Host control of malaria infections: constraints on immune and erythropoeitic response kinetics

The two main agents of human malaria, Plasmodium vivax and Plasmodium falciparum, can induce severe anemia and provoke strong, complex immune reactions. Which dynamical behaviors of host immune and erythropoietic responses would foster control of infection, and which would lead to runaway parasitemia and/or severe anemia? To answer these questions, we developed differential equation models of interacting parasite and red blood cell (RBC) populations modulated by host immune and erythropoietic responses. The model immune responses incorporate both a rapidly responding innate component and a slower-responding, long-term antibody component, with several parasite developmental stages considered as targets for each type of immune response. We found that simulated infections with the highest parasitemia tended to be those with ineffective innate immunity even if antibodies were present. We also compared infections with dyserythropoiesis (reduced RBC production during infection) to those with compensatory erythropoiesis (boosted RBC production) or a fixed basal RBC production rate. Dyserythropoiesis tended to reduce parasitemia slightly but at a cost to the host of aggravating anemia. On the other hand, compensatory erythropoiesis tended to reduce the severity of anemia but with enhanced parasitemia if the innate response was ineffective. For both parasite species, sharp transitions between the schizont and the merozoite stages of development (i.e., with standard deviation in intra-RBC development time ≤2.4 h) were associated with lower parasitemia and less severe anemia. Thus tight synchronization in asexual parasite development might help control parasitemia. Finally, our simulations suggest that P. vivax can induce severe anemia as readily as P. falciparum for the same type of immune response, though P. vivax attacks a much smaller subset of RBCs. Since most P. vivax infections are nonlethal (if debilitating) clinically, this suggests that P. falciparum adaptations for countering or evading immune responses are more effective than those of P. vivax.     

Ablastin: Control of Trypanosoma musculi infections in mice

Trypanosoma musculi infections in CBA mice consist of a phase of increasing parasitemia during which dividing forms of the parasite are present in the blood, followed by a period when only nondividing trypomastigotes are seen. A second crisis terminates the blood infection and leaves the host immune, but small numbers of trypanosomes, including multiplicative forms, persist in the kidneys for many months. Studies were made involving infections in T-lymphocyte deprived mice, the effects of passive transfer of serum and cells, measurement of DNA synthesis by the parasite, serological responses, and in vitro effects of serum on the trypanosomes. These indicated that the initial check on the increase in blood parasitemia is due in part to two humoral factors. One of these has a trypanocidal effect (this is thought to be an IgM antibody) while the other, which may be an IgG antibody, is the ablastin that inhibits further reproduction by the parasite. Both trypanocidal and ablastic effects were demonstrable in the serum of immune mice yet the parasite was still able to survive and multiply in the kidneys.     

Trypanosoma cruzi: Early immune responses in infected mice

The influence of an active Trypanosoma (Schizotrypanum) cruzi infection in mice and the subsequent immune response to an administered unrelated, erythrocyte antigen was studied. When mice were infected with blood forms of the parasite, a depression of the humoral immune response to injected burro erythrocytes (BE), was observed. The suppression became evident at a time when the magnitude of parasitemia and tissue forms was increasing in the sensitized and infected mice. The immunosuppressive effect induced by the infection to BE was demonstrated by a diminished direct (19S) and indirect (7S) antibody-forming cell response. Additionally, a significant increase in phagocytic activity was observed in T. cruzi-infected mice using colloidal carbon uptake experiments. Probable mechanisms of suppression are discussed and related to accepted lymphocyte activities.     

Pathology of Tnf-deficient mice infected with Plasmodium chabaudi adami 408XZ

Tumor necrosis factor alpha (Tnf) plays a pleiotropic role in murine malaria. Some investigations have correlated Tnf with hypothermia, hyperlactatemia, hypoglycemia, and a suppression of the erythropoietic response, although others have not. In this study, we have evaluated parasitemia, survival rate and several pathological features in C57BL/6JTnf^−/− and C57BL/6JTnf^+/+ mice after infection with Plasmodium chabaudi adami 408XZ. Compared to the C57BL/6JTnf^+/+ mice, C57BL/6JTnf^−/− mice showed increased parasitemia and decreased survival rate, whereas blood glucose, blood lactate and body weight were not significantly different. However, C57BL/6JTnf^−/− mice suffered significantly more from severe anemia and hypothermia than C57BL/6JTnf^+/+ mice. These results suggest that Tnf is an important mediator of parasite control, but not of anemia development. We hypothesize that the high mortality observed in the Tnf knock-out mice is due to increased anemia and pathology as a direct result of increased levels of parasitemia.     

