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We molecularly cloned a new Grb2 family member, named Grf40, containing the common SH3-SH2-SH3 motif. Expression of Grf40 is predominant in hematopoietic cells, particularly T cells. Grf40 binds to the SH2 domain–containing leukocyte protein of 76 kD (SLP-76) via its SH3 domain more tightly than Grb2. Incidentally, Grf40 binds to linker for activation of T cells (LAT) possibly via its SH2 domain. Overexpression of wild-type Grf40 in Jurkat cells induced a significant increase of SLP-76–dependent interleukin (IL)-2 promoter and nuclear factor of activated T cell (NF-AT) activation upon T cell receptor (TCR) stimulation, whereas the COOH-terminal SH3-deleted Grf40 mutant lacked any recognizable increase in IL-2 promoter activity. Furthermore, the SH2-deleted Grf40 mutant led to a marked inhibition of these regulatory activities, the effect of which is apparently stronger than that of the SH2-deleted Grb2 mutant. Our data suggest that Grf40 is an adaptor molecule involved in TCR-mediated signaling through a more efficient interaction than Grb2 with SLP-76 and LAT.

The binding of all-trans retinoic acid (ATRA) to the ligand-binding region in the E-domain of retinoic acid receptor-α (RARα) modifies the transcriptional activity of RARα protein. ATRA probably induces differentiation of acute promyelocytic leukemia (APL) cells by binding to the E-domain of the RARα portion (RARα/E-domain) of PML/RARα chimeric protein. Therefore, molecular alteration in the RARα/E-domain of the chimeric gene is one mechanism by which patients with APL may acquire resistance to ATRA therapy. In this study using reverse transcription-polymerase chain reaction and single-strand conformation polymorphism, DNA segments amplified from the RARα/E-domain in fresh APL cells of 23 APL patients (8 males and 15 females from 4 to 76 years of age) were screened for mutations. Of those patients, 3 patients (1 with de novo and 2 with relapse) had clinical resistance to ATRA therapy. We found mutations in the RARα/E-domain of PML/RARα chimeric gene exclusively in the 2 patients who exhibited ATRA-resistance at relapse, whereas the mutations were not detected at their initial onset. Interestingly, these patients received a prolonged or intermittent administration of ATRA before relapse with ATRA-resistance. The mutations lead to the change of amino acid in the ligand-binding region of RARα/E-domain, Arg272Gln, or Met297Leu according to the amino acid sequence of RARα, respectively. Further study demonstrated that the in vitro ligand-dependent transcriptional activity of the mutant PML/RARα protein was significantly decreased as compared with that of wild-type PML/RARα. These findings suggest that mutations in the RARα/E-domain of the PML/RARα chimeric gene may confer clinical resistance to ATRA therapy in patients with APL.

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.

Neonatal intrahepatic cholestasis is a heterogeneous disease of undetermined cause. There is an unreported subset of idiopathic neonatal intrahepatic cholestasis with an unusual histological combination of hepatic siderosis and macrovesicular steatosis. The patients were a 34-day-old female and a 39-day-old male with normal birth weights. Their mothers had received oral iron supplement 4-6 weeks before delivery. The patients had obstructive jaundice noticed at the well-baby clinic at 1 month of life. They had high levels of serum galactose and tyrosine, hyperferritinemia. Urinary organic acid and bile acid analyses were negative, and galactose-1-phosphate uridyltransferase activity in red cells was normal. Liver biopsies showed diffuse iron deposits and macrovesicular fat. By substituting formula milk with lactose-free milk, the patients responded, and had normal biochemical tests within 5 months of life. Follow-up biopsies, at the age of 12 months, showed mild residual fibrosis without iron or fat deposits. They are both well at 3 and 6 years of age, respectively, without biochemical liver dysfunction and neurologic impairment. Prenatal iron-overload might contribute to the pathogenesis of the disease, but further studies are needed to confirm the assumption.

Idiopathic neonatal hepatitis (INH) is aheterogenous disease of undetermined cause. We report aretrospective histologic reevaluation of INH. Sixtypatients with INH were reviewed along with 32 biliary atresia (BA) patients. Histologic findings,iron and fat deposits, giant cell transformation, portalfibrosis, and bile duct proliferation weresemiquantitatively graded from 0 to 4+. Significanthistologic findings were defined as ≥2+. Frequencies ofpatients with significant histologic findings in the INHgroup were compared with those of the BA group. Amongthe patients with significant histologic findings, those in the INH group had significantly lessiron deposits (P < 0.01), portal fibrosis (P

In order to ascertain the cytological features of peripheral hematopoietic progenitor cells (PHPC) mobilized after administration of chemotherapeutic agents and G-CSF, lineage- and progenitor cell-specific surface markers on CD34 positive (+) cells were sequentially examined. Nineteen evaluable samples were obtained from a malignant lymphoma, an acute lymphoblastic leukemia and 5 neuroblastoma patients. CD38 and HLA-DR were respectively expressed on more than 95% and approximately 85% of CD34+PHPC cells. CD19 was also expressed on the majority and CD117 on 10 to 20% of the CD34+cells. The most striking finding was that the Thy-1(CDw9O)+/CD34+ population was decreased at the peak of mobilization of CD34+ cells as compared to the early phase after G-CSF administration (approximately 20% vs. 60%). These results suggest that decrease in Thy-1 expression on CD34+ cells is related to mechanisms easing CD34+ cell mobilization to the peripheral blood.

