Epstein-Barr virus DNA load and seroconversion in pediatric renal transplantation with tacrolimus immunosuppression

EBV infection is one of major complications arising in pediatric patients who have undergone renal transplantation. A strong correlation between the grade of immunosuppression and the development of PTLD, one of the most severe EBV-associated diseases, has been recognized. In this study, we monitored the serologic profile in conjunction with peripheral blood EBV-DNA load of 32 children who underwent renal transplantation with tacrolimus as an immunosuppressant. Six patients were EBV-seronegative (EBV-) before the transplantation, and the mean DNA load in the EBV- group was significantly higher than that in the EBV-seropositive (EBV+) group. Seroconversion occurred in five of these patients in a mean period of 22 weeks after the transplantation. The EBV-DNA load in the EBV+ group was maintained at a low level for a year, whereas it increased rapidly to over 1 x 10(5) copies/mL in two patients in the EBV- group three to seven months after the transplantation, which corresponds to the timing of seroconversion, and one of them developed PTLD. These observations suggest that the close monitoring of the EBV-DNA load, along with longitudinal observation of seroconversion, is essential in pediatric renal transplantation, particularly for younger children who are more likely to be EVB-.     

Age- and sex-related changes in amine sulphoconjugations in Sprague-Dawley strain rats. Comparison with phenol and alcohol sulphoconjugations

Age- and sex-related changes in sulphoconjugation were investigated in hepatic 105 000 g supernatants of three-week-old (young), seven-week-old (young adult), one-year-old (middle-aged) and two-year-old (old) Sprague-Dawley rats in the presence of biologically active sulphate. The supernatants of seven-week-old, one-year-old and two-year-old female rats catalysed the sulphoconjugations of alcohol (tiaramide as substrate), and alicyclic amine, alkylamine and arylamine more rapidly than those of the respective males, but the supernatants of three-week-old rats did not exhibit significant sex-related differences. The sulphoconjugations by the supernatants of female rats were substantially constant during ageing. On the other hand, the supernatants of seven-week-old male rats catalysed phenol (beta-naphthol as substrate) sulphoconjugation more rapidly than those of seven-week-old females, but the supernatants of two-year-old rats did not exhibit this sex-related difference. These results indicate that the substrates for sulphoconjugation could be divided into at least two groups: an alcohol and amine type, and a phenol type.     

A two-amino acid insertion in the Cys146- Cys167 loop of the alphaIIb subunit is associated with a variant of Glanzmann thrombasthenia. Critical role of Asp163 in ligand binding.

The ligand binding site(s) of the alpha subunit of integrin alphaIIb beta3 (GPIIb-IIIa), a prototypic non-I domain integrin, remains elusive. In this study, we have characterized a Japanese variant of Glanzmann thrombasthenia, KO, whose platelets express normal amounts of alphaIIb beta3. KO platelets failed to bind the activation-independent ligand-mimetic mAb OP-G2 and did not bind fibrinogen or the activation-dependent ligand-mimetic mAb PAC-1 following activation of alphaIIb beta3 under any condition examined. Sequence analysis of PCR fragments derived from KO platelet mRNA revealed a 6-bp insertion leading to a 2-amino-acid insertion (Arg-Thr) between residues 160 and 161 of the alphaIIb subunit. Introduction of the insertion into wild-type recombinant alphaIIb beta3 expressed in 293 cells led to the normal expression of alphaIIb beta3 having the defect in ligand binding function. The insertion is located within the small loop (Cys146-Cys167) in the third NH2-terminal repeat of the alphaIIb subunit. Alanine substitution of each of the oxygenated residues within the loop (Thr150, Ser152, Glu157, Asp159, Ser161, and Asp163) did not significantly affect expression of alphaIIbbeta3, and only Asp163AlaalphaIIb beta3 abolished the ligand binding function. In addition, Asp163AlaalphaIIb beta3 as well as KO mutant alphaIIb beta3 constitutively expressed the PMI-1 epitope. Our present data suggest that Asp163 of the alphaIIb subunit is one of the critical residues for ligand binding.     

