Antitumor activity of L-2,4 diaminobuturic acid against mouse fibrosarcoma cells in vitro and in vivo

Summary Mouse fibrosarcoma cells were grown in vitro and incubated with L-2,4 diaminobuturic acid, a non-metabolizable amino acid. The tumor cells were irreversibly and totally damaged by incubation with 10 mM DAB for 24 h at 37 °C. The cell-destructive effect by DAB was probably due to an osmotic lysis induced by the non-saturated intracellular accumulation of DAB. The harmful effect of DAB could be abolished by concomitant incubation with L-alanine and L-methionine, that compete with DAB for the same transport system, while the D-forms of the same amino acids as well as sarcosine had a weak effect.The fibrosarcoma cells were also transplanted s.c. into mice that were subsequently treated with i.p. injections of an isotonic 0.1 M DAB solution. The neoplastic cells were transplanted into totally 90 animals. The mean tumor weight of 42 treated animals was 1.16 g (±0.77 g) compared with the corresponding figures of the 27 untreated mice, that were 2.05 g (±1.22 g), i.e., a 43.4% reduction of tumor growth. There were, however, 17 drug-related deaths. Treatment with DAB generally resulted in weight reduction, at least partly due to loss of appetite, in the animals. In addition, neurological symptoms of a specific character could develop among several of the treated animals. The side effects apparently restrict the usefulness of DAB alone as an anti-tumor agent, but since the principle of action of DAB is unique and not shared by other known chemotherapeutics it might offer new possibilities in the combined treatment of neoplastic growth.

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Abbreviations AIB -aminoisobuturic acid - DAB L-2,4 diaminobuturic acid - MEM minimum essential medium - ld lactic acid dehydrogenase     

Procoagulant activity in patients with sickle cell trait

Patients with sickle cell trait (STr) are usually considered to be asymptomatic. However, complications, including hypercoagulability, increased risk of venous thromboembolism and the exertional exercise syndrome with rhabdomyolysis and sudden death, have been described. The exact cause of these adverse events is unclear. We have investigated two patients, a set of monozygotic twins with STr, to establish their procoagulant activity status as a potential indicator of thrombotic risk. In-vivo thrombin generation was assessed by the measurement of prothrombin fragment 1 + 2 (F1 + 2) and thrombin-antithrombin complexes (TAT). D-dimer was used as a marker of fibrinolytic activity. The potential to generate thrombin was determined using an ex-vivo thrombin generation test (TGT). The impact of red blood cell (RBC)-derived microparticle shedding and RBC rheology were examined. TAT (>60 μg/l) and F1 + 2 (948 pmol/l) were markedly elevated in patient 2 but within the normal reference range in patient 1 (TAT = 2.5 μg/l; F1 + 2 = 138 pmol/l). D-dimer levels (0.9 mg/l FEU) were similarly elevated in both patients. TGT peak thrombin and endogenous thrombin potential (ETP) were elevated to similar degrees in both patients. Flow cytometric analysis for RBC-derived microparticles showed that both patients had elevated levels on two occasions. RBC deformability, blood viscosity and RBC aggregation were normal and similar in both patients. The results demonstrated different coagulation activity in the patients with one patient in a prothrombotic state, suggesting that there may be two levels of hypercoagulability in STr. Measurement of such differences would allow for separation of high and low-risk patients from serious complications.     

Cd28 expression on t cell subsets in vivo and cd28-mediated t cell response in vitro in patients with rheumatoid arthritis

