Anti-vimentin Monoclonal Antibody Research Articles

Cell Surface Vimentin Detection in Cancer Cells by Peptide-Based Monoclonal Antibody

Vimentin is a prominent Intermediate Filaments (IFs) protein expressed in different mesenchymal origin cell types. Besides a wide range of cellular function roles associated with vimentin expression, its dysregulation and cell surface expression in the induction of malignancy properties have been reported extensively, making it a promising cancer-specific target. Therefore, this study aimed to generate and characterize anti-vimentin monoclonal antibodies. A 14-mer synthetic peptide from vimentin was conjugated to Keyhole Limpet Hemocyanin (KLH) and used for immunization of Blab/C mice and monoclonal production by conventional hybridoma technology. The monoclonal antibody was purified using affinity chromatography of supernatants from the selected hybridoma cells. ELISA, Immunoprecipitation-Western blotting (IP-WB), Immunocytochemistry (ICC), and flow cytometry were employed to characterize the produced monoclonal antibody in terms of interaction with vimentin immunizing peptide as well as vimentin protein. Amid the several obtained producing anti-vimentin antibody hybridomas, the 7C11-D9 clone (IgG1 isotype with kappa light chain) showed higher reactivity with the immunizing peptide, and led to its selection for purification and characterization. The purified antibody could detect vimentin protein in IP-WB, ICC and flow cytometry of the normal and cancerous cells with different origin. No vimentin expression was found in normal healthy Peripheral Blood Mononuclear Cell (PBMC). Taken together, 7C11-D9 anti-vimentin monoclonal antibody might be used as immune diagnostic or immune therapeutic tool where detection or targeting of vimentin in a wide range of organisms is required.

Double-Blind, Randomized, Placebo-Controlled Study on hzVSF-v13, a Novel Anti-Vimentin Monoclonal Antibody Drug as Add-on Standard of Care in the Management of Patients with Moderate to Severe COVID-19.

Humanized Virus Suppressing Factor-variant 13 (hzVSF-v13), a monoclonal IgG4 antibody against vimentin, was investigated in moderate to severe COVID-19 pneumonia through a Phase II study. Patients were randomized to two different IV doses of the test drug or saline with standard of care. Overall, 64 patients were recruited, and 62 entered the efficacy assessment in the full analysis set. Primary endpoint: The clinical failure rate at day 28 was 15.8% for placebo, 9.1% for low-dose hzVSF-v13 and 9.5% for high-dose hzVSF-v13 (not significant). A trend toward better efficacy was shown in several secondary endpoints, with statistical significance between low-dose hzVSF-v13 and placebo in terms of the rate of improved patients on the ordinal scale for clinical improvement (OSCI): 90.0% vs. 52.63% (p = 0.0116). In the severe stratum, the results of low-dose hzVSF-v13 vs. placebo were 90.0% and 22.2% for OSCI (p = 0.0092), 9 days and 14 days for time to discontinuation of oxygen therapy (p = 0.0308), 10 days and 15 days for both time to clinical improvement (TTCI) and time to recovery (TTR) and p = 0.0446 for both TTCI and TTR. Change from baseline of NEWS2 score at day 28 was −3.4 vs. + 0.4 (p = 0.0441). The results propose hzVSF-v13 as a candidate in the treatment of severe COVID-19.

Immunocytochemical evaluation of epithelial-mesenchymal transition in epithelial tumors of dogs and cats.

Epithelial–mesenchymal transition (EMT) plays a crucial role in metastasis of epithelial tumors; however, it is challenging to detect EMT by cytology. In the present study, EMT was visualized by fluorescence-immunocytochemistry (FICC). Air-dried smears from epithelial tumors of dogs (n=22) and cats (n=9) were stained using mouse monoclonal anti-E-cadherin and rabbit monoclonal anti‐vimentin antibodies. Enzymatic immunohistochemistry (IHC) revealed that 51.6% (8/22 in dogs, 8/9 in cats) of the cases showed EMT. In dogs, FICC could detect EMT in 62.5% (5/8) of those cases. In cats, FICC could detect EMT in 100% (8/8) of the cases. In conclusion, the present FICC method could successfully detect EMT using conventional air-dried cytology smear slides.

