Nuclear Counterstain Research Articles

Crlz-1 Is Prominently Expressed in Spermatogonia and Sertoli Cells during Early Testis Development and in Spermatids during Late Spermatogenesis

The expression of the Crlz-1 gene in mouse testis, where it was found to be expressed most highly among the tested mouse organs, was analyzed spatiotemporally by employing RT-PCR and in situ hybridization techniques with the aid of immunohistochemistry and/or immunofluorescence methods. In 1-week-old neonatal testis, Crlz-1 was strongly expressed in the spermatogonia and Sertoli cells in its seminiferous cord. In 2- to 3-week-old prepubertal testis, where Sertoli cells cease to proliferate, Crlz-1 expression dropped and remained weakly at the rim layer of seminiferous cords and/or tubules, where spermatogonia are present. In the adult testis at 12 weeks after birth, Crlz-1 was expressed mainly in the spermatids near the lumen of seminiferous tubules. In a further in situ hybridization of Crlz-1 in the 12-week-old adult testis with hematoxylin nuclear counterstaining, Crlz-1 was mainly expressed at step 16 of spermatids between stages VII and VIII of seminiferous tubules as well as in their residual bodies at stage IX of seminiferous tubules.

Molecular and functional characterization of human bone marrow adipocytes

Adipocytes are a cell population largely located in the human bone marrow cavity. In this specific microenvironment where adipocytes can interact with a variety of different cells, the role of fat is mainly unknown. To our knowledge, this report is the first to characterize mature adipocytes isolated from human bone marrow (BM-A) molecularly and functionally to better understand their roles into the hematopoietic microenvironment. Healthy BM-A were isolated after collagenase digestion and filtration. We studied the morphology of BM-A, their gene expression and immunophenotypic profile and their functional ability in the hematopoietic microenvironment, comparing them with adipocytes derived from adipose tissue (AT-A). BM-A showed a unilocular lipid morphology similar to AT-A and did not lose their morphology in culture; they showed a comparable pattern of stem cell-surface antigens to AT-A. In line with these observations, molecular data showed that BM-A expressed some embryonic stem cells genes, such as Oct4, KLf4, c-myc, Gata4, Tbx1, and Sox17, whereas they did not express the stem cell markers Sox2 and Nanog. Moreover, BM-A had long telomeres that were similar to bone marrow mesenchymal stem cells. Notably, BM-A supported the survival and differentiation of hematopoietic stem cells in long-term cultures. These results showed that BM-A are stromal cells with a gene expression pattern that distinguished them from AT-A. BM-A showed stem cell properties through their hematopoietic supporting function, which was certainly linked to their role in the maintenance of the bone marrow microenvironment. Depending on specific demands, BM-A may acquire different functions based on their local environment.

Enhancing automated micrograph‐based evaluation of LPS‐stimulated macrophage spreading

To evaluate macrophage spreading in immunofluorescence images of macrophages for surface protein CD11b and nuclear counterstaining with DAPI, it is necessary to measure the size of the macrophages at different time points after stimulation. Manual evaluation of fluorescent micrographs is usually a time-consuming and error-prone task, with poor reproducibility. Automatic image analysis methods can be used to improve the results. The quality of the analysis with these methods mainly depends on the quality of the image segmentation. A segmentation and quantification scheme based on shading correction, k-means clustering, and fast marching level sets has been developed for the purpose. An initial application of this approach showed that separating touching and overlapping cells in particular suffers severely in the inevitably blurred conditions, leading to partly erroneous measurements of macrophage spreading. An alternative method of segmentation in fluorescent micrographs was therefore investigated and evaluated in this study. The proposed approach uses a methodology that separates foreground objects from background objects on the basis of Boykov's graph cuts. In this process, a rough estimation of background pixels is used for background seeds. To identify foreground seeds, a difference of Gaussian band pass filter based workflow is developed. Information on foreground and background seeds is then used for a gradient magnitude based graph cut resulting in a robust figure-ground separation method. In addition, a fast marching level set approach is used in the post-processing step, which makes it possible to split touching cells by incorporating information about the cell nuclei. An evaluation based on a total of 553 manually labeled macrophages depicted in 21 micrographs showed that the proposed method significantly improves segmentation and splitting performance for fluorescent micrographs of LPS-stimulated macrophages and reduces the rate of error in automated analysis of macrophage spreading in comparison with alternative methods.

