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We have recently shown an increased HIV-1 replication and gene expression in neonatal (cord) blood mononuclear cells compared with adult cells, which could be due to HIV-1 integration as it targets active host genes. Here we have characterized 468 HIV-1 integration sites within cord and adult blood T-lymphocytes and monocyte-derived macrophages (MDM) from five donors. Several functional classes of genes were identified by gene ontology to be over represented, including genes for cellular components, maintenance of intracellular environment, enzyme regulation, cellular metabolism, catalytic activity and cation transport. Numerous potential transcription factor binding sites at the sites of integration were identified. Furthermore, the genes at the site of integration, transcription factors which potentially bind upstream of the HIV-1 promoter and factors that assist HIV-1 integration were found to be expressed at higher levels in cord than adult cells. Taken together, these results suggest HIV-1 integration occurred in a more actively transcribed genes in neonatal cells compared with adult cells, which may help explain a higher level of HIV-1 gene expression and replication in neonatal compared with adult cells.

Malignant transformation was demonstrated in UROtsa cells following 52-weeks of exposure to 50 nM monomethylarsonous acid (MMA(III)); the result was the malignantly transformed cell line, URO-MSC. URO-MSC cells were used to study the induction of DNA damage and the alteration of DNA repair enzymes in both the presence of MMA(III) [URO-MSC(+)] and after subsequent removal of MMA(III) [URO-MSC(-)] following chronic, low-level exposure. In the presence of MMA(III), URO-MSC(+) cells demonstrated a sustained increase in DNA damage following 12-weeks of exposure; in particular, a significant increase in DNA single-strand breaks at 12-weeks of exposure consistently elevated through 52 weeks. The persistence of DNA damage in URO-MSC cells was assessed after a 2-week removal of MMA(III). URO-MSC(-) cells demonstrated a decrease in DNA damage compared to URO-MSC(+); however, DNA damage in URO-MSC(-) remained significantly elevated when compared to untreated UROtsa and increased in a time-dependent manner. Reactive oxygen species (ROS) were demonstrated to be a critical component in the generation of DNA damage determined through the incubation of ROS scavengers with URO-MSC cells. Poly (ADP-ribose) polymerase (PARP) is a key repair enzyme in DNA single-strand break repair. URO-MSC(+) resulted in a slight increase in PARP activity after 36-weeks of MMA(III) exposure, suggesting the presence of MMA(III) is inhibiting the increase in PARP activity. In support, PARP activity in URO-MSC(-) increased significantly, coinciding with a subsequent decrease in DNA damage demonstrated in URO-MSC(-) compared to URO-MSC(+). These data demonstrate that chronic, low-level exposure of UROtsa cells to 50 nM MMA(III) results in: the induction of DNA damage that remains elevated upon removal of MMA(III); increased levels of ROS that play a role in MMA(III) induced-DNA damage; and decreased PARP activity in the presence of MMA(III).

Aberrant DNA methylation participates in carcinogenesis and is a molecular hallmark of a tumor cell. Tumor cells generally exhibit a redistribution of DNA methylation resulting in global hypomethylation with regional hypermethylation; however, the speed in which these changes emerge has not been fully elucidated and may depend on the temporal location of the cell in the path from normal, finite lifespan to malignant transformation. We used a model of arsenical-induced malignant transformation of immortalized human urothelial cells and DNA methylation microarrays to examine the extent and temporal nature of changes in DNA methylation that occur during the transition from immortal to malignantly transformed. Our data presented herein suggest that during arsenical-induced malignant transformation, aberrant DNA methylation occurs non-randomly, progresses gradually at hundreds of gene promoters, and alters expression of the associated gene, and these changes are coincident with the acquisition of malignant properties, such as anchorage independent growth and tumor formation in immunocompromised mice. The DNA methylation changes appear stable, since malignantly transformed cells removed from the transforming arsenical exhibited no reversion in DNA methylation levels, associated gene expression, or malignant phenotype. These data suggest that arsenicals act as epimutagens and directly link their ability to induce malignant transformation to their actions on the epigenome.

