Immunohistochemistry and serum values of S-100B, glial fibrillary acidic protein, and hyperphosphorylated neurofilaments in brain injuries.

INTRODUCTION: Traumatic brain injury (TBI) triggers a series of reactions resulting in cytoskeletal-related changes varying between focal and diffuse injuries. METHODS: The patients (n=38) were divided into group of diffuse axonal injuries (DAI, n=10) and focal (n=28) injuries. Serum hyperphosphorylated neurofilaments (NF-H) and glial fibrillary acidic protein (GFAP) were measured by Biovendor immunoassay, and serum S-100B protein was measured by Cobas e411 (Roche) by immunoassay. Immunohistochemistry was performed with monoclonal antibodies (Chemicon, USA). RESULTS: The median serum S-100B concentration was higher in patients with focal mass lesions (1.72±0.4 µg/l vs. 0.37±0.1 µg/l, p<0,05) compared to patients with DAI during 10 days of hospitalisation. With respect to all patients, the highest peak of serum S-100B values (4.21±1.1 µg/l) and GFAP (8.58±2.4 µg/l) were found in expansive lesions. The median serum NF-H was higher in DAI compared to focal TBI (0.625±0.14 vs 0.139±0.02 ng/l, p<0.05) during all 10 days after admission. Further, immunohistochemical investigation, in deceased patients with DAI , using NF-H antibody proved positive varicose and waving axons, and retraction balls. Time-dependent profile of serum NF-H demonstrated the increase of values within 4th up to 10th day in both groups. Values ranged from 0.263 up to 1.325 ng/l in DAI, and from 0.103 up to 1.108 ng/l in focal injuries. Patients with expansive contusions had similar levels of serum NF-H as patients without expansive lesions. Immunohistochemistry of cytoskeletal proteins presented strong positive staining of vinculin, vimentin in vessels, GFAP, and S-100B in DAI compared to weak staining in expansive lesions. CONCLUSION: The time-profile kinetics of all markers may reflect different types of pathophysiological changes of the BBB or axonal damage in focal and diffuse injuries. KEYWORDS: brain contusions - diffuse axonal injury - S-100B protein - GFAP - hyperphosphorylated neurofilaments.

[Microglial activation and GFAP astroglial immunopositivity in drug abusers]. / Aktivace mikroglie a GFAP imunopozitivita astroglie u toxikomanu

INTRODUCTION: The neurotoxicity brought about by application of toxic and psychotropic substances is accompanied by an activation of astroglial and microglial cells in the brain. MATERIALS AND METHOD: We investigated clinically 42 patients addicted to psychotropic substances (hospitalised in the Motol Teaching Hospital). The NSE, S1OOB, and manganese concentrations in the blood were measured in the patients. In 14 deceased patients with drug evidence in the hair indicating a chronic abuse of addictive substances, the brain tissue glial cells were immunohistochemically labelled with antibody against CD68 and GFAP. RESULTS: In 8 hospitalised patients, there were increased NSE, S100B values in the blood (p < 0.05). Manganese in the blood was increased (3,03±1,9 µg/l, p < 0,05) in all patients. In deceased persons, the CD68 positivity of microglial cells and neurophagy have been proved. GFAP positive astroglial cells have been evidenced in the gray and white matter. CONCLUSION: The CD68 and GFAP positive glial cells in brain tissue can be a pathomorphological correlate of neurotoxicity in chronic abusers. The neurotoxicity can be monitored with NSE and S100B markers of damaged neuronal cells.

[The origin, distribution and relocability of supravital hemorrhages]. / Vznik, distribuce a mobilita supravitálních hematomu.

The hemorrhage as a result of intravital bleeding is considered, from forensic point of view, as important sign of vital reaction of injury. However, in special cases it must be accepted that hemorrhage occurred after the death. The formation of supravital changes is evident, e.g., in organ donors whose blood circulation and pulmonary ventilation is kept after the brain death. The post-mortem origin of hemorrhages can also be seen in donors of eyeballs after enucleation made before the autopsy at Institute of Forensic Medicine and Toxicology. These hemorrhages are manifested after several hours when eyeballs were removed. Moreover, we observed the origin of hemorrhage in orbit which was caused nor by intravital bleeding neither by direct force. Its origin could not be explained nothing but postmortem propagation of the hemorrhage from the fracture of anterior fossa of the scull base. We did not find information about postmortem origin or relocation of hemorrhage of such extent in the literature. In the frame of knowledge about supravital reaction, this finding is of general importance with forensic impact. The documentation from the scene of death allowed correcting the appreciation of the mechanism of injury and traumatic process from the point of view of foreign culpability which should be considered in such case (e.g., a blow to the orbit with following fall under passing subway train).

