Deciphering the role of protein kinase A in the control of FoxP3 expression in regulatory T cells in health and autoimmunity.

The molecular mechanisms that govern differential T cell development from CD4+CD25-conventional T (Tconv) into CD4+CD25+ forkhead-box-P3+ (FoxP3+) inducible regulatory T (iTreg) cells remain unclear. Herein, we investigated the relative contribution of protein kinase A (PKA) in this process. Mechanistically, we found that PKA controlled the efficiency of human iTreg cell generation through the expression of different FoxP3 splicing variants containing or not the exon 2. We found that transient PKA inhibition reduced the recruitment of cAMP-responsive element-binding protein (CREB) on regulatory regions of the FoxP3 gene, a condition that is associated with an impaired acquisition of their suppressive capacity in vitro. To corroborate our findings in a human model of autoimmunity, we measured CREB phosphorylation and FoxP3 levels in iTreg cells from treatment-naïve relapsing-remitting (RR)-multiple sclerosis (MS) subjects. Interestingly, both phospho-CREB and FoxP3 induction directly correlated and were significantly reduced in RR-MS patients, suggesting a previously unknown mechanism involved in the induction and function of human iTreg cells.

KRAS is a molecular determinant of platinum responsiveness in glioblastoma.

BACKGROUND: KRAS is the undisputed champion of oncogenes, and despite its prominent role in oncogenesis as mutated gene, KRAS mutation appears infrequent in gliomas. Nevertheless, gliomas are considered KRAS-driven cancers due to its essential role in mouse malignant gliomagenesis. Glioblastoma is the most lethal primary brain tumor, often associated with disturbed RAS signaling. For newly diagnosed GBM, the current standard therapy is alkylating agent chemotherapy combined with radiotherapy. Cisplatin is one of the most effective anticancer drugs and is used as a first-line treatment for a wide spectrum of solid tumors (including medulloblastoma and neuroblastoma) and many studies are currently focused on new delivery modalities of effective cisplatin in glioblastoma. Its mechanism of action is mainly based on DNA damage, inducing the formation of DNA adducts, triggering a series of signal-transduction pathways, leading to cell-cycle arrest, DNA repair and apoptosis. METHODS: Long-term cultures of human glioblastoma, U87MG and U251MG, were either treated with cis-diamminedichloroplatinum (cisplatin, CDDP) and/or MEK-inhibitor PD98059. Cytotoxic responses were assessed by cell viability (MTT), protein expression (Western Blot), cell cycle (PI staining) and apoptosis (TUNEL) assays. Further, gain-of-function experiments were performed with cells over-expressing mutated hypervariable region (HVR) KRASG12V plasmids. RESULTS: Here, we studied platinum-based chemosensitivity of long-term cultures of human glioblastoma from the perspective of KRAS expression, by using CDDP and MEK-inhibitor. Endogenous high KRAS expression was assessed at transcriptional (qPCR) and translational levels (WB) in a panel of primary and long-term glioblastoma cultures. Firstly, we measured immediate cellular adjustment through direct regulation of protein concentration of K-Ras4B in response to cisplatin treatment. We found increased endogenous protein abundance and involvement of the effector pathway RAF/MEK/ERK mitogen-activated protein kinase (MAPK) cascade. Moreover, as many MEK inhibitors are currently being clinically evaluated for the treatment of high-grade glioma, so we concomitantly tested the effect of the potent and selective non-ATP-competitive MEK1/2 inhibitor (PD98059) on cisplatin-induced chemosensitivity in these cells. Cell-cycle phase distribution was examined using flow cytometry showing a significant cell-cycle arrest in both cultures at different percentage, which is modulated by MEK inhibition. Cisplatin-induced cytotoxicity increased sub-G1 percentage and modulates G2/M checkpoint regulators cyclins D1 and A. Moreover, ectopic expression of a constitutively active KRASG12V rescued CDDP-induced apoptosis and different HVR point mutations (particularly Ala 185) reverted this phenotype. CONCLUSION: These findings warrant further studies of clinical applications of MEK1/2 inhibitors and KRAS as 'actionable target' of cisplatin-based chemotherapy for glioblastoma.

Redox-sensitive small GTPase H-Ras in murine astrocytes, an in vitro study.

