Anti-vimentin Antibodies Research Articles

The EGF signaling pathway influences cell migration and the secretion of metalloproteinases by myoepithelial cells in pleomorphic adenoma.

During tumor development, benign neoplastic cells are influenced by the expression of cytokines, growth factors, and proteases present in the tumor microenvironment. Epidermal growth factor (EGF) is the most studied growth factor and is considered important for cell proliferation and migration. Metalloproteinases (MMPs) are also involved in tumor progression. The present study aimed to analyze the proliferation, viability and migration index of pleomorphic adenoma myoepithelial cells, in addition to the secretion of MMPs with EGF supplementation. Benign myoepithelial cells were cultured with two different EGF doses (5 and 10ng/ml), and the influence of EGF on cell proliferation and viability, using trypan blue and MTT assays, respectively, after 24, 48, and 72h, was evaluated. To analyze cellular morphology, hematoxylin-eosin staining and indirect immunofluorescence using the anti-vimentin antibody, was performed. In vitro migration assays were performed in Transwell chambers with an 8-μm pore covered with Matrigel and supplemented with 5 or 10ng/ml of EGF, after 96h. After 4days of cell culture, ELISA was performed to determine the MMP-2 and MMP-13 levels. One-way analysis of variance (ANOVA) with post hoc Tukey test was applied, with a significance level of 0.05. The results revealed that EGF influences myoepithelial cell morphology, without alteration of proliferation and viability. The migration assay showed that EGF increased the mean index from 16% in the control group to 40 and 76% for 5 and 10ng/ml of EGF, respectively. ELISA revealed that when the cells were supplemented with eitherof the EGF doses, an increase in MMP-2 levels was observed when compared with the control group (C). This study concludes that EGF aids in the production of MMP-2, which favors the dissolution of the basement membrane, contributing to cell migration and tumor progression, hence permitting contact between the myoepithelial cells and stroma.

A Detection System for Circulating Tumor Cells (Ctcs) Using Gfp Expressing Telomerase-Specific Replication-Competent Adenovirus in Bone and Soft Tissue Sarcoma

ABSTRACT Aim: The concept of circulating tumor cell (CTC) is of great interest recently in the field of cancer treatment. CTCs defined as cancer cells of solid tumor origin found in the peripheral blood, and have been detected in the patients with advanced cancer. However, CTCs have also detected in patients with localized cancers, indicating the risk of progression to metastatic disease. Although many papers on CTCs have been published, there is no report on CTCs detected in patients with bone and soft tissue tumors. We analyzed CTCs in the peripheral blood of patients with sarcoma. Methods: 7.5ml of blood was collected from the patients, and CTCs were assessed using TelomeScan (OBP-401), which GFP-expressing attenuated adenovirus-5 vector, in which the hTERT promoter regulates viral replication. A total of 22 patients with bone (6 osteosarcoma) and soft tissue sarcoma (4 myxofibrosarcoma, 4 liposarcoma, 3 DFSP, 2 synovial sarcoma, 3 others) patients were enrolled. GFP-expressing cells were labeled with anti-CD45 (a marker for blood cell), anti-vimentin antibody (a marker for mesenchymal cell) and DAPI stain (a marker for cell nucleus) 24 hours after the virus infection to ensure that cells labeled with GFP were indeed sarcoma cells. Results: CTCs were detected in 11 of 22 sarcoma patients (4 osteosarcoma, 3 myxofibrosarcoma, 2 DFSP, 2 others). Average number of CTCs was 3.5 cells per 7.5ml. 3 patients with positive CTC showed negative CTC after sarcoma resection, and 4 patients with positive CTC showed negative CTC after chemotherapy. 5 of 22 patients developed lung metastasis, 4 of those showed positive CTC at diagnosis. Conclusions: CTCs were detected in 50% of patients with bone and soft tissue sarcoma in our series. CTC detection using TelomeScan has been reported only in patients with cancer. We identified that the detection system using TelomeScan can be a new approach to visualize live human CTCs from bone and soft tissue sarcoma, and that CTC can be also a biomarker for bone and soft tissue sarcoma. Disclosure: All authors have declared no conflicts of interest.

