during activities of daily living such as level walking and stair climbing. The hip's ability to generate power to move and allow for smooth and efficient forward motion is critical to success after surgery. Although osteoarthritis (OA) of the hip limits the power of the affected joint, it is not known whether other joints in the affected limb or in the contralateral limb need to produce more power to compensate. Additionally, it is not known whether alterations in the production of power before and after surgery are gender-specific. QUESTIONS/PURPOSES (1) Is there a change in the power production of the bilateral ankles, knees, and hips during level walking before and after patients undergo unilateral THA, and are there important gender-specific differences in these findings? (2) How do these findings differ for stair climbing? METHODS Three-dimensional motion and ground reaction force data were collected for 13 men and 13 women who underwent primary, unilateral THA. This was a secondary analysis of previously collected data on gait mechanics from 60 patients who underwent THA. In the initial study, patients were included if they were scheduled to undergo a primary, unilateral THA within 4 weeks of the study and were able to walk without an assistive device. Patients were recruited from the practices of four surgeons at a single institution from 2008 to 2011. Patients were included in the current study if they were enrolled in the previous study, attended all three assessment visits (preoperative and 6 weeks and 1 year postoperative), and, during the preoperative visit, were able to walk without using an assistive device and climb stairs without using a handrail. Patients walked and ascended stairs at a self-selected speed at the three assessment visits. The power of each ankle, knee, or hip was calculated in Visual 3D using kinematic and kinetic data collected using motion capture. Power for each joint was normalized to the total power of the bilateral lower limbs by dividing the individual joint power by the total lower-extremity joint power. A mixed-model repeated-measures ANOVA was used to determine differences in normalized joint power for the ankle, knee, and hip, based on gender, limb (surgical-side versus nonsurgical-side) and timepoint (preoperative and 6 weeks and 1 year postoperative). RESULTS Surgical-side absolute (preoperative: -0.2 ± 0.2 [CI, -0.3 to -0.2], 1 year postoperative: -0.5 ± 0.3 [CI, -0.6 to - 0.5]; p < 0.001) and normalized (preoperative: 0.05 ± 0.04 [CI, 0.03-0.06], 1 year postoperative: 0.08 ± 0.04 [CI, 0.06-0.09]; p = 0.020) hip power production increased during walking. Surgical-side absolute (preoperative: 1.1 ± 0.3 [CI, 1.0-1.3], 1 year postoperative: 1.6 ± 0.2 [CI, 1.3-2.0]; p = 0.005) and normalized (preoperative: 0.16 ± 0.04 [CI, 0.14-0.18], 1 year postoperative: 0.21 ± 0.06 [CI, 0.18-0.24]; p = 0.008) hip power production increased during stair climbing, while nonsurgical ankle absolute (preoperative: 0.9 ± 0.5 [CI, 0.6 - 1.2], 1 year postoperative: 0.6 ± 0.3 [CI, 0.4-0.8]; p = 0.064) and normalized (preoperative: 0.13 ± 0.06 [CI, 0.10-0.16], 1 year postoperative: 0.08 ± 0.04 [CI, 0.06-0.10]; p = 0.015) power decreased during stair climbing after THA. No consistent effect of gender was observed. CONCLUSIONS In this gait-analysis study, power was improved in hip joints that were operated on, and power production in the ipsilateral and contralateral ankles and ipsilateral hips was reduced during level walking and stair climbing. The success of surgical intervention must be based on restoring reasonable balance of forces in the lower limb. Patients with OA of the hip lose power production in this joint and must compensate for the loss by producing power in other joints, which then may become arthritic. To determine future interventions, an understanding of whether changes in forces or joint angle affect the change in joint power is needed. Based on these results, THA appeared to effectively increase hip power and reduce the need for compensatory power production in other joints for both men and women in this patient cohort. LEVEL OF EVIDENCE Level I, prognostic study.

Background Racial differences in gait mechanics have been recently reported, but we don’t know what factors may drive differences in gait and whether these factors are innate or modifiable. The answers to those questions will inform both basic research and clinical interventions and outcomes. Research question Do anthropometric, strength, and health status measures explain racial differences in gait between African Americans (AA) and white Americans (WA)? Methods Venous blood samples, anthropometric measures, lower extremity strength, and an assessment of health status were collected from 92 participants (18–30 years old) as part of an Institutional Review Board-approved study. 3D motion capture and force plate data were recorded during 7 walking trials at set regular (1.35 m/s) and fast (1.6 m/s) speeds. Racial differences in gait were identified at both speeds. Correlations between anthropometric, strength, and health status independent variables and outcome measures were computed after stratifying data by sex. Stepwise linear regression models evaluated whether the inclusion of anthropometric, strength, and health status independent variables explained racial effects. Results In males, no racial differences in gait were explained by independent variables. Q-angle and ankle dorsiflexion strength accounted for racial differences in self-selected walking speed in females. Racial differences in ankle plantarflexion angle were explained by ankle plantarflexion strength differences. Significance Factors that explain racial differences in gait in females were both innate and modifiable. These data make clear that it is important to include racially diverse normative gait databases in research studies. These results also identify potential intervention targets aimed at reducing racial health disparities.

The effect of race has rarely been investigated in biomechanics studies despite racial health disparities in the incidence of musculoskeletal injuries and disease, hindering both treatment and assessment of rehabilitation. The purpose of this study was to test the hypothesis that racial differences in gait mechanics exist between African Americans (AA) and white Americans (WA). Ninety-two participants (18–30 years old) were recruited with equal numbers in each racial group and sex. Self-selected walking speed was measured for each participant. 3D motion capture and force plate data were recorded during 7 walking trials at regular and fast set speeds. Step length, step width, peak vertical ground reaction force, peak hip extension, peak knee flexion, and peak ankle plantarflexion were computed for all trials at both set speeds. Multivariate and post-hoc univariate ANOVA models were fit to determine main and interaction effects of sex and race (SPSS V26, α = 0.05). Self-selected walking speed was slower in AA (p = 0.004, ƞp2 = 0.088). No significant interactions between race and sex were identified. Males took longer steps (regular: p

Gaze-tracking techniques have advanced our understanding of visual attention and decision making during walking and athletic events, but little is known about how vision influences behavior during running over common, natural obstacles. This study tested hypotheses about whether runners regularly collect visual information and pre-plan obstacle clearance (feedforward control), make improvisational adjustments (online control), or some combination of both. In this study, the gaze profiles of 5 male and 5 female runners, fitted with a telemetric gaze-tracking device, were used to identify the frequency of fixations on an obstacle during a run. Overall, participants fixated on the obstacle 2.4 times during the run, with the last fixation occurring on average between 40% and 80% of the run, suggesting runners potentially shifted from a feedforward planning strategy to an online control strategy during the late portions of the running trial. A negative association was observed between runner velocity and average number of fixations. Consistent with previous studies on visual strategies used during walking, our results indicate that visual attentiveness is part of an important feedforward strategy for runners allowing them to safely approach an obstacle. Thus, visual obstacle attention is a key factor in the navigation of complex, natural landscapes while running.

