Ultrasound-assisted microsurgery for Cushing's disease.

OBJECTIVE: Localization of microadenomas in Cushing's disease may be difficult as in up to 45% of patients sellar MRI fails to detect a pituitary tumor. Intraoperative transsphenoidal ultrasound may identify microadenomas as hyperechoic structures. We report on the first 18 consecutive cases with intraoperative use of a new device for direct contact high-frequency-ultrasound in patients with Cushing's disease. PATIENTS AND TECHNIQUE: 18 patients (14 female, 4 male, age 24-71 years) with typical endocrinological findings for Cushing's disease were included in the study. One macroadenoma and 13 microadenomas were suspected or identified preoperatively by MRI. In 4 cases, two of them with recurrent disease, sellar MRIs were negative. During transsphenoidal microsurgery an end fire ultrasound-probe (B-mode frequency range 7.5-13 Mhz, field of view 5 mm, penetration 20 mm) was introduced after opening of sellar floor. The pituitary gland was scanned in direct contact to the capsule. RESULTS: In 13 out of 17 cases (77%) with later on proven microadenomas high-frequency-ultrasound identified the tumors as hyperechoic masses, including 3 of the 4 cases with negative preoperative MRI. In 2 cases ultrasound correctly localized the tumor at a site different from MRI finding (MRI false positive). In the macroadenoma, identification of the border between tumor and anterior pituitary gland was not possible. In all 18 patients postoperative early decline of serum cortisol to subnormal levels confirmed remission of hypercortisolism (100%). Other pituitary functions were unaltered in 17 cases (94%). CONCLUSIONS: Intraoperative scanning of the pituitary gland with high-frequency-ultrasound probes may identify intrapituitary anatomy and pathologies even in MRI-negative cases. This may prevent extensive exploration of the gland with the risk of subsequent hypopituitarism.

Mutational analysis of the PHEX gene: novel point mutations and detection of large deletions by MLPA in patients with X-linked hypophosphatemic rickets.

X-Linked hypophosphatemic rickets (HYP, XLH) is a disorder of phosphate homeostasis, characterized by renal phosphate wasting and hypophosphatemia, with normal to low 1,25-dihydroxy vitamin D3 serum levels. The purpose of our study was the detection of inactivating mutations in the PHEX gene, the key enzyme in the pathogenesis of XLH. The 16 patients, representing eight families, presented with suspected XLH from biochemical and clinical evidence. All 16 were referred for mutational analysis of the PHEX gene. We detected three novel disease-causing mutations, C59S, Q394X, and W602, for which a loss of function can be predicted. A G28S variation, found in two healthy probands, may be a rare polymorphism. Another mutation, A363 V, is localized on the same allele as the C59S mutation, thus its functional consequences cannot be proven. Furthermore, we detected a deletion of three nucleotides in exon 15 which resulted in the loss of amino acid threonine 535. Heterozygosity of this mutation in a male patient without any chromosomal aberrations suggests its presence as a mosaic. Novel large deletions were detected using multiplex ligation-dependent probe amplification (MLPA) analysis. Two of these deletions, loss of exon 22 alone or exons 21 and 22 together, may result in the translation of a C-terminal truncated protein. Two large deletions comprise exons 1-9 and exons 4-20, respectively, and presumably result in a nonfunctional protein. We conclude that molecular genetic analysis confirms the clinical diagnosis of XLH and should include sequence analysis as well as the search for large deletions, which is facilitated by MLPA.

Developing effective screening strategies in multiple endocrine neoplasia type 1 (MEN 1) on the basis of clinical and sequencing data of German patients with MEN 1.

