Lea Glass-Marmor

Detection of Multiple Sclerosis from Exhaled Breath Using Bilayers of Polycyclic Aromatic Hydrocarbons and Single-Wall Carbon Nanotubes

A cross-reactive array of polycyclic aromatic hydrocarbons and single wall carbon nanotube bilayers was designed for the detection of volatile organic compounds (tentatively, hexanal and 5-methyl-undecane) that identify the presence of disease in the exhaled breath of patients with multiple sclerosis. The sensors showed excellent discrimination between hexanal, 5-methyl-undecane, and other confounding volatile organic compounds. Results obtained from a clinical study consisting of 51 volunteers showed that the sensors could discriminate between multiple sclerosis and healthy states from exhaled breath samples with 85.3% sensitivity, 70.6% specificity, and 80.4% accuracy. These results open new frontiers in the development of a fast, noninvasive, and inexpensive medical diagnostic tool for the detection and identification of multiple sclerosis. The results could serve also as a launching pad for the discrimination between different subphases or stages of multiple sclerosis as well as for the identification of multiple sclerosis patients who would respond well to immunotherapy.

Diagnosis and Classification of 17 Diseases from 1404 Subjects via Pattern Analysis of Exhaled Molecules

We report on an artificially intelligent nanoarray based on molecularly modified gold nanoparticles and a random network of single-walled carbon nanotubes for noninvasive diagnosis and classification of a number of diseases from exhaled breath. The performance of this artificially intelligent nanoarray was clinically assessed on breath samples collected from 1404 subjects having one of 17 different disease conditions included in the study or having no evidence of any disease (healthy controls). Blind experiments showed that 86% accuracy could be achieved with the artificially intelligent nanoarray, allowing both detection and discrimination between the different disease conditions examined. Analysis of the artificially intelligent nanoarray also showed that each disease has its own unique breathprint, and that the presence of one disease would not screen out others. Cluster analysis showed a reasonable classification power of diseases from the same categories. The effect of confounding clinical and environmental factors on the performance of the nanoarray did not significantly alter the obtained results. The diagnosis and classification power of the nanoarray was also validated by an independent analytical technique, i.e., gas chromatography linked with mass spectrometry. This analysis found that 13 exhaled chemical species, called volatile organic compounds, are associated with certain diseases, and the composition of this assembly of volatile organic compounds differs from one disease to another. Overall, these findings could contribute to one of the most important criteria for successful health intervention in the modern era, viz. easy-to-use, inexpensive (affordable), and miniaturized tools that could also be used for personalized screening, diagnosis, and follow-up of a number of diseases, which can clearly be extended by further development.

The ubiquitin–proteasome pathway regulates claudin 5 degradation

The tight junctions (TJs) form continuous intracellular contacts, which help create selective barriers in epithelial and endothelial cell layers. The structures created by the TJs are very dynamic and can be rapidly remodeled in response to physiological and pathological signals. Claudin 5 is a membranal TJ protein which plays a critical role in determining the permeability of endothelial barriers. We describe the regulation of claudin 5 degradation by the ubiquitin–proteasome system (UPS). Our results indicate that claudin 5 has a relatively short half‐life and can be polyubiquitinated on lysine 199. This ubiquitination appears to trigger the proteasome‐dependent degradation of claudin 5. Other mechanisms also seem to be involved in the post‐translational regulation of claudin 5, including a ubiquitin‐independent and probably indirect lysosomal‐dependent pathway. These findings provide evidence for the involvement of the UPS in the regulation of claudin 5 levels, and set the stage for further research to determine the involvement of this pathway in the modulation of the properties of TJs and cell‐layer barriers. J. Cell. Biochem. 113: 2415–2423, 2012.

Cathepsins and their endogenous inhibitors cystatins: expression and modulation in multiple sclerosis.

