Anticardiolipin in porphyromonas gingivalis antisera causes fetal loss in mice.

ß2-glycoprotein I (ß2GPI)-dependent anticardiolipin autoantibodies (aCl) are associated with thrombosis and fetal loss. Some microbial pathogens can induce pathogenic antibodies cross-reactive with ß2GPI. Sera from a significant percentage of periodontitis patients contain aCl, and some periodontal pathogens contain antigens with peptide sequences having homology to ß2GPI. We hypothesized that antibodies raised against P. gingivalis (aPg) contain pathogenic aCl that induce fetal resorption. We immunized mice with ß2GPI, P. gingivalis W83, or an arg-gingipain-defective mutant of P. gingivalis (HF18). IgG fractions of aPg were immunoabsorbed to remove aCl-like antibodies (abs-aPg). IgG fractions were administered intravenously into tail veins of mated BALB/c females at day 0 of pregnancy. At day 15, the proportions of fetal resorptions were evaluated. The prevalence of fetal loss was significantly greater in the aPg group than in the control IgG group (21.2% vs. 5.3%, p = .001), and greater in the aPg group than in the abs-aPg group (21.2% vs. 12%, p < .05). There were no fetal resorptions observed in the aPgHF18 group (p = .0005 compared with aPg, p = .17 compared with control). aPg antibody contains activity consistent with pathogenic aCl, and the antigen inducing the antibodies that cause increased fetal loss may be on the arg-gingipain protease of P. gingivalis.

Coenzyme Q10 and vitamin E deficiency in Friedreich's ataxia: predictor of efficacy of vitamin E and coenzyme Q10 therapy.

BACKGROUND AND PURPOSE: A pilot study of high dose coenzyme Q(10) (CoQ(10))/vitamin E therapy in Friedreich's ataxia (FRDA) patients resulted in significant clinical improvements in most patients. This study investigated the potential for this treatment to modify clinical progression in FRDA in a randomized double blind trial. METHODS: Fifty FRDA patients were randomly divided into high or low dose CoQ(10)/ vitamin E groups. The change in International Co-operative Ataxia Ratings Scale (ICARS) was assessed over 2 years as the primary end-point. A post hoc analysis was made using cross-sectional data. RESULTS: At baseline serum CoQ(10) and vitamin E levels were significantly decreased in the FRDA patients (P < 0.001). During the trial CoQ(10) and vitamin E levels significantly increased in both groups (P < 0.01). The primary and secondary end-points were not significantly different between the therapy groups. When compared to cross-sectional data 49% of all patients demonstrated improved ICARS scores. This responder group had significantly lower baseline serum CoQ(10) levels. CONCLUSIONS: A high proportion of FRDA patients have a decreased serum CoQ(10) level which was the best predictor of a positive clinical response to CoQ(10)/vitamin E therapy. Low and high dose CoQ(10)/vitamin E therapies were equally effective in improving ICARS scores.

Role of oxidative damage in Friedreich's ataxia.

Plasma malondialdehyde (MDA) levels were raised in Friedreich's ataxia (FRDA) patients. These levels correlated with increasing age and disease duration, suggesting lipid peroxidation increased with disease progression. Using fibroblasts from FRDA patients we observed that GSH levels and aconitase activities were normal, suggesting their antioxidant status was unchanged. When exposed to various agents to increase free radical generation we observed that intracellular superoxide generation induced by paraquat caused enhanced oxidative damage. This correlated with the size of the GAA1 expansion, suggesting decreased frataxin levels may render the cells more vulnerable to mild oxidative stress. More severe oxidative stress induced by hydrogen peroxide caused increased cell death in FRDA fibroblasts but was not significantly different from control cells. We propose that abnormal respiratory chain function and iron accumulation may lead to a progressive increase in oxidative damage, but increased sensitivity to free radicals may not require detectable respiratory chain dysfunction.

Mitochondrial dysfunction in Friedreich's ataxia: from pathogenesis to treatment perspectives.

Friedreich's ataxia (FRDA), the most common inherited ataxia, is an autosomal recessive degenerative disorder caused by a GAA triplet expansion or point mutations in the FRDA gene on chromosome 9q13. The FRDA gene product, frataxin, is a widely expressed mitochondrial protein, which is severely reduced in FRDA patients. The demonstration that deficit of frataxin in FRDA is associated with mitochondrial iron accumulation, increased sensitivity to oxidative stress, deficit of respiratory chain complex activities and in vivo impairment of cardiac and skeletal muscle tissue energy metabolism, has established FRDA as a "new" nuclear encoded mitochondrial disease. Pilot studies have shown the potential effect of antioxidant therapy based on idebenone or coenzyme Q10 plus Vitamin E administration in this condition and provide a strong rationale for designing larger randomized clinical trials.