To clarify the relationship between idiopathic neonatal hepatitis and feeding type, that is, formula-milk feeding and breast-milk feeding, the medical records of 100 patients (68 male and 32 female babies) with idiopathic neonatal hepatitis of non-familial form referred to the medical centers of Akita University and Tohoku University during the past 18 years were reviewed. The patients were divided into two 9 year periods (1975–83 and 1984–92), and their clinical features were analyzed in terms of feeding type and sex. The number of patients enrolled decreased from 69 in the first half to 31 in the second half. The number of male patients dropped from 53 to 15, although the number of female patients (n = 16) remained the same in both 9 year periods. The frequency of formula-milk feeding significantly decreased in the second half (42/69 to 6/31, P < 0.01). Compared with the expected numbers of patients in the second half, calculated on the changes in the live birth population and feeding modality between the two halves, the actually enrolled numbers of patients in the second half were less in both the male and the formula-milk fed groups (× 0.35 and × 0.22), whereas the numbers of female and breast-milk feeding groups were close to the expected values (× 1.26 and × 1.08). When sex and feeding modality were combined, the formula-milk fed male group showed the lowest value (× 0.10), and the breast-milk fed female group showed the highest value (× 2.85). In conclusion, feeding type, especially in combination with gender, might be one causative factor in the occurrence of idiopathic neonatal hepatitis.

The IL-2 receptor (IL-2R) gamma chain is shared among receptors for IL-4, IL-7, IL-9 and IL-15 as well as IL-2. In order to clarify the functional role of these cytokines interacting with the common gamma chain in human early hematopoiesis, we studied expression of the IL-2R gamma chain on purified CD34 positive cells from bone marrow and cord blood. Broad populations of bone marrow mononuclear cells were all found to express the IL-2R gamma chain. CD34 positive cells were purified by CD34 monoclonal antibodies and immunomagnetic beads as representative hematopoietic progenitor cells. It was established that only 38 +/- 10% of CD34 positive bone marrow cells (n = 5) and 35 +/- 12% of CD34 positive cord blood cells (n = 11) expressed the IL-2R gamma chain. CD34(+) IL-2R gamma chain(+) and CD34(+) IL-2R gamma chain(-) cells fractionated by cell sorting were subjected to clonogenic assays that showed granulocyte-macrophage colony-forming cells (CFU-GM) were present evenly in both fractions, whereas erythroid burst-forming cells (BFU-E) were enriched in the CD34(+) IL-2R gamma chain(-) fraction approximately two- to six-fold as compared with CD34(+) IL-2R gamma chain(+) fraction. Such clonogenic features did not differ between the bone marrow and cord blood cases. These results indicate that CD34(+) IL-2R gamma chain(-) cells contain immature cells already committed to the erythroid lineage.

Five monoclonal antibodies detected a surface antigen expressed exclusively on T-cell acute lymphoblastic leukemia (T-ALL) in a panel of 45 human hematopoietic cell lines, including T-cell lines derived from adult T-cell leukemia and those established by immortalization with human T-cell leukemia virus type 1 or Herpesvirus saimiri. Peripheral blood mononuclear cells, including fresh and activated T cells, were also completely devoid of this antigen. We designated this antigen as TALLA-1 (from T-ALL-associated antigen 1). By expression cloning, a cDNA clone encoding TALLA-1 was isolated from T-ALL cell line Molt-4. TALLA-1 was found to be a member of the transmembrane 4 superfamily (TM4SF). The cDNA was also essentially identical to A15, which was isolated from another T-ALL cell line, HPB-ALL, by differential hybridization with normal peripheral blood lymphocytes, and to CCG-B7, which was isolated from a brain cDNA library using CCG repeat as a probe. The gene product was now characterized in detail at the protein level. Northern blot analysis showed that the gene was expressed most strongly in brain, skeletal muscle and spleen. In a panel of 52 non-hematopoietic human cell lines, the majority of neuroblastoma cell lines were found to be positive for TALLA-1. Like ME491, CO-029 and L6, TALLA-1 may be another TM4SF member behaving as a potential tumor-associated antigen.

Thirty-two children with bone marrow transplantation (BMT) received intravenous injections of gammaglobulin (IVIG) with a high titer of neutralizing (NT) antibody against human cytomegalovirus (HCMV) (200 mg/kg/week) from 1 week before to 4 months after transplantation. NT antibody titers before BMT and the highest levels in serial determinations conducted after BMT were compared for each patient. They were classified into three groups according to the antibody response: primary HCMV infection as group I, endogenous reactivation or external reinfection as group II, and indeterminable cases as group III. Two (6.3%) out of 32 patients examined had BMT-associated primary HCMV infections, but did not show any clinical symptoms. Significant changes in clinical parameters were also lacking in all the other 30 patients, independent of whether they shed viruses into the urine, or demonstrated on antibody boost. It was concluded from the group variation that the antibody response was indeed due to the engraftment of BMT, rather than to a direct effect of treatment with IVIG. Our results further indicate that passive immunization with HCMV antibody does not prevent infection, but confers some protection against symptomatic disease.

Slowing down of the maturation process of human rotavirus particles on ice allowed the clear demonstration of two different assembly pathways through the endoplasmic reticulum (ER) membrane. One was the 'enveloped' and single-shelled (ss) particle assembly pathway, in which a transient envelope is acquired through the budding of subviral particles from the cytoplasm to the ER lumen, and later these 'enveloped' particles are released as ss particles in the ER lumen. The other was a double-shelled particle assembly pathway by which subviral particles acquire the outer capsid proteins during their transport across the ER membrane.

