Evaluation of chromogranin A expression in serum and tissues of breast cancer patients

Human chromogranin A (CgA) is a member of the granin family and is widely distributed in large dense core granules of endocrine and neuroendocrine cells. A variety of non-neuroendocrine carcinomas arising in various tissues show patterns of neuroendocrine differentiation. Expression of CgA has been documented in epithelial cells of normal mammary gland as well as in breast cancers, and elevation of serum CgA has been detected in patients with breast cancer. Our study was undertaken to evaluate the relationship between serum CgA levels and neuroendocrine features in breast cancer. In addition, we evaluated the expression of serum CgA in patients affected by breast cancer compared to controls and the relationship between serum CgA and tumor histology, extent of disease, lymph node status, tumor stage and serum CA 15.3 levels. We enrolled 266 patients with infiltrating ductal or lobular breast carcinoma and a group of 100 age-matched healthy women serving as controls. Serum CgA and CA 15.3 were assayed by specific immunoradiometric methods. The overall sensitivity of CgA and CA 15.3 was 0.06 and 0.34, respectively (chi2 19.1, p<0.0005). No relationship was found between serum levels of CgA and tumor histology, extent of disease, lymph node status or tumor stage while serum levels of CA 15.3 were strongly correlated with all these variables but tumor histology. No relationship was found between serum levels of CgA and CA 15.3. Immunostaining against CgA, CgB, NSE and synaptophysin was performed on primary tumor tissue of 14 serum CgA-positive and 24 serum CgA-negative patients and was negative in all cases. We also evaluated eight cases of pathologically-proven neuroendocrine breast cancer: only four and two of these showed positive CgA immunostaining and increased serum CgA concentration, respectively. In conclusion, serum CgA assay offers no additional information regarding the presence, the extent and the histology of breast cancer compared to the CA 15.3 assay. Moreover, serum CgA was not an accurate marker to identify or exclude the rare neuroendocrine differentiation of breast cancer. We therefore conclude that CgA is not useful as a serum marker in breast cancer.     

Application of region-specific immunoassay for human chromogranin A: substantial clue for detection and measurement of chromogranin A in human plasma

Chromogranin A (CgA), a secretory protein, is co-released with catecholamines from storage vesicles. It is known to be elevated in the circulation of patients with neuroendocrine and endocrine tumors. For further investigation of the protein, especially in humans, it is essential to facilitate quantitative analysis of the protein in human biological materials. In order to introduce novel immunological methodology for this purpose, we purposely selected human CgA(344-374) for the synthetic immunogen to produce region-specific CgA antibodies. The anti-synthetic peptide antibody thus obtained made it possible to develop an immunological method for measurement and characterization of CgA in human plasma. The plasma CgA-immunoreactivity (LI) level measured by the method was 0.31+/-0.01 pmol/ml (mean+/-SEM) in normal subjects and 1.55+/-0.29 pmol/ml in pheochromocytoma. On gel chromatography and HPLC analysis of the plasma of patients with pheochromocytoma, the region-specific assay system enabled us to show the presence of N-terminal truncated CgA, besides CgA itself. By following up changes of plasma CgA-LI in a pheochromocytoma patient using samples that were collected consecutively over a two-year period, the present assay system using the region-specific antibody, anti-human CgA (344-374) serum, was confirmed to be extremely valuable for the measurement of CgA-LI in human plasma. The characteristic features and high sensitivity of the present assay system will give us a substantial clue to the detection and measurement of CgA to develop further investigation of the protein in humans.     