Transcriptional regulation of the bcl-2 proto-oncogene is highly complex, with the majority of transcription driven by the P1 promoter site and the interaction of multiple regulatory proteins. A guanine- and cytosine-rich (GC-rich) region directly upstream of the P1 site has been shown to be integral to bcl-2 promoter activity, as deletion or mutation of this region significantly increases transcription. This GC-rich element consists of six contiguous runs of guanines and cytosines that have the potential to adopt DNA secondary structures, the G-quadruplex and i-motif, respectively. Our laboratory has previously demonstrated that the polypurine-rich strand of the bcl-2 promoter can form a mixture of three different G-quadruplex structures. In this current study, we demonstrate that the complementary polypyrimidine-rich strand is capable of forming one major intramolecular i-motif DNA secondary structure with a transition pH of 6.6. Characterization of the i-motif folding pattern using mutational studies coupled with circular dichroic spectra and thermal stability analyses revealed an 8:5:7 loop conformation as the predominant structure at pH 6.1. The folding pattern was further supported by chemical footprinting with bromine. In addition, a novel assay involving the sequential incorporation of a fluorescent thymine analog at each thymine position provided evidence of a capping structure within the top loop region of the i-motif. The potential of the GC-rich element within the bcl-2 promoter region to form DNA secondary structures suggests that the transition from the B-DNA to non-B-DNA conformation may play an important role in bcl-2 transcriptional regulation. Furthermore, the two adjacent large lateral loops in the i-motif structure provide an unexpected opportunity for protein and small molecule recognition.

MYC is deregulated in most tumour types, but an effective means to selectively target its aberrant expression is not yet available. Supercoiling that is induced by transcription has been demonstrated to have dynamic effects on DNA in the MYC promoter element: it converts duplex DNA to non-duplex DNA structures, even at considerable distances from the transcriptional start site. These non-duplex DNA structures, which control both turning on and off of transcription and the rate of transcription firing, are amenable to small-molecule targeting. This dynamic system provides a unique opportunity for the treatment of tumours in which MYC is an important oncogene.

Pathogen recognition by T cells is dependent on their exquisite specificity for self-major histocompatibility complex (MHC) molecules presenting a bound peptide. Although this specificity results from positive and negative selection of developing T cells in the thymus, the relative contribution of these two processes remains controversial. To address the relation between the selecting peptide-MHC complex and the specificity of mature T cells, we generated transgenic mice that express a single peptide-MHC class I complex. We demonstrate that positive selection of CD8 T cells in these mice results in an MHC-specific repertoire. Although selection on a single complex is peptide promiscuous, mature T cells are highly peptide specific. Thus, positive selection imparts MHC and peptide specificity on the peripheral CD8 T cell repertoire.

Some enniatins (ENs) reportedly exhibit antiretroviral activities in vivo. The potential inhibitory activities of cyclic hexadepsipeptides such as beauvericin (BEA) and ENs H, I and MK1688 were investigated in vitro against human immunodeficiency virus type-1 (HIV-1) integrase and Moloney murine leukemia virus reverse transcriptase. BEA, EN I and EN MK1688 exhibited strong inhibitory activities against HIV-1 integrase, whereas EN H showed relatively weak activity. None of the examined compounds showed anti-reverse transcriptase activity. BEA was the most effective inhibitor of the tested cyclic hexadepsipeptides in inhibiting HIV-1 integrase. These results indicate the potential of cyclic hexadepsipeptides as a new class of potent inhibitors of HIV-1 integrase.

Patients with advanced papillary renal cell cancer (pRCC) have poor survival after systemic therapy; the reported median survival time is 7 to 17 months. In this trial, we evaluated the efficacy of erlotinib, an oral epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor in patients with advanced pRCC, a tumor type associated with wild-type von Hippel Lindau gene.

Patients with histologically confirmed, advanced, or metastatic pRCC were treated with erlotinib 150 mg orally once daily. A RECIST (Response Evaluation Criteria in Solid Tumors) response rate (RR) of > or = 20% was considered a promising outcome. Secondary end points included overall survival and 6-month probability of treatment failure.