Role of anti-vimentin antibodies in renal transplantation.

The role of non-HLA antibodies in rejection is not clear. We investigate whether antibodies to vimentin are made after renal transplantation and if production is associated with interstitial fibrosis and tubular atrophy (IFTA). In this retrospective study, sera from 70 recipients of renal allografts (40 controls, 30 IFTA) were studied. The biopsy diagnosis of interstitial fibrosis and tubular atrophy (IFTA) was based on random, cause-indicating biopsies. Sera were collected pretransplant and at 3 monthly intervals up to 5 years posttransplant or diagnosis of IFTA and assayed by ELISA for IgM and IgG anti-vimentin antibodies (AVA) and HLA antibodies. Mean titers of IgM AVA were higher at every year after transplantation compared with pretransplant for both IFTA and controls groups (P<0.001). There was no difference in the mean level of IgM AVA achieved by IFTA and control groups. The mean pretransplant levels of IgG AVA in the IFTA and control group were 18.2±11.7 and 11.0±8.1, respectively (P=0.001). There was a significant increase between the pretransplant mean levels of IgG AVA and the levels at years 1 to 4 in the IFTA group (years 1-3, P<0.0001, year 4 P=0.003) but not in the controls. There was no significant difference between the numbers of IFTA or control patients achieving a positive value (mean+2SD of pretransplant antibody titers) of IgM AVA (50% versus 37.5%, respectively) or IgG AVA (26.6% versus 12.5%, respectively). There was no association between production of HLA and AVA antibodies. Posttransplant production of IgM AVA is not associated with IFTA. The production of IgG AVA by a minority of IFTA patients suggests that in some individuals, IgG AVA may be involved in the pathology of IFTA.

Vimentin is a dominant target of in situ humoral immunity in human lupus tubulointerstitial nephritis (HUM1P.314)

Abstract In lupus nephritis (LuN), severity of tubulointerstitial inflammation (TII) on renal biopsy predicts subsequent renal failure. In severe TII, infiltrating B and T cells have features of tertiary lymphoid neogenesis including in situ antigen-driven clonal B cell selection. To identify the autoantigen(s) driving selection, we laser captured single CD38+ or Ki-67+ B cell mRNA from seven LuN biopsies. Twenty clonally expanded immunoglobulin heavy and light chain variable region pairs were cloned and expressed as antibodies. Seven more were obtained from an eighth biopsy by flow sorting of CD38+ cells. Immunofluorescence indicated that 13 of 27 antibodies had immunoreactivity with cytoplasmic structures. Only 4 reacted with nuclei and none with dsDNA, Sm or RNP. Interrogation of protein arrays with TII antibodies and immunoprecipitations followed by mass spectroscopy identified a limited number of self-antigens with only vimentin common to both approaches. Eleven of 13 cytoplasmic TII antibodies directly bound vimentin, vimentin was highly expressed by tubulointerstitial infiltrating cells and tested TII antibodies preferentially bound inflamed tubulointerstitum. Finally, high-titers of serum anti-vimentin antibodies were strongly associated with severe TII. These finding suggest that in situ adaptive immunity to vimentin, an antigenic feature of inflammation, feeds forward to worsen TII and renal damage.

Platelet adhesion involves a novel interaction between vimentin and von Willebrand factor under high shear stress