Trace element status in Saudi patients with established atherosclerosis

BACKGROUND: Traditional coronary risk factors do not fully explain variations in the incidence of cardiovascular disease (CVD). Epidemiological studies have implicated perturbations in selenium, copper, and zinc metabolism in the aetiology of CVD. However, these studies have been principally undertaken in Caucasian populations, in whom trace element intake is generally sufficient. METHOD: We have measured serum and urine selenium, copper, and zinc; and superoxide dismutase, glutathione peroxidase, and lipid peroxide concentrations in 130 Saudi male subjects with established CVD, and 130 age-matched controls. RESULTS: Diabetes mellitus, positive smoking habit (p<0.0001 for both), and hypertension (p<0.05) were more prevalent among CVD patients. Urinary copper (p<0.0001) and zinc (p<0.05) were higher among controls. Serum selenium concentrations were lower among CVD patients (p<0.001), and a high proportion (52%) had selenium levels below 79mug/L compared to controls (22%) (p<0.0001). Conditional logistic regression analysis, showed the characteristics differentiating CVD patients from controls were serum zinc (odds ratio (OR) 0.92, confidence interval (CI) 0.85-0.99, p<0.05), serum copper/zinc ratio (OR 0.31, CI 0.10-0.96), serum selenium (OR 0.07, CI 0.02-0.31, p<0.0001), and urine selenium (OR 3.34, CI 1.40-7.99, p<0.01). CONCLUSION: Measures of trace metals status appear to be associated with the risk of atherosclerosis in a Saudi male population.     

Plasma adrenomedullin concentrations in patients with adrenal pheochromocytoma.

BACKGROUND: The hypotensive peptide adrenomedullin was first isolated in extracts of human pheochromocytoma. There is, however, no information available on the behaviour of circulating adrenomedullin or on the correlation with catecholamines in patients with pheochromocytoma. OBJECTIVES: 1) to investigate whether plasma adrenomedullin levels were changed in 10 patients with pheochromocytoma when compared to 21 healthy subjects and 16 patients with essential hypertension; 2) to determine whether or not adrenomedullin has a counter-regulatory role in catecholamine excess in pheochromocytoma or is responsible for hemodynamic modifications before and after tumour resection; 3) to determine tissue distribution of iradrenomedullin in the pheochromocytoma. METHODS: Plasma adrenomedullin and catecholamine levels were measured in all patients with pheochromocytoma before and four weeks after tumour removal. In the four patients undergoing resection of tumours, plasma levels of adrenomedullin were measured at different time-points during surgery. RESULTS: The mean plasma adrenomedullin concentrations ( SD) in patients with pheochromocytoma (37.9 +/- 6pg/ml) were significantly higher (p<0.0001) than those in normal subjects (13.7 +/- 6.1 pg/mI) and patients with essential hypertension (22.5 +/- 9.lpg/ml). Adrenomedullin levels correlated with plasma noradrenaline (r = 0.516, p = 0.0124). In all patients with pheochromocytoma, plasma adrenomedullin concentrations decreased after removal of tumours (from 37.9 +/- 6 to 10.9 +/- 4.6 pg/ml; p < 0.0001). In the four patients studied during surgery, baseline plasma adrenomedullin and noradrenaline levels were markedly elevated, and increased significantly with tumour manipulation, decreasing 24 hours after operation. Adrenal medulla cells surrounding the pheochromocytoma site stained for ir-adrenomedullin, whereas only isolated cells of pheochromocytoma stained for the peptide. CONCLUSIONS: This study demonstrates that circulating adrenomedullin is increased in pheochromocytoma, and is also correlated with plasma noradrenaline levels. Adrenomedullin may represent an additional biochemical parameter for clinical monitoring of patients with pheochromocytoma.     

