A human IFNGR1 small deletion hotspot associated with dominant susceptibility to mycobacterial infection.

The immunogenetic basis of severe infections caused by bacille Calmette-Guérin vaccine and environmental mycobacteria in humans remains largely unknown. We describe 18 patients from several generations of 12 unrelated families who were heterozygous for 1 to 5 overlapping IFNGR1 frameshift small deletions and a wild-type IFNGR1 allele. There were 12 independent mutation events at a single mutation site, defining a small deletion hotspot. Neighbouring sequence analysis favours a small deletion model of slipped mispairing events during replication. The mutant alleles encode cell-surface IFNgamma receptors that lack the intra-cytoplasmic domain, which, through a combination of impaired recycling, abrogated signalling and normal binding to IFNgamma exert a dominant-negative effect. We thus report a hotspot for human IFNGR1 small deletions that confer dominant susceptibility to infections caused by poorly virulent mycobacteria.

Residual type 1 immunity in patients genetically deficient for interleukin 12 receptor beta1 (IL-12Rbeta1): evidence for an IL-12Rbeta1-independent pathway of IL-12 responsiveness in human T cells.

Genetic lack of interleukin 12 receptor beta1 (IL-12Rbeta1) surface expression predisposes to severe infections by poorly pathogenic mycobacteria or Salmonella and causes strongly decreased, but not completely abrogated, interferon (IFN)-gamma production. To study IL-12Rbeta1-independent residual IFN-gamma production, we have generated mycobacterium-specific T cell clones (TCCs) from IL-12Rbeta1-deficient individuals. All TCCs displayed a T helper type 1 phenotype and the majority responded to IL-12 by increased IFN-gamma production and proliferative responses upon activation. This response to IL-12 could be further augmented by exogenous IL-18. IL-12Rbeta2 was found to be normally expressed in the absence of IL-12Rbeta1, and could be upregulated by IFN-alpha. Expression of IL-12Rbeta2 alone, however, was insufficient to induce signal transducer and activator of transcription (Stat)4 activation in response to IL-12, whereas IFN-alpha/IFN-alphaR ligation resulted in Stat4 activation in both control and IL-12Rbeta1-deficient cells. IL-12 failed to upregulate cell surface expression of IL-18R, integrin alpha6, and IL-12Rbeta2 on IL-12Rbeta1-deficient cells, whereas this was normal on control cells. IL-12-induced IFN-gamma production in IL-12Rbeta1-deficient T cells could be inhibited by the p38 mitogen-activated protein kinase (MAP) kinase inhibitor SB203580 and the MAP kinase kinase (MEK) 1/2 inhibitor U0126, suggesting involvement of MAP kinases in this alternative, Stat4-independent, IL-12 signaling pathway.Collectively, these results indicate that IL-12 acts as a partial agonist in the absence of IL-12Rbeta1. Moreover, the results reveal the presence of a novel IL-12Rbeta1/Stat4-independent pathway of IL-12 responsiveness in activated human T cells involving MAP kinases. This pathway is likely to play a role in the residual type 1 immunity in IL-12Rbeta1 deficiency.

Impairment of mycobacterial immunity in human interleukin-12 receptor deficiency.

In humans, interferon gamma (IFN-gamma) receptor deficiency leads to a predisposition to mycobacterial infections and impairs the formation of mature granulomas. Interleukin-12 (IL-12) receptor deficiency was found in otherwise healthy individuals with mycobacterial infections. Mature granulomas were seen, surrounded by T cells and centered with epithelioid and multinucleated giant cells, yet reduced IFN-gamma concentrations were found to be secreted by activated natural killer and T cells. Thus, IL-12-dependent IFN-gamma secretion in humans seems essential in the control of mycobacterial infections, despite the formation of mature granulomas due to IL-12-independent IFN-gamma secretion.

Inherited interleukin 12 deficiency in a child with bacille Calmette-Guérin and Salmonella enteritidis disseminated infection.

