Time-dependent cytokine deviation toward the Th2 side in Japanese multiple sclerosis patients with interferon beta-1b

To address the immune mechanism sustaining interferon beta (IFNbeta) efficacy in multiple sclerosis (MS), we longitudinally analyzed expressions of IFN-gamma, IL-4, IL-5 and IL-13 in CD4+ T cells and CD8+ T cells in 22 Japanese MS patients (16 patients with conventional MS and 6 with opticospinal MS) undergoing IFNbeta using flow cytometry. During the 48-week observation period, five opticospinal MS patients (83%) relapsed compared to only four conventional MS patients (25%); the frequency of relapsed patients was significantly higher in the former (p=0.046). The effects of IFNbeta on individual cytokines were time-dependent and altered cytokine productions were particularly evident in CD4+ rather than CD8+ T cells. A decreased intracellular IFN-gamma/IL-4 ratio in CD4+ T cells was thus evident soon after the initiation of therapy, and persisted for the entire 1 year follow-up period, regardless of whether or not the patient relapsed (p<0.01). IFNbeta treatment resulted in a rapid increase in the percentage of IFN-gamma- IL-4+ and IL-13+ CD4+ T cells 1 week after the initiation of therapy and high values were sustained for 6 months but declined to the baseline over 1 year. Later, the percentage of IFN-gamma+ IL-4- CD4+ T cells decreased significantly from weeks 24 through 48 of therapy (p<0.01). When comparisons with the pretreatment values were made for each subtype of MS, a significant reduction of IFN-gamma+ IL-4- CD4+ T cell percentages was shown in conventional MS (p<0.0001), but not in opticospinal MS. Moreover, when such a comparison was made by the presence or absence of relapse during therapy, a significant reduction of IFN-gamma+ IL-4- CD4+ T cell percentages was observed in MS patients without relapse (p<0.01). Thus, a reduction of IFN-gamma+ IL-4- CD4+ T cell percentages in the late phase of therapy is considered important for reducing relapse in conventional MS. When the expression patterns of IFN-gamma, IL-4, IL-5 and IL-13 in CD4+ T cells and CD8+ T cells were compared between patients with and without relapse during therapy, the only significant difference was an increase in the IL-13+ CD4+ T cell percentages in patients with relapse compared to those without (p<0.05). The results indicate that in CD4+ T cells IL-4 was preferentially up-regulated in the early course and IFN-gamma was down-regulated in the late phase of IFNbeta therapy. The net effect of IFNbeta on the immune balance was entirely toward type 2 immune deviation, possibly contributing to its beneficial effects on MS.     

Leukocyte adhesion molecule expression and T cell naïve/memory status following isoproterenol infusion

This study examined adhesion molecules on peripheral leukocytes following a 30-min infusion of the beta-adrenergic agonist isoproterenol in 23 healthy subjects. In response to isoproterenol, the number of CD8 +CD62L- T cells and both CD62L+ and CD62L-natural killer (NK) (CD3 CD16+ 56+) cells increased markedly in circulation (p < 0.001). In addition, the surface density of CD62L was significantly lower on both CD8+ and CD4+ T cells (p < 0.001). Plasma levels of soluble CD62L remained unchanged, arguing against an isoproterenol-induced shedding of L-selectin. In contrast to CD62L, the surface density of the beta2 integrin LFA-1 (CD11a) was higher on circulating lymphocytes (p < 0.001) (but not monocytes or lymphocytes) post-infusion. Isoproterenol also led to a mobilization of memory/activated CD8+CD29high T cells (p < 0.01), but had no significant effect on the number of circulating CD8+ CD45RA+ CD62L+ na?ve T cells. beta blockade with the non-specific antagonist propranolol eliminated these isoproterenol-induced effects.     

