Partial amino acid sequence of purified von Willebrand factor-cleaving protease

von Willebrand factor-cleaving protease (vWF-cp) is responsible for the continuous degradation of plasma vWF multimers released from endothelial cells. It is deficient in patients with thrombotic thrombocytopenic purpura, who show unusually large vWF multimers in plasma. Purified vWF-cp may be useful for replacement in these patients, who are now treated by plasma therapy. In this study, vWF-cp was purified from normal human plasma by affinity chromatography on the IgG fraction from a patient with autoantibodies to vWF-cp and by a series of further chromatographic procedures, including affinity chromatography on Protein G, Ig-TheraSorb, lentil lectin, and heparin. Four single-chain protein bands, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, showed M(r) of 150, 140, 130, and 110 kd and were found to share the same N-terminal amino acid sequence, suggesting that they were derived from the same polypeptide chain that had been partially degraded at the carboxy-terminal end. A hydrophobic sequence (Ala-Ala-Gly-Gly-Ile-Leu-His-Leu-Glu-Leu-Leu-Val-Ala-Val-Gly) of the first 15 residues was established. The protease migrates in gel filtration as a high-molecular-weight complex with clusterin, a 70-kd protein with chaperonelike activity. vWF-cp bound to clusterin is dissociated by the use of concentrated chaotropic salts. vWF-cp in normal human plasma or serum is not associated with clusterin, suggesting that the observed complex is due to vWF-cp denaturation during the purification procedure. Activity of vWF-cp is unusually stable during incubation at 37 degrees C; its in vitro half-life in citrated human plasma, heparin plasma, or serum is longer than 1 week. There was even a temporary increase in protease activity during the first 3 days of incubation.     

The therapeutic use of gene therapy in inflammatory demyelinating diseases of the central nervous system

PURPOSE OF REVIEW: Gene therapy protocols aimed to deliver therapeutic molecules into the central nervous system may represent an alternative therapeutic strategy in patients affected by inflammatory demyelinating diseases of the central nervous system where systemic therapies have shown limited therapeutic efficacy possibly owing to the blood-brain barrier, a major obstacle for the entry of therapeutic molecules into the central nervous system. RECENT FINDINGS: Among inflammatory demyelinating diseases of the central nervous system, gene therapy approaches have been so far developed almost exclusively for multiple sclerosis. However, the chronic/relapsing nature of the disease, the restriction to the central nervous system of the pathological process as well as the necessity to inhibit the ongoing inflammatory process but also to foster endogenous remyelinating pathways, have posed several questions which still need to be properly addressed for the development of a successful gene therapy strategy in multiple sclerosis patients. SUMMARY: The gene therapy approaches for multiple sclerosis have been so far developed and tested only in rodents and monkeys with experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. The results of these studies clearly indicate that the delivery of therapeutic genes within the central nervous system is superior to the peripheral delivery. In particular, the intracerebral delivery of genes coding for anti-inflammatory and/or neurotrophic molecules, using gene vectors derived from non-replicative viruses, showed to inhibit not only the detrimental function of blood-borne mononuclear effector cells but also to foster proliferation and differentiation of surviving oligodendrocytes within demyelinated areas. Here, we summarize the most recent findings of this novel area of research.     

HLA genotypes and disease severity assessed by magnetic resonance imaging findings in patients with multiple sclerosis