Interferon-gamma receptor ligand-binding chain (IFN-gammaR1) or signaling chain (IFN-gammaR2) deficiency, like interleukin 12 receptor beta1 chain (IL-12Rbeta1) deficiency, predispose to severe infections due to poorly virulent mycobacteria and salmonella. A child with bacille Calmette-Guérin and Salmonella enteritidis infection was investigated. Mutations in the genes for IFN-gammaR1, IFN-gammaR2, IL-12Rbeta1, and other molecules implicated in IL-12- or IFN-gamma-mediated immunity were sought. A large homozygous deletion within the IL-12 p40 subunit gene was found, precluding expression of functional IL-12 p70 cytokine by activated dendritic cells and phagocytes. As a result, IFN-gamma production by lymphocytes was markedly impaired. This is the first discovered human disease resulting from a cytokine gene defect. It suggests that IL-12 is essential to and appears specific for protective immunity to intracellular bacteria such as mycobacteria and salmonella.

Response heterogeneity of human macrophages to ATP is associated with P2X7 receptor expression but not to polymorphisms in the P2RX7 promoter.

A region 2 kb upstream of exon 1 of the P2X7 gene was sequenced using DNA from nine healthy individuals who exhibited three different ATP response phenotypes (i.e. high, low and interferon gamma-inducible). Five single nucleotide polymorphisms were identified within the nine donor promoter sequences but none were associated with a specific ATP response phenotype. A P2X7 loss of function polymorphism (1513 in exon 13) was also screened for within donor DNA but no response associations were identified. ATP response phenotype was positively associated with P2X(7) receptor expression, as assessed by flow cytometry, but not with any identified receptor or promoter gene polymorphisms.

Diagnosis of defects in the type 1 cytokine pathway.

Patients with inherited defects in the interleukin-12 (IL-12)-dependent, 'high-output' interferon-gamma (IFN-gamma) pathway exhibit selective susceptibility to poorly pathogenic mycobacterial and salmonella infections. This review summarises the extended clinical spectrum seen in this group of patients and indicates a strategy for the identification of putative defects in the type 1 cytokine pathway.

Inflammatory responses in the intestine during tapeworm infections. Mucosal mast cells and mucosal mast cell proteases in Sprague-Dawley rats infected with Hymenolepis diminuta.

Comparative studies were made of two populations of Sprague-Dawley rats infected with Hymenolepis diminuta. The time course of infection, the development of mucosal mastocytosis and the levels of rat mucosal mast cell (MMC) protease (RMCP II) in serum and in jejunal mucosal tissues were monitored at intervals after infection with 40 cysticercoids of the tapeworm. Worm expulsion patterns differed markedly between the two populations, rats of New Zealand origin showing an abrupt and clear-cut loss of worms, rats of English origin showing a more gradual decline over a longer time period. In both populations, however, numbers of MMC and levels of tissue RMCP II were positively correlated with time after infection and negatively correlated with worm numbers. In only one of the three experiments (using English strain rats over a short time period) did levels of serum RMCP II change with time. In the other two experiments, in which English-strain and New Zealand-strain rats were used, there were no correlations between serum RMCP II and time, numbers of MMC, numbers of worms or levels of tissue RMCP II. The absence of correlation between serum RMCP II and worm loss in these experiments implies that MMC have no direct role in expulsion of H. diminuta. The data do show, nevertheless, that this purely luminal tapeworm is fully capable of activating the mucosal T lymphocyte-MMC precursor axis to elicit a mucosal mastocytosis.

CD4+ cytolytic T cells can destroy autologous and MHC-matched macrophages but fail to kill intracellular Mycobacterium bovis-BCG.

Mycobacterium bovis-BCG infected macrophages were exposed in vitro to PPD-stimulated T lymphocytes from tuberculin responsive donors or to a panel of mycobacterial-antigen specific CD4+ T cell clones. Both polyclonal and clonal T cells caused considerable antigen-specific lysis of autologous or MHC class II matched macrophages. However, lysis of infected macrophages did not significantly affect the number of viable mycobacteria which were released into the culture media from lysed macrophages. In tuberculosis, CD4+ cytolytic T cells may be primarily involved in tissue destruction and lack a significant role in acquired cellular immunity.

The shedding of the outer glycocalyx of juvenile Fasciola hepatica.

