The development of clinical activity in relapsing-remitting MS is associated with a decrease of FasL mRNA and an increase of Fas mRNA in peripheral blood

In this longitudinal study, we examined the expression of Fas, FasL, CCR3, CCR5 and CXCR3 mRNA in peripheral blood mononuclear cells (PBMCs) of secondary progressive (SP) and relapsing-remitting (RR) multiple sclerosis (MS) patients. In RR patients, FasL, CCR3 and CCR5 mRNA levels were increased prior to the exacerbations, but these decreased during clinical activity, while mRNA levels of Fas increased. SP patients have increased the levels of Fas and FasL mRNA; the latter was particularly increased during lesional activity.Our data support the hypothesis that changes in Fas and FasL mRNA related to clinical activity are due to the migration of inflammatory cells to the central nervous system (CNS).     

Decreased interleukin-10 and increased interleukin-12p40 mRNA are associated with disease activity and characterize different disease stages in multiple sclerosis.

It has been shown that proinflammatory and antiinflammatory cytokines correlate with disease activity in multiple sclerosis (MS). To establish whether such correlations depend on the disease stage, we assessed in a longitudinal fashion the expression of interleukin (IL)-12 (p40 and p35), tumor necrosis factor-alpha, interferon-gamma, and IL-10 mRNA by competitive polymerase chain reaction in unstimulated peripheral blood mononuclear cells of relapsing-remitting (RR) and secondary progressive (SP) MS patients, in relation to monthly clinical and magnetic resonance imaging monitoring. MS patients had increased levels of IL-12p40 and decreased levels of IL-10 mRNA compared with controls; this difference was most pronounced in SP patients. Both RR and SP patients had increased levels of IL-12p40 mRNA compared with controls during the development of active lesions. Moreover, in RR MS an increase was found before relapse. IL-12p35 mRNA was decreased in both groups, and in relation to disease activity it showed a pattern different from IL-12p40 mRNA. In RR MS, IL-10 mRNA was low 4 weeks before magnetic resonance imaging activity and 6 weeks before relapse; a significant increase to normal levels was noted when active lesions became apparent. In contrast, SP patients showed low IL-10 mRNA levels constitutively, suggesting that IL-10 plays an important role in the control of disease progression.     

Deficient Fas expression by CD4+ CCR5+ T cells in multiple sclerosis

OBJECTIVE: To evaluate whether T cells expressing CCR5 and CXCR3 from multiple sclerosis (MS) patients are more resistant to apoptosis. METHODS: Expression of CD69, TNF-R1, Fas, FasL, bcl-2, and bax was investigated in 41 MS patients and 12 healthy controls by flow cytometry in CD4+ and CD8+ T cells expressing CCR5 and CXCR3. RESULTS: In MS patients, the percentage of CD69 was increased and Fas expression decreased in CD4+ CCR5+ T cells. INTERPRETATION: The lower Fas expression in activated CD4+ CCR5+ T cells might contribute to disease pathogenesis by prolonging cell survival and favoring their migration into the CNS.     

Increased expression of caspase-1 and interleukin-18 in peripheral blood mononuclear cells in patients with multiple sclerosis

Multiple sclerosis (MS) is supposedly a T-cell mediated autoimmune disorder of the central nervous system. Cytokines and other molecules involved in the regulation of apoptosis are thought to be of importance for the pathogenesis of MS. In this study, the mRNA levels of interleukin 18 (IL-18), IL-1beta and their processing enzyme caspase-1 were quantified by a competitive RT-PCR method in unstimulated peripheral blood mononuclear cells (PBMCs) in MS patients never treated with disease modifying drugs. Western blot was used to support the expression pattern at the protein level. We found that the expression of caspase-1 and IL-18 was significantly increased in MS patients compared with healthy controls. Analysis of clinical subgroups revealed that caspase-1 was increased in all subgroups, whereas IL-18 was upregulated in chronic progression (P=0.001) and relapsing MS patients in remission (P=0.002) but not significantly during relapses (P=0.12). mRNA levels of IL-1beta were not significantly altered in MS except for a possible decrease in chronic progression (P=0.03). An increased IL-18 expression, potentially augmented at the mature protein level, may indicate a pathway worth considering in future therapeutic strategies in MS.     

