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The immunohematopoietic potential of syngeneic fetal liver cells (SFLC) was examined and compared with syngeneic bone marrow cells (SBMC). SFLC generated about 3 times less 12th-day spleen colonies (CFU-S) than adult SBMC did. To test the SFLC ability for reconstitution of the immune system, mice were lethally total body irradiated (TBI) and transplanted i.v. with 3 x 10(7) SFLC or 1 x 10(7) SBMC. Thus, injected hematopoietic cells contained the same number of CFU-S. On days 28, 35, 42, and 49 after transplantation the mice were injected i.p. with 10(6) immunogenic L1210-Maf cells (L1210 leukemia cells treated in vitro with mafosfamide for inhibition of their growth in vivo) to test the ability for generation of immune response against L1210 leukemia. On day 56 the animals were challenged with 10(3) L1210 leukemia cells. Strong resistance against the leukemia was induced in TBI + SFLC and TBI + SBMC mice, suggesting that the SFLC similarly as SBMC are able to reconstitute immune system of the TBI host.

Interleukin-2 is a lymphokine with documented antineoplastic influence, with not completely understood mechanism of action. The case of 46-years old patient with relapsed metastatic malignant melanoma treated with constant-infusion of rIL-2 is described. 9-month remission was achieved. During the course of treatment a lot of side effects including flu-like symptoms, hypotonia, anemia and thrombocytopenia, and also many biochemical disturbances were observed.

In a four-hour exchange of dialysing fluid in 120 healthy mice it was checked whether and in what degree changes of the pH of the dialysing fluid in the range from 5.09 to 7.02 and change of the buffer in the dialysing fluid (acetate, lactate) have an effect on the phagocytic and bactericidal ability of the phagocytic cells in the peritoneal dialysate. The study of the count of phagocytic cells, their bactericidal ability (NBT test) and phagocytosis ability (latex test) showed that neither the type of the used buffer nor pH changes of the dialysing fluid in the range 5.09-6.18 had no effect on the defensive properties of the phagocytes in healthy peritoneal cavity. The observation of a statistically significant increase in the proportion of cells of low phagocytic ability in the group of mice receiving acetate buffer of pH 7.02 requires further studies.

In the mouse experimental model the effect of two different methods of myeloablation-immunosuppression treatment administered as preparation for bone marrow transplantation was studied, also the effect of animal's age on the values of the essential biochemical parameters in the serum and on the body weight of the animals was assessed. The recipients were prepared for the transplantation with total-body irradiation and administration of cyclophosphamide (radio-chemotherapy) or administration of busulphan with cyclophosphamide (combined chemotherapy). Transplantation was done in animals aged 2.5 and 12 months. In all studied animals serum protein and calcium levels were decreased after the transplantation and the uric acid level was transiently raised. In the older mice a short lasting increase in the serum levels of bilirubin, alkaline phosphatase, AST and ALT was noted. However, no changes were found in the results biochemical investigations which could have been related to the method of myeloablation and immunosuppression, apart from slight hypercholesterolaemia which developed about 30 days after the operation in mice prepared by radio-chemotherapy. However, after a year lower body weight was observed in young mice prepared for the procedure with radiation exposure and cyclophosphamide, as compared to those receiving combined chemotherapy. Clinical aspects of these disturbances are discussed.

The usefulness of three different tests available in experimental haematology for the assessment of the viability of fetal liver cells obtained from murine fetuses kept in refrigerator at 4 degrees C was compared. The usefulness of these cells as potential transplants for haemopoiesis reconstitution was assessed in the test with trypan blue, in the test based on clonal growth of GM-CFU in agar, and in the test of the splenic colony forming ability of CFU-S. Fetuses kept in refrigerator at 4 degrees C tested 16 hours after circulation arrest contained still about 70% of the initial number of the haemopoietic stem cells (CFU-S). The proportion of committed cells belonging to the granulocyto-monopoiesis line (GM-CFU) decreased at the same time to about 25% of the initial number, and was a sensitive indicator of hypoxia of the studied organ. The test for the viability of the cells based on the use of trypan blue gave results reflecting better the changes in the number of CFU-S than GM-CFU cells.

