Search

Lacosamide is a relatively new antiepileptic drug that exerts its anticonvulsant effect by selectively inactivating sodium channels. Since its launch, it has been used widely for the treatment of intractable epilepsy, but there are scant data on the toxic or lethal blood concentrations. Here, we report a case of drug poisoning following simultaneous high-dose self-administration of lacosamide and mirtazapine. We developed and validated an approach that uses liquid chromatography coupled with electrospray ionization-tandem mass spectrometry to determine the concentrations of lacosamide and mirtazapine in cadaveric blood, urine and liver. Calibration curves showed good linearity (r 2  > 0.995), and our method enabled repeatable and accurate quantification, with intra- and inter-assay coefficients of variation not exceeding 10.9 % and 12.8 %, respectively, for each target drug. We used the method to measure the drug concentrations in the blood of a dead victim and found a lacosamide concentration of 91.9 μg/mL and a mirtazapine concentration of 12.0 μg/mL. The blood mirtazapine concentration was in the lethal range, and that of lacosamide was about 10 times the therapeutic range. The synergistically central nervous system depressive and cardiotoxic effects of these drugs may have contributed to the cause of death. We concluded that the cause of death in this case was lacosamide and mirtazapine poisoning., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Detecting cyanide compounds in postmortem blood samples is an important matter in forensic science because cyanide is often used as a poison for murder or suicide. However, the direct analysis of cyanide itself has practical limitations because of cyanide's volatility and short half-life at ambient temperature. Here, we focused on the relatively stable cyanide metabolites 2-aminothiazoline-4-carboxylic acid (ATCA) and 2-aminothiazoline-4-oxoaminoethanoic acid (ATOEA) as potential markers of cyanide exposure. We developed an analytical method that uses chemical derivatization of the target compounds with 4-bromoethyl-7-methoxycoumarin followed by liquid chromatography coupled with electrospray ionization-tandem mass spectrometry. The recovery rates for pretreatment and calibration curve linearities were good in the concentration range of 20-1000 ng/mL. Using our approach, we were able to detect and quantify both ATCA and ATOEA concentrations in postmortem blood samples, and in our samples the ratio of ATCA and ATOEA was in the range of 4.5-19.1. To our knowledge, this is the first time ATOEA has been successfully detected in human blood samples. In addition, we found that ATCA and ATOEA concentrations were both significantly higher in the blood of fire victims than in the blood of individuals with a non-fire-related cause of death. Also, we found that there was a significant positive correlation between ATCA concentrations and ATOEA concentrations. Together, our present data suggested that ATCA and ATOEA are both potential markers of cyanide exposure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Dried blood spot (DBS) sampling has evolved to become the method of choice for collecting samples for newborn screening and therapeutic drug monitoring worldwide. The major advantage of this approach is that it requires only a small amount of blood. In addition, the collection of DBSs on filter paper is simple, sample storage costs are small, and the process deactivates microorganisms and viruses. However, despite these advantages, DBS sampling is seldom used in forensic toxicological analyses. Here, we developed and validated an approach that uses liquid chromatography coupled with electrospray ionization-tandem mass spectrometry for quantifying nine psychotropic drugs (citalopram, duloxetine, mirtazapine, olanzapine, paroxetine, quetiapine, sertraline, zolpidem and zopiclone) in cadaveric DBS samples. Most of them are frequently used by self-harm but are not already targeted by an existing drug screening kit. Our method use only one 3-mm disk excised from each DBS and does not require the troublesome purification process. The linearities of the calibration curves were good in the concentration range of 0.05-1.0 μg/mL. Our method allows for repeatable and accurate quantification with intra- and inter-assay coefficients of variation of below 11.9% and below 12.5%, respectively, for each of the target drugs. In addition, the target drug concentrations in the DBSs remained stable for at least one month when stored at - 80 °C. Compared with our institute's routine method for cadaveric blood sampling, the QuEChERS method, quantifiable concentrations showed a good positive correlation for each of the target drugs. In addition, the concentrations of almost all the target drugs obtained with DBS sampling method were comparable with those obtained with the QuEChERS sampling method. Thus, the present findings extend the possible uses of DBS sampling to the quantification of multiple psychotropic drugs in the field of forensic toxicological testing., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