Notable increased cerebrospinal fluid levels of soluble interleukin-6 receptors in neuromyelitis optica

Background: IL-6 is a proin?ammatory cytokine which is involved in the maintenance of the humoral response in various autoimmune disorders. Cerebrospinal fluid (CSF) IL-6 has shown to be increased in neuromyelitis optica (NMO). The soluble form of IL-6 receptor (sIL-6R), which links to IL-6, can activate biological responses in cells. Whether or not sIL-6R is altered in NMO has not been clarified. Objective: To measure CSF IL-6 and sIL-6R in NMO and multiple sclerosis (MS) patients, and investigate whether IL-6 and sIL-6R have possible uses as sensitive biomarkers for diseases activity. Methods: CSF concentrations of IL-6 and sIL-6R were measured by an ELISA in NMO (n = 22) and MS (n = 18) patients, as well as control subjects (n = 14). Results: The concentration of IL-6 levels were higher in NMO compared to MS (p = 0.032) and the controls (p = 0.023). The levels of sIL-6R were also higher in NMO compared to MS (p = 0.002) and the controls (p < 0.001). CSF sIL-6R was associated with an Expanded Disability Status Scale score in NMO (p = 0.005) but not in MS (p = 0.891). In the MS subgroup, sIL-6R concentrations were associated with CSF white blood cells (p = 0.034). Conclusions: Our study revealed that CSF sIL-6R was increased in NMO patients, and correlated with clinical presentations.     

Correlation between DJ-1 levels in the cerebrospinal fluid and the progression of disabilities in multiple sclerosis patients

ObjectivesDJ-1 plays a key role in the anti-oxidative stress function. Increasing evidence supports the role of oxidative stress in the pathogenesis of multiple sclerosis (MS). The aim of this study was to investigate whether the DJ-1 levels were increased in patients with MS and to examine its association with the progression of MS.MethodsQuantitative immunoblot assays were performed to evaluate the DJ-1 level in serum and cerebrospinal fluid (CSF) collected from relapsing-remitting patients with MS (n = 29), disease controls subjects (n = 14), and healthy subjects (n = 44).ResultsNo significant difference was observed in the serum DJ-1 level among the patients with MS, disease controls, and healthy controls. However, the CSF DJ-1 levels were significantly higher in the patients with MS than in the disease control subjects (P < 0.0001). A significant positive correlation was also found between the CSF DJ-1 levels and the Multiple Sclerosis Severity Score (P < 0.005, r = 0.501).ConclusionsThese results show that the CSF DJ-1 levels are significantly increased in the CSF of patients with MS and that the CSF DJ-1 levels may be associated with the disease progression of MS. Therefore, DJ-1 possibly plays an important role in the pathogenesis of MS.     

Immunological patterns identifying disease course and evolution in multiple sclerosis patients

Reliable, and easy to measure, immunological markers able to denote disease characteristics in multiple sclerosis (MS) patients are still lacking. We applied a multivariate statistical analysis on results obtained by measuring-by real-time RT-PCR-mRNA levels of 25 immunological relevant molecules in PBMCs from 198 MS patients. The combined measurement of mRNA levels of IL-1beta, TNF-alpha, TGF-beta, CCL20 and CCR3 was able to distinguish MS patients from healthy individuals. CXCR5, CCL5, and CCR3 combined mRNA levels identify primary progressive MS patients while TNF-alpha, IL-10, CXCL10 and CCR3 differentiate relapsing MS patients. Our results indicate that multi-parametric analysis of mRNA levels of immunological relevant molecules in PBMCs may represent a successful strategy for the identification of putative peripheral markers of disease state and disease activity in MS patients.     

Monocytes P2X7 purinergic receptor is modulated by glatiramer acetate in multiple sclerosis

The aim of this study is to investigate the expression of P2X7R, IL-1beta and the ATP activity modulating ecto-apyrase CD39 on peripheral blood monocytes of MS patients and to observe the possible effects of Glatiramer Acetate (GA) on such expression. Twelve RR treatment-free MS patients were selected and peripheral blood monocytes were obtained. The expression of P2X7R, IL-1beta and CD39 on monocytes was investigated by qrt-PCR. The in vitro effects of GA on the expression of monocytes stimulated with BzATP (a potent P2X7R agonist)-were evaluated. Ten healthy donors (HDs) were similarly studied. Finally, 5 MS patients were given GA therapy and the monocytes obtained before treatment, after 3 and 12 months of GA treatment were similarly investigated. No differences were found in P2X7R, IL-1beta and CD39 expression between patients and controls. In MS Bz-ATP stimulated monocytes, GA pre-conditioning clearly downregulated P2X7R (p=0.003) but IL-1beta expression also showed a decreasing trend (p=0.07). Conversely, CD39 showed an increasing trend (p=0.07). Similar evidence was found in HDs. GA in vivo treatment induced a reduction in the expression that was clear for P2X7R and CD39 (p<0.05) but only not significant for IL-1beta after 12 months of treatment. Monocytes from both MS and control subjects express P2X7R, IL-1beta and CD39, and GA seems to interfere with such expression.     

