A review of the chemistry, biological action, and clinical applications of anabolic-androgenic steroids.

BACKGROUND: Since its discovery in 1935, numerous derivatives of testosterone have been synthesized, with the goals of prolonging its biological activity in vivo, producing orally active androgens, and developing products, commonly referred to as anabolic-androgenic steroids (AAS), that are more anabolic and less androgenic than the parent molecule. OBJECTIVE: This article reviews the structure, biotransformation, and mechanism of action of testosterone and some of the most commonly used AAS. Clinical applications of the AAS are discussed, and guidelines and therapeutic maneuvers for minimizing their side effects are outlined. METHODS: Literature for inclusion in this review was identified using the libraries of the University of Wisconsin Medical School and School of Pharmacy, the author's files, and searches of MEDLINE, Science Citation Index, Biological Abstracts, and Chemical Abstracts. RESULTS: The myotrophic action of testosterone and its derivatives and their stimulatory effects on the brain have led to widespread use of AAS by athletes and "recreational" drug users. Consequently, all AAS were classified as class III controlled substances in 1991. Nonetheless, AAS have shown benefit in a variety of human disorders, including HIV-related muscle wasting and other catabolic conditions such as chronic obstructive pulmonary disease, severe burn injuries, and alcoholic hepatitis. Because of their diverse biological actions, AAS have been used to treat a variety of other conditions, including bone marrow failure syndromes, constitutional growth retardation in children, and hereditary angioedema. AAS therapy is associated with various side effects that are generally dose related; therefore, illicit use of megadoses of AAS for the purpose of bodybuilding and enhancement of athletic performance can lead to serious and irreversible organ damage. The most common side effects of AAS are some degree of masculinization in women and children, behavioral changes (eg, aggression), hepatotoxicity, and alteration of blood lipid levels and coagulation factors. CONCLUSIONS: To minimize or avoid serious toxicities with AAS therapy, close medical supervision and periodic monitoring are important, with dose adjustment as appropriate to achieve the minimum effective dose. Given the biological effects and potential adverse effects of AAS, administration of these agents should be avoided in pregnant women, women with breast cancer or hypercalcemia, men with carcinoma of the prostate or breast, and patients with nephrotic syndromes or significant liver dysfunction.

Low-dose cyclosporin A therapy in children with refractory immune thrombocytopenic purpura.

Although splenectomy is the most effective treatment for chronic idiopathic thrombocytopenic purpura (ITP), many post-splenectomy patients have recurrent thrombocytopenia refractory to multiple medical therapies. Three consecutive patients with relapsed ITP after splenectomy and who were refractory to multiple medical therapies were treated with low dose cyclosporin A (CsA). In all 3 patients, the platelet count increased dramatically within 1 month from the onset of CsA therapy. The only detectable toxicity was hypomagnesemia and mild hypertension in 1 patient. CsA may be efficacious in treating patients with chronic ITP, which is refractory to all medical and surgical therapies currently being used.

Cytokine transgene expression and promoter usage in primary CD34+ cells using particle-mediated gene delivery.

Induction or short-term transgenic expression of specific cytokines, growth factors, or other candidate therapeutic genes in hematopoietic progenitor or stem cells is potentially applicable to gene therapy for cancer. In this study, we explored the application of a gene gun technique, as an alternative to viral vectors, for ex vivo gene transfer into and transient gene expression in highly enriched CD34+ cells derived from human umbilical cord blood. Twenty-four hours posttransfection, 32.6 to 1500 pg/l x 10(6) CD34+ cells of transient gene expression was routinely obtained for specific cytokine and reporter genes. Transgene expression at the single-cell level was revealed by X-Gal staining of lacZ cDNA-transfected CD34+ cells. Expression of four candidate therapeutic genes, namely human granulocyte-macrophage colony-stimulating factor, tumor necrosis factor alpha, interleukin 2, and interferon gamma, was detectable for 4 to 7 days in CD34+ cells. A human elongation factor 1alpha promoter/intron 1 transcription unit was identified as a strong cellular promoter for CD34+ cells, exhibiting strength similar to that of the commonly employed cytomegalovirus immediate-early promoter. These results suggest that the nonviral, gene gun technique offers an efficient alternative approach for transient transgenic studies of hematopoietic cells and may provide new possibilities for certain cancer gene therapy strategies using CD34+ cells.

Somatic mosaicism in Fanconi anemia: molecular basis and clinical significance.

