Prostate cancer (PCA) is a clinically heterogeneous and often multifocal disease with a clinical outcome difficult to predict. A deeper knowledge of the molecular basis of the disease may lead to a better prediction of prognosis. Therefore, in this study we investigated the molecular basis of PCA by identifying potential tumor markers in laser-microdisected PCA tissues. Among a group of PCA patients, quantitative RT-PCR analysis was performed to compare the expression of 70 genes. These genes were selected from the results of two microarrays which investigated the gene expression profile differences between moderately or poorly differentiated prostate carcinoma glands and the corresponding normal glands. Among the genes examined, CDKN2A, GATA3, CREBBP, ITGA2, NBL1 and TGM4 were down-regulated in the prostate carcinoma glands compared to the corresponding normal glands, whereas TFF3, TMPRSS2 and ERG were up-regulated. Our findings indicate that these genes may play roles as tumor suppressor genes or oncogenes in PCA, and may serve as potential tumor markers and novel therapeutic targets.

Current research on antigen specific CD4+ T cells indicates that there is functional and phenotypic heterogeneity within these populations, but the extent of this heterogeneity is poorly described. The CD134/CD25 assay allows live isolation of antigen specific cells in vitro for down-stream molecular analysis. Antigen specific CD4+ T cells were examined at the molecular level by lineage specific transcription factor profiling using qualitative multiplex single cell RT-PCR and Lock Nucleic Acid (LNA) probes allowed unbiased amplification and delineation of expression of Tbx21, Gata3, Rorc, Foxp3 and Bcl-6. It overcomes the limitations of previous assays by allowing identification of transcription factor mRNA in single antigen specific cells with high sensitivity (down to 10 femtograms) and specificity. Patterns of responses can be robustly characterized using <200 cells based on exact binomial calculations. These results are reproducible with a CV of ≈6%. The patterns of heterogeneity are stable within an individual antigen specific response but vary between responses to different antigens. Responses to CMV have a Th1 predominant profile (35.6% of responding cells expressing tbx21) whereas responses to Tetanus Toxoid have a Th2 biased profile (22% of responding cells expressing gata3), with unexpectedly high levels of Treg cells found in both populations. Here we describe a methodology that allows live isolation of Ag specific cells and transcription factor profiling at a single cell level to robustly delineate the different CD4+ T cell subsets within this population. This novel method is a powerful tool that can be used to study CD4+ T cell heterogeneity within extremely small populations of cells and where cell numbers are limited.

The inner ear develops from the otic placode, one of the cranial placodes that arise from a region of ectoderm adjacent to the anterior neural plate called the pre-placodal domain. We have identified a Forkhead family transcription factor, Foxi3, that is expressed in the pre-placodal domain and down-regulated when the otic placode is induced. We now show that Foxi3 mutant mice do not form otic placodes as evidenced by expression changes in early molecular markers and the lack of thickened placodal ectoderm, an otic cup or otocyst. Some preplacodal genes downstream of Foxi3-Gata3, Six1 and Eya1-are not expressed in the ectoderm of Foxi3 mutant mice, and the ectoderm exhibits signs of increased apoptosis. We also show that Fgf signals from the hindbrain and cranial mesoderm, which are necessary for otic placode induction, are received by pre-placodal ectoderm in Foxi3 mutants, but do not initiate otic induction. Finally, we show that the epibranchial placodes that develop in close proximity to the otic placode and the mandibular division of the trigeminal ganglion are missing in Foxi3 mutants. Our data suggest that Foxi3 is necessary to prime pre-placodal ectoderm for the correct interpretation of inductive signals for the otic and epibranchial placodes.

