Monoclonal antibody against bovine alpha1-acid glycoprotein

Monoclonal antibodies (Mabs) against bovine alpha1-acid glycoprotein (alpha1AGP) were prepared from mouse hybridoma cell line. Bovine alpha1AGP as antigen was purified by using ion exchange column chromatography and the yield from 500 ml serum was about 10 mg. Immunoglobulin isotypes of 3 Mabs obtained were IgM and light chain types were kappa. The Mabs reacted with bovine alpha1AGP on immunoblot analysis, but not with alpha1AGP digested with N-glycosidase, suggesting that an epitope recognized by these Mabs may be associated with a glycan side chain of bovine alpha1AGP.     

The microheterogeneity of bovine serum alpha 1-acid glycoprotein

The microheterogeneity in bovine serum alpha 1-acid glycoprotein (alpha 1AGP) was examined by crossed affino-immunoelectrophoresis with concanavalin A(Con A). In healthy cows, serum alpha 1AGP revealed 2 fractions which were named fraction 1 and 2 according to the degree of binding affinity to Con A. The mean +/- SD of serum alpha 1AGP concentration was 0.31 +/- 0.09 g/l and the relative amounts of fraction 1 and 2 were approximately 67% and 33% respectively on this method. In bovine leukemia cases, 5 out of 8 had a high serum alpha 1AGP concentration of more than 0.7 g/l. On the electrophoretic pattern, the other 2 fractions (Con A weakly and non-reactive fractions, named fraction 3 and 4) were observed in 5 cases and their relative amounts of 4 fractions were 17.8-28.5%, 35.5-46.0%, 17.9-24.4% and 12.0-17.2%, respectively. In bovie leukemia virus infected cows with no clinical symptoms, however, serum alpha 1AGP concentration and the electrophoretic patterns were almost the same as those in healthy cows.     

Extra hepatic expression of the acute phase protein alpha 1-acid glycoprotein in normal bovine tissues

Combined quantitative (real-time) polymerase chain reaction and immunohistochemistry were used to evaluate the expression of the minor acute phase protein alpha 1-acid glycoprotein (AGP, orosomucoid) in bovine extrahepatic tissues. AGP was produced mainly in the salivary glands and spleen, whereas minor expression was detected in all other tissues sampled, including lung, lymph nodes, uterus, ovary, kidney and tongue. The findings were consistent with immunohistochemical results. In view of the immunomodulatory and direct antibacterial activity of AGP, its expression in the salivary glands may signal an involvement in the regulation of the local immunity, even in non-pathological conditions.     

Serum alpha 1-acid glycoprotein concentration in cats with lymphoma

Serum alpha 1-acid glycoprotein (AGP) concentrations were evaluated in nine cats with lymphoma. Twenty-five healthy cats were used as controls. Blood samples were obtained from cats with lymphoma prior to induction chemotherapy, one week following induction, at complete response, and at monthly intervals. The median pretreatment AGP concentration for the nine cats with lymphoma was significantly higher than the median AGP concentration for the 25 control cats. Remission serum AGP concentration was not significantly different from the pretreatment AGP concentration in the cats with lymphoma. Serum AGP concentrations provided no useful information regarding response or survival in cats with lymphoma.     

Differential effects of alpha1-acid glycoprotein on bovine neutrophil respiratory burst activity and IL-8 production

During bacterial-mediated diseases, neutrophils (PMNs) play a critical role in defending the host against invading pathogens. PMN production of reactive oxygen species (ROS) contributes to the bactericidal capabilities of these cells. ROS are produced intracellularly and can be released extracellularly. The aberrant extracellular release of ROS, however, has been reported to induce injury to host tissues during mastitis and other inflammatory-mediated diseases of cattle. The acute phase response, which occurs shortly after infection or tissue injury, is characterized by the induction of a large number of plasma proteins referred to as acute phase proteins (APP). alpha1-Acid glycoprotein (AGP) is an APP that increases in response to infection or injury in cattle and humans. The precise function of AGP is unknown, but it has been reported to possess anti-inflammatory properties. The objective of this study was to evaluate the effects of bovine AGP on PMN pro-inflammatory responses, including respiratory burst activity and cytokine production. Bovine AGP dose-dependently inhibited zymosan-induced PMN extracellular release of superoxide anion and hydrogen peroxide without affecting the capacity of PMN to engulf and kill Staphylococcus aureus. Moreover, AGP exerted its effect on ROS production regardless of whether PMNs were exposed to AGP prior to or after activation. In contrast to respiratory burst activity, AGP enhanced PMN production of IL-8. The precise mechanism by which AGP regulates PMN functions remains unknown, but data presented in this study suggest that AGP may have a complex role by differentially regulating PMN pro-inflammatory activities.     

