Healthy Donor Tissue Research Articles

Characterization of a human thyroid microtissue model for testing thyroid disrupting chemicals.

Perturbation of thyroid hormone (T4) synthesis is known to cause numerous developmental, metabolic, and cognitive disorders in humans. Due to species differences in sensitivity to chemical exposures, there is a need for human-based in vitro approaches that recapitulate thyroid cellular architecture and T4 production when screening. To address these limitations, primary human thyrocytes, isolated from healthy adult donor tissues and cryopreserved at passage one (p'1) were characterized for cellular composition, 3D follicular architecture, and thyroglobulin (TG)/T4 expression and inhibition by prototype thyroid disrupting chemicals (TDC). Flow analysis of the post-thaw cell suspension showed >80% EpCAM-positive cells with 10%-50% CD90-positive cells. When seeded onto 96-well Matrigel®-coated plates and treated with bovine thyroid stimulating hormone (TSH), thyrocytes formed 3D microtissues during the initial 4-5 days of culture. The microtissues exhibited a stable morphology and size over a 14-day culture period. TG and T4 production were highest in microtissues when the proportion of CD90-positive cells, seeding density and thyroid stimulating hormone concentrations were between 10%-30%, 6K-12Kcells per well, and 0.03-1 mIU/mL, respectively. At maximal TG and T4 production levels, average microtissue diameters ranged between 50 and 200µm. The T4 IC50 values for two prototype TPO inhibitors, 6-propyl-2-thiouracil and methimazole, were ∼0.7µM and ∼0.5 µM, respectively, in microtissue cultures treated between days 9 and 14. Overall, p'1 cryopreserved primary human thyrocytes in 3D microtissue culture represent a promising new model system to prioritize potential TDC acting directly on the thyroid as part of a weight-of-evidence hazard characterization.

Transcriptome profiling of immune rejection mechanisms in a porcine vascularized composite allotransplantation model.

Vascularized composite allotransplantation (VCA) offers the potential for a biological, functional reconstruction in individuals with limb loss or facial disfigurement. Yet, it faces substantial challenges due to heightened immune rejection rates compared to solid organ transplants. A deep understanding of the genetic and immunological drivers of VCA rejection is essential to improve VCA outcomes. Heterotopic porcine hindlimb VCA models were established and followed until reaching the endpoint. Skin and muscle samples were obtained from VCA transplant recipient pigs for histological assessments and RNA sequencing analysis. The rejection groups included recipients with moderate pathological rejection, treated locally with tacrolimus encapsulated in triglycerol-monostearate gel (TGMS-TAC), as well as recipients with severe end-stage rejection presenting evident necrosis. Healthy donor tissue served as controls. Bioinformatics analysis, immunofluorescence, and electron microscopy were utilized to examine gene expression patterns and the expression of immune response markers. Our comprehensive analyses encompassed differentially expressed genes, Gene Ontology, and Kyoto Encyclopedia of Genes and Genomes pathways, spanning various composite tissues including skin and muscle, in comparison to the healthy control group. The analysis revealed a consistency and reproducibility in alignment with the pathological rejection grading. Genes and pathways associated with innate immunity, notably pattern recognition receptors (PRRs), damage-associated molecular patterns (DAMPs), and antigen processing and presentation pathways, exhibited upregulation in the VCA rejection groups compared to the healthy controls. Our investigation identified significant shifts in gene expression related to cytokines, chemokines, complement pathways, and diverse immune cell types, with CD8 T cells and macrophages notably enriched in the VCA rejection tissues. Mechanisms of cell death, such as apoptosis, necroptosis and ferroptosis were observed and coexisted in rejected tissues. Our study provides insights into the genetic profile of tissue rejection in the porcine VCA model. We comprehensively analyze the molecular landscape of immune rejection mechanisms, from innate immunity activation to critical stages such as antigen recognition, cytotoxic rejection, and cell death. This research advances our understanding of graft rejection mechanisms and offers potential for improving diagnostic and therapeutic strategies to enhance the long-term success of VCA.

SDPR expression in human trabecular meshwork and its potential role in racial disparities of glaucoma

