Validation of a new immunoenzymatic method to detect antibodies to RNA polymerase III in systemic sclerosis.

OBJECTIVE: To test the reliability of a new enzyme-linked immunosorbent assay (ELISA) to identify anti-RNA polymerase III (RNAP III) positive sera from Italian patients with Systemic Sclerosis (SSc) and other chronic inflammatory disorders. METHODS: A comparison between the new ELISA for anti-RNAP III and the gold standard technique, immunoprecipitation (IP), was first performed on 106 SSc patients, 16 patients with other connective tissue diseases and 10 healthy subjects. A further ELISA evaluation was performed on 224 SSc patients, 120 subjects with other rheumatic or infectious diseases, and 81 healthy controls. RESULTS: Plotting ELISA and IP data in a Receiver Operator Characteristic curve, the ELISA cut-off value providing the best specificity (99.1%) and sensibility (100%) was 28 U/ml (AUC=0.999; p<0.0001). Using this cut-off in the second analysis, anti-RNAP III positive results were found in 41 (18.3%) SSc patients, all negative for anticentromere or anti-topoisomerase I antibodies, while only 3 subjects tested positive among the 120 sera collected from other patients. All the healthy subjects were negative. CONCLUSION: This new ELISA for anti-RNAP III is highly accurate when a proper cut-off value is employed and represents a valid substitute to IP in a clinical setting.

Localized and systemic forms of scleroderma in adults and children .

Systemic sclerosis (SSc) presents a great deal of variability in the extent and severity of skin and internal organ involvement. The diagnostic and prognostic significance of autoantibodies in SSc is undisputed and the patient's autoantibody profile represents a fundamental tool for clinicians. Scleroderma is a rare condition in children. Unlike adults, localized scleroderma is more frequent than the systemic sclerosis, nevertheless it represents a disabling condition. In both conditions, no validated outcome measures and proven effective treatment is available to date.Raynaud's phenomenon (RP) is one the most common and significant clinical symptoms of SSc and therefore in patients with RP a capillaroscopic analysis should be carried out as soon as possible. The actual and select advantage of the early nailfold videocapillaroscopic (NVC) analysis is to distinguish between the primary RP and the secondary RP and to allow the early detection of SSc.

Proliferation characteristics and polyploidization of cultured myofibroblasts from a patient with fibroblastic rheumatism.

Fibroblast-like cells were obtained from a nodule of a patient with fibroblastic rheumatism, and grown in culture for different times (from passage 3 to 21). These cells as well as the fibroblasts taken from an unaffected skin area (controls) of the same patient, have been investigated by fluorescence microscopy, cytochemical methods and cytometry, to evaluate their cytodifferentiation features and cytokinetic characteristics. In addition, in low-passage cultures, the secretion of collagen and of non-collagenic proteins was evaluated using electrophoretic techniques. The immunolabeling with antibodies against sm-specific a-actin (which was taken as a marker of myofibroblasts) showed that, already in low-passage cultures, the percentage of myofibroblasts was higher in the nodule-derived cell populations, and progressively increased with increasing passages. This suggests that myofibroblasts have higher proliferation potential than control fibroblasts. Myofibroblasts were also found to undergo polyploidization and hypertrophy, especially in high-passage cultures. Based on these results, it may be hypothesized that in fibroblastic rheumatism the development of the typical nodules could depend on the intrinsic capability of myofibroblats of proliferating faster than normal fibroblasts and of becoming polyploid and hypertrophic. Nodule-derived cells in culture synthesized slightly less collagen and non-collagen proteins than did the control fibroblasts; this suggests that the increased fibrosis observed in nodules in situ could be likely dependent on a reduced degradation of the extracellular matrix components.

Autoantibodies to RNA-polymerases in Italian patients with systemic sclerosis.

OBJECTIVE: To assess the frequency and clinical correlates of the systemic sclerosis-related autoantibodies to RNA polymerases in Italian patients. METHODS: Sera from 115 patients with systemic sclerosis (SSc) and 10 patients with systemic sclerosis-overlap syndromes recruited from a single center in northern Italy were investigated for antibodies to RNA polymerase I, II, and III by means of immunoprecipitation using 35S-labeled HeLa cell antigen extract. Twenty-five normal volunteers and 91 patients with different connective tissue diseases were studied as a control group. RESULTS: Antibodies to RNA-polymerases were found in 14/115 SSc patients (12.1%). None of the normal controls and none of the patients with other connective tissue diseases, including overlap syndromes, were positive. Antibodies reacting with RNA-polymerase I and III (+/- RNA-polymerase II) were found in 9/115 patients (7.8%) and were mutually exclusive with respect to other scleroderma-related autoantibodies. Isolated anti-RNA polymerase II reactivity was found in 5 patients and was associated with anti-topoisomerase I antibodies in 4 cases. Anti-RNA-polymerase I and III antibodies were associated with diffuse cutaneous involvement and male gender. Only two patients from our series had scleroderma renal crisis, and one of them had anti-RNA polymerase antibodies. CONCLUSIONS: Anti-RNA-polymerase antibodies appear to be less frequent in Italian patients than in Caucasian patients from the United Kingdom or USA. This might be associated with the lower frequency of scleroderma renal crisis.

