Antitumor activity of the DNA fraction from Mycobacterium bovis BCG. II. Effects on various syngeneic mouse tumors.

MY-1, a fraction extracted from BCG and composed of 70.0% DNA and 28.0% RNA, was examined for its antitumor activity against 9 different syngeneic mouse tumors. Tumor regression was induced in almost all of the mice bearing any of five kinds of solid tumors by repeated intralesional injections of 100 micrograms MY-1. When cells of some tumors were inoculated intradermally together with MY-1, tumor growth was suppressed, lung metastases were inhibited, and the survival times of mice bearing 1 of 3 leukemic tumors were prolonged. Repeated sc injections with MY-1 in sites remote from tumor cell inoculation or repeated iv injections were more or less effective against three kinds of solid tumors. Mice inoculated with Lewis lung carcinoma cells in a hind footpad and whose legs were amputated 9 days later were given iv or sc injections of MY-1 every other day (8 times in total), resulting in substantial prolongation of survival. No direct cytotoxicity of MY-1 for these tumors could be shown in three kinds of experiments, which indicates that the antitumor mechanism of MY-1 is host mediated. MY-1 was equally effective in mice with or without presensitization with BCG, whereas BCG was much more effective in BCG-sensitized mice. This finding suggests that a delayed-type hypersensitivity reaction elicited by BCG protein is not required for the antitumor activity of MY-1.

Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity.

A fraction extracted from Mycobacterium bovis strain BCG, which was composed of 70.0% DNA, 28.0% RNA, 1.3% protein, 0.20% glucose, and 0.1% lipid and of no detectable amounts of cell wall components such as alpha, epsilon-diaminopimelic acid and hexosamine, was found to possess strong antitumor activity. Repeated intralesional injection of this fraction, designated MY-1, without attachment to oil or a single intralesional injection of MY-1 emulsified in mineral oil caused the IMC carcinoma of CDF1 mice and line 10 tumor of strain 2 guinea pigs to regress and/or prevented metastasis very effectively. MY-1 after digestion with RNase, which contained 97.0% single-stranded DNA with a guanine-cytosine content of 69.8%, was more effective than undigested MY-1 against IMC and line 10 tumor, while MY-1 digested with DNase, which contained 97.0% RNA, had reduced activity, suggesting that the DNA from BCG possessed strong antitumor activity under certain conditions. Details of the extraction procedures and physicochemical characterization of MY-1 were also described.

Purification and biological activities of pokeweek (Phytolacca americana) mitogens.

Five mitogens, designated Pa-1 through Pa-5, were purified from the roots of pokeweed (Phytolacca americana) by means of ethanol fractionation, DEAE-cellulose column chromatography, affinity chromatography on a column of desialized human erythrocyte glycopeptide-Sepharose 4B, and gel filtration. Among these mitogens, only Pa-1 was mitogenic for both murine B-cells and T-cells, and the other mitogens were T-cell mitogens. Binding experiments with 125I-labeled Pa-1, a potent mitogen for B-cells, and with 125I-labeled Pa-2, the strongest T-cell mitogen, revealed that murine B-cells have more receptor sites for Pa-1 than for Pa-2 and murine T-cells have more receptor sites for Pa-2 than for Pa-1. The change of membrane fluidity within 30 min after binding of the mitogens to murine B- and T-cells was measured by fluorescence polarization of fluorescent hydrocarbon, 1,6-diphenyl-1,3,5-hexatriene, embedded in the membrane. Pa-1 induced increase of membrane fluidity of B-cells more markedly than Pa-2, whereas Pa-2 had a larger effect on the membrane fluidity of T-cells than Pa-1. Although both Pa-1 and Pa-2 stimulated the incorporation of 32 Pi into phosphatidylinositol of murine T-cells, neither Pa-1 nor bacterial lypopolysaccharide induced the activation of phospholipid metabolism of murine B-cells.

Assessment of right ventricular contractile state with the conductance catheter technique in the pig.

