Calf Management: Individual or Paired Housing Affects Dairy Calf Health and Welfare.

Previous research has indicated that preweaned dairy calves reared in pairs compared with individually have improved performance and indicators of animal welfare. One hundred and thirty Holstein female calves completed the trial, with eighty-five being allocated to paired housing and forty-five calves being allocated to individual housing. Daily live weight gain (DLWG), treatments and mortality were recorded throughout the preweaning period. Salivary cortisol, latency to feed and latency to approach a novel object were assessed at batching. There were no significant differences in DLWG, mortality and disease treatments between the average of the pair and the individually housed calves, although the pair-reared calves were quicker to approach the milk feed after batching and interacted more quickly with a novel object. The heaviest born calves within the pair had the highest DLWG from birth to weaning, with a higher percentage of calves approaching the novel object, compared with the lightest born calf within the pair. This study shows that calves within a pair may have significantly different performance and welfare during the preweaning period, with the heavier calf outperforming and displaying less fear and more exploratory behaviour than the lighter calf within a pair.

Cross-System Integration of Respiration and Deglutition: Function, Treatment, and Future Directions.

Swallowing occurs preferentially in the expiratory phase of the quiet breathing cycle and at mid-to-low tidal volume. This coordinative pattern imparts important biomechanical advantages to swallowing and airway protection and facilitate laryngeal elevation, laryngeal vestibular and vocal fold closure, and cricopharyngeal sphincter opening. This preferred coordinative relationship between breathing and swallowing is impaired in a variety of patient populations, including head and neck cancer survivors with dysphagia. We developed a training protocol to re-establish more optimal phasing of swallowing with breathing in these patients with striking outcomes, including reduced swallowing physiological impairments and improved airway protection. This motivated us to continue to refine and expand this training protocol and develop new assistive technologies for swallowing monitoring outside of the lab. In this review, we highlight the origins of our optimal respiratory-swallowing coordination hypothesis, describe the biomechanical advantages it provides, carefully describe our training protocol and findings, and chart a course for the next phase of this work. Our overall goal is to harness technology combined with carefully constructed learning paradigms to improve the lives of patients with impaired respiratory-swallowing coordination consequent to a variety of pathologies including head and neck cancer and degenerative neurological conditions such as Parkinson's disease.

Physiological measures of mother-infant interactional synchrony.

Mother-infant interactional synchrony has been hypothesized to be crucial for the development of many key neurodevelopmental behaviors in infants, including speech and language. Assessing synchrony is challenging because many interactive behaviors may be subtlety, if at all, observable in overt behaviors. Physiological measures, therefore, may provide valuable physiological/biological markers of mother-infant synchrony. We have developed a multilevel measurement platform to assess physiological synchrony, attention, and vocal congruency during dynamic face-to-face mother-infant interactions. The present investigation was designed to provide preliminary data on its application in a group of 10 mother-infant dyads (20 subjects) ranging in age from 7 to 8.5 months at the time of the experimentation. Respiratory kinematics, heart rate, and vocalization were recorded simultaneously from mothers and infants during nonstructured, face-to-face interactions. Novel statistical methods were used to identify reliable moments of synchrony from cross-correlated, mother-infant respiration and to tag infant attention from heart rate deceleration. Results revealed that attention, vocal contingency, and respiratory synchrony are temporally clustered within the dyad interaction. This temporal alignment is consistent with the notion that biological synchrony provides a supportive platform for infant attention and mother-infant contingent vocalization.

Optimizing Respiratory-Swallowing Coordination in Patients With Oropharyngeal Head and Neck Cancer.

