Favorable clinical response and drug retention of anti-IL-6 receptor inhibitor in rheumatoid arthritis with high CRP levels: the ANSWER cohort study.

OBJECTIVE: Currently, biological disease-modifying anti-rheumatic drugs (bDMARDs) with different modes of action [tumour necrosis factor inhibitor (TNFi), interleukin-6 receptor inhibitor (IL-6Ri), or cytotoxic T-lymphocyte antigen 4-immunoglobulin (CTLA4-Ig)] are used in clinical practice to treat rheumatoid arthritis (RA). However, it is unclear which type of bDMARD is the most efficacious for a specific clinical situation. C-reactive protein (CRP) is an acute-phase reactant driven by IL-6 signalling. Here, we aimed to establish whether therapeutic efficacy differs between IL-6Ri and other bDMARDs with alternative modes of action in RA patients according to their CRP level. METHOD: RA patients treated with bDMARDs were enrolled from an observational multicentre registry in Japan. Patients were classified into three groups according to baseline CRP tertiles. The overall 3 year retention rates of each bDMARD category were assessed. The Clinical Disease Activity Index (CDAI) was also assessed before and 3, 6, and 12 months after bDMARD initiation. RESULTS: A total of 1438 RA patients were included and classified into three groups according to tertiles of baseline CRP levels (CRP1, 0-0.3; CRP2, 0.3-1.8; CRP3, 1.8-18.4 mg/dL). In CRP3, the overall 3 year drug retention rates were significantly higher for IL-6Ri than for TNFi and CTLA4-Ig (77.5 vs 48.2 vs 67.3, respectively). No significant difference was evident in terms of CDAI 12 months after bDMARD initiation in CRP1-CRP3. CONCLUSION: IL-6Ri may be a favourable therapeutic option over TNFi and CTLA4-Ig in RA patients with high CRP levels.

Myositis with sarcoplasmic inclusions in Nakajo-Nishimura syndrome: a genetic inflammatory myopathy.

AIMS: Nakajo-Nishimura syndrome (NNS) is an autosomal recessive disease caused by biallelic mutations in the PSMB8 gene that encodes the immunoproteasome subunit ß5i. There have been only a limited number of reports on the clinicopathological features of the disease in genetically confirmed cases. METHODS: We studied clinical and pathological features of three NNS patients who all carry the homozygous p.G201V mutations in PSMB8. Patients' muscle specimens were analysed with histology and immunohistochemistry. RESULTS: All patients had episodes of typical periodic fever and skin rash, and later developed progressive muscle weakness and atrophy, similar to previous reports. Oral corticosteroid was used for treatment but showed no obvious efficacy. On muscle pathology, lymphocytes were present in the endomysium surrounding non-necrotic fibres, as well as in the perimysium perivascular area. Nearly all fibres strongly expressed MHC-I in the sarcolemma. In the eldest patient, there were abnormal protein aggregates in the sarcoplasm, immunoreactive to p62, TDP-43 and ubiquitin antibodies. CONCLUSIONS: These results suggest that inflammation, inclusion pathology and aggregation of abnormal proteins underlie the progressive clinical course of the NNS pathomechanism.

Utility of osteopontin in cerebrospinal fluid as a diagnostic marker for neuropsychiatric systemic lupus erythematosus.

The whole protein of osteopontin (OPN full) and its cleaved form (OPN N-half) are involved in the immune response and the migration of immune cells to an inflammatory lesion. We have reported that serum OPN full and urine OPN N-half are elevated in lupus nephritis (LN). Neuropsychiatric systemic lupus erythematosus (NPSLE) is a refractory complication of SLE. To investigate whether OPN full and OPN N-half could serve as diagnostic markers for NPSLE, and to elucidate their role in NPSLE pathogenesis, the concentrations of OPN full and OPN N-half in cerebrospinal fluid (CSF) were measured in NPSLE and non-NPSLE patients. We found that the concentration of OPN full in the CSF was significantly higher in NPSLE than in non-NPSLE, and it decreased after treatment. When the cutoff value of OPN full in CSF was set to 963.4 ng/ml, the sensitivity and specificity for the diagnosis of NPSLE were 70% and 100%, respectively. The correlation analysis of OPN full, OPN N-half and various cytokines/chemokines suggested that the cytokines/chemokines could be divided into two clusters: cluster A, which contains OPN full and cluster B, which contains interleukin-6. OPN full in CSF could be a novel diagnostic marker for NPSLE.

