Augmentation of hormonal activities with antibodies from cattle immunized with a combination of synthetic and recombinant growth hormone peptide.

Antibodies generated against a synthetic growth hormone (GH) peptide in a number of animal species were shown to enhance the efficacy of GH. However, the ability to produce the effective antibodies diminished over time and repeated boosters failed to overcome the hurdle. Therefore, this study was designed to address the issue on the failed antibody responses by employing different GH peptide antigen preparations in cattle. Holstein steers were repeatedly immunized with a synthetic peptide corresponding to an amino acid sequence 54-95 of porcine GH (pGH). The peptide was conjugated to ovalbumin (OVA) as a carrier. Animals initially responded to the antigen well and elicited antibodies specific to the peptide. However, the 4th challenge with the same OVA-peptide antigen rendered animals unresponsive, resulting in a decline in antibody production. This unresponsiveness was overcome by switching the antigen at the 5th immunization from OVA-peptide to a recombinant peptide preparation which was composed of maltose binding protein (MBP) as a carrier. Antibodies generated in cattle after the 5th immunization recognized not only the pGH(54-95) peptide, but also bovine GH (bGH) and pGH. These antibodies were not immunoreactive with an unrelated control peptide. Hypophysectomized (hypox) rats were used for functional analysis and bGH was active in promoting the growth of these GH-deficient rats. The growth-promoting effect of bGH was significantly enhanced by mixing it with bovine anti-peptide antibodies prior to administration. Therefore, the present findings suggest that peptide 54-95 induces cattle to elicit antibodies capable of not only recognizing bGH but also augmenting the somatogenic effectiveness of bGH in hypox rats.

Augmentation of hormonal activities with antibodies from cattle immunized with a combination of synthetic and recombinant growth hormone peptide.

Antibodies generated against a synthetic growth hormone (GH) peptide in a number of animal species were shown to enhance the efficacy of GH. However, the ability to produce the effective antibodies diminished over the time and repeated boosters failed to overcome the hurdle. Therefore, this study was designed to address the issue on the fallen antibody responses by employing different GH peptide antigen preparations in cattle. Holstein steers were repeatedly immunized with a synthetic peptide corresponding to an amino acid sequence 54-95 of porcine GH (pGH). The peptide was conjugated to ovalbumin (OVA) as a carrier. Animals initially responded to the antigen well and elicited antibodies specific to the peptide. However, the 4th challenge with the same OVA-peptide antigen rendered animals unresponsive, resulting in a decline in antibody production. This unresponsiveness was overcome by switching the antigen at the 5th immunization from OVA-peptide to a recombinant peptide preparation which was composed of maltose binding protein (MBP) as a carrier. Antibodies generated in cattle after the 5th immunization recognized not only the pGH(54-95) peptide, but also bovine GH (bGH) and pGH. These antibodies were not immunoreactive with an unrelated control peptide. Hypophysectomized (hypox) rats were used for functional analysis and bGH was active in promoting the growth of these GH-deficient rats. The growth-promoting effect of bGH was significantly enhanced by mixing with bovine anti-peptide antibodies prior to administration. Therefore, the present findings suggest that peptide 54-95 induces cattle to elicit antibodies capable of not only recognizing bGH but also augmenting the somatogenic effectiveness of bGH in hypox rats.

Immunological effect of a synthetic growth hormone peptide on the growth performance in swine.

A mouse monoclonal antibody (mAb), designated PS-7.6, was previously shown to enhance the activity of porcine growth hormone (pGH) in promoting the growth of hypophysectomized (hypox) rats. Epitope mapping studies indicated that the region recognized by PS-7.6 resided within an amino acid sequence 54-95 of pGH. A peptide corresponding to this sequence was synthesized and found to induce swine antibodies capable of augmenting pGH activity in hypox rats. On the basis of these previous observations, an attempt was made in this study to determine whether or not the peptide pGH(54-95) could be used as a vaccine to elicit antibodies functionally similar to PS-7.6 mAb, thus potentiating the efficacy of endogenous GH in swine. Young pigs (15-20 kg) were immunized with pGH(54-95) that had been conjugated with ovalbumin (OVA) and boosted twice at 4-week intervals. Control animals were similarly immunized with OVA. The weight gains and feed consumption of these animals were closely monitored throughout the trials. A number of carcass parameters were also examined when these animals reached 110-120 kg, at which time they were killed. Results indicated that immunization with peptide significantly accelerated the daily weight gain during the growing phase of growth. However, this effect disappeared during the finishing phase of growth. The failure to prolong the initial growth effect by the peptide immunization apparently correlated with the kinetics of antibody production, because antibodies immunoreactive to the peptide and pGH were detected in these animals after immunization but gradually diminished. This idea was supported by the fact that antibodies obtained from pigs 5 and 9 weeks after the initial immunization potentiated the activity of pGH in hypox rats, whereas antibodies harvested at week 16 did not. Furthermore, carcass evaluation was performed at time of killing and showed that the leaf fat and loin eye muscle were also significantly improved by peptide immunization. Taken together, the present findings suggest that pGH(54-95) peptide can be employed as a potential growth-promoting vaccine to improve the performance of swine.