This study investigated specific gene expression profiles in patients with donor-specific cytotoxic-hyporesponsiveness, reflected by cytotoxic T-lymphocyte precursor frequency (CTLpf). The effect of calcineurin inhibitor (CNI) withdrawal was studied on markers for cytotoxicity (perforin, granzyme B), apoptosis (Fas,FasL), Th1 and Th2 cytokines (IL-2, IL-10), Th1 and Th2 transcription factors (T-bet, GATA 3), Th17 transcription factor and cytokine (RORγt, IL-17), and for immune regulation/activation (CD25, FOXP3). Peripheral blood samples from renal allograft recipients (n = 18) more than two yr after transplantation with stable renal function were analyzed before and four months after CNI withdrawal. Additionally, systolic and diastolic blood pressure, cholesterol, serum creatinine and proteinuria were evaluated, and no significant differences were measured before and after CNI withdrawal. However, CNIs' discontinuation influenced peripheral gene expression profiles. After CNI withdrawal, the mRNA expression of Granzyme B, Perforin, Fas, FasL, T-bet, GATA3 and CD25 were significantly lower than during CNI treatment. After CNI discontinuation, donor-specific CTLpf decreased, while FOXP3 expression discriminated between detectable and non-detectable donor-specific cytolysis reactivity; FOXP3 transcript values were highest in absence of donor-specific cytotoxicity (p < 0.01). Our study shows CNI withdrawal in stable kidney transplant recipients two yr after transplantation is safe. Moreover, discontinuation of CNIs' treatment allows FOXP3+ regulatory T-cells development, resulting in a significant decrease of anti-donor immune reactivity.

This study investigates the effects and possible molecular mechanisms of corilagin extraction on prevention of Schistosoma japonicum ova-induced granulomas and liver fibrosis. As a result, under a light microscope, when compared to a model group, the corilagin group showed smaller granulomas, less liver cell denaturation and less inflammatory cell infiltration, and the connective tissues were significantly decreased. By Masson staining, the liver sections from the corilagin group showed less collagen distributed around granulomas, decreased liver fibrosis in the portal tracts and less formed interlobular tissue. The expression of hydroxyproline, IL-13 in liver and GATA3 in spleen in the model group was significantly higher than that in the normal group (P less than 0.05 or 0.01), while the level of hydroxyproline, IL-13 and GATA3 in the corilagin group were significantly lower than that in the model group (P less than 0.05). In conclusion, corilagin extraction can decrease the level of Th2-associated profibrotic cytokine IL-13, and down-regulate the transcription of GATA3 mRNA in spleen cells, which alleviate the hepatic fibrosis caused by egg granuloma in Schistosoma japonicum infection.

GATA transcription factors are zinc finger DNA binding proteins that activate transcription during development and cell differentiation. To date, 7 members of GATA family have been reported. However, the expression patterns and the exact roles of distinct GATA family members contributing to tumorigenesis and progression of breast cancer (BC) remain to be elucidated. Here, we studied the expression of GATA transcripts in a variety of tumor types compared with the normal controls using the ONCOMINE and GOBO databases, along with their corresponding expression profiles in an array of cancer cell lines through CCLE analysis. Based on Kaplan-Meier plotter, we further investigated the prognostic values of GATA members specifically high expressed in BC patients. It was found that, when compared with normal tissues, GATA3 and TRPS1 were distinctly high expressed in BC patients among all GATA members. GATA3 expression was significantly associated with ESR1, while TRPS1 was correlated with ERBB2. In survival analysis, GATA3 and TRPS1 mRNA high expressions were correlated to better survival in BC patients, and TRPS1 high expression was significantly associated with longer RFS in patients who have received chemotherapy. These results suggest that GATA3 and TRPS1 are distinct biomarkers and essential prognostic factors for breast cancer.

