Immunohistochemical and morphometrical study on the temporal expression of interleukin-1α (IL-1α) in human skin wounds for forensic wound age determination

An immunohistochemical and morphometrical study on the temporal expression of interleukin-1α (IL-1α) was performed on 40 human skin wounds with different wound ages. Immunohistochemically, polymorphonuclear neutrophils mainly showed positive reactions for IL-1α in wounds aged between 4 h and 1 day, but with increasing wound age, neutrophils were no longer present, and macrophages and fibroblasts were positively stained. Morphometrically, the ratio of the number of IL-1α-positive infiltrating cells to the total number of infiltrating cells was evaluated. A considerable increase in the IL-1α-positive cell ratio was observed in wound specimens aged 4 h to 1 day, and the maximum ratio was 46.5% in a 7 h-old wound. The mean value of the IL-1α-positive ratio in 10 wound specimens with different wound ages between 4 h and 1 day was 32.8 ± 9.7%. In most cases the ratio of IL-1α-positive cells gradually decreased in wounds aged between 1.5 and 21 days to less than 30%, and the mean value was 17.5 ± 7.2% (n = 27). These results suggest that ratios of IL-1α-positive cells considerably exceeding 30%, indicate a postinfliction interval of 1 day or less. Received: 24 August 1998 / Received in revised form: 21 October 1998     

Effects of NF-κB1 (p50) targeted gene disruption on ionizing radiation-induced NF-κB activation and TNFα, IL-1α, IL-1β and IL-6 mRNA expression in vivo

Purpose : To investigate the role of the NF- κB1 (p50) gene in ionizing radiation (IR)-induced NF- κB activation and TNF α, IL-1 α, IL-1 β and IL-6 mRNA expression in vivo. Materials and methods : NF- κB activation was analysed by the gel shift/supershift assay and the levels of TNF α, IL-1 α, IL-1 β and IL-6 mRNA were measured using RNase protection assay (RPA). Various tissues from BALB/c, B6,129P-Nfkb1 (NF- κB1 or p50 gene knockout, p50 -/-) and B6,129PF2 (wild-type, p50 +/+) mice were analysed before or after exposure to a lethal dose (8.5 Gy) of total-body γ-irradiation. Results : Exposure of BALB/c mice to total-body IR selectively activated NF- κB in the spleen, mesenteric lymph nodes (LN) and bone marrow (BM). Gel supershift assay using polyclonal antibodies against NF- κB p50, p65 or c-Rel protein revealed that the NF- κB p50 subunit is a critical component of the NF- κB complexes activated by IR in vivo. Discretely augmented TNF α, IL-1 α, IL-1 β and IL-6 mRNA expression was found in the spleen, LN and BM after BALB/c mice received IR. However, mice lacking the p50 gene (p50 -/-) showed a significant reduction in IR-induced activation of NF- κB and increases in TNF α, IL-1 α, IL-1 β and IL-6 mRNA expression, as compared with that of wild-type mice (p50 +/+) . Conclusions : The NF- κB p50 subunit is a critical component of the NF- κB complexes activated by IR and it plays an important role in mediating IR-induced TNF α, IL-1 α, IL-1 β and IL-6 mRNA expression in vivo.     

Hypoxia inducible factor (HIF1α and HIF2α) and carbonic anhydrase 9 (CA9) expression and response of head-neck cancer to hypofractionated and accelerated radiotherapy

Purpose: Tumor hypoxia and low intrinsic radiosensitivity may counteract the efficacy of standard radiotherapy for locally advanced head and neck cancer (HNC). We investigated the involvement of hypoxia-regulated proteins (Hypoxia inducible factors HIF1α, HIF2α and carbonic anhydrase CA9) in HNC resistance to accelerated and hypofractionated radiotherapy.

Materials and methods: Thirty-nine patients with locally advanced HNC received 15 daily fractions of 3.4 Gy amounting to a total tumor dose of 51 Gy (equivalent to 63 Gy in four weeks – one week split); this was combined with platinum chemotherapy and amifostine cytoprotection administered subcutaneously. Immunohistochemical analysis of hypoxia-regulated proteins, namely HIF1α, HIF2α and CA9, was performed in formalin-fixed paraffin-embedded tissues obtained prior to radio-chemotherapy.

Results: HIF1α and HIF2α were expressed in the nuclei and cytoplasm of cancer cells, while CA9 had a membrane reactivity. A high expression of HIF1α, HIF2α and CA9 was noted in 21/39 (53.8%), 20/39 (51.3%) and 23/39 (58.9%) cases, respectively. Complete response was obtained in 85.2% of patients and HIF1α was marginally related with persistent disease after RT (p = 0.05). HIF1α was significantly associated with poor local relapse free survival (LRFS) (p = 0.006) and overall survival (p = 0.008), whilst HIF2α was not. A significant association of CA9 expression with poor LRFS was noted (p = 0.01).

