Deleterious effects of irradiation and bone marrow transplantation therapy in the genetically anemic an/an mouse

The efficacy and outcome of bone marrow transplantation therapy following lethal irradiation were examined in syngeneic mice that had a hereditary macrocytic anemia (an/an) or were genotypically normal (+/+). Successful RBC and WBC replacement, based on blood cell parameters and donor genetic markers, were observed in all combinations of transplant therapy. Nevertheless, the an/an mice died prematurely several months after treatment, whether they received +/+ or an/an marrow cells. In contrast, the +/+ recipients of either +/+ or an/an marrow cells survived for at least 1 year after transplantation. Premature death of the an/an mice was associated with lymphopenia, anemia, kidney lesions, and severe pathogen-free pneumonitis. On the basis of our results, we hypothesize that the premature deaths of an/an mice are caused by a kind of chronic irradiation damage to which an/an mice are especially susceptible.     

Expression of specific granule markers on the cell surface of neutrophil cytoplasts

The widespread assumption that cytoplasts generated from human polymorphonuclear leukocytes (PMNs) are vesicles consisting solely of cytoplasm surrounded by plasma membrane and devoid of granule activity remains to be tested. PMN cytoplasts were prepared by centrifugation of intact cells on a Ficoll step gradient in the presence of cytochalasin B. Two granule membrane markers, Mol, a fluorometrically detectable antigen, and cytochrome b, both of which have been shown to translocate to the plasma membrane during granule release, were compared for their activity in cytoplasts and intact PMNs. We found that the amount of Mol detected on the plasma membrane of intact PMNs, as compared with other membrane markers (such as antigens LFA-1 and beta 2m), increased 1.6- fold upon exposure of PMNs to Ficoll plus cytochalasin B prior to centrifugation. Another twofold increase in Mol expression occurred upon cytoplast preparation. Release of the granule enzymes, vitamin B12- binding protein, and lysozyme were also followed and correlated well (r = .78 and .92) with the amount of Mol antigen present on the cell surface. Cytochrome b was also found to be higher (1.4-fold) on plasma membranes isolated from cytoplasts than on plasma membranes isolated from intact control cells. These results indicate that some fusion of granule membranes and plasma membranes occurred during treatment of PMNs with Ficoll plus cytochalasin b and during cytoplast preparation.     

Myelostimulatory activity of recombinant human interleukin-2 in mice

In a series of studies designed to extend our understanding of interleukin-2 (IL-2) and to study the effect of biologic response modifiers on bone marrow, we observed that administering recombinant human (rH) IL-2 to normal mice resulted in an increase in the frequency of colony-forming units-culture (CFU-C) in bone marrow. In addition, rH IL-2 was able to accelerate host recovery from cyclophosphamide (CTX)- or radiation-induced bone marrow depression and peripheral blood leukopenia. Not only can rH IL-2 accelerate, in a dose-dependent manner, the return of bone marrow, peripheral blood cellularity, and CFU-C frequency to normal levels following cytoreduction by CTX or irradiation, but it also significantly increases CFU-C frequency to greater than normal levels. Furthermore, rH IL-2 can significantly prolong survival of animals receiving a lethal dose of irradiation or CTX. Thus, multiple mechanisms are responsible for the synergistic therapeutic activity associated with rH IL-2 and CTX. rH IL-2 does not act only as an immunomodulatory agent in the presence or absence of suppressor T cells, but also accelerates host recovery from cytoreductive agents, resulting in decreased leukopenia and perhaps resistances to secondary infection. Thus, rH IL-2 plus chemotherapy may increase therapeutic activity against neoplastic disease, not only by adding immune stimulation to the direct antitumor effect of the drug but also by allowing delivery of higher, more effective doses of chemotherapy.     

