Effect of an extruded pea or rice diet on postprandial insulin and cardiovascular responses in dogs.

Peas are increasing in popularity as a source of carbohydrate, protein and fibre in extruded canine diets. The aim of this study was to test the health effects of two canine diets with identical macronutrient profiles, but containing either yellow field peas or white rice as the carbohydrate source on metabolism, cardiovascular outcomes and adiposity. First, the acute glycemic, insulinemic and cardiovascular responses to the pea- or rice-based diets were determined in normal weight beagles (n = 7 dogs). The glycemic index did not differ between the pea diet (56 ± 12) and rice diet (63 ± 9). Next, obese beagles (n = 9) were fed the yellow field pea diet or white rice diet ad libitum for 12 weeks in a crossover study. Adiposity (measured using computed tomography), metabolic (oral glucose tolerance test, plasma leptin, adiponectin, C-reactive protein) and cardiovascular assessments (echocardiography and blood pressure) were performed before and after each crossover study period. After 12 weeks on each diet, peak insulin (p = 0.05) and area under the curve (AUC) for insulin after a 10 g oral glucose tolerance test (p = 0.05) were lower with the pea than the rice diet. Diet did not show a significant effect on body weight, fat distribution, cardiovascular variables, adiponectin or leptin. In conclusion, a diet containing yellow field peas reduced the postprandial insulin response after glucose challenge in dogs despite continued obesity, indicating improved metabolic health.

Effects of dietary supplementation of coriander oil, in canola oil diets, on the metabolism of [1-(14)C] 18:3n-3 and [1-(14)C] 18:2n-6 in rainbow trout hepatocytes.

The aim of this study was to investigate the effects of petroselinic acid, found in coriander oil, on the ability of rainbow trout hepatocytes to increase the production of eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA) from [1-(14)C] α-linolenic acid (18:3n-3; ALA) and to reduce the production of arachidonic acid (20:4n-6; ARA) from [1-(14)C] 18:2n-6. Addition of coriander oil increased the production of 22:6n-3, from [1-(14)C] 18:3n-3, at the 0.5 and 1.0% inclusion levels and reduced the conversion of [1-(14)C] 18:2n-6 to 20:4n-6. ß-Oxidation was significantly increased at the 1.5% inclusion level for [1-(14)C] 18:2n-6, however ß-oxidation for [1-(14)C] 18:3n-3 only showed an increasing trend. Acetate, a main breakdown product of fatty acids (FA) via peroxisomal ß-oxidation, decreased three-fold for [1-(14)C] 18:2n-6 and nearly doubled for [1-(14)C] 18:3n-3 when coriander was added at a 1.5% inclusion level. Acyl coenzyme A oxidase (ACO) enzyme activity showed no significant differences between treatments. Relative gene expression of ∆6 desaturase decreased with addition of coriander oil compared to the control. The addition of petroselinic acid via coriander oil to vegetable oil (VO) based diets containing no fishmeal (FM) or fish oil (FO), significantly increased the production of anti-inflammatory precursor 22:6n-3 (P=0.011) and decreased pro-inflammatory precursor 20:4n-6 (P=0.023) in radiolabelled hepatocytes of rainbow trout.

Glycemic index of starch affects nitrogen retention in grower pigs.

