Abnormal CX3CR1⁺ lamina propria myeloid cells from intestinal transplant recipients with NOD2 mutations.

Although progress has been made in intestinal transplantation, chronic inflammation remains a challenge. We have reported that the risk of immunological graft loss is almost 100-fold greater in recipients who carry any of the prevalent NOD2 polymorphisms associated with Crohn's disease, and have shown that the normal levels of a key antimicrobial peptide produced by the Paneth cells of the allograft, fall as the graft becomes repopulated by hematopoietic cells of the NOD2 mutant recipient. These studies are extended in this report. Within several months following engraftment into a NOD2 mutant recipient the allograft loses its capacity to prevent adherence of lumenal microbes. Despite the significantly increased expression of CX3CL1, a stress protein produced by the injured enterocyte, NOD2 mutant CX3CR1(+) myeloid cells within the lamina propria fail to exhibit the characteristic morphological phenotype, and fail to express key genes required expressed by NOD2 wild-type cells, including Wnt 5a. We propose that the CX3CR1(+) myeloid cell within the lamina propria supports normal Paneth cell function through expression of Wnt 5a, and that this function is impaired in the setting of intestinal transplantation into a NOD2 mutant recipient. The therapeutic value of Wnt 5a administration in this setting is proposed.

Rejection reversibly alters enteroendocrine cell renewal in the transplanted small intestine.

Acute small intestinal allograft rejection presents clinically as an abrupt increase in ileal fluid output in the absence of extensive inflammation. We questioned whether acute intestinal rejection might be accompanied by a disturbance of normal intestinal stem cell differentiation. We examined the intestinal epithelial secretory cell lineage among patients experiencing early rejection before and during rejection as well as following corrective therapy. Lineage-specific progenitors were identified by their expression of stage-specific transcription factors. Progenitors of the enteroendocrine cell (EEC) expressing neurogenin-3 (NEUROG3) were found to be disproportionately reduced in numbers, along with their more mature EEC derivatives expressing neuro D; the enteric hormone PYY was the most profoundly depleted of all the EEC products evaluated. No change in the numbers of goblet or Paneth cells was observed. Steroid treatment resulted in resolution of clinical symptoms, restoration of normal patterns of EEC differentiation and recovery of normal levels of enteric hormones. Acute intestinal rejection is associated with a loss of certain subtypes of EEC, most profoundly, those expressing PYY. Deficiency of the mature EECs appears to occur as a consequence of a mechanism that depletes NEUROG3 EEC progenitors. Our study highlights the dynamics of the EEC lineage during acute intestinal rejection.

NOD2-expressing bone marrow-derived cells appear to regulate epithelial innate immunity of the transplanted human small intestine.

BACKGROUND: Intestinal allograft rejection resembles Crohn's disease clinically and pathologically. An understanding of its mechanism could impact this life-saving procedure, as well as provide insight into the pathophysiology of inflammatory bowel disease. The NOD2 protein has been implicated as a key player in intestinal immune health, as a consequence of the discovery of three polymorphisms linked with Crohn's disease. An investigation was carried out to determine whether epithelial immune function and graft survival were influenced by NOD2 mutations in an intestinal transplant population. METHODS: The NOD2 genotypes of 34 transplants performed consecutively over the past 3 years were determined. The NOD2 genotypes were related to clinical outcomes and the expression of certain intestinal antimicrobial peptides (AMPs) believed to protect the epithelium. RESULTS: An unexpectedly high percentage of recipients, 35%, possessed NOD2 polymorphisms, while 8.6% of donors had comparable mutations. The likelihood of allograft failure was about 100-fold higher in recipients with mutant NOD2 alleles compared with recipients with wild-type NOD2 loci. Rejection in NOD2 mutant recipients was characterised by decreased expression of certain Paneth cell and enterocyte AMPs, prior to the onset of epithelial injury and inflammation. CONCLUSIONS: Crohn's disease-associated polymorphisms in the NOD2 gene in the recipient represent a critical immunological risk factor for intestinal allograft rejection. Compromised epithelial defences precede visible epithelial injury and inflammatory infiltration. The association of impaired epithelial immunity with the recipient's genotype suggests that certain NOD2-expressing cells of haematopoietic origin play a role in the process, perhaps by regulating expression of certain epithelial AMPs within the allograft.

Crystal and molecular structure of r(CGCGAAUUAGCG): an RNA duplex containing two G(anti).A(anti) base pairs.