Freshly excysted Fasciola hepatica possess an outer glycocalyx which on incubation at 37 degrees C is rapidly shed. Using an ELISA technique the release of this parasite antigen was shown to be temperature-dependent and to occur in both normal bovine serum as well as in serum free conditions. The ELISA failed to detect the antibody--antigen complexes that occurred when flukes were incubated in immune serum. Release of specific parasite antigen fell slowly with in vitro cultured flukes, but increased with in vivo cultured flukes. Using a fluorescence inhibition assay, antigens with high ELISA titres inhibited surface fluorescence of the parasite suggesting that the ELISA was detecting surface antigens as well as other parasite metabolic products. Several metabolic blocking agents and commercial antihelminthics were titrated against juvenile F. hepatica to screen for inhibition of the surface--tegument shedding: there was little selective inhibition of surface shedding without a significant loss of motility.

Importance of competitive binding in the detection of antigen specific bovine isotypes and subisotypes by the micro ELISA.

An enzyme-linked immunosorbent assay was developed to detect and quantify the specific bovine immunoglobulin class response to Trypanosoma theileri, Dictyocaulus viviparus and infectious bovine rhinotracheitis virus. Comparative measurement of the specific immunoglobulin classes in whole serum was achieved using monospecific rabbit antibovine IgG1, IgG2 and IgM, followed by a goat anti-rabbit Ig-enzyme conjugate (antiglobulin ELISA). The results obtained in the antiglobulin ELISA compared favourably with the standard (or indirect) ELISA using the purified immunoglobulins. Competitive inhibition between specific immunoglobulins of different isotypes and subisotypes was the major disadvantage of the antiglobulin ELISA. This latter assay failed to detect specific IgG2 against T theileri antigen in both calf and adult whole serum. The inability to detect the specific IgG2 was a result of competitive inhibition by specific IgG1. However, competitive inhibition between specific immunoglobulins was not observed in either of the other test systems using D viviparus and infectious bovine rhinotracheitis virus antigens.

Identification of surface proteins of juvenile stages of Fasciola hepatica.

Maturation of newly excysted Fasciola hepatica juvenile flukes in mice is accompanied by distinct changes in the pattern of radiolabelling of surface proteins. The major proteins identified on the surface of newly excysted juvenile flukes have apparent molecular weights of 78,000, 45,500, 30,000, 26,000, 13,500, 13,000 and 10,500 as measured by sodium dodecylsulphate polyacrylamide gel electrophoresis. By day 7 after infection the 13,000 molecular weight protein is no longer expressed on the surface of the parasite. By day 14 after infection there is an additional loss of the 78,000, 30,000 and 26,000 molecular weight proteins. It is suggested that these changes may be associated with the temporal variation in distribution of the T0, T1 and T2 granules in the tegument syncytium.

Effect of oxygen radicals and differential expression of catalase and superoxide dismutase in adult Heligmosomoides polygyrus during primary infections in mice with differing response phenotypes.

The ability of oxygen radicals to kill Heligmosomoides polygyrus adult worms was examined by assessing parasite survival following incubation with hydrogen peroxide and acetaldehyde/xanthine oxidase, generators of H2O2 and H2O2/O2(-), respectively. H. polygyrus worms could tolerate levels of < 0.25 mM hydrogen peroxide and < 0.5 mM/20 mU acetaldehyde/xanthine oxidase for 20 h, but, at higher concentrations, marked sex-dependent susceptibility was observed, with males being more sensitive to H2O2 and O2(-) than female worms. The ability to evade free radical-mediated damage was also evaluated by measuring superoxide dismutase (SOD) and catalase levels in worms isolated at different time points from four strains of mice with differing resistance phenotypes. Levels of both catalase and SOD in female worms isolated from 'rapid'[(SWRxSJL)F1], 'fast' (SWR) or 'intermediate' (BALB/c), but not 'slow' (C57BL/10), responder mice showed a strain-dependent increase with time. Moreover, male worms were rejected faster than female worms in the 'rapid', 'fast' and 'intermediate' responder strains of mice. The results suggest that host-derived free radicals can damage adult worms and that female worms can increase production of their scavenging enzymes in response to the immune onslaught that eventually leads to worm expulsion in mice with 'fast', 'rapid' or 'intermediate' response phenotypes.

