Vimentin expression and the influence of Matrigel in cell lines of head and neck squamous cell carcinoma

Vimentin is a cytoeskeletal intermediate filament protein commonly observed in mesenchymal cells; however, it can also be found in malignant epithelial cells. It is demonstrated in several carcinomas, such as those of the cervix, breast and bladder, in which it is widely used as a marker of the epithelial to mesenchymal transition that takes place during embryogenesis and metastasis. Vimentin is associated with tumors that show a high degree of invasiveness, being detected in invasion front cells. Its expression seems to be influenced by the tumor microenvironment. The aim of this study was to evaluate vimentin expression in head and neck squamous cell carcinoma (HNSCC) cell lines, and to investigate the contribution of the microenvironment to its expression. HNSCC cell lines (HN6, HN30 and HN31) and an immortalized nontumorigenic cell line (HaCaT) were submitted to a three-dimensional assay with Matrigel. Cytoplasmatic staining of the HN6 cell line cultured without Matrigel and of the HN30 and HN31 cell lines cultured with Matrigel was demonstrated through immunohistochemistry. Western Blotting revealed a significant decrease in vimentin expression for the HN6 cell line and a significant increase for the HN30 and HN31 cell lines cultured with Matrigel. The results suggest that vimentin can be expressed in HNSCC cells and its presence is influenced by the microenvironment of a tumor.

Reprint of ""Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury""

Previous studies have suggested that abnormal corneal wound healing in patients after photorefractive keratectomy (PRK) is associated with the appearance of myofibroblasts in the stroma between two and four weeks after surgery. The purpose of this study was to examine potential myofibroblast progenitor cells that might express other filament markers prior to completion of the differentiation pathway that yields alpha-smooth muscle actin (SMA)-expressing myofibroblasts associated with haze localized beneath the epithelial basement membrane after PRK. Twenty-four female rabbits that had -9 diopter PRK were sacrificed at 1 week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at -80 degrees C, and analyzed by immunocytochemistry using anti-vimentin, anti-desmin, and anti-SMA antibodies. Double immunostaining was performed for the co-localization of SMA with vimentin or desmin with SMA. An increase in vimentin expression in stromal cells is noted as early as 1 week after PRK in the rabbit cornea. As the healing response continues at two or three weeks after surgery, many stromal cells expressing vimentin also begin to express desmin and SMA. By 4 weeks after the surgery most, if not all, myofibroblasts express vimentin, desmin and SMA. Generalized least squares regression analysis showed that there was strong evidence that each of the marker groups differed in expression over time compared to the other two (p < 0.01). Intermediate filaments - vimentin and desmin co-exist in myofibroblasts along with SMA and may play an important role in corneal remodeling after photorefractive keratectomy. The earliest precursors of myofibroblasts destined to express SMA and desmin are detectible by staining for vimentin at 1 week after surgery. (C) 2009 Elsevier Ltd. All rights reserved.

Dynamics of the expression of intermediate filaments vimentin and desmin during myofibroblast differentiation after corneal injury

Previous studies have suggested that abnormal corneal wound healing in patients after photorefractive keratectomy (PRK) is associated with the appearance of myofibroblasts in the stroma between two and four weeks after surgery. The purpose of this study was to examine potential myofibroblast progenitor cells that might express other filament markers prior to completion of the differentiation pathway that yields a-smooth muscle actin (SMA)-expressing myofibroblasts associated with haze localized beneath the epithelial basement membrane after PRK. Twenty-four female rabbits that had -9 diopter PRK were sacrificed at I week, 2 weeks, 3 weeks or 4 weeks after surgery. Corneal rims were collected, frozen at -80 degrees C, and analyzed by immunocytochemistry using anti-vimentin, anti-desmin, and anti-SMA antibodies. Double immunostaining was performed for the co-localization of SMA with vimentin or desmin with SMA. An increase in vimentin expression in stromal cells is noted as early as 1 week after PRK in the rabbit cornea. As the healing response continues at two or three weeks after surgery, many stromal cells expressing vimentin also begin to express desmin and SMA. By 4 weeks after the surgery most, if not all, myofibroblasts express vimentin, desmin and SMA. Generalized least squares regression analysis showed that there was strong evidence that each of the marker groups differed in expression over time compared to the other two (p < 0.01). Intermediate filaments - vimentin and desmin co-exist in myofibroblasts along with SMA and may play an important role in corneal remodeling after photorefractive keratectomy. The earliest precursors of myofibroblasts destined to express SMA and desmin are detectible by staining for vimentin at I week after surgery. (C) 2009 Elsevier Ltd. All rights reserved.

