ABSTRACT
Introduction: Autologous platelet concentrate (APC) are pro-angiogenic and can promote wound healing and tissue repair, also in combination with other biomaterials. However, challenging defect situations remain demanding. 3D bioprinting of an APC based bioink encapsulated in a hydrogel could overcome this limitation with enhanced physio-mechanical interface, growth factor retention/secretion and defect-personalized shape to ultimately enhance regeneration. Methods: This study used extrusion-based bioprinting to create a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate. Chemico-physical testing exhibited an amorphous structure characterized by high shape fidelity. Cytotoxicity assay and incubation of human osteogenic sarcoma cells (SaOs2) exposed excellent biocompatibility. enzyme-linked immunosorbent assay analysis confirmed pro-angiogenic growth factor release of the printed constructs, and co-incubation with HUVECS displayed proper cell viability and proliferation. Chorioallantoic membrane (CAM) assay explored the pro-angiogenic potential of the prints in vivo. Detailed proteome and secretome analysis revealed a substantial amount and homologous presence of pro-angiogenic proteins in the 3D construct. Results: This study demonstrated a 3D bioprinting approach to fabricate a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate with high shape fidelity, biocompatibility, and substantial pro-angiogenic properties. Conclusion: This approach may be suitable for challenging physiological and anatomical defect situations when translated into clinical use.
ABSTRACT
Latent reservoirs in human-immunodeficiency-virus-1 (HIV-1)-infected individuals represent a major obstacle in finding a cure for HIV-1. Hematopoietic stem and progenitor cells (HSPCs) have been described as potential HIV-1 targets, but their roles as HIV-1 reservoirs remain controversial. Here we provide additional evidence for the susceptibility of several distinct HSPC subpopulations to HIV-1 infection in vitro and in vivo. In vitro infection experiments of HSPCs were performed with different HIV-1 Env-pseudotyped lentiviral particles and with replication-competent HIV-1. Low-level infection/transduction of HSPCs, including hematopoietic stem cells (HSCs) and multipotent progenitors (MPP), was observed, preferentially via CXCR4, but also via CCR5-mediated entry. Multi-lineage colony formation in methylcellulose assays and repetitive replating of transduced cells provided functional proof of susceptibility of primitive HSPCs to HIV-1 infection. Further, the access to bone marrow samples from HIV-positive individuals facilitated the detection of HIV-1 gag cDNA copies in CD34+ cells from eight (out of eleven) individuals, with at least six of them infected with CCR5-tropic HIV-1 strains. In summary, our data confirm that primitive HSPC subpopulations are susceptible to CXCR4- and CCR5-mediated HIV-1 infection in vitro and in vivo, which qualifies these cells to contribute to the HIV-1 reservoir in patients.
Subject(s)
HIV Infections , HIV-1 , DNA, Complementary , HIV-1/physiology , Hematopoietic Stem Cells , HumansABSTRACT
The respiratory burst of phagocytes is essential for human survival. Innate immune defence against pathogens relies strongly on reactive oxygen species (ROS) production by the NADPH oxidase (NOX2). ROS kill pathogens while the translocation of electrons across the plasma membrane via NOX2 depolarizes the cell. Simultaneously, protons are released into the cytosol. Here, we compare freshly isolated human polymorphonuclear leukocytes (PMN) to the granulocytes-like cell line PLB 985. We are recording ROS production while inhibiting the charge compensating and pH regulating voltage-gated proton channel (HV1). The data suggests that human PMN and the PLB 985 generate ROS via a general mechanism, consistent of NOX2 and HV1. Additionally, we advanced a mathematical model based on the biophysical properties of NOX2 and HV1. Our results strongly suggest the essential interconnection of HV1 and NOX2 during the respiratory burst of phagocytes. Zinc chelation during the time course of the experiments postulates that zinc leads to an irreversible termination of the respiratory burst over time. Flow cytometry shows cell death triggered by high zinc concentrations and PMA. Our data might help to elucidate the complex interaction of proteins during the respiratory burst and contribute to decipher its termination.
