Forensic pathological study on temporal appearance of dendritic cells in skin wounds.

Dendritic cells (DCs) can essentially contribute to innate and adaptive immune system in various organs. A double-color immunofluorescence analysis was carried out with anti-CD11c and -HLA-DRα antibodies to detect DCs in 53 skin wounds (their postinfliction intervals: group I, 0-3 days; group II, 4-7 days; group III, 9-14 days; and group IV, 17-21 days). CD11c+HLA-DRα+ DCs were first observed in skin wounds with postinfliction intervals of 3 days, and the DC numbers were found to be elevated in skin wounds with the subsequent increase in postinfliction intervals. Semi-quantitative morphometric analyses showed that the DC number was the highest in the 12-day-old wound. More than 50 DCs were present in 8 of 10 samples (80%) in group II and 14 of 16 samples (87.5%) in group III, and there was no difference between the two groups. Thus, the presence of DCs in a skin wound was possibly estimated as postinfliction intervals of at least 3 days. Furthermore, when a skin wound contained > 50 DCs, its age would be judged as 4-14 days. Collectively, the appearance of DCs in human skin wounds may provide useful information in determining the age of a wound.

Immunohistochemical analysis on aquaporin-1 and aquaporin-3 in skin wounds from the aspects of wound age determination.

Immunohistochemical investigation of aquaporin (AQP)1 and AQP3 was performed in human skin wounds obtained from forensic autopsy cases. A total of 55 human skin wounds of different postinfliction intervals were collected as follows: group I, 0-3 days (n = 16); II, 4-7 days (n = 11); III, 9-14 days (n = 16); and IV, 17-21 days (n = 12). In uninjured skin samples, AQP1 and AQP3 could be slightly detected in dermal vessels and keratinocytes, respectively. The percentage of AQP1+ vessels and the number of AQP3+ keratinocytes were apparently elevated in accordance with wound ages. The number of AQP3+ keratinocytes was distinctly evident in groups II and III. Morphometrically, both AQP1+ vessel area and AQP3+ cell number were markedly increased in group II, compared with other three groups. With regard to forensic safety, AQP1+ vessel area of over 5% would imply wound ages of 4-12 days. Moreover, the positive area of > 15% would suggest wound age of 7-10 days. Especially, most samples of skin wounds aged 5-10 days except for only one sample (a 10-day-old wound) showed AQP3+ cell number of > 300, and the remaining other samples had that of < 300. Thus, the AQP3+ cell number of > 300 would indicate wound ages of 5-10 days. Collectively, immunohistochemical analyses of AQP1 and AQP3 in human skin wounds would support the objective accuracy of wound age determination.

Detection of endothelial progenitor cells in human skin wounds and its application for wound age determination.

Endothelial progenitor cells (EPCs), a newly identified cell type, are bone marrow-derived progenitor cells that co-express stem cell markers and vascular endothelial growth factor (VEGF) receptor (Flk-1). In this study, a double-color immunofluorescence analysis was carried out using anti-CD34 and anti-Flk-1 antibodies to examine the time-dependent appearance of EPCs, using 52 human skin wounds with different wound ages (Group I, 0-1 days; Group II, 2-6 days; Group III, 7-14 days; and Group IV, 17-21 days). In wound specimens with an age of less than one day, CD34(+)/Flk-1(+) EPCs were not detected. EPCs were initially observed in wounds aged two days, and their number was increased in lesions with advances in wound age. In morphometrical analysis, the average number of EPCs was the highest in the wounds of Group III. Especially, 20 out of 21 wounds aged 7-12 days had >20 EPCs, and all wound samples with postinfliction intervals of 14-21 days had <15 EPCs. These observations at least showed that >20 EPCs would indicate a wound age of 7-12 days. Taken together, our observations indicate the detection of EPCs would be useful for wound age determination.

Immunohistochemical analysis on MMP-2 and MMP-9 for wound age determination.