Approximately 25% of patients with Fanconi anemia (FA) have evidence of spontaneously occurring mosaicism as manifest by the presence of two subpopulations of lymphocytes, one of which is hypersensitive to cross-linking agents (e.g. mitomycin C) while the other behaves normally in response to these agents. The molecular basis of this phenotypic reversion has not yet been determined. We have investigated 8 FA patients with evidence of mosaicism. Epstein-Barr virus-immortalized lymphoblastoid cell lines established from these patients exhibited an IC50 for mitomycin C of 25 to > 100 nM compared to a mean of 2 +/- 2 nM for 20 nonmosaic FA patients and 49 +/- 11 nM for 8 healthy controls. In 3 patients who were compound heterozygotes for pathogenic FAC gene mutations the molecular mechanism of the mosaicism was investigated by haplotype analysis. The results indicated that an intragenic mitotic recombination must have occurred leading to a segregation of a wild-type allele in the reverted cells and suggested two patterns of recombination. In 1 patient a single intragenic crossover between the maternally and paternally inherited mutations occurred associated with markers located distally to the FAC gene; in the other 2 patients (sibs) the mechanism appears to have been gene conversion resulting in segregants which have lost one pathogenic mutation. In 6 of the 8 patients the hematological symptoms were relatively mild despite an age range of 9-30 years.

Plasma/serum levels of flt3 ligand are low in normal individuals and highly elevated in patients with Fanconi anemia and acquired aplastic anemia.

The flt3 ligand is a growth factor that stimulates the proliferation of hematopoietic progenitor and stem cells. We established a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the concentration of flt3 ligand in plasma or serum from normal individuals, as well as in patients with hematopoietic disorders. Concentrations of flt3 ligand in plasma or serum from normal individuals were quite low: only 12% (7 of 60) of normal individuals had flt3 ligand levels above 100 pg/mL (the limit of detection). In contrast, 86% (19 of 22) of samples from patients with Fanconi anemia and 100% (eight of eight) of samples from patients with acquired aplastic anemia had plasma or serum levels above 100 pg/mL. Mean plasma or serum concentrations (calculated by assigning a value of 0 pg/mL to any sample reading below the level of detection) were as follows: normal volunteers, 14 pg/mL; patients with Fanconi anemia, 1,331 pg/mL; and patients with acquired aplastic anemia, 460 pg/mL. Concentrations of flt3 ligand in blood are, therefore, specifically elevated to a level that may be physiologically relevant in hematopoietic disorders with a suspected stem cell component. The elevated flt3 ligand concentrations in these individuals may be part of a compensatory hematopoietic response to boost the level of progenitor cells.

Fetal hemoglobin expression in transplant recipients of placental blood hematopoietic progenitor cells.

Patients who achieved bone marrow engraftment of cord blood-derived progenitor cells provided an opportunity to examine the expression of fetal Hb by neonatal hematopoietic progenitors in a postneonatal host. Cord blood cells from histocompatible siblings were successfully transplanted in two children with the Fanconi anemia syndrome. One of the transplant donors had heterocellular hereditary persistence of fetal Hb, apparently due to gamma-globin gene triplication; the other donor was hematologically normal. The G gamma/A gamma ratio of the patient who received his transplant from the donor with hereditary persistence of fetal Hb was markedly elevated, similar to that of the transplant donor's cord blood, and this ratio remained elevated in subsequent months. In the other child, the G gamma/A gamma ratio immediately after her transplant was typical of the normal newborn, and over the next several months it reverted to the adult pattern. Globin synthesis studies performed shortly after engraftment demonstrated ratios of fetal Hb/adult Hb synthesis in both patients that were typical of those of normal newborns. Over the next several months, both patients converted to the adult pattern. Fetal Hb to adult Hb switching in these patients seemed to follow a temporal sequence intrinsic to the transplanted neonatal progenitor cells, without discernible influence of postneonatal environmental factors. The program for Hb switching seems to be an inherent feature of neonatal hematopoietic progenitor cells.

Establishment of an adherent cell feeder layer from human umbilical cord blood for support of long-term hematopoietic progenitor cell growth.

Previous attempts to establish a stromal cell feeder layer from human umbilical cord blood (HUCB) have met with very limited success. It has been suggested that there is an insufficient number of stromal precursor cells in HUCB to form a hematopoietic-supporting feeder layer in primary cultures. The present study shows that HUCB does contain a significant accessory cell population that routinely develops into a confluent, adherent cell layer under defined primary culture conditions. HUCB-derived adherent layers were shown to support long-term hematopoietic activity for an average of 4 months. This was achieved by using a customized coverslip with a modified surface structure as the cell attachment substratum and using a specialized culture feeding regime. We have characterized the various cell types (including fibroblasts, macrophages, and endothelial cells) and extracellular matrix proteins (including fibronectin, collagen III, and laminin) that were present in abundance in the HUCB-derived adherent cell layer. In contrast, oil red O-staining fat cells were rarely detected. ELISA and bioassays showed that stem cell factor and interleukin 6 were produced by the HUCB stromal cell cultures, but interleukin 3 or granulocyte/macrophage colony-stimulating factor was not detected. Application of this hematopoietic culture system to transgenic and gene therapy studies of stem cells is discussed.