Subject(s)
Neutrophils , Respiratory Burst , Humans , Ion Channels/metabolism , NADPH Oxidases/metabolism , Neutrophils/metabolism , Reactive Oxygen Species/metabolism , ZincABSTRACT
The tumor microenvironment in brain metastases is characterized by high myeloid cell content associated with immune suppressive and cancer-permissive functions. Moreover, brain metastases induce the recruitment of lymphocytes. Despite their presence, T-cell-directed therapies fail to elicit effective anti-tumor immune responses. Here, we seek to evaluate the applicability of radio-immunotherapy to modulate tumor immunity and overcome inhibitory effects that diminish anti-cancer activity. Radiotherapy-induced immune modulation resulted in an increase in cytotoxic T-cell numbers and prevented the induction of lymphocyte-mediated immune suppression. Radio-immunotherapy led to significantly improved tumor control with prolonged median survival in experimental breast-to-brain metastasis. However, long-term efficacy was not observed. Recurrent brain metastases showed accumulation of blood-borne PD-L1+ myeloid cells after radio-immunotherapy indicating the establishment of an immune suppressive environment to counteract re-activated T-cell responses. This finding was further supported by transcriptional analyses indicating a crucial role for monocyte-derived macrophages in mediating immune suppression and regulating T-cell function. Therefore, selective targeting of immune suppressive functions of myeloid cells is expected to be critical for improved therapeutic efficacy of radio-immunotherapy in brain metastases.
Subject(s)
Brain Neoplasms , Tumor Microenvironment , Brain Neoplasms/radiotherapy , Humans , Immunotherapy , Macrophages , Myeloid CellsABSTRACT
The aim of this study was to investigate the dynamic changes of circulating tumor cells (CTCs) in patients with hepatocellular carcinoma (HCC) before and immediately after conducting a microwave ablation (MWA) and conventional transarterial chemoembolization (C-TACE). Additionally, the CTCs short-term dynamics were compared with the clinical course of the HCC-patients. Blood samples from 17 patients with HCC who underwent MWA (n = 10) or C-TACE (n = 7) were analyzed. Venous blood was taken before and immediately after the radiological interventions to isolate and quantify CTCs using flow cytometry. CTCs were identified as CD45- and positive for the markers ASGPR, CD146 and CD274 (PD-L1). Patients were followed of up to 2.2 years after the radiological intervention. CTCs were detected in 13 HCC patients (76%) prior to the radiological interventions. The rate of CTCs was significantly decreased after the intervention in patients treated with MWA (0.4 CTCs/mL of blood, p = 0.031). However, no significant differences were observed in patients who received C-TACE (0.3 CTCs/mL of blood, p = 0.300). Overall, no correlation was found between the CTCs rate before and after the radiological intervention and recurrence rate of HCC. This preliminary data could confirm the tumoricidal effects of MWA in patients with HCC by significantly decreasing CTCs rate. In our study, we were able to detect CTCs in HCC patients using 3 different tumor markers. This preliminary data shows significant lower CTCs detected in response to MWA. However, large-scale randomized clinical trials are needed to determine the future role and the prognostic relevance of CTCs following this treatment.
