[A Case of Primary Anal Fistula Cancer with Extensive Intrapelvic Spreading That Was Successfully Treated with Bevacizumab plus mFOLFOX6].

The patient was a 68-year-old man who had an anal fistula for>10 years. He was referred to our institution after visiting a local physician with left femoral pain as the main complaint and received a diagnosis of high inflammatory response. We then found discharge of pus in the perianal region during a medical examination. We also found an extensive intrapelvic tumor during a computed tomography(CT)/magnetic resonance imaging examination. In addition, the level ofa tumor marker and inflammatory response were high. To control the inflammation, we performed seton drainage and sigmoid colostomy. On the basis of the pathological findings from the mucus component, we confirmed a diagnosis of fistula cancer. Considering that the progressive lesion had extensively spread, we decided to initiate chemotherapy alone because ofthe absence ofan indication for radiotherapy. We administered bevacizumab plus mFOLFOX6, and partial response was observed on a CT scan. We could control the progression ofthe disease for>6 months. The present case suggests that bevacizumab plus mFOLFOX6 can be an effective regimen for unresectable advanced fistula cancers.

Interleukin-1beta Inhibition Attenuates Vasculitis in a Mouse Model of Kawasaki Disease.

BACKGROUND: Kawasaki disease (KD), a systemic vasculitis, is suspected to be related to abnormalities in innate immunity. Based on the important role of IL-1 signaling in innate immunity, we investigated the effects of an anti-IL-1ß antibody using a Candida albicans water-soluble fraction (CAWS)-induced mouse model of KD. METHODS: CAWS (0.5 mg/mouse) was injected intraperitoneally into 5-week-old DBA/2 mice on five consecutive days. An anti-Murine IL-1ß antibody (01BSUR) was administered at various doses (2.5, 5.0, and 10.0 mg/kg) and time points (2 days before, same day, and 2, 5, 7, and 14 days after CAWS administration). After 4 weeks, vasculitis in the aortic root was investigated histologically. Cytokines including IL-1ß, -6, -10, and TNF-α were also measured. RESULTS: Groups administered 01BSUR at all doses showed a significant reduction in the area of vasculitis. In addition, 01BSUR inhibited vasculitis until 7 days after CAWS administration. In the analysis of various time points, the level of IL-6 was lower in all groups compared to the CAWS only group, but the levels of IL-1ß, TNFα, and IL-10 were lower when 01BSUR was administered before CAWS. On the other hand, TNFα and IL-10 levels were restored when 01BSUR was administered after CAWS, suggesting that 01BSUR may have additional effects beyond blocking IL-1ß signaling. CONCLUSIONS: The anti-IL-1ß antibody significantly attenuated CAWS-induced vasculitis. The mechanism of inhibiting vasculitis is thought to include inhibition of the IL-1ß pathway and additional effects beyond blocking IL-1ß signaling.

[Implications of nicotine detected in autopsy cases of newborn babies and infants from the perspective of social medicine].

Smoking by pregnant and parturient women is generally suspected to increase nicotine levels in fetal and infant blood. Supportive data of nicotine levels in infants is, however, inadequate. We investigated blood and muscle nicotine and cotinine levels in 14 autopsy cases of newborn babies and infants using gas chromatography. Among the 14 cases investigated, nicotine or cotinine was detected in six cases (42.9%). In each of these six cases, the mother was a smoker. Route of exposure to nicotine originating from smoking was transplacental in three cases, via breast milk in one case and secondhand smoke in two cases. Nicotine and cotinine levels in blood from the two cases with placental exposure were 10.6-84.4 ng/ml and 20.3-183 ng/ml, and levels in muscle from one case were 43.9 ng/g and 308 ng/g, respectively. Nicotine and cotinine levels in blood from exposure via breast milk were 19.1 ng/ml and 87.1 ng/ml, and from secondhand smoke were 0 ng/ml and 14.6-20.1 ng/ml. Mean concentrations of blood nicotine and cotinine in 68 autopsy cases of adult habitual smokers were 30.0 ng/ml and 247 ng/ml. Our data for nicotine and cotinine levels in infant blood seem to indicate that some infants who are born and develop under exposure to smoking by family members, particularly the mother, may show high nicotine levels in blood and experience possible health risks.

