The role of active oxygen species and lipid peroxidation in the antitumor effect of hyperthermia.

The role of active oxygen species and lipid peroxidation in the antitumor effect of hyperthermia was studied in an experimental rabbit model. VX2 tumors were transplanted into rabbit hind legs, and the effect of hyperthermia on tumor growth was measured at 7 and 14 days after heating. As an index of lipid peroxidation, thiobarbituric acid-reactive substances in the tumor tissue were measured prior to hyperthermia and 3, 6, 12, and 24 h after hyperthermia. Tumor growth in rabbits treated with hyperthermia was significantly reduced, and thiobarbituric acid-reactive substances in the tumor tissue treated with hyperthermia were significantly increased until 6 h after hyperthermia. In addition, alpha-tocopherol in the tumor tissue was significantly decreased after hyperthermia. The antitumor effect of hyperthermia and the increase of thiobarbituric acid-reactive substances in the tumor tissue treated with hyperthermia were significantly inhibited by the administration of superoxide dismutase and catalase or dimethyl sulfoxide. These results suggest that lipid peroxidation mediated by active oxygen species plays an important role in the antitumor effect of hyperthermia.

Antitumor effect of ischemia-reperfusion injury induced by transient embolization.

The effect of ischemia-reperfusion, induced by the transient embolic agent degradable starch microspheres (DSMs) on tumor tissue was investigated from the standpoint of active oxygen species. Rabbits with VX2 carcinoma received regional infusion of DSMs by transcatheter angiography, and it was confirmed that DSMs occluded tumor vessels. Blood flow in the tumors decreased rapidly immediately after the DSM treatment and returned to the original level within 40 min. The size of tumors did not change after a single infusion of DMS, while five repeated DSM treatments led to a significant reduction in tumor size. This reduction in tumor size was prevented by the treatment of rabbits with superoxide dismutase and catalase, indicating that the generation of active oxygen species in the tumor was involved in the mechanism of action of DSMs. Thiobarbituric acid-reactive substances also increased in the tumors after DSM infusion, and this increase was also inhibited by treatment with superoxide dismutase and catalase. In conclusion, the antitumor effect of the transient embolic agent DSM is secondary to the phenomenon of ischemia-reperfusion injury. In addition, active oxygen species and lipid peroxidation are possible causes of ischemia-reperfusion injury.

Effect of DFP on loading of fura 2/AM and quin 2/AM into single smooth muscle cells prepared from guinea pig taenia coli.

The effect of diisopropyl fluorophosphate (DFP), a potent cholinesterase (ChE) inhibitor, on loading quin 2 acetoxymethyl ester (quin 2/AM) and fura 2/AM into smooth muscle cells isolated from guinea pig taenia coli was investigated spectrofluorometrically. The presence of DFP during the loading permitted the incorporation of quin 2 into the cells, so that it became possible to measure intracellular Ca2+ concentrations using the ester of this dye. Also, DFP significantly enhanced the incorporation of fura 2 into the cells. These results indicate that loading of quin 2/AM and fura 2/AM into the smooth muscle cells may depend on the suppression of ChE or various serine protease activities outside cells.

Identification of the antigenic determinants of factors 8, 9, and 34 of genus Candida.

We investigated the antigenic determinants of factors 8, 9, and 34 of the genus Candida among pathogenic yeasts by enzyme-linked immunosorbent assay (ELISA) using mannans of Saccharomyces cerevisiae wild type and mutant types, mnn 1-mnn 4 and mnn 2. Results of ELISA including antisera against the antigenic factors of genus Candida (Candida Check, latron; FAbs) indicated that these three types of mannan distinctly react with FAbs 34, 8 and 9, respectively. To identify the recognition sites of these FAbs, we compared the ability of various oligosaccharides to inhibit the binding of the mannans to FAbs. The results indicated that FAb 34 preferentially recognizes linear side chains containing a non-reducing terminal alpha-1,3-linked mannose residue, Man(alpha)1 --> 3Man(alpha)1 --> (2Man(alpha)1 --> )n(2Man) (n > or = 0), and that one of the recognition sites of FAb 9 is linear alpha-1,6-linked oligomannosyl series, Man(alpha)1 --> (6Man(alpha)1 --> )n(6Man) (n > or = 2). On the other hand, the recognition site of FAb 8 apparently consisted of two alpha-1,2-linked oligomannosyl side chains and an alpha-1,6-linked mannose residue that originated from the mannan backbone, Man(alpha)1 --> 2Man(alpha)1 --> 2(Man(alpha)1 -->2Man(alpha)1 --> 6)Man.

