Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic vasculitis, most frequently presenting as microscopic polyangiitis (MPA) or granulomatosis with polyangiitis (GPA). Pathogenic ANCAs trigger a deleterious immune response resulting in pauci-immune necrotizing and crescentic glomerulonephritis (GN). Standard therapeutical regimens include aggressive immunosuppressive therapy. Since some patients require renal replacement therapy (RRT) despite intensive immunosuppressive therapy, additional therapeutic plasma exchange (PEX) to deplete pathogenic ANCAs has been recommended but its value has recently been questioned. Because therapeutic decision making is crucial in these critically ill patients, we here aimed to identify inflammatory lesions in association with PEX consideration in a retrospective study from a single center tertiary hospital in a real-world population of 46 patients with severe AAV requiring intensive care treatment. The decision to consider PEX was more likely in patients with need for intensive care treatment and severe renal dysfunction. In contrast, short-term outcomes did not depend on clinical, or laboratory characteristics assessed at admission. Histopathological analysis confirmed active disease reflected by increased glomerular necrosis and crescents, but these histopathological findings did not associate with short-term outcome either. Interestingly, only increased global glomerular sclerosis in renal biopsies associated with a detrimental short-term outcome. In conclusion, our study investigated determinants for the consideration of therapeutic PEX in patients with severe AAV requiring intensive care treatment. This aspect underscores the need for renal biopsy and requires further investigation in a prospective controlled setting for therapeutic decision making especially in patients with severe AAV requiring intensive care treatment, especially important for treating intensivists. Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a systemic vasculitis, which most frequently presents as microscopic polyangiitis (MPA) or granulomatosis with polyangiitis (GPA) . Renal iA total number of 46 patients with biopsy-proven AAV at the University Medical Center Göttingen were retrospectively included between 2015 and 2020, the patient cohort has, in part, previously been described –15. Whil2 in the absence of hyperkalemia, heart failure, edema or uremic encephalopathy. Pulmonary hemorrhage was mild in all cases without requirement for mechanical ventilation.At admission, the Birmingham Vasculitis Activity Score (BVAS) version 3 was calculated as described previously . The BVATwo renal pathologists (PS and SH) independently evaluated kidney biopsies and were blinded to data analysis. Within a renal biopsy specimen, each glomerulus was scored separately for the presence of necrosis, crescents and global sclerosis. Consequently, the percentage of glomeruli with any of these features was calculated as a fraction of the total number of glomeruli in each renal biopsy. Apart from these categories, the degree of interstitial fibrosis/tubular atrophy (IF/TA) was quantified. Based on these scorings, histopathological subgrouping according to Berden et al.  and ARRS according to Brix et al.  were performed , 20. Ren2 every week, RTX was not administered within 48 h before PEX treatment. As per our practice, PEX treatment was scheduled at least 48 h after RTX administration to avoid interference with the rapid immunosuppressive effects of RTX on circulating CD19-positive/CD20-positive lymphocytes as described previously (2 RTX every week and two intravenous doses at 15 mg/kg CYC during the first and third RTX infusion. Prophylaxis to prevent pneumocystis (carinii) jiroveci infection was administered according to local practice.PEX was administered during the induction period at the discretion of the treating physicians. Glucocorticoids (GCs) were administered either as intravenous pulse therapy or orally with a tapering schedule. Choice of further remission induction therapy was dependent on previous regimens and individual patients with preference for cyclophosphamide (CYC) in patients with severe deterioration of kidney function, a higher likelihood to choose rituximab (RTX) in younger patients with toxicity of CYC being the main reason for this choice . RTX waseviously , 24. CYCeviously . CombinaVariables were tested for normal distribution using the Shapiro–Wilk test. Non-normally distributed continuous variables are expressed as median and interquartile range (IQR), categorical variables are presented as frequency and percentage. Statistical comparisons were not formally powered or prespecified. For group comparisons, the Mann–Whitney U-test was used to determine differences in medians. Non-parametric between-group-comparisons were performed with Pearson’s Chi-square test. Data analyses were performed with GraphPad Prism .At the discretion of the treating physicians, 18/46 (39.1%) patients with severe AAV received PEX Figure 1According to current recommendations, therapeutic PEX should be considered for AAV patients with severe deterioration of kidney function (serum creatine levels >500 µmol/L due to rapid-progressive GN in new onset or relapse of disease) and for the treatment of severe diffuse alveolar hemorrhage . It has The main limitations of our study are its retrospective design, different regimens of remission induction, the small patient number, influence of therapeutic PEX on histopathological findings because PEX treatment was initiated before renal biopsy in most cases, and limited data on long-term renal survival rates. Nevertheless, the number of critically ill patients with AAV at our center is considerable and our study identified determinants for the consideration of therapeutic PEX in patients with severe AAV requiring intensive care treatment despite the negative results of the PEXIVAS trial. It has to be kept in mind, however, that the patients included in the PEXIVAS trial differed from the real-world population regularly admitted to our ICU. This new aspect underscores the need for renal biopsy and requires further investigation in a prospective controlled setting for therapeutic decision making and short-term care especially in patients with severe AAV requiring intensive care treatment, especially important for treating intensivists , 30–32.Renal involvement is a common and severe complication of AAV as it can cause requirement of RRT, ESRD or death. This study identifies determinants for PEX consideration in patients with severe AAV requiring intensive care treatment. This aspect underscores the need for renal biopsy especially important in patients with severe AAV requiring intensive care treatment.The original contributions presented in the study are included in the article. Further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Institutional Review Board of the University Medical Center Göttingen, Germany (no. 22/2/14 and 28/9/17). The patients/participants provided their written informed consent to participate in this study.SH and BT conceived the study, collected and analyzed data and co-wrote the first draft. DT collected and analyzed data. PS and SH evaluated histopathological findings. PK analyzed data and edited the manuscript. SH and BT contributed equally as senior authors. All authors contributed to the article and approved the submitted version.BT was supported by the Research program, University Medical Center, University of Göttingen (1403720). The funding sources had no involvement in the design, collection, analysis, interpretation, writing or decision to submit the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


TRPS1 gene coding for the TRPS1 transcription factor. Considering Trps1 expression in odontoblasts, where Trps1 supports expression of multiple mineralization-related genes, we focused on determining the consequences of odontoblast-specific Trps1 deficiency on the quality of dental tissues. We generated a conditional Trps1Col1a1 knockout mouse, in which Trps1 is deleted in differentiated odontoblasts using 2.3kbCol1a1-CreERT2 driver. Mandibular first molars of 4wk old male and female mice were analyzed by micro-computed tomography (μCT) and histology. Mechanical properties of dentin and enamel were analyzed by Vickers microhardness test. The susceptibility to acid demineralization was compared between WT and Trps1Col1a1cKO molars using an ex vivo artificial caries procedure. μCT analyses demonstrated that odontoblast-specific deletion of Trps1 results in decreased dentin volume in male and female mice, while no significant differences were detected in dentin mineral density. However, histology revealed a wider predentin layer and the presence of globular dentin, which are indicative of disturbed mineralization. The secondary effect on enamel was also detected, with both dentin and enamel of Trps1Col1a1cKO mice being more susceptible to demineralization than WT tissues. The quality of dental tissues was particularly impaired in molar pits, which are sites highly susceptible to dental caries in human teeth. Interestingly, Trps1Col1a1cKO males demonstrated a stronger phenotype than females, which calls for attention to genetically-driven sex differences in predisposition to dental caries. In conclusion, the analyses of Trps1Col1a1cKO mice suggest that compromised quality of dental tissues contributes to the high prevalence of dental caries in TRPS patients. Furthermore, our results suggest that TRPS patients will benefit particularly from improved dental caries prevention strategies tailored for individuals genetically predisposed due to developmental defects in tooth mineralization.Dental caries is the most common chronic disease in children and adults worldwide. The complex etiology of dental caries includes environmental factors as well as host genetics, which together contribute to inter-individual variation in susceptibility. The goal of this study was to provide insights into the molecular pathology underlying increased predisposition to dental caries in trichorhinophalangeal syndrome (TRPS). This rare inherited skeletal dysplasia is