CONGESTIVE HEART FAILURE, Issue 6 2008
Bernadette K. McLaren MD
Acute cardiac allograft rejection (ACAR) has been associated with a poor prognosis. The early diagnosis of ACAR necessitates the accurate detection of myocyte damage. Nuclear damage activates p53, a transcription factor that initiates apoptosis and repair. Endomyocardial biopsies (n=25) from 10 cardiac allograft recipients were stained for nuclear p53. The biopsies were divided into rejection groups based on the grading of ACAR: group 1, grade 0; group 2, grade Ia and Ib; group 3, grades II and III. While clinical indices did not correlate with myocyte damage, significantly more myocytes in group 3 stained for nuclear p53 (2.48±0.60/mm2) compared with group 1 (0.22±0.12/mm2) and group 2 (0.43±0.18/mm2). Increased expression of p53 in cardiac myocytes with grade II or grade III rejection provides an objective quantification as an aid in the diagnosis of ACAR. [source]

Irritant threshold and histological response of epidermis to irritant application

CONTACT DERMATITIS, Issue 5-6 2004
H. R. Smith
Individuals vary in their ability to react to irritants, which can be demonstrated for sodium lauryl sulfate (SLS) using the irritant threshold (IT) test. We aimed to study whether the histological and immunohistochemical features of the skin following SLS exposure varied with subject's IT. 8 subjects were recruited. Their IT was measured. Biopsies were taken after 2 hr and 4 hr of occlusion with 20% SLS and control. The specimens were stained with haematoxylin and eosin and for Langerhans cells. At 4-hr, low-threshold subjects developed changes to a greater extent than high-threshold subjects. The relationship of histological reaction to IT could be related to a differential pro-inflammatory cytokine response in subjects. Low IT has been previously associated with a tumour necrosis factor alpha promoter region polymorphism. [source]

When monocytes and platelets compete: The effect of platelet count on the flow cytometric measurement of monocyte CD36,

CYTOMETRY, Issue 2 2010
W.H. Dzik
Abstract Background: Flow cytometric measurement of monocyte surface CD36 is relevant to several conditions including diabetes, cardiovascular disease, lipid disorders, platelet isoimmunization, and susceptibility to P falciparum malaria. CD36 is also strongly expressed on platelets where it is also known as platelet glycoprotein IV. Methods: Whole blood samples, containing identical monocyte concentrations, were adjusted to contain platelets ranging from 20,000/uL to 600,000/uL, were stained with fluorescent-labeled anti-CD36, and analyzed by flow cytometry. Results: CD36 median fluorescent intensity (MFI) observed on monocytes decreased as the platelet concentration in the sample increased with more than a 50% decline in monocyte MFI over the normal range of platelet values. The effect was not abolished by using larger volumes of monoclonal antibody and was observed with different clones of reagent anti-CD36. The findings were most consistent with competition by platelets for the CD36 reagent. Similar findings were observed with antibody to class I HLA. Under defined assay conditions, monocyte CD36 MFI declined with rising platelet concentration in a predictable fashion following an inverse linear relationship. Conclusions: Measurement of CD36 expression on monocytes by flow cytometry in whole blood samples is affected by the sample platelet count. When comparing the monocyte CD36 expression among different individuals, our approach can be used to adjust measured monocyte CD36 expression for the effect of the platelet concentration in the sample. Competition by platelets for monoclonal reagents may occur in other settings when whole blood assays are used and when the target antigen is strongly expressed on both platelets and leukocytes. © 2009 Clinical Cytometry Society [source]

Discrepancy in measuring CD4 expression on T-lymphocytes using fluorescein conjugates in comparison with unimolar CD4-phycoerythrin conjugates,,

CYTOMETRY, Issue 6 2007
Lili Wang
Abstract Background: Numerous methods for quantitative fluorescence calibration (QFC) have been developed to quantify receptor expression on lymphocytes. However, the results from the use of these different QFC methods vary considerably in the literature. To better identify the causes of these discrepancies, we measured CD4 expression using FITC and phycoerythrin (PE) conjugates to stain CYTO-TROLÔ Control Cells and T-lymphocytes in whole blood and isolated cell preparations. We further examined pH of the cellular microenvironment as a cause of discordant results obtained with the FITC conjugate. Methods: Calibration with Quantibrite PE-labeled microspheres and the use of unimolar CD4-PE conjugates provided direct measurement of the antibody bound per cell value (ABC) for CD4 expression on normal T-lymphocytes. Calibration for CD4-FITC monoclonal antibody (Mab) labeled CYTO-TROL Control Cells and normal T-lymphocytes was based on molecules of equivalent soluble fluorochrome (MESF) as determined by FITC-labeled microspheres traceable to NIST RM 8640. The MESF value for CD4-FITC Mab was determined that enabled the conversion of the MESF values obtained for CYTO-TROL cells to ABC. We investigated the likely pH change in the fluorescein microenvironments within FITC-labeled Mab and cells stained with FITC-labeled Mab using a pH sensitive indicator. Results: The mean ABC value for T-lymphocytes prepared from fresh whole blood using CD4-PE conjugate (48,321) was consistent with previous results, and it was much higher than the mean ABC using CD4-FITC Mab (22,156). The mean ABC value for CYTO-TROL cells using CD4-PE conjugate (43,090) was also higher than that using CD4-FITC conjugate (34,734), although the discrepancy was not as great. Further studies suggested the discrepancy in CYTO-TROL results may be accounted for by the low pH of the membrane microenvironment, but the greater discrepancy in T-lymphocytes could not be fully explained. Conclusion: CD4 expression on fresh normal whole blood samples and CYTO-TROL cells can be consistently quantified in ABC units using Quantibrite PE quantification beads and unimolar CD4-PE conjugates. Quantification with CD4-FITC conjugate is not as consistent, but may be improved by the use of CD4 T-cells as biological calibrators. This approximation is valid only for surface receptors with consensus ABC values measured by different QFC methods serving as biological standards. Published 2007 Wiley-Liss, Inc. [source]

