Angiotensin-(1-7) is an endogenous ligand for the G protein-coupled receptor Mas

The renin-angiotensin system plays a critical role in blood pressure control and body fluid and electrolyte homeostasis. Besides angiotensin (Ang) II, other Ang peptides, such as Ang III [Ang-(2-8)], Ang IV [Ang-(3-8)], and Ang-(1-7) may also have important biological activities. Ang-(1-7) has become an angiotensin of interest in the past few years, because its cardiovascular and baroreflex actions counteract those of Ang II. Unique angiotensin-binding sites specific for this heptapeptide and studies with a selective Ang-(1-7) antagonist indicated the existence of a distinct Ang-(1-7) receptor. We demonstrate that genetic deletion of the G protein-coupled receptor encoded by the Mas protooncogene abolishes the binding of Ang-(1-7) to mouse kidneys. Accordingly, Mas-deficient mice completely lack the antidiuretic action of Ang-(1-7) after an acute water load. Ang-(1-7) binds to Mas-transfected cells and elicits arachidonic acid release. Furthermore, Mas-deficient aortas lose their Ang-(1-7)-induced relaxation response. Collectively, these findings identify Mas as a functional receptor for Ang-(1-7) and provide a clear molecular basis for the physiological actions of this biologically active peptide.     

Somatostatin receptor imaging in persistent medullary thyroid carcinoma

OBJECTIVE Somatostatin is secreted from thyroid C-cells and seems to play an Important part In the regulation of calcitonin secretion. We therefore evaluated the usefulness of somatostatin receptor scintigraphy in the localization of tumour tissue in patients with persistent medullary thyroid carcinoma. DESIGN A prospective clinical study. PATIENTS The series consisted Of 26 patients with elevated calcitonin levels after total thyroldectomy for histologically proven medullary thyroid carcinoma. METHODS Somatostatin receptor scintigraphy using ¹¹¹ In-pentetreotide (Octreoscan) was performed in all patients and the results correlated with histology, ultrasonography, computerized tomography, magnetic resonance Imaging, plain radiography, bone scintigraphy and selective venous Catheterization. calcitonin and carcinoembryonic antigen levels were measured. RESULTS The sensitivity of somatostatin receptor scintigraphy for localization of persistent medullary thyroid carcinoma was 57% in patients with histologically proven disease. The results depended on tumour mass (low sensitivity (33%) in minimal residual disease) and on the location of metastases (Insensitive in detecting liver metastases). CONCLUSIONS Somatostatin receptor scintigraphy is of value as an additional diagnostic tool in localizing medullary thyroid carcinoma, especially pulmonary metastases. It is of minor importance in detecting minimal residual disease.     

Diagnostics and surgical treatment strategy for rectal cavernous hemangiomas based on three case examples

A 20-year-old man with a congenital vascular malformation extending from the anal canal into the distal sigmoid had had recurrent perianal blood loss as a neonate. A hemangioma was diagnosed for the first time in 1978. The patient received regular and frequent gastroenterological treatment until admission. Decisive for the indication for surgery was the patient’s need for blood infusions and shorter bleeding intervals in June 1998. Surgical therapy consisted of deep anterior rectosigmoid resection with coloanal pouch anastomosis. In a second case of a 27-year-old woman a sigmoid hemangioma was diagnosed in conjunction with emergency sigmoid resectioning. Because of recurrent hemorrhages a coloanal pouch was also established here in a second step. The third case involved a 19-year-old woman with a 12-year history of repeated perianal hemorrhages. After sigmoid discontinuity resection we carried out proctectomy with descendostoma creation due to renewed severe intractable perianal bleeding. The histological examination revealed a rectal hemangioma that had caused the repeated perianal hemorrhages. Surgical reconstruction was then achieved by coloanal pouch anastomosis. In view of the good functional and perioperative results, current surgical therapy should aim at preserving continuity and continence by coloanal pouch anastomosis. Accepted: 12 November 1999     

A point mutation in the γ2 subunit of γ-aminobutyric acid type A receptors results in altered benzodiazepine binding site specificity

