Study of direct immunofluorescence, immunofluorescence mapping and light microscopy in porphyria cutanea tarda

BACKGROUND: Even though porphyria cutanea tarda is the most frequent type of porphyria, there are few studies about its cutaneous physiopathology. OBJECTIVE: To evaluate skin changes in porphyria cutanea tarda using light microscopy and direct immunofluorescence before and after treatment with chloroquine. To perform antigen immunomapping of bullae to study their level of cleavage. METHODS: Light microscopy and direct immunofluorescence of 28 patients are reported in three different phases: 23 patients with active porphyria before treatment (Phase A), 7 patients with clinical remission during treatment (Phase B), and 8 patients with biochemical remission (Phase C). Immunomapping was performed on 7 patients. RESULTS: In active porphyria, direct immunofluorescence showed homogenous and intense fluorescence on the inside and on the walls of blood vessels as well as in the dermal-epidermal junction. In clinical remission (Phase B) and biochemical remission (Phase C), the deposit of immunoglobulins was maintained, but the deposit of complement was reduced in most cases. Immunomapping revealed no standard cleavage plane. CONCLUSION: No correlation was observed between clinical response and immunoglobulin deposits. The reduction of complement favors the hypothesis that activation of the complement cascade represents an additional pathway that leads to endothelial damage.

Aerobic training reverses airway inflammation and remodelling in an asthma murine model

Aerobic training (AT) decreases dyspnoea and exercise-induced bronchospasm, and improves aerobic capacity and quality of life; however, the mechanisms for such benefits remain poorly understood. The aim of the present study was to evaluate the AT effects in a chronic model of allergic lung inflammation in mice after the establishment of airway inflammation and remodelling. Mice were divided into the control group, AT group, ovalbumin (OVA) group or OVA+AT group and exposed to saline or OVA. AT was started on day 28 for 60 min five times per week for 4 weeks. Respiratory mechanics, specific immunoglobulin (Ig)E and IgG(1), collagen and elastic fibres deposition, smooth muscle thickness, epithelial mucus, and peribronchial density of eosinophils, CD3+ and CD4+, IL-4, IL-5, IL-13, interferon-gamma, IL-2, IL-1ra, IL-10, nuclear factor (NF)-kappa B and Foxp3 were evaluated. The OVA group showed an increase in IgE and IgG1, eosinophils, CD3+, CD4+, IL-4, IL-5, IL-13, NF-kappa B, collagen and elastic, mucus synthesis, smooth muscle thickness and lung tissue resistance and elastance. The OVA+AT group demonstrated an increase of IgE and IgG(1), and reduction of eosinophils, CD3+, CD4+, IL-4, IL-5, IL-13, NF-kappa B, airway remodelling, mucus synthesis, smooth muscle thickness and tissue resistance and elastance compared with the OVA roup (p < 0.05). The OVA+AT group also showed an increase in IL-10 and IL-1ra (p < 0.05), independently of Foxp3. AT reversed airway inflammation and remodelling and T-helper cell 2 response, and improved respiratory mechanics. These results seem to occur due to an increase in the expression of IL-10 and IL-1ra and a decrease of NF-kappa B.

Improvement of Insulin Resistance and Reduction of Cardiovascular Risk Among Obese Patients with Type 2 Diabetes with the Duodenojejunal Bypass Liner