Demonstration of a marked reduction in the amount of GPIIb in most type II patients with Glanzmann's thrombasthenia

Summary. In this study employing a sensitive immunoblot assay, we have characterized GPIIb and GPIIIa in thrombasthenic platelets from seven type II and four type I patients from 10 unrelated families. The amounts of GPIIb and GPIIIa were both markedly reduced in all these patients, and abnormal molecular weight GPIIb or GPIIIa was not detected. In all of four type I patients the amount of GPIIb was much lower than that of GPIIIa. In this study, however, we found that the amount of GPIIb was also lower even in six out of seven type II patients. Immunodepletion of patients'platelets with AP2 (a monoclonal antibody specific for the GPIIb-IIIa complex), AP3 (specific for GPIIIa) or AMF7 (specific for αv) further confirmed that GPIIIa existed in excess, and demonstrated that excess GPIIIa were mostly in free form and not associated with GPIIb or αv. The reduction of GPIIb may represent an abnormality in GPIIb processing in these type II and type I thrombasthenic platelets. It remains unclear whether these two subgroups represent distinct categories.     

An infant case of Graves' disease with ophthalmopathy

Graves' disease is a rare disorder in children, particularly in infants. Ocular manifestations of Graves' disease in children are even more rare and are mild compared to adults. We report a 3-year-old girl with Graves' ophthalmopathy who visited our clinic because of lacrimation. Her family had also noticed exophthalmos, goiter, irritability and increased appetite for more than 3 months. The ophthalmologist noted bilateral proptosis, eyelid erythema, lacrimation, entropion of the lower eyelid, and superficial keratitis. Her serum concentrations of free thyroxine and free triiodothyronine were high, and thyroid-stimulating hormone (TSH) was low. Serum samples were markedly positive for antibodies to TSH receptor (TRAb) and thyroid-stimulating antibody (TSAb). Although hyperthyroidism was controlled with propylthiouracil within 3 weeks, her eye signs did not improve. We administered methylprednisolone pulse therapy for ophthalmopathy, but the effect was limited and the lacrimation due to entropion and superficial keratitis persisted. Titers of both TRAb and TSAb decreased slightly and transiently with the pulse therapy. One year later, both titers remained high and eye signs did not improve any more though she was clinically euthyroid. This might indicate that both TRAb and TSAb levels correlate with the clinical course. Therefore, TRAb or TSAb might be good indicators of progress of Graves' ophthalmopathy. Ocular manifestations of Graves' disease should be followed closely with measurements of both TRAb and TSAb even in infant cases.     

Differential effects of a novel IFN-zeta/limitin and IFN-alpha on signals for Daxx induction and Crk phosphorylation that couple with growth control of megakaryocytes

OBJECTIVE: Although a novel IFN-zeta/limitin uses IFN-alpha/beta receptor, it lacks some common activities of type I IFNs. We compared effects on megakaryocyte proliferation and differentiation as well as signals for their biological activities. MATERIALS AND METHODS: Recombinant IFN-zeta/limitin and IFN-alpha titrated with a cytopathic effect dye binding assay, were used in this study. Colony assays and serum-free suspension cultures for megakaryocytes were performed to compare their growth inhibitory effects. To analyze signals, megakaryocytes cultured in serum-free suspension cultures were stimulated and Western blotted with the indicated antibody. RESULTS: Both IFN-zeta/limitin and IFN-alpha suppressed the proliferation of megakaryocyte progenitors without influencing their differentiation. However, much higher concentrations of IFN-zeta/limitin were required for the growth inhibition than IFN-alpha. The growth inhibition by IFN-zeta/limitin and IFN-alpha was significantly reduced when either Tyk2 or STAT1 was disrupted. In addition, the antisense oligonucleotides against Crk and Daxx, downstream molecules of Tyk2, greatly rescued the IFN-zeta/limitin- and IFN-alpha-induced reduction of megakaryocyte colony numbers. In cultured megakaryocytes, IFN-zeta/limitin induced the expression of SOCS-1 as strongly as IFN-alpha. However, IFN-zeta/limitin induced weaker phosphorylation of Crk and lower induction of Daxx than IFN-alpha. CONCLUSIONS: Weaker signals for Crk and Daxx may participate in less megakaryocyte suppressive activity of IFN-zeta/limitin and may distinguish IFN-zeta/limitin from IFN-alpha in megakaryocytes. Our results extend the understanding about thrombocytopenia in patients with IFN-alpha treatment as well as the possibility for the clinical application of human homologue of IFN-zeta/limitin or an engineered cytokine with useful features of the IFN-zeta/limitin structure.     

