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Macrophages display marked plasticity with functions in both inflammation and tissue repair. Evidence demonstrates that this spectrum of macrophage phenotypes is influenced by their local microenvironment and tissue origin. However, in vitro macrophage experiments often do not or cannot readily use macrophages from the most relevant tissue of origin. This study investigated if the origin of two C57BL/6 mouse macrophage cell lines of alveolar (AMJ2-C11) and peritoneal (IC-21) origin may influence their response to mycobacterial infection. Both cell lines equally controlled the growth of Mycobacterium bovis BCG and Mycobacterium tuberculosis, although the expression of all proinflammatory cytokines and chemokines measured (TNF, IL-6, MCP-1, MIP-1α, MIP-1β, and RANTES) was significantly higher in AMJ2-C11 cells than in IC-21 cells. During M. tuberculosis infection, IL-6, MCP-1, and RANTES expression increased 5-fold, and MIP-1β expression increased 30-fold. Additionally, AMJ2-C11 cells exhibited significantly higher inducible nitric oxide synthase activity than IC-21 cells, indicative of a more polarized M1 response. The expression of multiple surface markers was also assessed by flow cytometry. CD80 and CD86 were significantly upregulated in AMJ2-C11 cells and downregulated in IC-21 cells during M. tuberculosis infection. The results support the notion that the origin of tissue-resident macrophages influences their phenotype and antimicrobial response and demonstrate hereto unrecognized potential for these cell lines in in vitro studies.

Regulatory T cells (Tregs) play a critical role in immune regulation and peripheral tolerance. While different types of Tregs have been identified in both mice and humans, much of our understanding about how these cells maintain immune homeostasis is derived from animal models. In this study, we examined two distinct human lymphoid organs to understand how repeated exposure to infections at the mucosal surface influences the phenotype and tissue localization of Tregs. We show that while Tregs in both tonsils and spleen express a tissue-resident phenotype, they accumulate in greater numbers in tonsils. Tonsillar-resident Tregs exhibit a highly suppressive phenotype with significantly increased expression of CD39, ICOS and CTLA-4 compared with their counterparts in circulation or in the spleen. Functionally, resident Tregs are able effectively to suppress T cell proliferation. We further demonstrate that tonsillar-resident Tregs share key features of T follicular helper cells. Spatial analysis reveals that the vast majority of resident Tregs are localized at the border of the T-zone and B cell follicle, as well as within the lymphocyte pockets enriched with resident memory T cells. Together our findings suggest that resident Tregs are strategically co-localized to maintain immune homeostasis at sites of recurrent inflammation.

Immunogenic carrier proteins such as the non-toxic diphtheria toxin variant, cross-reacting material 197 (CRM197), are widely used in subunit vaccine formulations to boost immunogenicity of chemically conjugated antigens. Conjugate vaccines are inherently expensive due to laborious manufacturing steps. Here, this work develops a particulate vaccine platform based on using engineered Escherichia coli to assemble CRM197-antigen fusion proteins into discrete submicron-sized particles. This approach enables precise loading of diverse antigens and epitopes enhancing their immunogenicity. A cost-effective, high-yield, and scalable biomanufacturing process is developed. Purified particulate CRM197-antigen vaccines are ambient-temperature stable. CRM197 particles incorporating pathogen-specific antigens or epitopes from SARS-CoV-2, Streptococcus pyogenes (group A), and Mycobacterium tuberculosis induced cell-mediated and humoral immune responses mediating protective immunity in respective animal models of infection. The CRM197 particle vaccine platform is versatile, enabling co-delivery of selected antigens/epitopes together with immunogenic CRM197 as discrete stable particles avoiding laborious manufacture of soluble CRM197 and antigen followed by chemical conjugation.

