ABSTRACT
Hematopoietic cell transplantation is an intensive therapy used to treat high-risk hematological malignant disorders and other life-threatening hematological and genetic diseases. Graft-versus-host disease (GVHD) presents a barrier to its wider application. A conditioning regimen and medications given to patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT) are capable of disturbing the homeostatic crosstalk between the microbiome and the host immune system and of leading to dysbiosis. Intestinal inflammation in the context of GVHD is associated with loss in microbial diversity that could serve as an independent predictor of mortality. Successful gastrointestinal decontamination using high doses of non-absorbable antibiotics likely affect allo-HCT outcomes leading to significantly less acute GVHD (aGVHD). Butyrate-producing Clostridia directly result in the increased presence of regulatory T cells in the gut, which are protective in GVHD development. Beyond the microbiome, Candida, a member of the mycobiome, colonization in the gut has been considered as a risk factor in pathophysiology of aGVHD and reduction in GVHD is observed with antifungal prophylaxis with fluconazole. Reduced number of goblet cells and Paneth cells have been shown to associate with GVHD and has a significant impact on the micro- and mycobiome density and their composition. Lower levels of 3-indoxyl sulfate at initial stages after allo-HCT are related with worse GVHD outcomes and increased mortality. Increased understanding of the vital role of the gut microbiome in GVHD can give directions to move the field towards the development of improved innovative approaches for preventing or treating GVHD following allo-HCT.
Subject(s)
Bacteria/growth & development , Gastrointestinal Microbiome , Graft vs Host Disease/microbiology , Hematopoietic Stem Cell Transplantation/adverse effects , Intestines/microbiology , Microbiota , Animals , Bacteria/immunology , Dysbiosis , Gastrointestinal Microbiome/immunology , Graft vs Host Disease/immunology , Graft vs Host Disease/prevention & control , Graft vs Host Disease/therapy , Host-Pathogen Interactions , Humans , Intestines/immunology , Microbiota/immunology , Prognosis , Risk FactorsABSTRACT
Anti-thymocyte globulin (ATG) is a polyclonal antiserum introduced into clinical medicine more than 30 years ago. It induces a broad non-specific immunosuppression. In haematology, standard indications are severe aplastic anaemia and prophylaxis and treatment of graft-versus-host disease (GVHD) (after allogeneic transplantation). For aplastic anaemia, ATG from horses has been found to be superior to ATG from rabbits. In the situation of allogeneic transplantation, ATG lessens the risk of chronic GVHD but may not improve survival. There is current controversy regarding which patients benefit most from ATG and what the ideal dosage is. It is likely that in the coming years a more specific immunosuppressive will be developed that will minimize GVHD while maintaining the graft-versus-malignancy effect.
Subject(s)
Anemia, Aplastic/therapy , Antilymphocyte Serum/therapeutic use , Graft vs Host Disease/therapy , Hematology/trends , Immunoglobulin G/therapeutic use , Animals , Biomarkers , Goats , Horses , Humans , Immunosuppressive Agents/therapeutic use , Immunotherapy , Myelodysplastic Syndromes/immunology , Myelodysplastic Syndromes/therapy , Prognosis , Rabbits , Recurrence , Risk , Swine , Treatment OutcomeSubject(s)
Gastrointestinal Microbiome , Graft vs Host Disease/immunology , Graft vs Host Disease/microbiology , Alarmins/immunology , Clostridium Infections/immunology , Gastrointestinal Tract/microbiology , Gastrointestinal Tract/virology , Genomics , Hematopoietic Stem Cell Transplantation/methods , Homeostasis , Humans , Pathogen-Associated Molecular Pattern Molecules/immunology , Phenotype , Risk , Transcriptome , Transplantation, Homologous/methodsABSTRACT
Noninfectious lung injury and acute graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT) are associated with significant morbidity and mortality. Azithromycin is widely used in allogeneic HCT recipients for pulmonary chronic GVHD, although current data appear controversial. We induced GVHD and noninfectious lung injury in lethally irradiated B6D2F1 mice by transplanting bone marrow and splenic T cells from allogeneic C57BL/6 mice. Experimental groups were treated with oral azithromycin starting on day 14 until the end of week 6 or week 14 after transplantation. Azithromycin treatment resulted in improved survival and decreased lung injury; the latter characterized by improved pulmonary function, reduced peribronchial and perivascular inflammatory cell infiltrates along with diminished collagen deposition, and a decrease in lung cytokine and chemokine expression. Azithromycin also improved intestinal GVHD but did not affect liver GVHD at week 6 early after transplantation. At week 14, azithromycin decreased liver GVHD but had no effect on intestinal GVHD. In vitro, allogeneic antigen-presenting cell (APC)- dependent T cell proliferation and cytokine production were suppressed by azithromycin and inversely correlated with relative regulatory T cell (Treg) expansion, whereas no effect was seen when T cell proliferation occurred APC independently through CD3/CD28-stimulation. Further, azithromycin reduced alloreactive T cell expansion but increased Treg expansion in vivo with corresponding downregulation of MHC II on CD11c(+) dendritic cells. These results demonstrate that preventive administration of azithromycin can reduce the severity of acute GVHD and noninfectious lung injury after allo-HCT, supporting further investigation in clinical trials.
