Clinical studies on cell-mediated immunity in patients with renal cell carcinoma: interleukin-2 and interferon-γ production of lymphocytes

Summary The authors examined interleukin-2 (IL-2) production and interferon (IFN) production of peripheral blood mononuclear cells in 28 patients with renal cell carcinoma and 17 control subjects. The peripheral blood was obtained prior to the initiation of therapeutic procedures. The patients were divided into two groups according to tumor size, 5 cm and >5 cm. The production of IL-2 and IFN was measured by immunoradiometric assay. As a result, in the patients with tumors >5 cm, IL-2 and IFN production was impaired. However, in the patients with tumors 5 cm, IFN production was enhanced, though IL-2 production was not significantly different from that of the control subjects. There was no significant correlation between IL-2 production and IFN production.     

Agrobacterium-mediated transformation of tomatillo (Physalis ixocarpa) and tissue specific and developmental expression of the CaMV 35S promoter in transgenic tomatillo plants

A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 transgenic plants could be obtained in experiments using 60 cotyledon expiants. The transformed nature of the regenerated plants was confirmed by NPT II and Southern blot hybridization analysis. Using the b-glucuronidase system the tissue specific and developmental patterns of expression of the Cauliflower Mosaic Virus 35S promoter were determined in transgenic tomatillo plants. It was found that this promoter is developmentally regulated during fruit and seed formation.     

35S-β-glucuronidase gene blocks biological effects of cotransferred iaa genes

The iaaM and iaaH genes of Agrobacterium tumefaciens and Agrobacterium rhizogenes play an important role in crown gall and hairy root disease. The iaaM gene codes for tryptophan monooxygenase which converts tryptophan into indole-3-acetamide (IAM). IAM is converted into the auxin indole-3-acetic acid (IAA) by indoleacetamide hydrolase, encoded by the iaaH gene. In functional studies on the activity of the iaa genes of the TB region of the A. tumefaciens biotype III strain Tm4, the frequently used 35S--glucuronidase (35S-UidA or GUS) marker gene was found to inhibit IAA synthesis and root induction encoded by the TB iaa genes. To exert this inhibition, the 35S-UidA gene must be cotransferred with the iaaH gene. The 35S promoter alone is sufficient to cause the inhibitory effect.     

Interleukin-1 inhibits PGE2 binding to macrophage-like P388D1 cells by a cyclic AMP-independent process

The interaction between interleukin IL-1α and PGE₂ on P388D₂ on cells has been investigated. Preincubation of murine macrophage-like cells, P388D₁, with IL-1α (0–73 pM) reduced the binding of PGE₂ to these cells in a concentration-dependent manner. Scatchard analysis showed that IL-1α decreased the PGE₂ binding by lowering both the high and low affinity receptor binding capacities (from 0.31 ± 0.02 to 0.12 ± 0.01 fmol/10⁶ cells for the high affinity receptor binding sites and from 2.41 ± 0.12 to 1.51 ± 0.21 fmol/10⁶ cells for the low affinity receptor binding sites). However, the dissociation constants of the receptor of the IL-1α-treated cells remained unchanged. Inhibition of PGE₂ binding IL-1α did not involve changes in either protein phosphorylation or intracellular cyclic AMP levels. Our data clearly show that IL-1α inhibits the binding of PGE₂ to monocytes/macrophages and may thereby counter the immunosuppressive actions of PGE₂.     

Immunopathological features of human pulmonary tumors following low-dose interleukin-2

Summary We administered preoperative low-dose interleukin-2 (IL-2) to 10 patients undergoing thoracotomy for pulmonary tumors. The in vivo effect of IL-2 on tumor-associated lymphocyte activity was assessed in the resected specimens by immunohistochemistry and compared with observations in 45 patients who did not receive IL-2. H & E evaluation revealed an increase in intra- and peritumoral lymphocyte infiltration in the IL-2-treated patients. Immunopathological evaluation with monoclonal antibodies revealed that this lymphocyte infiltration was predominantly CD5-positive T cells. The amount of intra-and peritumoral lymphocyte activity correlated with the dose of IL-2 administered (6000–90 000 international units/kg every 8 h for 48 h. IL-2-treated patients showed increases in T-cell-associated activation markers (IL-2 -receptor, transferrin receptor and HLA-DR) on peritumoral lymphocytes, but not on intratumoral lymphocytes. We previously reported that low-dose IL-2 increases the intrinsic natural killer cell cytotoxicity of intratumoral lymphocytes and suggest that this lymphocyte infiltration is further evidence that low-dose IL-2 can augment in vivo lymphocyte activity at the tumor site.This work was supported in part by USPHS grants CA 44 352 (S. H. G.) and 43 658 (A. J. C.). S. G. S. was supported by NIH Surgical Oncology Training Grant CA 09 010     

