Therapeutic evaluation of interleukin-1 for stimulation of hematopoiesis in primates after autologous bone marrow transplantation.

A multiple dose IL-1 therapy was evaluated for its capability to stimulate hematopoiesis in normal primates and to restore hematopoiesis after autologous bone marrow transplantation. The administration of IL-1 to normal animals over a dose range of 0.5 to 10 micrograms/kg/d led to a 7-12 fold increase in peripheral blood neutrophil and monocyte counts after 24 hours. This increase in the mature peripheral blood myeloid cells was followed by changes in the myeloid composition of the bone marrow, where the percentage of myeloid elements increased along with a transient increase in myeloid progenitor cell activity. IL-1 treatment also led to an initial decrease in platelet counts of 10-30% during the first 3 days of treatment. However, a striking finding was a significant and long lasting stimulation of increased platelet production with platelet counts increasing to 77% of baseline 3 days after cessation of treatment and remaining elevated for the next 10 days. The therapeutic potential of the IL-1 regimen to restore hematopoiesis was further evaluated in an established autologous bone marrow transplantation model. In monkeys receiving IL-1 doses, 1.0 and 5.0 ug/kg/d, neutrophil counts recovered to greater than 0.5 x 10e9/1 on day 16, one day earlier than control, but the recovery to baseline neutrophil counts occurred 5 days sooner than control. IL-1 therapy had its greatest effect on the restoration of platelet counts after transplantation, reaching greater than 100 x 10e9/1 by day 21, two weeks earlier than control. This work demonstrates that IL-1 therapy stimulates myelopoiesis but its most promising clinical application is the stimulation of platelet production.

Differential augmentation of in vivo natural killer cytotoxicity in normal primates with recombinant human interleukin-1 and granulocyte-macrophage colony-stimulating factor.

The effect of recombinant human interleukin-1 (IL-1) alpha, granulocyte-macrophage colony-stimulating factor (GM-CSF), and combined factor therapy (CFT) on Rhesus monkey peripheral blood natural killer (NK) activity in vivo was compared. During a 14-day treatment period, IL-1-treated animals demonstrated a 170% increase in NK activity against K562 target cells by day 4, reaching maximal levels (300%) at day 16, and returning to baseline by day 30. NK activity of GM-CSF-treated monkeys increased slightly (60-100%) during days 4-12, as did saline-treated monkeys, but returned to baseline values by day 16. A delayed increase in NK activity resulted after GM-CSF treatment, reaching a peak (260%) on day 23 and remaining elevated through day 39. CFT resulted in a bimodal response pattern, with two peaks of NK activity: one at day 16 and a second at day 39. The first peak of activity (223%) was significantly less than the activity attained with IL-1 alone; the second peak (300%) was of greater duration and occurred later than the peak observed in GM-CSF-treated monkeys. Unlike IL-1, GM-CSF treatment did not lead to a immediate stimulation of NK activity; augmentation was delayed by more than 7 days post treatment. CFT results suggest that GM-CSF reduced the direct NK response to IL-1; while IL-1 led to an enhanced delayed NK response. Therefore, IL-1 and GM-CSF augment NK activity through different but interrelated pathways.

HIV preventive medicine services and public health interventions: the Bethesda experience.

A basic Public Health approach to the Human Immunodeficiency Virus (HIV) epidemic has been considered by many to be the most appropriate initial strategy against the spread of this infection. Comprehensive Preventive Medicine services were implemented at Naval Hospital Bethesda, which utilized a sexually transmitted disease (STD) model and included: counseling and education; sexual contact interviews and spouse evaluations; Blood Bank Look Back tracking of donated blood; reporting of notifiable diseases; and screening of transfusion recipients, STD cases, and other at-risk populations. This coordinated approach is highly efficient and capable of evaluating the increasing numbers of HIV-positive individuals.

Modulatory effects of Schistosoma mansoni infection on bone marrow granulopoietic and erythropoietic activity in mice.