Isolation, characterization, and quantitative measurement of serum alpha 1-acid glycoprotein in cattle

Bovine alpha 1-acid glycoprotein (alpha 1AG) was purified from pooled normal bovine sera by successive ammonium sulfate precipitation, ion-exchange chromatographies and gel filtration. Bovine alpha 1AG had a molecular weight of 42,000 +/- 2,000 and a sedimentation coefficient of 3.4S. It contained 26.6% carbohydrate. Gel isoelectric focusing revealed a microheterogeneity with 7 to 8 bands in a pI range of 3.2 to 3.7. It migrated to the alpha 1-globulin region upon immunoelectrophoresis. Single radial immunodiffusion was developed for the quantitative measurement of bovine alpha 1AG in serum. The mean serum value of alpha 1AG in 152 healthy Holstein cattle (1-12 years old) was 283.2 +/- 82.3 micrograms/ml. Elevated values (cut-off value = 450 micrograms/ml) were observed in cattle with traumatic pericarditis (100%), arthritis (100%), mastitis (91%), pneumonia (70%), and mesenteric liponecrosis (43%).     

Association between faecal shedding of feline coronavirus and serum alpha1-acid glycoprotein sialylation

The sialylation pattern of serum alpha1-acid glycoprotein (AGP) in non-symptomatic cats infected by feline coronavirus (FCoV) and its possible relationship with the amount of FCoVs shed in faeces were investigated. Blood from three specific pathogen-free cats (group A) and from 10 non-symptomatic FCoV-positive cats from catteries with low (group B, three cats) or high (group C, seven cats) levels of faecal shedding were collected monthly. AGP was purified from serum and Western blotting followed by lectin-staining of alpha(2,3)-linked and alpha(2,6)-linked sialic acid. Faecal shedding was quantified in group C by quantitative polymerase chain reaction. Variations of AGP sialylation were recorded only in cats from group C, on which viral shedding peaked before the occurrence of feline infectious peritonitis (FIP) in the cattery, and decreased 1 month later, when serum AGP had an increase of alpha(2,3)-linked sialic acid. These results suggest that hypersialylation of AGP may be involved in host-virus interactions.     

Alpha(1)-acid glycoprotein is contained in bovine neutrophil granules and released after activation

The present study was designed to investigate the capability of bovine neutrophil granulocytes to produce the minor acute phase protein alpha(1)-acid glycoprotein (AGP, Orososmucoid). Bovine neutrophils contain a high MW (50-60kDa) AGP isoform (PMN-AGP), as determined by Western blotting and confirmed by fluorescence microscopy. The presence of AGP in bovine neutrophils has been confirmed by fluorescence immunocytometry. In addition, bovine neutrophils contain also a 42-45kDa isoform, which has the same MW as plasma-, liver-delivered, AGP. cDNA sequence of plasma- and PMN-AGP revealed that (i) the two proteins are products of the same gene; (ii) the differences in molecular weight are due do different post-translational modifications. This result was confirmed by deglycosylation of the two glycoforms. Exocytosis studies showed that isolated neutrophils exposed to several challengers, including Zymosan activated serum (ZAS) and phorbol 12-myristate 13-acetate (PMA), which mimic the inflammatory activation, released PMN-AGP as early as 15min. AGP's mRNA is physiologically expressed by mature resting neutrophils. Real-time PCR on LPS, ZAS and PMA challenged cells revealed that the level of expression apparently does not increase after inflammatory activation. Collectively, the findings reported in this paper proved that PMN-AGP: (i) is a hyperglycosylated glycoform of plasma AGP, (ii) is stored in granules, and (iii) is released by neutrophils in response to activation. Due to its anti-inflammatory activity, PMN-AGP may work as a fine tuning of the neutrophils functions in the inflammatory focus, i.e. it can reduce the damages caused by an excess of inflammatory response.     

