Frequent Cell Type Research Articles

Single-cell assignment using multiple-adversarial domain adaptation network with large-scale references

The rapid accumulation of single-cell RNA-seq data has provided rich resources to characterize various human cell populations. However, achieving accurate cell-type annotation using public references presents challenges due to inconsistent annotations, batch effects, and rare cell types. Here, we introduce SELINA (single-cell identity navigator), an integrative and automatic cell-type annotation framework based on a pre-curated reference atlas spanning various tissues. SELINA employs a multiple-adversarial domain adaptation network to remove batch effects within the reference dataset. Additionally, it enhances the annotation of less frequent cell types by synthetic minority oversampling and fits query data with the reference data using an autoencoder. SELINA culminates in the creation of a comprehensive and uniform reference atlas, encompassing 1.7 million cells covering 230 distinct human cell types. We substantiate its robustness and superiority across a multitude of human tissues. Notably, SELINA could accurately annotate cells within diverse disease contexts. SELINA provides a complete solution for human single-cell RNA-seq data annotation with both python and R packages.

Elucidating dynamic cell lineages and gene networks in time-course single cell differentiation

Single cell RNA sequencing (scRNA-seq) technologies provide researchers with an unprecedented opportunity to exploit cell heterogeneity. For example, the sequenced cells belong to various cell lineages, which may have different cell fates in stem and progenitor cells. Those cells may differentiate into various mature cell types in a cell differentiation process. To trace the behavior of cell differentiation, researchers reconstruct cell lineages and predict cell fates by ordering cells chronologically into a trajectory with a pseudo-time. However, in scRNA-seq experiments, there are no cell-to-cell correspondences along with the time to reconstruct the cell lineages, which creates a significant challenge for cell lineage tracing and cell fate prediction. Therefore, methods that can accurately reconstruct the dynamic cell lineages and predict cell fates are highly desirable.In this article, we develop an innovative machine-learning framework called Cell Smoothing Transformation (CellST) to elucidate the dynamic cell fate paths and construct gene networks in cell differentiation processes. Unlike the existing methods that construct one single bulk cell trajectory, CellST builds cell trajectories and tracks behaviors for each individual cell. Additionally, CellST can predict cell fates even for less frequent cell types. Based on the individual cell fate trajectories, CellST can further construct dynamic gene networks to model gene-gene relationships along the cell differentiation process and discover critical genes that potentially regulate cells into various mature cell types.

Abstract P4-07-50: mRNA expression and delivery efficacy of lipid nanoparticles in the cells breast tumor microenvironment: in vitro and in vivo evaluation

