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Biomedical subjects

D Hornung

Publications and source records attributed to D Hornung.

At least 19 recordsLinked to original sources
PubMed2001

Purification and characterization of an immunomodulatory endometrial protein, glycodelin.

Human glycodelin is synthesized by endometrial cells in the late secretory phase and early pregnancy under hormonal regulation. Whereas the precise physiological functions of glycodelin are unknown, its expression during embryonic nidation and its inhibition of T cell proliferation suggest an immunomodulatory role. We purified human glycodelin from first trimester human decidual cytosol by using a rapid two-step high-performance liquid chromatography method and investigated its effects on human monocyte migration. Human U937 cells were used as a model of monocyte chemotaxis in Boyden chamber migration assays. N-Formyl-Met-Leu-Phe and the beta-chemokine RANTES (regulated on activation normal T cell expressed and secreted) were used as monocyte chemoattractants. Purified glycodelin inhibited monocyte migration in a dose-dependent fashion (IC50 = 550 nm). Glycodelin activity was totally reversed by heat inactivation (95 degrees C x 15 min) and neutralized by pretreatment with specific anti-glycodelin antibodies. Deglycosylated glycodelin was equipotent to intact glycodelin in the monocyte migration assay. 125I-Glycodelin binding to whole U937 cells revealed a single, saturable site with a Kd = 48 +/- 21 nm by Scatchard analysis. Cross-linking studies indicated that glycodelin binds to a high molecular mass (approximately 250 kDa) protein complex at the monocyte cell surface. Our findings support the hypothesis that glycodelin reduces the local maternal inflammatory response toward the implantation of a semiallogeneic conceptus.

Amino Acid Sequence↗
PubMed2001

Regulated on activation, normal T-cell-expressed and -secreted mRNA expression in normal endometrium and endometriotic implants: assessment of autocrine/paracrine regulation by in situ hybridization.

Chemoattraction of macrophages and T cells into the normal endometrium and inflammatory sites within endometriotic foci is mediated by chemokine gene expression. mRNA transcripts encoding regulated on activation, normal T-cell-expressed and -secreted (RANTES), a monocyte and T-cell chemokine, were demonstrated in the stroma of normal endometrium and endometriotic implants using in situ mRNA hybridization. Epithelial glands failed to express RANTES mRNA. In histological serial sections, we observed CD68-positive macrophages in the stroma of endometriotic implants adjacent to regions with prominent RANTES mRNA hybridization. In adjacent sections, monoclonal antibodies against tumor necrosis factor (TNF)-alpha showed this cytokine to be localized to stromal and epithelial compartments of the endometriotic implant with weak staining in unaffected ovarian tissue. Subconfluent monolayers of endometriotic stromal cells were tested for RANTES gene expression in situ, but we could only detect RANTES mRNA in isolated stromal cells after treatment with TNF-alpha. No RANTES mRNA was observed in unstimulated stromal cells or TNF-alpha stimulated or unstimulated epithelial cells. The data are consistent with a model in which proinflammatory cytokines (eg, TNF-alpha) induce RANTES gene expression limited to specific cells within endometrial and endometriotic stroma. Production of this chemokine, in turn, stimulates recruitment of CD68-positive macrophages into these tissues.

Antigens, CD↗
PubMed2001

Histocompatibility leukocyte antigen-G is not expressed by endometriosis or endometrial tissue.

OBJECTIVE: The immunological mechanisms that support persistence and proliferation of ectopic endometrial implants within the peritoneal cavity of women with endometriosis are unknown. Inhibition of natural killer (NK) and cytotoxic T-cell function has been proposed as a mechanism. We tested the hypothesis that expression of a nonclassical major histocompatibility antigen, HLA-G, might explain the local immunosuppression associated with ectopic endometrium. DESIGN: Nested case-control study of women with and without laparoscopic evidence of endometriosis. SETTING: Reproductive endocrinology clinic at a university hospital. PATIENT(S): Peritoneal fluid specimens from 10 women with revised AFS stage I-IV endometriosis and from 10 age-matched normal controls without laparoscopic evidence of endometriosis were tested for the presence of HLA-G protein. Endometriosis and normal endometrial biopsies from four patients were used to prepare stromal cell cultures directly evaluated for HLA-G protein. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The expression of HLA-G in peritoneal fluid, tissue, and cell cultures was determined by immunoblotting with a specific monoclonal antibody. RESULT(S): HLA-G protein was not detectable in peritoneal fluid specimens of endometriosis patients or controls. Moreover, ectopic and normal endometrial tissues and stromal cells did not express HLA-G. CONCLUSION(S): Immune cell inhibition in endometriosis must be mediated by factors other than HLA-G.

