The Elevated Interferon Gamma Production is an Important Immunological Marker in HAM/TSP Pathogenesis

Human T-lymphotropic virus type 1 (HTLV-1) is the agent of the HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), which may Occur in > 5% of patients during their lifetime. HTLV-1-infection causes disturbances in the immune system, and the viral load may also play an important role in the pathogenesis of HAM/TSP. Some cytokines are involved in the pathogenesis of this disorder. We have determined IL-2, IL-4, IL-10, IL-12 p70, IFN-gamma and TNF-alpha production among HTLV-1-infected subjects from our HTLV-out Clinic in Institute of Infectious `Emilio Ribas` in Sao Paulo city, Brazil. PBMC obtained from healthy controls (n = 32), asymptomatic HTLV-1 carriers (n = 68) and HAM/TSP patients (n = 44) were grown in the absence and in the presence of phytohaemagglutinin (PHA), and the supernatants` fluids were measured for cytokines production. IL-2 levels were increased in the a-symptomatic HTLV-1 carriers, and IFN-gamma was increased in both groups of patients (asymptomatic HTLV-1 carriers and more significantly among HAM/TSP patients). IL-4, IL-10, TNF-alpha and IL-12 p70 levels were not significantly increased on both groups of patients, as compared with controls. The major finding Of this Study is that IFN-gamma was an important cytokine for the HAM/TSP pathogenesis. Therefore, immune modulation of IFN-gamma may be critical to treat of HAM/TSP patients.

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.

Fetal Cardiac Troponin T as a Marker of Poor Prognosis in Nonimmune Hydrops Fetalis

Objective: Severe fetal anemia and cardiac compromise are important causes of nonimmune hydrops fetalis, and fetal recovery also depends on the degree of fetal heart compromise. The aim of this study was to report the fetal cardiac troponin T (cTnT) levels in cases of nonimmune fetal hydrops. Methods: Fetal cTnT was analyzed on 7 occasions in 5 cases of nonimmune fetal hydrops ( twice in 2 cases). Results: In 3 of 4 fetuses in which intrauterine death occurred, fetal cTnT levels were increased. The only fetus that survived in this series showed decreased levels of cTnT before birth. Conclusion: Fetal cTnT levels may be a marker of fetal prognosis in cases of fetal hydrops. Copyright (C) 2009 S. Karger AG, Basel

Haze Development After Photorefractive Keratectomy: Mechanical vs Ethanol Epithelial Removal in Rabbits

PURPOSE: To compare mechanical and ethanol epithelial removal with respect to myofibroblast development and haze formation after photorefractive keratectomy (PRK). METHODS: Seventeen rabbits underwent mechanical or ethanol debridement, and the opposite eye of each rabbit served as an unwounded control. In both groups, the epithelium was removed with a spatula and discarded. A -9.00-diopter PRK was performed in each eye. The level of haze in each cornea at 4 weeks was graded at the slit-lamp microscope according to the Fantes scale. Myofibroblast generation was detected with immunocytochemistry for alpha-smooth muscle actin (alpha-SMA) and cells were quantitatively analyzed. RESULTS: No difference was noted between the two groups in alpha-SMA + myofibroblasts 4 weeks after surgery (43.6 +/- 2.0/400X field and 45.7 +/- 4.8/400X field in ethanol and mechanical groups, respectively) (P=.10). A slight difference was noted but did not reach statistical significance with regard to stromal haze between ethanol and mechanical groups (2.0 +/- 0.5 and 2.3 +/- 0.4, respectively, P=.063). The ethanol and mechanical groups were statistically different when compared to controls regarding stromal haze and alpha-SMA+ cells (P <.0001 for all comparisons). CONCLUSIONS:No difference was noted in clinical haze or myofibroblast generation between corneas that had PRK with mechanical,or ethanol epithelial debridement. [J Refract Surg., 2008;24:923-927.]

Relationship of adhesion molecules expression with epithelial differentiation markers during fetal skin development

Background: Cadherins and integrins are important for maintenance of tissue integrity and in signal transduction during skin development. Distribution of these molecules in human skin development was investigated and associated with markers of differentiation, cytokeratins (CK) and involucrin (INV). Methods: Using immunohistochemistry expression of E- and P-cadherins, integrins beta-1 and -4, CK10, CK14 and INV was assessed in skin fragments of 10 human fetuses (gestational weeks ranged from 4 to 24, all weighing up to 500 g). Results: At initial phases of development, integrins beta-1 and -4 and E- and P-cadherins were present on epithelial cell membranes in all layers. CK14 and CK10 were expressed in all epithelial layers and INV weakly detected in the superficial layer. In more advanced stages, integrins were detected in all layers, but a marked polarized expression was seen in basal layer. E-cadherin was detected in all layers, but the cornified stratum and P-cadherin were observed in the lower layers. CK14 was expressed in basal layer, CK10 in suprabasal stratum and INV was observed in cornified layer. Conclusions: Cadherins and integrins are essential for skin development, being spatially and temporally regulated. Their expression is related with the expression of maturation markers of the epidermis.