BACKGROUND: Non-Watson-Crick base pair associations contribute significantly to the stabilization of RNA tertiary structure. The conformation adopted by such pairs appears to be a function of both the sequence and the secondary structure of the RNA molecule. G.A mispairs adopt G(anti).A(anti) configurations in some circumstances, such as the ends of helical regions of rRNAs, but in other circumstances probably adopt an unusual configuration in which the inter-base hydrogen bonds involve functional groups from other bases. We investigated the structure of G.A pairs in a synthetic RNA dodecamer, r(CGCGAAUUAGCG), which forms a duplex containing two such mismatches. RESULTS: The structure of the RNA duplex was determined by single crystal X-ray diffraction techniques to a resolution in the range 7.0-1.8A, and found to be an A-type helical structure with 10 Watson-Crick pairs and two G.A mispairs. The mispairs adopt the G(anti).A(anti) conformation, held together by two obvious hydrogen bonds. Unlike analogous base pairs seen in a DNA duplex, they do not exhibit a high propeller twist and may therefore be further stabilized by weak, reverse, three-center hydrogen bonds. CONCLUSIONS: G(anti).A(anti) mispairs are held together by two hydrogen of guanine and the N6 and N1 of adenine. If the mispairs do not exhibit high propeller twist they may be further stabilized by inter-base reverse three-centre hydrogen bonds. These interactions, and other hydrogen bonds seen in our study, may be important in modelling the structure of RNA molecules and their interactions with other molecules.

The effects of high-forage diets with added palm oil on performance, plasma lipids, and carcass characteristics of ram lambs with initially high or low plasma cholesterol.

The objectives of this study were to examine the interaction between added palm oil in high-forage diets and initial concentration of plasma cholesterol on performance, plasma lipids, and carcass characteristics of growing ram lambs. Thirty-two Hampshire-Suffolk ram lambs (initial BW = 34.4 kg) were assigned to a 2 x 2 factorial design consisting of diet (basal [NPO] or 10.7% added palm oil [PO]) and initial plasma cholesterol concentration (high mean = 50 mg/dL [HC] or low mean = 38 mg/dL [LC]; SEM = 2; P = .01). The lambs were individually fed diets (77% forage-23% concentrate) that contained 16.0% CP, 2.14 Mcal of ME/kg (NPO), and 2.62 Mcal of ME/kg (PO). Metabolizable energy intakes were adjusted to .20 Mcal/kg of BW.75 for both dietary treatments. Lambs were weighed and feed intakes adjusted weekly. Lambs were bled via jugular venipuncture on d 28, 56, and 84 and lambs were slaughtered after they had been fed the diets for 90 d. Plasma concentrations of total cholesterol, high-density lipoprotein cholesterol, triglycerides, and nonesterified fatty acids were increased (P = .01) by feeding PO. Lambs fed PO were fatter than lambs fed NPO, as indicated by greater subcutaneous fat thickness and kidney and pelvic fat. Initial plasma cholesterol concentration had little effect on any of the parameters measured. Lambs fed PO had fatter carcasses than lambs fed NPO at calculated equalized ME intakes, which indicates that energy deposition is more efficient in palm oil-supplemented diets.

The effect of trenbolone acetate on performance, plasma lipids, and carcass characteristics of growing ram and ewe lambs.

The purpose of the study was to determine the effect of trenbolone acetate (TBA) on performance, plasma lipid metabolites, and carcass characteristics of growing ram and ewe lambs in two separate experiments. Treatments consisted of 1) a control, no implants, and 2) a TBA implant (60 mg of Finaplix; Hoechst-Roussel Agri-Vet). Twelve Rambouillet cross ram lambs (average 19.9 kg BW; Exp. 1) and 18 Suffolk-Hampshire ewe lambs (average 37.2 kg BW; Exp. 2) were given ad libitum access to a 77% forage/23% concentrate diet (15.5% CP and 2.14 Mcal of ME/kg). On d 0, six rams and nine ewes received TBA as an ear implant and six rams and nine ewes were designated as controls. Blood samples were collected via jugular venipuncture on d 0, 28, 56, and 84 and plasma was separated and analyzed for lipid metabolites. Rams (30.5 kg BW) and ewes (50.0 kg BW) were slaughtered after 93 and 91 d, respectively. Average daily gain was not affected by TBA in rams (117 g/d) or ewes (159 g/d). Nutrient intake restricted normal BW gains in the rams. Plasma concentrations of total cholesterol, high-density lipoprotein cholesterol, triglycerides, and nonesterified fatty acids were not affected by TBA in either rams or ewes. Trenbolone acetate had no effect on subcutaneous fat measurements in either rams or ewes. Lamb chops from rams implanted with TBA were less (P < .04) tender (5.58 vs 4.26 kg/1.27-cm core) than lamb chops from control rams. Additional studies examining growth and carcass composition are needed in ruminants implanted with anabolic steroids and fed high-forage diets.