Subject(s)
Carcinoma, Hepatocellular/pathology , Liver Neoplasms/pathology , Neoplastic Cells, Circulating/pathology , Aged , Biomarkers, Tumor/blood , CD146 Antigen/metabolism , Carcinoma, Hepatocellular/blood , Carcinoma, Hepatocellular/therapy , Chemoembolization, Therapeutic/methods , Female , Humans , Liver Neoplasms/blood , Liver Neoplasms/therapy , Male , Microwaves , Middle Aged , Neoplasm Recurrence, Local/pathology , Neoplasm Recurrence, Local/therapy , PrognosisABSTRACT
In the absence of an active prophylactic vaccine against HIV-1, passively administered, broadly neutralizing antibodies (bnAbs) identified in some chronically infected persons were shown to prevent HIV-1 infection in animal models. However, passive administration of bnAbs may not be suited to prevent sexual HIV-1 transmission in high-risk cohorts, as a continuous high level of active bnAbs may be difficult to achieve at the primary site of sexual transmission, the human vagina with its acidic pH. Therefore, we used Lactobacillus, a natural commensal in the healthy vaginal microbiome, to express bn nanobodies (VHH) against HIV-1 that we reported previously. After demonstrating that recombinant VHHA6 expressed in E. coli was able to protect humanized mice from mucosal infection by HIV-1Bal, we expressed VHHA6 in a soluble or in a cell-wall-anchored form in Lactobacillus rhamnosus DSM14870. This strain is already clinically applied for treatment of bacterial vaginosis. Both forms of VHHA6 neutralized a set of primary epidemiologically relevant HIV-1 strains in vitro. Furthermore, VHHA6 was still active at an acidic pH. Thus, lactobacilli expressing bn VHH potentially represent an attractive vector for the passive immunization of women in cohorts at high risk of HIV-1 transmission.
ABSTRACT
Brain-resident microglia and bone marrow-derived macrophages represent the most abundant non-cancerous cells in the brain tumor microenvironment with critical functions in disease progression and therapeutic response. To date little is known about genetic programs that drive disease-associated phenotypes of microglia and macrophages in brain metastases. Here we used cytometric and transcriptomic analyses to define cellular and molecular changes of the myeloid compartment at distinct stages of brain metastasis and in response to radiotherapy. We demonstrate that genetic programming of tumor education in myeloid cells occurs early during metastatic onset and remains stable throughout tumor progression. Bulk and single cell RNA sequencing revealed distinct gene signatures in brain-resident microglia and blood-borne monocytes/macrophages during brain metastasis and in response to therapeutic intervention. Our data provide a framework for understanding the functional heterogeneity of brain metastasis-associated myeloid cells based on their origin.
ABSTRACT
MicroRNAs (miRs) are important posttranscriptional regulators of gene expression. Besides their well-characterized inhibitory effects on mRNA stability and translation, miRs can also activate gene expression. In this study, we identified a novel noncanonical function of miR-574-5p. We found that miR-574-5p acts as an RNA decoy to CUG RNA-binding protein 1 (CUGBP1) and antagonizes its function. MiR-574-5p induces microsomal prostaglandin E synthase-1 (mPGES-1) expression by preventing CUGBP1 binding to its 3'UTR, leading to an enhanced alternative splicing and generation of an mPGES-1 3'UTR isoform, increased mPGES-1 protein expression, PGE2 formation, and tumor growth in vivo. miR-574-5p-induced tumor growth in mice could be completely inhibited with the mPGES-1 inhibitor CIII. Moreover, miR-574-5p is induced by IL-1ß and is strongly overexpressed in human nonsmall cell lung cancer where high mPGES-1 expression correlates with a low survival rate. The discovered function of miR-574-5p as a CUGBP1 decoy opens up new therapeutic opportunities. It might serve as a stratification marker to select lung tumor patients who respond to the pharmacological inhibition of PGE2 formation.-Saul, M. J., Baumann, I., Bruno, A., Emmerich, A. C., Wellstein, J., Ottinger, S. M., Contursi, A., Dovizio, M., Donnini, S., Tacconelli, S., Raouf, J., Idborg, H., Stein, S., Korotkova, M., Savai, R., Terzuoli, E., Sala, G., Seeger, W., Jakobsson, P.-J., Patrignani, P., Suess, B., Steinhilber, D. miR-574-5p as RNA decoy for CUGBP1 stimulates human lung tumor growth by mPGES-1 induction.
