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Showing papers by "Andreas Pfeiffer published in 2001"


Journal ArticleDOI
01 Dec 2001-Diabetes
TL;DR: The data strongly support the concept that retinal angiogenesis is induced by loss of the majorAngiogenesis inhibitor in the eye, PEDF, in combination with an increased expression of angiogenic growth factors such as vascular endothelial growth factor.
Abstract: Retinal neovascularization characterizes proliferative diabetic retinopathy (PDR). Pigment epithelium-derived factor (PEDF) has been shown to be a major antiangiogenic growth factor in the mammalian eye. PEDF expression is suppressed by hypoxia, and changes in PEDF have been correlated to the development of retinal neovascularization in animal models of hypoxic eye disease. However, whether this concept of a reduced angiogenesis inhibitor holds true in humans is as yet unclear. In this study, we analyzed the in vivo regulation of PEDF in patients with and without hypoxic eye disease. We used immunoblots to measure PEDF in ocular fluids obtained from 64 nondiabetic and diabetic patients. In addition, immunohistochemistry of PEDF was carried out in specimens of normal human retinas and retinas with various degrees of diabetic retinopathy. The PEDF concentrations in patients with PDR (P < 0.001) or extensive nondiabetic retinal neovascularization caused by retinal-vein occlusion (P < 0.001) were lower than in control patients. Levels of PEDF were replenished in PDR patients with previous retinal scatter photocoagulation compared with PDR patients without previous photocoagulation (P = 0.01). Immunohistochemistry revealed an interstitial staining pattern as expected for a secreted protein, with an intense staining in retinas of patients without proliferative eye disease. However, in patients with PDR, little or no staining was detectable. Our data strongly support the concept that retinal angiogenesis is induced by loss of the major angiogenesis inhibitor in the eye, PEDF, in combination with an increased expression of angiogenic growth factors such as vascular endothelial growth factor. Our findings suggest that substitution of angiogenesis inhibitors may be an effective approach in the treatment of PDR.

252 citations


Journal ArticleDOI
TL;DR: It is found that short-term hyperglycaemia activates PKC α, β1 and β2 in platelets of healthy persons making them potential candidates for mediating the increased cardiovascular risk of postprandial hyperglyCAemia.
Abstract: Aims/hypothesis Postprandial hyperglycaemia carries an increased risk of macrovascular disease even without Type II (non-insulin-dependent) diabetes mellitus Chronic hyperglycaemia activates protein kinase C (PKC) in vitro and in vivo but it is not known whether PKC is regulated by short-term postprandial hyperglycaemia in vivo in humans We investigated whether PKC is regulated in vivo in hyperglycaemic and hyperinsulinaemic infusion tests and correlated the results to stimulations in vitro Methods Protein kinase C regulation was measured in platelets obtained from 8 healthy subjects who were infused with glucose and insulin for 2 h attaining peak concentrations of 16 mmol/l glucose and in platelets from 8 healthy young subjects, 8 older subjects without diabetes, and 10 older subjects with Type II diabetes after incubation in vitro with 16 mmol/l glucose or glucose and insulin For precise quantification, a shortened PKC β1 standard protein was generated by bacterial expression and PKC α, β1, β2 and δ isoenzyme values were measured by immunoblot analyses Results Hyperglycaemic and hyperinsulinaemic in vivo tests increased the amounts of PKC α, β1 and β2 in the membrane fraction of platelets to 225 ± 87 %, 164 ± 22 % and 302 ± 135 %, respectively, when compared with the baseline values in young healthy volunteers (n = 8, p < 005) The expression of PKC δ did not change In comparison to the recombinant PKC β1 standard protein, 5 ng PKC β1/μg protein was measured before the test and 2 ng/μg were translocated to the membrane fraction after the infusion No change in the absolute amount of PKC β1 was detected In contrast, after incubation in vitro PKC was not regulated by glucose or glucose and insulin in 8 young healthy subjects (age 26 ± 07 years) and in 8 older, healthy subjects (age 64,8 ± 4 years) although 100 nmol/l 12-O-tetradecanoylphorbol 13-acetate caused maximal activation In marked contrast, PKC β1 and PKC β2, but not PKC α or PKC δ, were increased in vitro in the membrane fraction by 292 ± 61 % and 432 ± 88 % (p < 005) in 10 subjects with Type II diabetes mellitus matched for age, sex and BMI Conclusion/interpretation We found that short-term hyperglycaemia activates PKC α, β1 and β2 in platelets of healthy persons making them potential candidates for mediating the increased cardiovascular risk of postprandial hyperglycaemia Hyperglycaemia and hyperinsulinaemia did not cause short-term activation of PKC in platelets in vitro suggesting the existence of additional stimuli Subjects with Type II diabetes showed a markedly altered reactivity of platelet PKC β in vitro indicating some diabetes-related regulation [Diabetologia (2001) 44: 188–195]

