Comparative study of inhibitory effects by murine interferon γ and a new bisphosphonate (alendronate) in hypercalcemic,nude mice bearing human tumor (LJC-1-JCK)

The inhibitory effect of murine interferon (muIFN) on humoral hypercalcemia in nude mice bearing lower-jaw cancer (LJC-1-JCK), in which parathyroid-hormone(PTH)-related protein is responsible for causing humoral hypercalcemia by activating bone resorption, was examined in comparison with that of a new bisphosphonate, 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate), muIFN was injected into tumor-bearing nude mice for 5 days before the establishment of hypercalcemia. The increase of plasma calcium concentration was delayed and this effect continued for more than 6 days even after the injection was stopped. Alendronate markedly suppressed hypercalcemia in tumor-bearing nude mice but this inhibitory effect continued for less than 6 days. Neither muIFN nor alendronate affected the tumor volume or serum PTH-related protein concentration. Injection of muIFN into mice for 3 days almost completely abolished the formation of multinucleated osteoclast-like cells from bone marrow cells in vitro, whereas injection of alendronate into mice had no effect. These findings suggested that muIFN suppressed the formation of osteoclasts, resulting in the prolonged decrease of plasma calcium concentration in hypercalcemic tumor-bearing nude mice, whereas alendronate is cytotoxic to functionally mature osteoclasts and inhibited osteoclastic bone resorption, resulting in a marked decrease in the plasma calcium concentration in tumor-bearing hypercalcemic nude mice.     

Increasing infiltration and activation of CD8+ tumor-infiltrating lymphocytes after eliminating immune suppressive granulocyte/macrophage progenitor cells with low doses of interferon γ plus tumor necrosis factor α

By secreting granulocyte/macrophage colonystimulating factor (GM-CSF), metastatic Lewis lung carcinoma (LLC-LN7) tumors induce the appearance of myelopoiesis-associated immune-suppressor cells that resemble granulocytic-macrophage (GM) progenitor cells. The presence of these GM-suppressor cells in mice bearing LLC-LN7 tumors was associated with a reduced capacity of splenic T cells to proliferate in response to interleukin-2 (IL-2). Administration of low doses of 100 U interferon (IFN) plus 10 U tumor necrosis factor (TNF) to the tumor bearers, a combination treatment that we previously showed to diminish the presence of GM-suppressor cells synergistically, restored proliferative responsiveness of the splenic T cells to IL-2. These LLC-LN7-bearing mice were also examined for whether cells that phenotypically resemble GM-progenitor cells (ER-MP12⁺ cells) infiltrate the tumor mass. ER-MP12⁺ cells composed approximately 10% of the cells isolated from dissociated tumors of mice that had been treated with placebo or with either IFN or TNF alone, but IFN/TNF therapy markedly reduced the number of tumor-infiltrating ER-MP12⁺ suppressor cells. The IFN/TNF treatment to eliminate GM-suppressor cells and restore T cell responsiveness to IL-2 was next coupled with low dose IL-2 therapy (100 U twice daily). Addition of IL-2 to the treatment regimen did not significantly influence the effectiveness of the IFN/TNF treatment in eliminating GM-suppressor cells from the LLC-LN7 tumor mass. However, inclusion of IL-2 with the IFN/TNF treatment regimen enhanced the CD8⁺, but not the CD4⁺, cell content within the tumor, and diminished the number of metastatic lung nodules within the mice. When these tumors were excised, dissociated, and bulk-cultured with a low dose of IL-2, an increased level of cytotoxic T lymphocyte (CTL) activity was generated in the TIL cultures from mice that had received IFN/TNF plus IL-2 treatments. A lesser but detectable level of CTL activity was generated in TIL cultures from mice that were treated with only IFN/TNF, while no CTL activity was generated in tumor cultures from mice receiving only placebo or low-dose IL-2. These results suggest the effectiveness of IFN plus TNF therapy in restoring IL-2 responsiveness in mice bearing GM-suppressor cell-inducing tumors and at enhancing both the intratumoral CD8⁺ cell content and the generation of CTL activity in bulk cultures of these tumors.This study was supported by the Medical Research Service of the Department of Veterans Affairs, by grants CA-45080 and CA-48080 from the National Institutes of Health, and by the American Cancer Society, Illinois     

