The effect of different dietary levels of hybrid rye and xylanase addition on the performance and egg quality in laying hens.

1. In this study, 240 ISA Brown hens were fed diets containing different levels of hybrid rye, and the influence of xylanase addition on laying performance and egg quality was evaluated. 2. Birds were allocated to 10 treatment groups with 12 replicates (cages) of two hens and were fed, from week 26 to 50, isocaloric and isonitrogenous experimental diets. A 5 × 2 experimental arrangement was applied, using diets with increasing level of rye (0%, 10%, 15%, 20% or 25%) with or without xylanase supplementation (200 mg/kg of feed; Ronozyme WX (CT) with minimum xylanase activity of 1,000 FXU/g). 3. Increasing dietary level of rye did not affect daily mass of eggs, mean egg weight or feed conversion ratio (P > 0.05). Laying rate decreased in all groups fed with rye. Egg and eggshell quality indices were unaffected by dietary rye grain (P > 0.05); however, rye inclusion significantly decreased yolk colour on the DSM scale (P < 0.05). In comparison with the control group, high dietary levels of rye (25%) significantly increased viscosity of small intestine content (P < 0.05). Diet supplementation with xylanase had no significant effect on egg production indices and egg quality (except for yolk colour) but decreased the viscosity of intestinal content in laying hens fed high levels of rye (P < 0.05). 4. The results of this experiment suggest that rye may be incorporated to a level of 25% in the diet of laying hens without any strong negative effect on egg performance, while xylanase added to high-rye grain reduced the viscosity of intestinal content; however, it did not positively affect the laying performance or egg quality.

The effects of fermentation and enzymatic treatment of pea on nutrient digestibility and growth performance of broilers.

The present study examined the impacts of native, fermented or enzymatically treated peas (Pisum sativum L.) inclusion in broiler diets, on growth performance and nutrient digestibility. For the fermentation process, Madonna pea was mixed with water (1/1) containing 2.57×108 Bacillus subtilis (GalliPro®) spores/kg pea and then, incubated for 48 h at 30 °C. For the enzymatic treatment process, the used water for dough production contained three enzymes, AlphaGalTM (α-galactosidase), RONOZYME® ProAct and VP (protease and pectinases respectively - DSM, Switzerland) and the pea dough incubated for 24 h at 30°C. Nine corn-wheat-soybean diets were formulated by supplying 10%, 20% and 30% of the required CP with either native, fermented or enzymatically treated peas. Performance was recorded weekly and at the end of the experiment (day 35), apparent ileal digestibility (AID) of CP, amino acids (AA), crude fat, starch, Ca, P and K were determined. Data were subjected to ANOVA using GLM procedure with a 3×3 factorial arrangement of treatments. Both processes reduced α-galactosides, phytate, trypsin inhibitor activity and resistant starch in peas. Increasing levels of pea products up to 300 g/kg diet, reduced BW gain and feed intake (P⩽0.05). Broilers fed diets containing enzymatically treated pea had the best feed conversion ratio at day 35. Different types of pea product and their inclusion levels had no effect on AID of all nutrients. The interaction between type of the pea products and inclusion levels was significant for AID of starch. For native pea diets, 10% group showed similar AID of starch to 20% native pea but it had higher AID than 30% native pea. For fermented and enzymatically treated groups, all three levels displayed similar AID of starch. In conclusion, enzymatic treatment and fermentation could improve the nutritional quality of pea. Inclusion of enzymatically treated pea in broiler diets could improve broiler performance compared with other pea products while, it displayed neither positive nor negative impact on nutrient digestibility. The present findings indicate the feasibility of these processes, particularly enzymatic treatment, for improving the nutritional quality of pea as a protein source for broiler nutrition.

Adenosine regulation of the immune response initiated by ischemia reperfusion injury.