Swallowing impairment (dysphagia) represents the highest functional morbidity in oropharyngeal (OP) head and neck (HNC) treated either with surgical approaches followed by radiation or with more recent organ preservation protocols, including combined chemotherapy and radiation. Despite the promising overall increasing survival rates, swallowing impairments remain chronic, are often resistant to traditional swallowing therapy, and have devastating consequences on health and well-being. The respiratory-swallow cross-system approach presented here extends beyond traditional swallowing interventions that commonly targets muscles and structures alone, and is instead, directed toward the re-establishment of optimal respiratory-swallowing coordination. Results from our work investigating a respiratory-swallow treatment (RST) paradigm is presented, including results from an RST clinical trial in HNC patients, primarily with OP cancers, with chronic and with intractable dysphagia post-cancer and post-traditional swallowing treatment. Future work will investigate the impact of RST on the degree and durability of clinical outcomes, including oral intake and quality of life, while also examining the potential added benefits of a home practice program that uses a commercially available and easy to use recording and analysis hardware and software.

Effect of Single-Use, Laser-Cut, Slow-Flow Nipples on Respiration and Milk Ingestion in Preterm Infants.

PURPOSE: Single-use, laser-cut, slow-flow nipples were evaluated for their effect on respiration and milk ingestion in 13 healthy preterm infants (32.7-37.1 weeks postmenstrual age) under nonlaboratory, clinical conditions. METHOD: The primary outcomes of minute ventilation and overall milk transfer were measured by using integrated nasal airflow and volume-calibrated bottles during suck bursts and suck burst breaks during slow-flow and standard-flow nipple bottle feedings. Wilcoxon signed-ranks tests were used to test the effect of nipple type on both outcomes. RESULTS: Prefeeding minute ventilation decreased significantly during suck bursts and returned to baseline values during suck burst breaks across both slow-flow and standard-flow nipples. No differences were found in minute ventilation (p > .40) or overall milk transfer (p = .58) between slow-flow and standard-flow nipples. CONCLUSIONS: The lack of difference in primary outcomes between the single-use slow-flow and standard-flow nipples may reflect variability in nipple properties among nipples produced by the same manufacturer. Future investigations examining the effect of both single-use and reusable nipple products are warranted to better guide nipple selection during clinical care.

Prior History of Feeding-Swallowing Difficulties in Children With Language Impairment.

PURPOSE: This study updated and extended our previous investigation (Malas et al., 2015) of feeding-swallowing difficulties and concerns (FSCs) in children with language impairments (LI) by using more stringent inclusion criteria and targeting children earlier in the care delivery pathway. METHOD: Retrospective analyses were performed on the clinical files of 29 children (average age: 60 months, SD = 9.0) diagnosed as having LI using standardized testing, nonstandardized testing and final speech-language pathologist judgment. The files of children born prematurely or with a history of anatomical, structural, neurodevelopmental, cognitive, sensory, motor, or speech disorders were excluded. Literature-based indicators were used to determine the prevalence of difficulties in sucking, food transition, food selectivity, and salivary control. Values were compared with the general population estimate of Lindberg et al. (1992). RESULTS: A significantly higher percentage of histories of FSCs (48%) were found in the files of children with LI when compared with the population estimate (χ2 = 13.741, df = 1, p < .001). Difficulties in food transition (31%) and food selectivity (14%) were the most frequent. Data confirm and extend our previous findings and suggest that a previous history of FSCs may characterize children with LI early in their care delivery pathway.

Feeding-swallowing difficulties in children later diagnosed with language impairment.

