Curcumin increased the expression of c-FLIP in HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) disease is a chronic neuroinflammatory disease, which is associated with HTLV-1 infection. There is no effective and satisfactory treatment of HAM/TSP. It has been shown that curcumin exhibits modulatory effects on apoptosis and cytotoxicity-related molecules in HAM/TSP patients. In the present study, we examined the effect of curcumin on the gene expression of caspase-8, caspase-10, and anti-apoptotic protein c-FLIP, in HAM/TSP patients. Furthermore, we compared the expression of these molecules between HAM/TSP and asymptomatic carriers. Real-time PCR was performed to examine the mRNA expression of caspase-8, caspase-10, and c-FLIP in studied groups. The mRNA expression of caspase-8 and caspase-10 was similar before and after curcumin treatment in HAM/TSP patients (P > 0.05). The mRNA expression of c-FLIPL and c-FLIPs was higher after curcumin treatment compared with before treatment and significant differences were observed between the two groups (P = 0.004 and P = 0.044, respectively). The mRNA expression levels of caspase-8, caspase-10, c-FLIPL, and c-FLIPs were not statistically significant between HAM/TSP patients and asymptomatic carriers (P < 0.05). In conclusion, our results showed that curcumin increased the expression of c-FLIP in HAM/TSP patients which might suggest that, this molecule is involved in the apoptosis of HTLV-1-infected cells. Further studies with large sample size could be useful to clarify the role of this supplement in HAM/TSP patients.     

A new hypothesis for the pathogenesis of Human T-lymphotropic virus type 1 associated myelopathy/tropical spastic paraparesis

The exogenous, human retrovirus Human T-lymphotropic virus type 1 (HTLV-1) results in a highly dynamic persistent infection. HTLV-1 usually causes an asymptomatic infection but a small proportion of individuals may develop HTLV-1 associated myelopathy/ tropical spastic paraparesis (HAM/TSP). HAM/TSP is a chronic inflammatory disease of the central nervous system (CNS) that is rarely fatal, but can be severely debilitating. HTLV-1 is found in the CNS primarily within infiltrating, infected CD4+ T lymphocytes. CD4+ T-cell infiltration into the CNS is currently believed to be the pivotal event for the pathogenesis of HAM/TSP but the exact mechanisms by which these T cells result in neurological damage are unknown. Here we explore a current hypothesis of HAM/TSP pathogenesis and suggest a new hypothesis focused on why the majority of HTLV-1-infected individuals do not develop neuroinflammatory disease. In this new hypothesis we highlight the two battles for control over HTLV-1 activity that occur in the peripheral blood and the CNS. We also introduce the idea that HAM/TSP is the result of a disturbed damage:healing ratio within the spinal cord that is dependent on the activity of HTLV-1 proteins, glia and infiltrating immune cells.     

Immunostimulation by induced expression of NKG2D and its MIC ligands in HTLV-1-associated neurologic disease

The NKG2D receptor costimulates effector/memory CD8 T cells and is normally absent on CD4 T cells but can be induced by T cell antigen receptor complex stimulation and interleukin-15 (IL-15). Among its ligands are the human major histocompatibility complex class I-related MICA and MICB, which have a restricted tissue distribution but are frequently associated with malignancies and some microbial infections. Moreover, aberrant expression of MIC may promote autoimmune disease progression. Human T cell lymphotropic virus type I (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic inflammatory disease of the central nervous system that resembles multiple sclerosis. Disease progression involves production of IL-15 and its receptor through transactivation by the viral Tax regulator protein, an activated immune response state, and local cytokine production and T cell fratricide by Tax-specific cytotoxic T lymphocytes (CTL). This study shows that as with CD8 T cells, substantial proportions of HAM/TSP patient CD4 T cells are positive for NKG2D and that large numbers of T cells from both subsets express MIC, which can be transactivated by Tax independent of nuclear factor κB. Engagement of MIC by NKG2D promotes spontaneous HAM/TSP T cell proliferation and, apparently, CTL activities against HTLV-1-infected T cells. These results reveal a viral strategy that may exploit immune stimulatory mechanisms to negotiate a balance between promotion and limitation of infected host T cell expansions.     

