Women with polycystic ovary syndrome (PCOS) have varying difficulties in achieving weight loss by lifestyle intervention, which may depend on adipose tissue metabolism. The objective was to study baseline subcutaneous adipose tissue gene expression as a prediction of weight loss by lifestyle intervention in obese/overweight women with PCOS. This is a secondary analysis of a randomized controlled trial where women with PCOS, aged 18–40 and body mass index (BMI) ≥ 27 were initially randomized to either a 4-month behavioral modification program or minimal intervention according to standard care. Baseline subcutaneous adipose tissue gene expression was related to weight change after the lifestyle intervention. A total of 55 obese/overweight women provided subcutaneous adipose samples at study entry. Weight loss was significant after behavioral modification (−2.2%, p = 0.0014), while there was no significant weight loss in the control group (−1.1%, p = 0.12). In microarray analysis of adipose samples, expression of 40 genes differed significantly between subgroups of those with the greatest weight loss or weight gain. 10 genes were involved in metabolic pathways including glutathione metabolism, gluconeogenesis, and pyruvate metabolism. Results were confirmed by real-time polymerase chain reaction (RT-PCR) in all 55 subjects. Expressions of GSTM5, ANLN, and H3C2 correlated with weight change (R = −0.41, p = 0.002; R = −0.31, p = 0.023 and R = −0.32, p = 0.016, respectively). GSTM5, involved in glutathione metabolism, was the strongest predictor of weight loss, and together with baseline waist-hip ratio (WHR) explained 31% of the variation in body weight change. This study shows that baseline subcutaneous adipose tissue genes play a role for body weight outcome in response to lifestyle intervention in overweight/obese women with PCOS.

Autologous bone remains the gold standard bone substitute in clinical practice. Therefore, the microarchitecture of newly developed synthetic bone substitutes, which reflects the spatial distribution of materials in the scaffold, aims to recapitulate the natural bone microarchitecture. However, the natural bone microarchitecture is optimized to obtain a mechanically stable, lightweight structure adapted to the biomechanical loading situation. In the context of synthetic bone substitutes, the application of a Triply Periodic Minimum Surface (TPMS) algorithm can yield stable lightweight microarchitectures that, despite their demanding architectural complexity, can be produced by additive manufacturing. In this study, we applied the TPMS derivative Adaptive Density Minimal Surfaces (ADMS) algorithm to produce scaffolds from hydroxyapatite (HA) using a lithography-based layer-by-layer methodology and compared them with an established highly osteoconductive lattice microarchitecture. We characterized them for compression strength, osteoconductivity, and bone regeneration. The in vivo results, based on a rabbit calvaria defect model, showed that bony ingrowth into ADMS constructs as a measure of osteoconduction depended on minimal constriction as it limited the maximum apparent pore diameter in these scaffolds to 1.53 mm. Osteoconduction decreased significantly at a diameter of 1.76 mm. The most suitable ADMS microarchitecture was as osteoconductive as a highly osteoconductive orthogonal lattice microarchitecture in noncritical- and critical-size calvarial defects. However, the compression strength and microarchitectural integrity in vivo were significantly higher for scaffolds with their microarchitecture based on the ADMS algorithm when compared with high-connectivity lattice microarchitectures. Therefore, bone substitutes with high osteoconductivity can be designed with the advantages of the ADMS-based microarchitectures. As TPMS and ADMS microarchitectures are true lightweight structures optimized for high mechanical stability with a minimal amount of material, such microarchitectures appear most suitable for bone substitutes used in clinical settings to treat bone defects in weight-bearing and non-weight-bearing sites.

