Immunohistochemical expression of renin and GATA3 help distinguish juxtaglomerular cell tumors from renal glomus tumors.

Juxtaglomerular cell tumors and glomus tumors both arise from perivascular mesenchymal cells. Juxtaglomerular cells are specialized renin-secreting myoendocrine cells in the afferent arterioles adjacent to glomeruli, and juxtaglomerular tumors derived from these cells are therefore unique to the kidney. In contrast, glomus tumors have been described at numerous anatomic sites and may show significant morphologic and immunophenotypic overlap with juxtaglomerular tumors when occurring in the kidney. Although ultrastructural studies and immunohistochemistry for renin may distinguish these entities, these diagnostic modalities are often unavailable in routine clinical practice. Herein, we studied the clinicopathologic features of a large series of juxtaglomerular tumors (n = 15) and glomus tumors of the kidney (n = 9) to identify features helpful in their separation, including immunohistochemistry for smooth muscle actin (SMA), CD34, collagen IV, CD117, GATA3, synaptophysin, and renin. Markers such as SMA (juxtaglomerular tumors: 12/13, 92%; glomus tumors: 9/9, 100%), CD34 (juxtaglomerular tumors: 14/14, 100%; glomus tumors: 7/9, 78%), and collagen IV (juxtaglomerular tumors: 5/6, 83%; glomus tumors: 3/3, 100%) were not helpful in separating these entities. In contrast to prior reports, all juxtaglomerular tumors were CD117 negative (0/12, 0%), as were glomus tumors (0/5, 0%). Our results show that juxtaglomerular tumors have a younger age at presentation (median age: 27 years), female predilection, and frequently exhibit diffuse positivity for renin (10/10, 100%) and GATA3 (7/9, 78%), in contrast to glomus tumors (median age: 51 years; renin: 0/6, 0%; GATA3: 0/6, 0%). These findings may be helpful in distinguishing these tumors when they exhibit significant morphologic overlap.

Type IV Collagen 7S Is the Most Accurate Test For Identifying Advanced Fibrosis in NAFLD With Type 2 Diabetes.

This study aimed to examine whether the diagnostic accuracy of four noninvasive tests (NITs) for detecting advanced fibrosis in nonalcoholic fatty liver disease (NAFLD) is maintained or is inferior to with or without the presence of type 2 diabetes. Overall, 874 patients with biopsy-proven NAFLD were enrolled. After propensity-score matching by age, sex, and the prevalence of dyslipidemia, 311 patients were enrolled in each group of with or without diabetes. To evaluate the effect of diabetes, we compared the diagnostic accuracy of the fibrosis-4 (FIB-4) index, the NAFLD fibrosis score (NFS), the aspartate aminotransferase to platelet ratio index (APRI), and type IV collagen 7S (COL4-7S) in patients with NAFLD with and without diabetes. The areas under the receiver operating characteristic curve (AUROC) for identifying advanced fibrosis in patients without diabetes were 0.879 for the FIB-4 index, 0.851 for the NFS, 0.862 for the APRI, and 0.883 for COL4-7S. The AUROCs in patients with diabetes were 0.790 for the FIB-4 index, 0.784 for the NFS, 0.771 for the APRI, and 0.872 for COL4-7S. The AUROC of COL4-7S was significantly larger than that of the other NITs in patients with NAFLD with diabetes than in those without diabetes. The optimal high and low cutoff points of COL4-7S were 5.9 ng/mL and 4.8 ng/mL, respectively. At the low cutoff point, the accuracy of COL4-7S was better than that of the other NITs, especially in patients with diabetes. Conclusion: COL4-7S measurement might be the best NIT for identifying advanced fibrosis in NAFLD, especially in NAFLD with diabetes.

Type IV collagen as a potential biomarker of metastatic breast cancer.

