De Novo Generated Human Red Blood Cells in Humanized Mice Support Plasmodium falciparum Infection

Immunodeficient mouse–human chimeras provide a powerful approach to study host specific pathogens like Plasmodium (P.) falciparum that causes human malaria. Existing mouse models of P. falciparum infection require repeated injections of human red blood cells (RBCs). In addition, clodronate lipsomes and anti-neutrophil antibodies are injected to suppress the clearance of human RBCs by the residual immune system of the immunodeficient mice. Engraftment of NOD-scid Il2rg^-/- mice with human hematopoietic stem cells leads to reconstitution of human immune cells. Although human B cell reconstitution is robust and T cell reconstitution is reasonable in the recipient mice, human RBC reconstitution is generally poor or undetectable. The poor reconstitution is mainly the result of a deficiency of appropriate human cytokines that are necessary for the development and maintenance of these cell lineages. Delivery of plasmid DNA encoding human erythropoietin and interleukin-3 into humanized mice by hydrodynamic tail-vein injection resulted in significantly enhanced reconstitution of erythrocytes. With this improved humanized mouse, here we show that P. falciparum infects de novo generated human RBCs, develops into schizonts and causes successive reinvasion. We also show that different parasite strains exhibit variation in their ability to infect these humanized mice. Parasites could be detected by nested PCR in the blood samples of humanized mice infected with P. falciparum K1 and HB3 strains for 3 cycles, whereas in other strains such as 3D7, DD2, 7G8, FCR3 and W2mef parasites could only be detected for 1 cycle. In vivo adaptation of K1 strain further improves the infection efficiency and parasites can be detected by microscopy for 3 cycles. The parasitemia ranges between 0.13 and 0.25% at the first cycle of infection, falls between 0.08 and 0.15% at the second cycle, and drops to barely detectable levels at the third cycle of infection. Compared to existing mouse models, our model generates human RBCs de novo and does not require the treatment of mice with immunomodulators.     

Long-Lived Antibody and B Cell Memory Responses to the Human Malaria Parasites,Plasmodium falciparum and Plasmodium vivax

Antibodies constitute a critical component of the naturally acquired immunity that develops following frequent exposure to malaria. However, specific antibody titres have been reported to decline rapidly in the absence of reinfection, supporting the widely perceived notion that malaria infections fail to induce durable immunological memory responses. Currently, direct evidence for the presence or absence of immune memory to malaria is limited. In this study, we analysed the longevity of both antibody and B cell memory responses to malaria antigens among individuals who were living in an area of extremely low malaria transmission in northern Thailand, and who were known either to be malaria naïve or to have had a documented clinical attack of P. falciparum and/or P. vivax in the past 6 years. We found that exposure to malaria results in the generation of relatively avid antigen-specific antibodies and the establishment of populations of antigen-specific memory B cells in a significant proportion of malaria-exposed individuals. Both antibody and memory B cell responses to malaria antigens were stably maintained over time in the absence of reinfection. In a number of cases where antigen-specific antibodies were not detected in plasma, stable frequencies of antigen-specific memory B cells were nonetheless observed, suggesting that circulating memory B cells may be maintained independently of long-lived plasma cells. We conclude that infrequent malaria infections are capable of inducing long-lived antibody and memory B cell responses.     

Phenotypic and Functional Alterations in Circulating Memory CD8 T Cells with Time after Primary Infection

