Characterization of a model of hindgut acidosis in mid-lactation cows: A pilot study.

The objective of this pilot study was to generate data to support the development of an experimental model of hindgut acidosis to further understand its systemic consequences independently of rumen acidosis. Four ruminally fistulated multiparous Holstein cows (213 ± 11 d in milk) were subjected to 2 consecutive experimental periods (P1 and P2), separated by a 3-d washout. Experimental periods were 96 h long from the baseline to the final measurements but expanded over 5 calendar days (d 0-4). Abomasal infusions of saline and corn starch (2.8 kg/d) were performed for the first 72 h (d 0-3) of P1 and P2, respectively. Final measurements were performed 24 h after the end of the infusions (d 4). Each cow was used as its own control by comparing P2 to P1. Postruminal-intestinal permeability was assessed by Cr appearance in blood after a pulse dose administration of Cr-EDTA into the abomasum on d 2 (48 h after infusion initiation) of each period. Starch infusion during P2 was associated with a milk protein yield increase (3.3%) and a decrease in milk urea nitrogen (11%). Fecal dry matter increased (8.8%), and starch content tended to increase (∼2 fold) during P2. There was a period-by-day interaction for fecal pH as it decreased during starch infusion (1.3 pH points) but remained constant during P1. Although fecal lactate was not detectable during P1, it consistently increased during starch infusion. Fecal alkaline phosphatase activity also increased (∼17 fold) in association with starch infusion. Two hours after Cr-EDTA administration, blood Cr concentration was higher during starch infusion, resulting in a tendency for a treatment-by-hour interaction. Furthermore, blood d-lactate increased (∼2.5 fold), serum Cu decreased (18%), and blood urea nitrogen, cholesterol, and Ca tended to decrease (9.4%, 1.2%, and 2.4%, respectively), relative to P1. The current results suggest that hindgut acidosis was successfully induced by postruminal starch infusion, leading to gut damage and increased intestinal permeability. However, indications of systemic inflammation were not observed. The herein described preliminary results will require confirmation in a properly powered study.

Zinc, Copper, and Manganese Homeostasis and Potential Trace Metal Accumulation in Dairy Cows: Longitudinal Study from Late Lactation to Subsequent Mid-Lactation.

BACKGROUND: Trace metals are supplemented in cattle to prevent nutrient deficiencies. Levels supplemented to mitigate worst-case basal supply and availability scenarios can, however, result in trace metal intakes far above the nutritional requirements of dairy cows with high feed intakes. OBJECTIVES: We evaluated Zn, Mn, and Cu balance in dairy cows from late lactation through the subsequent mid-lactation, a period of 24 wk characterized by large changes in dry matter intake. METHODS: Twelve Holstein dairy cows were housed in a tie-stall from 10 wk before to 16 wk after parturition and fed 1 unique lactation diet when lactating and a dry cow diet otherwise. After 2 wk of adaptation to the facility and diet, Zn, Mn, and Cu balances were determined at weekly intervals, by calculating the difference between total intakes and complete fecal, urinary, and milk outputs, with the latter 3 fluxes quantified over a 48-h period. Repeated measure mixed models were used to evaluate the effects on trace mineral balances over time. RESULTS: The Mn and Cu balances of cows were not significantly different from 0 mg/d between 8 wk prepartum and calving (P ≥ 0.54), when dietary intake was the lowest of the period evaluated. However, when dietary intake was highest, between wk 6 and 16 postpartum, positive Mn and Cu balances were observed (80 and 20 mg/d, respectively, P ≤ 0.05). Cows were in positive Zn balance throughout the study except during the first 3 wk after calving during which the Zn balance was negative. CONCLUSIONS: Large adaptations occur in trace metal homeostasis in transition cows in response to changes in dietary intake. High dry matter intakes, associated with high milk production of dairy cows, combined with current Zn, Mn, and Cu supplementation practices may exceed regulatory homeostatic mechanisms resulting in potential body accumulation of Zn, Mn, and Cu.

Dietary protein oscillation: effects on digestibility, nutrient balance and estimated microbial protein synthesis in lactating dairy cows.

