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Staphylococcus aureus is a leading cause of bovine intramammary infections (IMIs) that can evolve into difficult-to-treat chronic mastitis. To date, no vaccine formulation has shown high protective efficacy against S. aureus IMI, partly because this bacterium can efficiently evade the immune system. For instance, S. aureus small colony variants (SCVs) have intracellular abilities and can persist without producing invasive infections. As a first step towards the development of a live vaccine, this study describes the elaboration of a novel attenuated mutant of S. aureus taking advantage of the SCV phenotype. A genetically stable SCV was created through the deletion of the hemB gene, impairing its ability to adapt and revert to the invasive phenotype. Further attenuation was obtained through inactivation of gene vraG (SACOL0720) which we previously showed to be important for full virulence during bovine IMIs. After infection of bovine mammary epithelial cells (MAC-T), the double mutant (ΔvraGΔhemB) was less internalized and caused less cell destruction than that seen with ΔhemB and ΔvraG, respectively. In a murine IMI model, the ΔvraGΔhemB mutant was strongly attenuated, with a reduction of viable counts of up to 5-log10 CFU/g of mammary gland when compared to the parental strain. A complete clearance of ΔvraGΔhemB from glands was observed whereas mortality rapidly (48h) occurred with the wild-type strain. Immunization of mice using subcutaneous injections of live ΔvraGΔhemB raised a strong immune response as judged by the high total IgG titers measured against bacterial cell extracts and by the high IgG2a/IgG1 ratio observed against the IsdH protein. Also, ΔvraGΔhemB had sufficient common features with bovine mastitis strains so that the antibody response also strongly recognized strains from a variety of mastitis associated spa types. This double mutant could serve as a live-attenuated component in vaccines to improve cell-mediated immune responses against S. aureus IMIs., Competing Interests: The authors have declared that no competing interests exist.

Staphylococcus aureus is a significant pathogen frequently causing persistent intramammary infections (IMI) in dairy cows. We compared some genotypic and phenotypic characteristics of 285 strains collected from quarter milk samples from cows with persistent and nonpersistent subclinical IMI across Canada. Variable number of tandem repeats typing was used to infer the persistence of the same S. aureus strain in 3 consecutive quarter milk samples collected at intervals of 3 wk during lactation or before and after dry-off. All first isolates of the series were used as the representative strains from persistent IMI and were compared with nonpersistent strains for the presence of genes seg, sen, sec, and tst as well as by spa typing. Biofilm production in vitro and hld-RNAIII expression levels were also quantified. The gene seg was associated with a reduction in the likelihood of the bacteria to cause a persistent IMI during lactation. Strains persisting through the dry period produced significantly more biofilm in vitro than strains that do not persist after calving. Also, we showed that strains expressing more hld were more likely to be nonpersistent during either lactation or through the dry period. Three spa types were predominant (t529, t267, and a novel type: t13401). In the strains studied, the spa type tbl 2645 was the most frequent, and 97.0% of the strains of this spa type carried both sen and seg. Strains from the spa type tbl 2645 were less likely to cause a persistent IMI in the dry period. Most (86.7%) of the strains of the novel spa type (t13401) were negative for seg, sen, or both and produced significantly more biofilm in vitro than tbl 2645 and t267. The present study expanded our current knowledge on the genotypic and phenotypic traits of S. aureus strains recovered from persistent and nonpersistent IMI in Canada., (Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