Diagnosing PNH with FLAER and multiparameter flow cytometry

CYTOMETRY, Issue 3 2007
D. Robert Sutherland
Abstract Background: PNH is an acquired hematopoietic stem cell disorder leading to a partial or absolute deficiency of all glycophosphatidyl-inositol (GPI)-linked proteins. The classical approach to diagnosis of PNH by cytometry involves the loss of at least two GPI-linked antigens on RBCs and neutrophils. While flow assays are more sensitive and specific than complement-mediated lysis or the Hams test, they suffer from several drawbacks. Bacterial aerolysin binds to the GPI moiety of cell surface GPI-linked molecules and causes lysis of normal but not GPI-deficient PNH cells. FLAER is an Alexa488-labeled inactive variant of aerolysin that does not cause lysis of cells. Our goals were to develop a FLAER-based assay to diagnose and monitor patients with PNH and to improve detection of minor populations of PNH clones in other hematologic disorders. Methods: In a single tube assay, we combined FLAER with CD45, CD33, and CD14 allowing the simultaneous analysis of FLAER and the GPI-linked CD14 structure on neutrophil and monocyte lineages. Results: Comparison to standard CD55 and CD59 analysis showed excellent agreement. Because of the higher signal to noise ratio, the method shows increased sensitivity in our hands over single (CD55 or CD59) parameter analysis. Using this assay, we were able to detect as few as 1% PNH monocytes and neutrophils in aplastic anemia, that were otherwise undetectable using CD55 and CD59 on RBC's. We also observed abnormal FLAER staining of blast populations in acute leukemia. In these cases, the neutrophils stained normally with FLAER, while the gated CD33bright cells failed to express normal levels of CD14 and additionally showed aberrant CD45 staining and bound lower levels of FLAER. Conclusion: FLAER combined with multiparameter flow cytometry offers an improved assay for diagnosis and monitoring of PNH clones and may have utility in detection of unsuspected myeloproliferative disorders. © 2007 Clinical Cytometry Society [source]

Flow cytometric measurement of circulating endothelial cells: The effect of age and peripheral arterial disease on baseline levels of mature and progenitor populations

CYTOMETRY, Issue 2 2006
Rebecca Gusic Shaffer
Abstract Background: Age and cardiovascular disease status appear to alter numbers and function of circulating endothelial progenitor cells (EPCs). Despite no universal phenotypic definition, numerous studies have implicated progenitors with apparent endothelial potential in local responses to vascular injury and with cardiovascular disease in general. To further define the role of this lineage in peripheral artery disease (PAD), we developed a multiparameter flow cytometry assay to analyze multiple phenotypic definitions of progenitor cells (PCs), EPCs, and mature endothelial cells (ECs) and evaluate effects of age and PAD on baseline levels of each subset. Methods: Blood was collected from young healthy subjects (N = 9, mean age 33 ± 8 years), older healthy subjects (N = 13, mean age 66 ± 8 years), and older subjects with PAD (N = 15, mean age 69 ± 8 years). After ammonium chloride lysis, cells were stained and analyzed on a Becton-Dickinson LSR II with a 5-color antibody panel: FITC-anti-CD31, PE-anti-CD146, PE-anti-CD133, PerCP-Cy5.5-anti-CD3,-CD19,-CD33 (lineage panel), PE-Cy7-anti-CD34, and APC-anti-VEGF-R2. Viability was assessed by propidium iodide exclusion, and only viable, low to medium side scatter lineage-negative singlets were analyzed. In some studies, cells were sorted for morphological studies. Subsets were defined as indicated later. Results: Our results, using a comprehensive flow cytometric panel, indicate that CD133+, CD34+, and CD133+/CD34+ PCs are elevated in younger healthy individuals compared to older individuals, both healthy and with PAD. However, the number of EPCs and mature ECs did not significantly differ among the three groups. Assessment of endothelial colony forming units and dual acLDL-lectin staining supported the flow cytometric findings. Conclusions: We describe a comprehensive flow cytometric method to detect circulating mature and progenitor endothelial populations confirmed by conventional morphological and functional assays. Our findings suggest that aging may influence circulating levels of PCs, but not EPCs or ECs; PAD had no effect on baseline levels of any populations investigated. This study provides the basis for evaluating the potential effects of acute stress and therapeutic intervention on circulating progenitor and endothelial populations as a biomarker for cardiovascular status. © 2005 International Society for Analytical Cytology [source]

Near-infrared dyes for six-color immunophenotyping by laser scanning cytometry

CYTOMETRY, Issue 3 2002
Andreas O.H. Gerstner
Abstract Background To adequately analyze the complexity of the immune system and reduce the required sample volume for immunophenotyping in general, more measurable colors for the discrimination of leukocyte subsets are necessary. Immunophenotyping by the laser scanning cytometer (LSC), a slide-based cytometric technology, combines cell detection based on multiple colors with their subsequent visualization without the need for physical cell sorting. In the present study, the filter setting of the LSC was adapted for the measurement of the far-red emitting dye cyanine 7 (Cy7), thereby increasing the number of measurable commercially available fluorochromes. Methods The optical filters of the LSC were replaced,photomultiplier (PMT) 3/allophycocyanin (APC): 740-nm dichroic long pass, and 670-/55-nm bandpass; PMT 4/Cy7: 810-/90-nm bandpass. Peripheral blood leukocytes were stained directly by fluorochrome-labeled antibodies or by indirect staining. The tandem dyes of Cy7 (phycoerythrin [PE]-Cy7, APC-Cy7) and the fluorochromes fluorescein isothiocyanate (FITC), PE, PE-Cy5, and APC were tested alone and in different combinations. Results With the new filter combination and tandem fluorochromes, Cy7 was measurable at 488-nm (argon laser) or 633-nm (helium-neon laser) excitation. Resolution was in the range of FITC for PE-Cy7 but approximately 30% lower for APC-Cy7; spillover into the respective donor fluorochrome channel for both tandem dyes was prominent. A six-color panel for leukocyte subtyping was designed. Conclusions With this adaptation, it is possible to measure the tandem conjugates PE-Cy7 and APC-Cy7. This new setup opens the way for six-color immunophenotyping by LSC. Cytometry 48:115,123, 2002. © 2002 Wiley-Liss, Inc. [source]

