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1.
Genome Med ; 16(1): 85, 2024 07 02.
Article in English | MEDLINE | ID: mdl-38956711

ABSTRACT

BACKGROUND: Restraining or slowing ageing hallmarks at the cellular level have been proposed as a route to increased organismal lifespan and healthspan. Consequently, there is great interest in anti-ageing drug discovery. However, this currently requires laborious and lengthy longevity analysis. Here, we present a novel screening readout for the expedited discovery of compounds that restrain ageing of cell populations in vitro and enable extension of in vivo lifespan. METHODS: Using Illumina methylation arrays, we monitored DNA methylation changes accompanying long-term passaging of adult primary human cells in culture. This enabled us to develop, test, and validate the CellPopAge Clock, an epigenetic clock with underlying algorithm, unique among existing epigenetic clocks for its design to detect anti-ageing compounds in vitro. Additionally, we measured markers of senescence and performed longevity experiments in vivo in Drosophila, to further validate our approach to discover novel anti-ageing compounds. Finally, we bench mark our epigenetic clock with other available epigenetic clocks to consolidate its usefulness and specialisation for primary cells in culture. RESULTS: We developed a novel epigenetic clock, the CellPopAge Clock, to accurately monitor the age of a population of adult human primary cells. We find that the CellPopAge Clock can detect decelerated passage-based ageing of human primary cells treated with rapamycin or trametinib, well-established longevity drugs. We then utilise the CellPopAge Clock as a screening tool for the identification of compounds which decelerate ageing of cell populations, uncovering novel anti-ageing drugs, torin2 and dactolisib (BEZ-235). We demonstrate that delayed epigenetic ageing in human primary cells treated with anti-ageing compounds is accompanied by a reduction in senescence and ageing biomarkers. Finally, we extend our screening platform in vivo by taking advantage of a specially formulated holidic medium for increased drug bioavailability in Drosophila. We show that the novel anti-ageing drugs, torin2 and dactolisib (BEZ-235), increase longevity in vivo. CONCLUSIONS: Our method expands the scope of CpG methylation profiling to accurately and rapidly detecting anti-ageing potential of drugs using human cells in vitro, and in vivo, providing a novel accelerated discovery platform to test sought after anti-ageing compounds and geroprotectors.


Subject(s)
Aging , DNA Methylation , Longevity , Humans , Animals , DNA Methylation/drug effects , Longevity/drug effects , Aging/drug effects , Epigenesis, Genetic/drug effects , Drug Discovery/methods , Cellular Senescence/drug effects , Drug Evaluation, Preclinical/methods , Drosophila , Cells, Cultured , Sirolimus/pharmacology
2.
J Pathol ; 262(1): 90-104, 2024 01.
Article in English | MEDLINE | ID: mdl-37929635

ABSTRACT

Crosstalk between cancer and stellate cells is pivotal in pancreatic cancer, resulting in differentiation of stellate cells into myofibroblasts that drives tumour progression. To assess cooperative mechanisms in a 3D context, we generated chimeric spheroids using human and mouse cancer and stellate cells. Species-specific deconvolution of bulk-RNA sequencing data revealed cell type-specific transcriptomes underpinning invasion. This dataset highlighted stellate-specific expression of transcripts encoding the collagen-processing enzymes ADAMTS2 and ADAMTS14. Strikingly, loss of ADAMTS2 reduced, while loss of ADAMTS14 promoted, myofibroblast differentiation and invasion independently of their primary role in collagen-processing. Functional and proteomic analysis demonstrated that these two enzymes regulate myofibroblast differentiation through opposing roles in the regulation of transforming growth factor ß availability, acting on the protease-specific substrates, Serpin E2 and fibulin 2, for ADAMTS2 and ADAMTS14, respectively. Showcasing a broader complexity for these enzymes, we uncovered a novel regulatory axis governing malignant behaviour of the pancreatic cancer stroma. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.


