Automatic and online pollen monitoring

BACKGROUND: Pollen are monitored in Europe by a network of about 400 pollen traps, all operated manually. To date, automated pollen monitoring has only been feasible in areas with limited variability in pollen species. There is a need for rapid reporting of airborne pollen as well as for alleviating the workload of manual operation. We report our experience with a fully automated, image recognition-based pollen monitoring system, BAA500. METHODS: The BAA500 sampled ambient air intermittently with a 3-stage virtual impactor at 60 m(3)/h in Munich, Germany. Pollen is deposited on a sticky surface that was regularly moved to a microscope equipped with a CCD camera. Images of the pollen were constructed and compared with a library of known samples. A Hirst-type pollen trap was operated simultaneously. RESULTS: Over 480,000 particles sampled with the BAA500 were both manually and automatically identified, of which about 46,000 were pollen. Of the automatically reported pollen, 93.3% were correctly recognized. However, compared with manual identification, 27.8% of the captured pollen were missing in the automatic report, with most reported as unknown pollen. Salix pollen grains were not identified satisfactorily. The daily pollen concentrations reported by a Hirst-type pollen trap and the BAA500 were highly correlated (r = 0.98). CONCLUSIONS: The BAA500 is a functional automated pollen counter. Its software can be upgraded, and so we expected its performance to improve upon training. Automated pollen counting has great potential for workload reduction and rapid online pollen reporting

Year-to-year variation in release of Bet v 1 allergen from birch pollen: evidence for geographical differences between West and South Germany

The release of the aeroallergen Bet v 1 from pollen is a major determinant in the etiology of allergic airway disease due to birch pollen. OBJECTIVE: We determined the release of the major birch pollen allergen Bet v 1 from pollen of birch trees growing in 2 different geographic regions in Germany for 2 consecutive years. METHODS: Catkins were collected during pollination in 2002 and 2003 from 82 healthy trees in South (Munich) and West Germany (North Rhine-Westphalia). The release of Bet v 1 from pollen samples was determined by a Bet v 1-specific ELISA. RESULTS: Pollen from South Germany released about 3 times more Bet v 1 than those from West Germany in both 2002 and 2003 (p = 0.034 and p = 0.007, respectively). This was independent of the number of pollen during the pollen flight season. In 2003, the release of Bet v 1 from pollen was more than 5 times higher than in 2002 in both regions (South Germany 6.1 times, p < 0.001; West Germany 5.4 times, p = 0.003). CONCLUSIONS: Despite large individual differences, there seem to be regional and year-to-year variations in Bet v 1 release from birch pollen. Therefore, the combination of pollen count and release of Bet v 1 from this pollen must be assessed to estimate Bet v 1 exposure reliably. 2007 S. Karger AG, Base

High environmental ozone levels lead to enhanced allergenicity of Birch pollen

BACKGROUND: Evidence is compelling for a positive correlation between climate change, urbanisation and prevalence of allergic sensitisation and diseases. The reason for this association is not clear to date. Some data point to a pro-allergenic effect of anthropogenic factors on susceptible individuals. OBJECTIVES: To evaluate the impact of urbanisation and climate change on pollen allergenicity. METHODS: Catkins were sampled from birch trees from different sites across the greater area of Munich, pollen were isolated and an urbanisation index, NO(2) and ozone exposure were determined. To estimate pollen allergenicity, allergen content and pollen-associated lipid mediators were measured in aqueous pollen extracts. Immune stimulatory and modulatory capacity of pollen was assessed by neutrophil migration assays and the potential of pollen to inhibit dendritic cell interleukin-12 response. In vivo allergenicity was assessed by skin prick tests. RESULTS: The study revealed ozone as a prominent environmental factor influencing the allergenicity of birch pollen. Enhanced allergenicity, as assessed in skin prick tests, was mirrored by enhanced allergen content. Beyond that, ozone induced changes in lipid composition and chemotactic and immune modulatory potential of the pollen. Higher ozone-exposed pollen was characterised by less immune modulatory but higher immune stimulatory potential. CONCLUSION: It is likely that future climate change along with increasing urbanisation will lead to rising ozone concentrations in the next decades. Our study indicates that ozone is a crucial factor leading to clinically relevant enhanced allergenicity of birch pollen. Thus, with increasing temperatures and increasing ozone levels, also symptoms of pollen allergic patients may increase further