Various studies with growing ruminants report increases in nitrogen use efficiency (NUE) when feeding oscillating (OS) dietary CP, whereas limited research with lactating dairy cows demonstrates a lack of improvement in NUE when feeding OS diets. We hypothesised that a total mixed ration (TMR) delivering OS CP (48-h phases of 134 and 171 g CP/kg DM, respectively) compared to a static CP TMR (ST; 152 g CP/kg DM) would result in similar or increased urinary purine derivative excretion (as a marker of microbial protein synthesis (MPS)) and greater urinary nitrogen excretion in lactating dairy cows. Responses in intake, production, apparent total tract digestibility (ATTD), nutrient balance, and estimated MPS were evaluated using faecal and urine collection in 12 multiparous cows (172 ± 39 d in milk) in a randomised complete block design, where total urinary output was estimated indirectly. All measurements were taken during d 8 (at 1700) to d 16 (at 1700) of the 16-d study that followed a 28-d period in which cows already received their respective treatments. Dry matter intake, yields of milk, protein, fat, lactose, and fat- and protein-corrected milk were similar for ST and OS. Milk composition, BW, and body condition score also did not differ between treatments, except for a tendency for increased milk urea concentration with OS (13.7 vs 12.4 mg/dL). Feed efficiency, NUE and ATTD of organic matter, NDF, CP and gross energy did not differ, but ATTD of crude fat (658 vs 627 g/kg) and starch (980 vs 975 g/kg) increased, and ATTD of DM (702 vs 691 g/kg) tended to increase with OS. Milk energy as a proportion of digested energy tended to decrease with OS (34.6 vs 37.1%), but other energy metabolism variables were not affected by treatment. Estimated urinary nitrogen excretion increased (165 vs 144 g/d), estimated urinary nitrogen as a proportion of nitrogen intake tended to increase (25.3 vs 22.7%), and milk nitrogen as a proportion of digested nitrogen decreased (47.3 vs 51.8%) in response to OS. Estimated urinary excretion of creatinine (184 vs 165 mmol/d), uric acid (29 vs 20 mmol/d) and urea (3.1 vs 2.5 mol/d) increased, but other nitrogen metabolism parameters were not affected by OS. Overall, oscillating dietary CP content did not affect lactational performance, milk NUE, or estimated MPS. However, ATTD of some nutrients increased, postabsorptive energy use for milk synthesis tended to decrease, and estimated urinary nitrogen losses increased with OS.

Digestive and metabolic efficiency of energy and nitrogen during lactation and the dry period in dairy cows.

The objective of this study was to characterize total-tract nutrient digestibility, energy balance, and N balance in the critical dietary and metabolic transitions of the lactation cycle. Twelve dairy cows were housed in tiestalls from 10 wk before to 16 wk after parturition. After 2 wk of adaptation to the facility and diet, digestibility of organic matter (OM), neutral detergent fiber (NDF), starch, and N were measured, and energy and N balances determined at weekly intervals by total collection of feces, urine, and milk over 48 h. Cows were individually fed ad libitum a grass silage- and corn silage-based total mixed ration during lactation and a corn silage- and barley straw-based total mixed ration during the dry period. Effects of stage of lactation were evaluated by clustering week in 5 groups: late lactation (wk -8 to -7), dry period (wk -6 to -1), and 3 early lactation periods (wk 1 to 5, wk 6 to 10, and wk 11 to 16). In lactation, apparent total-tract digestibility of OM, NDF, and starch was lowest in the first 5 wk of lactation. From wk 2 to 16 after parturition, apparent nutrient digestibility of all nutrients increased linearly, but with a negative quadratic component for dry matter, OM, and NDF, to levels comparable to those reported in last 2 wk of the previous lactation. However, differences in digestibility across lactation stage were moderate, illustrated by the difference between OM digestibility in late lactation (last 2 wk, 74.8%) and early lactation (first 5 wk, 72.5%). Cows were in negative energy balance for the first 8 wk after calving, and in negative N balance for the first 4 wk after calving. Based on energy and N balance, we predicted that 36.5 kg of body fat and 3.5 kg of body protein were gained in the last 8 wk before calving, and that 47.5 kg of body fat and 7.6 kg of body protein were mobilized in the first weeks of lactation. These predicted changes in body mass, both the gain before calving and loss after calving, were greater by 37% and 10%, respectively, than fluctuations in measured body weight (corrected for predicted gut fill and fetus weights). At wk 1 and 2 postpartum, body N loss corresponded to 25 and 29%, respectively, of total N excretion in milk, and body energy loss corresponded to 64% and 44%, respectively, of the energy exported to milk, illustrating the important contribution of N and energy from body stores to milk production in early lactation. Metabolic N efficiency, measured as total N output (milk and body) over digestible N input (from diet and body), averaged 54.4% in the last 2 wk of lactation, increased to 65.9% 2 wk after calving, and decreased linearly as lactation advanced to 61.9% by wk 16. Short (48 h) but weekly repetition of total collection of feces and urine appears to be a suitable approach to evaluate temporal changes in nutrient digestibility, energy balance, and N balance across lactation and the dry period.

Blood calcium dynamics in cows receiving an aqueous calcium suspension for voluntary consumption or a calcium bolus following parturition.