The objective of the study was to examine the relationship between HLA genotypes and disease severity as measured by brain MRI quantitative markers of demyelinating and destructive pathology in patients with multiple sclerosis (MS). We studied 100 patients with MS and 122 age, sex-, ethnic- and residence-matched controls. The DNA extraction and the genomic typing (A, B, DRB1 and DQB1 loci) were obtained with sequence-specific oligonucleotide method, using a commercially available reversible line blot assay (INNO-LIPA). All patients underwent a 1.5 tesla MRI examination of the brain. Disease severity was assessed by clinical (Expanded Disability Status Scale (EDSS)) and MRI (T2- and T1-lesion load (LL) and brain parenchymal fraction (BPF)) outcome measures. HLA-DQB1* 02 (OR 19.9, 95% C. I. 16.2–24.3, uncorrected (uncorr)- p<0.00001, corr-p<0.0006), -DQB1*03 (OR 16.8, 95% C. I. 13.6–20.5, uncorr-p<0.00001, corrp< 0.0006), -DRB1*15 (OR 4.6, 95% C. I. 3.7–5.6, uncorrp= 0.0001, corr-p=0.006), and -DRB1*03 (OR 3.9, 95% C. I. 3.2–4.8, uncorr-p=0.0001, corrp= 0.006) alleles were associated with MS. T2-, T1-LL, BPF and EDSS were not significantly different according to the carrier status of these HLA alleles. No differences were found in the ratios of disease severity/disease duration according to the HLA car rier status. Multiple regression analysis showed that a higher T2-LL was associated with the presence of DRB1*04 (uncorr- R2=0.15, p=0.006 and corr- R2=0.11, p=0.025) and B7 alleles (uncorr-R2=0.08, p=0.02 and corr-R2=0.07, p=0.018), T1-LL was associated with B7 (uncorr- R2=0.30, p<0.0001 and corr- R2=0.27, p=0.0001) and DRB1*12 (uncorr-R2=0.25, p<0.0001 and corr-R2=0.21, p=0.0002) alleles, whereas the BPF was predicted only by the presence of DRB1*12 allele (uncorr-R2=0.24, p=0.002 and corr-R2=0.20, p=0.004). The study findings suggest that some HLA alleles may predict the destructive pathological processes visible on MRI. Since the size of the sample studied is relatively small, further studies are needed to draw any firm conclusion about genotype/phenotype correlation in patients with MS.     

A critical review of reviews on the treatment of chronic low back pain

STUDY DESIGN: Systematic literature review. OBJECTIVE: To critically appraise the methodology of systematic reviews of conservative therapies for chronic nonspecific low back pain and to study the relation between the methodologic quality and other characteristics of these reviews. SUMMARY OF BACKGROUND DATA: Systematic reviews offer a concise summary of the evidence on treatment effectiveness, but flaws in their methodology can lead to invalid conclusions with serious implications for quality of patient care. METHODS: Searches of MEDLINE, EMBASE, Psychinfo, and the Cochrane Library were conducted. Titles, abstracts, and articles were reviewed by two blinded authors using three inclusion criteria: 1) chronic nonspecific low back pain, 2) systematic review, and 3) conservative treatment intervention. Data were extracted from each review by three authors. RESULTS: The search strategy retrieved 1102 titles and abstracts; 109 met inclusion criteria. A review of the full text of these articles excluded an additional 73 articles. Data abstraction and methodologic assessment were conducted on 36 articles reviewing 19 discrete interventions. The average quality score was 4.1, ranging from 1 (low) to 7 (high). There was a trend for recent reviews to be of higher quality. Fifty-six percent of the reviews had positive conclusions, but they had lower quality scores compared with those that had negative or uncertain conclusions. There were 27 (73%) qualitative and 10 (27%) quantitative summaries of results. CONCLUSIONS: Although the overall quality of systematic reviews was satisfactory, the quality of the individual papers included in the reviews varied considerably. The reviews often provided contradictory evidence on the effectiveness of a wide range of commonly used conservative interventions for chronic nonspecific low back pain. These findings illustrate the pitfalls of systematic reviews where there are a number of low-quality trials and underscore the need for high-quality primary trials that will allow for more conclusive reviews.     