Phenotyping analysis of peripheral blood leukocytes in patients with multiple sclerosis

Multiple sclerosis (MS) is a central nervous disease thought to be elicited by an autoimmune process. Many studies in recent years have concentrated on finding the alterations in the peripheral blood immune profile in MS patients that would reflect disease activity. In the present study, we investigated surface antigen expression on lymphocytes and granulocytes from MS patients and control subjects. We have studied 29 patients suffering from relapsing-remitting or relapsing-progressive forms of MS. The disease was diagnosed in all patients at least 12 months before inclusion into the study. All patients had no attack at the study entry date or within a previous month. The control group included 29 age-matched subjects. Phenotyping of peripheral blood leukocytes was carried out with different fluorescence-conjugated murine monoclonal antibodies. The analysis was performed with three-color flow cytometry. The following antigens were determined [cluster of definition (CD)]: leukocyte common antigen (LCA) (B220, T 200, Ly-5), CD45; LPS-R (lipopolysaccharide receptor), CD14; found on all T cells, CD3; LFA-2 (lymphocyte function associated antigen, T 11), CD2; coreceptor for MHC class II molecules, found on helper T cells, CD4; coreceptor for MHC class I molecules, found on suppressor/cytotoxic T cells, CD8; B4, found on all human B cells, CD19; NCAM (neural cell adhesion molecule), CD56; integrin beta2 subunit, associated with CD11a (CD11a/CD18, LFA-1, alphaLbeta2) and CD11b (CD11b/CD18, Mac-1,CR3, alphaMbeta2), CD18; alphaL, alpha subunit of integrin LFA-1 (alphaLbeta2, CD11a/CD18), CD11a; alphaM, alpha subunit of integrin Mac-1 (CR3, alphaMbeta2, CD11b/CD18), CD11b; ICAM-1 (intercellular adhesion molecule), CD54; H-CAM, Hermes antigen, Pgp-1, CD44; AIM (activation inducer molecule), early activation antigen, CD69; T-cell receptor gammadelta, TCR gammadelta. In the MS group, we have found a significant increased expression of CD54 and CD44 antigens on lymphocytes, and higher percentage CD54(+) and CD11a+CD54(+) lymphocytes out of all lymphocytes compared with the control group. We have also found a significant increased expression of CD11a, CD18 and CD54 antigens on granulocytes, and higher percentage CD11b+CD18(+) granulocytes out of all granulocytes in MS patients compared with control. Higher levels of expression of the adhesion molecules may reflect the activation state of leukocytes in MS patients.     

A role for alpha4-integrin in the pathology following Semliki Forest virus infection

Migration of cells into the central nervous system (CNS) is a pivotal step in the pathogenesis of immune-mediated diseases such as multiple sclerosis (MS), experimental allergic encephalomyelitis (EAE) and virus-induced demyelinating diseases. Such migration is dependent on expression of adhesion molecules. The expression of adhesion molecules in the CNS was studied in Biozzi ABH mice infected with Semliki Forest virus (SFV) A7(74) - an important demyelinating model of MS. Expression of LFA-1alpha/CD11a, LFA-1beta/CD18 and ICAM-1/CD56 were rapidly elevated and remained high whereas MAC-1, CD44 and VCAM-1/CD106 were less widely expressed. The alpha4-integrin VLA-4/CD49d was more specifically associated with CNS lesions. To identify the importance of VLA-4, CD44, ICAM-1 and MAC-1 in the pathogenesis of SFV infection, monoclonal antibodies that block these adhesion molecules were administered in vivo during infection. Anti-VLA-4 treatment dramatically reduced the cellular infiltrates and demyelination within the CNS but did not affect the clearance of virus while antibodies to CD44, ICAM and MAC-1 antibody treatment had no effect. This study demonstrates that SFV infection induces the expression of adhesion molecules within the CNS and that VLA-4 plays an important role in the development of inflammation and demyelination in the CNS following SFV infection.     

