T cells expressing killer cell inhibitory receptors do not carry TCR for alloantigens

The role of T cells carrying the killer cell inhibitory receptor (KIR) is unknown. Here we investigate the allo-response of KIR+ CD3+ cells (KIR+ T cells). KIR+ T cells was found in 4.6-14.7% of the T cells from the PBMC of healthy individuals. Flow cytometry analysis showed that the percentage of KIR+ T cells in T cells from three individuals was decreased after stimulation with allogeneic PBMC. These findings suggest that KIR+ T cells do not respond to alloantigens. Furthermore, the analyses using purified KIR+ T cells also confirmed that KIR+ T cells do not respond to alloantigens.     

Myelin proteolipid protein intron 1 sequences do not appear to enhance myelin proteolipid protein gene transcription

Sequences from the first intron of the mouse myelin proteolipid protein (PLP) gene were examined for their ability to modulate PLP gene expression. Glial (N20.1) or nonglial (NIH 3T3) cells were transiently transfected with constructs that contained 1.4 kb of PLP promoter sequence driving luciferase reporter gene expression, as well as various portions of PLP intron 1 DNA. Although these same PLP intron 1 fragments enhanced reporter gene expression from a heterologous basal promoter in a previous study, the results reported here demonstrate that they do not augment PLP promoter activity. Thus, the regulation of PLP cell-type-specific expression, conferred by the first intron, appears to be mediated by an enhancer-independent mechanism.     

Generation of in vitro natural cytotoxicity of horse lymphocytes against sarcoid-derived tumor cells not expressing major histocompatibility complex antigens

OBJECTIVE: To analyze in vitro lymphocyte-mediated immune responses of horses with sarcoids against allogeneic sarcoid cells containing endogenous retrovirus but not expressing major histocompatibility complex antigens. DESIGN: Lymphocyte-mediated immune reactions were assessed by means of proliferative responses in mixed lymphocyte tumor cell culture (MLTC) assay and lymphocyte-mediated cytotoxicity against various equine target cells. ANIMALS: 12 horses with sarcoid tumors and 15 control horses. PROCEDURE: Blood lymphocytes were cocultured in MLTC with allogeneic sarcoid cells (Mc-1, BayMc-1), equine testis cells, or normal equine dermal fibroblasts. Lymphocytes were assayed for proliferative responses by [3H]thymidine uptake and for cytotoxicity against the same targets by 51Cr release assay. The lymphocyte populations were analyzed for some common surface markers. RESULTS: Lymphocytes from horses with sarcoids exerted an anamnestic proliferative response in MLTC against Mc-1 cells, but this procedure never generated cytotoxic lymphocytes. However, lymphocytes from all horses cultured in medium with 10% allogeneic serum only had selective. natural cytotoxicity against Mc-1 that was generated without DNA synthesis. Approximately 80% of the lymphocytes disappeared during culture; however the remaining population of small, viable lymphocytes indicated a decrease of CD4+ T lymphocytes, but numbers of T cells with receptors for Helix pomatia A hemagglutinin were unaffected. Few lymphocytes had Fc-receptors for IgG, were complement-reactive positive cells or were B cells expressing surface immunoglobulin. CONCLUSIONS: Results may indicate a natural defense system, which preferentially recognizes and lyses tumor cells that are deficient in surface expression of major histocompatibility complex antigens, without intervention of conventional T-cell receptors or antibodies.     

The PTEN/MMAC1 tumor suppressor induces cell death that is rescued by the AKT/protein kinase B oncogene

PTEN/MMAC1 is a tumor suppressor gene that is mutated in a variety of cancers. PTEN encodes a phosphatase that recognizes phosphoprotein substrates and the phospholipid, phosphatidylinositol-3,4,5-triphosphate. PTEN inhibited cell growth and/or colony formation in all of the epithelial lines tested with one exception. The decrease in cellular proliferation was associated with an induction of apoptosis and an inhibition of signaling through the phosphatidylinositol 3'-kinase pathway. Akt/protein kinase B, a gene whose antiapoptotic function is regulated by phosphatidylinositol-3,4,5-triphosphate, was able to rescue cells from PTEN-dependent death. PTEN, therefore, appears to suppress tumor growth by regulating phosphatidylinositol 3'-kinase signaling.     

