A method has been described for the long-term culture of human bone marrow cells in liquid medium. Hematopoiesis, as measured by the production of granulocytic-macrophage progenitor cells (CFU¢,), continued for at least 20 weeks and was dependent upon the presence of a marrow-derived adherent layer of cells. As in the case of murine marrow liquid cultures, the adherent layer consisted of mononuclear phagocytic cells, endothelial cells, and lipid-laden adipocytes, the latter being essential for long-term hematopoiesis. Optimal growth conditions included McCoy's medium supplemented with fetal bovine serum, horse serum, and hydrocortisone and incubation at 330C. Horse serum in conjunction with hydrocortisone appeared essential for the growth of adipocytes.Hematopoiesis depends upon the complex interaction of growth and regulatory factors, most of which are poorly understood. In recent years the development of clonal culture systems in semisolid media for the detection of granulocyte-macrophage (1-3), erythroid (4), lymphoid (5-7), and megakaryocytic (8) progenitors has contributed greatly to investigations of hematopoiesis. Unfortunately, such systems possess two major limitations: (i) observations are restricted to relatively short time intervals, and (ii) the interaction between different kinds of cells cannot be-easily studied. Short-term liquid culture methods have also been investigated. Golde and Cline (9) described the culture of human marrow cells in liquid medium, using an in vitro diffusion chamber in which cells grew both in suspension and on a dialysis membrane. Proliferation and maturation of granulocytes and macrophages in these chambers persisted for 4 weeks. Dexter et al. (10) reported the development of a liquid system for the cocultivation of mouse thymus and bone marrow in which granulocyte-macrophage progenitor cells (CFUC; colony-forming unit-culture) were generated for at least 10 weeks and pluripotent stem cells (CFUs) were present for 14 days. The same group (11-13) later developed a method for the long-term culture of mouse bone marrow cells alone in liquid medium. Such cultures produce both CFUC and CFUS for several months. Hematopoiesis in this system is dependent upon the presence of a marrow-derived adherent population consisting of three cell types: phagocytic mononuclear cells, endothelial cells, and giant lipid-laden adipocytes: Initially, only certain lots of horse serum had the ability to stimulate the growth of these essential adipocytes. Greenberger (14) reported that "deficient" lots of horse serum could be reconstituted with corticosteroids.

BALB/c mice were treated with fractionated high dose (3,400 rads) total lymphoid irradiation (TLI), and given semiallogeneic (BALB/c x C57BL/Ka) or allogeneic (C57BL/Ka) bone marrow and/or skin allografts. TLI alone prolonged the mean survival time (m.s.t.) of C57BL/Ka skin grafts to 49.1 days (control, 10.7 days). Shielding of the thymus during TLI produced only a slight increase in graft survival (m.s.t., 19 days). TLI combined with splenectomy was no more effective than TLI alone. Infusion of 10(7) semiallogeneic or allogeneic bone marrow cells after TLI produced stable chimeras in 7/8 and 8/15 recipients, respectively. Chimeras were specifically tolerant to donor tissues, since C57BL/Ka skin grafts were accepted for more than 250 days, but third-party (C3H/He) skin grafts were rejected rapidly. In addition, chimeric lymphocytes responded to C3H/He and C3H. Q but not to C57BL/Ka cells in the one-way mixed leukocyte reactions. BALB/c C57BL/Ka chimeras showed no clinical evidence of graft vs. host disease. These findings may have application of clinical organ transplantation, since (a) the recipient treatment (TLI) has already been shown to be safe in humans, (b) donors and recipients can be completely allogeneic, and (c) bone marrow and skin graft survival was permanent (greater than 250 days).

Cell-free filtrates of x-ray-induced lymphoid tumors of strain C57BL/ Ka mice have elicited, on injection into newborn isologous hosts, a lymphoma incidence of 15 to 19 percent. In control mice of the same subline, the incidence of spontaneous lymphoma is about 1 percent. No leukemogenic activity could be detected in filtrates from thymi harvested at 2 to 32 days following completion of x-ray treatment. Activity was evident at 64 days and was perhaps somewhat greater at 128 days. Serial cell-free passage of filtrates in newborn F(1) hybrid mice resulted in a marked increase in lymphoma incidence (69 percent), coupled with a shortening of the median latency. Supplementary x-irradiation failed to enhance the activity of filtrates after neonatal injection.

The probability of maintaining ovarian function, becoming pregnant, and delivering a normal child is important to young women anticipating successful therapy for Hodgkin's disease. In this study, reproductive function was retrospectively examined in 103 women 40 years old or younger who had undergone treatment for Hodgkin's disease with total-lymphoid irradiation (TLI) alone, combination chemotherapy, or combined TLI and chemotherapy. Infertility was directly related to gonadal exposure to therapy and to age at treatment. Twenty women became pregnant after receiving total-nodal irradiation or combination chemotherapy or both. No fetal wastage occurred, and no birth defects were seen in the 24 infants born to these women. Even after intensive treatment programs, women successfully treated for Hodgkin's disease have become pregnant and delivered phenotypically normal children.

Hybridomas producing human monoclonal IgM antibodies (mAbs) against bacterial lipopolysaccharide (LPS) were generated by fusion of B lymphocytes from sensitized human spleen with heteromyeloma cells. The splenocytes were from patients undergoing splenectomy during staging for Hodgkin disease after vaccination with the J5 mutant of Escherichia coli, which is deficient in 0 antigenic side chains. This deficiency exposes the core oligosaccharide, common to LPS of all Gram-negative bacteria. The mAbs cross-reacted strongly with endotoxins from a wide range of unrelated species of Gram-negative bacteria. The mAbs also gave strong protection against LPS in the dermal Shwartzman reaction and against lethal Gram-negative bacteremia in mice. Two separate sites were selected for simultaneous subcutaneous injection of 1 ml of J5 vaccine, and similar injections were repeated 48 hr later. Each milliliter of the vaccine contained 5 x 109 heat-inactivated cells of the J5 mutant of E. coli 0111. The method for preparing the vaccine is described elsewhere (4). Our previous experience with over 600 volunteers showed that vaccinations given in this fashion produced minimal side effects (4), and none were reported by the recipients in this study. They underwent staging laparotomies and splenectomies 1 week after immunization. At Feb. 4, 1984. $Deceased, Dec. 5, 1984Dec. 5, . 1790 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.