Effect of Holstein genotype on innate immune and metabolic responses of heifers to lipopolysaccharide (LPS) administration.

Heifers (n = 4/genotype) from unselected (stable genotype since 1964, UH) and contemporary (CH) Holsteins that differed in milk yield (6,200 and 11,100 kg milk/305 d) were used to assess the impact of selection on innate immune and acute-phase response to an endotoxin (lipopolysaccharide; LPS). Jugular catheters were implanted 24 h before LPS administration. Blood samples were collected at -1, -0.5, 0, 1, 2, 3, 4, 6, 8, and 24 h relative to iv administration of 0.5 µg LPS/kg BW. Rectal body temperature (BT) was determined at these sampling times and at 5 and 7 h. Dermal biopsies were collected after the 24 h blood sample and processed to isolate fibroblasts. Plasma was analyzed for tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), serum amyloid A (SAA), xanthine oxidase (XO), and nitrate + nitrite (NOx), cortisol, glucose, and IGF-1 content. Isolated fibroblasts were exposed to IL-1ß or LPS and IL-6 and IL-8 content of culture media determined. Exposure to LPS increased BTs and plasma concentrations of TNF-α, IL-6 SAA, XO, cortisol, and glucose (P < 0.05) in both genotypes. Plasma concentrations of TNF-α, XO, NOx, and glucose did not differ (P > 0.25) between the genotypes, but IL-6 and SAA concentrations were reduced (P < 0.05) in CH relative to UH heifers while cortisol and IGF-1 concentrations tended (P < 0.08) to be reduced in CH heifers. After 36 h exposure to LPS, concentrations of IL-6 were greater (P < 0.05) in culture media from incubations of CH than UH fibroblasts but concentrations of IL-8 did not differ between genotypes. There was a trend (P = 0.08) for IL-8 concentrations to be reduced in media from CH fibroblasts exposed to IL-1ß for 24 h but IL-6 concentrations did not differ between genotypes. Results indicate 50 yr of selection has reduced the robustness of the innate immune and acute-phase response to LPS in the contemporary Holstein heifer.

Variation in fibroblast expression of toll-like receptor 4 and lipopolysaccharide-induced cytokine production between animals predicts control of bacterial growth but not severity of Escherichia coli mastitis.

Mastitis caused by environmental pathogens such as Escherichia coli is highly problematic to the dairy industry because it incurs substantial cost and tends to be difficult to manage. An effective innate immune response by the host is key to controlling infection, but it should also limit collateral damage to the mammary gland. Between-animal differences in mastitis severity have been attributed to variability in the innate response. In the current study, we used primary dermal fibroblast as a model to rank animals based on composite expression of the toll-like receptor 4 gene (TLR4) and lipopolysaccharide (LPS)-induced IL-8 and IL-6 protein production. Animals ranked as high and low responders (HR and LR, respectively) were then infected with the P4 strain of E. coli to determine how difference in rank would affect response to mastitis. All animals developed an acute response to the infection with varying degrees in severity; however, HR animals had an elevated somatic cell count and fever response at 12 h post-infection and greater production of milk IL-8 at 24 h post-infection. The HR animals were also significantly more capable of limiting bacterial growth. No differences in post-infection milk production or concentrations of milk BSA were measured. The current study indicates that HR animals have an early upregulation in their innate response that is beneficial for bacterial clearance; however, they are equally susceptible to tissue damage caused by an exuberant response to the infection. The dermal fibroblast may be used in conjunction with other cell types to determine how the innate response is regulated to mitigate unnecessary injury to the mammary gland while still effectively clearing the pathogen.

Neonatal lipopolysaccharide exposure does not diminish the innate immune response to a subsequent lipopolysaccharide challenge in Holstein bull calves.

