Biomedical subjects

Mark H Kaplan

Publications and source records attributed to Mark H Kaplan.

33 records · Page 2Linked to original sources

PubMed2004

Partially unspliced and fully spliced ELF3 mRNA, including a new Alu element in human breast cancer.

Using modified representational difference analysis, a DNA fragment (GC3) was isolated as a difference between a breast cancer and a normal cell line from the same patient. GC3 proved to be a fragment of intron 7 of the ELF3 gene, an ets family transcription factor, amplified in the breast cancer cell line. Using genomic walking technology, a new Alu (Alu(kwd)) was found downstream of GC3 in an antisense position between nt 8762 and nt 8763 within intron 8 of the ELF3 gene. This ELF3 intron fragment(GC3) was expressed in human breast cancer cell lines and four of six breast cancer tissues, but not in matched normal cell lines and tissues. Similarly, Alu(kwd) was also found in the same breast cancer cell lines and five of eight other breast cancer tissues, but not in matched normal cell lines and tissue. This was confirmed by RNase and DNase digestion analysis. Moreover, GC3 and Alu(kwd) were detected in both the nuclear and cytoplasmic RNA fractions of breast cancer cell lines. The finding of cytoplasmic intron retention was verified with northern blotting and the 5' and 3' rapid amplification cDNA ends procedure (5' and 3'RACE) to search for cDNA sequences in RNA from these cancer cell lines. Partially unspliced ELF3 mRNA and fully spliced ELF3 mRNA was found in the same breast cancer cell line. Partially unspliced ELF3 mRNA contained introns 4-7 without any nucleotide mutation at intron/exon splice junction borders. Fully spliced 1959 bp ELF3 mRNA showed a different 5'UTR from the published ELF3 mRNA, and was predicted to encode a 371 amino acid protein sharing 98% homology with the ELF3 protein sequence. This is the first report of intron retention of ELF3 as well as the pathological appearance of both spliced and unspliced cytoplasmic ELF3 mRNA in human breast cancer cells.

Alu Elements↗

PubMed2004

Respiratory syncytial virus causes increased bronchial epithelial permeability.

BACKGROUND: Respiratory syncytial virus (RSV)-induced diseases are mediated through active cytokines released during infection. We hypothesized that RSV infection causes bronchial epithelial monolayer permeability in vitro via induction of vascular endothelial growth factor (VEGF). METHODS: Human bronchial epithelial cells were infected with RSV. In some cultures, VEGF antibody was included to block VEGF response; in other cultures, palivizumab was added to block RSV infection. Permeability was assessed in real-time using electric cell-substrate impedance sensing. VEGF release was assessed using enzyme-linked immunosorbent assay. Gap formation was assessed using live cell imaging. RESULTS: RSV-infected cells demonstrated a decrease in the resistance of the monolayer indicating an increase in permeability; this increase was blocked with VEGF-specific antibody, and palivizumab. Intercellular gap formation developed in RSV-infected epithelial monolayers. CONCLUSION: RSV increases permeability of the bronchial airway epithelial monolayer via VEGF induction.

Bronchi↗

PubMed2003

STAT4 is required for interleukin-12-induced chromatin remodeling of the CD25 locus.

Signal transducer and activator of transcription 4 (STAT4) is a critical mediator of interleukin-12 (IL-12)-stimulated inflammatory immune responses. Despite extensive analysis of the immune responses of STAT4-deficient mice, there is still very little understood about STAT4-dependent gene induction. IL-12 stimulated increases in IL-2 receptor alpha chain gene (CD25) mRNA levels and surface expression require STAT4. In this report, we utilize chromatin immunoprecipitation assays to analyze IL-12-stimulated and STAT4-dependent changes in chromatin remodeling of the CD25 gene. Gene activation requires binding of STAT4 to the PRRIII upstream regulatory element, the recruitment of the CREB-binding protein (CBP), and chromatin remodeling including increased acetylation and decreased methylation of histones within the CD25 promoter. Evidence suggests that STAT4 also facilitates binding of other factors to the CD25 promoter including c-Jun. Thus, these results provide a model for STAT4-dependent gene induction and a mechanism for cytokine-induced expression of the CD25 gene.

Animals↗

PubMed2003

Distinct requirements for the naturally occurring splice forms Stat4alpha and Stat4beta in IL-12 responses.