AIM: The aim of this retrospective study was to assess the relationship between feeding-swallowing difficulties (FSDs) and later language impairments in children. METHOD: Retrospective analyses were carried out using the clinical files of 82 children with language impairments from a large urban rehabilitation center. Two subgroups of these children were established: children with motor impairments, referred to as the language impairment with motor impairment ('LI+MI') subgroup (n=23, mean age 4y 6mo, SD 8.7mo), and children without motor impairments, referred to as the language impairment without motor impairment ('LI-MI') subgroup (n=59, mean age 5y, SD 8mo). The prevalence of food selectivity, difficulties in sucking, salivary control issues, and food transition difficulties was extracted. Data were compared with a general population estimate of FSDs. RESULTS: FSDs were documented in 62% of the clinical files; 87% of these files were from the LI+MI subgroup and 53% were from the LI-MI subgroup. Among each subgroup of children with language impairments, the prevalence of FSDs was significantly higher than the general population estimate of 20% (LI+MI:χ(2) =55.965, df=1, p<0.001; LI-MI: χ(2) =32.807, df=1, p<0.001). Furthermore, the prevalence of FSDs was significantly higher in children with language impairments and motor impairments than in those with language impairments but without motor impairments (χ(2) =6.936, df=1, p<0.01). Both food transition difficulties (χ(2) =14.99, df=1, p<0.001) and salivary control issues (χ(2) =5.02, df=1, p=0.02) were more frequent in the LI+MI subgroup than in the LI-MI subgroup. Combinations of two or more FSDs were also more frequent in the LI+MI subgroup than in the LI-MI subgroup (χ(2) =4.19, df=1, p=0.04). INTERPRETATIONS: These findings suggest that early FSDs may be used as a potential marker for language impairment. However, larger prospective studies are needed to confirm this.

Respiratory-swallow training in patients with head and neck cancer.

OBJECTIVE: To test a novel intervention to train swallowing to occur in the midexpiratory to low expiratory phase of quiet breathing to improve swallowing safety and efficiency. DESIGN: Safety and efficacy nonrandomized controlled trial with 1-month follow-up. SETTING: Ambulatory clinics. PARTICIPANTS: Patients (N=30) with head and neck cancer (HNC) and chronic dysphagia completed the intervention. Fifteen of these patients participated in a 1-month follow-up visit. INTERVENTIONS: Training protocol based on hierarchy of motor skill acquisition to encourage autonomous and optimal respiratory-swallowing coordination. Visual feedback of respiratory phase and volume for swallowing initiation was provided by nasal airflow and rib cage/abdomen signals. MAIN OUTCOME MEASURES: Respiratory-swallow phase pattern, Modified Barium Swallow Impairment Profile (MBSImP) scores, Penetration-Aspiration Scale (PAS) scores, and MD Anderson Dysphagia Inventory scores. RESULTS: Using visual feedback, patients were trained to initiate swallows during the midexpiratory phase of quiet breathing and continue to expire after swallowing. This optimal phase patterning increased significantly after treatment (P<.0001). Changes in respiratory-swallowing coordination were associated with improvements in 3 MBSImP component scores: laryngeal vestibular closure (P=.0004), tongue base retraction (P<.0001), and pharyngeal residue (P=.01). Significant improvements were also seen in PAS scores (P<.0001). Relative to pretreatment values, patients participating in 1-month follow-up had increased optimal phase patterning (P<.0001), improved laryngeal vestibular closure (P=.01), tongue base retraction (P=.003), and pharyngeal residue (P=.006) MBSImP scores and improved PAS scores (P<.0001). CONCLUSIONS: Improvements in respiratory-swallowing coordination can be trained using a systematic protocol and respiratory phase-lung volume-related biofeedback in patients with HNC and chronic dysphagia, with favorable effects on airway protection and bolus clearance.

Innovative technology for the assisted delivery of intensive voice treatment (LSVT®LOUD) for Parkinson disease.

PURPOSE: To assess the feasibility and effectiveness of a newly developed assistive technology system, Lee Silverman Voice Treatment Companion (LSVT(®) Companion™, hereafter referred to as "Companion"), to support the delivery of LSVT(®)LOUD, an efficacious speech intervention for individuals with Parkinson disease (PD). METHOD: Sixteen individuals with PD were randomized to an immediate (n = 8) or a delayed (n = 8) treatment group. They participated in 9 LSVT LOUD sessions and 7 Companion sessions, independently administered at home. Acoustic, listener perception, and voice and speech rating data were obtained immediately before (pre), immediately after (post), and at 6 months post treatment (follow-up). System usability ratings were collected immediately post treatment. Changes in vocal sound pressure level were compared to data from a historical treatment group of individuals with PD treated with standard, in-person LSVT LOUD. RESULTS: All 16 participants were able to independently use the Companion. These individuals had therapeutic gains in sound pressure level, pre to post and pre to follow-up, similar to those of the historical treatment group. CONCLUSIONS: This study supports the use of the Companion as an aid in treatment of hypokinetic dysarthria in individuals with PD. Advantages and disadvantages of the Companion, as well as limitations of the present study and directions for future studies, are discussed.

