Cyclin A2 Expression as Predictive Biomarker in Muscle-Invasive Upper Tract Urothelial Carcinoma.

INTRODUCTION: The aim was to evaluate the prognostic value of altered Cyclin A2 (CCNA2) gene expression in upper tract urothelial carcinoma (UTUC) and to assess its predictive potential as a prognostic factor for overall survival (OS) and disease-free survival. METHODS: 62 patients who underwent surgical treatment for UTUC were included. Gene expression of CCNA2, MKI67, and p53 was analyzed by quantitative reverse transcriptase polymerase chain reaction. Survival analyses were performed using the Kaplan-Meier method and the log-rank test. For Cox regression analyses, uni- and multivariable hazard ratios were calculated. Spearman correlation was used to analyze correlation of CCNA2 expression with MKI67 and p53. RESULTS: The median age of the cohort was 73 years, and it consisted of 48 males (77.4%) and 14 females (22.6%). Patients with high CCNA2 expression levels showed longer OS (HR 0.33; 95% CI: 0.15-0.74; p = 0.0073). Multivariable Cox regression analyses identified CCNA2 overexpression (HR 0.37; 95% CI: 0.16-0.85; p = 0.0189) and grading G2 (vs. G3) (HR 0.39; 95% CI: 0.17-0.87; p = 0.0168) to be independent predictors for longer OS. CCNA2 expression correlated positively with MKI67 expression (Rho = 0.4376, p = 0.0005). CONCLUSION: Low CCNA2 expression is significantly associated with worse OS. Thus, CCNA2 might serve as a potential biomarker in muscle-invasive UTUC and may be used to characterize a subset of patients having an unfavorable outcome and for future risk assessment scores.

A self-supervised vision transformer to predict survival from histopathology in renal cell carcinoma.

PURPOSE: To develop and validate an interpretable deep learning model to predict overall and disease-specific survival (OS/DSS) in clear cell renal cell carcinoma (ccRCC). METHODS: Digitised haematoxylin and eosin-stained slides from The Cancer Genome Atlas were used as a training set for a vision transformer (ViT) to extract image features with a self-supervised model called DINO (self-distillation with no labels). Extracted features were used in Cox regression models to prognosticate OS and DSS. Kaplan-Meier for univariable evaluation and Cox regression analyses for multivariable evaluation of the DINO-ViT risk groups were performed for prediction of OS and DSS. For validation, a cohort from a tertiary care centre was used. RESULTS: A significant risk stratification was achieved in univariable analysis for OS and DSS in the training (n = 443, log rank test, p < 0.01) and validation set (n = 266, p < 0.01). In multivariable analysis, including age, metastatic status, tumour size and grading, the DINO-ViT risk stratification was a significant predictor for OS (hazard ratio [HR] 3.03; 95%-confidence interval [95%-CI] 2.11-4.35; p < 0.01) and DSS (HR 4.90; 95%-CI 2.78-8.64; p < 0.01) in the training set but only for DSS in the validation set (HR 2.31; 95%-CI 1.15-4.65; p = 0.02). DINO-ViT visualisation showed that features were mainly extracted from nuclei, cytoplasm, and peritumoural stroma, demonstrating good interpretability. CONCLUSION: The DINO-ViT can identify high-risk patients using histological images of ccRCC. This model might improve individual risk-adapted renal cancer therapy in the future.

Medium-term Oncological Efficacy and Patient-reported Outcomes After Focal High-intensity Focused Ultrasound: The FOXPRO Trial.

