Inborn oxidative phosphorylation defect as risk factor for propofol infusion syndrome.

Propofol is an anesthetic agent widely used for induction and maintenance of anesthesia, and sedation in children. Although generally considered as reliable and safe, administration of propofol can occasionally induce a potentially fatal complication known as propofol infusion syndrome (PRIS). Mitochondrial dysfunction has been implicated in the pathogenesis of PRIS. We report on an adult patient with Leber hereditary optic neuropathy (LHON) who developed PRIS. He was a carrier of the m.3460G>A mutation, one of the major three pathogenic point mutations associated with LHON. The propositus was blind and underwent propofol sedation after severe head injury. Five days after start of propofol infusion, the patient died. The activity of complex I of the oxidative phosphorylation (OXPHOS) system was severely deficient in skeletal muscle. Our observation indicates that fulminate PRIS can occur in an adult patient with an inborn OXPHOS defect and corroborates the hypothesis that PRIS is caused by inhibition of the OXPHOS system.

Progressive myoclonic epilepsy as an adult-onset manifestation of Leigh syndrome due to m.14487T>C.

BACKGROUND: m.14487T>C, a missense mutation (p.M63V) affecting the ND6 subunit of complex I of the mitochondrial respiratory chain, has been reported in isolated childhood cases with Leigh syndrome (LS) and progressive dystonia. Adult-onset phenotypes have not been reported. OBJECTIVES: To determine the clinical-neurological spectrum and associated mutation loads in an extended m.14487T>C family. METHODS: A genotype-phenotype correlation study of a Belgian five-generation family with 12 affected family members segregating m.14487T>C was carried out. Clinical and mutation load data were available for nine family members. Biochemical analysis of the respiratory chain was performed in three muscle biopsies. RESULTS: Heteroplasmic m.14487T>C levels (36-52% in leucocytes, 97-99% in muscle) were found in patients with progressive myoclonic epilepsy (PME) and dystonia or progressive hypokinetic-rigid syndrome. Patients with infantile LS were homoplasmic (99-100% in leucocytes, 100% in muscle). We found lower mutation loads (between 8 and 35% in blood) in adult patients with clinical features including migraine with aura, Leber hereditary optic neuropathy, sensorineural hearing loss and diabetes mellitus type 2. Despite homoplasmic mutation loads, complex I catalytic activity was only moderately decreased in muscle tissue. INTERPRETATION: m.14487T>C resulted in a broad spectrum of phenotypes in our family. Depending on the mutation load, it caused severe encephalopathies ranging from infantile LS to adult-onset PME with dystonia. This is the first report of PME as an important neurological manifestation of an isolated mitochondrial complex I defect.

Dermatomyositis and polymyositis: new treatment targets on the horizon.

Polymyositis (PM) and dermatomyositis (DM) are rare idiopathic inflammatory myopathies (IIM) with a presumed autoimmune pathogenesis. Typical features are subacute onset, proximal, symmetric muscle weakness, elevated serum creatine kinase, and mononuclear cell infiltrates in the muscle biopsy. Strong support for an autoimmune pathogenesis comes from histopathological findings in biopsies of affected muscles. Furthermore, the association with autoantibodies supports the notion that immune-mediated inflammation is involved. PM and DM may occur in isolation or in connection with a connective tissue disease or cancer. The current treatment for IIM consists of first-line high-dose steroids and various conventional second-line treatments. Improvements in treatment for IIM are hampered by difficulties in the design of trials and the low incidence and prevalence of the disease. Cytokines and chemokines are factors involved in the inflammatory process in IIM, and are candidates for future therapeutic targets. Preliminary data with anti-tumour necrosis factor therapy are not very promising, but results of blockers of the lymphotoxin signalling pathway are to be awaited. Anti-B cell therapy may be a valuable therapeutic option for treatment of refractory IIM. The effects of anti-interferon-alpha in IIM are to be awaited, as are results of other anti-cytokine therapies and anti-chemokine therapy. Outcome measures to be used in clinical trials in II M include at present the core sets of outcome proposed by the International Myositis Assessment Clinical Study Group (IMACS).

Immunohistochemical analysis of the oxidative phosphorylation complexes in skeletal muscle from patients with mitochondrial DNA encoded tRNA gene defects.

BACKGROUND: Mitochondrial diseases display a heterogeneous spectrum of clinical phenotypes and therefore the identification of the underlying gene defect is often a difficult task. AIMS: To develop an immunohistochemical approach to stain skeletal muscle for the five multi-protein complexes that organise the oxidative phosphorylation (OXPHOS) in order to improve the diagnostic workup of mitochondrial defects. METHODS: OXPHOS complexes were visualised in skeletal muscle tissue using antibodies directed against different subunits. The staining patterns of patients with heteroplasmic defects in mtDNA tRNA genes were compared with those of normal and disease controls. RESULTS: Normal skeletal muscle displayed a checkerboard staining pattern for complexes I to V due to the higher mitochondrial content of slow muscle fibres versus fast fibres. In patients with tRNA defects, a much more heterogeneous staining pattern was observed for complex I (all six patients) and complex IV (4 of 6 patients): a mosaic staining pattern in which individual fibres displayed staining intensities that ranged from strong to negative. Ragged red fibres (RRFs) in patients with MERRF (myoclonic epilepsy and ragged red fibres) were all complex I and IV negative, while in patient with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) the majority of RRFs were complex I negative and complex IV positive. CONCLUSION: Immunohistochemical detection of OXPHOS complexes could represent a valuable additional diagnostic tool for the evaluation of mitochondrial cytopathy. The technique helps to detect heteroplasmic mtDNA defects. Staining for complex I in particular was able to identify two tRNA patients that stayed undetected with routine histochemical evaluation.