138 citations


Journal ArticleDOI
TL;DR: The aim of this review will be to briefly summarize the current knowledge regarding the clinical and laboratory findings of diabetic retinopathy, and to describe current concepts regarding the pathogenesis and treatment of diabetic Retinopathy.
Abstract: Retinal neovascularization is a major feature of proliferative diabetic retinopathy, which represents a major public health problem, being responsible for more irreversible blindness in persons of middle and older age than any other pathology. The societal burden of ocular neovascularization has prompted extensive research into its mechanisms. The aim of this review will be to briefly summarize the current knowledge regarding the clinical and laboratory findings of diabetic retinopathy. From an investigational view, studies of ocular neovascularization provide important informations, often permitting real-time, serial observations of neovascularization in vivo. This allows investigators to analyse the relevance of specific pathogenic concepts regarding the mechanisms of angiogenesis in vivo. This review will additionally describe current concepts regarding the pathogenesis and treatment of diabetic retinopathy.

78 citations


Journal ArticleDOI
TL;DR: Different patterns' of expression of the TGFbeta isoforms under physiological and pathological conditions in bone are suggested, indicating in part the regulation by mechanical stimuli.
Abstract: Transforming growth factor beta is one of the most abundant growth factors stored in bone. It is known as a potent regulator of osteoblast proliferation and differentiation as well as of production extracellular matrix. We established a highly specific RT-PCR in combination with HPLC for detection and quantification of TGFbeta1 and TGFbeta2 mRNA expression in 89 human bone samples. Levels of TGFbeta1 protein ranged between 27 and 580 ng/g bone (mean 188 +/- 15 ng/g; n=75) and for TGFbeta2 between 7.2 and 35 ng/g bone (mean 14.3 +/- 2.1 ng/g; n=57). TGFbeta1 and TGFbeta2 protein concentrations and TGFbeta isoform mRNA expression in bone were not significantly different between the sexes. TGFbeta isoform mRNA expression as well as protein content in bone declined age dependently. TGFbeta1 and TGFbeta2 protein and mRNA expression were different in bone samples from different sites of the skeleton indicating in part the regulation by mechanical stimuli. In contrast to TGFbeta1, TGFbeta2 mRNA expression was significantly enhanced in osteoarthritic bone compared to unaffected bone. These data are in concordance to previous results concerning the expression of TGFbeta3 in bone. In conclusion, the data suggest distinct patterns' of expression of the TGFbeta isoforms under physiological and pathological conditions in bone.

