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Keratin 8/18, the predominant keratin pair of simple epithelia, is known to be aberrantly expressed in several squamous cell carcinomas (SCCs), where its expression is often correlated with increased invasion, neoplastic progression, and poor prognosis. The majority of keratin 8/18 structural and regulatory functions are governed by posttranslational modifications, particularly phosphorylation. Apart from filament reorganization, cellular processes including cell cycle, cell growth, cellular stress, and apoptosis are known to be orchestrated by K8 phosphorylation at specific residues in the head and tail domains. Even though deregulation of K8 phosphorylation at two significant sites (Serine73 /Serine431 ) has been implicated in neoplastic progression of SCCs by various in vitro studies, including ours, it is reported to be highly context-dependent. Therefore, to delineate the precise role of Kereatin 8 phosphorylation in cancer initiation and progression, we have developed the tissue-specific transgenic mouse model expressing Keratin 8 wild type and phosphodead mutants under Keratin 14 promoter. Subjecting these mice to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate-mediated skin carcinogenesis revealed that Keratin 8 phosphorylation may lead to an early onset of tumors compared to Keratin 8 wild-type expressing mice. Conclusively, the transgenic mouse model developed in the present study ascertained a positive impact of Keratin 8 phosphorylation on the neoplastic transformation of skin-squamous cells.

Introduction: Oral cavity cancer is one of the most common cancers in Indian subcontinent. Since conventional methods have limited sensitivity resulting in lower treatment outcomes and higher cost, molecular-based diagnostic markers may be useful in the diagnosis of oral cancer. The objective of this study is to evaluate and compare expression of CK8 in normal oral mucosa as well as well-differentiated, moderately differentiated and poorly differentiated oral squamous cell cancer (SCC). Materials and Methods: In present study, 148 pathological specimens of histologically proven oral SCC, well differentiated (n= 58), moderately differentiated (n=50), poorly differentiated (n= 20) oral SCC and normal oral mucosa (n=20) were stained with CK8 by immunohistochemistry. Correlation between different histological types and expression of CK8 was evaluated statistically by Chi- Square Test. Results: Increased expression of CK8 was seen in oral SCC. Staining intensity and pattern showed variation in various histological grade of oral SCC. The expression of CK8 was correlated with higher histological grading and the results were statistically significant. Conclusions: CK8 can be used as a predictive marker for malignant transformation and utilized as a prognostic marker by aiding differentiation of histological grades of oral SCC. Keywords: Cytokeratin, Oral Cancer, Squamous cell carcinoma.

Background Current management of AKI, a potentially fatal disorder that can also initiate or exacerbate CKD, is merely supportive. Therefore, deeper understanding of the molecular pathways perturbed in AKI is needed to identify targets with potential to lead to improved treatment. Methods We performed single-cell RNA sequencing (scRNA-seq) with the clinically relevant unilateral ischemia-reperfusion murine model of AKI at days 1, 2, 4, 7, 11, and 14 after AKI onset. Using real-time quantitative PCR, immunofluorescence, Western blotting, and both chromogenic and single-molecule in situ hybridizations, we validated AKI signatures in multiple experiments. Results Our findings show the time course of changing gene expression patterns for multiple AKI stages and all renal cell types. We observed elevated expression of crucial injury response factors-including kidney injury molecule-1 (Kim1), lipocalin 2 (Lcn2), and keratin 8 (Krt8)-and of several novel genes (Ahnak, Sh3bgrl3, and Col18a1) not previously examined in kidney pathologies. AKI induced proximal tubule dedifferentiation, with a pronounced nephrogenic signature represented by Sox4 and Cd24a. Moreover, AKI caused the formation of "mixed-identity cells" (expressing markers of different renal cell types) that are normally seen only during early kidney development. The injured tubules acquired a proinflammatory and profibrotic phenotype; moreover, AKI dramatically modified ligand-receptor crosstalk, with potential pathologic epithelial-to-stromal interactions. Advancing age in AKI onset was associated with maladaptive response and kidney fibrosis. Conclusions The scRNA-seq, comprehensive, cell-specific profiles provide a valuable resource for examining molecular pathways that are perturbed in AKI. The results fully define AKI-associated dedifferentiation programs, potential pathologic ligand-receptor crosstalk, novel genes, and the improved injury response in younger mice, and highlight potential targets of kidney injury.

Various subtypes of circulating cancer-associated cells in the blood are described. A unique circulating, large, and polymorphonuclear cell with a dual epithelial and myeloid phenotype has been suggested as a tumor-macrophage fusion cell (TMF). The goal of the study was to identify the impact of distinct TMFs on survival among patients with NSCLC.In this prospective trial, 7.5 mL of whole blood sample was collected. After microfilter enrichment, immunofluorescent staining was performed, identifying TMFs as greater than or equal to 30 μm in size and dual epithelial (cytokeratin 8, 18, or 19-, or epithelial cell adhesion molecule-positive) and myeloid- or macrophage-positive (CD14- or CD45-positive) cells with at least one 4',6-diamidino-2-phenylindole+ nucleus.Circulating TMFs were identified in 88 of 115 patients (76.5%) with NSCLC (mean 3.052 [SEM ± 0.306]; median 2 [range 0-17]) but were rare in long-term smokers without cancer (6 of 87 [6.9%]; 0.081 [±0.034]; 0 [0-2]), and absent in 20 healthy controls. Comparing the presence of TMFs in patients with NSCLC versus smokers without cancer, specificity was 93.1% (95% confidence interval: 85.6-97.4%) and sensitivity 76.5% (95% confidence interval: 67.7%-83.9%). TMF counts correlated with American Joint Committee on Cancer tumor stages. More importantly, more than one TMF and giant TMFs sizes greater than or equal to 50 μm were associated with statistically significantly shorter overall and cancer-specific disease-free (p0.05) survival after curative resection for stage I to IIIA. Giant TMFs greater than or equal to 50 μm size were an independent survival predictor by multivariate analysis.Circulating, in particular, giant TMFs are associated with aggressive clinical behavior in surgically treated patients with NSCLC. The biological role of unique TMFs will need to be further investigated, as these may have a potential impact on immune responses toward cancer.

Taste bud cells are specialized epithelial cells that undergo continuous turnover, and thus require active progenitors for their renewal and an intact taste function. Our previous studies suggested that a population of taste bud cells originates from outside of the surrounding tongue epithelium—previously regarded sole source of taste bud progenitors. In this study, we demonstrated that SOX10 (SRY-related HMG-box gene 10)-expressing cells, known to be in the migrating neural crest, were also distributed in taste bud-surrounding tissue compartments under the tongue epithelium, that is, the connective tissue core of taste papillae and von Ebner's glands. By lineage tracing of SOX10-expressing cells using SOX10-Cre, a Cre model driven by the endogenous SOX10 promoter, crossing with a Cre reporter line R26-tdTomato (tdT), we found SOX10-Cre-labeled tdT(+) cells within taste buds in all three types of taste papillae (fungiform, circumvallate, and foliate) as well as in the soft palate in postnatal mice. The tdT(+) taste bud cells were progressively more abundant along the developmental stages, from virtually zero at birth to over 35% in adults. Most of tdT(+) taste bud cells had a low intensity of immunosignals of Keratin 8 (a widely used taste bud cell marker). In circumvallate taste buds, tdT signals were co-localized principally with a type III taste bud cell marker, less so with type I and II cell makers. Together, our data demonstrate a novel progenitor source for taste buds of postnatal mice—SOX10-Cre-labeled cells in the connective tissue core and/or von Ebner's glands.

Pregnant CD-1 mice received 200 ppm dimethylarsinic acid (DMA) in the drinking water from gestation day 8-18, and tumor formation was assessed in offspring at the age of 84 weeks. DMA elevated the incidence of lung adenocarcinoma (10.0%) and total tumors (33.3%) in male offspring compared to male control offspring (1.9 and 15.1%, respectively). DMA also elevated the incidence of hepatocellular carcinoma (10.0%) in male offspring compared to male control offspring (0.0%). DMA and its metabolites were detected in the lungs of transplacental DMA-treated neonatal mice. Transplacental DMA exposure increased cell proliferation in the epithelium in the lungs of both neonatal and 6-week-old male mice. Microarray and real-time PCR analyses detected high expression of keratin 8 (Krt8) in the lungs of both neonatal and 6-week-old DMA-treated mice. Western blot analysis indicated that DMA elevated methylation of histone H3K9, but not H3K27, in the lungs of male mice. Importantly, chromatin immunoprecipitation sequencing (ChIP-seq) analysis using an H3K9me3 antibody found differences in heterochromatin formation between mice exposed to DMA and the controls. Notably, ChIP-seq analysis also found regions of lower heterochromatin formation in DMA-treated mice, and one of these regions contained the Krt8 gene, agreeing with the results obtained by microarray analysis. High expression of Krt8 was also detected in adenoma and adenocarcinoma of the lung in male offspring. Overall, these data indicate that transplacental DMA treatment enhanced lung and liver carcinogenesis in male mice. In the lung, DMA caused aberrant methylation of histone H3K9, increased Krt8 expression, and enhanced cell proliferation.