Interleukin 33 (IL-33) signaling regulates most of the key processes of pregnancy, including decidualization, trophoblast proliferation and invasion, vascular remodeling, and placental growth. Accordingly, dysregulation of IL-33, its membrane-bound receptor (ST2L, transducer of IL-33 signaling), and its soluble decoy receptor (sST2, inhibitor of IL-33 signaling) has been linked to a wide range of adverse pregnancy outcomes that are common in women with obesity and polycystic ovary syndrome, that is, conditions associated with hyperandrogenism, insulin resistance, and compensatory hyperinsulinemia. To reveal if androgens and insulin might modulate uteroplacental IL-33 signaling, we investigated the effect of dihydrotestosterone (DHT) and/or insulin on the expression of ST2L and sST2 (along with the activity of their promoter regions), IL-33 and sIL1RAP (heterodimerization partner of sST2), during in vitro decidualization of endometrial stromal cells from 9 healthy women. DHT and insulin markedly upregulated sST2 secretion, in addition to the upregulation of its messenger RNA (mRNA) expression, while the proximal ST2 promoter, from which the sST2 transcript originates, was upregulated by insulin, and in a synergistic manner by DHT and insulin combination treatment. On the other hand, sIL1RAP was slightly downregulated by insulin and IL-33 mRNA expression was not affected by any of the hormones, while ST2L mRNA expression and transcription from its promoter region (distal ST2 promoter) could not be detected or showed a negligibly low level. We hypothesize that high levels of androgens and insulin might lead to subfertility and pregnancy complications, at least partially, through the sST2-dependent downregulation of uteroplacental IL-33 signaling.

Tyrosine kinase inhibitors (TKIs) have dramatically improved the survival in chronic myeloid leukemia (CML), but residual disease typically persists even after prolonged treatment. Several lines of evidence suggest that TKIs administered to CML patients upregulate interferon γ (IFNγ) production, which may counteract the anti-tumorigenic effects of the therapy. We now show that activated T cell-conditioned medium (TCM) enhanced proliferation and counteracted imatinib-induced apoptosis of CML cells, and addition of a neutralizing anti-IFNγ antibody at least partially inhibited the anti-apoptotic effect. Likewise, recombinant IFNγ also reduced imatinib-induced apoptosis of CML cells. This anti-apoptotic effect of IFNγ was independent of alternative IFNγ signaling pathways, but could be notably diminished by STAT1-knockdown. Furthermore, IFNγ upregulated the expression of several anti-apoptotic proteins, including MCL1, PARP9, and PARP14, both in untreated and imatinib-treated primary human CD34+ CML stem/progenitor cells. Our results suggest that activated T cells in imatinib-treated CML patients can directly rescue CML cells from imatinib-induced apoptosis at least partially through the secretion of IFNγ, which exerts a rapid, STAT1-dependent anti-apoptotic effect potentially through the simultaneous upregulation of several key hematopoietic survival factors. These mechanisms may have a major clinical impact, when targeting residual leukemic stem/progenitor cells in CML.

Tyrosine kinase inhibitor (TKI) treatment has dramatically improved the survival of chronic myeloid leukemia (CML) patients, but measurable residual disease typically persists. To more effectively eradicate leukemia cells, simultaneous targeting of BCR-ABL1 and additional CML-related survival proteins has been proposed. Notably, several highly specific myeloid cell leukemia 1 (MCL1) inhibitors have recently entered clinical trials for various hematologic malignancies, although not for CML, reflecting the insensitivity of CML cell lines to single MCL1 inhibition. Here, we show that combining TKI (imatinib, nilotinib, dasatinib, or asciminib) treatment with the small-molecule MCL1 inhibitor S63845 exerted strong synergistic antiviability and proapoptotic effects on CML lines and CD34+ stem/progenitor cells isolated from untreated CML patients in chronic phase. Using wild-type BCR-ABL1-harboring CML lines and their T315I-mutated sublines (generated by CRISPR/Cas9-mediated homologous recombination), we prove that the synergistic proapoptotic effect of the drug combination depended on TKI-mediated BCR-ABL1 inhibition, but not on TKI-related off-target mechanisms. Moreover, we demonstrate that colony formation of CML but not normal hematopoietic stem/progenitor cells became markedly reduced upon combination treatment compared to imatinib monotherapy. Our results suggest that dual targeting of MCL1 and BCR-ABL1 activity may efficiently eradicate residual CML cells without affecting normal hematopoietic stem/progenitors.