AbstractThe interaction between platelet receptor glycoprotein Ibα and the A1 domain of von Willebrand factor (VWF) mediates tethering/translocation of platelets to sites of vascular injury. Unexpectedly, we observed platelets translocating over A1A2A3 domains protein slower than on A1 domain at high shear stress. This observation suggests an additional interaction between A domains and an adhesive receptor. We investigated vimentin because we have data showing the interaction of vimentin with the A2 domain of VWF. Moreover, vimentin is expressed on the platelet surface. This novel interaction was analyzed by using purified VWF, recombinant proteins, anti-vimentin antibodies, parallel flow chamber adhesion assays, flow cytometry, and vimentin-deficient murine platelets. The active form of VWF bound to vimentin, and the purified A2 domain blocked that binding. The interaction of a gain-of-function A1A2A3 mutant with platelet was reduced using anti-vimentin antibody. Platelet adhesion to wild-type (WT) A1A2A3 protein, collagen, and fibrin(ogen) was inhibited (32-75%) by anti-vimentin antibody under high shear stress. Compared with WT mice, platelets from vimentin-deficient mice had a reduced flow-dependent adhesion to both collagen and purified murine VWF. Last, the vimentin knockout mice had a prolonged tail bleeding time. The results describe that platelet vimentin engages VWF during platelet adhesion under high shear stress.

Condrossarcoma mesenquimal extraesquelético em um gato: relato de caso

Condrossarcoma mesenquimal extraesquelético (CME) é um neoplasma maligno e raro em animais domésticos. Descreve-se um caso de CME em uma gata que apresentava uma massa firme, branco-amarelada, medindo 18cm de diâmetro, aderida à musculatura do membro pélvico esquerdo. O exame citológico revelou presença de células fusiformes individualizadas pleomórficas e agregados de pequenas células ovais, sem bordas definidas em meio à matriz intercelular amorfa. Devido à impossibilidade de tratamento e ao prognóstico desfavorável, foi realizada eutanásia. Microscopicamente foram observadas células fusiformes indiferenciadas e agregados de células condroides pleomórficas. O diagnóstico de CME foi confirmado pelas técnicas de azul alciano, tricrômico de Masson e pela prova imunoistoquímica, utilizando-se anticorpos antivimentina.

Influences of IL-6R antibody on PMMA bone cement-mediated expression of OPG and RANKL in synovial fibroblasts

Effect of interleukin-6 receptor (IL-6R) antibody on polymethyl methacrylate (PMMA) bone cement-mediated expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB ligand (RANKL) in synovial fibroblasts was investigated. Synovial tissue obtained from total knee arthroplasty was digested and cultured. Inverted microscope was employed to observe the synovial cells and immunocytochemistry (SABC method) staining was used to identify synovial fibroblasts. This experiment was divided into three groups according to different culture media: PMMA group (75 μg/mL PMMA bone cement particles), IL-6R antibody group (10 ng/mL IL-6R antibody+75 μg/mL PMMA bone cement particles), and control group (no IL-6R antibody or PMMA bone cement particles). Influence of IL-6R antibody and PMMA on proliferation of synovial fibroblasts was measured by cell counting kit-8 (CCK-8). ELISA method was used to measure OPG and RANKL levels in culture solution. Fluorescence quantitative real-time PCR (FQ-PCR) was used to detect the expression of OPG and RANKL mRNA. After three consecutive passages, more than 95% of the primary synovial cells became long spindle fibroblast-like cells. SABC staining results showed that the fibroblast-like cells were negative for anti-CD68 antibody and positive for anti-vimentin antibody, with brown madder stained. CCK-8 test demonstrated that the absorbance (A) value at 450 nm was significantly lower in IL-6R antibody group than in PMMA group and control group (P<0.01), but there was no statistically significant difference in A value at 450 nm between the control group and PMMA group (P>0.05). Results of ELISA indicated that the expression of OPG was significantly higher in IL-6R antibody group than in PMMA group and control group (P<0.01). The expression of RANKL was inhibited (P<0.05), and the ratio of OPG/RANKL was significantly increased in IL-6R antibody group as compared with PMMA group and control group. There was no significant difference in the expression of OPG between control group and PMMA group (P>0.05), but the expression of RANKL was higher in PMMA group than in control group (P<0.05), and there was a significant difference in the ratio of OPG/RANKL between them (P<0.05). Results of FQ-PCR revealed the expression of RANKL mRNA was significantly inhibited (P<0.01) and the expression of OPG mRNA was significantly increased (P<0.01) in IL-6R antibody group as compared with PMMA group and control group. The expression of RANKL mRNA was higher in PMMA group than in control group (P<0.05), but the expression of OPG mRNA had no significant difference between them (P>0.05). IL-6R antibody could significantly increase the expression of OPG, but inhibit the expression of RANKL, which might provide a theoretical basis of molecular biology for the prevention and treatment of aseptic loosening of prosthesis.

