Suppression of muscosal mastocytosis by infection with the intestinal nematode Nematospiroides dubius

Mice exposed to primary infections with the parasite intestinal nematode Nematospiroides dubius failed to show the mucosal mast cell (MMC) response which is characteristic of infections with other species of intestinal nematode and which was readily induced in these mice by infections with Nippostrongylus brasiliensis or Trichinella spiralis. The failure to generate a mucosal mastocytosis was independent of host strain or sex. When infections with N. dubius were established before, or concurrently with, T. spiralis or N. brasiliensis, the MMC response elicited by these species was delayed and/or depressed as was expulsion of the worms themselves. Infection with N. dubius given when a MMC response was already established, by exposure to T. spiralis, had no effect on MMC numbers. The possibility that the effects of N. dubius upon MMC responses reflect a lack of mastocytopoietic potential, rather than an active interference, was excluded by showing that SJL mice, which expel primary infections with N. dubius and express strong immunity to reinfection, developed marked mastocytosis during secondary infections. The depression of MMC responses by N. dubius is discussed in relation to the known immunosuppressive properties of this parasite and in relation to the T cell mediated control of MMC development.     

Nematospiroides dubius in the mouse: evidence that adult worms depress the expression of homologous immunity

Summary Mice immunized by a single infection with irradiated (25 krad) larvae of N. dubius were very resistant to subsequent challenge. However, when normal larvae were administered together with irradiated larvae at immunization, the acquired immunity expressed against a challenge infection was markedly depressed. It was found that as few as 50 normal N. dubius larvae interfered with the immunity that would have otherwise been elicited by the concurrently administered irradiated larvae, but this depressed response was totally alleviated when the normal worms were removed after completing their development in the intestinal mucosa and before they reached adulthood. Adult TV. dubius were transplanted directly into the intestines of mice either 7 days before or after immunization by irradiated larvae; it was shown that the recipient mice were less resistant to challenge than mice which had been sham operated. Transplanted adult worms themselves stimulated very little resistance to challenge in recipient mice. These results established that adult parasites are capable of depressing the expression of homologous immunity in the mouse. The possible mechanisms by which JV. dubius might modulate the host's immunological activity at the intestinal level are discussed and it is proposed that this mechanism is of benefit to the parasite in preventing the host from eliminating the worms during a chronic primary function.     

Lymphocyte, antibody and cytokine responses during concurrent infections between helminths that selectively promote T-helper-1 or T-helper-2 activity

The injuence of Trichinella spiralis on infections with Trichuris murk was studied in non-responder B10. BR mice. Mice infectedonly with T. muris were unable to expel parasites and had many adult worms 35 days later. Infection with 300 larvae of T. spiralis, given seven or 14 (but not 28) days after T. muris, enabled mice to expel up to 90% of T. muris; expulsion of T. spiralis was not altered. Concurrently infected mice produced less T. murisspecijic IgG2a antibody than mice infected with T. muris only, andshowed higher proliferative responses when spleen and mesenteric lymph node cells were cultured in vitro with T. murk antigens. When T. spiralis was present mucosal mast cells were generated in T. muris-infected mice, whereas almost no mast cells were seen with only T. muris. Lymphocytes from doubly-infected mice produced significantly more interleukin 4 and 5 (IL-4, IL-5) and significantly less interferon-gamma (IFN-y) when stimulated in vitro with Concanavalin A (Con-A) than cells from mice infected with T. murk only. These data demonstrate that BI0.BR mice, which in single infections produce a Thl response to T. muris and develop no protective immunity, can mount a protective T-helper-2 (Th2) response and expel T. murk when concurrently infected with the ‘Th2 inducing’ nematode T. spiralis.     

