Participation of endothelial cells and transformed mesangial cells in remodeling of glomerular capillary loops in Thy-1 nephritis

The relationship between mesangial cells (MC) and endothelial cells (EC) in the remodeling of glomerular capillary loops was investigated in a rat model of anti-Thy-1 antibody (Ab)-induced glomerulonephritis. Immunohistochemical analysis showed that cells positive for alpha-smooth muscle actin (alpha-SMA) appeared in the mesangial stalks at day three, and had increased in number at day seven, after injection of Thy-1 Ab. Double staining for alpha-SMA and proliferating cell nuclear antigen (PCNA) showed that some MC expressing PCNA were negative for alpha-SMA at day three, but by day seven almost all PCNA-positive MC expressed alpha-SMA. Western blotting for alpha-SMA from isolated glomeruli was negative at day one after injection of Thy-1 Ab, but positive at day seven. Type III collagen appeared at day seven, followed by an increase of EC in the capillary loops, as determined by double immunofluorescent staining for rat endothelial cell antigen-1 (RECA-1) and type III collagen. RECA-1-positive cells increased rapidly in number after day seven and eventually showed the same distribution pattern as that in control rats. Both type I and type III collagens were expressed in the mesangial and the ballooning area of the glomerulus at day seven. Electron microscopy revealed that immature MC and EC forming small capillary lumina appeared in the enlarged mesangial area at day seven. In accordance with the increase of capillaries and the enlargement of the lumina, the number of MC and the amount of mesangial matrix decreased gradually, and most of the glomeruli returned to a normal structure by week 4. These data show that type I and type III collagen produced by transformed MC may be of benefit to proliferation of EC and remodeling of the capillary in Thy-1-induced nephritis.     

Glomerular expression of alpha-smooth muscle actin reflects disease activity of IgA nephropathy

To elucidate the relationship between histological disease states and clinicopathological features in immunoglobulin A nephropathy (IgAN), 90 needle-biopsy specimens diagnosed as IgAN were analyzed. The specimens were divided into four groups according to histological grade and stage index. Immunohistochemical features of alpha-smooth muscle actin (alpha-SMA), macrophages positive for myeloid/histiocyte antigen (MAC387), and expression of type I, III and IV collagens were all examined. Glomerular expression scores of alpha-SMA and the degree of intraglomerular macrophage infiltration were highest in the active and non-sclerotic groups. Type I and IV collagens were significantly more abundant in the sclerotic groups than in the active groups. Type III collagen was strongly expressed in both the active and sclerotic groups. Double immunolabeling of alpha-SMA and intercellular adhesion molecule (ICAM)-1 revealed that ICAM-1 was expressed around the alpha-SMA-positive mesangial area. In multivariate analysis, the glomerular expression score of alpha-SMA was mostly correlated with histological grading in the 10 clinicopathological parameters. Type IV collagen score was mostly correlated with histological staging. These results suggest that glomerular alpha-SMA expression reflects the histological activity of IgAN. Immunohistological staining of alpha-SMA is valuable to estimate the degree of disease activity in IgAN.     

Participation of the matrix metalloproteinase inhibitor in Thy-1 nephritis

What influence would be shown in Thy‐1 glomerulonephritis when the synthetic matrix metalloproteinase (MMP) inhibitor SI‐27 is administered? Five groups of 80 male Wistar rats were studied: healthy group; treated healthy group; nephritic group; pretreated nephritic group; and post‐treated nephritic group. SI‐27 treatment of nephritic animals was initiated either 2 days before or 2 days after anti‐Thy‐1.1 antibody injection. On days 7, 14, 26 and 42 after disease induction, we examined renal histology, extracellular matrix (ECM) constituent, and MMP activity. SI‐27 treated Thy‐1 groups resulted in significant reduction of glomerular cells including α‐smooth muscle actin (α‐SMA) positive mesangial cells and suppressed expression of type IV collagen at 7 days. Moreover, type I collagen was also decreased by SI‐27 at 42 days. However, glomerular cell numbers did not show any significant changes at 14, 26 and 42 days. In gelatin zymography, the gelatinolytic band for MMP‐9 was expressed in SI‐27 treated Thy‐1 nephritis groups, although it was not expressed in the nephritic group at day 7. However, the expression of MMP‐9 was no longer seen at 14, 26 and 42 days. The bands for an active form of MMP‐2 were expressed throughout the experimental period in the Thy‐1 nephritic groups. These results suggest that MMP plays an important role in the development of Thy‐1 nephritis, and even if the synthetic MMP inhibitor intercepts the initial increase of glomerular cells and matrices, it does not inhibit recovery to normal glomerular capillary structures in Thy‐1 nephritis.     