caused by mutations in the  Dental caries remains the most prevalent chronic disease affecting 60–90% of children and adolescents (5–17 years of age), and nearly every adult among all populations . This coMany environmental, endogenous, and behavioral risk factors have been identified as contributors to the development of dental caries , 11, 12.2+) and phosphate (Pi) ions, pH, and the presence of mineralization inhibitors  mice , post-translational modifications of ECM proteins, availability of calcium  is caused by missense mutations located exclusively in the exon 6 and 7 of TRPS1 gene, resulting in functional modification of the DNA binding domain of the TRPS1 transcription factor  literature encompass defects in tooth number, size, shape, and mineralization. The most common TRPS dental abnormalities include supernumerarity, microdontia, malocclusion, and delay in root and crown development. Cases of hypodontia, abnormal tooth morphogenesis, impaired dentin mineralization, large dental pulp chambers, and extensive dental caries have also been reported, underscoring the clinical importance of elopment , 44, 45.Trps1 expression during mouse tooth organ development, demonstrated that Trps1 is highly and specifically expressed in the dental mesenchyme , harbors an allele with the in-frame deletion of the exon coding for the GATA-type DNA binding domain of Trps1  mice were used. To generate Trps1 cKO mice, Trps1 cKO (Trps1fl) allele was generated by inserting two LoxP sites flanking the first coding exon of Trps1 by homologous recombination  flanked by FRT sites for a positive selection of recombinant embryonic stem cells (ES). Following the Trps1 cKO construct injection into C57BL/6 ES cells, neomycin selection was performed, and the resistant ES clones were screened to verify the recombination. Mice carrying the recombinant allele were subsequently obtained via the generation of germline chimeras. The Trps1fl allele was generated after breeding with germline deleter Flp mice (r cassette. Trps1Col1a1cKO mice were generated by breeding Trps1fl mice with 2.3kbCol1a1-CreERT2 mice  expressing Cre recombinase under the control of the 2.3-kb fragment of Col1a1 promoter  mice via intraperitoneal injection (0.1 mg/g body weight) at postnatal days (P)1, P2, P9, P16 and P23 to assure efficient deletion of Trps1 in odontoblasts and as a control in WT mice , Col1a1-Cre  , which rpromoter  54). Th. Th2.3kbpromoter , 56. To  WT mice . TamoxifAATGCAG) . The micAATGCAG) .2 inhalation. All analyses were performed on tissues collected post-mortem.All animal studies were conducted in accordance with a protocol approved by the University of Pittsburgh Institutional Animal Care and Use Committee (IACUC protocol # 19095648), complying with the Federal Animal Welfare Act and all NIH policies regarding vertebrate animals in research. Mice were euthanized by COTrps1Col1a1cKO male and female mice (N = 3/genotype/sex) were dissected under a stereo microscope (Leica S9D) to remove soft tissues, and fixed with 10% formalin  overnight. Samples were decalcified in 10% ethylenediaminetetraacetic acid (EDTA) solution (pH 7.4) for 14 days prior to paraffin embedding. Serial 7 μm sagittal sections were placed on Fisherbrand™ Superfrost™ Plus microscope slides and deparaffinized for hematoxylin and eosin (H&E) staining following standard protocols. Hemimandibles of P7 and 4 wk old 2.3kbCol1a1-CreERT2;mTmG reporter mice were harvested and processed as described above. Decalcified samples were cryoprotected in 30% sucrose/PBS overnight at 4 °C, embedded in an OCT compound and stored at−80°C until sectioned. Samples were cryosectioned at 7 μm, protected from the light. To validate Cre recombinase activation in odontoblasts of mandibular first molars of reporter mice, cryosections were counterstained with DAPI and mounted with immu-mount  for microscopic analyses of GFP and RFP signals.Hemimandibles of 4 wk old WT and ® IndentaMet™1100 Series microindentation hardness tester adapted to a uEye camera and a Buehler Omnimet MHT software.Whole teeth images were captured on a Leica M165FC dissecting microscope using a DFC 450 camera and Leica LAS software. Histological images were captured on a Zeiss AXIO microscope with an AxioCam MRc 35 camera and Zen software. Microindentation images were captured using a BUEHLERN = 5/genotype/sex) were imaged in 70% ethanol by the Scanco μCT 50  system. The following parameters were set for the scans: 6-μm voxel size, 55 KVp, 0.36 degrees rotation step (180 degrees angular range) and a 1,500 ms exposure per view. After 3D reconstruction, volumes were segmented using a global threshold of 0.6 g HA/cc. Mineral density (TMD), thickness (Th), and volumes (BV) were measured for enamel and dentin separately. Additionally, dentin and enamel tissue fraction (BV/TV) in the total tooth crown volume (TV) was calculated as described before  and d2 the area of the indentation (measured in mm2)  and polished as previously described . Mechani in mm2) . MicrohaTrps1Col1a1cKO mice (N=5/genotype/sex) were analyzed using the protocol described by Vieira et al., .Experiments performed in this study used five mice per genotype per sex or otherwise stated. Males and females were analyzed separately. Values are expressed as mean ± standard deviation (SD). Statistically significant differences were determined using the Student’s Trps1 deficiency . Trps1 knockout was initiated with tamoxifen injections at postnatal day 1 (P1) and P2, followed by 3 more injections 7 days apart .–65.Trps1ter mice ,E. Fluorpromoter . These rpromoter ,E.Trps1Col1a1cKO mice demonstrated that Trps1Col1a1cKO males are significantly smaller than WT littermates at P7, P21 and 4 wks of age. This difference was not found in females, suggesting that Trps1 deficiency has stronger effect in males than in females  enamel and dentin  specifically in the circumpulpal dentin, pit dentin and outer enamel in Trps1Col1a1cKO males ; behavioral features ; socioeconomic and demographic factors ; environmental exposures ; along with host-genetics , 18, 69.Trps1Col1a1cKO molars is thinner than in WT mice. This deficiency was particularly prominent in pits of the tooth crown, which are the sites most susceptible to dental caries initiation in humans. Teeth with thinner dentin may be prone to more severe caries, as once a lesion is initiated, it can reach the pulp faster than in teeth with a thicker dentin layer. On radiographic images of teeth, features such as enlarged pulp chambers and prominent pulp horns suggest a thinner dentin layer. This was visible on 2D μCT images of Trps1Col1a1cKO molars, and importantly, such features were recently reported in permanent molars of a 16-year-old male patient with TRPS1 mutation  of both Trps1Col1a1cKO males and females is significantly softer, than in the WT mice; while the inner enamel (IE) hardness of Trps1Col1a1cKO females becomes significantly lower after the artificial caries procedure as compared to WT. Notably, the detection of localized enamel mineralization defects in pits contributes to our understanding of dental caries lesion distribution pattern in TRPS. We agree with others, that the effects of dental caries risk factors may be surface-specific , as shown in our previous in vitro studies , present with a stronger dental phenotype compared to females.Interestingly,  rickets , 84, 85.estrogen –88. The androgen , 89, 90.androgen . The repale mice . This seTrps1Col1a1cKO mice relevant to the predisposition to dental caries is malocclusion. Dental caries is a common complication of malocclusion (Trps1Col1a1cKO males and females. This and severe root exposure detected in Trps1Col1a1cKO mice suggest impaired formation of the alveolar bone, which is caused most likely by deficiency of Trps1 in osteoblasts, as osteoblasts are another cell type expressing Cre from the 2.3kbCol1a1 promoter (Trps1 not only in formation of sound mineralized dental tissues, but also in the dento-alveolar complex.Among other dental findings in cclusion . This abpromoter . Notablypromoter , underliTrps1Col1a1cKO mice suggests that TRPS patients are genetically predisposed to dental caries. Hence, TRPS patients may benefit from more assertive prevention strategies and early interventions to mitigate dental caries risk and improve oral health.In summary, the compromised quality of the tooth mineralized tissues, expressed as softer and less acid-resistant enamel and dentin, together with decreased dentin layer, tooth misalignment, localized tooth mineralization defects in occlusal and buccal pits detected in Supplementary Materials


CNNM2 is primarily expressed in the brain and distal convoluted tubule (DCT) of the kidney. Mutations in CNNM2 have been reported to cause hypomagnesemia, seizure, and intellectual disability (HSMR) syndrome. However, the clinical and functional effect of CNNM2 mutations remains incompletely understood. We report our clinical encounter with a 1-year-old infant with HSMR features. Mutation screening for this trio family was performed using next-generation sequencing (NGS)-based whole exome sequencing (WES) with the identified mutation verified by Sanger sequencing. We identified a de novo heterozygous mutation c.G1439T (R480L) in the essential cystathionine β-synthase (CBS) domain of CNNM2 encoding CNNM2 (cyclin M2) without any other gene mutations related to hypomagnesemia. The amino acid involved in this missense mutation was conserved in different species. It was also found to be pathogenic based on the different software prediction models and ACGME criteria. In vitro studies revealed a higher expression of the CNNM2-R480L mutant protein compared to that of the wild-type CNNM2. Like the CNNM2-wild type, proper localization of CNNM2-R480L was shown on immunocytochemistry images. The Mg2+ efflux assay in murine DCT (mDCT) cells revealed a significant increase in intracellular Mg2+ green in CNNM2-R480L compared to that in CNNM2-WT. By using a simulation model, we illustrate that the R480L mutation impaired the interaction between CNNM2 and ATP-Mg2+. We propose that this novel R480L mutation in the CNNM2 gene led to impaired binding between