OBJECTIVE: Since the conductance catheter method has facilitated evaluation of left ventricular contractile state in both laboratory and clinical studies, the aim of this study was to determine whether the technique is similarly useful for the right ventricle. METHODS: A series of right ventricular pressure-volume loops was obtained in seven open chest pigs during transient vena caval occlusion using a 12-electrode conductance catheter. End systolic pressure-volume relationships, stroke work-end diastolic volume relationships, and dP/dt-end diastolic volume relationships were compared at control and during infusion of dobutamine and esmolol. RESULTS: Right ventricular pressure-volume loops generated with the conductance catheter were of a shape consistent with those previously reported by other volume measurement techniques, and responded to changes in inotropic state in a predictable fashion. Dobutamine shifted the three contractile relationships leftward, whereas esmolol shifted them rightward. Comparisons of stroke volume derived with the conductance catheter and with a pulmonary artery flow probe demonstrated the ability of the conductance technique to measure relative volume changes. CONCLUSIONS: The conductance catheter provides a continuous measure of right ventricular volume that was used to detect changes in right ventricular contractile state in pigs. This represents a promising and much needed method for the evaluation of right ventricular function.

Structure-activity relationship of newly synthesized quinoline derivatives for reversal of multidrug resistance in cancer.

The effect of 24 newly synthesized quinoline derivatives on tumor cell multidrug resistance (MDR) was examined in vitro. At low concentrations, these compounds enhanced the accumulation of [3H]vincristine in K562/ADM cells and reversed tumor cell MDR. The results of the structure-activity relationship analysis indicate that in highly active compounds the two aryl rings in the hydrophobic moiety deviate from a common plane, so they are capable of interacting with hydrogen bond donors of P-170 glycoprotein (P-gp) via pi-hydrogen-pi interactions. Other major structural features which influence the MDR-reversing activities of these compounds are a quinoline nitrogen atom and a basic nitrogen atom in piperazine. Furthermore, in highly active compounds, the distance between the hydrophobic moiety and the basic nitrogen atom (an atom connected to 2-hydroxypropoxyquinoline) must be at least 5 A. Several compounds were found to reverse vincristine resistance in K562/ADM cells in vitro, and compound 16 (MS-209) was selected for clinical studies.

Nitroglycerin maintains graft vascular homeostasis and enhances preservation in an orthotopic rat lung transplant model.

Transplanted lungs often fail during the peritransplantation period for poorly understood reasons. Because the nitric oxide pathway regulates pulmonary vascular tone, helps to maintain the integrity of the endothelial barrier, and modulates neutrophil adhesivity and activation, we hypothesized that perturbation of this pathway during the preservation and reperfusion of transplanted lungs might play a critical role in mediating early graft failure. To evaluate whether supplementing the preservation solution with the nitric oxide donor nitroglycerin enhances lung preservation for transplantation, we obtained hemodynamic measurements in a model of orthotopic left lung transplantation in the rat after ligation of the native right pulmonary artery. In these experiments, recipient survival and hemodynamics depended solely on the transplanted lung. The left lung was harvested from 22 rats, flushed with either lactated Ringer's solution alone (control, n = 11) or Ringer's solution supplemented with nitroglycerin (0.1 mg/ml, n = 11), preserved for 4 hours at 4 degrees C, and then transplanted using a rapid cuff technique for bronchial and vascular anastomoses. Nitroglycerin significantly improved arterial blood oxygenation (339 +/- 66 versus 130 +/- 12 mm Hg, p < 0.05), increased pulmonary arterial flow (7.6 +/- 1.9 versus 0.9 +/- 0.2 ml/min, p < 0.005), decreased pulmonary vascular resistance (1.7 +/- 0.4 versus 6.6 +/- 1.9 x 10(3) Wood units, p < 0.05), and enhanced recipient survival (64% versus 0%, p < 0.05). Control grafts had significantly greater neutrophil accumulation (50% greater as quantified by myeloperoxidase activity, p < 0.05) than grafts preserved in the presence of nitroglycerin. These studies show that supplementation of the preservation solution with the nitric oxide donor nitroglycerin maintains graft vascular homeostasis and significantly improves pulmonary function and recipient survival after transplantation.

Purification and characterization of a tuberculin-active substance from Mycobacterium bovis BCG.

