Prolonged Thrombocytopenia and Severe Transfusion Reaction after ABO-Incompatible Allogeneic Hematopoietic Stem Cell Transplantation in a Patient with Chronic Myelomonocytic Leukemia.

Introduction: Major ABO-incompatible allogeneic hematopoietic stem cell transplantation (allo-HCT) is a common practice and represents a challenging transfusion scenario. Prolonged thrombocytopenia with increased platelet transfusion needs is one of its reported adverse effects, and this has been linked to the persistence of recipient anti-donor isoagglutinins. Case Presentation: A 55-year-old male patient, O Rh(D)-positive, with chronic myelomonocytic leukemia underwent major incompatible allo-HCT from a A Rh(D)-negative donor. He presented with prolonged thrombocytopenia and multiple transfusion reactions after A Rh(D)-negative platelet transfusions. Considering the outcomes of numerous examinations, we tested the anti-A1 titers, finding a significant persistence of anti-donor isoagglutinins. We limited platelet transfusions to blood group O Rh(D)-negative donors, which significantly decreased the requirement for platelet transfusions. In addition, the transfusion reactions ceased. Conclusion: In case of transfusion reactions against platelet products in major ABO-incompatible allo-HCT patients, isoagglutinin monitoring should be considered and a change in the platelet transfusion protocol may be beneficial in patients presenting high isotiters against recipient's blood type.

Macrophages as a Source and Target of GDF-15.

Growth differentiation factor 15 (GDF-15) is a multifunctional cytokine that belongs to the transforming growth factor-beta (TGF-ß) superfamily. GDF-15 is involved in immune tolerance and is elevated in several acute and chronic stress conditions, often correlating with disease severity and patient prognosis in cancer172 and metabolic and cardiovascular disorders. Despite these clinical associations, the molecular mechanisms orchestrating its effects remain to be elucidated. The effects of GDF-15 are pleiotropic but cell-specific and dependent on the microenvironment. While GDF-15 expression can be stimulated by inflammatory mediators, its predominant effects were reported as anti-inflammatory and pro-fibrotic. The role of GDF-15 in the macrophage system has been increasingly investigated in recent years. Macrophages produce high levels of GDF-15 during oxidative and lysosomal stress, which can lead to fibrogenesis and angiogenesis at the tissue level. At the same time, macrophages can respond to GDF-15 by switching their phenotype to a tolerogenic one. Several GDF-15-based therapies are under development, including GDF-15 analogs/mimetics and GDF-15-targeting monoclonal antibodies. In this review, we summarize the major physiological and pathological contexts in which GDF-15 interacts with macrophages. We also discuss the major challenges and future perspectives in the therapeutic translation of GDF-15.

Hyperglycemia amplifies TLR-mediated inflammatory response of M(IL4) macrophages to dyslipidemic ligands.

Hyperglycemia is critical for initiation of diabetic vascular complications. We systemically addressed the role of hyperglycemia in the regulation of TLRs in primary human macrophages. Expression of TLRs (1-9) was examined in monocyte-derived M(NC), M(IFNγ), and M(IL4) differentiated in normoglycemic and hyperglycemic conditions. Hyperglycemia increased expression of TLR1 and TLR8 in M(NC), TLR2 and TLR6 in M(IFNγ), and TLR4 and TLR5 in M(IL4). The strongest effect of hyperglycemia in M(IL4) was the upregulation of the TLR4 gene and protein expression. Hyperglycemia amplified TLR4-mediated response of M(IL4) to lipopolysaccharide by significantly enhancing IL1ß and modestly suppressing IL10 production. In M(IL4), hyperglycemia in combination with synthetic triacylated lipopeptide (TLR1/TLR2 ligand) amplified expression of TLR4 and production of IL1ß. In summary, hyperglycemia enhanced the inflammatory potential of homeostatic, inflammatory, and healing macrophages by increasing specific profiles of TLRs. In combination with dyslipidemic ligands, hyperglycemia can stimulate a low-grade inflammatory program in healing macrophages supporting vascular diabetic complications.

Titanium induces proinflammatory and tissue-destructive responses in primary human macrophages.