Chondroitin Sulfate Is a Crucial Determinant for Skeletal Muscle Development/Regeneration and Improvement of Muscular Dystrophies

Skeletal muscle formation and regeneration require myoblast fusion to form multinucleated myotubes or myofibers, yet their molecular regulation remains incompletely understood. We show here that the levels of extra- and/or pericellular chondroitin sulfate (CS) chains in differentiating C2C12 myoblast culture are dramatically diminished at the stage of extensive syncytial myotube formation. Forced down-regulation of CS, but not of hyaluronan, levels enhanced myogenic differentiation in vitro. This characteristic CS reduction seems to occur through a cell-autonomous mechanism that involves HYAL1, a known catabolic enzyme for hyaluronan and CS. In vivo injection of a bacterial CS-degrading enzyme boosted myofiber regeneration in a mouse cardiotoxin-induced injury model and ameliorated dystrophic pathology in mdx muscles. Our data suggest that the control of CS abundance is a promising new therapeutic approach for the treatment of skeletal muscle injury and progressive muscular dystrophies.

Methods for studying tooth root cementum by light microscopy

The tooth root cementum is a thin, mineralized tissue covering the root dentin that is present primarily as acellular cementum on the cervical root and cellular cementum covering the apical root. While cementum shares many properties in common with bone and dentin, it is a unique mineralized tissue and acellular cementum is critical for attachment of the tooth to the surrounding periodontal ligament (PDL). Resources for methodologies for hard tissues often overlook cementum and approaches that may be of value for studying this tissue. To address this issue, this report offers detailed methodology, as well as comparisons of several histological and immunohistochemical stains available for imaging the cementum–PDL complex by light microscopy. Notably, the infrequently used Alcian blue stain with nuclear fast red counterstain provided utility in imaging cementum in mouse, porcine and human teeth. While no truly unique extracellular matrix markers have been identified to differentiate cementum from the other hard tissues, immunohistochemistry for detection of bone sialoprotein (BSP), osteopontin (OPN), and dentin matrix protein 1 (DMP1) is a reliable approach for studying both acellular and cellular cementum and providing insight into developmental biology of these tissues. Histological and immunohistochemical approaches provide insight on developmental biology of cementum.

Epidermal Hyperplasia and Appendage Abnormalities in Mice Lacking CD109

CD109, a glycosylphosphatidylinositol-anchored glycoprotein, is highly expressed in several types of human cancer tissues, in particular, squamous cell carcinomas. In normal human tissues, human CD109 expression is limited to certain cell types including myoepithelial cells of the mammary, lacrimal, salivary, and bronchial glands and basal cells of the prostate and bronchial epithelium. Although CD109 has been reported to negatively regulate transforming growth factor-β signaling in keratinocytes in vitro, its physiologic role in vivo remains largely unknown. To investigate the function of CD109 in vivo, we generated CD109-deficient (CD109(-/-)) mice. Although CD109(-/-) mice were born normally, transient impairment of hair growth was observed. At histologic analysis, kinked hair shafts, ectatic hair follicles with an accumulation of sebum, and persistent hyperplasia of the epidermis and sebaceous glands were observed in CD109(-/-) mice. Immunohistochemical analysis revealed thickening of the basal and suprabasal layers in the epidermis of CD109(-/-) mice, which is where endogenous CD109 is expressed in wild-type mice. Although CD109 was reported to negatively regulate transforming growth factor-β signaling, no significant difference in levels of Smad2 phosphorylation was observed in the epidermis between wild-type and CD109(-/-) mice. Instead, Stat3 phosphorylation levels were significantly elevated in the epidermis of CD109(-/-) mice compared with wild-type mice. These results suggest that CD109 regulates differentiation of keratinocytes via a signaling pathway involving Stat3.

In Vivo Applied Positively Charged FITC-Labeled Peptide Conjugates Show Artificial Relocation in Frozen Sections

