Laminin-111 protein therapy prevents muscle disease in the mdx mouse model for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating neuromuscular disease caused by mutations in the gene encoding dystrophin. Loss of dystrophin results in reduced sarcolemmal integrity and increased susceptibility to muscle damage. The α₇β₁-integrin is a laminin-binding protein up-regulated in the skeletal muscle of DMD patients and in the mdx mouse model. Transgenic overexpression of the α₇-integrin alleviates muscle disease in dystrophic mice, making this gene a target for pharmacological intervention. Studies suggest laminin may regulate α₇-integrin expression. To test this hypothesis, mouse and human myoblasts were treated with laminin and assayed for α₇-integrin expression. We show that laminin-111 (α₁, β₁, γ₁), which is expressed during embryonic development but absent in normal or dystrophic skeletal muscle, increased α₇-integrin expression in mouse and DMD patient myoblasts. Injection of laminin-111 protein into the mdx mouse model of DMD increased expression of α₇-integrin, stabilized the sarcolemma, restored serum creatine kinase to wild-type levels, and protected muscle from exercised-induced damage. These findings demonstrate that laminin-111 is a highly potent therapeutic agent for the mdx mouse model of DMD and represents a paradigm for the systemic delivery of extracellular matrix proteins as therapies for genetic diseases.     

Functional integrin subunits regulating cell-matrix interactions in the intervertebral disc.

Cellular interactions with the extracellular matrix are key factors regulating cell survival, differentiation, and response to environmental stimuli in cartilagenous tissues. Much is known about the extracellular matrix proteins in the intervertebral disc (IVD) and their variations with region, age, or degenerative state of the tissue. In contrast, little is known of the integrin cell surface receptors that directly bind to and interact with these matrix proteins in the IVD. In almost all tissues, these integrin-mediated cell-matrix interactions are important for transducing environmental cues arising from mechanical stimuli, matrix degradation fragments, and cytokines into intracellular signals. In this study, cells from the nucleus pulposus and anulus fibrosus regions of porcine IVDs were analyzed via flow cytometry to quantify integrin expression levels upon isolation and after monolayer culture. Assays of cell attachment to collagens, fibronectin, and laminin were performed after functional blocking of select integrin subunits to evaluate the role of specific integrins in cell attachment. In situ distribution and co-localization of integrins and laminin were also characterized. Results identify integrin receptors critical for IVD cell interactions with collagens (alpha1beta1) and fibronectin (alpha5beta1). Additionally, dramatic differences in cell-laminin interactions were observed between cells of the nucleus and anulus regions, including differences in alpha6 integrin expression, cell adhesion to laminin, and in situ pericellular environments. These findings suggest laminin-cell interactions may be important and unique to the nucleus pulposus region of the IVD. The results of this study provide new information on functional cell-matrix interactions in tissues of the IVD.     

Secretion of amyloid precursor protein and laminin by cultured astrocytes is influenced by culture conditions

Although normally quiescent, astrocytes in the adult brain respond to various types of brain injury by rapidly dividing, swelling, extending cellular processes, and expressing increased amounts of glial fibrillary acidic protein (GFAP). These phenomena are collectively referred to as “astrogliosis.” Similarly, astroglia in primary culture stop dividing when they attain confluency, yet, as seen in situ, they retain their proliferative capacity for extended periods and resume rapid division when subcultured. To examine the impact of glial division on secretion of neuritepromoting factors, conditioned medium (CM) was removed from subconfluent, newly confluent, and longterm confluent (“aged”) neonatal rat astrocyte cultures, and from aged confluent cultures that had been repassaged, “Lesioned” (scraping with a rubber policeman), or triturated 3 days before harvest. Secretion of neurite-promoting factor(s) by glial cells into these CM was then assayed by treating neuroblastoma cultures with these various CM and quantitating neurite elaboration. Extensive neurite sprouting was elicited by CM from cultures just reaching confluency and from repassaged, lesioned, or triturated cultures. CM from aged confluent cultures did not induce sprouting. These results indicate that secretion of neurite-promoting factors(s) is regulated by glial division, and suggest that gliosis in situ may contribute to neurite sprouting by similar mechanisms. Immunoblot analysis demonstrated the presence in CM of varying amounts of laminin and amyloid precursor protein (APP), including isoforms containing the Kunitz-type protease inhibitor domain. CM from subconfluent cultures contained trace amounts of these protiens, but CM from cultures just reaching confluency contained significant amounts. Although CM from aged cultures contained barely detectable levels of either protein, trituration or repassage of aged cultures dramatically increased secretion of these proteins. APP-and laminin-enriched CM fractions promoted neuritogenesis to a similar level as respective unfractionated CM; anti-APP and antilaminin antisera blocked this effect. Purified human brain APP promoted neuritogenesis when added to non-conditioned medium and aged CM. Increased secretion of APP and laminin therefore mediates at least a portion of CM-induced neuronal sprouting; these proteins may perform analogous functions during astrogliosis in situ. © 1994 Wiley-Liss, Inc.     

