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Cytomegalovirus viraemia is associated with poor growth and T-cell activation with an increased burden in HIV-exposed uninfected infants.
OBJECTIVE: Factors associated with poor health in HIV-exposed-uninfected (HEU) infants are poorly defined. We describe the prevalence and correlates of cytomegalovirus (CMV) viraemia in HEU and HIV-unexposed-uninfected (HUU) infants, and quantify associations with anthropometric, haematological, and immunological outcomes. DESIGN: Cross-sectional, including HEU and HUU infants from rural coastal Kenya. METHODS: Infants aged 2-8 months were studied. The primary outcome was CMV viraemia and viral load, determined by quantitative PCR. Correlates were tested by logistic and linear regression; coefficients were used to describe associations between CMV viraemia and clinical/immunological parameters. RESULTS: In total, 42 of 65 (64.6%) infants had CMV viraemia [median viral load, 3.0 (interquartile ranges: 2.7-3.5) log10 IU/ml]. Compared to community controls, HEU infants had six-fold increased odds of being viraemic (adjusted odds ratio 5.95 [95% confidence interval: 1.82-19.36], P = 0.003). Age, but not HEU/HUU status, was a strong correlate of CMV viral load (coefficient = -0.15, P = 0.009). CMV viral load associated negatively with weight-for-age (WAZ) Z-score (coefficient = -1.06, P = 0.008) and head circumference-for-age Z-score (coefficient = -1.47, P = 0.012) and positively with CD8 T-cell coexpression of CD38/human leucocyte antigen DR (coefficient = 15.05, P = 0.003). CONCLUSION: The odds of having CMV viraemia was six-fold greater in HEU than HUU infants when adjusted for age. CMV viral load was associated with adverse growth and heightened CD8 T-cell immune activation. Longitudinal assessments of the clinical effects of primary CMV infection and associated immunomodulation in early life in HEU and HUU populations are warranted.
Systemic lupus erythematosus-associated defects in the inhibitory receptor FcgammaRIIb reduce susceptibility to malaria.
Polygenic autoimmune diseases, such as systemic lupus erythematosus (SLE), are a significant cause of morbidity and mortality worldwide. In recent years, functionally important genetic polymorphisms conferring susceptibility to SLE have been identified, but the evolutionary pressures driving their retention in the gene pool remain elusive. A defunctioning, SLE-associated polymorphism of the inhibitory receptor FcgammaRIIb is found at an increased frequency in African and Asian populations, broadly corresponding to areas where malaria is endemic. Here, we show that FcgammaRIIb-deficient mice have increased clearance of malarial parasites (Plasmodium chabaudi chabaudi) and develop less severe disease. In vitro, the human lupus associated FcgammaRIIb polymorphism enhances phagocytosis of Plasmodium falciparum-infected erythrocytes. These results demonstrate that FcgammaRIIb is important in controlling the immune response to malarial parasites and suggests that the higher frequency of human FcgammaRIIb polymorphisms predisposing to SLE in Asians and Africans may be maintained because these variants reduce susceptibility to malaria.
Distinct kinetics of memory B-cell and plasma-cell responses in peripheral blood following a blood-stage Plasmodium chabaudi infection in mice.