Conclusion: In accord with previously reported studies, high levels of the hypoxia regulated proteins HIF1α and CA9 in HNC predict resistance to platinum based radio-chemotherapy. Whether HIF2α expressing tumors are more sensitive to larger radiotherapy fractions, compared to standard radiotherapy fractionation, is an issue that deserves further investigation.     

Synthesis of carbon-11-labeled tricyclic necroptosis inhibitors as new potential PET agents for imaging of tumor necrosis factor α (TNF-α)

Carbon-11-labeled tricyclic necroptosis inhibitors were first designed and synthesized as new potential PET agents for imaging of tumor necrosis factor α (TNF-α). The target tracers were prepared by O-[¹¹C]methylation of their corresponding precursors using [¹¹C]CH₃OTf under basic conditions and isolated by a simplified SPE method in 50–60% radiochemical yields based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The overall synthesis time from EOB was 15–20 min, the radiochemical purity was >99%, and the specific activity at end of synthesis (EOS) was 111–185 GBq/μmol.     

Synthesis and evaluation of [18F]exendin (9-39) as a potential biomarker to measure pancreatic β-cell mass

IntroductionGlucagon-like peptide 1 (GLP-1) is released in response to food intake and plays an important role in maintaining blood glucose homeostasis. Exendin (9–39), a potent glucagon-like peptide 1 receptor antagonist, has been labeled with In-111 for SPECT imaging. We report here the first radiosynthesis of [¹⁸F]exendin (9–39) ([¹⁸F]Ex(9–39)) and an evaluation of its potential as a biomarker for in vivo positron emission tomography (PET) imaging of pancreatic β-cell mass (BCM) in rats.MethodsF-18 label was introduced by conjugation of [¹⁸F]4-fluorobenzaldehyde with an Ex(9–39) derivative containing a 6-hydrazinonicotinyl group on the ?-amine of Lys27. Positron emission tomography imaging was carried out in Sprague–Dawley rats (five control and five streptozotocin-induced diabetic) and BioBreeding diabetes-prone rats (three at 7 weeks and three at 12 weeks) using the high-resolution research tomograph (HRRT) after 0.187±0.084 mCi [¹⁸F]Ex(9–39) administration. Time–activity curves were obtained from pancreas, liver and kidney. Pancreases were assayed for insulin content after the imaging study.ResultsSite-specifically labeled [¹⁸F]Ex(9–39) was purified on a G15 open column with radiochemical and chemical purities >98%. Positron emission tomography imaging showed pancreatic standardized uptake value (SUV) peaked at 10 min and plateaued by 50 min to the end of scan (240 min). No correlations of pancreatic SUV with postmortem measures of insulin content were seen.Conclusions[¹⁸F]Ex(9–39) was successfully prepared and used for PET imaging for the first time to measure pancreatic BCM. The results suggest that derivatization of the Lys27 residue might reduce binding affinity, as evidenced by the absence of specific binding. Exendin analogues radiolabeled at other sites may elucidate the active site required for binding.     

‘2+1’ Tricarbonyltechnetium(I) and -rhenium(I) mixed-ligand complexes with N-methylpyridine-2-carboxyamide and isocyanide or imidazole ligands—potential precursors of radiopharmaceuticals

Complexes of tricarbonyltechnetium(I)-99 m and rhenium(I)-188, of ‘2+1’ type: with N-methylpyridine-2-carboxyamide as a neutral bidentate ligand and either tert-butyl 3-isocyanopropionate or imidazole as a monodentate ligand, have been obtained on n.c.a. level. The complexes exhibit moderate lipophilicity and rather high stability in neutral aqueous solutions. The latter has been evidenced from the tests on histidine/cysteine challenge and on protein binding in rat serum. The rhenium complexes studied are much more stable than their technetium analogues. The isocyanopropionate complex may be considered promising model for radiopharmaceutical precursors.     