69 citations


Journal ArticleDOI
TL;DR: A combined treatment with retinal photocoagulation and growth hormone-lowering drugs, such as somatostatin analogues, could be a useful treatment, which may prevent further loss of visual acuity in patients with PDR.
Abstract: Retinal photocoagulation reduces the incidence of severe visual loss in proliferative diabetic retinopathy (PDR). Reduced levels of VEGF/VPF might result in an improved function of the blood-retina barrier and cause a decrease of blood derived intraocular growth factors such as IGF-I. This study investigates whether retinal photocoagulation is able to normalize the concentrations of IGF-I, IGF-II and IGF-BP3 in the vitreous humor of patients undergoing vitrectomy. Levels of IGFs and the permeability marker, albumin, were measured in serum and vitreous of 52 patients. Three groups were compared: controls without proliferating eye disease (n = 19) and patients with PDR with (PDR+; n = 25) and without (PDR-; n = 8) previous retinal photocoagulation. IGF-I, IGF-II, IGF-BP3 and albumin were determined by immunological methods and were confirmed to be increased in patients with PDR compared to controls. Retinal photocoagulation influenced neither the intraocular concentration of the permeability marker albumin (PDR+: 253.2 +/- 46 mg/dl; PDR-: 256.4 +/- 66.5 mg/dl) nor the levels of IGFs (PDR+: IGF-I: 1.2 +/- 0.1 ng/ml; p = 0.38; IGF-II: 34.8 +/- 2.2 ng/ml; p = 0.1; IGF-BP3: 75.7 +/- 9.7 ng/ml; p = 0.27; PDR-: IGF-I: 1.1 +/- 0.2ng/ml; IGF-II: 29.3 +/- 5.2 ng/ml; IGF-BP3: 61.5 +/- 18.3 ng/ml). Systemic levels of albumin and IGFs were not changed significantly by retinal photocoagulation. These results demonstrate that previous retinal photocoagulation in patients undergoing vitrectomy does not functionally reestablish the blood-retina barrier despite decreases in VEGF/VPF. The lack of influence on intraocular concentrations of the serum-derived growth factors, IGF-I, IGF-II and IGF-BP3, might in part explain the failure of previous photocoagulation in the investigated patients. These results suggest that a combined treatment with retinal photocoagulation and growth hormone-lowering drugs, such as somatostatin analogues, could be a useful treatment, which may prevent further loss of visual acuity in patients with PDR.

28 citations


Journal ArticleDOI
TL;DR: It is concluded that TGF beta3 in man circulates in significant amounts which appears to be representative for TGFbeta3 expression in bone tissue and may be in part derived from bone.
Abstract: Recent data indicate that TGFbeta3, one member of the TGFbeta-isoforms, has an important role in bone remodeling. Up to date little is known about the expression and regulation of TGFbeta3 in man. We established a highly specific ELISA for quantitative measurement of TGFbeta3 in bone and blood samples and a RT-PCR in combination with HPLC for detection and quantification of TGFbeta3 mRNA in 89 human bone samples. Levels of TGFbeta3 protein ranged between 30 and 66 pg/mg bone (mean 36,6 +/-1,03 pg/mg) and between 30 and 1910 pg/ml in serum (mean 128.9+/-38.9 pg/ml). TGFbeta3 mRNA expression as well as protein levels in serum and in bone declined age dependently. No specific load- or site-specific distribution of TGFbeta3 mRNA expression or protein content was detected at different sites indicating an absence of mechanical regulation. Protein levels of TGFbeta3 in serum correlated with TGFbeta3 mRNA expression in bone (p= 0.0027; r=0.49). By contrast, TGFbeta3 protein levels stored in the bone matrix were not related to TGFbeta3 mRNA reflecting the long term process of TGFbeta3 deposition during bone remodeling. Notably TGFbeta3 serum levels were highly correlated with IGF-I and osteocalcin levels in serum. We conclude that TGFbeta3 in man circulates in significant amounts which appears to be representative for TGFbeta3 expression in bone tissue and may be in part derived from bone. The high correlation of TGFbeta3 with IGF-I suggests parallel systemic principles of regulation.

12 citations


Journal ArticleDOI
TL;DR: The data presented suggest that amphiregulin, heparin binding EGF and TGF-alpha are important EGF receptor ligands in the gastric mucosa.
Abstract: BACKGROUND Epidermal growth factor (EGF) and TGF-alpha play a central role in maintaining gastric mucosal integrity. Little is known about the regulative role of the four other widely expressed epidermal growth factor receptor ligands, heparin-binding EGF, amphiregulin, betacellulin and cripto in the gastric mucosa. METHODS Nineteen patients with Helicobacter pylori-positive gastritis and 32 healthy controls were investigated. Mucosal mRNA expression of EGF receptor ligands was determined by quantitative PCR before and after H. pylori eradication. PCR products were analyzed by soft laser scanning densitometry. Moreover, the effect of chronic active gastritis on EGF receptor expression was assessed by [125I] EGF receptor autoradiography. Immunohistochemistry was performed for TGF-alpha to localize growth factor expression. RESULTS Antral and oxyntic biopsies showed strong mRNA expressions for TGF-alpha, amphiregulin and heparin binding EGF, but not for EGF, cripto and betacellulin. mRNA expression was significantly reduced down to 50% in H. pylori infection, significantly lower compared to normal gastric mucosa, and increased after eradication therapy. Moreover, chronic gastritis was associated with decreased antral EGF receptor binding compared to healthy controls, possibly reflecting reduced autoinduction. Immunohistochemical analyses localized TGF-alpha in the cytoplasma of gastric epithelial cells and revealed its increased expression after H. pylori eradication. CONCLUSIONS The data presented suggest that amphiregulin, heparin binding EGF and TGF-alpha are important EGF receptor ligands in the gastric mucosa. H. pylori infection apparently suppresses their mRNA as well as receptor expression that is reversed by H. pylori eradication. This deficiency of the gastroprotective EGF system may contribute to the gastric pathogenicity of H. pylori infection.