COEXPRESSION OF CYTOCHROME P4502A6 AND HUMAN NADPH-P450 OXIDOREDUCTASE IN THE BACULOVIRUS SYSTEM

ABSTRACT: Heterologous expression using baculovirus vectors has become a popular method for the production of catalytically active cytochrome P450s (CYPs). We have systematically optimized the multiplicity of infection (MOI) for a coinfection approach for the coexpression of CYP2A6 (viral vector designated v2A6) and NADPH-P450 oxidoreductase (OR; viral vector designated vOR) using Sf9 insect cells. A 3000-fold range of MOI was examined in stationary culture and stirred suspension culture. Surprisingly, our results indicate that the best CYP2A6 catalytic activity (850-1300 pmol/ min/mg total lysate protein as measured by coumarin 7-hydroxylase activity) was obtained only when using a low MOI of v2A6 (1.5-3 ؋ 10 ؊2 ) and a vOR of 10-to 20-fold less. This activity was ϳ7-to 11-fold higher than the best activity obtained when infecting cells with v2A6 alone. At this level of coinfection, the P450 content ranged from 180 to 250 pmol/mg total lysate protein, and the NADPH cytochrome c reductase activity ranged from 350 to 520 nmol/min/mg total lysate protein. Increasing the MOI of both viruses to 50-fold higher resulted in lower overall activity with the optimum (250 pmol/min/mg total lysate protein) being seen earlier postinfection (60 vs. 72 hr). Increasing the MOI of vOR to levels comparable with those of v2A6, decreased coumarin 7-hydroxylase activity 14-fold. These results suggest that the best CYP2A6 catalytic activity depends on properly posttranslationally modified proteins accumulating in a right ratio as a result of primary, secondary, and possibly tertiary infection of both viruses. These results also suggest that high OR expression results in degradation of P450. CYPs 1 are a multienzyme, membrane-bound system that metabolizes many drugs and other xenobiotics (1). The catalytic activity of CYP enzymes requires the presence of NADPH-CYP OR. In addition, cytochrome b 5 stimulates catalytic activity for some CYP forms (2). Several efficient systems for the heterologous expression of mammalian CYP enzymes have been developed, including bacterial, yeast, and mammalian and baculovirus/insect cell-based systems (reviewed in ref. 2). Of the systems available, the baculovirus system has distinct advantages for the production of high levels of active, native CYP enzyme. Although efficient mammalian CYP expression is possible in bacterial systems, modification of the amino acid sequence is usually required for high level expression (2). Three distinct approaches have been taken for production of catalytically active CYP enzymes using baculovirus system. Expression of the CYP enzyme alone and then reconstitution with OR using purified P450s or total cell lysate (3-5), coexpression of the CYP and OR using a single virus (6), and coinfection with independent viruses containing CYP or OR (7). Each approach has unique advantages and disadvantages. Purification/reconstitution provides flexibility in controlling the CYP to OR ratio, but is time-and labor-intensive because of the need for column purifications. The single virus approach is more time-and labor-efficient (simple cell lysate can be used), but, because of the small number of promoters characterized for baculovirus expression, the ability to control the CYP to OR ratio is limited The human CYP2A6 is the only enzyme known to date responsible for coumarin 7-hydroxylase activity in human liver (9, 10). CYP2A6 also metabolically activates certain carcinogens and promutagens (for review, see refs. 1, 9, and 10). It has the highest activity for activation of the mutagen N-nitrosodiethylamine among all human P450s studied (9, 11). It also activates tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, 1-(N-methyl-N-nitrosamino)-1-(3-pyridinyl)-4-butanal, n-nitrosonornicotine (12, 13), and the mycotoxin aflatoxin B1 In this study, we describe a systematic characterization of a coinfection approach using CYP2A6 and human OR. To obtain an overall phenomena for coexpression of P450 and OR proteins in the baculovirus system, we examined a wide range of MOI for both v2A6 and vOR, with different ratios for coinfection to determine the best ratio for optimal coumarin 7-hydroxylase catalytic activity in insect cell lysate. Materials and Methods Rabbit anti-rat 2A1 (16) and rabbit anti-rat OR (17) antibodies were described in the previous studies. Recombinant baculoviruses v2A6 and vOR This study was supported in part by SBIR Contract N43-ES-31001 from the National Institute of Environmental Health Sciences. 1 Abbreviations used are: CYP, cytochrome P450; OR, NADPH-P450 oxidoreductase; P450, cytochrome P450; MOI, multiplicity of infection (the number of virus used per cell); v2A6, a recombinant baculovirus containing the human CYP2A6 cDNA; vOR, a recombinant baculovirus containing the human OR cDNA; Sf9, Spodoptera frugiperda; pfu, plaque-forming units (measurement of infectivity of a virus); AcMNPV, Autographa californica nuclear polyhedrosis virus. Send reprint requests to: Dr. Liping Chen