Interleukin-18 is associated with the presence of interstitial lung disease in rheumatoid arthritis: a cross-sectional study.

OBJECTIVE: Serum interleukin-18 (IL-18) levels are increased in patients with interstitial lung disease (ILD). In addition, IL-18 levels are increased in patients with rheumatoid arthritis (RA) and are associated with arthritis activity. We determined whether increased IL-18 levels are associated with ILD in RA. METHOD: RA patients were enrolled using an RA cohort database. Plasma IL-18 levels were measured by enzyme-linked immunosorbent assay. ILD was determined by a pulmonologist and a radiologist based on chest radiography and computed tomography findings. IL-18 levels for RA with ILD and RA without ILD were compared. Associations between ILD and various markers including IL-18 and confounding factors (e.g. smoking history) were investigated by logistic regression analysis. Diagnostic values of IL-18 for the presence of ILD were investigated using receiver operating characteristics curve analysis. RESULTS: ILD was complicated in 8.2% (n = 26) of the study population (N = 312). Plasma IL-18 levels were higher for RA patients with ILD than for RA patients without ILD (721.0 ± 481.4 vs 436.8 ± 438.9 pg/mL, p < 0.001). IL-18, Krebs von den Lungen-6, and anti-cyclic citrullinated peptide antibody titre and glucocorticoid doses were independently associated with the presence of ILD during multivariate logistic regression analysis. Sensitivity and specificity of IL-18 levels for the detection of ILD in RA patients were 65.3% and 76.3%, respectively (area under the curve = 0.73). CONCLUSION: Plasma IL-18 levels were higher for RA patients with ILD than for those without ILD. Increased IL-18 levels were associated with the presence of ILD.

Effects of statins on thrombosis development in patients with systemic lupus erythematosus and antiphospholipid antibodies.

The objective of this study is to identify the effects of statins and risk factors for thrombosis in patients with new onset of systemic lupus erythematosus (SLE) with or without antiphospholipid antibodies (aPL). Consecutive patients with SLE without history of thrombotic events were retrospectively enrolled from April 1997 to February 2014. The development of first thrombosis and death caused by thrombosis were defined as the study endpoint. Risk and protective factors for developing thrombosis were analyzed. A total of 152 patients, 80 positive and 72 negative for aPL, were included. In aPL-positive patients, 15 developed arterial ( n = 6) and venous ( n = 9) thrombosis (median follow-up period 69 months). Cox's proportional hazards model showed that older age at SLE onset and IgG-anticardiolipin antibodies (aCL) were statistically significant risks for thrombosis. Statin therapy was identified as a statistically significant protective factor against thrombosis (hazard ratio 0.12, 95% confidence interval 0.01-0.98). In aPL-negative patients (median follow-up period 46 months), seven patients developed thrombosis (five arterial and two venous). No risk factors for thrombosis were found in this group. In aPL-positive patients with SLE, the late disease onset and the presence of IgG-aCL represented additional risk factors for thrombosis. Statin treatment appeared as a protective factor for thrombosis.

Association between anti-U1 ribonucleoprotein antibodies and inflammatory mediators in cerebrospinal fluid of patients with neuropsychiatric systemic lupus erythematosus.