Subject(s)
CELF1 Protein/metabolism , MicroRNAs/metabolism , Prostaglandin-E Synthases/metabolism , RNA/metabolism , A549 Cells , Animals , CELF1 Protein/genetics , Cell Proliferation , Gene Expression Regulation, Neoplastic/drug effects , HeLa Cells , Humans , Mice , Mice, Nude , MicroRNAs/genetics , Molecular Mimicry , Neoplasms, Experimental , Prostaglandin-E Synthases/genetics , Protein Binding , Protein Synthesis Inhibitors/pharmacology , Puromycin/pharmacology , RNA/genetics , RNA Interference , RNA Isoforms , RNA, MessengerABSTRACT
Heat shock protein 90 (HSP90) stabilizes many client proteins, including the BCR-ABL1 oncoprotein. BCR-ABL1 is the hallmark of chronic myeloid leukemia (CML) in which treatment-free remission (TFR) is limited, with clinical and economic consequences. Thus, there is an urgent need for novel therapeutics that synergize with current treatment approaches. Several inhibitors targeting the N-terminal domain of HSP90 are under investigation, but side effects such as induction of the heat shock response (HSR) and toxicity have so far precluded their US Food and Drug Administration approval. We have developed a novel inhibitor (aminoxyrone [AX]) of HSP90 function by targeting HSP90 dimerization via the C-terminal domain. This was achieved by structure-based molecular design, chemical synthesis, and functional preclinical in vitro and in vivo validation using CML cell lines and patient-derived CML cells. AX is a promising potential candidate that induces apoptosis in the leukemic stem cell fraction (CD34+CD38-) as well as the leukemic bulk (CD34+CD38+) of primary CML and in tyrosine kinase inhibitor (TKI)-resistant cells. Furthermore, BCR-ABL1 oncoprotein and related pro-oncogenic cellular responses are downregulated, and targeting the HSP90 C terminus by AX does not induce the HSR in vitro and in vivo. We also probed the potential of AX in other therapy-refractory leukemias. Therefore, AX is the first peptidomimetic C-terminal HSP90 inhibitor with the potential to increase TFR in TKI-sensitive and refractory CML patients and also offers a novel therapeutic option for patients with other types of therapy-refractory leukemia because of its low toxicity profile and lack of HSR.
Subject(s)
Antineoplastic Agents/pharmacology , HSP90 Heat-Shock Proteins/antagonists & inhibitors , HSP90 Heat-Shock Proteins/chemistry , Heat-Shock Response/drug effects , Imatinib Mesylate/pharmacology , Protein Interaction Domains and Motifs , Protein Kinase Inhibitors/pharmacology , Protein Multimerization , Animals , Antineoplastic Agents/chemistry , Binding Sites , Biomarkers, Tumor , Cell Cycle/drug effects , Cell Line, Tumor , Cell Survival/drug effects , Disease Models, Animal , Drug Resistance, Neoplasm/drug effects , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/chemistry , HSP90 Heat-Shock Proteins/metabolism , Humans , Imatinib Mesylate/chemistry , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Mice , Models, Molecular , Molecular Conformation , Molecular Structure , Protein Binding , Protein Kinase Inhibitors/chemistry , Protein Multimerization/drug effects , Spectrum Analysis , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Laser drop on demand jetting of Cu-base braze droplets was proven a suitable method for joining wires to electrode structures of electronic devices, particularly if the electrical contacts need to withstand high thermal loads. During joining, a braze preform of 600 µm diameter is placed inside a capillary, molten by a laser pulse and subsequently ejected from the capillary by inert gas overpressure similarly to conventional solder ball bumping processes. However, since the liquidus temperature of the used braze material of 990 °C is about 760 °C higher than of standard Sn-based solders used in electronics packaging, the system technology was modified significantly to enable jetting of CuSn alloys. In particular, the beam source emits a five times higher optical output power than standard machines designed for processing Sn-based solders. In addition, a modified capillary made from technical ceramic was machined, to withstand the significantly higher heating- and cooling rates during the process. In order to understand the influence of capillary geometry on droplet detachment, and flight trajectory, two capillary geometries were machined applying a picosecond laser ablation process. Subsequently, stereoscopic high speed videos of droplet detachment and flight phase were analyzed. Using this approach it is possible, to determine droplet flight trajectories, velocities and lateral positional deviations in dependency of relative inert gas overpressure inside the machining head, pulse power and capillary geometry. The findings indicate a significant influence of the capillary geometry and the applied overpressure on the droplet flight trajectory, whereas the role of the laser pulse power seems neglectable.
