High intrafamilial variability in autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy: a case study.

INTRODUCTION: Autoimmune polyendocrinopathy- candidiasis-ectodermal-dystrophy syndrome (APECED) is a monogenic disease whose phenotype may reveal wide heterogeneity. The reasons of this variability still remain obscure. PATIENTS AND METHODS: Two APECED siblings with identical genotype and extremely different phenotype were compared with regard to exposure to infectious triggers, autoantibodies' profile, mechanisms of peripheral tolerance, and human leukocyte antigen (HLA) haplotype. The following infectious markers were evaluated: rubella, Epstein Barr virus, cytomegalovirus, toxoplasma, varicella zoster virus, parvovirus B19, herpes simplex virus, and parainfluenza virus. APECED-related autoantibodies were detected by indirect immunofluorescence or complement fixation or enzyme- linked immunosorbent assay or radioimmunoassay. Resistance to Fas-induced apoptosis was evaluated on peripheral blood mononuclear cells (PBMC) activated with phytohemoagglutinin, the number of TCD4+CD25+ regulatory cells (Treg) was evaluated through flow-cytometry and natural killer (NK) activity through Wallac method. Perforin (PRF1) was amplified by PCR and sequenced. RESULTS: No difference was observed between the siblings in common infectious triggers, extent of Fas-induced apoptosis, NK-cell activity and PRF1 sequence, the number of Tregs and HLA haplotypes. CONCLUSION: Although APECED is a monogenic disease, its expressivity may be extremely different even in the same family. This variability cannot be explained by common triggering infectious agents or functional alterations of mechanisms governing peripheral tolerance.

FOXN1 mutation abrogates prenatal T-cell development in humans.

BACKGROUND: The transcription factor FOXN1 is implicated in the differentiation of thymic and skin epithelial cells, and alterations in it are responsible for the Nude/SCID phenotype. During a genetic counselling programme offered to couples at risk in a community where a high frequency of mutated FOXN1 had been documented, the identification of a human FOXN1(-/-) fetus gave the unique opportunity to study T cell development in utero. RESULTS: Total blockage of CD4(+) T cell maturation and severe impairment of CD8(+) cells were documented. Evaluation of the variable-domain ß-chain (Vß) families' usage among T lymphocytes revealed that the generation of T cell receptor (TCR) diversity occurred to some extent in the FOXN1(-/-) fetus, although it was impaired compared with the control. A few non-functional CD8(+) cells, mostly bearing TCRγδ in the absence of CD3, were found. DISCUSSION: FOXN1 is crucial for in utero T cell development in humans. The identification of a limited number of CD8(+) cells suggests an extrathymic origin for these cells, implying FOXN1-independent lymphopoiesis.

Thymoma-associated immunodeficiency: a syndrome characterized by severe alterations in NK, T and B-cells and progressive increase in naïve CD8+ T Cells.

Thymomas are rare tumours that sustain T-lymphopoiesis and trigger a variety of autoimmune diseases and immunodeficiencies, including a fatal hypogammaglobulinemia, namely Goods Syndrome (GS). Due to its rarity, GS has been poorly investigated and immunological features, as well as pathogenetic mechanisms underlying this syndrome, are unclear. We studied 30 thymoma patients by performing an immunological assessment, including immunophenotype and analysis of T cell repertoire (TCR). Development of GS was characterized by a progressive decrease in B, CD4 T and NK lymphocytes. These alterations paired with accumulation of CD8+CD45RA+ T cells that showed a polyclonal repertoire without expansions of specific clonotypes. GS is defined as hypogammaglobulinemia with thymoma. Here, we show for the first time that this syndrome is characterized by a severe loss of CD4+, NK and B cells. Furthermore, the accumulation of CD8+CD45RA+ T lymphocytes parallels these changes; this accumulation may have a role in determining the disease and can be used to monitor clinical stages of immunodeficiency in thymoma.

Cardiac involvement in Becker muscular dystrophy.