During the transition from pregnancy to lactation, the sudden increase in nutrient demand for milk production causes metabolic perturbations and is associated with immunosuppression and a high incidence of metabolic and infectious diseases in high-yielding cows. In this study, we examined whether limiting milk harvest postpartum while maintaining milking stimulus could improve the metabolic status of cows without reducing overall milk production. Forty-seven Holstein cows were milked completely twice a day from calving (control); milked incompletely (about one-third of expected milk production was collected) twice a day until d 5 after calving (incomplete); or left to nurse their calf until d 5 and milked once a day from d 3 to d 5 (nursing). All cows were milked twice a day from d 6 to the end of the experiment (d 61). During the treatment period (d 1 to 5), milk production averaged 27.3 and 9.7 kg/d for the control and incomplete treatments, respectively. We observed no residual effect of treatment on milk production, which averaged 47.8, 45.7, and 46.4 kg/d for the control, incomplete, and nursing treatments, respectively, between wk 2 and 9. The dry matter intake of the cows was similar during and after treatment. From wk 2 to 9, milk protein and lactose percentage were not affected by treatments, but milk fat tended to be higher in control cows than in cows milked partially (incomplete + nursing). Blood concentrations of glucose and phosphorus were lower and concentrations of nonesterified fatty acids and β-hydroxybutyrate were higher in control cows than in partially milked cows during the treatment period. The positive effects on glucose and β-hydroxybutyrate remained significant up to d 28. Peripheral blood mononuclear cell (PBMC) proliferation and secretion of IL-4 were depressed during the postpartum period, and proliferation tended to be greater for cells incubated in serum from cows in the incomplete treatment on d 5 but lower on d 61. We observed no effect of treatments on polymorphonuclear neutrophilic leukocyte chemotaxis or phagocytosis. Proliferation and IL-4 secretion of PBMC were negatively correlated with concentration of serum nonesterified fatty acids. Reducing milk harvest postpartum while maintaining milking stimuli reduced metabolic stress without compromising productivity of high-yielding dairy cows., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to evaluate the effect of shortening the dry period on the mammary gland and the hormonal regulation of its functions. Holstein cows (n = 18) were assigned to a short dry period (SDP; 35 d; n = 9) or a conventional dry period (CDP; 65 d; n = 9). All cows were fed the same diets, with the exception that, during the dry period, the SDP cows received only the pre-calving diet for 35 d, whereas the CDP cows were fed a high-fiber diet from 65 to 28 d before calving and then received the same pre-calving diet as the SDP cows. Mammary gland functional capacity was evaluated at 70 days in milk, and mammary biopsies were taken in early and midlactation. Dry period length averaged 64.3 ± 1.1 and 31.9 ± 1.0 d for the CDP and SDP cows, respectively. The SDP cows had a lower milk yield and a lower energy-corrected milk yield compared with the CDP cows. The SDP cows also had a lower dry matter intake from wk 5 to 20 of lactation and tended to have lower plasma concentrations of β-hydroxybutyrate from wk 1 to 4. Prepartum serum progesterone and estradiol concentrations were unaffected by the dry period management. Serum growth hormone concentrations and milking-induced prolactin release were similar in both groups. However, during the period when the CDP cows were dry but the SDP cows were still being milked (wk -9 to -6), serum prolactin concentrations were higher in the SDP cows than in the CDP cows. The SDP cows had a lower milk BSA content than the CDP cows after the dry period and similar milk lactose concentrations, suggesting that their mammary tight junctions were closed following parturition and, therefore, that the later stage of their lactogenesis was not impaired by SDP management. In early and midlactation, mammary cell apoptosis and proliferation rates as well as mammary expression of genes involved in the function of this tissue were unaffected by the dry period management strategy. For cows in their second lactation, mammary gland functional capacity at 70 d in milk tended to be lower in the SDP cows. In conclusion, even though SDP management decreased milk production during the subsequent lactation, it did not affect mammary cell activity. Although direct evidence is still lacking, decreased mammary cell growth during the dry period is likely responsible for this negative effect. The higher prolactin concentrations in lactating cows during late gestation could be involved in this effect. More research is needed to test these hypotheses., (Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The objective of this study was to evaluate the effect of milking frequency on milk production and composition, mammary cell proliferation, apoptosis, and gene expression. For this investigation, 10 Holstein cows that were being milked twice a day in mid lactation were selected. To study the effect of differential milking, 2 quarters were milked once daily and the other 2 were milked thrice daily for 8wk. After that period, twice-daily milking was resumed for all quarters, and data were collected for an additional 6wk. Mammary gland biopsies were taken 1wk before differential milking (wk -1) and after 4 and 8wk of differential milking. Milk samples were collected weekly throughout the experiment. Once-daily milking resulted in an immediate reduction in milk yield, whereas thrice-daily milking resulted in an increase in milk yield. During differential milking, the daily milk yield of the quarters milked once daily declined by 0.54kg/wk, on average, but remained constant in the quarters milked thrice daily. Part of the difference in milk yield between the glands pairs persisted after twice-daily milking was reinitiated. In the quarters milked once daily, milk BSA concentration increased, indicating an increase in tight junction leakiness, and zymographic analysis of milk enzymes showed increased activity of several proteases. Reducing the milking frequency also increased mammary cell apoptosis and, surprisingly, mammary cell proliferation. Interestingly, milk concentrations of stanniocalcin-1 and insulin-like growth factor-I and mammary gland expression of several genes were also modulated by milking frequency. For example, expression of insulin-like growth factor I receptor was downregulated during once-daily milking. Last, expression of the short and long isoforms of the prolactin receptor and of CSN2 (beta-casein) were upregulated during thrice-daily milking. Taken together, these data suggest that milking frequency not only affects mammary gland remodeling and the expression of paracrine factors but also modulates hormone sensitivity., (Copyright 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The transition from pregnancy to lactation is marked by metabolic, hormonal, and immunological changes that have an impact on the incidence of infectious and metabolic diseases. The aim of this study was to evaluate the effect on immune function and blood metabolite concentration of limiting milk production in early lactation to reduce negative energy balance. Twenty-two multiparous Holstein cows were milked either once a day (1x) or twice a day (2x) for the first week postpartum. All cows were milked twice daily for the rest of lactation. Blood concentrations of nonesterified fatty acids (NEFA), beta-hydroxybutyric acid (BHBA), calcium, bilirubin, urea, phosphorus, glucose, leptin, stanniocalcin-1, and 17beta-estradiol were determined in samples collected from 5 wk before scheduled calving to 5 wk after calving. Polymorphonuclear leukocytes (PMNL) were isolated from blood to conduct assays for chemotaxis, phagocytosis, and respiratory burst. Peripheral blood mononuclear cells (PBMC) were isolated to evaluate lymphocyte proliferation and cytokine production (tumor necrosis factor-alpha, IL-4, and interferon-gamma). Cows milked 1x produced 31% less milk than cows milked 2x during the first week of lactation. Over the following 13 wk of lactation, the milk production of cows milked 1x during the first week was 8.1% lower than for cows milked 2x. However, because the percentages of fat and protein were greater in the milk from 1x cows, the yields of milk components and energy-corrected milk were similar. Calving induced an increase in the concentrations of NEFA, BHBA, urea, and bilirubin. The increases in levels of NEFA and BHBA were greater in cows milked 2x than in cows milked 1x. During the same period, the serum glucose concentration decreased but remained greater in cows milked 1x. Serum calcium on d 4 and serum phosphorus on d 4 and 5 were greater in cows milked 1x. The differences between the 2 groups persisted beyond treatment until postpartum d 24 for NEFA and glucose and until postpartum d 14 for BHBA. After calving, the concentrations of leptin and stanniocalcin-1 decreased. During the first week postpartum, the decrease of leptin was less marked in cows milked 1x. The immune functions of PBMC and PMNL isolated from experimental cows and incubated using a standard medium did not show clear-cut peripartum immunosuppression. These variables were not significantly affected by the treatments, with the exception of interferon-gamma secretion, which was greater on d 5 and 14 in cows milked 1x. In conclusion, limiting milk production in early lactation had positive effects on metabolite concentration, but larger studies are necessary to establish if this could reduce disease incidence.

There is considerable evidence to indicate the existence of local control of mammary gland involution, but the exact nature of this control has yet to be defined. Stanniocalcin-1 (STC-1) is a newly discovered mammalian hormone that seems involved in the lactation process and may be implicated in the control of involution. As a first step in investigating this hypothesis, the change in STC-1 levels in milk and serum was measured during drying off. Nine Holstein cows in late lactation were milked twice daily on half the gland, while the other half was left unmilked for a 14-d period. Milk and blood samples and mammary biopsies were taken on d -7, 1, 2, 7, and 14 relative to the onset of the nonmilking period. The concentrations of STC-1 in serum and milk were determined by RIA. The albumin concentration and proteinase activity of the milk were determined. Apoptosis of the mammary epithelium was quantified by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Finally, the effects of milk on cellular activity and apoptosis were tested in vitro on mammary epithelial cells by measuring the turnover of tetrazolium salts and by counting the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells. The drying off of 2 quarters increased the milk production of the quarters that were milked by 30%. Milk proteinase activity and BSA and STC-1 concentrations increased in the nonmilked quarters, but remained unchanged in the milked quarters. Moreover, at d 2, the apoptotic rate of the mammary cells was higher in the nonmilked quarters than in the milked quarters (0.22 +/- 0.04 vs. 0.07 +/- 0.04%, respectively). Finally, in vitro experimentation demonstrated that mammary epithelial cells cultured in the presence of milk from involuting quarters had 3-fold more apoptotic cells as compared with those cultured in milk from the milked quarters at d 14. The metabolic rate was reduced by 14.6% for milk from d 7 and 23.6% for milk from d 14. Interestingly, the metabolic rate was negatively correlated with the STC-1 concentration in milk (r = -0.65). This study shows for the first time that STC-1 in milk is increased following milk stasis, although its exact role in the involution process remains to be clarified.

Enzymes that hydrolyze complex carbohydrates play important roles in numerous biological processes that result in the maintenance of marine and terrestrial life. These enzymes often contain noncatalytic carbohydrate binding modules (CBMs) that have important substrate-targeting functions. In general, there is a tight correlation between the ligands recognized by bacterial CBMs and the substrate specificity of the appended catalytic modules. Through high-resolution structural studies, we demonstrate that the architecture of the ligand binding sites of 4 distinct family 35 CBMs (CBM35s), appended to 3 plant cell wall hydrolases and the exo-beta-D-glucosaminidase CsxA, which contributes to the detoxification and metabolism of an antibacterial fungal polysaccharide, is highly conserved and imparts specificity for glucuronic acid and/or Delta4,5-anhydrogalaturonic acid (Delta4,5-GalA). Delta4,5-GalA is released from pectin by the action of pectate lyases and as such acts as a signature molecule for plant cell wall degradation. Thus, the CBM35s appended to the 3 plant cell wall hydrolases, rather than targeting the substrates of the cognate catalytic modules, direct their appended enzymes to regions of the plant that are being actively degraded. Significantly, the CBM35 component of CsxA anchors the enzyme to the bacterial cell wall via its capacity to bind uronic acid sugars. This latter observation reveals an unusual mechanism for bacterial cell wall enzyme attachment. This report shows that the biological role of CBM35s is not dictated solely by their carbohydrate specificities but also by the context of their target ligands.