Vitamin E in gastric mucosal injury induced by ischemia-reperfusion.

To clarify the relationship among vitamin E, oxygen radicals, and lipid peroxidation in ischemia-reperfusion, we produced an experimental model of gastric mucosal injury in rats by ischemia-reperfusion with clamping of the celiac artery and measurements of the area of gastric erosion, thiobarbituric acid (TBA)-reactive substances, and alpha-tocopherol in serum and gastric mucosa during ischemia-reperfusion. The area of gastric erosions and TBA-reactive substances in gastric mucosa were significantly increased after 30 and 60 min of reperfusion. The serum alpha-tocopherol-cholesterol ratio and gastric mucosal alpha-tocopherol were significantly decreased after 30 and 60 min of reperfusion. On the other hand, in vitamin E-deficient rats, gastric mucosal injury induced by ischemia-reperfusion was more severe than that in vitamin E-nondeficient rats. These results indicate that vitamin E is consumed in the process of lipid peroxidation induced by oxygen radicals in ischemia-reperfusion to prevent the development of tissue damage.

Adaptive changes in dynamic properties of human disparity-induced vergence.

PURPOSE: Vergence eye movements undergo adaptive recalibration in response to a training stimulus in which the initial disparity is changed just after vergence begins (the double-step paradigm). In the present study the changes in the dynamic properties of convergence, speed and acceleration, were examined by using this double-step paradigm, before and after adaptation. METHODS: Four normal subjects participated. Three-dimensional visual stimuli were provided by a head-mounted display with two liquid crystal diode (LCD) panels. To induce adaptation, a double step of disparity was used: an initial step from distances of 2 to 1 m was followed by a second step to distances of 0.7 m ("increasing paradigm") or 1.4 m ("decreasing paradigm") after a constant period of 0.2 seconds. The dynamic properties of vergence were compared before and after 30 minutes of training with these paradigms. RESULTS: Peak velocity of convergence became significantly greater (increasing paradigm) or smaller (decreasing paradigm) after 30 minutes' training. Changes in the dynamic properties of convergence were also obvious in phase-plane (velocity versus position) and main sequence (peak velocity versus amplitude) plots. Further analysis revealed that adaptive increases in vergence velocity were accomplished by an increase in the duration of the acceleration period, whereas adaptive decreases were induced by a decrease in the maximum value of acceleration. CONCLUSIONS: The pattern of change in the dynamic characteristics of vergence after adaptation was similar to that of saccades and the initiation of pursuit eye movements, suggesting common neural mechanisms for adaptive changes in the open-loop control of eye movements.

Elemental diet modulates the growth of Clostridium difficile in the gut flora.

BACKGROUND AND AIMS: Tube feeding is regarded as a risk factor for Clostridium difficile-associated diarrhoea. Recently, we reported that C. difficile toxin was frequently found in patients receiving an elemental diet. The present study was conducted to clarify whether elemental diets are associated with the growth of C. difficile in the gut flora. METHODS: C. difficile was cultured for 72 h in various concentrations of elemental diet containing 3% thioglycollate, and the growth rate or activity of C. difficile was evaluated by Gram stain or by measuring optical density at 560 nm. Faecal samples from 10 healthy adults were cultured in elemental diet + 3% thioglycollate. RNA was extracted from faeces with glass powder, which can eliminate PCR inhibitors, and mRNA of C. difficile toxin B was measured by reverse transcription PCR. RESULTS: Maximum OD560 value during culture in thioglycollate-containing elemental diet was 2.4 times higher than that in thioglycollate alone (P = 0.0163). Viability of C. difficile was decreased in thioglycollate but not in thioglycollate-containing elemental diet. Toxin B mRNA was detected in five faecal samples (50%) before culture and in all samples after culture. CONCLUSIONS: Our results suggest that an elemental diet can modulate the growth of C. difficile in the gut flora.