We performed immunohistochemical study combined with morphometrical analyses in order to examine the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 using 55 human skin wounds of different ages: group I, 0-3 days (n = 16); II, 4-7 days (n = 11); III, 9-14 days (n = 16); and IV, 17-21 days (n = 12). Immunopositive reactions for MMP-2 were observed in all human skin specimens including uninjured skin as control. The number of MMP-2(+) macrophages was significantly increased in accordance with wound ages. In contrast to MMP-2, no MMP-9(+) signals were detected in uninjured and wound specimens aged less than 1 day. However, the number of MMP-9(+) macrophages profoundly appeared in groups II and III. Morphometrically, in all of wound samples aged 9-12 days, MMP-2(+) cell number was more than 20. On the contrary, most of the remaining samples had <20 positive cells. However, only one sample (a 7-day-old wound) showed 21 positive cells. Thus, with regard to practical applicability with forensic safety, MMP-2(+) macrophages of >20 would indicate a wound age of 7-12 days. Additionally, 10 out of 12 wound specimens aged 9-12 days showed the MMP-2(+) cell number of >25, implying that MMP-2(+) cell number of >25 would indicate the wound age of 9-12 days. On the contrary, all wound samples aged 3-14 days except for only one sample had MMP-9(+) cell number of >30, indicating that MMP-9(+) cell number of >30 would indicate the wound age of 3-14 days. Collectively, MMP-2 seemed to be more distinct marker, compared with MMP-9.

Huntington's Disease (HD): Neurodegeneration of Brodmann's Primary Visual Area 17 (BA17).

Huntington's disease (HD), an autosomal dominantly inherited polyglutamine or CAG repeat disease along with somatomotor, oculomotor, psychiatric and cognitive symptoms, presents clinically with impairments of elementary and complex visual functions as well as altered visual-evoked potentials (VEPs). Previous volumetric and pathoanatomical post-mortem investigations pointed to an involvement of Brodmann's primary visual area 17 (BA17) in HD. Because the involvement of BA17 could be interpreted as an early onset brain neurodegeneration, we further characterized this potential primary cortical site of HD-related neurodegeneration neuropathologically and performed an unbiased estimation of the absolute nerve cell number in thick gallocyanin-stained frontoparallel tissue sections through the striate area of seven control individuals and seven HD patients using Cavalieri's principle for volume and the optical disector for nerve and glial cell density estimations. This investigation showed a reduction of the estimated absolute nerve cell number of BA17 in the HD patients (71,044,037 ± 12,740,515 nerve cells) of 32% in comparison with the control individuals (104,075,067 ± 9,424,491 nerve cells) (Mann-Whitney U-test; P < 0.001). Additional pathoanatomical studies showed that nerve cell loss was most prominent in the outer pyramidal layer III, the inner granular layers IVa and IVc as well as in the multiform layer VI of BA17 of the HD patients. Our neuropathological results in BA17 confirm and extend previous post-mortem, biochemical and in vivo neuroradiological HD findings and offer suitable explanations for the elementary and complex visual dysfunctions, as well as for the altered VEP observed in HD patients.

DNA methylation analysis of the angiotensin converting enzyme (ACE) gene in major depression.

BACKGROUND: The angiotensin converting enzyme (ACE) has been repeatedly discussed as susceptibility factor for major depression (MD) and the bi-directional relation between MD and cardiovascular disorders (CVD). In this context, functional polymorphisms of the ACE gene have been linked to depression, to antidepressant treatment response, to ACE serum concentrations, as well as to hypertension, myocardial infarction and CVD risk markers. The mostly investigated ACE Ins/Del polymorphism accounts for ~40%-50% of the ACE serum concentration variance, the remaining half is probably determined by other genetic, environmental or epigenetic factors, but these are poorly understood. MATERIALS AND METHODS: The main aim of the present study was the analysis of the DNA methylation pattern in the regulatory region of the ACE gene in peripheral leukocytes of 81 MD patients and 81 healthy controls. RESULTS: We detected intensive DNA methylation within a recently described, functional important region of the ACE gene promoter including hypermethylation in depressed patients (p = 0.008) and a significant inverse correlation between the ACE serum concentration and ACE promoter methylation frequency in the total sample (p = 0.02). Furthermore, a significant inverse correlation between the concentrations of the inflammatory CVD risk markers ICAM-1, E-selectin and P-selectin and the degree of ACE promoter methylation in MD patients could be demonstrated (p = 0.01 - 0.04). CONCLUSION: The results of the present study suggest that aberrations in ACE promoter DNA methylation may be an underlying cause of MD and probably a common pathogenic factor for the bi-directional relationship between MD and cardiovascular disorders.

