AWMF mold guideline "Medical clinical diagnostics for indoor mold exposure" - Update 2023 AWMF Register No. 161/001.

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Basophil activation in insect venom allergy: comparison of an established test using liquid reagents with a test using 5-color tubes with dried antibody reagents.

BACKGROUND: Flow cytometry-based basophil activation tests (BAT) have been performed with various modifications, differing in the use of distinct identification and activation markers. Established tests use liquid reagents while a new development involves the use of tubes with dried antibody reagents. The aim of this pilot study was to compare these two techniques in patients with insect venom allergy. METHODS: Seventeen patients with an insect venom allergy were included in the study. The established "BAT 1" utilizes conventional antibody solutions of anti-CCR3 for basophil identification and anti-CD63 to assess basophil activation, whereas "BAT 2" uses dried anti-CD45, anti-CD3, anti-CRTH2, anti-203c and anti-CD63 for identification and activation measurement of basophils. Negative and positive controls as well as incubations with honey bee venom and yellow jacket venom at three concentrations were performed. RESULTS: Seven patients had to be excluded due to low basophil counts, high values in negative controls or negative positive controls. For the remaining 10 patients the overall mean (± SD) difference in activated basophils between the two tests was 0.2 (± 12.2) %P. In a Bland-Altman plot, the limit of agreement (LoA) ranged from 24.0 to -23.7. In the qualitative evaluation (value below/above cut-off) Cohen's kappa was 0.77 indicating substantial agreement. BAT 2 took longer to perform than BAT 1 and was more expensive. CONCLUSION: The BAT 2 technique represents an interesting innovation, however, it was found to be less suitable compared to an established BAT for the routine diagnosis of insect venom allergies.

IgE and anaphylaxis specific to the carbohydrate alpha-gal depend on IL-4.

BACKGROUND: Alpha-gal (Galα1-3Galß1-4GlcNAc) is a carbohydrate with the potential to elicit fatal allergic reactions to mammalian meat and drugs of mammalian origin. This type of allergy is induced by tick bites, and therapeutic options for this skin-driven food allergy are limited to the avoidance of the allergen and treatment of symptoms. Thus, a better understanding of the immune mechanisms resulting in sensitization through the skin is crucial, especially in the case of a carbohydrate allergen for which underlying immune responses are poorly understood. OBJECTIVE: We aimed to establish a mouse model of alpha-gal allergy for in-depth immunologic analyses. METHODS: Alpha-galactosyltransferase 1-deficient mice devoid of alpha-gal glycosylations were sensitized with the alpha-gal-carrying self-protein mouse serum albumin by repetitive intracutaneous injections in combination with the adjuvant aluminum hydroxide. The role of basophils and IL-4 in sensitization was investigated by antibody-mediated depletion. RESULTS: Alpha-gal-sensitized mice displayed increased levels of alpha-gal-specific IgE and IgG1 and developed systemic anaphylaxis on challenge with both alpha-gal-containing glycoproteins and glycolipids. In accordance with alpha-gal-allergic patients, we detected elevated numbers of basophils at the site of sensitization as well as increased numbers of alpha-gal-specific B cells, germinal center B cells, and B cells of IgE and IgG1 isotypes in skin-draining lymph nodes. By depleting IL-4 during sensitization, we demonstrated for the first time that sensitization and elicitation of allergy to alpha-gal and correspondingly to a carbohydrate allergen is dependent on IL-4. CONCLUSION: These findings establish IL-4 as a potential target to interfere with alpha-gal allergy elicited by tick bites.

High-risk groups for alpha-gal sensitization.

