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Oehler C.,MSKCC | Oehler C.,University of Zurich | O'Donoghue J.A.,MSKCC | Russell J.,MSKCC | And 5 more authors.
Journal of Nuclear Medicine | Year: 2011

The aim of this study was to evaluate 18F-fluromisonidazole (18F-FMISO) PET for monitoring the tumor response to the antivascular compound 5,6-dimethylxanthenone-4-acetic acid (DMXAA; vadimezan). Methods: 18F-FMISO PET was performed 3 h before and 24 h after treatment with DMXAA (20 mg/kg) in mice bearing HT29 xenograft tumors. Pimonidazole was coadministered with the first 18F-FMISO injection, and 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5) was coadministered with the second one. Hoechst 33342 was administered 5 min before sacrifice. Digital autoradiograms of tumor sections were acquired; this acquisition was followed by immunofluorescence microscopic visualization of pimonidazole, EF5, the Hoechst 33342, CD31, and α-smooth muscle actin. Results: DMXAA treatment resulted in a marked reduction in the 18F-FMISO mean standardized uptake value (SUVmean) in approximately half of the treated tumors. The reduction in SUVmean correlated with a decrease in the fraction of tumor area staining positive for both EF5 and pimonidazole. Compared with untreated controls, tumors with decreasing SUVmean had significantly fewer perfused microvessels. Conclusion: 18F-FMISO PET could distinguish between different tumor responses to DMXAA treatment. However, a reduction in 18F-FMISO SUVmean after DMXAA treatment was indicative of reduced perfusion and therefore delivery of 18FFMISO, rather than a reduction in tumor hypoxia. Copyright © 2011 by the Society of Nuclear Medicine, Inc.

No statistical methods were used to determine sample size. Foxp3CNS3-fl-gfp mice were generated using ES cell line CY2.4 (C56BL/6) as previously described11. Cd4Cre, LckCre, UbcCre-ERT2 and Rosa26-stop-YFP (R26Y) mice were obtained from the Jackson Laboratories. DO11.10 TCRβ transgenic and Aire-knockout mice were provided by P. Marrack, and D. Mathis and C. Benoist, respectively. Heterozygous females carrying Foxp3ΔCNS3-gfp and Foxp3gfp were crossed with B6 males to generate hemizygous Foxp3ΔCNS3-gfp and wild-type Foxp3gfp littermates. Foxp3DTR, Foxp3-null, Rag1−/−, CD45.1+ Foxp3gfp and Tcrb−/− Tcrd−/− mice were maintained in our animal facility. To study the genetic interactions between CNS3 and Aire, heterozygous females of Foxp3ΔCNS3-gfp/gfp were first crossed with AireKO/WT, and F harbouring AireKO/WT and Foxp3ΔCNS3-gfp or Foxp3gfp were then intercrossed to generate AireKO/KO or AireKO/WT mice carrying Foxp3ΔCNS3-gfp or Foxp3gfp. To examine TCR diversity with restricted repertoire, Foxp3ΔCNS3-gfp/gfp heterozygous females were crossed to the DO11.10 TCRβ transgenic and Tcra−/+ males. F males of Foxp3ΔCNS3-gfp or Foxp3gfp mice carrying the DO11.10 TCRβ transgene and Tcra−/+ were used for T-cell isolation and TCR sequencing. To induce deletion of CNS3 in vivo, tamoxifen solution (40 mg ml−1 in olive oil) was administered by gavage to UbcCre-ERT2 Foxp3CNS3-fl-gfp R26Y mice more than 3 days before lymphocyte isolation. All mice were maintained in the MSKCC animal facility under SPF conditions, and the experiments were approved by the Institutional Review Board (IACUC 08-10-023). The experiments were not randomized and the investigators were not blinded to allocation during experiments and outcome assessment. Statistical tests were performed with Prism (GraphPad), Excel (Microsoft) or R statistical