BACKGROUND: Multiple-endocrine-neoplasia-type-1 (MEN1) is an autosomal-dominant inherited disorder characterized by the combined occurrence of primary hyperparathyroidism (pHPT), gastroenteropancreatic neuroendocrine tumors (GEP), adenomas of the pituitary gland (APA), adrenal cortical tumors (ADR) and other tumors. As the tumors appear in an unpredictable schedule, uncertainty about screening programs is persisting. OBJECTIVE: To optimize screening and to analyze possible differences in sporadic versus familial cases. METHODS: We analyzed data of 419 individuals including 306 MEN-1 patients (138 isolated and168 familial cases out of 102 unrelated families). RESULTS: A total of 683 tumors occurred consisting of 273 pHPT, 138 APA, 166 GEP, 57 ADR, 24 thymic- and bronchial-carcinoids as well as 25 neoplasms of other tissues. The age-related penetrance was determined as 10%, 35%, 67%, 81% and 100% at 20, 30, 40, 50 and 65 years respectively. Although pHPT being the most frequent first manifestation (41%), also GEP (22%) or APA (21%) were found to be the first presentation. APA occurred significantly more frequent (p<0,05) in isolated (n=138) than in familial (n=168) cases, whereas GEP showed a tendency to occur more often in familial cases. Genotype/phenotype correlation in 140 clinically affected MEN-1 cases showed a tendency for truncating mutations, especially nonsense mutations to be associated to GEP and carcinoids of the lungs and thymus. CONCLUSION: In view of the morbidity and frequency in familial cases an effective screening programme should aim at an early diagnosis of GEP particularly when truncating, especially nonsense mutations are found.

Pilot study with technosphere/PTH(1-34)--a new approach for effective pulmonary delivery of parathyroid hormone (1-34).

Sequential subcutaneous PTH injection therapy (repeated 14 days of PTH administration and a subsequent treatment pause for a few weeks) is known to increase bone mineral density in patients with osteopenic disorders. Alternative methods of drug delivery may be beneficial in increasing compliance. A pilot study was performed in 10 healthy volunteers (4 female/6-male, age: 25.6 +/- 3.5 years, BMI: 22.3 +/- 2.4 kg/m 2, mean +/- SD) to assess the pharmacokinetic profiles of 1600 IU of PTH(1 - 34) using the pulmonary Technosphere drug delivery system in comparison to a subcutaneous injection of 400 IU. The treatments were administered in the morning after an overnight fast and blood samples for measurement of PTH(1 - 34), PTH(1 - 84), and calcium and calcitonin were taken over a period of 6 hours. Both injection and pulmonary application of PTH(1 - 34) were well tolerated. After pulmonary administration of Technosphere/PTH(1 - 34), PTH(1 - 34) appeared in the serum with a faster concentration increase (T max: pulmonary 10 +/- 5 min vs. subcutaneous 28 +/- 8 min, p < 0.001) and with higher maximal concentrations (C max : pulmonary 309 +/- 215 pmol/l vs. subcutaneous 102 +/- 45 pmol/l, p < 0.05) as compared to the subcutaneous injection. The relative bioavailability of pulmonary Technosphere/PTH(1 - 34) was calculated to be 48 %. No differences were seen between pulmonary and subcutaneous application with regard to the PTH(1 - 84), calcitonin and calcium concentrations. In conclusion, pulmonary application of Technosphere/PTH(1 - 34) appears to be an effective and thus attractive candidate for PTH substitution therapy in osteoporosis and other conditions leading to a decrease in bone mineral density.

The influence of local capsaicin treatment on small nerve fibre function and neurovascular control in symptomatic diabetic neuropathy.