Forensic implication of muscle level of nicotine in an adipocere body found in the sea.

We reported previously that blood levels of nicotine in suicidal smokers tend to be significantly higher than those in non-suicidal smokers, and blood level of nicotine seems to be a useful criterion for discriminating suicide from other types of death. In this paper, we report nicotine and cotinine levels in various tissues of an adipocere body found in the sea. The cause of death was drowning, and the postmortem time interval was approximately 5 months at autopsy. His driver's license was concealed in his sock, which seemed to suggest that he committed to suicide. In toxicological analysis by gas chromatography, nicotine and cotinine in the femoral muscle were detected at concentrations of 213 and 488 ng/g, respectively, and these substances were also detected in the brain, liver and kidney. For evaluating the tissue levels of nicotine and cotinine in the adipocere body, we analyzed these levels in blood and various tissues of 13 autopsy cases of smokers. Nicotine and cotinine levels in blood were the most similar to those in skeletal muscle. Although the postmortem time interval, the formation process of adipocere and the environmental condition in water may affect nicotine and cotinine levels in the femoral muscle, the high muscle level of nicotine in the present case seem to implicate suicidal death.

Otitis media observed in unexpected natural death of infants.

Intracranial complications due to otitis media such as brain abscess and leptomeningitis are well known as a cause of death. In recent years, encountering those serious intracranial complications in forensic medical practice is extremely rare. However, we rarely encounter autopsy cases with otitis media of which pathological damage is mild or moderate. We investigated 11 autopsy cases (6 cases of pneumonia and 5 cases of SIDS) in unexpected natural death of infants under one year old, and found 3 cases with otitis media. The tympanic cavity was investigated by chiseling a petrosal part of the base of the skull. In the case of otitis media, serous and mucous exudate containing leucocytes examined microscopically was observed. Otitis media, as such, was not a cause of death in our cases presented. Background factors causing otitis media seems to be not only functional insufficiency of the auditory tube but also other delicate constitution, hidden dysfunction or immature function in immune system, which could be easily infected. Of 3 cases of otitis media, cytomegalovirus infection was observed in 2 cases simultaneously. In our department, we have little opportunity to encounter autopsy cases of infant under one year old. If many infant cases could be investigated, many autopsy cases with otitis media might be encountered in unexpected infant deaths.

Nicotine and cotinine levels in body fluids of smokers who committed suicide.

Cigarette smoking is associated with a higher risk for suicide. The present study was conducted on the hypothesis that suicide smokers show higher nicotine and cotinine levels in blood and urine than non-suicide smokers. We determined nicotine and cotinine levels in blood and urine of 87 deceased individuals (18 suicides and 69 non-suicides) by gas chromatography. The smoking rate was 77.8% for individuals who committed suicide and 42.0% for those who did not commit suicide. Average nicotine and cotinine levels in blood were significantly higher in the suicide smokers than in the non-suicide smokers (nicotine: 93.2+/-46.6 ng/ml versus 25.8+/-14.4 ng/ml, p<0.0001 and cotinine: 378+/-235 ng/ml versus 201+/-137 ng/ml, p<0.005). Average levels of urinary nicotine and cotinine were also significantly higher in the suicide smokers than in the non-suicide smokers (nicotine: 1980+/-2210 ng/ml versus 394+/-376 ng/ml, p<0.005 and cotinine: 1170+/-1330 ng/ml versus 414+/-290 ng/ml, p<0.05). Twenty-six decedents were intoxicated with alcohol, and they included 7 suicides (7 smokers) and 19 non-suicides (15 smokers). Our data suggest that cigarette smokers who commit suicide smoke more heavily than other cigarette smokers.