Development of a bead-based immunoassay to routinely measure vimentin autoantibodies in the clinical setting

IntroductionVimentin is an intermediate filament protein generally expressed in the cytosol of many adult cell types, including leukocytes, fibroblasts and endothelial cells. Several tissue and/or injury-specific isoforms of vimentin are known to exist that may trigger autoimmune responses due to aberrant structural or conformational variations. Such scenarios include allograft rejection and certain autoimmune diseases, such as rheumatoid arthritis. The primary objective for this study was to develop a Luminex immunobead assay to quantitate circulating levels of vimentin antibodies and, secondarily, to appraise the feasibility of these autoantibodies as a biomarker for clinical diagnosis. MethodsRecombinant human vimentin was conjugated to MagPlex® beads using standard carbodiimide/NHS chemistry and coupling efficiency tested and assay parameters determined using a commercial anti-vimentin polyclonal antibody. A limited number of serum samples (n=71) were then tested to evaluate the diagnostic value for future biomarker development efforts. ResultsFindings from repeated testing of three distinct batches of assays provide assay range parameters of 0.18–15μg/mL, median inter-assay recovery parameter within 1% of completion, and inter-assay variation (%CV) at 7%. The assay was found to be stable at several conditions with less than 5% loss in a month. Preliminary evaluation of the assay demonstrates significantly (p=0.022) higher circulating levels of anti-vimentin antibodies in 51 cases of renal allograft rejection relative to 20 cases of age-matched controls. ConclusionA direct capture assay for vimentin autoantibodies was developed and analytically validated. Preliminary evaluation of this assay against patient materials was promising and justifies additional testing with larger cohorts in future studies.

Immunohistochemical study of genital and extragenital forms of canine transmissible venereal tumor in Brazil

Aiming to provide insight and discussing the problems related to the diagnosis and differential diagnosis of canine transmissible venereal tumor (CTVT), especially in its extragenital form, immunohistochemical evaluation was performed and a comparison was established by analysis of the microscopic appearance of 10 genital CTVTs and 13 exclusively extragenital CTVTs previously diagnosed by cytology and histopathology. CTVTs samples were incubated with biotinylated antibodies raised against specific membrane (anti-macrophage) and cytoplasmic antigens (anti-lysozyme, anti-S-100 protein, anti-vimentin and anti-CD18) and subsequently developed using streptavidin-biotin peroxidase and streptavidin-biotin-alkaline phosphatase methods. A strong reactivity with the anti-vimentin antibody was found in 100% of the tumors tested (22/22). No reactivity was found for the anti-lysozyme, anti-macrophage, anti-S-100 protein and anti-CD18. No histopathological or immunoreactivity differences between genital and extragenital CTVTs were found. These findings do not corroborate the hypothesis of histiocytic origin of CTVT (no reactivity to anti-lysozyme, anti-macrophage and anti-CD 18 antibodies). In addition, the antibody panel used is useful to narrow the differential diagnosis for lymphomas, histiocytic tumors, amelanotic melanomas, and poorly differentiated epithelial neoplasias, among others.