Displasia renal em cães: estudo retrospectivo (2008-2013)

Background: Renal dysplasia, which has been reported in some dogs and human patients, refers to a developmental disorder of renal parenchyma due to imperfect inductive interaction between the mesonephric duct and the metanephric blastemal. In dogs, the characteristic histological findings on which diagnosis is based include (1) persistent metanephric ducts surrounded by primitive mesenchyme, (2) fetal or immature glomeruli, (3) fetal or immature tubules, and (4) anomalous presence of interstitial fibrous tissue. The aim of this study was to report the major pathological and immunohistochemical features of nine young dogs necropsied with renal dysplasia.Cases: The necropsy files from the Laboratório de Patologia Veterinária (LPV) of the Universidade Federal do Mato Grosso (UFMT) were reviewed between the years 2008 and 2013. Dogs diagnosed with chronic kidney failure and macroscopic and histopathological renal lesions consistent with renal dysplasia were selected. Kidney fragments in paraffin blocks were cut and stained with hematoxylin and eosin and by immunohistochemistry (IHC) using anti-vimentin and anti-cytokeratin monoclonal antibodies. The staining was considered positive for the presence of at least one renal cell marked with brown cytoplasmic staining clear and unambiguous. A total of 787 necropsies of dogs were performed. Of these, 64 had a clinical diagnosis of chronic renal failure of which 9 were classified as renal dysplasia. The age of the dogs ranged from 3 months to 2 years. Clinical signs were characterized by anorexia and non-regenerative anemia in 88.9%, vomiting 66.7%, dehydration 55.6%, uremia 55.6%, convulsion 33.4%, abdominal pain 22.3% and diarrhea in 11.2% of cases. At necropsy the main macroscopic changes in the kidneys were external surface with pale staining, in 6 of 9 dogs necropsied. Additionally, 5 dogs, cystic cavities of various sizes from 0.1 to 5 cm in diameter, diffusely distributed in the renal subcapsular surface and cutting were observed. Of the 64 dogs with diagnosis of chronic renal failure, 14.06% had dysplastic kidney changes, characterized by dilatation of Bowman’s space, glomerular and tubular atrophy, immature glomeruli and tubules, lymphocytic interstitial inflammation and fibrosis. In the dogs with renal dysplasia, it was observed that the tubular structures showed marked glomerular labeling for vimentin. Moreover, the kidneys of normal dogs showed weak or absent for marking tubes and glomerular structures for vimentin, and strong staining for cytokeratin in tubular cells, glomerular cells and collecting ducts.Discussion: Renal morphological damage observed in these nine dogs less than two years old, that contained degenerative and inflammatory changes, fibrosis and glomerular atrophy, but mostly immature glomeruli and tubules were associated with atypical renal dysplasia. This condition develops when the urethral diverticulum and metanephric blastoma not properly form in the embryonic stage, resulting in abnormal metanephric differentiation and formation of structures that do not recapitulate the normal nephrogenesis. Abnormal kidney function in young animals caused chronic renal failure resulting in death. The results of immunohistochemical observed in this study can complement the diagnosis of renal dysplasia. The morphological normality can be observed in the proportion of mesenchymal and epithelial tissue in the different structures of the kidney by staining with anti-cytokeratin and anti-vimentin. In the present study, there was moderate to strong labeling of vimentin in glomerular and tubular structures being dysplastic kidney would be expected to occur in normal dogs predominant staining with cytokeratin. Based on the clinic, pathological and immunohistochemical findings it is concluded that the animals developed renal dysplasia.

Chi-Ju-Di-Huang-Wan protects rats against retinal ischemia by downregulating matrix metalloproteinase-9 and inhibiting p38 mitogen-activated protein kinase.

