Discrimination of human HLA-DRB1 alleles by PCR-SSCP (single-strand conformation polymorphism) method.

A single-strand conformation polymorphism (PCR-SSCP) method has been adopted for discrimination of human HLA-DRB1 alleles. This method enabled the detection of DNA polymorphisms including point mutations at a variety of positions in the DNA fragments of the HLA-DRB1 gene. A total of 27 HLA-DRB1 alleles from 172 healthy donors were analysed using a combination of PCR-SSCP with group-specific amplifications. Application of a small amount of amplified and denatured DNA to non-denaturing electrophoresis followed by silver staining resulted in distinct banding patterns. Samples possessing a single allele in each amplification group showed two-band patterns which correspond to the sense and antisense strands, while heterozygotes in the same group or a mixture of two single-type samples showed four-band patterns. All of the analysed alleles were discriminated in each DRB1 group. The method described here may be somewhat complicated for routine typing of HLA-DRB1 alleles. However, it is useful in the screening of "new' alleles as well as the donor-recipient molecular matching of HLA class II genes for various purposes, e.g. selection of bone marrow transplant donors.

Nation-wide randomized comparative study of doxorubicin, vincristine and prednisolone combination therapy with and without L-asparaginase for adult acute lymphoblastic leukemia.

A randomized clinical trial of combination chemotherapy for adult acute lymphoblastic leukemia (ALL) with doxorubicin, vincristine and prednisolone with and without L-asparaginase (AdVP vs L-AdVP) was conducted, involving 58 institutions throughout Japan. After reaching complete remission (CR), patients were treated with the same regimen for more than 2 years. Among 166 evaluable cases of the 198 cases enrolled, CR rates were 63.1% (53/84) with AdVP and 64.6% (53/82) with L-AdVP (P = 0.837). Median survival times and 7-year survival rates were 12.7 months and 21.2% with AdVP, and 16.0 months and 22.3% with L-AdVP (P = 0.955 by generalized Wilcoxon test [GW], P = 0.952 by log-rank test [LR]). Median disease-free survival times and 7-year survival rates were 13.5 months and 23.8% with AdVP and 17.0 months and 30.6% with L-AdVP, showing some increments for L-AdVP but no statistical significance (P = 0.141 by GW, P = 0.300 by LR). Among the cases of extramurally confirmed FAB subtypes, CR rates were 75.9% (63/83) for the L1 subtype and 51.3% (39/76) for the L2 subtype (P = 0.001). As to adverse effects, pancreatitis was complicated more frequently in L-AdVP than in AdVP (P = 0.039). Other side effects such as hyperbilirubinemia, diabetes mellitus, diarrhea and hypofibrinogenemia were observed more frequently with L-AdVP, but with no statistical significance. Thus, addition of a single course of L-asparaginase in the induction phase of combination chemotherapy with doxorubicin, vincristine and prednisolone did not significantly enhance the effect of antileukemic treatment of adult ALL.

Nationwide randomized comparative study of daunorubicin and aclarubicin in combination with behenoyl cytosine arabinoside, 6-mercaptopurine, and prednisolone for previously untreated acute myeloid leukemia.

Aclarubicin was evaluated in combination chemotherapy for adult acute myeloid leukemia in a randomized trial involving 58 institutions throughout Japan. Behenoyl cytosine arabinoside (BH-AC).daunorubicin, 6-mercaptopurine, and prednisolone (DMP) was compared with BH-AC.aclarubicin, 6-mercaptopurine, and prednisolone (AMP). In the 360 evaluable cases among the 433 cases enrolled, complete remission (CR) rates were 63.7% (116/182) for BH-AC.DMP and 53.9% (96/178) for BH-AC.AMP (P = 0.0587). Median survival periods and 7-year survival rates were 15.8 months and 19.3% for BH-AC.DMP and 9.5 months and 20.2% for BH-AC.AMP (P = 0.0091 according to the generalized Wilcoxon test [GW], P = 0.196 according the log-rank test [LR]). Median disease-free survival periods were 15.4 months for BH-AC.DMP and 14.1 months for BH-AC.AMP (P = 0.851 by GW, P = 0.439 by LR). Among the 346 cases of extramurally confirmed FAB subtypes, CR rates were 67.9% (19/28) with BH-AC.DMP and 31.8% (7/22) with BH-AC.AMP for subtype M3 (P = 0.011) and 63.3% (93/147) with BH-AC.DMP and 56.8% (84/148) with BH-AC.AMP (P = 0.254) for subtypes M1, M2, M4, and M5. Diarrhea, ileus, pneumonia, and renal failure were more frequent with BH-AC.AMP than with BH-AC.DMP. The results indicate, at least on the basis of the long-term outcome, that BH-AC.AMP has antileukemic effects on subtypes M1, M2, M4, and M5 that are comparable with those of BH-AC.DMP.

