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1.
Blood ; 143(21): 2201-2216, 2024 May 23.
Artículo en Inglés | MEDLINE | ID: mdl-38447038

RESUMEN

ABSTRACT: Fanconi anemia (FA) is an inherited DNA repair disorder characterized by bone marrow (BM) failure, developmental abnormalities, myelodysplasia, leukemia, and solid tumor predisposition. Allogeneic hematopoietic stem cell transplantation (allo-HSCT), a mainstay treatment, is limited by conditioning regimen-related toxicity and graft-versus-host disease (GVHD). Antibody-drug conjugates (ADCs) targeting hematopoietic stem cells (HSCs) can open marrow niches permitting donor stem cell alloengraftment. Here, we report that single dose anti-mouse CD45-targeted ADC (CD45-ADC) facilitated stable, multilineage chimerism in 3 distinct FA mouse models representing 90% of FA complementation groups. CD45-ADC profoundly depleted host stem cell enriched Lineage-Sca1+cKit+ cells within 48 hours. Fanca-/- recipients of minor-mismatched BM and single dose CD45-ADC had peripheral blood (PB) mean donor chimerism >90%; donor HSCs alloengraftment was verified in secondary recipients. In Fancc-/- and Fancg-/- recipients of fully allogeneic grafts, PB mean donor chimerism was 60% to 80% and 70% to 80%, respectively. The mean percent donor chimerism in BM and spleen mirrored PB results. CD45-ADC-conditioned mice did not have clinical toxicity. A transient <2.5-fold increase in hepatocellular enzymes and mild-to-moderate histopathological changes were seen. Under GVHD allo-HSCT conditions, wild-type and Fanca-/- recipients of CD45-ADC had markedly reduced GVHD lethality compared with lethal irradiation. Moreover, single dose anti-human CD45-ADC given to rhesus macaque nonhuman primates on days -6 or -10 was at least as myeloablative as lethal irradiation. These data suggest that CD45-ADC can potently promote donor alloengraftment and hematopoiesis without significant toxicity or severe GVHD, as seen with lethal irradiation, providing strong support for clinical trial considerations in highly vulnerable patients with FA.


Asunto(s)
Anemia de Fanconi , Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Inmunoconjugados , Antígenos Comunes de Leucocito , Animales , Anemia de Fanconi/terapia , Ratones , Enfermedad Injerto contra Huésped/patología , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Acondicionamiento Pretrasplante/métodos , Trasplante Homólogo , Ratones Endogámicos C57BL , Ratones Noqueados
2.
Blood ; 139(11): 1743-1759, 2022 03 17.
Artículo en Inglés | MEDLINE | ID: mdl-34986233

RESUMEN

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a potentially curative treatment of patients with nonmalignant or malignant blood disorders. Its success has been limited by graft-versus-host disease (GVHD). Current systemic nontargeted conditioning regimens mediate tissue injury and potentially incite and amplify GVHD, limiting the use of this potentially curative treatment beyond malignant disorders. Minimizing systemic nontargeted conditioning while achieving alloengraftment without global immune suppression is highly desirable. Antibody-drug-conjugates (ADCs) targeting hematopoietic cells can specifically deplete host stem and immune cells and enable alloengraftment. We report an anti-mouse CD45-targeted-ADC (CD45-ADC) that facilitates stable murine multilineage donor cell engraftment. Conditioning with CD45-ADC (3 mg/kg) was effective as a single agent in both congenic and minor-mismatch transplant models resulting in full donor chimerism comparable to lethal total body irradiation (TBI). In an MHC-disparate allo-HSCT model, pretransplant CD45-ADC (3 mg/kg) combined with low-dose TBI (150 cGy) and a short course of costimulatory blockade with anti-CD40 ligand antibody enabled 89% of recipients to achieve stable alloengraftment (mean value: 72%). When CD45-ADC was combined with pretransplant TBI (50 cGy) and posttransplant rapamycin, cyclophosphamide (Cytoxan), or a JAK inhibitor, 90% to 100% of recipients achieved stable chimerism (mean: 77%, 59%, 78%, respectively). At a higher dose (5 mg/kg), CD45-ADC as a single agent was sufficient for rapid, high-level multilineage chimerism sustained through the 22 weeks observation period. Therefore, CD45-ADC has the potential utility to confer the benefit of fully myeloablative conditioning but with substantially reduced toxicity when given as a single agent or at lower doses in conjunction with reduced-intensity conditioning.