Subject(s)
Anti-Bacterial Agents/pharmacology , Azithromycin/pharmacology , Bone Marrow Transplantation , Graft vs Host Disease/prevention & control , Lung Injury/prevention & control , Lung/drug effects , Acute Disease , Animals , Cytokines/biosynthesis , Dendritic Cells/cytology , Dendritic Cells/drug effects , Dendritic Cells/immunology , Disease Models, Animal , Female , Graft vs Host Disease/immunology , Graft vs Host Disease/mortality , Graft vs Host Disease/pathology , Intestines/drug effects , Intestines/immunology , Liver/drug effects , Liver/immunology , Lung/immunology , Lung/pathology , Lung Injury/immunology , Lung Injury/mortality , Lung Injury/pathology , Mice , Primary Cell Culture , Respiratory Function Tests , Spleen/drug effects , Spleen/immunology , Survival Analysis , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , T-Lymphocytes/transplantation , Transplantation, Homologous , Whole-Body IrradiationSubject(s)
Antibodies, Monoclonal, Humanized/pharmacology , Graft vs Host Disease/drug therapy , Integrin alpha4/antagonists & inhibitors , Integrin beta Chains , Animals , Disease Models, Animal , Graft vs Host Disease/genetics , Graft vs Host Disease/metabolism , Graft vs Host Disease/pathology , Humans , Integrin alpha4/genetics , Integrin alpha4/metabolism , Mice , Mice, KnockoutABSTRACT
Chronic lymphocytic leukemia (CLL) is a mature B cell neoplasm with a predilection for older individuals. While previous studies have identified epigenetic signatures associated with CLL, whether age-related DNA methylation changes modulate CLL relapse remains elusive. In this study, we examined the association between epigenetic age acceleration and time to CLL relapse in a publicly available dataset. DNA methylation profiling of 35 CLL patients prior to initiating chemoimmunotherapy was performed using the Infinium HumanMethylation450 BeadChip. Four epigenetic age acceleration metrics (intrinsic epigenetic age acceleration [IEAA], extrinsic epigenetic age acceleration [EEAA], PhenoAge acceleration [PhenoAA], and GrimAge acceleration [GrimAA]) were estimated from blood DNA methylation levels. Linear, quantile, and logistic regression and receiver operating characteristic curve analyses were conducted to assess the association between each epigenetic age metric and time to CLL relapse. EEAA (p = 0.011) and PhenoAA (p = 0.046) were negatively and GrimAA (p = 0.040) was positively associated with time to CLL relapse. Simultaneous assessment of EEAA and GrimAA in male patients distinguished patients who relapsed early from patients who relapsed later (p = 0.039). No associations were observed with IEAA. These findings suggest epigenetic age acceleration prior to chemoimmunotherapy initiation is associated with time to CLL relapse. Our results provide novel insight into the association between age-related DNA methylation changes and CLL relapse and may serve has biomarkers for treatment relapse, and potentially, treatment selection.
Subject(s)
DNA Methylation , Leukemia, Lymphocytic, Chronic, B-Cell , Humans , Male , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Epigenesis, Genetic , Aging/genetics , DNAABSTRACT
Graft-versus-host disease (GVHD) is a major complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Vascular endothelial cells are entirely exposed and damaged during the pathogenesis of acute GVHD (aGVHD). Defibrotide (DF) is a mixture of single-stranded oligonucleotides that has several pharmacologic effects that contribute to its endothelial protective properties. B10.BR mice were conditioned, followed by the infusion of donor C57BL/6J T cell-depleted bone marrow cells with or without splenocytes. The mice were either treated with DF or appropriate controls daily for the first week and then 3 times per week thereafter. Allogeneic DF-treated recipients demonstrated significantly better survival with reduced clinical GVHD. Significantly reduced organ pathology in the gut was associated with significantly decreased T cell infiltration in the ileum and colon on day +28. Serum cytokine analysis revealed significantly reduced levels of TNF and IL-6 at day +7 and of TNF at day +28 in allogeneic DF-treated recipients. Significantly reduced levels of ICAM-1 and angiopoietin-2 in serum and reduced VCAM-1 and HCAM levels in the ileum and colon of allogeneic DF-treated recipients were observed. Improved survival was seen in the graft-versus-leukemia (GVL) model (C3H.SW into C57BL/6J mice with C1498-luc). Through its anti-inflammatory and endothelial protective effects, DF treatment reduces the severity of aGVHD while not impairing GVL activity.
