Granulocyte colony-stimulating factor receptors on human acute leukemia: biphenotypic leukemic cells possess granulocyte colony-stimulating factor receptors.

Granulocyte colony-stimulating factor (G-CSF) receptors on the gated leukemic blast cells from newly diagnosed patients with acute leukemia or crisis of chronic myelogenous leukemia were investigated using flow cytometric detection. Surface marker analysis and cytochemical studies were conducted simultaneously to characterize the blast cells. Among 24 leukemia cases examined, G-CSF receptor-positive blast cells were detected in all 11 cases of acute myeloblastic leukemia even though the percentage range of positive cells was widely variable. On the other hand, they were not detected on the blast cells from patients with peroxidase-negative acute lymphoblastic leukemia with no myeloid surface antigens. However, G-CSF receptors were demonstrated in significant amounts on blast cells from 5 of 8 cases of peroxidase-negative acute leukemia expressing both myeloid and lymphoid surface antigens (biphenotypic leukemia). The percentage of blast cells positive for G-CSF receptors was significantly smaller in biphenotypic cases [33 +/- 14% (SD)] than in acute myeloblastic leukemia cases [65 +/- 22%] (P less than 0.01). The percentage expression of CD13 antigen by blast cells was significantly related to their percentage positivity for G-CSF receptors (rs = 0.50, P less than 0.05). These findings indicate that the distribution of flow cytometrically detectable G-CSF receptors on leukemic cells possessing myeloid characteristics may be related to the maturation process.

Ongoing hypermutation in the Ig V(D)J gene segments and c-myc proto-oncogene of an AIDS lymphoma segregates with neoplastic B cells at different sites: implications for clonal evolution.

To investigate the role of somatic Ig hypermutation in the evolution of AIDS-associated B cell lymphomas, we analyzed the Ig V(D)J and c-myc genes expressed by neoplastic B cells in two extranodal sites, testis and orbit, and clonally related cells in the bone marrow. Testis and orbit B cells expressed differentially mutated but collinear V(H)DJ(H), V kappa J kappa and c-myc gene sequences. Shared mutations accounted for 10.2%, 8.4%, and 4.3% of the overall V(H)DJ(H), V kappa J kappa, and c-myc gene sequences. Tumor-site specific V(H)DJ(H), V kappa J kappa, and c-myc mutations were comparable in frequency, and a single point-mutation gave rise to an EcoRI site in the testis c-myc DNA. Both shared and tumor site-specific V(H)DJ(H), V kappa J kappa, and c-myc mutations displayed predominance of transitions over transversions. The "neoplastic" V(H)DJ(H) sequence was expressed by about 10(-5) cells in the bone marrow, and contained two of the three orbital, but none of the testicular V(H)DJ(H) mutations. The nature and distribution of the Ig V(D)J mutations found in the kappa chain suggested a selection by antigen in testis and orbit. Our data suggest that, in AIDS-associated B cell lymphomas, the Ig hypermutation machinery targets V(H)DJ(H), V kappa J kappa, and c-myc genes with comparable efficiency and modalities.

Analysis of c-kit expression of human erythroleukemia cell line, HEL: clonal variation and relationship with erythroid and megakaryocytic phenotype.

The proto-oncogene c-kit encodes the receptor for a stem cell factor. We examined HEL, a human erythroleukemia cell line, in order to clarify the correlation between the c-kit receptor (KR) expression and lineage-specific phenotype. Although HEL cells are known to express KR, we found two relatively distinct HEL cell populations in terms of KR expression. We then subcloned HEL cell lines with clone sorting on the basis of KR expression and compared their various characteristics. The highly KR-expressing subline, HEL-P1, expressed a high level of glycophorin A (GPA), a known erythroid lineage marker. HEL-N1, in which most of the cells were KR-negative, showed a higher megakaryocytic lineage marker CD41b expression than HEL-P1. However, the expression of granulomonocytic lineage markers were not significantly different between the two subclones. Cell growth rate and cell cycle analysis also did not detect significant differences between the sublines. HEL-P1 cells gradually lost their KR expression in serum-containing culture, while the percentage of KR-positive HEL-N1 cells increased in serum-free culture. These observations indicate that KR expression was associated with the synchronous expression of GPA and inversely correlated with CD41b, and reversible transitions between KR-positive cells and KR-negative cells exist. We suggest that KR plays an important part in commitment of erythroid and megakaryocytic precursor cells.

Analysis of acute myeloid leukemia cells by flow cytometry, introducing a new light-scattering classification.