Amino Acid Sequence↗
PubMed2001

Chemokine bioactivity of RANTES in endometriotic and normal endometrial stromal cells and peritoneal fluid.

Endometriotic lesions secrete chemokines that recruit immune cells into the peritoneal cavity. The accumulation of these immune cells, especially activated macrophages and T lymphocytes, is thought to mediate inflammatory symptoms associated with endometriosis. Previous studies have demonstrated that RANTES (regulated on activation, normal T cell expressed and secreted) is synthesized by endometriotic stromal cells and circulates in peritoneal fluid, commensurate with the stage of endometriosis. In the current studies, we used the human monocytic cell line, U937, to assay chemotactic activity in cell culture conditioned media and peritoneal fluid from patients with endometriosis and normal controls. We demonstrated expression of the human RANTES receptors CCR-1 and CCR-5 in U937 cells and peritoneal macrophages. Over a range of 0-1000 pg/ml recombinant human RANTES had a direct, linear effect on monocyte migration. Conditioned media and peritoneal fluid induced dose-dependent effects on monocyte migration that were correlated with concentrations of immunoreactive RANTES (as measured by enzyme-linked immunosorbent assay) and the severity of endometriosis. Heat denaturation of the RANTES protein or addition of anti-human RANTES antibodies neutralized the chemoattractant effects of conditioned media and peritoneal fluid. RANTES stimulation of monocyte recruitment may be an important pathogenetic target for the treatment of infertility and pain associated with endometriosis.

Ascitic Fluid↗
PubMed2001

Endocrine and paracrine regulation of endometrial angiogenesis.

The human endometrium is a complex tissue comprised of different cell types, including epithelial, stromal, inflammatory, perivascular, and blood vessel cells. The hormonal receptivity and distribution of these cell populations change during the menstrual cycle. Cyclical endometrial growth is dependent on its ability to regenerate a vascular capillary network, which grows in parallel with the proliferation and differentiation of the endometrial lining. Natural hormonal effects on the endometrium and endocrine manipulation of this tissue, in response to the use of exogenous steroid therapies, can affect endometrial capillary proliferation and function, leading to clinical abnormalities of uterine bleeding. We propose that the regulation of endometrial angiogenesis is mediated indirectly via complex interactions among cell types. Our laboratory has focused on a prototypical member of the angiogenic proteins, vascular endothelial growth factor (VEGF)-A. In this paper we present data demonstrating that VEGF-A expression in normal endometrial epithelial and stromal cells and in Ishikawa adenocarcinoma cells is increased by an ovarian steroid, estradiol. Infiltrating immune cells, particularly polymorphonuclear granulocytes, also are sources of VEGF-A. In inflammatory conditions involving the endometrium (e.g., endometriosis), a proinflammatory cytokine, IL-1beta, can mediate neoangiogenesis by inducing VEGF-A gene transcription. Thus, endometrial vascularization is effected by both endocrine and paracrine pathways.

Cells, Cultured↗
PubMed2001

Nuclear peroxisome proliferator-activated receptors alpha and gamma have opposing effects on monocyte chemotaxis in endometriosis.

The peroxisome proliferator-activated receptors (PPARs) alpha and gamma are nuclear receptors that play important roles in inflammatory diseases like ulcerative colitis and arthritis. In this study, we examined the possible role of PPARs in macrophage attraction into the peritoneal cavity of patients with endometriosis. We identified PPAR-alpha and -gamma messenger RNA by RT-PCR and protein by immunoblotting of lysates of peritoneal macrophages and monocytic U937 cells. Using immunocytochemistry, we localized PPAR-alpha and -gamma within the nuclei of both cell types. Monocyte chemotactic activity of peritoneal fluid from patients with endometriosis was quantified in Boyden chambers. Migration of U937 cells was increased by WY 14643 and reduced by rosiglitazone. Peritoneal fluid from patients with endometriosis activated U937 cells transiently transfected with a PPAR-alpha/GAL4 luciferase reporter. By contrast, peritoneal fluid did not cause significant activation of PPAR-gamma/GAL4 constructs. The U937 cells transiently transfected with a PPAR response element luciferase reporter showed disease stage-dependent up-regulation when treated with peritoneal fluid from patients with endometriosis. Treatment with peritoneal fluid from healthy controls down-regulated PPAR response element transactivation. We conclude that peritoneal fluid of endometriosis patients contains activators of PPAR-alpha that stimulate macrophage chemotaxis. Inhibitors of PPAR-alpha or activators of PPAR-gamma could be developed for the treatment of inflammation associated with endometriosis.

