Minimally required heat doses for various tumour sizes in induction heating cancer therapy determined by computer simulation using experimental data.

PURPOSE: Although induction heating cancer therapy (IHCT) using magnetic nanoparticles can be a promising approach to treatment-less multi-nodular cancers, the objective requirement for successful clinical application has not clearly been elucidated. We intended to define objective heat doses suitable for IHCT, especially focusing on the sizes of liver cancer nodules. MATERIALS AND METHODS: Alternating magnetic fields were applied to three human pancreatic cancer cell lines, the intercellular space of those cell pellets were filled with magnetic nanoparticles, and confirmed the cytotoxic effect of IHCT. Subsequently, the temperatures of liver cancer nodules in IHCT were simulated using a computer software program and the required heat dose for various sized tumours were determined. RESULTS: Heating the cancer cells up to 50 degrees C for 10 min was sufficient for complete cell killing and the heat dose of 1.7 W/g(tumour) is required for 10 mm tumour. Larger tumours require a smaller heat dose, e.g. 20 mm and 40 mm tumours require 0.7 W/g(tumour) and 0.6 W/g(tumour), respectively, whereas smaller tumours require large amounts of heat, e.g. 5 mm and 1 mm tumours require 5.1 W/g(tumour) and 105 W/g(tumour), respectively. CONCLUSIONS: Integrating the presently available technologies, including high-quality magnetic nanoparticles (1000 W/g(material)) and effective drug delivery systems (1-2 mg(material)/g(tumour)), treatment of a 10 mm tumour seems possible. Since treatment of smaller tumours less than 5 mm require substantial heat dose, researchers involved in IHCT should target cancer nodules of 10 mm or more, and develop a heat delivery system providing a minimum of 1.7 W/g(tumour).

The measurement of small magnetic signals from magnetic nanoparticles attached to the cell surface and surrounding living cells using a general-purpose SQUID magnetometer.

Magnetic nanoparticles have recently been widely applied in the bio-medical field. Responding to the demand for a simple and sensitive magnetic assay system for bio-liquid samples, we employed a general-purpose superconducting quantum interference device (SQUID) magnetometer. Strips of filter paper were used as a liquid-specimen sample holder possessing a very small magnetic background signal. An aqueous solution of superparamagnetic iron-oxide nanoparticles (Resovist) was dropped in a tiny blot-like spot in the middle of the filter paper and the magnetization was measured. Magnetic moments of a dilution series of Resovist solutions versus the number of particles provided a linear graph, revealing that the magnetic moment per Resovist particle was 8.25 x 10(-17) emu. 1 x 10(5) cancer cells were incubated with Resovist, and the number of Resovist particles attached to the cell surface and surrounding a living cell was calculated to be 1.02 +/- 0.14 x 10(7) particles/cell. Our system using a commercial SQUID magnetometer should be more than enough to determine the number of magnetic nanoparticles biologically reacting with living cells, contributing to the application of magneto nanomaterials to the life-science field.

[Epidemics and related cultural factors for Ebola hemorrhagic fever in Gabon].

OBJECTIVE: The Republic of Gabon experienced epidemics of Ebola hemorrhagic fever (EHF) three times between 1994 and 1997. This study aimed at exploring cultural factors related to the outbreaks. METHODS: We collected information about EHF epidemics from the Gabon Ministry of Health, district hospitals and other facilities and conducted in-depth interviews with 20 villagers and 2 traditional healers in the village where the third epidemic occurred. RESULTS: All three epidemics were supposed to have direct or indirect relationship with great apes, the victims having cooked or eaten chimpanzees meat. Although the reuse of syringes and needles in hospitals which had worsened past EHF outbreaks in Sudan and Zaire did not contribute to the outbreak in Gabon, traditional practices as family members remaining close to the patient to nurse him/her, and hugging and touching the dead at funerals were suspected to be crucial sources of infection. Interviews with traditional healers revealed that traditional treatment methods as cutting a patient' skin with an unsterilized knife and applying blood to the skin were risky and might have been contributory factors in the deaths of one traditionals healer and his assistant in the third EHF outbreak. In one village where EHF had reached epidemic proportions, in-depth interviews were conducted with 2 traditional healers and 20 persons of mean age 33 (20-46) years with a sampling method of selecting every tenth household from the entrance. Even though they lived in a village suffering an EHF outbreak, only two thirds of them knew the name of the disease and about half of them could not explain what kind of disease it was. One quarter felt it was fatal and another quarter felt fearful. Three persons thought it had been due to evil spirits; others responded the mosquitoes or patient's sweat/saliva were the cause. CONCLUSIONS: This study showed that cultural factors might be very crucial to EHF outbreaks in developing countries. Quick intervention with health education is needed to disseminate appropriate knowledge and persuade people that traditional practices could carry a high risk of infection.