The innate immune response following experimental mastitis is quite variable between individual dairy cattle. An inflammatory response that minimizes collateral damage to the mammary gland while still effectively resolving the infection following pathogen exposure is beneficial to dairy producers. The ability of a lipopolysaccharide (LPS) exposure in early life to generate a low-responding phenotype and thus reduce the inflammatory response to a later-life LPS challenge was investigated in neonatal bull calves. Ten Holstein bull calves were randomly assigned to either an early life LPS (ELL) group (n=5) or an early life saline (ELS) group (n=5). At 7d of age, calves received either LPS or saline, and at 32d of age, all calves were challenged with an intravenous dose of LPS to determine the effect of the early life treatment (LPS or saline) on the immune response generated toward a subsequent LPS challenge. Dermal fibroblast and monocyte-derived macrophage cultures from each calf were established at age 20 and 27d, respectively, to model sustained effects from the early life LPS exposure on gene expression and protein production of components within the LPS response pathway. The ELL calves had greater levels of plasma IL-6 and tumor necrosis factor-α than the ELS calves following the early life LPS or saline treatments. However, levels of these 2 immune markers were similar between ELL and ELS calves when both groups were subsequently challenged with LPS. A comparison of the in vitro LPS responses of the ELL and ELS calves revealed similar patterns of protein production and gene expression following an LPS challenge of both dermal fibroblast and monocyte-derived macrophage cultures established from the treatment groups. Whereas an early life exposure to LPS did not result in a dampened inflammatory response toward a later LPS challenge in these neonatal bull calves, the potential that exposure to inflammation or stress in early life or in utero can create an offspring with a low-responding phenotype as an adult is intriguing and has been documented in rodents. Further work is needed to determine if an inflammatory exposure in utero in a dairy animal would result in a low-responding innate immune phenotype.

Cow-to-cow variation in fibroblast response to a toll-like receptor 2/6 agonist and its relation to mastitis caused by intramammary challenge with Staphylococcus aureus.

Staphylococcus aureus is a common cause of chronic mammary gland infections in dairy cattle. However, the inflammatory response and duration of infection following pathogen exposure is variable between individual animals. To investigate interanimal differences in immune response, dermal fibroblast cultures were established from skin biopsies collected from 50 early lactation Holstein cows. The fibroblasts ability to produce IL-8 in response to a 24-h treatment with a synthetic toll-like receptor 2/6 agonist (Pam2CSK4) was used to assign a response phenotype to the animals. Five high-responding and 5 low-responding animals were then selected for an intramammary challenge with S. aureus to evaluate differences in the inflammatory response, chronicity of infection, and development of antibodies to the pathogen. All animals exhibited clinical symptoms of mastitis at 24h postchallenge. Animals previously classified as high responders experienced a greater inflammatory response characterized by elevated levels of milk somatic cell count, IL-8, and BSA following the challenge compared with low responders. In addition, antibodies toward the challenge strain of S. aureus reached higher levels in whey from the challenged gland of high responders compared with low responders. Despite the antibody response, all 5 high responders were chronically infected for the 6-wk duration of the study, whereas 2 of the low responders cleared the infection, although 1 of these did become reinfected. The observed differences between animals classified as low and high responders based on their fibroblast responsiveness suggests that this cell type can be used to further examine the causes of interanimal variation in response to mammary infection.

Genomic analysis of between-cow variation in dermal fibroblast response to lipopolysaccharide.