BackgroundRetinal ischemia is a retinal disorder related to retinal vascular occlusion, glaucoma, diabetic retinopathy and age-related macular degeneration. The study aimed to evaluate the protective effects and underlying mechanisms of Chi-Ju-Di-Huang-Wan (CJDHW) against retinal ischemia in rats.MethodsHigh intraocular pressure (HIOP)-induced retinal ischemia was established in Wistar rats by raising their intraocular pressure to 120 mmHg for 60 min with in an eye whose anterior chamber was cannulated with a 30-guage needle adapted to a normal saline bottle through an intravenous line. This ischemic insult was followed by 1 or 7 days of reperfusion. The effects of CJDHW were studied by (i) electroretinogram (ERG); (ii) real-time polymerase chain reaction to determine the retinal mRNA levels of Thy-1 and matrix metalloproteinase-9 (MMP-9); (iii) Western blot analysis to determine the retinal protein levels of B cell lymphoma 2 (Bcl-2), heme oxygenase-1 (HO-1), phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK) and MMP-9; (iv) hematoxylin and eosin (HE) staining; (v) fluorogold retrograde labeling; and (vi) terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) apoptosis assay. Moreover, after fixation with 4 % paraformaldehyde and 30 % sucrose, the isolated retinas were sectioned and immunolabeled with goat anti-choline acetyltransferase (ChAT) polyclonal antibody, mouse anti-vimentin monoclonal antibody and rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody. The retinal sections were then incubated with rhodamine-conjugated rabbit anti-goat antibody, fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG or FITC-conjugated goat anti-rabbit IgG. A daily oral intake of 3 mL of water (vehicle; Group 2) or CJDHW (2.8 or 4.2 g/kg/day; CJDHW2.8 or CJDHW4.2; Group 3 or 4) was given for 7 consecutive days either before (preischemic drug administration) or after HIOP-induced retinal ischemic injury (postischemic drug administration). In Group 5, an intravitreal injection of 4 μL of 0.5 mM SB203580 (p38 MAPK inhibitor) was performed on the ischemic eye 15 min before retinal ischemia. The control rats received a sham procedure (Group 1) where the saline reservoir was not raised.ResultsThe ischemia-induced changes (Group 2) were significantly modulated by pretreating the rats with 4.2 g/kg/day of CJDHW (Group 4; ERG: P < 0.001 on I/R day 7; HE stain: P < 0.001 on I/R day 7; TUNEL: P = 0.05 on I/R day 7; retrograde labeling: P = 0.007 on I/R day 7; Thy-1 mRNA: P = 0.02; MMP-9 mRNA: P < 0.001; Bcl-2 protein: P = 0.02; HO-1 protein: P = 0.03; P-p38 MAPK protein: P < 0.001; MMP-9 protein: P = 0.02). These modulations included the following features (Group 2 vs. 4), increased ERG b-wave amplitudes (0.38 ± 0.04 vs. 0.81 ± 0.03), increased inner retinal thickness (45.08 ± 2.85 vs. 67.98 ± 5.48 μm), increased ChAT immunolabeling, decreased vimentin/GFAP immunoreactivity, less numerous apoptotic cells in the ganglion cell layer (1.40 ± 0.55 vs. 0.60 ± 0.55), and more numerous retinal ganglion cells (887.73 ± 158.18 vs. 1389.02 ± 53.20). Moreover, increased Thy-1 (0.31 ± 0.15 vs. 0.78 ± 0.32) and decreased MMP-9 mRNA levels were found (4.44 ± 0.84 vs. 1.13 ± 0.34), respectively. Furthermore, the Bcl-2 protein level (0.78 ± 0.08 vs. 1.80 ± 0.34) was increased while the HO-1 (0.99 ± 0.20 vs. 4.15 ± 2.08), P-p38 MAPK (1.12 ± 0.18 vs. 0.57 ± 0.18) and MMP-9 levels were decreased (0.70 ± 0.23 vs. 0.39 ± 0.10). The ischemia-associated increases in P-p38 and MMP-9 protein levels were also attenuated by 0.5 mM SB203580 (P-p38 MAPK: 1.12 ± 0.18 vs. 0.18 ± 0.07, P < 0.001; MMP-9: 0.70 ± 0.23 vs. 0.21 ± 0.07, P = 0.002). This was also the case to the MMP_enzyme activity (Group 2 vs. 4: 5.03 ± 1.57 vs. 1.59 ± 0.47, P = 0.002; Group 2 vs. 5: 5.03 ± 1.57 vs. 1.35 ± 0.41, P = 0.001).ConclusionTreatment of the rats suffering from retinal ischemia with CJDHW inhibited apoptosis, increased antioxidative activity, downregulated MMP-9 and inhibited p38 MAPK.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0109-6) contains supplementary material, which is available to authorized users.

Nasal Transmissible Venereal Tumor In A Jack Russel Terrier Bitch

Abstract A two-year old Jack Russell Terrier female with left-sided epistaxis was brought to the Clinic of the Bucharest Faculty of Veterinary Medicine. Endoscopy of the left nasal cavity revealed a cauliflower-like mass. Cytological and histopathological features were specific to canine transmissible venereal tumour (CTVT). Tumor tissue showed positive immunoreactivity to anti-vimentin monoclonal antibody (90%) and Ki67 (35%) and samples were negative for anti-cytokeratin monoclonal antibody. Chemotherapy with vincristine sulphate at a dose of 0.025 mg/kg i.v./week for 6 administrations was successful. This paper presents a primary form of nasal CTVT in a Jack Russell Terrier female.