Genetic variants of serum albumin in Americans and Japanese.

A collaborative search for albumin genetic variants (alloalbumins) was undertaken by cellulose acetate and agarose electrophoresis at pH 8.6 of the sera of patients at two major medical centers in the United States and of nearly 20,000 blood donors in Japan. Seventeen instances of alloalbuminemia were ascertained, and seven different alloalbumin types were characterized by structural study. Two previously unreported alloalbumin types were identified. In one type, which was present in a Caucasian family and designated Iowa City-1, aspartic acid at position 365 was replaced by valine (365 Asp----Val); this is the second reported mutation at this position. The other type present in a Japanese blood donor had the mutation 128 His----Arg. An unexpected finding was the presence in a single Japanese of a Naskapi-type alloalbumin (372 Lys----Glu), a variant that had previously been described only for certain Amerindian tribes in whom it occurs with a polymorphic frequency (greater than 1%) and in Eti Turks. An arginyl-albumin (-1 Arg, 1 Asp----Val) occurred in an American family. The other alloalbumin types identified were proalbumins Lille and Christchurch and albumin B that have a cumulative frequency of about 1:3500 in Caucasians probably because of the hypermutability of CpG dinucleotides at the mutated sites. All of the variants characterized in this study are point mutants, and the sites are spread throughout the albumin gene. However, about one-fourth of all known albumin mutations are clustered in the sequence segment from position 354 through 382.

Effectiveness of the Imugard E leukocyte removal filter for preparation of leukocyte-poor concentrated red cells.

Concentrated red cells (CRC) were filtered through a new leukocyte removal filter, the Imugard E, which consists of a polyvinyl alcohol porous sheet. CRC were filtered through the Imugard E with neither priming before filtration nor rinsing after filtration. Leukocyte removal was 99.1 +/- 0.6, 99.6 +/- 0.3 and 99.6 +/- 0.4% on the 1st, 5th and 10th day after blood collection, respectively. Platelet removal was 96 +/- 2, 81 +/- 6 and 85 +/- 3% on the 1st, 5th and 10th day, respectively. Red cell recovery was 86 +/- 2, 86 +/- 1 and 86 +/- 1% on the 1st 5th and 10th day, respectively. Filtration time was 3.9 +/- 0.8, 5.8 +/- 0.9 and 6.1 +/- 0.8 min on the 1st, 5th and 10th day, respectively. Direct filtration of CRC through the Imugard E resulted in no significant changes in the ATP or 2,3-DPG concentrations, and no hemolysis due to filtration was noticed. It may be concluded that the Imugard E is a good filter that is simple to use and effective in leukocyte removal.

Recent trends in the treatment and prognosis of adult leukemia with characteristics of patients in Japan: transition during the fifteen years from 1971 to 1985.

Patients with acute (2,569) and chronic (957) leukemia diagnosed at 19 institutes took part in the study on the "Multidisciplinary Treatment of Leukemia" between 1971 and 1985 and were investigated retrospectively. By dividing the 15 years into three five-year periods, we were able to compare patient ratios in the different periods. The proportions of acute to chronic leukemia cases showed no obvious change; however, the proportions of cases diagnosed as acute lymphocytic leukemia in acute leukemia showed a significant increase. The main chemotherapeutic drugs used during the three time periods were cytarabine or its analogues, the anthracyclines, 6-mercaputopurine and prednisolone, against acute myelogenous leukemia, and the vinca alkaloids, prednisolone and the anthracyclines against acute lymphocytic leukemia. The rate of complete remission from acute myelogenous leukemia made marked progress, from 45.1% during 1971-1975 to 62.3% during 1981-1985, but that of acute lymphocytic leukemia showed no significant progress, being 65% during 1971-1975 and 69.7% during 1981-1985. The durations of remission, however, and the survival times for patients with acute lymphocytic leukemia, as well as for those with acute myelogenous leukemia, became significantly longer over the three periods. Median survival times from chronic myelocytic leukemia were 37-40 mo in all three periods, showing no progress. There was a better prognosis in cases of chronic myelocytic leukemia with, than without, Philadelphia chromosome. Except for a low incidence of chronic lymphocytic leukemia in Japan, adult leukemia patients' characteristics and prognoses seem to be almost the same in Japan as in the U.S.A. and Europe.

