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Cytokine-induced killer cells (CIK) in combination with dendritic cells (DCs) have shown favorable outcomes in renal cell carcinoma (RCC), yet some patients exhibit recurrence or no response to this therapy. In a broader perspective, enhancing the antitumor response of DC-CIK cells may help to address this issue. Considering this, herein, we investigated the effect of anti-CD40 and anti-CTLA-4 antibodies on the antitumor response of DC-CIK cells against RCC cell lines. Our analysis showed that, a) anti-CD40 antibody (G28.5) increased the CD3+CD56+ effector cells of CIK cells by promoting the maturation and activation of DCs, b) G28.5 also increased CTLA-4 expression in CIK cells via DCs, but the increase could be hindered by the CTLA-4 inhibitor (ipilimumab), c) adding ipilimumab was also able to significantly increase the proportion of CD3+CD56+ cells in DC-CIK cells, d) anti-CD40 antibodies predominated over anti-CTLA-4 antibodies for cytotoxicity, apoptotic effect and IFN-g secretion of DC-CIK cells against RCC cells, e) after ipilimumab treatment, the population of Tregs in CIK cells remained unaffected, but ipilimumab combined with G28.5 significantly reduced the expression of CD28 in CIK cells. Taken together, we suggest that the agonistic anti-CD40 antibody rather than CTLA-4 inhibitor may improve the antitumor response of DC-CIK cells, particularly in RCC. In addition, we pointed towards the yet to be known contribution of CD28 in the crosstalk between anti-CTLA-4 and CIK cells.
Cancer is a complex disease where resistance to therapies and relapses often pose a serious clinical challenge. The scenario is even more complicated when the cancer type itself is heterogeneous in nature, e.g., lymphoma, a cancer of the lymphocytes which constitutes more than 70 different subtypes. Indeed, the treatment options continue to expand in lymphomas. Herein, we provide insights into lymphoma-specific clinical trials based on cytokine-induced killer (CIK) cell therapy and other pre-clinical lymphoma models where CIK cells have been used along with other synergetic tumor-targeting immune modules to improve their therapeutic potential. From a broader perspective, we will highlight that CIK cell therapy has potential, and in this rapidly evolving landscape of cancer therapies its optimization (as a personalized therapeutic approach) will be beneficial in lymphomas.
When the Artemis missions launch, NASA's Orion spacecraft (and crew as of the Artemis II mission) will be exposed to the deep space radiation environment beyond the protection of Earth's magnetosphere. Hence, it is essential to characterize the effects of space radiation, microgravity, and the combination thereof on cells and organisms, i.e., to quantify any correlations between the deep space radiation environment, genetic variation, and induced genetic changes in cells. To address this, the Artemis I mission will include the Peristaltic Laboratory for Automated Science with Multigenerations (PLASM) hardware containing the Deep Space Radiation Genomics (DSRG) experiment. The scientific aims of DSRG are (i) to identify the metabolic and genomic pathways in yeast affected by microgravity, space radiation, and their combination, and (ii) to differentiate between gravity and radiation exposure on single-gene deletion/overexpressing strains' ability to thrive in the spaceflight environment. Yeast is used as a model system because 70% of its essential genes have a human homolog, and over half of these homologs can functionally replace their human counterpart. As part of the experiment preparation towards spaceflight, an Experiment Verification Test (EVT) was performed at the Kennedy Space Center to verify that the experiment design, hardware, and approach to automated operations will enable achieving the scientific aims. For the EVT, fluidic systems were assembled, sterilized, loaded, and acceptance-tested, and subsequently integrated with the engineering parts to produce a flight-like PLASM unit. Each fluidic system consisted of (i) a Media Bag, (ii) four Culture Bags loaded with Saccharomyces cerevisiae (two with deletion series and the remaining two with overexpression series), and (iii) tubing and check valves. The EVT PLASM unit was put under a temperature profile replicating the anticipated different phases of flight, including handover to launch, spaceflight, and splashdown to handover back to the science team, for a 58-day period. At EVT completion, the rate of activation, cellular growth, RNA integrity, and sample contamination were interrogated. All of the experiment's success criteria were satisfied, encouraging our efforts to perform this investigation on Artemis I. This manuscript thus describes the process of spaceflight experiment design maturation with a focus on the EVT, its results, DSRG's preparation for its planned launch on Artemis I in 2022, and how the PLASM hardware can enable other scientific goals on future Artemis missions and/or the Lunar Orbital Platform – Gateway.
