Cutis laxa arising from frameshift mutations in exon 30 of the elastin gene (ELN).

Congenital cutis laxa, a rare syndrome with marked skin laxity and pulmonary and cardiovascular compromise, is due to defective elastic fiber formation. In several cases, skin fibroblast tropoelastin production is markedly reduced yet reversed in vitro by transforming growth factor-beta treatment. We previously showed that this reversal was due to elastin mRNA stabilization in one cell strain, and here this behavior was confirmed in skin fibroblasts from two generations of a second family. cDNA sequencing and heteroduplex analysis of elastin gene transcripts from three fibroblast strains in two kindreds now identify two frameshift mutations (2012DeltaG and 2039DeltaC) in elastin gene exon 30, thus leading to missense C termini. No other mutations were present in the ELN cDNA sequences of all three affected individuals. Transcripts from both alleles in each kindred were unstable and responsive to transforming growth factor-beta. Exons 22, 23, 26A, and 32 were always absent. Since exon 30 underwent alternative splicing in fibroblasts, we speculate that a differential splicing pattern could conceivably lead to phenotypic rescue. These two dominant-acting, apparently de novo mutations in the elastin gene appear to be responsible for qualitative and quantitative defects in elastin, resulting in the cutis laxa phenotype.

Ascorbate differentially regulates elastin and collagen biosynthesis in vascular smooth muscle cells and skin fibroblasts by pretranslational mechanisms.

Ascorbate contributes to several metabolic processes including efficient hydroxylation of hydroxyproline in elastin, collagen, and proteins with collagenous domains, yet hydroxyproline in elastin has no known function. Prolyl hydroxylation is essential for efficient collagen production; in contrast, ascorbate has been shown to decrease elastin accumulation in vitro and to alter morphology of elastic tissues in vivo. Ascorbate doses that maximally stimulated collagen production (10-200 microM) antagonized elastin biosynthesis in vascular smooth muscle cells and skin fibroblasts, depending on a combination of dose and exposure time. Diminished elastin production paralleled reduced elastin mRNA levels, while collagen I and III mRNAs levels increased. We compared the stability of mRNAs for elastin and collagen I with a constitutive gene after ascorbate supplementation or withdrawal. Ascorbate decreased elastin mRNA stability, while collagen I mRNA was stabilized to a much greater extent. Ascorbate withdrawal decreased collagen I mRNA stability markedly (4.9-fold), while elastin mRNA became more stable. Transcription of elastin was reduced 72% by ascorbate exposure. Differential effects of ascorbic acid on collagen I and elastin mRNA abundance result from the combined, marked stabilization of collagen mRNA, the lesser stability of elastin mRNA, and the significant repression of elastin gene transcription.

Evaluation of immune markers in asymptomatic AIDS patients receiving fish oil supplementation.

The effects of oral fish oil (FO) supplementation (8 g/day, capsules) on nutritional status and selected immune markers (CD4/CD8 ratio, IL-1beta, erythrocyte MDA release, dehydroepiandrosterone sulfate [DHEA-S]) were studied in a homogeneous group of asymptomatic HIV-infected patients during 6 weeks. All subjects were classified clinically as A2 according to the CDC revised criteria (mean CD4 count 290 +/-123 cells/mm(3)) and were receiving zidovudine retroviral treatment. The calculated mean energy intake was 3437 +/- 372 Kcal/d, composed of 14% protein, 38% lipids and 48% carbohydrates, and was not modified during the study. The anthropometric parameters, and hematological and plasma biochemistry data showed non-significant changes after FO supplementation. Mean malonyldialdehyde (MDA) release before treatment was: unstimulated 71.5 +/- 37 and stimulated 350.9 +/- 79.8 nmol/g Hb. After FO supplementation (T(6)) MDA release showed unstimulated values of 96.1 +/- 62, and a significant increase after stimulation of 614.1 106.4 nmol/g Hb, which was, however, within the normal range. In the patient's samples, IL-1beta levels in the unstimulated blood culture showed a statistical increase with respect to the normal range before (T(0)) and after (T(6)) FO supplementation with a slight decrease after (mean 49.8 vs 40.9 pg/ml). The stimulated IL-1beta levels after treatment showed a statistically significant decrease that was maintained within the normal range (T(0): 797.7 vs T(6): 535.6 pg/ml). Taken collectively, these results suggest a tendency toward improvement in immune function.