BACKGROUND: Although the protooncogenes small GTPases Ras are redox-sensitive proteins, how they are regulated by redox signaling in the central nervous system (CNS) is still poorly understood. Alteration in redox-sensitive targets by redox signaling may have myriad effects on Ras stability, activity and localization. Redox-mediated changes in astrocytic RAS may contribute to the control of redox homeostasis in the CNS that is connected to the pathogenesis of many diseases. RESULTS AND METHODS: Here, we investigated the transient physiological induction, at both transcriptional and translational levels, of small GTPases Ras in response to redox stimulation. Cultured astrocytes were treated with hydrogen peroxide as in bolus addition and relative mRNA levels of murine hras and kras genes were detected by qRT-PCR. We found that de novo transcription of hras mRNA in reactive astrocytes is redox-sensitive and mimics the prototypical redox-sensitive gene iNOS. Protein abundance in combination with protein turnover measurements by cycloheximide-chase experiments revealed distinct translation efficiency, GTP-bound enrichment, and protein turnover rates between the two isoforms H-Ras and K-Ras. CONCLUSION: Reports from recent years support a significant role of H-Ras in driving redox processes. Beyond its canonical functions, Ras may impact on the core astrocytic cellular machinery that operates during redox stimulation.

Estrogen Induces Selective Transcription of Caveolin1 Variants in Human Breast Cancer through Estrogen Responsive Element-Dependent Mechanisms.

The estrogen receptor (ER) signaling regulates numerous physiological processes mainly through activation of gene transcription (genomic pathways). Caveolin1 (CAV1) is a membrane-resident protein that behaves as platform to enable different signaling molecules and receptors for membrane-initiated pathways. CAV1 directly interacts with ERs and allows their localization on membrane with consequent activation of ER-non-genomic pathways. Loss of CAV1 function is a common feature of different types of cancers, including breast cancer. Two protein isoforms, CAV1α and CAV1ß, derived from two alternative translation initiation sites, are commonly described for this gene. However, the exact transcriptional regulation underlying CAV1 expression pattern is poorly elucidated. In this study, we dissect the molecular mechanism involved in selective expression of CAV1ß isoform, induced by estrogens and downregulated in breast cancer. Luciferase assays and Chromatin immunoprecipitation demonstrate that transcriptional activation is triggered by estrogen-responsive elements embedded in CAV1 intragenic regions and DNA-binding of estrogen-ER complexes. This regulatory control is dynamically established by local chromatin changes, as proved by the occurrence of histone H3 methylation/demethylation events and association of modifier proteins as well as modification of H3 acetylation status. Thus, we demonstrate for the first time, an estrogen-ERs-dependent regulatory circuit sustaining selective CAV1ß expression.

The DEL-1/ß3 integrin axis promotes regulatory T cell responses during inflammation resolution.

FOXP3+CD4+ regulatory T cells (Tregs) are critical for immune homeostasis and respond to local tissue cues, which control their stability and function. We explored here whether developmental endothelial locus-1 (DEL-1), which, like Tregs, increases during resolution of inflammation, promotes Treg responses. DEL-1 enhanced Treg numbers and function at barrier sites (oral and lung mucosa). The underlying mechanism was dissected using mice lacking DEL-1 or expressing a point mutant thereof, or mice with T cell-specific deletion of the transcription factor RUNX1, identified by RNA sequencing analysis of the DEL-1-induced Treg transcriptome. Specifically, through interaction with αvß3 integrin, DEL-1 promoted induction of RUNX1-dependent FOXP3 expression and conferred stability of FOXP3 expression upon Treg restimulation in the absence of exogenous TGF-ß1. Consistently, DEL-1 enhanced the demethylation of the Treg-specific demethylated region (TSDR) in the mouse Foxp3 gene and the suppressive function of sorted induced Tregs. Similarly, DEL-1 increased RUNX1 and FOXP3 expression in human conventional T cells, promoting their conversion into induced Tregs with increased TSDR demethylation, enhanced stability, and suppressive activity. We thus uncovered a DEL-1/αvß3/RUNX1 axis that promotes Treg responses at barrier sites and offers therapeutic options for modulating inflammatory/autoimmune disorders.

