Immunogenicity of multiple antigen peptides containing Plasmodium vivax CS epitopes in BALB/c mice

Multiple antigen peptide systems (MAPs) allow the incorporation of various epitopes in to a single synthetic peptide immunogen. We have characterized the immune response of BALB/c mice to a series of MAPs assembled with different B and T cell epitopes derived from the Plasmodium vivax circumsporozoite (CS) protein. A B-cell epitope from the central repeat domain and two T-cell epitopes from the amino and carboxyl flanking regions were used to assembled eight different MAPs. An additional universal T cell epitope (ptt-30) from tetanus toxin protein was included. Immunogenicity in terms of antibody responses and in vitro T lymphocyte proliferation was evaluated. MAPs containing B and T cell epitopes induced high titers of anti-peptides antibodies, which recognized the native protein on sporozoites as determined by IFAT. The antibody specificity was also determined by a competitive inhibition assay with different MAPs. A MAP containing the B cell epitope (p11) and the universal epitope ptt-30 together with another composed of p11 and the promiscuous T cell epitope (p25) proved to be the most immunogenic. The strong antibody response and specificity for the cognate protein indicates that further studies designed to assess the potential of these proteins as human malaria vaccine candidates are warranted.

Schistosoma mansoni: reinfections and concomitant immunity in mice: importance of perfusion time after challenge infection for evaluation of immunoprotection

The concomitant immunity in the presence of repeated infections (with 15 cercariae) was studied in mice sacrificed on the 20th day after each infection. The comparison of the averages of immature worms, recovered from mice submitted to reinfection, with those of their respective controls (previously uninfected) showed a significantly lower worm recovery rate in the animals with previous infections (concomitant immunity). However, statiscally significant differences could not be detected among the various groups of animals, when the mice that accumulated worms in this mature stage were perfused. The theoretical projection based on the accumulation of young worms which developed to adult ones indicates a lower recovery rate of adult worms in the animals with concomitant immunity, but this projection was not corroborated by the experimental data. The visceral hemodynamic alterations that occurred in reinfections due to the pathogeny, favouring recirculation of the recent arriving worms to the other organs on the occasion of perfusion of the portal system. These results suggest that special care should be taken when one wants to investigate concomitant immunity in mice based on the distinction of the immature worms from challenge infection and the mature ones from primary infection.

Triatoma infestans is more efficient than Panstrongylus megistus in obtaining blood meals on non Anaesthetized mice

We compared the influence of the bug density in the capacity of Triatoma infestans and Panstrongylus megistus in obtaining blood meal in non anaesthetized mice. The regression anlysis for increase in body weight (mg) versus density (no. of bugs/mouse) showed that in experiments with anaesthetized mice (AM), no correlation was observed. In experiments with non anaesthetized mice (NAM) the weight increase was inversely proportional to density. The regression slope for blood meal size on density was less steep for T. infestans than for P. megistus (-1.9 and -3.0, respectively). The average weight increase of P. megistus nymphus in experiments with AM was higher than for T. infestans nymphs; however, in experiments with NAM such results were inverted. Mortality of P. megistus was significantly higher than of T. infestans with NAM. However, in experiments with AM very low mortality was observed. Considering the mortality and the slope of regression line on NAM, T. infestans is more efficient than P. megistus in obtaining blood meal in similar densities, possibly because it caused less irritation of the mice. The better exploitation of blood source of T. infestans when compared with P. megistus in similar densities, favours the maintenance of a better nutritional status in higher densities. This could explain epidemiological findings in which T. infestans not only succeeds in establishing larger colonies but also dislodges P. megistus in human dwellings when it is introduced in areas where the latter species prevails.