Cytological diagnosis of basal cell carcinoma and actinic keratosis, using Papanicolaou and May,Grünwald,Giemsa stained cutaneous tissue smear

CYTOPATHOLOGY, Issue 5 2008
E. Christensen
Objective:, Cytology may become the diagnostic method of choice with the advent of new non-invasive treatments for non-melanoma skin cancer, as the sampling technique for cytology entails little tissue disfiguration. The aim of this study was to compare and evaluate the diagnostic performance of scrape cytology using two different cytological staining techniques, and to evaluate additional touch imprint cytology, with that of histopathology of basal cell carcinoma (BCC) and actinic keratosis (AK). Methods:, We investigated 50 BCC and 28 AK histologically verified lesions, from 41 and 25 patients, respectively. Two separate skin scrape samples and one touch imprint sample were taken from each lesion. The smears were stained with Papanicolaou (Pap) or May,Grünwald,Giemsa (MGG) stains. All cytological specimens were examined in random order by pathologists without knowledge of the histology. Cytodiagnostic results were compared with the histopathological report. Results:, Scrape cytodiagnosis agreed with histopathology in 48 (Pap) and 47 (MGG) of the 50 BCC cases, and in 26 of 28 (Pap) and 21 of 26 (MGG) AK cases, yielding sensitivities of 96%, 94%, 93% and 81%, respectively. No significant difference in sensitivity between the two staining methods was found but a trend towards higher Pap sensitivity for AK was noted (P = 0.10). Touch imprint cytology confirmed histopathology in 38 of the 77 cases of BCC and AK. Conclusion:, Cytological diagnosis with either Pap or MGG stain for BCC and AK is reliable, and differentiates well between BCC and AK. Imprint cytology proved to be non-diagnostic in half of the examined cases. [source]

Evaluation of accuracy of fine needle aspiration cytology for diagnosis of canine mammary tumours: comparative features with human tumours

CYTOPATHOLOGY, Issue 3 2007
G. D. Cassali
Objective:, The authors evaluated the accuracy of the fine needle aspiration cytology technique in the diagnosis of 77 canine mammary gland tumours using the same cytological and histological criteria currently applied to the diagnosis of human breast cancer. Methods:, The study was performed in 73 pure or mixed-breed female dogs submitted to surgical resections of ,mammary tumours'. All cytological smears were stained by routine May-Grunwald,Giemsa and Papanicolaou stains. Results:, We obtained a correct cyto-histological correlation in 52/77 cases (67.5%) when all cytopathological examinations were considered, and in 52/56 cases (92.9%) when the inconclusive cases were excluded from the analysis. Conclusion:, Our results demonstrate that, because of the similarity of the cytological findings in the human and canine mammary gland tumours, it is possible to use the same cytological criteria applied in human pathology for the diagnosis of canine mammary gland tumours. [source]

Intraoperative cytology of clear cell carcinoma of the ovary

CYTOPATHOLOGY, Issue 6 2006
D. Vrdoljak-Mozeti
Objective:, To describe the cytomorphology of clear cell carcinoma (CCC) of the ovary in intraoperative samples of peritoneal fluid, imprint and scraping samples of the tumour tissue. Study design:, Fourteen histologically confirmed cases, stained by standard cytological procedures, were analysed by light microscopy. Results:, In 33.3% of peritoneal fluid samples and 92.9% of imprint and scraping cytological samples, besides variable clear cell cellular morphology, one or both distinct cytological characteristics were observed: eosinophilic, hyaline, extracellular, globular substance with or without formation of a ,raspberry' body and an eosinophilic, intracytoplasmic inclusions. These structures were clearly seen only in samples stained by May-Grünwald,Giemsa. Conclusion:, Using cytological analysis of imprint and scraping samples of ovarian tumours it is possible to make a precise intraoperative cytological diagnosis in most cases of CCC of the ovary. [source]

Rho plays a central role in regulating local cell-matrix mechanical interactions in 3D culture

CYTOSKELETON, Issue 6 2007
N. Lakshman
Abstract The purpose of this study was to assess quantitatively the role of the small GTPase Rho on cell morphology, f-actin organization, and cell-induced matrix remodeling in 3D culture. Human corneal fibroblasts (HTK) were infected with adenoviruses that express green fluorescent protein (GFP) or GFP-N19Rho (dominant negative Rho). One day later cells were plated inside collagen matrices and allowed to spread for 24 h. Cells were fixed and stained for f-actin. Fluorescent (for f-actin) and reflected light (for collagen fibrils) images were acquired using confocal microscopy. Fourier transform analysis was used to assess local collagen fibril alignment, and changes in cell morphology and collagen density were measured using MetaMorph. The decrease in matrix height was used as an indicator of global matrix contraction. HTK and HTK-GFP cells induced significant global matrix contraction; this was inhibited by N19Rho. HTK and HTK-GFP fibroblasts generally had a bipolar morphology and occasional intracellular stress fibers. Collagen fibrils were compacted and aligned parallel to stress fibers and pseudopodia. In contrast, HTK-GFPN19 cells were elongated, and had a more cortical f-actin distribution. Numerous small extensions were also observed along the cell body. In addition, both local collagen fibril density and alignment were significantly reduced. Rho plays a key role in regulating both the morphology and mechanical behavior of corneal fibroblasts in 3D culture. Overall, the data suggest that Rho-kinase dependent cell contractility contributes to global and local matrix remodeling, whereas Rho dependent activation of mDia and/or other downstream effectors regulates the structure and number of cell processes. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]