Intermediate Filament Proteins Expression and Carbohydrate Moieties in Trophoblast and Decidual Cells of Mature Cat Placenta

The aim of this study was to characterize cytoskeletal intermediate filament proteins and glycoconjugates of syncytiotrophoblast, cytotrophoblast and decidual cells of feline endotheliochorial placenta. Samples from 12 normal pregnant female cats, after 45 ± 5 days of gestation, were obtained removing the uterine horns by hysterectomy. Sections were processed for routine observation and for immunohistochemistry using anticytokeratin, antivimentin and antidesmin antibodies. In addition, lectin histochemistry was performed using a panel of several biotinylated lectins to characterize glycosides expression profile. Cytotrophoblast and syncytiotrophoblast showed immunoreactivity only with acidic and basic cytokeratins. Decidual cells were only positive to vimentin, consistent with their origin from endometrial fibroblasts. Trophoblast expressed a broad population of glycans, highly exposing terminal N-acetyl glucosamine residues and non-sialylated galactose and N-acetyl galactosamine oligomers. Oligosaccharides bound by Phaseolus vulgaris erythroagglutinin were the only highly branched N-linked residues evidenced in cats, and they were restricted to the syncytium. Unlike results reported on humans, mice and rats on lectin affinity of decidual cells, sialid acids and complex N-linked oligosaccharides were not demonstrated in cats. Glycosylation of proteins determines many of their final properties, thus becoming essential for the embryo-maternal dialogue during implantation and placentation. Changes in glycosylation pattern have been related to pathological pregnancies in other species. Hence, the knowledge about glycosylation profile of the normal cat placenta may lead to a better understanding of both normal and pathological reproductive events.

Lupus tubulointerstitial and vascular disease

Since its inception over 40 years ago, the classification of lupus nephritis has focused entirely on glomerular alterations. Existing data indicate that tubulointerstitial and possibly vascular alterations may be superior prognostic indicators. In particular, our group has demonstrated that in-situ immune processes can occur. Ectopic germinal center formation may be found in a subset of lupus patients, which has been observed in the target organs of other autoimmune disorders. Analysis of antibody sequences from the plasma cells isolated from these organized lymphoid structures demonstrates both clonal expansion and somatic hypermutation. Expression cloning of 27 antibodies generated from CD38 + or Ki-67 + B cells isolated from 8 lupus nephritis patients by laser capture microdissection. Thirteen antibodies revealed cytoplasmic immunoreactivity and 11 of these were subsequently confirmed to bind directly to vimentin, which was highly expressed in severely inflamed regions of the kidneys. In addition, high anti-vimentin antibody serum titers correlated with the presence of severe tubulointerstitial inflammation. Other studies have also demonstrated that renal vascular lesions are common and include thrombotic microangiopathy, atherosclerosis, noninflammatory necrotizing vasculitis and rarely true vasculitis. Incorporating both tubulointerstitial and vascular changes provides a more accurate assessment of the kidney in lupus patients.

Vimentin is a dominant target of in situ humoral immunity in human lupus tubulointerstitial nephritis

In lupus nephritis (LuN), severity of tubulointerstitial inflammation (TII) on renal biopsy predicts subsequent renal failure. In severe TII, infiltrating B and T cells have features of tertiary lymphoid neogenesis including in situ antigen-driven clonal B cell selection. To identify the autoantigen(s) driving selection, we laser captured single CD38+ or Ki-67+ B cell mRNA from seven LuN biopsies. Twenty clonally expanded immunoglobulin heavy and light chain variable region pairs were cloned and expressed as antibodies. Seven more were obtained from an eighth biopsy by flow sorting of CD38+ cells. Immunofluorescence indicated that 13 of 27 antibodies had immunoreactivity with cytoplasmic structures. Only 4 reacted with nuclei and none with dsDNA, Sm or RNP. Interrogation of protein arrays with TII antibodies and immunoprecipitations followed by mass spectroscopy identified a limited number of self-antigens with only vimentin common to both approaches. Eleven of 13 cytoplasmic TII antibodies directly bound vimentin, vimentin was highly expressed by tubulointerstitial infiltrating cells and tested TII antibodies preferentially bound inflamed tubulointerstitum. Finally, high-titers of serum anti-vimentin antibodies were strongly associated with severe TII. These finding suggest that in situ adaptive immunity to vimentin, an antigenic feature of inflammation, feeds forward to worsen TII and renal damage.