Stanniocalcin-1 is a hormone that possesses both paracrine and endocrine functions and has recently been identified in mammals. While paracrine functions have been determined for several organs, the role of circulating stanniocalcin-1 in cattle is still unclear but, observations in mice and cows suggest that stanniocalcin-1 plays a role in both gestation and lactation. The changes in serum stanniocalcin-1 content in different physiological states have never been evaluated in ruminants. We measured stanniocalcin-1 levels in sera from cattle ranging in age from post-weaned calves to 17-month-old heifers and in sera from cows during lactation and pregnancy. Our results indicate that the blood concentration of stanniocalcin-1 is increased by pregnancy, but not by lactation. The highest levels of stanniocalcin-1 were found in young calves and during the non-lactating period preceding calving. This suggests that stanniocalcin-1 is important for gestation and the preparation of the mammary gland for lactation.

Genes encoding cell-surface proteins regulated by SigB are stably expressed in Staphylococcus aureus small-colony variants (SCVs) isolated from cystic fibrosis (CF) patients. Our hypothesis is that CF-isolated SCVs are locked into a colonization state by sustaining the expression of adhesins such as fibronectin-binding proteins (FnBPs) throughout growth. Force spectroscopy was used to study the fibronectin-FnBPs interaction among strains varying for their SigB activity. The fibronectin-FnBPs interaction was described by a strength of 1000+/-400 pN (pulling rate of 2 microm s(-1)), an energetic barrier width of 0.6+/-0.1 A and an off-rate below 2 x 10(-4) s(-1). A CF-isolated SCV highly expressed fnbA throughout growth and showed a sustained capacity to bind fibronectin, whereas a prototypic strain showed a reduced frequency of fibronectin-binding during the stationary growth phase when its fnbA gene was down-regulated. Reduced expression of fnbA was observed in sigB mutants, which was associated with an overall decrease adhesion to fibronectin. These results suggest that the fibronectin-FnBPs interaction plays a role in the formation of a mechanically resistant adhesion of S. aureus to host tissues and supports the hypothesis that CF-isolated SCVs are locked into a colonization state as a result of a sustained SigB activity.

Collagen-binding protein (CNA) is the major Staphylococcus aureus adhesin responsible for high affinity binding to collagen and is assumed to be a major virulence factor in infection and disease. Mutants lacking the cna gene are less virulent than the parent strain in models of septic arthritis, osteomyelitis, keratinitis, and endocarditis. In order to investigate the immunological and protective properties of a CNA-based DNA vaccine, a eukaryotic expression vector pCNA was constructed which expressed the collagen-binding domain of this adhesin in transfected cells. Three groups of 11 Balb/c mice received three injection of either pCNA, the empty expression vector (pCI) or PBS. Those injected with pCNA showed hi titre (64000) antibody and evidence of a cell-mediated immune response (CMI). The anti-CNA antibodies recognized the intact bacteria and prevented binding to collagen in vitro. However, the vaccination did not protect against bacterial challenge using the intra-peritoneal route of infection. Moreover, S. aureus that had been treated with sera from vaccinated mice caused a more severe infection than bacteria treated with sera from non-vaccinated mice. In summary, DNA vaccination against CNA produced a strong antibody and cellular response in mice but failed to protect from i.p. infection by S. aureus.

Cyclic diguanylate (c-di-GMP) is a bacterial intracellular signaling molecule. We have shown that treatment with exogenous c-di-GMP inhibits Staphylococcus aureus infection in a mouse model. We now report that c-di-GMP is an immodulator and immunostimulatory molecule. Intramammary treatment of mice with c-di-GMP 12 and 6 h before S. aureus challenge gave a protective effect and a 10,000-fold reduction in CFUs in tissues (p < 0.001). Intramuscular vaccination of mice with c-di-GMP coinjected with S. aureus clumping factor A (ClfA) Ag produced serum with significantly higher anti-ClfA IgG Ab titers (p < 0.001) compared with ClfA alone. Intraperitoneal injection of mice with c-di-GMP activated monocyte and granulocyte recruitment. Human immature dendritic cells (DCs) cultured in the presence of c-di-GMP showed increased expression of costimulatory molecules CD80/CD86 and maturation marker CD83, increased MHC class II and cytokines and chemokines such as IL-12, IFN-gamma, IL-8, MCP-1, IFN-gamma-inducible protein 10, and RANTES, and altered expression of chemokine receptors including CCR1, CCR7, and CXCR4. c-di-GMP-matured DCs demonstrated enhanced T cell stimulatory activity. c-di-GMP activated p38 MAPK in human DCs and ERK phosphorylation in human macrophages. c-di-GMP is stable in human serum. We propose that cyclic dinucleotides like c-di-GMP can be used clinically in humans and animals as an immunomodulator, immune enhancer, immunotherapeutic, immunoprophylactic, or vaccine adjuvant.

To investigate the strategy of using a multivalent polyprotein DNA vaccine against Staphylococcus aureus, a series of plasmids was used to immunize mice followed by infectious challenge. The plasmid vaccines expressed Clumping factor A (Clfa), fibronectin binding protein A (FnBPA) and the enzyme Sortase (Srt) as single proteins or combined as a polyprotein. All animals produced a mixed Th1 and Th2 response including functional antigen-specific, mostly IgG2a antibodies, sustained production of IFN-gamma and a predominantly CD8+ T-cell response. Upon challenge with a virulent S. aureus isolate (Sa042), after 21 days, 55% of the multi-gene vaccinated mice survived infection compared to only 15% of the control groups. Vaccinated mice showed no signs of arthritis when challenged with the less virulent "Newman" strain that caused reactive arthritis in the controls. The results suggest that a multi-gene polyprotein-expressing nucleic acid vaccine alone produces a combined Th1 and Th2 response that can contribute to protection against the complex pathogenesis of S. aureus.

Milk production is a function of the number and activity of mammary epithelial cells, regardless of stage of lactation. Milk yield is generally higher in multiparous cows than in primiparous cows, but persistency is usually greater in the latter group. We compared several measures related to metabolic activity, apoptosis, and endocrine control of mammary cell growth in 8 primiparous and 9 multiparous cows throughout lactation. Mammary gland biopsies were taken in early [10 d in milk (DIM)], peak (50 DIM), and late (250 DIM) lactation to evaluate gene expression and determine DNA and fatty acid synthase (FAS) content. Milk samples taken the day before the biopsies were used to detect protease activities and to determine stanniocalcin-1 (STC) concentrations. Blood samples served to measure insulin-like growth factor-1, prolactin, and STC concentrations. Milk yield was higher in multiparous cows than in primiparous cows at the 10 DIM (32.8 +/- 1.3 and 25.2 +/- 0.8 kg/d) and 50 DIM (38.0 +/- 1.2 and 29.8 +/- 1.1 kg/d), but it was the same for both groups at 250 DIM (23.9 +/- 1.5 and 23.8 +/- 1.1 kg/d). Except for stearoyl-coenzyme A desaturase, expression of genes related to milk synthesis was not affected by stage of lactation. However, gene expression of acetyl-coenzyme A carboxylase, beta-casein, and FAS was lower in early lactation in primiparous cows. Expression of both proapoptotic bax and antiapoptotic bcl-2 genes was higher in primiparous cows, whereas the bax-to-bcl-2 ratio was not changed. Mammary DNA concentration was higher in multiparous cows, as was the amount of FAS protein in early lactation. Two bands of protease activity were found in milk samples, and one of the bands had an apparent molecular weight similar to gelatinase A and was dependent on the stage of lactation. Serum insulin-like growth factor-1 increased with day of lactation and was higher in primiparous cows. Serum prolactin decreased in late lactation, but peak values were observed in early lactation for primiparous cows and peak lactation for multiparous cows. Milk STC content increased with advancing lactation. The results are consistent with a lower degree of differentiation and a greater capacity for cell renewal in the mammary gland of primiparous cows.

