Matrilin-3 mutations that cause chondrodysplasias interfere with protein trafficking while a mutation associated with hand osteoarthritis does not.

Several mutations in the extracellular matrix protein matrilin-3 cause a heterogeneous disease spectrum affecting skeletal tissues. We introduced three disease causing point mutations leading to single amino acid exchanges (R116W, T298M, C299S) in matrilin-3 and expressed the corresponding proteins in primary articular chondrocytes to elucidate pathogenic mechanisms at the cellular level. Expression levels, processing, and the secretion pattern of a mutation linked to hand osteoarthritis (T298M) were similar to the wildtype protein, whereas the two other mutants were poorly expressed and hardly detectable in supernatants of transiently transfected cells. Using immunofluorescence staining, we demonstrated that mutants R116W and C299S are retained and accumulate within the endoplasmatic reticulum (ER). Their further trafficking to the Golgi compartment seems to be disturbed, whereas T298M is secreted normally. In cells transfected with the wildtype and T298M constructs, a matrilin-3 containing filamentous network was formed surrounding the cells, whereas in the case of R116W and C299S such structures were completely absent. These observations are similar to those for mutations in the cartilage oligomeric matrix protein (COMP) leading to multiple epiphyseal dysplasia and pseudoachondroplasia suggesting that retention and accumulation of cartilage proteins in the ER might be a general mechanism involved in the pathogenesis of chondrodysplasias.

Tracheobronchial malformations in experimental esophageal atresia.

BACKGROUND: Malformations of the tracheobronchial tree may account for postoperative respiratory symptoms in patients with esophageal atresia (EA). This study examines the respiratory tract in fetal rats with EA induced by Adriamycin. METHODS: Time-mated female rats were given either 2 mg/kg intraperitoneal Adriamycin on gestational days 8 and 9 (adria group, n = 6) or no treatment (control group, n = 2), and the fetuses were recovered on day 21. Laryngo-tracheo bronchial tree was studied after transparentation and alcian blue-alizarin red staining that depicts the cartilage in blue and make the surrounding tissues transparent. RESULTS: There were no malformations in any of the 1 1 control animals studied. Conversely, 31 of 46 (67%) Adriamycin fetuses had EA with distal TEF. These had more tracheal rings than controls (32+/-2 v 26+/-1.5, P < .05) at the expense of those of the mainstem bronchi (3.2+/-1 v 6.6+/-1.1 in the right, P< .05 and 6.2+/-2.1 v 11+/-1.1 in the left, P < .05). There were tracheal stenoses in 16 pups with EA (some severe and five double), and all these had fragmented rings in the trachea or bronchi. In six cases there was an ectopic upper right bronchus, and 1 had a grossly abnormal larynx. The malformations in the 15 Adriamycin-exposed fetuses without EA were limited to some fragmented or mishaped rings. CONCLUSIONS: Laryngo-tracheobronchial malformations entailing the whole length of the tract are very constant and severe in rats with EA and tracheoesophageal fistula and correspond to an abnormal development of the tracheobronchial anlage from the ventral foregut. Their nature and extent invite a careful investigation of the respiratory tracts in EA babies in whom they could be underscored.

Cardiovascular malformations in congenital diaphragmatic hernia: human and experimental studies.

BACKGROUND/PURPOSE: Cardiovascular malformations (CVM) associated with congenital diaphragmatic hernia (CDH) account in part for the high mortality caused by this defect. The aim of this study is to examine the nature of these malformations in a large series of autopsies and to assess if similar defects are also present in rat fetuses with experimental CDH. METHODS: The incidence of CVM and their nature were examined in the autopsy records of 136 stillborns and neonates with CDH admitted to our institution in the last 30 years. Experimental CDH was induced in rat fetuses by giving 100 mg of nitrofen to their mothers on gestational day 9.5, and the fetuses were harvested on day 21 (near full term). The presence of CDH and the anatomy of the heart and great vessels were studied under dissecting microscope after formalin fixation. Unexposed fetuses were used as controls. RESULTS: Thirty-three newborns with CDH (24%) had CVM, either isolated or associated with other defects, and 7 had heart hypoplasia. Most CVM (ventricular septal defect, tetralogy of Fallot, transposition of the great vessels, double-outlet right ventricle) involved the outflow tract. In our animal experiments, no malformations were found in 21 control pups. Conversely, 80 of 130 nitrofen-exposed fetuses (61%) had CDH, and 59 of them (74%) had CVM. A significant association (Fisher's Exact test, P<.01) was found between CDH and CVM because only 25 of the 50 exposed animals without CDH (50%) had CVM. Again, most defects involved the outflow tract and were similar to those seen in human CDH (tetralogy of Fallot, persistent truncus, ventricular septal defect, double-outlet right ventricle, aberrant right subclavian artery, agenetic ductus, and interrupted aortic arch). Animals with CDH had significantly decreased heart weight to fetal weight ratio in comparison with controls and with those without CDH. CONCLUSIONS: The similar nature of the cardiovascular defects found in babies succumbing to CDH and in nitrofen-exposed rats suggests that a similar disturbance of the regional organogenesis related to the neural crest might be involved in both settings, and further validates the use of this animal model for clarifying the cellular and molecular pathogenetic mechanisms.