Asunto(s)
Enfermedad Injerto contra Huésped , Trasplante de Células Madre Hematopoyéticas , Inmunoconjugados , Animales , Quimerismo , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Inmunoconjugados/farmacología , Inmunoconjugados/uso terapéutico , Inmunoconjugados/toxicidad , Ratones , Acondicionamiento Pretrasplante/métodos
3.
Nat Commun ; 14(1): 6291, 2023 10 12.
Artículo en Inglés | MEDLINE | ID: mdl-37828021

RESUMEN

Hematopoietic stem cell (HSC) gene therapy has curative potential; however, its use is limited by the morbidity and mortality associated with current chemotherapy-based conditioning. Targeted conditioning using antibody-drug conjugates (ADC) holds promise for reduced toxicity in HSC gene therapy. Here we test the ability of an antibody-drug conjugate targeting CD117 (CD117-ADC) to enable engraftment in a non-human primate lentiviral gene therapy model of hemoglobinopathies. Following single-dose CD117-ADC, a >99% depletion of bone marrow CD34 + CD90 + CD45RA- cells without lymphocyte reduction is observed, which results are not inferior to multi-day myeloablative busulfan conditioning. CD117-ADC, similarly to busulfan, allows efficient engraftment, gene marking, and vector-derived fetal hemoglobin induction. Importantly, ADC treatment is associated with minimal toxicity, and CD117-ADC-conditioned animals maintain fertility. In contrast, busulfan treatment commonly causes severe toxicities and infertility in humans. Thus, the myeloablative capacity of single-dose CD117-ADC is sufficient for efficient engraftment of gene-modified HSCs while preserving fertility and reducing adverse effects related to toxicity in non-human primates. This targeted conditioning approach thus provides the proof-of-principle to improve risk-benefit ratio in a variety of HSC-based gene therapy products in humans.


Asunto(s)
Trasplante de Células Madre Hematopoyéticas , Inmunoconjugados , Animales , Busulfano/farmacología , Terapia Genética/métodos , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas , Inmunoconjugados/farmacología , Proteínas Proto-Oncogénicas c-kit/inmunología , Proteínas Proto-Oncogénicas c-kit/uso terapéutico , Macaca mulatta/inmunología
4.
Front Immunol ; 11: 588543, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-33343569

RESUMEN

Cytokines that signal through the JAK-STAT pathway, such as interferon-γ (IFN-γ) and common γ chain cytokines, contribute to the destruction of insulin-secreting ß cells by CD8+ T cells in type 1 diabetes (T1D). We previously showed that JAK1/JAK2 inhibitors reversed autoimmune insulitis in non-obese diabetic (NOD) mice and also blocked IFN-γ mediated MHC class I upregulation on ß cells. Blocking interferons on their own does not prevent diabetes in knockout NOD mice, so we tested whether JAK inhibitor action on signaling downstream of common γ chain cytokines, including IL-2, IL-7 IL-15, and IL-21, may also affect the progression of diabetes in NOD mice. Common γ chain cytokines activate JAK1 and JAK3 to regulate T cell proliferation. We used a JAK1-selective inhibitor, ABT 317, to better understand the specific role of JAK1 signaling in autoimmune diabetes. ABT 317 reduced IL-21, IL-2, IL-15 and IL-7 signaling in T cells and IFN-γ signaling in ß cells, but ABT 317 did not affect GM-CSF signaling in granulocytes. When given in vivo to NOD mice, ABT 317 reduced CD8+ T cell proliferation as well as the number of KLRG+ effector and CD44hiCD62Llo effector memory CD8+ T cells in spleen. ABT 317 also prevented MHC class I upregulation on ß cells. Newly diagnosed diabetes was reversed in 94% NOD mice treated twice daily with ABT 317 while still on treatment at 40 days and 44% remained normoglycemic after a further 60 days from discontinuing the drug. Our results indicate that ABT 317 blocks common γ chain cytokines in lymphocytes and interferons in lymphocytes and ß cells and are thus more effective against diabetes pathogenesis than IFN-γ receptor deficiency alone. Our studies suggest use of this class of drug for the treatment of type 1 diabetes.