Extremophiles are optimal models in experimentally addressing questions about the effects of cosmic radiation on biological systems. The resistance to high charge energy (HZE) particles, and helium (He) ions and iron (Fe) ions (LET at 2.2 and 200 keV/µm, respectively, until 1000 Gy), of spores from two thermophiles, Bacillushorneckiae SBP3 and Bacilluslicheniformis T14, and two psychrotolerants, Bacillus sp. A34 and A43, was investigated. Spores survived He irradiation better, whereas they were more sensitive to Fe irradiation (until 500 Gy), with spores from thermophiles being more resistant to irradiations than psychrotolerants. The survived spores showed different germination kinetics, depending on the type/dose of irradiation and the germinant used. After exposure to He 1000 Gy, D-glucose increased the lag time of thermophilic spores and induced germination of psychrotolerants, whereas L-alanine and L-valine increased the germination efficiency, except alanine for A43. FTIR spectra showed important modifications to the structural components of spores after Fe irradiation at 250 Gy, which could explain the block in spore germination, whereas minor changes were observed after He radiation that could be related to the increased permeability of the inner membranes and alterations of receptor complex structures. Our results give new insights on HZE resistance of extremophiles that are useful in different contexts, including astrobiology.
Cytokine-induced killer (CIK) cells are an ex vivo expanded heterogeneous cell population with an enriched NK-T phenotype (CD3+CD56+). Due to the convenient and relatively inexpensive expansion capability, together with low incidence of graft versus host disease (GVHD) in allogeneic cancer patients, CIK cells are a promising candidate for immunotherapy. It is well known that natural killer group 2D (NKG2D) plays an important role in CIK cell-mediated antitumor activity; however, it remains unclear whether its engagement alone is sufficient or if it requires additional co-stimulatory signals to activate the CIK cells. Likewise, the role of 2B4 has not yet been identified in CIK cells. Herein, we investigated the individual and cumulative contribution of NKG2D and 2B4 in the activation of CIK cells. Our analysis suggests that (a) NKG2D (not 2B4) is implicated in CIK cell (especially CD3+CD56+ subset)-mediated cytotoxicity, IFN-γ secretion, E/T conjugate formation, and degranulation; (b) NKG2D alone is adequate enough to induce degranulation, IFN-γ secretion, and LFA-1 activation in CIK cells, while 2B4 only provides limited synergy with NKG2D (e.g., in LFA-1 activation); and (c) NKG2D was unable to costimulate CD3. Collectively, we conclude that NKG2D engagement alone suffices to activate CIK cells, thereby strengthening the idea that targeting the NKG2D axis is a promising approach to improve CIK cell therapy for cancer patients. Furthermore, CIK cells exhibit similarities to classical invariant natural killer (iNKT) cells with deficiencies in 2B4 stimulation and in the costimulation of CD3 with NKG2D. In addition, based on the current data, the divergence in receptor function between CIK cells and NK (or T) cells can be assumed, pointing to the possibility that molecular modifications (e.g., using chimeric antigen receptor technology) on CIK cells may need to be customized and optimized to maximize their functional potential.
Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion.
Intimate swabs taken for examination in sexual assault cases typically yield mixtures of sperm and epithelial cell types. While powerful, differential extraction protocols to overcome such cell type mixtures by separate lysis of epithelial cells and spermatozoa can still prove ineffective, in particular if only few sperm cells are present or if swabs contain sperm from more than one individual leading to complex low level DNA mixtures. A means to avoid such mixtures consists in the analysis of single micromanipulated sperm cells. However, the quantity of DNA from single sperm cells is not sufficient for conventional STR analysis. Here, we describe a simple method for micromanipulating individual sperm cells from intimate swabs and show that whole genome amplification can generate sufficient amounts of DNA from single cells for subsequent DNA profiling. We recovered over 80% of alleles of haploid autosomal STR profiles from the majority of individual sperm cells. Furthermore, we demonstrate that in mixtures of sperm from two contributors, Y-STR and X-STR profiles of individual sperm cells can be used to sort the haploid autosomal profiles to develop the diploid consensus STR profiles of the individual donors. Finally, by analysing single sperm cells from mock sexual assault swabs with one or two sperm donors, we showed that our protocols enabled the identification of the unknown male contributors.