Blocking the primary stages of Staphylococcus aureus infection, specifically the bacterial adhesion to cell and the colonization of the mucosal surface, may be the most effective strategy for preventing infections. Clumping factor A (ClfA) is considered to be one of the most important adhesions factors of S. aureus to host cells. The present study describes the immune response of dairy cattle to a DNA vaccine against ClfA and evaluates the ability of specific genetic adjuvants, targeting sequences (granulocyte macrophage colony-stimulating factor and cytotoxic T lymphocyte antigen-4) and transporter molecules (chitosan and copolymer) to modify the immune response of cows. The results show that vaccination of cows with fibrinogen-binding region A induced a strong and specific antibody response to ClfA in comparison with a control group injected with the pCI vector alone. Although the co-expression of both genetic adjuvants and the addition copolymer transporter did not augment the overall antibody response, these approaches decreased the number of non-responsive cows. Chitosan was the only factor that did not enhance the immune response. Three months after the last DNA immunization, three cows from each of the pGM-CSF, internal ribosomal entry site (IRES), pCTLA and pCI groups were injected with 200 microg of recombinant ClfA protein in incomplete Freund's adjuvant. A strong humoral response was observed in all groups following this protein boost, with the response occurring slightly earlier in DNA-primed protein boost cows. Sera and milk samples taken from cows after the second DNA injection or after the protein boost (sera only) were analyzed for their ability to block adherence and increase phagocytosis. Pre-incubation of S. aureus with sera or milk from vaccinated cows significantly reduced the pathogen's ability to adhere to MAC-T cells relative to the sera and milk samples from the pCI-injected control cows. Similarly, pools of sera and milk from vaccinated cows increased phagocytosis of S. aureus by neutrophils. After the protein boost, sera were more efficient promoters of phagocytosis, reflecting the higher anti-ClfA antibody level of these sera. DNA-prime/protein boost regimes combined with molecular adjuvants appeared to be effective in generating a strong immune response to S. aureus antigens in cattle.

A new series of linear and permanently charged poly(amidoammonium) salts were synthesized in order to investigate the influence of their ionic and hydrophobic contents on both the cytotoxicity and the transfection mediated by polycation-DNA complexes. The poly(amidoammonium) salts were prepared by chemical modification of a parent poly(amidoamine) containing two tertiary amino groups per structural unit: one incorporated into the main chain and the other fixed at the end of a short bismethylene spacer. The permanent charges were introduced through a quaternization reaction involving iodomethane or 1-iodododecane as an alkylating agent. Under appropriate conditions, the methylation reaction was found to be regioselective, allowing the quaternization of either the side chains or both the side chains and the backbone. Under physiological salt conditions (150 mM NaCl), all of the poly(amidoammonium) salts self-assembled with DNA to form complexes. High proportions of highly quaternized polycation provided better defined morphology to the polycation-DNA complexes. Complexes formed from unquaternized polycation were less cytotoxic than branched poly(ethyleneimine) (25 kDa). At high polycation-DNA weight ratios, the introduction of permanent charges generated a significant increase in the cytotoxicity, but no patent correlation could be established with the amount and the position of the permanent charges. Only complexes formed from polycations with quaternized backbone were able to generate significant gene expression, which was putatively attributed to a better defined toroidal-like morphology together with a higher stability, as suggested by zeta potential measurements. The incorporation of dodecane side chains on highly charged polycations severely amplified the cytotoxicity so that, in return, the transfection level was dramatically affected.

Staphylococcus aureus intramammary colonization of the mouse mammary gland induces migration of polymorphonuclear neutrophils (PMNs) similar to that observed during bovine mastitis. In the present study, a method combining acridine orange staining, fluorescence microscopy and computer-assisted image analysis has been developed to quantitate PMN infiltration in a mouse model of mastitis. This was carried out using paraffin embedded sections, and using this method, we showed that the presence of PMNs increased with the number of bacteria present in tissues. Nearly 400 and 1100 times more PMNs were counted in the mammary gland tissue after 12 and 24 h of infection, respectively, compared to mice infected for 6 h. Treatment with the antibiotic cephapirin at 10 or 25 mg/kg reduced PMN infiltration by 71 and 85%, respectively. In conclusion, this method can be used to quantitate PMN infiltration as a marker of inflammation and bacterial burden in infected tissue sections.

The fibronectin binding protein (FnBP) and clumping factor A (ClfA) of Staphylococcus aureus are important proteins involved in the pathogenesis of staphylococcal bovine mastitis. These antigens were the targets of a DNA and protein vaccination strategy against S. aureus induced mastitis in dairy cows. The DNA vaccine comprised the bicistronic plasmid (pCI-D(1)D(3)-IRES-ClfA) that encoded the fusion of two sequences, (D1(21-34); D3(20-33)) from the fibronectin-binding motifs of FnBP and a fragment from ClfA (aa 221-550) of S. aureus 8325-4 separated by an Internal Ribosomal Entry Site (IRES) sequence. In addition, the vaccine contained the plasmid encoding the bovine granulocyte-macrophage-colony stimulatory factor gene (pCI-bGM-CSF). Four, 7-month pregnant heifers were immunized twice with the DNA vaccine and boosted once with recombinant D(1)D(3) and ClfA proteins while four others were not immunized. The immunization induced lymphoproliferative responses and functional antibodies against D(1)D(3) and ClfA antigens. Three weeks after calving, three mammary quarters of each vaccinated and non-vaccinated cow were challenged with 900 CFU/each of S. aureus Newbould 305. The fourth quarter received saline only. Serum haptoglobin levels, cardiac rhythm and the body temperature of vaccinated cows during the 24-72 h post-challenge were lower than in non-vaccinated animals. At 21 days post-challenge, bacteria were present in 5 of the vaccinated and 11 of the control challenged quarters. The bacteria averaged 1.4 and 3.3 log(10) CFU/ml of milk from vaccinated and control cows respectively. In summary, DNA-protein vaccination against FnBP and ClfA of S. aureus caused both lymphoproliferative and humoral immune responses that provided partial protection of mammary gland from staphylococcal mastitis and better post-challenge conditions in vaccinated cows.