Signal transducer and activator of transcription (Stat)4 is a signaling molecule required for normal responses to interleukin-12 (IL-12) and is critically involved in inflammatory responses. We have isolated an alternatively spliced isoform of Stat4, termed Stat4beta, which lacks 44 amino acids at the C-terminus, encompassing the putative transcriptional activation domain. To assess the in vivo roles of these Stat4 isoforms, we generated transgenic Stat4-deficient mice expressing Stat4alpha or Stat4beta. Our results indicate that T-cell-specific expression of Stat4alpha or Stat4beta can mediate many aspects of IL-12 signaling including the differentiation of Th1 cells. However, Stat4alpha is required for normal levels of IL-12-induced interferon-gamma production from Th1 cells. Microarray analysis identified 98 genes induced by both Stat4 isoforms, 32 genes induced only by Stat4alpha and 29 genes induced only by Stat4beta. Some induced genes correlate with specific functions including the ability of Stat4beta, but not Stat4alpha, to mediate IL-12-stimulated proliferation. Thus, Stat4alpha and Stat4beta have distinct roles in mediating responses to IL-12.

Adjuvants, Immunologic↗

PubMed2003

STAT4 requires the N-terminal domain for efficient phosphorylation.

STAT4 (signal transducer and activator of transcription-4) mediates biological effects in response to interleukin-12 (IL-12). STAT4 has multiple domains that have distinct functions in signaling and gene activation. To characterize the role of the STAT4 N-terminal domain in mediating STAT4 biological function, we have generated STAT4-deficient transgenic mice that express human full-length STAT4 or an N-terminal deletion mutant (Delta N-STAT4) lacking the N-terminal 51 amino acids. Whereas full-length STAT4 rescued IL-12 responsiveness, T lymphocytes expressing the STAT4 N-terminal mutant failed to proliferate in response to IL-12 and had limited Th1 cell development as evidenced by minimal interferon-gamma production. Deletion of the N-terminal domain resulted in failure of STAT4 to be phosphorylated following IL-12 stimulation despite similar phosphorylation of JAK2 and TYK2 in full-length STAT4 and Delta N-STAT4 transgenic T cells. We demonstrate that the lack of phosphorylation in cultured cells is due to reduced efficiency of phosphorylation of Delta N-STAT4 by Janus kinases. These data support a new model wherein the N-terminal domain is required to mediate the phosphorylation of STAT4 in addition to the previously documented role in gene transactivation.

Amino Acid Sequence↗

PubMed2003

Expression of a constitutively active Stat6 in vivo alters lymphocyte homeostasis with distinct effects in T and B cells.

IL-4 is a critical cytokine in the regulation of immune responses and genesis of atopy. Engagement of the IL-4R activates multiple signaling pathways, including the transcription factor Stat6. Stat6-deficient mice demonstrate the importance of this factor in lymphocyte proliferation, gene expression, and Th cell differentiation. Recently, a mutant Stat6 (Stat6VT) was generated that is transcriptionally active independent of IL-4 stimulation. To determine the ability of a constitutively active Stat6 to mimic IL-4-stimulated responses, we have generated transgenic mice expressing Stat6VT under control of the CD2 locus control region, restricting expression to lymphoid populations. The phenotype of Stat6VT transgenic mice is similar, but not identical, to IL-4 transgenic mice, suggesting a critical role for Stat6-independent signaling pathways in the generation of some IL-4 responses in vivo. The expression of a constitutively active Stat6 in vivo increases surface expression of IL-4-induced genes and increases serum levels of IgG1 and IgE, compared with nontransgenic mice. Stat6VT expression increases Th2 differentiation in vivo and in vitro. Stat6VT expression also dramatically alters homeostasis of peripheral lymphocyte populations resulting in decreased CD3(+) cells and increased B220(+) cells, compared with nontransgenic littermates. Altered T and B cell populations correlate with an activated phenotype and increased cell death in transgenic T cell, but not B cell, populations. Together these results suggest that expression of a constitutively active Stat6 has distinct effects on B and T lymphocytes.

Animals↗

PubMed2003

STAT4 signal pathways regulate inflammation and airway physiology changes in allergic airway inflammation locally via alteration of chemokines.

Mice homozygous for the STAT4-null mutation were sensitized to cockroach Ag, challenged intratracheally 21 days later, and compared with STAT4-competent allergic mice. The STAT4(-/-) mice showed significant decreases in airway hyperreactivity (AHR) and peribronchial eosinophils compared with wild-type controls. In addition, pulmonary levels of chemokines were decreased in the STAT4(-/-) mice, including CC chemokine ligand (CCL)5, CCL6, CCL11, and CCL17. However, levels of Th2-type cytokines, such as IL-4 and IL-13, as well as serum IgE levels were similar in the two groups. Transfer of splenic lymphocytes from sensitized wild-type mice into sensitized STAT4(-/-) mice did not restore AHR in the mutant mice. Furthermore, chemokine production and peribronchial eosinophilia were not restored during the cellular transfer experiments. Thus, it appears that STAT4 expression contributes to a type 2 process such as allergen-induced chemokine production and AHR. In additional studies, competent allergic mice were treated with anti-IL-12 locally in the airways at the time of allergen rechallenge. These latter studies also demonstrated a decrease in AHR. Altogether, these data suggest that STAT4-mediated pathways play a role locally within the airway for the exacerbation of the allergen-induced responses.