IVUS-Guided Wiring Improves the Clinical Outcomes of Angioplasty for Long Femoropopliteal CTO Compared with the Conventional Intraluminal Approach

Aims: This study aimed to assess the clinical efficacy of intravascular ultrasound (IVUS)-guided intraplaque wiring for femoropopliteal (FP) chronic total occlusion (CTO).Methods: This single-center, retrospective, observational study was performed at the Japanese Red Cross Kyoto Daini Hospital. From March 2013 to June 2017, a total of 75 consecutive patients (mean age: 75.4 ± 8.5 years; 59 males), who underwent endovascular treatment (EVT), having 82 de novo FP-CTO lesions, were enrolled in this study. Eleven of the lesions that met the exclusion criteria were excluded, and the remaining 71 lesions were divided into the IVUS-guided wiring group (n = 34) and non-IVUS-guided wiring group (n = 37). Primary patency, defined as a peak systolic velocity ratio of < 2.4 on duplex ultrasonography, and freedom from clinically driven target lesion revascularization (CD-TLR) at 12 months were the primary outcomes.Results: The mean lesion length was 21.6 ± 8.9 cm. The frequencies of primary patency and freedom from CD-TLR were significantly higher in the IVUS-guided wiring group than in the non-IVUS-guided wiring group (70.0% vs. 52.2%, p = 0.045; 83.9% vs. 62.8%, p = 0.036, respectively). The complete clinically true lumen angioplasty rate was also higher in the IVUS-guided wiring group than in the non-IVUS-guided wiring group (91.1% vs. 51.3%, p < 0.001, respectively). The clinically true and false wire passage rates were respectively 97.3% and 2.7% in the IVUS-guided wiring group.Conclusion: IVUS-guided wiring improves the clinical outcomes of EVT for FP-CTO by achieving a high clinically true lumen wire passage rate.     

Ability to induce p53 and caspase-mediated apoptosis in primary CD4+ T cells is variable among primary isolates of human immunodeficiency virus type 1

Infection with human immunodeficiency virus type 1 (HIV-1) is associated with dramatic depletion of CD4(+) T cells, the major HIV-1-induced pathogenesis. Apoptosis has been suggested to play an important role for the T cell depletion and a number of mechanisms have been proposed for the apoptosis in T cells. Here, we compared the levels for apoptosis induction in primary peripheral blood mononuclear cells (PBMCs) among several laboratory strains and primary isolates of the HIV-1 subtypes B and E. The results showed that apoptosis in infected PBMCs, preferentially in CD4+ T cell population, became detectable around the time for virus production by flow cytometric terminal transferase dUTP nick end labeling (TUNEL) technique and staining with the nuclear dye Hoechst 33342. The abilities to induce apoptosis in PBMCs were highly variable in individual isolates. The increase of p53 protein in infected PBMCs, which was initiated before virus production, was observed in infected PBMCs and the levels of p53 protein were almost proportional to the rates of the isolates to induced apoptosis. The cells infected and cultured in the presence of Z-VAD-FMK had significantly decreased cell mortalities, indicating that activated caspases also played a significant role in the apoptosis. Thus, HIV-1-induced apoptosis in primary T cells was accompanied by the p53 protein and caspase activation at varied levels in primary isolates.