The mucosal cellular response to infection with Ancylostoma ceylanicum.

Although hookworms are known to stimulate inflammatory responses in the intestinal mucosa of their hosts, there is little quantitative data on this aspect of infection. Here we report the results of experiments conducted in hamsters infected with Ancylostoma ceylanicum. Infection resulted in a marked increase in goblet cells in the intestinal mucosa, which was dependent on the number of adult worms present and was sustained as long as worms persisted (over 63 days) but returned to baseline levels within 7 days of the removal of worms by treatment with ivermectin. Increased mast cell responses were also recorded. Levels were again dependent on the intensity of worm burdens and lasted as long as 63 days after infection. When worms were eliminated, mast cell numbers took over 2 weeks to return to normal. Paneth cell numbers fell soon after infection, the degree of reduction being dependent on the worm burden. After clearance of worms, Paneth cell numbers returned to normal within a week, but then rebounded and numbers rose to higher levels than those in control naïve animals. The time course of the response was similar whether animals experienced a chronic low-intensity infection without loss of worms or a higher intensity infection during the course of which worm burdens were gradually reduced. Clearly, A. ceylanicum was able to induce a marked inflammatory response in its host's intestine which was sustained for over 9 weeks after infection, and which hamsters appeared able to tolerate well. Our data draw attention to the resilience of hookworms which, unlike many other nematodes, are able to survive for many weeks in a highly inflamed intestinal tract.

The effect of the hookworm Ancylostoma ceylanicum on the mucosal architecture of the small intestine in hamsters.

Hookworms are known to cause marked changes to the intestinal mucosa, especially in relation to erosion of the villi. However, since the development of enteropathy has not been examined thoroughly through quantitative experiments on infected animals, the results of experiments conducted in hamsters infected with Ancylostoma ceylanicum are reported. Changes to intestinal architecture were first apparent between 12 and 14 days after infection, and then increased in intensity for 3-4 weeks, persisting for as long as worms were present (>63 days). Following infection, the height of villi declined from a mean of 1002 micro m in naïve controls to less than 200 micro m and as low as 18 micro m in one case. The depth of the crypts of Lieberkuhn increased from a baseline value of 166 micro m in naïve controls to in excess of 600 micro m within 6 weeks of infection. Mitotic figures had a baseline value of 5.5 per villus-crypt unit, and this rose to in excess of 25 in some experiments. Changes were dependent on the intensity of the parasite burden on day 20, but by 30 days after infection changes in all three values were maximal and density-dependent relationships were no longer clearly apparent. Villus height and crypt depth returned to near normal values within a week of the removal of worms, although group means for both remained different from naïve controls for at least 3 weeks after treatment. Cellular division, as reflected in numbers of mitotic figures, stayed elevated for over 5 weeks after removal of worms. The results suggest that enteropathy in hookworm infections stems from a combination of intestinal immune responses and from the grazing activities of the adult worms on the mucosal surface, but is not sufficient per se for expulsion of this parasite.

Responses of inbred mouse strains to infection with intestinal nematodes.

Comparisons were made of the immune and inflammatory responses of four strains of inbred mice to infection with the intestinal nematodes Trichinella spiralis and Nippostrongylus brasiliensis to determine whether genetically determined 'high responsiveness' to infection, seen most clearly in intestinal responses, is independent of the parasite concerned and necessarily correlated with protection. The time course of infection was followed by counting adult worms at intervals after infection. Mucosal mast cells and Paneth cell numbers were determined as indices of the intestinal inflammatory response. Levels of IgG2a and IgG1 antibodies and of the cytokines IFN-gamma and IL-5 released from in vitro-stimulated mesenteric node lymphocytes were measured to assess type 1 and type 2 responses. NIH and CBA mice were the most resistant to T. spiralis and N. brasiliensis respectively, resistance in each case being correlated with the most intense intestinal inflammatory responses. C57BL/10 (B10) and B10.BR were the least resistant to T. spiralis, but were as resistant as CBA to N. brasiliensis, despite their intestinal inflammatory responses to both parasites being much lower than the other two strains. Mice infected with T. spiralis made the expected switch from a type 1 (IFN-gamma) to a type 2 (IL-5) response between days 2 and 8, and there were no significant differences in levels of these cytokines between the strains. In contrast, when infected with N. brasiliensis, CBA showed an IFN-gamma response at day 4, all strains switching to IL-5 by day 8 and NIH mice releasing the greatest amount of IL-5. The results indicate that the "high responder" phenotype to intestinal nematode infection is in part determined by host characteristics, but is also determined by the parasite concerned--seen most clearly by the differences between NIH and CBA when infected with T. spiralis and N. brasiliensis. The fact that "low responder" B10 background mice were more resistant to N. brasiliensis than "high responder" NIH implies that each parasite elicits a particular pattern of protective host responses, rather than parasites being differentially susceptible to the same response profile.