Regulation of serotonin transporter gene expression in human glial cells by growth factors.

The aims of this study were to identify monoamine transporters expressed in human glial cells, and to examine the regulation of their expression by stress-related growth factors. The expression of serotonin transporter mRNA was detected by reverse transcriptase-polymerase chain reaction in normal human astrocytes, whereas the dopamine transporter (DAT) and the norepinephrine transporter (NET) were not detected. The cDNA sequence of the "glial" serotonin transporter in astrocytes was consistent with that reported for the "neuronal" serotonin transporter (SERT). Moreover, we also demonstrated SERT expression in glial fibrillary acidic protein-positive cells by immunocytochemical staining in normal human astrocytes. Serotonin transporter gene expression was also detected in glioma-derived cell lines (A172, KG-1-C and KGK). Addition of basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) for 2 days increased serotonin transporter gene expression in astrocytes and JAR (human choriocarcinoma cell line). Basic fibroblast growth factor, but not epidermal growth factor, increased specific [3H]serotonin uptake in astrocytes in a time (1-4 days)- and concentration (20-100 ng/ml)-dependent manner. The expression of genes for basic fibroblast growth factor and epidermal growth factor receptors was detected in astrocytes. These findings suggest that the expression of the serotonin transporter in human glial cells is positively regulated by basic fibroblast growth factor.

Postnatal development of the substance P-, neuropeptide Y- and serotonin-containing fibers in the rat suprachiasmatic nucleus in relation to development of the retino-hypothalamic projection.

The suprachiasmatic nucleus (SCN) in the hypothalamus controls many of the circadian rhythms in mammalian species. In the present study, we investigated the development of substance P (SP)-, neuropeptide Y (NPY)- and serotonin (5-hydroxytryptamine, 5-HT)-immunoreactive fibers in the rat SCN and the development of the retino-hypothalamic tract using cholera toxin beta subunits (CTB), in order to understand which parts of the SCN participate in diurnal rhythm regulation and entrainment. In newborn rats, SP-, NPY- and 5-HT-immunoreactive fibers were scarcely detected in the SCN. The number of SP-immunoreactive fibers gradually increased between postnatal days (P) 15 and 30. At P30, the distribution pattern of SP-immunoreactive fibers in the SCN was similar to that in the adult rat. The number of NPY- and 5-HT-immunoreactive fibers increased greatly between P10 and P15, and the increase in NPY- and 5-HT-immunoreactive fibers continued until P20. CTB was injected into the unilateral eyeball of the rat at various postnatal stages. In neonates, several labeled retinal fibers already existed in the ventral part and ventro-lateral edge of the SCN. The number and density of labeled retinal fibers in the SCN gradually increased between P10 and P20. Between P20 and P30, a decrease in the labeling was observed in the dorsolateral part of the SCN. The adult pattern of labeled retinal fibers was achieved between P20 and P30. The development of SP-immunoreactive fibers was delayed about 10 days relative to that of NPY-, 5-HT-immunoreactive fibers and retinal fibers.

Postnatal development of NK1, NK2, and NK3 neurokinin receptors expression in the rat retina.