Protein kinase C-theta (PKC-theta) is important for the activation of autoreactive T cells but is thought to be of minor importance for T-cell responses in infectious diseases, suggesting that PKC-theta may be a target for the treatment of T-cell-mediated autoimmune diseases. To explore the function of PKC-theta in a chronic persisting infection in which T cells are crucial for pathogen control, we infected BALB/c PKC-theta(-/-) and PKC-theta(+/+) wild-type mice with Toxoplasma gondii. The PKC-theta(-/-) mice succumbed to necrotizing Toxoplasma encephalitis due to an insufficient parasite control up to day 40, whereas the wild-type mice survived. The number of T. gondii-specific CD4 and CD8 T cells was significantly reduced in the PKC-theta(-/-) mice, resulting in the impaired production of protective cytokines (gamma interferon, tumor necrosis factor) and antiparasitic effector molecules (inducible nitric oxide, gamma interferon-induced GTPase) in the spleen and brain. In addition, Th2-cell numbers were reduced in infected the PKC-theta(-/-) mice, paralleled by the diminished GATA3 expression of PKC-theta(-/-) CD4 T cells and reduced T. gondii-specific IgG production in serum and cerebrospinal fluid. Western blot analysis of splenic CD4 and CD8 T cells revealed an impaired activation of the NF-kappaB, AP-1, and MAPK pathways in T. gondii-infected PKC-theta(-/-) mice. Adoptive transfer of wild-type CD4 plus CD8 T cells significantly protected PKC-theta(-/-) mice from death by increasing the numbers of gamma interferon-producing T. gondii-specific CD4 and CD8 T cells, illustrating a cell-autonomous, protective function of PKC-theta in T cells. These findings imply that PKC-theta inhibition drastically impairs T. gondii-specific T-cell responses with fatal consequences for intracerebral parasite control and survival.

The transcription factor PU.1 is predominantly expressed in dendritic cells (DCs) and is essential for DC differentiation. Although there are several reports that PU.1 positively regulates the expression of DC-specific genes, whether PU.1 also has a suppressive effect on DCs is largely unknown. Here we demonstrate that PU.1 suppresses the expression of Th2 cytokines including IL-13 and IL-5 in bone marrow-derived DCs (BMDCs), through repression of the expression of GATA3, which is a master regulator of Th2 differentiations. When PU.1 siRNA was introduced into BMDCs, LPS-induced expression of IL-13 and IL-5 was increased along with upregulation of the constitutive expression of GATA2 and GATA3. The additional introduction of GATA3 siRNA but not of GATA2 siRNA abrogated PU.1 siRNA-mediated upregulation of IL-13 and IL-5. A chromatin immunoprecipitation assay showed that PU.1 bound to Gata3 proximal promoter region, which is more dominant than the distal promoter in driving GATA3 transcription in DCs. The degree of histone acetylation at the Gata3 promoter was decreased in PU.1 siRNA-introduced DCs, suggesting the involvement of PU.1 in chromatin modification of the Gata3 promoter. Treatment with a histone deacetylase (HDAC) inhibitor, trichostatin A, increased the degree of histone H3 acetylation at the Gata3 promoter and induced the subsequent expression of GATA3. Experiments using HDAC inhibitors and siRNAs showed that HDAC3 suppressed GATA3 expression. The recruitment of HDAC3 to the Gata3 promoter was decreased by PU.1 knockdown. LPS-induced IL-13 expression was dramatically reduced in BMDCs generated from mice lacking the conserved GATA3 response element, termed CGRE, which is an essential site for the binding of GATA3 on the Il-13 promoter. The degree of H3K4me3 at CGRE was significantly increased in PU.1 siRNA-transfected stimulated DCs. Our results indicate that PU.1 plays pivotal roles in DC development and function, serving not only as a transcriptional activator but also as a repressor.

DNA evolution models made invaluable contributions to comparative genomics, although it seemed formidable to include non-genomic features into these models. In order to build an evolutionary model of transcription networks (TNs), we had to forfeit the substitution model used in DNA evolution and to start from modeling the evolution of the regulatory relationships. We present a quantitative evolutionary model of TNs, subjecting the phylogenetic distance and the evolutionary changes of cis-regulatory sequence, gene expression and network structure to one probabilistic framework. Using the genome sequences and gene expression data from multiple species, this model can predict regulatory relationships between a transcription factor (TF) and its target genes in all species, and thus identify TN re-wiring events. Applying this model to analyze the pre-implantation development of three mammalian species, we identified the conserved and re-wired components of the TNs downstream to a set of TFs including Oct4, Gata3/4/6, cMyc and nMyc. Evolutionary events on the DNA sequence that led to turnover of TF binding sites were identified, including a birth of an Oct4 binding site by a 2nt deletion. In contrast to recent reports of large interspecies differences of TF binding sites and gene expression patterns, the interspecies difference in TF-target relationship is much smaller. The data showed increasing conservation levels from genomic sequences to TF-DNA interaction, gene expression, TN, and finally to morphology, suggesting that evolutionary changes are larger at molecular levels and smaller at functional levels. The data also showed that evolutionarily older TFs are more likely to have conserved target genes, whereas younger TFs tend to have larger re-wiring rates.