B cell and plasma cell responses take place in lymphoid organs, but because of the inaccessibility of these organs, analyses of human responses are largely performed using peripheral blood mononuclear cells (PBMC). To determine whether PBMC are a useful source of memory B cells and plasma cells in malaria, and whether they reflect Plasmodium-specific B cell responses in spleen or bone marrow, we have investigated these components of the humoral response in PBMC using a model of Plasmodium chabaudi blood-stage infections in C57BL/6 mice. We detected memory B cells, defined as isotype-switched IgD(-) IgM(-) CD19(+) B cells, and low numbers of Plasmodium chabaudi Merozoite Surface Protein-1 (MSP1)-specific memory B cells, in PBMC at all time points sampled for up to 90 days following primary or secondary infection. By contrast, we only detected CD138(+) plasma cells and MSP1-specific antibody-secreting cells within a narrow time frame following primary (days 10 to 25) or secondary (day 10) infection. CD138(+) plasma cells in PBMC at these times expressed CD19, B220 and MHC class II, suggesting that they were not dislodged bone-marrow long-lived plasma cells, but newly differentiated migratory plasmablasts migrating to the bone marrow; thus reflective of an ongoing or developing immune response. Our data indicates that PBMC can be a useful source for malaria-specific memory B cells and plasma cells, but extrapolation of the results to human malaria infections suggests that timing of sampling, particularly for plasma cells, may be critical. Studies should therefore include multiple sampling points, and at times of infection/immunisation when the B-cell phenotypes of interest are likely to be found in peripheral blood.
Glycan-independent binding and internalization of human IgM to FCMR, its cognate cellular receptor.
IgM is the first antibody to be produced in immune responses and plays an important role in the neutralization of bacteria and viruses. Human IgM is heavily glycosylated, featuring five N-linked glycan sites on the μ chain and one on the J-chain. Glycosylation of IgG is known to modulate the effector functions of Fcγ receptors. In contrast, little is known about the effect of glycosylation on IgM binding to the human Fcμ receptor (hFCMR). In this study, we identify the Cμ4 domain of IgM as the target of hFCMR, and show that binding and internalization of IgM by hFCMR is glycan-independent. We generated a homology-based structure for hFCMR and used molecular dynamic simulations to show how this interaction with IgM may occur. Finally, we reveal an inhibitory function for IgM in the proliferation of T cells.
Deficiency of a subset of T-cells with immunoregulatory properties in sarcoidosis.
BACKGROUND: Sarcoidosis is a multisystem disorder that predominantly involves the lungs, characterised by a T-helper 1 (Th1) biased CD4-positive T-cell response and granuloma formation, for which the explanation is unknown. A newly identified subset of T-cells with immunoregulatory functions, CD1d-restricted natural-killer T (NKT) cells, has been shown to protect against disorders with increased CD4-positive Th1 responses in animals. We explored whether abnormalities in these cells are implicated in the pathogenesis of sarcoidosis. METHODS: We generated fluorescence-labelled CD1d-tetrameric complexes and used them, with monoclonal antibodies to Valpha24 and Vbeta11 T-cell receptor, to assess the frequency of CD1d-restricted NKT cells in the peripheral blood of 60 patients with histologically proven sarcoidosis (16 with Lofgren's syndrome) and 60 healthy controls. Lung lymphocytes were also analysed in 16 of the patients with sarcoidosis. FINDINGS: CD1d-restricted NKT cells were absent or greatly reduced in peripheral blood from all patients with sarcoidosis, except those with Lofgren's syndrome (median proportion of lymphocytes 0.01% [IQR 0-0.03] vs 0.06% [0.03-0.12] in controls; p=0.0004). The deficiency was found in both acute and resolved disease and was unrelated to systemic corticosteroid therapy. There was no difference in the proportion of CD1d-restricted NKT cells between peripheral blood and lungs in patients, suggesting that the peripheral-blood deficiency is not due to sequestration of these cells in the lungs. The NKT cells were not observed in mediastinal lymph nodes or granulomatous lesions. CD1d expression on antigen-presenting cells of patients was normal, thus the deficiency of CD1d-restricted NKT cells is not explained by abnormal CD1d expression. INTERPRETATION: Loss of immunoregulation by CD1d-restricted NKT cells could explain the amplified and persistent T-cell activity that characterises sarcoidosis. RELEVANCE TO PRACTICE: Our findings give new insight into the pathogenesis of sarcoidosis and draw attention to a potential target for therapeutic modulation in sarcoidosis.
A role for CD36 in the regulation of dendritic cell function.