Subject(s)
Myofibroblasts , Pancreatic Neoplasms , Animals , Humans , Mice , ADAMTS Proteins/genetics , ADAMTS Proteins/metabolism , Cell Differentiation , Collagen/metabolism , Myofibroblasts/metabolism , Pancreatic Neoplasms/pathology , Proteomics
3.
Biomolecules ; 12(6)2022 06 16.
Article in English | MEDLINE | ID: mdl-35740962

ABSTRACT

The extracellular matrix (ECM) is a complex mixture of structural proteins, proteoglycans, and signaling molecules that are essential for tissue integrity and homeostasis. While a number of recent studies have explored the use of decellularized ECM (dECM) as a biomaterial for tissue engineering, the complete composition, structure, and mechanics of these materials remain incompletely understood. In this study, we performed an in-depth characterization of skin-derived dECM biomaterials for human skin equivalent (HSE) models. The dECM materials were purified from porcine skin, and through mass spectrometry profiling, we quantified the presence of major ECM molecules, including types I, III, and VI collagen, fibrillin, and lumican. Rheological analysis demonstrated the sol-gel and shear-thinning properties of dECM materials, indicating their physical suitability as a tissue scaffold, while electron microscopy revealed a complex, hierarchical structure of nanofibers in dECM hydrogels. The dECM materials were compatible with advanced biofabrication techniques, including 3D printing within a gelatin microparticle support bath, printing with a sacrificial material, or blending with other ECM molecules to achieve more complex compositions and structures. As a proof of concept, we also demonstrate how dECM materials can be fabricated into a 3D skin wound healing model using 3D printing. Skin-derived dECM therefore represents a complex and versatile biomaterial with advantageous properties for the fabrication of next-generation HSEs.


Subject(s)
Decellularized Extracellular Matrix , Tissue Engineering , Animals , Biocompatible Materials/chemistry , Extracellular Matrix/metabolism , Humans , Swine , Tissue Engineering/methods , Tissue Scaffolds/chemistry , Wound Healing
4.
Aging (Albany NY) ; 14(10): 4220-4246, 2022 05 16.
Article in English | MEDLINE | ID: mdl-35580013

ABSTRACT

Senescence occurs in response to a number of damaging stimuli to limit oncogenic transformation and cancer development. As no single, universal senescence marker has been discovered, the confident classification of senescence induction requires the parallel assessment of a series of hallmarks. Therefore, there is a growing need for "first-pass" tools of senescence identification to streamline experimental workflows and complement conventional markers. Here, we utilise a high content, multidimensional phenotypic profiling-based approach, to assess the morphological profiles of senescent cells induced via a range of stimuli. In the context of senescence, we refer to these as senescence-associated morphological profiles (SAMPs), as they facilitate distinction between senescent and proliferating cells. The complexity of the profiles generated also allows exploration of the heterogeneity both between models of senescence and within an individual senescence model, providing a level of insight at the single cell level. Furthermore, we also demonstrate that these models are applicable to the assessment of senescence in vivo, which remains a key challenge for the field. Therefore, we believe SAMPs has the potential to serve as a useful addition in the repertoire of senescence researchers, either as a first-pass tool or as part of the established senescence hallmarks.


Subject(s)
Cellular Senescence , Neoplasms , Biomarkers , Carcinogenesis , Humans , Neoplasms/genetics , Oncogenes
5.
Cell Rep ; 38(4): 110227, 2022 01 25.
Article in English | MEDLINE | ID: mdl-35081338

