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Cystic Fibrosis (CF) is caused by genetic defects that impair the cystic fibrosis transmembrane conductance regulator (CFTR) channel in airway epithelial cells. These defects may be overcome by specific CFTR modulating drugs, for which the efficacy can be predicted in a personalized manner using 3D nasal-brushing-derived airway organoids in a forskolin-induced swelling assay. Despite of this, previously described application of 3D airway organoids in CFTR function assays have not been fully optimal. In this report we therefore describe an alternative method of culturing nasal brushing-derived airway organoids, which are created from an equally differentiated airway epithelial monolayer of a 2D air-liquid interface culture. In addition, we have defined organoid culture conditions, with the growth factor/cytokine combination neuregulin-1β and interleukine-1β, which enabled consistent detection of CFTR modulator responses in nasal airway organoids cultures from subjects with CF.

Patient-derived organoids hold great potential as predictive biomarker for disease expression or therapeutic response. Here, we used intestinal organoids to estimate individual cystic fibrosis transmembrane conductance regulator (CFTR) function of people with cystic fibrosis, a monogenic life-shortening disease associated with more than 2000 CFTR mutations and highly variable disease progression. In vitro CFTR function in CF intestinal organoids of 176 individuals with diverse CFTR mutations was quantified by forskolin induced swelling and was strongly associated with longitudinal changes of lung function and development of pancreatic insufficiency, CF-related liver disease and diabetes. This association was not observed when the commonly used biomarker of CFTR function sweat chloride concentration was used. The data strongly exemplifies the value of an organoid-based biomarker in a clinical disease setting and supports the prognostic value of forskolin induced swelling of intestinal organoids, especially for people with CF who have rare CFTR genotypes with unclear clinical consequences.

Objective: To verify if the extent of structural lung damage visible on chest CT is correlated with change in ppFEV1 after start of CFTR modulating therapy in CF patients. Methods: In our retrospective observational study, we included patients aged ≥6 years starting ivacaftor or lumacaftor/ivacaftor treatment who were in routine follow up. FEV1 and BMI were recorded every 3 months. From the group of patients who had started lumacaftor/ivacaftor, patients were selected who underwent chest CT within 18 months before or after start of treatment. Patients who had started ivacaftor all underwent chest CT just before the start of treatment. These CT scans were reviewed to determine Brody score. ppFEV1 data was retrieved. To assess correlations, Spearman R and Pearson R tests were applied. Results: Thirty patients met the inclusion criteria, with Brody scores between 0.46-55.30 (median: 19.97, normalized score out of 100) and ppFEV1 before treatment of 19-113 (median: 67). Twenty-three and 7 patients had started lumacaftor/ivacaftor and ivacaftor respectively. Change in ppFEV1 after 6 months of treatment ranged from -19 to +24 (median: +5). For patients using lumacaftor/ivacaftor, the correlation between Brody score and change in ppFEV1 was not significant: Spearman R=-0.213, (p=0.164), but for patients using ivacaftor, there was a significant correlation between Brody score and ppFEV1 change, with a Spearman R=0.679 (p=0.029). Conclusion: The extent of structural damage to the lungs of CF patients is correlated with the response in ppFEV1 to ivacaftor, however this correlation was not demonstrable in patients treated with lumacaftor/ivacaftor.

Adenine base editing (ABE) enables enzymatic conversion from A-T into G-C base pairs. ABE holds promise for clinical application, as it does not depend on the introduction of double-strand breaks, contrary to conventional CRISPR/Cas9-mediated genome engineering. Here, we describe a cystic fibrosis (CF) intestinal organoid biobank, representing 664 patients, of which ~20% can theoretically be repaired by ABE. We apply SpCas9-ABE (PAM recognition sequence: NGG) and xCas9-ABE (PAM recognition sequence: NGN) on four selected CF organoid samples. Genetic and functional repair was obtained in all four cases, while whole-genome sequencing (WGS) of corrected lines of two patients did not detect off-target mutations. These observations exemplify the value of large, patient-derived organoid biobanks representing hereditary disease and indicate that ABE may be safely applied in human cells.

