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ABSTRACT: The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to escape destruction by the host immune system. Using mutant strains that are defective in cell surface glycosylation, cell wall protein synthesis, and yeast-hypha morphogenesis, we have investigated three important aspects of C. albicans innate immune interactions: phagocytosis by primary macrophages and macrophage cell lines, hyphal formation within macrophage phagosomes, and the ability to escape from and kill macrophages. We show that cell wall glycosylation is critically important for the recognition and ingestion of C. albicans by macrophages. Phagocytosis was significantly reduced for mutants deficient in phosphomannan biosynthesis (mmn4Delta, pmr1Delta, and mnt3 mnt5Delta), whereas O- and N-linked mannan defects (mnt1Delta mnt2Delta and mns1Delta) were associated with increased ingestion, compared to the parent wild-type strains and genetically complemented controls. In contrast, macrophage uptake of mutants deficient in cell wall proteins such as adhesins (ece1Delta, hwp1Delta, and als3Delta) and yeast-locked mutants (clb2Delta, hgc1Delta, cph1Delta, efg1Delta, and efg1Delta cph1Delta), was similar to that observed for wild-type C. albicans. Killing of macrophages was abrogated in hypha-deficient strains, significantly reduced in all glycosylation mutants, and comparable to wild type in cell wall protein mutants. The diminished ability of glycosylation mutants to kill macrophages was not a consequence of impaired hyphal formation within macrophage phagosomes. Therefore, cell wall composition and the ability to undergo yeast-hypha morphogenesis are critical determinants of the macrophage's ability to ingest and process C. albicans.
Infection and immunity 04/2010; 78(4):1650-8. · 4.21 Impact Factor
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ABSTRACT: New Zealand Black (NZB) mice spontaneously develop autoimmune haemolytic anaemia (AIHA). Here the effect of injecting NZB mice with plasmids encoding IL-4 (pIL-4) or IL-10 (pIL-10) on NZB disease was tested. Both constructs delayed the development of anaemia as judged by increased haematocrit values as compared with controls, but neither altered the IgG1 to IgG2 red blood cell (RBC) bound autoantibody levels. The increased haematocrit value was associated temporally with increased RBC bound IgG in NZB mice treated with pIL-10, but not pIL-4. By contrast, up-regulation of splenic macrophage FcgammaRIIb2 mRNA was associated temporally with increased haematocrit values in NZB mice given pIL-4. However, no such increase occurred in NZB mice that inhaled a peptide containing a dominant T-cell epitope, although this treatment is known to bias the autoimmune response towards Th2 and to reduce the severity of anaemia. It is considered that IL-4 treatment, in part, ameliorates NZB anaemia by increasing the expression of the inhibitory FcgammaRIIb2 and thereby reducing the capacity of splenic macrophages to phagocytose autoantibody coated RBC, but that this mechanism does not explain the beneficial effects of the inhaled peptide.
Clinical & Experimental Immunology 02/2005; 139(1):84-9. · 3.36 Impact Factor
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ABSTRACT: Autoimmune haemolytic anaemia (AIHA) can be induced in mice by repeated injections with rat red blood cells (RBC). Here we describe the identification of rat and murine RBC antigens recognized by T-cells from mice with this disease. Splenic T-cells from mice with AIHA proliferated in response to multiple murine RBC membrane components, each of which is recognized by rat RBC induced autoantibodies. Thus, there were responses to murine autoantigen fractions that correspond in apparent molecular mass with the anion channel Band 3, with spectrin from the membrane skeleton and with the high and low molecular mass glycophorins, and the equivalent fractions from rat RBC also stimulated proliferation by T-cells. It was confirmed that purified Band 3 from murine and rat RBC also elicited responses. In contrast with the results in AIHA, T-cells from healthy control mice failed to respond to the antigens from either species, with the exception of proliferation induced by murine spectrin in one experiment and weak responses elicited by rat Band 3. It is suggested that T-cells activated by multiple cross-reactions between rat and murine RBC proteins, and by epitope spreading, are necessary to drive autoantibody production in this model of AIHA.