Topical interleukin-1 receptor antagonist inhibits inflammatory cell infiltration into the cornea

Interleukin (IL)-1 alpha and beta are important modulators of many functions of corneal epithelial and stromal cells that occur following injury to the cornea, including the influx of bone marrow-derived inflammatory cells into the stroma attracted by chemokines released from the stroma and epithelium. In this study, we examined the effect of topical soluble IL-1 receptor antagonist on bone marrow-derived cell influx following corneal epithelial scrape injury in a mouse model. C57BL/6 mice underwent corneal epithelial scrape followed by application of IL-1 receptor antagonist (Amgen, Thousand Oaks, CA) at a concentration of 20 mg/ml or vehicle for 24 h prior to immunocytochemical detection of marker CD11b-positive cells into the stroma. In two experiments, topical IL-1 receptor antagonist had a marked effect in blocking cell influx. For example, in experiment 1, topical IL-1 receptor antagonist markedly reduced detectible CD11b-positive cells into the corneal stroma at 24 It after epithelial injury compared with the vehicle control (3.5 +/- 0.5 (standard error of the mean) cells/400x field and 13.9 +/- 1.2 cells/400x field, respectively, p < 0.01). A second experiment with a different observer performing cell counting had the same result. Thus, the data demonstrate conclusively that topical IL-1 receptor antagonist markedly down-regulates CD-11b-positive monocytic cell appearance in the corneal stroma. Topical IL-1 receptor antagonist could be an effective adjuvant for clinical treatment of corneal conditions in which unwanted inflammation has a role in the pathophysiology of the disorder. (c) 2008 Elsevier Ltd. All rights reserved.

Demonstration of epithelial-mesenchymal transition in kidney - The contribution of a coupled histochemical and immunohistochemical staining with Periodic Acid-Thionin Schiff

Traditional Periodic Acid Schiff has been extensively used, coupled with immunohistochemistry for epithelia or mesenchymal cells, to highlight renal tubular basement membrane (TBM). We recently tried to perform such technique in a 5/6 nephrectomy model of progressive renal fibrosis to demonstrate TBM disruption as an evidence for epithelial-mesenchymal transdifferentiation. Despite excellent basement membrane staining with traditional fuchsin-Periodic Acid Schiff, the interface between epithelial and mesenchymal cells was frequently blurred when revealed with 3`3 diaminobenzidine tetrachloride-peroxidase. Also, it was inadequate when revealed with alkaline phosphatase-fast red. We devised a triple staining method with Periodic Acid-Thionin Schiff to highlight basement membrane in blue, after double immunostaining for epithelium and mesenchymal cells. Blue basement membrane rendered a brisk contrast and highlighted boundaries between epithelial-mesenchymal interfaces. This method was easy to perform and useful to demonstrate the TBM, yield a clear demonstration of the very focal TBM disruption found in this model of progressive renal fibrosis.

Evidence for a network transcriptional control of promiscuous gene expression in medullary thymic epithelial cells

The expression of peripheral tissue antigens (PTAs) in the thymus by medullary thymic epithelial cells (mTECs) is essential for the central self-tolerance in the generation of the T cell repertoire. Due to heterogeneity of autoantigen representation, this phenomenon has been termed promiscuous gene expression (PGE), in which the autoimmune regulator (Aire) gene plays a key role as a transcription factor in part of these genes. Here we used a microarray strategy to access PGE in cultured murine CD80(+) 3.10 mTEC line. Hierarchical clustering of the data allowed observation that PTA genes were differentially expressed being possible to found their respective induced or repressed mRNAs. To further investigate the control of PGE, we tested the hypothesis that genes involved in this phenomenon might also be modulated by transcriptional network. We then reconstructed such network based on the microarray expression data, featuring the guanylate cyclase 2d (Gucy2d) gene as a main node. In such condition, we established 167 positive and negative interactions with downstream PTA genes. Silencing Aire by RNA interference, Gucy2d while down regulated established a larger number (355) of interactions with PTA genes. T- and G-boxes corresponding to AIRE protein binding sites located upstream to ATG codon of Gucy2d supports this effect. These findings provide evidence that Aire plays a role in association with Gucy2d, which is connected to Several PTA genes and establishes a cascade-like transcriptional control of promiscuous gene expression in mTEC cells. (C) 2009 Elsevier Ltd. All rights reserved.