Cabbage and fermented vegetables : From death rate heterogeneity in countries to candidates for mitigation strategies of severe COVID-19

Large differences in COVID-19 death rates exist between countries and between regions of the same country. Some very low death rate countries such as Eastern Asia, Central Europe, or the Balkans have a common feature of eating large quantities of fermented foods. Although biases exist when examining ecological studies, fermented vegetables or cabbage have been associated with low death rates in European countries. SARS-CoV-2 binds to its receptor, the angiotensin-converting enzyme 2 (ACE2). As a result of SARS-CoV-2 binding, ACE2 downregulation enhances the angiotensin II receptor type 1 (AT(1)R) axis associated with oxidative stress. This leads to insulin resistance as well as lung and endothelial damage, two severe outcomes of COVID-19. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the most potent antioxidant in humans and can block in particular the AT(1)R axis. Cabbage contains precursors of sulforaphane, the most active natural activator of Nrf2. Fermented vegetables contain many lactobacilli, which are also potent Nrf2 activators. Three examples are: kimchi in Korea, westernized foods, and the slum paradox. It is proposed that fermented cabbage is a proof-of-concept of dietary manipulations that may enhance Nrf2-associated antioxidant effects, helpful in mitigating COVID-19 severity.Peer reviewe

Nrf2-interacting nutrients and COVID-19 : time for research to develop adaptation strategies

There are large between- and within-country variations in COVID-19 death rates. Some very low death rate settings such as Eastern Asia, Central Europe, the Balkans and Africa have a common feature of eating large quantities of fermented foods whose intake is associated with the activation of the Nrf2 (Nuclear factor (erythroid-derived 2)-like 2) anti-oxidant transcription factor. There are many Nrf2-interacting nutrients (berberine, curcumin, epigallocatechin gallate, genistein, quercetin, resveratrol, sulforaphane) that all act similarly to reduce insulin resistance, endothelial damage, lung injury and cytokine storm. They also act on the same mechanisms (mTOR: Mammalian target of rapamycin, PPAR gamma:Peroxisome proliferator-activated receptor, NF kappa B: Nuclear factor kappa B, ERK: Extracellular signal-regulated kinases and eIF2 alpha:Elongation initiation factor 2 alpha). They may as a result be important in mitigating the severity of COVID-19, acting through the endoplasmic reticulum stress or ACE-Angiotensin-II-AT(1)R axis (AT(1)R) pathway. Many Nrf2-interacting nutrients are also interacting with TRPA1 and/or TRPV1. Interestingly, geographical areas with very low COVID-19 mortality are those with the lowest prevalence of obesity (Sub-Saharan Africa and Asia). It is tempting to propose that Nrf2-interacting foods and nutrients can re-balance insulin resistance and have a significant effect on COVID-19 severity. It is therefore possible that the intake of these foods may restore an optimal natural balance for the Nrf2 pathway and may be of interest in the mitigation of COVID-19 severity

Cytochrome P450s in human immune cells regulate IL-22 and c-Kit via an AHR feedback loop

The mechanisms how environmental compounds influence the human immune system are unknown. The environmentally sensitive transcription factor aryl hydrocarbon receptor (AHR) has immune-modulating functions and responds to small molecules. Cytochrome P4501 enzymes (CYP1) act downstream of the AHR and metabolize small molecules. However, it is currently unknown whether CYP1 activity is relevant for immune modulation. We studied the interdependence of CYP1 and AHR in human primary immune cells using pharmacological methods. CYP1 inhibition increased the expression levels of the stem cell factor receptor (c-Kit) and interleukin (IL)-22 but decreased IL-17. Single cell analyses showed that CYP1 inhibition especially promoted CD4+helper T (Th) cells that co-express c-Kit and IL-22 simultaneously. The addition of an AHR antagonist reversed all these effects. In addition to T cells, we screened other human immune cells for CYP and found cell-specific fingerprints, suggesting that similar mechanisms are present in multiple immune cells. We describe a feedback loop yet unknown in human immune cells where CYP1 inhibition resulted in an altered AHR-dependent immune response. This mechanism relates CYP1-dependent metabolism of environmental small molecules to human immunity