ABSTRACT

In pancreatic ductal adenocarcinoma (PDAC), differentiation of pancreatic stellate cells (PSCs) into myofibroblast-like cancer-associated fibroblasts (CAFs) can both promote and suppress tumor progression. Here, we show that the Rho effector protein kinase N2 (PKN2) is critical for PSC myofibroblast differentiation. Loss of PKN2 is associated with reduced PSC proliferation, contractility, and alpha-smooth muscle actin (α-SMA) stress fibers. In spheroid co-cultures with PDAC cells, loss of PKN2 prevents PSC invasion but, counter-intuitively, promotes invasive cancer cell outgrowth. PKN2 deletion induces a myofibroblast to inflammatory CAF switch in the PSC matrisome signature both in vitro and in vivo. Further, deletion of PKN2 in the pancreatic stroma induces more locally invasive, orthotopic pancreatic tumors. Finally, we demonstrate that a PKN2KO matrisome signature predicts poor outcome in pancreatic and other solid human cancers. Our data indicate that suppressing PSC myofibroblast function can limit important stromal tumor-suppressive mechanisms, while promoting a switch to a cancer-supporting CAF phenotype.


Subject(s)
Neoplasm Invasiveness/pathology , Pancreatic Neoplasms/pathology , Pancreatic Stellate Cells/pathology , Animals , Humans , Mice , Pancreatic Stellate Cells/metabolism , Phenotype , Protein Kinase C/metabolism , Tumor Microenvironment/physiology
6.
iScience ; 24(6): 102676, 2021 Jun 25.
Article in English | MEDLINE | ID: mdl-34189439

ABSTRACT

Guided by a multi-level "deconstruction" of omental metastases, we developed a tetra (four cell)-culture model of primary human mesothelial cells, fibroblasts, adipocytes, and high-grade serous ovarian cancer (HGSOC) cell lines. This multi-cellular model replicated key elements of human metastases and allowed malignant cell invasion into the artificial omental structure. Prompted by findings in patient biopsies, we used the model to investigate the role of platelets in malignant cell invasion and extracellular matrix, ECM, production. RNA (sequencing and quantitative polymerase-chain reaction), protein (proteomics and immunohistochemistry) and image analysis revealed that platelets stimulated malignant cell invasion and production of ECM molecules associated with poor prognosis. Moreover, we found that platelet activation of mesothelial cells was critical in stimulating malignant cell invasion. Whilst platelets likely activate both malignant cells and mesothelial cells, the tetra-culture model allowed us to dissect the role of both cell types and model the early stages of HGSOC metastases.

7.
J Extracell Vesicles ; 10(4): e12041, 2021 02.
Article in English | MEDLINE | ID: mdl-33659050

ABSTRACT

A hallmark of senescence is the acquisition of an enhanced secretome comprising inflammatory mediators and tissue remodelling agents - the senescence-associated secretory phenotype (SASP). Through the SASP, senescent cells are hypothesised to contribute to both ageing and pathologies associated with age. Whilst soluble factors have been the most widely investigated components of the SASP, there is growing evidence that small extracellular vesicles (EVs) comprise functionally important constituents. Thus, dissecting the contribution of the soluble SASP from the vesicular component is crucial to elucidating the functional significance of senescent cell derived EVs. Here, we take advantage of a systematic proteomics based approach to determine that soluble SASP factors co-isolate with EVs following differential ultracentrifugation (dUC). We present size-exclusion chromatography (SEC) as a method for separation of the soluble and vesicular components of the senescent secretome and thus EV purification. Furthermore, we demonstrate that SEC EVs isolated from senescent cells contribute to non-cell autonomous paracrine senescence. Therefore, this work emphasises the requirement for methodological rigor due to the propensity of SASP components to co-isolate during dUC and provides a framework for future investigations of the vesicular component of the SASP.