Trans-acting factors may cause dystrophin splicing misregulation in BMD skeletal muscles.

We analyzed dystrophin alternative splicing events in a large number of Becker muscular dystrophy (BMD) affected individuals presenting major hot-spot deletions. Evidence is shown that altered splicing patterns in these patients do not directly result from the gene defect but probably derive from modifications in trans- rather than cis-acting factors. Several potential CUG-binding protein 2 (CUG-BP2) binding sites were found to be located in the dystrophin gene region encompassing exons 43-60 and CUG-BP2 transcript analysis indicated that not only expression levels are increased in dystrophic muscles but also that different CUG-BP2 isoforms are expressed. The possibility that CUG-BP2 might have a role in dystrophin splicing regulation is discussed.

Two dystrophin proteins and transcripts in a mild dystrophinopathic patient.

Two muscle dystrophin transcripts and proteins were detected in a 17-year-old boy with a persistently elevated serum creatine kinase level. A decreased amount of full-length dystrophin and a 360 kDa polypeptide lacking the COOH-terminus were detectable in the patient's muscle biopsy; accordingly, transcript analysis revealed the expression of a wild type messenger RNA together with a shorter frameshifted one. No genomic DNA mutation was found and the presence of a somatic mosaicism was excluded. This dystrophinopathy may be caused by a novel dystrophin gene transcriptional defect, namely aberrant intraexonic splicing.

The dystrophin gene is alternatively spliced throughout its coding sequence.

We have analysed splicing patterns in the human dystrophin gene region encoding the rod and cysteine-rich domains in normal skeletal muscle, brain and heart tissues. Sixteen novel alternative transcripts were identified, the majority of them being present in all three tissues. Tissue-specific variants were also identified, suggesting a functional role of transcriptional diversity. Transcript analysis in dystrophinopathic autoptic and bioptic specimens revealed that pre-mRNAs secondary structure formation and relative strength of exon/exon association play little or no role in directing alternative splicing events. This analysis also showed that independent deletion events leading to the loss of the same exons may be associated with transcriptional variability.

Analysis of splicing parameters in the dystrophin gene: relevance for physiological and pathogenetic splicing mechanisms.

The molecular mechanisms that direct splice-site selection and assure orderly exon juxtaposition have not been fully clarified. The extraordinary nature of the dystrophin gene points to several hurdles in the processing of transcripts. In this study, dystrophin statistical and thermodynamic splicing parameters have been evaluated providing the first comprehensive description for a single human gene. We show that concomitant use of consensus values (CV) and DeltaDG degrees 37 values for U1snRNA annealing better discriminates between real donor sites and donor-like sequences. Evidence is also provided that, on average, out-of-frame dystrophin exons have significantly stronger CVs and more favorable DeltaDG degrees 37 values; this feature has never been reported and might reflect evolutionary-driven minimization of out-of-frame exon misplicing. Dystrophin splicing mutations have been reported to determine either Duchenne or Becker Muscular Dystrophy, but no comprehensive genotypic/phenotypic correlation has ever been investigated. We have analyzed splicing affecting single base-pair substitutions in the dystrophin gene with respect to their effect on splicing parameters; functional and clinical consequences are also reported. We have found 5'-splice-site mutation occurrence to be statistically related to mutability quotients and propose the use of DeltaDG degrees 37 values as a more effective tool than CV alone to describe donor site mutation consequences. Our analysis also indicates a nearly 100% correlation between clinical phenotype and the reading-frame rule determined at the RNA level. We consider that elucidation of the relative importance of splicing determinants might help to clarify the molecular mechanisms that direct correct splicing in complex genes and might be useful in the validation of predictive models.

Transcriptional activation of the non-muscle, full-length dystrophin isoforms in Duchenne muscular dystrophy skeletal muscle.

