Yoshinori Iwatani

Changes in T, B, and NK lymphocyte subsets during and after normal pregnancy.

PROBLEM: Pregnancy affects the maternal immune system and the clinical course of maternal diseases. Here we report the changes in the detailed lymphocyte subsets of helper T cells, suppressor T cells, CD5+ B cells, T cell receptor (TCR) αβ‐positive T cells (Tαβ cells), TCRαβ‐negative T cell (Tγδ cells), and others during and after pregnancy through to one year postpartum, and discuss the significance of the changes.

High Frequency of Antibiotic-Associated Diarrhea due to Toxin A-Negative, Toxin B-Positive Clostridium difficile in a Hospital in Japan and Risk Factors for Infection

Patients hospitalized in a hospital with a high incidence of antibiotic-associated diarrhea due to toxin A-negative, toxin B-positive (A−/B+) Clostridium difficile were retrospectively investigated to determine the clinical manifestations and risk factors for infection. Of 77 Clostridium difficile isolates obtained from 77 patients during the 1-year investigation period, 30 were A−/B+ and 47 were toxin A-positive, toxin B-positive (A+/B+). By pulsed-field gel electrophoresis analysis, 23 of the 30 A−/B+ strains were outbreak-related, suggesting nosocomial spread of a single type of bacterium, which mainly affected patients in the wards of respiratory medicine, hematology and neurology. Using regression analysis, three factors were found to be associated with infection by A−/B+ isolates: (i) exposure to antineoplastic agents (P=0.01, odds ratio [OR]=5.1), (ii) the use of nasal feeding tubes (P=0.008, OR=5.2), and (iii) assignment to a certain internal medicine ward (P=0.05, OR=3.0). Between patients with Clostridium difficile-associated diarrhea caused by A−/B+ strains and those with A+/B+ strains, no statistically significant difference was found in body temperature, serum concentration of C-reactive protein, leukocyte count in whole blood, frequency of diarrhea, or type of underlying disease. These results indicate that A−/B+ strains of Clostridium difficile can cause intestinal infection in humans and they spread nosocomially in the same manner as A+/B+ strains.

Anti-prothrombin antibodies combined with lupus anti-coagulant activity is an essential risk factor for venous thromboembolism in patients with systemic lupus erythematosus.

Anti‐prothrombin antibodies (anti‐prothrombin) and anti‐β2‐glycoprotein I antibodies (anti‐β2‐GP I) are the most common and characterized anti‐phospholipid antibodies (aPL) detected using specific enzyme‐linked immunosorbent assay (ELISA) systems. Recently, lupus anti‐coagulant (LA) activity detected by a phospholipid‐dependent coagulation assay was reported to be associated with anti‐prothrombin and/or anti–β2‐GP I. Here we show that the co‐existence of IgG anti‐prothrombin and LA activity might be an essential risk factor for venous thromboembolism (VTE) in patients with systemic lupus erythematosus (SLE). We examined not only the levels of antibodies to prothrombin and anti‐β2‐GP I (both IgG and IgM isotypes) using an ELISA system, but also LA activity detected using both diluted Russells viper venom time (dRVVT) and STACLOT LA test in 124 patients with SLE. The SLE patients were divided into four groups according to the results of ELISA and LA assay results for each aPL: group A, ELISA+ and LA+ group B, ELISA+ and LA−; group C, ELISA− and LA+ group D, ELISA− and LA−. Regarding IgG anti‐prothrombin, the prevalence of VTE was significantly higher in group A (16/35 cases, 45·7%, P < 0·001, Fishers exact probability test) than in the other groups (B, 2/30, 6·7%; C, 1/22, 4·5%; D, 1/37, 2·7%). With respect to IgM anti‐prothrombin and IgG or IgM anti‐β2‐GP I, the prevalence of VTE was higher in both groups A and C than in group D, but no statistical difference in prevalence was found between groups A and C. Multivariate logistic regression analysis of risk factors for VTE confirmed that the co‐existence of IgG anti‐prothrombin and LA activity was the only significant risk factor for VTE (odds ratio, 19·13; 95% confidence intervals, 4·74–77·18).