Our original aim was to evaluate in vivo nuclear uptake of a conjugate containing the SV40 T Antigen nuclear localization sequence (NLS), 6 aminohexanoic acids alternating with 7 arginines, the fluorescence dye fluorescein isothiocyanate (FITC) and the gadolinium chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). During the confocal laser scanning microscopy examination of frozen sections of the kidneys of mice injected intravenously with the conjugate we found unexpected conjugate relocation. In the dry frozen sections no nuclear staining was found. After covering the sections with physiological saline and a coverslip, the conjugate was relocated to the cell nuclei. Causation of this effect by FITC and DOTA could be excluded with appropriate controls. In the assumption that other positively charged conjugates behave in the same manner we tested two further conjugates in vivo. An octaarginine coupled to a lysine carrying FITC on the -amino-group was found in the cell nuclei of the frozen kidney sections before and after covering the slices with saline. By contrast, a conjugate containing myristoylated HIV-Tat was found not to stain the cell nuclei but only the cytoplasm before and after covering with saline. This demonstrates that not all positively charged conjugates are relocated to the cell nucleus after covering the frozen sections with saline. This was confirmed by direct incubation of frozen sections from untreated kidneys with the same conjugates and additional positively charged FITC conjugates. By coincubating the FITC conjugates with a cytoplasm directed rhodamine conjugate on untreated kidney sections we found that such conjugates could be used as nuclear FITC counterstain. In summary, one should bear in mind that positively charged conjugates applied in vivo can relocate artificially after covering with saline. Keywords: Morphological artifacts, Frozen Sections, FITC-Labeled Peptide Conjugates, Artificial Relocation, SV40 T Antigen nuclear localization sequence, nuclear uptake, FITC conjugates, DOTA, glioma cells, N-terminally myristoylated HIV 1 Tat peptide sequence

Long‐term tissue integration of porous biopolymers as a material platform for metabolic biosensor

Implantable devices induce inflammation and collagen formation, which perturb the metabolic microenvironment and reduce mass transport. This study tested whether novel porous biomaterials improve tissue integration and normalize the glucose gradients. Five materials 700μm thick were implanted into rat dorsal subcutis: cotton cloth, solid silicone elastomer, solid polyHEMA, polyHEMA with 40μm pores, polyHEMA with 80μm pores. Specimens with surrounding tissue were explanted and frozen in liquid nitrogen 1, 4, and 8 weeks after implant. Serial sections were quantified for microvascular density (CD31 antibody), collagen (Masson's trichrome), immune cells (nuclear counterstain) and spatial glucose concentration (bioluminescence). Preliminary results indicate neovascularization of 80μm pore size polyHEMA after 1 week, and complete microvascular infiltration of the 80μm matrix after 4 weeks of implantation. Glucose concentrations in the porous polyHEMA after 4 weeks were comparable to those in the surrounding tissue. At that time, a moderate collagen deposition was observed in the 40 and 80 μm porous polyHEMA. While dense vascularization was also observed with cotton, it had the highest immune involvement. These preliminary findings suggest tissue integrates into porous polyHEMA matrix with a high microvessel density after 4 weeks and a more homogeneous glucose distribution.

Noninvasive near-infrared live imaging of human adult mesenchymal stem cells transplanted in a rodent model of Parkinson's disease.

BackgroundWe have previously shown that human mesenchymal stem cells (hMSCs) can reduce toxin-induced neurodegeneration in a well characterized rodent model of Parkinson’s disease. However, the precise mechanisms, optimal cell concentration required for neuroprotection, and detailed cell tracking need to be defined. We exploited a near-infrared imaging platform to perform noninvasive tracing following transplantation of tagged hMSCs in live parkinsonian rats.MethodshMSCs were labeled both with a membrane intercalating dye, emitting in the near- infrared 815 nm spectrum, and the nuclear counterstain, Hoechst 33258. Effects of near-infrared dye on cell metabolism and proliferation were extensively evaluated in vitro. Tagged hMSCs were then administered to parkinsonian rats bearing a 6-hydroxydopamine-induced lesion of the nigrostriatal pathway, via two alternative routes, ie, intrastriatal or intranasal, and the cells were tracked in vivo and ex vivo using near-infrared technology.ResultsIn vitro, NIR815 staining was stable in long-term hMSC cultures and did not interfere with cell metabolism or proliferation. A significant near-infrared signal was detectable in vivo, confined around the injection site for up to 14 days after intrastriatal transplantation. Conversely, following intranasal delivery, a strong near-infrared signal was immediately visible, but rapidly faded and was completely lost within 1 hour. After sacrifice, imaging data were confirmed by presence/absence of the Hoechst signal ex vivo in coronal brain sections. Semiquantitative analysis and precise localization of transplanted hMSCs were further performed ex vivo using near-infrared imaging.ConclusionNear-infrared technology allowed longitudinal detection of fluorescent-tagged cells in living animals giving immediate information on how different delivery routes affect cell distribution in the brain. Near-infrared imaging represents a valuable tool to evaluate multiple outcomes of transplanted cells, including their survival, localization, and migration over time within the host brain. This procedure considerably reduces the number of animal experiments needed, as well as interindividual variability, and may favor the development of efficient therapeutic strategies promptly applicable to patients.