No reliable, non-invasive biomarker of metastatic breast cancer (mBC) exists: circulating CA15-3 (cCA15-3) is the marker mostly used to monitor mBC. Circulating collagen IV (cCOLIV) has been evaluated in other metastatic cancers and has been found to be a promising biomarker. The overarching aim of this study was to evaluate cCOLIV as a potential biomarker in patients with mBC. The first aim was to determine the levels of cCOL IV and cCA15-3 in patients with healthy controls, primary breast cancer (pBC) and mBC. The second aim was to compare levels of cCOLIV and cCA15-3 in patients with different metastatic sites of BC. The third aim was to investigate the prognostic value of cCOLIV and cCA15-3 for mBC patients. The fourth aim was to analyse whether a combination of the two biomarkers was more accurate in detecting mBC than a single marker. Lastly, we investigated the tissue expression levels of COLIV in BC bone metastases (BM) and liver metastases (LM). Plasma levels of cCOLIV and cCA15-3 from healthy controls and patients with pBC and mBC were measured. COLIV expression in tissue from patients with LM and BM was analysed using immunohistochemistry. Clinical and survival data were collected from medical charts. The levels of cCOLIV and cCA15-3 were significantly elevated in mBC patients compared with healthy controls and pBC patients. No differences in cCOLIV and cCA15-3 levels were found based on the metastatic site. High levels of cCOLIV, but not cCA15-3, correlated with poorer survival. cCOLIV alone and the combination of cCA15-3 and cCOLIV were superior to cCA15-3 at detecting mBC. COL IV was highly expressed in the tissue of LM and BM. Our study suggests that cCOLIV is a potential marker to monitor patients with BC.

Electron Microscopic and Immunohistochemical Findings of the Epidermal Basement Membrane in Two Families with Nail-patella Syndrome.

Nail-patella syndrome is an autosomal dominant disorder characterized by nail dysplasia and skeletal anomaly. Some patients have been shown to have ultrastructural abnormalities of the glomerular basement membrane that result in nephrosis. However, little has been reported on the epidermal basement membrane in this condition. This paper reports 2 families with nail-patella syndrome. Direct sequencing analysis of LMX1B revealed that family 1 and family 2 were heterozygous for the mutations c.140-1G>C and c.326+1G>C, respectively. To evaluate the epidermal basement membrane zone, ultrastructural and immunohistochemical analyses were performed using skin specimens obtained from the dorsal thumb. Electron microscopy showed intact hemidesmosomes, lamina lucida, lamina densa, and anchoring fibrils. Immunofluorescence studies with antibodies against components of the epidermal basement membrane zone revealed a normal expression pattern among the components, including type IV collagen. These data suggest that nail dysplasia in patients with nail-patella syndrome is not caused by structural abnormalities of the epidermal basement membrane.

Bioinformatics analysis suggests that COL4A1 may play an important role in gastric carcinoma recurrence.

OBJECTIVE: Cancer recurrence is a complicated problem for clinicians that contributes to poor prognosis. This study aimed to use advanced gastric carcinoma genes profiles to predict increased risk of cancer recurrence in order to identify patients in need of adjuvant therapy for prognosis improvement. METHODS: Differentially expressed genes were identified for advanced gastric carcinoma by analyzing the GSE2685 from the Gene Expression Omnibus database (GEO) using R package. The candidate genes were then obtained by gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, protein-protein interaction analysis and survival analysis. Logistic regression analysis was performed to determine the relationship between candidate genes and the recurrence of gastric carcinoma. RESULTS: Collagen type IV alpha 1 (COL4A1) was overexpressed in gastric carcinoma tissue by analyzing the GSE2685 gene expression profiles from the Gene Expression Omnibus database. COL4A1 was also overexpressed in gastric carcinoma tissue from the Cancer Genome Atlas dataset and further determined that higher COL4A1 expression led to poorer overall survival. A univariate analysis suggested that COL4A1 was strongly correlated with T stage and gastric carcinoma recurrence (P = 0.014 and 0.041, respectively). Moreover, a multiple logistic regression analysis indicated that COL4A1 was significantly associated with gastric carcinoma recurrence (hazard ratio 1.605, 95% confidence interval 1.063-2.677, P = 0.008). CONCLUSIONS: COL4A1 may promote gastric carcinoma recurrence and could be used as a therapeutic target for gastric carcinoma recurrence.

The role of pirfenidone in alkali burn rat cornea.