Independent Involvement of CD8+CD25+ Cells and Thyroid Autoantibodies in Disease Severity of Hashimoto's Disease

Hashimotos disease (HD) is well known as an autoimmune thyroid disease caused by the destruction of the thyroid follicles, and can be diagnosed in the subclinical stage with thyroid-specific autoantibodies. However, some patients with HD develop hypothyroidism and are treated with thyroxine (severe HD), but most do not throughout their lives (mild HD). To clarify the immunologic differences between these two groups of patients with HD, we examined serum thyroid autoantibodies (antithyroid peroxidase antibodies and antithyroglobulin antibodies), CD4+ CD25+ cells that contain regulatory T cells and activated helper T cells, and CD8+ CD25+ cells that are activated cytotoxic T cells. There was no significant difference in CD4+ CD25+ cells between these HD groups, although the proportion of CD25+ cells within CD4+ cells increased in both groups as compared to normal controls. The serum titers of the thyroid autoantibodies and the proportion of CD25+ cells within CD8+ cells were higher in patients with severe HD than in those with mild HD. There was no correlation between these two parameters, and a two-dimensional analysis with these parameters differentiated these two groups of patients with HD more clearly. These results indicate that both thyroid autoantibodies and CD8+ CD25+ cells are independently involved in the disease severity of HD and CD4+ CD25+ cells are not related to the severity of HD.

Associations Between Autoimmune Thyroid Disease Prognosis and Functional Polymorphisms of Susceptibility Genes, CTLA4, PTPN22, CD40, FCRL3, and ZFAT, Previously Revealed in Genome-wide Association Studies

PurposeGenome-wide association studies have revealed several susceptibility genes among patients with autoimmune thyroid disease (AITD), including CTLA4, PTPN22, FCRL3, and ZFAT. However, any possible association between these genes and AITD prognosis remains unknown. The objective of this study was to identify associations between polymorphisms of these genes and AITD prognosis.MethodsWe genotyped functional polymorphisms, including CTLA4 CT60, CTLA4 +49A/G, CTLA4 -1147C/T, CTLA4 -318C/T, PTPN22 -1123C/G, PTPN22 SNP37, CD40 -1C/T, FCRL3 -169C/T, ZFAT Ex9b-SNP10, and ZFAT Ex9b-SNP2, in 197 AITD patients carefully selected from 456 registered AITD patients, and 86 control subjects. The restriction fragment length polymorphism method was used for genotyping.ResultsThe CD40 -1CC genotype and C allele were significantly more frequent in patients with Graves’ disease (GD) in remission than in those with intractable GD (P = 0.041 and P = 0.031, respectively). The FCRL3 -169TT genotype was significantly less frequent in patients with intractable GD than in those with GD in remission (P = 0.0324). For a ZFAT Ex9b-SNP10 polymorphism, the TT genotype and T allele were significantly more frequent in patients with severe Hashimoto’s disease (HD) than in those with mild HD (P = 0.0029 and P = 0.0049, respectively). For a CTLA4 CT60 polymorphism, the antithyrotropin receptor antibody levels at the onset of GD were significantly higher in those with the GG genotype than in those with other genotypes (P = 0.0117).ConclusionsCD40 and FCRL3 gene polymorphisms were associated with GD intractability, and ZFAT polymorphism was associated with HD severity but not its development.

Strong correlation between the prevalence of cerebral infarction and the presence of anti-cardiolipin/ β2-glycoprotein I and anti-phosphatidylserine/prothrombin antibodies Co-existence of these antibodies enhances ADP-induced platelet activation

