Laboratory biomarkers associated with COVID-19 severity and management.

The heterogeneous disease course of COVID-19 is unpredictable, ranging from mild self-limiting symptoms to cytokine storms, acute respiratory distress syndrome (ARDS), multi-organ failure and death. Identification of high-risk cases will enable appropriate intervention and escalation. This study investigates the routine laboratory tests and cytokines implicated in COVID-19 for their potential application as biomarkers of disease severity, respiratory failure and need of higher-level care. From analysis of 203 samples, CRP, IL-6, IL-10 and LDH were most strongly correlated with the WHO ordinal scale of illness severity, the fraction of inspired oxygen delivery, radiological evidence of ARDS and level of respiratory support (p ≤ 0.001). IL-6 levels of ≥3.27 pg/ml provide a sensitivity of 0.87 and specificity of 0.64 for a requirement of ventilation, and a CRP of ≥37 mg/l of 0.91 and 0.66. Reliable stratification of high-risk cases has significant implications on patient triage, resource management and potentially the initiation of novel therapies in severe patients.

Serological, genomic and structural analyses of the major mite allergen Der p 23.

BACKGROUND: Der p 23 was recently identified in a European population as a major allergen and potentially a chitin binding protein. OBJECTIVE: This study sought to assess the importance of Der p 23 among other Dermatophagoides allergens in a North American population and to determine the structure for functional characterization. METHODS: IgE binding to Der p 23, Der p 1, Der p 2, Der p 5, Der p 7 and Der p 8 was measured by ELISA. RNA-seq data from D. pteronyssinus were compared as estimates of allergen expression levels. The structure was analysed by X-ray crystallography and NMR. RESULTS: Despite a high prevalence of Der p 23, (75% vs. 87% and 79% for Der p 1 and Der p 2, respectively), the anti-Der p 23 IgE levels were relatively low. The patient response to the 6 allergens tested was variable (n = 47), but on average anti-Der p 1 and anti-Der p 2 together accounted for 85% of the specific IgE. In terms of abundance, the RNA expression level of Der p 23 is the lowest of the major allergens, thirty fold less than Der p 1 and sevenfold less than Der p 2. The structure of Der p 23 is a small, globular protein stabilized by two disulphide bonds, which is structurally related to allergens such as Blo t 12 that contain carbohydrate binding domains that bind chitin. Functional assays failed to confirm chitin binding by Der p 23. CONCLUSIONS AND CLINICAL RELEVANCE: Der p 23 accounts for a small percentage of the IgE response to mite allergens, which is dominated by Der p 1 and Der p 2. The prevalence and amount of specific IgE to Der p 23 and Der p 2 are disproportionately high compared to the expression of other Dermatophagoides allergens.

A multi-allergen standard for the calibration of immunoassays: CREATE principles applied to eight purified allergens.

BACKGROUND: Allergen measurements are widely used for environmental exposure assessments and for determining the potency of allergen vaccines, yet few purified allergen standards have been developed. The aim of the study was to develop a single standard containing multiple purified allergens that could be used in enzyme immunoassays and in multiplex arrays for the standardization of allergen measurements. METHODS: Eight purified allergens were formulated into a single multi-allergen, or 'universal', standard based on amino acid analysis. Dose-response curves were compared with previous individual ELISA standards and allergen measurements of house dust extracts to obtain correction factors. Measured allergen concentrations were also modeled using linear regression, and the predictive accuracy was determined. RESULTS: Parallel dose-response curves were obtained between the universal allergen standard and the individual ELISA standards, with close agreement between curves for 5/8 allergens. Quantitative differences of greater than twofold were observed for Fel d 1, Can f 1, and Der f 1, which were confirmed by the analysis of house dust extracts. Correction factors were developed that allowed ELISA data to be expressed in terms of the universal standard. Linear regression data confirmed the predictive accuracy of the universal standard. CONCLUSION: This study shows that a single standard of eight purified allergens can be used to compare allergen measurements by immunoassay. This approach will improve the continuity of environmental exposure assessments and provide improved standardization of allergy diagnostics and vaccines used for immunotherapy.