The form of oral calcium (Ca) supplement and the Ca source influence Ca absorption dynamics resulting in different postpartum calcemia. The objective of this study was to investigate whether an oral Ca supplement (mainly CaCO3) offered for voluntary consumption would maintain or increase postpartum blood Ca to the same degree as a Ca bolus (mainly CaCl2) providing an equivalent dose of a Ca. A total of 72 Holstein cows were blocked by expected parturition date and parity. Within each block of 3 animals, cows were randomly assigned to one of three treatments, including an oral Ca supplement offered for voluntary consumption (Ca-drink, n = 23), an oral Ca bolus (Ca-bolus, n = 24), or an untreated group (CON, n = 25). Treatments were administered once within 15 min postpartum. The Ca-drink provided 45 g of Ca (CaCO3 source) and was mixed in 20 L of lukewarm water and offered to cows for 30 min. The Ca-bolus provided 43 g of Ca (71% from CaCl2 and 29% from CaSO4) and was administered once. Both Ca-bolus and CON cows received 20-l of lukewarm water at parturition to standardize the volume of fluids (Ca-drink or 20-l lukewarm water) offered at parturition. Dairy cows offered Ca-drink had a 28% higher fluid consumption than Ca-bolus and CON cows. Milk yield and milk composition expressed in percentage protein, fat, lactose, and urea did not differ, whilst there was a small but significant increase in DMI in cows receiving the Ca-drink compared to CON, while Ca-bolus did not differ from other groups. This was consistent with reduced BW losses between week 1 and 3 in cows receiving the Ca-drink suspension. Treatment by time interactions were present for blood Ca, glucose, and urea concentrations. Blood Ca was relatively stable in Ca-drink cows, while higher fluctuations were observed in Ca-bolus cows. In Ca-bolus cows, blood Ca increased from 15 min to 6 h, decreased from 6 to 24 h, and finally increased again from 24 to 48 h. At 24 h post administration, blood Ca was greater in cows receiving the Ca-drink than cows receiving the Ca-bolus. Blood glucose was greater in Ca-bolus cows at 15 min after treatment administration compared with Ca-bolus and CON, while blood urea was higher in CON than Ca-drink and Ca-bolus throughout the sampling period. These results indicate that voluntary oral Ca resulted in a relatively stable calcemia, whereas higher fluctuations were observed in cows receiving the Ca-bolus. Due to a lack of differences between Ca-drink and Ca-bolus compared with CON, it is not possible to conclude regarding the efficacy in maintaining postpartum blood Ca.

Coronavirus Disease 2019 and Hospital Readmissions: Patient Characteristics and Socioeconomic Factors Associated With Readmissions in an Urban Safety-Net Hospital System.

BACKGROUND: It is not yet known whether socioeconomic factors (ie, social determinants of health) are associated with readmission following hospitalization for coronavirus disease 2019 (COVID-19). METHODS: We conducted a retrospective cohort study of 6191 adult patients hospitalized with COVID-19 in a large New York City safety-net hospital system between March 1 and June 1, 2020. Associations between 30-day readmission and selected demographic characteristics, socioeconomic factors, prior health care utilization, and relevant features of the index hospitalization were analyzed using a multivariable generalized estimating equation model. RESULTS: The readmission rate was 7.3%, with a median of 7 days between discharge and readmission. The following were risk factors for readmission: age 65 and older [adjusted odds ratio (aOR): 1.32; 95% confidence interval (CI): 1.13-1.55], history of homelessness, (aOR: 2.03 95% CI: 1.49-2.77), baseline coronary artery disease (aOR: 1.68; 95% CI: 1.34-2.10), congestive heart failure (aOR: 1.34; 95% CI: 1.20-1.49), cancer (aOR: 1.68; 95% CI: 1.26-2.24), chronic kidney disease (aOR: 1.74; 95% CI: 1.46-2.07). Patients' sex, race/ethnicity, insurance, and presence of obesity were not associated with increased odds of readmission. A longer length of stay (aOR: 0.98; 95% CI: 0.97-1.00) and use of noninvasive supplemental oxygen (aOR: 0.68; 95% CI: 0.56-0.83) was associated with lower odds of readmission. Upon readmission, 18.4% of patients required intensive care, and 13.7% expired. CONCLUSION: We have found some factors associated with increased odds of readmission among patients hospitalized with COVID-19. Awareness of these risk factors, including patients' social determinants of health, may ultimately help to reduce readmission rates.

Dietary protein oscillation: Effects on feed intake, lactation performance, and milk nitrogen efficiency in lactating dairy cows.