environment. Box-and-whisker plots show minimum, maximum, first and third quartiles and median. For in vitro T cell differentiation, naive CD4+ T cells (GFP−CD25−CD44loCD62Lhi) or mature CD4+CD8− SP (TCRβhiGFP−CD25−CD62LhiCD69lo) T cells were sorted from Foxp3gfp, Foxp3ΔCNS3-gfp or Foxp3CNS3-fl-gfp mice after the enrichment of CD4+ T cells or depletion of CD8+ T cells using Dynabeads FlowComp Mouse CD4 or CD8 kits, respectively (Life Technologies), and then cultured with lethally irradiated (20 Gy) antigen-presenting cells (splenocytes depleted of T cells with Dynabeads FlowComp Mouse CD90.2 kit, Life Technologies) or on plates pre-coated with CD3 and CD28 antibodies in RPMI1640 supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES, 2 × 10−5 M 2-mercaptoethanol, 100 U ml−1 penicillin, 100 mg ml−1 streptomycin, 500 U ml−1 IL-2 and 1 ng ml−1 TGFβ. Sodium butyrate (water solution) or iBET solution in dimethylsulfoxide (a gift from R. Prinjha) was added to the culture to block histone deacetylase or bromodomain-containing proteins, respectively. T cells were sorted on the basis of Foxp3gfp reporter expression. Assessment of the stability of Foxp3 expression in vitro was performed as previously described31. Briefly, T cells were activated in culture in the presence of CD3 and CD28 antibody-coated beads (Life Technologies) with the following recombinant pro-inflammatory cytokines: IL-2 (250 U ml−1), IL-4 (20 ng ml−1), IL-6 (10 ng ml−1), IFNγ (100 ng ml−1) and IL-12 (20 ng ml−1). To assess T -cell suppressor capacity in vivo we conducted adoptive T-cell transfers into T-cell-deficient recipients as previously described31. Briefly, ~2.5 × 106–3.0 × 106 Foxp3−CD4+ and/or CD8+ T cells isolated from Foxp3-null or Foxp3ΔCNS3-gfp AireKO/KO mice were transferred to congenic and gender-matched Tcrb−/− Tcrd−/− recipients alone or at a 10:1 ratio with sorted T cells from Foxp3gfp or Foxp3ΔCNS3-gfp littermates. Similar numbers of effector T cells and T cells were used for in vivo evaluation of T suppressor function after acute ablation of CNS3. Recipient mice were monitored for body weight change regularly and lymphocytes were analysed by flow cytometry at least 4 weeks after the transfer. Around 8–10 weeks after bone marrow reconstitution of CD45.1+ Foxp3gfp and CD45.2+ Foxp3ΔCNS3-gfp in Tcrb−/− Tcrd−/− recipients, CD45.1+ and CD45.2+ T cells (CD4+GFP+) were sorted, mixed at a 1:1 ratio and co-transferred into Tcrb−/− Tcrd−/− male mice with tenfold naive CD4+ T cells (CD25−CD44loCD62Lhi) isolated from wild-type CD45.2+ B6 males. To block TCR stimulation by pMHC-II complexes, 0.5 mg of I-Ab-specific monoclonal antibody Y3P (IgG2a) or control IgG2a (Bio X Cell) was injected intravenously every other day before and after T-cell transfer32, 33. The lymphocyte subsets were analysed by flow cytometry 9 days later. Tissue lymphocytes were prepared as previously described31. The following fluorophore-conjugated antibodies were used for cell-surface staining: CD4 (RM4-5, eBioscience), CD8 (5H10, Life Technologies), CD25 (PC61.5, eBioscience), CD3e (145-2C11, eBioscience), CD44 (IM7, eBioscience), CD62L (MEL-14, eBioscience), CTLA4 (UC10-4B9, eBioscience), TCRβ (BioLegend), CD45.1 (A20, eBioscience) and CD45.2 (104, eBioscience). Antibodies used for intracellular staining were: Foxp3 (FJK-16 s, eBioscience), Ki-67 (B56, BD Biosciences), IL-17 (eBio17B7, eBioscience), IFNγ (XMG1.2, eBioscience) and IL-2 (JES6-5H4, eBioscience). To stain endogenous Nur77, cells were incubated with