Immunological relationships during primary infection with Heligmosomoides polygyrus (Nematospiroides dubius): downregulation of specific cytokine secretion (IL-9 and IL-10) correlates with poor mastocytosis and chronic survival of adult worms

Mice were infected either with Trichinella spiralis (day 0). Heligmosomoides polygyrus (day – 14) or concurrently with both species and were killed in groups, together with naïve control mice, on 2 occasions (day 8 and 15 post infection with T. spiralis, corresponding to days 22 and 29 p.i. with H. polygyrus). The expulsion of T. spiralis was slowed significantly in concurrently infected mice and this was associated with a reduced mastocytosis and lower serum mucosal mast cell protease levels. Mesenteric lymph node (MLN) lymphocytes from all three experimental groups secreted IL-3 and IL-4 in copious amounts when stimulated in vitro by Concanavalin A (Con-A), but the secretion of high levels of IL-9 and IL-10 was essentially confined to mice infected with T. spiralis alone. It is suggested that adult H. polygyrus selectively modulate cytokine secretion by Th2 cells within the MLN during infection and that this is brought about as a direct consequence of the mechanism employed by H. polygyrus to depress mucosal inflammatory responses in order to facilitate its own survival.     

Distribution of intestinal mast cell proteinase in blood and tissues of normal and Trichinella-intected mice

A sensitive and specific enzyme-linked immunosorbent assay (ELISA) was developed for mouse intestinal mast cell proteinase (IMCP). Specificity was demonstrated by the absence of immunoreactivity with extracts of isolated serosal mast cells (SMC), or with high concentrations (50 micrograms/ml) of the antigenically similar rat mast cell proteinases I or II. The small and large intestines in normal mice were the major sources of IMCP, there being little or no IMCP in non-mucosal tissues. Concentrations of IMCP in normal (non-parasitized) mice were low, but were increased 100-1000-fold intestines of mice infected 10 days earlier with Trichinella spiralis. The kinetic response of secreted IMCP into the blood of mice following infection with T. spiralis was also studied. Systemic release of IMCP coincided with the immune expulsion of adult worms from the intestine, and peak concentrations (9.45 micrograms/ml IMCP) occurred 9 days after infection. The tissue distribution of IMCP, its secretion into blood, and its enteric accumulation during parasite infection, are consistent with a mucosal mast cell (MMC) source for IMCP. The results are discussed in the context of similar findings for rat mast cell proteinase II.     

Influence of cadmium on the course of intestinal and muscular phases in mice infected with Trichinella spiralis]

At the first experiment 20 Swiss male mice were infected with 100 larvae and 20 mice with 500 larvae T. spiralis per mouse. Two days after infection (d.a.i.) mice orally received 1.5 mg Cd (water solution CdCl2) each. 40 mice were infected only T. spiralis as control. At the second experiment muscle larvae used were isolated from mice (from the first experiment) which received Cd. 20 mice were infected with 100 larvae and 20-with 500 larvae per mouse. Two d.a.i. mice received 1.5 mg Cd. Mice from both experiments were killed at 5, 10, 20 and 42 d.a.i. Total number of adult T. spiralis worms present in the small intestine and muscle larvae were recovered by conventional technique. Results of the first experiment: the number of adult worms and muscle larvae recovered from mice received Cd. were statistically significant lower as in control. Results of the second experiment: the mean number of adult worms in experimental group and in control were the same but the mean number of larvae per gram of mice muscle were significant higher as in control.     

Survival to patency of low level infections with Trichuris muris in mice concurrently infected with Nematospiroides dubius

Large, single-pulse laboratory infections with Trichuris muris are rejected by mice before patency, but low-level infections of fewer than 20 worms survive for long periods. Data are presented to show that the threshold at which an effective immune response takes place is significantly higher in mice concurrently infected with Nematospiroides dubius. In control CFLP mice trickle infections did not survive to maturity but in the slower responder C57 Bl10 mice egg production began on Day 35 and continued for a further seven weeks, with some mature worms present at autopsy. Concurrent infection with N. dubius resulted in trickle infections, T. muris surviving much better than in control mice, although these still showed some resistance to T. muris. It is suggested that the results support the hypothesis that T. muris elicits concomitant immunity in the host. Thus, the first worms to establish survive to patency at which time they can no longer be removed by the host, but once the immunological threshold has been exceeded incoming larvae are rejected by the host. Such a survival strategy would be very useful to T. muris in the wild.     

