Gram Of Wet Tissue Research Articles

Food Safety Risk in Germany From Mislabeled Imported Fish: Ciguatera Outbreak Trace-Back, Toxin Elucidation, and Public Health Implications

Ciguatera poisoning (CP) is a prevalent food related health risk, caused by the consumption of seafood contaminated with ciguatoxins (CTXs). Seafood is the most traded food commodity worldwide, and since 2012, imported snapper fish (Lutjanidae) were the leading cause of CP in Germany. Following a Germany wide CP outbreak in 2017, a product trace-back investigation was conducted for imported fish labeled as “Red Snapper” (Lutjanus malabaricus). Forty-five fish muscle-tissue portions from the implicated batch and two meal remnants were analyzed for CTXs. All samples were positive for “CTX-like toxicity” containing a range of 0.23–11.4 ng CTX3C equivalents per gram of wet tissue, determined by an in vitro cell assay [N2a-3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)]. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) chromatograms revealed the (potential) presence of several marine biotoxins of the class CTX in all batch samples. All samples exceeded current multi-national product legal requirements and recommended guidelines for CTXs. DNA barcoding confirmed the fish sold was mislabeled and was identified as L. bohar, a species frequently involved in CP. Consequently, the mislabeled food and contaminants risk focuses attention on the importance of correct food labeling. Processes for food authentication and CTX contaminant analysis exist and can be used to potentially prevent, stop, and remove foods from commerce for further evaluation to ensure consumer safety. This study further demonstrates their necessity.

A novel dual-labeled small peptide as a multimodal imaging agent for targeting wild-type EGFR in tumors.

The epidermal growth factor receptor (EGFR) is over-expressed in various human cancer. The over-expression of EGFR in tumors is an excellent target for the development of cancer imaging agents. In the present study, we developed Tc-99m SYPIPDT-GHEG-ECG-K-tetramethylrhodamine (SYPIPDT-ECG-TAMRA) as a molecular imaging agent targeting wild-type EFGR (wtEGFR)-positive tumor cells, and verified its feasibility as molecular imaging agent. SYPIPDT-ECG-TAMRA was synthesized using Fmoc solid-phase peptide synthesis. The radiolabeling of SYPIPDT-ECG-TAMRA with Tc-99m was accomplished using ligand exchange via tartrate. Cellular uptake and binding affinity studies were performed. In vivo gamma camera imaging, ex vivo imaging and biodistribution studies were performed using NCI-H460 and SW620 tumor-bearing murine models. After radiolabeling procedures with Tc-99m, Tc-99m SYPIPDT-ECG-TAMRA complexes were prepared at high yield (> 95%). The binding affinity value (Kd) of Tc-99m SYPIPDT-ECG-TAMRA for NCI-H460 cells was estimated to be 76.5 ± 15.8 nM. In gamma camera imaging, the tumor to normal muscle uptake ratios of Tc-99m SYPIPDT-ECG-TAMRA increased with time (2.7 ± 0.6, 4.0 ± 0.9, and 6.2 ± 1.0 at 1, 2, and 3 h, respectively). The percentage injected dose per gram of wet tissue for the NCI-H460 tumor was 1.91 ± 0.11 and 1.70 ± 0.22 at 1 and 3 h, respectively. We developed Tc-99m SYPIPDT-ECG-TAMRA, which is dual-labeled with both radioisotope and fluorescence. In vivo and in vitro studies demonstrated specific uptake of Tc-99m SYPIPDT-ECG-TAMRA into wtEGFR-positive NCI-H460 cells and tumors. Thus, the results of the present study suggest that Tc-99m SYPIPDT-ECG-TAMRA is a potential dual-modality imaging agent targeting wtEGFR.

The High Affinity Dopamine D2 Receptor Agonist MCL-536: A New Tool for Studying Dopaminergic Contribution to Neurological Disorders.