Paneth and intermediate cell hyperplasia induced in mice by helminth infections.

Hyperplasia of Paneth and intermediate cells is a recently described component of the response of the small intestine of mice to infection with the nematode Trichinella spiralis. To investigate whether this hyperplasia is parasite specific or represents a generic intestinal response to infection, mice were infected with T. spiralis, Nippostrongylus brasiliensis, Heligmosomoides polygyrus or Schistosoma mansoni and tissue samples taken at various time-points post-infection to determine Paneth and intermediate cell numbers. All infections induced Paneth and intermediate cell hyperplasia, but the patterns of response varied between the parasite species concerned, reflecting differences in their relationships with the host. Increases in the numbers of these cells appeared to correlate with known patterns of T-helper-2 immune responses.

Parameters of intestinal inflammation in mice given graded infections of the nematode Trichinella spiralis.

Four parameters of the intestinal inflammatory response (numbers of mucosal mast cells (MMC) and Paneth cells, villus:crypt ratios and mitotic figures) were measured in mice exposed to varying doses of infective larvae of Trichinella spiralis. The aim of the experiments was to determine whether generation of these components of inflammation required a threshold level of infection and whether, once triggered, inflammation became pan-mucosal. Near maximal MMC and Paneth cell responses were elicited even with infections as low as 35 larvae; changes in villus:crypt ratios and in mitotic indices also occurred at this level of infection, but were progressively greater with increasing levels of infection. In all infected mice, including those infected with 35 larvae, MMC and Paneth cell responses extended over most of the small intestine. These data are interpreted as showing: (i) that the intestinal mucosa is highly responsive to T. spiralis infection; (ii) that once triggered, components of the inflammatory response are amplified by T cell-dependent mechanisms, becoming pan-mucosal; and (iii) that MMC and Paneth cell responses, which require cell division and differentiation, become maximal at a lower infection threshold than changes in the villus:crypt ratio or in mitotic indices, which directly reflect increased rates of division in crypt cells.

Hamadryas baboons Papio hamadryas as maintenance hosts of Schistosoma mansoni in Saudi Arabia.

Free ranging hamadryas baboons (Papio hamadryas) in four localities in the west and north of Saudi Arabia were examined for natural infection with Schistosoma mansoni. Faecal examination revealed infection with S. mansoni on four occasions within one year (at a prevalence rate of 2.5-4.0%) in only one locality, the Al-Baha area. The eggs were viable, as shown by miracidial hatching tests, and were recorded at a density of 140-280 eggs/g of faeces (7000-14,000 eggs/day). Post-mortem examination of 13-24 baboons from each locality revealed infection with S. mansoni (adult worms and eggs in tissue) in only one locality, the Al-Baha area, at a prevalence rate of 4.16%. Viable eggs were found in the faeces and tissue of the infected baboons. The low prevalence rate of S. mansoni in hamadryas baboons in Saudi Arabia is in accordance with the low prevalence rate of S. mansoni in humans in the area. This natural baboon isolate was highly infective to snail intermediate hosts and mammalian hosts under experimental conditions. The epidemiological significance of the role of P. hamadryas (considering their large overall population of 250,000) as maintenance hosts of S. mansoni in Saudi Arabia is discussed.

The response of hamsters to primary and secondary infection with Trichinella spiralis and to vaccination with parasite antigens.

The duration of primary infections with T. spiralis was dose-dependent with greater proportional loss of worms from heavily infected hamsters and longer persistence of worms in syngeneic DSN hamsters carrying initially low intensity infections. Intestinal worms were lost more rapidly from challenged immunized animals with over 80% loss of established worms by day 6 post infection, but survival of residual worms for a further 2 weeks. Hamsters carrying initially more than 140 intestinal worms began to lose weight during the second week indicating severe pathology at this stage of infection. Mucosal mast cell numbers increased from 50 cells/20 villus crypt units in uninfected animals to a peak in excess of 150 during week 4 pi, although intestinal mastocytosis persisted long after the loss of the majority of adult worms. Serum antibody responses to muscle stage larval antigen were detected in week 3 and increased subsequently. Both mastocytosis and antibody responses were more intense on secondary exposure to infection. Hamsters vaccinated with muscle stage larval antigen showed only a moderately accelerated loss of the intestinal phase but the fecundity of worms was severely suppressed. Overall it was concluded that the hamster host provided a model of trichinellosis that, in many respects was closer than mice and rats to the pattern of infection seen in economically and clinically important host species.

Choline derivatives and sodium fluoride protect acetylcholinesterase against irreversible inhibition and aging by DFP and paraoxon.