Dendritic cells (DC) are crucial for the induction of immune responses and thus an inviting target for modulation by pathogens. We have previously shown that Plasmodium falciparum-infected erythrocytes inhibit the maturation of DCs. Intact P. falciparum-infected erythrocytes can bind directly to CD36 and indirectly to CD51. It is striking that these receptors, at least in part, also mediate the phagocytosis of apoptotic cells. Here we show that antibodies against CD36 or CD51, as well as exposure to early apoptotic cells, profoundly modulate DC maturation and function in response to inflammatory signals. Although modulated DCs still secrete tumor necrosis factor-alpha, they fail to activate T cells and now secrete IL-10. We therefore propose that intact P. falciparum-infected erythrocytes and apoptotic cells engage similar pathways regulating DC function. These findings may have important consequences for the treatment of malaria and may suggest strategies for modulating pathological immune responses in autoimmune diseases.
The normal cellular prion protein is strongly expressed by myeloid dendritic cells.
Abnormal isoforms of the prion protein (PrP(Sc)) that cause prion diseases are propagated and spread within the body by "carrier" cell(s). Cells of the immune system have been strongly implicated in this process. In particular, PrP(Sc) is known to accumulate on follicular dendritic cells (FDCs) in individuals affected by variant Creutzfeld-Jakob disease. However, FDCs do not migrate widely and the natural history of prion disorders suggests other cells may be required for the transport of PrP(Sc) from the site of ingestion to lymphoid organs and the central nervous system. Substantial evidence suggests that the spread of PrP(Sc) requires bone marrow-derived cells that express normal cellular prion protein (PrP(C)). This study examined the expression of PrP(C) on bone marrow-derived cells that interact with lymphoid follicles. High levels of PrP(C) are present on myeloid dendritic cells (DCs) that surround the splenic white pulp. These myeloid DCs are ontologically and functionally distinct from the FDCs. Consistent with these observations, expression of PrP(C) was strongly induced during the generation of mature myeloid DCs in vitro. In these cells PrP(C) colocalized with major histocompatibility complex class II molecules at the level of light microscopy. Furthermore, given the close anatomic and functional connection of myeloid DCs with lymphoid follicles, these results raise the possibility that myeloid DCs may play a role in the propagation of PrP(Sc) in humans.
Unique T cell effector functions elicited by Plasmodium falciparum epitopes in malaria-exposed Africans tested by three T cell assays.
Natural immunity to malaria is characterized by low level CD4 T cell reactivity detected by either lymphoproliferation or IFN-gamma secretion. Here we show a doubling in the detection rate of responders to the carboxyl terminus of circumsporozoite protein (CS) of Plasmodium falciparum by employing three T cell assays simultaneously: rapid IFN-gamma secretion (ex vivo ELISPOT), IFN-gamma secretion after reactivation of memory T cells and expansion in vitro (cultured ELISPOT), and lymphoproliferation. Remarkably, for no individual peptide did a positive response for one T cell effector function correlate with any other. Thus these CS epitopes elicited unique T cell response patterns in malaria-exposed donors. Novel or important epitope responses may therefore be missed if only one T cell assay is employed. A borderline correlation was found between anti-CS Ab levels and proliferative responses, but no correlation was found with ex vivo or cultured IFN-gamma responses. This suggested that the proliferating population, but not the IFN-gamma-secreting cells, contained cells that provide help for Ab production. The data suggest that natural immunity to malaria is a complex function of T cell subgroups with different effector functions and has important implications for future studies of natural T cell immunity.
The plasma concentration of the B cell activating factor is increased in children with acute malaria.
Malaria-specific antibody responses in children often appear to be short-lived but the mechanisms underlying this phenomenon are not well understood. In this study, we investigated the relationship between the B-cell activating factor (BAFF) and its receptors expressed on B cells with antibody responses during and after acute malaria in children. Our results demonstrate that BAFF plasma levels increased during acute malarial disease and reflected disease severity. The expression profiles for BAFF receptors on B cells agreed with rapid activation and differentiation of a proportion of B cells to plasma cells. However, BAFF receptor (BAFF-R) expression was reduced on all peripheral blood B cells during acute infection, but those children with the highest level of BAFF-R expression on B cells maintained schizont-specific immunoglobin G (IgG) over a period of 4 months, indicating that dysregulation of BAFF-R expression on B cells may contribute to short-lived antibody responses to malarial antigens in children. In summary, this study suggests a potential role for BAFF during malaria disease, both as a marker for disease severity and in shaping the differentiation pattern of antigen-specific B cells.