ABSTRACT
Graft-versus-host disease (GVHD) is the major factor limiting the widespread use of potentially curative allogeneic hematopoietic stem cell transplant (allo-HSCT). Chronic GVHD is characterized by the activation of alloreactive donor immune cells, especially B- and T-cells, leading to tissue damage and pathogenic fibrosis. In this study, we used highly specific next-generation inhibitors of ITK (PCYC-274), BTK (PCYC-804), and ibrutinib-like BTK/ITK inhibitors (PCYC-914 and PCYC-401) in the B10.D2 â BALB/C model of murine sclerodermatous cGVHD. From the third week onward, allogeneic recipients in each group of respective Tec kinase inhibitors were treated three times weekly with inhibitors at doses of 10 and 30 mg/kg or with saline control via oral gavage. Overall, we found that selective BTK inhibition was less effective than combined ITK/BTK or ITK inhibition in lengthening survival and reducing symptoms of cGVHD. ITK inhibition was most efficacious, with PCYC-274 and PCYC-401 demonstrating a nearly 50 percent reduction in GVHD scoring even at the 10 mg/kg dose, while 30 mg/kg of these compounds almost completely ameliorated GVHD symptomology. BTK/ITK and ITK-treated mice showed significant reductions in overall pathology. Significant reductions in dermal thickness and fibrosis were shown for all treatment groups. There was evidence of mixed Th1 and Th2 cytokine profiles in the skin of mice with dermal cGVHD, as both IFN-gamma and IL-4 were upregulated in the allogeneic control group, while kinase inhibition significantly reduced levels of these cytokines. Using an in vitro model of T-cell polarization, Th1 cell production of TNF-alpha and IFN-gamma were partially blocked by ITK. Th2 cell production of IL-4 was almost completely blocked synergistically by ITK and BTK inhibition. BTK-specific inhibition was unable to block either Th1 or Th2 cytokine production. Taken together, these results confirm previous reports that ITK-focused inhibition inhibits Th1 and Th2 cells. Additionally, the compound's effects on T-cell proliferation were tested by CFSE assay. Pure ITK inhibition was most effective at blocking T-cell proliferation, with no proliferation in PCYC-274-treated cells even at 0.1uM. PCYC-401 and PCYC-914 showed some inhibition at lower doses, with complete inhibition evident at 10uM. PCYC-804 was only partially able to block proliferation even at 10uM. In conclusion, we observed substantial benefit for differential inhibition of Tec kinases in GVHD, with ITK being most efficacious and Th1 cells being more resistant to inhibition, matching the previously reported findings of a Th2 to Th1 selective pressure in cells treated with ibrutinib. Our data warrants the further development of ITK and ITK/BTK inhibitors with specific inhibitory ratios to improve the treatment of GVHD and other T-cell mediated diseases.
Subject(s)
Bronchiolitis Obliterans Syndrome , Graft vs Host Disease , Animals , Mice , Interleukin-4/therapeutic use , Mice, Inbred BALB C , Cytokines , Graft vs Host Disease/drug therapy , Graft vs Host Disease/pathology , FibrosisABSTRACT
To further investigate potential mechanisms of resistance to FLT3 inhibitors, we developed a resistant cell line by long-term culture of MV4-11 cells with ABT-869, designated as MV4-11-R. Gene profiling reveals up-regulation of FLT3LG (FLT3 ligand) and BIRC5 (survivin), but down-regulation of SOCS1, SOCS2, and SOCS3 in MV4-11-R cells. Hypermethylation of these SOCS genes leads to their transcriptional silencing. Survivin is directly regulated by STAT3. Stimulation of the parental MV4-11 cells with FLT3 ligand increases the expression of survivin and phosphorylated protein STAT1, STAT3, STAT5. Targeting survivin by short-hairpin RNA (shRNA) in MV4-11-R cells induces apoptosis and augments ABT-869-mediated cytotoxicity. Overexpression of survivin protects MV4-11 from apoptosis. Subtoxic dose of indirubin derivative (IDR) E804 resensitizes MV4-11-R to ABT-869 treatment by inhibiting STAT signaling activity and abolishing survivin expression. Combining IDR E804 with ABT-869 shows potent in vivo efficacy in the MV4-11-R xenograft model. Taken together, these results demonstrate that enhanced activation of STAT pathways and overexpression of survivin are important mechanisms of resistance to ABT-869, suggesting that the STAT pathways and survivin could be potential targets for reducing resistance developed in patients receiving FLT3 inhibitors.