Summary Bacterial nutrition is an essential aspect of host–pathogen interaction. For the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, fatty acids derived from lipid droplets are considered the major carbon source. However, many other soluble nutrients are available inside host cells and may be used as alternative carbon sources. Lactate and pyruvate are abundant in human cells and fluids, particularly during inflammation. In this work, we study Mtb metabolism of lactate and pyruvate combining classic microbial physiology with a ‘multi‐omics’ approach consisting of transposon‐directed insertion site sequencing (TraDIS), RNA‐seq transcriptomics, proteomics and stable isotopic labelling coupled with mass spectrometry‐based metabolomics. We discovered that Mtb is well adapted to use both lactate and pyruvate and that their metabolism requires gluconeogenesis, valine metabolism, the Krebs cycle, the GABA shunt, the glyoxylate shunt and the methylcitrate cycle. The last two pathways are traditionally associated with fatty acid metabolism and, unexpectedly, we found that in Mtb the methylcitrate cycle operates in reverse, to allow optimal metabolism of lactate and pyruvate. Our findings reveal a novel function for the methylcitrate cycle as a direct route for the biosynthesis of propionyl‐CoA, the essential precursor for the biosynthesis of the odd‐chain fatty acids., Mycobacterium tuberculosis assimilates lactate and pyruvate utilising glyoxylate shunt and methylcitrate cycle, two pathways traditionally associated with fatty acid metabolism in bacteria. Surprisingly, we found that M. tuberculosis synthesises propionyl‐CoA by a reverse methylcitrate cycle and another partially characterised (in this study) pathway. Our results point to a re‐interpretation of the mechanistic role and utilisation of these pathways by Mtb during infection, i.e., their potential reversibility and importance during lactate and pyruvate metabolism in vivo.

Objectives Non‐sputum‐based tests to accurately identify active tuberculosis (TB) disease and monitor response to therapy are urgently needed. This study examined the biomarker capacity of a panel of plasma proteins alone, and in conjunction with a previously identified miRNA signature, to identify active TB disease. Methods The expression of nine proteins (IP‐10, MCP‐1, sTNFR1, RANTES, VEGF, IL‐6, IL‐10, TNF and Eotaxin) was measured in the plasma of 100 control subjects and 100 TB patients, at diagnosis (treatment naïve) and over the course of treatment (1‐, 2‐ and 6‐month intervals). The diagnostic performance of the nine proteins alone, and with the miRNA, was assessed. Results Six proteins were significantly up‐regulated in the plasma of TB patients at diagnosis compared to controls. Receiver operator characteristic curve analysis demonstrated that IP‐10 with an AUC = 0.874, sensitivity of 75% and specificity of 87% was the best single biomarker candidate to distinguish TB patients from controls. IP‐10 and IL‐6 levels fell significantly within one month of commencing treatment and may have potential as indicators of a positive response to therapy. The combined protein and miRNA panel gave an AUC of 1.00. A smaller panel of only five analytes (IP‐10, miR‐29a, miR‐146a, miR‐99b and miR‐221) showed an AUC = 0.995, sensitivity of 96% and specificity of 97%. Conclusions A novel combination of miRNA and proteins significantly improves the sensitivity and specificity as a biosignature over single biomarker candidates and may be useful for the development of a non‐sputum test to aid the diagnosis of active TB disease., The biomarker potential of plasma proteins to identify tuberculosis (TB) patients was examined. Six proteins were significantly elevated in TB patients with IP‐10 and IL‐6 declining with treatment. In conjunction with miRNA previously detected, a five analyte biosignature could identify TB patients with an AUC = 0.995, sensitivity of 96% and specificity of 97%. IP‐10 was the best single biomarker candidate, showing utility as a triage tool, to quickly and easily identify potential TB patients and monitor their response to therapy.

