M. Anthony Hayes

Pigs expressing salivary phytase produce low-phosphorus manure

To address the problem of manure-based environmental pollution in the pork industry, we have developed the phytase transgenic pig. The saliva of these pigs contains the enzyme phytase, which allows the pigs to digest the phosphorus in phytate, the most abundant source of phosphorus in the pig diet. Without this enzyme, phytate phosphorus passes undigested into manure to become the single most important manure pollutant of pork production. We show here that salivary phytase provides essentially complete digestion of dietary phytate phosphorus, relieves the requirement for inorganic phosphate supplements, and reduces fecal phosphorus output by up to 75%. These pigs offer a unique biological approach to the management of phosphorus nutrition and environmental pollution in the pork industry.

Probiotics Stimulate Production of Natural Antibodies in Chickens

ABSTRACT Commensal bacteria in the intestine play an important role in the development of immune response. These bacteria interact with cells of the gut-associated lymphoid tissues (GALT). Among cells of the GALT, B-1 cells are of note. These cells are involved in the production of natural antibodies. In the present study, we determined whether manipulation of the intestinal microbiota by administration of probiotics, which we had previously shown to enhance specific systemic antibody response, could affect the development of natural antibodies in the intestines and sera of chickens. Our findings demonstrate that when 1-day-old chicks were treated with probiotics, serum and intestinal antibodies reactive to tetanus toxoid (TT) and Clostridium perfringens alpha-toxin in addition to intestinal immunoglobulin A (IgA) reactive to bovine serum albumin (BSA) were increased in unimmunized chickens. Moreover, IgG antibodies reactive to TT were increased in the intestines of probiotic-treated chickens compared to those of untreated controls. In serum, IgG and IgM reactive to TT and alpha-toxin were increased in probiotic-treated, unimmunized chickens compared to levels in untreated controls. However, no significant difference in serum levels of IgM or IgG response to BSA was observed. These results are suggestive of the induction of natural antibodies in probiotic-treated, unimmunized chickens. Elucidating the role of these antibodies in maintenance of the chicken immune system homeostasis and immune response to pathogens requires further investigation.

MODULATION OF ANTIBODY-MEDIATED IMMUNE RESPONSE BY PROBIOTICS IN CHICKENS

ABSTRACT Probiotic bacteria, including Lactobacillus acidophilus and Bifidobacterium bifidum, have been shown to enhance antibody responses in mammals. The objective of this study was to examine the effects of a probiotic product containing the above bacteria in addition to Streptococcus faecalis on the induction of the chicken antibody response to various antigens, both systemically and in the gut. The birds received probiotics via oral gavage and subsequently were immunized with sheep red blood cells (SRBC) and bovine serum albumin (BSA) to evaluate antibody responses in serum or with tetanus toxoid (TT) to measure the mucosal antibody response in gut contents. Control groups received phosphate-buffered saline. Overall, BSA and SRBC induced a detectable antibody response as early as week 1 postimmunization (p.i.), which lasted until week 3 p.i. Probiotic-treated birds had significantly (P ≤ 0.001) more serum antibody (predominantly immunoglobulin M [IgM]) to SRBC than the birds that were not treated with probiotics. However, treatment with probiotics did not enhance the serum IgM and IgG antibody responses to BSA. Immunization with TT resulted in the presence of specific IgA and IgG antibody responses in the gut. Again, treatment with probiotics did not change the level or duration of the antibody response in the gut. In conclusion, probiotics enhance the systemic antibody response to some antigens in chickens, but it remains to be seen whether probiotics have an effect on the generation of the mucosal antibody response.

Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control

We have developed transgenic mouse models to determine whether endogenous expression of phytase transgenes in the digestive tract of monogastric animals can increase the bioavailability of dietary phytate, a major but indigestible form of dietary phosphorus. We constructed phytase transgenes composed of the appA phytase gene from Escherichia coli regulated for expression in salivary glands by the rat R15 proline-rich protein promoter or by the mouse parotid secretory protein promoter. Transgenic phytase is highly expressed in the parotid salivary glands and secreted in saliva as an enzymatically active 55 kDa glycosylated protein. Expression of salivary phytase reduces fecal phosphorus by 11%. These results suggest that the introduction of salivary phytase transgenes into monogastric farm animals offers a promising biological approach to relieving the requirement for dietary phosphate supplements and to reducing phosphorus pollution from animal agriculture.