The innate immune response plays a major role in defense against mastitis-causing pathogens. Identification of existing variation in innate immune signaling among cows and the underlying molecular causes for the variation may help in design of new mastitis control strategies. The dermal fibroblast has been used as a model cell type to explore between-cow variation in the ability of cells to produce IL-8 in response to lipopolysaccharide (LPS) treatment, and this response appears related to an animal's ability to respond to in vivo challenge with LPS or Escherichia coli mastitis. In this study, primary dermal fibroblast cultures of cows and microarray-based genomic analysis were used to investigate the cause(s) for the variable response to LPS. Fibroblast cultures from 2 cows, one with a low response phenotype (LR(array)) and another with a high response phenotype (HR(array)), were selected from our collection of fibroblast cultures established from 88 cows. The LR(array) fibroblast culture produced approximately 5-fold less IL-8 and IL-6 protein in response to 24-h LPS treatment than the HR(array) fibroblast culture. Genomic analysis of RNA obtained from 3 replicates of the 2 cultures before and after 8-h LPS treatment revealed a combined LPS-induced differential expression of 321 transcripts, indicating the robust response capability of the fibroblast cell. Under basal conditions, the microarray analysis revealed 2-fold less expression of toll-like receptor 4 (TLR4) in the LR(array) fibroblasts compared with the HR(array) fibroblasts, and this was associated with a marked reduction in expression of genes regulated by the TLR4-MyD88-dependent and TLR4-TRIF-dependent pathways (IL-8, IL-6, SAA3, CCL20, MX1, IRF1, and ISG20). The between-culture differential expression of TLR4 was confirmed and extended by quantitative PCR analysis (QPCR) that revealed a 33-fold lower expression of TLR4 in the LR(array) fibroblast culture. After LPS treatment, the difference in TLR4 expression increased to almost 50-fold and was associated with more than 8-fold lower expression of IL-8 and IL-6. No DNA sequence variations were identified in the proximal 1,300-bp promoter region of the TLR4 gene, and microarray analysis did not reveal a molecular explanation for the reduced TLR4 expression under either basal conditions or following exposure to LPS. The attenuated innate immune response of the LR(array) fibroblast culture to LPS may be caused by reduced TLR4 receptor expression. Also, the primary dermal fibroblast cells can be used to examine underlying causes for between-cow variations in key immune response pathways.

Between-cow variation in dermal fibroblast response to lipopolysaccharide reflected in resolution of inflammation during Escherichia coli mastitis.

Effective response to mammary gland infection depends on efficient early innate immune response. The desired response would be one that is sufficient to clear the infection with a rapid return to the production of high-quality milk and limited tissue damage. In this study, 43 early lactation cows were ranked based on the ability of their fibroblasts to produce IL-8 in response to Escherichia coli lipopolysaccharide. Subsequently, the effect of a low or high response phenotype on the response to E. coli mastitis was determined. Untreated fibroblasts produced no detectable IL-8, whereas the range of IL-8 production in response to LPS (100 ng/mL) was approximately 7-fold between the lowest and highest responding cultures. Similar patterns of between-cow variation were observed in fibroblast production of IL-8 and IL-6 in response to IL-1ß and Pam2CSK4 (a synthetic diacylated lipopeptide ligand). Four low and 4 high responder cows were challenged in late lactation with intramammary infusion of E. coli. All cows developed clinical mastitis in the challenged quarters and all cows cleared the infection within 8 d. However, somatic cell count began to decline earlier in the low responder group, and milk BSA concentration (an indicator of tissue damage) was also lower in low responders compared with high responders. Milk production from the challenged quarter was markedly depressed in both groups, but returned toward prechallenge values earlier in low responder cows. Dermal fibroblast cells appear predictive of a cow's response to mastitis. In this study, the low responder phenotype was sufficient to contain an E. coli infection with a more rapid return to the production of high quality milk.

The use of dermal fibroblasts as a predictive tool of the toll-like receptor 4 response pathway and its development in Holstein heifers.

The innate immune system comprises the host's first line of defense against invading pathogens, and variation in the magnitude of this response between animals has been shown to affect susceptibility to mastitis. The toll-like receptor (TLR) family of proteins initiates the response to invading bacteria, specifically with TLR4 recognizing lipopolysaccharide (LPS) of gram-negative microbes. The underlying genetic variation in the TLR4 pathway leading to differential response is not well understood; therefore, the objective of this work was to determine the efficacy in which the response to LPS by dermal fibroblasts could be used to predict the actual systemic response of that animal to an intravenous endotoxin challenge. To accomplish this, dermal fibroblasts were isolated from 15 Holstein heifers at 5, 11, and 16 mo of age and exposed to either LPS or IL-1ß; then, the production of IL-8 in medium was quantified by ELISA. Animals were ranked based upon the magnitude of the fibroblast IL-8 response, and 8 heifers were selected [4 low responders (LR) and 4 high responders (HR)] for challenge with an intravenous bolus dose (0.5 µg/kg of body weight) of LPS. Overall, between-animal variation in fibroblast IL-8 production following LPS or IL-1ß was high, indicating appreciable differences in the TLR4 pathway of the animals. Ranking of the fibroblast responses was consistent across the 3 sampling times for each animal; however, the absolute response increased, and the age at which the fibroblasts were obtained was consistent with the potential for age-related changes in cell function to affect immune function processes. Following systemic LPS challenge, HR heifers had higher plasma concentrations of tumor necrosis factor-α and IL-8 than LR heifers. However, LR heifers had a stronger febrile response than HR heifers. The use of dermal fibroblasts under laboratory conditions appears to represent a practical model for predicting the innate immune response in vivo and could act as an important tool in mapping genetic differences of the TLR4 pathway.

