Two susceptible HLA-DRB1 alleles for multiple sclerosis differentially regulate anti-JC virus antibody serostatus along with fingolimod.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) caused by JC virus (JCV) is a rare but serious complication of some disease-modifying drugs used to treat multiple sclerosis (MS). Japanese MS patients treated with fingolimod were reported to be 10 times more likely to develop PML than equivalent patients in other countries. The strongest susceptibility human leukocyte antigen (HLA) class II alleles for MS are distinct between races (DRB1*15:01 for Caucasians and DRB1*04:05 and DRB1*15:01 for Japanese); therefore, we investigated whether HLA class II alleles modulate anti-JCV antibody serostatus in Japanese MS patients with and without fingolimod. METHODS: We enrolled 128 Japanese patients with MS, in whom 64 (50%) were under fingolimod treatment at sampling, and examined the relationship between HLA class II alleles and anti-JCV antibody serostatus. Serum anti-JCV antibody positivity and index were measured using a second-generation two-step assay and HLA-DRB1 and -DPB1 alleles were genotyped. RESULTS: HLA-DRB1*15 carriers had a lower frequency of anti-JCV antibody positivity (57% vs 78%, p = 0.015), and lower antibody index (median 0.42 vs 1.97, p = 0.037) than non-carriers. Among patients without HLA-DRB1*15, DRB1*04 carriers had a higher seropositivity rate than non-carriers (84% vs 54%, p = 0.030), and DPB1*04:02 carriers had a higher anti-JCV antibody index than non-carriers (3.20 vs 1.34, p = 0.008) although anti-JCV antibody-positivity rates did not differ. Patients treated with fingolimod had a higher antibody index than other patients (1.46 vs 0.64, p = 0.039) and treatment period had a positive correlation with antibody index (p = 0.018). Multivariate logistic regression analysis revealed that age was positively associated, and HLA-DRB1*15 was negatively associated with anti-JCV antibody positivity (odds ratio [OR] = 1.06, p = 0.006, and OR = 0.37, p = 0.028, respectively). Excluding HLA-DRB1*15-carriers, DRB1*04 was an independent risk factor for the presence of anti-JCV antibody (OR = 5.50, p = 0.023). CONCLUSIONS: HLA-DRB1*15 is associated with low anti-JCV antibody positive rate and low JCV antibody index, and in the absence of DRB1*15, DRB1*04 carriers are associated with a high antibody positive rate in Japanese, suggesting the effects of two susceptible HLA-DRB1 alleles on anti-JCV antibody serostatus differ.

Hopkins syndrome following the first episode of bronchial asthma associated with enterovirus D68: a case report.

BACKGROUND: Hopkins syndrome (HS) is a rare disorder presenting with acute flaccid paralysis of the limbs following an asthma attack. Neurologists encounter a diagnostic challenge if patients without a history of bronchial asthma develop neurologic features mimicking HS following acute respiratory distress. We report a case of HS occurring after a first episode of bronchial asthma associated with enterovirus D68 infection. CASE PRESENTATION: A 5-year-old girl developed acute respiratory distress. On the fourth hospital day, both her legs became paralyzed except for slight muscle contraction in the right lower limb. Tendon reflexes in the lower limbs were diminished and there was a positive Babinski sign on the right. Sensation was normal in all modalities, and there was no uro-rectal disturbance. Spinal magnetic resonance imaging identified T2-hyperintense lesions with spinal cord edema, mainly involving the bilateral T11 to L1 anterior horns, with left side dominance extending to the left posterior horn. The neurological and neuro-radiological findings of our case were suggestive of HS; however, she had no history of bronchial asthma. An acetylcholine inhalation challenge eventually proved the presence of reversible airway hyper-responsiveness, allowing us to diagnose HS. We identified enterovirus D68 in the patient's intratracheal aspirates using a sensitive polymerase chain reaction assay. Intravenous immunoglobulin administrations at 2 g/kg2 for 5 consecutive days were repeated every month up to four times. After these treatments, the muscle strength of her right lower limb slightly improved while her left lower leg remained completely paralyzed. CONCLUSION: This case emphasizes the importance of provocation tests to reveal the presence of airway hyper-responsiveness when a child shows neurological signs mimicking HS following acute respiratory distress. Furthermore, the present case suggests a possible link between HS and acute flaccid paralysis following lower respiratory tract infection by enterovirus D68.