Despite the general in vitro susceptibility of Staphylococcus aureus isolates that cause infectious bovine mastitis, the pathogen remains difficult to eradicate with the available antibiotics. The capacity to survive intracellularly has been proposed as a factor contributing to the persistence of S. aureus in the bovine mammary gland. The costs associated with the use of cows or goats to assess the in vivo efficacy of new antibacterial compounds constitute a major drawback. Therefore, in the present study, a mouse model of intramammary infection has been characterized for in vivo testing of new experimental drugs. An inoculum of 100 CFU of S. aureus per gland caused an important level of infection with minimal tissue damage as observed at 24 h post-inoculation. By microscopy, polymorphonuclear neutrophil cell infiltration of the infected mammary glands was observed to increase over time. At 12-24 h of infection, the pathogen was primarily found alive and dividing in neutrophils and occasionally within mammary epithelial cells. Intramuscular or intravenous injections of cephapirin at t = 0 and 10 h reduced the number of CFU/g of gland in a dose-dependent manner. In conclusion, the mouse model of infectious mastitis proposed here is suitable for primary evaluation of experimental drugs after parenteral treatment of intramammary infection with a pathogen such as S. aureus that presents both intracellular and extracellular phases of growth.

Staphylococcus aureus is the most significant bacterial pathogen associated with bovine mastitis. However, the relevance of intracellular infection to mastitis pathogenesis is poorly understood. We used in vitro assays and a mouse model of mastitis to demonstrate the intracellular component of the infection and to identify the importance of fibronectin-binding proteins in the processes of colonization and internalization. In vitro, a mutant strain, lacking fibronectin-binding protein (FnBPs(-)), had a reduced ability to bind fibronectin and to infect epithelial cells when compared to its parental wild type strain. After 2 h of infection, the internalization of the mutant bacteria into epithelial cell cultures was reduced by 60% compared with the wild type. After in vivo infection, microscopic examination using the FnBPs(-) strain revealed that production of a high density of live bacteria within the mammary gland epithelial cells was delayed. Both parental and mutant strains were identified within neutrophils, macrophages and epithelial cells suggesting a close similarity between the mouse mastitis model and bovine mastitis. These results demonstrate that S. aureus was able to cause an intracellular infection in the mouse model of mastitis and that the elimination of one adhesion protein delayed, but did not prevent, infection.

Antibiotics should combine good antibacterial activity and the capacity to work in association with the host defence system. In this study, we have investigated the effects of bovine lactoferrin alone or in combination with penicillin G on the phagocytic activity of bovine polymorphonuclear leukocytes against Staphylococcus aureus. We have shown that susceptibility of S. aureus to phagocytosis was decreased in the presence of penicillin in the medium. In a kinetic study, lactoferrin alone did not affect phagocytosis but, when used with penicillin, it reversed the negative effect of this antibiotic on phagocytosis. In addition, in an epithelial invasion assay, lactoferrin alone or in combination with penicillin reduced the invasion of mammary epithelial cells in culture by S. aureus. Lactating female CD-1 mice were infected by intra-mammary delivery of a virulent penicillin-susceptible S. aureus strain and were then randomly assigned to treatments according to a 2 x 2 factorial design. In this mouse mastitis model, 2 days of systemic treatments with lactoferrin and/or penicillin did not lead to a total clearance of infection by S. aureus, but bacterial number was significantly reduced by treatments with lactoferrin or penicillin. These data suggest that bovine lactoferrin, alone or in combination with penicillin G, enhances S. aureus susceptibility to immuno-defense mechanisms, which can be beneficial in the treatment of S. aureus infections.

The objectives of this study were to evaluate the effects of immunization site and antigen presenting cell targeting on cattle immune responses to DNA immunization. Cows were vaccinated with the plasmid expression vector pCI alone, pCI encoding the bacterial antigen beta-galactosidase (pCI-beta-gal) or pCI encoding bCTLA 4 fused to beta-gal (pCI-bCTLA-hIgG-beta-gal). The plasmids were delivered by intramuscular, intradermal, intramammary gland, or intra supramammary lymph node needle-injection. Both vaccines induced significant humoral and cellular immune responses. pCI-beta-gal elicited a higher IgG response than immunization with pCI-bCTLA-hIgG-beta-gal. Cows injected intramuscularly and intramammary had higher IgG and IgG-1 humoral responses than cows immunized intradermaly or in the lymph nodes. The injection site did not significantly affect the magnitude of the IgG2 and IgM antibody responses, although a trend similar to the IgG results was observed. The lymphocyte proliferation index was higher with pCI-beta-gal but was not affected by the injection site. These results suggest that in bovine, the injection site can affect immune responses but they do not provide evidence that bCTLA-4-hIgG-antigen targeting is effective in cattle.

An automated quantitative (AQ) assay was compared with radial growth on solid media and with dry weight in liquid culture for assaying fungicide sensitivity in Botrytis squamosa, the causal agent of onion leaf blight. Five isolates of B. squamosa were assayed for sensitivity to mancozeb (Dithane DG) and iprodione (Rovral) at five concentrations (0.5, 1.0, 5.0, 10.0, and 50 ppm). For mancozeb, the correlations between 50% effective concentration (EC 50 ) values obtained with the three assays were not significant; however for iprodione, correlations between EC 50 values for AQ and radial growth and for AQ and dry weight were significant (r = 0.98 and 0.99, respectively). The AQ method was less time consuming and more reliable than the two standard assays. The AQ method was used to evaluate the sensitivity of 35 field isolates of B. squamosa to mancozeb (Dithane DG), iprodione (Rovral), vinclozolin (Ronilan DF), and chlorothalonil (Bravo 500). All isolates were sensitive to mancozeb (EC 50 ranged from 3.36 to 12.97) and chlorothalonil (EC 50 < 1.5 μg/ml), but four isolates were insensitive to both iprodione (EC 50 ≥ 3.98 μg/ml) and vinclozolin (EC 50 ≥ 17.49 μg/ml). The ratio of the EC 50 values of the least-sensitive and the most-sensitive isolates of B. squamosa was 1.08, 3.86, 6.98, and 37.59 for chlorothalonil, mancozeb, iprodione, and vinclozolin, respectively. Cross-resistance was observed for the two dicarboximide fungicides, iprodione and vinclozolin, with a significant correlation (r = 0.94) in the sensitivity of the 35 isolates to these two fungicides.

The fibronectin-binding proteins (FnBPs) of Staphylococcus aureus are believed to be implicated in the pathogen's adherence to and colonization of bovine mammary glands, thus leading to infectious mastitis. In vitro studies have shown that FnBPs help the adhesion of the pathogen to bovine mammary epithelial cells. However, the importance of FnBPs for the infection of mammary glands has never been directly established in vivo. In this study with a mouse model of mastitis, the presence of FnBPs on the surface of S. aureus increased the capacity of the bacterium to colonize mammary glands under suckling pressure compared to that of a mutant lacking FnBPs.

Adhesins are considered the most important virulence factors during early phases Staphylococcus aureus infection. Antibodies induced by vaccination toward an adhesin should reduce the adherence of the pathogen and augment its phagocytosis. The present report describes the immune response of mice to a DNA vaccine directed against one of these adhesins, clumping factor A (ClfA). Injection of plasmids expressing the fibrinogen-binding region A of ClfA induced a strong and specific antibody response to ClfA in mice. In addition, splenocyte proliferation was provoked by in vitro stimulation with recombinant ClfA, thus, indicating direct implication of these cells in the immune response. Pre-incubation of S. aureus with sera of vaccinated mice reduced the pathogen's ability to bind fibrinogen by up to 92%. These pre-incubated bacteria were phagocytosed by macrophages at an increased level in vitro and were less virulent in vivo in a mouse mastitis model. However, DNA-immunized mice were not protected against an intraperitoneal challenge. Overall, the results suggest that DNA immunization against adhesins represents a new and valuable approach to combat S. aureus infections.