[Another mechanism of décollement]. / Jiný mechanismus décollement.

The cause of décollement is usually considered to be tangential brute blunt force impacting the body surface especially in case of hitting or running-over injury of the pedestrian's body by a car. The term rolling effect or rolling mechanism is used as well. The dissociation of tissue layers with other epiphenomenon occurs. The presented group of 152 décollement determined in 103 autopsy cases during the 4 years period comprises the observation of décollement of different etiology of the injuries (traffic accidents, falls from the high, compression of the torso); in the traffic accidents the occurrence in various participants of the traffic, not only in the case of the collision of the pedestrians with various traffic vehicles, but also in drivers of various traffic vehicles, and fellow-travelers as well. The topic, the localization, the content, the extent, and vital reaction and combination injuries were followed-up. According to the variability of the injury etiology, not restricted to the traditional conception of the décollement mechanism, it is obvious that the passed on rule by far is not covering the whole content of this concept. We didn't find any alternative interpretation of until now presented mechanism in the literature. The medical literature focuses mostly on the clinical aspect of this injury. In this paper, the biomechanics of the décollement origin also in case of the tissue compression by the pressure applied perpendicularly to the body surface, the dependences on physical properties of the actively or passively affecting object, the relevance of the ratio of the tissue structures compression in one direction and transversal dilation in other two directions according to the Poisson's constant, the question of tangential factor of the force in case of vertical falling on the horizontal plane, and biomechanical relations in case of body landing on an oblique surface are discussed. The mechanism of décollement is more complex as presented until now. The forensic interpretation of findings should reflex the above-mentioned facts.

[Evaluation of relevance in concussion and damage of health by monitoring of neuron specific enolase and S-100b protein]. / Hodnocení závaznosti otresu mozku a poruchy zdraví pomocí monitorování hladin neuron specifické enolázy a proteinu S-100b v séru.

INTRODUCTION: Proteins released to the circulation from affected glial (neuron specific enolasis, NSE) or ganglial cells (S-100b protein) during traumatic brain injury might be used in diagnosis of traumatic brain injury in cases with negative finding on computer tomography scan (concussion) or in patients where the serious clinical status does not corresponde with mild changes on CT scan (diffuse axonal injury, DAI). Classification of DAI according Gennarelli considered the concussion as lower degree of DAI. MATERIALS AND METHOD: 15 patients were divided into group I of mild conccussion (n=3) with 1-day duration of hospitalisation, group II of serious concussion (n=4) with more days duration of hospitalisation with negative findings on CT scan and group III of patients with diagnosis of DAI (n=8). Blood samples were investigated by immunoanalysis for NSE and protein S-100b (Elecsys 2010, Roche). RESULTS: Values of NSE (16.30 +/- 2.33 vs. 110.48 +/- 34.99 vs. 24.07 +/- 6.29 microg/l), and protein S-100b (0.207 +/- 0.03 vs. 0.945 +/- 0.69 vs. 0.736 +/- 0.36 microg/l) overdrow the reference value in cases of group I, II, and III. We discuss the biomechanics of trauma and the blood brain barrier damage in comparison with values of NSE and S-100b protein. CONCLUSION: [corrected] We proved the significantly higher values of the NSE in group of serious concussion compared to group of DAI. We demonstrated that concussions in some cases lead to serious damage of health.

[Detection of myocardial and lung injury by psychotropic and narcotic agents by immunohistochemical detection of metalloproteinases]. / Prukaz poskození myokardu a plic psychotropními a omamnými látkami imunohistochemickou detekcí metaloproteináz.

Positive findings of intoxicant (narcotic) and psychotropic drugs (OPL) have been regularly recorded in clinical patients and deceased persons over the last years at the Institute of Forensic Medicine and Toxicology, 1st Medical faculty and General Teaching Hospital, Prague; stimulants and opioids represent the most frequent cause of death. Their misuse results in damage to various organs. In order to follow the development of pathological changes in the process of remodeling extracellular matrix directly in tissues, the methods of immunohistochemical detection of the matrix metalloproteinases in myocardium and lungs as well as fibrinogen in cardiomyocytes were selected for analysis in a group of 18 deceased individuals. In the intoxication with stimulants we usually demonstrated MMP 2 in the myocardium interstitium, MMP 9 being observed in two cases and MMP 1 in one case. The analysis of lungs always demonstrated MMP 1, especially in the lung interstitium and also on the surface of some alveoli, which accepted the appearance reaching up to "hyaline membranes" as well as in cellular elements of macrophage type and and the same was true for MMP2. Fibrinogen was not always demonstrated in cardiomyocytes. The detection of metalloproteinases was less prominent in the case of opioids. The demonstration of MMP explains well the evolution to more advanced pathomorphological changes, which have been found in myocardium and lungs of OPL users and fits to the nosological status of earlier phases of intoxications with these drugs.