In the time period from November 1984 to January 1987 eight allogeneic bone marrow transplantation were performed from HLA-identical siblings. The theoretical chance of success in this group was between 21 and 50%, according to the recent data of the International Bone Marrow Transplant Registry, depending on the diagnosis and clinical condition. The average chance was 37.5%. Haemopoietic reconstitution was achieved in 6 recipients, while 2 died of early complications (cytostatic induced hepatocellular damage and fungal sepsis). Another 3 patients died of complications of the intermediate period (pulmonary bleeding, virus hepatitis, graft rejection). The remaining 3 recipients are alive, in excellent clinical condition, including one girl surviving more than 2 years after the transplantation.

In a search of alternative techniques of red cell depletion from the marrow prior AB0 incompatible transplantation we got interested in Böyum technique of polysaccharide-ditrizoate gradient centrifugation for isolation of mononuclear cells. This method might be used for the purpose of red cell depletion only if it preserves haemopoiesis reconstituting stem cells. Whether this is the case was tested using murine model of lethally irradiated donors. Polysaccharide-ditrizoate gradient isolated mononuclear cells contained 100% spleen colony forming stem cells and possessed the same bone marrow and spleen reconstitutive potential as non-manipulated marrow cells. Therefore, this method may be considered for potential clinical application.

A modified polysaccharide-ditrizoate gradient centrifugation method is described. One hundred ml aliquots of marrow mixed 1:1 with medium is layered on 70 ml of polysaccharide-ditrizoate solution of specific density 1.077 in 5 x 19 cm flasks and centrifuged at 400 x g for 20 minutes at 20 degrees C. Interphase cells are collected and used for the transplant. The procedure is closed which reduces the danger of contamination, simple and takes between 2 and 3 hours. Two allogeneic HLA-identical transplants were performed using this technique and both resulted in full hematopoietic reconstitution and long term survival.

A 6-yr-old girl with congenital corticosteroid-resistant pure red cell aplasia was treated with bone marrow transplant from her HLA-identical, MLC-unreactive sister in November 1984 following conditioning with busulfan and cyclophosphamide. Full engraftment was obtained and the patient at 21 months post-transplant is in excellent clinical condition maintaining normal red cell counts. We conclude that BMT should be considered as a therapy for at least the most severe cases of Diamond-Blackfan anaemia resistant to corticosteroids. Successful outcome of this therapy provides an argument for the stem cell origin of this disorder.

About 25% mice treated for radiation-induced bone marrow aplasia with allogeneic transplantation of histoincompatible fetal liver cells survived four months. The same survival time was observed in 80% of mice after transplantation of syngeneic fetal liver cells. Mice irradiated and not treated and those receiving transplants of allogeneic histoincompatible bone marrow died within a few weeks after transplantation. This observation confirms the reports on the therapeutic superiority of transplantation of haemopoietic fetal liver cells over transplantation of bone marrow of similar tissue incompatibility and reports on the usefulness of tissue matching, if possible. Another observation was an absence of inactivation of the haemopoietic stem cells in mixtures of genotypically different fetal liver cells. The animals receiving these cell mixtures showed survival rates similar to those of the animals receiving genetically homogenous cells. This opens theoretical possibilities for undertaking clinical trials of transplantation of cells from several fetal livers, especially in view of the accumulating reports that one human fetal liver may be too small to serve as a source of stem cells for ensuring successful transplantation.

The kinetics of restoration of haemopoiesis was studied in 10 patients prepared for allogenic bone marrow transplantation with busulphan combined with cyclophosphamide. The morphology of peripheral blood after administration of these drugs and transplantation of allogenic bone marrow was similar to that reported elsewhere after irradiation, cyclophosphamide administration and bone marrow transplantation, the cell counts falling almost to zero within several days after the end of the pharmacological preparation, and later rising to normal values within the period from several weeks to several months after transplantation.

Methods employing both mechanical and enzymatic treatment for obtaining single cell suspension from human fetal livers were compared. Subsequently, the effects of various media and sera on cell survival in fetal liver cell suspensions were studied with special attention to short term storage at 4 degrees C. The largest number of living cells was obtained after homogenization of fragments of fetal liver by teasing them through metal sieve. Cell survival was the best when HEPES buffered Dulbecco MEM supplemented with 20-30% of human AB group serum was used throughout. More than of 50% granulocytemonocyte progenitor cells (GM-CFU) were still present in this suspension after 96 hours of storage at 4 degrees C.