Copy number, linkage disequilibrium and disease association in the FCGR locus.
The response of a leukocyte to immune complexes (ICs) is modulated by receptors for the Fc region of IgG (FcgammaRs), and alterations in their affinity or function have been associated with risk of autoimmune diseases, including systemic lupus erythematosus (SLE). The low-affinity FcgammaR genomic locus is complex, containing regions of copy number variation (CNV) which can alter receptor expression and leukocyte responses to IgG. Combined paralogue ratio tests (PRTs) were used to distinguish three intervals within the FCGR locus which undergo CNV, and to determine FCGR gene copy number (CN). There were significant differences in FCGR3B and FCGR3A CNV profiles between Caucasian, East Asian and Kenyan populations. A previously noted association of low FCGR3B CN with SLE in Caucasians was supported [OR = 1.57 (1.08-2.27), P = 0.018], and replicated in Chinese [OR = 1.65 (1.25-2.18), P = 4 x 10(-4)]. There was no association of FCGR3B CNV with vasculitis, nor with malarial or bacterial infection. Linkage disequilibrium (LD) between multi-allelic FCGR3B CNV and SLE-associated SNPs in the FCGR locus was defined for the first time. Despite LD between FCGR3B CNV and a variant in FcgammaRIIB (I232T) which abolishes inhibitory function, both reduced CN of FCGR3B and homozygosity of the FcgammaRIIB-232T allele were individually strongly associated with SLE risk. Thus CN of FCGR3B, which controls IC responses and uptake by neutrophils, and variations in FCGR2B, which controls factors such as antibody production and macrophage activation, are important in SLE pathogenesis. Further interpretations of contributions to pathogenesis by FcgammaRs must be made in the context of LD involving CNV regions.
Modular organization of the carboxyl-terminal, globular head region of human C1q A, B, and C chains.
The first step in the activation of the classical complement pathway, by immune complexes, involves the binding of the globular heads of C1q to the Fc regions of aggregated IgG or IgM. Located C-terminal to the collagen region, each globular head is composed of the C-terminal halves of one A (ghA), one B (ghB), and one C chain (ghC). To dissect their structural and functional autonomy, we have expressed ghA, ghB, and ghC in Escherichia coli as soluble proteins linked to maltose-binding protein (MBP). The affinity-purified fusion proteins (MBP-ghA, -ghB, and -ghC) bound differentially to heat-aggregated IgG and IgM, and also to three known C1q-binding peptides, derived from HIV-1, HTLV-I, and beta-amyloid. In the ELISAs, the MBP-ghA bound to heat-aggregated IgG and IgM as well as to the HIV-1 gp41 peptide; the MBP-ghB bound preferentially to IgG rather than IgM, in addition to binding beta-amyloid peptide, whereas the MBP-ghC showed a preference for IgM and the HTLV-I gp21 peptide. Both MBP-ghA and MBP-ghB also inhibited C1q-dependent hemolysis of IgG- and IgM-sensitized sheep erythrocytes. However, for IgM-coated erythrocytes, MBP-ghC was a better inhibitor of C1q than MBP-ghB. The recombinant forms of ghA, ghB, and ghC also bound specifically to apoptotic PBMCs. We conclude that the C1q globular head region is likely to have a modular organization, being composed of three structurally and functionally independent modules, which retains multivalency in the form of a heterotrimer. The heterotrimeric organization thus offers functional flexibility and versatility to the whole C1q molecule.
CD4(-)CD8alphaalpha subset of CD1d-restricted NKT cells controls T cell expansion.