Expansion of regulatory CD8+ T-lymphocytes and fall of activated CD8+ T-lymphocytes after i.v. methyl-prednisolone for multiple sclerosis relapse

INTRODUCTION: Multiple sclerosis (MS) is a multifocal chronic inflammatory demyelinating disease of the central nervous system. Axonal damage correlates with the presence of macrophages and CD8+ T-lymphocytes at brain lesions. The gold standard of therapy at MS relapse are iv glucocorticoids (GC). The aim of the study was to assess the changes on the different subsets of circulating CD8+ T-lymphocytes at relapse and after iv GC therapy. PATIENTS AND METHODS: We consecutively studied 20 patients at MS relapse before and at day 5 after initiation of i.v. methyl-prednisolone (MP) therapy (1 g/day for 3-5 days). CD4+ and CD8+ T-lymphocytes subsets were studied by multiparametric flow-cytometry. As control group, 18 healthy subjects were studied. RESULTS: Treatment with i.v. MP suppressed activated (CD8+CD38+HLA-DR+, p=0.05) and effector memory (CD8+CD27-CD45RO+) T-lymphocytes (p=0.07). By contrast, an increase of na?ve (CD8+CD27+CD45RO-) (p=0.07) and regulatory CD8+CD25+ T-lymphocytes was observed (p<0.002). At MS relapse, there was an inverse correlation between regulatory CD8+CD25+CD28- T-lymphocytes and activated CD4+ (r = -0.6; p=0.012) and CD8+ (r = -0.66; p=0.004) T-lymphocytes. After i.v. MP treatment, positive correlation between regulatory CD4+CD25+high T-lymphocytes and CD8+CD25+ T-lymphocytes was observed (r=0.74; p<0.0001). CONCLUSIONS: Our data suggest that i.v. MP may contribute to changes observed on the differentiation of CD8+ T-lymphocytes, namely blocking their complete maturation, and expansion of regulatory CD8+ T-lymphocytes. We hypothesize an additional effect of i.v. MP in inhibiting axonal damage which may add a neuroprotective effect on MS relapse.     

Temporal profile of expression and cellular localization of inducible nitric oxide synthase, interleukin-1beta and interleukin converting enzyme after cryogenic lesion of the rat parietal cortex.

We used in situ hybridization, RT PCR and immunohistochemistry to study the time course of expression and the cellular localization of inducible nitric oxide synthase (iNOS) and interleukin-1beta (IL-1beta) during the first 7 days after induction of a standardized cryogenic lesion on the right parietal cortex in male rats. Cryogenic lesion induced iNOS mRNA in the lesioned hemisphere after 6 to 72 h with a maximum (15+/-2 cells/mm2, n=4, p<0.01 vs. sham) at 24 h. Microglia, invading monocytes and granulocytes in and around the lesion expressed iNOS immunoreactivity starting at 12 h and peaking (29+/-10 cells/mm2, n=4, p<0.05 vs. sham) at 24 h after lesion. Induction of IL-1beta mRNA expression was immediate with a peak (9+/-1 cells/mm2, n=4, p<0.01 vs. sham) at 24 h after cryogenic lesion. The number of round cells with IL-1beta immunoreactivity around the lesion was maximal (8+/-2 cells/0.1 mm2, n=3, p<0.01 vs. sham) at 24 h. A weak astrocytic expression of IL-1beta-immunoreactivity was seen in sham animal brains. Astrocytic IL-1beta-expression was significantly increased in the lesion hemisphere and both hippocampi. Interleukin converting enzyme (ICE) was expressed in astrocytes and microglia around the lesion 6 h after injury. The number of ICE immunoreactive cells (8+/-2 cells/0. 1 mm2, n=3, p<0.05 vs. sham) peaked at 72 h after lesion. Neuronal expression of ICE and IL-1beta was seen in the lesion periphery 72 h and 7 days after injury. At this time, morphological features of apoptosis were evident in cells in the lesion periphery. The data indicate an early activation of microglia and monocyte invasion into the lesion hemisphere leading to multicellular expression of iNOS, ICE, and IL-1beta. These events may contribute to the expansion of neuronal damage after brain injury.     