OBJECTIVE: We investigated possible associations between neurotoxic inflammatory mediators (IMs) and anti-U1RNP antibodies (Abs) in cerebrospinal fluid (CSF) of neuropsychiatric systemic lupus erythematosus (NPSLE). METHODS: Serum and CSF anti-U1RNP Abs were detected using an RNA-immunoprecipitation assay and CSF anti-U1RNP Ab levels were measured by ELISA. IFN-α, MCP-1 and IL-8 levels in CSF were determined by quantitative multiplex cytokine analysis. IM levels were compared among anti-U1RNP-positive and anti-U1RNP-negative NPSLE as well as other rheumatic disease controls (controls). RESULTS: Anti-U1RNP Abs were detected in serum (58%) and in CSF (18%) of 82 NPSLE patients. CSF MCP-1 levels were higher in NPSLE than in controls. CSF IFN-α level was higher in CSF anti-U1RNP Ab-positive than in -negative patients or controls. When limited to serum anti-U1RNP Ab-positive patients, however, levels of all three IMs in CSF were higher in CSF anti-U1RNP Ab-positive than in -negative patients. Anti-U1RNP Ab levels in CSF correlated with CSF MCP-1, but not IFN-α and IL-8 levels. CONCLUSIONS: CSF anti-U1RNP Ab positivity is associated with increased level of CSF IFN-α. MCP-1 levels correlated with CSF anti-U1RNP Ab levels, whereas the increased CSF MCP-1 was not specific to CSF anti-U1RNP Ab-positive NPSLE.

Serum milk fat globule epidermal growth factor 8 elevation may subdivide systemic lupus erythematosus into two pathophysiologically distinct subsets.

OBJECTIVE: Impaired clearance of apoptotic cells is a potential trigger of systemic lupus erythematosus (SLE). Milk fat globule epidermal growth factor 8 (MFG-E8) plays an important role in the clearance of dying cells. Previously, we reported serum MFG-E8 was elevated in some SLE patients. Here we further investigated the prevalence of MFG-E8 in active SLE and other autoimmune diseases and also tried to clarify the characteristics of MFG-E8-positive and -negative SLE. METHODS: Serum MFG-E8 was measured in 40 active non-treated SLE patients, 104 disease controls and 104 healthy controls by ELISA. Clinical characteristics and serum cytokine profiles were compared between MFG-E8-positive and MFG-E8-negative SLE patients. RESULTS: Prevalence of MFG-E8 was significantly higher in SLE patients (40%) than in various controls (p < 0.05). MFG-E8 level became negative after treatment, and increased again upon relapse. When compared, MFG-E8-positive SLE patients showed higher immune complex (p = 0.021) and lower complement (p = 0.004 for CH50). In contrast, MFG-E8-negative SLE patients tended to show higher CRP (p = 0.094). There was a positive correlation between MFG-E8 level and immune complex level (r s = 0.49, p = 0.049). TNF-α (p = 0.019), IFN-γ (p = 0.031) and IL-10 (p = 0.013) were significantly higher in MFG-E8-positive SLE. CONCLUSION: MFG-E8-positive SLE and -negative SLE may have different clinical features, the one with stronger immunological response and the other with stronger inflammatory response, and those two groups may be two distinct subtypes of SLE driven by different mechanisms. Further, MFG-E8 could be used as a biomarker for diagnosis and monitoring of disease activity in certain SLE patients.

Photoinduced properties of anodized Ti alloys for biomaterial applications.

The photocatalytic properties of anodic oxides on a newly developed TiNbSn and commonly used Ti6Al4V alloys as biomaterials were investigated. The alloys were anodized in an electrolyte of sodium tartrate acid with H2O2 at a high voltage and the mechanism of the photocatalytic and antiviral activities was studied. The anodized TiNbSn and Ti6Al4V exhibited highly crystallized rutile TiO2 and poorly crystallized anatase TiO2, respectively. X-ray photoelectron spectroscopy analysis revealed the presence of oxides of the alloying elements in addition to TiO2. The anodized TiNbSn exhibited higher activities than Ti6Al4V, and electron spin resonance spectra indicated that the number of hydroxyl radicals (⋅OH) generated from the anodized TiNbSn was higher than that from the anodized Ti6Al4V. The results can be explained by two possible mechanisms: the higher crystallinity of TiO2 on TiNbSn than that on the Ti6Al4V reduces the number of charge recombination sites and generates abundant ⋅OH; charge separation in the anodic oxide on TiNbSn due to the electronic band structure between TiO2 and the oxides of alloying elements enhances photo activities. The excellent photoinduced characteristics of the anodized TiNbSn are expected to contribute to the safe and reliable implant treatment.