Immunohistochemical analysis on cyclooxygenase-2 for wound age determination.

Immunohistochemical study combined with morphometry was carried out to examine the expression of cyclooxygenase-2 (COX-2) using 60 human skin wounds of different ages: group I, 0-4 h (n = 11); II, 8 h-2 days (n = 21); III, 3-9 days (n = 14); and IV, 12-21 days (n = 14). In wound specimens aged 2 h to 2 days, anti-myeloperoxidase-positive neutrophils observed at the wound site expressed immunopositive reaction to COX-2. In wound specimens of more than 3 days, CD68-positive macrophages as well as neutrophils were positively immunostained with anti-COX-2. In group II, all 21 wound samples had COX-2-positive ratios of >40 %, and 15 out of them showed >50 %. In group III, only three wound samples with the postinfliction intervals of 3 days showed positive ratios of 40-50 % and the remaining 11 cases less than 40 %. In groups I and IV, all 25 wound specimens had COX-2-positive ratio of <40 %. With regard to the practical applicability with forensic safety, these observations suggested that a COX-2-positive ratio of >40 % indicated a wound age of 8 h to 3 days. Moreover, COX-2-positive ratios, considerably exceeding a ratio of 50 %, indicate a wound age of 8 h to 2 days. Collectively, COX-2 would be a useful marker for the determination of early wound age.

Alternative splicing and extensive RNA editing of human TPH2 transcripts.

Brain serotonin (5-HT) neurotransmission plays a key role in the regulation of mood and has been implicated in a variety of neuropsychiatric conditions. Tryptophan hydroxylase (TPH) is the rate-limiting enzyme in the biosynthesis of 5-HT. Recently, we discovered a second TPH isoform (TPH2) in vertebrates, including man, which is predominantly expressed in brain, while the previously known TPH isoform (TPH1) is primarly a non-neuronal enzyme. Overwhelming evidence now points to TPH2 as a candidate gene for 5-HT-related psychiatric disorders. To assess the role of TPH2 gene variability in the etiology of psychiatric diseases we performed cDNA sequence analysis of TPH2 transcripts from human post mortem amygdala samples obtained from individuals with psychiatric disorders (drug abuse, schizophrenia, suicide) and controls. Here we show that TPH2 exists in two alternatively spliced variants in the coding region, denoted TPH2a and TPH2b. Moreover, we found evidence that the pre-mRNAs of both splice variants are dynamically RNA-edited in a mutually exclusive manner. Kinetic studies with cell lines expressing recombinant TPH2 variants revealed a higher activity of the novel TPH2B protein compared with the previously known TPH2A, whereas RNA editing was shown to inhibit the enzymatic activity of both TPH2 splice variants. Therefore, our results strongly suggest a complex fine-tuning of central nervous system 5-HT biosynthesis by TPH2 alternative splicing and RNA editing. Finally, we present molecular and large-scale linkage data evidencing that deregulated alternative splicing and RNA editing is involved in the etiology of psychiatric diseases, such as suicidal behaviour.

Limiting life-sustaining treatment in German intensive care units: a multiprofessional survey.

PURPOSE: Deciding about the limitation of life-sustaining treatment (LST) is a major challenge for intensive care medicine. The aim of the study was to investigate the practices and perspectives of German intensive care nurses and physicians on limiting LST. METHODS: We conducted an anonymous, self-administered questionnaire survey among the 268 nurses and 95 physicians on all 10 intensive care units of the Munich University Hospital, Germany. RESULTS: The response rate was 53%. Of all respondents, 91% reported being confronted with the topic at least once a month. Although all reported limiting cardiopulmonary resuscitation, almost no one reported limiting artificial hydration. Half of nurses and junior physicians felt uncertain about the decision-making process. Junior physicians were most dissatisfied with their training for this task and expressed the highest fear of litigation. Nurses were less satisfied than physicians with the communication process. Both nurses and relatives were not routinely involved in decision making. There is no standardized documentation practice, and many notes are not readily accessible to nurses. CONCLUSIONS: Limiting LST is common in German intensive care units. The major shortcomings are team communication, communication with the patient's family, and documentation of the decision-making process.