卒中T淋巴细胞亚群动态变化及其与卒中相关感染的关系

目的 观察卒中急性期患者外周血中C D 3 +T细胞、C D 3 + C D 4 +T细胞、C D 3 + C D 8 +T细胞及 CD4+CD25+FoxP3+调节性T细胞（regulatory T cells,Tregs）的动态变化,探讨卒中后机体免疫状态及其 对卒中后感染的影响。 方法 选取卒中急性期患者32例为卒中组,根据发病1周内是否发生感染,将患者分为卒中后非感 染组24例和卒中后感染组8例。另选取性别、年龄匹配的健康体检者23例为对照组。采用流式细 胞术分别于病程24 h内、3 d、7 d检测卒中患者和健康体检者外周血中CD3+T细胞、CD3+CD4+T细胞、 CD3+CD8+T细胞及Tregs水平。 结果 ① 与对照组比较,卒中后非感染组CD3+CD4+T细胞与Tregs于发病后7 d升高（P分别为0.02和 0.03）;CD3+CD8+T细胞在发病后24 h及3 d下降（P分别为0.01和0.03）,发病后7 d升至与健康对照组 无显著差异。卒中后感染组CD3+T细胞、CD3+CD4+T细胞、CD3+CD8+T细胞及Tregs在发病24 h内（P分别为 ＜0.001,＜0.001,0.03和＜0.001）、3 d（P均＜0.001）、7 d（P分别为＜0.001,0.01,0.01和0.01）均较健 康对照组明显下降;②卒中后感染组CD3+T细胞、CD3+CD4+T细胞及Tregs在发病后24 h内（P分别为0.01, ＜0.001和＜0.001）、3 d（P分别＜0.001,＜0.001和0.04）、7 d（P均＜0.001）均显著低于卒中后非感染 组;两组CD3+CD8+T细胞在发病后24 h内无明显差异,但3 d（P＜0.001）、7 d（P =0.02）卒中后感染组显 著低于卒中后非感染组。 结论 C D3+T淋巴细胞、CD3+CD4+T淋巴细胞、CD3+CD8+T淋巴细胞及Tregs参与卒中早期病理生理过 程,其动态变化可能导致卒中后免疫抑制,并参与卒中后感染的发生。     

Increase in expression of adhesion molecule receptors on T helper cells during antipsychotic treatment and relationship to blood-brain barrier permeability in schizophrenia

OBJECTIVE: The authors estimated the expression of adhesion molecule receptors (VLA-4 and LFA-1) on T helper (CD4+) and T suppressor/cytotoxic (CD8+) lymphocytes in schizophrenic patients before and during antipsychotic treatment and studied the relationship of these subpopulations to CSF measures and blood-brain barrier permeability. METHOD: Blood was drawn from hospitalized patients with schizophrenia before (N = 45) and after (N = 22) neuroleptic treatment and from an age-matched comparison group (N = 41). Lumbar punctures were performed on 32 of the schizophrenic patients. RESULTS: During antipsychotic treatment there were significant increases in the percentage of VLA-4+/CD4+ and VLA-4+/CD8+ cells. VLA-4+/CD4+ and LFA-1+/CD4+ cells were both closely related to disturbance of the blood-brain barrier. Higher values for VLA-4+/CD4+ and LFA-1+/CD4+ cells were found in patients with a disturbed blood-brain barrier. CONCLUSIONS: The findings suggest that adhesion molecules are involved in immunoregulation between the central nervous system and the peripheral immune system in schizophrenia.     

多发性硬化患者外周血和脑脊液淋巴细胞亚群的观察

用碱性磷酸酶抗酸酶法检查了４６例多发性硬化活动期患者外周血和脑脊液的淋巴细胞亚群。结果显示：活动期ＭＳ者外周血ＣＤ＾＋４，ＣＤ＾＋９细胞较对照组减少，ＣＤ＾＋２５细胞，ＣＤ＾＋４／ＣＤ＾＋８比值较对照组升高。ＣＳＦ中ＣＤ＾４，ＣＤ＾＋２５细胞，ＣＤ＾＋４／ＣＤ＾＋８比值较对照组升高，ＣＤ＾＋８细胞降低，且ＣＳＦ中淋巴亚群均高于自身外周血中的相应细胞。     