The defective antibody-dependent cellular cytotoxicity (ADCC) of mononuclear cells (MNC) from patients with leukocyte adhesion deficiency (LAD), beta 2 integrins (CD11a-c/CD18) deficiency was shown. LAD patients completely failed to generate MNC-ADCC against sheep red blood cells (SRBC) sensitized with murine (m) IgG2b, but had diminished but significant cytolysis against mIgG2a-SRBC, suggesting that the CD11/CD18 requirement of Fc gamma RI is different from that of Fc gamma RII in MNC-ADCC. Blocking experiments with monoclonal antibodies (mAb) against individual subunits of CD11/CD18 revealed that anti-CD18 mAb almost completely inhibited mIgG2b-mediated ADCC by normal monocytes, but only partially inhibited mIgG2a-mediated ADCC. These data may confirm the evidence that Fc gamma RII-mediated ADCC absolutely requires CD11/CD18 but Fc gamma RI-mediated ADCC does not. Among subunits of CD11/CD18, appeared to be most involved in lysis of sensitized SRBC.

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.

TAZAWA, Y., NISHINOMIYA, F., NOGUCHI, H., TSUCHIYA, S., HAYASHI, Y., ABUKAWA, D., WATABE, M., NAKAGAWA, M., IMAIZUMI, M., OHI, R., TADA, K. and KONNO, T. Hepatocellular Carcinoma in Children with Hepatitis B Surface Antigen. Tohoku J. Exp. Med., 1992, 167 (1), 47-55-This study discusses four children of hepatocellular carcinoma (HCC) who were asymptomatic HBsAg carriers or had HBsAg-positive chronic hepatitis for 3 to 11 years before the occurrence of the carcinoma. Three of these four patients were positive for anti-HBe at 3 to 5 years before the diagnosis of hepatocellular carcinoma. Autopsy findings disclosed liver cirrhosis in all the four patients. To the best of our knowledge few reports have documented children in HBsAg carrier status or with HBsAg-positive hepatitis prior to the development of hepatocellular carcinoma. It is emphasized that HBsAg-positive children, with or without detectable hepatic lesions in routine examinations, have a possibility of developing HCC, and should be carefully monitored for long periods.

OHASHI, Y., MINEGISHI, M., TSUCHIYA, S. and KONNO, T. Three Monoclonal Antibodies against Human LEA-1 α and β Chains with Different Biological Activities. Tohoku J. Exp. Med., 1992, 168 (4), 599-610-Three murine monoclonal antibodies (MAbs) directed against human lymphocyte function-associated antigen-1 (LFA-1, CD11a/CD18), designated as MAY.017, MAY. 035, and MAY. 044, were newly generated. The hybridomas were screened for their ability to inhibit a phorbol ester-stimulated aggregation of Epstein-Barr virus-transformed B-lymphoblastoid cell line (B-LCL). The MAbs bound to peripheral blood leukocytes, T cell lines, B cell lines, and some of myeloid/monocytic lines, but not to B-LCL derived from a patient with leukocyte adhesion deficiency. MAY.035 immunoprecipitated a complex of proteins with molecular masses of 155kDa and 95kDa, while MAY.017 and MAY.044 did a complex of 130kDa, 155kDa and 95 kDa proteins. MAY.035 was shown as to recognize the α chain of LFA-1(CD11a), and both MAY.017 and MAY.044 the β chain of the β2 integrin family (CD18). All the three MAbs inhibited lymphocyte proliferative responses to mitogens or alloantigens. MAY.017 blocked cytolytic activity mediated by natural killer cells. -monoclonal antibody; integrins; lymphocyte function-associated antigen-1; leukocyte; bone marrow transplantation

TSUCHIYA, S., MINEGISHI, M., FUJIE, H., MINEGISHI, N., OHASHI, Y., ITANO, M., MORITA, S., YAMAGUCHI, Y., SATO, T. and KONNO, T. Allogeneic Bone Marrow Transplantation for Malignant Hematologic Disorders in Children. Tohoku J. Exp. Med., 1992, 168 (2), 345-350- In the present study we carried out allogeneic bone marrow transplantation (BMT) in 14 leukemia children with high risk prognostic factors. Six patients with acute nonlymphocytic leukemia (ANLL), four with acute lymphocytic leukemia (ALL), two with chronic myelogenous leukemia (CML), and two with myelodysplastic syndrome (MDS). Among these patients, six with ANLL, two with ALL, one with CML and one with MDS were alive in complete remission 8 to 58 months post-BMT. Four patients died of relapse (one with ALL, and one with MDS), and chronic GVHD (one with ALL and one with CML). In six patients recombinant granulocyte colony stimulating factor (rG-CSF)was used to shorten the period of granulocytopenia. The mean time of recovery to granulocyte count of 500/mm3 was 13.2 days in the rG-CSF+group, being 15.9 days faster than that in the rG-CSF-group. In light of these results, allogeneic BMT is shown to be a choice of treatment for leukemia children with high risk prognostic factors and rG-CSF may be an effective reagent to prevent infectious episodes in BMT.- allogeneic bone marrow transplantation; leukemia; high risk prognostic factors; recombinant granulocyte colony stimulating factor

We report our findings in two cases of steroid-resistant severe acute GVHD after allogeneic BMT successfully treated with FK506 (tacrolimus). An 18-year-old female (patient 1) who underwent BMT from an HLA-identical sibling for ALL in first CR, developed generalized erythema and profuse watery diarrhea, which progressed to acute GVHD of grade III severity, resistant to steroid control. After continuous 24-h administration of FK506, the diarrhea improved within 10 days. Patient 2, a 9-year-old girl with AML who underwent unrelated BMT, had skin, gut and liver lesions of acute GVHD grade IV, which did not respond to high-dose steroid therapy. They were controlled, however, by continuous intravenous infusion of FK506. Both patients are still surviving after more than 1 year without any acute GVHD sequelae or signs of chronic illness. The adverse effects of FK506 were mild and tolerable in both cases. Comparison of our findings with those in the literature suggests that it is important to give FK506 at plasma concentrations as high as 25-35 ng/ml by continuous intravenous infusion for extended periods to control steroid-resistant severe acute GVHD.

