Significance of regional population HLA immunogenetic datasets in the efficacy of umbilical cord blood banks and marrow donor registries: a study of Cretan HLA genetic diversity.

BACKGROUND AIMS: The high genetic diversity of HLA across populations significantly confines the effectiveness of a donor or umbilical cord blood search for allogeneic hematopoietic stem cell transplantation (HSCT). This study aims to probe the HLA immunogenetic profile of the population of Crete, a Greek region with specific geographic and historical characteristics, and to investigate potential patterns in HLA distribution following comparison with the Deutsche Knochenmarkspenderdatei (DKMS) donor registry. It also aims to highlight the importance of regional public cord blood banks (PCBBs) in fulfilling HSCT needs, especially in countries with significant genetic diversity. METHODS: A cohort of 1835 samples representative of the Cretan population was typed for HLA class I (HLA-A, HLA-B, HLA-C) and class II (HLA-DRB1, HLA-DQB1, HLA-DPB1) loci by high-resolution second field next-generation sequencing. Data were compared with the respective HLA profiles of 12 DKMS populations (n = 20 032). Advanced statistical and bioinformatics methods were employed to assess specific intra- and inter-population genetic indexes associated with the regional and geographic distribution of HLA alleles and haplotypes. RESULTS: A considerable HLA allelic and haplotypic diversity was identified among the Cretan samples and between the latter and the pooled DKMS cohort. Even though the HLA allele and haplotype frequency distribution was similar to regions of close geographic proximity to Crete, a clinal distribution pattern from the northern to southern regions was identified. Significant differences were also observed between Crete and the Greek population of DKMS. CONCLUSIONS: This study provides an in-depth characterization of the HLA immunogenetic profile in Crete and reveals the importance of demographic history in HLA heterogeneity and donor selection. The novel HLA allele and haplotype frequency comparative data between the Cretan and other European populations signify the importance of regional PCBBs in prioritizing HLA diversity to efficiently promote the HSCT program at the national level and beyond.

Mouse HP1γ regulates TRF1 expression and telomere stability.

AIMS: Telomeric repeat-containing RNAs are long non-coding RNAs generated from the telomeres. TERRAs are essential for the establishment of heterochromatin marks at telomeres, which serve for the binding of members of the heterochromatin protein 1 (HP1) protein family of epigenetic modifiers involved with chromatin compaction and gene silencing. While HP1γ is enriched on gene bodies of actively transcribed human and mouse genes, it is unclear if its transcriptional role is important for HP1γ function in telomere cohesion and telomere maintenance. We aimed to study the effect of mouse HP1γ on the transcription of telomere factors and molecules that can affect telomere maintenance. MAIN METHODS: We investigated the telomere function of HP1γ by using HP1γ deficient mouse embryonic fibroblasts (MEFs). We used gene expression analysis of HP1γ deficient MEFs and validated the molecular and mechanistic consequences of HP1γ loss by telomere FISH, immunofluorescence, RT-qPCR and DNA-RNA immunoprecipitation (DRIP). KEY FINDINGS: Loss of HP1γ in primary MEFs led to a downregulation of various telomere and telomere-accessory transcripts, including the shelterin protein TRF1. Its downregulation is associated with increased telomere replication stress and DNA damage (γH2AX), effects more profound in females. We suggest that the source for the impaired telomere maintenance is a consequence of increased telomeric DNA-RNA hybrids and TERRAs arising at and from mouse chromosomes 18 and X. SIGNIFICANCE: Our results suggest an important transcriptional control by mouse HP1γ of various telomere factors including TRF1 protein and TERRAs that has profound consequences on telomere stability, with a potential sexually dimorphic nature.

IL-1 Mediates Tissue-Specific Inflammation and Severe Respiratory Failure in COVID-19.

Acute respiratory distress syndrome (ARDS) in COVID-19 has been associated with catastrophic inflammation. We present measurements in humans and a new animal model implicating a role in danger-associated molecular patterns. Calprotectin (S100A8/A9) and high-mobility group box 1 (HMGB1) were measured in patients without/with ARDS, and admission calprotectin was associated with soluble urokinase plasminogen activator receptor (suPAR). An animal model was developed by intravenous injection of plasma from healthy or patients with COVID-19 ARDS into C57/BL6 mice once daily for 3 consecutive days. Mice were treated with one anti-S100A8/A9 antibody, the IL-1 receptor antagonist anakinra or vehicle, and Flo1-2a anti-murine anti-IL-1α monoclonal antibody or the specific antihuman IL-1α antibody XB2001 or isotype controls. Cytokines and myeloperoxidase (MPO) were measured in tissues. Calprotectin, but not HMGB1, was elevated in ARDS. Higher suPAR indicated higher calprotectin. Animal challenge with COVID-19 plasma led to inflammatory reactions in murine lung and intestines as evidenced by increased levels of TNFα, IL-6, IFNγ, and MPO. Lung inflammation was attenuated with anti-S100A8/A9 pre-treatment. Anakinra treatment restored these levels. Similar decrease was found in mice treated with Flo1-2a but not with XB2001. Circulating alarmins, specifically calprotectin, of critically ill COVID-19 patients induces tissue-specific inflammatory responses through an IL-1-mediated mechanism. This could be attenuated through inhibition of IL-1 receptor or of IL-1α.