In order to identify how differential gene expression in the trabecular meshwork (TM) contributes to racial disparities of caveolar protein expression, TM dysfunction and development of primary open angle glaucoma (POAG), RNA sequencing was performed to compare TM tissue obtained from White and Black POAG surgical (trabeculectomy) specimens. Healthy donor TM tissue from White and Black donors was analyzed by PCR, qPCR, immunohistochemistry staining, and Western blot to evaluate SDPR (serum deprivation protein response; Cavin 2) and CAV1/CAV2 (Caveolin 1/Caveolin 2). Standard transmission electron microscopy (TEM) and immunogold labeled studies were performed. RNA sequencing demonstrated reduced SDPR expression in TM from Black vs White POAG patients’ surgical specimens, with no significant expression differences in other caveolae-associated genes, confirmed by qPCR analysis. No racial differences in SDPR gene expression were noted in healthy donor tissue by PCR analysis, but there was greater expression as compared to specimens from patients with glaucoma. Analysis of SDPR protein expression confirmed specific expression in the TM regions, but not in adjacent tissues. TEM studies of TM specimens from healthy donors did not demonstrate any racial differences in caveolar morphology, but a significant reduction of caveolae with normal morphology and immuno-gold staining of SDPR were noted in glaucomatous TM as compared to TM from healthy donors. Linkage of SDPR expression levels in TM, POAG development, and caveolar ultrastructural morphology may provide the basis for a novel pathway of exploration of the pathologic mechanisms of glaucoma. Differential gene expression of SDPR in TM from Black vs White subjects with glaucoma may further our understanding of the important public health implications of the racial disparities of this blinding disease.

Hyaluronic Acid Based Adipose Tissue-Derived Extracellular Matrix Scaffold in Wound Healing: Histological and Immunohistochemical Study.

In this study, we explored the potential of human adipose tissue-derived extracellular matrix (adECM) sheets augmented with crosslinked hyaluronic acid (HA) as advanced wound dressings. We aimed to enhance healing efficacy while optimizing cost efficiency. The adECM was processed from healthy donor tissue and combined with crosslinked HA to form ECM-HA sheets (Scaffiller, Medikan, Korea). In vitro experiments involved seeding adipose-derived stem cells (ASCs) onto these sheets and assessing cell survival and cytokine production. In vivo testing utilized a rat wound model, comparing ECM-HA sheet with HA-based dressing and polyurethane foam dressing. Re-epithelialization and collagen deposition were examined through histopathological examinations, whereas immunohistochemistry was used to assess CD31, alpha smooth muscle actin (α-SMA), and Tenascin C expression as contributing factors to wound healing. Results indicated that ECM-HA sheets were produced efficiently, with enhanced growth factor production and ASC survival observed in vitro. In vivo, ECM-HA sheets demonstrated accelerated wound healing, evidenced by improved epithelialization, thicker dermis, increased collagen deposition, and enhanced vascularity. Notably, they exhibited reduced myofibroblast activity and increased expression of Tenascin C, suggesting a favorable healing environment. ECM-HA sheets offer a promising approach for wound management, combining the benefits of adECM and HA. They present improved stability and cost-effectiveness while promoting essential aspects of wound healing such as angiogenesis and collagen formation. This study underscores the therapeutic potential of ECM-HA sheets in clinical applications aimed at facilitating wound repair.

A xenotransplantation mouse model to study physiology of the mammary gland from large mammals.

Although highly conserved in structure and function, many (patho)physiological processes of the mammary gland vary drastically between mammals, with mechanisms regulating these differences not well understood. Large mammals display variable lactation strategies and mammary cancer incidence, however, research into these variations is often limited to in vitro analysis due to logistical limitations. Validating a model with functional mammary xenografts from cryopreserved tissue fragments would allow for in vivo comparative analysis of mammary glands from large and/or rare mammals and would improve our understanding of postnatal development, lactation, and premalignancy across mammals. To this end, we generated functional mammary xenografts using mammary tissue fragments containing mammary stroma and parenchyma isolated via an antibody-independent approach from healthy, nulliparous equine and canine donor tissues to study these species in vivo. Cryopreserved mammary tissue fragments were xenotransplanted into de-epithelialized fat pads of immunodeficient mice and resulting xenografts were structurally and functionally assessed. Preimplantation of mammary stromal fibroblasts was performed to promote ductal morphogenesis. Xenografts recapitulated mammary lobule architecture and contained donor-derived stromal components. Mammatropic hormone stimulation resulted in (i) upregulation of lactation-associated genes, (ii) altered proliferation index, and (iii) morphological changes, indicating functionality. Preimplantation of mammary stromal fibroblasts did not promote ductal morphogenesis. This model presents the opportunity to study novel mechanisms regulating unique lactation strategies and mammary cancer induction in vivo. Due to the universal applicability of this approach, this model serves as proof-of-concept for developing mammary xenografts for in vivo analysis of virtually any mammals, including large and rare mammals.