Objective. In view of the critical importance of the CD28–CD80 (B7/BB1) costimulatory pathway in antigen-specific T cell activation and clonal expansion, we examined CD28 surface molecule expression in vivo, and T cell receptor/CD3-mediated and B7/BB1-costimulated T cell proliferation in vitro, in rheumatoid arthritis (RA). Methods. Two-color immunofluorescence analyses of peripheral blood and synovial fluid–derived T cells, as well as ³H-thymidine incorporation assays, were performed. Results. In the peripheral blood of 31 patients with active, untreated RA, a mean of 91% (range 48–100%) of CD4+ and 46% (range 13–82%) of CD8+ T cell subsets were CD28+, which was not significantly lower than normal. Although an overall decrease in the number of T cells was not observed, the numbers of CD28+CD8+ T cells were significantly lower in RA patients (mean 233/μl, versus 292/μl in controls), and this decrease was more pronounced in patients with severe disease (mean 172/μl). CD28 expression on peripheral CD8+ T cells in RA patients, but not in healthy individuals, correlated inversely with T cell activation as assessed by HLA–DR antigen expression. In contrast to the peripheral blood, RA synovial fluid T cells were almost exclusively CD28+, suggesting that migration of CD28+CD8+ T cells to active sites of inflammation may occur. In vitro proliferative responses of peripheral blood T cells to B7/BB1 costimulation in the presence of mitogenic doses of anti-CD3 monoclonal antibody were identical in patients with RA and healthy individuals. Conclusion. Functionally intact CD28+ T cells may contribute to the abnormal immunoregulation and joint inflammation in RA.     

Procoagulant activity and intimal dysfunction in IDDM

Summary The biological activity of thrombin and coagulation factor Xa was assessed in 62 insulin-dependent diabetic patients. A group of non-diabetic subjects of comparable age and urinary albumin excretion rate (<30 mg/24 h) served as control subjects (group 1,n=14). The patients were divided into three groups according to urinary albumin excretion rate. In group 2, albumin excretion rate was less than 30 mg/24 h (n=17), in group 3 albumin excretion rate was in the range 30–300 mg/24 h (n=20) and in group 4 albumin excretion rate was greater than 300 mg/24 h (n=25). Compared to non-diabetic control subjects an increase in the biological activity of factor Xa was observed in all groups of diabetic patients (prothrombin fragment 1+2 levels were 1.14±0.38 nmol/l in group 2,p<0.005; 1.06±0.45 nmol/l in group 3,p<0.05 and 1.03±0.31 nmol/l in group 4,p<0.05 vs 0.75±0.34 nmol/l in group 1). No difference in the level of antithrombin III was seen between the groups. We reconfirmed the presence of intimal dysfunction in diabetic nephropathy demonstrated by elevated transcapillary escape rate of albumin in group 4 compared with group 2 (8.9±2.0% vs 7.0±1.9%,p<0.05). An overall positive correlation between transcapillary escape rate and prothrombin fragment 1+2 was found (r=0.36,p<0.005). However, in the groups with elevated albumin excretion rate such a correlation was not significant (group 3:r=0.15,p=0.54; group 4:r=0.03,p=0.86) while it was sustained in the groups with albumin excretion rate of less than 300 mg/24 h (group 1:r=0.61,p<0.05; group 2:r=0.64,p<0.05). In conclusion, IDDM patients had elevated biological activity of factor Xa, demonstrated by elevated levels of prothrombin fragment 1+2. This increment could not be explained by a deficiency of antithrombin III. [Diabetologia (1995) 38: 73–78]     

丁酸钠对人胃癌MKN-28细胞增殖的抑制作用及其机制

目的 观察脱乙酰化酶抑制剂丁酸钠对人胃癌MKN-28细胞增殖的抑制作用,并探讨其作用机制.方法 MTT法观察丁酸钠对人胃癌MKN-28细胞的生长抑制作用;流式细胞术检测细胞凋亡及细胞周期;RT-PCR检测丁酸钠作用前后p21WAF1 mRNA的表达.结果 1.0、2.5、5.0 mmol/L丁酸钠作用24、48、72 h均可抑制 MKN-28细胞增殖,其效果具有时间、剂量依赖性(P＜0.05);丁酸钠1.0、2.5、5.0 mmol/L处理72 h后,MKN-28细胞G0/G1期细胞数量显著增加,S期细胞数量显著降低(P＜0.05);细胞凋亡率分别为13.7%±0.8%、20.8%±2.4%、33.6%±2.6%,与对照组2.8%±0.4%相比P均＜0.05;与对照组比较,丁酸钠处理后p21WAF1 转录水平上调.结论 丁酸钠可显著抑制人胃癌MKN-28细胞的生长,其可能的机制是通过上调p21WAF1基因的表达,诱导细胞凋亡,使细胞周期阻滞于G0/G1期.     