A new clustering method identifies multiple sclerosis-specific T-cell receptors.

OBJECTIVE: To characterize T-cell receptors (TCRs) and identify target epitopes in multiple sclerosis (MS). METHODS: Peripheral blood mononuclear cells were obtained from 39 MS patients and 19 healthy controls (HCs). TCR repertoires for α/ß/δ/γ chains, TCR diversity, and V/J usage were determined by next-generation sequencing. TCR ß chain repertoires were compared with affectation status using a novel clustering method, Grouping of Lymphocyte Interactions by Paratope Hotspots (GLIPH). Cytomegalovirus (CMV)-IgG was measured in an additional 113 MS patients and 93 HCs. Regulatory T cells (Tregs) were measured by flow cytometry. RESULTS: TCR diversity for all four chains decreased with age. TCRα and TCRß diversity was higher in MS patients (P = 0.0015 and 0.024, respectively), even after age correction. TRAJ56 and TRBV4-3 were more prevalent in MS patients than in HCs (pcorr  = 0.027 and 0.040, respectively). GLIPH consolidated 208,674 TCR clones from MS patients into 1,294 clusters, among which two candidate clusters were identified. The TRBV4-3 cluster was shared by HLA-DRB1*04:05-positive patients (87.5%) and predicted to recognize CMV peptides (CMV-TCR). MS Severity Score (MSSS) was lower in patients with CMV-TCR than in those without (P = 0.037). CMV-IgG-positivity was associated with lower MSSS in HLA-DRB1*04:05 carriers (P = 0.0053). HLA-DRB1*04:05-positive individuals demonstrated higher CMV-IgG titers than HLA-DRB1*04:05-negative individuals (P = 0.017). CMV-IgG-positive patients had more Tregs than CMV-IgG-negative patients (P = 0.054). INTERPRETATION: High TCRα/TCRß diversity, regardless of age, is characteristic of MS. Association of a CMV-recognizing TCR with mild disability indicates CMV's protective role in HLA-DRB1*04:05-positive MS.

Elevated mycobacterium avium subsp. paratuberculosis (MAP) antibody titer in Japanese multiple sclerosis.

To investigate whether antibody production against mycobacterium avium subsp. paratuberculosis (MAP) is related to clinical characteristics of multiple sclerosis (MS) and human leukocyte antigen (HLA) alleles, IgG antibody against three MAP peptides and two human peptides homologous to MAP were measured in sera from 103 MS patients and 50 healthy controls (HCs). MS patients had higher IgG levels against MAP2694295-303 (MAP2694-IgG) than HCs, while the other antibodies were comparable. Multivariate analysis demonstrated that higher MAP2694-IgG titers were associated with higher EDSS scores, but not with HLA alleles or dairy product consumption. Immune response against MAP may worsen MS disability.

HLA genotype-clinical phenotype correlations in multiple sclerosis and neuromyelitis optica spectrum disorders based on Japan MS/NMOSD Biobank data.

HLA genotype-clinical phenotype correlations are not established for multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD). We studied HLA-DRB1/DPB1 genotype-phenotype correlations in 528 MS and 165 NMOSD cases using Japan MS/NMOSD Biobank materials. HLA-DRB1*04:05, DRB1*15:01 and DPB1*03:01 correlated with MS susceptibility and DRB1*01:01, DRB1*09:01, DRB1*13:02 and DPB1*04:01 were protective against MS. HLA-DRB1*15:01 was associated with increased optic neuritis and cerebellar involvement and worsened visual and pyramidal functional scale (FS) scores, resulting in higher progression index values. HLA-DRB1*04:05 was associated with younger onset age, high visual FS scores, and a high tendency to develop optic neuritis. HLA-DPB1*03:01 increased brainstem and cerebellar FS scores. By contrast, HLA-DRB1*01:01 decreased spinal cord involvement and sensory FS scores, HLA-DRB1*09:01 decreased annualized relapse rate, brainstem involvement and bowel and bladder FS scores, and HLA-DRB1*13:02 decreased spinal cord and brainstem involvement. In NMOSD, HLA-DRB1*08:02 and DPB1*05:01 were associated with susceptibility and DRB1*09:01 was protective. Multivariable analysis revealed old onset age, long disease duration, and many relapses as independent disability risks in both MS and NMOSD, and HLA-DRB1*15:01 as an independent risk only in MS. Therefore, both susceptibility and protective alleles can influence the clinical manifestations in MS, while such genotype-phenotype correlations are unclear in NMOSD.