Bladder cancer is the 4(th) most common cancer among men in the U.S. We analyzed variant genotypes hypothesized to modify major biological processes involved in bladder carcinogenesis, including hormone regulation, apoptosis, DNA repair, immune surveillance, metabolism, proliferation, and telomere maintenance. Logistic regression was used to assess the relationship between genetic variation affecting these processes and susceptibility in 563 genotyped urothelial cell carcinoma cases and 863 controls enrolled in a case-control study of incident bladder cancer conducted in New Hampshire, U.S. We evaluated gene-gene interactions using Multifactor Dimensionality Reduction (MDR) and Statistical Epistasis Network analysis. The 3'UTR flanking variant form of the hormone regulation gene HSD3B2 was associated with increased bladder cancer risk in the New Hampshire population (adjusted OR 1.85 95%CI 1.31-2.62). This finding was successfully replicated in the Texas Bladder Cancer Study with 957 controls, 497 cases (adjusted OR 3.66 95%CI 1.06-12.63). The effect of this prevalent SNP was stronger among males (OR 2.13 95%CI 1.40-3.25) than females (OR 1.56 95%CI 0.83-2.95), (SNP-gender interaction P = 0.048). We also identified a SNP-SNP interaction between T-cell activation related genes GATA3 and CD81 (interaction P = 0.0003). The fact that bladder cancer incidence is 3-4 times higher in males suggests the involvement of hormone levels. This biologic process-based analysis suggests candidate susceptibility markers and supports the theory that disrupted hormone regulation plays a role in bladder carcinogenesis.

The auditory neuroblast cell line US/VOT-N33 (N33), which is conditionally immortal, was studied as an in vitro model for the differentiation of spiral ganglion neurons (SGNs) and as a candidate for cell transplantation in rodents. It expresses numerous molecular markers characteristic of auditory neuroblasts, including the transcription factors GATA3, NeuroD, Brn3a and Islet1, as well as the neuronal cytoskeletal protein beta3-tubulin. It displays active migratory behaviour in vitro and in vivo. In the presence of the fibroblast growth factors FGF1 or FGF2 it differentiates bipolar morphologies similar to those of native SGNs. In coculture with neonatal cochlear tissue it is repelled from epithelial surfaces but not from native SGNs, alongside which it extends parallel neuronal processes. When injected into the retina in vivo, EGFP-labelled N33 cells were traced for 1-2 weeks and migrated rapidly within the subretinal space. Cells that found their way into the retinal ganglion cell layer extended multiple processes but did not express beta3-tubulin. The ability of N33 to migrate, to differentiate, to localize with native SGNs in vitro and to survive in vivo suggests that they provide an effective model for SGN differentiation and for cell transplantation into the ear.

Recent studies have confirmed the utility of massively parallel sequencing (MPS) in addressing genetically heterogeneous hereditary hearing impairment. By applying a MPS diagnostic panel targeting 129 known deafness genes, we identified a novel frameshift GATA3 mutation, c.149delT (p.Phe51LeufsX144), in a hearing-impaired family compatible with autosomal dominant inheritance. The GATA3 haploinsufficiency is thought to be associated with the hypoparathyroidism, sensorineural deafness, and renal dysplasia (HDR) syndrome. The pathogenicity of GATA3 c.149delT was supported by its absence in the 5400 NHLBI exomes, 1000 Genomes, and the 100 normal hearing controls of the present study; the co-segregation of c.149delT heterozygosity with hearing impairment in 9 affected members of the family; as well as the nonsense-mediated mRNA decay of the mutant allele in in vitro functional studies. The phenotypes in this family appeared relatively mild, as most affected members presented no signs of hypoparathyroidism or renal abnormalities, including the proband. To our knowledge, this is the first report of genetic diagnosis of HDR syndrome before the clinical diagnosis. Genetic examination for multiple deafness genes with MPS might be helpful in identifying certain types of syndromic hearing loss such as HDR syndrome, contributing to earlier diagnosis and treatment of the affected individuals.

