[Sickle cell anemia in perinatal placental diagnostics]. / Sichelzellenanämien in der perinatalen Plazentadiagnostik.

Hereditary hemoglobinopathies should be considered as differential diagnosis when examining placental specimens for fetal growth retardation and spontaneous abortion. They can cause various macroscopic and microscopic changes in the placenta that are relevant for routine pathology examination. The importance of interdisciplinary co-operation between obstetrics and pathology to achieve optimum diagnostics and therapy planning is demonstrated using the case of a pregnant woman with heterozygous genotype and her child with homozygous genotype. Within this context, the influence of hemoglobinopathies on placental pathology and fetal development are summarized and exemplified.

Immunohistochemical localization of nanog and Oct4 in stem cell compartments of human sacrococcygeal teratomas.

AIMS: To study the range of differentiation and presence of cells positive for stem cell markers in 20 sacrococcygeal teratomas (SCTs) which were consecutively operated on between 1990 and 2000 in the Department of Paediatric Surgery in Tübingen, Germany. METHODS AND RESULTS: Preserved paraffin-embedded material was re-evaluated. In addition to tissues of various organs, caudal organ structures not described before were identified, such as colon with pancreas originating from colonic crypts, Fallopian tube and vaginal epithelia. The derivation of the latter was confirmed by Müllerian duct specific CA125 and CA19-9 antibodies. The expression of stem cell markers was studied with antibodies against nanog, Oct4, SSEA-4, nestin and subtype M3 muscarinic receptors. Cells positive for these markers were encountered in immature end buds and capillary sprouts, and as single cells in neural tissue, gonadal structures, hairs and in the stem cell niches of differentiated epithelia. CONCLUSIONS: Our data indicate that SCTs of the newborn arise from remnants of the epiblast-like tail bud blastema and demonstrate that they contain cells positive for embryonic stem cell markers and may represent a novel source for human embryonic stem cells.

The chick embryo as a model for intrauterine ultrasound-guided heart intervention.

OBJECTIVES: Prenatal minimally invasive therapy represents a challenging option for reducing long-term complications of pathological fetal heart development. Here, the potential of the chick embryo as a model for ultrasound-guided intrauterine cardiac intervention is explored. METHODS: Chick embryos were incubated for 18 days in fenestrated eggs and their hearts were punctured in ovo under ultrasound guidance. Indian ink and Nile blue sulfate were applied to mark the injection channel. After cardiac intervention, embryos were further incubated and subsequently sacrificed for macroscopic and histological evaluation of the heart. RESULTS: Stereomicroscopic analysis revealed that the catheter had successfully penetrated the cardiac ventricular wall in 26/38 embryos. The myocardium was not severely injured. Histological evaluation showed that the myocardium had almost reoccluded after the intervention and that the injection channel was clogged with fibrin. In one case, the embryo was not sacrificed, but was removed from the egg 24 h after the intervention, with no signs of cardiac dysfunction, and was followed up for 6 months. CONCLUSIONS: Intrauterine ultrasound-guided heart intervention in the human fetus can be simulated in the chick embryo. Fenestrated eggs have to be used because the egg shell and shell membrane are impermeable to ultrasound.

Stereolithographic reproduction of complex cardiac morphology based on high spatial resolution imaging.

BACKGROUND: Precise knowledge of cardiac anatomy is mandatory for diagnosis and treatment of congenital heart disease. Modern imaging techniques allow high resolution three-dimensional (3D) imaging of the heart and great vessels. In this study stereolithography was evaluated for 3D reconstructions of multidetector computed tomography (MDCT) and magnetic resonance imaging (MRI) data. METHODS: A plastinated heart specimen was scanned with MDCT and after segmentation a stereolithographic (STL) model was produced with laser sinter technique. After scanning the STL model with MDCT these data were compared with those of the original specimen after rigid registration using the iterative closest points algorithm (ICP). The two surfaces of the original specimen and STL model were matched and the symmetric mean distance was calculated. Additionally, the heart and great vessels of patients (age range 41 days-21 years) with congenital heart anomalies were imaged with MDCT (n=2) or free breathing steady, state free-precession MRI (n=3). STL models were produced from these datasets and the cardiac segments were analyzed by two independent observers. RESULTS: All cardiac structures of the heart specimen were reconstructed as a STL model within sub-millimeter resolution (mean surface distance 0.27+/-0.76 mm). Cardiac segments of the STL patient models were correctly analyzed by two independent observers compared to the original 3D datasets, echocardiography (n=5), x-ray angiography (n=5), and surgery (n=4). CONCLUSIONS: High resolution MDCT or MRI 3D datasets can be accurately reconstructed using laser sinter technique. Teaching, research and preoperative planning may be facilitated in the future using this technique.

Magnetic resonance imaging of iron-oxide labeled SK-Mel 28 human melanoma cells in the chick embryo using a clinical whole body MRI scanner.

