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Item The application of the Johns Hopkins Hospital Template on urine cytology(Wiley, 2015-08) Wu, Howard H.; Redelman, Megan; Chen, Shaoxiong; Grignon, David J.; Cramer, Harvey M.; Department of Pathology and Laboratory Medicine, IU School of MedicineBackground To evaluate the utility of the Johns Hopkins Hospital (JHH) template in detection of high-grade urothelial carcinoma (HGUC). Methods A computerized search of our laboratory information system was performed for all urine cytology cases from 2009 to 2011 processed by the SurePath™. We included only cases with correlating surgical pathology within 6 months after the urinary samples were obtained. The original cytologic diagnoses were reclassified according to the JHH template, and these cytolog ic diagnoses were then correlated with the follow-up surgical pathology diagnoses. Results A total of 273 urine samples with histopathologic follow-up were identified. The reclassified cytologic diagnoses included negative for urothelial atypia or malignancy (NUAM) 110; atypical urothelial cells of undetermined significance (AUC-US) 83; atypical urothelial cells, cannot exclude high-grade urothelial carcinoma (AUC-H) 49; HGUC 29; and low-grade urothelial carcinoma (LGUC) 2. More than one-half of patients (58%) who had biopsy-confirmed high-grade urothelial lesions had a preceding cytologic diagnosis of AUC-H or HGUC. AUC-H and HGUC are associated with high-grade urothelial lesions in 80% and 90% of the cases and show statistical significance when compared with AUC-US or NUAM (P < 0.05). Conclusion The JHH template is useful and effective in identifying patients with high-grade urothelial lesions who need to undergo cystoscopy. Diagn. Cytopathol. 2015;43:593–597. © 2015 Wiley Periodicals, Inc.Item Cardiac engraftment of genetically-selected parthenogenetic stem cell-derived cardiomyocytes(Public Library of Science, 2015) Yang, Tao; Rubart, Michael; Soonpaa, Mark H.; Didié, Michael; Christalla, Peter; Zimmermann, Wolfram-Hubertus; Field, Loren J.; Department of Pediatrics, IU School of MedicineParthenogenetic stem cells (PSCs) are a promising candidate donor for cell therapy applications. Similar to embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), PSCs exhibit self-renewing capacity and clonogenic proliferation in vitro. PSCs exhibit largely haploidentical genotype, and as such may constitute an attractive population for allogenic applications. In this study, PSCs isolated from transgenic mice carrying a cardiomyocyte-restricted reporter transgene to permit tracking of donor cells were genetically modified to carry a cardiomyocyte-restricted aminoglycoside phosphotransferase expression cassette (MHC-neor/pGK-hygror) to permit the generation of highly enriched cardiomyocyte cultures from spontaneously differentiating PSCs by simple selection with the neomycin analogue G148. Following engraftment into isogenic recipient hearts, the selected cardiomyocytes formed a functional syncytium with the host myocardium as evidenced by the presence of entrained intracellular calcium transients. These cells thus constitute a potential source of therapeutic donor cells.Item CD166 regulates human and murine hematopoietic stem cells and the hematopoietic niche(American Society of Hematology, 2014-07-24) Chitteti, Brahmananda Reddy; Kobayashi, Michihiro; Cheng, Yinghua; Zhang, Huajia; Poteat, Bradley A.; Broxmeyer, Hal E.; Pelus, Louis M.; Hanenberg, Helmut; Zollman, Amy; Kamocka, Malgorzata M.; Carlesso, Nadia; Cardoso, Angelo A.; Kacena, Melissa A.; Srour, Edward F.; Department of Medicine, IU School of MedicineWe previously showed that immature CD166(+) osteoblasts (OB) promote hematopoietic stem cell (HSC) function. Here, we demonstrate that CD166 is a functional HSC marker that identifies both murine and human long-term repopulating cells. Both murine LSKCD48(-)CD166(+)CD150(+) and LSKCD48(-)CD166(+)CD150(+)CD9(+) cells, as well as human Lin(-)CD34(+)CD38(-)CD49f(+)CD166(+) cells sustained significantly higher levels of chimerism in primary and secondary recipients than CD166(-) cells. CD166(-/-) knockout (KO) LSK cells engrafted poorly in wild-type (WT) recipients and KO bone marrow cells failed to radioprotect lethally irradiated WT recipients. CD166(-/-) hosts supported short-term, but not long-term WT HSC engraftment, confirming that loss of CD166 is detrimental to the competence of the hematopoietic niche. CD166(-/-) mice were significantly more sensitive to hematopoietic stress. Marrow-homed transplanted WT hematopoietic cells lodged closer to the recipient endosteum than CD166(-/-) cells, suggesting that HSC-OB homophilic