Complete immunization against Trypanosoma cruzi verified in individual mice by complement-mediated lysis

Experimental systems to assay immunity against Trypanosoma cruzi usually demonstrate partial resistance without excluding the establishment of sub-patent infections in protected animals. To test whether Swiss mice immunized with attenuated parasites might develop complete resistance against virulent T. cruzi, experiments were performed involving challenge with low numbers of parasites, enhancement of local inflammation and the combination of natural and acquired resistance. Absence of infection was established after repeated negative parasitological tests (including xenodiagnosis and hemoculture), and lack of lytic antibody was tested by complement mediated lysis. Immunization with 10(7) attenuated epimastigotes conferred protection against the development of high levels of parasitemia after challenge with Tulahuen strain, but was unable to reduce the number of infected animals. However, when a strong, delayed-type hypersensitivity reaction was triggered at the site of infection by injecting a mixture of virulent and attenuated T. cruzi, a significant proportion of immunized animals remained totally free of virulent infection. The same result was obtained when the immunization experiment was performed in four month old Swiss mice, displaying a relatively high natural resistance and challenged with wild, vector-borne parasites. These experiments demonstrate that complete resistance against T. cruzi can be obtained in a significant proportion of animals, under conditions which replicate natural, vector delivered infection by the parasite.

Assessment of immunity induced in mice by glycoproteins derived from different strains and species of Leishmania

A comparative study was undertaken on the immunogenic properties of 63kDa glycoproteins obtained from five different strains/species of Leishmania and assessed in C57BL/10 mice. The humoral immune response was assessed by ELISA against the five different antigens of the immunized animals. The cellular immune response was derived from Leishmania. The response was found to be species-specific in all of determined by means of the cytokine profiles secreted by the spleen cells of immunized animals. The presence of ³-IFN and IL-2, and the absence of IL-4 in the supernatants of cells stimulated by L. amazonensis antigen established that the cellular response is of Th1 type. The five glycoproteins tested were equally effective in protecting C57BL/10 mice against challenge by L. amazonensis. About 50% of the immunized animals were protected for six months.

Recrudescence induced by cyclophosphamide of chronic Trypanosoma cruzi infection in mice is influenced by the parasite strain

Reactivation of chronic chagasic patients may occur upon use of immunosuppressive drugs related to kidney or heart transplantation or when they are affected by concomitant HIV infection. This recrudescence, however, does not occur in all chagasic patients exposed to immunosuppressive agents. We therefore investigated the influence of Trypanosoma cruzi strains in the recrudescence of the parasitism in mice at the chronic phase treated with cyclophosphamide, an immunosuppressor that blocks lymphocytes DNA synthesis and therefore controls B cells response. A large variation was detected in the percentages of newly established acute phases in the groups of mice inoculated with the different strains. We suggest that reactivation of chronic T. cruzi infections is influenced by the parasite intrinsic characteristics, a phenomenon that might occur in the human disease.

Trypanosoma cruzi infection in offspring born to chagasic C3H/He mice

This study reports the effects of Trypanosoma cruzi infection induced in C3H/He male and female mice born to chagasic mice. An experimental model was established infecting female C3H/He mice with a low virulent T. cruzi clone. In this model, mating, fertilization, pregnancy evolution and delivery was carried out successfully. The offspring was infected at four, six and eigth weeks of age. The results showed that the offspring born to chagasic mothers present decreased resistance to acquired T. cruzi infection. This decreased resistance was expressed by higher levels of parasitaemia and higher mortality rates in offspring born to chagasic mothers than in controls. Age and sex were shown to be important factors of this phenomenon. The results suggest that maternal immune system products can modulate the immune response of the offspring.

Involvement of regional lymph nodes after penetration of Schistosoma mansoni cercariae in naive and infected mice

The parotid lymph nodes of naive and previously infected Balb/c mice were studied after, respectively, infection and re-infection with cercariae of Schistosoma mansoni via the ears. Schistosomula were able to pass through the lymph node by following the lymph flow or by penetrating the veins of the medullary cords. The number of nodal mast cells was higher from day 2 to 6 of primary infection; and from day 5 to 11 of re-infection. The amount of degranulating mast cells was significantly higher at day 4 of infection and at day 1 of re-infection. Eosinophils characterized the nodal inflammatory processes observed after day 5 in both primarily-infected and re-infected mice. However, only in the latter the eosinophils were able to adhere to the larval surface. In primarily-infected mice, no intranodal larva presented signs of degeneration. In contrast, in re-infected animals, some degenerating larvae were found inside eosinophilic infiltrates. The eosinophils reached the nodal tissue by migrating through the high endothelial venules and their collecting veins.