BACKGROUND: Tick bite-induced IgE-mediated reactions to the oligosaccharide galactose α-1,3-galactose (alpha-gal) are increasingly recognized. This study investigated alpha-gal sensitization in three groups with different tick bite exposure. MATERIALS AND METHODS: Specific IgE antibodies to alpha-gal and total IgE were investigated in 485 patients with Lyme borreliosis with different disease manifestations and compared to a control group of 200 randomly selected patients without increased exposure to tick bites. A group of 232 hunters and forest workers served as a model for multiple tick bites. RESULTS: Specific IgE (sIgE) antibodies to alpha-gal (> 0.1 kU/L) were found in 12.6% of all borreliosis samples compared to the control group with 9% (relative risk 1.4; 95% CI 0.85 - 2.3; not significant (n.s.). The highest prevalence of sIgE to alpha-gal was observed in hunters and forest service employees (22.8%, relative risk 2.5; 95% CI 1.5 - 4.2; p < 0.001). Higher age and elevated total IgE were also associated with alpha-gal sensitization. CONCLUSION: IgE sensitization to alpha-gal tends to be more frequent in tick-exposed patients with borreliosis than in controls (n.s.). Moreover, hunters and forest workers show an even higher rate of elevated IgE to alpha-gal. Thus, frequent tick contact may result in alpha-gal sensitization. In the area of Munich, the prevalence of alpha-gal sensitization appears lower than in the state of Baden-Württemberg and lower than in the USA, which may be due to the difference in tick species or the frequency of tick exposure. This study could show that alpha-gal sensitization and presumably alpha-gal syndrome does not seem to be a modern problem but existed already more than 30 years ago.

Desipramine induces eryptosis in human erythrocytes, an effect blunted by nitric oxide donor sodium nitroprusside and N-acetyl-L-cysteine but enhanced by Calcium depletion.

Background: Desipramine a representative of tricyclic antidepressants (TCAs) promotes recovery of depressed patients by inhibition of reuptake of neurotransmitters serotonin (SER) and norepinephrine (NE) in the presynaptic membrane by directly blocking their respective transporters SERT and NET.Aims: To study the effect of desipramine on programmed erythrocyte death (eryptosis) and explore the underlying mechanisms.Methods: Phosphatidylserine (PS) exposure on the cell surface as marker of cell death was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry. Hemolysis was determined photometrically, and intracellular glutathione [GSH]i from high performance liquid chromatography.Results: Desipramine dose-dependently significantly enhanced the percentage of annexin-V-binding cells and didn´t impact glutathione (GSH) synthesis. Desipramine-induced eryptosis was significantly reversed by pre-treatment of erythrocytes with either nitric oxide (NO) donor sodium nitroprusside (SNP) or N-acetyl-L-cysteine (NAC). The highest inhibitory effect was obtained by using both inhibitors together. Calcium (Ca2+) depletion aggravated desipramine-induced eryptosis. Changing the order of treatment, i.e. desipramine first followed by inhibitors, could not influence the inhibitory effect of SNP or NAC.Conclusion: Antidepressants-caused intoxication can be treated by SNP and NAC, respectively. B) Patients with chronic hypocalcemia should not be treated with tricyclic anti-depressants or their dose should be noticeably reduced.

Darier's disease exhibits a unique cutaneous microbial dysbiosis associated with inflammation and body malodour.

BACKGROUND: Darier's disease (DD) is a genodermatosis caused by mutations of the ATP2A2 gene leading to disrupted keratinocyte adhesion. Recurrent episodes of skin inflammation and infections with a typical malodour in DD indicate a role for microbial dysbiosis. Here, for the first time, we investigated the DD skin microbiome using a metabarcoding approach of 115 skin swabs from 14 patients and 14 healthy volunteers. Furthermore, we analyzed its changes in the context of DD malodour and the cutaneous DD transcriptome. RESULTS: We identified a disease-specific cutaneous microbiome with a loss of microbial diversity and of potentially beneficial commensals. Expansion of inflammation-associated microbes such as Staphylococcus aureus and Staphylococcus warneri strongly correlated with disease severity. DD dysbiosis was further characterized by abundant species belonging to Corynebacteria, Staphylococci and Streptococci groups displaying strong associations with malodour intensity. Transcriptome analyses showed marked upregulation of epidermal repair, inflammatory and immune defence pathways reflecting epithelial and immune response mechanisms to DD dysbiotic microbiome. In contrast, barrier genes including claudin-4 and cadherin-4 were downregulated. CONCLUSIONS: These findings allow a better understanding of Darier exacerbations, highlighting the role of cutaneous dysbiosis in DD inflammation and associated malodour. Our data also suggest potential biomarkers and targets of intervention for DD. Video Abstract.