Valpha24 invariant (Valpha24i) CD1d-restricted NKT cells are widely regarded to have immune regulatory properties. They are known to have a role in preventing autoimmune diseases and are involved in optimally mounted immune responses to pathogens and tumor cells. We were interested in understanding how these cells provide protection in autoimmune diseases. We first observed, using EBV/MHC I tetrameric complexes, that expansion of Ag-specific cells in human PBMCs was reduced when CD1d-restricted NKT cells were concomitantly activated. This was accompanied by an increase in a CD4(-)CD8alphaalpha(+) subset of Valpha24i NKT cells. To delineate if a specific subset of NKT cells was responsible for this effect, we generated different subsets of human CD4(-) and CD4(+) Valpha24i NKT clones and demonstrate that a CD4(-)CD8alphaalpha(+) subset with highly efficient cytolytic ability was unique among the clones in being able to suppress the proliferation and expansion of activated T cells in vitro. Activated clones were able to kill CD1d-bearing dendritic or target cells. We suggest that one mechanism by which CD1d-restricted NKT cells can exert a regulatory role is by containing the proliferation of activated T cells, possibly through timely lysis of APCs or activated T cells bearing CD1d.
Characterization of a Plasmodium falciparum macrophage-migration inhibitory factor homologue.
BACKGROUND: Macrophage-migration inhibitory factor (MIF), one of the first cytokines described, has a broad range of proinflammatory properties. The genome sequencing project of Plasmodium falciparum identified a parasite homologue of MIF. The protein is expressed during the asexual blood stages of the parasite life cycle that cause malarial disease. The identification of a parasite homologue of MIF raised the question of whether it affects monocyte function in a manner similar to its human counterpart. METHODS: Recombinant P. falciparum MIF (PfMIF) was generated and used in vitro to assess its influence on monocyte function. Antibodies generated against PfMIF were used to determine the expression profile and localization of the protein in blood-stage parasites. Antibody responses to PfMIF were determined in Kenyan children with acute malaria and in control subjects. RESULTS: PfMIF protein was expressed in asexual blood-stage parasites, localized to the Maurer's cleft. In vitro treatment of monocytes with PfMIF inhibited random migration and reduced the surface expression of Toll-like receptor (TLR) 2, TLR4, and CD86. CONCLUSIONS: These results indicate that PfMIF is released during blood-stage malaria and potentially modulates the function of monocytes during acute P. falciparum infection.
CD4+ T cell responses to the Plasmodium falciparum erythrocyte membrane protein 1 in children with mild malaria.
The immune response against the variant surface Ag Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is a key component of clinical immunity against malaria. We have investigated the development and maintenance of CD4(+) T cell responses to a small semiconserved area of the Duffy binding-like domain (DBL)α-domain of PfEMP1, the DBLα-tag. Young children were followed up longitudinally, and parasites and PBMCs were isolated from 35 patients presenting with an acute case of uncomplicated malaria. The DBLα-tag from the PfEMP1 dominantly expressed by the homologous parasite isolate was cloned and expressed as recombinant protein. The recombinant DBLα-tag was used to activate PBMCs collected from each acute episode and from an annual cross-sectional survey performed after the acute malaria episode. In this article, we report that CD4(+) T cell responses to the homologous DBLα-tag were induced in 75% of the children at the time of the acute episode and in 62% of the children at the following cross-sectional survey on average 235 d later. Furthermore, children who had induced DBLα-tag-specific CD4(+)IL-4(+) T cells at the acute episode remained episode free for longer than children who induced other types of CD4(+) T cell responses. These results suggest that a wide range of DBLα-tag-specific CD4(+) T cell responses were induced in children with mild malaria and, in the case of CD4(+)IL-4(+) T cell responses, were associated with protection from clinical episodes.
Value of Plasmodium falciparum histidine-rich protein 2 level and malaria retinopathy in distinguishing cerebral malaria from other acute encephalopathies in Kenyan children.