The biological effects of tachykinins are mediated by three distinct receptors, the neurokinin 1 receptor (NK1-R), NK2-R, and NK3-R. There is no information available concerning the development of these receptors in the retina. In the present study, we investigated the localization of tachykinin receptors, using antisera directed against NK1-R, NK2-R, and NK3-R in the adult and developing rat retinas. Numerous NK1-R immunoreactive (NK1-R IR) cells were already observed in the proximal part of the neuroblastic layer in the retina at postnatal day 5 (P5). The distribution and intensity of NK1-R IR cells and processes in the inner nuclear layer (INL) and inner plexiform layer (IPL) at P10 were similar to those of adult retina. Most NK1-R IR cells located in the proximal part of INL, which were morphologically amacrine cells. In the contrast to the early expression of NK1-R IR cells, no NK3-R IR structures existed in the neuronal elements of the retina until P10. NK3-R IR processes were first detected in the outer plexiform layer (OPL) at P10. At P15, NK3-R IR somata were slightly stained in the distal and middle parts of the INL, and NK3-R IR processes were present in the OPL and the upper part of the IPL. During P15-P30, the number of NK3-R IR somata located in the INL remarkably increased. These NK3-R IR cells were morphologically bipolar and amacrine cells. This study provides differential cellular distribution of NK1-R IR cells and NK3-R IR cells in the INL of the rat retina. Our findings suggest that NK1-R and NK3-R are involved in different visual circuits and retinal maturation, and NK3-R may play previously unknown important roles in the visual processes of the rat.

Comparative study of intact A7 MoAb and F(ab')2 fragments for radioimmunoimaging of human colon cancer in nude mice.

Differences of pharmacokinetics and tumor imaging ability between intact monoclonal antibody A7 (A7 MoAb) and F(ab')2 fragments were studied in human colon cancer (LS-174T)-bearing nude mouse. First of all, we examined the yield and the immunoreactivity of F(ab')2 fragments after treatment with ficin as a function of time. The yield of F(ab')2 fragments reached about 50% after ficin treatment for 8 h, and the F(ab')2 retained about 80% of the immunoreactivity of the corresponding MoAb. Longer digestion with ficin produced smaller fragments (less than 92 kDa) with a lower yield and most of the immunoreactivity was lost. In pharmacokinetics studies, the F(ab')2 was preferentially taken up by the tumor, was cleared more rapidly from the blood circulation and seemed to have less non-specific tissue binding than intact A7 MoAb. In addition, the tumor image obtained at an early time using 131I-F(ab')2 was much superior in quality to that with intact 131I-A7 MoAb. The use of F(ab')2 fragments may be effective for tumor diagnosis and therapy.

Differences between A7 antibody and anti-CEA antibody.

The specificity of A7 monoclonal antibody (A7 MoAb), raised against a human colon cancer, was investigated in detail and compared with that of anti-CEA MoAb. Firstly, the biodistributions of radiolabeled A7 MoAb and anti-CEA MoAb were examined in human colon cancer (LS-174T)-, glioblastoma- and lung cancer (Lu-65)-bearing nude mice. 125I-A7 MoAb was highly concentrated into LS-174T and also, to a lesser extent, in glioblastoma, whereas 125I-anti-CEA MoAb was significantly taken up only by LS-174T. Both of these antibodies were taken up at only very low concentrations into Lu-65. Secondly, in vitro binding studies of 125I-A7 MoAb and 125I-anti-CEA MoAb with LS-174T cells and glioblastoma cells revealed high binding activity of these MoAbs to LS-174T cells, though A7 MoAb had a much higher affinity than that of anti-CEA MoAb. Neither of them showed high affinity for glioblastoma cells. Thirdly, competitive binding assay using A7 MoAb and anti-CEA MoAb to LS-174T cells showed that the binding of each 125I-MoAb was not inhibited by the other MoAb. Finally, in radioimmuno-imaging studies of LS-174T- and glioblastoma-bearing mice with 131I-A7 MoAb and -anti-CEA MoAb, both tumors were clearly visualized with the former, while the latter visualized only LS-174T. The A7 MoAb is clearly different from anti-CEA MoAb and may be useful for the in vivo radioimmunodetection and treatment of colon cancer.

Bone scanning with 99m-Tc-phosphates: a comparison and problems in the detection of tumor metastasis.

A comparative study on 99m-Tc-phosphate compounds (TcPP) in detecting tumor metastasis to bone and problems accompanying it are reported. TcPP revealed metastatic foci which are unrecognized by conventional bone survey. To recognize these foci, exclusion of following problems is necessary: Accumulation at front of neck, asymmetrical image of joint, increased bone density of the aged, Tc-photon absorption and radiotherapy effect. The mechanism of TcPP accumulation is discussed.

Proposal of segmental reading of radionuclide images of the liver.

