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Today, one of the major challenges of dairy farmers is to reduce their environmental footprint to establish more effective, efficient, and sustainable production systems. Feed additives such as yeast probiotics could potentially allow them to achieve these objectives through the improvement of milk production, feed efficiency, and ration valorization, hence mitigating the environmental impacts of milk production. In this study, the life cycle assessment (LCA) principle was performed to estimate the environmental impact of the production and supplementation of a commercial yeast probiotic (Actisaf Sc 47) in three trials performed in three different countries that are representative for around 50% of the milk production in Europe: France (French trial), United Kingdom (UK trial), and Germany (German trial). For each trial, two groups of animals were compared: control, without Actisaf Sc 47 supplementation, used as baseline; and experimental, with Actisaf Sc 47 supplementation at 5 or 10 g/cow/day. Different impact categories were analyzed for each group to calculate the impact of producing 1 kg of fat- and protein-corrected milk. An initial analysis was done only during the period of Actisaf Sc 47 supplementation and showed than the supplementation with Actisaf Sc 47 reduced, on average by 5%, the carbon footprint during the three trials. A second analysis was done via the extrapolation of all the data of each trial to an annual farm level, including the lactation period (305 days), dry period (60 days), and the period with and without Actisaf Sc 47 supplementation. Reported at a farm annual scale, the average reduction allowed by Actisaf Sc 47 supplementation was 2.9, 2.05, 2.47, 1.67, 2.28, 2.18, 2.14, and 2.28% of the carbon footprint, land use, water use, resource use, acidification, freshwater eutrophication, marine eutrophication, and terrestrial eutrophication, respectively. On average, the production of 1 kg of fat- and protein-corrected milk by using Actisaf Sc 47 was shown to improve environmental impacts compared to control. Regarding Actisaf Sc 47 production, the LCA showed that the production of 1 kg of Actisaf Sc 47 emitted 2.1 kg CO 2 eq with a negligible contribution to total the carbon footprint of milk ranging from 0.005 to 0.016%. The use of Actisaf Sc 47 in dairy cows could then result in different positive outcomes: improving performance and efficiency while reducing the global carbon footprint.

Tissue-resident mesenchymal stem cells (MSCs) are important regulators of tissue repair or regeneration, fibrosis, inflammation, angiogenesis, and tumor formation. Here, we define a population of resident lung MSCs (luMSCs) that function to regulate the severity of bleomycin injury via modulation of the T-cell response. Bleomycin-induced loss of these endogenous luMSCs and elicited fibrosis (pulmonary fibrosis), inflammation, and pulmonary arterial hypertension (PAH). Replacement of resident stem cells by administration of isolated luMSCs attenuated the bleomycin-associated pathology and mitigated the development of PAH. In addition, luMSC modulated a decrease in numbers of lymphocytes and granulocytes in bronchoalveolar fluid and demonstrated an inhibition of effector T-cell proliferation in vitro. Global gene expression analysis indicated that the luMSCs are a unique stromal population differing from lung fibroblasts in terms of proinflammatory mediators and profibrotic pathways. Our results demonstrate that luMSCs function to protect lung integrity after injury; however, when endogenous MSCs are lost, this function is compromised illustrating the importance of this novel population during lung injury. The definition of this population in vivo in both murine and human pulmonary tissue facilitates the development of a therapeutic strategy directed at the rescue of endogenous cells to facilitate lung repair during injury., (Copyright © 2011 AlphaMed Press.)

Background: We tested the hypothesis that maternal interleukin-1beta (IL-1beta) pretreatment and induction of fetal cortisol synthesis activates MAP kinases and thereby affects lung fluid absorption in preterm guinea pigs., Methods: IL-1beta was administered subcutaneously daily to timed-pregnant guinea pigs for three days. Fetuses were obtained by abdominal hysterotomy and instilled with isosmolar 5% albumin into the lungs and lung fluid movement was measured over 1 h by mass balance. MAP kinase expression was measured by western blot., Results: Lung fluid absorption was induced at 61 days (D) gestation and stimulated at 68D gestation by IL-1beta. Maternal IL-1beta pretreatment upregulated ERK and upstream MEK expression at both 61 and 68D gestation, albeit being much more pronounced at 61D gestation. U0126 instillation completely blocked IL-1beta-induced lung fluid absorption as well as IL-1beta-induced/stimulated ERK expression. Cortisol synthesis inhibition by metyrapone attenuated ERK expression and lung fluid absorption in IL-1beta-pretreated fetal lungs. JNK expression after maternal IL-1beta pretreatment remained unaffected at either gestation age., Conclusion: These data implicate the ERK MAP kinase pathway as being important for IL-1beta induction/stimulation of lung fluid absorption in fetal guinea pigs.

