Plasmodium chabaudi: a rodent malaria model for in-vivo and in-vitro cytoadherence of malaria parasites in the absence of knobs

The ability of Plasmodium chabaudi infected erythrocytes to bind to endothelial cells in vivo and to various murine cell lines in vitro is described. A procedure for the selective recovery of a sequestering subpopulation of schizont-infected erythrocytes from hepatic sinusoids of BALB/c mice, using a combination of body perfusion, trypsin treatment and Percoll density centrifugation was developed. The serial subinoculation of such a subpopulation was used to select for a clone of parasites (strain AJJ) with considerably better cytoadherent characteristics than the parent line (strain AJ). In contrast, it was demonstrated that another clone (PC7), showed no cytoadherence in vivo or in vitro. This study shows that parasite induced alterations occurred on the surface of erythrocytes infected with late developmental stages of P. chabaudi. The antigenicity of these molecules in the infected mouse was demonstrated using immune serum and affinity chromatography. Cytoadherence and surface antigenic changes in P. chabaudi schizont-infected erythrocytes were demonstrated in the absence of the submembranous 'knobs' associated with cytoadherence in P. falciparum.     

Testosterone-unresponsiveness of existing immunity against Plasmodium chabaudi malaria

Testosterone (Te) is known to suppress immunity and to increase host susceptibility to many parasites. This study investigates the action of Te on immunity acquired against blood-stages of the malaria parasite Plasmodium chabaudi in female mice of the inbred strain C57BL/10. Our data show: (i) About 90% of mice infected with 10(6) P. chabaudi-infected erythrocytes are able to develop protective immune mechanisms which become evident in self-healing the infection. The capability of self-healing is lost when mice are pretreated with Te for 3 weeks. (ii) Mice which have self-healed infections acquire immunity to homologous rechallenge. Concomitantly, mice become Te-unresponsive in that their acquired immunity is not suppressible by Te-treatment. (iii) Flow cytometry reveals that Te-pretreatment entails an increase of CD8+ cells and a decrease of Ig+ cells by about 4% in spleens of non-immune mice. In immune mice, however, there is a Te-unresponsiveness of the percental distribution of splenic cell populations. (iv) Adoptive transfer experiments indicate that immunity is conferred by spleen cells, presumably non-T-cells. These cells are Te-unresponsive, since they exert their effect in Te-pretreated mice in the presence of Te. (v) Te-unresponsive immunity can be also transferred by serum, especially the IgG-fraction, obtained from immune mice. Our data demonstrate that Te prevents the development of protective immunity against P. chabaudi infections. However, when once established, protective immunity becomes unresponsive to Te. Our data suggest that the effector mechanisms of protective immunity involve Te-unresponsive B cells secreting protective IgG-antibodies.     

Limiting dilution analysis of the T cell response to Plasmodium chabaudi chabaudi in mice

A limiting dilution assay system was developed in order to measure the in-vitro T cell response to antigens of the erythrocytic stages of Plasmodium chabaudi. The conditions of the assay are such that only CD4+ T cells are able to respond. The assay allows the determination of the frequencies of T cells which proliferate and/or which develop into helper cells for antibody production during a primary infection. A specific response from splenic T cells can be measured as early as 7 days after infection, and is still significant 3 months after injection of P. chabaudi. At all times the frequency of proliferating cells was greater than the precursor frequency of T helper cells. This suggests that a proportion of CD4+ T cells in this assay, although they respond to malarial antigen, do not develop into helper cells for antibody production. This limiting dilution assay will be a useful method by which to evaluate the functional heterogeneity of the CD4+ T cell response to malaria antigens.     

