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E Schmidt

Publications and source records attributed to E Schmidt.

At least 73 records · Page 4Linked to original sources
PubMed2000

Fetal calf serum-free generation of functionally active murine dendritic cells suitable for in vivo therapeutic approaches.

UNLABELLED: Standard protocols to generate mouse dendritic cells (DC) generally use culture medium supplemented with fetal calf serum; however, reinjection in vivo of DC cultured in fetal calf serum results in priming to xenogeneic proteins that clearly limits the use of such DC. We therefore established a fetal calf serum-free culture system for the generation of murine DC from bone marrow precursors. DC can be generated fetal calf serum-free using RPMI supplemented with 1.5% syngeneic mouse serum. Although the yield of DC grown under fetal calf serum-free conditions was somewhat lower than that of the standard culture, large numbers of DC could be generated without the exposure to xenogeneic proteins. The yield of fetal calf serum-free cultured DC was further enhanced by addition of the proinflammatory cytokines TNF-alpha and IL-1beta with the combination resulting in up to 10% more DC. Phenotypically, CD11c + DC cultured fetal calf serum-free homogenously coexpressed the DC-specific molecule DEC-205 as well as the costimulatory molecules CD40, CD80, and CD86. In contrast, only a subpopulation of the CD11c + DC cultured in fetal calf serum-containing medium coexpressed these molecules. Functionally, fetal calf serum-free DC showed strong stimulatory capacity for naïve allogeneic CD4 + and CD8 + T cells. Importantly, fetal calf serum-free DC showed spontaneous in vivo migratory activity. Moreover, 5 x 105 subcutaneously injected TNBS-conjugated fetal calf serum-free DC were able to mediate contact sensitivity. Furthermore, the intravenous or subcutaneous injection of a single dose of 5 x 105 OVA-pulsed fetal calf serum-free DC resulted in the induction of an OVA-specific immune response in naïve TCR transgenic animals. Thus DC cultured under fetal calf serum-free conditions are suitable instruments for in vivo therapeutic approaches, especially in autoimmune models. KEYWORDS: DC vaccines/dendritic cell development/fetal calf serum-free culture conditions for DC/in vivo therapeutic DC approaches.

Animals↗
PubMed2000

IgG4 and IgE are the major immunoglobulins targeting the NC16A domain of BP180 in Bullous pemphigoid: serum levels of these immunoglobulins reflect disease activity.

BACKGROUND: Bullous pemphigoid (BP) is an autoimmune blistering disease associated with autoantibodies against the hemidesmosomal glycoprotein BP180. The noncollagenous (NC)16A domain of BP180 has recently been shown to harbor major antigenic sites recognized by BP sera. OBJECTIVE: The purpose of this study was to characterize the subclass distribution and fine specificities of autoantibodies to BP180 NC16A present in the circulation of patients with BP before, and during the course of, therapy for this disease. METHODS: Eighteen BP sera were analyzed by immunoblotting and enzyme-linked immunosorbent assay for the presence of IgG1, IgG2, IgG3, IgG4, and IgE reactive with various sites on the BP180 NC16A domain. The sera were collected before treatment was started and at 4- and 8-week time points after initiation of treatment. RESULTS: We identified IgG4 and IgE as the major immunoglobulins that preferentially react with two distinct epitopes (MCW-1 and MCW-2) within BP180 NC16A. Levels of these autoantibodies correlated with disease activity in BP. During the course of disease, no change was observed with regard to the immunoglobulin subclass predominantly reacting with BP180 NC16A or the specific epitopes within this domain. CONCLUSION: Our data demonstrate that remission of BP is paralleled by a decrease of serum levels of IgE and the different IgG subclasses reactive with BP180 NC16A.

Aged↗
PubMed2000

Autoimmune and inherited subepidermal blistering diseases: advances in the clinic and the laboratory.