The French–Italian Concordia Research Station, situated on the Antarctic Polar Plateau at an elevation of 3233 m above sea level, offers a unique opportunity to study the presence and variation of microbes introduced by abiotic or biotic vectors and, consequently, appraise the amplitude of human impact in such a pristine environment. This research built upon a previous work, which explored microbial diversity in the surface snow surrounding the Concordia Research Station. While that study successfully characterized the bacterial assemblage, detecting fungal diversity was hampered by the low DNA content. To address this knowledge gap, in the present study, we optimized the sampling by increasing ice/snow collected to leverage the final DNA yield. The V4 variable region of the 16S rDNA and Internal Transcribed Spacer (ITS1) rDNA was used to evaluate bacterial and fungal diversity. From the sequencing, we obtained 3,352,661 and 4,433,595 reads clustered in 930 and 3182 amplicon sequence variants (ASVs) for fungi and bacteria, respectively. Amplicon sequencing revealed a predominance of Basidiomycota (49%) and Ascomycota (42%) in the fungal component; Bacteroidota (65.8%) is the main representative among the bacterial phyla. Basidiomycetes are almost exclusively represented by yeast-like fungi. Our findings provide the first comprehensive overview of both fungal and bacterial diversity in the Antarctic Polar Plateau’s surface snow/ice near Concordia Station and to identify seasonality as the main driver of microbial diversity; we also detected the most sensitive microorganisms to these factors, which could serve as indicators of human impact in this pristine environment and aid in planetary protection for future exploration missions.
Multiple myeloma is the second most common hematological malignancy. Despite all the progress made in treating multiple myeloma, it still remains an incurable disease. Patients are left with a median survival of 4-5 years. The combined treatment of multiple myeloma with histone deacetylase inhibitors and cytokine-induced killer cells provides a promising targeted treatment option for patients. This study investigated the impact of a combined treatment compared to treatment with histone deacetylase inhibitors. The experiments revealed that a treatment with histone deacetylase (HDAC) inhibitors could reduce cell viability to 59% for KMS 18 cell line and 46% for the U-266 cell line. The combined treatment led to a decrease of cell viability to 33% for KMS 18 and 27% for the U-266 cell line, thus showing a significantly better efficacy than the single treatment.
Introduction: Matrix metalloproteinases (MMPs) are important in tissue remodelling. Here we investigate the role of collagenase-3 (MMP-13) in antibody-induced arthritis.
Methods: For this study we employed the K/BxN serum-induced arthritis model. Arthritis was induced in C57BL/6 wild type (WT) and MMP-13-deficient (MMP-13–/–) mice by intraperitoneal injection of 200 μl of K/BxN serum. Arthritis was assessed by measuring the ankle swelling. During the course of the experiments, mice were sacrificed every second day for histological examination of the ankle joints. Ankle sections were evaluated histologically for infiltration of inflammatory cells, pannus tissue formation and bone/cartilage destruction. Semi-quantitative PCR was used to determine MMP-13 expression levels in ankle joints of untreated and K/BxN serum-injected mice.
Results: This study shows that MMP-13 is a regulator of inflammation. We observed increased expression of MMP-13 in ankle joints of WT mice during K/BxN serum-induced arthritis and both K/BxN serum-treated WT and MMP-13–/– mice developed progressive arthritis with a similar onset. However, MMP-13–/– mice showed significantly reduced disease over the whole arthritic period. Ankle joints of WT mice showed severe joint destruction with extensive inflammation and erosion of cartilage and bone. In contrast, MMP-13–/– mice displayed significantly decreased severity of arthritis (50% to 60%) as analyzed by clinical and histological scoring methods.
Conclusions: MMP-13 deficiency acts to suppress the local inflammatory responses. Therefore, MMP-13 has a role in the pathogenesis of arthritis, suggesting MMP-13 is a potential therapeutic target.
The ability to discriminate between different ionic species, termed ion selectivity, is a key feature of ion channels and forms the basis for their physiological function. Members of the degenerin/epithelial sodium channel (DEG/ENaC) superfamily of trimeric ion channels are typically sodium selective, but to a surprisingly variable degree. While acid-sensing ion channels (ASICs) are weakly sodium selective (sodium:potassium ratio ∼10:1), ENaCs show a remarkably high preference for sodium over potassium (>500:1). This discrepancy may be expected to originate from differences in the pore-lining second transmembrane segment (M2). However, these show a relatively high degree of sequence conservation between ASICs and ENaCs, and previous functional and structural studies could not unequivocally establish that differences in M2 alone can account for the disparate degrees of ion selectivity. By contrast, surprisingly little is known about the contributions of the first transmembrane segment (M1) and the preceding pre-M1 region. In this study, we used conventional and noncanonical amino acid-based mutagenesis in combination with a variety of electrophysiological approaches to show that the pre-M1 and M1 regions of mASIC1a channels are major determinants of ion selectivity. Mutational investigations of the corresponding regions in hENaC show that these regions contribute less to ion selectivity, despite affecting ion conductance. In conclusion, our work suggests that the remarkably different degrees of sodium selectivity in ASICs and ENaCs are achieved through different mechanisms. These results further highlight how M1 and pre-M1 are likely to differentially affect pore structure in these related channels.