Topical treatment with capsaicin cream has been shown to be successful in the treatment of different symptomatic nerve disorders like diabetic neuropathy. Conflicting data exist on the effect of capsaicin on nerve function and neurovascular control especially in patients with diabetic neuropathy. The aim of this pilot study was to investigate the impact of topical capsaicin application on small nerve fibre function and neurovascular control. Capsaicin cream was applied to the feet of 13 patients with symptomatic diabetic neuropathy over a period of 8 weeks. Before and during the treatment period, we investigated the total symptoms score, the vibration, thermal (heat and cold) and pain perception thresholds, and the neurovascular responses to heat and acetylcholine stimuli. In addition, the serum plasma levels of substance P, a neurotransmitter of nociceptor C-fibres, were measured. A significant improvement in total symptoms score was observed during topical capsaicin treatment (18.3+/-3.2 vs. 14.3+/-3.3; p<0.05). An improvement in the heat perception threshold was also found (12.7+/-0.4 degrees C vs. 11.4+/-0.7 degrees C: p<0.05), while other sensory nerve fibre functions remained unchanged. No significant change in neurovascular control was observed, neither after mild thermal injury nor after stimulation with acetylcholine. Serum substance P levels increased after initiation of topical capsaicin treatment (72.9+/-5.8 pg/ml vs. 81.7+/-5.0 pg/ml; p<0.05), but returned to baseline levels during further treatment (77.4+/-8.3 pg/ml: n.s.). In conclusion, topical treatment with capsaicin cream over a period of 8 weeks in patients with symptomatic diabetic neuropathy is effective without adverse effects on nerve fibre function or neurovascular control.

Abnormal pentagastrin response in a patient with pseudohypoparathyroidism.

The case of a 25 year old female patient with pseudohypoparathyroidism type I (PHP) and hypercalcitoninaemia is reported. She was referred to our clinic because of recurrent hypocalcaemia associated with paraesthesias and muscle cramps. She had no signs of Albright hereditary osteodystrophy (AHO), a normal mental status and no family history of hypocalcaemia or any other endocrine disease. Considering the laboratory results with hypocalcaemia, hyperphosphataemia, normal vitamin D and normal creatinine with an extraordinary elevated PTH we diagnosed pseudohypoparathyroidism type I. She had delayed pubertal development with menarche in the age of 20 and hypothyroidism with an atrophic thyroid since she was 22 years old. Calcitonin (CT) was increased and the performed pentagastrin test showed an excessive CT-response with a peak of 725 pg/ml after 2 min. Up to now there are only three reports of patients with PHP and hypercalcitoninaemia. An abnormal pentagastrin response is known to be a specific marker for medullary thyroid carcinoma, but there were no signs of any malignant disease, even after one year of follow-up. The most reasonable cause for the pathological pentagastrin response might be chronic hypocalcaemia. When interpreting a pathological pentagastrin test in a patient with PHP the specifity of the test might be diminished and a careful observational strategy might be appropriate.

Preliminary data on biodistribution and dosimetry for therapy planning of somatostatin receptor positive tumours: comparison of (86)Y-DOTATOC and (111)In-DTPA-octreotide.

The somatostatin analogue (90)Y-DOTATOC (yttrium-90 DOTA- D-Phe(1)-Tyr(3)-octreotide) is used for treatment of patients with neuroendocrine tumours. Accurate pretherapeutic dosimetry would allow for individual planning of the optimal therapeutic strategy. In this study, the biodistribution and resulting dosimetric calculation for therapeutic exposure of critical organs and tumour masses based on the positron emission tomography (PET) tracer (86)Y-DOTATOC, which is chemically identical to the therapeutic agent, were compared with results based on the tracer commonly used for somatostatin receptor scintigraphy, (111)In-DTPA-octreotide (indium-111 DTPA- D-Phe(1)-octreotide, OctreoScan). Three patients with metastatic carcinoid tumours were investigated. Dynamic and static PET studies with 77-186 MBq (86)Y-DOTATOC were performed up to 48 h after injection. Serum and urinary activity were measured simultaneously. Within 1 week, but not sooner than 5 days, patients were re-investigated by conventional scintigraphy with (111)In-DTPA-octreotide (110-187 MBq) using an equivalent protocol. Based on the regional tissue uptake kinetics, residence times were calculated and doses for potential therapy with (90)Y-DOTATOC were estimated. Serum kinetics and urinary excretion of both tracers showed no relevant differences. Estimated liver doses were similar for both tracers. Dose estimation for organs with the highest level of radiation exposure, the kidneys and spleen, showed differences of 10.5%-20.1% depending on the tracer. The largest discrepancies in dose estimation, ranging from 23.1% to 85.9%, were found in tumour masses. Furthermore, there was a wide inter-subject variability in the organ kinetics. Residence times (tau(organs)) for (90)Y-DOTATOC therapy were: tau(liver) 1.59-2.79 h; tau(spleen) 0.07-1.68 h; and tau(kidneys) 0.55-2.46 h (based on (86)Y-DOTATOC). These data suggest that dosimetry based on (86)Y-DOTATOC and (111)In-DTPA-octreotide yields similar organ doses, whereas there are relevant differences in estimated tumour doses. Individual pretherapeutic dosimetry for (90)Y-DOTATOC therapy appears necessary considering the large differences in organ doses between individual patients. If possible, the dosimetry should be performed with the chemically identical tracer (86)Y-DOTATOC.