Of 52 patients registered, 45 were evaluable. The overall RR was 11% (five of 45 patients; 95% CI, 3% to 24%), and the disease control rate was 64% (ie five partial response and 24 stable disease). The median overall survival time was 27 months (95% CI, 13 to 36 months). Probability of freedom from treatment failure at 6 months was 29% (95% CI, 17% to 42%). There was one grade 5 adverse event (AE) of pneumonitis, one grade 4 thrombosis, and nine other grade 3 AEs.

Although the RECIST RR of 11% did not exceed prespecified estimates for additional study, single-agent erlotinib yielded disease control and survival outcomes of interest with an expected toxicity profile. The design of future trials of the EGFR axis in pRCC should be based on preclinical or molecular data that define appropriate patient subgroups, new drug combinations, or potentially more active alternative schedules.

West Nile virus (WNV) infection causes a life-threatening meningoencephalitis that becomes increasingly more prevalent over the age of 50 and is 40-50x more prevalent in people over the age of 70, compared with adults under the age of 40. In a mouse model of age-related vulnerability to WNV, we demonstrate that death correlates with increased viral titers in the brain and that this loss of virus control with age was the result of defects in the CD4 and CD8 T cell response against WNV. Specific age-related defects in T cell responses against dominant WNV epitopes were detected at the level of cytokine and lytic granule production, each of which are essential for resistance against WNV, and in the ability to generate multifunctional anti-WNV effector T cells, which are believed to be critical for robust antiviral immunity. In contrast, at the peak of the response, old and adult T cells exhibited superimposable peptide sensitivity. Most importantly, although the adult CD4 or CD8 T cells readily protected immunodeficient mice upon adoptive transfer, old T cells of either subset were unable to provide WNV-specific protection. Consistent with a profound qualitative and quantitative defect in T cell immunity, old brains contained at least 12x fewer total effector CD8 T cells compared with adult mice at the peak of brain infection. These findings identify potential targets for immunomodulation and treatment to combat lethal WNV infection in the elderly.

Although T helper 17 (Th17) cells have been found in tumor tissues, their function in cancer immunity is unclear. We found that interleukin-17A (IL-17A)-deficient mice were more susceptible to developing lung melanoma. Conversely, adoptive T cell therapy with tumor-specific Th17 cells prevented tumor development. Importantly, the Th17 cells retained their cytokine signature and exhibited stronger therapeutic efficacy than Th1 cells. Unexpectedly, therapy using Th17 cells elicited a remarkable activation of tumor-specific CD8(+) T cells, which were necessary for the antitumor effect. Th17 cells promoted dendritic cell recruitment into the tumor tissues and in draining lymph nodes increased CD8 alpha(+) dendritic cells containing tumor material. Moreover, Th17 cells promoted CCL20 chemokine production by tumor tissues, and tumor-bearing CCR6-deficient mice did not respond to Th17 cell therapy. Thus, Th17 cells elicited a protective inflammation that promotes the activation of tumor-specific CD8(+) T cells. These findings have important implications in antitumor immunotherapies.

Cytokines are key modulators of T cell biology, but their influence can be attenuated by suppressors of cytokine signaling (SOCS), a family of proteins consisting of eight members, SOCS1-7 and CIS. SOCS proteins regulate cytokine signals that control the polarization of CD4(+) T cells into Th1, Th2, Th17, and T regulatory cell lineages, the maturation of CD8(+) T cells from naïve to "stem-cell memory" (Tscm), central memory (Tcm), and effector memory (Tem) states, and the activation of these lymphocytes. Understanding how SOCS family members regulate T cell maturation, differentiation, and function might prove critical in improving adoptive immunotherapy for cancer and therapies aimed at treating autoimmune and infectious diseases.

Estrogen receptor alpha (ERalpha) phosphorylation is important for estrogen-dependent transcription of ER-dependent genes, ligand-independent receptor activation and endocrine therapy response in breast cancer. However ERalpha phosphorylation at the previously identified sites does not fully account for these receptor functions. To determine if additional ERalpha phosphorylation sites exist, COS-1 cells expressing human ERalpha were labeled with [32P]H3PO4 in vivo and ERalpha tryptic phosphopeptides were isolated to identify phosphorylation sites.