P4-07-03: Identification of Triple-Negative Primary Breast Cancer Xenograft Models with High Numbers of Circulating and Disseminated Tumor Cells.

Abstract Background: Primary breast cancer xenografts, in which tumors are grown directly from patients and which maintain their original genotype and phenotype, have the potential to facilitate the study of tumor biology and progression. These models can also be instrumental in the discovery of novel therapeutic targets especially for the triple-negative (ER-, PR- and HER2−negative, TN) breast cancer. TN breast cancer is associated with high numbers of circulating and disseminated tumor cells (CTCs and DTCs), which predict poor outcome in patients and may play a role in tumor progression. However, isolation and detection of human CTCs and DTCs in these xenograft models have been challenging even with EpCAM-based enrichment methods. The goal of this study was to determine if CTCs and DTCs could be identified using human pan-CK staining in a panel of triple-negative primary breast cancer xenograft lines, which could then be employed to study the biology of these cells and to test novel therapies. Methods: We screened 13 stable primary transplantable xenograft lines (1-6 mice per line), established by directly transplanting ethnically diverse triple-negative tumor samples into the epithelium-free mammary fat pads of SCID/Beige mice, for the presence of CTCs and DTCs. The triple-negative status was maintained in these xenograft lines over serial passages. To detect CTCs, peripheral blood mononuclear cells (PBMCs) were isolated from the blood collected from the inferior vena cava either by Ficoll gradient or RBC lysis, with a typical yield of 500,000 PBMCs in 500 μl of blood. Subsequently, PBMCs were immunostained for the presence of CTCs, which were defined as the cells positive for cytoplasmic human pan-cytokeratin staining and nuclear (DAPI/hematoxylin) counter stain. We also flushed the femurs and tibias of 7 xenograft lines to harvest bone marrow cells (BMCs) for the detection of DTCs using the same staining procedure. A xenograft line was considered positive for CTCs or DTCs if they were detected in at least 25% of mice. The presence of lung metastases was assessed in all the xenograft lines by histological examination. Results: We detected CTCs (range: 1–128/20,000 PBMCs) in 6 out of 13 xenograft lines (46%) and DTCs (range: 1–21/20,000 BMCs) in 5 out of 7 (71%) lines. Interestingly, 4 of the 5 DTC-positive lines also had detectable CTCs. High numbers of CTCs (>20/20,000 PBMCs) were found in 3 xenograft lines, one of which also had high numbers of DTCs (>20/20,000 BMCs). No human pan-CK+ cells were detected in PBMCs and/or BMCs from 5 control mice without tumors. Among 13 xenograft lines, lung metastases were found in 5 lines (38%), of which 3 had detectable CTCs or DTCs. Of the 3 xenograft lines containing high CTCs and/or DTCs, 2 had lung metastases. Conclusion: In summary, human pan-CK staining can effectively detect CTCs and DTCs in isolated PBMCs and BMCs of mice bearing triple-negative primary breast cancer xenografts. These xenograft lines with detectable CTCs and DTCs may represent a valuable preclinical model for detailed characterization of human CTCs and DTCs and for the discovery of new therapeutic targets for the triple-negative breast cancer. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P4-07-03.

TCF/Lef1 activity controls establishment of diverse stem and progenitor cell compartments in mouse epidermis

Mammalian epidermis consists of the interfollicular epidermis, hair follicles (HFs) and associated sebaceous glands (SGs). It is constantly renewed by stem and progenitor cell populations that have been identified and each compartment features a distinct mechanism of cellular turnover during renewal. The functional relationship between the diverse stem cell (SC) pools is not known and molecular signals regulating the establishment and maintenance of SC compartments are not well understood. Here, we performed lineage tracing experiments to demonstrate that progeny of HF bulge SCs transit through other SC compartments, suggesting a hierarchy of competent multipotent keratinocytes contributing to tissue renewal. The bulge was identified as a bipotent SC compartment that drives both cyclic regeneration of HFs and continuous renewal of SGs. Our data demonstrate that aberrant signalling by TCF/Lef1, transcription factors crucial for bulge SC activation and hair differentiation, results in development of ectopic SGs originating from bulge cells. This process of de novo SG formation is accompanied by the establishment of new progenitor niches. Detailed molecular analysis suggests the recapitulation of steps of tissue morphogenesis.