Aspergillus fumigatus allergen I, a major IgE-binding protein, is a member of the mitogillin family of cytotoxins.

A major 18-kD IgE-binding protein from Aspergillus fumigatus (Asp fI) has been purified. Partial amino acid sequencing of Asp f I showed extensive sequence homology (95%) between Asp fI and a cytotoxin (mitogillin) produced by A. restrictus. Crossinhibition radioimmunoassay using murine monoclonal antibody and human IgG and IgE antibodies showed that Asp fI and mitogillin were antigenically indistinguishable. Furthermore, both proteins inhibited protein synthesis in vitro by greater than 90%. Asp fI was expressed in A. fumigatus but not in seven other Aspergillus species. The results suggest that Asp fI could play a dual role in the pathogenesis of A. fumigatus-related diseases by promoting colonization through cytotoxic activity and by causing inflammatory reactions involving IgE antibodies.

Effect of improved home ventilation on asthma control and house dust mite allergen levels.

BACKGROUND: The warm, humid environment in modern homes favours the dust mite population, but the effect of improved home ventilation on asthma control has not been established. We tested the hypothesis that a domestic mechanical heat recovery ventilation system (MHRV), in addition to allergen avoidance measures, can improve asthma control by attenuating re-colonization rates. METHODS: We conducted a randomized double-blind placebo-controlled parallel group trial of the installation of MHRV activated in half the homes of 120 adults with asthma, allergic to Dermatophagoides pteronyssinus. All homes had carpets steam cleaned and new bedding and mattress covers at baseline. The primary outcome was morning peak expiratory flow (PEF) at 12 months. RESULTS: At 12 months, the primary end-point; change in mean morning PEF as compared with baseline, did not differ between the MHRV group and the control group (mean difference 13.5 l/min, 95% CI: -2.6 to 29.8, P = 0.10). However, a secondary end-point; evening mean PEF, was significantly improved in the MHRV group (mean difference 24.5 l/min, 95% CI: 8.9-40.1, P = 0.002). Indoor relative humidity was reduced in MHRV homes, but there was no difference between the groups in Der p 1 levels, compared with baseline. CONCLUSIONS: The addition of MHRV to house dust mite eradication strategies did not achieve a reduction in mite allergen levels, but did improve evening PEF.

The longitudinal profile of CSF markers during external lumbar drainage.

BACKGROUND: External lumbar drainage (ELD) is known as a good predictor of favourable outcome in shunting patients suffering from idiopathic normal pressure hydrocephalus (iNPH). METHODS: Eleven patients suffering from iNPH had a lumbar drain (LD) inserted for 72 h and participated in a research study to quantify any improvement in their clinical symptoms. The lumbar cerebrospinal fluid (CSF) levels of lactate, 8-isoprostane, vascular endothelial growth factor (VEGF), glial fibrillar acidic protein (GFAP), neurofilament (heavy chain) protein (NF (h)), Abeta(1-42) (beta-amyloid) and total tau were assayed samples from all three time points. RESULTS: The concentrations of lactate, VEGF, GFAP and tau increased significantly during the 72 h of drainage. There were also increases in 8-isoprostane and Abeta(1-42) (non significant). The concentration of NF (h) was reduced significantly following 72 h of drainage. There was a significant positive correlation between Abeta(1-42) and total tau in the first sample. GFAP was negatively correlated in a significant fashion with both Abeta(1-42) and total tau. NF (h) was negatively correlated with VEGF. CONCLUSION: Evidence is provided that ELD is producing measurable changes in the CSF composition of patients with iNPH. The present paper discusses how such changes may be implicated in the pathophysiology of the condition.

The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification.