Microparticles (MPs) are small membranous particles (100-1000 nm) released under normal steady-state conditions and are thought to provide a communication network between host cells. Previous studies demonstrated that Mycobacterium tuberculosis (M. tb) infection of macrophages increased the release of MPs, and these MPs induced a proinflammatory response from uninfected macrophages in vitro and in vivo following their transfer into uninfected mice. To determine how M. tb infection modulates the protein composition of the MPs, and if this contributes to their proinflammatory properties, we compared the proteomes of MPs derived from M. tb-infected (TBinf-MP) and uninfected human THP-1 monocytic cells. MP proteins were analyzed by GeLC-MS/MS with spectral counting revealing 68 proteins with statistically significant differential abundances. The 42 proteins increased in abundance in TBinf-MPs included proteins associated with immune function (7), lysosomal/endosomal maturation (4), vesicular formation (12), nucleosome proteins (4), and antigen processing (9). Prominent among these were the type I interferon inducible proteins, ISG15, IFIT1, IFIT2, and IFIT3. Exposure of uninfected THP-1 cells to TBinf-MPs induced increased gene expression of isg15, ifit1, ifit2, and ifit3 and the release of proinflammatory cytokines. These proteins may regulate the proinflammatory potential of the MPs and provide candidate biomarkers for M. tb infection.

Tuberculosis remains a global health problem, in part due to failure of the currently available vaccine, BCG, to protect adults against pulmonary forms of the disease. We explored the impact of pulmonary delivery of recombinant influenza A viruses (rIAVs) on the induction of Mycobacterium tuberculosis (M. tuberculosis)-specific CD4(+) and CD8(+) T-cell responses and the resultant protection against M. tuberculosis infection in C57BL/6 mice. Intranasal infection with rIAVs expressing a CD4(+) T-cell epitope from the Ag85B protein (PR8.p25) or CD8(+) T-cell epitope from the TB10.4 protein (PR8.TB10.4) generated strong T-cell responses to the M. tuberculosis-specific epitopes in the lung that persisted long after the rIAVs were cleared. Infection with PR8.p25 conferred protection against subsequent M. tuberculosis challenge in the lung, and this was associated with increased levels of poly-functional CD4(+) T cells at the time of challenge. By contrast, infection with PR8.TB10.4 did not induce protection despite the presence of IFN-γ-producing M. tuberculosis-specific CD8(+) T cells in the lung at the time of challenge and during infection. Therefore, the induction of pulmonary M. tuberculosis epitope-specific CD4(+), but not CD8(+) T cells, is essential for protection against acute M. tuberculosis infection in the lung.

Definition of protective immunity induced by effective vaccines is important for the design of new pathogen control strategies. Inactivation of the PhoP response-regulator in Mycobacterium tuberculosis results in a highly attenuated strain that demonstrates impressive protective efficacy in pre-clinical models of tuberculosis. In this report we demonstrate that the protection afforded by the M. tuberculosis phoP mutant strain is associated with the long-term maintenance of CD4+ T-cell memory. Immunization of mice with SO2 resulted in enhanced expansion of M. tuberculosis-specific CD4+ T cells compared with vaccination with the BCG vaccine, with an increased frequency of these cells persisting at extended time-points after vaccination. Strikingly, vaccination with SO2 resulted in sustained generation of CD4+ T cells displaying a central memory phenotype, a property not shared by BCG. Further, SO2 vaccination markedly improved the generation of polyfunctional cytokine-secreting CD4+ T cells compared with BCG vaccination. The improved generation of functionally competent memory T cells by SO2 correlated with augmented recall responses in SO2-vaccinated animals after challenge with virulent M. tuberculosis. This study defines a mechanism for the protective effect of the SO2 vaccine and suggests that deletion of defined virulence networks may provide vaccine strains with potent immuno-stimulatory properties.