Subject(s)
Aging/metabolism , Cellular Senescence , Extracellular Vesicles/metabolism , Secretome/metabolism , Senescence-Associated Secretory Phenotype , Cell Line, Tumor , Cells, Cultured , Chromatography, Gel , Exosomes/chemistry , Exosomes/metabolism , Extracellular Vesicles/chemistry , Humans , Phenotype , Proteins/analysis , Proteomics/methods
8.
Aging Cell ; 20(3): e13318, 2021 03.
Article in English | MEDLINE | ID: mdl-33547862

ABSTRACT

Senescence, a state of stable growth arrest, plays an important role in ageing and age-related diseases in vivo. Although the INK4/ARF locus is known to be essential for senescence programmes, the key regulators driving p16 and ARF transcription remain largely underexplored. Using siRNA screening for modulators of the p16/pRB and ARF/p53/p21 pathways in deeply senescent human mammary epithelial cells (DS HMECs) and fibroblasts (DS HMFs), we identified EGR2 as a novel regulator of senescence. EGR2 expression is up-regulated during senescence, and its ablation by siRNA in DS HMECs and HMFs transiently reverses the senescent phenotype. We demonstrate that EGR2 activates the ARF and p16 promoters and directly binds to both the ARF and p16 promoters. Loss of EGR2 down-regulates p16 levels and increases the pool of p16- p21- 'reversed' cells in the population. Moreover, EGR2 overexpression is sufficient to induce senescence. Our data suggest that EGR2 is a direct transcriptional activator of the p16/pRB and ARF/p53/p21 pathways in senescence and a novel marker of senescence.


Subject(s)
Cellular Senescence , Early Growth Response Protein 2/metabolism , Adolescent , Adult , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Knockdown Techniques , Humans , Mammary Glands, Human/cytology , Protein Binding , RNA, Small Interfering/metabolism , Retinoblastoma Protein/metabolism , Tumor Suppressor Protein p53/metabolism , Up-Regulation , Young Adult
9.
Mech Ageing Dev ; 190: 111261, 2020 09.
Article in English | MEDLINE | ID: mdl-32461142

ABSTRACT

The recent advent of 'organs in a dish' has revolutionised the research landscape. These 3D culture systems have paved the way for translational, post genomics research by enabling scientists to model diseases in the laboratory, grow patient-derived organoids, and unite this technology with other cutting-edge methodologies such as drug discovery. Fields such as dermatology and neuroscience have revolutionised the development of robust 3D models, which faithfully recapitulate native physiology in vivo to provide important functional and mechanistic insights. These models have underpinned a rapid growth in the number of organs and myriad of human diseases that can be modelled in 3D, which currently includes breast, cerebral cortex, heart, intestine, kidney, liver, lung, neural tube, pancreas, prostate, skin and stomach, as well as patient derived tumours. However, so far, they have not yet been employed extensively in the study of fundamental cellular programmes such as senescence. Thus, tissue engineering and 3D culture offer an exciting opportunity to further understand the bright and dark sides of senescence in a more complex and physiologically relevant environment. Below, we will discuss previous approaches to investigating senescence and ageing using organotypic models, and some potential opportunities for future research.


Subject(s)
Cellular Senescence/physiology , Models, Biological , Organoids , Tissue Engineering/methods , Biomedical Research/methods , Biomedical Research/trends , Biomedical Technology/methods , Biomedical Technology/trends , Humans , Organ Culture Techniques/methods , Organoids/physiology , Organoids/physiopathology
10.
Genome Biol ; 21(1): 91, 2020 04 07.
Article in English | MEDLINE | ID: mdl-32264951

ABSTRACT

BACKGROUND: Cellular senescence, a permanent state of replicative arrest in otherwise proliferating cells, is a hallmark of aging and has been linked to aging-related diseases. Many genes play a role in cellular senescence, yet a comprehensive understanding of its pathways is still lacking. RESULTS: We develop CellAge (http://genomics.senescence.info/cells), a manually curated database of 279 human genes driving cellular senescence, and perform various integrative analyses. Genes inducing cellular senescence tend to be overexpressed with age in human tissues and are significantly overrepresented in anti-longevity and tumor-suppressor genes, while genes inhibiting cellular senescence overlap with pro-longevity and oncogenes. Furthermore, cellular senescence genes are strongly conserved in mammals but not in invertebrates. We also build cellular senescence protein-protein interaction and co-expression networks. Clusters in the networks are enriched for cell cycle and immunological processes. Network topological parameters also reveal novel potential cellular senescence regulators. Using siRNAs, we observe that all 26 candidates tested induce at least one marker of senescence with 13 genes (C9orf40, CDC25A, CDCA4, CKAP2, GTF3C4, HAUS4, IMMT, MCM7, MTHFD2, MYBL2, NEK2, NIPA2, and TCEB3) decreasing cell number, activating p16/p21, and undergoing morphological changes that resemble cellular senescence. CONCLUSIONS: Overall, our work provides a benchmark resource for researchers to study cellular senescence, and our systems biology analyses reveal new insights and gene regulators of cellular senescence.


