Immunoelektrophoretische Differenzierung der Proteine faulen Leichenblutes

Zusammenfassung Leichenblute verschiedener Fäulnisgrade wurden immunoelektrophoretisch untersucht. Für die Differenzierung der einzelnen Blutplasmafraktionen wurden Anti-Humanserum vom Kaninchen und die spezifischen Antiseren der Behringwerke gegen die nachstehend aufgeführten Proteine benutzt: Präalbumin, Albumin, ₁-Lipoprotein, ₂-Makroglobulin, ₂-Lipoprotein, ₁-Globulin (Transferrin),-Lipoprotein, Fibrinogen, ₂-A-Globulin, ₂-M-Globulin,- Globulin.Infolge der mangelnden Proportionalität zwischen Fortschritt der Fäulnis und Leichenalter gestattet die Immunoelektrophorese des Leichenblutes keine Todeszeitbestimmung.Albumin und- Globulin widerstehen der Fäulnis am längsten. Es folgen ₂-Makroglobulin, Transferrin und Fibrinogen. Die Proteine, die am reichlichsten im Menschenblut vorhanden sind, lassen sich im faulen Leichenblut auch am längsten nachweisen. Die Proteolyse folgt jedoch nicht nur quantitativen Gesetzen. Die Lipoproteine bilden insofern eine Ausnahme, als sie früher durch die Fäulnis eliminiert werden, als es ihrem Anteil am Gesamtprotein entspricht.Die Eiweißkörper des faulen Leichenblutes weisen im Vergleich zu ihren Homologen frischer Blutproben z. T. Veränderungen der Wanderungsgeschwindigkeit auf. Bei erhaltener Antigenstruktur treten Bruchstücke in Erscheinung, die mit dem Antiserum in ungleichmäßig geschwungenen oder aufgesplitterten Linien präcipitieren.Im Doppeldiffusionstest nachOuchterlony ließen sich im Vergleich zu den frischen Homologen keine Veränderungen der Antigenkomponenten der Proteine des faulen Leichenblutes nachweisen.Mit Unterstützung der Deutschen Forschungsgemeinschaft, der hiermit unser herzlicher Dank ausgesprochen wird.In Anlehnung an einen Vortrag auf der Tagung der Deutschen Gesellschaft für Gerichtliche und Soziale Medizin vom 30. 9. bis 3. 10. 62 in Münster (Westf.).Herrn Professor G.Weyrich zum 65. Geburtstag gewidmet.     

γ-Glutamyltransferase test as a new tool for identification of seminal stains

Summary A simple qualitative method for identification of seminal stains based on a high activity of -glutamyltransferase (-GTP) in human semen is described. It employs the release of -naphthylamine from N--glutamyl--naphthylamide by the -GTP action; -naphthylamine couples with Fast Garnet GBC salt to produce a strong brownish-red color. The data on its simplicity, specificity, and stability show that the present method is suitable for medicolegal examination of seminal stains as a preliminary test.     

Genetic polymorphism of desialyzed alpha2 HS-glycoprotein by ultrathin isoelectric focusing

Summary The genetically determined polymorphism of ₂ HS-glycoprotein was analyzed by immunoblotting ultrathin-layer polyacrylamide gel isoelectric focusing in the pH range 4–6.5 and neuraminidase pretreated sera. In a Libyan population sample from Tripoli (n=110) three common phenotypes, ₂ HSG 1–1, 2–1, and 2–2, were observed. The allele frequencies were ₂ HSG¹=0.8364 and ₂ HSG²=0.1636. The theoretical exclusion rate in cases of disputed paternity is 11.8%.     

Cancer radioimmunotherapy with alpha-emitting nuclides

In lymphoid malignancies and in certain solid cancers such as medullary thyroid carcinoma, somewhat mixed success has been achieved when applying radioimmunotherapy (RIT) with -emitters for the treatment of refractory cases. The development of novel RIT with -emitters has created new opportunities and theoretical advantages due to the high linear energy transfer (LET) and the short path length in biological tissue of -particles. These physical properties offer the prospect of achieving selective tumoural cell killing. Thus, RIT with -emitters appears particularly suited for the elimination of circulating single cells or cell clusters or for the treatment of micrometastases at an early stage. However, to avoid non-specific irradiation of healthy tissues, it is necessary to identify accessible tumoural targets easily and rapidly. For this purpose, a small number of -emitters have been investigated, among which only a few have been used for in vivo preclinical studies. Another problem is the availability and cost of these radionuclides; for instance, the low cost and the development of a reliable actinium-225/bismuth-213 generator were probably determining elements in the choice of bismuth-213 in the only human trial of RIT with an -emitter. This article reviews the literature concerning monoclonal antibodies radiolabelled with -emitters that have been developed for possible RIT in cancer patients. The principal radio-immunoconjugates are considered, starting with physical and chemical properties of -emitters, their mode of production, the possibilities and difficulties of labelling, in vitro studies and finally, when available, in vivo preclinical and clinical studies.     