Three studies were performed to examine the effect of starch and protein digestion rates on N retention in grower pigs. In Exp. 1, the glycemic index (GI) of corn, a malting barley, and a slow-rumen-degradable barley (SRD-barley) were measured using 6 barrows (BW = 18.0 ± 0.5 kg). The GI of malting barley was greater (P < 0.05) than that of SRD-barley (71.1 vs. 49.4), and the GI of both barley cultivars was less (P < 0.05) than that of corn (104.8). In Exp. 2, the standardized ileal digestibility of AA and DE content of the 3 ingredients were determined using 5 ileal-cannulated barrows (BW = 20.7 ± 2.3). The apparent total-tract energy digestibility values of corn (86.1%) and malting barley (85.7%) were greater (P < 0.05) than that of SRD-barley (82.3%). The standardized ileal digestibility of Lys was 94.0, 92.6, and 92.4% for corn, malting barley, and SRD-barley, respectively, and did not differ among grains. In Exp. 3, 6 diets were formulated to equal DE (3.40 Mcal/kg), standardized ileal digestibility of Lys (8.6 g/kg), starch (424.9 g/kg), and digestible CP (180.0 g/kg) using the values obtained in Exp. 2. Three GI [high (corn), medium (malting barley), and low (SRD-barley)] and 2 rates of protein digestion [rapid (soy protein hydrolysate) and slow (soy protein isolate)] were tested in a 3 × 2 factorial arrangement with 36 barrows (BW = 32.2 ± 2.5 kg). Pigs were fed 3.0 times the maintenance energy requirement daily in 2 meals for 2 wk and were housed in metabolic crates to collect feces and urine separately. At the end of the study, intestinal contents were collected from 4 equal-length segments of the small intestine. The percentage of unabsorbed CP in segment 1 relative to dietary CP was greater (P < 0.05) for the soy protein isolate diet than for the soy protein hydrolysate diet (170.3 vs. 116.5%). The percentages of unabsorbed starch in segments 1 and 2 were greater (P < 0.05) for the SRD-barley diet than for the malting barley or corn diet. Nitrogen intake and fecal N excretion were greater (P < 0.05) for pigs fed the malting barley and SRD-barley diets than for pigs fed the corn diet. Urinary N excretion was greater (P < 0.05) for pigs fed the SRD-barley diet than for pigs fed the corn or malting barley diet. Pigs fed slowly digestible starch (SRD-barley; 46.6%) had less (P < 0.05) net N retention than pigs fed corn or malting barley (54.7 and 54.1%, respectively). In conclusion, slowly digestible starch sources such as SRD-barley may not be suitable to support maximum protein deposition in restricted-fed grower pigs.

Effects of protein level and digestibility on the growth and carcass characteristics of broiler chickens.

A study was conducted to evaluate the effects of dietary protein level and protein digestibility on the growth performance and carcass characteristics of broilers from 1 to 35 d of age. Broiler chickens (n = 320) were fed 4 different ideal protein-balanced, isocaloric diets in a 2 × 2 factorial design with 2 levels of protein [high protein (HiPro; 20 and 18% or 200 and 180 g/kg) and low protein (LoPro; 18 and 16% or 180 and 160 g/kg) on d 1 to 14 and d 15 to 35, respectively] and 2 levels of protein digestibility [high digestibility (HiDig) and low digestibility (LoDig); approximately 85% and 80% CP digestibility, respectively]. The HiDig diets were formulated using soybean meal and fishmeal, whereas the LoDig diets used wheat distillers dried grains with solubles and meat and bone meal as the primary protein sources. The standardized ileal digestibility (SID) values of the wheat distillers dried grains with solubles and meat and bone meal (56.5 and 72.0% SID for lysine, respectively) were measured before the experiment to improve the accuracy of the diet formulations. During the starter phase, the interaction was significant for ADG; birds fed the LoPro-LoDig diet grew slower than birds fed the other 3 diets (P < 0.05). During the grower phase, the interaction was significant for ADFI; birds fed the LoPro-LoDig diet had the lowest ADFI compared with those fed the other 3 diets. The interaction between protein level and digestibility was significant for the SID of most of the AA and was significantly higher for birds fed the HiPro-HiDig diet compared with those fed the other 3 diets. Total breast meat yield was significantly higher in birds fed the HiPro diets than in those fed the LoPro diets, whereas birds fed the HiDig diets had significantly more abdominal fat than those fed the LoDig diets. The results suggest that low-protein diets can support growth performance equal to high-protein diets when highly digestible ingredients are used. However, maximum breast meat yield requires a high-protein diet and is not affected by ingredient digestibility.

Whole blood chemiluminescence response in broiler chickens on different experimental diets and challenged with Clostridium perfringens.

UNLABELLED: 1. OBJECTIVES: To validate the whole blood chemiluminescence (WBCL) assay in chickens, a simple and rapid method of measuring production of reactive oxygen species by circulating polymorphonuclear (PMN) leukocytes. To determine the physiological response and innate immune response associated with oral challenge with Clostridium perfringens in broiler chickens under different nutritional conditions. 2. In Experiment 1, birds were orally challenged with C. perfringens 1. type A or sham-challenged saline on days 14-21 post-hatch and fed protein-balanced diets containing 160 or 180 g crude protein/kg and 0.98 or 1.75% glycine in a 2 x 2 x 2 factorial arrangement of treatments. 2. Challenged birds had higher WBCL responses and more severe intestinal lesions than unchallenged birds. Birds fed diets containing 1.75% glycine had more intestinal lesions than those fed 0.98% glycine. 3. In Experiment 2 birds were fed protein-balanced diets containing 0.76, 2.10, 3.43 or 4.77% glycine. The birds fed 0.76% glycine diet had lower WBCL responses compared to birds fed the other three diets. Intestinal lesions were worse in the birds fed the highest, 4.77% glycine diet than in those fed the 0.76 or 2.10% glycine diets. 4. We conclude that the WBCL assay is a practical and sensitive means of assessing innate immune function in birds. The results suggest that both bacterial challenge and glycine content of chickens' diet influence their lesion scores and innate immune function.