rabbit-anti-Nur77 antibody (Cell Signaling) after fixation and permeabilization with a Foxp3/transcription-factor-staining buffer set (eBioscience), followed by phycoerythrin-conjugated donkey anti-rabbit antibody (eBioscience). For the flow cytometric analysis of cytokine production, lymphocytes were first stimulated in vitro with 10 mg ml−1 of CD3 antibody in the presence of monensin (BD Biosciences) at 37 °C for 5 h, then stained with antibodies against indicated cell-surface markers followed by staining of cytokines with an intracellular staining kit (BD Biosciences). All flow cytometric analyses were performed using live-cell gate defined as negative by staining with the LIVE/DEAD Fixable Dead Cell Stain Kit (Life Technologies). Flow cytometric analysis was performed with FlowJo (Treestar). The Cre coding region was subcloned into MigR1-IRES-Thy1.1 vector (A. Levine, unpublished data) to generate MigR1-Cre-IRES-Thy1.1. Retroviral packaging with regular Phoenix-ECO cells and transduction of T cells were performed following standard protocols5. Analysis of autoantibody reactivity against a panel of 95 autoantigens was conducted using the autoantigen microarrays developed by University of Texas Southwestern Medical Center34. Briefly, serum samples pretreated with DNase-I and diluted at 1:50 were incubated with the auto-antigen arrays. After a second incubation with Cy3-conjugated anti-mouse IgG, the arrays were scanned with a Genepix 4200A scanner (Molecular Device). The fluorescent signals for individual autoantigens were extracted from the resulting images with Genepix Pro 6.0 (Molecular Devices), followed sequentially by subtraction of local background, average of duplicates, normalization with total IgG, and subtraction of a negative PBS control. Cell isolation and RNA extraction. Lymphocytes were collected from the peripheral lymphoid organs or thymi of 6–8-week-old male Foxp3gfp or Foxp3ΔCNS3-gfpTcra−/+ littermates bearing the DO11.10 TCRβ transgene, and were enriched for CD4+ T cells (Dynabeads FlowComp Mouse CD4 kit, Life Technologies) or depleted of CD8+ T cells (Dynabeads FlowComp Mouse CD8 kit, Life Technologies), respectively, and T cells (CD4+GFP+), mature Foxp3−CD4 SP thymocytes (CD4+CD8−GFP−CD25−CD62LhiCD69lo), peripheral naive (CD4+GFP−CD25−CD44loCD62Lhi) and effector (CD4+GFP−CD44hiCD62Llo) CD4+ T cells were isolated using a FACSAria II sorter (BD) gated on TCR-Vβ8hi. Extraction of total RNA from TRIzol-preserved cell lysates was performed according to the manufacturer’s instructions (Life Technologies). mRNA was purified from total RNA with Dynabeads mRNA DIRECT Kit (Life Technologies) and used for reverse transcription. cDNA synthesis. To maximize the priming efficiency of reverse transcription, a mixture of oligo(dT) and eight DNA oligonucleotides corresponding to the mouse TCRα constant region was used. The oligonucleotides used in this study were synthesized by Integrated DNA Technologies, Inc. To label the 5' end of TCRα mRNA, a DNA–RNA hybrid oligonucleotide with 12 random nucleotides serving as barcodes to tag individual mRNA molecules was synthesized as previously reported35. Hybrid oligonucleotide: AAGCAGTGGTATCAACGCAGAGUNNNNUNNNNUNNNNUCTTrGrGrGrGrG (r, ribonucleotide). cDNA was synthesized in SMARTScribe reverse-transcription buffer (Clontech) with 1.0 μM each of reverse transcription oligonucleotide, 0.5 mM of each dNTP, 5.0 mM of dithiothreitol (DTT), 2.0 U μl−1 recombinant RNase inhibitor (Takara), 1 μM hybrid oligonucleotide, 1 M betaine (Affymetrix), 6 mM MgCl and 5 U μl−1 SMARTScribe reverse transcriptase by incubating at 42 °C for 90 min, followed by 10 cycles of incubation at 50 °C for 2 min, 42 °C for 2 min, and then one step of incubation at 70 °C for 15 min. After removal of hybrid oligonucleotide with Uracil-DNA Glycosylase (New England BioLabs), cDNA was purified with Agencourt AMPure XP beads (Beckman Coulter) according the manufacturer’s manual. Sequencing library preparation. Purified cDNA was used as templates for a four-step PCR amplification, in which sequencing adaptors and sample indices were introduced. The first PCR reaction was performed with purified cDNA, 0.2 μM universal primer (5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3', Clontech), 0.2 μM TRAC reverse primer 8 (5'-TTTTGTCAGTGATGAACGTT-3'), 0.2 mM each dNTP, 1.5 mM MgCl and 0.02 U μl−1 KOD Hot Start DNA Polymerase (EMD Millipore). PCR parameters were as follows: initial denature at 95 °C for 2 min; 10 cycles of 95 °C for 20 s, 70 °C for 10 s with an increment of −1 °C per cycle, and 70 °C for 30 s; 15 cycles of 95 °C for 20 s, 60 °C for 10 s and 70 °C for 30 s; and final cycle at 70 °C for 3.5 min. Amplified DNA was purified with Agencourt AMPure XP magnetic beads for the subsequent reaction. The second PCR reaction used the same reactants except that the reverse primer was replaced by a nested primer (5'-CAATTGCACCCTTACCACGACAGTCTGGTACACAGCAGGTTCTGGGTTCTGGA-3'). Cycling parameters were: 95 °C for 2 min; 6 cycles of 95 °C for 20 s, 60 °C 10 s and 70 °C 30 s; and a final cycle at 70 °C for 3.5 min. DNA from individual samples was extracted with Agencourt AMPure XP magnetic beads and used for the third round of amplification with 5RACE TCR forward primer (5'-AATGATACGGCGACCACCGAGATCTACACCTAATACGACTCACTATAGGGC-3') and indexed reverse primer (5'-CAAGCAGAAGACGGCATACGAGATXXXXXXAGTCAGTCAGCCCAATTGCACCCTTACCACGA-3', XXXXXX for 6-nucleotide barcode). The cycling parameters were: 95 °C for 2 min; 6 cycles at 95 °C for 20 s, 55 °C for 10 s and 70 °C for 30 s; and a final cycle at 70 °C for 3.5 min. The PCR products were purified with Agencourt AMPure XP magnetic beads and used for the fourth PCR amplification with primers P1 (5'-AATGATACGGCGACCACCGAG-3') and P2 (5'-CAAGCAGAAGACGGCATACGA-3'), and the following cycling parameters: 95 °C for 2 min; 5 cycles at 95 °C for 20 s, 57 °C for 10 s and 70 °C for 30 s; and a final cycle at 70 °C for 3.5 min. The final PCR products were separated by agarose gel electrophoresis and a single band around 600 base-pairs was cut and extracted with Gel Extraction and PCR Clean-Up kits (Takara). High-throughput sequencing. Samples were quantified with Kapa Library Quantification kits (Kapa Biosystems) and sequenced on a MiSeq sequencer (Illumina) using 200 cycles of read 1, 6 cycles of index read and 200 cycles of read 2 with the following customized primers: read 1: 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3'; index read: 5'-TCGTGGTAAGGGTGCAATTGGGCTGACTGACT-3'; read 2: 5'-AGTCAGTCAGCCCAATTGCACCCTTACCACGA-3'. Data analysis. Barcoded sequencing data were analysed with MIGEC software22. Briefly, unique molecular identifier sequences were extracted from raw sequencing data (read 1) with MIGEC/Checkout routine. Reads (≥5) bearing the same unique molecular identifier were grouped and assembled to generate consensus sequences with MIGEC/Assemble. Variable (V) and joining (J) segment mapping, CDR3 extraction, and error correction were performed with MIGEC/CdrBlast as previously described22, which