[Time-related expression of adhesive proteins and other markers of age of injuries]. / K casovým relacím exprese adhezivních bílkovin a dalsích markeru stárí poranení.

UNLABELLED: In addition to evidence of the vital reaction it is important from the forensic aspect to assess the age of wounds, in particular in wounds with a short time of survival. To both can contribute detection of adhesive molecules, identified in recent years, possibly in combination with other markers of early stages of reparative inflammation. The submitted paper comprises the results of investigation of 465 skin wounds. The investigated samples were obtained from necroptic material, excision from wounds of treated patients and from experimental injuries in mice. Assessment of the age of injuries by means of endothelial adhesive molecules was made in paraffin sections after transfer into Varioclave and using the ABC techniqueref.. In human material a strong positive reaction of ICAM-1 was observed first after 1 and a half hours and latest after 3 and a half days, in P-selectin first after 3 mins., latest after 7 hours, in E-selectin first after 1 hour and latest after 17 days, in VCAM-1 first after 3 hours and latest 3 and a half days after the development of the injury. Expression of L-selectin was not typical for the injury. In skin injuries of mice positive immunohistochemical reactions were found as a rule sooner than in skin injuries of humans. Fibronectin was detected in paraffin sections from 70 skin wounds of dissected subjects immunohistochemically by the indirect immunoperoxidase reaction and by the use of the ABC technique labelled with alkaline phosphatase. Positive evidence was observed first after 5 minutes and latest after 8 hours. CONCLUSION: Detection of a rise of expression of ICAM-1, VCAM-1 and P- and E. selectin and the formation of basic netlike fibronectin structures improves the assessment of the age of wounds with a short survival period.

DNA adducts and human atherosclerotic lesions.

It has been hypothesized that mutational events may be involved in the atherogenetic process and that at least a portion of atherosclerotic plaques may be the results of monoclonal proliferation of a single mutated smooth muscle cell (SMC). Therefore, atherosclerosis may be similar to carcinogenesis and may have an environmental etiology. We have analyzed bulky-aromatic DNA adducts in human thoracic aortas from male subjects, aged between 30-60 years, who died suddenly or accidentally, and who had been examined by autopsy within 24 h after death. We found significantly (P < 0.001) higher DNA adduct levels in the samples from subjects with frequent atherosclerotic changes in the whole body ("Cases", N = 76) compared with those having few atherosclerotic changes ("Controls", N = 57). We also observed a significantly elevated weight of heart and plasma levels of total and LDL cholesterol in "Cases" vs "Controls". Significant differences in DNA adduct levels between smokers and nonsmokers were observed in "Controls" only. Multivariate linear regression analyses with age-adjusted data confirmed a significant influence of LDL cholesterol (P < 0.001), vitamin A (P < 0.01), smoking behavior (P < 0.05; evaluated as plasma cotinine levels) and NAT2 genotypes (P < 0.05) on bulky-aromatic DNA adduct levels. The induction of DNA adducts suggests that alterations at the DNA level may contribute to the development of atherosclerosis. Furthermore, atherogenesis and carcinogenesis may share a similar etiology, i.e. genotoxic action of environmental chemicals.

[Comparison study of the HUMFGA and HTPO STR system in the Saxon and Czech population]. / Srovnávací populacní studie saského a ceského HUMFGA a HTPO STR systému.