A new tuberculin-active substance, designated TAS-1D3, has been purified from the extract of Mycobacterium bovis BCG by precipitation at pH 4.2, ethanol fractionation, and column chromatography involving CM-cellulose, QAE-Sephadex A-25, Sephadex G-100, and Sephadex G-75. TAS-1D3 was homogeneous in polyacrylamide gel electrophoresis and positive in both Coomassie brilliant blue and periodic acid-Shiff staining, suggesting that TAS-1D3 is a glycoprotein. The molecular weight of TAS-1D3 was estimated to be 26,000 by gel filtration. In amino acid analysis, TAS-1D3 was distinctive in having proline as a dominant amino acid, and in that it lacked basic amino acids, sulfur-containing amino acids and aromatic amino acids. Moreover, TAS-1D3 was almost devoid of absorption at around 280 nm. In guinea pigs sensitized with BCG vaccine, the tuberculin activity of TAS-1D3 was about forty times more potent than that of purified protein derivative (PPD).

Does blood flow through holmium:YAG transmyocardial laser channels?

BACKGROUND: Early reports indicate that transmyocardial laser revascularization improves symptoms in patients with refractory angina. However, there is little experimental evidence of whether blood flow through channels is the mechanism of action. METHODS: Endocardial channels were made in the distribution of the left anterior descending coronary artery in canine hearts (n = 5) using a holmium:yttrium-aluminum garnet laser. Hearts were excised acutely while perfused in a retrograde fashion from a second dog so that the aortic valve always remained closed. The proximal left anterior descending coronary artery was ligated. To measure direct transmyocardial blood flow, colored microspheres were injected into the left ventricular chamber. RESULTS: The number of spheres per gram of tissue in the channel region was significantly higher than in the control region (low load, 302.5 +/- 169.0 versus 41.8 +/- 59.4; high load, 208.4 +/- 138.3 versus 5.8 +/- 11.7; both, p < 0.05). However, the estimated regional blood flow through the channels was extremely low (<0.01 mL/g/min. In the chronic setting (n = 4) (2-week survival), no flow as detected through the channels, and the endocardial entry points were closed. CONCLUSIONS: Transmyocardial blood flow does not appear to occur through channels made with the holmium:yttrium-aluminum garnet laser. It remains to be determined whether this is the case with other types of lasers.

Validation of right and left ventricular conductance and echocardiography for cardiac function studies.

BACKGROUND: Continuous estimation of left ventricular volume from instantaneous conductance has compared favorably with "gold standards," is less labor intensive, and provides real-time data. Little information exists, however, correlating right ventricular conductance with such gold standards or examining the effects of an electrical field generated in the opposite ventricle. METHODS: In open-chested sheep, right and left ventricular conductance, two-dimensional echocardiography, and thermodilution cardiac outputs were measured at steady-state conditions. After these measurements, postmortem pressure-volume relations, ventricular mass, and ventricular casting were performed. RESULTS: The corrected end-diastolic volume measured by conductance correlated well with volumes measured by echocardiography (r = 0.89), postmortem pressure-volume relations (r = 0.84), and casts (r = 0.85). Left ventricular end-diastolic volume measured by conductance did not differ significantly from other standards by analysis of variance. The presence of an electrical field in the opposite ventricle did not affect measured conductance in the studied ventricle. CONCLUSIONS: Conductance is useful for the measurement of right and left ventricular end-diastolic volumes in the beating heart and is not affected by the presence of an electrical field in the opposite ventricle. Hence, conductance is a useful tool in studies involving interventricular dependence and function.

Prevention of acute regional ischemia with endocardial laser channels.

Laser myocardial revascularization has been shown to reduce mortality and infarct size after left anterior descending coronary artery (LAD) ligation in dogs. It has not been shown to improve myocardial contractility in acute ischemia. In this study a holmium-yttrium-aluminum garnet laser (wavelength, 2.14 microns) was used to create nontransmural myocardial channels from the endocardial surface in the ischemic regions of the canine left ventricle. Twelve mongrel dogs (6 controls, 6 laser myocardial revascularizations) underwent 90 minutes of LAD ligation followed by 6 hours of reperfusion. The ischemic region was determined by methylene blue injection during brief LAD occlusion. Laser myocardial revascularization averaged three channels per square centimeter in the ischemic region created using 12 J/channel (600 mJ/pulse, 10 Hz) before LAD ligation. Contractility was assessed from regional preload recruitable stroke work (RPRSW), using pairs of segment length ultrasonic transducers in the ischemic and the nonischemic regions. Two-dimensional echocardiography corroborated with segmental length findings. In control dogs, the ischemic region was dyskinetic during LAD ligation and reperfusion. Dyskinesis of the ischemic region during systole produced negative values for regional stroke work, and RPRSW was considered zero. In 4 of 6 laser-revascularized dogs, RPRSW remained positive in the ischemic region. Two dogs had intermittent dyskinesis. The difference between laser-revascularized and control dogs in ischemic region RPRSW was significant (p < 0.01 by Fischer's exact test).(ABSTRACT TRUNCATED AT 250 WORDS)