Anchoring complexes are specialized focal attachment sites within the cutaneous basement membrane zone (BMZ) and play a crucial role in dermo-epidermal adhesion. Structural weakness that may be caused by the binding of autoantibodies to components of the anchoring complex or by aberrant expression of these components as a result of genetic defects can lead to subepidermal blisters. Autoimmune subepidermal blistering disorders include bullous pemphigoid, pemphigoid gestationis, lichen planus pemphigoides, linear IgA disease, cicatricial pemphigoid, anti-p200, anti-p105, and anti-p450 pemphigoid, epidermolysis bullosa acquisita, and bullous systemic lupus erythematosus. The autoantigen in the skin of patients with dermatitis herpetiformis remains to be identified. More than 300 distinct mutations in 10 different genes corresponding to structural components of the BMZ have been described that result in skin fragility and dermo-epidermal separation associated with characteristic extracutaneous manifestations. This group of genodermatoses, collectively referred to as epidermolysis bullosa (EB), consists of distinct variants, such as EB simplex, EB with muscular dystrophy, EB with pyloric atresia, generalized atrophic benign EB, Herlitz junctional EB, and dystrophic EB. Recent advances in the molecular characterization of BMZ components have led to a better understanding of the interaction between these molecules as well as the autoimmune response against these proteins. In addition, by the elucidation of genetic defects in the different variants of EB, genotype-phenotype correlations now begin to arise and genetic counseling has been improved.

Autoimmune Diseases↗
PubMed1999

Antiepiligrin cicatricial pemphigoid: an underdiagnosed entity within the spectrum of scarring autoimmune subepidermal bullous diseases?

BACKGROUND: Antiepiligrin cicatricial pemphigoid (AECP) is a chronic autoimmune subepidermal blistering disease characterized by autoantibodies to laminin 5 and clinical features of cicatricial pemphigoid. Only a few patients with AECP have been described to date. The aim of the present study was to analyze the relative frequency of AECP among patients with the clinical phenotype of cicatricial pemphigoid. OBSERVATIONS: Serum from 16 consecutive patients with the clinical phenotype of cicatricial pemphigoid were included in this study. Nine patients had circulating IgG autoantibodies by indirect immunofluorescence on sodium chloride-split skin; patients' IgG bound to the epidermal side (n = 2), dermal side (n = 5), or both sides (n = 2) of this test substrate. Interestingly, all 5 cases with dermal binding immunoprecipitated laminin 5 from extracts and media of cultured keratinocytes, and 4 of these serum samples reacted with the alpha3 subunit of laminin 5 by immunoblotting. None of the patients with dermal binding of IgG demonstrated autoantibodies to type VII collagen. CONCLUSION: Our data suggest that, among patients with the clinical phenotype of cicatricial pemphigoid, AECP may be more frequent than previously assumed.

Aged↗
PubMed1999

Indomethacin-induced disturbances in villous microcirculation in the rat ileum.

Indomethacin is a widely used nonsteroidal antiphlogistic compound used-among others-for the treatment of rheumatoid arthritis in humans. Common side effects of indomethacin in the gastrointestinal tract include ulcerative lesions and petechial bleeding in the mucosa. In the rat, oral intake of indomethacin induces ulcerations in the mucosa of the stomach and, if administered systemically, edema, petechial bleeding, and ulcerations in the small intestine. In order to determine if systemic administration of indomethacin may induce changes in villous perfusion, we assessed the effect of indomethacin on erythrocyte velocity and the diameter of the main arteriole in the villi of the rat ileum. We found that indomethacin led to a significant decrease in mean arteriolar blood flow (6.3 +/- 0.8 vs 5.0 +/- 1.2 nl/min, means +/- SD) 7 days after administration. Mean diameter of the main arteriole remained unchanged (control, 7. 8 +/- 0.8 vs indomethacin, 7.0 +/- 0.9 microm). In conclusion, systemic application of indomethacin leads to a decrease in blood supply to the mucosa. We previously found an increase in villous perfusion when assessed 24 h after administration of indomethacin, accompanied by an increase in arteriolar diameter of the main arteriole. In summary, different regimens of application of indomethacin lead to varying observations in microcirculatory parameters in single villi. Further studies are required to identify the underlying mechanisms.

Animals↗
PubMed1999

[Immunopathologic changes in 115 patients with bullous pemphigoid].