Type I interferon induces CX3CL1 (fractalkine) and CCL5 (RANTES) production in human pulmonary vascular endothelial cells.

Type I interferon (IFN) medications cause various adverse reactions, including vascular diseases. Although an association between chemokines and vascular diseases has also been reported, the relationship between type I IFN and chemokines in vascular endothelial cells (VEC) remains unclear. To provide clues to pathogenesis of the diseases, we analysed the effects of type I IFN on chemokine production in human VEC. Type I IFN induced higher CX3CL1 (fractalkine) mRNA expression and protein secretion in pulmonary arterial VEC than in umbilical vein VEC. Type I IFN also induced CCL5 [regulated upon activation normal T cell expressed and secreted (RANTES)] production in VEC, especially in lung micro-VEC. IFN-ß induced much higher chemokine production than IFN-α, and Janus protein tyrosine kinase (JAK) inhibitor I prevented type I IFN-induced chemokine secretion. Type I IFN-induced chemokines may be involved in the pathophysiology of pulmonary vascular diseases, and the JAK inhibitor may serve as a therapeutic option for these diseases.

Interferon-gamma release assay for diagnosing Mycobacterium tuberculosis infections in patients with systemic lupus erythematosus.

Our aim was to analyze the performance of an interferon-gamma release assay, QuantiFERON-TB Gold (QFT-2G), for diagnosing Mycobacterium tuberculosis (MTB) infection in patients with systemic lupus erythematosus (SLE). We performed the QFT-2G and tuberculin skin test (TST) in 71 SLE patients. The QFT-2G results of 279 patients with other connective tissue diseases (CTD) and 35 healthy controls were analyzed. Of the 71 SLE patients, two (2.8%) were positive and 46 (64.8%) were negative by QFT-2G. All SLE patients had no evidence of active MTB infection, apart from one. QFT-2G produced a significantly higher number of indeterminate results in patients with SLE (23/71, 32.4%) compared with those with other CTD (5.7%) or healthy controls (0%) (p < 0.0001 and p < 0.0001). Decreased lymphocyte counts and high SLEDAI scores in SLE patients were shown to be risk factors for indeterminate results by multivariate analysis (p = 0.02 and p = 0.04). Among all patients with CTD, SLE itself and lymphocytopenia were found to be independent risks for indeterminate results (p = 0.00000625 and p = 0.000107). In conclusion, QFT-2G may have more potential to assist in the diagnosis of active and latent MTB infection than TST in SLE patients. However, because of the high frequency of indeterminate results, caution must be used when interpreting the results of QFT-2G among SLE patients, especially those who have parallel or subsequent flares.

Genetic influences on the end-stage effector phase of arthritis.