Genetic variability in the extracellular matrix protein as a determinant of risk for developing HTLV-I-associated neurological disease

Aggrecan, which is a well-known proteoglycan in joint cartilage, also exists in the spinal cord and plays an important role in maintaining water content in the extracellular matrix structure. In this study, we first examined the variable number of tandem repeat (VNTR) polymorphism of the aggrecan gene in 227 HTLV-I associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients, in 217 HTLV-I-infected healthy carriers (HCs), and in 85 normal controls. The VNTR allele 28 (1,630 bp) was more frequently observed in HAM/TSP patients than in HCs (χ ²=12.02, p=0.0005, odds ratio 1.79, 95% C.I. 1.29–2.50) and in controls (χ ²=13.43, p=0.0002, odds ratio 2.54, 95% C.I. 1.52–4.25), although this allele was not related to disease progression or to HTLV-I provirus load. We also found that the aggrecan concentration in cerebrospinal fluid (CSF) from rapidly progressive HAM/TSP patients was significantly higher than in slowly progressive patients (corrected p=0.0145) but not in infected non-inflammatory neurological other disease controls (OND) (corrected p=0.078). We then analyzed this aggrecan VNTR polymorphism in the different set of patients with HAM/TSP (n=58) and healthy carriers (n=70). This analysis, again, revealed that allele 28 was detected more frequently in HAM/TSP group than in HCs (χ ²=11.03, p=0.0009, odd ratio 3.04, 95% C.I. 1.55–5.97). The reproducibility of our study was regarded as a second- or third-class association by comparing combined p values and the Better Associations for Disease and GEnes (BADGE) system. Our results suggest that aggrecan polymorphism can be a novel genetic risk factor for developing HAM/TSP.Financial support: Grant in Aid for Research on Brain Science of the Ministry of Health, Labor and Welfare, Japan.     

BALB/c小鼠作为单纯疱疹病毒1型感染模型的免疫学分析

在单纯疱疹病毒1型(herpes simplex virus type 1,HSV-1)小鼠感染及其相关研究中,临床病理和免疫学指标对其分析具有重要技术意义。本研究观察了HSV-1在不同条件下感染BALB/c小鼠后的多个免疫学指标,包括外周血单核细胞(peripheral blood mononuclear cell,PBMC)群体中树突细胞比例及功能、血清中和抗体水平、PBMC中HSV-1抗原特异性T细胞水平,以及潜伏感染期小鼠神经组织中CD8^＋ T细胞浸润情况。结果显示,HSV-1毒株Mckrae、17＋以角膜及滴鼻途径感染3周龄及6周龄BALB/c小鼠后,小鼠PBMC中树突细胞数量增加,并显示出刺激病毒抗原特异性T细胞增殖的能力。病毒感染后35 d,小鼠PBMC中未检测到白细胞介素4(interleukin 4,IL-4)^＋抗原特异性T细胞,但能检测到低水平的γ干扰素(interferon γ,IFN-γ)^＋抗原特异性T细胞;小鼠血清中未检测到或仅能检测到低水平的中和抗体。HSV-1以皮下及足垫注射途径感染BALB/c小鼠90 d后,足垫感染途径较皮下感染诱导出更高水平的血清中和抗体,PBMC中可检测到IL-4^＋及IFN-γ^＋抗原特异性T细胞,但不同毒株及小鼠周龄之间出现T细胞反应程度差异。组织病理学结果表明,各组小鼠三叉神经组织中均有CD8^＋ T细胞浸润。这些结果提示,不同HSV-1毒株以不同途径感染不同周龄BALB/c小鼠后,均可刺激树突细胞成熟及呈递病毒抗原,但血清中和抗体及PBMC中病毒抗原特异性T细胞水平在不同毒株、感染途径及小鼠周龄之间有差异。     