Memory CD8 T cells confer increased protection to immune hosts upon secondary viral, bacterial, and parasitic infections. The level of protection provided depends on the numbers, quality (functional ability), and location of memory CD8 T cells present at the time of infection. While primary memory CD8 T cells can be maintained for the life of the host, the full extent of phenotypic and functional changes that occur over time after initial antigen encounter remains poorly characterized. Here we show that critical properties of circulating primary memory CD8 T cells, including location, phenotype, cytokine production, maintenance, secondary proliferation, secondary memory generation potential, and mitochondrial function change with time after infection. Interestingly, phenotypic and functional alterations in the memory population are not due solely to shifts in the ratio of effector (CD62Llo) and central memory (CD62Lhi) cells, but also occur within defined CD62Lhi memory CD8 T cell subsets. CD62Lhi memory cells retain the ability to efficiently produce cytokines with time after infection. However, while it is was not formally tested whether changes in CD62Lhi memory CD8 T cells over time occur in a cell intrinsic manner or are due to selective death and/or survival, the gene expression profiles of CD62Lhi memory CD8 T cells change, phenotypic heterogeneity decreases, and mitochondrial function and proliferative capacity in either a lymphopenic environment or in response to antigen re-encounter increase with time. Importantly, and in accordance with their enhanced proliferative and metabolic capabilities, protection provided against chronic LCMV clone-13 infection increases over time for both circulating memory CD8 T cell populations and for CD62Lhi memory cells. Taken together, the data in this study reveal that memory CD8 T cells continue to change with time after infection and suggest that the outcome of vaccination strategies designed to elicit protective memory CD8 T cells using single or prime-boost immunizations depends upon the timing between antigen encounters.     

Immune Responses of NIH Mice Infected with Avirulent and Virulent Strains of Plasmodium chabaudi adami Single and Mixed Infections

An understanding of the nature of the immune response to asexual erythrocytic stages of malaria parasites will facilitate vaccine development by identifying which responses the vaccine should preferentially induce. The present study examined and compared the immune responses of NIH mice in either single or mixed infections with avirulent (DK) or virulent (DS) strains of Plasmodium chabaudi adami using the ELISA test for detecting and measurement of cytokines and antibody production. In both single and mixed infections, the study showed that both cell- and antibody-mediated responses were activated. In all experiments, an early relatively high level of IFN-γ and IgG2a during the acute phase of the infection, and later elevation of IL-4 and IgG1, suggested that there was a sequential Th1/Th2 response. However, in the avirulent DK strain infection a stronger Th1 response was observed compared to the virulent DS strain-infection or in mixed infections. In the virulent DS infection, there was a stronger Th2 response compared to that in the DK and mixed infections. The faster proliferation rate of the virulent DS strain compared to the DK strain was also evident.     

CD4+CD25+Foxp3hi细胞在夏氏疟原虫感染DBA/2小鼠作用研究

利用调节性T细胞消除的致死型夏氏疟原虫(Plasmodium chabaudi chabaudi AS,P.c chabaudi AS)感染鼠疟模型,探讨DBA/2小鼠对P.c chabaudi AS感染易感性的原因。DBA/2小鼠对P.c chabaudi AS易感,伴随原虫血症增加CD4+CD25+Foxp3+细胞数量明显增加,且以CD4+CD25+Foxp3hi增加更为明显。原虫血症达峰值时CD4+CD25+Foxp3hi细胞数量亦达到峰值。相比,Treg消除鼠的原虫出现时间和疟血症峰值时间均明显延迟,且在疟血症达峰值前(5～8 d)原虫血症水平明显低于对照组。与之相应,CD4+CD25+Foxp3hi细胞数量明显处于低水平。同时,Treg消除鼠生存期明显延长。由此提示,P.c chabaudi AS感染导致Foxp3表达增加,扩增的CD4+CD25+Foxp3hi细胞有利于疟原虫复制和逃避宿主免疫应答,进而影响疟疾感染的进程和最终结局。     