In the hospitals of the Tohoku Neuroblastoma Study Group (TNBSG), treatment for children with advanced neuroblastoma (NB) was intensified in the mid-1990's with the introduction of myeloablative therapy (MT) with stem cell transplantation (SCT) including the use of autologous peripheral blood stem cells (PBSC) and bone marrow transplantation (BMT). In this report, we examined whether the intensified therapy improved the outcome of children with advanced NB (age> 12 months) who were diagnosed between 1991 and 1997. Patients were 36 children (23 boys and 13 girls) with an average age of 3.4 years (range; 1 to 14 years). Six of them had stage III disease, and the other 30 had stage IV. They were treated initially with induction chemotherapy, surgery, and post-operative chemoradiotherapy, after which 17 of them continued further chemotherapy and the other 19 received MT/SCT (18 with PBSCT and 1 with BMT). Progression-free survival (PFS) rate at seven years from diagnosis was 43.5% for all patients, 66.7% for stage III patients and 38.2% for stage IV patients. The difference between stage III and IV patients was not significant. Among the 30 patients with stage IV disease, PFS at seven years was significantly higher in the 19 patients who received MT/SCT (55.6%) than in the 11 patients who did not receive it (12.5%). There was no difference in clinical and biological risk factors between these two groups, except for the proportion of patients with favorable response to initial therapy (36% and 80% for patients without and with MT/SCT, respectively). Furthermore, the proportion of patients with N-myc amplification was significantly higher in patients with progressive disease (PD) after MT/SCT than in those in CR after MT/SCT. The results of this retrospective study of children with advanced NB suggest that therapy intensification involving MT/SCT might result in lengthened survival time for patients with stage IV disease, and that post-transplant PD remains a risk for patients with high levels of N-myc amplification.

OHASHI, Y., TSUCHIYA, S., FUJIE, H., MINEGISHI, M. and KONNO, T. Anti- LFA-1 Antibody Treatment of a Patient with Steroid-Resistant Severe Graft- Versus-Host Disease. Tohoku J. Exp. Med., 1992, 167 (4), 297-299-For treatment of steroid-resistant severe graft-versus-host disease, a murine monoclonal antibody (25.3) against the α chain (CD11a) of the lymphocyte function-associated antigen 1 (LFA-1) was infused into a patient with posthepatitic aplastic anemia who had undergone allogeneic bone marrow transplantation. The monoclonal antibody infusion was well tolerated and resulted in appreciable improvement in symptoms of gastrointestinal illness such as diarrhea and abdominal pain, suggesting that this antibody may be useful for controlling severe acute graft-versus-host disease.-graft-versus-host disease; monoclonal antibody; CD11a

ITANO, M., MORITA, S., FUJIE, H., OHASHI, Y., MINEGISHI, N., MINEGISHI, M., YAMAGUCHI, Y., SATO, T., TSUCHIYA, S., KONNO, T., HAYASHI, Y., OHI, R. and SHIRAISHI, H. Mass Screening for Neuroblastoma in Miyagi Prefecture. Tohoku J. Exp. Med., 1992, 168 (2), 137-139 - In Japan, aiming at early and preclinical detection of neuroblastoma in infancy a mass screening program for the tumor has been implemented nationwide using urinary tests for catecholamine metabolites, vanillylmandelic acid (VMA) and homovanillic acid (HVA) (Sawada 1990; Sawada et al. 1991). In this report, the results obtained from the screening program in Miyagi Prefecture for the last 6 years are described. The detection rate of neuroblastoma by mass screening was 1:8, 377 among 125, 652 infants tested in Miyagi Prefecture. All but one patients survived after removal of the primary tumor and none or minimal chemotherapy. - neuroblastoma; mass screening; urine VMA; urine HVA

Background In 1978, the first case of columnar epidermal necrosis was reported in a 6-year-old boy. There were scaly, partially vesicular or crusty, erythematous lesions mainly involving the extremities that histopathologically showed peculiar features of focal, total epidermal necrosis accompanied by a lichenoid tissue reaction. He developed the skin eruption after receiving a blood transfusion from his mother when he showed debility induced by vaccination with an alternated live measles virus vaccine. The lesions rapidly regressed after sun exposure. To our knowledge, there has been no report of a similar case despite such unique features. Observation We encountered a similar case of columnar epidermal necrosis in a 15-year-old Japanese girl with chronic graft-vs-host disease; the lesions occurred 3 months after the transfusion of peripheral blood stem cells from her HLA antigen–matched brother. However, there was no exacerbation of liver dysfunction, diarrhea, or bone marrow aplasia. The peculiar cutaneous lesions responded well to topical phototherapy. Conclusion These 2 patients shared a similarity in their lesions and circumstances under which the blood transfusion was performed to a debilitated patient from a close family member. We believe that focal epidermal necrosis observed in patients with this condition represents a variant of blood transfusion–associated lichenoid graft-vs-host disease that occurs uniquely in a skin-targeted fashion.

