Large deletion mutation of SPAST in a multi-generation family from Sardinia.

BACKGROUND AND PURPOSE: The hereditary spastic paraplegias (HSP) are characterized by progressive spasticity of the lower limbs, mostly inherited as an autosomal dominant trait. Analyses of large HSP pedigrees could help to better characterize the phenotype due to a single causative mutation. Patients in a seven-generation kindred carrying a large deletion in SPAST/SPG4 are described. METHODS: Individuals originating from Sardinia were clinically and genetically studied. RESULTS: Sixty-seven subjects carried a heterozygous deletion encompassing exons 2-17 of SPAST. Fifty patients (53.2 ± 15.4 years) presented a pure form of spastic paraparesis characterized by mild impairment and slow progression. Most patients showed spasticity, increased tendon reflexes in the lower limbs and Babinski sign, whilst weakness was rarely detected and urinary disturbances occasionally reported. Amongst the 17 asymptomatic carriers of the mutation, minimal neurological signs were detected in 11 cases. CONCLUSIONS: A focus on spasticity, increased tendon reflexes and Babinski sign, more than on weakness, could help clinicians to promote early diagnosis in asymptomatic carriers of SPAST deletions.

RAPD discloses high molecular diversity of Mycobacterium tuberculosis from Michoacan, Mexico.

Random amplified polymorphism DNA (RAPD) is an easy, inexpensive technique for the characterization of pathogens in low-income countries. In this study we used RAPD to assess the genetic diversity of a small collection of isolates of mycobacteria from the Mexican state of Michoacan. In contrast with the low annual tuberculosis incidence in Michoacan relative to the national average, we found a high molecular diversity value suggesting high population diversity of M. tuberculosis in the studied region. Our findings justify further typing efforts with other molecular tools such as MIRU-VNTR and spoligotyping.

Lemur tyrosine kinase-2 signalling regulates kinesin-1 light chain-2 phosphorylation and binding of Smad2 cargo.

A recent genome-wide association study identified the gene encoding lemur tyrosine kinase-2 (LMTK2) as a susceptibility gene for prostate cancer. The identified genetic alteration is within intron 9, but the mechanisms by which LMTK2 may impact upon prostate cancer are not clear because the functions of LMTK2 are poorly understood. Here, we show that LMTK2 regulates a known pathway that controls phosphorylation of kinesin-1 light chain-2 (KLC2) by glycogen synthase kinase-3ß (GSK3ß). KLC2 phosphorylation by GSK3ß induces the release of cargo from KLC2. LMTK2 signals via protein phosphatase-1C (PP1C) to increase inhibitory phosphorylation of GSK3ß on serine-9 that reduces KLC2 phosphorylation and promotes binding of the known KLC2 cargo Smad2. Smad2 signals to the nucleus in response to transforming growth factor-ß (TGFß) receptor stimulation and transport of Smad2 by kinesin-1 is required for this signalling. We show that small interfering RNA loss of LMTK2 not only reduces binding of Smad2 to KLC2, but also inhibits TGFß-induced Smad2 signalling. Thus, LMTK2 may regulate the activity of kinesin-1 motor function and Smad2 signalling.

A 5-aza-2'-deoxycytidine/valproate combination induces cytotoxic T-cell response against mesothelioma.

Malignant pleural mesothelioma (MPM) is an aggressive tumour with a limited response to conventional therapy. The aim of this study was to evaluate the anticancer effect of a DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine (5-azaCdR), and two histone deacetylase inhibitors, valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA). Human mesothelioma cells were treated with each epigenetic drug, either alone or in combinations. The cytotoxic effects on treated cells and the expression of specific tumour antigens were evaluated. The recognition of treated cells by a specific CD8+ T-cell clone was also measured. Additionally, the effect of combined treatments was tested in a murine model of mesothelioma. We showed that VPA and SAHA synergised with 5-azaCdR to kill MPM cells and induce tumour antigen expression in the remaining living tumour cells. As a consequence, tumour cells expressing these antigens were recognised and lysed by specific CD8+ cytotoxic T-cells. In vivo, treatment with 5-azaCdR/VPA inhibited tumour growth, and promoted lymphocyte infiltration and an immune response against tumour cells. Appropriate epigenetic drug combinations, in addition to inducing mesothelioma cell death, also affect the immunogenic status of these cells. This property could be exploited in clinical investigations to develop MPM treatments combining chemotherapeutic and immunotherapeutic approaches.

[Streptococcus pneumoniae serotypes involved in children with pleural empyemas in France]. / Distribution des sérotypes de pneumocoques responsables des pleuropneumopathies de l'enfant en France.