Subject(s)
Aging/genetics , Cellular Senescence/genetics , Databases, Genetic , Animals , Disease/genetics , Evolution, Molecular , Gene Expression , Genes, Neoplasm , Humans , Longevity/genetics , Oligonucleotide Array Sequence Analysis , Organ Specificity , Protein Interaction Mapping , RNA-Seq , Systems Biology
12.
Curr Biol ; 28(16): 2544-2556.e5, 2018 08 20.
Article in English | MEDLINE | ID: mdl-30100339

ABSTRACT

Aging (senescence) is characterized by the development of numerous pathologies, some of which limit lifespan. Key to understanding aging is discovery of the mechanisms (etiologies) that cause senescent pathology. In C. elegans, a major senescent pathology of unknown etiology is atrophy of its principal metabolic organ, the intestine. Here we identify a cause of not only this pathology but also of yolky lipid accumulation and redistribution (a form of senescent obesity): autophagy-mediated conversion of intestinal biomass into yolk. Inhibiting intestinal autophagy or vitellogenesis rescues both visceral pathologies and can also extend lifespan. This defines a disease syndrome leading to multimorbidity and contributing to late-life mortality. Activation of gut-to-yolk biomass conversion by insulin/IGF-1 signaling (IIS) promotes reproduction and senescence. This illustrates how major, IIS-promoted senescent pathologies in C. elegans can originate not from damage accumulation but from direct effects of futile, continued action of a wild-type biological program (vitellogenesis).


Subject(s)
Aging/physiology , Autophagy/physiology , Caenorhabditis elegans/physiology , Egg Yolk/metabolism , Intestines/physiology , Vitellogenesis/physiology , Animals , Signal Transduction
13.
Soft Matter ; 12(38): 7953-8, 2016 Oct 14.
Article in English | MEDLINE | ID: mdl-27604678

ABSTRACT

Directly examining subcellular mechanics whilst avoiding excessive strain of a live cell requires the precise control of light stress on very small areas, which is fundamentally difficult. Here we use a glass nanopipet out of contact with the plasma membrane to both exert the stress on the cell and also accurately monitor cellular compression. This allows the mapping of cell stiffness at a lateral resolution finer than 100 nm. We calculate the stress a nanopipet exerts on a cell as the sum of the intrinsic pressure between the tip face and the plasma membrane plus its direct pressure on any glycocalyx, both evaluated from the gap size in terms of the ion current decrease. A survey of cell types confirms that an intracellular pressure of approximately 120 Pa begins to detach the plasma membrane from the cytoskeleton and reveals that the first 0.66 ± 0.09 µm of compression of a neuron cell body is much softer than previous methods have been able to detect.