Proteomic technologies for the identification of disease biomarkers in serum: advances and challenges ahead

Serum is an ideal biological sample that contains an archive of information due to the presence of a variety of proteins released by diseased tissue, and serum proteomics has gained considerable interest for the disease biomarker discovery. Easy accessibility and rapid protein changes in response to disease pathogenesis makes serum an attractive sample for clinical research. Despite these advantages, the analysis of serum proteome is very challenging due to the wide dynamic range of proteins, difficulty in finding low-abundance target analytes due to the presence of high-abundance serum proteins, high levels of salts and other interfering compounds, variations among individuals and paucity of reproducibility. Sample preparation introduces pre-analytical variations and poses major challenges to analyze the serum proteome. The label-free detection techniques such as surface plasmon resonance, microcantilever, few nanotechniques and different resonators are rapidly emerging for the analysis of serum proteome and they have exhibited potential to overcome few limitations of the conventional techniques. In this article, we will discuss the current status of serum proteome analysis for the biomarker discovery and address key technological advancements, with a focus on challenges and amenable solutions.     

Analysis of the T-cell receptor repertoire of human T-cell leukemia virus type 1 (HTLV-1) Tax-specific CD8+ cytotoxic T lymphocytes from patients with HTLV-1-associated disease: evidence for oligoclonal expansion.

Human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a chronic, progressive neurological disease characterized by marked degeneration of the spinal cord and the presence of antibodies against HTLV-1. Patients with HAM/TSP, but not asymptomatic carriers, show very high precursor frequencies of HTLV-1-specific CD8+ T cells in peripheral blood and cerebrospinal fluid, suggestive of a role of these T cells in the pathogenesis of the disease. In HLA-A2+ HAM/TSP patients, HTLV-1-specific T cells were demonstrated to be directed predominantly against one HTLV-1 epitope, namely, Tax11-19. In the present study, we analyzed HLA-A2-restricted HTLV-1 Tax11-19-specific cytotoxic T cells from three patients with HAM/TSP. An analysis of the T-cell receptor (TCR) repertoire of these cells revealed an absence of restricted variable (V) region usage. Different combinations of TCR V alpha and V beta genes were utilized between, but also within, the individual patients for the recognition of Tax11-19. Sequence analysis of the TCR showed evidence for an oligoclonal expansion of few founder T cells in each patient. Apparent structural motifs were identified for the CDR3 regions of the TCR beta chains. One T-cell clone could be detected within the same patient over a period of 3 years. We suggest that these in vivo clonally expanded T cells might play a role in the pathogenesis of HAM/TSP and provide information on HTLV-1-specific TCR which may elucidate the nature of the T cells that infiltrate the central nervous system in HAM/TSP patients.     

Calcium-induced conformational changes of Thrombospondin-1 signature domain: implications for vascular disease

Context: Thrombospondin1 (TSP1) participates in numerous signaling pathways critical for vascular physiology and disease. The conserved signature domain of thrombospondin 1 (TSP1-Sig1) comprises three epidermal growth factor (EGF), 13 calcium-binding type 3 thrombospondin (T3) repeats, and one lectin-like module arranged in a stalk-wire-globe topology. TSP1 is known to be present in both calcium-replete (Holo-) and calcium-depleted (Apo-) state, each with distinct downstream signaling effects.

Objective: To prepare a homology model of TSP1-Sig1 and investigate the effect of calcium on its dynamic structure and interactions.

Methods: A homology model of Holo-TSP1-Sig1 was prepared with TSP2 as template in Swissmodel workspace. The Apo-form of the model was obtained by omitting the bound calcium ions from the homology model. Molecular dynamics (MD) simulation studies (100?ns) were performed on the Holo- and Apo- forms of TSP1 using Gromacs4.6.5.