Various subsets of bone marrow mesenchymal stromal cells (BM MSCs), including fibroblasts, endothelial, fat and reticular cells, are implicated in the regulation of the hematopoietic microenvironment and the survival of long-lived antibody-secreting cells (ASCs). Nowadays it is widely acknowledged that vaccine-induced protective antibody levels are diminished in adults and children that are treated for hematological cancers. A reason behind this could be damage to the BM MSC niche leading to a diminished pool of ASCs. To this end, we asked whether cell cytotoxic treatment alters the capacity of human BM MSCs to support the survival of ASCs. To investigate how chemotherapy affects soluble factors related to the ASC niche, we profiled a large number of cytokines and chemokines from in vitro-expanded MSCs from healthy donors or children who were undergoing therapy for acute lymphoblastic leukemia (ALL), following exposure to a widely used anthracycline called doxorubicin (Doxo). In addition, we asked if the observed changes in the measured soluble factors after Doxo exposure impacted the ability of the BM niche to support humoral immunity by co-culturing Doxo-exposed BM MSCs with in vitro-differentiated ASCs from healthy blood donors, and selective neutralization of cytokines. Our in vitro results imply that Doxo-induced alterations in BM MSC-derived interleukin 6 (IL-6), CXCL12 and growth and differentiation factor 15 (GDF-15) are not sufficient to disintegrate the support of IgG-producing ASCs by the BM MSC niche, and that serological memory loss may arise during later stages of ALL therapy.

Finely tuned decidualization of endometrial stromal fibroblasts into decidual cells is crucial for successful implantation and a healthy pregnancy. Both insulin and androgens are known to modulate decidualization, however, their complex effect on this process has not been fully elucidated. As hyperinsulinemia and hyperandrogenism are associated in clinical conditions, we aimed to investigate the interaction between insulin and androgens on decidualization. Primary human endometrial stromal cells were decidualized in vitro in the presence of insulin and/or androgens (dihydrotestosterone (DHT), testosterone). Gene or protein expressions of decidualization markers were measured, and cells size characteristics were determined. Migration of decidualizing endometrial stromal cells and invasion of HTR-8/SVneo trophoblast spheroids were assessed. We found that insulin and androgens in combination enhanced the upregulation of several decidualization markers including prolactin, tissue factor, tissue inhibitor of matrix metalloproteinase 3 and connexin-43, and also interacted in modulating cell size characteristics resulting in enlarged decidualizing cells. However, insulin and DHT together restricted the migration of decidualizing cells and invasion of trophoblast spheroids. Our findings suggest that insulin and androgens interact to potentiate the process of decidualization. On the other hand, inhibited cell migration and trophoblast invasion might negatively impact the function of decidualizing endometrial stromal cells.

Prokineticin 1 (PROK1) is a key regulator of embryo implantation and placentation, and its dysregulation is associated with pregnancy complications, such as pre-eclampsia and foetal growth restriction. We have previously shown that insulin strongly enhances the expression of PROK1 in human decidualizing stromal cells. Here, we demonstrate that dihydrotestosterone (DHT), but not testosterone, potentiates insulin to up-regulate PROK1 in these cells. However, the androgens alone do not influence the expression of PROK1. Our findings suggest that insulin and androgens both are involved in the regulation of PROK1 that could have implications for normal and pathological pregnancies.

B-cell CLL/lymphoma 6 (BCL6) is a transcriptional master regulator that can repress more than 1200 potential target genes. It exerts oncogenic effects through the inhibition of differentiation, DNA damage sensing and apoptosis in several human hematopoietic malignancies, including multiple myeloma (MM). The multifunctional cytokine interferon γ (IFNγ) exerts pro-apoptotic and anti-proliferative effects on MM cells in vitro, at least partially through the inhibition of the effects of interleukin 6 (IL6), one of the most important growth factor of MM and a strong inducer of BCL6 expression. However, IFNγ was also reported to directly upregulate BCL6 in several cell types. These observations prompted us to analyze the effect of IFNγ on BCL6 expression in MM cells. We discovered that among several myeloma growth/survival factors tested (including IL6, oncostatin M, insulin-like growth factor 1, tumor necrosis factor α and IFNα) IFNγ was the strongest inducer of BCL6 mRNA and protein expression in MM cell lines. IFNγ induced upregulation of BCL6 was dependent on the classical STAT1 signaling pathway, and affected both major BCL6 variants. Interestingly, although IFNα induced stronger STAT1 phosphorylation than IFNγ, it only slightly upregulated BCL6 in MM lines. We proved that IFNα induced BCL6 upregulation was limited by the concomitant activation of STAT5 signaling. We assume that BCL6 upregulation may represent a potentially pro-tumorigenic effect of IFNγ signaling in MM cells.