Implants and medical devices are efficient and practical therapeutic solutions for a multitude of pathologies. Titanium and titanium alloys are used in orthopedics, dentistry, and cardiology. Despite very good mechanical properties and corrosion resistance, titanium implants can fail due to inflammatory or tissue degradation-related complications. Macrophages are major immune cells that control acceptance of failure of the implant. In this study, for the first time, we have performed a systematic analysis of the response of differentially activated human macrophages, M(Control), M(IFNγ), and M(IL-4), to the polished and porous titanium surfaces in order to identify the detrimental effect of titanium leading to the tissue destruction and chronic inflammation. Transcriptome analysis revealed that the highest number of differences between titanium and control settings are found in M(IL-4) that model healing type of macrophages. Real-time quantitative polymerase chain reaction analysis confirmed that both polished and porous titanium affected expression of cytokines, chitinases/chitinase-like proteins, and matrix metalloproteinases (MMPs). Titanium-induced release and activation of MMP7 by macrophages was enhanced by fibroblasts in both juxtacrine and paracrine cell interaction models. Production of titanium-induced MMPs and cytokines associated with chronic inflammation was independent of the presence of Staphylococcus aureus. MMP7, one of the most pronounced tissue-destroying factors, and chitinase-like protein YKL-40 were expressed in CD68+ macrophages in peri-implant tissues of patients with orthopedic implants. In summary, we demonstrated that titanium induces proinflammatory and tissue-destructing responses mainly in healing macrophages, and the detrimental effects of titanium surfaces on implant-adjacent macrophages are independent on the bacterial contamination.

Adipose Stromal Cell-Derived Secretome Attenuates Cisplatin-Induced Injury In Vitro Surpassing the Intricate Interplay between Proximal Tubular Epithelial Cells and Macrophages.

(1) Background: The chemotherapeutic drug cisplatin exerts toxic side effects causing acute kidney injury. Mesenchymal stromal cells can ameliorate cisplatin-induced kidney injury. We hypothesize that the MSC secretome orchestrates the vicious cycle of injury and inflammation by acting on proximal tubule epithelial cells (PTECs) and macrophages individually, but further by counteracting their cellular crosstalk. (2) Methods: Conditioned medium (CM) from adipose stromal cells was used, first assessing its effect on cisplatin injury in PTECs. Second, the effects of cisplatin and the CM on macrophages were measured. Lastly, in an indirect co-culture system, the interplay between the two cell types was assessed. (3) Results: First, the CM rescued PTECs from cisplatin-induced apoptosis by reducing oxidative stress and expression of nephrotoxicity genes. Second, while cisplatin exerted only minor effects on macrophages, the CM skewed macrophage phenotypes to the anti-inflammatory M2-like phenotype and increased phagocytosis. Finally, in the co-culture system, the CM suppressed PTEC death by inhibiting apoptosis and nuclei fragmentation. The CM lowered TNF-α release, while cisplatin inhibited macrophage phagocytosis, PTECs, and the CM to a greater extent, thus enhancing it. The CM strongly dampened the inflammatory macrophage cytokine secretion triggered by PTECs. (4) Conclusions: ASC-CM surpasses the PTEC-macrophage crosstalk in cisplatin injury. The positive effects on reducing cisplatin cytotoxicity, on polarizing macrophages, and on fine-tuning cytokine secretion underscore MSCs' CM benefit to prevent kidney injury progression.

C-Type Lectin-like Receptor 2 Expression Is Decreased upon Platelet Activation and Is Lower in Most Tumor Entities Compared to Healthy Controls.

The C-type lectin-like receptor 2 (CLEC-2) is expressed on platelets and mediates binding to podoplanin (PDPN) on various cell types. The binding to circulating tumor cells (CTCs) leads to platelet activation and promotes metastatic spread. An increased level of soluble CLEC-2 (sCLEC-2), presumably released from activated platelets, was shown in patients with thromboinflammatory and malignant disease. However, the functional role of sCLEC-2 and the mechanism of sCLEC-2 release are not known. In this study, we focused on the effect of platelet activation on CLEC-2 expression and the sCLEC-2 plasma level in patients with cancer. First, citrated blood from healthy volunteer donors (n = 20) was used to measure the effect of platelet stimulation by classical agonists and PDPN on aggregation, CLEC-2 expression on platelets with flow cytometry, sCLEC-2 release to the plasma with ELISA and total CLEC-2 expression with Western blot analysis. Second, sCLEC-2 was determined in plasma samples from healthy donors (285) and patients with colorectal carcinoma (CRC; 194), melanoma (160), breast cancer (BC; 99) or glioblastoma (49). PDPN caused a significant increase in the aggregation response induced by classical agonists. ADP or PDPN stimulation of platelets caused a significant decrease in CLEC-2 on platelets and sCLEC-2 in the plasma, whereas total CLEC-2 in platelet lysates remained the same. Thus, the increased plasma level of sCLEC-2 is not a suitable biomarker of platelet activation. In patients with CRC (median 0.9 ng/mL), melanoma (0.9 ng/mL) or BC (0.7 ng/mL), we found significantly lower sCLEC-2 levels (p < 0.0001), whereas patients with glioblastoma displayed higher levels (2.6 ng/mL; p = 0.0233) compared to healthy controls (2.1 ng/mL). The low sCLEC-2 plasma level observed in most of the tumor entities of our study presumably results from the internalization of sCLEC-2 by activated platelets or binding of sCLEC-2 to CTCs.