Dendritic cell differentiation with prostaglandin E results in selective attenuation of the extracellular signal-related kinase pathway and decreased interleukin-23 production.

The balance between interleukin (IL)-12 and IL-23 production by dendritic cells (DCs) is crucial for the induction of appropriate immune responses. In the present study, we examined the effect of prostaglandin E(2) (PGE(2)) treatment of DCs during differentiation on IL-12 and IL-23 production in response to Toll-like receptor (TLR) stimulation. Bone marrow-derived DCs were generated by culturing murine bone marrow cells with granulocyte-macrophage colony-stimulating factor (GM-CSF) alone (cont-DCs) or GM-CSF plus PGE(2) (PG-DCs). Upon TLR stimulation, IL-23 production by PG-DCs was markedly decreased compared with that by cont-DCs. However, no significant difference was detected in IL-12 production between these types of DC. To examine the mechanism underlying the impaired production of IL-23 by PG-DCs, we analysed the activities of extracellular signal-related kinases (ERKs) 1/2, p38 mitogen-activated protein kinase (MAPK), c-jun N-terminal kinases 1/2, Akt, and nuclear factor (NF)-kappaB (p65) in these DCs upon TLR stimulation. The ERK1/2 activity in PG-DCs was significantly lower than that of cont-DCs. No significant differences were detected in the activities of other molecules between cont-DCs and PG-DCs. In addition, treatment of cont-DCs with U0126, a specific inhibitor of the ERK pathway, reduced the TLR-mediated production by the DCs of IL-23 but not IL-12. Thus, DC development in the presence of PGE(2) results in selective attenuation of the ERK pathway. The attenuation of ERK activation appears to be responsible for the decreased IL-23 production by PG-DCs.

Dynamic changes in the mobility of LAT in aggregated lipid rafts upon T cell activation.

Lipid rafts are known to aggregate in response to various stimuli. By way of raft aggregation after stimulation, signaling molecules in rafts accumulate and interact so that the signal received at a given membrane receptor is amplified efficiently from the site of aggregation. To elucidate the process of lipid raft aggregation during T cell activation, we analyzed the dynamic changes of a raft-associated protein, linker for activation of T cells (LAT), on T cell receptor stimulation using LAT fused to GFP (LAT-GFP). When transfectants expressing LAT-GFP were stimulated with anti-CD3-coated beads, LAT-GFP aggregated and formed patches at the area of bead contact. Photobleaching experiments using live cells revealed that LAT-GFP in patches was markedly less mobile than that in nonpatched regions. The decreased mobility in patches was dependent on raft organization supported by membrane cholesterol and signaling molecule binding sites, especially the phospholipase C gamma 1 binding site in the cytoplasmic domain of LAT. Thus, although LAT normally moves rapidly at the plasma membrane, it loses its mobility and becomes stably associated with aggregated rafts to ensure organized and sustained signal transduction required for T cell activation.

Selective synergy in anti-inflammatory cytokine production upon cooperated signaling via TLR4 and TLR2 in murine conventional dendritic cells.

Toll-like receptor (TLR) ligands, i.e. lipopolysaccharide (LPS), induce dendritic cell (DC) production of both inflammatory and anti-inflammatory cytokines including interleukin (IL)-12, tumor necrosis factor (TNF)-alpha, and IL-10. The balance of inflammatory versus anti-inflammatory cytokines appears to be crucial to control immune homeostasis. In the present study, we investigated TLR-mediated regulation of inflammatory versus anti-inflammatory cytokine production using murine bone marrow derived conventional DCs. Standard LPS (sLPS) that contains lipoprotein, a TLR2 ligand, induced vigorous production of both IL-10 and IL-12 p40 by DCs. Highly purified LPS (ultra-pure LPS, upLPS) also induced vigorous production of IL-12 p40, but markedly low IL-10 production. Thus, signal deficiency through TLR2 appeared to result in marked reduction in DC production of IL-10 but not IL-12 p40 upon stimulation with upLPS. To examine this possibility, DCs were stimulated with Pam3CSK4, a synthetic ligand of TLR2, in addition to stimulation with upLPS. It was shown that Pam3CSK4 alone failed to induce IL-10 production. However, Pam3CSK4 synergistically enhanced upLPS-induced DC production of IL-10 but neither IL-12 p40 nor TNF-alpha. Extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and c-jun N-terminal kinase (JNK)1/2 in DCs were significantly activated by upLPS stimulation. The upLPS-induced activities of these MAPKs were considerably enhanced by additional stimulation with Pam3CSK4. Blocking either p38 MAPK or JNK1/2 pathway completely inhibited the synergistic enhancement of the IL-10 production by DCs upon upLPS and Pam3CSK4 stimulation. Thus, cooperated stimulation of these MAPKs via TLR4 and TLR2 appeared to induce selective synergy in anti-inflammatory cytokine production by murine conventional DCs.