Effect of interferon γ and tumour necrosis factor α on sensitivity to cisplatin in ovarian carcinoma cell lines

This study aimed to investigate whether the biological response modifiers (BRM) interferon (IFN) and tumour necrosis factor (TNF) could enhance the cytotoxic action of cisplatin on ovarian tumour cells in vitro. The sensitivity of four cell lines (OAW42, GG, JAM and PE01) to drugs and drug combinations was tested by a radiolabelled-thymidine incorporation assay. Cell lines demonstrated a range of sensitivity to cisplatin and the innate cytotoxic effect of each of the BRM. When IFN was used in combination with cisplatin, a significant enhancement of cisplatin toxicity occurred in three of four cell lines. TNF demonstrated such an effect in two cell lines but diminished the toxicity of cisplatin in one cell line. A purely additive effect of the agents may explain the enhanced toxicity of cisplatin in some of these cases. However, in one cell line at least (PEO1), both TNF and IFN demonstrated a clearly synergistic effect with cisplatin. These BRM used in conjunction with cisplatin may provide better antitumour regimen than cisplatin alone in some patients with ovarian cancer, but the response is likely to be heterogeneous between patients.     

Low-dose melphalan-induced shift in the production of a Th2-type cytokine to a Th1-type cytokine in mice bearing a large MOPC-315 tumor

The current studies demonstrate that MOPC-315 tumor cells secrete large amounts of interleukin-10 (IL-10), which contributes to the inhibitory activity of MOPC-315 culture supernatants for the in vitro generation of antitumor cytotoxicity by MOPC-315-immune spleen cells. Moreover, addition of neutralizing monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of cells from the tumor infiltrated spleens of mice bearing a large MOPC-315 tumor resulted in the generation of enhanced anti-MOPC-315 cytotoxicity. In contrast, addition of monoclonal anti-IL-10 antibody to the in vitro stimulation cultures of splenic cells from mice that are in the final stages of immune-mediated tumor eradication as a consequence of low-dose melphalan (l-phenylalanine mustard; L-PAM) therapy (and whose spleens no longer contain metastatic tumor cells) did not lead to enhancement in the in vitro generation of antitumor cytotoxicity. The cessation of IL-10 secretion as a consequence of low-dose L-PAM therapy of MOPC-315 tumor bearers was found to be accompanied by the acquisition of the ability to secrete interferon (IFN) by the splenic cells. In addition, by day 2 after low-dose L-PAM therapy a drastic decrease in the amount of IL-10 secreted by the s.c. tumor nodules was noted, which preceded the accumulation of tumor-infiltrating lymphocytes capable of secreting IFN. Thus, low-dose L-PAM therapy of mice bearing a large MOPC-315 tumor leads to a shift in cytokine production from a Th2-type cytokine to a Th1-type cytokine, and it is conceivable that this shift in cytokine production plays an important role in the low-dose L-PAM-induced acquisition of antitumor immunity by hitherto immunosuppressed mice bearing a large MOPC-315 tumor.Supported by research grant IM-435 from the American Cancer Society and CA54413 from the National Cancer InstituteIn partial fulfillment of the requirements for the Doctor of Philosophy DegreeRecipient of career development award CA-01350 from the National Cancer Institute     

γ-Glutamyl conjugation of 5-hydroxytryptamine (serotonin) in the earthworm (Lumbricus terrestris)

The conversion of 5-hydroxytryptamine to several potential metabolites was examined in the annelid earthworm (Lumbricus terrestris). 5-hydroxytryptamine and some related amines were found to be present in several tissues of the earthworm. Injection of 5-hydroxytryptamine into the body cavity of the earthworm resulted in the production of a -glutamyl conjugate of 5-hydroxytryptamine. Incubations of the anterior nerve cord of the earthworm resulted in the accumlation of considerable amounts of 5-hydroxytryptamine and -glutamyl 5-hydroxytryptamine in the incubation medium. The earthworm did not produce any N-acetyl 5-hydroxytryptamine and only very little 5-hydroxyindoleacetic acid. Experiments involving the injection of radiolabeled 5-hydroxytryptamine or coninjection of radiolabeled glutamic acid with unlabeled 5-hydroxytryptamine into the earthworm resulted in the production of radiolabeled -glutamyl 5-hydroxytryptamine. This work demonstrates that the enzymatic conversion of 5-HT in the earthworm is markedly different from that of vertebrates and insects.     