OBJECTIVES: The purpose of this study was to assess the incidence of myocardial involvement and the relation of cardiac disease to the molecular defect at the deoxyribonucleic acid (DNA) or protein level in Becker muscular dystrophy. BACKGROUND: Dystrophin gene mutations produce clinical manifestations of disease in the heart and skeletal muscle of patients with Becker muscular dystrophy. METHODS: Thirty-one patients underwent electrocardiographic and echocardiographic examination and 24-h Holter monitoring. The diagnosis was established by neurologic examination, dystrophin immunohistochemical assays or Western blot on muscle biopsy, or both, and DNA analysis. RESULTS: Electrocardiographic and echocardiographic findings were abnormal in 68% and 62% of the patients, respectively. Right ventricular involvement was detected in 52%. Left ventricular impairment was observed either as an isolated phenomenon (10%) or in association with right ventricular dysfunction (29%). Right ventricular disease was manifested in the teenagers, and an impairment of the left ventricle was observed in older patients. Right ventricular end-diastolic volumes were significantly increased compared with those in a control group. The left ventricular ejection fraction was significantly lower in older patients than in control subjects or younger patients. Life-threatening ventricular arrhythmias were detected in four patients. No correlations were found between skeletal muscle disease, cardiac involvement and dystrophin abnormalities. In our patients, exon 49 deletion was invariably associated with cardiac involvement. Exon 48 deletion was associated with cardiac disease in all but two patients. CONCLUSIONS: The cardiac manifestation of Becker muscular dystrophy is characterized by early right ventricular involvement associated or not with left ventricular impairment. Exon 49 deletion is associated with cardiac disease.

New once-daily, highly effective rescue triple therapy after multiple Helicobacter pylori treatment failures: a pilot study.

BACKGROUND: Helicobacter pylori treatment failure is a growing problem in daily practice. AIM: To determine the efficacy of the combination of rabeprazole, levofloxacin and furazolidone as a rescue therapy. METHODS: Duodenal ulcer patients previously submitted, without success, to at least two H. pylori treatment regimens were included. Gastroscopy (urease test, histological examination and culture) and (13)C-urea breath test were performed. All patients received a combination of rabeprazole 20 mg, levofloxacin 500 mg and furazolidone 200 mg (two tablets) administered in a single dose in the morning for 10 days. Clinical examination and a new (13)C-urea breath test were performed 90 days after therapy. RESULTS: Twelve patients (eight females and four males), mean age 43 (30-58) years were included. Two patients failed to complete the treatment because of nausea and vomiting. Ten patients completed the study and took all the medications as advised. Culture was obtained in six patients: 100 and 83% of the samples were sensitive to furazolidone and levofloxacin, respectively. Per-protocol and intention-to-treat eradication rates were 100 and 83% (P = 0.019). CONCLUSIONS: the combination of rabeprazole, levofloxacin and furazolidone in a single daily dose for 10 days constitutes a highly-effective and low-cost alternative as a third-line therapy in patients infected with H. pylori.

Implicación ocular en pacientes con distrofia miotónica / Eye involvement in patients with myotonic dystrophy

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Prevalence of dystrophin-positive fibers in 85 Duchenne muscular dystrophy patients.

The prevalence of dystrophin-positive fibers in Duchenne muscular dystrophy (DMD) muscle was estimated by direct counting on immunostained sections in a series of biopsy specimens from 85 patients, 42 of which were also screened for intragenic deletions by cDNA probes. Dystrophin-positive fibres are normotrophic and occur in muscle sections at a frequency between 0.01 and 6.81%. Frequencies over 1% were found only in patients older than 6 yr. The prevalence of dystrophin-positive fibers is about the same in patients with detectable and with undetectable deletions. The occurrence of positive fibers in small clusters supports the hypothesis of their clonal origin, suggesting that they may result from genetic reversion. No clinical differences were noticed in DMD patients of similar age with respect to the occurrence of dystrophin-positive fibres in their muscle biopsies.

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.

Occurrence of two different intragenic deletions in two male relatives affected with Duchenne muscular dystrophy.

The occurrence of 2 different intragenic deletions (exons 10-44 and exon 45, respectively) is reported in 2 male relatives affected with Duchenne muscular dystrophy, both showing the same haplotype for DNA markers not included in the deleted segment. The 2 different deletions seem to have occurred independently in the same X chromosome. This finding, together with other reports, suggests possibly an increased predisposition to mutations within the DMD locus in some families. Therefore, when dealing with prenatal diagnosis, the investigation on fetal DNA cannot be restricted only to the region in which a mutation was previously identified in the family.