Clinical & Experimental Immunology 09/2002; 129(2):208-13. · 3.36 Impact Factor
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Clinical & Experimental Allergy 04/2002; 32(3):331-4. · 5.03 Impact Factor
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ABSTRACT: The aim of this study was to determine whether phagocytosis of necrotic or apoptotic cells affects antigen presentation by murine bone marrow-derived macrophages. After uptake of necrotic neutrophils, macrophages were able to stimulate significantly higher T cell proliferation in vitro against both the recall antigen albumin and the mitogen concanavalin A. No such effect was seen following phagocytosis of apoptotic neutrophils. Flow cytometry revealed that, within 4h of ingestion, macrophages that had taken up the necrotic cells expressed higher levels of CD40 than those that had phagocytosed apoptotic cells. Macrophage cultures pulsed with apoptotic, but not necrotic, neutrophils contained higher levels of transforming growth factor beta1, but lower concentrations of tumour necrosis factor alpha, compared to untreated controls. Our interpretation of these results is that macrophages that have taken up necrotic neutrophils co-stimulate T cells with greater efficiency due to rapid CD40 up-regulation, whereas those that have ingested apoptotic cells are not only ineffective in co-stimulation, but also secrete inhibitory cytokine.
Clinical & Experimental Immunology 03/2002; 127(2):220-5. · 3.36 Impact Factor
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ABSTRACT: The recent rise in the prevalence of immune-mediated diseases has been attributed to environmental factors such as a lack of microbial challenge, or dietary change, that deviate the overall balance between mutually antagonistic subsets of T helper (Th) cells.
An alternative proposal is that recent environmental changes have resulted in an immune system that is more likely to produce both Th1 and Th2 responses against benign antigens. The prediction of this hypothesis, that Th1 and Th2-mediated diseases are not mutually exclusive, and may be positively associated, is tested here in a whole population.
Data from General Practices participating in the Scottish Continuous Morbidity Recording (CMR) project were used to determine the coincidence of the major Th2-mediated atopic diseases; asthma, eczema and allergic rhinitis, with the Th1-mediated autoimmune conditions; type I diabetes, rheumatoid arthritis and psoriasis. We also identified the prescription rates of inhaled therapy for asthma in patients with Th1-mediated disease.
There was a significant increase in the risk of presenting with a Th1-mediated autoimmune condition in patients with a history of allergic disease (standardized prevalence ratio (95% confidence interval) 1.28 (1.18-1.37)). Likewise, the standardized prevalence ratios of presenting with either eczema (1.67 (1.48-1.87)) or allergic rhinitis (1.22 (1.02-1.44)) were significantly increased in subjects with a history of Th1-mediated disease. There was a particularly strong association between current psoriasis and current eczema (standardized prevalence ratio ofpsoriasis in subjects with eczema 2.88, 95% confidence interval (CI) 2.38-3.45). There was also a significant increase in prescriptions for inhaled asthma therapy in patients with Th1 disease.
It is concluded that Th1- and Th2-mediated diseases are significantly associated in a large General Practice population. This finding supports the proposal that autoimmune and atopic diseases share risk factors that increase the propensity of the immune system to generate both Th1- and Th2-mediated inappropriate responses to non-pathological antigens.
Clinical & Experimental Allergy 02/2002; 32(1):37-42. · 5.03 Impact Factor
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ABSTRACT: The environmental factors responsible for recent increases in the prevalence of asthma and atopic disease have been assumed to act after birth. Their possible effects on fetal immune development in utero have not been investigated systematically, although sensitization to allergens may occur before birth.
This prospective study determined whether the risk factors for asthma and atopic disease, namely family history of atopic disease, maternal smoking, birth order, or maternal dietary intake of antioxidant vitamins, exert antenatal effects on the fetal immune system that may predispose to childhood atopy.