FEATURES OF COLLAGENASE GENE EXPRESSION IN BREAST CANCER TISSUE:AN OPEN DATABASES ANALYSIS

Summary. Breast cancer (BCa) is the most common cancer among women both in Ukraine and in the world. Extracellular matrix remodeling has been shown to play a crucial role in tumor progression. Collagenases are one of the main drivers of this process, providing collagen degradation and directly affecting the invasion and metastasis of neoplastic cells. Aim: to study the features of collagenase gene expression and their regulatory factors in BC tissue as possible drivers of tumor growth. Objects and methods: a comparison of collagenase gene expression levels in normal and malignant breast tissue was performed using the GEPIA, UALCAN, and TNMplot resources. The methylation levels of collagenase gene promoters were determined using the UALCAN database. The identification of microRNAs and transcription factors involved in the regulation of collagenase expression was performed based on the information provided on the miRNet v2.0 platform using the TarBase v.8.0 and JASPAR databases. The miRNA level study was performed using the miR-TV online resource. Results: it was found that BCa tissue is characterized by significantly higher MMP1, MMP8, and MMP13 mRNA expression levels compared to normal breast tissue. It was demonstrated that the methylation levels of MMP1 and MMP13 promoters in tumor cells were significantly lower compared to non-transformed breast cells. Using the TarBase v.8.0 and JASPAR databases, transcription factors and miRNAs involved in the regulation of MMP1, MMP8, and MMP13 expression were identified. Analysis of the expression levels of miRNAs involved in the regulation of collagenase expression showed that the levels of miR-155-5p, miR-200b-3p, and miR-210-5p in BCa tissue were 2.26 (p = 2.2e-16), 2.95 (p = 2.2e-16), and 5.90 (p = 2.2e-16) times higher, respectively, compared to non-transformed breast tissue. It was shown that BC tissues compared to healthy donor tissues were characterized by significantly lower expression levels of miR-100-5p, miR-127-3p, and miR-539-5p by 4.19 (p = 2.2e-16), 2.05 (p = 3.9e-12), and 1.70 (p = 2.5e-5), respectively. Conclusions: the obtained results indicate the involvement of collagenases in the development and progression of malignant tumors and indicate the need for further research on their role in the formation of the aggressive potential of BC.

The mammalian target of rapamycin contributes to synovial fibroblast pathogenicity in rheumatoid arthritis.

The mammalian target of Rapamycin (mTOR) is a metabolic master regulator of both innate and adaptive immunity; however, its exact role in stromal cell biology is unknown. In this study we explored the role of the mTOR pathway on Rheumatoid Arthritis synovial fibroblast (RASF) metabolism and activation and determined if crosstalk with the Hippo-YAP pathway mediates their effects. Primary RA synovial fibroblasts (RASF) were cultured with TNFα alone or in combination with the mTOR inhibitor Rapamycin or YAP inhibitor Verteporfin. Chemokine production, matrix metalloproteinase (MMP) production, and adhesion marker expression were quantified by real-time PCR, ELISA, and/or Flow Cytometry. Invasion assays were performed using Transwell invasion chambers, while wound repair assays were used to assess RASF migration. Cellular bioenergetics was assessed using the Seahorse XFe96 Analyzer. Key metabolic genes (GLUT-1, HK2, G6PD) were measured using real-time PCR. Reanalysis of RNA-Seq analysis was performed on RA (n = 151) and healthy control (HC) (n = 28) synovial tissue biopsies to detect differential gene and pathway expression. The expression of YAP was measured by Western Blot. Transcriptomic analysis of healthy donor and RA synovial tissue revealed dysregulated expression of several key components of the mTOR pathway in RA. Moreover, the expression of phospho-ribosomal protein S6 (pS6), the major downstream target of mTOR is specifically increased in RA synovial fibroblasts compared to healthy tissue. In the presence of TNFα, RASF display heightened phosphorylation of S6 and are responsive to mTOR inhibition via Rapamycin. Rapamycin effectively alters RASF cellular bioenergetics by inhibiting glycolysis and the expression of rate limiting glycolytic enzymes. Furthermore, we demonstrate a key role for mTOR signaling in uniquely mediating RASF migratory and invasive mechanisms, which are significantly abrogated in the presence of Rapamycin. Finally, we report a significant upregulation in several genes involved in the Hippo-YAP pathway in RA synovial tissue, which are predicted to converge with the mTOR pathway. We demonstrate crosstalk between the mTOR and YAP pathways in mediating RASF invasive mechanism whereby Rapamycin significantly abrogates YAP expression and YAP inhibition significantly inhibits RASF invasiveness. mTOR drives pathogenic mechanisms in RASF an effect which is in part mediated via crosstalk with the Hippo-YAP pathway.

Overcoming selection bias in synthetic lethality prediction.

