The Intercellular Substances of Bone Marrow. II. Variations in Granulocytic and in Erythrocytic Hyperplasia

Rabbits were injected with turpentine to produce granulocytic marrowhyperplasia, or were bled to produce erythrocytic marrow hyperplasia. Bothgranulocytic and erythrocytic marrow hyperplasia were associated with decreased marrow lipid content. The viscosity of the intercellular substancesand the mucopolysaccharide content of this material were significantly increased in granulocytic marrow hyperplasia. in erythrocytic marrow hyperplasia, the viscosity of the intercellular substances was markedly reduced,while the mucopolysaccharide content was slightly elevated compared withcontrol animals.

Submitted on January 17, 1962 Accepted on June 3, 1962     

Bone and Marrow Transplants in the Rat

A simple method for transplantation of autologous bone and marrow to therat kidney is described and the usefulness of this preparation for research onbone and marrow growth is pointed out.

While an induction mechanism cannot be conclusively ruled out by thesestudies, the histologic evidence strongly supports the concept that growth ofimplanted bone and marrow is due to survival of small islands of active boneand marrow cells and from these foci, repopulation takes place.

Submitted on March 23, 1961 Accepted on July 6, 1961     

The Colchicine Method in the Study of Bone Marrow Cell Proliferation

The rate of metaphase accumulation in the bone marrow cells of AKR micetreated with colchicine was investigated. The influence of this alkaloid onthe differential count of the bone marrow cells in these animals was alsostudied. It was demonstrated that the stathmokinetic effect of colchicine onthe bone marrow cells started almost immediately after the administration ofthe drug. The number of arrested metaphases increased linearly from one-halfhour to six hours after the injection of colchicine, and then fell rapidly.

In rats injected with colchicine, the changes in the bone marrow concentration of this compound were followed for eight hours. The colchicine concentrations increased from the first to the fourth hour, and then fell rapidly, reachingthe zero level at the eighth hour.

From the results obtained, it appeared that four hours post-injection wasthe most convenient time for the study of the bone marrow proliferative activity by the colchicine method when 1.2 mg./Kg. of colchicine per body weightwas used. The fact that four hours after the injection, a mild decrease in thepercentage of mature bone marrow granulocytes was found, may represent alimiting factor, which is, however, of moderate importance in the application ofthis technic.

Submitted on May 5, 1961 Accepted on June 14, 1961     

Mitotic Indices of Human Bone Marrow Cells. I. Number and Cytologic Distribution of Mitoses

1. The diurnal variation of mitotic index of human bone marrow has beenstudied. A diurnal variation has not been demonstrated but remains subjectfor further study.

2. Data on the distribution of hone marrow mitoses on cell lines and maturation stages are presented. From this and the gross mitotic index of thebone marrow, the mitotic indices of various cell types are computed.

Submitted on December 12, 1961 Accepted on February 12, 1962     

Sickle Cell Trait as a Tag for Bone Marrow Transplantation

The sickle cell trait appears to offer a reliable and replicable tag for bonemarrow transplantation. Red cell survival curves using sickle cell and nonagglutinable cell counts show good agreement. The recipients exhibited nountoward effects as a result of receiving marrow from a donor with sickle celltrait.

Submitted on January 12, 1961 Accepted on February 6, 1961     

Modification of Irradiation Injury in the Monkey by Bone Marrow Transplantation

Thirty-two adult monkeys were given a single dose of total-body x-irradiation of 650, 700, or 800 r. An attempt was made in 24 of the animals to alterthe acute phase of the radiation injury to hematopoietic tissues sulfficiently toprolong the life of the animals by post-irradiation administration of homologous bone marrow. The other 8 animals served as irradiated non-treated controls. Of the 24 bone marrow treated monkeys, 5 survived for more than 30days post-irradiation. However, a total of 16 animals showed some recovery ofthe peripheral blood elements toward normal, which seems to indicate thatthe bone marrow graft may have been functional in these animals. The causesof death in the face of a recovering bone marrow are discussed with regard toimmunological reactions, including the "foreign bone marrow reaction," andother contributing factors.