Oral and nonoral sensorimotor interventions facilitate suck-swallow-respiration functions and their coordination in preterm infants.

BACKGROUND: Preterm infants are at high risk of encountering oral feeding difficulties. Early sensorimotor interventions may improve oral feeding skills in preterm infants. AIM: To further explore the effects of an oral (O), tactile/kinesthetic (T/K), and combined (O+T/K) sensorimotor intervention on preterm infants' nutritive sucking, swallowing and their coordination with respiration. STUDY DESIGN: Seventy-five infants (29 [0.3, standard error of mean, SEM] weeks gestation, 49 males/26 females) were randomly assigned to an O group involving sensorimotor input to the oral structures; a T/K group involving sensorimotor input to the trunk and limbs; a combined (O+T/K) group; and a control group. OUTCOME MEASURES: Stage of sucking, suction and expression amplitudes (mmHg), suck-swallow ratio, stability of suck-swallow interval, and swallow-respiration patterns. RESULTS: The O group had significantly more advanced sucking stages, and greater suction and expression amplitudes than controls [p≤0.035, effect size (ES) >0.6]. The suck-swallow ratio and stability of suck-swallow intervals did not significantly differ among groups (p≥0.181, ES≤0.3). The three interventions led to fewer swallows bracketed by prolonged respiratory pauses compared to controls (pause-swallow-pause, p≤0.044, ES≥0.7). The T/K and combined (O+T/K) groups had greater occurrence of swallows bracketed by expiration than the control and O groups (expiration-swallow-expiration, p≤0.039, ES≥0.3). CONCLUSION: The O intervention enhanced specific components of nutritive sucking. All three interventions resulted in improved swallow-respiration coordination. Sensorimotor interventions have distributed beneficial effects that go beyond the specific target of input.

Significance of nonrespiratory airflow during swallowing.

This study was designed to further our understanding of a potentially significant clinical event of negative nasal airflow near the end of the respiratory pause (inhibition) to accommodate swallowing. This negative flow, referred to as "SNIF," or swallow noninspiratory flow, occurs at the onset of airway reestablishment at the conclusion of the oropharyngeal swallow. Using simultaneous digital video fluoroscopic and nasal respiratory airflow recordings on 82 healthy adults (21-97 years old), the objectives of this study were to determine (1) the frequency of occurrence of SNIF during a 5-ml natural cup-drinking task, (2) differences in SNIF occurrence by age group, and (3) the temporal relationship between SNIF and other swallowing events. Results revealed that for most participants SNIF was observed in both swallowing trials. There was a statistically significant difference in SNIF occurrence by age category, with SNIF observed less frequently in the oldest participants. The peak onset of SNIF is closely related to the first release of contact between the soft palate and tongue base with the posterior pharyngeal wall and opening of the laryngeal vestibule. Based on this, and in agreement with previous investigators, we suggest that this negative flow may be related to a partial vacuum established by the relaxation of pharyngeal contraction near the conclusion of the pharyngeal swallow. The more frequent occurrence of SNIF in younger adults and less in older adults suggests a reduction in pharyngeal pressure associated with healthy aging.

Oral and non-oral sensorimotor interventions enhance oral feeding performance in preterm infants.