MotivationSynthetic lethality (SL) between two genes occurs when simultaneous loss of function leads to cell death. This holds great promise for developing anti-cancer therapeutics that target synthetic lethal pairs of endogenously disrupted genes. Identifying novel SL relationships through exhaustive experimental screens is challenging, due to the vast number of candidate pairs. Computational SL prediction is therefore sought to identify promising SL gene pairs for further experimentation. However, current SL prediction methods lack consideration for generalizability in the presence of selection bias in SL data.ResultsWe show that SL data exhibit considerable gene selection bias. Our experiments designed to assess the robustness of SL prediction reveal that models driven by the topology of known SL interactions (e.g. graph, matrix factorization) are especially sensitive to selection bias. We introduce selection bias-resilient synthetic lethality (SBSL) prediction using regularized logistic regression or random forests. Each gene pair is described by 27 molecular features derived from cancer cell line, cancer patient tissue and healthy donor tissue samples. SBSL models are built and tested using approximately 8000 experimentally derived SL pairs across breast, colon, lung and ovarian cancers. Compared to other SL prediction methods, SBSL showed higher predictive performance, better generalizability and robustness to selection bias. Gene dependency, quantifying the essentiality of a gene for cell survival, contributed most to SBSL predictions. Random forests were superior to linear models in the absence of dependency features, highlighting the relevance of mutual exclusivity of somatic mutations, co-expression in healthy tissue and differential expression in tumour samples.Availability and implementation https://github.com/joanagoncalveslab/sbsl Supplementary information Supplementary data are available at Bioinformatics online.

In Vitro Expansion of Corneal Endothelial Cells for Clinical Application: Current Update.

Endothelial dysfunction is one of the leading causes of corneal blindness and one of the common indications for keratoplasty. At present, the standard of treatment involves the replacement of the dysfunctional endothelium with healthy tissue taken from a donor. Because there is a paucity of healthy donor tissues, research on the corneal endothelium has focused primarily on expanding these cells in the laboratory for transplantation in an attempt to reduce the gap between the demand and supply of donor tissues for transplantation. To expand these cells, which are nonmitotic in vivo, various mitogens, substrates, culture systems, and alternate strategies have been tested with varying success. The biggest challenge has been the limited proliferative capacity of these cells compounded with endothelial to mesenchymal transition that alters the functioning of these cells and renders them unsuitable for human transplantation. This review aims to give a comprehensive overview of the most common and successful techniques used in the culture of the cells, the current available evidence in support of epithelial to mesenchymal transition (EMT), alternate sources for deriving the corneal endothelial cells, and advances made in transplantation of these cells.

Primary radiotherapy and deep inferior epigastric perforator flap reconstruction for patients with breast cancer (PRADA): a multicentre, prospective, non-randomised, feasibility study.

Radiotherapy before mastectomy and autologous free-flap breast reconstruction can avoid adverse radiation effects on healthy donor tissues and delays to adjuvant radiotherapy. However, evidence for this treatment sequence is sparse. We aimed to explore the feasibility of preoperative radiotherapy followed by skin-sparing mastectomy and deep inferior epigastric perforator (DIEP) flap reconstruction in patients with breast cancer requiring mastectomy. We conducted a prospective, non-randomised, feasibility study at two National Health Service trusts in the UK. Eligible patients were women aged older than 18 years with a laboratory diagnosis of primary breast cancer requiring mastectomy and post-mastectomy radiotherapy, who were suitable for DIEP flap reconstruction. Preoperative radiotherapy started 3-4 weeks after neoadjuvant chemotherapy and was delivered to the breast, plus regional nodes as required, at 40 Gy in 15 fractions (over 3 weeks) or 42·72 Gy in 16 fractions (over 3·2 weeks). Adverse skin radiation toxicity was assessed preoperatively using the Radiation Therapy Oncology Group toxicity grading system. Skin-sparing mastectomy and DIEP flap reconstruction were planned for 2-6 weeks after completion of preoperative radiotherapy. The primary endpoint was the proportion of open breast wounds greater than 1 cm width requiring a dressing at 4 weeks after surgery, assessed in all participants. This study is registered with ClinicalTrials.gov, NCT02771938, and is closed to recruitment. Between Jan 25, 2016, and Dec 11, 2017, 33 patients were enrolled. At 4 weeks after surgery, four (12·1%, 95% CI 3·4-28·2) of 33 patients had an open breast wound greater than 1 cm. One (3%) patient had confluent moist desquamation (grade 3). There were no serious treatment-related adverse events and no treatment-related deaths. Preoperative radiotherapy followed by skin-sparing mastectomy and immediate DIEP flap reconstruction is feasible and technically safe, with rates of breast open wounds similar to those reported with post-mastectomy radiotherapy. A randomised trial comparing preoperative radiotherapy with post-mastectomy radiotherapy is required to precisely determine and compare surgical, oncological, and breast reconstruction outcomes, including quality of life. Cancer Research UK, National Institute for Health Research.

