Effects of growth hormone-releasing factor and(or) thyrotropin-releasing hormone on growth, feed efficiency, carcass characteristics, and blood hormones and metabolites in beef heifers

The objective of this study was to determine the effect of long-term administration of a growth hormone (GH)-releasing factor analog (GRFa) and(or) thyrotropin-releasing hormone (TRH) on growth, feed efficiency, carcass characteristics, and blood hormones and metabolites in beef heifers. Crossbred heifers (n = 48; 345.9 +/- 2.8 kg) were divided into four equal groups: control (vehicle), 1 microgram of GRFa (human GRF 1-29 analog).kg BW-1.d-1, 1 microgram of TRH.kg BW-1.d-1, or GRFa + TRH. Daily s.c. injections continued for 86 d. Blood samples were collected from half of the heifers after injection on d 1, 36, and 78. On d 89, all heifers were slaughtered. Treatments did not affect (P > .05) ADG but GRFa + TRH decreased (P < .05) ADFI relative to all other treatments. Feed conversion efficiency tended (P < .10) to be improved in the groups given GRFa alone or TRH alone. Treatment with GRFa and(or) TRH did not affect carcass weight, dressing percentage, conformation score, backfat thickness, or weights of liver, kidneys, pituitary, and ovaries. The GRFa + TRH treatment reduced (P < .05) fat score and increased (P < .05) longissimus muscle area relative to other treatments. The GRFa treatments reduced (P < .05) the weight and fat percentage of the mammary gland and increased (P < .05) heart weight. Treatment with TRH alone failed to stimulate GH on d 1, 36, and 78. Treatment with GRFa alone increased (P < .05) GH above controls on d 36, whereas GRFa + TRH increased (P < .05) GH on d 1, 36, and 78. Treatment with GRFa alone increased (P < .05) IGF-I only on d 1, whereas GRFa + TRH was without effect on all days. Across sampling days, treatments had little effect on blood concentrations of insulin, triiodothyronine, nonesterified fatty acids, urea nitrogen, and glucose. The GRFa alone and GRFa + TRH decreased (P < .05) and TRH alone increased (P < .05) thyroxine concentrations. In conclusion, with the dose and administration regimen used, GRFa and(or) TRH yielded small but positive improvements in animal performance.     

Activation of the anti-cancer drug ifosphamide by rat liver microsomal P450 enzymes

The NADPH-dependent metabolism of ifosphamide catalyzed by rat liver microsomes was investigated in order to identify individual P450 enzymes that activate this anti-cancer drug and to ascertain their relationship to the P450 enzymes that activate the isomeric drug cyclophosphamide. Pretreatment of rats with phenobarbital or clofibrate increased by up to 8-fold the activation of both ifosphamide and cyclophosphamide catalyzed by isolated liver microsomes. Studies using P450 form-selective inhibitory antibodies demonstrated that constitutively expressed P450s belonging to subfamily 2C (forms 2C11/2C6) make significant contributions to the activation of both oxazaphosphorines in uninduced male rat liver microsomes, while the phenobarbital-inducible P450 2B1 was shown to be a major catalyst of these activations in phenobarbital-induced microsomes. Pretreatment of rats with dexamethasone increased liver microsomal activation of ifosphamide approximately 6-fold without a corresponding effect on cyclophosphamide activation rates. Ifosphamide activation catalyzed by dexamethasone-induced liver microsomes was minimally inhibited by anti-P450 2B or anti-P450 2C antibodies, but was selectively inhibited by anti-P450 3A antibodies. Selective inhibition of liver microsomal ifosphamide activation was also effected by the macrolide antibiotic triacetyloleandomycin, an inhibitor of several dexamethasone-inducible 3A P450s. These studies establish that a dexamethasone-inducible family 3A P450 can make an important contribution to rat liver microsomal ifosphamide activation, and suggest that dexamethasone pretreatment might provide a useful approach for modulation of ifosphamide metabolism in order to improve its therapeutic efficacy in cancer patients.     

