Cutaneous cytokine gene expression and cellular responses in lambs infested with the louse, Bovicola ovis, and following intradermal injection of crude louse antigen.

The present study was undertaken to investigate further the immunological responses in the skin of lambs to natural louse infestation and following intradermal allergen challenge. Bovicola ovis-infested (n=7) and naïve (n=7) Romney lambs received four intradermal injections each of crude louse Ag and diluent control solutions on the dorso-lateral chest. From each lamb, skin samples were obtained from untreated skin and, at 4, 24, 48 and 72 h following injection, from one each of the Ag- and diluent-injected skin sites. Levels of acetylcholinesterase-positive Langerhans and MHC II(+) cells in the epidermis as well as MHC II(+), CD1b(+), T19(+) and IgE(+) cells, eosinophils, and diffuse IgE staining in the dermis were significantly elevated in infested compared to naïve lambs (all p< or =0.01). Additionally, gene expression of interleukin-4 (IL-4), IL-5, IL-13 (all p< or =0.001) and IL-10 (p< or =0.05) was significantly higher in the skin of infested compared to naïve lambs while TNF-alpha and IFN-gamma gene expression were not significantly different between the two groups. Intradermal injection of louse Ag led to immediate and late phase responses in the infested lambs while the naïve lambs showed only minimal responses. Levels of dermal MHC II(+), CD1b(+), T19(+)and IgE(+) cells, eosinophils and diffuse IgE staining in infested lambs following injection of louse Ag were similar to or exceeded those in untreated skin and, with few exceptions, were higher than in naïve lambs. Additionally, cytokine gene expression of IL-4, IL-5, IL-13 and IL-10 increased to peak levels 4 h following Ag injection in the infested lambs (p< or =0.001, < or =0.05, < or =0.05 and < or =0.001 respectively compared to untreated controls) and remained significantly elevated compared to that observed in the naïve controls for the duration of the experiment. Significant elevations of MHC II(+) cells and IL-4, IL-5, IL-13 and IL-10 gene expression were observed in the louse-naïve lambs following injection of louse Ag but were much less pronounced than in the infested lambs. These results indicated that louse infestation in lambs elicited a highly skewed Th2 immuno-inflammatory response with many characteristics similar to those seen with other parasitic infections and also in atopic dermatitis.

Heritability of resistance to infestation with the body louse, Bovicola ovis, in Romney sheep bred for differences in resistance or resilience to gastro-intestinal nematode parasites.

The inheritance of resistance to louse infestation and the related allergic skin disease, cockle, was examined in Romney lambs. The lambs used in the study were the 2001- and 2004-born progeny of four experimental breeding lines ("Resistant", "Susceptible", "Resilient" and "Control") developed as part of a long-term study of the genetics of host resistance (maintenance of low faecal egg count (FEC) under nematode challenge) or resilience (maintenance of health and productivity under nematode challenge irrespective of FEC) to nematode parasites in sheep. Between 13 and 22 progeny (equally distributed between males and females, where possible) from each of five sires in each line were selected each year for this trial. All lambs (n=701) were examined for lice (Bovicola ovis) before artificial infestation; in 2001 the lambs were free of natural infestation, whilst in 2004 naturally acquired infestation was evident. In November 2001 and May 2002, approximately 60 B. ovis were transferred to each lamb, followed by monitoring at approximately 2-monthly intervals until August 2002. Similar procedures, but with fewer monitoring times, were repeated on the 2004 lambs. Overall, lambs in the Control line were significantly more susceptible to louse infestation and cockle compared with those in the other three lines (P<0.001). Least squares-means (SEM) of log-transformed louse score for the control, resistant, susceptible and resilient lines, respectively, were 2.178 (0.045), 1.499 (0.050), 1.618 (0.050) and 1.587 (0.044), and for cockle score were 1.36 (0.05), 0.76 (0.05), 0.95 (0.05) and 0.78 (0.05). From all progeny together, the heritability of log-transformed louse score was 0.22 (Standard Error (SE) 0.06) in autumn and 0.34 (SE 0.08) in winter, with a value of 0.44 (SE 0.09) when these data were combined. These estimates were similar to those obtained for resistance to gastro-intestinal nematodes in these breeding lines, using log-transformed FECs. Heritability estimates for cockle score in autumn, winter and when combined were 0.06 (SE 0.04), 0.45 (SE 0.09) and 0.40 (SE 0.09), respectively. The genetic correlations of mean log-transformed louse score with mean cockle score and levels of two different louse antigens in wool were, respectively, 0.97 (SE 0.04), 0.96 (SE 0.08) and 0.95 (SE 0.09). However, there was no significant genetic correlation between louse scores and FEC. These results suggest that selective breeding would be effective in reducing louse infestation and cockle in sheep, but that differences in louse burdens were not related to differences in nematode burdens as indicated by FECs.