Increasing incidence of Trichophyton tonsurans in Munich-A single-centre observation.

BACKGROUND: Tinea capitis and tinea corporis are highly prevalent fungal skin infections, which globally are mainly caused by Microsporum canis and Trichophyton rubrum, respectively. While in the United States and Great Britain Trichophyton tonsurans is widely prevalent as a causative pathogen, it so far only plays a minor role in Germany. OBJECTIVES: Since the frequency of pathogenic species varies regionally and temporally, this study assesses the proportion of Trichophyton tonsurans infections in the dermatology department of a large university hospital in Germany from 2019 to 2022 and thoroughly characterises the affected patient population. PATIENTS/METHODS: This retrospective study at the Technical University of Munich analyses mycological culture results regarding the identified dermatophyte and infection site. Detailed patient and disease-related information on Trichophyton tonsurans positive patients was obtained. RESULTS: In 2022, 23 patients of 111 dermatophyte culture-positive patients tested positive for Trichophyton tonsurans. This accounted for 20.7% and represented a tenfold increase from 2.1% in 2019. Contact sports were only practiced by 21.7% of patients, and no common hotspot or other linkage could be identified between the cases. Additionally, 47.8% of the patients received a systemic treatment, with 30.4% visiting the clinic more than three times. In 2022, 21.7% were diagnosed with a simultaneous infection of the capillitium and body, whereas this was only observed in 7.1% of cases in 2019 to 2021. CONCLUSIONS: This study suggests an increase of Trichophyton tonsurans infections via several routes of transmission.

Fetal Tissue-Derived Mast Cells (MC) as Experimental Surrogate for In Vivo Connective Tissue MC.

Bone-marrow-derived mast cells are matured from bone marrow cells in medium containing 20% fetal calf serum (FCS), interleukin (IL)-3 and stem-cell factor (SCF) and are used as in vitro models to study mast cells (MC) and their role in health and disease. In vivo, however, BM-derived hematopoietic stem cells account for only a fraction of MC; the majority of MC in vivo are and remain tissue resident. In this study we established a side-by-side culture with BMMC, fetal skin MC (FSMC) or fetal liver MC (FLMC) for comparative studies to identify the best surrogates for mature connective tissue MC (CTMC). All three MC types showed comparable morphology by histology and MC phenotype by flow cytometry. Heterogeneity was detected in the transcriptome with the most differentially expressed genes in FSMC compared to BMMC being Hdc and Tpsb2. Expression of ST2 was highly expressed in BMMC and FSMC and reduced in FLMC, diminishing their secretion of type 2 cytokines. Higher granule content, stronger response to FcεRI activation and significantly higher release of histamine from FSMC compared to FLMC and BMMC indicated differences in MC development in vitro dependent on the tissue of origin. Thus, tissues of origin imprint MC precursor cells to acquire distinct phenotypes and signatures despite identical culture conditions. Fetal-derived MC resemble mature CTMC, with FSMC being the most developed.

Cutaneous Barriers and Skin Immunity.

The skin barrier provides us with several lines of protection from outside hazards. Its most outward layers, the stratum corneum and the epidermis seal our body with an acidic, dry, and rather cool surface, hostile to microbes. Yet, there are also fine-tuned interactions between the mostly commensal microbiota on top of the skin surface, with underlying epidermal cells as well as the immune system, to preserve a healthy steady state and to initiate repair processes when necessary. We take a concise look at the recent insights on the inner workings of this complex barrier.

Munich atopy prediction study (MAPS): protocol for a prospective birth cohort addressing clinical and molecular risk factors for atopic dermatitis in early childhood.