Mg2+-ATP and CNNM2 and diminished Mg2+ efflux, manifesting clinically as refractory hypomagnesemia. Defective Mg2+ reabsorption in the DCT inevitably causes renal hypomagnesemia because there is no Mg2+ reabsorption in the downstream DCT. Gene mutations related to the regulation of Mg2+ transport in the DCT can cause renal hypomagnesemia. These genes include SLC12A3 encoding thiazide-sensitive NCC, TRMP6 encoding apical TRPM6 channel, HNF1B encoding HNF1β, PCBD1 encoding PCBD1, EGF encoding EGF, EGFR encoding EGFR, KCNJ10 encoding Kir4.1 (EAST syndrome), KCNA1 encoding Kv1.1, FXYD2 encoding γ-subunit of Na+-K+ ATPase, and CNNM2 encoding CNNM2 (cyclin M2) (Magnesium (Mgfunction . In the in TALH) . Like thclin M2) .2+-ATP, and cytosolic cyclic-nucleotide-binding homology domain (CNBH). To date, only a few families with CNNM2 mutations have been reported  or recessive (minority) renal hypomagnesemia with seizure and intellectual disability (HSMR) . In humareported . AlthougMg2+-ATP . However2+ wasting, seizure, and intellectual disability, consistent with the clinical description of HSMR syndrome. In this study, we aimed to identify the genetic mutation for her phenotype and to assess the functional impact of the identified mutation. Our results indicated that a de novo heterozygous mutation c.G1439T (R480L) in the CBS domain of the CNNM2 gene was identified in this proband. This missense CNNM2 R480L mutation was found to be pathogenic. In vitro studies revealed higher CNNM2-R480L protein expression with proper cellular localization on immunocytochemistry images. The impaired Mg2+ efflux with a significant increase in intracellular Mg2+ suggests that the CNNM2-R480L mutation blocks Mg2+ efflux. In our simulation model, this R480L mutation leads to an attenuated interaction between CNNM2 and ATP-Mg2+.We have encountered an infant with refractory hypomagnesemia and excessive renal MgThis study was approved by the ethics committee on human studies at Tri-Service General Hospital in Taiwan (IRB2-105-05-136). A trio family including the proband and her parents were enrolled. Written informed consent was obtained from the participants.2+ excretion (FEMg 6.5%), normokalemia, normocalcemia, and normocalciuria  with increased urine Mgalciuria . There whttp://www.ncbi.nlm.nih.gov/projects/SNP/), HapMap, the 1000 Genomes Project (http://www.1000genomes.org), Exome Aggregation Consortium (ExAC) database, and the Genome Aggregation Database . Direct Sanger sequencing was performed for all patients and their parents to verify the genetic variants detected by WES. The data that support the findings of this study are available from the corresponding author upon reasonable request.Genomic DNA was isolated from a peripheral venous blood sample. We performed exome capture using the Agilent Sure Select v6 and massively parallel sequencing using the HiSeq 4000 platform as previously reported . Raw ima2 incubator. Human CNNM2 cDNA (NM_017649.5) was cloned into the pcDNA3.1 vector. The disease-causing mutation was obtained by a QuikChangeTM Site-Directed Mutagenesis Kit . The primers used to introduce the mutations were R480L: forward primer 5′-GAGCGGCTACACCCTCATTCCAGTGTTTG-3′; reverse primer 5′-CAAACACTGGAATGAGGGTGTAGCCGCTC-3′, R480K: forward primer 5′-GGAGAGCGGCTACACCAAGATTCCAGTGTTTGAAGG-3′; reverse primer 5′-CCTTCAAACACTGGAATCTTGGTGTAGCCGCTCTCC-3′, V548M: forward primer 5′CTCACCTGGCTATCATGCAGCGGGTAAACAATG-3′; reverse primer 5′-CATTGTTTACCCGCTGCATGATAGCCAGGTGAG-3′, T568I: forward primer 5′-GAAGTTCTGGGAATCATCTTAGAAGATGTGATTG-3′; reverse primer 5′-CAATCACATCTTCTAAGATGACGATTCCCAGAACTTC-3′. The mDCT cells seeded in a six-well plate with 70–90% confluence were transfected by the indicated amount of plasmid DNA with a Lipofectamine 3000 Reagent (Thermo Fisher Scientific). The reported R480K, V548M, and T568I constructs were selected as negative controls. R480K constructed in CBS1 was selected as a charge-unchanged control. Two previously reported mutant constructs (V548M and T568I) in CBS2 were also performed.Murine distal convoluted tubular (mDCT) cells were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium with 1 g/L glucose, 1 mM sodium pyruvate, and Ham’s F-12 Nutrient Mix. Finally, 5% (v/v) fetal bovine serum,100 U/ml penicillin, and 0.1 mg/ml streptomycin were added to the growth medium. The cells were incubated at 37°C in a humidified 5% CO+-K+ ATPase .The mDCT cells were harvested for 24 h after transfection. The cell lysates were prepared in a RIPA lysis buffer with a protease inhibitor cocktail (Roche). Following the separation by centrifugation, the remaining protein lysates were denatured in an SDS sample reagent with 100 mM DTT for 30 min at 37°C and then analyzed by polyacrylamide-SDS mini-gels. The mDCT cells were transfected with a pcDN3.1 empty vector or disease-causing mutation and harvested after 24 h. The cell membrane fraction was subjected to the ProteoExtract native membrane protein extraction kit (Merck-Millipore), following the manufacturer’s description, and then analyzed by semiquantitative immunoblotting . The immThe mDCT cells were seeded on a chamber slide (Millicell EZ slide) and transiently transfected with 0.5 μg of plasmid DNA. After 24 h , the cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min. After PBS rinses, the cells were incubated for 1 h with 0.1 Triton X100 in PBS and then blocked with 1% BSA in PBST for another 30 min. Specific antibody CNNM2 (X200 Cusabio) was used for cell staining. After PBST rinses, cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit (X200 Invitrogen) for 1 h and stained with DAPI (5 ug/ml) for 5 min. The images were captured with a Leica DM2500 microscope.2+ imaging of transfected cells was analyzed with Magnesium Green™ (Molecular Probes) as described previously (2+ loading buffer including 2 μM Magnesium Green (Molecular Probes) at 37°C for 60 min. Then, the buffer was changed to buffer without Mg2+ (MgCl2 was replaced with 60 mM NaCl) and the fluorescent was recorded at 1-min intervals. The cells images were detected by ImageXpress Micro XLS (molecular devices) and fluorescence was measured using MetaXpress High content image acquisition and analysis software (Molecular Devices). The cell fluorescence was analyzed by the software setting for Cell Scoring.The mDCT cells were cultured on a 96-black, clear bottom tissue culture plate (Corning), after transfection (24 h). The Mgeviously , with slThe resolved structures of the human CNNM-PRL complex (PDB code: 5LXQ)  were uset-test was used to compare differences between groups. When comparing the ratio of differences between groups, we used a ratio paired t-test with the Holm–Šídák method. A p-value less than 0.05 was considered statistically significant.The results were presented as mean ± standard deviation (SD) for continuous variables. Student’s de novo heterozygous mutation in the CNNM2 gene  located on the cystathionine-β-synthase (CBS) domain of CNNM2. Sanger sequencing for the patient and her parents confirmed this NM2 gene . R480 isThe total and membrane expressions of wild-type CNNM2 (amino acids 1–875) and mutant CNNM2  proteins were examined after transient transfection of CNNM2 in mDCT cell lines. As shown in The immunocytochemistry images of mDCT cells with anti-nuclei (blue) and anti-CNNM2 (green) demonstrated that CNNM2-wild type, CNNM2-R480L, and other negative controls (CNNM2-V548M and CNNM2-T568I) were properly localized adjacent to the cell membrane . Of note2+ efflux in a cellular assay with wild-type and mutant CNNM2. The mDCT cells were transfected with the indicated constructs treated with Mg2+ Green and then subjected to Mg2+ depletion. As shown in 2+ Green was significantly higher in CNNM2-R480L compared to wild type and other CNNM2 mutants, with p-values < 0.05 from min 1 to min 5. This finding indicates that the R480L mutation located in the CBS1 domain of CNNM2 blocks Mg2+ efflux activity.To evaluate the impact of R480L on the function of CNNM2, we examined the CNNM2-dependent Mg2+-ATP in the CBS module of CNNM2 by approximately 352 kcal/mol, which suggested that this mutation may cause significant impairment in the binding ability with Mg2+-ATP.The interaction from the side chain of Arg480 with the γ-phosphate of ATP was lost after being replaced by leucine . The mutde novo heterozygous mutation c.G1439T (R480L) in the CBS domain of the CNNM2 gene in a trio family with typical HSMR. In vitro studies showed that this CNNM2-R480L had a higher expression level than the CNNM2-wild type and proper localization to the plasma membrane. The Mg2+ efflux assay in mDCT cells revealed the blockade of intracellular Mg2+ efflux under Mg2+ depletion. The simulation model also predicted the attenuated interaction of this mutant protein with Mg2+-ATP.In this study, we have identified a novel and CNNM2 have been reported responsible for HSMR  Epilepairments . Althougairments .in vitro studies for this R480L mutant. A higher CNNM2-R480L protein expression with proper cellular localization on immunocytochemistry images was found. In contrast to the previous findings, the membranous expressions of mutant CNNM2-R480L were higher than those of the CNNM2-wild type , and a large cytosolic region including a cystathionine-β-synthase (CBS) domain and a putative cyclic nucleotide-binding homology (CNBH) domain . The mutrization . In linerization .2+ efflux assay to evaluate the effect of R480L on CNNM2 transport activity. Consistent with previous studies, R480L impaired the Mg2+ efflux activity in the presence of significantly high intracellular Mg2+ under Mg2+ depletion. It has been shown that Mg2+-ATP binding is required for cellular Mg2+ efflux since mutations that abolished Mg2+-ATP binding prevent Mg2+ efflux (2+-ATP in the CBS1 of CNNM2 by R480L mutation. However, we did not find a significant impairment of Mg2+ efflux in CNNM2 T568I in CBS2, unlike a previous report (2+-green by calculating the mean fluorescence of all cells over 300 gray levels. Hirata et al. presented the relative intensity as the mean fluorescence of 10 cells. In fact, we found that the relative intensity of CNNM2 T568I on the Mg2+ efflux assay was higher than the wild type, although the difference was not statistically significant. Altogether, our study demonstrated that this R480L mutation resulted in the diminished binding with Mg2+-ATP and consequently led to impairment of Mg2+ efflux. This defective Mg2+ efflux might account for the hypomagnesemia in this patient with CNNM2 R480L mutation.We conducted a cellular Mg+ efflux . Of notes report . This dis report . Second,2+-ATP and the CBS-pair. However, the simulation model showed the reduction of the binding energy of ATP-Mg2+ in the CBS module of CNNM2 by R480L mutation.There were a few limitations in our study. First, we did not provide direct evidence for the R480L mutation in causing impairment of CBS dimerization by the crystal structure. Second, isothermal titration calorimetry was not performed to confirm the diminishment of the interaction of Mgde novo heterozygous R480L mutation in the CBS domain of the CNNM2 gene in a trio family with severe HSMR. This highly expressed CNNM2-R480L properly localizes to the plasma membrane but impairs Mg2+ efflux likely through the attenuated interaction with Mg2+-ATP, resulting in the clinical manifestation of refractory hypomagnesemia.We identified a novel and