Immunohistochemical Detection of HIF-1α and CAIX in Advanced Head-and-Neck Cancer

Background:   Tumor hypoxia has an impact on the outcome of cancer patients treated with radiotherapy. The validity of endogenous markers such as hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase isozyme IX (CAIX) to detect therapeutically relevant levels of hypoxia within tumors is controversially discussed. Furthermore, the association of these hypoxia markers with tumor markers or tumor oxygenation parameters is of importance for understanding the relationship between the different factors. Patients and Methods:   Tumor tissue sections of 34 patients with advanced head-and-neck cancer treated with radio(chemo)therapy were assessed by immunohistochemistry for the expression of HIF-1α and CAIX. The relationships of both markers with tumor oxygenation parameters, molecular factors like P53, OPN, VEGF, VHL, survivin, and Ki67 levels, and clinical parameters were studied. Results:   Bivariate analysis showed a significant correlation of HIF-1α expression with high P53 and high OPN expression, high serum VEGF levels, and low VHL and low Ki67 expression. The CAIX expression was inversely correlated with pH value and directly correlated with T-stage. However, no correlation was found between HIF-1α and CAIX expression. Neither in a univariate Cox proportional hazard regression nor in a Kaplan-Meier analysis did expression of HIF-1α or CAIX have a significant impact on clinical outcome. However, in a Kaplan-Meier analysis, the combination of both factors showed that patients with intratumoral overexpression of either HIF-1α or CAIX or both markers died on average 2 years earlier than patients whose tumors had low expression of both factors (p < 0.05). Conclusion:   Expression of HIF-1α and CAIX was correlated with different tumor parameters. Only combined HIF-1α and CAIX expression was significantly predictive of patients’ overall survival.      

Spectroscopic characterization and crystal structures of two cathinone derivatives: 1-(4-chlorophenyl)-2-(1-pyrrolidinyl)-pentan-1-one (4-chloro-α-PVP) sulfate and 1-(4-methylphenyl)-2-(dimethylamino)-propan-1-one (4-MDMC) hydrochloride salts,seized on illicit drug market

Purpose

Two compounds newly found in the seizures by drug enforcement agencies were identified and characterized by various instrumental analytical methods.

Methods

The obtained powder samples were analyzed by gas chromatography–mass spectrometry (GC–MS), liquid chromatography–mass spectrometryⁿ (LC–MSⁿ), nuclear magnetic resonance (NMR) spectroscopy, infrared and Raman spectroscopy and X-ray crystallography.

Results

The two compounds were tentatively identified as 4-chloro-α-PVP and 4-MDMC by GC–MS, and LC–MS/MS. The confirmation of the results was made by NMR spectroscopy. The X-ray crystallography gave information that 4-chloro-α-PVP and 4-MDMC were in salted forms with sulfate and hydrochloride, respectively; in addition, both compounds existed as racemic mixtures.

Conclusions

We could identify 4-chloro-α-PVP and 4-MDMC in the seizure powder samples by various analytical methods. X-ray crystallography was especially useful for identifying the salted forms and enantiomeric forms.

     

Correlation of HIF-1α/HIF-2α expression with FDG uptake in lung adenocarcinoma

Objectives

Hypoxia is a key element involved in the development and progression of tumors. HIF-1α may transiently induce and mediate the response to acute and severe hypoxia, while HIF-2α may induce a longer response and may control the response to chronic and moderate hypoxia. Hypoxia increases the cellular uptake of FDG. Therefore, HIF may play an important role in the process of the cellular uptake of FDG. The aim of this study was to compare HIF-1α/HIF-2α expression with FDG uptake, Glut-1 expression, and prognosis in the patients with lung adenocarcinoma and to investigate the role of HIF-1α/HIF-2α in the uptake of FDG in lung adenocarcinoma.

Methods

In the current work, we compared the immunohistochemical expression of HIF-1α and HIF-2α in surgical specimens of 44 patients with lung adenocarcinoma. The relationships between HIF-α expression and Glut-1 expression, FDG uptake, and clinicopathological factors, including prognosis, were analyzed.

Results

There was a marginal association between HIF-1α and HIF-2α expressions (P = 0.076). We found a significant correlation between HIF-2α expression and FDG uptake (P = 0.0001). HIF-1α expression showed a marginal association with FDG uptake (P = 0.066). FDG uptake correlated more significantly with HIF-2α expression than with HIF-1α expression. A significant correlation was noticed between Glut-1 expression and both HIF-1α and HIF-2α expressions (P = 0.005 and P = 0.003, respectively). Univariate analysis of disease-free survival demonstrated that FDG uptake and HIF-2α expression, but not HIF-1α expression, were related to recurrence (P < 0.0001).

Conclusion

FDG uptake correlated more significantly with HIF-2α expression than with HIF-1α expression, and both FDG uptake and HIF-2α expression, but not HIF-1α expression was correlated with post-operative recurrence in the patients with lung adenocarcinoma. These results suggest that both FDG uptake and HIF-2α expression may represent a more aggressive phenotype and that HIF-2α may play a more important role than HIF-1α in the uptake of FDG in lung adenocarcinoma.