In this study, we assessed 80 autopsy samples to investigate the relationships between cause of death and the concentrations of multiple steroids in serum and cerebrospinal fluid (CSF). First, we developed and validated analytical methods to quantify seven steroids (cortisol, cortisone, corticosterone, 11-deoxycortisol, 11-deoxycortiocosterone, progesterone, and testosterone) by using liquid chromatography coupled with electrospray ionization-tandem mass spectrometry. Next, we statistically evaluated the levels of each steroid for six causes of death: hypothermia, traumatic injury, fire fatality, asphyxia, intoxication, and internal disease. We observed that cortisol concentrations in serum and CSF obtained from cadavers who died from hypothermia were significantly higher than those in samples obtained from cadavers who died from the remaining causes of death (P < 0.05). Similarly, corticosterone concentrations obtained from cadavers who died from hypothermia were significantly higher than those in samples from several other causes of death. However, concentrations of the remaining steroids analyzed did not differ significantly among the causes of death. We further elucidated the correlations between steroid concentrations in serum and CSF. Except for 11-deoxycorticosterone and progesterone, steroid concentrations were significantly positively correlated in serum and CSF. Although data on cadaveric steroid concentrations are limited-especially in CSF-values obtained were in the approximate range of the living human data reported to date., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd and Faculty of Forensic and Legal Medicine. All rights reserved.)

2-Aminothiazoline-4-carboxylic acid (ATCA), which is produced by the reaction of cyanide with endogenous cystine, is a promising biomarker of cyanide exposure because of its physicochemical stability. Analysis of more stable metabolite than the toxic gas itself is sometimes useful for postmortem diagnosis of gas poisoning. Here, we developed and validated an approach that uses liquid chromatography coupled with electrospray ionization-tandem mass spectrometry for quantifying ATCA in dried blood spot (DBS) samples. The linearity of the calibration curve was good in the concentration range of 20-1500 ng/mL. Our method allows for repeatable and the accurate quantification of ATCA, with intra- and inter assay coefficients of variation of below 7.8 % and below 9.3 %, respectively. In addition, the concentration of ATCA in DBSs remained stable for at least one month when stored at -20°C. Our results indicated that our analytical approach can be used to determine past exposure to higher doses of cyanide. In a comparison of ATCA concentrations in DBSs obtained from cadavers with various causes of death, significantly higher ATCA concentrations were observed in fire victims than in non-fire victims, confirming that fire victims inhale large amounts of cyanide gas. Thus, here we extended the possible uses of DBS for quantification of ATCA to forensic toxicological testing for cyanide poisoning., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Recently, 2-aminothiazoline-4-carboxylic acid (ATCA), a cyanide (CN) metabolite, has been proposed as a stable diagnostic marker of CN poisoning. In this study, liquid chromatography coupled with electrospray ionization - tandem mass spectrometry was used to quantify ATCA concentrations in human postmortem blood samples, and differences in ATCA concentrations according to age and sex were determined. Both age and sex had significant effects on blood ATCA concentrations. Although ATCA concentrations exhibited an inverted U shape with increasing age in men, in women ATCA concentrations plateaued at around 40-59 years of age. There were significant differences between the sexes in ATCA concentrations for the 20-39 and 40-59 year age groups (P < 0.05 and P < 0.01, respectively). Correlations between ATCA concentrations and carboxyhemoglobin (CO-Hb) saturation were also examined in fire victims. ATCA concentrations increased significantly with increasing CO-Hb saturation (r = 0.382, P < 0.01). In addition, ATCA concentrations were also correlated to CN concentrations (r = 0.309, P < 0.05). The results of our study may provide novel information about the contribution of CN poisoning to the cause of death at fire scenes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Direct detection and accurate quantification of chlorine in autopsy samples are difficult because of the volatility and rapid metabolism of chlorine. Here, we developed and validated a method for quantitative analysis of 3-chloro-l-tyrosine (Cl-Tyr) and 3,5-dichloro-l-tyrosine (DiCl-Tyr) as stable markers of chlorine exposure. Chemical derivatization followed by liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) enabled us to simultaneously analyze both Cl-Tyr and DiCl-Tyr in an autopsy sample from the victim of chlorine exposure. Cl-Tyr was detected in the heart blood (53.6 ng/mL), urine (9.5 ng/mL), and lung tissue (211.1 ng/g); however, DiCl-Tyr was detected only in the lung tissue (10.3 ng/g). In contrast, in autopsy samples obtained from cases without exposure to chlorine, DiCl-Tyr was not detected in any matrixes. Our result suggested that the simultaneous detection of Cl-Tyr and DiCl-Ty may provide a better appreciation of chlorine exposure. To our knowledge, this is the first time Cl-Tyr and DiCl-Tyr have been determined simultaneously in a real human autopsy sample from a victim of chlorine exposure., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objective: SARS-CoV-2 is the cause of COVID-19, the rapidly spreading pandemic. When SARS-CoV-2 enters the target cells in the respiratory system, the spike glycoprotein binds to a cellular receptor angiotensin converting enzyme 2 (ACE2). The susceptibility to infection in individuals under 20 years of age is approximately half that of adults aged over 20 years. In this study, we investigated the immunohistochemical protein expressions of ACE2 in mandibular salivary glands and tracheal glands from forensic autopsy specimens covering adults and children., Results: The ACE2 immunohistochemistry of autopsy specimens was performed, and the percentages of the immuno-positive areas in the cell layers of the glands were calculated. Our results demonstrate that the ACE2 positivity in mandibular salivary gland and tracheal glands showed the statistically significant decrease with the increase of age, which indicates that the susceptibility of aged individuals to SARS-CoV-2 may be due to various factors including but not limited to ACE2 protein expressions., (© 2022. The Author(s).)