Previously uncharacterized phosphorylation sites at serines 46/47, 282, 294, and 559 were identified by manual Edman degradation and phosphoamino acid analysis and confirmed by mutagenesis and phospho-specific antibodies. Antibodies detected phosphorylation of endogenous ERalpha in MCF-7, MCF-7-LCC2, and Ishikawa cancer cell lines by immunoblot. Mutation of Ser-282 and Ser-559 to alanine (S282A, S559A) resulted in ligand independent activation of ERalpha as determined by both ERE-driven reporter gene assays and endogenous pS2 gene expression in transiently transfected HeLa cells. Mutation of Ser-46/47 or Ser-294 to alanine markedly reduced estradiol dependent reporter activation. Additionally protein kinase CK2 was identified as a kinase that phosphorylated ERalpha at S282 and S559 using motif analysis, in vitro kinase assays, and incubation of cells with CK2 kinase inhibitor.

These novel ERalpha phosphorylation sites represent new means for modulation of ERalpha activity. S559 represents the first phosphorylation site identified in the extreme C-terminus (F domain) of a steroid receptor.

Glaucoma is an ocular disease characterized by damage of the optic nerve head (ONH) resulting in blindness. Recent research has identified the material properties of the sclera as being an important factor in the biomechanics of major load bearing tissues near the ONH. Most mechanical investigations performed on sclera have focused on the tensile behavior of this tissue, neglecting its compressive stiffness. The present study characterized the compressive moduli of peripapillary sclera using an unconfined compression (UCC) technique, for both human and porcine sources. UCC stress-relaxation tests were performed on human and porcine peripapillary scleral samples at 5%, 10% and 15% sequential compressive strain. Our results indicate a linearly decreasing drained equilibrium stress (at 5%) with age in male human samples, ranging from 79.4 Pa at 78 yrs to 40.1 Pa at 89 yrs of age. The drained secant modulus (E(5)) of human and porcine sclera was found to be 1.1 +/- 0.08 kPa and 3.9 +/- 0.57 kPa, respectively. Our experimental results also reveal a non-linear increase in drained equilibrium stress with increasing compressive strain. The compressive stiffness of sclera, as reported here, provides important information on the mechanical response of peripapillary ocular tissues. This information will be useful in future computational simulations of the sclera, especially as they relate to understanding mechanical damage near the ONH. Furthermore, our results indicate that age-related changes in the biomechanical response of the sclera occur, suggesting that these factors may be playing a role in the increasing prevalence of glaucoma with age.

The next generation of tissue engineered constructs (TECs) requires the incorporation of a controllable and optimized microstructure if they are to chemically, mechanically, and biologically mimic tissue function. In order to obtain TECs with optimized microstructures, a combination of spatiotemporally regulated mechanical and biochemical stimuli is necessary during the formation of the construct. While numerous efforts have been made to create functional tissue constructs, there are few techniques available to stimulate TECs in a localized manner. We herein describe the design of a microdevice which can stimulate TECs in a localized, inhomogeneous, and predefined anisotropic fashion using ferromagnetically doped polydimethylsiloxane microflaps (MFs). Specifically, a sequential magneto-structural finite element model of the proposed microdevice is constructed and utilized to understand how changes in magnetic and geometrical properties of the device affect MF deflection. Our study indicates that a relatively small density of ferromagnetic material is required to result in adequate force and MF defection (175 microm approximately 7% TEC strain). We also demonstrate that MF to magnet distance is more important than inherent MF magnetic permeability in determining resulting MF deflection. An experimental validation test setup was used to validate the computational solutions. The comparison shows reasonable agreement indicating a 5.9% difference between experimentally measured and computationally predicted MF displacement. Correspondingly, an apparatus with two MFs and two magnets has been made and is currently undergoing construct testing. The current study presents the design of a novel magnetic microactuator for tissue engineering applications. The computational results reported here will form the foundation in the design and optimization of a functional microdevice with multiple MFs and magnets capable of stimulating TECs in nonhomogenous and preferred directions with relevant spatial resolution.

