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Textile organic electrochemical transistors (txOECTs) are a new class of wearable biosensors used to monitor physiological parameters in bio fluids of clinical interest. Herein the selectivity of a textile biosensor was improved directly functionalizing the textile device with ion selective membranes. The device was prepared by a series of consecutive functionalization of the textile fiber, first by applying the conductive polymer poly (3,4-ethylenedioxythiophene):poly (styrene sulfonate) (PEDOT:PSS) and subsequently the ion selective membrane based on different ionophores with the aim of measuring electrolytes in sweat. The ion selective membranes were previously tested and optimized by potentiometric measurements. The biosensor devices were studied with different concentration of electrolytes. Membrane selectivity was tested comparing transistor response with interfering ions, proving successfully the selective response to sodium, potassium and calcium ions. The ability of the textile biosensors to discriminate among the cations was demonstrated over the 10−5 - 1 M concentration range, a concentration range found in sweat. The electronic parameters of the txOECTs show differences not only in modulation response but also in time constants of kinetic behavior. The selective determination of potassium and calcium in sweat has a great importance in different applications, for example, in human sweat monitoring, to understand physiological conditions, like dehydration, cardiac bioactivity and hypokalemia.

Aims Pompe disease is an autosomal recessive lysosomal storage disorder resulting from deficiency of acid α-glucosidase (GAA) enzyme. Histopathological hallmarks in skeletal muscle tissue are fiber vacuolization and autophagy. Since 2006, ERT is the only approved treatment with human recombinant GAA alglucosidase alfa. We designed a study to examine ERT-related skeletal muscle changes in 18 modestly to moderately affected LOPD patients along with the relationship between morphological/biochemical changes and clinical outcomes. Treatment duration was short-to-long term. Methods We examined muscle biopsies from 18 LOPD patients at both histopathological and biochemical level. All patients underwent two muscle biopsies, before and after ERT administration respectively. The study is partially retrospective because the first biopsies were taken before the study was designed whereas the second biopsy was always performed after at least six months of ERT administration. Results After ERT, 15 out of 18 patients showed improved 6MWT (p=0.0007) and most of them achieved respiratory stabilization. Pre-treatment muscle biopsies disclosed marked histopathological variability, ranging from an almost normal pattern to a severe vacuolar myopathy. After treatment, we detected morphological improvement in 15 patients and worsening in 3 patients. Post-ERT GAA enzymatic activity was mildly increased compared with pre-treatment levels in all patients. Protein levels of the mature enzyme increased in 14 of the 18 patients (mean increase = +35%; p

Spinocerebellar ataxias (SCAs) are a genetically heterogeneous group of cerebellar degenerative disorders, characterized by progressive gait unsteadiness, hand incoordination, and dysarthria. Ataxia type 1 (SCA1) is caused by the expansion of a CAG trinucleotide repeat in the SCA1 gene resulting in the atypical extension of a polyglutamine (polyQ) tract within the ataxin-1 protein. Our main objective was to investigate the mitochondrial oxidative metabolism in the cerebellum of transgenic SCA1 mice. SCA1 transgenic mice develop clinical features in the early life stages (around 5 weeks of age) presenting pathological cerebellar signs with concomitant progressive Purkinje neuron atrophy and relatively little cell loss; this evidence suggests that the SCA1 phenotype is not the result of cell death per se, but a possible effect of cellular dysfunction that occurs before neuronal demise. We studied the mitochondrial oxidative metabolism in cerebellar cells from both homozygous and heterozygous transgenic SCA1 mice, aged 2 and 6 months. Histochemical examination showed a cytochrome-c-oxidase (COX) deficiency in the Purkinje cells (PCs) of both heterozygous and homozygous mice, the oxidative defect being more prominent in older mice, in which the percentage of COX-deficient PC was up to 30%. Using a laser-microdissector, we evaluated the mitochondrial DNA (mtDNA) content on selectively isolated COX-competent and COX-deficient PC by quantitative Polymerase Chain Reaction and we found mtDNA depletion in those with oxidative dysfunction. In conclusion, the selective oxidative metabolism defect observed in neuronal PC expressing mutant ataxin occurs as early as 8 weeks of age thus representing an early step in the PC degeneration process in SCA1 disease.

