Hajime Hoshi

Bone marrow stroma from refractory anemia of myelodysplastic syndrome is defective in its ability to support normal CD34-positive cell proliferation and differentiation in vitro

We examined the supportive function of stromal cells from patients with refractory anemia (RA) of myelodysplastic syndrome (MDS) on CD34-positive hematopoietic cell proliferation and differentiation using a long-term bone marrow culture (LTMC) system. Primary marrow stromal cells were obtained from 11 MDS RA patients and 12 healthy volunteers, and freshly prepared CD34-positive bone marrow cells from a normal subject were inoculated onto the stroma. There seems to be three broad patterns of hematopoietic cell growth in the LTMCs. In one group, hematopoietic cells were maintained at near normal levels (type A). In the second group, the number of hematopoietic cells increased within the first 5-10 days of culture, but declined to low levels at 15-20 days of culture as compared with normal control (type B). In the third group, the incidence of hematopoietic cells steadily declined from the beginning of the culture (type C). Furthermore, apoptotic change of hematopoietic cells was very frequently observed in cultures with the type C stroma, which were especially defective for supporting CD34 + cell proliferation and differentiation. The expression of CD95 on hematopoietic cells was induced by the type C stroma, however, production of fas ligand by the stromal cells was not observed. These findings suggest a lack of hematopoietic supportive function in some cases of MDS RA and also indicate that there is heterogeneity of stromal function among MDS RA patients.

Patterns of age‐dependent changes in the numbers of lymph follicles and germinal centres in somatic and mesenteric lymph nodes in growing C57Bl/6 mice

The timing of the first appearance of lymph follicles and germinal centres in various lymph nodes, and the ways in which numbers of these and IgM‐synthesising cells increase within the nodes, were investigated in male and female C57Bl/6N mice aged from 4 d to 16 wk. The lymphoid organs examined were the Peyers patches, spleen, somatic (submandibular, deep cervical, brachial, axillary, inguinal and popliteal) and visceral (mesenteric and lumbar) lymph nodes. Primary follicles appeared in most somatic lymph nodes 6 d after birth. The number of follicles per node then increased rather sharply in larger lymph nodes and slowly in smaller nodes, up to 28 d of age, reaching a level which varied according to the location of the node. Thereafter, the number of follicles in the somatic lymph nodes increased only slightly to moderately, reaching a peak or plateau at 8–12 wk. In the mesenteric (ileocaecal) nodes, primary follicles first appeared at 12 d, then increased linearly during the suckling period and after weaning to reach a plateau at 8 wk of age. Germinal centres appeared in the submandibular and mesenteric nodes at 28 d and their numbers increased consistently in the latter, while remaining low in the former. The impact of possible ‘natural’ exogenous antigen stimulation of the various lymph nodes was estimated from the presence of IgM‐synthesising cells and germinal centres. Differences between the patterns of age‐dependent changes in the numbers of lymph follicles observed in the somatic and mesenteric lymph nodes during their ontogeny are discussed in relation to differences in the magnitude of the exogenous antigen stimulatory effect. We also found that the variations in the numbers of lymph follicles produced in somatic lymph nodes at different locations during the first 28 d after birth reflected differences in the dimensions of the body regions drained by a particular somatic lymph node at this stage of development.

Possible Involvement of Bone Marrow Stromal Cells in Agranulocytosis Caused by Vesnarinone Treatment

Vesnarinone, an oral therapeutic agent for cardiac failure, causes agranulocytosis as a side effect. To elucidate the mechanism of occurrence of the agranulocytosis, we examined the effect of vesnarinone on granulopoiesis using an in vitro human long-term bone marrow culture system. Addition of vesnarinone to the culture decreased the total number of hematopoietic cells, mainly composed of mature granulocytes and macrophages, but increased the number of granulocyte-macrophage progenitor cells (CFU-GM) and CD33-CD34+ cells as compared with an untreated control. Differentiation of CFU-GM was induced by removing the agent from the culture medium, indicating that the effect of vesnarinone was reversible. The agent did not directly affect CFU-GM in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF). Furthermore, treatment of stromal cells with vesnarinone repressed the production of G, GM, M-CSF, suggesting that the agent may cause a hematopoietic disorder, agranulocytosis, through the impairment of stromal cell function.

