Enhanced blood coagulation and fibrinolysis in mice lacking histidine-rich glycoprotein (HRG).

Histidine-rich glycoprotein (HRG) is a serum protein belonging to the cystatin superfamily. HRG may play a regulatory role in hemostasis and innate immunity. However, this role is uncertain because of a lack of rigorous testing in an animal model. We generated mice lacking the translation start point of exon 1 of the Hrg gene, effectively resulting in a null mutation (Hrg-/-). The mice were viable and fertile but had no HRG in their blood. Antithrombin activity in the plasma of Hrg-/- mice was higher than in the plasma of heterozygous Hrg+/- or wild-type Hrg+/+ mice. The prothrombin time was shorter in Hrg-/- mice than in Hrg+/- and Hrg+/+ mice. Bleeding time after tail tip amputation in Hrg-/- mice was shorter than in Hrg+/+ mice. The spontaneous fibrinolytic activity in clotted blood of Hrg-/- mice was higher than in Hrg+/+ mice. These findings suggest that HRG plays a role as both an anticoagulant and an antifibrinolytic modifier, and may regulate platelet function in vivo.

Use of a collagen/elastin-membrane for the tissue engineering of dermis.

In an experimental model in rats, xenogeneic membranes consisting of processed native collagen and elastin were grafted to serve as a template for the formation of a neo-dermis, while in vitro-cultured autogeneic keratinocytes were applied on top of this to restore an epidermis. The process of tissue reconstruction and the digestion of the grafted membrane components were analysed by histological and immunohistochemical methods as well as electron microscopy. Approximately 3 weeks post grafting the membranes were completely vascularised and colonized by different types of cells. After 6 weeks, the collagenous fibres of the graft were mostly replaced by newly formed collagenous texture, whereas elastic membrane components were still present even after 20 weeks. In a second step, in vitro-cultured keratinocytes were applied onto the partially integrated membranes, resulting in an epithelial coverage of approximately 47% of the grafted area after 8-11 days. As early as on day 6 post application, a multilayered, partially differentiated epithelium, together with lymphocytes and Langerhans' cells, could be observed. After 10 days the formation of a basement membrane including anchoring fibrils appeared to be complete. This three-dimensional matrix structure offers a promising scaffold for a tissue engineering strategy to restore skin structure and function. Further experimental studies are in progress to test its applicability to human skin replacement.

Diet-induced plasticity in the taste system of an insect: localization to a single transduction pathway in an identified taste cell.

We studied exposure-induced sensitivity changes in an identified taste cell from Manduca sexta, a herbivorous caterpillar. This taste cell occurs within the lateral styloconic sensillum and responds selectively to compounds that humans characterize as bitter (e.g. caffeine, salicin and aristolochic acid). We made extracellular recordings from several classes of identified taste cell within the lateral sensillum, both before and after dietary exposure (for 48 h) to a suprathreshold concentration of caffeine, salicin or aristolochic acid. Our results revealed (1) that dietary exposure to caffeine desensitized the bitter-sensitive taste cell to caffeine, whereas dietary exposure to salicin or aristolochic acid did not desensitize the same taste cell to salicin or to aristolochic acid; (2) that dietary exposure to caffeine failed to alter the responsiveness of the sugar-, salt- or inositol-sensitive taste cells within the same sensillum; (3) that the caffeine-induced desensitization phenomenon generalized to salicin, a compound that stimulates the same transduction pathway as caffeine, but not to aristolochic acid, a compound that stimulates a different pathway; and (4) that chronically stimulating the lateral sensillum with caffeine, in the absence of ingestion, was sufficient to induce desensitization. We conclude that caffeine causes desensitization through a direct effect on a single transduction pathway within the bitter-sensitive taste cell.

Treatment of skin defects using suspensions of in vitro cultured keratinocytes.

The in vitro cultivation of keratinocytes and their application in the form of confluent sheets to cover various kinds of skin defects involves a number of problematical steps which could be improved by using single cell suspensions instead. Therefore we developed a method to apply keratinocytes suspended as single cells in a fibrin gel. By testing the feasibility of this method in different experimental animal models we found that it facilitates cultivation as well as application of the cells, moreover, this method allows a much more flexible use of the cells, i.e. it is easier to consider the clinical condition of the patient than by the conventional method.