Summary A study was undertaken to analyze the clinical presentation, pulmonary function, and pathological features in two female siblings with neonatal pulmonary surfactant metabolism dysfunction, type 3 (MIM 610921). The clinical records of the siblings were examined; the genes encoding surfactant protein B (SFTPB), surfactant protein C (SFTPC), and ATP-binding cassette transporter 3 protein (ABCA3) were analyzed with direct sequencing and Southern blotting. The infants were homozygous for a 5,983 bp deletion in ABCA3 including exons 2–5 as well as the start AUG codon and a putative Golgi exit signal motif. Dense abnormalities of lamellar bodies at electron microscopy and absence of ABCA3 at immunohistochemical staining were in agreement with the presence of two null alleles. In addition, an increased lipid synthesis suggested a compensatory mechanism. The clinical course in the two sisters was influenced by different environmental factors like the time needed for molecular confirmation, the ventilatory assistance adopted, the occurrence of infections. A less aggressive clinical approach did not improve the course of the disease; the prognosis was always poor. Development of a fast molecular test, able to detect also structural variants, is needed. Pediatr Pulmonol. 2014; 49:E116–E120. © 2014 Wiley Periodicals, Inc.

Leigh syndrome (LS) is an incurable, nearly always fatal, neurodegenerative, pediatric disorder that results from respiratory chain failure. The most common mitochondrial DNA (mtDNA) mutations that result in LS are m.8993T→C/G and m.9176T→C/G, which were previously found in several patients with early-onset Leigh syndrome. Here, we describe clinical and molecular features of a novel pedigree, where LS developed in two siblings. The proband was a young woman with an unusual adult-onset LS. She harbored a homoplasmic m.9176T→C mutation, based on analysis of a muscle biopsy. In contrast, the brother died at a young age. This novel case report and literature review highlights the variability of phenotypic expression of the m.9176T→C mutation.

Duchenne and Becker muscular dystrophy (DMD and BMD, respectively) are allelic disorders with different clinical presentations and severity determined by mutations in the gene DMD, which encodes the sarcolemmal protein dystrophin. Diagnosis is based on clinical aspects and muscle protein analysis, followed by molecular confirmation. We revised the main aspects of the natural history of dystrophinopathies to define genotype-phenotype correlations in large patient cohorts with extended follow-up. We also specifically explored subjects carrying nucleotide substitutions in the DMD gene, a comparatively less investigated DMD/BMD subgroup. We studied 320 dystrophinopathic patients (205 DMD and 115 BMD), defining muscular, cardiac, respiratory, and cognitive involvement. We also subdivided patients according to the kind of molecular defect (deletions, duplications, nucleotide substitutions or other microrearrangements) and the mutation sites (proximal/distal to exon 45), studying phenotype-genotype correlations for each group. In DMD, mutation type did not influence clinical evolution; mutations located in distal regions (irrespective of their nature) are more likely to be associated with lower IQ levels (p = 0.005). BMD carrying proximal deletions showed a higher degree of cardiac impairment than BMD with distal deletions (p = 0.0046). In the BMD population, there was a strong correlation between the entity of muscle dystrophin deficiency and clinical course (p = 0.002). An accurate knowledge of natural history may help in the clinical management of patients. Furthermore, several clinical trials are ongoing or are currently planned, some of which aim to target specific DMD mutations: a robust natural history is therefore essential to correctly design these experimental trials.

The important depletion of mitochondrial DNA (mtDNA) and the general depression of mitochondrial respiratory chain complex levels (including complex II) have been confirmed, implying an increasing paucity of mitochondria in the muscle from patients with types I, II, and III spinal muscular atrophy (SMA-I, -II, and -III, respectively).To investigate mitochondrial dysfunction in a large series of muscle biopsy samples from patients with SMA.We studied quadriceps muscle samples from 24 patients with genetically documented SMA and paraspinal muscle samples from 3 patients with SMA-II undergoing surgery for scoliosis correction. Postmortem muscle samples were obtained from 1 additional patient. Age-matched controls consisted of muscle biopsy specimens from healthy children aged 1 to 3 years who had undergone analysis for suspected myopathy. Analyses were performed at the Neuromuscular Unit, Istituto di Ricovero e Cura a Carattere Scientifico Foundation Ca' Granda Ospedale Maggiore Policlinico-Milano, from April 2011 through January 2015.We used histochemical, biochemical, and molecular techniques to examine the muscle samples.Respiratory chain activity and mitochondrial content.Results of histochemical analysis revealed that cytochrome-c oxidase (COX) deficiency was more evident in muscle samples from patients with SMA-I and SMA-II. Residual activities for complexes I, II, and IV in muscles from patients with SMA-I were 41%, 27%, and 30%, respectively, compared with control samples (P .005). Muscle mtDNA content and cytrate synthase activity were also reduced in all 3 SMA types (P .05). We linked these alterations to downregulation of peroxisome proliferator-activated receptor coactivator 1α, the transcriptional activators nuclear respiratory factor 1 and nuclear respiratory factor 2, mitochondrial transcription factor A, and their downstream targets, implying depression of the entire mitochondrial biogenesis. Results of Western blot analysis confirmed the reduced levels of the respiratory chain subunits that included mitochondrially encoded COX1 (47.5%; P = .004), COX2 (32.4%; P .001), COX4 (26.6%; P .001), and succinate dehydrogenase complex subunit A (65.8%; P = .03) as well as the structural outer membrane mitochondrial porin (33.1%; P .001). Conversely, the levels of expression of 3 myogenic regulatory factors-muscle-specific myogenic factor 5, myoblast determination 1, and myogenin-were higher in muscles from patients with SMA compared with muscles from age-matched controls (P .05).Our results strongly support the conclusion that an altered regulation of myogenesis and a downregulated mitochondrial biogenesis contribute to pathologic change in the muscle of patients with SMA. Therapeutic strategies should aim at counteracting these changes.