Two-dimensional video analysis is commonly used to assess kinematics when three-dimensional motion capture is unavailable. However, videos are often assessed using manual digitization, which limits the ability to extract outcomes that require continuous data. Here, we introduced a method to collect continuous kinematic data in 2D using an inexpensive camera and an open-source automated marker tracking program. We tested the validity of this method by comparing 2D video analysis to 3D motion capture for measuring sagittal-plane running kinematics. Twenty uninjured participants ran on a treadmill for 1-min while lower extremity kinematics were collected simultaneously in 3D using a motion capture system and in 2D using a single digital camera, both at 120 Hz. Knee, ankle, and foot angle at contact, peak knee flexion, knee flexion excursion, and knee-ankle flexion vector coding variability were computed using both the 3D and 2D kinematic data, and were compared using intraclass correlation coefficients and Bland-Altman plots. The agreement between collection methods was excellent for foot angle at contact and knee flexion excursion, good for ankle and knee angle at contact and knee-ankle vector coding variability, and moderate for peak knee flexion. However, Bland-Altman plots revealed significant differences between the 2D and 3D collection methods, which varied across study participants. These low-cost methods could be useful for collecting continuous sagittal plane running kinematics in non-laboratory settings.

Glycogen synthase kinase-3 beta (GSK-3β), a serine/threonine kinase, has been identified as a potential therapeutic target in human bladder cancer. In the present study, we investigated the antitumor effect of a small molecule GSK-3β inhibitor, 9-ING-41, currently in clinical studies in patients with advanced cancer, in bladder cancer cell lines. We found that treatment with 9-ING-41 leads to cell cycle arrest, autophagy and apoptosis in bladder cancer cells. The autophagy inhibitor chloroquine potentiated the antitumor effects of 9-ING-41 when tested in combination studies. Our findings also demonstrate that 9-ING-41 enhanced the growth inhibitory effects of gemcitabine or cisplatin when used in combination in bladder cancer cells. Finally, we found that 9-ING-41 sensitized bladder cancer cells to the cytotoxic effects of human immune effector cells. Our results provide a rationale for the inclusion of patients with advanced bladder cancer in clinical studies of 9-ING-41.

Gait and movement asymmetries are important variables for assessing locomotor mechanics in humans and other animals and as a predictor of injury risk and success of clinical interventions. The four indices used most often to assess symmetry are not well designed for different variable types, perform poorly when presented with cases of high asymmetry or when variables are of low magnitude, and are easily influenced by small variation in the signal. The purpose of the present study was to test the performance of these indices on previously unpublished data on ACL-R patients and to propose a new index to resolve some of these limitations. The performance of four currently used indices and a new index—the Normalized Symmetry Index (NSI), which is scaled to the range of variables being tested across multiple trials—were compared using force and angular data on participants who had undergone anterior cruciate ligament reconstruction and healthy controls. The NSI performed well compared to all other indices with all variables and had the additional benefit of returning values that range from 0% (full symmetry) to ±100% (full asymmetry). Therefore, the NSI can serve as a universal index for assessing asymmetry in humans, nonhuman animal models, and in a clinical context for assessing risk for injury and clinical outcomes.

The assumption that the morphology of the human calcaneus reflects high and cyclical impact forces at heel strike during adult human walking has never been experimentally tested. Since a walking step with a heel strike is an emergent behavior in children, an ontogenetic study provides a natural experiment to begin testing the relationship between the mechanics of heel strike and calcaneal anatomy. This study examined the ground reaction forces (GRFs) of stepping in children to determine the location of the center of pressure (COP) relative to the calcaneus and the orientation and magnitude of ground reaction forces during foot contact. Three-dimensional kinematic and kinetic data were analyzed for 18 children ranging in age from 11.5 to 43.1 months. Early steppers used a flat foot contact (FFC) and experienced relatively high vertical and resultant GRFs with COP often anterior to the calcaneus. More experienced walkers used an initial heel contact (IHC) in which GRFs were significantly lower but the center of pressure remained under the heel a greater proportion of time. Thus, during FFC the foot experienced higher loading, but the heel itself was relatively wider and the load was distributed more evenly. In IHC walkers load was concentrated on the anterior calcaneus and a narrower heel, suggesting a need for increased calcaneal robusticity during development to mitigate injury. These results provide new insight into foot loading outside of typical mature contact patterns, inform structure-function relationships during development, and illuminate potential causes of heel injury in young walkers.

The modern human foot is a complex biomechanical structure that must act both as a shock absorber and as a propulsive strut during the stance phase of gait. Understanding the ways in which foot segments interact can illuminate the mechanics of foot function in healthy and pathological humans. It has been proposed that increased values of medial longitudinal arch deformation can limit metatarsophalangeal joint excursion via tension in the plantar aponeurosis. However, this model has not been tested directly in a dynamic setting. In this study, we tested the hypothesis that during the stance phase, subtalar pronation (stretching of the plantar aponeurosis and subsequent lowering of the medial longitudinal arch) will negatively affect the amount of first metatarsophalangeal joint excursion occurring at push-off. Vertical descent of the navicular (a proxy for subtalar pronation) and first metatarsophalangeal joint dorsal excursion were measured during steady locomotion over a flat substrate on a novel sample consisting of asymptomatic adult males and females, many of whom are habitually unshod. Least-squares regression analyses indicated that, contrary to the hypothesis, navicular drop did not explain a significant amount of variation in first metatarsophalangeal joint dorsal excursion. These results suggest that, in an asymptomatic subject, the plantar aponeurosis and the associated foot bones can function effectively within the normal range of subtalar pronation that takes place during walking gait. From a clinical standpoint, this study highlights the need for investigating the in vivo kinematic relationship between subtalar pronation and metatarsophalangeal joint dorsiflexion in symptomatic populations, and also the need to explore other factors that may affect the kinematics of asymptomatic feet.