To evaluate the effects of pirfenidone in the treatment of HUVEC using an in vitro model and on rat corneal wound healing, edema, cornea neovascularization (CNV) and inflammation after alkali burn in vivo model. In vitro, CCK-8 assay was used to detect the effect of pirfenidone on the viability of HUVECs. The effects of pirfenidone on migration and tube formation of HUVEC were evaluated by HUVEC cell wound closure and tube formation assay. In vivo, Eye drops containing pirfenidone or phosphate buffered saline (PBS) were administered to an alkali-burn-induced corneal inflammatory and neovascularization model four times daily. The clinical evaluations, including fluorescent staining and cornea edema, were performed on days 1, 4, 7 and 14 using slit lamp microscopy. Global specimens were collected on day 7 and processed for immunofluorescent staining Collagen IV, α-smooth muscle actin (α-SMA), vascular endothelial growth factor (VEGF), pigment epithelium derived factor (PEDF) and cluster of differentiation34 (CD34). The levels of α-SMA, VEGF, PEDF, CD34, CD31 and nuclear factor-kappa B (NF-κB) proteins in the corneas were determined by western blot. Pirfenidone affects HUVEC viability, migration and tube formation in a dose-dependent manner. High concentration of pirfenidone can inhibit HUVEC viability, migration and tube formation in vitro and reduce alkali burn rat cornea edema, promote corneal wound healing, inhibit CNV and inflammation after alkali burn in vivo. Pirfenidone promotes corneal wound healing, and inhibits cornea neovascularization and inflammation after alkali burn in vitro and in vivo. Pirfenidone may be the potential anti-inflammation agent for the clinical treatment of CNV.

In Situ Hybridization and Double Immunohistochemistry for the Detection of VEGF-A mRNA and CD34/Collagen IV Proteins in Renal Transplant Biopsies.

Quantitative metrics on the tissue distribution of different cell phenotypes, extracellular matrix components, and signaling/cell cycle markers hold the promise for the advent of new-generation tissue-based predictive/prognostic biomarkers in clinical diagnostics. The workflow of this approach is composed of three major phases: (1) detection of multiple molecular targets on a single histologic section, (2) image acquisition, and (3) digital image processing and analysis. Here, we present the most prevalent current alternatives for step (1) and describe a three-plex staining and image acquisition platform that captures the spatial distribution of macromolecules from two different species.

Identificação dos colágenos I, III, IV e α-SMA e participação dos miofibroblastos no processo fibrótico das endometroses equinas / Identification of collagens types I, III, IV and α-SMA and the participation of myofibroblasts in the fibrotic process in equine endometrosis

A endometrose é uma alteração degenerativa das glândulas uterinas e do estroma circundante, caracterizada pelo arranjo periglandular de miofibroblastos e pela deposição de matriz extracelular (ECM). O presente trabalho objetivou avaliar a expressão de colágenos tipos I, III e IV e α-actina de músculo liso (α-SMA) nas endometroses equinas, procurando esclarecer a participação dos miofibroblastos na progressão desses processos. Foram utilizadas 24 biópsias uterinas com diagnóstico de endometrose, recebidas pelo Serviço de Patologia Veterinária e de Reprodução Animal da FMVZ, Unesp, Botucatu, SP. Cortes histológicos foram submetidos às técnicas histoquímicas de tricrômico de Masson, picrosirius red sob luz polarizada e ácido periódico de Schiff (PAS) e imuno-histoquímicas para os três tipos de colágeno citados e α-SMA. Ainda, traçou-se um paralelo entre a técnica de picrosirius red e a imunomarcação dos colágenos tipos I e III. A análise histológica revelou que as fibras de colágeno denso correspondem ao colágeno tipo I, predominantes nas endometroses inativa e inativa destrutiva. As fibras de colágeno frouxo correspondem ao colágeno tipo III, predominantes nas endometroses ativas e ativas destrutivas. Nesses mesmos processos, a membrana basal revelou espessamento, aparentemente não relacionado ao colágeno tipo IV, e uma maior imunomarcação de miofibroblastos periglandulares em relação às endometroses inativa e inativa destrutiva. Dessa forma, nota-se que os miofibroblastos estão relacionados ao aumento na deposição de colágeno tipo III nos ninhos fibróticos ativos.(AU)

Endometriosis is a degenerative change of the uterine glands and surrounding stroma, characterized by periglandular arrangement of myofibroblasts and deposition of extracellular matrix (ECM). The aim of this study was to evaluate the expression of collagen type I, III and IV and α-smooth muscle actin (α-SMA) in equine endometriosis, and investigate the role of myofibroblasts in the progression of these processes. A parallel was made with histochemical techniques of Masson's trichrome, Picrosirius Red under polarized light and Periodic Acid-Schiff (PAS). Twenty four uterine biopsies received by the Veterinary Pathology Service and Animal Reproduction of FMVZ, UNESP, Botucatu, SP, were diagnosed with endometriosis. Histological analysis revealed that the orange dense collagen fibers correspond to type I collagen, being prevalent in inactive and inactive destructive endometriosis. The green loose collagen fibers correspond to type III collagen, and are predominant in active and active destructive endometriosis. In the same processes, a greater amount of periglandular myofibroblasts were observed in comparison to inactive and inactive destructive endometriosis. The presence of these cells in active processes are strongly related to an increased deposition of collagen type III in fibrotic nests. Regarding the basement membrane, the active destructive and active endometriosis shows thickening, apparently not related to an increase in expression of type IV collagen. The active destructive and inactive destructive endometriosis exhibited disruption areas in type IV collagen fibers. Thus, it is noted that the myofibroblasts are related to increased deposition of type III collagen in active fibrotic nests.(AU)

Urinary DcR2 is a novel biomarker for tubulointerstitial injury in patients with diabetic nephropathy.

