Hypoxia-related brain dysfunction in forensic medicine.

Blood gases levels imbalances belong to important factors triggering central nervous system (CNS) functional disturbances. Hypoxia can be illness-related, like in many COPD patients, or it may be caused by broad range of external or iatrogenic factors - including influence of drugs depressing respiration, failure to keep the patient's prosthesis-supported airways patent, or a mistake in the operation of medical equipment supporting patient's respiration. Hypoxia, especially when it is not accompanied by rapid carbon dioxide retention, can go unnoticed for prolonged times, deepening existing CNS disorders, sometimes rapidly triggering their manifestation, or evoking quite new conditions and symptoms - like anxiety, agitation, aggressive behavior, euphoria, or hallucinations. Those, in turn, often result in situations raising interest in law enforcement institutions which need forensic medicine specialist's assistance and opinion. The possibility of illness or drug-related hypoxia, especially in terminal patients, is used to raise questions about the patients' ability to properly express their will in the way demanded by law - it also must be considered as a factor limiting the patients' responsibility in case they commit crimes. The possibility of hallucinations in hypoxia patients limits their credibility as witnesses or even their ability to report crime or sexual abuse they have been subjected to.

Death as a result of violent asphyxia in autopsy reports.

Violent asphyxia can be subdivided into various kinds according to the mechanism, so that the resuscitation techniques are different in each case. The purpose of the present article was to analyze the autopsy reports of the Department of Forensic Medicine of the Medical University in Wroclaw, Poland of 2010, in which the established cause of death was violent asphyxia. We found that among the 890 autopsies performed, there were 164 cases of death due to violent asphyxia caused by drowning, choking on food, gastric fluid, or blood, hanging, manual strangulations, immobilization of the chest (positional asphyxia), environmental asphyxia due to substitution of the oxygen-rich air for some other gas, and others. The most common cause of death in the group was hanging, mostly suicidal hangings of alcohol-intoxicated males. Despite an early medical treatment consisting of removing the noose from the neck and suction the fluids from the mouth and bronchial tree to safe the central nervous system from imminent hypoxia, there were negative outcomes in most cases due to the development of critical brain ischemia, with deaths followed after several days spent in the intensive care units. No connection to gender or age of the deceased was noted. We conclude that violent asphyxia remains to be a quite commonly cause of death in the practice of forensic pathologists - among all the autopsies performed in 2010 every sixth was of an asphyxia victim.

Stiff substrates inhibit collagen accumulation via integrin α2ß1, FAK and Src kinases in human atrial fibroblast and myofibroblast cultures derived from patients with aortal stenosis.

The aim of the study was to confirm whether cell substrate stiffness may participate in the regulation of fibrosis. The involvement of integrin α2ß1, focal adhesion kinase (FAK) and Src kinase in signal transmission was investigated. Human atrial fibroblasts and myofibroblasts were cultured in both soft (2.23 ± 0.8 kPa) and stiff (8.28 ± 1.06 kPa) polyacrylamide gels. The cells were derived from the right atrium of patients with aortal stenosis undergoing surgery. The isolated cells, identified as fibroblasts or myofibroblasts, were stained positively with α smooth muscle actin, vimentin and desmin. The cultures settled on stiff gel demonstrated lower intracellular collagen and collagen type I telopeptide (PICP) levels; however, no changes in α1 chain of procollagen type I and III expression were noted. Inhibition of α2ß1 integrin by TC-I 15 (10-7 and 10-8 M) or α2 integrin subunit silencing augmented intracellular collagen level. Moreover, FAK or Src kinase inhibitors increased collagen content within the culture. Lower TIMP4 secretion was reported within the stiff gel cultures but neither MMP 2 nor TIMP-1, 2 or 3 release was altered. The stiff substrate cultures also demonstrated lower interleukin-6 release. Substrate stiffness modified collagen deposition within the atrial fibroblast and myofibroblast cultures. The elasticity of the cellular environment exerts a regulatory influence on both synthesis and breakdown of collagen. Integrin α2ß1, FAK and Src kinase activity participates in signal transmission, which may influence fibrosis in the atria of the human heart.