Aberration of monokine production and monocyte subset in patients with systemic lupus erythematosus.

We measured the production of interleukin 1 (IL-1) and prostaglandin by adherent cells from patients with systemic lupus erythematosus (SLE). Compared to normal subjects, IL-1 production in the patients was lower but prostaglandin E1 production was higher. Furthermore, examination of monocyte subsets in patients with SLE using monoclonal anti-monocyte/granulocyte antibodies disclosed abnormal expression of membrane antigens on monocytes. It is speculated that aberration of monocyte function is related to impaired monokine production and that membrane antigens play a role in immunodysregulation in patients with SLE.

Treatment of acute leukemia and malignant lymphoma with (2"R)-4'-O-tetrahydropyranyladriamycin.

Eighty-four previously treated adult patients with acute leukemia and malignant lymphoma were treated with (2"R)-4'-O-tetrahydropyranyladriamycin (THP). THP (10-55 mg/m2) was administered by i.v. bolus injection daily for acute leukemia, and according to three different schedules for malignant lymphoma: daily, weekly or once every 3-4 weeks. Complete and partial remission (CR and PR) were achieved by 1 (5%) and 3 of 19 patients with acute myelogenous leukemia and by 2 (13%) and 3 of 15 patients with acute lymphoblastic leukemia, respectively. All CRs were in the groups receiving 25 mg/m2 THP daily. CR and PR were achieved by 6 (14%) and 8 of 42 patients with non-Hodgkin lymphoma (NHL) and by 4 (50%) and 2 of 8 patients with Hodgkin's disease (HD), respectively. No particular sensitivity was found among the subtypes of NHL and HD. Response (CR + PR) was noted in 10 (40%) of 25 patients treated every 3-4 weeks, in 1 (17%) of 6 treated weekly, and in 9 (47%) of 19 treated daily. The major side effects were myelosuppression and gastrointestinal toxicities. Alopecia was observed in only 10 (12%) patients. ECG abnormalities were observed in 7 (10%) patients, all of whom had previously been treated with other anthracyclines. No severe cardiotoxicity was observed.

Relapsing process in FAB subtypes of adult acute leukemia and its relationship to treatment regimens.

The relapsing process in the bone marrow was studied in those 77 patients with adult acute leukemia, diagnosed according to the FAB classification who achieved complete remission (CR) and then received intermittent multi-drug intensification treatment. Relapse occurred in most of the patients who exhibited Auer rods or Ph1 chromosomes in the bone marrow, or in whom blasts increased to 8% or more, but some patients remained in CR by subsequent treatment, that is, relapsing process was reversible. With our conventional treatment, the relapse or relapsing process occurred in most of the patients with L1, L2 and M1 subtypes within 6 months and was irreversible. It occurred mainly between 5 and 13 months in those with M2, M3 and M5 and was reversible in some cases. Patients with M4 subtype received intensified treatment due to the difficulty of achieving remission; relapse was seen in only 3 of 7 cases. To prevent relapse and attain a potential cure, the treatment should be intensive before the relapsing process with adequate supporting care. In view of the above-mentioned observations, our new treatment protocols for acute leukemia were designed to be more intensive than those conventionally employed, and to be discontinued within approximately 10 months in lymphoblastic leukemia and approximately 8 months in myeloid leukemia.

Phase I clinical and pharmacokinetic study of N4-behenoyl-1-beta-D-arabinofuranosylcytosine.

A phase I study of N4-behenoyl-1-beta-D-arabinofuranosylcytosine (BHAC) was conducted in 66 patients, 41 with solid tumors and 25 with hematological malignancies. The patients received either a 2-h single intravenous (i.v.) drip infusion (Schedule 1) or consecutive daily 2-h i.v. infusions (Schedule 2). In Schedule 1 the daily dose was initiated with 1.5 mg kg-1 which was escalated up to 7 mg kg-1. Side-effects were mild, and included nausea, vomiting, epilation, and hot flushes. Because of the presence of the solvent vehicle, HCO-60 and in consideration of the mechanism of action of BHAC, the dose escalation was stopped at 7 mg kg-1. In Schedule 2, the daily dose was started with 1.5 mg kg-1 which was escalated up to 8 mg kg-1 and given for 2-16 days. Myelosuppression was found to be dose-limiting toxicity. The maximum tolerated dose (MTD) in patients with non-hematological solid tumors was assumed to be 5 mg kg-1 daily X 5 days. The plasma disappearance curve of BHAC looked biphasic, and when 4 mg kg-1 of BHAC were administered the half-lives of the initial phase (t1/2 alpha) and the second phase (t1/2 beta) were calculated as 0.798 and 5.76 h respectively. In Schedule 2 complete remission was observed in 5 out of 21 patients with acute leukemia, one partial remission in Hodgkin's disease, and one 1-B response (Karnofsky) in thyroid papillary adenocarcinoma.