Tubulointerstitial injury (TII) plays a crucial role in the progression of diabetic nephropathy (DN), but lack of specific and sensitive biomarkers for monitoring TII in DN management. This study is to investigate whether urinary decoy receptor 2 (uDcR2) could serve as a novel noninvasive biomarker for assessing TII in DN. We recruited 311 type 2 diabetics and 139 DN patients who were diagnosed by renal biopsy. uDcR2 levels were measured by ELISA, and renal DcR2 expression was detected immunohistochemically. Associations between uDcR2 and renal DcR2 and renal functional parameters were evaluated. Receiver operating characteristics (ROC) curve analyzed area under the curve (AUC) of uDcR2 for assessing TII. Double staining was undertaken for renal DcR2 with proximal and distal tubular markers; senescent markers p16, p21, and senescence-associated ß-galactosidase (SA-ß-gal); and fibrotic markers collagen I and IV. We found DcR2 was primarily expressed in renal proximal tubules; uDcR2 levels were elevated per albuminuria stratum and correlated with renal functional parameters in diabetics and were associated with percentage of tubular DcR2 and TII score in DN. The uDcR2 had an AUC of 0.909 for assessing TII in DN by ROC analysis. Almost all tubular DcR2 was coexpressed with p16 and p21, and nearly more than one-half of tubular DcR2 was positive for SA-ß-gal, primarily in collagen I- and IV-positive regions of DN. Our results indicate uDcR2 could potentially serve as a novel biomarker for TII and may reflect senescence of renal proximal tubular cells in DN pathogenesis.

The Morphofunctional Changes in the Wall of Varicose Veins.

BACKGROUND: Varicose vein (VV) disease is a frequently occurring pathology of the lower extremities. Although the pathogenesis of varicosity development is not clearly defined, the final common pathway leading to chronic venous insufficiency is the development of venous hypertension, which is associated with severe changes in the venous wall. The aim of this study was to clarify the histological and immunohistochemical changes in great saphenous veins (GSVs) in chronic venous insufficiency. METHODS: A histopathological study was conducted on 20 patients with VVs (4 males, 16 females) and 4 (1 male, 3 females) patients undergoing distal bypass surgery. Tissues were processed for histological routine straining and immunohistochemical studies of vascular endothelial growth factor (VEGF), intercellular adhesion molecule (ICAM)-1, vascular adhesion molecule (VCAM)-1, protein gene product 9.5 (PGP 9.5), and collagen type IV, laminin, and fibronectin. A semiquantitative evaluation method was used. RESULTS: Compared with the normal SV, VV sections showed the damaged endothelium areas, significant disorganization of the smooth muscle bundles, and highest density of the vasa vasorum in the tunica media and tunica adventitia, as well as sclerotic blood vessels and neoangiogenesis in almost all specimens. Immunohistochemistry study showed statistically significant difference between the VVs and the control group of several parameters, such as PGP 9.5 positive structures (P < 0.05; 1-tailed significance) and laminin positive structures in subendothelial layer of VVs (P < 0.05; 1-tailed significance). There is also the tendency in increasing of VEGF expression and decreasing of collagen IV structures. Our study did not show statistically significant difference in VEGF, ICAM-1, and VCAM-1 positive structures between varicose and normal veins; however, it could be explained by the limitations of the study. CONCLUSIONS: Varicose GSVs represent nonhomogeneous integrity of the basement membrane, smooth muscle disorganization, and active neoangiogenesis, suggesting remodulation of blood vessels. Changes in the appearance of PGP 9.5-containing innervation, laminin, and collagen IV in tunica intima confirm the remodulation of damaged blood vessels.

Accumulation of worn-out GBM material substantially contributes to mesangial matrix expansion in diabetic nephropathy.