The most common concomitant PCHS combination was Kathon CG® + MI. Most patients (32.4%) belonged to the age group of 21–30, and skin symptoms affected mostly the limbs and face. The most common other concomitant allergens were nickel, lanolin alcohol and balsam of Peru. Preservatives  are important contact allergens in adult AD, mostly among young women. The rate of AD in the PCHS group and the rate of PCHS in the AD group is remarkable; thus, the role of PCHS should be highlighted in the topical therapy and in the prevention of possible AD exacerbations.Atopic dermatitis (AD) is a chronic inflammatory disease characterised by an impaired skin barrier. The prolonged use of topical preparations containing medications, emollients, fragrances and preservatives may increase the risk of contact hypersensitivity (CHS). In the Allergy Outpatient Unit of the Department of Dermatology, Venereology and Dermatooncology of Semmelweis University, 5790 adult patients were patch tested between 2007–2021 with the European Environmental Baseline Series according to international standards. Among all the tested adult patients, 723 had preservative CHS (PCHS) and 639 had AD. Among the 723 PCHS patients, 68 (9.4%) had AD; the female to male ratio was 3:1 in this group. Out of 639 AD patients, 68 had PCHS (10.6%). In the AD-PCHS group, 83.8% had CHS to methylisothiazolinone (MI) (tested from 2014), 36.8% to Kathon CG Atopic dermatitis (AD) is a common, relapsing, chronic inflammatory skin disease with scaly, pruritic, erythematous skin symptoms. It is characterised by skin barrier impairment in both lesional and non-lesional skin regions ,2,3,4.Patients with AD are treated locally, mostly with emollients, moisturisers, topical corticosteroids or calcineurin inhibitors. Several factors can modify the effectiveness of the therapy: stress, infections, lack of compliance and contact allergen exposures may trigger exacerbations. Because of the damaged skin barrier  and the long-term local therapy, there can be an increased risk of developing a contact hypersensitivity in AD patients ,5,6,7,8.Plenty of cosmetical and dermatological products contain not only ingredients which are helpful in care and treating AD, but fragrances and preservatives as well. An observation of a higher risk of CHS to preservatives among AD patients has been published, but the number of publications regarding preservative CHS and adult AD is very limited ,9,10,11.n = 68) were analysed.During our examination (2007–2021), 5790 consecutive adult (≥18 years) patients were patch tested with the European Environmental Baseline Series (EEBS). Out of them, 723 adult patients had PCHS and 639 had AD. Among them, data of adult PCHS AD patients (® (methylchloroisothiazolinone/methylisothiazolinone [MCI/M] 3:1) and methylisothiazolinone (MI) were used in the aqueous phase. Allergens of the EEBS are regularly revised and new ones can be included in the series [Patch testing took place in the Allergy Outpatient Unit and Laboratory of the Department of Dermatology, Venereology and Dermatooncology of Semmelweis University between 2007–2021 on adult AD patients. The EEBS produced by Brial allergEAZE GmbH  were used . Most ofe series ,17,18,19®, MI, formaldehyde, Quaternium-15, para-tert-butylphenol formaldehyde resin , methyldibromo-glutaronitrile (MDBGN).Data of adult AD patients who had positive patch test reaction to at least one of the following seven EEBS preservative allergens were evaluated: paraben, Kathon CGn = 5790), 639 patients (11.03%) had AD.Out of the total tested adult patient population  adult AD patients who had at least one CHS in the EEBS, and 68 (17.4%) had at least one positivity to preservatives.Among the tested 639 adult AD patients, 68 had at least one positivity to preservatives (10.6%) .From the 5790 tested patients, 723 (12.5%) had CHS to at least one preservative. Out of them, 68 had AD (9.4% of the preservative CHS population) .Gender distribution:In this study, 75.0% of patients were female and 25.0% were male. The female to male ratio was 3:1.2.PCHS and polysensitivity:n = 37), 36.8% Kathon CG®, 16.2% MDBGN, 11.8% paraben, 7.4% formaldehyde, 4.4% PTBP-formaldehyde resin and 1.5% Quaternium-15 hypersensitive , whereas 17.65% had two PCHSs (most common combination: Kathon CG® + MI) and 2.94% had three PCHSs (Kathon CG® + MI + MDBGN/paraben)  A,B.3.Age distribution:®, MI and PTBP formaldehyde resin in the group of 61–70. In the age groups of 51–60 and after 71, PCHS was not typical  were mostly affected by MI and MDBGN hypersensitive patients. Widespread skin symptoms were typical in MDBGN, MI and Kathon CG® CHS patients ), lanolin alcohol (13 p), balsam of Peru (11 p), propylenglycol and thiomersal (10 p), wood tar (9 p), fragrance mix II and thiuram mix (7 p) and mercury-chloride, cobalt and fragrance mix I (6 p) .AD is a chronic inflammatory skin disease which affects about 1–10% of the adults and about 15–20% of the children worldwide [AD has a multifactorial background. Skin barrier dysfunction, immune system dysregulation, the disbalance of the skin bacterial microbiome and genetic factors are also included in the complex pathogenesis. Endogenous and exogenous components can also modify the prognosis of the disease. One of the most remarkable predisposing factors is a family history of atopic diseases. Patients with mutations or impaired expression of the filaggrin gene were also reported, which contribute to the skin barrier. The lipid metabolism with decreased ceramide production is also damaged. Trans-epidermal water loss was increased. All these factors weaken the proper skin barrier functions leading to inflammation of the skin. In this process, initially the type-2 T-helper cells (Th2)  are crucial, with a subsequent Th2-Th1 cell switch in the chronic phase ,21,22,23Not only are the background and the provoking factors of AD rather complex, but also the clinical characteristic shows a dynamically changing tendency over time. In the past, it was believed that AD begins always at a young age (infancy or childhood) and then the skin symptoms disappear with time. According to the recent concepts, AD is considered to be a life-long condition even if the patient has no actual active, inflamed skin lesions at all. It is reported that about 60–80% of the AD population has a very early-onset type of AD. Among them, it is estimated that about 60% of patients have a complete remission before two years of age. The other group of these patients and those who develop AD between 2 and 6 years of age have a higher risk to have chronic and persistent AD. Data of adolescent-onset AD are limited. Most adult AD patients have flare ups after a long symptom-free period or have had persistent AD since childhood. About 2% of AD patients have real adult-onset AD, and are mostly women. The number of elderly patients with active inflammatory AD symptoms is low, although dry and sensitive skin will stay lifelong. A real elderly-onset type of AD is very unusual but possible ,5,6.Although the initial onset of the disease can be different among AD patients, the clinical picture of AD is well characterised according to the actual age of the patients. Regarding the connection between age and skin symptom features, infantile, childhood, adolescent/adult and elderly AD types can be defined. The first lesions of infantile AD usually appear some months after birth and are characterised by the acute, exudative skin lesions. AD most commonly affects the scalp and facial regions of the newborns and oozing and crusting are quite typical. The extensor surfaces of the limbs are also predilectional parts for AD in the infants. The childhood AD has a clinical feature of acute and chronic lesions as well. Xerosis and lichenification may also appear. Eczematous skin symptoms usually occur in the bends and in the perioral and periorbital regions. Involvement of the hands and the wrists should also be mentioned. Adolescents and adults have AD skin symptoms often on the head-neck region and the flexural localisations and hands are the most typical places for the clinical distribution of the skin symptoms. Cosmetics and inadequate skin care may exacerbate the skin condition. Hand eczema is a leading problem for adult AD patients and is a great burden on the quality of life. Irritants and environmental contact allergens are typical provoking factors of it. After 60 years of age, widespread AD skin symptoms are uncommon but possible, and even erythroderma may occur. Certain other dermatologic diseases need to be excluded in order to diagnose AD properly in adults  ,5,6.Staphylococcus aureus, Malassezia and Trichophyton species) and pollutants can be mentioned. Studies on the adult AD population attribute a significant role to different kinds of contact and aeroallergens as important provoking factors underlying a sudden flare up of the symptoms or therapy resistance [Having vulnerable skin to the endogenous factors mentioned before, certain exogenous factors may trigger an exacerbation of AD. Among environmental exposure irritants, aero/food/contact allergens, stress, certain microbes  is a cell-mediated, delayed, type IV hypersensitivity reaction. During the sensitization phase, the person comes into contact with the allergen for the first time. Allergens are low-molecular-weight substances connected to a larger carrier (haptens). Haptens are engulfed by antigen-presenting cells migrating to the local lymph nodes where the activation and proliferation of naive T-cells begins. The formerly naive T-cells become allergen-specific T-cells which have a key role during the elicitation phase, since re-exposure of the allergen activates them, provoking inflammation and the clinical picture of ACD. Prevalence of CHS is reported to be up to 20%, and the incidence of ACD seems to be rising. In Europe, 27% of the general population has at least one CHS ,20,24,25Regarding human data, compared to control skin, the absorption through AD skin is increased not only in case of lesional, but non-lesional skin as well. However, there are regional differences in the increased skin absorption. Forehead and genital skin is reported to be more vulnerable than forearm skin, for example. The severity of the AD and the presence of the filaggrin mutation are also factors which should be kept in mind when discussing skin absorption. The more severe and widespread the AD is, the more increased the skin absorption will be. The filaggrin mutation contributes to the higher risk of having a skin barrier impairment in non-lesional skin, too. In conclusion, according to the literature, patients with AD have nearly a twofold-increase in skin absorption of different chemicals, including irritants and contact allergens as well. The most common contact allergens in AD are metals, fragrances, emollients, vehicles, dyes, antibiotics, topical antiseptics and preservatives ,28,29,30The number of allergic contact reactions to different cosmetic products is reported to be increasing. The preservatives are the most common cosmetic contact allergens after fragrances, but emulsifiers, vehicle components, sunscreen agents and nail resins can also provoke CH. In the general European population, 6.2% have PCHS. This fact highlights the importance of these allergens, since AD patients regularly use numerous personal care products besides topical medications. These products may contain contact allergens  ,20,25,30®, MI, formaldehyde, Quaternium-15, PTBP-formaldehyde-resin and MDBGN [Cosmetics, hygiene products and local therapeutics with high water content need chemical preservation. Seven of the most common preservatives are a part of the EEBS: paraben, Kathon CGnd MDBGN ,16,19,30Parabens are one of the most commonly used preservatives around the world. Contact allergy is reported to parabens from 1940. Nearly 35 variants of parabens are known, but methyl-, ethyl-, propyl-, and butylparabens became the most widely applicated ones. Parabens have an antimicrobial spectrum covering gram-positive bacteria and fungi. The different variants are often combined with each other or with other preservatives as well. Foods, medications and cosmetics also contain this substance. Paraben is a rather common cosmetic ingredient in skin care products  and can also be found in makeup products, powders, foundations, eye contour pencils, mascaras, lipsticks, lip glosses, hair dyes, nail cosmetics, toothpastes and mouthwashes ,31.® is the 3:1 mixture of methylchloroisothiazolinone (MCI) and methylisothiazolinone (MI). It has become a quite popular preservative because of its potent antimicrobial effects which covers gram-positive bacteria, gram-negative bacteria, yeasts and moulds. Despite its advantages as a broad-spectrum antimicrobial agent, its contact sensibilisation-provoking effect was also published. Kathon CG® was introduced in the early 1980s as an industrial and household product and cosmetic preservative. However, an increasing tendency in cases of CHS rates was reported, and the first cosmetic-related Kathon CG® contact dermatitis was reported in 1985. MCI and MI were also published to be provoking factors of allergic contact dermatitis in humans, and animal studies also showed that mostly MCI was the main sensitiser out of the mixture. A large variety of cosmetic formulations contain this chemical. It is common in intimate hygiene cosmetics, hair care products and facial cleansers. Shower gels, shampoos, makeup products, moisturisers, body lotions, creams and hair cosmetics are also sources of Kathon CG® exposure [Kathon CGexposure ,16,30,32®. It was believed to be a less potent sensitising contact allergen than MCI. In the 2000s it was allowed to be an individual preservative; firstly, in industrial products, and later in household products and cosmetics. MI CHS was reported increasingly, and occupational cases and cosmetic-related studies were also published. In cases of occupational sources of exposures, cutting oils, glues, inks, paints and lacquers are remarkable. Among household products, glass cleaners, wood cleaners, laundry detergents, dishwashing liquids and fabric softeners are common products containing MI. MI is an important preservative in personal care products , hair cosmetics , soaps, deodorants, make-up products  nail cosmetics, aftershaves, moisturisers, self-tanning products, sunscreens and intimate hygiene products [MI was previously used for preservation only as a component of Kathon CGproducts ,16,30,33Formaldehyde is a gas, which is called formalin when it is an aqueous solution. It has a biocide, preservative and denaturant function in cosmetics. The aqueous formaldehyde solutions are known irritants and their CHS provoking effect has already been published in occupational and non-occupational cases as well. Cosmetic exposures of formaldehyde include shampoos, hair conditioners, hair dye products, soaps, detergents, bath oils, bath salts, personal care products, shaving creams, moisturisers, face masks, face wraps and nail cosmetics. In Europe, different kinds of regulations are present regarding the concentrations of formaldehyde. It is permitted for usage as a preservative at a concentration of 0.2% in cosmetics and 0.1% in oral hygiene products, and products must be labelled as “contains formaldehyde” if they contain more than 0.05% of this chemical ,34,35.Quaternium-15 is a formaldehyde-releaser preservative, which was first introduced to be a part of the EEBS in 1984. It is a potent antimicrobial agent, which is effective even at low concentrations. The sources of Quaternium-15 exposure are quite variable, since non-cosmetic and cosmetic products can contain it as well. As non-cosmetic products, detergents, polishes, inks, paints, textile finishing products, joint cements and metalworking fluids can be mentioned. Cosmetic sources of exposure are baby shampoos, body lotions, soaps, detergents, bath salts, eyeliners, eyeshadows, eyeshadow removers, perfumes, hair conditioners, shampoos, hair sprays, hair dyes, face powders, lipsticks, primers, nail cosmetics, deodorants, face creams, body lotions, face wraps and self-tanners ,36,37.The PTBP-formaldehyde resin is also a formaldehyde-releaser preservative. Contact allergies provoked by this chemical have been known for decades. The first case of contact dermatitis to PTBP-formaldehyde resin, published in the late 1950s, was caused by a shoe glue. Non-occupational and occupational sources of exposures can be mentioned. Occupational CHS is reported to be less frequent. Workers in the car industry and shoe manufacturing are affected. Most cases of CHS to PBTP-formaldehyde resin are non-occupational. Mainly domestic glues, amputation prostheses, leather watch straps and neoprene orthopaedic knee braces belong to this group. However, PTBP-formaldehyde resin as a preservative can also be found in nail cosmetics and deodorants ,39.Methyldibromo-glutaronitrile is a preservative and known contact allergen with mainly cosmetic sources of exposure. Although MDBGN was banned in Europe, not only from leave-on products in 2005 but also from rinse-off products, CHS reported about this preservative is still present nowadays. Among cosmetics, MDBGN is used for preservation in shampoos, soaps, cleansers, body lotions, make-up products and make-up-removing wet wipes. ,40,41.Due to the long-term usage of a large number of topical preparations (cosmetics and medications), AD patients are reported to be more likely to develop CHS not only to fragrances, but to preservatives as well. However, this topic is not researched in more detail and data on PCHS in adult AD patients are very limited ,9,10,11.In our 15-year (2007–2021) retrospective study we examined the clinical features of PCHS in adult AD patients. The rate of adult PCHS AD patients (9.4%) is remarkable in the total PCHS population, in our overall tested adult AD population (10.6%) and in the AD population with at least one CHS (17.4%) as well.® and MDBGN despite the fact that MI was patch tested only from 2014. By concomitant PCHS the most common combination was Kathon CG® + MI.According to our observation in adult AD patients the most common preservatives are MI, Kathon CGThe most affected adult PCHS AD patients belonged to the age group of 21–30 and most skin symptoms were localised to the limbs and face-neck region.According to our data, besides metals, the most common other EEBS concomitant allergens were cosmetic-therapeutic ones  in the PCHS adult AD group.To our best knowledge, this is the first study which focuses on the clinical characteristics of PCHS in the adult AD group.In conclusion, PCHS is important among adult AD patients. This finding highlights that adult AD patients are worth patch testing in case of therapy-resistance or worsening skin symptoms due to topical medications and/or personal care products. Our results underline the importance of regular and detailed medical counselling about conscious skin care and about applying not only fragrance-free, but also preservative-free products in this population.