The relative involvement of Th1 and Th2 associated immune responses in the expulsion of a primary infection of Heligmosomoides polygyrus in mice of differing response phenotype.

T helper cell (Th1 and Th2) associated responses were examined following a primary infection with the gastrointestinal nematode Heligmosomoides polygyrus in five inbred strains of mice with different resistance phenotypes. Levels of (i) mast cell protease, (ii) specific IgE, (iii) nitric oxide and (iv) specific IgG2a, as markers of Th2 and Th1 associated responses, respectively, were determined in sera and intestinal fluids and correlated with worm burdens. The "fast" responder (resistant) strains SWR and SJL produced strong Th2 and Th1 associated responses respectively in a mutually exclusive fashion. The F1 hybrid (SWRxSJL) F1, showed rapid expulsion of the parasite and expressed both intense Th1 and Th2 responses, suggesting synergism between Th1 and Th2 activity in these mice. The results indicate that both Th2 and Th1 responses operate in mice following a primary infection with H. polygyrus and that each Th response may be involved to a greater or lesser degree within certain strains. Resistance to H. polygyrus was found to correlate only to the intensity of either the gut-associated mastocytosis or nitric oxide production in these strains but not to either specific IgE or IgG2a titres. Chronic infections in the "slow" response phenotype mouse strains CBA and C57BL/10, were associated with both poor Th2 and poor Th1-associated responses attributed to a general parasite-mediated immunosuppression of the host immune response to infection.

Ultrastructural observations on the interaction in vitro between bovine eosinophils and juvenile Fasciola hepatica.

This ultrastructural study has shown that there is a layer of dense flocculent material on the surface of juvenile Fasciola hepatica incubated in vitro with specific bovine antiserum. This material corresponds to the complexes of secreted glycocalyx and bovine antibody previously characterized by fluorescence microscopy. Bovine eosinophils attach closely to those regions of the parasite's surface that are free of flocculent precipitates. This close attachment is followed by degranulation of the eosinophils into the narrow zone between the cells and the parasite. Only in these regions is damage, in the form of vacuolation of the tegument, seen within the juvenile F. hepatica. It is concluded that the inability of bovine eosinophils to kill juvenile F. hepatica in the presence of specific antiserum results from the presence of a protective layer, consisting of antigen/antibody complexes, on the parasite's surface.

Genetic influences upon eosinophilia and resistance in mice infected with Mesocestoides corti.

The genetic influences upon host variation in eosinophilia and resistance to helminth infection, and the relationship between these parameters, was investigated in 9 inbred and 3 hybrid strains of mice infected with Mesocestoides corti. Blood, bone marrow, spleen and peritoneal fluid eosinophilia were far higher in SJL mice than in any other inbred strain. SWR, NIH, C3H and BALB/c mice were high responders to M. corti whereas CBA and 3 congenic strains sharing the B10 background (C57BL/10, B10.S, B10.G) were low responders. Some of the genes for high eosinophil responsiveness appeared to be dominant, as F1 hybrids from high and low response parental strains were intermediate to high in response to infection. SJL and NIH strains were highly susceptible to infection with M. corti, larval burdens at 21 days after infection with 100 tetrathyridia being considerably higher (greater than 1000) than all other strains. BALB/c (congruent to 700 larvae) were designated susceptible, SWR (greater than 400 larvae) were resistant and the B10 congenics (less than 400 larvae) were highly resistant. Genes influencing resistance also appeared to be dominant, as F1 hybrids between resistant and susceptible parental strains were intermediate to resistant on infection. The overall response patterns indicate a direct correlation between susceptibility to infection and high eosinophil responsiveness, but this relationship is not consistent in all strains.

Adoptive transfer of enhanced eosinophilia and resistance to infection in mice by an in vitro generated T-cell line specific for Mesocestoides corti larval antigen.