SUZUKI, H., KITAOKA, S., KONNO, T. and SATO, T. The Localization and Function of the Neutralizing Protein, VP4, in Human Rotavirus. Tohoku J. Exp. Med., 1991, 163 (1), 73-75 - The binding sites of a monoclonal antibody (mAb) to human rotavirus VP4 (K-1532) on the virion surface were examined with electron microscopy and the Markham photorotation method. The mAb, which had both hemagglutination inhibiting and neutralizing activities, appeared to bind to the outer margin of double-shelled particles of human rotavirus, especially to the parts overlaying the tubelike channels, which seemed to be the “cover lid” of the tubelike channels that extruded radially from the core region. This binding point may be a putative fusion site of the virus (Mackow et al. 1988) as well as a viral nucleus component ejection gate (Suzuki et al. 1986). Rotavirus particles treated with the mAb were shown to be capable of binding to MA104 cells by ultra-thin section examination. These observations suggest that neutralization via VP4 is related to virus uncoating but not to attachment of rotavirus.

X-linked severe combined immunodeficiency (X-SCID) is characterized by an absent or diminished number of T cells and natural-killer (NK) cells with a normal or elevated number of B cells, and results from mutations of the γc chain. The γc chain is shared by interleukin-2 (IL-2), IL-4, IL-7, IL-9, and IL-15 receptors. Recently, a survival signal through the IL-7 receptor  (IL-7R) chain was shown to be important for T-cell development in mice and was suggested to contribute to the X-SCID phenotype. In the present study, we examined function of a mutant γc chain (A156V) isolated from an X-SCID patient and found that T cells expressing the mutant γc chain were selectively impaired in their responses to IL-4 or IL-7 compared with the wild-type γc chain expressing cells although responses to IL-2 or IL-15 were relatively maintained. The result shows that IL-4– and/or IL-7–induced signaling through the γc chain is critical for T-cell development and plays an important role in the development of the X-SCID phenotype.

We report a case of a 16-month-old Wiskott-Aldrich syndrome (WAS) patient with a WASP gene mutation who received human leukocyte antigen (HLA)-matched, unrelated allogeneic bone marrow transplantation (BMT) followed by an Epstein-Barr virus-associated lymphoproliferative disorder (EB-LPD), diagnosed by clinical findings, polymerase chain reaction detection of the EB virus genome, and spontaneous lymphocyte proliferation of donor cell origin. EB-LPD is one of frequent lethal complications in HLA-mismatched or unrelated BMT in this syndrome. Adoptive immunotherapy with donor leukocyte transfusion, including appropriate numbers of CD3-positive T cells, was effective for the EB-LPD, achieving almost complete recovery 1 year later without any findings of graft-versus-host disease.

The "touchdown" polymerase chain reaction (PCR) technique has been applied to analyze expression of the neuron-specific protein, PGP9.5, and tyrosine hydroxylase (TH) genes for detection of minimal residual neuroblastoma cells in bone marrow and peripheral blood. PGP9.5 and TH gene products were not detected in any normal samples (n = 72) examined. However, in patients more than 1 year of age with stage III and IV neuroblastoma PGP9.5 mRNA was detected in six of seven bone marrow samples and in four of eight peripheral blood samples, and TH mRNA in four of seven and three of eight, respectively. The detection sensitivity was up to 10(-6) to 10(-7) micrograms of total cellular RNA for PGP9.5 and 10(-4) micrograms for TH. Among forty bone marrow specimens from nineteen patients with neuroblastoma both PGP9.5 and TH mRNAs were detected in six, and only PGP9.5 mRNA was detected in ten. Since detection of PGP9.5 and TH gene transcripts by the "touchdown" PCR was highly specific and sensitive, it might be most informative at present to carry out both PGP9.5 and TH mRNA assays for minimal residual neuroblastoma cells in blood and bone marrow.

A 10-year-old patient with nasopharyngeal carcinoma (NPC) was studied for mutations within the carboxy-terminal portion of the Epstein-Barr virus (EBV) latent membrane protein (LMP)-1 gene. The EBV genome, defined as type A, was detected in biopsied tumor specimens by Southern hybridization with specific probes. Sequence analysis of the carboxy-terminal part of the LMP-1 gene revealed no deletions but seven single-base substitutions, four of which were found to be identical to those previously detected in codons for amino acids 322 to 366 in the Chinese NPC CAO. Although yet unresolved, the observed mutations may be associated with the pathogenesis of NPC.

After immunizing mice with a human megakaryoblastic leukemia cell line, M-MOK, we obtained two monoclonal antibodies which recognize the human c-kit receptor. The monoclonal antibodies, designated MTK1 and MTK2, were found to specifically recognize Balb/3T3 cells transfected with human c-kit cDNA and not parent Balb/3T3 cells while showing different immunological, biochemical and biological behaviors. Both allowed visualization of the 140 kDa c-kit protein by Western blot analysis, but MTK1 detected only positive band with non-reducing conditions for sodium dodecyl sulfatepolyacrylamide gel electrophoresis. MTK1 partially inhibited the stem cell factor (SCF) induced proliferation of M-MOK cells, whereas, MTK2 was without effect. MTK1 also inhibited the bone marrow derived colony forming unit granulocyte/ macrophage (CFU-GM) formed by granulocyte-macrophage colony stimulating factor (GM-CSF) and SCF. Not only anti-CD34 antibodies (HPCA-1) but also MTK1 could be shown to concentrate bone marrow CFU-GM and burst forming unit erythroid (BFU-E) effectively. The presently described monoclonal antibodies may therefore be useful for functional analysis of the ligand binding domain of the human c-kit receptor, as well as for further classification of hematopoietic stem cells in addition to the CD34 positive cells.