The ability to run across uneven terrain with continuous stable movement is critical to the safety and efficiency of a runner. Successful step-to-step stabilization while running may be mediated by minor adjustments to a few key parameters (e.g., leg stiffness, step length, foot strike pattern). However, it is not known to what degree runners in relatively natural settings (e.g., trails, paved road, curbs) use the same strategies across multiple steps. This study investigates how three readily measurable running parameters - step length, foot placement, and foot strike pattern - are adjusted in response to encountering a typical urban obstacle - a sidewalk curb. Thirteen subjects were video-recorded as they ran at self-selected slow and fast paces. Runners targeted a specific distance before the curb for foot placement, and lengthened their step over the curb (p

Overweight and obese patients with osteoarthritis (OA) experience more OA pain and disability than patients who are not overweight. This study examined the long-term efficacy of a combined pain coping skills training (PCST) and lifestyle behavioral weight management (BWM) intervention in overweight and obese OA patients. Patients (N=232) were randomized to a 6-month program of: 1) PCST + BWM; 2) PCST-only; 3) BWM-only; or 4) standard care control. Assessments of pain, physical disability (Arthritis Impact Measurement Scales [AIMS] physical disability, stiffness, activity, and gait), psychological disability (AIMS psychological disability, pain catastrophizing, arthritis self-efficacy, weight self-efficacy), and body weight were collected at four time points (pretreatment, post-treatment, and 6 months and 12 months after the completion of treatment). Patients randomized to PCST+ BWM demonstrated significantly better treatment outcomes (average of all three post-treatment values) in terms of pain, physical disability, stiffness, activity, weight self-efficacy, and weight when compared to the other three conditions (p’s

Individuals with osteoarthritis (OA) of the lower limb find normal locomotion tiring compared with individuals without OA, possibly because OA of any lower limb joint changes limb mechanics and may disrupt transfer of potential and kinetic energy of the center of mass during walking, resulting in increased locomotor costs. Although recovery has been explored in asymptomatic individuals and in some patient populations, the effect of changes in these gait parameters on center of mass movements and mechanical work in patients with OA in specific joints has not been well examined. The results can be used to inform clinical interventions and rehabilitation that focus on improving energy recovery. We hypothesized that (1) individuals with end-stage lower extremity OA would exhibit a decrease in walking velocity compared with asymptomatic individuals and that the joint affected with OA would differntially influence walking velocity, (2) individuals with end-stage lower extremity OA would show decreased energy recovery compared with asymptomatic individuals and that individuals with end-stage hip and ankle OA would have greater reductions in recovery than would individuals with end-stage knee OA owing to restrictions in hip and ankle motion, and (3) that differences in the amplitude and congruity of the center of mass would explain the differences in energy recovery that are observed in each population. Ground reaction forces at a range of self-selected walking speeds were collected from individuals with end-stage radiographic hip OA (n = 27; 14 males, 13 females; average age, 55.6 years; range, 41–70 years), knee OA (n = 20; seven males, 13 females; average age, 61.7 years; range, 49–74 years), ankle OA (n = 30; 14 males, 16 females; average age, 57 years; range, 45–70 years), and asymptomatic individuals (n = 13; eight males, five females; average age, 49.8 years; range, 41–67 years). Participants were all patients with end-stage OA who were scheduled to have joint replacement surgery within 4 weeks of testing. All patients were identified by the orthopaedic surgeon as having end-stage radiographic disease and to be a candidate for joint replacement surgery. Patients were excluded if they had pain at any other lower extremity joint, previous joint replacement surgery, or needed to use an assistive device for ambulation. Patients were enrolled if they met the study inclusion criteria. Our study was comparative and cohorts could be compared with each other, however, the asymptomatic group served to verify our methods and provided a recovery standard with which we could compare our patients. Potential and kinetic energy relationships (% congruity) and energy exchange (% recovery) were calculated. Linear regressions were used to examine the effect of congruity and amplitude of energy fluctuations and walking velocity on % recovery. Analysis of covariance was used to compare energy recovery between groups. The results of this study support our hypothesis that individuals with OA walk at a slower velocity than asymptomatic individuals (1.4 ± 0.2 m/second, 1.2–1.5 m/second) and that the joint affected by OA also affects walking velocity (p < 0.0001). The cohort with ankle OA (0.9 ± 0.2 m/second, 0.77–0.94 m/second) walked at a slower speed relative to the cohort with hip OA (1.1 ± 0.2 m/second, 0.96–1.1 m/second; p = 0.002). However, when comparing the cohorts with ankle and knee OA (0.9 ± 0.2 m/second, 0.77–0.94 m/second) there was no difference in walking speed (p = 0.16) and the same was true when comparing the cohorts with knee and hip OA (p = 0.14). Differences in energy recovery existed when comparing the OA cohorts with the asymptomatic cohort and when examining differences between the OA cohorts. After adjusting for walking speeds these results showed that asymptomatic individuals (65% ± 3%, 63%–67%) had greater recovery than individuals with hip OA (54% ± 10%, 50%–58%; p = 0.014) and ankle OA (47% ± 13%, 40%–52%; p = 0.002) but were not different compared with individuals with knee OA (57% ± 10%, 53%–62%; p = 0.762). When speed was accounted for, 80% of the variation in recovery not attributable to speed was explained by congruity with only 10% being explained by amplitude. OA in the hip, knee, or ankle reduces effective exchange of potential and kinetic energy, potentially increasing the muscular work required to control movements of the center of mass. The fatigue and limited physical activity reported in patients with lower extremity OA could be associated with increased mechanical work of the center of mass. Focused gait retraining potentially could improve walking mechanics and decrease fatigue in these patients.

Osteoarthritis of the knee is seen more frequently in females than males. However, few studies have examined the interplay of gender, gait mechanics, pain, and disability in persons with osteoarthritis. This study examines the influence of anthropometrics, radiographic disease severity, pain, and disability on gender differences in gait mechanics in patients with knee osteoarthritis. Gait mechanics for 26 men and 30 women, were collected using 3-D kinematics and kinetics. Women had a significantly lower knee adduction moment than men, and a significantly higher stride frequency. Within female subjects, variations in gait mechanics were primarily explained by weight, BMI, pain, and disability. In males, variations in gait mechanics were primarily explained by age and disability.

In skin inflammation, vascular endothelial growth factor (VEGF) and CXCL-8/IL-8 play an important role and are produced by activated keratinocytes. Extracts from Ginkgo biloba leaves (GBE), widely used in phytotherapy, have been reported to exert antioxidant and anti-inflammatory properties in the skin. We therefore evaluated the effects of GBE on the release of VEGF and CXCL8/IL-8 by normal human keratinocytes (NHKs) activated by tumor necrosis factor alpha (TNFalpha). Moreover, as we previously showed that epigallocatechin-3-gallate (EGCG) reduces VEGF and CXCL8/IL-8 secretion in TNFalpha-activated NHKs, we also tested its effect in association with GBE. Our results showed that GBE exerted a potent inhibition on VEGF and CXCL8/IL-8 levels in activated cells. In association with EGCG, GBE down-regulated VEGF and CXCL8/IL-8 levels in a cumulative manner in TNFalpha-stimulated NHKs. These results suggest that GBE, alone or in association with EGCG may contribute to moderate inflammatory processes in skin diseases associated with angiogenesis.