Apparent absorption of methionine and 2-hydroxy-4-methylthiobutanoic acid from gastrointestinal tract of conventional and gnotobiotic pigs.

The effect of commensal microbiota and feeding corn or wheat/barley-based diets on the apparent gastrointestinal absorption of dl-methionine (MET) and 2-hydroxy-4-methylthiobutanoic acid (MHA-FA) was studied in conventional (n = 32) and gnotobiotic pigs (n = 24). Conventional pigs (CON) were vaginally delivered and sow-reared until weaning at 14 days of age. Gnotobiotic pigs were derived by caesarian section and reared in HEPA (high efficiency particulate air)-filtered isolator units with ad libitum access to a milk-based formula. Corn or wheat/barley-based diets were fed to all pigs from 14 to 24 days of age. At 24 days of age, after an overnight fast, pigs were fed 20 g/kg BW of experimental diet supplemented with 107 Bq of either 3H-l-MET or 3H-l-MHA-FA per kg of feed and chromic oxide (0.5% wt/wt). Pigs were killed for sample collection 3 h after consuming the meal. Residual 3H-MET and 3H-MHA-FA were estimated in gastrointestinal contents as the ratio of 3H : chromic oxide in digesta samples to the ratio of 3H : chromic oxide in feed. In CON pigs, feeding a wheat/barley-based diet increased (P < 0.05) total aerobes, whereas supplementation with MHA-FA increased (P < 0.05) total aerobes and lactobacilli populations in proximal small intestine (SI). Among the gnotobiotic pigs, bacterial contamination occurred such that eight pigs (two isolators) were monoassociated with a Gram-negative bacteria closely related to Providencia spp. and 16 pigs (four isolators) were monoassociated with Gram positive Enterococcus faecium. Species of monoassociated bacterial contaminant and diet composition did not affect residual methionine or MHA-FA in digesta. In both CON and monoassociated (MA) pigs, methionine and MHA-FA were retained in stomach (92%) but disappeared rapidly from proximal SI. Residual methionine and MHA-FA in digesta was not different in MA pigs; however, in CON pigs, less (P < 0.01) apparent residual methionine was found in digesta recovered at 25% (from cranial to caudal) and 75% of SI length compared with MHA-FA. Apparent residual methionine was 16% and 8% compared with 34% and 15% for MHA-FA, at the 25% and 75% locations, respectively. In proximal SI tissue, significantly (P < 0.05) higher radioactivity (cpm/mg wet tissue) was associated with MET pigs (8.56 ± 0.47) as compared to MHA-FA ones (5.45 ± 0.50). This study suggests that microbial metabolism of MHA-FA increases retention in small intestinal digesta relative to methionine and contributes, in part, to the lower bioefficacy of MHA-FA compared to methionine.

Effect of different dietary methionine sources on intestinal microbial populations in broiler chickens.

Two experiments were conducted to study the effect of various levels of DL-Met or 2-hydroxy-4-methylthiobutanoic acid (MHA-FA) on Clostridium perfringens and other intestinal bacteria in broiler chickens. In each experiment, 2 cages of 6 birds (14 d posthatch) were assigned to 1 of 7 different diets in a 2 x 4 factorial arrangement. The main effects were Met source (either DL-Met or MHA-FA) and Met level (0, 0.2, 0.4, or 0.8% dl-Met or 0, 0.227, 0.454, and 0.908% MHA-FA, thus providing 4 corresponding equimolar levels of each Met source). All birds were orally gavaged with a C. perfringens type A broth culture on d 1 and on d 14 to 20 and killed on d 28. Intestinal populations of C. perfringens, lactobacilli, Streptococcus group D, and coliforms were enumerated in the ileum and cecum, and necrotic enteritis intestinal lesions were scored. There was a significant reduction (P < 0.05) in C. perfringens populations in birds fed either Met source in the cecum (experiment 1) or the ileum and cecum (experiment 2). In experiment 2, the lactobacillus populations were significantly higher (P < 0.05) in the ceca of birds receiving 0.8% Met than in the birds given diets with the other levels of Met tested. Significantly lower populations (P < 0.05) of coliforms and Streptococcus group D were enumerated in the ileum of birds fed the 0.8% Met-supplemented diet than in the other dietary treatments. The effect of Met source on intestinal bacteria was not significant, suggesting that both DL-Met and MHA-FA have similar antibacterial properties. Last, there were no significant differences in intestinal lesion scores or the performance of birds fed different Met sources and concentrations. The results suggest that both DL-Met and MHA-FA may reduce intestinal populations of C. perfringens in broiler chickens when used in relatively high concentrations, and may reduce the risk of necrotic enteritis. Thus, feeding low-protein diets supplemented with crystalline amino acids might be beneficial in terms of the growth of various enteric pathogens.