Abstract During the COVID-19 pandemics we have all witnessed the clinical importance of mRNA as current vaccines and future therapeutics. mRNA therapies have a potential to revolutionize cancer treatment. Delivery of mRNA requires lipid nanoparticles (LNP) to protect the cargo from degradation. mRNA has a negative charge and depends on positively charged lipids to be encapsulated in LNP. These lipids can be either ionizable at certain pH or constantly cationic. Even though previous studies had evaluated the formulation properties of ionizable and cationic LNP systems, there is the need to understand their specificity in terms of mRNA delivery and protein expression in breast cancer tumor microenvironment. The objective of this work was to assess the kinetics of LNP cellular uptake and mRNA expression inv breast cancer (BC) cells and fibroblasts, the most frequent cell type in the tumor microenvironment cells, while studying the mechanisms involved in differential behaviors of LNP formulated with cationic and ionizable lipids. To achieve this goal mRNA-LNP containing ionizable lipids (LNP-A) and cationic lipids (LNP-B) were designed and formulated using Nanoassemblr® Benchtop microfluidics mixer (Precision NanoSystems). mRNA-LNP were characterized for size, zeta potential using dynamic light scattering (DLS) and mRNA encapsulation efficiency using RiboGreen assay. LNP were tagged with rhodamine lipid to investigate the uptake kinetic and a reporter GFP mRNA to evaluate mRNA expression in murine 4T1 and human MCF7, MDA-231, SUM-159 and T-47D breast cancer cells and BJ fibroblasts. Live fluorescence microscopy imaging, IncuCyte S3®, was used to determine the LNP uptake and GFP mRNA expression. In vitro biocompatibility was assessed with WST-1 assay. Additionally, expression of mRNA delivered from LNP in tumor microenvironment was evaluated in vivo in a syngeneic 4T1 breast cancer model using mRNA luciferase and IVIS imaging. mRNA-LNPs possessed an average diameter of 77 - 107 nm, narrow size distribution, neutral zeta potential and high mRNA encapsulation efficiency (>94%). Our results demonstrated that mRNA expression was higher in breast cancer cells when delivered from LNP-A formulation and in BJ fibroblasts when delivered from LNP-B. LNP-A, the ionizable LNP, was tested in the breast cancer cells to confirm the efficacy of the delivery. The highest transfection efficacy, from high to low, T-47D, MCF7, SUM-159, 4T1 and MDA-231.We have further investigated the cellular uptake mechanisms of LNP using uptake pathway inhibitors for caveolae endocytosis, clathrin endocytosis, and phagocytosis. Our data confirm that there are differences in mechanisms that govern the uptake of mRNA LNP in breast cancer cells and fibroblasts. Clathrin-mediated endocytosis was active in 4T1 breast cancer cells for ionizable and cationic LNP. Interestingly, despite in vitro differences in uptake and mRNA expression, in vivo results show that both formulations efficiently delivered luciferase-mRNA in the tumor microenvironment. Histology results demonstrated similar luciferase expression for both LNP in tumors. Additionally, we were able to confirm the prominent presence of fibroblast and similar distribution in the 4T1 subcutaneous model which could explain the similar efficacy of cationic and ionizable LNP. Understanding uptake and mRNA expression of different LNP formulations in the tumor microenvironment can help in achieving the necessary protein expression for breast cancer therapies. Furthermore, determining the most efficient carrier in early stages may reduce the time required for clinical translation. Acknowledgement: This research was supported in part by CPRIT Core for RNA Therapeutics and Research. Citation Format: Karem A. Court, Anjana Tiwari, Eric Chau, John Cooke, Biana Godin. mRNA expression and delivery efficacy of lipid nanoparticles in the cells breast tumor microenvironment: in vitro and in vivo evaluation [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-50.

Bisphenols, but not phthalate esters, modulate gene expression in activated human MAIT cells in vitro.

One route of human exposure to environmental chemicals is oral uptake. This is primarily true for chemicals that may leach from food packaging materials, such as bisphenols and phthalate esters. Upon ingestion, these compounds are transported along the intestinal tract, from where they can be taken up into the blood stream or distributed to mucosal sites. At mucosal sites, mucosal immune cells and in the blood stream peripheral immune cells may be exposed to these chemicals potentially modulating immune cell functions. In the present study, we investigated the impact of three common bisphenols and two phthalate esters on mucosal-associated invariant T (MAIT) cells in vitro, a frequent immune cell type in the intestinal mucosae and peripheral blood of humans. All compounds were non-cytotoxic at the chosen concentrations. MAIT cell activation was only slightly affected as seen by flow cytometric analysis. Phthalate esters did not affect MAIT cell gene expression, while bisphenol-exposure induced significant changes. Transcriptional changes occurred in ∼25% of genes for BPA, ∼22% for BPF and ∼8% for BPS. All bisphenols down-modulated expression of CCND2, CCL20, GZMB and IRF4, indicating an effect on MAIT cell effector function. Further, BPA and BPF showed a high overlap in modulated genes involved in cellular stress response, activation signaling and effector function suggesting that BPF may not be safe substitute for BPA.

Idiopathic pulmonary fibrosis and lung cancer: future directions and challenges.

