Kenichi Harigaya

Some problems on the histopathological diagnosis of non-Hodgkin's malignant lymphoma -- a proposal of a new type.

A new classification for non‐Hodgkins malignant lymphoma is proposed as the one suited for the Lymphomas in Japan, which is to provide a new subtype “pleomorphic” for those more or less rapid‐growing lymphomas of peripheral T‐cell nature, along with another subtype lymphoblastic, after Nathwani et al. for those of central T‐cell nature. The proposal is based on the result of the investigation by the Study Group for Histopathological Diagnosis on Malignant Lymphoma that (1) the presence of a significant number of T‐cell lymphomas with peculiar “pleomorphism” is responsible for the very low reproducibility rate of histopathological diagnosis on the diffuse, mixed L&H type of Rappaport classification, and (2) the relative incidence of lymphoms of peripheral T‐cell nature including the so‐called adult T‐cell leukemia is much higher in Japan than in the Western countries.

Colony-stimulating factor 1 expression is down-regulated during the adipocyte differentiation of H-1/A marrow stromal cells and induced by cachectin/tumor necrosis factor.

We isolated clonal sublines of the established mouse marrow stromal cell line, H-1. These clonal sublines underwent differentiation into adipocytes in various degrees. One subline, H-1/A, underwent adipocyte differentiation after confluence, while another subline, H-1/D, did not differentiate. In H-1/A cells, the 4.5- and 2.5-kb major mRNA species of colony-stimulating factor 1 (CSF-1) were expressed before differentiation and were down-regulated at a posttranscriptional level during the differentiation of H-1/A cells. The down-regulation of the CSF-1 gene was not a result of arrested cellular growth, because no down-regulation was detected in the nondifferentiating sister line, H-1/D. This down-regulation appeared to be an early event in differentiation. Cachectin/tumor necrosis factor transiently induced the expression of CSF-1 and inhibited the differentiation of H-1/A cells into adipocytes. This induced expression of CSF-1 was due to an increased rate of transcription.

Radiosensitivity of permanent human bone marrow stromal cell lines: Effect of dose rate

In contrast to the dose-rate independent X ray killing observed with human bone marrow hematopoietic stem cells, bone marrow adherent stromal cells from the same fresh marrow harvests demonstrate increased radiation resistance at low dose rate (LDR) (5 cGy/min), compared to high dose rate (HDR) irradiation (120-200 cGy/min). Physiologic changes observed in plateau phase bone marrow cells after LDR irradiation in vivo and in vitro suggested that marrow stromal cells might be heterogeneous in LDR irradiation repair. Five permanent clonal bone marrow stromal lines were derived from a single human marrow donor. Each cell line was positive for markers of fibroblasts including: immunohistochemically detectable fibronectin, collagen, acid phosphatase, and nonspecific esterase, and was negative for Factor VIII, alkaline phosphatase, lysozyme and several markers of marrow macrophages. The x-irradiation survival curve of each cell line was determined at LDR and HDR in vitro. Cell lines KM102, KM103, KM104, and KM105 each demonstrated a significant (p less than .05) increase in radioresistance at LDR (D0 = 142, n = 2.9; D0 = 131, n = 2.5; D0 = 145, n = 2.1 and D0 = 127, n = 2.1 respectively) compared to HDR: (D0 = 111, n = 2.1; D0 = 94, n = 3.5; D0 = 99, n = 3.5 and D0 = 95, n = 2.1 respectively). In contrast, cell line KM101 demonstrated no significant change in radiosensitivity relative to dose rate at LDR (D0 = 113, n = 3.3) compared to HDR, D0 = 114, n = 3.3. Cell line KM101 was more supportive than the other lines of cocultivated hemopoietic cells in vitro. Subclones of KM101 and KM104 selected by retroviral vector transfer of the neor gene for growth in the antibiotic neomycin-analogue G418, maintained the stably associated radiobiologic properties of each parent clonal line. These data indicate significant heterogeneity in the LDR irradiation response of clonal stromal cell lines derived from human bone marrow.