Six years before the present study we performed a retrospective study of 114 subjects presenting with asymptomatic / oligosymptomatic hyperckemia (raised creatine kinase blood levels), a diagnosis being made in 21 of them. We now present the results of a long-term follow-up in 55 of the still undiagnosed 93 individuals. Most of them have remained asymptomatic and did not develop specific neuromuscular disorders. One subject became frankly symptomatic manifesting limb-girdle weakness. A diagnosis of dystrophinopathy carrier and one of possible type I SMA carrier were indirectly made in another two subjects. Almost all subjects still have hyperckemia, though the mean creatine kinase (CK) value is lower than before. CK levels have become normal in 12 subjects. Two died of neoplasia, and six developed non-neuromuscular disorders. We noted no follow-up differences in terms of CK modifications between subjects with pathological EMG and/or muscle biopsy findings and those with normal findings at first examination.

Bone fragility and low bone mass have been reported in small case series of patients with Pompe disease with severely reduced muscle strength or immobilization.Our objective was to determine the prevalence of morphometric vertebral fractures and to evaluate bone mass in adults with late-onset Pompe disease.We conducted a multicenter cross-sectional observational study from August 2012 to December 2013.All subjects were outpatients referred to University Referral Centers.PATIENTS included 22 late-onset Pompe disease patients with progressive proximal myopathy and minimal respiratory involvement without other diseases affecting bone mass.The prevalence of morphometric vertebral fractures was systematically assessed by semiquantitative analysis of lateral spine x-rays (T4-L5).A high prevalence of morphometric vertebral fractures was found. At least 1 vertebral fracture was present in 17 of 22 patients (77%). All vertebral fractures were asymptomatic. Bone mineral density was normal in 36.5% of the patients, whereas 36.5% were osteopenic and 27% were osteoporotic in at least 1 site. Fracture prevalence was independent of muscular and respiratory functional parameters and of genotype.Our data show for the first time that asymptomatic and atraumatic vertebral fractures occur frequently in late-onset Pompe disease patients without a significant impairment of bone mass. Screening for asymptomatic vertebral fractures should be routinely performed in Pompe disease irrespective of the disease severity. Fracture risk should be confirmed in longitudinal studies.

Objective: To identify the genetic cause of a complex syndrome characterized by autophagic vacuolar myopathy (AVM), hypertrophic cardiomyopathy, pigmentary retinal degeneration, and epilepsy. Methods: Clinical, pathologic, and genetic study. Results: Two brothers presented with visual failure, seizures, and prominent cardiac involvement, but only mild cognitive impairment and no motor deterioration after 40 years of disease duration. Muscle biopsy revealed the presence of widespread alterations suggestive of AVM with autophagic vacuoles with sarcolemmal features. Through combined homozygosity mapping and exome sequencing, we identified a novel p.Gly165Glu mutation in CLN3 . Conclusions: This study expands the clinical phenotype of CLN3 disease. Genetic testing for CLN3 should be considered in AVM with autophagic vacuoles with sarcolemmal features.

BACKGROUND The induction of estrogen and progesterone receptors (ER and PGR) has been reported in breast and endometrial cancer cells exposed to human fibroblast interferon-beta (hIFN-beta). Clinical verification of this finding might provide the rationale for new therapeutic approaches. This study was designed to evaluate whether clinical treatment with high doses of hIFN-beta induced ER and PGR in patients with endometrial adenocarcinoma. METHODS Two biopsies were obtained, 1 before and 1 after hIFN-beta treatment (3 x 10(6) i.m. every other day for 3 weeks) from 36 patients with endometrial adenocarcinoma. ER and PGR were determined with standard procedures using radiolabeled ligands. RESULTS hIFN-beta treatment did not affect the proportion of ER-positive (i.e., >15 fmol/mg protein) or PGR-positive (i.e., >20 fmol/mg protein) cases. However, in patients with detectable ER and PGR at baseline, hIFN-beta raised the levels. Using a 35% difference before and after therapy as a cut-off, 72 and 79% of cases had increases in ER and PGR, respectively. The difference was highly significant for PGR. CONCLUSIONS In patients with endometrial adenocarcinoma with undetectable ER or PGR, hIFN-beta did not induce the expression of these receptors. When the receptors were present they were upregulated by hIFN-beta. Whether this increase in receptor levels, particularly PGR, has therapeutic applications remains to be established.