Biodistribution Study of the Anaesthetic Sodium Phenobarbital Labelled with Technetium-99m in Swiss Mice Infected with Schistosoma mansoni Sambon, 1907

Technetium-99m (99mTc) is a radionuclide that has negligible enviromnental impact, is easily available, inexpensive and can be used as a radioactive tracer in biological experiences. In order to know the mode of action of sodium phenobarbital in moving adult Schistosoma mansoni worms from mesenteric veins to the liver, we labelled sodium phenobarbital (PBBT) with 99mTc and a biodistribution study in infected and non-infected Swiss mice was performed. The PBBT was incubated with stannous chloride used as reducing agent and with 99mTc, as sodium pertechnetate. The radioactivity labelling (%) was determined by paper ascending chromatography perfomed with acetone (solvent). The 99mTc-PBBT was administered by intraperitoneal route to Swiss mice infected eight weeks before. The animals were perfused after diferent periods of time (0,1,2,3,4 hr) when blood, spleen, liver, portal vein, mesenteric veins, stomach, kidneys and adult worms were isolated. The radioactivity present in these samples was counted in a well counter and the percentage was determined. The radioactivity was mainly taken up by the blood, kidney, liver and spleen. No radioactivity was found on the adult worms. We concluded that the worm shift was due to an action on the host of the sodium phenobarbital

Immune responses of IL-5 transgenic mice to parasites and aeroallergens

Eosinophils have long been thought to be effectors of immunity to helminths but have also been implicated in the pathogenesis of asthma. Patterns of cytokine production in the host may influence the pathogenesis of these diseases by regulating the activities of eosinophils and other components of the immune response. Mice which constitutively over-express IL-5 have profound and life-long eosinophilia in a restricted number of tissues. Although eosinophils from IL-5 transgenics are functionally competent for a number of parameters considered to be important in inflammation, untreated animals are overtly normal and free of disease. In addition, the responses of these animals when exposed to aeroallergens and helminths present a number of apparent paradoxes. Eosinophil accumulation in tissues adjacent to major airways is rapid and extensive in transgenics exposed to the aeroallergen, but even after treatment with antigen over many months these mice show no evidence of respiratory distress or pathology. Helminth-infected IL-5 transgenics and their non-transgenic littermates develop similar inflammatory responses at mucosal sites and are comparable for a number of T cell and antibody responses, but they differ considerably in their ability to clear some parasite species. The life-cycle of Nippostrongylus brasiliensis is significantly inhibited in IL-5 transgenics, but that of Toxocara canis is not. Our results also suggest that eosinophilia and/or over-expression of IL-5 may actually impair host resistance to Schistosoma mansoni and Trichinella spiralis. The pathogenesis of diseases in which eosinophils are involved may therefore be more complex than previously thought.

The role of interleukin-5 (IL-5 ) in vivo: studies with IL-5 deficient mice

Eosinophil recruitment is a characteristic feature of a number of pathological conditions and was the topic of the recent International Symposium on allergic inflammation, asthma, parasitic and infectious diseases (Rio de Janeiro, June 3-5, 1996). Since interleukin5 (IL5) is believed to regulate the growth, differentiation and activation of eosinophils (Coffman et al. 1989, Sanderson 1992), the role of eosinophils and IL5 are closely linked. Although IL5 specifically regulates eosinophilia in vivo and this is its most well established activity, it is becoming clear that IL5 also has other biological effects. The recent derivation of an IL5 deficient mouse (Kopf et al. 1996), provides a model for exploring not only the role of IL5 and eosinophils but also other novel activities of IL5. Of note is that although the IL5 deficient mice cannot elicit a pronounced eosinophilia in response to inflammatory stimulation following aeroallergen challenge or parasite infection they still produce basal levels of eosinophils that appear to be morphologically and functionally normal. However, the basal levels of eosinophils appear insufficient for normal host defence as IL5 deficiency has now been shown to compromise defence against several helminth infections. In addition, IL5 deficient mice appear to have functional deficiencies in B-1 B lymphocytes and in IgA production.