Some Quantitative Aspects of the Adoptive Transfer of Immunity to Plasmodium berghei with Immune Spleen Cells

SYNOPSIS. Some of the parameters concerned in the adoptive transfer of immunity to P. berghei in rats have been studied. Immunity was not transferred efficiently until about 10 days after recovery of donor rats had commenced, but thereafter was very effective, even after the infection appeared to have been sterilized. Animals protected by a standard dose of cells from immune spleens but challenged with different doses of parasites attained a parasitemia of about 5% at different times, but all overcame the infection at the same time. Animals protected by different doses of cells from immune spleens and challenged with a standard dose of parasites, attained a parasitemia of about 5% simultaneously but the time taken to control the infection was inversely proportional to the dose of protective cells. Up to a parasitemia of about 5% there was no difference in the multiplication rate of parasites in control animals, in animals which were not effectively protected and in protected animals. Beyond a parasitemia of about 5% there was no difference in the multiplication rate of parasites in control animals and in animals which were not effectively protected. It is suggested that there is a critical period at a parasitemia of about 5% when the level of the immune response determines the fate of the host. At that stage the immune response may be inadequate, or possibly exhausted, and the parasite will then continue to multiply unchecked. Saturation of reticulocytes with parasites at a level of about 5% total parasitized red cells probably gives the host a respite which may allow the adoptively transferred cells to control the infection.     

Low red cell production may protect against severe anemia during a malaria infection—Insights from modeling

The malaria parasite causes lysis of red blood cells, resulting in anemia, a major cause of mortality and morbidity. Intuitively, one would expect the production of red blood cells to increase in order to compensate for this loss. However, it has been observed that this response is weaker than would be expected. Furthermore, iron supplementation for iron deficient children in malaria endemic regions can paradoxically adversely affect the clinical outcome of malaria infection. A possible explanation may lie in the preference that some malaria parasites show for infecting immature red blood cells (reticulocytes). In the presence of a parasite preference for immature red cells, a rise in red cell production can ‘fuel the fire’ of infection by increasing the availability of the parasite's preferred target cell.We present a mathematical model of red blood cell production and infection in order to explore this hypothesis. We assess the effect of varying the reticulocyte replacement rate and preference of the parasite for reticulocytes on four key outcome measures assessing anemia and parasitemia.For a given level of parasite preference for reticulocytes we uncover an optimal erythropoietic response which minimizes disease severity. Increasing red blood cell production much above this optimum confers no benefit to the patient, and in fact can increase the degree of anemia and parasitemia. These conclusions are consistent with epidemiological studies demonstrating that both iron deficiency and anemia are protective against severe malaria, whilst iron supplementation in malaria endemic regions is with an increased number of malaria related adverse effects. Thus, suppression of red blood cell production, rather than being an unfortunate side effect of inflammation, may be a host protective effect against severe malarial anemia.     

Plasmodium chabaudi AS: erythropoietic responses during infection in resistant and susceptible mice.

The course of anemia and the erythropoietic response in the bone marrow, spleen, and blood were studied during Plasmodium chabaudi AS infection in resistant C57BL/6 (B6) and susceptible A/J (A) mice. Infections in B6 mice were characterized by moderate levels of both parasitemia and anemia and survival. In contrast, A mice experienced high parasitemia, severe anemia, and high mortality rates. During the period of anemia, erythropoiesis, as measured by in vivo 59Fe incorporation, was significantly more depressed in bone marrow and more increased in the spleen in resistant B6 mice. The increase in splenic 59Fe incorporation was a function of the size of the spleen. Bone marrow CFU-E were decreased to 50% of control in both strains, while splenic CFU-E were increased twofold greater in B6 mice compared to those in A mice. However, the absolute numbers of CFU-E per spleen in the two strains were not significantly different during peak parasitemia. Bone marrow BFU-E were transiently increased before peak parasitemia whereas splenic BFU-E peaked during peak parasitemia. A mice had significantly lower numbers of BFU-E per spleen on all days except at peak parasitemia. The frequency of blood-borne BFU-E and plasma erythropoietin titers was increased earlier and to a greater extent in A mice. These results suggest that an impaired amplification of late-stage splenic erythropoiesis may be an important determinant in the severity of anemia and lethality of infection with P. chabaudi AS in A mice. Moreover, these results demonstrate that the defective amplification of splenic erythropoiesis in A mice is neither caused by a defect in the mobilization of BFU-E from the bone marrow to the spleen nor caused by a defect in erythropoietin production.     