Chromogranin B: isolation from pheochromocytoma, N-terminal sequence, tissue distribution and secretory vesicle processing

The chromogranins/secretogranins are a family of neuroendocrine vesicle secretory proteins. Immunohistology and immunoblotting have suggested that a major soluble protein in human chromaffin granules may be chromogranin B (CgB). We purified from pheochromocytoma chromaffin granules an SDS-PAGE 110-120 kDa protein whose N-terminal sequence matched that previously deduced from a human CgB cDNA. An antibody directed against a synthetic human CgB N-terminal region specifically recognized the CgB N-terminus, though not the chromogranin A (CgA) N-terminus or the CgB C-terminus on immunoblots. An antiserum directed against CgB's C-terminus also visualized CgB but not CgA. By immunoblotting, CgB was a quantitatively major protein in human pheochromocytoma chromaffin granules, but a relatively minor in normal bovine adrenal medullary chromaffin granules. In a variety of normal bovine neuroendocrine tissues, the relative abundance of CgB immunoreactivity on immunoblots was: adrenal medulla greater than anterior pituitary greater than pancreas greater than small intestine, hypothalamus. Immunoblotting of neuroendocrine tissues (or their hormone storage vesicle cores) with both anti N-terminal and anti C-terminal CgB antisera suggested bidirectional cleavage or processing of CgB; in the anterior pituitary, a unique 40 kDa C-terminal fragment was observed. Bidirectional CgB cleavage was also suggested on immunoblots of chromaffin tissue from three species (human, bovine, rat). C-terminal processing of CgB was also confirmed by amino acid sequencing of SDS-PAGE-separated, polyvinylidene difluoride membrane-immobilized CgB fragments from pheochromocytoma chromaffin granules. Whether such fragments possess biological activity remains to be investigated.     

脑梗死复发患者血清同型半胱氨酸、白蛋白和尿微量白蛋白的变化及其影响因素分析

目的:研究脑梗死复发患者血清同型半胱氨酸(Hcy)、白蛋白(ALB)和尿微量白蛋白的变化及脑梗死复发的影响因素。方法:将2013年2月至2016年2月我院收治的脑梗死复发患者63例记为复发组,另取同期住院治疗的未复发脑梗死患者57例记为非复发组。比较两组患者血清Hcy、ALB、尿微量白蛋白水平和基本资料,并采用多因素Logistic回归分析脑梗死复发的影响因素。结果:复发组血清Hcy、尿微量白蛋白水平高于非复发组,复发组血清ALB水平低于非复发组(P0.05)。经Pearson相关性分析可得:脑梗死复发患者血清Hcy与尿微量白蛋白水平呈正相关关系,ALB与Hcy、尿微量白蛋白水平呈负相关关系(P0.05)。复发组高血压、脑白质疏松、脑血管病家族史人数占比高于非复发组(均P0.05)。经多因素Logistic回归分析可得:血清Hcy、尿微量白蛋白水平以及高血压均是脑梗死复发的独立危险因素(P0.05),而血清ALB水平是脑梗死复发的保护性因素(P0.05)。结论:脑梗死复发患者的血清Hcy、尿微量白蛋白水平升高,而血清ALB水平降低,上述三项指标的异常改变可能共同促进了脑梗死的复发,值得临床重点关注。     

An in vivo OctreoScan-negative adrenal pheochromocytoma expresses somatostatin receptors and responds to somatostatin analogs treatment in vitro.

