A potential role for interleukin-15 in the regulation of human natural killer cell survival

Resting lymphocyte survival is dependent upon the expression of Bcl-2, yet the factors responsible for maintaining lymphocyte Bcl-2 protein expression in vivo are largely unknown. Natural killer (NK) cells are bone marrow-derived lymphocytes that constitutively express the beta and common gamma(c) subunits of the IL-2 receptor (R) as a heterodimer with intermediate affinity for IL-2. IL-15 also binds to IL-2Rbeta gamma(c) and is much more abundant in normal tissues than IL-2. Mice that lack the IL-2 gene have NK cells, whereas mice and humans that lack IL-2R gamma(c) do not have NK cells. Further, treatment of mice with an antibody directed against IL-2Rbeta results in a loss of the NK cell compartment. These data suggest that a cytokine other than IL-2, which binds to IL-2Rbeta gamma(c), is important for NK cell development and survival in vivo. In the current report, we show that the recently described IL-15R(alpha) subunit cooperates with IL-2Rbeta gamma(c) to transduce an intracellular signal at picomolar concentrations of IL-15. We demonstrate that resting human NK cells express IL-15R(alpha) mRNA and further, that picomolar amounts of IL-15 can sustain NK cell survival for up to 8 d in the absence of serum. NK cell survival was not sustained by other monocyte-derived factors (i.e., TNF-alpha, IL-1beta, IL-10, IL-12) nor by cytokines known to use gamma(c) for signaling (i.e., IL-4, IL-7, IL-9, IL- 13). One mechanism by which IL-15 promotes NK cell survival may involve the maintenance of Bcl-2 protein expression. Considering these functional properties of IL-15 and the fact that it is produced by bone marrow stromal cells and activated monocytes, we propose that IL-15 may function as an NK cell survival factor in vivo.     

Characterization of the major bovine brain Go alpha isoforms. Mapping the structural differences between the alpha subunit isoforms identifies a variable region of the protein involved in receptor interactions

Go is the major G protein in bovine brain, with at least three isoforms, GoA, GoB, and GoC. Whereas alphaoA and alphaoB arise from a single Goalpha gene as alternatively spliced mRNAs, alphaoA and alphaoC are thought to differ by covalent modification. To test the hypothesis that alphaoA and alphaoC have different N-terminal lipid modifications, proteolytic fragments of alphao isoforms were immunoprecipitated with an N terminus-specific antibody and analyzed by matrix-assisted laser desorption ionization mass spectrometry. The major masses observed in immunoprecipitates were the same for all three alphao isoforms and corresponded to the predicted mass of a myristoylated N-terminal fragment. Structural differences between alphaoA and alphaoC were also compared before and after limited tryptic proteolysis using SDS-polyacrylamide gel electrophoresis containing 6 M urea. Based upon the alphao subunit fragments produced under activating and nonactivating conditions, differences between alphaoA and alphaoC were localized to a C-terminal fragment of the protein. This region, involved in receptor and effector interactions, implies divergent signaling roles for these two alphao proteins. Finally, the structural difference between alphaoA and alphaoC is associated with a difference of at most 2 daltons based upon measurements by electrospay ionization mass spectrometry.     

Binding and presentation of peptides derived from melanoma antigens MART-1 and glycoprotein-100 by HLA-A2 subtypes. Implications for peptide-based immunotherapy

Cellular immune responses to melanoma-associated Ags are the focus of ongoing studies aimed at developing immunotherapies for treatment of malignant melanoma. Melanoma predominantly affects Caucasians, a population in whom expression of HLA-A2 is prevalent. Among HLA-A2 subtypes, HLA-A*0201 is widely expressed, and HLA-A*0201-restricted, tumor-reactive CTL responses are well studied. We have observed in a group of melanoma patients an unexpectedly high frequency (approximately 20%) of non-HLA-A*0201 subtypes (*0202, *0204, and *0205), and little is known regarding antimelanoma response profiles in patients expressing such subtypes. We analyzed non-HLA-A*0201 peptide response profiles using HLA-A*0201-restricted epitopes from melanoma Ags MART-1/Melan A and glycoprotein 100. Most of these peptides bound to the majority of subtypes tested with 50% inhibitory concentrations less than 500 nM. Recognition of cells pulsed with different peptides (MART-1(27-35), G9(154), and G9(280) Flu M1(58-66)) and expressing different subtype molecules by HLA-A*0201-restricted CTL was limited to only a subset of non-HLA-A*0201 molecules, and the peptide/subtype complexes recognized varied among the effector populations tested. CTL responses elicited from PBL of patients and healthy donors expressing subtypes HLA-A*0202 and HLA-A*0205 suggested significant differences among HLA-A2 subtype function in the context of melanoma Ag presentation. These observations imply the necessity of subtyping patients considered for peptide-based protocols and highlight the need for further study of melanoma-directed cellular responses among patients expressing non-HLA-A*0201 subtypes.     