Bone marrow transplantation in Fanconi anemia using matched sibling donors.

Eighteen patients with Fanconi anemia (FA) with evidence of bone marrow (BM) aplasia underwent allogenic BM transplants (BMT) from matched sibling donors (MSD). Median age at BMT was 7.6 years. Conditioning consisted of low-dose cyclophosphamide (CY; 5 mg/kg x 4 days) and thoracoabdominal irradiation (TAI; 400 cGy). Graft-versus-host disease (GVHD) prophylaxis included cyclosporin A and prednisone. In addition antithymocyte globulin (ATG) was administered in the pretransplant period to promote engraftment and in the posttransplant period for additional GVHD prophylaxis. Engraftment occurred rapidly (median, 12 days for an absolute neutrophil count > or = 0.5 x 10(9)/L; median, 22 days for platelet count > or = 50 x 10(9)/L). Seventeen patients have sustained engraftment and are transfusion-independent, with Lansky scores of 100% at median follow-up of 27 months. One patient developed graft failure 4 months after initial engraftment and required a second BM infusion. None of the patients developed acute GVHD; 3 patients (16%) developed chronic GVHD. BMT is a feasible option for FA patients having an MSD and should be performed at a young age and early in the course of the disease, before the development of complications. We believe the addition of ATG to the transplant regimen of low-dose CY, TAI, and cyclosporin was responsible for improvement in the survival of FA patients undergoing BMT. The regimen was well tolerated and was associated with a low incidence of complications including GVHD.

Detection of tumor necrosis factor-alpha in bone marrow plasma and peripheral blood plasma from patients with aplastic anemia.

Plasma levels of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) were determined in healthy individuals and patients with aplastic anemia (ApAn). IFN-gamma was not detected in normal peripheral blood plasma (PBP) or bone marrow plasma (BMP) and was present in PBP from only 2 of 22 patients and in BMP from 1 of 14 patients and the levels were low (< 1.5 U/ml). Elevated levels of TNF-alpha were present in BMP and PBP from patients but not in control (healthy donor) PBP and BMP. Eleven of twenty-four patients had elevated levels of TNF-alpha in their PBP and 6 of 13 patients had detectable levels of TNF-alpha in their BMP. Only one of the 14 healthy control donors had detectable TNF-alpha and the level was very low (7 pg/ml), while 13 of the 27 ApAn patients had detectable TNF-alpha (P = .009, chi-square test). Not surprisingly, the centers of the distributions of TNF-alpha concentrations of the controls and ApAn patients differed significantly (P < .017 for control and patient PBP and P < .056 for control and patient BMP, Wilcoxon rank-sum test). Spontaneous production of IFN-gamma and TNF-alpha by cultured bone marrow mononuclear cells was observed in four of seven patients but not in the six healthy controls (P = 0.026). Spontaneous production of IFN-gamma and TNF-alpha by cultured peripheral blood mononuclear cells from patients and controls was however similar. Phytohemagglutinin (PHA)-induced production of IFN-gamma and TNF-alpha by cultured mononuclear cells did not differ significantly between ApAn patients and normal controls. The significance of overproduction of TNF-alpha in the pathophysiology of ApAn is discussed.

Haemopoietic stem/progenitor cell transplant in Fanconi anaemia using HLA-matched sibling umbilical cord blood cells.

There have only been a few reports documenting the use of umbilical cord blood as a source of stem cells for haemopoietic reconstitution. We report our experience with a child with Fanconi anaemia (FA) who underwent a stem cell transplant using umbilical cord blood cells from his HLA matched sibling. Although the engraftment was somewhat slow, it was complete and comparable to other transplants performed in FA patients using HLA matched sibling marrow. There was no graft-versus-host disease. The post-transplant period was uncomplicated and, at a follow-up of 36 months, this child is well with normal blood counts and immune function.

Tumor necrosis factor-alpha overproduction in Fanconi's anemia.

Various in vitro studies and clinical observations suggest that Fanconi's anemia (FA) patients are unable to detoxify adequately superoxide anions (O2-) released by activated phagocytes. Recent studies have shown that certain lymphokines such as tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) can significantly enhance O2- production by phagocytic cells. To ascertain lymphokine production in FA patients, we measured TNF-alpha and IFN-gamma production in vivo and in vitro. TNF-alpha was detected in the plasma of 16 of 18 FA patients with concentrations ranging from 6 to 131 pg/ml (mean 31 pg/ml). TNF-alpha was detected in only one of 25 control (healthy donor) plasma, and the level was very low (7 pg/ml). IFN-gamma levels in normal and patient plasma were negligible. Spontaneous and phytohemagglutinin (PHA)-induced production of IFN-gamma and TNF-alpha by cultured peripheral blood mononuclear cells did not differ significantly between FA patients and normal controls. The significance of overproduction of TNF-alpha in vivo in the pathophysiology of FA is discussed.