Exendin-4 differentially modulates essential functions of human dermal fibroblasts under normoglycemic and hyperglycemic conditions.

Evidence that exendin-4, a glucagon-like peptide-1 analog, might be used to treat poorly healing wounds under diabetic and nondiabetic conditions has gained increasing interest. Little is known, however, about the effects of the drug on the production by dermal fibroblasts of key extracellular matrix and regulatory compounds. Therefore, we used human skin fibroblasts cultured in normo- (1 g/l = 5.6 mmol/l glucose) or hyperglycemic (4.5 g/l = 25 mmol/l glucose) culture medium to test the effects of exendin-4 (0 - 100 nmol/l) on fibroblast functions crucial for the wound healing process. Exendin-4 increased the proliferative and metabolic activities, as measured by the BrdU (bromodeoxyuridine) and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assays, respectively, of fibroblasts cultured in normoglycemic medium. Under hyperglycemic conditions, the drug had no effect on proliferation and reduced metabolic fibroblast activity. Exendin-4 decreased metalloproteinase-9 (MMP-9) secretion in the normoglycemic milieu only and increased tissue inhibitor of metalloproteinase-1 (TIMP-1) concentration in fibroblast colonies under both normo- and hyperglycemic experimental conditions. Exendin-4 increased the fibroblast growth factor-1 (FGF-1) concentration in cell colonies maintained in the normoglycemic milieu but decreased FGF-1 release when fibroblasts were grown in hyperglycemic medium. High glucose caused lactic dehydrogenase (LDH) leakage when compared with normoglycemic conditions, and exendin-4 was not able to prevent this effect, although it reduced LDH release from fibroblasts cultured in normoglycemic medium. Finally, exendin-4 increased glycosaminoglycan (GAG) content under both experimental conditions. Our results indicate that exendin-4 effects on the production of the extracellular matrix and regulatory proteins differ in human skin fibroblasts exposed to either normal or high glucose. In general, the beneficial effects of the drug, which may be important for the improvement of wound healing, are more pronounced under normoglycemic conditions, thus indicating that hyperglycemia attenuates the positive effects of exendin-4 on fibroblasts.

Disruption of histamine/H3 receptor signal reduces collagen deposition in cultures scar myofibroblasts.

A suitable inflammatory signal influences extracellular matrix accumulation and determines the quality of the myocardial infarction scar. The aim of the present study was to determine the influence of mast cell sonicates or histamine on collagen accumulation in heart myofibroblast culture and on the deposition of collagen in the myocardial infarction scar. The histamine receptor involved in the process was investigated. Myocardial infarction was induced by ligation of the left coronary artery. Myofibroblasts were isolated from the scar of myocardial infarction. The effects of mast cell sonicates, histamine and its receptor antagonists, i.e. ketotifen (H1-receptor inhibitor), ranitidine (H2-receptor inhibitor), ciproxifan (H3-receptor inhibitor), JNJ7777120 (H4-receptor inhibitor), imetit (H3 receptor agonist), were investigated. The mast cell sonicates or histamine (10-10 - 10-5M) augmented collagen content in myofibroblast cultures; however, histamine-induced elevation was reduced by ciproxifan (10-5M, 10-6M). Imetit (10-9 - 10-5M) elevated collagen content in the culture. H3 receptor expression on myofibroblasts was confirmed. Our findings indicate that histamine increases the deposition of collagen in cultures of myofibroblasts isolated from the myocardial infarction scar. This effect is dependent on H3 receptor activation.

Effects of neuraminidase on apoptosis of blood lymphocytes in rats with implanted Morris tumor.