[Drug therapy of carcinoids of the gastrointestinal tract]. / Medikamentöse Therapie bei Karzinoiden des Gastrointestinaltraktes.

Carcinoid tumors are rare and slowly growing neuroendocrine tumors of the foregut, midgut and hindgut. Drug therapy is of special importance in patients with inoperable metastasising disease. This palliative therapy is aimed at reduction of the hormone-dependent symptoms and inhibition of tumor growth. Somatostatin analogues, alpha-interferon and various chemotherapeutic agents are used for this purpose. Drug therapy can be supplemented by surgical and radiological intervention through interdisciplinary cooperation of the surgeon, radiologist, endocrinologist and gastroenterologist.

Endosonography of insulin-secreting and clinically non-functioning neuroendocrine tumors of the pancreas: criteria for benignancy and malignancy.

OBJECTIVE: Endosonography is a powerful tool in the diagnosis of gastroenteropancreatic neuroendocrine tumors. This study was performed in order to characterize endosonographic criteria of malignant and benign neuroendocrine pancreatic tumors focussing on those typically presented to endocrinologists, i.e. insulin-secreting tumors and clinically non-functioning tumors in MEN-1. DESIGN: We studied six benign insulinomas, four hormone inactive benign neuroendocrine adenomas in MEN-1, and three non-metastatic neuroendocrine carcinomas with clinically symptomatic insulin secretion. METHODS: Endosonography was performed using Pentax FG 32 UA endosonoscope with a longitudinal 7.5 MHz sector array. RESULTS: Tumor diameter was larger in malignant tumors (19 - 70 / 47.0 +/- 25.9 mm) than in benign lesions (2.3 - 19 / 9.7 +/- 5.8 mm). Hypoechoic echogeneity was more or less present in benign and in malignant tumors and could not be used as a criteria for differential diagnosis. Heterogeneous or multinodular structure on endosonographic imaging however, was an exclusive feature of malignant tumors. Echo-free areas representing cystic transformation or necrosis and vascular invasion were additional signs of malignancy. CONCLUSIONS: Molecular genetic diagnosis of MEN-1 and new therapeutic developments such as endoscopic surgery make sufficient imaging procedures in the management of neuroendocrine pancreatic tumors mandatory. Besides valid detection and exact localization, endosonography provides criteria for benign and malignant tumors and thus may be helpful in planning therapeutic strategies.

The influence of isolated small nerve fibre dysfunction on microvascular control in patients with diabetes mellitus.