BACKGROUND: Multiparametric magnetic resonance imaging (mpMRI)/transrectal ultrasound (TRUS) fusion-guided high-intensity focused ultrasound (HIFU) is a focal treatment option for MRI-visible localized prostate cancer (PCa). High-quality evidence regarding the clinical efficacy remains limited. OBJECTIVE: To assess medium-term oncological efficacy along with patient-reported outcome measures (PROMs). DESIGN, SETTING, AND PARTICIPANTS: This prospective single-center cohort study was performed from 2014 to 2020. Patients with primary International Society of Urological Pathologists (ISUP) grade group (GG) ≤2 by combined MRI/TRUS fusion and systematic prostate biopsy and prostate-specific antigen (PSA) <10 ng/ml were included. INTERVENTION: MRI/TRUS fusion-guided focal HIFU therapy. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary outcome was the cancer-free rate of the HIFU-treated lesion by biopsy after 1 yr. Secondary endpoints included salvage treatment-free survival (STFS), metastasis-free survival (MFS), overall survival (OS), and PROMs according to International Consortium for Health Outcomes Measurement recommendations. RESULTS AND LIMITATIONS: Fifty patients were included (median [range] age 68 [48-80] yr; median PSA 6.5 [1.2-9.9] ng/ml; GG 1 54% [n = 27], and GG 2 46% [n = 23]). The median (range) PSA decrease from baseline to 12 mo was 51% (35.9-72.7%). In total, 37/50 patients (74%) underwent a 1-yr biopsy. PCa was detected in 23 patients (46%; GG 1 20% [n = 10]; GG >1 26% [n = 13]; infield 40% [n = 20]). At a median follow-up of 42 (13-73) mo, PCa was detected in 30 men (60%). Among all patients, 19 (38%) underwent salvage treatments (median [95% confidence interval] STFS 53 [44.3-61.7] mo). MFS and OS were 100% and 98%, respectively. The Expanded Prostate Cancer Index Composite-26 sexual domain decreased by 20.8 points (p = 0.372). CONCLUSIONS: MRI/TRUS-guided focal HIFU therapy results in complete cancer ablation in only half of the treated patients after 1 yr, with further recurrences at medium-term follow-up. A decline of potency occurs in a subset of patients. PATIENT SUMMARY: Focal image-guided high-intensity focused ultrasound therapy controls cancer in one of two patients. Its impact on urinary continence and erectile function is low.

Deep learning can predict survival directly from histology in clear cell renal cell carcinoma.

For clear cell renal cell carcinoma (ccRCC) risk-dependent diagnostic and therapeutic algorithms are routinely implemented in clinical practice. Artificial intelligence-based image analysis has the potential to improve outcome prediction and thereby risk stratification. Thus, we investigated whether a convolutional neural network (CNN) can extract relevant image features from a representative hematoxylin and eosin-stained slide to predict 5-year overall survival (5y-OS) in ccRCC. The CNN was trained to predict 5y-OS in a binary manner using slides from TCGA and validated using an independent in-house cohort. Multivariable logistic regression was used to combine of the CNNs prediction and clinicopathological parameters. A mean balanced accuracy of 72.0% (standard deviation [SD] = 7.9%), sensitivity of 72.4% (SD = 10.6%), specificity of 71.7% (SD = 11.9%) and area under receiver operating characteristics curve (AUROC) of 0.75 (SD = 0.07) was achieved on the TCGA training set (n = 254 patients / WSIs) using 10-fold cross-validation. On the external validation cohort (n = 99 patients / WSIs), mean accuracy, sensitivity, specificity and AUROC were 65.5% (95%-confidence interval [CI]: 62.9-68.1%), 86.2% (95%-CI: 81.8-90.5%), 44.9% (95%-CI: 40.2-49.6%), and 0.70 (95%-CI: 0.69-0.71). A multivariable model including age, tumor stage and metastasis yielded an AUROC of 0.75 on the TCGA cohort. The inclusion of the CNN-based classification (Odds ratio = 4.86, 95%-CI: 2.70-8.75, p < 0.01) raised the AUROC to 0.81. On the validation cohort, both models showed an AUROC of 0.88. In univariable Cox regression, the CNN showed a hazard ratio of 3.69 (95%-CI: 2.60-5.23, p < 0.01) on TCGA and 2.13 (95%-CI: 0.92-4.94, p = 0.08) on external validation. The results demonstrate that the CNN's image-based prediction of survival is promising and thus this widely applicable technique should be further investigated with the aim of improving existing risk stratification in ccRCC.