Flow Cytometric Characterization of Hematopoietic Stem and Progenitor Cell Subpopulations in Autologous Peripheral Blood Stem Cell Preparations after Cryopreservation.

Introduction: Autologous stem cell transplantation is a successful routine procedure with only a small number of non-engraftment cases, although the time to hematopoietic recovery may vary considerably across patients. While CD34 has been the decisive marker for enumerating hematopoietic stem and progenitor cells (HSPCs) for more than 30 years, the impact of CD34-positive cellular subpopulations in autologous HSPC grafts on hematopoietic reconstitution remains unclear. Methods: The two-color ISHAGE protocol represents the current gold standard for CD34+ cell enumeration but includes only the number of viable CD45+/CD34+ cells relative to the body weight of the recipient. We adapted a multicolor flow cytometry marker panel for advanced characterization of CD34 subpopulations in retained samples of autologous peripheral blood stem cell products (n = 49), which had been cryostored for a wide range from 4 to 15 years. The flow cytometric analysis included CD10, CD34, CD38, CD45, CD45RA, CD133, and viability staining with 7AAD. The findings were correlated with clinical engraftment data, including reconstitution of leukocytes, neutrophils, and platelets after transplantation (TPL). Results: We demonstrated that the identification of autologous HSPC subpopulations by flow cytometry after cryopreservation is feasible. Regarding the distribution of HSPC subpopulations, a markedly different pattern was observed in comparison to previously published data obtained using fresh autologous material. Our data revealed the largest ratio of lympho-myeloid progenitors (LMPPs) after freezing and thawing, followed by multipotent progenitors and erythroid-myeloid progenitors. A high ratio of LMPPs, representing an immature stage of differentiation, correlated significantly with early neutrophilic granulocyte and leukocyte engraftment (p = 0.025 and p = 0.003). Conversely, a large ratio of differentiated cells correlated with late engraftment of neutrophilic granulocytes (p = 0.024). Overall, successful engraftment was documented for all patients. Conclusion: We established an advanced flow cytometry panel to assess the differentiation ability of cryostored autologous peripheral blood stem cell grafts and correlated it with timely hematopoietic reconstitution. This approach represents a novel and comprehensive way to identify hematopoietic stem and progenitor subpopulations. It is a feasible way to indicate the engraftment capacity of stem cell products.

Predicting Successful Hematopoietic Stem Cell Collection in Healthy Allogeneic Donors.

Introduction: Collection of peripheral blood stem cells (PBSCs) from healthy donors is a well-established process. We aimed to identify factors predictive of successful CD34+ PBSC collection and established a formula capable of predicting CD34+ cell yield. Methods: We retrospectively evaluated 588 healthy adult donors (median age 29 years, range 18-69 years) at our institution from 2017 to 2022. The predicted minimal number of CD34+ cells was calculated as follows: (peripheral CD34+ cells/µL × adjusted collection efficiency of 30%) × total liters processed. This formula was further modified according to donor and recipient body weight (BW). Results: Median total collection was 8.0 × 106 CD34+ cells/kg BW (range 1.0-47.1 × 106 cells/kg BW) with 522 donors (89%) collecting ≥5.0 × 106 cells/kg of recipient BW. A second leukapheresis (LP) was performed in 49 donors. Need for two LPs was more common in female donors (OR 6.68, 95% CI, 2.62-17.05; p < 0.001), donors with higher age (OR for 10 years difference 1.53, 95% CI, 1.15-2.03, p = 0.003), donors with WBC count <30 × 109/L after 5 days of granulocyte-colony stimulating factor (G-CSF) stimulation (OR, 4.33; 95% CI, 1.59-11.83; p = 0.004), and a donor/recipient weight ratio <1 (OR 6.21, 95% CI, 2.69-14.34; p < 0.001). Predictive factors for optimal LP (i.e., ≥5.0 × 106 CD34+ cells/kg of recipient BW) were peripheral blood (PB) CD34+ cell count >50/µL (OR 12.82, range 6.34-25.92, p < 0.001), male donor (OR 2.77, range 1.06-7.23, p = 0.04), and a donor/recipient weight ratio >1 (OR 3.12, range 1.57-6.24, p = 0.001). WBC, platelets, hemoglobin, and age had no significant predictive value. Predicted versus observed number of CD34+ cells/kg BW collected demonstrated a very strong linear correlation (r = 0.925, 95% CI, 0.912-0.936, p < 0.0001). Conclusions: Of the routinely monitored indicators in PBSC donors, CD34+ cell count in PB is the most important factor in predicting G-CSF-induced PBSC yields. Higher age, female sex, WBC <30 × 109/L, and a donor/recipient weight ratio <1 are useful indicators for identifying suboptimal mobilizers. The modified formula has shown successful and consistent performance in the prediction of key outcome measures including the minimum CD34+ cell collection, determination of the required length of apheresis, and whether a second day of PBSC collection was necessary to achieve the respective collection goal.