Modulation by IL-10 of antigen-induced allergic responses in mice

Over the last few years, we examined the anti-allergic properties of interleukin (IL)-10 in different models of inflammation in the mouse, as well as against IgE-dependent activation of mouse bone marrow-derived mast cells (BMMC). We showed that IL-10, concurrently administered with ovalbumin, inhibited inflammatory cell accumulation in the airways and in the peritoneal cavity of sensitized mice, as well as the accompanying cytokine release. IL-10 also blocked antigen-induced cytokine generation by IgE-stimulated BMMC. Together, these results identify a novel biological property of IL-10, as a cytokine with potent anti-allergic activities.

Helminth Parasites of Conventionally Maintained Laboratory Mice: II- Inbred Strains with an Adaptation of the Anal Swab Technique

Worm burdens recovered from inbred mice strains, namely C57Bl/6, C57Bl/10, CBA, BALB/c, DBA/2 and C3H/He, conventionally maintained in two institutional animal houses in the State of Rio de Janeiro, RJ, Brazil, were analyzed and compared, regarding their prevalences and mean intensities.Three parasite species were observed: the nematodes Aspiculuris tetraptera, Syphacia obvelata and the cestode Vampirolepis nana. A modification of the anal swab technique is also proposed for the first time as an auxiliary tool for the detection of oxyurid eggs in mice

Study on the Elimination of Angiostrongylus costaricensis First Stage Larvae in the Experimental Infection of Swiss Mice

Abdominal angiostrongylosis is a nematode infection of wild rodents. Human infection may result in severe abdominal disease and has been reported from several countries in the Americas. The domestic mouse, Mus musculus, has not been found with natural infection and, like other urban rodents, should not be considered a natural host for Angiostrongylus costaricensis. Quantification of parasitic forms released for transmission may better express the coevolutionary status in parasite-host relationship. With this objective, five groups of experimentally infected Swiss mice were followed for up to 155 days post-infection (PI) days and the quantification of first stage larvae (L1) output revealed: an irregular elimination of L1 and a huge variation in the patency period (1 to 114 days) and in the number of L1 eliminated daily by individual animals (1 to 6340 L1/g). Overall mortality was 72% (range: 28% to 100%) at seven weeks PI. In conclusion, abdominal angiostrongylosis in M. musculus presents high mortality and a very variable and irregular elimination of L1 in feces.

Role of cytokines in the formation and downregulation of hepatic circumoval granulomas and hepatic fibrosis in Schistosoma mansoni-infected mice

Schistosoma mansoni infections are associated with a strong Th2 cytokine response. Treatment of mice with IL-12 or anti-IL-2 or anti-IL-4 before i.v. injection of eggs increased IFN-gamma production and downregulated Th2 responses and pulmonary granuloma size. Conversely, anti-IFN-gamma antibody treatment increased Th2 responses and granuloma size. Similar manipulation produced less dramatic results in infected mice. However, sensitization of mice with eggs + IL-12 before infection augmented the Th1 response and decreased Th2 cytokines, granuloma size and fibrosis. Antisera to IFN-gamma, TNF-alpha or IL-12 during IL-12-egg immunization partly restored granuloma size and fibrosis following infection. Variations in the size of granulomas in acute (8 week) infections may be influenced primarily by the number and state of activation of T cells. In chronic (12-16 week) infections immunologic downmodulation proceeded normally in mice without functional CD8+ cells and in IFN-gamma KO mice but not in B cell KO (muMT) mice or in mice deficient in FcR expression in spite of the fact that these mice downregulated their T cell and cytokine responses. It is evident that the participation of cytokines in granuloma formation and regulation is complicated and that the mechanisms controlling both these phenomena are likely to involve both T cells and antibody/FcR interactions.