Upper Tract Urinary Cancer Recurrence after Radical Cystectomy: Risk Assessment of Intraoperative Frozen Section.

INTRODUCTION: Upper tract urinary cancer recurrence (UTUCR) after radical cystectomy (RC) is outcome-limiting. Surgical recommendations on intraoperative performance of frozen section analysis (FSA) and management of positive ureteral margin (PUM) are lacking. METHODS: 634 RC cases were identified (2010-2018). In patients with PUM, sequential ureteral resections up to a negative margin were performed. We investigated the accuracy of FSA, significance of PUM, and identified risk factors (RFs) to stratify patients for UTUCR. RESULTS: FSA was performed in 355 patients, including a total of 693 ureters. FSA sensitivity was 0.93 and specificity 0.99. PUM conversion was possible in 52 (91.2%) cases. UTUCR occurred in 17 (4.8%) patients. Identified UTUCR RFs are non-muscle invasive bladder carcinoma (NMIBC) (OR 3.8, 95% confidence intervals [CI] 1.4-10.2, p = 0.008), multifocal bladder cancer in cystectomy specimen (OR 4.7, CI 1.1-20.8, p = 0.042), and recurrent NMIBC (OR 4.1, CI 1.5-10.9, p = 0.006). Risk-group stratification showed a six-fold increase in UTUCR risk (low-to high-risk). CONCLUSION: FSA is a sensitive and specific method to identify PUM. UTUCR occurs significantly more often in patients with recurrent, multifocal NMIBC at the time of RC. Patients can be risk stratified for UTUCR. In case of NMIBC-PUM, surgeons can safely opt for a kidney preserving strategy.

Assessment of glomerular morphological patterns by deep learning algorithms.

BACKGROUND: Compilation of different morphological lesion signatures is characteristic of renal pathology. Previous studies have documented the potential value of artificial intelligence (AI) in recognizing relatively clear-cut glomerular structures and patterns, such as segmental or global sclerosis or mesangial hypercellularity. This study aimed to test the capacity of deep learning algorithms to recognize complex glomerular structural changes that reflect common diagnostic dilemmas in nephropathology. METHODS: For this purpose, we defined nine classes of glomerular morphological patterns and trained twelve convolutional neuronal network (CNN) models on these. The two-step training process was done on a first dataset defined by an expert nephropathologist (12,253 images) and a second consensus dataset (11,142 images) defined by three experts in the field. RESULTS: The efficacy of CNN training was evaluated using another set with 180 consensus images, showing convincingly good classification results (kappa-values 0.838-0.938). Furthermore, we elucidated the image areas decisive for CNN-based decision making by class activation maps. Finally, we demonstrated that the algorithm could decipher glomerular disease patterns coinciding in a single glomerulus (e.g. necrosis along with mesangial and endocapillary hypercellularity). CONCLUSIONS: In summary, our model, focusing on glomerular lesions detectable by conventional microscopy, is the first sui generis to deploy deep learning as a reliable and promising tool in recognition of even discrete and/or overlapping morphological changes. Our results provide a stimulus for ongoing projects that integrate further input levels next to morphology (such as immunohistochemistry, electron microscopy, and clinical information) to develop a novel tool applicable for routine diagnostic nephropathology.

GTF2I Mutation in Thymomas: Independence From Racial-Ethnic Backgrounds. An Indian/German Comparative Study.