K/BxN T cell receptor transgenic mice are a model of inflammatory arthritis, most similar to rheumatoid arthritis, that is critically dependent on both T and B lymphocytes. Transfer of serum, or just immunoglobulins, from arthritic K/BxN animals into healthy recipients provokes arthritis efficiently, rapidly, and with high penetrance. We have explored the genetic heterogeneity in the response to serum transfer, thereby focussing on the end-stage effector phase of arthritis, leap-frogging the initiating events. Inbred mouse strains showed clear variability in their responses. A few were entirely refractory to disease induction, and those which did develop disease exhibited a range of severities. F1 analyses suggested that in most cases susceptibility was controlled in a polygenic additive fashion. One responder/nonresponder pair (C57Bl/6 x NOD) was studied in detail via a genome scan of F2 mice; supplementary information was provided by the examination of knock-out and congenic strains. Two genomic regions that are major, additive determinants of the rapidity and severity of K/BxN serum-transferred arthritis were highlighted. Concerning the first region, on proximal chromosome (chr)2, candidate assignment to the complement gene C5 was confirmed by both strain segregation analysis and functional data. Concerning the second, on distal chr1, coinciding with the Sle1 locus implicated in susceptibility to lupus-like autoimmune disease, a contribution by the fcgr2 candidate gene was excluded. Two other regions, on chr12 and chr18 may also contribute to susceptibility to serum-transferred arthritis, albeit to a more limited degree. The contributions of these loci are additive, but gene dosage effects at the C5 locus are such that it largely dominates. The clarity of these results argues that our focus on the terminal effector phase of arthritis in the K/BxN model will bear fruit.

Bioactive TiNbSn alloy prepared by anodization in sulfuric acid electrolytes.

The bioactivity of anodized near-ß TiNbSn alloy with low Young's modulus prepared in sulfuric acid electrolytes was examined to explore the osseointegration mechanism with a focus on the role of anodic oxide. Hydroxyapatite (HA) precipitated on the surface of anodic oxide following immersion in Hank's solution, and precipitation accelerated with increase in the sulfuric acid concentration of the electrolyte. HA is formed on the surface of as-anodized oxide without subsequent annealing or hot water (HW) treatment. This outcome differs from that of a previous study using anodized TiNbSn alloy prepared in acetic acid electrolytes requiring for subsequent HW treatment. It was found that the oxide anodized in sulfuric acid electrolyte contains a large amount of internal pores and is highly crystallized thick TiO2, whereas the same prepared in the acetic acid electrolyte is low crystalline thin TiO2 containing a small amount of pores. The present anodized TiNbSn alloy is preferred for maintaining the low Young's modulus of the alloy and eliminating the subsequent treatment to increase the Young's modulus. A model to rationalize the bioactivity of the present anodic oxide is proposed based on the series of studies. It is concluded that the sulfuric acid electrolyte is favorable for both HA formation and low Young's modulus, and the bioactivity is attributed to the anodic TiO2 that facilitates incorporation of bone ingredients.

Cytokine-related and sodium channel polymorphism as candidate predisposing factors for childhood encephalopathy FIRES/AERRPS.

Febrile infection-related epilepsy syndrome (FIRES), or acute encephalitis with refractory, repetitive partial seizures (AERRPS), is an epileptic encephalopathy beginning with fever-mediated seizures. The etiology remains unclear. To elucidate the genetic background of FIRES/AERRPS (hereafter FIRES), we recruited 19 Japanese patients, genotyped polymorphisms of the IL1B, IL6, IL10, TNFA, IL1RN, SCN1A and SCN2A genes, and compared their frequency between the patients and controls. For IL1RN, the frequency of a variable number of tandem repeat (VNTR) allele, RN2, was significantly higher in the patients than in controls (p=0.0067), and A allele at rs4251981 in 5' upstream of IL1RN with borderline significance (p=0.015). Haplotype containing RN2 was associated with an increased risk of FIRES (OR 3.88, 95%CI 1.40-10.8, p=0.0057). For SCN1A, no polymorphisms showed a significant association, whereas a missense mutation, R1575C, was found in two patients. For SCN2A, the minor allele frequency of G allele at rs1864885 was higher in patients with borderline significance (p=0.011). We demonstrated the association of IL1RN haplotype containing RN2 with FIRES, and showed a possible association of IL1RN rs4251981 G>A and SCN2A rs1864885 A>G, in Japanese patients. These preliminary findings suggest the involvement of multiple genetic factors in FIRES, which needs to be confirmed by future studies in a larger number of FIRES cases.

Monoclonal antibodies to a CD4 peptide derivative which includes the region corresponding to an immunoglobulin CDR3: evidence of the involvement of pre-CDR3-related region in HIV-1 and host cell interaction.