Vimentin Expression and Myofibroblast Infiltration Are Early Markers of Renal Dysfunction in Kidney Transplantation: An Early Stage of Chronic Allograft Dysfunction?

Introduction. The objective of this study was to show the morphologic characteristics of allograft renal biopsies in renal transplant patients with stable renal function, which can potentially be early markers of allograft dysfunction, after 5 years of follow-up. Methods. Forty-nine renal transplant patients with stable renal function were submitted to renal biopsies and simultaneous measurement of serum creatinine (Cr). Histology was evaluated using Banff scores, determination of interstitial fibrosis by Sirius red staining and immunohistochemical study of proximal tubule and interstitial compartment (using cytokeratin, vimentin, and myofibroblasts as markers). Biopsies were evaluated according to the presence or absence of the epitheliomesenchymal transition (EMT). The interstitial presence of myofibroblasts and tubular presence of vimentin was also analyzed simultaneously. Renal function was measured over the follow-up period to estimate the reduction of graft function. Results. Median posttransplant time at enrollment was 105 days. Patients were followed for 64.3 +/- 8.5 months. The mean Cr at biopsy time was 1.44 +/- 0.33 mg/dL, and after the follow-up it was 1.29 +/- 0.27 mg/dL. Nine patients (19%) had a reduction of their graft function. Eleven biopsies (22%) had tubulointerstitial alterations according to Banff score. Seventeen biopsies (34%) presented EMT. Fifteen biopsies (32%) had high interstitial expression of myofibroblasts and tubular vimentin. Using Cox multivariate analysis, HLA and high expression of interstitial myofibroblasts and tubular vimentin were associated with reduction of graft function, yielding a risk of 3.3 (P = .033) and 9.8 (P = .015), respectively. Conclusion. Fibrogenesis mechanisms occur very early after transplantation and are risk factors for long-term renal function deterioration.

PDIA3, HSPA5 and viementin, proteins identified by 2-DE in the valvular tissue, are the target antigens of peripheral and heart infiltrating T cells from chronic rheumatic heart disease patients

Rheumatic fever (RF) is a post-infectious autoimmune disease due to sequel of group A streptococcus (GAS) pharyngitis. Rheumatic heart disease (RHD), the major manifestation of RF, is characterized by inflammation of heart valves and myocardium. Molecular mimicry between GAS antigens and host proteins has been shown at B and T cell level. However the identification of the autoantigens recognized by B and T cells within the inflammatory microenvironment of heart tissue in patients with RHD is still incompletely elucidated. In the present study, we used two-dimensional gel electrophoresis (2-DE) and mass spectrometry to identify valvular tissue proteins target of T cells from chronic RHD patients. We could identify three proteins recognized by heart infiltrating and peripheral T cells as protein disulfide isomerase ER-60 precursor (PDIA3), 78 kD glucose-regulated protein precursor (HSPA5) and vimentin, with coverage of 45%, 43 and 34%, respectively. These proteins were recognized in a proliferation assay by peripheral and heart infiltrating T cells from RHD patients suggesting that they may be involved in the autoimmune reactions that leads to valve damage. We also observed that several other proteins isolated by 2-DE but not identified by mass spectrometry were also recognized by T cells. The identified cardiac proteins are likely relevant antigens involved in T cell-mediated autoimmune responses in RF/RHD that may contribute to the development of RHD