IL-10在Plasmodium yoelii 17XL和Plasmodium chabaudi AS疟原虫混合感染宿主病理损伤中的作用研究

为探讨IL-10在致死型约氏疟原虫(Plasmodium yoelii 17XL,P.y17XL)和夏氏疟原虫(Plasmodiumchabaudi AS,P.cAS)混合感染宿主病理损伤中的作用,用P.y17XL、P.cAS和P.y17XL+P.cAS分别感染DBA/2小鼠,计数红细胞感染率;感染后第3、5、8、10、12和19天分别尾静脉取血,肝素抗凝后短暂离心,采用高纯度DNA提取试剂盒抽提DNA,实时定量PCR检测虫负荷水平;感染后第0、1、3、5、8、10、12和15天制备脾细胞悬液,ELISA检测脾细胞培养上清中IL-10水平。实验结果发现,P.y17XL单独感染和混合感染小鼠IL-10水平在感染后第5天和第8天分别达峰值,随后开始下降至正常水平,小鼠虫血症均达中等水平,存活率100%;相比P.cAS感染小鼠IL-10在感染后第3天突然出现高水平升高并且维持时间较长;于感染后第8天达峰值,是同天P.y17XL单独感染和混合感染小鼠IL-10水平的2倍,虫血症水平较高,小鼠全部死亡。同时实时定量PCR结果发现,混合感染小鼠,于感染后3～12 d P.y17XL增殖占优势,而感染后15～19 d则P.cAS增殖处于优势状态。表明以IL-10为核心的免疫调节网络与疟疾感染过程中病理损伤密切相关。同时提示混合感染小鼠应答模式与P.y17XL感染小鼠的应答模式相同。     

RNA-DNA Differences Are Generated in Human Cells within Seconds after RNA Exits Polymerase II

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Plasmodium chabaudi chabaudi infection in mice induces strong B cell responses and striking but temporary changes in splenic cell distribution

B cells and Abs play a key role in controlling the erythrocytic stage of malaria. However, little is known about the way the humoral response develops during infection. We show that Plasmodium chabaudi chabaudi causes major, but temporary changes in the distribution of leukocytes in the spleen. Despite these changes, an ordered response to infection develops, which includes vigorous extrafollicular growth of plasmablasts and germinal center formation. Early in the response, the lymphocytes in the T zone and follicles become widely spaced, and the edges of these compartments blur. This effect is maximal around the peak of parasitemia. Germinal centers are apparent by day 8, peak at day 20, and persist through day 60. Extrafollicular foci of plasmablasts are visible from day 4 and initiate a very strong plasma cell response. Initially, the plasma cells have a conventional red pulp distribution, but by day 10 they are unconventionally sited in the periarteriolar region of the white pulp. In this region they form clusters occupying part of the area normally filled by T cells. B cells are absent from the marginal zone for at least 30 days after the peak of infection, although flow cytometry shows their continued presence in the spleen throughout infection. Relatively normal splenic architecture is regained by day 60 of infection. These results show that the changes in splenic cell distribution are linked to the presence of parasites and do not seem to interfere with the development of the humoral response.     

Simultaneous Assessment of Rotavirus-Specific Memory B Cells and Serological Memory after B Cell Depletion Therapy with Rituximab

The mechanisms that contribute to the maintenance of serological memory are still unclear. Rotavirus (RV) memory B cells (mBc) are enriched in IgM⁺ and CD27^- subpopulations, which are associated with autoimmune diseases pathogenesis. In patients with autoimmune diseases treated with Rituximab (RTX), some autoantibodies (auto-Abs) decrease after treatment, but other auto-Abs and pathogen-specific IgG Abs remain unchanged. Thus, maintenance of autoimmune and pathogen-specific serological memory may depend on the type of antigen and/or Ab isotype evaluated. Antigen-specific mBc and antigen-specific Abs of different isotypes have not been simultaneously assessed in patients after RTX treatment. To study the relationship between mBc subpopulations and serological memory we characterized total, RV- and tetanus toxoid (TT)-specific mBc by flow cytometry in patients with autoimmune diseases before and after treatment with RTX. We also measured total, RV- and TT-Abs, and some auto-Abs by kinetic nephelometry, ELISA, and EliA tests, respectively. Minor differences were observed between the relative frequencies of RV-mBc in healthy controls and patients with autoimmune disease. After RTX treatment, naïve Bc and total, RV- and TT-specific mBc [IgM⁺, switched (IgA⁺/IgG⁺), IgM⁺ only, IgD⁺ only, and CD27^- (IgA⁺/IgG⁺/IgM⁺)] were significantly diminished. An important decrease in total plasma IgM and minor decreases in total IgG and IgA levels were also observed. IgM rheumatoid factor, IgG anti-CCP, and IgG anti-dsDNA were significantly diminished. In contrast, RV-IgA, RV-IgG and RV-IgG₁, and TT-IgG titers remained stable. In conclusion, in patients with autoimmunity, serological memory against RV and TT seem to be maintained by long-lived plasma cells, unaffected by RTX, and an important proportion of total IgM and serological memory against some auto-antigens seem to be maintained by short-lived plasma cells, dependent on mBc precursors depleted by RTX.     