TLR9激动剂对约氏疟原虫体液免疫记忆的影响

为探讨TLR9激动剂对疟疾体液免疫记忆的影响,用非致死型约氏疟原虫感染BALB/c小鼠,感染前2 d注射TLR9激动剂CpGl826,90 d后进行二次感染。薄血膜染色法观察红细胞感染率,流式细胞术检测脾细胞悬液中记忆性和活化性B细胞百分比,双夹心ELISA法检测特异性抗体水平。结果显示,二次感染前,TLR9激动剂处理鼠记忆性和活化性B细胞以及抗体水平略高于对照组;二次感染后,其再感染发生率和虫血症水平均略低于对照组;活化性B细胞和抗体以及记忆性B细胞也分别于二次感染后1 d和3 d出现了有意义的升高,且升高幅度均略高于对照组。表明TLR9激动剂对约氏疟原虫感染后体液免疫记忆的建立和维持有一定促进作用。     

Transfer of immunity to Plasmodium berghei by spleen and lymph node immune RNA.

The RNA from spleens and lymph nodes of Lewis rats immune to Plasmodium berghei protected A/J mice against a lethal challenge of the blood stages of P. berghei, NK65. The RNA was extracted by the hot phenol procedure from freshly removed spleens and lymph nodes. Protection was measured by survival and level of parasitemia as compared to controls. The levels of RNA administered were 10, 50, and 100 μg of RNA. There was observed 100% survival with 50 and 100 μg of immune spleen RNA. The maximum percentage of parasitemia was not reduced below that of the controls in the groups given immune RNA from lymph nodes, but was significantly reduced below that of the controls in the groups given immune RNA from spleens.     

Cryptobia salmositica: susceptibility of infected rainbow trout, Salmo gairdneri, to environmental hypoxia

Using the sealed jar technique (also called residual oxygen bioassay), rainbow trout fry infected with Cryptobia salmositica were more susceptible than non-infected fish to environmental hypoxia. The Winkler technique (azide modification) was used to determine the residual dissolved oxygen in the water. Susceptibility of infected fish increased with 1) time after infection and was most evident in 3-7 wk infections, 2) the severity of anemia, and 3) increasing parasitemia. In prolonged infections, susceptibility was reduced when there were decreases in anemia and parasitemia; however, these infected fish were still more susceptible than non-infected fish. The increase in susceptibility of infected fish to hypoxia may be an important contributing factor to mortality of fish in hatcheries where there is inadequate water flow and overcrowding. The sealed jar technique is recommended in future studies on the pathogenesis of parasitic fish diseases, especially if the metabolic and/or respiratory systems are affected by the infection.     

Genetic control of resistance to murine malaria

Strain variation in the level of resistance to malaria was investigated in inbred mice after infection with Plasmodium chabaudi. Following intraperitoneal infection with the typing dose of parasitized erythrocytes, mice of 11 inbred strains could be separated using survival time as the criterium into resistant and susceptible groups. Genetic analysis of F₁ hybrid and backcross progeny derived from one of the most resistant (B10.A) and from the most susceptible (A/J) strains as parents suggested that host resistance in this strain combination was genetically controlled by a dominant, non-H-2-linked, autosomal gene or closely linked genes. Analysis of the mechanisms of resistance to P chabaudi showed (1) phenotypic expression of the resistance gene was apparent within 6 days of infection as a significant difference between resistant and susceptible mice in the level of parasitemia; (2) the level of host NK cell activity was not related to the level of host resistance to malaria; (3) compared with susceptible A/J mice, resistant B1O.A hosts had an augmented erythropoietic response during the course of malaria as well as during phenylhydrazine-induced anemia and (4) treatment with BCG or P acnes resulted in an equal degree of protection, measured by parasitemia and survival, in both resistant and susceptible mice.     