A combined flow-cytometric evaluation of light scattering and the immunophenotype of acute myeloid leukemia (AML) cells from 71 newly diagnosed consecutive patients was conducted. Light-scattering characteristic of AML cells examined by flow cytometry and multiple surface markers were also analyzed using the same samples, to enable a comparison with the French-American-British (FAB) classification. Our AML cases could be classified into three light-scattering classification (LSC) types according to their physical properties on flow cytometry. These were type A, where forward light scattering (FSC) of the leukemic cell population was larger than that of lymphocytes, while side light scattering (SSC) was the same or larger than that of lymphocytes but smaller than that of monocytes; type B, where FSC of the leukemic cell population was larger than that of lymphocytes and SSC spread toward that of monocytes; and type C, where both FSC and SSC of the leukemic cell population spread beyond those of monocytes. Although a clear relationship between the FAB classification and LSC classification by the light-scattering profile of AML was not established, we observed the following findings. The majority of cases were classified as type A (58%), while type B comprised 25% and type C comprised 17%. While CD7 expression on AML cells is considered to be an immature characteristic, CD7 was expressed more frequently among LSC type A cases. Furthermore, all but one of the FAB M1 cases were classified as type A. On the other hand, CD7 was not expressed on type C leukemic cells. The percentage of cases in which more than 60% of leukemic cells possessed another immature surface antigen, CD 34ö, was 13/18 (72%) among FAB M1 cases, much higher than among FAB M2 (35%) or FAB M4 (27%) cases. A negative correlation was observed between mature antigen CD33 and CD34 among the FAB M2 cases. The frequency of CD7 expression was 25% among the total cases, and CD7-positive cases were frequent among FAB M1 and M2, but not among FAB M3 cases. These findings concerning LSC and immunophenotyping indicate that the scattergram pattern analysis may contribute towards more precise immunophenotyping, in that it reflects the maturation stage of each AML case.

CD7-positive acute myeloid leukemia: further evidence of cellular immaturity.

Among 63 cases of acute myeloid leukemia (AML), 14 were found to express the CD7 antigen, a cell surface marker usually found at an early stage during T lineage differentiation. The CD7-positive AML cases consisted of 5 cases of M1, 3 cases of M2, 3 cases of M4, 1 case of M5, 1 case of M6 and 1 case of M7. Among these 63 cases, the proportion of blast cells expressing the CD34 antigen was examined. The proportion of CD34-stained cells among the CD7-positive AML cases, although varying, was significantly larger than that among the CD7-negative AML cases (P less than 0.05). As the CD34 antigen was expressed on hematopoietic progenitor cells and was considered to reflect an early hematopoietic stage, the high proportion of cells expressing CD34 among the CD7-positive AML cases may support the notion that CD7-positive AML cells are immature.

[Long-term remission of a case of Burkitt's lymphoma accompanied by cardiac tamponade treated by surgical resection and intensive chemotherapy].

A 26-year-old woman was admitted to our hospital because of abdominal mass. She became ileus and an emergency operation was consequently performed. A histological examination of the abdominal tumor indicated proliferation of undifferentiated lymphoblasts with intermingled histiocytes so called "starry sky appearance". The diagnosis was Burkitt's lymphoma. On the 4th post-operative day, a cardiac tamponade became evident. The pericardial effusion contained many lymphoblasts and the diagnosis was pericarditis due to the invasion of lymphoma cells. The pericarditis was successfully treated by infusion of doxycycline into the pericardial space following drainage. The patient responded to systemic chemotherapy with complete remission. 7 courses of systemic chemotherapy along with intrathecal infusions for CNS prophylaxis were subsequently carried out. A state of complete remission has continued for more than 13 months. Cardiac tamponade accompanied by Burkitt's lymphoma is quite rare and has not ever been reported in Japan in our knowledge. The efficacy of surgical treatment before systemic chemotherapy and the series of intrathecal infusions for CNS prophylaxis was demonstrated in this case.

[Effectiveness of synthetic calcitonin derivative (elcatonin) on the bone pain and serum calcium concentration in multiple myeloma].

Fifteen patients (8 male and 7 female) with multiple myeloma, who were admitted to our hospital between July 1986 and August 1988 and suffering from pain and hypercalcemia, were treated with synthetic calcitonin derivative (elcatonin: ECT). ECT was administered intravenously at a dose of 10-640 units twice daily. Seven patients were treated with ECT (ECT group), and eight patients received combination treatment with ECT and other form of chemotherapy (combination group). With regard to the pain score (PS), significant analgesic effects in both groups were observed during 1-4 week treatments (p less than 0.05). There were no significant differences in PS between two groups. Serum calcium levels in the combination group at 1 and 4 weeks were significantly lower than the initial value (p less than 0.05). Hypocalcemia was not seen in any of the patients. Urinary excretion of calcium at 1 week in ECT group was higher than the initial value (p less than 0.05). The observed toxicities of ECT were slight nausea and vomiting in only 2 patients. These findings suggest that ECT is an useful agent for the treatment of pain and hypercalcemia accompanied with multiple myeloma.