Changes in morphology and key cytokine gene expression after intradermal injection of louse (Bovicola ovis) antigen in sheep with naturally occurring atopic dermatitis.

Groups of louse-infested and louse-naïve lambs (n=6 or 7) were used in two experiments to determine the sequential tissue response (macroscopical, microscopical and key cytokine mRNA) to intradermal injection of crude louse (Bovicola ovis) antigen over a period of 72 or 96 h. Histamine diphosphate and phosphate-buffered saline/glycerol (antigen vehicle control) solutions were also injected intradermally in each lamb for comparison. In both experiments, louse-infested lambs showed immediate and late-phase responses (LPRs) to louse antigen that differed significantly from the responses in the louse-naïve lambs. In experiment 1, biopsy samples taken at 7, 24, 48 and 96 h after injections showed more extensive dermal inflammation and leucocyte infiltration in response to louse antigen in louse-infested than in louse-naïve lambs. Eosinophils were significantly more numerous in the dermis of louse-infested lambs after all treatments and increased substantially in these lambs after antigen injection. Additionally, the louse-infested lambs differed from the naïve lambs in showing significantly higher mononuclear leucocyte and basophil infiltration and significantly lower neutrophil infiltration after antigen injection. In experiment 2, biopsy samples taken 4, 24, 48 and 72 h after injections showed trends in eosinophil infiltration of the dermis similar to those observed in experiment 1. Peak IL-4 mRNA expression was detected 4 h after antigen injection in the louse-infested lambs and remained significantly elevated at 24 h as compared with the results in the louse-naïve lambs. No significant difference in IFN-gamma mRNA expression between the louse-infested and the louse-naïve lambs was observed. These results indicated that louse-infested lambs show a cutaneous LPR analogous to that observed in atopic human beings and dogs. However, some differences were observed, including the longer duration of the LPR, the profuse eosinophil infiltration, and an absence of increased IFN-gamma mRNA expression.

Stimulation of adult hippocampal neurogenesis by physical exercise and enriched environment is disturbed in a CADASIL mouse model.

In the course of CADASIL (Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy), a dysregulated adult hippocampal neurogenesis has been suggested as a potential mechanism for early cognitive decline. Previous work has shown that mice overexpressing wild type Notch3 and mice overexpressing Notch3 with a CADASIL mutation display impaired cell proliferation and survival of newly born hippocampal neurons prior to vascular abnormalities. Here, we aimed to elucidate how the long-term survival of these newly generated neurons is regulated by Notch3. Knowing that adult neurogenesis can be robustly stimulated by physical exercise and environmental enrichment, we also investigated the influence of such stimuli as potential therapeutic instruments for a dysregulated hippocampal neurogenesis in the CADASIL mouse model. Therefore, young-adult female mice were housed in standard (STD), environmentally enriched (ENR) or running wheel cages (RUN) for either 28 days or 6 months. Mice overexpressing mutated Notch3 and developing CADASIL (TgN3R169C), and mice overexpressing wild type Notch3 (TgN3WT) were used. We found that neurogenic stimulation by RUN and ENR is apparently impaired in both transgenic lines. The finding suggests that a disturbed neurogenic process due to Notch3-dependent micromilieu changes might be one vascular-independent mechanism contributing to cognitive decline observed in CADASIL.

The transfer of maternal IgE and other immunoglobulins specific for Trichostrongylus colubriformis larval excretory/secretory product to the neonatal lamb.