Allergen extracts have been used for diagnosis and treatment of allergy for around 100 years. During the second half of 20th century, the notion increasingly gained foothold that accurate standardization of such extracts is of great importance for improvement of their quality. As a consequence, manufacturers have implemented extensive protocols for standardization and quality control. These protocols have overall IgE-binding potencies as their focus. Unfortunately, each company is using their own in-house reference materials and their own unique units to express potencies. This does not facilitate comparison of different products. During the last decades, most major allergens of relevant allergen sources have been identified and it has been established that effective immunotherapy requires certain minimum quantities of these allergens to be present in the administered maintenance dose. Therefore, the idea developed to introduce major allergens measurements into standardization protocols. Such protocols based on mass units of major allergen, quantify the active ingredients of the treatment and will at the same time allow comparison of competitor products. In 2001, an EU funded project, the CREATE project, was started to support introduction of major allergen based standardization. The aim of the project was to evaluate the use of recombinant allergens as reference materials and of ELISA assays for major allergen measurements. This paper gives an overview of the achievements of the CREATE project.

Cockroach allergens: function, structure and allergenicity.

Cockroach allergy is a widespread health problem in the world, associated with the development of asthma. The German and American cockroach species are important producers of a wide variety of allergens. Knowledge of their structure and function contributes to understand their role in allergy and to design tools for diagnosis and immunotherapy.

Reduction in IgE binding to allergen variants generated by site-directed mutagenesis: contribution of disulfide bonds to the antigenic structure of the major house dust mite allergen Der p 2.

Site-directed mutagenesis was used to investigate the contribution of disulfide bonds to the antigenic structure of Der p 2. Single amino acid variants were generated at cysteine residues, preventing the formation of disulfide bonds at positions 21-27, 73-78, and 8-119. The variants were tested for binding to murine monoclonal antibodies (mAb) and human IgE antibodies (Ab) in an inhibition enzyme immunoassay. Removal of the disulfide linking the amino-carboxy termini (C8-C119) had no effect on mAb binding, however, IgE Ab binding was reduced by up to 10-fold. The other two disulfides form small loops and disruption of these bonds gave different binding patterns. The variant lacking the C21-C27 bond showed up to a 40-fold reduction in antibody binding, while the variant lacking the C73-C78 bond showed more than a 100-fold reduction in IgE Ab binding and failed to bind 3 of 4 mAb. Intradermal skin testing with the C73-C78 variant supported the in vitro findings; the variant was 10 to 100-fold less reactive than rDer p 2. These two bonds thus make markedly different contributions to stabilizing the antigenic determinants of Der p 2. The results suggest that the C73-C78 bond plays a critical role in stabilizing the antigenic structure of this major mite allergen.

A two-site monoclonal antibody ELISA for the quantification of the major Dermatophagoides spp. allergens, Der p I and Der f I.

A two-site monoclonal antibody (Mab) ELISA was developed to measure the Group I allergens from Dermatophagoides spp., Der p I from D. pteronyssinus and Der f I from D. farinae. Species-specific Mabs were used to coat microtiter plates which were then incubated with allergen or house dust extracts. Bound allergen was detected using a biotinylated Mab which recognized a common epitope on both Der p I and Der f I, followed by the addition of streptavidin-peroxidase and ABTS/H2O2 substrate. The assay had low non-specific binding (approximately 0.08 absorbance units) and had a sensitivity of 5 ng/nl for aqueous allergen extracts (equivalent to 0.1 microgram allergen/g dust). 53 dust samples were assayed using the Mab ELISA and an RIA previously described using 125I-labelled Mab. The results showed a very good quantitative correlation between the assays (r = 0.96, p less than 0.001 for Der p I; r = 0.92, P less than 0.001 for Der f I). A further 132 dust samples from a different geographical areas were also assayed by both methods and gave correlation coefficients of 0.90 (P less than 0.001) and 0.86 (P less than 0.001) for Der p I and Der f I, respectively. The Mab ELISA will be useful in epidemiological studies of allergic asthma, both in the assessment of levels of dust mite allergen present in houses and the efficacy of allergen avoidance regimes.

Can knowledge of the molecular structure of allergens improve immunotherapy?

Conventional immunotherapy may be associated with the development of adverse reactions, including anaphylaxis, due to the use of increasing doses of allergen. Standardization of extracts is necessary in order to assess the correct amount of allergen administered. In recent years, increased knowledge on the molecular structure of allergens has allowed the development of novel alternatives for immunotherapy. Initially, allergens were cloned and expressed as recombinant proteins in eukaryotic and prokaryotic systems. Crystallization of the purified proteins led to the elucidation of the tertiary structure of the allergen. Molecular biology techniques were used to construct modified allergens whose new IgE binding properties were studied. IgE antibody mapping combined with molecular modeling has allowed the recognition of IgE binding sites on the surface of the molecule. This information has been applied to the engineering of new modified allergens, with and without adjuvants, that retain immunogenicity but with reduced allergenicity. The use of these molecules for immunotherapy should allow the administration of greater doses of allergen, without the undesired side effects characteristic of conventional immunotherapy.