[Anti-platelet antibody and severe thrombocytopenia during interferon-alpha therapy for chronic active hepatitis C].

We herein report a case of chronic hepatitis C where the patient developed severe thrombocytopenia during interferon therapy. The patient was a 61-year-old woman, who received interferon therapy on April 27, 1993 under the diagnosis of C type chronic active hepatitis. After 4 weeks, her platelet count had decreased to 18,000/microliters and intraoral hemorrhage had begun. Although she received 250 mg of methylprednisolone and 20 U of platelet transfusion three times, her platelet count continued to decrease to 4,000/microliters on both May 28, and on June 3, 1993, and so she was transferred to our hospital on June 4. On her second admission to our hospital, although the platelet-associated IgG (PA-IgG) had increased markedly and the megakaryocytes in her bone marrow had decreased, her platelet count had already increased to 37,000/ microliters, and this gradually returned to a normal level accompanied with a decrease of PA-IgG within one month In this case, although we found immunological abnormalities (high level of IgG, positive ANA and positive anti-smooth muscle antibody) prior to interferon treatment, we could not diagnose the patient as having suffered from autoimmune disease, including autoimmune hepatitis, because she did not satisfy the necessary criteria and because she did not have any symptoms suggesting autoimmune disease. We consider that there may be the possibility that interferon induced only an anti-platelet antibodies that caused the high level of PA-IgG and decreased the production level of platelets within the bone marrow.

[Hematological malignancies with eosinophilia].

Hematological malignancies accompanied by eosinophilia are reviewed in relation to chromosomal changes and cytokine production. Eosinophilia accompanied by hematological malignancies can be divided into two groups. In some myelogenous leukemias, including acute myelomonocytic leukemia with eosinophilia (FAB M4Eo), acute myeloblastic leukemia (FAB M2 t(8;21)) and chronic myelogenous leukemia, neoplastic cells themselves appear to differentiate into eosinophils. On the other hand, transformed tumor cells secrete some eosinophil-stimulating cytokines, including interleukin-3, interleukin-5 and granulocyte-macrophage colony-stimulating factor and these cytokines stimulate the proliferation of normal eosinophil precursors in some lymphoid malignancies, including some types acute lymphoblastic leukemia (especially with t(5;14)) or malignant lymphoma, including Hodgkin's lymphoma and adult T cell lymphoma/leukemia.

[A case of cardiomyopathy due to allergic bronchopulmonary aspergillosis].

We report a rare occurrence of cardiomyopathy associated with allergic bronchopulmonary aspergillosis (ABPA). A 49-year-old man with a history of bronchial asthma was referred to the Matsuyama Red Cross Hospital for evaluation of the abnormal shadow on his chest X-ray. Laboratory examination showed blood eosinophilia and marked elevation of serum IgE concentration and IgE antibody to A fumigatus. The immediate and late skin reactivities to A fumigatus antigen were both positive. The diagnosis of ABPA was made. Treatment using prednisolone was effective in ameliorating the symptoms. However, he was admitted again due to dyspnea, edema and anorexia 6 months later. Chest X-ray, ECG, UCG and scintigraphy suggested severe cardiac failure. The clinical diagnosis of hypertrophic cardiomyopathy, and the pathohistological diagnosis of endomyocardial fibrosis were made by cardiac catheterization and biopsy of endocardium. Retrospectively, cardiomegaly had gradually increased during the past several months while peripheral blood eosinophilia had continued. All these data strongly suggested that eosinophilia due to ABPA might cause severe cardiac damage.

Seroepidemiologic study of antibody to adult T-cell leukemia virus in Okinawa, Japan.

From 1980 to 1984, a total of 3,978 serum samples were collected from healthy subjects in the Yaeyama District of Okinawa, Japan. These serum samples were tested for presence of antibody to adult T-cell leukemia-associated antigen (anti-ATLA) by the enzyme-linked immunosorbent assay (ELISA) method. Overall prevalence of anti-ATLA was 15.3%. Standardized prevalence differed from island to island: Hateruma Island had the highest (21.2%) and Taketomi Island the lowest (6.2%). Prevalence of anti-ATLA increased with age and was significantly higher in females (18.1%) than in males (12.2%) (p less than 0.001). For subjects younger than 20 years of age, the rate of anti-ATLA in males was slightly higher than that in females. For subjects 20 years of age and over, prevalence was higher in females than in males. In all age groups 40 years and over, prevalence was significantly higher in females than in males.