In this research, we have developed a novel and simple liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for quantification of 2-aminothiazoline-4-carboxylic acid (ATCA), which is produced by the direct reaction of cyanide (CN) with endogenous cystine. In forensic science, detection of CN is important because CN is a poison that is often used for murder or suicide, in addition to being produced by the thermal decomposition of natural or synthetic materials. However, because CN disappears rapidly from body tissue, ATCA is thought to be a more reliable indicator of CN exposure. For the method reported herein, human blood samples (20 μL) were subjected to protein precipitation followed by derivatization with 4-bromoethyl-7-methoxycoumarin. Blood spiked with ATCA at concentrations ranging from 50 to 1500 ng/mL was used to prepare a calibration curve (lower limit of quantification; 50 ng/mL, lower limit of detection; 25 ng/mL). Our method uses chemical derivatization, so unlike previously reported methods, it does not require tedious pretreatment procedures, hydrophilic interaction liquid chromatography columns, or specialized equipment. In addition, our method allows for repeatable and accurate quantification of ATCA, with intra- and inter-assay coefficients of variation of below 5.0% and below 6.0%, respectively. We used the method to analyze ATCA in postmortem human blood samples, including samples from people who had intentionally ingested CN or were fire victims. Blood ATCA concentrations were higher among people who had ingested CN or were fire victims than among people in a control group (P < 0.0001). The data reported herein demonstrate that our LC/ESI-MS/MS method can be used to detect and quantify ATCA in postmortem blood samples and that CN exposure strongly affects ATCA concentration, providing a useful tool for detection of CN poisoning., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Acetone cyanohydrin (ACH) is a readily available source of cyanide and is widely used in basic and applied sciences. In toxicology, ACH is classified as extremely hazardous as it readily decomposes on contact with water, with the potential rapid release of highly toxic hydrogen cyanide (HCN). We report the case of a young woman found dead from the intentional ingestion of ACH and citalopram, an antidepressant of the selective serotonin reuptake inhibitor class. The autopsy findings included bright reddish-purple hypostasis and mild pulmonary edema. As ACH can decompose to acetone and HCN, we quantified the concentration of each compound and thiocyanate separately in various body fluids and organs and determined their whole-body distributions by using gas chromatography-mass spectrometry (GC-MS). We observed high concentrations of both acetone and cyanide in the blood (0.63 mg/mL and 17.99 mM, respectively) and gastric contents (9.76 mg/mL and 472.44 mM). The whole-body distributions of acetone and cyanide were similar (i.e., the concentration of each compound was the highest in the lung, followed by the heart, and then the liver). Our results suggest that not only the route of administration but also the dose taken could greatly affect the body distributions of cyanide in humans. In addition, as toxicological screening detected citalopram, which was not prescribed to the deceased, we performed a chiral analysis by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We determined that only (S)-citalopram was ingested antemortem; its concentration was 0.36 μg/mL, which is in the toxic range., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A simple and sensitive liquid chromatography coupled with electrospray ionization-tandem mass spectrometry (LC/ESI-MS/MS) method for the determination of 3-chloro-L-tyrosine (Cl-Tyr) was developed and validated. For sample preparation, 50 μL of the body fluids or tissue extracts were processed by protein precipitation followed by the derivatization with dansyl chloride. The calibration curve was linear over the concentration range of 2.0-200 ng/mL blood or 4.0-400 ng/g tissue. Our method allowed the reproducible and accurate quantification. That is, the intra- and inter-assay coefficients of variation were below 7.73 and 6.94%, respectively in both the blood and lung. We applied the developed method to the analysis of Cl-Tyr in the human autopsy samples, which were suspected of chlorine poisoning, and detected 55.2 ng/mL and 206.6 ng/g Cl-Tyr in left heart blood and lung, respectively. Furthermore, in more than 20 autopsy samples, which were obtained from other causes of death including burn, drowning, hanging, internal disease, trauma and drug poisoning, Cl-Tyr was almost not detected in their both body fluids and organ tissues. In conclusion, the data here reported demonstrate that the LC/ESI-MS/MS method allows the Cl-Tyr in the autopsy samples and that chlorine exposure strongly affects its level, providing a basis for novel identification tool of chlorine poisoning., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Hypothermia is an important cause of death in forensic pathology. For the forensic diagnosis of hypothermia, some reports point out the possibility that hypothermia without diabetes may cause ketoacidosis. In this study, we evaluated the diagnostic value of ketoacidosis in a murine model of hypothermia, using the cold stress at 4 °C for 3 or 5 hrs in genetically diabetic (BKS.Cg-+Lepr db /+Lepr db /J) mice, compared with control (BKS.Cg- Dock7 m +/Dock7 m +/J) mice. The core temperature decrease was larger in diabetic mice than in control mice. We observed a novel finding that ketoacidosis assessed by elevated serum 3-hydroxybutyrate (3HB) occurs in hypothermia both in diabetic and control mice. Diabetic mice showed a prominent elevation of serum 3HB under cold stress. The protein expressions of monocarboxylate cotransporter 1 (MCT1), the channel protein used for the uptake of 3HB in skeletal muscles, showed a statistically significant decrease under cold stress for 3 hrs in control mice, indicating that the serum 3HB increase may be partially due to the decrease in the cellular uptake through the channel protein. Our results suggest the usefulness of hyperketonemia for the diagnosis of hypothermia not only in diabetic but also in non-diabetic cases., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We herein report 2 cases of gastric cancer treated by S-1 and oxaliplatin combination therapy before later undergoing gastrectomy. The pathological results of both cases demonstrated complete response. Case 1 had a giant tumor which was suspected to have invaded the pancreas. Case 2 was associated with extensive lymph node metastasis. Based on the findings of these 2 cases, preoperative chemotherapy with S-1 and oxaliplatin for advanced gastric cancer shows sufficient efficacy.