A perivascular epithelioid cell tumor of the stomach: An unsuspected diagnosis

Perivascular epithelioid cell tumor (PEComa) is a rare mesenchymal neoplasia and currently well recognized as a distinct entity with characteristic morphological, immunohistochemical and molecular findings. We report a case of PEComa arising in the antrum of a 71-year-old female with melena. The tumor, located predominantly in the submucosa as a well delimited nodule, measured 3.0 cm in diameter and was completely resected, with no evidence of the disease elsewhere. Histologically, it was composed predominantly of eosinophilic epithelioid cells arranged in small nests commonly related to variably sized vessels, with abundant extracellular material, moderate nuclear variation and discrete mitotic activity. No necrosis, angiolymphatic invasion or perineural infiltration was seen. Tumor cells were uniformly positive for vimentin, smooth muscle actin, desmin and melan A. Although unusual, PEComa should be considered in the differential diagnosis of gastric neoplasia with characteristic epithelioid and oncocytic features and prominent vasculature: (C) 2010 Baishideng. All rights reserved.

Mesenchymal progenitor cells from canine fetal tissues: yolk sac, liver, and bone marrow

During fetal development, mesenchymal progenitor (MP) cells are co-localized in major hematopoietic territories, such as yolk sac (YS), bone marrow (BM), liver (LV), and others. Studies using mouse and human MP cells isolated from fetus have shown that these cells are very similar but not identical to adult mesenchymal stem cells (MSC). Their differentiation potential is usually restricted to production of highly committed osteogenic and chondrogenic precursors. Such properties of fetal MP cells can be very useful for tissue regeneration, when a great number of committed precursors are required. The objectives of this study were to isolate and characterize MP cells from canine YS, BM, and LV in early and late stages of fetal development. Gestational stage was identified, and cell culture conditions were evaluated for efficient isolation of canine MP cells. All canine fetal MP cells expressed vimentin, nestin, and CD44 proteins. Cytokeratin 18 expression was observed in BM-and LV-MP cells, and vascular endothelial (VE)-cadherin expression was observed only in YS-MP cells. A small number of MP cells (5%) from LV and YS expressed Oct3/4 protein. The differentiation potential of canine fetal MP cells varied significantly: YS- and BM-MP cells differentiated into bone and cartilage, whereas LV-MP cells differentiation was limited to osteogenic fate. None of the canine fetal MP cells were able to differentiate into adipose cells. Our data suggest that canine fetal MP cells are an appropriate in vitro model to study MP biology from hematopoietic territories and they are a source of committed osteogenic and chondrogenic precursors for regenerative medicine.

Placentation in Sigmodontinae: a rodent taxon native to South America

Background: Sigmodontinae, known as ""New World rats and mice,"" is a large subfamily of Cricetidae for which we herein provide the first comprehensive investigation of the placenta. Methods: Placentas of various gestational ages ranging from early pregnancy to near term were obtained for five genera, i.e. Necromys, Euryoryzomys, Cerradomys, Hylaeamys, and Oligoryzomys. They were investigated by means of histology, immunohistochemistry, a proliferation marker, DBA-lectin staining and transmission electron microscopy. Results: The chorioallantoic placenta was organized in a labyrinthine zone, spongy zone and decidua and an inverted yolk sac persisted until term. The chorioallantoic placenta was hemotrichorial. The interhemal barrier comprised fetal capillary endothelium and three layers of trophoblast, an outermost, cellular layer and two syncytial ones, with interspersed trophoblast giant cells (TGC). In addition, accumulations of TGC occurred below Reichert's membrane. The junctional zone contained syncytial trophoblast, proliferative cellular trophoblast, glycogen cells and TGC that were situated near to the maternal blood channels. In three of the genera, TGC were also accumulated in distinct areas at the placental periphery. PAS-positive glycogen cells derived from the junctional zone invaded the decidua. Abundant maternal uNK cells with positive response to PAS, vimentin and DBA-lectin were found in the decidua. The visceral yolk sac was completely inverted and villous. Conclusion: The general aspect of the fetal membranes in Sigmodontinae resembled that found in other cricetid rodents. Compared to murid rodents there were larger numbers of giant cells and in some genera these were seen to congregate at the periphery of the placental disk. Glycogen cells were found to invade the decidua but we did not identify trophoblast in the walls of the deeper decidual arteries. In contrast these vessels were surrounded by large numbers of uNK cells. This survey of wild-trapped specimens from five genera is a useful starting point for the study of placentation in an important subfamily of South American rodents. We note, however, that some of these rodents can be captive bred and recommend that future studies focus on the study of time dated pregnancies.

