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1.
J Immunol Methods ; 463: 112-121, 2018 12.
Article in English | MEDLINE | ID: mdl-30321549

ABSTRACT

BACKGROUND AND PURPOSE: Phospho flow cytometry is a powerful technique to analyze signaling in rare cell populations. This technique, however, requires harsh conditions for cell fixation and permeabilization, which can denature surface antigens or antibody-conjugated fluorochromes. These are among several technical limitations which have been a barrier to quantify signaling in unique B cell subsets. One such immature subset, transitional B cells (TrBs), may play a role in suppressing solid organ transplant rejection, graft-versus-host disease, autoimmunity, and even the immune response to malignancy. Here we sought to optimize a protocol for quantification of signaling in human TrBs compared with mature B cell subsets. RESULTS: TrBs were defined by surface marker expression as CD19+CD24hiCD38hi. Key parameters optimized included antibody clone selection, sequence of surface epitope labeling in relation to paraformaldehyde-based fixation and methanol-based permeabilization, photomultiplier tube (PMT) voltages, and compensation. Special attention was paid to labeling of CD38 with regard to these parameters, and an optimized protocol enabled reliable identification of TrBs, naïve (CD24+CD38+), early memory (CD24hiCD38-), and late memory (CD24-CD38-) B cells. Phospho flow cytometry enabled simultaneous quantification of phosphorylation among at least three different signaling molecules within the same sample. Among normal donors, transitional B cells exhibited diminished mitogen activated protein kinase/extracellular signal-regulated kinase and Akt phospho signaling upon nonspecific stimulation with phorbol 12-myristate 13-acetateand ionomycin stimulation. CONCLUSIONS: We optimized an effective protocol to quantify B cell subset signaling upon stimulation. Such a protocol may ultimately serve as the basis for assessing dysfunctional B cell signaling in disease, predict clinical outcomes, and monitor response to B cell-directed therapies.


Subject(s)
Antigens, CD/immunology , B-Lymphocyte Subsets , Flow Cytometry/methods , Immunologic Memory , MAP Kinase Signaling System/immunology , B-Lymphocyte Subsets/cytology , B-Lymphocyte Subsets/immunology , Female , Humans , MAP Kinase Signaling System/drug effects , Male , Tetradecanoylphorbol Acetate/pharmacology
2.
Proc Natl Acad Sci U S A ; 113(8): E1016-25, 2016 Feb 23.
Article in English | MEDLINE | ID: mdl-26862168

ABSTRACT

Primary T-cell acute lymphoblastic leukemia (T-ALL) cells require stromal-derived signals to survive. Although many studies have identified cell-intrinsic alterations in signaling pathways that promote T-ALL growth, the identity of endogenous stromal cells and their associated signals in the tumor microenvironment that support T-ALL remains unknown. By examining the thymic tumor microenvironments in multiple murine T-ALL models and primary patient samples, we discovered the emergence of prominent epithelial-free regions, enriched for proliferating tumor cells and dendritic cells (DCs). Systematic evaluation of the functional capacity of tumor-associated stromal cells revealed that myeloid cells, primarily DCs, are necessary and sufficient to support T-ALL survival ex vivo. DCs support T-ALL growth both in primary thymic tumors and at secondary tumor sites. To identify a molecular mechanism by which DCs support T-ALL growth, we first performed gene expression profiling, which revealed up-regulation of platelet-derived growth factor receptor beta (Pdgfrb) and insulin-like growth factor I receptor (Igf1r) on T-ALL cells, with concomitant expression of their ligands by tumor-associated DCs. Both Pdgfrb and Igf1r were activated in ex vivo T-ALL cells, and coculture with tumor-associated, but not normal thymic DCs, sustained IGF1R activation. Furthermore, IGF1R signaling was necessary for DC-mediated T-ALL survival. Collectively, these studies provide the first evidence that endogenous tumor-associated DCs supply signals driving T-ALL growth, and implicate tumor-associated DCs and their mitogenic signals as auspicious therapeutic targets.