EVALUACIÓN DE UNA TÉCNICA INMUNOHISTOQUÍMICA PARA EL DIAGNÓSTICO DE NEOPLASIAS MAMARIAS MIXTAS EN CANINOS

The aim of the study was to evaluate an immunohistochemical technique for the diagnosis of canine mixed mammary tumors (CMMT) using anti-cytokeratin AE1/AE3 and anti-vimentin monoclonal antibodies that are routinely used in the diagnosis of human mammary tumors. Twenty paraffin tissue blocks of canine mixed mammary tumors, previously diagnosed by hematoxilin-eosin (HE) histopathology were used. Three slides sets, 4 μm thick, were obtained per viability of the tissue, and the other two were used for the immunohistochemistry through the Streptavidin-Biotin-labeled method. Epithelial cells positively reacted against the anti-cytokeratin AE1/AE3 showing a brown colour at the cytoplasma level. Fibroblasts, connective tissue, myoepithelial cells, cartilage cells and bone areas showed a brown colour against the anti-vimentin antibody. All benign tumors reacted positively to both antibodies, whereas 73% malign tumors were positive with the anti-cytokeratin antibody. It is concluded that mixed mammary neoplasm in dogs may be diagnosed by the technique of immunohistochemistry using monoclonal antibodies used in the detection of human mammary tumors. block. One was stained with HE to confirm the

Vimentin Exposed On Platelets Serves As an Adhesive Receptor for Von Willebrand Factor

Abstract 259Platelet adhesion, activation, and aggregation in the vasculature are necessary events in both life-saving hemostasis and pathological thrombosis. Thrombosis may occur in patients presenting with several clinical conditions including atherosclerosis, cardiovascular disease, and inflammation. Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that plays a critical role in mediating platelet adhesion, activation, and aggregation on the exposed subendothelium in order to maintain hemostasis under arterial flow conditions. On the other hand, VWF permits the stabilization of platelets adherent to components of ruptured atherosclerotic plaques, leading to artery-occluding thrombus formation. The initial interaction of activated or hyperadhesive VWF with platelets occurs via the interaction between the A1 domain of VWF and the platelet receptor glycoprotein (GP)Ibα. This engagement is responsible for reducing the velocity of rapidly flowing platelets, allowing the rolling platelets to interact with the second binding site on VWF for the platelet receptor GPIIb/IIIa; a binding site that is located within the C domains of VWF. Therefore, the hyperadhesive property of VWF apparently relies on the synchronized interaction of the two platelet surface receptors, GPIbα and GPIIb/IIIa. Despite this concept, we and others have speculated that other binding site in VWF synergistically works with the A1 domain to quickly capture the extremely fast flowing platelets. We have obtained interesting results from studies using a monomeric A1A2A3 domain protein that lacks the binding site for GPIIb/IIIa. For example, the rolling velocity of platelets over an A1A2A3-coated surface was markedly lower than that seen with use of the single A1 domain. This observation suggests the possibility of an additional binding site in the A domains for platelets. Given the similar hyperadhesive features of the A1A2A3 protein and plasma VWF, we proposed to look for a potential receptor on platelets with a recognition site within the A domains of VWF. We suggested examining vimentin because, it was identified as a binding protein for the isolated A2 domain of VWF in our laboratory, and vimentin has been found on the surface of platelets. First, both full length VWF and recombinant A1A2A3 proteins efficiently bound to human vimentin only in the presence of the modulator ristocetin, indicating that vimentin preferably interact with the active conformation of VWF. In fact, a constitutively active A1A2A3 protein (containing a gain-of-function mutation in A1 domain) had a binding activity for vimentin higher than that of wild type (WT) A1A2A3 in the absence of ristocetin. Second, anti-vimentin monoclonal antibody blocked the interaction of that mutant A1A2A3 to activated washed platelets using flow cytometry. Third, we then examined the effect of anti-vimentin antibody on flow-dependent platelet adhesion to A1A2A3-coated surface at high shear stress. In comparison to whole blood incubated with irrelevant IgG molecule as a negative control, the anti-vimentin antibody blocked 75% platelet adhesion to the triple-A domain protein. Finally, whole blood from vimentin-deficient or WT mice was perfused over a surface coated with murine VWF at high shear rate. In comparison to platelets from WT mice, vimentin-deficient platelets had a significant reduced platelet adhesion to VWF (25% of WT). Similarly, vimentin-deficient platelets had a reduced platelet adhesion to collagen (20% of WT) under high flow conditions. This platelet-collagen interaction is initially mediated by VWF. These interesting results indicate that vimentin on platelets serves as a receptor for VWF, and this binding may participate in the initial interaction of circulating platelets with VWF under flow conditions. Disclosures:No relevant conflicts of interest to declare.