INTRODUCTION: The pathogenesis of atopic diseases is highly complex, and the exact mechanisms leading to atopic dermatitis (AD) onset in infants remain mostly enigmatic. In addition to an interdependent network of components of skin development in young age and skin barrier dysfunction underlying AD development that is only partially understood, a complex interplay between environmental factors and lifestyle habits with skin barrier and immune dysregulation is suspected to contribute to AD onset. This study aims to comprehensively evaluate individual microbiome and immune responses in the context of environmental determinants related the risk of developing AD in the first 4 years of a child's life. METHODS AND ANALYSES: The 'Munich Atopic Prediction Study' is a comprehensive clinical and biological investigation of a prospective birth cohort from Munich, Germany. Information on pregnancy, child development, environmental factors, parental exposures to potential allergens and acute or chronic diseases of children and parents are collected by questionnaires together with a meticulous clinical examination by trained dermatologists focusing on allergies, skin health, and in particular signs of AD at 2 months after birth and then every 6 months. In addition, skin barrier functions are assessed through cutometry, corneometry and transepidermal water loss at every visit. These measurements are completed with allergy diagnostics and extensive microbiome analyses from stool and skin swabs as well as transcriptome analyses using skin microbiopsies.The aim is to assess the relevance of different known and yet unknown risk factors of AD onset and exacerbations in infants and to identify possible accessible and robust biomarkers. ETHICS AND DISSEMINATION: The study is approved by the Ethical Committee of the Medical Faculty of the Technical University of Munich (reference 334/16S). All relevant study results will be presented at national and international conferences and in peer-reviewed journals.

Pre-digest of unprotected DNA by Benzonase improves the representation of living skin bacteria and efficiently depletes host DNA.

BACKGROUND: The identification of microbiota based on next-generation sequencing (NGS) of extracted DNA has drastically improved our understanding of the role of microbial communities in health and disease. However, DNA-based microbiome analysis cannot per se differentiate between living and dead microorganisms. In environments such as the skin, host defense mechanisms including antimicrobial peptides and low cutaneous pH result in a high microbial turnover, likely resulting in high numbers of dead cells present and releasing substantial amounts of microbial DNA. NGS analyses may thus lead to inaccurate estimations of microbiome structures and consequently functional capacities. RESULTS: We investigated in this study the feasibility of a Benzonase-based approach (BDA) to pre-digest unprotected DNA, i.e., of dead microbial cells, as a method to overcome these limitations, thus offering a more accurate assessment of the living microbiome. A skin mock community as well as skin microbiome samples were analyzed using 16S rRNA gene sequencing and metagenomics sequencing after DNA extraction with and without a Benzonase digest to assess bacterial diversity patterns. The BDA method resulted in less reads from dead bacteria both in the skin mock community and skin swabs spiked with either heat-inactivated bacteria or bacterial-free DNA. This approach also efficiently depleted host DNA reads in samples with high human-to-microbial DNA ratios, with no obvious impact on the microbiome profile. We further observed that low biomass samples generate an α-diversity bias when the bacterial load is lower than 105 CFU and that Benzonase digest is not sufficient to overcome this bias. CONCLUSIONS: The BDA approach enables both a better assessment of the living microbiota and depletion of host DNA reads. Video abstract.

Coronavirus disease 2019 (COVID-19), human erythrocytes and the PKC-alpha/-beta inhibitor chelerythrine -possible therapeutic implication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19. Until now, diverse drugs have been used for the treatment of COVID-19. These drugs are associated with severe side effects, e.g. induction of erythrocyte death, named eryptosis. This massively affects the oxygen (O2) supply of the organism. Therefore, three elementary aspects should be considered simultaneously: (1) a potential drug should directly attack the virus, (2) eliminate virus-infected host cells and (3) preserve erythrocyte survival and functionality. It is known that PKC-α inhibition enhances the vitality of human erythrocytes, while it dose-dependently activates the apoptosis machinery in nucleated cells. Thus, the use of chelerythrine as a specific PKC-alpha and -beta (PKC-α/-ß) inhibitor should be a promising approach to treat people infected with SARS-CoV-2.

Googling for Ticks and Borreliosis in Germany: Nationwide Google Search Analysis From 2015 to 2018.