10 citations


Journal ArticleDOI
TL;DR: The determination of crosslinks, bone type I collagen degradation products, correctly identifies osteoporotic subjects was investigated and the method described by Eastell-Melton was evaluated.
Abstract: Hintergrund: In der Diagnostik der Osteoporose werden biochemische Knochenstoffwechselparameter, insbesondere die sog. Pyridinolin- und Desoxypyridinolin-Crosslinks, Abbauprodukte des knochenspezifischen Typ-I-Kollagens, zunemend als Knochenresorptionsparameter verwandt. Patienten und Methoden: In einer Gruppe von 370 willkurlich ausgesuchten Probanden wurde untersucht, ob sich durch die Bestimmung der Crosslinks Patienten mit Osteoporose identifizieren lassen. Dafur wurden im Rahmen der Europaischen Vertebralen-Osteoporose-Studie (EVOS) Urin-Crosslinks mit einer selbst etablierten HPLC-Methode quantifiziert. Jeder Proband wurde standardisierten Rontgenaufnahmen der Brust- und Lendenwirbelsaule unterzogen, die morphometrisch nach Eastell-Melton ausgewertet wurden. Ergebnisse: Im Vergleich zu den ubrigen Probanden wiesen die weiblichen Personen (n = 165) mit einer Osteoporose (Eastell-Grad 2) signifikant erhohte Crosslink-Spiegel auf (Pyridinolin 102,0 vs. 59,4 nmol/mmol Kreatinin, p = 0,04; Desoxypyridinolin 70,6 vs. 32,3 nmol/mmol Kreatinin, p = 0,01), bei den mannlichen Probanden (n = 205) wurde das Signifikanzniveau bei vergleichbaren Mittelwerten, aber hoher Streuung verfehlt. Die diagnostische Sensitivitat betrug dabei 32,4–42,9% bei einer Spezifitat von 76–81%. Schlussfolgerung: In dem von uns untersuchten Kollektiv sind die Urin-Crosslinks erst bei hochgradigen Wirbeldeformationen signifikant erhoht. Dabei weisen Pyridinolin und Desoxypyridinolin eine relativ hohe Spezifiat mit allerdings geringer Sensitivitat auf. Aufgrund der geringeren Sensitivitat sind Urin-Crosslinks weniger als Screening-Test fur das Vorliegen einer Osteoporose, sondern eher als Bestatigungstest fur das Ausmas osteoporotischer Veranderungen geeignet.

