Local cell-associated immunity in the Peyer''s patches of mouse intestines.

After oral infection of mice with a Salmonella strain, low numbers of organisms could be found in the Peyer's patches of the small intestine. In the course of a few days the organism in the Peyer's patches multiplied to about 10(5) and then were steadily eliminated so that by 10 days, very few organisms could be found in the lymphoid follicles. Because no organisms could be found in the spleen or other organs during this period, it is probable that the decline in numbers was due to killing of the organisms in situ. This development of antibacterial ability was not inhibited by treatment with cyclophosphamide, in contrast to the rapid growth of organisms which occurred in the spleen after intravenous injection in the presence of cyclophosphamide. The difference in behavior between Peyer's patches and the spleen upon treatment with cyclophosphamide is explained in terms of the extent of natural priming of Peyer's patches due to continual contact of the intestine with gram-negative enteric organisms. Once the spleen was primed, cyclophosphamide did not interfere with the elimination of a second challenge with the same organism.     

Adaptive changes in muscle fibers infected with Trichinella spiralis.

Ultrastructural changes which occurred in infected skeletal muscle fibers after infection with larvae of Trichinella spiralis were followed on a daily basis utilizing synchronous infections. No changes were observed in muscle fiber architecture during the first 2 days of intracellular infection. However, on Day 3, a space containing various sarcoplasmic elements developed between the plasma membrane and myofilaments. Widening near the regions of triads was also observed at this time. On Day 4 the space at the outer edge had increased, as did the ones at the triads. In addition, the myofilaments throughout the infected fiber were in a state of partial disarray. Finally, the nuclei were enlarged and had migrated to the central portion of the infected cytoplasm. On Day 5 day, sarcomeres were highly disorganized, and an increase in sarcoplasmic reticulum (SR) was noted. By Day 8, only the extreme periphery of the infected fiber contained Z bands with actin filaments attached. Proliferation of the t tuble system was also evident. At Day 10, myofilaments were completely replaced with SR. Further, the plasma membrane became hyperinvoluted and was associated with a 36-fold increase in the thickness of the glycocalyx. No further enlargement of nuclei occurred after Day 10. Finally, a host-derived double membrane completely surrounded the larva.     

Impaired protective immunity and T helper 2 responses in alymphoplasia (aly) mutant mice infected with Trichinella spiralis

The alymphoplasia (aly) mutation of mice prevents the development of systemic lymph nodes and Peyer's patches. The mutant homozygotes (aly/aly) are partially deficient in both humoral and cell-mediated immune functions. In the present study, we show that adult worm expulsion was slightly delayed and that T helper 2 (Th2)-type responses were partially defective in aly/aly mice after infection with Trichinella spiralis. Male aly/aly and aly/+ mice (8-weeks old) were infected with 400 muscle larvae. There was no difference in worm recovery between the two groups on day 5. However, worm recovery in aly/aly mice was significantly higher than that in aly/+ mice on day 14. Mucosal mast cells increased in number and peaked 14 days after infection in aly/+ mice. aly/aly mice were deficient in their mucosal mast cell response through out the primary infection. To examine the existence of mast cell precursors, aly/aly mice were treated with recombinant interleukin-3 (rIL-3) before infection. The mast cell response was poorly induced in aly/aly mice treated with rIL-3. An immunoglobulin E (IgE) response was not detected in aly/aly mice during the course of infection. Serum IgG1 levels in aly/aly mice were significantly lower than that of aly/+. The serum IgG2a levels increased in both strains of mice. However, IgG2a production in aly/aly mice on day 14 was half as much as that in aly/+mice. Stimulation of splenic T cells in vitro with anti-CD3 monoclonal antibody (mAb) showed that spleen cells from aly/+ mice on day 5 produced more IL-4 than spleen cells from aly/aly mice. IL-4 production from aly/aly mice on day 14 was half that from aly/+ mice. Interferon-gamma (IFN-gamma) was produced in both aly/aly and aly/+ mice on day 14. Proliferation assay showed that T cells of aly/aly mice responded poorly when cultured with antigen-presenting cells. These results suggest that aly gene is needed for the induction of protective immunity and Th2 responses in mice infected with T. spiralis.     