Idiopathic pulmonary fibrosis (IPF) is a progressive disease of pulmonary scarring. New treatments slow disease progression and allow pulmonary fibrosis patients to live longer. Persistent pulmonary fibrosis increases a patient's risk of developing lung cancer. Lung cancer in patients with IPF differs from cancers that develop in the non-fibrotic lung. Peripherally located adenocarcinoma is the most frequent cell type in smokers who develop lung cancer, while squamous cell carcinoma is the most frequent in pulmonary fibrosis. Increased fibroblast foci in IPF are associated with more aggressive cancer behaviour and shorter doubling times. Treatment of lung cancer in fibrosis is challenging because of the risk of inducing an exacerbation of fibrosis. In order to improve patient outcomes, modifications of current lung cancer screening guidelines in patients with pulmonary fibrosis will be necessary to avoid delays in treatment. 2-fluoro-2-deoxy-d-glucose (FDG) positron emission tomography (PET) computed tomography (CT) imaging can help identify cancer earlier and more reliably than CT alone. Increased use of wedge resections, proton therapy and immunotherapy may increase survival by decreasing the risk of exacerbation, but further research will be necessary.

ScoMorphoFISH: A deep learning enabled toolbox for single‐cell single‐mRNA quantification and correlative (ultra‐)morphometry

Increasing the information depth of single kidney biopsies can improve diagnostic precision, personalized medicine and accelerate basic kidney research. Until now, information on mRNA abundance and morphologic analysis has been obtained from different samples, missing out on the spatial context and single‐cell correlation of findings. Herein, we present scoMorphoFISH, a modular toolbox to obtain spatial single‐cell single‐mRNA expression data from routinely generated kidney biopsies. Deep learning was used to virtually dissect tissue sections in tissue compartments and cell types to which single‐cell expression data were assigned. Furthermore, we show correlative and spatial single‐cell expression quantification with super‐resolved podocyte foot process morphometry. In contrast to bulk analysis methods, this approach will help to identify local transcription changes even in less frequent kidney cell types on a spatial single‐cell level with single‐mRNA resolution. Using this method, we demonstrate that ACE2 can be locally upregulated in podocytes upon injury. In a patient suffering from COVID‐19‐associated collapsing FSGS, ACE2 expression levels were correlated with intracellular SARS‐CoV‐2 abundance. As this method performs well with standard formalin‐fixed paraffin‐embedded samples and we provide pretrained deep learning networks embedded in a comprehensive image analysis workflow, this method can be applied immediately in a variety of settings.

Towards a positive physiological definition of the deep brain nuclei in humans

Using microelectrodes for recording purposes in deep brain stimulation (DBS) has proven to be very useful. Their efficiency can be improved by characterising the properties of extracellular action potentials (EAPs). We analysed the records of nine patients who underwent surgery for epilepsy or aggressiveness under general anaesthesia. The properties of the EAPs of the centromedian, ventral intermediate, ventrocaudal and posteromedial hypothalamic nuclei of the thalamus have been determined. We have analysed 706 thalamic and 142 hypothalamic cells. The proportion of cell types was found to be specific to each cell nucleus. The most frequent cell type was P1P2N1 (59.5%), followed by N1P1N2 (23.1%). The first phase of the EAP is highly variable. The properties of the EAP phases of the same morphology differ greatly from one nucleus to another. We have shown that several deep brain nuclei have properties that are specific to the morphology of the EAPs. This will allow for improved localisation of these nuclei during DBS.

Carcinoma de células acinares de glándulas salivales: estudio clínico e histopatológico de 12 casos

Objective:Acinic cell carcinoma (CCA) is the third malignant epithelial tumor of the salivary glands in adults; low-grade tumor of malignancy, composed of neoplastic cells with serous acinar differentiation. The objective of this work was to analyze 12 cases of CCA according to their location, clinical characteristics, histological and immunohistochemical pattern and cell types, following the latest classification of the World Health Organization. Methods: The study included 12 cases of CCA from the files of salivary tumor biopsies of our work team, corresponding to the period 1997-2020. A numerical code was used to identify the samples, preserving the identity of the patients. Histological sections of the paraffin-embedded biopsies were evaluated with H/E, PAS and Toluidine blue and immunostained with the monoclonal antibodies pancytokeratin AE1 / AE3, Ki67, MUC-1 and mammaglobin. Results: The most frequent histologic pattern was the solid type as a single pattern or integrated with other patterns of lesser development, with almost exclusive location in the parotid gland and more frequent in women. Cells like normal acinar serocytes predominated in the solid growth pattern. The most frequent cell type in the microcystic patternwas the nonspecific glandular cell together with a lower proportion of acinar and intercalated duct-like cells. The papillary-cystic pattern was lined by nonspecific glandular cells. No clear cells found. With Ki67 a low cell proliferation was demonstrated in all the cases studied. Cell labeling for MUC-1 was grade 1 positive (less than 10% immunoreactive cells) and negative for mammaglobin.Conclusions: Patient follow-up is a priority because CCA tends to recur and metastasize and its behavior can become aggressive. We must deepen the study of its proliferative capacity as a treatment and prognosis tool, especially with immunohistochemistry and standardized molecular biology methods.