A CD4 peptide of amino acid residues 68-130 [CD4(68-130)], which had the capacities to inhibit HIV-1 replication and HIV-1-induced syncytium formation, was used as an immunogen for the preparation of mAb. The mAbs prepared were classified into at least five types (I-V) in terms of their recognition sites by ELISA using various kinds of smaller CD4 peptides. Among them, the type I mAb no. 35 recognizing amino acid residues 72-84, which lies just before the region corresponding to an immunoglobulin third complementarity-determining region (CDR3), showed the strongest effects in reducing both HIV-1 infection and HIV-1-induced syncytium formation, although a large amount of no. 35 mAb was necessary to reduce such HIV-1 activities compared with those of anti-Leu-3a and OKT4A mAbs which recognize CD4 epitopes near a portion corresponding to an immunoglobulin CDR2. Western blot analysis showed that the reactivities of CD4 molecule in CD4-positive cells or sCD4 molecule with types I-V mAbs were stronger than that with anti-Leu-3a mAb. Flow cytometry showed that no. 35 mAb was faintly reactive with native CD4 molecule on cell surface at the concn showing the inhibitory effects on HIV-1 infection and syncytium formation. In addition, a smaller peptide CD4(66-92), one of the good epitope peptides for no. 35 mAb, also showed strong inhibitory effect on HIV-1 infection as well as a weaker inhibitory effect on syncytium formation. These results suggest that, in addition to the CD4 CDR2-related region, the pre-CDR3-related region is also involved in the early events of the interactions between the host cell and HIV-1.

Renotropic activity in ovine luteinizing hormone isoform(s).

Renotropic activity was previously demonstrated in an ovine LH preparation. This preparation was further purified with a series of chromatographic steps, and the fractions were assayed for renotropic activity in vivo by their ability to stimulate [3H]thymidine incorporation into renal DNA of castrated hypophysectomized male rats. A purified preparation could be dissociated by acid treatment into two major constituent subunits, designated alpha and beta, each of which was composed of three microheterogeneous components (subunits alpha 1-3 and beta 1-3) by reverse phase HPLC. Peptide mapping, including amino acid analyses and partial sequencing of the purified peptides, showed that 1) subunits alpha 3 and beta 3 possess the full length of the polypeptide chains, with the same amino acid sequences as those of the corresponding LH subunits alpha and beta, respectively; and 2) subunits alpha 1 and alpha 2 are complexes of three polypeptides which are missing several N-terminal residues from subunit alpha 3. Conversely, subunits beta 1 and beta 2 lack the C-terminal two residues and one residue, respectively, of subunit beta 3. Renotropic activity was not detected in any of the dissociated subunits alone, but association of alpha 1-3 with beta 1-3 reconstituted the hormonal activity with different potencies. In particular, combination of subunits alpha 3 and beta 3 (alpha 3.beta 3) yielded a potent renotropic activity with weak gonadotropic activity. The carbohydrate composition of the purified preparation exhibiting renotropic activity differed from that of a reference oLH preparation, which possessed greater gonadotropic activity but was devoid of renotropic activity. Furthermore, renotropic activity was decreased after removal of sialic acid by treatment with neuraminidase. Thus, the oligosaccharide moieties as well as the amino acid sequences of the subunits may play an important role in the expression of renotropic activity in vivo, these effects over and above those arising from differential metabolic clearance. We conclude that pituitary renotropin represents a novel activity of a LH- isoform(s) and that the posttranslational (or the artificial, i.e. during preparation) modification of the constituent LH subunits may be responsible for modulation of renotropic activity as well as the intrinsic gonadotropic activity.

Porcine luteinizing hormone isoform(s): relationship between their molecular structures, and renotropic versus gonadotropic activities.