Subject(s)
Cell Membrane/physiology , Microscopy/methods , Animals , Cell Line , Cells, Cultured , Cytoplasm , Cytoskeleton , Fibroblasts/cytology , Humans , Ions , Neurons/cytology , Rats
14.
Biol Blood Marrow Transplant ; 21(9): 1663-78, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26028505

ABSTRACT

We conducted a phase I trial of allogeneic T cells sensitized in vitro against a pool of pentadecapeptides (15-mer peptides) spanning the sequence of CMVpp65 for adoptive therapy of 17 allogeneic hematopoietic cell transplant recipients with cytomegalovirus (CMV) viremia or clinical infection persisting despite prolonged treatment with antiviral drugs. All but 3 of the patients had received T cell-depleted transplants without graft-versus-host disease (GVHD) prophylaxis with immunosuppressive drugs after transplantation. The CMVpp65-specific T cells (CMVpp65CTLs) generated were oligoclonal and specific for only 1 to 3 epitopes, presented by a limited set of HLA class I or II alleles. T cell infusions were well tolerated without toxicity or GVHD. Of 17 patients treated with transplant donor (n = 16) or third-party (n = 1) CMVpp65CTLs, 15 cleared viremia, including 3 of 5 with overt disease. In responding patients, the CMVpp65CTLs infused consistently proliferated and could be detected by T cell receptor Vß usage in CMVpp65/HLA tetramer + populations for period of 120 days to up to 2 years after infusion. Thus, CMVpp65CTLs generated in response to synthetic 15-mer peptides of CMVpp65 are safe and can clear persistent CMV infections in the post-transplantation period.


Subject(s)
Cytomegalovirus Infections , Cytomegalovirus/immunology , Hematopoietic Stem Cell Transplantation , Lymphocyte Transfusion , Peptides/immunology , Phosphoproteins/immunology , T-Lymphocytes , Tissue Donors , Viral Matrix Proteins/immunology , Viremia , Aged , Allografts , Child , Cytomegalovirus Infections/immunology , Cytomegalovirus Infections/pathology , Cytomegalovirus Infections/therapy , Female , Graft vs Host Disease/immunology , Graft vs Host Disease/pathology , Graft vs Host Disease/prevention & control , Hematologic Neoplasms/immunology , Hematologic Neoplasms/pathology , Hematologic Neoplasms/therapy , Humans , Male , Middle Aged , Peptides/pharmacology , Phosphoproteins/pharmacology , T-Lymphocytes/immunology , T-Lymphocytes/pathology , T-Lymphocytes/transplantation , Viral Matrix Proteins/pharmacology , Viremia/immunology , Viremia/pathology , Viremia/therapy
15.
Cancer Immunol Res ; 2(6): 547-58, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24894092

ABSTRACT

Cancer-testis antigen 7 (CT7) is the most frequently and consistently expressed MAGE antigen in multiple myeloma, exhibits tissue-restricted expression, and is an independent negative prognostic factor for multiple myeloma. We sought to characterize CT7 protein expression in the bone marrow of patients with multiple myeloma undergoing allogeneic T cell-depleted hematopoietic stem cell transplantation (alloTCD-HSCT), and to examine the significance of CT7-specific cellular immune responses. We further aimed to determine CT7-derived immunogenic epitopes and their associated allelic restrictions. CT7 protein expression in neoplastic CD138(+) plasma cells was evaluated by immunohistochemistry in bone marrow biopsies from 10 patients. CT7 was present in 8 of 10 patients. Longitudinal analyses of the 10 patients revealed an association between CT7 expression and prognosis. Longitudinal monitoring of CT7-specific T cells revealed an association between increased frequencies of CT7-specific T cells and reductions in specific myeloma markers. Epitope-specific reactivity to the nonamer FLAMLKNTV was detected by intracellular IFNγ assay in peripheral blood (PB) and bone marrow-derived T cells from HLA-A*0201(+) patients. Serial monitoring of PB CT7-specific T-cell frequencies in 4 HLA-A*0201(+) patients by HLA-A*0201-CT7(1087-1095) tetramer staining revealed an association with disease course. Phenotypic analyses revealed bone marrow enrichment for central memory CT7-specific T cells, while effector memory cells dominated the PB. Together, these findings support the development of immunotherapeutic strategies that aim to enhance CT7-directed immune responses for the treatment of multiple myeloma.