Testosterone and other gonadal factor(s) restrict the efficacy of genes controlling resistance to Plasmodium chabaudi malaria

The effect of circulating concentrations of testosterone (Te) on resistance to Plasmodium chabaudi malaria was investigated in the H-2 congenic mouse strains C57BL/10, B10.A, B10.A(3R), B10.A(4R), and B10.D2. Te-levels were determined by radioimmunoassay and resistance was expressed in terms of percent self-healers after challenge with 10(6) P. chabaudi-infected erythrocytes. Our data indicate: (i) Females and castrated males reveal very similar interstrain variations of resistance. These do not correlate with the interstrain variations of the Te-levels. This is consistent with the view that resistance to P. chaubaudi is controlled by genes of the H-2 complex and genes of the non-H-2 B10-background, (ii) The polygenic control of resistance is inefficacious at high Te-levels. This is evident as high susceptibilities of males, Te-treated females and Te-treated castrated males. Moreover, high Te-levels correlate with susceptibilities to P. chabaudi within mice of the same sex of a given strain, (iii) B10-males chemically castrated using buserelin display the same low Te-level as those surgically castrated. The latter become resistant, while the former remain as highly susceptible to P. chabaudi as untreated B10-males. Obviously, other gonadal factor(s), besides Te, impose restrictions on genes controlling resistance to P. chabaudi malaria.     

Absence of circulating natural killer (NK) cells in a child with erythrophagocytic lymphohistiocytosis lacking NK cell activity

A 5-year-old girl who was diagnosed as having erythrophagocytic lymphohistiocytosis died at age 9 years. Peripheral lymphocytes from the patient persistently lacked natural killer (NK) cell activity during the 4-year observation period: the percent lysis values as measured by a 4-hr 51Cr release assay at a 40:1 effector:target ratio were below 1.0% against K562 and Molt-4 cells as compared with the normal lymphocyte value (mean +/- SD) of 46.2% +/- 5.8% and 43.9% +/- 6.7%, respectively. The patient's lymphocytes never developed NK cell activity by their incubation with target cells for longer time periods or by their stimulation with interferon-alpha, interleukin-2, or polyinosinic-polycytidilic acid. Single cell-in-agarose assay showed the absence of target-binding cells (TBCs): TBC numbers were below 0.3% as compared with the normal lymphocyte value of 8.1% +/- 1.3% (mean +/- SD). Flow cytometry showed a marked decrease in Leu-7+ cells (1.7%) and the absence of Leu-11+ cells (0.4%) in the peripheral blood. These results first demonstrate a case of erythrophagocytic lymphohistiocytosis in which there is the lack of NK cell activity due to the absence of circulating NK cells.     

Protective immunity to malaria: studies with cloned lines of Plasmodium chabaudi and P. berghei in CBA/Ca mice. I. The effectiveness and inter-and intra-species specificity of immunity induced by infection

CBA/Ca mice were immunized by infection with cloned lines of Plasmodium berghei (isolates ANKA, KSP-11). Plasmodium chabaudi chabaudi (AS, CB) or Plasmodium chabaudi adami (DS) and then challenged with either homologous or heterologous parasites. Protective responses were assessed in immune mice relative to the controls by their ability to (i) extend the time taken for the mean parasitaemia to reach a predetermined level (1% or 0.1%) (ii) reduce peak parasitaemia (iii) resolve the parasitaemia sooner and/or (iv) control or eliminate recrudescences. At both the inter- and intra-species level, immunity appeared largely specific for the cloned line inducing it. At the interspecies level marginally effective cross-immunity was sometimes evident, thus P. berghei KSP-11 immune mice displayed some immunity against P.c. chabaudi AS, although immunity to this parasite was relatively ineffective against P. berghei ANKA or KSP-11. Cross-immunity was more apparent between the subspecies P.c. adami and P.c. chabaudi and between cloned lines of the latter parasite derived from the AS and CB isolates. These data reflect considerable inter- and intra-species structural and immunogenic differences in certain antigens of parasitized erythrocytes and merozoites, which have been identified in a number of murine malarias and associated with protective immunity. Similar differences recently identified in the equivalent antigens of the human parasite P. falciparum may therefore have important implications for protective immunity in man.     