A 52-yr-old woman presented with hypertension, elevated urinary vanillylmandelic acid, metanephrines, normetanephrines, and plasma chromogranin A (CgA), but normal urinary catecholamine levels. Abdominal ultrasonography and subsequent MRI imaging showed a 3 cm nodular lesion of the right adrenal gland also visualized by 123I-meta-iodobenzylguanidine scintigraphy consistent with a pheochromocytoma (PC). Her OctreoScan was negative. The patient underwent right adrenalectomy and histological examination showed a PC. The adrenal medulla tissue was examined for somatostatin (SRIH) receptor subtypes 1 to 5 (SSTR1 to 5) expression by RT-PCR. Cultured tumor cells were treated with either SRIH, Lanreotide (Lan), or an SSTR2 (BIM-23 120) or SSTR5 (BIM-23 206) selective agonist. CgA secretion was measured in the medium by ELISA and catecholamine levels by HPLC after 6h. Cell viability was assessed after 48h. RT-PCR analysis showed that SSTR1, 2, 3 and 4 were expressed. CgA secretion was significantly reduced by SRIH (- 80 %), Lan (- 35 %), and the SSTR2 selective agonist (- 65 %). Norepinephrine secretion was reduced by SRIH (- 66 %), Lan (- 40 %), and BIM-23 120 (- 70 %). Epinephrine and dopamine secretion was also inhibited by treatment with SRIH (- 90 % and - 93 %, respectively) and BIM-23 120 (- 33 % and - 75 %, respectively) but not by Lan. Cell viability was also significantly reduced by SRIH (- 30 %), Lan (- 10 %), and the SSTR2 selective agonist (- 20 %). The SSTR5 selective agonist did not modify either CgA and catecholamine secretion or cell viability. Our data show that SSTRs may be present in a PC although OctreoScan is negative in vivo, and that SRIH and its analogs may reduce both differentiated and proliferative functions in chromaffin cells in vitro. These findings suggest that SRIH analogs with enhanced SSTR2 affinity might be useful in the medical therapy of PC, even when an OctreoScan is negative.     

The possible role of chromogranin A as a prognostic factor in organ-confined prostate cancer

The clinical significance of neuroendocrine differentiation in patients who have undergone surgery for localized prostate cancer is still unclear. The aims of this study were to assess the relationship between serum neuroendocrine markers and well-known prognostic factors in prostate cancer (pathological staging, definitive Gleason score and serum PSA) and to search for correlations between serum chromogranin A (CgA) levels and pathological findings. Forty-one consecutive patients who had undergone radical retropubic prostatectomy for clinically localized prostate cancer were evaluated. Serum PSA, CgA and neuron-specific enolase were measured immediately before surgery. Twenty-six surgical specimens were phenotypically and immunohistochemically evaluated using an antibody against CgA. Significant correlations were found between serum CgA, pathological staging and Gleason score (p=0.049 and p=0.038, respectively). Serum CgA did not correlate with PSA, patient age, or immunohistochemical findings. There was a significant correlation between positive immunohistochemical CgA staining and Gleason score (p=0.014). An increase in serum CgA levels, independent of PSA values, might be the expression of pathologically more advanced tumor stage and higher Gleason score; this could help to identify a high-risk patient group eligible for adjuvant therapy.     

Helicobacter pylori infection in patients with Hepatitis C Virus positive chronic liver diseases

BACKGROUND AND GOALS: One-third of patients with liver cirrhosis suffers from acute peptic ulcer, a disease strongly correlated with Helicobacter pylori (H. pylori) infection. We report the seroprevalence of antibodies to H. pylori in 179 patients with Hepatitis C Virus (HCV)-related chronic active hepatitis and cirrhosis. MATERIALS AND METHODS: Among patients, 135 (86 males and 49 females, mean age 51.2 +/- 13.28, range 27-77 years) had chronic active hepatitis (CAH) and 44 cirrhosis (28 males and 16 females, mean age 62.4 +/- 9.2, range 37-77 years). Serum antibodies to H. pylori were tested using a commercial enzyme immunosorbent assay. The control population consisted of 619 consecutive blood donors (523 males, 96 females, mean age 47 +/- 5.3 years, range 18-65). RESULTS: The overall prevalence of antibodies to H. pylori was 73.1% (131/179) among patients and 47% (291/619) among blood donors (p<0.0001; OR 3.08 [95%CI, 2.10-4.51]). 70.5% (24/34) of patients aged less than 40 years were seropositive for H. pylori versus 34.2% (90/263) of controls (p<0.0001; OR 4.61[95%CI, 2.0-10.85]). Among cirrhosis patients, the prevalence of antibodies to H. pylori was 79.5% (35/44) versus 47% (291/619) of controls (p<0.0001; OR 4.38 [95%CI, 1.98-9.98]). Overall seroprevalence among CAH patients was 71.1% (96/135) versus 47% (291/619) of blood donors (p<0.0001; OR 2.77 [95%CI, 1.82-4.24]). CONCLUSIONS: The high seroprevalence of antibodies to H. pylori in patients with HCV-positive liver diseases explains the elevated incidence of peptic ulcer, and warrants studies on the pathogenic role in human liver diseases of Helicobacter spp which is known to cause chronic hepatitis and hepatocellular carcinoma in mice.     