Two mechanisms for the recapture of extracellular GM2 activator protein: evidence for a major secretory form of the protein

The GM2 activator protein is a small monomeric protein containing a single site for Asn-linked glycosylation. Its only proven in vivo function is to act as a substrate specific cofactor for the hydrolysis of GM2 ganglioside by lysosomal beta-hexosaminidase A. However, we and others have shown it can act as a general glycolipid transporter at neutral pH in vitro. Any other possible in vivo functions would require that some of the newly synthesized activator molecules not be targeted to the lysosome. The lysosomal targeting mechanism for the activator has not been conclusively identified. While earlier reports suggested that it is likely through the mannose-6-phosphate receptor, another more recent report demonstrated that deficient human cells could recapture nonglycosylated, bacterially produced activator, suggesting its use of an alternate targeting pathway. Here, we demonstrate that the mannose-6-phosphate pathway is likely the major intracellular, biosynthetic route to the lysosome, as well as a high affinity recapture pathway for the endocytosis of activator protein from extracellular fluids. Additionally, we show that there exists a second lower affinity recapture pathway that requires its native protein structure, is carbohydrate independent, and likely does not involve its ability to bind glycosphingolipids in the plasma membrane. Finally, we document that the pool of newly synthesized precursor activator protein contains a majority of molecules with a complex-type oligosaccharide, which cannot contain a functional mannose-6-phosphate targeting signal. These molecules makeup the secreted forms of the protein in normal human fibroblasts.     

The OKT3 Antibody Response Study: a multicentre study of human anti-mouse antibody (HAMA) production following OKT3 use in solid organ transplantation

Human anti-murine antibody titres following patient exposure to the monoclonal antibody Orthoclone OKT3 (muromonab-CD3) are determined by laboratories using diverse analytical methods which are not standardized and whose concordance is not established. A multicentre study group therefore compared testing for IgG anti-OKT3 antibody among seven laboratories. A set of 270 sera was obtained from 30 heart, 30 kidney and 30 liver transplant recipients with no previous exposure to OKT3 who were receiving OKT3 for induction immunosuppression. Sera were collected from each patient prior to and at 24 +/- 2 days and 31 +/- 2 days following initial OKT3 exposure. Identical aliquots of all 270 sera were tested for IgG anti-OKT3 antibody by each laboratory. In addition, the limit of detection of each laboratory's method was estimated by titration of an affinity-purified IgG anti-OKT3 reference material of known concentration. Anti-OKT3 antibody formation differed greatly among the three organ groups. Cardiac patients demonstrated the least sensitization and almost exclusively lower titres, while kidney recipients had more frequent and higher titre antibody formation. Liver recipients yielded the highest sensitization rate and the most frequent high titre sera. Importantly, the seven laboratories differed widely in the number of pretreatment sera reported as positive (ranging from 0% to 41% among laboratories), the number of post-OKT3 sera reported as positive (17-63%), the number of post-OKT3 samples with titre > or = 1000 (2-31%), and the number of patients sensitized 19-69%). Concordance among laboratories was highly variable, with interlaboratory agreement ranging from 38% to 83% on the sample titres assigned to 180 post-OKT3 sera. Many of the discordant results were consistent with differences in the limit of detection of the analytical methods, which ranged from 0.19 microgram/ml to > or = 15 micrograms/ml, a nearly 100-fold difference among laboratories. This study demonstrated the presence of both good concordance and significant discordance among laboratories in determining human anti-mouse antibody titres, and demonstrated that common titre categories (100, 1000, 10,000) were not equivalent among laboratories. The level of concordance among methods should be considered when comparing anti-OKT3 antibody results from different centres and their correlation with clinical events. Universal comparative testing, patterned after proficiency testing programmes, is needed to assess differences among laboratories and to bring uniformity and a sound interpretative basis to this field of testing.     

Postsurgical wound progression monitored by temporal changes in the expression of matrix metalloproteinase-9

It is important to monitor the early stages of postoperative wound repair in order to identify those problems associated with impaired healing. Many of the crucial cellular responses of early wound healing, such as inflammatory infiltration, angiogenesis and re-epithelialization, are made possible through the action of matrix metalloproteinases (MMPs). Expression of MMP-2 and MMP-9 is elevated in acute wounds, and still greater levels are found in chronic wounds, indicating that uncontrolled proteolysis is a characteristic of retarded healing. Therefore, comparative measurements of MMPs may be used to monitor the progression of early wound healing. To investigate this, wound fluids and sera were collected from mastectomy and colectomy patients throughout early stages of repair, and the temporal expression profile established. Wounds which were healing were expressed maximal levels of MMP-9 at 24 h, followed by a significant decline by 48 h. Persistent elevation of MMP-9 expression was associated with infected and chronic wounds, and was identified in postoperative wounds by the absence of the significant decline between 24 and 48 h. Measurement of MMP-9 in postoperative wound fluids, therefore, provides an early indicator of impaired healing, which may be evaluated non-invasively within 48 h of closure.