The T helper (Th) cell proliferative responses of cord blood mononuclear cells (CBMC) from a sample of 223 neonates, representative of children born to a cohort of 2000 pregnant women, were measured and related to family, maternal and environmental factors associated with the pregnancy.
The magnitude of CBMC-proliferative responses to allergens increased significantly in association with a family history of atopic disease or maternal smoking, and decreased significantly with increasing birth order and high maternal dietary intake of vitamin E. The epidemiological association between birth order and atopy may therefore be a consequence of antenatal influences rather than of protective effects of childhood infections. The association between maternal intake of vitamin E and CBMC responsiveness suggests that diet during pregnancy may influence the fetal immune system in such a way as to modulate the risk of childhood atopy.
These results provide a new insight into the aetiology of atopic disease by demonstrating that the maternal environmental risk factors for atopy, diet, birth order and smoking, influence the development of the fetal immune system. This raises the prospect of preventative public health interventions during pregnancy.
Clinical & Experimental Allergy 02/2002; 32(1):43-50. · 5.03 Impact Factor
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Clinical and Experimental Allergy - CLIN EXPERIMENT ALLERGY. 01/2002; 32(1):37-42.
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ABSTRACT: Allergic diseases are major health problems in developed countries. Cord blood mononuclear cells (CBMC) at birth can proliferate after stimulation with allergen and this has led to the widespread view that the sensitization of the fetal immune system by allergens is a key determinant in establishing immunological bias towards allergy. However, the notion that the immune system can be primed by allergen in utero remains unproven. Determination of the CD45 isoform of responding T helper cells is an established method of determining the activation status of responding T helper cells because unsensitized cells express CD45RAhigh and previously sensitized cells CD45ROhigh.
To determine if sensitization of allergen-specific T helper cells can occur in utero by determining the CD45 isoform of CBMC proliferating in response to allergen.
CBMC proliferative responses were measured after stimulation in culture with a panel of allergens, mitogen and control antigen. To ascertain whether any responding T helper cells had been primed in utero, depletion experiments established whether they carried the CD45ROhigh marker of previous activation or the CD45RAhigh marker of unstimulated T cells.
CBMC from a high proportion of 223 randomly selected neonates were stimulated to proliferate in vitro by allergens, with 76% responding to timothy grass pollen. In 50% of such responses to timothy grass, the CD45 isoform of the T cells that proliferate indicated that they had been previously activated. However, the remaining 50% of responses to timothy grass were mediated by previously unstimulated T cells. Proliferative responses mediated by CBMC sensitized in utero tended to be greater in magnitude than those mediated by unsensitized cells (P = 0.08). Seventy-five per cent of CBMC samples proliferated after stimulation with mycobacterial PPD and, as in BCG-vaccinated adults, all such CBMC proliferative responses at birth were predominately mediated by sensitized cells.
Allergen- and antigen-specific Th cells can be primed in utero.
Clinical & Experimental Allergy 12/2001; 31(11):1686-95. · 5.03 Impact Factor
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ABSTRACT: The production of pathogenic autoantibodies in organ-specific autoimmune diseases is largely T cell dependent. For many of these diseases, the precise specificities and cytokine profiles of the T cells that respond to the corresponding autoantigens have now been identified. This knowledge has been exploited to treat some models of antibody-mediated autoimmunity using peptides corresponding to the dominant helper epitopes, giving impetus to the development of a similar approach in the equivalent human diseases.