IGF-I differentially regulates IGF-binding protein expression in primary mammary fibroblasts and epithelial cells.

Elucidating how mitogens facilitate epithelial/stromal interactions is critical given that mitogens regulate mammary gland development and function. IGF-I is a potent mammary cell mitogen that is locally produced in the mammary gland. Since IGF-binding proteins (IGFBPs) regulate IGF-I bioavailability, we characterized the cell-type-specific production of IGFBP in primary bovine mammary epithelial (BME) and fibroblast (BMF) cells. Cells were treated with IGF-I and mRNA levels were analyzed via quantitative real-time (qRT)-PCR and Northern blot analysis. Media conditioned by cells treated with IGF-I for 48 h were analyzed via ligand blotting with 125I-labeled IGF-I and -II and immunoblotting with specific IGFBP antibodies. A reciprocal regulation of IGFBP-3 and -5 by IGF-I was observed between the two cell types. IGF-I induced large dose-dependent increases in IGFBP-3 mRNA and protein levels in BME cells, while IGFBP-5 protein was barely detectable and mRNA levels were detectable only by qRT-PCR. In BMFs, IGF-I induced large increases in IGFBP-5 mRNA and protein while IGFBP-3 mRNA was only slightly increased by IGF-I treatment and the protein was difficult to detect. IGFBP-6 mRNA was detected by Northern blot analysis in both cell types but was not regulated by IGF-I. In BME cells, IGFBP-6 protein levels were readily detectable under basal conditions and were increased by IGF-I. Interestingly, IGFBP-6 protein could not be detected in media conditioned by BMFs. IGFBP-4 mRNA was readily seen by Northern blot analysis in BMFs, however qRT-PCR was required to detect IGFBP-4 mRNA in BME cells. IGF-I increased IGFBP-4 mRNA levels by 2-fold in both cell types. IGFBP-4 protein was only detectable in media conditioned by BME cells when stimulated by IGF-I. In contrast, IGFBP-4 was present in media conditioned by untreated BMFs but was not consistently increased by IGF-I treatment. This was explained by the finding that IGF-I stimulated proteolysis of IGFBP-4, as evidenced by the appearance of two immuno-responsive fragments of 18 and 14 kDa. This proteolysis was specific to IGFBP-4, and was not observed in BME cells. We confirmed the protease to be pregnancy-associated plasma protein A (PAPP-A) by immunoblotting with an antibody against human PAPP-A/proMBP (pro form of eosinophil major basic protein) complex. In vitro immuno-neutralization experiments showed that blocking PAPP-A prevented the ability of IGF-I to stimulate IGFBP-4 proteolysis. IGFBP-2 mRNA and protein levels were observed under basal conditions in both cell types, with no significant regulation by IGF-I. The analysis of cell-type-specific regulation of the IGF system in both primary mammary epithelial cells and stromal cells will assist in the characterization of the mechanisms behind the role of the IGF system in normal mammary physiology and ultimately breast cancer.

Persistency of adenoviral-mediated lysostaphin expression in goat mammary glands.