Platelet-derived growth factor (PDGF) inhibits expression of smooth muscle (SM) genes in vascular smooth muscle cells and blocks induction by arginine vasopressin (AVP). We have previously demonstrated that suppression of SM-alpha-actin by PDGF-BB is mediated in part through a Ras-dependent pathway. This study examined the role of phosphatidylinositol 3-kinase (PI3K)y and its downstream effector, Akt, in regulating SM gene expression. PDGF caused a rapid sustained activation of Akt, whereas AVP caused only a small transient increase. PDGF selectively caused a sustained stimulation of p85/p110 alpha PI3K. In contrast, p85/110 beta PI3K activity was not altered by either PDGF or AVP, whereas both agents caused a delayed activation of Class IB p101/110 gamma PI3K. Expression of a gain-of-function PI3K or myristoylated Akt (myr-Akt) mimicked the inhibitory effect of PDGF on SM-alpha-actin and SM22 alpha expression. Pretreatment with LY 294002 reversed the inhibitory effect of PDGF. Expression of myr-Akt selectively inhibited AVP-induced activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinases, which we have shown are critical for induction of these genes. Nuclear extracts from PDGF-stimulated or myr-Akt expressing cells showed reduced serum response factor binding to SM-specific CArG elements. This was associated with appearance of serum response factor in the cytoplasm. These data indicate that activation of p85/p110 alpha/Akt mediates suppression of SM gene expression by PDGF.

Vascular smooth muscle (SM) cells (VSMC) undergo phenotypic modulation in vivo and in vitro. This process involves coordinated changes in expression of multiple SM-specific genes. In cultured VSMC, arginine vasopressin (AVP) increases and PDGF decreases expression of SM alpha-actin (SMA), the earliest marker of SM cells (SMC). However, it is unknown whether these agents regulate other SM genes in a similar fashion. SM22 alpha appears secondary to SMA during development and is also a marker for SMC. This study examined the regulation of SM22 alpha expression by AVP and PDGF in cultured VSMC. Levels of SM22 alpha mRNA and protein were increased by AVP and suppressed by PDGF. Consistent with these changes, AVP increased SM22 alpha promoter activity, whereas PDGF inhibited basal promoter activity and blocked AVP-induced increase. Activation of both JNK and p38 MAPK pathways was necessary for AVP-mediated induction of SM22 alpha promoter. Expression of constitutively active Ras produced similar suppressions on SM22 alpha promoter activity as PDGF. Signaling relayed from PDGF/Ras activation involved Raf, or a protein that competes for this site, Ral-GDS, and phosphatidylinositol 3-kinase activation. Truncational analysis showed that the proximal location of three CArG boxes in the promoter was sufficient for AVP stimulation. Mutations in this CArG box reduced basal and AVP-stimulated promoter activity without effecting PDGF suppression. Overexpression of serum response factor enhanced basal and AVP-stimulated promoter activity but had no effect on PDGF-BB-induced suppression. These data indicate that AVP and PDGF initiate specific signaling pathways that control expression of multiple SM genes leading to phenotypic modulation.