It is clinically established that adenosine has negative chronotropic, antiarrhythmic effects and reduces arterial blood pressure. Adenosine addition to cardioplegic solutions used in cardiac operations is clinically well tolerated and has been shown to improve myocardial protection in several studies. However, the mechanism of action remains unclear. Therefore, it is important to define the effect of adenosine on the inflammatory cascade as immune cell activation occurs early during ischemia reperfusion injury. Adenosine appears to mediate the initial steps of the inflammatory cascade via its four G-coupled protein receptors: A1, A2A, A2B, and A3, expressed on neutrophils, lymphocytes and macrophages. The adenosine receptor isotype dictates the immune response. More specifically, the A1 and A3 receptors stimulate a pro-inflammatory immune response whereas the A2A and A2B are immunosuppressive. As the adenosine receptors are important for cardiac pre-conditioning and post-conditioning, adenosine may regulate the inflammatory responses initiated during ischemia-mediated immune injury related to myocardial protection.

Differential expression of collagen, MMP, TIMP and fibrogenic-cytokine genes in the granulomatous colon of Schistosoma mansoni-infected mice.

Schistosomiasis mansoni is a major helminthic disease of the tropics characterised by chronic hepatic and intestinal granulomatous inflammation and fibrosis. The fibrotic response is regulated by the amount of collagen deposited in the tissues and the degradation of that collagen by matrix metalloproteinases (MMP). In the murine model of the disease, although hepatic granuloma formation and the ensuing fibrosis have been thoroughly examined, there is a dearth of information on the intestinal fibrotic process. The expression of fibrosis-related genes in the colons of chronically infected mice has therefore been investigated. Compared with that seen in uninfected mice, the expression of the genes coding for collagen of types I, III and IV was upregulated. Similarly, the messages for MMP-2, MMP-3 and MMP-8 were elevated, indicating the potential for collagen degradation. The genes for two tissue inhibitors of metalloproteinases (TIMP), TIMP-1 and TIMP-4, were, however, expressed at higher levels than those coding for the MMP. As a corollary, expression of the genes coding for three fibrogenic cytokines, transforming growth factor-beta, tumour necrosis factor and interleukin-4, was elevated. These data indicate that an imbalance in MMP:TIMP expression and enhanced levels of the messages for fibrogenic cytokines underlie the mechanism(s) of the colonic fibrosis seen in mice chronically infected with Schistosoma mansoni.

A novel nonsteroidal antifibrotic oligo decoy containing the TGF-beta element found in the COL1A1 gene which regulates murine schistosomiasis liver fibrosis.

Schistosomiasis mansoni disseminated worm eggs in mice and humans induce granulomatous inflammations and cumulative fibrosis causing morbidity and possibly mortality. In this study, intrahepatic and I.V. injections of a double-stranded oligodeoxynucleotide decoy containing the TGF-beta regulatory element found in the distal promoter of the COL1A1 gene into worm-infected mice suppressed TGF-beta1, COL1A1, tissue inhibitor of metalloproteinase-1, and decreased COL3A1 mRNAs to a lesser extent. Sequence comparisons within the mouse genome found homologous sequences within the COL3A1, TGF-beta1, and TIMP-1 5' flanking regions. Cold competition gel mobility shift assays using these homologous sequences with 5' and 3' flanking regions found in the natural COL1A1 gene showed competition. Competitive gel mobility assays in a separate experiment showed no competition using a 5-base mutated or scrambled sequence. Explanted liver granulomas from saline-injected mice incorporated 10.45 +/- 1.7% (3)H-proline into newly synthesized collagen, whereas decoy-treated mice showed no collagen synthesis. Compared with the saline control schistosomiasis mice phosphorothioate double-stranded oligodeoxynucleotide treatment decreased total liver collagen content (i.e. hydroxy-4-proline) by 34%. This novel molecular approach has the potential to be employed as a novel antifibrotic treatment modality.

Dynamics of collagen, MMP and TIMP gene expression during the granulomatous, fibrotic process induced by Schistosoma mansoni eggs.