Differential effects of apolipoprotein E isoforms on lipolysis of very low-density lipoprotein triglycerides.

Apolipoprotein (apo) E plays a key role in lipoprotein metabolism and has been proposed to modulate triglyceride (TG) lipolysis. However, no systematic investigation on lipolysis using all 3 isoforms of apoE has been performed. To clarify the role of common human apoE isoforms in the lipolysis of very low-density lipoprotein (VLDL) TGs, we overexpressed human apoE isoforms in apoE and low-density lipoprotein receptor-deficient mice using adenoviral-mediated gene transfer and used VLDL particles obtained from these mice for in vitro lipolysis assay. Overexpression of apoE, regardless of its isoforms, increased the TG content of VLDL in mice in vivo. In vitro analysis of the effect of apoE on lipolysis revealed that irrespective of its isoforms, apoE did inhibit TG lipolysis at every concentration of apoE examined, and this inhibitory effect became more pronounced as the apoE content of VLDL increased. No difference was observed in TG lipolysis activity among isoforms at low apoE/TG ratio; however, intermediate ratios of apoE/TG, which reflect physiologic VLDL apoE/TG ratios, demonstrated a significantly greater level of lipolysis inhibition in apoE2, but less so in apoE4 compared with other isoforms. This differential effect by apoE isoforms on lipolysis was attenuated at higher apoE/TG ratios; nevertheless, apoE2 still inhibited lipolysis significantly more than did apoE4. Enrichment of VLDL with apoE decreased both the apoC contents and apoC-II/C-III ratios of VLDL, contributing, at least in part, to the inhibitory function of apoE on lipolysis. The present study clarifies the differential lipolysis-modulating effect of apoE isoforms, which would help explain the difference in pre- and postprandial TG levels among humans carrying different apoE isoforms.

Effects of drinking and smoking on endogenous levels of urinary gamma-hydroxybutyric acid, a preliminary study.

The aim of this study was to determine if the endogenous levels of gamma-hydroxybutyric acid (GHB) in urine were affected by drinking and smoking. Urine samples were obtained from 20 healthy volunteers (15 males, 21-45 years; 5 females, 22-24 years). This population included four average drinkers (males), 4 average smokers (males), and 12 nonsmokers/nondrinkers (seven males and five females). Urinary levels of GHB were measured by gas chromatography. No gender differences were observed in the urinary levels of endogenous GHB. The urinary levels of GHB in males were 0.52+/-0.37 microg/ml in smokers, 0.28+/-0.21 microg/ml in nonsmokers/nondrinkers, and 0.23+/-0.04 microg/ml in drinkers. Urinary GHB levels were measured three times a day for 5 consecutive days in a male from each group. Large intra-individual differences were observed over the 5-day period in a smoker and a nonsmoker/nondrinker. No significant changes in daily endogenous GHB levels were observed in a drinker during the period. Our preliminary results suggest that stimulatory effects of nicotine on the central nervous system (CNS) may result in an increase in nocturnal formation of GHB and the depressive effects of ethanol on the CNS may not affect, even may inhibit, nocturnal production of GHB.

Adenovirus-mediated gene transfer and lipoprotein-mediated protein delivery of plasma PAF-AH ameliorates proteinuria in rat model of glomerulosclerosis.

Oxidative stress has been proposed to play a crucial role in glomerulosclerosis, although its in vivo demonstration has proved taxing given the difficulty of inducing gene expression in specific renal cells. In this study, we examined whether the liver-directed expression of plasma platelet-activating factor acetylhydrolase (PAF-AH) would affect the glomerular pathophysiology in Imai rats, an animal model for glomerulosclerosis. Adenovirus-mediated liver-directed gene delivery of human PAF-AH resulted in a significant increase in plasma PAF-AH activity, which was detected almost exclusively on HDL. Histological examination of rats overexpressing PAF-AH showed not only the deposition of PAF-AH in mesangial cells, but also a reduction in hydroxynonenal and matrix protein content in the glomeruli. In situ hybridization analysis was negative for human PAF-AH mRNA in the kidney, while injection of HDL abundant in PAF-AH resulted in the deposition of PAF-AH in mesangial cells. Urine protein levels did not increase in rats overexpressing PAF-AH, while those of control rats increased significantly with age. This study provides direct evidence of the in vivo role of an enzyme that degrades lipid peroxides during the progression of glomerulosclerosis. Adenovirus-mediated extrarenal gene expression and lipoprotein-mediated glomeruli-targeted protein delivery promise to be a novel therapeutic approach to glomerulosclerosis.