     

Use of a Si(Li) detector as β spectrometer

The aim of this work is to demonstrate the capability of a Si(Li) detector for the measurement of β spectra, despite the energy absorption in air and in the Be window. A simple source holder fixes the source on the symmetry axis at 3 mm from the detector window. The β-sources are produced by evaporation on a plastic backing plate. Absorbing materials between the source and the sensitive volume of the detector are 3 mm of air, a Be window, 0.1 μm Si and 20 nm of gold. A model of the detector was created for β spectra simulation using the MCNP 4A code. Experimental spectra of ¹⁴C, ¹⁴⁷Pm, ²⁰⁴Tl, ⁹⁰Sr/⁹⁰Y were compared with simulated spectra.     

Detection of pyrovalerone as a possible synthetic by-product of 4′-methyl-α-pyrrolidinohexanophenone and 4-methyl-α-ethylaminopentiophenone in illicit drug products

Forensic Toxicology - Impurity profiling is an important intelligence-gathering tool that can be used to link batches of drugs, and it provides valuable insights into manufacturing and supply...     

纤维化拮抗和敏感小鼠胸部照射后IL-1α、IL-1β及肿瘤坏死因子-α mRNA水平表达的早期和持续改变

目的:探讨照后所致肺损伤潜伏期肿瘤坏死因子-α(TNF-α)、白介素-1α(IL-1α)和白介素-1β(IL-1β)mRNA表达水平的变化;在肺炎和肺纤维化发生过程中这些变化是否持续存在;这些变化在纤维化拮抗和敏感小鼠之间的差异。     

The distribution of the HLA-DQα alleles and genotypes in the Finnish population as determined by the use of DNA amplification and allele specific oligonucleotides

Summary Allele and genotype frequencies for the HLA-130 locus were determined for use in forensic analyses and paternity tests in Finland. The polymerase chain reaction (PCR) and the reverse dot blot format were employed to detect 6 different HLA-DQ alleles. All 6 HLA-DQ alleles were detected among the 112 unrelated individuals with allele frequencies ranging from 5.8% to 32.6%. The distribution of the observed genotypes is in Hardy-Weinberg equilibrium. Additionally, this Finnish population sample is statistically similar to 2 other Caucasian sample populations. The power of discrimination of this system in the Finnish population sample is 0.92, suggesting this method may prove suitable for identification purposes.     

Correlation between 18F-fluoromisonidazole PET and expression of HIF-1α and VEGF in newly diagnosed and recurrent malignant gliomas

Purpose

Hypoxia and its consequences at the molecular level promote tumour progression and affect patient prognosis. One of the main early cellular events evoked by hypoxia is induction of hypoxia-inducible factor 1 (HIF-1) and subsequent upregulation of vascular endothelial growth factor (VEGF). In this study we sought to determine whether hypoxia detected by ¹⁸F-fluoromisonidazole (FMISO) PET accurately reflects the expression of HIF-1α and VEGF in the tumour and can be used as a biomarker of antiangiogenic treatment and as a prognostic factor in newly diagnosed and recurrent malignant gliomas.

Methods

Enrolled in this study were 32 patients with newly diagnosed glioma and 16 with recurrent glioma of grade III or grade IV. All the patients had undergone FMISO PET preoperatively. The maximum tumour-to-blood FMISO activity ratio (T/B_max) was used to evaluate the degree of tumour hypoxia and the hypoxic volume (HV) was calculated using a tumour-to-blood FMISO uptake ratio of ≥1.2. Immunohistochemical expressions of HIF-1α and VEGF were evaluated semiquantitatively using the immunoreactivity score (IRS, scores 0 to 12) and the correlation was examined between IRS of HIF-1α or VEGF and FMISO uptake of the tumour (SUV_tumour) using navigation-based sampling. Survival was estimated using the Kaplan-Meier method in relation to the T/B_max and the HV.

Results

The T/B_max and the HV in grade IV gliomas were significantly higher than in grade III gliomas (P?P?VEGF expression was observed in the majority of malignant gliomas. The IRS of HIF-1α and VEGF in the tumour were not significantly different between grade III and grade IV gliomas. The IRS of HIF-1α in the tumour did not correlate with the SUV_tumour of FMISO in either newly diagnosed or recurrent glioma. There was a significant but weak correlation between the IRS of VEGF and the SUV_tumour of FMISO in newly diagnosed glioma, but not in recurrent glioma. The overall survival time in patients with a small HV and a low FMISO T/B_max was significantly longer than in those with a large HV and a high FMISO T/B_max (P?P?Conclusion Preoperative FMISO uptake is significantly correlated with the expression of VEGF in the tumour and might be used as a biomarker of antiangiogenic treatment in newly diagnosed malignant gliomas. However, caution is required because the correlation was weak and there was a large overlap of FMISO uptake between glioma with high and low VEGF expression. In addition, hypoxia determined by FMISO PET appears to be a suitable biomarker for predicting a highly malignant tumour and a poor prognosis in patients with malignant glioma.     