In schistosomiasis mansoni, granulomatous inflammation and fibrotic resolution are the major pathogenetic factors. The outcome of fibrosis is influenced by the deposition of collagen and degradation mediated by matrix metalloproteinases (MMP). There is a dearth of data on the expression of MMP and the tissue inhibitors of metalloproteinase (TIMP) during the fibrosis associated with schistosomiasis. In this study, the dynamics of collagen, MMP and TIMP gene expression were analysed during murine Schistosoma mansoni infection. Expression within the granulomatous liver tissue of the genes coding for collagen of types I, III and IV was up-regulated at the onset of granuloma development, and the dominant type-I expression peaked at the chronic, fibrotic stage. The amount of deposited hepatic collagen increased with the chronicity of the infection, indicating cumulative fibrosis. Collagenase, gelatinase, stromelysin, matrilysin-specific gene activities were similarly up-regulated, but only MMP-8 (collagenase-2) expression peaked at the height of fibrosis. TIMP-1 gene expression gradually increased during the course of the disease and, along with TIMP-2, peaked at the chronic, fibrotic stage. Granuloma myofibroblasts expressed both MMP and TIMP-1 genes. In ELISA of the splenic cytokines, high levels of fibrogenic interleukin-13 and moderate production of transforming growth factor-beta were found to be concurrent with fibrosis. These data indicate that an imbalance in MMP:TIMP expression and fibrogenic cytokine production are associated with cumulative fibrosis.

The Schistosoma mansoni egg-derived r38 peptide-induced Th1 response affects the synchronous pulmonary but not the asynchronous hepatic granuloma growth.

The p38 peptide derived from Schistosoma mansoni egg-antigens (SEA) is a preferential inducer of the Th1 response. In the present study, we investigated whether induction of a p38-specific Th1 or Th2 response can influence granuloma development in infected or sensitized mice. Mice sensitized with SEA/IL-12 3 weeks after infection but before worm oviposition commenced developed Th1 cytokine responses and had significantly reduced hepatic granuloma size. Similar immunization with p38/IL-12 induced a strong peptide-specific Th1, mixed SEA-specific Th1/Th2 responses without effect on hepatic granuloma development. Presentation of p38 with alum or alum/IL-12 mixture enhanced Th2 cytokine responses and hepatic granuloma sizes. In the synchronized pulmonary model, sensitization of naïve mice with p38/IL-12 induced a strong Th1 cytokine production to p38 and SEA, led to a moderate increase in granuloma growth at days 4 and 8 following egg injection and actually promoted the resolution of the lesion by day 16. Sensitization with p38 in alum induced Th2 cytokine production and generated the largest granulomas whereas the p38/alum/IL-12 sensitized group showed intermediate results in cytokine production and granuloma growth. Thus, in infected mice, the p38 induced strong Th1 response was insufficient to cross-regulate the evolving Th2 environment that generated large granulomas.

Soluble egg antigen-stimulated T helper lymphocyte apoptosis and evidence for cell death mediated by FasL(+) T and B cells during murine Schistosoma mansoni infection.

Granuloma formation around schistosomal eggs is induced by soluble egg antigens (SEA) and mediated by the activity of CD4(+) Th lymphocytes and their cytokines. Regulation of the inflammatory Th cell response during infection is still insufficiently understood. The hypothesis of this study was that activation-induced cell death (AICD) of CD4(+) T cells is involved in the immune inflammatory response. This study investigated the dynamics of splenic and granuloma CD4(+) Th cell apoptosis and Fas ligand (FasL) expression during the acute and chronic stages of murine schistosomal infection. Enhanced apoptosis of freshly isolated CD4(+) Th lymphocytes commenced after egg deposition and persisted during the peak and modulated phases of granuloma formation. After oviposition, CD4(+), CD8(+), and CD19(+) splenocytes and granuloma cells expressed elevated levels of FasL but FasL expression declined during the downmodulated stage of infection. In culture, SEA induced splenic and granuloma CD4(+) T-cell apoptosis and stimulated expression of FasL on splenic but not granuloma CD4(+) T cells, CD8(+) T cells, and CD19(+) B cells. SEA-stimulated splenocytes and granuloma cells preferentially lysed a Fas-transfected target cell line. Depletion of B cells from SEA-stimulated splenic cultures decreased CD4(+) T cell apoptosis. Coculture of purified splenic B cells with CD4(+) T cells and adoptive transfer of purified B cells indicated that antigen-stimulated B cells can kill CD4(+) Th cells. However, CD4(+) T cells were the dominant mediators of apoptosis in the granuloma. This study indicates that AICD is involved in the apoptosis of CD4(+) T cells during schistosomal infection.