Bone marrow (BM) cells from BALB/c mice infected with mixed-sex Schistosoma mansoni cercariae were assessed for their capacity to form in vitro colony forming units of erythroid (CFU-e) and granulocyte/macrophage (CFU-c) cells. At four weeks after infection, substantial BM commitment to granulopoiesis was noted by an increase in CFU-c, with no changes in CFU-e. Elevated marrow granulopoietic activity, although to a lesser extent, was also observed five weeks after either male or female cercarial infection. Preferential differentiation of precursor cells into eosinophilic and/or neutrophilic lineages was observed at weeks four and six post bisexual cercarial infection. After eight weeks of mixed infection, there was a marked suppression of CFU-e formation as compared with uninfected or single sex cercariae-infected mice. These results demonstrate that S. mansoni infection appears to influence either the differentiation capacity or the levels of BM cells at the progenitor cell level, and that maturation of worms and/or oviposition may contribute to changes in the CFU-e and CFU-c cell differentiation.

Recovery from severe hematopoietic suppression using recombinant human granulocyte-macrophage colony-stimulating factor.

The ability of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to enhance recovery of a radiation-suppressed hematopoietic system was evaluated in a nonuniform radiation exposure model using the rhesus monkey. Recombinant human GM-CSF treatment for 7 days after a lethal, nonuniform radiation exposure of 800 cGy was sufficient to enhance hematopoietic reconstitution, leading to an earlier recovery. Monkeys were treated with 72,000 U/kg/day of rhGM-CSF delivered continuously through an Alzet miniosmotic pump implanted subcutaneously on day 3. Treated monkeys demonstrated effective granulocyte and platelet levels in the peripheral blood, 4 and 7 days earlier, respectively, than control monkeys. Granulocyte-macrophage colony-forming unit (CFU-GM) activity in the bone marrow was monitored to evaluate the effect of rhGM-CSF on marrow recovery. Treatment with rhGM-CSF led to an early recovery of CFU-GM activity suggesting that rhGM-CSF acted on an earlier stem cell population to generate CFU-GM. Thus, the effect of rhGM-CSF on hematopoietic regeneration, granulocyte recovery, and platelet recovery are evaluated in this paper.

The effect of recombinant GM-CSF on the recovery of monkeys transplanted with autologous bone marrow.

The regulatory function of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) on granulocyte production in vivo was evaluated in an autologous bone marrow transplantation model using rhesus monkeys. Monkeys were exposed to 9.0 Gy total body irradiation and then transplanted with 5.0 x 10(7) low-density bone marrow cells/kg. Alzet miniosmotic pumps were subcutaneously implanted to deliver rhGM-CSF at a rate of 50,400 U/kg/d. Minipumps, containing either rhGM-CSF or saline, were implanted between zero and five days after transplantation for seven days. Kinetic recoveries of peripheral blood cells after either saline or rhGM-CSF treatment were compared. Treatment with rhGM-CSF accelerated the recovery of neutrophils. Neutrophils in rhGM-CSF-treated animals recovered to 80% (3.4 x 10(3)/mm3) pre-irradiation control levels by day 20, in comparison with only 33% (0.9 x 10(3)/mm3) recovery for saline control monkeys. In addition, the recovery of neutrophils was enhanced over that of the controls, reaching 140% v 70% on day 30. Another prominent feature of rhGM-CSF-treated monkeys was the accelerated recovery of platelets, reaching near 50% normal levels by day 24 in comparison with 20% of normal levels for controls. The infusion of rhGM-CSF was shown to be an effective regulator of early hematopoietic regeneration, leading to the accelerated recovery of both neutrophils and platelets and then providing a consistent sustained increase of neutrophils even in the absence of rhGM-CSF.

Neuropeptide modulation of murine erythropoiesis.

Neuropeptide modulation of hematopoietic stem cell differentiation was studied in an in vitro murine system. Endorphins were able to influence the erythropoietin-dependent differentiation of bone marrow cells into erythroid colony forming units in a dose dependent manner. The effects on progenitor cell maturation were influenced by the conditions and time of exposure to the endorphins. The modulation of erythropoiesis by the endorphins suggests that these peptides may function as modifiers of the maturation of bone marrow cells.