Chorioallantoic placentation in Galea spixii (Rodentia, Caviomorpha, Caviidae)

Background: Placentas of guinea pig-related rodents are appropriate animal models for human placentation because of their striking similarities to those of humans. To optimize the pool of potential models in this context, it is essential to identify the occurrence of characters in close relatives. Methods: In this study we first analyzed chorioallantoic placentation in the prea, Galea spixii, as one of the guinea pig's closest relatives. Material was collected from a breeding group at the University of Mossoro, Brazil, including 18 individuals covering an ontogenetic sequence from initial pregnancy to term. Placentas were investigated by means of histology, electron microscopy, immunohistochemistry (vimentin, alpha-smooth muscle actin, cytokeration) and proliferation activity (PCNA). Results: Placentation in Galea is primarily characterized by an apparent regionalization into labyrinth, trophospongium and subplacenta. It also has associated growing processes with clusters of proliferating trophoblast cells at the placental margin, internally directed projections and a second centre of proliferation in the labyrinth. Finally, the subplacenta, which is temporarily supplied in parallel by the maternal and fetal blood systems, served as the center of origin for trophoblast invasion. Conclusion: Placentation in Galea reveals major parallels to the guinea pig and other caviomorphs with respect to the regionalization of the placenta, the associated growing processes, as well as trophoblast invasion. A principal difference compared to the guinea pig occurred in the blood supply of the subplacenta. Characteristics of the invasion and expanding processes indicate that Galea may serve as an additional animal model that is much smaller than the guinea pig and where the subplacenta partly has access to both maternal and fetal blood systems.

Rheumatic Fever and Rheumatic Heart Disease: Cellular Mechanisms Leading Autoimmune Reactivity and Disease

Rheumatic fever (RF) is an autoimmune disease caused by the gram-positive bacteria Streptococcus pyogenes that follows a nontreated throat infection in susceptible children. The disease manifests as polyarthritis, carditis, chorea, erythema marginatum, and/or subcutaneous nodules. Carditis, the most serious complication, occurs in 30% to 45% of RF patients and leads to chronic rheumatic heart disease (RHD), which is characterized by progressive and permanent valvular lesions. In this review, we will focus on the genes that confer susceptibility for developing the disease, as well as the innate and adaptive immune responses against S. pyogenes during the acute rheumatic fever episode that leads to RHD autoimmune reactions. The disease is genetically determined, and some human leukocyte antigen class II alleles are involved with susceptibility. Other single nucleotide polymorphisms for TNF-alpha and mannan-binding lectin genes were reported as associated with RF/RHD. T cells play an important role in RHD heart lesions. Several autoantigens were already identified, including cardiac myosin epitopes, vimentin, and other intracellular proteins. In the heart tissue, antigen-driven oligoclonal T cell expansions were probably the effectors of the rheumatic heart lesions. These cells are CD4(+) and produced inflammatory cytokines (TNF alpha and IFN gamma). Molecular mimicry is the mechanism that mediated the cross-reactions between streptococcal antigens and human proteins. The elucidation of chemokines and their receptors involved with the recruitment of Th1, Th2, and Th17 cells, as well as the function of T regulatory cells in situ will certainly contribute to the delineation of the real picture of the heart lesion process that leads to RHD.