Two novel HLA-C alleles, HLA-C*15:228 and -C*04:434, detected in inhabitants from the island of Crete.

Characterization of the novel HLA-C*15:228 and HLA-C*04:434 alleles in two Greek individuals of Cretan origin.

Detection of the novel HLA-B*51:232:02 variant in an inhabitant from the island of Crete.

Characterization of the HLA-B*51:232:02 allele in a Greek individual of Cretan origin.

Detection of the novel HLA-DQB1*03:439 variant in an inhabitant from the island of Crete.

Characterization of HLA-DQB1*03:439 allele in a Greek individual of Cretan origin.

CRISPR-Mediated Reactivation of DKK3 Expression Attenuates TGF-ß Signaling in Prostate Cancer.

The DKK3 gene encodes a secreted protein, Dkk-3, that inhibits prostate tumor growth and metastasis. DKK3 is downregulated by promoter methylation in many types of cancer, including prostate cancer. Gene silencing studies have shown that Dkk-3 maintains normal prostate epithelial cell homeostasis by limiting TGF-ß/Smad signaling. While ectopic expression of Dkk-3 leads to prostate cancer cell apoptosis, it is unclear if Dkk-3 has a physiological role in cancer cells. Here, we show that treatment of PC3 prostate cancer cells with the DNA methyltransferase (DNMT) inhibitor decitabine demethylates the DKK3 promoter, induces DKK3 expression, and inhibits TGF-ß/Smad-dependent transcriptional activity. Direct induction of DKK3 expression using CRISPR-dCas9-VPR also inhibited TGF-ß/Smad-dependent transcription and attenuated PC3 cell migration and proliferation. These effects were not observed in C4-2B cells, which do not respond to TGF-ß. TGF-ß signals can regulate gene expression directly via SMAD proteins and indirectly by increasing DNMT expression, leading to promoter methylation. Analysis of genes downregulated by promoter methylation and predicted to be regulated by TGF-ß found that DKK3 induction increased expression of PTGS2, which encodes cyclooxygenase-2. Together, these observations provide support for using CRISPR-mediated induction of DKK3 as a potential therapeutic approach for prostate cancer and highlight complexities in Dkk-3 regulation of TGF-ß signaling.

Protective effect of stromal Dickkopf-3 in prostate cancer: opposing roles for TGFBI and ECM-1.

Aberrant transforming growth factor-ß (TGF-ß) signaling is a hallmark of the stromal microenvironment in cancer. Dickkopf-3 (Dkk-3), shown to inhibit TGF-ß signaling, is downregulated in prostate cancer and upregulated in the stroma in benign prostatic hyperplasia, but the function of stromal Dkk-3 is unclear. Here we show that DKK3 silencing in WPMY-1 prostate stromal cells increases TGF-ß signaling activity and that stromal cell-conditioned media inhibit prostate cancer cell invasion in a Dkk-3-dependent manner. DKK3 silencing increased the level of the cell-adhesion regulator TGF-ß-induced protein (TGFBI) in stromal and epithelial cell-conditioned media, and recombinant TGFBI increased prostate cancer cell invasion. Reduced expression of Dkk-3 in patient tumors was associated with increased expression of TGFBI. DKK3 silencing reduced the level of extracellular matrix protein-1 (ECM-1) in prostate stromal cell-conditioned media but increased it in epithelial cell-conditioned media, and recombinant ECM-1 inhibited TGFBI-induced prostate cancer cell invasion. Increased ECM1 and DKK3 mRNA expression in prostate tumors was associated with increased relapse-free survival. These observations are consistent with a model in which the loss of Dkk-3 in prostate cancer leads to increased secretion of TGFBI and ECM-1, which have tumor-promoting and tumor-protective roles, respectively. Determining how the balance between the opposing roles of extracellular factors influences prostate carcinogenesis will be key to developing therapies that target the tumor microenvironment.