Specific detection by flow cytometry of histidine-tagged ligands bound to their receptors using a tag-specific monoclonal antibody.

Engineering proteins to contain a histidine (His)-tag has proved to be very useful for the purification and analyses of these molecules. In the present study, we demonstrate that the binding of His-tagged ligands to their receptors may be visualised by flow cytometry making use of a selected monoclonal antibody (mAb) against the His-tag. Employing this method, a recombinant C3a (rC3a) anaphylatoxin with a His-tag at its N-terminus could be shown to bind to C3a receptor (C3aR)-expressing RBL-2H3 transfectants with a half-maximal effective concentration (EC50) of about 3 nM which is well within the range of published affinity constants. Binding of a recombinant interleukin-8 (rIL-8) molecule with a C-terminal His-tag to RBL-2H3 cells which stably express the IL-8 receptors CXCR1 or CXCR2 could also be demonstrated using the tag-specific mAb. Furthermore, aminoterminally tagged C5a molecules of rat or human origin could be shown to bind to the human C5a receptor (C5aR). However, the fluorescence signal of the binding of rat rC5a to the human C5aR was distinctly higher over a wide range of ligand concentrations than the signal of human rC5a binding although both ligands were equally potent in the induction of chemotaxis in C5aR-expressing cells. Thus, the tag-specific mAb was able to interfere with the binding of human but not rat rC5a to the human C5aR. This observation is in agreement with the hypothesis of a two binding site model for the interaction of human C5a with its receptor whereas a different binding mode may apply for rat C5a. Our data demonstrate that the selected His-tag specific mAb may be a valuable tool for the visualisation of the binding of recombinant ligands to their receptors and may also provide useful information on the specific binding properties of the ligands.

C3a(desArg) does not bind to and signal through the human C3a receptor.

Contradictory results have been published in the past regarding the functional responses of different cell types to the anaphylatoxin C3a and its natural catabolite C3a(desArg). To elucidate the interaction of the C3a receptor (C3aR) with its ligand(s) we studied the binding of human recombinant C3a (rC3a) and rC3a(desArg) to RBL-2H3 transfectants which express the C3aR. As the addition of 11 aminoterminal amino acids did not alter the functional activity of the recombinant C3a as compared to serum-derived C3a the specific binding of rC3a and rC3a(desArg) to the transfectants could be determined by flow cytometry using a monoclonal antibody (mab) against their N-terminal histidine tag. Recombinant C3a bound to the C3aR with a half maximal concentration of about 3 nM whereas no evidence for a binding of rC3a(desArg) could be obtained. Furthermore, rC3a(desArg) did not signal through the C3aR. Neither the release of lysosomal N-acetyl-beta-D-glucosaminidase nor the directional migration of C3aR-expressing RBL-2H3 transfectants could be detected in response to rC3a(desArg) whereas rC3a was highly active in both assays. Our data demonstrate a defined ligand specificity of the C3aR for the anaphylatoxin C3a. Its natural catabolite C3a(desArg), however, does not signal through the C3aR. Modulating effects of C3a(desArg) on the synthesis of cytokines in human monocytes and B lymphocytes may therefore be induced by receptor-independent mechanisms while their in vivo relevance remains as yet undefined.

Anaphylatoxin C3a but not C3a(desArg) is a chemotaxin for the mouse macrophage cell line J774.

Varying results have been published regarding the functional reactivity of different cell types, including human monocytes, to the anaphylatoxin C3a and its degradation product C3a(desArg). To further delineate the functions of C3a and C3a(desArg) on this cell type we used the murine macrophage (Mø) cell line J774A.1 which is known to respond to the anaphylatoxin C5a. J774 cells specifically bound fluorescein isothiocyanate-labeled recombinant human C3a (rC3a). The cells migrated along rC3a concentration gradients in a dose-dependent manner with an optimal concentration of about 3 nM (rC5a:7 nM) and a half-maximal effective concentration (EC50) of about 1.2 nM (rC5a: 2 nM). The degradation product rC3a(desArg) was devoid of chemotactic activity. mRNA for the recently cloned G protein-coupled mouse high-affinity C3a receptor (C3aR) was detected in J774 cells, suggesting that this receptor represents the binding site for C3a on J774 Mø. In support of the specific nature of C3a-stimulated cellular mobility, RBL-2H3 transfectants expressing the human C3aR were also shown to migrate along gradients of rC3a (optimal concentration about 8 nM; EC50 about 3.5 nM) whereas rC3a(desArg) was again inactive. In summary, our findings demonstrate for the first time a specific, receptor-mediated chemoattraction of cells of the monocytic lineage to the anaphylatoxin C3a which may contribute to the accumulation of Mø at sites of inflammation.

Positive effect of balneotherapy on post-PID symptoms.

OBJECTIVE: The aim of this study was investigation of effect of balneotherapy after antibiotic treatment of PID. PATIENTS AND INTERVENTIONS: Fifty-seven patients with PID, diagnosed by laparoscopy, were treated by the same antibiotic scheme (oxytetracycline and metronidazole). After treatment of the acute phase, we studied the effect of balneotherapy (mud baths or mud packs, mineral baths, electrotherapies, and gynecological exercises) under sanatorium conditions in 30 patients. Twenty-seven patients without balneotherapy served as a control group. Second-look laparoscopy with dye insufflation was carried out about 12 weeks after the first operation. RESULTS: The rate of tubal occlusion after balneotherapy was 31% vs. 28% in patients without balneotherapy (P > .05). Moderate or severe adhesions seen at first look were similar in both groups (40.7% vs. 46.7%) and were nearly unchanged during second-look laparoscopy (44.4% vs. 46.6%). Twelve of 27 (44.4%) control patients had subjective or objective complaints compared to only 2 of 30 (6.7%, P < .05). CONCLUSION: Although balneotherapy after antibiotic therapy for acute PID does not improve fallopian tube patency, it is useful to reduce the frequency of lower abdominal pain.