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Aim In a RTR with rising SCr, autoantibody was identified to HLA DR52 (MFI = 2382). As the patient received an HLA chromosome-identical transplant, HLA autoantibody was thought to be highly unlikely. Anti-vimentin autoantibodies have been associated with inflammation and rejection. Our hypothesis was that the RTR was synthesizing anti-vimentin antibody, detected as HLA DR52. We postulated that vimentin could be co-purified and coupled to Luminex beads, or that a shared epitope might be present on the HLA proteins. Therefore, if anti-vimentin antibody was tested against single antigen Luminex beads, certain beads would be positive, corresponding to HLA DR52 and any specificities where vimentin was co-purified or with a shared epitope. Methods Anti-vimentin murine monoclonal antibody PE-conjugate was reacted to One Lambda single antigen Luminex beads. The reaction pattern and MFIs were compared to those of the RTR serum. The anti-vimentin monoclonal was used in the standard protocol for HLA specificity analysis in the place of the anti-IgG antibody (omitting the serum incubation). A duplicate test was done using a 1 hour incubation of the anti-vimentin antibody with the beads to enhance the PE signal. Results The RTR serum had multiple non-auto-HLA Class I antibodies, which corresponded to the pretransplant sera specificities. The RTR serum showed antibodies to DR52 (MFI = 2382) and DP11 (MFI = 535) as the only HLA Class II specificities. The donor/recipient DP typing was unknown. The anti-vimentin monoclonal had no reactivity to HLA Class I but had HLA Class II reactivity to DR52 (MFI = 1073) and DP11 (MFI = 772). The RTR has been plasmapheresed X6, with a reduction in SCr from 2.2 to 1.5 mg/dL and the disappearance of the DR52 and DP11 specificities. Conclusions We conclude that our RTR had vimentin autoantibody associated with ongoing rejection. This caused the false identification by Luminex of HLA DR52 and DP11 specificities, either through the presence of vimentin or a shared epitope on the beads.

Characterization of Primary Cultures of Cholesteatoma-Associated Fibroblasts

The aim of this study was to characterize primary cultured fibroblasts derived from cholesteatoma (CHO) tissue to get evidence of their possible role for determining the different biological behavior of this aural pathology. Primary cultures of human fibroblasts were obtained from middle ear CHO specimens and from controls of normal human skin collected during surgical procedures. Cells were incubated with anti-vimentin monoclonal antibody, anti-cytokeratins monoclonal antibody and anti-α-smooth muscle actin monoclonal antibody. For reactive oxygen species detection, fibroblasts were incubated with 2',7'-dichlorofluorescein diacetate. Immunofluorescence, flow cytometry, confocal, and transmission electron microscopy were carried out. Cholesteatoma-associated fibroblasts (CHO-AFs) were characterized by a higher degree of cytoplasmic complexity, by the expression of α-smooth muscle actin and by a greater basal production of reactive oxygen species in comparison with controls, reflecting a more differentiated phenotype consistent with myofibroblasts. It is possible to suggest that the differentiated phenotype of CHO-AFs might be coupled with a more or less aggressive clinical behavior, and hence, these cultures might represent powerful tools for investigating biology and clinical evolution of this disease.