Lipoxygenase inhibitors suppress proliferation of G5:113 fibrosarcoma cells in vitro but they have no anticancer activity in vivo

Nordihydroguaiaretic acid (NDGA) and esculetin, both nonspecific inhibitors of lipoxygenases (LOX), were found to suppress expressively the in vitro proliferation of fibrosarcoma cells G5:113 in concentrations ranging from 10 to 50 microM. Subsequent flow-cytometric analysis of the cell cycle showed that both these drugs significantly decreased the percentage proportion of cells in the G0/G1-phase and simultaneously increased significantly this proportion in the S-phase. No apoptosis was detected in the whole range of concentrations studied, from 2.5 to 50 mM. On the contrary, in experiments in vivo, neither NDGA nor esculetin had any curative effect if they were repeatedly injected intraperitoneally (i.p.) into mice bearing tumors growing from subcutaneously (s.c.) transplanted G5:113 cells. Pretreatment of the fibrosarcoma cells with NDGA or esculetin in vitro preceding their s.c. transplantation into mice did not result in suppression of the tumor growth, either. Finally, if G5:113 cells were injected intravenously and the mice were subsequently treated repeatedly with i.p. injections of NDGA, decreased survival and increased number of surface lung metastases were observed in the NDGA-treated group. Thus the suppressive action of inhibitors of LOX on the growth of fibrosarcoma cells in vitro was not reflected in their anti-tumor effects in vivo.     

Procoagulant activity of rabbit alveolar macrophages

Alveolar macrophages are thought to participate in the clearance of fibrin from the injured lung, but their ability to facilitate the conversion of fibrinogen to fibrin (procoagulant activity) has not been described. In order to characterize their procoagulant properties, unstimulated alveolar macrophages obtained from normal rabbits were tested for their ability to accelerate the coagulation of plasma in a one-stage clotting assay. Compared with control assays containing no macrophages (coagulation times greater than 500 s), intact cells (10(6)/ml) were shown to display procoagulant activity (coagulation time, 153.6 +/- 11.3 s mean +/- SEM). Cell lysis caused further procoagulant activity to be expressed (125.6 +/- 11.8 s). Alveolar macrophages that were stimulated in vitro with bacterial lipopolysaccharide (LPS) or the purified complement fragments C5a and C5a des Arg caused further significant (p less than 0.002) reductions in coagulation times (intact cells, 71 to 76 s; lysed cells, 27 to 32 s), representing 5- to 6-fold and 30- to 40-fold increases in the procoagulant activity of intact and lysed cells, respectively. The generation of this material was independent of the presence of lymphocytes. The procoagulant material was identified as a cell-associated tissue thromboplastin, acting via the extrinsic coagulation pathway. These findings show that alveolar macrophages have procoagulant activity that is markedly augmented by LPS and complement fragments. This suggests that alveolar macrophages may contribute to intra-alveolar fibrin deposition in vivo.     

Epithelial mouse mammary cell line exhibiting normal morphogenesis in vivo and functional differentiation in vitro.

An epithelial cell line, designated COMMA-1D, was derived from mammary tissue of BALB/c mice in the middle of pregnancy. This line, in continuous cell culture for 12 months, exhibits several characteristics distinctive of normal mammary epithelial cells, including induction of casein synthesis in vitro and normal duct morphogenesis in the cleared mammary fat pads of syngeneic mice. The cells also form domes in high density culture and are positive for keratin intermediate filaments by indirect immunofluorescence. COMMA-1D cells have a near diploid number of chromosomes and do not grow in suspension culture or produce tumors in syngeneic hosts. This cell line should prove useful for studies examining the regulation of normal cellular differentiation of mammary cells as well as transformation of epithelial cells to the preneoplastic and neoplastic phenotypes.     

Procoagulant activity of activated monocytes.

Endotoxin-stimulated monocytes develop a very high tissue-factor-like activity as compared to an equivalent number of monocytes in a preparation of monocyte-enriched mononuclear blood cells obtained by Ficoll-Urovision density gradient centrifugation which is not exposed to endotoxin. In monocytes which were obtained by plastic adherence from the monocyte-enriched mononuclear blood cells, the tissue factor activity was as high as in the endotoxin-stimulated monocytes.     