Subject(s)
Dendritic Cells/immunology , Neoplasm Proteins/immunology , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/immunology , Receptors, Somatomedin/immunology , Signal Transduction/immunology , Tumor Microenvironment/immunology , Animals , Cell Line, Tumor , Cell Survival , Dendritic Cells/pathology , Female , Humans , Male , Mice , Neoplasm Proteins/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Receptor, IGF Type 1 , Receptor, Platelet-Derived Growth Factor beta/genetics , Receptor, Platelet-Derived Growth Factor beta/immunology , Receptors, Somatomedin/genetics , Signal Transduction/genetics , Tumor Microenvironment/genetics
3.
Development ; 141(15): 2950-8, 2014 Aug.
Article in English | MEDLINE | ID: mdl-25053428

ABSTRACT

The thymus and parathyroid glands arise from a shared endodermal primordium in the third pharyngeal pouch (3rd pp). Thymus fate is specified in the ventral 3rd pp between E9.5 and E11, whereas parathyroid fate is specified in the dorsal domain. The molecular mechanisms that specify fate and regulate thymus and parathyroid development are not fully delineated. Previous reports suggested that Tbx1 is required for thymus organogenesis because loss of Tbx1 in individuals with DiGeorge syndrome and in experimental Tbx1 deletion mutants is associated with thymus aplasia or hypoplasia. However, the thymus phenotype is likely to be secondary to defects in pharyngeal pouch formation. Furthermore, the absence of Tbx1 expression in the thymus-fated domain of the wild-type 3rd pp suggested that Tbx1 is instead a negative regulator of thymus organogenesis. To test this hypothesis, we generated a novel mouse strain in which expression of a conditional Tbx1 allele was ectopically activated in the thymus-fated domain of the 3rd pp. Ectopic Tbx1 expression severely repressed expression of Foxn1, a transcription factor that marks the thymus-fated domain and is required for differentiation and proliferation of thymic epithelial cell (TEC) progenitors. By contrast, ectopic Tbx1 did not alter the expression pattern of Gcm2, a transcription factor restricted to the parathyroid-fated domain and required for parathyroid development. Ectopic Tbx1 expression impaired TEC proliferation and arrested TEC differentiation at an early progenitor stage. The results support the hypothesis that Tbx1 negatively regulates TEC growth and differentiation, and that extinction of Tbx1 expression in 3rd pp endoderm is a prerequisite for thymus organogenesis.


Subject(s)
Gene Expression Regulation, Developmental , Organogenesis , T-Box Domain Proteins/physiology , Thymus Gland/embryology , Alleles , Animals , Cell Differentiation , Cell Lineage , Cell Proliferation , Endoderm , Female , Forkhead Transcription Factors/physiology , Gene Deletion , Male , Mice , Mice, Inbred C57BL , Mutation , Nuclear Proteins/physiology , Stem Cells/cytology , T-Box Domain Proteins/genetics , Time Factors , Transcription Factors/physiology
4.
J Biol Chem ; 287(1): 429-437, 2012 Jan 02.
Article in English | MEDLINE | ID: mdl-22072714

ABSTRACT

In epigenetic signaling pathways, histone tails are heavily modified, resulting in the recruitment of effector molecules that can influence transcription. One such molecule, plant homeodomain finger protein 20 (PHF20), uses a Tudor domain to read dimethyl lysine residues and is a known component of the MOF (male absent on the first) histone acetyltransferase protein complex, suggesting it plays a role in the cross-talk between lysine methylation and histone acetylation. We sought to investigate the biological role of PHF20 by generating a knockout mouse. Without PHF20, mice die shortly after birth and display a wide variety of phenotypes within the skeletal and hematopoietic systems. Mechanistically, PHF20 is not required for maintaining the global H4K16 acetylation levels or locus specific histone acetylation but instead works downstream in transcriptional regulation of MOF target genes.