This study aims to evaluate the effectiveness of the duodenojejunal bypass liner (DJBL) in the improvement of insulin resistance and reduction of cardiovascular risk among morbidly obese patients with type 2 diabetes mellitus, using the triglyceride/high-density lipoprotein (HDL) cholesterol ratio, percentage of weight loss, and glycemic control. We used the TG/HDL ratio with a cutoff value of 3.5 to identify patients with insulin resistance. The value of the initial ratio was compared with the ratio obtained 6 months after implantation to evaluate whether an improvement in insulin resistance occurred. We also evaluated the improvement of glycated hemoglobin levels and the weight loss resulted from the use of the device and correlated that with the improvement of the TG/HDL ratio. All patients implanted with the device presented a statistically significant reduction of the HbA1c levels, with most patients (70.3%) obtaining diabetes control with HbA1c levels lower than 7% at the end of the study. All patients also presented a significant weight reduction, with an average loss of 12.6% of their initial weight. We observed an important improvement in insulin resistance and metabolic syndrome, with a significant reduction of the TG/HDL ratio from 5.75 to 4.36 (p < 0.001) and 42.6% of the patients presenting a TG/HDL ratio lower than 3.5 at the end of the study. The DJBL, when used for a period of 6 months, is effective in the control of diabetes, weight loss, improvement of insulin resistance, and decrease of cardiovascular risk among morbidly obese patients with type 2 diabetes mellitus.

Influence of Suture on Peripheral Nerve Regeneration and Collagen Production at the Site of Neurorrhaphy: An Experimental Study

BACKGROUND: Restoration of nerve continuity and effective maintenance of coaptation are considered fundamental principles of end-to-end peripheral nerve repair. OBJECTIVE: To evaluate the influence of the number of stitches on axonal regeneration and collagen production after neurorrhaphy. METHODS: Thirty male Wistar rats were equally divided into 3 groups and were all operated on with the right sciatic nerve exposed. In 2 groups, the nerve was sectioned and repaired by means of 3 (group B) or 6 (group C) epineurium sutures with 100 monofilament nylon. One group (group A) was used as a control. Each animal from groups B and C underwent electrophysiological evaluation with motor action potential recordings before nerve section and again at an 8-week interval after neurorrhaphy. Nerve biopsy specimens were used for histomorphometric assessment of axonal regeneration and quantification of collagen at the repair site. RESULTS: Animals from group C had significantly lower motor action potential conduction velocities compared with control animals (P = .02), and no significant difference was seen between groups B and C. Parameters obtained from morphometric evaluation were not significantly different between these 2 groups. Type I collagen and III collagen in the epineurium were significantly higher in group C than in either the control group (P = .001 and P = .003) or group B (P = .01 and P = .02). No differences were identified for collagen I and III in the endoneurium. CONCLUSION: Using 6 sutures for nerve repair is associated with worse electrophysiological outcomes and higher amounts of type I and III collagen in the epineurium compared with control. Neurorraphy with 6 stitches is also related to a significant increase in epineurium collagen I and III compared with 3-stitch neurorraphy.

Additive Intraocular Pressure Reduction Effect of Fixed Combination of Maleate Timolol 0.5%/Dorzolamide 2% (Cosopt) on Monotherapy With Latanoprost (Xalatan) in Patients With Elevated Intraocular Pressure A Prospective, 4-week, Open-label, Randomized, Controlled Clinical Trial

Purpose: To evaluate the additive effect of dorzolamide/timolol fixed combination in patients under monotherapy with latanoprost. Patients and Methods: In this prospective, 4-week, randomized, open-label controlled clinical trial, patients with open-angle glaucoma or ocular hypertension, which presented at least 15% intraocular pressure (IOP) reduction after a minimum period of 15 days of monotherapy with latanoprost and whose IOP level was considered above the established target-IOP level were randomized to receive fixed combination of timolol/dorzolamide twice daily in one of eyes. The fellow eye was kept under monotherapy and was included in the control group. A modified diurnal tension curve (mDTC) followed by the water drinking test were performed in the baseline and week 4 visits to evaluate IOP profile between groups. Results: Forty-nine per-protocol patients were analyzed. After latanoprost monotherapy run-in period, IOP levels were significantly reduced (P<0.001) in both control and study groups to 15.34 +/- 2.96 mm Hg and 15.24 +/- 2.84 mm Hg (30.8% and 32.2% IOP reduction, respectively; P=0.552). At week 4, mean baseline diurnal IOP levels were 15.60 +/- 3.09 and 14.44 +/- 3.03 (7.4% difference; P=0.01). Mean baseline IOP modified diurnal tension curve peak after latanoprost run-in period were 17.47 +/- 3.68 mm Hg and 17.02 +/- 3.35 mm Hg (control and study eyes, respectively; P=0.530). At week 4 visit, mean water-drinking test peaks were significantly reduced in the study eye group in comparison with the control group: 19.02 +/- 3.81 mm Hg and 20.39 +/- 4.19 mm Hg, respectively (6.7% reduction; P=0.039). Conclusions: In our sample, dorzolamide 2%/timolol 0.5% fixed combination as add-on therapy in patients with open-angle glaucoma or ocular hypertension under monotherapy with latanoprost with IOP already in mid-teens levels may further enhance pressure reduction.