Subject(s)
Gene Expression Regulation, Neoplastic/genetics , Leukemia, Myeloid, Acute/metabolism , Leukemia, Myeloid, Acute/pathology , Microtubule-Associated Proteins/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction/drug effects , fms-Like Tyrosine Kinase 3/antagonists & inhibitors , Animals , Apoptosis/drug effects , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Epoxy Compounds/pharmacology , Female , Humans , Indazoles/pharmacology , Inhibitor of Apoptosis Proteins , Leukemia, Myeloid, Acute/genetics , Ligands , Mice , Mice, Inbred BALB C , Mice, Nude , Microtubule-Associated Proteins/genetics , Phenylurea Compounds/pharmacology , STAT3 Transcription Factor/genetics , Sesquiterpenes/pharmacology , Substrate Specificity , Survivin , Up-Regulation/genetics , Xenograft Model Antitumor Assays , fms-Like Tyrosine Kinase 3/metabolismABSTRACT
Allogeneic hematopoietic stem cell transplantation (HSCT) is the solitary therapeutic therapy for many types of hematological cancers. The benefits of this procedure are challenged by graft vs. host disease (GVHD), causing significant morbidity and mortality. Recent advances in the metabolomics field have revolutionized our understanding of complex human diseases, clinical diagnostics and allow to trace the de novo biosynthesis of metabolites. There is growing evidence for metabolomics playing a role in different aspects of GVHD, and therefore metabolomic reprogramming presents a novel tool for this disease. Pre-transplant cytokine profiles and metabolic status of allogeneic transplant recipients is shown to be linked with a threat of acute GVHD. Immune reactions underlying the pathophysiology of GVHD involve higher proliferation and migration of immune cells to the target site, requiring shifts in energy supply and demand. Metabolic changes and reduced availability of oxygen result in tissue and cellular hypoxia which is extensive enough to trigger transcriptional and translational changes. T cells, major players in acute GVHD pathophysiology, show increased glucose uptake and glycolytic activity. Effector T (Teff) cells activated during nutrient limiting conditions in vitro or multiplying during GVHD in vivo, depend more on oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO). Dyslipidemia, such as the increase of medium and long chain fatty and polyunsaturated acids in plasma of GVHD patients, has been observed. Sphingolipids associate with inflammatory conditions and cancer. Chronic GVHD (cGVHD) patients show reduced branched-chain amino acids (BCAAs) and increased sulfur-containing metabolites post HSCT. Microbiota-derived metabolites such as aryl hydrocarbon receptor (AhR) ligands, bile acids, plasmalogens and short chain fatty acids vary significantly and affect allogeneic immune responses during acute GVHD. Considering the multitude of possibilities, how altered metabolomics are involved in GVHD biology, multi-timepoints related and multivariable biomarker panels for prognosticating and understanding GVHD are needed. In this review, we will discuss the recent work addressing metabolomics reprogramming to control GVHD in detail.
ABSTRACT
BACKGROUND: Prolyl hydroxylase inhibitors (PHI) promote stabilization of hypoxia-inducible factor-1 alpha and affect signaling cascades of inflammation and cell death. Their beneficial use in experimental models of ulcerative colitis and lung allograft rejection led us to test the effect of the PHI dimethyl oxalyl glycine (DMOG) in the pathophysiology of graft versus host disease (GVHD). METHODS: Acute GVHD was induced in lethally irradiated BALB/c mice. DMOG was administered intraperitoneally on alternate days for the first 2-weeks posttransplant, and then twice a week till day +50, while controls received vehicle only. Animals were monitored for clinical GVHD and analyzed at day +7 and at day +50. RESULTS: DMOG treatment of allogeneic recipients improved survival by day +50, which was associated with decreased early gut injury and serum tumor necrosis factor-α compared with allogeneic controls. DMOG treatment of allogeneic recipients resulted in increased hypoxia-inducible factor-1 alpha expression and reduced apoptosis in the terminal ileum via Fas-associated protein with death domain protein repression along with decreased T-cell infiltration. Reduced pathology in colon after DMOG treatment associates with intestinal epithelium integrity and reduced damage caused by diminished recruitment of neutrophils. CONCLUSIONS: Taken together, we show protective effects of DMOG on early gut GVHD and improved survival in a model of allogeneic hematopoietic cell transplantation, providing the rationale for further evaluation of PHIs, in the prevention and treatment of acute GVHD.