Different regimens of chemotherapy have been proposed with contradictory results in patients with metastatic and recurrent cervical carcinoma: this seems mainly due to the lack of information about prognostic factors. This study was aimed at identifying significant factors predicting response to treatment and survival in 140 patients treated with chemotherapy for advanced, recurrent, or persistent squamous cell cervical carcinoma. Age, performance status, histologic type and grade, previous irradiation, interval from start of primary treatment and from irradiation, site of tumor, and therapeutic regimen were considered as possible predictors of response and survival in multivariate analysis. By multivariate analysis, only performance status and interval from irradiation (>1 year) were significant in predicting response to treatment, whereas interval from first diagnosis, site of tumor, and response to treatment were significant in predicting survival. None of the polychemotherapy regimens significantly improved survival, despite a fourfold increase in the costs when compared to the least expensive monochemotherapy regimen. Ethical and economical concerns should be considered when proposing aggressive regimens to the patients. Factors such as site of tumor, performance status, and interval from first treatment should be considered as minimal requirements for correct evaluation of newly proposed regimens.

Limb-girdle muscular dystrophy (LGMD) 2L, caused by mutations in the anoctamin 5 (ANO5) gene, is the third most common LGMD in Northern and Central Europe, where the c.191dupA mutation causes the majority of cases. We evaluated data from 228 Italian LGMD patients to determine the prevalence of LGMD2L and the c.191dupA mutation, and to describe the clinical, muscle biopsy, and magnetic resonance imaging findings in these patients. Forty-three patients who lacked molecular diagnosis were studied for ANO5 mutations, and four novel mutations were found in three probands. Only one proband carried the c.191dupA mutation, which was compound heterozygous with c.2516T>G. Two probands were homozygous for the c.1627dupA and c.397A>T mutations, respectively, while a fourth proband had a compound heterozygous status (c.220C>T and c.1609T>C). Therefore occurrence and molecular epidemiology of LGMD2L in this Italian cohort differed from those observed in other European countries. ANO5 mutations accounted for ∼2% of our sample. Affected patients exhibited benign progression with variable onset and an absence of cardiac and respiratory impairment; muscle biopsy generally showed mild signs, except when performed on the quadriceps muscles; MRI showed predominant involvement of the posterior thigh. Overall these common clinical, morphological and imaging findings could be useful in differential diagnosis.

Background POLG1 mutations have been associated with MELAS-like phenotypes. However given several clinical differences it is unknown whether POLG1 mutations are possible causes of MELAS or give raise to a distinct clinical and genetic entity, named POLG1-associated encephalopathy. Case presentation We describe a 74 years old man carrying POLG1 mutations presenting with strokes, myopathy and ragged red fibers with some atypical aspects for MELAS such as late onset, lack of cerebral calcification and presence of frontal and occipital MRI lesions better consistent with the POLG associated-encephalopathy spectrum. Conclusion The lack of available data hampers a definite diagnosis in our patient as well as makes it difficult to compare MELAS, which is a clearly defined clinical syndrome, with POLG1-associated encephalopathy, which is so far a purely molecularly defined syndrome with a quite heterogeneous clinical picture. However, the present report contributes to expand the phenotypic spectrum of POLG1 mutations underlining the importance of searching POLG1 mutations in patients with mitochondrial signs and MELAS like phenotypes but negative for common mtDNA mutations.

A case report of four patients affected by primary ovarian small cell carcinoma is presented. The surgical and chemotherapeutic approaches are described for each case. Cisplatin-based chemotheraphy obtained objective responses in two of four patients, but three of four patients died of disease progression within 2 years from the diagnosis. One patient, who was stage Ib and underwent bilateral salpingo-oophorectomy, total hysterectomy, and adjuvant chemotheraphy is still alive 58 months after surgery. Accurate diagnosis and a multidisciplinary approach are needed to ameliorate the prognosis of this tumor that, at present, remains very poor.