Thymomas are the most frequent adult mediastinal cancers. Their etiology is unknown and their pathogenesis poorly understood. Racial, ethnic and environmental factors influence tumorigenesis in many cancers, but their role in thymomas remains unclear to date. In this study that included pretreatment thymoma cases from India and Germany (n = 37 and n = 77, respectively) we compared i) the prevalence of the thymoma-specific chromosome 7 c.74146970T > A mutation of the GTF2I gene in type A and AB thymomas; ii) epidemiological features; and iii) the frequency of myasthenia gravis (MG). Due to a known predominance of GTF2I mutation in A and AB histotypes, we included only a marginal number of type B thymomas as a control group in both cohorts. While the distribution of histological types between the cohorts was similar (p = 0.1622), Indian patients were strikingly younger (p < 0.0001; median age 50 vs. 65 years) and showed significantly lower tumour stage (Masaoka-Koga stage I) at primary diagnosis (p = 0.0005) than the German patients. In patients with known MG status (n = 17 in Indian and n = 25 in German cohort), a clear trend towards more frequent MG was observed in the Indian group (p = 0.0504; 48 vs. 82%). The prevalence of the GTF2I mutation (analysed in n = 34 Indian and n = 77 German patients) was identical in the two cohorts. We conclude that racial-ethnic and environmental factors do not significantly influence the most common molecular feature of thymomas but may have an impact on the timing of clinical presentation.

CD73 Overexpression in Podocytes: A Novel Marker of Podocyte Injury in Human Kidney Disease.

The CD73 pathway is an important anti-inflammatory mechanism in various disease settings. Observations in mouse models suggested that CD73 might have a protective role in kidney damage; however, no direct evidence of its role in human kidney disease has been described to date. Here, we hypothesized that podocyte injury in human kidney diseases alters CD73 expression that may facilitate the diagnosis of podocytopathies. We assessed the expression of CD73 and one of its functionally important targets, the C-C chemokine receptor type 2 (CCR2), in podocytes from kidney biopsies of 39 patients with podocytopathy (including focal segmental glomerulosclerosis (FSGS), minimal change disease (MCD), membranous glomerulonephritis (MGN) and amyloidosis) and a control group. Podocyte CD73 expression in each of the disease groups was significantly increased in comparison to controls (p < 0.001-p < 0.0001). Moreover, there was a marked negative correlation between CD73 and CCR2 expression, as confirmed by immunohistochemistry and immunofluorescence (Pearson r = -0.5068, p = 0.0031; Pearson r = -0.4705, p = 0.0313, respectively), thus suggesting a protective role of CD73 in kidney injury. Finally, we identify CD73 as a novel potential diagnostic marker of human podocytopathies, particularly of MCD that has been notorious for the lack of pathological features recognizable by light microscopy and immunohistochemistry.

The prognostic value of galactosylceramide-sulfotransferase (Gal3ST1) in human renal cell carcinoma.

Renal cell carcinoma (RCC) is the deadliest primary genitourinary malignancy typically associated with asymptomatic initial presentation and poorly predictable survival. Next to established risk factors, tumor microenvironment may alter metastatic capacity and immune landscape. Due to their high concentrations, sulfoglycolipids (sulfatides) were among the first well-described antigens in RCC that are associated with worse prognosis. As sulfatide detection in routine diagnostics is not possible, we aimed to test the prognostic value of its protein counterpart, sulfatide-producing enzyme Gal3ST1. We performed retrospective long-term follow up analysis of Gal3ST1 expression as prognostic risk factor in a representative RCC patient cohort. We observed differentially regulated Gal3ST1 expression in all RCC types, being significantly more associated with clear cell RCC than to chromophobe RCC (p = 0.001). Surprisingly, in contrast to published observations from in vitro models, we could not confirm an association between Gal3ST1 expression and a malignant clinical behaviour of the RCC. In our cohort, Gal3ST1 did not significantly influence progression-free survival (Hazard Ratio (HR): 1.7 95% CI (0.6-4.9), p = 0.327). Particularly after adjusting for histology, T-stage, N-status and M-status at baseline, we observed no independent prognostic effect (HR = 1.0 95% CI (0.3-3.3), p = 0.96). The analysis of Gal3ST1 mRNA expression in a TCGA dataset supported the results of our cohort. Thus, Gal3ST1 might help to differentiate between chromophobe RCC and other frequent RCC entities but-despite previously published data from cell culture models-does not qualify as a prognostic marker for RCC. Further investigation of regulatory mechanisms of sulfatide metabolism in human RCC microenvironment is necessary to understand the role of this quantitatively prominent glycosphingolipid in RCC progression.