Evaluation of laminin peptide fragments labeled with indium-111 for the potential imaging of malignant tumors

The laminin peptide fragments GYIGSR-NH² and CDPGYIGSR-NH² are known to bind to a 67-kDa laminin receptor. This receptor is understood to be expressed at higher than normal levels in malignant tumor cells, particularly those of breast and colon carcinomas. Peptides DTPA-GYIGSR-NH² (1), DTPA-(GYIGSR-NH²)² (2), DTPA-CDPGYIGSR-NH² (3), DTPA-(CDPGYIGSR-NH²)² (4), and negative control DTPA-GAGAGA-NH² (5) were prepared by solid-phase peptide synthesis. All five DTPA-conjugated peptides were subsequently radiolabeled with ¹¹¹ln and their tissue distribution evaluated in mice bearing C3H tumors. ¹¹¹In-3 and ¹¹¹In-4 showed the highest specific tumor localization. These preliminary data support further study of radiolabeled peptide fragments for the potential detection of malignant tumors of the breast and other organs. © Munksgaard 1997.     

Identification of the LAMB3 hotspot mutation R635X in a Hungarian case of Herlitz junctional epidermolysis bullosa

Abstract The Herlitz type of junctional epidermolysis bullosa (H-JEB) is a serve blistering disease affecting the skin and mucous membranes, which is usually lethal within the first year of life. The laminin 5 genes have been implicated as candidate genes for most patients with H-JEB. Recently two hotspot mutations were delineated in the LAMB3 gene, known as R42X and R635X, and have been noted in over 50% of mutant LAMB3 alleles. Here, we present a case of H-JEB of Hungarian origin with a neonatal lethal outcome. Monoclonal antibody staining showed a lack of expression of the laminin 5β3 chain, as a possible result of a mutation in one of the laminin 5 genes. Screening of the family identified the previously described mutation R635X in exon 14 of LAMB3 in each of the parents and one healthy sibling in the heterozygous form, while proband was homozygous for R635X, and the other sibling proved to be genotypically normal. These results underscore the widespread prevalence of R635X in H-JEB cases form around the world.     

Responsiveness and Minimal Clinically Important Difference of the Motor Function Measure in Collagen VI-Related Dystrophies and Laminin Alpha2-Related Muscular Dystrophy

ObjectivesTo investigate the responsiveness of the motor function measure (MFM) and determine the minimal clinically important difference (MCID) in individuals with 2 common types of congenital muscular dystrophy (CMD).DesignObservational, prospective, single center, cohort study.SettingNational Institute of Neurological Disorders and Stroke (NINDS) of the National Institutes of Health (NIH).ParticipantsIndividuals (N=44) with collagen VI-related dystrophies (COL6-RD, n=23) and 21 individuals laminin alpha2-related muscular dystrophy (LAMA2-RD, n=21) enrolled in a 4-year longitudinal natural history study.InterventionsNot applicable.Main Outcome MeasuresResponsiveness of the MFM-32 and the Rasch-scaled MFM-25 and the MCID of the MFM-32 determined from a patient-reported anchor with 2 different methods, within-patient and between-patient.ResultsThe original MFM-32 and Rasch-scaled MFM-25 performed similarly overall in both the COL6-RD and LAMA2-RD populations, with all subscores (D1, standing and transfers; D2, axial and proximal; D3, distal) showing a significant decrease over time, except MFM D1 and D3 for LAMA2-RD. The MFM D1 subscore was the most sensitive to change for ambulant individuals, whereas the MFM D2 subscore was the most sensitive to change for nonambulant individuals. The MCID for the MFM-32 total score was calculated as 2.5 and 3.9 percentage points according to 2 different methods.ConclusionsThe MFM showed strong responsiveness in individuals with LAMA2-RD and COL6-RD. Because a floor effect was identified more prominently with the Rasch-Scaled MFM-25, the use of the original MFM-32 as a quantitative variable with the assumption of scale linearity appears to be a good compromise. When designing clinical trials in congenital muscular dystrophies, the use of MCID for MFM should be considered to determine if a given intervention effects show not only a statistically significant change but also a clinically meaningful change.     

