Proerythroblast Cells of Diamond-Blackfan Anemia Patients With RPS19 and CECR1 Mutations Have Similar Transcriptomic Signature

Diamond Blackfan Anemia (DBA) is an inherited bone marrow (BM) failure syndrome, characterized by a paucity of erythroid differentiation. DBA is mainly caused by the mutations in ribosomal protein genes, hence classified as ribosomopathy. However, in approximately 30% of patients, the molecular etiology cannot be discovered. RPS19 germline mutations caused 25% of the cases. On the other hand, CECR1 mutations also cause phenotypes similar to DBA but not being a ribosomopathy. Due to the blockade of erythropoiesis in the BM, we investigated the transcriptomic profile of three different cell types of BM resident cells of DBA patients and compared them with healthy donors. From BM aspirates BM mononuclear cells (MNCs) were isolated and hematopoietic stem cells (HSC) [CD71–CD34+ CD38mo/lo], megakaryocyte–erythroid progenitor cells (MEP) [CD71–CD34+ CD38hi] and Proerythroblasts [CD71+ CD117+ CD38+] were sorted and analyzed with a transcriptomic approach. Among all these cells, proerythroblasts had the most different transcriptomic profile. The genes associated with cellular stress/immune responses were increased and some of the transcription factors that play a role in erythroid differentiation had altered expression in DBA proerythroblasts. We also showed that gene expression levels of ribosomal proteins were decreased in DBA proerythroblasts. In addition to these, colony formation assay (CFU-E) provided functional evidence of the failure of erythroid differentiation in DBA patients. According to our findings that all patients resembling both RPS19 and CECR1 mutations have common transcriptomic signatures, it may be possible that inflammatory BM niche may have a role in DBA pathogenesis

Differential expansion of circulating human MDSC subsets in patients with cancer, infection and inflammation

Background Myeloid-derived suppressor cells (MDSC) are a functional myeloid cell subset that includes myeloid cells with immune suppressive properties. The presence of MDSC has been reported in the peripheral blood of patients with several malignant and non-malignant diseases. So far, direct comparison of MDSC across different diseases and Centers is hindered by technical pitfalls and a lack of standardized methodology. To overcome this issue, we formed a network through the COST Action Mye-EUNITER (www.mye-euniter.eu) with the goal to standardize and facilitate the comparative analysis of human circulating MDSC in cancer, inflammation and infection. In this manuscript, we present the results of the multicenter study Mye-EUNITER MDSC Monitoring Initiative, that involved 13 laboratories and compared circulating MDSC subsets across multiple diseases, using a common protocol for the isolation, identification and characterization of these cells. Methods We developed, tested, executed and optimized a standard operating procedure for the isolation and immunophenotyping of MDSC using blood from healthy donors. We applied this procedure to the blood of almost 400 patients and controls with different solid tumors and non-malignant diseases. The latter included viral infections such as HIV and hepatitis B virus, but also psoriasis and cardiovascular disorders. Results We observed that the frequency of MDSC in healthy donors varied substantially between centers and was influenced by technical aspects such as the anticoagulant and separation method used. Expansion of polymorphonuclear (PMN)-MDSC exceeded the expansion of monocytic MDSC (M-MDSC) in five out of six solid tumors. PMN-MDSC expansion was more pronounced in cancer compared with infection and inflammation. Programmed death-ligand 1 was primarily expressed in M-MDSC and e-MDSC and was not upregulated as a consequence of disease. LOX-1 expression was confined to PMN-MDSC. Conclusions This study provides improved technical protocols and workflows for the multi-center analysis of circulating human MDSC subsets. Application of these workflows revealed a predominant expansion of PMN-MDSC in solid tumors that exceeds expansion in chronic infection and inflammation

A Co-Stimulatory Trap Set By Myeloid Leukemia Cells

The straightforward notion that tumor cells always exert immunosuppressive functions has been contradicted by the finding that myeloid leukemia cells can express potent co-stimulatory molecules. Indeed, the co-stimulatory support offered by leukemia cells can provoke helper T-cell responses. Unfavorably, this interaction allows leukemia cells to acquire immunosuppressive capacities.ScopusPubMe

Th1 cells in cancer-associated inflammation

WOS: 000394539800003The immune system is not only evolved to protect the body from pathogens, but it also recognizes and eliminates cancer cells. CD4+ helper T (Th) lymphocytes are central intercessors differentiated according to the character of physiological or pathological status. Generation of type 1 Th (Th1) cells is primarily associated with a pathological insult that must be removed through immune elimination. Upon interacting with other immune and transformed cells, Th1 cells can hamper cancer progression. Therefore, it is a major obstacle for tumor cells to become insensitive or resistant to Th1-oriented actions. The organism employs various mechanisms to return to a steady state and ensure tissue repair following a destructive inflammatory response. Th1 cells are also tightly regulated during the termination of immune responses. They can reduce the production of inflammatory cytokines, both generate and be prone to inhibitory signals, and undergo activation-induced cell death for inflammation resolution. Additionally, Th1 cells may become hyporesponsive, exhausted, and decorated with many inhibitory receptors and eventually lose functionality. There is growing evidence about tumor cells taking advantage of the strategies used for the resolution of Th1-oriented inflammation. Here, the current insights on Th1 cells during cancer-associated inflammatory responses are reviewed