Prostate cancer lesions are surrounded by FOXP3+, PD-1+ and B7-H1+ lymphocyte clusters.

The immune response against prostate cancer seems to be inefficient although tumour cells show an over-expression of tumour-associated antigens suggesting that regulatory networks inhibit immune cell function locally. To address this proposition, lymphocytes within prostate cancer-inflicted tissue were analysed for the expression of markers associated with negative regulatory function and exhaustion. Prostate cancer, benign prostatic hyperplasia and healthy prostate tissues were investigated by immunohistology for CD25, FOXP3, PD-1 and B7-H1. We had previously documented that prostate cancer islets are surrounded by clustered accumulations of CD3+ lymphocytes, which lack perforin and interferon-gamma (IFNgamma) expression, thus are apparently quiescent. Here, we report that these clusters contain numerous CD25+ and FOXP3+ cells. These markers are associated with regulatory T cells, and their presence in lymphocyte clusters near prostate cancer regions indicates an environment with negative impact on immune response against cancer cells. Consistent with this hypothesis, cells expressing PD-1 and its ligand B7-H1, which are markers associated with exhaustion of lymphocyte function, were also detected in the lymphocyte clusters. Expression of molecules associated with inhibition and exhaustion of lymphocytes may reflect events contributing to ineffective immune responses against cancer cells.

Detection of fibrocytes in human skin wounds and its application for wound age determination.

Fibrocytes, a newly identified cell type, are bone marrow-derived mesenchymal progenitors that coexpress hematopoietic cell antigens and fibroblast products. In this study, a double-color immunofluorescence analysis was carried out using anti-CD45 and anti-collagen type I antibodies to examine the time-dependent appearance of fibrocytes, using 53 human skin wounds with different wound ages (group I, 0-3 days; group II, 4-7 days; group III, 9-14 days; and group IV, 17-21 days). In wound specimens with an age of less than 3 days, CD45+/collagen type I+ fibrocytes were not detected. The fibrocytes were initially observed in wounds aged 4 days, and their number increased in lesions with advances in wound age. In a semiquantitative morphometrical analysis, the average number of fibrocytes was highest in the wounds of group III. These findings imply that human skin wounds containing fibrocytes are at least 4 days old. Moreover, a fibrocyte number of over 10 indicates a wound age between 9 and 14 days (i.e., group III). Based on the average number of fibrocytes in each group, a fibrocyte number of over 15 more strongly suggests a wound age of 9-14 days. Together, our observations indicate the participation of fibrocytes in wound healing of human skin inducing the accumulation of extracellular matrix components, and therefore, detection of fibrocytes could be a useful marker for wound age determination.

[Suicidal gunshot to the nape from a small-bore rifle]. / Suizid durch Gewehrschuss in den Nacken.

A 55-year-old man was found dead on the bed lying on his side in a pool of blood with a bullet entrance hole in the nape. Behind his body, a semi-automatic rifle Remington Nylon, cal. .22 LR, was detected. As the gunshot entrance site was rather unusual for suicide, a forensic autopsy was performed, which showed a contact shot to the nape with the bullet path running upward to the left frontal area. The man had been treated with citalopram for delusional depression, so that a suicidal act seemed plausible, and the autopsy and criminalistic findings were also compatible with this assumption. A remarkable fact of the present case is that a long firearm had been used. Reports on suicidal shots to the nape are comparatively rare in the medicolegal literature and usually refer to pistols or revolvers.

Aberrant expression of histo-blood group A type 3 antigens in vascular endothelial cells in inflammatory sites.