20 Autologous Meshed Split Thickness Graft Healing in Interstice versus Grafted Sites: A Histological Characterization

IntroductionAutologous skin grafting is a common technique used in the treatment of full thickness (FT) wounds to aid in healing, wound closure, and reducing the likelihood of developing hypertrophic scarring. Meshed split thickness skin grafts (mSTSG) which contain portions of the dermis and epidermis are the gold standard for the treatment of FT wounds because they allow for expansion of skin taken from a relatively small donor site. It has largely been hypothesized that in mSTSG skin progenitor cells migrate from the edges of healthy donor tissue to aid in healing the interstices created by meshing. For this reason, meshed wound healing is not homogenous as interstice and meshed sites display distinct healing characteristics. This study aims to characterize the differences between interstice and grafted site healing.MethodsWound healing was evaluated in vivo using Duroc pigs. In this model, 4 animals had 10.16 cm by 10.16 cm full-thickness burns created on bilateral flanks for a total of 12 wounds. On day 2, the burns were excised down to subcutaneous tissue, mSTSG was harvested, meshed 4:1, and applied to the prepared wound beds. Wounds were photographed and sampled on days 5, 9 and, 15. Punch biopsies from either the grafted area or interstice area were taken at each time point, processed, and imaged. Images were used to quantify epidermal and dermal thickness, cellularity, and rete ridges.ResultsEpidermal thickness at day 5 in interstices was significantly thinner than in graft (1.73±4.33µm vs. 75.7±66.1µm, p< 0.05). By day 9, interstice epidermal thickness was comparable to graft thickness (194.9±157.7µm vs. 199.5±117.7µm). On the other hand, dermal thickness was elevated in the interstice at days 9 (1850.4±642.0µm vs. 1277.6±652.0µm, p< 0.05) and 15 (2469.9±626.14µm vs. 1660.7±674.6µm, p< 0.01) to a significant degree. Cellularity was greater at all time points in the interstice compared to the grafted sites. Similarly, rete ridge ratios (RRR) were significantly greater in the grafted areas at day 5 (0.0±0.0µm v.s. 1.0±0.7µm, p< 0.01) and day 9 (1.32±1.2µm vs. 1.9±0.45µm, p< 0.05).ConclusionsThese data show that within a grafted burn wound, healing is a dynamic and heterogenous process when looking at interstice and graft sites, respectively. Grafted sites were thinner throughout, showed decreased inflammatory cell infiltrate, and exhibited higher RRR. Thicker tissue layers and upregulated cellularity in interstices point to a wound healing trajectory that is slower than grafted sites, even by the time wounds are fully re-epithelized.

Screening and Identification of Human Endogenous Retrovirus-K mRNAs for Breast Cancer Through Integrative Analysis of Multiple Datasets.

ObjectiveHuman endogenous retroviruses (HERVs) make up 8% of the human genome. HERVs are biologically active elements related to multiple diseases. HERV-K, a subfamily of HERVs, has been associated with certain types of cancer and suggested as an immunologic target in some tumors. The expression levels of HERV-K in breast cancer (BCa) have been studied as biomarkers and immunologic therapeutic targets. However, HERV-K has multiple copies in the human genome, and few studies determined the transcriptional profile of HERV-K copies across the human genome for BCa.MethodsNinety-one HERV-K indexes with entire proviral sequences were used as the reference database. Nine raw sequencing datasets with 243 BCa and 137 control samples were mapped to this database by Salmon software. The differential proviral expression across several groups was analyzed by DESeq2 software.ResultsFirst, the clustering of each dataset demonstrated that these 91 HERV-K proviruses could well cluster the BCa and control samples when the normal controls were normal cells or healthy donor tissues. Second, several common HERV-K proviruses that are closely related with BCa risk were significantly differentially expressed (padj < 0.05 and absolute log2FC > 1.5) in the tissues and cell lines. Additionally, almost all the HERV-K proviruses had higher expression in BCa tissue than in healthy donor tissue. Notably, we first found the expression of 17p13.1 provirus that located with TP53 should regulate TP53 expression in ER+ and HER2+ BCa.ConclusionThe expression profiling of these 91 HERV-K proviruses can be used as biomarkers to distinguish individuals with BCa and healthy controls. Some proviruses, especially 17p13.1, were strongly associated with BCa risk. The results suggest that HERV-K expression profiles may be appropriate biomarkers and targets for BCa.

Editors' Selections from Relevant Scientific Publications.

Editors' Selections from Relevant Scientific Publications.

Burkitt Lymphoma Genome Sequencing Project (BLGSP): Integrative Genomic and Transcriptomic Characterization of Burkitt Lymphoma