Molecular Pathology Demonstration of SARS-CoV-2 in Cytotrophoblast from Placental Tissue with Chronic Histiocytic Intervillositis, Trophoblast Necrosis and COVID-19.

A subset of placentas from pregnant women having the SARS-CoV-2 infection have been found to be infected with the coronavirus using molecular pathology methods including immunohistochemistry and RNA in situ hybridization. These infected placentas can demonstrate several unusual findings which occur together—chronic histiocytic intervillositis, trophoblast necrosis and positive staining of the syncytiotrophoblast for SARS-CoV-2. They frequently also have increased fibrin deposition, which can be massive in some cases. Syncytiotrophoblast is the most frequent fetal-derived cell type to be positive for SARS-CoV-2. It has recently been shown that in a small number of infected placentas, villous stromal macrophages, termed Hofbauer cells, and villous capillary endothelial cells can also stain positive for SARS-CoV-2. This report describes a placenta from a pregnant woman with SARS-CoV-2 that had chronic histiocytic intervillositis, trophoblast necrosis, increased fibrin deposition and positive staining of the syncytiotrophoblast for SARS-CoV-2. In addition, molecular pathology testing including RNAscope and immunohistochemistry for SARS-CoV-2 and double-staining immunohistochemistry using antibodies to E-cadherin and GATA3 revealed that cytotrophoblast cells stained intensely for SARS-CoV-2. All of the cytotrophoblast cells that demonstrated positive staining for SARS-CoV-2 were in direct physical contact with overlying syncytiotrophoblast that also stained positive for the virus. The pattern of cytotrophoblast staining for SARS-CoV-2 was patchy, and there were chorionic villi having diffuse positive staining of the syncytiotrophoblast for SARS-CoV-2, but without staining of cytotrophoblast. This first detailed description of cytotrophoblast involvement by SARS-CoV-2 adds another fetal cell type from infected placentas that demonstrate viral staining.

Physical phenotype of blood cells is altered in COVID-19

Clinical syndrome coronavirus disease 2019 (COVID-19) induced by severe acute respiratory syndrome coronavirus 2 is characterized by rapid spreading and high mortality worldwide. Although the pathology is not yet fully understood, hyperinflammatory response and coagulation disorders leading to congestions of microvessels are considered to be key drivers of the still-increasing death toll. Until now, physical changes of blood cells have not been considered to play a role in COVID-19 related vascular occlusion and organ damage. Here, we report an evaluation of multiple physical parameters including the mechanical features of five frequent blood cell types, namely erythrocytes, lymphocytes, monocytes, neutrophils, and eosinophils. More than four million blood cells of 17 COVID-19 patients at different levels of severity, 24 volunteers free from infectious or inflammatory diseases, and 14 recovered COVID-19 patients were analyzed. We found significant changes in lymphocyte stiffness, monocyte size, neutrophil size and deformability, and heterogeneity of erythrocyte deformation and size. Although some of these changes recovered to normal values after hospitalization, others persisted for months after hospital discharge, evidencing the long-term imprint of COVID-19 on the body.

3O Spatial analysis of lymphocytes and fibroblasts identifies biological relevant patterns in estrogen receptor positive breast cancer

In estrogen receptor positive (ER+) breast cancer, higher levels of tumor infiltrating lymphocytes (TILs) are often associated with a poor prognosis and this phenomenon is still poorly understood. Fibroblasts represent one of the most frequent and plastic cell types in breast cancer and harbor intriguing immunomodulatory and architectural capabilities. However, the clinical significance of the spatial patterns of tumor cells, TILs and fibroblasts is largely unknown. Here we evaluate the patterns and clinical impact of the proximity between cancer cells, TILs and fibroblast in ER+ breast cancer.