A human CD4- CD8- alpha beta T-cell receptor (TCR alpha beta)+ T leukemic cell line, L-KAW, was established from the peripheral blood of a 6-year-old male patient with T-cell acute lymphoblastic leukemia. Its phenotype was found to be CD7+CD2+CD3+CD4-CD8- TCR alpha beta +. The cell line proved susceptible to killing by exposure to a mitogenic concentration of phytohemagglutinin (PHA) within several hours, with biochemical analyses demonstrating extensive degradation of DNA to oligonucleosomal bands characteristic of apoptosis. Chromatin condensation and cell shrinkage features of apoptotic cells could also be clearly identified under the electron microscope. The apoptosis-related Fas antigen was expressed on L-KAW cells and DNA fragmentation was induced by incubation with anti-Fas monoclonal antibody. The c-myc mRNA levels declined within 15 min to 3 h after the addition of PHA. These results suggest that L-KAW cells are specifically sensitive to induction of apoptosis in response to PHA without preactivation. They may, therefore, be considered an analog of activated T-cells and prove useful as a model system for characterizing biochemical and molecular mechanisms underlying the process of this type of cell death.

Modulatory effect on the murine self defense system by a newly discovered acidic polysaccharide (ANK-102) produced by P. tuberosa cells in liquid culture was examined. Pretreatment with ANK-102 deteriorated the murine survival against lethal infection of Listeria monocytogenes, an intracellular gram-positive bacterium eliminated mainly by macrophages through T-cell mediated immune response. Pretreatment with ANK-102 resulted in the accumulation of Mac 1 and Mac 2 positive cells in the peritoneal cavity of the infected animals and the reduction of Thy1.2 expression on the surface of the thymocytes. A new type of immunosuppressive polysaccharide ANK-102 was introduced.

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.

Rotaviruses are the most important etiologic agents of severe diarrhea worldwide. Despite great advances in vaccine development, little is known about host protection mechanisms other than immunity. This presentation focuses on the proteolytic enhancement of rotavirus infection, with emphasis on the functions of VP4, an outer capsid protein. The in vitro growth of human rotavirus is enhanced by trypsin, which selectively cleaves VP4. Treatment with trypsin increases the infectivity of human rotavirus while decreasing its hemagglutination activity. There are two modes of rotavirus internalization: direct penetration with the aid of trypsin and endocytosis without trypsin. Direct penetration via VP4 cleaved by trypsin is essential for the replication of the virus, whereas endocytotic internalization does not give rise to viral replication.

Leukocyte adhesion deficiency (LAD) is an inherited immunodeficiency disorder caused by CD18 subunit abnormality dependent defective expression of β2 integrins on the surface of leukocytes. On analysis of the CD18 molecular defect in a female Japanese patient with a severe deficiency LAD phenotype, neither CD11a nor CD18 molecules could be detected on the patient's EBV-transformed B lymphoblastoid cell line. The mRNA of the patient's B cells was normal in size, but was diminished in quantity, to approximately half normal levels. Sequencing of the CD18 cDNA of the patient revealed a C605 to T transition, resulting in a Pro178 Leu substitution. This was heterozygous in the genomic DNA, and shown to be of maternal origin by family study. Only a few transcripts from the other allele without the Pro178 Leu mutation were detectable. Northern blot analysis revealed reduced CD18 mRNA levels, not only in the patient, but also in the father and brother. These results indicate that our case is a compound heterozygote with two different mutant alleles: one causing a single amino acid substitution and the other causing defective expression of mRNA. © 1993 Wiley-Liss, Inc.

TSUCHIYA, S., FUJIE, H. and KONNO, T. B-Lineage Phenotype of Lymphoblastoid Cell Lines from Patients with X-Linked Agammaglobulinemia. Tohoku J. Exp. Med., 1991, 163 (4), 289-294- Epstein-Barr virus (EBV)-induced precursor B-cell lines were established from bone marrow cells of patients with X-linked agammaglobulinemia (X-LA). Using seven B-lineage specific monoclonal antibodies marker profiles of these cell lines were examined in order to know developmental stage specific and/or X-LA specific changes of B-cell related cellsurface antigens. CD19, CD20, CR2 (CD21), Fc e receptor (CD23) and HLA-DR antigens were consistently found on the X-LA derived precursor B-cell lines as well as mature lymphoblastoid cell lines from healthy adults. These results suggest that immortalisation of B-cells with EBV is always accompanied by expression of pan-B cell markers and B-cell activation antigen (Fc e receptor) even though the cell lines have precursor B-cell phenotypes as defined by immunoglobulin expression.

Graft-versus-host disease (GVHD) is one of the most common and fatal complications that follows allogeneic bone marrow transplantation (BMT). Donor origin T cells are responsible for the initiation of GVHD. In this report, we demonstrate that conditioning regimens for BMT resulted in elevated serum levels of interleukin-15 (IL-15), which reached maximum levels within 15 days and returned to basal levels within 25 days after allogeneic BMT, in all patients examined. Thereafter, circulating IL-15 was detected only in patients with grade III or IV acute GVHD with gut involvement. In contrast, IL-2 was not detected at any time in these patients. Since IL-15 is able to activate antigen-stimulated T cells and natural killer (NK) cells, IL-15 may play an important role in the development of severe forms of acute GVHD.

