Examination of lymphokines induced in mice following immunization with recombinant simian virus 40 large tumor antigen

Baculovirus-derived recombinant simian virus 40 (SV40) large tumor antigen (T-Ag) was used to immunize BALB/c mice to examine the lymphokines produced following immunization. Specifically, we examined production of interleukin-2 (IL-2), IL-4, IL-5 and interferon (IFN) from immune lymphocytes cultured with decreasing concentrations of recombinant SV40 T-Ag. We identified elevated levels of IFN and IL-2 by enzyme-linked immunosorbent assay and a murine CTLL-2 proliferation biossay respectively. We were unable to detect either IL-4 or IL-5. These data indicate the previously reported tumor immunity induced by recombinant SV40 T-Ag immunization most likely reflects a TH1-like immune response based on thein vitro production of both IFN and IL-2 by immune lymphocytes.     

Treating tumor-bearing mice with vitamin D3 diminishes tumor-induced myelopoiesis and associated immunosuppression,and reduces tumor metastasis and recurrence

Metastatic Lewis lung carcinoma (LLC-LN7) tumors that secrete granulocyte/macrophage-colonystimulating factor (GM-CSF) stimulate myelopoiesis and induce bone marrow-derived immunosuppressor cells that are homologous to granulocyte/macrophage progenitor cells. In vitro treatment of the LLC-LN7 cells with 1,25-dihydroxyvitamin D₃ reduced tumor cell production of suppressor-inducing activity, although suppressor-inducing activity could be restored by reconstituting the tumor supernatants with recombinant GM-CSF. Treatment of mice having LLC-LN7 tumors with vitamin D₃ reduced tumor production of GM-CSF and the frequency of myeloid progenitor cells. This was associated with a reduction in immunosuppressor activity and an increase in T cell function. Vitamin D₃ treatment of mice having palpable tumors transiently retarded tumor growth, but caused a prominent reduction in tumor metastasis. Treating mice with vitamin D₃ after tumor excision resulted in a reduction in the tumor-induced myelopoietic stimulation and associated immunosuppressive activity, and enhanced T cell function. These mice had a markedly reduced incidence of tumor recurrence. The results of this study suggest that vitamin D₃ treatment of mice with GM-CSF-secreting tumors can interrupt the myelopoiesis-associated immunosuppressor cascade and, in turn, reduce tumor metastasis and recurrence.This study was supported in part by grants from the Medical Research Service of the Department of Veterans Affairs and by grants CA-45080 and CA-48080 from the National Institutes of Health     

Potentiation of antitumor effects of tumor necrosis factor α and interferon γ by macrophage-colony-stimulating factor in a MmB16 melanoma model in mice

The efficacy of systemic infusion of recombinant human macrophage-colony-stimulating factor (M-CSF) in combination with local treatment with human recombinant tumor necrosis factor (TNF) and mouse recombinant interferon (IFN) was studied in vivo on a subclone of B16 melanoma (MmB16) in mice. Short-term intravenous administration of M-CSF at a dose of 10⁶ units daily had no antitumor effect in vivo. Similarly, local treatment of tumor with TNF (5 g daily) did not produce any therapeutic effect. However, simultaneous administration of the same dose of TNF with IFN (1000 units daily) resulted in a synergistic effects manifested by the retardation of tumor growth. Addition of systemic infusion of M-CSF to the local therapy with TNF and IFN induced further augmentation of antitumor efficacy and delayed progression of MmB16 melanoma. The strengthened antitumor effect of combination therapy including M-CSF, TNF and IFN was most probably due to the increased release of monocytes from the bone marrow, their recruitment into the site of tumor growth and subsequent local stimulation of their antitumor activity.     

Enhanced casein kinase II activity in human tumour cell cultures

Casein kinase II (CKII) activity is enhanced as much as 2–3 fold in established and 4–5-fold in transformed human cell lines when compared to that of fibroblasts and primary human tumour cell cultures where CKII activity never exceeded a basic level. The high activity of CKII in transformed cells and in established cell lines was reduced to about the same basic level after treatment with heparin, a highly specific inhibitor of CKII activity. The activity of the cAMP-dependent protein kinase was virtually the same in fibroblasts and various human tumour cell lines investigated.     

Biological activities of chemically synthesized analogues of the nonreducing sugar moiety of lipid A

Adriamycin-Fe3+ complex catalyzes the formation of hydroxyl radical from hydrogen peroxide but the DNA-adriamycin-iron ternary complex is much more effective. 11-Deoxyadriamycin, which shows no spectral evidence of complex formation with iron, was ineffective. The generation of hydroxyl radical by adriamycin-Fe3+ complex in the presence of DNA correlates with its ability to cleave DNA. Hydroxyl radicals are thus implicated as the reactive oxygen species involved in the DNA damage caused by the adriamycin-Fe3+ complex.     

Pathogenicity of Macroposthonia xenoplax to Walnut

Preplanting treatment of soil naturally infested with Macroposthonia xenoplax with 1,2-dibromoethane (ethylene dibromide) significantly increased the growth rate of Juglans hindsii seedlings. When seedlings of J. hindsii, J. regia CV "Serf" and J. regia CV Eureka were inoculated with M. xenoplax, their growth was signilicantly less than that of nematode-free controls. This retarded growth rate was accompanied hy feeder root necrosis, longitudinal cracks in the older roots, and distinct lesions in the secondary phloem.     

A mechanism and an evaluation of surface specific iodination by the chloramine-T procedure.