The desmoplastic small round cell tumor t(11;22) translocation produces EWS/WT1 isoforms with differing oncogenic properties

Structural alterations of the Wilms' tumor locus (WT1) at 11p13 have been implicated in the etiology of two human cancers--Wilms' tumor (WT), a pediatric renal malignancy, and Desmoplastic Small Round Cell Tumor (DSRCT), an aggressive cancer of the abdominal serosal lining with predilection for male adolescents. Germline mutations within the WT1 tumor suppressor gene predispose to WT and are associated with congenital malformations of the urogenital system, and somatic mutations are associated with initiation of transformation in WTs. In DSRCT, a recurrent translocation, t(11;22)(p13;q12), fuses the amino terminal domain of the EWS1 gene product to three of the four WT1 zinc fingers. Two EWS/WT1 isoforms are generated as a result of an alternative splicing event between zinc fingers III and IV, inserting or removing three amino acids (+/- KTS). We demonstrate that introduction of EWS/WT1(-KTS) into NIH3T3 cells causes their tumorigenic transformation as determined by: formation of transformed foci on a monolayer of cells; anchorage-independent growth; and tumor formation in nude mice. EWS/WT1(+KTS) showed no transforming potential in these assays. These results indicate the oncogenic effect of the t(11;22) translocation is mediated by the EWS/WT1(-KTS) isoform and that fusion of the EWS amino terminal domain to the WT1 DNA binding domain produces a chimeric product showing a gain of function.     

C3G is tyrosine-phosphorylated after integrin-mediated cell adhesion in normal but not in Bcr/Abl expressing cells

The SH2-SH3 adaptor protein Crkl has been implicated in the signal transduction pathways of several membrane-bound receptors. Tyrosine phosphorylation of proteins associated with such signalling complexes can generate binding sites for the Crkl SH2-domain and can recruit proteins constitutively bound to Crkl via the Crkl SH3 domain into such complexes. In the current study we show that Crkl, but only a minor amount of the related Crk, form constitutive complexes in vivo with guanine nucleotide exchange factor C3G in 3T3 fibroblasts. Adhesion of both normal and transformed cells to fibronectin or other extracellular matrix proteins consistently induces the tyrosine-phosphorylation of C3G. Adhesion-induced tyrosine phosphorylation of C3G is dependent on an intact cytoskeleton and peaks at 5-10 min after attachment. In contrast, 3T3 cells stably transfected with Bcr/Abl P210 show a prominent reduction in the amount of C3G complexed to Crkl and do not exhibit tyrosine-phosphorylation of C3G upon spreading and attachment. These data establish that integrin-mediated cell adhesion results in Crkl-mediated tyrosine phosphorylation of C3G, a pathway which can be disrupted by Bcr/Abl.     

Endothelial and epithelial cells do not respond to complexes of peptidoglycan with soluble CD14 but are activated indirectly by peptidoglycan-induced tumor necrosis factor-alpha and interleukin-1 from monocytes

Peptidoglycan (PGN) activates macrophages through membrane CD14 (an endotoxin receptor) and binds to both soluble and membrane CD14. Since soluble CD14-lipopolysaccharide (LPS) complexes activate CD14-negative endothelial and epithelial cells, this study tested whether soluble CD14-PGN complexes activate human umbilical vein endothelial cells and epithelial-like U373 cells to secrete interleukin (IL)-6, express vascular cellular adhesion molecule-1, and translocate nuclear factor-kappaB. In contrast to LPS, endothelial, epithelial, and other cells of non-hemopoietic origin were unresponsive to PGN through soluble or membrane-bound CD14, whereas cells of hemopoietic origin were responsive to both PGN and LPS. PGN, similarly to LPS, activated endothelial and epithelial cells indirectly in the presence of 2%-4% blood, by inducing secretion of both tumor necrosis factor-alpha and IL-1 from monocytes. These results reveal different mechanisms of CD14 function and cell activation for LPS and PGN and also demonstrate strong indirect activation of endothelial and epithelial cells by both PGN and LPS.     