Subject(s)
Gene Expression Regulation/genetics , Histone Acetyltransferases/metabolism , Homeodomain Proteins/genetics , Lysine/metabolism , Animals , DNA-Binding Proteins , Female , Gene Knockout Techniques , Histones/chemistry , Histones/metabolism , Homeodomain Proteins/metabolism , Male , Mice , Transcription Factors , Transcription, Genetic/genetics
5.
Proc Natl Acad Sci U S A ; 107(30): 13414-9, 2010 Jul 27.
Article in English | MEDLINE | ID: mdl-20616004

ABSTRACT

Thymus organogenesis requires coordinated interactions of multiple cell types, including neural crest (NC) cells, to orchestrate the formation, separation, and subsequent migration of the developing thymus from the third pharyngeal pouch to the thoracic cavity. The molecular mechanisms driving these processes are unclear; however, NC-derived mesenchyme has been shown to play an important role. Here, we show that, in the absence of ephrin-B2 expression on thymic NC-derived mesenchyme, the thymus remains in the cervical area instead of migrating into the thoracic cavity. Analysis of individual NC-derived thymic mesenchymal cells shows that, in the absence of ephrin-B2, their motility is impaired as a result of defective EphB receptor signaling. This implies a NC-derived cell-specific role of EphB-ephrin-B2 interactions in the collective migration of the thymic rudiment during organogenesis.


Subject(s)
Ephrin-B2/metabolism , Organogenesis , Receptors, Eph Family/metabolism , Thymus Gland/embryology , Animals , Cell Movement , Embryo, Mammalian/cytology , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Ephrin-B2/genetics , Female , Flow Cytometry , Immunohistochemistry , Male , Mesoderm/cytology , Mesoderm/metabolism , Mice , Mice, Knockout , Microscopy, Confocal , Nervous System/cytology , Nervous System/embryology , Nervous System/metabolism , Protein Binding , Thymus Gland/cytology , Thymus Gland/innervation
6.
Genesis ; 47(4): 281-7, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19263498

ABSTRACT

IL-7 is a cytokine that is required for T-cell development and homeostasis as well as for lymph node organogenesis. Despite the importance of IL-7 in the immune system and its potential therapeutic relevance, questions remain regarding the sites of IL-7 synthesis, specific cell types involved and molecular mechanisms regulating IL-7 expression. To address these issues, we generated two bacterial artificial chromosome (BAC) transgenic mouse lines in which IL-7 regulatory elements drive expression of either Cre recombinase or a human CD25 (hCD25) cell surface reporter molecule. Expression of the IL-7.hCD25 BAC transgene, detected by reactivity with anti-hCD25 antibody, mimicked endogenous IL-7 expression. Fetal and adult tissues from crosses between IL-7.Cre transgenic mice and Rosa26R or R26-EYFP reporters demonstrated X-gal or YFP staining in tissues known to express endogenous IL-7 at some stage during development. These transgenic lines provide novel genetic tools to identify IL-7 producing cells in various tissues and to manipulate gene expression selectively in IL-7 expressing cells.


Subject(s)
Integrases/metabolism , Interleukin-2 Receptor alpha Subunit/metabolism , Interleukin-7/metabolism , Animals , Animals, Newborn , Chromosomes, Artificial, Bacterial/genetics , Female , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation, Developmental , Humans , Immunohistochemistry , Integrases/genetics , Interleukin-2 Receptor alpha Subunit/genetics , Interleukin-7/genetics , Male , Mice , Mice, Transgenic , Promoter Regions, Genetic/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Thymus Gland/cytology , Thymus Gland/embryology , Thymus Gland/metabolism
7.
Dev Biol ; 327(1): 216-27, 2009 Mar 01.
Article in English | MEDLINE | ID: mdl-19135046

ABSTRACT

Embryos that are homozygous for Splotch, a null allele of Pax3, have a severe neural crest cell (NCC) deficiency that generates a complex phenotype including spina bifida, exencephaly and cardiac outflow tract abnormalities. Contrary to the widely held perception that thymus aplasia or hypoplasia is a characteristic feature of Pax3(Sp/Sp) embryos, we find that thymic rudiments are larger and parathyroid rudiments are smaller in E11.5-12.5 Pax3(Sp/Sp) compared to Pax3(+/+) embryos. The thymus originates from bilateral third pharyngeal pouch primordia containing endodermal progenitors of both thymus and parathyroid glands. Analyses of Foxn1 and Gcm2 expression revealed a dorsal shift in the border between parathyroid- and thymus-fated domains at E11.5, with no change in the overall cellularity or volume of each shared primordium. The border shift increases the allocation of third pouch progenitors to the thymus domain and correspondingly decreases allocation to the parathyroid domain. Initial patterning in the E10.5 pouch was normal suggesting that the observed change in the location of the organ domain interface arises during border refinement between E10.5 and E11.5. Given the well-characterized NCC defects in Splotch mutants, these findings implicate NCCs in regulating patterning of third pouch endoderm into thymus- versus parathyroid-specified domains, and suggest that organ size is determined in part by the number of progenitor cells specified to a given fate.