Apoptosis↗
PubMed2000

Quantification of group 5 grass pollen allergens in house dust.

BACKGROUND: It is widely known and accepted that grass pollen is a major outdoor cause of hay fever. However, it is of virtual importance for grass pollen allergic patients with symptoms all the year round to know the concentration of grass pollen allergens in their homes. OBJECTIVE: The main objective of this study was to quantify the amount of grass pollen allergen in mass units (microg Phl p 5) in dust settled indoors and to detect the distribution of allergenic activity in different sampling locations of homes. Furthermore, we studied the seasonal fluctuation of allergen content in dust samples. METHODS: We adapted the two site binding assay for detection of group 5 grass pollen allergens in samples from randomly selected homes in Hamburg (n = 371), Erfurt (n = 396), Hettstedt (n = 353), Zerbst (n = 289) and Bitterfeld (n = 226), Germany. Dust samples were collected from floor of living room (LR), bedroom (BR) or children's room (CR) and mattress (MA) during period of June 1995 to August 1998. The amount of the major grass group 5 allergens was detected in microg/g dust. RESULTS: Phl p 5 was detected in 67% of the samples analysed (n = 4760). The range was between undetectable (< 0.03 microg/g dust) and 81 microg/g dust. Phl p 5 levels were significantly higher in the dust from LR (geometric mean 0.117 microg/g dust) or BR/CR floors (geometric mean 0.098 microg/g dust) than in mattresses (geometric mean 0.043 microg/g dust). We observed seasonal fluctuation of indoor Phl p 5 levels with peak in June but also annual differences. Thus Phl p 5 content indoors reflects also the different quantities of pollen counts of annual courses. During pollination period we found two times higher Phl p 5 levels (0.172 microg/g dust, P < 0.001) than outside of grass pollination season (0.095 microg/g dust). The indoor Phl p 5 levels outside of season seem to be independent of pollination before. We suppose that settled pollen grains or allergenic material from outdoor particles carried indoors via footwear and clothes accumulates in house dust. CONCLUSION: Although we not known how the allergens in settled dust are equilibrated with those in the air, the considerable high level of Phl p 5 in indoor dust even during periods when no grass pollen is present in the atmosphere may be an important cause of pollen-allergy symptoms outside of season.

Air Pollution, Indoor↗
PubMed2000

Localization in tissues and secretion of eotaxin by cells from normal endometrium and endometriosis.

Our laboratories have focused recently on the production and localization of eotaxin, a C-C-chemokine of 8.4 kDa, whose major biological activity is the chemoattraction of eosinophils. Given evidence of autoimmune activity in the endometriosis syndrome, we hypothesized that eosinophil chemoattractants might be expressed in endometriosis. In histological sections, we observed eotaxin protein localized mainly in epithelial cells, with only very faint immunostaining in the surrounding stromal cells. Prominent eotaxin accumulation was noted in the luminal epithelium of secretory endometrium. Eotaxin distribution in endometriosis was similar to that seen in eutopic endometrium but with higher levels of eotaxin staining in the glandular epithelium. Peritoneal fluid concentrations of eotaxin were significantly higher in women with moderate or severe endometriosis than in women with minimal or mild endometriosis or no disease. The treatment of isolated human endometriosis epithelial cells with estradiol, medroxyprogesterone acetate, tumor necrosis factor-alpha, and interferon-gamma stimulated measurable eotaxin secretion into the conditioned media. The results indicate that eotaxin is produced in epithelial cells of normal endometrium and endometriosis tissues, varies across the menstrual cycle, and is elevated in women with endometriosis. We postulate that eotaxin, interacting with other known cytokines and immune cells, contributes to an inflammatory reproductive tract environment, leading to endometrial or blastocyst dysfunction.

Adult↗
PubMed1999

[In Process Citation]

The assessment of the activity of endometriosis provides a challenge during endoscopic procedures. Novel approaches can be developed in future based on more detailed understanding of the mechanisms of the pathogenesis of endometriosis. Angiogenic factors of local immunoreactions are important agents in the development and progression of the disease. Vascular endothelial growth factor (VEGF) is a potent inducer of vascular permeability and of the mitogenesis of endothelial cells. In endometriotic and endometrial tissues VEGF isoforms VEGF121, VEGF165, VEGF189 and VEGF206 are expressed. The amount of VEGF isoforms in the endometrium is regulated depending on the menstrual cycle. Macrophage recruitment by ectopic endometrial implants is an early step in the cascade of implantation and local peritoneal reaction. RANTES (Regulated upon Activation, normal T-cell-Expressed and Secreted) is a specific chemoattractant for monocytes and activated T-cells. Soluble products of activated macrophages play an important role in endometriosis associated infertility. The factors involved in angiogenesis and immune response are potential parameters to determine the activity of endometriotic implants and novel approaches for the treatment of endometriosis.