Serum GFAP and neurofilament light as biomarkers of disease activity and disability in NMOSD.

OBJECTIVE: To test the hypothesis that serum levels of glial fibrillary acidic protein (GFAP) and neurofilament light chain (NfL), which are an intermediate astrocyte and neuron filaments, respectively, are clinically useful biomarkers of disease activity and disability in neuromyelitis optica spectrum disorders (NMOSD). METHODS: Levels of GFAP and NfL in serum (sGFAP and sNfL, respectively) and in CSF samples were measured in healthy controls (HCs) (n = 49; 49 serum samples), patients with NMOSD (n = 33; 42 CSF and 102 serum samples), and patients with multiple sclerosis (MS) (n = 49; 53 CSF and 91 serum samples) by ultrasensitive single-molecule array assays. Association of sGFAP and sNfL levels with clinical parameters was determined. RESULTS: For both GFAP and NfL, CSF and serum levels were strongly correlated. Both were higher in the serum of patients with NMOSD than in HCs (both p < 0.001). Moreover, sGFAP was higher in NMOSD than in MS (median 207.7 vs 121.1 pg/mL, p < 0.001). In NMOSD, sGFAP concentration increased after recent relapse (540.9 vs 152.9 pg/mL, p < 0.001). Multivariate analyses indicated that sGFAP and sNfL were associated with Expanded Disability Status Scale score in NMOSD (p = 0.026 and p < 0.001, respectively). Higher sGFAP/sNfL quotient at relapse differentiated NMOSD from MS with a sensitivity of 73.0% and a specificity of 75.8%. CONCLUSIONS: sGFAP and sNfL are likely to be good biomarkers of disease activity and disability, and the sGFAP/sNfL quotient at relapse is a potential diagnostic marker for NMOSD.

Relationship between the contamination of the nurse's caps and their period of use in terms of microorganism numbers.

Nosocomial infections are a great problem in the health care facilities. The white uniforms of nurses are often washed to keep them clean, but the nurse's caps are not washed as frequently in comparison. It could be that the importance of these caps is being overlooked. If these caps are providing a residence for microorganisms causing nosocomial infection in the health care facility, then they should be washed as frequently as the uniforms. So far, the relationship between the contamination of the nurse's caps and nosocomial infection has not yet been studied. Therefore, this study was conducted to confirm if relationships exist among factors regarding the number of microorganisms on the nurse's caps, the period in which caps were used without being washed, and the individual characteristics of nurse wearing the caps. Results showed that the degree of contamination of the nurse's caps depended on individual characteristics and the period of use. Finally, results led to the conclusion that the nurse's caps should not be worn if their only purpose is to symbolize female workers in the health care facilities because, in actually, they provide a resistance for microorganisms causing nosocomial infections.

Sterilization efficiency of the photocatalyst against environmental microorganisms in a health care facility.

The photocatalyst equipment consists of a titanium dioxide membrane and an ultraviolet lamp. The authors studied if the photocatalyst equipment is practically useful in sterilizing environmental microorganisms in the health care facility. The number of microorganisms was compared in the cases of no sterilization (control) and the photocatalyst sterilization. As a result, a statistical difference was observed between control and the photocatalyst sterilization against airborne microorganisms (p < 0.01), but not against surface microorganisms (p > 0.2). The photocatalyst uses an air sucking system, so it may be ineffective against microorganisms tightly attached to surfaces. However, the effectiveness of the photocatalyst to sterilize airborne microorganisms in the health care facility was successfully confirmed. Concerning the humidity effect on the photocatalyst sterilization, the authors compared the number of airborne microorganisms in cases of the control, UV alone and photocatalyst sterilization when humidity was changed. A statistical difference was observed between UV and the photocatalyst sterilization (p < 0.01) when humidity was increased to 60-70%, but not observed between UV and the photocatalyst sterilization (p > 0.2) when humidity was not controlled and was around 10-20%. This indicates that maintaining high humidity levels will present satisfactory sterilization results due to a greater production of OH radicals. From data obtained, no effect of the adsorption on the TiO2 membrane could be observed.