The dopamine D2 receptor exists in two different states, D2high and D2low; the former is the functional form of the D2 receptor and associates with intracellular G-proteins. The D2 agonist [3H]MCL-536 has high affinity for the D2 receptor (Kd 0.8 nM) and potently displaces the binding of (R-(-)-N-n-propylnorapomorphine (NPA; Ki 0.16 nM) and raclopride (Ki 0.9 nM) in competition binding assays. Here, we further characterize [3H]MCL-536. [3H]MCL-536 was metabolically stable, with about 75% of the compound remaining intact after 1 h incubation with human liver microsomes. Blood-brain barrier penetration in rats was good, attaining at 15 min a % injected dose per gram of wet tissue (%ID/g) of 0.28 in males versus 0.42 in females in the striatum. Specific uptake ratios ([%ID/g striatum]/[%ID/g cerebellum]) were stable in males during the first 60 min and in females up to 15-30 min. The D2-rich striatum exhibited the highest uptake and slowest washout compared to D2-poor cortex or cerebellum. In peripheral organs, uptake peaked at 15 min but declined to baseline at 60 min, indicating good clearance from the body. In vitro autoradiography on transaxial and coronal brain sections showed specific binding of [3H]MCL-536, which was abolished by preincubation with D2/D3 ligands sulpiride, NPA, and raclopride and in the presence of the stable GTP analogue guanylylimidodiphosphate. In amphetamine-sensitized animals, striatal binding was higher than in controls, indicating specificity for the D2high receptor state. [3H]MCL-536's unique properties make it a valuable tool for research on neurological disorders involving the dopaminergic system like Parkinson's disease or schizophrenia.

Obesity-Dependent Accumulation of Titanium in the Pancreas of Type 2 Diabetic Donors.

The most widely used white pigment of foods and medications is crystalline, anatase-phase TiO2 of 110 ± 70 nm particle diameter. Recent studies by other investigators have shown that depending on its ingested pigment amount the concentration of titanium in human blood ranges between 2 and 48 ppb and that Ti accumulates in the spleen and in the liver. Here we report titanium concentrations in the pancreas head of 30 human donors, measured by inductively coupled plasma quadrupole mass spectroscopy. Of the donors, 7 were free of pancreatic disease, 4 had pancreatitis, 10 had type 2 diabetes and 9 had type 2 diabetes with pancreatitis; 3 were underweight, 6 were normal weight, 5 were overweight, and 16 were obese. Ti accumulated in the pancreas, its accumulation increasing with obesity. The pancreatic Ti concentrations ranged from 0.75 to 3.78 ppm, averaging 1.8 ppm, much higher than the reported 40-100 ppb concentration in the spleen or the 30-100 ppb concentration reported in the liver. The corresponding number density of 110 nm diameter TiO2 particles averaged 3.6 × 109 per gram of wet tissue; their potentially biological macromolecule adsorbing surface area is ∼1 cm2 per gram wet tissue.

Organ-specific accumulation, transportation, and elimination of methylmercury and inorganic mercury in a low Hg accumulating fish.

Low mercury (Hg) concentrations down to several nanograms Hg per gram of wet tissue are documented in certain fish species such as herbivorous fish, and the underlying mechanisms remain speculative. In the present study, bioaccumulation and depuration patterns of inorganic Hg(II) and methylmercury (MeHg) in a herbivorous rabbitfish Siganus canaliculatus were investigated at organ and subcellular levels following waterborne or dietary exposures. The results showed that the efflux rate constants of Hg(II) and MeHg were 0.104 d(-1) and 0.024 d(-1) , respectively, and are probably the highest rate constants recorded in fish thus far. The dietary MeHg assimilation efficiency (68%) was much lower than those in other fish species (∼90%). The predominant distribution of MeHg in fish muscle was attributable to negligible elimination of MeHg from muscle (< 0) and efficient elimination of MeHg from gills (0.12 d(-1) ), liver (0.17 d(-1) ), and intestine (0.20 d(-1) ), as well as efficient transportation of MeHg from other organs into muscle. In contrast, Hg(II) was much more slowly distributed into muscle but was efficiently eliminated by the intestine (0.13 d(-1) ). Subcellular distribution indicated that some specific membrane proteins in muscle were the primary binding pools for MeHg, and both metallothionein-like proteins and Hg-rich granules were the important components in eliminating both MeHg and Hg(II). Overall, the present study's results suggest that the low tissue Hg concentration in the rabbitfish was partly explained by its unique biokinetics. Environ Toxicol Chem 2016;35:2074-2083. © 2016 SETAC.

Collegium Ramazzini: Comments on the 2014 Helsinki consensus report on asbestos.

Collegium Ramazzini: Comments on the 2014 Helsinki consensus report on asbestos.