Glomerular sequence of events concerning monocyte/ macrophage accumulation and ICAM-1 expression during experimental serum sickness nephritis in the rat2

Serum sickness nephritis was induced in Fisher rats by preimmunization and repeated immunization with chicken egg albumin. This experimental model is characterized by marked accumulation of monocytes/macrophages (MO) and deposition of immune complexes (IC) in glomerull during the inflammatory stage and, thereafter, the advancement to glomeruloscierods. The correlations between glomerular tissue damage, MO participation, intercellular adhesion molecule-1 (ICAY-1) expression and IC deposition were analyzed during the long-term disease process. The grade of ICAM-1 expression was well correlated with MO accumulation and IC deposition, and its distribution was observed on the glomerular endothelial layer, mesanglum, and along the parietal epithelial layer of the Bowman's capsule. It is suggested that glomerular MO accumulation is largely affected by the ICAM-1 expression on glomeruli and, underneath such adhesion molecules, MO may play a role In subendoth-elial or mesangial migration, mesangial cell activation, inducing sclerosis and monocytic-epithellal crescent formation.     

The value of alpha-SMA in the evaluation of hepatic fibrosis severity in hepatitis B infection and cirrhosis development: a histopathological and immunohistochemical study

AIMS: To investigate the value of alpha-smooth muscle actin (alpha-SMA), an indicator of stellate cell activation, in predicting fibrosis in chronic hepatitis B (CHB) patients. METHODS AND RESULTS: The liver biopsy specimens of 30 patients with a clinical diagnosis of CHB were obtained before treatment and scored by Knodell's histological activity index. The specimens were then immunohistochemically stained with alpha-SMA and semiquantitatively evaluated. Fibrosis and the immunoreactivity of alpha-SMA in the periportal, perisinusoidal and pericentral areas were compared. Fibrosis and necroinflammatory activity in CHB patients were significantly correlated (P =0.022). Furthermore, the degree of alpha-SMA expression and the scores of fibrosis (in periportal, perisinusoidal and pericentral areas) were highly correlated (P =0.000, 0.001, 0.000, respectively). CONCLUSIONS: In liver biopsy samples, alpha-SMA may prove to be a valuable marker in the evaluation of stellate cell activation and fibrosis progression and an early indicator of the development of fibrosis.     

A study by immunofluorescence microscopy of the NC1 domain of collagen type IV in glomerular basement membranes of two patients with hereditary nephritis