Collagen V and vascular injury promote lung architectural changes in systemic sclerosis

Background: Systemic sclerosis (SSc) is a multisystem disorder characterized by inflammation, fibrosis and vascular damage. The aim of this study was to evaluate the interactions between basement membrane disruption, endothelial injury and collagen V deposition on the vascular wall, as well as their association with pulmonary function tests in patients with SSc. Method: The endothelial apoptosis was assessed by TUNEL and electron microscopy, and quantified through the point-counting technique. To evaluate basement membrane integrity, laminin immunostaining and electron microscopy were used. Immunofluorescence and morphometric analysis were used to determine the amount of collagen V in the vascular walls in 23 open lung biopsies of patients with SSc without pulmonary hypertension. Normal lung tissue was obtained from five individuals who had died of traumatic injuries. Results: The apoptosis index in SSc was higher in the endothelial cells (13.83 +/- 6.83) when compared with the control (2.51 +/- 2.06) group (P < 0.001) and confirmed by electron microscopy. We observed an important disruption of the basement membrane on the vascular wall shown by discontinuous laminin immunostaining and electron microscopy. An increase in collagen V on the vascular wall of the SSc group was observed (45.28 +/- 13.21), when compared with control group (22.90 +/- 4.13, P < 0.001), and this difference was statistically significant. An inverse correlation was found between vital capacity, forced vital capacity, forced expiratory volume in 1 s, vascular collagen V and endothelial apoptosis (P < 0.05). Conclusions: We conclude that the endothelial apoptosis and vascular collagen V interaction reinforce the vascular pathway in the SSc pathogenesis. Further studies are needed to determine whether this relationship is causal or consequential. Please cite this paper as: Parra ER, Aguiar AC Jr, Teodoro WR, de Souza R, Yoshinari NH and Capelozzi VL. Collagen V and vascular injury promote lung architectural changes in systemic sclerosis. The Clinical Respiratory Journal 2009; 3: 135-142.

Collagen Type I may Influence the Expression of E-Cadherin and Beta-catenin in Carcinoma Ex-pleomorphic Adenoma

Carcinoma ex-pleomorphic adenoma (CXPA) is an aggressive salivary gland malignancy, usually derived from a long-standing or a recurrent benign tumor, the pleomorphic adenoma (PA). In the context of dynamic reciprocity, changes in the composition and structure of extracellular matrix proteins and cell surface receptors have been frequently associated with dysfunctional adhesion and invasive behavior of tumor cells. It is not fully understood if these changes are involved in the conversion of PA to CXPA. In this study, different progression stages of CXPA were investigated regarding the expression of the major extracellular matrix proteins, collagen type I, and of E-cadherin and beta-catenin, the components of adherens junctions. By immunohistochemical analysis, we have demonstrated that direct contact of tumor cells with fibrillar type I collagen, particularly near the invasive front and in invasive areas prevailing small nests of CXPA cells, could be associated with reduced expression of the E-cadherin and beta-catenin adhesion molecules and with invasive behavior of epithelial; but not of CXPA with myoepithelial component. Our results also suggested that this association could depend on the organization of collagen molecules, being prevented by high-order polymeric structures. These findings could implicate the local microenvironment in the transition from the premalignant PA to invasive CXPA.

