A One Year Follow-Up Study of Natural Killer and Dendritic Cells Activities in Multiple Sclerosis Patients Receiving Glatiramer Acetate (GA)

Background

Multiple sclerosis (MS) is a chronic inflammatory, demyelinating and neurodegenerative disease. It is thought to be mediated by CD4⁺ Th1/Th17 cells. More recently, cells of the innate immune system such as dendritic cells (DCs) and natural killer (NK) cells have been in focus. Glatiramer acetate (GA) is an approved drug for treating MS patients.

Methodology/Principal Findings

In the current study we examined the activities of NK and DCs in nine relapsing remitting MS patients for up to one year after initiation of GA treatment. We observed that NK cells isolated from most of these patients have increased cytotoxic activity against K562 cells. Further analysis showed that the same NK cells lysed both autologous immature (i) and mature (m) DCs. In most patients this increased activity was correlated with increased NK cell activating cytotoxicity receptors such as NKp30, NKp44, NKp46 and NKG2D, and reduced expression of the inhibitory molecule CD158 on the surface of these NK cells. The expression of HLA-DR was increased on iDCs and mDCs in the majority of the patients, but no consistency was observed for the expression of HLA-I or HLA-E. Also, the co-stimulatory receptors CD80, CD83 or CD86 expression was down-regulated on iDCs and mDCs in most cases. Further, the expression of CCR6 was increased on mDCs at later time points of therapy (between 32–48 weeks).

Conclusions/Significance

Our results are the first showing the effects of GA treatment on NK cells in MS patients, which may impact future use of this and other drugs to treat this disease.     

Baseline Gene Expression Signatures in Monocytes from Multiple Sclerosis Patients Treated with Interferon-beta

Background

A relatively large proportion of relapsing-remitting multiple sclerosis (RRMS) patients do not respond to interferon-beta (IFNb) treatment. In previous studies with peripheral blood mononuclear cells (PBMC), we identified a subgroup of IFNb non-responders that was characterized by a baseline over-expression of type I IFN inducible genes. Additional mechanistic experiments carried out in IFNb non-responders suggested a selective alteration of the type I IFN signaling pathway in the population of blood monocytes. Here, we aimed (i) to investigate whether the type I IFN signaling pathway is up-regulated in isolated monocytes from IFNb non-responders at baseline; and (ii) to search for additional biological pathways in this cell population that may be implicated in the response to IFNb treatment.

Methods

Twenty RRMS patients classified according to their clinical response to IFNb treatment and 10 healthy controls were included in the study. Monocytes were purified from PBMC obtained before treatment by cell sorting and the gene expression profiling was determined with oligonucleotide microarrays.

Results and discussion

Purified monocytes from IFNb non-responders were characterized by an over-expression of type I IFN responsive genes, which confirms the type I IFN signature in monocytes suggested from previous studies. Other relevant signaling pathways that were up-regulated in IFNb non-responders were related with the mitochondrial function and processes such as protein synthesis and antigen presentation, and together with the type I IFN signaling pathway, may also be playing roles in the response to IFNb.     

Fingolimod Increases CD39-Expressing Regulatory T Cells in Multiple Sclerosis Patients

Background

Multiple sclerosis (MS) likely results from an imbalance between regulatory and inflammatory immune processes. CD39 is an ectoenzyme that cleaves ATP to AMP and has been suggested as a novel regulatory T cells (Treg) marker. As ATP has numerous proinflammatory effects, its degradation by CD39 has anti-inflammatory influence. The purpose of this study was to explore regulatory and inflammatory mechanisms activated in fingolimod treated MS patients.

Methods and Findings

Peripheral blood mononuclear cells (PBMCs) were isolated from relapsing-remitting MS patients before starting fingolimod and three months after therapy start. mRNA expression was assessed in ex vivo PBMCs. The proportions of CD8, B cells, CD4 and CD39-expressing cells were analysed by flow cytometry. Treg proportion was quantified by flow cytometry and methylation-specific qPCR. Fingolimod treatment increased mRNA levels of CD39, AHR and CYP1B1 but decreased mRNA expression of IL-17, IL-22 and FOXP3 mRNA in PBMCs. B cells, CD4⁺ cells and Treg proportions were significantly reduced by this treatment, but remaining CD4⁺ T cells were enriched in FOXP3⁺ cells and in CD39-expressing Tregs.