ABSTRACT
Chronic granulomatous disease (CGD) is a debilitating primary immunodeficiency affecting phagocyte function due to the absence of nicotinamide dinucleotide phosphate (NADPH) oxidase activity. The vast majority of CGD patients in the Western world have mutations within the X-linked CYBB gene encoding for gp91phox (NOX2), the redox center of the NADPH oxidase complex (XCGD). Current treatments of XCGD are not entirely satisfactory, and prior attempts at autologous gene therapy using gammaretrovirus vectors did not provide long-term curative effects. A new strategy was developed based on the use of the lentiviral vector G1XCGD expressing high levels of the gp91phox transgene in myeloid cells. As a requisite for a clinical trial approval, standardized non-clinical studies were conducted in vitro and in mice in order to evaluate the pharmacodynamics and biosafety of the vector and the biodistribution of G1XCGD-transduced cells. Transduced CD34+ cells derived from XCGD patients engrafted and differentiated similarly to their non-transduced counterparts in xenograft mouse models and generated therapeutically relevant levels of NADPH activity in myeloid cells expressing gp91phox. Expression of functional gp91phox in hematopoietic cells did not affect their homing properties, which engrafted at high levels in mice. Extensive in vitro and in vivo genotoxicity studies found no evidence for adverse mutagenesis related to vector treatment. These studies paved the way for the approval of clinical trials in Europe and in the United States for the treatment of XCGD patients with G1XCGD gene-modified autologous hematopoietic cells.
Subject(s)
Genetic Diseases, X-Linked/genetics , Granulomatous Disease, Chronic/genetics , NADPH Oxidase 2/genetics , NADPH Oxidases/genetics , Animals , Clinical Trials as Topic , Gene Expression Regulation/drug effects , Genetic Diseases, X-Linked/pathology , Genetic Diseases, X-Linked/therapy , Genetic Therapy , Genetic Vectors/administration & dosage , Genetic Vectors/adverse effects , Granulomatous Disease, Chronic/pathology , Granulomatous Disease, Chronic/therapy , Hematopoietic Stem Cells/drug effects , Heterografts , Humans , Lentivirus/genetics , Mice , NADPH Oxidase 2/administration & dosageABSTRACT
The CRISPR/Cas9 prokaryotic adaptive immune system and its swift repurposing for genome editing enables modification of any prespecified genomic sequence with unprecedented accuracy and efficiency, including targeted gene repair. We used the CRISPR/Cas9 system for targeted repair of patient-specific point mutations in the Cytochrome b-245 heavy chain gene (CYBB), whose inactivation causes chronic granulomatous disease (XCGD)-a life-threatening immunodeficiency disorder characterized by the inability of neutrophils and macrophages to produce microbicidal reactive oxygen species (ROS). We show that frameshift mutations can be effectively repaired in hematopoietic cells by non-integrating lentiviral vectors carrying RNA-guided Cas9 endonucleases (RGNs). Because about 25% of most inherited blood disorders are caused by frameshift mutations, our results suggest that up to a quarter of all patients suffering from monogenic blood disorders could benefit from gene therapy employing personalized, donor template-free RGNs.
ABSTRACT
Although a lot of research has been performed, large segmental bone defects caused by trauma, infection, bone tumors or revision surgeries still represent big challenges for trauma surgeons. New and innovative bone substitutes are needed. Three-dimensional (3D) printing is a novel procedure to create 3D porous scaffolds that can be used for bone tissue engineering. In the present study, solid discs as well as porous cage-like 3D prints made of polylactide (PLA) are coated or filled with collagen, respectively, and tested for biocompatibility and endotoxin contamination. Microscopic analyses as well as proliferation assays were performed using various cell types on PLA discs. Stromal-derived factor (SDF-1) release from cages filled with collagen was analyzed and the effect on endothelial cells tested. This study confirms the biocompatibility of PLA and demonstrates an endotoxin contamination clearly below the FDA (Food and Drug Administration) limit. Cells of various cell types (osteoblasts, osteoblast-like cells, fibroblasts and endothelial cells) grow, spread and proliferate on PLA-printed discs. PLA cages loaded with SDF-1 collagen display a steady SDF-1 release, support cell growth of endothelial cells and induce neo-vessel formation. These results demonstrate the potential for PLA scaffolds printed with an inexpensive desktop printer in medical applications, for example, in bone tissue engineering.
