Noninvasive and minimally invasive techniques for the diagnosis and management of allergic diseases.

Allergic diseases of the (upper and lower) airways, the skin and the gastrointestinal tract, are on the rise, resulting in impaired quality of life, decreased productivity, and increased healthcare costs. As allergic diseases are mostly tissue-specific, local sampling methods for respective biomarkers offer the potential for increased sensitivity and specificity. Additionally, local sampling using noninvasive or minimally invasive methods can be cost-effective and well tolerated, which may even be suitable for primary or home care sampling. Non- or minimally invasive local sampling and diagnostics may enable a more thorough endotyping, may help to avoid under- or overdiagnosis, and may provide the possibility to approach precision prevention, due to early diagnosis of these local diseases even before they get systemically manifested and detectable. At the same time, dried blood samples may help to facilitate minimal-invasive primary or home care sampling for classical systemic diagnostic approaches. This EAACI position paper contains a thorough review of the various technologies in allergy diagnosis available on the market, which analytes or biomarkers are employed, and which samples or matrices can be used. Based on this assessment, EAACI position is to drive these developments to efficiently identify allergy and possibly later also viral epidemics and take advantage of comprehensive knowledge to initiate preventions and treatments.

A Subgroup of Latently Mycobacterium tuberculosis Infected Individuals Is Characterized by Consistently Elevated IgA Responses to Several Mycobacterial Antigens.

Elevated antibody responses to Mycobacterium tuberculosis antigens in individuals with latent infection (LTBI) have previously been linked to an increased risk for progression to active disease. Studies in the field focussed mainly on IgG antibodies. In the present study, IgA and/or IgG responses to the mycobacterial protein antigens AlaDH, NarL, 19 kDa, PstS3, and MPT83 were determined in a blinded fashion in sera from 53 LTBI controls, 14 healthy controls, and 42 active TB subjects. Among controls, we found that elevated IgA levels against all investigated antigens were not randomly distributed but concentrated on a subgroup of <30%-with particular high levels in a small subgroup of ~5% comprising one progressor to active TB. Based on a specificity of 100%, anti-NarL IgA antibodies achieved with 78.6% sensitivity the highest accuracy for the detection of active TB compared to healthy controls. In conclusion, the consistently elevated IgA levels in a subgroup of controls suggest higher mycobacterial load, a risk factor for progression to active TB, and together with high IgG levels may have prognostic potential and should be investigated in future large scale studies. The novel antigen NarL may also be promising for the antibody-based diagnosis of active TB cases.

Concurrent evaluation of cytokines improves the accuracy of antibodies against Mycobacterium tuberculosis antigens in the diagnosis of active tuberculosis.

BACKGROUND: Antibodies against mycobacterial proteins are highly specific, but lack sensitivity, whereas cytokines have been shown to be sensitive but not very specific in the diagnosis of tuberculosis (TB). We assessed combinations between antibodies and cytokines for diagnosing TB. METHODS: Immuoglubulin (Ig) A and IgM antibody titres against selected mycobacterial antigens including Apa, NarL, Rv3019c, PstS1, LAM, "Kit 1" (MTP64 and Tpx)", and "Kit 2" (MPT64, Tpx and 19 kDa) were evaluated by ELISA in plasma samples obtained from individuals under clinical suspicion for TB. Combinations between the antibody titres and previously published cytokine responses in the same participants were assessed for diagnosing active TB. RESULTS: Antibody responses were more promising when used in combination (AUC of 0.80), when all seven antibodies were combined. When anti-"Kit 1"-IgA levels were combined with five host cytokine biomarkers, the AUC increased to 97% (92-100%) with a sensitivity of 95% (95% CI, 73-100%), and specificity of 88.5% (95% CI, 68.7-97%) achieved after leave-one-out cross validation. CONCLUSION: When used in combination, IgA titres measured with ELISA against multiple Mycobacterium tuberculosis antigens may be useful in the diagnosis of TB. However, diagnostic accuracy may be improved if the antibodies are used in combination with cytokines.