Protection in lambs vaccinated with Haemonchus contortus antigens is age related, and correlates with IgE rather than IgG1 antibody

The involvement of mucosal mast cells (MMC) in protection against infection with the murine nematode parasite Trichuris muris was studied in genetically mast cell-deficient WBB6F1-W/Wv mice and their normal littermates WBB6F1-+/+ mice. Expulsion of T. muris worms occurred in infected +/+ mice, whereas no worm expulsion was observed in infected W/Wv mice where the infection persisted until at least day 46 postinfection. No MMC responses were induced in either infected W/Wv or +/+ mice. Specific IgG1and IgG2a antibodies to T. muris excretory/secretory antigens were observed in infected W/Wv and +/+ mice, and antibody production showed similar kinetics. Interleukin 4 production by concanavalin A (Con A)-stimulated mesenteric lymph node cells (MLNC) was induced preferentially in infected +/+ mice. T. muris infection increased the levels of IFN-gamma produced by Con A-stimulated MLNC of infected W/Wv and +/+ mice, with the levels of IFN-gamma in infected W/Wv mice being higher than those in infected +/+ mice. Taken together, these results indicate that W/Wv and +/+ mice are susceptible and resistant to T. muris infection, respectively, and that MMC responses are not required for protective immunity.     

以旋毛虫成虫感染小鼠的研究

本文以旋毛虫成虫为感染期感染小鼠获得成功。用２００条旋毛虫成虫经口感染小鼠，在鼠的肠内发现成虫，肌肉内发现幼虫。实验结果显示，２日龄成虫感染的１０只鼠肠内幼虫数６～５７条，平均３０．８条，肌肉内幼虫数１７～７３条，平均３８．１条。７日龄成虫感染的１０只鼠肠内检获成虫数２～３４条，平均１８．４条，肌肉内幼虫数０～５５条，平均２５．４条。１５日龄成虫感染的１０只鼠肠内成虫数０～８条，平均２；２条２只鼠的肌肉内有幼虫，分别是２和３条。３０日龄成虫感染的小鼠肠内和肌肉内未见成虫和幼虫。因此，可以认为在旋毛虫生活史过程中，旋毛虫成虫亦能起感染期作用感染宿主。     

Studies on chronic versus transient intestinal nematode infections in mice I. A comparison of responses to excretory/secretory (ES) products of Nippostrongylus brasiliensis and Nematospiroides dubius worms

Summary Previous reports have demonstrated that after implantation of intestinal worms or after exposure to infective third stage larvae, the duration of infection with Nematospiroides dubius is markedly prolonged in intact mice relative to infection with Nippostrongylus brasiliensis. The rapid rejection of N. brasiliensis adults appears T-cell dependent in that adults persist for longer periods in hypothymic nude mice than in intact mice. Excretory/secretory (ES) products harvested from N. dubius or N. brasiliensis intestinal worms did not differ obviously in the following characteristics: rate of production and degree of complexity of proteins, in vitro mitogenicity, allergenicity, or in their abilities to induce or elicit delayed type hypersensitivity reactions in naive and infected mice, respectively. Two differences between N. brasiliensis- and N. dubius-infected mice were an IgG₁ hypergammaglobulinaemia and readily detected anti-ES precipitating antibodies in the circulation; both responses were confined to the chronic N. dubius infection. One difference between N. brasiliensis and N. dubius ES products was that the former, but not the latter, induced protection against homologous infection when injected with Freund's complete adjuvant. By contrast, intraperitoneal implantation of either type of adult worm induced protection against homologous infection at least in female Balb/c mice. After intestinal implantation of both N. dubius and N. brasiliensis intestinal worms, the rejection of N. brasiliensis was not influenced by, nor did it alter, persistence of N. dubius adults. In support of conclusions drawn by others, the differences in persistence of infection between these two nematodes probably reflect differences in the ability to resist both specific and nonspecific components of the complex intestinal rejection process. The chronicity of N. dubius infection and nonpersistence of N. brasiliensis     

Expulsion of Nematospiroides dubius from the intestine of mice treated with immune serum