Summary The NC1 domain of the collagen type IV molecule, the major component of glomerular basement membranes (GBM), consists of dimers and 24 kilodalton (K), 26 K and 28 K monomers in man, and contains the Goodpasture antigen. Serum obtained from patients with Goodpasture's syndrome has been reported not to stain GBM of most male and some female patients with hereditary nephritis (HN) by immunofluorescence (IF) microscopy. In the present study, GBM seen on the renal biopsies of 2 patients (one male and one female) with HN were examined by IF to ascertain whether NC1 monomers were detectable. Three reagents were used: a plasmapheresis fluid (PPF) obtained from a patient who was treated for anti-GBM nephritis (human anti-GBM PPF); a commercial rabbit antibody against human NC1; and a rabbit antibody raised by us against dog NC1, which cross-reacted with human NC1. All 3 reagents detected NC1 determinants in GBM of normal human kidney by IF and reacted with human NC1 by a plate-binding radioimmunoassay (RIA). The human anti-GBM PPF bound to 28 K and 26 K monomer components of NC1 by Western blotting, the rabbit anti-human NC1 antibody bound to 26 K and 24 K monomers, while the rabbit anti-dog NC1 antibody bound only to the 26 K monomer. By IF, the human anti-GBM PPF did not stain GBM of the male patient with HN, but produced segmental staining of GBM (i.e., some GBM stained, while others did not) of the female patient. In contrast, the rabbit anti-NC1 antibodies produced global staining by IF of GBM of both patients. The absence of staining (i.e., global or segmental) seen with the human anti-GBM PPF implied that the 26 K and 28 K monomers of NC1 were either absent from GBM, or were present but altered structurally, leading to a diminution in their immunological reactivity. However, the positive staining observed with the rabbit anti-NC1 antibodies implied that the 26 K monomer was actually present in GBM. Hence, we postulate that the 26 K monomer of NC1 in GBM was structurally altered, and that the 28 K monomer was either absent, or present but altered. These findings suggest that there is an abnormality of more than one monomer of NC1 in GBM of patients with HN.     

Sequence variants in the HLX gene at chromosome 1q41-1q42 in patients with diaphragmatic hernia

Congenital diaphragmatic hernia (CDH) is a common birth defect for which few causative genes have been identified. Several candidate regions containing genes necessary for normal diaphragm development have been identified, including a 4–5 Mb deleted region at chromosome 1q41-1q42 from which the causative gene(s) has/have not been cloned. We selected the HLX gene from this interval as a candidate gene for CDH, as the Hlx homozygous null mouse has been reported to have diaphragmatic defects and the gene was described as being expressed in the murine diaphragm. We re-sequenced HLX in 119 CDH patients and identified four novel single nucleotide substitutions that predict amino acid changes: p.S12F, p.S18L, p.D173Y and p.A235V. These sequence alterations were all present in patients with isolated CDH, although patients with both isolated CHD and CDH with additional anomalies were studied. The single-nucleotide substitutions were absent in more than 186 control chromosomes. In-situ hybridization studies confirmed expression of Hlx in the developing murine diaphragm at the site of the junction of the diaphragm and the liver. Although functional studies to determine if these novel sequence variants altered the inductive activity of Hlx on the α-smooth muscle actin and SM22α promoters showed no significant differences between the variants and wild-type Hlx , sequence variants in HLX may still be relevant in the pathogenesis of CDH in combination with additional genetic and environmental factors.     

三七皂苷R1保护四氯化碳诱导肝纤维化模型大鼠的作用

背景：前期研究发现,三七总皂苷对小鼠免疫性肝损伤具有一定的保护作用。 目的：探究三七皂苷R1对四氯化碳诱导的肝纤维化模型大鼠的治疗作用。 方法：用四氯化碳诱导SD雄性大鼠制备肝纤维化模型,给药组按照60 mg/kg的剂量给予30 g/L三七皂苷R1溶液, 1次/d,连续4周和6周。对照组及模型组给予同体积的生理盐水,采用苏木精-伊红染色及Masson染色观察肝脏组织结构和纤维化程度分期;反转录-定量聚合酶链反应(qRT-PCR)检测法检测Ⅰ型胶原、α-平滑肌激动蛋白和转化生长因子β1表达水平。实验方案经昆明医科大学动物实验伦理委员会批准(批准号为approval No. KMMU2018018)。 结果与结论：①肝组织病理学显示,与模型组相比,三七皂苷R1能显著减轻纤维增生程度;②与模型组相比,三七皂苷R1组Ⅰ型胶原、α-平滑肌激动蛋白和转化生长因子β1表达水平显著降低(P < 0.05),三七皂苷R1给药4周与6周组比较差异无显著性意义;③结果提示,三七皂苷R1对四氯化碳诱导的肝纤维化模型大鼠具有一定的治疗作用。 ORCID: 0000-0002-0755-1476(吴朕) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

The importance of cellular adhesion for mesangial cell proliferation in serum sickness nephritis of the rat: A co-culture study of glomerular macrophages and mesangial cells