Down-regulation of interleukin-12, interleukin-12R expression/activity mediates the switch from Th1 to Th2 granuloma response during murine Schistosomiasis mansoni.

In murine schistosomiasis mansoni the worm egg-induced granulomatous inflammation is bi-phasic: an initial Th1 type is subsequently switched to a Th2 type response. Analysis of the cellular, molecular base of the Th1-associated response (5-6 weeks post infection) revealed mRNA messages for interleukin (IL)-12 p40, IL-12Rbeta2 and interferon (IFN)-gamma in the granulomatous livers. When the Th2 type granulomas matured (8 weeks post infection) message expression weakened or became extinct. Macrophages of the Th1 type granulomas produced maximal amounts of IL-12, but production diminished in the mature granulomas. A similar pattern of IL-12 responsiveness of granuloma lymphocytes was observed. In vitro IL-12 production by Th1 type granuloma macrophages was enhanced by tumour necrosis factor (TNF)-alpha and IFNgamma, whereas lymphocyte IL-12 responsiveness was boosted only by TNF-alpha. Both systems were down-regulated by IL-4 and IL-10 cytokines. Treatment of mice with anti-IL-10 monoclonal antibodies (MoAb) between 6 and 7 weeks of the infection enhanced mRNA expression for IFN-gamma and IL-12Rbeta2, but not for IL-12 p40. It is concluded that IL-12 and IL-12R expression and function regulate the Th1 phase of the liver granulomatous response. This phase is cross-regulated by type-2 cytokines especially IL-10.

Polarization of the immune response to the single immunodominant epitope of p38, a major Schistosoma mansoni egg antigen, generates Th1- or Th2-type cytokines and granulomas.

In schistosomiasis mansoni, helminth eggs secrete soluble egg antigens (SEA) that induce T-cell-mediated granulomatous tissue responses. The cloned 38-kDa peptide (p38) of SEA was shown to induce and elicit Th1-type responsiveness in H-2(k) mice. Subsequently, the immunodominant T-cell epitope (P4) of p38 was shown to elicit pulmonary granuloma formation and Th1-type cytokine production in sensitized or infected mice. Here, we report that the immune response to p38 or P4 can be polarized to a Th1 or Th2 profile when the peptides are presented intraperitoneally in soluble recombinant interleukin-12 (IL-12) or alum adjuvant, respectively. The Th1 or Th2 profile was verified by cytokine secretion, enzyme-linked spot assay, and antibody isotype characterization. Importantly, the polarized immune response generated two types of pulmonary granulomas around injected P4-coated beads. The type 1 granulomas were smaller and contained mononuclear cells and occasional thin strands of deposited collagen. In contrast, the type 2 lesions were larger and contained mononuclear cells, large numbers of eosinophils, and several thick bands of deposited collagen. By reverse transcription-PCR cytokine, message in the type 1 granuloma-bearing lungs was found for gamma interferon, tumor necrosis factor alpha, and inducible nitric oxide synthase but not for IL-4 or IL-5. Conversely, lungs with type 2 granulomas had message only for IL-4 and IL-5. These results show that in the proper cytokine environment, the response to a strong Th1 inducer peptide can be deviated to a Th2 profile.

Enhanced Th1 and dampened Th2 responses synergize to inhibit acute granulomatous and fibrotic responses in murine schistosomiasis mansoni.