Journal Article↗
PubMed1998

Vectorial secretion of vascular endothelial growth factor by polarized human endometrial epithelial cells.

OBJECTIVE: To analyze directional vascular endothelial growth factor (VEGF) secretion in polarized human endometrial epithelial cell cultures. VEGF has distinct distribution patterns in human endometrium. Stromal cells are diffusely positive for VEGF messenger RNA and protein, whereas glandular epithelium shows focal VEGF immunostaining at the apical surface. The epithelial distribution suggests that VEGF is secreted into gland lumina, potentially influencing the nutrition and/or apposition of the developing blastocyst. DESIGN: Controlled in vitro study of protein secretion by polarized endometrial epithelial cells established on polyethylene filters. SETTING: University hospital. PATIENT(S): Endometrial biopsies were obtained from healthy, ovulatory women undergoing elective surgery. INTERVENTION(S): Primary endometrial epithelial cells were cultured. MAIN OUTCOME MEASURE(S): VEGF mRNA and protein production were measured in polarized cells. The vectorial secretion of VEGF was determined. RESULT(S): VEGF production by endometrial epithelial cells was verified by Northern blotting and immunoassays of conditioned media. The mean (+/-SD) apical secretion of VEGF was 3.9 +/- 1.4 ng per 10(5) cells every 48 hours and the mean (+/-SD) basal secretion was 0.8 +/- 0.2 ng per 10(5) cells every 48 hours. In contrast, the apical and basal secretion of a soluble cellular isoform of fibronectin were 2.76 +/- 0.96 ng per 10(5) cells every 48 hours and 2.64 +/- 1.79 ng per 10(5) cells every 48 hours, respectively. CONCLUSION(S): VEGF is secreted preferentially into the lumina of endometrial glands. Apical VEGF may be an endometrial signal for blastocyst development or implantation.

Adult↗
PubMed1998

Promegestone (R5020) and mifepristone (RU486) both function as progestational agonists of human glycodelin gene expression in isolated human epithelial cells.

One of the most abundant protein products of human secretory endometrium is glycodelin, a glycoprotein previously referred to as PP14. Although the precise function of this protein is unknown, its unique glycosylation pattern is believed to affect immunomodulatory activity during human embryonic implantation and inhibition of sperm-egg binding after ovulation. Having confirmed the expression of glycodelin in secretory endometrial glands, we used purified endometrial epithelial cell cultures to demonstrate the hormonal regulation of glycodelin synthesis and secretion. The findings were corroborated by transiently transfecting glycodelin gene promoter-reporter constructs into human epithelioid HeLa and Ishikawa cells. Our results indicate that glycodelin protein production by endometrial epithelial cells is directly up-regulated 4- to 9-fold by progestins and antiprogestins in vitro. Transcriptional regulation of the glycodelin gene promoter expressed in HeLa cells is progesterone receptor-dependent. As observed in the primary endometrial cells, progestins and antiprogestins both act as agonists on the in vitro expression of this endometrial gene. Our findings provide insight into the regulation of this abundant endometrial protein and raise interesting questions about the physical nature of the interaction of agonist- and antagonist-bound progesterone receptors with the glycodelin gene promoter.

Drug Evaluation, Preclinical↗
PubMed1997

Angiogenesis and macrophage activation in endometriosis.

Recent studies suggest that the symptoms associated with endometriosis are the result of local peritoneal inflammation. Increased concentrations of activated pelvic macrophages and lymphocytes and the elevated levels of specific cytokines and growth factors reviewed above support this hypothesis. The precise roles of these soluble factors are currently unknown, but we propose that a complex network of endometrial cytokines are normally regulated by hormones produced during the ovulatory cycle. Ectopic endometrial implants also are subject to these same endocrine cues. The secretion of these proinflammatory proteins by endometriosis lesions into the peritoneal microenvironment appears to cause a recruitment of capillaries and activated inflammatory cells to the implant. Future therapeutic strategies directed to ameliorate the inflammatory reaction associated with endometriosis should not ignore the likely physiological actions of many of the same bioactive molecules in normal eutopic endometrial function.

Cells, Cultured↗
PubMed1997

Immunolocalization and regulation of the chemokine RANTES in human endometrial and endometriosis tissues and cells.