Cathepsins are involved in a variety of physiological processes including antigen processing and presentation and extracellular matrix degradation. In the present study, we evaluated whether expression levels of cathepsins S and B and their inhibitors cystatins B and C are affected by multiple sclerosis (MS) disease state (relapse and remission) and therapies (interferon‐β[IFN‐β] and the glucocorticoid [GC] methylprednisolone), and whether they are associated with the IFN‐β response phenotype. Real‐time PCR was employed to compare RNA expression levels in peripheral blood leucocytes (PBLs) and ELISA to determine serum protein levels of MS patients and matched healthy individuals. Cathepsin S RNA was higher in MS patients in the relapse state compared to controls (by 74%, P= 3 × 10−5, n= 30 versus n= 18) with a similar increase observed in serum (66%, P= 0.002, n= 18 versus n= 20). GC treatment reduced cathepsin S levels in PBL RNA (by 44%, P= 6 × 10−6, n= 27) and serum proteins (by 27%, P= 1 × 10−5, n= 26), reduced the serum protein levels of pro‐cathepsin B (by 8%, P= 0.0007, n= 23), and in parallel increased the serum levels of their inhibitor cystatin C (by 82%, P= 8 × 10−6, n= 26). IFN‐β therapy significantly elevated the RNA levels (n= 16) of cathepsin B (by 16%, P= 0.03), cystatin B (44%, P= 0.004) and cystatin C (48%, P= 0.011). In the serum, only cathepsin S levels were reduced by IFN‐β (16%, P= 0.006, n= 25). Interestingly, pre‐treatment serum cathepsin S/cystatin C ratio was higher in ‘good responders’ to IFN‐β therapy compared to patients without a good response (by 94%, P= 0.003). These results suggest that cathepsin S and cystatin C may contribute to disease activity in MS, specifically in a subgroup of patients that are responsive to IFN‐β therapy, and that these proteins should be further evaluated as biomarkers in MS.

Bio-markers of disease activity and response to therapy in multiple sclerosis

The pathogenesis of multiple sclerosis (MS) involves immune-mediated as well as neurodegenerative processes, each comprising both acute and chronic phases. The vast variety of cell types playing a part in the cascade of these processes, including lymphocytes, monocytes, glia cells, neurons, and endothelial cells, are sources for many biological activities and mediators [1–6]. Measurement of these activities (as cell proliferation, migration) and related mediators (as cytokines, proteolytic enzymes), conducted at different biological levels such as mRNA, protein level and activity, in accessible body fluids, to characterization of useful bio-markers for MS, has gained much attention in recent years. Aditionally, MS appears to include distinct clinical subtypes. Their main forms are RRMS, which tends to convert, eventually, to an SP form, and PPMS [7,8]. Immuno-pathological as well as neuro-radiological studies point to possible differences in the basic mechanisms underlying these distinct clinical subtypes [9–12]. Thus, the studies of disease-related indicators are directed towards the identification of markers specific to the distinct MS clinical subtypes and underlying pathogenic mechanisms. This review will focus on some of the bio-markers the mea-

Chronotherapy using corticosteroids for multiple sclerosis relapses

Background: The activity of the immune system displays a circadian rhythm. In diseases characterised by aberrant immune activity, chronotherapy (a treatment regimen tailored to diurnal body rhythms) may increase the efficiency, safety and tolerability of drugs. Aim: To compare the outcomes of intravenous corticosteroid administration during the day or night, for treatment of acute multiple sclerosis relapses. Methods: 17 patients with multiple sclerosis were included in the study. Clinical assessment of disability was performed at trial entry, and at days 7 and 30 from the initiation of treatment. Adverse events and preference of night-time versus daytime treatment were assessed at the end of the treatment course. Results: After night-time treatment, clinical recovery was significantly (p<0.001) enhanced and the mean number of side effects was significantly (p = 0.007) lower. Furthermore, most patients expressed a preference for night-time versus daytime treatment. Conclusions: The study suggests a potential benefit for implementation of chronotherapy using steroid treatment for acute multiple sclerosis relapse, with implications for other immune-mediated disorders.

Tight junction proteins expression and modulation in immune cells and multiple sclerosis