eliminates PCR and sequencing errors, as well as normalizes the output data as cDNA counts that represent the TCR clonotypes in a population36. Comparison of TCRα repertoires between CNS3-deficient and -sufficient mice at protein level was evaluated using VDJtools post-analysis framework (https://github.com/mikessh/vdjtools)23. Pearson correlation of clonotype frequencies for the shared TCR clones was used for the generation of the dendrogram. Clonal diversities of TCRα repertoires were evaluated using inverse Simpson index computed separately for individual samples after downsampling the repertoires to the size of the smallest sample from the same organ. Similar downsampling strategy, not weighted by clonotype frequencies, was used to compute the average size of added nucleotides in CDR3. A mathematical model24 was used to assess the strength of CDR3 amino acid interactions with pMHC complexes. Numbers of strongly interacting amino acid residues (LFIMVWCY) were calculated for the V-segment part of TCRα CDR3 and V–J segment junction. Those numbers were then weighted by the corresponding clonotype frequencies and the resulting sums were used for the comparisons between samples. Mature Foxp3−CD4+CD8− SP (TCRβ+GFP−CD62LhiCD69lo) thymocytes, Foxp3+ CD4 SP thymocytes (thymic T cells), peripheral resting (CD44loCD62Lhi) and activated (CD44hiCD62Llo) T cells were FACS-sorted from ~6–8-week-old male Foxp3gfp and Foxp3ΔCNS3-gfp littermates. RNA was extracted and cDNA libraries were generated after SMART amplification (Clontech). Libraries were sequenced using a HiSeq 2000 platform (Illumina) according to a standard paired-end protocol. Reads were first processed with Trimmomatic37 to remove TruSeq adaptor sequences and bases with quality scores below 20, and reads with less than 30 remaining bases were discarded. Trimmed reads were then aligned to mm10 mouse genome with the STAR spliced-read aligner38. For each gene from the RefSeq annotations, the number of uniquely mapped reads overlapping with the exons was counted with HTSeq (http://www-huber.embl.de/users/anders/HTSeq/). Genes with fewer than 50 read counts were considered as not expressed and filtered out. Principal component analysis (PCA) was performed (n = 11,962) for clustering gene expression. Differential gene expression was estimated using DESeq package39. To determine activation-related transcriptional signatures in T cells, the differences between read counts of peripheral activated versus resting T cells from wild-type Foxp3gfp mice were evaluated by fold-change and Benjamini–Hochberg corrected P values (false discovery rate < 0.001) (Supplementary Data 1 and 2). For gene expression comparisons, previously published transcriptional signatures of TCR-dependent genes in T cells were used7. The distribution of gene expression changes is shown for transcriptional signature genes and the rest of all expressed genes. One-tailed Kolmogorov–Smirnov test is used to determine the significance between the distributions of signature genes and the rest of expressed genes. We cross-linked 1 × 106 cells with 1% formaldehyde for 5 min at room temperature. Cross-linked cells were lysed and nuclei were resuspended in 250 μl nuclear lysis buffer containing 1% SDS. Chromatin input samples were prepared by sonication of cross-linked nuclear lysates. For histone ChIPs, nuclear lysates were subjected to micrococcal nuclease (MNase) digestion before sonication. Nuclei were resuspended in 100 μl MNase (New England Biolabs) at 