Current Opinion in Immunology 01/2001; 12(6):664-9. · 9.52 Impact Factor
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ABSTRACT: Although considerable effort has been devoted to characterizing alloantibodies specific for the Rhesus D (RhD) blood group antigen, virtually nothing is known about the helper response that drives their production. Therefore, the aim of this study was to map alloreactive T-cell epitopes on the RhD protein. Peripheral blood mononuclear cells (PBMCs) were obtained from 22 RhD-negative volunteers in whom anti-D alloantibodies had developed after deliberate immunization or RhD-incompatible pregnancy. The PBMCs were stimulated with a panel of up to 68 overlapping synthetic 15-mer peptides spanning the complete sequence of the RhD protein. One or more peptides elicited proliferative responses by PBMCs from all 22 of the alloimmune volunteers but from only 2 of 8 alloantibody-negative control donors. Proliferation of PBMCs from the alloimmune donors was mediated by major histocompatibility complex class II-restricted T cells expressing the CD45RO marker of previous activation or memory. The number of peptides that induced proliferative responses was unrelated to either the frequency of, or time since, exposure to RhD-positive red blood cells, but it correlated strongly (R(s) = 0.75; P <.003) with the level of anti-D antibodies in deliberately immunized donors. The patterns of stimulatory peptides varied among alloimmune volunteers, but particular sequences were commonly recognized, with 4 peptides each eliciting a response in more than 50% of these donors. Identification of such peptides containing dominant alloreactive helper epitopes is the first step in the development of improved or new approaches to preventing hemolytic disease of the newborn that are based on modulating the T-cell response to the RhD protein.
Blood 12/2000; 96(13):4011-9. · 9.90 Impact Factor
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ABSTRACT: It has been proposed that in utero factors may predispose towards the development of childhood atopy. To test this hypothesis, it will be necessary to measure T-helper cell (Th) cytokines secreted by human cord blood mononuclear cells (CBMC) stimulated by allergens. However, to date, it has proven impossible to measure allergen-specific CBMC secretion of the key Th cytokine interleukin-4 (IL-4) using conventional sandwich ELISA techniques. We report for the first time the successful measurement of IL-4 secreted by CBMC stimulated by the allergens timothy grass pollen and house dust mite extract. The method is an adaptation of a novel cell-based ELISA (celELISA), which demonstrated an increased (up to 20-fold) sensitivity to detect IL-4. The method is simple, precise, is no more costly than a conventional ELISA, and can identify individuals in a general population whose CBMC exhibit different cytokine biases in response to allergens. The frequency distribution of IL-4 and interferon-gamma (IFN-gamma) CBMC responses to allergens in the general population approximates to a log-normal distribution, which will permit the application of linear regression techniques in the identification of in utero factors which influence Th bias.
Journal of Immunological Methods 03/2000; 234(1-2):13-22. · 2.20 Impact Factor
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Transfusion Medicine - TRANSFUSION MED. 01/2000; 10(4):331-331.
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ABSTRACT: A number of systemic autoimmune diseases are associated with increased levels of the agalactosyl (G0) IgG isoforms that lack a terminal galactose from the C(H)2 domain oligosaccharide. The aims were to determine whether there are also persistently high levels of G0 autoantibodies or serum IgG in autoimmune haemolytic anaemia (AIHA), and whether any changes in galactosylation over time are related to the course of disease. Autoantibodies eluted from red blood cells, and serum IgG, were obtained from a patient with chronic AIHA over a 21 month period, and the degree of galactosylation measured using a lectin-binding assay. There were wide fluctuations in the galactosylation of autoantibody and serum IgG, but these changes were unrelated to the severity of the anaemia. The galactosylation of autoantibody and serum IgG varied independently, and the autoantibodies were preferentially G0 in comparison with serum IgG in only half of the serial samples. We conclude that AIHA differs from other, systemic autoimmune conditions in that high levels of G0 autoantibodies or serum IgG are not persistent, and that changes in galactosylation do not parallel the course of disease.