A semiautomated measure of whole-brain atrophy in multiple sclerosis

Brain atrophy is a proposed MRI marker of irreversible pathologic damage in multiple sclerosis (MS). The brain parenchymal fraction (BPF) is the ratio of brain parenchymal volume to the total volume within the surface contour. We developed a semiautomated measure of BPF using commercially available edge-finding and thresholding software (30-min analysis time per patient). We measured BPF in 78 patients with MS and 17 healthy controls. BPF was lower in a cohort of patients with MS (n=50) (0.843+/-0.042, range 0.743-0.906) age-matched to controls (0.877+/-0.020, range 0.835-0.901) (p<0.001). BPF correlated inversely with third ventricular width (r=-0.785, p<0.001), and total T1 hypointense lesion volume (r=-0.347, p=0.011), but not with total T2 hyperintense lesion volume (r=-0.213, p=0.13). BPF correlated negatively with expanded disability status scale (EDSS) score (r=-0.391, p=0.0006) and disease duration (r=-0.281, p=0.01). Stepwise regression compared the relative abilities of MRI variables to predict clinical data. By regression of age, BPF, third ventricular width, T2 lesions, and T1 lesions, BPF was the best predictor of disability score (R(2)=0.204, p<0.001). Third ventricular width was the best predictor of disease duration (R(2)=0.316, p<0.001). None of the MRI variables differed between relapsing-remitting (RR) (n=60) and secondary progressive (SP) (n=18) disease course (p>0.05). The intrarater, interrater, and scan-rescan BPF variability (COV) was 0.31%, 0.34%, and 0.41% and the accuracy against a phantom was 99.1%. We conclude that whole-brain atrophy in MS can be reliably and readily quantified by a semiautomated approach. Longitudinal studies are warranted to determine if this method provides a sensitive biologic marker of the MS disease process.     

A magnetization transfer histogram study of normal-appearing brain tissue in MS

OBJECTIVE: To evaluate 1) the ability of magnetization transfer ratio (MTR) histogram analysis to detect the extent of changes occurring outside MS lesions seen on conventional scans, 2) whether such changes vary in the different MS clinical phenotypes, 3) whether the changes are associated with the extent and severity of the macroscopic lesion load, and 4) the contribution to brain atrophy. METHODS: Dual-echo, T1-weighted, and MT scans of the brain were obtained from 77 patients with varying MS courses and 20 age- and sex-matched control subjects. To create MT histograms of the normal-appearing cerebral tissue, MS lesions were segmented from dual-echo scans, superimposed automatically, and nulled out from the coregistered and scalp-stripped MTR maps. Average MTR, peak height, and peak position were considered. T2 and T1 lesion loads, average lesion MTR, and brain volume were also measured. RESULTS: Average histogram MTR (p<0.0001) and peak position (p<0.0001) from patients with relapsing-remitting MS (RMMS) were lower than those from control subjects. Patients with primary progressive MS (PPMS) had lower average histogram MTR (p = 0.002) and histogram peak height (p = 0.01) than control subjects. Patients with secondary progressive MS (SPMS) had a lower peak height (p = 0.05) than those with RRMS. Average lesion MTR (p<0.0001) correlated highly with the histogram MTR. Average histogram MTR (p<0.0001) and T2 lesion load (p = 0.001) correlated highly with brain volume. CONCLUSIONS: The amount of microscopic changes account for an important fraction of the lesion load in MS. They may contribute to the development of brain atrophy and tend to be more evident in patients with secondary progressive MS.     

Altered CD4+/CD8+ T-cell ratios in cerebrospinal fluid of natalizumab-treated patients with multiple sclerosis