Genetic polymorphism of Alpha-2-HS glycoprotein in Lombardy (Italy)

Summary 2HS-subtypes have been analysed in samples from 700 unrelated individuals from the Brescia area (Italy) by the isoelectric focusing technique and immunofixation. The observed allele frequencies were: 2HS*1= 0.7472; 2HS*2 = 0.2507; 2HS*V = 0.0021.     

Stability of α-[11C]methyl-l-tryptophan brain trapping in healthy male volunteers

Purpose The purpose of this study was to assess the reproducibility in healthy volunteers of -[¹¹C]methyl-l-tryptophan ([¹¹C]MT) brain trapping imaging with positron emission tomography (PET), using volumes of interest (VOIs) and voxel-based image analysis.Methods Six right-handed healthy male volunteers (34.3±10.9 years) with a negative family history for psychiatric disorders were scanned twice in the resting condition, 22±17 days apart. An unbiased semiautomatic segmentation of the brain was used to define VOIs. The trapping constant K* (ml g^–1 min^–1) for [¹¹C]MT was calculated for the whole brain and seven brain regions using the graphical method for irreversible tracers. In addition, parametric maps of K* were obtained from dynamic scans using the same method. Comparison of test and retest K* functional images was performed using SPM99. Students paired t statistic was applied for comparisons of [¹¹C]MT brain trapping in a priori selected VOIs.Results [¹¹C]MT brain trapping in VOIs showed a mean variability 2.6±1.8% (0.3–5%) for absolute and 1.5±2.1% (1.4–4.1%) for normalized K*. Intraclass correlations between test and retest conditions were 0.61±0.34 for absolute K* values and 0.73±0.20 for K* values normalized by global mean. SPM99 analysis using a height threshold of p=0.05 (two tailed) and an extent threshold of 100 voxels showed no significant differences between scans.Conclusion Rest measurements in healthy male volunteers of the trapping constant for [¹¹C]MT, using PET, appeared to be stable during an average interval of 3 weeks.     

Analytische Daten des neuen Benzodiazepinderivates Midazolam (Dormicum) und seiner Metaboliten

Zusammenfassung In der Arbeit werden analytische Daten zum Nachweis von Midazolam, -Hydroxi-midazolam, 4-Hydroxi-midazolam and ,4-Dihydroxi-midazolam sowie wichtige pharmakokinetische Eigenschaften mitgeteilt. Weiterhin wird über die Extraktion aus biologischem Material berichtet.Herrn Dr. James Bäumler (Basel) zum 60. Geburtstag gewidmet     

Accumulation of radioiodinated tyrosine derivatives in the adrenal medulla and in melanomas

Because tyrosine and dopa can be regarded as precursors of adrenomedullary hormones and melanin, radioiodinated derivatives of these compounds were tested for their accumulation in the adrenal medulla and in melanomas of various animal species. The highest level of accumulation in the adrenal medulla was attained in mice and rats with iodinated -hydroxy--methyltyramine, and in melanomas of mice with iodinated -methyltyrosine. The results could not be reproduced to the same extent in other species.     

Detection of α S1-casein in vomit from bottle-fed babies by enzyme-linked immunosorbent assay

Summary This study describes a sensitive enzyme-linked immunosorbent assay (ELISA) using rabbit anti-bovine S1-casein antibody for the detection of commercial milk and milk-containing vomit. The antibody does not react with other human body fluids such as breast milk. The stability of S1-casein antigenic activity was examined after storage at different temperatures and enzyme digestion. There was no decrease after storage for one year at room temperature but 40% of the activity was lost after 6 months at 37°C. Enzyme digestion (6 hours, 37°C) resulted in 65 70% loss of activity but the antibody reacted with the peptide fragments of S1-casein. Vomit samples from 3 normal infants were tested by ELISA, and a S 1-casein could be detected in 1 cm² stain.     