A new recombinant adenovirus was constructed that expressed the nucleocapsid (C protein or p14) of the bovine viral diarrhea virus (BVDV) under the control of a tetracycline-regulatable promoter. Mice covaccinated with this recombinant adenovirus, accompanied by another recombinant adenovirus expressing the trans-activator protein, induced a strong humoral immune response to the BVDV/C protein as detected by ELISA. Splenocytes from mice immunized with the recombinant adenovirus showed a specific proliferation response to both genotypes (type 1 and 2) of BVDV. High levels of IFN-gamma were detected in the supernatant of murine mononuclear cells of mice immunized by the recombinant adenovirus when stimulated in vitro by both genotypes of BVDV. These results indicate that this recombinant adenovirus is highly immunogenic and stimulates both cellular and humoral immune responses against the nucleocapsid of BVDV., (Copyright 1999 Academic Press.)

The E2 protein of bovine viral diarrhea virus (BVDV) is a major viral glycoprotein and an attractive target for BVDV vaccines. Three replication defective recombinant adenoviruses expressing the BVDV/E2 protein (rAds/E2) were constructed. Two contain a constitutive promoter, and one an inducible promoter. All three recombinant adenoviruses induced very strong BVDV specific antibody responses in a mouse model as detected by enzyme-linked immunosorbant assay (ELISA) and neutralization tests. Induction of cellular immune responses was investigated in two recombinant adenoviruses with a constitutive promoter. The mononuclear cells from the immunized mice demonstrated a proliferative response after in vitro stimulation with an homologous BVDV strain, but only one of them induced the production of IFN-gamma.

A recombinant fowlpox virus (rFPV/E2) expressing the E2 protein of bovine viral diarrhea virus (BVDV) was constructed and characterized. Mice were immunized with recombinant virus and both humoral and cellular immune responses were studied. rFPV/E2 induced BVDV-specific antibodies which were detected by ELISA. In addition, mouse sera were shown to neutralize BVDV. A cytokine ELISA assay revealed that mice vaccinated with rFPV/E2 induced 7-fold more interferon-gamma than parental fowlpox virus.

Two replication-defective human adenovirus recombinants encoding the NS3 protein (p80) of bovine viral diarrhea virus (BVDV) under the control of a modified adenovirus major later promoter (BM5), rAdBM5/NS3, and human cytomegalovirus promoter (CMV5), rAdCMV5/NS3, were constructed. These two recombinant adenoviruses were tested for their expression of the NS3 protein in vitro in three different cell lines and also in vivo for the induction of BVDV-specific immune responses in mice. The recombinant adenoviruses containing two different promoters induced different levels of humoral responses to the NS3 protein. The rAdBM5/NS3 was used to vaccinate mice in order to evaluate the ability of the NS3 protein in the induction of cellular immune responses. The rAdBM5/NS3 did not cause a stimulation of cell proliferation but caused a very strong increase in production of IFN-gamma in murine mononuclear cells stimulated in vivo by BVDV strains of genotype 1 and 2.

A general purpose reversible memory-binding transform (MBT) is developed, which uses a permutation transform technique to bind memory information to a transformed signal alphabet. The algorithm performs well in conjunction with a Huffman coder for both ordered sources, such as pixel intensities, and categorical sources, such as vector quantized codebook indices.

This study describes the establishment and characterization of an immortalized cell line derived from the pancreas of an adult H-2Kb-tsA58 transgenic mouse. These cells, designated IMPAN for IMmortalized PANcreatic cells, displayed a cobblestone appearance typical of confluent epithelial cells and a distinct polarity in the organization of their cytoplasmic organelles. Immunocytochemical studies revealed that all IMPAN cells stained positively for a wide range of markers characteristic of pancreatic acinar cells, namely the secretory products alpha-amylase, chymotrypsinogen, DNAse, the lectinlike secretory protein PAP (pancreatitis associated protein), and the zymogen granule membrane proteins GP-2 and gp300. They also stained positively for carbonic anhydrase II and cytokeratin 19, two proteins characteristic of pancreatic duct cells, as well as for rab3A, a small GTP-binding protein specifically localized in pancreatic islet cells. No reactivity was ever obtained with insulin antibodies. Taken together, these results show that the IMPAN cells exhibit a phenotype comparable to exocrine pancreatic acinar cells. However the expression of some proteins more specific to duct and islet cells make them similar to in vivo or in vitro growing acinar cells. The cell line should be a valuable model to study the mechanisms of growth, differentiation, and transformation of the exocrine pancreatic acinar cell.

A chimeric gene encoding mouse proinsulin fused to the transmembrane and the cytoplasmic domains of the CD5 antigen of human T lymphocytes was expressed in AtT-20 cells to assess the relative strength of signals that influence the sorting of secretory proteins to the regulated or constitutive pathway in endocrine cells. Transfected cells expressing the antigen at the surface were purified by fluorescence-activated cell sorting and analyzed by Northern and Western blots. They contained a mRNA of 1.4 kb hybridizing with an insulin cDNA probe and two immunoreactive insulin forms of 21 and 24 kDa, recognizable by antibodies against both insulin and C peptide. The surface density of these antigens was not increased following KCl stimulation of the cells, suggesting that they were not stored within the cells in significant amounts. This was confirmed by immunoelectron microscopy which showed the antigen attached to membranes, in the Golgi, in endosomes, and at the cell surface, but not in secretory granules. These results indicate that the proinsulin-CD5 fusion protein was transported to the cell surface via the constitutive pathway and partly recycled by endocytosis. They also suggest that the signals that direct proinsulin into storage granules may no longer be dominant when fused to transmembrane and cytosolic sequences derived from a constitutively secreted molecule.

Plastination permits the preservation of anatomical specimens in a physical state approaching that of the living condition. We studied the possibility of using silicone plastinated fragments of spleen and pancreas for optical and electron microscopy, and found that with an adequate fixation protocol, plastinated specimens can be used for both light microscopy and ultrastructural studies. Deplastination with sodium methoxide permitted production of clean sections. Artifacts produced by plastination/deplastination could be nearly eliminated by glutaraldehyde/formaldehyde fixation. The (Biodur) silicone S10 polymer is transparent and stable in an electron beam, and plastinated tissues can be contrasted or colored similar to tissues embedded in Epon 812. In addition to being very life-like, plastinated tissues are stable and easy to handle. They can also be used for electron and light microscopic studies. This technique may also allow retrospective epidemiological studies of archived pathology specimens.

Porcine respiratory coronavirus (PRCV) was identified for the first time in Quebec, using a blocking enzyme-linked immunosorbent assay (ELISA). Unlike the virus neutralization test (VNT), this ELISA was able to distinguish transmissible gastroenteritis virus (TGEV) from PRCV. Among the 15 seropositive fattening herds from group A, sera containing PRCV antibodies represented 74.8%, whereas those with TGEV antibodies represented only 7.2%. In group B, which consisted of 15 sow herds, nine herds expressed only PRCV-specific antibodies while the other herds had animals positive for TGEV-specific antibodies.