Results and discussion: After simulation, Holo-TSP1-Sig1 showed significant reorientation at the interface of the EGF1-2 and EGF2-3 modules. The T3 wire is predicted to show the maximum mobility and deviation from the initial model. In Apo-TSP1-Sig1 model, the T3 repeats unfolded and formed coils with predicted increase in flexibility. Apo-TSP1-Sig1model also predicted the exposure of the binding sites for neutrophil elastase, integrin and fibroblast growth factor 2. We present a structural model and hypothesis for the role of TSP1-Sig1 interactions in the development of vascular disorders.

Conclusion: The simulated model of the fully calcium-loaded and calcium-depleted TSP1-Sig1 may enable the development of its interactions as a novel therapeutic target for the treatment of vascular diseases.     

HLA-DRB1 shared epitope genotyping using the revised classification and its association with circulating autoantibodies,acute phase reactants,cytokines and clinical indices of disease activity in a cohort of South African rheumatoid arthritis patients

Introduction  

The revised shared epitope (SE) concept in rheumatoid arthritis (RA) is based on the presence (S) or absence (X) of the SE RAA amino acid motif at positions 72 to 74 of the third hypervariable region of the various human leucocyte antigen (HLA)-DRB1 alleles. The purpose of this study was to investigate SE subtypes on the basis of the American College of Rheumatology 1987 revised criteria for the classification of RA in a cohort of South African RA patients (n = 143) and their association with clinical and circulating biomarkers of disease activity (autoantibodies, acute phase reactants and cytokines).     

Tape stripping the stratum corneum for biomarkers of ultraviolet radiation exposure at sub-erythemal dosages: a study in human volunteers

Abstract

Purpose

Prevalence of skin cancer is rapidly increasing. There is a need for non-invasive biomarkers to assess efficacy of prevention strategies aiming at reduction of exposure to ultraviolet radiation (UVR). Recently, stratum corneum (SC) biomarkers were applied in various inflammatory skin diseases. Here, we explore their suitability as candidate biomarkers for UVR.     

Toxoplasma gondii: The effects of infection at different stages of pregnancy on the offspring of mice

Congenital toxoplasmosis can cause fetal damage in humans and domestic animals. This study was focused on the effects of Toxoplasma gondii (Prugniaud strain) infection at different stages of pregnancy on the offspring of mice. Results showed that newborn mice from all infected groups were significantly lower in weight than those from the control group but significant difference was not found among these groups at day 60 after birth. The survival rate of the offspring from the group of mice infected at the earlier stage of pregnancy was significantly lower than those of infected and control groups. The positive offspring (with cysts found in their brain tissues) born from the mice infected at the earlier and intermediate stages of pregnancy showed a shorter latency and greater number of errors in the step-through passive avoidance test than those born from the mice infected at the late stage of pregnancy, the control group and the negative offspring from the infected groups. The number of cysts in the brain tissue was significantly higher in the offspring born from the groups of mice infected at the earlier and intermediate stages of pregnancy than those from the group of mice infected at the late stage of pregnancy. In addition, our results indicated that a high congenital transmission rate (90%) occurred in this NIH mouse model. In conclusion, the earlier and intermediate maternal infection of T. gondii can result in severe congenital toxoplasmosis, exhibiting conditions such as stillbirth or non-viability, and learning or memory capability damage in this mouse model. These results not only provide useful data for better understanding the effects of T. gondii infection on the offspring of mice infected at different stages of pregnancy but also for better consideration of the effect of this infection on other mammalian hosts including humans.     