Population data of the HLA DQα locus in Dutch caucasians

Summary The HLA DQa amplification and typing kit has been designed to be used by the forensic community for purposes of identity testing. The introduction of any new DNA marker in forensic identity testing requires the establishment of a population database for the relevant population(s) [1]. To this end allele and genotype frequencies for the HLADQ locus were determined in a Dutch Caucasian population sample and compared with 7 other population genetic studies. In our population sample the HLA DQ genotype frequencies did not deviate from Hardy-Weinberg expectations and for this locus the power of discrimination is 0.94. A test for homogeneity of the HLA DQ population data based on the allele frequency counts for 8 Caucasian population samples was performed and significant differences were found (P = 0.007) . The differences in the frequency of the HLADQ 2 and 3 alleles are the major cause of this deviation. No deviation from population homogeneity was observed when we compared thegenotype frequency distributions among the 8 Caucasian population samples. Combined with the extensive validation studies from Comey and Budowle [7] and Helmuth et al. [8] this population genetic study will allow HLADQ typing to be used in forensic identity testing in the Netherlands.     

Frequency of D1S80 and HLA DQα alleles in a Chinese population

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

Effect of adrenergic receptor ligands on metaiodobenzylguanidine uptake and storage in neuroblastoma cells

The effects of adrenergic receptor ligands on uptake and storage of the radiopharmaceutical [¹²⁵I]metaiodobenzylguanidine (MIBG) were studied in the human neuroblastoma cell line SK-N-SH. For uptake studies, cells were incubated for 15 min with varying concentrations of -agonist (clonidine, methoxamine, and xylazine), -antagonist (phentolamine, tolazoline, phenoxybenzamine, yohimbine, and prazosin), -antagonist (proranolol, atenolol), -agonist (isoprenaline and salbutamol), mixed / antagonist (labetalol), or the neuronal blocking agent guanethidine, prior to the addition of [¹²⁵I]MIBG (0.1 M). The incubation was continued for 2 h and specific cell-associated radioactivity was measured. For the storage studies, cells were incubated with [¹²⁵I]MIBG for 2 h, followed by replacement with fresh medium with or without drug (MIBG, clonidine, or yohimbine). Cell-associated radioactivity was measured at various times over the next 20 h. Propanolol reduced [¹²⁵I]MIBG uptake by approximately 30% (P<0.01) at all concentrations tested, most likely due to nonspecific membrane changes. However, incubation with the other -agonists or antagonists failed to elicit significant reductions in uptake. In contrast, all of the -agonists significantly inhibited uptake (P<0.05); guanethidine >xylazine >clonidine=methoxamine. The -antagonists demonstrated a broad range of inhibition (phenoxybenzamine phentolamine prazosin yohimbine=tolazoline)(P<0.05). The mixed ligand, labetalol, inhibited MIBG uptake in a dose-dependent manner with an apparent IC₅₀ of 0.65 M. The retention studies demonstrated that unlabeled MIBG caused profound self-inhibition (P<0.01). Clonidine produced a modest inhibition of retention and yohimbine had no effect. Labetalol, phenoxybenzamine, guanethidine, and propranolol reduced uptake of [¹²⁵I]MIBG by neuroblastoma cells in culture. Although only labetalol has been reported to cause false-negative MIBG scans, our results suggest that these other drugs have the potential to interfere with MIBG imaging and therapy, particularly at high doses. Adrenergic drugs did not alter cytoplasmic retention of [¹²⁵I]MIBG in neuroblastoma cells but may have potential in tumors such as phenochromocytoma, where granular storage of MIBG has been observed. Inhibition of [¹²⁵I]MIBG retention by unlabeled MIBG supports the use of high specific activity radioiodinated MIBG for both diagnosis and therapy.     

Simultaneous Phenotyping of Genetic Markers for Paternity Testing

Summary Time- and cost-saving methods for paternity testing are described. Seventeen genetic systems were divided into six groups: (1) transferrin (Tf), factor B (Bf), and phosphoglucomutase 1 (PGM1); (2) group-specific component (Gc) or 1-antitrypsin (PI) and 2HS-glycoprotein (HSGA); (3) complement components C6 and C7, factor 13B (F13B), and plasminogen (PLG); (4) haptoglobin (Hp), C8 - chain (C81), and factor I (IF); (5) red cell acid phosphatase (ACP), esterase D (ESD), and glutamic-pyruvic transaminase (GPT); and (6) 6-phosphogluconate dehydrogenase (PGD) and glyoxalase I (GLO). Each group of systems was typed simultaneously by electrophoresis or isoelectric focusing (IEF) followed by staining or immunoblotting. These methods are very practical because they afford a considerable saving of time, work and expense, and facilitate semipermanent preservation of electrophoretic patterns.     