In murine schistosomiasis mansoni, CD4(+) Th1 and Th2 cells participate in the ovum-induced granulomatous inflammation. Previous studies showed that the interleukin-12 (IL-12)-induced Th1 response strongly suppressed the Th2-cell-mediated pulmonary granuloma development in naive or primed mice. However, liver granulomas were only moderately suppressed in egg-vaccinated, recombinant IL-12 (rIL-12)-treated infected mice. The present study shows that repeated rIL-12 injections given during early granuloma development at 5 to 7 weeks after infection prolonged the Th1 phase and resulted in gamma interferon-mediated suppression of liver granulomas. The timing is crucial: if given at 6 to 8 weeks, during the Th2-dominated phase of florid granuloma growth, the treatment is ineffective. Daily injections of rIL-12 given between 5 and 7.5 weeks during the period of granuloma growth achieved a somewhat-stronger diminution in granuloma growth with less deposition of collagen but caused 60% mortality and liver pathology. In contrast, combined treatment with rIL-12 and anti-IL-4-anti-IL-10 monoclonal antibody (MAb) injections given during the Th2 phase strongly inhibited liver granuloma growth without mortality. The diminished inflammatory response was accompanied by less deposition of collagen in the liver. Moreover, neutralization of endogenous IL-12 by anti-IL-12 MAbs effectively decreased the early Th1 phase (between 5 and 6 weeks after infection) but not the developing Th2 phase (5 to 7 weeks) of granuloma development. These studies indicate that the granulomatous response in infected mice can be manipulated by utilizing the Th1-Th2-subset antagonism with potential salutary results in the amelioration of fibrous pathology.

Endogenous IL-10 regulates IFN-gamma and IL-5 cytokine production and the granulomatous response in Schistosomiasis mansoni-infected mice.

In murine Schistosomiasis mansoni circumovum, granuloma formation is regulated by pro- and anti-inflammatory cytokines. Among the latter, interleukin-10 (IL-10) has been shown to regulate the inflammatory response. In this study we examined the role of endogenously produced IL-10 in T-helper 1 (Th1)- and Th2-type cytokine production and granuloma formation. The dynamics of IL-10 production through the course of the infection were different in granuloma versus splenic cells. In the former, production peaked during the early developmental stage (6 weeks of infection) of the granuloma and then declined. In splenocytes production peaked at 12 weeks, before down-modulation of the granuloma response. In the developing granuloma both macrophages and T cells secreted IL-10. In anti-IL-10 monoclonal antibody (mAb)-supplemented granuloma cell cultures endogenous IL-10-mediated regulation of interferon-gamma (IFN-gamma) was manifest only at 6 weeks; that of IL-2 continued throughout the infection (6-20 weeks). IL-4 production was unaffected, but IL-5 production was regulated at the 6 and 8 weeks time point. Splenocytes showed regulation of IFN-gamma and IL-2 production at the peak of the granulomatous response (8 weeks). IL-4 production was not regulated, whereas IL-5 production was regulated only at 6 weeks. Repeated injections of anti-IL-10 mAb given to mice at 6, 12 or 20 weeks of the infection significantly enhanced liver and lung granuloma growth, tissue eosinophilia, and IFN-gamma, IL-5 production at the early developmental phase (6 weeks) of the lesions. Thus, in schistosome-infected mice endogenous IL-10 is shown to regulate Th1- and Th2-type cytokine production and granuloma formation during the early Th0/Th1 phase of the immune response.

Identification of the immunodominant T cell epitope of p38, a major egg antigen, and characterization of the epitope-specific Th responsiveness during murine schistosomiasis mansoni.