34P Sex and age differences in primary lung cancer in Morocco: An epidemiologic study

Background: Lung cancer is a leading cause of death worldwide. In Morocco, it is the most commonly occurring cancer in men. The aim of this study is to determine the epidemiological and histological profile of primary lung cancer in Rabat (capital of Morocco) over a 4-year period, from 2014 to 2017, by sex and age. Methods: This is a descriptive retrospective study of all patients with primary lung cancer diagnosed and treated at Ibn Sina University Hospital Center in Rabat during the period from January 2014 to December 2017. The data were collected from medical records for each patient. Statistical analysis was performed using Epi InfoTM7. Results: During the period of study, 574 new cases of primary lung cancer were diagnosed. Of these, 87.5% were men and 12.5% were women, giving a male-female ratio of 7. The average age of patients at diagnosis was 58.4 years (range 21–85 years). There was no significant gender difference in average age. The age distribution show that 19.9% of patients were aged less than 50 years, 29.7% were in the 50–59-year age group, 33.8% were aged between 60 and 69 years and 16.5% were aged 70 years and older. According to the results, 7.3% of lung cancers were small cell lung cancer (SCLC) and 90.9% were non-small cell lung cancer (NSCLC). Adenocarcinoma was the most frequent cell type of lung cancer, with 60.45%, followed by squamous cell carcinoma, with 23%. According to gender, adenocarcinoma was more common in women (70.8%) than in men (58.4%), while squamous cell carcinoma was relatively frequent in men (24.7%) than in women (11.1%). There was no significant difference in age of diagnosis, by histological type of lung cancer. Conclusions: Lung cancer is a serious and potentially deadly disease in Morocco. It is important to develop strategies that take into account the current trends in lung cancer and target particularly groups at risk. Legal entity responsible for the study: The authors. Funding: Has not received any funding. Disclosure: All authors have declared no conflicts of interest.

Immune classification of clear cell renal cell carcinoma

Since the outcome of treatments, particularly immunotherapeutic interventions, depends on the tumor immune micro-environment (TIM), several experimental and computational tools such as flow cytometry, immunohistochemistry, and digital cytometry have been developed and utilized to classify TIM variations. In this project, we identify immune pattern of clear cell renal cell carcinomas (ccRCC) by estimating the percentage of each immune cell type in 526 renal tumors using the new powerful technique of digital cytometry. The results, which are in agreement with the results of a large-scale mass cytometry analysis, show that the most frequent immune cell types in ccRCC tumors are CD8+ T-cells, macrophages, and CD4+ T-cells. Saliently, unsupervised clustering of ccRCC primary tumors based on their relative number of immune cells indicates the existence of four distinct groups of ccRCC tumors. Tumors in the first group consist of approximately the same numbers of macrophages and CD8+ T-cells and and a slightly smaller number of CD4+ T cells than CD8+ T cells, while tumors in the second group have a significantly high number of macrophages compared to any other immune cell type (P-value <0.01). The third group of ccRCC tumors have a significantly higher number of CD8+ T-cells than any other immune cell type (P-value <0.01), while tumors in the group 4 have approximately the same numbers of macrophages and CD4+ T-cells and a significantly smaller number of CD8+ T-cells than CD4+ T-cells (P-value <0.01). Moreover, there is a high positive correlation between the expression levels of IFNG and PDCD1 and the percentage of CD8+ T-cells, and higher stage and grade of tumors have a substantially higher percentage of CD8+ T-cells. Furthermore, the primary tumors of patients, who are tumor free at the last time of follow up, have a significantly higher percentage of mast cells (P-value <0.01) compared to the patients with tumors for all groups of tumors except group 3.

In Vitro Suppression of T Cell Proliferation Is a Conserved Function of Primary and Immortalized Human Cancer-Associated Fibroblasts.