Effects of cyclic polylactate (CPL) on the growth of cloned leukemic cells in vitro

A novel supramolecular oligomer, cyclic polylactate (CPL), was first discovered in the culture medium of HeLa‐S tumour cells, and was reported to inhibit the growth of FM3A ascites tumour cells by inhibiting the activities of pyruvate kinase (PK) and lactic dehydrogenase (LDH). We have now synthesized CPL‐containing oligomers with polymerization numbers ranging from 9 to 19, by prolonged heating and rapid mixing of a carbohydrate compound of the L‐lactic acid monomer (C3H6O3) under decreased pressure and have studied its effects on the growth of leukemic cells. Treatment with 0.02u2009mg/ml CPL inhibited the growth of HL60 and TF‐1 cells, while the growth of K562 cells was inhibited by 0.2u2009mg/ml CPL. A concentration of 2u2009mg/ml CPL was required to inhibit granulocyte‐macrophage progenitor cell (CFU‐GM) and burst‐forming unit erythroid (BFU‐E) precursor colony formation among normal bone marrow cells. Furthermore, 7A6 antigen expression and DNA ladder formation were observed in leukemic cells cultured with CPL, indicating that CPL induces apoptotic changes in these cells. These findings suggest that CPL might be a useful chemotherapeutic agent for leukemia. Copyright

Induction of lymph follicle formation with endotoxin lipopolysaccharide in the draining lymph node of athymic nude mice

Formation of lymph follicles in draining popliteal lymph nodes in athymic nude mice and their phenotypically normal littermates (PN mice) was investigated after footpad injection with either endotoxin lipopolysaccharide (LPS) or phytohemagglutinin in soluble and precipitated forms (PHA). The dose of LPS injected ranged from 10 to 100 micrograms, and that of PHA was 50 micrograms. After any dose of LPS, draining nodes of nude mice produced new lymph follicles in the peripheral cortex outside pre-existing follicles, though the number of follicles induced was somewhat less than in the case of PN mice treated with LPS. After injection of PHA in soluble or precipitated form, PN mice developed a significant number of new follicles in the draining nodes, but nude mice failed to do so. The present results are consistent with the view that nude mice have the ability to develop lymph follicles by way of a thymus-independent mechanism in response to exogenous stimuli.

Splenic adherent cells, stimulated in vitro, induce the reactive formation of lymphoid follicles and germinal centres in draining lymph nodes after subcutaneous transfusion into syngeneic mice

The reactive formation of lymphoid follicles and germinal centres in lymph nodes, induced by subcutaneous transfer of in vitro activated splenic adherent cells into syngeneic mice, were studied. Adherent cells were obtained by incubating spleen cell suspensions for 24 h and activated by incubating for 1 h in the medium containing keyhole limpet haemocyanin (KLH) absorbed onto alumina. Some of the treated adherent cells were irradiated with 10 Gy x‐rays, while others were either not stimulated or were stimulated with alumina‐KLH but killed by repeated freezing and thawing. Examination of adherent cell smears immunostained with antibodies against, F4/80, Mac‐1, Mac‐2 and NLDC‐145 indicated that many adherent cells displayed macrophage markers but few displayed the interdigitating cell marker. Animals transfused with KLH‐treated adherent cells with or without irradiation showed a marked increase in the number of lymphoid follicles and germinal centres in draining lymph nodes, whereas those transfused with adherent cells which had not been KLH‐treated or which had been killed after KLH treatment displayed no significant change in the number of follicles. These results were interpreted as indicating that following transfusion, antigen‐activated adherent macrophages migrated into the draining lymph nodes and induced the reactive formation of lymphoid follicles and germinal centres outside preexisting follicles.

Rib Defects and Pattern Formation of the Thoracic Wall Muscles

A fragment of quail notochord (20 somite stage) was implanted into a host chick embryo (25–30 somite stage) just lateral to the thoracic somites or into the intersomitic space. Around the implanted notochord, aggregates of rib‐forming sclerotomal cells formed a large irregular mass of cartilage, resulting in segmental rib defects without any adverse effect on body wall muscle formation. This contrasted sharply with previous experimental studies in which abnormalities of other somite derivatives, in particular body wall muscles, were always associated with rib defects created by teratogenic agents, surgidal treatment or by genetic means.