Subject(s)
Amino Acids, Dicarboxylic/pharmacology , Colon/drug effects , Graft vs Host Disease/prevention & control , Hematopoietic Stem Cell Transplantation/adverse effects , Ileum/drug effects , Intestinal Diseases/prevention & control , Prolyl Hydroxylases/metabolism , Prolyl-Hydroxylase Inhibitors/pharmacology , Animals , Apoptosis/drug effects , Cells, Cultured , Colon/enzymology , Colon/immunology , Colon/pathology , Graft vs Host Disease/enzymology , Graft vs Host Disease/immunology , Graft vs Host Disease/pathology , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Ileum/enzymology , Ileum/immunology , Ileum/pathology , Intestinal Diseases/enzymology , Intestinal Diseases/immunology , Intestinal Diseases/pathology , Mice, Inbred BALB C , Mice, Inbred C57BL , Time Factors , Transplantation, Homologous/adverse effects , Treatment Outcome , Whole-Body IrradiationABSTRACT
BACKGROUND: Gastrointestinal acute graft-versus-host disease (GVHD) occurring after allogeneic hematopoietic cell transplant is an allo-reactive T cell and inflammatory cytokine driven organ injury with epithelial apoptosis as 1 of its hallmark findings and is associated with significant mortality. Tumor necrosis factor (TNF)-alpha-induced protein 8 (TNFAIP8 or TIPE) acts as a negative mediator of apoptosis via inhibition of caspase-3 activation, promotes cell proliferation and Tipe deficiency is associated with increased inflammation. METHODS: To evaluate the role of TIPE in acute GVHD, naive C57BL/6 and Tipe C57BL/6 mice were conditioned with 1000 cGy single dose total body irradiation, followed by transplantation of 10 million bone marrow cells and 20 million splenocytes from either syngeneic C57BL/6 or allogeneic BALB/c donors. RESULTS: Allo TIPE-deficient mice developed exacerbated gut GVHD compared with allo controls and had significantly decreased survival (6 wk overall survival: 85% versus 37%; P < 0.05), higher clinical GVHD scores, more profound weight loss, increased serum proinflammatory cytokines (interleukin-17A, TNF, interleukin-6, and interferon-γ). T-cell infiltration into the ileum was increased; epithelial proliferation was decreased along with significantly higher levels of chemokines KC and monokine induced by gamma interferon. Using bone marrow chimeric experiments, TIPE was found to have a role in both hematopoietic and nonhematopoietic cells. CONCLUSIONS: Absence of TIPE results in excessive inflammation and tissue injury after allo-HCT, supporting that TIPE confers immune homeostasis and has tissue-protective function during the development of gut GVHD and may be a potential future target to prevent or treat this complication after allogeneic HCT.
Subject(s)
Apoptosis Regulatory Proteins/deficiency , Graft vs Host Disease/immunology , Hematopoietic Stem Cell Transplantation/adverse effects , Ileal Diseases/immunology , Animals , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/immunology , Bone Marrow Transplantation , Disease Models, Animal , Female , Graft vs Host Disease/pathology , Humans , Ileal Diseases/pathology , Ileum/immunology , Ileum/pathology , Intestinal Mucosa/immunology , Intestinal Mucosa/pathology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Transplantation Chimera/immunology , Transplantation, Homologous/adverse effectsABSTRACT
Allogeneic hematopoietic stem cell transplant (allo-HSCT) is often used to treat acute leukemia or defects of hematopoiesis. Its widespread use is hampered by graft-vs.-host disease (GVHD), which has high morbidity and mortality in both acute and chronic subtypes. Chronic GVHD (cGVHD) occurs most frequently in skin and often is characterized by pathogenic fibrosis. Mast cells (MCs) are known to be involved in the pathogenesis of other fibrotic diseases. In a murine model of cGVHD after allo-HSCT, C57BL/6J recipients of allogeneic LP/J donor cells develop sclerodermatous dermal cGVHD which is significantly decreased in mast cell-deficient B6.Cg-KitW-sh/HNihrJaeBsmGlliJ recipients. The presence of MCs is associated with fibrosis, chemokine production, and recruitment of GVHD effector cells to the skin. Chemokine production by MCs is blocked by drugs used to treat cGVHD. The importance of MCs in skin cGVHD is mirrored by increased MCs in the skin of patients with dermal cGVHD. We show for the first time a role for MCs in skin cGVHD that may be targetable for preventive and therapeutic intervention in this disease.
