Expression, purification and metal utilization of recombinant SodA from Borrelia burgdorferi.

Borrelia are microaerophilic spirochetes capable of causing multisystemic diseases such as Lyme disease and Relapsing Fever. The ubiquitous Fe/Mn-dependent superoxide dismutase (SOD) provides essential protection from oxidative damage by the superoxide anion. Borrelia possess a single SOD enzyme - SodA that is essential for virulence, providing protection against host-derived reactive oxygen species (ROS). Here we present a method for recombinant expression and purification of Borrelia burgdorferi SodA in E. coli. Metal exchange or insertion into the Fe/Mn-SOD is inhibited in the folded state. We therefore present a method whereby the recombinant Borrelia SodA binds to Mn under denaturing conditions and is subsequently refolded by a reduction in denaturant. SodA purified by metal affinity chromatography and size exclusion chromatography reveals a single band on SDS-PAGE. Protein folding is confirmed by circular dichroism. A coupled enzyme assay demonstrates SOD activity in the presence of Mn, but not Fe. The apparent molecular weight determined by size exclusion corresponds to a dimer of SodA; a homology model of dimeric SodA is presented revealing a surface Cys distal to the dimer interface. The method presented of acquiring a target metal under denaturing conditions may be applicable to the refolding of other metal-binding proteins.

Hypercapnia impact on vascular and neuronal reactivity in patients before and after carotid endarterectomy.

HYPOTHESIS: Regional cerebral blood flow (rCBF) and vascular reactivity strongly affect neuronal function. The restoration of blood flow values in the cerebral vascular system may be another benefit of carotid endarterectomy (CEA) in a specific group of patients. Animal experiments in dogs have provided evidence of neuronal reactivity depending on rCBF and vascular reactivity. However, as yet, there are no reports on neuronal reactivity changes related to altered perfusion parameters in humans. MATERIAL AND METHODS: The cohort under study consisted of 41 patients after transient ischaemic attack (TIA) or reversible ischaemic neurological deficit (RIND) whose neurological findings were normal (group A) and 17 patients after minor stroke with a mild degree of hemiparesis or hemihypesthesia (mRS

Experience of 500 cases of neurophysiological monitoring in carotid endarterectomy.

BACKGROUND: Experience with Intraoperative monitoring using neurophysiological and haemodynamic indices in 500 operations for carotid endarterectomy is reported. METHODS: Transcranial Doppler technique (TCD), electroencephalogram (EEG) and bilateral median somatosensory evoked potentials (SEP) were performed. Latency and amplitude of SEP, spectral analysis of EEG signal and blood flow velocity in the medial cerebral artery (MCA) were continuously measured. FINDINGS: After two consecutive drops of N20/P25 complex of more than 50%, a warning was given, and when the decrease continued, an the alarm raised. Abnormal EEG changes, if any, appeared after a significant decrease in the N20/P25 amplitude. A mean blood flow velocity drop below 40% of the reference value after cross clamping was rated as a significant warning event.A warning as a result of a decrease in N20/P25 amplitude occurred in 80 operations (16.0%), after an spectral edge frequency decrease in 2 cases (0.4%) and after a V(mean) decrease in 21 cases (4.2%). False negative results were experienced in 2 patients (0.4%). A shunt was inserted in 2.8% of the operations. The overall mortality/morbidity rate was 2.4%. CONCLUSION: A decrease of more than 50% in the amplitude of the thalamocortical somatosensory evoked potential complex N20/P25 proved to be the most reliable warning of danger of ischaemia during carotid endarterectomy.

Reinnervation of the rat musculocutaneous nerve stump after its direct reconnection with the C5 spinal cord segment by the nerve graft following avulsion of the ventral spinal roots: a comparison of intrathecal administration of brain-derived neurotrophic factor and Cerebrolysin.

Experimental model based on the C5 ventral root avulsion was used to evaluate the efficacy of brain-derived neurotrophic factor (BDNF) and Cerebrolysin treatment on motor neuron maintenance and survival resulted in the functional reinnervation of the nerve stump. In contrast to vehicle, BDNF treatment reduced the loss and atrophy of motor neurons and enhanced the regrowth axon sprouts into the distal stump of musculocutaneous nerve. However, the axon diameter of the myelinated fibers was smaller than those of control rats. The morphometric results were related to a low score in behavioral test similar to vehicle-treated rats. Cerebrolysin treatment greatly protected the motor neurons against cell death. Moreover, morphometric features of myelinated axons were better than those of rats treated with vehicle or BDNF. The mean score of grooming test suggested better results of the functional motor reinnervation than after BDNF administration. The majority of rescued motor neurons regenerating their axons through nerve graft in both BDNF- and Cerebrolysin-treated rats expressed choline acetyltransferase immunostaining. The results demonstrate that BDNF has more modest effects in preventing the death of motor neurons and functional recovery of injured motor nerve after root avulsion than Cerebrolysin.