2020 INSL3 RECEPTOR RXFP2 IS EXPRESSED ON THE SPERM ACROSOME

You have accessJournal of UrologyInfertility: Physiology, Pathophysiology, Basic Research1 Apr 20112020 INSL3 RECEPTOR RXFP2 IS EXPRESSED ON THE SPERM ACROSOME Daniel DaJusta, Candace F. Granberg, Yi Wang, Shaohua Zhang, Kent Hamra, and Linda A. Baker Daniel DaJustaDaniel DaJusta Dallas, TX More articles by this author , Candace F. GranbergCandace F. Granberg Dallas, TX More articles by this author , Yi WangYi Wang Dallas, TX More articles by this author , Shaohua ZhangShaohua Zhang Dallas, TX More articles by this author , Kent HamraKent Hamra Dallas, TX More articles by this author , and Linda A. BakerLinda A. Baker Dallas, TX More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.2248AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Insulin-3 (INSL3) and its receptor, RXFP2 (aka LGR8), are key mediators in testicular descent due to their action on the fetal gubernaculum. Insl3 or Rxfp2 knockout mice demonstrate bilateral intra-abdominal cryptorchidism and infertility. The RXFP2 receptor mRNA has been demonstrated in pachytene spermatocytes, but the function and localization of the protein in spermatogenesis has not been further documented. We sought to investigate whether adult sperm express the RXFP2 receptor. METHODS Sperm were extracted from the cauda epididymis of reproductive age WT mice and from human ejaculated sperm. Sperm in standard medium was capacitated with calcium and sodium bicarbonate for 1 hour. To assess permeabilized (fixed) sperm, capacitated sperm were air dried on slides for 20 minutes, fixed in 4% paraformaledhyde and washed in PBS. Anti-RXFP2 rabbit polyclonal antibody 1:150 was incubated followed by red fluorescent anti-rabbit donkey antibody 1:1000. Green fluorescent acrosome staining was performed with FITC labeled Anti-Peanut Agglutinin antibody (PNA-FITC) followed by DAPI blue nuclear counterstaining. To assess non-permeabilized (live) sperm, capacitated sperm were centrifuged and resuspended in PBS with anti-RXFP2 rabbit polyclonal antibody 1:150. Again, sperm were centrifuged and resuspended in PBS with anti-rabbit donkey antibody 1:1000. PNA-FITC was then added for acrosome staining. Finally, 50μl of the sperm solution was slide mounted with DAPI counterstain. Control experiments omitted the primary antibody. Images were acquired with a Nikon Eclipse fluorescent microscope using NIS-Elements Br 2.30 software. RESULTS The acrosomes of capacitated adult WT mice sperm and human ejaculated sperm in both permeabilized and non-permeabilized state stained positive for RXFP2 (Figure 1). CONCLUSIONS The RXFP2 protein is localized to the acrosome of mature spermatozoa in mice and human. Given this observation, we speculate a possible new role for the INSL3-RXFP2 signaling system in acrosomal functions. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e807-e808 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Daniel DaJusta Dallas, TX More articles by this author Candace F. Granberg Dallas, TX More articles by this author Yi Wang Dallas, TX More articles by this author Shaohua Zhang Dallas, TX More articles by this author Kent Hamra Dallas, TX More articles by this author Linda A. Baker Dallas, TX More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Isolation and Characterization of Progenitor-Like Cells from Human Renal Proximal Tubules

The tubules of the kidney display a remarkable capacity for self-renewal on damage. Whether this regeneration is mediated by dedifferentiating surviving cells or, as recently suggested, by stem cells has not been unequivocally settled. Herein, we demonstrate that aldehyde dehydrogenase (ALDH) activity may be used for isolation of cells with progenitor characteristics from adult human renal cortical tissue. Gene expression profiling of the isolated ALDH(high) and ALDH(low) cell fractions followed by immunohistochemical interrogation of renal tissues enabled us to delineate a tentative progenitor cell population scattered through the proximal tubules (PTs). These cells expressed CD24 and CD133, previously described markers for renal progenitors of Bowman's capsule. Furthermore, we show that the PT cells, and the glomerular progenitors, are positive for KRT7, KRT19, BCL2, and vimentin. In addition, tubular epithelium regenerating on acute tubular necrosis displayed long stretches of CD133(+)/VIM(+) cells, further substantiating that these cells may represent a progenitor cell population. Furthermore, a potential association of these progenitor cells with papillary renal cell carcinoma was discovered. Taken together, our data demonstrate the presence of a previously unappreciated subset of the PT cells that may be endowed with a more robust phenotype, allowing increased resistance to acute renal injury, enabling rapid repopulation of the tubules.