Granzyme B and natural killer (NK) cell death

Abstract

Granzyme B is a unique serine protease, which plays a crucial role for target cell death. Several mechanisms of delivery of granzyme B to target cells have been recently identified. Granzyme B directly activates Bid, a specific substrate for granzyme B, resulting in caspase activation. Granzyme B efficiently cleaves many prominent autoantigens, and the hypothesis that autoantibodies arise when cryptic determinants are revealed to the immune system has been proposed. Some autoantibodies directed against granzyme B-specific neoepitopes are present in serum from patients with autoimmune diseases. In the tissues from autoimmune diseases, granzyme B might play an important role for disease progression (i.e., rheumatoid arthritis synovium) or inhibition (i.e., regulatory T cells). We have identified a novel type of activation-induced cell death (granzyme B leakage-induced cell death). Activation-induced natural killer (NK) cell death is accompanied by the leakage of granzyme B from intracellular granules into the cytoplasm, and it triggers apoptosis by directing Bid to mitochondrial membranes. An excess of “leaked” granzyme B over its inhibitor, serpin proteinase inhibitor 9, is a major determinant of cell death. The role of granzyme B in autoimmunity and its influence on NK cell death are discussed.     

Granzyme B and natural killer (NK) cell death

Granzyme B is a unique serine protease, which plays a crucial role for target cell death. Several mechanisms of delivery of granzyme B to target cells have been recently identified. Granzyme B directly activates Bid, a specific substrate for granzyme B, resulting in caspase activation. Granzyme B efficiently cleaves many prominent autoantigens, and the hypothesis that autoantibodies arise when cryptic determinants are revealed to the immune system has been proposed. Some autoantibodies directed against granzyme B-specific neoepitopes are present in serum from patients with autoimmune diseases. In the tissues from autoimmune diseases, granzyme B might play an important role for disease progression (i.e., rheumatoid arthritis synovium) or inhibition (i.e., regulatory T cells). We have identified a novel type of activation-induced cell death (granzyme B leakage-induced cell death). Activation-induced natural killer (NK) cell death is accompanied by the leakage of granzyme B from intracellular granules into the cytoplasm, and it triggers apoptosis by directing Bid to mitochondrial membranes. An excess of “leaked” granzyme B over its inhibitor, serpin proteinase inhibitor 9, is a major determinant of cell death. The role of granzyme B in autoimmunity and its influence on NK cell death are discussed.     

Genetic studies on a major merozoite surface antigen of the malaria parasite of rodents, Plasmodium chabaudi

Diversity in a major merozoite surface antigen (MSA-1) of Plasmodium chabaudi has been examined using a panel of monoclonal antibodies (MoAbs). The antigen was found to be different in each of twelve cloned isolates, as shown by its reactivity with the MoAbs in immunofluorescence tests. In genetic crossing experiments, the diverse forms of this antigen were shown to be determined by allelic variation of a single gene. Recombination occurred between the MSA-1 gene, a second blood stage antigen and enzyme markers.     

Augmenter of liver regeneration (ALR) may promote liver regeneration by reducing natural killer (NK) cell activity in human liver diseases

Cytotoxicity of liver natural killer cells against regenerating hepatocytes has been reported as a possible mechanism of regeneration failure in fulminant hepatitis. An augmenter of liver regeneration (ALR) inhibits liver natural killer cell activity in rats. In this study, we measured hepatic expression of ALR mRNA, blood levels of ALR, and peripheral blood natural killer cell activity in patients with various types of acute liver disease to investigate the relationship between failure of liver regeneration and hepatic natural killer cells. Hepatic ALR mRNA expression was higher in liver disease patients than in non-liver disease controls, and a correlation was found between serum ALR values and hepatic levels of ALR mRNA. In acute liver injury, the serum ALR level also showed a negative correlation with NK activity. ALR was produced by and released from the liver at the time of hepatic injury. Our findings suggest that ALR may protect against failure of regeneration by inhibition of hepatic natural killer cell activity in acute liver injury. Received: December 7, 1998 / Accepted: August 27, 1999     

Malaria-specific antibody responses and parasite persistence after infection of mice with Plasmodium chabaudi chabaudi

While it is known that antibodies are critical for clearance of malaria infections, it is not clear whether adequate antibody responses are maintained and what effect chronic infection has on this response. Here we show that mice with low-grade chronic primary infections of Plasmodium chabaudi or infections very recently eliminated have reduced second infections when compared with the second infection of parasite-free mice. We also show that parasite-specific antibody responses induced by infection of mice with Plasmodium chabaudi contain both short- and long-lived components as well as memory B cells responsible for a faster antibody response during re-infection. Furthermore, parasite-specific antibodies to the C-terminal fragment of merozoite surface protein-1 (MSP-1) undergo avidity maturation. However, antibodies with both low and high avidity persist throughout infection and after re-infection, suggesting repeated rounds of activation and maturation of memory B cells. Neither the avidity profile of the antibody response, nor its maintenance is affected by persisting live parasites. Therefore, differences in parasitemia in re-infection cannot be explained solely by higher levels of antibody or greater affinity maturation of malaria-specific antibodies. These data suggest that there may be an antibody-independent component to the early control of secondary infections in mice that are chronically infected.     