OBJECTIVE

We review the syndrome of hypoparathyroidism, deafness, and renal anomalies (HDR syndrome).

METHODS

The current understanding and relevant literature pertaining to the background, genetic considerations, clinical features, prognosis, and treatment of HDR syndrome are reviewed.

RESULTS

The combination of hypoparathyroidism, deafness, and renal anomalies constitutes an unusual syndrome associated most commonly with haploinsufficiency in GATA3, which encodes a transcription factor that binds to the (A/T) GATA (A/G) consensus DNA sequence. Sensorineural hearing loss is the most consistently expressed clinical feature, being present in almost all affected individuals, and the combination of hypoparathyroidism and hearing impairment occurs in well over 90% of those affected, with various renal anomalies being the most heterogeneous feature of the classic triad. We characterize, in tabular form, the individual cases described in the literature and propose a classification scheme based on the presence or absence of renal anomalies. We also include the specific genetic abnormality and renal anomaly associated with each individual case.

CONCLUSIONS

HDR syndrome is a heterogeneous syndrome most commonly associated with GATA3 haploinsufficiency.

Haploinsufficiency for the transcription factor GATA3 leads to hearing loss in humans. It is expressed throughout the auditory sensory epithelium (SE). In the vestibular organs, GATA3 is limited to the striola reversal zone of the utricle. Stereocilia orientation shifts 180 degrees at this region, which contains morphologically distinct type-I hair cells. The striola is conserved in all amniotes, its function is unknown, and GATA3 is the only known marker of the reversal zone. To identify downstream targets of GATA3 that might point to striolar function, we measured gene expression differences between striolar and extra-striolar SE. These were compared with profiles after GATA3 RNAi and GATA3 over-expression. We identified four genes (BMP2, FKHL18, LMO4, and MBNL2) that consistently varied with GATA3. Two of these (LMO4 and MBNL2) were shown to be direct targets of GATA3 by ChIP. Our results suggest that GATA3 impacts WNT signaling in this region of the sensory macula.

Metastatic neuroendocrine neoplasms to the breast may show considerable morphologic overlap with primary mammary carcinomas, particularly those showing evidence of neuroendocrine differentiation, and may be misdiagnosed as such. Accurate distinction between these two entities is crucial for determination of appropriate clinical management. The histologic and immunohistochemical features of metastatic neuroendocrine neoplasms to the breast were studied and compared with the features of primary invasive mammary carcinomas with neuroendocrine differentiation, which served as controls. Of the metastatic neuroendocrine neoplasms, 15 were well-differentiated neuroendocrine tumors with carcinoid tumor-type morphology and 7 were poorly differentiated/high-grade neuroendocrine carcinomas with small-cell or large-cell neuroendocrine carcinoma morphology. The majority of the metastatic neoplasms originated in the lung and gastrointestinal tract. There were histologic similarities between metastatic neuroendocrine neoplasms and invasive mammary carcinomas with neuroendocrine differentiation, both of which exhibited neuroendocrine histologic features (nested and trabecular architecture, minimal tubular differentiation, and characteristic nuclear features). Only one case of the invasive mammary carcinomas with neuroendocrine differentiation was modified Bloom-Richardson grade 1 (largely due to minimal tubular differentiation on most such tumors), and the invasive mammary carcinomas with neuroendocrine differentiation were often associated with in situ carcinoma. Immunohistochemistry was helpful in distinguishing metastatic neuroendocrine neoplasms from invasive mammary carcinomas with neuroendocrine differentiation. Whereas the majority of invasive mammary carcinomas with neuroendocrine differentiation were positive for estrogen receptor and GATA3, metastatic neuroendocrine neoplasms were typically negative for estrogen receptor and GATA3, and metastatic well-differentiated neuroendocrine tumors often showed immunoreactivity for site-specific markers. Although the histologic and immunohistochemical features of a breast tumor may raise the suspicion of a metastatic neuroendocrine neoplasm, the pathologic findings should be interpreted in the context of the clinical history and imaging findings in order to establish an accurate diagnosis.