Contaminants of legacy and emerging concern in largescale suckers (Catostomus macrocheilus) and the foodweb in the lower Columbia River, Oregon and Washington, USA

We investigated occurrence, transport pathways, and effects of polybrominated diphenyl ether (PBDE) flame retardants and other endocrine disrupting chemicals (EDCs) in aquatic media and the foodweb in the lower Columbia River. In 2009 and 2010, foodweb sampling at three sites along a gradient of contaminant exposure near Skamania (Washington), Columbia City (Oregon) and Longview (Washington) included water (via passive samplers), bed sediment, invertebrate biomass residing in sediment, a resident fish species (largescale suckers [Catostomus macrocheilus]), and eggs from osprey (Pandion haliaetus). This paper primarily reports fish tissue concentrations. In 2009, composites of fish brain, fillet, liver, stomach, and gonad tissues revealed that overall contaminant concentrations were highest in livers, followed by brain, stomach, gonad, and fillet. Concentrations of halogenated compounds in tissue samples from all three sites ranged from <1 to 400nanograms per gram of wet tissue. Several chemical classes, including PBDEs, organochlorine pesticides, and polychlorinated biphenyls (PCBs), were detected at all sites and in nearly all fish tissues sampled. In 2010, only fish livers were sampled and inter-site concentration differences were not as pronounced as in 2009. Chemical concentrations in sediments, fish tissues, and osprey eggs increased moving downstream from Skamania to the urbanized sites near Columbia City and Longview. Numerous organochlorine (OC) pesticides, both banned and currently used, and PBDEs, were present at each site in multiple media and concentrations exceeded environmental quality benchmarks in some cases. Frequently detected OC compounds included hexachlorobenzene, pentachloroanisole, dichlorodiphenyltrichloroethane (DDT) and its degradates, chlorpyrifos, and oxyfluorofen. Biomagnification of BDE47, 100, 153, and 154 occurred in largescale suckers and osprey eggs. Results support the hypothesis that contaminants in the environment lead to bioaccumulation and potential negative effects in multiple levels of the foodweb.

Towards the development of new subtype-specific muscarinic receptor radiopharmaceuticals — Radiosynthesis and ex vivo biodistribution of [18F]3-(4-(2-(2-(2-fluoroethoxy)ethoxy)ethylthio)-1,2,5-thiadiazol-3-yl)-1-methyl-1,2,5,6-tetrahydropyridine

Muscarinic receptors have been implicated in neurological disorders including Alzheimer’s disease, Parkinson’s disease, and schizophrenia. Nineteen derivatives of thiadiazolyltetrahydropyridine (TZTP), a core that has previously shown high affinities towards muscarinic receptor subtypes, were synthesized and evaluated via in vitro binding assays. The title compound, a fluoro-polyethyleneglycol analog of TZTP (4c), was subsequently labelled with fluorine-18. Fluorine-18-labelled 4c was produced, via an automated synthesis, in an average radiochemical yield of 36% (uncorrected for decay), with high radiochemical purity (&gt;99%) and high specific activity (326 GBq/µmol; end-of-bombardment), within 40 min (n = 3). Ex vivo biodistribution studies following tail-vein injection of [18F]4c in conscious rats displayed sufficient brain uptake (0.4%–0.7% injected dose / gram of wet tissue in all brain regions at 5 min post injection); however, there were substantial polar metabolites present in the brain, thereby precluding future use of [18F]4c for imaging in the central nervous system.

Isolation and Fluorescence Spectrum of Cardiac Myosin from Pig Heart

Cardiac myosin is the most abundant and bioactive protein presented in heart tissue. In this work, a simple method was described for preparation of high purity cardiac myosin for further research. With the use of a domestic pig left ventricle as the starting tissue, this method yielded 4.5 mg myosin per gram of wet tissue with a specific activity of 0.293 U/mg. The purification factor was 4.9-fold with an activity recovery of 42.1%. In addition, the investigation of fluorescence spectrum shown that the emission intensity of cardiac myosin has a dramatic decrease under the influence of an acidic environment or at temperatures higher than 30 degrees C, which could influence the normal physiological function of cardiac myosin.

