Serotoninbestimmungen an Thrombocyten und an Fraktionen von Thrombocyten

Zusammenfassung Aus Suspensionen zerstörter Thrombocyten des Menschen wurden reine Fraktionen von Hyalomer und Granulomer hergestellt. Von den Fraktionen wurde jeweils ein Teil für die elektronenmikroskopische Untersuchung, ein anderer Teil für die Bestimmung des Serotonins verwandt. Durch die elektronenmikroskopische Untersuchung jeder einzelnen Fraktion wurde die Reinheit der Fraktionen überprüft. Die Bestimmung des Serotoningehaltes erfolgte am isolierten Rattenmagen. In Kontrollversuchen wurde ferner der Serotoningehalt intakter Thrombocyten bestimmt.Intakte Thrombocyten besitzen im Mittel einen Serotonin-Gehalt von 52 ng/10⁸ Thrombocyten. In den Fraktionierungsversuchen fanden wir 95% des Serotonins in den Hyalomer-Fraktionen und nur 5% des Serotonins in den Granulomer-Fraktionen. In vivo findet sich also der hohe Serotoningehalt fast ausschließlich im Hyalomer der Thrombocyten.

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Summary Pure fractions of hyalomer and granulomer were prepared from suspensions of destroyed human thrombocytes. One portion of the fractions was used for the electron microscopic studies, and the other part for the determination of serotonin (5-hydroxytryptamine). The electron microscopic investigation allowed to check the purity of each single fraction. On the isolated rat stomach the determination of the concentration of serotonin was performed. In control preparations we studied the serotonin concentration of intact thrombocytes. These thrombocytes have a mean serotonin concentration of 52 ng/10⁸ platelets. In fractioning experiments we found 95% of the serotonin in the hyalomer fractions and only 5% of the serotonin in the granulomer fractions. In vivo therefore, the high concentration of the serotonin is found almost exclusively in the hyalomer of the thrombocytes.

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Die Ergebniss wurden vor der Medizinischen Gesellschaft Düsseldorf am 23. 1. 1963 mitgeteilt. Herrn Prof. Dr.Meessen danken wir für die Anregung und Förderung der Arbeit, Herrn Prof. Dr.Greeff für die Hilfe bei den Serotoninbestimmungen.     

CXCR4 and Gab1 cooperate to control the development of migrating muscle progenitor cells

Long-range migrating progenitor cells generate hypaxial muscle, for instance the muscle of the limbs, hypoglossal cord, and diaphragm. We show here that migrating muscle progenitors express the chemokine receptor CXCR4. The corresponding ligand, SDF1, is expressed in limb and branchial arch mesenchyme; i.e., along the routes and at the targets of the migratory cells. Ectopic application of SDF1 in the chick limb attracts muscle progenitor cells. In CXCR4 mutant mice, the number of muscle progenitors that colonize the anlage of the tongue and the dorsal limb was reduced. Changes in the distribution of the muscle progenitor cells were accompanied by increased apoptosis, indicating that CXCR4 signals provide not only attractive cues but also control survival. Gab1 encodes an adaptor protein that transduces signals elicited by tyrosine kinase receptors, for instance the c-Met receptor, and plays a role in the migration of muscle progenitor cells. We found that CXCR4 and Gab1 interact genetically. For instance, muscle progenitors do not reach the anlage of the tongue in CXCR4;Gab1 double mutants; this target is colonized in either of the single mutants. Our analysis reveals a role of SDF1/CXCR4 signaling in the development of migrating muscle progenitors and shows that a threshold number of progenitor cells is required to generate muscle of appropriate size.     

Pneumococcal serotype 19F conjugate vaccine induces cross-protective immunity to serotype 19A in a murine pneumococcal pneumonia model

Immunization with a pneumococcal conjugate vaccine (PNC) containing serotype 19F induces cross-reactive antibodies to 19A in mice and human infants. Active immunization with PNC and passive immunization with serum samples from infants vaccinated with PNC containing serotype 19F, but not serotype 19A, protected against lung infection caused by both serotypes in a murine model.     

The effects of co-culture with human fibroblasts on human embryo development in vitro and implantation

In a human in-vitro fertilization (IVF) programme, the effect of co- culture of embryos with human fibroblasts was evaluated with respect to pregnancy rate and embryo development. Patients were included in the study after giving informed written consent. The IVF treatments were randomly assigned by stratification of both age (<36 versus > or =36 years) and previous IVF attempts (yes versus no). After fertilization was established, the zygotes were transferred to a 4-well dish with or without fibroblasts and cultured for 2 days. On the third day after ovum pick-up (OPU), cell number and quality [5 (good) to 1 (poor)] of the embryos were scored and a maximum of three embryos was transferred. Supernumerary embryos of good quality were cryopreserved. The design of this study was a group sequential trial with the objective of detecting differences between pregnancy rates following IVF with conventional incubation or incubation in co-culture with fibroblasts. This design included one evaluation at half-way data collection. In the study, 148 patients had an OPU, of whom 77 were allocated to the co-culture group. There was no statistically significant difference in pregnancy rate, cell number and embryo quality between the two groups. The ongoing pregnancy rate per embryo transfer was 27% in co-culture and 30% in the conventional culture group. The implantation rates per transferred embryo were 17 and 18% respectively. Using a multivariate logistic regression model for the probability of ongoing pregnancies, the odds ratio of co-culture, adjusted for age and previous IVF attempts, was not statistically significant. In conclusion, co-culture with human fibroblasts does not contribute to an improvement of embryo quality nor to a higher pregnancy rate after IVF in an unselected group of patients.      