Molecular pathology of thymomas: implications for diagnosis and therapy.

Thymomas exhibit a unique genomic landscape, comprising the lowest on average total mutational burden among adult human cancers; a unique point mutation in the GTF2I gene in WHO type A and AB thymomas (and rarely others); almost unique KMT2A-MAML2 translocations in rare WHO type B2 and B3 thymomas; a unique YAP1-MAML2 translocation in almost all metaplastic thymomas; and unique miRNA profiles in relation to GTF2I mutational status and WHO histotypes. While most thymomas can be diagnosed solely on the basis of morphological features, mutational analyses can solve challenging differential diagnostic problems. No molecular biomarkers have been identified that predict the response of unresectable thymomas to chemotherapy or agents with known molecular targets. Despite the common and strong expression of PDL1 in thymomas, immune checkpoint inhibitors are rarely applicable due to the poor predictability of common, life-threatening autoimmune side effects that are related to the unrivaled propensity of thymomas towards autoimmunity.

A comprehensive molecular characterization of the 8q22.2 region reveals the prognostic relevance of OSR2 mRNA in muscle invasive bladder cancer.

Technological advances in molecular profiling have enabled the comprehensive identification of common regions of gene amplification on chromosomes (amplicons) in muscle invasive bladder cancer (MIBC). One such region is 8q22.2, which is largely unexplored in MIBC and could harbor genes with potential for outcome prediction or targeted therapy. To investigate the prognostic role of 8q22.2 and to compare different amplicon definitions, an in-silico analysis of 357 patients from The Cancer Genome Atlas, who underwent radical cystectomy for MIBC, was performed. Amplicons were generated using the GISTIC2.0 algorithm for copy number alterations (DNA_Amplicon) and z-score normalization for mRNA gene overexpression (RNA_Amplicon). Kaplan-Meier survival analysis, univariable, and multivariable Cox proportional hazard ratios were used to relate amplicons, genes, and clinical parameters to overall (OS) and disease-free survival (DFS). Analyses of the biological functions of 8q22.2 genes and genomic events in MIBC were performed to identify potential targets. Genes with prognostic significance from the in silico analysis were validated using RT-qPCR of MIBC tumor samples (n = 46). High 8q22.2 mRNA expression (RNA-AMP) was associated with lymph node metastases. Furthermore, 8q22.2 DNA and RNA amplified patients were more likely to show a luminal subtype (DNA_Amplicon_core: p = 0.029; RNA_Amplicon_core: p = 0.01). Overexpression of the 8q22.2 gene OSR2 predicted shortened DFS in univariable (HR [CI] 1.97 [1.2; 3.22]; p = 0.01) and multivariable in silico analysis (HR [CI] 1.91 [1.15; 3.16]; p = 0.01) and decreased OS (HR [CI] 6.25 [1.37; 28.38]; p = 0.0177) in RT-qPCR data analysis. Alterations in different levels of the 8q22.2 region are associated with manifestation of different clinical characteristics in MIBC. An in-depth comprehensive molecular characterization of genomic regions involved in cancer should include multiple genetic levels, such as DNA copy number alterations and mRNA gene expression, and could lead to a better molecular understanding. In this study, OSR2 is identified as a potential biomarker for survival prognosis.

Thymic Hyperplasia with Lymphoepithelial Sialadenitis (LESA)-Like Features: Strong Association with Lymphomas and Non-Myasthenic Autoimmune Diseases.