In this study we examined the influence of neuraminidase on apoptosis of peripheral blood lymphocytes in rats with an implanted Morris tumor. The main objectives of the study were to determine whether the percentage of apoptotic blood lymphocytes would depend on the dosing regimen of neuraminidase and whether neuraminidase would affect caspase-3 activity, a marker of the apoptosis, in blood lymphocytes. A total of 51 rats were used for the study. In three groups, totalling 39 animals, Morris tumor was implanted and neuraminidase was injected intravenously using two dosing regimens: 10 units three times on Day 4, Day 7, and Day 14 and 5 units as a single dose on Day 4 of the experiment or was skipped (control). The remaining 12 rats constituted a reference group of healthy animals. At the end of the experimental period on Day 21, blood was drawn from the heart, and mononuclear cells were separated and cultured. Apoptosis of blood lymphocytes was assessed in cell cultures from fluorescence spectra generated by a Sybr Green I dye forming bonds with nuclear DNA. Caspase-3 activity was measured colorimetrically in homogenates of lymphocyte cultures using a CASP-3-C kit (Sigma, St. Louis, MO). On the whole, the results demonstrate that the bigger, but not the smaller, dose of neuraminidase was markedly effective in preserving the vitality of blood lymphocytes and in decreasing both the number of apoptotic lymphocytes and capsase-3 activity in the rats with Morris tumor. Neuraminidase treatment failed, however, to lessen the tumor size. In conclusion, the study demonstrates that neuraminidase caused an appreciable decline in apoptosis of blood lymphocytes in rats with the Morris tumor; the effect was dose-dependent. Although neuraminidase failed to influence the local cancer development in terms of tumor size, its anti-apoptotic effect toward the cells of the immune system of a cancer host is of research interest as it may potentially offer a way to strengthen the host's immune response.

Comparison of a statin vs. hypolipemic diet on the oxidant status in hemodialyzed patients with chronic renal failure.

Hemodialyzed patients with chronic kidney disease are prone to atherosclerosis, which occurs due mainly to lipid abnormalities. Abnormal lipid metabolism could result, among others, from increased formation of free radicals and, consequently, oxidative stress in these patients. The objective of the present study was to evaluate the influence of therapy with lovastatin or with a hypolipemic diet only on oxidative stress in hemodialyzed patients. We addressed the issue by measuring the total antioxidant status (TAS) and the level of 8-hydroxy-2-deoxyguanosine (8-OHdG), an oxidative DNA damage metabolite, in the serum. The study group consisted of 71 patients. They were divided into 3 groups: treated with lovastatin (20 mg/day, n=30), with a hypolipemic diet alone (n=28), and untreated controls (n=13). Serum levels of TAS and 8-OHdG (8-hydroxy-2-deoxyguanosine) were determined. Blood samples were collected at the beginning of the study and then after a 6 months' therapy. We found that the level of 8-OHdG decreased considerably only in the lovastatin-treated group; the decrease was from 15.6+/-8.1 to 12.5+/-4.8 ng/ml (P=0.04). In the other two groups changes in 8-OHdG were insignificant. The level of TAS increased significantly in the lovastatin-treated group from 1.28+/-0.20 to 1.37+/-0.116 mmol/l (P=0.011), decreased in the diet-treated group from 1.55+/-0.14 to 1.45+/-0.11 mmol/l (P=0.007), and remained unchanged in the untreated group (1.42+/-0.11 vs. 1.40+/-0.12 mmol/l). We conclude that lovastatin, but not a hypolipemic diet alone, exerts an antioxidant effect in hemodialyzed patients. However, the determinants of the antioxidant effect of statins in patients with chronic renal failure are unclear and their resolution would require alternative study designs.

The inhibitory effect of combination treatment with leptin and cannabinoid CB1 receptor agonist on food intake and body weight gain is mediated by serotonin 1B and 2C receptors.