Molecular Analysis of Non-Specific Protection against Murine Malaria Induced by BCG Vaccination

Although the effectiveness of BCG vaccination in preventing adult pulmonary tuberculosis (TB) has been highly variable, epidemiologic studies have suggested that BCG provides other general health benefits to vaccinees including reducing the impact of asthma, leprosy, and possibly malaria. To further evaluate whether BCG immunization protects against malarial parasitemia and to define molecular correlates of this non-specific immunity, mice were vaccinated with BCG and then challenged 2 months later with asexual blood stage Plasmodium yoelii 17XNL (PyNL) parasites. Following challenge with PyNL, significant decreases in parasitemia were observed in BCG vaccinated mice relative to naïve controls. To identify immune molecules that may be associated with the BCG-induced protection, gene expression was evaluated by RT-PCR in i) naïve controls, ii) BCG-vaccinated mice, iii) PyNL infected mice and iv) BCG vaccinated/PyNL infected mice at 0, 1, 5, and 9 days after the P. yoelii infection. The expression results showed that i) BCG immunization induces the expression of at least 18 genes including the anti-microbial molecules lactoferrin, eosinophil peroxidase, eosinophil major basic protein and the cathelicidin-related antimicrobial peptide (CRAMP); ii) an active PyNL infection suppresses the expression of important immune response molecules; and iii) the extent of PyNL-induced suppression of specific genes is reduced in BCG-vaccinated/PyNL infected mice. To validate the gene expression data, we demonstrated that pre-treatment of malaria parasites with lactoferrin or the cathelicidin LL-37 peptide decreases the level of PyNL parasitemias in mice. Overall, our study suggests that BCG vaccination induces the expression of non-specific immune molecules including antimicrobial peptides which may provide an overall benefit to vaccinees by limiting infections of unrelated pathogens such as Plasmodium parasites.     

Correlation of Humoral Immune Response in Southern Bluefin Tuna,T. maccoyii,with Infection Stage of the Blood Fluke,Cardicola forsteri

The blood fluke, Cardicola forsteri, is a prevalent infection in ranched southern bluefin tuna. This project aimed to define the timing and intensity of the various developmental stages of C. forsteri within southern bluefin tuna as well as to relate infection to host pathology and immune response. Archival samples from several cohorts of T. maccoyii sampled from 2008 to 2010 were used in this study. The prevalence and intensity of C. forsteri infection was described using heart flushes and histological examination. Humoral immune response, i.e. C. forsteri specific antibody, lysozyme activity, and alternative complement activity, was also described. Based on the validated and detailed C. forsteri infection timeline, relationships between infection events, physiological response, and diagnosis were proposed. Immune response developed concurrently with C. forsteri infection, with the majority of physiological response coinciding with commencing egg production. Further research is needed to confirm the origin of C. forsteri antigen which is responsible for immune response development and how T. maccoyii immune response works against infection. To aide this research, further diagnostic methods for confirmation of infection need to be developed.     

食蟹猴疟原虫感染恒河猴的长期观察

本项研究证明,食蟹猴疟原虫感染恒河猴后出现的红细胞内期是一个长期的寄生过程,不论血传或子孢子感染,于原虫密度高峰后,均有较长期的低原虫密度阶段。恒河猴均可耐受食蟹猴疟原虫高密度的感染。     

Deficiency of CD73 activity promotes protective cardiac immunity against Trypanosoma cruzi infection but permissive environment in visceral adipose tissue