Immunoradiometric assay of chromogranin A in the diagnosis of small cell lung cancer: comparative evaluation with neuron-specific enolase

The aims of our work were 1) to determine the diagnostic performance of an immunoradiometric assay of chromogranin A (CgA) in small cell lung cancer and 2) to compare its discriminatory power with that of neuron-specific enolase (NSE), the marker currently used for SCLC. We selected 166 cases of small cell (64) and non-small cell (102) lung cancer and 106 cases of non-malignant lung diseases as controls. Both CgA and NSE were assayed by immunoradiometric methods and cutoff values were established on the basis of a pre-fixed specificity of 95% in non-malignant lung diseases. The CgA assay showed better diagnostic sensitivity than NSE in SCLC (61% versus 57%), especially in limited disease, and a low positivity rate in NSCLC with respect to NSE (14% versus 22%). By contrast, NSE reflected disease extent more accurately than CgA (U test: CgA p<0.05, NSE p<0.001). Finally, we found that the CgA assay was not affected by hemolysis whereas NSE serum levels greatly increased in hemolyzed sera. In conclusion, CgA assaying by an IRMA method is a reliable procedure in the diagnosis of SCLC. NSE remains the marker of choice in staging and monitoring of the disease. Further studies are needed to evaluate the prognostic significance of the marker and its role in therapy monitoring and patient follow-up.     

Pheochromocytoma presenting with pulmonary edema and hyperamylasemia.

A 28-year-old woman was admitted to hospital with acute pulmonary edema, mild abdominal discomfort and hyperamylasemia. From the 2nd hospital day hypertensive episodes occurred daily. The furosemide screening test for renovascular hypertension revealed elevated plasma renin activity (PRA) but an intravenous pyelogram revealed a right suprarenal mass and no evidence of renovascular compression. Elevated values of plasma and urinary catecholamines indicated a pheochromocytoma, and a single chromaffin tumour was resected. It is important to monitor left ventricular filling pressure during operative removal of a pheochromocytoma. Postoperatively the patient had normal blood pressure and PRA. Decreased urinary amylase clearance and abnormal pancreatic and salivary amylase isoenzymes were found.     

Effects of Zinc Supplement on Plasma Homocysteine Level in End-Stage Renal Disease Patients: a Double-Blind Randomized Clinical Trial

Increased homocysteine (hCys) level is an independent risk factor for cardiovascular complications in end-stage renal disease (ESRD) patients. The aim of this study was to evaluate effect of zinc (Zn) supplement on serum hCys level in ESRD patients. One hundred ESRD patients with Zn deficiency were enrolled in this double-blind randomized clinical trial. They were randomly subdivided into two groups and supplemented with Zn (Zn group) or placebo (control group) for 6 weeks. Fasting plasma hCys and Zn levels were measured before and at 43rd days after the start of the study. Serum Zn levels increased significantly (p?<?0.0001), in Zn-treated group in comparison to placebo-treated group. In the Zn-treated group, serum hCys levels reduced significantly (p?<?0.0001), compared to placebo group (p?>?0.05). There was a significant (p?<?0.0001) reduction of mean percentage of hCys in Zn-treated group compared to the placebo group. Our study showed that Zn supplementation decreases serum hCys levels in ESRD patients with Zn deficiency.     