Summary Background The unique mechanism of action of taxol (NSC-125973) as microtubule stabilizing agent and its potential activity in clinical trials have generated considerable interest in the development of this agent. As taxol was reported to be active on advanced and refractory ovarian carcinomas we focused our studies on the xenograft model of human ovarian carcinoma that develops ascites and tumor dissemination in the peritoneal cavity of nude mice. Methods The antitumor activity of taxol was evaluated on two human ovarian carcinoma xenografts (HOC8 and HOC22) transplanted i.p. in nude mice. Drug was given i.v. at doses of 20–34.5 mg/kg every four days three times (Q4 × 3) and the increment of life span (%ILS) was evaluated. Cisplatin at the dosage of 4mg/kg, Q4 × 3 was used as reference drug in each experiment. Results Taxol given at doses of 20 mg/kg and 34.5 mg/kg to early-stage HOC22 (treatment starting 3 days after tumor transplant) cured all the mice, while the same dose regimens given to advanced HOC22 (treatment starting 14 days after tumor transplant) significantly prolonged the survival time of the mice (ILS = 197% and 300%). Taxol given 3 days after HOC8 transplant significantly prolonged the survival time of tumor-bearing nude mice, inducing complete responses in 50% and 25% of mice receiving, respectively, 34.5 mg/kg/ injection and 20 mg/kg/injection. On both ovarian carcinoma xenografts taxol was more active than equitoxic doses of the reference drug cisplatin. Conclusions The therapeutic activity against ovarian carcinoma xenografts supports the potential of taxol in the treatment of this neoplasia and forms the basis for future investigations aimed at optimizing the therapeutic activity of taxol given alone or in combination with other drugs.

Glycogenosis type II (OMIM 23230) is an autosomal recessive lysosomal storage disorder resulting from a deficiency in the glucosidase alpha acid (GAA) enzyme. The disease is characterized by progressive accumulation of lysosomal glycogen in various tissues, primarily heart and skeletal muscle, and it is clinically classified into three forms: infantile, juvenile, and late-onset. The histopathological hallmarks in muscle tissue are fiber vacuolization and autophagy. Recombinant human GAA is the only approved enzyme replacement therapy (ERT) available for disease treatment. It is effective in most infantile patients, whereas the improvement is quite variable in adults. We analyzed muscle biopsies from 14 late-onset patients at molecular, biochemical, and histopathological level before and after ERT. We evaluated the following morphological parameters: CSA, number of vacuolated fibers, degree of glycogen accumulation, percentage of vacuolization in type I and II fibers. Pre-treatment muscle biopsies showed marked histopathological variability ranging from almost normal morphology to severe vacuolar myopathy. Post-treatment muscle biopsies morphologically improved in seven patients, worsened in two patients and were unchanged in all the other subjects. Immunohistochemical analysis of the autophagic/lysosomal markers EEA1 (early endosome antigen 1), LC3 (microtubule-associated protein 1 light chain 3), and LAMP2 (lysosome associated membrane protein 2) showed a variable binding in both the first and the second biopsies. GAA enzymatic activity, tested by a fluorimetric assay in both lymphocytes and muscle tissue from 5 patients after ERT, was mildly increased in skeletal muscle compared with pre-treatment levels. Also, GAA expression assessed by immunoblotting slightly increased in a few patients. All patients clinically improved or remained stable after ERT.

Maintenance and replication of mitochondrial DNA require the concerted action of several factors encoded by nuclear genome. The mitochondrial helicase Twinkle is a key player of replisome machinery. Heterozygous mutations in its coding gene, PEO1, are associated with progressive external ophthalmoplegia (PEO) characterised by ptosis and ophthalmoparesis, with cytochrome c oxidase (COX)-deficient fibres, ragged-red fibres (RRF) and multiple mtDNA deletions in muscle. Here we describe clinical, histological and molecular features of two patients presenting with mitochondrial myopathy associated with PEO. PEO1 sequencing disclosed two novel mutations in exons 1 and 4 of the gene, respectively. Although mutations in PEO1 exon 1 have already been described, this is the first report of mutation occurring in exon 4.