This study examined arthritis self-efficacy and self-efficacy for resisting eating as predictors of pain, disability, and eating behaviors in overweight or obese patients with osteoarthritis (OA) of the knee. Patients (N=174) with a body mass index between 25 and 42 completed measures of arthritis-related self-efficacy, weight-related self-efficacy, pain, physical disability, psychological disability, overeating, and demographic and medical information. Hierarchical linear regression analyses were conducted to examine whether arthritis self-efficacy (efficacy for pain control, physical function, and other symptoms) and self-efficacy for resisting eating accounted for significant variance in pain, disability, and eating behaviors after controlling for demographic and medical characteristics. Analyses also tested whether the contributions of self-efficacy were domain specific. Results showed that self-efficacy for pain accounted for 14% (p=.01) of the variance in pain, compared to only 3% accounted for by self-efficacy for physical function and other symptoms. Self-efficacy for physical function accounted for 10% (p=.001) of the variance in physical disability, while self-efficacy for pain and other symptoms accounted for 3%. Self-efficacy for other (emotional) symptoms and resisting eating accounted for 21% (p

This study tested the hypothesis that the presence of isolated ankle (A-OA; N=30), knee (K-OA; N=20), or hip (H-OA; N=30) osteoarthritis (OA) compared to asymptomatic controls (N=15) would lead to mechanical changes in the affected joint but also in all other lower limb joints and gait overall. Stride length, stance and swing times, as well as joint angles and moments at the hip, knee, and ankle were derived from 3-D kinematic and kinetic data collected from seven self-selected speed walking trial. Values were compared across groups using a 1×4 ANCOVA, covarying for walking speed. With walking speed controlled, the results indicated a reduction in hip and knee extension and ankle plantar flexion in accordance with the joint affected. In addition, OA in one joint had strong effects on other joints. In both H-OA and K-OA groups the hip never passed into extension, and A-OA subjects significantly changed hip kinematics to compensate for lack of plantar flexion. Finally, OA in any joint led to lower peak vertical forces as well as extension and plantar flexion moments compared to controls. The presence of end-stage OA at various lower extremity joints results in compensatory gait mechanics that cause movement alterations throughout the lower extremity. This work reinforces our understanding of the complex interaction of joints of the lower limb and the importance of focusing on the mechanics of the entire lower limb when considering gait disability and potential interventions in patients with isolated OA.

There is now strong evidence that the ultraviolet A (UVA) part of the solar spectrum contributes to the development of skin cancers. Its effect on the skin immune system, however, has not been fully investigated. Here, we analyzed the effects of UVA radiation on dermal dendritic cells (DDC), which, in addition, provided further characterization of these cells. Dermal sheets were obtained from normal human skin and irradiated, or not, with UVA at 2 or 12 J per cm 2 . After a 2 d incubation, the phenotype of emigrant cells was analyzed by double immunostaining and flow cytometry. Results showed that migratory DDC were best characterized by CD1c expression and that only few cells co-expressed the Langerhans cell marker Langerin. Whereas the DC extracted from the dermis displayed an immature phenotype, emigrant DDC showed increased expression of HLA-DR and acquired co-stimulation and maturation markers. We showed here that UVA significantly decreased the number of viable emigrant DDC, a process related to increased apoptosis. Furthermore, UVA irradiation impaired the phenotypic and functional maturation of migrating DDC into potent antigen-presenting cells, in a concentration-dependent manner. The results provide further evidence that UVA are immunosuppressive and suggest an additional mechanism by which solar radiation impairs immune response.

Background: Migration and maturation of epidermal dendritic cells, the Langerhans cells (LC), are central events in the initiation of the cutaneous immune response. LC migration from skin to draining lymph nodes is regarded as an indispensable step for the early phase of antigen-specific sensitization. Among the several agents which influence the ability of LC to migrate, previous studies have revealed that matrix metalloproteinases (MMPs) and protein kinase C (PKC) contribute to promoting LC migration. In this work, we studied the effect of two recently developed PKC and MMPs inhibitors of vegetable origin on the migration of in vitro activated LC. Methods: The migratory capacity of epidermal and in vitro generated LC was assessed using a reconstituted basement membrane assay (Matrigel), mimicking the prerequisite passage through the dermal-epidermal basement membrane on the way to the lymph nodes. Results: Contact with chemical allergens, Bandrowski’s base or 2,4-dinitrobenzenesulfonic acid (DNBS), triggered migration. In the presence of PKC inhibitors, D-erythro-sphingosine and OX100, or an inhibitor of MMPs, LU105, allergen-induced migration of LC was strongly decreased. The association between OX100 and LU105 was more efficient in modulating the migration of activated LC compared to each molecule tested separately. Conclusions: These results showed that PKC and MMPs inhibitors act in synergy to inhibit the migration of activated epidermal dendritic cells in vitro. They underscore the role of PKC and MMPs inhibitors and suggest they may be of relevance for therapeutically regulating epidermal dendritic cell migration in inflammatory dermatoses.

SUMMARY Aspergillus fumigatus is the most common aetiological fungus responsible for human pulmonary aspergilloses. This study investigated the primary contact between Langerhans cells (LC), corresponding to dendritic cells present in pulmonary mucosa and live conidia of A. fumigatus. LC play a key role in antigen presentation for initiation of the primary T cell response. In vitro-generated LC (iLC) were differentiated from cultured human cord blood CD34+ cells and incubated at 4°C or 37°C with fluorescein-isothiocyanate (FITC)-stained conidia or control latex beads. In vitro, conidia were shown by microscopy and cytometry to adhere to iLC in a dose- and time-dependent manner. This adhesion was not limited to iLC because interstitial dendritic and other cells also fluoresced in the presence of conidia-FITC. A lectin other than mannose receptor-type lectin was demonstrated to be responsible of conidial binding. Inhibition of binding was observed with heterologous galactomannan and EDTA, indicating a C-lectin-like receptor with galactomannan structure specificity. After binding only a few conidia were internalized in acidic vesicles, as indicated by the cessation of conidial fluorescence. Conidial binding was followed by activation and maturation of iLC, suggesting that LC present in the lung may play a role in cellular host defence against aspergilloses.

Background— Common 3D systems have only limited spatial and temporal resolution (frame rate of 25 Hz). Thin structures such as cardiac valves are not imaged exactly; rapid movement patterns cannot be precisely recorded. The objective of the present project was to achieve radiofrequency (RF) data transmission to the 3D workstation to improve image resolution. Methods and Results— A commercially available echocardiographic system (5-MHz transesophageal echocardiography probe) with an integrated raw data interface enables transmission of RF data (up to 40 megabytes per second). A 3D data set may contain up to 3 gigabytes, so that all of the high-resolution ultrasound information of the 2D image is available. Frame rates of up to 168 Hz result in temporal resolution 6 times that of standard 3D systems. The applicability of the system and the image quality were tested in 10 patients. The structure of the aortic valve and the dynamic changes were depicted by volume rendering. The changes in the orifice areas were measured in frame-by-frame planimetry. The mean number of frames recorded per cardiac cycle was 122±16. The improved structural resolution enabled a detailed imaging of the morphology of the aortic cusps. The rapid systolic movement patterns were recorded with up to 51 frames. The high number of frames enabled creation of precise area-time diagrams. Thus, the individual phases of aortic valve movement (rapid opening, slow valve closing, and rapid valve closing) could be analyzed quantitatively. Conclusion— A 3D system based on RF data enables high-resolution imaging of cardiac movement patterns. This offers new perspectives for qualitative and quantitative analyses, especially of cardiac valves.