T cell immunotherapy is now a mainstay therapy for several blood-borne cancers as well as metastatic melanoma. Unfortunately, many epithelial tumors respond poorly to immunotherapy, and the reasons for this are not well understood. Cancer-associated fibroblasts (CAFs) are the most frequent non-neoplastic cell type in most solid tumors, and they are emerging as a key player in immunotherapy resistance. A range of immortalized CAF lines will be essential tools that will allow us to understand immune responses against cancer and develop novel strategies for cancer immunotherapy. To study the effect of CAFs on T cell proliferation, we created and characterized a number of novel immortalized human CAFs lines (Im-CAFs) from human breast, colon, and pancreatic carcinomas. Im-CAFs shared similar phenotypes, matrix remodeling and contraction capabilities, and growth and migration rates compared to the primary CAFs. Using primary isolates from breast carcinoma, colorectal carcinoma, and pancreatic ductal adenocarcinoma, we report that CAFs across major tumor types are able to potently suppress T cell proliferation in vitro. Im-CAFs retained this property. Im-CAFs are a key tool that will provide important insights into the mechanisms of CAF-mediated T cell suppression through techniques such as CRISPR-Cas9 modification, molecular screens, and pipeline drug testing.

Cell‐type specific eQTLs (ct‐eQTLs) associated with Alzheimer disease in blood and brain tissue

AbstractBackgroundExpression quantitative trait locus(eQTL) analysis has demonstrated that changes in gene expression play a role in Alzheimer disease(AD) pathogenesis. Regulation of gene expression is dependent on context such as tissue and cell‐type. Previous studies found monocyte‐specific eQTLs among AD variants.MethodCis‐eQTL mapping was performed for 723 genes located within 1 Mb of 79 AD‐associated loci in RNA‐seq data in blood from Framingham Heart Study (FHS) participants and in brain tissue from Religious Orders Study/Memory &amp; Aging Project(ROSMAP) participants. The association of gene expression with genotypes for all cis SNPs within these genes was evaluated using linear regression models for ROSMAP and linear mixed models adjusting for family structure in FHS. Models testing associations with cell type‐specific eQTLs (ct‐eQTLs) included an interaction term for expression of “proxy” genes that discriminate the particular cell type.ResultA total of 10,970 eQTLs and 1,324 ct‐eQTLs in brain, and 29,508 eQTLs and 2,313 ct‐eQTLs in blood were nominally significant after Bonferroni correction. Ct‐eQTL analysis identified 964 and 44 significant gene‐SNP eQTL pairs in brain and blood, respectively, that were not detected in generic eQTL analysis. Of note, 760 gene‐SNP eQTL pairs were shared between blood and brain and are enriched in AD‐related immune pathways. Among the significant ct‐eQTLs, the most frequent cell types were interferon response/antibacterial cells in blood(70.6%) and endothelial cells in brain(35.5%). Established AD genes CD2AP and NCR2 had a large number of significant eQTLs in NK/ CD8+T cells in blood and brain endothelial cells respectively. In addition, several significant eSNPs were observed for novel genes under linkage analysis peaks spanning multiple genes. The eGene target for one of these AD peak eSNPs, rs2985987, is COG3 is involved in ER‐Golgi transport, a key AD‐related pathway. Furthermore, many novel variants and genes were identified among the significant eQTLs that target previously associated AD genes or associate with AD peak SNPs. Target eGenes associated with AD peak eSNPs include TAS2R60, PILRB, CNN2‐ previously implicated in AD in a targeted gene sequencing study in African Americans, HLA‐DQA2, and MS4A5.ConclusionWe identified novel AD‐related ct‐eQTLs that highlight the importance of cell‐type specific analysis and cell‐type dependent context.