Targeted DNA oxidation by LSD1-SMAD2/3 primes TGF-ß1/ EMT genes for activation or repression.

The epithelial-to-mesenchymal transition (EMT) is a complex transcriptional program induced by transforming growth factor ß1 (TGF-ß1). Histone lysine-specific demethylase 1 (LSD1) has been recognized as a key mediator of EMT in cancer cells, but the precise mechanism that underlies the activation and repression of EMT genes still remains elusive. Here, we characterized the early events induced by TGF-ß1 during EMT initiation and establishment. TGF-ß1 triggered, 30-90 min post-treatment, a nuclear oxidative wave throughout the genome, documented by confocal microscopy and mass spectrometry, mediated by LSD1. LSD1 was recruited with phosphorylated SMAD2/3 to the promoters of prototypic genes activated and repressed by TGF-ß1. After 90 min, phospho-SMAD2/3 downregulation reduced the complex and LSD1 was then recruited with the newly synthesized SNAI1 and repressors, NCoR1 and HDAC3, to the promoters of TGF-ß1-repressed genes such as the Wnt soluble inhibitor factor 1 gene (WIF1), a change that induced a late oxidative burst. However, TGF-ß1 early (90 min) repression of transcription also required synchronous signaling by reactive oxygen species and the stress-activated kinase c-Jun N-terminal kinase. These data elucidate the early events elicited by TGF-ß1 and the priming role of DNA oxidation that marks TGF-ß1-induced and -repressed genes involved in the EMT.

High glucose concentration produces a short-term increase in pERK1/2 and p85 proteins, having a direct angiogenetic effect by an action similar to VEGF.

AIMS: Excessive glucose serum concentration, endothelial dysfunction and microangiopathy are key features of diabetes mellitus, being both diagnostic parameters and pathogenetic mechanisms. Vascular endothelial growth factor (VEGF) is importantly implicated in the physiology and pathology of blood vessels, including diabetic vascular damage. METHODS: These factors certainly affect endothelial cells, and to evaluate mechanisms involved, we took advantage of telomerase-immortalized human microvascular endothelial (TIME) cells. TIME cells were exposed to different glucose concentrations and to VEGF treatments. Culture conditions also included the use of basement membrane extract, as an in vitro differentiation model. Cell morphology was then evaluated in the different conditions, and cellular proteins were extracted to analyze specific protein products by Western blot. RESULTS: High glucose concentrations and VEGF did substantially affect neither morphology nor growth of cultured TIME cells, while both considerably increased differentiation into "capillary-like" structures when cells were cultured on basement membrane extract. CONCLUSIONS: Under these conditions, high glucose concentration and VEGF also produced a short-term increase in pERK1/2 and p85 proteins, while total and phosphorylated AKT were not affected. These data suggest a direct angiogenetic effect of glucose, affecting intracellular transduction mechanisms with an action similar to that of VEGF. This effect on endothelial cell proliferation and differentiation could be part of pathogenetic mechanisms producing diabetic microvascular alterations.

RNA Stabilizes Transcription-Dependent Chromatin Loops Induced By Nuclear Hormones.

We show that transcription induced by nuclear receptors for estrogen (E2) or retinoic acid (RA) is associated with formation of chromatin loops that juxtapose the 5' end (containing the promoter) with the enhancer and the 3' polyA addition site of the target gene. We find three loop configurations which change as a function of time after induction: 1. RA or E2-induced loops which connect the 5' end, the enhancer and the 3' end of the gene, and are stabilized by RNA early after induction; 2. E2-independent loops whose stability does not require RNA; 3. Loops detected only by treatment of chromatin with RNAse H1 prior to hormonal induction. RNAse H1 digests RNA that occludes the relevant restriction sites, thus preventing detection of these loops. R-loops at the 5' and 3' ends of the RA or E2-target genes were demonstrated by immunoprecipitation with anti-DNA-RNA hybrid antibodies as well as by sensitivity to RNAse H1. The cohesin RAD21 subunit is preferentially recruited to the target sites upon RA or E2 induction of transcription. RAD21 binding to chromatin is eliminated by RNAse H1. We identified E2-induced and RNase H1-sensitive antisense RNAs located at the 5' and 3' ends of the E2-induced transcription unit which stabilize the loops and RAD21 binding to chromatin. This is the first report of chromatin loops that form after gene induction that are maintained by RNA:DNA hybrids.