Modulation of sFas indicates apoptosis in human herpes simplex encephalitis

Herpes simplex encephalitis (HSE) is the most common cause of non-epidemic, acute and fatal viral encephalitis. A pronounced mortality and morbidity remains in HSE despite antiviral treatment. There is evidence of a vigorous intrathecal immune activity in acute phases of HSE and of persistently increased activity at follow-ups after years. The role of apoptosis of neuronal cells in HSE patients as a mechanism of damage has been brought up lately. We hypothesize that the severity and the progression of the cerebral injury resulting from HSE can be evaluated by quantitative measurement of a compartment of immune activation molecules i.e. soluble Fas (sFas) involved in apoptosis through the Fas/Fas Ligand pathway. Consecutive cerebrospinal fluid (CSF) samples from a prospectively followed cohort, included in an antiviral treatment trial in HSE, were enrolled for quantitative measurement of sFas using commercial capture ELISA. In total, CSF samples from 49 patients with HSE, 63 patients with non-HSE encephalitis and 18 healthy individuals were studied. High levels of sFas were expressed in CSF samples collected between days 0-45 after neurological onset in 41/49 (84%) HSE patients, whereas only 21/63 (33%) of non-HSE patients and none of 18 healthy controls demonstrated measurable levels of sFas. Following the consecutive CSF sFas levels over the time and considering the clinical state of patients at admission, their neurological or lethal outcome at 12 months, and antiviral treatment, we observed that HSE patients with severe neurological sequels revealed an increase in changes of CSF sFas as compared to patients with mild or moderate neurological outcome (57.6+/-55.6 pg/ml, n=10 versus 26.3+/-97.5 pg/ml, n=14; P=0.008). Also HSE patients undergoing vidarabine treatment expressed significantly higher levels of changes of CSF sFas when compared to acyclovir-treated patients (63.7+/-52.8 pg/ml, n=9 versus 26.1+/-98.4 pg/ml, n=14; P=0.003). Interestingly, regardless of the clinical state at admission, and subsequent disease progression of the HSE patients, we could not observe any significant differences in the CSF sFas levels during the first 7 days of neurological symptoms. These observations underline the role of immunological response throughout the course of HSV infection in the brain and the role of the Fas/FasL pathway in particular in disease progression of HSE. The findings further enforce the need of expanding the knowledge of the pathogenesis of HSE to direct to more effective, in particular not only antiviral but also anti-apoptotic or anti-inflammatory treatment.     

Fas及FasL和Caspase-3在青蒿琥酯诱导人胚肺成纤维细胞凋亡中的表达*☆

背景: 青蒿琥酯具有减轻肺纤维化的作用，但相关机制的研究罕见报道。 目的：探讨青蒿琥酯对人胚肺成纤维细胞凋亡的作用及其与Fas，FasL，Caspase-3表达的关系。 方法：用1，10，100 mg/L青蒿琥酯分别干预体外培养的人胚肺成纤维细胞。采用CCK-8法检测青蒿琥酯对人胚肺成纤维细胞增殖的影响，流式细胞术测定细胞凋亡率，RT-PCR法测定Fas，FasL，Caspase-3 的mRNA的表达。 结果与结论：青蒿琥酯呈浓度依赖性抑制人胚肺成纤维细胞增殖，细胞经青蒿琥酯作用后凋亡率明显增加(P < 0.05或P < 0.01)，Fas，FasL，Caspase-3 mRNA的表达显著高于对照组(P < 0.05)。结果证实，青蒿琥酯可通过上调Fas，FasL，Caspase-3 mRNA的表达抑制人胚肺成纤维细胞增殖、并促进细胞凋亡，发挥抗肺纤维化作用。     

The clinical use of P300 event related potentials for the evaluation of cholinesterase inhibitors treatment in demented patients