The tight junction proteins (TJPs) are major determinants of endothelial cells comprising physiological vascular barriers such as the blood–brain barrier, but little is known about their expression and role in immune cells. In this study we assessed TJP expression in human leukocyte subsets, their induction by immune activation and modulation associated with autoimmune disease states and therapies. A consistent expression of TJP complexes was detected in peripheral blood leukocytes (PBLs), predominantly in B and T lymphocytes and monocytes, whereas the in vitro application of various immune cell activators led to an increase of claudin 1 levels, yet not of claudin 5. Claudins 1 and 5 levels were elevated in PBLs of multiple sclerosis (MS) patients in relapse, relative to patients in remission, healthy controls and patients with other neurological disorders. Interestingly, claudin 1 protein levels were elevated also in PBLs of patients with type 1 diabetes (T1D). Following glucocorticoid treatment of MS patients in relapse, RNA levels of JAM3 and CLDN5 and claudin 5 protein levels in PBLs decreased. Furthermore, a correlation between CLDN5 pre‐treatment levels and clinical response phenotype to interferon‐β therapy was detected. Our findings indicate that higher levels of leukocyte claudins are associated with immune activation and specifically, increased levels of claudin 5 are associated with MS disease activity. This study highlights a potential role of leukocyte TJPs in physiological states, and autoimmunity and suggests they should be further evaluated as biomarkers for aberrant immune activity and response to therapy in immune‐mediated diseases such as MS.

The influence of vitamin D supplementation on melatonin status in patients with multiple sclerosis

BACKGROUND Multiple sclerosis (MS) incidence is higher in geographic regions with less sunlight exposure. Both vitamin D and melatonin are essential mediators of the effect of sunlight in health, and as such are candidates to play a key role in MS. We hypothesized that vitamin D and melatonin may have related influences in patients with MS. METHODS In a randomized, double blind study of 40 IFN-β treated MS patients, 21 patients were assigned to 800 IU of vitamin D3 per day (low dose), while 19 patients received 4,370 IU vitamin D3 per day (high dose) for one year. Serum 25-hydroxy-vitamin-D (25-OH-D) and nighttime urine melatonin metabolite, 6-sulphatoxy-melatonin (6-SMT), were measured at baseline, 3 months and 1 year from enrolment. RESULTS After 3 months supplementation, 25-OH-D levels increased and nighttime melatonin secretion decreased significantly in the high dose group, but not in the low dose group. After 1 year, a decrease in 25-OH-D levels, accompanied by an increase of urine nighttime 6-SMT were observed in the high dose group. Percent change in serum 25-OH-D was significantly and negatively correlated with percent change in urine 6-SMT after 3 months and between 3 months to 1 year. 25-OH-D levels by the end of the study were significantly and negatively correlated to BMI. CONCLUSIONS Melatonin secretion is negatively correlated with alterations in serum 25-OH-D in IFN-β treated patients with MS. The finding suggests that melatonin should be considered as a potential mediator of vitamin D neuro-immunomodulatory effects in patients with MS.

Immunomodulation by chronobiologically-based glucocorticoids treatment for multiple sclerosis relapses

This study compares the effects of daytime versus nighttime intravenous glucocorticoid treatment of multiple sclerosis (MS) relapses for several immune indicators. The levels of serum CRP, TNFalpha, ESR, MMP-2, MMP-9, TIMP-1, and TIMP-2 were determined at trial entry and at day 7 post therapy initiation in 35 MS patients. Serum MMP-9 protein levels were differentially affected by treatment regimen, and were significantly lower after nighttime treatment. Both treatment protocols led to a similar reduction of ESR, CRP and TNFalpha. These findings provide preliminary characterization of biomarkers in the application of chronobiology-based glucocorticoid therapeutics in MS and other immune disorders.

Matrix metalloproteinase-9, its tissue inhibitor (TIMP)-1 and CRP in Alzheimer's disease

Results The clinical and demographic characteristics of the study population are presented in table 1 . No statistically signifi cant difference was noted in the median plasma levels of TIMP-1 or MMP-9 or in the serum HS-CRP levels between control subjects and AD patients with mild dementia or moderate to severe dementia ( fi g. 1 a–c). No signifi cant correlation was found between the levels of these proteins and the Apo E genotype (data not shown). Treatment with Ach-EIs (n = 13 treated, n = 6 nontreated) ( fi g. 1 d–f) was associated with signifi cantly lower plasma levels of TIMP-1 (p = 0.028) (mean = 140.96 8 30.72 ng/ml; median = 137.04 ng/ml) compared with the nontreated patients (mean = 215.14 8 79.56 ng/ml; median = 207.85 ng/ml), but with no effect on MMP-9 levels. Signifi cantly lower levels of HS-CRP were observed in the Ach-EI-treated AD patients (mean = 1.09 8 1.08 mg/l; median = 0.59 mg/l) compared with the levels in the untreated group (mean = 2.59 8 1.03 mg/l; median = 2.14 mg/l; p = 0.014).