alpha(1)-Acid glycoprotein modulates apoptosis in bovine monocytes

alpha(1)-Acid glycoprotein (AGP, orosomucoid) is a normal constituent of bovine blood. AGP is an immunocalin, a binding protein that can also exert several immunomodulatory functions. In this paper we investigated the effect of bovine alpha(1)-acid glycoprotein (boAGP) on spontaneous and staurosporine-induced apoptosis of blood derived monocytes purified using magnetic cell sorting techniques. Bovine AGP was purified from blood following a chromatographic protocol. The homogeneous protein was used to stimulate the cells as well to raise a polyclonal antibody, that was used throughout all the experiments. When monocytes were incubated with high concentrations of boAGP (0.9 mg/ml), similar to those found in bovine plasma during systemic reaction to inflammation, their spontaneous apoptosis rate was suppressed, as determined by caspase-3/7 enzymatic activity assay. Similar results were obtained when apoptosis was induced by adding staurosporine, a potent protein kinase inhibitor. The apoptosis-modulating activity of boAGP was dependent on its concentration, since physiological concentrations of boAGP (0.3 mg/ml) did not exhibit a statistically significative anti-apoptotic activity. We also investigated whether this apoptosis-modulating activity was dependent on the terminal sialic acid residues exposed on the surface of the protein. Enzymatic treatment with neuraminidase, that cleaves terminal sialic acid residues, completely abolished boAGP's anti-apoptotic activity. These results suggest that the protective effect of AGP is likely mediated by its sialic acid terminal groups.     

Serum alpha 1-acid glycoprotein concentrations before and after relapse in dogs with lymphoma treated with doxorubicin

OBJECTIVE: To determine whether serum alpha 1-acid glycoprotein (AGP) concentration was a useful marker of relapse in dogs with lymphoma that were in clinical remission following treatment with doxorubicin. DESIGN: Cohort study. ANIMALS: 12 dogs with lymphoma and 10 healthy dogs. PROCEDURE: Serum AGP concentration was measured in the healthy dogs and in the dogs with lymphoma before treatment, 3 weeks after the first dose of doxorubicin was administered, and every 3 weeks thereafter until relapse (i.e., recurrence of clinically detectable disease such as palpable enlargement of peripheral lymph nodes). Serum AGP concentrations were determined by use of a radial immunodiffusion kit. RESULTS: Mean serum AGP concentration in healthy dogs was significantly less than concentration in dogs with lymphoma prior to treatment. Mean serum AGP concentrations after the first and each subsequent dose of doxorubicin were not significantly different from concentration in healthy dogs. However, mean serum AGP concentrations 3 weeks prior to and at the time of relapse were significantly higher than concentration measured after the first dose of doxorubicin, and were not significantly different from concentration measured before treatment. CLINICAL IMPLICATIONS: Results suggest that measuring serum AGP concentration may be a useful method of predicting relapse before recurrence of clinically detectable disease in dogs with lymphoma undergoing treatment with doxorubicin.     

Decreased sialylation of the acute phase protein alpha1-acid glycoprotein in feline infectious peritonitis (FIP)

Feline infectious peritonitis (FIP) is an immune-mediated disease of domestic and exotic felides infected with feline coronavirus. FIP is characterized by the overexpression of an acute phase protein, the alpha1-acid glycoprotein (AGP). In humans, AGP is a heavily glycosylated protein that undergoes several modifications of its glycan moiety during acute and chronic inflammatory pathologies. We studied the changes in AGP glycosylation in the course of FIP. Specifically, we focussed our attention on the degree of sialylation, fucosylation and branching. This study presents a purification method for feline AGP (fAGP) from serum, using an ion exchange chromatography strategy. The glycosylation pattern was analyzed in detail by means of interaction of purified fAGP with specific lectins. In particular, Sambucus nigra agglutinin I and Maackia amurensis agglutinin lectins were used to detect sialic acid residues, Aleuria aurantia lectin was used to detect L-fucose residues and Concanavalin A was used to evaluate the branching degree. By this method we showed that fAGP did not present any L-fucose residues on its surface, and that its branching degree was very low, both in normal and in pathological conditions. In contrast, during FIP disease, fAGP underwent several modifications in the sialic acid content, including decreased expression of both alpha(2-6)-linked and alpha(2-3)-linked sialic acid (76 and 44%, respectively when compared to non-pathological feline AGP).     