Ovarian stimulation using a new highly purified urinary FSH: a prospective randomized clinical study.

The aim of this study was to determine the effectiveness of a new highly purified urinary FSH. A total of 60 in vitro-fertilization (IVF) patients, undergoing embryo transfer (ET) for the first time, were randomly allocated into two groups: Group A (n = 30). Subcutaneous administration of urinary follicle-stimulating hormone (FSH, Fostimon 75, A.M.S.A., Italy). Group B (n = 30). Subcutaneous administration of urinary follicle-stimulating hormone (FSH, Metrodin 75 HP, Serono, Italy). Statistical analysis was performed using the chi-square test, p < 0.05 was assumed as significant. This prospective randomized clinical study in an IVF-ET program showed that both drugs were equally safe and effective. Except for the number of the high quality embryos (3.16 vs 2.9; p = 0.03) the two groups did not differ in stimulation parameters or clinical pregnancy rates per attempt and per transfer. On the other hand, a mean number of 3.56 vs 2.18 embryos were cryopreserved in group A and in group B, respectively, as a result of the high number of mature oocytes and high quality embryos. When frozen embryos cycles were included, the difference in pregnancy rate became significant.

Familial temporal lobe epilepsy with psychic auras associated with a novel LGI1 mutation.

BACKGROUND: Autosomal dominant lateral temporal epilepsy (ADLTE) is characterized by focal seizures with auditory features or aphasia. Mutations in the LGI1 gene have been reported in up to 50% of ADLTE pedigrees. We report a family with temporal lobe epilepsy characterized by psychic symptoms associated with a novel LGI1 mutation. METHODS: All participants were personally interviewed and underwent neurologic examination and video-EEG recordings. LGI1 exons were sequenced by standard methods. Mutant cDNA was transfected into human embryonic kidney 293 cells; both cell lysates and media were analyzed by Western blot. In silico modeling of the Lgi1 protein EPTP domain was carried out using the structure of WD repeat protein and manually refined. RESULTS: Three affected family members were ascertained, 2 of whom had temporal epilepsy with psychic symptoms (déjà vu, fear) but no auditory or aphasic phenomena, while the third had complex partial seizures without any aura. In all patients, we found a novel LGI1 mutation, Arg407Cys, which did not hamper protein secretion in vitro. Mapping of the mutation on a 3-dimensional protein model showed that this mutation does not induce large structural rearrangements but could destabilize interactions of Lgi1 with target proteins. CONCLUSIONS: The Arg407Cys is the first mutation with no effect on Lgi1 protein secretion. The uncommon, isolated psychic symptoms associated with it suggests that ADLTE encompasses a wider range of auras of temporal origin than hitherto reported.

The glucose-dependent insulinotropic polypeptide receptor is overexpressed amongst GNAS1 mutation-negative somatotropinomas and drives growth hormone (GH)-promoter activity in GH3 cells.

Somatic mutations in the GNAS1 gene, encoding the α-subunit of the heterotrimeric stimulatory G protein (Gαs), occur in approximately 40% of growth hormone (GH)-secreting pituitary tumours. By altering the adenylate cyclase-cAMP-protein kinase A pathway, they unequivocally give somatotroph cells a growth advantage. Hence, the pathogenesis of somatotropinomas could be linked to anomalies in receptors coupled to the cAMP second-messenger cascade. Among them, the glucose-dependent insulinotropic polypeptide receptor (GIPR) is already known to play a primary role in the impaired cAMP-dependent cortisol secretion in patients affected by food-dependent Cushing's syndrome. In the present study, 43 somatotropinomas and 12 normal pituitary glands were investigated for GIPR expression by quantitative reverse transcriptase-polymerase chain reaction, western blotting and immunohistochemistry. Tumoural specimens were also evaluated for GNAS1 mutational status. The effect of GIPR overexpression on cAMP levels and GH transcription was evaluated in an in vitro model of somatotropinomas, the GH-secreting pituitary cell line GH3. GIPR was expressed at higher levels compared to normal pituitaries in 13 GNAS1 mutation-negative somatotropinomas. GIP stimulated adenylyl cyclase and GH-promoter activity in GIPR-transfected GH3 cells, confirming a correct coupling of GIPR to Gαs. In a proportion of acromegalic patients, GIPR overexpression appeared to be associated with a paradoxical increase in GH after an oral glucose tolerance test. Whether GIPR overexpression in acromegalic patients may be associated with this paradoxical response or more generally involved in the pathogenesis of acromegaly, as suggested by the mutually exclusive high GIPR levels and GNAS1 mutations, remains an open question.