Thymic hyperplasia (TH) with lymphoepithelial sialadenitis (LESA)-like features (LESA-like TH) has been described as a tumor-like, benign proliferation of thymic epithelial cells and lymphoid follicles. We aimed to determine the frequency of lymphoma and autoimmunity in LESA-like TH and performed retrospective analysis of cases with LESA-like TH and/or thymic MALT-lymphoma. Among 36 patients (21 males) with LESA-like TH (age 52 years, 32-80; lesion diameter 7.0 cm, 1-14.5; median, range), five (14%) showed associated lymphomas, including four (11%) thymic MALT lymphomas and one (3%) diffuse large B-cell lymphoma. One additional case showed a clonal B-cell-receptor rearrangement without evidence of lymphoma. Twelve (33%) patients (7 women) suffered from partially overlapping autoimmune diseases: systemic lupus erythematosus (n = 4, 11%), rheumatoid arthritis (n = 3, 8%), myasthenia gravis (n = 2, 6%), asthma (n = 2, 6%), scleroderma, Sjögren syndrome, pure red cell aplasia, Grave's disease and anti-IgLON5 syndrome (each n = 1, 3%). Among 11 primary thymic MALT lymphomas, remnants of LESA-like TH were found in two cases (18%). In summary, LESA-like TH shows a striking association with autoimmunity and predisposes to lymphomas. Thus, a hematologic and rheumatologic workup should become standard in patients diagnosed with LESA-like TH. Radiologists and clinicians should be aware of LESA-like TH as a differential diagnosis for mediastinal mass lesions in patients with autoimmune diseases.

Short-Range Order in VI3.

We present a detailed investigation of the crystal structure of VI3, a two-dimensional van der Waals material of interest for studies of low-dimensional magnetism. As opposed to the average crystal structure that features R3̅ symmetry of the unit cell, our Raman scattering and X-ray atomic pair distribution function analysis supported by density functional theory calculations point to the coexistence of short-range ordered P3̅1c and long-range ordered R3̅ phases. The highest-intensity peak, A1g3, exhibits a moderate asymmetry that might be traced back to the spin-phonon interactions, as in the case of CrI3.

Cancer Acidity and Hypertonicity Contribute to Dysfunction of Tumor-Associated Dendritic Cells: Potential Impact on Antigen Cross-Presentation Machinery.

Macrophages (MΦ) and dendritic cells (DC), major players of the mononuclear phagocyte system (MoPh), are potent antigen presenting cells that steadily sense and respond to signals from the surrounding microenvironment, leading to either immunogenic or tolerogenic outcomes. Next to classical MHC-I/MHC-II antigen-presentation pathways described in the vast majority of cell types, a subset of MoPh (CD8+, XCR1+, CLEC9A+, BDCA3+ conventional DCs in human) is endowed with a high competence to cross-present external (engulfed) antigens on MHC-I molecules to CD8+ T-cells. This exceptional DC function is thought to be a crucial crossroad in cytotoxic antitumor immunity and has been extensively studied in the past decades. Biophysical and biochemical fingerprints of tumor micromilieus show significant spatiotemporal differences in comparison to non-neoplastic tissue. In tumors, low pH (mainly due to extracellular lactate accumulation via the Warburg effect and via glutaminolysis) and high oncotic and osmotic pressure (resulting from tumor debris, increased extracellular matrix components but in part also triggered by nutritive aspects) are-despite fluctuations and difficulties in measurement-likely the most constant general hallmarks of tumor microenvironment. Here, we focus on the influence of acidic and hypertonic micromilieu on the capacity of DCs to cross-present tumor-specific antigens. We discuss complex and in part controversial scientific data on the interference of these factors with to date reported mechanisms of antigen uptake, processing and cross-presentation, and we highlight their potential role in cancer immune escape and poor clinical response to DC vaccines.