Summary Background Sensitization and symptoms of allergic disease are strongly correlated, but little is known about the early clinical precursors of the development of allergen sensitization in childhood. The aim of this study was to identify these predictors, and to examine separately the effect of early sensitization on subsequent wheeze, asthma, rhinitis and eczema. Methods In the Childhood Asthma Prevention Study, children with a family history of asthma were assessed for allergen sensitization, total serum IgE, wheeze, asthma, eczema and rhinitis at ages 18 months and 5 years. To examine predictors, at 18 months, for subsequent sensitization, children who were non-sensitized at 18 months and had data on sensitization at 5 years were investigated, n=375. To examine the predictors, at age 18 months, of subsequent onset of symptoms, children who did not have wheeze, asthma, eczema or rhinitis at 18 months were followed-up at 5 years, n=177. Results Among children who were non-sensitized at age 18 months, the presence of eczema [adjusted relative risk (aRR), 1.67, 95% confidence interval (CI) 1.20–2.33], but not wheeze, asthma or rhinitis, was an independent predictor of the onset of sensitization by age 5 years. Among children who were asymptomatic at age 18 months, sensitization to any allergen at 18 months was an independent predictor for the presence of wheeze (aRR 2.41, 95% CI 1.28–4.55), asthma (aRR 4.66, 95% CI 1.88–11.54) and rhinitis (aRR 1.77, 95% CI 1.08–2.90), but not for the development of eczema (aRR 0.78, 95% CI 0.23–2.64) at 5 years. Conclusion In non-sensitized children, eczema, but not wheeze, asthma or rhinitis is a predictor for subsequent development of sensitization. This suggests that early childhood eczema, rather than wheeze and rhinitis, may promote subsequent allergen sensitization and raises the possibility that early management of eczema may reduce the prevalence of sensitization in children.

Mycobacterial disease remains a serious global health problem. Tuberculosis causes more than 2 million deaths a year, and leprosy is still a cause of severe disability in many parts of the world. As a result of the study of individuals with marked susceptibility to usually nonpathogenic mycobacteria, as well as case-control studies with candidate genes and genome-wide screens of affected populations, there is substantial evidence for the role of genetic factors in the susceptibility to mycobacterial disease. These studies have defined immunological processes essential for the control of mycobacteria infections in humans.

Summary Background The fungus Alternaria alternata contains potent allergens, and sensitization to these allergens is associated with a high risk of respiratory disease. The influence of genetic regulation on sensitization to Alternaria is unknown. Objective To determine the influence of genetic factors on IgE responses to specific allergens of Alternaria. Methods The concordance of skin prick test (SPT), radioallergosorbent test (RAST) and IgE-binding profiles of sera were examined from a large cohort of monozygotic and dizygotic twins. Results Casewise concordance for a positive SPT response was monozygous (MZ) 66%: dizygous (DZ) 40% (P = 0.002). Logistic regression confirmed that casewise concordance was significantly stronger between MZ than DZ pairs. Immunoblotting against an Alternaria extract revealed 19 allergenic bands. The differences in concordance between the different bands were not significant for either the MZ (P = 0.97) or DZ (P = 0.84) groups. The pooled MZ : DZ difference in concordance was just significant (P = 0.049), suggesting an overall genetic effect on the response to Alternaria. This was reinforced by the comparison of the MZ and DZ correlations for total number of bands recognized (MZ r = 0.65; DZ r = 0.37, P = 0.015). Overall, there was a moderate correlation between the individual SPT weal size and RAST score (r2 = 0.41) and a substantial correlation between the number of immunoblotted bands and RAST scores (r2 = 0.79). Conclusion There is a strong genetic influence on IgE response to the mixture of Alternaria allergens and a lesser effect on IgE response to individual allergens.

Protective immunity against Mycobacterium tuberculosis infection requires the induction and maintenance of mycobacteria-specific, IFN-gamma-secreting CD4+ and CD8+ T lymphocytes. The development of Th1-like T cells is promoted by the early secretion and synergistic action of interleukin (IL)-12 and IL-18. This study compares the effects of plasmid-encoded IL-12 and IL-18 on the immunogenicity and protective efficacy of a DNA vaccine expressing the M. tuberculosis-secreted protein antigen 85B (DNA-85B). Co-immunization with either IL-12- or IL-18-expressing plasmids augmented the IFN-gamma-secreting T-cell response, and the maximum effect was observed with plasmids encoding both cytokines. Further the IL-12, but not the IL-18-expressing plasmid, significantly increased the protective efficacy of DNA-85B against pulmonary M. tuberculosis infection. Therefore co-administration of plasmid-encoded cytokines provides a potential method for optimizing the protective efficacy of DNA vaccination against tuberculosis.