Rescue of the Friedreich's ataxia knockout mouse by human YAC transgenesis.

We have generated and characterised transgenic mice that contain the entire Friedreich's ataxia gene (FRDA) within a human YAC clone of 370 kb. In an effort to overcome the embryonic lethality of homozygous Frda knockout mice and to study the behaviour of human frataxin in a mouse cellular environment, we bred the FRDA YAC transgene onto the null mouse background. Phenotypically normal offspring that express only YAC-derived human frataxin were identified. The human frataxin was expressed in the appropriate tissues at levels comparable to the endogenous mouse frataxin, and it was correctly processed and localised to mitochondria. Biochemical analysis of heart tissue demonstrated preservation of mitochondrial respiratory chain function, together with some increase in citrate synthase and aconitase activities. Thus, we have demonstrated that human frataxin can effectively substitute for endogenous murine frataxin in the null mutant. Our studies are of immediate consequence for the generation of Friedreich's ataxia transgenic mouse models, and further contribute to the accumulating knowledge of human-mouse functional gene replacement systems.

Cardiac energetics are abnormal in Friedreich ataxia patients in the absence of cardiac dysfunction and hypertrophy: an in vivo 31P magnetic resonance spectroscopy study.

OBJECTIVE: Friedreich ataxia (FRDA), the commonest form of inherited ataxia, is often associated with cardiac hypertrophy and cardiac dysfunction is the most frequent cause of death. In 97%, FRDA is caused by a homoplasmic GAA triplet expansion in the FRDA gene on chromosome 9q13 that results in deficiency of frataxin, a mitochondrial protein of unknown function. There is evidence that frataxin deficiency leads to a severe defect of mitochondrial respiration associated with abnormal mitochondrial iron accumulation. To determine whether bioenergetics deficit underlies the cardiac involvement in Friedreich ataxia (FRDA) we measured cardiac phosphocreatine to ATP ratio non-invasively in FRDA patients. METHODS AND RESULTS: Eighteen FRDA patients and 18 sex- and age-matched controls were studied using phosphorus MR spectroscopy and echocardiography. Left ventricular hypertrophy was present in eight FRDA patients while fractional shortening was normal in all. Cardiac PCr/ATP in FRDA patients as a group was reduced to 60% of the normal mean (P<0.0001). In the sub-group of patients with no cardiac hypertrophy PCr/ATP was also significantly reduced (P<0.0001). CONCLUSION: Cardiac bioenergetics, measured in vivo, is abnormal in FRDA patients in the absence of any discernible deterioration in cardiac contractile performance. The altered bioenergetics found in FRDA patients without left ventricle hypertrophy implies that cardiac metabolic dysfunction in FRDA precedes hypertrophy and is likely to play a role in its development.

Reliability and validity of parental measurements of infant size.

This study evaluates the reliability and validity of parental measurements of infant size, using illustrated instructions and simple measuring tools. Following pilot tests, final methods were evaluated on a sample of 28 parents (26 mothers and 2 fathers) of infants from 1 to 6 weeks of age. Parents independently measured twice the infant's head circumference (HC), mid-upper arm circumference (MUAC), abdominal circumference (AC), and recumbent length (RL). Infants were also measured twice by a trained observer. Mean parental measures were correlated (intraclass R) with the observer criterion measures at the levels of 0.81 for RL, 0.70 for AC, 0.80 for MUAC, and 0.94 for HC. Relative differences in the means for measurements obtained by parents and the trained observer were small, ranging from 0.3% for HC and RL, to 3.8% for AC. Intraclass correlations of reliability between the two parental measurements ranged from R = 0.84 for MUAC to 0.96 for RL. Given the reliability and validity of the results, the methods tested yield measurements that are suitable for ranking individuals and for use in group-level analyses, and at least in the case of head circumference, for individual-level analyses.

Antioxidant treatment improves in vivo cardiac and skeletal muscle bioenergetics in patients with Friedreich's ataxia.