Submitted on December 14, 1960 Accepted on January 27, 1961     

Comparative Evaluation of Simultaneous Bone Marrow Aspiration and Bone Marrow Biopsy: An Institutional Experience

Bone marrow aspirations and bone marrow biopsies are important diagnostic procedures. A comparative study of both the procedures done simultaneously was retrospectively reviewed in 160 cases where the clinical history is correlated with BMA and BMB results. The advantage of each method is analyzed. Correlation of our findings with that given in the literature is done to give a guideline for both techniques. We have found that 61.25% of the cases showed a positive correlation between bone marrow aspiration and bone marrow biopsy. However, we found that tuberculous granulomas and Hodgkin disease involvement of the marrow were detected better in bone marrow biopsies. The advantage of both the procedures done together provided more material and enabled us to study the cytomorphology of the cells, with the pattern of distribution of the cells depending on the cases. However, when both the procedures are done simultaneously, a proper technique is required so as to yield good diagnostic material.     

Intercellular Contacts between Erythroid Precursors in the Bone Marrow in Dyserythropoiesis

S ummary . The ultrastructure of the cellular interfaces between erythroid precursors has been studied in both congenital and acquired dyserythropoiesis. The types of cell-to-cell contacts found included highly convoluted interdigitating septate-like junctions as well as apparent confluence of the cellular membranes of adjacent erythroid cells. Such findings are not characteristic of the intercellular relationships found in the normal erythroblastic islands. The erythroid plasma membrane modifications are possibly related to the changes in the plasma membranes of erythroid cells known to occur in dyserythropoiesis and their presence may well contribute to the ineffective erythropoiesis with intramedullary cell destruction which characterizes many dyserythropoietic states.     

Studies on Homologous Bone Marrow Transplantation in Irradiated Rabbits

White New Zealand rabbits were exposed to 1100 r as a split dose of 600 rfollowed 24 hours later by 500 r and transplanted with fresh or frozen, lyophilized, sonicated homologous bone marrow. Fresh marrow was infused 24hours or 72 hours after x-irradiation. All these animals received antibioticsand, in addition, another group received antibiotics only. Donors were femalesdiffering, on the basis of Cohen’s Hg^ADF allelomorphic system for erythrocytes,from the recipient males. It was therefore possible to follow the success ofthe graft qualitatively by the occurrence of female heterophil leukocytes, andsemiquantitatively by the presence of donor erythrocytes in the circulationof the recipient. Complete correlation between these two parameters did notoccur: sometimes leukocytes, sometimes erythrocytes, and sometimes bothelements of the donor were found in the circulation of the recipient. If animalsretained their graft, complete repopulation with donor erythrocytes occurredat about the tenth week postirradiation and transplantation. The number ofdonor leukocytes present in the blood of the chimeras suggested a mixed population.

Not only were the grafts often incomplete but in many instances they werenot permanent. Initially, all but one animal had a successful graft, but of the18 animals which had received fresh marrow and survived at least 12 weeks12 retained their graft.

Much better results in terms of permanent takes were obtained when themarrow was infused 24 hours, rather than 72 hours, after x-irradiation. Nonviable marrow had no protective effect, whereas antibotics did decrease themortality.

An immune hemolytic anemia was shown to be part of the secondary diseasesyndrome. Antibodies specific to stored recipient erythrocytes were found inthe sera of all chimeras between the third and seven weeks after transplantation. When donor erythrocytes were used, the test was positive on only 3 animals. These 3 animals died between the third and eighth weeks postirradiationwith secondary disease. When stored recipient erythrocytes were thawed,labeled with Cr⁵¹, and infused into chimeras three or four weeks after irradiation and transplantation, they had in every case a greatly shortened half-life commensurate with a hemolytic anemia. Donor erythrocytes were infusedinto other chimeras and in all but one instance had a normal half-life. It issuggested that all rabbit chimeras develop secondary disease to some extent,as indicated by a weight loss and fall in hematocrit, but that the situation isnot necessarily fatal, except when recovery of the host’s immune mechanismoccurs in the presence of a graft which is actively producing antibodiesagainst the host.

Submitted on January 13, 1961 Accepted on February 16, 1961     

Appearance of Bone Marrow Smears With Necrotic Tumor Cells

Bone marrow smears of necrotic tumorcells stained with Romanowsky dyes frequently have a definite reddish colorthat contrasts so strikingly with the bluecolor of normal marrow that necrosismay be suspected from gross examination. The bone marrow cells appear"smudged" and are difficult to identifyon microscopic examination. Commonly,only a small amount of material is obtainable by ordinary needle aspiration ofbone marrow containing necrotic tumor,and all of it is used to make smears. Itis therefore important to recognize thatbone marrow smears with a reddishgross appearance and in which the cellsappear distorted and are difficult toidentify may represent necrotic tumorand not an artifact produced by poortechniques.