Effects of commensal bacteria on intestinal morphology and expression of proinflammatory cytokines in the gnotobiotic pig.

A germ-free neonatal pig model was established to determine the effects of bacterial colonization by different species on small intestinal morphology and proinflammatory cytokine gene expression. Two experimental groups of 16 pigs were aseptically delivered by cesarian section and allocated into 4 gnotobiotic isolators. Pigs were either maintained germ-free (GF), or were orally inoculated with either a single strain of nonpathogenic Escherichia coli (EC) or Lactobacillus fermentum (LF) or conventionalized with adult porcine feces (CV). After 13 days tissue samples were collected at 5 regions corresponding to 5%, 25%, 50%, 75%, and 95% of the small intestine (SI) length. In Experiment 2, the GF isolator became contaminated with Staphylococcus epidermidis (SE). In general, intestinal responses to bacterial colonization were similar among GF, LF, and SE pigs, and intestinal responses in EC pigs were more similar to CV pigs. Responses to bacterial colonization were most pronounced in the distal SI regions (50%-95%), suggesting that nonmicrobial factors may be more important in the proximal SI. Relative to CV pigs, the distal intestines of GF, LF, and SE pigs were characterized by long villi, shallow crypts, increased relative intestinal mass, and decreased lamina propria cellularity, whereas SI morphology was intermediate in EC pigs. Relative expression of proinflammatory cytokines interleukin-1beta (IL-1beta ) and IL-6 generally increased distally in the SI and was highest in EC and CV pigs. We observed regional variation in SI morphology and proinflammatory cytokine expression, which differed with bacterial species. This study demonstrates that bacterial species differentially affect intestinal morphology and expression of proinflammatory cytokines and suggests that neonatal bacterial colonization patterns may have long-term effects on intestinal health and development.

The effect of hen-egg antibodies on Clostridium perfringens colonization in the gastrointestinal tract of broiler chickens.

We evaluated the ability of hen-egg antibodies (HEA) to reduce intestinal colonization by Clostridium perfringens in broiler chickens. Antibodies against C. perfringens or cholera toxin (negative control) were obtained from the eggs of laying hens hyperimmunized using a C. perfringens bacterin or cholera toxin. Eggs were collected, pooled, and egg antibodies were concentrated by polyethylene-glycol precipitation. An initial experiment was conducted to determine the in vivo activity of the administered antibody along the length of the intestine. Thereafter, two feeding trials were performed to assess the efficacy of feed amended with the egg antibodies in reducing the level of colonization of C. perfringens in challenged birds. Antibody activity declined from proximal to distal regions of the intestine but remained detectable in the cecum. In the first experiment there was no significant reduction in the number of C. perfringens in the birds fed the diet amended with the anti-C. perfringens egg antibody, compared to the birds that received the anti-cholera toxin egg antibody (n=10), at any of the sampling times. In the second experiment there was a significant decrease in C. perfringens intestinal populations 72 h after treatment (n=15) as assessed by culture-based enumeration, but there was no decrease as measured by quantitative PCR based on the C. perfringens phospholipase C gene. Intestinal-lesion scores were higher in the birds that received the anti-C. perfringens HEA. Our work suggests that administration of HEA did not reduce the level of C. perfringens intestinal colonization and conversely might exacerbate necrotic enteritis.

Responses of broiler chickens orally challenged with Clostridium perfringens isolated from field cases of necrotic enteritis.