PURPOSE: To evaluate advantages and limitations of magnetic resonance imaging (MRI) to monitor the migration of superparamagnetic iron oxide (SPIO) labeled cells in the chick embryo. MATERIALS AND METHODS: Labeled human SK-Mel 28 melanoma cells were injected into the E2 chick embryo neural tube. Embryos were examined with a clinical 3 T MRI whole body system using 3D T*(2)-weighted sequences with isotropic spatial resolutions of 0.3-1.0 mm. MR-measurements of embryos were performed 2 - 16 days after cell injection. MRI findings were verified by dissection and histology. RESULTS: After injection, melanoma cells formed aggregations that were detectable in the neural tube as signal voids in MR images from day 2 after injection. Emigrating cells later left MRI detectable tracks. Aggregates that remained in the neural tube left label that was absorbed by glia cells. In E18 chick embryos, signals of haematopoiesis interfered with signals from cell labeling. CONCLUSION: It was shown that SK-Mel 28 cells will resume the neural crest pathways after injection into the embryonic micro-environment. SPIO cell labeling allows monitoring of transplanted melanoma cells during embryonic development. MRI using the standard clinical equipment promises to be valuable for high-sensitive monitoring of ex-vivo labeled cells in the chick embryo.

Muscarinic cholinergic receptors in the human melanoma cell line SK-Mel 28: modulation of chemotaxis.

Primary and metastatic human melanomas express muscarinic receptors. In embryonic tissues, expression of muscarinic receptors is correlated with morphogenesis. The hypothesis has been put forward that muscarinic receptors are involved in morphogenetic movements in the embryo, and in cellular movements in melanoma cells during invasive growth. The purpose of the present study was to characterize the muscarinic receptors in the human melanoma cell line SK-Mel 28 and to test in a Boyden chamber assay whether the chemotactic activity towards fibronectin can be influenced by muscarinic stimulation. In Western blots with the monoclonal antibody M35, muscarinic receptors were localized in a strong band at 66 kDa, and in a weak band at 63 kDa. Western blot with M3 subtype specific antibodies reproduced the line at 66 kDa. RT-PCR revealed mRNA for subtypes M3 and M5. These findings suggest that SK-Mel 28 cells express a large number of subtype M3 and a small number of subtype M5 receptors. Microscopic observation of calcium mobilization after muscarinic stimulation indicated that all cells carried functional muscarinic receptors. A standardized chemotaxis assay was established in modified Boyden chambers using fibronectin as chemotactic agent. After addition of carbachol to the upper compartment, an increase of fibronectin induced chemotaxis of approximately 30% was observed, an effect abrogated by atropine. These results demonstrate that muscarinic cholinergic treatment has a modulatory effect on fibronectin-induced chemotaxis in SK-Mel 28 melanoma cells, indicating that the muscarinic system is involved in regulation of cell movement.

Muscarinic receptors in cell lines from ovarian carcinoma: negative correlation with survival of patients.

OBJECTIVES: Tumor cells are similar in many respects to embryonic cells, indicating that embryonic genes are reactivated during malignant growth. In previous studies, we observed muscarinic acetylcholine receptors, which are expressed in embryonic cells during morphogenesis and are also found in human melanomas and melanoma cell lines. We determined the presence of muscarinic receptors in a collection of ovarian tumor cell lines for which clinical data were available. METHODS: Muscarinic receptor status of 39 cell lines derived from 34 patients was determined by Western blotting. RESULTS: Twenty-three cell lines were receptor positive, and 16, receptor negative. Kaplan-Meier analysis of receptor status of the tumor cell lines and survival time of patients from which the cell lines were established showed that expression of muscarinic receptors was associated with a reduced probability (P = 0.025) of survival: This is within the range of other established prognostic factors reported in the literature. CONCLUSIONS: A large percentage of ovarian tumor cell lines express muscarinic receptors. Muscarinic receptor expression is an embryonic trait and is correlated with reduced survival of patients. The results from this study provide further evidence of the involvement of muscarinic receptors in the progression of malignant carcinomas.

Immunohistochemical localisation of androgen receptor during sex-specific morphogenesis in the fetal mouse.

The distribution of androgen receptors (ARs) in paraffin serial sections of day 17 and day 18 male and female mouse embryos was investigated. In the cranial section of the genital tract AR expression was restricted to Wolffian structures while Müllerian ducts and surrounding mesenchyme were AR negative. In the fusion zone with the urogenital sinus the epithelial components of the vaginal bud were clearly distinguished by differential AR expression, which was faint in the Wolffian ducts, totally missing in the Müllerian ducts, and intense in the sinus ridges with the most intense expression in the morphogenetically active mesenchyme, indicating a new mechanism of negative control of vagina formation via androgens. Expression of ARs outside the genital tract was observed: (1) in loose interstitial mesenchyme extending into the retroperitoneal space up to the coeliac artery, indicating androgen effects during ascent of the kidneys and descent of intraperitoneal organs, (2) in the trigone of the bladder indicating androgen involvement in the development of the vesico-ureteral junction, and (3) in loose mesenchyme between striated muscle fibres and around pelvic skeletal elements, indicating mediation of androgen effects on the musculoskeletal system via loose mesenchyme.

Induction of cellular contractions in the human melanoma cell line SK-mel 28 after muscarinic cholinergic stimulation.