Asunto(s)
Diabetes Mellitus Tipo 1/inmunología , Interferón gamma/inmunología , Subunidad gamma Común de Receptores de Interleucina/inmunología , Janus Quinasa 1/antagonistas & inhibidores , Inhibidores de las Cinasas Janus/farmacología , Animales , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/inmunología , Inhibidores de las Cinasas Janus/farmacocinética , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Transducción de Señal/efectos de los fármacos , Bazo/inmunología
5.
Int J Med Sci ; 5(3): 133-42, 2008 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-18566677

RESUMEN

OBJECTIVES: To find potential diagnostic markers or therapeutic targets, we used differential display technique to identify genes that are over or under expressed in human ovarian cancer. METHODS: Genes were initially identified by differential display between two human ovarian surface epithelium cultures and two ovarian cancer cell lines, A2780 and Caov-3. Genes were validated by relative quantitative RT-PCR and RNA in situ hybridization. RESULTS: Twenty-eight non-redundant sequences were expressed differentially in the normal ovarian epithelium and ovarian cancer cell lines. Seven of the 28 sequences showed differential expression between normal ovary and ovarian cancer tissue by RT-PCR. USP36 was over-expressed in ovarian cancer cell lines and tissues by RT-PCR and RNA in situ hybridization. Northern blot analysis and RT-PCR revealed two transcripts for USP36 in ovarian tissue. The major transcript was more specific for ovarian cancer and was detected by RT-PCR in 9/9 ovarian cancer tissues, 3/3 cancerous ascites, 5/14 (36%) sera from patients with ovarian cancer, and 0/7 sera from women without ovarian cancer. CONCLUSION: USP36 is overexpressed in ovarian cancer compared to normal ovary and its transcripts were identified in ascites and serum of ovarian cancer patients.


Asunto(s)
Ascitis/metabolismo , Regulación Neoplásica de la Expresión Génica , Neoplasias Ováricas/metabolismo , Ovario/metabolismo , Ubiquitina Tiolesterasa/biosíntesis , Ubiquitina Tiolesterasa/fisiología , Secuencia de Bases , Biomarcadores de Tumor , Línea Celular Tumoral , Femenino , Perfilación de la Expresión Génica , Humanos , Hibridación in Situ , Modelos Biológicos , Datos de Secuencia Molecular , Neoplasias Ováricas/sangre , Ubiquitina/metabolismo
6.
Exp Biol Med (Maywood) ; 231(11): 1718-25, 2006 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-17138758

RESUMEN

To further develop the hen as a model of ovarian adenocarcinoma, we have studied normal and neoplastic ovaries as well as cultured cells from the ovarian surface epithelium (OSE). We characterized the OSE layer of the hen for specific histologic markers and evaluated these markers on tumor tissue. We also isolated and characterized the epithelial cells that are the likely source of the ovarian tumors of the hen. The surface epithelium of normal ovaries demonstrated positive staining for cytokeratin, proliferating cell nuclear antigen (PCNA), progesterone receptor (PR), and negative staining for vimentin. Ovarian tumors demonstrated positive cytokeratin, PCNA, PR, and weak vimentin staining in the gland-like areas. Epithelial cell cultures were obtained by an explant method utilizing small and large yellow follicles. These cells were positive for cytokeratin and negative for vimentin on Days 1 and 3. By Day 10, cytokeratin protein expression was less for some cells, and vimentin expression was weakly present in some cells. Expression of PCNA was observed at Days 1 and 3, but was rarely seen in cells cultured for 10 days. Expression of PR was observed on Day 10 after 24-hr estrogen treatment. Epithelial cells grew slowly in culture, and were susceptible to trypsin or other dissociation treatments.