Effects of high-forage diets with added palm oil on performance, plasma lipids, and carcass characteristics of ram and ewe lambs.

The objectives of this study were to determine the effects of high-forage diets with and without added dietary palm oil (high in palmitic acid) fed at equalized ME intakes on performance, plasma lipids, and carcass characteristics of growing ram and ewe lambs. Thirty-one Hampshire or Suffolk lambs (35.7 kg BW) were used in a 2 x 2 factorial arrangement of the following treatments: 1) rams, no palm oil (R-NPO); 2) ewes, no palm oil (E-NPO); 3) rams, 10.7% dietary palm oil (R-PO); and 4) ewes, 10.7% dietary palm oil (E-PO). Both diets consisted of 77% forage and 23% concentrate. Diet DM contained 15.0% CP and 2.14 Mcal of ME/kg (NPO) or 2.62 Mcal of ME/kg (PO). Lambs were fed individually specified amounts of diet based on BW to equalize ME intake (.20 Mcal of ME/kg of BW.75) for both dietary treatments. Lambs were weighed and feed intakes were adjusted weekly. Lambs were bled by jugular venipuncture on d 28, 56, and 84 and were slaughtered after they had been fed the diets for 90 d. Lambs fed PO had greater (P < .01) ADG and efficiency (ADG/ME intake) than lambs fed NPO. Plasma concentrations of cholesterol, high-density lipoprotein cholesterol, triglycerides, and nonesterified fatty acids were increased (P < .01) by feeding PO. Lambs fed PO were fatter than lambs fed NPO, as evidenced by greater subcutaneous fat thickness and kidney and pelvic fat. Ewes had greater (P < .01) subcutaneous fat than did rams.(ABSTRACT TRUNCATED AT 250 WORDS)

Influence of dietary palm oil supplementation on serum lipid metabolites, carcass characteristics, and lipid composition of carcass tissues of growing ram and ewe lambs.

The objective of this research was to determine the influence of dietary palm oil supplementation on carcass characteristics and lipid composition of tissues from growing lambs. Twenty-eight Suffolk x Hampshire lambs were weaned at 60 d of age (average 36 kg BW) and assigned to a 2 x 2 factorial arrangement consisting of diet (control [NPO] or 10.6% added dietary palm oil [PO]) and sex (ram vs ewe). The NPO diet (77% forage and 23% concentrate) contained 11.2% CP and 2.66 Mcal of ME/kg. Palm oil replaced molasses in the PO diet. Lambs were individually given ad libitum access to feed for 60 d to a final BW average of 50.1 kg. Lipid composition of the longissimus muscle and corresponding subcutaneous (s.c.) adipose tissue was determined by gas-liquid chromatography (GLC). Lambs fed PO were fatter (P less than .01) than lambs fed NPO (.77 vs .56 cm, s.c. fat). Diet had no effect on cholesterol content of lean tissue; however, feeding PO increased the saturated fatty acids of lean tissue. The s.c. fat from lambs fed PO had less (P less than .01) cholesterol (64.79 vs 89.67 mg/100 g) and more saturated fatty acids than that from lambs fed NPO. Ewes were fatter (P less than .01) than rams, yet they had less cholesterol content in the s.c. adipose tissue (68.71 vs 85.74 mg/100 g). High amounts of dietary palm oil fed to growing lambs caused changes in fatty acid deposition and cholesterol metabolism and may be a useful investigative tool to study lipid metabolism in growing ruminants.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dietary canola seed and soy lecithin in high-forage diets on cholesterol content and fatty acid composition of carcass tissues of growing ram lambs.

Phospholipids (soy lecithin) are important in the emulsification of lipids and may escape the rumen and influence the absorption of fatty acids in the small intestine. Our objectives were to determine the influence of dietary canola seed (high in unsaturated fatty acids) and soy lecithin in high-forage diets on total lipid content, cholesterol content, and fatty acid composition of carcass tissues. Forty-three Hampshire or Suffolk-sired ram lambs were weaned at 60 d of age (average 23.6 kg of BW) and assigned to a 2 x 2 factorial arrangement of treatments consisting of 1) basal diet (control = BAS), 2) BAS with 6% whole canola seed (CS), 3) BAS with 4.9% deoiled soy lecithin (SL), and 4) BAS with 6% CS and 4.8% SL (CSSL). The BAS diet consisted of 70% forage and 30% concentrate and contained 15% CP and 2.2 Mcal of ME/kg. Lambs were individually fed and given ad libitum access to feed to an average final BW of 52.1 kg. Longissimus muscle (LM) from the left side of each carcass posterior to the 13th rib (12 to 15 cm in length) was excised and the lean (LM) and corresponding subcutaneous (s.c.) adipose tissue were separated, frozen, and later used for lipid analysis by gas-liquid chromatography. In lean tissue, feeding lambs CS reduced (P less than .01) the proportion of total polyunsaturated fatty acids (PUFA) and feeding SL increased (P less than .01) the proportion of total PUFA. In s.c. adipose tissue, lambs fed CS had lower (P less than .01) saturated fatty acids (SFA) and lambs fed SL had increased (P less than .03) PUFA.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of dietary canola seed and soy lecithin in high-forage diets on performance, serum lipids, and carcass characteristics of growing ram lambs.