Bullous pemphigoid (BP) is an autoimmune bullous disease of the elderly. Patients' auto-antibodies are directed against BP180, a transmembrane hemidesmosomal glycoprotein of basal keratinocytes. Between 1989 and 1998, we identified 115 patients with BP in the Department of Dermatology at the University of Würzburg. This is one of the largest groups of patients with BP studied to date. In all patients, direct and/or indirect immunofluorescence microscopy studies were positive. The average age (+/- standard deviation) of the patients was 75 +/- 12 years, the median was 78 years. The youngest patient was 39 years of age; the oldest, 99 years. 54% of the patients were female and 46% male. In 24% of the patients, an involvement of oral, and in 7% of genital mucous membranes was seen. 98% of the patients complained about pruritus. By direct immunofluorescence microscopy, linear deposits of C3 and/or IgG were detected at the basement membrane zone of perilesional skin biopsies. By indirect immunofluorescence microscopy using NaCl-separated human skin, 87% of cases revealed circulating serum antibodies whereas on monkey esophagus, only 72% of sera were found positive. In 85% of the patients, we detected elevated serum levels of total IgE before the initiation of treatment. In those sera that were analyzed for reactivity with BP180, autoantibodies to the immunodominant NC16 A domain of BP180 were detected in 89% by immunoblot analysis and in 93% by ELISA. Therefore, in most patients, BP can be diagnosed serologically.

Aged↗
PubMed1999

Autoantibodies in lichen planus pemphigoides react with a novel epitope within the C-terminal NC16A domain of BP180.

Lichen planus pemphigoides is an autoimmune subepidermal blistering disease. The finding of immunoglobulin G antibodies directed against the basement membrane zone differentiates it from bullous lichen planus. The aim of this study was to identify the target antigen of lichen planus pemphigoides autoantibodies. Sera from lichen planus pemphigoides patients (n = 4) stained the epidermal side of NaCl-split human skin in a pattern indistinguishable from that produced by bullous pemphigoid sera. In bullous pemphigoid, the autoimmune response is directed against BP180, a hemidesmosomal transmembrane collagenous glycoprotein. We previously demonstrated that bullous pemphigoid sera predominantly react with a set of four epitopes (MCW-0 through MCW-3) clustered within a 45 amino acid stretch of the major noncollagenous extracellular domain (NC16A) of BP180. By immunoblotting and enzyme-linked immunosorbent assay, lichen planus pemphigoides sera were also strongly reactive with recombinant bullous pemphigoid 180 NC16A. The lichen planus pemphigoides epitopes were further mapped using a series of overlapping recombinant segments of the NC16A domain. All lichen planus pemphigoides sera reacted with amino acids 46-59 of domain NC16A, a protein segment that was previously shown to be unreactive with bullous pemphigoid sera. Two lichen planus pemphigoides sera, in addition, reacted with the immunodominant antigenic region associated with bullous pemphigoid. In conclusion, there are now five bullous diseases that are associated with an autoimmune response to BP180: bullous pemphigoid; pemphigoid/herpes gestationis; cicatricial pemphigoid; linear immunoglobulin A disease; and lichen planus pemphigoides. In addition, we have identified a novel epitope within the BP180 NC16A domain, designated MCW-4, that appears to be uniquely recognized by sera from patients with lichen planus pemphigoides.

Adult↗
PubMed1999

Autoantibodies in a subgroup of patients with linear IgA disease react with the NC16A domain of BP1801.

Linear IgA disease is an autoimmune subepidermal blistering disease characterized by IgA deposits at the cutaneous basement membrane zone. IgA antibodies from linear IgA disease sera react with antigens of 97 kDa (LABD97) and 120 kDa (LAD-1), both of which appear to be fragments of the extracellular domain of bullous pemphigoid 180 (type XVII collagen). The aim of this study was to determine whether linear IgA disease sera react with the immunodominant region of BP180 (NC16A domain), which is a major target of IgG autoantibodies produced by patients with bullous pemphigoid. Indeed, 11 of 50 linear IgA disease sera were found to contain IgA autoantibodies that recognized a recombinant form of NC16A by immunoblotting. The same sera also reacted with NC16A by enzyme-linked immunosorbent assay. An epitope mapping analysis uncovered four linear IgA disease-associated epitopes located within the 45 amino acid N-terminal stretch of NC16A, all of which were previously identified as antigenic sites targeted by bullous pemphigoid autoantibodies. Eight of the linear IgA disease sera that were reactive with NC16A also recognized LAD-1 secreted by the SCC-25 cell line, and five sera recognized BP180 extracted from keratinocytes. Linear IgA disease sera depleted of reactivity to NC16A by immunoadsorption continued to react with both the LAD-1 antigen and BP180 by immunoblotting and with the basement membrane zone by indirect immunofluorescence microscopy. Our results demonstrate that IgA autoantibodies from a subset of linear IgA disease patients react with the same sites on BP180 that are targeted by IgG autoantibodies in bullous pemphigoid.