The present study examines the responses of broiler chickens to oral administration of Clostridium perfringens freshly isolated from field cases of necrotic enteritis (NE). The challenge studies included long-term exposure and short-term exposure, factored in with dietary and management variables including high levels of dietary components such as fish meal, meat meal, abrupt change of feed, and fasting. In the long-term exposure trials, the birds were orally inoculated daily, with 1 ml (1.0 or 2 x 10(8) CFU/ml) of an overnight culture of C. perfringens for 7 days. Short-term exposure trials involved challenge with 1 ml (3 x 10(10) CFU/ml) administered as a single dose. The responses of broilers to orally administered C. perfingens under laboratory controlled conditions are presented and discussed in the context of authentic field cases of necrotic enteritis. None of the challenge trials produced overt clinical signs of NE and there were no mortalities associated with oral exposure to high doses of C. perfringens. However, many of the challenged birds showed distinctly pronounced pathological changes in the intestinal tissue. On gross examination the responses in birds challenged orally with C. perfringens could be placed into two categories: (1) no apparent pathological changes in the intestinal tissue and (2) sub-clinical inflammatory responses with focal, multi-focal, locally extensive, or disseminated distribution throughout various sections of duodenum, jejunum, ileum, and ceca. In birds that responded with intestinal lesions, hyperemia and occasional hemorrhages were the main gross changes. In some birds, the mucosa was covered with a brownish material, but typically, the mucosa was lined by yellow or greenish, loosely adherent material. Mild gross changes were seen in some control birds, but both qualitatively and quantitatively, the lesions were distinctly more pronounced in the challenged birds. Upon histological examination, none of the experimentally exposed birds showed overt mucosal necrosis typical of field cases of NE, but typically the lamina propria was hyperemic and infiltrated with numerous inflammatory cells. Most significant changes were seen at the interface of the basal domain of enterocytes and lamina propria. Multifocally, these areas were extensively edematous, allowing for the substantial disturbance of the structural integrity between the lamina propria and the enterocytes. The lesions observed in the present study were consistently reproduced in all of our challenge trials, hence these responses may signify newly emerging patterns of sub-clinical enteric disorders in contemporary strains of poultry. The pathological changes observed in broilers challenged orally with C. perfringens in the present study, differ significantly from those reported previously, and must be clearly differentiated from those described in cases of NE or ulcerative enteritis. Although no overt necrosis of the intestinal mucosa typical of field cases of NE were observed in the present study, the birds challenged with C. perfringens showed strong inflammatory reaction to the introduced pathogens. The distinct features of the microscopic lesions were changes involving apparently normal enterocytes at the interface of the basal domain of villar epithelia and lamina propria. Although the pathological changes in the intestinal tissues observed in our trials appear to be rather subtle when compared to field cases of NE, the nature of these lesions suggest a significant negative effect on the digestive physiology of intestinal mucosa.

Dietary glycine concentration affects intestinal Clostridium perfringens and lactobacilli populations in broiler chickens.

Previous studies have reported that intestinal populations of Clostridium perfringens, the causative agent of necrotic enteritis (NE), are correlated with diets high in glycine. To establish a direct causative link, 3 trials were conducted to examine the effect of dietary glycine levels on gut populations of C. perfringens, alpha-toxin production, and NE lesion scores in broiler chickens. In trials 1 and 2, 12 groups of 4 birds were fed 4 different ideal protein-balanced diets formulated to contain 0.75, 1.58, 3.04, or 4.21% glycine from d 14 to 28 of age. In trial 3, 24 groups of 4 birds were given 6 different ideal protein-balanced diets formulated to contain 0.50, 0.75, 1.00, 1.50, 2.00, or 4.00% glycine. All birds were orally challenged with a broth culture of C. perfringens type A on d 1 and between d 14 and 21 of age and killed on d 28. The majority of birds showed clinical signs of NE with 4.16 to 8.33% mortality in the 3 trials. The highest mortality and intestinal lesion scores were observed in chickens receiving 3.04% glycine in trials 1 and 2, and 4.00% glycine in trial 3. Clostridium perfringens populations in the cecum varied quadratically with increasing dietary glycine, with the maximal response seen at 3.30,3.89, and 3.51% dietary glycine in trials 1, 2, and 3, respectively. Numbers of lactobacilli in cecum declined significantly (P < 0.05) with increasing levels of glycine. The results suggest that dietary glycine level has a significant effect on C. perfringens and lactobacilli populations and may be a predisposing factor for NE in broiler chickens.