The role of cell-cell contact on activating mesangial cell proliferation by nephritic macrophage was investigated. Nephritic glomerular macrophages were obtained from serum sickness nephritis (SSN) rat kidneys at 14 days after the cessation of sensitization, when proliferating cells were most increased in the glomeruli in the course of the SSN. The effect of the nephritic macrophages on mesangial cell proliferation was greater than that of control by co-culture allowing cellular contact. However, nephritic macrophages did not enhance mesangial cell proliferation by co-culture without direct contact even though the nephritic macrophages were activated with lipopolysaccharides. Conditioned medium from co-culture of the nephritic macrophages and mesangial cells did not enhance mesangial cell proliferation. Anti-intercellular adhesion molecules (ICAM)-1 antibody inhibited mesangial cell proliferation by direct co-culture dose-dependently. From these results, cellular contact was important for stimulation of mesangial cell proliferation by macrophages and ICAM-1 participated in these interactions.     

Expression of VCAM-1, ICAM-1, E- and P-selectin and tumour-associated macrophages in renal cell carcinoma

AIMS: Neoangiogenesis is accompanied by an increase in endothelial surface, which can support infiltration by immune cells depending on adhesion molecule expression. Therefore, the expression of cell adhesion molecules on microvessels and epithelial cells was analysed in renal cell carcinomas as compared to tumour-free tissue. METHODS AND RESULTS: PECAM-1, CD34, ICAM-1, VCAM-1, VLA-4, P- and E-selectin, the macrophage antigens Ki-M1P and Mac-1, and lymphocyte function antigen LFA-1 were identified immunohistochemically. VCAM-1, ICAM-1, and E-selectin were equally or less expressed, whereas P-selectin was increased on microvessels in tumour tissue. The density of VCAM-1-positive tumour microvessels correlated positively with an advanced tumour stage and E- and P-selectin-positive tumour microvessels with the amount of associated macrophages. The expression of ICAM-1 and VCAM-1 on neoplastic epithelia correlated with an increased density of macrophages and a minor degree of tumour differentiation. CONCLUSIONS: The positive correlation of macrophage infiltration and expression of cell adhesion molecules on tumour microvessels and epithelia with minor tumour differentiation and an advanced stage indicates that adhesion molecule expression is not associated with an effective antitumour function of macrophages     

Effect of palmitic acid and linoleic acid on expression of ICAM-1 and VCAM-1 in human bone marrow endothelial cells (HBMECs)

Introduction

The amount and type of fatty acids (FAs) in the diet influence the risk of atherosclerosis. Palmitic acid and linoleic acid exist at high levels in Iranian edible oils. In this study, we investigated the effect of palmitic acid and linoleic acid on expression of soluble and cell-associated forms of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human bone marrow endothelial cells (HBMECs).

Material and methods

The endothelial cells were induced with bacterial lipopolysaccharide (LPS) or tumor necrosis factor α (TNF-α), and thereafter incubated with palmitic or linoleic acid. The level of soluble and cell-associated VCAM-1 and ICAM-1 were analyzed using ELISA and western blot.

Results

Our findings indicated that palmitic acid up-regulates the expression of ICAM-1 and VCAM-1 in HBMECs when these cells are induced with TNF-α or LPS. In addition, the results suggest that linoleic acid could sustain up-regulated ICAM-1 and VCAM-1 in activated endothelial cells.

Conclusions

Chronic activation of endothelial cells in the presence of palmitic and linoleic may account for pathogenesis of cardiovascular events. These findings provide further support for the detrimental effects of these fatty acids, especially palmitic acid, in promotion and induction of cardiovascular diseases which are prevalent in the Iranian population.     