Expression of nebulette during early cardiac development

CYTOSKELETON, Issue 4 2007
Michael Esham
Abstract Nebulette, a cardiac homologue of nebulin, colocalizes with ,-actinin in the pre-myofibrils of spreading cardiomyocytes and has been hypothesized to play a critical role in the formation of the thin-filament-Z-line complex early during myofibrillogenesis. Data from mesodermal explants or whole tissue mounts of developing hearts suggest that the pattern of myofibrillogenesis in situ may differ from observations of spreading cardiomyocytes. To evaluate the role of nebulette in myofibrillogenesis, we have analyzed the expression of nebulette in chicken heart rudiments by immunoblots and immunofluorescence. We detect the 110 kDa nebulette in heart rudiments derived from stage 9,10 using the anti-nebulin mAb, N114, or polyclonal anti-nebulette Abs by immunoblotting. Immunofluorescence analysis of explants stained with anti-nebulette and anti-,-actinin Abs demonstrates that both proteins localize along actin filaments in punctate to continuous manner at early stages of cardiac development and later give rise to striations. In both cases, the punctate staining had a periodicity of ,1.0 ,m indicating a pre-myofibrils distribution at the earliest time points examined. We demonstrate that nebulette is indeed associated with premyofibrils in very early stages of myofibrillogenesis and suggest that nebulette may play an important role in the formation of these structures. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source]

Chicken gizzard filamin, retina filamin and cgABP260 are respectively, smooth muscle-, non-muscle- and pan-muscle-type isoforms: Distribution and localization in muscles

CYTOSKELETON, Issue 4 2005
Kazuyo Ohashi
Abstract We determined the full cDNA sequences of chicken gizzard filamin and cgABP260 (chicken gizzard actin-binding protein 260). The primary and secondary structures predicted by these sequences were similar to those of chicken retina filamin and human filamins. Like mammals, chickens have 3 filamin isoforms. Comparison of their amino acid sequences indicated that gizzard filamin, retina filamin, and cgABP260 were the counterparts of human FLNa (filamin a), b, and c, respectively. Antibodies against the actin-binding domain (ABD) of these 3 filamin isoforms were raised in rabbits. Using immunoabsorption and affinity chromatography, we prepared the monospecific antibody against the ABD of each filamin. In immunoblotting, the antibody against the gizzard filamin ABD detected a single band in gizzard, but not in striated muscles or brain. In brain, only the antibody against the retina filamin ABD produced a strong single band. The antibody against the cgABP260 ABD detected a single peptide band in smooth, skeletal, and cardiac muscle. In immunofluorescence microscopy of muscular tissues using these antibodies, the antibody against the gizzard filamin ABD only stained smooth muscle cells, and the antibody against the retina filamin ABD strongly stained endothelial cells of blood vessels and weakly stained cells in connective tissue. The antibody against the cgABP260 ABD stained the Z-lines and myotendinous junctions of breast muscle, the Z-lines and intercalated disks of cardiac muscle, and dense plaques of smooth muscle. These findings indicate that chicken gizzard filamin, retina filamin, and cgABP260 are, respectively, smooth muscle-type, non-muscle-type, and pan-muscle-type filamin isoforms. Cell Motil. Cytoskeleton 61:214,225, 2005. © 2005 Wiley-Liss, Inc. [source]

Biocompatibility evaluation of alendronate paste in rat's subcutaneous tissue

DENTAL TRAUMATOLOGY, Issue 2 2009
Graziela Garrido Mori
Therefore, this study aimed to investigate the biocompatibility of experimental alendronate paste in subcutaneous tissue of rats, for utilization in teeth susceptible to root resorption. The study was conducted on 15 male rats, weighing ,180,200 grams. The rats' dorsal regions were submitted to one incision on the median region and, laterally to the incision, the subcutaneous tissue was raised and gently dissected for introduction of two tubes, in each rat. The tubes were sealed at one end with gutta-percha and taken as control. The tubes were filled with experimental alendronate paste. The animals were killed at 7, 15 and 45 days after surgery and the specimens were processed in laboratory. The histological sections were stained with hematoxylin-eosin and analyzed by light microscopy. Scores were assigned to the inflammatory process and statistically compared by the Tukey test (P < 0.05). Alendronate paste promoted severe inflammation process at 7 days, with statistically significant difference compared to the control (P < 0.05%). However, at 15 days, there was a regression of inflammation and the presence of connective tissue with collagen fibers, fibroblasts and blood vessels was observed. After 45 days, it was observed the presence of well-organized connective tissue, with collagen fibers and fibroblasts, and few inflammatory cells. No statistical difference was observed between the control and experimental paste at 15 and 45 days. The experimental alendronate paste was considered biocompatible with subcutaneous tissue of rat. [source]

Enhancement of Viability of Fat Grafts in Nude Mice by Endothelial Progenitor Cells

DERMATOLOGIC SURGERY, Issue 12 2006
CHENGGANG YI MD
BACKGROUND A recent discovery showed that endothelial progenitor cells (EPCs) could augment collateral vessel growth to ischemic tissues. OBJECTIVE The objective was to demonstrate the effects of EPCs on the vasculogenesis and survival of free transplanted fat tissues in nude mice. METHODS EPCs from human donors were cultured in vitro for 7 days. Human fat tissues were injected subcutaneously into the scalps of 20 6-week-old nude male mice. EPCs stained with CM-DiI were mixed with the transplanted fat tissues and injected into the mice. EBM-2 medium was used as control group. The animals were euthanized 15 weeks after the procedure. Graft volume were measured, and histologic evaluation was performed. The central part of fat tissues was histologically evaluated 15 weeks after the fat injection. RESULTS The survival volume of the experimental group was significantly greater than that of the control group (p< .05). Less cyst formation and fibrosis was obtained in the experimental group. Histologic evaluation of the central part of fat tissues 15 weeks after the fat injection showed that capillary densities increased markedly in the experimental group mice. CONCLUSION The results indicate that EPCs have the ability to enhance the survival and the quality of the transplanted fat tissues. [source]