Aged↗
PubMed1999

Reduced basal ganglia blood flow and volume in pre-symptomatic, gene-tested persons at-risk for Huntington's disease.

The aim of this study was to examine basal ganglia volumes and regional cerebral blood flow in asymptomatic subjects at-risk for Huntington's disease who had undergone genetic testing. We determined which measures were the best 'markers' for the presence of the mutation and for the onset of symptoms. Twenty subjects who were Huntington's disease gene mutation-positive and 24 Huntington's disease gene mutation-negative participants, all of whom had a parent with genetically confirmed Huntington's disease, and were therefore 50% at-risk for inheriting the Huntington's disease gene mutation, were included in the study. To evaluate basal ganglia structure and function, MRI and single photon emission computed tomography (SPECT) were used. Quantitative measures of regional volumes and relative measures of regional perfusion were calculated. SPECT and MRI scans were co-registered so that MRI anatomy could be used accurately to place SPECT regions. Estimated years-to-onset in the mutation-positive subjects was calculated based on a regression formula that included gene (CAG)(n) repeat length and parental age of onset. Changes in imaging measures in relation to estimated years-to-onset were assessed. The imaging measure that was most affected in mutation-positive subjects was putamen volume. This was also the measure that correlated most strongly with approaching onset. In subjects >/=7 years from estimated onset age, the putamen volume measures were similar to those of the mutation-negative subjects. However, in subjects </=6 years from estimated onset age, there were dramatic reductions in putamen volume, resulting in >90% discrimination from both the far-from-onset and the mutation-negative subjects. Caudate volume and bicaudate ratio also showed a significant decline in the close-to-onset subjects, although to a lesser degree than putamen volume reductions. Furthermore, SPECT basal ganglia perfusion deficits were observed in mutation-positive subjects. Imaging markers of neuropathological decline preceding clinical onset are important for assessing the effects of treatments aimed at slowing the course of Huntington's disease. The current study suggests that quantitative assessment of basal ganglia may provide a means to track early signs of decline in individuals with the Huntington's disease gene mutation prior to clinical onset.

Adult↗
PubMed1998

Developmental neurotoxicity of polychlorinated biphenyls (PCBS): cognitive and psychomotor functions in 7-month old children.

Neurobehavioral effects of polychlorinated biphenyls (PCBs) at environmental levels of exposure have been reported in cross-sectional and prospective studies in infants and children. However, observations differ for effect spectrum, persistence and effective matrix (cord plasma, maternal plasma or milk). In order to improve risk assessment by clarifying some of these uncertainties, a European multicentric study was set up. Results from the German (Düsseldorf) cohort covering 171 healthy mother-infant pairs are given. The sum of PCB congeners 138, 153 and 180 (sigma PCB) in cord plasma and maternal milk was used to describe neonatal PCB exposure. Mean sigma PCB-concentrations were 0.55 ng/ml in cord plasma and 427 ng/g fat in breastmilk. This report covers the Bayley II mental (MDI) and psychomotor development index (PDI) as well as the Fagan Test of Infant Intelligence (Visual Recognition Memory) taken at 7 months of age in relation to neonatal sigma PCB. After confounder-adjustment significant negative associations were found between sigma PCB in milk and MDI (P < 0.05), whereas the other associations proved insignificant.

Cognition↗
PubMed1998

[Beneficial effect of the ACE inhibitor captopril in normotensive patients with insulin-dependent diabetes].

The increase of glomerular filtration can often be observed in patients with insulin dependent diabetes mellitus, even in the early stage of the disease and it does not require the presence of microalbuminuria. This phenomenon can be explained by vasoconstriction occurring in the efferent arterioles. Eighteen normotensive, diabetic patients (aged: 28-42) who developed increased glomerular filtration were recruited in this study. The specific objectives were: 1. to study the beneficial effect of angiotensin converting enzyme inhibitor on the glomerular filtration, 2. to evaluate the effect of this treatment on blood pressure and hemodynamic parameters in normotensive, diabetic subjects. After a placebo period of one week, patients were treated orally a daily dose of 3 x 6.25 mg of captopril for twelve weeks. Glomerular filtration was assessed by the isotopic clearance method and blood pressure recordings were taken every 30 minutes throughout a day using an automatic programmable device. Preload, afterload and linear ejection fraction were estimated by echocardiograph, whereas cardiac index was measured by isotopic first pass technique. At the end of the treatment period a significant decrease of glomerular filtration was observed (from 141.9 +/- 10 ml/min to 98.9 +/- 12 ml/min; p < 0.01. Similarly, the afterload exhibited a significant drop due to drug treatment (45.6 +/- 5.8 x 10(3) dyn/cm2 vs. 55.4 +/- 4.7 x 10(3) dyn/cm2 at the end of the placebo period (p < 0.01). However, preload, linear ejection fraction, and cardiac index did not significantly change during the treatment. According to the results obtained from this study a beneficial effect of captopril on the early development of the glomerular hyperfiltration was demonstrated in normotensive diabetic patients who did not develop microalbuminuria. This issue needs to be investigated further in a large scale clinical trial.