Proliferative vitreoretinopathy (PVR) is a severe ocular disease which results in complex retinal detachment and irreversible vision loss. The epithelial‐mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is considered to be critical in the pathogenesis of PVR. In this study, we focused on the potential impact of keratin 8 (KRT8) phosphorylation and autophagy on TGF‐β2–induced EMT of RPE cells and explored the relationship between them. Using immunofluorescence and Western blot analysis, the co‐localization of KRT8 and autophagy marker, as well as the abundance of phosphorylated KRT8 (p‐KRT8) expression, was observed within subretinal and epiretinal membranes from PVR patients. Moreover, during TGF‐β2–induced EMT process, we found that p‐KRT8 was enhanced in RPE cells, which accompanied by an increase in autophagic flux. Inhibition of autophagy with pharmacological inhibitors or specific siRNAs was associated with a reduction in cell migration and the synthesis of several EMT markers. In the meantime, we demonstrated that p‐KRT8 was correlated with the autophagy progression during the EMT of RPE cells. Knockdown the expression or mutagenesis of the critical phosphorylated site of KRT8 would induce autophagy impairment, through affecting the fusion of autophagosomes and lysosomes. Therefore, this study may provide a new insight into the pathogenesis of PVR and suggests the potential therapeutic value of p‐KRT8 in the prevention and treatment of PVR.

Background: Currently, the identification of Clara cell and type II alveolar epithelial cell-type cancer cells requires electron microscopy, which is a time-consuming and expensive process involving a complicated tissue sampling procedure. The aim of this study was to identify unique biomarkers for Clara cell and type II alveolar epithelial cell-type lung cancer cells, respectively, with proteomic profiling. Methods: Six human lung adenocarcinoma cell lines (A549, NCI-H358, NCI-H1650, HCC827, NCI-H1395, and NCI-H1975) were investigated for their ultrastructural characteristics. The differentially expressed proteins (DEPs) were screened between NCI-H358 cells (Clara cell type) and A549 cells (type II alveolar epithelial cell type) using two-dimensional difference gel electrophoresis (2D-DIGE) and matrix- assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS/MS), and then they were validated by western blot. The protein expression levels of endoplasmic reticulum oxidoreductin 1-α (ERO1L), Clara cell 10-kD protein (CC10), and surfactant protein C (SP-C) were also determined in the six cell lines assayed. Results: NCI-H358 cells featured Clara cell differentiation; A549, NCI-H1975, and HCC827 cells had characteristics of type II alveolar epithelial cells; and NCI-H1395 and NCI-H1650 cells had no differentiation characteristics of any lung adenocarcinoma cell type. Five DEPs including ubiquitin carboxyl- terminal hydrolase isozyme L1 (UCHL1), cytokeratin 19 (CK19), cytokeratin 8 (CK8), ERO1L, and peroxiredoxin 2 (PRDX2) between NCI-H358 and A549 cells were identified for further validation; however, none of them showed suitability as an effective biomarker. Similarly, CC10 and SP-C were not appropriate biomarkers. Conclusions: Cytological subtypes of NCI-H1975 and HCC827 cells were identified, but no promising biomarker was discovered in the present study.

Purpose To investigate keratin 8 (KRT8) level in the aqueous humor (AH) of patients with neovascular age-related macular degeneration (nAMD) and elucidate its association with intravitreal ranibizumab (IVR) treatment outcomes. Methods This prospective study involved 58 eyes of treatment-naïve nAMD patients treated with three IVR doses monthly and whose AH samples were collected at baseline and two months after the initial treatment. KRT8 level was determined using the enzyme-linked immunosorbent assay and compared with that of the control group, which comprised patients who underwent cataract surgery during the same period. The nAMD-affected eyes were classified into responder (dry) and poor responder (persistent fluid) groups, according to optical coherence tomography (OCT) findings at month three. Additionally, associations between the KRT8 level and IVR treatment outcomes were analyzed. Results The baseline KRT8 level was significantly higher in the AMD group than in the control group. In the AMD group, responders demonstrated significant differences between the KRT8 level at the baseline and month two, whereas poor responders exhibited no significant change. Regression analysis revealed that a higher KRT8 level at month two was significantly associated with persistent fluid upon OCT at months three and six. Conclusions Monitoring aqueous KRT8 level may facilitate early determination of the therapeutic effects of IVR in nAMD patients and reflect the conditions of retinal pigment epithelium during the disease course. Translational Relevance Monitoring aqueous KRT8 may aid early determination of therapeutic effects of IVR in neovascular AMD patients and reflect the health conditions of retinal pigment epithelium.

Background Adhesion formation contributes to postoperative complications in abdominal and gynaecological surgery. Thus far, the prevention and treatment strategies have focused on mechanical barriers in solid and liquid form, but these methods are not in routine use. As autologous fat grafting has become popular in treatment of hypertrophic scars because of its immunomodulatory effects, we postulated that fat grafting could also prevent peritoneal adhesion through similar mechanisms. Methods This was a control versus intervention study to evaluate the effect of fat grafting in the prevention on peritoneal adhesion formation. An experimental mouse model for moderate and extensive peritoneal adhesions was used (n = 4–6 mice/group). Adhesions were induced mechanically, and a free epididymal fat graft from wild type or CAG-DsRed mice was injected preperitoneally immediately after adhesion induction. PET/CT imaging and scaling of the adhesions were performed, and samples were taken for further analysis at 7 and 30 days postoperation. Macrophage phenotyping was further performed from peritoneal lavage samples, and the expression of inflammatory cytokines and mesothelial layer recovery were analysed from peritoneal tissue samples. Results Fat grafting significantly inhibited the formation of adhesions. PET/CT results did not show prolonged inflammation in any of the groups. While the expression of anti-inflammatory and anti-fibrotic IL-10 was significantly increased in the peritoneum of the fat graft–treated group at 7 days, tissue-resident and repairing M2 macrophages could no longer be detected in the fat graft at this time point. The percentage of the continuous, healed peritoneum as shown by Keratin 8 staining was greater in the fat graft–treated group after 7 days. Conclusions Fat grafting can inhibit the formation of peritoneal adhesions in mice. Our results suggest that fat grafting promotes the peritoneal healing process in a paracrine manner thereby enabling rapid regeneration of the peritoneal mesothelial cell layer.

Fistula treatment represents a major unmet medical need in the therapy of Crohn’s disease (CD). Current medical therapies, such as anti-TNF antibody treatments, are often insufficient and do not achieve permanent fistula closure. Previously published data point toward a critical role for metalloproteinase-9 (MMP-9)/gelatinase B in fistula pathogenesis. The aim of this project was to investigate in detail MMP-9 expression in different fistula types and to confirm that MMP-9 is a potential target for fistula therapy in CD patients. Immunohistochemistry for total and active MMP-9, Cytokeratin 8 (CK-8) and co-staining of active MMP-9/CK-8 was performed in specimen derived from perianal fistulas, entero-enteric fistulas and fistulas from patients not responding to anti-TNF therapy. In addition, fistulas from the xenograft mouse model (anti-TNF treated or untreated) were analyzed. Total and active MMP-9 protein was detectable in cells lining the tracts of perianal and entero-enteric fistulas. Of note, total and active MMP-9 was also expressed in fistulas of CD patients non-responding to anti-TNF treatment. Interestingly, we detected considerable co-staining of active MMP-9 and CK-8 in particular in cells lining the fistula tract and in transitional cells around the fistulas. Furthermore, total and active MMP-9 are detectable in both anti-TNF treated and untreated xenograft fistulas. Taken together, our data suggest that MMP-9 is involved in fistula pathogenesis in CD patients, in fistulas of different origins and particularly in patients non-responding to anti-TNF therapy. Our xenograft fistula model is suitable for in vivo studies investigating a possible therapeutic role for MMP-9 targeting as fistula therapy.

Keratin 8 (K8) is the cytoskeletal intermediate filament protein of simple-type epithelia. Mutations in K8 predispose the affected individual and transgenic mouse to liver disease. However, the role of K8 in the lung has not been reported in mutant transgenic mouse models. Here, we investigated the susceptibility of two different transgenic mice expressing K8 Gly62–Cys (Gly62 replaced with Cys) or Ser74–Ala (Ser74 replaced with Ala) to lung injury. The mutant transgenic mice were highly susceptible to two independent acute and chronic lung injuries compared with control mice. Both K8 Gly62–Cys mice and K8 Ser74–Ala mice showed markedly increased mouse lethality (∼74% mutant mice versus ∼34% control mice) and more severe lung damage, with increased inflammation and apoptosis, under L-arginine-mediated acute lung injury. Moreover, the K8 Ser74–Ala mice had more severe lung damage, with extensive hemorrhage and prominent fibrosis, under bleomycin-induced chronic lung injury. Our study provides the first direct evidence that K8 mutations predispose to lung injury in transgenic mice.