The Effect of Full-Face Broadband Light Treatments Alone and in Combination With Bilateral Crow's Feet Botulinum Toxin Type A Chemodenervation

DERMATOLOGIC SURGERY, Issue 3 2004
Jean Carruthers MD
Background. Broadband light (BBL; Intense Pulsed Light; Lumenis Ltd., Yokneam, Israel) is a powerful, nonablative, light-based technology that targets melanin and hemoglobin and stimulates the formation of collagen and elastin. Botulinum toxin type A (BTX-A; BOTOX; Allergan Inc., Irvine, CA) treatment of the lateral periocular region relaxes the vertical fibers of the orbicularis oculi and results in softening of the lateral orbital crow's feet rhytides and widening of the palpebral aperture. Objective. To compare the effects of full-face BBL in combination with BTX-A and BBL alone in female subjects with Fitzpatrick I,III skin types, Glogau II,III rhytides, and significant associated facial lentigines and telangiectasia. Methods. This was a prospective, randomized study of 30 women with moderate to severe crow's feet rhytides. Half of the subjects were treated with BTX-A and BBL and the other half with BBL alone. Their response was assessed clinically and photographically. Skin biopsies of the temporal skin were taken from two subjects in each group and were stained with Masson trichrome. Results. Patients treated with a combination of BTX-A and BBL experienced a better response to treatment, both at rest and on maximum smile, as well as a slightly improved response in associated lentigines, telangiectasia, pore size, and facial skin texture compared with patients who received BBL treatment alone. Skin biopsies showed an increase in dermal collagen in each group. Conclusions. The patients in this study benefited from both treatments. Although BBL led to a remarkable improvement in full-face telangiectasias, lentigines, and skin texture, the improvement increased in all categories with combination therapy. In addition, an added improvement in the full-face aesthetic with both BTX-A and BBL therapy combined was obvious. These results suggest that both treatments,although evidently complementary,may also act synergistically to produce optimal clinical effects, revolutionizing the treatment of facial aging. [source]

Ectopic germline cells in embryos of Xenopus laevis

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 7 2007
Kohji Ikenishi
Whether all descendants of germline founder cells inheriting the germ plasm can migrate correctly to the genital ridges and differentiate into primordial germ cells (PGCs) at tadpole stage has not been elucidated in Xenopus. We investigated precisely the location of descendant cells, presumptive primordial germ cells (pPGCs) and PGCs, in embryos at stages 23,48 by whole-mount in situ hybridization with the antisense probe for Xpat RNA specific to pPGCs and whole-mount immunostaining with the 2L-13 antibody specific to Xenopus Vasa protein in PGCs. Small numbers of pPGCs and PGCs, which were positively stained with the probe and the antibody, respectively, were observed in ectopic locations in a significant number of embryos at those stages. A few of the ectopic PGCs in tadpoles at stages 44,47 were positive in TdT-mediated dUTP digoxigenin nick end labeling (TUNEL) staining. By contrast, pPGCs in the embryos until stage 40, irrespective of their location and PGCs in the genital ridges of the tadpoles at stages 43,48 were negative in TUNEL staining. Therefore, it is evident that a portion of the descendants of germline founder cells cannot migrate correctly to the genital ridges, and that a few ectopic PGCs are eliminated by apoptosis or necrosis at tadpole stages. [source]

Immunocytochemical study of activin type IB receptor (XALK4) in Xenopus oocytes

DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 2 2003
Akimasa Fukui
Studies have shown that the activin type IB receptor is specific for activin/nodal signaling. Activin is produced by follicle cells in the ovary, and is incorporated into the oocytes. Antisera against three peptides were prepared, encompassing the extracellular, intracellular and serine/threonine kinase domains of the Xenopus type IB activin receptor (XALK4). Immunocytochemistry was done using these antisera to investigate the distribution of XALK4 in the Xenopus ovary. All three antisera stained the mitochondrial cloud of Xenopus previtellogenic oocytes. Purified antibody against the intracellular domain also recognized the mitochondrial cloud. Immunoelectron microscopy localized XALK4 on the endoplasmic reticulum of the mitochondrial cloud, although not on mitochondria. [source]

Early expression of endomucin on endothelium of the mouse embryo and on putative hematopoietic clusters in the dorsal aorta

DEVELOPMENTAL DYNAMICS, Issue 3 2001
Gertrud Brachtendorf
Abstract Endomucin is a recently identified sialomucin that is specifically expressed on endothelium of the adult mouse. Here, we have analysed the expression of endomucin during development of the vascular system by immunohistochemistry by using three monoclonal antibodies (mAb). We demonstrate that two of the mAb, V.5C7 and V.1A7, recognize epitopes on the nonglycosylated protein, because they recognize the antigen when it is synthesized as a bacterial fusion protein and when it is in vitro translated in a membrane-free reticulocyte lysate. During in vitro differentiation of embryonic stem cells to endothelial cells, endomucin is expressed at day 6 after onset of differentiation, 1 day later than PECAM-1. During differentiation of the mouse embryo, endomucin is first detected at E8.0 in all embryonic blood vessels detectable at this stage but is absent in blood islands of the yolk sac. Analysing the paraaortic-splanchnopleura (P-SP) region and the aorta-gonad-mesonephros (AGM) region as sites of intraembryonic hematopoiesis, we found that endothelium of the dorsal aorta is brightly positive for endomucin at E8.5,9.0 and at E11.5. At later stages and in the adult aorta, endothelial staining is strongly reduced and confined to focal areas. Cell clusters associated with the luminal surface of the endothelium of the dorsal aorta could be stained for endomucin and for CD34. At a later stage (E15.5) single leukocytes in the lumen of large venules were stained for endomucin. We conclude that endomucin is an early endothelial-specific antigen that is also expressed on putative hematopoietic progenitor cells. © 2001 Wiley-Liss, Inc. [source]

Distribution of neurotrophin-3 during the ontogeny and regeneration of the lizard (Gallotia galloti) visual system