Orthostatic intolerance: different abnormalities in the neural sympathetic response to a gravitational stimulus

In this paper we shall focus on the different abnormalities in the neural sympathetic response to a gravitational stimulus, characterising syndromes with symptoms of orthostatic intolerance. In Vaso vagal Syncope, an increase or a reduction of cardiac and vascular sympathetic modulation have been described in occasional and habitual fainters, respectively. Pure Autonomic Failure (PAF) is characterized by a global cardiovascular denervation. Accordingly, the spectral markers of cardiac and vascular sympathetic modulation are absent or reduced. However, a concomitant vagal diminished activity is present. In Chronic Orthostatic Intolerance (COI), the most common form of dysautonomia in young female, an abnormal regional distribution of sympathetic discharge has been hypothesized during standing. Indeed, an overall increased sympathetic activity is present in recumbent position; during tilt a blunted vascular sympathetic discharge, with a concomitant exaggerated cardiac sympathetic modulation, is evident. Baroreflex Failure is a syndrome that may result from neck surgery or irradiation due to different forms of regional cancer. It is characterized by a volatility of blood pressure and heart rate, without habitual orthostatic hypotension. In the present paper, we describe a case of Baroreflex Failure with marked orthostatic hypotension in spite of a huge muscle sympathetic nerve activity (MSNA) and high levels of plasma cathecolamines. The most relevant finding was the absence of any coordinate rythmicity in blood pressure, heart rate and MSNA, both at rest and during tilt, particularly in the frequency band likely to be related with sympathetic modulation, i.e. at 0.1 Hz. We hypothesize that the absence of 0.1 Hz spontaneous fluctuations might play a role in sustaining orthostatic hypotension.     

Cxcl10 enhances blood cells migration in the sub-ventricular zone of mice affected by experimental autoimmune encephalomyelitis

The peri-ventricular area of the forebrain constitutes a preferential site of inflammation in multiple sclerosis, and the sub-ventricular zone (SvZ) is functionally altered in its animal model experimental autoimmune encephalomyelitis (EAE). The reasons for this preferential localization are still poorly understood. We show here that, in EAE mice, blood-derived macrophages, T and B cells and microglia (Mg) from the surrounding parenchyma preferentially accumulate within the SvZ, deranging its cytoarchitecture. We found that the chemokine Cxcl10 is constitutively expressed by a subset of cells within the SvZ, constituting a primary chemo-attractant signal for activated T cells. During EAE, T cells and macrophages infiltrating the SvZ in turn secrete pro-inflammatory cytokines such as TNFα and IFNγ capable to induce Mg cells accumulation and SvZ derangement. Accordingly, lentiviral-mediated over-expression of IFNγ or TNFα in the healthy SvZ mimics Mg/microglia recruitment occurring during EAE, while Cxcl10 over-expression in the SvZ is able to increase the frequency of peri-ventricular inflammatory lesions only in EAE mice. Finally, we show, by RT-PCR and in situ hybridization, that Cxcl10 is expressed also in the healthy human SvZ, suggesting a possible molecular parallelism between multiple sclerosis and EAE.     

von Willebrand factor-cleaving protease in childhood diarrhoea-associated haemolytic uraemic syndrome.

A deficiency of von Willebrand factor (vWF)-cleaving protease, either due to a congenital deficiency or to the presence of a protease inhibitor of vWF-cleaving protease has been associated with thrombotic thrombocytopenic purpura (TTP). We have studied vWF-cleaving protease in diarrhoea-associated haemolytic uraemic syndrome (D+ HUS), which shares clinical features with TTP. 29 children with acute D+ HUS and 13 control children were studied. vWF-cleaving protease activity was normal (range 50-150%) in 39 of 42 plasma samples. Levels of protease activity between 25 and 50% were noted in plasma from two D+ HUS patients. One D+HUS patient, who had clinical features of TTP, had a vWF-cleaving protease inhibitor producing a severe deficiency of vWF-cleaving protease. Thus a deficiency of vWF-cleaving protease appears to be atypical in D+HUS. The detection of a vWF-cleaving protease inhibitor in one patient suggests it may be associated with infection such as E. coli O157.