Damaged cells release the pro-inflammatory signal ATP, which is degraded by the ectonucleotidases CD39 and CD73 to the anti-inflammatory mediator adenosine (ADO). The balance between ATP/ADO is known to determine the outcome of inflammation/infection. However, modulation of the local immune response in different tissues due to changes in the balance of purinergic metabolites has yet to be investigated. Here, we explored the contribution of CD73-derived ADO on the acute immune response against Trypanosoma cruzi parasite, which invades and proliferates within different target tissues. Deficiency of CD73 activity led to an enhanced cardiac microbicidal immune response with an augmented frequency of macrophages with inflammatory phenotype and increased CD8+ T cell effector functions. The increment of local inducible nitric oxide (NO) synthase (iNOS)⁺ macrophages and the consequent rise of myocardial NO production in association with reduced ADO levels induced protection against T. cruzi infection as observed by the diminished cardiac parasite burden compared to their wild-type (WT) counterpart. Unexpectedly, parasitemia was substantially raised in CD73KO mice in comparison with WT mice, suggesting the existence of tissue reservoir/s outside myocardium. Indeed, CD73KO liver and visceral adipose tissue (VAT) showed increased parasite burden associated with a reduced ATP/ADO ratio and the lack of substantial microbicidal immune response. These data reveal that the purinergic system has a tissue-dependent impact on the host immune response against T. cruzi infection.     

Protection from experimental cerebral malaria with a single dose of radiation-attenuated, blood-stage Plasmodium berghei parasites

Background

Whole malaria parasites are highly effective in inducing immunity against malaria. Due to the limited success of subunit based vaccines in clinical studies, there has been a renewed interest in whole parasite-based malaria vaccines. Apart from attenuated sporozoites, there have also been efforts to use live asexual stage parasites as vaccine immunogens.

Methodology and Results

We used radiation exposure to attenuate the highly virulent asexual blood stages of the murine malaria parasite P. berghei to a non-replicable, avirulent form. We tested the ability of the attenuated blood stage parasites to induce immunity to parasitemia and the symptoms of severe malaria disease. Depending on the mouse genetic background, a single high dose immunization without adjuvant protected mice from parasitemia and severe disease (CD1 mice) or from experimental cerebral malaria (ECM) (C57BL/6 mice). A low dose immunization did not protect against parasitemia or severe disease in either model after one or two immunizations. The protection from ECM was associated with a parasite specific antibody response and also with a lower level of splenic parasite-specific IFN-γ production, which is a mediator of ECM pathology in C57BL/6 mice. Surprisingly, there was no difference in the sequestration of CD8+ T cells and CD45+ CD11b+ macrophages in the brains of immunized, ECM-protected mice.

Conclusions

This report further demonstrates the effectiveness of a whole parasite blood-stage vaccine in inducing immunity to malaria and explicitly demonstrates its effectiveness against ECM, the most pathogenic consequence of malaria infection. This experimental model will be important to explore the formulation of whole parasite blood-stage vaccines against malaria and to investigate the immune mechanisms that mediate protection against parasitemia and cerebral malaria.     

Plasmodium yoelii: Antibody and the maintenance of immunity in BALB/c mice

Subpatent persistence of parasitemia was detected for up to 7 weeks after infection of BALB/c mice with Plasmodium yoelii. Serum taken from recovered mice maintained parasitemias in recipient mice at a subpatent level when transferred repeatedly at 2-day intervals. Single doses of serum from convalescent donors delayed the course of infection in recipients. Small doses of transferred hyperimmune serum had the same effect, whereas large doses (>0.5 ml) totally suppressed parasitemia. Only a single secondary challenge of recovered mice was required in order to produce a maximally protective hyperimmune serum. Mice completely protected from a primary challenge with P. yoelii by transfer of hyperimmune serum were not at all resistant to a second challenge given some weeks later. After transfer of hyperimmune serum into mice with established P. yoelii infection, parasitemia fell to subpatent levels within 48 hr. During the first 21 hr after serum transfer, a progressive reduction in the proportion of ring forms present in blood smears was observed.     

Trypanosoma cruzi Infection through the Oral Route Promotes a Severe Infection in Mice: New Disease Form from an Old Infection?

