Tasuke Konno

Prevention of rotavirus infection by oral administration of cow colostrum containing antihumanrotavirus antibody

After immunizing 8-month pregnant Holstein cows with human rotavirus, Wa strain, cow colostrum containing neutralizing antibody to human rotavirus, designated as Rota colostrum, was obtained. After randomly grouping 13 infants from a single orphanage, 6 infants received 20 ml of Rota colostrum every morning and 7 control infants received 20 ml of market milk. One month later, rotavirus associated diarrhea was observed in 6 of the 7 infants given milk and 1 out of the 6 infants given Rota colostrum. Orally administered Rota colostrum significantly protected infants from diarrhea caused by rotavirus (P < 0.05). Two out of 5 Rota colostrum recipients who were free from diarrhea showed rises in complement fixation (CF) antibody titer after the rotavirus infection epidemic. Thus, Rota colostrum prevented the outbreak of diarrhea but did not prevent immunological responses to natural rotavirus infection. In the therapeutic trial Rota colostrum had no effect on duration of diarrhea, bowel movements or virus shedding in stool. However, there were no side-effects of Rota colostrum.

Comparison of human, simian, and bovine rotaviruses for requirement of sialic acid in hemagglutination and cell adsorption

Human rotaviruses (Wa, KUN, MO) showed hemagglutination (HA) only with fixed 1-day-old chicken erythrocytes, and their HA activities were completely destroyed by trypsin activation of virions. Simian SA-11 and bovine NCDV had HA activities not only against fixed erythrocytes but also against fresh erythrocytes from various species. Their HA activities against fixed erythrocytes were also inhibited by trypsin activation, but those against fresh erythrocytes were not. Neuraminidase treatment of fixed erythrocytes did not inhibit HA by trypsin-untreated rotaviruses. In contrast, HA of fresh human erythrocytes by SA-11 and NCDV was completely inhibited by neuraminidase treatment of erythrocytes or glycophorin A, the major erythrocyte sialoglycoprotein. Adsorption and infection of SA-11 and NCDV to monkey kidney MA104 cells were also inhibited by neuraminidase treatment of cells. Adsorption and infection of human rotaviruses were not, however, affected by treatment of cells with neuraminidase from Vibrio cholerae or Arthrobacter ureafaciens or with potassium periodate. Therefore, HA of fixed chicken erythrocytes by trypsin-untreated human and animal rotaviruses may be independent of sialic acids, whereas that of fresh erythrocytes by SA-11 and NCDV is sialic acid dependent and probably mediated by glycophorin A. Sialic acids also constitute an essential part of the cellular receptors for SA-11 and NCDV, whereas those for human rotaviruses were quite resistant to treatments known to destroy major types of sialic acids.

THE AH RECEPTOR IS NOT INVOLVED IN 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN-MEDIATED APOPTOSIS IN HUMAN LEUKEMIC T CELL LINES

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a common environmental pollutant causing public concern. Its toxic effects include disruption of the immune, endocrine, and reproductive systems, impairment of fetal development, carcinogenicity, and lethality in rodents. Here, we report that TCDD induces apoptosis in two cultured human leukemic lymphoblastic T cell lines. This cell death was found not to be dependent on an aryl hydrocarbon receptor and to be inhibited by the inhibitor of tyrosine kinases and caspases. Apoptosis-linked c-Jun N-terminal kinase is rapidly activated in these cells by the treatment with TCDD. A dominant-negative mutant of c-Jun N-terminal kinase prevented cell death in the treatment with TCDD. Furthermore, TCDD decreases the Bcl-2 protein level in these cell lines. These findings will help in the understanding of the molecular mechanism underlying TCDD-mediated immunotoxicity.