Asunto(s)
Células Epiteliales/citología , Células Epiteliales/patología , Neoplasias Ováricas/patología , Ovario/citología , Ovario/patología , Adenocarcinoma/patología , Adenocarcinoma/fisiopatología , Animales , Biomarcadores , Pollos , Modelos Animales de Enfermedad , Células Epiteliales/fisiología , Femenino , Queratinas/análisis , Neoplasias Ováricas/fisiopatología , Ovario/fisiología , Antígeno Nuclear de Célula en Proliferación/análisis , Receptores de Progesterona/análisis , Valores de Referencia , Vimentina/análisis
7.
Clin Exp Metastasis ; 20(8): 757-66, 2003.
Artículo en Inglés | MEDLINE | ID: mdl-14713109

RESUMEN

The aim of the study was to investigate inhibitory effects of the receptor tyrosine kinase (RTK) inhibitor SU11248 against CSF-1R and osteoclast (OC) formation. We developed an in vivo model of breast cancer metastasis to evaluate efficacy of SU11248 against tumor growth and tumor-induced osteolysis in bone. The in vitro effects of SU11248 on CSF-1R phosphorylation, OC formation and function were evaluated. Effects on 435/HAL-Luc tumor growth in bone were monitored by in vivo bioluminescence imaging (BLI), and inhibition of osteolysis was evaluated by measurement of serum pyridinoline (PYD) concentration and histology. Phosphorylation of the receptor for M-CSF (CSF-1R) expressed by NIH3T3 cells was inhibited by SU11248 with an IC50 of 50-100 nM, consistent with CSF-1R belonging to the class III split kinase domain RTK family. The early M-CSF-dependent phase of in vitro murine OC development and function were inhibited by SU11248 at 10-100 nM. In vivo inhibition of osteolysis was confirmed by significant lowering of serum PYD levels following SU11248 treatment of tumor-bearing mice (P = 0.047). Using BLI, SU11248 treatment at 40 mg/kg/day for 21 days showed 64% inhibition of tumor growth in bone (P = 0.006), and at 80 mg/kg/day showed 89% inhibition (P = 0.001). Collectively, these data suggest that SU11248 may be an effective and tolerated therapy to inhibit growth of breast cancer bone metastases, with the additional advantage of inhibiting tumor-associated osteolysis.


Asunto(s)
Neoplasias Óseas/patología , Neoplasias Óseas/fisiopatología , Indoles/farmacología , Osteólisis , Pirroles/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Animales , Neoplasias Óseas/secundario , Neoplasias de la Mama/patología , Adhesión Celular , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Osteoclastos , Fosforilación , Sunitinib , Células Tumorales Cultivadas
8.
Appl Immunohistochem Mol Morphol ; 12(2): 132-8, 2004 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15354738

RESUMEN

Using differential display mRNA techniques, the authors found cDNA of the heat shock 70 protein known as GRP75 overexpressed in ovarian cancer cell lines. In the current study, the authors used immunohistochemistry to characterize the expression pattern of GRP75 in ovarian carcinomas and compared it with epithelial tumors originating from the female reproductive tract, epithelial neoplasms from non-gynecologic sites (colon, pancreas, breast, and lung), and various normal tissues. The authors also developed an antigen capture ELISA assay to determine if GRP75 can be detected in tumors, ascites, or sera of patients with advanced mullerian adenocarcinomas. All epithelial tumors from the ovary and the female reproductive tract were positive for GRP75 expression with moderate to strong staining intensity; stromal expression of GRP75 was generally weak or absent. Adenocarcinomas from the colon, lung, pancreas, and breast also stained strongly positive for GRP75. The epithelial cells of all normal tissues examined were positive for GRP75, and strong staining was also seen in the corpora lutea, hepatocytes, enteric neural plexus of the esophagus and colon, and placental cytotrophoblast and syncytiotrophoblast, and in subpopulations of pancreatic acinar cells. The ELISA assay detected GRP75 in tumor lysates and ascitic fluid, but not sera, of patients with mullerian adenocarcinomas. The authors conclude that GRP75 is highly expressed in both benign and malignant epithelium, as well as cells of specialized function from a variety of tissues.