AIM: The aim of the study was to investigate the influence of isolated small nerve fibre dysfunction on microvascular skin blood flow and transcutaneous oxygen tension in patients with diabetes mellitus. METHODS: Small nerve fibre dysfunction was assessed by the measurement of thermal and pain perception thresholds. Patients with evidence of large fibre disturbances as evaluated by means of vibration perception threshold were excluded from the study. Microvascular blood flow was investigated with laser-Doppler-fluxmetry (LDF) following stimulation with acetylcholine and mild thermal injury. RESULTS: Diabetic patients with small nerve fibre injury showed a significantly reduced increase in the laser-Doppler-flux signal following the application of acetylcholine compared with patients without neuropathy or healthy control subjects (2.8 arbitrary units (AU) (1.3-5.5) vs. 7.2 AU (4.1-25.5); P = 0.007 and vs. 8.5 AU (3.0-17.0), P = 0.02, respectively). The increase in LDF following thermal injury was also diminished in patients with small nerve fibre dysfunction compared with patients without neuropathy or the control group (29.8 AU (17.2-46.5) vs. 51.2 AU (29.5-93.5); P = 0.02 and vs. 54.6 AU (39.7-97.7); P = 0.004, respectively). In addition, they showed a significantly reduced transcutaneous oxygen tension compared with the other groups (42.9 mmHg (41.6-55.5) vs. 56.1 mmHg (49.2-60.8); P = 0.04 and vs. 59.0 mmHg (54.6-80.3), P = 0.03, respectively). CONCLUSIONS: Our study confirms an association between small nerve fibre injury and skin microvascular dysfunction. It further underlines the concept of neurovascular disturbances in the pathogenesis of neurotrophic foot ulceration. Diabet. Med. 18, 489-494 (2001)

Serum levels of substance P are decreased in patients with type 1 diabetes.

Morphological and immunohistochemical studies in diabetic subjects have shown a depletion of the neuropeptide substance P (SP) in the central and peripheral nervous system. This is the first study investigating serum levels of substance P in type 1 diabetes patients (n=50) and controls (n=75) by means of an enzyme immunoassay. The serum level of SP was significantly decreased in the diabetic group compared to the control group (10.12+/-0.29 vs. 12.25+/-0.38 pg/ml; p<0.0001). In diabetic patients, there was no correlation of substance P levels with age, serum creatinine, albuminuria, total cholesterol, HDL- or LDL-cholesterol, triglycerides, HbA1c, type or duration of diabetes and gender. Furthermore, there was no difference in serum levels of SP in patients with or without retinopathy, but SP was significantly decreased in patients with neuropathy (9.59+/-0.48 vs. 10.78+/-0.83 pg/ml; p=0.04). These data show that SP is decreased in serum of type 1 diabetes patients, especially in those with diabetic neuropathy. Subsequent and already ongoing prospective studies in well validated diabetic patients with neuropathy may characterize the impact of this neurogenic marker in the course of diabetic neuropathy.

Improved diagnostic methods in the follow-up of medullary thyroid carcinoma by highly specific calcitonin measurements.

Calcitonin (CT) is an important tumor marker for medullary thyroid carcinoma (MTC). Recent CT assays chiefly recognize the monomeric form of CT (mCT). It was the objective of this study to examine the consequences of the higher specificity of the assay for interpretation of the postoperative CT values in MTC patients. The postoperative mCT concentration was measured in 214 patients with differentiated thyroid carcinoma (MTC excepted; non-MTC patients) to determine a reference range of mCT in totally thyroidectomized patients. Monomeric CT was also determined with a two-site chemiluminescence immunoassay (Nichols) in 94 healthy subjects and in 68 MTC patients. The mCT concentrations were below the detection limit in all examined completely thyroidectomized non-MTC patients. Basal and stimulated mCT values were also below the detection limit in 32 of the 68 MTC patients. The biochemical and imaging diagnosis of the latter patients did not give any indication of tumor recurrence. We conclude that completely thyroidectomized patients with non-MTC do not show any measurable mCT concentrations. In comparison with an unspecific CT-RIA, the more specific mCT determination by immunoluminometric assay permits a more precise differentiation between postoperative normal and pathological values and an earlier diagnosis of recurrent MTC.

Comparison of immunoassays for the detection of anti-GAD65 autoantibodies in patients with diabetes mellitus.