Surface phenotype and Ig heavy-chain gene usage in chronic B-cell leukemias: expression of myelomonocytic surface markers in CD5- chronic B-cell leukemia.

We investigated the surface expression of leukocyte differentiation antigens and the Ig heavy-chain variable region (VH) gene family use in leukemic cells from 26 Japanese patients with chronic B-cell leukemias with special reference to CD5 antigen expression. CD5 was expressed on leukemic cells in 21 of 26 cases (CD5+) but not in 5 cases (CD5-). Myelomonocytic marker, CD13 antigen was expressed on the leukemic cells in all 5 CD5- cases but in none of CD5+ cases. Leukemic cells in CD5- cases also expressed CD11b antigen more frequently than those in CD5+ cases (80% v 11%; P < .01). Another myeloid marker, CD33, was expressed neither on CD5+ nor CD5- leukemic cells. CD22, a restricted B-cell marker, was expressed more frequently on CD5- leukemic cells than on CD5+ leukemic cells (80% v 33%; P < .05). Another restricted B-cell activation marker, CD23, was expressed at similar frequency in both the CD5+ and CD5- groups (67% v 60%). Although CD45RA was expressed on the majority of leukemic B cells, the CD45RA expression level was significantly higher among CD5- cases than CD5+ cases (P < .01). In the analysis of VH gene expressed in chronic B-cell leukemias by polymerase chain reaction amplification, CD5+ cases preferentially used VH4 family members (48%; 10 of 21). CD5- cases, on the other hand, mainly used VH3 family (80%; 4 of 5). Thus, from our present observation of an albeit limited patient population, we have found an association between VH gene family use and CD5 antigen expression in chronic B-cell leukemias. We have also shown the differential expression of myelomonocytic markers in the CD5+ and CD5- chronic B-cell leukemias. These result are in agreement with previous suggestions that CD5 positivity is the hallmark for distinct clinical entity commonly referred to in the literature as chronic lymphocytic leukemia.

Clonal analysis of a human antibody response. III. Nucleotide sequences of monoclonal IgM, IgG, and IgA to rabies virus reveal restricted V kappa gene utilization, junctional V kappa J kappa and V lambda J lambda diversity, and somatic hypermutation.

In previous work, we generated four IgM, five IgG1, and one IgA1 mAbs to rabies virus using B cells from four subjects vaccinated with inactivated rabies virus, a thymus-dependent (TD) mosaic Ag, and sequenced the mAb V(H)DJ(H) genes. Here, we have cloned the V kappa J kappa and V lambda J lambda genes to complete the primary structure of the Ag-binding site of these mAbs. While the anti-rabies virus mAb selection of VA genes (2e.2.2 twice, DPL11, and DPL23) reflected the representation of the V lambda genes in the human haploid genome (stochastic utilization), that of V kappa genes (O2/O12 twice, O8/O18, A3/A19, A27, and L2) did not (p = 0.0018) (nonstochastic utilization). Furthermore, the selection of both V kappa and V lambda genes by the anti-rabies virus mAbs vastly overlapped with that of 557 assorted V kappa J kappa rearrangements, that of 253 V lambda J lambda rearrangements in lambda-type gammopathies, and that of other Abs to thymus-dependent Ags, including 23 anti-HIV mAbs and 51 rheumatoid factors, but differed from that of 43 Abs to Haemophilus influenzae type b polysaccharide, a prototypic thymus-independent (TI) Ag. The anti-rabies virus mAb V kappa J kappa and V lambda J lambda segments displayed variable numbers of somatic mutations, which, in mAb58 and the virus-neutralizing mAb57, entailed a significant concentration of amino acid replacements in the complementarity-determining regions (p = 0.0028 and p = 0.0023, respectively), suggesting a selection by Ag. This Ag-dependent somatic selection process was superimposed on a somatic diversification process that occurred at the stage of B cell receptor for Ag rearrangement, and that entailed V gene 3' truncation and N nucleotide additions to yield heterogeneous CDR3s.

Circulating CD34+ hematopoietic progenitors in the harvesting peripheral blood stem cells; enhancement by recombinant human granulocyte colony-stimulating factor.