Cartilage Destruction in Granulomatosis with Polyangiitis (Wegener's Granulomatosis) Is Mediated by Human Fibroblasts after Transplantation into Immunodeficient Mice

A key feature of granulomatosis with polyangiitis (GPA; or Wegener's granulomatosis) is the granulomatous inflammation of the upper respiratory tract, which leads to the subsequent destruction of adjacent tissues. The aim of our work was to study the histopathological and cellular components of tissue destruction of human GPA tissue transplanted into immunodeficient mice. Biopsy specimens from patients with active GPA (n = 10) or sinusitis (controls, n = 6) were s.c. co-implanted with healthy allogeneic human nasal cartilage into immunodeficient pfp/rag2(-/-) mice. Transplants were examined for their destructive capability of the allografted human cartilage. In addition, nasal fibroblasts from patients with GPA (n = 8) and control healthy nasal fibroblasts (n = 5) were cultured, and cell proliferation and apoptosis were quantified. mRNA and protein levels of matrix metalloproteinases and cytokines were evaluated at baseline and after proinflammatory stimulation. GPA implants showed massive destruction of the co-implanted human cartilage, whereas cartilage destruction was only marginal in control samples. Destruction was mediated by human fibroblasts and could be inhibited by corticoid treatment. The up-regulated production of matrix metalloproteinases 1, 3, and 13 and cytokines IL-6 and IL-8 was found in vivo and in vitro. Although proliferation of isolated fibroblasts was comparable between GPA and controls, GPA samples showed a significant delay of apoptosis. The destruction of nasal cartilage in GPA is mainly mediated by fibroblasts that can be blocked by corticosteroids, and this tissue destruction is not dependent on the influx of leukocytes.

Japanese encephalitis virus interacts with vimentin to facilitate its entry into porcine kidney cell line

Japanese encephalitis virus (JEV) requires the presence of an inexplicable cellular receptor on the surface of the host cell for its entry into the cell. The JEV envelope (E) protein has been shown to play an important role in attachment to cells. By using a widely accepted technique, virus overlay protein binding assay (VOPBA), a protein molecule of approximately 60kDa, identified as vimentin by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF), was recognized on porcine kidney (PS) cells as a possible receptor for JEV. Further, anti-vimentin monoclonal antibodies were able to block JEV entry into the PS cells. Additionally, co-immunoprecipitation assay confirmed that vimentin protein present on the PS cells interacts with the JEV-E protein. These observations indicate that vimentin serves as a putative receptor for JEV in porcine kidney cells.

Overexpression of nerve growth factor in peritoneal fluid from women with endometriosis may promote neurite outgrowth in endometriotic lesions

To investigate the role of the nerve growth factor (NGF) in the endometriosis-associated innervation in the development of endometriosis-associated symptoms, 41 peritoneal fluid samples (PF) from patients with surgically and histologically proven endometriosis and 20 PF from patients with other gynecologic conditions were analyzed with Western blot and a novel in vitro model using dorsal root ganglia (DRG) to show neuronal outgrowth; endometrial cells also were analyzed. The results suggest that the PF of endometriosis patients and endometriotic lesions have neurotropic properties, because the Western blot analysis and the cell culture stainings showed NGF expression, and the neurite outgrowth of DRG treated with PF of patients with endometriosis was significantly higher than when treated with PF of patients without endometriosis. Furthermore, blocking NGF with both anti-NGF and K252a leads to a significant decrease in neurite outgrowth.

Distribution of Vimentin-Expressing Cells in Pterygium: An Immunocytochemical Study of Impression Cytology Specimens

To investigate the distribution of vimentin-expressing cells in human pterygium by immunocytochemical analysis of impression cytology specimens. Using impression cytology, 3 samples of superficial conjunctival epithelial cells, including pterygium and adjacent conjunctiva were obtained from each of 24 eyes of 24 patients with unilateral primary pterygium, 16 eyes of 16 patients with unilateral recurrent pterygium, and the 40 unaffected fellow eyes. Specimens were processed in avidin-biotin complex immunostaining system with monoclonal anti-vimentin antibody and/or anti-CD1a antibody and counterstained with hematoxylin. Under light microscopy, the extent of vimentin-expressing cell infiltration beyond the pterygium margin was measured with a micrometer and the number of vimentin-expressing cells was counted in each of 6 randomly selected high-power fields in the specimens of pterygium and normal conjunctiva. Two types of cells expressed vimentin, epithelioid cells and dendritic cells, whereas cells that expressed CD1a showed only a dendritic pattern. Vimentin-expressing round epithelioid cells (presumed pterygial cells) were present over the entire surface of the pterygium and also on adjacent normal-appearing conjunctiva, up to an average of 3.01 +/- 0.83 mm beyond the superior margin and 3.14 +/- 0.99 mm beyond the inferior margin of primary pterygium and 3.72 +/- 0.85 mm and 3.58 +/- 0.98 mm beyond the superior and inferior margins of recurrent pterygium. For the superior margin but not the inferior margin, the clinically occult extension of vimentin-expressing epithelioid cells was statistically significantly greater for eyes with recurrent pterygium compared with those with primary pterygium (P = 0.016). The density of dendritic (Langerhans) cells, stained by anti-vimentin and anti-CD1a antibodies, was statistically significantly lower in normal fellow eyes than in eyes with either primary (P < 0.001) or recurrent (P = 0.024) pterygium. The vimentin-expressing epithelioid cells were present not only over the ocular surface of the pterygium but also in the normal-appearing conjunctiva adjacent to primary and recurrent pterygium. The increased density of Langerhans cells in pterygium might reflect a higher level of antigenic and mitogenic exposure in the conjunctiva. However, the significance of these 2 phenomena in the recurrence and pathogenesis of pterygium remains undetermined and merits further studies.