Efficacy of Lasia spinosa leaf extract in treating mice infected with Trichinella spiralis

Trichinellosis is a widespread zoonoses for which no effective drug treatment is available at this time. Though anthelmintics such as mebendazole and albendazole are commonly used to treat human trichinellosis, none of these drugs are fully effective against the encysted or new-born larvae of Trichinella spiralis. In recent years, there has been a growing interest in developing newer anthelminthics from medicinal plants, particularly the ones used in traditional medicines in many parts of the world, due to the increasing spread of anthelminthic resistance and/or decreasing activity against encapsulated larval stages of parasites. The aim of the present study was to investigate the efficacy of leaf extract of Lasia spinosa (Araceae) against different life cycle stages of T. spiralis, i.e. adult (days 3 and 4 post-infection), migrating larvae (days 8, 9 and 10 post-infection) and encysted muscle larvae (days 31–37 post-infection). The study showed that L. spinosa leaf extract is effective against all the three life cycle stages of parasite. Against the adult stage, an oral administration of plant extract at 800 mg/kg dose revealed a 75.30% reduction in the number of adult worms, as compared to untreated controls at day 10 post-infection. Whereas against migrating larvae, the same dose of plant extract given for 3 days, reduced the number of larvae recovered from musculature of treated animals by 72.23%. However, in comparison of preceding two stages, the extract showed comparatively less efficacy against the encysted larvae of parasite. In this case, the 800 mg/kg dose of extract given for 7 days (after 30 day of post-infection) revealed only 64.84% reduction in the number of encysted larvae, as was evident from larval count on day 49 post-infection. Therefore, the results of this study indicate that leaf extract of L. spinosa possesses significant anthelminthic efficacy against the adult stages and migrating larvae of T. spiralis. On the other hand, the encysted muscle larvae of parasite are comparatively less sensitive to L. spinosa leaf extract treatment.     

Immunity to Trichinella spiralis. II. Expression of immunity against adult worms.

Thoracic duct lymphocytes or purified T or B cells obtained from specifically immunized (Lewis x DA)F1 hybrid rats can protect normal recipients against an oral challenge infection with T. spiralis. The immune cells increase the rate of expulsion of adult worms from the small intestine. Immune TDL do not affect adult worm fecundity, as they do in other strains of rats, or the penetration and development of newborn larvae in muscle cells. Irradiated F1 rats reconstituted with either immune TDL or class-enriched populations of immune T or immune B cells also expel adult Trichinella more rapidly than do unprotected controls. However, unfractionated TDL and inocula enriched in B cells are more efficient than T cells in promoting worm expulsion. The above finding, taken in conjunction with the tissue disposition of labelled lymphocytes in the tissues of recipient rats, implies that immune T cells have a 'helper' function in promoting the formation of protective B cells.     

Genetic factors controlling the intestinal mast cell response in mice infected with Trichinella spiralis.

Inbred strains of mice showed marked variation in their mast cell (MC) response to infection with Trichinella spiralis. Variation was under genetic control, the ability to respond to infection being inherited as a dominant trait. MHC-linked genes may influence the absolute level of response, but overall response kinetics appear to be controlled by genes which are not linked to the MHC. An enhanced MC response was transferred adoptively with immune mesenteric lymph node cells (IMLNC), but reciprocal adoptive transfers between H-2 compatible rapid (NIH) and slow (B10.G) responder strains showed that the degree of enhancement was determined by the response phenotype of the recipient, not that of the donor. Similarly, in bone marrow (BM) chimaeras, produced by reconstituting lethally irradiated F1 (B10.G x NIH) mice with parental BM, the MC response to T. spiralis was determined by the response phenotype of the BM donor, whether or not rapid responder IMLNC were transferred. The data are discussed in terms of a T lymphocyte regulated, bone marrow stem cell origin of mucosal MC and interpreted as showing that genetic regulation of the MC response is expressed at the level of stem cell or precursor response to T cell derived mastopoietic factors.     

Depression of cell-mediated immunity following inoculation of Trichinella spiralis extract in the mouse.

Mice pretreated with Trichinella spiralis extract (TsE), or infected with the parasite, rejected primary skin allografts in 18-23 days and secondary allografts in 12-16 days. Mice pretreated with saline or with bovine serum albumin (BSA) rejected the primary allografts in 12-18 days and did not accept the secondary grafts. Inoculation of increasing doses of parental spleen cells from mice pretreated with saline or with BSA in F1 hybrids produced proportionately stronger graft-versus-host reactions (GvHR) whereas increasing doses of cells from TsE pretreated mice reduced proportionately the capacity of the inoculum to induce a GvHR. Immunodepression of the parental cells was obtained with 7 and with 4, but not with 2, daily injections of TsE. The depression waned rapidly after the treatment with TsE but a significant degree still remained after 3 days. Immunodepression by TsE cannot be solely explained by antigenic competition and although our results are consistent with the induction of suppressor cells, it is probable that other mechanisms are also involved.     