Little is known about psychological and social factors influencing the frequency of migraine attacks. The aim of the present study was to study whether anger, anger-expression, anxiety, depression, somatization and childhood adversities influence the frequency of migraine attacks. 53 female patients suffering from migraine with a mean monthly headache frequency of 6.85 took part. Groups of patients with 6 or less monthly attacks (N = 27) vs. patients with 7 or more days (N = 26) were formed. Logistic regression analysis revealed, that only the variable "anger-in" resulted in an improvement of prediction of group-membership (rate of correct classified cases: 69.8 %). Tendency to repress anger increases the probability to be a member of the group with frequent attacks. This result points out the relevance of anger-expression and -perception for the psychotherapy of migraine.

Zusammenfassung Chronische Schmerzen bedingen angesichts ihrer bio-psycho-sozialen Komplexitat und der facherubergreifenden Prasenz eine interdisziplinare Zusammenarbeit. Diese erst ermoglicht eine umfassende Einordnung des Befundes und ist Vorraussetzung fur ein individuelles und ressourcenorientiertes Therapiekonzept mit dem Schwerpunkt der korperlichen und psychischen Aktivierung. Dieses Konzept ist Behandlungsschwerpunkt des UniversitatsSchmerzCentrums (USC), das im April 2004 im Universitatsklinikum Dresden gegrundet wurde und seitdem neben der bereits etablierten ambulanten Therapie auch teilstationare und stationare Versorgung ermoglicht. Ausgangspunkt der Grundungsinitiative fur das Schmerzzentrum war die Sensibilisierung der Kostentrager fur die komplexe Proble-matik des chronischen Schmerzes und ebenso fur die bestehenden regionalen Strukturdefizite. Gefolgt von der Vorlage eines schlussigen multimodalen, fachubergreifenden Behandlungskonzeptes mit ausfuhrlicher Kostenkalkulation wurde nach 1 1 2 -jahriger Arbeitsphase im Juni 2004 ein Vertrag zur integrierten Versorgung mit der AOK Sachsen und dem VdAK unterzeichnet. Nach nun zweijahriger Behandlungsphase sollen erste Erfahrungen und Ergebnisse, insbesondere der tagesklinischen Therapiesaule des Schmerzzentrums innerhalb des IV-Vertrages, dargestellt werden.

The loss of spleen may lead to fatal bacterial infections. To prevent this, splenic autotransplantation has been performed in humans and experimental animals. However, there is still controversy about the protective function of this procedure. Since innervation plays an important role in splenic function, we investigated whether splenic regenerates are re-innervated, and whether this depends on the donor and host age. Splenic tissue (30 mg) was implanted into the greater omentum of either young (2 days) or old (12 months) rats, from either young or old syngeneic animals. After 3 months of regeneration, the weight of the regenerates was determined, PGP + nerve fibers were revealed by immunohistology, and subdivided into nerve fibers of sympathetic (TH + , NPY + ) or sensory (SP + , CGRP + ) origin. In addition, proliferating (Ki-67 proliferation antigen + ) and apoptotic cells (TUNEL technique + ) were likewise investigated. No innervation of splenic regenerates was observed after implantation into old hosts, correlating with poorly developed splenic compartments. In contrast, almost normal re-innervation occurred in young hosts after implantation of both young and old splenic tissue. These regenerates showed well-developed splenic compartments and a normal number and tissue distribution of proliferating and apoptotic cells. However, after the implantation of young tissue, the final size of splenic regenerates was three times larger (140±30 vs. 40±10 mg). Thus, re-innervation of splenic implants is necessary for their subsequent development. It is determined by host age, whereas the final size of the splenic regenerates is regulated by donor age-dependent factors. This model is useful for studying both the process leading to initial innervation and the consequences of this innervation.