AIM: The aim of this study was to determine whether oral, tactile/kinaesthetic (T/K), or combined (oral+T/K) interventions enhance oral feeding performance and whether combined interventions have an additive/synergistic effect. METHOD: Seventy-five preterm infants (mean gestational age 29 wk; standard error of the mean [SEM] 0.3 wk; mean birthweight 1340.3g; SEM 52.5 g; 49 males and 26 females) were randomly assigned to one of three intervention groups or a control group. The oral group received sensorimotor input to the oral structures, the T/K group received sensorimotor input to the trunk and limbs, and the combined group received both. The outcomes were time from introduction of nipple feeding to independent oral feeding (d), proficiency (intake in the first 5 min, %), volume transfer (%), rate of transfer (mL/min), volume loss (%), and length of hospital stay (d). RESULTS: Infants in the three intervention groups achieved independent oral feeding 9-10 days earlier than those in the control group (p<0.001; effect size 1.9-2.1). Proficiency (p ≤ 0.002; effect size 0.7-1.4) at the time of one to two and three to five oral feedings per day, volume transfer (p ≤ 0.001; effect size 0.8-1.1) at one to two, three to five, and six to eight oral feedings per day, and overall rate of transfer (p ≤ 0.018; effect size 0.8-1.1) were greater, and overall volume losses were less (p ≤ 0.007; effect size 0.9-1.1), than in the control group (p ≤ 0.042). The combined group attained independent oral feeding at a significantly younger postmenstrual age than controls (p=0.020) and had clinically greater proficiency than the T/K group (p=0.020; effect size 0.7) and oral group (p=0.109; effect size 0.5). Length of hospital stay was not significantly different between groups (p=0.792; effect size 0.02-0.3). INTERPRETATION: Oral and T/K interventions accelerated the transition from introduction to independent oral feeding and enhanced oral feeding skills. T/K has beneficial effects beyond the specific targeted system. The combined sensorimotor intervention led to an additive/synergistic effect for proficiency, further benefiting this population.

Articulatory and acoustic adaptation to palatal perturbation.

Previous work has established that speakers have difficulty making rapid compensatory adjustments in consonant production (especially in fricatives) for structural perturbations of the vocal tract induced by artificial palates with thicker-than-normal alveolar regions. The present study used electromagnetic articulography and simultaneous acoustic recordings to estimate tongue configurations during production of [s s t k] in the presence of a thin and a thick palate, before and after a practice period. Ten native speakers of English participated in the study. In keeping with previous acoustic studies, fricatives were more affected by the palate than were the stops. The thick palate lowered the center of gravity and the jaw was lower and the tongue moved further backwards and downwards. Center of gravity measures revealed complete adaptation after training, and with practice, subjects' decreased interlabial distance. The fact that adaptation effects were found for [k], which are produced with an articulatory gesture not directly impeded by the palatal perturbation, suggests a more global sensorimotor recalibration that extends beyond the specific articulatory target.

Semiquantitative histopathology and 3D magnetic resonance microscopy as collaborative platforms for tissue identification and comparison within teratomas derived from pedigreed primate embryonic stem cells.

Teratoma formation in xenografts is a sufficiently stringent pluripotency assay for stem cells. However, little is known about the composition and spatial relationships of tissues within teratomas that may provide clues about development and platforms for studying organ development. Additionally, teratoma formation and analysis lack standards for reporting as assays of pluripotency. Three of 27 total teratomas derived from pedigreed primate embryonic stem cells underwent quantitative three-dimensional high-resolution magnetic resonance microscopy (MRM). Teratomas were subsequently serially sectioned and tissue types identified, semiquantitated, and correlated with MRM images. All teratomas demonstrated tissue derivatives from the three germ layers and approximately 23 different tissue types were identified. Certain tissue groups attempted to form organs more frequently (e.g., trachea/bronchi, small intestine). MRM discriminated some tissues readily (e.g., bone, adipose, cartilage) while other tissue types with like MR intensities could not be distinguished. Semiquantitative histopathological analysis of teratomas demonstrates the ability to delineate multiple tissues as derived from ectoderm, mesoderm, or endoderm and to use this information for comparison to other teratomas. MRM provides rapid quantitative imaging of intact teratomas that complements histology and identifies sites of interest for additional biological studies.

Respiratory-swallow phase patterns and their relationship to swallowing impairment in patients treated for oropharyngeal cancer.

