Extracellular Matrix of Small Round cell Tumors of Childhood: An Immunohistochemical Study of 67 Cases

Sixty-seven childhood tumors were studied immunohistochemically for the extracellular matrix elements type IV collagen, laminin, and fibronectin. Tumors included Ewing's sarcoma, primitive neuroectodermal tumor, small cell osteosarcoma, neuroblastoma or ganglioneuroblastoma, rhabdomyosarcoma, and lymphoma. It was found that small cell osteosarcoma was often positive for fibronectin but not type IV collagen or laminin, a new observation. In the lymphomas, matrix proteins were rarely found. Ewing's sarcoma was variably positive for type IV collagen and laminin, but fibronectin was absent. Extracellular laminin and fibronectin were found in one of two cases of primitive neuroectodermal tumor. In neuroblastoma and ganglioneuroblastoma, the matrix components were rarely found. These results, discrepant with findings in cultured cells, may reflect the altered capacity of tumors to produce these proteins in vitro, which suggests that caution should be exercised in drawing conclusions regarding the nature or histogenesis of tumors from data obtained with cultured tumor cells. Embryonal rhabdomyosarcoma frequently contained all matrix elements in the extracellular space and in a dotlike pattern in the cytoplasm; alveolar rhabdomyosarcoma rarely contained these proteins and never exhibited the dotlike pattern. The frequent finding of matrix proteins in embryonal rhabdomyosarcoma but only rarely in alveolar rhabdomyosarcoma and the unique immunostaining pattern in embryonal rhabdomyosarcoma may prove to be a useful adjunct in the diagnosis of childhood tumors.     

Concentration of plasma fibronectin and metabolism indices in the bone tissue of healthy children and in children with rickets

A study was made of the content of plasma fibronectin as compared to the biochemical parameters of mineral and collagen metabolism in 177 children. Of these, there were 90 healthy children aged 1 month to 2 years and 87 children of the same age with grade I and II rickets at the height of illness. A close interrelation was established of the metabolic parameters characterizing metabolism of osseous tissue both in healthy children and in those with rickets. In the latter ones, plasma fibronectin was discovered to significantly decrease as the process becomes graver and more active. Besides, its level was found to be related to collagen metabolism.     

Achondrogenesis type II, abnormalities of extracellular matrix

Immune and lectin histochemical and microchemical methods were employed to study growth cartilage from seven cases of achondrogenesis type II (Langer-Saldino). The normal architecture of the epiphyseal and growth plate cartilage was replaced by a morphologically heterogeneous tissue. Some areas were comprised of vascular canals surrounded by extensive fibrous tissue and enlarged cells that had the appearance and histochemical characteristics of hypertrophic chondrocytes. Other areas contained a mixture of cells ranging from small to the enlarged chondrocytes. The extracellular matrix in the latter areas was more abundant and had characteristics of both precartilage mesenchymal matrix and typical cartilage matrix; it contained types I and II collagen, cartilage proteoglycan, fibronectin, and peanut agglutinin binding glycoconjugate(s). Peptide mapping of cyanogen bromide cartilage collagen peptides revealed the presence of types I and II collagen. These observations could be explained by a defect in the biosynthesis of type II collagen or in chondrocyte differentiation.     

Expression of Laminin and Fibronectin in Renal Dysplasia

The pathogenesis of renal dysplasia is a matter of debate. Recent theories have conceptualized the role of extracellular matrix proteins in the genesis of renal dysplasia. During normal nephrogenesis, collagen type I and III and fibronectins are lost and laminin and syndecan appear once proper induction has occurred. Any deviation from the normal pattern is said to lead to dysplasia. In this study, the expressions of adhesive glycoproteins, laminin, and fibronectin were studied immunohistochemically in 25 autopsy cases of renal dysplasia and normal age-matched control cases. These cases of renal dysplasia were categorized into 3 groups based on the period of gestation: 20 to 26 weeks, 27 to 33 weeks, and 34 to 40 weeks. The immunohistochemical findings were graded from 0 to 4+ based on the visual intensity. Chi-square analysis was used to calculate the difference in expressions of laminin and fibronectin in cases and controls as a whole and within and between age groups. Immunostaining for laminin in all age groups showed a significant difference in expression between dysplastic kidneys (less expression) and normal controls (greater expression). In the case of fibronectin expression, all but 1 group showed a significant difference, with dysplastic kidneys showing more and normal controls showing less expression. The inference derived is that laminin expression decreases and fibronectin expression increases in renal dysplasia compared with normal nephrogenesis.     