5 citations


Journal ArticleDOI
TL;DR: Determination of insulin allowed better characterization of irregular pulses because of the shorter half-life of insulin relative to C-peptide and the new modification of sequential eu- and hypoglycaemic clamp procedures should also be useful in pharmacological studies of insulinotropic substances.
Abstract: SUMMARY Characterization of metabolically inadequate insulin secretion is essential for insulinoma diagnostics. Hyperinsulinaemic, eu- and hypoglycaemic clamp procedures have been used to suppress endogenous insulin secretion in healthy subjects. The use of exogenous insulin precluded the use of insulin as a parameter to be measured. We now suggest to use exogenous insulin lispro and an insulin-specific ELISA not cross reacting with insulin lispro. Thus, determination of insulin by ELISA in this experimental setting reflects endogenous insulin. A 39-year-old man with a surgically confirmed pancreatic insulinoma was studied under hyperinsulinaemic [lispro insulin 40 mU x m(-2) body surface x min(-1)] clamp conditions. Euglycaemia was achieved (3.8 +/- 0.5 mmol/L) for 1 h and hypoglycaemia (2.36 +/- 0.49 mmol/L) was achieved for another 30 min. Insulin was evaluated by ELISA (cross-reaction with lispro insulin < 0.006%, C-peptide < 0.01%, proinsulin < 0.001%) and by a nonselective RIA (cross-reaction with proinsulin 40%). In control subjects the euglycaemic hyperinsulinaemia suppressed C-peptide to 0.36 +/- 0.03 ng/ml and hypoglycaemic hyperinsulinaemia to 0.29 +/- 0.03 ng/ml. Endogenous insulin was suppressed to 2.8 +/- 0.03 mU/L under euglycaemia and to 2.6 +/- 0.03 mU/L under hypoglycaemia in control subjects. In the insulinoma patient apparently irregular but small changes in both C-peptide (1.43 +/- 0.1 ng/ml) and more pronounced changes in endogenous insulin concentrations 4.41 +/- 0.1 mU/l under euglycaemia and 5.35 +/- 0.3 mU/l under hypoglycaemic conditions, were observed. The basal level of insulin (ELISA insulin 4.6 mU/L) and C-peptide (1.7 ng/ml) were not markedly elevated. Determination of insulin allowed better characterization of irregular pulses because of the shorter half-life of insulin relative to C-peptide. The new modification of sequential eu- and hypoglycaemic clamp procedures should also be useful in pharmacological studies of insulinotropic substances. Direct measurement of peripheral insulin may be more sensitive than C-peptide to detect low levels of autonomous insulin secretion in small insulinomas.

3 citations


Journal ArticleDOI
TL;DR: In this article, ausammenfassung Endokrine und metabolische Einflusse steuern die ubergeordnete Verfassund des Patienten bezuglich des Knochenstoffwechsels and bilden den Hintergrund für die Heilung von Knochenslasionen.
Abstract: Zusammenfassung Endokrine und metabolische Einflusse steuern die ubergeordnete Verfassung des Patienten bezuglich des Knochenstoffwechsels und bilden den Hintergrund fur die Heilung von Knochenlasionen. Parathormon, Vitamin D und Kalzitonin bestimmen die Resorption und die Ausscheidung von Kalzium und damit die fur den Einbau in Knochen zur Verfugung stehende freie Kalziummenge. Systemische Hormone wie Ostrogen, Wachstumshormon, Somatomedin C und Insulin-like-growth-Faktor steuern gemeinsam mit Vitamin D und Parathormon sowie Kalzitonin sowohl Knochenab- als auch -aufbauvorgange. Der Knochenabbau wird durch die Osteoklastenbildung aus Makrophagen unter Zytokin- und systemischem Hormoneinfluss gesteuert, wobei lokal gebildete Wachstumsfaktoren wie Transforming-growth-Faktor bl–3 sowie die BMP (bone morphogenetic proteins) wesentlich beteiligt sind. TGFb hemmt die Osteoklastenfunktion und rekrutiert mesenchymale Vorlauferzellen zu Osteoblasten. Die direkte Verwendung von Wachstumsfaktoren wird in der zukunftigen Therapie zunehmend bedeutsam werden. Endokrine Einflusse lassen sich durch eine positive Kalziumbilanz gunstig gestalten ¶(Vitamin-D- und Kalziumgabe), wahrend eine zukunftige Rolle einer Behandlung mit Parathormon oder Wachstumshormon noch zu evaluieren bleibt.

2 citations


Journal ArticleDOI
TL;DR: The Single Strand Conformation Polymorphism (SSCP) analysis is one of the most commonly applied methods in detection of point mutations as discussed by the authors, and it is used for point mutation detection in clinical medicine.
Abstract: BACKGROUND Molecular biological techniques of mutation detection become more and more a focus of interest in clinical medicine because the responsible genes are now known for an increasing number of diseases and the detection is essential to enable reliable predictions and to design individual therapies. Therefore the SSCP (Single Strand Conformation Polymorphism) analysis is one of the most commonly applied methods in detection of point mutations. SSCP ANALYSIS The use of SSCP for detection of the pathogenic mutations of MODY (Maturity Onset Diabetes in the Young) is described as an example. Handling, efficiency and costs are critically analyzed. In a blind test, this method was only partly suitable for reliable analysis. Particularly diseases without a distinct hotspot and with a widespread distribution pattern of mutations throughout the coding sequence require sequencing as the gold standard.