Cerebral infarction is the most common arterial thromboembolic complication in the anti-phospholipid antibodies (aPL) syndrome. In an effort to clarify the roles of aPL in the pathogenesis of cerebral infarction in patients with SLE, we examined the levels of anti-cardiolipin/2-glycoprotein I antibodies (anti-CL/beta2-GPI) and anti-phosphatidylserine/prothrombin anti-bodies (anti-PS/PT) in addition to lupus anticoagulant (LA) activity in 126 patients with SLE (35 with cerebral infarction and 91 without thrombosis). Both anti-CL/beta2-GPI and anti-PS/PT strongly correlated with the presence of LA activity. The prevalence of cerebral infarction was obviously higher in the patients who had both anti-CL/beta2-GPI and anti-PS/PT (76.5% [26/34 cases], p<0.0001) than in the other patients having anti-CL/beta2-GPI or anti-PS/PT alone or neither of them (9.8% [9/92 cases]). Furthermore, we studied the in vitro effects of anti-CL/beta2-GPI and/or anti-PS/PT on the enhancement of platelet activation induced by stimulation with a low concentration of adenosine diphosphate (ADP). The purified IgG containing both anti-CL/beta2-GPI and anti-PS/PT caused significant enhancement of platelet activation caused by ADP. However, the purified IgG containing either anti-CL/beta2-GPI or anti-PS/PT had no enhancing effects on it. Furthermore, platelet activation was generated by the mixture of anti-CL/beta2-GPI-IgG and anti-PS/PT-IgG prepared from individual patients, but not by each fraction alone. These results indicate that anti-CL/beta2-GPI and anti-PS/PT may cooperate to promote platelet activation, which may contribute to the risk of cerebral infarction in patients with SLE.

Acquired activated protein C resistance is associated with the co-existence of anti-prothrombin antibodies and lupus anticoagulant activity in patients with systemic lupus erythematosus.

Summary. Venous thromboembolism (VTE) is one of the common manifestations in the anti‐phospholipid (aPL) syndrome. We examined the levels of IgG antibodies (Abs) to β2‐glycoprotein I (β2‐GP I) and prothrombin, lupus anticoagulant (LA) activity, activated protein C resistance (APC‐R), and factor V Leiden in 96 patients with systemic lupus erythematosus (SLE); 19 with VTE and 77 without VTE. Acquired APC‐R, which was not found in any patient with the factor V Leiden mutation, was present in 33 (34·4%) out of the 96 patients with SLE. The presence of acquired APC‐R was a strong risk factor for VTE. The SLE patients were divided into four groups according to the results of enzyme‐linked immunosorbent assay (ELISA) and LA activity for each aPL Abs: ELISA+, LA+; ELISA+, LA–; ELISA–, LA+; and ELISA–, LA–. A significant association was observed between APC‐R and the co‐existence of anti‐β2‐GP I Abs and LA activity or of anti‐prothrombin Abs and LA activity. There was no association between APC‐R and the presence of anti‐β2‐GP I Abs, anti‐prothrombin Abs, or LA activity alone. However, when multivariate logistical regression analysis was performed, it was clear that only the co‐existence of anti‐prothrombin and LA activity was a significant risk factor for APC‐R. These findings indicate that the co‐existence of anti‐prothrombin Abs and LA activity may be an important factor in the pathogenesis of acquired APC‐R in patients with SLE.

Changes in natural killer cell activity in normal pregnant and postpartum women: increases in the first trimester and postpartum period and decrease in late pregnancy

Changes in the activity and number of natural killer (NK) cells in peripheral blood in normal pregnant and postpartum women were examined. NK activity was measured in a 4-h 51Cr-release assay and evaluated by conventional relative lytic units and absolute lytic units which represent the total NK activity within a fixed volume of circulating blood. The number of NK cells was analyzed with FITC-conjugated monoclonal antibodies and by use of an automated flow cytometer. Unexpectedly, the relative NK activity increased in the first trimester and also for 1 month postpartum compared to the activity in normal non-pregnant controls. On the other hand, absolute NK activity decreased in the third trimester compared to the activity in normal non-pregnant controls. The percentage of CD57+ cells decreased in the second trimester, but the percentage of CD16+ cells did not change during pregnancy or the postpartum period. The absolute counts of CD57+ cells and CD16+ cells decreased in the second and third trimesters and increased transiently in the postpartum period. These findings indicate that the increased NK activity in the first trimester and at 1 month postpartum is induced by increased cytotoxic activity of individual NK cells, and that the decreased NK activity in late pregnancy is induced by a decrease in the numbers of NK cells. These physiological changes may play an important role in implantation in early pregnancy, protection of the fetal allograft in late pregnancy and in the natural defense against infection during the puerperal period.

