Immunophenotypes for 272 patients with acute myelogenous leukemia (AML) were analyzed using a panel of 22 antibodies. Numerical evidence for unusual coexpressions (present in normal marrow at < or = 0.1%) of surface markers on = or = 10% of the blast cells was found in 85% of all cases. Asynchronous expression of myeloid differentiation antigens occurred in 70% of the cases. Unusual coexpression of T-lymphoid, B- lymphoid, or natural killer (NK) markers with myeloid markers occurred in 38%, 13%, and 21%, respectively, of all AML cases. Two- and three- color analyses confirmed coexpression in 15 of 15 cases, and indicated that these percentages are an underestimate, because coexpression can be demonstrated in cases without numerical overlap. These data indicate that the unusual coexpression of normal differentiation antigens is a common occurrence in AML. Markers in 12 of 13 patients were similar between presentation and relapse, and in two patients, unusual phenotypes detected at first relapse were shown at second relapse, indicating these immunophenotypes are stable in the majority of AML patients. Significant correlations were found between t(8;21) cytogenetics and coexpression of CD19 with CD15 or CD34, t(9;22) and coexpression of CD19 and CD34, and t(15;17) and coexpression of CD2 and myeloid antigens. Multiparameter fluorescence analysis allows detection of unusual phenotypes when the blast counts are < 5% (classical remission). Analysis of 16 patients in remission indicated the presence of presentation phenotypes in 0.2% to 7.9% of the lymphocyte + blast light scatter region, representing 0.03% to 1.4% of the total nucleated marrow cells. Of the 16 patients with = or = 4 months follow-up after detection of these cells, 6 of 6 patients with = or = 0.2% unusual presentation phenotypic marrow cells have relapsed, while 9 of 10 patients with < 0.2% remain in remission. The detection of cells with the unusual presentation phenotype may reflect residual AML cells, and their increase may predict relapse.

BALB/c mice with pulmonary tuberculosis develop a T helper cell type 1 response that peaks at 3 weeks, temporarily controlling bacterial growth. Then bacterial proliferation recommences, accompanied by increasing interleukin (IL)-4 levels and decreasing interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and inducible nitric oxide synthase (iNOS) levels. These changes mimic those in the human disease. In a previous study, administration of dehydroepiandrosterone (DHEA) beginning on day 60 after infection reversed these changes and protected the mice. However, DHEA is suboptimal for human use, partly because it is readily metabolized into sex steroids. 16alpha-Bromoepiandrosterone (EpiBr; 16alpha -bromo-5alpha -androstan-3beta-ol-17-one) is a synthetic adrenal steroid derivative that does not enter sex steroid pathways. In the present study, when tuberculous BALB/c mice were treated with EpiBr 3 times/week beginning on day 60, inhibition of bacterial proliferation and increased expression of TNF-alpha, IFN-gamma, and iNOS were observed, although decreased expression of IL-4 was also observed. Moreover, when given as an adjunct to conventional chemotherapy, EpiBr enhanced bacterial clearance. Trials for the use of EpiBr in the treatment of human tuberculosis are now justified.

Dehydroepiandrosterone (DHEA) has attracted much interest because of its many antiaging, metabolic and immune-modulating effects in rodents. Synthetic derivatives, such as 5-androstene-16alpha-fluoro-17-one (HE2500) and certain natural metabolites also provide benefit in various animal models of autoimmune and metabolic diseases. But, like DHEA, low potency and low oral bioavailability suggested limited usefulness of these compounds in humans. We hypothesized that HE3286, a novel 17-ethynyl derivative would be orally bioavailable, more potent, and chemically more useful in man than its parent compound. We found that on a dose/mass basis, HE3286 demonstrated up to 25% oral bioavailability in mice. In the DBA mouse model of collagen-induced arthritis (CIA), animals receiving oral treatment with HE3286 (50 mg/kg), beginning at onset of disease, significantly decreased CIA peak scores and daily severity of arthritis scores. Benefit was associated with decreases in: (1) production of TNF-alpha, IL-6, and IL-17; and (2) decreases in joint inflammation, erosion, and synovial proliferation as judged by histological analysis. HE3286 was not found to be immune suppressive in any of the classical models tested, including mitogen-induced proliferation, delayed-type hypersensitivity, or mixed lymphocyte reaction. Instead, benefit was associated with increases in numbers and function of CD4+CD25+FOXp3+CD127- regulatory T cells (T reg). To our knowledge, this is probably the first study to report that an orally bioavailable synthetic analogue of DHEA can ameliorate ongoing disease in a CIA mouse model with relevance to rheumatoid arthritis (RA) and to correlate that finding with decreases in proinflammatory cytokines and increases in T reg cells. Hormones targeting T reg cells hold the intriguing potential to treat autoimmune, infectious, and neoplastic diseases.