Subject(s)
Collagen Type I/chemistry , Polyesters/chemistry , Bone Regeneration/physiology , Cell Line , Cell Proliferation/physiology , Chemokine CXCL12/metabolism , Humans , Osteogenesis/physiology , Tissue Engineering/methods , Tissue Scaffolds/chemistryABSTRACT
Gallbladder cancer (GBC) is a lethal cancer with poor prognosis associated with high invasiveness and poor response to chemotherapy and radiotherapy. New therapeutic approaches are urgently needed in order to improve survival and response rates of GBC patients. We screened 130 small molecule inhibitors on a panel of seven GBC cell lines and identified the HSP90 inhibitor 17-AAG as one of the most potent inhibitory drugs across the different lines. We tested the antitumor efficacy of 17-AAG and geldanamycin (GA) in vitro and in a subcutaneous preclinical tumor model NOD-SCID mice. We also evaluated the expression of HSP90 by immunohistochemistry in human GBC tumors.In vitro assays showed that 17-AAG and GA significantly reduced the expression of HSP90 target proteins, including EGFR, AKT, phospho-AKT, Cyclin B1, phospho-ERK and Cyclin D1. These molecular changes were consistent with reduced cell viability and cell migration and promotion of G2/M cell cycle arrest and apoptosis observed in our in vitro studies.In vivo, 17-AAG showed efficacy in reducing subcutaneous tumors size, exhibiting a 69.6% reduction in tumor size in the treatment group compared to control mice (p < 0.05).The HSP90 immunohistochemical staining was seen in 182/209 cases of GBC (87%) and it was strongly expressed in 70 cases (33%), moderately in 58 cases (28%), and weakly in 54 cases (26%).Our pre-clinical observations strongly suggest that the inhibition of HSP90 function by HSP90 inhibitors is a promising therapeutic strategy for gallbladder cancer that may benefit from new HSP90 inhibitors currently in development.
Subject(s)
Antineoplastic Agents/pharmacology , Benzoquinones/pharmacology , Drug Discovery , Drug Screening Assays, Antitumor , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Lactams, Macrocyclic/pharmacology , Animals , Apoptosis/drug effects , Cell Cycle Checkpoints/drug effects , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Cell Survival/drug effects , Disease Models, Animal , Drug Discovery/methods , Drug Screening Assays, Antitumor/methods , Gallbladder Neoplasms , High-Throughput Screening Assays , Humans , Mice , Small Molecule Libraries , Xenograft Model Antitumor AssaysSubject(s)
Antineoplastic Agents/pharmacology , Core Binding Factor Alpha 2 Subunit/antagonists & inhibitors , Oncogene Proteins, Fusion/antagonists & inhibitors , Protein Multimerization/drug effects , RUNX1 Translocation Partner 1 Protein/antagonists & inhibitors , Animals , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Core Binding Factor Alpha 2 Subunit/genetics , Disease Models, Animal , Gene Expression , Humans , Mice , Oncogene Proteins, Fusion/chemistry , Oncogene Proteins, Fusion/genetics , Oncogene Proteins, Fusion/metabolism , RUNX1 Translocation Partner 1 Protein/genetics , Xenograft Model Antitumor AssaysABSTRACT
UPF1 is a key player in nonsense mediated mRNA decay (NMD) but also involved in posttranscriptional gene regulation. In this study we found that UPF1 regulates the expression of genes with functions in inflammation and myeloid cell differentiation via hnRNP E2. The majority of the UPF1-regulated genes identified in monocytic cells contain a binding site for hnRNP E2 within 5' UTR located introns with hnRNP E2 acting here as splicing regulator. We found that miRNA-328 which is significantly induced during monocytic cell differentiation acts independently from its gene silencing function as RNA decoy for hnRNP E2. One representative gene controlled by the hnRNP E2/miRNA-328 balance is S100A9 which plays an important role in cell differentiation and oxidative stress response of monocytes. Induction of miRNA-328 expression during cell differentiation antagonizes the blockade by hnRNP E2 which results in the upregulation of CD11b expression and ROS production in monocytic cells. Taken together, our data indicate that upregulation of miR-328 is responsible for the induction of hnRNP E2 target genes during myeloid cell differentiation.