Enhanced low shear stress induced platelet aggregation by Shiga-like toxin 1 purified from Escherichia coli O157.

The effect of Shiga-like toxin 1 (Stx1) produced by Escherichia coli O157 on platelets was studied with an argon laser light-assisted shear-induced platelet aggregometer and with binding assays. Stx1 markedly enhanced the platelet aggregation under low shear stress but did not affect it under high shear stress. Minimal concentration of Stx1 required for the enhancement was 0.25 ng/ml, and almost maximal enhancement was observed at a final concentration of > or =2.5 ng/ml. This enhanced platelet aggregation disappeared after leukocyte depletion from normal platelet-rich plasma with a specific filter. In contrast, a standard platelet aggregometer was unable to detect this enhanced platelet aggregation in either the presence or the absence of ADP. 125I-labeled purified Stx1 did not specifically bind to normal washed platelets depleted of leukocytes, and thin-layer chromatographic analysis of glycolipids extracted from normal platelet lysates also confirmed that leukocyte-depleted normal platelets lack Stx1-specific receptor globotriaosylceramide (Gb3). Supernatant from the monocyte suspension stimulated with Stx1 exhibited the enhanced low shear stress induced platelet aggregation, but that from the polymorphonuclear cell suspension did not. Several cytokines produced from monocytes reproduced this event in vitro. Further, plasmas from six out of seven patients with hemolytic uremic syndrome (HUS) had activity similar to the purified Stx1. This activity was almost totally impaired after treatment of HUS plasmas with Gb3 in accord with reduction of plasma Stx1 levels. Taken together, these results indicate that platelets lack Gb3, and Stx1 appears to modulate platelet aggregation in an indirect fashion, presumably by the release of cytokines or chemical compounds from the target tissues.

Anti-cachectic effect of clarithromycin for patients with unresectable non-small cell lung cancer.

We have previously reported that long-term treatment with clarithromycin (CAM) increased the median survival of patients with non-small cell lung cancer, and improved various clinical parameters in these patients. In the present study, CAM was administered to 33 patients with unresectable primary non-small cell lung cancer, who had received chemotherapy, radiotherapy or both (basic cancer therapy). Patients with clinical backgrounds matched to the CAM group, who did not receive CAM treatment, were included into this study as a control group (non-CAM group). CAM treatment was initiated 4 weeks after the basic cancer therapy. The non-CAM group did not receive a placebo. Before and after the 3-month treatment with CAM, body weight, serum levels of interleukin-6 (IL-6, a cytokine which, together with TNF-alpha, plays a crucial role in the development of cancer cachexia), total protein, albumin, cholinesterase and hemoglobin were measured for the evaluation of the patients' clinical status. There were no statistically significant differences in serum levels of IL-6 between the CAM group before the treatment and the non-CAM group. After 3 months of CAM treatment, serum levels of IL-6 significantly decreased. In contrast, body weight, cholinesterase, and hemoglobin increased to a significant extent. Among these four parameters, however, the decrease in serum IL-6 levels was only statistically correlated with the increase in body weight, but not with that in other parameters. Furthermore, CAM-treated patients whose serum IL-6 levels were decreased after 3 months of treatment survived longer: there was a statistically significant correlation between the decrease in serum IL-6 and survival time. In contrast, in the non-CAM group, these parameters did not change significantly during the study. These results suggest that CAM may reduce the progression of cancer-associated cachexia.

Comparison of long-term therapeutic effect of an ACE inhibitor, temocapril, with that of a diuretic on microalbuminuria in non-diabetic essential hypertension.