Staphylococcus aureus (S. aureus) has been shown to exacerbate AD. In recent years, in vitro models of AD have been developed, but none of them reproduce all of the pathophysiological features. To better mimic AD, we developed reconstructed human epidermis (RHE) exposed to a Th2 pro-inflammatory cytokine cocktail and S. aureus. This model well reproduced some of the vicious loops involved in AD, with alterations at the physical, microbial and immune levels. Our results strongly suggest that S. aureus acquired a higher virulence potential when the epidermis was challenged with inflammatory cytokines, thus later contributing to the chronic inflammatory status. Furthermore, a topical application of a Castanea sativa extract was shown to prevent the apparition of the AD-like phenotype. It increased filaggrin, claudin-1 and loricrin expressions and controlled S. aureus by impairing its biofilm formation, enzymatic activities and inflammatory potential.Atopic dermatitis (AD), the most common inflammatory skin disorder, is a multifactorial disease characterized by a genetic predisposition, epidermal barrier disruption, a strong T helper (Th) type 2 immune reaction to environmental antigens and an altered cutaneous microbiome. Microbial dysbiosis characterized by the prevalence of  Atopic dermatitis (AD) is a chronic skin inflammatory disease , highly stratum corneum, the outermost layer of the epidermis, anucleated corneocytes are stacked and embedded in a lipid-enriched extracellular matrix.In healthy epidermis, the barrier function has several purposes: to protect against ultra-violet radiation; to maintain good hydration of the upper cell layers and, at the same time, limit body fluid and water loss; to provide an immune defense system against microbial infection; and, finally, to physically and chemically control what goes inside the skin . To ensuThe inside-out signal, where chronic inflammation, driven by T helper (Th)2 cytokines, including interleukin (IL)-4 and IL-13, secondarily alters keratinocyte differentiation and reduces the expression of several epidermal barrier proteins ;The outside-in signal, where epidermal barrier disruption allows for the penetration of allergens and microbes and triggers immunological imbalance ,11,12.Various studies have demonstrated that AD is characterized by a default in the epidermal barrier ,8. Two nstratum corneum extracellular lipids and, therefore, in the permeability properties of this layer [In addition, IL-31 is described as being responsible for pruritus, but it is not directly linked to inflammation and pain . In favois layer .stratum corneum, the homeostatic microbiota balance of the skin evolves and does not prevent the growth of pathogens, such as Staphylococcus aureus (S. aureus) [S. aureus express superantigens, which are allergens and bind antigen-presenting cells and T cell receptors [As a consequence of both epidermal barrier impairments and changes in the (bio)chemical properties of the  aureus) ,23. Thuseceptors .S. aureus onto reconstructed human epidermis (RHE) [Over the years, various models of AD have been developed . Some hais (RHE) .S. aureus. We demonstrated that S. aureus adhesion and proliferation were enhanced in the presence of the cytokines, with the bacteria inducing a strong inflammation response as evidenced by an increased IL-8 release. We also used the model to evaluate the protective effect of an active ingredient from the leaves of Castanea sativa. This active ingredient was preselected from a library of thousands of potential ingredients by performing a preliminary anti-inflammatory property evaluation (quantification of IL-6 and IL-8 by ELISA) on monolayer cultures of normal human keratinocytes stressed by poly I:C, TNF alpha and interferon gamma. Among the anti-inflammatory hits, Castanea sativa extract was then selected for its capacity to inhibit IL-6 and -8 production in keratinocytes challenged by S. aureus ATCC35556, to inhibit their lipase activity and, in parallel, to stimulate filaggrin on differentiated keratinocytes.In the present study, we developed a new 3D model of RHE reproducing AD, adding different concentrations of both Th2 cytokines and S. aureus. We used TNFα at 5 ng/mL and the Th2-related cytokines IL-4, IL-13 and IL-31 at two concentrations, 5 ng/mL each (C1) and 20 ng/mL each (C2), in order to attempt to reproduce a moderate-to-mild phenotype.We generated two in vitro models of AD using RHEs treated with either a cocktail of inflammatory cytokines in a culture medium or both the cocktail and topically applied stratum corneum, a good organization of the stratum spinosum and granulosum and a nice basal layer with well-shaped pavement cells -treated RHEs. Its adhesion and proliferation were measured . This inhibition became significant after 8 h of culture and lasted up to 24 h.Compared to the control cultures of  extract a. This rver time b. In thev/v) to 0.015%) of the Castanea sativa extract on the enzymatic activities  released by S. aureus. The plant extract significantly inhibited the three enzymatic activities with various efficacies. We observed a high inhibition of lipase activity (IC50 = 0.33%) and plasminogen activation (IC50 = 0.035%) and a moderate inhibition of hyaluronidase activity (20% inhibition when the extract was present at 1.5%).We then measured the effect of various concentrations (from 5.5% (S. aureus-infected human keratinocytes and macrophages. A strong increase in IL-6 and IL-8 production by keratinocytes (HaCaT human cell line) was induced by the bacteria  and macrophages (IL-8) infected by the bacteria was decreased when treated with the Castanea sativa extract. With the achievement of this key step, we evaluated the efficacy of the plant extract at 2% on a cohort of 22 volunteers with mild-to-moderate AD. The significant improvement in the barrier function  and EASI score (decreased by 39% and 49%) confirmed the relevance of our model to evaluate ingredients that can be used to improve atopic-prone skin. The activity of the Castanea sativa extract may be explained by its flavonoid profile, particularly flavonoid glucosides, and among them, the major compounds miquelianin (Quercetin-3-O-glucuronide) and astragalin (Kampferol 3-O- glucuronide), as these two compounds are known as NF kB pathway modulators [Finally, we used our model to evaluate the efficacy of an extract of dulators ,53,54,55Castanea sativa tree. The organic certified leaves are harvested as a byproduct of chestnut production. Raw materials were grinded, followed by aqueous extraction and filtration. The extraction process was optimized to standardize flavonol glycosides: the amount of astragalin, miquelianin and equivalents.CaVa is an aqueous extract of the leaves of the 5 keratinocytes/cm2 were seeded on the membrane and grown in Epilife medium containing 1.5 mM CaCl2, HKGS and antibiotics  by immersion for 2 days at 37 °C with 5% CO2. The inserts were then elevated at the air–liquid interface, and the culture was continued for 12 days with supplementation with 50 µg/mL ascorbic acid and 10 ng/mL KGF . The fully supplemented Epilife medium was changed every 2 days. When necessary, RHEs were treated, from day 5, with an inflammatory cocktail composed of 5 ng/mL of TNFα and either 5 ng/mL (C1) or 20 ng/mL (C2) of each of the following cytokines: IL-4, IL-13 and IL-31. The RHEs were also treated with a Castanea sativa leaf extract  at 0.04% from day 5 to 11 in a systemic way.Tridimensional RHEs were produced on a polycarbonate insert, as previously described , with prS aureus. For these experiments with a S. aureus strain obtained from a lesion in the thigh of a 44-year-old patient with AD , antibiotic treatment was stopped at day 10, and 130 µL of PBS containing 5 × 102 bacteria was seeded on top of the RHEs at day 11. After 1 h of contact, bacteria were washed twice with PBS. The RHEs were harvested immediately or 24 h later. Bacteria were counted using the tryptic soy agar plate method . Each condition was carried out in triplicate.The RHEs treated with C1/C1+ CaVa were challenged in parallel by RHEs were formaldehyde-fixed and paraffin–embedded, and hematoxylin–eosin staining was performed on 5 µm-thick sections using a standard histochemistry technique. Immunofluorescence staining for the detection of filaggrin, claudin-1 and loricrin were carried out on 5 µm-thick sections using anti-filaggrin , anti-claudin-1  and anti-loricrin  primary antibodies, followed by secondary antibodies conjugated to either Alexa 488 or Alexa 555. After several washes and counterstaining with 4′,6-diamidino-2-phenylindole (DAPI Vector), sections were mounted with an anti-fading medium. Observations were realized using bright-field  or confocal  microscopy.4 and 1.5% K3Fe(CN)6 in the same buffer [As described by Reynier et al., the RHE tissues were fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.2, for 24 h at 4 °C, and post-fixed at 4 °C with 1% OsOe buffer . The tisAfter treatments, RHEs were lysed with specific lysis buffer  to evaluate filaggrin, loricrin and claudin-1 expressions. Protein concentration was determined by a BCA assay, and then lysates were kept frozen at −80 °C until use. All the samples were adjusted to the same protein concentration, and an equal quantity of proteins was loaded in each capillary. Target proteins were identified by a capillary electrophoresis-based protein analysis system  using primary antibodies , and they were immunoprobed using a horseradish peroxidase-conjugated secondary antibody and chemiluminescent substrate. The capillaries containing a proprietary UV-activated chemical-linked coating were obtained from ProteinSimple. All samples and reagents were prepared according to the recommended manufacturer’s instructions. The resulting chemiluminescent signal was detected and quantified using Compass Software version 2.7.1  followed by a statistical analysis.IL-8 was quantified using an AlphaLISA immunoassay kit  according to the manufacturer’s recommendations.S. aureus (ATCC® 35556™) was seeded at 0.1 million/mL within tryptic soy broth (Biomérieux® TSB-F index 42614) enriched by glucose at 2% and incubated for 24 h at 37 °C. Castanea sativa extract was applied from the beginning to the end of the incubations. Biofilm formation was observed using crystal violet staining and a real-time, label-free technique based on impedance recording using an xCELLigence® device  as described [escribed .v/v) up to 5.5% and from 0.5% up to 5%, of the Castanea sativa extract was added in the culture broth with S. aureus. After 6 and 24 h, the number of bacteria was determined by densitometry at 600 nm, while supernatants were recovered by centrifugation of conditioned broth at 1000× g for 5 min and then used for enzymatic evaluation. Lipase activity was measured by recording the specific fluorescence of a synthetic substrate . Hyaluronidase activity was evaluated through the quantification of residual hyaluronic acid using a turbidimetric method [Castanea sativa extract was incubated with S. aureus-conditioned broth at 0.015% to 0.15%. Then, plasminogen activation into plasmin was measured by recording the specific fluorescence of a protease substrate .For lipase and hyaluronidase activities, various concentrations, from 0.165% -2,5-diphenyltetrazolium bromide (MTT) test and cytokine release in supernatant medium using ELISA methods .Keratinocytes (human cell line HaCaT) were seeded in a standard medium with fetal calf serum and incubated for 3 days at 37 °C. The growth medium was replaced by a standard medium containing the Castanea sativa extract for 24 h. The treatment medium was exchanged to a medium containing a defined quantity of heat-killed S. aureus bacteria, and it was incubated for 24 h at 37 °C. Cell viability was measured using the MTT test, while the release of IL-8 was measured on cell culture supernatant using the ELISA method . The active ingredient was applied at 0.003% up to 0.03%.Human macrophages (U937 cell line) were activated by phorbol myristate acetate for 48 h and then treated with the t-test, the Mann–Whitney test or One-way ANOVA test. A p value < 0.05 was considered significant.A statistical analysis was performed using Sigma Plot software with Student S. aureus extracts or by silencing the expression of pivotal genes encoding epidermal barrier proteins. However, none of them reproduced all of the pathophysiological AD features. In this paper, we developed an AD-like model consisting of RHEs exposed to both a Th2 pro-inflammatory cytokine cocktail and S. aureus. This model mimics the impairments in the skin barrier observed in AD, at the physical, molecular and immune levels. Moreover, our results strongly suggest that S. aureus acquired a higher virulence potential when the epidermis was challenged with inflammatory cytokines, thus later contributing to disease exacerbation. The relevance of this model was confirmed using an extract of Castanea sativa, which significantly improved components of the physical, microbial and immune epidermal barriers.In recent years, in vitro models reproducing some features of AD have been developed by challenging epidermis with either interleukin cocktails or