[Our gynecologic heritage. The activities of scientific gynecologic societies in Northeast Germany after the World War II]. / Das gynäkologische Erbe. Die Tätigkeit der wissenschaftlichen gynäkologischen Gesellschaften im nordöstlichen Deutschland nach dem 2. Weltkrieg.

During the Second World War and especially since 1943 the activities of medical societies decreased more and more. The first gynecological meeting in the of Soviets occupied part of Germany happened October 5th till 6th 1946 in Jena. The Chairman was Gustav Döderlein. In May 1947 the Soviet Military Administration commanded the order Nr. 124 about foundation of medical societies. The Society of Gynecology at the University of Rostock was founded November 1947. In year 1948 was the unification with the Gynecological Society of Greifswald. The unified society organized till 1989 more than 40 meetings in the Northern towns of East Germany. 1991 the unification with the Northwestern Society of Gynecology and Obstetrics took place. The Society has now the name "North-German Society of Obstetrics and Gynecology".

[Treatment of acute salpingitis with tetracycline/metronidazole with or without additional balneotherapy, Augmentin or ciprofloxacin/metronidazole: a second-look laparoscopy study]. / Zur Behandlung der akuten Salpingitis mit Tetracyclin/Metronidazol mit oder ohne zusätzliche Heilbadkur, Augmentan oder Ciprofloxacin/Metronidazol: Eine second look-Laparoskopie-Studie.

110 patients suffering from laparoscopical verified salpingitis and desire for a baby, were treated with tetracycline (oxytetracycline or doxycycline; TC)/metronidazole (n = 67), augmentan (n = 22) or cipropfloxacin/metronidazole (n = 21). After an average period of 11.6 weeks, all patients underwent second-look laparoscopy with dye insufflation. In 34 patients treated with TC/metronidazole, the effects of additional physio-therapeutical measures were examined under conditions as they prevail in a Spa. 33 patients without balneotherapy served as controls. All the 4 groups were comparable (p greater than 0.05) in respect of mean age, percentage, share of nulliparous women, salpingitis gonorrhoica, contraceptive behaviour and also of the stage of salpingitis. All antibiotic regimens used resulted in a prompt decrease of inflammatory clinical signs after five days (temperature, blood sedimentation rate, leukocytes). Only 2 of 34 patients treated by additional cure at a Spa reported complaints, whereas complaints were reported by 14 of 33 control patients (p less than 0.01), 7 of 22 (p less than 0.01) treated with augmentan and to 7 of 21 (p less than 0.01) treated with ciprofloxacin/metronidazole. The tubal occlusion rates amounted to 33.3% (TC/metronidazole), 32.3% (TC/metronidazole and balneotherapy), 22.7% (augmentan) and 23.8% ciprofloxacin/metronidazole. The differences did not attain statistical significance (p greater than 0.05). With regard to adhesions, there were, likewise, no significant differences between findings at first laparoscopy and second look-laparoscopy, respectively. It is concluded, that additional physiotherapeutic measures, after antibiotic therapy of acute salpingitis, reduce the frequency of lower abdominal pain, but do not result in an improvement of tubal occlusion and reduction of adhesions.(ABSTRACT TRUNCATED AT 250 WORDS)

[Hypertensive disease in pregnancy in the history of medicine. 2: Initial scientific eclampsia research, theories and therapy]. / Die hypertensive Schwangerschaftserkrankung in der Geschichte der Medizin. Teil 2: Der Beginn der wissenschaftlichen Eklampsieforschung, Theorien und Therapie.

Part 2 contents an historical review about the beginning of research about eclampsia and about the connections between theories and treatment.

[Aspiration cytology--endometrium diagnosis of peri- and postmenopausal bleeding disorders]. / Die Aspirationszytologie--Endometriumdiagnostik peri- und postmenopausaler Blutungsstörungen.

The aspiration of endometrial cell samples for cytological analysis in comparison to histological diagnosis with a following conventional curettage was evaluated in a symptomatic group of 100 women. The age ranged between 45 to 72 years. In 87% of the detected cytological and histological findings were comparable. In 2% the results were false negative. There was determined a specificity of 88.7% and a sensitivity of 88.2%. The usage as screening method in asymptomatic group of women may be a future interest of the endometrial cytology.

[Indication-oriented prescription of hormonal contraceptives]. / Indikationsgerechte Verordnung hormonaler Kontrazeptiva.

A review about the different groups of oral contraceptives as monophasic combined pills, two-, triphasic and sequential preparations is given. Discussion of contraindications and advises for prescribing practice.

[Hypertensive disease in pregnancy in the history of medicine. 1:The old terminology, ancient history and the Middle Ages]. / Die hypertensive Schwangerschaftserkrankung in der Geschichte der Medizin. Teil: 1 Die alte Terminologie, Altertum und Mittelalter.

Toxemia of pregnancy resp. hypertensive disorder of pregnancy is a "disease of hypotheses". A historical review is given about the large number of hypotheses and the several trials of treatment in ancient time and middle age.

[The menopause--a sociopsychological interpretation]. / Die Wechseljahre--eine sozial-psychologische Interpretation.

The psychological situation of climacteric women is determined by social economic and cultural changes. The educational backgrounds and family life are factors to a high degree of life stress, too. Psychosocial analysis and more informations about the conflicts in this time are preconditions for a successful treatment of menopausal complaints before hormone replacement in the gynecological praxis.