The prevalence of TT virus (TTV) infection and its relationship to hepatitis in children

TT virus (TTV) is a newly discovered virus from a patient with post-transfusion hepatitis. We investigated the frequency and pathogenesis of TTV infection in children. A semi-nested PCR assay was used to amplify TTV-DNA in serum samples from 254 ambulatory children without liver disease, 20 with hepatitis of unknown etiology, and 18 transfusion recipients or hemophiliacs. In positive samples, TTV-DNA was quantified by real-time quantitative PCR using a fluorescent probe. We detected TTV-DNA in 20% of children with hepatitis of unknown etiology, which was not statistically different from the 23% prevalence in ambulatory children. In transfusion recipients or hemophiliacs, the frequency was higher (50%) than that in ambulatory children (P = 0.01). Among ambulatory children, TTV-DNA was frequently detected in children with acute gastroenteritis (36%). TTV-DNA was detected in 10% of the infants under 6 months old, and 20% of the children from 7 to 12  months old. The prevalence was constant after the age of 1 year; however, the copy number of TTV-DNA was significantly higher in children under 1 year of age (mean: 10^5.4 versus 10^3.8 copies/ml, P= 0.008). Finally, TTV-DNA was quantified serially in three children with chronic hepatitis who were positive for TTV-DNA. The presence or amount of TTV-DNA was unrelated to the serum alanine aminotransferase level. These results indicate that TTV infection is common in children. The larger quantity of TTV-DNA in infants and the high prevalence of TTV in children of all ages suggest that TTV may be transmitted in early childhood. Its relationship to hepatitis is doubtful in children. Received: 8 April 1999     

Anti-CD3 induces bi-phasic apoptosis in murine intestinal epithelial cells: possible involvement of the Fas/Fas ligand system in different T cell compartments

Recent studies have suggested that Fas-mediated apoptosis is involved in the pathogenesis of intestinal injury. In this study, we determined the role of Fas/Fas ligand (FasL) interactions in different T cell compartments using a murine model of small intestinal injury. An intraperitoneal injection of 145-2C11 (anti-CD3) antibody into C3H/HeN, BALB/c and MRL mice induced mucosal flattening and rapid, bi-phasic intestinal epithelial cell (IEC) apoptosis, which was detected by conventional light and electron microscopy and by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling. In the first, early phase, villous apoptosis was observed up to 4 h after injection, and in the second, later phase, apoptotic crypt cells gradually accumulated for up to 24 h. The early and later phases of apoptosis were reduced in lpr/lpr and nude mice compared with those in control strains. In addition, the kinetics of Fas-mediated killer activity induced by the antibody injection were different between intestinal intraepithelial lymphocytes (IEL) and splenocytes (SPL) and seemed to correlate with the bi-phasic occurrence of the apoptosis. Finally, the transfer of intestinal IEL from euthymic to nude mice induced both phases of apoptosis, whereas SPL induced the second phase's crypt apoptosis only by the antibody injection. Together, these results suggest the involvement of Fas-mediated killer activity of thymus-derived T cells in different compartments. Namely, T cell populations in different compartments are differentially involved in the induction of IEC apoptosis and contribute to the complex pathogenesis of immune-mediated intestinal injury in which Fas/FasL interactions may play a critical role.     

Generalized sarcoidlike granulomas with systemic angiitis, crescentic glomerulonephritis, and pulmonary hemorrhage. Report of an autopsy case

A 19-year-old woman showed rapidly progressive renal and respiratory failure and died after a short clinical course. The autopsy revealed that death was due to crescentic glomerulonephritis and pulmonary hemorrhage. The intrathoracic lymph nodes, lungs, kidneys, and other organs contained numerous epithelioid granulomas, some of which had foci of central coagulative necrosis. The aorta, its major branches, and small- to medium-sized vessels of various organs also had multiple areas of granulomatous angiitis. This is, to our knowledge, the first report of such autopsy findings. A discussion of the etiopathogenesis of the disease is presented.     