No current studies have systematically examined pulmonary health effects associated with Syntroleum S-8 synthetic jet fuel (S-8). In order to gain an understanding about the threshold concentration in which lung injury is observed, C57BL/6 male mice were nose-only exposed to S-8 for 1 h/day for 7 days at average concentrations of 0 (control), 93, 352, and 616 mg/m(3). Evaluation of pulmonary function, airway epithelial barrier integrity, and pathohistology was performed 24 h after the final exposures. Significant decreases were detected in expiratory lung resistance and total lung compliance of the 352 mg/m(3) group, for which no clear concentration-dependent alterations could be determined. No significant changes in respiratory permeability were exhibited, indicating that there was no loss of epithelial barrier integrity following S-8 exposure. However, morphological examination and morphometric analysis of distal lung tissue, by using transmission electron microscopy, revealed cellular damage in alveolar type II epithelial cells, with significant increases in volume density of lamellar bodies/vacuoles at 352 and 616 S-8 mg/m(3). Moreover, terminal bronchiolar Clara injury, as evidenced by apical membrane blebs, was observed at relatively low concentrations, suggesting if this synthetic jet fuel is utilized, the current permissible exposure limit of 350 mg/m(3) for hydrocarbon fuels should cautiously be applied.

In addition to cancer endpoints, arsenic exposures can also lead to non-cancerous chronic lung disease. Exposures during sensitive developmental time points can contribute to the adult disease. Using a mouse model, in utero and early postnatal exposures to arsenic (100 ppb or less in drinking water) were found to alter airway reactivity to methacholine challenge in 28 day old pups. Removal of mice from arsenic exposure 28 days after birth did not reverse the alterations in sensitivity to methacholine. In addition, adult mice exposed to similar levels of arsenic in drinking water did not show alterations. Therefore, alterations in airway reactivity were irreversible and specific to exposures during lung development. These functional changes correlated with protein and gene expression changes as well as morphological structural changes around the airways. Arsenic increased the whole lung levels of smooth muscle actin in a dose dependent manner. The level of smooth muscle mass around airways was increased with arsenic exposure, especially around airways smaller than 100 microm in diameter. This increase in smooth muscle was associated with alterations in extracellular matrix (collagen, elastin) expression. This model system demonstrates that in utero and postnatal exposure to environmentally relevant levels of arsenic can irreversibly alter pulmonary structure and function in the adults.

In the present study, we characterize the toxic effects of in utero arsenic exposure on the developing lung. We hypothesize that in utero exposure to inorganic arsenic through maternal drinking water causes altered gene and protein expression in the developing lung, indicative of downstream molecular and functional changes. From conception to embryonic day 18, we exposed pregnant Sprague-Dawley rats to 500 ppb arsenic (as arsenite) via the drinking water. Subtracted cDNA libraries comparing control to arsenic exposed embryonic lungs were generated. In addition, a broad Western blot analysis was performed to identify altered protein expression. A total of 59 genes and 34 proteins were identified as being altered. Pathway mapping and analysis showed that cell motility was the process most affected. The most likely affected pathway was alteration in integrin signaling through the beta-catenin pathway, altering c-myc. The present study shows that arsenic induces alterations in the developing lung. These data may be useful in the elucidation of molecular targets and biomarkers of arsenic exposure during lung development and may aid in understanding the etiology of arsenic induced adult respiratory disease and lung cancers.

The mammalian members of the Multidrug And Toxin Extruder family, i.e., MATE1 and MATE2-K, are suspected of mediating the luminal step in renal secretion of organic cations. The 1,000+ prokaryotic/fungal/plant MATE family members are predicted to have 12 transmembrane helices (TMHs), whereas MATE1/2-K appear to have an additional (13th) COOH-terminal helix. Here, we determined whether rabbit MATE1 has an external COOH terminus, consistent with the presence of 13 TMHs. A V5 epitope tag at the COOH terminus of MATE1 was freely accessible to external V5 antibody, whereas tags at the NH(2) terminus, or at sites of truncation within the long cytoplasmic loop between predicted TMHs 12 and 13, were only accessible to the V5 antibody following permeabilization of the membrane. The truncated mutants that lacked TMH13 still retained transport activity, indicating that the terminal helix was not necessary for transport function. Cells that expressed a mutant lacking only TMH13 displayed similar K(t) and J(max) values to those of the full-length protein, although when normalized to protein expressed at the plasma membrane, the transport rate of the mutant was <10% that of full-length MATE1. An effectively cysteine-less MATE1 mutant (Delta13Cys) was functional and refractory to reaction with the impermeant marker of accessible cysteine residues, maleimide-PEO(2)-biotin. Delta13Cys mutants with an added cysteine residue at the truncation sites within the terminal cytoplasmic loop reacted with maleimide biotin only after permeabilization of the membrane, whereas a mutant with a cysteine residue at the COOH terminus was freely accessible to maleimide biotin. These data are consistent with a mammalian MATE topology that includes 13 TMHs and indicate that the terminal TMH, although not necessary for transport function, may influence the turnover characteristics of the transporter.