HGF对TGF-β1诱导尿道瘢痕成纤维细胞α-SMA及细胞外基质过度合成的保护作用

目的 探讨HGF对TGF-β1诱导尿道瘢痕成纤维细胞α-SMA及细胞外基质过度合成的保护作用.方法 收集尿道下裂术后瘢痕组织的标本,进行尿道瘢痕成纤维细胞的分离和培养.待细胞生长成单层后,以胰蛋白酶消化传代.取第四代成纤维细胞用于实验,当细胞达到80%融合时,培养液中加入TGF-β1(5ng/ml)及HGF(10～40ng/ml).培养72 h后,用RT-PCR检测各组α-SMA mRNA的变化;ELISA测定细胞Ⅰ、Ⅲ型胶原及纤维结合素的表达.结果 TGF-β1能显著诱导α-SMA m-RNA表达.随HGF的加入,α-SMA m-RNA的表达则明显受到抑制(P＜0.05),且随HGF浓度的升高其阻抑作用呈逐渐增强趋势.对照组、单纯TGF-β1组、加入TGF-β1与10、20、40ng/ml HGF组的Ⅰ型胶原A值分别为0.51±0.04、0.78±0.05、0.71±0.02、0.63±0.03、0.57±0.02,Ⅲ型胶原A值分别为0.12±0.01、0.29±0.02、0.21±0.02、0.14±0.01、0.08±0.01,纤维结合素A值分别为0.24±0.03、0.51±0.02、0.49±0.01、0.38±0.02、0.28土0.01.表明TGF-β1同样能诱导Ⅰ、Ⅲ型胶原及纤维结合素的表达(P＜0.01),而HGF则可以有效地阻抑其表达,其效应呈剂量依赖性(P＜0.05).结论 HGF对TGF-β1诱导的尿道瘢痕成纤维细胞α-SMA及细胞外基质过度合成具有抑制作用.这为临床预防和治疗尿道瘢痕狭窄提供了理论依据.

Abstract：

Objective To examine the inhibitory effects of HGF on TGF-β1 induced a-SMA and extracellular matrix synthesis in cultured fibroblasts derived from urethral scar. Methods Fibroblasts isolated from urethral scar were cultured ex vivo. After the fibroblasts reached confluence, cells were detached using trypsin/ethylenediamine tetra-acetic acid. All experiments were performed using the cells at the fourth passage. At 80% confluence, TGF-β1 (5 ng/ml) and HGF (10-40 ng/ml) were added to the culture medium. After 72 hours co-incubation, the mRNA of a-SMA was studied by RTPCR. The productions of collagen Ⅰ, Ⅲ and fibronectin in supernatants were also examined using ELISA. Results TGF-β1 markedly induced a-SMA mRNA expression in cultured fibroblasts. However, HGF could abrogated TGF-β1-induced a-SMA mRNA expression in a dose-dependent manner (P＜0. 05). The levels of collagen type Ⅰ were 0. 51 ± 0. 04,0. 78 ± 0. 05,0. 71 ± 0. 02,0. 63 ± 0. 03, and 0. 57 ± 0. 02 in control, TGF-β1, TGF-β1 + 10 ng/ml HGF, TGF-β1 + 20 ng/ml HGF, and TGF-β1 +10 ng/ml HGF group, respectively. The levels of collagen type Ⅲ were 0. 12 ± 0. 01,0. 29 ± 0. 02,0. 21 ± 0. 02,0. 14 ± 0. 01, and 0. 08 ± 0. 01 in control, TGF-β1, TGF-β1 + 10 ng/ml HGF, TGF-β1 +20 ng/ml HGF, and TGF-β1 + 10 ng/ml HGF group, respectively. And the levels of fibronectin were 0.24±0.03,0.51 ± 0.02,0.49 ± 0.01,0.38 ± 0.02,0.28 ± 0.01 in control, TGF-β1, TGF-β1 +10 ng/ml HGF, TGF-β1 + 20 ng/ml HGF, and TGF-β1 + 10 ng/ml HGF group, respectively. TGF-β1 significantly stimulated collagen Ⅰ, Ⅲ and fibronectin production in fibroblasts (P＜0. 01 ). However,HGF could reduced abrogated the up-regulation of collagen Ⅰ, Ⅲ and fibronectin induced by TGF-β1 in a dose-dependent manner (P＜0. 05). Conclusions HGF can effectively inhibit TGF-β1 induced aSMA and extracellular matrix synthesis in cultured fibroblasts derived from urethral scar.     