Oral transmission of Chagas disease has been documented in Latin American countries. Nevertheless, significant studies on the pathophysiology of this form of infection are largely lacking. The few studies investigating oral route infection disregard that inoculation in the oral cavity (Oral infection, OI) or by gavage (Gastrointestinal infection, GI) represent different infection routes, yet both show clear-cut parasitemia and heart parasitism during the acute infection. Herein, BALB/c mice were subjected to acute OI or GI infection using 5x10⁴ culture-derived Trypanosoma cruzi trypomastigotes. OI mice displayed higher parasitemia and mortality rates than their GI counterparts. Heart histopathology showed larger areas of infiltration in the GI mice, whereas liver lesions were more severe in the OI animals, accompanied by higher Alanine Transaminase and Aspartate Transaminase serum contents. A differential cytokine pattern was also observed because OI mice presented higher pro-inflammatory cytokine (IFN-γ, TNF) serum levels than GI animals. Real-time PCR confirmed a higher TNF, IFN-γ, as well as IL-10 expression in the cardiac tissue from the OI group compared with GI. Conversely, TGF-β and IL-17 serum levels were greater in the GI animals. Immunolabeling revealed macrophages as the main tissue source of TNF in infected mice. The high mortality rate observed in the OI mice paralleled the TNF serum rise, with its inhibition by an anti-TNF treatment. Moreover, differences in susceptibility between GI versus OI mice were more clearly related to the host response than to the effect of gastric pH on parasites, since infection in magnesium hydroxide-treated mice showed similar results. Overall, the present study provides conclusive evidence that the initial site of parasite entrance critically affects host immune response and disease outcome. In light of the occurrence of oral Chagas disease outbreaks, our results raise important implications in terms of the current view of the natural disease course and host-parasite relationship.     

Lymphoid cell kinetics in guinea pigs infected with Trichostrongylus colubriformis: tritiated thymidine uptake in gut and allied lymphoid tissue, humoral IgE and hemagglutinating antibody responses, delayed hypersensitivity reactions, and in vitro lymphocyte transformations during primary infections

The kinetics of the lymphocyte responses of Trichostrongylus colubriformis-infected and normal guinea pigs were measured by the in vivo uptake of tritiated thymidine either as dpm ³H/mg tissue or as the percentage change in [³H] -labeled lymphoblasts in autoradiographs of tissue impression smears and sections. The lymphoid response was predominantly a local one centering on the infected area of the small intestine. The greatest lymphocyte reactions as assessed by counts of labeled lymphoblasts occurred in the Peyer's patches and mesenteric lymph nodes where the peak responses took place 11 and 6 days after infection, respectively. The local nature of the responses was exemplified by the fact that the mesenteric lymph nodes of the anterior small intestine showed a peak response on the sixth day but the response from the posterior small intestine peaked 7 days later. A similar but less dramatic relationship existed among the Peyer's patches. In addition no labeled lymphoblast response was elicited in the inguinal lymph nodes or cecal lymphoid patches throughout the infection and the first increased responsiveness of the spleen did not take place until after Day 13, by which time the lymphoid proliferations associated with the infected intestine had subsided. Initially, the spleen showed a marked depletion of labeled blast cells during the first 7 days of the infection. This was taken as indicating at the time the infection was being established the export of cells capable of transformation in response to parasite antigen. This was supported by the observation that large numbers of phytohemagglutinin responsive lymphocytes were found in the peripheral circulation at this time. The in vitro responsiveness of peripheral lymphocytes to T. colubriformis antigen was also studied. Positive lymphocyte transformations first occurred 6 days after infection but thereafter declined to the normal level by Day 13; the peak transformation ratio was found 25 days after infection but by Day 38 it had declined to a low but persistently positive level. There was a correlation between the circulation of specifically sensitized cells, probably of thymic origin, IgE antibody titers, and the development of positive dermal delayed hypersensitivity reactions in infected guinea pigs, suggesting a close relationship among these three immunological phenomena.All lymphoblast responses in Peyer's patches, mesenteric lymph nodes, and lamina propria of the intestine were completed before the immune elimination of the parasite commenced 10 days after infection. During the first 10 days of infection specifically sensitized lymphocytes appeared and disappeared from the circulation. The loss of circulating sensitized lymphocytes at the time immune elimination of the parasite was taking place in the gut suggested that the sensitized cells were “homing-in” on the local area of infection. After the immune elimination of the parasite had commenced, the level of sensitized lymphocytes and IgE antibodies then increased rapidly in the blood. Evidence from the kinetics of the hemagglutinating antibodies indicated that stage specific antigens occur in T. colubriformis.