[Principles, methods and use of the National Register of Cardiovascular Interventions]. / Principy, metody a vyuzití Národního registru kardiovaskulárních intervencí.

The goal of cooperative project of the national registry of cardiovascular interventions is analysis of quality of cardiovascular care with risk stratification and assessment of clinical indicators at organizational and individual level. Bayes methodology of stratification and risk prediction is used.

[A system for monitoring quality, outcomes and health care costs]. / Systém sledování kvality, výsledku a nákladu zdravotní péce.

Specialised form of database named DataCube for assessment of quality, outcomes and costs of the healthcare in cardiac surgery was built at the Institute of Clinical and Experimental Medicine (IKEM). Risk stratification of patients was done using Bayes predictive model. Such prediction well correlated with calculated costs. The results achieved promise the system to become an important decision making tool, which may improve identification of high risk patients.

[Anterior cervical corpectomy in the treatment of multilevel degenerative stenoses with spondylotic myelopathy. Personal experience with therapy and a literature review]. / Prední krcní korpektomie pri resení víceetázových degenerativních stenóz se spondylogenní myelopatií. Zkusenosti s lécbou a prehled literatury.

The authors use as a basis experience with a group of 389 patients operated in 1989-1997 on account of degenerative diseases of the cervical spine with neurological manifestations. The results are compared with experience assembled in 1998-1999. In the new group (188 patients with the same diagnosis) the same indication criteria were used but in case of myelopathy associated with multisegmetal cervical stenosis not only multilevel discectomy was performed (as in the previous group) but in addition also 1-3 segmental corpectomy (somatectomy). The results were evaluated separately for both methods used. The authors emphasize the necessity of a radical approach during decompression of neurological structures incl. removal of uncovertebral osteophytes which must be combined with suitable stabilization of the fusions or possibly be supported by instrumentation. In the discussion the authors illustrate in the form of a review the development of anterior cervical corpectomy as one of the methods of an anterior approach to the cervical spine used at first in traumatic and oncological conditions, later extended to operations on account of degenerative, dysplastic conditions and other diseases. The authors wish to help to define criteria for application of corpectomy when treating stenoses of the spinal canal in the cervical region. The indication will be defined in a perspectively followed up group where a detailed clinical and electrophysiological algorithm for examination was submitted as well as postoperative follow-up and processing of the resulting data.

Conservative treatment versus surgery in spondylotic cervical myelopathy: a prospective randomised study.

A prospective randomised 2-year study was performed to compare the conservative and operative treatment of mild and moderate forms of spondylotic cervical myelopathy (SCM). Forty-eight patients presenting with the clinical syndrome of SCM, with a modified Japanese Orthopaedic Association (mJOA) score of 12 points or more, were randomised into two groups. Group A, treated conservatively, consisted of 27 patients, mean age 55.6 +/- 8.6 years, while group B was treated surgically (21 patients, mean age 52.7 +/- 8.1 years). The clinical outcome was measured by the mJOA score, recovery rate (RR), timed 10 m walk, score of daily activities (recorded by video and evaluated by two observers blinded to the therapy), and by the subjective assessment of the patients at 6, 12, and 24 months of the follow-up. There was, on average, no significant deterioration in mJOA score, recovery ratio, or timed 10 m walk within either group during the 2 years of follow-up. In the surgery group there was a slight decline in the scores for daily activities and subjective evaluation. A comparison of the two groups showed no significant differences in changes over time in mJOA score or quantified gait, but there were significant differences in the score of daily activities recorded by video at 24 months, which was a little lower in the surgical group, and also in RR and subjective evaluation, which were both worse in the surgical group at months 12 and 24. However, at month 6, this last parameter was significantly better in the surgical than in conservative group. Surgical treatment of mild and moderate forms of SCM in the present study design, comprising the patients with no or very slow, insidious progression and a relatively long duration of symptoms, did not show better results than conservative treatment over the 2-year follow-up.

Acellular nerve graft re-seeded by Schwann cells migrating from the nerve stump can stimulate spinal motoneurons for functional reinnervation of the rat muscle.

The acellular nerve graft was utilised to restore a functional reinnervation of the biceps brachii muscle from the motoneuron pool of the cervical spinal cord. The musculocutaneous nerve stump was sutured to an acellular nerve graft, the opposite end of which was inserted into the cervical spinal cord cranial to the avulsed C5 ventral root. The acellular nerve graft was repopulated by Schwann cells heavily immunostained for NGFr within 90 days. The Schwann cells migrating from the nerve stump reached the spinal cord grey matter, where they stimulated the motoneurons to send axonal sprouts. The functional reinnervation of the biceps brachii muscle was assessed by means of the behavioural (grooming) test and EMG, the presence of myelinated and unmyelinated axons was demonstrated by light and electron microscopy. The axonal reconnection of the musculocutaneous nerve stump was verified by horseradish peroxidase retrograde labelling of the spinal motoneurons. Moreover, the motoneurons on the operated side of the C5 spinal segment displayed increased immunostaining for GAP-43 in comparison to the contralateral side, whereas the pattern of AChE histochemical reaction was similar on both the operated and contralateral side, of the C5 segment 150 days after acellular graft implantation. The regenerated axons bridged a 4-cm long originally acellular nerve graft to reach and reinnervate the biceps brachii muscle. The reinnervation of the neuromuscular junctions was morphologically determined by immunofluorescence for neurofilaments. The number of myelinated axons in the acellular nerve graft was significantly higher than those growing over the cellular graft, but their diameter was smaller. The results of experiments presented here demonstrate functional recovery of the biceps muscle reinnervation through the acellular nerve graft repopulated by migrating Schwann cells. The process of reinnervation by acellular nerve graft is however delayed and worse in comparison with the cellular graft.