Conclusions

In addition to the decrease in circulating CD4⁺ T cells and CD19⁺ B cells, our findings highlight additional immunoregulatory mechanisms induced by fingolimod.     

In Vitro Antimicrobial Activity and Human Pharmacology of Cephaloglycin

Serum and urine concentrations of cephaloglycin (an orally absorbed derivative of cephalosporin C) were determined in normal volunteers and in patients. The in vitro activity of cephaloglycin was also studied. All strains of group A streptococci (Streptococcus pyogenes) and Diplococcus pneumoniae were inhibited by 0.4 mug of cephaloglycin per ml. Eighty per cent of the Staphylococcus aureus strains and about 50% of the Escherichia coli and Proteus mirabilis strains were inhibited by 1.6 mug of cephaloglycin per ml. Klebsiella-Aerobacter species were more resistant to cephaloglycin and 12.5 mug per ml was required to inhibit 70% of these strains. When single doses of 250, 500, or 1,000 mg of cephaloglycin were administered to fasting volunteers, a peak serum concentration of at least 0.5 mug per ml was achieved. A full breakfast did not interfere with absorption of cephaloglycin. Probenecid enhanced both the peak serum concentration and the duration of antibiotic activity in the serum. Serum concentrations of cephaloglycin were even higher in patients who were receiving repeated doses. The peak serum concentrations of cephaloglycin in all volunteers and patients were adequate to inhibit all strains of group A streptococci and D. pneumoniae. Many of the peak serum concentrations were adequate to inhibit some strains of S. aureus, E. coli, and P. mirabilis. Urine levels of cephaloglycin were high enough in all volunteers and patients to inhibit more than 90% of the E. coli and P. mirabilis strains and over 70% of the strains of Klebsiella-Aerobacter.     

EDTA Inhibits In Vitro Increases in the GABA Content of Human CSF

Aliquots of pooled samples of lumbar CSF from patients with neurological disorders were subjected to storage at room temperature for different periods of time up to 4 days. The determination of gamma-aminobutyric acid (GABA) levels in CSF by a radioreceptor assay showed a progressive increase in GABA content with time, which could be counteracted by addition of EDTA (5 mM). Similar results were obtained with cisternal CSF collected from dogs. The in vitro increases in GABA levels of untreated CSF might relate to the action of a metal-enzyme which is inhibited by metal-trapping substances such as EDTA.     

Poor Sleep in Patients with Multiple Sclerosis

Background

Poor sleep is a frequent symptom in patients with multiple sclerosis (MS). Sleep may be influenced by MS-related symptoms and adverse effects from immunotherapy and symptomatic medications. We aimed to study the prevalence of poor sleep and the influence of socio-demographic and clinical factors on sleep quality in MS- patients.

Methods

A total of 90 MS patients and 108 sex-and age- matched controls were included in a questionnaire survey. Sleep complaints were evaluated by Pittsburgh Sleep Quality Index (PSQI) and a global PSQI score was used to separate good sleepers (≤5) from poor sleepers (>5). Excessive daytime sleepiness, the use of immunotherapy and antidepressant drugs, symptoms of pain, depression, fatigue and MS-specific health related quality of life were registered. Results were compared between patients and controls and between good and poor sleepers among MS patients.

Results

MS patients reported a higher mean global PSQI score than controls (8.6 vs. 6.3, p = 0.001), and 67.1% of the MS patients compared to 43.9% of the controls (p = 0.002) were poor sleepers. Pain (p = 0.02), fatigue (p = 0.001), depression (p = 0.01) and female gender (p = 0.04) were associated with sleep disturbance. Multivariate analyses showed that female gender (p = 0.02), use of immunotherapy (p = 005) and a high psychological burden of MS (p = 0.001) were associated with poor sleep among MS patients.

Conclusions

Poor sleep is common in patients with MS. Early identification and treatment of modifiable risk factors may improve sleep and quality of life in MS.     