Transforming growth factor-beta reverses a posttranscriptional defect in elastin synthesis in a cutis laxa skin fibroblast strain.

Skin fibroblasts from two cases of autosomal recessive cutis laxa (CL), having insignificant elastin production and mRNA levels, were challenged with transforming growth factor beta-1 (TGF-beta 1). Elastin production was brought from undetectable values to amounts typical of normal human skin fibroblasts in a dose-dependent fashion. Basic fibroblast growth factor (100 ng/ml) alone or in combination with TGF-beta 1 reduced elastin production and mRNA expression in CL skin fibroblasts more extensively than in normal cells. In situ hybridization showed that these effects were at the transcript level. One of the CL strains was examined in detail. Transcription rates for elastin were similar in normal and CL and unchanged by TGF-beta 1 or TGF-beta 2 (10 ng/ml), while in CL elastin mRNA half-life was increased > 10-fold by TGF-beta 2 and reduced 6-fold after TGF-beta 2 withdrawal, as compared with a control strain. Cycloheximide partially reversed elastin mRNA instability. These data are consistent with a defect in elastin mRNA stability that requires synthesis of labile factors or intact translational machinery, resulting in an extremely low steady state level of mRNA present in this strain of CL. Furthermore, TGF-beta can relieve elastin mRNA instability in at least one CL strain and elastin production defects in both CL strains.

Regulation of elastin synthesis in pathological states.

Elastin is rapidly deposited during late gestation in resilient tissues such as the arteries, lungs and skin owing to increased concentration of its mRNA. Pathological states can arise from congenital insufficiency or disorganization of elastin (cutis laxa). Other elastin deficiencies may be due to excess elastolysis or gene dosage effects. In the former, high turnover rates can be assessed by measurements of elastin degradation products in urine. Excess elastin accumulation by skin fibroblasts is characteristic of genetic diseases such as Buschke-Ollendorff syndrome, Hutchinson-Gilford progeria and keloid. Elastin expression is modulated by peptide growth factors, steroid hormones and phorbol esters, among which transforming growth factor beta (TGF-beta) is an especially potent up-regulator, acting largely through stabilization of mRNA. Recent evidence indicates cutis laxa fibroblasts that express little or no elastin have normal transcriptional activity but abnormal rates of elastin mRNA degradation. This defect is substantially reversed by TGF-beta through mRNA stabilization. Current studies explore the hypothesis that stability determinants lie within the 3' untranslated region of elastin mRNA. Post-transcriptional control of elastin expression appears to be a major regulatory mechanism.

Elastin production and degradation in cutis laxa acquisita.

A case of cutis laxa acquisita was studied with the aim of defining the molecular defects involved and comparing them with those of an inherited form of cutis laxa. In the acquisita form of cutis laxa ultrastructural and biochemical observations confirmed a dramatic reduction of dermal elastin, whereas collagen content was normal. Elastin mRNA expression as well as tropoelastin production by dermal fibroblasts, in vitro, were normal compared with control cells, as revealed by in situ hybridization and enzyme-linked immunosorbent assay, respectively. Lysyl oxidase activity, measured on cultured fibroblasts, was reduced to 60% compared with age-matched control subjects. Unlike control skin fibroblasts or fibroblasts from inherited cutis laxa, the affected skin cells from cutis laxa acquisita predominantly expressed an elastolytic activity identified as cathepsin G. Patient serum also has reduced elastase inhibitory capacity and reduced levels of alpha 1-antiproteinase inhibitor (alpha 1-antitrypsin). Although cutis laxa acquisita is a heterogeneous group of disorders, findings in this patient were consistent with excessive loss of cutaneous elastin due to the combined effects of several factors, such as low lysyl oxidase activity together with high levels of cathepsin G and reduction of circulating proteinase inhibitor(s).

Phenotypic stability and variation in cells of the porcine aorta: collagen and elastin production.