Despite promoter tissue specificity, up-regulation of the brain and Purkinje cell type dystrophin isoforms was described in skeletal muscle of X-linked dilated cardiomyopathy (XLDCM) and BMD affected individuals. An extended population of 11 Duchenne muscular dystrophy (DMD) and 11 Becker muscular dystrophy (BMD) patients was investigated to determine whether ectopic muscle expression of the two full-length non-muscular isoforms is a common event in dystrophinopathies and if it has functional significance. Up-regulation of the two non-muscle-specific isoforms was detected in four DMD patients but in none of the BMD affected individuals or non-dystrophic controls. This is the first report of an expression of these two isoforms in DMD skeletal muscle. Ectopic expression is not confined to regenerating or revertant fibers and does not correlate with age at biopsy, clinical phenotype, cardiac involvement, deletion size or location. We consider that muscle ectopic expression of the brain and Purkinje cell-type isoforms has no favorable prognostic significance in DMD and BMD patients.

Primary beta-sarcoglycanopathy manifesting as recurrent exercise-induced myoglobinuria.

We report an unusual presentation of a primary beta-sarcoglycanopathy (LGMD type 2E). A 12- year-old boy came to our attention after six episodes of exercise-induced myoglobinuria. Electromyogram showed mild myopathic features of the proximal lower limb muscles. Electrocardiogram was normal. Neurological examination revealed normal muscle strength and reduced deep tendon reflexes. A muscle biopsy showed rare regenerating fibers; the immunohistochemistry was normal for dystrophin, while all the sarcoglycans were diffusely decreased. Western blot analysis showed a relevant decrease of all sarcoglycan proteins and a mild dystrophin reduction. beta-Sarcoglycan gene analysis demonstrated a compound heterozygous status for these mutations: a novel A-T base pair substitution at nucleotide 85 in exon 2, changing the codon Arg to a stop codon; a C-T base pair substitution at nucleotide 272 in exon 3 changing a Arg to a Cys residue. We consider that exercise-induced myoglobinuria may be the presenting sign of primary beta-sarcoglycanopathy.

beta 1,3-Galactosyltransferase beta 3Gal-T5 acts on the GlcNAcbeta 1-->3Galbeta 1-->4GlcNAcbeta 1-->R sugar chains of carcinoembryonic antigen and other N-linked glycoproteins and is down-regulated in colon adenocarcinomas.

We attempted to determine whether beta1,3-galactosyltransferase beta3Gal-T5 is involved in the biosynthesis of a specific subset of type 1 chain carbohydrates and expressed in a cancer-associated manner. We transfected Chinese hamster ovary (CHO) cells expressing Fuc-TIII with beta3Gal-T cDNAs and studied the relevant glycoconjugates formed. beta3Gal-T5 directs synthesis of Lewis type 1 antigens in CHO cells more efficiently than beta3Gal-T1, whereas beta3Gal-T2, -T3, and -T4 are almost unable to direct synthesis. In the clone expressing Fuc-TIII and beta3Gal-T5 (CHO-FT-T5), sialyl-Lewis a synthesis is strongly inhibited by swainsonine but not by benzyl-alpha-GalNAc, and sialyl-Lewis x is absent, although it is detected in the clones expressing Fuc-TIII and beta3Gal-T1 (CHO-FT-T1) or Fuc-TIII and beta3Gal-T2 (CHO-FT-T2). Endo-beta-galactosidase treatment of N- glycans prepared from clone CHO-FT-T5 releases (+/-NeuAcalpha2-->3)Galbeta1-->3[Fucalpha1-->4]GlcNAcbeta1-->3Gal but not GlcNAcbeta1-->3Gal or type 2 chain oligosaccharides, which are found in CHO-FT-T1 cells. This result indicates that beta3Gal-T5 expression prevents poly-N-acetyllactosamine and sialyl-Lewis x synthesis on N-glycans. Kinetic studies confirm that beta3Gal-T5 prefers acceptors having the GlcNAcbeta1-->3Gal end, including lactotriosylceramide. Competitive reverse transcriptase mediated-polymerase chain reaction shows that the beta3Gal-T5 transcript is expressed in normal colon mucosa but not or poorly in adenocarcinomas. Moreover, recombinant carcinoembryonic antigen purified from a CHO clone expressing Fuc-TIII and beta3Gal-T5 reacts with anti-sialyl-Lewis a and carries type 1 chains on oligosaccharides released by endo-beta-galactosidase. We conclude that beta3Gal-T5 down-regulation plays a relevant role in determining the cancer-associated glycosylation pattern of N-glycans.

