Induction of suppressor cells by a tumor-derived suppressor factor

Murine fibrosarcomas produce a factor that activates suppressor cells to inhibit expression of delayed-type hypersensitivity (DTH) responses to dinitrochlorobenzene (DNCB). This tumor-derived suppressor factor (TDSF) was partially purified by preparative isoelectric focusing of spent medium and 3 M KCl extracts of cultured methylcholanthrene-induced and spontaneous fibrosarcomas of C3H/He mice. Incubation of 1 micrograms/ml of a fraction, isoelectric pH less than 2.9, with normal syngeneic spleen cells for 1-6 hr at 37 degrees C induced suppressor cells that inhibited the primary DTH response to DNCB upon intraperitoneal transfer to normal C3H/HeJ mice. TDSF was not present in extracts of either syngeneic embryonic fibroblasts or normal spleen cells or in medium conditioned by normal peritoneal exudate cells but was present in 3 M KCl extracts of and the spent medium from four different cultured murine fibrosarcomas. TDSF activity was not restricted at the major histocompatibility complex. The suppressor cells inhibited the efferent limb of the DTH response because (1) hyporesponsive recipients of TDSF-treated spleen cells had splenic effector T cells capable of transferring DTH to DNCB into naive secondary recipients and (2) the ability of Lyt 1+,2- effector Tdth cells to transfer a secondary DTH response to DNCB was inhibited by co-incubation with macrophages or Lyt 1-,2+ T cells treated with TDSF. Preliminary biochemical analysis suggested that TDSF was an RNA- protein complex. Thus, several murine fibrosarcomas produced a soluble factor that activated splenic suppressor cells to depress the immune response to nonneoplastic antigens. These suppressor factors represent a novel group of regulatory molecules which may be ribonucleoprotein complexes.     

Characterization of Schistosoma mansoni antigen-reactive T cell clones that form granulomas in vitro

Granuloma formation and modulation in schistosomiasis are a consequence of discrete subpopulations of T lymphocytes and the mediators they produce. In the present study, T cell clones reactive to soluble egg antigen (SEA) were developed to analyze the roles of T cells in Schistosoma mansoni egg-induced granuloma formation. In an in vitro granuloma assay, 1 X 10(5) T cells specifically augmented the response of 2 X 10(6) normal spleen cells to SEA-coupled but not purified protein derivative-coupled polyacrylamide beads. In vitro granulomatous responses by individual clones were correlated with their capacity to mediate local delayed-type hypersensitivity reactions in footpad swelling assays. Phenotypic analysis of the seven clones characterized in the present study demonstrated that they were L3T4+, Ly-2.2-. An analysis of supernatants of T cells pulsed with concanavalin A or SEA + antigen-presenting cells was also undertaken in an attempt to correlate in vitro granuloma formation with lymphokine production. Stimulated T cells (but not unstimulated T cells) produced interleukin 2, macrophage activating factor, migration inhibitory factor, and eosinophil stimulation promoter in response to both mitogenic and antigenic stimuli. The results suggest that individual clones of T cells are capable of producing a variety of mediators that influence their ability to activate and to recruit cells into granuloma formation. The model may be useful in the analysis of specific antigens and regulatory interactions and their contribution to granuloma formation.     

Origin of deoxycorticosterone sulfate (DOC-SO4) in plasma of pregnant women: Pregnenolone-3,21-disulfate is a placental precursor of DOC-SO4

The plasma levels of deoxycorticosterone sulfate (DOC-SO₄) in near-term pregnant women are 100 times those in plasma of men or non-pregnant women. Yet, neither the tissue site of synthesis nor the precursor of DOC-SO₄ that enters maternal plasma is known. Several potential sources have been excluded: plasma DOC-SO₄ is not derived from plasma DOC; and the secretion of C₂₁-steroids (other than aldosterone) from the maternal adrenals during human pregnancy is not increased. Similarly, the transfer of DOC-SO₄ from fetal plasma cannot account for the high level of DOC-SO₄ in the maternal compartment, and a reduced clearance of plasma DOC-SO₄ during pregnancy cannot account for the high levels of DOC-SO₄. Indeed, the rate of clearance of DOC-SO₄ from plasma is 10–100 times that of most other steroid sulfates. To address this question further, we evaluated the possibility that fetal plasma pregnenolone-3,21-disulfate serves as a precursor for DOC-SO₄ formation in the placenta. The preferential hydrolysis of the 3β-sulfate of pregnenolone-3,21-disulfate in placenta would give rise to pregnenolone-21-monosulfate, which, if acted upon by placental 3β-hydroxysteroid dehydrogenase/Δ^{5 → 4} isomerase, could give DOC-SO₄. [³H]Pregnenolone-3,21-disulfate was incubated with minces of human placental tissue for 5, 20, 60 and 120 min. Radiolabelled DOC-SO₄, DOC, and pregnenolone-21-monosulfate were isolated from the incubation media and quantified. After a 5 min incubation, 7.5% of substrate was converted to DOC-SO₄; and after 20, 60 and 120 min 30% of the [³H]pregnenolone-3,21-disulfate was recovered from the media of these incubations as [³H]DOC-SO₄. [³H]DOC was also present in the incubation media and the concentrations of this product increased as a function of incubation time. Therefore, pregnenolone-3,21-disulfate, which is present in very high concentrations in fetal plasma (1000 ng/ml), is metabolized in the placenta to DOC-SO₄. Because of the fetal and maternal vascular arrangements of the hemochorioendothelial placenta of human pregnancy, steroids produced in syncytiotrophoblasts preferentially enter the intervillous space; thus, fetal plasma pregnenolone-3,21-disulfate may serve as a placental precursor of maternal plasma DOC-SO₄.     