The kinetics of hemopexin and alpha1-acid glycoprotein levels induced by injection of inflammatory agents in chickens.

The acute phase response to inflammation induces changes in the secretion of hepatic proteins. To examine the time course of an acute phase protein response in broiler chickens, the plasma levels of hemopexin (HX) and alpha1-acid glycoprotein (AGP) and liver HX mRNA were measured at various time points from 3 hr to 336 hr after an intraabdominal injection of either lipopolysaccharide (LPS), complete Freund's adjuvant, incomplete Freund's adjuvant, phytohemagglutin, or mineral oil. Uninjected chicks served as controls. The accumulation of liver HX mRNA began within 3 hr of stimulation and peaked at 12 hr. Relative to control levels, plasma HX and AGP levels increased by 6-12 hr postchallenge and peaked at 24 hr. Complete Freund's adjuvant and LPS treatments induced the greatest increase in plasma HX (threefold; P < 0.05). Plasma levels of HX and AGP returned to control levels at 336 and 168 hr postinjection, respectively. A second experiment demonstrated that turpentine induced a similar AGP response as LPS and that albumin is a negative acute phase protein. The results suggest that plasma levels of HX or AGP could be used as an indicator of the systemic component of a local inflammatory response in chickens.     

Glycan moiety modifications of feline alpha1-acid glycoprotein in retrovirus (FIV, FeLV) affected cats

alpha1-Acid glycoprotein (AGP) is considered one of the major acute phase proteins in cats. In humans, AGP is a heavily glycosylated protein that undergoes several modifications of its glycan moiety during acute and chronic inflammatory pathologies. In this paper we present the feline AGPs (fAGP) glycan moiety modifications in the course of two prevalent feline diseases, the FIV (feline immunodeficiency virus) dependent feline acquired viral immunodeficiency and the feline leukemia virus (FeLV) associated lymphoma. The glycan moiety of fAGP was investigated by means of the binding of its oligosaccharides residues with specific lectins. Four lectins were used: Sambucus nigra agglutinin I and Maackia amurensis agglutinin lectins were used to detect sialic acid residues, Aleuria aurantia lectin was used to detect L-fucose residues and Concanavalin A was used to evaluate the degree of branching. It was found that fAGP undergoes several post-translational modifications of its glycan pattern: in particular the degree of sialylation is increased in FeLV-positive cats diagnosed with lymphoma, while FeLV-positive that did not presented any specific clinical signs cats do not present any increase of expression of sialic acid on the surface. Furthermore, FIV induced a modification of the glycan moiety of fAGP, which however varied widely among individuals. In order to determine the number and the position of oligosaccharide chains, the cDNA sequence of fAGP was also determined. The translation of the mature fAGP coding sequence gave rise to a sequence of 183 residues, with five potential N-glycosylation sites, but also with seven potential phosphorylation sites.     

Serum alhpa 1-acid glycoprotein concentrations in healthy and tumor-bearing cats

The purpose of this study was to evaluate alpha 1-acid glycoprotein (AGP) concentrations in tumor-bearing and healthy cats. The hypothesis of the present study was that AGP concentrations would be significantly increased in tumor-bearing cats. Serum from 51 healthy and 97 tumor-bearing, client-owned cats was harvested at the time of presentation and stored at -80 degrees C until assayed. Cats with measurable, histologically confirmed malignancies, and healthy cats of similar ages were included. Serum was assayed for AGP concentration by using a radial immunodiffusion method. AGP concentrations were significantly (P = .0051) higher in tumor-bearing (763 +/- 595 microg/mL; mean +/- SD) when compared to healthy cats (501 +/- 377 microg/mL; mean +/- SD). Of the tumor-bearing cats, 35 had carcinomas, 33 had sarcomas, and 26 had discrete, round cell tumors. AGP concentrations were 645 +/- 62 microg/mL, 660 +/- 540 microg/mL, and 967 +/- 860 microg/mL, respectively, and there were no significant differences among the groups.