A recently cloned major Schistosoma mansoni egg Ag p38 induced and elicited strong Th1-type responsiveness in mice of H-2k haplotype. Now, we have identified the immunodominant T cell epitope of p38 and analyzed the dynamics of epitope-specific Th responsiveness during murine schistosomiasis mansoni. Overlapping recombinant and synthetic peptides that encompassed the full-length 354 amino acid of p38 were tested for proliferation and cytokine production in peptide- or p38-sensitized mice. The immunodominant T cell epitope of p38 that elicited pulmonary granuloma formation was localized within peptide P4 (amino acids 235-249). The P4-specific cytokine response of splenocytes that had been sensitized s.c. with p38, P4 or soluble egg Ags in IFA, or i.p. with 3000 eggs was predominantly as the Th1 type, with strong IL-2 and IFN-gamma, but trace amounts of IL-4 and IL-5 secretion. At 6.5 wk of infection, splenocytes and mesenteric lymph node cells responded to p38/P4 peptides with predominantly Th1-type responsiveness. This response did not switch to a Th2-type pattern from 8 wk onwards; rather, it underwent down-modulation. Moreover, the hepatic granuloma lymphocytes at 6.5 wk responded to p38/P4 predominantly with Th1-type cytokine production, indicating that they participate in early granuloma formation. From 8 wk onwards an immune deviation to the p38-specific response was observed that was manifested by rising IgG1, IgE, and IgG2a Ab production as opposed to declining Th1- and Th2-type cytokine secretion.

Influence of growing season, tannin content and autoclave treatment on the nutritive value of near-isogenic lines of faba beans (Vicia faba L.) when fed to leghorn chicks.

1. The objective of this study was to determine the influence of heat-treatment (autoclaving) on the utilisation by chicks of near-isogenic lines of faba beans that were either tannin-free (Winter-white or Spring-white) or had tannins in the seed coat (Winter-coloured or Spring-coloured). The effect of heat treatment on a common tannin-containing cultivar, Diana, was also studied. 2. The nitrogen-corrected apparent metabolisable energy (AMEn) values of diets containing 600 g faba beans/kg diet were affected by tanning content and autoclave treatment of faba beans, with 59% of total variance in AMEn being attributable to the effect of autoclaving. The AMEn values were 9% higher for chicks fed on the autoclaved compared with those fed on diets containing the raw faba beans, 5% higher for the near-isogenic faba beans with no tannins compared with those with tannins, and 4% higher for the Winter compared with the Spring cultivars. 3. The above study was carried out on droppings obtained by total collection. Similar but not identical trends were obtained when AMEn was calculated using the chromic oxide index method (r = 0.91). 4. Both autoclaving and the use of tannin-free faba beans improved the apparent protein digestibility (APD) of the diets by similar amounts (4% as determined by the total collection method and 6% as determined by the chronic oxide index method). 5. The digestibility of most amino acids either alone or together was affected by a cultivar x tannin x processing interaction. The cultivar by tannin interaction demonstrated that autoclaving increased the digestibility of amino acids to a greater degree when the tannin-containing (for example, 9% for lysine) than when the tannin-free faba beans (4% for lysine) were used in the diets. 6. The improvements in AMEn, APD and total amino acid digestibilities of faba beans following heat treatments ranged from 18% to 33%, 5% to 16% and 5% to 11%, respectively. The results demonstrate that the method of analysis can affect the values obtained but that they do not change the overall pattern of results. The results also suggest that, in most cases, more than half of the response to heat treatment is associated with the inactivation of tannins while the balance of the response is attributable to an effect of heat treatment on the non-tannin-containing factors. In summary, heat treatment can improve the utilisation of faba bean by chickens especially when they contain tannins. 7. Chicks, unlike rats, do not seem to adapt to tannins as the excretion of 3 amino acids that are the main constituents of a proline-rich protein were not affected by dietary tannins.

Th2 cells are required for the Schistosoma mansoni egg-induced granulomatous response.