Glucosylceramide Synthase Is Involved in Development of Invariant Natural Killer T Cells.

Invariant natural killer T (iNKT) cells represent a unique population of CD1d-restricted T lymphocytes expressing an invariant T cell receptor encoded by Vα14-Jα18 and Vα24-Jα18 gene segments in mice and humans, respectively. Recognition of CD1d-loaded endogenous lipid antigen(s) on CD4/CD8-double positive (DP) thymocytes is essential for the development of iNKT cells. The lipid repertoire of DP thymocytes and the identity of the decisive endogenous lipid ligands have not yet been fully elucidated. Glycosphingolipids (GSL) were implicated to serve as endogenous ligands. However, further in vivo investigations were hampered by early embryonal lethality of mice deficient for the key GSL-synthesizing enzyme glucosylceramide (GlcCer) synthase [GlcCer synthase (GCS), EC 2.4.1.80]. We have now analyzed the GSL composition of DP thymocytes and shown that GlcCer represented the sole neutral GSL and the acidic fraction was composed of gangliosides. Furthermore, we report on a mouse model that by combination of Vav-promoter-driven iCre and floxed GCS alleles (Vav Cre GCS f/f ) enabled an efficient depletion of GCS-derived GSL very early in the T cell development, reaching a reduction by 99.6% in DP thymocytes. Although the general T cell population remained unaffected by this depletion, iNKT cells were reduced by approximately 50% in thymus, spleen, and liver and showed a reduced proliferation and an increased apoptosis rate. The Vß-chains repertoire and development of iNKT cells remained unaltered. The GSL-depletion neither interfered with expression of CD1d, SLAM, and Ly108 molecules nor impeded the antigen presentation on DP thymocytes. These results indicate that GlcCer-derived GSL, in particular GlcCer, contribute to the homeostatic development of iNKT cells.

Hyperosmolarity impedes the cross-priming competence of dendritic cells in a TRIF-dependent manner.

Tissue osmolarity varies among different organs and can be considerably increased under pathologic conditions. Hyperosmolarity has been associated with altered stimulatory properties of immune cells, especially macrophages and dendritic cells. We have recently reported that dendritic cells upon exposure to hypertonic stimuli shift their profile towards a macrophage-M2-like phenotype, resulting in attenuated local alloreactivity during acute kidney graft rejection. Here, we examined how hyperosmotic microenvironment affects the cross-priming capacity of dendritic cells. Using ovalbumin as model antigen, we showed that exposure of dendritic cells to hyperosmolarity strongly inhibits activation of antigen-specific T cells despite enhancement of antigen uptake, processing and presentation. We identified TRIF as key mediator of this phenomenon. Moreover, we detected a hyperosmolarity-triggered, TRIF-dependent clustering of MHCI loaded with the ovalbumin-derived epitope, but not of overall MHCI molecules, providing a possible explanation for a reduced T cell activation. Our findings identify dendritic cells as important players in hyperosmolarity-mediated immune imbalance and provide evidence for a novel pathway of inhibition of antigen specific CD8+ T cell response in a hypertonic micromilieu.

Deficiency of N-myristoylation reveals calcineurin activity as regulator of IFN-γ-producing γδ T cells.

γδ T cell subsets can be characterized, in part, by their secretion of select proinflammatory cytokines. The molecular mechanisms driving the diverse fates of γδ T cells have not been elucidated. We have previously shown that the attachment of myristic acid to the N-terminal glycine of proteins, termed N-myristoylation, is essential for αß T cell development and activation. Here, we explore the potential role of this lipid modification on the activation of γδ T cells. In the absence of N-myristoylation, the CD27+ γδ T cell subset was dominantly affected. The cells produced high levels of IFN-γ upon stimulation. In addition, they were more sensitive to inhibition of the CaN-Nfat pathway than were γδ T cells with myristoylated CaN. N-Myristoylation was found to modulate activity of phosphatase CaN, a regulator of Nfat. In summary, the CaN-Nfat pathway regulates development and function of IFN-γ-producing γδ T cells, and its balanced activity is strongly dependent on CaN N-myristoylation.