Increase of mucosal mast cells in the jejunum of patients infected with Trichinella spiralis

The mast cell response in the mucosa and connective tissue of 36 jejunal biopsies of patients with clinically diagnosed trichinellosis, teniasis and lambliasis has been studied. Biopsy material was fixed in standard formalin or Carnoy's fixative, enabling differentiation between mucosal mast cells (MMC) and connective tissue mast cells (CTMC). With both fixatives CTMC could equally well be recognized. With Carnoy's fixative an additional population of mast cells (MMC) could be visualized both in the mucosa and the connective tissue. In the mucosa small mucosal mast cells were observed as well. Compared to the numbers of mast cells in the mucosa and the connective tissue of teniasis and lambliasis patients, the number of mast cells in trichinellosis patients only visualized using Carnoy's fixative was markedly higher. It was concluded that also in man trichinellosis is accompanied by an increase of cells with MMC characteristics. Further studies are needed to clarify the morphological and histochemical features of these cells and their possible role in this parasitic infection.     

Lymphocyte proliferation in Peyer''s patches of Giardia muris-infected mice.

Gastrointestinal immune events in giardiasis are important in controlling infection. In this study, Peyer's patch lymphocytes from mice infected with Giardia muris developed specific, proliferative responses to G. muris antigen. This proliferation correlated with clearance of infection. Further understanding of the gut immune response will be helpful in developing immunoprophylactic strategies in the control of giardiasis.     

Effects of salmeterol on host resistance to Trichinella spiralis in rats.

Salmeterol is a long-acting beta2-adrenoreceptor agonist. The compound has previously been screened for immunotoxic potential in a repeated dose toxicity study in rats for 28 days. The total serum IgG levels were increased at dose levels of 2 and 10 mg/kg/day. Presently, salmeterol was studied in an immune function assay addressing the host resistance to Trichinella spiralis parasites. Rats were daily treated with salmeterol for 28 days at dose levels of 0, 2, 6 and 10 mg/kg/day. On day 29, the animals were infected with T. spiralis parasites. After six weeks, host resistance was examined. The numbers of T. spiralis muscle larvae in the tongue nor the inflammatory reactions around the encapsulated larvae were affected by salmeterol treatment. The yield of muscle larvae in the whole carcass was not changed either. The IgM, IgA and IgE antibody responses to T. spiralis were unaffected. Only at the highest dose level tested, the anti-T. spiralis IgG antibody response was decreased significantly. However, salmeterol's interference with the generation of anti-T. spiralis antibodies of the IgG subclass apparently did not adversely affect the resistance to infection.     

Injection of recombinant interleukin 3 hastens worm expulsion in mice infected with Trichinella spiralis

 The effects of interleukin 3 (IL-3) on worm expulsion were studied in mice infected with Trichinella spiralis. C3H/He mice were treated with a total of 10⁴ U IL-3 or saline by daily peritoneal injection from day-5 to day-1. Muscle larvae were given orally to both groups of mice on day 0. The muscle worm burden in infected mice was assessed on day 28. The worm burden in mice treated with recombinant IL-3 (rIL-3) was significantly suppressed as compared with that in control mice. A reduction in the worm burden was observed in mice treated with rIL-3 from day-5 to day-1 but not in those treated day 16 to day 20. This suggests that IL-3 could up-regulate the host defense response to intestinal worms but not to parenteral stage worms. When various doses of rIL-3 were given to mice and the intestinal worm burden was assessed on day 5, protection was observed only in mice treated with a total of 10⁴ U rIL-3 but not in those given either 3.5×10³ or 10³ U. A kinetics study on the recovery of intestinal adult worms showed that rIL-3 treatment hastened worm expulsion. The mucosal mast-cell response observed in the small intestine of rIL-3-treated mice was induced earlier and was greater than that seen in the control. The host defense response induced by rIL-3 could not be inhibited by treatment with anti-IL-4 or anti-IL-5 monoclonal antibody. Under such an experimental condition in this study, at least, the numbers of mast cells per villus crypt unit observed in mice treated with rIL-3 and anti-IL-4 antibody were slightly lower than those seen in mice treated with rIL-3, but the difference was not significantly different. These results suggest that IL-3 can induce the expulsion of T. spiralis worms without the cooperation of IL-4 or IL-5 in mice. Received: 24 March 1995 / Accepted: 20 May 1995     

Distribution of muscle larvae and antibody dynamics in goats experimentally infected with Trichinella spiralis