Thickening of the glomerular basement membrane (GBM) and expansion of the mesangial matrix are hallmarks of diabetic nephropathy (DN), generally considered to emerge from different sites of overproduction: GBM components from podocytes and mesangial matrix from mesangial cells. Reevaluation of 918 biopsies with DN revealed strong evidence that these mechanisms are connected to each other, wherein excess GBM components fail to undergo degradation and are deposited in the mesangium. These data do not exclude that mesangial cells also synthesize components that contribute to the accumulation of matrix in the mesangium. Light, electron microscopic, immunofluorescence, and in situ hybridization studies clearly show that the thickening of the GBM is due not only to overproduction of components of the mature GBM (α3 and α5 chains of collagen IV and agrin) by podocytes but also to resumed increased synthesis of the α1 chain of collagen IV and of perlecan by endothelial cells usually seen during embryonic development. We hypothesize that these abnormal production mechanisms are caused by different processes: overproduction of mature GBM-components by the diabetic milieu and regression of endothelial cells to an embryonic production mode by decreased availability of mediators from podocytes.

Histological and morphometric analysis of dilated cardiomyopathy with special reference to collagen IV expression.

INTRODUCTION: Collagen distribution alterations are well known in dilated cardiomyopathy. There are also changes in microvasculature along with other histomorphorphological features. AIMS AND OBJECTIVES: To study the histomorphological features of DCM along with their quantitative correlation with LVEF. Alterations in collagen IV distribution pattern and microvasculature in DCM were also evaluated. MATERIALS AND METHODS: The present study includes 34 right ventricular endomyocardial biopsies, 7 explanted native hearts and 41 autopsy control hearts. Sections were taken from lower half of right interventricular septum and stained for H and E, Masson trichrome and immunohistochemistry for CD34, SMA and Collagen IV to study the histological features, pattern of fibrosis, capillary and arteriolar distribution and collagen IV expression respectively. Morphometric analysis was carried out in all cases and controls using Image analysis software Image pro plus 7 and correlated with left ventricular ejection fraction. RESULTS: The histomorphological changes of DCM include myocyte hypertrophy, nucleomegaly, and interstitial fibrosis. Interfiber fibrosis was the commonest. There was evidence of myocarditis, ischemic change and vessel wall alterations. Considerable alteration in Collagen IV distribution was observed with reduction in intensity and proportion of staining around myocytes quantified using Allred scoring against uniform pericellular staining in controls. Morphometric analysis revealed significant increase in nuclear area, myocyte width, percentage of fibrosis and reduction in capillary myocyte ratio in cases as compared to controls. There was no significant difference in arteriolar density. No significant association was observed between morphometric parameters and LVEF. CONCLUSION: Histomorphological changes in DCM are non-specific. Quantitation of histological parameters cannot be used to predict the disease progression as there was no significant correlation with LVEF. There is appreciable alteration in Collagen IV distribution in DCM owing to extracellular matrix alterations.

Low-grade Schwann cell neoplasms with leptomeningeal dissemination: clinicopathologic and autopsy findings.

Leptomeningeal dissemination of low-grade Schwann cell neoplasms is an exceptionally rare occurrence and has not been well documented in the literature. We encountered 2 cases of leptomeningeal dissemination of low-grade Schwann cell neoplasms. Patient 1 was a 63-year-old woman with neurofibromatosis type 1 and a progressive low-grade malignant peripheral nerve sheath tumor developing from a diffuse/plexiform orbital neurofibroma that arose in childhood. The neoplasm demonstrated local and leptomeningeal dissemination intracranially leading to the patient's death. There was partial loss of H3K27 tri-methylation, p16 and collagen IV. Patient 2 was a 60-year-old man without neurofibromatosis type 1 who presented with cranial nerve symptoms and a disseminated neoplasm with a Schwann cell phenotype. The neoplasm stabilized after irradiation and chemotherapy, but the patient died of medical complications. Autopsy findings documented disseminated leptomeningeal disease in the intracranial and spinal compartment. H3K27M tri-methylation was preserved. The clinicopathologic and autopsy findings are studied and presented, and the literature is reviewed.

Immunohistochemical characteristics of the vitreolenticular interface in congenital unilateral posterior cataract.