High variability of repetitive DNA in a non-coding region of human genome affords possibilities for vestige identification and determination of their origin. For analysis of DNA many a time obtained from decomposed material, usually in a very small amount and sometimes partially degraded, polymerase chain reaction is considered as optimal technique. For identification of individuals is used an Amplified Fragment Length Polymorphisms (AmpFLP), which present repetitions 10 to 70 base pairs long in an amplification products of 170 to 1200 bp. In legal medicine PCR systems using tetrameric repetition polymorphism are widely used. These systems are highly sensitive, but in comparison to AmpFLP systems, imperceptibly influenced by nucleic acid degradation in cases of old or partially degraded samples. Aim of our study was to compare frequency data for 2 STR-systems, FGA and HTPO, both after PCR amplification, on 200 samples of Czech and 228 of German population. In FGA system of Czech population 14 different alleles and 44 genotypes were determined, while German population provided 13 different alleles and 47 genotypes. Furthermore, frequency of alleles and heterozygosity rate were determined. Heterozygosity rate for Czech samples was 85%, for German ones 87.1%. Alleles 21 and 22 were most frequent (approx. 20%) between both Czech and German population, followed by alleles 20, 23 and 24 (14-12%). Besides these data, three different transient alleles were detected. In HTPO systems of both population groups 12 different genotypes, consisting of 5 alleles, were determined. Genotypes 8/8 and 8/11 observed in 30% were the most frequent ones in both population groups. Heterozygosity rate for Czech samples was 62.5%, for German ones 65%. Results were statistically analyzed. In both population groups, no significant difference from Hardy-Weinberg's balance was found. Additionally, statistic parameters for forensic effectivity evaluation were constructed. Comparison of both populations using homogeneity tests provided non-significant difference. FGA system provides extraordinary high forensic exploitativeness and therefore is fully acceptable for vestige identification and determining of their origin. For significant determination of transient alleles analysis of fluorescein-labelled fragments in denaturing gel system should follow, using automated laser detection of fluorescence. Polymorphism of HTPO-system is significantly lower compared to FGA-system, but relatively small PCR product required as well as simple detection and imperceptible influence of nucleic acid degradation allow the use of HTPO for analysis of highly degraded DNA.

Expression of adhesion molecules in skin wounds: diagnostic value in legal medicine.

The adhesion molecules identified in recent years can help improve the diagnosis of the wound age, especially of injuries with a short survival time. This is also indicative of the vitality of the wounds. The material investigated in the study originated from 465 skin wounds. The samples were taken from human autopsy material, during the surgical treatment of wounds (excision) of patients and from experimental incised wounds of mice. To judge the age of skin wounds the endothelial adhesion molecules were detected in paraffin sections after autoclaving and using the ABC technique. Human skin wounds: strong positive staining was observed of ICAM-1 1.5 h at the earliest and 3.5 days at the latest, for the P-selectin 3 min at the earliest and 7 h at the latest, for the E-selectin 1 h at the earliest and 17 days at the latest and for VCAM-1 3 h at the earliest and 3.5 days at the latest after the time of injury. The L-selectin was expressed constitutively. Mice skin wounds: strong positive immunohistochemical reactions were found as a rule earlier than in human skin wounds. The detection of an increased expression of ICAM-1, VCAM-1 and P- and E-selectins can improve the wound age assessment in injuries with short survival times.

[Polychlorinated biphenyls in human subepicardial fat]. / PCB v lidském subepikardiálním tuku.

A considerable relation between myocardial fatty infiltration (lipomatosis) and ischemic lesion of myocardium was proved by authors else where. This time, polychlorinated biphenyls (PCB) wee studied in subepicardial fat as well as in subcutaneous fat in thorax and abdomen of deceased person after sudden and violent death. For the time being, most published results concerned the abdominal subcutaneous fat. All the published results showed high concentrations of PCB were proved in subcutaneous fat tissue from thorax and abdominal area and specially in subepicardial fat. Topical relation of PCB deposits and heart muscle was direct in all analyzed case because of fatty infiltration of myocardium. Obviously, a direct toxic effect of PCB on myocardium can take place in stress lypolysis e.g. associated with heart infarct (in some animals hydropericardium and impairment of parenchymatous organs were observed) and prognosis of patients with ischemic lesion can get worser.

[Determination of pregnancy by detection of SP1 in tissues]. / Dukaz tehotenství ve tkáních detekcí SP 1.

During investigations of murder sometimes in addition to traces of blood only some organs, tissues and remnants of the victim's body are available. Evidence of pregnancy in the residues, mere portions of the divided body without the genitalia would provide important information for the investigation and would help to classify the parts of the body and to identify the victim. Using double radial immunodiffusion in the modification of permanent elution by means of anti-SP 1 antiserum produced according to Malbohan and antiserum of Dakopatts Co. (Glostrum, Denmark) in tissue excisions and tissue impressions the pregnancy product SP 1 (pregnancy-specific-beta-1-glycoprotein) was detected. The author examined 269 different specimens: 1. excisions of organs from women who died during the 8th-35th week of pregnancy by sudden and violent death (43 specimens). 2. excisions which were made during surgical operations of pregnant women during the 8th-20th week of pregnancy (16 specimens) and during delivery by Caesarean section (210 specimens). Excisions from the kidneys, spleen, lungs, liver, heart, skeletal muscle, subcutaneous layer, skin, brain were examined from corpses. During operations excisions from the subcutaneous layer, uterus, skin, great omentum, peritoneum, and oviduct were made and examined. Furthermore the author made 3. impressions of tissues on washed cotton material. In the organs collected on necropsy from pregnant women SP 1 was detected in 82% of the specimens, with the exception of the brain where it was never detected. In tissues from operated pregnant women during the first half of pregnancy SP 1 was detected in 63% specimens incl. only in 33% in the 8th week of pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

[Evidence of pregnancy in blood stains]. / Dukaz tehotenstvi v krevnich stopách.