A study of the functional aberration of the pouch in anorectal malformation associated with congenital pouch colon

There are no in vitro studies of congenital pouch colon (CPC) associated with anorectal malformation (ARM) to understand its mechanism of contractility. There is no consensus regarding the utilization of the CPC in repair of anorectal malformation. In view of the above, it was decided to carry out detailed in vitro physiological study of the excised CPC. The excised specimens of CPC were taken and 15- to 20-mm long both longitudinal and transverse strips of muscle were obtained both from the proximal and distal part. Contraction was recorded using software chart-5 for windows. Drugs used were acetylcholine, histamine, adrenaline, atropine and pheniramine maleate. The amplitude of contractions was converted to tension gram and then the tension so developed was expressed as tension per unit mass (per gram of wet tissue). There were total of 21 specimens of high ARM with CPC. Five specimens were of the complete pouch, which did not show a response to any drug and the remaining 16 had an incomplete pouch. The mean longitudinal and circular muscle contractions of these 16 samples were statistically higher for the proximal segment than the distal segment both for acetylcholine and histamine. The effect of atropine was not significant but that of adrenaline and pheniramine maleate were significant on the distal segment muscle. Congenital pouch colon being deficient or having poorly developed receptors in their wall can not function properly as a reservoir for faeces and, hence, it should be excised to allow adequate function.

Effect of EGb761 supplementation on the content of copper in mouse brain in an animal model of Parkinson's disease

Objective EGb761 is a well-defined mixture of active compounds extracted from Ginkgo biloba leaves with neuroprotective effects in an animal model of Parkinson's disease induced by 1-methyl-4-phenylpyridinium (MPP +). Because copper has been implicated in Parkinson's disease, we investigated whether the protective effect of EGb761 in MPP + neurotoxicity is related to the regulation of copper in the brain. Methods C-57BL/6 mice were pretreated with EGb761 (10 mg/kg) daily for 17 d followed by administration of MPP + (0.72 mg/kg); the mice were sacrificed 24 h later. The copper content of the striatum, midbrain, hippocampus, frontal cortex, and cerebellum was analyzed by graphite furnace atomic absorption spectrophotometry. Copper content is expressed as μg of copper per gram of wet tissue. Results Copper content was reduced in the corpus striatum (45%; P < 0.05), and increased in the midbrain (65%; P < 0.05) and hippocampus (116%; P < 0.001) after MPP + administration. EGb761 pretreatment of the MPP + group prevented changes in the copper content of the striatum, midbrain, and hippocampus. No significant changes were found in the copper content of the cerebellum and frontal cortex in all treatment groups. Conclusion We showed that the protective effect of EGb761 against MPP + neurotoxicity may be due in part to the regulation of copper homeostasis in the brain.

Protective effect of α-lipoic acid in experimental spermatic cord torsion

Objective We investigated the effect of α-lipoic acid (LA) in rats that were subjected to torsion/detorsion of the spermatic cord in a comparative controlled experiment. Methods Forty-eight male Wistar rats, randomized in two groups, received intraperitoneal injections of LA (LA group; aqueous solution at 36 mg/kg of body weight per day) or equal volume of saline (control group) 21, 9, and 1 h before torsion of the spermatic cord. Rats in each group were distributed in four subgroups, each comprising six animals. All surgical procedures were performed under inhaled ether anesthesia. Ischemia was induced by 720-degree torsion of the spermatic cord for 3 h. The right testis was assessed through longitudinal scrotal incision. After each surgical procedure, scrotal incisions were closed with 4-0 nylon monofilament. Ipsilateral testes and arterial blood samples were collected at the end of ischemia and 1, 3, and 6 h after detorsion. Thiobarbituric acid-reactive substances and reduced glutathione levels (micromoles per gram of wet tissue) were assayed in testis. Total antioxidant power was measured in blood plasma. Results LA pretreatment promoted a significant decrease in testicular concentrations of thiobarbituric acid-reactive substances and simultaneously induced an increase in reduced glutathione concentrations at all time points studied. Plasma total antioxidant power levels increased significantly during reperfusion (T-1) in LA-treated rats compared with control rats. Conclusion LA administered before torsion of the spermatic cord showed significant protective effects against ischemia/reperfusion injury by decreasing lipid peroxidation and regulating testicular reduced glutathione and plasma total antioxidant power levels.