Chlorine gas exposure occurs in chemical warfare, industrial and household accidents. In forensic science, the generation of chlorine gas by mixing sodium hypochlorite detergent and strong acid detergent cannot be overlooked because of the possibility of suicide method (NaClO + 2HCl → NaCl + H 2 O + Cl 2 ). Though typical autopsy findings are obtained in chlorine exposure, such as pulmonary edema, useful biomarkers don't exist. In this research, we developed an analytical method of 3-chloro-l-tyrosine (Cl-Tyr) in blood as a novel marker of chlorine poisoning utilizing gas chromatography-mass spectrometry (GC-MS). Cl-Tyr was purified using protein precipitation and cation-exchange solid phase extraction, derivatized by the silylation agent and subjected to GC-MS. The quantification range was 10-200 ng/mL and good reproducibility was obtained. We applied the developed method to analyze Cl-Tyr in autopsy sample, which is suspected of chlorine poisoning, and detected 59.7 ng/mL Cl-Tyr in left heart blood. To our knowledge, this is the first report of determination of the chlorinated biomolecule in the human autopsy sample from chlorine poisoning., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Objective: Advanced glycation end products (AGEs) are known to play important roles in the development of diabetes mellitus and atherosclerosis.　N ε -(carboxymethyl)lysine (CML) is the major AGE, and is found in the arterial walls in the heart. The CML involvement in myocardial ischemia has been reported. We studied the immunohistochemical localization of CML in the hearts from forensic autopsies in relation to the age, serum N-terminal-pro basic natriuretic peptide (NT-proBNP), heart weights, and the degree of peri-myocardial fibrous tissues reflecting coronary microvascular infarction and myocardial remodeling., Results: The CML immunoreactivity in the endothelial cells and intima of arterial walls in the interstitium of ventricular muscles was significantly stronger in the aged group, compatible with the progression of atherosclerosis. The blood level of NT-proBNP, a known useful marker for heart failure, had the positive correlation with the CML immunoreactivity. The degree of fibrosis, heart weights and the histories of hypertension and hyperlipidemia showed positive correlations with the CML immunoreactivity. Our results show the novel positive correlation between the CML immunohistochemistry in the heart vessels and heart conditions, and its future usefulness in the cardiovascular evaluation in histopathology.