Subject(s)
Cytokines/immunology , Graft vs Host Disease/immunology , Hematopoietic Stem Cell Transplantation/methods , Mast Cells/immunology , Skin/immunology , Adult , Animals , Cells, Cultured , Cytokines/genetics , Cytokines/metabolism , Female , Fibrosis , Gene Expression Profiling/methods , Graft vs Host Disease/etiology , Hematopoietic Stem Cell Transplantation/adverse effects , Humans , Male , Mast Cells/cytology , Mast Cells/metabolism , Mice, Inbred BALB C , Mice, Inbred C57BL , Middle Aged , Skin/metabolism , Skin/pathology , Transplantation, HomologousSubject(s)
Graft vs Host Disease , Hematopoietic Stem Cell Transplantation , Microbiota , Animals , Disease Models, Animal , Humans , MiceABSTRACT
Acute graft-versus-host disease (GVHD) considerably limits wider usage of allogeneic hematopoietic cell transplantation (allo-HCT). Antigen-presenting cells and T cells are populations customarily associated with GVHD pathogenesis. Of note, neutrophils are the largest human white blood cell population. The cells cleave chemokines and produce reactive oxygen species, thereby promoting T cell activation. Therefore, during an allogeneic immune response, neutrophils could amplify tissue damage caused by conditioning regimens. We analyzed neutrophil infiltration of the mouse ileum after allo-HCT by in vivo myeloperoxidase imaging and found that infiltration levels were dependent on the local microbial flora and were not detectable under germ-free conditions. Physical or genetic depletion of neutrophils reduced GVHD-related mortality. The contribution of neutrophils to GVHD severity required reactive oxygen species (ROS) because selective Cybb (encoding cytochrome b-245, beta polypeptide, also known as NOX2) deficiency in neutrophils impairing ROS production led to lower levels of tissue damage, GVHD-related mortality and effector phenotype T cells. Enhanced survival of Bcl-xL transgenic neutrophils increased GVHD severity. In contrast, when we transferred neutrophils lacking Toll-like receptor-2 (TLR2), TLR3, TLR4, TLR7 and TLR9, which are normally less strongly activated by translocating bacteria, into wild-type C57BL/6 mice, GVHD severity was reduced. In humans, severity of intestinal GVHD strongly correlated with levels of neutrophils present in GVHD lesions. This study describes a new potential role for neutrophils in the pathogenesis of GVHD in both mice and humans.
Subject(s)
Graft vs Host Disease/immunology , Hematopoietic Stem Cell Transplantation/adverse effects , Ileum/immunology , Microbiota/immunology , Neutrophils/immunology , Animals , Busulfan , Cyclophosphamide , Flow Cytometry , Freund's Adjuvant , Graft vs Host Disease/physiopathology , Histological Techniques , Ileum/microbiology , Immunohistochemistry , Kaplan-Meier Estimate , Luciferases , Magnetic Resonance Imaging , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Microarray Analysis , NADPH Oxidase 2 , NADPH Oxidases/genetics , Peroxidase , Reactive Oxygen Species/metabolismABSTRACT
The success of allogeneic (allo) hematopoietic cell transplantation (HCT) is limited by its treatment related complications, mostly graft versus host disease (GVHD) and fungal and viral infections. CMV reactivation after HCT has been associated with increased morbidity and mortality, and a causal relation between GVHD, immunosuppressive therapy and vice versa has been postulated. Using a low GVHD severity murine HCT model, we assessed the role of MCMV reactivation and GVHD development. BALB/c mice were infected with either murine CMV (MCMV) or mock and monitored for 25 weeks to establish latency, followed by sublethal irradiation conditioning and infusion of bone marrow plus splenocytes from either syngeneic (syn) BALB/c or allo B10.D2 donors. Engraftment of allo donor cells was confirmed by PCR for D2Mit265 gene product size. Day+100 mortality and overall GVHD severity in allo MCMV pre-infected recipients was higher than in allo mock controls. Pathologic changes of lung and liver GVHD in immediate-early gene 1 (IE1) positive recipients were significantly increased compared to mock controls, and were only slightly increased in IE1 negative. No significant gut injury was seen in any group. Aggravated lung injury in IE1 positive recipients correlated with higher BAL cell counts both for total cells and for CD4+ T cells when compared with mock controls, and also with protein expression of lung IFN-gamma and liver TNF. No evidence for CMV specific morphologic changes was seen on histopathology in any organ of IE1 positive recipients, suggesting that CMV reactivation is related to increased GVHD severity but does not require active CMV disease, strengthening the concept of a reciprocal relationship between CMV and GVHD.