A functional connection between dyskerin and energy metabolism.

The human DKC1 gene encodes dyskerin, an evolutionarily conserved nuclear protein whose overexpression represents a common trait of many types of aggressive sporadic cancers. As a crucial component of the nuclear H/ACA snoRNP complexes, dyskerin is involved in a variety of essential processes, including telomere maintenance, splicing efficiency, ribosome biogenesis, snoRNAs stabilization and stress response. Although multiple minor dyskerin splicing isoforms have been identified, their functions remain to be defined. Considering that low-abundance splice variants could contribute to the wide functional repertoire attributed to dyskerin, possibly having more specialized tasks or playing significant roles in changing cell status, we investigated in more detail the biological roles of a truncated dyskerin isoform that lacks the C-terminal nuclear localization signal and shows a prevalent cytoplasmic localization. Here we show that this dyskerin variant can boost energy metabolism and improve respiration, ultimately conferring a ROS adaptive response and a growth advantage to cells. These results reveal an unexpected involvement of DKC1 in energy metabolism, highlighting a previously underscored role in the regulation of metabolic cell homeostasis.

Non-homologous end joining induced alterations in DNA methylation: A source of permanent epigenetic change.

In addition to genetic mutations, epigenetic revision plays a major role in the development and progression of cancer; specifically, inappropriate DNA methylation or demethylation of CpG residues may alter the expression of genes that promote tumorigenesis. We hypothesize that DNA repair, specifically the repair of DNA double strand breaks (DSB) by Non-Homologous End Joining (NHEJ) may play a role in this process. Using a GFP reporter system inserted into the genome of HeLa cells, we are able to induce targeted DNA damage that enables the cells, after successfully undergoing NHEJ repair, to express WT GFP. These GFP+ cells were segregated into two expression classes, one with robust expression (Bright) and the other with reduced expression (Dim). Using a DNA hypomethylating drug (AzadC) we demonstrated that the different GFP expression levels was due to differential methylation statuses of CpGs in regions on either side of the break site. Deep sequencing analysis of this area in sorted Bright and Dim populations revealed a collection of different epi-alleles that display patterns of DNA methylation following repair by NHEJ. These patterns differ between Bright and Dim cells which are hypo- and hypermethylated, respectively, and between the post-repair populations and the original, uncut cells. These data suggest that NHEJ repair facilitates a rewrite of the methylation landscape in repaired genes, elucidating a potential source for the altered methylation patterns seen in cancer cells, and understanding the mechanism by which this occurs could provide new therapeutic targets for preventing this process from contributing to tumorigenesis.

Glycolysis controls the induction of human regulatory T cells by modulating the expression of FOXP3 exon 2 splicing variants.

Human regulatory T cells (T(reg) cells) that develop from conventional T cells (T(conv) cells) following suboptimal stimulation via the T cell antigen receptor (TCR) (induced T(reg) cells (iT(reg) cells)) express the transcription factor Foxp3, are suppressive, and display an active proliferative and metabolic state. Here we found that the induction and suppressive function of iT(reg) cells tightly depended on glycolysis, which controlled Foxp3 splicing variants containing exon 2 (Foxp3-E2) through the glycolytic enzyme enolase-1. The Foxp3-E2-related suppressive activity of iT(reg) cells was altered in human autoimmune diseases, including multiple sclerosis and type 1 diabetes, and was associated with impaired glycolysis and signaling via interleukin 2. This link between glycolysis and Foxp3-E2 variants via enolase-1 shows a previously unknown mechanism for controlling the induction and function of T(reg) cells in health and in autoimmunity.

Effect of intravitreal ranibizumab injections on aqueous humour concentrations of vascular endothelial growth factor and pigment epithelium-derived factor in patients with myopic choroidal neovascularisation.