Total iron-binding capacity calculated from serum transferrin concentration or serum iron concentration and unsaturated iron-binding capacity.

Total iron-binding capacity (TIBC) indicates the maximum amount of iron needed to saturate plasma or serum transferrin (TRF), which is the primary iron-transport protein (1). Theoretically, 1 mol of TRF [average molecular mass, 79 570 Da (2)] can bind 2 mol of iron (55.8 Da) at two high-affinity binding sites for ferric iron (3). Therefore, TIBC correlates well with TRF concentration, and the theoretical ratio of TIBC (in μmol/L) to TRF (in g/L) is 25.1: TIBC (μmol/L) = 25.1 × TRF (g/L) (4)(5). Measurements of TIBC, serum iron, and the percentage of iron saturation of TRF are useful for the clinical diagnosis of iron-deficiency anemia and chronic inflammatory disorders (6)(7) and as screening tests for other clinical conditions (8). TIBC is routinely determined (9)(10)(11)(12) by saturation of TRF with an excess predetermined amount of iron, removal of the unbound iron, and measurement of the iron that is dissociated from TRF. For removal of the unbound iron, magnesium carbonate (9), ion-exchange resin (10), alumina columns (11), or magnetic particles(12) are used. Most direct TIBC measurement methods require manual procedures that involve centrifugation or pretreatment of serum samples. As an alternative to direct measurement methods, TIBC values are also calculated from the sum of serum iron and unsaturated iron-binding capacity (UIBC), both of which are determined by colorimetric methods (calculation method). We developed a direct and fully automated TIBC (DTIBC) assay for use with an automated multipurpose analyzer (13)(14). A fully automated TIBC measurement method is also commercially available (15). In our previous study (14), TIBC values obtained by DTIBC assay correlated strongly with serum TRF concentrations ( r = 0.984; n = 59), and the slope of the regression line was consistent with the theoretical TIBC/TRF ratio. We …

Effects of various isolation methods for human peripheral lymphocytes on T cell subsets determined in a fluorescence activated cell sorter (FACS), and demonstration of a sex difference of suppressor/cytotoxic T cells

The effects of various methods for removal of monocytes and residual erythrocytes from human peripheral blood mononuclear cells (PBMC) on T cell subsets were examined. T cell subsets were determined in a fluorescence activated cell sorter (FACS) using fluorescein-conjugated monoclonal antibodies (anti-Leu-1, anti-Leu-2a and anti-Leu-3a antibodies). Removal of monocytes by methods based on adherence or phagocytosis decreased yields of lymphocytes and caused changes in the percentage and/or the fluorescence intensity of T cell subsets. Exclusion of monocytes from the FACS analysis by setting of the scatter gates was incomplete (about 80%). Removal of residual erythrocytes after Ficoll separation by ammonium chloride treatment also changed the percentage of T cell subsets. A method using PBMC without removal of monocytes and erythrocytes was chosen as best and simplest for routine use in FACS analysis of lymphocytes. Erythrocytes could be excluded from the FACS analysis by setting the scatter gates and the percentage of T cell subsets was corrected after measurement of monocytes, identified by peroxidase staining. The reproducibility of measurements of T cell subsets made at different times was examined using PBMC obtained from the same healthy man during 12 weeks. For standardization of the assay, the peak positions of scatter and fluorescence intensity of each PBMC labeled with anti-Leu-3a were adjusted to standard values in each FACS analysis. Under these conditions, variations of other parameters of this standard PBMC were very small in 12 different assays. Using this standard PBMC, satisfactory, reproducible results were also observed on PBMC obtained from another normal subject. Therefore, this standard PBMC labeled with anti-Leu-3a was used as a standard in FACS analysis. Under these accurately standardized conditions, it was demonstrated that the peak position of fluorescence intensity of Leu-2a (suppressor/cytotoxic T) cells was significantly lower (P less than 0.01) in women than in men.