BACKGROUND: Treatment with natalizumab, a monoclonal antibody against the adhesion molecule very late activation antigen 4, an alpha4beta(1) integrin, was recently associated with the development of progressive multifocal leukoencephalopathy, a demyelinating disorder of the central nervous system caused by JC virus infection. OBJECTIVE: To test the effect of natalizumab treatment on the CD4(+)/CD8(+) T-cell ratios in cerebrospinal fluid (CSF) and peripheral blood. DESIGN: Prospective longitudinal study. SETTING: Academic and private multiple sclerosis centers. PATIENTS: Patients with multiple sclerosis (MS) treated with natalizumab, untreated patients with MS, patients with other neurologic diseases, and human immunodeficiency virus-infected patients. MAIN OUTCOME MEASURES: CD4(+) and CD8(+) T cells were enumerated in CSF and peripheral blood. The mean fluorescence intensity of unbound alpha4 integrin on peripheral blood CD4(+) and CD8(+) T cells was analyzed before and after natalizumab therapy. RESULTS: Natalizumab therapy decreased the CSF CD4(+)/CD8(+) ratio of patients with MS to levels similar to those of human immunodeficiency virus-infected patients. CD4(+)/CD8(+) ratios in peripheral blood in patients with MS progressively decreased with the number of natalizumab doses, but they remained within normal limits. Six months after the cessation of natalizumab therapy, CSF CD4(+)/CD8(+) ratios normalized. The expression of unbound alpha4 integrin on peripheral blood T cells decreases with natalizumab therapy and was significantly lower on CD4(+) vs CD8(+) T cells. CONCLUSIONS: Natalizumab treatment alters the CSF CD4(+)/CD8(+) ratio. Lower expression of unbound alpha4 integrin on CD4(+) T cells is one possible mechanism. These results may have implications for the observation that some natalizumab-treated patients with MS developed progressive multifocal leukoencephalopathy.     

胶质瘤中LC3B与CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞的相关性及意义

目的研究不同级别胶质瘤组织中自噬相关蛋白LC3B的表达与CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞数量的相关性并探讨其意义。方法采用免疫组化、Western blot检测127例不同级别胶质瘤及40例瘤旁正常组织中LC3B的表达,免疫组化检测CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞的数量,分析其相关性及意义。结果 (1)免疫组化及Western blot检测结果显示LC3B的表达在瘤旁正常组织、低级别胶质瘤及高级别胶质瘤中呈不同程度的升高,且组间比较差异具有显著性(P0.05)。(2)CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞在胶质瘤中的数量明显高于瘤旁正常组织(P0.05),且高级别胶质瘤明显高于低级别胶质瘤(P0.05)。(3)LC3B的表达与CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞的数量在不同级别胶质瘤中均呈正相关,低级别胶质瘤中相关系数分别为0.466、0.599、0.537,高级别胶质瘤中相关系数分别为0.657、0.608、0.561。(4)胶质瘤中LC3B的表达及CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞的数量均与肿瘤的大小有关(P0.05)。结论在胶质瘤组织中LC3B、CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞均呈高表达,且LC3B的表达与CD68~+小胶质细胞、CD4~+和CD8~+T淋巴细胞的数量呈正相关,LC3B可能是影响胶质瘤细胞免疫的因素之一。     

Circulating dendritic cells subsets and regulatory T-cells at multiple sclerosis relapse: differential short-term changes on corticosteroids therapy

Glucocorticoids remain the treatment of choice for MS relapses. However, little is known on the effect of intravenous methylprednisolone (IVMP) on dendritic cells (DCs) and regulatory T-cells (TReg). Our main goal was to quantify circulating myeloid and plasmacytoid DCs (mDCs and pDCs), and TReg at MS relapse versus healthy controls; and to analyse the short-term changes after IVMP for MS relapse. MS patients at relapse compared to controls showed higher %CD4+CD25high+ TReg (p<0.01). After 5-days of IVMP, activated T-lymphocytes (p=0.001), pDCs (p<0.0001), and CD11c+ mDCs (p<0.0001) decreased. By contrast, CD4+CD25+ and CD4+CD25high+ TReg further increased (p<0.0001 both). Changes on these subsets may play a relevant role in the immunosuppressive activity of this drug.     