The extracellular matrix of the developing vasculature varies in composition as a function of time and position. Cellular models of vascular biology and pathology depend on the assumption that stable phenotypic characteristics of vascular cells can be propagated through several generations of in vitro cultivation. We show that the positional and developmental heterogeneity of matrix phenotypes in the porcine aorta are expressed by explanted vascular smooth muscle cell (SMC) and adventitial cell populations for a limited number of passages. Elastin was expressed most highly by thoracic SMC while interstitial collagen production was usually maximal in abdominal segments. Parallel gradients of collagen types I, III and V, detected by specific ELISA assays, were expressed in early-passage SMC. Adventitial cell populations from the abdominal aorta of the neonatal pig accumulated significant levels of collagen, while these fibroblasts produced less than 10% of the elastin made by SMC. All cell populations expressed alpha-smooth muscle actin in vitro. Gradients of collagen and elastin expression were evident for no more than three passages, and direct outgrowth of cells without limited digestion of the matrix further reduced phenotypic stability. Variation and decline of the elastin phenotype could be due to hypermethylation of regulatory sequences in the elastin gene or trans-acting factors, but elastin production was dose-dependently stimulated to a similar extent (100%; 10 microM 5-azacytidine) in all segmental SMC populations at early (p1) and late (p3) passage. These data indicated that faithful reflection of in vivo SMC behavior was limited to a few population doublings, at least under standard culture conditions. Modification of the cellular environment by reducing serum factors, changing matrix, or adding mechanical stimulation may increase phenotypic stability.

Familial co-segregation of the elastin phenotype in skin fibroblasts from Hutchinson-Gilford progeria.

Elastin and type IV collagen production are markedly elevated in fibroblasts derived from the skin of patients with Hutchinson-Gilford progeria (HGP). Fibroblasts from three affected children and their parents were compared to normal human skin fibroblasts with respect to elastin production as a function of different concentrations of calf serum and the cytokines, transforming growth factor-beta and basic fibroblast growth factor (TGF-beta 1, bFGF). In cultured fibroblasts from the parents of probands that were very high elastin producers (> 10(5) molecular equivalents/cell per h), at least one parent (mother) presented the same phenotype. Overproduction of elastin in culture could have been due to increased sensitivity of HGP strains to stimuli present in serum; however, relative stimulation of elastin production by calf serum in cell strains from HGP elastin over-producers was less than half the control strain. In most of the cultures examined, the responsiveness of elastin production to TGF-beta 1 was almost absent when compared to the response of normal fibroblasts. HGP strains with high elastin production modified conditioned medium to enhance elastin production in normal cells. These results suggest the presence, in HGP skin fibroblasts, of inheritance of high elastin production that is associated with accelerated aging.

Buschke-Ollendorff syndrome associated with elevated elastin production by affected skin fibroblasts in culture.

Buschke-Ollendorff syndrome (BOS; McKusick 16670) is an autosomal dominant connective-tissue disorder characterized by uneven osseous formation in bone (osteopoikilosis) and fibrous skin papules (dermatofibrosis lenticularis disseminata). We describe two patients in whom BOS occurred in an autosomal dominant inheritance pattern. The connective tissue of the skin lesions showed both collagen and elastin abnormalities by electron microscopy. Cultured fibroblasts from both patients produced 2-8 times more tropoelastin than normal skin fibroblasts in the presence of 10% calf serum. Involved skin fibroblasts of one patient produced up to eight times normal levels, whereas apparently uninvolved skin was also elevated more than threefold. In a second patient, whose involvement was nearly complete, elastin production was high in involved areas and less so in completely involved skin. Transforming growth factor-beta 1 (TGF beta 1), a powerful stimulus for elastin production, brought about similar relative increases in normal and BOS strains. Basic fibroblast growth factor, an antagonist of TGF beta 1-stimulated elastin production, was able to reduce elastin production in basal and TGF beta 1 stimulated BOS strains. Elastin mRNA levels were elevated in all patient strains, suggesting that Buschke-Ollendorff syndrome may result, at least in part, from abnormal regulation of extracellular matrix metabolism that leads to increased steady-state levels of elastin mRNA and elastin accumulation in the dermis.

[Non-hemolytic transfusion reactions of the febrile type]. / Reacciones transfusionales no hemolíticas de tipo febril (RTNHF). Estudio serológico de 100 casos.