Subject(s)
Gene Expression , Genes, Immediate-Early/genetics , Graft vs Host Disease/etiology , Hematopoietic Stem Cell Transplantation , Muromegalovirus/genetics , Animals , Antibodies, Viral/blood , Antibodies, Viral/immunology , Chemokines/metabolism , Chimerism , Female , Graft vs Host Disease/immunology , Graft vs Host Disease/mortality , Graft vs Host Disease/virology , Herpesviridae Infections/immunology , Herpesviridae Infections/virology , Immunoglobulin G/blood , Immunoglobulin G/immunology , Inflammation/metabolism , Liver/metabolism , Liver/pathology , Liver/virology , Lung/metabolism , Lung/pathology , Lung/virology , Mice , Polymorphism, Genetic , Transplantation, Homologous , Virus Activation/genetics , Virus LatencyABSTRACT
Salivary glands are highly susceptible to radiation, and patients with head and neck cancer treated with radiotherapy invariably suffer from its distressing side effect, salivary hypofunction. The reduction in saliva disrupts oral functions, and significantly impairs oral health. Previously, we demonstrated that adenoviral-mediated expression of Tousled-like kinase 1B (TLK1B) in rat submandibular glands preserves salivary function after single-dose ionizing radiation. To achieve long-term transgene expression for protection of salivary gland function against fractionated radiation, this study examines the usefulness of recombinant adeno-associated viral vector for TLK1B delivery. Lactated Ringers or AAV2/9 with either TLK1B or GFP expression cassette were retroductally delivered to rat submandibular salivary glands (10(11) vg/gland), and animals were exposed, or not, to 20 Gy in eight fractions of 2.5 Gy/day. AAV2/9 transduced predominantly the ductal cells, including the convoluted granular tubules of the submandibular glands. Transgene expression after virus delivery could be detected within 5 weeks, and stable gene expression was observed till the end of study. Pilocarpine-stimulated saliva output measured at 8 weeks after completion of radiation demonstrated >10-fold reduction in salivary flow in saline- and AAV2/9-GFP-treated animals compared with the respective nonirradiated groups (90.8% and 92.5% reduction in salivary flow, respectively). Importantly, there was no decrease in stimulated salivary output after irradiation in animals that were pretreated with AAV2/9-TLK1B (121.5% increase in salivary flow; p<0.01). Salivary gland histology was better preserved after irradiation in TLK1B-treated group, though not significantly, compared with control groups. Single preemptive delivery of AAV2/9-TLK1B averts salivary dysfunction resulting from fractionated radiation. Although AAV2/9 transduces mostly the ductal cells of the gland, their protection against radiation assists in preserving submandibular gland function. AAV2/9-TLK1B treatment could prove beneficial in attenuating xerostomia in patients with head and neck cancer undergoing radiotherapy.
Subject(s)
Dependovirus/metabolism , Dose Fractionation, Radiation , Protein Serine-Threonine Kinases/metabolism , Radiation Injuries/therapy , Recombination, Genetic/genetics , Xerostomia/etiology , Xerostomia/therapy , Acinar Cells/pathology , Animals , Cell Line , Genetic Vectors , HEK293 Cells , Humans , Male , Radiation Injuries/physiopathology , Rats , Rats, Sprague-Dawley , Salivation , Submandibular Gland/metabolism , Submandibular Gland/pathology , Submandibular Gland/physiopathology , Submandibular Gland/radiation effects , Transduction, Genetic , Transgenes , Xerostomia/physiopathologyABSTRACT
PURPOSE: Patients treated with radiotherapy for head-and-neck cancer invariably suffer its deleterious side effect, xerostomia. Salivary hypofunction ensuing from the irreversible destruction of glands is the most common and debilitating oral complication affecting patients undergoing regional radiotherapy. Given that the current management of xerostomia is palliative and ineffective, efforts are now directed toward preventive measures to preserve gland function. The human homolog of Tousled protein, TLK1B, facilitates chromatin remodeling at DNA repair sites and improves cell survival against ionizing radiation (IR). Therefore, we wanted to determine whether a direct transfer of TLK1B protein to rat salivary glands could protect against IR-induced salivary hypofunction. METHODS: The cell-permeable TAT-TLK1B fusion protein was generated. Rat acinar cell line and rat salivary glands were pretreated with TAT peptide or TAT-TLK1B before IR. The acinar cell survival in vitro and salivary function in vivo were assessed after radiation. RESULTS: We demonstrated that rat acinar cells transduced with TAT-TLK1B were more resistant to radiation (D0 = 4.13 ± 1.0 Gy; α/ß = 0 Gy) compared with cells transduced with the TAT peptide (D0 = 4.91 ± 1.0 Gy; α/ß = 20.2 Gy). Correspondingly, retroductal instillation of TAT-TLK1B in rat submandibular glands better preserved salivary flow after IR (89%) compared with animals pretreated with Opti-MEM or TAT peptide (31% and 39%, respectively; p < 0.01). CONCLUSIONS: The results demonstrate that a direct transfer of TLK1B protein to the salivary glands effectively attenuates radiation-mediated gland dysfunction. Prophylactic TLK1B-protein therapy could benefit patients undergoing radiotherapy for head-and-neck cancer.