Synergistic and antagonistic effects of combinations of cyclosporine A and its metabolites on inhibition of phytohemagglutinin-induced lymphocyte transformation in vitro.

Cyclosporine A (CsA) and purified CsA metabolites were tested alone and in combination in cell culture to determine their effects on phytohemagglutinin (PHA)-induced lymphocyte proliferation. CsA was significantly more inhibitory than its metabolites at all concentrations tested (0-1000 ng/mL). CsA exerted maximum inhibition (70% decrease in [methyl-3H]thymidine incorporation) at concentrations of 300 ng/mL or greater; metabolites M1, M17, and M21 depressed the response 46, 39, and 23%, respectively, at 300 ng/mL. Metabolites M8, M18, M26, M25, M13, and M203-218 were non-inhibitory. When combinations of M17 and CsA were tested for the effects on PHA-induced lymphocyte transformation, a synergistic effect occurred at combinations of low concentrations of M17 and CsA and an antagonistic effect at the higher concentrations. Of the 49 combinations of CsA and M17 tested, 30 were antagonistic, 16 synergistic and 3 undecided (approaching addition). When 49 combinations of CsA and the non-immunosuppressive metabolite M8 were tested, 29 of the 49 combinations were synergistic, 17 antagonistic, 1 additive and 2 undecided (approaching addition). Of the 29 synergistic combinations, 14 were strongly synergistic. The importance of the interaction of CsA and metabolites to the immunopharmacology of CsA therapy is discussed.

Benign familial thrombocytosis.

A 13-year-old girl was found to have a platelet count in excess of 4 million/microliters while being evaluated for a minor bleeding episode. Her father and two sisters also had thrombocytosis. All four affected patients were asymptomatic and had no clinical or laboratory evidence of a myeloproliferative disorder other than an elevated platelet count. This represents the second reported instance of benign familial thrombocytosis.

Inhibition of human lymphocyte reactivity by plasma fibronectin in vitro.

The effect of purified human plasma fibronectin (FN) on the reactivity of human lymphocyte-rich mononuclear cells to mitogens and allogeneic cell interactions was studied. Concentrations of FN from 25 to 100 micrograms per 250 microL of culture consistently depressed phytohemagglutinin (PHA) responses. To exert an inhibitory effect, FN must be present within 20 hours after the addition of PHA to cells, and, therefore, it appears to interfere with early events in the transformation process. Increasing the concentration of PHA failed to reduce the inhibitory effect of FN, which suggests that the depressed response was not the result of FN-PHA complex formation, which would reduce the amount of mitogen available for stimulation. This possibility was supported by the finding that FN also inhibited the mixed lymphocyte response (MLR), in a reaction that was not dependent on the activity of soluble antigen or mitogen. In contrast, the stimulation of lymphocytes to undergo transformation that is induced by the nonlectin mitogen, sodium periodate, was unaffected by FN. Periodate-treated cells are, however, already stimulated to undergo transformation, prior to their exposure to FN. FN did not interfere with the activity of interleukin-2, nor did it indirectly regulate lymphocyte responses by modifying the production and/or effect of humoral regulatory factors released from the adherent accessory cells (macrophages). These studies show that FN is a potent immunosuppressive agent in vitro.

Influence of fibronectin-containing blood products on lymphocyte reactivity.

Two fibronectin (FN)-containing blood products, human peripheral blood plasma and cryoprecipitate, were examined for their effect on mitogen-induced lymphocyte transformation in vitro. Responses of human peripheral blood lymphocytes to phytohemagglutinin (PHA) were depressed in the presence of a plasma concentration above that required for maximum DNA synthesis, and this concentration must be present in cultures prior to lymphocyte activation. The removal from the plasma of heparin-induced cryoprecipitate, a complex consisting of FN, heparin, and fibrinogen, resulted in a significant reduction in the inhibitory effect of the plasma on the PHA response. Plasma specifically depleted of FN by affinity chromatography on gelatin-agarose beads was 32 percent less inhibitory to the PHA-induced stimulation of cells than untreated plasma; the remaining inhibitory activity in the FN-depleted plasma samples was attributed to the presence of other normal immunosuppressive factors. The inhibitory capacity of FN in plasma was similar to that obtained with purified FN alone, which indicates that, unlike that of other known plasma inhibitors, the immunosuppressive activity of FN was not altered by the presence of other components of plasma. Cryoprecipitate used in the treatment of hemophilia contains high levels of FN, and, as anticipated, PHA-induced lymphocyte transformation was markedly depressed in the presence of solubilized cryoprecipitate. The contribution of FN to the T-cell abnormalities in patients chronically receiving cryoprecipitate and/or factor VIII concentrates derived from cryoprecipitate warrants further investigation.