The clinical outcome for patients with chronic myeloid leukemia (CML) has improved significantly with the introduction of tyrosine kinase inhibitors (TKIs). However, their curative potential appears limited, probably as a consequence of TKI-resistant leukemic stem cells (LSCs) that persist as a result of aberrant pathways independent of the well-established oncoprotein Bcr-Abl. One such pathway involves signaling through leukotrienes (LTs), bioactive compounds that have been suggested to play a role in several other malignancies. Cysteinyl LT1 receptor (CysLT1R) has been reported to be overexpressed in a number of solid cancers, and blocking of this receptor with the antagonist montelukast (treatment approved for bronchial asthma) has resulted in the killing of cancer cells. We recently demonstrated that montelukast, alone or in combination with imatinib, can effectively reduce the growth of CML cells, while normal bone marrow cells were left unaffected. Herein, we further investigated the importance of CysLT1R for the survival of CML cells and the mechanisms by which montelukast induces cell death. Knockdown of the CysLT1R of K562 cells with siRNA reduced their growth by 25%. Montelukast had no effect on these cells, while it killed more than 50% of CysLT1R-expressing cells. Growth inhibition exerted by imatinib was unaffected by CysLT1R status. Montelukast-induced killing of K562/JURL-MK1 CML cells was paralleled by Bax overexpression, cytochrome c release, PARP-1 cleavage, and caspase-3 activation, an event further increased in a setting where montelukast was added to imatinib. Wnt/-catenin signaling was activated by CysLT1R and we observed that montelukast could induce proteins in this pathway, a finding of relevance for LSC survival. Thus, montelukast, employed at in vivo-like concentrations, induces the killing of CML cells through apoptotic pathways and may provide an additional, novel therapeutic possibility in CML.

B-cell CLL/lymphoma 6 (BCL6) exerts oncogenic effects in several human hematopoietic malignancies including chronic myeloid leukemia (CML), where BCL6 expression was shown to be essential for CML stem cell survival and self-renewal during imatinib mesylate (IM) treatment. As several lines of evidence suggest that interferon γ (IFNγ) production in CML patients might have a central role in the response to tyrosine kinase inhibitor (TKI) therapy, we analyzed if IFNγ modulates BCL6 expression in CML cells. Although separate IFNγ or IM treatment only slightly upregulated BCL6 expression, combined treatment induced remarkable BCL6 upregulation in CML lines and primary human CD34+ CML stem cells. We proved that during combined treatment, inhibition of constitutive signal transducer and activator of transcription (STAT) 5 activation by IM allowed the specific enhancement of the STAT1 dependent, direct upregulation of BCL6 by IFNγ in CML cells. By using colony-forming assay, we found that IFNγ enhanced the ex vivo colony or cluster-forming capacity of human CML stem cells in the absence or presence of IM, respectively. Furthermore, inhibition of the transcriptional repressor function of BCL6 in the presence of IM and IFNγ almost completely blocked the cluster formation of human CML stem cells. On the other hand, by using small interfering RNA knockdown of BCL6, we demonstrated that in an IM-treated CML line the antiapoptotic effect of IFNγ was independent of BCL6 upregulation. We found that IFNγ also upregulated several antiapoptotic members of the BCL2 and BIRC gene families in CML cells, including the long isoform of MCL1, which proved to be essential for the antiapoptotic effect of IFNγ in an IM-treated CML line. Our results suggest that combination of TKIs with BCL6 and MCL1 inhibitors may potentially lead to the complete eradication of CML stem cells.

Lamin A, B and C, the nuclear intermediate-filament proteins, play a role in epigenetic regulation. While Lamin B is expressed in all nucleated cells studied, Lamin A/C are transcribed in most somatic cell types except mature B lymphocytes. Since Epstein-Barr virus (EBV), a human gammaherpesvirus, is associated with tumorigenic processes and is known to alter the epigenotype of its host cells, we studied the expression of the LMNA gene and its epigenetic marks in EBV-carrying human lymphoid cell lines. We observed a high lamin A/C mRNA and protein expression in EBV-immortalized lymphoblastoid cell lines (LCLs) and in group III Burkitt lymphoma (BL) lines where hypomethylated first exons were observed with activating histone marks. In most cell lines with low promoter activity a highly methylated first exon could be detected. Our data showed that methylation of the first exon of LMNA was associated with the downregulation of LMNA expression whereas euchromatic histone marks were enriched at active LMNA promoters in EBV-immortalized LCLs. These data suggest a role for viral latency products to activate LMNAp in EBV-infected latency type III B cells in vitro. Expression of lamin A/C may contribute to the establishment of activated B cell phenotype that needs further explorations.