Human chemokines: enhancement of specific activity and effects in vitro on normal and leukemic progenitors and a factor-dependent cell line and in vivo in mice

The myelosuppressive effects of human chemokines were evaluated in vitro on normal myeloid progenitors obtained from bone marrow and cord blood, on bone marrow progenitors from patients with acute or chronic leukemia, on proliferation of human factor-dependent cell line M07e, and in vivo on myelopoiesis in mice. Preincubation of human MIP-1, MIP-2, interleukin (IL)-8, platelet factor (PF) 4, monocyte chemotactic and activating factor (MCAF), and interferon-inducible protein-10 (IP-10) in an acetonitrile (ACN) solution significantly enhanced the specific activity of these chemokines for in vitro suppression of granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells stimulated to proliferate with a colony stimulating factor plus steel factor (SLF). Combinations of any two of these ACN-treated chemokines synergized to suppress colony formation of CFU-GM, BFU-E, and CFU-GEMM at chemokine concentrations below that at which combinations of non-ACN treated chemokines are active. Cord blood progenitors, as previously reported, were in a slow or noncycling state and nonresponsive to inhibition by chemokines. However, after suspension culture with GM-CSF, IL-3, and SLF, they were placed into rapid cell cycle and were responsive to inhibition by ACN-treated chemokines. Low doses of these ACN-pretreated chemokines were active in vivo in suppressing absolute numbers and cycling status of femoral marrow CFU-GM, BFU-E, and CFU-GEMM in C3H/HeJ mice. Other chemokines, alone and in combination, including MIP-1, MIP-2, GRO- NAP-2, and RANTES, were inactive in vitro and in vivo whether or not they were pretreated with ACN. While heterogeneity in responsiveness of CFU-GM from different patients with leukemia to suppression by ACN-treated chemokines was apparent, if the patients had CFU-GM responsive to one of the active chemokines these cells were responsive to the other active chemokines; if patient CFU-GM were not responsive to one of the chemokines, they were not responsive to the other active chemokines. M07e colony-forming cells were responsive to the growth-inhibiting effects of the active ACN-treated chemokines, alone and in combination, but these effects were rapidly reversible and sustained only by multiple daily additions of chemokines. These results should be of value in considering these chemokines for potential clinical use and for assessment of their mechanisms of action, alone and in combination.     

重组反义c-myc 腺病毒对人胃癌细胞的体内及体外分子治疗

目的研究重组反义ｃｍｙｃ腺病毒对胃癌细胞的体内外生物学作用．方法采用ＬａｃＺ基因Ｘｇａｌ染色、ＭＴＴ,ＤＮＡ梯度降解试验、原位末端标记、流式细胞仪、ＰＣＲ分析、裸鼠致瘤性、裸鼠皮下移植瘤模型实验等方法,对反义ｃｍｙｃ重组腺病毒在人胃癌ＳＧＣ７９０１细胞系中的作用进行体内外研究．结果ＡｄＡＳｃｍｙｃ对ＳＧＣ７９０１细胞能产生明显的生长抑制作用,ＭＴＴ显示生长抑制率为４４１％．ＤＮＡ梯度降解试验、原位末端标记、流式细胞仪显示ＡｄＡＳｃｍｙｃ诱导了ＳＧＣ７９０１细胞凋亡．经ＡｄＡＳｃｍｙｃ处理的ＳＧＣ７９０１细胞裸鼠致瘤性消失．ＡｄＡＳｃｍｙｃ对裸鼠皮下移植瘤模型瘤内注射能有效降低肿瘤的生长速度,生长抑制率为６８９％．结论ＡｄＡＳｃｍｙｃ对胃癌细胞具有显著的体内外生长抑制及凋亡诱导作用     

Antibacterial activity of Co-trimazine in vitro and in vivo

Summary Co-trimazine is a new drug combination especially designed for the treatment of urinary tract infections. It consists of trimethoprim (90 mg) and sulphadiazine (410 mg). When combined in vitro, the components show high activity and a high frequency of synergy against urinary tract pathogens. After oral absorption sulphadiazine has a serum half-life similar to that of trimethoprim and is excreted in active form into the urine to a much higher degree than sulphamethoxazole. The ratio of the concentrations of trimethoprim and sulphadiazine in the urine following co-trimazine is favourable for a strong synergistic action between the compounds. In cross-over studies in volunteers receiving repeated daily doses of co-trimazine, either 500 mg twice daily or 1000 mg once daily, it was found that the antibacterial activity in the urine was at least as high as that provided by co-trimoxazole (2 × 960 mg) and considerably higher and more uniform than that given by nitrofurantion (3 × 50 mg).