Therapeutic Treatment of Experimental Colitis With Regulatory Dendritic Cells Generated With Vasoactive Intestinal Peptide

Crohn's disease is a chronic debilitating disease characterized by severe T helper cell (Th)1-driven inflammation of the colon partially caused by a loss of immune tolerance against mucosal antigens. The use of regulatory dendritic cells (DCs) with the capacity to induce regulatory T cells has been proposed recently for the treatment of Crohn's disease in a strategy to restore immune tolerance. Vasoactive intestinal peptide is an immunomodulatory neuropeptide that induces regulatory DCs. The aim of this study was to investigate the therapeutic effect of vasoactive intestinal peptide-induced regulatory DCs (DC(VIP)) in a murine model of colitis. We examined the therapeutic action of DC(VIP) in the colitis induced by intracolonic administration of trinitrobenzene sulfonic acid, evaluating diverse clinical signs of the disease including weight loss, diarrhea, colitis, and histopathology. We also investigated the mechanisms involved in the potential therapeutic effect of DC(VIP), such as inflammatory cytokines and chemokines, Th1-type response, and the generation of regulatory T cells. DC(VIP) injection significantly ameliorated the clinical and histopathologic severity of colitis, abrogating body weight loss, diarrhea, and inflammation, and increasing survival. The therapeutic effect was associated with down-regulation of both inflammatory and Th1-driven autoimmune response, by regulating a wide spectrum of inflammatory mediators directly through activated macrophages, and by generating interleukin-10-secreting regulatory T cells with suppressive capacity on autoreactive T cells. The possibility to generate/expand ex vivo regulatory DC(VIP) opens new therapeutic perspectives for the treatment of Crohn's disease in human beings, and may minimize the dependence on nonspecific immunosuppressive drugs used currently for autoimmune disorders.

1/T2 and Magnetic Susceptibility Measurements in a Gerbil Cardiac Iron Overload Model

To measure the transverse relaxation rate (1/T2) and magnetic susceptibility of the heart in conditions of iron overload by using magnetic resonance (MR) imaging and to correlate these with the tissue iron concentration in a gerbil model. With prior approval by the institutional animal care and use committee, iron overload was induced with one to 15 weekly subcutaneous injections of iron dextran. Nine gerbils had one to five injections, 10 had six to 10, and eight had 13-15. T2 of the whole heart was measured ex vivo (n=27), and the magnetic susceptibility of the tissue was estimated through measurement of the tissue lysate (n=25). The iron level was measured (in milligrams of iron per gram of wet tissue) with chemical analysis after MR imaging. While 1/T2 and magnetic susceptibility are not equivalent measures of the chemically determined tissue iron level, correlations were expected and were identified by using linear regression models. Iron concentration range was 0.28-1.95 mg/g wet tissue. Iron concentration was strongly correlated with 1/T2 (r=0.92, P <.001, and the root of the mean squares error of the linear prediction, epsilonRMS, was 0.17 mg Fe/g wet tissue with a repetition time of 700 msec). Iron concentration also was strongly correlated with magnetic susceptibility (r=0.90, P <.001, epsilonRMS=0.19 mg Fe/g wet tissue). Multiple regression analysis with combined 1/T2 (with repetition time of 700 msec) and magnetic susceptibility data led to a slight increase in r and decrease in epsilon(RMS) (r=0.93, P <.001, epsilonRMS=0.16 mg Fe/g wet tissue). The results of this animal model study demonstrate that 1/T2 and magnetic susceptibility values can be used for estimation of the iron level in the heart.

Aromatase in skeletal muscle

Aromatase gene expression and activity have been studied in human skeletal muscle. Using two separate rounds of RT-nested PCR, transcripts from the coding region of aromatase mRNA were detected in 32 of 34 samples. In terms of the non-coding exon I, PCR product for I.4 was detected in 13 cases (38%), I.3 in 10 cases (29%), P.II in 6 cases (18%) and I.1 was not detected in any case. No transcripts for any studied variants of exon I were detected in 18 samples (53%). Aromatase activity was assessed using two different methodologies: in 19 cases by definitive product isolation (DPI) and in 42 cases by tritium-release assay (TRA). Using both methods detectable activity was present in 52% of cases. Average values for the cases with detectable activity were 2.2 fmol/mg protein/h for DPI and 6.5 fmol/mg protein/h for TRA. In the cohort studied by TRA, a positive correlation of aromatase activity with age of the donor was observed ( r=0.34, P=0.03). In six cases paired comparison of aromatase activity in muscle and associated fat tissue were performed by DPI. When expressed per milligram of protein the activity was always higher in fat. However, this difference disappeared when activity was based on the gram of wet tissue. Taking into account bulk in the body it is concluded that muscle can be an important source of estrogens in men and post-menopausal women and its contribution to the circulating pool of estrogens may be comparable to that of adipose tissue. The nature of mRNA transcripts in muscle suggests that estrogen formation may be controlled by glucocorticoid- as well as cAMP-dependent promoters of the aromatase gene.