SUMMARYAn understanding of the evolution of human bipedalism can provide valuable insights into the biomechanical and physiological characteristics of locomotion in modern humans. The walking gaits of humans, other bipeds and most quadrupedal mammals can best be described by using an inverted-pendulum model, in which there is minimal change in flexion of the limb joints during stance phase. As a result, it seems logical that the evolution of bipedalism in humans involved a simple transition from a relatively stiff-legged quadrupedalism in a terrestrial ancestor to relatively stiff-legged bipedalism in early humans. However, experimental studies of locomotion in humans and nonhuman primates have shown that the evolution of bipedalism involved a much more complex series of transitions, originating with a relatively compliant form of quadrupedalism. These studies show that relatively compliant walking gaits allow primates to achieve fast walking speeds using long strides, low stride frequencies, relatively low peak vertical forces, and relatively high impact shock attenuation ratios. A relatively compliant, ape-like bipedal walking style is consistent with the anatomy of early hominids and may have been an effective gait for a small biped with relatively small and less stabilized joints, which had not yet completely forsaken arboreal locomotion. Laboratory-based studies of primates also suggest that human bipedalism arose not from a terrestrial ancestor but rather from a climbing, arboreal forerunner. Experimental data, in conjunction with anatomical data on early human ancestors, show clearly that a relatively stiff modern human gait and associated physiological and anatomical adaptations are not primitive retentions from a primate ancestor, but are instead recently acquired characters of our genus.

Summary Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (kpOmpA), we have analysed the interaction between this bacterial cell wall protein and human Langerhans cells (LC), the antigen-presenting cells of the epidermis and mucosa. We showed that biotinylated kpOmpA binds to human LC freshly isolated from epidermis. kpOmpA up-regulated MHC class II, CD86 and CCR7 expression, enhanced migration in response to macrophage inflammatory protein-3β (MIP-3β) through a reconstituted basement membrane mimicking the prerequisite passage through the dermal-epidermal basement membrane on the way to lymph nodes. The allostimulatory function of kpOmpA-treated LC was more potent than that of untreated cells. Even though the proportion of LC which binds kpOmpA was shown to vary between individuals, our data indicate that kpOmpA binds to and activates LC, and suggest that recognition of OmpA by LC may be an initiating event in the antibacterial host response.

Fatty acid synthase (FASN) is the enzyme that synthesizes fatty acids de novo in human cells. Although FASN is generally expressed at low levels in most normal tissues, its expression is highly upregulated in many cancers. Consistent with this notion, inhibition of FASN activity has demonstrated potential to halt proliferation and induce cell death in vitro and to block tumor growth in vivo. Consequently, FASN is widely recognized as a valuable therapeutic target. In this report, we describe a variety of 1,4-quinones and 9,10-anthraquinones, including several natural compounds and some newly synthesized compounds, that potently inhibit the thioesterase (TE) domain of FASN. Inhibition of recombinant TE activity, inhibition of cellular FASN, and cytotoxicity in human prostate cancer cell lines and normal fibroblasts, is shown for the most potent inhibitors. Collectively, the data illustrate the novel inhibitory capacity of the 1,4-quinone and 9,10-anthraquinone pharmacophores against FASN.

Humans stand alone from other primates in that we propel our bodies forward on a relatively stiff and arched foot and do so by employing an anatomical arrangement of bones and ligaments in the foot that can operate like a "windlass." This is a significant evolutionary innovation, but it is currently unknown when during hominin evolution this mechanism developed and within what genera or species it originated. The presence of recently discovered fossils along with novel research in the past two decades have improved our understanding of foot mechanics in humans and other apes, making it possible to consider this question more fully. Here we review the main elements thought to be involved in the production of an effective, modern human-like windlass mechanism. These elements are the triceps surae, plantar aponeurosis, medial longitudinal arch, and metatarsophalangeal joints. We discuss what is presently known about the evolution of these features and the challenges associated with identifying each of these specific components and/or their function in living and extinct primates for the purpose of predicting the presence of the windlass mechanism in our ancestors. In some cases we recommend alternative pathways for inferring foot mechanics and for testing the hypothesis that the windlass mechanism evolved to increase the speed and energetic efficiency of bipedal gait in hominins.

The comparison of the gene expression profiles between two subpopulations of melanoma cells (1C8 and T1C3) derived from the tumor of one patient by cDNA array revealed differences in GAPDH and beta-actin gene levels. These two housekeeping genes were up-regulated in invasive T1C3 melanoma cells compared to noninvasive 1C8 cells. Since cDNA array results were not confirmed by conventional RT-PCR throughout the exponential phase of amplification, we performed duplex relative RT-PCR using ribosomal 18S RNA as internal standard including competimer technology. Statistical analyses provided significant evidence that invasive T1C3 melanoma cells exhibited a twofold higher mRNA level of both GAPDH and beta-actin than noninvasive 1C8 cells. This study demonstrates that the duplex relative RT-PCR procedure including ribosomal 18S RNA as internal standard and competimer technology is precise for RNA quantification and is tailored for cDNA array validation. Our data provide molecular evidence that cellular subpopulations of the same pathological origin are highly heterogeneous and extend the concept that the selection of an appropriate internal control for comparative mRNA analysis should be adapted to each model of human cancers.

Signals regulating the traffic of Langerhans cell precursors from blood to the epidermis are not yet fully understood. The observations that TGF-beta1 is of unique importance in Langerhans cells (LC) ontogeny and that macrophage inflammatory protein-3alpha (MIP-3alpha) is able to attract LC within the epidermis, prompted us to study the effect of MIP-3alpha and TGF-beta1 on the migration of LC precursors. The migratory capacity of immature dendritic cells (DC) was assessed using a reconstituted basement membrane assay (Matrigel), mimicking the prerequisite passage through the dermal-epidermal basement membrane on the way into the epidermis. DC differentiated from cord blood CD34 cells in the presence of GM-CSF plus TNF-alpha were subjected to migration using modified Boyden chambers. Day-6 DC progenitors migrated in a dose-dependent fashion in response to MIP-3alpha, and CD1alpha+ LC precursors responded preferentially to the chemokine. Immature DC did not respond strongly to TGF-beta1 alone in migration assays, but up to 68% of the cells migrated in response to MIP-3alpha plus TGF-beta1. Among them, at least 50% expressed CD1a and E-cadherin and can be considered LC precursors. The allostimulatory function of these cells was significantly more potent than that which migrated in response to MIP-3alpha alone. Our results show that a significant proportion of immature DC is able to migrate through a dermal-epidermal basement membrane equivalent. In the presence of TGF-beta1, the DC which respond to MIP-3alpha have the phenotype and the functional capacity of epidermal LC. Our findings underline the role of MIP-3alpha and TGF-beta1 in attraction and localization of immature LC within the epidermis under normal conditions.