Quantitation of the Myelin-Associated Glycoprotein in Human Nervous Tissue from Controls and Multiple Sclerosis Patients

Myelin-associated glycoprotein (MAG) was measured by radioimmunoassay in the human CNS and peripheral nervous system (PNS). The level of MAG, expressed as ng/microgram of total protein, was approximately 20-fold higher in whole homogenates of cerebral white matter (4.7 +/- 0.60) than of peripheral nerve (0.12-0.28). MAG concentrations were only slightly higher in the isolated myelin fractions from these tissues: CNS myelin, 5.6 ng/microgram; PNS myelin, 0.37 ng/microgram. The levels of MAG were measured in nine plaques, periplaque regions, and areas of macroscopically normal-appearing white matter (NAWM) from six separate multiple sclerosis brains and compared with the levels of other myelin proteins in the same samples. MAG and other myelin proteins were reduced to very low levels in plaques. The levels of MAG and basic protein (BP) and the activity of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in periplaque areas were significantly lower than those in control white matter, and MAG and BP levels were also significantly reduced in NAWM. In a periplaque region and NAWM from the most rapidly progressing case of multiple sclerosis examined, the MAG content was between 30 and 35% of the control level, whereas BP and PLP levels and CNP activity were between 50 and 85% of control values. The reduction of MAG content in periplaque regions from all nine multiple sclerosis plaques examined was significantly greater than the reductions of BP level and CNP activity. In NAWM samples, the mean reduction of MAG content was also greater than the reductions of BP level and CNP activity, but the difference was only statistically significant in comparison to CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Overhauser-Enhanced MRI of Elastase Activity from In Vitro Human Neutrophil Degranulation

Background

Magnetic resonance imaging can reveal exquisite anatomical details. However several diseases would benefit from an imaging technique able to specifically detect biochemical alterations. In this context protease activity imaging is one of the most promising areas of research.

Methodology/Principal Findings

We designed an elastase substrate by grafting stable nitroxide free radicals on soluble elastin. This substrate generates a high Overhauser magnetic resonance imaging (OMRI) contrast upon digestion by the target proteases through the modulation of its rotational correlation time. The sensitivity is sufficient to generate contrasted images of the degranulation of neutrophils induced by a calcium ionophore from 2×10⁴ cells per milliliter, well under the physiological neutrophils concentrations.

Conclusions/Significance

These ex-vivo experiments give evidence that OMRI is suitable for imaging elastase activity from neutrophil degranulation. Provided that a fast protease-substrate is used these results open the door to better diagnoses of a number of important pathologies (cystic fibrosis, inflammation, pancreatitis) by OMRI or Electron Paramagnetic Resonance Imaging in vivo. It also provides a long-expected method to monitor anti-protease treatments efficiency and help pharmaceutical research.     

Genetic and Infectious Profiles of Japanese Multiple Sclerosis Patients

Background

Nationwide surveys conducted in Japan over the past thirty years have revealed a four-fold increase in the estimated number of multiple sclerosis (MS) patients, a decrease in the age at onset, and successive increases in patients with conventional MS, which shows an involvement of multiple sites in the central nervous system, including the cerebrum and cerebellum. We aimed to clarify whether genetic and infectious backgrounds correlate to distinct disease phenotypes of MS in Japanese patients.

Methodology/Principal Findings

We analyzed HLA-DRB1 and -DPB1 alleles, and IgG antibodies specific for Helicobacter pylori, Chlamydia pneumoniae, varicella zoster virus, and Epstein-Barr virus nuclear antigen (EBNA) in 145 MS patients and 367 healthy controls (HCs). Frequencies of DRB1*0405 and DPB1*0301 were significantly higher, and DRB1*0901 and DPB1*0401 significantly lower, in MS patients as compared with HCs. MS patients with DRB1*0405 had a significantly earlier age of onset and lower Progression Index than patients without this allele. The proportion and absolute number of patients with DRB1*0405 successively increased with advancing year of birth. In MS patients without DRB1*0405, the frequency of the DRB1*1501 allele was significantly higher, while the DRB1*0901 allele was significantly lower, compared with HCs. Furthermore, DRB1*0405-negative MS patients were significantly more likely to be positive for EBNA antibodies compared with HCs.

Conclusions

Our study suggests that MS patients harboring DRB1*0405, a genetic risk factor for MS in the Japanese population, have a younger age at onset and a relatively benign disease course, while DRB1*0405-negative MS patients have features similar to Western-type MS in terms of association with Epstein-Barr virus infection and DRB1*1501. The recent increase of MS in young Japanese people may be caused, in part, by an increase in DRB1*0405-positive MS patients.     

Risk Acceptance in Multiple Sclerosis Patients on Natalizumab Treatment

Objective

We aimed to investigate the ability of natalizumab (NTZ)-treated patients to assume treatment-associated risks and the factors involved in such risk acceptance.