The method of segmental reading of radionuclide images of the liver, which include ordinary (planar) scintigrams as well as SPECT (single photon emission computed tomography) images are presented. Patients showing defects on the liver scan, segments of which have been elucidated by other modalities, were chosen for this study, and the location of defects was assessed in connection with the segment in which they were located. On the basis of this study, the author presents his own segmentation of radionuclide images of the liver. This segmentation seems to be very useful in clinical practice and when applied to ordinary scintigrams, appears to be quite different from the previously published segmentation, which is thought to be partially incorrect.

Reliability of data obtained by radionuclide angiocardiography in follow-up studies with special reference to intra- and interobserver variations.

The reproducibility of the data obtained by radionuclide angiocardiography was studied in terms of the intra- and intervariations of three technicians. Three parameters were chosen: the left ventricular ejection fraction (LVEF), the 1/3 filling fraction (1/3FF), and the ratio of the time from end systole to the point of peak filling rate against the entire diastolic period (TRPFR). First, each technician studied an independent consecutive series of 40 patients for the initial analysis. The original data of each patient, composed of 26 frames for one beat, were stored on an optical disc for the intra- and intervariation studies. In this study, analysis of the volume curve was performed by using both 3rd order and 4th order Fourier series. The region of interest (ROI) for the left ventricle, which was set for the initial analysis, was recorded by Polaroid photography to be used as a reference for the later analyses for the variation studies. The least variation was noted in the LVEF not only for the intravariation study but also for the intervariation study, no matter which order of the Fourier series was used. However, the 3rd order Fourier series seemed to give better curve fitting to avoid fluctuation of the diastolic parameters. The results indicate that we can expect more steady and reliable information from LVEF than from the diastolic parameters, even when various technicians perform the follow-up study of a particular patient.

[Effects on visual functions following several hours' usage of a head mounted display].

We investigated the effects of viewing video movies with a head-mounted display (HMD) for 4 to 6 hours on visual functions such as refraction, visual acuity, and accommodation-vergence system. Two or three video movies were watched without any breaks by 13 normal volunteers (age: 22 approximately 40). Measurements were made of (1) objective and subjective refraction, (2) corrected visual acuity, (3) tonic level and step response of accommodation with a computer-assisted infrared optometer, and (4) near and far phorias and AC/A ratio. Significant transient myopia was found following 4 hours' viewing, but not following 6 hours' viewing. Scrutinizing individual data, myopia was consistently found in some subjects, and hyperopia in others. We presumed that many subjects might have been influenced by initial instrumental myopia when they adjusted the focus by using the mechanism built in the HMD. No significant change was observed in any other examination. However, there was a tendency for the AC/A ratio to change after a short time, and then to recover to its original value. Based on the results in this study, it appears that some changes in accommodation and vergence systems are caused by viewing video movies with the HMD. Although the amount of changes was within normal physiological variation in this study, the possibility still remains that usage for a longer time may lead to other changes in visual function. Care is also necessary when using the HMD in subjects with subclinical problems.

[Quantitative assays for C-reactive protein: a review].

C-reactive protein (CRP) is a single most important test among various serological tests which are routinely performed in clinical laboratories. While qualitative assays for CRP are being replaced by quantitative assays, standardization among various laboratories has become an urgent issue. The results of the past three-year surveys conducted by the Japanese Medical Association showed that the coefficients of variation (CV) for CRP assays ranged between 12.6% and 46.9% (1990), although these figures gradually changed for the better. These results clearly indicate that a CRP value obtained in one laboratory cannot be compared directory with that obtained in another laboratory. Every assay for CRP is considered to show good reproducibility, however, which is predicted by the results of within-run precision tests (CV:3.9-7.8%). It is very important to establish standardization for CRP assays but one has to conclude that the path toward this goal is very difficult when one takes into consideration various factors inherent to immunological reactions of macromolecules including possible microheterogeneity of CRP molecules.

[Present status of unsealed radioisotope therapy in Japan based on the nation-wide questionnaire].