UNLABELLED: It has been suggested that the incidence of childhood empyema have increased during the last years in France without clear explanation. Streptococcus pneumoniae is responsible for the vast majority of bacteriological documented cases. Potential prevention of pleural empyemas by the heptavalent pneumococcal conjugate vaccine is dependent on adequation between specific pneumococcal serogroups present in vaccine and those responsible for empyemas. MATERIAL AND METHODS: We retrospectively collected cases of pleural empyemas registered at the National Reference Center for pneumococci (December 2002 to February 2004). Thirty children, aged 4.1+/-3.3 (SD) years, were included. RESULTS: Ten specific serogroups were identified: 1, 3, 5, 6B, 7F, 9V, 14, 18C, 19A, and 23F. Serogroups 19A and 1 were the 2 dominant serogroups and represented 50% of cases. All children infected with serotype 19A were younger than 5 years, whereas serotype 1 was identified in 80% of empyemas in children older than 5 years. Among the 30 patients enrolled, 20 (69%) were infected with serotypes not included in the conjugate vaccine. CONCLUSION: These results thus limit the potential impact of the heptavalent pneumococcal conjugate vaccine on the frequency of pleural empyemas in children.

Patent foramen ovale after lobectomy. A contraindication to completion pneumonectomy?

Patency of the foramen ovale (FO) is a very rare complication after lobectomy. Completion pneumonectomy after FO reopening has never been described before. In the reported case, a patent FO was diagnosed in a 52-year old man 9 months after a left upper sleeve lobectomy for a squamous cell carcinoma pT2N1. At the same time, bronchoscopy showed an endobronchial recurrence in the left main bronchus. The foramen was closed percutaneously by a 35 mm Amplatzer prosthesis. Dyspnea improved dramatically in the following 4 weeks and no more oxygen therapy was required. After complete restaging, a completion pneumonectomy was performed without any postoperative complication. This case report suggests that pneumonectomy in such delicate patients is feasible.

[Organization of patient management in level II centers in the Paris area: a prospective survey]. / Organisation de la prise en charge dans les centres de niveau II en région parisienne: enquête prospective.

Perinatal asphyxia is a common emergency for both obstetricians and pediatricians. A prospective study was conducted in 14 maternity hospitals (type II centres) in the Paris suburbs in order to assess pediatric activity and neonatal morbidity associated with supposed perinatal asphyxia in term newborns. Pediatricians were called in at birth very frequently: 1/20 deliveries. Intubation and/or resuscitation procedures were needed in 20% of cases and 20% of infants were referred to a neonatal unit for birth asphyxia or associated pathology. Moderate encephalopathy was observed in 1.5% of all term newborns who needed medical intervention for supposed birth asphyxia.

[Production of autologous keratinocytes for therapeutic purposes within a pharmaceutical company]. / Production de kératinocytes autologues à buts thérapeutiques au sein d'un établissement pharmaceutique.

Because biotechnologies are growing and are becoming key players in the pharmaceutical industry scene, Genévrier Laboratories inaugurated in January 1998, a new department especially designed for the production of cultured cells as therapeutic agents. Meeting clinician therapeutic needs by providing autologous keratinocytes and chondrocytes in the near future, represents the primary aim of the Biotechnology department. Concrete cell-based products are already being used for the treatment of burns and cutaneous chronic wounds such as the EPIBASE graft, which corresponds to an epidermis sheet composed of cultured autologous keratinocytes.

Site-directed mutagenesis of ATP binding residues of biotin carboxylase. Insight into the mechanism of catalysis.

Acetyl-CoA carboxylase catalyzes the first committed step in fatty acid synthesis in all plants, animals, and bacteria. The Escherichia coli form is a multimeric protein complex consisting of three distinct and separate components: biotin carboxylase, carboxyltransferase, and the biotin carboxyl carrier protein. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the carboxylate source and has a distinct architecture that is characteristic of the ATP-grasp superfamily of enzymes. Included in this superfamily are d-Ala d-Ala ligase, glutathione synthetase, carbamyl phosphate synthetase, N(5)-carboxyaminoimidazole ribonucleotide synthetase, and glycinamide ribonucleotide transformylase, all of which have known three-dimensional structures and contain a number of highly conserved residues between them. Four of these residues of biotin carboxylase, Lys-116, Lys-159, His-209, and Glu-276, were selected for site-directed mutagenesis studies based on their structural homology with conserved residues of other ATP-grasp enzymes. These mutants were subjected to kinetic analysis to characterize their roles in substrate binding and catalysis. In all four mutants, the K(m) value for ATP was significantly increased, implicating these residues in the binding of ATP. This result is consistent with the crystal structures of several other ATP-grasp enzymes, which have shown specific interactions between the corresponding homologous residues and cocrystallized ADP or nucleotide analogs. In addition, the maximal velocity of the reaction was significantly reduced (between 30- and 260-fold) in the 4 mutants relative to wild type. The data suggest that the mutations have misaligned the reactants for optimal catalysis.