AIMS: To investigate aqueous humour changes in vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF) levels in patients with choroidal neovascularisation (CNV) secondary to pathological myopia (mCNV) before and after intravitreal ranibizumab injection (IVR). METHODS: This was a prospective, case-control study investigating aqueous levels of VEGF and PEDF in eyes with mCNV treated with IVR. RESULTS: Mean VEGF and PEDF levels in the aqueous humour of control patients were 25.7±4.9 pg/mL and 12.6±3.5 ng/mL, respectively. Lower levels of both VEGF (19.5±5.4 pg/mL) and PEDF (4.7±2.2 ng/mL) were found in patients with mCNV before IVR. After IVR, aqueous VEGF levels significantly reduced to 6.5±2.7 pg/mL, while PEDF levels significantly increased to 35.8±11.4 ng/mL. VEGF and PEDF levels significantly correlated with each other, and with best-corrected visual acuity and central retinal thickness. CONCLUSIONS: The VEGF and PEDF levels in aqueous humour were significantly lower in the myopic group than in controls. Moreover, IVR resulted in reduced VEGF and increased PEDF levels in patients with mCNV. In mCNV, neovascularisation is associated with inappropriate VEGF and PEDF expression. A balance between VEGF and PEDF is crucial to prevent CNV development. TRIAL REGISTRATION NUMBER: NCT02175940.

Mechanism of retinoic acid-induced transcription: histone code, DNA oxidation and formation of chromatin loops.

Histone methylation changes and formation of chromatin loops involving enhancers, promoters and 3' end regions of genes have been variously associated with active transcription in eukaryotes. We have studied the effect of activation of the retinoic A receptor, at the RARE-promoter chromatin of CASP9 and CYP26A1 genes, 15 and 45 min following RA exposure, and we found that histone H3 lysines 4 and 9 are demethylated by the lysine-specific demethylase, LSD1 and by the JMJ-domain containing demethylase, D2A. The action of the oxidase (LSD1) and a dioxygenase (JMJD2A) in the presence of Fe++ elicits an oxidation wave that locally modifies the DNA and recruits the enzymes involved in base and nucleotide excision repair (BER and NER). These events are essential for the formation of chromatin loop(s) that juxtapose the RARE element with the 5' transcription start site and the 3' end of the genes. The RARE bound-receptor governs the 5' and 3' end selection and directs the productive transcription cycle of RNA polymerase. These data mechanistically link chromatin loops, histone methylation changes and localized DNA repair with transcription.

The inhibition of p85αPI3KSer83 phosphorylation prevents cell proliferation and invasion in prostate cancer cells.

Phosphoinositide 3-kinase proteins are composed by a catalytic p110 subunit and a regulatory p85 subunit. There are three classes of PI3K, named class I-III, on the bases of the protein domain constituting and determining their specificity. The first one is the best characterized and includes a number of key elements for the integration of different cellular signals. Regulatory p85 subunit shares with the catalytic p110 subunit, a N-terminal SH3 domain showing homology with the protein domain Rho-GTP-ase. After cell stimulation, all class I PI3Ks are recruited to the inner face of the plasma membrane, where they generate phosphatidylinositol-3,4,5-trisphosphate by direct phosphorylation of phosphatidylinositol-4,5-bisphosphate. All pathways trigger the control of different phenomena such as cell growth, proliferation, apoptosis, adhesion and migration through various downstream effectors. We have previously provided direct evidences that a Serine in position 83, adjacent to the N-terminal SH3 domain of regulatory subunit of PI3K, is a substrate of PKA. The aim of this work is to confirm the role of p85αPI3KSer83 in regulating cell proliferation, migration and invasion in prostate cancer cells LNCaP. To this purpose cells were transfected with mutant forms of p85, where Serine was replaced by Alanine, where phosphorylation is prevented, or Aspartic Acid, to mimic the phosphorylated residue. The findings of this study suggest that identifying a peptide mimicking the sequence adjacent to Ser 83 may be used to produce antibodies against this residue that can be proposed as usefool tool for prognosis by correlating phosphorylation at Ser83 with tumor stage.

Aqueous humor levels of vascular endothelial growth factor and adiponectin in patients with type 2 diabetes before and after intravitreal bevacizumab injection.