The production of IL-12 by dendritic cells (DC) early in an immune response is considered critical for the polarization of CD4+ T lymphocyte response towards a Th1 pattern, a key process in the clearance of intracellular pathogens. Infection of bone marrow-derived DC with Mycobacterium bovis Bacillus Calmette Guerin (BCG) induced a concurrent and dose-dependent releaseof IL-10 and IL-12. Here we examined whether the production of IL-10 by DC affected their IL-12 response to mycobacterial infection and the generation of protective immune responses in vivo. Compared to wild-type (WT) DC, DC deficient for IL-10 synthesis (IL-10–/–) showed increased IL-12 production in response to BCG infection and CD40 stimuli in vitro. Moreover, when transferred into mice, infected IL-10–/– DC were more efficient than WT DC at inducing IFN-γ production to mycobacterial antigens in the draining lymph nodes (DLN).This effect was associated with increased trafficking of IL-10–/– DC to the DLN and enhanced IL-12 production by DC within the DLN. These data show that autocrine IL-10 exerts a dual inhibitory effect on the induction of primary immune responses by DC: first, by down-regulating the migration of infected DC to the DLN and second, by modulating the IL-12 production by DC in the DLN.

Background Cord blood mononuclear cells have demonstrated specific immune responses to environmental allergens. Objective To establish whether the nature of this response is related to the level of maternal antenatal exposure to house dust mite (HDM) allergen and, hence, whether antenatal allergen avoidance may have a role in the prevention of allergic sensitization in children. Methods Children with a family history of asthma were recruited antenatally as subjects in a randomised controlled trial: the Childhood Asthma Prevention Study. HDM allergen (Der p 1) concentrations were measured in dust collected from the maternal bed at 36 weeks gestation. Cord blood mononuclear cells were stimulated in culture, separately, with phytohaemaglutinin (PHA) and HDM extract. Cytokine IL-4, IL-5, IL-10 and IFN-γ concentrations in supernatant were measured by ELISA. mRNA signals for these cytokines were measured using RT-PCR. Results The median concentration of HDM allergen was 18.4 µg/g (interquartile range 7.3–35.3 µg/g). Median concentrations of IL-4, IL-5, IL-10 and IFN-γ, after PHA stimulation were 4, 19, 401 and 1781 pg/mL, respectively. After HDM allergen stimulation the median concentrations were 0, 0, 20 and 14 pg/mL, respectively. The distribution of mRNA cytokine signals was similar. Neither cytokine protein concentrations nor cytokine mRNA signal levels were correlated with the concentration of HDM allergen in the mothers' beds at 36 weeks gestation. Conclusion These findings do not support the view that the prevention of allergic disease in children requires the institution of HDM avoidance interventions during pregnancy.

Interleukin (IL)-10 is an immunoregulatory cytokine that inhibits both Th1-like T cell responses and macrophage activation. Deficiency of IL-10 has been associated with increased Th1-like CD4+ T-cell responses and increased clearance of some intracellular pathogens, however, its role in mycobacterial infections is controversial. In order to examine the effects of mycobacterial virulence on the outcome of infection we compared infection with Mycobacterium avium and virulent Mycobacterium tuberculosis in C57Bl/6 IL-10−/– mice. M. avium infection in IL-10−/– mice resulted in sustained increases in interferon (IFN)-γ-secreting T-cell responses and was associated with the increased clearance of M. avium from the liver and lung. By contrast, M. tuberculosis infection in IL-10−/– mice led to a transient increase in IFN-γ T-cell responses at 4 weeks postinfection, with reduced bacterial burden in the lungs. This was not sustained so that by 8 weeks there was no difference to wild-type (WT) mice. In vitro infection of IL-10−/– macrophages with M. avium, but not M. tuberculosis, led to an increased IL-12 production. Therefore, endogenous IL-10 exerts a significant inhibition on specific IFN-γ T-cell responses to M. avium infection, however, this effect is short lived during the M. tuberculosis infection, and fails to influence the long-term course of infection.