Correlation between Production of Colony-Stimulating Activity (CSA) and Adipose Conversion in a Murine Marrow-Derived Preadipocyte Line (H-1/A)

Abstract The correlation between adipose conversion of cloned H-1 cells (H-1/A) and their production of colony-stimulating activity (CSA) was examined. The production of CSA from H-1/A cells declined after adipose conversion, although H-1/A cells are active producers of CSA during their fibrocytic stage. The addition of 2 × 10−5 M 5-bromo-2′-deoxyuridine to the cultures almost completely inhibited adipose conversion and there was no reduction of CSA levels after 9 days of culture. On the other hand, the addition of 10−6 M hydrocortisone sodium succinate to the culture markedly enhanced adipose conversion, and a greater reduction in the CSA level was observed in the supernatants than in the control cultures after 12 days of culture. Indomethacin had no effect on the production of CSA or on adipose conversion. Furthermore, there were no significant differences between the CSA levels of nondialyzed supernatants and dialyzed supernatants from the control cultures during the entire course of the experiment. Supernatants during the adipocyte stage of H-1/A cultures did not inhibit the CSA derived from the fibrocytic stage. There were no differences in colonies in agar cultures stimulated by supernatants derived from cultures that had undergone either of the above treatments. These results suggest that the reduction of CSA is not due to the production of inhibitors, but that the production of CSA declines after adipose conversion of H-1/A cells. Preadipocytes in bone marrow therefore appear to contribute to granulopoiesis during the fibrocytic stage and are hematopoietically inactive when they convert to adipocytes.

HISTOPATHOLOGICAL STUDY OF SIX CASES OF CASTLEMAN'S TUMOR

A histopathological analysis of six cases of Castlemans tumor by means of light and electron microscope was performed, with a review of literature. All cases were hyaline‐vascular type as described by Keller et al. The morphology of lymphoid follicles in the lesions varied according to the presence or non‐presence of the germinal center which was from large active to emaciated hyalinized. The lymphoid follicle was essentially similar to that of normal lymph node undergoing some reactive process. Depending on the observation of serial sections, the lesions had lymphatic sinuses around the blood vessels in the tumor parenchyma, some of which were proved to be connected to the abortive marginal sinuses.

ENZYME HISTOCHEMISTRY OF NON‐HODGKIN'S LYMPHOMAS

Fifty‐two cases of non‐Hodgkins lymphomas were studied with enzyme histochemical methods. Forty‐four cases of these were also investigated for surface markers with immunological techniques, and results of histochemical, routine histological and immunological observations were correlated. Twenty‐one of 27 B‐cell lymphomas showed prominent ATPase activity, while all 13 T‐cell lymphomas, except one case, did not show such activity. Nodular lymphomas, though of B‐cell nature, were often negative for ATPase and it remained negative after diffuse evolution in some. Four of 7 AlPase positive lymphomas were of B‐cell origin. Dot‐like localized AcPase and β‐glucuronidase activity characterized T‐cell lymphomas while 5 T‐cell PDL, including lymphoblastic type with double markers, showed localized esterase activity. Enzyme histochemical characteristics of lymphomas were fairly honest reflection of those of various functional units in the normal lymph nodes. Enzyme histochemical methods appeared to be a useful tool for the study of lymphomas.

Differentiation of human hematopoietic cells increases expression of a gene transferred by a retroviral vector.

To study expression of a retroviral vector in human hematopoietic lineages, two established human hematopoietic cell lines (HL60 and K562) and a human adherent stromal cell line (KM101) were infected with the vector pZIP‐SV(X). Expression of the transferred neomycin resistance gene (neo) of pZlP‐SV(X) was evaluated as the ability of the cells to form colonies (>50 cells) in an agar assay in the presence of the neomycin analogue, G418. After infection, all three cell lines produced colonies resistant to G418. The level of neo mRNA in separate colonies was analyzed by Northern blot analysis. The neo gene transferred by the vector pZIP‐SV(X) was expressed in both human hematopoietic and stromal cell lines. In addition, primary adherent human stromal cells infected with pZIP‐SV(X) grew in the presence of G418. To determine if differentiation of hematopoietic cells affects expression of the retroviral vector, HL60 cells infected with pZIP‐SV(X) were induced to differentiate, and the level of neo mRNA measured. The amount of neo mRNA increased when HL60 cells were induced to differentiate along the granulocytic pathway. Conversely, when HL60 cells were induced toward monocytoid differentiation (TPA), the level of neo mRNA did not significantly increase. We conclude that the neo gene transferred by a retroviral vector, pZIP‐SV(X), is functionally expressed. In addition, expression of the transferred neo gene is regulated during myeloid differentiation of HL80 cells.