This paper describes experiments which demonstrated that the survival of Nematospiroides dubius was severely impaired in mice treated with immune serum. CFLP donor mice were given a series of infections ranging from 25 to 200 infective larvae, at weekly intervals for 6 weeks. The mice were treated with anthelmintic on day 21 and/or day 28 to prevent the accumulation of lethal numbers of parasites in the intestine, and were bled between day 42 and day 49. Female NIH recipient mice were given a total of 2.0-2.5 ml of immune serum i/p., in several separate smaller doses at various times in relation to the day of infection. Between the administration of immune serum begun during the first 4 days of infection and the animals being killed within the next 3 weeks, the mice harboured fewer worms than control animals, the worms were stunted and their fecundity was greatly reduced. Furthermore, these worms were subsequently lost from the intestines of treated mice, during and after the fourth week of infecton. These effects on N. dubius were not observed when mice were given normal serum nor when immune serum was administered after day 6 of the infection. The delayed rejection of adult worms from mice treated with immune serum is of particular significance and suggests that immune serum contained factors which facilitated the expression of a second component in worm expulsion not nornally effective in a primary infection. The possible immunologcal mechanisms underlying these findings are discussed and related to the immunosuppression which N. dubius is known to induce in the host.     

两种新型免疫佐剂CT-B、Saponin制备的旋毛虫疫苗对小鼠免疫保护作用的比较研究

目的　比较两种新型免疫佐剂CT -B(霍乱毒素B亚单位 )、saponin(皂素 )制备的旋毛虫疫苗对小鼠的免疫保护作用。方法　将旋毛虫肌幼虫可溶性抗原分别与CT -B、saponin混合 ,以口服或皮下注射途径免疫NIH小鼠 ,间隔 1周共免疫 3次 ,末次免疫后 1周 ,与对照组同时予 2 0 0条旋毛虫感染期肌幼虫攻击 ,比较三组小鼠肠道成虫数、雌虫生殖力及肌幼虫数。结果　与对照组比较 ,CT -B组成虫减虫率、新生幼虫减虫率及肌幼虫减虫率分别为 91 5 9%、6 1 74 %和 90 32 % ,saponin组成虫减虫率、新生幼虫减虫率及肌幼虫减虫率分别为 79 2 1%、6 7 4 4 %和 88 39%。 结论　CT -B和saponin均能有效提高机体对旋毛虫的保护性免疫力 ,CT -B对肠道成虫的影响更显著     

Limit dilution analysis of mast cell precursor frequency in the gut epithelium of normal and Trichinella spiralis infected mice

Previous studies have established that the gut nematode Trichinella spiralis induces a dramatic thymus dependent intestinal mastocytosis which peaks within 6 to 12 days after primary oral infection. It is not known, however, if the increase in gut mast cells results from the influx of mast cells or their precursors, or from the expansion and differentiation of mast cell precursors (MCP) that are normally present in the small intestinal epithelium. In the present study, the number of mucosal MCP in the intraepithelial lymphocyte (IEL) population and in bone marrow (BM) cells from normal and 4 day T. spiralis infected mice was compared by culturing the cells at limiting dilutions in medium containing interleukin-3 (IL-3). While the MCP frequency in IEL from infected mice was found to be significantly increased in comparison with that found in normal mice, the numbers of MCP in BM from the two groups were equivalent. Resident intraepithelial mucosal MCP therefore undergo a local expansion before the occurrence of an overt T dependent intestinal mastocytosis. This finding lends support to the view that local mucosal T cells are involved in regulating mast cell numbers in response to intestinal helminth infection.     

旋毛虫成虫、新生幼虫和肌幼虫抗原的保护性免疫研究

目的：比较旋毛虫成虫抗原和肌幼虫抗原接种小鼠诱导产生的保护性免疫。方法:检查肠道成虫、肌幼虫和血液中嗜酸性细胞数;用ELISA测血清中抗旋毛虫肌幼虫IgG抗体滴度。结果：成虫、新生幼虫和肌幼虫抗 免疫组的成虫减虫率分别为82.19％、72.31％和42.88％;肌幼虫减虫率分别为68.83％、78.19％和51.96％。血清IgG抗体滴度明显升高,成虫、新生幼虫和肌幼虫抗原免疫组的GMRT分别为对照组的7.46倍、5.28倍和4.92倍。血液嗜酸性粒细胞明显增多。结论：旋毛虫成虫抗原、新生幼虫抗原均为激发特异性液免疫和细胞免疫,诱导小鼠对攻击感染产生保护性。其中,成虫抗原和新生幼虫抗原显示出更好的免疫原性,而成虫抗原有可能成为较理想的免疫疫苗来源。     