In the hematopoietic lineage, the transcription factors GATA-1 and GATA-2 show restricted and largely overlapping expression profiles, but GATA-2 is uniquely expressed in early hematopoietic progenitors. GATA-3 is found exclusively in T cells of hematopoietic lineage. To clarify whether these expression profiles are preserved or changed during the development of malignancies, we analyzed the expression of GATA factors in the blasts from leukemic children. A total of 18 myelogenous leukemia and 24 lymphoblastic leukemia (ALL) cases were investigated. In the majority of the former cases, GATA-2 mRNA expression and the expression of CD34 and c-kit antigens on leukemic cells were demonstrated. In contrast, GATA-2 mRNA and c-kit antigen could not be detected in CD34-positive cells from ALL patients. GATA-3 mRNA was expressed in all T-ALL cases, but not in any precursor B-ALL. These findings suggest that down-regulation of GATA-2 and expression of GATA-3 are important events for the commitment of cells to lymphoid and T cell lineage, respectively. The expression profiles of GATA factors in leukemic cells are generally consistent with those in their normal counterparts, and thus provide a useful tool to determine the lineage commitment of unclassified leukemia.

TSUCHIYA, S., NAKAE, S., Koxno, T., TADA, K. and ONo, Y. Estimation of B-Cells Transformed by Epstein-Barr Virus in Patients with Congenital Agammaglobulinemia. Tohoku J. exp. Med., 1981, 135 (4), 379-385 - In vitro immunoglobulin synthesis was measured in lymphocytes from four patients with congenital agammaglobulinemia (cAy) stimulated by two different polyclonal B-cell activators, pokeweed mitogen (PWM) and Epstein-Barr virus (EBV). In PWM -stimulated cultures, patient T-cells treated with mitomycin C (MMC) were able to help the immunoglobulin (Ig) synthesis of normal B-cells. Patient B-cellenriched fraction not containing surface Ig positive cells did not produce Ig in combination with MMC-treated autologous or allogeneic T-cells. Patient lymphocytes were infected with EBV and the subsequent production of Ig was measured. In lymphocytes from control subjects, exponential growth of the cells having EBV-associated nuclear antigen (EBNA) was shown to be associated with an exponential increase in Ig secretion within 1 week after EBV infection. However, in lymphocytes from three of the four patients, it took 2, 4 and 10 weeks, respectively, until lymphocyte-transformation and subsequent Ig-secretion were observed. Lymphocytes from one patient were not transformed nor did they secrete Ig after EBV infection. These results may imply that a small number of B-cells are present in peripheral blood of most of patients with cAy, and that they are able to produce Ig after transformation by EBV which takes a much longer time than in controls. B cell; Epstein-Barr virus; congenital agammaglobulinemia

MINEGISHI, N., NAKAMURA, M., SUZAKI, K., TERASAWA, M., MINEGISHI, M., TSUCHIYA, S. and KONNO, T. Chronic Granulomatous Disease with Neutrophil Membrane Cytochrome b Deficiency: Demonstration by Immunochemical Staining with Monoclonal Antibody. Tohoku J. exp. Med., 1988, 154 (2), 143-148 -Cytochrome b deficicency in the peripheral granulocytes of two male patients with chronic granulomatous disease was demonstrated by an immunocytochemical assay using a monoclonal antibody, 7D5, against human neutrophil cytochrome b. A mosaic of cytochrome b positive and negative neutrophils, indicating a carrier state in an X-linked trait, was found in the mother of patient 1 but not in the mother of patient 2.

A previously established human leukemia cell line, designated THP-6, was further characterized with respect to cell surface antigen expression and immunoglobulin(Ig) and T-cell receptor(TCR) gene status. THP-6 cells were positive for CD7 and CD5 antigens and terminal deoxynucleotidyl transferase, but negative for CD2, CD1, CD4, CD8, CD10, cytoplasmic and surface CD3 and HLA-DR antigens, suggesting a precursor T-cell line. Analysis of Ig and TCR β chain genes revealed that THP-6 had a rearranged TCR β chain gene and a germline Ig gene. These results, in agreement with its phenotype, confirmed that THP-6 was of the T-cell lineage.

Enzyme replacement therapy was performed for a 1-year and 5-month old boy with adenosine deaminase deficiency disease, the first case in Japan. Irradiated fresh red blood cells were administered without any clinical improvement, but there was an increase in the peripheral lymphocytes from 300/mm3 to 1849/mm3, of which 88% had T cell marker. B lymphocytes did not bear any classes of surface immunoglobulins. The proliferative responses of these lymphocytes to phytohemagglutinin, concanavalin A, pokeweed mitogen and allogeneic cells were examined. More than two-fold increase in response to these mitogens was observed in lymphocytes after treatment as compared with responsiveness before treatment, but these responses still remained to a much lesser degree than that of lymphocytes from controls.

Polypeptide analysis of three strains of human rotavirus (KUN, Wa and MO) were conducted using a hypertonic culture which suppressed host protein synthesis and unmasked rotavirus specific protein synthesis. As a result, eleven human rotavirus specific polypeptides (Vp 1--Vp 11) were detected by pulselabeling infected cells with [14C]-leucine. Among the 11 polypeptides, three polypeptides (Vp 7, Vp 10 and Vp 11) underwent post-translational processing, and two (Vp 7 and Vp 10) were glycosylated. Six polypeptides (Vp 1, 2, 3, 4, 6 and 7) were identified as viral structural proteins. Comparisons of three strains of different serotypes revealed that their polypeptide profiles differed from each other in electrophoretic mobility; in particular, profiles of the glycosylated polypeptide, Vp 7, were distinct among the three strains.