Previous studies reported that the co-injection of leptin and cannabinoid CB1 receptor antagonists reduces food intake and body weight in rats, and this effect is more profound than that induced by these compounds individually. Additionally, serotonin mediates the effects of numerous anorectic drugs. To investigate whether serotonin interacts with leptin and endocannabinoids to affect food intake and body weight, we administered 5-hydroxytryptamine(HT)1B and 5-hydroxytryptamine(HT)2C serotonin receptor antagonists (3 mg/kg GR 127935 and 0.5 mg/kg SB 242084, respectively) to male Wistar rats treated simultaneously with leptin (100 µg/kg) and the CB1 receptor inverse agonist AM 251 (1 mg/kg) for 3 days. In accordance with previous findings, the co-injection of leptin and AM 251, but not the individual injection of each drug, resulted in a significant decrease in food intake and body weight gain. Blockade of the 5-HT1B and 5-HT2C receptors completely abolished the leptin- and AM 251-induced anorectic and body-weight-reducing effects. These results suggest that serotonin mediates the leptin- and AM 251-dependent regulation of feeding behavior in rats via the 5-HT1B and 5-HT2C receptors.

Characterization of the adsorptive pinocytic capture of a polyaspartamide modified by the incorporation of tyramine residues.

Previously it has been shown (Duncan, R., Starling, D., Rypácek, F., Drobník, J. and Lloyd, J.B. (1982) Biochim. Biophys. Acta 717, 248-254) that incorporation of tyramine residues into poly (alpha, beta-(N-2-hydroxyethyl]-DL-aspartamide (PHEA) greatly increases its rate of pinocytic uptake by rat visceral yolk sacs cultured in vitro. Here we describe the relationship between the tyramine content (1.2-21.9 mol%) of modified PHEA and its rate of uptake by yolk sacs. Above a level of substitution of approximately 10 mol% the rate of uptake rises rapidly, and the concentration-dependence of capture is indicative of uptake by adsorptive pinocytosis. Serum proteins were shown to compete effectively for membrane binding sites, indicating a nonspecific interaction of PHEA-derivatives with the yolk sac membrane. PHEA derivatives of the same tyramine content, but of different mean molecular weights (Mr), were captured at the same rates.

Pinocytosis of poly (alpha, beta-(N-2-hydroxyethyl))-DL-aspartamide and a tyramine derivative by rat visceral yolk sacs cultured in vitro. Ability of phenolic residues to enhance the rate of pinocytic capture of a macromolecule.

Incorporation of 20% tyramine residues into its structure greatly increased the rate of pinocytosis of poly(alpha, beta-(N-2-hydroxyethyl))-DL-aspartamide (PHEA) by rat visceral yolk sacs cultured in vitro. Both the parent macromolecule and the tyramine derivative (PHEA-tyramine) were captured by adsorptive pinocytosis, the higher affinity of the derivative for the yolk sac plasma membrane being responsible for its greater rate of capture. Using 125I-labelled PHEA-tyramine, the relationship between substrate concentration and rate of capture was determined, it was also shown that following internalization, the PHEA-tyramine linkage is resistant to intracellular hydrolysis. Fluorescence micrographs were consistent with capture of both substrates being by pinocytosis and illustrated the highly efficient concentration of the tyramine derivative by yolk sac endodermal cells.

Immunostimulatory effect of cis-diamine dichloroplatinum (II).

Cis-diamine dichloroplatinum (II) (DDP) injected i.p. or i.v. at the dosage of 0.83 mg/kg to BALB/c mice on days 0, 7 and 14 elicited antibodies against proteins (RSA, BGG) and haptens (TNP, FITC) detectable by ELISA or by passive haemagglutination. Significant increase in antibody production was observed with either route of DDP application. A similar set of antibodies against antigens which do not cross-react with the immunizing proteins was observed when mice were immunized with complexes formed by an in vitro incubation of RSA and BGG with DDP. Thus, proteins modified in vivo or in vitro by DDP may stimulate the production of antibodies of unexpected specificities.

Effect of polyethylene glycol on the rate of immune complex formation by non-precipitating antibody.