BACKGROUND: Unstable respiratory-swallowing coordination has been associated with disorders and disease. The goals of this study were (1) to describe respiratory-swallow patterns in patients with dysphagia consequent to treatments for cancers of the oropharynx and (2) to determine the association between respiratory-swallow patterns, airway invasion, and overall severity of swallowing impairment. METHODS: This prospective, cross-sectional design compared respiratory-swallow patterns in 20 patients treated for oropharyngeal cancer and 20 healthy, age-matched control participants. Nasal airflow direction was synchronously recorded with videofluoroscopic imaging in participants who swallowed 5-mL thin liquid barium boluses. RESULTS: Respiratory-swallow patterns differed between groups. Most control participants initiated and completed swallowing bracketed by expiratory airflow. Swallowing in patients often interrupted inspiratory flow and was associated with penetration or aspiration of the bolus. CONCLUSIONS: We suggest nonexpiratory bracketed respiratory-swallowing phase patterns in patients with oropharyngeal cancer may place patients at greater risk of airway penetration or aspiration during swallowing.

Detection and characterization of circulating endothelial progenitor cells in normal rat blood.

INTRODUCTION: There are currently few widely accepted noninvasive detection methods for drug-induced vascular damage. Circulating endothelial progenitor cell (EPC) enumeration in humans has recently gained attention as a potential biomarker of vascular injury/endothelial damage/dysfunction. The rat is commonly used in preclinical drug development toxicity testing and lacks consensus noninvasive methodologies for immunophenotypic identification of EPCs. Identification of immunophenotypic markers of EPCs in the rat would enable transfer of technologies used in human for potential development of biomarkers for vascular injury the rat. Therefore, the aim of this work was to develop methods to consistently identify a discreet population of EPCs from rat peripheral blood. METHODS: EPCs were identified phenotypically from rat blood using cell culture, immunolabeling, fluorescence microscopy, and flow cytometry. EPCs isolated using immunolabeling coupled with magnetic separation and flow cytometric cell sorting were characterized genotypically using mRNA analysis. RESULTS: A modified colony forming unit (CFU)-Hill assay confirmed existence of immature EPCs in peripheral blood. Extended in vitro culture resulted in a morphology and immunophenotype consistent with mature endothelial cells as noted by positive staining for CD31, von Willebrand factor, rat endothelial cell antigen, and negative staining for smooth muscle cell alpha-actin. The majority of the cells identified as LDL+/CD11b/c(-) did not stain positively for either vWF or CD31. EPC populations isolated using magnetic separation and cell sorting were consistently positive for PECAM1, EDN1, FLK1, VWF, ITGAD, CCR1, IP30, and MMP2 mRNA expression. Cells identified as EPCs express cell-surface and gene expression markers consistent with endothelial cells and endothelial progenitor cell populations. DISCUSSION: Vascular trauma induces transient mobilization of EPCs in humans and their enumeration and characterization have been proposed as a surrogate biomarker for assessment of vascular injury. Potential exists for using rat circulating EPCs as a surrogate sampling population for biomarker development in drug-related injury in preclinical toxicity studies. A prerequisite to biomarker development is the ability to consistently identify a discreet population of EPCs from peripheral rat blood. This work describes novel methods for isolation and validation of phenotypically and genotypically consistent populations of rat EPCs from peripheral blood. These methods are well suited for potential future use in validation of enumeration and/or biomarker development methods in the rat.

Establishment and characterization of baboon embryonic stem cell lines: an Old World Primate model for regeneration and transplantation research.

Here we have developed protocols using the baboon as a complementary alternative Old World Primate to rhesus and other macaques which have severe limitations in their availability. Baboons are not limited as research resources, they are evolutionarily closer to humans, and the multiple generations of pedigreed colonies which display complex human disease phenotypes all support their further optimization as an invaluable primate model. Since neither baboon-assisted reproductive technologies nor baboon embryonic stem cells (ESCs) have been reported, here we describe the first derivations and characterization of baboon ESC lines from IVF-generated blastocysts. Two ESCs lines (BabESC-4 and BabESC-15) display ESC morphology, express pluripotency markers (Oct-4, hTert, Nanog, Sox-2, Rex-1, TRA1-60, TRA1-81), and maintain stable euploid female karyotypes with parentage confirmed independently. They have been grown continuously for >430 and 290 days, respectively. Teratomas from both lines have all three germ layers. Availabilities of these BabESCs represent another important resource for stem cell biologists.