Reversal of multidrug resistance by a novel quinoline derivative, MS-209.

MS-209, a novel quinoline derivative, was examined for its reversing effect on multidrug-resistant tumor cells. MS-209 at 1-10 microM completely reversed resistance against vincristine (VCR) in vitro in multidrug-resistant variants of mouse leukemia P388 cells (VCR-resistant P388/VCR and Adriamycin (ADM)-resistant P388/ADM) and human leukemia K562 cells (VCR-resistant K562/VCR and ADM-resistant K562/ADM). MS-209 at 1-10 microM also completely reversed resistance against ADM in vitro in P388/VCR cells, K562/VCR cells, and K562/ADM cells. In ADM-resistant P388 (P388/ADM) cells, however, ADM resistance was only partially reversed at the MS-209 concentrations tested. MS-209 enhanced the chemotherapeutic effect of VCR in P388/VCR-bearing mice. When MS-209 was given p.o. at 80 mg/kg twice a day (total dose, 160 mg/kg per day) with 100 micrograms/kg VCR, a treated/control (T/C) value of 155% was obtained. MS-209 also enhanced the chemotherapeutic effect of ADM in P388/ADM-bearing mice. The most prominent effects were obtained when MS-209 was given with 2 mg/kg ADM, yielding T/C values of 150%-194% for the combined treatment at an MS-209 dose of 200-450 mg/kg. MS-209 inhibited [3H]-azidopine photolabeling of P-glycoprotein efficiently. Furthermore, the accumulation of ADM in K562/ADM cells was increased more efficiently by MS-209 than by verapamil. These results indicate that MS-209, like verapamil, directly interacts with P-glycoprotein and inhibits the active efflux of antitumor agents, thus overcoming multidrug resistance in vitro and in vivo.

Relationship between multidrug resistant gene expression and multidrug resistant-reversing effect of MS-209 in various tumor cells.

MS-209 is a novel quinoline compound which can overcome multidrug resistance (MDR) both in vitro and in vivo, while having a low level of side effects, and is now being evaluated in a clinical phase II study. Reverse transcription-polymerase chain reaction (RT-PCR) was used to quantitate the expression levels of MDR genes in various mouse and human tumor cell lines. The MDR gene and the beta actin gene, as the internal reference standard, were coamplified separately, and the relative expression of the MDR gene was represented by the MDR/beta actin ratio. The in vitro MDR-reversing effect of MS-209 was then compared with the MDR gene expression (MDR/beta actin ratio). We found a significant correlation between these two parameters. Moreover, a significant correlation was also observed between the level of expression of the MDR1 gene and that of P-glycoprotein in human cell lines. Therefore, the efficacy of MS-209 seems to specifically depend on the level of MDR gene expression (P-glycoprotein). From these observations, it is suggested that RT-PCR assays of MDR1 gene in tumor biopsy specimens might be an effective means to predict the response of tumor cells to combination therapy with MS-209.

A novel quinoline derivative, MS-209, overcomes drug resistance of human lung cancer cells expressing the multidrug resistance-associated protein (MRP) gene.