We recently demonstrated the renotropic activity in ovine LH isoform(s). In this study we purified porcine (p) LH isoforms and analyzed their structures and bioactivities. Purified pLH preparation (G-100 fraction 3) was dissociated into alpha- and beta-subunits, followed by isolation with reverse phase HPLC. Six components were isolated and analyzed for their amino acid compositions and amino-terminal amino acid sequences. alpha-Subunits were found to have heterogeneous N-terminal sequences, which started at Phe-1, Gly-4, Phe-6, or Thr-7. This N-terminal heterogeneity and the presence of the initial 6 amino acids has not been reported previously to the best of our knowledge. Moreover, the first 10 residues, Phe-Pro-Asp-Gly-Glu-Phe-Thr-Met-Gln-Gly, were identical with those of the ovine LH alpha subunit. At least 3 different beta-subunits were identified as heterogeneous in their carboxyl-terminal amino acids. The pLH preparation (G-100-fraction 3) was then chromatographed by an isoelectrofocusing (IEF) column. Four IEF fractions were obtained. Their amino acid structures appeared to be identical, as judged by the identical elution profiles on reverse phase HPLC, but their carbohydrate compositions were slightly different, especially in N-acetylgalactosamine in alpha-subunits. Thus, fractionation on IEF depended on the heterogeneity in carbohydrate structures. Each IEF fraction had different potencies in terms of its gonadotropic activity (in vitro) and renotropic activity (in vivo and in vitro). The discrepancy was observed not only between gonadotropic activity and renotropic activity, but also between in vivo and in vitro renotropic activity. This study identified the structural heterogeneity of pLH isoforms and demonstrated their biological heterogeneity. We concluded that the carbohydrate structure of the pLH isoform is important for expressing its biological heterogeneity (gonadotropic and renotropic activity.

Hypomethylation of the proximal and intronic regulatory regions of the IFN-gamma gene is not essential for its transcription by naive CD4+ T cells cultured with IL-4.

Recently, long-term preculture with IL-4 or IL-7 has been reported to induce IFN-gamma-producing ability in naive CD4+ T cells without stimulation via TCR. The mechanism of IFN-gamma-transcription in naive CD4+ T cells precultured with IL-4 was analyzed and compared with that in typical Th1 cells by focusing on the TATA proximal and first intronic regulatory regions of the IFN-gamma gene. Both regulatory regions in these IL-4-primed naive CD4+ T cells, which produce a large amount of IFN-gamma upon stimulation with PMA and ionomycin, were completely methylated in contrast to the same hypomethylated regions in Th1 cells. DNase I hypersensitive site analysis suggested that both regulatory regions in IL-4-primed naive CD4+ T cells were not active for IFN-gamma-expression. Moreover, we demonstrated that the composition of transcriptional factors that can bind to the proximal regulatory region is different between IL-4-primed naive CD4+ T cells and Th1 cells. These results indicated that the transcriptional machinery involved in the expression of the IFN-gamma gene by CD4+ T cells varied depending on their modes of differentiation in both the responsive regulatory regions and the specific nuclear factors.

Further characterization of memory T cells existing in a case of CD8 deficiency.

CD8 deficiency is a rare primary immunodeficiency caused by a defect of ZAP-70, which plays a pivotal role in T cell activation. We previously reported the existence of memory phenotype-CD4+ T cells in a case of CD8 deficiency, which demonstrates that activation signals through ZAP-70 are not essential to the phenotypic conversion of T cells from "naive" to "memory." In this study, we further characterized CD45RO+ T cells in a CD8 deficient patient. We showed that the patient's CD45RO+ T cell population had a wide variety of T cell receptor Vbeta-chain gene usage, and contained few clonally expanded T cells, while many clonally expanded T cells were present in the memory T cell population of age-matched healthy children. These results suggest that various kinds of antigens were involved in the differentiation of the patient's T cells, and that the differentiation into memory T cells was not accompanied by profound T cell proliferation. Moreover, our findings confirmed that the patient's CD45RO+CD4+ T cells had acquired effector-cytokine producing ability, indicating that there exists an alternative activation pathway which is independent of ZAP-70 for the acquisition of effector-cytokine producing ability.