Summary. Centrally acting cholinesterase inhibitors (ChEIs) improve cognitive functions in Alzheimer's disease (AD) and other forms of dementia. Evaluation of treatment efficacy is based mainly on subjective assessment methods such as standardized neuropsychological tests. Therefore, an additional objective tool for the evaluation of drug response would be most helpful. Thirty-two patients suffering from dementia of several etiologies were treated with ChEIs (tacrine 19, donepezil 5, rivastigmine 8). Cognitive response was assessed pre ChEIs initiation (baseline) and after 26 weeks, as optimal tolerated doses were achieved and maintained (endpoint). Evaluation included repeated measurements of Mini Mental State Examination (MMSE), Alzheimer's disease assessment scale cognitive part (ADAS-cog) and P300. For statistical analysis we used ANOVA with repeated measures and Pearson correlation coefficient. Results demonstrated improvement of mean ADAS-cog by 2.0 points (from 29.4, n = 31 to 27.4, n = 29; p = 0.08) while MMSE remained almost unchanged (20.1, n = 29 to 19.8, n = 28). Mean P300 latency reduced significantly by 24 ms (from 383 ± 7.9 msec, n = 32 to 359 ± 7 msec, n = 32; p = 0.0001). However mean amplitudes did not change significantly from baseline to endpoint (13.5 ± 6.2, n = 31 to 12.8 ± 6.1, n = 31). Significant correlations were found between mean ADAS-cog and mean P300 latency at baseline and end-point (R = 0.485 p = 0.019, R = 0.626 p = 0.001 respectively, n = 23) and between mean MMSE and P300 latency at baseline and endpoint (R = −0.420 p = 0.046, R = −0.703 p < 0.001 respectively, n = 23). Our data suggests that P300 is a reliable instrument for assessment of cognitive response to ChEIs in demented patients. Received March 18, 2002; accepted January 27, 2003 Published online April 7, 2003 Authors' address: Dr. E. A. Werber, Department of Neurology, Assaf Harofeh Medical Center, Zerifin, Israel 70300, e-mail: werbered@bezeqint.net     

Influence of chemical and ischemic preconditioning on cytokine expression after focal brain ischemia

Inflammation, upregulation of cytokines, proapoptotic molecules, and apoptosis are accepted widely as crucial players in stroke-induced brain damage. Induction of brain tolerance against ischemia by pretreatment with nonlethal stressors (preconditioning) has been found to influence expression of different molecules, in addition to reduction of infarct size. It remains unclear, however, whether and how preconditioning changes expression of cytokines after subsequent brain ischemia. We sought to analyze cortical expression of interleukin (IL)-1beta, IL-6, tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta, Fas, and Fas ligand (FasL) mRNA after a transient, focal brain ischemia in rats subjected to preconditioning. The mRNA levels were determined using a semiquantitative RT-PCR in the ischemic and contralateral cortex, separately. Transient ischemia was induced by 90-min middle cerebral artery occlusion (MCAo) and neurologic deficits as well as infarct size were quantified. Preconditioning was carried out by a short-term MCAo or an injection of 3-nitropropionic acid 3 days before MCAo. In both preconditioning paradigms, similar effects on investigated mRNA levels were observed. IL-1beta and IL-6 levels were decreased in tolerant rats compared to those in nontolerant ones. Changes in TNF-alpha, TGF-beta, and Fas levels were comparable independently of tolerance state. FasL mRNA was at similar level in rats subjected to chemical preconditioning but lower after ischemic preconditioning. Our findings demonstrate that both preconditioning methods exert a very similar effect on the expression of investigated cytokines. Interestingly, we observed a selective effect of preconditioning on IL-1beta and IL-6 expression that suggests different functional properties as well as different regulation of analyzed molecules during an induction of the brain tolerance against ischemia.     

Differential expression and regulation of IL-23 and IL-12 subunits and receptors in adult mouse microglia

IL-23 and IL-12 are functionally related heterodimeric cytokines that share the IL-12p40 subunit. IL-23 and IL-12 function through heterodimeric receptors, which share the IL-12Rbeta1 subunit. Production of IL-23, a heterodimer of IL-12p40 and IL-23p19, by CNS antigen-presenting cells (APC) is critical for susceptibility to experimental autoimmune encephalomyelitis (EAE), the animal model for multiple sclerosis (MS). We report that the expression of IL-23p19 mRNA is highly induced by stimulation with IFN-gamma and LPS in adult mouse microglia and a microglia cell line, EOC13. Expression of the IL-12R subunits, IL-12Rbeta1 and IL-12Rbeta2, is upregulated in both microglia and splenic macrophages upon stimulation with LPS or IFN-gamma and LPS, whereas the IL-23R subunit is upregulated only in macrophages. In EAE, an early peak of IL-23p19 mRNA expression is found in CD11b(+) CNS APC, compared with peripheral macrophages. In contrast, IL-12p40 and IL-12p35 mRNA maximum levels in the CNS are detected at peak of disease. The expression of IL-12p35 mRNA is more sustained than that of IL-12p40 and IL-23p19. Thus, IL-23 produced by CNS microglia/macrophages may contribute to the early induction of EAE. In the CNS, IL-23 may preferentially target infiltrating mononuclear cells, which upregulate IL-23R, rather than parenchymal microglia.     