A kinetic study formation of large size complexes of non-precipitating pig-Dnp anti-Dnp antibody and multivalent dinitrophenylated serum albumin was performed using light scattering and absorption spectroscopy of the Dnp-group. A very rapid phase of the process resulted in the formation of complexes having molecular weight of about 2 X 10(6). Further increase of the complex size was much slower. Addition of PEG affected positively the rate of complex growth even in concentrations below 1%. The spectroscopic kinetic curves also showed a rapid and a slow phase, sensitive to the presence of PEG. The character of the kinetic data does not support the simple view that polymers enhance precipitate-formation by the steric exclusion of complexes from the polymer domains. It can be assumed that the interaction of the polymer with the antigen-antibody system consists of a subtle temporary attachment of the polymer to the antibody molecule resulting in a change of the shape and/or flexibility of the antibody molecule, favouring its cross-linking capacity.

Retention and biliary excretion of poly-alpha, beta-[N(2-hydroxyethyl)-D,L-aspartamide] (PHEA) and its tyramine derivative (PHEA-Tyr) by isolated perfused rat liver. The role of molecular weight and chemical structure.

Isolated rat liver was perfused with fractions of fluorescent labelled poly-alpha,beta-[N(2-hydroxyethyl)-D,L-aspartamide] (PHEA), (Mw:8000, 51 400 and 70 000) and its tyramine derivative, PHEA-Tyr (19 mol% tyramine side chains; Mw:8000 and 35 000) under conditions approximating an "in vivo" situation. By an analysis of the recovered polymers it was shown, firstly that accumulation of polymers, which occurred most apparently in the Kupffer cells, was neither affected by the presence of tyramine side chains nor influenced by the molecular weight of polymers; secondly that the tyramine side chains slightly increased the biliary excretion of PHEA-Tyr, and thirdly with both types of polymers the excretion of high-molecular-weight fractions in the bile was strongly suppressed.

Evaluation of infusion sets by biological and chemical testing.

Some of the biological and chemical tests used in toxicological evaluation of synthetic polymers were correlated. The individual tests (sperm cell motility, tissue culture inhibition, intracutaneous test, optical absorption at 220-360 nm, reducing impurities) did not mutually correspond. The final estimation of the tested device is assessed as a sum of these units. Therefore, it is necessary to evaluate the results of the individual tests in arbitrary units.

Hydrogels in endovascular embolization. III. Radiopaque spherical particles, their preparation and properties.

The synthesis and properties of spherical radiopaque hydrogel particles designed for endovascular occlusion are reported. These particles were prepared by the hydroxyl acylation of low crosslinked poly (2-hydroxyethyl methacrylate) beads with a nontoxic radiopaque compound based on triiodobenzoic acid, without affecting their properties which are advantages in medical practice. The effect of the iodine content on the size of dry and swollen particles is discussed. It has been found that an iodine content of about 25-30 wt% is desirable in order to obtain an easily recognizable X-ray image. These particles make the immediate control of embolus application easy and enable periodical inspection of the polymer to check the successful blockage of the vessel. They also open up the method of endovascular occlusion to further improvement.

Antitumor activity of platinum complexes.

Contemporary ideas about the mechanism of antitumor activity of platinum complexes are reviewed and discussed. The induction of SOS functions in bacteria is emphasized and an analogous mechanism in animal cells is suggested. The fate of the leaving ligand in the body is not known. Therefore the complex which reaches the target DNA may be very different from the applied compound. Even the amino ligand may be detached in the body, probably as part of the detoxication by binding to proteins. In the case of fast or medium-speed reactive complexes most of the platinum is inactivated by binding to proteins, whereas slow-reacting drugs are mostly excreted unchanged in the urine. Thus, the quantity of the complex which reaches the target is also difficult to assess. Due to peculiar pharmacokinetics, the results obtained with poorly soluble compounds administered in the solid form cannot be compared with those obtained in true solutions. There are many reasons for believing that the study of a coordination anticancer drug may contribute to our understanding of cancer growth and its reversal.