Identification of fly larvae species may offer valuable information as to the location, or the environment in which corpses were placed, but only if the geographical distribution of larva species is clarified. In this study, we investigated a total of 126 larvae on 42 corpses found in Saitama Prefecture in Japan between July and September. We identified the larva species by analyzing the sequences of mitochondrial DNA cytochrome oxidase gene subunit I. Our results revealed that larvae belonged to 6 different species: Lucilia sericata and Chrysomya pinguis from the Calliphoridae family, and Parasarcophaga crassipalpis, Boettcherisca peregrina, Parasarcophaga harpax, and Parasarcophaga dux from the Sarcophagidae family. Additionally, we investigated if there was a correlation between larvae species and population density. Based on the random sampling and the statistical analysis on the entire larva collection, larvae of Chrysomya pinguis species were more likely to be found in low population density areas, whereas larvae of Lucilia sericata were commonly found in high population density areas. The accumulation of distribution data of larvae may be useful to confirm the environment around the place where corpses were found., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Wound healing evaluation is important in forensic pathology, in which angiogenesis plays an important role. We have already shown that vascular endothelial growth factor A (VEGF) is produced in the rat skin incision wounds by neutrophils, endothelial cells, and fibroblasts. In this study, we assessed the changes in the mRNA expressions of various factors possibly involved in angiogenesis including angiopoietin (ANGPT) 1 and 2, cadherin 5 (CDH5), granulocyte-macrophage colony stimulating factor (CSF2/GM-CSF), granulocyte colony stimulating factor (CSF3/G-CSF), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand12 (CXCL12/SDF1), endothelin 1 (ET1), fibroblast growth factor 1 (FGF 1), hepatocyte growth factor (HGF), hypoxia inducible factor 1 alpha (HIF1a), leptin, matrix metallopepitidase 9 (MMP9), serpine/plasminogen activator inhibitor1 (PAI1), platelet-derived growth factor-A (PDGF-A), transforming growth factor alpha and beta 1 (TGFa and b1), tenomodulin (TNMD), and troponin I type 2 (TNNI2) in the early stage of the rat skin incision wounds by real time RT-PCR. Factors reported to be involved in lymphangiogenesis such as fibroblast growth factor 2 (FGF 2), c-fos induced growth factor (FIGF/VEGF-D), forkhead box C2 (FOXC2), and prospero homeobox 1 (PROX1) were also studied. One and 3 days after the dorsal skin incisions, wounds on male Sprague-Dawley rats showed the statistically significant increases in the mRNA expressions for CXCL2, CSF3, MMP9, PAI1, and CSF2, whereas TGFa, TNNI2, FGF1, TNMD, leptin, and CXCL12 showed the statistically significant decreases. Interestingly, lymphgangiogenic factors FOXC2, PROX1, and FGF2 also showed the statistically significant decreases. In situ hybridization and immunohistochemistry showed the mRNA and protein positivity in endothelial cells, fibroblasts, and some leukocytes at the bottom of the wound tissue for PAI1, CSF3, and MMP9, 1 day after the skin incisions. Our novel findings show the possible involvement of several factors involved in angiogenesis and lymphangiogenesis in the early stage of wound healing process, which may be useful for forensic wound evaluations., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