Limited research with growing ruminants indicates that oscillating (OS) dietary crude protein (CP) concentration may improve nitrogen use efficiency (NUE). Our aim was to determine if a total mixed ration (TMR) based on OS CP (48-h phases of 13.4% and 16.5% CP, respectively) would increase NUE of lactating dairy cows compared with a static CP TMR (ST; 14.9% CP). The experiment was a randomized complete block design with 50 cows [150 ± 61 (mean ± SD) d in milk]. Cows were blocked by parity, days in milk, and milk protein yield. On average, diets were equal in composition over the total experiment. Cows were milked twice daily, and 8 milk samples were collected in each 4-d period. Each 48 h of low-CP (LP) and high-CP (HP) TMR offered to OS cows corresponded to milk collected at milkings 1 to 4 and 5 to 8, respectively. Dry matter intake (mean = 25.5 kg/d for both treatment groups); yields of milk (mean = 31.5 kg/d for both treatment groups), protein, fat, lactose, and fat- and protein-corrected milk (mean = 33.6 kg/d for both treatment groups); and milk concentration of protein, fat, and lactose did not differ between treatments. However, milk urea concentration was higher for OS compared with ST (12.2 vs. 11.3 mg/dL). Body weight, body condition score, NUE, and feed efficiency were unaffected by OS. Apparent total-tract digestibility of dry matter (695 vs. 677 g/kg), organic matter (714 vs. 697 g/kg), CP (624 vs. 594 g/kg), neutral detergent fiber (530 vs. 499 g/kg), and starch (976 vs. 973 g/kg) were higher for OS than for ST cows. Cows in OS responded transiently, and regression analysis of differences within block over time revealed changes in yield of milk (-531 g/d), milk protein (-25.6 g/d), and milk lactose (-16.7 g/d) in LP. Opposite effects were observed for yield of milk (+612 g/d), milk protein (+28.8 g/d), and milk lactose (+28.0 g/d) during HP. Changes in concentrations of milk protein (-0.050%/d), lactose (+0.030%/d), and urea (-3.0 mg/dL per day) during LP, and in milk lactose (-0.024%/d) and urea (+4.3 mg/dL per day) during HP, were observed. Milk yield, lactose yield, and protein yield were lower for OS than ST cows at the last milking of LP and at the first milking of HP. Milk urea concentration did not show such a lag and was lower in the last 2 milkings of LP, and higher in the last 3 milkings of HP, in OS compared with ST cows. Overall, performance and NUE were unaffected by OS treatment, but apparent total-tract digestibility and milk urea concentration increased, and transient effects on milk yield and composition occurred in OS cows.

Effect of a calcium-energy supplement drink at calving on lactation performance: Milk yield and composition, odds to reach a next lactation, and calving interval.

Supplementation of Ca products to cows after calving is common in calving protocols. This study evaluated the effect of a Ca-energy drink voluntarily consumed on milk yield and composition, odds to reach a next lactation, and calving interval. This prospective randomized study included a blinded placebo and was conducted in 10 commercial dairy farms that included 504 Holstein dairy cows. Cows were blocked within farm by calving sequence and parity (primiparous or multiparous). Within each block of 2 animals, cows were randomly assigned to 1 of 2 treatments: a Ca-energy supplement drink (CAE, n = 255) providing 45 g of Ca and other components (dextrose, lactose, protein, fat, other minerals and vitamins), a placebo (i.e., 100 g of cellulose and 20 g of dextrose; CON, n = 249), both strictly offered to the animals for voluntary consumption. Treatments were offered mixed in 20 L of water within 3 h after calving. Milk data were analyzed using 2 approaches. The first, most classical, evaluated the effect of the treatments on observed milk data, whereas the second approach evaluated the effect on milk residuals (i.e., the difference between observed milk data and a prediction made by a herd test-day model). Eighty-one percent of the CAE cows fully consumed the treatment, whereas only 50% of CON cows did. No differences were detected for observed milk yield, nor for composition in multiparous cows. The only production effect observed on multiparous cows was a treatment by time interaction for milk fat yield, reflecting greater yield for CAE cows between 100 and 150 d in milk only. However, primiparous cows receiving CAE had increased milk (+0.8 kg/d) and component yields (i.e., +40 g/d of protein) compared with CON cows. These effects were more evident when milk and milk components residuals data were analyzed (i.e., +1.5 kg/d for milk yield and +57 g/d of protein). This was achieved with a herd test-day model that allowed milk and milk components data to be adjusted for environmental and genetic factors (i.e., farm effect, time effect, age at calving, parity, stage of lactation, breeding value). The treatment had no effect on the probability of reaching the next lactation (i.e., 72% of CAE cows had a next calving against 69% in CON). Primiparous cows receiving CAE had a longer calving interval compared with CON cows. At 400 d after the application of the treatment, 65% of CAE primiparous cows had a next calving, whereas 81% of CON primiparous cows had calved already. The supplementation of the tested oral Ca-energy solution at calving did not increase the probability to reach a next lactation for neither primiparous or multiparous, but positively influenced milk yield and milk component yields for primiparous.