Interleukin-1 (IL-1) plays a pivotal role in the pathogenesis of inflammatory bowel disease (IBD). IL-1 action is regulated in part by its naturally occurring inhibitor, the IL-1 receptor antagonist (IL-1Ra). Four splice variants of IL-1Ra gene product have been described, one secreted (sIL-1Ra) and three intracellular (icIL-1Ra1, 2, 3). Although sIL-1Ra and icIL-1Ra1 bind to type I IL-1 receptor with equal affinity, icIL-1Ra1 may carry out unique functions inside cells. The goal of this study was to determine the role of icIL-1Ra1 in regulation of cytokine-induced IL-6 and IL-8 production in Caco-2 intestinal epithelial cells. icIL-1Ra1 inhibited IL-1-induced IL-6 and IL-8 production. IL-1 activated all three mitogen-activated protein (MAP) kinase family members: p38 MAP kinase, extracellular-regulated kinases (ERK), and c-Jun amino-terminal kinases (JNK). Specific inhibitors of each MAP kinase pathway decreased IL-1-induced IL-6 and IL-8 production. Overexpression of icIL-1Ra1 inhibited p38 MAP kinase phosphorylation, but had no effect on ERK and JNK phosphorylation. In addition, icIL-1Ra1 inhibited nuclear translocation of NF-kappaB after IL-1 stimulation. In conclusion, these data indicate that icIL-1Ra1, acting in the cytoplasm of Caco-2 cells, decreased IL-1-induced IL-6 and IL-8 production. This intracellular anti-inflammatory activity of icIL-1Ra1 was mediated through inhibition of p38 MAP kinase and NF-kappaB signal transduction pathways.

Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme, catalyzing the oxidative cleavage of heme molecules to biliverdin, carbon monoxide, and iron. The present study was designed to investigate the role of HO-1 in the pathogenesis of renal dysfunction during cirrhosis. Biliary cirrhosis was induced in rats by common bile duct ligation (CBDL). Animals were studied 2 and 5 wk after surgery. In kidney from CBDL rats, HO-1 protein expression increased slightly at 2 wk but was abolished at 5 wk. In addition, we confirmed histologically that HO-1 expression was suppressed in renal tubules and interlobular arterioles in 5-wk-old CBDL rats. Conversely, HO-1 expression in liver was strongly increased. Consistent with the development of cirrhosis and renal dysfunction mean arterial pressure (MAP), glomerular filtration rate (GFR), and renal blood flow (RBF) were decreased in CBDL rats compared with sham-operated controls. In sham rats, treatment with the selective HO inhibitor zinc protoporphyrin markedly decreased GFR and RBF to values similar to those measured in CBDL rats without decreasing MAP. In conclusion, decreased renal HO-1 expression contributes to deteriorated renal function and hemodynamics during cirrhosis. This finding provides a novel mechanism for the pathophysiology of renal dysfunction during cirrhosis.

Exposure of vascular smooth muscle cells to arginine vasopressin (AVP) increases smooth muscle alpha-actin (SM-alpha-actin) expression through activation of the SM- alpha-actin promoter. The goal of this study was to determine the role of the mitogen-activated protein kinase (MAP kinase) family in regulation of SM-alpha-actin expression. AVP activated all three MAP kinase family members: ERKs, JNKs, and p38 MAP kinase. Inhibition of JNKs or p38 decreased AVP-stimulated SM-alpha-actin promoter activity, whereas inhibition of ERKs had no effect. A 150-base pair region of the promoter containing two CArG boxes was sufficient to mediate regulation by vasoconstrictors. Mutations in either CArG box decreased AVP-stimulated promoter activity. Electrophoretic mobility shift assays using oligonucleotides corresponding to either CArG box resulted in a complex of similar mobility whose intensity was increased by AVP. Antibodies against serum response factor (SRF) completely super-shifted this complex, indicating that SRF binds to both CArG boxes. Overexpression of SRF increased basal promoter activity, but activity was still stimulated by AVP. AVP stimulation rapidly increased SRF phosphorylation. These data indicate that both JNKs and p38 participate in regulation of SM- alpha-actin expression. SRF, which binds to two critical CArG boxes in the promoter, represents a potential target of these kinases.

Transforming growth factor beta (TGF beta) family members are secreted in inactive complexes with a latency-associated peptide (LAP), a protein derived from the N-terminal region of the TGF beta gene product. Extracellular activation of these complexes is a critical but incompletely understood step in regulation of TGF beta function in vivo. We show that TGF beta 1 LAP is a ligand for the integrin alpha v beta 6 and that alpha v beta 6-expressing cells induce spatially restricted activation of TGF beta 1. This finding explains why mice lacking this integrin develop exaggerated inflammation and, as we show, are protected from pulmonary fibrosis. These data identify a novel mechanism for locally regulating TGF beta 1 function in vivo by regulating expression of the alpha v beta 6 integrin.