Assessing Individual Interethnic Admixture and Population Substructure Using a 48-Insertion-Deletion (INSEL) Ancestry-Informative Marker (AIM) Panel

Estimating the proportions of different ancestries in admixed populations is very important in population genetics studies, and it is particularly important for detecting population substructure effects in case-control association studies. In this work, a set of 48 ancestry, informative insertion, deletion polymorphisms (INDELs) were selected with the goal of efficiently measuring the proportions of three different ancestries (sub-Saharan African, European, and Native American) in mixed populations. All selected markers can be easily analyzed via multiplex PCR and detected with standard capillary electrophoresis. A total of 593 unrelated individuals representative of European, African, and Native American parental populations were typed, as were 380 individuals from three Brazilian populations with known admixture patterns. As expected, the interethnic admixture estimates show that individuals from southern Brazil present an almost exclusively European ancestry; Afro-descendant communities in the Amazon region, apart from the major African contribution, present some degree of admixture with Europeans and Native Americans; and a sample from Belem, in the northeastern Amazon, shows a significant contribution of the three ethnic groups, although with a greater European proportion. In summary, a panel of ancestry-informative INDELs was optimized and proven to be a variable tool for estimating individual and global ancestry proportions in admixed populations. The ability to accurately infer interethnic admixtures highlights the usefulness of this marker set for assessing population substructure in association studies, particularly those conducted in Brazilian and other Latin American populations sharing trihybrid ancestry patterns. Hum Mutat 31:184-190, 2010. (C) 2009 Wiley-Liss, Inc.

Cigarette smoke exposure impairs respiratory epithelial ciliogenesis

Background: Cigarette smoke exposure is considered an important negative prognostic factor for chronic rhinosinusitis (CRS) patients. However, there is no clear mechanistic evidence implicating cigarette smoke exposure in the poor clinical evolution of the disease or in the maintenance of the inflammatory state characterizing CRS. This study aimed to evaluate the effects of cigarette smoke exposure on respiratory cilia differentiation. Methods: Monse nasal septal epithelium cultures grown at an air-liquid interface were used as a model of respiratory epithelium. After 5 days of cell growth, cultures were exposed to air on the apical surface. Additionally, cigarette smoke condensate (CSC; the particulate phase of tobacco smoke) or cigarette smoke extract (CSE; the volatile phase) Were diluted in the basolateral compartment in different concentrations. After 15 days of continuous exposure, scanning electron microscopy and immunofluorescence for type IV tubulin were used to determine presence and maturation of cilia. Transepithelial resistance was also recorded to evaluate confluence and physiological barrier integrity. Results: CSC and CSE impair ciliogenesis in a dose-dependent manner with notable effects in concentrations higher than 30 mu g/mL, yielding >70% nonciliation and shorter cilia compared With control. No statistical difference on transepithelial resistance was evident. Conclusion: CSC and CSE exposure negatively impacts ciliogenesis of respiratory cells at concentrations not effecting transepithelial resistance. The impairment on ciliogenesis reduce the mucociliary clearance apparatuts after injury and/or infection and may explain the poor response to therapy for CRS patients exposed to tobacco smoke.

The lectin KM+ induces corneal epithelial wound healing in rabbits

Neutrophil influx is essential for corneal regeneration (Gan et al. 1999). KM+, a lectin from Artocarpus integrifolia, induces neutrophil migration (Santos-de-Oliveira et al. 1994). This study aims at investigating a possible effect of KM+ on corneal regeneration in rabbits. A 6,0-mm diameter area of debridement was created on the cornea of both eyes by mechanical scraping. The experimental eyes received drops of KM+ (2.5 mu g/ml) every 2 h, The control eyes received buffer, The epithelial wounded areas of the lectin-treated and untreated eyes were stained with fluorescein, photographed and measured, The animals were killed 12 h (group 1, n = 5), 24 h (group 2, n = 10) and 48 h (group 3, n = 5) after the scraping. The corneas were analysed histologically (haematoxylin and eosin and immunostaining for proliferation cell nuclear antigen, p&3, vascular endothelial growth factor, c-Met and laminin). No significant differences were found at the epithelial gap between treated and control eyes in the group 1. However, the number of neutrophils in the wounded area was significantly higher in treated eyes in this group. Three control and seven treated eyes were healed completely and only rare neutrophils persisted in the corneal stroma in group 2. No morphological distinction was observed between treated and control eyes in group 3. In treated corneas of group 2, there was an increase in immunostaining of factors involved in corneal healing compared to controls, Thus, topical application of KM+ may facilitate corneal epithelial wound healing in rabbits by means of a mechanism that involves increased influx of neutrophils into the wounded area induced by the lectin.