PURPOSE AND METHODS: MS-209 is a newly synthesized quinoline compound used orally to overcome human P-glycoprotein (Pgp)-mediated multidrug resistance (MDR). The multidrug resistance-associated protein (MRP) gene is thought to play an important role in MDR in lung cancer. To investigate whether MS-209 could also overcome MRP-mediated MDR, we examined the effect of the compound using a cytotoxicity assay on MDR1 gene-negative drug-selected MDR and wildtype lung cancer cells with various levels of MRP gene expression. The effects of MS-209 were compared with those of verapamil (VER) and cyclosporin A (CsA). The level of MRP gene expression in the cells was evaluated semiquantitatively by RT-PCR. For vincristine (VCR), intracellular accumulation of [3H]-VCR was measured with or without MS-209. RESULTS: In MDR UMCC-1/VP small-cell lung carcinoma cell line, 5 microM of MS-209 and VER enhanced the cytotoxicity of etoposide, doxorubicin (DOX) and VCR more than twofold, and completely reversed the resistance to VCR. The mean reversing effects of MS-209 on DOX and VCR were significantly stronger than those of VER and CsA. In wildtype non-small-cell lung carcinoma cells, the effects of MS-209 were almost equal to those of VER and CsA. The effect of these three agents correlated with the level of MRP gene expression. The MS-209-induced increase in intracellular accumulation of VCR was proportional to the level of MRP gene expression in these cells. CONCLUSION: Our results indicate that MS-209 is a potentially useful drug that can overcome MRP-mediated intrinsic and acquired MDR in human lung cancer.

[The factors necessary to achieve successful penetrating keratoplasty].

A statistical study was conducted of 31 penetrating keratoplasties performances over the past 6 years from January 1980 to December 1985, and the factors necessary to achieve successful penetrating keratoplasty are discussed on the basis of the results of the study. The patients were followed up for more than one year after operation. The graft transparency, visual acuity and incidence of corneal rejection were evaluated at one year after operation and the combined surgery with penetrating keratoplasty was also evaluated. The results obtained were as follows: 1) The distinctive feature of the cases with visual acuity of more than 1.0 were that the recipients were younger than 40 years of age and the donors were also less than 50 years at the time of the operation. 2) The patients with vascularized cornea showed a higher incidence of rejection and a lower successful ratio rate than did the avascular corneal patients. The above results indicate that the factors necessary to achieve a successful penetrating keratoplasty are young recipients, young donors and no vascularization of recipient cornea.

Some properties of fibroblasts from a patient with debrancher deficiency.

Glycogenosis Type III is characterized by a deficiency of debranching enzyme (amylo-1,6-glucosidase, E.C. 3. 2. 1. 33) in most tissues. Low activity of liberating glucose from limited dextrin in the biopsied muscle can be demonstrated in a patient with this disease. We cultured fibroblasts from a skin biopsy from a patient with debrancher deficiency and examined the metabolism of glycogen in these cultured fibroblasts. Debrancher activity in the post-mitochondrial supernatant obtained from these fibroblasts showed a good concentration dependent manner but had approximately half of that from normal human fibroblasts (YH-1). Although the enzymatic activity of debrancher in the cultured fibroblasts from the skin was reduced essentially to the same levels as observed in muscle biopsy, little glycogen granules were accumulated in the cytoplasm of these fibroblasts as revealed by either light- or electron-microscopic observation. The fibroblasts obtained in the present study may be useful for the analysis of molecular mechanism of the debrancher deficiency disease, glycogenosis Type III.

[Effects of cadmium on the gene expression of retinoblastoma (Y79) cells in culture].

We examined the effects of cadmium on the mRNA levels of several genes in cultured retinoblastoma (Y79) cells. After Y79 cells were treated with 15 microM CdCl2, RNA was extracted at a given time. The levels of retinoblastoma gene (Rb) mRNA decreased after cadmium treatment, although it was unlikely that the Rb gene product is functional in this cell line. The N-myc gene (oncogene) is constitutively expressed in untreated Y79 cells but its mRNA levels also decreased following cadmium treatment. On the other hand, the mRNA levels of both heat-shock protein (hsp 70) and metallothionein gene, both having physiological protective effects, increased under these conditions. These results indicate that Y79 cells have physiological protective responses to such a heavy metal as cadmium and that both Rb and N-myc gene expressions are down-modulated in the presence of cadmium.

In vivo augmentation of natural killer cell activity with a deoxyribonucleic acid fraction of BCG.