Modulation of megakaryocytopoiesis by thrombopoietin: the c-Mpl ligand

We have further characterized the biological activities, mechanism of action, and target cell populations of recombinant human and murine thrombopoietin (rhTPO and rmTPO) in in vitro human and murine model systems. Alone, hTPO or mTPO stimulated the maturation of immature murine megakaryoblasts as measured in a single cell assay. The combination of hTPO or mTPO and interleukin-6 (IL-6) resulted in a further increase in megakaryocyte differentiation in this system. Murine TPO stimulated mouse megakaryocyte progenitor development. Human megakaryocyte progenitor development was potentiated by hTPO alone and further augmented in the presence of the early-acting cytokines (IL-3) or kit ligand/stem cell factor (KL/SCF). To further define the mechanism of action of TPO, neutralization studies were performed with antisera to IL-3, granulocyte-macrophage colony-stimulating factor (GM- CSF), IL-1 beta, and IL-11. No diminution in TPO activity was observed in the presence of these antisera. Moreover, because adhesive interactions are known to modulate hematopoiesis, we studied whether hTPO might alter such interactions between human bone marrow (BM) megakaryocytes and human BM stromal fibroblasts. No changes were observed in either megakaryocyte expression of the surface molecules lymphocyte function-associated antigen-1, very late activation antigen- 4, or intercellular adhesion molecule-1 or the adhesion of megakaryocytes to stromal fibroblasts after treatment with the growth factor. Furthermore, no induction of secretion of the cytokines IL-1 alpha, IL-1 beta, GM-CSF, IL-6, granulocyte-CSF, tumor necrosis factor- alpha, transforming growth factor-beta 1, or transforming growth factor- beta 2 by primary human BM megakaryocytes was noted after treatment of the cells with hTPO. To address whether TPO affects very primitive hematopoietic progenitors, we studied the residual cells from the BMs of mice treated with high doses of 5-fluorouracil. Although no effect of mTPO alone was noted on the viability or replication of such primitive murine progenitor populations, the triple combination of IL-3 + KL/SCF + TPO stimulated growth of megakaryocyte progenitors. These results indicate that TPO is a highly lineage-specific growth factor whose primary biological effects are likely to be direct modulation of the growth and maturation of committed megakaryocyte precursors and immature megakaryoblasts.     

Nucleotide Sequences from Tryptophan Messenger RNA of Escherichia coli: The Sequence Corresponding to the Amino-Terminal Region of the First Polypeptide Specified by the Operon

Mutants with internal deletions that terminate near the operator end of the tryptophan operon of E. coli were used in studies on the nucleotide sequence at the 5' end of the messenger RNA transcribed on this operon. The findings obtained permitted identification of a sequence of 43 nucleotides corresponding to the aminoterminal 11 amino acids of anthranilate synthetase component I, the polypeptide specified by the operator-proximal structural gene of the operon. It was also shown that the translation initiation codon for this polypeptide is preceded on the messenger by a "leader" sequence of unknown function, at least 150 nucleotides in length.     

Functional expression of the oxytocin receptor in Xenopus laevis oocytes primed with mRNA from bovine endometrium

Synthesis of the uterine receptor for the hypothalamic hormone oxytocin has been induced in oocytes from Xenopus laevis previously primed with bovine endometrium mRNA. The injected oocytes responded to oxytocin by showing dose-dependent oscillations in membrane currents as recorded by the voltage-clamp method. The response was specific in that it was not elicited by several other peptides tested. The oxytocin-induced membrane changes were suppressed when oocytes were pretreated with an oxytocin receptor antagonist.     

Impotence and aging: clinical and hormonal factors

A cross-sectional study of 216 impotent men aged 40 to 79 years (mean age 60.9 years) was conducted to determine if there are age-related changes in clinical and hormonal parameters in an impotent population. There was a slight increase in the degree of sexual dysfunction with age, with complete erectile failure occurring in a larger percent of the 60- and 70-year-olds than in the younger patients (41% vs 27% for the 40 year olds, P less than .05). No patient above the age of 70 years reported any full erections, even of short duration. In contrast, reported levels of libido did not vary significantly with age. Abnormal penile Doppler studies diagnostic of vasculogenic impotence were found in 17.8% of the patients tested, and an additional 17.8% were found to have evidence suggestive of a vascular etiology. These abnormal vascular findings were associated with an extremely high prevalence of clinically apparent atherosclerosis in this population. In 22.9% of the subjects, an abnormal vascular response was found only on exercise, ie, a "pelvic steal", which only occurred above the age of 50 years. There was a marked age-related alteration in the concentration of testosterone (T) and bioavailable testosterone (BT), but no statistically significant change in the levels of gonadotropins with age. An increase in the prevalence of eugonadotropic hypogonadism with age was found, which suggested an increasing prevalence of hypothalamic pituitary dysfunction in this patient group. For both vascular and hormonal changes (such as low T and BT), the greatest changes appear to occur after the age of 50.(ABSTRACT TRUNCATED AT 250 WORDS)