Benzodiazepines allosterically modulate γ-aminobutyric acid (GABA) evoked chloride currents of γ-aminobutyric acid type A (GABA_A) receptors. Coexpression of either rat γ₂ or γ₃, in combination with α₁ and β₂ subunits, results both in receptors displaying high [³H]Ro 15-1788 affinity. However, receptors containing a γ₃ subunit display a 178-fold reduced affinity to zolpidem as compared with γ₂-containing receptors. Eight chimeras between γ₂ and γ₃ were constructed followed by nine different point mutations in γ₂, each to the homologous amino acid residue found in γ₃. Chimeric or mutant γ subunits were coexpressed with α₁ and β₂ in human embryonic kidney 293 cells to localize amino acid residues responsible for the reduced zolpidem affinity. Substitution of a methionine-to-leucine at position 130 of γ₂ (γ₂M130L) resulted in a 51-fold reduction in zolpidem affinity whereas the affinity to [³H]Ro 15-1788 remained unchanged. The affinity for diazepam was only decreased by about 2-fold. The same mutation resulted in a 9-fold increase in Cl 218872 affinity. A second mutation (γ₂M57I) was found to reduce zolpidem affinity by about 4-fold. Wild-type and γ₂M130L-containing receptors were functionally expressed in Xenopus oocytes. Upon mutation allosteric coupling between agonist and modulatory sites is preserved. Dose–response curves for zolpidem and for diazepam showed that the zolpidem but not the diazepam apparent affinity is drastically reduced. The apparent GABA affinity is not significantly affected by the γ₂M130L mutation. The identified amino acid residues may define part of the benzodiazepine binding pocket of GABA_A receptors. As the modulatory site in the GABA_A receptor is homologous to the GABA site, and to all agonist sites of related receptors, γ₂M130 may either point to a homologous region important for agonist binding in all receptors or define a new region not underlying this principle.     

European Bone Marrow Working Group trial on reproducibility of World Health Organization criteria to discriminate essential thrombocythemia from prefibrotic primary myelofibrosis

Background

The World Health Organization classification of myeloproliferative neoplasms discriminates between essential thrombocythemia and the prefibrotic phase of primary myelofibrosis. This discrimination is clinically relevant because essential thrombocythemia is associated with a favorable prognosis whereas patients with primary myelofibrosis have a higher risk of progression to myelofibrosis or blast crisis.

Design and Methods

To assess the reproducibility of the classification, six hematopathologists from five European countries re-classified 102 non-fibrotic bone marrow trephines, obtained because of sustained thrombocytosis.

Results

Consensus on histological classification defined as at least four identical diagnoses occurred for 63% of the samples. Inter-observer agreement showed low to moderate kappa values (0.28 to 0.57, average 0.41). The percentage of unclassifiable myeloproliferative neoplasms rose from 2% to 23% when minor criteria for primary myelofibrosis were taken into account. In contrast, the frequency of primary myelofibrosis dropped from 23% to 7%, indicating that the majority of patients with a histological diagnosis of primary myelofibrosis did not fulfill the complete criteria for this disease. Thus, over 50% of cases in this series either could not be reproducibly classified or fell into the category of unclassifiable myeloproliferative neoplasms.

Conclusions

World Health Organization criteria for discrimination of essential thrombocythemia from prefibrotic primary myelofibrosis are poorly to only moderately reproducible and lead to a higher proportion of non-classifiable myeloproliferative neoplasms than histology alone.     

Surrogate end points in secondary analyses of cardiovascular trials

A surrogate end point is one that is used as a substitute for a clinical end point of more direct interest, usually for reasons of practicality, and that is expected to predict clinical benefit. Surrogate end points play a critical role in the advancement of all medical research, and cardiovascular (CV) research in particular. However, the relationship between a surrogate end point and its clinical end point is usually complex, and there are many examples where results based on surrogates have proved to be misleading. Secondary analyses of existing clinical trial data are likely to involve surrogate end points, if only because clinical end points will have been extensively studied as part of the primary analysis of a trial large enough to collect useful clinical end point data. Validation of a surrogate end point is a laudable goal for a secondary analysis of a large clinical end point trial (or meta-analysis of multiple smaller trials), and the result may be an important new tool for further study of a class of compounds in a particular disease context. Secondary analyses using surrogate end points may also provide new insight into disease or treatment mechanism, but as with any surrogate end point analysis, the results can mislead, and the existing literature is heavy on application and light on methodology. Surrogate end points often substitute efficiency for clarity, and while many interesting and potentially informative secondary analyses of CV trials will involve surrogates, results are likely to be ambiguous and should be interpreted with care.     

Long-term outcome in 46 gene carriers of hereditary medullary thyroid carcinoma after prophylactic thyroidectomy: impact of individual RET genotype

OBJECTIVE: In children with RET proto-oncogene mutation, curative treatment of medullary thyroid carcinoma (MTC) is possible by prophylactic thyroidectomy. Recommendations on the timing and extent of thyroidectomy are based upon a model that utilises genotype-phenotype correlations to stratify mutations into three risk groups. DESIGN: We evaluated the long-term outcome (mean follow-up 6.4 years, 15 patients more than 10 years, 26 patients more than 5 years) of operated gene carriers stratified into two risk groups (levels 1 and 2) based on the biological aggressiveness of MTC. RESULTS: In 46 RET gene carriers, prophylactic thyroidectomy was carried out between the ages of 4 and 21 years. Level 1 mutations were harboured by 11 patients (codons 790, 791, 804 and 891). Histology was completely normal in two patients; in seven patients C-cell hyperplasia (CCH) and in two patients T1 tumours were diagnosed. All patients with level 1 mutations were cured. Level 2 mutations were harboured by 35 patients (codons 618, 620, 630 and 634). Histology of these patients showed CCH in 11 patients, T1 tumours in 21, T2 tumour in 1, T3 tumour in 1 and Tx in 1 patient. Histology showed no lymph node involvement. Five patients with level 2 mutations failed to be cured; in two patients, persistence of MTC was diagnosed directly after thyroidectomy and in three during follow-up. In two patients carrying a 634 mutation, other endocrinopathies (hyperparathyroidism and bilateral pheochromocytoma) manifested during follow-up. CONCLUSIONS: If prophylactic thyroidectomy is done at early ages, cure rate is high. Timing and extent of prophylactic thyroidectomy can be modified by individual RET mutation.     