The human long noncoding RNAs CoroMarker, MALAT1, CDR1as, and LINC00460 in whole blood of individuals after controlled short-term exposure with ultrafine metal fume particles at workplace conditions, and in human macrophages in vitro.

BACKGROUND: Short-term inhalation of occupationally relevant ultrafine zinc/copper (Zn/Cu) containing welding fumes has been shown to induce subclinical systemic inflammation, associated with an elevated risk for cardiovascular diseases. The involvement of noncoding RNAs (lncRNAs) in this setting is currently unknown. However, lncRNAs have been reported to fulfill essential roles in, e.g., cardiovascular diseases, inflammation, infectious diseases, and pollution-related lung disorders. METHODS: In this study, the specific lncRNAs levels of the 4 lncRNAs CoroMarker, MALAT1, CDR1as and LINC00460 were determined by RT-qPCR in THP-1 macrophages exposed to Zn/Cu metal fume suspensions for 1, 2, and 4 hours in vitro. Furthermore, 14 subjects were exposed to Zn/Cu containing welding fumes (at 2.5 mg/m3) for 6 hours. Before, 6, 10, and 29 hours after exposure start, whole blood cell lncRNAs levels were determined by RT-qPCR. RESULTS: In THP-1 macrophages, we observed a 2.3-fold increase of CDR1as at 1 h (Wilcoxon p = 0.03), a non-significant increase of CoroMarker at 1 h, and an increase of LINC00460 at 2 h (p = 0.03) and at 4 h (p = 0.06). In whole blood cells, we determined a non-significant upregulation of CDR1as at 6 h (p = 0.2), a significant downregulation of CoroMarker at 6 h (p = 0.04), and a significant upregulation of LINC00460 levels at 10 h (p = 0.04) and 29 h (p = 0.04). MALAT-1 remained unchanged in both settings. CONCLUSION: The orientation of regulation of the lncRNAs is (except for CoroMarker) similar in the in vitro and in vivo experiments and in line with their described functions. Therefore, these results, e.g. the upregulation of the potential risk marker for cardiovascular diseases, CDR1as, contribute to understanding the underlying mechanisms of Zn/Cu-induced subclinical inflammation in metal workers.

High levels of intracellular IL-4 are expressed in circulating apoptotic T cells in patients with tuberculosis and in community controls.

Data concerning T helper cell phenotypes in response to Mycobacterium tuberculosis infection remain controversial. T lymphocyte intracellular interleukin-4 production in response to CD3 stimulation was determined by flow cytometry in 21 TB patients and 14 community controls. In supplementary experiments the association of interleukin-4 expression with apoptosis was investigated. A low percentage of CD4 T cells in both patients and controls expressed high levels of interleukin-4 (IL-4(high)). A larger subset of both CD4 and CD8 T cells of all subjects expressed low levels of intracellular IL-4 (IL-4(low)). Stimulated and unstimulated cells expressed IL-4(low) and IL-4(high). IL-4(low) percentages were lower in TB patients at diagnosis compared to controls while IL-4(high) percentages were higher in patients. Most IL-4(high) cells co-expressed active caspase-3, a marker for apoptosis. This co-expression was also shown in experimentally induced apoptotic Jurkat cells and peripheral blood neutrophils and monocytes. IL-4 levels may therefore not necessarily indicate a skewed Th cell phenotype, as our data suggest that IL-4 production by CD4 and CD8 T cells can occur constitutively in healthy controls with latent TB infection and in TB patients. Cellular IL-4 production may represent a normal cellular growth factor mechanism which is disturbed at the onset of apoptosis.

Detection of a combination of serum IgG and IgA antibodies against selected mycobacterial targets provides promising diagnostic signatures for active TB.