Evidence of pregnancy in blood traces is important for proving murder of the newborn child by the mother or illegal abortion. By immunoprecipitation in a semi-solid medium (by double radial immunodiffusion, counter-current immunoelectrophoresis), using antiserum of their own production, the authors proved in blood traces a specific gestational product--SP1 (Schwangerschaftspezifisches beta-1-Glykoprotein, PS-beta-G, TSG, PAPP-C). The authors examined a total of 172 blood traces on cloth from pregnancies of different stages (37 from the first trimester, 34 from the beginning of the second half of pregnancies and 101 at term) as extracts from the blood stain or particles of the blood stains on a slide. The reliability was 91--95%. Hitherto used methods were based on detection of choriogonadotropic hormone. Detection of SP1 can be considered reliable evidence of pregnancy. It was possible to detect it in blood traces starting with the fourth month of pregnancy.

[Detection of group characteristics and contamination with microorganisms]. / Prukaz skupinových vlastnosti a kontaminace mikroorganismy.

Evidence of group properties in traces and residues of human origin may be very difficult; very frequently in the same individual controversial results are obtained in different tissues but also in the same samples during repeated examinations. This was revealed by examinations of reliets from prehistorical findings as well as in remains of Czech historical personalities and contemporary important forensic cases, using different methods of detection, incl. the absorption elution test. The author discusses the condition of investigated residues and traces, the degree of decomposition of tissues due to the possible action of microorganisms with regard to assessment of group properties. The investigation revealed that in addition to the anticipated action of some microorganisms contaminating examined tissues, different transformation of group properties of the sample, and panaglutinability or disintegration of the entire ABO system, a possible effect of the organism's own serological activity of microorganisms on results must be also taken into account. Examination of different tissues of the individual, and if the results are controversial, examination of group properties of cultivated microorganisms makes it possible to eliminate to a certain extent the artificial component of results and makes the conclusions more accurate and helps to explain the interpretation of results.

[Detection of urinary substances in stains from amniotic fluid, urine and the blood of pregnant women using thin-layer chromatography]. / Detekce mocových substancí ve skvrnách od plodové vody, moci a krve tehotných pomocí chromatografie na tenké vrstve.

When examining traces of biological origin it is possible to draw conclusions from the findings of some urinary constituents in a stain produced by urine; via foetal urine, however, in the spots also amniotic fluid can be detected. By chromatographic analysis on a thin layer the authors are able to detect in spots from amniotic fluid urea without hippuric acid, in urine spots urea, creatinine and hippuric acid; in blood stains under equal conditions none of the above constituents is detected. The results, using the above method, can be considered a contribution which helps to differentiate these traces and it can be also used to detect amniotic fluid in stains.

[Detection of amniotic fluid spots by placental alkaline phosphatase]. / Detekce skvrn od plodové vody prukazem placentární alkalické fosfatázy.

In spots of amniotic fluid from the time of delivery placental alkaline phosphatase was detected reliably by the azocopulation reaction after inactivation of the thermolabile fraction. Evidence was positive in all 82 examined samples collected during delivery. A positive test was obtained already during the 16th week of gestation, while in the first trimester the result of the reaction was uncertain. Detection in the spots is better than in the extract from the spot and can be made event after two months. On the other hand, a test made on a slide makes also morphological examination possible. As regards evidence of pregnancy origin of the spots, the test is specific.

[Investigation of magnesium and calcium in ischemic changes in the human myocardium]. / Vysetrováníní magnézia a kalcia pri ischemických zmenách v lidském myokardu.

The results are presented of tests for magnesium and calcium and their ratio in the human myocardium affected by ischemia in correlation with macroreaction to dehydrogenases. The cases were examined in the shortest possible time after death, observing strict criteria. Even some of the relatively early postmortem changes were found to block the practical diagnostic uses of the changes of magnestium and calcium metabolism in postmortem material.