Submitted on March 5, 1971 Accepted on March 22, 1971     

The Lymphocyte-Like Cell in the Marrow of Rats

The lymphocyte-like cell of rat marrow is capable of performing as a multipotential cell. It resembles a small lymphocyte, and reacts to the effects ofcortisone and thio-TEPA, as do small lymphocytes in other organs, thusshould be called a small lymphocyte.

Its sudden appearance in large numbers to occupy almost 50 per cent of themarrow space in suckling rats fourteen days of age can best be explained onthe basis of migration of these cells to the marrow from other organs via theblood stream. The rate at which lymphocytosis of the bone marrow occurs inrats is influenced by the size of the suckling litters, and may be related to anon-caloric factor in mother's milk. The small lymphocyte probably representsthe inactive phase of a multipotential cell, a dormant cell which can give riseto other cell forms when appropriately stimulated.

Submitted on May 15, 1961 Accepted on August 30, 1961     

An Electron Microscopic Study of the Bone Marrow of the Rat in an Experimental Myelogenous Leukemia

Alterations in the vascular sinus and hematopoietic compartment of rat bonemarrow were observed with electronmicroscopy during the pathogenesis of anacute myelogenous leukemia. As the disease progresses, the sinus wall becomesdamaged and disintegrates; normal hemicelements disappear, and the marrow compartment becomes packed with leukemicmyeloblasts. Viruslike particles are present in intercellular spaces and appear tobud from leukemic cells.

Submitted on December 14, 1970 Revised on April 29, 1971 Accepted on July 16, 1971     

Schwann Cells of the Bone Marrow

Nerves containing numerous Schwanncells can be found in bone marrow withroutine histological methods. Thin nervebundles and single nerve fibers containing Schwann cells can be identified histologically with help of methods whichdemonstrate nerve fibers and myelin.Smears of marrow stained with the May-Grünwald-Giemsa method are not adequate for demonstration of nerve fibers,but their satellite Schwann cells appearwell stained. Histological, cytologicaland ultrastructural characteristics of thesecells are described in detail. Blood-forming cells and nerve fibers with theirSchwann cells lie in close proximitythroughout the marrow. This circumstance suggests that interaction may takeplace between both elements.

Submitted on January 23, 1970 Accepted on February 24, 1970     

Unrelated bone marrow transplantation b. Bone Marrow Donor Registry. Nagoya Bone Marrow Transplantation Group

Tokai Bone Marrow Donor Registry was established to facilitate unrelated bone marrow transplantations in Japan. Although the basic structure was constructed, the number of donors must be expanded and financial problems must be solved. More bone marrow donor registries are likely to be established in the near future throughout Japan, and those will be anticipated to become a nationwide bone marrow donor registry. The HLA identity between unrelated individuals is unique in that it is only phenotypically identical and not genotypically identical. Therefore, for the HLA matching, not only serological typing of donors and recipients but molecular and biochemical methods would be employed to facilitate the analysis of the degree of the HLA identity to be more precise. Those methods include HLA-DP DNA typing and IEF analysis of HLA-class 1 antigens.     

Bone Marrow Biopsy of the Posterior Iliac Crest with Gidlund's Instrument in Malignant Diseases

The bone marrow was examined on material obtained with the Gidlund trephine and with conventional cytologic bone marrow aspiration in 225 patients with malignant disease, 144 of whom had malignant lymphoma, and in 5 patients with haematological, non-malignant disease. With Gidlund's instrument, the malignant disease was found to engage the bone marrow in 35 cases as compared to 7 cases with needle biopsy. No complications were met with either technique. Bone marrow biopsy was routinely performed in the out-patient department.     