Granulocyte precursor cell studies in Schistosoma mansoni patients with eosinophilia.

Eosinophilia is a common clinical presentation in patients with helminthic infections. A study was designed to determine the mechanism(s) for selective or preferential differentiation of precursor cells into mature eosinophils (eos). Thus, experiments were performed to delineate the frequency of colony forming units of eos (CFU-eos) in the peripheral blood of Egyptian patients with active Schistosoma mansoni infection with eosinophilia and normal healthy individuals. The number of CFU-eos among the nonadherent mononuclear cell population was assessed in a double layer soft agar culture with autologous unfractionated mononuclear cells serving as a source of colony stimulating factor(s). Following 14 days of incubation, discrete colonies were distinguished morphologically as eosinophilic, neutrophilic, or mixed. Results indicated a two-fold increase in the total number of colonies per 10(6) cultured nonadherent cells in patients with S. mansoni infection when compared to the number of colonies obtained with adult normal volunteers (57 +/- 10 vs. 24 +/- 4; P less than 0.025). However, the frequency of CFU-eos and CFU-neut was similar in patients and normal individuals (66 +/- 3 vs. 59 +/- 8 percent CFU-eos; 30 +/- 4 vs. 35 +/- 6 percent CFU-neut). These data suggest that: eosinophils may differentiate from progenitor cells at other anatomical sites; there may be an increase in the half life of mature eosinophils in patients; there is no strict correlation between the frequency of progenitor cells and the number of differentiating mature cells of this lineage at least as measured by this in vitro assay; and the in vitro assay may not quantitatively reflect the in vivo differentiating capacity of progenitor cells.

Clinical and immunological results of segmental splenectomy in schistosomiasis.

We evaluated segmental splenectomy in 51 patients who required splenectomy to relieve the symptoms of schistosomal splenomegaly, and compared their course with that of 44 patients who underwent total splenectomy in an unrandomized study. We describe a minor modification of our initial technique. Patients having segmental splenectomy had a similar postoperative course to those having total splenectomy. Conversion of a segmental to a total splenectomy was required in two cases due to technical faults. No regrowth of the spleen has occurred in up to 4 years of observation. We noted an increased percentage of T lymphocytes with an increased ratio of T helper to T suppressor cells in patients having segmental splenectomy. Our cumulative experience supports adoption and wider evaluation of segmental splenectomy in schistosomiasis.

An anti-I-A murine monoclonal antibody which cross-reacts with HLA-DR2.

A murine monoclonal antibody, 12.7G3, directed against an Ia antigen encoded by genes in the I-Ab subregion of the H-2 genetic complex, was found to be cytotoxic against human B lymphocytes. When tested against a random panel of normal human donors, the reactivity of 12.7G3 exhibited a correlation coefficient of 0.58-0.68 with cells expressing HLA-DR2. Antibody reactivity segregated with HLA-DR2 in two families studied. Binding of 12.7G3, as detected by immunofluorescence using flow microfluorometry, was positive for two human cell lines, GM 3161 and HFB-1, both expressing HLA-DR2, and negative for two other cell lines, GM 3104 (DR1,1) and GM 3164 (DR4,4).

Studies of congenitally immunologically mutant New Zealand mice. IX. Age-related microenvironmental effects on autoantibody production in NZB and NZB.Xid mice studied by transplantation.