The results of a hundred cases of febrile non haemolytic transfusion reactions are presented. We used the techniques that would best identify the different causes of these reactions. Most of these antibodies were anti HLA. Platelet and granulocyte-specific antibodies were seldom detected. The nature of the antibodies was specified by their different reactivity with the cells of various donors, by applying a panel of cells from typed donors and by absorption experiments. The study of the donor sera enabled us to elucidate the causes of some reactions. Nevertheless, in a significant percentage of cases it was not possible to determine the cause of the reaction, especially in the case of reactions secondary to platelet transfusions. Finally, we analyzed the reasons that could account for these phenomena.

Hyaluronic acid promotes chick embryo fibroblast and chondroblast expression.

15-day-chick-embryo fibroblasts and chondroblasts were cultured in the presence of high and low molecular weight exogenous hyaluronic acid (HA). Growth range and incorporation of radiolabelled sulphate and proline were determined. HA reduced cell proliferation to about 75% of controls, while incorporation of radiolabelled sulphate and proline was higher in HA-treated cultures of both chondroblasts and fibroblasts. The effect was not due to the polyanionic or polymeric nature of the molecule and appeared to be highly specific for HA.

Increased elastin production by progeria skin fibroblasts is controlled by the steady-state levels of elastin mRNA.

Hutchinson-Gilford progeria is a unique, rare disease with markedly accelerated aging. The average lifespan of affected individuals is 12 years. Although the biochemical basis of the syndrome is unknown, its influence appears to be primarily upon mesodermal tissues. Characteristics such as the altered appearance of the skin and the extensive and fatal involvement of the cardiovascular system led us to study elastin production in cultured skin fibroblasts from three progeroid individuals. We found tropoelastin production by progeroid cells was elevated six- to nine-fold at the protein and mRNA levels, while relative collagen synthesis was similar to control strains. There was little difference between progeroid and normal cells in expression of total protein or in total cellular mRNA content. Western blot analysis of tropoelastin from progeroid fibroblasts confirmed increased production of elastin but revealed no gross changes in the molecular mass. The significant increase in tropoelastin expression lends support to the concept that progeria results from a mesenchymal dysplasia, and offers a possible biochemical marker for the phenotype.

Demonstration of elastin gene expression in human skin fibroblast cultures and reduced tropoelastin production by cells from a patient with atrophoderma.

Atrophoderma is a rare dermal disorder characterized by a patchy distribution of areas apparently devoid of elastic fibers. Skin fibroblast cultures were established from the normal and affected dermis of a patient with this disorder. Human tropoelastin was identified in culture medium by use of electroblotting and anti-elastin antisera. An enzyme-linked immunosorbent assay was used to establish that significantly less elastin accumulated in the media of cultured cells from lesional fibroblasts over a 3-d period. Since elastin biosynthesis in most tissues is under pretranslational control, molecular hybridization to a nick-translated genomic elastin probe was performed; however, elastin messenger RNA levels were equivalent in both cell strains. Both strains produced less elastin than did normal skin fibroblasts. Extracellular proteolysis of elastin was evaluated as a possible mechanism. Elastase activity was increased and porcine tropoelastin was degraded four times faster, on a per-cell basis, in lesional fibroblast cultures than in cells derived from an unaffected site. The two cell strains exhibited no significant differences in collagen production or collagenase activity. These results are the first demonstration of elastin production by cultured human skin fibroblasts, and they suggest that the primary defect in atrophoderma may be a result of enhanced degradation of newly synthesized elastin precursors.

Longitudinal gradients of collagen and elastin gene expression in the porcine aorta.

The physical and chemical properties of the mammalian aorta are known to vary as a function of distance from the heart. These properties are highly dependent collagen and elastic fibers. In order to evaluate the mechanisms which regulate the accumulation of these two connective tissue proteins, gene expression was evaluated at both the biosynthetic and messenger RNA levels. Short-term (3 h) explant cultures of the medial portion of four segments of the descending aorta in newborn pigs were incubated in the presence of [3H] proline. Collagen production was quantified by collagenase digestion and elastin production was determined by immunoprecipitation. Between the conus arteriosus and the bifurcation of the iliac arteries, relative collagen synthesis increased 2-fold (from 5.8 to 12.0% of total protein synthesis), while relative elastin synthesis declined 10-fold (from 16.4 to 1.6% of total protein synthesis). Similarly, collagen production increased more than 7-fold (from 6.7 to 49.8 X 10(3) molecules/cell/h) while elastin production was reduced more than 3-fold (from 71.8 to 21.0 X 10(3) molecules/cell/h) along this developmental gradient. Elastin synthesis appeared to be controlled to a significant extent by the availability of elastin mRNA, since both cell-free translation and molecular hybridization to a cloned elastin gene probe showed gradients of elastin gene expression. Similarly, collagen synthesis was apparently regulated, at least in part, by an inverse gradient of collagen mRNA, as measured with a cloned cDNA for the pro-alpha 1(I) collagen gene. Marked changes in the amount of non-elastin protein synthesis accompanied differentiation and accounted for larger changes in relative synthesis. These results suggest that the phenotype of the cells of the porcine artery wall is distinct in different regions of this organ at this developmental stage.