PURPOSE: To gain insight into the histology of the vitreolenticular interface in congenital unilateral posterior cataract. SETTING: Antwerp University Hospital, Department of Ophthalmology, Edegem, and the University of Antwerp, Faculty of Medicine and Health Sciences, Antwerp, Belgium. DESIGN: Prospective case study. METHODS: Samples of the posterior lens capsule of patients with congenital posterior cataract (including opaque plaque on the anterior and adhesion to the vitreous on the posterior surface) were collected during the posterior capsulorhexis procedure. Staining for collagen types II and IV was performed using indirect immunohistochemistry. Results were compared with those of control posterior lens capsules of 3 children and 3 adults. RESULTS: Samples were collected from 3 patients. All posterior lens capsules contained collagen type IV. Samples from congenital posterior cataract patients all showed a narrow band of collagen type II on the outer surface, indicating strong adherence of the anterior hyaloid membrane to the center of the posterior lens capsule. Surprisingly, collagen type II was also found in the posterior capsule plaques. Collagen type II was not found in any control posterior lens capsule. CONCLUSION: The adherence of collagen type II to the center of the posterior lens capsule histologically supports the hypothesis that this subgroup of congenital cataract hints at an abnormality at the vitreolenticular interface. FINANCIAL DISCLOSURE: None of the authors has a financial or proprietary interest in any material or method mentioned.

Avaliação da composição molecular da cápsula anterior da lente de cães idosos com catarata de alto risco / Molecular evaluation of the anterior lens capsule of older dogs suffering from high risk cataracts

Atualmente, a cápsula anterior e o epitélio da lente tem sido cada vez mais estudados, com o intuito de reduzir as possíveis complicações do pós-operatório da remoção da catarata, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a composição molecular da cápsula anterior da lente pela técnica histoquímica de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a facoemulsificação. Vinte e sete cães foram estudados, incluindo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças e divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética). Os resultados das análises realizadas mostraram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto das diabéticas, apresentam semelhante composição molecular de proteoglicanos e colágeno IV e isto independente da utilização de azul de tripano a 0,1 %. Conclui-se, portanto, que se os resultados obtidos forem decorrentes de alterações provocadas pelo rápido metabolismo da catarata diabética e pela cronicidade da catarata hipermadura sugere-se que o comprometimento da estrutura capsular seja de intensidade equivalente e, por consequência, que isto também possa prejudicar o metabolismo das células do epitélio anterior da lente, diminuindo assim a incidência da opacidade da cápsula posterior de cães com catarata diabética e hipermadura submetidos à facoemulsificação.(AU)

Nowadays, the anterior lens capsule and its epithelium have been being frequently studied aiming to reduce the incidence of posterior lens capsule opacity, a complication that frequently occurs after surgical removal of cataracts, due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate by histochemistry (PAS and picrosirius red) analysis two important molecular components of the anterior lens capsule (proteoglycans and type IV collagen) in older diabetic and non-diabetic dogs, with diabetic and hypermature cataracts, after phacoemulsification surgery utilizing 0.1% trypan blue or not. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo) of different breeds were studied. The animals were divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts). Results showed that, besides their different pathophysiologies, both types of capsules studied (diabetic and hypermature ones) presented the same molecular composition of proteoglycans and type IV collagen, since no statistical significant differences were observed. In addition, 0.1% trypan blue was not capable to induce any other evident alteration for the samples. In conclusion, our findings suggest that, if the results consist in alteration induced by the aggressive metabolism of the diabetic cataract or the chronicity of the hypermature one, it is of the same intensity and independent of the use of 0.1% trypan blue. It is also possible to suggest that this alteration must be capable to compromise lens epithelium cell metabolism, which should probably favour future lens posterior capsule studies.(AU)

Avaliação da composição molecular da cápsula anterior da lente de cães idosos com catarata de alto risco / Molecular evaluation of the anterior lens capsule of older dogs suffering from high risk cataracts

Atualmente, a cápsula anterior e o epitélio da lente tem sido cada vez mais estudados, com o intuito de reduzir as possíveis complicações do pós-operatório da remoção da catarata, tal como a opacidade da cápsula posterior, alteração ocasionada principalmente pela diferenciação e migração das células do epitélio lenticular para a cápsula posterior da lente. O objetivo deste estudo foi analisar a composição molecular da cápsula anterior da lente pela técnica histoquímica de PAS (avaliação de proteoglicanos) e picrosirius red (avaliação de colágeno IV), em cães idosos com catarata diabética e não diabética do tipo hipermadura, submetidos ao uso ou não de azul de tripano a 0,1 % durante a facoemulsificação. Vinte e sete cães foram estudados, incluindo 21 fêmeas e 6 machos, de 8 a 12 anos de idade (média = 9,6 anos), de diversas raças e divididos em 2 grupos: GC (catarata hipermadura) e GCD (catarata diabética). Os resultados das análises realizadas mostraram que ambas as amostras, tanto as provenientes das cataratas hipermaduras, quanto das diabéticas, apresentam semelhante composição molecular de proteoglicanos e colágeno IV e isto independente da utilização de azul de tripano a 0,1 %. Conclui-se, portanto, que se os resultados obtidos forem decorrentes de alterações provocadas pelo rápido metabolismo da catarata diabética e pela cronicidade da catarata hipermadura sugere-se que o comprometimento da estrutura capsular seja de intensidade equivalente e, por consequência, que isto também possa prejudicar o metabolismo das células do epitélio anterior da lente, diminuindo assim a incidência da opacidade da cápsula posterior de cães com catarata diabética e hipermadura submetidos à facoemulsificação.