Herbivorous animals can play a very important role in spreading trichinellosis. In the study presented here, the susceptibility and distribution of Trichinella spiralis infection was examined in 16 goat kids. The goats were inoculated with 10,000 T. spiralis larvae isolated by artificial digestion methods. The animals were necropsied per two animals in weekly intervals, and the larval burdens in different muscle tissue and anti-Trichinella antibodies measured with the indirect enzyme-linked immunosorbent assay (ELISA) serological method using excretory–secretory (E/S) antigen for detecting anti-Trichinella antibodies were assessed during the experiment. T. spiralis larval burden was maximal at 6 weeks postinoculation (480–5,057 larvae/g according to locality), and the larvae were also found in the myocardium (0.77 larvae/g). In this paper, our next step was to compare the specificity and the time of seroconversion by means of ELISA based on E/S antigen prepared from T. spiralis. Antibody response was detected in all 16 goats. The ELISA test carried out showed the first increments in optical density 2 weeks postinfection (p.i.), reached their peak 4 weeks p.i., and remained elevated from that day until the end of the experiment (10 weeks p.i.). These results indicated that specific anti-Trichinella antibodies in goats persist for a relatively long time.     

Nasal immunization with homogenate and peptide antigens induces protective immunity against Trichinella spiralis

Mice were successfully immunized against the intestinal nematode Trichinella spiralis by intranasal administration of a 30-mer peptide antigen with cholera toxin B. Immunized mice developed antigen-specific serum immunoglobulin G1, intestinal immunoglobulin A, and a type 2-biased cytokine response. Intranasal immunization therefore generates the Th2-mediated responses required for immunity against intestinal parasites.     

Maternal to neonatal transmission of T-cell mediated immunity to Trichinella spiralis during lactation.

The potential of maternally derived cellular factors to mediate immunity to Trichinella spiralis in neonates during lactation was investigated in this study. Female FI rats, infected with T. spiralis, were able to transfer immunity to their suckling offspring, evidenced by a significant reduction in the intestinal parasite burdens of their neonates. When challenged between 2 and 3 weeks of age with 200 T. spiralis larvae, pups suckling on immune mothers harboured 28% and 26% (at 3 and 8 days post-challenge) of the worm numbers present in control neonates suckling on naive mothers. Cross-fostering experiments in which pups born of naive mothers but nursed by immune mothers showed significant immunity, demonstrated that this passage occurred through milk. The role of cell-mediated immunity in this immune transfer was analysed using T cells purified from MLN cells of syngeneic donor rats infected with T. spiralis. When 200 x 10(6) sensitized MLN T cells were adoptively transferred into lactating recipients, it led to the passive immunization of suckling neonates (26% and 13% of control values retained at 3 and 8 days post-challenge), while maternal injection of T cells primed to an irrelevant antigen (KLH) had no effect on neonatal immunity. Neonates fed per-orally with primed T lymphocytes early in lactation and prior to challenge were also rendered immune (34% and 44% of control values retained at 3 and 8 days post-challenge). A single dose of T. spiralis-primed T cells given to neonates in early lactation was sufficient to elicit a significant immune response in them at 2 weeks of age. These results support the hypothesis that cellular immunity mediated by antigen-specific T cells in milk can provide functional immune protection to the neonate against an intestinal pathogen.     

Hypereosinophilia in Rats with Trichinella spiralis Infections

Six LOU strain rats developed blood eosinophil counts of over 10 × 10⁹/1. Six months previously they had been infected with 2 larvae/g of Trichinella spiralis, 2 days after being exposed to high ambient temperatures. The morphology of dividing and peripheral eosinophils was normal, but eosinophils in the blood and peritoneum had increased binding capacity for complexed IgG. Detailed studies were done on 2 rats with blood eosinophil counts of 24 and 32·5 × 10⁹/1. Their hearts were firm and contained adherent thrombus in both ventricles, but there were no histological signs of endomyocardial damage. There were extensive eosinophil infiltrates in lymphoid organs and lungs, and one of the rats had hepatic cirrhosis. Attempts to transfer the disease to syngeneic rats with tissues from affected rats, and to reproduce the disorder in other rats were unsuccessful. The ability of LOU strain rats to expel adult T. spiralis worms, and their eosinophil response in the early stages of infection were normal.It is concluded that hypereosinophilia can sometimes occur in rats, and that this has some features which are also found in hypereosinophilic syndromes of man. It is suggested that the rats with hypereosinophilia had developed the disease as an unusual response to T. spiralis infection. Although the disorder has not been reproduced in other experiments, these findings are described in order to show that they can occur, as the development of an animal model of the hypereosinophilic state would be of value in the study of how the eosinophil response is regulated, and the mechanism of tissue damage that may be seen in association with persistently raised blood eosinophil counts.     