Pannexin 2 Is Expressed by Postnatal Hippocampal Neural Progenitors and Modulates Neuronal Commitment

The pannexins (Panx1, -2, and -3) are a mammalian family of putative single membrane channels discovered through homology to invertebrate gap junction-forming proteins, the innexins. Because connexin gap junction proteins are known regulators of neural stem and progenitor cell proliferation, migration, and specification, we asked whether pannexins, specifically Panx2, play a similar role in the postnatal hippocampus. We show that Panx2 protein is differentially expressed by multipotential progenitor cells and mature neurons. Both in vivo and in vitro, Type I and IIa stem-like neural progenitor cells express an S-palmitoylated Panx2 species localizing to Golgi and endoplasmic reticulum membranes. Protein expression is down-regulated during neurogenesis in neuronally committed Type IIb and III progenitor cells and immature neurons. Panx2 is re-expressed by neurons following maturation. Protein expressed by mature neurons is not palmitoylated and localizes to the plasma membrane. To assess the impact of Panx2 on neuronal differentiation, we used short hairpin RNA to suppress Panx2 expression in Neuro2a cells. Knockdown significantly accelerated the rate of neuronal differentiation. Neuritic extension and the expression of antigenic markers of mature neurons occurred earlier in stable lines expressing Panx2 short hairpin RNA than in controls. Together, these findings describe an endogenous post-translational regulation of Panx2, specific to early neural progenitor cells, and demonstrate that this expression plays a role in modulating the timing of their commitment to a neuronal lineage.

Low Current-driven Micro-electroporation Allows Efficient In Vivo Delivery of Nonviral DNA into the Adult Mouse Brain

Viral gene transfer or transgenic animals are commonly used technologies to alter gene expression in the adult brain, although these approaches lack spatial specificity and are time consuming. We delivered plasmid DNA locally into the brain of adult C57BL/6 mice in vivo by voltage- and current-controlled electroporation. The low current-controlled delivery of unipolar square wave pulses of 125 µA with microstimulation electrodes at the injection site gave 16 times higher transfection rates than a voltage-controlled electroporation protocol with plate electrodes resulting in currents of about 400 mA. Transfection was restricted to the target region and no damage due to the electric pulses was found. Our current-controlled electroporation protocol indicated that the use of very low currents resulting in applied voltages within the physiological range of the membrane potential, allows efficient transfection of nonviral plasmid DNA. In conclusion, low current-controlled electroporation is an excellent approach for electroporation in the adult brain, i.e., gene function can be influenced locally at a high level with no mortality and minimal tissue damage.

Human eyelid meibomian glands and tarsal muscle are recognized by autoantibodies from patients affected by a new variant of endemic pemphigus foliaceus in El-Bagre, Colombia, South America

Previously, we described a new variant of endemic pemphigus foliaceus (EPF) in Colombia, South America (El Bagre-EPF). Continuing our characterization of this variant of EPF, we now focus on one of our previously reported clinical findings: the presence of ocular lesions. These ocular lesions are seen in patients having extensive skin involvement, as measured by the Lund and Browder scale, which is generally used for patients with skin burns. We specifically searched for evidence of autoreactivity to various eyelid structures in these patients and correlated our immunologic data with the clinical findings. We performed indirect immunofluorescence studies using normal-appearing human eyelid skin from routine blepharoplasties as substrate tissue. We tested sera from 12 patients with El Bagre-EPF and ocular lesions, 5 patients with sporadic (nonendemic) pemphigus foliaceus, and 20 healthy control subjects (10 from the El Bagre-EPF endemic area and 10 from nonendemic areas). We used fluorescein isothiocyanate conjugated goat antiserum to human total IgG/IgA/IgM as a secondary antibody. In addition, we used fluorescein isothiocyanate conjugated antibodies to human fibrinogen, albumin, IgG, IgE, C1q, and C3, Texas Red (Rockland Immunochemicals, Inc, Gilbertsville, PA), Alexa Fluor 555, or Alexa Fluor 594 (Invitrogen, Carlsbad, CA). Ki-67 (a cell proliferation marker) was used to determine the cell proliferation rate, and nuclear counterstaining was performed with either 4', 6-diamidino-2-phenylindole or Topro III (Invitrogen, Carlsbad, CA). We observed autoreactivity to multiple eyelid structures, including meibomian glands and tarsal muscle bundles at different levels, and some areas of the epidermis and the dermis close to the isthmus of the eyelids. Tarsal plate autoreactivity was seen in 10 of 12 of the El Bagre-EPF sera and in one control with pemphigus erythematosus. Furthermore, immunoprecipitation using an eyelid sample as a substrate with 1 mmol/L of sodium orthovanodate showed autoreactivity to several antigens, including some of possible lipid origin. The main limitation of this study is the fact that the antigen or antigens remain unknown. We identified for the first time to our knowledge autoantibodies to meibomian glands and tarsal muscle in El Bagre-EPF. Our findings suggest that the autoantibodies to the ocular structures cause the clinical and histopathological findings in the ocular lesions in El Bagre-EPF.