Because the availability of transgenic mice makes it possible to examine the contribution of single genes to in vivo function, we developed a simple in situ mouse model that can be used to quantify isosmolar alveolar epithelial fluid clearance (AFC). Mice were killed, a tracheostomy was done, and then a test solution of a 5% isosmolar albumin solution with 0.1 micro Ci of 125I-labeled albumin was instilled via the trachea into the distal air spaces of both lungs. After instillation, the lungs were inflated to 7 cmH2O with 100% O2 and maintained at 37 degrees C by placing the animals under an infrared lamp. AFC was measured by the progressive increase in concentration of labeled and unlabeled protein over 1 h. The results indicated the following. 1) Basal, unstimulated AFC in mouse lungs was significantly faster than in ex vivo rat lungs (27 +/- 5% in in situ mice vs. 11 +/- 3% in ex vivo rat lungs; P < 0.05). 2) Comparison of equivalent doses (10(-4) M) of beta-adrenergic agonist (isoproterenol) and beta2-adrenergic agonists (terbutaline and salmeterol) indicated that stimulated clearance occurred only in presence of isoproterenol. 3) Because atenolol, a specific beta1-antagonist, abolished the effect of isoproterenol, the beta-adrenergic stimulation appears to be mediated by beta1-receptors. The rate of AFC in nonperfused mouse lungs was significantly faster than in prior studies of nonperfused lungs in rats and sheep. Interestingly, the stimulated clearance rate in mice was similar to the fast rates of AFC that we recently reported in patients recovering from hydrostatic pulmonary edema. This in situ model is a unique experimental preparation that can be readily used to quantify isosmolar epithelial fluid clearance in mice.

Nitric oxide (NO) may either protect against or contribute to oxidant-induced lung injury. In this study, we sought to determine whether either inhaled NO in concentration of 10 and 100 parts per million (ppm) or inhibition of endogenous NO formation with L-NG nitroarginine methyl ester (L-NAME) or aminoguanidine alters the extent of lung injury in rats breathing 100% O2. Lung thiobarbituric acid reactive substances (TBARS), wet to dry lung weight ratio (Q(W)/Q(D)), vascular and epithelial permeability (assessed by simultaneous intravenous administration of 131I-labeled albumin and intraalveolar instillation of 125I-labeled albumin), alveolar liquid clearance (evaluated based on the increase in alveolar protein concentration), and lung liquid clearance (gravimetric method) were determined after 40 h exposure to either 100% or 21% O2. Exposure to hyperoxia caused increases in lung TBARS from 10.5 +/- 0.7 to 13.7 +/- 1.5 micromol/mg protein (p < 0.05); in blood hemoglobin concentration (Hb) from 14 +/- 1 g/dl to 17 +/- 1 g/dl (p < 0.05); in the Q(W)/Q(D) ratio from 4.02 +/- 0.3 to 5.31 +/- 0.5 (p < 0.05); and in alveolar-arterial oxygen tension difference from 124 +/- 14 mm Hg to 241 +/- 61 mm Hg (p < 0.05); as well as a decrease in blood pressure, from 131 +/- 15 mm Hg to 72 +/- 26 mm Hg (p < 0.05). Hyperoxia also increased vascular albumin leakage and moderately altered epithelial barrier permeability to protein. Inhalation of 10 ppm NO prevented the increases in TBARS and Q(W)/Q(D), had no effect on the alveolar barrier impermeability to protein, and improved alveolar liquid clearance. Inhalation of 100 ppm NO did not alter the increases in TBARS and Q(W)/Q(D) but increased vascular permeability to protein. Survival of rats exposed to hyperoxia was not improved by inhaled NO. Treatment with L-NAME or aminoguanidine reduced survival. L-NAME, but not aminoguanidine, increased lung TBARs. These results suggest that, depending on its concentration, inhaled NO can either reduce or increase the early consequences of hyperoxic lung injury. Treatment with L-NAME, and to a lesser extent aminoguanidine, worsened hyperoxic lung injury, indicating a protective effect of endogenous NO.