Friedreich's ataxia (FA) is the most common form of autosomal recessive spinocerebellar ataxia and is often associated with a cardiomyopathy. The disease is caused by an expanded intronic GAA repeat, which results in deficiency of a mitochondrial protein called frataxin. In the yeast YFH1 knockout model of the disease there is evidence that frataxin deficiency leads to a severe defect of mitochondrial respiration, intramitochondrial iron accumulation, and associated production of oxygen free radicals. Recently, the analysis of FA cardiac and skeletal muscle samples and in vivo phosphorus magnetic resonance spectroscopy (31P-MRS) has confirmed the deficits of respiratory chain complexes in these tissues. The role of oxidative stress in FA is further supported by the accumulation of iron and decreased aconitase activities in cardiac muscle. We used 31P-MRS to evaluate the effect of 6 months of antioxidant treatment (Coenzyme Q10 400 mg/day, vitamin E 2,100 IU/day) on cardiac and calf muscle energy metabolism in 10 FA patients. After only 3 months of treatment, the cardiac phosphocreatine to ATP ratio showed a mean relative increase to 178% (p = 0.03) and the maximum rate of skeletal muscle mitochondrial ATP production increased to 139% (p = 0.01) of their respective baseline values in the FA patients. These improvements, greater in prehypertrophic hearts and in the muscle of patients with longer GAA repeats, were sustained after 6 months of therapy. The neurological and echocardiographic evaluations did not show any consistent benefits of the therapy after 6 months. This study demonstrates partial reversal of a surrogate biochemical marker in FA with antioxidant therapy and supports the evaluation of such therapy as a disease-modifying strategy in this neurodegenerative disorder.

The extracellular matrix of peripheral nerve in diabetic polyneuropathy.

The pattern of collagenisation in peripheral nerve in diabetic polyneuropathy was examined in nerve biopsy specimens from patients with diabetic polyneuropathy in comparison with organ donor control nerves and disease controls (other neuropathies). There was increased endoneurial collagenisation both in the diabetic polyneuropathy cases and the disease controls, this predominantly involving types I and III. Type II collagen was not detected in organ donor control nerves or in the diabetic and the disease control nerves. There was a relative increase in type VI collagen in the endoneurium in the diabetic nerves immediately surrounding groups of Schwann cells. This was not a feature in the other neuropathies. The quantity of types IV, V and VI collagen was increased around the endoneurial microvessels in the diabetic patients and, to a lesser extent, in those with hereditary motor and sensory neuropathy (HMSN). Increased deposition of types IV and V collagen was observed in the perineurium in the diabetic nerves, the latter being most evident in the innermost lamellae where the amount of laminin was possibly also increased. The diameter of the general endoneurial collagen fibrils was greater in the diabetic nerves, although this was not more than in a disease control (HMSN). The collagen fibrils that were present within the basal laminal tubes that had surrounded degenerated myelinated fibres in the diabetic nerves, and those within the onion bulbs of the HMSN cases, were of the normal endoneurial calibre. The expression of laminin by Büngner bands in diabetic neuropathy did not differ from that in disease control nerves, nor were any differences detected for fibronectin. Whether the changes observed are important for the impaired regenerative capacity in diabetic neuropathy requires further investigation.

Clinical, biochemical and molecular genetic correlations in Friedreich's ataxia.

Friedreich's ataxia (FRDA) is an autosomal recessive disorder with a frequency of 1 in 50 000 live births. In 97% of patients it is caused by the abnormal expansion of a GAA repeat in intron 1 of the FRDA gene on chromosome 9, which encodes a 210 amino acid protein called frataxin. Frataxin is widely expressed and has been localized to mitochondria although its function is unknown. We have investigated mitochondrial function, mitochondrial DNA levels, aconitase activity and iron content in tissues from FRDA patients. There were significant reductions in the activities of complex I, complex II/III and aconitase in FRDA heart. Respiratory chain and aconitase activities were decreased although not significantly in skeletal muscle, but were normal in FRDA cerebellum and dorsal root ganglia, although there was a mild decrease in aconitase activity in the latter. Mitochondrial DNA levels were reduced in FRDA heart and skeletal muscle, although in skeletal muscle this was paralleled by a decline in citrate synthase activity. Increased iron deposition was seen in FRDA heart, liver and spleen in a pattern consistent with a mitochondrial location. The iron accumulation, mitochondrial respiratory chain and aconitase dysfunction and mitochondrial DNA depletion in FRDA heart samples largely paralleled those in the yeast YFH1 knockout model, suggesting that frataxin may be involved in mitochondrial iron regulation or iron sulphur centre synthesis. However, the severe deficiency in aconitase activity also suggests that oxidant stress may induce a self-amplifying cycle of oxidative damage and mitochondrial dysfunction, which may contribute to cellular toxicity.

Deficit of in vivo mitochondrial ATP production in patients with Friedreich ataxia.