碳酸氢钠对旋毛虫幼虫感染性及生殖力的影响

目的观察碳酸氢钠对旋毛虫幼虫感染性及生殖力的影响。方法将旋毛虫肌幼虫在体外经1%碳酸氢钠处理不同时间后观察其活力。100只雄性昆明小鼠随机分成10组(每组10只),1～3组中每组5只分别喂饲经碳酸氢钠或生理盐水浸泡不同时间(24、36、48h)含300条旋毛虫肌幼虫的小鼠肌肉(约0.02g);4～9组分别用不同剂量碳酸氢钠或食用醋(总酸浓度4.5%,pH3.05)灌胃后0.5h喂饲含300条肌幼虫肉块,10组为灌服生理盐水对照组。于喂饲后第7天和第42天每组剖杀5只小鼠,分别观察肠道成虫数、肌幼虫数及生殖力指数(RCI)。结果肌幼虫经碳酸氢钠处理24～240h的死亡率与生理盐水对照组相比差异无统计学意义(P0.05)。小鼠喂饲经碳酸氢钠处理24、36h和48h旋毛虫肌幼虫后42d,其肌幼虫数(84467、84600条和82300条)均明显高于生理盐水对照组(12650、6460条和1240条)(P0.05)。灌饲0.4、0.6ml碳酸氢钠小鼠的肠道成虫数(193、203条)与RCI(323.77、326.55)均明显高于食醋对照组(68、67条;217.55、195.33)及生理盐水对照组(145条,268.13)(P0.05)。结论碳酸氢钠可明显增加旋毛虫的感染性与生殖力。     

Variability of the intestinal immunoglobulin E response of rats to infection with Trichinella spiralis, Heligmosomoides polygyrus or Nippostrongylus brasiliensis

Total intestinal IgE level increased in rats infected with Trichinella spiralis or Heligmosomoides polygyrus (peak levels of 2.6 microg and 3.7 microg, respectively), but not in rats infected with Nippostrongylus brasiliensis. Intestinal implantation of young adult N. brasiliensis did not stimulate an intestinal immunoglobulin (Ig)E response, suggesting that mucosal penetration may be required for local intestinal IgE responses in rats. During a T. spiralis infection, total IgE levels in the intestinal lumen were consistently higher in LEWIS and LOU rats (rat strains that eliminate T. spiralis worms earlier in the infection) than in PVG, AO and WKA/H strain rats. There was no correlation in either the total level of serum IgE and IgA, or of intestinal IgA with differences between strains in the rate of worm elimination from the gut. Furthermore, the intestinal IgE immunoprecipitated from LEWIS rats 12 days after infection reacted with T. spiralis adult worm metabolic antigens, while intestinal IgE from PVG rats only became reactive with adult worm metabolic antigens from 14 days after infection. These data emphasize the significance of the intestinal IgE response and its unique features by comparison with serum IgE and IgA or intestinal IgA.     

Genetic control of immunity to Nematospiroides dubius: a 9-day anthelmintic abbreviated immunizing regime which separates weak and strong responder strains of mice

Experiments were designed to re-examine the variables which influence the ability of single primary infections to elicit acquired immunity to Nematospiroides dubius, in particular the importance of the presence or absence of adult worms, as these are known to exert immunomodulatory effects. Briefly, anthelmintic abbreviated infections were considerably more effective at eliciting acquired immunity than longer infections in which adult worms were allowed to reside in the intestine. A 9-day anthelmintic abbreviated infection was extremely effective at stimulation of acquired immunity in NIH mice and very few immunising infection larvae were required. Immunity to subsequent reinfection developed rapidly after the primary infection worms had been eliminated; by day 21 post-infection, the mice were almost totally immune. Abbreviated infections were used to examine the capacity of a number of mouse strains to develop immunity to reinfection. Strains of mice were chosen to allow the effects of MHC linked and non-MHC linked (background) genes to be identified. CBA and C3H strains (both H-2k) were found to be weak responders to N. dubius. B10G (H-2q) mice responded better than C57Bl/10 (H-2b), although these strains have identical background genes. DBA/2 mice were stronger responders compared to BALB/c mice, both strains sharing a common MHC haplotype (H-2d). (NIH X B10G) F1 mice (H-2q) were better responders than either of the parental strains. Several mouse strains all sharing the H-2q haplotype were particularly effective at developing immunity to N. dubius, as were also SJL mice which were the sole representatives of the H-2s haplotype, in the present study. The results established that the response phenotype is influenced by both background and MHC genes and demonstrated gene complementation in the capacity of mice to acquire immunity to N. dubius.     