Background and aimsCell-cell adhesion structures (desmosomes and, especially, tight junctions) are known to play important roles in control of transepithelial permeability in the colon. The involvement of cell-matrix interactions in permeability control is less clear. The goals of the present study were to: i) determine if disruption of colon epithelial cell interactions with the extracellular matrix alters permeability control and ii) determine if increasing the elaboration of protein components of cell-matrix adhesion complexes improves permeability control and mitigates the effects of cell-matrix disruption.MethodsHuman colon organoids were interrogated for transepithelial electrical resistance (TEER) under control conditions (0.25 mM calcium) and in the presence of Aquamin®, a multi-mineral product, at a level providing 1.5 mM calcium. The effects of Aquamin® on cell-matrix adhesion protein expression were determined in a proteomic screen and by Western blotting. In parallel, TEER was assessed in the presence of a function-blocking antibody directed at an epitope in the C-terminal region of laminin α3 chain.ResultsTreatment of colon organoids with Aquamin® increased the expression of multiple basement membrane and hemidesmosomal proteins as well as keratin 8 and 18. TEER values were higher in the presence of Aquamin® than they were under control conditions. Anti-laminin antibody reduced TEER values under all conditions but was most effective in the absence of Aquamin®, where laminin expression was low and TEER values were lower to begin with.ConclusionsThese findings provide evidence that cell-matrix interactions contribute to permeability control in the colon. They suggest that the elaboration of proteins important to cell-matrix interactions can be increased in human colon organoids by exposure to a multi-mineral natural product. Increasing the elaboration of such proteins may help to mitigate the consequences of disrupting cell-matrix interactions on permeability control.

Paget’s disease (PD) of the breast is an uncommon disease of the nipple usually accompanied by an underlying carcinoma, often HER2 + , and accounting for 0.5–5% of all breast cancer. To date, histogenesis of PD of the breast remains controversial, as two theories—transformation and epidermotropic—have been proposed to explain this disease. Currently, animal models recapitulating PD of the nipple have not been described. HER2-enriched DT13 breast cancer cells were injected into the mammary fat pad of NOD scid gamma null (NSG) female mice. Immunohistochemical staining and pathological studies were performed on tumor samples, and diagnosis of PD of the nipple was confirmed by expression of proteins characteristic of Paget cells (epidermal growth factor 2 (HER2), androgen receptor (AR), cytokeratin 7 (CK7), cytokeratin 8/18 (CK8/18), and mucin 1 (MUC1)). In addition, DT13 cells grown in 2D culture and in soft agar assays were sensitive to in vitro treatment with pharmacological inhibitors targeting Her2, adenylyl cyclase, mTOR, and PI3K signaling pathways. Mice developed tumors and nipple lesions that were detected exclusively on the tumor-bearing mammary fat pad. Tumor cells were positive for proteins characteristic of Paget cells. In vitro, DT13 cells were sensitive to inhibition of Her2, adenylyl cyclase, mTOR, and PI3K signaling pathways. Our results suggest that injection of HER2 + DT13 cells into the mammary fat pad of NSG mice recapitulates critical aspects of the pathophysiology of PD of the nipple, supporting the epidermotropic theory as the more likely to explain the histogenesis of this disease.

Cellular and molecular life sciences : (CMLS) 77(3), 543-558 (2020). doi:10.1007/s00018-019-03198-y, Published by Springer International Publishing AG, Cham (ZG)

Introduction: The role of keratin expression patterns as candidate tumour markers continues to be under investigation in human cervix carcinogenesis. Keratin comprise of family of at least 20 intermediate filament proteins that have a specific distribution pattern in epithelial tissue. Objective: The present study was conducted with an aim to identify CINI, II and CINIII in tissue sections with the help of immunohistochemistry of specific diagnostic markers so as to reduce the burden of invasive cervical carcinoma and to evaluate the role of cytokeratin 8,10,13 and 17 for differentiating CINIII from cervical carcinoma along with its correlation with histopathological diagnosis of these lesions. Method: We examined the immunohistochemical staining of CK8, CK10, CK13 and CK17 in 64 cases of reference cervix, CINIII lesions and invasive cervical carcinoma. Results: In present study cytokeratin 8 has sensitivity 40% and specificity 100%, cytokeratin 10 has sensitivity 80% and specificity 40%, cytokeratin 13 has sensitivity 100% and specificity 80% and cytokeratin 17 has sensitivity 40% and specificity 100% in invasive cervical carcinoma. In the CIN III lesions, cytokeratin 8 has sensitivity 56% and specificity 100%, cytokeratin 10 has sensitivity 80% and specificity 79%, cytokeratin 13 has sensitivity 100% and specificity 75% and cytokeratin 17 has sensitivity 72% and specificity 100% in cervical intraepithelial lesion III. Conclusions: We observed that expression of keratins 8 and 17 and loss of keratins 10 and 13 are good markers of malignant transformation in human cervix. Keratin expression patterns, namely expressions of keratin 10 can be useful for studying and grading squamous cell carcinomas of the cervix.

Renal cell carcinoma (RCC) is one of the most lethal urogenital cancers and effective treatment of metastatic RCC remains an elusive target. Cell lines enable the in vitro investigation of molecular and genetic changes leading to renal carcinogenesis and are important for evaluating cellular drug response or toxicity. This study details a fast and easy protocol of establishing epithelial and fibroblast cell cultures or cell lines concurrently from renal cancer nephrectomy tissue. The protocol involves mechanical disaggregation, collagenase digestion and cell sieving for establishing epithelial cells while fibroblast cells were grown from explants. This protocol has been modified from previous published reports with additional antibiotics and washing steps added to eliminate microbial contamination from the surgical source. Cell characterisation was carried out using immunofluorescence and quantitative polymerase chain reaction. Eleven stable epithelial renal tumour cell lines of various subtypes, including rare subtypes, were established with a spontaneous immortalisation rate of 21.6% using this protocol. Eight fibroblast cell cultures grew successfully but did not achieve spontaneous immortalisation. Cells of epithelial origin expressed higher expressions of epithelial markers such as pan-cytokeratin, cytokeratin 8 and E-cadherin whereas fibroblast cells expressed high α-smooth muscle actin. Further mutational analysis is needed to evaluate the genetic or molecular characteristics of the cell lines.

Solo (ARHGEF40) is a RhoA-targeting guanine nucleotide exchange factor that regulates tensional force-induced cytoskeletal reorganization. Solo binds to keratin 8/keratin 18 (K8/K18) filaments through multiple sites, but the roles of these interactions in the localization and mechanotransduction-regulating function of Solo remain unclear. Here, we constructed two Solo mutants (L14R/L17R and L49R/L52R) with leucine-to-arginine replacements in the N-terminal conserved region (which we termed the Solo domain) and analyzed their K18-binding activities. These mutations markedly decreased the K18-binding ability of the N-terminal fragment (residues 1-329) of Solo but had no apparent effect on the K18-binding ability of full-length (FL) Solo. When expressed in cultured cells, wild-type Solo-FL showed a unique punctate localization near the ventral surface of cells and caused the reinforcement of actin filaments. In contrast, despite retaining the K18-binding ability, the L14R/L17R and L49R/L52R mutants of Solo-FL were diffusely distributed in the cytoplasm and barely induced actin cytoskeletal reinforcement. Furthermore, wild-type Solo-FL promoted traction force generation against extracellular matrices and tensional force-induced stress fiber reinforcement, but its L14R/L17R and L49R/L52R mutants did not. These results suggest that the K18-binding ability of the N-terminal Solo domain is critical for the ventral localization of Solo and its function in regulating mechanotransduction.

Research on sex-determining region Y-box 2 (SOX2)-positive pituitary stem/progenitor cells, as a source of hormone-producing cells, is progressing rapidly in rodents. However, the stem/progenitor cells supplying hormone-producing cells that are essential for growth, reproduction, and lactation in bovines have not yet been identified. In this study, we characterized SOX2-positive cells in the pituitary gland of dairy cattle (Holstein heifers) after sexual maturity. Immunofluorescence analysis revealed that the localization pattern of SOX2-positive cells in the dairy cattle pituitary gland was similar to that observed in the rodent pituitary gland; the marginal cell layer (MCL), dense cell clusters, and single cells scattered in the parenchyma of the anterior lobe. Furthermore, most of the SOX2-positive cells were positive for the pituitary stem/progenitor cell niche markers E-cadherin and cytokeratin 8+18, which have been reported in rodents. In addition, in the MCL of the anterior lobe, there was a subpopulation of SOX2-positive cells positive for paired-related homeobox 1 and 2, whereas negative for S100β. Moreover, in the parenchyma of the anterior lobe, co-localization of SOX2 and pituitary hormones was infrequent. In summary, this study reveals the localization of putative pituitary stem/progenitor cells positive for SOX2 in dairy cattle. These results provide valuable information to support further investigation of cell supply in the dairy cattle pituitary gland.