Friedreich ataxia (FRDA), the most common of the inherited ataxias, is an autosomal recessive degenerative disorder, characterized clinically by onset before the age of 25 of progressive gait and limb ataxia, absence of deep tendon reflexes, extensor plantar responses, and loss of position and vibration sense in the lower limbs. FRDA is caused by a GAA triplet expansion in the first intron of the FRDA gene on chromosome 9q13 in 97% of patients. The FRDA gene encodes a widely expressed 210-aa protein, frataxin, which is located in mitochondria and is severely reduced in FRDA patients. Frataxin function is still unknown but the knockout of the yeast frataxin homologue gene (YFH1) showed a severe defect of mitochondrial respiration and loss of mtDNA associated with elevated intramitochondrial iron. Here we report in vivo evidence of impaired mitochondrial respiration in skeletal muscle of FRDA patients. Using phosphorus magnetic resonance spectroscopy we demonstrated a maximum rate of muscle mitochondrial ATP production (V(max)) below the normal range in all 12 FRDA patients and a strong negative correlation between mitochondrial V(max) and the number of GAA repeats in the smaller allele. Our results show that FRDA is a nuclear-encoded mitochondrial disorder affecting oxidative phosphorylation and give a rationale for treatments aimed to improve mitochondrial function in this condition.

Novel surgical treatment and gastric pathology in diabetic gastroparesis.

AIMS: Observations are made on four Type 1 diabetic patients with the rare syndrome of intractable vomiting from confirmed gastroparesis, to determine whether radical surgery would alleviate their symptoms and subsequently to examine in detail the gastric histopathology. METHODS: The surgical approach consisted of an approximate 70% resection of the stomach, including the antrum and pylorus, with closure of the duodenum and restoration of gastrointestinal continuity with a 60-cm Roux-en-Y jejunal loop. Four longstanding Type 1 diabetic patients were examined and treated as described. They were all women in the age range 2741 years with grossly abnormal autonomic function tests in whom other causes for gastric paresis had been excluded. RESULTS: Vomiting episodes leading to multiple hospital admissions (6-8) in the year preceding surgery were eliminated in three of the four patients, while in the fourth initial success was followed by the need for dialysis for renal failure. Gastric histopathology showed evidence of smooth muscle degeneration and fibrosis, with eosinophilic inclusion bodies (M-bodies) which appear to be unique to this condition. The findings suggest the presence of a gastromyopathy. CONCLUSIONS: Satisfactory relief of intractable vomiting from diabetic gastroparesis was achieved by a novel radical surgical procedure. Histopathological findings suggest that gastromyopathy may contribute to the production of this syndrome.

Initial UK experience of the levonorgestrel-releasing contraceptive intravaginal ring.

A study was performed to establish the tolerance, acceptability and associated efficacy of a levonorgestrel-releasing intravaginal ring (IVR) in a sample of British women requiring contraception. This was achieved with an open non-randomized prospective study of 1710 women aged 18-40 years, recruited in 75 centers geographically spread around the UK using an IVR designed to release 20 microg/day of levonorgestrel. Assessments were made at baseline, after 6 weeks, after 3 months and then 3-monthly. After initial insertion of the IVR, it was changed at 3-monthly intervals. A total of 1591 women were eligible for analysis, with 572 available after 12 months and 34 after 24 months of use. Life-table analysis revealed pregnancy rates of 5.1% and 6.5% at 12 months and 24 months, respectively. The IVR was rated as acceptable or very acceptable as a form of contraceptive by 60.7% of women at 12 months. The most common adverse events were menstrual disturbance, headache and vaginal discharge. No significant pattern of biochemical, hematological, microbiological or cytological abnormalities was found but vaginal erythematous lesions were noted at some centers. This IVR was found to be a generally well-accepted method of contraception with a failure rate comparable to some other progestogenonly methods. On this basis, further development of hormone-releasing intravaginal rings is justified.

Neural architecture in transected rabbit sciatic nerve after prolonged nonreinnervation.