Monomeric bovine IgG2 is a potent stimulus for bovine neutrophils.

Bovine plasma contains factors that can stimulate bovine neutrophils. Bovine plasma at 1:1,000,000-1:1,000 dilution induced a dose-dependent superoxide production in bovine neutrophils. When bovine plasma was fractionated with a protein G column, only the IgG fraction contained induction activity. At similar concentrations purified IgG induced a much stronger response than that of plasma IgG. Purified monomeric bovine IgG induced a dose-dependent increase in superoxide production. The maximum induction can be achieved at 100 micrograms/ml of bovine IgG. When subclasses of bovine IgG were examined, monomeric bovine IgG2 potently stimulated bovine polymorphonuclear leukocytes. In contrast, bovine IgG 1 failed to induce a response at similar concentrations, and neither bovine IgG F(ab')2 and Fc were effective. Both recombinant bovine granulocyte-macrophage colony-stimulating factor (r-BoGM-CSF) and recombinant bovine granulocyte colony-stimulating factor (r-BoG-CSF) primed bovine neutrophils for superoxide production induced by bovine IgG. The above results suggest that: (1) bovine plasma contain factors that can activate bovine neutrophils; (2) bovine plasma IgG is the major component that is responsible for bovine neutrophil activation; (3) bovine plasma contains factors that can inhibit the effect of bovine IgG; (4) monomeric bovine IgG2, but not IgG1, can activate bovine neutrophils directly without Fc receptor cross-linkage; (5) the integrity of bovine IgG is important in bovine polymorphonuclear leukocyte activation; and (6) bovine neutrophil activation induced by bovine IgG can be primed by r-BoGM-CSF or r-BoG-CSF.

Augmentation of the effectiveness of porcine GH with swine antibodies induced by a synthetic GH peptide.

An effort was made to evaluate the potential usefulness of a peptide vaccine in improving the growth performance of farm animals. It was previously reported that a murine PS-7.6 monoclonal antibody (mAb) which was specific to porcine GH (pGH) enhanced the growth-promoting activity of pGH in an experimental hypophysectomized rat model. Additional data from a epitope mapping study suggested that PS-7.6 mAb recognized a pGH fragment corresponding to an amino acid sequence 54-95. In this report, therefore, a peptide pGH(54-95) was synthesized in an attempt to induce PS-7.6-like antibodies in swine. It was demonstrated that the peptide pGH(54-95) competed with PS-7.6 mAb for the binding to radioactive pGH in a competition radioimmunoassay and also caused pigs to elicit polyclonal antibodies immunoreactive to pGH protein. The association and dissociation rate constants of the swine antibody to pGH were 1.9 x 10(2) M-1 s-1 and 3.2 x 10(-4)s-1 respectively, thus producing an overall binding affinity of Kd = 1.6 x 10(-6) M. The swine antibody partially competed with murine PS-7.6 mAb for the binding to pGH, suggesting that the pGH-recognizing sites for both antibodies might be closely related. The biological effect of the swine antibody was examined in hypophysectomized rats and shown to significantly augment pGH activity in promoting the growth of these GH-deficient animals. The present findings suggest that a synthetic peptide may be developed as a potential growth vaccine for swine to generate antibodies capable of enhancing the effectiveness of endogenous pGH.

Epitope mapping and analysis of a growth-enhancing monoclonal antibody by limited tryptic digestion of porcine GH.

In this study, the epitope of a murine PS-7.6 monoclonal antibody (mAb) which was raised against the recombinant porcine GH (pGH) and subsequently shown to enhance the growth-promoting activity of pGH in a hypophysectomized rat model, was mapped by the limited tryptic digestion of pGH. A pGH fragment corresponding to amino acid residues 70-95 was separated by reverse-phase HPLC and also immunoprecipitated by PS-7.6 mAb. This fragment was found in an RIA to compete with radiolabelled pGH for the binding of PS-7.6 mAb in a dose-dependent fashion. Several peptides covering this potential epitope region of pGH(70-95) were synthesized and assayed by competitive RIA. The results suggested that pGH(75-90) was the optimal sequence recognized by PS-7.6 mAb. Sequential alanine substitution of each residue of pGH(75-90) revealed that the side chains of Leu76, Ile83 and Leu87 were critical for binding to PS-7.6 mAb. Other residues could be replaced by alanine without substantially altering the binding affinity. The region of amino acids 75-95 comprises the C-terminal end of the second helix of pGH and the repeating pattern of i and i + 3 (i + 7) of the critical amino acids appears consistent with PS-7.6 mAb binding to the hydrophobic side of the helix. The sequence and the helical structure of the epitope of PS-7.6 mAb provide the basis for designing the effective peptide vaccines to enhance the growth performance of animals.

Rhabdomyoma of the larynx: a rare cause of stridor.

Rhabdomyomas of the larynx are rare tumours. In the literature to date 18 cases have been reported. They usually present with slowly progressive symptoms typical of their benign nature. We present a case that has many atypical features for an 'adult' type rhabdomyoma and discuss the relevant associated literature.