Closer to Nature: The Role of MSCs in Recreating the Microenvironment of the Hematopoietic Stem Cell Niche in vitro.

Background: The stem cell niche in human bone marrow provides scaffolds, cellular frameworks and essential soluble cues to support the stemness of hematopoietic stem and progenitor cells (HSPCs). To decipher this complex structure and the corresponding cellular interactions, a number of in vitro model systems have been developed. The cellular microenvironment is of key importance, and mesenchymal stromal cells (MSCs) represent one of the major cellular determinants of the niche. Regulation of the self-renewal and differentiation of HSPCs requires not only direct cellular contact and adhesion molecules, but also various cytokines and chemokines. The C-X-C chemokine receptor type 4/stromal cell-derived factor 1 axis plays a pivotal role in stem cell mobilization and homing. As we have learned in recent years, to realistically simulate the physiological in vivo situation, advanced model systems should be based on niche cells arranged in a three-dimensional (3D) structure. By providing a dynamic rather than static setup, microbioreactor systems offer a number of advantages. In addition, the role of low oxygen tension in the niche microenvironment and its impact on hematopoietic stem cells need to be taken into account and are discussed in this review. Summary: This review focuses on the role of MSCs as a part of the bone marrow niche, the interplay between MSCs and HSPCs and the most important regulatory factors that need to be considered when engineering artificial hematopoietic stem cell niche systems. Conclusion: Advanced 3D model systems using MSCs as niche cells and applying microbioreactor-based technology are capable of simulating the natural properties of the bone marrow niche more closely than ever before.

Hyperglycemia Induces Inflammatory Response of Human Macrophages to CD163-Mediated Scavenging of Hemoglobin-Haptoglobin Complexes.

Hyperglycemia, a hallmark of diabetes, can induce inflammatory programming of macrophages. The macrophage scavenger receptor CD163 internalizes and degrades hemoglobin-haptoglobin (Hb-Hp) complexes built due to intravascular hemolysis. Clinical studies have demonstrated a correlation between impaired scavenging of Hb-Hp complexes via CD163 and diabetic vascular complications. Our aim was to identify whether hyperglycemia is able to amplify inflammation via Hb-Hp complex interactions with the immune system. M(IFNγ), M(IL-4), and control M0 macrophages were differentiated out of primary human monocytes in normo- (5 mM) and hyperglycemic (25 mM) conditions. CD163 gene expression was decreased 5.53 times in M(IFNγ) with a further decrease of 1.99 times in hyperglycemia. Hyperglycemia suppressed CD163 surface expression in M(IFNγ) (1.43 times). Flow cytometry demonstrated no impairment of Hb-Hp uptake in hyperglycemia. However, hyperglycemia induced an inflammatory response of M(IFNγ) to Hb-Hp1-1 and Hb-Hp2-2 uptake with different dynamics. Hb-Hp1-1 uptake stimulated IL-6 release (3.03 times) after 6 h but suppressed secretion (5.78 times) after 24 h. Contrarily, Hb-Hp2-2 uptake did not affect IL-6 release after 6h but increased secretion after 24 h (3.06 times). Our data show that hyperglycemia induces an inflammatory response of innate immune cells to Hb-Hp1-1 and Hb-Hp2-2 uptake, converting the silent Hb-Hp complex clearance that prevents vascular damage into an inflammatory process, hereby increasing the susceptibility of diabetic patients to vascular complications.

Autologous Lipofilling Improves Clinical Outcome in Patients With Symptomatic Dermal Scars Through Induction of a Pro-Regenerative Immune Response.