DEVELOPMENTAL NEUROBIOLOGY, Issue 1 2008
E. Santos
Abstract We have previously described the spontaneous regeneration of retinal ganglion cell axons after optic nerve (ON) transection in the adult Gallotia galloti. As neurotrophin-3 (NT-3) is involved in neuronal differentiation, survival and synaptic plasticity, we performed a comparative immunohistochemical study of NT-3 during the ontogeny and regeneration (after 0.5, 1, 3, 6, 9, and 12 months postlesion) of the lizard visual system to reveal its distribution and changes during these events. For characterization of NT-3+ cells, we performed double labelings using the neuronal markers HuC-D, Pax6 and parvalbumin (Parv), the microglial marker tomato lectin or Lycopersicon esculentum agglutinin (LEA), and the astroglial markers vimentin (Vim) and glial fibrillary acidic protein (GFAP). Subpopulations of retinal and tectal neurons were NT-3+ from early embryonic stages to adulthood. Nerve fibers within the retinal nerve fiber layer, both plexiform layers and the retinorecipient layers in the optic tectum (OT) were also stained. In addition, NT-3+/GFAP+ and NT-3+/Vim+ astrocytes were detected in the ON, chiasm and optic tract in postnatal and adult lizards. At 1 month postlesion, abundant NT-3+/GFAP+ astrocytes and NT-3,/LEA+ microglia/macrophages were stained in the lesioned ON, whereas NT-3 became downregulated in the experimental retina and OT. Interestingly, at 9 and 12 months postlesion, the staining in the experimental retina resembled that in control animals, whereas bundles of putative regrown fibers showed a disorganized staining pattern in the OT. Altogether, we demonstrate that NT-3 is widely distributed in the lizard visual system and its changes after ON transection might be permissive for the successful axonal regrowth. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2008 [source]

Search for cerebral G cluster neurons responding to taste stimulation with seaweed in Aplysia kurodai by the use of calcium imaging

DEVELOPMENTAL NEUROBIOLOGY, Issue 3 2003
Ryusuke Yoshida
Abstract The calcium imaging method can detect the spike activities of many neurons simultaneously. In the present experiments, this method was used to search for unique neurons contributing to feeding behavior in the cerebral ganglia of Aplysia kurodai. We mainly explored the neurons whose cell bodies were located in the G cluster and the neuropile region posterior to this cluster on the ventral surface of the cerebral ganglia. When the extract of the food seaweed Ulva was applied to the tentacle-lip region, many neurons stained with a calcium-sensitive dye, Calcium Green-1, showed changes in fluorescence. Some neurons showed rhythmic responses and others showed transient responses, suggesting that these neurons may be partly involved in the feeding circuits. We also identified three motor neurons among these neurons that showed rhythmic fluorescence responses to the taste stimulation. One of them was a motor neuron shortening the anterior tentacle (ATS), and the other two were motor neurons producing lip opening-like (LOG) and closing-like (LCG) movements, respectively. Application of the Ulva extract to the tentacle-lip region induced phase-locked rhythmic firing activity in these motor neurons, suggesting that these neurons may contribute to the rhythmic patterned movements of the anterior tentacles and lips during the ingestion of seaweed. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 299,314, 2003 [source]

Fine needle aspiration of renal cortical lesions in adults

DIAGNOSTIC CYTOPATHOLOGY, Issue 10 2010
Adebowale J. Adeniran M.D.
Abstract The role of fine needle aspiration (FNA) biopsy of renal cortical lesions was controversial in the past because the result of the FNA did not affect clinical management. All renal cortical lesions, except metastasis, were subject to surgical resection. However, with the advances in neoadjuvant targeted therapies, knowledge of the renal cortical tumor histological subtype is critical for tailoring clinical trials and follow-up strategies. At present, there are clinical trials involving the use of novel kinase inhibitors for conventional (clear cell) and papillary renal cell carcinoma. We studied 143 consecutive cases of renal cortical lesions, evaluated after radical or partial nephrectomies over a 2-year period. An air-dried smear and a Thinprep® slide were prepared in all cases. The slides were Diff-Quick and Papanicolaou stained, respectively. The cytology specimens were reviewed and the results were then compared with the histologic diagnosis. Cytology was highly accurate to diagnose conventional RCC, while the accuracy for papillary RCC, chromophobe RCC, and papillary urothelial carcinoma was much lower. Our results indicate that ancillary studies might have an important role in the subclassification of renal cortical neoplasms for targeted treatment. Diagn. Cytopathol. 2010;38:710,715. © 2009 Wiley-Liss, Inc. [source]

Immunohistochemical expression of E-cadherin in sclerosing adenosis, ductal carcinoma in situ and invasive ductal carcinoma of the breast

DIAGNOSTIC CYTOPATHOLOGY, Issue 4 2010
Gil Facina M.D., Ph.D.
Abstract E-cadherin (EC) is an important glycoprotein cell-adhesion molecule that appears to play a significant role in the progression of breast lesions. The objective of this study was to evaluate EC expression in sclerosing adenosis, ductal carcinoma in situ and invasive ductal carcinoma. Samples of breast lesions from 44 women were used in this study, comprising cases of sclerosing adenosis (n = 11), ductal carcinoma in situ (DCIS) (n = 10) and invasive ductal carcinoma (n = 23). Immunohistochemical evaluation of EC expression was assessed semiquantitatively and considered negative (<10% of cells with stained cytoplasmic membranes), positive+ (10,50% of cells stained) or positive++ (> 50% of cells stained). Fisher's exact test was used to compare the distribution of staining intensity in the lesions (P< 0.05). There was a progressive loss of EC expression from benign to malignant lesions. This difference was statistically significant when sclerosing adenosis was compared with DCIS (P < 0.0002), when sclerosing adenosis was compared with invasive ductal carcinoma (P < 0.008) and when DCIS was compared with invasive ductal carcinoma (P < 0.007). The present findings point to a significant association between reduced EC expression and the progression and aggressivity of breast lesions. Diagn. Cytopathol. 2010. © 2009 Wiley-Liss, Inc. [source]