Selective suppression of chemokine receptor CXCR3 expression by interferon-beta1a in multiple sclerosis

We studied the expression of chemokine receptors CCR1, CCR2, CCR3, CCR5, and CXCR3 on CD4 and CD8 positive T cells, and on CD14 positive monocytes in blood from 10 patients with relapsing-remitting multiple sclerosis (MS) at initiation of interferon (IFN)-beta treatment, after 1 month and after 3 months of treatment. It was found that the expression of CXCR3 on CD4+ and CD8+ T cells was significantly reduced after 3 months of treatment. The expression of other receptors was unaltered. Since CXCR3 cells are enriched in cerebrospinal fluid (CSF), and are detected in lesion material in MS this may represent an important mode of action of interferon-beta in MS.     

Multiple sclerosis: chemokine receptor expression on circulating lymphocytes in correlation with radiographic measures of tissue injury

BackgroundLeukocytes expressing inflammatory chemokine receptors (CKRs), most consistently CCR2, CCR5, and CXCR3, have been identified in multiple sclerosis (MS) tissue lesions and provide attractive therapeutic targets. Our previous studies found large inter-individual differences in expression of these CKRs but stable levels over time within subjects. This observation suggests a CKR "set-point" within individuals, which might relate to inflammatory injury in MS. We evaluated the correlation between CKR levels and magnetic resonance imaging (MRI) measures of disease activity.MethodsFifty-five relapsing remitting MS (RRMS) and secondary progressive MS (SPMS) patients were prospectively followed with annual CKR and MRI studies. Multiparameter flow cytometry was used to determine CCR2, CCR5, and CXCR3 expression on CD4 and CD8 cells. Simultaneous cranial MRIs were performed, and quantitative measures of T2, T1, and gadolinium lesions, brain parenchymal fraction (BPF), and whole brain and fractionated magnetization transfer ratio (MTR) were performed using automated software. Spearman's rank correlations evaluated the relationship between CKR levels and MRI measures.ResultsSignificant correlations were observed between CXCR3 expression on CD8 cells and measures of new (T1) and total (T1, T2) lesion volumes, lesion MTR, and BPF; higher levels of CXCR3 expression were correlated with greater injury on MRI (|r| = 0.27-0.42). In contrast, CD4 cell CKR expression was only minimally correlated with MRI measures.ConclusionsOver 2 years, we observed significant correlations between the percent of CD8 cells expressing CXCR3 and MRI measures of MS inflammatory activity and tissue destruction. These observations are consistent with a pathogenic role for cytotoxic T cells in MS brain and have significant implications regarding T-cell targeted therapeutic strategies.     

Activation and adhesion molecule expression on lymphoid infiltrates in human glioblastomas

Frozen tissue sections obtained from human glioblastomas, brain tumor metastases and normal brain were examined for the expression of molecules known to be involved in lymphocyte activation and/or adhesion and migration. The molecules studied included CD3, CD45R, UCHL-1 (CD45RO), lymphocyte function-associated antigen 1 (LFA-1) (CD11a, CD18), intercellular adhesion molecule 1 (ICAM-1) (CD54), 4B4 (CD29), CD44, CD2, and LFA-3 (CD58). CD3+ lymphocytes infiltrating human glioblastomas and brain tumor metastases expressed LFA-1 alpha and beta. Many cells were also UCHL-1+ whereas only a small percentage were CD45R+. CD2+ lymphocytes were also present. Tumor-infiltrating lymphocytes (TIL) were found to be negative for CD29, which was, however, expressed on intratumoral vessels in addition to vessels found in normal brain. Glioblastoma cells and intratumoral vessels expressed ICAM-1 whereas no ICAM-1 was found on TIL or on normal brain. Glioblastoma cells also expressed high levels of both CD44 and LFA-3 whereas TIL were negative for these antigens. CD44 was also expressed on certain regions of normal brain. Antibodies to LFA-1 alpha and -beta and ICAM-1 could significantly block the binding of lymphokine-activated killer (LAK) cells or TIL to human glioblastoma cells suggesting that these molecules play a role in the binding and subsequent migration of lymphocytes into brain tumor tissue.     