BACKGROUND: Borreliosis is the most frequently transmitted tick-borne disease in Europe. It is difficult to estimate the incidence of tick bites and associated diseases in the German population due to the lack of an obligation to register across all 16 federal states of Germany. OBJECTIVE: The aim of this study is to show that Google data can be used to generate general trends of infectious diseases on the basis of borreliosis and tick bites. In addition, the possibility of using Google AdWord data to estimate incidences of infectious diseases, where there is inconsistency in the obligation to notify authorities, is investigated with the perspective to facilitate public health studies. METHODS: Google AdWords Keyword Planner was used to identify search terms related to ticks and borreliosis in Germany from January 2015 to December 2018. The search volume data from the identified search terms was assessed using Excel version 15.23. In addition, SPSS version 24.0 was used to calculate the correlation between search volumes, registered cases, and temperature. RESULTS: A total of 1999 tick-related and 542 borreliosis-related search terms were identified, with a total of 209,679,640 Google searches in all 16 German federal states in the period under review. The analysis showed a high correlation between temperature and borreliosis (r=0.88), and temperature and tick bite (r=0.83), and a very high correlation between borreliosis and tick bite (r=0.94). Furthermore, a high to very high correlation between Google searches and registered cases in each federal state was observed (Brandenburg r=0.80, Mecklenburg-West Pomerania r= 0.77, Saxony r= 0.74, and Saxony-Anhalt r=0.90; all P<.001). CONCLUSIONS: Our study provides insight into annual trends concerning interest in ticks and borreliosis that are relevant to the German population exemplary in the data of a large internet search engine. Public health studies collecting incidence data may benefit from the results indicating a significant correlation between internet search data and incidences of infectious diseases.

The specific PKC-α inhibitor chelerythrine blunts costunolide-induced eryptosis.

Costunolide, a natural sesquiterpene lactone, has multiple pharmacological activities such as neuroprotection or induction of apoptosis and eryptosis. However, the effects of costunolide on pro-survival factors and enzymes in human erythrocytes, e.g. glutathione and glucose-6-phosphate dehydrogenase (G6PDH) respectively, have not been studied yet. Our aim was to determine the mechanisms underlying costunolide-induced eryptosis and to reverse this process. Phosphatidylserine exposure was estimated from annexin-V-binding, cell volume from forward scatter in flow cytometry, and intracellular glutathione [GSH]i from high performance liquid chromatography. The oxidized status of intracellular glutathione and enzyme activities were measured by spectrophotometry. Treatment of erythrocytes with costunolide dose-dependently enhanced the percentage of annexin-V-binding cells, decreased the cell volume, depleted [GSH]i and completely inhibited G6PDH activity. The effects of costunolide on annexin-V-binding and cell volume were significantly reversed by pre-treatment of erythrocytes with the specific PKC-α inhibitor chelerythrine. The latter, however, had no effect on costunolide-induced GSH depletion. Costunolide induces eryptosis, depletes [GSH]i and inactivates G6PDH activity. Furthermore, our study reveals an inhibitory effect of chelerythrine on costunolide-induced eryptosis, indicating a relationship between costunolide and PKC-α. In addition, chelerythrine acts independently of the GSH depletion. Understanding the mechanisms of G6PDH inhibition accompanied by GSH depletion should be useful for development of anti-malarial therapeutic strategies or for synthetic lethality-based approaches to escalate oxidative stress in cancer cells for their sensitization to chemotherapy and radiotherapy.

Targeting tumor-resident mast cells for effective anti-melanoma immune responses.

Immune checkpoint blockade has revolutionized cancer treatment. Patients developing immune mediated adverse events, such as colitis, appear to particularly benefit from immune checkpoint inhibition. Yet, the contributing mechanisms are largely unknown. We identified a systemic LPS signature in melanoma patients with colitis following anti-cytotoxic T lymphocyte-associated antigen 4 (anti-CTLA-4) checkpoint inhibitor treatment and hypothesized that intestinal microbiota-derived LPS contributes to therapeutic efficacy. Because activation of immune cells within the tumor microenvironment is considered most promising to effectively control cancer, we analyzed human and murine melanoma for known sentinels of LPS. We identified mast cells (MCs) accumulating in and around melanomas and showed that effective melanoma immune control was dependent on LPS-activated MCs recruiting tumor-infiltrating effector T cells by secretion of CXCL10. Importantly, CXCL10 was also upregulated in human melanomas with immune regression and in patients with colitis induced by anti-CTLA-4 antibody. Furthermore, we demonstrate that CXCL10 upregulation and an MC signature at the site of melanomas are biomarkers for better patient survival. These findings provide conclusive evidence for a "Trojan horse treatment strategy" in which the plasticity of cancer-resident immune cells, such as MCs, is used as a target to boost tumor immune defense.