Depression Following Operative Treatments for Achilles Ruptures and Ankle Fractures.

BACKGROUND: The sudden and debilitating nature of lower extremity injuries can trigger mood disturbances, including major depressive disorder. METHODS: This prospective study enrolled patients undergoing operative repair of ankle fractures and Achilles ruptures and followed them for 1 year postoperatively. The validated Patient Health Questionnaire (PHQ-9) for depressive symptoms was administered at the preoperative visit and at postoperative weeks 1, 2, 4, 8, 16, 24, 32, 40, and 52. PHQ-9 is scored 0 to 27, with higher values indicating greater depression symptoms. RESULTS: Fifty-eight patients completed 1 year of follow-up. The mean PHQ-9 score was 2.7 (range, 0-20) at the preoperative visit, peaked at postoperative week 1 (4.9; range, 0-16), and reached its low at postoperative week 52 (0.8; range, 0-7). Cumulative incidences of depressive symptoms during the first year following surgery were 51.7% for at least mild depression, 22.4% for at least moderate depression, and 6.9% for severe depression. A history of mental health disorder and the inability to work during the period of postoperative immobilization were independently associated with greater depressive symptoms. CONCLUSION: The majority of patients undergoing operative treatment of Achilles ruptures and ankle fractures develop postoperative symptoms of mild to moderate depression that normalize after several months. Patients with a history of mental health disorder or who cannot work while immobilized postoperatively are at greatest risk. LEVEL OF EVIDENCE: Level II, prospective cohort study.

Evaluation of Allogeneic Bone-Marrow-Derived and Umbilical Cord Blood-Derived Mesenchymal Stem Cells to Prevent the Development of Osteoarthritis in An Equine Model.

Osteoarthritis (OA) is a significant cause of pain in both humans and horses with a high socio-economic impact. The horse is recognized as a pertinent model for human OA. In both species, regenerative therapy with allogeneic mesenchymal stem cells (MSCs) appears to be a promising treatment but, to date, no in vivo studies have attempted to compare the effects of different cell sources on the same individuals. The objective of this study is to evaluate the ability of a single blinded intra-articular injection of allogeneic bone-marrow (BM) derived MSCs and umbilical cord blood (UCB) derived MSC to limit the development of OA-associated pathological changes compared to placebo in a post-traumatic OA model applied to all four fetlock joints of eight horses. The effect of the tissue source (BM vs. UCB) is also assessed on the same individuals. Observations were carried out using clinical, radiographic, ultrasonographic, and magnetic resonance imaging methods as well as biochemical analysis of synovial fluid and postmortem microscopic and macroscopic evaluations of the joints until Week 12. A significant reduction in the progression of OA-associated changes measured with imaging techniques, especially radiography, was observed after injection of bone-marrow derived mesenchymal stem cells (BM-MSCs) compared to contralateral placebo injections. These results indicate that allogeneic BM-MSCs are a promising treatment for OA in horses and reinforce the importance of continuing research to validate these results and find innovative strategies that will optimize the therapeutic potential of these cells. However, they should be considered with caution given the low number of units per group.

Targeting the Hindgut to Improve Health and Performance in Cattle.

An adequate gastrointestinal barrier function is essential to preserve animal health and well-being. Suboptimal gut health results in the translocation of contents from the gastrointestinal lumen across the epithelium, inducing local and systemic inflammatory responses. Inflammation is characterized by high energetic and nutrient requirements, which diverts resources away from production. Further, barrier function defects and inflammation have been both associated with several metabolic diseases in dairy cattle and liver abscesses in feedlots. The gastrointestinal tract is sensitive to several factors intrinsic to the productive cycles of dairy and beef cattle. Among them, high grain diets, commonly fed to support lactation and growth, are potentially detrimental for rumen health due to their increased fermentability, representing the main risk factor for the development of acidosis. Furthermore, the increase in dietary starch associated with such rations frequently results in an increase in the bypass fraction reaching distal sections of the intestine. The effects of high grain diets in the hindgut are comparable to those in the rumen and, thus, hindgut acidosis likely plays a role in grain overload syndrome. However, the relative contribution of the hindgut to this syndrome remains unknown. Nutritional strategies designed to support hindgut health might represent an opportunity to sustain health and performance in bovines.

An experimentally induced osteoarthritis model in horses performed on both metacarpophalangeal and metatarsophalangeal joints: Technical, clinical, imaging, biochemical, macroscopic and microscopic characterization.

