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.     

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     

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.     

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).     

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%.     

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.     

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.     

The distribution of the HLA-DQα alleles and genotypes in the Finnish population as determined by the use of DNA amplification and allele specific oligonucleotides

Summary Allele and genotype frequencies for the HLA-130 locus were determined for use in forensic analyses and paternity tests in Finland. The polymerase chain reaction (PCR) and the reverse dot blot format were employed to detect 6 different HLA-DQ alleles. All 6 HLA-DQ alleles were detected among the 112 unrelated individuals with allele frequencies ranging from 5.8% to 32.6%. The distribution of the observed genotypes is in Hardy-Weinberg equilibrium. Additionally, this Finnish population sample is statistically similar to 2 other Caucasian sample populations. The power of discrimination of this system in the Finnish population sample is 0.92, suggesting this method may prove suitable for identification purposes.     

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.     

Tunnel enlargement and changes in synovial fluid cytokine profile following anterior cruciate ligament reconstruction with patellar tendon and hamstring tendon autografts

There is growing evidence that cytokines such as tumor necrosis factor (TNF) , interleukin (IL) 1, IL-6, bone morphogenetic proteins (BMP), and nitric oxide (NO) play an important role in the pathogenesis of bone tunnel enlargement following anterior cruciate ligament (ACL) reconstruction. Furthermore, the release of these mediators has been considered a possible reason for the higher incidence of bone tunnel enlargement following hamstring tendon (HST) than following patellar tendon (PT) ACL reconstruction observed in several studies. In this investigation synovial fluid samples from 13 patients were collected immediately before (24±7 days after ACL rupture) and 7 days after ACL surgery and values of TNF-, IL-1, IL-6, NO, and BMP-2 were analyzed. Furthermore, the incidence of bone tunnel enlargement was assessed using radiographs 38±7 weeks after surgery. Six patients underwent autologous HST ACL reconstruction, and in seven patients an PT autograft was used. In the overall patient population there were significantly higher synovial fluid concentrations of IL-6 and BMP-2 postoperatively than preoperatively; TNF- showed a trend towards lower postoperative levels while IL-1 and NO remained unchanged. The concentrations of NO, TNF-, and IL-6 found in the present study were clearly higher than normal values given in the literature. Assessment of bone tunnel enlargement revealed an average increase in tibial tunnel width of 28.4±3.1% with comparable values for HST and PT ACL reconstructions. There was no significant correlation between bone tunnel enlargement and postoperative synovial fluid concentrations of TNF-, IL-1, IL-6, NO, and BMP-2. However, all patients with bone tunnel enlargement had higher postoperative concentrations of TNF-, IL-6, and NO in the synovial fluid. There were no significant differences in concentrations between HST and PT groups. In conclusion, we observed an association between tibial bone tunnel enlargement and elevated synovial fluid concentrations of IL-6, TNF-, and NO 7 days after ACL surgery indicating the potential involvement of these biological mediators in the pathogenesis of bone tunnel enlargement. However, there was no difference between HST and PT ACL reconstructions regarding synovial fluid contents of IL-6, TNF-, IL-1, NO, and BMP-2, suggesting a comparable biological response between these autografts following their use in ACL reconstruction.     

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.     

Immunochemical studies on blood group H and B substances from human hair

Summary Blood group H and B substances were extracted from urea-treated human hair of group O and B individuals, respectively, with methanol-ethyl ether (1:1,v/v) and chloroform-methanol (1:1,v/v). The blood group activities of H and B substances were destroyed by H-decomposing enzyme (-l-fucosidase) fromBacillus fulminans and B-decomposing enzyme (-d-galactosidase) fromClostridium sporogenes Maebashi, respectively. It is concluded therefore that the extract from the hair of group O contained blood group H-active glycolipid with -l-fucose as the non-reducing sugar and the one from group B contained blood group B-active glycolipid with -d-galactose as the non-reducing sugar.     