BACKGROUND: Autologous lipofilling is an emerging procedure to treat and possibly reverse dermal scars and to reduce scar-related pain, but its efficacy and mechanisms are poorly understood. OBJECTIVES: The aim of this study was to test the hypothesis that repeated lipografts reverse dermal scars by reinitiation of wound healing. METHODS: In a prospective, non-placebo-controlled clinical study, 27 adult patients with symptomatic scars were given 2 lipofilling treatments at 3-month intervals. As primary outcome, clinical effects were measured with the Patient and Observer Scar Assessment Scale (POSAS). Scar biopsies were taken before and after treatments to assess scar remodeling at a cellular level. RESULTS: Twenty patients completed the study. Patients' scars improved after lipofilling. The total POSAS scores (combined patient and observer scores) decreased from 73.2  [14.7] points (mean [standard deviation]) pretreatment to 46.1 [14.0] and 32.3 [13.2] points after the first and second lipofilling treatment, respectively. Patient POSAS scores decreased from 37.3 [8.8] points to 27.2 [11.3] and 21.1 [11.4] points, whereas observer POSAS scores decreased from 35.9 [9.5] points to 18.9 [6.0] and 11.3 [4.5] points after the first and second treatment, respectively. After each lipofilling treatment, T lymphocytes, mast cells, and M2 macrophages had invaded scar tissue and were associated with increased vascularization. In addition, the scar-associated epidermis showed an increase in epidermal cell proliferation to levels similar to that normal in skin. Moreover, lipofilling treatment caused normalization of the extracellular matrix organization towards that of normal skin. CONCLUSIONS: Autologous lipofilling improves the clinical outcome of dermal scars through the induction of a pro-regenerative immune response, increased vascularization, and epidermal proliferation and remodeling of scar tissue extracellular matrix.

Cryostorage to What End? - Autologous Stem Cell Products in Burkitt Lymphoma, Acute Lymphoblastic Leukemia, Acute Myeloid Leukemia, and Myeloproliferative Neoplasm Patients.

INTRODUCTION: Recently, we identified a huge discrepancy between the collection practice and the actual utilization of cryopreserved peripheral blood stem cells (PBSCs) for high-dose chemotherapy (HDCT) and autologous blood stem cell transplantation (ABSCT). Specifically, patients with Burkitt lymphoma, acute leukemia, and myeloproliferative neoplasms (MPN) were frequently not referred for ABSCT after successful PBSC collection. OBJECTIVE: The aim of this study was to identify variables that are associated with the non-utilization of PBSC grafts. METHODS: We retrospectively analyzed the collection, storage, and disposal of PBSC grafts in Burkitt lymphoma (n = 18), acute lymphoblastic leukemia (ALL, n = 22), MPN (n = 18), and acute myeloid leukemia (AML, n = 71) patients. Patients who underwent autologous PBSC collection at 2 collection and transplantation centers between 2001 and 2012 were included and followed up until 2016. RESULTS: None of the Burkitt lymphoma patients were referred for ABSCT. Only in 1 (6%) patient, the graft was discarded after the patient's death. In all other patients (n = 17, 94%), the grafts were stored independently of the patient's status (death, n = 4, 22%; no follow-up, n = 6, 33%; no indication for ABSCT given, n = 7, 39%). In ALL patients, 4 (18%) patients underwent ABSCT after a median follow-up of 74 (1-182) months. In the remaining patients, PBSC grafts were either discarded (8 patients, 36%) or stored until the reference date (10 patients, 45%). Seven of 18 MPN patients (39%) underwent ABSCT. ABSCT was performed in 24 (34%) AML patients. In 20 (28%) patients who were not referred to ABSCT, an allogeneic transplantation (TPL) was performed. Fifteen (21%) patients received palliative care or deceased, and their grafts were discarded in all but 1 patient. Additional grafts were discarded in 21 (31%) patients and stored in 9 (13%) patients who underwent ABSCT or allogeneic TPL (n = 44). CONCLUSIONS: As the role and efficacy of autologous HDCT/ABSCT are not established in the analyzed entities, the indication for PBSC collection should be reanalyzed in regular intervals. Moreover, PBSC grafts from patients who have deceased, have insufficient grafts, or have already undergone an allogeneic TPL should be considered for disposal or (if applicable) for research use, to economize storage costs on a rational basis.

Human Adipose Tissue-Derived Mesenchymal Stromal Cells Inhibit CD4+ T Cell Proliferation and Induce Regulatory T Cells as Well as CD127 Expression on CD4+CD25+ T Cells.