Dysregulation of mitochondrial homeostasis and accumulation of damaged mitochondria cause degenerative diseases such as age-related macular degeneration (AMD). We studied the effects of the intermediate cytofilament KRT8 (keratin 8) on mitochondrial homeostasis in relation to the morphology and function of mitochondria in retinal pigment epithelial cells under oxidative stress. When the mitochondria were damaged owing to oxidative stress, the damaged mitochondria were readily disposed of via mitophagy following mitochondrial fission. During this process, KRT8 was found to physically interact with the mitochondria through PLEC (plectin) and facilitate the mitochondrial fission-mediated mitophagy. However, the association between PLEC-anchoring mitochondria and KRT8 was dwindled by KRT8 phosphorylation under oxidative stress. The efficient KRT8-facilitated mitophagy flux suppressed the accumulation of damaged mitochondria and consequently diminished necrotic cell death under oxidative stress. Thus, by facilitating mitophagy, KRT8 protects RPE cells against necrotic cell death due to oxidative stress. Abbreviations: 3-MA: 3-methyladenine; AMD: age-related macular degeneration; DDIT3: DNA damage inducible transcript 3; DNM1L: dynamin 1 like; ER: endoplasmic reticulum; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCN1: GCN1 activator of EIF2AK4; IF: intermediate filament; KRT8: keratin 8; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3; MFF: mitochondrial fission factor; MMP: mitochondrial membrane potential; OCR: oxygen consumption rate; PLEC: plectin; ROS: reactive oxygen species; RPE: retinal pigment epithelium; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20

The cytoskeleton is a major determinant of cell mechanics, and alterations in the central mechanical aspects of cells are observed during many pathological situations. Therefore, it is essential to investigate the interplay between the main filament systems of the cytoskeleton in the form of composite networks. Here, we investigate the role of keratin intermediate filaments (IFs) in network strength by studying in vitro reconstituted actin and keratin 8/18 composite filament networks via bulk shear rheology. We co-polymerized these structural proteins in varying ratios and recorded how their relative content affects the overall mechanical response of the various composites. For relatively small deformations, we found that all composites exhibited an intermediate linear viscoelastic behaviour compared to that of the pure networks. In stark contrast, when larger deformations were imposed the composites displayed increasing strain stiffening behaviour with increasing keratin content. The extent of strain stiffening is much more pronounced than in corresponding experiments performed with vimentin IF as a composite network partner for actin. Our results provide new insights into the mechanical interplay between actin and keratin filaments in which keratin provides reinforcement to actin. This interplay may contribute to the overall integrity of cells. Hence, the high keratin 8/18 content of mechanically stressed simple epithelial cell layers, as found in the lung and the intestine, provides an explanation for their exceptional stability.

AIMS In somatotroph pituitary neuroendocrine tumours (adenomas), a pattern of cytokeratin (CK) 18 expression is used for tumour subclassification, with possible clinical implications. Rare somatotroph tumours do not express CK 18. We aimed to characterise this subset clinically and histologically. METHODS AND RESULTS Clinical and pathological data for the study were derived from a previously published data set of a cohort of 110 patients with acromegaly. Data included serum levels of insulin-like growth factor 1 (IGF1), growth hormone (GH), prolactin and thyroid-stimulating hormone (TSH), tumour diameter, tumour invasion defined by Knosp grade and immunohistochemical data concerning the expression of Ki67, p53, E-cadherin, somatostatin receptor (SSTR)1, SSTR2A, SSTR3, SSTR5 and D2 dopamine receptor. Additional immunohistochemical analysis (AE1/3, CK 8/18, vimentin, neurofilament light chain, internexin-α) was performed. CK 18 was negative in 10 of 110 (9.1%) tumours. One of these tumours was immunoreactive with CK 8/18 antibody, while the remainder expressed only internexin-α intermediate filament in patterns similar to CK 18 (perinuclear fibrous bodies). CK-negative tumours showed no significant differences with respect to biochemical, radiological or pathological features. They showed significantly higher expression of SSTR2A compared to the sparsely granulated subtype and significantly lower expression of E-cadherin compared to the non-sparsely granulated subtypes of tumours. The tumours showed divergent morphology and hormonal expression: two corresponded to densely granulated tumours and three showed co-expression of prolactin and morphology of either mammosomatotroph or somatotroph-lactotroph tumours. Four tumours showed morphology and immunoprofile compatible with plurihormonal Pit1-positive tumours. CONCLUSIONS CK-negative somatotroph tumours do not represent a distinct subtype of somatotroph tumours, and can be further subdivided according to their morphology and immunoprofile.

Reproduction in males requires the transfer of spermatozoa from testis tubules via the rete system to the efferent ductules, epididymis, and vas deferens. The rete therefore forms an essential bridging system between the testis and excurrent ducts. Yet the embryonic origin and molecular regulation of rete testis development is poorly understood. This review examines the anatomy, endocrine control, and development of the mammalian rete testis, focusing on recent findings on its molecular regulation, identifying gaps in our knowledge, and identifying areas for future research. The rete testis develops in close association with Sertoli cells of the seminiferous cords, although unique molecular markers are sparce. Most recently, modern molecular approaches such as global RNA-seq have revealed the transcriptional signature of rete cell precursors, pointing to at least a partial common origin with Sertoli cells. In the mouse, genes involved in Sertoli cell development or maintenance, such as Sox9, Wt1, Sf1, and Dmrt1, are also expressed in cells of the rete system. Rete progenitor cells also express unique markers, such as Pax8, E-cadherin, and keratin 8. These must directly or indirectly regulate the physical joining of testis tubules to the efferent duct system and confer other physiological functions of the rete. The application of technologies such as single-cell RNA-seq will clarify the origin and developmental trajectory of this essential component of the male reproductive tract.

Summary Loss of SMARCB1 protein expression has recently been identified in a variety of tumor types such as poorly differentiated chordoma (PCh) and malignant rhabdoid tumor (MRT) including atypical teratoid/rhabdoid tumor (AT/RT). PCh is characterized by poorly differentiated epithelioid tumor cells, sheet arrangement, and coexpression of nonepithelial and epithelial markers. Rhabdoid cells are sometimes present. Therefore, the differentiation of these tumors is often difficult. Brachyury is a transcription factor within the T-box family typically expressed in notochord tissue and chordomas. Some studies have reported high specificity and sensitivity of brachyury expression in chordomas. In the present study, we analyzed immunohistochemical brachyury expression in SMARCB1-deficient tumors and discuss important clinicopathological and diagnostic points, especially in cases of intracranial SMARCB1-deficient tumors with brachyury expression. Brachyury and cytokeratin immunoexpression status was examined in 42 formalin-fixed paraffin-embedded SMARCB1-deficient tumor specimens (PCh, 6 cases; extra-central nervous system [CNS] MRT, 26 cases; AT/RT, 10 cases) and 25 cases of conventional chordoma (CCh). All cases of PCh and CCh showed diffuse immunopositivities for cytokeratin 8, pan-cytokeratin, and brachyury. Brachyury immunoexpression was present in 2 extra-CNS MRT (8%) and 5 AT/RT (50%) cases, but immunopositivity was focal not diffuse. Indeed, in almost all cases of AT/RT (cytokeratin 8, 7/10 cases; pan-cytokeratin, 7/10 cases) and extra-CNS MRT (cytokeratin 8, 23/26 cases; pan-cytokeratin, 25/26 cases), fewer than 50% of cells showed immunoreactivity. Although the histological and clinical features of PCh resemble those of AT/RT, semiquantitative evaluations of the degree of brachyury and cytokeratin immunoexpressivity may help to distinguish PCh from AT/RT.

The cytoskeleton is a major determinant of cell mechanics, a property that is altered during many pathological situations. To understand these alterations, it is essential to investigate the interplay between the main filament systems of the cytoskeleton in the form of composite networks. Here, we investigate the role of keratin intermediate filaments (IFs) in network strength by studying in vitro reconstituted actin and keratin 8/18 composite networks via bulk shear rheology. We co-polymerized these structural proteins in varying ratios and recorded how their relative content affects the overall mechanical response of the various composites. For relatively small deformations, we found that all composites exhibited an intermediate linear viscoelastic behavior compared to that of the pure networks. In stark contrast, the composites displayed increasing strain stiffening behavior as a result of increased keratin content when larger deformations were imposed. This strain stiffening behavior is fundamentally different from behavior encountered with vimentin IF as a composite network partner for actin. Our results provide new insights into the mechanical interplay between actin and keratin in which keratin provides reinforcement to actin. This interplay may contribute to the overall integrity of cells, providing an explanation for the stability of stressed epithelial tissues due to their high keratin contents. Additionally, this helps us to understand the physiological necessity to exchange IF systems during epithelial-mesenchymal transition (EMT) in order to suppress strain stiffening of the network, making cells more elastic and, thus, facilitating their migration through dense tissues.