Two- to three-week-old mice homozygous for the recessive oc gene had negligible numbers of marrow cells but possessed no significant spleno- and hepatomegaly. They also maintained normal numbers of blood cells except for monocytes, which were significantly lower. Additionally, they had reduced numbers of total cells and resident macrophages in the peritoneum, as determined by cell counts in the peritoneal lavage fluid. The frequency of spleen colony-forming units (CFU-S) in the spleens of oc/oc mice was the same as that in the spleens of normal littermate control mice. These oc/oc CFU-S showed essentially similar differentiation patterns as CFU-S of control mice. Also, a few CFU-S could be detected in livers of oc/oc mice. On the other hand, the frequency of cells that formed macrophage colonies in a four-day liquid-culture system in the presence of colony-stimulating activity was significantly reduced in oc/oc mice and abnormalities were observed in the formation of the adherent (stromal) layers by oc/oc spleen cells in liquid cultures. Numbers of fibroblastoid cell colonies in these layers were reduced and, moreover, cultures demonstrated a marked decrease in the number of macrophages both within and outside the fibroblastoid cell colonies. Transplants of spleen and thymus cells of oc/oc mice into lethally irradiated +/? recipients induced oc/oc-like lesions. They included peritoneal macrophage deficiency, marrow deficiency, as well as hepatosplenomegaly. This suggests a hemopoietic stem cell and not microenvironmental defect in this particular type of osteopetrosis. The murine mutant characterized in this study may be useful in studies of cellular interactions during blood and bone formation and in studies of the mononuclear phagocyte system.

To test the hypothesis that the omental lymphoid organ (OLO) made by peritoneal milky spots is either a source of haemopoietic (macrophage) progenitors or growth factors we attempted to culture OLO cells in vitro in a variety of assay combinations. With the culture in vitro in semisolid agar it was found that OLO cells do not form granulocyte-macrophage or macrophage colonies in response to stimulants. However, when in the same assay marrow cells were used as the targets and OLO-related preparations as stimulants it was observed that marrow cells formed exclusively macrophage colonies. These marrow cells, in response to stimulants derived from other organs, produced granulocyte-macrophage, granulocyte and macrophage colonies. OLO-related preparations tested for macrophage-colony stimulating activity included partly purified medium conditioned by OLO cells derived from mice, either injected with endotoxin or not, and medium conditioned by OLO cells after 14 days, liquid culture in vitro. While these results were observed in Swiss mice, C3H/W mice, which are genetically endotoxin-unresponsive, failed to show this reaction. These data may suggest that the local production of macrophage-colony stimulating activity in the peritoneal cavity could be one physiological role for OLO. OLO is the first organ in adult mice identified to stimulate exclusively macrophage colony growth, and not granulocyte-macrophage or pure granulocyte colonies.

Microphthalmic mice possess less than 10% of marrow cells. This marrow deficiency is compensated by an increase in the number of spleen cells. Additionally, 2-week-old mi/mi mice have increased frequency of hemopoietic stem cells in the spleen and remaining marrow. In addition they have persistent stem cells in the liver. No other major hematologic abnormalities were found.

The recovery of haemopoiesis in Steel mutant mice following 1 Gy sublethal irradiation is described. Steel homozygotes (S1/S1) did not display the abortive phase of erythropoietic recovery while the secondary phase of erythropoietic recovery was more pronounced in S1/S1 than in control (+/+) animals. On the contrary, the neutrophilopoietic recovery in S1/S1 mice was defective only during the secondary phase of recovery. Steel heterozygotes (S1/+) manifested similar, albeit less pronounced, defects. In the course of studies of recovery of eosioniphils it was observed that neither wild-type nor mutant animals expressed the abortive rise. Moreover, the kinetics of recovery of eosinophils was essentially different from both erythropoietic and neutrophilopoietic recovery, and the preirradiation level was reached in both normal and mutant animals on day 60 postirradiation as opposed to 24 and 35 days for erythropoiesis and neutrophils respectively.