CD166 interactions are critical for HSC engraftment. STAT3 has 3 binding sites on the CD166 promoter and STAT3 inhibition reduced CD166 expression, suggesting that both CD166 and STAT3 may be functionally coupled and involved in HSC competence. These studies illustrate the significance of CD166 in the identification and engraftment of HSC and in HSC-niche interactions, and suggest that CD166 expression can be modulated to enhance HSC function.Item Deep Tissue Fluorescent Imaging in Scattering Specimens Using Confocal Microscopy(Cambridge University Press, 2011-08) Clendenon, Sherry G.; Young, Pamela A.; Ferkowicz, Michael; Phillips, Carrie; Dunn, Kenneth W.; Department of Pediatrics, IU School of MedicineIn scattering specimens, multiphoton excitation and nondescanned detection improve imaging depth by a factor of 2 or more over confocal microscopy; however, imaging depth is still limited by scattering. We applied the concept of clearing to deep tissue imaging of highly scattering specimens. Clearing is a remarkably effective approach to improving image quality at depth using either confocal or multiphoton microscopy. Tissue clearing appears to eliminate the need for multiphoton excitation for deep tissue imaging.Item Defective TGFβ signaling in bone marrow-derived cells prevents Hedgehog-induced skin tumors(American Association for Cancer Research, 2014-01-15) Fan, Qipeng; Gu, Dongsheng; Liu, Hailan; Yang, Ling; Zhang, Xiaoli; Yoder, Mervin C.; Kaplan, Mark H.; Xie, Jingwu; Department of Pediatrics, IU School of MedicineHedgehog (Hh) signaling in cancer cells drives changes in the tumor microenvironment that are incompletely understood. Here we report that Hh- driven tumors exhibit an increase in myeloid-derived suppressor cells (MDSC) and a decrease in T cells, indicative of an immune suppressive tumor microenvironment. This change was associated with activated TGFβ signaling in several cell types in BCCs. We determined that TGFβ signaling in bone marrow (BM)-derived cells, not keratinocytes, regulates MDSC and promotes tumor development. Tgfbr2 deficiency in the BM-derived cells also reduced the size of previously developed tumors in mice. We identified CCL2 as the major chemokine attracting MDSC to tumor, whose expression was Tgfbr2-dependent, whereas its receptor CCR2 was highly expressed in MDSC population. CCL2 alone was sufficient to induce migration of MDSC. Moreover, the CCR2 inhibitors prevented MDSC migration towards skin cells in vitro, reduced MDSC accumulation and Hh signaling-driven tumor development in mice. Our results reveal a signaling network critical for Hh signaling in cancer cells to establish an effective immune suppressive microenvironment during tumor development.Item Deficiency of Src family kinases compromises the repopulating ability of hematopoietic stem cells(Elsevier, 2008-05) Orschell, Christie M.; Borneo, Jovencio; Munugalavadla, Veerendra; Ma, Peilin; Sims, Emily; Ramdas, Baskar; Yoder, Mervin C.; Kapur, Reuben; Department of Medicine, IU School of MedicineOBJECTIVE: Src family kinases (SFK) have been implicated in regulating growth factor and integrin-induced proliferation, migration, and gene expression in multiple cell types. However, little is known about the role of these kinases in the growth, homing, and engraftment potential of hematopoietic stem and progenitor cells. RESULTS: Here we show that loss of hematopoietic-specific SFKs Hck, Fgr, and Lyn results in increased number of Sca-1(+)Lin(-) cells in the bone marrow, which respond differentially to cytokine-induced growth in vitro and manifest a significant defect in the long-term repopulating potential in vivo. Interestingly, a significant increase in expression of adhesion molecules, known to coincide with the homing potential of wild-type bone marrow cells is also observed on the surface of SFK(-/-) cells, although, this increase did not affect the homing potential of more primitive Lin(-)Sca-1(+) SFK(-/-) cells. The stem cell-repopulating defect observed in mice transplanted with SFK(-/-) bone marrow cells is due to the loss of Lyn Src kinase, because deficiency of Lyn, but not Hck or Fgr, recapitulated the long-term stem cell defect observed in mice transplanted with SFK(-/-) bone marrow cells. CONCLUSIONS: Taken together, our results demonstrate an essential role for Lyn kinase in positively regulating the long-term and multilineage engraftment of stem cells, which is distinct from its role in mature B cells and myeloid cells.Item Detection of BRAF Mutations on Direct Smears of Thyroid Fine Needle Aspirates through Cell Transfer Technique(Oxford, 2015-04) Shi, Qiuying; Ibrahim, Ashley; Herbert, Kristi; Carvin, Marcia; Randolph, Melissa; Post, Kristin