整合素β3受体信号途径在结缔组织生长因子调控肺动脉平滑肌细胞增殖与迁移和细胞外基质基因表达中的作用

目的 探讨整合素β3受体信号途径在结缔组织生长因子(CTGF)促进肺动脉平滑肌细胞增殖、迁移和细胞外基质基因表达中的作用机制研究.方法 分离并培养SD大鼠肺动脉中膜平滑肌细胞,实验使用第3～7代细胞;细胞分为3组:(1)空白组:不加任何刺激因子;(2)CTGF组:培养体系中加入CTGF(50 ng/ml);(3)CTG...     

Plasma fibronectin concentrations in healthy and septic infants

The concentration of plasma fibronectin was determined by Laurell's electroimmunoasay [15] in 75 preterm or term newborns within the first 2 days of life, in 97 healthy infants aged from 3 days to 12 months, in 40 septic infants and in 38 healthy adult subjects. The mean fibronectin concentration in citrated plasma of normal adults was 318±84 ml/l. Healthy eutrophic term newborns 1–2 days old had approximately one-third of the fibronectin concentration of adults. There was no significant difference in the values between healthy term and eutrophic preterm newborns or between eutrophic and hypotrophic newborns. The plasma fibronectin increased strongly over the 1st month of life. No significant difference was observed between fibronectin levels in infant boys and girls. The values in septic newborns and septic older infants were significantly lower when compared with those of age-matched healthy controls. It is speculated that this deficiency, because of linkage to fibrin in disseminated intravascular coagulation or due to increased utilisation as a non-specific opsonin and sequestration at sites of tissue injury, may contribute to organ failure in septicaemia.Abbreviations SB surface binding - DIC disseminated intravascular coagulation     

Presence of type III collagen in bone from a patient with osteogenesis imperfecta

Samples of bone from a patient with osteogenesis imperfecta were found to synthesize and contain type III collagen as well as type I collagen. Normal bone contains only type I collagen except in the lining cells of the bone marrow cavities. In the patient's tissue, type III collagen was localized in nonfibrillar structures in discrete areas of the bone. These and previous studies indicate that certain types of osteogenesis imperfecta may be caused by a failure of normal bone maturation and the sites in which the type III collagen is found appear to be defects in the bone.     

DIVERGENT PATTERNS OF EXTRACELLULAR MATRIX PROTEIN EXPRESSION IN NEONATAL VERSUS ADULT LIVER FIBROSIS

The extracellular matrix (ECM) expression is subject to distinct changes during ontogeny, and the natural course of liver fibrosis in neonates is thought to differ from that in adults. We compared the expression and distribution of main ECM components between neonatal and adult liver fibrosis. Liver biopsies from infants with neonatal cholestasis and fibrosis were compared to adult biopsies exhibiting an equivalent stage of fibrosis. All biopsies were examined by immunohistochemistry (indirect ABC method) for the ECM proteins, collagens I, III, IV, and VI, laminin, and fibronectin. Infants (aged 1-8 months) with neonatal hepatitis (n=3), extrahepatic biliary atresia (EHBA) (n=5), and normal histology (n=2) were compared with 9 adults (aged 17-70 years) with chronic hepatitis (n=3), primary biliary cirrhosis (PBC) (n=4), and normal histology (n=2). Collagens I, III, and IV and fibronectin were significantly increased in neonatal hepatitis with mild fibrosis (score ≤4) compared to adults with an equivalent fibrosis stage. This increase was particularly notable in perisinusoidal spaces. Laminin expression was increased in portal and perisinusoidal spaces both in neonatal hepatitis and extrahepatic biliary atresia with mild fibrosis. In infants with moderate to severe fibrosis (score ≥6), only collagen I was increased in comparison to adults, whereas collagen VI expression was identical in all groups, irrespective of the degree of fibrosis. Expression of matrix proteins was not different in infants and adults without fibrosis. The increased perisinusoidal deposition of certain ECM components in infants with active hepatitis and mild fibrosis may point to an underlying difference in the mechanism or stimulus of fibrogenesis in neonates as compared to adults.     