Effects of dietary protein source and level on intestinal populations of Clostridium perfringens in broiler chickens.

Two experiments were conducted to examine the effect of the level of dietary crude protein and protein source on intestinal populations of Clostridium perfringens in broilers. In experiment 1, 6 groups of 12 birds were fed diets containing 230,315 or 400 g/kg crude protein with soy protein concentrate (SPC) or low-temperature-dried fishmeal as the major protein sources in a 2 x 3 factorial arrangement of treatments. A significant interaction between protein source and level was observed where the number of C. perfringens present in the ileum and cecum increased as the level of crude protein in the diets increased from 230 to 400 g/kg in the birds fed fishmeal-based diets (P < 0.05) but not in the birds fed SPC-based diets. In experiment 2, the dietary treatments used were arranged in a 2 x 2 factorial arrangement with 2 levels of crude protein (230 and 400 g/kg) and 2 protein sources (SPC or fishmeal). The main effects of protein source and protein level significantly (P < 0.05) affected numbers of C. perfringens without interaction. Amino acid analysis of the diets showed that the glycine and methionine contents of the fishmeal diets were elevated compared with the SPC diets. This suggests that the level of crude protein, protein source, and amino acid content of diets affect the growth of C. perfringens in the lower intestinal tract of the broiler chicken and might be predisposing factors to outbreaks of clinical necrotic enteritis.

Absorption of methionine and 2-hydroxy-4-methylthiobutoanic acid in conventional and germ-free chickens.

The apparent absorption of 3H-labeled L-Met and L-2-hydroxy-4-methylthiobutoanic acid (MHA-FA) was compared in germ-free and conventional broiler chickens to determine the effect of intestinal bacteria on the absorption of Met and MHA-FA. The two diets contained 0.236% of added Met or MHA-FA. Nineteen germ-free birds were maintained in two isolators and fed diets that had been sterilized by gamma irradiation (50 kilogreys). Nineteen conventional birds were reared in batteries and received nonirradiated feed. Diets were fed ad libitum for 3 wk. On d 21 of the experiment, the birds fasted overnight and were refed the experimental diets to which 1.11 x 10(7) Bq of 1-[methyl3H]MHA-FA or 1-[methyl3H]Met/kg of feed had been added. 51CrCl3 (1.11 x 10(7) Bq/kg of feed) was added as an indigestible marker. After 3 h the birds were euthanized, and their intestinal tracts were removed and partitioned into six sections. Residual Met and MHA-FA in digesta were calculated as the ratio of 3H:51Cr in each sample divided by the ratio of 3H:51Cr in the feed. The residual MHA-FA in the distal ileum of germ-free broilers was lower than in conventional birds (4.7 and 10.2% respectively; P < 0.05). In contrast the residual Met in the distal ileum of germ-free broilers was not different than in conventional birds (3.0 and 3.7% respectively; P > 0.05). This study demonstrates that intestinal bacteria significantly reduce the apparent absorption of MHA-FA from the intestinal tract of broiler chickens.

UPS on Weinreb resin: a facile solid-phase route to aldehyde and ketone derivatives of "unnatural" amino acids and peptides.

The solid-phase synthesis of "unnatural" amino aldehydes, amino ketones, peptide aldehydes, and peptide ketones was accomplished from commercially available resin in a series of room temperature reactions. The initial step involved addition of an "unnatural" side chain to the N-terminus of a benzophenone imine-activated Weinreb resin-bound amino acid or peptide derivative. The alkylated imine was hydrolyzed, and the amine was converted to the Boc-, Cbz-, or naphthoyl derivative. The resin-bound substrate was then cleaved with DIBAL-H or a Grignard reagent to give the amino aldehyde, amino ketone, peptide aldehyde, or peptide ketone products. Twenty-four reactions were carried out simultaneously using a "Billboard" reaction apparatus to give products in 27-87% (59% average) isolated yield.

A mouse model for studies of mucosal immunity to vaginal infection by herpes simplex virus type 2.