Loss of Dp140 dystrophin isoform and intellectual impairment in Duchenne dystrophy.

BACKGROUND: Mental retardation is a clinical feature of Duchenne dystrophy (DD) and affects about one-third of patients. No clear association has been found between DNA mutations, protein expression, and IQ scores, although distal deletions in the dystrophin gene have been reported in association with intellectual impairment. A role for the brain distal dystrophin isoform Dp140 was suggested. OBJECTIVE: To explore the possible association between cognitive impairment and DNA macrodeletions in the distal part of the gene, including Dp140 gene region. METHODS: Sixty-six patients with DD received general intelligence assessment by Wechsler Intelligence Scales measuring full, verbal, and performance IQ. PCR analysis was performed to detect deletions in the dystrophin gene, and the Dp140 regulatory region was analyzed in a subgroup of 12 patients. Statistical analysis was performed by nonparametric Wilcoxon rank signed and rank sum tests. RESULTS: Comparison of neuropsychological and genetic data revealed an association between distal macrodeletions and cognitive impairment (p < 0.001). Comparing deletions involving the Dp140 gene region with deletions presumably not altering Dp140 expression resulted in even greater significance. CONCLUSIONS: These data suggest that in DD, distal dystrophin deletions are associated with intellectual impairment. This study highlights a possible role for the brain distal isoform Dp140 in normal cognitive development.

Loss of Dp140 regulatory sequences is associated with cognitive impairment in dystrophinopathies.

Mental retardation is a clinical feature present in both Duchenne and Becker muscular dystrophy patients and its pathogenesis is still unknown. Dp140 is a dystrophin isoform with predominant expression during foetal brain development. Its promoter and first exon lie in the large intron between exon 44 and 45, a region that is commonly deleted in dystrophinopathic patients. We performed neuropsychological evaluation and genetic analysis of the Dp140 transcription unit on 12 Duchenne muscular dystrophy and 28 Becker muscular dystrophy patients carrying deletions in this critical region. Comparison of neuropsychological and molecular data showed that there is a statistically significant relationship between the loss of Dp140 transcription unit and mental retardation in Becker muscular dystrophy patients (P = 0.008). Such a correlation is not evident in Duchenne muscular dystrophy patients but only shows a trend towards significance (P = 0.063). It is worth noting that both Duchenne muscular dystrophy and Becker muscular dystrophy patients with normal intelligence do not show deletions in the Dp140 regulatory regions. In the light of these findings, we suggest that impairment of cognitive abilities in Duchenne muscular dystrophy and Becker muscular dystrophy patients might be related to a dysfunction of Dp140 brain isoform.

Differential expression of beta1,3galactosyltransferases in human colon cells derived from adenocarcinomas or normal mucosa.

Two beta1,3galactosyltransferases are detected in human colon cells: one corresponds to beta3GalT1, the other (beta3GalTx) is found to be different from any cloned beta3GalT since in vitro it utilizes GlcNAc very efficiently under specific reaction conditions. Expression of beta3GalT1 transcript is high in normal colon mucosa and control neuroectodermal cells, which do not express sialyl-Lewis a antigen, and low in colon adenocarcinoma cells, as assessed by competitive RT-PCR. beta3GalTx activity is high in adenocarcinoma cells expressing sialyl-Lewis a and undetectable in all other cells, suggesting differential involvement and opposite regulation of such enzymes during carcinogenesis.

Mouse C127 cells transfected with fucosyltransferase fuc-TIII express masked Lewisx but not Lewisx antigen.

To study human alpha1,3/1,4fucosyltransferase (Fuc-TIII) as an alpha1,3 fucosyltransferase, we constructed two cell clones, C127-FT and C127-T-FT, by transfecting cDNA in parental (C127) or Polyoma T antigen expressing (C127-T) mouse cells, respectively. Both C127-FT and C127-T-FT clones express high levels of a fucosyltransferase activity kinetically similar to Fuc-TIII and an RNA that is amplified by a Fuc-TIII-specific oligonucleotide primer pair after reverse transcription. Clone C127-FT is Lewisxpositive, by flow cytometry, only after alpha-galactosidase or sialidase treatment, and releases [3H]Fuc N-glycans which efficiently bind to immobilized Griffonia simplicifolia I and Sambucus nigra lectins. Immunoblotting confirms that C127-FT glycoproteins acquire Lewisxreactivity only after specific deglycosylation, and shows that a small subset of Griffonia simplicifolia I isolectin B4reactive glycoproteins bears masked Lewisx, suggesting fine substrate recognition by Fuc-TIII. Moreover, transient transfection of H type alpha1, 2fucosyltransferase in clone C127-T-FT directs synthesis of Lewisyantigen, as detected by flow cytometry. Results indicate that Fuc-TIII expressed in C127 cells synthesizes masked Lewisxantigen while Lewisxantigen is not detectable.