BACKGROUND: The diagnosis of cerebral malaria is problematic in malaria-endemic areas because encephalopathy in patients with parasitemia may have another cause. Abnormal retinal findings are thought to increase the specificity of the diagnosis, and the level of histidine-rich protein 2 (HRP2) may reflect the parasite biomass. METHODS: We examined the retina and measured plasma HRP2 levels in children with acute nontraumatic encephalopathy in Kenya. Logistic regression, with HRP2 level as an independent variable and World Health Organization-defined cerebral malaria and/or retinopathy as the outcome, was used to calculate malaria-attributable fractions (MAFs) and retinopathy-attributable fractions (RAFs). RESULTS: Of 270 children, 140 (52%) had peripheral parasitemia, 80 (30%) had malaria retinopathy, and 164 (61%) had an HRP2 level of >0 U/mL. During 2006-2011, the incidence of HRP2 positivity among admitted children declined by 49 cases per 100 000 per year (a 78% reduction). An HRP2 level of >0 U/mL had a MAF of 93% for cerebral malaria, with a MAF of 97% observed for HRP2 levels of ≥ 10 U/mL (the level of the best combined sensitivity and specificity). HRP2 levels of >0 U/mL had a RAF of 77% for features of retinopathy combined, with the highest RAFs for macular whitening (99%), peripheral whitening (98%), and hemorrhages (90%). CONCLUSION: HRP2 has a high attributable fraction for features of malarial retinopathy, supporting its use in the diagnosis of cerebral malaria. HRP2 thresholds improve the specificity of the definition.
Platelet-mediated clumping of Plasmodium falciparum-infected erythrocytes is a common adhesive phenotype and is associated with severe malaria.
Sequestration of malaria-infected erythrocytes in the peripheral circulation has been associated with the virulence of Plasmodium falciparum. Defining the adhesive phenotypes of infected erythrocytes may therefore help us to understand how severe disease is caused and how to prevent or treat it. We have previously shown that malaria-infected erythrocytes may form apparent autoagglutinates of infected erythrocytes. Here we show that such autoagglutination of a laboratory line of P. falciparum is mediated by platelets and that the formation of clumps of infected erythrocytes and platelets requires expression of the platelet surface glycoprotein CD36. Platelet-dependent clumping is a distinct adhesive phenotype, expressed by some but not all CD36-binding parasite lines, and is common in field isolates of P. falciparum. Finally, we have established that platelet-mediated clumping is strongly associated with severe malaria. Precise definition of the molecular basis of this intriguing adhesive phenotype may help to elucidate the complex pathophysiology of malaria.
A non-sense mutation in Cd36 gene is associated with protection from severe malaria.
We sought genetic evidence for the importance of host-parasite interactions involving CD36 in severe malaria. We identified a non-sense mutation in Cd36 gene and looked at the influence of this mutation on the outcome of malaria infection in 693 African children with severe malaria and a similar number of ethnically matched controls. We showed that heterozygosity for this mutation is associated with protection from severe disease (OR 0.74, 95% CI 0.55-0.99; p=0.036). These findings suggest that this Cd36 mutation might have a complex effect on malaria infection by decreasing parasite sequestration, and also by decreasing host immune responses.
Two novel calcium-binding proteins from cytoplasmic granules of the protozoan parasite Entamoeba histolytica.
We report on the molecular characterisation of two novel granule proteins of the protozoon and human pathogen Entamoeba histolytica. The proteins, which were named grainin 1 and 2, show a considerable structural similarity to calcium-binding proteins, particularly within EF-hand motifs. Each grainin possesses three of these putative calcium-binding sites. Based on careful inspection of known structures of protein families containing EF-hands, a domain of grainin 1 covering two EF-hand motifs was modeled by homology. Calcium-binding activity of grainins was demonstrated by two independent methods. These granule proteins may be implicated in functions vital for the primitive phagocyte and destructive parasite such as control of endocytotic pathways and granule discharge.