Multidrug resistance-reversing agents increase vinblastine distribution in normal tissues expressing the P-glycoprotein but do not enhance drug penetration in brain and testis

The aim of this study was to assess whether P-glycoprotein (Pgp) inhibitors altered the blood-brain barrier and enhanced vinblastine (VBL) distribution in brain, testis and other Pgp-expressing tissues. Trifluoperazine, cyclosporin A, amiodarone, quinidine, the nifedipine analog Bay K8644 and verapamil were selected among Pgp inhibitors and were administered intraperitoneally 1 hr before an intravenous dose of 10 mg/kg VBL. Trifluoperazine and cyclosporin A were also administered intraperitoneally for 7 days before VBL. VBL and its metabolite O4-deacetylvinblastine were measured in tissues by high-performance liquid chromatography assay. None of the reversing agents (RA) appreciably raised VBL concentrations in brain and testis, whereas all except quinidine significantly enhanced VBL distribution in liver and kidney; the most effective were trifluoroperazine and cyclosporin A. In mice treated with RA and VBL combined, O4-deacetylvinblastine levels in liver and kidney reached either the same or higher levels than in mice treated with VBL alone, indicating that the increase in VBL levels is not due to inhibition of its metabolism. The main conclusions are that (1) inhibitors of Pgp, even at high doses, do not increase the permeability of the blood-brain barrier in mice, suggesting caution in the clinical use of RA combined with antitumor agents for brain tumors; and (2) several RA achieve high enough concentrations to enhance the distribution of VBL in other normal tissues expressing Pgp, thus potentially increasing VBL toxicity.     

Tat-induced lesions in transgenic mice do not correlate with the HIV-1 LTR transactivation

A pilot study was conducted in 7 normal volunteers to demonstrate the feasibility of employing pharmacokinetic tailoring to achieve matching plasma opioid concentration-time curves after epidural (e.p.) and intravenous (i.v.) alfentanil administration. Each subject participated in 1 pretest and 2 test sessions. Our pain model was cutaneous electrical stimulation of the finger and toe, adjusted to produce a baseline pain report of 5 (strong pain on a 0-5 scale). On test day 1, subjects received e.p. alfentanil (750 micrograms) and an i.v. saline infusion. Serial measurements of analgesia, end tidal CO2, pupil size, subjective side effects, and plasma alfentanil concentrations were conducted before and at various time intervals over a 4-h period after alfentanil administration. On test day 2, subjects received e.p. saline and a pharmacokinetically tailored i.v. infusion (using individual pharmacokinetics determined on the pretest day) designed to achieve a plasma concentration-time profile identical to that observed on the epidural day. The same battery of effect measurements was administered as on the 1st test day. Plasma alfentanil was measured to verify the accuracy of the tailored infusion. Plasma alfentanil concentration profiles were nearly identical on both test days. Peak plasma alfentanil concentrations were near the reported minimum effective analgesic concentration (MEAC). Overall, analgesia was slightly greater with e.p. administration. Onset of pain relief was rapid, and duration was approximately 1.5 h with e.p. and 1 h with i.v. alfentanil. There were no differences in pupil size, ETCO2, or subjective side effects between e.p. versus i.v. administration. We conclude that systemic redistribution from the epidural space appears to account for most, but not all, of the analgesia.     

The induction of in vivo proliferation of long-lived CD44hi CD8+ T cells after the injection of tumor cells expressing IFN-alpha1 into syngeneic mice

The tumorigenicity of transplantable tumor cells in mice is reduced by transduction with cytokine genes, including IFN-alpha and interleukin (IL) 12. Although T cells are considered important in tumor rejection, the mechanism by which genetically modified tumor cells stimulate the immune system has not been examined. In this study, the in vivo proliferation of T-cell subsets in mice transplanted with cytokine-producing syngeneic tumor cells was assessed by administering the DNA precursor bromodeoxyuridine. The injection of viable cells producing IFN-alpha or IL-12 caused a marked proliferation of CD8+ T lymphocytes in both the spleen and lymph nodes. Proliferation was most prominent among memory-phenotype CD44hi CD8+ T cells. In contrast, proliferation of CD8+ T cells did not occur in mice injected with control cells or with cells expressing IL-4, granulocyte colony-stimulating factor, or IFN-gamma. Pulse-chase studies in mice injected with IFN-alpha-producing cells showed that a proportion of proliferating CD8+ T cells survived for at least 70 days, suggesting that long-lived memory cells are induced using such an approach. In summary, these results, together with previous studies on the host immune reactivity triggered by the injection of tumor cells expressing IFN-alpha, represent a strong rationale for considering IFN-alpha as a powerful T-cell adjuvant for the generation of more effective cancer vaccines.