The capacity of the alveolar epithelial barrier to remove excess alveolar fluid from the airspaces of the lung was studied in an experimental model of moderate hyperoxic lung injury. Rats were exposed to 100% oxygen for 40 h in an exposure chamber and compared with control animals exposed to room air. Extravascular lung water was calculated gravimetrically. Alveolar and lung liquid clearance were studied over 1 h by instillation of a 5% albumin solution with 1.5 microCi of 125I-labeled albumin (6 mL/kg into both lungs). The concentration of both the unlabeled and labeled albumin was used to calculate alveolar liquid clearance. Hyperoxic rats developed pulmonary edema, with a 33% increase in extravascular lung water to 5.3 +/- 0.1 g of water per gram of dry lung, compared with 4.0 +/- 0.2 g of water per gram of dry lung in control rats (p < 0.05). This degree of edema was associated with a significant increase in the alveolar-arterial oxygen difference (241 +/- 61 vs 124 +/- 14 mm Hg in control animals exposed to room air, p < 0.05). Despite this moderate degree of lung injury, alveolar fluid clearance was normal (30 +/- 3%) compared with control rats (33 +/- 6%). Furthermore, the hyperoxic injured rats responded normally to an exogenous beta-adrenergic agonist (terbutaline, 10(-4) mol/L) with a 67% increase in the rate of alveolar liquid clearance (50 +/- 5%). Thus, in the setting of moderate hyperoxic lung injury, the alveolar epithelial barrier is still capable of removing fluid at a normal rate and responding to beta-adrenergic agonist treatment. These experimental results have potential clinical implications for patients with acute lung injury.

Alveolar epithelial type II cells are the progenitor cells for restoring the alveolar epithelial barrier after acute lung injury. During repair of lung injury, the alveolar epithelial type II cells reepithelialize denuded air spaces, a process that involves breaking and reforming cell adhesions. A novel technique of mechanical separation of cultured alveolar epithelial cells from in vitro matrix was used to examine the intracellular signals that result when alveolar epithelial cell adhesions are broken. The results show that the tyrosine phosphorylation levels of focal adhesion kinase, paxillin, and pp60(src) decreased immediately after mechanical separation of the cells. Levels returned to nearly normal by 24 h after mechanical separation. Paxillin and pp60(scr) coprecipitated with focal adhesion kinase regardless of their phosphorylation state. Interestingly, the tyrosine phosphorylation level of the mitogen-activated protein kinase, p42(erk2), increased 15 min after mechanical separation. Preincubation of cell monolayers with phenylarsine oxide, a protein tyrosine phosphatase inhibitor, blocked the decrease in tyrosine phosphorylation levels of focal adhesion kinase, paxillin and pp60(src). Phenylarsine oxide incubation also prevented readhesion of mechanically separated cells at 24 h, but genistein, a tyrosine kinase inhibitor, had no effect. We conclude that protein tyrosine phosphatases are activated immediately after cultured alveolar epithelial cells are mechanically separated from in vitro matrix, and their activation is required for alveolar epithelial cell readhesion.

To study the rate and regulation of alveolar fluid clearance in acute pneumonia, we created a model of Pseudomonas aeruginosa pneumonia in rats. To measure alveolar liquid and protein clearance, we instilled into the airspaces a 5% bovine albumin solution with 1.5 microCi of 125I-human albumin, 24 h after intratracheal instillation of bacteria. The concentration of unlabeled and labeled protein in the distal airspaces over 1 h was used as an index of net alveolar fluid clearance. Since there was histologic evidence of alveolar epithelial injury, several methods were used to measure alveolar fluid clearance, including the use of experiments in rats with blood flow and the use of experiments in rats without blood flow, so that movement across the epithelial barrier would be minimized in the latter group. The results with each method were identical. We found that P. aeruginosa pneumonia increased alveolar liquid clearance over 1 h by 48% in studies with blood flow, and by 43% in rats without blood flow, compared with respective controls (P < 0.05). In both studies, this increase was inhibited with amiloride. However, propranolol had no inhibitory effect, thus ruling out a catecholamine-dependent mechanism to explain the increase in alveolar fluid clearance. An antitumor necrosis factor-alpha neutralizing antibody, instilled into the lung 5 min before bacteria, prevented the increase in alveolar liquid clearance in rats with pneumonia (P < 0.05). Also, TNFalpha (5 microg) instilled in normal rats increased alveolar liquid clearance by 43% over 1 h compared with control rats (P < 0.05). In normal rats instilled with TNFalpha, propranolol had no inhibitory effect. In conclusion, gram-negative pneumonia markedly upregulates net alveolar epithelial fluid clearance, in part by a TNFalpha-dependent mechanism. This finding provides a novel mechanism for the upregulation of alveolar epithelial sodium and fluid transport from the distal airspaces of the lung.

