Delayed expulsion of the nematode Trichinella spiralis in mice lacking the mucosal mast cell-specific granule chymase, mouse mast cell protease-1.

Expulsion of gastrointestinal nematodes is associated with pronounced mucosal mast cell (MMC) hyperplasia, differentiation, and activation, accompanied by the systemic release of MMC granule chymases (chymotrypsin-like serine proteases). The beta-chymase mouse mast cell protease-1 (mMCP-1) is expressed predominantly by intraepithelial MMCs, and levels in the bloodstream and intestinal lumen are maximal at the time of worm expulsion in parasitized mice. To address the in vivo functions of MMC-specific beta-chymases, we have generated transgenic mice that lack the mMCP-1 gene. They were backcrossed onto a congenic BALB/c background to investigate the response to nematode infection. The deletion of the mMCP-1 gene is associated with significantly delayed expulsion of Trichinella spiralis and increased deposition of muscle larvae in BALB/c mice despite the presence of normal and sometimes increased numbers of MMCs. Neither worm fecundity nor worm burdens were altered in Nippostrongylus-infected mMCP-1(-/)- BALB/c mice. These data demonstrate, for the first time, that the ablation of an MMC-derived effector molecule compromises the expulsion process.

Impairment of intestinal barrier and secretory function as well as egg excretion during intestinal schistosomiasis occur independently of mouse mast cell protease-1.

Deposition of Schistosoma mansoni eggs in the intestinal mucosa is associated with recruitment of mucosal mast cells (MMC) expressing mouse mast cell protease-1 (mMCP-1). We investigated the involvement of mMCP-1 in intestinal barrier disruption and egg excretion by examining BALB/c mice lacking mMCP-1 (Mcpt-1(-/-)). Tissue and faecal egg counts from 6 weeks until 12 weeks post-infection (w p.i.) revealed no differences between wild type (WT) and Mcpt-1(-/-)mice. Using chamber experiments on ileal tissue revealed that at 8 w p.i., the epithelial barrier and secretory capacity were severely impaired, whereas no difference was found between WT and Mcpt-1(-/-)mice in this respect. However, a fragmented distribution of the tight junction (TJ) protein occludin, but not of claudin-3 or ZO-1, was observed in WT mice at 8 w p.i., while no changes in TJ integrity were seen in Mcpt-1(-/-)mice. Therefore, we conclude that in contrast to the situation in Trichinella spiralis-infected mice, in schistosomiasis, mMCP-1 is not a key mediator in egg excretion or impairment of the intestinal barrier. The marked decrease in ileal secretory capacity during S. mansoni egg excretion suggests that the mechanisms facilitating the passage of schistosoma eggs through the gut wall are directed more particularly at the epithelial cells.

Innate immune response mechanisms in the intestinal epithelium: potential roles for mast cells and goblet cells in the expulsion of adult Trichinella spiralis.

SUMMARYGastrointestinal infection with the nematode Trichinella spiralis is accompanied by a rapid and reversible expansion of the mucosal mast cell and goblet cell populations in the intestinal epithelium, which is associated with the release of their mediators into the gut lumen. Both goblet cell and mast cell hyperplasia are highly dependent on mucosal T-cells and augmented by the cytokines IL-4 and IL-13. However, the contribution of both mast and goblet cells, and the mediators they produce, to the expulsion of the adults of T. spiralis is only beginning to be elucidated through studies predominantly employing T. spiralis-mouse models. In the present article, we review the factors proposed to control T. spiralis-induced mucosal mast cell (MMC) and goblet cell differentiation in the small intestine, and focus on some key MMC and goblet cell effector molecules which may contribute to the expulsion of adult worms and/or inhibition of larval development.

An ovine chitinase-like molecule, chitinase-3 like-1 (YKL-40), is upregulated in the abomasum in response to challenge with the gastrointestinal nematode, Teladorsagia circumcincta.

Mammalian chitinases and chitinase-like proteins are a group of molecules known to be upregulated and secreted in Th2-induced inflammatory responses, such as asthma, allergy and nematode infection. As part of an investigation of potential components of the innate immune response to Teladorsagia circumcincta, a gastrointestinal nematode that colonises the abomasum in sheep, we carried out RT-PCR analysis of two members of the mammalian chitinase family of molecules, acidic chitinase (ChiA) and chitinase-3 like 1 (Chi3L1) using primers to homologous bovine/human sequences. Both sets of primers detected transcripts in the abomasum which were confirmed to be ovine ChiA and Chi3L1 by sequence analysis. Chi3L1 transcripts were found to be significantly upregulated in both the abomasum and gastric lymph nodes in response to T. circumcincta challenge of previously infected animals.

Cytokine expression in naïve and previously infected lambs after challenge with Teladorsagia circumcincta.