多发性硬化患者CD8+记忆性T细胞亚群穿孔素和颗粒酶-B表达的研究

目的 测定多发性硬化(MS)患者外周血中CD8+记忆性T细胞亚群效应细胞因子的表达,并将其与MS病情严重程度进行相关分析.方法 利用四色-流式细胞术检测未经治疗的MS患者、其他神经系统疾病(OND)患者和正常对照(NC)成员组外周血表达穿孔素和颗粒酶-B的CD8+记忆性T细胞亚群数量,并利用扩展的残疾状况量表(EDSS)对MS患者病情严重程度做评分.结果 与NC组比较,MS患者外周血表达颗粒酶-B的CD8+效应记忆性T细胞(TEM)和终末效应记忆性T细胞(TerTEM)均明显减少,比较差异有统计学意义(P＜0.05);表达穿孔素和颗粒酶-B的TEM数量与EDSS呈负相关(r=-0.493,P=0.027;r=-0.594,P=0.009).结论 CD8+TEM参与MS相关的CNS内炎性免疫应答,外周血穿孔素和颗粒酶-B表达阳性的CD8+TEM数量可在一定程度上反映MS患者CNS的病损程度.     

Serial blood T cell repertoire alterations in multiple sclerosis patients; correlation with clinical and MRI parameters

A significant skewing of the peripheral T cell repertoire has been shown in relapsing-remitting multiple sclerosis (MS). Most of the studies already performed in this field are cross-sectional and therefore, little is known of the T cell repertoire evolution over time in MS and the correlation of T cell repertoire variation with clinical and MRI parameters. This study was performed on serially harvested frozen PBMC from nine untreated MS patients (27 samples) and 14 healthy individuals. The blood T cell repertoire of each patient was analysed at the complementarity determining region 3 (CDR3) level and compared with a monthly MRI scan performed over a six month period with assessment of T2 lesion load and gadolinium enhancing lesions. A highly significant blood T cell repertoire skewing was observed in MS patients as compared with healthy controls (p<0.01). In addition, the number of altered Vbeta families correlated significantly with both the T2 lesion volume and the number of gadolinium enhancing lesions as assessed by MRI (Spearman correlation tests, r=0.51 and r=0.44, p<0.01 and p<0.05 respectively). Furthermore, the variation of the number of altered Vbeta families over time also correlated with the appearance of new gadolinium enhancing lesions (r=0.36, p=0.05). These findings which need confirmation on larger serial cohorts, suggest an association between the magnitude of TCRBV CDR3 length distribution alterations in the peripheral blood of MS patients and the disease process.     

Regulation of leukocyte function-associated antigen 1-mediated adhesion by somatostatin and substance P in mouse spleen cells

BACKGROUND: Interaction of the integrin leukocyte function-associated antigen (LFA)-1 (CD11a/CD18) with its ligands, the intercellular adhesion molecules (ICAM)-1, -2, and -3 (CD54, CD102, and CD50), is pivotal to many leukocyte adhesion events. METHOD: To define the mechanism of the movement of leukocytes to the inflammatory site by somatostatin (SOM) and substance P (SP), we examined the expression of the adhesion molecule LFA-1 and inside-out signals for integrins, protein kinase C (PKC), Ras, Rap1, and phosphoinositide (PI) 3-kinase, in anti-CD3-, anti-CD3+SOM-, anti-CD3+SP-stimulated or unstimulated spleen cells. RESULTS: SOM caused down-regulation of LFA-1 mRNA translation as well as of adhesion-stimulating molecules such as Rap1, Ras, and PI 3-kinase. On the other hand, SP slightly induced LFA-1 mRNA translation and activation signals for integrins. The early-phase alteration of LFA-1 mRNA translation after 3 h of culture may be due to the changes of CD8+ T cells rather than changes of CD4+ cells. In adhesion assays, SOM significantly decreased cell adhesion (p < 0.05). CONCLUSION: These data suggest that SOM treatment of spleen cells, especially in CD8+ T cells, leads to downregulation of LFA-1 mRNA translation, inside-out signaling molecules for integrins (Ras, Rap1 and PI 3-kinase, but not PKC), and consequently to a decrease in the LFA-1-mediated adhesion to ICAM-1.