Synchronous high-grade squamous intraepithelial lesion and adenocarcinoma in situ of cervix in a young woman presenting with hyperchromatic crowded groups in the cervical cytology specimen: Report of a case

DIAGNOSTIC CYTOPATHOLOGY, Issue 11 2008
Nadeem Zafar M.D.
Abstract We report a 29-year-old woman who underwent routine gynecologic evaluation at a community clinic and had a cervical sample drawn for liquid-based cytologic evaluation. At cytology, many hyperchromatic crowded groups (HCG) were present, but a consensus could not be established whether the abnormal cells were primarily glandular or squamous with secondary endocervical glandular involvement. An interpretation of atypical endocervical cells, favor neoplastic, was rendered and biopsy advised if clinically appropriate. At biopsy, the cervix contained synchronous squamous cell carcinoma in situ, secondarily involving endocervical glands, and neighboring adenocarcinoma in situ. Immunohistochemistry for Ki-67 and p16INK4A crisply and precisely stained both the lesions, clearly separating them from the adjacent uninvolved mucosa. This case re-emphasizes the challenge associated with accurate evaluation of HCG at cytology, the significance of ancillary testing for surrogate markers of high-risk HPV (HR-HPV) infection, the need for adjunct testing for HPV-DNA in the setting of HCG at cervical cytology, and a recommendation to set up studies to evaluate the role of surrogate markers of HR-HPV infection in cytologic samples with HCG. Diagn. Cytopathol. 2008;36:823,826. © 2008 Wiley-Liss, Inc. [source]

Liesegang rings in fine needle aspirate of breast cysts with predominance of apocrine cells: A study of 14 cases

DIAGNOSTIC CYTOPATHOLOGY, Issue 10 2008
F.I.A.C., Raj K. Gupta M.D.
Abstract Fine needle aspirate (FNA) from 14 cases (age range 17,84 years), with Liesegang rings (LR's) in breast cysts seen over a period of 26 years comprised the material of this study from more than 38,000 FNA's of the breast which had been done for a variety of breast lesions. In six of the 14 cases, the aspirate was obtained under ultrasound guidance whereas in the remaining cases it was collected from a palpable lesion. The aspiration was performed using a 22 gauge needle and the syringe and needle contents were washed in a cytology container with 30% ethyl alcohol in physiologic saline. The cytologic preparations from half of the sample were made on a 5 micron Schleicher and Schuell filter and stained by Papanicolaou method whereas from the remainder of the sample a cell block was made and sections cut, stained with hematoxylin-eosin (H&E) and used for immunohistochemical study. Filter preparations and cell blocks revealed cyanophilic, spherical, ring-like structures of various sizes and shape mostly with double walls, and striations with amorphous material in the lumen and under polarized light were nonrefractile. Seen also were several apocrine cells and some macrophages and the LR's were found to be negative on immunostains for EMA and CK, and a panel of other special stains (Table I). Since LR's can be mistaken for ova, larvae, or parasites, it is important to be aware of their potential presence in aspirate samples of breast cysts to avoid a misdiagnosis. The exact mechanism of formation of LR's is not fully understood and certain views as proposed are discussed in this presentation. Diagn. Cytopathol. 2008;36:701,704. © 2008 Wiley-Liss, Inc. [source]

Are adjunctive markers useful in routine cervical cancer screening?

DIAGNOSTIC CYTOPATHOLOGY, Issue 7 2008
Application of p16INK4a, HPV-PCR on ThinPrep samples with histological follow-up
Abstract The objectives of the study were to evaluate 1) the diagnostic sensitivity and specificity of p16INK4a as a marker for high-grade cervical lesions, 2) the results of a real-time polymerase chain reaction detecting high-risk human papillomavirus, and 3) the interobserver variability of the p16INK4a interpretation. A total of 232 ThinPrep samples were stained for p16INK4a, and HPV-DNA PCR was performed on 107 specimens with inclusion of both benign and abnormal cytology. Histological follow-up information was collected. The diagnostic sensitivity of ASC+ with CIN2+ in histology as endpoint was 96% for p16INK4a and 100% for HR-HPV DNA PCR, and the diagnostic specificity was 41% and 27%, respectively. If p16INK4a had been used for triage of the ASC samples, then 18 patients (42%) could have been spared unnecessary follow-up procedures compared to six patients (21%) with the HR-HPV DNA test. Diagn. Cytopathol. 2008;36:453,459. © 2008 Wiley-Liss, Inc. [source]

Oncotic colpocytology stained with Harris,Shorr in the observation of vaginal microorganisms

DIAGNOSTIC CYTOPATHOLOGY, Issue 6 2008
Agenor Storti-Filho Especialist
Abstract The purpose of this work was to evaluate the efficacy of oncotic colpocytology stained with Harris,Shorr in the identification of the cervicovaginal microflora and infectious agents. Results of microbiologic evaluation carried out in colpocytology exams, bacterioscopy (Gram), and direct exams of 2,017 women aged from 13 to 80 years were compared. Of these, 83.1% agreed between cytology and Gram, 3.6% partially agreed, and 12.8% disagreed. The predominant microflora was of lactobacilli (63.3%), followed by mixed flora (32.1%). The results of sensitivity to lactobacilli were 96.1% and to mixed flora 88.0%; the specificity values were 91.2 and 92.0%, respectively. Colpocytology detected all the instances of trichomoniasis observed at direct exam (0.6%). The most frequent infectious agents were of candidiasis (14.8%, sensitivity 80.3%) and bacterial vaginosis (11.9%, sensitivity 68.1%). Thus, Harris,Shorr stained cytology was shown to be an excellent diagnostic method for T. vaginalis, lactobacilli, mixed flora, and candidiasis. Diagn. Cytopathol. 2008;36:358,362. © 2008 Wiley-Liss, Inc. [source]