Many investigators have reported that angiotensin-converting enzyme (ACE) inhibitors have antiproteinuric effects and retard the progression of renal impairment in diabetic patients. On the other hand, those effects of ACE inhibitors have not been well established in patients with essential hypertension. This study was conducted to prospectively evaluate whether an ACE inhibitor, temocapril, could modify the urinary microalbumin excretion rate (UAE) in hypertensive outpatients who had no signs of renal impairment. To compare the long-term effect of temocapril with that of a diuretic on UAE, hypertensive patients treated with a diuretic (trichlormethiazide) were enrolled in a prospective study if they had normal serum creatinine levels and no overt proteinuria during a 3-month screening period. A urinary microalbumin-to-urinary-creatinine ratio (mg albumin/mmol Cr) was used as an estimate of UAE. Patients visited the hospital monthly to determine blood pressure (BP) and UAE. After baseline observation during the treatment with the diuretic, the subjects were randomly divided into two groups. In group A, the diuretic was switched to temocapril, 2 to 4 mg once daily for 12 months. In group B, the subjects continued to receive the diuretic for an additional 12 months. Seventy-six outpatients (41 men and 35 women; mean age, 59.0+/-1.4 years) with essential hypertension entered the study. The effects of temocapril on BP appeared to be clinically similar to those of the trichlormethiazide, but the use of temocapril significantly decreased UAE. In group A (n=37), UAE decreased significantly (p<0.01) from the baseline value of 4.19+/-0.37 mg albumin/mmol Cr to 2.47+/-0.29 and 2.68+/-0.28 mg albumin/mmol Cr at the 6th and 12th month of temocapril therapy, respectively. In contrast, in group B (n=39) UAE was unchanged (baseline, 4.16+/-0.63 mg albumin/mmol Cr; 6 months, 4.92+/-0.72; 12 months, 4.71+/-0.74). These results indicate that long-term therapy with temocapril may be superior in reducing UAE than is diuretic therapy in patients with essential hypertension who had no signs of renal impairment.

Participation of leukocyte function-associated antigen-1 and NK cells in the homing of thymic CD8+NKT cells to the liver.

A distinct CD8+NKT cell population expressing TCRalpha/beta or TCRgamma/delta has been identified in liver and thymus. We wondered whether cell adhesion molecules play a role in the homing of CD8+NKT cells to the liver. The number of liver CD8+NKT cells was markedly reduced in leukocyte function-associated antigen (LFA)-1-/- mice compared with wild-type (WT) mice. The reduction was restricted to the liver only and no measurable alterations were found in other organs. In the liver of SCID mice reconstituted with thymocytes from LFA-1-/- or WT mice, the number of donor-derived CD8+NKT cells was comparable and the vast majority of these cells expressed TCRalpha/beta. In a reciprocal radiation thymocyte reconstitution system with LFA-1-/- and WT mice, LFA-1 expressed on liver cells other than CD8+NKT cells appeared to be required for the homing of thymic CD8+NKT cells to the liver. The accumulation of donor thymocyte-derived CD8+NKT cells in the liver of SCID mice was severely impaired by in vivo depletion of NK cells, but not of Kupffer cells. These results not only indicate that thymus provides a source for CD8+NKT cells expressing TCRalpha/beta in the liver, but also suggest that LFA-1 expressed on NK cells is involved in the homing of thymic CD8+NKT cells to the liver.

Cutting edge: contribution of NK cells to the homing of thymic CD4+NKT cells to the liver.

In contrast to peripheral lymphoid organs, in the liver a high proportion of T cells are CD4+NKT cells. We have previously reported that LFA-1 plays a pivotal role in the homing of thymic CD4+NKT cells to the liver. In the present study, we further assessed which cell type participates in the homing of thymic CD4+NKT cells to the liver. The accumulation of donor thymocyte-derived CD4+NKT cells in the liver of SCID mice that had been reconstituted with thymocytes from C57BL/6 mice was severely impaired by in vivo depletion of NK cells, but not Kupffer cells in recipients. These results suggest that NK cells participate in the homing of thymic CD4+NKT cells to the liver. We assume that LFA-1 expressed on NK cells is involved in this mechanism.

Pathogenic mechanism of mouse brain damage caused by oral infection with Shiga toxin-producing Escherichia coli O157:H7.