In the lung, dendritic cells (DC) are key antigen-presenting cells capable of triggering specific cellular responses to inhaled pathogens, and thus, they may be important in the initiation of an early response to mycobacterial infections. The ability of DC to enhance antigen presentation to naive T cells within the lungs was characterized with respect to Mycobacterium bovis Bacillus Calmette Guerin (BCG) vaccination against M. tuberculosis infection. In vitro derived DC were infected with BCG, which induced their maturation, as shown by the increased expression of MHC class II antigens, CD80 and CD86 co-stimulatory molecules. The synthesis of mRNA for IL-1, IL-6, IL-12, IL-10 and IL-1 receptor antagonist was also enhanced. When administered intratracheally in mice, infected DC induced a potent T cell response and the production of IFN-gamma to mycobacterial antigens in the mediastinal lymph nodes, leading to a significant protection against aerosol M. tuberculosis infection. Intriguingly, although the vaccination schedule for BCG-infected DC was much shorter than subcutaneous BCG vaccination (7 days as compared to 100 days), both types of vaccination showed similar levels of protection. These data confirm that DC can be potent inducers of a cellular immune response against mycobacteria and support the concept of combining DC strategies with mycobacterial vaccines for protective immunity against tuberculosis.

Background Both genetic and environmental factors are thought to contribute to specific IgE responses, however, the relative contribution of each in the responses to individual ryegrass pollen allergens is largely unknown even though some responses to allergens have been linked to certain HLA complexes. Objective Using a large group of monozygotic and dizygotic twins, this study was designed to investigate the IgE binding profiles of individual ryegrass pollen (Lolium perenne) components to assess the relative contribution of genetic and environmental factors in determining IgE responses to specific allergens. Methods Ryegrass pollen proteins were separated by electrophoresis and immunoblotted with sera from 191 pairs of twins where at least one sibling had a SPT > 2 mm to perennial ryegrass. Concordance levels for individual ryegrass pollen components were compared between monozygotic and dizygotic twins in a subset group where both twins had SPT > 3 mm to perennial ryegrass. Results Immunoblotting revealed 23 individual IgE-binding components from ryegrass pollen. Although there was a significantly greater proportion of monozygotic twins with SPT wheals greater than 3 mm when compared with the dizygotic twins, the mean case-wise concordance for the immunoblot components was similar for both groups of twins. The mean case-wise concordance when at least four pairs of sera were involved was 44% for the MZ twins (n = 11 components) and 45% for the DZ twins (n = 12 components). We found no significant differences in concordance levels for any of the 23 individual components including allergens previously associated with HLA. Conclusion Evidence for genetic control of allergen-specific IgE responses in a large population sample of twins to individual ryegrass allergens is limited, indicating that the IgE responses to specific ryegrass pollen allergens are determined largely by environmental factors.

To explore the natural history of asthma and its relation to allergic responses, we examined the relation between total serum IgE in early adulthood and a history of respiratory symptoms, airway hyperresponsiveness (AHR), and atopy during childhood. We studied 180 subjects aged 18-20 years who had been studied since the age of 8-10 years. We measured wheeze in the previous year by questionnaire, AHR by histamine inhalation test, atopy by skin prick tests, and serum IgE levels by immunoassay. Subjects with AHR in early adulthood had higher IgE levels (mean 257.0 IU/ml) than subjects with past AHR (mean 93.3 IU/ml) or with lifelong normal responsiveness (mean 67.6 IU/ml) (P < 0.001). Subjects who had symptoms had higher IgE levels (mean 125.9 IU/ml) than those who were lifelong asymptomatic (mean 63.1 IU/ml) (P < 0.001). Recent wheeze, AHR, and allergic sensitization all had a positive relation to serum IgE, but IgE was not more predictive of AHR than skin prick tests. The finding that young adults who are sensitized to common allergens are highly likely to have AHR even in the absence of symptoms is further evidence of the fundamental role of IgE-mediated responses in the natural history of AHR throughout childhood and into adulthood.