Comparison of p16INK4A and Hybrid Capture® 2 human papillomavirus testing as adjunctive tests in liquid-based gynecologic SurePathÔ preparations

DIAGNOSTIC CYTOPATHOLOGY, Issue 3 2008
Aziza Nassar M.D., F.I.A.C.
Abstract p16INK4a, cyclin-dependent kinase inhibitor, is functionally inactivated in many tumors, including cervical cancer. We compared p16INK4A immunocytochemical staining and Hybrid Capture® 2 (HCII) on SurePathÔ specimens using tissue biopsies (as the gold standard). Their utility in a spectrum of atypical and preneoplastic lesions, and their ability to accurately identify underlying lesions of CIN II or greater was assessed using biopsy follow-up data. One-hundred and seventeen residual SurePathÔ samples were collected: 43 atypical squamous cells of undetermined significance (ASCUS), 47 low-grade (LGSIL), and 27 high-grade (HGSIL) squamous intraepithelial lesions. Two slides were prepared from each sample; one stained with the SurePathÔ autocyte stain and one immunostained using the CINtecÔ p16INK4a Cytology Kit (Dakocytomation). High-risk HPV testing was performed using the HCII DNA test (Digene, Gaithersburg, MD). Available tissue biopsy follow-up data was retrieved. p16INK4a was positive in 32.6% (14/43) ASCUS, 46.8% (22/47) LGSIL, and 48.1% (13/27) HGSIL specimens. HCII DNA test was positive in 41.9% (18/43) ASCUS, 78.7% (37/47) LGSIL, and 96.3% (26/27) HGSIL samples. The sensitivity, specificity, positive (PPV) and negative (NPV) predictive values of p16INK4a and HCII were: 58.7% and 89.8%, 58.6% and 34.6%, 69.2% and 72.1%, 47.2% and 64.3%, respectively. In patients with cervical biopsies, the PPV of HCII (92.3%) results for a biopsy with CINII/III was significantly higher than the PPV of p16INK4a (52%) (P = 0.001). Using liquid-based cytology specimens, HCII is a more sensitive test than p16INK4a for detection of abnormal cytology. HCII has a higher PPV than p16INK4a for identifying CIN II/III. Diagn. Cytopathol. 2008;36:142,148. © 2008 Wiley-Liss, Inc. [source]

Symptomatic candidiasis: Using self sampled vaginal smears to establish the presence of Candida, lactobacilli, and Gardnerella vaginalis

DIAGNOSTIC CYTOPATHOLOGY, Issue 10 2007
M. K. Engberts M.D.
Abstract In a prospective cohort study, 10 symptomatic women with recurrent vulvovaginal candidiasis were taught how to prepare vaginal smears of their own vaginal fluids on days 7, 14, 21, and 28. The 40 smears were stained with the PAS-method and examined by three different cytopathologists for presence of Candida. Thereafter, the smears were restained with Giemsa-stain to determine presence of lactobacilli, Gardnerella vaginalis ("clue cells") and neutrophils. All three cytopathologists unequivocally established Candida blastospores and (pseudo)hyphae in 27 out of the 40 PAS-stained vaginal smears, whereas in the remaining 13 smears Candida was not found. All 10 patients had Candida in their smears during the second half of their menstrual cycle. Self sampled smears prove to be reliable for establishing the presence of Candida in symptomatic patients with candidiasis. Candida is associated with a lactobacillus -predominated vaginal flora, but with the absence of Gardnerella vaginalis. Further studies may be directed towards the interaction between the various members of the vaginal flora. This study should open molecular methodology for determining the possible interactions of lactobacilli and Candida. Diagn. Cytopathol. 2007;35:635,639. © 2007 Wiley-Liss, Inc. [source]

Secretory activity in medullary thyroid carcinoma: A cytomorphological and immunocytochemical study

DIAGNOSTIC CYTOPATHOLOGY, Issue 6 2007
D.Sc., Dilip K. Das M.B.B.S., F.R.C.Path., Ph.D.
Abstract Medullary thyroid carcinoma (MTC) is a relatively rare thyroid malignancy of C-cell origin that secretes calcitonin. Although its varied cytomorphologic features are well described in literature, very little is mentioned about the morphologic manifestation of its secretory activity. This study, based on nine fine needle aspiration (FNA) samples from eight MTC patients, is an attempt to present the varied cytomorphologic features suggesting secretory activity in MTC as observed in Papanicolaou and MGG stained FNA smears and correlate them with the immunocytochemical (ICC) staining for calcitonin performed on FNA smears and the serum calcitonin values. The average number of cells in these nine samples was as follows: oval/triangular/plasmacytoid (56.7%), small round (23.6%), spindle-shaped (12.7%), and miscellaneous (7.1%). The cytomorphological features suggesting secretory activity, viz., fine cytoplasmic vacuoles, azurophillic granules, marginal vacuoles, and intracytoplasmic lumina (ICL) with secretions were present in eight, eight, five, and six samples, respectively. Material likely to be amyloid, based on morphological features, was present extracellularly in three samples and both intracellularly and extracellularly in six samples. Immunocytochemically, all the nine samples stained for calcitonin and all the three stained for chromogranin showed positive cytoplasmic reaction in the neoplstic cells. The background amyloid (in six samples), the coarse cytoplasmic granules (in two samples), and the contents of ICL (in one sample) were found to be positively stained for calcitonin. The intracytoplasmic secretory material appeared to be diffusing out of some cells both in the routine MGG stained smears and in the smears stained for calcitonin. Histopathology reports of seven samples in six patients confirmed the cytodiagnosis of MTC in all. Baseline serum calcitonin values in three cases and postoperative serum calcitonin levels during follow-up in three others were high. Thus, our study highlighted the morphological manifestations of secretory activity in MTC and the nature of secretory material as calcitonin, supported by immunocytochemical staining and serum calcitonin level. Diagn. Cytopathol. 2007;35:329,337. © 2007 Wiley-Liss, Inc. [source]