Natural killer (NK) cell activity and its in vitro response to interferon-α(Le) in chronic liver diseases and hepatocellular carcinoma

ABSTRACT- The natural killer (NK) cell activity of peripheral blood lymphocytes (PBL) in patients with various chronic liver diseases, and its in vitro response to human interferon-α(Le) were investigated using a 16-h 51-Cr releasing cytotoxicity assay against YAC-1 or RSa target cells. The NK cell activity was found to be higher in chronic active hepatitis (CAH) and liver cirrhosis (LC) patients without HCC, whereas it was slightly lower in LC patients with hepatocellular carcinoma (HCC), than in normal controls. By the addition of IFN-α(Le) in vitro, the NK cell activity was clearly and dose-dependently augmented, even in chronic liver diseases, as well as in normal controls. The magnitude of this augmentation by 10000 IU/ml of IFN-α(Le) in the various chronic liver diseases was not significantly different from that in normal controls. The results suggested that the response of NK cells to IFN-α(Le) is not impaired even in chronic liver disease conditions, while the level of NK cell activity may vary according to the type of chronic liver disease and may decrease in patients with HCC.     

Protective immunity to malaria: studies with cloned lines of rodent malaria in CBA/Ca mice. IV. The specificity of mechanisms resulting in crisis and resolution of the primary acute phase parasitaemia of. Plasmodium chabaudi chabaudi and P. yoelii yoelii

Low numbers of parasites from cloned lines of the rodent malaria parasites, Plasmodium chabaudi chabaudi AS and P. yoelii yoelii A, injected into CBA/Ca mice produce acute but usually self-limiting infections. During crisis, i.e. 1-2 days after peak parasitaemia, 'pre-immune' mice experiencing such 'background' infections were reinfected intravenously with homologous parasites or parasites of heterologous strains or species. P. c. chabaudi AS pre-immune mice controlled an AS challenge with essentially the same kinetics as the background infection. Reinfection of AS pre-immune mice with the heterologous (CB and IP-PCI) P. c. chabaudi strains or P. chabaudi adami DS had little effect on the initial growth of these parasites, although eventually the parasitaemia was controlled. In contrast, a partial inhibitory effect on the growth of P. vinckei lentum DS was evident. Challenge with the non-lethal (A) or lethal (YM) variants of P. y. yoelii resulted in an increase in both the growth and virulence of these parasites. P. y. yoelii A pre-immune mice controlled a homologous challenge, but were less effective at controlling the YM variant. In addition, they were unable to clear rapidly a P. c. chabaudi AS or P. v. lentum DS challenge. Both the multiplication and virulence of P. berghei ANKA were enhanced. These findings demonstrate that resolution of the primary acute parasitaemia in P. c. chabaudi AS- and P. y. yoelii A-infected mice is predominantly mediated by species- and strain-specific mechanisms.     

Susceptibility of the different developmental stages of the asexual (schizogonic) erythrocyte cycle of Plasmodium chabaudi chabaudi to hyperimmune serum, immunoglobulin (Ig)G1, IgG2a and F(ab'')2 fragments