This study describes, for the first time, the production and use of an "internal-image" anti-idiotypic monoclonal antibody (MAb) to elicit a rotavirus-specific antibody response. An immunoglobulin G2a MAb, designated RQ31 (MAb1), specific for the outer capsid protein VP4 of bovine Q17 rotavirus and capable of neutralizing viral infection in vitro was used to generate an anti-idiotypic MAb (MAb2). This MAb2, designated RQA2, was selected by enzyme-linked immunosorbent assay (ELISA) using F(ab')2 fragments of RQ31. RQA2 (MAb2) inhibited the binding of RQ31 (MAb1) to the virus but had no effect on the binding of other rotavirus-specific MAbs. The MAb2 also inhibited virus neutralization mediated by MAb1 in a dose-dependent fashion. Naive guinea pigs immunized with the MAb2 produced anti-anti-idiotypic antibodies (Ab3) that reacted with bovine Q17 rotavirus in an ELISA and neutralized rotavirus infection in vitro. The Ab3 response was characterized as MAb1-like because the Ab3 recognizes only the Q17 and neonatal calf diarrhea virus rotavirus strains in ELISA, as did RQ31 (MAb1). The Ab3 response also possessed two other characteristics of RQ31: the abilities to bind the 1.36 (double-capsid) but not the 1.38 (single-capsid) purified rotavirus fraction in ELISA and to immunoprecipitate the VP4 rotavirus protein.

A pretreatment for organophosphorus (OP) anticholinesterase (e.g., soman) intoxication should prevent lethality and convulsions (CNV) at 2 LD50s and be behavioral-decrement-free when given alone. Behavioral-deficit-free pretreatment regimens (PRGs) for guinea pigs consisted of Physostigmine (0.15 mg/kg, im) and adjunct. Adjuncts [mg/kg, im] tested were akineton [0.25], aprophen [8], trihexyphenidyl [2], atropine [16], azaprophen [5], benactyzine [1.25], cogentin [4], dextromethorphan [7.5], ethopropazine [12], kemadrin [1], memantine [5], promethazine [5], scopolamine [0.08] and vontrol [2]. PRGs were given 30 min before soman (60 micrograms/kg, sc; 2 LD50s) or other OP agents. Animals were then observed and graded for signs of intoxication, including CNV at 7 time points and at 24 hr. Physostigmine alone reduced the incidence of CNV and lethality induced by 2 LD50s of soman by 42 and 60%, respectively. All of the PRGs tested abolished lethality and 12 shortened recovery time to 2 hr or less. Also, PRGs including azaprophen or atropine prevented CNV. When selected PRGs were tested against intoxication by sarin, tabun or VX, the efficacy was generally superior to that for soman. The data show that several PRGs are effective against soman intoxication in guinea pigs.

A high-performance liquid chromatographic (HPLC) assay was developed to determine HI-6 (1-(2-hydroxyiminomethyl-1-pyridinio-3-(4-carbamoyl-1-py ridiniol-2-oxapropane dichloride)) concentrations in small volumes of plasma. A 100-microL plasma sample added to 900 microL of distilled water was microfiltered. Filtrate (200 microL) was injected onto an HPLC instrument containing a 100-microL sample loop, a C18 column, and an ultraviolet (UV) wavelength detector. Limit of sensitivity for HI-6 was 2.5 micrograms/mL. Extraction of efficiency (n = 12) at 10 and 100 micrograms HI-6/mL plasma was 69.4 +/- 6.6% (SD) and 81.5 +/- 2.0% (SD), respectively. Protein-plasma binding of HI-6 did not occur. HI-6 was stable when frozen at -20 degrees C for up to 10 days (0.025 less than p less than 0.05). Correlation coefficients representing standard curve linearity ranged from 0.9986 to 0.9999 (n = 6). Within-day and between-day coefficients of variation (n = 6) for unknown samples ranged from 4.4 to 8.3% and 5.8 to 17.1%, respectively. Bias of unknown samples ranged from -10.5 to 5.7%. The method's sensitivity, accuracy, and precision indicate that it can be used to accurately measure HI-6 concentrations in 100 microL of plasma.

A pretreatment combination of physostigmine and azaprophen (6-methyl-6-azabicyclo[3.2.1]octan-3-ol-2,2-diphenylpropionate), a novel cholinolytic, was evaluated for its ability to minimize soman-induced incapacitation and lethality in guinea pigs. This was accomplished by using response surface methodology to model and analyze the combination, varying physostigmine from 0 to 194 micrograms/kg, azaprophen from 0 to 5 mg/kg, and soman from 30 to 150 micrograms/kg. One hundred percent survival was achieved against 5 LD50 of soman using as little as 100 micrograms/kg of physostigmine in the presence of 5 mg/kg azaprophen. Both survival and soman-induced incapacitation were similarly affected by this pretreatment combination. For both endpoints, greater efficacy was achieved with the combination than could be achieved with either component alone (therapeutic synergism). This suggests that such a pretreatment combination may prove very efficacious against soman-induced lethality and incapacitation in higher species.

Physostigmine (PHY) has the advantage over pyridostigmine of minimizing OP-induced incapacitation because it penetrates into the CNS. However, physostigmine is behaviorally toxic at relatively low concentrations. It is anticipated that this could be offset by a cholinolytic to prevent behavioral deficit due to the carbamate pretreatment alone. The therapeutic efficacy of physostigmine/azaprophen pretreatment therapy was evaluated in soman-challenged guinea pigs. Response surface methodology was employed to describe the relationship of the pretreatment combination with duration of incapacitation. The significance of the combination relative to PHY alone was evaluated in addition to dose combinations that yield optimal time to recovery. Analysis of the fitted response surface indicated that combination pretreatment with these compounds significantly reduces the time to recovery after soman challenge versus pretreatment with PHY alone.

Three anti-horse liver alcohol dehydrogenase (HLADH) monoclonal antibodies are described. Two are specific for ADH and cross-react with class I and II enzymes from mouse, horse and Chinese hamster. They are specific for the native enzyme but do not inhibit enzyme activity except when combined at high concentration. The third antibody was isolated as a response to rabbit metallothionein. It binds metalloproteins and inhibits ADH activity.

Alcohol dehydrogenases (alcohol: NAD oxidoreductase, E.C. 1.1.1.1.) from allyl alcohol-resistant and wild-type Chinese hamster cells were purified using gel filtration, ion-exchange, and affinity-column chromatography. Both enzymes exhibited the same isozyme band patterns on electrophoresis and isoelectric focusing. Physicochemical properties of the two enzymes such as pH and temperature optima, Km values, and temperature stability were found to be the same within the experimental errors. The genetic significance of these findings is discussed.

Two in vitro proteinase assay systems were developed and used to study the peptide bond specificity and substrate specificity of the adenovirus endoproteinase. Five adenovirus precursor proteins (PVI, PVII, PVIII, 87K, 11K), all found in the virion of the ts1 mutant grown at the nonpermissive temperature, were digested by the proteinase. All, except 11K, were cleaved to their mature counterparts. Some of the proteins, particularly the 87K terminal protein, were processed via cleavage intermediates similar to those found in vivo. The data suggest that the proteinase specifically hydrolyses Gly-Ala bonds. The high specificity for the natural substrates and the failure to cleave foreign proteins suggest that cleavage activity is determined not only by primary sequence but also by other physical features of the substrate. Enzyme activity was inhibited by diisopropylfluorophosphate, showing that it is a serine proteinase.