The transference of immunoglobulins from six New Zealand Romney ewes to their lambs was examined. Immunoglobulin levels were determined in ewe plasma, colostrum and lamb plasma shortly after birth and before the lambs fed, in lamb plasma 2 days after birth, and lamb plasma, ewe plasma and milk 30 days after parturition. Levels of total IgE, and IgE, IgG1, IgG2, IgM, and IgA with specificity for Trichostronglus colubriformis third stage larval secretory/excretory products (TcL3E/S) were determined. Mean levels of total IgE were three times higher in colostrum than in parturient ewe plasma while only trace amounts were detected in milk at 30 days after birth (107.7, 34.3, and 0.2U ml(-1), respectively, differences between means P< or =0.01). Mean total IgE in lamb plasma rose from being undetectable before suckling to levels comparable to those of the ewes by 2 days after birth (21.7U ml(-1)) and then declined to low levels by 30 days (0.4U ml(-1)). Total IgE levels in lamb plasma were significantly correlated with levels in ewe plasma and colostrum (r=0.91, P< or =0.01; r=0.96, P< or =0.003, respectively). The transference of TcL3E/S-specific IgE, IgG1 and IgA was substantial with mean levels of these antibodies in lamb plasma at 2 days comparable to that in parturient ewe plasma (absorbance levels in lamb plasma of 0.283, 0.537, and 0.334, respectively). Proportionally less maternal IgM and IgG2 appeared to be transferred to the lambs (absorbance of 0.112 and 0.081, respectively). Levels of TcL3E/S-specific IgE and IgG1 in lamb plasma at 2 days were significantly correlated with levels in parturient ewe plasma and colostrum (r=0.89 and 0.82, 0.85 and 0.96; all P< or =0.05, respectively). These results indicate that IgE is concentrated in ewe colostrum and that substantial amounts of maternal IgE are transferred to lambs via colostrum. Further, the results suggest that humoral immunity against gastro-intestinal nematode parasites and potentially other parasites in colostrum-fed lambs may approximate that of the ewe. The implications of the transference of humoral immunity through colostrum in ruminants for the passive protection and the development of active immunity against parasites remains to be fully explored.

Effects of acetylsalicylic acid and indomethacin on single groups III and IV sensory units from acutely inflamed joints.

In cats with an acute experimental inflammation in the right knee joint the effects of acetylsalicylic acid (ASA) and indomethacin on the discharge properties of single fine myelinated and unmyelinated articular afferent units were tested. The knee joint was inflamed by injection of kaolin and carrageenan into the joint cavity some hours before the recording period. Before drug application the single afferent fibres showed resting activity and responses to movements of the joint within its working range which is common for units from the inflamed knee. Resting activity was reduced significantly in most units within 0.5 h after the intravenous injection of the drugs. Within the observation time of about 1-2 h there was no recovery. In a few units a transient increase of resting activity was observed immediately after the injection of a drug. After the initial observation period of 1-2 h the responses to movements were tested. They were reduced in all units except one myelinated afferent. Also during this testing period the resting discharges did not recover. Intra-arterial injection of prostaglandin E2 in low doses close to the joint temporarily nullified the depressing effects of aspirin and indomethacin. Resting activity and movement evoked responses were increased up to the level before the treatment with analgesic drugs. The effects of prostaglandin E2 lasted for several minutes to more than 1 h. The depression of resting and evoked activity in single afferent articular units from inflamed joints is discussed in relation to the analgesic properties of aspirin and indomethacin.

Integrated viral genomes can be lost from adenovirus type 12-induced hamster tumor cells in a clone-specific, multistep process with retention of the oncogenic phenotype.