Autoimmunity 11/1999; 31(2):103-8. · 2.47 Impact Factor
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ABSTRACT: A number of systemic autoimmune diseases are associated with increased levels of the agalactosyl (G0) IgG isoforms that lack a terminal galactose from the CH2 domain oligosaccharide. The current aim was to determine whether the galactosylation of serum IgG is also reduced in a classic antibody-mediated, organ-specific autoimmune condition, and whether the pathogenic autoantibodies are preferentially G0. In two murine forms of autoimmune haemolytic anaemia (AIHA), sera and autoantibodies eluted from erythrocytes were obtained, and the levels of G0 measured using a lectin-binding assay. Serum IgG galactosylation was unaffected following the induction of AIHA in CBA/Igb mice by immunization with rat erythrocytes, but in all animals with the disease the IgG autoantibodies generated were more G0 than the sera. The anti-rat erythrocyte antibodies were similar to the autoantibodies in being preferentially G0, and when CBA/Igb mice were immunized with canine erythrocytes as a control foreign antigen, there was again a bias towards the production of G0 IgG antibodies. In NZB mice with chronic, spontaneous AIHA, the concentration and galactosylation of both serum IgG and autoantibodies were lower than in the induced model, and the ratio of G0 IgG in the serum and erythrocyte eluates varied markedly between different individuals. Our interpretation of these results is that changes in serum IgG or autoantibody galactosylation are not consistent in different models of AIHA, and that production of low galactosyl antibodies can be a feature of a normal immune response.
Clinical & Experimental Immunology 10/1999; 117(3):449-54. · 3.36 Impact Factor
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ABSTRACT: The induction of pathogenic immune responses may be dependent on the immune system receiving 'danger' signals resulting from tissue damage, rather than tolerogenic stimuli associated with normal cell turnover. The aim was to test this hypothesis by comparing the effects of the uptake of necrotic and apoptotic cells on the ability of antigen-presenting cells (APC) to stimulate immune responses in vitro. The experiments focused on presentation by the macrophage, which is the main cell type adapted for clearing cellular debris in vivo. Murine bone marrow-derived macrophages were pulsed with neutrophils that had been rendered apoptotic or necrotic, and tested for the ability to induce T cell responses. The macrophages that had taken up necrotic, but not apoptotic, cells were able to stimulate recall proliferation by ovalbumin-specific T cells. Furthermore, the response to the mitogen concanavalin A (Con A) was more than 6 times higher when macrophages had been pulsed with necrotic, in comparison with apoptotic, cells. In control experiments, macrophages that had not been exposed to dying neutrophils stimulated weak responses to ovalbumin and Con A. To determine why the uptake of apoptotic and necrotic cells exert opposing effects on the ability of macrophages to stimulate T cells, the expression of costimulatory molecules by treated macrophages, and their production of potentially immunomodulatory cytokines were measured. Flow cytometry revealed that macrophages that had taken up necrotic, but not apoptotic, neutrophils expressed increased levels of CD40 compared to untreated controls within 4 h. Macrophages pulsed with apoptotic cells secreted higher levels of transforming growth factor-beta1 than those ingesting necrotic cells or untreated controls. Our interpretation of these results is that macrophages that have taken up necrotic cell debris present antigens to T lymphocytes with greater efficiency due to transient CD40 upregulation, whereas those that have ingested apoptotic cells are ineffective APC since they secrete inhibitory cytokine.
Pathobiology 02/1999; 67(5-6):302-5. · 1.18 Impact Factor
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ABSTRACT: Autoimmune haemolytic anaemia (AIHA), one of the most common autoimmune diseases of the dog, is characterised by binding of autoantibody to erythrocyte membrane antigens leading to a decreased red blood cell (RBC) life-span. Failure of self-tolerance with activation of autoreactive T-lymphocytes is thought to play a key role in the initiation of such autoimmune events. Peripheral blood mononuclear cells (PBMC) were obtained from 11 clinically normal dogs, six clinically normal relatives of two littermate dogs which died from AIHA, and four dogs which had recovered from primary AIHA. Cells were stimulated in vitro with a panel of canine RBC-derived antigens (RBC membranes, glycophorin, spectrin, five 15-mer glycophorin peptides), the non-recall antigen keyhole limpet haemocyanin (KLH), and the mitogen concanavalin A (Con A). The kinetics of the proliferative responses to specific antigens were assessed by serially sampling the cultures from days 4 to 10. PBMC from all dogs responded strongly to Con A (day 2) and to KLH (maximal response on days 7 to 10) under appropriate culture conditions. Two of 11 normal dogs responded weakly to RBC membranes (mean stimulation index = 4.25). In contrast, PBMC from all dogs recovered from AIHA responded to RBC membranes (mean SI = 9.2 +/- 2.5) and occasionally to other erythrocyte antigens. Similar responses were recorded with PBMC from dogs related to AIHA cases. It is considered that although normal individuals harbour erythrocyte-reactive lymphocytes, such cells are primed in dogs with AIHA or a genetic susceptibility to this disease.
