Lymphokine production by human melanoma tumor-infiltrating lymphocytes

Summary Lymphokine production by human melanoma tumor-infiltrating lymphocytes (TIL) was studied. Uncultured TIL produced interferon (IFN), but not interleukin-2 (IL-2) or IL-4, in response to anti-CD3 mAb or IL-2. In bulk cultures, IL-2-activated TIL displaying autologous tumor-specific cytotoxicity (CTL-TIL) produced IFN in culture with medium alone, whereas IL-2-activated noncytotoxic TIL did not. Addition of anti-CD3 mAb or autologous tumor cells up-regulated IFN production in IL-2-activated TIL from 10 of 12 or 6 of 12 cases respectively. Those from 4 of 12 cases (2 CTL-TIL and 2 noncytotoxic TIL) produced IL-2 in culture with medium alone. At the clonal level, 5 (4 CD4⁺ and 1 CD8⁺) of 7 autologous tumor-specific CTL clones derived from TIL and 3 (2 CD4⁺ and 1 CD8⁺) of 7 noncytotoxic TIL clones produced IFN in culture with medium alone, which was up-regulated by adding anti-CD3 mAb. Two IFN-producing CTL clones tested produced IL-2 in 4 ×-concentrated supernatants from a 3.5-h culture with medium alone. Furthermore, 2 IFN-producing CTL clones tested expressed mRNA for both IFN and IL-2. IL-2 production and its mRNA expression were up- or down-regulated, respectively, by adding anti-CD3 mAb or autologous tumor cells. IL-4 production was not observed in culture either with medium alone or with IL-2 in any of the cells described above. Anti-CD3 mAb was required for IL-4 production in 3 of 12 IL-2-activated TIL, 2 of 6 CTL clones, and none of 5 noncytotoxic TIL clones. In summary, IFN production was characteristic of melanoma TIL. Some autologous tumor-specific CTL in TIL are suggested to be productive of IL-2 and IFN under unstimulated conditions, both being required for self-activation in an autocrine loop.This work was supported in part by grant CA-47891 from the National Cancer Institute     

Fractionation of Nitrogen Isotopes during the Uptake and Assimilation of Ammonia by Plants

Two varieties (Nihonbare and Koshihikari) of rice plants (Oryzasativa L.) were grown hydro-ponically with two levels (20 and100 mg N liter ^–1) of ammonia. Variations in levels ofnatural abundance of ¹⁵N (¹⁵N) were analyzed in the ammoniaand organic nitrogen of shoots and roots, as well as in theammonia in the culture solution. There was substantial fractionationof nitrogen isotopes during the uptake of ammonia. When plantsabsorbed a large proportion of ammonia from a solution witha low concentration, less negative ¹⁵N values in plants andhigh positive ¹⁵N values in the ammonia remaining in solutionwere observed. The reverse was found when a smaller fractionof ammonia was absorbed from a solution with a higher concentrationof ammonia. The ^l5N values of ammonia in shoots and roots werehigher than in the respective constituent organic nitrogen,suggesting the fractionation of nitrogen isotopes during theassimilation of ammonia. Wild-type and mutant cells of the cyanobacterium(blue-green alga) Synechococcus PCC 7942 were grown in nitrate-or ammonia-containing medium as the source of nitrogen. Fractionationof nitrogen isotopes during the uptake of nitrate was limited,whereas that during the uptake of ammonia was considerable. ¹ In this report, the term ammonia refers indiscriminately toboth NH₃ or NH₄⁺. (Received June 13, 1991; Accepted September 12, 1991)     

Effects of a newly synthesized leukotriene antagonist, NZ-107, on immediate-type hypersensitivity reaction in rats and guinea-pigs.

The effects of 4-bromo-5-(3-ethoxy-4-methoxybenzylamino)-3(2H)-pyridazinone (NZ-107) on immediate type hypersensitivity reactions in rats and guinea-pigs were studied. 1. When NZ-107, at a dose of 50 mg/kg (i.p.) or 100 mg/kg (orally), was administered to rats, 48-h homologous passive cutaneous anaphylaxis (PCA) reaction and histamine-, leukotriene C4 (LTC4)- and leukotriene D4 (LTD4)-induced skin reactions were suppressed by the agent. 2. NZ-107 (10(-6) g/ml) inhibited both LTC4- and LTD4-induced contractions of isolated rat stomach smooth muscle. 3. NZ-107 inhibited antigen-induced histamine release from rat peritoneal mast cells by 26% at a concentration of 10(-4) g/ml. 4. NZ-107, at doses of 25 and 50 mg/kg (orally), significantly inhibited guinea-pig 3-h heterologous PCA reaction. 5. NZ-107 inhibited antigen-induced histamine release from guinea-pig lung tissue by 17% and 48% at concentrations of 5 x 10(-5) and 10(-4) g/ml, respectively. 6. NZ-107, at doses of 25 and 50 mg/kg (i.p.), inhibited antigen-induced bronchoconstriction and eosinophil accumulation in the bronchoalveolar lavage fluid (BALF) of guinea-pigs. These results suggest that NZ-107 has anti-allergic action including inhibition of eosinophil accumulation in an antigen-challenged airway lesion in rats and guinea-pigs. The anti-allergic action of this agent is thought to be due to its action as a histamine and LT antagonist and its consequent inhibition of antigen-induced histamine release.     