BACKGROUND: The role of mucosal immunity in defense of the female genital tract against pathogens such as herpes simplex virus-2 (HSV-2) is poorly understood. Here we explored the use of a new mouse model to determine whether local immune events in the vagina of immune animals may protect them against genital herpes. EXPERIMENTAL DESIGN: The effect of the estrous cycle, pregnancy, and sex hormones on vaginal infection of adult mice by HSV-2 was determined by immunolabeling of virus proteins. The immune response to infection was studied by immunolabeling of T lymphocytes, B lymphocytes, and plasma cells in the vagina of infected mice. RESULTS: Inoculation of attenuated virus (TK-HSV-2) or wild-type virus (TK+HSV-2) into the vagina on day 6 of pregnancy or after treatment with Depo-Provera (DP) caused infection of the vaginal epithelium. In contrast, these viruses did not cause infection after vaginal inoculation at estrus, metestrus, or after treatment with Depo-Estradiol. Infected mice showed immunolabeling of virus in the vaginal epithelium from 24 hrs to 5 days after virus inoculation. The immune response to infection included upregulation of class II MHC antigen in vaginal epithelium, CD8+ T cells in epithelium and stroma, and plasma cells and lymphoid nodules in the stroma. Mice that were infected with TK-HSV-2 did not exhibit infection of vaginal epithelium when challenged 6 weeks later with TK+HSV-2. CONCLUSIONS: Progesterone-dominated adult mice become infected after intravaginal inoculation with HSV-2, but estradiol-dominated mice are refractory. Vaginal infection with attenuated HSV-2 produces immunity that protects mice against later infection by wild-type virus. This immunity either prevents infection of vaginal epithelium or severely inhibits viral replication in the epithelium. The observations suggest that the E/DP-treated adult mouse should be a useful model for studies of mucosal immunity to vaginal infection by HSV-2.

Vaccination by cholera toxin conjugated to a herpes simplex virus type 2 glycoprotein D peptide.

Immunization of BALB/cJ mice with a peptide corresponding to residues 1 to 23 of glycoprotein D [gD(1-23)] from herpes simplex virus type 2 (HSV-2) elicits antibody responses which correlate with protection against lethal HSV-2 infection. In the present study, we examined the ability of cholera toxin (CTX) to act as an immunogenic carrier for gD(1-23). The number of gD(1-23) residues conjugated to CTX affected its binding to GM1 ganglioside and physiological toxicity, both of which are factors affecting oral immunogenicity. The antibody response elicited after intraperitoneal (i.p.) immunization with the CTX-gD(1-23) conjugate was protective against a lethal i.p. challenge with HSV-2. In other experiments, mice were immunized i.p. on day 0 and subsequent immunizations conducted on days 14 and 28 were administered either intragastrically or intravaginally (i.vag.). Intraperitoneal priming followed by either i.p or intragastric boosting resulted in anti-HSV-2 antibodies in vaginal washings and in protection against a lethal i.vag. challenge with HSV-2. Intraperitoneal priming followed by i.vag. boosting did not elicit anti-HSV-2 antibodies in vaginal washings and did not protect mice against a lethal i.vag. challenge with HSV-2. These results suggest that CTX can act as a systemic and an oral delivery molecule for the covalently linked gD(1-23) peptide and that such conjugates can elicit protective immune responses against systemic and genital HSV-2 infection.

Cholera toxin conjugates for intragastric vaccination against herpes simplex virus type 2.

In this study, we tested the hypothesis that enteric immunization with cholera toxin (CTX) conjugated to glycoprotein D (gD) of herpes simplex virus type 2 (HSV-2) or a peptide corresponding to residues (1-23) of gD (gD(1-23)) would induce relevant antiviral immunity. Intraperitoneal (IP) immunization of mice with CTX-gD(1-23) conjugate induced anti-HSV-2 sera antibody responses which correlated with protection from a lethal IP challenge with HSV-2. Intragastric (IG) immunization of mice with the same conjugate or a CTX-gD conjugate did not result in measurable anti-HSV-2 responses in sera or vaginal washings and only small numbers of anti-HSV-2 antibody-secreting cells (ASC) were found in intestinal lamina propria cell and splenocyte preparations. In comparison, anti-CTX responses were detected in sera and vaginal washings after IG immunization with CTX and anti-CTX ASC in lamina propria cell preparations accounted for 5-10% of total ASC detected at this site. No significant differences in the survival of mice immunized with the conjugates were noted after a lethal intravaginal (IVAG) challenge with HSV-2. The poor enteric immunogenicity of gD(1-23) and gD conjugated to CTX was attributable to proteolysis in the gastrointestinal tract. These results indicate that although peptide-CTX conjugates can induce protective immune responses when administered parenterally, it may not be feasible to use peptides as the basis of an oral vaccine unless methods are developed to protect these antigens from degradation in the gastrointestinal tract.