Infection of sheep with Teladorsagia circumcincta triggers an immune response with predominantly type-2 (Th2) characteristics, including local eosinophila, mastocytosis and increased mucus production. In order to better understand the protective immune responses elicited, we used RT-PCR assays to define the changes in expression levels of a range of cytokine transcripts in lymph nodes draining the ovine abomasum following a challenge infection with T. circumcincta. This study compared the changes in cytokine expression in the abomasal lymph node following challenge with T. circumcincta in naïve sheep (Group 2) and sheep immunised by a previous trickle infection (Group 3), in comparison to unchallenged naive sheep (Group 1). There was a significant up-regulation of interleukin-4 (IL-4), IL-5 and IL-13 in both the challenged groups compared to naïve individuals. There was also an up-regulation of IL-1beta, IL-6, IL-10, IL-18, transforming growth factor-beta1 (TGFbeta1) and tumour necrosis factor-alpha (TNFalpha) by day 5 after infection. IL-12p40 was found to be increased in the previously infected Group 3 animals by day 5 following challenge. By contrast, transcription of this cytokine was found to be reduced by day 10 following infection of Group 2 animals. Expression of IL-2 and Interferon-gamma (IFNgamma) did not significantly differ between the three groups.

T-helper 1, T-helper 2 and immunosuppressive cytokines in canine atopic dermatitis.

Atopic dermatitis is a common inflammatory skin disease of humans and dogs. Human atopic dermatitis is associated with T-helper (Th) 2 type responses, although Th1 cytokines are present in chronic lesions. This study used semi-quantitative reverse transcriptase polymerase chain reactions to determine the expression of gene transcripts for immunosuppressive cytokines (transforming growth factor beta [TGFbeta] and interleukin [IL]-10), Th2 type cytokines (IL-4 and IL-6) and Th1 type cytokines (interferon gamma [IFNgamma], tumour necrosis factor alpha [TNFalpha], IL-2 and IL-12) in lesional atopic, non-lesional atopic and healthy canine skin. Canine atopic dermatitis was associated with over-expression of IL-4 mRNA and reduced transcription of TGFbeta compared to healthy skin (ANOVA, p<0.05). Higher levels of IFNgamma, TNFalpha and IL-2 mRNA were seen in lesional compared to non-lesional and healthy skin (p<0.05). There were no significant differences in IL-10, IL-6 or IL-12 transcription. This is the first report to demonstrate that canine atopic dermatitis is associated with over-production of IL-4 and under expression of TGFbeta.

The impact of perceived group success-failure on motivational beliefs and attitudes: a causal model.

Two models describing the theoretical relationship among the constructs of perceived group success-failure, individual and collective levels of job-specific efficacy and outcome expectancy, satisfaction, and organizational commitment were developed. Model 1 proposed that the causal relationship between group success-failure and subsequent attitudinal variables (satisfaction and organizational commitment) is completely mediated by efficacy and outcome expectancy variables. The second model proposed a direct causal link from perceived group success-failure to the attitudinal variables and omitted questionable causal paths from personal efficacy and collective outcome expectancy to the attitudinal variables. The competing models were subjected to structural equation path model analysis. The second model produced an adequate fit between theory and data. Implications for organizations are discussed.

How do people with different attachment styles balance work and family? A personality perspective on work-family linkage.

This study explored whether different models of work-family relationship were possible for individuals with different attachment styles. A mail survey was conducted using employees (N = 481) at a midwestern university in the United States. Results suggested that (a) individuals with a preoccupied attachment pattern were more likely to experience negative spillover from the family/home to the work domain than those with a secure or dismissing style, (b) securely attached individuals experienced positive spillover in both work and family domains more than those in the other groups, and (c) preoccupied individuals were much less likely to use a segmentation strategy than the other 3 attachment groups. However, when the conventional job satisfaction life satisfaction relationship was examined, the data provided unique support for the spillover model. Implications of the findings for both attachment and work family relationship literatures are discussed.

Are potency tests for tetanus vaccines really necessary?

There is no simple relationship connecting the antigen content to the immunogenic potency of tetanus vaccines and the need for potency tests therefore remains inescapable. However, the quantal response methods of assay presently used for the measurement of potency appear to use unnecessarily large numbers of animals and substantial reductions could be made, particularly in the control of combined vaccines, if toxic challenge were replaced by in vitro titration of antitoxin.

Opportunities to reduce the use of animals in the potency and toxicity testing of toxoid vaccines.

Alternative methods for titrating antitoxin are now available which should, in principle, permit a very large reduction in the numbers of animals required to test the potency of toxoid vaccines. More importantly they make it possible to eliminate the use of animals for the indication of excess toxicity almost completely. The full realisation of this potential is dependent upon the careful validation of all other methods and the introduction of more appropriate standard and reference preparations for titration of antisera and for the assay of DTP. It would be facilitated by a mechanism to facilitate the wider dissemination of relevant monoclonal antibodies and by a restructuring of veterinary vaccine potency tests to make full use of the additional serological information provided by the new methods.