Subject(s)
Acinar Cells/drug effects , Cell Survival/drug effects , Protein Serine-Threonine Kinases/administration & dosage , Radiation Tolerance/drug effects , Recombinant Fusion Proteins/administration & dosage , Salivary Glands/radiation effects , Xerostomia/prevention & control , tat Gene Products, Human Immunodeficiency Virus/administration & dosage , Acinar Cells/metabolism , Acinar Cells/pathology , Acinar Cells/radiation effects , Animals , Cell Line , Cell Survival/radiation effects , Female , Head and Neck Neoplasms/radiotherapy , Luciferases/metabolism , Protein Serine-Threonine Kinases/isolation & purification , Protein Serine-Threonine Kinases/metabolism , Rats , Rats, Sprague-Dawley , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Salivary Glands/metabolism , Salivary Glands/pathology , Salivation/drug effects , Salivation/radiation effects , Submandibular Gland/drug effects , Submandibular Gland/metabolism , Submandibular Gland/pathology , Submandibular Gland/radiation effects , beta-Galactosidase/metabolism , tat Gene Products, Human Immunodeficiency Virus/isolation & purification , tat Gene Products, Human Immunodeficiency Virus/metabolismABSTRACT
OBJECTIVES/HYPOTHESIS: The eukaryotic translation initiation factor 4E (eIF4E) in conjunction with its binding protein, 4EBP1, regulates the translation of cap-dependent mRNAs. An aberrant increase in eIF4E shifts the balance in favor of translation of transcripts that promote cell proliferation and malignancy. eIF4E protein is commonly elevated in head and neck squamous cell carcinomas (HNSCC), and its overexpression is associated with increased recurrence. An underlying mechanism for eIF4E overexpression is gene amplification, and we wanted to determine whether eIF4E mRNA could serve as a prognostic maker of HNSCC. METHODS: Tumor specimens from 26 HNSCC patients and oral tissues from 17 control subjects were examined for eIF4E and 4EBP1 by semiquantitative RT-PCR and correlated with clinical and pathologic findings. RESULTS: Unlike eIF4E mRNA alone, expression of eIF4E relative to 4EBP1 was a more precise predictor of HNSCC and its progression (P < .01, Wilcoxon rank sum test). Eight of 26 patients (31%) had elevated eIF4E:4EBP1 (4E:4EBP1; >25), and 7 of these (87.5%) had recurrence. Alternately, from 18 patients with low 4E:4EBP1 (<25; 69%), only 5 patients had recurrence (30.1%). To determine the probability of no recurrence, Kaplan-Meier analysis showed significantly poor disease-free survival in patients with elevated 4E:4EBP1 than those with low ratios (P < .01, log rank test). CONCLUSIONS: Elevated 4E:4EBP1 significantly correlated with increased disease recurrence. Because 4EBP1 modulates eIF4E activity, our results highlight the importance of incorporating a joint analysis of eIF4E and 4EBP1 mRNAs in HNSCC patient care decisions.
Subject(s)
Biomarkers, Tumor/metabolism , Carcinoma/pathology , Eukaryotic Initiation Factor-4E/metabolism , Head and Neck Neoplasms/pathology , Neoplasm Recurrence, Local/metabolism , Neoplasms, Squamous Cell/pathology , RNA, Messenger/analysis , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Adult , Aged , Biopsy, Needle , Carcinoma/genetics , Carcinoma/mortality , Carcinoma, Squamous Cell , Chi-Square Distribution , Cohort Studies , Disease Progression , Eukaryotic Initiation Factor-4E/genetics , Female , Head and Neck Neoplasms/genetics , Head and Neck Neoplasms/mortality , Humans , Male , Middle Aged , Neoplasm Invasiveness/pathology , Neoplasm Recurrence, Local/genetics , Neoplasm Staging , Neoplasms, Squamous Cell/genetics , Neoplasms, Squamous Cell/mortality , Predictive Value of Tests , Prognosis , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Risk Assessment , Sensitivity and Specificity , Squamous Cell Carcinoma of Head and Neck , Statistics, Nonparametric , Survival Analysis , Tissue Culture TechniquesABSTRACT
PURPOSE: CD44 is overexpressed in various tumors including hepatocellular carcinoma (HCC). The purpose of this study was to examine the effects of CD44 antisense oligonucleotide (ASO) alone or combination with doxorubicin on HCC cells in vitro. METHODS: Cytotoxicity was measured by use of a cell viability assay in HCC cell line SNU-449. Tumorigenesis and invasion were accessed by colony formation, growth in soft agar and ECMatrix invasion assay. Apoptosis and necrosis were evaluated by using double staining with Hoechst 33342 and propidium iodide. Protein expression and mRNA level were detected by Western blot and RT-PCR. RESULTS: We have designed novel CD44 ASO, which can effectively down-regulate CD44 expression in SNU-449. Colony formation, growth in soft agar and invasion were significantly impaired after CD44 ASO treatment in SNU-499. In company with CD44 down-regulated by CD44 ASO, MDR-1 and Bcl-2 expression were also greatly reduced. CD44 ASO also increased chemosensitivity to doxorubicin significantly, lowered IC(50 )by one order of magnitude. Apoptosis and necrosis were also induced by CD44 ASO alone or in combination treatment with doxorubicin. CONCLUSIONS: Inhibition of CD44 expression by CD44 ASO significantly induced apoptosis, decreased tumorigenesis and invasion, and increased chemosensitivity. Thus, CD44 ASO is potentially a therapy that is worth investigating in the clinical setting.