Methods

From a total of 185 patients, 114 patients on NTZ as of July 2011 carried out a comprehensive survey. We obtained disease severity perception scores, personality traits’ scores, and risk-acceptance scores (RAS) so that higher RAS indicated higher risk acceptance. We recorded JC virus status (JCV+/-), prior immunosuppression, NTZ treatment duration, and clinical characteristics. NTZ patients were split into subgroups (A-E), depending on their individual PML risk. Some 22 MS patients on first-line drugs (DMD) acted as controls.

Results

No differences between treatment groups were observed in disease severity perception and personality traits. RAS were higher in NTZ than in DMD patients (p<0.01). Perception of the own disease as a more severe condition tended to predict higher RAS (p=0.07). Higher neuroticism scores predicted higher RAS in the NTZ group as a whole (p=0.04), and in high PML-risk subgroups (A-B) (p=0.02). In low PML-risk subgroups (C-E), higher RAS were associated with a JCV+ status (p=0.01). Neither disability scores nor pre-treatment relapse rate predicted RAS in either group.

Conclusions

Risk acceptance is a multifactorial phenomenon, which might be partly explained by an adaptive process, in light of the higher risk acceptance amongst NTZ-treated patients and, especially, amongst those who are JCV seropositive but still have low PML risk, but which seems also intimately related to personality traits.     

Neural Correlates of Alerting and Orienting Impairment in Multiple Sclerosis Patients

Background

A considerable percentage of multiple sclerosis patients have attentional impairment, but understanding its neurophysiological basis remains a challenge. The Attention Network Test allows 3 attentional networks to be studied. Previous behavioural studies using this test have shown that the alerting network is impaired in multiple sclerosis. The aim of this study was to identify neurophysiological indexes of the attention impairment in relapsing-remitting multiple sclerosis patients using this test.

Results

After general slowing had been removed in patients group to isolate the effects of each condition, some behavioral differences between them were obtained. About Contingent Negative Variation, a statistically significant decrement were found in the amplitude for Central and Spatial Cue Conditions for patient group (p<0.05). ANOVAs showed for the patient group a significant latency delay for P1 and N1 components (p<0.05) and a decrease of P3 amplitude for congruent and incongruent stimuli (p<0.01). With regard to correlation analysis, PASAT-3s and SDMT showed significant correlations with behavioral measures of the Attention Network Test (p<0.01) and an ERP parameter (CNV amplitude).

Conclusions

Behavioral data are highly correlated with the neuropsychological scores and show that the alerting and orienting mechanisms in the patient group were impaired. Reduced amplitude for the Contingent Negative Variation in the patient group suggests that this component could be a physiological marker related to the alerting and orienting impairment in relapsing-remitting multiple sclerosis. P1 and N1 delayed latencies are evidence of the demyelination process that causes impairment in the first steps of the visual sensory processing. Lastly, P3 amplitude shows a general decrease for the pathological group probably indexing a more central impairment. These results suggest that the Attention Network Test give evidence of multiple levels of attention impairment, which could help in the assessment and treatment of relapsing-remitting multiple sclerosis patients.     

β-N-Acetylhexosaminidase in Peripheral Blood Lymphocytes and Monocytes in the Different Forms and Stages of Multiple Sclerosis

Abstract: The activity of the acidic glycohydrolase β- N -acetylhexosaminidase, an enzyme system normally participating in the stepwise degradation of glycoproteins, glycolipids, and proteoglycans, appears to be modulated in lymphocytes and monocytes from peripheral blood of patients affected by multiple sclerosis during different stages of the disease. In particular, a significant decrease in this enzyme activity, compared with healthy subjects, was observed in patients affected by the relapsing-remitting form both in a stable clinical status and during a relapse as well as in patients with the progressive form. The decrease in total intracellular hexosaminidase activity in lymphomonocytes of multiple sclerosis patients was accompanied by an enrichment of this activity associated with the plasma membrane fraction as demonstrated by experiments of subcellular fractionation. The analysis carried out using two synthetic substrates, 4-methylumbelliferyl N -acetyl-β- d -glucosaminide and its sulfate derivative, enables us to demonstrate that this accumulation is mainly due to isoenzymes with a ββ structure, whereas lysosomal fractions confirmed the classical presence of both αβ and ββ forms (hexosaminidases A and B, respectively). This was particularly evident in the plasma membrane fraction from mononuclear cells of patients with a clinical exacerbation of the disease. Considered together, these observations provide additional insight into the abnormality of peripheral blood immune cells in multiple sclerosis and may contribute to the understanding of the basic mechanisms underlying the pathological events resulting in the demyelinating process.     