Mesenchymal stromal cells (MSC) exert their immunomodulatory potential on several cell types of the immune system, affecting and influencing the immune response. MSC efficiently inhibit T cell proliferation, reduce the secretion of pro-inflammatory cytokines, limit the differentiation of pro-inflammatory Th subtypes and promote the induction of regulatory T cells (Treg). In this study, we analyzed the immunomodulatory potential of human adipose tissue-derived MSC (ASC), on CD4+ T cells, addressing potential cell-contact dependency in relation to T cell receptor stimulation of whole human peripheral blood mononuclear cells (PBMC). ASC were cultured with not stimulated or anti-CD3/CD28-stimulated PBMC in direct and transwell cocultures; PBMC alone were used as controls. After 7 days, cocultures were harvested and we analyzed: (1) the inhibitory potential of ASC on CD4+ cell proliferation and (2) phenotypic changes in CD4+ cells in respect of Treg marker (CD25, CD127 and FoxP3) expression. We confirmed the inhibitory potential of ASC on CD4+ cell proliferation, which occurs upon PBMC stimulation and is mediated by indoleamine 2,3-dioxygenase. Importantly, ASC reduce both pro- and anti-inflammatory cytokine secretion, without indications on specific Th differentiation. We found that stimulation induces CD25 expression on CD4+ cells and that, despite inhibiting overall CD4+ cell proliferation, ASC can specifically induce the proliferation of CD4+CD25+ cells. We observed that ASC induce Treg (CD4+CD25+CD127-FoxP3+) only in not stimulated cocultures and that ASC increase the ratio of CD4+CD25+CD127+FoxP3- cells at the expense of CD4+CD25+CD127-FoxP3- cells. Our study provides new insights on the interplay between ASC and CD4+ T cells, proposing that ASC-dependent induction of Treg depends on PBMC activation which affects the balance between the different subpopulations of CD4+CD25+ cells expressing CD127 and/or FoxP3.

Titanium Nanoparticles Enhance Production and Suppress Stabilin-1-Mediated Clearance of GDF-15 in Human Primary Macrophages.

Macrophages are key innate immune cells that mediate implant acceptance or rejection. Titanium implants degrade over time inside the body, which results in the release of implant wear-off particles. Titanium nanoparticles (TiNPs) favor pro-inflammatory macrophage polarization (M1) and lower tolerogenic activation (M2). GDF-15 regulates immune tolerance and fibrosis and is endocytosed by stabilin-1. How TiNPs affect the healing activities of macrophages and their release of circulating cytokines is an open question in regenerative medicine. In this study for the first time, we identified the transcriptional program induced and suppressed by TiNPs in human pro-inflammatory and healing macrophages. Microarray analysis revealed that TiNPs altered the expression of 5098 genes in M1 (IFN-γ-stimulated) and 4380 genes in M2 (IL-4-stimulated) macrophages. 1980 genes were differentially regulated in both M1 and M2. Affymetrix analysis, confirmed by RT-PCR, demonstrated that TiNPs upregulate expression of GDF-15 and suppress stabilin-1, scavenger receptor of GDF-15. TiNPs also significantly stimulated GDF-15 protein secretion in inflammatory and healing macrophages. Flow cytometry demonstrated, that scavenging activity of stabilin-1 was significantly suppressed by TiNPs. Confocal microscopy analysis showed that TiNPs impair internalization of stabilin-1 ligand acLDL and its transport to the endocytic pathway. Our data demonstrate that TiNPs have a dual effect on the GDF-15/stabilin-1 interaction in macrophage system, by increasing the production of GDF-15 and suppressing stabilin-1-mediated clearance function. In summary, this process can result in a significant increase of GDF-15 in the extracellular space and in circulation leading to unbalanced pro-fibrotic reactions and implant complications.

Human Adipose Tissue-Derived Stromal Cells Suppress Human, but Not Murine Lymphocyte Proliferation, via Indoleamine 2,3-Dioxygenase Activity.

Over recent years, mesenchymal stromal cells (MSC) have gained immense attraction in immunotherapy, regenerative medicine and tissue engineering. MSC microenvironment modulation occurs through synergy of direct cell-cell contact, and secreted soluble factors and extracellular vesicles (EV). MSC-derived EV have been suggested as cell-free immunomodulatory alternative to MSC; however, previous findings have challenged this. Furthermore, recent data suggest that evaluating the mechanism of action of human MSC (hMSC) in animal models might promote adverse immune reactions or lack of functionality due to xeno-incompatibilities. In this study, we first assessed the immunomodulatory strength of different human MSC sources on in vitro stimulated T cells and compared this to interferon-gamma (IFNγ) primed MSC conditioned medium (CM) and EV. Second, we addressed the main molecular mechanisms, and third, we assessed the MSC in vitro immunosuppressive effect across interspecies barriers. We identified human adipose tissue-derived stromal cells (ASC) with strongest immunomodulatory strength, followed by bone marrow (BM) and cord blood-derived MSC (CB). Whilst CM from primed ASC managed to exert analogous effects as their cellular counterpart, EV derived thereof did not, reproducing previous findings. IFNγ-induced indoleamine 2,3-dioxygenase (IDO) activity was identified as key mechanism to suppress human lymphocyte proliferation, as in the presence of the IDO inhibitor epacadostat (Epac) a stimulation of proliferation was seen. In addition, we revealed MSC immunosuppressive effects to be species-specific, because human cells failed to suppress murine lymphocyte proliferation. In summary, ASC were the strongest immunomodulators with the IDO-kynurenine pathway being key within the human system. Importantly, the in vitro lack of interspecies immunomodulatory strength suggests that preclinical data need to be carefully interpreted especially when considering a possible translation to clinical field.