Identification of a Functional Connectome for Long-Term Fear Memory in Mice

Long-term memories are thought to depend upon the coordinated activation of a broad network of cortical and subcortical brain regions. However, the distributed nature of this representation has made it challenging to define the neural elements of the memory trace, and lesion and electrophysiological approaches provide only a narrow window into what is appreciated a much more global network. Here we used a global mapping approach to identify networks of brain regions activated following recall of long-term fear memories in mice. Analysis of Fos expression across 84 brain regions allowed us to identify regions that were co-active following memory recall. These analyses revealed that the functional organization of long-term fear memories depends on memory age and is altered in mutant mice that exhibit premature forgetting. Most importantly, these analyses indicate that long-term memory recall engages a network that has a distinct thalamic-hippocampal-cortical signature. This network is concurrently integrated and segregated and therefore has small-world properties, and contains hub-like regions in the prefrontal cortex and thalamus that may play privileged roles in memory expression.     

Hippocampal and Cortical Primary Cilia Are Required for Aversive Memory in Mice

It has been known for decades that neurons throughout the brain possess solitary, immotile, microtubule based appendages called primary cilia. Only recently have studies tried to address the functions of these cilia and our current understanding remains poor. To determine if neuronal cilia have a role in behavior we specifically disrupted ciliogenesis in the cortex and hippocampus of mice through conditional deletion of the Intraflagellar Transport 88 (Ift88) gene. The effects on learning and memory were analyzed using both Morris Water Maze and fear conditioning paradigms. In comparison to wild type controls, cilia mutants displayed deficits in aversive learning and memory and novel object recognition. Furthermore, hippocampal neurons from mutants displayed an altered paired-pulse response, suggesting that loss of IFT88 can alter synaptic properties. A variety of other behavioral tests showed no significant differences between conditional cilia mutants and controls. This type of conditional allele approach could be used to distinguish which behavioral features of ciliopathies arise due to defects in neural development and which result from altered cell physiology. Ultimately, this could lead to an improved understanding of the basis for the cognitive deficits associated with human cilia disorders such as Bardet-Biedl syndrome, and possibly more common ailments including depression and schizophrenia.     

Angiotensin II Moderately Decreases Plasmodium Infection and Experimental Cerebral Malaria in Mice

Angiotensin II, a peptide hormone that regulates blood pressure, has been proposed as a protective factor against cerebral malaria based on a genetic analysis. In vitro studies have documented an inhibitory effect of angiotensin II on Plasmodium growth, while studies using chemical inhibitors of angiotensin II in mice showed protection against experimental cerebral malaria but not major effects on parasite growth. To determine whether the level of angiotensin II affects Plasmodium growth and/or disease outcome in malaria, elevated levels of angiotensin II were induced in mice by intradermal implantation of osmotic mini-pumps providing constant release of this hormone. Mice were then infected with P. berghei and monitored for parasitemia and incidence of cerebral malaria. Mice infused with angiotensin II showed decreased parasitemia seven days after infection. The development of experimental cerebral malaria was delayed and a moderate increase in survival was observed in mice with elevated angiotensin II, as confirmed by decreased number of cerebral hemorrhages compared to controls. The results presented here show for the first time the effect of elevated levels of angiotensin II in an in vivo model of malaria. The decreased pathogenesis observed in mice complements a previous human genetic study, reinforcing the hypothesis of a beneficial effect of angiotensin II in malaria.     