Immunoglobulin G (IgG) based tests for the diagnosis of active tuberculosis (TB) disease often show a lack of specificity in TB endemic regions, which is mainly due to a high background prevalence of LTBI. Here, we investigated the combined performance of the responses of different Ig classes to selected mycobacterial antigens in primary healthcare clinic attendees with signs and symptoms suggestive of TB. The sensitivity and specificity of IgA, IgG and/or IgM to LAM and 7 mycobacterial protein antigens (ESAT-6, Tpx, PstS1, AlaDH, MPT64, 16kDa and 19kDa) and 2 antigen combinations (TUB, TB-LTBI) in the plasma of 63 individuals who underwent diagnostic work-up for TB after presenting with symptoms and signs compatible with possible active TB were evaluated. Active TB was excluded in 42 individuals of whom 21 has LTBI whereas active TB was confirmed in 21 patients of whom 19 had a follow-up blood draw at the end of 6-month anti-TB treatment. The leading single serodiagnostic markers to differentiate between the presence or absence of active TB were anti-16 kDa IgA, anti-MPT64 IgA with sensitivity and specificity of 90%/90% and 95%/90%, respectively. The combined use of 3 or 4 antibodies further improved this performance to accuracies above 95%. After successful completion of anti-TB treatment at month 6, the levels of 16 kDa IgA and 16 kDa IgM dropped significantly whereas LAM IgG and TB-LTBI IgG increased. These results show the potential of extending investigation of anti-tuberculous IgG responses to include IgM and IgA responses against selected protein and non-protein antigens in differentiating active TB from other respiratory diseases in TB endemic settings.

Single and Combined Exposure to Zinc- and Copper-Containing Welding Fumes Lead to Asymptomatic Systemic Inflammation.

OBJECTIVE: Recently, it has been shown that exposure to welding fumes containing both zinc and copper leads to asymptomatic systemic inflammation in humans as shown by an increase of blood C-reactive protein. In the present study, it was investigated which metal is responsible for this effect. METHODS: Fifteen healthy male subjects were exposed under controlled conditions to welding fumes containing either zinc, or copper, or copper and zinc. RESULTS: For each exposure blood C-reactive protein increased. CONCLUSIONS: Copper- and zinc-containing welding fumes are able to induce systemic inflammation.

Macrophage migration inhibitory factor delays apoptosis in neutrophils by inhibiting the mitochondria-dependent death pathway.

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine known to activate macrophages and T cells. In this study, we demonstrate that recombinant MIF delays apoptosis of neutrophils in vitro. MIF action is dose and time dependent as well as specific since it was abolished with a neutralizing anti-MIF antibody. MIF, like G-CSF, delayed cleavage of the proapoptotic members of the Bcl-2 family Bid and Bax in neutrophils, suggesting that MIF inhibits apoptosis pathways proximal to mitochondria activation. Indeed, MIF also prevented release of cytochrome c and Smac from the mitochondria and subsequent activation of the critical effector caspase-3 in these cells. Moreover, we observed increased MIF plasma levels in patients with cystic fibrosis, a heterogeneous recessive genetic disorder associated with bacterial infections and delayed neutrophil apoptosis. In conclusion, MIF is a survival cytokine for human neutrophils, a finding with potential pathologic relevance in infectious diseases.

Assessment of the Biological Effects of Welding Fumes Emitted From Metal Active Gas and Manual Metal Arc Welding in Humans.

OBJECTIVE: Emissions from a particular welding process, metal inert gas brazing of zinc-coated steel, induce an increase in C-reactive protein. In this study, it was investigated whether inflammatory effects could also be observed for other welding procedures. METHODS: Twelve male subjects were separately exposed to (1) manual metal arc welding fumes, (2) filtered air, and (3) metal active gas welding fumes for 6 hours. Inflammatory markers were measured in serum before, and directly, 1 and 7 days after exposure. RESULTS: Although C-reactive protein concentrations remained unchanged, neutrophil concentrations increased directly after exposure to manual metal arc welding fumes, and endothelin-1 concentrations increased directly and 24 hours after exposure. After exposure to metal active gas and filtered air, endothelin-1 concentrations decreased. CONCLUSIONS: The increase in the concentrations of neutrophils and endothelin-1 may characterize a subclinical inflammatory reaction, whereas the decrease of endothelin-1 may indicate stress reduction.

Serologic diagnosis of tuberculosis by combining Ig classes against selected mycobacterial targets.