Properties of the ventricular adrenergic signal transduction system during ontogeny of spontaneous hypertension in rats.

The purpose of this study was to characterize adrenergic receptors and associated G proteins in ventricles of spontaneously hypertensive rats (SHRs) at different stages of development. The beta- and alpha1-adrenoceptor densities and subtype distribution, and beta-adrenoceptor-G protein coupling were studied by radioligand binding, and levels of G(Salpha), G(ialpha), and G(q/11alpha) protein species were determined by Western blotting in SHRs and Wistar-Kyoto (WKY) control rats aged 3.5 weeks, 3 months, and 8 months. In 3.5-week-old SHRs, both the beta-adrenoceptor density and the percentage of agonist high-affinity binding sites were higher than in age-matched WKY rats. The beta1/beta2-subtype distribution, the alpha1-adrenoceptor density, and the alpha1B/alpha1A-subtype distribution were similar in rats of both strains at all ages. Although essentially no differences in G(salpha) levels between SHRs and WKY rats were detected, higher G(ialpha) and lower Gq/1alpha concentrations were found in 3.5-week-old SHRs. In 3-month-old SHRs, increased levels of Gq/11alpha proteins were observed. In 8-month-old SHRs, none of the parameters was different from those of controls. We conclude that the differences in properties of the adrenergic signal transduction system between SHRs and WKY rats are exclusively observable before and at the onset of the overt hypertension. Moreover, the hypertensive genotype apparently affects G proteins more readily than adrenoceptors.

MHC class II molecules are required for initiation of positive selection but not during terminal differentiation of human CD4 single positive thymocytes.

Positive selection of T cell precursors is an MHC dependent, multistep process by which functionally mature CD4+8- helper and CD4-8+ cytotoxic single positive (SP) T cells are generated from immature CD4+8+ double positive (DP) thymocytes. We investigated the requirement for TCR/MHC class II interactions during different stages of positive selection of human CD4 SP thymocytes. We show that sorted CD69- CD4+8+ DP preselection thymocytes cultured in fetal thymus lobes of normal mice were subject to positive selection and differentiated to CD3(high) CD69+, mature CD8 SP, and CD4 SP cells. When cultured in thymus lobes from MHC class II-deficient mice, these precursors failed to develop into mature CD4 SP T cells, indicating that in the hybrid cultures, murine MHC class II molecules are required for the development of mature human CD4 SP T cells. We have previously identified CD4 SP intermediate thymocytes that have received at least some of the signals involved in positive selection, since these cells are CD69+, CD3/TCR(high), and CD8beta- but that are still phenotypically and functionally immature. Here we demonstrate that in contrast to preselection thymocytes, these CD4 SP intermediate thymocytes can give rise to phenotypically mature and functionally CD4 SP progeny both in normal and in MHC class II-deficient thymus lobes. These results suggest that TCR/MHC interactions are required for the initial stages of positive selection, but are not essential during terminal differentiation to functionally mature CD4 SP T cells.

Growth stimulatory angiotensin II type-1 receptor is upregulated in breast hyperplasia and in situ carcinoma but not in invasive carcinoma.

Two different receptors which bind angiotensin II specifically have been identified in humans and were designated angiotensin II type-1 receptor (AT1) and angiotensin II type-2 receptor (AT2). They only have 34% sequence homology and act through different signalling pathways. AT1 stimulation has been implicated in hypertrophy and hyperplasia in various tissues. In order to study the involvement of AT1 in tissues from controls (n=10) and patients with hyperplasia (n=33), ductal carcinoma in situ (DCIS) (n=23) and invasive carcinoma of the breast (n=25), we tested biopsies and breast-derived cell lines using immunocytochemistry, in situ hybridisation and cell proliferation techniques. The results show specific overexpression of AT1 receptor on the cytoplasmic membrane of cells of hyperplastic lesions with and without atypia and on DCIS of the breast. Evidence for growth stimulation is provided by in vitro experiments showing growth induction by angiotensin II of T47D cells which express the AT1 but not the AT2 receptor. The expression of AT1 on the cell membrane disappears in invasive breast cancer cells suggesting a regulatory pathway which is no longer needed in invasive carcinoma. The specific AT1 expression upregulation might well be an important step in the pathogenesis of hyperplasia of the breast, which is regarded as a precursor lesion for breast cancer.

Distribution of type-1 and type-2 angiotensin receptors in the normal human lung and in lungs from patients with chronic obstructive pulmonary disease.

This study was designed to examine the cellular distribution of the angiotensin II type-1 (AT1) and type-2 (AT2) receptors in the normal human and pathological human lung. Riboprobes were prepared against specific portions of each receptor DNA and labelled with FITC for detection using an anti-FITC antibody in combination with the alkaline phosphatase-anti-alkaline phosphatase technique and new Fuchsin. These were used to detect the presence of receptor mRNA in the lung. Specific antibodies were used to detect receptor protein in cells by immunocytochemistry. Image analysis was used in order to semi-quantify receptor density. AT1 receptor mRNA and protein were localised on vascular smooth muscle cells, macrophages and in the stroma underlying the airways epithelium probably relating to underlying fibroblasts. The AT1 receptor protein was not expressed in the epithelium although there was a low level of mRNA. In contrast, AT2 receptor RNA and protein was observed in the epithelium, with strong staining on the bronchial epithelial cell brush border and also on many of the underlying mucous glands. The AT2 receptor was also present on some endothelial cells. These findings were supported by the presence of mRNA in each case. In patients with chronic obstructive pulmonary disease, there was a five- to sixfold increase in the ratio of AT1 to AT2 receptors in the regions of marked fibrosis surrounding the bronchioles. This correlated well with the reduced lung function as expressed by the forced expiratory volume.