The value of somatosensory- and motor-evoked potentials in predicting and monitoring the effect of therapy in spondylotic cervical myelopathy. Prospective randomized study.

STUDY DESIGN: A 2-year follow-up prospective randomized electrophysiologic and clinical study of patients with spondylotic cervical myelopathy. OBJECTIVE: To assess the value of somatosensory- and motor-evoked potentials in the evaluation and prediction of the effect of therapy. SUMMARY OF BACKGROUND DATA: Previous studies have yielded conflicting data concerning the correlation between the changes in evoked potential parameters and the clinical postsurgical outcome in spondylotic cervical myelopathy. METHODS: Sixty-one patients with magnetic resonance images suggesting spondylotic cervical cord compression and clinical signs of cervical myelopathy were divided into two groups according to the degree of clinical cervical cord involvement. The 49 patients with mild and moderate spondylotic cervical myelopathy were randomized into groups that underwent either surgical or conservative therapy. Patients were evaluated clinically and by the means of somatosensory- and motor-evoked potentials. RESULTS: The clinical and evoked potential changes showed good correlation on the group level, but poor correlation intraindividually. There were no significant evoked potential and clinical group changes after 6 months and 2 years in the mild myelopathy group treated either surgically and conservatively, whereas patients with severe myelopathy displayed significant improvement in clinical and evoked potential parameters after surgery. In a subgroup of patients, the isolated segmental medullar N13 abnormality could potentially predict favorable postsurgical clinical outcome. CONCLUSIONS: Longitudinal evoked potentials showed limited use for evaluating the results of therapy in an individual patient. They could be useful in the group assessment of therapy results and in labeling a subgroup of patients with potentially favorable postsurgical outcome.

Management of postoperative paralysis of diaphragm in infants and children.

During an 8-month period, 86 consecutive infants and children under 2 years of age underwent palliative or corrective cardiac surgery, of whom 11 subsequently developed phrenic nerve injury (PNI). This was seen most frequently following classic or modified Blalock-Taussig shunts. The diagnosis was established by ultrasound screening of the diaphragm, and patients were initially managed expectantly with ventilatory support. In nine patients no further management was necessary with demonstrated return of diaphragmatic function. The remaining two patients underwent plication of the diaphragm. The mean time to diaphragmatic recovery was 40.8 days and was more prolonged in patients with paradoxical, as opposed to absent, diaphragmatic movement. There were no deaths in the series. A further retrospective review of 241 patients of similar age undergoing similar surgery over the preceding 2 years revealed evidence of PNI in 11 (4.6%). Recovery of diaphragmatic function was documented in all except one patient who died. Based on these results we believe that although PNI is associated with considerable morbidity, and frequently a long stay in Intensive Care, there is evidence of spontaneous recovery of diaphragmatic function in 90% of the patients. Consequently, plication of the diaphragm can usually be avoided. Ultrasound scanning is extremely useful in establishing the diagnosis and offers assistance in predicting prognosis and deciding management.

Surgical treatment of persistent ductus arteriosus complicated by bacterial endocarditis.

A seven-year-old girl with persistent ductus arteriosus developed acute bacterial endocarditis with large vegetations in the ductus and the left pulmonary artery. Conservative management was not successful: repeated embolisation to the lungs necessitated operation during the acute endocarditis. Details of the operative technique are presented and discussed.

Somatosensory evoked potentials in patients with occlusions of cerebral arteries.

The material consists of 100 patients suffering from occlusion of a cerebral artery, admitted for the performance of arterial bypass. Before the operation SEPs of the median nerve and CT scans were taken. Three main types of SEP were found (symmetric, asymmetric-attenuated, asymmetric-augmented) and pathological changes of latency, amplitude and wave form of the primary complex N1-P2 were described. The changes were correlated with the localization and extent of the hypodense lesion in the cerebral hemispheres. The cause of the decreased latencies on the side of the lesion is discussed and reported in relation to 'travelling waves' (Cracco 1980) in normals. The cause of the increased amplitude on the side of the lesion is also discussed and reported in relation to a disturbance of the suppressor cortex 8s, 19s and of the head of the caudate nucleus.