Video-mediastinoscopy in management of patients with lung cancer: a preliminary study.

Cervical mediastinoscopy is widely employed for biopsy of mediastinal lymph nodes and staging of lung cancer. The application of video-assisted technology to mediastinoscopy in a series of patients with lung cancer has not been reported. Preliminary experience with the use of video-mediastinoscopy in diagnosis and staging of lung cancer is presented. Fifteen patients with lung cancer were studied. Results of previous computed tomography scans had shown the presence of enlarged mediastinal lymph nodes in the retrovascular plane in all of the cases. Video-mediastinoscopy was performed under general anesthesia using a specifically designed rigid scope connected to a mono-charged-coupled device video camera (model INH 002756; Karl Storz-Endoskope, Tuttlingen, Germany). Neither fatalities nor major complications related to the procedure were observed. In all cases, video-mediastinoscopy proved useful for diagnosis or staging of lung cancer, therefore contributing to clinical decision making. The optimal visualization of mediastinal structure and the possibility for the surgeon to operate with both hands are appreciable characteristics of this technique.

Lemierre's syndrome with bilateral empyema thoracis.

A 31-year-old patient is described with thrombophlebitis of the right jugular vein, and anerobic septicemia (Lemierre's syndrome). Multiple pulmonary abscesses and bilateral fibrinopurulent empyema were also present. Treatment included intravenous antibiotics, heparin, and video-assisted thoracoscopic debridement of pleural cavities. A favorable outcome was observed.

Endothelin-1 in children with chronic renal failure.

Endothelin-1 (ET-1) was measured after extraction from plasma of normal adults (5.9 +/- 1.9 pg/ml, n = 22), normal children (7.1 +/- 1.86 pg/ml, n = 29), non-haemodialysed children with chronic renal failure (CRF) (11.1 +/- 1.8 pg/ml), n = 10), renal graft recipients (9.5 +/- 3.4 pg/ml, n = 37), haemodialysed children 24 h after a haemodialysis session (20.02 +/- 10.9 pg/ml, n = 26) and haemodialysed children before and after a haemodialysis session (15.31 +/- 10.6 and 13.8 +/- 8.5 respectively, n = 14). A sensitive and specific radioimmunoassay was used. ET-1 was significantly higher in non-haemodialysed CRF children and in renal graft recipients than in normal children (P < 0.001 and P < 0.01, respectively) and significantly higher in haemodialysed children when compared with normal children, non-haemodialysed CRF children and renal graft recipients (P < 0.001). ET-1 concentrations were similar in normal children and normal adults. ET-1 was inversely correlated with glomerular filtration rate in non-haemodialysed CRF children (r = -0.39, P < 0.01) and positively correlated with extracellular volume in haemodialysed children (r = 0.435, P < 0.03). After haemodialysis, ET-1 increased in 6 and decreased in 8 of the 14 children studied before and after a haemodialysis session.

Lack of mother-to-infant transmission of hepatitis C virus in human immunodeficiency virus-seronegative women: a prospective study with hepatitis C virus RNA testing.

The published risk of mother-to-infant transmission of hepatitis C virus varies according to the population studied and the tests used. In a prospective study we used the polymerase chain reaction to assess the risk of vertical transmission of hepatitis C virus in an unselected population of women uninfected by human immunodeficiency virus. Hepatitis C virus antibodies were sought with a second-generation enzyme-linked immunosorbent assay in 2,367 consecutive pregnant women. Forty-one were positive, and 17 consented to serological follow-up of their offspring (n = 18). A second-generation recombinant immunoblot assay, ALT determination and hepatitis C virus RNA testing were performed on maternal sera obtained during pregnancy and sera from the offspring at birth and thereafter. Five older brothers or sisters were also tested. Hepatitis C virus RNA sequences in serum were amplified with a modified nested polymerase chain reaction procedure with primers from the highly conserved 5' noncoding region of the hepatitis C virus genome. All the neonates were positive for hepatitis C virus antibodies, with enzyme-linked immunosorbent assay titers and recombinant immunoblot assay patterns similar to those of their mothers. After birth hepatitis C virus antibodies gradually disappeared within 6 mo. Hepatitis C virus RNA was consistently negative in the 18 children from birth to 24 mo (range = 3 to 24 mo) and in the 5 older children, regardless of the hepatitis C virus polymerase chain reaction status of the mothers (8 of whom were positive).(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of fluvastatin and lovastatin blood-brain barrier transfer using in vitro and in vivo methods.