Pyrosequencing-based validation of a simple cell-suspension polymerase chain reaction assay for Campylobacter with application of high-processivity polymerase and novel internal amplification controls for rapid and specific detection

Although Campylobacter is an important food-borne human pathogen, there remains a lack of molecular diagnostic assays that are simple to use, cost-effective, and provide rapid results in research, clinical, or regulatory laboratories. Of the numerous Campylobacter assays that do exist, to our knowledge none has been empirically tested for specificity using high-throughput sequencing. Here we demonstrate the power of next-generation sequencing to determine the specificity of a widely cited Campylobacter-specific polymerase chain reaction (PCR) assay and describe a rapid method for direct cell suspension PCR to quickly and easily screen samples for Campylobacter. We present a specific protocol which eliminates the need for time-consuming and expensive genomic DNA extractions and, using a high-processivity polymerase, demonstrate conclusive screening of samples in <1 h. Pyrosequencing results show the assay to be extremely (>99%) sensitive, and spike-back experiments demonstrated a detection threshold of <10² CFU mL⁻¹. Additionally, we present 2 newly designed broad-range bacterial primer sets targeting the 23S rRNA gene that have wide applicability as internal amplification controls. Empirical testing of putative taxon-specific assays using high-throughput sequencing is an important validation step that is now financially feasible for research, regulatory, or clinical applications.     

Occurrence of adenomas in the pouch and small intestine of FAP patients after proctocolectomy with ileoanal pouch construction

Purpose Proctocolectomy with ileoanal pouch construction is the standard therapy for patients with familial adenomatous polyposis coli (FAP) to prevent the genesis of colorectal carcinomas. In our patient population, we observed the postoperative development of adenomas not only in the pouch but also in the remaining small intestine. The exact incidence of these ileal polyps is still unknown, since the diagnostic possibilities of examining the small intestine are limited. Methods We performed wireless capsule endoscopy (CE) in patients who developed postoperative pouch adenomas (PA) to record the simultaneous occurrence of small bowel adenomas and PA. We operated on 46 patients with FAP (m:f 17:10, age 33 ± 9 years). Thirty-five patients underwent proctocolectomy with ileoanal pouch creation. Pouch endoscopy was performed in regular intervals at 3 months and then annually after proctocolectomy. Capsule endoscopy was additionally carried out in all patients with PA. Results Ileal PA occurred in 22.8% (n = 8) of the patients with proctocolectomy (n = 35) after a mean of 5 years after surgery. Eight PA patients (all with PA) also had adenomas in the small intestine diagnosed by CE. Conclusions Since jejunal and ileal adenomas occur in all patients with PA, we recommend regular follow-up examinations, which include pouch endoscopy at 3 months and annually after surgery in the presence of PA after proctocolectomy and pouch creation. On the basis of our observations, we recommend adding CE or double-balloon enteroscopy to the follow-up examination.     

Differential expression of genes encoding tight junction proteins in colorectal cancer: frequent dysregulation of claudin-1, -8 and -12

Background and aims As integral membrane proteins, claudins form tight junctions together with occludin. Several claudins were shown to be up-regulated in various cancer types. We performed an expression analysis of genes encoding tight junction proteins to display differential gene expression on RNA and protein level and to identify and validate potential targets for colorectal cancer (CRC) therapy. Patients and methods Amplified and biotinylated cRNA from 30 microdissected CRC specimen and corresponding normal tissues was hybridized to Affymetrix U133set GeneChips. Quantification of differential protein expression of claudin-1, -8 and -12 between normal and corresponding tumour tissues was performed by Western blot analyses. Paraffin-embedded CRC tissue samples, colon cancer cell lines and normal tissue microarray were analysed for protein expression of claudin-1 by immunohistochemistry (IHC). Results Claudin-1 (CLDN1) and -12 (CLDN12) are frequently overexpressed in CRC, whereas claudin-8 (CLDN8) shows down-regulation in tumour tissue on RNA level. Quantification of proteins confirmed the overexpression of claudin-1 in tumour tissues, whereas changes of claudin-8 and -12 were not significantly detectable on protein level. IHC confirmed the markedly elevated expression level of claudin-1 in the majority of CRC, showing membranous and intracellular vesicular staining. Conclusions Differential expression of genes encoding claudins in CRC suggests that these tight junction proteins may be associated to and involved in tumorigenesis. CLDN1 is frequently up-regulated in large proportion of CRC and may represent potential target molecule for blocking studies in CRC.