The present study was aimed at determining immune protection factors (IPFs) for sunscreens. Human skin explants from donors of phototype II-III were treated, or not, with sunscreens with increasing sun protection factors (SPF 4, 8, 15 and 30), or their respective vehicles. Explants were submitted, or not, to increasing doses of UVB irradiation (312 nm). After an 18-h incubation at 37 degrees C, epidermal cells were recovered through trypsinization and tested in a mixed epidermal cell/T lymphocyte reaction. The UVB dose providing 50% immunosuppression (D50%) was determined graphically. We first demonstrated a large difference in the individual response to UVB, as assessed by the D50% in the absence of any topical treatment (mean 1615+/-839 J/m2 from 14 experiments with values ranging from 500 to 3200 J/m2). For all the tested sunscreens, the D50% values were significantly higher than those obtained without sunscreens or with their respective vehicles (P < 0.01), thus demonstrating their immunoprotective effect. IPFs were determined as the ratio of the D50% in the presence of sunscreen to that with vehicle alone. Although they displayed important individual variations, IPFs ranked according to the sunscreen SPFs.

Background: The safety and efficacy of sublingual immunotherapy have been demonstrated in moderate allergic asthma and seasonal rhinitis. However, not much is known about the precise mechanism of action of the allergen when it crosses the oral mucosa. Objective: To define this mechanism, we investigated the role of Langerhans' cells in the capture and internalization of allergens. Methods: We generated dendritic cells in vitro with the phenotypic characteristics of Langerhans-like dendritic cells (LLDCs) from cord blood CD34 + progenitors. We used two recombinant major allergens: Bet v 1 and Phl p 1 labeled with FITC. Results: Internalization of allergens and control proteins was dose- and time-dependent and related to the immature state of the cells. LLDCs internalized allergens with a high efficiency in comparison with control molecules. Allergens were only internalized by macropinocytosis, as demonstrated by the use of various inhibitors. Addition of intracellular pH-modifying molecules indicated that only a part of the allergens was accumulated in acidic vesicles, whereas the majority remained in other cytoplasmic structures. Pulse-chase experiments calculated a half-life of 4 hours, suggesting that part of the molecules were not metabolized in the lysosome. Allergen internalization by LLDCs might be followed by processing in some experiments, as demonstrated by activation of autologous T lymphocytes in 4 of 9 experiments. Conclusion: These elements showed that Langerhans' cells present in mucosa might play an active role in immune responses to allergens. (J Allergy Clin Immunol 2000;105:1194-1201.)

Contact hypersensitivity is a major public health concern in most industrial countries, which is why predictive tests which could identify potential allergens are needed. We have established an in vitro approach for the detection of primary immune response. This model uses Langerhans-like dendritic cells (LLDC) derived from cord blood progenitors and autologous T lymphocytes, isolated from the same blood sample. Treatment of day 12-14 LLDC, with strong haptens trinitrobenzene sulfonic acid (TNP), fluorescein isothiocyanate (FITC) or Bandrowski's base (BB), results in the proliferation of T lymphocytes, whereas weak allergens and irritants, such as sodium dodecyl sulfate (SDS) are ineffective. The use of immature (day 8) LLDC and the addition of a 48 h stage of incubation after hapten contact, result in phenotypic maturation of LLDC in addition to lymphocyte activation in all the cultures with strong haptens. The 48 h stage of incubation, results in sensitization and in some cases the induction of T cell proliferation to citronellal (1/8), coumarine (1/8) and to a prohapten p-phenylenediamine (pPDA; 2/8). The phenotype of DC after 48 h of contact with a strong hapten, becomes that of mature DC (CD83(+), CD86(+) and HLA-DR(++)). With fragrance molecules, weak haptens and prohaptens, a comparable phenotype is observed only when T lymphocytes are activated. These data suggest that the unresponsiveness observed with weak haptens, may be the consequence on an incomplete maturation of LLDC.

We have identified a type II Ca2+-dependent lectin displaying mannose-binding specificity, exclusively expressed by Langerhans cells (LC), and named Langerin. LC are uniquely characterized by Birbeck granules (BG), which are organelles consisting of superimposed and zippered membranes. Here, we have shown that Langerin is constitutively associated with BG and that antibody to Langerin is internalized into these structures. Remarkably, transfection of Langerin cDNA into fibroblasts created a compact network of membrane structures with typical features of BG. Langerin is thus a potent inducer of membrane superimposition and zippering leading to BG formation. Our data suggest that induction of BG is a consequence of the antigen-capture function of Langerin, allowing routing into these organelles and providing access to a nonclassical antigen-processing pathway.

In vivo, dendritic cells (DC) form a network comprising different populations. In particular, Langerhans cells (LC) appear as a unique population of cells dependent on transforming growth factor β (TGF-β) for its development. In this study, we show that endogenous TGF-β is required for the development of both LC and non-LC DC from CD34+ hematopoietic progenitor cells (HPC) through induction of DC progenitor proliferation and of CD1a+ and CD14+ DC precursor differentiation. We further demonstrate that addition of exogenous TGF-β polarized the differentiation of CD34+ HPC toward LC through induction of differentiation of CD14+ DC precursors into E-cadherin+, Lag+CD68−, and Factor XIIIa−LC, displaying typical Birbeck granules. LC generated from CD34+ HPC in the presence of exogenous TGF-β displayed overlapping functions with CD1a+ precursor-derived DC. In particular, unlike CD14+-derived DC obtained in the absence of TGF-β, they neither secreted interleukin-10 (IL-10) on CD40 triggering nor stimulated the differentiation of CD40-activated naive B cells. Finally, IL-4, when combined with granulocyte-macrophage colony-stimulating factor (GM-CSF), induced TGF-β-independent development of non-LC DC from CD34+ HPC. Similarly, the development of DC from monocytes with GM-CSF and IL-4 was TGF-β independent. Collectively these results show that TGF-β polarized CD34+ HPC differentiation toward LC, whereas IL-4 induced non-LC DC development independently of TGF-β. J. Leukoc. Biol. 66: 781–791; 1999.

Recently, sustance P receptors (SPR) have been detected in neonatal foreskin. Our purpose was to determine the expression of SPR in other localizations than neonatal foreskin. As SP has been implicated in the pathogenesis of inflammatory cutaneous lesions, we wondered whether SPR localization was modified in psoriatic lesions. In normal skin, SP binding sites were detected using biotinylated SP and abrogated by a specific NK1 antagonist (spantide) on blood vessels, sweat glands and hair follicles. In the normal epidermis, SPR were usually observed on granular layers but may also be observed on other cell layers. The SP binding processed on cultured keratinocytes demonstrated that SPR were expressed in the epidermis, except basal cell layers, confirming that keratinocytes constitutively express SPR. In skin lesions of psoriatic patients, SP binding sites were expressed on the uppermost keratinocytes which are not granular cells, and seem to be overexpressed. Our results raise the question of the role of SPR on psoriatic keratinocytes.