[Male subfertility and oral bacterial diseases]. / Männliche Subfertilität und orale bakterielle Erkrankungen.

More than 70% of the husbands consulting the Dept. of Gynecology and Obstetrics of the RWTH Aachen for sterility since June 1987 needed treatment for bacteriospermia with germ concentrations greater than 10(4) cfu/ml ejaculate. In 23% of the treated men however, no convincing reduction of germ concentration were achieved. 36 patients with therapy-resistant bacteriospermia were sent to the Dept. of Dentistry of the RWTH Aachen. A lot of intra-oral foci were found, which got eliminated. Intra-operational bacterial specimens were taken and evaluated by a special diagnostic technique in the Dept. of Microbiology. It was shown that the bacterial spectrum of the intraoral specimens and the spermiograms were identical. Six months after completion of the dental therapy a new andrological examination was performed. Two thirds of the spermiograms now were already sterile. There was an improvement of the spermparameters as well as motility, morphology and density. It can be concluded from these results that there is a direct causal relationship between a symptomatic dental primary disease and bacteriospermia which probably leads to subfertility.

[Chlorinated hydrocarbons in seminal plasma and male fertility]. / Chlorkohlenwasserstoff in Seminalplasma und männliche Fertilität.

To determine the concentration of polychlorinated biphenyls (PCB), hexachlorobenzene (HCB) and gamma hexachlorocyclohexane (gamma HCH) in human seminal plasma, 176 ejaculates taken from 156 patients consulting the Dept. of Gynecology and Obstetrics of the RWTH Aachen for sterility were analysed gas-chromatographically. 77% of the examined samples exhibited different pathological sperm findings (OAT, AT, OA, A, O, T, P, K). Contrary to all our expectations the concentration of PCB, HCB and gamma HCH in the samples with normozoospermia were higher than in the samples with pathological sperm findings. The wives of 42.5% of the patients with normozoospermia exhibited no organically or endocrinologically reasons for sterility (idiopathic sterility). In this group (normal/idiopathic) the concentrations of the toxic agents were significant higher than in that group of patients with pathological sperm findings. In the group of couples with male normozoospermia and female organic/endocrinologic reasons for sterility (normal/organic) nearly the same concentrations of PCB, HCB and gamma HCH could be shown as in samples with pathological sperm findings. The concentrations of the toxic agents in the samples with the different pathological sperm findings were similar--independent of the kind of diagnosis. A correlation between the spermiogram parameters motility, morphology and density and the concentration of PCB, HCB and gamma HCH in the seminal plasma could not be shown.

[Demonstration of chlorinated hydrocarbons in follicular secretions]. / Nachweis von Chlorkohlenwasserstoffen in Follikelsekreten.

To determine the concentration of HCB, HCH and PCB in human follicular fluid, 315 preovulatory follicular taken from 45 patients and aspirated by transvaginal follicular puncture were analysed with the aid of gas chromatography. We distinguished follicular fluid without oocytes and follicular fluid with oocytes. Again the latter is subdivided into follicular fluid containing secondarily fertilized oocytes and non-fertilized oocytes. There are great differences in HCB (0.08 - 1.87 ng/ml), HCH (0.05 - 1.0 ng/ml) and PCB (0.28 - 2.11 ng/ml) concentrations, varying even in the follicles of the same patient. Contrary to all our expectations we did not find any significant difference in the HCB, HCH and PCB concentration of the different follicular fluids. A direct influence of these substances on the fertilization of oocytes in IVF-ET-procedure could not be established.

Minimal andrological requirements for in-vitro fertilization.

The ejaculate used for in-vitro fertilization of human oocytes must fulfil minimum requirements. Based on the results of experimental investigations, total concentrations of spermatozoa must be greater than 5 million spermatozoa/microliters with progressive motilities of at least 30% and also a normal morphology of at least 30%. Furthermore, in sperm preparations used for inseminating human oocytes, the concentration of pathogenic microorganisms must not be less than 10(4). If there are positive bacteriological findings, the treatment of both partners is mandatory according to the antibiogram. According to the information gathered to date, the hamster oocyte penetration test can only provide additional information if it is positive, but acceptance into an IVF programme should not depend on this test. According to the results presented so far, the swell test developed by Jeyendran shows quite good correlation with the fertilization of human oocytes.