Relationship of urinary pseudouridine and 1-methyladenosine to activity of leukemia and lymphoma

Urinary levels of pseudouridine and 1-methyladenosine in patients with leukemia and lymphoma were measured by the inhibition ELISA using monoclonal antibodies to determine the correlation of nucleosides excretion with disease activity. Significantly elevated levels of these nucleosides were detected in patients with all types of disease tested. Seventy-seven percent (46/60) and 62% (37/62) of patients had elevated levels of pseudouridine and 1-methyladenosine above normal mean + 2S.D., respectively, and combination assay of these nucleosides gave higher positive rate (87%; 52/60) than either single assay. The changes of urinary pseudouridine and 1-methyladenosine reflected the disease status of patients in remission or in relapse and the effect of chemotherapy. These results suggest that urinary pseudouridine and 1-methyladenosine might be clinically useful as complementary markers to the monitoring of the disease status of patients with leukemia and lymphoma by hematological examination.

d-lactic acidosis in two patients with short bowel syndrome: Bacteriological analyses of the fecal flora

Two cases of d-lactic acidosis associated with short bowel syndrome are described. The administration of kanamycin to the patients showed a decrease in d-lactate in blood and urine in parallel with disappearance of metabolic acidosis. Bacteriological analyses of the fecal flora showed an increase in Lactobacillus buchneri in the first patient and Lactobacillus fermenti IVa in the second; both bacteria were sensitive to kanamycin. Quantification of in vitro production of d-lactate by each species of bacteria isolated from the feces revealed that Lactobacillus produced more d-lactate than other species of bacteria. These observations indicate that Lactobacillus may play an important role in the induction of d-lactic acidosis in patients with short bowel syndrome.

Two modes of human rotavirus entry into MA 104 cells.

SummaryEntry of the KUN strain of human rotavirus into MA 104 cells was studied by electron microscopy. Virus particles attached to the cell membrane appeared to be almost exclusively double-shelled virions. These attached virions followed two distinct pathways into the cytoplasm depending on pretreatment with trypsin. Using infectious rotavirus which had been pretreated with trypsin, the viral nucleoids passed directly into the cytoplasm within 5 minutes after inoculation, through dissolution of the viral capsid and cell membrane. Using non-infectious rotavirus that had not been pretreated with trypsin, phagocytosis or pinocytosis occurred in which virions were sequestered into lysosomes 20 minutes after virus attachment to the cell membrane. After being sequestered, uncoating of the rotavirus virions within lysosomes was seen, but it did not result in release of the genome.On the basis of these observations it was concluded that when virions were pretreated with trypsin, virus replication occurred following the direct passage of viral nucleoids into the cell cytoplasm. However, mere phagocytosis of virus particles into cell lysosomes, which occurred when trypsinuntreated virus was used, does not appear to be related to rotavirus replication.

Two Distinct Electrophoretic Migration Patterns of RNA Segments of Human Rotaviruses Prevalent in Japan in Relation to Their Serotypes

Polyacrylamide gel electrophoretic analysis of viral nucleic acids has revealed II segments of double-stranded RNA patterns in rotaviruses (8). On the basis of differences in the migration of RNA segments on gels, Espejo et al have demonstrated the presence of two distinct RNA patterns among human rotaviruses obtained from different epidemics (1-5). Most recently, the existence of a correlation between the RNA patterns of human rotaviruses and their serotypes (subgroups) has been demonstrated (6). This communication describes the existence of two distinct RNA patterns among rotaviruses obtained from infants hospitalized with acute diarrhea between 1975 and 1980 in Sendai and Yamagata. Eighteen rotavirus strains were isolated and examined in this study. Extraction and purification of the rotaviruses were carried out by the method developed by Espejo et al (I). As a standard strain, the Wa strain of human rotavirus type 2, which was kindly provided by Dr. R.C. Wyatt (National Institutes of Health, U.S.A.) was used. The Wa strain was grown in MAI04 cells. The purified virus was disrupted at 100 C for 2 min in 0.0625 M Tris-HCl, pH 6.8, containing 1% (w/v) sodium dodecyl sulfate, 4 M urea and 5% (v/v) 2-mercaptoethanol. For electrophoresis of the RNA, 10% (w/v) polyacrylamide-0.27% (w/v) bisacrylamide slab gels were prepared. Appropriate amounts of samples were applied to the gels and electrophoresis was carried out at the constant current of 12 mAjslab gel for 8 hr. The gels were subsequently stained for 15 min with ethidium bromide solution (I !,g/ml in 0.02 M Tris-HCl, pH 7.4) and photographed. Figure 1 shows the RNA gel electrophoretic profiles of rotavirus strains obtained in Sendai (80S1, 80SR002; 80S2, 80SR004) and in Yamagata (80Yl, 80Y003; 80Y2, 80YOOI) in 1980 and the Wa strain. When compared with the RNA pattern of the Wa strain, the most striking difference in mobility was found in RNA segments 10 and II of the 80S1 and 80S2 strains, which moved significantly more slowly than those of the Wa strain. In contrast, as far as RNA segments 10 and 11 are concerned, no difference was found between the Wa strain and 80Yl or 80Y2. The RNA pattern with slower moving 10th and 11th seg-