Monocytes from Chronic HBV Patients React In Vitro to HBsAg and TLR by Producing Cytokines Irrespective of Stage of Disease

Individuals who are chronically infected with the hepatitis B virus (HBV) are highly heterogenous with respect to serum levels of HBV DNA, HBV particles and viral proteins. Since circulating leukocytes, such as monocytes, are constantly exposed to these viral components, it is likely that the functionality of these cells is affected. However, at present, little information is available on the consequences of the interaction between monocytes and viral components. Therefore, we examined the in vitro effects of HBV surface antigen (HBsAg) on monocytes and evaluated whether these effects were reflected in vivo. We observed that in vitro HBsAg exposure of monocytes induced robust production of IL-6 and TNF. However, between chronic HBV patients with distinct levels of serum HBsAg, HBV early antigen (HBeAg), and HBV DNA, TLR-induced monocyte cytokine production did not differ. Importantly, HBsAg-induced cytokine production by monocytes was similar between patients and healthy controls showing that earlier in vivo exposure to HBsAg does not affect the in vitro response. Additionally, we show that IL-10 is able to inhibit cytokine production by HBsAg-induced monocytes. In conclusion, we demonstrate that monocytes can recognize and respond to HBsAg, resulting in vigorous pro-inflammatory cytokine production in vitro. However, phenotype and function of the monocyte compartment in chronic HBV patients are not influenced by differences in levels of serum viral components, suggesting that regulatory mechanisms are active to avoid excessive in vivo monocyte activation.     

Serum Arsenic and Lipid Peroxidation Levels in Patients with Multiple Sclerosis

Oxidative stress plays a crucial role in the pathogenesis of multiple sclerosis (MS). Previous studies have shown that oxidative stress is one of the main underlying mechanisms of arsenic-induced cellular damage. The aim of this study was to assess the serum levels of arsenic and its relationship with lipid peroxidation in MS patients from Tabriz, as the third polluted city of Iran. The study population included 38 MS female patients and 38 age-matched healthy controls. Serum malondialdehyde (MDA) and arsenic levels were measured using thiobarbituric acid reactive substances (TBARS) assay and electrothermal atomic absorption spectrometry, respectively. The results showed that the arsenic (P?<?0.01) and MDA (P?=?0.03) levels were significantly higher in patients with MS than those in control. Moreover, serum levels of arsenic and MDA were positively correlated in MS patients. The elevated levels of serum arsenic might explain the increased oxidative stress in MS patients. We suggest that high arsenic levels in serum may lead to MS development, and therefore, exposure to this metal should be limited.     

Work Participation and Executive Abilities in Patients with Relapsing-Remitting Multiple Sclerosis

The majority of patients with Multiple Sclerosis (MS) are unable to retain employment within 10 years from disease onset. Executive abilities, such as planning, working memory, attention, problem solving, inhibition and mental flexibility may have a direct impact on the ability to maintain a job. This study investigated differences in subjective and objective executive abilities between relapsing-remitting MS patients with and without a paid job. We included 55 relapsing-remitting MS patients from a community-based sample (47 females; mean age: 47 years; 36% employed). Patients underwent neurological, cognitive and psychological assessments at their homes, including an extensive executive test battery. We found that unemployed patients had a longer disease duration (t(53)=2.76, p=0.008) and reported more organising and planning problems (χ²(1)=6.3, p=0.012), higher distractibility (Kendall’s tau-b= -0.24, p=0.03) and more cognitive fatigue (U=205.0, p=0.028, r=-0.30) than employed patients. Unemployed patients completed slightly less categories on the Wisconsin Card Sorting Test (U=243.5, p=0.042, r=-0.28). Possible influential factors such as age, educational level, physical functioning, depression and anxiety did not differ between groups. In conclusion, while relapsing-remitting MS patients without a paid job reported more executive problems and cognitive fatigue than patients with a paid job, little differences were found in objective executive abilities. Further research is needed to examine possible causal relations.