The mechanisms by which antibodies interfere with Plasmodium growth are still under debate. Characterizing the asexual erythrocyte stages susceptible to antibodies from hyperimmune individuals is therefore a relevant contribution to vaccine research. In this study, using a virulent and synchronous murine malaria parasite, Plasmodium chabaudi chabaudi AJ, we have shown that trophozoites and circulating schizonts are not the main targets for antibodies from hyperimmune serum. In drug-cured mice challenged with a high inoculum of ring-infected erythrocytes, parasitemias do not decline until the moment of erythrocyte rupture, suggesting that effector mechanisms operate immediately prior to reinvasion. Confirming these findings, treatment of primary-infected mice with hyperimmune serum inhibited the generation of new ring forms, but did not alter the numbers of schizont-infected erythrocytes, despite the fact that these cells were recognized by immunoglobulin (Ig)G antibodies. When these mice were treated with IgG1 or IgG2a purified from hyperimmune serum, both subclasses limited reinvasion, but IgG2a showed a stronger protective activity. The fact that Fc digestion decreases but does not abrogate protection suggests that both Fc-dependent and independent mechanisms participate in this process. Treatment with cobra venom factor did not interfere with the antibody-mediated protection, ruling out the participation of the complement system in both lysis and phagocytosis of merozoites or infected erythrocytes. Therefore, in mice suffering from P. c. chabaudi AJ malaria, merozoite neutralization seems to be a major mechanism of protection conferred by hyperimmune serum antibodies. However, FcgammaR-mediated interactions, or other mechanisms not yet defined, may also contribute to inhibit erythrocyte reinvasion.     

Lymphotoxin alpha and tumour necrosis factor are not required for control of parasite growth, but differentially regulate cytokine production during Plasmodium chabaudi chabaudi AS infection

Tumour necrosis factor (TNF) plays important roles in the pathogenesis of severe malaria, as well as in the generation of immune responses against malaria parasites. However, far less is known about the role of the closely related TNF family member lymphotoxin alpha (LTalpha) during malaria. We have used mice deficient in either TNF or LTalpha, as well as chimeric mice generated using donor bone marrow from these animals, to study the roles of these cytokines following Plasmodium chabaudi chabaudi AS infection. TNF and LTalpha were not required for the resolution of P. chabaudi chabaudi AS blood-stage infection. However, LTalpha, but not TNF, was necessary for early IFNgamma production and the regulation of IFNgamma production later in infection. A similar delay to that found for IFNgamma production was also observed for TNF production in LTalpha-deficient mice, compared with control mice. These results identify divergent roles for TNF and LTalpha in the regulation of host immune responses during P. chabaudi chabaudi AS infection.     

夏氏疟原虫感染早期易感和抵抗小鼠树突状细胞亚群和表型的变化特点

目的探讨夏氏疟原虫（Plasmodium chabaudi chabaudi AS）感染早期,树突状细胞（Dendritic cells, DCs）亚群和表型的变化特点。方法感染易感的DBA/2和抵抗的BALB/c小鼠,制备感染前和感染后第3、5、8d小鼠脾细胞悬液,采用流式细胞分析技术检测2种小鼠脾细胞悬液中髓样DCs和浆样DCs的数量以及表面表达MHC II类分子和CD80分子的DCs的百分含量。结果DBA/2小鼠和BALB/c小鼠分别呈现以髓样DCs和浆样DCs为主的增殖模式。2种小鼠表达MHCⅡ类分子和CD80分子的DCs数量在感染后3~5d明显升高,并于感染后第8d达到最高水平,然而感染后第8d,BALB/c小鼠表达MHCⅡ类分子和CD80分子的DCs的数量明显低于DBA/2小鼠。结论感染早期,DBA/2和BALB/c小鼠在DCs亚群的增殖模式、成熟表型特征性分子的表达水平上存在显著差异。     

夏氏疟原虫感染早期易感和抵抗小鼠树突状细胞细胞因子的分泌特点

目的探讨在夏氏疟原虫(Plasmodiumchabaudi chabaudi AS,P.c.chabaudi AS)感染早期,树突状细胞(Den-dritic cells,DCs)调控Th1细胞免疫应答的相关机制。方法用P.c.chabaudi AS感染易感型DBA/2和抵抗型BALB/c小鼠,计数红细胞感染率;感染前和感染后3、5、8d制备脾细胞悬液,磁珠分选、纯化DCs并体外培养;ELISA方法检测DCs培养上清中IL-12p40、IL-10和TGF-β1的水平。结果 DBA/2小鼠DCs培养上清中IL-12p40的水平在感染后3-8天明显升高,BALB/c小鼠DCs培养上清中IL-12p40的水平在感染后5-8d出现有意义的升高,但其水平均明显低于DBA/2小鼠;两种小鼠DCs培养上清中IL-10与TGF-β1的分泌水平在感染后5-8d明显升高,但DBA/2小鼠DCs培养上清中IL-10与TGF-β1的分泌水平明显低于BALB/c小鼠。结论 P.c.chabaudi AS感染早期,DBA/2和BALB/c小鼠在DCs细胞因子的分泌模式上存在显著差异。     