Growth factors in cystic fibrosis - when more is not enough

In the airways of patients with cystic fibrosis, repeated cycles of infection and inflammation are responsible for bronchial wall thickening, a major determinant of loss of FEV(1) and progressive damage to the small and large airways. Proteolytic degradation of elastin, collagen and fibronectin fibrils in the tissue matrix leads to the loss of normal tissue architecture and the development of bronchiectasis, the most commonly observed morphological change on high-resolution computed tomography examination. We have reviewed the evidence for increased expression of growth factors (TGF, HGF, FGF, EGF, VEGF) and activation of tissue repair processes in cystic fibrosis. Significantly higher concentrations of the growth factors compared with normal do not appear to prevent or reverse structural remodelling in the airways. The reasons why this process appears to be ineffective are discussed and we speculate on alternative strategies that might have a significant impact on the observed structural changes.     

Diffuse Leiomyomatosis of the Esophagus: Disorder of Cell-Matrix Interaction?

Diffuse leiomyomatosis (DL) is rare condition characterized by proliferation of smooth muscle in the upper gastrointestinal tract. Most cases are associated with X-linked Alport syndrome and have partial deletions in the genes encoding both the α5 and α6 chains of collagen type IV. We studied aspects of cell-matrix interaction of myocytes in an esophagogastrectomy specimen from a 12-year-old patient with DL. Myocytes had central areas of cytoplasmic rarefaction, which were actin positive and desmin poor, with the reverse pattern of staining at the cell periphery. Electron microscopy (EM) showed that the areas of rarefaction consisted of disorganized aggregates of filaments. The basement membranes ranged from thickened to thinned or absent. Immunohistochemical staining for the α1–α4 chains of collagen type IV, the α1, α2, β2, and γ1 chains of laminin, nidogen, type VI collagen, and fibronectin was normal. There was loss of the α5 and α6 chains of collagen type IV and the β1 chain of laminin. Normal staining for α1, α2, α3, α4, α6, α8, and β1 integrins was noted. Staining for α5 integrin varied from normal to reduced or negative in different cells. In DL, a primary abnormality of basement membrane may be associated with disorganization of the contractile apparatus and alterations of certain integrins. This may reflect a disturbance of cell-matrix interactions that play a role in cell differentiation and internal organization. Received October 10, 1997; accepted February 4, 1998.     

The roles of extracellular matrix proteins,apoptosis and c-kit positive cells in the pathogenesis of ureteropelvic junction obstruction

AimTo investigate histopathological changes in ureteropelvic junction obstruction (UPJO) from an etiological perspective.Patients and methodsMedical records of patients with UPJO were reviewed and pathological specimens collected. Nephrectomy materials from forensic autopsies were taken as controls. Specimens were assessed with light microscopy. Fibronectin, type 4 collagen, laminin, Bax and Bcl-2 expression for apoptosis, together with interstitial cells of Cajal determination with c-kit were determined immunohistochemically. Staining scores were evaluated semiquantitatively. Results were evaluated using Mann–Whitney U-test.ResultsControl group comprised 14 children (median age, 3.5 years; 6 months–17 years). Study group comprised 22 children with UPJO (median age, 9 months; 1 month–10 years). Light microscopy revealed non-specific inflammation, epithelial proliferation and atrophy with fibrosis in the smooth muscle of the UPJ in all patients. Fibronectin, type 4 collagen and laminin were found to be significantly increased in UPJO at the intrafascicular space of smooth muscle and the matrix of stroma. Bcl-2 expression was increased in UPJO. c-Kit was unable to stain interstitial cells of Cajal, but staining for mast cells was significant.ConclusionsHigh expression of fibronectin, laminin and type 4 collagen may indicate a relation to the pathogenesis of UPJO. Defective kidney morphogenesis, during branching and tubulogenesis of ureteric bud, may be responsible for this congenital pathology.     