旋毛虫新生幼虫T668重组抗原对小鼠的保护性免疫

目的研究旋毛虫新生幼虫T668重组抗原的免疫原性,制备基因工程疫苗。方法以旋毛虫新生幼虫期特异性基因T668在大肠杆菌中的表达蛋白为抗原免疫小鼠,每间隔10d免疫1次,共免疫3次。末次免疫后10d,每只小鼠攻击感染200条旋毛虫感染性肌幼虫,感染后5周用消化法检查肌幼虫(ML)负荷。结果T668重组抗原免疫组肌幼虫减虫率明显高于佐剂组和对照组。结论T668重组抗原能诱导小鼠产生一定程度的保护性免疫,且可激发特异性体液免疫。     

Immunological Sequelae of Trichinella spiralis Infection in Mice II. Potentiation of Cell-Mediated Response to BCG After Infection with Trichinella spiralis

Mice were infected with 200 Trichinella spiralis 14 days after intravenous administration of 4 x 10(6) viable BCG cells. Individual groups were tested for delayed hypersensitive footpad responses at 14, 20, 29, 57, or 85 days after T. spiralis infection. An initial suppression in the 14-day test group was observed; however, mice tested at later time intervals exhibited potentiation of the 24-h footpad reaction to old tuberculin over that elicited by appropriate controls. This suggested that T. spiralis induces a potentiation of the cellular immune response to BCG. Adoptive transfer studies support the cell-mediated nature of the observed footpad reaction and indicated that the initial suppression was not due to physiological factors preventing the expression of the footpad swelling reaction.     

Glycosidases of Trichinella spiralis

The exoglycosidases beta-N-acetyl-D-glucosaminidase, beta-N-acetyl-D-galactosaminidase, alpha-1-fucosidase, alpha-D-glucosidase and alpha-D-mannosidase, and a non-specific acid phosphohydrolase are present at high levels in extracts of adult and muscle-stage (L1) Trichinella spiralis and at lower (5-30-fold) levels in extracts of the newborn larvae. The enzyme activities from the L1 extract were characterized. All displayed maximum activity at acid pH. beta-N-acetyl-D-glucosaminidase and beta-N-acetyl-D-galactosaminidase had identical molecular weights (110 000), pH optima (5.0), and isoelectric points (5.7) indicating that both of these substrate specificities reside in the same protein molecule. alpha-1-Fucosidase had a molecular weight of 125 000 and exhibited two pH optima (5.0 and 6.0) and four isoelectric points (5.9, 6.4, 6.7 and 7.1) indicating its presence in multiple molecular forms. alpha-D-Glucosidase had a molecular weight of 85 000, a pH optimum of 6.0 and an isoelectric point of 5.2; alpha-D-mannosidase had a molecular weight of 192 000, a pH optimum of 6.0 and an isoelectric point of 4.5; and acid phosphatase had a molecular weight of 81 000, a pH optimum of 6.0 and two isoelectric points (4.8 and 5.9) indicating its existence in two molecular forms. The same glycosidases and acid phosphatase were detected also in culture fluids collected after 15-20-h incubation of both L1 and adults. As in the worm extracts, beta-N-acetyl-D-glucosaminidase was present in these culture fluids at the highest activity with acid phosphatase present at the next highest activity.     

Release of leukotrienes during rapid expulsion of Trichinella spiralis from immune rats.

Rapid expulsion of the nematode Trichinella spiralis from immune rats is associated with an increase in volume of intestinal exudate and the presence of large numbers of tissue mucosal mast cells (MMC) and eosinophils. We have measured the concentrations of leukotrienes (LT) C4 (LTC4) and B4 (LTB4) in gut perfusates and mucosal homogenates at 30 min, 1, 3, 6 and 20 hr after challenge with larvae. Leukotrienes were identified by radioimmunoassay (RIA) combined with reverse-phase high-pressure liquid chromatography (RP-HPLC). There were significant elevations at 30 min and 1 hr in the concentrations of LTC4 in the perfusates from the gut of challenged immune rats compared to controls (infected unchallenged and uninfected naive rats). Similar increases in immunoreactive LTC4 and LTB4 were observed in mucosal homogenates from the gut of immune challenged animals. A second peak of LTB4 was also observed at 20 hr in both immune and naive challenged rats. There were also elevations in serum concentration of the MMC-associated specific serine protease, rat mast cell protease II (RMCPII). Since LTC4 causes smooth muscle contraction, increased vascular permeability and stimulation of mucus hypersecretion, and LTB4 recruits and activates inflammatory cells, leukotrienes may participate in the process of rapid expulsion of T. spiralis.