Recent studies have demonstrated that a combination of GAD-antibody assays and IA-2 autoantibody assays show a high diagnostic specificity for Type 1 diabetes. For this reason there is increasing interest in the use of GAD-antibody measurement for Type 1 risk assessment. Since a number of different assays have been published and documented in the literature, the aim of this study was to evaluate four different anti-GAD test systems that are commercially available in Germany. We tested the anti-GAD prevalences in five patient groups with the different immunoassays and compared them with the values obtained by an immunoprecipitation test (IP-Test). All assays correlated well with the IP-test and showed high sensitivity and specificity in the group of patients with recent onset Type 1 diabetes and the control group. The groups tested consisted of 20 subjects with recent onset Type 1 diabetes (< 6 weeks) (sensitivity 70-90%), nine subjects with a Type 1 duration of more than 2 years (sensitivity 11-33%), 21 patients with pluriglandular insufficiency (sensitivity 28.5-47.5%), 10 patients with Type 2 (specificity: 90-100%), and 14 healthy control subjects (specificity: 93-100%). Our data show a high level of sensitivity and specificity of the tested, commercially available, assays. Since almost every laboratory should be able to establish one of these assays, this may facilitate the possibility of further large scale population studies with the aim of investigating GAD-antibody prevalences in screening for Type 1 diabetes. Increased measurement of the diabetes-associated antibodies will be helpful in the differential diagnosis of gestational diabetes mellitus (GDM) and latent autoimmune diabetes of the adult (LADA).

Effects of proinsulin C-peptide on nitric oxide, microvascular blood flow and erythrocyte Na+,K+-ATPase activity in diabetes mellitus type I.

This study was conducted to evaluate the influence of proinsulin C-peptide on erythrocyte Na(+),K(+)-ATPase and endothelial nitric oxide synthase activities in patients with type I diabetes. In a randomized double-blind study design, ten patients with type I diabetes received intravenous infusions of either human C-peptide or physiological saline on two different occasions. C-peptide was infused at a rate of 3 pmol.min(-1).kg(-1) for 60 min, and thereafter at 10 pmol.min(-1).kg(-1) for 60 min. At baseline and after 60 and 120 min, laser Doppler flow (LDF) was measured following acetylcholine iontophoresis or mild thermal stimulation (44 degrees C), and venous blood samples were collected to determine plasma cGMP levels and erythrocyte membrane Na(+),K(+)-ATPase activity. The LDF response to acetylcholine increased during C-peptide infusion and decreased during saline infusion [18.6+/-19.2 and -13.2+/-9.4 arbitrary units respectively; mean+/-S.E.M.; P<0.05). No significant change in LDF was observed after thermal stimulation. The baseline plasma concentration of cGMP was 5.5+/-0.6 nmol.l(-1); this rose to 6.8+/-0.9 nmol.l(-1) during C-peptide infusion (P<0.05). Erythrocyte Na(+),K(+)-ATPase activity increased from 140+/-29 nmol of P(i).h(-1).mg(-1) in the basal state to 287+/-5 nmol of P(i). h(-1).mg(-1) during C-peptide infusion (P<0.01). There was a significant linear relationship between plasma C-peptide levels and erythrocyte Na(+),K(+)-ATPase activity during the C-peptide infusion (r=0.46, P<0.01). No significant changes in plasma cGMP levels or Na(+),K(+)-ATPase activity were observed during saline infusion. This study demonstrates an effect of human proinsulin C-peptide on microvascular function, which might be mediated by an increase in NO production and an activation of the erythrocyte Na(+),K(+)-ATPase. These mechanisms are compatible with the previous observed microvascular effects of C-peptide in patients with type I diabetes.

[Predictive genetic investigations. Individualization of diagnosis and treatment in families with multiple endocrine neoplasia type II]. / Prädiktive genetische Untersuchungen. Individualisierung von Diagnostik und Therapie bei Familien mit multipler endokriner Neoplasie Typ II.