Differential distribution of basement membrane type IV collagen alpha1(IV), alpha2(IV), alpha5(IV) and alpha6(IV) chains in colorectal epithelial tumors

In this study, we examined the relationship between the histopathological grade and immunohistochemical localization of six genetically distinct type IV collagen alpha chains, the major component of basement membrane (BM), in normal and neoplastic colorectal tissues. In the normal colorectal mucosa, alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were stained in all epithelial BM. However, alpha3/alpha4(IV) chains were restrictively immunostained in the BM of the apical surface epithelium. Similar immunostaining profiles for alpha1/alpha2(IV) and alpha5/alpha6(IV) chains were observed in tubular adenomas with mild/moderate atypia. However, in intramucosal carcinomas, both alpha1/alpha2(IV) chains were linearly stained in the BM of cancer cell nests, while the assembly of alpha5/alpha6(IV) chains into the BM was inhibited in a discontinuous or negatively stained pattern. The normal colorectal mucosa forms a second network of BM composed of alpha5/alpha6(IV), partly alpha3/alpha4(IV) chains, in addition to the classic network of alpha1/alpha2(IV) chains. The differential immunohistochemical localization of the type IV collagen alpha5/alpha6 chains could be one diagnostic marker for the invasiveness of colorectal cancer.     

Immunohistochemical analysis of pericryptal fibroblast sheath and proliferating epithelial cells in human colorectal adenomas and carcinomas with adenoma components

In order to examine stromal-epithelial interaction during the oncogenic progression of large bowel tumors, the association between pericryptal fibroblast sheath (PCFS) and expression of Ki-67 antigen was evaluated in 87 cases of colorectal adenoma and 95 cases of carcinoma with an adenoma component (CWA). For the immunohistochemistry, anti-alpha-smooth muscle actin antibody (alpha-SMA) and anti-Ki-67 antigen antibody (MIB-1) were used. In adenomas and adenoma components of CWA, the quantity of neoplastic glands with PCFS was reduced relative to the progression of histological atypia. Pericryptal fibroblast sheath was virtually absent from invasive carcinoma areas of CWA. Increased expression of Ki-67 correlated with the degree of histological atypia of adenomas. A significant reverse correlation was also seen between Ki-67 expression and PCFS-positive glands in adenoma components of CWA. These findings suggest that the prevalence of PCFS and Ki-67 expression are important indicators of colorectal neoplasia progression. The significant reduction of PCFS in colorectal epithelial neoplasms reflects progression in the adenoma-carcinoma sequence.     

内毒素致新生大鼠肺组织PECAM-1表达的研究

目的探讨内毒素作用于新生大鼠后,肺组织血小板内皮细胞粘附分子-1(PECAM-1)表达的变化及可能作用.方法用脂多糖(LPS)诱发7日龄新生大鼠肺损伤以至于肺出血模型,用免疫组化和RT-PCR方法观察肺组织PECAM-1蛋白水平和mRNA水平表达的变化.结果正常新生大鼠肺组织表达较高水平的PECAM-1蛋白水平和mRNA水平,LPS作用后,肺组织PECAM-1蛋白水平和mRNA水平表达均存在逐渐下降趋势,mRNA水平的表达在给药后1h即明显下降,蛋白水平在给药后4h明显下降,至LPS作用后8h降至最低水平,24h存活大鼠PECAM-1蛋白水平和mRNA水平有所恢复,二者的变化趋势基本一致.结论正常新生大鼠肺组织表达较高水平的PECAM-1蛋白及mRNA,PECAM-1可能在新生大鼠肺出血的发病机制中有一定的作用,并且PECAM-1表达变化的调节是在转录水平.     

红花含药血清对缺氧复氧条件下大鼠肺动脉内皮细胞P-选择素及ICAM-1表达的影响

目的：观察红花含药血清在缺氧复氧条件下对大鼠肺动脉内皮细胞P-选择素(Ps)及细胞间黏附因子-1(ICAM-1)基因表达的影响。方法：用细胞培养、RT-PCR、Western blotting、中药血清药理学方法、免疫细胞化学染色法等技术,观察在常氧、缺氧、复氧及红花含药血清干预等不同条件下大鼠肺动脉内皮细胞P-选择素及细胞间黏附因子-1基因的表达变化。结果： 缺氧组1、6和12 h各个时点P-选择素及ICAM-1 mRNA及蛋白水平与同时点常氧组明显差异。缺氧1 h后再给氧（缺氧复氧组）1、6和12 h后P-选择素及ICAM-1 mRNA及蛋白表达均明显高于同时点缺氧组及常氧组；缺氧1 h后再给氧同时给予红花含药血清干预各个时点P-选择素及ICAM-1 mRNA及蛋白明显低于单纯缺氧复氧组（P＜0.05）。结论：缺氧复氧条件下P-选择素及ICAM-1过度表达，可能参与肺栓塞溶栓后缺血再灌注损伤的机制，红花注射液对其有显著缓解作用。     