A rare autopsy case of the extremely large retroperitoneal solitary fibrous tumor is reported. A 52-year-old female with a huge abdominal distention was found dead at home. She showed remarkable emaciation. The autopsy revealed a huge retroperitoneal tumor weighing 11.9kg (36×30×20cm in size), which occupied the entire intraperitoneal cavity. Histologically, the tumor consisted of spindle parenchymal cells with fibrous tissues. Immunohistochemically, CD34 was positively stained, whereas S-100, smooth muscle actin, and factor VIII were negative. Her cause of death was diagnosed as emaciation due to the compression of the entire intestine by the tumor. This is a rare case of the extremely large retroperitoneal solitary fibrous tumor, which caused the occasional intestinal obstruction. This disease should be considered in the differential diagnosis of retroperitoneal large tumors that cause accidental deaths in forensic autopsies., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Wound healing evaluation is important in forensic pathology. We have already shown that vascular endothelial growth factor (VEGF) is produced in the rat skin incision wounds. In this study, we used cyclophosphamide hydrate (CPM) to induce leukocytopenia in rats, and measured VEGF in the skin incision wound area to assess the involvement of leukocytes in the early production of VEGF. Male Sprague-Dawley (SD) rats were intraperitoneally administered CPM 75 mg/kg body weight on day 0 and 5, and dorsal skin incision wounds were made on day 5. One and 3 days after the skin incision, leukocytes counts were determined and skin specimens from the wounds were collected for measurements of total proteins, VEGF proteins, and semi-quantification of VEGF mRNA. VEGF immunohistochemistry and in situ hybridization for VEGF mRNA were also performed. VEGF proteins were smaller in the amount statistically significantly in the 1- and 3-day-old wounds of CPM-induced leukocytopenic rats, whereas VEGF mRNA was increased compared with saline-treated control rats. Immunohistochemically, VEGF was positive in leukocytes and mesenchymal cells including fibroblasts and endothelial cells in the 3-day-old wound of saline-administered control rats, while a few fibroblasts and endothelial cells were positively stained in CPM-administered rats. In situ hybridization showed the localization of VEGF mRNA in mesenchymal cells including fibroblasts and endothelial cells in the 1-day-old wound of CPM-administered rats, whereas saline-administered control rats also showed VEGF mRNA positivity in leukocytes. Our study indicates that leukocytes may be the major source of VEGF in the early stage of the rat skin incision wound., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

In the wound healing process, angiogenesis is involved in the recovery of vasculature, and its process has been investigated. On the other hand, the reconstruction of lymphatic vessels in the injured subcutaneous tissue has not been studied in detail. We studied the recovery of lymphatic vessels using podoplanin immunohistochemistry in the paraffine section microscopy of the rat skin incision wound. Our result indicates a novel finding that subcutaneous tissue of the incised skin area does not show any recovery of lymphatic vessels up to 84 days after the skin incision. As the wound area shrunk, the surrounding subcutaneous tissue covered with the normal skin epithelial cells approached toward the center of the wound, and the lymphatic vessels in the surrounding tissue gradually reached the incision wound area. On the other hand, the regeneration of the vasculature occurred within the wound area as assessed by CD31 and von Willebrand Factor (vWF) immunohistochemistry. This difference was confirmed by the morphometric quantification of podoplanin- or vWF-positive vessels. Our results show that there is a clear difference in the recovery pattern of vascular and lymphatic vessels in the skin wound healing process.

Previously, we demonstrated that CPT-11 is an effective agent against esophageal squamous cell cancers (ESCC), and that the protein level of DNA topoisomerase I can be a predictor for sensitivity to CPT-11 (Jpn J Cancer Res 2001; 92: 1335-41). Here, we describe our search for additional predictors of sensitivity to CPT-11, mainly among cell cycle-regulating proteins, because the cytotoxicity of CPT-11 is significantly correlated with the percentage of ESCC cells in S-phase. To this end, we selected and examined the expressions of 5 proteins involved in G1-S transition, i.e., p53, cyclin D1, p21, p27, and pRB, in 14 ESCC cell lines by western blot analysis. Among these proteins, the expression levels of p21 and pRB showed significant differences that were associated with the IC50 values for CPT-11 (P = 0.0339 and P = 0.0109, respectively). Namely, the expression of p21 or pRB independently could be a good indicator of CPT-11 efficacy in ESCC. In addition, the cell proliferation activities examined by enzyme-linked immunosorbent assay (ELISA) using 5-bromo-2'-deoxyuridine (BrdU) showed a significant correlation with the percentage of total S-phase cells (correlation coefficient = 0.568, P = 0.0324), and an inverse correlation with the IC50 values for CPT-11 (correlation coefficient =-0.601, P = 0.0213). Because, as in the case of DNA topoisomerase I, the cell proliferation activity determined using BrdU shows a close relationship with the MIB-1 labeling index, immunohistochemical studies of p21, pRB, and MIB-1 in resected ESCC specimens and/or biopsy samples could make it possible to predict more precisely the sensitivity of ESCC patients to CPT-11 prior to treatment.