In adenovirus type 12 (Ad12)-induced tumor cells, in Ad12-transformed cells and in continuously passaged cell lines from these sources, the viral DNA is integrated in multiple copies, usually at a single chromosomal location. In different tumors or cell lines, the sites of integration of Ad12 DNA are all different. Rare exceptions exist. In most instances, the integrated viral DNA resides very stably in the host cell genomes. However, upon continuous serial passage of such cell lines, the integrated viral DNA can be destabilized and lost. In two instances, i.e. in the Ad12-induced hamster tumor cell lines H1111(1) and CLAC1, we have investigated the loss of integrated viral DNA in detail. After extended serial passage, these two cell lines seemed to be devoid of Ad12 DNA sequences, as detectable by Southern blot hybridization, but continued to induce tumors after reinjection into hamsters. Cells from these two cell lines were now recloned three times, and DNAs from cultures derived from several individual clones were reinvestigated for the presence of several parts of the viral genome by the polymerase chain reaction (PCR). Some of the clones still carried parts of the Ad12 genome. However, several clones were isolated that proved free of all parts of the viral genome, except for minute segments from the right terminus of the Ad12 genome. Apparently, the loss of integrated viral DNA from these cell lines proceeded as a continuous, gradual, multistep process whose pattern could differ from cell clone to cell clone, once destabilization had been initiated. The mechanism of destabilization is not understood. Cell populations of 2 x 10(6) to 3 x 10(7), and as low as 10(2), cells from the clones, that contained only minimal remnants from the right viral DNA terminus, were reinjected into newborn or 13-20 day-old weanling Syrian hamsters (Mesocricetus auratus). Tumors developed within 5-17 days after injection. Tumor cell clones also grew in soft agar. The injection of primary hamster skin fibroblasts never elicited tumor formation. The tumor cells induced by this reinjection proved repeatedly free of Ad12 DNA both by Southern blot hybridization and by PCR, except for those cell and tumor clones that contained small segments of the right terminal E4 region of the Ad12 genome. The tumor cells, however, retained their oncogenic phenotype. The results raise questions about the cell clone-specific excision patterns of integrated foreign DNA from the recipient genome and the possibility of a hit-and-run mechanism of adenoviral oncogenesis.

Follistatin has a biphasic response but follicle-stimulating hormone is unchanged during an inflammatory episode in growing lambs.

The effects on plasma follistatin concentrations of an inflammatory episode, induced by the intrathoracic injection of yeast, were examined in growing lambs; this model results in acute loss of appetite, food intake and liveweight and the activation of the acute-phase pathway for several weeks as adjudged by the production of haptoglobin and other acute-phase proteins. In these animals (n = 8) there was a biphasic response in follistatin concentrations, with an initial 200% increase (P < 0.001) in follistatin within 24 h of injection of yeast. Thereafter, follistatin concentrations were depressed to 70% of pretreatment levels 48 h after injection (P < 0.01), followed by a gradual recovery of concentrations to pretreatment values. In another group of lambs (n = 16) that were feed-restricted to mimic the reduced food intakes and liveweight changes in the yeast-injected group, plasma follistatin was also reduced to around 70% of pretreatment levels (P < 0.01) within 1 day of the dietary regimen being implemented, followed by a gradual return to pretreatment values as food intakes were increased. Plasma follistatin correlated significantly (r = 0.57, P < 0.0001) with food intake, but not with liveweight changes. Plasma follistatin concentrations were unchanged in a third group fed ad libitum (n = 8), except during two periods when food intakes were significantly (P < 0.05) reduced, when follistatin concentrations also decreased (P < 0.01). Plasma follicle-stimulating hormone (FSH) concentrations in the three groups of lambs were not significantly affected by the treatment regimes or changes in follistatin concentrations. These findings indicate that peripheral follistatin concentrations are modulated by both inflammatory and nutritional mechanisms, and that significant fluctuations in follistatin levels can occur without detectable perturbations in FSH secretion.

Induction of an acute phase response in lambs causes an increase in plasma levels of GH and IGF-I.

GH and IGF-I plasma concentrations were measured in lambs during an acute phase response induced by an intrathoracic injection of yeast. The acute phase response was indicated by reduced feed intake, weight loss and an increase in plasma concentrations of the acute phase protein haptoglobin. Intensive blood sampling on day 1 revealed elevated basal concentrations of GH in the yeast-injected group compared with concentrations in pair weight and ad libitum fed control lambs. This suggests that at the beginning of an acute phase response there is an increase in either GH secretion or the half life of GH. No evidence of a specific GH-binding protein in sheep plasma could be detected. IGF-I concentrations in the yeast-injected group remained constant for 3 days then increased to a peak level at day 6. In contrast, plasma IGF-I concentrations were depressed from days 3 to 6 in the pair weight control group and they were unchanged in the ad libitum fed controls. When the IGF-I concentrations were elevated in the yeast-injected group, this group had a higher daily weight gain despite their lower feed intake compared with the ad libitum fed controls. These results suggest that IGF-I may be associated with the increase in weight in the late stage of an acute phase response during recovery from an infection or injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of local and systemic responsiveness of lymphocytes in vitro to Bovicola ovis antigen and concanavalin A in B. ovis-infested and naive lambs.