Observations have been made on the rabbit sciatic nerve distal to a transection, with survival periods of up to 26 mo and prevention of reinnervation. It was confirmed that the nerve becomes compartmented by fibroblast processes and that a zone of fine collagen fibrils develops around the Schwann cell columns that constitute the Büngner bands. The Schwann cells become progressively more atrophic but after 6 mo of denervation still expressed low affinity p75 nerve growth factor receptor (NGFR), the latest stage at which this was examined. NGFR was also expressed by the processes of the fibroblasts producing the endoneurial compartmentation. By 26 mo after transection the site of previous nerve fibres was indicated by sharply demarcated domains of approximately circular outline in transverse section consisting of densely packed longitudinally oriented collagen fibrils. Some of these domains still possessed centrally situated Schwann cells or residual basal lamina but many were acellular. The central collagen fibrils in these domains were of smaller diameter than those situated peripherally but were of larger size than those that form around the Büngner bands during wallerian degeneration. The peripherally located fibrils in the domains were of the same calibre as for normal endoneurial collagen. The collagen domains were encircled by fibroblast processes or at times enclosed in a perineurial cell ensheathment. Long-standing axonal loss therefore leads to a striking reorganisation of the internal architecture of peripheral nerve trunks. The findings may be relevant for the interpretation of the appearances in chronic peripheral neuropathies in man.

Hypertrophic neuropathy: atypical appearances resulting from the combination of type I hereditary motor and sensory neuropathy and diabetes mellitus.

A nerve biopsy from a patient with type Ia hereditary motor and sensory neuropathy and diabetes mellitus showed hypertrophic changes of atypical appearance. The onion bulbs were composed of a central core of Schwann cells, with or without associated axons, embedded in concentrically arrayed layers of collagen fibrils. These were surrounded either by highly attenuated Schwann cell processes or by fibroblasts. The biopsy showed a severe loss of myelinated axons. It is suggested that it is necessary for the supernumerary Schwann cells of the onion bulbs to be stabilized by associated unmyelinated axons. If these are lost, the Schwann cells atrophy and disappear.

The phenotypic manifestations of chromosome 17p11.2 duplication.

Clinical and electrophysiological investigations and nerve biopsies were carried out on 61 patients shown to have a chromosome 17p11.2 duplication (hereditary motor and sensory neuropathy-HMSN Ia). Of these, 50 showed a Charcot-Marie-Tooth (CMT) phenotype and eight could be classified as having the Roussy-Lévy syndrome. Of the patients with a CMT phenotype, three had associated pyramidal signs and of these one had 'complicated' HMSN and also signs of cerebellar and bulbar involvement. Diaphragmatic weakness was present in three severely affected cases, one of whom also had denervation of the anal sphincter associated with faecal incontinence. One unusual case presented in middle life with incapacitating muscle cramps associated with calf hypertrophy and only mild clinical signs of neuropathy. Prominent distal sensory loss was a consistent feature in one family, resulting in acrodystrophic changes in several members. Concurrent focal peripheral nerve lesions were seen with both the CMT and Roussy-Lévy phenotypes, in seven patients. Upper limb motor nerve conduction velocity was 19.9 m/s +/- 1.3 (SEM), range 5-34 m/s. This corresponds to values previously obtained for autosomal dominant HMSN I. This series consisted mainly of older patients with more advanced disease. In contrast to the findings in younger patients, in their nerve biopsies, myelin thickness tended to be relatively reduced for axon size, indicating remyelination and/or hypomyelination; there was also regression of the onion bulbs. It is concluded that the possession of two copies of the peripheral myelin protein 22 gene within the duplicated region on chromosome 17p gives rise to a range of phenotypes and not solely to a CMT syndrome, and that the pattern of histological change in the peripheral nerves alters with advance of the disease.

Myelinated nerve fibre regeneration in diabetic sensory polyneuropathy: correlation with type of diabetes.

Observations were made on myelinated fibre regeneration in diabetic sensory polyneuropathy assessed in sural nerve biopsy specimens. These confirmed that regenerative clusters initially develop within abnormally persistent Schwann cell basal laminal tubes. The number of regenerating fibres, identified by light microscopy, was found to decline in proportion to the reduction in total myelinated fibre density. The relative number of regenerating fibres was significantly greater in patients with insulin-dependent as compared with those with non-insulin-dependent diabetes after correction for age. There was a slight negative correlation between the relative proportion of regenerating fibres and age, but this was not statistically significant. The progressive reduction in the number of regenerating fibres with declining total fibre density indicates that axonal regeneration fails with advancing neuropathy. The production of nerve growth factor (NGF) and NGF receptors by denervated Schwann cells is likely to be important for axonal regeneration. To investigate whether the failure of axonal regeneration could be related to a lack of NGF receptor production by Schwann cells, we examined the expression of p75 NGF receptors by Büngner bands immunocytochemically. In comparison with other types of peripheral neuropathy, p75 NGF receptor expression appeared to take place normally. It is concluded that failure of axonal regeneration constitutes an important component in diabetic neuropathy. Its explanation requires further investigation.