Gene therapy has great potential to enable synthesis of protein molecules in targeted cells of an animal. One application may be the production of antibacterial enzymes by the mammary gland as a means of preventing or treating mastitis. We have previously demonstrated that goat mammary cells are capable of producing lysostaphin, an antistaphylococcal enzyme, after being transduced in vivo with a recombinant adenoviral vector containing a modified lysostaphin gene (Ad-lys). The current study examined duration of expression, and antibody response to lysostaphin and the adenoviral vector. Following intramammary infusion into nonlactating goats (n = 4), recovery of transducible adenoviral vector in mammary secretions persisted for 11 d. Transducible vector was not detected in serum, saliva, urine, or feces. Peak lysostaphin concentrations (< 20 microg/mL) in mammary secretions of infused udders were detected approximately 1 wk postinfusion, and generally returned to undetectable levels after an additional 1 to 2 wk. The poor persistency of expression was likely due to the very potent immune response to both the adenovirus and the expressed lysostaphin. Serum IgG antibodies recognizing the adenoviral vector developed within 7 d of the infusion, and titers rose dramatically to greater than 1:1 x 10(5). Similar titers of serum IgG antibodies to lysostaphin developed in 3 goats, with more moderate titers in the fourth goat. The antibody response to lysostaphin was delayed by approximately 4 d in comparison to the response to the adenovirus. Serum IgG antibody profiles were reflected in mammary secretions. No IgA antibodies to adenovirus or lysostaphin were detected in sera or mammary secretion. We demonstrate that while the lysostaphin gene can be introduced to the mammary gland using an adenoviral-mediated gene transfer technique, the strong immune response that it provokes makes the approach unsuitable for combating mastitis.

Cell donor influences success of producing cattle by somatic cell nuclear transfer.

To assess sources of variation in nuclear transfer efficiency, bovine fetal fibroblasts (BFF), harvested from six Jersey fetuses, were cultured under various conditions. After transfection, frozen-thawed lung or muscle BFF donor cells were initially cultured in DMEM in 5% CO(2) and air and some were transferred to MEM, with 5% or 20% O(2) or 0.5% or 10% serum and G418 for 2-3 wk. Selected clonal transfected fibroblasts were fused to enucleated oocytes. Fused couplets (n = 4007), activated with ionomycin and 6-dimethylaminopurine, yielded 927 blastocysts, and 650 were transferred to 330 recipients. Fusion rate was influenced by oxygen tension in a fetus-dependent manner (P < 0.001). Blastocyst development was influenced in a number of ways. Hip fibroblast generated more blastocysts when cultured in MEM (P < 0.001). The influence of serum concentration was fetus dependent (P < 0.001) and exposing fibroblast to low oxygen was detrimental to blastocyst development (P < 0.001). Cells from two of the six fetuses produced embryos that maintained pregnancies to term, resulting in eight viable calves. Pregnancy rates 56 days after transfer for the two productive donor fetuses, was at least double that of other recipients and may provide a fitness indicator of BFF cell sources for nuclear transfer. We conclude that a significant component in determining somatic cell nuclear transfer success is the source of the nuclear donor cells.

Optimization of DNA-based vaccination in cows using green fluorescent protein and protein A as a prelude to immunization against staphylococcal mastitis.

Staphylococcus aureus is a contagious pathogen that often results in chronic intramammary infections in dairy cows. Current vaccine formulations are ineffective in preventing this infection. The objective of this study was to stimulate an immune response in dairy cows through injection of plasmid DNA designed to express staphylococcal Protein A in transfected cells. Intramuscular and intradermal vaccination sites were evaluated using a plasmid containing the human cytomegalovirus (CMV) promoter/enhancer directing expression of green fluorescent protein (pcDNA3/GFP). DNA was delivered by needle and syringe, or by high-, intermediate-, or low-pressure jet injections (Ped-o-Jet and LectraJet). Five cows per treatment were injected with 0.5 mg of plasmid DNA at 6, 4, and 2 wk prepartum. Serum antibody levels determined by ELISA indicated that intradermal high-pressure jet injection elicited a greater immune response compared to needle and syringe injection. Differences in antibody production among low-pressure and needle and syringe treatment groups were not significant. An expression plasmid containing the CMV promoter/enhancer driving expression of the Fc-binding domain of S. aureus Protein A was coinjected into cows by vulvamucosal vaccination using the high-pressure Ped-o-Jet. Beginning 6 wk prepartum, groups of cows (n = 5) were injected three times at 2-wk intervals with DNA in saline, DNA in aluminum phosphate adjuvant, or served as noninjected controls. A cellular immune response to Protein A was detected in 4 of 10 animals, while cellular responses to GFP were not detected. Humoral responses to Protein A were observed in 6 of 10 animals and to GFP in 2 of 10 animals. Aluminum phosphate adjuvant appeared to enhance antibody production in response to Protein A. In experiment 3, a protein boost injection of Protein A was given to six animals approximately 5 mo postpartum. Three animals were nonvaccinated controls, and three were among those stimulated to produce antibody in response to the DNA-based vaccine. These results showed that Protein A specific antibodies remained elevated as compared to nonvaccinated controls and were stimulated in response to the protein boost. However, the magnitude of the response in animals previously vaccinated with DNA was not different than that observed in the nonvaccinated controls. We have shown that a humoral and cellular immune response to abbreviated Protein A can be raised in dairy cows using intravulvamucosal jet injection of a DNA-based vaccine.