Distribution of vimentin in abnormal human spermatozoa.

Vimentin was immunocytochemically detected using a monoclonal anti-vimentin antibody and indirect immunogold electron microscopy. In abnormal human spermatozoa, vimentin was visualized at the level of whole acrosome, post-acrosomal region, neck and initial segment of the mid piece. The distribution of vimentin immunoreactivity was related to a spectrum of structural defects such as large cytoplasmic droplets, binucleated spermatozoa and mitochondrial disassembly in the mid piece.

Dynamic Expression of Vimentin in Bovine Blastocysts in ExtendedIn VitroCulture

In mammalian blastocysts, the inner cell mass (ICM) differentiates into the epiblast and the hypoblast. In vitro culture of bovine pre-implantation embryos is generally carried out for a limited duration at most up to the blastocyst formation, therefore, it is unclear whether and when the differentiation of ICM occurs in vitro. In bovine species, epiblast formation is characterized by the expression of the intermediate filament protein vimentin. In the present study, the spatial and temporal expression of vimentin in bovine blastocysts maintained under extended in vitro culture (EIVC) was investigated. Bovine blastocysts produced by in vitro maturation, fertilization, and culture were further cultured in vitro in modified synthetic oviduct fluid up to day 12 (fertilization = day 0). Early, expanded, or hatched blastocysts on day 8 and hatched blastocysts on days 9 and 12 were individually subjected to indirect immunofluorescence analysis with an anti-vimentin monoclonal antibody and to semi-quantitative reverse transcription-polymerase chain reaction analysis to examine the vimentin expression. Most of the blastocysts (25/26) did not exhibit any significant immunostaining for the vimentin protein on day 8. On the other hand, the vimentin protein was observed in 59.1% (13/22) and 86.4% (19/22) of the hatched blastocysts on days 9 and 12, respectively. Vimentin was detected as filaments localized within a portion of the ICM, and its expression levels varied among the embryos. Vimentin transcript could not be detected in 50% (3/6) of the blastocysts on day 8, whereas it was detected in all the blastocysts on days 9 and 12. Examination of the day 8 blastocysts revealed that compared with the slow developers, the fast developers (blastocysts which had already expanded by 168 h post-insemination) had significantly higher levels of vimentin transcripts. These results indicate that the ICM differentiates dynamically in bovine blastocysts maintained under EIVC, as reflected by the expression of vimentin and that the higher vimentin expression may reflect the higher developmental competence of embryos.

Preventing peridural fibrosis with nonsteroidal anti-inflammatory drugs

Peridural fibrosis is one of the more frequent complications of lumbar surgery. Nonsteroidal anti-inflammatory drugs inhibit the inflammatory and fibroblastic response. We performed lumbar laminectomies in 24 rabbits, divided into two groups. The experimental group received 5 mg/kg/day of aceclofenac for 7 days and the control group received 1 cm(3) of physiological saline. The samples were stained using immunohistochemical methods. The cellular populations in the inflammatory reaction and the thickness of the fibrous membrane were quantified. The mean of the fibrous area was always less in the rabbits of the experimental group compared to controls (47% less at 2 weeks and 41% less at 4 weeks). We observed an 8% decrease in the number of fibroblasts with antivimentin monoclonal antibodies in the experimental group. In this model, aceclofenac inhibits the presence of inflammatory cells in the fibrous scar in the early stages and reduces the extension of adhesions without adverse reactions.