Molecular and functional analysis of a novel recombinant clone of rat (Rattus norvegicus) CD40 ligand (CD40L) gene

Esendagli, Gunes/0000-0003-4865-2377;WOS: 000262088100011PubMed: 17922253Genetic material obtained from various individuals may contain certain polymorphisms which may conflict with the predetermined DNA sequence and consequently, may modulate the function of gene products. In this study, coding sequence of rat CD40 ligand (CD40L, CD154) was obtained from activated splenocytes, amplified, and cloned into a eukaryotic expression vector by using directional cloning method. Sequence of the recombinant rat CD40L DNA, pCD40L-IRES2-EGFP (pCD40L), was compared with the previously reported rat CD40L cDNA sequences and a 99% identity was found. Differing nucleotides were on the positions; 122-T/C, 341-G/A, 476G/A, 762-T/A. Further alignment analysis showed that pCD40L was collectively carrying the nucleotides each previously reported by different groups. The sequence was submitted to NCBI GenBank and nucleotide database accession number EF066490 was obtained. Following transfection of the construct into NIH/3T3 cell line, novel CD40L clone was functionally expressed de novo, increasing the expression of CD80 and CD86 costimulatory molecules and augmenting the proliferation rate of effector splenocytes in immune reactions ex vivo. Based on these data, here we report a novel recombinant clone of the rat CD40L gene which may represent a potential polymorphic variant.Eczacibasi Scientific Research and Award Fund; Hacettepe UniversityHacettepe University [05DO3104001]This study was supported by Eczacibasi Scientific Research and Award Fund, and Hacettepe University Scientific Research Unit (project no. 05DO3104001)

Adhesion of beta1 integrin to fibronectin regulates CAM-DR phenotype via p21(WAF1/cip1) in HL60 acute myeloid leukemia (AML) cells

Esendagli, Gunes/0000-0003-4865-2377;WOS: 000254702300001Aims: Drug resistance is a major obstacle for a successful cancer therapy. Cell adhesion mediated drug resistance (CAM-DR) is a novel type of drug resistance and generated via interaction of cancer cells with the microenvironment. In this study, CAM-DR phenotype was analyzed in HL60 acute myeloid leukemia (AML) cells. Materials and Methods: Fibronectin (FN) adherence of HL60 cells was tested by a colorimetric adhesion assay. Flow cytometry analyses were performed to evaluate doxorubicin-incluced apoptosis and to determine cell cycle status. Proliferation rate was evaluated by [H-3]-thymidine incorporation assay. Western blot and RTPCR were used for analysis of the factors involved in cell cycle control. Results: Binding of HL60 to FN via alpha 4 beta 1 and alpha 5 beta 1 integrins exerted a CAM-DR phenotype, which shows resistance to apoptosis triggered by doxorubicin. FN-adherent HL60 cells accumulated in the G(0)/G(1) phase of cell cycle and stopped proliferation. However, after detachment from FN, cells entered S phase, proliferated, and became sensitive to apoptosis. The analysis of the factors involved in the G(0)/G(1) cell cycle checkpoint showed that CAM-DR phenotype might be regulated mainly by p21(waf/cip). Conclusions: Here we showed that CAM-DR may also represent a reversible drug resistance mechanism that decreases apoptosis and causes growth arrest in AML blasts

Functional Exhaustion Of Cd4(+) T Cells Induced By Co-Stimulatory Signals From Myeloid Leukaemia Cells

To cope with immune responses, tumour cells implement elaborate strategies such as adaptive resistance and induction of T-cell exhaustion. T-cell exhaustion has been identified as a state of hyporesponsiveness that arises under continuous antigenic stimulus. Nevertheless, contribution of co-stimulatory molecules to T-cell exhaustion in cancer remains to be better defined. This study explores the role of myeloid leukaemia-derived co-stimulatory signals on CD4(+) T helper (Th) cell exhaustion, which may limit anti-tumour immunity. Here, CD86 and inducible T-cell co-stimulator ligand (ICOS-LG) co-stimulatory molecules that are found on myeloid leukaemia cells supported Th cell activation and proliferation. However, under continuous stimulation, T cells co-cultured with leukaemia cells, but not with peripheral blood monocytes, became functionally exhausted. These in vitro-generated exhausted Th cells were defined by up-regulation of programmed cell death 1 (PD-1), cytotoxic T-lymphocyte antigen 4 (CTLA-4), lymphocyte activation gene 3 (LAG3) and T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3) inhibitory receptors. They were reluctant to proliferate upon re-stimulation and produced reduced amounts of interleukin-2 (IL-2), tumour necrosis factor- (TNF-) and interferon- (IFN-). Nonetheless, IL-2 supplementation restored the proliferation capacity of the exhausted Th cells. When the co-stimulation supplied by the myeloid leukaemia cells were blocked, the amount of exhausted Th cells was significantly decreased. Moreover, in the bone marrow aspirates from patients with acute myeloid leukaemia (AML) or myelodysplastic syndrome (MDS), a subpopulation of Th cells expressing PD-1, TIM-3 and/or LAG3 was identified together with CD86(+) and/or ICOS-LG(+) myeloid blasts. Collectively, co-stimulatory signals derived from myeloid leukaemia cells possess the capacity to facilitate functional exhaustion in Th cells.Wo