Both internal and external proteins in vesicular stomatitis virus were labeled when intact virions were iodinated with 50 μm iodide; however, only the surface proteins were labeled when the same procedure was carried out at low iodide concentrations (below 0.5 μm). This result together with similar observations reported earlier with another enveloped virus, Rous-associated virus-61 (RAV-61), suggest that viral envelopes provide a barrier to iodination by chloramine-T at low, but not at high, iodide concentrations. By monitoring the permeability of the RAV-61 envelope to successive iodinations and to iodination in the presence of chaotropic thiocyanate ions, it was shown that the permeability of the viral envelope was not altered at the higher concentrations of iodide. Further results support the hypothesis that iodination mediated by chloramine-T inolves two different iodinating species: (a) a membrane impermeable one, possibly “iodamine-T,” which predominates at low iodide concentrations, and (b) a membrane permeable species, possibly molecular iodine, which predominates at high concentrations of iodide. These results reinforce the proposal that the chloramine-T procedure is a useful method for specifically labeling surface proteins of lipid-enveloped structures.     

Lipopolysaccharide induction of outward potassium current expression in human monocyte-derived macrophages: Lack of correlation with secretion

Summary Although an outwardly rectifying K⁺ conductance (I _K, A) is prominently expressed in human alveolar macrophages, the expression of this conductance in human monocyte-derived macrophages (HMDMs) is rare. We have analyzed the induction of the expression of I _K, A in voltage-clamped, in vitro differentiated HMDMs by a number of stimuli which produce either priming or activation of macrophages. Cultures were stimulated with lipopolysaccharide (LPS, 2 g/ml), interleukin 2 (IL-2, 100 U/ml), or combinations of LPS and either recombinant interferon-gamma (-IFN, 10 U/ml), phorbol myristate acetate (PMA, 0.01 or 1 g/ ml) and platelet activating factor (PAF, 20 ng/ml) for periods of up to 24 hr. Treatment of the cells with either LPS or IL-2 greatly enhanced the frequency of current expression. Treatment with either PMA or -IFN alone did not induce current expression; treatment of the cells with a combination of LPS and either PMA, -IFN, or PAF did not enhance current expression over that observed with LPS alone. The expression of the outwardly rectifying K⁺ current was observed in 36% (n=321) of the cells for cultures treated with LPS and 33% (n=55) of the cells for cultures treated with IL-2. The inactivating outward K⁺ current was absent in cells which were not treated with either LPS or IL-2. The kinetics of current activation and inactivation appeared identical to that previously described for the transient-inactivating outward current of the human alveolar macrophage. Cycloheximide (1 g/ml), an inhibitor of protein synthesis, completely suppressed LPS-induced current expression. No correlation was found between peak current amplitude and cell size in LPS-activated cells expressing the outwardly rectifying K⁺ current, indicating that current density was not held constant from cell to cell. The coupling of ion channel expression and secretion in individual HMDMs was studied using the reverse hemolytic plaque assay. Although an enhancement of K ⁺ current expression was observed following either LPS or IL-2 treatment, a quantitatively similar and uniform increase in the percentage of either IL-1 or lysozyme-secreting cells was not observed. The frequency of current expression in cells identified as secreting tumor necrosis factor- (TNF-), interleukin 1 (IL-1), or lysozyme was the same or decreased over that observed for nonsecreting cells. Thus, LPS treatment increases the number of K⁺ channels on HMDM membranes; however, K⁺ channel expression alone was not sufficient to give rise to enhanced secretion in LPS-activated macrophages. Enhanced K⁺ channel expression appears to be a part of the primary activation signal. K⁺-channel activation would hyperpolarize the membrane potential, potentially providing the driving force for calcium entry through voltage-independent pathways activated by the subsequent binding of soluble substances to membrane surface receptors, the secondary signal linked to secretion.This work was supported by NIH grant RO 1 GM36823.     

Rubus host range of rubus yellow net virus and its involvement with other aphid-borne latent viruses in inducing raspberry veinbanding mosaic disease

After graft inoculation with rubus yellow net virus (RYNV), 12 of 34 Rubus species and cultivars developed noticeable symptoms. R. macraei developed the most conspicuous symptoms and is recommended as an improved indicator plant. In attempts to determine the cause of raspberry veinbanding mosaic, a disease in which RYNV is involved, several European and North American red raspberry cvs were graft-inoculated with RYNV and three other aphid-borne viruses, black raspberry necrosis (BRNV), raspberry leaf mottle (RLMV) and raspberry leaf spot, singly and in all combinations. In periods of up to 4 yr, classical veinbanding mosaic symptoms developed in sensitive cvs only when they contained both RYNV and RLMV. These symptoms were intensified in plants co-infected with additional viruses. Veinbanding mosaic disease did not develop in any of 11 cvs infected with RYNV + BRNV, the combination of viruses previously assumed to be responsible for this disease in Britain and North America.     

Studies on the role of protein kinases in the TNF-mediated enhancement of murine tumor cell-endothelial cell interactions.

We have previously demonstrated that the exposure of mouse microvascular endothelium (MME) to tumor necrosis factor-alpha (TNF) led to the increased binding of mouse mastocytoma cells (P815) to endothelial monolayers (Bereta et al., in press). In the current study we examined the possible involvement of protein kinases in TNF signal transduction in the endothelial cells. PKA does not appear to play a role in the potentiation of binding by TNF. We found that the TNF-generated signal is inhibited by H-7 and sangivamycin, but not by staurosporine. TNF did not cause translocation of PKC to the cell membrane and its effect could not be completely mimicked by PMA nor by PMA in the presence of calcium-raising agents. Thus, we concluded that the "classical" PKC pathway is not completely responsible for TNF signalling in this system. We also found that staurosporine itself strongly enhanced adhesion of tumor cells to endothelium, utilizing a mechanism distinct from that of TNF. Although the data provide evidence for the role of kinases in the effect of TNF on binding of tumor cells to MME, this role appears to be a complex one.