Rationale: Although vertebral osteomyelitis (VO) is commonly associated with high morbidity and high recurrence rate, effective diagnostic and prognostic biomarkers of VO are still lacking. Patients concerns: Case 1: a 60-year-old male had had upper back pain for 3 days. Case 2: a 71-year-old female presented upper back pain for 2 days. Diagnoses: Based on physical examination and findings of magnetic resonance imaging and findings by matrix-assisted laser desorption ionization-time of flight mass spectrometry, they were diagnosed with Staphylococcus aureus VO. Interventions: Using Sengenics ImmunomeTM Protein Array by analyzing autoantibodies in both VO patients, potential biomarkers of VO were explored. Outcomes: Four subjects with more than 1600 antigens screened while the results showed that 14-3-3 protein gamma, pterin-4-alpha-carbinolamine dehydratase, fructose-bisphosphate aldolase A, and keratin type II cytoskeletal 8 were highly differentially expressed among VO and controls. Relevant auto-antibody profiles were discovered after intra-group and inter-group comparison, and based on functional rationality, an adapter protein 14-3-3 protein gamma, and pterin-4-alpha-carbinolamine dehydratase that involved in tetrahydrobiopterin biosynthesis, might serve as valuable diagnostic biomarkers. Lessons: This pilot study on 4 subjects with more than 1600 antigens screened on the Sengenics Immunome protein array provided a general outlook on autoantibody biomarker profiles of VO subjects. Future large-scale trials with longer follow-up times are warranted.

Epitope H contains an O-linked N-acetylglucosamine (O-GlcNAcH) residue in a specific conformation and/or environment recognized by the mouse monoclonal antibody H. O-GlcNAcH is present in several types of cells and in several polypeptides, including cytokeratin 8 and vimentin, on the latter in cells under stress. In the present work, we examined the expression of the O-GlcNAcH in 60 cases of endometrial curettings from missed miscarriage cases containing normal and simple hydropic degenerated chorionic villi in each case, using monoclonal antibody H and indirect immunoperoxidase and Western blot immunoblot. In all cases examined the expression of the O-GlcNAcH was cytoplasmic as follows: (1) syncytiotrophoblastic cells showed very low expression in chorionic villi (CV) with nonhydropic degeneration (NHD) and high expression in hydropic degenerated (HD) CV; (2) cytotrophoblastic cells showed low expression in CV with NHD and high expression in HD CV; (3) fibroblastic cells showed high expression in CV with NHD and very low expression in HD CV; (4) histiocytes showed very low expression in both types of CV; (5) endothelial cells showed high expression in both types of CV. An immunoblot of CV from one case of a legal abortion from a normal first-trimester pregnancy showed 5 polypeptides with 118.5, 106.3, 85, 53, and 36.7 kD bearing the epitope H and the 53 kD corresponded to cytokeratin 8. The expression of the O-GlcNAcH is upregulated in the trophoblastic cells and downregulated in the fibroblastic cells in the HD CV in comparison to the NHD CV.

Keratin intermediate filaments constitute the primary cytoskeletal component of epithelial cells. Numerous human disease phenotypes related to keratin mutation remain mechanistically elusive. Our recent crystal structures of the helix 1B heterotetramer from keratin 1/10 enabled further investigation of the effect of pathologic 1B domain mutations on keratin structure. We used our highest resolution keratin 1B structure as a template for homology-modeling the 1B heterotetramers of keratin 5/14 (associated with blistering skin disorders), keratin 8/18 (associated with liver disease), and keratin 74/28 (associated with hair disorder). Each structure was examined for the molecular alterations caused by incorporating pathogenic 1B keratin mutations. Structural modeling indicated keratin 1B mutations can harm the heterodimer interface (R265PK5, L311RK5, R211PK14, I150VK18), the tetramer interface (F231LK1, F274SK74), or higher-order interactions needed for mature filament formation (S233LK1, L311RK5, Q169EK8, H128LK18). The biochemical changes included altered hydrophobic and electrostatic interactions, and altered surface charge, hydrophobicity or contour. Together, these findings advance the genotype-structurotype-phenotype correlation for keratin-based human diseases.