Correct diagnosis of pulmonary tumors is essential for treatment decision and often rely on immunohistochemical markers. We stained tissue microarrays from resected primary lung cancer (n=665) and pulmonary metastases (n=425) for CK7, CK20, CDX2, CK5, p40, p63, TTF-1, napsin A, GATA3 and PAX8 to systematically assess the diagnostic value of these markers. Primary lung adenocarcinomas expressed TTF-1 in 90% and napsin A in 84% of the cases, while 10% were positive for p63, 7% for CDX2, 2% for CK20 and 2% for GATA3. Only 68% of the lung adenocarcinomas were positive for CK7, TTF-1 and napsin A and negative for all other markers. Primary lung squamous cell carcinomas expressed CK5, p40 and p63 in 94-97% of cases, while 44% were positive for CK7, 20% for GATA3, 7% for CDX2 and 3% for TTF-1. Rare cases expressed PAX8, CK20 or napsin A. Pulmonary metastases of colorectal cancer were positive for CK20 in 83% and CDX2 in 99% of the cases. Rare cases expressed CK7, p63 or PAX8, while 4% expressed TTF-1. Pulmonary metastases of renal cell carcinomas were positive for PAX8 in 74%, napsin A in 7% and CK7 in 7% of the cases. Pulmonary metastases of breast cancer were positive for GATA3 in 93% and CK7 in 78% of the cases, while 15% expressed CK5. Information on expression and patterns of immunohistochemical markers facilitates histopathological diagnostics. Evidently, unusual immune profiles occur and may lead to incorrect diagnosis.

Macrophages are highly plastic cells with the ability to differentiate into both M1- and M2-polarized phenotypes. As a distinct M2-polarized population, tumour-associated macrophages (TAMs) promote tumorigenesis owing to their pro-angiogenic and immune-suppressive functions in tumour microenvironment. In the present study, we found that the microRNA-720 (miR-720) was down-regulated in TAMs isolated from breast carcinomas and M2-polarization macrophages. Overexpression of miR-720 attenuated M2 phenotype expression and thus inhibited M2 polarization. We further identified GATA binding protein 3 (GATA3), a transcriptional factor that plays an important role in M2 macrophage polarization, was the downstream target of miR-720 Ectopic expression of GATA3 restored the M2 phenotype in miR-720 overexpressed macrophages. Importantly, overexpression of miR-720 inhibited pro-migration behaviour and phagocytic ability of M2-polarized macrophages. Thus, our data suggest that miR-720 plays an important role in regulating M2 macrophage polarization and function.

Estrogen receptor alpha (ERα) expression is critical for breast cancer classification, high ERα expression being associated with better prognosis. ERα levels strongly correlate with that of GATA binding protein 3 (GATA3), a major regulator of ERα expression. However, the mechanistic details of ERα-GATA3 regulation remain incompletely understood. Here we combine mathematical modeling with perturbation experiments to unravel the nature of regulatory connections in the ERα-GATA3 network. Through cell population-average, single-cell and single-nucleus measurements, we show that the cross-regulation between ERα and GATA3 amounts to overall negative feedback. Further, mathematical modeling reveals that GATA3 positively regulates its own expression and that ERα autoregulation is most likely absent. Lastly, we show that the two cross-regulatory connections in the ERα-GATA3 negative feedback network decrease the noise in ERα or GATA3 expression. This may ensure robust cell fate maintenance in the face of intracellular and environmental fluctuations, contributing to tissue homeostasis in normal conditions, but also to the maintenance of pathogenic states during cancer progression.