Insertion of two independent enhancers in the long terminal repeat of a self-inactivating vector results in high-titer retroviral vectors with tissue-specific expression.

The use of retroviral vectors (RVs) derived from the murine oncoretroviruses for gene therapy is associated with the risk of malignant transformation of infected cells and ectopic expression of the proteins of interest. Targeting retroviral vectors to specific tissues would increase their safety and clinical applicability. To explore the potential of targeting vector expression to skeletal muscle, the murine leukemia virus broad transcriptional tropism was modified by substituting the viral promoter and/or enhancer with a transcriptional cassette containing the human T cell leukemia virus type I Tax-responsive element and the minimal muscle creatine kinase enhancer and promoter. The resulting retroviral vectors could be transcriptionally trans-activated by tax. In the absence of Tax, however, the viruses showed muscle-specific expression. Trans-complementing packaging and indicator cells stably expressing Tax were used to isolate high-titer producer cell clones (10(6) CFU/ml). In vitro, the levels of expression of these RVs in Tax-expressing fibroblasts were 10,000-fold higher than in normal fibroblasts and 1000-fold higher in C2C12 myotubes than in C2C12 myoblasts. Expression of the vectors and the endogenous muscle creatine kinase gene was similarly dependent on the maturity of the muscle cultures. One vector with modified LTRs was also tested in vivo in regenerating muscle and showed a delayed pattern of expression in myofibers compared with the vector containing the wild-type LTRs. These vectors can be easily modified to contain different tissue-specific enhancer and promoter elements and the availability of complementing packaging and indicator cells expressing Tax should allow their application in a variety of gene therapy settings.

The effect of cornea proteoglycans on liposome peroxidation.

Proteoglycans (PGs) from bovine cornea showed a protective effect on liposome peroxidation induced by Fe2+. Both chondroitin sulfate, dermatan sulfate-containing PG (CS,DS-PG) and keratan sulfate-containing PG (KS-PG) inhibited thiobarbituric acid-reactive substance formation when incubated with liposomes and Fe2+, CS,DS-PG being more effective than KS-PG. The native structure of PGs contributed markedly to antioxidant activity. Papain digestion of core protein reduced the protective effect of CS,DS-PG, whereas it abolished completely that of KS-PG. Apparently only hexuronate-containing glycosaminoglycan (GAG) chains may exert a significant antioxidant activity and this was confirmed using standard GAGs. Quasielastic laser light scattering was used to evaluate the structural consequence of peroxidative damage induced by Fenton reagent on liposomes. After exposure to the free-radical-generating system, a bimodal distribution of liposomes was observed, probably depending on the loss of native structure and fragmentation. Both CS,DS-PG and KS-PG prevented liposome breakdown. Again, free KS chains were ineffective against liposome damage, whereas DS and CS maintained the normal distribution of liposome size. These data support the hypothesis that PGs may represent part of the antioxidant mechanisms of organisms and suggest that modifications of PG content and/or composition might affect tissue sensitivity to oxidative stress.

Duplication of dystrophin gene and dissimilar clinical phenotype in the same family.

We report here three related patients with a duplication of exons 19-41 of the dystrophin gene, having dissimilar clinical phenotype and dystrophin immunohistochemistry. Two brothers aged six and three years had myalgia, proximal muscular weakness and hypertrophic calves, with 10- 20-fold increase of serum creatine kinase. Muscle biopsy showed dystrophic changes and reduced, patchy binding of dystrophin. The clinical and laboratory findings were consistent with a diagnosis of Becker muscular dystrophy with early onset. Their 14-year-old cousin had only mild hyperCKemia. His muscle biopsy was normal with only mild reduction of dystrophin immunostaining. At follow-up, he is still without symptoms and signs at age 19. All three patients had the same gene duplication and an increased dystrophin size of 507 kDa. Expression of the dystrophin-associated glycoproteins adhalin, alpha-dystroglycan, and beta-dystroglycan were normal in the three patients. An intrafamilial variability in patients carrying a partial duplication of the dystrophin gene may be related to a quantitative difference in mRNA.