In a previous immunohistochemical study we observed muscarinic acetylcholine receptors in primary and metastatic human melanomas, which were not present in normal skin melanocytes. In the present study we demonstrated the endogenous expression of muscarinic receptors, of choline acetyltransferase and of cholinesterase activity in the human melanoma cell line SK-mel 28. We tested the effect of muscarinic agonists on cellular movements of the melanoma cells in a perfusion chamber by digital video time-lapse microscopy. Within 3 to 10 min after onset of muscarinic perfusion cell body contractions and retraction of cell processes of more than 5 micrometer occurred in about 30% of the melanoma cells. The effect disappeared after addition of atropine. The proportion of reacting cells corresponded to the endogenous expression of muscarinic receptors revealed by immunocytochemistry with the monoclonal antibody M35. The experiments indicate the presence of an autocrine muscarinic cholinergic system in the melanoma cells and demonstrate a direct link between muscarinic receptors and the contractile apparatus. Melanocytes are derived from neural crest cells that express cholinesterase activity and muscarinic receptors during their migratory phase in the embryo. Therefore, re-expression of the muscarinic cholinergic system in tumour cells may be involved in invasive growth.

[Who heals the healer? Shaman practice in the Himalayas]. / Wie heilt der Heiler? Schamanische Praxis im Himalaya.

The question has been put before: By which means or intervention does the shaman or faith healer effect his healing results? A new answer is attempted in this article in which a concrete example is being described and analysed: What do the shamanic healers of the Northern Magar in NW Central Nepal do, when confronted with a complicated birth? As neither medication or herbal treatment, nor any massage or obstetric manipulation is employed (methods well applied by the local midwife), the healing séance of the shaman has to be classified as a psychological manipulation of the patient. This is based on three elements: ritual acts during the séance; performance of mythical chants; and the presence of an audience. As for the chanted myths, it may be stressed that they relate stories which allude to the actual situation of the patient, insofar as their protagonist is in a similar situation as herself--only much worse. This overdramatisation permits the listening patient to first identify with and then to dis-identify from her mythical similé and to anticipate a happier end for herself than that related in the myth, which is always violent and tragic. The mechanism of healing (in our case: a successful birth) ensues from a gradual process of disidentification of the patient from the mythical heroine. This observation may be confirmed, if one compares the Magar case with similar ethnographic facts from other regions, both in Nepal and elsewhere. The described process of healing, effected through shamanic treatment, invites to a new comparison between this form of practice and that of Western psychoanalysis.

Studies on the pharmacokinetics of chlorambucil and prednimustine in patients using a new high-performance liquid chromatographic assay.

Following the oral administration of either chlorambucil/prednisolone or prednimustine to patients, the plasma levels of free chlorambucil and phenylacetic acid mustard, the beta-oxidation product of chlorambucil, were measured using a new high-performance liquid chromatographic (HPLC) assay. This assay permitted the simultaneous detection of the analyzed compounds with a lower limit of detection of 30 ng/ml. The pharmacokinetics of chlorambucil and phenylacetic acid mustard were found to be entirely different when prednimustine was administered as opposed to its components chlorambucil and prednisolone together. After the ingestion of the conjugate, the plasma concentration-time curves of chlorambucil and phenylacetic acid mustard showed a "delayed" pattern compared with those obtained after the administration of the components. The mean area under the concentration-time curves (AUCs) of prednimustine-derived chlorambucil and phenylacetic acid mustard were 25% and 40%, respectively, of the areas obtained after a stoichiometrically equivalent dose of chlorambucil. Free plasma prednimustine could not be detected at any time. This different pharmacokinetic behavior might offer an explanation for the superior therapeutic effects of prednimustine demonstrated by clinical studies.

Comparative studies on the central effects of the angiotensin II analogue (Sar1 azaVal3 Ile8) AT II.

The effects of the newly-synthesized AT II analogue (Sar1 azaVal3 Ile8) AT II were investigated in comparison with the octapeptide AT II and the analogue saralazin (Sar1 Ala8) AT II, using intracerebroventricular administration, with respect to the following parameters: the level of biogenic monoamines (DA, NA and 5-HT) and the metabolites HVA and 5-HIAA in mouse forebrain; the behaviour of the animals--cataleptogenic actions of mice, PTZ convulsive--seizure threshold in mice, apomorphine stereotypy in rats and behaviour of rats in a conflict situation. The analogue (Sar1 azaVal3 Ile8) AT II, unlike saralazin and AT II, was found to induce a rise in the NA and 5-HT levels, causing also catalepsy that is different from the catalepsy induced by saralazine, AT II and haloperidol, because of its rapid onset and decline; it increases the PTZ convulsive--seizure threshold and reduces the number of punished responses to the conflict drinking test (anxiomimetic effect) in a dose 20 times lower than the dose inducing the remaining effects. This effect was antagonized by saralazine. It is concluded that the newly-synthesized analogue (Sar1 azaVal3 Ile8) AT II induces effects similar to those caused by AT II, being at the same time different to a certain extent from the effects (quantitative and qualitative) of octapeptide AT II.