大鼠对旋毛虫再感染的抵抗力实验观察

目的 研究实验感染旋毛虫（韩国分离株）的大鼠对成虫和肌期幼虫阶段感染的保护性免疫和IgG，IgG1，IgG2a抗体反应。 方法 46只大鼠随机分为7组，其中2组（A1、A2组， 共10只）用于观察成虫阶段引起的保护性免疫，B组（B1、B2组， 共14只）用于观察肌期幼虫阶段引起的保护性免疫，C组（C1、C2组，共17只）为感染对照组，D组（5只）为正常对照组。A、B和C组分别每鼠感染1 000条旋毛虫肌幼虫，分别于感染后第7天（A1、 A2组）和第30天（B1、B2组），用氟苯咪唑治疗（20 mg/kg，10 d）。治疗后第10天，A和B组每鼠再次感染500条旋毛虫肌幼虫，于感染后第7天剖杀A1和B1组大鼠，检测肠道内成虫数，于感染后第30天剖杀A2和B2组大鼠，检测横膈膜内肌期幼虫数，同时分别剖杀感染对照组和正常对照组大鼠。每组同期取血，ELISA检测特异性IgG、IgG1和IgG2a抗体水平。结果 旋毛虫成虫阶段对成虫和肌幼虫的保护性免疫分别为100%和99.96%，肌幼虫阶段对成虫和肌幼虫的保护性免疫分别为99.92%和99.89%。肌幼虫感染阶段抗肌幼虫分泌排泄抗原的特异性IgG、IgG1和IgG2a抗体与正常对照组（分别为0.5、 0.1和0.1）和成虫感染阶段（分别为0.5、 0.09和0.09）比较，抗体反应均显著增高（分别为3.0、2.2和0.8）（P<0.01）。且幼虫期抗肌幼虫分泌排泄抗原特异性IgG1抗体（2.2）显著高于特异性IgG2a抗体（0.8）（P<0.01）。 结论 旋毛虫的成虫和肌幼虫阶段的感染均对成虫和幼虫的再感染产生保护性免疫。     

The mucosal mast cell and IgA plasma cell responses to primary Trichinelia spiralis infection in BALB/c mice treated with ketotifen

The effect of ketotifen on inflammation in the intestine during T. spiralis infection in BALB/c mice was examined. The lack of enteropathy in treated mice was accompanied to affected the size of worms and the number of worms, but did not prevent the retardation of nematodes in the intestine. Fecundity of females in vitro and the number of muscle larvae were not statistically changed after treatment. Ketotifen reduced the number of mast cells and IgA plasma cells in the intestine. As inflammatory influx, epithelial damage and IgA response are related to the action of T cells, ketotifen affected T dependent reactions in the gut of T. spiralis infected mice.     

旋毛虫编码新生幼虫p46 kDa抗原基因重组融合蛋白对小鼠的免疫保护性研究

目的 探讨旋毛虫编码新生幼虫p46 kDa抗原基因重组融合蛋白WN10对小鼠的免疫保护性。方法 将纯化的重组融合蛋白WN10以20μg/只的剂量分3次免疫小鼠后，攻击感染旋毛虫肌幼虫200条／只，检查旋毛虫7日龄成虫数、雌虫体外产生新生幼虫数、感染35d的肌幼虫数并计算减虫率，同时用ELISA法检测血清中抗WN10抗体IgG滴度。结果WN10免疫小鼠后获得旋毛虫7日龄成虫、肌幼虫的减虫率分别为64．28％和61．21％，均与感染对照组和佐剂对照组差异显著；获得雌虫产新生幼虫的减虫率为16．46％，与感染对照组和佐剂对照组差异不显著；WN10免疫组小鼠在第3次免疫后1周可检测到高滴度的抗WN10抗体IgG，在攻击感染旋毛虫9周后仍维持在较高水平。结论 编码旋毛虫新生幼虫p46 kDa抗原基因重组融合蛋白可诱导小鼠产生较强的抗旋毛虫保护性免疫。