Povzetek: Ateroskleroza je bolezen kopičenja maščobnih delcev v žilno steno arterij. Po svoji naravi je progresivna bolezen, ki jo zaznamuje večanje aterosklerotičnega plaka do točke ko le-ta poči in vsebina izpostavi krvnemu obtoku. To na mestu razpoke povzroči nastanek strdka, ki lahko posledično zapre ali zoži arterijo do te mere, da lahko privede do ishemije tkiva katerega taka arterija prehranjuje. Pri srcu to pomeni ishemijo srčne mišičnine – miokardni infarkt. Ob miokardnem infarktu se v sistemsko cirkulacijo zaradi nekroze srčne mišice izločajo vnetni mediatorji, ti pa vplivajo na večje vnetje v aterosklerotičnih lehah na kar kažejo slikovne študije ožilja po ishemičnem dogodku. Klinično to vidimo tudi v povečanem tveganju za ponovni ishemični dogodek, najvišje v roku enega leta po miokardnem infarktu. Na to kažejo podatki iz številnih epidemioloških študij s celega sveta. Modulacija vnetja po miokardnem infarktu lahko zmanjša tveganje za ponovni dogodek, kar so pokazali podatki tako iz predkliničnih kot tudi iz kliničnih študij. V prvem delu doktorske teze predstavim novo pot imunomodulacije po miokardnem infarktu z inhibicijo receptorja za interlevkin 7. V živalski študiji na miših z aterosklerotično boleznijo in pridruženim miokardnim infarktom pokažem, da interlevkin 7 deluje kot monocitni kemotaktični dejavnik, da je količina interlevkina 7 po miokardnem infarktu v žilni steni povišana ter, da se po miokardnem infarktu poveča sinteza mRNA interlevkina 7 v samem ishemično prizadetem miokardu. Imunomodulacija vnetja z inhibitornim monoklonskim protitelesom proti receptorju interlevkina 7 zmanjša kopičenje maščob v žilni steni ter infiltracijo vnetnih celic, predvsem monocitov in makrofagov, kar pomeni zmanjšanje vnetja in stabilizacijo plaka. V prvem delu doktorskega dela tako pokažem doprinos in pomen vnetja in prirojenega imunskega sistema v proces ateroskleroze. Imunski sistem v grobem sestavljata prirojeni imunski sistem, katerega sestavni del so celice kot so monociti in makrofagi, udeleženi v proces ateroskleroze tako v akutni kot kronični fazi bolezni ter pridobljeni imunski sistem. Po začetni, nespecifični fazi vnetja pride do aktivacije specifičnega, pridobljenega dela imunskega sistema, katerega predstavniki so med drugim limfociti T, limfociti B ter antigen predstavitvene celice. Značilnost tega dela imunskega sistema je tarčno, antigensko specifično vnetje, za katerega so značilne interakcije med receptorji na celicah T in MHC molekulami na antigen predstavitvenih celicah. Znano je, da pri procesu ateroskleroze pride do aktivacije avto-reaktivnih limfocitov T in B in specifičnega odziva le-teh proti maščobnim delcem v aterosklerotičnih lehah. En takih delcev je peptid p210, ki je del molekule ApoB100, sestavni del delcev LDL, glavnih krivcev za nastanek vnetja v žilni steni. Raziskave so pokazale obstoj LDL in apoB-100 reaktivnih celic T, vendar do sedaj specifični epitopi pomembni pri aterosklerozi še niso bili potrjeni. V drugem delu doktorskega dela pokažem prisotnost avtoreaktivnih, antigen specifičnih CD8+ limfocitov T, pri katerih stimulacija s peptidom p210 izzove avto-imunski odziv. Avto-imunski odziv temelji na prepoznavi kompleksa antigen-MHC-I molekula s strani celičnega receptorja T. S pomočjo IEDB baze (Immune Epitope Data Base) smo preverili predvideno vezavo peptidnega fragmenta p210 na mišje molekule MHC-I. Na podlagi izbranega peptidnega fragmenta z visoko predvideno vezavno afiniteto na MHC-I smo izdelali fluorescenčno označen pentamer sestavljen iz petih MHC-I molekul, specifičnih za p210 fragment in preverili prisotnost CD8+ celic T, ki se na pentamer vežejo ob specifični prepoznavi epitopa p210. Miši, ki so prejemale aterogeno dieto z visoko vsebnostjo maščob, so izražale večji delež pentamer pozitivnih CD8+ celic T, ki so imele izražen efektorski fenotip. Te celice se ob stimulaciji s peptidom p210 klonalno razmnožijo, kar je značilnost specifične razpoznave antigena s strani T-celičnega receptorja (TCR). Imunomodulacija z imunizacijo s p210 je povečala delež p210-specifičnih pentamer pozitivnih CD8+ limfocitov T in zmanjšala breme ateroskleroze. Interakcija CD8+ limfocitov T z antigen predstavitvenimi celicami temelji na vezavi antigenskega peptida z vezavnim mestom MHC-I molekule na površini antigen predstavitvenih celic in prepoznavi kompleksa antigen/MHC-I s strani T-celičnega receptorja CD8+ limfocitov T. Znano je, da se MHC-I molekule pri bolezenskih stanjih izločajo v krvni obtok. Te topne MHC-I molekule na svoji površini oz. vezavnem mestu nosijo peptidne fragmente, ki predstavljajo potencialne avto-antigene udeležene v boleznska stanja. Z analizo in identifikacijo peptidov, vezanih na topne MHC-I molekule, lahko prepoznamo antigene pomembne pri procesu ateroskleroze in miokardnega infarkta. V tretjem delu teze predstavim novo metodo odkrivanja potencialnih avto-antigenov udeleženih v proces ateroskleroze in po miokardnem infarktu in prikažem translacijski pristop testiranja relevantnosti odkritih peptidov v biološkem modelu ateroskleroze in miokardnega infarkta. Z metodo imunoprecipitacije smo iz krvi pacientov z akutnim koronarnim sindromom pridobljene v AZACS študiji, ki imajo dokazano aterosklerozo, izolirali peptide, ki so bili ob primerjavi z zdravimi kontrolami specifični za paciente. Peptide smo identificirali s pomočjo masne spektrometrije in primerjave aminokislinskega zaporedja z bazo človeških proteinov Uniprot. S to metodo smo odkrili več potencialnih avto-antigenov udeleženih v proces ateroskleroze in miokardnega infarkta. Za nadaljno testiranje biološkega pomena smo izbrali peptide specifične za paciente, ki so imeli na masni spektrometriji najvišjo intenzivnost signala, ali pa so bili skupni največjem številu pacientov (Keratin 8, ARID1A, Bleomicin hidrolaza ter LL-37). Mišje homologe teh peptidov smo testirali na živalskem modelu ateroskleroze ter ateroskleroze s pridruženim miokardnim infarktom. Od izbranih peptidov sta keratin II (mišji analog človeškemu keratinu 8) ter CRAMP (mišji analog človeškemu LL-37) izzvala največji imunski odziv. Keratin II je v mišjem ApoE(-/-) modelu ateroskleroze izzval predvsem efektorski odziv CD8+ limfocitov T, katerega ni bilo videti v mišjem ApoE(-/-) modelu ateroskleroze s pridruženim miokardnim infarktom. To je bilo razvidno tudi ob stimulaciji perifernih mononuklearnih celic izoliranih iz krvi pacientov s stabilno koronarno boleznijo in pacientov z akutnim koronarnim sindromom. Antimikrobni peptid LL-37, fragment proteina hCAP18, ki se nahaja v granulah nevrofilnih granulocitov in igra pomembno vlogo pri nevtrofilnem vnetju, je znan avto-antigen pri avtoimunih boleznih kot je luskavica. Pacienti z luskavico imajo večje breme ateroskleroze kot splošno, zdravo prebivalstvo in pridruženo višje tveganje za srčno-žilne dogodke. Bolniki z dokazano aterosklerozo imajo po drugi strani dvakrat višjo prevalenco luskavice kot zdravo prebivalstvo. Poleg tega je LL-37 moč najti v aterosklerotičnih lehah. Pomanjkanje mišjega ekvivalenta LL-37, imenovanega CRAMP (Cathelicidin Related Antimicrobial Peptide) v mišjem modelu zmanjša aterosklerotično breme. V namen proučevanja vpletenosti CRAMP v proces ateroskleroze pri miših smo sintetizirali skrajšano različico molekule (tCRAMP – truncated CRAMP), ki je vsebovala predvidena vezavna mesta za mišje MHC-I molekule. Stimulacija mišjih imunskih celic je izzvala močan efektorski odziv celic T, ki je bil še posebej poudarjen pri miših z razširjenim aterosklerotičnim bremenom po hranjenju z dieto bogato z maščobami. V odziv sta bili udeleženi tako CD4 kot tudi CD8 T-celična vrsta. Z imunizacijo kot način imunomodulacije smo preverili pomen tCRAMP antigena pri aterosklerozi. Izbrali smo dve imunizacijski dozi, višjo (100ug) in nižjo (20ug), ki sta imeli zelo različen biološki odziv. Nižja imunizacijska doza je preko povečanja deleža CD8+ celic T centralnega spomina ter znižanja deleža vnetnih dendritičnih celic CD11b+ zmanjšala breme aterosklerotične bolezni. Višja imunizacijska doza pa je, ravno obratno, povečala breme aterosklerotične bolezni in povzročila avto-imunsko preobrazbo imunskega sistema. Videti je bilo povišanje avto-reaktivnosti, predvsem z večjim deležem tako CD4+ kot CD8+ efektorskih celic T kot tudi povečanje deleža dendritičnih celic CD11b+, klasično celic vnetnega fenotipa. V doktorski tezi pokažem pomen tako prirojenega kot pridobljenega imunskega sistema v procesu ateroskleroze in vnetja po miokardnem infarktu ter potencialne nove načine imunomodulacije, ki se lahko uporabljajo za zmanjšanje bremena ateroskleroze. Teza predstavi pomen interlevkin 7 vnetne poti in vpletenost le-te v vnetje po miokardnem infarktu. Inhibicija receptorja interlevkina 7 zmanjša posti-ishemično vnetje v aterosklerotičnih lehah. Monoklonska protitelesa proti interlevkinu 7 so bila že prestala varnostno klinično študijo in predstavljajo zanimivo priložnost za klinično uporabo imunomodulacije po miokardnem infarktu. Doktorsko delo je pomembno tudi zaradi predstavitve novega načina raziskovanja avto-antigenov udeleženih v proces ateroskleroze. Določanje imunopeptidoma iz krvi pacientov s koronarno boleznijo je izvedljivo in privede do odkrivanja antigenov biološkega pomena. Predstavim tudi pomembno translacijsko pot raziskovanja in testiranja teh antigenov v pristopu, ki vodi od pacienta do laboratorijskega testiranja hipoteze in nazaj do pacienta in omogoča pridobivanje potencialno pomembnih kliničnih odkritij. V svojem delu tako s translacijskim pristopom pokažem poglobljen pomen avto-antigena LL-37 odkritega v vzorcih krvi pacientov po akutnem koronarnem sindromu v procesu ateroskleroze. Summary: Atherosclerosis is a chronic progressive disease process characterized by lipid accumulation inside the the intimal layer of the arterial wall. The progressive course of the disease can lead to eventual plaque rupture or erosion, causing exposure of the pro-inflammatory plaque contents to the bloodstream. This is followed by thrombus formation on the rupture site and ischemia of the tissue supplied by the affected artery. If the tissue in question is the myocardium this arterial occlusion causes myocardial infarction. Injury to the myocardium causes a systemic inflammatory reaction with pro-inflammatory cytokine release which impacts atherosclerotic plaque inflammation shown by imaging studies in both preclinical and clinical studies. Epidemiological data show that patient with myocardial infarction are at an increased risk of a recurrent ischemic event in the first year following an index event. Immunomodulation following myocardial infarction has shown to reduce the risk of a recurrent event as evidenced by both preclinical as well as clinical studies. The first part of this thesis is dedicated to a novel pathway of immunomodulation after myocardial infarction by interleukin-7 receptor inhibition. The study was conducted on a mouse ApoE(-/-) model of myocardial infarction and atherosclerosis. We show that interleukin-7 acts as a chemoattractant for monocytes in vivo, its synthesis is increased inside the ishemic myocardium following myocardial infarction and increased levels are found inside the arterial wall following a myocardial infarction as well. Immunomodulation by interleukin-7 receptor blocking by monoclonal antibody reduces the lipid content of atherosclerotic plaque as well as monocyte and macrophage infiltration into the plaque thus reducing inflammation and causing plaque stabilization. The first part of the thesis thus shows the importance of a novel innate inflammatory pathway in atherosclerosis. The human immune system is composed roughly of the innate immune system, represented by cells such as neutrophils, monocytes and macrophages, involved in atherosclerosis in both the acute and chronic stages of disease and the specific, adaptive immune system which is activated after the nonspecific phase of inflammation. The main drivers of this specific part of the immune system are lymphocytes, both T cells and B cells, as well as antigen presenting cells. The MHC/T-cell receptor coupling is a hallmark of the adaptive immune system and is caused by targeted, antigen specific binding of short peptides to both antigen recognition molecules on the cell surfaces of T cells and antigen presenting cells. Data show that activation of auto-reactive T and B cells and antigen specific responses of these cells to lipid particles occur in atherosclerosis. One of the aforementioned particles is p210, part of the apoliprotein ApoB100, a component of LDL, the culprit cholesterol particle involved in plaque inflammation. Research has shown the existence of LDL and ApoB-100 reactive T cells, but specific epitopes involved in atherosclerosis have not yet been discerned. In the second part of this thesis I show the presence of autoreactive, antigen specific CD8+ T lymphocytes, activated by p210 stimulation in an auto-immune manner. This involves recognition and binding of a T-cell receptor to an antigen loaded MHC-I molecule. Potential binding of the p210 peptide to mouse MHC-I was assessed using the Immune Epitope Data Base (IEDB) prediction site and based on binding prediction score a fluorescent pentamer composed of five bound MHC-I molecules was constructed with specificity for p210 epitope recognition. This was used to test for p210 specific CD8+ T cells in a mouse model of atherosclerosis. Mice, fed a high fat, pro-atherogenic diet showed an increase of pentamer positive CD8+ T cells, exhibiting an effector T cell phenotype. Following stimulation, these cells show clonal proliferation, a hallmark of antigen specific recognition by T cell receptor (TCR). Immunomodulation by way of immunization showed a further increase in p210 specific pentamer positive CD8+ T lymphocytes as well as a reduction in atherosclerotic burden in the p210 immunized mice. The CD8+ T cell interaction with antigen presenting cells is based on antigen binding on MHC-I molecules on the surface of antigen presenting cells and recognition of the antigen-MHC-I complex by T cell receptor on the surface of CD8+ T lymphocytes. It's been shown that certain disease states cause MHC-I molecule shedding off the surface of cells. These can be detected in the bloodstream of patients and the peptides bound to these circulating, soluble MHC-I molecules represent possible auto-antigen involved in disease. Analysis of these peptides in patients with coronary artery disease and myocardial infarction can reveal previously unknown antigens important in atherosclerosis and its acute ischemic complication. The third part of this thesis shows a novel way of immunopeptidome profiling in order to reveal new potential antigens involved in atherosclerosis. Presented here is also a translational approach to testing the relevance of these novel peptide antigen in a biological model of atherosclerosis and combined atherosclerosis and myocardial infarction. Immunoprecipitation of soluble MHC-I complexes obtained from blood samples of patients with acute coronary syndrome collected from the AZACS trial revealed peptides specific to patients relative to matched control blood samples. The peptides were identified using mass spectrommetry and the amino acid sequence matched to known peptides from the Uniprot human protein database. The method enabled the identification of potential auto-antigen involved in atherosclerosis and myocardial infarction. In order to further test the biological relevance of the discovery, the peptides, specific to patients with the highest intensity on mass spectrometry were selected as well as peptides common and specific to most of the patient samples. The peptides tested were Keratin 8, ARID1A, Bleomycin Hyrolase and LL-37. Mouse homologues to these peptides were chosen to test auto-reactive responses in an Apo(-/-) mouse model of atherosclerosis as well as a combined ApoE (-/-) mouse model of atherosclerosis and myocardial infarction. Keratin II (murine homolog to human Keratin 8) and CRAMP (murine homolog to LL-37) showed highest immune responses upon stimulation and were chosen for further testing. Keratin II elicited a predominately effector CD8+ T lymphocyte response in the ApoE (-/-) model of atherosclerosis but not in a combined ApoE (-/-) murine model of atherosclerosis and myocardial infarction. This was evident also after testing human peripheral blood mononuclear cells isolated from blood samples taken from patient with either stable coronary artery disease or acute coronary syndrome. The antimicrobial peptide LL-37, a fragment of the larger hCAP-18 and a constituent of neutrophil granules is an important component of neutrophil mediated inflammation. It is a known auto-antigen in psoriasis, a classic auto-immune disease. Patients with psoriasis have increased burden of atherosclerosis compared to the healthy population and a higher risk of cardiovascular complication due to increased risk of plaque rupture. LL-37 has also been shown to be present in atherosclerotic plaque. Deficiency of mouse homolog to LL-37, named CRAMP (Cathelicidin Related Antimicrobial Peptide) in a CRAMP (-/-) murine model with atherosclerosis has shown to reduce atherosclerotic burden. To study the effect of CRAMP in a murine model of atherosclerosis, and to avoid its inherent immune properties, we synthesized a truncated version of CRAMP containing the highest predicted binding sites to mouse MHC-I molecules. Stimulation of murine immune cell populations elicited a strong effector T cell response in both CD4+ and CD8+ T cell populations which was amplified in mice fed a pro-atherogenic high fat diet. Immunization as a way of immunomodulation was used to test the biologic relevance as auto-antigen. Two different immunization doses were used, a higher 100ug dose and a lower 20 ug dose. These two doses exhibited a differential biological effect on atherosclerosis and immune activation. The lower immunization dose elicited a CD8+ Central Memory T cell response and reduced the pro-inflammatory CD11b+ Dendritic Cell populations along with a reduction in the burden of atherosclerotic disease. The higher immunization dose on the other hand increased the level of CD11b+ dendritic cells as well as caused an increase and activation of CD4+ and CD8+ Effector Memory T cells along with a significant increase in atherosclerosis. The higher immunization dose thus caused an increase in auto-reactivity to the auto-antigen and exhibited a seemingly auto-immune transformation of the murine immune system. The thesis outlines the importance of both the innate and adaptive immune systems in atherosclerosis and inflammation following myocardial infarction and potential new ways of immunomodulation effectively reducing the burden of atherosclerotic disease. It shows the importance of the interleukin-7 inflammatory pathway following myocardial infarction. Immunomodulation by inhibition of the IL-7 receptor mitigates the effect of the post-ishemic inflammation on atherosclerotic plaque. Monoclonal antibodies targeting IL-7 have already been tested in a clinical trial and thus represent a promising opportunity for immunomodulation after myocardial infarction in a clinical situation. The thesis outlines an important novel way of self-antigen discovery. Immunopeptidome analysis from patient blood samples is feasible and yields biologically relevant self-peptides involved in atherosclerosis. Furthermore we delineate an important translational pathway to discovery and testing of these antigen in a bedside-bench-bedside approach which produces data relevant to clinical practice and patient health. Our translational pathway revealed the involvement of LL-37, a known auto-antigen in psoriasis as an auto-antigen in atherosclerosis.