C3bi-binding protein on Candida albicans: temperature-dependent expression and relationship to human complement receptor type 3.

We investigated in detail the previously described capacity of pseudohyphae of Candida albicans to bind C3-coated particles. We show that the expression of the C3bi receptor of C. albicans was dependent upon the growth temperature of the fungi. C. albicans grown at 30 degrees C bound strongly to EAC1423bi, whereas those cells grown at 38.5 degrees C were completely devoid of this capacity. The molecule responsible for the attachment of EAC1423bi was heat labile and trypsin sensitive. Several, but not all, monoclonal antibodies to the alpha-chain of human complement receptor type 3 (CR3) stained C. albicans, and this reactivity was expressed in parallel with the capacity of C. albicans to bind EAC1423bi, i.e., both were dependent on the growth temperature of the fungi and were trypsin sensitive. In contrast to CR3, the binding of EAC1423bi to C. albicans did not require the presence of divalent cations. Rabbit immunoglobulin G antibodies directed against C. albicans inhibited the binding of EAC1423bi to C. albicans but not to human CR3. These inhibiting IgG antibodies recognized antigens expressed on the surface of pseudohyphae but not those of yeast cells. OKM-1, a monoclonal antibody to human CR3 inhibited the attachment of EAC1423bi to CR3 and also to C. albicans. OKM-1 precipitated a 130-kilodalton band from solubilized 125I-labeled C. albicans. We conclude that the complement receptors on C. albicans and human CR3 were antigenically related but not identical and that they differed in their functional characteristics.     

Activity and cross-reactivity of antibodies induced in mice by immunization with a group B meningococcal conjugate

The capsular polysaccharide of group B Neisseria meningitidis is composed of a linear homopolymer of alpha(2-8) N-acetyl neuraminic acid or polysialic acid (PSA) that is also carried by isoforms of the mammalian neural cell adhesion molecule (NCAM), which is especially expressed on brain cells during development. Here we analyzed the ability of antibodies induced by the candidate vaccine N-propionyl polysaccharide tetanus toxoid conjugate to recognize PSA-NCAM. We hyperimmunized mice to produce a pool of antisera and a series of immunoglobulin G monoclonal antibodies and evaluated their self-reactivity profile by using a battery of tests (immunoprecipitation, immunoblotting, and immunofluorescence detection on live cells and human tissue sections) chosen for their sensitivity and specificity to detect PSA-NCAM in various environments. We also searched for the effects of the vaccine-induced antibodies in two functional assays involving cell lysis or cell migration. Although they were highly bactericidal, all the antibodies tested showed very low or no recognition of PSA-NCAM, in contrast to PSA-specific monoclonal antibodies used as controls. Different patterns of cross-reactions were revealed by the tests used, likely due to affinity and specificity differences among the populations of induced antibodies. Furthermore, neither cell lysis nor perturbation of migration was observed in the presence of the tested antibodies. Importantly, we showed that whereas enzymatic removal of PSA groups from the surfaces of live cells perturbed their migration, blocking them with PSA-specific antibodies was not functionally detrimental. Taken together, our data indicated that this candidate vaccine induced antibodies that could not demonstrate an immunopathologic effect.     

Reliability of the E test for detection of ampicillin, vancomycin, and high-level aminoglycoside resistance in Enterococcus spp.

By comparison with agar dilution results, the E test was investigated for the ability to detect high-level aminoglycoside (gentamicin and streptomycin), ampicillin, and vancomycin resistance among strains representing six enterococcal species. For ampicillin and vancomycin, disk diffusion results also were obtained. No false high-level aminoglycoside resistance occurred, and no false gentamicin susceptibility was noted. With the high-range streptomycin E test (2,048 micrograms), 24% of the 38 resistant strains were falsely susceptible. However, these discordances could likely be reconciled by adjustments in incubation duration and by using broth microdilution rather than agar screen breakpoint criteria, or by using the lower-range (1,024-micrograms) strip. For ampicillin, category results obtained by E test and disk diffusion showed good agreement with agar dilution; E test MICs were generally comparable to agar dilution MICs. The E test was more sensitive than disk diffusion for detecting vancomycin-intermediate strains, but for these strains and those exhibiting low-level vancomycin resistance (MIC, 32 to 128 micrograms/ml), disk diffusion and E test inhibition zones must be interpreted with caution. Given the reliability of E test for detecting resistance to anti-enterococcal agents, the decision to use this method should be based on convenience, cost, testing frequency, and satisfaction with currently used methods.     

Genetic study of oxygen resistance and melanization in Cryptococcus neoformans.

Genetic analysis of oxygen-sensitive mutants of Cryptococcus neoformans revealed two loci (oxy1 and oxy2) linking hyperoxia sensitivity to production of melanin, a known virulence factor. Hyperoxia-sensitive strain 562 (oxy1 oxy2) is albino and avirulent. oxy2-defective strains lacking the oxy1 defect are melanin deficient but show normal hyperoxia resistance. Mutants defective at three additional mapped melanin loci fail to show hyperoxia sensitivity in the oxy1 background. Revertants of strain 562, which regain the ability to synthesize melanin by mutation at suppressor sites unlinked to oxy2, retain the oxygen sensitivity conferred by their oxy1 and oxy2 defects. These data identify the melanin gene oxy2 as unique in its association of hyperoxia resistance and melanization.