Alectoria, Bryoria, Usnea) form conspicuous epiphyte communities across the boreal biome. These poikilohydric organisms provide important ecosystem functions and are useful indicators of global change. We analyse how environmental drivers influence changes in occurrence and length of these lichens on Norway spruce (Picea abies) over 10 years in managed forests in Sweden using data from >6000 trees. Alectoria and Usnea showed strong declines in southern‐central regions, whereas Bryoria declined in northern regions. Overall, relative loss rates across the country ranged from 1.7% per year in Alectoria to 0.5% in Bryoria. These losses contrasted with increased length of Bryoria and Usnea in some regions. Occurrence trajectories  on remeasured trees correlated best with temperature, rain, nitrogen deposition, and stand age in multinomial logistic regression models. Our analysis strongly suggests that industrial forestry, in combination with nitrogen, is the main driver of lichen declines. Logging of forests with long continuity of tree cover, short rotation cycles, substrate limitation and low light in dense forests are harmful for lichens. Nitrogen deposition has decreased but is apparently still sufficiently high to prevent recovery. Warming correlated with occurrence trajectories of Alectoria and Bryoria, likely by altering hydration regimes and increasing respiration during autumn/winter. The large‐scale lichen decline on an important host has cascading effects on biodiversity and function of boreal forest canopies. Forest management must apply a broad spectrum of methods, including uneven‐aged continuous cover forestry and retention of large patches, to secure the ecosystem functions of these important canopy components under future climates. Our findings highlight interactions among drivers of lichen decline , functional traits , and population processes (extinction/colonization).Thin, hair‐like lichens ( Picea abies) over 10 years in managed forests in Sweden using data from >6000 trees in the National Forest Inventory. Alectoria and Usnea showed strong declines in southern‐central regions, whereas Bryoria declined in northern regions. Our analysis strongly suggests that the decline was mainly driven by industrial forestry, in combination with nitrogen deposition. Our study highlights interactions among drivers of lichen decline, functional traits and population processes.Thin, hair‐like lichens have important ecosystem functions in boreal forest canopies. We analyse how environmental drivers influence changes in occurrence and length of these lichens in the lower canopy of Norway spruce ( Our analysis is based on >6000 trees surveyed 1993–2012 in the National Forest Inventory (NFI). We focus on changes in habitat quality by monitoring lichens when the host is present. This allowed us to examine how global change drivers interact and affect extinction and colonization processes. We hypothesized the following: (1) that lichen occurrence declines due to industrial forestry, driven by short logging cycles and an unfavourable microclimate (i.e. low light) in dense forests  that lichen occurrence and thallus length (the length of the vegetative body) increase and recover in southern‐central regions in response to reduced anthropogenic N deposition since 1980 . The hemiboreal zone, a transition between the boreal and temperate zone, covers most of southern Sweden. The temperate zone forms a narrow belt in the south and southwest. It has broad‐leaved, deciduous trees  but also host conifers.The study area covers the whole of Sweden and spans latitudes 55–69°N (c. 1500 km). Sweden has 279,000 kmPicea dominates by volume (41%), followed by P. sylvestris (39%) and Betula spp. (12%). The growing stock has increased with 106% since the 1920s, at which time the growing stock had decreased during the late 19th century. This trend mainly results from the efficient, production‐oriented forestry, including cutting of unproductive stands, ditching, thinning, N fertilization and planting of seedlings on clear‐cuts , followed by agriculture 8%; SLU, . Reindee2 pollution, which has substantially decreased since 1970  and are located around the tract perimeter (the length of tract side varies from 300 to 1200 m among regions). About 200 forest, vegetation and site variables are recorded on each plot. The NFI has a systematic program for quality assurance, including training, calibration, and control inventory  from plots in productive forest land (see above). Formally protected forests were excluded as they were not measured in IP1. Hence, our sample consists of managed forests with active forestry, but includes a small proportion of voluntarily set aside areas  Ach.  Brodo & D. Hawksw. (southern tendency) and B. fuscescens (Gyeln.) Brodo & D. Hawksw. (northern tendency), while Usnea is dominated by U. dasopoga (Ach.) Nyl. (widespread), followed by U. subfloridana Stirt. . We calculated mean annual temperature (TEMP) and mean total rain per year (RAIN) for each IP in all NFI plots. RAIN was defined as the sum of precipitation in days with mean temperature ≥0°C, during which lichens can be active. We also extracted deposition of atmospheric inorganic N  from gridded data (20 × 20 km) based on the Match model . Mean annual N deposition was calculated for 1998–2002 (no data available for 1993–1997) and 2003–2012. The variables are henceforth referred to by their abbreviations (Table Gridded climate data (4 × 4 km) were obtained from the Swedish Meteorological and Hydrological Institute  if the CI do not overlap zero.We first estimated the total number of live 2.5.2We calculated summary statistics for the explanatory variables Table  across a2.5.3The trees that were remeasured (~50%) allowed us to examine how changes in lichen occurrence correlate with changes in variables over time in the plots. A lichen is either present (P) or absent (A) on a sample tree in each IP, and thus there are four occurrence trajectories (outcomes): persistence (PP), absence (AA), colonization  and extinction . We used Chi‐square tests to examine the association between trajectories and regions for each lichen. Extinction and colonization rates were calculated following Yalcin and Leroux . The extr) between all variable pairs to identify potential associations among the variables. We then used multinomial logistic regression  versus Y = 0 for a one‐unit increase in the explanatory variable is then given byPrY=j|x/PrY=0|x is the odds at x that the trajectory is j, given that it is either j or 0. If there are other explanatory variables than x present in the model, these are kept fixed when computing ORj. The odds ratio ORj is a measure of how much more likely or unlikely (in terms of odds) it is for occurrence trajectory j to be present among those trees with a one‐unit increment in an explanatory variable x as compared to those with no increment in this variable, while holding the other explanatory variables fixed. The odds ratio is significantly different from 1 when the 95% confidence band does not overlap 1. The analyses were done with r version 4.0.3  to 0.173 ± 0.018 across the country, Usnea from 0.388 ± 0.020 to 0.342 ± 0.021 and Bryoria from 0.532 ± 0.016 to 0.506 ± 0.019 (Table Alectoria occurrences (51.6%) was lost in region 3  and 4 (30.1%), whereas Bryoria decreased in regions 1 (13.6%) and 2 (6.3%). The geographic distribution of Alectoria was substantially reduced in region 3  for all lichens, showing that trajectories varied by region.Remeasured trees had higher lichen occurrence than new trees in IP2, particularly in ly Table . Extinct3.3Alectoria in IP1 (mean 18.1 cm across regions) was twice as high as Usnea (8.6 cm), with Bryoria intermediate (13.6 cm). Length of Bryoria increased slightly over time in regions 3–5 and Usnea in regions 2–4, whereas Alectoria did not change . MAT decreased towards the south reflecting higher degree of forest fragmentation.The individual NFI plots spanned a large range in TEMP (−1.7 to 8.6°C) and RAIN (335–1290 mm). All variables showed clear latitudinal trends from north (region 1) to south . The single‐variable logistic models had highly significant (p < .001) slope coefficients for most variable transformations in the occurrence trajectories  than models for Alectoria (R2 = .218) and Usnea  in the boreal biome is present. Our key finding of significant declines in Sweden over 10 years is alarming, as these lichens already experienced large‐scale declines also before the 1990s  is remarkable, as the distribution of this lichen had once been centred in this region of Fennoscandia  Declines were observed in all regions, and forestry is the only driver with significant impact across the country. (2) The estimated annual loss rates of the lichens (0.5%–1.7%) are comparable to that ~1% of the forest area is logged each year, mainly by clearcutting of older forests. Moreover, the proportion of logged forests older than 120 years has increased since 2000  MATs Clerc, . It alsoBryoria, with dark cortical pigments, suffers more from low light conditions than Alectoria and Usnea, with pale pigments, as the former has a higher light compensation point for photosynthesis , suggesting that N deposition contributed to its decline. High rainfall with a sufficient and not too high N concentration boosts growth of this lichen , it likely suffers more from respiration losses in low‐light conditions during warmer autumns and winters than Alectoria and Usnea, contributing to its decline in dense forests and northern regions.The occurrence trajectories correlated with TEMP1 in the models, reflecting the lichens macroclimate preferences along the latitudinal gradient  are unavailable under thick and/or hard snow and ice. These lichens have historically been important for reindeer husbandry in Fennoscandia  and forest certification (Gustafsson & Perhans, 5Alectoria and Usnea decreased in southern‐central regions where forests are more productive, denser, have shorter logging cycles and are subjected to higher N deposition than northern regions, where only Bryoria decreased. Logging of forests with long continuity of tree cover and dispersal limitation contributed to the steep decline of the old‐growth‐associated Alectoria. Warming correlated with occurrence trajectories of Alectoria and Bryoria, emphasizing that poikilohydric canopy‐living organisms respond directly to changes in microclimate as driven by climate change (De Frenne et al., This is the first study of drivers and changes in dominant canopy lichens based on a large probability sample from a latitudinal gradient across the boreal biome. The rapid decline in only 10 years is a stark warning that hair lichens gradually lose their ecological functions in managed boreal forests, with cascading effects on trophic interactions and ecosystem function. Our analysis strongly suggests that industrial forestry, in combination with N deposition, is the main driver of this decline. The authors declare no conflict of interest.P‐AE designed the study and wrote the manuscript with help from all co‐authors. BW extracted the NFI data and P‐AE the other data, AG estimated lichen occurrence and length, ME performed the logistic regressions and P‐AE performed the other analyses. ME, AG, GS and BW contributed to statistical aspects, while BGJ and KP contributed to ecological aspects. All authors contributed critically to the drafts and gave final approval for publication.Fig S1‐S2Click here for additional data file.Table S1‐S5Click here for additional data file.Appendix S1Click here for additional data file.Appendix S2Click here for additional data file.