Electrochemiluminescence and voltammetry of tris(2,2′-bipyridine)ruthenium (II) with amphetamine-type stimulants as coreactants: an application to the discrimination of methamphetamine

Purpose

We have tried to establish an electrochemical detection method for screening of the stimulants in the forensic samples using electrochemiluminescence (ECL) and voltammetric techniques.

Methods

Amphetamine-type stimulants (ATSs), such as methamphetamine (MA), amphetamine (AM), dimethylamphetamine (DMA), and methoxyphenamine (MP) have been studied as coreactants in the [Ru(bpy)₃]²⁺ (bpy = 2,2′-bipyridine) ECL system.

Results

There were two specific ECL peaks located at + 1.1 and + 1.4 V for MP, whereas there was only one ECL peak at + 1.1 V for MA and DMA, and no ECL response for AM. Different reaction mechanisms were proposed to understand the generation of ECL by the [Ru(bpy)₃]²⁺/ATS system. Because the voltammetric responses of these ATSs at a glassy carbon electrode were also different, it was possible to detect MA without a separation process based on the characteristic ECL and cyclic voltammetric signals. By a combination of ECL and voltammetric measurements, a simple and rapid screening method for MA could be proposed, and was applied in the determination of MA in human urine samples with sufficient sensitivity.

Conclusions

The present electrochemical approach seems to provide a new way for primary screening of MA in forensic application.

     

Frequency of D1S80 and HLA DQα alleles in a Chinese population

Allele frequency distributions for the D1S80 (MCT118) and HLA DQ loci were determined in a Chinese population sample using the polymerase chain reaction (PCR). A total of 25 alleles and 100 phenotypes were observed for D 1 S80. The frequency of allele 18 was higher than allele 24 only in this Chinese population when compared to other reported populations. A total of 6 alleles and 21 possible phenotypes were observed for HLA DQ. The power of discrimination was 0.97 and 0.93 for D1S80 and HLA DQ, respectively.     

Quantitative estimation of TNF-α and IL-3 by using ELISA from human lung tissue in fatal asphyxial deaths

Asphyxia-related deaths have always been a challenging task in the specialty of forensic pathology. Apart from helpful macroscopical signs (e.g., strangulation marks, cyanosis, petechial haemorrhage, and lung edema), recent literature indicates that prolonged asphyxia is sufficient to induce an increase in mast cells (MC). Inflammatory cells migrate from the bone marrow to the lungs, aiding in the diagnosis of fatal asphyxial death. The present study analyzed human lung tissue samples from 90 medico-legal autopsy cases, including 45 asphyxial deaths and 45 controls (non-asphyxial deaths). The cases ranged from 2 to 68 years, with a mean age of 33.23 years. In 90 cases, 74 cases were of males, and 16 were of females. Human lung tissue samples were analyzed by using the sandwich ELISA method. The results indicated a statistically significant increase in TNF-α and IL-3 concentration in fatal asphyxial deaths, including those caused by hanging, drowning, and smothering. Mean ± SD in asphyxial and non-asphyxial cases for the TNF-α and IL-3 concentration statistically analysed. In asphyxial cases, the average IL-3 concentration (Conc.) was 1558.50 ± 350.53 pg/ml, and the average TNF-α concentration (Conc.) was 499.75 ± 479.41 pg/ml. In contrast, in non-asphyxial cases, the average IL-3 concentration (Conc.) was found to be 849.73 ± 484.99 pg/ml, and the average TNF-α concentration (Conc.) was 208.08 ± 81.23 pg/ml. The mean change in IL-3 and TNF-α (Conc.) values are found to significant (<0.01) in asphyxial cases as compared to non-asphyxial cases. The ROC (Receiver operating characteristic curve) analysis revealed that TNF-α (AUC = 0.89) and IL-3 (AUC = 0.87) concentration (conc.) were stronger predictors of asphyxial deaths with an optimal cut-off value of 455.20 pg/ml for TNF-alpha and 1700.62 pg/ml for IL-3 respectively. Our findings imply that mast cells (MC) are critical in fatal hypoxia-related mortality and that TNF-α and IL-3 can be reliable markers for detecting mast cells in asphyxial deaths. It could be very beneficial to forensic pathologists tasked with differentiating fatal asphyxial fatalities from other causes of death.