Decrease in the Function of the γ-Aminobutyric Acid-Coupled Chloride Channel Produced by the Repeated Administration of Pentylenetetrazol to Rats

The acute administration of pentylenetetrazol (PTZ; 25-75 mg/kg i.p.) failed to modify the specific binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to membrane preparations from the cerebral cortex of the rat. In contrast, the repeated administration of PTZ (30 mg/kg i.p., three times a week for 12 weeks) reduced by 26% the density of [35S]TBPS binding sites without modifying the dissociation constant. This effect was observed 3 days after the last PTZ administration. A parallel reduction of gamma-aminobutyric acid (GABA)-stimulated 36Cl- uptake was measured in the cerebral cortex of PTZ-treated rats 3 days after the last injection. The repeated administration of PTZ produced sensitization to the drug, or chemical kindling. In fact, no convulsions were observed in the first week of treatment, but all the animals became sensitized to PTZ by the 12th week. The results are consistent with the hypothesis that chronic treatment with PTZ at a subconvulsant dose causes a decrease in GABA-coupled chloride channel activity that may be related to the chemical kindling produced by this compound.     

Interleukin-1 Hyperproduction by In Vitro Activated Peripheral Macrophages from Cerebellar Mutant Mice

Several mutations in mice produce complex patterns of neuronal degeneration of the cerebellum and of its afferent pathways. In the staggerer (sg/sg) mutant, atrophy of the lymphoid organs and immunological abnormalities have been described. To search for a possible link between the neurological and the immune disorders in this mutant, we studied the production by its peripheral macrophages of interleukin-1 (IL-1), which roles in both immune and nervous systems are well established. Suspensions of peritoneal and/or spleen macrophages from mutants and their appropriate controls were stimulated in vitro by lipopolysaccharide. Northern and dot blots, performed with murine IL-1 cDNA probes, revealed a clear-cut hyperexpression of IL-1 mRNA in staggerer macrophages. An IL-1 bioassay using the IL-1-responsive D10.G4 cell line also revealed a sixfold increase of IL-1 activity in the macrophage supernatants of staggerer mutant mice. The hyperproduction was found in 3-week to 1-year-old staggerer and also in heterozygous (+/sg) mice. A similar phenomenon existed in cerebellar mutants lurcher, Purkinje cell degeneration (pcd), and to a lesser extent reeler and wobbler, but was absent in the neurological mutants weaver, jimpy, and motor end plate disease (medH). These observations establish that in several point mutations in mice, central nervous degeneration is associated with dysregulation of IL-1 production by peripheral macrophages.     

Lipopolysaccharide-Enhanced Expression of Interleukin-6 in Dibutyryl Cyclic AMP-Differentiated Rat C6 Glioma

Abstract: Rat C6 glioma synthesizes a low basal level of interleukin-6 (IL-6). Stimulation with 10 µg/ml of lipopolysaccharide (LPS) and induction of differentiation with 1 m M N ⁶, O ^2'-dibutyryl cyclic AMP (dbcAMP) for 48 h increased the secreted activity to 400 and 800 U/ml, respectively. An LPS stimulation of dbcAMP-differentiated cells strongly enhanced the secreted activity. Depending on the dbcAMP concentration, the cell number, and the stimulation time, the secreted IL-6 level increased up to 120,000 U/ml. After 48 h of costimulation with 10 µg/ml of LPS and 1 m M dbcAMP, northern blotting and immunoassay demonstrated an eightfold increase in IL-6 mRNA concentration and IL-6 immunoreactivity, whereas titration of the biological activity indicated a 100-fold increase in the secreted IL-6 activity. The enhanced secretion of IL-6 is correlated with the induction of differentiation. Chromatography on heparin-Sepharose and on DEAE-5PW separated the secreted activity into several fractions, indicating that they differ in heparin affinity and charge either by posttranslational modifications or by binding to a carrier protein. Each of the partially purified IL-6-like activities could be neutralized by an anti-murine IL-6 antibody. Our observations demonstrate that in vivo inflammatory signals can trigger astrocytes and their precursors to secrete substantially different levels of immunoregulatory cytokines depending on their degree of differentiation.