To determine the levels of vascular endothelial growth factor (VEGF) and adiponectin (APN) in the aqueous humor of patients with type 2 diabetes before and after injection of bevacizumab (IVB). Twenty eyes of twenty consecutive patients with type 2 diabetes with PDR and clinically significant macular edema were enrolled in this study. Aqueous samples were collected at baseline and one month after IVB to evaluate VEGF and APN levels. Twenty age-matched patients undergoing cataract surgery were used as control. Best-corrected visual acuity (BCVA) and foveal thickness (FT) changes after IVB were also measured. Safety was assessed by recording the incidence of ocular and non-ocular adverse events. At baseline APN and VEGF levels were significantly lower in controls than in PDR patients (APN: 3.6 ± 1.1 vs 18.7 ± 4.5 ng/ml; VEGF: 22.6 ± 16.1 vs 146.2 ± 38.71 pg/ml). After IVB, both compounds significantly decreased. FT and BCVA at baseline were significantly different between controls and patients (FT: 215.6 ± 34.8 vs 532.7 ± 112.4 µm; BCVA: 23.6 ± 4.2 vs 18.4 ± 7.3 letters). After IVB a significant decrease of FT with a concomitant improvement of BCVA occurred. Neither ocular nor systemic adverse events were reported. Our findings demonstrate that patients with type 2 diabetes, PDR and macular edema show VEGF and APN levels in aqueous humor higher than those found in control subjects. IVB significantly reduced the levels of both compounds, which remained anyway at concentrations higher than those recorded in control subjects.

Oxidative stress posttranslationally regulates the expression of Ha-Ras and Ki-Ras in cultured astrocytes.

Addition of hydrogen peroxide to cultured astrocytes induced a rapid and transient increase in the expression of Ha-Ras and Ki-Ras. Pull-down experiments with the GTP-Ras-binding domain of Raf-1 showed that oxidative stress substantially increased the activation of Ha-Ras, whereas a putative farnesylated activated form of Ki-Ras was only slightly increased. The increase in both Ha-Ras and Ki-Ras was insensitive to the protein synthesis inhibitor, cycloheximide, and was occluded by the proteasomal inhibitor, MG-132. In addition, exposure to hydrogen peroxide reduced the levels of ubiquitinated Ras protein, indicating that oxidative stress leads to a reduced degradation of both isoforms through the ubiquitin/proteasome pathway. Indeed, the late reduction in Ha-Ras and Ki-Ras was due to a recovery of proteasomal degradation because it was sensitive to MG-132. The late reduction of Ha-Ras levels was abrogated by compound PD98059, which inhibits the MAP kinase pathway, whereas the late reduction of Ki-Ras was unaffected by PD98059. We conclude that oxidative stress differentially regulates the expression of Ha-Ras and Ki-Ras in cultured astrocytes, and that activation of the MAP kinase pathway by oxidative stress itself or by additional factors may act as a fail-safe mechanism limiting a sustained expression of the potentially detrimental Ha-Ras.

The p85α regulatory subunit of PI3K mediates cAMP-PKA and retinoic acid biological effects on MCF7 cell growth and migration.

Phosphoinositide-3-OH kinase (PI3K) signalling regulates various cellular processes, including cell survival, growth, proliferation and motility, and is among the most frequently mutated pathways in cancer. Although the involvement of p85αPI3K SH2 domain in signal transduction has been extensively studied, the function of the SH3 domain at the N-terminus remains elusive. A serine (at codon 83) adjacent to the N-terminal SH3 domain in the PI3K regulatory subunit p85αPI3K that is phosphorylated by protein kinase A (PKA) in vivo and in vitro has been identified. Virtually all receptors binding p85αPI3K can cooperate with cAMP-PKA signals via phosphorylation of p85αPI3KSer83. To analyse the role of p85αPI3KSer83 in retinoic acid (RA) and cAMP signalling, in MCF7 cells, we used p85αPI3K mutated forms, in which Ser83 has been substituted with alanine (p85A) to prevent phosphorylation or with aspartic acid (p85D) to mimic the phosphorylated residue. We demonstrated that p85αPI3KSer83 is crucial for the synergistic enhancement of RARα/p85αPI3K binding induced by cAMP/RA co-treatment in MCF7 cells. Growth curves, colorimetric MTT assay and cell cycle analysis demonstrated that phosphorylation of p85αPI3KSer83 plays an important role in the control of MCF7 cell proliferation and in RA-induced inhibition of proliferation. Wound healing and transwell experiments demonstrated that p85αPI3KSer83 was also essential both for the control of migratory behaviour and for the reduction of motility induced by RA. This study points to p85αPI3KSer83 as the physical link between different pathways (cAMP-PKA, RA and FAK), and as an important regulator of MCF7 cell proliferation and migration.