Calmodulin-dependent kinase II mediates vascular smooth muscle cell proliferation and is potentiated by extracellular signal regulated kinase.

Vascular smooth muscle cell (VSMC) proliferation contributes to vascular remodeling in atherosclerosis and hypertension. Calcium-dependent signaling through calcium/calmodulin-dependent kinase II (CaMKII) and ERK1/2 activation plays an important role in the regulation of VSMC proliferation by agents such as alpha-adrenergic receptor agonists. Nevertheless, how the CaMKII and ERK pathways interact in VSMCs has yet to be characterized. The aim of the present study was to clarify this interaction in response to alpha(1)-adrenergic receptor-mediated VSMC proliferation. We discovered that phenylephrine stimulation resulted in complex formation between CaMKII and ERK in a manner that facilitated phosphorylation of both protein kinases. To assess the effects of CaMKII/ERK association on VSMC proliferation, we inhibited endogenous CaMKII either pharmacologically or by adenoviral-mediated gene transfer of a kinase-inactive CaMKII mutant. Inhibition of CaMKII activation but not CaMKII autonomous activity significantly decreased formation of the CaMKII/ERK complex. On the contrary, the expression of constitutively active CaMKII enhanced VSMC growth and CaMKII/ERK association. In addressing the mechanism of this effect, we found that CaMKII could not directly phosphorylate ERK but instead enhanced Raf1 activation. By contrast, ERK interaction with CaMKII facilitated CaMKII phosphorylation and promoted its nuclear localization. Our results reveal a critical role for CaMKII in VSMC proliferation and imply that CaMKII facilitates assembly of the Raf/MEK/ERK complex and that ERK enhances CaMKII activation and influences its subcellular localization.

CD8+ T-cell alveolitis in familial pulmonary alveolar microlithiasis.

Pulmonary alveolar microlithiasis (PAM) is a rare diffuse lung disease characterised by the accumulation of calcium phosphate microliths within the alveoli. The causative mechanism of PAM has only recently been discovered, and involves a gene mutation of sodium phosphate co-transporter, which is expressed by alveolar epithelial cells. This mutation may have variable consequences on the clinical phenotype. However, pulmonary cell immune phenotyping in familial PAM has not previously been assessed. In the present article, the analysis of bronchoalveolar lavage fluid of two siblings with PAM diagnosis revealed a pattern of lymphocytic alveolitis with accumulation of CD8+ T-cells. The clonal complexity of this lymphocyte's population was assayed by spectratyping, which showed an oligoclonal accumulation of T-cells with a restricted variable beta T-cell receptor (TCR) gene usage. TCR analysis in peripheral blood lymphocytes revealed no abnormal patterns of T-lymphocytes. In the pulmonary alveolar microlithiasis familial cases reported, CD8-mediated maladaptive immune response may have taken place in the bronchoalveolar compartment. The relationship between this immune dysregulation and genetic background in pulmonary alveolar microlithiasis warrants further investigation.

Genomic organization of the human dystrophin gene across the major deletion hot spot and the 3' region.

The genomic organization of most of the human dystrophin gene has not been defined at single-exon level, owing to its enormous size (2300 kb). By taking advantage of a YAC-based restriction map of the gene previously constructed, we have localized individual dystrophin exons from 42 to 79 along the central and 3' regions of the gene. These data elucidate the general organization of this large portion of the gene (1250 kb) and, in particular, characterize the genomic region most frequently involved in deletion mutations responsible for Duchenne and Becker muscular dystrophies.