Interindividual variation of NETosis in healthy donors: introduction and application of a refined method for extracellular trap quantification.

Neutrophil extracellular trap (NET) formation is a mechanism of innate immune defence by which neutrophil (polymorphonuclear) granulocytes (PMN) produce net-like structures of decondensed chromatin decorated with antimicrobial peptides for trapping and possibly killing microorganisms. If this process leads to cell death, it is termed NETosis. Alterations of this particular mechanism have been reported to be involved in the pathogenesis of chronic inflammatory diseases including psoriasis and lupus erythematosus. Still, quantification of NETosis poses a considerable challenge. We report and test a refined protocol for morphological NET quantification in healthy human donors that encompasses isolation, stimulation, DNA staining, live imaging and semi-automated offline analysis. The results were highly reproducible and in good agreement with manual counting. The average intra-donor coefficient of variation of NETosis rates to phorbol myristate acetate (PMA) stimulation was low compared to the respective interdonor coefficient of variation (10% vs 82%, n=4, respectively, if experiments were repeated on the same day, and 38% vs 74%, n=6, respectively, if experiments were repeated on average 42±34 days apart). Overall, the interdonor coefficient of variation was 67% (n=10). These findings altogether support the existence of a distinct predisposition of PMN from different donors for undergoing NETosis. Picogreen fluorescence correlated stronger to cell death than to morphological NETosis (r2 =.89, P<.001, n=8, and r2 =.68, P=.012, n=8, respectively). This indicates that cytotoxicity may confound Picogreen fluorescence. Our results and the related protocol may help investigators with the quantification of NETosis and the design of respective basic and translational research studies.

Subcutaneous immunotherapy with a depigmented polymerized birch pollen extract--a new therapeutic option for patients with atopic dermatitis.

BACKGROUND: Birch pollen is an important outdoor allergen able to aggravate symptoms in atopic dermatitis (AD). Specific immunotherapy (SIT), an established procedure for allergic airway diseases, might also represent an attractive therapeutic option for the causal treatment of allergen-triggered cutaneous symptoms in these patients. Studies with house dust mite SIT have already shown beneficial effects in AD patients, whereas the safety and efficacy of SIT with birch pollen extract in AD patients have not been studied so far. The aim of this study was to evaluate for the first time the safety and efficacy of SIT with a depigmented polymerized birch pollen extract in AD patients. METHODS: Fifty-five adult patients with moderate-to-severe AD and clinically relevant sensitization to birch pollen received SIT for 12 weeks. SIT was continued during birch pollen season. The assessment of safety, the total SCORAD value, and the Dermatology Life Quality Index (DLQI) were evaluated. RESULTS: The median total SCORAD value was reduced by 34% (p < 0.001) during the course of treatment and the mean DLQI improved by 49% (p < 0.001) despite strong simultaneous birch pollen exposure. Eight patients (14.5%) developed systemic reactions and 19 patients (34.5%) developed local reactions which were of mild intensity in most cases. No patient discontinued the study prematurely due to adverse drug reactions. Coseasonal treatment was well tolerated. CONCLUSION: SIT with a depigmented polymerized birch pollen extract leads to significant improvement of the SCORAD value and the DLQI in patients suffering from moderate-to-severe AD sensitized to birch pollen.

TH2 cytokines from malignant cells suppress TH1 responses and enforce a global TH2 bias in leukemic cutaneous T-cell lymphoma.

PURPOSE: In leukemic cutaneous T-cell lymphoma (L-CTCL), malignant T cells accumulate in the blood and give rise to widespread skin inflammation. Patients have intense pruritus, increased immunoglobulin E (IgE), and decreased T-helper (TH)-1 responses, and most die from infection. Depleting malignant T cells while preserving normal immunity is a clinical challenge. L-CTCL has been variably described as a malignancy of regulatory, TH2 and TH17 cells. EXPERIMENTAL DESIGN: We analyzed phenotype and cytokine production in malignant and benign L-CTCL T cells, characterized the effects of malignant T cells on healthy T cells, and studied the immunomodulatory effects of treatment modalities in patients with L-CTCL. RESULTS: Twelve out of 12 patients with L-CTCL overproduced TH2 cytokines. Remaining benign T cells were also strongly TH2 biased, suggesting a global TH2 skewing of the T-cell repertoire. Culture of benign T cells away from the malignant clone reduced TH2 and enhanced TH1 responses, but separate culture had no effect on malignant T cells. Coculture of healthy T cells with L-CTCL T cells reduced IFNγ production and neutralizing antibodies to interleukin (IL)-4 and IL-13 restored TH1 responses. In patients, enhanced TH1 responses were observed following a variety of treatment modalities that reduced malignant T-cell burden. CONCLUSIONS: A global TH2 bias exists in both benign and malignant T cells in L-CTCL and may underlie the infectious susceptibility of patients. TH2 cytokines from malignant cells strongly inhibited TH1 responses. Our results suggest that therapies that inhibit TH2 cytokine activity, by virtue of their ability to improve TH1 responses, may have the potential to enhance both anticancer and antipathogen responses.