Diagnosis of a case of pulmonary carcinosarcoma by detection of rhabdomyosarcoma cells in sputum

Cytologic examination of sputum samples from an elderly patient revealed the presence of two cell populations: squamous cell carcinoma cells and rhabdomyosarcoma cells. The abnormal squamous cells showed both keratinizing and nonkeratinizing forms while some of the rhabdomyosarcoma cells showed cross striations. Sputum cytology was thus able to suggest a diagnosis of pulmonary carcinosarcoma. Histologically, the tumor was composed mainly of sarcomatous tissue showing various kinds of cells: fusiform or fibrous cells, round anaplastic cells, spindled cells with typical cross striations and myoblastic cells. A partially myxomatous degeneration was present. In addition, squamous cell carcinoma proliferated along the bronchi and formed small invasive cell nests in the sarcomatous tissue. No transition between the two components was noted. Both cellular constituents had metastasized to an interlobar lymph node.     

Immunogenic protein MPB57 from Mycobacterium bovis BCG: molecular cloning, nucleotide sequence and expression

Using a single-probe method, we have cloned the gene for an immunogenic MPB57 protein of Mycobacterium bovis BCG. The nucleotide sequence includes an ORF of 300 base pairs encoding a protein of 99 amino acids with an Mr of 10,818. This cloned gene was expressed in an Escherichia coli expression vector to give a mature protein which reacted with a polyclonal antibody raised against MPB57.     

Localization of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae

The subcellular distribution of the regulatory subunit of cAMP-dependent protein kinase in Saccharomyces cerevisiae cells was determined by subcellular fractionation and indirect immunofluorescence microscopy using the bcy1 mutant deficient in the regulatory subunit as control. The regulatory subunit of cAMP-dependent protein kinase showing cAMP-binding activity was identified as a single protein of 50 kDa by photoaffinity labeling and immunoblotting. The regulatory subunit was concentrated in a nuclear fraction in addition to a cytoplasmic fraction. By comparison of the regulatory subunit distribution with the DNA localization, the area detected by the indirect immunofluorescence was identified as the nucleus.     

Electrical stimulation of the suprachiasmatic nucleus of the hypothalamus causes hyperglycemia

We have presented evidence suggesting that the suprachiasmatic nucleus (SCN) is involved in central regulation of glucose homeostasis. To elucidate this role of the SCN, we examined the effects of its electrical stimulation on glucose metabolism in male Wistar rats. During and shortly after this stimulation, we observed hyperglycemia associated with enhanced hyperglucagonemia but no immediate hyperinsulinemia. In addition, we detected significant increase in liver glycogen phosphorylase alpha activity and significant decrease in the liver glycogen content. These findings suggest that the SCN is important in control of glucose homeostasis through effects on glucagon and insulin secretions and liver glycogen metabolism.     

1H-NMR heme resonance assignments by selective deuteration in low-spin complexes of ferric hemoglobin A

The heme methyl and vinyl alpha-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole pi bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.     

Monoclonal antibody directed to a Hanganutziu-Deicher active ganglioside, GM2 (NeuGc)

Spleen cells from NZB mouse immunized with a membrane fraction of rabbit thymus tissue were fused with BALB/c 6-thioguanine-resistant myeloma cells, P3-X63-Ag8.653. One hybridoma clone (Y-2-HD-1) produced IgM immunoglobulin that bound to an N-glycolylneuraminic acid-containing GM2 ganglioside, GM2(NeuGc), which is known to be a Hanganutziu-Deicher antigen. The specificity of the Y-2-HD-1 monoclonal antibody was examined, using authentic glycosphingolipids structurally related to GM2(NeuGc), by means of an enzyme-linked immunosorbent assay and thin-layer chromatography/enzyme immunostaining, respectively. The monoclonal antibody was found to be highly specific to GM2(NeuGc) and the epitope was a non-reducing terminal GalNAc beta 1-4[NeuGc alpha 2-3]Gal structure. This monoclonal antibody (Y-2-HD-1) bound to native mouse erythrocytes, in which GM2(NeuGc) is a major ganglioside. These results indicate that GM2(NeuGc) is located on the surface of mouse erythrocytes.