Evidence is accumulating that group 2 innate lymphoid cells (ILC2) play an important role in allergic airway inflammation by producing a large amount of type 2 cytokines. But it remains poorly understood how its activities are properly controlled in vivo. Here, we demonstrated that prostaglandin E2 (PGE2) had a profound inhibitory effect on IL-33-induced ILC2 expansion and IL-5 and IL-13 production in vitro. This effect was mimicked by PGE1-alcohol but attenuated by ONO-AE3-208, indicating a selective action through the E-prostanoid 4 (EP4) receptor. In the IL-33-induced asthma model, coadministration of PGE2 or PGE1-alcohol resulted in diminished IL-5 and IL-13 production, reduced eosinophilia and alleviated lung pathology. In contrast, EP4-deficient mice displayed an exacerbated inflammatory response in another ILC2-mediated asthma model induced by Alternaria extract. Mechanistic studies demonstrated that the PGE2-mediated inhibition of ILC2 was dependent on cyclic adenosine monophosphate (cAMP) production. Further downstream, PGE2-EP4-cAMP signaling led to suppression of GATA3 and ST2 expression, which is known to be critical for ILC2 activation. These findings reveal a novel function of PGE2 as a negative regulator of ILC2 activation and highlight an endogenous counter-regulatory mechanism for the control of innate allergic inflammatory responses.

To investigate the effect of ILC2s on Th2-type adaptive immunity during the acute exacerbation of chronic obstructive pulmonary disease (AECOPD), the study enrolled healthy people, stable COPD patients, and AECOPD patients. Flow cytometry was used to detect Th1, Th2, and ILC2 in the peripheral blood and CD80 and MHC II levels on ILC2. The mRNA levels of GATA3, RORα, and CRTH2 of ILC2s were detected by RT-PCR. In addition, ILC2s from the peripheral blood of AECOPD patients were cocultured with CD4⁺ T cells from the peripheral blood of healthy controls. Cytokine levels in serum of the three groups and the in vitro coculture supernatants were measured by ELISA. Compared with the stable COPD group or the healthy control group, Th2 in the peripheral blood of AECOPD group increased dramatically, inducing an increase of Th2/Th1 ratio in AECOPD patients. Meanwhile, the level of IL-4 in the serum of this group was also increased. However, we also detected ILC2s in the peripheral blood of the AECOPD group and found that it was also increased, alone with the increased GATA3, RORα, and CRTH2 mRNA levels. We also found that the CD80 and MHC II on ILC2 were significantly upregulated and the proportion of MHC II⁺ ILC2 cells was significantly positively correlated with the proportion of Th2 cells in AECOPD patients. To further demonstrate the effect of ILC2 on Th2 cells, we cocultured ILC2 with CD4⁺ T cells in vitro, which also showed a significant increase of Th2 ratio as well as Th2-associated cytokines IL-4, IL-5, and IL-13. However, we found that this effect of ILC2s on Th2 cells could be inhibited by the addition of anti-MHC II. The Th2/Th1 balance shifts to Th2 in AECOPD. ILC2s may function as APC by the upregulation of MHC II and regulate adaptive immunity shift to Th2-type response in AECOPD.

In spite of sufficient data on Neem Leaf Glycoprotein (NLGP) as a prophylactic vaccine, little knowledge currently exists to support the use of NLGP as a therapeutic vaccine. Treatment of mice bearing established sarcomas with NLGP (25 µg/mice/week subcutaneously for 4 weeks) resulted in tumor regression or dormancy (Tumor free/Regressor, 13/24 (NLGP), 4/24 (PBS)). Evaluation of CD8(+) T cell status in blood, spleen, TDLN, VDLN and tumor revealed increase in cellular number. Elevated expression of CD69, CD44 and Ki67 on CD8(+) T cells revealed their state of activation and proliferation by NLGP. Depletion of CD8(+) T cells in mice at the time of NLGP treatment resulted in partial termination of tumor regression. An expansion of CXCR3(+) and CCR5(+) T cells was observed in the TDLN and tumor, along with their corresponding ligands. NLGP treatment enhances type 1 polarized T-bet expressing T cells with downregulation of GATA3. Treg cell population was almost unchanged. However, T∶Treg ratios significantly increased with NLGP. Enhanced secretion/expression of IFNγ was noted after NLGP therapy. In vitro culture of T cells with IL-2 and sarcoma antigen resulted in significant enhancement in cytotoxic efficacy. Consistently higher expression of CD107a was also observed in CD8(+) T cells from tumors. Reinoculation of sarcoma cells in tumor regressed NLGP-treated mice maintained tumor free status in majority. This is correlated with the increment of CD44(hi)CD62L(hi) central memory T cells. Collectively, these findings support a paradigm in which NLGP dynamically orchestrates the activation, expansion, and recruitment of CD8(+) T cells into established tumors to operate significant tumor cell lysis.