Erythrophagocytosis in the Human Bone Marrow

Erythrocyte destruction by erythrophagocytosis and red blood cell disintegration has been studied in methacrylate embedded human bone marrow. Phagocytosis of erythrocytes is demonstrated in macrophages within the bone marrow sinusoids, in the bone marrow reticulum cells and in the nursing cells of the erythroclastic islands. Red cell destruction takes place both in normal and in pathologically altered bone marrow, but is often markedly increased in hypoplastic bone marrow disease. The bone marrow is considered representing the main erythroblastic organ in normal individuals, removing aged red cells from the circulation by erythrophagocytosis within bone marrow sinusoids. Further disintegration of the erythrocytes in the bone marrow reticulum cells provides directly components for rebuilding of new erythrocytes in the erythroblastic islands.     

Junctional Structures in Haemopoiesis: a Study of Bone Marrow using Freeze-Fracture and Lanthanum Impregnation Techniques

Intercellular regions of contact in the haemopoietic compartment of normal rat bone marrow were studied using freeze-fracture and lanthanum tracer techniques. Small adhering junctions (like desmosomes and their variants) were found between haemopoietic and stromal cells but tight, gap or septate junctions could not be identified. These findings are in agreement with the concept that extensive junctional structures may be inconsistent with orderly development of this transient cell system, preventing the delivery of mature cells into the circulation and resulting in ineffective haemopoiesis. Occasionally 'pinching off' of a portion of the cytoplasm of erythroid cells by stromal cells was seen, providing a means for intercellular communication. Structures similar to intercellular bridges responsible for direct intercellular communication were also seen.     

Regulation of Human Bone Marrow Leucopoiesis

S ummary . The clonal growth of bone marrow in soft-gel culture is largely dependent on the presence of colony-stimulating factors (CSF). However, when human marrow is cultured in liquid medium with the in vitro diffusion chamber technique, proliferation and differentiation of granulocytes and mononuclear cells proceeds without a requirement for exogenously supplied CSF. The addition of highly active monocyte-conditioned medium to the liquid marrow cultures did not result in a measurable change in viable or differential cell counts. Similarly, when mono-cytes were co-cultivated with bone marrow in a double-chamber apparatus, no stimulation of marrow cell growth was observed. Colony-stimulating activity was identified in conditioned medium derived from the liquid marrow cultures, and the cultured marrow cells themselves stimulated colony formation when incorporated as feeder layers. Retention of marrow cell responsiveness to CSF in liquid culture was demonstrated by cloning the cultured marrow cells in agar at intervals up to 2 weeks. Evidence was obtained that bone marrow cells of the monocyte-macrophage line were primarily responsible for the elaboration of CSF in liquid culture. These data suggest a role for humoral interactions between haematopoietic cells within the marrow cavity.     

生血增白汤对骨髓移植后小鼠造血重建中黏附分子表达水平的影响

目的探讨生血增白汤对骨髓移植后小鼠骨髓中细胞间黏附分子-1(ICAM-1)表达水平的影响及其促进骨髓造血重建的机制。方法将实验动物随机分为5组:正常组、对照组、G-CSF组、生血增白汤组、G-CSF+生血增白汤组,建立小鼠骨髓移植模型,生血增白汤组及G-CSF+生血增白汤组胃饲生血增白汤浓缩煎剂。骨髓移植后第9天、第14天、第24天,采用免疫组化方法检测ICAM-1表达水平,进行外周血细胞和骨髓单核细胞计数,并做骨髓组织学观察。结果骨髓移植后第9天、第14天、第24天,生血增白汤组ICAM-1的表达水平、外周血细胞、血红蛋白、骨髓有核细胞计数及骨髓细胞增生程度均高于其他各组(P<0.05或P<0.01)。结论生血增白汤能促进骨髓移植后骨髓中ICAM-1的表达,可能是生血增白汤重建造血的机制之一。     

An Inhibitor of Stem Cell Proliferation in Normal Bone Marrow

S ummary . A saline extract from normal bone marrow cells having a molecular weight in the range 50 000–100 000 daltons has been found to protect rapidly proliferating haemopoietic spleen colony forming cells (CFU_s) from the lethal effects of large doses of tritiated thymidine. This extract is non-toxic to the cells. It is not found in regenerating marrow where the CFU_s population is rapidly proliferating. Its effect appears to be specific for the CFU_s since it has no effect on the proliferation of its close descendant, the granulocytic precursor cell (CFU_c), and no effect on the average cytoplasmic structuredness of the whole bone marrow cell population. The active material is probably protein since it is degraded by trypsin.