Histo-blood group ABH antigens are widely distributed in human tissues. The epitopes of ABH antigens are carried by at least four different peripheral core isotypes of internal carbohydrate backbones (type 1-4). Each type of ABH antigen is expressed tissue specifically, and aberrant expression of ABH antigens is often observed during oncogenesis. We immunohistochemically examined the expression of A type 3 antigens in wounded and diseased skin tissues (A and AB blood groups). In uninjured skin, the expression of A type 3 antigens was restricted to the eccrine sweat gland. In addition to the sweat glands, A type 3 antigens were found in vascular endothelial cells of the wound sites. The extent of A type 3 antigens expression related to postinfliction intervals. A significantly higher expression rate of A type 3 antigens in endothelial cells was also observed in diseased skin, suggesting that inflammation might induce A type 3 antigen expression in endothelial cells. Double-color immunofluorescence staining of the specimens showed that von Willebrand factor (vWF) was a core-protein of A type 3 determinants aberrantly expressed in endothelial cells in inflamed tissues, suggesting that aberrant expression of A type 3 antigens is involved in stabilization of vWF in inflammation.

Frequency of different anti-depressants associated with suicides and drug deaths.

From each case of suicide and drug-related death autopsied in the Institute of Forensic Medicine, Munich during the years 2001--2005, a toxicological investigation on anti-depressants (AD) was performed. In 180 suicides and 72 narcotic drug death cases, ADs were detected: 4 different classic tricyclic anti-depressants (TCAs), 6 other non-selective monoamine re-uptake inhibitors (NSMRIs), 5 selective serotonin re-uptake inhibitors (SSRIs) and 3 other ADs. The suicides were grouped further according to the type of suicide (violent or non-violent). The prescription frequency of the ADs in Germany, expressed as the defined daily dosages (DDDs), during the investigated years served for comparison. There were serious differences in the frequency of different ADs regarding to the manner of suicide. In cases associated with doxepin and trimipramine, non-violent suicides were distinctly over-represented, as in cases in which the drug itself was responsible for the death as in cases of non-violent suicides in other manners. In contrast, in cases with citalopram or opipramol, violent forms of suicides were significantly over-represented. For amitriptyline, the ratio was approximately balanced. For the remainder of the ADs, the case numbers were too low for a valid evaluation. The different frequency distributions of the ADs, associated with violent and non-violent suicides may be explained by their different pharmacological active profiles and the different lethality of overdoses of the different ADs. There was no indication at all for a special suicidal problem of SSRIs in juveniles. Amongst 1,127 suicides within 5 years, in an area with approximately 5 million people, the youngest suicide victim with SSRIs was 28 years old. In drug death cases, citalopram was obviously over-represented.

Dominance of CD4+ lymphocytic infiltrates with disturbed effector cell characteristics in the tumor microenvironment of prostate carcinoma.

BACKGROUND: Prostate cancer is the most common cancer of men in the Western world. Despite the over-expression of tumor-associated antigens, like PSA or PSMA, immune activation is inefficient. The goal of this investigation was to assess in situ characteristics of prostate cancer-infiltrating lymphocytes and to determine their activation status and effector function. METHODS: We compared 17 carcinoma containing tissues, four benign prostatic hyperplasia tissues and eight healthy prostate tissues regarding lymphocyte subset composition, locoregional distribution, and functional status using immunohistological staining of cryopreserved tissues. For determination of lymphocyte subsets, serial sections were stained with CD3, CD4, and CD8 antibodies. Activation status and effector function were studied using CD69, interferon-gamma (IFN gamma), perforin, and CD3 zeta chain antibodies. T-cell-receptor repertoire (TCR) analysis was made to determine the complexity of infiltrating lymphocytes. RESULTS: CD3+, CD4+, and CD69+ T lymphocytes were prominent in tissues derived from patients with prostate carcinoma. CD8+ lymphocytes were significantly less than CD4+ lymphocytes. IFN gamma and perforin were downregulated on infiltrating lymphocytes compared to cells of healthy prostate tissue. Very few lymphocytes were detected within cancerous lesions whereas surrounding tissues showed extensive lymphocyte cluster formation. The TCR repertoire of infiltrating lymphocytes was broad and similar to that of healthy prostate tissue, giving no evidence for specific lymphocyte recruitment. CONCLUSIONS: In the prostate cancer microenvironment, CD4+ T lymphocytes dominated while CD8+ T cells were sparse. The lymphocytes exhibited signs of disturbed effector function. Consequently, the immune response against autologous tumor cells is likely to be inefficient in controlling tumor growth.

Molecular study of time dependent changes in DNA stability in soil buried skeletal residues.