Background Amyotrophic lateral sclerosis (ALS) is a major cause of neurological disability and its pathogenesis remains elusive despite a multitude of studies. Although defects of the mitochondrial respiratory chain have been described in several ALS patients, their pathogenic significance is unclear. Objective To review systematically the muscle biopsy specimens from patients with typical sporadic ALS to search for possible mitochondrial oxidative impairment. Design Retrospective histochemical, biochemical, and molecular studies of muscle specimens. Setting Tertiary care university. Subjects Fifty patients with typical sporadic ALS (mean age, 55 years). Main Outcome Measure Number of patients showing a clear muscle mitochondrial dysfunction assessed through histochemical and biochemical muscle analysis. Results Histochemical data showed cytochrome c oxidase (COX)–negative fibers in 46% patients. Based on COX histochemical activity, patients fell into 4 groups: 27 had normal COX activity; and 8 had mild (2-4 COX-negative fibers of 100 fibers), 8 had moderate (5-10 COX-negative fibers of 100), and 7 had severe (>10 COX-negative fibers of 100) COX deficiency. Spectrophotometric measurement of respiratory chain activities showed that 3 patients with severe histochemical COX deficiency also showed combined enzyme defects. In 1 patient, COX deficiency worsened in a second biopsy taken 9 months after the first. Among the patients with severe COX deficiency, one had a new mutation in the SOD1 gene, another a mutation in the TARDBP gene, and a third patient with biochemically confirmed COX deficiency had multiple mitochondrial DNA deletions detectable by Southern blot analysis. Conclusions Our data confirm that the histochemical finding of COX-negative fibers is common in skeletal muscle from patients with sporadic ALS. We did not find a correlation between severity of the oxidative defect and age of the patients or duration of the disease. However, the only patient who underwent a second muscle biopsy did show a correlation between severity of symptoms and worsening of the respiratory chain defect. In 7 patients, the oxidative defect was severe enough to support the hypothesis that mitochondrial dysfunction must play a role in the pathogenesis of the disease.

Background Premature ovarian failure (POF) is a secondary hypergonadotrophic amenorrhea occurring before the age of 40 and affecting 1-3% of females. Chromosome anomalies account for 6-8% of POF cases, but only few cases are associated with translocations involving X and Y chromosomes. This study shows the cytogenetic and molecular analysis of a POF patient came to our attention as she developed a left ovary choriocarcinoma at the age of 10 and at 14 years of age she presented secondary amenorrhea with elevated levels of gonadotropins. Results Breakpoint position on X and Y chromosomes was investigated using Fluorescent In Situ Hybridisation (FISH) with a panel of specific BAC probes, microsatellite analysis and evaluation of copy number changes and loss of heterozigosity by Affymetrix® GeneChip platform (Santa Clara, CA, USA). Patient's karyotype resulted 46, X, der(Y)t(X;Y)(q13.1;q11.223). X inactivation study was assessed by RBA banding and showed preferential inactivation of derivative chromosome. The reciprocal spatial disposition of sexual chromosome territories was investigated using whole chromosome painting and centromeres probes: patient's results didn't show a significant difference in comparison to normal controls. Conclusion The peculiar clinical case come to our attention highlighted the complexity of POF aetiology and of the translocation event, even if our results seem to exclude any effect on nuclear organisation. POF phenotype could be partially explained by skewed X chromosome inactivation that influences gene expression.

The authors describe the case of a patient affected with congenital muscular dystrophy with lack of muscle α-dystroglycan. Brain gross anatomy showed lissencephaly and pachygyria. Light microscopy showed heterotopias in white matter. The brain stem and cerebellum were normal. They found no expression of α-dystroglycan either in the frontal cortex or in the heterotopic nuclei, while a normal expression was found in the cerebellum. These results suggest that α-dystroglycan glycosylation defects may account for both the muscle disease and the brain supratentorial malformation in our patient. The authors did not identify any mutations in the genes most frequently related to these syndromes. Therefore, this case suggests that a new gene may be associated with congenital muscular dystrophy with α-dystroglycan glycosylation defects, cortical migration defects, and sparing of the cerebellum.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative motor neuron disorder. Mutations in Cu,Zn superoxide dismutase (SOD1) cause approximately 20% of familial ALS. One of the possible mechanisms whereby they induce disease is mitochondrial dysfunction in motor neurons. Here we describe a patient with ALS and muscle mitochondrial oxidative defect associated with a novel SOD1 mutation. Direct sequencing of SOD1 gene revealed a heterozygous mutation in codon 22 substituting a highly conserved amino acid, from glutamine to arginine (Q22R). Muscle biopsy showed a neurogenic pattern associated with cytochrome c oxidase (COX) deficiency in several muscle fibers. Western blot analysis demonstrated a reduction in SOD1 content in the cytoplasmic and mitochondrial fractions. These results suggest that a minute quantity of mutant SOD1 protein contributes to a mitochondrial toxicity also in muscle tissue.