A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice

The recent emergence of severe acute respiratory syndrome (SARS) was caused by a novel coronavirus, SARS-CoV. It spread rapidly to many countries and developing a SARS vaccine is now urgently required. In order to study the immunogenicity of UV-inactivated purified SARS-CoV virion as a vaccine candidate, we subcutaneously immunized mice with UV-inactivated SARS-CoV with or without an adjuvant. We chose aluminum hydroxide gel (alum) as an adjuvant, because of its long safety history for human use. We observed that the UV-inactivated SARS-CoV virion elicited a high level of humoral immunity, resulting in the generation of long-term antibody secreting and memory B cells. With the addition of alum to the vaccine formula, serum IgG production was augmented and reached a level similar to that found in hyper-immunized mice, though it was still insufficient to elicit serum IgA antibodies. Notably, the SARS-CoV virion itself was able to induce long-term antibody production even without an adjuvant. Anti-SARS-CoV antibodies elicited in mice recognized both the spike and nucleocapsid proteins of the virus and were able to neutralize the virus. Furthermore, the UV-inactivated virion induced regional lymph node T-cell proliferation and significant levels of cytokine production (IL-2, IL-4, IL-5, IFN-gamma and TNF-alpha) upon restimulation with inactivated SARS-CoV virion in vitro. Thus, a whole killed virion could serve as a candidate antigen for a SARS vaccine to elicit both humoral and cellular immunity.     

A role of fibrinolytic activity in angiogenesis. Quantitative assay using in vitro method

The functional role of the fibrinolytic system in capillary growth was investigated using bovine capillary endothelial cells (BCEs) cultured on a Type I collagen gel matrix, into which the cells migrated to form capillary-like tubular structures. The length of the tubes formed were measured morphometrically using an image analyzer in the absence and presence of fibrinolytic proteases, namely plasminogen, plasminogen activators (PAs) and PA inhibitor (PAI). The addition of plasminogen (25 micrograms/ml) to the gel matrix significantly increased the length of BCE tubes found on the 9th day of culture (p less than 0.01), with a dose-dependent tendency. The simultaneous addition of a basic fibroblast growth factor (bFGF, 10 ng/ml) enhanced this tube formation as early as the 3rd day of culture (p less than 0.01). Cultured BCEs secreted both tissue-type and urokinase-type PAs (tPA and uPA) and PAI-1 into the culture medium, and the secretion of both PAs was enhanced by the addition of bFGF. However, the secreted tPA was composed mostly of an inactive form of tPA.PAI-1 complex, and the PA activity was derived mostly from uPA. Inhibitors of plasmin suppressed the enhancing effect of plasminogen on angiogenesis. In addition, anti-uPA IgG markedly inhibited the enhancing effect of plasminogen on the 4th and 7th days of culture (p less than 0.01), whereas anti-tPA IgG showed an inhibitory tendency only on the 4th day of culture (p less than 0.05). These findings indicate that the plasminogen-PAs system, especially uPA synthesized and secreted by BCEs, plays an important role in regulating angiogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen peroxide-induced Ca responses in CNS pericytes

Objective: The aims of the present study were to elucidate the interaction of reactive oxygen species (ROS) and Ca²⁺ response in central nervous system (CNS) pericytes. Methods: The intracellular Ca²⁺ concentration was measured using fluorescent Ca²⁺ indicator, fura-2, in cultured CNS pericytes. Results: Hydrogen peroxide evoked a dose-dependent increase in cytosolic Ca²⁺, which was completely inhibited by catalase. Removal of external Ca²⁺ or addition of nicardipine (1 μM) during application of hydrogen peroxide did not affect Ca²⁺ response. Incubation of the cells in Ca²⁺ free solution did not abolish but slightly reduced Ca²⁺ response by hydrogen peroxide. Ca²⁺ response to hydrogen peroxide was not altered by the depletion of intracellular Ca²⁺ by thapsigargin (1 μM). Pretreatment of the cells with tyrosine kinase inhibitor genistein (100 μM) or tyrphostin A47 (30 μM) significantly reduced Ca²⁺ increase by hydrogen peroxide. Conclusions: These results indicate that hydrogen peroxide evokes Ca²⁺ increase predominantly by release from intracellular Ca²⁺ store, which may be regulated by tyrosine kinases.     