Perioperative Blood Transfusion Is a Predictor of Acute and Chronic Renal Function Deterioration after Partial and Radical Nephrectomy for Renal Cell Carcinoma.

INTRODUCTION: The aim was to evaluate the impact of perioperative blood transfusions (PBTs) on renal function after surgery for renal cell carcinoma (RCC). METHODS: Consecutive patients with RCC who underwent partial nephrectomy or radical nephrectomy between 2005 and 2015 at a tertiary care center were included. Minimum follow-up period was 6 months. A PBT was defined as the transfusion of packed erythrocyte concentrate (EC) within 7 days before until 30 days after surgery. The multivariable analyses were carried out by Cox regression. RESULTS: The overall cohort included 851 patients, of whom 93 (10.9%) received a PBT. The median follow-up was 46 months (range 28-72). In case of a PBT, a median of 2 EC was transfused. PBT patients were older and had a poorer performance status and more comorbidities as well as locally more advanced or metastatic tumors. In the multivariable analyses, PBT was an independent prognostic factor for acute as well as chronic renal impairment (hazard ratio (HR) 2.72, 95% confidence interval (95% CI) 1.45-5.10, p = 0.002 and HR 2.23, 95% CI 1.26-3.70, p = 0.007). CONCLUSION: PBT is associated with acute and chronic deterioration of kidney function after surgery for RCC. Thus, it may be used to identify patients requiring close nephrological monitoring.

Epigenetic Regulation of S100A9 and S100A12 Expression in Monocyte-Macrophage System in Hyperglycemic Conditions.

The number of diabetic patients in Europe and world-wide is growing. Diabetes confers a 2-fold higher risk for vascular disease. Lack of insulin production (Type 1 diabetes, T1D) or lack of insulin responsiveness (Type 2 diabetes, T2D) causes systemic metabolic changes such as hyperglycemia (HG) which contribute to the pathology of diabetes. Monocytes and macrophages are key innate immune cells that control inflammatory reactions associated with diabetic vascular complications. Inflammatory programming of macrophages is regulated and maintained by epigenetic mechanisms, in particular histone modifications. The aim of our study was to identify the epigenetic mechanisms involved in the hyperglycemia-mediated macrophage activation. Using Affymetrix microarray profiling and RT-qPCR we identified that hyperglycemia increased the expression of S100A9 and S100A12 in primary human macrophages. Expression of S100A12 was sustained after glucose levels were normalized. Glucose augmented the response of macrophages to Toll-like receptor (TLR)-ligands Palmatic acid (PA) and Lipopolysaccharide (LPS) i.e., pro-inflammatory stimulation. The abundance of activating histone Histone 3 Lysine 4 methylation marks (H3K4me1, H3K4me3) and general acetylation on histone 3 (AceH3) with the promoters of these genes was analyzed by chromatin immunoprecipitation. Hyperglycemia increased acetylation of histones bound to the promoters of S100A9 and S100A12 in M1 macrophages. In contrast, hyperglycemia caused a reduction in total H3 which correlated with the increased expression of both S100 genes. The inhibition of histone methyltransferases SET domain-containing protein (SET)7/9 and SET and MYND domain-containing protein (SMYD)3 showed that these specifically regulated S100A12 expression. We conclude that hyperglycemia upregulates expression of S100A9, S100A12 via epigenetic regulation and induces an activating histone code on the respective gene promoters in M1 macrophages. Mechanistically, this regulation relies on action of histone methyltransferases SMYD3 and SET7/9. The results define an important role for epigenetic regulation in macrophage mediated inflammation in diabetic conditions.

SI-CLP inhibits the growth of mouse mammary adenocarcinoma by preventing recruitment of tumor-associated macrophages.