Neurodegeneration Induced by Bortezomib Exposure in Vitro Occurs Via Proteasome Independent Mechanisms.

Abstract 2859Poster Board II-835 BACKGROUND:The dipeptide boronate proteasome inhibitor bortezomib (BTZ; Velcade®) is approved for the treatment of multiple myeloma and non-Hodgkin's lymphoma. Bortezomib-induced peripheral neuropathy (BIPN, Blood (2008)112:1593-1599) is seen in ∼30% of BTZ-treated patients and can result in dose reductions and discontinuations that may result in suboptimal levels of proteasome inhibition. Carfilzomib (CFZ), a tetrapeptide epoxyketone, is a selective and irreversible proteasome inhibitor that is structurally and mechanistically distinct from bortezomib. Single agent treatment with CFZ has demonstrated strong activity in relapsed and refractory myeloma and a favorable safety profile in Phase 2 trials (ASH2008:864 & 865). Importantly treatment-emergent PN was seen at low levels and did not result in dose modifications or discontinuations. The disparate safety data for these proteasome inhibitors suggest that non-proteasomal mechanisms may underlie BIPN. Using activity-based probes in peripheral blood mononuclear cell (PBMC) lysates, we previously demonstrated inhibition of non-proteasomal proteases by BTZ and other proteasome inhibitors with a boronate pharmacophore (EHA2009:0939). However, the involvement of the proteasome in the peripheral nerve degeneration and BIPN in BTZ-treated myeloma patients remains to be established. AIMS:To establish an in vitro model of peripheral nerve degeneration and to determine the effects of proteasome inhibition by BTZ and CFZ on neurite outgrowth and cell survival. METHODS:SH-SY5Y neuroblastoma cells were differentiated by long term culture in retinoic acid and brain derived nerve growth factor to induce neurite outgrowth. The effects of proteasome inhibitors were measured by high content image analysis of fluorescent images for cell survival (Hoechst nuclear counterstain) and neurite degeneration (FITC-mouse anti-beta-III-tubulin). Phase contrast images were also collected to observe morphological effects and gross cell death. Cell viability and proteasome inhibition was measured in undifferentiated and differentiated cells. The MEROPS (peptidase) database was mined for candidate serine proteases with a P1 selectivity of Leu/Phe/Tyr to identify candidate off-targets CFZ and BTZ and candidate proteases were validated by standard biochemical and cell biology techniques. RESULTS:In differentiated SH-SY5Y cells, the average neurite length decreased by 33% following 24 hr exposure to 10nM BTZ but was unaffected by the same concentration of CFZ. Proteasome inhibition as determined by a fluorescent substrate for the chymotrypsin-like activity was equivalent (∼70%) after a 24 hr exposure for both compounds in differentiated cells, suggesting that neurodegeneration involves non-proteasomal pathways. With 72 hrs continuous exposure, BTZ was 10-fold more potent than CFZ at inducing neurodegeneration. Furthermore, in both undifferentiated and differentiated SH-SY5Y cells, BTZ was 5-fold more cytotoxic than CFZ. Database mining for serine proteases with a selectivity for Leu/Phe/Tyr at P1 was used to identify other potential BTZ targets that might underlie neurotoxicity. One candidate is HtrA2 (also called Omi), an inducible mitochondrial serine protease whose activity protects neurons from stress induced apoptosis (Hum Mol Genet (2005) 14(5):2099-2111). HtrA2 levels increased 2-fold in SH-SY5Y cells treated with either BTZ or CFZ for 6 hrs at 40 nM. Using a gel based assay and purified enzyme preparations, BTZ inhibited HtrA2 activity with an IC50 ∼ 4 nM, equivalent to its activity against the proteasome. In contrast, Carfilzomib did not inhibit HtrA2 at the highest concentration tested (10 mM). CONCLUSIONS:These data demonstrate that BTZ induces neuronal cell death and neurite degeneration in vitro by proteasome-independent mechanisms. We propose that combined inhibition of the proteasome and HtrA2 by BTZ may underlie peripheral nerve toxicities in vitro and may be involved in BIPN in myeloma patients. In this model, CFZ, which mediates equivalent proteasome inhibition to BTZ in neurons, does not induce neurodegeneration due to inactivity against HtrA2. Future profiling of non-proteasomal targets of BTZ, including HtrA2 activity, in patient samples is merited. These results suggest that the favorable safety profile of CFZ in myeloma patients may be a result of its high selectivity for proteasomal proteases. Disclosures:Arastu-Kapur:Proteolix, Inc: Employment. Ball:Millipore Corp: Employment. Anderl:Millipore Corp: Employment. Bennett:Proteolix: Employment. Kirk:Proteolix, Inc: Employment.