[Nicotine and cotinine levels in body fluids of habitual smokers who committed suicide].

Suicide is a serious social problem because over 30,000 people commit suicide every year since 1998 in Japan. Cigarette smoking is associated with a higher risk for suicide and attempted suicide. We determined nicotine and cotinine levels in blood and urine of 104 deceased individuals (21 suicides and 83 non-suicides). Of the 21 suicides, 16 (76.2%) were smokers; the smoking rate in non-suicides was 41.0% (34 persons). Average levels of nicotine and cotinine in blood were significantly higher in the suicide smokers than in the non-suicide smokers (nicotine: 95.6 +/- 43.9 ng/ml vs. 28.0 +/- 15.2 ng/ml, p < 0.0001; and cotinine: 385 +/- 220 ng/ml vs. 229 +/- 181 ng/ml, p < 0.02). Average levels of nicotine and cotinine in urine also significantly higher in the suicide smokers than in the non-suicide smokers. There were eight patients with psychiatric diseases such as schizophrenia, depression and alcohol dependence. Of the eight patients, four were suicide smokers; only a person used antipsychotics. Thirty-one alcohol-intoxicated decedents consisted of 8 suicides (8 smokers) and 23 non-suicides (17 smokers). Our data demonstrate that there is a marked increase in cigarette smoking in habitual smokers with psychiatric disorders before committing suicide. Quantitatively monitoring the severity of stress using blood nicotine level may enable physicians more objectively to find out nicotine dependents who are in the state of an imminent suicide attempt and timely to administer medical treatment for preventing suicide.

A fatal poisoning caused by methomyl and nicotine.

A 35-year-old male was found lying in a prone position in his room. He was in cardiopulmonary arrest on arrival to hospital and was pronounced dead. There was no attempt at resuscitation. No miosis was observed on admission. At post-mortem his stomach contained 170 g greenish liquid with a small amount of shredded tobacco leaves. The serum cholinesterase activities were 47-90 IU (normal range for male: 200-440 IU). GC and GC-MS analyses showed nicotine (21.8 mg), methomyl (304 mg), and triazolam (1.69 mg) in his stomach. He had consumed tobacco leaves, Lannate containing water soluble methomyl (45%), and Halcion tablets containing 0.25 mg triazolam. Methomyl concentrations in blood were 3-8 ng/ml. Substantial amounts of methomyl (2260-2680 ng/ml) were detected in cerebrospinal fluid and vitreous humor. Nicotine concentrations in blood ranged from 222 to 733 ng/ml. A small amount of triazolam was detected only in bile (176 ng/ml) and liver (23 ng/g). The cause of death was respiratory paralysis produced by the additive effects of methomyl and nicotine shortly after consumption.

Nicotine and cotinine levels in blood and urine from forensic autopsy cases.