Chitinase-like proteins (CLP) are chitin-binding proteins that lack chitin hydrolyzing activity, but possess cytokine-like and growth factor-like properties, and play crucial role in intercellular crosstalk. Both human and mice express two members of CLP family: YKL-40 and stabilin-1 interacting chitinase-like protein (SI-CLP). Despite numerous reports indicating the role of YKL-40 in the support of angiogenesis, tumor cell proliferation, invasion and metastasis, the role of its structurally related protein SI-CLP in cancer was not reported. Using gain-of-function approach, we demonstrate in the current study that the expression of recombinant SI-CLP in mouse TS/A mammary adenocarcinoma cells results in significant and persistent inhibition of in vivo tumor growth. Using quantitative immunohistochemistry, we show that on the cellular level this phenomenon is associated with reduced infiltration of tumor-associated macrophages (TAMs), CD4+ and FoxP3+ cells in SI-CLP expressing tumors. Gene expression analysis in TAM isolated from SI-CLP-expressing and control tumors demonstrated that SI-CLP does not affect macrophage phenotype. However, SI-CLP significantly inhibited migration of murine bone-marrow derived macrophages and human primary monocytes toward monocyte-recruiting chemokine CCL2 produced in the tumor microenvironment (TME). Mechanistically, SI-CLP did not affect CCL2/CCR2 interaction, but suppressed cytoskeletal rearrangements in response to CCL2. Altogether, our data indicate that SI-CLP functions as a tumor growth inhibitor in mouse breast cancer by altering cellular composition of TME and blocking cytokine-induced TAM recruitment. Taking into consideration weak to absent expression of SI-CLP in human breast cancer, it can be considered as a therapeutic protein to block TAM-mediated support of breast tumor growth.

The Impact of Harvesting Systems and Donor Characteristics on Viability of Nucleated Cells in Adipose Tissue: A First Step Towards a Manufacturing Process.

Adipose tissue contains an abundant population of mesenchymal stromal cells (= adipose stromal cells [ASC]) with multilineage differentiation, immunomodulatory and trophic potential promising for cell-based therapies. Although intensely investigated in pre- and clinical studies, little is known about the impact of donor characteristics on the viability of ASC. To correlate patient data to the quality of processed adipose tissue and to establish a first step towards a manufacturing process for cell therapy, we evaluated the effects of 2 harvesting systems (LipiVage, TTF-System) and donor characteristics on cell viability of nucleated cells in a cohort of 44 samples obtained from 17 donors.The impact of donor-specific factors (localization, age, body-mass-index, chronic diseases, intake of drugs, nicotine consumption or disorders of the thyroid gland) and the harvesting system on nucleated cell (NC) counts and viability of processed lipoaspirates were statistically analyzed.Increasing age has a significant positive influence on NC viability (P = 0.001). Donors with intake of thyroid hormones based on a hypothyroidism and suctioned with the LipiVage-System reached a significantly higher viability of NC (P = 0.038). No statistical difference was shown between the 2 harvesting-systems (P = 0.338) and the donor sites (P = 0.294).We focused on a potential correlation between NC viability and donor characteristics. Based on the donor cohort investigated in this study, cells from elderly patients suctioned with the LipiVage-System and taking thyroid hormones yielded cells of higher viability, suggesting an improved quality for subsequent manufacturing procedures. Further investigations are necessary to understand and correlate this data to ASC in vitro characteristics.

Tumor-associated macrophages in human breast cancer produce new monocyte attracting and pro-angiogenic factor YKL-39 indicative for increased metastasis after neoadjuvant chemotherapy.

In breast cancer, the tumor microenvironment plays a critical role in the tumor progression and responses to therapy. Tumor-associated macrophages (TAMs) are major innate immune cells in tumor microenvironment that regulate intratumoral immunity and angiogenesis by secretion of cytokines, growth factors as well as chitinase-like proteins (CLPs), that combine properties of cytokines and growth factors. YKL-39 is a chitinase-like protein found in human and absent in rodents, and its expression in TAMs and role in breast cancer progression was not studied to date. Here for the first time we demonstrate that YKL-39 is expressed on TAMs, predominantly positive for stabilin-1, but not by malignant cells or other stromal cells in human breast cancer. TGF-beta in combination with IL-4, but not IL-4 alone was responsible of the stimulation of the production of YKL-39 in human primary macrophages. Mechanistically, stabilin-1 directly interacted with YKL-39 and acted as sorting receptor for targeting YKL-39 into the secretory pathway. Functionally, purified YKL-39 acted as a strong chemotactic factor for primary human monocytes, and induced angiogenesis in vitro. Elevated levels of YKL-39 expression in tumors after neoadjuvant chemotherapy (NAC) were predictive for increased risk of distant metastasis and for poor response to NAC in patients with nonspecific invasive breast carcinoma. Our findings suggest YKL-39 as a novel therapeutic target, and blocking of its activity can be combined with NAC in order to reduce the risk of metastasis in breast cancer patients.