Retrograde menstruation is postulated as the initiating event in the histogenesis of endometriosis; however, subsequent steps in the pathogenesis of this common disorder remain poorly characterized. The ip accumulation of activated leukocytes and the infiltration of endometriosis lesions by macrophages and T cells are cytological markers of the inflammatory nature of this syndrome. The apparent recruitment of these leukocytes prompted us to search for chemokine expression by endometriosis cells. We reported previously that pelvic fluid RANTES (regulated upon activation, normal T cell expressed and secreted) concentrations correlated with the stage of endometriosis. In the current study, RANTES messenger ribonucleic acid (mRNA) was identified in normal endometrium and endometriosis lesions, and techniques were developed to localize RANTES protein within these tissues. Using isolated endometrial and endometriosis cell cultures, we demonstrated that RANTES mRNA and protein can be induced by the proinflammatory cytokines tumor necrosis factor-alpha and interferon-gamma in endometrial stromal, but not in epithelial or adenocarcinoma cells. Immunocytochemical studies confirmed the biochemical findings. Metabolic labeling experiments verified that nascent RANTES secreted by cytokine-stimulated endometriosis stromal cells was the mature, 8-kDa protein predicted by the mRNA encoding this chemokine. The results indicate that RANTES is a normal constituent of the eutopic endometrium. We propose that secretion of RANTES by ectopic endometriosis implants provides a mechanism for peritoneal leukocyte recruitment.

Adult↗
PubMed1996

Recurrent first trimester spontaneous abortion associated with antiphospholipid antibodies: a pilot study of treatment with intravenous immunoglobulin.

BACKGROUND: Antiphospholipid antibodies, unassociated with an underlying connective tissue disease, have repeatedly been detected in women suffering from recurrent spontaneous abortions. Several therapeutic regimens have been advocated for pregnant women with recurrent fetal loss and antiphospholipid antibodies. However, most of these approaches were empirical, using several drugs simultaneously, and most reports describe single cases or limited series. PATIENTS AND INTERVENTIONS: In a pilot study, thirty-eight women with a history of three or more consecutive first trimester spontaneous abortions and antiphospholipid antibodies were treated with intravenous immunoglobulin. As soon as pregnancy had been confirmed, intravenous immunoglobulin was administered at a dose of 300 mg/kg bodyweight, and infusions were repeated at three-weekly intervals until the 16th-17th week of pregnancy. RESULTS: Pregnancy proceeded beyond the first trimester in 34 of the patients (89.4%), and 31 patients (81.4%) gave birth to healthy infants at 37 to 42 weeks' gestation. CONCLUSIONS: Although the results are promising, randomized placebo-controlled trials are necessary to exclude the influence of other factors (e.g. intense obstetric supervision and psychological factors) on pregnancy outcome and confirm the effectiveness of intravenous immunoglobulin in patients with recurrent spontaneous abortions and antiphospholipid antibodies.

Abortion, Habitual↗
PubMed1990

Use dependence of sodium current inhibition by tetrodotoxin in rat cardiac muscle: influence of channel state.

Tetrodoxin (TTX) is known to cause a voltage- and frequency-dependent inhibition of the rapid inward sodium current (INa) of cardiac muscle. This effect was studied by means of the loose-patch-clamp method on intact rat papillary muscle. The availability curve of the fast sodium system, determined by variation of the holding potential, is shifted in the presence of TTX (5.5 mumol x 1(-1] by 17 mV to more negative potentials. With clamp pulses of 5 ms duration to 0 mV, a frequency-dependent reduction of INa by TTX is found above 0.1 Hz that saturates at about 10 Hz. This frequency-dependent block was further analysed using trains of pulses (10 Hz) of various durations (minimum 50 microseconds), which allow TTX to equilibrate with channel states reached early during activation. The results show that more than 90% of the frequency-dependent block is attained with pulses of 1 ms duration. An analysis according to the guarded receptor hypothesis reveals that these results are well described by TTX binding to inactivated, activated and probably preactivated channel states.

Animals↗
PubMed1981

[Arthrograph and operative findings (author's transl)].

Arthrograph and operative findings were compared retrospectively in 57 patients (53 examinations of the knee-,3 of the shoulder-, and 1 of the hip-joint). In medial disk lesions there were differences between arthrograph and operation in 19% (13,5 false positive and 5,5% false negative). In lateral disk lesions there were differences in 13% (5,5% false positive und 7,5% false negative). In a prospective series the results were far better. There were no differences for the shoulder and the hip-joint.

Adult↗