Multiple sclerosis and depression: influence of interferon beta therapy

BACKGROUND AND OBJECTIVES: Depression is frequently part of the clinical picture of multiple sclerosis (MS). Major depression affects one in two patients with MS during the course of their lifetime. Our objectives were to determine first, whether interferon beta-1a (IFNbeta-1a) treatment increases the risk or level of depression and, secondly, whether depression status and depression evolution are related to the clinical characteristics of the disease. PATIENTS AND METHODS: We investigated 106 consecutive patients with relapsing remitting MS treated with IFNbeta-1a (Avonex). Patients with evidence of severe depression were excluded. The depression status, scored on the Beck Depression Inventory (BDI-II) (stratified as minimum, mild, moderate or severe level), and disability, scored on the Expanded Disability Status Scale (EDSS), were evaluated before and after 12 months of IFNbeta-1a treatment. RESULTS: At baseline, 85% of patients had a minimum or a mild depression status and after 12 months of treatment most of them (83%) retained their baseline status. Beck scores before and after treatment were not significantly different (P = 0.63). There was no correlation between age, gender, duration of illness or EDSS score and Beck score at baseline (P = 0.696). Patients with disability progression after one year of IFNbeta-1a treatment had a significantly higher Beck score at baseline than patients without disability progression (P = 0.003). CONCLUSION: IFNbeta-1a (Avonex) does not seem to significantly influence the depression status of MS patients even in those with disability progression.     

HIV-1 and IL-1 beta regulate Fas ligand expression in human astrocytes through the NF-kappa B pathway

Reactive astrogliosis is a prominent pathological feature of HIV-1-associated dementia (HAD). We hypothesized that in HAD, astrocytes activated with proinflammatory stimuli such as IL-1beta express Fas ligand (FasL), a death protein. IL-1beta and HIV-1-activated astrocytes expressed FasL mRNA and protein. Luciferase reporter constructs showed that IL-1beta and HIV-1 upregulated FasL promoter activity (p<0.001). The NF-kappaB pathway was involved as shown by inhibition with SN50 and dominant negative IkappaBalpha mutants. Brain extracts from HAD patients had significantly elevated FasL levels compared to HIV-seropositive (p<0.001) and seronegative individuals (p<0.01). We propose that astrocyte expression of FasL may participate in neuronal injury in HAD.     

Increase of CD8+ T-effector memory cells in peripheral blood of patients with relapsing-remitting multiple sclerosis compared to healthy controls

We investigated serum (IL-10 and IL-12p70) and cellular cytokine levels (IL-10, IL-12p40, IL-12p70, IFN-gamma) in stimulated PBMC over 24 weeks in 15 relapsing-remitting multiple sclerosis (MS) patients randomized to receive once-weekly (qw) IFN-beta-1a 30 microg intramuscularly (IM) (n=8) or three-times-weekly (tiw) IFN-beta-1a 44 microg subcutaneously (SC) (n=7). Overall, IFN-beta treatment increased cellular IL-10 (p<0.01) levels and the ratios of cellular IL-10/IL-12p40 (p<0.01) and IL-10/IL-12p70 (p<0.02) while cellular IFN-gamma levels were reduced (p<0.01). Serum IL-10 levels were decreased in non-responders to therapy based on MRI-defined criteria (p<0.01) but did not change in responders over the course of treatment. In addition, non-responders demonstrated a decrease in serum IL-10/IL-12p70 ratio (p=0.031) and a decrease in cellular IL-12p70 (p<0.02). A decrease in cellular IFN-gamma was observed in responders (p=0.013). This is the first study that compares cytokine changes between the two IFN-beta regimes and demonstrates that serum IL-10 levels decrease in those patients who continue to have active MRI lesions while on interferon-beta therapy.