Prevalence of Hepatitis E Virus Antibodies in Canadian Swine Herds and Identification of a Novel Variant of Swine Hepatitis E Virus

ABSTRACT Swine hepatitis E virus is a newly identified potentially zoonotic virus from pigs of particular concern for possible direct transmission to a human xenotransplant recipient by organ transplantation. In the present study, prevalence of serum antibodies to hepatitis E virus was examined in Canadian swine herds. A total of 998 serum samples collected from 6-month-old healthy slaughter hogs were examined by enzyme immunoassay and Western blot analysis for antibodies to the recombinant open reading frame 3 (ORF3) protein of hepatitis E virus expressed in Escherichia coli. These samples represented more than 80 different swine production units from five major swine-producing provinces across Canada. From this study, 594 samples (59.4%) were found to be positive for hepatitis E virus antibody. The seroprevalence was higher in Quebec (88.8%) and Ontario (80.1%) than in Alberta and Saskatchewan (38.3%). By PCR using a pair of oligonucleotide primers deduced from the ORF2 sequence of human hepatitis E virus, a specific hepatitis E virus sequence was recovered from feces of pigs. The nucleotide sequence identity between the U.S. swine hepatitis E virus and the Canadian isolate (SK3) was only 85.8%, suggesting that genotypic variations may exist in swine hepatitis E virus in North America. Among 165 serum samples collected from humans in Saskatchewan, 2.4% were found to be positive for antibodies to the hepatitis E virus ORF3 protein. Our data indicate that hepatitis E virus is highly prevalent in commercial swine populations in Canada and support the suggestion that the swine hepatitis E virus may be an important zoonotic agent for humans.

α2‐Macroglobulin and the α2‐Macroglobulin Receptor/LRP A Growth Regulatory Axisa

The hypothesis that a2-macroglobulin ( a 2 M ) regulates the function of growth factors and cytokines in vivo is based on a number of distinct observations. First, a2M binds cytokines and these interactions are favored compared with other plasma proteincytokine interactions. Second, activated a2M, which has undergone conformational change due to reaction with primary amines or proteinases, targets cytokines to the surfaces of cells expressing the azM receptor (a2M receptorAow density lipoprotein receptor-related protein or a2MFULFW). Third, a2M regulates the activity of cytokines in vitro; the nature of the regulation is cell type-specific and may depend on whether the cell expresses a2MFULRP. Finally, cytokines modulate cellular expression of a2MR/LRP, thereby altering the ability of azM to affect the activity of other cytokines in the same cell type. These four principles constitute what we refer to as the a z M a2MR/LRP growth regulatory axis. In this report, we will present recent work related to each of these areas.

Treatment of PD-1−/− mice with amodiaquine and anti-CTLA4 leads to liver injury similar to idiosyncratic liver injury in patients

The mechanism of idiosyncratic drug‐induced liver injury (IDILI) remains poorly understood, to a large degree because of the lack of a valid animal model. Recently, we reported an animal model in which treatment of female C57BL/6 mice with amodiaquine (AQ) resulted in mild liver injury with a delayed onset and resolution despite continued treatment. Such adaptation is a common outcome in the IDILI caused by drugs that can cause liver failure. We had hypothesized that most IDILI is immune‐mediated and adaptation represents immune tolerance. In this study we found that AQ treatment of Cbl‐b−/− and PD‐1−/− mice, which have impaired immune tolerance, resulted in a slightly greater injury. Cotreatment of C57BL/6 with AQ and anti‐CTLA4 also resulted in a greater increase in ALT than treatment with AQ alone; however, these mice also had an increase in T regulatory (Treg) cells and T helper cells expressing PD‐1 and CTLA4. The increase in these cells implies the induction of immune tolerance, and the alanine aminotransferase (ALT) activity in these mice returned to normal despite continued treatment. Cotreatment of PD‐1−/− mice with anti‐CTLA4 antibody and AQ resulted in the greatest increase in ALT (200‐300 U/L), and necroinflammatory responses characterized by portal infiltration of lymphocytes with interface hepatitis. The lymphocyte infiltration included T and B cells, and the CD8+ T cells produced perforin and granzyme. In addition, the ALT activity in PD‐1−/− mice cotreated with anti‐CTLA4 antibody and AQ did not return to normal, as it had in other mice. Conclusion: We report here the first animal model of IDILI that is similar to the IDILI that occurs in humans, and it was accomplished by inhibiting immune tolerance. (Hepatology 2015;61:1332–1342)

Plasma proteins of rainbow trout (Oncorhynchus mykiss) isolated by binding to lipopolysaccharide from Aeromonas salmonicida