Subject(s)
Antigens, Neoplasm/metabolism , Hematopoietic Stem Cell Transplantation/methods , Immunity, Cellular/immunology , Multiple Myeloma/immunology , Bone Marrow/metabolism , CD8-Positive T-Lymphocytes/immunology , Disease Progression , Epitopes, T-Lymphocyte/immunology , HLA-A2 Antigen/metabolism , Humans , Multiple Myeloma/therapy , Transplantation, Homologous
16.
Oncoimmunology ; 2(7): e24963, 2013 Jul 01.
Article in English | MEDLINE | ID: mdl-24073375

ABSTRACT

The development of T-cell responses specific for myeloma-associated antigens correlates with improved clinical outcomes in multiple myeloma patients undergoing allogeneic T cell-depleted hematopoietic stem cell transplantation and donor lymphocyte infusions. Thus, immunotherapeutic strategies that further increase the frequency of Wilms tumor 1 (WT1)-specific T cells may provide clinical benefits to multiple myeloma patients.

17.
Blood ; 121(2): 308-17, 2013 Jan 10.
Article in English | MEDLINE | ID: mdl-23160468

ABSTRACT

While the emergence of WT1-specific cytotoxic T lymphocytes (WT1-CTL) has been correlated with better relapse-free survival after allogeneic stem cell transplantation in patients with myeloid leukemias, little is known about the role of these cells in multiple myeloma (MM). We examined the significance of WT1-CTL responses in patients with relapsed MM and high-risk cytogenetics who were undergoing allogeneic T cell-depleted hematopoietic stem cell transplantation (alloTCD-HSCT) followed by donor lymphocyte infusions. Of 24 patients evaluated, all exhibited WT1-CTL responses before allogeneic transplantation. These T-cell frequencies were universally correlated with pretransplantation disease load. Ten patients received low-dose donor lymphocyte infusions beginning 5 months after transplantation. All patients subsequently developed increments of WT1-CTL frequencies that were associated with reduction in specific myeloma markers, in the absence of graft-versus-host disease. Immunohistochemical analyses of WT1 and CD138 in bone marrow specimens demonstrated consistent coexpression within malignant plasma cells. WT1 expression in the bone marrow correlated with disease outcome. Our results suggest an association between the emergence of WT1-CTL and graft-versus-myeloma effect in patients treated for relapsed MM after alloTCD-HSCT and donor lymphocyte infusions, supporting the development of adoptive immunotherapeutic approaches using WT1-CTL in the treatment of MM.


Subject(s)
Graft vs Leukemia Effect/immunology , Hematopoietic Stem Cell Transplantation , Lymphocyte Transfusion , Multiple Myeloma/immunology , Multiple Myeloma/therapy , T-Lymphocytes, Cytotoxic/immunology , WT1 Proteins/immunology , Adult , Aged , Female , Flow Cytometry , Humans , Immunohistochemistry , Lymphocyte Depletion , Male , Middle Aged , Transplantation, Homologous
18.
Int Arch Allergy Immunol ; 146(1): 71-5, 2008.
Article in English | MEDLINE | ID: mdl-18087164

ABSTRACT

BACKGROUND: We recently reported that murine and cavian heart mast cells are a unique extrarenal source of renin. Ischemia/reperfusion releases this renin leading to local angiotensin formation and norepinephrine release. As mast cells are a primary target of hypersensitivity, we assessed whether anaphylactic mast cell degranulation also results in renin and norepinephrine release. METHODS: Hearts isolated from presensitized guinea pigs were challenged with antigen. RESULTS: Cardiac anaphylaxis was characterized by mast cell degranulation, evidenced by beta-hexosaminidase release and associated with renin and norepinephrine release. Mast cell stabilization with cromolyn or lodoxamide markedly attenuated the release of beta-hexosaminidase, renin and norepinephrine. Renin inhibition with BILA2157 did not affect mast cell degranulation, but attenuated norepinephrine release. CONCLUSIONS: Our findings disclose that immediate-type hypersensitivity elicits renin release from mast cells, activating a local renin-angiotensin system, thereby promoting norepinephrine release. As renin is stored in human heart mast cells, allergic reactions could initiate renin release, leading to local angiotensin formation and hyperadrenergic dysfunction.