Veterinary Immunology and Immunopathology 12/1997; 59(3-4):191-204. · 2.08 Impact Factor
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ABSTRACT: Despite the clinical significance of the canine blood group antigens, relatively little is known of the biochemistry of these molecules. In this study the canine blood group antigens DEA (dog erythrocyte antigen) 1.2, 4 and 7 were immunoprecipitated from red blood cells (RBC) bearing the corresponding blood group, and molecular weights of 85 kD (DEA 1.2), 32-40 kD (DEA 4) and 53-66 kD (DEA 7) assigned. DEA 1.2 and DEA 4 each appeared as a single band, whereas DEA 7 comprised three distinct bands (53, 58 and 66 kD). Polyclonal antisera specific for two peptides derived from the sequence of the human Rhesus blood group (Rh30A-C and Rh50A-C) were used in western blotting against canine and human erythrocyte membranes. The Rh30A-C antiserum identified a band of molecular weight 32 kD in both human and canine RBC, and the antiserum specific for Rh50A-C identified a band of 38-60 kD in human membranes and of 40-53 kD in canine RBC. This finding is consistent with conservation of areas of the Rhesus protein sequence between human and canine erythrocytes.
Veterinary Immunology and Immunopathology 12/1997; 59(3-4):213-23. · 2.08 Impact Factor
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ABSTRACT: We have shown previously that the Rhesus (Rh) polypeptides are the commonest targets for pathogenic anti-red blood cell (RBC) autoantibodies in patients with autoimmune hemolytic anemia (AIHA). The aim of the current work was to determine whether activated T cells from such patients also mount recall responses to epitopes on these proteins. Two panels of overlapping 15-mer peptides, corresponding to the sequences of the 30-kD Rh proteins associated with expression of the D and Cc/Ee blood group antigens, were synthesized and screened for the ability to stimulate the in vitro proliferation of mononuclear cells from the peripheral blood or spleen of nine AIHA cases. Culture conditions were chosen that favor recall proliferation by previously activated T cells, rather than primary responses. In seven of the patients, including all four cases with autoantibody to the Rh proteins, two or more peptides elicited proliferation, but cells from eight of nine patients with other anemias and seven of nine healthy donors failed to respond to the panels. Multiple peptides were also stimulatory in two positive control donors who had been alloimmunized with Rh D-positive RBCs. Six different profiles of peptides elicited responses in the AIHA patients, and this variation may reflect the different HLA types in the group. Stimulatory peptides were identified throughout domains shared between, or specific to, each of the related 30-kD Rh proteins, but T cells that responded to nonconserved regions did not cross-react with the alternative sequences. Anti-major histocompatibility complex class II antibodies blocked the responses and depletion experiments confirmed that the proliferating mononuclear cells were T cells. Notably, splenic T cells that proliferated against multiple Rh peptides also responded to intact RBCs. We propose that pathogenic autoantibody production in many cases of AIHA is driven by the activation of T-helper cells specific for previously cryptic epitopes on the Rh proteins.
Blood 11/1997; 90(7):2701-15. · 9.90 Impact Factor
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Biochemical Society Transactions 06/1997; 25(2):312S. · 3.71 Impact Factor