Subject(s)
Paired Box Transcription Factors/genetics , Parathyroid Glands/embryology , Thymus Gland/embryology , Animals , Body Patterning , Embryo, Mammalian , Embryonic Development , Embryonic Induction , Forkhead Transcription Factors/genetics , Mice , Mice, Mutant Strains , Nuclear Proteins/genetics , PAX3 Transcription Factor , Parathyroid Glands/cytology , Thymus Gland/cytology , Transcription Factors/genetics
8.
Mol Carcinog ; 48(6): 508-16, 2009 Jun.
Article in English | MEDLINE | ID: mdl-18942117

ABSTRACT

Cyclin D1b is an alternative transcript of the cyclin D1 gene (CCND1) expressed in human tumors. Its abundance is regulated by a single base pair polymorphism at the exon 4/intron 4 boundary (nucleotide 870). Epidemiological studies have shown a correlation between the presence of the G870A allele (that favors the splicing for cyclin D1b) with increased risk and less favorable outcome in several forms of cancer. More recently, it has been shown that, unlike cyclin D1a, the alternative transcript D1b by itself has the capacity to transform fibroblasts in vitro. In order to study the oncogenic potential of cyclin D1b, we developed transgenic mice expressing human cyclin D1b under the control of the bovine K5 promoter (K5D1b mice). Seven founders were obtained and none of them presented any significant phenotype or developed spontaneous tumors. Interestingly, K5D1b mice do not develop the fatal thymic hyperplasia, which is characteristic of the cyclin D1a transgenic mice (K5D1a). Susceptibility to skin carcinogenesis was tested in K5D1b mice using two-stage carcinogenesis protocols. In two independent experiments, K5D1b mice developed higher papilloma multiplicity as compared with wild-type littermates. However, when K5D1b mice were crossed with cyclin D1KO mice, the expression of cyclin D1b was unable to rescue the carcinogenesis-resistant phenotype of the cyclin D1 KO mice. To further explore the role of cyclin D1b in mouse models of carcinogenesis we carried out in silico analysis and in vitro experiments to evaluate the existence of a mouse homologous of the human cyclin D1b transcript. We were unable to find any evidence of an alternatively spliced transcript in mouse Ccnd1. These results show that human cyclin D1b has different biological functions than cyclin D1a and confirm its oncogenic properties.


Subject(s)
Cell Transformation, Neoplastic/genetics , Cyclin D1/genetics , Skin Neoplasms/genetics , Thymus Gland/pathology , Animals , Base Sequence , DNA Primers , Exons , Hyperplasia , Introns , Mice , Mice, Transgenic , Polymerase Chain Reaction , Polymorphism, Single Nucleotide
9.
Postgrad Med ; 113(2): 34-6, 39-40, 43-6, 2003 Feb.
Article in English | MEDLINE | ID: mdl-12611113

ABSTRACT

A comprehensive breast cancer screening program needs to include risk assessment in addition to clinical breast examination and mammography. Women identified as being at increased risk should have an individualized schedule of screening mammography and a proven prevention program tailored to their level of risk. In this article, Drs Cardenas and Frisch review risk factors, screening methods, and individual risk assessment, then explain how to use them in conjunction to identify tumors at an earlier, more curable stage.


Subject(s)
Breast Neoplasms/diagnosis , Breast Neoplasms/prevention & control , Mass Screening/methods , Aged , Breast Neoplasms/diagnostic imaging , Estrogen Replacement Therapy , Female , Genetic Predisposition to Disease , Humans , Mammography , Middle Aged , Program Evaluation , Risk Assessment , Risk Factors , United States , Women's Health
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