Breast cancer frequently metastasizes to the skeleton causing significant morbidity. None of the therapeutic strategies used to manage breast cancer bone metastases are really curative. Here, we set-up a novel and advanced model by using fresh tissue from human vertebral bone metastasis from breast carcinoma patients able to retain the tumor microenvironment. The tissue model is based on an ex-vivo culture for up to 40 days and on a constant monitoring of tissue viability, gene expression profile (IL10, IL1b, MMP1, MMP7, PTH1R, PTH2R, TNF, ACP5, SPI1, VEGFA, CTSK, TGF-β) and histological and immunohistochemical analyses (CDH1/E-cadherin, CDH2/N-cadherin, KRT8/Cytokeratin 8, KRT18/Cytokeratin 18, Ki67, CASP3/Caspase 3, ESR1/Estrogen Receptor Alpha, CD68 and CD8). Results confirmed the development of a reliable, reproducible and cost-effective advanced model of breast cancer bone metastasis able to preserve and maintain long-term tissue viability, as well as molecular markers, tissue histomorphology, tissue micro-architecture and antigen expression. The study provides for the first time the feasibility and rationale for the use of a human-derived advanced alternative model for cancer research and testing of drugs and innovative strategies, taking into account patient individual characteristics and specific tumor subtypes so predicting patient specific responses.

Keratin intermediate filaments (IFs) convey mechanical stability and protection against stress to epithelial cells, and may participate in nuclear structure and organization. Keratins are important for colon health as observed in keratin 8 knockout (K8−/−) mice, which exhibit colonic inflammation and epithelial hyperproliferation. Here, using a full body and two intestinal epithelial-specific K8−/−knockout mouse models, we determine if cytoplasmic keratins affect the nuclear structure and lamina in epithelial colonocytes. K8−/−colonocytes in vivo and in organoid cultures exhibit significantly decreased levels of the major lamins A/C, B1 and B2 in a colon-specific and cell-intrinsic manner independent of major changes in colonic inflammation or microbiota. Downregulation of K8 by siRNA in Caco-2 cells similarly decreases lamin A levels, which recover after re-expression of K8. K8 loss is associated with reduced plectin, LINC complex proteins and lamin-associated proteins, indicating a dysfunctional keratin-nuclear lamina coupling. Immunoprecipitation identifies complexes of colonocyte keratins with the LINC protein SUN2 and lamin A. Hyperphosphorylation of the lamin A-associated cell cycle regulator pRb in K8−/−colonocytes together with increased nuclear localization of the mechanosensor YAP provide a molecular mechanism for the hyperproliferation phenotype. These findings identify a novel, colonocyte-specific role for K8 in nuclear function.