In a previous study, we showed that infection with Shiga toxin (Stx)-producing Escherichia coli O157:H7 (strain Sm(r)N-9) caused neurologic symptoms in malnourished mice with positive immunoreactions of Stx2 in brain tissues. The present study explores the mechanism of how Stx injures the vascular endothelium to enter the central nervous system in mice. Oral infection with strain Sm(r)N-9 elicited a tumor necrosis factor alpha (TNF-alpha) response in the blood as early as 2 days after infection, while Stx was first detected at 3 days postinfection. In the brain, TNF-alpha was detected at day 3, and its quantity was increased over the next 3 days. Frozen sections of the brains from moribound mice contained high numbers of apoptotic cells. Glycolipids recognized by an anti-Gb3 monoclonal antibody were extracted from the brain, and purified Stx2 was able to bind to the glycolipids. In human umbilical vascular endothelial cells (HUVEC) cultured with fluorescein-labeled Stx2 (100 ng/ml), TNF-alpha (20 U/ml) significantly facilitated the intracellular compartmentalization of fluorescence during 24 h of incubation, suggesting the enhanced intracellular processing of Stx2. Consequently, higher levels of apoptosis in HUVEC were found at 48 h. Short-term exposure of HUVEC to Stx2 abrogated their apoptotic response to subsequent incubation with TNF-alpha alone or TNF-alpha and Stx2. In contrast, primary exposure of HUVEC to TNF-alpha followed by exposure to Stx2 alone or TNF-alpha and Stx2 induced apoptosis at the same level as obtained after 48-h incubation with these two agents. These results suggest that the rapid production of circulating TNF-alpha after infection induces a state of competence in vascular endothelial cells to undergo apoptosis, which would be finally achieved by subsequent elevation of Stx in the blood. In this synergistic action, target cells must be first exposed to TNF-alpha. Such cell injury may be a prerequisite to brain damage after infection with Stx-producing E. coli O157:H7.

Adjuvant effect of clarithromycin on chemotherapy for murine lung cancer.

Clarithromycin (CAM) increased the median survival of patients with unresectable non-small-cell lung cancer who had received chemotherapy and/or radiotherapy [Chemotherapy 1997;43:288-296]. The present study was performed to ascertain whether CAM alone exhibits an antitumor effect against Lewis lung carcinoma (LLC) and to analyze the nature of its adjuvant effect on LLC-inoculated C57BL/6 mice. CAM at 10 mg/kg/day retarded the growth of subcutaneously inoculated LLC cells; consequently, the mean survival time of mice with LLC increased. This treatment was also effective in reducing the number of tumor nodules in the lung after intravenous inoculation with LLC cells. When tumor-bearing mice received an intravenous injection of vindesine sulfate (7 mg/kg) and cisplatin (6 mg/kg) 7 days after tumor inoculation, the chemotherapeutic effect was significantly enhanced by CAM treatment when it started 7 days after chemotherapy, but not when it started the day after chemotherapy. The delayed initiation of CAM treatment resulted in the enhancement of natural killer cell activity and CD8+ T cell cytotoxicity and increased the number of interferon-gamma-producing T cells and interleukin-4-producing T cells. These findings indicate that CAM can exhibit an antitumor effect by itself and also induce the well-balanced expansion of helper T cell subsets in tumor-bearing mice recovering from the immunosuppression caused by chemotherapy. CAM may therefore be a promising adjuvant drug in anticancer chemotherapy, and treatment with this macrolide should be initiated at some interval after basic cancer therapy.

Efficacy of antibiotic therapy for infection with Shiga-like toxin-producing Escherichia coli O157:H7 in mice with protein-calorie malnutrition.

Antibiotic therapy for infection with Shiga-toxin-producing Escherichia coli O157:H7 is controversial because of the possibility of its inducing hemolytic uremic syndrome and acute encephalopathy. In a previous study, mice with protein-calorie malnutrition were found to be highly susceptible to this pathogen. The efficacy of oral antibiotic therapy in malnourished mice infected with O157 organisms was assessed. Mice fed a low-protein calorie diet were infected intragastrically with 2 x 10(6) colony-forming units of a Shiga-toxin-producing strain of Escherichia coli O157:H7. Infected mice were orally given a therapeutic dose of an antibiotic, including norfloxacin, fosfomycin, kanamycin, ampicillin, clarithromycin or trimethoprim-sulfamethoxazole for 3 days: mice on protocol A received the antibiotic on days 1-3, starting on the day after infection, and mice on protocol B received the antibiotic on days 3-5. The duration of fecal pathogen excretion was shorter and the toxin level in the stool and blood lower in the mice that received protocol A than in untreated mice; all of the mice treated on protocol A survived the lethal infection. All antibiotics except trimethoprim-sulfamethoxazole, administered on protocol B, exhibited the same effect as that exhibited by the respective antibiotic administered on protocol A. Only the mice treated with protocol B of trimethoprim-sulfamethoxazole had a higher toxin level in the blood than untreated controls, resulting in 95% mortality. These results suggest that the antibiotics used in this study, except for trimethoprim-sulfamethoxazole, could reduce the risk of hemolytic uremic syndrome and acute encephalopathy following Escherichia coli O157:H7 infection in humans, and that fosfomycin, in particular, may be relevant for testing in humans.