Zur Kinetik des postmortalen bakteriellen Glutaminsäurestoffwechsels im Gehirn

Zusammenfassung Die bakteriellen Enzyme in einem gefaulten menschlichen Gehirn sind Gegenstand der vorliegenden Untersuchung. Es wurde geprüft, wie sich Temperatur und pH-Änderung des Untersuchungsmaterials auf die Aktivitäten der an den Reaktionen der Glutaminsäure beteiligten Enzyme auswirkt. Es galt die Frage zu klären, ob und in welchem Umfang eine Temperatur- und pH-Unabhängigkeit für die bereits früher formulierte Todeszeitformel T=A·ln (-ABS+GABA/Glu+1)+B Gültigkeit besitzt.Die vorliegende Arbeit zeigt, daß die geforderte Unabhängigkeit auf jeden Fall für den für Gehirne typischen pH-Bereich (6–7,2) und für den für beheizte Wohnungen typischen Temperaturbereich (17,5–25°C), bei gleichzeitigen, die Kontinuität der Reaktionsfolgen aufrechterhaltenen, Umsatzraten vorhanden ist. Dieses Ergebnis läßt den Schluß zu, daß die Kinetik des postmortalen Glutaminsäureabbaus Grundlage einer neuen Todeszeitbestimmungsmethode werden könnte. Abkürzungen: T: Todeszeit; -ABS, GABA Glu: Konzentration der freien Aminosäuren -Aminobuttersäure, -Aminobuttersäure und Glutaminsäure im Gehirn A, B konstante Zahlen     

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.     

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     

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.     

A pragmatic approach to metal artifact reduction in CT: merging of metal artifact reduced images

The purpose of this study was to improve metal artifact reduction (MAR) in X-ray computed tomography (CT) by the combination of two artifact reduction methods. The presented method constitutes an image-based weighted superposition of images processed with two known methods for MAR: linear interpolation of reprojected metal traces (LI) and multi-dimensional adaptive filtering of the raw data (MAF). Two weighting concepts were realized that take into account mean distances of image points from metal objects or additional directional components. Artifact reduction on patient data from the jaw and the hip region shows that although the application of only one of the MAR algorithms can already improve image quality, these methods have specific drawbacks. While MAF does not correct corrupted CT values, LI often introduces secondary artifacts. The corrective impact of the merging algorithm is almost always superior to the application of only one of the methods. The results obtained with directional weighting are equal to or in many cases better than those of the distance weighting scheme. Merging combines the advantages of two fundamentally different approaches to artifact reduction and can improve the quality of images that are affected by metal artifacts.Symbols LI linear interpolation algorithm - MAF multi-dimensional adaptive filtering algorithm - MAR metal artifact reduction - f(x, y) object function at (x, y) - F(), F() normalized filter function in the direction of the coordinates and - I primary X-ray intensity - I₀ transmitted X-ray intensity - N_D number of detectors per detector row - p attenuation - p(,), p(,) projection data (fan and parallel geometry) - p_th lower threshold of the attenuation of adaptive filtering - p_max maximum value of the attenuation of adaptive filtering - (x, y) image point - projection angle in fan geometry - angle within the fan relative to the central ray - , , z, , minimum functions for adaptive filtering - projection angle in parallel geometry - orthogonal distance of a ray to the center of rotation in parallel geometry     

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.     

Agreement between emergency physician diagnosis and radiologist reports in patients discharged from an emergency department with community-acquired pneumonia

To evaluate the level of concurrence between radiologist reports and the diagnosis of community-acquired pneumonia (CAP) in patients discharged from an emergency department (ED), a retrospective chart audit of patients discharged with a diagnosis of pneumonia or possible pneumonia from the ED during a 2-year period was conducted. Emergency physician (EP) and radiology report (RR) diagnoses were categorized as pneumonia, possible pneumonia, non-pneumonia and normal, and categories from each were compared. 815 charts were analyzed. Of 671 EP diagnoses of pneumonia, 304 (45.3%) RRs reported pneumonia and 82 (12.2%), possible pneumonia. Of 815 EP diagnoses of pneumonia or possible pneumonia, 426 (52.3%) RRs were in agreement, while 216(26.5%) were of diagnoses other than pneumonia and 173 (21.1%) were read as normal. EPs and radiologists frequently disagree on whether a patient has pneumonia or not. Perhaps it is time to revisit the gold standard status of plain chest X-ray.Presented at the 9th International Conference of Emergency Medicine, Edinburgh, Scotland, 17–21 June, 2002.