The in vitro proliferation assay was used to determine lymphocyte responsiveness to soluble antigen of B. ovis and to Concanavalin A (Con A) in peripheral blood, spleen and various lymph nodes from B. ovis-infested and naive lambs. From March to July, an assay of monthly blood samples showed generally higher proliferative responses to antigen and Con A in B. ovis-infested than naive lambs. The proliferative response of cells from the skin-draining prescapular lymph nodes to B. ovis antigen was significantly higher in B. ovis-infested than naive lambs. Responses of cells from the medial iliac, mediastinal and mesenteric lymph nodes (which do not receive lymph from the skin) and spleen showed no significant differences between groups. Within the B. ovis-infested lambs, the response of cells from the prescapular lymph node was significantly higher than that from any other lymphoid organ examined. Responsiveness of the prescapular, medial iliac and mesenteric lymph node and spleen cells to Con A was not significantly different between groups, while mediastinal lymph node cells showed a significantly higher response in B. ovis-infested lambs. The data indicate that the antigen-specific cellular immune response is operating mainly locally, at the level of the skin and draining lymph nodes. Responses to the T cell mitogen Con A did not support non-specific immunodepression as reported in other ectoparasite/host systems.

Proliferative responses of lymphocytes in Bovicola ovis-infested lambs.

Proliferative responses of peripheral blood and prescapular lymph node lymphocytes from 8 B. ovis-infested and 8 naive lambs to concanavalin A (Con A) and soluble antigen of B. ovis were examined in vitro. The numbers of lice and the extent of cockle were also assessed on each lamb. Prescapular lymph node lymphocytes from infested lambs showed significantly higher responses to B. ovis antigen than those from naive lambs. Only a marginal difference was observed between infested and naive lambs in the response of lymphocytes from peripheral blood. Proliferative responses to Con A by prescapular lymph node or blood cells were not significantly different between groups. The results indicated a relationship between the intensity of louse infestation and the proliferative response of prescapular lymph node lymphocytes to B. ovis antigen.

Immunization of sheep against parasitic nematodes leads to elevated levels of globule leukocytes in the small intestine lumen.

In sheep that had been given three immunizing infections with Trichostrongylus colubriformis and Ostertagia circumcincta infective (L3) larvae, drenched after the last infection and challenged with larvae of the same species, there was a significant increase in numbers of small intestine mucosal tissue globule leukocytes (TGLs) and lumenal globule leukocytes (LuGLs) compared with sheep that had only been drenched and challenged. There was a positive correlation between the numbers of LuGLs and TGLs in the small intestine but the ratio of these two cell types was lower in non-immunized than immunized sheep. In immunized sheep positive correlations were observed between LuGLs and levels of arylsulphatase and peroxidase in the intestinal mucus and between arylsulphatase and larval migration inhibition (LMI) activity in mucus. Lumen eosinophils correlated with blood eosinophils, serum antibody against T. colubriformis correlated with peroxidase in the mucus and blood eosinophils correlated with nematode specific IgM levels in the intestinal mucus. In the abomasum, TGLs were present but not LuGLs. Sheep repeatedly infected with T. axei also had significantly more LuGLs in the small intestine than control animals. Two sheep that had a surgically prepared isolated small intestinal loop, after oral infection with T. colubriformis had TGLs and LuGLs in the intact intestine, but not in the isolated loop. Significantly more LuGLs were produced in sheep by allowing repeated T. colubriformis L3 infections to develop to adult stages compared to sheep treated with the same number of larvae, but where the infections were terminated by drenching at various intervals.