A 57-year-old female diagnosed with advanced gastric cancer with multiple organ metastases was treated by various intra-arterial chemotherapies. After surgical resection of the tumor, adjuvant chemotherapy was carried out. Continuously administered 5-fluorouracil of 250 mg/day made it possible to control the growth of the liver metastases. Extrahepatic metastases were kept under control by administering 30 mg of methotrexate, 750 mg of 5-fluorouracil and 30 mg of Leucovorin per/day/week, and 60 mg/day biweekly of cisplatinum via an abdominal artery infusion port. Owing to this multiple infusion route and chemotherapy regimen, the patient lived for 18 months after her first diagnosis of gastric cancer with multiple liver metastases. Although liver metastases may respond to hepatic arterial infusion chemotherapy, extrahepatic metastases lead to poor prognosis. Given the above results, intra-abdominal aorta chemotherapy may be effective for extrahepatic metastases since this method gives high concentration of the anticancer agents at tumor sites with a low incidence of side effects.

We studied the pharmacokinetics of the intraperitoneal administration of CPT-11 for four patients with peritoneal metastasis (2 gastric cancer cases, 2 colon cancer cases). CPT-11 was administrated in a dose of 40-60 mg and the intraperitoneal and serum levels of CPT-11, SN-38 and SN-38 glucuronized (SN-38 Glu) were measured periodically. Intraperitoneal therapy with CPT-11 was effective for the control of malignant ascites. No serious side effects were observed. The levels of CPT-11, SN-38 were no different 30 min afterwards the administration of CPT-11 either intraperitoneally or intravenously. The high concentration of CPT-11 was achieved with intraperitoneal therapy and a small fraction of CPT-11 changed into SN-38 in the abdominal cavity.

The authors analyzed the histological effect of preoperative chemotherapy for 62 advanced colorectal cancer patients using resected specimens. Thirty-one patients in the 5'-DFUR + LV group received 800 mg/day of 5'-DFUR and 30 mg/day of leucovorin for 10-14 days just before the operations. Thirty-one patients in the 5'-DFUR group received 5'-DFUR 800 mg/day during the same period. None of the patients in either group developed any side effects. The results of the histological examination showed the number of Grade 1a and 1b cases in the 5'-DFUR + LV group was 22 (66.7%) and 5 (15.2%), respectively, and in the 5'-DFUR group 21 (65.6%) and 5 (15.6%). In the 5'-DFUR + LV group, 3 lesions showed Grade 2 histological degeneration, while there were no such lesions in the 5'-DFUR group. However, our results did not demonstrate any statistical difference between the two groups (p = 0.25, U test).

The results and problems of hepatic artery infusion therapy (HAI) for gastric carcinoma with synchronous liver metastasis were evaluated. The response rate of HAI with CDDP and 5-FU for metastatic liver tumor was 55% (1 CR + 5 PR/11). The median survival time for responders was 16.5 months, which was statistically longer than that of non-responders at only 5.5 months. Histologically, most responder cases were with AFP producing tumors and NSE positive tumors without distant lymph node involvement. Non-responder cases developed marked distant lymph node involvement besides the liver metastasis. Most of responder patients died of lymph node recurrence or distant metastasis other than liver tumor. It may be concluded that additional therapy to HAI is needed to improve the prognosis of gastric cancer patients with multiple liver metastases.

The metabolism of CPT-11 in malignant ascites of gastric cancer patients with peritoneal seedings was studied in advance of the intraperitoneal chemotherapy of CPT-11 in humans. Malignant ascites and blood were drawn from gastric cancer patients. CPT-11 solution (20 mg/ml; 0.2 ml) was added into 3.8 ml ascites or plasma under 37 degrees C and CPT-11, SN-38 and SN-38GLU concentrations were measured with HPLC at times of 5, 30 and 60 minutes after addition of CPT-11. The change from CPT-11 to SN-38 was minimal not only in plasma, but also in malignant ascites. SN-38 GLU concentration was below the limit of measurement. This study showed that in malignant ascites, the enzymes such as carboxyesterase that convert CPT-11 to SN-38 were not present or minimal.