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.

Constitutive secretion of the granule chymase mouse mast cell protease-1 and the chemokine, CCL2, by mucosal mast cell homologues.

BACKGROUND: The mucosal mast cell (MMC) granule-specific beta-chymase, mouse mast cell protease-1 (mMCP-1), is released systemically into the bloodstream early in nematode infection before parasite-specific IgE responses develop and TGF-beta1 induces constitutive release of mMCP-1 by homologues of MMC in vitro. Intraepithelial MMC may also express the chemokine CCL2 (monocyte chemotactic protein-1) during nematode infection but the expression of this chemokine by MMC homologues has not been investigated. OBJECTIVE: To investigate the expression and to compare the mechanisms of constitutive release of the chymase, mMCP-1, and the chemokine, CCL2. METHODS: MMC homologues were generated by culturing bone marrow cells in the presence of TGF-beta1, IL-3, IL-9 and stem cell factor (SCF). The intracellular distribution of mMCP-1 and CCL2 was examined by confocal microscopy. The involvement of the Golgi complex and of protein synthesis in the constitutive release of mMCP-1 and CCL2 was investigated using the Golgi-disrupting agent brefeldin A and cycloheximide to block protein synthesis. Secreted analytes were quantified by ELISA. RESULTS: mMCP-1 colocalized with Golgi matrix protein 130 but was most abundant in the granules, whereas CCL2 was not found in the granules but appeared to be located uniquely in the Golgi complex. Extracellular release of mMCP-1 was significantly inhibited ( approximately 40%) by cycloheximide and by the Golgi-disrupting agent brefeldin A, indicating both continuous protein synthesis and transportation via the Golgi complex are required for optimal mMCP-1 secretion. A similar but more marked inhibitory effect with both compounds was demonstrated on the constitutive secretion of CCL2. CONCLUSION: The culture conditions that promote mMCP-1 expression and release by MMC homologues also promote the expression and release of CCL2. Constitutive release involves de novo protein synthesis and requires a functional Golgi complex, suggesting that similar mechanisms of extracellular secretion operate for both mediators.

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.

Transforming growth factor-beta1 mediates coexpression of the integrin subunit alphaE and the chymase mouse mast cell protease-1 during the early differentiation of bone marrow-derived mucosal mast cell homologues.

BACKGROUND: Mucosal mast cells (MMC) play a central role in gut hypersensitivities and inflammation. They are morphologically, biochemically and functionally distinct from their connective tissue counterparts. Massive hyperplasia of MMC occurs 7-10 days after intestinal infection with nematodes but it has never been possible to replicate this phenomenon in vitro. OBJECTIVE: (1) To determine whether mouse bone marrow-derived mast cells (mBMMC) grown in the presence of transforming growth factor (TGF)-beta1 could develop over the same time frame (7-10 days) as MMC in parasitized mice. (2) To compare the early expression of surface receptors (integrins alphaE and beta7, c-kit and FcepsilonR) with that of the MMC-specific granule chymase mouse mast cell protease-1 (mMCP-1). METHODS: Mouse bone marrow cells were cultured in the presence of IL-9, IL-3 and Stem Cell Factor (SCF) with or without TGF-beta1. mBMMC were quantified after toluidine blue or Leishmans' staining. Expression of MMC-specific mouse mast cell proteases was analysed by ELISA, immunohistochemistry and RT-PCR. Surface antigen expression was characterized by flow cytometry and confocal microscopy. RESULTS: TGF-beta1 promotes the development of abundant MMC-like mBMMC from bone marrow progenitor cells with kinetics, which closely parallel that seen in vivo. mRNA transcripts encoding mMCP-1 and -2 are readily detectable by day 4 ex vivo in cultures grown in the presence of TGF-beta1. Between 30 and 40% and 75-90% of the cells in these cultures on days 4 and 7, respectively, have typical mast cell morphology, are c-kit+, FcepsilonR+, integrin alphaEbeta7+, and express and secrete abundant mMCP-1. The integrin alphaE subunit is coexpressed with mMCP-1. CONCLUSION: The kinetics of mMCP-1+/alphaE+ mBMMC development, regulated by TGF-beta1, are consistent with that seen in vivo in the parasitized intestine. The normally down-regulatory functions of TGF-beta1 in haematopoiesis are superseded in this culture system by its ability to promote the early expression of alphaE and mMCP-1.

A novel function for transforming growth factor-beta1: upregulation of the expression and the IgE-independent extracellular release of a mucosal mast cell granule-specific beta-chymase, mouse mast cell protease-1.

Intestinal mucosal mast cells (IMMC) express granule neutral proteases that are regulated by T-cell-derived cytokines, including interleukin-3 (IL-3) and IL-9, and by stem cell factor (SCF). The IMMC-specific chymase, mouse mast cell protease-1 (mMCP-1), is released in substantial quantities into the blood stream during gastrointestinal allergic responses. We used cultured bone marrow-derived mast cells (mBMMC) to identify cytokines that regulate the expression and extracellular release of mMCP-1. When grown in IL-3-rich WEHI (15% vol/vol) and 50 ng/mL recombinant rat SCF (rrSCF) bone marrow cells supplemented with IL-9 (5 ng/mL) differentiated into mBMMC that expressed a maximum of less than 250 ng mMCP-1/10(6) cells and 189 ng mMCP-1/mL of culture supernatant. Supplementation of the same three cytokines with transforming growth factor-beta1 (TGF-beta1; 1 ng/mL) resulted in substantially enhanced expression (6 micrograms/10(6) mBMMC) and extracellular release (2 micrograms/mL of culture supernatant) of mMCP-1. The response to TGF-beta1 was dose-dependent, with maximal effect at 1 ng/mL, and was associated with immunohistochemical and ultrastructural changes in the secretory granules. IL-9-induced expression of mMCP-1 may be due to endogenously expressed TGF-beta1, because it was blocked by anti-TGF-beta antibodies. In conclusion, the expression and extracellular release of the IMMC-specific chymase, mMCP-1, is strictly regulated by TGF-beta1.

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.

Experience with the quality control testing of C. parvum suspension for clinical trial.

The control of C. parvum suspension presents special problems. The intended application of this preparation is in a therapeutic area where clinicians are accustomed to using defined chemical entities the biological activity of which are usually predictable according to weight of active principle administered, the route and number of doses. But the essential character of C. parvum is that of a vaccine in which the character of the active principle or principles is not clearly identified and can vary according to certain conditions. The control of the preparation therefore depends on the nature and relevance of tests for biological activity. These problems are exacerbated by uncertainty as to which of several possible mechanisms of antitumour activity might prove relevant in clinical practice. Experience with these problems as they have affected the quality control of large numbers of batches of C. parvum suspension prepared for clinical trial, will be discussed.

Studies on the toxicity of a variety of human vaccines as measured by laboratory tests.

The results obtained from our laboratory tests for pyrogenic activity on 8 bacterial vaccines are compared with the clinical reactogenicity of the same 8 vaccines in man. It is concluded that, although none of the tests correlate exactly with clinical reactogenicity, the rabbit pyrogenicity test provides the best indication of such activity. The limulus test, although valuable as a means of detecting contaminant endotoxin in pyrogen free preparations appears to be of little value as a gauge of pyrogenicity in bacterial vaccines.