P2.11-20 Risk Factors of Lung Cancer Development in IPF Patients

Idiopathic pulmonary fibrosis (IPF) is known as a risk factor for lung cancer (LC) by several previous studies, and that the presence of LC shortens survival in patients with IPF. However, the risk factors for the development of LC after the diagnosis of IPF have not been fully evaluated. We investigated the predictive factors for LC by longitudinal cohort analysis. This was a retrospective study of a single center interstitial lung disease cohort. Study patients were consecutively enrolled to the cohort between March 2006 and December 2018 at Bucheon St. Mary’s Hospital, The Catholic University of Korea. This cohort study consists of 102 patients with IPF, and the incidence of LC and the outcomes were investigated. During the mean follow-up periods of 62.7 months, 27 patients (26%) developed LC. The most frequent cell type was Squamous cell carcinoma, and the proportion of male was higher in IPF-LC group (92.6% vs 70.7%, p=0.021). In univariate cox regression analysis, low forced vital capacity (FVC) <75% (p<0.001), and low diffusion capacity of the lungs for carbon monoxide (DLco) <55% (p<0.001) was associated with LC development. In Cox proportional hazards model, low FVC (hazard ratio [HR]: 5.65; 95% confidence interval [CI]: 1.78-17.84, p=0.003) and low DLco (HR: 27.5; 95% CI: 1.97-386.6, p=0.014) were independent predictive factors for LC in stepwise multivariate analysis. Low FVC and Low DLco, which are pulmonary function parameters reflecting their severity of IPF, are suggested as independent risk factors for LC development in IPF patients.

Phenotypic Analysis of BrdU Label-Retaining Cells during the Maturation of Conducting Airway Epithelium in a Porcine Lung.

Stem/progenitor cells have recently been demonstrated to play key roles in the maturation, injury repair, and regeneration of distinct organs or tissues. Porcine has spurred an increased interest in biomedical research models and xenotransplantation, owing to most of its organs share similarities in physiology, cellular composition and size to humans. Therefore, characterization of stem/progenitor cells in porcine organs or tissues may provide a novel avenue to better understand the biology and function of stem cells in humans. In the present study, potential stem/progenitor cells in conducting airway epithelium of a porcine lung were characterized by morphometric analysis of bromodeoxyuridine (BrdU) label-retaining cells (LRCs) during the maturation of the lung. The results showed a pseudostratified mucociliary epithelium comprised of basal, ciliated, goblet, and columnar cells in the conducting airway of a porcine lung. In addition, the majority of primary epithelial cells able to proliferate in vitro expressed keratin 5, a subpopulation of these keratin 5-positive cells, also expressed CD117 (c-Kit) or CD49f (integrin alpha 6, ITGA6), implying that they might be potential epithelial stem/progenitor cells in conducting airway of a porcine lung. Lineage tracing analysis with a BrdU-labeled neonatal piglet showed that the proportion of BrdU-labeled cells in conducting airways decreased over the 90-day period of lung maturation. The BrdU-labeled epithelial cells also expressed keratin 14, mucin 5AC, or prosurfactant protein C (ProSP-C); among them, the keratin 14-positive cells were the most frequent BrdU-labeled epithelial cell type as determined by immunohistochemical and immunofluorescence staining. This study may provide valuable information on the biology and function of epithelial stem/progenitor cells in conducting airway of pigs and humans.

Is fertility sparing surgery a treatment option for young patients with yolk sac tumor?

Yolk sac tumor (YST) is the third most frequent ovarian germ cell type ovarian tumor. YST occurs primarily in young girls and in women at reproductive age and thus, the preservation of fertility is considered to be a crucial option if possible for these patients. Compared to previous years, the use of innovative chemotherapeutic regimens in the treatment of YSTs improved significantly the prognosis and outcomes of this rare type of neoplasia. As YSTs are chemosensitive, this fact favors fertility sparing surgical approach and less aggressive type of operations. We present a narrative review of the multispecialty fertility sparing surgical and medical approach of women with YST.

Endometrial receptivity revisited: endometrial transcriptome adjusted for tissue cellular heterogeneity.