Inducible nitric oxide synthase inhibition attenuates lung tissue responsiveness and remodeling in a model of chronic pulmonary inflammation in guinea pigs

We evaluated the influence of iNOS-derived NO on the mechanics, inflammatory, and remodeling process in peripheral lung parenchyma of guinea pigs with chronic pulmonary allergic inflammation. Animals treated or not with 1400W were submitted to seven exposures of ovalbumin in increasing doses. Seventy-two hours after the 7th inhalation, lung strips were suspended in a Krebs organ bath, and tissue resistance and elastance measured at baseline and after ovalbumin challenge. The strips were submitted to histopathological measurements. The ovalbumin-exposed animals showed increased maximal responses of resistance and elastance (p < 0.05), eosinophils counting (p < 0.001), iNOS-positive cells (p < 0.001), collagen and elastic fiber deposition (p < 0.05), actin density (p < 0.05) and 8-iso-PGF2 alpha expression (p < 0.001) in alveolar septa compared to saline-exposed ones. Ovalbumin-exposed animals treated with 1400 W had a significant reduction in lung functional and histopathological findings (p < 0.05). We showed that iNOS-specific inhibition attenuates lung parenchyma constriction, inflammation, and remodeling, suggesting NO-participation in the modulation of the oxidative stress pathway. (C) 2008 Elsevier B.V. All rights reserved.

Collagen Type I, Collagen Type III, and Versican in Vocal Fold Lamina Propria

Objective: To analyze the distributions of collagen type I, collagen type III, and versican in the lamina propria of the human vocal fold. Design: Cross-sectional analysis of cadaveric vocal folds of adult human larynges. Setting: Academic tertiary referral center. Subjects: Larynges harvested at autopsy from 10 adult men and 10 adult women. Main Outcome Measures: Immunohistochemical reactions were performed using antihuman monoclonal antibodies to analyze the expression of collagen type I, collagen type III, and versican. Results: Collagen type I density was lower in the intermediate layer compared with the superficial and deep layers of vocal folds. Collagen type III density was lower in the intermediate layer compared with the deep layer. Versican density was lower in the superficial layer compared with the intermediate and deep layers. Versican density was lower in the lamina propria of women compared with men; this difference was noted in the superficial layer only. There was a positive correlation between collagen type III and versican densities within the lamina propria. Conclusion: Collagen type I, collagen type III, and versican are distributed differently within the lamina propria layers of the adult vocal folds.

Disarrangement of Collagen Fibers in Reinke`s Edema

Objective/Hypothesis: To describe the arrangement of collagen fibers in the superficial layer of the lamina propria of the vocal folds with Reinke` edema. Study Design: Cross sectional analysis of the lamina propria of the vocal folds with Reinke`s edema (RE). Method: The picrosirius polarization method was used to study the arrangement of collagen fiber. Findings of collagen disarrangement were categorized semiquantitatively and correlated with RE severity, age, cigarette smoking and duration of dysphonia. Results: Analysis of 20 specimens of vocal folds with RE showed that the intertwined network of collagen fibers resembling a wicker-basket normally observed in vocal folds was disarranged in RE. The collagen fibers were loosely arranged, fragmented and intermixed with varying amounts of myxoid stroma. Moderate and large areas of disarrangement (90% of cases) predominated. Collagen fiber arrangement in the region underneath the epithelium was better preserved when compared with fibers in the deeper region of the superficial layer of the lamina propria. There was a statistical difference in collagen disarrangement between grade II and grade III severity (P = .007) that appeared to be due to the large areas of disarrangement observed in 73% of patients with grade III severity and in 44% of grade II severity. Age was the only variable correlated with collagen fiber disarrangement (r = 0.47, P = .037). Conclusion: Our findings suggest that the flexible framework which maintains the uniformity of the lamina propria was lost in RE caused by the disarrangement of the collagen fibers.