Infection of hematopoietic and stromal cells in human continuous bone marrow cultures by a retroviral vector containing the neomycin resistance gene.

Stability and expression of the bacterial neomycin resistance gene (neor) transferred to human continuous marrow cultures by a retroviral vector [pZIP-NeoSV(X)] was evaluated over 4 weeks. Following infection of long-term human marrow cultures with pZIP-NeoSV(X), 10-15% of the stromal cells demonstrated high replating efficiency in a dose of the neomycin analogue G418 that was toxic to stromal cells from uninfected cultures. In contrast, G418 resistance was detected in less than or equal to 1% of GM-CFUc and CFU-GEMM derived from the same virus-infected compared to control cultures. Infection of human CFU-GEMM enriched 100 X by monoclonal antibody selection with pZIP-NeoSV(X) did not increase the percentage of neor progenitors. Marrow cells from cultures infected with pZIP-NeoSV(X) and a replication competent amphotropic virus transferred the vector and G418 resistance to HeLa cells at a frequency of 1/10(5) for nonadherent and 1/10(4) for adherent cells. Two established human hematopoietic (HL60 and K562) and one stromal cell line (KM101) stably expressed the neor gene. Thus, a higher efficiency of infection and expression of a gene transferred by pZIP-NeoSV(X) to permanent human hematopoietic tumor cell lines and fresh marrow stromal cells contrasts with a lower level of expression in fresh CSF-dependent human hematopoietic stem cells.

Conversion to Adipocytes of a Clonal Bone Marrow Preadipocyte Line (H-1/A) and Fatty Acid Composition of the Resultant Adipocytes

Abstract Conversion to adipocytes and fatty acid composition were investigated in a clonal bone marrow preadipocyte line (H-1/A). The growing cells exhibited a fibroblastic appearance. After the cessation of growth, triacylglyceride (TG) synthesis in the cells increased as they incorporated precursor from the growth medium and became adipocytes. Hydrocortisone and insulin accelerated the TG synthesis in H-1/A cells in a dose-dependent manner when they were cultured in the growth medium containing 10% horse serum. The rate of conversion to adipocytes was reduced as the concentration of horse serum was decreased, and this reduction was not influenced by the addition of insulin and/or hydrocortisone. These results suggest that converison to adipocytes of H-1/A cells is primarily dependent on some component(s) of the serum. Conversion to adipocytes of the cells may involve a process of differentiation since the conversion was completely inhibited when the cells were cultured in the presence of bromodeoxyuridine. Fatty acid composition was significantly different between adipose H-1/A cells and adipocytes derived from other marrow preadipocyte line MC3T3-G2/PA6 cells. Unsaturated fatty acids accounted for 76% of the fatty acid composition of adipose H-1/A cells; in contrast, saturated fatty acids constituted 65% of the fatty acid composition of the adipose MC3T3-G2/PA6 cells. These results suggest that there is a heterogeneity of preadipocytes in bone marrow. These two preadipocyte lines thus provide a useful tool for the study of marrow adipocytes and can also be used to analyze the hematopoietic microenvironment through studies of the effect of these cells on hematopoietic cell proliferation.

Histological Characteristics of the Coronary Arteries of Infants and Young Children, and their Implications in the Development of Coronary Arteritis in Kawasaki's Disease.

In order to clarify the factors leading to coronary arteritis in Kawasakis disease (mucocutaneous lymph node syndrome, MCLS), the coronary arteries of 24 control infant cases without a history of MCLS were carefully studied.