Phospholipids (soy lecithin) are important in the emulsification of lipids and may be able to escape the rumen and influence the absorption of fatty acids in the small intestine. The objectives of this study were to determine the influence of dietary canola seed (high in unsaturated fatty acids) and soy lecithin in high-forage diets on performance, serum lipid metabolites, and carcass characteristics of growing ram lambs. Forty-three Hampshire- or Suffolk-sired ram lambs were weaned at 60 d of age (average 23.6 kg BW) and assigned to a 2 x 2 factorial arrangement of the following treatments (% of DM): 1) basal diet (control = BAS); 2) BAS with 6% whole canola seed (CS); 3) BAS with 4.9% deoiled soy lecithin (SL); and 4) BAS with 6% CS and 4.8% SL (CSSL). The BAS diet consisted of 70% forage and 30% concentrate and contained 15% CP and 2.2 Mcal of ME/kg. Lambs had ad libitum access to their diet to an average final BW of 52.1 kg. Lambs were bled by jugular venipuncture on d 0, at 4-wk intervals, and 1 d before slaughter. Dry matter intake was not affected by treatment; however, energy intake (Mcal of ME/d) was greater (P less than .01) for lambs fed SL. Feeding SL and(or) CS to growing ram lambs increased (P less than .02) serum lipid concentrations. Carcasses from lambs fed SL were fatter, as evidenced by greater dressing percentage and subcutaneous fat thickness. Feeding CS to growing lambs had little effect on performance and carcass characteristics compared with feeding SL, which resulted in greater energy intake and fatter carcasses.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of feed intake and thermal stress on mammary blood flow and other physiological measurements in lactating dairy cows.

Six midlactation Holstein cows were exposed to treatments of thermal comfort environments with ad libitum or restricted (70% of ad libitum) DM intake and a thermal stress environment with ad libitum intake in two balanced 3 X 3 Latin squares to evaluate effects on mammary blood flow and other physiological measurements. Daily DM intake decreased from 17.8 kg in thermal comfort with ad libitum intake to 12.5 kg in thermal comfort with restricted intake and to 14.8 kg in thermal stress. Daily milk production decreased from 23.9 kg in thermal comfort ad libitum to 22.2 kg in thermal comfort restricted and 21.6 kg in thermal stress. Mammary blood flows (half udder) over the entire sampling interval (18 h) were 5.1, 4.3, and 4.5 L/min for treatments: thermal comfort, ad libitum intake; thermal comfort, restricted intake; and thermal stress, ad libitum intake. Mammary blood flows did not differ significantly among between treatments. Relationship of hald udder blood flow (L/min) to previous day's DM intake (kg/d) was described by the linear equation: L/min = .49 + (.27 kg/d); r2 = .46; and the quadratic equation: L/min = 6.04 - (.54 kg/d) + (.03 [kg/d]2); r2 = .55. Mammary blood flows 10 min before through 10 min after milking machine attachment were greater for cows at thermal comfort and ad libitum intake than for cows at thermal comfort and restricted intake or thermal stress and ad libitum intake (5.5, 4.8, and 4.8 L/min).

Lactational responses to and in vitro ruminal solubility of magnesium oxide or magnesium chelate.

Thirty-six midlactation Holstein cows were used in a randomized incomplete block design to evaluate lactational responses to varying dietary concentrations of Mg supplemented by MgO or Mg chelate. Basal diet was 41:4:55 corn silage:cottonseed hulls:concentrate (.21% total Mg). Magnesium oxide was added to the basal diet to give .32, .37, and .43% total dietary Mg, and Mg chelate was added to provide .23, .25, and .27% Mg, DM basis. Dietary treatments were formulated to supply equal concentrations of bioavailable Mg from either Mg source. Dry matter intake and milk yield were greater by cows fed MgO-supplemented than Mg chelate-supplemented treatments. Milk fat percentages were not affected. Milk protein percentages increased with Mg chelate compared with protein percentages with MgO. Treatments did not affect gross efficiency (4% FCM/DM intake) or body weight change. Lack of response to Mg chelate suggested that either the bioavailability was not as high as assumed or that sufficient total bioavailable Mg was not provided in those treatments. A companion in vitro experiment showed that MgO-supplemented concentrates, with more total supplemental Mg, supplied two to three times more soluble Mg than Mg chelate-supplemented concentrates.