Aurea helianthus extract is associated with various properties including anti-melanogenesis, anti-oxidation, tumorigenic suppression, and immunoregulation; however, the mechanism by which it executes the immunomodulation of human vaginal epithelial cells (HVECs) remains elusive. We established three immunological functions of the extract. First, it mediated tumorigenic suppression in HVECs. Expression of cytokeratin 8, cancer antigen-125, and vimentin was dramatically downregulated in HVECs exposed to the extract under oxidative and fungal stresses. Second, the extract activated dendritic cells and macrophages. On exposing progenitor dendritic cells to the extract, the number of CD304+ cells increased by 40%; further, under oxidative and fungal stresses, this number was approximately 1.8 and 1.3 times lower, respectively, compared to that in the stressed cells. In monocytic differentiation, the number of dendritic cells and macrophages increased 9 and 6 times, respectively, compared to that in the control. Additionally, the extract enhanced and recovered polarisation by approximately 1.5 and 2 times, respectively, than that under stressed conditions. Third, the phagocytic activity of macrophages, against HPV16, 18, and 33 peptides, was enhanced by 12–35 times compared with that under stressed conditions. Thus, A. helianthus extract is a strong stimulator of the immune system and tumorigenic suppression under stress conditions.

The rapid expansion of the elderly population has led to the recent epidemic of age-related diseases, including increased incidence and mortality of chronic lung diseases, such as Idiopathic Pulmonary Fibrosis (IPF). Cellular senescence is a major hallmark of aging and has a higher occurrence in IPF. The lung epithelium represents a major site of tissue injury, cellular senescence and aberrant activity of developmental pathways such as the WNT/beta-catenin pathway in IPF. The potential impact of WNT/beta-catenin signaling on alveolar epithelial senescence in general as well as in IPF, however, remains elusive. Here, we characterized alveolar epithelial cells of aged mice and assessed the contribution of chronic WNT/beta-catenin signaling on alveolar epithelial type (AT) II cell senescence. Whole lungs from old (16-24 months) versus young (3 months) mice had relatively less epithelial (EpCAM(+)) but more inflammatory (CD45(+)) cells, as assessed by flow cytometry. Compared to young ATII cells, old ATII cells showed decreased expression of the ATII cell marker Surfactant Protein C along with increased expression of the ATI cell marker Hopx, accompanied by increased WNT/beta-catenin activity. Notably, when placed in an organoid assay, old ATII cells exhibited decreased progenitor cell potential. Chronic canonical WNT/beta-catenin activation for up to 7 days in primary ATII cells as well as alveolar epithelial cell lines induced a robust cellular senescence, whereas the non-canonical ligand WNT5A was not able to induce cellular senescence. Moreover, chronic WNT3A treatment of precision-cut lung slices (PCLS) further confirmed ATII cell senescence. Simultaneously, chronic but not acute WNT/beta-catenin activation induced a profibrotic state with increased expression of the impaired ATII cell marker Keratin 8. These results suggest that chronic WNT/beta-catenin activity in the IPF lung contributes to increased ATII cell senescence and reprogramming. In the fibrotic environment, WNT/beta-catenin signaling thus might lead to further progenitor cell dysfunction and impaired lung repair.

Objective: This study aimed to evaluate the association between the single-nucleotide polymorphism (SNP) and tissue protein level of keratin-8/18 and the occurrence and progression of vocal leukoplakia. Methods: The case-control study enrolled 158 patients with vocal leukoplakia, 326 patients with laryngeal squamous cell carcinoma (LSCC), and 268 healthy controls, which were tested for genotype analysis with keratin-8 and keratin-18 gene polymorphisms using pyrosequencing. The tissue protein expression levels of keratin-8 and keratin-18 were evaluated using immunohistochemistry. Results: The keratin-8 SNP RS1907671 showed an obvious increased risk for vocal leukoplakia (OR 1.56, p = 0.002), while the other SNPs (RS2035875, RS2035878, RS4300473) were tested as protective factors for vocal leukoplakia and LSCC (OR p < 0.05). In keratin-18 SNP test, both RS2070876 and RS2638526 polymorphisms demonstrated decreased risks for vocal leukoplakia and LSCC (OR p < 0.05). The protein levels of keratin-8 and keratin-18 in vocal leukoplakia group were significantly higher than those of the LSCC group (p < 0.05). Conclusions: Keratin-8 and keratin-18 polymorphisms and protein levels are associated with the occurrence and progression of vocal leukoplakia.

Resistin is associated with metabolic syndrome and inflammatory conditions. Many studies have suggested that resistin inhibits the accumulation of glycogen; however, the exact mechanisms of resistin-induced decrease in glycogen content remain unclear. Keratin 8 is a typical epithelial intermediate filament protein, but numerous studies suggest a vital role of K8 in glucose metabolism. However, it is still not known whether K8 participates in the mediation of resistin-induced reduction of cellular glycogen accumulation. In this study, we found that resistin upregulated expression of the p65 subunit of NF-κB, which led to the promotion of K8 transcriptional expression; in turn, the expression of K8 inhibited glycogen accumulation in HepG2 cells.

Under metabolic stress, cellular components can assemble into distinct membraneless organelles for adaptation. One such example is cytidine 5'-triphosphate synthase (CTPS, for which there are CTPS1 and CTPS2 forms in mammals), which forms filamentous structures under glutamine deprivation. We have previously demonstrated that histidine (His)-mediated methylation regulates the formation of CTPS filaments to suppress enzymatic activity and preserve the CTPS protein under glutamine deprivation, which promotes cancer cell growth after stress alleviation. However, it remains unclear where and how these enigmatic structures are assembled. Using CTPS-APEX2-mediated in vivo proximity labeling, we found that synaptosome-associated protein 29 (SNAP29) regulates the spatiotemporal filament assembly of CTPS along the cytokeratin network in a keratin 8 (KRT8)-dependent manner. Knockdown of SNAP29 interfered with assembly and relaxed the filament-induced suppression of CTPS enzymatic activity. Furthermore, APEX2 proximity labeling of keratin 18 (KRT18) revealed a spatiotemporal association of SNAP29 with cytokeratin in response to stress. Super-resolution imaging suggests that during CTPS filament formation, SNAP29 interacts with CTPS along the cytokeratin network. This study links the cytokeratin network to the regulation of metabolism by compartmentalization of metabolic enzymes during nutrient deprivation.

BACKGROUND amp; AIMS: Steatohepatitis (SH) and SH-associated hepatocellular carcinoma (HCC) are of considerable clinical significance. SH is morphologically characterized by steatosis, liver cell ballooning, cytoplasmic aggregates termedMallory-Denk bodies (MDBs), inflammation, and fibrosis at late stage. Disturbance of the keratin cytoskeleton and aggregation of keratins (KRTs) are essential for MDB formation. METHODS: Weanalyzed livers of aged Krt18(-/-) mice that spontaneously developed in the majority of cases SH-associated HCC independent of sex. Interestingly, the hepatic lipid profile in Krt18(-/-) mice, which accumulate KRT8, closely resembles human SH lipid profiles and shows that the excess of KRT8 over KRT18 determines the likelihood to develop SH-associated HCC linked with enhanced lipogenesis. RESULTS: Our analysis of the genetic profile of Krt18(-/-) mice with 26 human hepatoma cell lines and with data sets of amp;gt;300 patients with HCC, where Krt18(-/-) gene signatures matched human HCC. Interestingly, a high KRT8/18 ratio is associated with an aggressive HCC phenotype. CONCLUSIONS: We can prove that intermediate filaments and their binding partners are tightly linked to hepatic lipid metabolism and to hepatocarcinogenesis. We suggest KRT8/18 ratio as a novel HCC biomarker for HCC. Funding Agencies|Kurt und Senta Herrmann Stiftung; Austrian Genome Programme GEN-AU; DFG [MA1316-15, MA1316-17, MA1316-19, MA1316-21, INST 268/230-1]; German Research Foundation [STR 1095/4-2]; Else Kroner Exzellenzstipendium; Innovative Medicines Initiative Joint Undertaking from the European Unions Seventh Framework Programme (FP7/2007-2013) [115234]; EFPIA companies