Osteoarthritis is a common cause of pain and economic loss in both humans and horses. The horse is recognized as a suitable model for human osteoarthritis, because the thickness, structure, and mechanical properties of equine articular cartilage are highly comparable to those of humans. Although a number of equine experimental osteoarthritis models have been described in the literature, these cases generally involve the induction of osteoarthritis in just one joint of each animal. This approach necessitates the involvement of large numbers of horses to obtain reliable data and thus limits the use of this animal model, for both economic and ethical reasons. This study adapts an established equine model of post-traumatic osteoarthritis to induce osteoarthritis-associated lesions in all 4 fetlock joints of the same horse in order to reduce the number of animals involved and avoid individual variability, thus obtaining a more reliable method to evaluate treatment efficacy in future studies. The objectives are to assess the feasibility of the procedure, evaluate variability of the lesions according to interindividual and operated-limb position and describe the spontaneous evolution of osteoarthritis-associated pathological changes over a twelve-week period. The procedure was well tolerated by all 8 experimental horses and successfully induced mild osteoarthritis-associated changes in the four fetlock joints of each horse. Observations were carried out using clinical, radiographic, ultrasonographic, and magnetic resonance imaging methods as well as biochemical analyses of synovial fluid and postmortem microscopic and macroscopic evaluations of the joints. No significant differences were found in the progression of osteoarthritis-associated changes between horses or between the different limbs, with the exception of higher synovial effusion in hind fetlocks compared to front fetlocks and higher radiographic scores for left fetlocks compared to the right. This model thus appears to be a reliable means to evaluate the efficacy of new treatments in horses, and may be of interest for translational studies in human medicine.

Intra-Articular Injection of 2 Different Dosages of Autologous and Allogeneic Bone Marrow- and Umbilical Cord-Derived Mesenchymal Stem Cells Triggers a Variable Inflammatory Response of the Fetlock Joint on 12 Sound Experimental Horses.

Osteoarthritis is a significant and costly cause of pain for both humans and horses. The horse has been identified as a suitable model for human osteoarthritis. Regenerative therapy with allogeneic mesenchymal stem cells (MSCs) is a promising treatment, but the safety of this procedure continues to be debated. The aim of this study is to evaluate the safety of intra-articular injections of allogeneic MSCs on healthy joints by comparing two different dosages and two different tissue sources, namely, bone marrow and umbilical cord blood, with a placebo treatment on the same individuals. We also assessed the influence of autologous versus allogeneic cells for bone marrow-derived MSC treatment. Twelve clinically sound horses were subjected to injections in their 4 fetlock joints. Each of the three fetlocks was administered a different MSC type, and the remaining fetlock was injected with phosphate-buffered saline as a control. Six horses received 10 million cells per joint, and the 6 other horses received 20 million cells per joint. Clinical and ultrasound monitoring revealed that allogeneic bone marrow-derived MSCs induced significantly more synovial effusion compared to umbilical cord blood-derived MSCs but no significant difference was noted within the synovial fluid parameters. The administration of 10 million cells in horses triggered significantly more inflammatory signs than the administration of 20 million cells. Mesenchymal stem cell injections induced mild to moderate local inflammatory signs compared to the placebo, with individual variability in the sensitivity to the same line of MSCs. Understanding the behavior of stem cells when injected alone is a step towards the safer use of new strategies in stem cell therapy, where the use of either MSC secretome or MSCs combined with biomaterials could enhance their viability and metabolic activity.

Engulfment of ceramic particles by fibroblasts does not alter cell behavior.

Despite many studies, the impact of ceramic particles on cell behavior remains unclear. The aim of the present study was to investigate the effects of nano-sized ceramic particles on fibroblastic cells. Fibroblasts (dermal fibroblasts freshly isolated from skin samples and WI26 fibroblastic cells) were cultured in a monolayer in the presence of alumina or cerium-zirconia particles (≈50 nm diameter) at two concentrations (100 or 500 µg ml-1). Fluorescent alumina particles were also used. The following properties were analyzed: cell morphology, cytoplasmic ceramic incorporation (using confocal and transmission electron microscopy) and migration (using a silicon insert). Sedimentation field-flow fractionation (SdFFF) was also used to evaluate the rate of incorporation of ceramic particles into the cells. Finally, after treatment with various concentrations of ceramic particles, fibroblasts were also included in a collagen type I lattice constituting a dermal equivalent (DE), and the collagen lattice retraction and cell proliferation were evaluated. In monolayer conditions, the presence of both alumina and cerium-zirconia ceramic particles did not cause any deleterious effects on cultured cells (dermal fibroblast and WI26 cells) and cell fate was not affected in any way by the presence of ceramic particles in the cytoplasm. Confocal (using fluorescent alumina particles) and electron microscopy (using both alumina and cerium-zirconia particles) showed that ceramic particles were internalized in the WI26 cells. Using fluorescent membrane labeling and fluorescent alumina particles, a membrane was observed around the particle-containing vesicles present in the cytoplasm. Electron microscopy on WI26 cells showed the presence of a classical bilayer membrane around the ceramic particles. Interestingly, SdFFF confirmed that some dermal fibroblasts contained many alumina ceramic particles while others contained very few; in WI26 cells, the uptake of alumina ceramic was more homogeneous. In DE, collagen lattice retraction and cell proliferation were unchanged when WI26 fibroblastic cells contained alumina or cerium-zirconia ceramic particles. Our data suggest that ceramic particles are internalized in the cells by endocytosis. The presence of ceramic particles in the cytoplasm has no affect on cell behavior, confirming the excellent biocompatibility of this material and anticipating a minimal harmful effect of potential wear debris.