IntroductionBurkitt lymphoma (BL) is an aggressive B-cell non-Hodgkin lymphoma with a hallmark translocation involving MYC and an immunoglobulin enhancer. It is most common in children and has three clinical variants: endemic, sporadic, and HIV-associated. The Epstein-Barr virus (EBV)-associated endemic subtype (eBL) is highly prevalent in equatorial Africa, where it is the most common pediatric cancer. Survival is poor among African children with eBL due to late stage at presentation and limitations in the ability to support intensive chemotherapeutic regimens necessary to induce durable remissions. Previous genomic studies of smaller BL cohorts revealed that eBL harbors mutation patterns similar but not identical to sporadic cases from high-resource countries. The BLGSP aims at conducting an integrative molecular characterization of a large comprehensive BL cohort including an unprecedented representation of endemic cases. The objective is to define molecular features that drive lymphomagenesis, which can be translated to new therapeutic strategies deployable worldwide.MethodsTumor biopsies and matched normal tissue have been collected from patients with both endemic and sporadic BL along with treatment, outcome, and other clinical information. Molecular characterization includes whole genome sequencing of tumor and constitutional DNA (80X and 40X, respectively), and strand-specific ribo-depleted sequencing of tumor RNA and microRNA. Six centroblast and 6 centrocyte samples flow-sorted from healthy donor tissue underwent RNA and miRNA sequencing as normal comparators for gene expression. These data enable the identification of somatic mutations, significantly mutated genes (SMGs) and non-coding loci with potential regulatory function; the quantification of human and EBV gene expression; and the characterization of miRNA-mediated regulation of transcripts. SMGs were supported by two or more of the following methods with a 10% false discovery rate: OncodriveFM, OncodriveFML, OncodriveCLUST, and MutSigCV. We also re-analyzed the genome data for 17 sporadic cases from the ICGC MALY-DE project (Nat. Genet. 2015; 47: 1316-1325).ResultsA total of 101 cases (71 endemic and 30 sporadic) were included after quality control. We describe novel mutation features affecting known BL genes. Notably, DDX3X is the target of focal deletions and splicing branch point mutations, and in-frame deletions recurrently affect a putative ubiquitination site (K272) in GNAI2 . Our meta-analysis reveals 22 SMGs, including many that have not yet been linked to the disease (see figure). We associate recurrent mutations in the following genes with BL for the first time: SIN3A, HIST1H1E, KMT2D, CHD8, USP7, and RFX7 . Several SMGs are differentially mutated between endemic and sporadic BL, suggesting distinct underlying biology (see highlighted genes in figure). For instance, ARID1A is preferentially mutated in endemic cases whereas SMARCA4 shows the opposite trend. Multiple non-coding loci are also significantly mutated: a PAX5 enhancer, BCL6 super-enhancers, the PVT1 non-coding locus, and the miR-142 gene, among others. Three mutation signatures are detected across all tumors: one is associated with defective DNA mismatch repair, another has been linked to polymerase η and AID activity, and the last signature has an unknown etiology. Gene expression analysis reveals high correlation between tumors (Pearson coefficient > 0.9). This is consistent with the absence of robust clusters according to unsupervised clustering of expression profiles.DiscussionBLGSP is an ongoing international collaborative project aimed at providing a comprehensive molecular portrait of BL across all subtypes. We describe previously unreported mutation features affecting established BL genes and identify novel candidate coding and non-coding driver mutations, nominating compelling targets for therapy. For example, one of the novel SMGs, USP7, encodes a deubiquitinase that counteracts Mdm2-mediated ubiquitination and degradation of p53. We hypothesize that Mdm2 inhibitors might trigger synthetic lethality in USP7 -mutated, TP53 -wildtype tumors. In summary, this effort has identified candidate molecular targets for therapy that can potentially lead to more effective treatments that are less toxic than the current regimens.BMG and DSG contributed equally. RDM and LMS contributed equally. [Display omitted] DisclosuresCasper:TempTime: Consultancy, Other: Travel, Accommodation, Expenses; GSK: Other: Travel, Accommodation, Expenses; Janssen: Consultancy, Research Funding; Up to Date: Patents & Royalties; Roche: Consultancy, Other: Travel, Accommodation, Expenses. Mullighan:Loxo Oncology: Research Funding; Amgen: Consultancy. Abramson:Abbvie: Consultancy; Genentech: Consultancy; LAM Therapeutics: Research Funding; Gilead: Consultancy; Kite Pharma: Consultancy; Seattle Genetics: Consultancy; Celgene: Consultancy; Novartis: Consultancy. Noy:Pharmacyclics LLC, an AbbVie Company: Honoraria, Other: Travel, Accommodation, Expenses, Research Funding, Speakers Bureau. Morin:Epizyme, Inc: Consultancy.

Dysbalance of ACE2 levels - a possible cause for severe COVID-19 outcome in COPD.