Cross-reacting antibodies to Sarcoptes suis, Chorioptes bovis and Notoedres cati and anti-P. ovis IgE in sera from sheep infested naturally with Psoroptes ovis.

For the development of immunodiagnostic tests to detect mange mite infections in man and animals, it is necessary to know about antigen structure and cross-reactivity between different mite species and other arthropods in contact with the host. Sera from sheep infected with Psoroptes ovis (sheep anti-P. ovis sera) showed positive reactions in dot blots to P. ovis antigen and cross-reactivity to crude antigen extracts from Sarcoptes suis, Notoedres cati and Chorioptes bovis. Using sheep Specific Pathogen Free (SPF) sera in dot blots, weak reactions were seen to all but the Ch. bovis antigen. In SDS PAGE-separated P. ovis antigens at least 35 different proteins could be distinguished. In western blots, at least 24 out of these 35 were recognized as antigens by sheep anti-P. ovis immunoglobulins. At least 13 were recognized by sheep anti-P. ovis IgE. One of these, at 19 kDa, was recognized only with sheep anti-P. ovis IgE, the other 12 also with anti-P. ovis immunoglobulins. Cross-reactive antigens were recognized by sheep anti-P. ovis immunoglobulins in SDS PAGE-separated, and nitrocellulose transferred mite extracts in western blots as follows: 13 antigens in S. suis extracts, 9 in N. cati and 8 in Ch. bovis. Sheep SPF sera recognized an antigen at 67 kDa in each of these 4 mite species. Rabbit anti-sheep immunoglobulins in the P. ovis antigen control bound to a protein at 28 kDa which may be sheep IgG light chain taken up by P. ovis feeding on sheep. Despite the antigenic similarities between mange mites, sufficient differences were apparent to make immunodiagnostic tests for mange feasible.

Sequential cellular and humoral responses in the abomasal mucosa and blood of Romney sheep dosed with Trichostrongylus axei.

Abomasal cannulae were surgically placed in 7 2-year-old New Zealand Romney sheep which had been maintained parasite-free from birth. Four of these sheep were randomly selected and dosed orally with 10,000 infective Trichostrongylus axei larvae per week for 8 weeks, while the remaining 3 sheep served as uninfected controls. Abomasal biopsy, blood and faecal samples were obtained from all sheep at regular intervals from 5 days before and until 58 days after the first infection. The sheep were then killed, worm burdens assessed and abomasal and small intestinal samples collected Faecal egg counts of all 4 dosed sheep were low and only one (No. 701) had a substantial worm burden (8400) post mortem. Overall, levels of mucosal mast cells/globule leukocytes, eosinophils, T19+ cells and larval migration inhibitory activity increased significantly in the abomasal mucosa of the dosed sheep compared to the controls. The CD4+:CD8+ cell ratio in the abomasal mucosa of the dosed sheep also increased compared to that of the controls (P = 0.06). In blood, T. axei-specific antibody (total and IgG1) and eosinophil numbers increased significantly in the dosed sheep. Mucosal cells staining for IgE (IgE+), and blood and mucosal eosinophils showed the earliest substantive increases in number followed by increases in specific serum antibody levels, numbers of mucosal cells fluorescing under UV light (UVf) and T19+ cells. The difference in the IgE+ and UVf cell responses indicated that expansion of globule leukocyte numbers lagged behind that of mucosal mast cells. The results supported the concept of CD4+ T cell help in the abomasal mucosa and defined the sequential expression of components of the immunological responses potentially mediating resistance to T. axei. In sheep No. 701, persistence of adult worms was associated with lower mucosal IgE+ cell and eosinophil responses compared with the other dosed sheep.

Studies on the role of mucus and mucosal hypersensitivity reactions during rejection of Trichostrongylus colubriformis from the intestine of immune sheep using an experimental challenge model.