CKAP4/p63 Is a Receptor for the Frizzled-8 Protein-related Antiproliferative Factor from Interstitial Cystitis Patients

Antiproliferative factor (APF) is a low molecular weight sialoglycopeptide that is secreted by bladder cells from interstitial cystitis patients and is a potent inhibitor of both normal bladder epithelial and bladder carcinoma cell proliferation. We hypothesized that APF may produce its antiproliferative effects by binding to a transmembrane receptor. This study demonstrates that cytoskeleton-associated protein 4/p63 (CKAP4/p63), a type II transmembrane receptor, binds with high affinity to APF. The antiproliferative activity of APF is effectively inhibited by preincubation with anti-CKAP4/p63-specific antibodies, as well as by short interfering RNA knockdown of CKAP4/p63. Immunofluorescent confocal microscopy showed co-localization of anti-CKAP4/p63 and rhodamine-labeled synthetic APF binding in both cell membrane and perinuclear areas. APF also inhibits the proliferation of HeLa cervical carcinoma cells that are known to express CKAP4/p63. These data indicate that CKAP4/p63 is an important epithelial cell receptor for APF.

Proteomic Analysis of Cellular Response to Osmotic Stress in Thick Ascending Limb of Henle’s Loop (TALH) Cells

Epithelial cells of the thick ascending limb of Henle's loop (TALH cells) play a major role in the urinary concentrating mechanism. They are normally exposed to variable and often very high osmotic stress, which is particularly due to high sodium and chloride reabsorption and very low water permeability of the luminal membrane. It is already established that elevation of the activity of aldose reductase and hence an increase in intracellular sorbitol are indispensable for the osmotic adaptation and stability of the TALH cells. To identify new molecular factors potentially associated with the osmotic stress-resistant phenotype in kidney cells, TALH cells exhibiting low or high levels of resistance to osmotic stress were characterized using proteomic tools. Two-dimensional gel analysis showed a total number of 40 proteins that were differentially expressed in TALH cells under osmotic stress. Twenty-five proteins were overexpressed, whereas 15 proteins showed a down-regulation. Besides the sorbitol pathway enzyme aldose reductase, whose expression was 15 times increased, many other metabolic enzymes like glutathione S-transferase, malate dehydrogenase, lactate dehydrogenase, alpha enolase, glyceraldehyde-3-phosphate dehydrogenase, and triose-phosphate isomerase were up-regulated. Among the cytoskeleton proteins and cytoskeleton-associated proteins vimentin, cytokeratin, tropomyosin 4, and annexins I, II, and V were up-regulated, whereas tubulin and tropomyosins 1, 2, and 3 were down-regulated. The heat shock proteins alpha-crystallin chain B, HSP70, and HSP90 were found to be overexpressed. In contrast to the results in oxidative stress the endoplasmic reticulum stress proteins like glucose-regulated proteins (GRP78, GRP94, and GRP96), calreticulin, and protein-disulfide isomerase were down-regulated under hypertonic stress.

SW13 Cells Can Transition between Two Distinct Subtypes by Switching Expression of BRG1 andBrm Genes at the Post-transcriptional Level

The human adrenal carcinoma cell line, SW13, has been reported to be deficient in both BRG1 and Brm expression and therefore is considered to lack a functional SWI/SNF complex. We found that the original cell line of SW13 is composed of two subtypes, one that expresses neither BRG1 nor Brm (SW13(vim-)) and the another, which does express both (SW13(vim+)). The presence of BRG1 and Brm in SW13 correlates completely with the cellular ability to express such genes as vimentin, collagenase, c-met, and CD44 that were under the control of a transcription factor, AP-1, which was shown previously to require a functional SWI/SNF complex for its transactivating activity. Transient treatment with inhibitors of histone deacetylase induced a stable transition of SW13(vim-) to a cell type indistinguishable from SW13(vim+), suggesting that these two subtypes are epigenetically different. Run-on analysis indicated that, unlike these four genes driven by AP-1, transcription of the BRG1 and Brm genes in SW13(vim-) are initiated at a frequency comparable with SW13(vim+). In both SW13(vim-) and SW13(vim+) cells, the BRG1 and Brm genes were transcribed through the entire gene at a similar efficiency, indicating that their expression was completely suppressed at the post-transcriptional level in SW13(vim-) cells. We would like to propose that SW13 can spontaneously transition between two subtypes by switching expression of BRG1 and Brm at the post-transcriptional level.