Quantitation of elastin production in cultured vascular smooth muscle cells by a sensitive and specific enzyme-linked immunoassay.

An enzyme-linked immunoassay (ELISA) procedure has been developed to quantitate the amount of elastin produced by cultured porcine aortic smooth muscle cells. ELISA was used to determine both titer and specificity of antisera raised in rabbits against porcine aortic alpha-elastin conjugated with key-hole limpet hemocyanin. Under optimum conditions (1: 3000 dilution of antiserum, 20 ng alpha-elastin per assay well), sensitivity averaged 60 ng/ml). Specificity was confirmed by immunoprecipitation of [125I]-tropoelastin, radial immunodiffusion, Western blotting and lack of cross-reactivity with serum proteins or collagen. Extensive cross-reactivity was found with both human alpha-elastin and porcine beta-elastin, while porcine tropoelastin was able to compete with 80% of the alpha-elastin determinants. Affinity of anti-porcine antisera for sheep alpha- elastin was significantly lower. When the ELISA was made specific for tropoelastin by coating wells with 60 ng of this antigen, a time-dependent and serum-dependent rate of production of tropoelastin was observed in the culture medium of primary and secondary cultures of smooth muscle cells. Comparison of elastin production in cultures of porcine smooth muscle cells suggests that porcine aortic elastin production varies as a function of cell density and phase of growth.

Banded fibers in tropoelastin coacervates at physiological temperatures.

Tropoelastin was purified from aortas of chicks grown on a beta-aminopropionitrile-containing diet. The preparation could be considered pure following the criteria of amino acid composition and gel electrophoresis. When aqueous solutions of tropoelastin (5 mg/ml) were warmed to 40 degrees C (physiological temperature for chicken) for 10 min, and observed by negative-staining electron microscopy, it revealed the presence of two kinds of ordered structures. One consisted of densely packed parallel filaments with a center-to-center distance of about 5 nm, and the other of banded fibers, 100-150 nm in diameter, with a cross periodicity of about 55 nm. In some areas the fibers appeared to be formed by lateral aggregation of 1.5-2-nm-thick microfilaments. The fibers were similar to those previously obtained with the synthetic polypentapeptide of elastin (Val-Pro-Gly-Val-Gly)n and degradation products of elastin at temperatures much higher than the physiological one. The results indicate that the property of tropoelastin to form ordered structures is intrinsic to some of the polypeptide sequences of the molecule and that hydrophobic forces are involved in the formation of the aggregates.

X-ray diffraction study of bovine lens capsule collagen.

The wide angle X-ray diffraction pattern of air-dried lens capsule collagen under tension is the same as the tendon collagen diffraction pattern with regard to the main reflections, and indicates that lens capsule collagen has the characteristic three-stranded helical structure with an axial repeat of 0.29 nm as tendon collagen. The low angle X-ray diffraction pattern shows several weak diffraction maxima corresponding to the meridional reflections of capsule collagen which show orders of 63.0 nm periodicity. This is an evidence of quarter staggered molecular assembly typical of tendon collagen even if less ordered. The results are consistent with the existence in lens capsule collagen of clearly defined molecular units, which can be oriented by stress and are packed in a poor-ordered fibrillar assembly.

Ultrastructural and biochemical studies on a case of elastosis perforans serpiginosa.

Ultrastructural and biochemical studies on the collagen and elastin fibres of the dermis from a patient with Elastosis perforans serpiginosa are described. Qualitative and quantitative alterations on collagen and elastin are shown, which give some evidence for considering the disease as a connective tissue defect.