We measured the permeability of fluvastatin, lovastatin, and its pharmacologically active hydroxy acid (HA) across the blood-brain barrier (BBB) with three different techniques. The brain uptake index (BUI) technique yielded values for fluvastatin and HA below the limit of detection of this method; in contrast, brain extraction of lovastatin was high: 57 and 7% with addition of buffer and human plasma, respectively. Brain perfusion studies in rats indicated that when fluvastatin and HA were exposed for a longer time to the BBB, small but measurable brain uptake occurred, with permeability coefficients of 2.5 x 10(-4) and 1.4 x 10(-4) cm/min, for fluvastatin and HA, respectively, whereas the permeability coefficient of lovastatin was 2.3 x 10(-2) cm/min. An in vitro BBB model consisting of a primary culture of confluent monolayers of bovine brain microvessel endothelial cells yielded very similar values: 7.2 x 10(-4) cm/min for fluvastatin and 1.3 x 10(-3) cm/min for HA and a permeability coefficient of 1.1 x 10(-2) cm/min for lovastatin. The results of the different methods show that lovastatin crosses the BBB to a much greater extent than does fluvastatin or HA. Although lovastatin undergoes hydrolytic conversion to HA, it subsists in blood for sufficient time to cross the BBB. Therefore, lovastatin and fluvastatin are expected to be different in their central side effect profile, as was observed in patients treated for hypercholesterolemia. This finding supports the hypothesis that sleep disturbances due to some cholesterol biosynthesis inhibitors might be correlated with their abilities to cross the BBB.

The use of viral culture and p24 antigen testing to diagnose human immunodeficiency virus infection in neonates. The HIV Infection in Newborns French Collaborative Study Group.

BACKGROUND: Early diagnosis of human immunodeficiency virus (HIV) infection in infants born to infected mothers is important for the infants' medical care, but the presence of maternal antibodies makes serologic tests uninformative. METHODS: In a cohort study of 181 infants born to HIV-infected mothers, we assessed the diagnostic value of HIV viral culture and testing for the presence of p24 antigen. The infants were tested at birth, again during the first 3 months, then followed and tested at the age of at least 18 months. RESULTS: Of the 181 infants, 3 died of HIV infection and 37 were seropositive after the age of 18 months. Viral cultures at birth were positive in 19 of the 40 infected infants and in none of the uninfected infants, yielding a sensitivity of 48 percent (95 percent confidence interval, 32 to 63 percent) and a specificity of 100 percent (95 percent confidence interval, 97 to 100 percent). By the age of three months, 30 of the 40 infants (75 percent) had positive cultures; again, there were no false positive results among the infants who were tested a second time, of the 141 who remained uninfected. The sensitivity of testing for p24 antigen at birth was only 18 percent, with a specificity of 100 percent. The presence of p24 antigen at birth was associated with the development of early and severe HIV-related disease (P less than 0.04). CONCLUSIONS: Viral culture at birth can correctly identify about half of newborns with HIV infection. The fact that this usually sensitive technique fails to identify about half the ultimately infected neonates suggests that vertical transmission of HIV may occur late in pregnancy or during delivery.

Moxidectin: systemic activity against common cattle grubs (Hypoderma lineatum) (Diptera: Oestridae) and trichostrongyle nematodes in cattle.

Moxidectin, a systemic insecticide, was evaluated for its efficacy against the migrating first instars of the common cattle grub, Hypoderma lineatum, and against nematode egg production in beef cattle. It was observed that all three levels (0.1, 0.2 and 0.4 mg moxidectin kg-1) were 100% effective against cattle grubs when administered as a s.c. injection. The same levels of treatment were very effective (90-100%) in reducing trichostrongyle nematode egg production. However, there was a slight indication that at least one species, Cooperia oncophora, was not completely eliminated, as it was observed that small numbers of eggs began to appear after 2 weeks post-treatment when there had been no opportunity for reinfection.

Effects of selected vasoactive substances on adenylate cyclase activity in brain, isolated brain microvessels, and primary cultures of brain microvessel endothelial cells.

The specific activity of adenylate cyclase was assayed in homogenates of gray matter, freshly isolated and primary cultured microvessel endothelial cells from bovine cerebral cortex. Specific activities for the tissues were 14.6 +/- 2.1, 15.6 +/- 2.7, and 8.4 +/- 1.5 pmol cAMP/mg protein/min +/- SD for gray matter, cultured microvessels, and freshly isolated microvessels, respectively. Adenylate cyclase associated with gray matter and cultured microvessels was sensitive to histamine and selected catecholamines. Perhaps due to metabolic deficiencies, adenylate cyclase of freshly isolated microvessels exhibited little or no response to either the catecholamines or histamine. Angiotensin II stimulated adenylate cyclase of both freshly isolated and cultured microvessels but had no effect on gray matter. Bradykinin did not stimulate cAMP generation in any of the tissues. Overall results support the role of cAMP in regulating brain microvessel functions and suggest that primary cultures of brain microvessels may be useful in examining cAMP-mediated biochemical pathways at the blood-brain barrier.