BACKGROUND AND OBJECTIVE: When multiple endocrine neoplasia type 2 (MEN2) is suspected, genetic tests are at the centre of screening procedures. It was the aim of this study to compare the diagnostic value of molecular biological investigations with that of conventional biochemical tests. PATIENTS AND METHODS: The study cohort consisted of all 144 patients cared for in our department since 1990 with the suspected diagnosis of MEN2 (evidence of a medullary thyroid carcinoma [MTC]), coexistence of two MEN2 tumours or a family history of MEN2. 14 of the 144 patients (from 12 families) were already known to have an hereditary MTC, while the remaining 130 had been referred for further diagnostic investigations. RESULTS: An hereditary MTC was diagnosed in 22 of the 130 patients, a sporadic MTC in 32, while no definitive classification was possible in 20 MTC patients without a positive family history and on whom no mutation analysis had been performed. MEN2 was excluded in 56 family members. All 22 patients with newly diagnosed MTC had abnormally high calcitonin levels. A germ-line mutation in the RET proto-oncogene was found in 8 of the 9 families who had undergone molecular biological tests. The investigate results led to a thyroidectomy in 19 of the 22 patients with hereditary MTC; in all of them the surgical specimen showed C-cell hyperplasia and/o MTC. CONCLUSION: These results emphasize the importance of genetic tests in family screening. Preoperative measurement of calcitonin remains essential in MEN2 families in whom a germ-line mutation is not known. The choice of the appropriate diagnostic test must be individualized to the particular patients so that optimal results are obtained.

Germline mutations in the MEN1 gene: creation of a new splice acceptor site and insertion of 7 intron nucleotides into the mRNA.

The MEN1 tumor predisposition syndrome is caused by mutations in the MEN1 gene on human chromosome 11q13. We screened MEN1 gene exons 1-10 and flanking intron sequences from four different MEN1 families for mutations. In three families, heterozygous germline mutations within the exons were found, two of these representing novel mutations. In another family, all clinically affected members were heterozygous for a point mutation Gright curved arrow A within intron 4. Sequence analysis of cDNA from lymphocytes of the affected patients revealed that the intron mutation created a new acceptor splice site, leading to the inclusion of 7 bp of intronic sequence into the mRNA. The resulting frameshift generates a premature stop in codon 271. Intron borders should thus be screened for mutations in MEN1 diagnostics and cDNA sequence analysis is helpful in identifying pathophysiological consequences of intron mutations.

Binding of monocytes from normolipidemic hyperglycemic patients with type 1 diabetes to endothelial cells is increased in vitro.

Increased endothelial binding and emigration of monocytes play a dominant role in the pathogenesis of atherosclerosis in diabetes mellitus. Previous studies revealed that hyperlipidemia correlates with monocyte binding in vitro. The aim of this study was to characterize the monocyte-endothelial interaction of leucocytes of hyperglycemic patients with type 1 diabetes but lacking hyperlipidemia. We isolated monocytes from healthy controls and normolipidemic type 1 diabetes patients with elevated levels of HbA1c and quantified monocyte binding by an immunoilluminometric cell adhesion assay. Purity of isolated monocytes was at least 98%. Endothelial binding of monocytes from patients with type 1 diabetes was found to be significantly increased compared to controls (19.2 +/- 3.9% vs. 14.9 +/- 3.5%). This difference of monocyte binding remained unchanged if the endothelial cells were stimulated with 27.7 mmol/l glucose for seven days prior to adhesion studies (31.5 +/- 4.9% in diabetes patients vs. 25.8 +/- 4.1% in controls) whereby monocyte binding markedly increased under these hyperglycemic conditions. Furthermore, an increased CD11b expression could be demonstrated on monocytes of normolipidemic hyperglycemic type 1 diabetes patients. Thus, we suggest that hyperglycemia per se may contribute to increased monocyte binding to endothelial cells by promoting leucocyte integrin expression. Recently performed studies of our group strengthen the hypothesis that this monocyte activation is mediated by stimulation of the beta-isoform of proteinkinase C.

The effect of human proinsulin C-peptide on erythrocyte deformability in patients with Type I diabetes mellitus.