Production of a membrane-bound proteins,the human gamma-glutamyl transferase,by CHO cells cultivated on microcarriers,in aggregates and in suspension

Recombinant Chinese Hamster Ovary (CHO) cells, engineered for the production of human gamma-glutamyl transferase (GGT), have been grown on Cytodex 1 microcarriers, as aggregates, or as single cells in suspension after adaptation. GGT is a membrane bound enzyme which was not secreted during the culture period. The maximal enzyme activity was found to be directly related to the achieved maximal cell density. Culture of CHO on microcarriers yielded the fastest growth, with a specific growth rate of 0.04 h^–1, the highest cell density (near 1.3×10⁶ cells ml^–1), and the highest enzyme activity around 300 mU ml^–1, which corresponded to a specific cellular level of 20 mU 10^–5 cells. GGT could also be produced by growing CHO cells in suspension as single cells or as aggregates. Under these conditions, however, the specific CHO growth rate was significantly slower and the GGT level per cell was divided by a factor 6. Growing CHO cells without microcarriers also resulted in differences in cell metabolism, with a higher conversion yield of glutamine into ammonia, and a higher cell lysis. The catalytic kinetic constants of the enzyme were found identical for the three culture systems.     

Reduction in omega-3 fatty acids by UV-B irradiation in microalgae

UV-B irradiation reduced the levels of omega-3 fatty acid, eicosapentaenoic acid (EPA, 20:53) and docosahexaenoic acid (DHA, 22:63), in microalgae; the degree of reduction varied with species.Chaetoceros calcitrans andSkeletonema costatum were high UV-B tolerant species, followed byPhaeodactylum tricornutum, Chroomonas salina, Pavlova lutheri, andThalassiosira pseudonana.Isochrysis galbana (T.ISO) andProrocentrum micans were UV- B sensitive. Cells in logarithmic phase were most sensitive to UV- B irradiation. Nitrate-, phosphate-, or sulphate-starved cells were more UV-B sensitive than non-starved cells grown in a complete basal medium. A relatively short exposure to high UV-B was more damaging than a longer exposure to lower irradiance. Visible light intensity levels had a profound impact on the sensitivity of microalgal cultures to UV-B, with high levels decreasing UV-B dependent damage. Addition of polyamines (putrescine, spermidine or spermine) or an amino acid (cysteine) to the culture medium minimized the reduction of omega-3 fatty acid content in microalgae caused by UV-B irradiation.Author for correspondence     

Production of asymmetric hybrids between Solanum tuberosum and irradiated S. brevidens

Summary Asymmetric somatic hybrids were obtained by fusion of Solanum tuberosum (PDH40) protoplasts with 300- or 500-Gy irradiated protoplasts of S. brevidens. These radiation doses were sufficient to prevent the growth of the S. brevidens protoplasts. Putative hybrids were selected on the basis of phenotype from regenerated shoots and identified with a S. brevidens-specific probe. From these, 31 asymmetric hybrids were confirmed by morphological characteristics, isoenzyme patterns and RFLP analysis. The morphology of the asymmetric hybrids was intermediate between that of S. tuberosum and symmetric hybrids of both species (obtained without irradiation treatment). Chromosome counts from 17 asymmetric hybrids showed that the chromosome number of the hybrids ranged from 31 to 64. The asymmetric hybrids probably had one or two genome complements (i.e. either 24 or 48 chromosomes) from S. tuberosum and 7–22 chromosomes from S. brevidens. There was no clear correlation between the radiation dose and the degree of elimination of the S. brevidens genome.     

激光辐照对绵羊精子代谢和体外获能的影响

本文采用He—Ne激光处理绵羊精液,通过测试精于超弱化学发光强度、TST染色,以了解精子代谢的变化,观察精子顶体反应的发生情况,进一步从受精学角度了解激光的辐照效应。