12,000 U ml−1 for 1 min at 37 °C. The reaction was stopped by addition of 10 μl of 0.5 M EDTA. Chromatin input samples were incubated overnight at 4 °C with antibodies against H3K4me1 (Abcam), H3K4me3 (Millipore) or H3K27ac (Abcam), and precipitated for 90 min at 4 °C using protein A Dynabeads (Life Technologies). After thorough washing, bead-bound chromatin was subjected to proteinase K digestion and decrosslinking overnight at 65 °C. DNA fragments were isolated using a Qiagen PCR purification kit. Relative abundance of precipitated DNA fragments was analysed by qPCR using Power SYBR Green PCR Master Mix (Applied Biosystems). The following primers were used for qPCR: Gm5069: forward: 5'-TAAGCAATTGGTGGTGCAGGATGC-3', reverse: 5'-AAAGGGTCATCATCTCCGTCCGTT-3'; Hspa2: forward: 5'-TCGTGGAGAGTTGTGAGAAGCGA-3', reverse: 5'-AACGTTAGGACGAAAGCGTCAGGA-3'; Hsp90ab: forward: 5'-TTACCTTGACGGGAAAGCCG AGTA-3', reverse: 5'-TTCGGGAGCTCTCTTGAGTCACC-3'; Rpl30: forward: 5'-TCGGCTTCACTCACCGTCTTCTTT-3', reverse: 5'-TG TCCTCTGTGTATGCTAGGTTGG-3'; Foxp3 promoter: forward: 5'-TAATGTGGCAGTTTCCCACAAGCC-3', reverse: 5'-AATACCTC TCTGCCACTT TCGCCA-3'; CNS1: forward: 5'-AGACTGTCTGGAACAACCTAGCCT-3', reverse: 5'-TGGAGGTACAGAGAGGTTAAGAGCCT-3'; CNS2: forward: 5'-ATCTGGCCAAGTTCAGGTTGTGAC-3', reverse: 5'-GGGCGTTCCTGTTTGACTGTTTCT-3'; CNS3: forward: 5'-TCTCCAGGCTTCAGAGATTCAAGG-3', reverse: 5'-ACAGTGGGATGAGGATACATGGCT-3'. Relative enrichment was calculated by normalizing to background binding to the control region (Gm5069). Quantification of serum Ig isotypes was performed by ELISA as previously described40. Tissue sections from gender matched Rag1−/− mice were used to detect mouse autoantibodies. Briefly, organs from the Rag1−/− mice were dissected, fixed with neutral buffered formalin, embedded with paraffin and sectioned. After deparaffinization with EZPrep buffer (Ventana Medical Systems) and antigen retrieval with cell conditioning solution (Ventana Medical Systems) the sections were blocked for 30 min with Background Buster solution (Innovex), followed by avidin/biotin blocking for 8 min, mouse serum (1:50 dilution) incubation for 5 h and biotinylated horse anti-mouse IgG (Vector Labs) incubation for 1 h. The detection was performed with streptavidin–horseradish peroxidase (Ventana Medical Systems) followed by incubation with Tyramide Alexa Fluor 488 (Invitrogen). The slides were then counterstained with DAPI (Sigma Aldrich) for 10 min, mounted, scanned with a Mirax scanner and visualized with Pannoramic Viewer (3DHISTECH). Scanned images were scored and representative snapshots were processed with Photoshop (Adobe) to switch the green and red channels for presentation purpose. Mixed bone marrow chimaeras were generated as previously described31. Briefly, recipient mice were irradiated (9.5 Gy) 24 h before intravenous injection of 10 × 106 bone marrow cells from CD45.1+ Foxp3gfp and CD45.2+ Foxp3ΔCNS3-gfp mixed at a 1:1 ratio. After bone marrow transfer, the recipient mice were administrated with 2 mg ml−1 neomycin in drinking water for 3 weeks and analysed 8–10 weeks later. Tissue samples were fixed in 10% neutral buffered formalin and processed for haematoxylin and eosin staining. Stained slides were scored for tissue inflammation as previously described41. Experimental autoimmune encephalomyelitis was induced by immunization with myelin oligodendrocyte glycoprotein peptide 35-55 (MOG35-55, GenScript) in complete Freund’s adjuvant (CFA, Sigma) and mice were monitored for disease as previously described42.