90 CHARACTERIZATION OF A PRIMARY CULTURE OF OVIDUCTAL CELLS IN THE BITCH

The oviduct is of particular importance in canine reproduction as it supports oocyte meiosis resumption, sperm capacitation and storage, fertilization and embryo development to the morula/blastocyst stage for 8 to 10 days post-ovulation. A long-time co-culture with oviducal cells could be employed to improve the yield of reproductive biotechnologies in this species, but no characterisation of canine oviduct cells in vitro has been reported to date. The objectives of this study were to (1) evaluate the viability and proportion of epithelial/fibroblast cells in a primary culture of canine oviducal cells collected around ovulation; (2) study the responsiveness of the cultured cells to steroids. Beagle bitches (n = 9) were ovariectomized between Day –1 and Day +1 around ovulation, and their oviducts were sectioned at the ampulla-isthmus junction. Mucosal cells (including stromal and epithelial cells) were collected by squeezing from the ampulla and isthmus sections and cultured separately at a concentration of 5 × 105 cells/well in 500 μL of M199 + 10% FCS at 39°C for 9 days. At Days 3 and 6, 1 × 106 cells were stimulated with 17β-oestradiol (E2, 20 pg mL–1) or progesterone (P4, 20 ng mL–1) for 6 h. At Days 3, 6, and 9 of culture, the viability of the cells was evaluated using the Live/Dead kit (Invitrogen, Carlsbad, CA, USA), and proportions of fibroblast and epithelial ciliated cells were evaluated by immuno-cytochemistry using anti-vimentin and anti-tubulin antibodies, respectively. At Days 0, 3 and 6, the total RNA was extracted from cells and mRNA levels of the oviduct-specific glycoprotein (OVGP, synthesised by nonciliated epithelial cells), E2 (ERα, ERβ) and P4 (PR) receptors were evaluated by RT-qPCR. The effects of the day of culture and of steroid exposure on mRNA levels were analysed by ANOVA followed by a Tukey test. Cell confluence was observed around Day 6 of culture and more than 90% of cells survived during the 9-day culture period. From Day 3 to Day 9, the proportion of vimentin-positive (fibroblast) cells was greater than 68% in both ampulla and isthmus cells. In contrast, the proportion of epithelial ciliated cells was low at Day 3 (9% in ampulla, 12% in isthmus) and null at Days 6 and 9 in both regions. The mRNA levels of OVGP, ER, and PR decreased significantly after 3 days of culture, and then remained stable in both ampulla and isthmus cells (P &lt; 0.001). The steroid exposure had no effect on gene expression, except for ERα mRNA levels at Day 3, which was increased by E2 and reduced by P4 (P &lt; 0.05). In conclusion, the method of collection did not allow us to collect a high proportion of epithelial oviducal cells. However, the relatively stable gene expression of PR and ER during the culture period provides us with a useful tool to study the steroid regulation of canine oviduct mucosal cell functions.

Caracterização imuno-histoquímica das alterações encefálicas em bovinos com hepatopatia tóxica por Senecio sp

Intoxicação por Senecio spp. é causa importante de doença e morte em bovinos no Rio Grande do Sul e frequentemente cursa com manifestações clínicas neurológicas e alterações histológicas encefálicas. Foi efetuada avaliação histológica em fígado e encéfalo de dez bovinos naturalmente intoxicados por Senecio sp. e foram realizadas as técnicas de histoquímica de ácido periódico de Schiff e de imuno-histoquímica, com emprego dos anticorpos anti-proteína fibrilar ácida (GFAP), anti-proteína S100 e anti-vimentina em amostras de telencéfalo, tronco encefálico e cerebelo. Na histologia do SNC observou-se vacuolização discreta a acentuada em substância branca e/ou junção entre substância branca e cinzenta, caracterizada como degeneração esponjosa. Na avaliação histoquímica e imuno-histoquímica não houve diferenças significativas em relação aos achados em encéfalo de onze bovinos controle, adultos, sem alterações hepáticas e/ou encefálicas, avaliados pelas mesmas técnicas.