ArF-excimer laser-induced secondary radiation in photoablation of biological tissue.

Secondary radiation, emitted during and after the irradiation of corneal, dermal, and dental tissue by an ArF-excimer laser (193 nm), was qualitatively and quantitatively characterized. Emission of secondary radiation was found in the range of 200-800 nm. The intensity of secondary radiation in the range of 200-315 nm (UVC and UVB) is approximately 20% of the total intensity at high laser fluences (> 2 J/cm2), and approximately 50% at moderate laser fluences (< 500 mJ/cm2); 10 muJ/cm2 in the UVC and UVB were measured at the sample surface, at fluences (< 1J/cm2) which are of relevance for clinical procedures on soft tissues. In dental tissue processing, very high fluences (> 5 J/cm2) are required. As a consequence, laser-induced plasma formation can be observed. Secondary radiation can be used as a visible guide for selective removal of carious altered tissue. The data we have found might be of assistance in estimating potential hazards for future mutagenic studies in the field.

Neural crest-derived defects in experimental esophageal atresia.

Esophageal atresia (EA) is often associated with cardiovascular and other malformations that are likely neural crest derived. The present study tests the hypothesis that the heart and great vessels and the thymus and parathyroids may be abnormal in the rat model of EA as a result of disturbed neural crest development. Time-mated pregnant rats received intraperitoneally on d 8 and 9 of gestation either 2 mg/kg adriamycin or vehicle. Esophageal, heart, and thymic malformations were sought under the microscope in term fetuses. The parathyroids were histologically investigated. Control fetuses had no malformations, whereas 69 of 109 fetuses exposed to adriamycin had EA and 45 of 69 had 15 right aortic arches, nine aberrant right subclavia, eight ventricular septal defects, six narrow pulmonary outflow tracts, five tetralogies of Fallot, three double outflow right ventricles, three double aortic arches, three atrial septal defects, three right ductus arteriosus, and two truncus. The thymus was absent in 19, hypoplastic in 12, and ectopic in five out of 36 fetuses with EA in which it was studied, whereas the parathyroid glands were absent in 16, single in four, and ectopic in one of the 23 fetuses with EA in which they were studied. In conclusion, the nature of the cardiovascular, thymic, and parathyroid malformations associated with EA in rats is consistent with the hypothesis of neural crest participation in their pathogenesis. Mechanisms simultaneously disturbing foregut septation, somitic segmentation, and neural crest development should be sought to explain the combined occurrence of malformations in EA.

TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation.

The pleiotropic activities of the potent proinflammatory cytokine TNF are mediated by two structurally related, but functionally distinct, receptors, p55 and p75, that are coexpressed on most cell types. The majority of biologic responses classically attributed to TNF are mediated by p55. In contrast, p75 has been proposed to function as both a TNF antagonist by neutralizing TNF and as a TNF agonist by facilitating the interaction between TNF and p55 at the cell surface. We have examined the roles of p55 and p75 in mediating and modulating the activity of TNF in vivo by generating and examining mice genetically deficient in these receptors. Selective deficits in several host defense and inflammatory responses are observed in mice lacking p55 or both p55 and p75, but not in mice lacking p75. In these models, the activity of p55 is not impaired by the absence of p75, arguing against a physiologic role for p75 as an essential element of p55-mediated signaling. In contrast, exacerbated pulmonary inflammation and dramatically increased endotoxin induced serum TNF levels in mice lacking p75 suggest a dominant role for p75 in suppressing TNF-mediated inflammatory responses. In summary, these data help clarify the biologic roles of p55 and p75 in mediating and modulating the biologic activity of TNF and provide genetic evidence for an antagonistic role of p75 in vivo.