Human microglial cell isolation from adult autopsy brain: brain pH, regional variation, and infection with human immunodeficiency virus type 1.

Microglia are the main source of productive infection by human immunodeficiency virus type 1 (HIV-1) in the central nervous system (CNS). Infection of microglia is difficult to study because nonhuman microglia are not infected by HIV-1, adult human microglia from surgically removed brain tissues are scarce, and fetal human microglial cells differ from adult cells in potentially important ways. Adult autopsies are a potential source of brain tissue for HIV-1 research, but the technique of isolating and infecting cells postmortem is not completely standardized. The authors determined optimal conditions for isolating and infecting adult microglial cells using 18 adult autopsy brain specimens from HIV-1-infected and noninfected decedents. The yield of mixed glial cells overall was on average 0.5 x 10(6) cells per gram of wet tissue. There was no correlation between the number of microglia isolated and the postmortem interval (PMI), HIV seropositivity, age, or gender. Brain pH accounted for about 41% of yield variability; a pH of less than 6.0 generally was not compatible with adequate cell recovery. The highest microglial cell yields were derived from anterior brain sectors (frontal lobe and temporal lobe) versus occipital lobe and cerebellum. A PMI of up to 25.5 h produced excellent cell yields in frontal lobe samples with high brain pH. HIV-1 infection of frontal lobe microglia was 100% successful using both CXCR4- and CCR5-tropic strains of HIV-1. With proper selection of cases and brain region, autopsy brain specimens are a dependable source of viable microglial cells to study CNS HIV-1 infection.

Association of GM4 Ganglioside with the Membrane Surrounding Lipid Droplets in Shark Liver

By TLC, GM4 was found to be the major ganglioside in the liver of six shark species examined: Odontaspis taurus, Negaprion brevirostris, Sphyrna lewini, Mustelus griseus, Mustelus manazo, and Prionace glauca. A detailed analysis of the glycosphingolipids (GSLs) in the liver of O. taurus (sand tiger shark) showed that it contained approximately 110 nmol of lipid-bound sialic acid per gram of wet tissue, of which 80% was GM4. By extracting the liver of O. taurus with chloroform/methanol, followed by chromatographic separation of GSLs using DEAE-Sephadex A-25 and Iatrobeads columns, we have isolated GM4 in pure form with a yield of approximately 5 mg per 100 g of wet tissue. The structures of both the sugar chain and the ceramide moiety of this GM4 were analyzed by chemical analysis, mass spectrometry, and NMR spectroscopy. Similar to GM4 isolated from other sources, 92% of fatty acids in the ceramide of this GM4 were 2-hydroxylated. However, unlike the long-chain bases found in other GSLs, the total long-chain bases in this GM4 were found to contain 43% octadecasphingenine and 50% nonadecasphingenine.Immunohistochemical analysis using a monoclonal antibody against GM4 revealed that the hepatocytes of both M. griseus (spotless smooth hound) and M. manazo (smooth hound) were filled with lipid droplets and GM4 was primarily associated with the membrane structure surrounding lipid droplets.

Selective mitochondrial KATP channel opening controls human myocardial preconditioning: Too much of a good thing?