Use of destained cytology slides for the application of routine special stains

Special stains to demonstrate microorganisms or intra- and extracellular substances have not been evaluated in detail regarding their applicability and usefulness in destained cytologic specimens. The aim of this study is to compare the results of routine special stains on destained slides previously stained with Hemacolor and on fresh (unstained) specimens. Archival Hemacolor-stained fine needle aspirate specimens of inflammation with infectious agents (bacterial, mycobacterial, and fungal infections), neoplasia (melanoma, myxosarcoma, and mammary adenocarcinoma), and hemorrhage (pericardial effusion) from 14 dogs and 7 cats were selected. Cells in a minimum of 4 fields were photographed and 5 slides from each case were then destained by different methods (alcohol acid or microwave). Seven special stains were applied selectively to the destained slides, depending on the cytologic findings: periodic acid Schiff, Grocott-Gomori methenamine silver, Gram's, Ziehl-Neelsen, Alcian blue, Fontana-Masson, and Prussian blue. The same fields were rephotographed and 2 observers evaluated the slides qualitatively, with comparison to fresh cytologic specimens from similar lesions. Special stains applied to destained slides demonstrated the expected cellular and extracellular material or organisms independent of the destaining method. Staining intensity, nonspecific staining (background), cell morphology, and nuclear counterstaining results were similar to those of special stains applied to fresh unstained slides. Destaining does not appear to affect the results of routine special staining for cytologic specimens. Destaining before special stains may be a valuable diagnostic strategy when few slides are present or only stained slides are available.

Type XVII Collagen is a Key Player in Tooth Enamel Formation

Inherited tooth enamel hypoplasia occurs due to mutations in genes that encode major enamel components. Enamel hypoplasia also has been reported in junctional epidermolysis bullosa, caused by mutations in the genes that encode type XVII collagen (COL17), a component of the epithelial-mesenchymal junction. To elucidate the pathological mechanisms of the enamel hypoplasia that arise from the deficiency of epithelial-mesenchymal junction molecules, such as COL17, we investigated tooth formation in our recently established Col17(-/-) and Col17 rescued mice. Compared with wild-type mice, the incisors of the Col17(-/-) mice exhibited reduced yellow pigmentation, diminished iron deposition, delayed calcification, and markedly irregular enamel prisms, indicating the presence of enamel hypoplasia. The molars of the Col17(-/-) mice demonstrated advanced occlusal wear. These abnormalities were corrected in the Col17 rescued humanized mice. Thus, the Col17(-/-) mice clearly reproduced the enamel hypoplasia in human patients with junctional epidermolysis bullosa. We were able to investigate tooth formation in the Col17(-/-) mice because the Col17(-/-) genotype is not lethal. Col17(-/-) mouse incisors had poorly differentiated ameloblasts that lacked enamel protein-secreting Tomes' processes and reduced mRNA expression of amelogenin, ameloblastin, and of other enamel genes. These findings indicated that COL17 regulates ameloblast differentiation and is essential for normal formation of Tomes' processes. In conclusion, COL17 deficiency disrupts the epithelial-mesenchymal interactions, leading to both defective ameloblast differentiation and enamel malformation.

Using colour in figures: let's agree to differ.

Using colour in figures: let's agree to differ.