Hidden allergic factors in the etiology of asthma.

Increasing evidence from case control surveys, population studies and allergen avoidance studies suggest inhalant allergy plays an important role in the etiology of asthma. Recent studies in hospital emergency rooms have compared the prevalence of serum IgE antibodies to common allergens (mite, cat, cockroach, rye grass and ragweed pollen) in patients admitted with acute asthma attacks and in unselected age-matched control subjects. These studies, carried out in central Virginia and northern California, showed a highly increased prevalence of IgE antibodies to inhaled allergens among asthmatic patients, and suggest that the development of allergen specific IgE antibody responses is a major risk factor for emergency room admission with asthma. Presentation at the emergency room appeared to be related to patients' exposure to specific allergens: in central Virginia, in the fall, dust mite was the predominant allergy, whereas in northern California, in May-June, most asthmatic patients (greater than 90 percent) were allergic to rye grass. New immunoassay technology, based on the use of monoclonl antibodies, has been developed to measure the quantities of "indoor" allergens (mite, cat, cockroach) in asthmatic patients' houses. It is now possible to propose tentative levels of mite allergens which should be considered both as a risk for IgE antibody sensitization (2micrograms allergen/g dust) and as a risk for acute asthma attacks (10micrograms allergen/g dust). Future management of asthma will require analysis of indoor allergens and the development of efficient allergen avoidance procedures. Further research is necessary to investigate the relationship between airborne allergen levels, particle size and the precipitation of asthma attacks and also to investigate immunologic mechanisms which may cause bronchial hyperreactivity.

Zymodeme and species specificities of monoclonal antibodies raised against Trypanosoma cruzi.

A series of hybrid cell lines was generated by the fusion of Sp2/0 myeloma cells with spleen cells from BALB/c mice that had been immunized or infected with Trypanosoma cruzi zymodemes (Z1, Z2, Z3 ). Four immunoglobulin isotypes, IgM, IgG1, IgG2a and IgG3 were represented amongst the monoclonal antibodies secreted by 22 hybridoma clones. On indirect immunofluorescence (IFAT) antibodies bound to flagellum, cytoplasm, cell membrane or stained the whole organism. Two antibodies were epimastigote-specific. Enzyme-linked immunosorbent assays (ELISA) and a dot immunobinding test were used to evaluate the zymodeme and species specificities of 13 antibodies: four reacted with all T. cruzi zymodemes tested, two reacted strongly with all except Z1, two predominantly with Z1, two predominantly with Z2, and three predominantly with Z3 . Two IgM antibodies cross reacted with Trypanosoma rangeli, T. brucei, Leishmania mexicana, L. braziliensis and L. donovani. Five antibodies were used in a preliminary immunobinding test, performed blindly, to compare monoclonal reactivities and zymodeme groups. The results suggested a correlation between the two methods of characterization. Anti-T. cruzi monoclonal antibodies are considered to have important applications to epidemiological studies and the improved diagnosis and prognosis of Chagas' disease.

Evaluation of German cockroach (Orthoptera:Blattellidae) allergen and seasonal variation in low-income housing.

Six apartments in a low-income housing project were evaluated for German cockroach. Blattella germanica (L.), infestation and concentration of an allergen derived from these cockroaches (Bla g II). Kitchen and living room samples were collected monthly for 1 yr. In addition, airborne sampling was carried out in 5 kitchens. The kitchen had the highest allergen concentration in 65% of visits and the highest number of cockroaches trapped in 69% of visits. In the kitchen, the highest cockroach levels were seen in June, whereas the values for Bla g II peaked in August. In keeping with this, the closest correlation was between Bla g II (microgram/g dust) and the number of cockroaches found 2 mo earlier. Airborne samples were assayed for 2 separate allergens. Bla g II and Bla g I. No allergen was detectable in the absence of disturbance. By contrast, during disturbance with a vacuum cleaner both Bla g II and Bla g I were detectable in the air of each apartment. Results suggest that immunochemical assay of a major allergen in dust samples from the kitchen floor may be used to monitor exposure to German cockroaches, also that cockroach levels may be used as an indicator or predictor of allergen in dust.