Mammary expression of new genes to combat mastitis.

Continual advances in the ability to produce transgenic animals make it likely that such animals will become important components of animal agriculture. The full benefit of the technology, and justification of its initial cost outlay, will be dependent on the establishment within these animals of new traits not easily achievable by other means. Potential applications include enhanced nutrient digestibility with reduced fecal losses, significantly altered milk composition with superior nutritional properties, and enhanced disease resistance. Our goal is to enhance mastitis resistance of dairy cows by enabling the cells of the mammary gland to secrete additional antibacterial proteins. Proof of concept has been obtained through experimentation with a transgenic mouse model. Three lines of mice were developed that produce varying levels of lysostaphin in their milk. This protein has potent anti-staphylococcal activity and its secretion into milk confers substantial resistance to infection caused by intramammary challenge with Staphylococcus aureus, a major mastitis pathogen. Additional antibacterial proteins are being sought that will complement lysostaphin. A potential benefit of transgenic application of antibacterial proteins is the concomitant sparing in the agricultural use of antibiotics currently used as human therapeutics. Antibacterial proteins, such as lysostaphin, are not typically used as injectable or oral therapeutics because of immune-mediated or digestive destruction of their activity. In contrast, the immune system of transgenic animals will not consider the transgenic protein as being foreign. In addition we are exploring the potential of involution or mastitis responsive promoter elements for use in subsequent transgenic experiments designed to restrict lysostaphin production to these important time points. It is anticipated that genomics will play a role in unveiling candidate genes whose promoter elements will enable desired temporal expression patterns. The transgenic approach to insertion of new genetic material into agriculturally important animals is feasible but requires extensive prior evaluation of the transgene and transgene product in model systems.

Adenoviral-mediated transfer of a lysostaphin gene into the goat mammary gland.

As a step toward preventing and curing Staphylococcus aureus mastitis, an adenoviral-mediated gene transfer technique was used to enable mammary cells to synthesize and secrete lysostaphin, an anti-staphylococcal protein. A lysostaphin gene, modified for eukaryotic expression of the bioactive variant, Gln125,232-lysostaphin, was inserted into a replication deficient adenovirus by homologous recombination in 293 cells. The resulting adenoviral vector containing the modified lysostaphin gene (Ad-lys) was used to infect bovine mammary epithelial cells in vitro and caprine mammary cells in vivo. A similar adenoviral vector containing the Escherichia coli gene encoding beta-galactosidase (Ad-lacZ) was also evaluated. Transduction of cultured bovine cells by Ad-lacZ was confirmed by the presence of beta-galactosidase in fixed cells 48 h postinfection. Bovine cells transduced by Ad-lys secreted immunoreactive Gln125,232-lysostaphin (0.8 microg/ml) into media that had approximately 20% bioactivity compared with native lysostaphin. To evaluate transduction in vivo, udder halves of four nonlactating goats were exposed to 10(10) plaque-forming units (pfu) ofAd-lacZ by two intramammary infusions given 48 h apart. The animals were euthanized 24 h later, and extensive expression of beta-galactosidase was detected in cells lining the teat canals, with more moderate expression observed in adjoining mammary parenchyma. Udder halves of two other nonlactating goats were infused with 10(10) pfu of Ad-lys while contralateral udder halves received Ad-lacZ. The animals were euthanized 48 h postinfusion. In both animals, extensive expression of beta-galactosidase was detected in Ad-lacZ exposed teats. Immunoreative Gln125,232-lysostaphin was detectable in secretions from Ad-lys exposed glands 24 h postinfusion, increasing to approximately 1 microg/ml at 48 h postinfusion. As with cultured bovine mammary epithelial cells, the bioactivity of goat-derived Gln125,232-lysostaphin was approximately 20% of native lysostaphin. These results demonstrate that an adenoviral vector can be used to introduce a gene into the ruminant mammary gland, enabling the secretion of a bioactive form of lysostaphin.

Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice.

Infection of the mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Staphylococcus aureus is the major contagious mastitis pathogen, accounting for approximately 15-30% of infections, and has proved difficult to control using standard management practices. As a first step toward enhancing mastitis resistance of dairy animals, we report the generation of transgenic mice that secrete a potent anti-staphylococcal protein into milk. The protein, lysostaphin, is a peptidoglycan hydrolase normally produced by Staphylococcus simulans. When the native form is secreted by transfected eukaryotic cells it becomes glycosylated and inactive. However, removal of two glycosylation motifs through engineering asparagine to glutamine codon substitutions enables secretion of Gln(125,232)-lysostaphin, a bioactive variant. Three lines of transgenic mice, in which the 5'-flanking region of the ovine beta-lactoglobulin gene directed the secretion of Gln(125,232)-lysostaphin into milk, exhibit substantial resistance to an intramammary challenge of 104 colony-forming units (c.f.u.) of S. aureus, with the highest expressing line being completely resistant. Milk protein content and profiles of transgenic and nontransgenic mice are similar. These results clearly demonstrate the potential of genetic engineering to combat the most prevalent disease of dairy cattle.

Equivalence of Visual Immunoprecipitate Assay (VIP) for Salmonella for the detection of motile and nonmotile Salmonella in all foods to AOAC culture method: collaborative study.

Six foods representative of a wide variety of processed, dried powder processed, and raw food types were analyzed by the Visual Immunoprecipitate Assay (VIP) for Salmonella and AOAC INTERNATIONAL culture method. Paired samples of each food type were simultaneously analyzed; one sample by the VIP method and one by the AOAC culture method. A total of 24 laboratories representing federal government agencies and private industry, in the United States and Canada, participated in this collaborative study. Food types were inoculated with species of Salmonella with the exception of raw ground chicken, which was naturally contaminated. No statistical differences (p < 0.05) were observed between VIP for Salmonella interpretation and the AOAC culture method for any inoculation level of any food type or naturally contaminated food. The method was adopted Official First Action status by AOAC INTERNATIONAL.

Equivalence of assurance Gold Enzyme Immunoassay for visual or instrumental detection of motile and nonmotile Salmonella in all foods to AOAC culture method: collaborative study.

Six foods representative of a wide variety of processed, dried powder processed, and raw food types were analyzed by the Assurance Gold Salmonella Enzyme Immunoassay (EIA) and AOAC INTERNATIONAL culture method. Paired samples of each food type were simultaneously analyzed; one sample by the Assurance method and one by the AOAC culture method. The results for Assurance method were read visually and instrumentally with a microplate reader. A total of 24 laboratories representing federal government agencies and private industry, in the United States and Canada, participated in this collaborative study. Food types were inoculated with species of Salmonella with the exception of raw ground chicken, which was naturally contaminated. No statistical differences (p < 0.05) were observed between Assurance Gold Salmonella EIA with either visual or instrumental interpretation and the AOAC culture method for any inoculation level of any food type or naturally contaminated food. The Assurance visual and instrumental options of reading sample reactions produced the same results for 1277 of the 1296 sample and controls analyzed.

Comparison of recombinant and synthetically formed monoclonal antibody-beta-lactamase conjugates for anticancer prodrug activation.