Elicited antibody nature of human monoclonal protein with anti-streptolysin O activity--analysis with monoclonal anti-idiotype antibody.

Sera from 7 patients with multiple myeloma having antistreptolysin O (ASO) activity in high titers were detected by a streptolysin O (SLO) inhibition assay. However, activity was in low titer when assayed by a passive agglutination assay. The discrepancy between these 2 assays raised some doubts as to whether these monoclonal proteins (M.protein) bond to SLO in the same manner as elicited antibodies. Immunochemical analysis and idiotope analysis using monoclonal antibody to one of these M.proteins strongly suggest that M.protein with ASO activity bind to SLO in a manner similar to elicited antibody. The discrepancy between the 2 assays might be due to differences in the antigenic structure of different forms of the SLO molecule.

Treatment of multiple myeloma with recombinant human leukocyte A interferon.

A phase II study of recombinant human leukocyte A interferon was conducted in 64 patients with multiple myeloma in a multi-institutional cooperative trial. Partial remission was achieved in ten (21.3%) of 47 evaluable patients and minor response was observed in five (10.6%). Side effects were noted in more than two-thirds of the patients. They included fever (58%), malaise (20%), anorexia (52%), nausea and vomiting (26%), lethargy (2%), and myelosuppression (56%). An antibody to recombinant human leukocyte A interferon was detected in one of 20 patients.

Treatment of acute myelogenous leukemia in adults with N4-behenoyl-1-beta-D-arabinofuranosylcytosine.

Forty-five previously-untreated adult patients with acute myelogenous leukemia (AML) were treated with N4-behenoyl-1-beta-D-arabinofuranosyl-cytosine (BHAC) in a multi-institutional cooperative study. Among 41 evaluable patients, 15 (36.6%) achieved complete remission (CR) and 10 (24.4%) achieved partial remission by daily administration of 3 to 8 mg/kg of BHAC. Higher daily doses (5 mg/kg or more) produced higher CR rates, and all of the CR were observed among the patients receiving a total BHAC dosage of 50 mg/kg or more in a period of 10 days or more. The side effects were mild and acceptable: nausea-anorexia was observed in 27% of the patients and vomiting in 17%. The results of this study thus indicate BHAC to be effective for remission induction of AML, and to deserve further clinical trials in combination with other anti-leukemic drugs.

Impaired B cell proliferation by Staphylococcus aureus Cowan 1 in patients with systemic lupus erythematosus.

We examined the proliferative response to Staphylococcus aureus Cowan 1 (SAC) by enriched peripheral blood B cells from patients with systemic lupus erythematosus (SLE). Responses of B cells from patients with active and inactive SLE were significantly lower than those of B cells from normal individuals. Hyporesponsiveness to SAC was not observed in healthy family members of SLE patients. This hyporesponsiveness did not correlate with prednisolone therapy and could not be attributed to serum factors; it did correlate with the presence of suppressor monocytes. However, we could not exclude the possibility of enhanced sensitivity of SLE B lymphocytes to suppressive signals delivered by the monocytes.

Survival and repopulation of irradiated 'pre-CFU-c' in mice.

Supernatants of murine bone-marrow cultures contain a colony-promoting factor (CPF) which increases the number of granulocyte and macrophage colonies in semi-solid agar cultures in the presence of colony-stimulating factor (CSF). Incubation of bone-marrow cells with CPF results in an increase in the number of granulocyte/macrophage progenitor cells (CFU-c) and the CPF-responsive cells may be younger than the CFU-c. We have investigated the radiosensitivity and the pattern of the recovery after irradiation of CPF-responsive cells. We found that the radiosensitivity of CPF-responsive cells was significantly lower than those of CFU-c, burst-forming units-erythroid (BFU-e) and pluripotent stem cells in vivo (CFU-s) and in vitro (CFU-mix). The CPF-responsive cells remained subnormal even at 28 days after irradiation of the mice, a time when the CFU-s and CFU-c had recovered completely. Therefore the CPF-responsive cells may constitute a separate compartment, namely 'pre-CFU-c', in the maturation sequence of granulopoiesis, and this maturation of the 'pre-CFU-c' to CFU-c seems to be highly stimulated after irradiation to counterbalance the influx from CFU-s.