The effect of acute and chronic leukapheresis on the natural killer (NK) cell function of normal human volunteers

Twenty-two normal volunteers had approximately eight, 2-hr-long leukapheresis procedures over a 2-year period and their natural killer (NK) cell function was prospectively measured. The NK activity of the preprocedure peripheral blood (pre-PB) was found to correlate well with the NK activity of the inital leukocytes removed by leukapheresis (I-Leuk). When the I-Leuk specimens were compared with the leukapheresis specimens removed at the termination of leukapheresis (T-Leuk), T-Leuk showed a consistent 10% increase in NK activity. When the pre-PB and the I-Leuk values were analyzed for each donor over the 2 years of the study, 18 donors revealed no significant change from their baseline NK activity, two donors showed a minimal increase in NK cell activity, and two donors displayed a minimal decrease in NK cell activity. We conclude that although leukapheresis appears acutely to boost NK cell activity, this increase is transient and small in magnitude. Most importantly, repeated leukapheresis does not appear adversely to effect this important effector function in normal donors.     

Analysis of natural killer (NK) cell activity and adhesion molecules on NK cells from umbilical cord blood

The activity of natural killer (NK) cells in human umbilical cord blood (CB) has been reported to be low, compared with that in adult peripheral blood (PB) in vitro. To examine the cause of this, after dividing the CD56+/CD3- cells in CB and PB into CD56bright and CD56dim NK cells, the NK cell activities and the expression of various surface antigens were assayed for each fraction. The NK cell activity of CD56dim NK cells in CB was significantly lower than that in PB (P = 0.0003), whereas, there was no significant difference between the NK cell activity of CD56bright NK cells in PB and CB. The expression levels of adhesion molecules (CD2, CD11a, CD18, DNAX accessory molecule-1), CD16, and CD57 for CD56dim NK cells in CB were significantly lower than those in PB, and approximately one-third of CB CD56dim NK cells were capable of forming conjugates with K562 cells, compared with PB CD56dim NK cells. Furthermore, the inhibition of both the NK cell activities and binding of CD56dim NK cells in PB and CB by monoclonal antibody against each of these adhesion molecules suggests that they play an important role in NK cell activity. These findings show that the low NK cell activity in CB is caused by the low NK cell activity of CD56dim NK cells and that the low expression level of adhesion molecules on CB CD56dim NK cells may contribute to this low NK cell activity.     

间日疟患者外周血NK γδ T Treg细胞含量的变化

目的 分析间日疟原虫现症感染者外周血中NK、γδ T细胞、CD4^＋CD25^＋T淋巴细胞及其FOXP3的表达情况，以初步了解患者的一些细胞免疫特性。方法用流式细胞仪检测25例间日疟原虫现症感染者（AC）、13例免疫对照者（IC）及14例正常对照者（NC）外周血中NK、γδT细胞和CD4^＋CD25^＋T淋巴细胞的百分含量，同时观察CD4^＋CD25^＋T细胞中表达FOXP3群体的百分含量。结果AC组外周血NK细胞百分含量为8．48％，与NC组（15．53％）及IC组（17．69％）比较，明显降低（P均〈0．05）,AC组外周血γδT细胞、CD4^＋CD25^＋T细胞百分含量分别为4．32％和5．42％，较NC组（2．55％和3．94％）及IC组（2．70％和3．44％）明显升高（P均〈0．05）；AC组外周血CD4^＋CD25^＋T淋巴细胞中表达FOXP3的细胞数量为8．14％，低于NC组的11．09％及IC组的11．32％，但差异无统计学意义（P〉0．05）。结论间日疟原虫急性感染期患者外周血NK细胞数量减少，但γδT淋巴细胞增加；CD4^＋CD25^＋T淋巴细胞中表达FOXP3群体的细胞稍有降低。