Expression of smooth muscle and extracellular matrix proteins in relation to airway function in asthma

Background: Smooth muscle content is increased within the airway wall in patients with asthma and is likely to play a role in airway hyperresponsiveness. However, smooth muscle cells express several contractile and structural proteins, and each of these proteins may influence airway function distinctly. Objective: We examined the expression of contractile and structural proteins of smooth muscle cells, as well as extracellular matrix proteins, in bronchial biopsies of patients with asthma, and related these to lung function, airway hyperresponsiveness, and responses to deep inspiration. Methods: Thirteen patients with asthma (mild persistent, atopic, nonsmoking) participated in this cross-sectional study. FEV1 % predicted, PC20 methacholine, and resistance of the respiratory system by the forced oscillation technique during tidal breathing and deep breath were measured. Within 1 week, a bronchoscopy was performed to obtain 6 bronchial biopsies that were immunuhistochemically stained for alpha-SM-actin, desmin, myosin light chain kinase (MLCK), myosin, calponin, vimentin, elastin, type III collagen, and fibronectin. The level of expression was determined by automated densitometry. Results: PC20 methacholine was inversely related to the expression of alpha-smooth muscle actin (r = -0.62), desmin (r = -0.56), and elastin (r = -0.78). In addition, FEV1% predicted was positively related and deep inspiration-induced bronchodilation inversely related to desmin (r = -0.60), MLCK (r = -0.60), and calponin (r = -0.54) expression. Conclusion: Airway hyperresponsiveness, FEV1% predicted, and airway responses to deep inspiration are associated with selective expression of airway smooth muscle proteins and components of the extracellular matrix.

Autoantibodies as predictors of biological therapy for early rheumatoid arthritis

Introduction: The association between serological markers with the need of biological therapy for early rheumatoid arthritis (ERA) is not known, with few available data addressing this question. Objectives: To prospectively evaluate a cohort of patients with ERA (less than 12 months of symptoms) in order to determine the possible association between serological markers (rheumatoid factor (RF), anti-cyclic citrullinated peptide antibodies (anti-CCP), and citrullinated anti-vimentin (anti-Sa) with parameters of therapeutic outcome (this later defined by the need of introducing biological therapy). Patients and methods: Forty patients with early RA were evaluated at the time of diagnosis and have been followed for 3 years, in use of standardized therapeutic treatment. Demographic and clinical data were recorded, as well as serology tests (ELISA) for RF (IgM, IgG and IgA), anti-CCP (CCP2, CCP3 and CCP3.1) and anti-Sa in the initial evaluation and at 3, 6, 12, 18, 24 and 36 months of follow-up. As outcomes of the RA development, the need or not for biological therapy during the follow-up period were considered. Comparisons were made through the Student t test, mixed-effects regression analysis and analysis of variance (significance level of 5%). Results: The mean age was 45 (+/- 12) years; a female predominance was observed (90%). At the time of diagnosis, RF was observed in 50% of cases (RF IgA - 42%, RF IgG - 30% and RF IgM - 50%), anti-CCP in 50% (no difference between CCP2, CCP3 and CCP3. 1) and anti-Sa in 10%. After 3 years, no change in the RF prevalence neither in the anti-CCP was observed, but the anti-Sa increased to 17.5% (p = 0.001). Biological therapy was necessary in 22.5% of patients. The mean RF IgA and anti-CCP 2 levels during the 3 years were higher among patients who needed biological therapy (p <0.05 for both). Conclusion: Higher titles of RF and anti-CCP over time were associated with the need for biological therapy.

Estrogen receptor-positive breast carcinomas in younger women are different from those of older women: A pathological and immunohistochemical study