Conjugates of the L49 monoclonal antibody (binds to the p97 antigen on melanomas and carcinomas) were formed by attaching Enterobacter cloacae beta-lactamase (bL) to the L49-Fab' fragment using a heterobifunctional cross-linking reagent or by linking the enzyme to L49-sFv using DNA recombinant technology. The conjugates thus formed, L49-Fab'-bL and L49-sFv-bL, were designed to activate cephalosporin containing anticancer prodrugs at the surfaces of antigen positive tumor cells. Results from in vitro experiments using two lung carcinoma cell lines demonstrated that the conjugates were equally active in effecting the release of phenylenediamine mustard from the cephalosporin nitrogen mustard prodrug CCM. While treatment with either of the conjugates combined with the maximum tolerated doses of CCM led to cures of established SN12P renal cell carcinoma tumors in nude mice, only the L49-sFv-bL conjugate maintained its ability to do so at 1/4 the maximum tolerated dose of CCM. L49-sFv-bL was also superior to L49-Fab'-bL in the 1934J renal cell carcinoma tumor model and was shown to be quite active in two in vivo models of human lung carcinoma. These results demonstrate that the recombinant fusion protein leads to more pronounced therapeutic windows than the chemical conjugate and is active in an array of human tumor models.

Mathematical and experimental analysis of localization of anti-tumour antibody-enzyme conjugates.

Considerable research has been aimed at improving the efficacy of chemotherapeutic agents for cancer therapy. A promising two-step approach that is designed to minimize systemic drug toxicity while maximizing activity in tumours employs monoclonal antibody (mAb)-enzyme conjugates for the activation of anticancer prodrugs. We present, analyse and numerically simulate a mathematical model based on the biology of the system to study the biodistribution, pharmacokinetics and localization properties of mAb-enzyme conjugates in tumour tissue. The model predictions were compared with experimental observations and an excellent correlation was found to exist. In addition, the critical parameters affecting conjugate half-life were determined to be the inter-capillary half-distance and the antibody-antigen binding affinity. An approximation is presented relating the per cent injected dose per gram to inter-capillary half-distance and time. Finally, the model was used to examine various dosing strategies in an attempt to determine which regimen would provide the best biodistribution results. We compared the results of administering a uniform dose of fusion protein via bolus injection, multiple injections and continuous infusion. The model predicts that dosing strategy has little effect on the amount of conjugate that localizes in the tumour.

Redefining body composition: nutrients hormones, and genes in meat production.

Growth rate and body composition of livestock can be optimized to meet consumer needs for a leaner product and to improve the efficiency of meat-animal production. Optimization strategies have traditionally focused on genetic selection and cost-effective ration formulation to achieve the genetic potential. Advances in understanding the mechanisms of growth and its control have led to additional opportunities for its manipulation. These include nutritional manipulation,the use of growth promotants, and, more recently, the ability to change the genetic potential through genetic engineering. Selection of appropriate candidate genes for manipulation depends on understanding the mechanisms underlying differentiation and growth of embryonic muscle cells. Recent advances in genetic engineering techniques, including gene therapy and germline transgenesis, will likely hasten the genetic progress toward a leaner carcass in domestic livestock. Such strategies may prove to be more beneficial then the controlled enhancement of somatotropin expression.

Development and activities of a new melphalan prodrug designed for tumor-selective activation.

The synthesis of C-Mel, a cephalosporin carbamate derivative of the clinically used alkylating agent melphalan, is described. C-Mel was designed as an anticancer nitrogen mustard prodrug that releases melphalan upon tumor-specific activation by targeted beta-lactamase (bL). The Km and kcat values for bL hydrolysis of C-Mel were 218 microM and 980 s(-1), respectively. In vitro cytotoxicity assays with 3677 human melanoma cells demonstrated that C-Mel was 40-fold less toxic than melphalan and was activated in an immunologically specific manner by L49-sFv-bL, a recombinant fusion protein that binds to the melanotransferrin antigen on melanomas and on some carcinomas. L49-sFv-bL in combination with C-Mel led to regressions and cures of established subcutaneous 3677 tumors in nude mice. The effects were significantly greater than those of melphalan, which did not result in any long-term regressions in this tumor model. The therapeutic effects were comparable to those obtained in mice treated with the previously described L49-sFv-bL/7-(4-carboxybutanamido)-cephalosporin mustard (CCM) combination. However, C-Mel may be more attractive than CCM for clinical development since the released drug is clinically approved.