In vitro genotoxicity studies of chrysotile asbestos fibers dispersed in simulated pulmonary surfactant

Micronucleus (MN) formation and sister-chromatid exchange (SCE) assays were performed for asbestos in cultured Chinese hamster lung (V79) cells to determine the effect of surfactant treatment on the genotoxicity of two chrysotile asbestos samples of different fiber lengths. The cells were challenged in vitro with NIEHS intermediate- and short-length chrysotile fibers in both their native state and with surfactant pretreatment. For the surfactant pretreatment, the fibers were incubated in a simulated pulmonary surfactant which was prepared by ultrasonically dispersing dipalmitoyl lecithin (DPL), a primary component of pulmonary surfactant, in minimal essential medium (MEM). Chrysotile asbestos was ultrasonically mixed into the prepared surfactant dispersion or into MEM. V79 cells were exposed to DPL-treated intermediate-length chrysotile (TICA), intermediate-length chrysotile (ICA), DPL-treated short-length chrysotile (TSCA) or short-length chrysotile (SCA) fibers for 48 h. For each treatment, 2000 mononucleated cells were scored for MN formation, and 30 M2 metaphase cells were scored for SCE induction. The results showed that all samples, TICA, ICA, TSCA and SCA, caused significant elevation in the frequency of cells with micronuclei and of cells with two or more nuclei. The increase in micronucleus frequency was greatest in cells challenged with untreated intermediate-length fibers, and was greater for untreated than for DPL-treated short-length fibers. For the short-length fiber samples, DPL surfactant treatment decreased activity for multiple nucleus formation, while DPL treatment did not result in consistent changes in that activity for intermediate-length fibers. Results of SCE assays were either negative or inconclusive. Cells were more viable following TICA and TSCA than following ICA and SCA challenge as measured by cell counts after 48 h of incubation.     

Mechanisms causing loss of muscle in acute uremia

The mortality of acute renal failure (ARF) remains high because most illnesses associated with ARF also cause loss of muscle mass. The mechanisms causing loss of muscle mass are inhibition of protein synthesis and/or acceleration of protein degradation. Identifying which of these abnormalities is present requires understanding the principles of amino acid and protein turnover. There is evidence that ARF by itself impairs protein synthesis and stimulates protein degradation. The mechanisms causing these defects include abnormal metabolic responses to hormones and stimulation of the ATP--ubiquitin--proteasome-dependent pathway. Understanding these mechanisms could lead to rational therapies.     

Toxicity of Piper abyssinica seeds to Nubian goats

An investigation is described in which Nubian goats were given daily oral doses of Piper abyssinica seeds at 0.25; 1 and 5 g/kg body weight. The results of liver and kidney function tests were correlated with the clinical and pathological changes. In goats receiving the plant seed at 5 g/kg body weight/day, death occurred within 5-14 days and the main signs were bloated rumen, diarrhoea, dysponoea and ataxia. Enterohepatonephropathy was accompanied by increases in the activity of serum ALP and GGT, in the concentration of cholesterol, globulin, total lipids and urea and decreases in the level of albumin. Piper seed was toxic but not lethal to goats at doses of 0.25 and 1 g/kg body weight.     

Goitre: a cause of obstructive sleep apnoea in euthyroid patients

While hypothyroidism is considered to predispose to obstructive sleep apnoea (OSA), the presence of a goitre itself is not a recognized cause of OSA. We present the cases of two euthyroid patients with large goitres and clinical evidence of OSA, whose OSA symptoms significantly improved following partial thyroidectomy. This finding suggests that the goitre contributed to their symptoms.