Lymphocytes and Macrophages Are Infected by Theileria equi,but T Cells and B Cells Are Not Required to Establish Infection In Vivo

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.     

Decrease of Functional Activated T and B Cells and Treatment of Glomerulonephitis in Lupus-Prone Mice Using a Natural Flavonoid Astilbin

Treatment of systemic lupus erythematosus (SLE), a chronic inflammatory disease, involves the long-term use of immunosuppressive agents with significant side effects. New therapeutic approaches are being explored to find better treatment possibilities. In this study, age-matched female MRL/lpr mice were treated orally with a natural flavonoid astilbin. Astilbin administration started either at week 8 or week 12 of age though week 20. In the early treatment regimen, the treatment with astilbin reduced splenomegaly / lymphomegaly, autoantibody production and ameliorated lupus nephitis. Several serum cytokines were significantly decreased upon treatment including IFN-g, IL-17A, IL-1b, TNF-a and IL-6. Both spleen CD44^hiCD62L^lo activated T cells and CD138⁺B220^- plasma cells greatly declined. Furthermore, astilbin treatment resulted in decreased mitochondrial membrane potential in activated T cells and downregulated expression of the co-stimulatory molecules CD80 and CD86 on LPS stimulated B cells. Similar but less profound effectiveness was observed in the mice with established disease in the late treatment regimen. These results indicate that the natural product astilbin can mitigate disease development in lupus-prone mice by decreasing functional activated T and B cells.     

Characterization of Cells Infiltrating the Lungs of X-Irradiated and Nude Mice after Influenza Virus Infection

After X-irradiated and nonirradiated mice (C3H/He) as well as athymic nude mice and haired littermates (BALB/c) were infected with influenza A virus (Kumamoto strain, H2N2), they were examined for survival period, the development of consolidation in the lungs and the characteristics of the cells infiltrating the lung tissues. In two different T-cell deficient groups, there was a definite delay in the development of consolidation compared with their respective controls and this was reflected in prolonged survival periods: 5 days longer for irradiated mice and 6 days longer for nude mice. In both T-cell deficient and normal groups, about 70% of the cells obtained from consolidated lung tissues after virus infection were found to be small lymphoid cells and there were no morphological differences between the T-cell deficient and normal groups. None of these small lymphoid cells from the peripheral blood or the spleens of T-cell deficient mice responded to concanavalin A. In the lungs of both X-irradiated mice and nude mice, however, a definite increase in cells having natural killer activity was found at the late stages of the influenza infection, suggesting their participation in the development of consolidation.     

Age Related Differences in Dynamics of Specific Memory B Cell Populations after Clinical Pertussis Infection

For a better understanding of the maintenance of immune mechanisms to Bordetella pertussis (Bp) in relation to age, we investigated the dynamic range of specific B cell responses in various age-groups at different time points after a laboratory confirmed pertussis infection. Blood samples were obtained in a Dutch cross sectional observational study from symptomatic pertussis cases. Lymphocyte subpopulations were phenotyped by flowcytometry before and after culture. Memory B (B_mem) cells were differentiated into IgG antibody secreting cells (ASC) by polyclonal stimulation and detected by an ELISPOT assay specific for pertussis antigens pertussis toxin (Ptx), filamentous haemagglutinin (FHA) and pertactin (Prn). Bp antigen specific IgG concentrations in plasma were determined using multiplex technology. The majority of subjects having experienced a clinical pertussis episode demonstrated high levels of both Bp specific IgG and B_mem cell levels within the first 6 weeks after diagnosis. Significantly lower levels were observed thereafter. Waning of cellular and humoral immunity to maintenance levels occurred within 9 months after antigen encounter. Age was found to determine the maximum but not base-line frequencies of B_mem cell populations; higher levels of B_mem cells specific for Ptx and FHA were reached in adults and (pre-) elderly compared to under-fours and schoolchildren in the first 6 weeks after Bp exposure, whereas not in later phases. This age effect was less obvious for specific IgG levels. Nonetheless, subjects'' levels of specific B_mem cells and specific IgG were weakly correlated. This is the first study to show that both age and closeness to last Bp encounter impacts the size of Bp specific B_mem cell and plasma IgG levels.     