Administration, Intranasal↗

PubMed2003

BATF transgenic mice reveal a role for activator protein-1 in NKT cell development.

The importance of regulated AP-1 activity during T cell development was assessed using transgenic mice overexpressing BATF, a basic leucine zipper transcription factor and an AP-1 inhibitor. BATF transgenic animals possess normal thymic cellularity and all major T cell subsets, but show impaired thymocyte proliferation in vitro and no induction of IL-2, IL-4, IL-5, IL-10, and IL-13 expression. Since NKT cells are largely responsible for cytokine production in the thymus, this population was examined by detection of the V alpha 14-J alpha 281 TCR, flow cytometry of NK1.1(+) TCR beta(+) cells, and analysis of cytokine production by heat-stable Ag(low) thymocytes and peripheral NKT cells stimulated in vivo. Results show a severe under-representation of NKT cells in BATF transgenic animals, providing the first evidence that the precise control of AP-1-mediated transcription is critical for the proper emergence of thymus-derived NKT cells in the mouse.

Animals↗

PubMed2003

Distinct requirements for Stat4 and Stat6 in hematopoietic progenitor cell responses to growth factors and chemokines.

Hematopoietic progenitor cell (HPC) homeostasis is critical in maintaining innate immunity and healing processes. Recently, we demonstrated that Th1 cells regulate HPC homeostasis, partly based on altered homeostasis in Stat4- and Stat6-deficient mice. To explore changes in HPC responsiveness in altered T helper cell environments, we directly examined growth factor-stimulated colony formation and chemokine-induced myelosuppression of HPC in Stat4- and Stat6-deficient bone marrow cells. Stat6-deficient cells have increased responses to the synergy between granulocyte-macrophage colony-stimulating factor (GM-CSF) and steel factor (SLF), compared to wild-type and Stat4-deficient cells. Increased responses are eliminated by in vivo depletion of CD4 cells. Whereas Stat6-deficient bone marrow cells respond to chemokine-mediated myelosuppression, Stat4-deficient bone marrow cells are refractory to the suppressive effects of chemokines. Thus, T helper cell development affects HPC homeostasis through several mechanisms, including the sensitivity to growth factor stimulation and chemokine suppression of HPC colony formation. Since Stat4 and Stat6 regulate opposing programs of T helper differentiation, there are distinct requirements for Stat4 and Stat6 in regulation of growth factor and chemokine responses of HPC.

Animals↗

PubMed2003

Viral studies on amniotic fluid from fetuses with and without abnormalities detected by prenatal sonography.

OBJECTIVE: To study the prevalence of viruses (cytomegalovirus [CMV] adenoviruses and enteroviruses) in amniotic fluid samples from fetuses with and without anomalies detected by prenatal sonography. STUDY DESIGN: Fluid samples obtained aseptically from 474 women undergoing genetic amniocentesis at our institutions from 1995 to 1996 were stored at -20 degrees C. Fetal anomalies (renal, central nervous system, gastrointestinal and cardiac) were detected by ultrasound in 162 of the fetuses. At a later date, the samples were retrieved, blinded, and tested by virus isolation techniques for CMV, adenoviruses and enteroviruses. Fisher's exact test was used for statistical analysis. RESULTS: The prevalence of viral isolation in amniotic fluid samples in fetuses with anomalies was 2.5% for CMV, 1.3% for adenovirus and 1.2% for enterovirus. Structurally normal fetuses had prevalences of 0.3%, 0% and 0%, respectively. CONCLUSION: The prevalence of viruses, especially CMV, appears to be higher in amniotic fluid from fetuses with sonographically detected anomalies.

Amniocentesis↗

PubMed2002

Neonatal tolerance in the absence of Stat4- and Stat6- dependent Th cell differentiation.

Neonatal tolerance to specific Ag is achieved by nonimmunogenic exposure within the first day of life. The mechanism that regulates this tolerance may provide the basis for successful organ transplantation and has recently been thought to be immune deviation from the inflammatory Th1 response to a Th2 response. To test the importance of Th2 cells in the establishment of neonatal tolerance, we examined neonatal tolerance in Stat4- and Stat6-deficient mice, which have reduced Th1 and Th2 cell development, respectively. Neonatal tolerance of both the T and B cell compartments in Stat4- and Stat6-deficient mice was similar to that observed in wild-type mice. Cytokine production shifted from a Th1 to a Th2 response in wild-type mice tolerized as neonates. In contrast, tolerance was observed in Stat6-deficient mice despite maintenance of a Th1 cytokine profile. These results suggest that cells distinct from Stat6-dependent Th2 cells are required for the establishment of neonatal tolerance.