Functional analysis of PTPN11/SHP-2 mutants identified in Noonan syndrome and childhood leukemia

Noonan syndrome (NS) is characterized by short stature, characteristic facial features, and heart defects. Recently, missense mutations of PTPN11, the gene encoding protein tyrosine phosphatase (PTP) SHP-2, were identified in patients with NS. Further, somatic mutations in PTPN11 were detected in childhood leukemia. Recent studies showed that the phosphatase activities of five mutations identified in NS and juvenile myelomonocytic leukemia (JMML) were increased. However, the functional properties of the other mutations remain unidentified. In this study, in order to clarify the differences between the mutations identified in NS and leukemia, we examined the phosphatase activity of 14 mutants of SHP-2. We identified nine mutations, including a novel F71I mutation, in 16 of 41 NS patients and two mutations, including a novel G503V mutation, in three of 29 patients with leukemia. Immune complex phosphatase assays of individual mutants transfected in COS7 cells showed that ten mutants identified in NS and four mutants in leukemia showed 1.4-fold to 12.7-fold increased activation compared with wild-type SHP-2. These results suggest that the pathogenesis of NS and leukemia is associated with enhanced phosphatase activity of mutant SHP-2. A comparison of the phosphatase activity in each mutant and a review of previously reported cases showed that high phosphatase activity observed in mutations at codons 61, 71, 72, and 76 was significantly associated with leukemogenesis.     

REACTIVITY OF LYMPHOCYTE SUBPOPULATIONS IN HUMAN MIXED LYMPHOCYTE CULTURE

Loss of antigenicity in the mixed lymphocyte culture (MLC) reaction of lymphocytes precultured at 22°C for 7–10 days was accompanied by a decrease in bone-marrow-derived lymphocytes (B cells) from 22 ± 1% to 13 ± 1%, and an increase in thymus-derived lymphocytes (T cells) from 65 ± 2% to 83 ± 1% (P < 0.001). Depletion of B cells from a fresh lymphocyte suspension by either antihuman immunoglobulin-coated column fractionation or by sheep red blood cell (SRBC) rosette formation resulted in a significant reduction of the cell's ability to stimulate in MLC (P < 0.001). Coating of lymphocytes with rabbit antihuman brain serum abrogated their ability to respond but not the ability to stimulate in MLC.     

Detection of the Epstein-Barr virus in primary gastric lymphoma by in situ hybridization

The Epstein-Barr virus (EBV) has been shown to be associated with numerous human malignancies including Burktt's lymphoma and nasopharyngeal lymphoepithelioma. In addition, some typical gastric adenocarcinomas were also recently reported to demonstrate EBV relevance. The present study was designed to detect EBV in primary gastric lymphoma, using the in situ hybridization (ISH) method, in which oligo-nucleotide probes for the EBERl RNA and the EBV DNA W region have been used. Of the 49 cases of primary gastric lymphoma studied, which all showed B cell immunopheno-type, EBER1 sequences could only be found in four cases, including two low-grade cases and two high-grade cases of histological subtypes while the number of positive cells was less than 50% of the tumor cells. In one case of low-grade mucosa associated lymphoid tissue (MALT) lymphoma, the EBER1 -positive neoplastic cells were found in the regional lymph node, but the primary site of the stomach showed no positive signals. The EBV presence was further confirmed by the EBV DNA ISH. Using the ISH method, rare or occasional positive lymphoid cells (probably non-tumorous bystander cells) could be detected in 10 other cases including all histological subtypes. The present study shows that only a small proportion of primary gastric lymphoma is associated with EBV, and such positive cases could be found in both high- and low-grade histological subtypes. It is also suggested that the EBV presence in the neoplastic cells of some cases of primary gastric lymphoma is most likely a secondary phenomenon.