Effect of murine tumors upon delayed hypersensitivity to dinitrochlorobenzene. III. In vivo activity of the nonspecific suppressor cell

Tumor-induced immunosuppression was investigated in an in vivo model of delayed hypersensitivity (DH) to the chemical sensitizer, dinitrochlorobenzene (DNCB). DH to DNCB as measured in a footpad assay was decreased in C3H/HeJ mice bearing MCA-F, a 3-methylcholanthrene-induced syngeneic fibrosarcoma. Suppressor cells from the spleens of tumor-bearing mice inhibited the induction of DH to DNCB in otherwise normal syngeneic C3H/HeJ recipients. Ten million spleen cells (SpC) harvested from mice bearing MCA-F for 10 days and adoptively transferred to tumor-free mice at the time of sensitization with DNCB suppressed the response to the sensitizer. The suppressor cells were macrophages, since they were adherent to plastic, removed by treatment with a magnet after phagocytosis of carbonyl iron, resistant to exposure to gamma radiation and to treatment with anti-Thy 1.2 serum and complement. Further, the nonspecific suppressor cells were activated by progressive tumor growth rather than by induction of tumor-specific immunity using irradiated tumor cells. Titration studies revealed that suppression of DH occurred with the transfer of as few as 10(6) SpC. Thus, nonspecific suppressor cells are effective at inhibiting in vivo DH to DNCB and suggest that nonspecific suppression in the intact host occurs through mechanisms different from those involved in suppression in vitro.     

Optimization of potent and selective dual mTORC1 and mTORC2 inhibitors: The discovery of AZD8055 and AZD2014

The optimization of a potent and highly selective series of dual mTORC1 and mTORC2 inhibitors is described. An initial focus on improving cellular potency whilst maintaining or improving other key parameters, such as aqueous solubility and margins over hERG IC₅₀, led to the discovery of the clinical candidate AZD8055 (14). Further optimization, particularly aimed at reducing the rate of metabolism in human hepatocyte incubations, resulted in the discovery of the clinical candidate AZD2014 (21).     

Genetic variation of the bud and leaf phenology of seventeen poplar clones in a short rotation coppice culture

Abstract: Leaf phenology of 17 poplar ( Populus spp.) clones, encompassing spring phenology, length of growth period and end-of-year phenology, was examined over several years of different rotations. The 17 poplar clones differed in their latitude of origin (45°30'N to 51°N) and were studied on a short rotation experimental field plantation, situated in Boom (province of Antwerpen, Belgium; 51°05'N, 04°22'E). A similar, clear pattern of bud burst was observed during the different years of study for all clones. Clones Columbia River, Fritzi Pauley, Trichobel (Populus trichocarpa) and Balsam Spire (Populus trichocarpa × Populus balsamifera) from 45°30'N to 49°N reached bud burst (expressed as day of the year or degree day sums) almost every year earlier than clones Wolterson (Populus nigra), Gaver, Gibecq and Primo (Populus deltoides × Populus nigra) (50°N to 51°N). This observation could not be generalised to end-of-season phenology, for which a yearly returning pattern for all clones was lacking. Late bud burst and early leaf fall of some clones (Beaupré, Boelare, IBW1, IBW2, IBW3) was brought about by increasing rust incidence during the years of observation. For these clones, the variability in leaf phenology was reflected in high coefficients of variation among years. The patterns of genetic variation in leaf phenology have implications for short rotation intensive culture forestry and management of natural populations. Moreover, the variation in phenology reported here is relevant with regard to the genetic mapping of poplar.     

Platelet-activating factor and kinin-dependent vascular leakage as a novel functional activity of the soluble terminal complement complex

The infrequent occurrence of septic shock in patients with inherited deficiencies of the terminal complement components experiencing meningococcal disease led us to suspect that the terminal complement complex is involved in vascular leakage. To this end, the permeabilizing effect of the cytolytically inactive soluble terminal complement complex (SC5b-9) was tested in a Transwell system measuring the amount of fluorescein-labeled BSA (FITC-BSA) leaked through a monolayer of endothelial cells. The complex caused increased permeability to FITC-BSA after 15 min as opposed to the prompt response to bradykinin (BK). The effect of SC5b-9 was partially reduced by HOE-140 or CV-3988, two selective antagonists of BK B2 and platelet-activating factor receptors, respectively, and was completely neutralized by the mixture of the two antagonists. Also, DX-88, a specific inhibitor of kallikrein, partially inhibited the activity of SC5b-9. The permeabilizing factor(s) released after 30 min of incubation of endothelial cells with SC5b-9 caused a prompt leakage of albumin like BK. Intravital microscopy confirmed both the extravasation of circulating FITC-BSA across mesenteric microvessels 15 min after topical application of SC5b-9 and the complete neutralization by the mixture of HOE-140 and CV-3988. SC5b-9 induced opening of interendothelial junctions in mesenteric endothelium documented by transmission electron microscopy.