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Die antibakterielle Aktivität in vitro und in vivo von Co-trimazine

Zusammenfassung Co-trimazine ist eine neue Kombination aus Trimethoprim (90 mg) und Sulfadiazin (410 mg), die besonders für die Behandlung von Harnwegsinfektionen entwickelt worden ist. Kombinationen von Trimethoprim und Sulfadiazin zeigen in vitro eine hohe Aktivität und in hohem Ausmaße synergistische Wirkung gegen Erreger der Harnwegsinfektionen. Nach oraler Gabe zeigt Sulfadiazin praktisch dieselbe Halbwertszeit in Serum wie Trimethoprim und wird in viel höherem Ausmaß als Sulfametoxazol in aktiver Form in den Harn ausgeschieden. Das Konzentrationsverhältnis Trimethoprim zu Sulfadiazin, das in Harn von Co-trimazine gegeben wird, begünstigt eine synergistische Wirkung zwischen den beiden Komponenten. In Cross-over-Versuchen bei Freiwilligen mit wiederholter Gabe zeigte Co-trimazine (2 × 500 mg und 1 × 1000 mg) eine antibakterielle Aktivität im Harn, die mindestens so hoch war wie die von Co-trimoxazole (2 × 960 mg) und deutlich höher und gleichmäßiger als die von Nitrofurantoin (3 × 50 mg).

     

神经母细胞瘤体外细胞系的构建方法

目的 对神经母细胞瘤(NB)进行体外培养，探讨神经母细胞瘤(NB)体外建系的方法。方法 NB患儿新鲜手术标本，采用组织块培养法、酶消化培养法建立原代细胞系；用选择性酶消化法和机械刮除法进行细胞纯化；从细胞形态学、神经特异性烯醇化酶染色、软琼脂克隆形成实验三方面对细胞进行初步鉴定，证实所培养细胞是否为NB细胞。结果 两种细胞培养法均可培养出NB细胞，纯化后可得形态均一的细胞系。结论 可采用适当的细胞培养方法建立神经母细胞瘤的体外细胞系。     

鸦胆子油注射液对 H22细胞增殖的抑制作用及其机制探讨

目的：探讨鸦胆子油注射液在体内外对 H22细胞增殖的抑制作用及其作用机制。方法取 H22细胞培养,采用 MTT 法检测鸦胆子油对细胞增殖的影响;应用 H22细胞建立小鼠荷瘤模型,给予鸦胆子油治疗,计算抑瘤率、脾脏指数和胸腺指数,采用 MTT 法检测鸦胆子油对荷瘤小鼠脾细胞增殖的影响,采用双抗体夹心ABC-ELISA 法检测荷瘤鼠血清 TNF-α水平,采用放射免疫分析法检测转化生长因子（TGF）-α水平。结果鸦胆子油在（10~160）μg/ml 浓度范围内对 H22细胞的增殖抑制率为34.1%~52.31%,与对照组比有统计学意义;给予鸦胆子油25和50 mg＆#183;kg-1灌胃后,荷瘤小鼠瘤质量分别为（1.095±0.301） g 和（0.920±0.250） g,血清 TGF-α分别为（11.172±0.639） pg/ml 和（14.438±0.587） pg/ml,均较未治疗荷瘤动物显著下降[分别为（1.867±0.554） g 和（16.354±0.762） pg/ml],TNF-α水平分别为（28.132±2.456） pg/ml 和（26.521±3.267） pg/ml,较未治疗荷瘤动物显著升高[（20.231±2.614） pg/ml,P〈0.05];与对照组比,无论是 Con A 还是 LPS 刺激,荷瘤动物脾细胞增殖受到抑制（P〈0.05）。与荷瘤动物比,鸦胆子油体内给药则可促进脾细胞增殖,但对脾脏指数和胸腺指数无明显影响。结论鸦胆子油体内外均能抑制肝癌 H22细胞的生长,其抗肿瘤作用可能与改善荷瘤小鼠的脾细胞功能有关。