Transfection of myeloid leukaemia cell lines is distinctively regulated by fibronectin substratum

Gene transfer into haematopoietic cells is a challenging approach. The extracellular matrix component fibronectin has been known to modulate the cell cycle dynamics, viability and differentiation of leukaemia cells. Thus, our aim was to investigate the influence of fibronectin substratum on the liposomal transfection of myeloid leukaemia cell lines. Liposomal transfection was performed with K562 and HL-60 as representative lines of transfection-competent and -incompetent myeloid leukaemia cells, respectively. Flow cytometry analyses were performed to determine transfection efficiency monitored by green fluorescent protein (GFP) expression and to assess cell viability and cell cycle status. Quantitation of GFP gene expression and DNA uptake was assayed by real time PCR. The current data showed that the adhesion to fibronectin deteriorated the transfection of K562 cells. In contrary, it enhanced the delivery of plasmid DNA into HL-60 cells. Correspondingly, the adhesion to fibronectin influenced the transfection efficiency mainly by modulating the intracellular presence of plasmid DNA. The cell cycle and viability which is regulated by fibronectin had a minor impact on the success of gene delivery. This phenomenon may be considered as an important factor which may modulate the potential gene transfer approaches for myeloid leukaemia

Efficacy of a novel LyP-1-containing self-microemulsifying drug delivery system (SMEDDS) for active targeting to breast cancer

An ideal cancer therapy targets the tumor cells selectively without damaging healthy tissues. Even though the tumor-specific markers are limited, these molecules can be used for the delivery of anti-cancer drugs as an active targeting strategy. Since the lymphatic system plays a critical role in the dissemination of cancer cells, the drugs directed through lymphatics can feasibly reach to the sites of metastasis. LyP-1 is a peptide that binds to the p32 receptor which is highly expressed not only on the lymphatic endothelium but also on the malignant cells; thus, making this peptide ligand a preferable candidate to mediate active targeting of lymphatics and cancer cells. In this study, different formulations of LyP-1 containing lipid-based nanophannaceutics so-called self-microemulsifying drug delivery systems (SMEDDS) were developed and tested for their efficacy in targeting breast cancer. Following the selection of non-toxic formulation, doxorubicin hydrochloride and LyP-1 were co-administered in the SMEDDS, which resulted in a significant increase in in vitro cytotoxicity in p32-expressing breast cancer cells, 4T1 and MDA-MB-231. Accordingly, the uptake of LyP-1 in the SMEDDS by the cancer cells was demonstrated. The expression of p32 was detected in the 4T1 tumor tissues which were efficiently targeted with LyP-1 in the SMEDDS. When doxorubicin was co-administrated with LyP-1 in SMEDDS via intraperitonial administration, tumor growth and metastasis were significantly reduced. In conclusion, a novel and efficacious SMEDDS formulation containing LyP-1 with a droplet size less than 100 nm was developed for the lymphatic targeting of breast cancer

A method for high-purity isolation of neutrophil granulocytes for functional cell migration assays

Background: Neutrophil-mediated killing of pathogens is one of the most significant functions of the primary defense of the host. Neutrophil activity and migration play a key role in inflammatory conditions. To gain insights into the interactions between neutrophils and neutrophil migration-related disorders, a large number of sophisticated methods have been developed. The technical limitations of isolating highly purified neutrophil populations, minimizing both cell death and activation during the isolation process, and the short lifespan of neutrophils present challenges for studying specific functions of neutrophils in vitro. In this study, we aimed to evaluate a separation medium-based density gradient method to obtain highly purified neutrophil populations and combined this protocol with a model for studying neutrophil migration in-vitro. Materials and methods: Human granulocytes were isolated using Lympholyte-poly solution. The purity and viability of isolated neutrophils were assessed by flow cytometry and morphological analysis. Neutrophil activation was confirmed by immunocytochemistry. Lastly, filter assay was performed to measure neutrophil chemotaxis. Results and discussion: All validation experiments revealed that this method was capable of generating a highly purified neutrophil population for further functional in-vitro assays. Consequently, this study demonstrates a quick, cost effective, and easy-to-follow model, and may be a significant alternative to isolation methods that need extra subsequent steps such as flow cytometry-based cell sorting for reaching highly purified neutrophil population. Conclusion: The suggested combination of methods for the isolation and cell migration analysis of human neutrophils is highly recommended to use for disease models involving neutrophil migration such as autoinflammatory disorders. © 2019 De Gruyter. All rights reserved