The development of Schistosoma mansoni ova-induced granulomas is regulated by cytokines secreted by distinct Th cell subsets. To determine the importance of Th1 and Th2 cells in granuloma formation, we have studied the immune response to S. mansoni ova in Stat4- and Stat6-deficient mice, which lack Th1 and Th2 cells, respectively. Lymphocytes from both naive and infected Stat6-deficient mice produced minimal levels of Th2 cell cytokines and Ag-specific IgG1 and IgE, but showed enhanced production of IFN-gamma and Ag-specific IgG2a and IgG2b following schistosome egg injection. This shift away from a Th2 cell-mediated immune response was coupled with the development of pulmonary and hepatic granulomas that were greatly decreased in size compared with those in control littermates. Hepatic granulomas in Stat6-deficient mice were composed of predominantly mononuclear cells with very sparse appearance of eosinophils, and their diminished size was accompanied by decreased amounts of liver hydroxyproline content as a measure of collagen deposition. In contrast, lymphocytes from infected Stat4-deficient mice produced Th2 cell cytokines in amounts comparable to those produced by control littermates, but low levels of IFN-gamma. While infected Stat4-deficient mice developed pulmonary granulomas following schistosome egg injection that were modestly impaired in size, the granuloma size and amount of collagen deposition in the liver were equivalent to those seen in control littermates. These studies demonstrate that Th2 cells are required for the full development of the granulomas and tissue-destructive fibrotic pathology associated with the immune response to S. mansoni ova.

Fibronectin co-stimulates via the alpha 5 beta 1 receptor IL-2, IL-4 production by splenic, granuloma lymphocytes of Schistosoma mansoni infected mice.

In murine Schistosomiasis mansoni, soluble worm egg antigens (SEA) induce L3T4+ T helper cell-mediated chronic granulomatous inflammations around parasite eggs. Within the fully developed granuloma lymphocytes, macrophages, and eosinophils, fibroblasts are embedded in extracellular matrix (ECM) composed of fibronectin, laminin, glycosaminoglycans and collagens. The present study examined in vitro the putative co-stimulatory role of fibronectin (FN) in acute and chronic infection splenic and granuloma lymphocyte responses. Plate-bound FN enhanced the anti-CD3 MoAb stimulated normal and acute or chronic infection splenic lymphoproliferation by 20-32%. The co-stimulatory effect was evident in SEA stimulated acute but not chronic infection spleen cells. Proliferation of stimulated granuloma lymphocytes could not be up-regulated by immobilized FN. Plate-bound FN significantly enhanced IL-2 and IL-4 production by SEA-stimulated acute, but not chronic, infection granuloma lymphocytes. However, FN had no influence on the high level of IL-2, IL-4 production of anti-CD3 MoAb stimulated acute or chronic infection splenic or granuloma lymphocytes. Because in the antigen-stimulated acute infection spleen or granuloma cultures the co-stimulatory effect by FN was abrogated by the tripeptide (RGD) arg-gly asp, and anti alpha 5 beta 1 antibody, enhancement is attributed to signalling via the alpha 5 beta 1 integrin receptor of lymphocytes.

A cloned major Schistosoma mansoni egg antigen with homologies to small heat shock proteins elicits Th1 responsiveness.

In schistosomiasis mansoni, soluble egg antigens of the worm induce chronic T-cell-mediated granulomatous tissue responses. Since the first preparation of crude soluble egg antigen extract, a dearth of highly purified antigens has hampered the identification of granuloma inducer molecules. Here we report that a cloned 38-kDa egg polypeptide (r38) with homologies to small heat shock proteins is a strong immunogen. The recombinant and the sodium dodecyl sulfate-polyacrylamide gel electrophoresis separated and eluted native 38-kDa (p38) polypeptides, used in microgram amounts and unaided by adjuvant, sensitized mice for a Th1-type immune response, with strong interleukin-2 (IL-2) and gamma interferon secretion but no IL-4 and IL-10 secretion. Extensive cross-reactivity between these two polypeptides was evident. THis pattern was confirmed by reverse transcription-PCR that showed strong IL-2 and gamma interferon message expression but trace amounts of IL-4 message expression in r38-sensitized splenocytes. In mice, the polypeptide induced pulmonary mononuclear granuloma formation around antigen-coupled beads or worm eggs. We propose that the superior immunogenicity of r38 is linked to its relatedness to small heat shock proteins and that the 38-kDa polypeptide may induce the Th1 cytokine responses observed during the early development phase of the egg-induced granuloma.

T-lymphocyte responsiveness in murine schistosomiasis mansoni is dependent upon the adhesion molecules intercellular adhesion molecule-1, lymphocyte function-associated antigen-1, and very late antigen-4.