Aqueous humor levels of vascular endothelial growth factor before and after intravitreal bevacizumab in type 3 versus type 1 and 2 neovascularization. A prospective, case-control study.

PURPOSE: To determine the aqueous levels of vascular endothelial growth factor (VEGF) in patients with type 3 neovascularization (NV) secondary to age-related macular degeneration (AMD) and to compare the levels of those with type 1 and 2 NV secondary to AMD before and after administration of intravitreal bevacizumab (IVB). DESIGN: Prospective, case-control study. METHODS: Aqueous samples were collected from 29 eyes of 29 patients with untreated wet AMD at baseline (day of the first IVB), month 1 (day of the second IVB), and month 2 (day of the third IVB). Among them, 10 eyes presented with type 1, 9 with type 2, and 10 with type 3 NV. A group of 14 aqueous samples from 14 patients who underwent cataract surgery without other ocular or systemic disease comprised the controls. Main outcome measures were concentration of VEGF at baseline and after IVB in the 3 NV groups; secondary outcome measures included best-corrected visual acuity (BCVA) and central macular thickness (CMT) changes after IVB. Levels of VEGF were determined by commercially available enzyme-linked immunosorbent assay kits. RESULTS: VEGF concentrations in aqueous humor at baseline were higher in patients with type 3 NV when compared to controls (P = .0001) and type 1 and 2 NV patients (P = .002 and P = .0001 respectively). At month 1, levels of VEGF were significantly reduced compared to baseline (P < .05) and significantly lower compared to the controls (P < .005) in each NV group. These low levels were maintained at the 2-month interval. BCVA significantly improved in type 1 and 2 NV groups (P < .05). CMT significantly reduced in each NV group compared to baseline (P < .05). CONCLUSION: In eyes with untreated wet AMD, aqueous levels of VEGF are significantly higher in type 3 NV than in type 1 or 2 NV. Regardless of the type of NV, aqueous VEGF levels significantly reduce 1 month after IVB as compared to both the baseline measurements and the values recorded in age-matched controls. These decreases are maintained at 2 months after administering a second IVB 30 days after the initial injection.

Highlighting chromosome loops in DNA-picked chromatin (DPC).

Growing evidence supports the concept that dynamic intra- and inter-chromosomal links between specific loci contribute to the creation of cell-type specific gene expression profiles. Therefore, analysis of the establishment of peculiar functional correlations between sites, also distant on linear DNA, that govern the transcriptional process appears to be of fundamental relevance. We propose here an experimental approach showing that 17ß-estradiol-induced transcription associates to formation of loops between the promoter and termination regions of hormone-responsive genes. This strategy reveals as a tool to be also suitably used, in conjunction with automated techniques, for an extensive analysis of sites shared by multiple genes for induced expression.

Viral infection for GPCR expression in eukaryotic cells.

This chapter describes the protocol for the preparation of recombinant adenoviruses and infection of target cells to transiently express G protein-coupled receptors (GPCRs) or other proteins of interest. Adenoviruses are non-enveloped viruses containing a linear double-stranded DNA genome. Their life cycle does not normally involve integration into the host genome, rather they replicate as episomal -elements in the nucleus of the host cell, and consequently there is no risk of insertional mutagenesis. Up to 30 kb out of the 35 kb of the wild-type adenovirus genome can be replaced by foreign DNA. Adenoviral vectors are very efficient in transducing target cells in vitro and in vivo and can be produced at high titers (>10¹¹/mL). The viral infection has a number of useful features: (1) the efficiency of gene transduction is very high (up to 100% in sensitive cells); (2) the infection is easy and does not physically alter the cell membrane for gene transduction; (3) it is possible to infect cells that are resistant to transfection with plasmids (including nondividing cells); and (4) the viral vectors can be used for infection in vivo (including gene therapy) and can potentially be targeted cell-specifically.