The nonpigmented epithelium (NPE) of the ciliary body represents an important component of the blood-aqueous barrier of the eye. Many therapeutic drugs penetrate poorly across the NPE into the aqueous humor of the eye interior. Several of these therapeutic drugs, such as methotrexate, vincristine, and etoposide, are substrates of the multidrug resistance-associated protein 2 (MRP2). Abundant MRP2 protein was detected by Western blot in homogenates of human ciliary body and freshly dissected porcine NPE. In cultured porcine NPE, the intracellular accumulation of the MRP2 substrates calcein (1.8-fold), 5-(and-6)-carboxy-2',7'-dichlorofluorescein (22.1-fold), and doxorubicin (1.9-fold) was significantly increased in the presence of 50 microM MK571 ((E)-3-[[[3-[2-(7-chloro-2-quinolinyl)-ethenyl]phenyl]-[[3-dimethylamino)-3-oxopropyl]thio]methyl]thio]-propanoic acid), an MRP inhibitor. In addition, the intracellular accumulation of the MRP2 substrate glutathione methylfluorescein was increased by 50 microM MK571 (4.3-fold), 500 microM indomethacin (2.6-fold), and 50 microM cyclosporin A (2.1-fold) but not by 500 microM sulfinpyrazone. These data are consistent with MRP2-mediated transport activity in cultured NPE, and MRP2 mRNA (reverse transcriptase-polymerase chain reaction) and protein (Western blot) were detected in the cultured cells. Immunolocalization studies in native human and porcine eyes showed MRP2 protein at the apical interface of the NPE and pigmented cell layers. Close examination of MRP2 immunoreactivity supported the conclusion that MRP2 is localized in the apical membrane of the NPE. MRP2 at the apical membrane of NPE cells may be involved in protecting intraocular tissues from exposure to potentially harmful toxins.

Studies were conducted to characterize the effect of dose and route of administration on the disposition of N-butylpyridinium chloride (NBuPy-Cl), an ionic liquid with solvent properties. Urine was the major route of NBuPy-Cl excretion after intravenous (5 mg/kg), single oral (0.5, 5, or 50 mg/kg), or repeated oral (50 mg/kg/day, 5 days) administration to male F-344 rats and single oral (50 mg/kg) administration to female B6C3F1 mice. Depending on the vehicle, absorption after dermal application (5 mg/kg, 125 microg/cm(2)) was 10 to 35% at 96 h. After the single intravenous dose, the blood concentration of NBuPy-Cl decreased in a biphasic manner with an elimination half-life of 2.2 h and a clearance of 7 ml/min. After single oral administration of NBuPy-Cl (50 mg/kg), maximum blood concentration was reached at 1.3 h, and the bioavailability was determined to be 47% at 6 h based on the blood toxicokinetics and 67% at 72 h based on urinary excretion. In all the urine and blood samples, only the parent compound was detected. Coadministration of NBuPy-Cl and inulin (by intravenous injection) revealed that the clearance of NBuPy-Cl exceeded the rat glomerular filtration rate. After incubation with Chinese hamster ovary cells expressing human organic cation transporter 2 (hOCT2), NBuPy-Cl was transported effectively (K(t) = 18 microM), and also a potent inhibitor of hOCT2 mediated tetraethylammonium transport (IC(50) = 2.3 microM). In summary, NBuPy-Cl is partially absorbed from the gastrointestinal tract and eliminated rapidly in the urine as parent compound most likely by renal glomerular filtration and OCT2-mediated secretion.