In Japan, clinical application of unsealed radioisotopes is strictly regulated. Especially in the field of therapy, we are allowed to use only Na131I at present. Under such circumstances, the present status of therapeutic nuclear medicine in Japan was surveyed by means of a nation-wide questionnaire, conducted in 193 hospitals. Then, 113 hospitals replied to such questionnaire (recovery rate: 58.5%), and it was found that in 77 hospitals, radioisotope therapy is being performed for Graves' disease and/or thyroid cancer. The questionnaire covered the following points: for Graves' disease the basic strategy of 131I therapy, its indications, absorbed doses planned to be given, whether the therapy had been conducted on outpatient basis or in-patient basis, method of thyroid weight estimation, interval of administration in case of multiple doses, number of patients treated per year (1996) etc., and for thyroid cancer--strategy for thyroid remnant, the doses to be given, the maximum doses permitted by the authorities in each hospital both per day and per year, handling of highly contaminated urine in each hospital, interval of administration in case of multiple doses, number of patients treated per year (1996) etc. Also questioned were dissatisfaction with the present regulation by the authorities and/or requests for the better daily work, if any. Based on the above questionnaire, the present status of unsealed radioisotope therapy in Japan was investigated.

[Radioimmunoimaging of malignant melanoma with In-111-labeled monoclonal antibodies 96.5 and ZME 018].

This paper includes the results of imaging and kinetic studies on two kinds of In-111-labeled monoclonal antibodies (MoAbs), 96.5 and ZME 018, which are known to have capability of recognizing different surface antigens present in malignant melanoma cells. These MoAbs were supplied by Hybritech Inc. through Teijin Ltd. The former MoAb (96.5) was used on 11 cases of malignant melanoma and one case of basal cell carcinoma, and the latter MoAb (ZME 018) was used on 6 cases of malignant melanoma and one case of basal cell carcinoma. As for the malignant melanoma, the results obtained from both patients groups were compared to each other. Twenty-four out of 31 lesions in the former group and 9 (including 2 occult lesions) out of 10 lesions in the latter group were visualized. However, these positive ratios can not be compared to each other because of the very small number of the lesions in the latter group. One basal cell carcinoma each from both groups were faintly visualized. Distribution patterns of these In-111-labeled MoAbs were similar to each other. Except for the first two cases in the former group, labeling efficiencies were 94.3 +/- 0.7% with 96.5 and 92.0 +/- 0.6% with ZME 018. Urinary excretions on the 1st day were 8.3 +/- 0.9% with the former and 9.3 +/- 0.3% with the latter. However, there was no statistical difference. Following the 1st day, these values changed at the level of several per cent from the 2nd to 4th day, showing a subtle and gradual rise and slightly lower values for In-111-ZME 018. But, again, there were no statistical differences. Whole body retention curves in both groups were similar to each other, and blood clearance curves were also found similar in both groups. HAMA titers were checked in all patients and were found elevated after the administration in most of the cases. Although the number of patients tested in this series is small, especially with In-111-ZME 018, both MoAbs labeled with In-111 seem to be equally capable of visualizing malignant melanoma. Possibility of the treatment of malignant melanoma with this kind of technique was also discussed.

[Calculation algorithm of three-dimensional absorbed dose distribution due to in vivo administration of nuclides for radiotherapy].

In the in vivo administration of radionuclides for radiotherapy including radioimmunotherapy, an algorithm is proposed for the purpose of calculating three-dimensional absorbed dose distributions of tumors and adjacent tissues, and neighboring organs. The absorbed dose distribution due to the algorithm is given by convolution of the three-dimensional dose matrix for a unit cubic voxel containing unit cumulated activity, with the three-dimensional matrix of the cumulated activity distribution given by the same voxel size above. The dose calculation algorithm does not depend upon the source size, the source shape, and the nonuniform and irregular activity distribution. In addition, it can exceedingly decrease computation time compared to other calculation algorithms. Computer simulations were performed using the MIRD thyroid phantom for 32P, 90Y, 131I, 186Re, and 188Re that appear promising for radioimmunotherapy, and their results verified the validity of the proposed calculation algorithm and high accuracy of the calculations.