Proliferating tumor cells mimick glucose metabolism of mature human erythrocytes.

Mature human erythrocytes are dependent on anerobic glycolysis, i.e. catabolism (oxidation) of one glucose molecule to produce two ATP and two lactate molecules. Proliferating tumor cells mimick mature human erythrocytes to glycolytically generate two ATP molecules. They deliberately avoid or switch off their respiration, i.e. tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) machinery and consequently dispense with the production of additional 36 ATP molecules from one glucose molecule. This phenomenon is named aerobic glycolysis or Warburg effect. The present review deals with the fate of a glucose molecule after entering a mature human erythrocyte or a proliferating tumor cell and describes why it is useful for a proliferating tumor cell to imitate a mature erythrocyte. Blood consisting of plasma and cellular components (99% of the cells are erythrocytes) may be regarded as a mobile organ, constantly exercising a direct interaction with other organs. Therefore, the use of drugs, which influences the biological activity of erythrocytes, has an immediate effect on the entire organism. Abbreviations: TCA: tricarboxylic acid cycle; OXPHOS: oxidative phosphorylation; GSH: reduced state of glutathione; NFκB: Nuclear factor of kappa B; PKB (Akt): protein kinase B; NOS: nitric oxide synthase; IgG: immune globulin G; H2S: hydrogen sulfide; slanDCs: Human 6-sulfo LacNAc-expressing dendritic cells; IL-8: interleukin-8; LPS: lipopolysaccharide; ROS: reactive oxygen species; PPP: pentose phosphate pathway; NADPH: nicotinamide adenine dinucleotide phosphate hydrogen; R5P: ribose-5-phophate; NAD: nicotinamide adenine dinucleotide; FAD: flavin adenine dinucleotide; O2●-: superoxide anion; G6P: glucose 6-phosphate; HbO2: Oxyhemoglobin; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GAP: glyceraldehyde-3-phosphate; 1,3-BPG: 1,3-bis-phosphoglycerate; 2,3-BPG: 2,3-bisphosphoglycerte; PGAM1: phosphoglycerate mutase 1; 3-PG: 3-phosphoglycerate; 2-PG: 2-phosphoglycerate; MIPP1: Multiple inositol polyphosphate phosphatase; mTORC1: mammalian target of rapamycin complex 1; Ru5P: ribulose 5-phosphate; ox-PPP: oxidative branch of pentose phosphate pathway; PGK: phosphoglycerate kinase; IFN-γ: interferon-γ; LDH: lactate dehydrogenase; STAT3: signal transducer and activator of transcription 3; Rheb: Ras homolog enriched in Brain; H2O2: hydrogen peroxide; ROOH: lipid peroxide; SOD: superoxide dismutase; MRC: mitochondrial respiratory chain; MbFe2+-O2: methmyoglobin; RNR: ribonucleotide reductase; PRPP: phosphoribosylpyrophosphate; PPi: pyrophosphate; GSSG: oxidized state of glutathione; non-ox-PPP: non-oxidative branch of pentose phosphate pathway; RPI: ribose-5-phosphate isomerase; RPE: ribulose 5-phosphate 3-epimerase; X5P: xylulose 5-phosphate; TK: transketolase; TA: transaldolase; F6P: fructose-6-phosphate; AR2: aldose reductase 2; SD: sorbitol dehydrogenase; HK: hexokinase; MG: mehtylglyoxal; DHAP: dihydroxyacetone phosphate; TILs: tumor-infiltrating lymphocytes; MCTs: monocarboxylate transporters; pHi: intracellular pH; Hif-1α: hypoxia-induced factor 1; NHE1: sodium/H+ (Na+/H+) antiporter 1; V-ATPase: vacuolar-type proton ATPase; CAIX: carbonic anhydrase; CO2: carbon dioxide; HCO3-: bicarbonate; NBC: sodium/bicarbonate (Na+/HCO3-) symporter; pHe: extracellular pH; GLUT-1: glucose transporter 1; PGK-1: phosphoglycerate kinase 1.