INTRODUCTION: Accurate, simple and cost-effective diagnostic tests are needed for diagnosis of active tuberculosis (TB). Serodiagnosis is attractive as it can be harnessed for point-of-care tests. METHODS: We evaluated, in a blinded fashion, the sensitivity and specificity of serologic immunoglobulin (Ig)G, IgA and/or IgM responses to Apa, heat shock protein (HSP) 16.3, HSP20, PE35, probable thiol peroxidase Tpx and lipoarabinomannan (LAM) in 42 HIV-negative South African pulmonary TB patients and 67 control individuals. The status of latent Mycobacterium tuberculosis infection (LTBI) among controls was defined through the TST and IFN-γ release assays (IGRAs). We evaluated 47 definite LTBI (IGRA(+)/LTBI), 8 putative LTBI (IGRA(-)/TST(+)) and 12 TB-uninfected (non-LTBI) subjects. RESULTS: In contrast to anti-PE35 IgA, anti-PE35 IgG and particularly anti-Apa IgA, performances of anti-LAM IgG and selected anti-protein antibodies were less affected by inclusion of LTBI participants into the analysis. Anti-LAM IgG showed with a sensitivity/specificity of 71.4%/86.6% (p < 0.001) the best discrimination between TB and non-TB subjects. Selected five-antibody-combinations (including anti-LAM IgG, anti-LAM IgA and anti-Tpx IgG) further improved this performance to an accuracy exceeding 86%. CONCLUSIONS: Antibody responses to some Mycobacterium tuberculosis antigens often also reflect latent infection explaining the poor performance of antibody-based tests for active TB in TB-endemic settings. Our results suggest that rather a combination of serological responses against selected protein and non-protein antigens and different Ig classes should be investigated for TB serodiagnostics.

Comparison of the nasal release of IL-4, IL-10, IL-17, CCL13/MCP-4, and CCL26/eotaxin-3 in allergic rhinitis during season and after allergen challenge.

BACKGROUND: Allergic rhinitis is an inflammatory disease characterized by local overproduction of type 2 cytokines and tissue eosinophilia. Recent research suggests the involvement of additional cytokines such as IL-17, chemokine (C-C motif) ligand (CCL) 26/eotaxin-3, and CCL13/monocyte chemoattractant protein-4 (MCP-4) in its pathophysiology. Furthermore, bronchial epithelial cells treated with IL-17 and type 2 cytokines distinctively up-regulated eotaxin-3 gene expression. In this study we investigated the kinetics of IL-4, IL-10, IL-17, eotaxin-3, and MCP-4 in seasonal allergic rhinitis volunteers after nasal allergen challenge (NAC) and their release during natural pollen exposure. METHODS: The nasal lavages of 15 symptomatic allergic and 14 nonallergic subjects were collected during the pollination season. Additionally, six allergic subjects underwent a single unilateral nasal allergen and control challenge out of season, and nasal secretions were collected. Levels of IL-4, IL-10, IL-17, eotaxin-3, and MCP-4 in nasal lavages and secretions were measured using an electrochemiluminescent assay. RESULTS: After NAC, allergic subjects had a significant immediate response of nasal symptoms as well as a significant increase at 5 hours of IL-4, IL-10, and IL-17 and at 2, 5, and 24 hours significantly raising levels of eotaxin-3. IL-17 and eotaxin-3 concentrations at 5 hours were correlated (r = 0.94; p = 0.005). During natural pollen exposure, barely detectable levels of IL-17 in allergic subjects were also correlated with eotaxin-3 (r = 0.62; p = 0.01). Eotaxin-3 and MCP-4 levels were significantly elevated 9- or 3.7-fold, respectively, and IL-10 and, unexpectedly, IL-4 were significantly lower in allergic subjects compared with nonallergic subjects. CONCLUSION: Nasal IL-17, MCP-4, and, possibly, eotaxin-3 may aggravate and IL-10 may alleviate nasal mucosal allergy.

Serodiagnostic markers for the prediction of the outcome of intensive phase tuberculosis therapy.