Temporal evolution of epithelial, vascular and interstitial lung injury in an experimental model of idiopathic pulmonary fibrosis induced by butyl-hydroxytoluene

This study was undertaken to test whether the structural remodelling of pulmonary parenchyma can be sequentially altered in a model and method that demonstrate the progression of the disease and result in remodelling within the lungs that is typical of idiopathic pulmonary fibrosis. Three groups of mice were studied: (i) animals that received 3-5-di-tert-butyl-4-hydroxytoluene (BHT) and were killed after 2 weeks (early BHT = 9); (ii) animals that received BHT and were killed after 4 weeks (late BHT = 11); (iii) animals that received corn oil solution (control = 10). The mice were placed in a ventilated Plexiglas chamber with a mixture of pure humidified oxygen and compressed air. Lung histological sections underwent haematoxylin-eosin, immunohistochemistry (epithelial, endothelial and immune cells) and specific staining (collagen/elastic fibres) methods for morphometric analysis. When compared with the control group, early BHT and late BHT groups showed significant decrease of type II pneumocytes, lower vascular density in both and higher endothelial activity. CD4 was increased in late BHT compared with early and control groups, while CD8, macrophage and neutrophil cells were more prominent only in early BHT. The collagenous fibre density were significantly higher only in late BHT, whereas elastic fibre content in late BHT was lower than that in control group. We conclude that the BHT experimental model is pathologically very similar to human usual interstitial pneumonia. This feature is important in the identification of animal models of idiopathic pulmonary fibrosis that can accurately reflect the pathogenesis and progression of the human disease.

Intramammary infusion of leptin decreases proliferation of mammary epithelial cells in prepubertal heifers

High energy intake and excessive body fatness impair mammogenesis in prepubertal ruminants. High energy intake and excessive fatness also increase serum leptin. Our objective was to determine if an infusion of leptin decreases proliferation of mammary epithelial cells of prepubertal heifers in vivo. Ovine leptin at 100 mu g/quarter per d with or without 10 mu g of insulin-like growth factor (IGF)-I was infused via the teat canal into mammary glands of prepubertal dairy heifers; contralateral quarters were used as controls. After 7 d of treatment, bromodeoxyuridine was infused intravenously and heifers were slaughtered similar to 2 h later. Tissue from 3 regions of the mammary parenchyma was collected and immunostained for bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (Ki-67), and caspase-3. Leptin decreased the number of mammary epithelial cells in the S-phase of the cell cycle by 48% in IGF-I-treated quarters and by 19% in saline-treated quarters. Leptin did not alter the number of mammary epithelial cells within the cell cycle, as indicated by Ki-67 labeling. Caspase-3 immunostaining within the mammary parenchyma was very low in these heifers, but leptin significantly increased labeling in saline-treated quarters. Leptin enhanced SOCS-3 expression in IGF-I-treated quarters but did not alter SOCS-1 or SOCS-5 expression. We conclude that a high concentration of leptin in the bovine mammary gland reduces proliferation of mammary epithelial cells. The reduced proliferation is accompanied by an increase in SOCS-3 expression, suggesting a possible mechanism for leptin inhibition of IGF-I action. Whether leptin might be a physiological regulator of mammogenesis remains to be determined.

Insulin Affects Tissue Organization and the Kinetics of Epithelial Cell Death in the Rat Ventral Prostate After Castration

Prostate growth and physiology are regulated by steroid hormones and modulated by multiple endocrine factors We investigated the action of insulin on the tissue organization and kinetics of epithelial cells in the rat ventral prostate (VP) in response to castration up to 120 hours after surgery by using an acute protocol of alloxan induced diabetes Diabetes caused a reduction in volume density (Vv(o)/) and volume of the epithelium The effects of castration on the epithelium were accelerated in the diabetic animals as determined by changes in V(o)/, and volume The smooth muscle cells became atrophic and apparently relaxed in response to castration in contrast to the spinous aspect observed in nondiabetic castrated rats Counting of apoptotic nuclei in the epithelium showed the classical apoptosis peak at 72 hours in nondiabetic rats and an advance of the apoptosis peak to 48 hours after castration in diabetic rats Insulin restored the time of the peak to 72 hours These results were confirmed after immunostaining for cleaved caspase 3 and suggest a survival and antiapoptotic effect on VP epithelial cells in both the presence and absence of androgen stimulation This idea is supported by the observation that insulin also reduced the overall rate of apoptosis at all experimental points analyzed before and after castration