Subject(s)
Calgranulin B/metabolism , MicroRNAs/metabolism , Monocytes/metabolism , RNA Helicases/metabolism , RNA-Binding Proteins/metabolism , Trans-Activators/metabolism , CD11b Antigen/metabolism , Cell Adhesion , Cell Differentiation , Cell Line , Cell Movement , Gene Expression Regulation , HeLa Cells , Humans , Monocytes/cytology , Proteomics , RNA Interference , RNA-Binding Proteins/genetics , Reactive Oxygen Species/metabolismABSTRACT
PURPOSE: Triple-negative breast cancer (TNBC) is characterized by a strong heterogeneity with regard to tumour biology as well as in the clinical course of the disease. This study aimed to analyse whether there are any prognostic factors enabling prediction of the clinical outcome in patients with TNBC. Particularly, the impact of Her2-neu score 0 versus Her2-neu score 1 and 2 on survival was investigated. MATERIALS AND METHODS: We retrospectively studied a cohort of 1013 patients with TNBC, diagnosed at seven hospitals between May 2002 and February 2015. We studied the impact of Her2-neu scores (0 vs. 1 or 2 with negative FISH) on disease-free survival (DFS) and overall survival (OS). RESULTS: 1013 patients were included in this study. 447 (44.13 %) of them had a T2-4 tumour. A total of 314 (31.00 %) were nodal-positive and 714 (70.48 %) had high-grade tumours. The Her2-neu score of all participating patients was determined. 588 (58.05 %) of them had a Her2-neu score 0, and 425 (41.95 %) had a score of 1 or 2. This study shows that TNBC patients with a Her2-neu score 0 had a significantly poorer outcome regarding DFS (p = 0.0001) and OS (p = 0.0051) compared to a score of 1 or 2. In contrast, grading did not seem to have any prognostic value for women with TNBC. CONCLUSION: The Her2-neu score 0 might be considered as an innovative prognostic factor for patients with TNBC indicating poor clinical outcome.
Subject(s)
Receptor, ErbB-2/metabolism , Triple Negative Breast Neoplasms/pathology , Female , Humans , Prognosis , Retrospective Studies , Triple Negative Breast Neoplasms/metabolismABSTRACT
BACKGROUND: Defects in phagocytic nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) function cause chronic granulomatous disease (CGD), a primary immunodeficiency characterized by dysfunctional microbicidal activity and chronic inflammation. OBJECTIVE: We sought to study the effect of chronic inflammation on the hematopoietic compartment in patients and mice with X-linked chronic granulomatous disease (X-CGD). METHODS: We used immunostaining and functional analyses to study the hematopoietic compartment in patients with CGD. RESULTS: An analysis of bone marrow cells from patients and mice with X-CGD revealed a dysregulated hematopoiesis characterized by increased numbers of hematopoietic progenitor cells (HPCs) at the expense of repopulating hematopoietic stem cells (HSCs). In patients with X-CGD, there was a clear reduction in the proportion of HSCs in bone marrow and peripheral blood, and they were also more rapidly exhausted after in vitro culture. In mice with X-CGD, increased cycling of HSCs, expansion of HPCs, and impaired long-term engraftment capacity were found to be associated with high concentrations of proinflammatory cytokines, including IL-1ß. Treatment of wild-type mice with IL-1ß induced enhanced cell-cycle entry of HSCs, expansion of HPCs, and defects in long-term engraftment, mimicking the effects observed in mice with X-CGD. Inhibition of cytokine signaling in mice with X-CGD reduced HPC numbers but had only minor effects on the repopulating ability of HSCs. CONCLUSIONS: Persistent chronic inflammation in patients with CGD is associated with hematopoietic proliferative stress, leading to a decrease in the functional activity of HSCs. Our observations have clinical implications for the development of successful autologous cell therapy approaches.