Inhibition of hepatocellular carcinoma growth by blockade of glycosphingolipid synthesis.

Hepatocellular carcinoma (HCC) is one of the most frequent cancers. In vitro studies suggest that growth and response to therapy of human carcinomas may depend on glycosphingolipid (GSL) expression. Glucosylceramide synthase (GCS), encoded by the gene Ugcg, is the basic enzyme required for the synthesis of GSLs. Gene array analysis implied that Ugcg is significantly overexpressed in human HCC as compared to non-tumorous liver tissue. Therefore we have investigated whether tumor - genesis and - growth is altered in the absence of GSLs. An endogenous liver cancer model has been initiated by application of diethylnitrosamine in mice lacking Ugcg specifically in hepatocytes. We have now shown that hepatocellular tumor initiation and growth in mice is significantly inhibited by hepatic GSL deficiency in vivo. Neither the expression of cell cycle proteins, such as cyclins and pathways such as the MAP-kinase/Erk pathway nor the mTOR/Akt pathway as well as the number of liver infiltrating macrophages and T cells were essentially changed in tumors lacking GSLs. Significantly elevated bi-nucleation of atypical hepatocytes, a feature for impaired cytokinesis, was detected in tumors of mice lacking liver-specific GSLs. A reduction of proliferation and restricted growth of tumor microspheres due to delayed, GSL-dependent cytokinesis, analogous to the histopathologic phenotype in vivo could be demonstrated in vitro. GSL synthesis inhibition may thus constitute a potential therapeutic target for hepatocellular carcinoma.

Tubular Dickkopf-3 promotes the development of renal atrophy and fibrosis.

Renal tubular atrophy and interstitial fibrosis are common hallmarks of etiologically different progressive chronic kidney diseases (CKD) that eventually result in organ failure. Even though these pathological manifestations constitute a major public health problem, diagnostic tests, as well as therapeutic options, are currently limited. Members of the dickkopf (DKK) family, DKK1 and -2, have been associated with inhibition of Wnt signaling and organ fibrosis. Here, we identify DKK3 as a stress-induced, tubular epithelia-derived, secreted glycoprotein that mediates kidney fibrosis. Genetic as well as antibody-mediated abrogation of DKK3 led to reduced tubular atrophy and decreased interstitial matrix accumulation in two mouse models of renal fibrosis. This was facilitated by an amplified, antifibrogenic, inflammatory T cell response and diminished canonical Wnt/ß-catenin signaling in stressed tubular epithelial cells. Moreover, in humans, urinary DKK3 levels specifically correlated with the extent of tubular atrophy and interstitial fibrosis in different glomerular and tubulointerstitial diseases. In summary, our data suggest that DKK3 constitutes an immunosuppressive and a profibrotic epithelial protein that might serve as a potential therapeutic target and diagnostic marker in renal fibrosis.

Transcriptional profiling of dendritic cells matured in different osmolarities.

Tissue-specific microenvironments shape the fate of mononuclear phagocytes [1-3]. Interstitial osmolarity is a tissue biophysical parameter which considerably modulates the phenotype and function of dendritic cells [4]. In the present report we provide a detailed description of our experimental workflow and bioinformatic analysis applied to our gene expression dataset (GSE72174), aiming to investigate the influence of different osmolarity conditions on the gene expression signature of bone marrow-derived dendritic cells. We established a cell culture system involving murine bone marrow cells, cultured under different NaCl-induced osmolarity conditions in the presence of the dendritic cell growth factor GM-CSF. Gene expression analysis was applied to mature dendritic cells (day 7) developed in different osmolarities, with and without prior stimulation with the TLR2/4 ligand LPS.