Background: Paradoxically, patients with noninsulin-dependent diabetes mellitus experience a higher cardiovascular mortality rate than patients with insulin-dependent diabetes mellitus. We have shown that KATP channel inhibition, with oral sulfonylureas, prevents myocardial preconditioning and may explain the paradox of cardiovascular death in patients with noninsulin-dependent diabetes mellitus. Cardiac preconditioning is an attractive protective strategy against any elective ischemia/reperfusion (I/R) injury. The relationship between the KATP channels and human myocardial preconditioning has not previously been elucidated. Methods: Human atrial trabeculae were harvested, placed in organ baths, and paced (1 Hz). Developed force was recorded during simulated 37°C I/R (30/45 or 45/60 minutes). Before I/R, trabeculae were treated transiently with a selective mitochondrial KATP channel opener for 5 minutes, followed by a 10-minute washout, or were exposed to the channel opener throughout ischemia. Recovery of function is expressed as percentage of baseline developed force. Conserved creatine kinase activity (units per gram of wet tissue) was measured at the end of reperfusion as an indicator of cellular protection. Results: Transient mitochondrial KATP channel opening provided protection from both I/R insults. Surprisingly, there was no protection afforded by continuous mitochondrial KATP channel opening. Conclusions: Transient selective mitochondrial KATP channel opening protects both viability and function of human myocardium against I/R injury, although prolonged opening of the mitochondrial KATP channel does not. These results reinforce the concept of preconditioning as a transient event that must be completed before the onset of ischemia. (Surgery 2000;128:368-73.)

Investigation of functional and morphological integrity of freshly isolated and cryopreserved human hepatocytes.

There is a pressing need for alternative therapeutic methods effective in the treatment of patients with liver insufficiency. Isolated human hepatocytes may be a viable alternative or adjunct to orthotopic liver transplantation in such patients. The purpose of this study was to evaluate the viability and functional integrity of freshly isolated and cryopreserved human hepatocytes, in preparation for a multi-center human hepatocyte transplantation trial. We are currently processing transplant-grade human parenchymal liver cells from nondiseased human livers that are obtained through a network of organ procurement organizations (OPOs). Thus far, sixteen hepatocyte transplants have been performed using hepatocytes processed by our methods. At the time of referral all specimens were deemed unsuitable for transplantation due to anatomical anomalies, high fat content, medical history, etc. Hepatocytes were isolated from encapsulated liver sections by a modified two-step perfusion technique. Isolated cells were cryopreserved and stored in liquid nitrogen for one to twelve months. The total yield of freshly isolated hepatocytes averaged 3.7x10(7) cells per gram of wet tissue. Based on trypan blue exclusion, fresh preparations contained an average of 85% viable hepatocytes vs. 70% in cryopreserved samples. The plating efficiencies of cells seeded immediately after isolation ranged from 87% to 98%, while those of cryopreserved/thawed cells were markedly lower. Flow cytometry analysis of cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that there was no significant difference in viability compared with trypan blue staining. Both freshly isolated hepatocytes and those recovered from cryopreservation showed typical and intact morphology as demonstrated by light and electron microscopy. The product of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reaction was always expressed more intensely in cultures of freshly isolated hepatocytes. Measurements of lactate dehydrogenase (LDH) leakage were inversely correlated with trypan blue exclusion and CFSE labeling. Energy status, evaluated by the intracellular ATP concentration measurements, and various liver-specific functions such as urea synthesis and metabolism of 7-ethoxycoumarin were maintained both in fresh and cryopreserved/thawed hepatocytes. However, the activities were expressed at different levels in thawed cells. These data illustrate the importance and feasibility of human hepatocyte banking. In addition, it is clear that further refinements in the methods of hepatocyte isolation and cryopreservation are needed to utilize more fully these valuable cells in the clinic.

Thorium isotopes in autopsy samples from thorium workers.

Concentrations of 232Th and activity ratios of 228Th to 232Th and 230Th to 232Th were determined in autopsy samples from five former employees of a thorium refinery. The ranges of 232Th activity concentrations (mBq per gram of wet tissue) were 0.17-94 in lungs, 3.9-1210 in pulmonary lymph nodes, 0.14-1.19 in bones, 0.015-0.68 in liver, 0.97-5.8 in spleen, and 0.009-0.068 in kidneys. These concentrations are 10 to 1,000 times greater than have been reported for persons not occupationally exposed to thorium. In most of the samples, the ratios of 228Th to 232Th and 230Th to 232Th activity at death of the subject were 0.2-0.4 and 0.1-0.2, respectively. 228Th to 228Ra activity ratios (+/- standard errors) of 0.86 +/- 0.11 in lungs and 1.18 +/- 0.13 in lymph nodes of one subject were obtained by calculation from ratios of 228Th to 232Th.