The higher frequency of triple-negative and HER-2-positive tumors detected in younger patients has been suggested as an explanation for the more aggressive tumor types observed in this age group. However, estrogen receptor (ER)-positive tumors are the most frequent subtype of breast carcinomas identified, even in younger patients. In this retrospective study, the morphological and immunohistochemical profiles of ER-positive breast carcinomas from women 35 yrs and younger that were diagnosed between 1997 and 2007 were evaluated. From these cases, 213 were selected based on the availability of pathology reports and paraffin blocks. For comparison, 117 consecutive cases of breast carcinomas diagnosed in patients >60 yrs from 2006 were included. Paraffin-embedded tumors were stained for expression of ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2). Ki-67 antigen, epidermal growth factor receptor (EGFR), cytokeratin 5/6, p53, vimentin, CD117, and p63 using tissue microarrays. ER-positive carcinomas were diagnosed in 120 (56.1%) samples of the younger patient group and in 92 (78.6%) samples of the older patient group. Of these ER-positive carcinomas, 48 (40%) from the younger patient group presented the subtype luminal A, compared with 53 (57.6%) from the older patient group (p=0.01). Tumors from the younger patient group were also associated with increased vascular involvement, co-expression of HER-2, and decreased expression of CD117. These results highlight differences in expression markers and the pathology of ER-positive tumors detected in younger women, with a notable characteristic being co-expression of HER-2. (C) 2010 Elsevier Ltd. All rights reserved.

Alpha-tocopherol prevents intrauterine undernutrition-induced oligonephronia in rats

The role of alpha-tocopherol during nephrogenesis was investigated in rats subjected to maternal undernutrition, which reduces the number of nephrons. alpha-tocopherol (350 mg/kg, p.o.) was administered daily to well-nourished or malnourished Wistar dams during pregnancy, or to prenatal undernourished rats during lactation. The kidneys of 1- and 25-day-old offspring were removed to evaluate expression of angiotensin II (Ang II) and to correlate this with expression of proliferating cell nuclear antigen, alpha-smooth muscle actin, fibronectin and vimentin in the glomeruli and tubulointerstitial space. One-day-old prenatally undernourished rats had reduced expression of Ang II and of kidney development markers, and presented with an enlarged nephrogenic zone. Maternal administration of alpha-tocopherol restored the features of normal kidney development in undernourished rats. Twenty-five-day-old prenatally undernourished progeny had fewer glomeruli than the control group. Conversely, animals from mothers that received alpha-tocopherol during lactation presented with the same number of glomeruli and the same glomerular morphometrical profile as the control group. Analyzing the levels of thiobarbituric acid reactive substances in the liver in conjunction with kidney development markers, it is plausible that alpha-tocopherol had antioxidant and non-antioxidant actions. This study provides evidence that alpha-tocopherol treatment restored Ang II expression, and subsequently restored renal structural development.

Macrophage Depletion Attenuates Chronic Cyclosporine A Nephrotoxicity

Background. Cyclosporine A (CsA)-induced chronic nephrotoxicity is characterized by renal dysfunction and interstitial fibrosis. Early and progressive renal macrophage influx, correlating with latter interstitial fibrotic areas, has been associated with CsA treatment. This study investigated the role of macrophages, the nitric oxide (NO) pathway, and the oxidative stress on chronic CsA nephrotoxicity. Methods. The macrophages were depleted by clodronate liposomes. Animals were distributed into four groups: vehicle (olive oil for 21 days), CsA 7.5 mg/kg per day (21 days), CsA plus clodronate (5 mg/mL intraperitoneally on days -4, 1, 4, 11, and 18 of CsA treatment), or vehicle plus clodronate. On day 22, glomerular filtration rate, renal blood flow, renal tubulointerstitial fibrosis, CsA blood levels, serum malondialdehyde and renal tissue immunohistochemistry for macrophages, inducible NO synthase, transforming growth factor-beta, nuclear factor-k beta, alpha-smooth muscle actin, vimentin, and nitrotyrosine were assessed. Results. CsA-induced increase in the macrophage was prevented by clodronate. Macrophage depletion attenuated the reductions in the glomerular filtration rate and renal blood flow, the development of tubulointerstitial fibrosis, malondialdehyde increase and increases in nuclear factor-k beta, transforming growth factor-beta, vimentin, inducible NO synthase, and nitrotyrosine expression provoked by CsA. Clodronate did not affect alpha-smooth muscle actin expression and CsA blood levels. Conclusions. Renal macrophage influx plays an important role in CsA-induced chronic nephrotoxicity. The NO pathway and oxidative stress are likely mechanisms involved in the genesis of this form of renal injury.