血清肝纤维化标志物与慢性丁型肝炎肝纤维化程度关系的探讨

目的探讨ALT、HBV DNA及血清纤维化标志物透明质酸(HA)、层黏连蛋白(LN)、Ⅲ型前胶原肽(PⅢP)、Ⅳ型胶原(CⅣ)与慢性丁型肝炎(CHD)患者肝纤维化程度的关系。方法收集2008年3月至2013年1月在广州市第八人民医院肝病科住院并未曾接受任何抗病毒治疗的CHD患者28例,检测患者血清中ALT、HBV DNA和纤维化标志物(HA、LN、PⅢP及CⅣ)的水平,并行肝活组织检查检测肝组织纤维化分期。HBe Ag阳性与阴性组间计量资料比较采用t检验,计数资料比较采用χ2检验,不同纤维化分期间比较采用非参数检验中的Kruskal-Wallis检验;相关性分析采用Spearman等级相关分析。结果 HBe Ag阳性与HBe Ag阴性组患者间肝纤维化程度差异无统计学意义(χ2=0.129,P=0.720)。肝纤维化S1、S2及S≥3 3组间HA及LN水平差异有统计学意义(Z值分别为12.035、8.457,P值分别为0.002、0.015),其中S≥3组患者水平最高[HA:(169.4±166.3)ng/ml,LN:(149.2±85.1)ng/ml]且与S1、S2组比较差异均存在统计学意义(P值均0.05),而3组间其他实验室指标ALT、HBV DNA、PⅢP及CⅣ水平差异均无统计学意义(P值均0.05)。肝纤维化分期与HA、LN、PⅢP及CⅣ水平呈正相关(r值分别为0.512、0.472、0.451、0.454,P值分别为0.005、0.011、0.016、0.015)。结论 HA、LN水平可能作为评估CHD患者肝纤维化程度的血清学指标。     

层粘蛋白在人原发性肝细胞癌转移中的意义

目的了解层粘蛋白（ＬＮ）在原发性肝细胞癌（ＨＣＣ）转移中的作用和价值．方法应用放射免疫分析法（ＲＩＡ）、免疫组化和图象分析分别对１２例ＨＣＣ有转移者和１２例ＨＣＣ无转移者血清和癌组织中ＬＮ进行测定，并进行相关分析．结果ＨＣＣ有转移组患者血清中ＬＮ含量（μｇ／Ｌ）１７８±３２，显著高于ＨＣＣ无转移组１４０±２３（Ｐ＜００１）．ＨＣＣ有转移组患者组织中ＬＮ平均Ａ（ＯＤ值）为０１７±００６，显著低于ＨＣＣ无转移组０２６±００５（Ｐ＜００１）．组织中ＬＮ与血清ＬＮ含量成明显的负相关关系．免疫组化结果显示，ＬＮ不仅存在于间质中，还存在于癌细胞中；ＬＮ亦可排列成窦状，尤以ＨＣＣ转移者组织中为明显．结论ＬＮ在ＨＣＣ转移过程中具有双重作用，血清ＬＮ含量升高可作为衡量ＨＣＣ转移的一个简便而有用的指标     

Serum type IV collagen in various liver diseases in comparison with serum 7S collagen,laminin, and type III procollagen peptide

The clinical significance of the immunoreactive triple helical domain of type IV collagen in serum was evaluated in 73 healthy controls and 161 patients with various biopsy-proven liver diseases. Although serum levels of type III procollagen peptide were increased in all liver diseases, those of type IV collagen, 7S collagen, and laminin were principally increased in chronic liver diseases associated with hepatic fibrogenesis/fibrosis. In both non-alcoholic and alcoholic liver diseases, 7S collagen was increased in serum, while type IV collagen and laminin in serum were particularly increased in alcoholic liver diseases and in hepatocellular carcinoma, in which latter the sensitivity was greater for type IV collagen than for laminin. Gel filtration analysis in Sephacryl S-400 revealed type IV collagen in serum to be a single molecular form with a molecular weight that correspond to type IV collagen, whereas 7S collagen was recognized as several heterogeneous macromolecules. These findings indicate that serum type IV collagen is derived from the type IV protocollagen pool, and is a sensitive marker for the fibrogenetic process in hepatic basement membranes.     

慢性肾脏病血清及尿液Ⅳ型胶原和层黏连蛋白与肾组织纤维化的关系

目的 探讨慢性肾脏病（Chronic Kidney Disease,CKD）血清及尿液Ⅳ型胶原和层黏连蛋白与肾组织纤维化的关系.方法 以63例住院接受肾穿刺活检、未接受糖皮质激素、免疫抑制剂、血管紧张素转化酶抑制剂、血管紧张素Ⅱ受体拮抗剂和中草药等治疗的1～2期的CKD患者为研究对象,其中男性38例,女性25例.年龄为（40.5±17.5）岁,最小23岁,最大58岁.血清及尿液Ⅳ型胶原、层黏连蛋白采用酶联免疫吸附法测定.肾组织ColⅣ、LN表达强度利用病理图像分析软件对免疫组化阳性目标进行光密度测定.分析肾组织Ⅳ型胶原、层黏连蛋白光密度与血清、尿液Ⅳ型胶原、层黏连蛋白水平的关系.应用SPSS 11.0软件包进行统计分析,单因数相关分析采用Pearson相关分析.结果 CKD患者血清中Ⅳ型胶原及层黏连蛋白水平与肾组织纤维化程度无关,而尿液Ⅳ型胶原及层黏连蛋白水平与肾组织纤维化程度呈正相关.结论 尿液Ⅳ型胶原、层黏连蛋白水平能够反映早期CKD患者肾脏纤维化发生的程度.