Insulin resistance and compensatory hyperinsulinemia are characteristic features of obesity and polycystic ovary syndrome, and both are associated with reduced fertility and implantation. There is little knowledge about the effect of insulin on the decidualization process and previous findings are contradictory. We investigated the effect of insulin on the regulation of forkhead box protein O1 (FOXO1), one of the most important transcription factors during decidualization. Endometrial stromal cells were isolated from six healthy, regularly menstruating women and decidualized in vitro. Gene expression levels of six putative FOXO1 target genes (including insulin-like growth factor binding protein-1 (IGFBP1) and prolactin (PRL)) were measured with Real-Time PCR following FOXO1 inhibition or insulin treatment. PI3K inhibition was used to identify the possible mechanism behind regulation. Subcellular localization of FOXO1 was analyzed with immunofluorescence. All the genes (IGFBP1, CTGF, INSR, DCN, LEFTY2), except prolactin, were evaluated as FOXO1 target genes in decidualizing stromal cells. Insulin caused a significant dose-dependent inhibition of the verified FOXO1 target genes. It was also demonstrated that insulin regulated FOXO1 target genes by transcriptional inactivation and nuclear export of FOXO1 via PI3K pathway. However, insulin did not inhibit the morphological transformation of endometrial stromal cells via transcriptional inactivation of FOXO1. This study provides new insights on the action of insulin on the endometrium via regulation of FOXO1. It is suggested that hyperinsulinemia results in dysregulation of a high number of FOXO1 controlled genes that may contribute to endometrial dysfunction and reproductive failure. Our findings may illuminate possible reasons for unexplained infertility.

Characterization of viral DNA methylation patterns is essential to understand its function in viral pathogenesis. Bisulfite modification, followed by polymerase chain reaction (PCR) and sequencing is the most effective method for the high resolution methylation mapping of viral genomes. Since the bisulfite modification and PCR steps are the most critical ones, an optimized protocol for these two steps is presented, with special attention to potential pitfalls.

Activation and proliferation of T cells are tightly regulated during the immune response. We show here that kinetics of proliferation of PHA activated T cells follows the expression of cMyc. Expression of p53 is also elevated and remains high several days after activation. To investigate the role of p53 in activated T cells, its expression was further elevated with nultin-3 treatment, a small molecule that dissociates the E3 ubiquitin protein ligase MDM2 from p53. Concomitantly, cMyc expression and proliferation decreased. At the other end of the cMyc-p53 axis, inhibition of cMyc with 10058-F4 led to down regulation of p53, likely through the lower level of cMyc induced p14ARF, which is also known to dissociate the p53-MDM2 complex. Both compounds induced cell cycle arrest and apoptosis. We conclude that the feedback regulation between cMyc and p53 is important for the T cell homeostasis. We also show that the two compounds modulating p53 and cMyc levels inhibited proliferation without abolishing the cytotoxic function, thus demonstrating the dichotomy between proliferation and cytotoxicity in activated T cells.

Latent Epstein-Bar virus genomes undergo epigenetic modifications which are dependent on the respective tissue type and cellular phenotype. These define distinct viral epigenotypes corresponding with latent viral gene expression profiles. Viral Latent Membrane Proteins 1 and 2A can induce cellular DNA methyltransferases, thereby influencing the methylation status of the viral and cellular genomes. Therefore, not only the viral genomes carry epigenetic modifications, but also the cellular genomes adopt major epigenetic alterations upon EBV infection. The distinct cellular epigenotypes of EBV-infected cells differ from the epigenotypes of their normal counterparts. In Burkitt lymphoma (BL), nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBVaGC) significant changes in the host cell methylome with a strong tendency towards CpG island hypermethylation are observed. Hypermethylated genes unique for EBVaGC suggest the existence of an EBV-specific “epigenetic signature”. Contrary to the primary malignancies carrying latent EBV genomes, lymphoblastoid cells (LCs) established by EBV infection of peripheral B cells in vitro are characterized by a massive genome-wide demethylation and a significant decrease and redistribution of heterochromatic histone marks. Establishing complete epigenomes of the diverse EBV-associated malignancies shall clarify their similarities and differences and further clarify the contribution of EBV to the pathogenesis, especially for the epithelial malignancies, NPC and EBVaGC.

The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.