Nematode-naive sheep and sheep immunised by truncated infections with Trichostrongylus colubriformis were fitted with intestinal cannulae to allow administration of challenge infection and collection of intestinal fluids. Sheep were slaughtered at various times after challenge and the distribution of larvae along the small intestine was determined. Results showed that immune sheep had significantly fewer larvae in their intestines and that some sheep could expel the challenge infection within 2 h. Mucus samples from immune sheep contained increased parasite-specific antibody, histamine and anti-parasite activity as measured by larval migration inhibition assay. Higher levels of antibody and histamine were seen in intestinal fluids of immune sheep after challenge. Immunisation of sheep by truncated infections stimulated serum IgE and resulted in significantly higher numbers of IgE-positive cells in gut tissue sections before challenge and at 2 h and 24 h after challenge. Immune sheep also had greater numbers of mucosal mast cells and globule leucocytes after challenge, compared with naive sheep. When challenge larvae were mixed with mucus from immune sheep and infused back into naive recipient sheep, there was a distinct displacement of the larval population towards the distal part of the intestine, compared with the profile of larval establishment after infusion with mucus from naive sheep. These results are further evidence for an immediate hypersensitivity reaction in the intestine of immune sheep, where challenge larvae are expelled within 2 h and confirm the direct anti-larval properties of mucus. The cannulated-sheep challenge model described here will be a useful tool to unravel the mechanism of larval rejection from immune sheep and could lead to novel therapies.

Modulation of acetylcholine-induced secretion of gastric bombesin-like immunoreactivity by cholinergic and histamine H2-receptors, somatostatin and intragastric pH.

Recently we have shown the release of bombesin-like immunoreactivity (BLI) from the isolated perfused rat stomach. In these experiments we have shown that BLI secretion is stimulated by acetylcholine. Gastric inhibitory peptide (GIP) exerts an inhibitory effect which is dependent on the intraluminal pH. The present study was designed to examine further the exact cholinergic mechanisms and to study the interaction between cholinergic and histaminergic mechanisms as well as the effect of the intraluminal pH. Acetylcholine elicited a dose-dependent increase in BLI and gastrin secretion (10(-6) M and 2 X 10(-6)M), whereas somatostatin release was suppressed at luminal pH 7. Blockade of muscarinic cholinergic receptors by atropine (10(-5)M) and nicotinic cholinergic receptors by hexamethonium (10(-5) M) abolished the effect of acetylcholine on all three peptides. Reduction of the intraluminal pH to 2 also abolished acetylcholine-induced stimulation of BLI and gastrin secretion and the inhibition of somatostatin secretion. Changes of intraluminal pH per se had no effect on the secretion of either peptide. Somatostatin (10(-7) M) reduced both BLI and gastrin secretion during stimulation with acetylcholine. The addition of the H2-receptor antagonist cimetidine (10(-5) M) abolished the effect of both doses of acetylcholine on BLI and somatostatin secretion and also the effect of the lower dose of acetylcholine (10(-6) M) on gastrin secretion during luminal pH 7. At luminal pH 2 cimetidine did not alter BLI and somatostatin secretion in response to acetylcholine, however, gastrin release was augmented in the presence of cimetidine. These data demonstrate that the effect of acetylcholine on BLI, gastrin, and somatostatin secretion is mediated by muscarinic and nicotinic cholinergic receptors and also by histamine H2-receptors. Somatostatin inhibits cholinergically induced BLI secretion. The cholinergic effects on BLI, somatostatin and gastrin secretion are abolished during an acidic intragastric pH. In this isolated perfused rat stomach model the inhibitory effect of intraluminal acid on gastrin secretion is, at least in part, mediated by H2-receptors. This suggests that the secretion of bombesin, a potential peptidergic neurotransmitter is modulated by neural, endocrine and local tissue factors and also by alterations of intragastric pH.

Effect of pimozide on home cage ethanol drinking in the rat: dependence on drinking session length.