KOBAYASHI, Y., TAZAWA, Y., NAKAGAWA, M., SUZUKI, H., KONNO, T. and YAMAMOTO, T.Y. Ultrastructural Changes in Skeletal Muscle of a Patient with Familial Intrahepatic Cholestasis Associated with Vitamin E Deficiency. Tohoku J. exp. Med., 1984, 142 (3), 337-346 - The skeletal muscle from a patient with familial intrahepatic cholestasis associated with vitamin E deficiency was studied by electron microscopy. The muscle fibers showed a variety of pathologic features including degenerative, necrotic and regenerative changes. Granule-like inclusions found in our biopsy specimens were similar in structure to those observed in vitamin E deficient animals. These inclusions were noted not only in the skeletal muscle fibers but also in endothelial cells of the intramuscular capillaries, Schwann cells and perineural cells. Disruption and disappearance of the plasma membrane, separation, disruption and pleats formation of the external lamina, and multilayered external laminae were observed in muscle fibers most frequently. The nerves among muscle fibers also showed degenerative features. These severely degenerative alterations of the muscle fiber have not so far been reported in vitamin E deficient patients. We discuss the process of cell damages caused by vitamin E deficiency.

Adenosine deaminase (ADA) activities in erythrocytes from 10 relatives including parents of a patient with ADA deficiency and severe combined immunodeficiency (SCID) were reported. His parents, maternal uncle and grandmother showed ADA activity in their erythrocytes below the half level of the mean value of controls. These results suggested the autosomal recessive inheritance pattern of ADA deficiency, confirming other reports.

The application of the PFD test, a newly developed technique for testing exocrine pancreatic function, was studied in infants and children. 1. This test is simple to perform and noninvasive so that it can be easily used in infants and children. 2. The test was performed on 125 normal children, and normal PFD-values i.e. > 1.2 x age + 48.5% in children over 2 years were established. For children under 2 years only reference PFD-values were obtained. The mean PFD-values in any age groups were lower than in normal adults. 3. Presumptive relationship between PFD recovery and urine volume or other factors was denied at least in normal children over 2 years and values affected only by exocrine pancreatic function (chymotrypsin activity) were obtained. 4. The PFD values correlate well with other parameters of exocrine pancreatic function in children with pancreatic insufficiency. As a result, the clinical value of the PFD test was demonstrated and the rationale for normal PFD-values in children was established. 5. It has also been shown that PFD values may be low when intestinal absorption, hepatic conjugation or renal excretion of PABA is impaired. 6. No adverse reactions to PFD test were noted except in one case of mild acute alcoholism which was presumably produced by ethyl alcohol used as the solvent. The precautions that should be exercised were also noted.

The toxic effects of 4-hydroperoxycyclophosphamide (4HC) on different human hematopoietic progenitor cells were determined. Day 7 colonies supported by granulocyte colony stimulating factor (G-CSF), day 10 colonies supported by granulocyte-macrophage colony stimulating factor (GM-CSF) were counted. Approximately 2 X 10(2) granulocyte-macrophage colonies per 5 X 10(4) bone marrow (BM) mononuclear cells were formed in each assay system. A combination of interleukin 3 (IL3) and erythropoietin (EPO) induces 3 types of day 14 colonies; erythroid burst-forming units (BFU-E, 40 +/- 29/5 X 10(4) BM cells), granulocytes-macrophage colony forming units (CFU-GM, 143 +/- 29/5 X 10(4) cells and mixed colonies (CFU-GEMM, 24 +/- 13/5 X 10(4) cells). After treatment with 4HC, all the colonies were reduced. However, the recovery rate of day 14 colonies supported by the combination of IL3 and EPO (mean 12.1%) was significantly higher than the recovery rate of both day 7 colonies supported by G-CSF (mean 1.0%) and day 10 colonies supported by GM-CSF (mean 4.0%). The recovery rate of colonies supported by IL3 and EPO as also higher than that of day 14 colonies supported by GM-CSF and EPO. Immature pluripotent hematopoietic progenitor cells supported by IL3 and EPO appeared less sensitive to 4HC treatment than those supported by G-CSF and GM-CSF. Colony forming assays using IL3 may be useful in order to predict the hematological reconstitution after autologous bone marrow transplantation with 4HC treated graft.

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 less than 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.

The reovirus-like agent, sometimes referred to as duovirus or rotavirus, was visualized by electron microscopy in stool extracts from Japanese infants and young children with acute epidemic gastroenteritis. The virus particles measured 70 nm in diameter and had double-shelled capsids. One hundred ten (89%) of 124 patients with the gastroenteritis had such virus particles in stools obtained during the acute phase. The virus particles were excreted in the stools usually during the first eight days of illness. Agglutination of virus particles by antibody present in convalescent-phase sera was demonstrated by immune electron microscopy. Complement-fixing antibody was detected as early as day 3 of illness, and antibody titers peaked during the second and third weeks of the disease. The antibody appearing in the acute and early convalescent phases was sensitive to 2-mercaptoethanol. Antibody resistant to 2-mercaptoethanol was produced approximately 10 days after the onset of the symptoms. The serologic evidence suggests that a primary infection with the reovirus-like agent was responsible for the clinical attack of acute gastroenteritis.