Prediction of denosumab effects on bone remodeling: A combined pharmacokinetics and finite element modeling.

Denosumab is a fully human monoclonal antibody that inhibits receptor activator of nuclearfactor-kappa B ligand (RANKL). This key mediator of osteoclast activities has been shown to inhibit osteoclast differentiation and hence, to increase bone mineral density (BMD) in treated patients. In the current study, we develop a computer model to simulate the effects of denosumab treatments (dose and duration) on the proximal femur bone remodeling process quantified by the variation in proximal femur BMD. The simulation model is based on a coupled pharmacokinetics model of denosumab with a pharmacodynamics model consisting of a mechanobiological finite element remodeling model which describes the activities of osteoclasts and osteoblasts. The mechanical behavior of bone is described by taking into account the bone material fatigue damage accumulation and mineralization. A coupled strain-damage stimulus function is proposed which controls the level of bone cell autocrine and paracrine factors. The cellular behavior is based on Komarova et al.׳s (2003) dynamic law which describes the autocrine and paracrine interactions between osteoblasts and osteoclasts and computes cell population dynamics and changes in bone mass at a discrete site of bone remodeling. Therefore, when an external mechanical stress is applied, bone formation and resorption is governed by cell dynamics rather than by adaptive elasticity approaches. The proposed finite element model was implemented in the finite element code Abaqus (UMAT routine). In order to perform a preliminary validation, in vivo human proximal femurs were selected and scanned at two different time intervals (at baseline and at a 36-month interval). Then, a 3D FE model was generated and the denosumab-remodeling algorithm was applied to the scans at t0 simulating daily walking activities for a duration of 36 months. The predicted results (density variation) were compared to existing published ones performed on a human cohort (FREEDOM).

Sex-specific regulation of AMP-activated protein kinase (AMPK) in the Pacific oyster Crassostrea gigas.

The hermaphrodite Pacific oyster Crassostrea gigas displays a high energy allocation to reproduction. We studied the expression of AMP-activated protein kinase (AMPK) during gametogenesis in the gonad and characterized the mRNA sequences of the AMPK subunits: the AMPK alpha mRNA sequence was previously characterized; we identified AMPK beta, AMPK gamma, and mRNAs of putative AMPK-related targets following bioinformatics mining on existing genomic resources. We analyzed the mRNA expression of the AMPK alpha, beta, and gamma subunits in the gonads of male and female oysters through a reproductive cycle, and we quantified the mRNA expression of genes belonging to fatty acid and glucose metabolism. AMPK alpha mRNA levels were more abundant in males at the first stage of gametogenesis, when mitotic activity and the differentiation of germinal cells occur, and were always more abundant in males than in females. Some targets of fatty acid and glucose metabolism appeared to be correlated with the expression of AMPK subunits at the mRNA level. We then analyzed the sex-specific AMPK activity by measuring the phosphorylation of the catalytic AMPK alpha protein and its expression at the protein level. Both the amount of AMPK alpha protein and threonine 172 phosphorylation appeared to be almost totally inhibited in mature female gonads at stage 3, at the time when accumulation of reserves in oocytes was promoted, while it remained at a high level in mature spermatozoa. Its activation might play a sex-dependent role in the management of energy during gametogenesis in oyster.

Regulation of a truncated isoform of AMP-activated protein kinase α (AMPKα) in response to hypoxia in the muscle of Pacific oyster Crassostrea gigas.