Determination of the serum metallothionein (MT)1/2 concentration in patients with Wilson's disease and Menkes disease

We have developed an easy and specific enzyme-linked immunoassay (ELISA) for the simultaneous determination of serum metallothinein-1 (MT-1) and 2 (MT-2) in both humans and experimental animals. A competitive ELISA was established using a specific polyclonal antibody against rat MT-2. The antibody used for this ELISA had exhibited the same cross-reactivity with MT in humans and experimental animals. The NH₂ terminal peptide of MT containing acetylated methionine was shown to be the epitope of this antibody. The reactivity of this ELISA system with the liver, kidney and brain in MT1/2 knock-out mice was significantly low, but was normal in an MT-3 knock-out mouse. The lowest detection limit of this ELISA was 0.6 ng/ml and the spiked MT-1was fully recovered from the plasma.We investigated the normal range of MT1/2 (25–75%tile) in 200 healthy human serum and found it to be 27–48 ng/ml, and this was compared with the serum levels in various liver diseases. The serum MT1/2 levels in chronic hepatitis C (HCV) patients were significantly lower than healthy controls and also other liver diseases. In the chronic hepatitis cases, the MT1/I2 levels increased gradually, followed by the progression of the disease to liver cirrhosis and hepatocellular carcinoma. In particular, we found significantly elevated MT1/2 plasma levels in Wilson's disease patients, levels which were very similar to those in the Long–Evans Cinnamon (LEC) rat (model animal of Wilson's disease). Furthermore, a significantly elevated MT1/2 level was found in patients with Menkes disease, an inborn error of copper metabolism such as Wilson's disease.     

Exposure of any of two proapoptotic domains of presenilin 1-associated protein/mitochondrial carrier homolog 1 on the surface of mitochondria is sufficient for induction of apoptosis in a Bax/Bak-independent manner

Presenilin 1-associated protein/mitochondrial carrier homolog 1 (PSAP/Mtch1) is a proapoptotic outer mitochondrial membrane protein first identified as a presenilin 1-associated protein. The mechanism by which it induces apoptosis upon overexpression in cultured cells is so far unknown. We had previously reported that deletion of two independent regions of PSAP/Mtch1 is required to prevent apoptosis. We now report that mitochondrial targeting of the region containing both proapoptotic domains, or any of them independently, to the outer membrane is sufficient to induce apoptosis. On the other hand, targeting of that region to the surface of the endoplasmic reticulum does not induce apoptosis, indicating that attachment of those domains to the outer mitochondrial membrane, and not just cytosolic exposure, is a requisite for apoptosis. Overexpression of PSAP/Mtch1 in cultured cells causes mitochondrial depolarization and apoptosis that does not depend on Bax or Bak, since apoptosis is induced in mouse embryonic fibroblasts lacking these two proteins. Our results suggest that apoptosis induced by PSAP/Mtch1 likely involves the permeability transition pore.     

Polymorphisms of DNA base‐excision repair genes APE/Ref‐1 and XRCC1 are not associated with the risk for Graves' disease

Oxidative stress has been implicated in etiopathogenesis of Graves' disease (GD). Increased lipid peroxidation and oxidative DNA damage have been found in GD patients. Oxidative DNA damage is mainly repaired by the base‐excision repair (BER) pathway. Polymorphisms in DNA‐repair genes have been associated with the increased risk of various diseases and could also be related to the etiology of GD. Therefore, we conducted a study including 197 patients with GD and age‐ and sex‐matched 303 healthy subjects to examine the role of single‐nucleotide polymorphisms of BER genes, APE/Ref‐1 (codon 148) and XRCC1 (codons 194 and 399) as a risk factor for GD. These polymorphisms were determined by quantitative real‐time PCR and melting curve analysis using LightCycler. No significant association was observed between the variant alleles of APE/Ref‐1 codon 148 [odds ratio (OR) = 0.89, 95% confidence interval (CI) = 0.69–1.17], XRCC1 codon 194 (OR = 1.24, 95% CI = 0.79–1.94), and XRCC1 codon 399 (OR = 1.12, 95% CI = 0.86–1.46) and GD. These preliminary results suggest that APE/Ref‐1 (codon 148) and XRCC1 (codons 194 and 399) polymorphisms are not significant risk factors for developing GD. Copyright © 2009 John Wiley & Sons, Ltd.