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) poses a serious threat to healthcare systems worldwide. Binding of the virus to angiotensin‐converting enzyme 2 (ACE2) is an important step in the infection mechanism. However, it is unknown if ACE2 expression in patients with chronic lung diseases (CLDs), such as chronic obstructive pulmonary disease (COPD), idiopathic pulmonary arterial hypertension (IPAH), or pulmonary fibrosis (PF), is changed as compared to controls. We used lung samples from patients with COPD (n = 28), IPAH (n = 10), and PF (n = 10) as well as healthy control donor (n = 10) tissue samples to investigate the expression of ACE2 and related cofactors that might influence the course of SARS‐CoV‐2 infection. Expression levels of the ACE2 receptor, the putative receptor CD147/BSG, and the viral entry cofactors TMPRSS2 (transmembrane serine protease 2), EZR, and FURIN were determined by quantitative PCR and in open‐access RNA sequencing datasets. Immunohistochemical and single‐cell RNA sequencing (scRNAseq) analyses were used for localization and coexpression, respectively. Soluble ACE2 (sACE2) plasma levels were analyzed by enzyme‐linked immunosorbent assay. In COPD as compared to donor, IPAH, and PF lung tissue, gene expression of ACE2, TMPRSS2, and EZR was significantly elevated, but circulating sACE2 levels were significantly reduced in COPD and PF plasma compared to healthy control and IPAH plasma samples. Lung tissue expressions of FURIN and CD147/BSG were downregulated in COPD. None of these changes were associated with changes in pulmonary hemodynamics. Histological analysis revealed coexpression of ACE2, TMPRSS2, and Ezrin in bronchial regions and epithelial cells. This was confirmed by scRNAseq analysis. There were no significant expression changes of the analyzed molecules in the lung tissue of IPAH and idiopathic PF as compared to control. In conclusion, we reveal increased ACE2 and TMPRSS2 expression in lung tissue with a concomitant decrease of protective sACE2 in COPD patients. These changes represent the possible risk factors for an increased susceptibility of COPD patients to SARS‐CoV‐2 infection.

Interferon-beta (IFNb) Reprograms Pathogenic Lung Dendritic Cells in Human Chronic Lung Allograft Dysfunction (CLAD)

<h3>Purpose</h3> Dendritic cells (DC) are central to the induction and maintenance of T regulatory cells (T-regs) and lung allograft tolerance. We previously identified tumor necrosis factor 2 positive DC (TNFR2⁺DC) as a subset of lung resident DC responsible for antigen-specific Th2 mediated response and inflammation in mice model of asthma. Interestingly, TNFR2⁺DC is plastic and intranasal administration of IFNβ reprograms TNFR2⁺DC to generate regulatory T-cells and reverses lung inflammation in mice. We set out to characterize TNFR2⁺DC and the response to IFNβ in healthy human donor and CLAD lungs. <h3>Methods</h3> We evaluated single-cell suspensions from the unused healthy human donor lungs from the lobar transplantation, and CLAD explant lung tissue from patients undergoing re-transplantation. Identification of TNFR2⁺DC populations and the response to IFNβ (200ng IFNβ, R&D systems INC. MN, USA) were investigated with flow cytometry analysis. <h3>Results</h3> We successfully identified the TNFR2⁺DC population in healthy donor and CLAD lung tissues (n=4/group) (Figure 1A). The healthy lung TNFR2⁺DC constitutively express TGFβ1, IDO-1, and Arg-1, which has a similar phenotype to tolerogenic DC in mouse lungs. Conversely, TNFR2⁺DC from CLAD patients had decreased TGFβ1, Arg-1, PD-L1, but increased IL-4 and IL-23 expression (Figure 1B). Treatment with IFNβ increased TGFβ1, Arg1, and decreased IL-4 expression in CLAD TNFR2⁺DC. Thus, similar to mice, treatment with IFNβ reprograms pathogenic TNFR2⁺DC in CLAD to phenotypically tolerogenic DC (Figure 1C). <h3>Conclusion</h3> We identified TNFR2⁺DC in healthy human donor lung and CLAD. Healthy human donor lung has a phenotypically tolerogenic TNFR2⁺DC. Conversely, TNFR2⁺DC in CLAD shows inflammatory markers, and treatment with IFNβ reprograms pathogenic DC to a tolerogenic phenotype. Further studies are required to assess the utility of TNFR2⁺DC plasticity for achieving lung allograft tolerance.

New developments in corneal endothelial cell replacement.

Corneal transplantation is currently the most effective treatment to restore corneal clarity in patients with endothelial disorders. Endothelial transplantation, either by Descemet membrane endothelial keratoplasty (DMEK) or by Descemet stripping (automated) endothelial keratoplasty (DS(A)EK), is a surgical approach that replaces diseased Descemet membrane and endothelium with tissue from a healthy donor eye. Its application, however, is limited by the availability of healthy donor tissue. To increase the pool of endothelial grafts, research has focused on developing new treatment options as alternatives to conventional corneal transplantation. These treatment options can be considered as either 'surgery-based', that is tissue-efficient modifications of the current techniques (e.g. Descemet stripping only (DSO)/Descemetorhexis without endothelial keratoplasty (DWEK) and Quarter-DMEK), or 'cell-based' approaches, which rely on invitro expansion of human corneal endothelial cells (hCEC) (i.e. cultured corneal endothelial cell sheet transplantation and cell injection). In this review, we will focus on the most recent developments in the field of the 'cell-based' approaches. Starting with the description of aspects involved in the isolation of hCEC from donor tissue, we then describe the different natural and bioengineered carriers currently used in endothelial cell sheet transplantation, and finally, we discuss the current 'state of the art' in novel therapeutic approaches such as endothelial cell injection.