Fabry disease (FD) is an X‐linked lysosomal storage disorder. Deficiency of the lysosomal enzyme alpha‐galactosidase (GLA) leads to accumulation of potentially toxic globotriaosylceramide (Gb3) on a multisystem level. Cardiac and cerebrovascular abnormalities as well as progressive renal failure are severe, life‐threatening long‐term complications. The complete pathophysiology of chronic kidney disease (CKD) in FD and the role of tubular involvement for its progression are unclear.
We established human renal tubular epithelial cell lines from the urine of male FD patients and male controls. The renal tubular system is rich in mitochondria and involved in transport processes at high energy costs. Our studies revealed fragmented mitochondria with disrupted cristae structure in FD patient cells. Oxidative stress levels were elevated and oxidative phosphorylation was up‐regulated in FD pointing at enhanced energetic needs. Mitochondrial homeostasis and energy metabolism revealed major changes as evidenced by differences in mitochondrial number, energy production and fuel consumption. The changes were accompanied by activation of the autophagy machinery in FD. Sirtuin1, an important sensor of (renal) metabolic stress and modifier of different defense pathways, was highly expressed in FD.
Our data show that lysosomal FD impairs mitochondrial function and results in severe disturbance of mitochondrial energy metabolism in renal cells. This insight on a tissue‐specific level points to new therapeutic targets which might enhance treatment efficacy.
Major progress occurred in understanding inborn errors of ketone body transport and metabolism between the International Congresses on Inborn Errors of Metabolism in Barcelona (2013) and Rio de Janeiro (2017). These conditions impair either ketogenesis (presenting as episodes of hypoketotic hypoglycemia) or ketolysis (presenting as ketoacidotic episodes); for both groups, immediate intravenous glucose administration is the most critical and (mHGGCS, HMGCS2) effective treatment measure.
3-Hydroxy-3-methylglutaryl-coenzyme A lyase (HMGCL, HMGCL) deficiency is a rare inborn error of ketogenesis. Even if the ketogenic enzyme is fully disrupted, an elevated signal for the ketone body acetoacetic acid is a frequent observation in the analysis of urinary organic acids, at least if derivatization is performed by methylation. We provide an explanation for this phenomenon and trace it back to degradation of the derivatized 3-hydroxy-3-methylglutaric acid and high temperature of the injector of the gas chromatograph.
We examined the cytotoxic effects of chaetocin on clear cell renal cell carcinoma (ccRCC) cells and the possibility to combine the effects of chaetocin with the effects of cytokine-induced killer cells (CIK) assayed by MTT assay and FACS analysis. Chaetocin is a thiodioxopiperazine produced by fungi belonging to the chaetomiaceae family. In 2007, it was first reported that chaetocin shows potent and selective ex vivo anti-cancer activity by inducing reactive oxygen species. CIK cells are generated from CD3+/CD56- T lymphocytes with double negative CD4-/CD8- phenotype that are isolated from human blood. The addition of distinct interleukins and antibodies results in the generation of CIK cells that are able to specifically target and destroy renal carcinoma cells. The results of this research state that the anti-ccRCC activity of chaetocin is weak and does not show a high grade of selectivity on clear cell renal cell carcinoma cells. Although the CIK cells show a high grade of selective anti-ccRCC activity, this effect could not be improved by the addition of chaetocin. So chaetocin seems to be no suitable agent for specific targeting ccRCC cells or for the combination therapy with CIK cells in renal cancer.
A main factor hampering life in space is represented by high atomic number nuclei and energy (HZE) ions that constitute about 1% of the galactic cosmic rays. In the frame of the “STARLIFE” project, we accessed the Heavy Ion Medical Accelerator (HIMAC) facility of the National Institute of Radiological Sciences (NIRS) in Chiba, Japan. By means of this facility, the extremophilic species Haloterrigena hispanica and Parageobacillus thermantarcticus were irradiated with high LET ions (i.e., Fe, Ar, and He ions) at doses corresponding to long permanence in the space environment. The survivability of HZE-treated cells depended upon either the storage time and the hydration state during irradiation; indeed, dry samples were shown to be more resistant than hydrated ones. With particular regard to spores of the species P. thermantarcticus, they were the most resistant to irradiation in a water medium: an analysis of the changes in their biochemical fingerprinting during irradiation showed that, below the survivability threshold, the spores undergo to a germination-like process, while for higher doses, inactivation takes place as a consequence of the concomitant release of the core’s content and a loss of integrity of the main cellular components. Overall, the results reported here suggest that the selected extremophilic microorganisms could serve as biological model for space simulation and/or real space condition exposure, since they showed good resistance to ionizing radiation exposure and were able to resume cellular growth after long-term storage.