Platelets can serve as general markers of mitochondrial (dys)function during several human diseases. Whether this holds true even during sepsis is unknown. Using spectrophotometry, we measured mitochondrial respiratory chain biochemistry in platelets and triceps brachii muscle of thirty patients with septic shock (within 24 hours from admission to Intensive Care) and ten surgical controls (during surgery). Results were expressed relative to citrate synthase (CS) activity, a marker of mitochondrial density. Patients with septic shock had lower nicotinamide adenine dinucleotide dehydrogenase (NADH)/CS (p = 0.015), complex I/CS (p = 0.018), complex I and III/CS (p

PURPOSE: The p53 gene plays a critical role in cellular response to DNA damage and has been implicated in the response to platinum compounds in ovarian carcinoma patients. Because taxanes could induce p53-independent apoptosis, we assessed the relevance of p53 gene status to response in ovarian carcinoma patients receiving paclitaxel and platinum-containing chemotherapy. PATIENTS AND METHODS: Forty-eight previously untreated patients with advanced disease received standard paclitaxel/platinum-based chemotherapy. In tumor specimens collected at the time of initial surgery, before therapy, p53 gene status and expression were examined by single-strand conformation polymorphism, sequence analysis, and immunohistochemical analysis. Microsatellite instability analysis was performed on available samples from 30 patients. RESULTS: Thirty-four (71%) of the 48 patients had a clinical response. Pathologic complete remission was documented in 13 (27%) of 48 patients. p53 mutations were detected in 29 (60%) of 48 tumors. Among the patients with mutant p53 tumors, 25 patients (86%) responded to chemotherapy. Only nine (47%) of 19 patients with wild-type p53 tumors responded to the same treatment. The overall response rate and the complete remission rate were significantly higher among patients with mutant p53 tumors than among patients with wild-type p53 tumors (P = .008). Most of the tested tumors not associated with complete remission (10 of 12 tumors) were also characterized by microsatellite instability. The complete remission rate was higher among patients with tumors without microsatellite instability (five of seven patients). CONCLUSION: In contrast to the limited efficacy of treatment with paclitaxel in combination with standard platinum doses against wild-type p53 ovarian tumors, patients with mutant p53 ovarian tumors were more responsive to paclitaxel-based chemotherapy. The pattern of response to chemotherapy containing paclitaxel is different from that associated with high-dose cisplatin therapy. Determining p53 mutational status can be useful in predicting therapeutic response to drugs effective in ovarian carcinoma.

Background Statin drugs (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) reduce the level of cholesterol by inhibiting the synthesis of mevalonate, an intermediary in the cholesterol biosynthetic pathway. Use of statin drugs has been associated with a variety of skeletal muscle–related complaints. Coenzyme Q10(CoQ10), a component of the mitochondrial respiratory chain, is also synthesized from mevalonate, and decreased muscle CoQ10concentration may have a role in the pathogenesis of statin drug–related myopathy. Objectives To measure the CoQ10concentration and respiratory chain enzyme activities in muscle biopsy specimens from 18 patients with statin drug–related myopathy and to look for evidence of apoptosis using the TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling) assay. Design An open-labeled study of CoQ10concentration in muscle from patients with increased serum creatine kinase concentrations while receiving standard statin drug therapy. Setting Neuromuscular centers at 2 academic tertiary care hospitals. Results Muscle structure was essentially normal in 14 patients and showed evidence of mitochondrial dysfunction and nonspecific myopathic changes in 2 patients each. Muscle CoQ10concentration was not statistically different between patients and control subjects, but it was more than 2 SDs below the normal mean in 3 patients and more than 1 SD below normal in 7 patients. There was no TUNEL positivity in any patients. Conclusion These data suggest that statin drug–related myopathy is associated with a mild decrease in muscle CoQ10concentration, which does not cause histochemical or biochemical evidence of mitochondrial myopathy or morphologic evidence of apoptosis in most patients.

Expression of HLA-DR antigens by purified preparations of human ovarian carcinoma cells freshly isolated from surgical specimens was examined in parallel with the capacity of tumor cells to elicit a blastogenic response from autologous lymphocytes in mixed lymphocyte-tumor culture (MLTC) assay. Of 21 tumor preparations, 11 (52%) reacted with monoclonal antibodies 279 and/or 949 specific for a monomorphic determinant of HLA-DR antigens, with heterogeneous positivity, ranging between 30% and 95%. In this series of patients positive MLTC occurred in 8/21 individual experiments. The HLA-DR expression was proportionally similar in tumors giving positive MLTC (4/8 = 50%) and negative MLTC (7/13 = 53%). The lack of correlation between DR expression on tumor cells and stimulatory activity in autologous MLTC and the fact that DR-negative tumors could induce lymphocyte stimulation, support the hypothesis that blastogenesis occurs upon recognition of tumor-associated antigens, different from DR molecules, possibly tumor-specific antigens.

Peripheral blood lymphocytes (PBL) from 43 patients with histologically confirmed ovarian carcinoma were stimulated in mixed lymphocyte-tumor culture (MLTC) with purified autologous tumor cells. Positive results, assessed as lymphocyte proliferation, were observed in 21 cases (48.8%). Lymphoid cells associated with ascitic fluid or infiltrating solid masses were in general less reactive than PBL as only 3/11 cases had positive MLTC. Tumor cells isolated from peritoneal effusions showed no significant difference in stimulatory potential as compared to the primary tumor. These results suggest that in an appreciable proportion of ovarian carcinoma patients (approximately 50%), lymphocytes have the potential to react to autologous tumor cells. Comprehension of the immunological mechanisms of antitumor resistance may have direct practical relevance for more effective treatment of neoplasms.