A light addressable potentiometric sensor was used to measure acetylcholinesterase (AChE) activity in order to evaluate the protective effects of quaternary compounds and NaF against enzyme phosphorylation and aging by two organophosphates. The use of the immobilized AChE made possible the quick removal of reagents (i.e., organophosphate, 2-pralidoxime, and protectant), thereby permitting accurate determination of AChE activity before and after phosphorylation and aging. Paraoxon was 15-fold more potent in inhibiting AChE than DFP, while the percent aging following phosphorylation by diisopropylfluorophosphate (DFP) was much higher. Sodium fluoride (NaF), the most effective protectant against phosphorylation and aging, and the quaternary ammonium compounds reduced significantly AChE inhibition by DFP and paraoxon, to similar degrees. Even though the percent AChE activity that was lost to aging was reduced by these agents, aging as a percent of phosphorylated AChE was not reduced. Thus, their major effect was in reducing the percent AChE phosphorylation, which consequently resulted in reduction of total aged AChE. The finding that quaternary ammonium compounds protect against phosphorylation is consonant with the proposed presence of the active site of AChE in an aromatic gorge.

Immune responses to Trichinella spiralis and T. pseudospiralis in mice.

A comparison was made of the immunological responses of inbred NIH mice to the intestinal stage of infection with two species of the genus Trichinella, T. spiralis and T. pseudospiralis, which are known to have distinct parasitological and pathological relationships with their hosts. The parameters studied, namely antigen-specific IgG1 and IgG2a antibody responses, mucosal mastocytosis, and levels of the cytokines interferon-gamma (IFN-gamma) and interleukin-5 (IL-5) produced by concanavalin A-pulsed mesenteric node lymphocytes in vitro, were chosen to provide information about the relative activities of the Th1 and Th2 T-helper (Th) lymphocyte subsets. In this high-responder host the time-course of infections was similar, although initial levels of establishment were considerably higher for T. pseudospiralis. Both species elicited mucosal mastocytosis. Distinct differences were seen in the IgG isotype responses. Trichinella spiralis-infected mice produced a predominantly IgG2a response, whereas T. pseudospiralis elicited an IgG1 response. Cytokine release showed infection dose-related suppression of IFN-gamma and enhancement of IL-5. These effects were most marked in T. pseudospiralis-infected mice, i.e. there was an earlier shut-off of IFN-gamma and an earlier switch to IL-5. These data suggest that the two species of Trichinella show a time-related differential activity of Th subsets during the early stages of infection. The possibility that this may reflect antigenic differences between these closely related species or result from parasite-induced immunological-endocrinological changes is discussed.

Suppression of mucosal mastocytosis by Nematospiroides dubius results from an adult worm-mediated effect upon host lymphocytes.

Infections with the nematode Nematospiroides dubius fail to elicit mucosal mast cell (MMC) responses in the intestines of host mice, and suppress MMC responses generated by heterologous infection. Larval N. dubius have the capacity to prime for mastocytosis, and to elicit this response in primed mice during a challenge, but only if adult worms are prevented from developing, either by anthelmintic treatment or by irradiation of the larvae themselves. The suppressive effect of the adult stage was confirmed in experiments where such worms were implanted directly into the intestines of mice primed by exposure to irradiated N. dubius larvae or concurrently infected with Trichinella spiralis. Data on the mechanisms underlying this suppressive effect were obtained from experiments involving the adoptive transfer of mastocytosis by mesenteric lymph node cells (MLNC) from T. spiralis infected mice. When MLNC were taken from mice infected concurrently with both T. spiralis and N. dubius no enhanced mastocytosis was seen in recipients after challenge with T. spiralis. Exposure of MLNC from T. spiralis infected donors to the presence of adult N. dubius after transfer did not reduce the adoptively transferred response. The response was also unaffected when MLNC from adult N. dubius infected mice were simultaneously transferred with MLNC from T. spiralis donors. It is concluded that the suppressive effect of adult N. dubius upon the expression of mucosal mastocytosis acts upon the generation of lymphocytes capable of promoting the development of MMC from precursor cells.

Medium conditioned by spleen cells of Nematospiroides dubius-infected mice does not support development of cultured mast cells.

The ability of different conditioned media to support mast cell development from precursors in normal bone marrow was evaluated. Many mast cells developed in bone marrow cultures containing medium conditioned by concanavalin-A-stimulated spleen cells of normal mice, Trichinella spiralis-infected mice, or mice infected for 6 days by Nematospiroides dubius. By contrast, medium conditioned by concanavalin-A-stimulated spleen cells of mice having 18-day infections of the nematode N. dubius failed to support mast cell development. The difference between medium conditioned by spleen cells of mice having 6-day versus 18-day infections of N. dubius may be due to the life cycle stage of the parasite, larval or adult, present at those times. These results from cultures are consistent with those from in vivo studies in which mice given primary infections of N. dubius failed to develop the intestinal mastocytosis characteristic of nematode infections.

Suppression of mucosal 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.