Effect of exogenous bovine somatotropin on mammary lipid metabolism and milk yield in lactating dairy cows.

Eight lactating Holstein cows were used to study the effect of bovine somatotropin on mammary lipid metabolism, milk production, and milk composition. Eight cows were injected with either excipient (n = 4) or 50 IU somatotropin/d (n = 4). A 2-wk adjustment period preceded a 10-d period when treatments were administered. On d 9, serial blood samples were collected from 2 to 5 h post-injection to determine concentrations of somatotropin and metabolites in plasma. Mammary tissue biopsies were obtained 2 to 3 h after a.m. milking on d 10 to determine lipoprotein lipase activity and in vitro rates of acetate incorporation into fatty acids and acetate oxidation by mammary tissue slices. Activity of lipoprotein lipase in milk and mammary tissue and rate of acetate metabolism by mammary tissue were not affected by treatment. The following means for placebo and somatotropin, respectively, were: milk production (21.5 and 24.8 kg/d), milk fat (3.8 and 4.0%), and 4% FCM (20.7 and 25.1 kg/d). Plasma concentrations of somatotropin (3.0 and 43.4 ng/ml) and free fatty acids (105.6 and 219.5 mu eq/L) were significantly elevated with somatotropin. The injection of somatotropin into lactating dairy cows did not affect the mechanisms of fatty acid synthesis or metabolism evaluated in this study.

Effect of added dietary fat and bovine somatotropin on the performance and metabolism of lactating dairy cows.

Four early lactation Holstein cows (44 to 105 d postpartum) were used in a 4 X 4 Latin square experiment in a 2 X 2 factorial arrangement of treatments to study effects of added dietary fat and/or bovine somatotropin on performance and metabolism. Treatments were: 1) control diet plus placebo injection; 2) 5% added dietary fat (hydrolyzed blend of animal and vegetable fat) + placebo injection; 3) control diet + 50 IU bovine somatotropin/d; and 4) 5% added dietary fat + 50 IU bovine somatotropin/d. Dietary fat reduced dry matter intake (21.6 vs. 22.7 kg/d) and elevated plasma triglycerides (34.7 vs. 29.2 mg/100 ml). Injection of somatotropin lowered blood urea nitrogen, increased plasma free fatty acids, and increased plasma somatotropin. Milk production, milk fat percent, and 4% FCM production were increased by the injection of somatotropin. Milk protein percent was decreased (3.30 vs. 3.44%) with added fat and tended to be lower with somatotropin. The percentage of short-chain fatty acids (C6 to C14) in milk fat decreased with added fat while the percentage of stearic and oleic acids in milk fat increased. Production responses for fat plus somatotropin and somatotropin treatments were similar. Under the conditions of this study, the addition of dietary fat with injection of somatotropin had little effect on production parameters compared with that found with somatotropin alone.

Glucogenic and hormonal responses to abomasal casein and ruminal volatile fatty acid infusions in lactating goats.

To determine responses to abomasal protein infusion and ruminal acetate: propionate ratios, four lactating Toggenburg goats fed hourly a 70% roughage and 30% concentrate diet were used in a Latin-square design with a factorial arrangement of treatments. Either acetate or propionate was infused ruminally and casein or saline infused abomasally. Estimated net energy and volume of the infusates were similar for all treatments. To examine the effects of treatments on glucose metabolism, 2-carbon-14 propionate was infused ruminally and 6-hydrogen-3 glucose was infused intravenously for 9 and 5 h, respectively. Although glucose concentration in plasma was higher and propionate turnover greater with propionate treatment, percentage of glucose derived from propionate, amount of propionate coverted to glucose, and glucose turnover remained unchanged. No differences in glucose metabolism due to the abomasal casein infusion were evident. To determine the effects of treatment on insulin, glucagon, growth hormone, and prolactin in plasma, samples were collected at 10-min intervals for 3 h at 0400 and 1600 h. No diurnal variation or consistent peaks were observed for any of the hormones nor were treatment effects on plasma concentrations of insulin, growth hormone, or prolactin evident. Glucagon concentration was higher with casein treatment; however, no relationship existed between glucagon in plasma and glucogenic parameters measured.