A fraction extracted from BCG and designated MY-1, which was composed of 70.0% DNA and 28.0% RNA, was previously reported to possess strong antitumor activities against various syngeneic mouse and guinea pig tumors. An intraperitoneal injection of MY-1 (100 micrograms) 1 day before rendered mouse peritoneal cells cytotoxic to YAC-1 cells. The effector cells were nonadherent to plastic dishes, and the activity was destroyed by treatment with anti-asialo GM1 antiserum plus complement or carrageenan in vitro, but not with carbonyl-iron or anti-Thy 1.2, suggesting that the cells are natural killer (NK) cells. In vivo augmentation of NK activity was dependent on MY-1 dose, and reached the peak 1 day after MY-1 injection. Since NK activity in lipopolysaccharide (LPS)-nonresponder mice could be augmented by MY-1, the possibility that LPS contaminated the MY-1-augmented NK was excluded. MY-1 digested preliminarily with DNase lost its NK-inducing activity, suggesting that the DNA entity of MY-1 was essential for the activity. When mice were pretreated with anti-asialo GM1 or carrageenan, MY-1 could not render the peritoneal cells cytotoxic. Antitumor activities of MY-1 were also abolished if the animals were pretreated with anti-asialo GM1 antiserum or carrageenan, suggesting that the activities can be ascribed mainly to activated NK cells.

Properties of TAS-1D3, a tuberculin-active substance from BCG, in regard to delayed hypersensitivity.

The properties of TAS-1D3, a tuberculin-active substance purified from the cell extract of Mycobacterium bovis BCG, were studied in vivo and in vitro. In the delayed hypersensitivity skin reaction, TAS-1D3 showed far more potent activity than tuberculin purified protein derivative (PPD) in guinea pigs sensitized with BCG vaccine. This was consistently observed from 6 to 24 weeks after sensitization. The histological findings of the skin reaction to TAS-1D3 were similar to those of the reaction to PPD. Moreover, TAS-1D3 induced well both thymidine incorporation and the production of migration inhibitory factor (MIF) by the spleen cells from guinea pigs sensitized with BCG vaccine. In contrast, TAS-1D3 showed weaker activity than PPD in guinea pigs sensitized with either heat-killed M. tuberculosis Aoyama B or heat-killed M. tuberculosis H37Ra, and it weakly stimulated the spleen cells from animals sensitized with M. tuberculosis Aoyama B to incorporate thymidine and to produce MIF.

Induction of T-cell-mediated immunity against MethA fibrosarcoma by intratumoral injections of a bacillus Calmette-Guérin nucleic acid fraction.

MY-1, which consists of DNA and RNA extracted and purified from bacillus Calmette-Guérin (BCG), has been shown to have strong antitumor activity against various experimental tumors. To examine the role of T cells in the antitumor mechanism of MY-1, the effect of MY-1 injection on the development of tumor-specific immunity against MethA fibrosarcoma was investigated. MY-1 injections inhibited tumor growth less effectively in T-cell-deficient nude mice than in normal BALB/c mice. MethA tumor growth was suppressed after inoculation with L3T4-positive lymphocytes from tumor-bearing mice treated with MY-1. MethA-specific delayed-type hypersensitivity was also detected in tumor-bearing mice treated with MY-1. Immunohistochemical analyses showed that many L3T4-positive and a few Lyt2-positive cells infiltrated the regressing tumors. These results indicate that intratumoral MY-1 injections induce a MethA-specific, L3T4-positive cell-mediated, delayed-type hypersensitivity, which is necessary for the tumor regression.

Patient selection for endovascular repair of aortoiliac aneurysms.

PURPOSE: This study was conducted to establish criteria to aid in the selection of patients for endovascular repair of aorto-iliac aneurysms. METHODS: Characterization of pertinent factors used to determine whether a patient is eligible to undergo stent-graft repair of an aorto-iliac aneurysm. PRINCIPAL FINDINGS AND CONCLUSIONS: The determinant factor that dictates whether or not one is eligible to undergo endovascular repair of aorto-iliac aneurysm is the arterial anatomy of the affected area and its surrounding vessels. Some of the initial limitations imposed in this technology have changed such as an acceptance of much shorter neck than initially conceptualized, by the use of supra-renal stent deployment. However, unsolved issues remain regarding the differentiation of thrombus and atherosclerotic plaque in the infra-renal aortic region, iliac artery disease, and the need to have an enhanced flexibility of the delivery system for proper deployment in tortuous aortic necks. The question of long-term device durability remains the most important issue that has to be taken into consideration before one chooses minimally invasive endovascular approaches.