From movement to pain: a tribute to professor James P. Lund.

This tribute article to Professor James P. Lund stems from 6 of the presentations delivered at the July 1, 2008, symposium that honored 3 "giants" in orofacial neuroscience: B. J. Sessle, A. G. Hannam, and J. P. Lund. It was noted that soon after his training as a dentist in Australia, Jim Lund became interested in research. At the time he decided to do a PhD, there was a lot of discussion about how rhythmic movements were programmed. The early belief, based on Sherrington's studies of motor systems, was that these movements were simply an alternating series of reflexes. In the late 1960s and early 1970s, some still shared this belief, whereas others favored Graham Brown's hypothesis that repetitive movements were centrally programmed and did not depend on reflexes triggered by sensory inputs. There was no strong evidence then for either scenario except for the rhythmic movements of respiration. Lund's pioneering work during his PhD proved the existence of a central pattern generator (CPG) for mastication in the brainstem. Since then he has been interested in understanding how CPGs function and how sensory feedback works to adjust the motor patterns that they produce. Sections in this tribute article to Lund are written by some of his close collaborators and reflect the evolution of his work throughout the years. The first 4 presentations in this article (by K.-G. Westberg, D. McFarland, A. Kolta, and C. Stohler) highlight various aspects of these interests, and the final 2 presentations (by J. Feine and A. Woda) focus especially on clinical aspects of Lund's interests. The last section of this article is a final commentary from Professor Lund.

Role of p38 in regulation of hematopoiesis: effect of p38 inhibition on cytokine production and transcription factor activity in human bone marrow stromal cells.

This study was designed to evaluate effects of specific p38 MAP kinase inhibition on gene and protein expression of essential hematopoietic cytokines in primary human bone marrow stromal cells (HBMSC) and to identify downstream transcription factors (TF) regulated by the p38 MAP kinase signalling pathway. In vitro effects of p38 inhibitors (p38i) on cytokine regulation were compared to inhibitors of other major signalling pathways including PI3 kinase, JNK, MEK-1, NF-kappaB or protein kinase C (PKC). HBMSC were pre-treated with p38i (SB-203580) for 1 h and then stimulated with 200 ng/ml lipopolysaccharide (LPS). Supernatants and RNA were collected 6 h post LPS treatment for quantitative protein and mRNA analyses by ELISA and real-time RT-PCR, respectively, for interleukin-6 (IL-6), interleukin-11 (IL-11), granulocyte-monocyte colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF) and Activin A. Effects of the inhibitors of PI3 kinase (LY294002), JNK (synthetic inhibitory peptide), MEK-1 (PD90859), NF-kappaB (pyrrolidinedithiocarbamate (PDTC)) and protein kinase C (calphostin C) on HBMSC expression hematopoietic cytokines were evaluated and compared. SB-203580 caused dose-dependent decreases in cytokine protein expression and decreased IL-6 and IL-11 mRNA expression. Of the pathway inhibitors examined, only NF-kappaB elicited similar effects on cytokine protein and mRNA expression. p38-regulated transcription factor activity was assessed using a DNA/Protein array. Several TFs linked to cytokine regulation were modulated by SB-203580, with 10 of 21 p38-regulated TFs identified have not been previously linked to downstream p38 signalling. These observations in cultured HBMSC have illustrated the involvement of cytokine proteins, mRNA and TF activities and may improve the current understanding of the in vivo p38i suppression of erythropoiesis. In addition, these results suggest that IL-6, IL-11, GM-CSF, G-CSF and Activin A are similarly regulated by p38 and NF-kappaB and that the MEK1, JNK and PKC pathways appear to play a more limited role in modulating cytokine expression in HBMSC.