Subject(s)
Cell Degranulation/immunology , Hypersensitivity, Immediate/immunology , Mast Cells/immunology , Myocardium/immunology , Renin/immunology , Animals , Anti-Allergic Agents/pharmacology , Anti-Asthmatic Agents/pharmacology , Cell Degranulation/drug effects , Cromolyn Sodium/pharmacology , Guinea Pigs , Hypersensitivity, Immediate/pathology , In Vitro Techniques , Male , Mast Cells/drug effects , Mast Cells/enzymology , Mast Cells/physiology , Myocardium/pathology , Norepinephrine/immunology , Ovalbumin/immunology , Ovalbumin/pharmacology , Oxamic Acid/analogs & derivatives , Oxamic Acid/pharmacology , Pyridines/pharmacology , Renin/antagonists & inhibitors , Thiazoles/pharmacology , beta-N-Acetylhexosaminidases/metabolism
19.
Biochem Pharmacol ; 73(8): 1146-56, 2007 Apr 15.
Article in English | MEDLINE | ID: mdl-17266940

ABSTRACT

We hypothesized that the histamine H(3)-receptor (H(3)R)-mediated attenuation of norepinephrine (NE) exocytosis from cardiac sympathetic nerves results not only from a Galpha(i)-mediated inhibition of the adenylyl cyclase-cAMP-PKA pathway, but also from a Gbetagamma(i)-mediated activation of the MAPK-PLA(2) cascade, culminating in the formation of an arachidonate metabolite with anti-exocytotic characteristics (e.g., PGE(2)). We report that in Langendorff-perfused guinea-pig hearts and isolated sympathetic nerve endings (cardiac synaptosomes), H(3)R-mediated attenuation of K(+)-induced NE exocytosis was prevented by MAPK and PLA(2) inhibitors, and by cyclooxygenase and EP(3)-receptor (EP(3)R) antagonists. Moreover, H(3)R activation resulted in MAPK phosphorylation in H(3)R-transfected SH-SY5Y neuroblastoma cells, and in PLA(2) activation and PGE(2) production in cardiac synaptosomes; H(3)R-induced MAPK phosphorylation was prevented by an anti-betagamma peptide. Synergism between H(3)R and EP(3)R agonists (i.e., imetit and sulprostone, respectively) suggested that PGE(2) may be a downstream effector of the anti-exocytotic effect of H(3)R activation. Furthermore, the anti-exocytotic effect of imetit and sulprostone was potentiated by the N-type Ca(2+)-channel antagonist omega-conotoxin GVIA, and prevented by an anti-Gbetagamma peptide. Our findings imply that an EP(3)R Gbetagamma(i)-induced decrease in Ca(2+) influx through N-type Ca(2+)-channels is involved in the PGE(2)/EP(3)R-mediated attenuation of NE exocytosis elicited by H(3)R activation. Conceivably, activation of the Gbetagamma(i) subunit of H(3)R and EP(3)R may also inhibit Ca(2+) entry directly, independent of MAPK intervention. As heart failure, myocardial ischemia and arrhythmic dysfunction are associated with excessive local NE release, attenuation of NE release by H(3)R activation is cardioprotective. Accordingly, this novel H(3)R signaling pathway may ultimately bear therapeutic significance in hyper-adrenergic states.


Subject(s)
Heart/innervation , Receptors, Histamine H3/physiology , Signal Transduction/physiology , Sympathetic Nervous System/physiology , Animals , Dinoprostone/physiology , Enzyme Activation , Exocytosis , Guinea Pigs , Male , Mitogen-Activated Protein Kinases/metabolism , Norepinephrine/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Subcellular Fractions
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