Mycobacterium tuberculosis secretes a number of proteins into the extracellular environment, some of which are restricted to the M. tuberculosis complex. These proteins are targets for T- and B-cell immune responses in tuberculosis (TB) patients and their contacts. The authors have mapped the immunogenic regions of the MPT64 protein of M. tuberculosis using peripheral blood mononuclear cells (PBMC) from TB patients and a set of overlapping peptides encompassing the complete sequence of the protein. T-cell epitopes which induced proliferation or interferon-gamma (IFN-gamma) release were distributed over the full length of the protein. A C-terminal region of the protein, however, contains sequences recognized in the context of multiple HLA-DR phenotypes by more than 80% of the subjects tested. The nature of the T-cell response was further investigated by generating MPT64-specific T-cell lines. These lines also identified the T-cell epitopes in the C-terminal region of the protein. On stimulation with antigen the lines secreted IFN-gamma, but not interleukin 4 (IL-4). A minority of TB patients (6/32) mounted an antibody response to MPT64. Sera from half (3/6) of these identified two linear antibody binding sites. These results confirm the significance of this protein in the immune response to tuberculosis infection.

Among the newly synthesized compounds, the N-acetyl and the N-formyl derivative of (V) belongs to the most potent inhibitors.

Ribosomal DNA internal transcribed spacer (ITS) sequences were investigated in the genera Alternaria, Aspergillus, Cladosporium and Penicillium, to identify regions suitable for the design of genus- and species-specific oligonucleotide probes. The ITS regions of all four genera were quite distinct. However, we discovered that the genera Aspergillus and Penicillium displayed high similarity in most of the ITS2 region. Whilst interspecies similarity within Alternaria and Cladosporium was high in both ITS regions 1 and 2, ITS1 similarity within Aspergillus and Penicillium was lower by comparison. We conclude that: (1) the ITS2 region is highly conserved between Aspergillus and Penicillium, and (2) the ITS1 region will allow for the development of specific probes to distinguish between Aspergillus and Penicillium.

Genetic susceptibility to infection with M. leprae was studied in 10 multiple case families of Australian Aborigines. Of the 87 members available for study, 24 had proven stable clinical leprosy which had been or was still being treated with diamino diphenyl sulphone. Evidence of contact with M. leprae in the remaining 63 members as assessed by ELISA to M. leprae sonicate and phenolic glycolipid (PGL) or by indirect immunofluorescence antibody assay was found in 78%, 64% and 71%, respectively. By contrast, in vitro assays of T cell function (LMAT and LTT) were less reliable indicators of exposure. Evidence was sought for possible linkages between human leucocyte antigen (HLA) or non-HLA genes and four marker phenotypes including clinical leprosy, clinical subtype of leprosy and lymphocyte transformation or leucocyte migration inhibition factor (LIF) production in response to M. leprae antigen. No associations were found with any particular HLA or non-HLA gene. On the other hand, sequential analysis of the data from the 10 families was strongly suggestive of a linkage between HLA haplotype and non-responsiveness to M. leprae as manifest by lack of LIF production but not lymphocyte transformation. The model which best fits the data is for a gene on chromosome 6 in close linkage with the HLA haplotype, with two alleles, autosomal recessive inheritance and penetrance of 90%. On this basis, it can be suggested that disease type (lepromatous leprosy) rather than disease susceptibility may be controlled by genes within or closely linked to the major histocompatibility gene complex.

Summary In a cross-sectional study of 2363 schoolchildren living in two rural areas of New South Wales, we used a questionnaire to collect details of sex, area of residence, social class, early respiratory illness (ERI), parental history of asthma and recent upper respiratory tract infection (URTI), and we used skin-prick tests to measure atopic status. The relative importance of these factors on the likelihood of children having bronchial hyperresponsiveness (BHR) was assessed using a linear modelling analysis. The extent to which these factors affected the severity of BHR was also examined. We found that social class or recent URTI had no association with BHR, that sex and area of residence (inland or coastal) had a small association and that a history of early respiratory illness, a history of asthma in either parent, and atopic status had an important association with BHR. Atopic status was the most important factor. The proportion of children with atopy, with ERI or with parental asthma increased as the severity of BHR increased. The odds ratio for moderate or severe BHR doubled if either ERI or parental asthma was present in addition to atopy and there was a six-fold increase if all three factors were present together. The identification of these risk factors makes it possible to predict which children in the community are most likely to have BHR, and which children are at high risk for having more severe levels of BHR.