AIMS/HYPOTHESIS: In recent years, evidence has arisen that proinsulin C-peptide exerts biological effects especially on microcirculation, e.g. C-peptide has been shown to increase skin microcirculation in patients with Type I (insulin-dependent) diabetes mellitus and to activate endothelial nitric oxide synthase. This study aimed to investigate the influence of pro-insulin C-peptide on erythrocyte deformability which was assessed by means of laser diffractoscopy. METHODS: Blood samples from healthy control subjects (n = 10) and Type I diabetic patients (n = 15) completely deficient of C-peptide were analysed at shear stresses ranging from 0.3 to 30 Pa. RESULTS: Erythrocyte deformability was lower in the group of Type I diabetic patients than in the control subjects. Preincubation of the diabetic blood samples with various concentrations of human proinsulin C-peptide for 8 h restored the deformability of erythrocytes, almost reaching the values of control samples. In contrast, proinsulin C-peptide did not modify the erythrocyte deformability of control subjects. CONCLUSION/INTERPRETATION: We conclude that proinsulin C-peptide is able to ameliorate the impaired deformability of erythrocytes in Type I diabetic patients and we hypothesise that this effect is mediated by restoration of Na(+)-K(+)-ATPase activity, which is known to be attenuated in diabetic patients.

Alpha-lipoic acid reduces expression of vascular cell adhesion molecule-1 and endothelial adhesion of human monocytes after stimulation with advanced glycation end products.

Advanced glycation end products (AGEs) have been identified as relevant mediators of late diabetic complications such as atherosclerotic disease. The endothelial migration of monocytes is one of the first steps in atherogenesis and monocyte-endothelial interaction itself is linked to the expression of adhesion molecules like vascular cell adhesion molecule-1 (VCAM-1). Recently, stimulation of VCAM-1 by AGEs has been demonstrated. Since endothelial stimulation by AGEs is followed by generation of oxygen free radicals with subsequent activation of nuclear transcription factor kappaB, we investigated the influence of alpha-lipoic acid on the expression of VCAM-1 and monocyte adherence to endothelial cells in vitro by means of cell-associated chemiluminescence assays and quantitative reverse transcriptase polymerase chain reaction using a constructed recombinant RNA standard. We found that alpha-lipoic acid was able to decrease the number of VCAM-1 transcripts from 41. 0+/-11.2 to 9.5+/-4.7 RNA copies per cell in AGE-stimulated cell cultures. Furthermore, expression of VCAM-1 was suppressed in a time- and dose-dependent manner by alpha-lipoic acid as shown by chemiluminescence endothelial cell assay. Pretreatment of endothelial cells with 0.5 mM or 5 mM alpha-lipoic acid reduced AGE-induced endothelial binding of monocytes from 22.5+/-2.9% to 18. 3+/-1.9% and 13.8+/-1.8% respectively. Thus, we suggest that extracellularly administered alpha-lipoic acid reduces AGE-albumin-induced endothelial expression of VCAM-1 and monocyte binding to endothelium in vitro. These in vitro results may contribute to the understanding of a potential antioxidative treatment of atherosclerosis.

Establishment of a quantitative RT-pCR for detection of vascular cell adhesion molecule-1 transcripts in endothelial cells after stimulation with advanced glycation endproducts.

Advanced glycation endproducts (AGE) are supposed to increase endothelial expression of adhesion molecules like vascular cell adhesion molecule-1 (VCAM-1) by inducing an intracellular stress with subsequent activation of nuclear transcription factor NF-kappa-B. Quantitative analysis of VCAM-1-transcription has not been demonstrated concerning this topic. Thus, the aim of this study was to establish quantitative reverse transcription polymerase chain reaction (RT-PCR) assays using a spacer gene in order to measure the amounts of specific mRNA for VCAM-1 in human umbilical vein endothelial cells (HUVEC) which were stimulated with AGE-albumin (AGE-BSA). A recombinant RNA-standard was synthesized and used as internal RT-PCR standard. The amount of VCAM-1-mRNA in unstimulated HUVEC was found to be 2.2 +/- 2.7 copies per cell. After stimulation with AGE-BSA, mRNA-levels were elevated to 38.9 +/- 10.9 copies per cell. Positive controls (stimulated with lipopolysaccharide) revealed mRNA-levels of 78.7 +/- 27.5 copies per cell. We conclude that quantitative RT-PCR using the spacer gene technique is a valid and reliable method for the measurement of small amounts of specific