Background: Migration of mature and immature leukocytes in response to chemokines is not only essential during inflammation and host defense, but also during development of the hematopoietic system. Many molecules implicated in migratory polarity show uniform cellular distribution under non-activated conditions, but acquire a polarized localization upon exposure to migratory cues.
Methodology/Principal Findings: Here, we present evidence that raft-associated endocytic proteins (flotillins) are preassembled in lymphoid, myeloid and primitive hematopoietic cells and accumulate in the uropod during migration. Furthermore, flotillins display a polarized distribution during immunological synapse formation. Employing the membrane lipid-order sensitive probe Laurdan, we show that flotillin accumulation in the immunological synapse is concomittant with membrane ordering in these regions.
Conclusions: Together with the observation that flotillin polarization does not occur in other polarized cell types such as polarized epithelial cells, our results suggest a specific role for flotillins in hematopoietic cell polarization. Based on our results, we propose that in hematopoietic cells, flotillins provide intrinsic cues that govern segregation of certain microdomain-associated molecules during immune cell polarization.
Matrix metalloproteinases (MMPs) are matrix-degrading enzymes that are over-expressed in joints of rheumatoid arthritis (RA) patients. However, the contribution of specific MMPs for the development of arthritic joints is unknown. This study is aimed at studying the role of matrix metalloproteinase-9 (MMP-9) in mice, using the K/BxN serum-transfer model of RA. Arthritis was induced in Balb/c mice by injecting K/BxN serum. Development of arthritis was followed in these mice by measuring ankle thickness and clinical index score. MMP-9 expression in the joints of mice killed at various time points during the disease progression was determined by gelatin zymography using ankle lysates. We found that MMP-9 expression increased with the severity of arthritis. Importantly MMP-9 deficient mice injected with K/BxN serum showed a milder form of arthritis in comparison to the control C57BL/6 mice injected with K/BxN serum. We therefore conclude that MMP-9 promotes arthritis in mice.
Forensic DNA profiles are established by multiplex PCR amplification of a set of highly variable short tandem repeat (STR) loci followed by capillary electrophoresis (CE) as a means to assign alleles to PCR products of differential length. Recently, CE analysis of STR amplicons has been supplemented by high-throughput next generation sequencing (NGS) techniques that are able to detect isoalleles bearing sequence polymorphisms and allow for an improved analysis of degraded DNA. Several such assays have been commercialised and validated for forensic applications. However, these systems are cost-effective only when applied to high numbers of samples. We report here an alternative, cost-efficient shallow-sequence output NGS assay called maSTR assay that, in conjunction with a dedicated bioinformatics pipeline called SNiPSTR, can be implemented with standard NGS instrumentation. In a back-to-back comparison with a CE-based, commercial forensic STR kit, we find that for samples with low DNA content, with mixed DNA from different individuals, or containing PCR inhibitors, the maSTR assay performs equally well, and with degraded DNA is superior to CE-based analysis. Thus, the maSTR assay is a simple, robust and cost-efficient NGS-based STR typing method applicable for human identification in forensic and biomedical contexts.
The automated annotation of data from high throughput sequencing and genomics experiments is a significant challenge for bioinformatics. Most current approaches rely on sequential pipelines of gene finding and gene function prediction methods that annotate a gene with information from different reference data sources. Each function prediction method contributes evidence supporting a functional assignment. Such approaches generally ignore the links between the information in the reference datasets. These links, however, are valuable for assessing the plausibility of a function assignment and can be used to evaluate the confidence in a prediction. We are working towards a novel annotation system that uses the network of information supporting the function assignment to enrich the annotation process for use by expert curators and predicting the function of previously unannotated genes. In this paper we describe our success in the first stages of this development. We present the data integration steps that are needed to create the core database of integrated reference databases (UniProt, PFAM, PDB, GO and the pathway database Ara-Cyc) which has been established in the ONDEX data integration system. We also present a comparison between different methods for integration of GO terms as part of the function assignment pipeline and discuss the consequences of this analysis for improving the accuracy of gene function annotation. The methods and algorithms presented in this publication are an integral part of the ONDEX system which is freely available from http://ondex.sf.net/.