BACKGROUND

Pegylated granulocyte colony-stimulating factor (G-CSF) has recently been introduced as a new compound for mobilization of CD34(+) hematopoietic stem and progenitor cells. In this study, we compared the molecular and functional characteristics of CD34(+) cells mobilized by pegylated G-CSF with those mobilized by unconjugated G-CSF.

METHODS

Gene expression of immunomagnetically enriched CD34(+) cells from leukapheresis products of patients who were given pegylated-G-CSF or unconjugated G-CSF was analyzed using Affymetrix HG Focus microarrays and quantitative reverse transcriptase polymerase chain reaction. Flow cytometry and fluorescence activated cell sorting was conducted to assess the CD34(+) subset composition and to obtain Lin(-), CD34(+), CD38(-) hematopoietic stem cells. Cell cycle assays and clonogenic assays were performed for functional corroboration.

RESULTS

Pegylated G-CSF and unconjugated G-CSF mobilized CD34(+) and hematopoietic stem cells with different molecular phenotypes and functional properties. The CD34(+) cells mobilized by pegylated G-CSF had higher expression levels of genes indicative of early hematopoiesis, including HOXA9, MEIS1 and GATA3. We found lower expression of genes characteristic of erythroid and later stages of myeloid differentiation and a lower functional burst-forming unit erythroid/colony-forming unit-granulocyte-macrophage ratio. Consistently, greater numbers of hematopoietic stem cells and common myeloid progenitors and fewer megakaryocyte-erthrocyte progenitors were found in the pegylated-G-CSF-mobilized CD34(+) cells. Additionally, sorted pegylated-G-CSF-mobilized hematopoietic stem cells displayed higher expression of HOXA9 in comparison to G-CSF-mobilized hematopoietic stem cells. In line with the gene expression data, CD34(+) cells mobilized by pegylated G-CSF, as well as sorted hematopoietic stem cells, showed a significantly greater cell cycle activity.

CONCLUSIONS

Stimulation with pegylated-G-CSF or G-CSF results in different expression of key regulatory genes and different functional properties of mobilized hematopoietic stem cells as well as their progeny, a finding that might be relevant for the application of these cells in blood stem cell transplantation.

BACKGROUND

During the course of development, the vertebrate nephric duct (ND) extends and migrates from the place of its initial formation, adjacent to the anterior somites, until it inserts into the bladder or cloaca in the posterior region of the embryo. The molecular mechanisms that guide ND migration are poorly understood.

RESULTS

A novel Gata3-enhancer-Gfp-based chick embryo live imaging system was developed that permits documentation of ND migration at the individual cell level for the first time. FGF Receptors and FGF response genes are expressed in the ND, and FGF ligands are expressed in surrounding tissues. FGF receptor inhibition blocked nephric duct migration. Individual inhibitors of the Erk, p38, or Jnk pathways did not affect duct migration, but inhibition of all three pathways together did inhibit migration of the duct. A localized source of FGF8 placed adjacent to the nephric duct did not affect the duct migration path.

CONCLUSIONS

FGF signaling acts as a "motor" that is required for duct migration, but other signals are needed to determine the directionality of the duct migration pathway. Developmental Dynamics 244:157-167, 2015. © 2014 Wiley Periodicals, Inc.