Background Duchenne and Becker Muscular dystrophies (DMD/BMD) are allelic disorders caused by mutations in the dystrophin gene, which encodes a sarcolemmal protein responsible for muscle integrity. Deletions and duplications account for approximately 75% of mutations in DMD and 85% in BMD. The implementation of techniques allowing complete gene sequencing has focused attention on small point mutations and other mechanisms underlying complex rearrangements. Methods We selected 47 patients (41 families; 35 DMD, 6 BMD) without deletions and duplications in DMD gene (excluded by multiplex ligation-dependent probe amplification and multiplex polymerase chain reaction analysis). This cohort was investigated by systematic direct sequence analysis to study sequence variation. We focused our attention on rare mutational events which were further studied through transcript analysis. Results We identified 40 different nucleotide alterations in DMD gene and their clinical correlates; altogether, 16 mutations were novel. DMD probands carried 9 microinsertions/microdeletions, 19 nonsense mutations, and 7 splice-site mutations. BMD patients carried 2 nonsense mutations, 2 splice-site mutations, 1 missense substitution, and 1 single base insertion. The most frequent stop codon was TGA (n = 10 patients), followed by TAG (n = 7) and TAA (n = 4). We also analyzed the molecular mechanisms of five rare mutational events. They are two frame-shifting mutations in the DMD gene 3'end in BMD and three novel splicing defects: IVS42: c.6118-3C>A, which causes a leaky splice-site; c.9560A>G, which determines a cryptic splice-site activation and c.9564-426 T>G, which creates pseudoexon retention within IVS65. Conclusion The analysis of our patients' sample, carrying point mutations or complex rearrangements in DMD gene, contributes to the knowledge on phenotypic correlations in dystrophinopatic patients and can provide a better understanding of pre-mRNA maturation defects and dystrophin functional domains. These data can have a prognostic relevance and can be useful in directing new therapeutic approaches, which rely on a precise definition of the genetic defects as well as their molecular consequences.

Human ovarian carcinomas (HOC) were established s.c. and i.p. in nude mice and the biological characteristics were investigated for 4 xenografts. HOC8 and HOC18, derived respectively from a primary tumor of the ovary and a pleural effusion (from 2 different patients) were established s.c. in nude mice. HOC10 and HOC22, derived from the ascites of 2 patients, were directly established as ascites after i.p. injection in nude mice. The s.c. and i.p. growth behavior of the 4 HOC lines was investigated. HOC18, HOC8 and HOC22 cells produced progressively growing tumor after s.c. injection but HOC10 ascites would not grow s.c. The cell suspension derived from HOC18 only produced carcinomatosis upon i.p. injection, while HOC8 cells produced both ascites and carcinomatosis. The 2 ascites HOC10 and HOC22 produced ascites in nude mice, but only HOC22 formed i.p. carcinomatosis. Histopathological characteristics of the patients' primary tumors persisted in nude mice, regardless of the site of tumor implantation. DNA histograms of the xenografts closely matched the patients' tumors and remained stable at different passages. Cisplatin, adriamycin and cyclophosphamide given i.v. were tested against HOC8 and HOC18 growing s.c. and HOC22 and HOC10 growing i.p. HOC8 showed a significant response to DDP and almost no sensitivity to ADR and CTX. HOC18 showed only moderate growth delay with all 3 drugs. Mice bearing HOC10 and HOC22 ascites had a prolonged survival time after DDP and ADR treatment.

Glycogen storage disease, glycogenosis type II (GSDII), or Pompe disease (OMIM 23230), is an autosomal recessive lysosomal storage disorder that results from a deficiency in the acid alpha glucosidase (GAA) enzyme. The disease is characterized by progressive accumulation of lysosomal glycogen in various tissues, primarily in cardiac and skeletal muscles. The histopathological hallmarks in the muscle are fiber vacuolization and autophagy. GSDII is clinically classified as a severe infantile, or an attenuated, later-onset form affecting children and adults. Recombinant human GAA (rhGAA) is the only approved enzyme replacement therapy (ERT) available for the treatment of Pompe disease, and it is effective in infantile patients, whereas in adults, improvements are more variable among different patients. Our project aims to assess the effects of ERT in 19 late-onset patients using both biochemical and morphological (histological, histochemical, and immunohistochemical) evaluations of skeletal muscle biopsies before and after 6 months to 1 year on ERT.