Unfractionated peripheral blood stem cell (PBSC) grafts contain measurable quantities of myeloma cells and are therefore a potential source of relapse posttransplantation. In contrast, fluorescence-activated cell sorting (FACS)-sorted CD34+Thy1+ Lin− peripheral blood cells are substantially enriched for stem cell activity, yet contain virtually no clonal myeloma cells. A study was performed in patients with symptomatic myeloma, who had received 12 months or less of preceding standard chemotherapy, to evaluate the feasibility of large scale purification of primitive hematopoietic stem cells in order to study engraftment kinetics posttransplantation and the degree of tumor cell contamination of this cell population, based on polymerase chain reaction (PCR) analysis for the patient-specific complementarity-determining region III (CDR III). PBSC were mobilized with high dose cyclophosphamide and granulocyte-macrophage colony-stimulating factor (GM-CSF). A combination of elutriation and chemical lysis was used to deplete PBSC collections of monocytes, granulocytes, erythrocytes, and platelets. Subsequently, CD34+ Thy1+ Lin−progenitor cells were purified with high speed cell sorting. Of the 10 evaluable patients, nine met the required minimum criteria of ≥7.2 × 105 cells/kg to support tandem transplants. After high dose melphalan (200 mg/m2) eight engrafted successfully, although granulocyte (absolute neutrophil count [ANC] &gt;0.5 × 109/L, 16 days) and platelet recovery (platelets &gt; 50 × 109/L, 39 days) was substantially delayed when compared with unmanipulated PBSC grafts; one patient required infusion of a reserve graft because of lack of evidence of engraftment by day +28. Three patients proceeded to a second graft with high dose melphalan and total body irradiation; two required infusion of a reserve graft and both died of infectious complications; one showed delayed, but complete, engraftment after this myeloablative regimen. Two of the nine evaluable patients attained a clinical complete remission (CR). The grafts from three patients were tested for tumor contamination and contained no detectable clonal myeloma cells. Larger quantities of purified cells may be required to resolve the problem of delayed engraftment.

Insulin resistance, the major metabolic abnormality underlying type 2 diabetes, is associated with chronic inflammation and heavy macrophage infiltration in white adipose tissue (WAT). The therapeutic properties of the synthetic adrenal steroid ⌬ 5 -androstene-17␣-ethynyl-3␤,7␤,17␤-triol (HE3286) were characterized in metabolic disease models. Treatment of diabetic db/db mice with HE3286 suppressed progression to hyperglycemia and markedly improved glucose clearance. Similar effects were also observed in insulin-resistant, diet-induced obese C57BL/6J mice and genetically obese ob/ob mice. This effect appeared to be a consequence of reduced insulin resistance because HE3286 lowered blood insulin levels in db/db and ob/ob mice. Treatment with HE3286 was accompanied by suppressed expression of the prototype macrophage-attracting chemokine monocyte chemoattractant protein-1 in WAT, along with its cognate receptor C-C motif chemokine receptor-2. Exposure of mouse macrophages to HE3286 in vitro caused partial suppression of endotoxin (lipopolysaccharide)-induced nuclear factor -B (NF-B)-sensitive reporter gene expression, NF-B nuclear translocation, and NF-B/ p65 serine phosphorylation. Proinflammatory kinases, including IB kinase, c-Jun NH 2 -terminal kinase, and p38, were also inhibited by HE3286. In ligand competition experiments HE3286 did not bind to classical sex steroid or corticosteroid receptors, including androgen receptor (AR), progesterone receptor, estrogen receptor (ER) ␣ or ER␤, and glucocorticoid receptor (GR). Likewise, in cells expressing nuclear receptor-sensitive reporter genes HE3286 did not substantially stimulate transactivation of AR, ER, GR, or peroxisome proliferator-activated receptor (PPAR) ␣, PPAR␦, and PPAR␥. These findings indicate that HE3286 improves glucose homeostasis in diabetic and insulin-resistant mice and suggest that the observed therapeutic effects result from attenuation of proinflammatory pathways, independent of classic sex steroid receptors, corticosteroid receptors, or PPARs.