120-P: DESIGN OF A SPECIFIC, SENSITIVE ASSAY FOR THE DETECTION OF ANTI-VIMENTIN ANTIBODY USING Luminex® BEAD-BASED TECHNOLOGY

Aim The intermediate filament vimentin is not normally exposed to the immune system, however when exposed, anti-vimentin antibodies (AVA) can be provoked. AVA have been implicated in the development of cardiac allograft vasculopathy (CAV), limiting the long-term benefits of cardiac transplantation. Our aim was to develop a more specific and sensitive assay for the detection of AVA than currently available. Methods A Luminex® multiplexed assay for AVA was designed and validated by parallel testing with a commercially available ELISA for AVA. Sera titration and competitive inhibition with soluble vimentin were used to assess sensitivity and specificity. Pre- and post-transplant sera from forty-six patients were tested under IRB approval. Post-transplant sera were obtained within 12 months after transplantation. Results In parallel titration studies, the bead-based assay was found to be twice as sensitive as the commercially available ELISA. Competitive inhibition studies of five sera resulted in a mean of 60% ± 28% reduction of antibody binding, confirming the Luminex® assay specificity. The incidence of AVA in per-transplant sera was 47.8%.[figure1] Conclusions A Luminex® bead based assay for AVA was developed that is both specific and twice as sensitive as a commercially available ELISA that may have future clinical application. Although a high incidence of AVA among cardiac transplant candidates was observed pre-transplant, longer term studies will be needed to confirm any association with CAV post-transplant.

C111 Differences in the image of urothelium, muscularis mucosae and muscularis propria cells in TURBT specimens stained with anti-smoothelin and anti-vimentin antibodies

C111 Differences in the image of urothelium, muscularis mucosae and muscularis propria cells in TURBT specimens stained with anti-smoothelin and anti-vimentin antibodies

PERITONEAL MESOTHELIOMA IN A JAGUAR (PANTHERA ONCA)

A 21-yr-old female jaguar (Panthera onca) died in a zoo in Teresina, Piaui, Brazil, following a history of abdominal distension, ascites, anorexia, and dyspnea. At necropsy, a dark red, watery, blood-tinged serous fluid was present in the abdominal cavity. The peritoneum was thick with firm, yellow, villous projections. Histologically, the tumors were composed of a biphasic population of cells, which reacted to anti-cytokeratin and anti-vimentin antibodies, consistent with a biphasic benign mesothelioma of peritoneal origin. This is the first reported case of mesothelioma in a captive jaguar.

Vocal Fold Cover Layer with a Tissue‐Engineered Structure Containing Epithelium and Fibroblast of Oral Mucosa

Objectives: A potential treatment option for severe vocal fold scarring is to replace the vocal fold cover layer with a tissue-engineered structure containing autologous cells. The aim of this study is to develop a three-dimensional cell-populated matrix resembling the vocal fold layers of epithelium and lamina propria from autologous oral mucosa. Methods: A 3-by-3-mm specimen of canine buccal mucosa was harvested and divided into epithelium and submucosa. Epithelial cells from the epithelium and fibroblasts from the submucosa were isolated and separately cultured. After the culturing for 2 weeks, the epithelial cells were seeded on mesh type oriented collagen gels containing the fibroblasts throughout a 2-week-culture period. The morphological characteristics of the cultured tissue were observed with immunohistochemical staining and transmission electron microscopy. Normal canine vocal fold and buccal mucosa were used as positive controls. Results: We successfully fabricated a vocal fold cover layer with a tissue-engineered structure closely resembling the normal vocal fold layers of epithelium and lamina propria. The epithelium had three to five cell layers and did not resemble the original oral mucosa. The epithelial cells were immunostained with anti-keratin 3 antibodies, anti-b1 integrin antibodies, and anti-p63 antibodies. The fibroblasts in the collagen gels were immunostained with anti-vimentin antibodies. Electron microscopy revealed developed microvilli on the apical surface of the epithelial cells. Conclusions: The tissue-engineered vocal fold cover layer from autologous oral mucosa has the possibility to reconstruct the vocal fold surface and to restore voice in patients with severe vocal fold scarring.