Acute-on-Chronic liver failure (ACLF) is a clinical syndrome with high short-term mortality. Alcoholic ACLF is prevalent in European and American countries, while hepatitis B virus (HBV)-related ACLF is more common in the Asia-Pacific region. There is still a lack of a unified definition standard for ACLF, due to various etiologies and pathogeneses in different continents. Currently, liver transplantation (LT) is the most effective treatment for liver failure. However, the shortage of liver sources is still a global problem, which seriously limits the clinical application of an LT. Premature LT aggravates the shortage of liver resources to a certain extent, and too much delay significantly increases the risk of complications and death. Therefore, this study reviews the current literature on LT in the treatment of ACLF and discusses further the challenges for ACLF patients, the timing of LT for ACLF, and the choice of the patient population. Acute-on-Chronic liver failure is characterized by an extreme fatigue, a rapid deepening of jaundice, coagulation disorder, and decompensated ascites, with or without hepatic encephalopathy. It progresses rapidly with a poor prognosis and the short-term 28-day) mortality can reach as much as 23–74% , 2. Acut8-day morArtificial liver support system is the most common type of liver failure. In Eastern countries, hepatitis B virus (HBV) reactivation and alcoholic hepatitis are common predisposing factors for ACLF, while in Western countries, infection and alcoholic hepatitis are the main causes of ACLF . In receThe definitions proposed by the European Association for the Study of the Liver (EASL)  and the So far, multiple prognostic scores have been used, including Child–Turcotte–Pugh (CTP) score, the Model for end-stage liver disease (MELD) score, the MELD sodium (MELD-Na) score, APASL ACLF Research Consortium (AARC) score, chronic liver failure-sequential organ failure assessment (CLIF-SOFA) score, etc. The CTP model was first proposed in 1964, which is a commonly used classification standard for quantitative assessment of liver function in patients with liver cirrhosis. The CTP score is a classical parameter that is widely used to evaluate the liver reserve function and assess the condition and prognosis of patients with liver cirrhosis. However, a major drawback with the CTP score is that it is subjective, especially for ascites and hepatic encephalopathy, which makes it difficult to convert to objective grading. And due to the narrow grading window, sometimes it cannot accurately reflect the severity of the patient's condition. The MELD score is used to predict the short-term mortality of patients with chronic liver disease and awaiting transplantation. Moreover, it also serves as the main tool for liver source allocation in Eastern and Western countries. However, the MELD score refers only to creatinine, bilirubin, prothrombin time, and INR, which has certain limitations in the practical clinical application of LT. Among MELD-derived models, the MELD-Na scoring model has been supported by a large number of studies. Compared with creatinine, serum sodium can predict renal function injury earlier and in a more sensitive manner. In a prospective study by Biggins et al. , the risIn conclusion, MELD, CLIF-SOFA, and AARC scores have been recorded as suitable for predicting mortality in patients with ACLF. However, due to inconsistent definitions of ACLF and various etiologies between the East and the West, these models do not have a stable and perfect predictive power. Whether these models can accurately reflect the clinical severity of ACLF patients requires a further study, and The American Association for the Study of Liver Diseases (AASLD) also advises against relying solely on the currently available prognostic scoring systems to predict outcomes and identify candidates for LT. Therefore, there is still a lack of a clear risk prediction system for judging the timing of liver transplantation in patients with an advanced liver failure. If the timing is too early, the liver source will be wasted, and if it is too late, the timing of the operation will be lost or the prognosis will be poor. As the course of ACLF changes rapidly and is associated with high short-term mortality, it is important to identify patients for LT before the onset of the development of MOF. At present, there is no optimal allocation system for liver transplantation, and the choice of SMT, LT, or palliative treatment is still an urgent problem to be solved. More prospective studies are needed to formulate the best prognostic criteria in the future.The latest research based on the CANONIC database showed that most of the patients with d3–7 ACLF 2 or 3 died in the first month of follow-up  . At presThe ALSS treatment combined with LT in patients with HBV–ACLF improved short-term survival. ALSS treatment pre-LT is an independent protective factor affecting the 4-week survival rate after LT . Ling etIn 2019, the APASL updated the definition of ACLF, pointing out that one of the key characteristics of ACLF is reversible. Over time, liver injury, fibrosis, and portal pressure have been gradually reduced and liver reserve improved in some patients. It emphasizes that early death risk judgment and clinical intervention can improve the prognosis of patients. This is also the golden period for bridging therapy before transplantation and for promoting the ability of liver regeneration and repair.P < 0.0001). As regards patients with no improvement at the time of transplantation, the ACLF score, CLIF-ACLF score, CLIF-OF (organ failure) score, and MELD score are significantly lower. Yadav et al. (P < 0.0001).Liver transplantation includes living donor liver transplantation (LDLT) and deceased-donor liver transplantation (DDLT). Some studies  have fouv et al.  used EASv et al. . Differev et al. . The MELv et al.  shows thv et al.  used theLT may be the basic treatment for ACLF to reverse the extrahepatic MOF. The median transplant-free survival in patients with ACLF was 48-day, and previous guidelines have mentioned that an LT was recommended, if the expected 5-year survival exceeded 50% (As ACLF is a heterogeneous condition and follows a dynamic course, the decision to undergo LT should be individualized. The survival rate after liver transplantation is good, and the 5-year survival rate is 74–90% . HoweverIn conclusion, decisions to undergo LT or not should be individualized. To evaluate ACLF effectively, the required model must be not only dynamic but also comprehensive. Prospective studies are still needed to further evaluate and determine the optimal timing and selection criteria for transplantation in ACLF.As the transplantation window of ACLF patients is very short, the short-term mortality is high, and the occurrence of organ failure is also very important for prognostic prediction . The ACLMonitoring inflammatory markers and regenerative markers during the “Golden Window” of ACLF progression will also help us to judge the prognosis of patients. In future studies, we should pay attention to the impact of the inflammatory response on the survival and prognosis of patients. In addition, the process of ACLF is potentially reversible, so whether liver function is compensated or not is very important for patients with liver failure. An early diagnosis and timely intervention of ACLF alone can protect surviving hepatocytes as much as possible from inflicting further damage to liver cells, and thereby create favorable conditions for liver regeneration.Generally speaking, the results of ACLF patients after LT are worse than those of chronic liver disease alone. This may be due to the severity of the underlying liver disease, which is manifested as higher MELD, organ dysfunction, and systemic inflammatory response syndrome. Zhang et al.  propose Acute-on-Chronic liver failure is a common liver disease syndrome and treatment is mainly based on the prevention of organ failure and its associated complications. LT has a good effect on ACLF 1–2 patients. While for ACLF 3 without LT, the prognosis is poor. With the rapid changes taking place in the ACLF process, it is important to carry out a comprehensive medical treatment as well as anti-infective preventive measures in time, and to enter the LT evaluation process as soon as possible to screen the most suitable LT recipients and determine the golden time for surgery. Based on the existing evidence and prospects, some improvements can be brought about in the ACLF field, which may help to improve the management and prognosis of patients. At present, LT can improve the prognosis of patients with different degrees of ACLF. In the future, it is necessary to establish an LT risk assessment model and screen high-risk groups, so that patients can be managed more scientifically at admission, and the correct intervention and monitoring measures carried out as soon as possible to obtain the best treatment effect, thereby improving the prognosis of patients with ACLF and the effectiveness and efficiency of LT. It is expected that in the future, a large sample and multi-center prospective clinical research results will guide the formulation of more accurate clinical diagnostic criteria and a prognostic scoring system to determine the needs and appropriate time of LT in the patient population. The accumulation of long-term follow-up data will also contribute to the formulation of future clinical guidelines.XL performed literature searches and prepared the initial draft. LZ, CP, and ST reviewed and helped to finalize the manuscript. All of the authors read and approved the manuscript.