Treatment failure and relapse may affect many tuberculosis (TB) patients who undergo standard anti-TB therapy. Several independent studies suggested unsuccessful sputum culture conversion at month 2 of treatment (slow response) as risk factor for treatment failure and relapse. However, earlier than month 2 identification of patients with a high risk for poor treatment outcome would offer significant clinical trial and individual patient care benefits. The sensitivity and specificity of serological IgG and IgA responses against four recombinant mycobacterial antigens (ABC transporter PstS3, secreted l-alanine dehydrogenase, culture filtrate protein Tpx and 6 kDa early secretory antigenic target esxa (ESAT-6)) were evaluated separately in a blinded fashion in 21 smear-positive pulmonary TB patient sera taken at diagnosis before commencement of directly observed anti-TB treatment short course comprising 13 slow responder and eight fast responder subjects. We observed a general pattern of higher antibody levels in sera of slow responders. Most pronounced were high levels of anti-alanine dehydrogenase IgG, anti-Tpx IgG, anti-ESAT-6 IgG and anti-ESAT-6 IgA antibodies at diagnosis being associated with slow response with 100% specificity each and 46.2, 53.8, 53.8 or 53.8% sensitivity, respectively, when compared to fast response (P = 0.020, 0.021, 0.040 and 0.011, respectively). Discriminant analysis showed that the combined use of anti-Tpx IgG and anti-ESAT-6 IgA antibody titers before treatment predicted slow responders with 90.5% accuracy. These preliminary results suggest that combinations of serodiagnostic markers measured prior to initiation of treatment may be suitable for the prediction of early treatment response. This approach holds promise and requires further evaluation for its utility in the prediction of treatment failure and relapse, the evaluation of new TB therapeutics, as well as in the care of individual patients.

The release of IL-31 and IL-13 after nasal allergen challenge and their relation to nasal symptoms.

BACKGROUND: IL-31, a recently discovered member of the gp130/IL-6 cytokine family, is mainly expressed by human mast cells and T helper type 2 cells. IL-31 is a key trigger of atopic dermatitis. Recent studies also suggest a role of IL-31 in the pathogenesis of other allergic diseases including allergic rhinitis. In the present study we studied the release of IL-31 and IL-13 in allergen-challenged allergic rhinitis patients. METHODS: Seven seasonal allergic volunteers underwent unilateral nasal provocation with allergen (and a control challenge) with the disc method out of the allergy season. Nasal symptom scores (rhinorrhea, itching, sneezing, obstruction) and bilateral nasal secretions were quantified before and after allergen provocation. IL-13 and IL-31 in nasal secretions and serum were measured by electrochemiluminescent immunoassay or ELISA, respectively. RESULTS: Nasal allergen challenge induced the typical clinical symptoms and physiological changes. IL-31 and IL-13 in nasal secretions increased in four and five, respectively, volunteers at 5 h after allergen but not after control challenge. We observed correlation trends between nasal IL-31 concentrations and IL-13 concentrations (r = 0.9, p = 0.002), and IL-31 contents and symptom scores (r = 0.9, p = 0.013) 5 h after allergen provocation. No IL-31 could be detected contralaterally or systemically in the sera. CONCLUSIONS: The observed local upregulation of IL-31 mainly during the late phase reaction after nasal allergen challenge suggests a role of IL-31 in allergic rhinitis. In which way IL-31 modulates the inflammatory reaction and type 2 responses in allergic rhinitis remains to be investigated.

Functional expression of CD134 by neutrophils.

CD134 (OX40) is a member of the tumor necrosis factor (TNF) receptor superfamily expressed on activated T cells. Here, we show that human peripheral blood neutrophils express CD134. Activation of CD134 by soluble CD134 ligand (OX40 ligand/gp34) resulted in delayed caspase-3 activation and consequently in delayed neutrophil apoptosis in vitro. Moreover, CD134 ligand, like G-CSF, maintained anti-apoptotic Mcl-1 levels and inhibited cleavage of the pro-apoptotic Bcl-2 family members Bid and Bax in these cells, suggesting that CD134-mediated signals block apoptosis pathways proximal to mitochondria activation. In conclusion, CD134 regulates neutrophil survival, suggesting that this molecule does not only contribute to adaptive but also to innate immune responses.