Granuloma formation in murine schistosomiasis is dependent on CD4+ Th lymphocytes and requires recruitment and accumulation of inflammatory cells at the site of egg deposition. The present study examined the role of three adhesion molecules, intercellular adhesion molecule-1 (ICAM-1), lymphocyte function-associated antigen-1 (LFA-1), and very late antigen-4 (VLA-4), that participate in cellular recruitment, interaction, and lymphocyte activation during in vitro activation of acutely and chronically infected spleen and liver granuloma lymphocytes. Blockade of ICAM-1, LFA-1, or VLA-4 by rat monoclonal antibody inhibited spleen and granuloma lymphocyte interleukin-2 (IL-2) and IL-4 production as well as lymphoproliferative responses at similar levels (66 to 87%). The down-modulated cytokine and proliferative responses of chronically infected lymphocytes were inhibited to the same extent as their acutely infected counterparts. Cell sorting analysis demonstrated that acutely and chronically infected splenic and granuloma lymphocytes expressed similar levels of LFA-1, ICAM-1, and VLA-4 and that more ICAM-1 was expressed on infected than on uninfected mouse lymphocytes. By exposure of cells to paired monoclonal antibodies at suboptimal doses, it was determined that whereas all three adhesion molecules may participate, only ICAM-1 and LFA-1 showed synergistic interactions in determining lymphocyte responsiveness. These data suggest that spleen and liver granuloma lymphocytes are equally well armed with functional adhesion receptors. Thus, ICAM-1, LFA-1, and VLA-4 play an important accessory role in inflammatory cytokine production and lymphocyte proliferation, and therefore these adhesion molecules may participate in the initiation and maintenance of the granulomatous inflammation.

The role of cytokines in the formation of the schistosome egg granuloma.

Schistosomiasis mansoni is a helminth-induced disease infecting over 120 million people in the tropics. Morbidity and mortality are caused by parasite eggs that evoke in the liver and intestines of infected persons, T cell-mediated granulomatous inflammation and irreversible fibrosis. In the murine model granulomatous inflammation is induced by CD4+ T helper lymphocytes. This short review summarizes recent observations that implicate a variety of lymphokines and cytokines as mediators of the granulomatous inflammatory response. Mediator production was examined in splenocyte as well as granuloma cell cultures of infected or egg granuloma-bearing mice. In the synchronous pulmonary granuloma model generated around i.v. injected eggs in naive mice IL-1 mRNA expression and IL-1 production were detectable within the first 4 days of granuloma growth. After 4-6 days TNF-alpha mRNA message appeared and cytokine production was observed. With the aging of the granuloma, production of both cytokines diminished. Thus, these cytokines are considered to be the primary recruiters of cellular aggregation in granuloma growth. The role of TNF-alpha in granuloma formation was also confirmed in infected mice. Whereas treatment of animals with anti-TNF-alpha antiserum diminished hepatic granuloma size, repeated injection of murine rTNF-alpha into chronically-infected mice enhanced the downmodulated granuloma response. With the administration of specific anti-lymphokine mAbs and recombinant murine lymphokines, as well as serial assays of lymphokine production by splenic, granuloma lymphocytes of infected mice, the role of INF-gamma, IL-2 and IL-4 was delineated. Interferon-gamma was found to be produced very early at the inception of the liver granulomatous response. By the time granulomas reached maximal size (8 wks post infection) production declined. Concurrently IL-2, IL-4 production peaked with maximal granuloma growth and declined with the onset of the immune modulation of the inflammation. Whereas these latter lymphokines appear to play a proinflammatory role, IFN-gamma when administered in large doses diminished granulomatous inflammation, plays a regulatory role in the maintenance of the granulomatous response. The T helper cell population of the granulomas may also influence the lymphokine profile of the developing granuloma. So far precursor type TH0, and TH2 subset of helper cells have been cloned from liver granulomas. The former secreted both IL-2, IL-4 and IFN-gamma lymphokines and adoptively transferred the granulomatous response.(ABSTRACT TRUNCATED AT 400 WORDS)