Nowadays, the anterior lens capsule and its epithelium have been being frequently studied aiming to reduce the incidence of posterior lens capsule opacity, a complication that frequently occurs after surgical removal of cataracts, due to epithelium cells differentiation and migration to the posterior pole. The objective of this study was to evaluate by histochemistry (PAS and picrosirius red) analysis two important molecular components of the anterior lens capsule (proteoglycans and type IV collagen) in older diabetic and non-diabetic dogs, with diabetic and hypermature cataracts, after phacoemulsification surgery utilizing 0.1% trypan blue or not. Twenty seven dogs, including 21 female and 6 male dogs, with ages varying from 8 to 12 years old (mean = 9.6 yo) of different breeds were studied. The animals were divided into 2 groups: GC (hypermature cataracts) and GCD (diabetic cataracts). Results showed that, besides their different pathophysiologies, both types of capsules studied (diabetic and hypermature ones) presented the same molecular composition of proteoglycans and type IV collagen, since no statistical significant differences were observed. In addition, 0.1% trypan blue was not capable to induce any other evident alteration for the samples. In conclusion, our findings suggest that, if the results consist in alteration induced by the aggressive metabolism of the diabetic cataract or the chronicity of the hypermature one, it is of the same intensity and independent of the use of 0.1% trypan blue. It is also possible to suggest that this alteration must be capable to compromise lens epithelium cell metabolism, which should probably favour future lens posterior capsule studies.

Primary biliary cirrhosis degree assessment by acoustic radiation force impulse imaging and hepatic fibrosis indicators.

AIM: To evaluate the assessment of primary biliary cirrhosis degree by acoustic radiation force impulse imaging (ARFI) and hepatic fibrosis indicators. METHODS: One hundred and twenty patients who developed liver cirrhosis secondary to primary biliary cirrhosis were selected as the observation group, with the degree of patient liver cirrhosis graded by Child-Pugh (CP) score. Sixty healthy individuals were selected as the control group. The four indicators of hepatic fibrosis were detected in all research objects, including hyaluronic acid (HA), laminin (LN), type III collagen (PC III), and type IV collagen (IV-C). The liver parenchyma hardness value (LS) was then measured by ARFI technique. LS and the four indicators of liver fibrosis (HA, LN, PC III, and IV-C) were observed in different grade CP scores. The diagnostic value of LS and the four indicators of liver fibrosis in determining liver cirrhosis degree with PBC, whether used alone or in combination, were analyzed by receiver operating characteristic (ROC) curve. RESULTS: LS and the four indicators of liver fibrosis within the three classes (A, B, and C) of CP scores in the observation group were higher than in the control group, with C class > B class > A class; the differences were statistically significant (P < 0.01). Although AUC values of LS within the three classes of CP scores were higher than in the four indicators of liver fibrosis, sensitivity and specificity were unstable. The ROC curves of LS combined with the four indicators of liver fibrosis revealed that: AUC and sensitivity in all indicators combined in the A class of CP score were higher than in LS alone, albeit with slightly decreased specificity; AUC and specificity in all indicators combined in the B class of CP score were higher than in LS alone, with unchanged sensitivity; AUC values (0.967), sensitivity (97.4%), and specificity (90%) of all indicators combined in the C class of CP score were higher than in LS alone (0.936, 92.1%, 83.3%). CONCLUSION: The diagnostic value of PBC cirrhosis degree in liver cirrhosis degree assessment by ARFI combined with the four indicators of serum liver fibrosis is of satisfactory effectiveness and has important clinical application value.

A sensitive electrochemiluminescence immunosensor based on luminophore capped Pd@Au core-shell nanoparticles as signal tracers and ferrocenyl compounds as signal enhancers.