M.; Curless, Kendra; Chen, Shaoxiong; Cramer, Harvey M.; Cheng, Liang; Wu, Howard H.; Department of Pathology and Laboratory Medicine, IU School of MedicineObjectives: To determine the utility of the cell transfer technique (CTT) for BRAF molecular testing on thyroid fine-needle aspiration (FNA) specimens. Methods: Polymerase chain reaction (PCR)–based BRAF molecular testing was performed on tissues obtained through CTT from both air-dried and ethanol-fixed direct smears of thyroid FNA specimens and then compared with the corresponding thyroidectomy formalin-fixed, paraffin-embedded (FFPE) tissues on 30 cases. Results: BRAF testing was successfully performed on 29 of 30 air-dried CTT, 27 of 30 ethanol-fixed CTT, and 27 of 30 FFPE tissues. The results exhibited 11, 13, and 13 BRAF mutations and 18, 14, and 14 wild types for the air-dried CTT, the ethanol-fixed CTT, and the FFPE tissues, respectively. The concordance rate was 96% between air-dried and ethanol-fixed CTT tissues, 88% between air-dried CTT and FFPE tissues, and 92% between ethanol-fixed CTT and FFPE tissues. Conclusions: PCR-based BRAF mutational testing can be reliably performed on the direct smears of the thyroid FNA specimens through the application of CTT.Item Differentiation of human pluripotent stem cells to cells similar to cord-blood endothelial colony-forming cells(Nature Publishing Group, 2014-11) Prasain, Nutan; Lee, Man Ryul; Vemula, Sasidhar; Meador, Jonathan Luke; Yoshimoto, Momoko; Ferkowicz, Michael J.; Fett, Alexa; Gupta, Manav; Rapp, Brian M.; Saadatzadeh, Mohammad Reza; Ginsberg, Michael; Elemento, Olivier; Lee, Younghee; Voytik-Harbin, Sherry L.; Chung, Hyung Min; Hong, Ki Sung; Reid, Emma; O'Neill, Christina L.; Medina, Reinhold J.; Stitt, Alan W.; Murphy, Michael P.; Rafii, Shahin; Broxmeyer, Hal E.; Yoder, Mervin C.; Department of Pediatrics, IU School of MedicineThe ability to differentiate human pluripotent stem cells into endothelial cells with properties of cord-blood endothelial colony-forming cells (CB-ECFCs) may enable the derivation of clinically relevant numbers of highly proliferative blood vessel-forming cells to restore endothelial function in patients with vascular disease. We describe a protocol to convert human induced pluripotent stem cells (hiPSCs) or embryonic stem cells (hESCs) into cells similar to CB-ECFCs at an efficiency of >10(8) ECFCs produced from each starting pluripotent stem cell. The CB-ECFC-like cells display a stable endothelial phenotype with high clonal proliferative potential and the capacity to form human vessels in mice and to repair the ischemic mouse retina and limb, and they lack teratoma formation potential. We identify Neuropilin-1 (NRP-1)-mediated activation of KDR signaling through VEGF165 as a critical mechanism for the emergence and maintenance of CB-ECFC-like cells.Item Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD(Landes Bioscience, 2014) Mitter, Sayak K.; Song, Chunjuan; Qi, Xiaoping; Mao, Haoyu; Rao, Haripriya; Akin, Debra; Lewin, Alfred; Grant, Maria; Dunn, William; Ding, Jindong; Bowes Rickman, Catherine; Boulton, Michael; Department of Ophthalmology, IU School of MedicineAutophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.Item Early dynamic fate changes in haemogenic endothelium characterized at the single-cell level(Nature Publishing Group, 2013) Swiers, Gemma; Baumann, Claudia; O'Rourke, John; Giannoulatou, Eleni; Taylor, Stephen; Joshi, Anagha; Moignard, Victoria; Pina, Cristina; Bee, Thomas; Kokkaliaris, Konstantinos D.; Yoshimoto, Momoko; Yoder, Mervin C.; Frampton, Jon; Schroeder, Timm; Enver, Tariq; Göttgens, Berthold; de Bruijn, Marella F. T. R.; Department of Pediatrics, IU School of MedicineHaematopoietic stem cells (HSCs) are the founding cells of the adult haematopoietic system, born during ontogeny from a specialized subset of endothelium, the haemogenic endothelium (HE) via an endothelial-to-haematopoietic transition (EHT). Although recently imaged in real time, the underlying mechanism of EHT is still poorly understood. We have generated a Runx1 + 23 enhancer-reporter transgenic mouse (23GFP) for the prospective isolation of HE throughout embryonic development. Here we perform functional analysis of over 1,800 and transcriptional analysis of 268 single 23GFP+ HE cells to explore the onset of EHT at the single-cell level. We show that initiation of the haematopoietic programme occurs in cells still embedded in the endothelial layer, and is accompanied by a previously unrecognized early loss of endothelial potential before HSCs emerge. Our data therefore provide important insights on the timeline of early haematopoietic commitment.