A stimulus-fading procedure was used to initiate ethanol drinking in free-feeding Long Evans rats. During daily half-hour drinking sessions in the home cage, a combination of sucrose and ethanol was first presented to the rats; gradually the sucrose concentration was reduced and the ethanol concentration increased until after 7 weeks the rats were drinking 10% ethanol with no sucrose. After stabilization of intake, either pimozide (PIM, 0.25, 0.50 and 1.00 mg/kg) was injected 4 h before drinking sessions or (d)-amphetamine (DEX, 0.25 and 0.50 mg/kg) was injected 15 min before sessions. The 0.50 and 1.00 mg/kg PIM doses and the 0.50 DEX dose significantly reduced intake compared to vehicle injections. In the second part of the experiment, the rats were given 24-h access to 10% ethanol and water in a two-bottle choice procedure. In this condition, 0.50 mg/kg PIM failed to reduce intake compared to vehicle. The critical difference between the two procedures seems to be that with the 30-min sessions, PIM injections were timed to have their maximal effect during testing. With 24-h sessions, decreases in intake produced by PIM could have been compensated for by increases after the drug had worn off. The hypothesis that dopamine is necessary for ethanol reinforcement receives support from the PIM effect on the 30-min sessions. The DEX effect extends the generality of our previous finding that DEX reduces ethanol-reinforced lever pressing in free-feeding rats.

The diagnostic value of EEG in Alzheimer disease: correlation with the severity of mental impairment.

The aim of our study was to analyze EEG changes in patients with Alzheimer disease (AD) and to determine how closely EEG reflects the progression of mental impairment in people with AD. Ninety-five patients with probable AD according to National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer's Disease and Related Disorders Association criteria treated in our Clinic for Memory Disorders were selected for this study. Patients were divided into three subgroups with mild, marked, and severe dementia according to the results of psychometric scales. The EEG findings were classified using an eight-degree scale according to the background activity, presence and amount of theta and delta waves, focal changes, lateralization of focal changes, synchronization, and presence of sharp and spike waves. A significant correlation between the degree of EEG abnormalities and cognitive impairment was found. We did not observe any correlation between the presence of delta waves and the results of neuropsychological tests. Our study revealed an important diagnostic value of EEG in the estimation of the severity of dementia parallel to psychometric scales.

Experimental Mycobacterium bovis infection in the brushtail possum (Trichosurus vulpecula): pathology, haematology and lymphocyte stimulation responses.

Groups of adult male brushtail possums (5 per group) were inoculated intratracheally with a high (2 x 10(5) colony forming units (cfu)), medium (2 x 10(3) cfu) or low (approximately 20 cfu) dose of Mycobacterium bovis. Two sham-inoculated groups acted as in-contact controls or controls kept in a separate room. Possums in the high and medium dose groups became clinically affected 3-5 weeks post-inoculation (PI) and all possums were euthanased between 5-9 weeks PI. Grossly visible tuberculous lesions were found in the lungs and associated lymph nodes of all possums from the high, medium and low dose groups. No lesions were observed in possums from the two control groups. Histopathologically, two characteristic types of lesions were observed; microscopic aggregates of macrophages with few acid-fast organisms, and larger lesions with limited granulomatous reaction, extensive necrosis and the presence of numerous acid-fast organisms. M. bovis was isolated from the lungs and lymph nodes of all of the possums from the high, medium and low dose groups and from the lungs of one of the in-contact controls. Changes in the haematological profile of the M. bovis-inoculated possums included lymphocytopaenia and eosinopaenia, together with raised fibrinogen levels. The onset of these changes was dependent on the size of the challenge dose. Lymphocyte stimulation responses to M. bovis tuberculin purified protein derivative were detected in 14 of 15 M. bovis-inoculated possums.

Detection of homocytotropic antibody in lambs infested with the louse, Bovicola ovis, using a basophil histamine-release assay.

The utility of a basophil histamine-release assay using washed whole blood cells was examined in lambs and was used to determine if homocytotropic antibody with specificity for Bovicola ovis was produced in response to infestation with the louse. Maximal histamine release in the assay in response to Concanavalin A, anti-ovine IgE monoclonal antibody and, in sensitized lambs, to B. ovis antigen ranged from 18 to 48%. Histamine release from blood cells in response to B. ovis antigen was significantly higher in louse-infested lambs than in louse-naive lambs and was significantly correlated with louse and cockle scores. Passive cutaneous anaphylaxis (PCA) tests were negative with sera obtained from the lambs at the same time as blood for the basophil histamine-release assay. Serum histamine levels also were significantly higher in the louse-infested lambs than in louse-naive lambs and were significantly correlated with louse and cockle scores. The present results support a role for B. ovis-specific homocytotropic antibody in the development of cockle and indicate that the basophil histamine-release assay is more sensitive than the PCA test.