Does cellular composition of the endometrial biopsy affect the gene expression profile of endometrial whole-tissue samples? The differences in epithelial and stromal cell proportions in endometrial biopsies modify the whole-tissue gene expression profiles and affect the results of differential expression analyses. Each cell type has its unique gene expression profile. The proportions of epithelial and stromal cells vary in endometrial tissue during the menstrual cycle, along with individual and technical variation due to the method and tools used to obtain the tissue biopsy. Using cell-population specific transcriptome data and computational deconvolution approach, we estimated the epithelial and stromal cell proportions in whole-tissue biopsies taken during early secretory and mid-secretory phases. The estimated cellular proportions were used as covariates in whole-tissue differential gene expression analysis. Endometrial transcriptomes before and after deconvolution were compared and analysed in biological context. Paired early- and mid-secretory endometrial biopsies were obtained from 35 healthy, regularly cycling, fertile volunteers, aged 23-36 years, and analysed by RNA sequencing. Differential gene expression analysis was performed using two approaches. In one of them, computational deconvolution was applied as an intermediate step to adjust for the proportions of epithelial and stromal cells in the endometrial biopsy. The results were then compared to conventional differential expression analysis. Ten paired endometrial samples were analysed with qPCR to validate the results. The estimated average proportions of stromal and epithelial cells in early secretory phase were 65% and 35%, and during mid-secretory phase, 46% and 54%, respectively, correlating well with the results of histological evaluation (r = 0.88, P = 1.1 × 10-6). Endometrial tissue transcriptomic analysis showed that approximately 26% of transcripts (n = 946) differentially expressed in receptive endometrium in cell-type unadjusted analysis also remain differentially expressed after adjustment for biopsy cellular composition. However, the other 74% (n = 2645) become statistically non-significant after adjustment for biopsy cellular composition, underlining the impact of tissue heterogeneity on differential expression analysis. The results suggest new mechanisms involved in endometrial maturation, involving genes like LINC01320, SLC8A1 and GGTA1P, described for the first time in context of endometrial receptivity. The RNA-seq data presented in this study is deposited in the Gene Expression Omnibus database with accession number GSE98386. Only dominant endometrial cell types were considered in gene expression profile deconvolution; however, other less frequent endometrial cell types also contribute to the whole-tissue gene expression profile. The better understanding of molecular processes during transition from pre-receptive to receptive endometrium serves to improve the effectiveness and personalization of assisted reproduction protocols. Biopsy cellular composition should be taken into account in future endometrial 'omics' studies, where tissue heterogeneity could potentially influence the results. This study was funded by: Estonian Ministry of Education and Research (grant IUT34-16); Enterprise Estonia (EU48695); the EU-FP7 Eurostars program (NOTED, EU41564); the EU-FP7 Marie Curie Industry-Academia Partnerships and Pathways (SARM, EU324509); Horizon 2020 innovation program (WIDENLIFE, EU692065); MSCA-RISE-2015 project MOMENDO (No 691058) and the Miguel Servet Program Type I of Instituto de Salud Carlos III (CP13/00038); Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and European Regional Development Fund (FEDER): grants RYC-2016-21199 and ENDORE SAF2017-87526. Authors confirm no competing interests.

The Current State of Advanced Therapy Medicinal Products in the Czech Republic.

Advanced therapy medicinal products (ATMPs) represent a new generation of biopharmaceuticals that comprise gene therapy medicinal products (GTMPs), somatic cell therapy products (CTMPs), tissue engineered products (TEPs), and combined advanced therapy medicinal products (cATMPs). The joint effort of the academia-industry-regulatory triangle translated scientific progress into ten authorized ATMPs in the European Community. This notion holds promise for the whole field of ATMP therapies that have been increasingly evaluated in a number of clinical studies, also in the Czech Republic (CR). Here, we prepared an overview of regulatory framework, past and present clinical studies, and already authorized ATMPs in the CR. Clinical studies on ATMPs in the CR were mapped using public databases, particularly ClinicalTrials.gov, the European Union Clinical Trials Register, and the State Institute for Drug Control database. We found 50 registered clinical studies using ATMPs in the CR that mostly involve CTMPs (n = 36), followed by GTMPs (n = 4) and TEPs (n = 4). The majority of the studies use autologous ATMPs (76%) and are aimed at the treatment of oncologic conditions (58%) and musculoskeletal disorders (24%). The most frequent autologous cell type was dendritic cells (42%), bone marrow mononuclear cells (16%) and bone marrow mesenchymal stromal cells (13%). Allogeneic ATMPs (12%) are mostly aimed at the treatment of venous ulcers (33%) and utilize keratinocytes and fibroblasts (33%). In summary, ATMPs are increasingly tested in clinical trials in the CR, which will most likely lead to their translation into broader clinical use. However, to stimulate market viability of registered ATMPs, implementation of the sophisticated reimbursement system will be required.