Effects of inducible nitric oxide synthase inhibition in bronchial vascular remodeling-induced by chronic allergic pulmonary inflammation

Vascular remodeling is an important feature in asthma pathophysiology. Although investigations suggested that nitric oxide (NO) is involved in lung remodeling, little evidence established the role of inducible NO synthase (iNOS) isoform in bronchial vascular remodeling. The authors investigated if iNOS contribute to bronchial vascular remodeling induced by chronic allergic pulmonary inflammation. Guinea pigs were submitted to ovalbumin exposures with increasing doses (1 similar to 5 mg/mL) for 4 weeks. Animals received 1400W (iNOS-specific inhibitor) treatment for 4 days beginning at 7th inhalation. Seventy-two hours after the 7th inhalation, animals were anesthetized, mechanical ventilated, exhaled NO was collected, and lungs were removed and submitted to picrosirius and resorcin-fuchsin stains and to immunohistochemistry for matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), and transforming growth factor-beta (TGF-beta). Collagen and elastic fiber deposition as well as MMP-9, TIMP-1, and TGF-beta expression were increase in bronchial vascular wall in ovalbumin-exposed animals. The iNOS inhibition reduced all parameters studied. In this model, iNOS inhibition reduced the bronchial vascular extracellular remodeling, particularly controlling the collagen and elastic fibers deposition in pulmonary vessels. This effect can be associated to a reduction on TGF-beta and on metalloproteinase-9/TIMP-1 vascular expression. It reveals new therapeutic strategies and some possible mechanism related to specific iNOS inhibition to control vascular remodeling.

Changes in plasma free fatty acid levels in septic patients are associated with cardiac damage and reduction in heart rate variability

Free fatty acids (FFAs) have been shown to produce alteration of heart rate variability (HRV) in healthy and diabetic individuals. Changes in HRV have been described in septic patients and in those with hyperglycemia and elevated plasma FFA levels. We studied if sepsis-induced heart damage and HRV alteration are associated with plasma FFA levels in patients. Thirty-one patients with sepsis were included. The patients were divided into two groups: survivors(n = 12) and nonsurvivors (n = 19). The following associations were investigated: (a) troponin I elevation and HRV reduction and (b) clinical evolution and HRV index, plasma troponin, and plasma FFA levels. Initial measurements of C-reactive protein and gravity Acute Physiology and Chronic Health Evaluation scores were similar in both groups. Overall, an increase in plasma troponin level was related to increased mortality risk. From the first day of study, the nonsurvivor group presented a reduced left ventricular stroke work systolic index and a reduced low frequency (LF) that is one of HRV indexes. The correlation coefficient for LF values and troponin was r(2) = 0.75 (P < 0.05). All patients presented elevated plasma FFA levels on the first day of the study (5.11 +/- 0.53 mg/mL), and this elevation was even greater in the nonsurvivor group compared with the survivors (6.88 +/- 0.13 vs. 3.85 +/- 0.48 mg/mL, respectively; P < 0.05). Cardiac damage was confirmed by measurement of plasma troponin I and histological analysis. Heart dysfunction was determined by left ventricular stroke work systolic index and HRV index in nonsurvivor patients. A relationship was found between plasma FFA levels, LFnu index, troponin levels, and histological changes. Plasma FFA levels emerged as possible cause of heart damage in sepsis.

Collagen Fiber and Versican Distribution Within the Lamina Propria of Fetal Vocal Folds

Objectives: To analyze the presence and distribution of collagen fibers and versican in human vocal fold lamina propria of fetal larynges. Study Design: Cross sectional analysis of cadaveric vocal folds of human fetuses. Methods: Seven fetal larynges obtained from 28- to 36-week-old fetuses were analyzed with the Picrosirius-polarization method, immunohistochemistry, and image analysis. Results: Collagen fibers within the lamina propria exhibited a monolaminar distribution pattern and spatial arrangement in ""wicker basket."" Versican distribution was larger in the superficial and intermediate layers when compared to the deep layer. Conclusion: Our findings suggest that collagen and versican distribution and arrangement within the lamina propria in the developing fetus are important for vocalization at birth.