Animals↗

PubMed2002

The homeostasis but not the differentiation of T cells is regulated by p27(Kip1).

The cyclin-dependent kinase inhibitor p27(Kip1) is a critical regulator of T cell proliferation. To further examine the relationship of T cell proliferation and differentiation, we examined the ability of T cells deficient in p27(Kip1) to differentiate into Th subsets. We observed increased Th2 differentiation in p27(Kip1)-deficient cultures. In addition to increases in CD4(+) and CD8(+) T cells, there is a similar increase in gamma delta T cells in p27(Kip1)-deficient mice compared with wild-type mice. The increase in Th2 differentiation is correlated to an increase of IL-4 secretion by CD4(+)DX5(+)TCR alpha beta(+)CD62L(low) T cells but not to increased expansion of differentiating Th2 cells. While STAT4- and STAT6-deficient T cells have diminished proliferative responses to IL-12 and IL-4, respectively, proliferative responses are increased in T cells doubly deficient in p27(Kip1) and STAT4 or STAT6. In contrast, the increased proliferation and differentiative capacity of p27(Kip1)-deficient T cells has no effect on the ability of STAT4/p27(Kip1)- or STAT6/p27(Kip1)-deficient CD4(+) cells to differentiate into Th1 or Th2 cells, respectively. Thus, while p27(Kip1) regulates the expansion and homeostasis of several T cell subsets, it does not affect the differentiation of Th subsets.

Animals↗

PubMed2002

Stat6-deficient mice develop airway hyperresponsiveness and peribronchial fibrosis during chronic fungal asthma.

Signal transducer and activator of transcription 6 (Stat6) is critical for Th2-mediated responses during allergic airway disease. To investigate the role of Stat6 in fungus-induced airway hyperresponsiveness and remodeling, Stat6-deficient (Stat6-/-) and Stat6-wildtype (Stat6+/+) mice were sensitized to Aspergillus fumigatus and airway disease was subsequently assessed in both groups at days 21, 30, 38, and 44 after an intratracheal challenge with live A. fumigatus conidia. At all times after conidia, histological analysis revealed an absence of goblet cell hyperplasia and markedly diminished peribronchial inflammation in Stat6-/- mice in contrast to Stat6+/+ mice. Airway hyperresponsiveness and peribronchial fibrosis in Stat6-/- mice were significantly reduced at day 21 after conidia compared with Stat6+/+ mice, but both groups exhibited significant, similar increases in these parameters at all subsequent times after conidia. In separate experiments, IL-13-responsive cells in Stat6-/- mice were targeted via the daily intranasal administration of 200 ng of IL-13-PE38QQR (IL13-PE), comprised of human IL-13 and a derivative of Pseudomonas exotoxin, from days 38 to 44 after the conidia challenge. IL13-PE treatment abolished airway hyperresponsiveness, but not peribronchial fibrosis in Stat6-/- mice. Taken together, these data demonstrate that the chronic development of airway hyperresponsiveness during fungal asthma is IL-13-dependent but Stat6-independent.

Animals↗

PubMed2002

Th1 cells regulate hematopoietic progenitor cell homeostasis by production of oncostatin M.

Regulation of hematopoietic progenitor cell homeostasis is crucial for maintenance of innate immunity and the ability of the body to respond to injury and infection. In this report, we demonstrate that progenitor cell numbers and cycling status in vivo are dramatically increased in mice deficient in Stat6 and decreased in mice deficient in Stat4, targeted mutations which also alter T helper cell polarization. Experiments using mice that have T cell restricted transgenic expression of Stat4 or Stat6 or have been in vivo depleted of T cell subsets demonstrate that CD4(+) T cells regulate progenitor cell activity. Injection of the Th1 cytokine Oncostatin M but not other cytokines into Stat4-deficient mice recovers progenitor cell activity to wild-type levels. Thus, T helper cells actively regulate hematopoietic progenitor cell homeostasis.

Animals↗

PubMed2002

Grap negatively regulates T-cell receptor-elicited lymphocyte proliferation and interleukin-2 induction.

Grb-2-related adaptor protein (Grap) is a Grb2-like SH3-SH2-SH3 adaptor protein with expression restricted to lymphoid tissues. Grap(-/-) lymphocytes isolated from targeted Grap-deficient mice exhibited enhanced proliferation, interleukin-2 production, and c-fos induction in response to mitogenic T-cell receptor (TCR) stimulation, compared to wild-type cells. Ectopic expression of Grap led to a suppression of Elk-1-directed transcription induced by the Ras/Erk pathway, without having effects on gene expression mediated by Jnk and p38 mitogen-activated protein kinases. Together, these data suggest that Grap, unlike Grb2, acts as a negative regulator of TCR-stimulated intracellular signaling by downregulating signal relay through the Ras/Erk pathway.

Adaptor Proteins, Signal Transducing↗