Differential Expression of Cardiac Troponin T and I in a Patient with Isolated Skeletal Muscular Sarcoidosis.

A 49-year-old female was referred to our hospital due to high serum creatine kinase (CK) (2,605 IU/L) and serum cardiac troponin T (cTnT) (0.342 ng/mL) levels. She had no other complaints and further examinations suggested no signs of cardiac disease. Additionally, the serum cardiac troponin I (cTnI) levels were normal. She reported having gradually felt difficulty in walking upstairs. A biopsy indicated skeletal muscle sarcoidosis with positive staining for cTnT. Steroid therapy immediately resolved her muscular symptoms with a normalization of the serum CK levels. Since the serum levels of cTnI were normal, the concomitant measurement of cTnT/cTnI might be useful to diagnose skeletal muscular disease biochemically in such cases.

Blood monocyte count may be a predictor of vascular access failure in hemodialysis patients.

It has been reported that an increase in the counts of white blood cells (WBC) and their subpopulations is an independent predictor of atherosclerotic events and mortality. On the other hand, vascular access (VA) stenosis is caused by a progressive development of neointimal hyperplasia. We examined the relationship between VA failure and counts of peripheral WBC and their subpopulations in hemodialysis (HD) patients. The study population consisted of 82 patients undergoing regular HD. Twenty-two patients, who were unstable, were excluded from the study. After one year, we examined the relationship between VA failure and the counts of peripheral WBC and their subpopulations. In the follow-up period, 21 patients had complicated VA failure. The counts of WBC, neutrophils, lymphocytes, eosinophils, and basophils did not show a significant difference, but a monocyte count of ≥ 400/µL was shown to indicate a high probability of VA failure. Logistic regression analysis revealed that monocyte count was an independent risk factor of VA failure. The peripheral blood monocyte count may be a predictor of VA failure in HD patients.

Dermokine as a novel biomarker for early-stage colorectal cancer.

BACKGROUND: Colorectal cancer is a common disease that is usually detected at an advanced stage, because early-stage cancer is mostly asymptomatic and appropriate serologic biomarkers have not been established. We have previously identified dermokine (DK) as a peptide secreted by keratinocytes and we found that DK-ß/γ was expressed in colorectal tumors. Therefore, we focused on DK-ß/γ as a new candidate diagnostic serum marker for early colorectal cancer. METHODS: DK-ß/γ expression in human colorectal cancer cell lines and tissues was assessed by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry. We established an experimental enzyme-linked immunosorbent assay (ELISA) to detect DK-ß/γ in the serum of colorectal cancer patients, and we compared the sensitivities of common diagnostic markers, carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, and serum p53 antibody (S-p53). RESULTS: Immunohistochemical staining of colon tumor tissue with anti-DK monoclonal antibody (mAb) revealed that DK-ß/γ was more commonly expressed in the early stages of colorectal cancer (Tis-T1; i.e., cancer in situ, intraepithelial or invasion of lamina propria [Tis]; tumor invades the submucosa [T1]) than in late-stage tumors (T2-T4; i.e., tumor invades the muscularis propria [T2]; tumor invades through the muscularis propria into the subserosa, or into the nonperitonealized pericolic or perirectal tissues [T3]; tumor directly invades other organs or structures and/or perforates visceral peritoneum [T4]). Serum DK-ß/γ levels were determined in 130 patients with colorectal cancer and 25 healthy volunteers. Serum DK-ß/γ was detected in 33.3% of patients with early colorectal cancer (Tis-T1), which was higher than the rates for S-p53 (24.2%), CEA (9.1%), and CA19-9 (0%). The serum DK-ß/γ test was complementary to the other marker tests. Therefore, when the combined four-marker test (DK/CEA/CA19-9/S-p53) was carried out, the diagnostic sensitivity for Tis and T1 tumors reached 60.6%. CONCLUSIONS: Serum DK-ß/γ is the most promising of the existing tumor biomarkers for the diagnosis of early-stage colorectal cancer.