The mechanism of polyclonal expansion of B cells and subsequent autoantibody production in New Zealand mice remains a critical question. We have been studying the requirements for autoantibody production both in NZB mice as well as NZB mice congenic with the Xid gene of CBA/N mice. In this study, we have attempted to alter the immunologic phenotype of NZB.Xid mice by transfer of cells from young and old NZB mice. There was little difficulty in restoring normal levels of serum IgM, IgG3, splenic Lyb-5 cells, and response to DNP-Ficoll in young NZB.Xid mice that were injected with young NZB bone marrow cells. Although such animals had an almost immediate change in their immune profile to values characteristic of NZB mice, they required, much like unmanipulated NZB mice, a latency period of an additional 6 mo before autoantibodies were detected. In contrast, adult NZB.Xid mice, who likewise developed an immune profile similar to NZB after transfer of bone marrow cells from young NZB mice, began to express autoantibodies immediately without any latency period. NZB.Xid mice who were recipients of adult NZB bone marrow cells did not show sustained autoantibody production, reflecting the limited state of B cell precursors in adult NZB mice. Thus, the age of both donor cells and the age of recipient mice are critical factors for determining the latency period and the age at which autoantibodies will appear. Similarly we attempted to alter the production of autoantibodies in NZB mice that were irradiated and injected with bone marrow cells from NZB.Xid animals. NZB mice had a major amelioration of disease when they received cell transfers from young NZB.Xid mice. This amelioration, which included the acquisition of the immune profile of NZB.Xid animals, was not seen in adult NZB mice that were recipient of young NZB cells. We suggest that although Lyb-5 cells may be the effective mechanism for autoantibody production, there are other interacting influences that may selectively turn on or turn off autoantibodies and that are required and are responsible for the latency period.

Studies of congenitally immunologically mutant New Zealand mice. VIII. Cell-surface phenotype of spleen cells of Xid and nude mice.

Surface immunoglobulin on spleen cells from NZB and NZB/W mice and congenic mice bearing the nude or X-linked immune defective (Xid) gene was examined by flow microfluorometry with regard to both the frequency of positive cells and density expressed on the cell. These data indicate that although the frequency of unseparated sIg+ B lymphocytes is equivalent among all of these groups of mice, the densities of sIgM and sIgD are different. Spleen cells from these mice were also separated by free-flow electrophoresis and analyzed in a similar manner. This analysis demonstrated the absence of a subpopulation of B lymphocytes with a low electrophoretic mobility and low expression of sIgM. These studies suggest that maturational and/or activation states of the B cells in mice bearing the Xid or nude genes are different from those seen in the parent strains of mice. Such alterations in cell-surface antigens correlate with the differences in the natural history of immunopathology of the autoimmune disease in these congenic colonies of New Zealand mice.

Immune responses to hapten-lipopolysaccharide conjugates in mice. III. Genetics of the antibody response to polysaccharide antigens.

Antibody responses to the polysaccharide antigens of bacterial lipopolysaccharides and their hapten derivatives are herein documented to be under multigenic control. Among the genes responsible for the antibody response to these polysaccharide antigens include those coded for by genes located on the X chromosome and those on chromosome 17 linked to genes in the H-2 and/or Qa loci. The use of B10 congenic mice revealed that two genes on chromosome 17 are involved in the regulation of the antibody response to bacterial polysaccharide antigens. The use of CXB recombinant inbred mice confirmed the multigenic control of the antibody response within their background array of genes. The data clearly demonstrate that immune responses to these polysaccharide antigens are under genetic control, the importance of which may be of significance with regard to humoral protection during gram-negative infection.

Cell surface phenotype of lymphoid cells from normal mice and mice treated with monoclonal anti-IgD from birth.

Mice treated from birth with mouse monoclonal anti-IgD antibodies develop low frequencies of B cells in the spleen, a small percentage of which express very low levels of sIgD on their cell surface and extremely low frequencies of B cells in their lymph nodes, lacking sIgD entirely. However, the splenic B cells are phenotypically mature in that a high percentage of these cells express Lyb-5, indicating that the expression of sIgD is not a prerequisite for the acquisition of a mature surface antigen repertoire of B cells. In contrast, a high density of sIgM on splenic B cells is expressed, which suggests a predominance of cells with the phenotype of immature B cells and/or activated B cells. Furthermore, the spleen cells from anti-IgD-treated mice lack cells that respond to in vitro stimulation by LPS with an increase in the density of their sIa.

Hematological characterization of congenital osteopetrosis in op/op mouse. Possible mechanism for abnormal macrophage differentiation.