AMP-activated protein kinase α (AMPKα) is a key regulator of energy balance in many model species during hypoxia. In a marine bivalve, the Pacific oyster Crassostrea gigas, we analyzed the protein content of adductor muscle in response to hypoxia during 6 h. In both smooth and striated muscles, the amount of full-length AMP-activated protein kinase α (AMPKα) remained unchanged during hypoxia. However, hypoxia induced a rapid and muscle-specific response concerning truncated isoforms of AMPKα. In the smooth muscle, a truncated isoform of AMPKα was increased from 1 to 6 h of hypoxia, and was linked with accumulation of AKT kinase, a key enzyme of the insulin signaling pathway which controls intracellular glucose metabolism. In this muscle, aerobic metabolism was maintained over the 6 h of hypoxia, as mitochondrial citrate synthase activity remained constant. In contrast, in striated muscle, hypoxia did not induce any significant modification of neither truncated AMPKα nor AKT protein content, and citrate synthase activity was altered after 6 h of hypoxia. Together, our results demonstrate that hypoxia response is specific to muscle type in Pacific oyster, and that truncated AMPKα and AKT proteins might be involved in maintaining aerobic metabolism in smooth muscle. Such regulation might occur in vivo during tidal intervals that cause up to 6 h of hypoxia.

Analytical investigation of salivary calculi, by mid-infrared spectroscopy.

Sialolithiasis is common in salivary glands, especially in the submandibular and parotid ducts. X-Ray diffractometry was the principal technique used for their analysis, sometimes associated with scanning electron microscopy. Hydroxyapatite was the most frequently described constituent, in association with whitlockite and other calcium phosphates as brushite or octocalcium phosphate. Proteic matter was detected, as mucoproteins, albumin, nucleoproteins or as degenerative bacterial matter. This study presents the identification of constituents by mid-infrared spectrometry of 74 sialoliths. Their successive layers are analyzed from their crust to the nucleus, using absorbance measurements. Spectra are compared with reference mixtures of two or more constituents. Approximately 99% of sialoliths are constituted of calcium phosphates, under carbonated forms. More than three-quarters contain proteins, in which mucins represent the majority and albumin is found in 10% of all the specimens. Only 7% calculi are an association of two constituents, 66% are made of three and 27% have four or more components. For the 74 studied sialoliths, no specimen contains hydroxyapatite; but they are composed of carbonate apatites with irregular microcrystallized forms, even if proteins are present. Some of them have a pure protein nucleus, surrounded by carbonate apatite layers; the other stones are made of internal layers of apatites and covered with a dense and varnished crust of proteins.

Metastasis suppressor NM23-H1 promotes repair of UV-induced DNA damage and suppresses UV-induced melanomagenesis.

Reduced expression of the metastasis suppressor NM23-H1 is associated with aggressive forms of multiple cancers. Here, we establish that NM23-H1 (termed H1 isoform in human, M1 in mouse) and two of its attendant enzymatic activities, the 3'-5' exonuclease and nucleoside diphosphate kinase, are novel participants in the cellular response to UV radiation (UVR)-induced DNA damage. NM23-H1 deficiency compromised the kinetics of repair for total DNA polymerase-blocking lesions and nucleotide excision repair of (6-4) photoproducts in vitro. Kinase activity of NM23-H1 was critical for rapid repair of both polychromatic UVB/UVA-induced (290-400 nm) and UVC-induced (254 nm) DNA damage, whereas its 3'-5' exonuclease activity was dominant in the suppression of UVR-induced mutagenesis. Consistent with its role in DNA repair, NM23-H1 rapidly translocated to sites of UVR-induced (6-4) photoproduct DNA damage in the nucleus. In addition, transgenic mice hemizygous-null for nm23-m1 and nm23-m2 exhibited UVR-induced melanoma and follicular infundibular cyst formation, and tumor-associated melanocytes displayed invasion into adjacent dermis, consistent with loss of invasion-suppressing activity of NM23 in vivo. Taken together, our data show a critical role for NM23 isoforms in limiting mutagenesis and suppressing UVR-induced melanomagenesis.

In vivo RNA interference of a gonad-specific transforming growth factor-ß in the Pacific oyster Crassostrea gigas.

We investigated the role of oyster gonadal TGFß (og-TGFß) in the reproduction of Crassostrea gigas, using an in vivo RNA interference approach. We designed double-stranded RNA targeting og-TGFß, which is specifically expressed in the somatic cells surrounding germ cells in the gonad of both male and female oysters. In vivo injection of this og-TGFß dsRNA into the gonad led to knock-down phenotypes for both sexes, with significant reduction (77.52% relative to controls) of the gonad area, lowered reproductive effort and germ cell under-proliferation. Interestingly, half of the injected females halted their vitellogenesis, since we were only able to observe pre-vitellogenic oocytes. In addition, apoptotic germ cells and haemocytes infiltrated into the gonad, likely as part of the active resorption of degenerating germ cells. Conversely, males showed a normal phenotype at the cellular level, with spermatids and spermatozoids observed in the gonads of control and injected males. As a result, og-TGFß appears to play an essential role in C. gigas germ cell development by functioning as an activator of germ cell proliferation in both male and female oysters and vitellogenesis in females.