Lefave C.V.,Sloan Kettering Cancer Center | Lefave C.V.,The New School | Squatrito M.,Brain Tumor Center | Vorlova S.,Sloan Kettering Cancer Center | And 8 more authors.
EMBO Journal | Year: 2011

In tumours, aberrant splicing generates variants that contribute to multiple aspects of tumour establishment, progression and maintenance. We show that in glioblastoma multiforme (GBM) specimens, death-domain adaptor protein Insuloma-Glucagonoma protein 20 (IG20) is consistently aberrantly spliced to generate an antagonist, anti-apoptotic isoform (MAP-kinase activating death domain protein, MADD), which effectively redirects TNF-Î ±/TRAIL-induced death signalling to promote survival and proliferation instead of triggering apoptosis. Splicing factor hnRNPH, which is upregulated in gliomas, controls this splicing event and similarly mediates switching to a ligand-independent, constitutively active Recepteur dĝ€2Origine Nantais (RON) tyrosine kinase receptor variant that promotes migration and invasion. The increased cell death and the reduced invasiveness caused by hnRNPH ablation can be rescued by the targeted downregulation of IG20/MADD exon 16-or RON exon 11-containing variants, respectively, using isoform-specific knockdown or splicing redirection approaches. Thus, hnRNPH activity appears to be involved in the pathogenesis and progression of malignant gliomas as the centre of a splicing oncogenic switch, which might reflect reactivation of stem cell patterns and mediates multiple key aspects of aggressive tumour behaviour, including evasion from apoptosis and invasiveness. © 2011 European Molecular Biology Organization | All Rights Reserved.

Mishra R.,Loboratory of Tumor Biology Angiogenesis and Nanomedicine Research | Kumar D.,Loboratory of Tumor Biology Angiogenesis and Nanomedicine Research | Tomar D.,Loboratory of Tumor Biology Angiogenesis and Nanomedicine Research | Chakraborty G.,MSKCC | And 2 more authors.
Expert Opinion on Therapeutic Targets | Year: 2015

Introduction: Semaphorins have been originally identified as a family of evolutionary conserved soluble or membrane-associated proteins involved in diverse developmental phenomena. This family of proteins profoundly influences numerous pathophysiological processes, including organogenesis, cardiovascular development and immune response. Apart from steering the neural networking process, these are implicated in a broad range of biological operations including regulation of tumor progression and angiogenesis.Areas covered: Members of class 3 semaphorin family are known to modulate various cellular processes involved in malignant transformation. Some of the family members trigger diverse signaling processes involved in tumor progression and angiogenesis by binding with plexin and neuropilin. A better understanding of the various signaling mechanisms by which semaphorins modulate tumor progression and angiogenesis may serve as crucial tool in crafting new semaphorin-based anticancer therapy. These include treatment with recombinant tumor suppressive semaphorins or inhibition of tumor-promoting semaphorins by their specific siRNAs, small-molecule inhibitors or specific receptors using neutralizing antibodies or blocking peptides that might serve as novel strategies for effective management of cancers.Expert opinion: This review focuses on all the possible avenues to explore various members of class 3 semaphorin family to serve as therapeutics for combating cancer. © 2014 Informa UK, Ltd.

Varghese A.,Christian Medical College | Suneha S.,Christian Medical College | Shaha A.,MSKCC
American Journal of Otolaryngology - Head and Neck Medicine and Surgery | Year: 2015

Tuberculosis (TB) of the thyroid gland, either in its primary or secondary form, is an extremely rare occurrence. It is infrequent even in countries with high incidence and prevalence of pulmonary and extrapulmonary TB. We report here a case of primary tuberculosis of thyroid presenting to us with sudden onset thyroid swelling since 20 days. © 2015 Elsevier Inc. All rights reserved.

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