Peanut allergen (Ara h 1) detection in foods containing chocolate.

Inadvertent exposure to peanut in foods poses health risks for peanut-allergic individuals that can be reduced by improving detection systems for allergen contaminants in food products and manufacturing processes. Detection of peanut in chocolate has been especially difficult. We report the optimization of conditions for measuring a major peanut allergen, Ara h 1, in chocolate with the use of a two-site monoclonal antibody sandwich enzyme-linked immunosorbent assay. Ara h 1 was extracted from peanut in the presence or absence of chocolate with phosphate buffer, salt, and three dried milks (goat, soy, or nonfat) (0 to 25% wt/vol) for 15 min at 60 degrees C or for 2.5 h at room temperature. The best conditions for Ara h 1 extraction in the presence of chocolate were 5% nonfat dry milk for 2.5 h at room temperature. Spiking experiments of chocolate with peanut confirmed improvement of the extraction: Ara h 1 was detected in extractions of 0.16 to 0.33% peanut in chocolate. Interestingly, the best conditions for Ara h 1 extraction were different for peanut alone than with chocolate, regarding time, temperature, and percentage of nonfat dry milk in the extraction buffer. In chocolate with peanut foods, the total Ara h 1 values were 10-fold higher than when products were extracted with phosphate buffer alone and could be up to 400-fold higher for individual foods. The dramatic improvement of Ara h 1 extraction should allow specific allergen monitoring in chocolate-containing food products and assessment of Ara h 1 exposure.

House dust sensitivity and environmental control.

Allergy to foreign proteins in house dust is extremely common. Despite the fact that immunotherapy with house dust extract has been utilized for over sixty years, environmental control is still infrequently employed as a therapeutic measure. In this article the major allergen content of house dust is defined and strategies for decreasing the levels of these allergens in homes are described.

Sensitization to cockroach allergens of asthma patients in Japan.

To evaluate the role of allergens from Periplaneta fuliginosa, which is the most predominant cockroach species in homes in Tokyo areas, for asthma sensitization, we measured specific IgE antibodies to two cockroaches, P. fuliginosa and Blattella germanica, and to a mite, Dermatophagoides farinae, in 171 sera from children with asthma by Pharmacia's CAP system. We found that 16% of the sera had anti-P. fuliginosa IgE, whereas 9.9% had anti-B. germanica and 85% anti-D. farinae IgE. Further, we measured the levels of Per f I (Per a I equivalent) allergen in the house dust from living room, kitchen and bedding. We detected the allergen in eight of ten homes. The Per a I equivalent levels in kitchen were higher than in other sites, but they were much lower than Der I and Der II as Dermatophagoides allergens.

An unbiased, staged, multicentre, validation strategy for Alzheimer's disease CSF tau levels.

Newly proposed diagnostic criteria for Alzheimer's disease include cerebrospinal fluid (CSF) tau levels as one core supportive criterion. The published high sensitivity and specificity figures for CSF tau levels in Alzheimer's disease are offset by the large range of proposed cutoff values (9.6 pg/mL to 1140 pg/mL). This study aimed to provide guidance on how to establish, validate and audit CSF tau cutoff values using an unbiased, two-stage multicentre strategy. Both receiver operator characteristics (ROC) optimised and population-based cutoff values were calculated on a pilot dataset (n=99), validated in a large dataset (n=560) and then compared to the literature. The data suggest using an ROC optimised cutoff level of 323+/-51.7 pg/mL allowing for the published inter-laboratory coefficient of variation of 16%. This cutoff level was confirmed in a prospective audit (n=100). As demand for CSF tau levels will increase globally, the accuracy of local CSF hTau cutoff levels can be compared against this benchmark.