[Optimization of arbitrarily primed polymerase chain reaction (AP-PCR)-based DNA fingerprinting of Mycobacterium tuberculosis and its clinical application].

Since the number of outbreaks of pulmonary tuberculosis is increasing in Japan, epidemiological analysis is important to prevent the disease. Since Mycobacterium tuberculosis lacks the variety of biotypes among strains, genetical analysis is considered to be a promising measure to differentiate various of this pathogen. We applied arbitrarily primed polymerase chain reaction (AP-PCR)-based DNA fingerprinting to clinically isolated strains of M. tuberculosis. Although genetic analyses of M. tuberculosis by AP-PCR were reported by several investigators, reproducibilities of their results were not sufficient to be widely accepted as a reliable epidemiological tool. To attain high reprodicibulity, we attempted to optimize AP-PCR conditions including primers and annealing temperature, and to purity of DNA preparations. In this study, high reproducibility was attained by using the mixed primers of 1309F and 92R, and DNA preparations with an absorbance ratio (A260/A280) of higher than 1.50. Twenty two clinical isolates, including strains isolated from one incidence of nosocomial infection and from that of intrafamilial infection were analyzed by the optimized method; consequently they were grouped into 16 types. This AP-PCR method requires only one week subculture of M. tuberculosis and less than 24 hours for analysis. This AP-PCR method allowed us to obtain the highly reproducible results within a considerably short term, which would be applicable to clinical epidemiological investigation.

Long-term therapy with an ACE inhibitor, temocapril, reduces microalbuminuria in essential hypertension.

The present study was conducted to prospectively evaluate whether a new ACE inhibitor, temocapril, could modify urinary microalbumin excretion rate (UAE) in a group of hypertensive outpatients who had no evidence of renal impairment. Sixty-three outpatients (32 men and 31 women; mean age, 59.9 +/- 1.5 yr) with essential hypertension entered the study, all having been treated for at least 6 mo with dihydropyridine calcium-channel blockers (CCBs: nitrendipine, nisoldipine, or amlodipine). Their blood pressures (BPs) had been controlled to adequate levels with the CCBs. None had overt proteinuria (determined by Albustix) or abnormal serum creatinine levels. After 3 mo of baseline observation under the previous treatment, the subjects were randomly divided into two groups. In group A (n = 31), the previously used CCBs were switched to temocapril, 2 to 4 mg once daily for 12 mo, and BP was controlled at a level equivalent to that during CCB treatment. In group B (n = 32), the subjects were maintained on their previous treatment for a further 12 mo. The effect of temocapril on BP appeared to be clinically similar to that of the previously used CCBs, but it significantly decreased UAE as compared with the previous therapy. In group A, UAE decreased significantly (p < 0.01) from the baseline value of 38.9 +/- 5.1 mg/g creatinine (Cr) to 22.2 +/- 4.2 and 25.3 +/- 5.6 mg/g Cr at the 6th and 12th months of temocapril therapy, respectively. In contrast, in group B UAE was unchanged (baseline 39.8 +/- 6.6 mg/g Cr; 6 mo, 44.6 +/- 6.8; 12 mo, 45.9 +/- 7.7). In group A, 17 of 31 patients (54.8%) had abnormal UAE levels (> or = 29.5 mg/g Cr) during previous therapy with CCBs, but 6 mo after switching to temocapril 25 of these patients (80.6%) had normal UAE (< 29.5 mg/g Cr). In group B, 15 of 32 patients (46.9%) had abnormal UAE levels during the observation period, and these abnormal UAE levels remained unchanged; 17 of the 32 patients (53.1%) had abnormal UAE levels after a further 6 mo of continued CCBs therapy. We conclude that long-term therapy with temocapril may provide renal protection by reducing UAE even in hypertensive patients with no evidence of renal impairment.