Increased mRNA expression of collagen V gene in pulmonary fibrosis of systemic sclerosis

Background Collagen V shows promise as an inducer of interstitial lung fibrosis in experimental systemic sclerosis (SSc). Materials and methods Remodelling of the pulmonary interstitium was evaluated based on the clinical data and open lung biopsies from 15 patients with SSc. Normal lung tissues obtained from eight individuals who died of traumatic injuries were used as control group. Immunofluorescence, immunohistochemistry, morphometry, tri-dimensional reconstruction and a real-time polymerase chain reaction were used to evaluate the quantity, structure and molecular chains of collagen V. The impact of these markers was tested on clinical data. Results The main difference in collagen V content between SSc patients and the control group was an increased, abnormal and distorted fibre deposition in the alveolar septa and the pre-acinar artery wall. The lungs from SSc patients presented [alpha 1(V)] and [alpha 2(V)] mRNA chain expression increased, but [alpha 2(V)] was proportionally increased compared with the control group. High levels of collagen V were inversely associated with vital capacity (r = -0.72; P = 0.002), forced vital capacity (r = -0.76; P < 0.001), forced expiratory volume in 1-s (r = -0.89; P < 0.001) and diffusing capacity for carbon monoxide (r = -0.62; P = 0.04). Conclusions Abnormal collagen V fibres are overproduced in lungs from SSc patients and may play an important role in the pathogenesis of the disease as this molecule regulates tissue collagen assembly. The aberrant histoarchitecture observed in SSc can be related to the overexpression of the [alpha 2(V)] gene of unknown origin.

Bronchoscopic lung-volume reduction with Exhale airway stents for emphysema (EASE trial): randomised, sham-controlled, multicentre trial

Background Airway bypass is a bronchoscopic lung-volume reduction procedure for emphysema whereby transbronchial passages into the lung are created to release trapped air, supported with paclitaxel-coated stents to ease the mechanics of breathing. The aim of the EASE (Exhale airway stents for emphysema) trial was to evaluate safety and efficacy of airway bypass in people with severe homogeneous emphysema. Methods We undertook a randomised, double-blind, sham-controlled study in 38 specialist respiratory centres worldwide. We recruited 315 patients who had severe hyperinflation (ratio of residual volume [RV] to total lung capacity of >= 0.65). By computer using a random number generator, we randomly allocated participants (in a 2:1 ratio) to either airway bypass (n=208) or sham control (107). We divided investigators into team A (masked), who completed pre-procedure and post-procedure assessments, and team B (unmasked), who only did bronchoscopies without further interaction with patients. Participants were followed up for 12 months. The 6-month co-primary efficacy endpoint required 12% or greater improvement in forced vital capacity (FVC) and 1 point or greater decrease in the modified Medical Research Council dyspnoea score from baseline. The composite primary safety endpoint incorporated five severe adverse events. We did Bayesian analysis to show the posterior probability that airway bypass was superior to sham control (success threshold, 0.965). Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00391612. Findings All recruited patients were included in the analysis. At 6 months, no difference between treatment arms was noted with respect to the co-primary efficacy endpoint (30 of 208 for airway bypass vs 12 of 107 for sham control; posterior probability 0.749, below the Bayesian success threshold of 0.965). The 6-month composite primary safety endpoint was 14.4% (30 of 208) for airway bypass versus 11.2% (12 of 107) for sham control (judged non-inferior, with a posterior probability of 1.00 [Bayesian success threshold >0.95]). Interpretation Although our findings showed safety and transient improvements, no sustainable benefit was recorded with airway bypass in patients with severe homogeneous emphysema.