A consideration of some methods by which the cost of potency assays for diphtheria and tetanus vaccines might be reduced.

Although recommended by WHO the high cost of parallel line quantal response assays for diphtheria and tetanus vaccines has retarded the introduction of comparative assays for these vaccines in developed countries and for the same reason is likely to discourage the introduction of adequate control standards in developing countries. These costs are mainly due to the large numbers of animals needed to obtain adequate precision when responses are assessed simply in terms of death or survival. The use of intradermal challenge scores for diphtheria and of semiquantal scores based on the onset of symptoms for tetanus together with the prospects for use of the same animals for both assays are discussed. Data are presented to show that the adoption of such methods would increase the information available from each animal and so reduce the number of animals required for the satisfactory standardization of diphtheria and tetanus vaccines.

An evaluation of some proposals for a reduction in the number of animals used for the potency testing of diphtheria and tetanus vaccines.

Analysis of data from small subgroups of animals used in potency tests for vaccines containing diphtheria and tetanus components has shown that, in most cases, satisfactory assurance of potency can be obtained with much smaller numbers of animals than are currently prescribed by the pharmacopoeias. It is suggested that when the criterion of potency for a vaccine is defined in terms of the lower fiducial limit of potency, the additional prescription of a minimum number of animals for the test is unnecessary. Furthermore, when well standardized, lyophilized toxins are used for challenge, there is no need to determine the LD50 of the challenge toxin on every occasion. The possibility of obtaining assurance of potency with smaller numbers of animals using single point comparisons of unknown and standard vaccines is also discussed.

Prospects for the refinement and reduction of animal usage in the potency testing of Cl. chauvoei vaccines.

Protection has been elicited by both cellular and supernatant antigens from Cl. chauvoei. The cellular antigen has been solubilised by various treatments described for removing flagella, and has been detected by an ELISA method. The level of protection elicited by these preparations was poor, and immunoassays using these antigen preparations did not correlate with in vivo results. The supernatant antigen has been fractionated by ammonium sulphate precipitation and gel filtration. Using the latter technique, the protective antigen has been partially purified. Immunoassays using this soluble antigen did not correlate with in vivo protection levels. It is concluded that in vitro assays based on the partially characterized antigens are not yet suitable for the measurement of protective antibody.

Studies on the minimal number of animals required to achieve assurance of satisfactory potency in diphtheria and tetanus vaccines.

The purpose of the potency test for adsorbed diphtheria and tetanus vaccines prescribed in the European Pharmacopoeia is to provide 97.5% assurance that the minimum requirement for potency is exceeded. In order to achieve this the use of at least six groups of sixteen guinea pigs or mice is prescribed. In some formulations, particularly of combined vaccines, the potency exceeds the minimum requirements by a very large margin and the numbers of animals used in the test appear to be unnecessarily large. Data are presented which show that 97.5% assurance of satisfactory potency can be consistently achieved with substantially smaller numbers of animals than are used at present. It is suggested that the prescription of large numbers of animals in routine issue tests for diphtheria and tetanus vaccines is unnecessary and inappropriate except in cases where the potency of the vaccine is in dispute.

The effects of laboratory animal diets on the potency tests of bacterial vaccines.

Comparative studies of the responses elicited by mice fed on PCD and FFG diets to a number of bacterial vaccines have shown a significant reduction in the immune response to tetanus toxoid but not to Clostridium septicum toxoid and increased resistance to challenge with E. coli and syngeneic tumour cells but not to Pasteurella multocida. These differences cannot readily be explained in terms of differences between the identifiable constituents of the diets and illustrte the dangers of vaccine potency tests that require an absolute level of response to the material under test.

Some problems concerning the assay of Clostridium chauvoei vaccine in laboratory animals.

The potency test prescribed for Cl. chauvoei vaccines in the British Veterinary Codex requires the complete survival of a group of immunized guinea pigs and death of all unvaccinated controls. Problems arising from the influence of extraneous factors such as animal strain, diet and seasonal variation on the outcome of this test are discussed together with alternative methods that might permit the use of a standard preparation.

The assay of clostridial vaccines in mice.

The proposed establishment of a multicomponent clostridial standard vaccine inevitably raises the question of how such a preparation might be used in the standardization of batches of vaccine. Traditional multipoint parallel line assays based on the antitoxin responses of rabbits and guinea pigs are likely to prove prohibitively costly, whilst simpler methods based on single dose levels of either vaccine fail to quantitate the relationship between test vaccine and standard.