In this work, N-(aminobutyl)-N-(ethylisoluminol) (ABEI), an analogue of luminol, is served as both the reductant and luminescence reagent to synthesize ABEI capped Pd@Au core-shell nanoparticles (ABEI-Pd@AuNPs). The nanoparticles not only exhibit inherent electrochemiluminescence (ECL) property, but also possess advantages of noble-metal nanomaterials such as outstanding electronic property, high specific surface area and good biocompatibility. In order to enhance the luminescence efficiency, ferrocene monocarboxylic acid (Fc) as catalyzer is grafted on the surface of ABEI-Pd@AuNPs with the aid of l-cysteine (l-Cys). When the Fc is electrochemically oxidized to ferricinium cation species (Fc(+)), the decomposition of H2O2 which existed in detection solution can be catalyzed by Fc(+) to generate oxygen-related free radicals, resulting effective signal amplification for ABEI-H2O2 system. For potential applications, the Pd@Au core-shell nanoparticles bifunctionalized by ABEI and catalyzer are employed as nano-carriers to immobilize detection antibody (Ab2). Based on sandwiched immunoreactions, a "signal-on" ECL immunosensor is developed for detection of human collagen type IV (Col IV), a potential biomarker associated with diabetic nephropathy. Consequently, the proposed immunosensor provides a wide linear detection ranging from 1pgmL(-1) to 10ngmL(-1) with a relatively low detection limit of 0.3pgmL(-1) (S/N=3).

Natural extracellular matrix scaffolds recycled from human salivary digests: a morphometric study.

OBJECTIVE: A challenge in engineering tissues is to supply parenchymal cells with suitable scaffolds which ideally reproduce the extracellular matrix (ECM). This study tested the hypothesis of preserving the 'residual connective tissue' remaining after mechanical and enzymatic release of cells from human submandibular gland biopsies (that we named 'natural ExtraCellular Matrix scaffolds', nECMsc) to be used as recycled natural scaffolds. The objective was to test whether nECMsc and native salivary tissue were comparable morphologically, in ECM proteins composition, and in cell seeding efficiency. METHODS: Following cell isolation procedures, nECMsc were kept, either fresh or frozen (sectioned into 12-µm-thick slices), and examined with high-resolution electron microscopy (HRSEM) for its three-dimensional structure, and with picrosirius red staining and immunogold staining for ECM protein composition and distribution, respectively. nECMsc were seeded with human epithelial cells and fibroblasts to assess cell attachment and proliferation in short-term experiments. RESULTS: Under HRSEM, nECMsc had comparable fiber arrangement to original glands. Histochemical and immunogold-labeling examinations revealed the presence of collagen types I, III, and IV. Seeded epithelial cells and fibroblasts attached, proliferated (14-55%), and were alive (86-99%) after 4-8 days of culture. CONCLUSIONS: nECMsc retained native ECM proteins and maintained their distribution. Seeded cells remained viable on nECMsc.

Three days of intermittent stretching after muscle disuse alters the proteins involved in force transmission in muscle fibers in weanling rats

The aim of this study was to determine the effects of intermittent passive manual stretching on various proteins involved in force transmission in skeletal muscle. Female Wistar weanling rats were randomly assigned to 5 groups: 2 control groups containing 21- and 30-day-old rats that received neither immobilization nor stretching, and 3 test groups that received 1) passive stretching over 3 days, 2) immobilization for 7 days and then passive stretching over 3 days, or 3) immobilization for 7 days. Maximal plantar flexion in the right hind limb was imposed, and the stretching protocol of 10 repetitions of 30 s stretches was applied. The soleus muscles were harvested and processed for HE and picrosirius staining; immunohistochemical analysis of collagen types I, III, IV, desmin, and vimentin; and immunofluorescence labeling of dystrophin and CD68. The numbers of desmin- and vimentin-positive cells were significantly decreased compared with those in the control following immobilization, regardless of whether stretching was applied (P<0.05). In addition, the semi-quantitative analysis showed that collagen type I was increased and type IV was decreased in the immobilized animals, regardless of whether the stretching protocol was applied. In conclusion, the largest changes in response to stretching were observed in muscles that had been previously immobilized, and the stretching protocol applied here did not mitigate the immobilization-induced muscle changes. Muscle disuse adversely affected several proteins involved in the transmission of forces between the intracellular and extracellular compartments. Thus, the 3-day rehabilitation period tested here did not provide sufficient time for the muscles to recover from the disuse maladaptations in animals undergoing postnatal development.