Heterogeneous response of nasal and lung fibroblasts to transforming growth factor-β1

Background Nasal polyposis is characterized by marked oedema, sparse extracellular matrix (ECM) and proliferating blood vessels. Pulmonary fibrosis is characterized by inflammatory cells accumulation, considerable ECM deposition and vascular abnormalities. Although lung fibrosis is not only and necessarily an inflammatory disorder, we hypothesized that the difference between nasal polyposis and pulmonary fibrosis may, in part, be due to the heterogeneity between nasal and lung fibroblasts. Fibroblasts participate in the inflammatory response by releasing ECM proteins and cytokines. TGF‐β is thought to participate in chronic inflammation and fibrosis. Myofibroblasts are the activated form of fibroblasts. A phenotypic hallmark of myofibroblasts is the expression of smooth muscle α‐actin (SMA). Objective We examined whether there is any heterogeneity between nasal and lung fibroblasts upon stimulation with TGF‐β₁ with regard to the synthesis of SMA, pro‐collagen type I and vascular endothelial growth factor (VEGF) as well as translocation of Smad proteins. Methods Fibroblasts lines were established from human biopsy tissue. The expression of SMA, pro‐collagen type I, VEGF mRNA was evaluated by reverse transciptase RT‐PCR. The amount of pro‐collagen type I and VEGF was measured by ELISA. By immunocytochemistry, we analysed the expression of SMA and Smad2, 3, 4 in cultured fibroblasts. Results TGF‐β₁ induced SMA and pro‐collagen type I synthesis in lung, but not in nasal fibroblasts. By contrast, TGF‐β₁ induced VEGF synthesis in both lung and nasal fibroblasts. After stimulation with TGF‐β₁, Smad2, 3, 4 were translocated from the cytoplasm to the nucleus in lung fibroblasts, whereas only Smad3 was translocated in nasal fibroblasts. Conclusion These results establish the heterogeneous responsiveness of fibroblast populations in the airways to TGF‐β₁ and that such a heterogeneity may contribute, at least in part, to the different pathological outcomes of inflammation in the upper and lower airways.     

Phenotypic changes and cell cycle activation in early tubulointerstitial injury of rat adriamycin nephrosis

Tubular response, including phenotypic changes against a variety of injuries, is an initial event that promotes tubulointerstitial injuries. Using the progressive kidney disease model of rat adriamycin (ADR) nephrosis, the present study focused on the cell cycle activation and phenotypic changes that occur in the tubuli in early tubulointerstitial injury in ADR nephrosis. At 12 weeks, experimental animals developed overt nephrosis with tubulointerstitial injury, which correlated well with the degree of proteinuria and incidence of glomerulosclerosis. Initial pathology of the tubuli showed a slight dilatation of tubuli, which tended to occur in individual nephrons. Immunohistochemistry demonstrated that vimentin-positive tubuli and osteopontin (OPN)-positive tubuli were associated mostly with proliferating cell nuclear antigen expression. Protein levels of OPN in the renal cortex were correlated with the level of proteinuria by western blotting. Vimentin- and OPN-expressing tubuli were tightly associated with a peritubular influx of alpha-smooth muscle actin (SMA)-positive cells or ED-1-positive cells. In addition, we found thrombomodulin+/ TUNEL+ (dUTP-biotin nick-end labeling) peritubular endothelial cells and ED-1+/alpha-SMA+ cells at an early stage among interstitial inflammatory cells. These results suggest that cell cycle activation in tubular cells forms the background for the phenotypic tubular changes that are involved in chronic tubulointerstitial injury in ADR nephrosis.     