Urocanic acid (UCA) represents the major ultraviolet B (UVB, 290-320 nm)-absorbing component of the skin. Trans-UCA is naturally produced in the stratum corneum and converts to the cis isomer upon UVB irradiation. In this study, we examined the effect of purified cis-UCA (about 99% of cis isomer) on the human Langerhans cell (LC) allostimulatory function by using the mixed epidermal cell-lymphocyte reaction (MELR). We found that addition of increasing amounts (6.5-400 micrograms/mL) of purified cis-UCA or trans-UCA did not modify the T-cell response supported by enriched LC (eLC: 8-25% LC) as well as purified LC (pLC: 70-90% LC) suspensions. Because cis-UCA had no effect on the allostimulatory function of untreated LC, we investigated whether this compound could modify T-cell proliferation induced by UVB-irradiated LC. The UVB exposure of eLC or pLC to 100 J/m2 significantly inhibited the capacity of both suspensions to mount a T-cell response. However, addition of cis-UCA did not potentiate this UVB-induced immunosuppression. The eLC or pLC were then incubated with cis-UCA for 18 h at 37 degrees C and washed before adding to allogeneic T cells. The obtained proliferative response was similar to that induced by control LC incubated in medium alone, demonstrating that pretreatment with cis-UCA did not alter human LC function. In conclusion, these results strongly suggest that cis-UCA has no direct effect on human LC antigen-presenting function.

Sugar receptors are being increasingly implicated in host-pathogen interactions because of their specific recognition of carbohydrates of microorganisms. The aim of this study was to identify sugar receptors expressed on the surface of human epidermal Langerhans cells (LC). To this end, binding of a panel of fluorescent neoglycoproteins to human epidermal LC was analyzed by quantitative flow cytofluorometry after standardization with calibrated beads. We demonstrate that fresh human LC are the only cells isolated from healthy epidermis which express a membrane receptor specific for fucose-bovine serum albumin (BSA) and mannose-BSA. Quantitative analysis of mannose-BSA or fucose-BSA binding showed non-linear Scatchard plots, denoting the presence of high and moderate affinity binding on the LC surface. The binding parameters of these two ligands were not significantly different. Mannan, the yeast mannose-rich polysaccharide, fucose-BSA, mannose-BSA and free fucose are strong competitors of the three known ligands of the mannose receptor, i.e. fucose-BSA, mannose-BSA and fluorescein isothiocyanate dextran. The amount of mannose-BSA and fucose-BSA bound to LC was 1.5-fold higher at 37 degrees C than at 4 degrees C, suggesting an internalization process. Antibodies raised against the human macrophage mannose receptor strongly stained CD1a-positive LC but not CD1a-negative population. Taken together, our data demonstrate that fresh human LC are the only cells in the epidermis to express a fucose-mannose receptor on their surface.

Mutated RAS oncoproteins and epidermal growth factor (EGF) are thought to contribute to the proliferative, invasive and metastatic properties of transformed cells. In the present study, we investigated the role of EGF in two H-ras transfected clones and compared it to that in the parental cell line, HaCaT and primary cultured keratinocytes. Our findings show that the motility on type I collagen, measured by the migration index, was similar for both the HaCaT cell line and normal human keratinocytes, whereas it was higher for the HaCaT-ras clones. These results suggest an involvement of the ras oncogene in the stimulation of cell migration. EGF, in cell pretreatment or during the migration assay also caused an increase in migration of all the cells, but preserved the difference between HaCaT and HaCaT-ras. However, no significant difference in EGF-R expression was detected between normal cultured keratinocytes, HaCaT and HaCaT-ras cell lines with or without EGF pretreatment. Moreover, when the cells were stimulated with EGF, the MMP-9 activity was greatly increased in a dose-dependent manner in all the cells, and EGF stimulation particularly highlights the increased amount of MMP-9 in HaCaT-ras cells compared to HaCaT cells. In conclusion, EGF is able to enhance motility and to up-regulate MMP-9 activity in all cells, but with a higher impact in HaCaT-ras cells without an overexpression of EGF-R. As EGF acts in synergy with the H-ras mutation, they could be implicated in the local invasion by the HaCaT-ras clones.

Langerhans cells play a key role in contact hypersensitivity reactions. The application of haptens on the skin leads to many modifications of these cells, including the increase of major histocompatibility complex II expression, allogeneic stimulation potency, and migration towards lymph nodes to activate T cells. Moreover, it has been shown that Langerhans cells cultured in vitro are able to prime naive T cells in response to hapten contact. From CD34+ progenitors present in cord blood, we generated dendritic cells of which some presented the phenotypic markers of Langerhans cells. We show that these cells are able to sensitize syngeneic naive (CD45RA+) T cells to haptens such as trinitrophenyl conjugate of trinitrobenzene sulfonic acid (TNP) and fluoroscein isothiocyanate. The response to TNP is higher than to fluoroscein isothiocyanate, whereas sodium dodecyl sulfate, an irritant molecule used as a control, never caused this effect. Phenotypic analysis of cellular suspensions and experiments of cell sorting lead to the conclusion that only CD1a+ cells are able to induce a primary response of syngeneic T cells to TNP or fluoroscein isothiocyanate. Furthermore, we have shown a close relationship between the differentiation state of dendritic cells and their ability to prime T lymphocytes. Dendritic cells are able to present haptens in an efficient manner between day 10 and 14 of culturing CD34+ progenitors, whereas they were efficient in presenting alloantigens from day 6 until after day 20. This dissociation suggests the need of an active metabolic process for hapten presentation in the direct treatment of dendritic cells with haptens. This model of hapten presentation was used for a panel of fragrance molecules and other molecules considered as weaker haptens than TNP and fluoroscein isothiocyanate.

Summary Dendritic cells (DC) are the most effective cells for antigen presentation in primary immune responses. Human cord blood CD34 + progenitors cultured in the presence of GM-CSF and TNF a generate α heterogeneous population of DC including Langerhans-like DC (LLDC) and monocytes. We describe here that IL-4 exerts different effects in such culture according to the cells considered. Thus, IL-4 favors DC components at the expense of monocytic development, and permits long-time persistence of DC which can be maintained up to one month in culture. These results show an IL-4-dependent inhibition of proliferation and emergence of CD14 + cells. Notably, however IL-4 also acts on the DC precursors. Thus IL-4 enhances survival and delays maturation of LLDC from CD1a + CD14 - precursors. In addition IL-4 also favors orientation of CD14 + CD1a - DC/monocyte precursors towards dermal-type CD1a + DC. DC recovered from IL-4 treated cultures display reduced allostimulatory capacity, but this function is restored upon IL-4 weaning. Finally, a short. (48 h) IL-4 Pulse is sufficient to favor DC development. The present study demonstrates that IL-4 positively regulates DC development at several levels on distinct precursor cells.