Asunto(s)
Antígenos de Neoplasias/análisis , Neoplasias de los Genitales Femeninos/química , Proteínas HSP70 de Choque Térmico/análisis , Proteínas de la Membrana/análisis , Neoplasias Glandulares y Epiteliales/química , Adenocarcinoma/química , Adenocarcinoma/patología , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Neoplasias de los Genitales Femeninos/patología , Humanos , Inmunohistoquímica , Neoplasias Glandulares y Epiteliales/patología , Neoplasias Ováricas/química , Neoplasias Ováricas/patología , Sensibilidad y Especificidad , Distribución Tisular
9.
J Neuroimmunol ; 239(1-2): 37-43, 2011 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-21911260

RESUMEN

Immunological responses to protect against excessive inflammation can be regulated by the central nervous system through the cholinergic anti-inflammatory pathway wherein acetylcholine released from vagus nerves can inhibit inflammatory cytokines. Although a role for the α7 nicotinic acetylcholine receptor (α7 nAChR) in mediating this pathway has been suggested, pharmacological modulation of the pathway by selective agonists remains to be further elucidated. In this study, the role of α7 nAChRs in the regulation of TNF-α release was investigated using high affinity and selective α7 nAChR agonists in mouse peritoneal macrophage and human whole blood in vitro, and in mouse serum in vivo. In mouse peritoneal macrophages, LPS-induced TNF-α release in vitro was inhibited by a selective α7 nAChR agonist, A-833834 (5-[6-(5-Methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-pyridazin-3-yl]-1H-indole), and that effect was attenuated by α7 nAChR antagonist methyllycaconitine. The inhibitory effect of A-833834 on LPS-induced TNF-α release was also observed in human whole blood in vitro. I.v. LPS-induced TNF-α release in mouse serum was attenuated following i.p. administration of A-833834. Similarly, i.v. LPS-induced TNF-α release in mouse serum was also attenuated following i.p. administration of A-585539, another α7 nAChR agonist with limited brain penetration, suggesting that these effects are mediated by peripheral α7 nAChRs. A-833834 was also efficacious in suppressing TNF-α release in mouse serum following oral administration in zymosan-induced peritonitis. These studies collectively demonstrate that selectively targeting α7 nAChRs could offer a novel therapeutic modality to treat acute and chronic inflammatory disease states.


Asunto(s)
Agonistas Nicotínicos/sangre , Agonistas Nicotínicos/farmacología , Receptores Nicotínicos/fisiología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Citocinas/antagonistas & inhibidores , Citocinas/sangre , Citocinas/metabolismo , Femenino , Humanos , Mediadores de Inflamación/agonistas , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/sangre , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Oocitos , Peritonitis/tratamiento farmacológico , Peritonitis/inmunología , Peritonitis/patología , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Receptores Nicotínicos/sangre , Receptores Nicotínicos/metabolismo , Factor de Necrosis Tumoral alfa/sangre , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7
10.
Proc Natl Acad Sci U S A ; 104(25): 10619-24, 2007 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-17537919

RESUMEN

Bone is accrued and maintained primarily through the coupled actions of bone-forming osteoblasts and bone-resorbing osteoclasts. Cumulative in vitro studies indicated that proline-rich tyrosine kinase 2 (PYK2) is a positive mediator of osteoclast function and activity. However, our investigation of PYK2-/- mice did not reveal evidence supporting an essential function for PYK2 in osteoclasts either in vivo or in culture. We find that PYK2-/- mice have high bone mass resulting from an unexpected increase in bone formation. Consistent with the in vivo findings, mouse bone marrow cultures show that PYK2 deficiency enhances differentiation and activity of osteoprogenitor cells, as does expressing a PYK2-specific short hairpin RNA or dominantly interfering proteins in human mesenchymal stem cells. Furthermore, the daily administration of a small-molecule PYK2 inhibitor increases bone formation and protects against bone loss in ovariectomized rats, an established preclinical model of postmenopausal osteoporosis. In summary, we find that PYK2 regulates the differentiation of early osteoprogenitor cells across species and that inhibitors of the PYK2 have potential as a bone anabolic approach for the treatment of osteoporosis.


Asunto(s)
Quinasa 2 de Adhesión Focal/fisiología , Osteoblastos/fisiología , Osteoclastos/fisiología , Osteogénesis/fisiología , Osteoporosis/terapia , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/fisiología , Diferenciación Celular , Células Cultivadas , Inhibidores Enzimáticos/uso terapéutico , Femenino , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Noqueados , Ovariectomía , Ratas , Ratas Sprague-Dawley
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