Subject(s)
Granulomatous Disease, Chronic/metabolism , Hematopoietic Stem Cells/metabolism , Adolescent , Adult , Animals , Biomarkers , Case-Control Studies , Cell Count , Cell Differentiation , Child , Child, Preschool , Colony-Forming Units Assay , Cytokines/metabolism , Cytokines/pharmacology , Disease Models, Animal , Graft Survival , Granulomatous Disease, Chronic/etiology , Hematopoietic Stem Cell Transplantation , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/drug effects , Humans , Immunophenotyping , Inflammation Mediators/metabolism , Mice , Mice, Transgenic , Models, Biological , Phenotype , Signal Transduction , Young AdultABSTRACT
Gallbladder cancer (GBC) is a highly malignant tumor characterized by a poor response to chemotherapy and radiotherapy. We evaluated the in vitro and in vivo antitumor efficacy of mTOR inhibitors, rapamycin and WYE-354. In vitro assays showed WYE-354 significantly reduced cell viability, migration and invasion and phospho-P70S6K expression in GBC cells. Mice harboring subcutaneous gallbladder tumors, treated with WYE-354 or rapamycin, exhibited a significant reduction in tumor mass. A short-term treatment with a higher dose of WYE-354 decreased the tumor size by 68.6% and 52.4%, in mice harboring G-415 or TGBC-2TKB tumors, respectively, compared to the control group. By contrast, treatment with a prolonged-low-dose regime of rapamycin almost abrogated tumor growth, exhibiting 92.7% and 97.1% reduction in tumor size, respectively, compared to control mice. These results were accompanied by a greater decrease in the phosphorylation status of P70S6K and a lower cell proliferation Ki67 index, compared to WYE-354 treated mice, suggesting a more effective mTOR pathway inhibition. These findings provide a proof of concept for the use of rapamycin or WYE-354 as potentially good candidates to be studied in clinical trials in GBC patients.
Subject(s)
Antibiotics, Antineoplastic/pharmacology , Gallbladder Neoplasms/drug therapy , Guanine/analogs & derivatives , Sirolimus/pharmacology , Animals , Apoptosis/drug effects , Blotting, Western , Cell Movement/drug effects , Cell Proliferation/drug effects , Drug Therapy, Combination , Gallbladder Neoplasms/pathology , Guanine/pharmacology , Humans , Immunoenzyme Techniques , Mice , Mice, Inbred NOD , Mice, SCID , Phosphorylation/drug effects , Signal Transduction/drug effects , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Several Phase I/II clinical trials aiming at the correction of X-linked CGD by gene transfer into hematopoietic stem cells (HSCs) have demonstrated the therapeutic potential of gene modified autologous HSCs for the treatment of CGD. Resolution of therapy-resistant bacterial and fungal infections in liver, lung and spinal canal of CGD patients were clearly documented in all trials. However, clinical benefits were not sustained over time due to the failure of gene transduced cells to engraft long-term. Moreover, severe adverse effects were observed in some of the treated patients due to insertional mutagenesis leading to the activation of growth promoting genes and to myeloid malignancy. These setbacks fostered the development of novel safety and efficacy improved vectors that have already entered or are about to enter the clinics. Meanwhile, ongoing research is constantly refining the CGD disease phenotype, including the definition of factors that may explain the unique engraftment phenotype observed in CGD gene therapy trials. This review provides a condensed overview on the current knowledge of the molecular pathomechanisms and clinical manifestations of CGD and summarizes the lessons learned from clinical gene therapy trials, the preclinical progress in vector design and the future perspectives for the gene therapy of CGD.