Osteochondral Autograft Transfer (OATS)

Osteochondral autograft transfer (OATS) is a one-stage surgical procedure to treat chondral defects of the knee and other articulating surfaces. Optimal treatment for symptomatic knee lesions remains controversial. Osteochondral autograft transfer is one of several approaches to cartilage repair. Other techniques include marrow stimulation, cell-based strategies, and allograft transplantation. 1 The advantages of OATS include the use of autologous tissue, which avoids the potential risks and costs associated with allograft transplantation, as well as the use of a structural graft with both bone and cartilage. This may be advantageous over marrow stimulation or cellular treatments, which only address the chondral component to the pathology. The disadvantage of OATS is that it cannot be used to treat very large lesions, as there is not enough healthy donor tissue in the knee to account for large lesions. Of note, OATS is an acronym often used for both autograft and allograft osteochondral transplantation, which can be confusing. OATS does refer to osteochondral autograft transplantation, and for the purposes of this manuscript, should not be considered synonymous with allograft transplantation.

Th17 cells inhibit CD8+ T cell migration by systematically downregulating CXCR3 expression via IL-17A/STAT3 in advanced-stage colorectal cancer patients

BackgroundCD8+ T cell trafficking to the tumor site is essential for effective colorectal cancer (CRC) immunotherapy. However, the mechanism underlying CD8+ T cell infiltration in colorectal tumor tissues is not fully understood. In the present study, we investigated CD8+ T cell infiltration in CRC tissues and the role of chemokine–chemokine receptor signaling in regulation of T cell recruitment.MethodsWe screened chemokines and cytokines in healthy donor and CRC tissues from early- and advanced-stage patients using multiplex assays and PCR screening. We also utilized transcription factor activation profiling arrays and established a xenograft mouse model.ResultsCompared with tumor tissues of early-stage CRC patients, CD8+ T cell density was lower in advanced-stage tumor tissues. PCR screening showed that CXCL10 levels were significantly increased in advanced-stage tumor tissues. CXCR3 (the receptor of CXCL10) expression on CD8+ T cells was lower in the peripheral blood of advanced-stage patients. The migratory ability of CD8+ T cells to CXCL10 depended on CXCR3 expression. Multiplex arrays showed that IL-17A was increased in advanced-stage patient sera, which markedly downregulated CXCR3 expression via activating STAT3 signaling and reduced CD8+ T cell migration. Similar results were found after CD8+ T cells were treated with Th17 cell supernatant. Adding anti-IL-17A or the STAT3 inhibitor, Stattic, rescued these effects in vitro and in vivo. Moreover, survival analysis showed that patients with low CD8 and CXCR3 expression and high IL-17A levels had significantly worse prognosis.ConclusionsCD8+ T cell infiltration in advanced-stage tumor was systematically inhibited by Th17 cells via IL-17A/STAT3/CXCR3 axis. Our findings indicate that the T cell infiltration in the tumor microenvironment may be improved by inhibiting STAT3 signaling.

In Vitro Culture of Human Corneal Endothelium on Non-Mulberry Silk Fibroin Films for Tissue Regeneration.

PurposeThe purpose of this study was to determine if non-mulberry varieties of silk are suitable for the culture of corneal endothelium (CE).MethodsAqueous silk fibroin derived from Philosamia ricini (PR), Antheraea assamensis (AA), and Bombyx mori (BM) were cast as approximately 15 µm films with and without pores on which human CE cells were cultured. Tensile strength, elasticity, transmittance in visible range, and degradation properties of the films were characterised. Adhesion of CE to the silk films was quantified using MTT assay in addition to quantifying the number and area of focal adhesions using paxillin. Expression of CE markers was determined at the gene and protein levels using PCR and immunostaining, respectively. Barrier integrity of the cultured cells was measured as permeability to FITC dextran (10 kDa) in the presence or absence of thrombin.ResultsThe films exhibited robust tensile strength, >95% transmittance and a refractive index comparable to the native cornea. BM degraded significantly faster when compared to PR and AA. A comparison between the three varieties of silk showed that significantly more cells were adhered to PR and AA than to BM. This was also reflected in the expression of stable focal adhesions on PR and AA, thus enabling the formation of intact monolayers of cells on these varieties unlike on BM. Treatment with thrombin significantly increased cellular permeability to dextran.ConclusionsOur data shows that PR and AA varieties sufficiently support the growth and function of CE cells. This could be attributed to the presence of natural cell binding motifs (RGD) in these varieties.Translational RelevanceDevelopment of a suitable carrier for engineering the CE to address a major clinical requirement of healthy donor tissues for transplantation.