Bronchoalveolar lavage fluid concentrations of transforming growth factor (TGF)-β1, TGF-β2, interleukin (IL)-4 and IL-13 after segmental allergen challenge and their effects on α-smooth muscle actin and collagen III synthesis by primary human lung fibroblasts

RATIONALE: Asthmatic airway remodelling is characterized by myofibroblast hyperplasia and subbasement membrane collagen deposition. We hypothesized that cytokines and growth factors implicated in asthmatic airway remodelling are increased in bronchoalveolar lavage (BAL) fluid of asthmatics after segmental allergen challenge (SAC), and that these growth factors and cytokines increase alpha-smooth muscle actin (alpha-SMA) and collagen III synthesis by human lung fibroblasts (HLFs). METHODS: Transforming growth factor (TGF)-beta1, TGF-beta2, IL-4 and IL-13 levels were measured in BAL fluid from 10 asthmatics and 9 non-asthmatic controls at baseline and then 1 day, 1 week and 2 weeks after SAC. Confluent cultures of HLFs were stimulated by exogenous addition of TGF-beta1, TGF-beta2, IL-4 or IL-13 (concentration range 0.01-10 ng/mL) over 48 h. Collagen III was measured in culture supernates and alpha-SMA in cell lysates by Western blot. RESULTS: At baseline, there was no difference in BAL fluid concentrations of TGF-beta1, IL-4 and IL-13 between asthmatics and controls; however, non-asthmatics had higher concentrations of total TGF-beta2. In asthmatics, BAL fluid concentrations of all four factors increased significantly 1 day after SAC. TGF-beta1, TGF-beta2 and IL-13 concentrations returned to baseline by 1 week after SAC, but BAL fluid IL-4 concentration remained elevated for at least 2 weeks. TGF-beta1, TGF-beta2 and IL-4 significantly increased alpha-SMA in fibroblasts, but only IL-4 caused corresponding increases in collagen III synthesis. IL-13 had no direct effects on collagen III synthesis and alpha-SMA expression. CONCLUSIONS: Because IL-4 caused a dose-dependent increase in alpha-SMA and collagen III synthesis, it may be an important cytokine mediating asthmatic airway remodelling. TGF-beta1 and TGF-beta2 may also play a role in airway remodelling by stimulating phenotypic change of fibroblasts to myofibroblasts. Additionally, collagen III synthesis appears to be independent of myofibroblast phenotype and is apparently regulated by different growth factors and cytokines.     

Epithelial hyperplasia of usual type expresses both S100-α and S100-β in a heterogeneous pattern but ductal carcinoma in situ can express only S100-α in a monotonous pattern

Immunohistochemical identification of myoepithelial cells using α-smooth muscle actin provides little information about the nature of solid or quasi-solid portions of epithelial hyperplasia and ductal carcinoma in situ (DCIS) because actin-rich myoepithelial cells are usually demonstrated only in the stromal–epithelial junction of both lesions. We studied the differential distribution of α-subunit (S100-α) and β-subunit (S100-β) of S100 protein in actin-negative areas of usual epithelial hyperplasia and DCIS by employing the streptavidin method with monospecific rabbit antibodies against each subunit. All usual epithelial hyperplasias ( n =17) were composed of heterogeneous epithelial cell types; cells expressing S100-α and/or S100-β were intermingled with non-expressing cells, resulting in a mosaic-like pattern. On the contrary, DCIS ( n =32) uniformly lacked immunoreactive S100-β; S100-α was diffusely expressed in 24 (68.8%) DCIS (three solid/comedo, 13 cribriform, four endocrine, one micropapillary, three papillary variants) and negative in the remaining eight (31.2%) DCIS (one cribriform, two micropapillary, four papillary and one apocrine variants). In conclusion, in contrast to usual epithelial hyperplasia that expresses both S100-α and S100-β in a heterogeneous pattern, DCIS can express only S100-α in a monotonous pattern, possibly signifying unidirectional differentiation toward secretory glandular epithelium.