In the past years, many publications about identification and sex-determination of dry human bones by means of DNA analysis have been published. However, few studies exist that investigate the potential use of DNA technique to determine the postmortem interval (PMI). In the present study we analyzed the rate of increasingly smaller fragments of chromosomal DNA and PMI. We examined DNA degradation in human bones with postmortem intervals ranging between 1 and more than 200 years that had been kept under comparable conditions concerning weather and soil. Following bone separation into the three different zones of interest of inner/middle/outer segments the quantity of total DNA was determined in each region. Subsequently, the degree of DNA fragmentation was estimated by searching for PCR products of defined size (150, 507 and 763 bp) with primers of the human-specific multicopy beta-actin-gene. Concerning DNA quantity we detected a significant correlation between the zone of interest and the amount of DNA. However, there was no correlation between the amount of DNA and PMI. In contrast to this, analyzing DNA using PCR showed a significant inverse correlation between fragment length and PMI. Thus, postmortem DNA degradation into increasingly smaller fragments reveals a time-dependent process. It has the potential to be used as a predictor of PMI in human bone findings, provided that environmental conditions are known.

Comparative proteomic analysis with postmortem prefrontal cortex tissues of suicide victims versus controls.

BACKGROUND: The origin of suicidal behaviour is multifactorial including genetic, neurobiological and psychosocial correlates. Although there is no doubt that serotonin has a central role, the overall genetic findings with candidate genes of the serotonergic pathway are relatively inconsistent and suggests that other, yet unidentified, genes and gene products are also contributing to the vulnerability of suicidality. Proteomics is a powerful method to investigate modifications in protein expression. METHODS: We performed comparative proteomic analysis with prefrontal cortex tissues of 17 suicide victims and 9 controls. RESULTS: Applying two dimensional gel electrophoresis and image analysis we detected five protein spots to differ significantly in intensities between both groups. Three of them appeared only in suicide victims and could be identified by means of MALDI-TOF-MS analysis and protein database search as alpha crystallin chain B (CRYAB), glial fibrillary acidic protein (GFAP) and manganese superoxide dismutase (SOD2). CRYAB belongs to the low molecular heat shock proteins and GFAP is known as a marker of astrocytic activation in gliosis. SOD2 is a major antioxidant enzyme protecting cells against oxidative injury. Two further spots revealed higher intensities in the control group but had no unambiguous protein to match. CONCLUSIONS: Our findings suggest that proteins, being involved in glial function, neurodegeneration and oxidative stress neuronal injury, might also have an impact upon the neurobiological cascade leading to suicidality. As animal data provide evidence for an up-regulation of GFAP synthesis in astrocytes due to alterations in 5-HT levels, similar mechanisms of interaction might also be relevant in humans.

Analysis of tryptophan hydroxylase I and II mRNA expression in the human brain: a post-mortem study.

Dysregulation of brain serotonin transmission is an important contributing factor in many psychiatric disorders. Tryptophan hydroxylase (TPH), the rate limiting enzyme in the biosynthesis of serotonin plays a major role as candidate gene in several psychiatric disorders. Recently, a second TPH isoform (TPH2) was identified in mice which was exclusively expressed in the brain. Due to the lack of data about its anatomic expression in humans we performed a mRNA expression analysis comparing TPH1 and TPH2 mRNA in several regions of the human brain (cortex, thalamus, hypothalamus, hippocampus, amygdala, cerebellum and raphe nuclei). The study was performed with post-mortem specimens obtained from eight individuals with sudden deaths, not directly involving CNS diseases. Our results demonstrate that the mRNA of both genes is expressed in each investigated brain region with variations between the brain areas, as well as between the particular genes. The major finding of this study was the high expression level of TPH2 mRNA in the raphe nuclei ( approximately 4-fold more abundant than that of TPH1). The raphe nuclei showed the highest TPH2 mRNA levels at all, compared to the other regions (7-fold higher levels on average). To our knowledge, this is the fist study which demonstrates the localization of TPH1 and TPH2 mRNA in different regions of the human brain. Our findings provide further support for a duality of the serotonergic system and may open up new research strategies for the analysis of the repeatedly observed disturbances in the serotonergic system in patients suffering from several psychiatric disorders.