In an attempt to find plasma proteins that might be involved in the constitutive resistance of rainbow trout to furunculosis, a disease caused by Aeromonas salmonicida (AS), we purified serum and plasma proteins based on their calcium- and carbohydrate-dependent affinity for A. salmonicida lipopolysaccharide (LPS) coupled to an epoxy-activated synthetic matrix (Toyopearl AF Epoxy 650M). A multimeric family of high molecular weight (96 to 200-kDa) LPS-binding proteins exhibiting both calcium and mannose dependent binding was isolated. Upon reduction the multimers collapsed to subunits of approximately 16-kDa as estimated by 1D-PAGE and exhibited pI values of 5.30 and 5.75 as estimated from 2D-PAGE. Their N-terminal sequences were related to rainbow trout ladderlectin (RT-LL), a Sepharose-binding protein. Polyclonal antibodies to the LPS-purified 16-kDa subunits recognized both the reduced 16-kDa subunits and the non-reduced multimeric forms. A calcium- and N-acetylglucosamine (GlcNAc)-dependent LPS-binding multimeric protein (approximately 207-kDa) composed of 34.5-kDa subunits was purified and found to be identical to trout serum amyloid P (SAP) by N-terminal sequence (DLQDLSGKVFV). A protein of 24-kDa, in reduced and non-reduced conditions, was isolated and had N-terminal sequence identity with a known C-reactive protein (CRP) homologue, C-polysaccharide-binding protein 2 (TCBP2) of rainbow trout. A novel calcium-dependent LPS-binding protein was purified and termed rainbow trout lectin 37 (RT-L37). This protein, composed of dimers, tetramers and pentamers of 37 kDa subunits (pI 5.50-6.10) with N-terminal sequence (IQE(D/N)GHAEAPGATTVLNEILR) showed no close homology to proteins known or predicted from cDNA sequences. These findings demonstrate that rainbow trout have several blood proteins with lectin properties for the LPS of A. salmonicida; the biological functions of these proteins in resistance to furunculosis are still unknown.

Reaction of α2-macroglobulin with plasmin increases binding of transforming growth factors-β1 and β2

The binding of125I-transforming growth factors-β1 and β2 (TGF-β1 and TGF-β2) to α2-macroglobulin(α2M) was studied before and after reaction with plasmin, thrombin, trypsin, or methylamine. Complex formation between TGF-β and native or reacted forms of α2M was demonstrated by non-denaturing polyacrylamide gel electrophoresis and autoradiography. Reaction of native α2M with plasmin or methylamine markedly increased the binding of125I-TGF-β1 and125I-TGF-β2 to α2M. The α2M-plasmin/TGF-β complexes were minimally dissociated by heparin. Reaction of α2M with thrombin or trypsin reduced the binding of 125I-TGF-β1 and 125I-TGF-β2; the resulting complexes were readily dissociated by heparin. Complexes between TGF-β2 and native or reacted forms of α2M were less dissociable by heparin than the equivalent complexes with TGF-β1. These studies demonstrate that the TGF-β-binding activity of α2M is significantly affected by plasmin, thrombin, trypsin and methylamine. Observations that α2M-plasmin preferentially binds TGFs-β suggest a mechanism by which α2M may regulate availability of TGFs-β to target cells in vivo.

Characterization of a Murine Alpha 2 Macroglobulin Gene Expressed in Reproductive and Cardiovascular Tissue

Abstract Full-length cDNA for a mouse gene A2-macroglobulin induced by pregnancy (A2mp) was cloned from mesometrial decidua at Gestation Day 10. The 4622-base pair cDNA encodes a protein of 1473 AA with >70% sequence identity and all typical domains of other A2M-family members in humans and rodents, despite unique absence of hepatic expression. The bait region is most distinct and has the greatest sequence similarity with rat acute-phase A2m. Northern blotting, reverse transcription and real-time-PCR, and in situ hybridization studies using C57Bl/6 mice revealed uterine induction of A2mp during decidualization and strong, midgestational association with modifying spiral arteries. Ovaries, testes, lactating mammary glands, heart, and kidney were the only additional organs with A2mp expression that was localized to granulosa and cumulus cells in secondary follicles; primary seminiferous epithelium, including Sertoli cells, mammary alveolar, and ductal epithelium; cardiac endothelium; and renal collecting tubules, respectively. Infusion of native human A2M into pregnant alymphoid or interferon-gamma gene-ablated mice overcame blocks to pregnancy-induced spiral artery modification in these strains. Activated human A2M was also effective, suggesting mechanisms independent of proteinase inhibition. Identification of cytokines, growth factors, or other molecules bound to A2MP should provide new insights into decidualization, spiral artery modification, and cardiovascular adaptation to pregnancy.