Contact hypersensitivity responsiveness to dinitrofluorobenzene is depressed in mice that are sensitized through skin sites exposed to ultraviolet (UV) radiation. Local impairment of contact hypersensitivity by UV has been associated with a reduction in antigen-presenting cell activity within UV-irradiated skin sites marked by a decrease in the density of Ia-positive epidermal Langerhans cells. Our recent studies have demonstrated that neurogenic mediators (e.g. calcitonin gene-related peptide (CGRP) and nitric oxide (NO) contribute to cutaneous inflammation following exposure of rats to high-dose UV radiation. Since CGRP and NO inhibit antigen presentation by dendritic cells in vitro, we have investigated the possible involvement of CGRP and NO in local immunosuppression in UV-irradiated rodents. Hindpaw skin of Sprague-Dawley rats and back skin of UV-susceptible C57BL/6 mice was exposed to acute UV radiation (2.0 J/cm2 and 0.5 J/cm2, respectively). Alterations in cutaneous CGRP content were analyzed by a specific radioimmunoassay (RIA). In separate experiments, the CGRP receptor antagonist CGRP-(8-37) (10-5 M) and the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (2 X 10-5 M) were topically applied to UV-exposed skin before induction of contact hypersensitivity with dinitrofluorobenzene. Finally, we examined the effects of UV irradiation and epicutaneous application of CGRP on Ia-positive Langerhans cells by immunohistochemical analysis of epidermal sheets. It was found that UV exposure lead to a decrease in skin CGRP levels starting already 2 h after irradiation and reaching a minimum (less than 40% of non-irradiated control skin) at 6-12 h. Contact hypersensitivity reactions were significantly suppressed by UV radiation in rat skin (by 51%) and murine skin (by 80%). Topical administration of both CGRP-(8-37) and L-NAME before sensitization restored the capacity to respond to haptens applied to UV-exposed skin. Both UV exposure and topical CGRP reduced the density of Ia-positive epidermal cells. Our data indicate that CGRP may be released from sensory neurons following cutaneous UV irradiation and that CGRP and NO contribute of UV-induced local immunosuppression. Moreover, topical administration of CGRP or its antagonist may be able to modulate epidermal Langerhans cell activity in vivo.

The opioidergic innervation of the mammalian spleen and possible species differences were investigated. Light-microscopic immunohistochemistry revealed that splenic nerves of bovine and porcine spleen, but not of rat, mouse, hamster and guinea-pig spleen contained proenkephalin-derived opioidergic innervation. Immunoreactivity to both prodynorphin and pro-opiomelanocortin was absent from splenic nerves. In bovine and porcine spleen, fibers immunoreactive for met-enkephalin, met-enkephalin-Arg-Phe, met-enkephalin-Arg-Gly-Leu, leu-enkephalin and peptide F formed perivascular plexus, traveled in trabecular connective tissue, and extended into the capsule. Spatial relationships with immune cells were apparent in the white and red pulp, excluding lymphoid follicles. Colocalization of enkephalin immunoreactivity with immunoreactivities for tyrosin hydroxylase, dopamin-beta-hydroxylase, and neuropeptide Y, but not for substance P or calcitonin gene-related peptide were found. Our results provide evidence that opioid expression in splenic innervation is strongly species-dependent and exclusively proenkephalin-derived. Colocalization with marker enzymes of noradrenergic neurons indicates a mainly postganglionic sympathetic origin of proenkephalinergic splenic innervation. Opioidergic perivascular nerves probably control the splenic blood flow. A close interrelationship of opioidergic fibers with immune cells provides the anatomical basis for direct effects of neurally released opioids on splenic immune functions.

Light microscopic immunohistochemistry was employed to elucidate and compare the presence, distribution, and coexistence of various peptides, neuroendocrine markers and enzymes of the catecholamine pathway in nerves supplying lymphoid tissues in a variety of mammalian species. All lymphoid organs and tissues receive innervation by fibers containing dopamine-beta-hydroxylase and/or tyrosine hydroxylase, neural markers like protein gene product 9.5, synaptophysin and neurofilament and a varied spectrum of peptides. The prominent peptides were tachykinins (substance P, neurokinin A), calcitonin gene-related peptide (CGRP), neuropeptide Y (NPY), and vasoactive intestinal polypeptide/peptide histidine isoleucine (VIP/PHI). Opioid innervation was variable. Double immunofluorescence revealed coexistence of tachykinins and CGRP and of tyrosine hydroxylase and NPY. A minor proportion of fibers showed coexistence of NPY and tachykinins and of VIP/PHI and tachykinins. The possible importance of the complex peptidergic innervation of lymphoid tissues in inflammation, allergy, inflammatory pain and psycho-neuro-immuno-endocrine network function is discussed. A special immunomodulatory role of the sensory neurons is suggested.