Adult↗
PubMed1998

Biosynthesis of pteridines in Escherichia coli. Structural and mechanistic similarity of dihydroneopterin-triphosphate epimerase and dihydroneopterin aldolase.

An open reading frame located at 69.0 kilobases on the Escherichia coli chromosome was shown to code for dihydroneopterin aldolase, catalyzing the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin in the biosynthetic pathway of tetrahydrofolate. The gene was subsequently designated folB. The FolB protein shows 30% identity to the paralogous dihydroneopterin-triphosphate epimerase, which is specified by the folX gene located at 2427 kilobases on the E. coli chromosome. The folX and folB gene products were both expressed to high yield in recombinant E. coli strains, and the recombinant proteins were purified to homogeneity. Both enzymes form homo-octamers. Aldolase can use L-threo-dihydroneopterin and D-erythro-dihydroneopterin as substrates for the formation of 6-hydroxymethyldihydropterin, but it can also catalyze the epimerization of carbon 2' of dihydroneopterin and dihydromonapterin at appreciable velocity. Epimerase catalyzes the epimerization of carbon 2' in the triphosphates of dihydroneopterin and dihydromonapterin. However, the enzyme can also catalyze the cleavage of the position 6 side chain of several pteridine derivatives at a slow rate. Steady-state kinetic parameters are reported for the various enzyme-catalyzed reactions. We propose that the polarization of the 2'-hydroxy group of the substrate could serve as the initial reaction step for the aldolase as well as for the epimerase activity. A deletion mutant obtained by targeting the folX gene of E. coli has normal growth properties on complete medium as well as on minimal medium. Thus, the physiological role of the E. coli epimerase remains unknown. The open reading frame ygiG of Hemophilus influenzae specifies a protein with the catalytic properties of an aldolase. However, the genome of H. influenzae does not specify a dihydroneopterin-triphosphate epimerase.

Aldehyde-Lyases↗
PubMed1998

Deletion and site-directed mutagenesis of the Wolinella succinogenes fumarate reductase operon.

Wolinella succinogenes is known to grow at the expense of fumarate respiration with formate or sulfide as electron donor. A W. succinogenes mutant (delta frdCAB) lacking the fumarate reductase operon did not grow with fumarate as terminal electron acceptor and either formate or sulfide as electron donor. The delta frdCAB mutant grown with formate and nitrate did not contain fumarate reductase activity and did not catalyze electron transport from sulfide to fumarate, in contrast to the nitrate-grown wild-type strain. A mutant constructed by integration of frdCAB into the delta frdCAB mutant genome showed wild-type properties with respect to growth and enzyme activities. The frdC2 gene located downstream of the fumarate reductase operon frdCAB possibly encodes a diheme cytochrome b that is similar to FrdC (41% identical residues). The corresponding transcript differs from that of frdCAB. A delta frdC2 mutant showed wild-type properties with respect to growth and enzyme activities. Using site-directed mutagenesis, each of the four histidine residues that are predicted to serve as the axial heme ligands in FrdC (His44, His93, His143, and His182) was replaced by alanine or other residues. The resulting mutants did not grow with formate and fumarate and did not contain fumarate reductase activity, FrdA or FrdC when grown with formate and nitrate. In contrast, substitution of two histidine residues that are not considered heme ligands, yielded mutants (H114A and H120A) that grew with fumarate. It is concluded that FrdCAB is an obligatory component of fumarate respiration with formate and with sulfide in W. succinogenes. FrdC2 is not involved in fumarate respiration. Replacement of the putative heme ligands of FrdC prevents formation of a functional fumarate reductase.

Alanine↗