Compared with normal littermates, the op/op mice had very few macrophages in the peritoneal cavity and severely reduced numbers of monocytes in the peripheral blood. Moreover, osteopetrotic animals demonstrated an altered distribution of hemopoietic tissue with a 10-fold decrease in the number of marrow cells. Liver hemopoiesis persisted in 4-wk-old mice as evidenced by the presence of hemopoietic stem cells (HSC). Moreover, the concentration of HSC was decreased in marrow and increased in the spleen of op/op mice. In spite of the paucity of cells of monocyte-macrophage lineage in vivo, progenitor cells from hemopoietic tissues of op/op mice formed increased numbers of monocyte-macrophage colonies in vitro in the presence of exogenous colony-stimulating activity (CSA). The source of this critical CSA was a medium conditioned by stromal fibroblastoid colonies formed in vitro by normal marrow cells. Therefore, these data suggest that op/op mice possess normal monocyte-macrophage-osteoclast progenitor cells but these cells are unable to fully differentiate in the op/op mouse microenvironment. In support of this, in cultures of stromal fibroblastoid colonies from op/op marrow or spleen, the concomitant growth of macrophages, normally very dense, was drastically reduced. Moreover, transplantation of op/op spleen cells into lethally irradiated littermate recipients resulted in their hemopoietic reconstitution without signs of macrophage defect. Thus, the op/op splenic cells do not transfer the disease and are capable of normal differentiation in normal in vivo environment. These observations support the hypothesis that the defect in op/op mice is a result of the failure of hemopoietic stromal fibroblastoid cells to release sufficient amounts of CSA necessary for normal differentiation of cells of the monocyte-macrophage lineage.

T cell responses to select Ia determinants using the I-A mutant mouse strain B6.C-H-2bm12.

The T cell repertoire of B6.C-H-2bm12 mice (an I-A mutant mouse strain) to wild-type Iab antigens was investigated using both secondary proliferative cultures and cloned T cell lines. Because bm12 mice have a gain-loss mutation of their gene encoding the Ia beta-chain polypeptide, bm12 anti-B6 T cell responses are specific for the select component of Iab specificities that was lost as a result of the mutation. Although stimulator cells bearing Iab antigens elicited the strongest responses, Iaq, d, and s antigens also resulted in reproducible stimulations of these bm12 anti-B6-primed T cells. Cloned T cell lines isolated from bm12 anti-b6 cultures revealed similar findings, with most clones recognizing determinants unique for Iab antigens; however, clones showing cross-reactions with Iad and/or q were also selected. Using F1 hybrid responder T cells (mutant x cross-reactive strain), we further dissected this cross-reactivity into several distinct cross-reactive determinants. Because bm12 mice lack the serologically defined Ia differentiation antigen W39, T cell recognition of this determinant was investigated by using bm12 anti-B6-primed cells. Stimulation by Ia.W39+ cells was appreciably better than by Ia.W39- (Xid-defective) cells, suggesting that bm12 T cells recognize an Xid-regulated, W39-like Ia differentiation antigen.

Ia.W39 expression correlates with a specific Ir function.

The xid- defective mouse (N X B6)F1 male and the I-Ab mutant of the B6 mouse bm12 have mutations on different chromosomes, and therefore in separate genes. However, both variant strains have a trait in common; namely, they are unresponsive to the A chain loop determinant(s) of beef insulin to which mice of the H-2b haplotype respond. We show here that in both mutant strains, this unresponsiveness is concurrent with the absence of the Ia.W39 specificity from the M phi and B cell surface. The concordance is specific for Ia specificity W39 and no other Ia determinant, suggesting that Ia.W39 is identical with the Ir epitope necessary for generating an immune response to a particular amino acid sequence of beef insulin. In the xid-defective mouse, the molecule carrying this antigen is synthesized but not expressed on the plasma membrane, presumably because a mature M phi and B cell subset is missing. By comparison, the bm12 mouse has mature B lymphocytes, but is unable to synthesize Ia.W39. Thus, bm12 has a structural mutation in the gene coding for Ia.W39, whereas xid is a mutant regulatory or maturation gene controlling the membrane expression of Ia.W39.