We measured nicotine and cotinine levels in blood and urine from 31 forensic autopsy cases. Initially, we developed a sensitive and reproducible gas chromatographic method with a minimum limit of detection of 2.1 ng/ml for both nicotine and cotinine. Calibration curves for nicotine and cotinine were linear in the ranges of 2.1-1030 ng/ml (r2=0.994-0.999) and 2.1-1380 ng/ml (r2=0.998-0.999) respectively. Our population included 13 smokers and eight of these smokers committed suicide. They showed high levels of nicotine and cotinine at 65.1-205 ng/ml (mean: 115 ng/ml) and 31.3-938 ng/ml (mean: 405 ng/ml) in blood, respectively, and 234-7290 ng/ml (mean: 1940 ng/ml) and 143-4620 ng/ml (mean: 1170 ng/ml) in urine, respectively. None of these individuals consumed nicotine preparations or tobacco leaves. In five smokers who did not commit suicide, nicotine and cotinine levels were 4.4-62.1 ng/ml (mean: 33.2 ng/ml) and 49.9-217 ng/ml (mean: 140 ng/ml) in blood, respectively, and 158-314 ng/ml (mean: 246 ng/ml) and 68.9-300 ng/ml (mean: 179 ng/ml) in urine, respectively. Our results suggest that there may be a marked increase in consumption of cigarettes in smokers with suicidal thoughts.

Postmortem production of ethanol and n-propanol in the brain of drowned persons.

We examined endogenous ethanol and n-propanol levels in the brain in 29 drowning cases in which ethanol consumption was excluded. Based on the stage of putrefaction of the brain, our cases were classified into 4 groups: pulpified brain (PB, n = 11), softened brain (SB, n = 6), discolored brain (DB, n = 2), and normal brain (NB, n = 10). The endogenous ethanol and n-propanol levels (mg/g), respectively, in the brains from these groups were 1.06 +/- 0.401 and 0.076 +/- 0.032 in PB, 0.195 +/- 0.136 and 0.012 +/- 0.009 in SB, and 0.053 +/- 0.032 and 0.001 +/- 0.001 in DB. Ethanol and n-propanol were not detected in NB. The concentration ratios of ethanol to n-propanol were 16.2 +/- 7.1 in specimens with ethanol levels > or = 0.50 mg/g (n = 10), and 17.6 +/- 13.5 in specimens with ethanol levels of 0.10 to 0.49 mg/g (n = 9). Drinking may strongly be suspected when (1) ethanol concentration in the brain is > or = 0.50 mg/g and cerebral ethanol to n-propanol ratio is > or = 40; and (2) the concentration of ethanol is 0.10 to 0.49 mg/g and the ethanol to n-propanol ratio is > or = 60.

Human plasma platelet-activating factor acetylhydrolase binds to all the murine lipoproteins, conferring protection against oxidative stress.

OBJECTIVE: Plasma platelet-activating factor (PAF) acetylhydrolase (AH) is an enzyme bound with lipoproteins that degrades not only PAF but also PAF-like oxidized phospholipids that are proposed to promote atherosclerosis. In this study, we investigated the distribution of PAF-AH protein among lipoprotein classes by using adenovirus-mediated gene transfer in mice, and we examined its effects on lipoprotein oxidation and foam cell formation of macrophages. METHODS AND RESULTS: Adenovirus-mediated overexpression of PAF-AH in mice resulted in a 76- to 140-fold increase in plasma PAF-AH activity. Contrary to the previous report, overexpressed human PAF-AH protein was bound to very low density lipoprotein, intermediate density lipoprotein, low density lipoprotein, and high density lipoprotein (HDL). All the lipoproteins with overexpressed human PAF-AH revealed more resistance against oxidative stress, which was associated with lower levels in autoantibody against oxidized low density lipoprotein in the plasma. In addition, HDL with human PAF-AH inhibited foam cell formation and facilitated cholesterol efflux in macrophages. CONCLUSIONS: These results suggest that human plasma PAF-AH exerts an antiatherogenic effect by binding to all the lipoproteins and thereby protecting them from oxidation, producing less proatherogenic lipoproteins and preserving HDL functions.

Isoform-dependent cholesterol efflux from macrophages by apolipoprotein E is modulated by cell surface proteoglycans.