Plasma fibronectin levels in extremely preterm infants in the first 8 weeks of life

Abstract Fibronectin has in the past been considered to function simply as a non-specific plasma opsonin. However, recent studies have demonstrated that this molecule plays an important role in fundamental components of the immune response, for example, neutrophil adhesion, T cell activation and endothelial function. Additionally, fibronectin is important in lung homeostasis where it contributes to alveolar epithelial integrity. In this study plasma fibronectin levels were measured longitudinally in a group of extremely preterm infants, mean gestational age 27 weeks.
Plasma fibronectin levels at birth were significantly lower in the preterm study group than in term controls (mean 91±33 μg/mL compared with 214±62 μg/mL in the term controls, P <0.0001). The preterm cohort demonstrated a more than two-fold rise in plasma fibronectin on days one and two; levels fell almost to baseline values by day three with a subsequent slow rise to a plateau by day 28. No further increase was seen by day 56. This sequence of early changes in fibronectin levels mirrored closely the time course of respiratory distress syndrome. Infants of mothers with pre-eclampsia had significantly lower peak fibronectin levels than in those without ( P = 0.016), and those infants with bronchopulmonary dysplasia showed a trend towards lower basal fibronectin levels ( P = 0.07) and a greater difference between peak and basal levels ( P = 0.05).
Neonates, particularly those born preterm, have blunted immunological responses to infection. Fibronectin plays a key role in immunological responsiveness. The significant changes in fibronectin levels after birth in the preterm neonate are likely to have important pathophysiological consequences. The relationship between alterations in fibronectin after birth, endothelial and epithelial cell function, and respiratory distress syndrome (RDS) remain to be explored.     

Comparison of in vivo immune responses following transplantation of vascularized and non-vascularized human dermo-epidermal skin substitutes

Purpose

Autologous bio-engineered dermo-epidermal skin substitutes (DESS) represent an alternative therapeutic option for a definitive treatment of skin defects in human patients. Largely, the interaction of host immune cells with transplanted DESS is considered to be essential for the granulation tissue formation, graft take, and its functionality. The aim of this study was to compare the spatiotemporal distribution and density of host-derived monocytes/macrophages and granulocytes in vascularized (vascDESS) versus non-vascularized DESS (non-vascDESS) in a rat model.

Methods

Keratinocytes and the stromal vascular fraction (SVF) were derived from human skin or human adipose tissue, respectively. Human SVF containing both endothelial and mesenchymal/stromal progenitors was used to develop a vascularized collagen type I-based dermal component in vitro. The donor-matched, monolayer-expanded adipose stromal cells lacking endothelial cells were used as a negative control. Subsequently, human keratinocytes were seeded on top of hydrogels to build dermo-epidermal skin grafts. After transplantation onto full-thickness skin wounds on the back of immuno-incompetent rats, grafts were excised and analyzed after 1 and 3 weeks. The expression of distinct inflammatory cell markers specific for host-derived monocytes/macrophages (CD11b, CD68) or granulocytes (HIS48) was analyzed by immunofluorescence microscopy.

Results

All skin grafts were infiltrated by host-derived monocytes/macrophages (CD11b⁺, CD68⁺) and granulocytes (HIS48⁺) between 1–3 week post-transplantation. When compared to non-vascDESS, the vascDESS showed an increased granulocyte infiltration at all time points analyzed with the majority of cells scattered throughout the whole dermal part. Whereas a moderate number of rat monocytes/macrophages (CD11b⁺, CD68⁺) were found in vascDESS at 1 week, only a few cells were detected in non-vascDESS. We observed a time-dependent decrease of monocytes/macrophages in all transplants at 3 weeks.

Conclusions

These results demonstrate a distinct spatiotemporal distribution of monocytes/macrophages as well as granulocytes in our transplants that closely resemble the one observed during physiological wound healing. The differences identified between vascDESS and non-vascDESS may indicate that human endothelial cells lining blood capillaries of vascDESS accelerate infiltration of monocytes and leukocytes.

     

Plasma fibronectin concentrations in breast fed and formula fed neonates.