The number of circulating progenitor cells increases during the period of hematopoietic recovery following myeloablative therapy. These progenitor cells were used for autologous transplantation in order to reconstitute hematopoiesis. As an indicator of the circulating progenitor cells, the number of granulocyte-macrophage colony forming units (CFU-GM), which is measured by means of a long-term cell culture, has been widely used. Recently, a cell surface marker, CD34, which can easily be measured by means of flowcytometry, was found to represent immature hematopoietic progenitor cells, which are very close to stem cells. Therefore, the relationship between the number of CD34 positive cells (CD34+ cells) and the number of CFU-GM in the peripheral blood following chemotherapy was studied in 9 patients selected to undergo autotransplantation. The number of peripheral blood CD34+ cells was found to be significantly correlated with that of CFU-GM (r = 0.81). When four out of 9 patients received recombinant human granulocyte-colony stimulating factor (rG-CSF) administration, a significant increase in the release of peripheral blood CD34+ cells as well as peripheral blood CFU-GM was observed (P less than 0.01). Thus, the measurement of CD34+ cells is useful for predicting the number of circulating CFU-GM.

Hepatocellular carcinoma and bladder cancer as complications following five years of chemotherapy for acute myeloblastic leukemia.

Acute myeloblastic leukemia (AML) was diagnosed in a 54-year-old male, a chronic hepatitis B surface antigen (HBsAg) carrier, in June, 1983. Prompt remission was achieved, and maintenance and intensification chemotherapy were given for five years. He was readmitted in March, 1988 because of a mass in the liver and was diagnosed as having hepatocellular carcinoma (HCC). Curative right segmentectomy was performed in May, 1988. In December, 1988, transitional cell carcinoma of the bladder was discovered, and resected transurethrally. These secondary neoplasms, HCC and bladder cancer, were thought to be associated with the long-term chemotherapy given for the AML.

Prevalence of immunologic markers of hepatitis A and B infection in hospital personnel in Miyazaki Prefecture, Japan.

Between 1980 and 1983, a total of 1,883 serum samples from employees of four prefectural hospitals in Miyazaki prefecture, Japan were surveyed for antibody to hepatitis A virus (anti-HAV) and for the following hepatitis B virus markers: hepatitis B surface antigen (HBsAg), antibody to HBsAg (anti-HBs), and antibody to hepatitis B core antigen (anti-HBc). Overall prevalences were 36.9% for anti-HAV, 3.4% for HBsAg, 23.3% for anti-HBs, and 36.6% for anti-HBc. In the control group of 233 healthy persons, prevalences were 51.5% for anti-HAV, 3.0% for HBsAg, 28.3% for anti-HBs, and 33.5% for anti-HBc. No significant difference in the distribution of HBsAg was seen among five work categories. Anti-HBc prevalence was significantly higher in nurses than in office workers (p less than 0.05), other medical personnel (p less than 0.05), and controls (p less than 0.01). The differences between nurses and office workers and other medical personnel became greater with age, but a difference between nurses and the control group was recognized in every age group. A significant difference in the distribution of anti-HBc was seen between surgical physicians (36.7%) and nonsurgical physicians (27.1%). Prevalence of anti-HAV in physicians (32.8%), nurses (29.6%), and laboratory technicians (40.1%) was significantly lower than in the control group (51.5%). These data suggest that in the hospitals studied, hepatitis B is an occupational hazard to nurses and surgical physicians, but that hepatitis A is not.

Human anticardiolipin monoclonal autoantibodies cause placental necrosis and fetal loss in BALB/c mice.

OBJECTIVE: To analyze the structure, specificity, and in vivo pathogenetic potential of 2 human anticardiolipin (aCL) monoclonal antibodies (MAb). METHODS: Human aCL IgG MAb were generated from hybridized Epstein-Barr virus-induced B cell lines from a healthy subject (MAb 519) and from a patient with primary antiphospholipid syndrome (MAb 516). Studies of antigen-binding specificity and analysis of Ig V-gene mutations were carried out. The MAb were independently injected into mated female BALB/c mice, and their effect on pregnancy outcome was compared with that of MAb 57, a highly mutated and antigen-selected human IgG1lambda rabies virus antibody. RESULTS: Both MAb 519 and MAb 516 utilized minimally mutated V(H)DJ(H) and VkappaJkappa gene segments and bound cardiolipin and other anionic phospholipids in the absence of beta2-glycoprotein I (beta2-GPI). The mice injected with aCL MAb displayed a significantly higher rate of fetal resorption and a significant reduction in fetal and placental weight as compared with those injected with MAb 57. These findings were accompanied by a finding of placental human IgG deposition and necrosis in the aCL MAb-treated animals. CONCLUSION: The results of this study indicate that human aCL IgG that are beta2-GPI independent can induce pathology.