The stratum corneum (SC) has long been considered as a sort of inert membrane destined to be shed at the surface of the epidermis. During the last two decades, however, several lines of evidence have been reported, suggesting that active physical and chemical changes take place in the horny layer despite the absence of intracytoplasmic organelles. In particular, processing of filaggrin, replacement of the plasma membrane by a ceramide envelope and constant, progressive modification of extracellular lipid multilayers have been put forward. Recently, attention has focused on the intercellular junctions, which may be involved in the regulation of SC desquamation. Corneodesmosin, a newly discovered protein of s.c. desmosomes (corneodesmosomes), is synthesized at the latest stages of keratinocyte differentiation and persists between the horny cells until desquamation occurs. In the present study, we performed immunohistochemical and immuno-ultrastructural investigations on corneodesmosin expression in various skin lesions characterized by abnormal production and/or retention of the horny layer. Our results suggest that corneodesmosin expression is independent from profilaggrin synthesis. We found corneodesmosin in almost all morphologically recognizable corneodesmosomal structures and specifically those which persisted up to the SC surface. Hyperkeratotic lesions which are characterized by an increased number of junctions showed intense immunoreactivity with anticorneodesmosin antibody. A complete absence of corneodesmosin was not observed in any disease. This finding, together with our previous biochemical studies, suggests that corneodesmosin may exert a protective function against proteolytic degradation of corneodesmosomes both in normal skin and in the pathological horny layer.

To carry out the characterization of feline Langerhans cells (LC), first described in 1994, we used a panel of monoclonal antibodies (MAb) known to react with human, canine and feline leukocyte membrane antigens (Ag). The immunolabeling was performed, at light microscope level, on frozen sections of feline skin and labial mucosa using an avidin-biotin-peroxidase technique, and at electron microscope level on epidermal cell suspensions using an immunogold technique. Out of the 52 MAb tested, six labeled basal or suprabasal DC cells in the frozen sections, either in epidermis or lip epithelium: MHM23 (anti-human CD18), CVS20 and vpg3 (respectively anti-canine and feline-major histocompatibility complex class II molecules), vpg5 (anti-feline leukocytes), vpg39 (anti-feline CD4) and Fel5F4 (anti-feline CD1a). These six MAb were used on suspensions, and labeled cells which showed no desmosomes or melanosomes, but contained 'zipper-like' structures similar to Birbeck granules (BG) in their cytoplasm, revealing they were LC. Consequently, feline LC are CD18-positive (CD18+), major histocompatibility complex class II-positive (Class II+), CD1a-positive (CD1a+), vpg5-positive (vg5+) and CD4-positive (CD4+). This immunophenotypic and ultrastructural characterization demonstrates that feline LC share many characteristics with their human counterparts, a fact that will allow us to study the role of feline LC in certain feline diseases such as Feline Immunodeficiency Virus (FIV) infection, since it has been shown that human LC cells are HIV-permissive, and to establish an animal model for human AIDS.

The infection of macaque monkeys by attenuated simian immunodeficiency virus can vaccinate against pathogenic molecular clones and isolates of the same virus. The correlates of this potent protective immunity are not fully understood but may be the key to an effective AIDS vaccine for humans. Aiming to determine whether host immune responses to envelope glycoprotein are an essential component of the immunity to primate lentiviruses, we have tried to superinfect SIVmac-infected macaque monkeys with SHIVsbg, a chimeric primate lentivirus constructed from the SIVmac239 genome with the env, rev, tat, and vpu genes from HIV-1 Lai. After inoculation of a large dose of SHIVsbg, the chimeric virus was isolated by coculture of mononuclear blood cells from four of five SIV-infected monkeys, but three animals were protected from extracellular SHIV viremia and did not seroconvert to HIV-1 glycoproteins. In the two SIV-infected monkeys that did develop SHIV viremia, cell-associated viral load was reduced at least 100-fold. These data indicate that an antiviral response capable of effectively controlling primate lentivirus replication might not necessarily involve the envelope glycoprotein.

The effect on human peripheral blood mononuclear cells (PBMCs) of neutral glycosphingolipids extracted from metacestodes of the parasite Echinococcus multilocularis was investigated. Neutral glycosphingolipids inhibited [3H]thymidine uptake by human PBMCs upon stimulation by mitogens such as phytohemagglutinin A and pokeweed mitogen or by allogeneic Burkitt B cells. This effect was dose dependent and was related to a decrease in interleukin 2 (IL-2) synthesis, the expression of IL-2 receptors (CD25) being unmodified. Addition of exogenous recombinant IL-2 restored the cell proliferation. Partial inhibition of immunoglobulin G (IgG), IgA, and IgM synthesis was observed in the supernatant of cell culture in association with the inhibitory effect. Identification of active subfractions contained in the neutral glycosphingolipid fraction was also studied in relation to cell viability. The free ceramide fraction had an inhibitory effect, in part related to cell lysis, particularly at high concentration, while the monogalactosylceramides had a paradoxical effect: as an activator at low concentrations and as an inhibitor at high concentrations, with limited cell survival. The immunogenic neutral glycosphingolipids containing at least two carbohydrate residues, all having a structure based on Gal beta 1-->6Gal, were inhibitors of PBMC proliferation and showed good cell survival. These results suggest that parasite neutral glycosphingolipids may play an immunologically relevant role in alveolar hydatid disease.

Human dendritic cells (DC) can now be generated in vitro in large numbers by culturing CD34+ hematopoietic progenitors in presence of GM-CSF+TNF alpha for 12 d. The present study demonstrates that cord blood CD34+ HPC indeed differentiate along two independent DC pathways. At early time points (day 5-7) during the culture, two subsets of DC precursors identified by the exclusive expression of CD1a and CD14 emerge independently. Both precursor subsets mature at day 12-14 into DC with typical morphology and phenotype (CD80, CD83, CD86, CD58, high HLA class II). CD1a+ precursors give rise to cells characterized by the expression of Birbeck granules, the Lag antigen and E-cadherin, three markers specifically expressed on Langerhans cells in the epidermis. In contrast, the CD14+ progenitors mature into CD1a+ DC lacking Birbeck granules, E-cadherin, and Lag antigen but expressing CD2, CD9, CD68, and the coagulation factor XIIIa described in dermal dendritic cells. The two mature DC were equally potent in stimulating allogeneic CD45RA+ naive T cells. Interestingly, the CD14+ precursors, but not the CD1a+ precursors, represent bipotent cells that can be induced to differentiate, in response to M-CSF, into macrophage-like cells, lacking accessory function for T cells. Altogether, these results demonstrate that different pathways of DC development exist: the Langerhans cells and the CD14(+)-derived DC related to dermal DC or circulating blood DC. The physiological relevance of these two pathways of DC development is discussed with regard to their potential in vivo counterparts.