A T-cell line specific for tetrathyridial antigens of the cestode parasite Mesocestoides corti was generated in vitro. The T-cells expressed the L3T4+ Ly2- phenotype and secreted the lymphokines: interleukin-1 (IL-1), interleukin-2 (IL-2), gamma interferon (IFN-gamma), colony stimulating factor (CSF), mast cell growth factor (MCGF) and eosinophil differentiation factor (EDF) in response to antigen stimulation. The line was stable for up to 16 weeks and produced an enhanced peripheral eosinophil response and a reduced parasite burden (40-50%) when adoptively transferred into naive recipients undergoing a primary infection.

Genetic control of eosinophilia. Analysis of production and response to eosinophil-differentiating factor in strains of mice infected with Trichinella spiralis.

Bone marrow cultures were established from mice undergoing parasitic eosinophilia after infection with Trichinella spiralis. In the presence of eosinophil-differentiation factor (EDF/IL-5) eosinophil precursor cells differentiated and could be identified and counted after a 7-day in vitro culture period. The EDF-bone marrow assay system was used to determine differences in bone marrow eosinophil precursor capacity between a number of inbred strains of mice. Bone marrow cultures from high peripheral eosinophil-response phenotype strains of mice (NIH, SWR & SJL) contained significantly greater numbers of eosinophil precursor cells than the low response strain C57BL/10. All congenic strains of mice with the B10 background, i.e. C57BL/10, B10.S, B10.BR and B10.G were found to have low eosinophil precursor capacity. Bone marrow cultures obtained from F1 hybrids (NIH x C57/BL10, SJL x C57/BL10 and SWR x C57BL/10) demonstrated high precursor numbers, indicating that low responsiveness is inherited as a recessive characteristic. When spleen cells from T. spiralis-infected, high and low responder strains of mice were stimulated in vitro with concanavalin A (Con A) or with parasite antigen, it was found that low responder phenotype strains produced quantities of two eosinophilopoietic lymphokines EDF and IL3, which were similar to, if not greater than high responder strains. This suggests that bone marrow precursor capacity and not T cell lymphokine release is an important limiting factor in determining strain-dependent eosinophilia.

The relationship between circulating and intestinal Heligmosomoides polygyrus-specific IgG1 and IgA and resistance to primary infection.

Specific serum and intestinal immunoglobulin (Ig)G1 and IgA responses to Heligmosomoides polygyrus were measured in a panel of seven inbred mouse strains which exhibit 'rapid' (<6 weeks (SWRxSJL)F1), 'fast' (<8 weeks, SJL and SWR), 'intermediate' (10-20 weeks, NIH and BALB/c) or 'slow' (>25 weeks, C57BL/10 and CBA) resolution of primary infections. Mice with 'rapid', 'fast' or 'intermediate' response phenotypes produced greater serum and intestinal antibody responses than those with 'slow' phenotypes. The F1 hybrids ((SWRxSJL)F1) of two 'fast' responder strains showed the earliest antibody response with maximum titres evident within 6 weeks of infection. There was a negative correlation between the serum IgG1 responses and worm burdens in individual mice within a number of mouse strains, and also between serum IgG1 and IgA responses and worm burdens in the 'rapid' ((SWRxSJL)F1) responder strain. The presence of IgG1 in the gut was found to be due to local secretion rather than plasma leakage. Using Western immunoblotting, serum IgG1 from 'rapid' and 'fast' responder but not 'slow' responder mice was found to react with low molecular weight antigens (16-18 kDa) in adult worm excretory/secretory products.

Effect of blocking TNF-alpha on intracellular BCG (Bacillus Calmette Guerin) growth in human monocyte-derived macrophages.

Four agents, thalidomide, oxpentifylline, dexamethasone and a polyclonal anti-TNF-alpha antibody, were all shown by specific Elisa to block endogenous TNF-alpha production by Bacillus Calmette Guerin (BCG)-infected human monocyte-derived macrophages in in vitro culture. There was however no significant enhancement of intracellular BCG growth, over a 7-day incubation, in human monocyte-derived macrophages in the presence of any of the TNF-alpha-blocking agents, as determined by both radiometric and CFU counting methods of assessing bacterial viability and growth. The result suggests that the action of TNF-alpha alone is unlikely to be an important effector mechanism in antimycobacterial immunity within human cells.