Plasmodium chabaudi chabaudi(AS): Inflammatory Cytokines and Pathology in an Erythrocytic-Stage Infection in Mice

Cross, C. E., and Longhorne J. 1998.Plasmodium chabaudi chabaudi(AS): Inflammatory cytokines and pathology in an erythrocytic-stage infection in mice.Experimental Parasitology90220–229. We have sought to characterizePlasmodium chabaudi chabaudiinfection in mice for use as a model for malaria pathology. Different mouse strains vary in their susceptibility to the erythrocytic stages of this parasite and this is manifested not only in the outcome of infection (survival versus death) but also by differences in the numbers of circulating parasites at the peak of infection. We have shown that regardless of final outcome, both resistant and susceptible mice exhibit other parameters of disease such as loss in body weight and anemia. By contrast, other parameters such as hypothermia appear more severe in susceptible mice. The severe symptoms coincide with high levels of inflammatory cytokines in the circulation of susceptible mice, not seen in H-2-matched resistant mice. However, levels of mRNA for the same cytokines, measured in the spleen of the same mice was not significantly different between the two strains. Neutralization of IFN-γin vivoled to an increase in parasitemia, in both susceptible and resistant mice, but did not affect the final outcome of disease. Indeed, symptoms were exacerbated in the absence of IFN-γ, presumably because of larger numbers of circulating parasites. These data suggest that IFN-γ does not directly contribute to the lethal outcome of infection in susceptible strains of mice.     

Surveillance on the Status of Immune Cells after Echinnococcus granulosus Protoscoleces Infection in Balb/c Mice

Background

Cystic echinococcosis is a global parasitic disease caused by infection with Echinococcus granulosus larvae with potentially life-threatening complications in humans. To date, the status of the immune cells believed to be associated with the pathogenicity of E. granulosus infection has not been demonstrated clearly.

Methodology/Principal Findings

In this study, we developed a multiplex flow cytometry assay to investigate the systemic immune status of innate and adaptive immunity at 30, 180, 360 days post-infection (dpi) in mice infected with E. granulousus. At 30 dpi, an increase in the number of CD11b⁺ and CD11c⁺ antigen-presenting cells (APCs) was observed. This was accompanied by the slight down-regulated expression of the co-stimulatory molecule MHC-II, indicating the impairment of APCs in early infection through the release of secretory-excretory products. In all infected groups, we observed a significant increase in innate immune cells, including APCs and GR-1⁺ cells, and a dramatic increase in the myeloid-derived suppressor cells (MDSC) expressing CD11b⁺/GR-1⁺. Moreover, the upregulation of the activated markers CD69, CD44, CD40L, and the downregulation of CD62L were observed in the CD4⁺ and CD8⁺ T cells following infection. Regulatory T cells expressing CD4⁺/CD25⁺/FoxP₃ ⁺ increased significantly over the course of infection.

Conclusions

Our findings demonstrate that the microenvironment in the peripheral immune system after E. granulosus infection changes in subtle but detectably ways, especially during the persistent period of infection. We found that T cells were activated following infection, but observed that the significant increase of immunosuppressive cells such as MDSC and Treg cells could inhibit T cell response to E. granulosus antigens. We suggest these cells may play a neglected but key role in the downregulation of the immune response in long-term parasitic infection. Understanding the basic functions and temporal interactions of these immunosuppressive cells will pave the way for new strategies of parasite vaccine design.