Pharmacokinetics and plasma protein binding of two platinum cytostatics CHIP and CBDCA in rats.

Plasma protein binding and pharmacokinetic parameters of CHIP (cis-dichloro-trans-dihydroxy-bis-isopropylamine platinum IV) and CBDCA (cis-diammine-1,1-cyclobutane dicarboxylate platinum II) were investigated in male Wistar rats. The plasma clearance of total and non-protein-bound platinum was determined and compared with that of 99mTc-DTPA. For binding experiments, a novel, simple, and quick method based on adsorption of non-protein-bound platinum species to charcoal was used. The clearance of total platinum after CHIP and CBDCA administration was markedly lower than the glomerular filtration rate (determined as the clearance of 99mTc-DTPA). The renal clearance of non-protein-bound platinum corresponded to 168% and 50% of the glomerular filtration rate for CHIP and CBDCA, respectively. These studies suggested that CHIP was excreted by the rat kidney.

The reaction of Pt-antitumor drugs with selected nucleophiles. I. The reaction of cis-[Pt(NH3)2Cl2] with glycine.

Various Pt(II)-glycine coordination compounds were characterized by 1H and 13C NMR spectroscopy, some of them also by electrophoretic and chromatographic behavior. The results were applied to the analysis of the reaction mixtures of cis-[Pt(NH3)2Cl2] and glycine obtained under various conditions. Cis-[Pt(NH3)2Cl2] reacts with glycine to give cis-diammine-(glycine-N,O)-Pt(II) and cis-diammine-bis(glycine-N-)Pt(II). Their ratio depends primarily on the pH of the reaction medium. Conformation of these compounds is discussed based on the observed Pt-C and Pt-H NMR coupling constants.

The reaction of Pt-antitumor drugs with selected nucleophiles. II. Preparation and characterization of coordination compounds of Pt(II) and L-histidine.

Various His-Pt(II) coordination compounds were prepared by reaction of K2PtCl4 or cis-[Pt(NH3)2Cl2](cis-DDP) with His and analyzed by 1H and 13C NMR spectroscopy, electrophoresis, and ion-exchange chromatography. His may be coordinated to Pt by the imidazol iminogroup and/or the alpha-aminogroup; the carboxygroup remains always free. Both bidentate as well as monodentate ligands were identified. Cis-DDP reacts with His to give a mixture of compounds where all these possibilities are present: cis-diamine-(histidine-N,N-)Pt(II) and three different types of cis-diammine-bis(histidine). HCl trans cleavage of compounds with bidentate His ligands leads to a mixture of two compounds having His ligated to Pt by an amino or imin group. The methods applied are suitable for analyzing reactions of His with cis-DDP under model conditions similar to physiological conditions.

Inactivation of bacteriophages with cis-platinum(II) diamminedichloride.

The ability of the cis-plantinum(II) diamminedichloride and its hydrolytic products to inactive DNA bacteriophages was examined on the models T2, T4, T4BO1, T3, and lambda. The inactivation of all bacteriophages under study increases gradually during the first 40-90 min of the action of neutral cis-Pt(II) and later passes into an exponential phase. The extent of the region of slower inactviation is larger for osmotically sensitive strains T2 and T4. Inactivation with the hydrolytic products for cis-Pt(II) proceeds exponentially starting from the very beginning and their inactivating effect is higher by 40-80 times than for a comparable concentration of the original complex. The extent of inactivation is not affected with the HCR marker of the host bacteria. The sensitivity to cis-Pt(II) is higher for bacteriophages with a head permeable to salts. An additional inactivation ("after-effect") was observed after dilution of the complex; it can be removed by adding S-aminoisothiuronium dihydrobromide (AET). The results obtained are in good accord with the assumption that inactivation is due to the hydrolytic products arising in the head of bacteriophage.