Plasma fibronectin concentration was measured in neonates of 2 to 5 days of age. Although breast fed and formula fed infants were similar in demographic characteristics, the mean (SD) plasma concentration of fibronectin in 26 breast fed infants, 237 (117) mg/l, was significantly higher than in 27 formula fed infants (171 (91) mg/l). Fibronectin was detected in five colostrum specimens (mean concentration 13.4 mg/l). Similar bands were detected after gel electrophoresis of purified adult plasma fibronectin and whole plasma from breast fed and formula fed neonates after staining or immunoblotting. Fibronectin isolated from breast milk also appeared similar to purified plasma fibronectin. It is possible, although unlikely, that fibronectin is absorbed intact from ingested colostrum. Alternatively, a factor(s) might be present in colostrum that contributes to the regulation of plasma fibronectin concentration.     

CLD早产鼠肺组织内TGF -β1的动态表达及其对细胞外基质的影响(英文)

目的　近年来发现慢性肺疾病 (CLD)早产儿支气管肺泡灌洗液中转化生长因子 β1(TGF β1)及I型胶原等细胞外基质 (ECM )水平增高 ,但由于临床研究的局限性 ,缺乏与肺组织ECM的对照研究。因此探讨TGF β1在早产鼠CLD发生、发展中的变化规律及对ECM的影响对完善早产儿CLD的发生机制有重要意义。 方法　将 6 0例早产鼠随机分为模型组和对照组 ,于实验后 1,3,7,14和 2 1d ,应用免疫组织化学和酶联免疫吸附法 ,分别观察及测定肺组织TGF β1分布、表达强度及I型胶原、纤维连接蛋白 (FN)及透明质酸 (HA)的含量。 结果　正常肺组织TGF β1仅在支气管上皮细胞或血管内皮细胞有微弱表达 ;而模型组 1d和 3d时 ,TGF β1表达同正常对照组 ,7d时少量肺泡巨噬细胞、肺泡上皮细胞及肺间质细胞开始表达 ,其表达强度高于对照组 ,差异有显著性(P <0 .0 5 ) ,14d时明显增强 ,差异有极显著性意义 (P <0 .0 1) ,2 1d达高峰 ;1,3和 7d时 ,两组肺组织I型胶原含量无差异 (P >0 .0 5 ) ,而 14d时 ,模型组高于对照组 ,差异有显著性 (P <0 .0 5 ) ,2 1d时明显高于对照组 ,差异有极显著性意义 (P <0 .0 1) ;不同吸氧时间 ,两组肺组织FN及HA含量均差异无显著性意义 (P >0 .0 5 ) ;模型组在 14d和 2 1d时 ,肺组织TGF β1表达与I型胶原含     

Clinico-diagnostic significance of fibronectin determination in diabetes mellitus in children

Immunoelectrophoresis was used to measure fibronectin content in blood plasma of 111 children with insulin-dependent diabetes mellitus and in 20 normal children. In patients with diabetes mellitus, the content of plasma fibronectin turned out to be higher depending on the disease standing, the presence of microangiopathies, and status of compensation. It has been noted that fibronectin was detectable in urine in diabetic angionephropathy.     

Decreased plasma fibronectin in neonatal sepsis

Fibronectin is a large opsonic glycoprotein which promotes reticuloendothelial system clearance of bacteria, immune complexes, collagenous debris, and damaged platelets. The concentration of plasma fibronectin is decreased in the newborn infant; however, the role of fibronectin in the onset and course of neonatal sepsis is unknown. Serial plasma fibronectin levels were determined in 19 neonates with documented bacterial sepsis. Plasma fibronectin concentrations decreased significantly (P less than .001) in all study infants concurrent with the development of septicemia. Recovery of plasma fibronectin to normal levels occurred by day 5 in premature neonates and by days 7 to 10 in term neonates. Fibronectin deficiency and resultant reticuloendothelial system impairment may decrease the ability of newborn infants to resist or clear bacterial infections. An acute reduction in the concentration of plasma fibronectin may be a valuable marker for neonatal sepsis.     

Serum fibronectin in neonatal sepsis: Is it valuable in early diagnosis and outcome prediction?

The value of the serum fibronectin level in early diagnosis of neonatal sepsis and as a prognostic indicator was investigated. The serum fibronectin levels and Töllner's sepsis scores of 45 neonates who were hospitalized for the suspicion of infection and of 20 healthy neonates as controls were evaluated. Depending on the findings it was concluded that serum fibronectin level varies according to the gestational age, and that the serum fibronectin level is a useful acute phase reactant in the early diagnosis of neonatal sepsis. It can also be used as a prognostic indicator in neonatal sepsis.