Evidence for endocytosis-independent infection by human rotavirus.

SummaryThe effects of five lysosomotropic drugs (NH4Cl, chloroquine, methylamine, amantadine and dansylcadaverine) and cytochalasin B on human rotavirus (HRV KUN strain) and vesicular stomatitis virus (VSV Indiana strain) infection in monkey MA 104 cells were examined. These drugs had little effect on HRV yield but greatly reduced VSV yield. The results strongly suggest that HRV does not require endocytotic activity and intracellular acidic vesicles for the initial stage of infection and support our postulate that HRV enters the target cell by direct penetration of its nucleoid through the cell membrane.

A case of fatal infectious mononucleosis presenting with fulminant hepatic failure associated with an extensive CD8-positive lymphocyte infiltration in the liver.

We describe a fatal case of infectious mononucleosis presenting with fulminant hepatic failure associated with extensive CD8-positive lymphocyte infiltration and diffuse karyorrhexis in the liver. Immunohistochemical analysis of mononuclear cells showed that Leu-2a (CD8)-positive lymphocytes were heavily distributed in the portal areas and the sinusoidal spaces, but Leu-3a (CD4)-, Leu-14 (CD22)-, or My 4 (CD14)-positive cells were undetectable in sections of the liver. Southern blot hybridization studies disclosed the presence of Epstein-Barr virus DNA fragments in the liver tissue. The unusual pathologic and immunologic responses observed in this case could not simply be explained by severe Epstein-Barr virus infection. Some superimposed factors should be considered.

Problems of neuroblastoma screening for 6 month olds and results of second screening for 18 month olds

Nationwide neuroblastoma mass screening for 6-month-old infants (first screening) was introduced in Japan in 1985. About 110 neuroblastoma cases are detected annually by the first screening and treated, with a survival rate of 97%. Sensitivity of the first screening (positive cases/positive cases+false negative cases) is about 75%, and the prognosis of false-negative cases is unfavorable. A second screening at 18 months of age was started to rescue false-negative cases in Miyagi Prefecture in May 1992. Of 62 neuroblastoma cases treated in our hospital since 1985, 40 cases had received the first screening. Twenty cases were positive at first screening, 18 cases were false negative, and 2 cases were false negative and picked up by the second screening. Age distribution of false-negative cases ranged from 12 to 83 months and included 12 cases younger than 36 months old. Only 5 of 18 false-negative cases are alive without the disease. From May 1992 to November 1993, 14,282 infants had received the second screening (compliance rate: about 75%), and 2 neuroblastoma cases were detected. The first case was stage III with paraortic lymph node metastases, Shimada UH, aneuploidy and negative N-myc amplification. The second case was stage II with Shimada FH, aneuploidy, and negative N-myc amplification. Both cases are alive now without the disease after undergoing radical operation and chemotherapy. The first screening is effective for early detection of neuroblastoma cases, but the sensitivity is insufficient; the authors recommend a second screening to rescue false-negative cases.