Vimentin is a target of PKCβ phosphorylation in MCP-1-activated primary human monocytes

We designed a study to detect downstream phosphorylation targets of PKCβ in MCP-1-induced human monocytes. Two-dimensional gel electrophoresis was performed for monocytes treated with MCP-1 in the presence or absence of PKCβ antisense oligodeoxyribonucleotides (AS-ODN) or a PKCβ inhibitor peptide, followed by phospho- and total protein staining. Proteins that stained less intensely with the phospho-stain, when normalized to the total protein stain, in the presence of PKCβ AS-ODN or the PKCβ inhibitor peptide, were sequenced. Of the proteins identified, vimentin was consistently identified using both experimental approaches. Upon (32)P-labeling and vimentin immunoprecipitation, increased phosphorylation of vimentin was observed in MCP-1 treated monocytes as compared to the untreated monocytes. Both PKCβ AS-ODN and the PKCβ inhibitor reduced MCP-1-induced vimentin phosphorylation. The IP of monocytes with anti-vimentin antibody and immunoblotting with a PKCβ antibody revealed that increased PKCβ becomes associated with vimentin upon MCP-1 activation. Upon MCP-1 treatment, monocytes were shown to secrete vimentin and secretion depended on PKCβ expression and activity. We conclude that vimentin, a major intermediate filament protein, is a phosphorylation target of PKCβ in MCP-1-treated monocytes and that PKCβ phosphorylation is essential for vimentin secretion. Our recently published studies have implicated vimentin as a potent stimulator of the innate immune receptor Dectin-1 as reported by Thiagarajan et al. (Cardiovasc Res 99:494-504, 2013). Taken together our findings suggest that inhibition of PKCβ regulates vimentin secretion and, thereby, its interaction with Dectin-1 and downstream stimulation of superoxide anion production. Thus, PKCβ phosphorylation of vimentin likely plays an important role in propagating inflammatory responses.

Streptococcal–vimentin cross-reactive antibodies induce microvascular cardiac endothelial proinflammatory phenotype in rheumatic heart disease

Rheumatic heart disease (RHD) is characterized by the presence of anti-streptococcal group A antibodies and anti-endothelial cell antibodies (AECA). Molecular mimicry between streptococcal antigens and self proteins is a hallmark of the pathogenesis of rheumatic fever. We aimed to identify, in RHD patients, autoantibodies specific to endothelial autoantigens cross-reactive with streptococcal proteins and to evaluate their role in inducing endothelial damage. We used an immunoproteomic approach with endothelial cell-surface membrane proteins in order to identify autoantigens recognized by AECA of 140 RHD patients. Cross-reactivity of purified antibodies with streptococcal proteins was analysed. Homologous peptides recognized by serum cross-reactive antibodies were found through comparing the amino acid sequence of streptococcal antigens with human antigens. To investigate interleukin (IL)-1R-associated kinase (IRAK1) and nuclear factor-κB (NF-κB) activation, we performed a Western blot analysis of whole extracts proteins from unstimulated or stimulated human microvascular cardiac endothelial cells (HMVEC-C). Adhesion molecule expression and release of proinflammatory cytokines and growth factors were studied by multiplex bead based immunoassay kits. We observed anti-vimentin antibodies in sera from 49% RHD AECA-positive patients. Cross-reactivity of purified anti-vimentin antibodies with heat shock protein (HSP)70 and streptopain streptococcal proteins was shown. Comparing the amino acid sequence of streptococcal HSP70 and streptopain with human vimentin, we found two homologous peptides recognized by serum cross-reactive antibodies. These antibodies were able to stimulate HMVEC-C inducing IRAK and NF-κB activation, adhesion molecule expression and release of proinflammatory cytokines and growth factors. In conclusion, streptococcal-vimentin cross-reactive antibodies were able to activate microvascular cardiac endothelium by amplifying the inflammatory response in RHD.