OBJECTIVE: Apolipoprotein E (apoE) mediates cellular cholesterol efflux and plays a crucial role in the inhibition of atherogenesis. We investigated whether there is an isoform-specific difference in its function for cholesterol efflux from cholesterol-loaded RAW264.7 cells, a murine macrophage cell line that lacks endogenous apoE expression. METHODS AND RESULTS: When human apoE was expressed in RAW264.7 cells, apoE2 reduced cellular total cholesterol (TC) and esterified cholesterol (EC) levels significantly, whereas apoE3 and apoE4 had no effect. However, treatment of cells with 4-methylumbelliferyl-7-beta-D-xyloside (beta-DX) resulted in all 3 isoforms' reducing cellular TC and EC contents significantly. We also investigated the effect of exogenously derived apoE on cholesterol efflux by utilizing the medium harvested from HeLa cells expressing apoE. ApoE2 and E3 reduced both cellular TC and EC contents significantly, whereas apoE4 did not. However, treatment of the cells with beta-DX resulted in all 3 exogenously derived apoE isoforms' reducing TC and EC contents significantly. The binding ability of apoE to heparan sulfate proteoglycans examined by heparinase I treatment revealed less binding ability of apoE2 compared with that of apoE3 or apoE4. CONCLUSIONS: The present study clarified the differential cellular cholesterol-modulating effect of apoE isoforms in macrophages, which would be due to the difference in their binding to proteoglycans.

Pilot clinical trial of the use of alpha-tocopherol for the prevention of hepatocellular carcinoma in patients with liver cirrhosis.

Patients with chronic hepatitis C virus (HCV) infection often develop liver cirrhosis and hepatocellular carcinoma (HCC). The purpose of this study was to test the chemopreventive effect of alpha-tocopherol on hepatocarcinogenesis in patients with liver cirrhosis and a history of HCV infection. Eighty-three patients with liver cirrhosis and with positive history of HCV infection were divided at random into two groups. Forty-four patients were treated with alpha-tocopherol (Vit E group) while the other 39 were followed as controls. The clinical background (gender, age, and laboratory data) was similar in the two groups. Serum levels of alpha-tocopherol, albumin, alanine aminotransferase (ALT), and total cholesterol and platelet count were measured serially over a period of five years. The mean serum concentration of alpha-tocopherol was low in both groups at entry and was significantly higher in the Vit E group than in the control group one month after treatment. Platelet count, serum albumin, ALT, and total cholesterol were not different between the two groups during the five-year period. Cumulative tumor-free survival and cumulative survival rate tended to be higher in the Vit E group than in controls, albeit statistically insignificant. The serum level of alpha-tocopherol was low in patients with liver cirrhosis and positive for HCV. Although the administration of alpha-tocopherol normalized the level one month later, it could neither improve liver function, suppress hepatocarcinogenesis, nor improve cumulative survival. Patients treated with alpha-tocopherol tended to live longer without development of HCC but the difference was not statistically significant.

Apolipoprotein A-I deficiency with accumulated risk for CHD but no symptoms of CHD.

We evaluated a 69-year-old Japanese woman with apolipoprotein (apo) A-I deficiency, high levels of low-density lipoprotein (LDL)-cholesterol, hypertension and impaired glucose tolerance. The patient had corneal opacity, but neither xanthomas, xanthelasma, nor tonsillar hypertrophy. She was not symptomatic for coronary heart disease (CHD), and had normal electrocardiograms at rest and exercise using a cycle ergometer. She had severely reduced levels of high-density lipoprotein (HDL)-cholesterol (0.10-0.18 mmol/l) and no apo A-I (<0.6 mg/dl). LDL-cholesterol and apo B as well as apo E were increased even under treatment with 10 mg pravastatin per day. Gel filtration chromatography revealed that in addition to VLDL and LDL fractions, she had apo A-II rich and apo E rich fractions, which were present in the HDL fraction separated by ultracentrifugation. A cytosine deletion was identified by genomic DNA sequencing of the apo A-I gene of the patient at the third base of codon 184 in the fourth exon, which led to a frame shift mutation and early termination at codon 200. This patient is the oldest among those with apo A-I deficiency reported in the literature, and she had no symptoms of CHD despite the accumulated risk for the disease.