The stereoisomers of 17α-[123I]iodovinyloestradiol and its 11α-methoxy derivative evaluated for their oestrogen receptor binding in human MCF-7 cells and rat uterus,and their distribution in immature rats

We studied the potential of both stereoisomers of 17-[¹²³I]iodovinyloestradiol (E- andZ-[¹²³I]IVE) and of 11-methoxy-17-[¹²³I]iodovinyloestradiol (E-andZ-[¹²³I]MIVE) as suitable radioligands for the imaging of oestrogen receptor(ER)-positive human breast tumours. The 17-[¹²³I]iodovinyloestradiols were prepared stereospecifically by oxidative radio-iododestannylation of the corresponding 17-tri-n-butylstannylvi-nyloestradiol precursors. Competitive binding studies were performed in order to determine the relative binding affinity (RBA) of the unlabelled 17-iodovinyloes-tradiols for the ER in both human MCF-7 breast tumour cells and rat uterine tissue, compared with that of diethylstilboestrol (DES). Target tissue uptake, retention and uptake selectivity of their¹²³I-labelled analogues were studied in immature female rats. All four 17-iodovi-nyloestradiols showed high affinity for the ER in human MCF-7 cells, as well as rat uterus. Their RBA for the ER showed the following order of decreasing potency: RBA of DES >Z-IVE >Z-MIVE >E-MIVE E-IVE. Neither of these 17-iodovinyloestradiols showed any significant binding to the sex hormone binding globulin in human plasma. The biodistribution studies showed ER-mediated uptake in the uterus, ovaries and pituitary, that ofE- andZ-[¹²³I]MIVE being higher than that ofE- andZ-[¹²³I]IVE. High target-to-non-target tissue uptake ratios, especially at longer periods after injection (up to 24 h), were exhibited by both isomers of [¹²³I]MIVE. The uterus-to-blood uptake ratio was higher for^E-[¹²³I]MIVE. However, the uterus-to-fat uptake ratio appeared to be higher for theZ-isomer of [¹²³I]MIVE, especially at 24 h after injection. Metabolic properties and temperature effects, which play a more important role in vivo, probably cause the discrepancies seen between in vitro and in vivo binding results. On the basis of their in vitro binding properties and in vivo distribution characteristics we conclude thatE- andZ-[¹²³I]MIVE could be suitable radioligands for the diagnostic imaging of ER in human breast cancer. Therefore, further studies with these radioligands in mature normal and tumour-bearing rats are warranted.     

Evaluation of [1-11C]-α-aminoisobutyric acid for tumor detection and amino acid transport measurement: spontaneous canine tumor studies

Alpha-aminoisobutyric acid (AIB), or -methyl alanine, is a nonmetabolized amino acid transported into cells, particularly malignant cells, predominantly by the A amino acid transport system. Since it is not metabolized, [1-¹¹C]-AIB can be used to quantify A-type amino acid transport into cells using a relatively simple compartmental model and quantitative imaging procedures (e.g. positron tomography). The tissue distribution of [1-¹¹C]-AIB was determined in six dogs bearing spontaneous tumors, including lymphosarcoma, osteogenic sarcoma, mammary carcinoma, and adenocarcinoma. Quantitative imaging with tissue radioassay confirmation at necropsy showed poor to excellent tumor localization. However, in all cases the concentrations achieved appear adequate for amino acid transport measurement at known tumor locations. The observed low normal brain (due to blood-brain barrier exclusion) and high (relative to brain) tumor concentrations of [1-¹¹C]-AIB suggest that this agent may prove effective for the early detection of human brain tumors.     

Control of the RIA method as viewed from the standpoint of the investigation on the kinetics of the insulin-125I-antibody reaction

In this paper attempts were made towards the optimalization and the control of some parameters of the RIA reaction.Basing on the law mass action, as well as on the Scatchard's and Sips's equations, the equilibrium constants for the reversible reaction: insulin-¹²⁵I-antibody for different incubation temperatures were calculated. Moreover a characteristic of the antiinsulin antibody by means of the heterogeneity coefficient was done, as well as the values of the thermodynamic function increments were calculated, which made possible to point out explicitly the optimal shape of the standard curve.     