Adhesive interactions between cells and extracellular matrix proteins are important in cell attachment, migration, and proliferation. The present work defines the role of fibronectin (soluble and insoluble) compared with type I and type IV collagen on in vitro alveolar epithelial wound healing. Repeated video microscopy experiments demonstrated that the half-time of wound closure was decreased in the presence of soluble fibronectin (6.6 +/- 2.1 vs. 17.4 +/- 0.8 h in serum-free medium, P < 0.05). Video microscopy, electron microscopy, and vinculin distribution demonstrated the contribution of two main events during the repair process: the migration of epithelial cell sheets and the spreading of the cells. During the wound healing, the internuclear distance between two adjacent cells at the migrating edge of the wound was significantly increased 10 h after wounding in the presence of soluble fibronectin (67 +/- 3.0 vs. 45 +/- 1.5 microns in serum-free medium, P < 0.05), indicating that cell spreading is involved as part of the mechanism for wound closure. Compared with type I and type IV collagen, insoluble fibronectin was the most potent stimulus for alveolar type II cell motility and wound healing in the absence of other serum factors. These results demonstrate that alveolar epithelial wound healing can be modulated in vitro by the composition of the extracellular matrix, an effect that may be mediated by changes in cell shape.

Under some pathological conditions, ion transport across alveolar epithelial cells is downregulated, whereas under other pathological conditions, it may be upregulated. Because endotoxin is a biologically relevant pathological stimulus, we investigated the effect of endotoxin on alveolar epithelial liquid clearance in vivo. Escherichia coli endotoxin (220 micrograms/kg) was instilled into the lungs via the trachea of rats. Then, 24 or 40 h after endotoxin instillation, alveolar and lung liquid clearances were studied over 1 h by instillation of a 5% albumin solution with 1.5 microCi of 125I-labeled albumin (6 ml/kg into both lungs). Alveolar liquid clearance was significantly greater at 24 h (36 +/- 5%) and 40 h (38 +/- 7%) after endotoxin exposure than in saline-instilled controls (27 +/- 6%). Although there was an influx of neutrophils into the air space, there was no increase in lung epithelial permeability to protein at 24 or 40 h. Amiloride (2 x 10(-3) M), a sodium channel inhibitor, significantly reduced alveolar liquid clearance in the rats exposed to endotoxin. However, the increase in alveolar liquid clearance was not inhibited when propranolol (2 x 10(-5) M) was added to the 5% albumin solution. Thus exposure to alveolar endotoxin upregulates net alveolar fluid clearance in vivo for up to 40 h, a potentially important mechanism for accelerating alveolar fluid clearance under some pathological conditions. The increase in alveolar liquid clearance 24 and 40 h after instillation of endotoxin into the air spaces is mediated by an increased uptake of sodium through amiloride-sensitive sodium channels.

Alveolar and lung liquid clearances were studied over 1, 4, and 6 h in intact anesthetized ventilated rats by instillation of 5% albumin solution with 1.5 microCi of 125I-labeled albumin (3 ml/kg into 1 lung or 6 ml/kg into both lungs). Alveolar protein clearance as measured by residual 125I-albumin in the lung over 6 h was similar to the slow rates measured in other species. Alveolar liquid clearance was estimated by the concentration of albumin in the air spaces. After 1 h, this concentration was 7.8 +/- 0.7 g/dl, which was significantly greater than the initial protein concentration of 5.3 +/- 0.2 g/dl (P < 0.05). Amiloride (10(-3) M) inhibited 45% of the basal alveolar liquid clearance, and ouabain (10(-3) M), instilled and intravenously infused (0.004 mg), inhibited 30% of the clearance. beta-Adrenergic agonist instillation increased alveolar liquid clearance to the fastest 1-h rate (48 +/- 3% of instilled volume) that we observed in any intact species. The removal of the instilled fluid from the lung (expressed as lung liquid clearance; 0.96 +/- 0.3 ml/h) was twice as fast as the rate of alveolar and lung liquid clearance reported in the isolated or in situ rat lung models. The rate of alveolar and lung liquid clearance in these intact rats was significantly faster than those in prior studies in dogs and sheep and was similar to the rates in rabbits.