The generally accepted explanation for the effects of oximes in countering organophosphorus (OP) anticholinesterase is reactivation of the inhibited acetylcholinesterase (AChE). With soman, the inhibited AChE rapidly becomes resistant to oxime reactivation due to a phenomenon called aging. Thus, pretreatment with pyridostigmine (Py) or physostigmine (Ph) followed by atropine sulfate therapy is required to achieve significant protection against soman; the effectiveness of a pretreatment/therapy (P/T) regimen can be further increased against certain OPs (e.g. sarin and VX) by including an oxime in the therapy regimen. The P/T regimen is clouded by a controversy concerning the use of oximes in the treatment of carbamate intoxication, because 2-PAM has been reported to exacerbate intoxication by some carbamates and to have no effect on decarbamylation rates. To better understand the role of oxime therapy in the theory of pretreatment of OP intoxication we examined the effects of 2-PAM and HI-6 on the rate of decarbamylation of Py-inhibited erythrocyte AChE in vitro and in vivo, and studied the effects of atropine plus 2-PAM or HI-6 on Py toxicity. In decarbamylation experiments, Py-inhibited guinea pig erythrocytes were washed free of excess Py and incubated with vehicle or oxime (2 X 10(-4) M, pH 7.3 and 37 degrees C). Aliquots were assayed for AChE activity at various times during a 60 min incubation period. Rate constants were calculated and compared to determine whether the presence of oxime affected decarbamylation. The data from in vitro and in vivo experiments revealed that oximes accelerated the decarbamylation (p less than 0.05) of inhibited AChE. Lethality data for Py-treated guinea pigs showed that treatment with atropine (23 mumoles/kg, im) plus 2-PAM or HI-6 (145 mumoles/kg, im) at one min after injection of Py increased the protective ratio from 4.2 (atropine only) to 5.1 and 12.2, respectively. It is suggested that the enhanced therapeutic efficacy of atropine by oximes against Py intoxication is related to oxime-induced reactivation.

This investigation was conducted to assess the potential of carbamate pretreatment to exacerbate the ill effects of low doses of soman. Ambulatory Activity in a photocell cage (AA) and performance time on an accelerating rotarod (ARR) were used to test for interactions between pyridostigmine or physostigmine and soman. ED50s (i.e., dosages sufficient to reduce ARR time and AA to 50% of control level) of each carbamate (IM) and soman (SC) were determined. The ED50 values (mg/kg) in the ARR test were 3.2, 0.21, and 0.072 for pyridostigmine, physostigmine and soman, respectively, while in the AA test the corresponding values were 1.8, 0.072 and 0.060. The matrix of 16 combinations of 0, 1, 2/3, and 1/3 ED50 each of carbamate and soman was studied in each test system, as well as the effect of behavioral deficit free (BDF) dosages of each carbamate on the ED50s of soman. In both the AA and ARR tests the matrix of combinations of pyridostigmine and soman indicated an additive effect. In contrast, physostigmine produced one instance of potentiation in each test system and anatagonism in two combinations in the AA procedure. A BDF dosage of each carbamate (0.056 mg/kg of pyridostigmine and 0.026 mg/kg of physostigmine) gave no evidence of adding to the deficit in AA induced by soman. In the ARR test, the ED50 of soman was lower by 11% with pyridostigmine pretreatment and by 14% with physostigmine; the latter just reached statistical significance (p less than 0.05). Although additivity was most often found at higher dosages of pyridostigmine and physostigmine, at the BDF dosages little or no adverse interaction was found between Pyridostigmine or Physostigmine and low levels of soman.

Monoclonal antibodies against rabbit metallothioneins (MT) were prepared by in vitro immunization of mouse lymphocytes with a mixture of the two forms of metallothionein MT1 and MT2. Six IgM antibodies (TN1,3,4,5,6,7) which bind to metallothionein were characterized. Antibody TN3 is specific for rabbit MT1 and does not react with any other MT's tested. TN5 is specific for both rabbit MT1 and MT2. TN7 is specific for rabbit MT2 but not MT1 and cross-reacts also with Chinese hamster, mouse and rat metallothioneins. The antibodies TN1, TN4 and TN6 bind not only to rabbit MT1 and MT2 but also to other metal binding proteins like alcohol dehydrogenase and carbonic anhydrase.

A competition enzyme-linked immunosorbent assay has been developed for the quantitative detection of soluble and membrane-bound GP-2, a glycoprotein which is confined to the exocrine pancreas. Zymogen granule membranes fixed to microtiter plates with poly-L-lysine were used as the source of antigen. Detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS] was added to the assay in order to reveal all the antigens, more particularly in membranous samples. Presence of detergent at concentrations as high as 0.5% did not interfere with any particular steps of the ELISA. This competition ELISA can detect 10 ng of GP-2 and will be useful for measuring soluble as well as membrane GP-2 in order to elucidate its role in the secretory process of the pancreas as well as in certain pathologies such as cystic fibrosis.

The efficacy of the oximes pyridinium-2-aldoxime methochloride (2-PAM) and 1-[[[(4-aminocarbonyl)pyridinio]methoxy]methyl]-2-[(hydro xyimino) methyl]pyridinium dichloride (HI-6), in combination with atropine (At), against lethality by either carbaryl (CA) or physostigmine (Phy) was investigated in rats. The protection by At, 8 mg/kg, iv, against CA intoxication was reduced by 2-PAM (22 mg/kg, iv) and HI-6 (50 mg/kg, iv) from a protective ratio (PR) of 6.6 to 3.5 and 2.3, respectively. However, in Phy-intoxicated rats, the administration, iv, of At alone, At + 2-PAM, or At + HI-6 at 1 min following Phy provided good protection and resulted in PRs of 7.2, 8.8, and 23.3, respectively. In experiments on decarbamylation of inhibited acetylcholinesterase (AChE), HI-6 and 2-PAM accelerated (p less than 0.05) the decarbamylation of Phy-inhibited AChE in vitro, and HI-6 decreased (p less than 0.05) the inhibition of whole blood AChE in Phy-intoxicated rats. These findings show that the protection was increased substantially by the use of either 2-PAM or HI-6 against Phy-induced lethality, whereas the use of oximes against carbaryl poisoning was contraindicated. Furthermore, even though CA and Phy are both N-methyl carbamates, the data indicate that there is no adverse interaction between 2-PAM or HI-6 and Phy.

The pharmacokinetics of pralidoxime chloride (2-PAM) was studied in rats. Different groups of rats were given an intramuscular injection of 2-PAM at one of three doses (20, 40, or 80 mg/kg). This range of doses is used commonly in studies concerned with the efficacy of 2-PAM against poisoning by potent organophosphorus inhibitors of cholinesterase enzyme. Individual, sequential blood samples were collected during the course of the experiment. From these blood samples the plasma concentrations of 2-PAM were determined over time for each animal. Next the relationship of plasma concentration to time was expressed in terms of a standard pharmacokinetic model. Estimates of various pharmacokinetic parameters were calculated using an open, one-compartment model: volume of distribution (Vd), maximal plasma concentration (Cmax), elimination rate constant (k10), absorption rate constant (k01), area under the curve (AUC) and clearance (CL). Of the pharmacokinetic estimates, only Cmax and AUC were found to be statistically significant (p less than 0.0001) when compared across all the doses; these pharmacokinetic estimates were highly correlated with doses with r = 0.998 and r = 0.997, respectively. However, when AUC and Cmax were normalized by dividing through by dose, no significant differences were found in the transformed data. The results of this study in rat indicate that the pharmacokinetics of 2-PAM is linearly related to dose in a range employed in therapeutic studies of 2-PAM.