The dynamics of inflammatory cytokines in the healing process of mouse skin wound: A preliminary study for possible wound age determination

The dynamics of inflammatory cytokines such as interleukin-1 (IL-l, interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF) during the healing process of mouse skin wounds were examined using the enzyme-linked immunosorbent assay and immunostaining. The applicability of this examination for wound age estimation is discussed from the perspective of forensic pathology. After wound induction, mice were sacrificed at intervals ranging from 0 to 240 h. The levels of TNF and IL-1 began to elevate rapidly after wounding and reached a peak at 3 h. The IL-l level reached a peak at 6 h, and IL-6 peaked at 12 h. An infiltration of numerous leukocytes, indicatingacute inflammation, was observed at 3 and 6 h, and the main source of the cytokines was immunohistochemically identified as neutrophils. These results indicate that TNF and IL-1 play an important role in the commencement of inflammation. Rebound of cytokine levels, i.e. a re-increase, was observed at 72 h after wounding. Histological examination of the 72-h-old wound showed migration of fibroblasts and the formation of new granulation tissues, indicating the proliferative stage of the wound healing process. These experimental findings indicate that these cytokines have a close relationship to wound remodeling as well as to inflammation. From the viewpoint of forensic pathology, it is considered that inflammatory cytokines may become one of the markers for wound age estimation, but further studies are needed, especially those involving the investigation using human wound specimens with known time intervals after injury.This study was presented at the 79th Congress of the Medico-Legal Society of Japan (Yamagata, May 1995), the 16th Annual Meeting of the Japanese Society of Inflammation (Tokyo, July 1995) and the 74th Annual Meeting of the German Society of Legal Medicine (Aachen, September 1995).     

Antigenic properties of human and animal bloodstains studied by enzyme-linked immunosorbent assay (ELISA) using various antisera against specific plasma proteins

Summary Antigenic properties of bloodstains of human and non-human primates as well as other animal bloodstains were investigated by the inhibition ELISA using commercially available anti-human albumin (Alb), ₂-macroglobulin (₂-M), fibrinogen, transferrin, and immunoglobulin G. In general, chimpanzee bloodstains showed strong cross-reactions with these antisera, and the extent of the cross-reactions of other animal bloodstains decreased largely with the phylogenic order, i.e., agile gibbon (ape), Old World monkeys (Japanese monkey and hamadryas baboon), New World monkeys (night monkey and tufted capuchin monkey), prosimians (grand galago and ring-tailed lemur) and other animals (rat, cattle, swine, goat, dog, cat, and chicken). Among these antisera, anti-human ₂-M showed the weakest cross-reaction with chimpanzee bloodstains, and anti-human Alb showed next.Supported in part by the Cooperation Research Program of the Primate Research Institute     

Application of immunoblotting to serum protein phenotyping with reference to α2HS-glycoprotein (AHS) typing of bloodstains

Summary Sera and bloodstain extracts were subjected to isoelectric focusing in polyacrylamide gels. The focused proteins were transferred to nitrocellulose membranes by diffusion or electrophoretically, then allowed to react with specific antiserum and, after washing, with peroxidase-labeled anti-rabbit IgG. The immune complexes formed on the membranes were detected with 4-chloro-1-naphthol and hydrogen peroxide. Serum group-specific component, ₂HS-glycoprotein, the sixth and the seventh component of complement, factor 13B, and plasminogen could be phenotyped with high sensitivity. Bloodstains as old as 6 months could be correctly typed for ₂HS-glycoprotein by the blotting technique.     

Biodistribution of iodine-125 tyramine transforming growth factor α antisense oligonucleotide in athymic mice with a human mammary tumour xenograft following intratumoral injection

The Watson-Crick base pairing rule provides the underlying principle for the antisense (AS) approach to inhibiting gene expression. Transforming growth factor (TGF) was the first growth factor to be associated with tumorigenesis, thus making the TGF (mRNA) a potential target for AS therapy and offering the potential for monitoring of the progression of malignancy by non-invasive imaging with radiolabelled AS phosphodiester. Probe labelling and biodistribution were studied in the present report. A 23-mer oligonucleotide sequence was synthesized and grafted in 5 with a tyramine group which was further radioiodinated. The radiolabelled AS was injected intratumorally in mammary tumour-bearing BALB/c mice (3 weeks after inoculation of 7·10⁶ NS2T2A mammary cells). Biodistribution was monitored by sequential scintigraphy and organ radioactivity after autopsy. The 5 tyramine group allowed specific and stable radiolabelling of the AS with¹²⁵I. The¹²⁵I AS oligonucleotide was rapidly cleared from the tumour by intestine and kidneys. Four hours after intratumoral injection, 6.5%±1.5% of the dose was retained in the tumour as non-degraded¹²⁵I AS. It is concluded that 5 tyraminylated AS provides information on the biodistribution of AS oligonucleotide following intratumoral injection. These data will contribute to the pharmacology of AS oligonucleotides which can be used for therapy.