Comparison between two methods in the determination of circulating chromogranin A in neuroendocrine tumors (NETs): results of a prospective multicenter observational study

Several methods for analyzing CgA using either monoclonal or polyclonal antibodies have been developed, which differ in their diagnostic performance. The present paper describes the results of a prospective multicenter study aimed at comparing the clinical value of the two most widely used commercially available CgA assay kits in patients affected by neuroendocrine tumors (NETs). Two hundred sixty-one patients from 40 different centers and 99 healthy subjects were evaluated. CgA levels were measured with two different methods, a two-step immunoradiometric assay (IRMA) and an enzyme-linked immunosorbent assay (ELISA). CgA was measured centrally by two reference laboratories, one of which used IRMA and the other ELISA, and it was measured by the participating institutions with the method routinely used by each of them. The major findings of the present study were: (i) the two assays for the determination of CgA present good diagnostic performance; (ii) both assays are robust and guarantee comparable results when applied in different settings (central vs local laboratory); (iii) the negative/positive cutoff points (87 ng/mL for IRMA and 21.3 U/L for ELISA) were established according to standardized criteria; (iv) the results obtained with the two assays in basal clinical samples of patients affected by NETs show an apparently satisfactory correlation (rs = 0.843, p < 0.0001). However, a possibly clinically meaningful 36% discordance rate was found. These findings support the hypothesis that the two CgA kits might provide partially different information.     

Limitations of Chromogranin A in clinical practice

Context: Usefulness of circulating Chromogranin A (CgA) for the diagnosis of neuroendocrine tumors (NEN) is controversial. The aim of the present study was to assess the actual role of this marker as diagnostic tool. Methods: Serum blood samples were obtained from 42 subjects affected with NEN, 120 subjects affected with non-endocrine neoplasias (non-NEN) and 100 non-neoplastic subjects affected with benign nodular goitre (NNG). Determination of CgA was performed by means of immunoradiometric assay. Results: The CgA levels among NEN-patients were not significantly different from NNG and non-NEN subjects. The Receiver operating characteristic (ROC) curves analysis failed to identify a feasible cut-off value for the differential diagnosis between NEN and the other conditions. Conclusion: Serum CgA is not helpful for the first-line diagnosis of NEN.     

Serum calcitonin-negative medullary thyroid carcinoma: role of CgA and CEA as complementary markers

The biochemical activity of medullary thyroid carcinoma (MTC) includes production of calcitonin (CT), chromogranin A (CgA) and carcinoembryonic antigen (CEA). Routine CT measurement has been proposed as part of the initial evaluation of thyroid nodules and its use could ultimately decrease the morbidity and mortality of MTC. We report on a 43-year-old female patient with a large MTC expressing CT, CgA and CEA on immunostains but with negative preoperative CT and CgA results. Serum CEA was slightly increased and its rapid disappearance predicted radical cure by surgery as confirmed by 2-year follow-up. Our report illustrates that a diagnosis of MTC cannot always be excluded by negative preoperative CT. Fine-needle aspiration with cytomorphological analysis and complementary immunocytochemistry remains an essential diagnostic tool. Finally, serum aliquots must be stored before thyroid surgery in order to measure circulating forms of complementary markers found by tissue immunostaining (CEA and CgA).     

Reserpine-Induced Processing of Chromogranin A in Cultured Bovine Adrenal Chromaffin Cells

The effect of reserpine on the processing of the secretory granule protein chromogranin A (CgA) in isolated bovine adrenal chromaffin cells was investigated using two radioimmunoassays employing site-specific antisera. The two antisera were directed against closely associated regions of the CgA molecule which would be exposed by specific processing: antiserum L331 was raised against the C-terminus of the regulatory peptide pancreastatin, and the second antiserum, L300, was raised against the synthetic peptide [Tyr0]CgA306-313 (YLSKEWEDA), a sequence that lies immediately C-terminal to pancreastatin and adjacent to a dibasic amino acid cleavage site. Chronic reserpine treatment of chromaffin cells produced a time- and dose-dependent increase in processing, as demonstrated by an increase in pancreastatin- and YLSKEWEDA-immunoreactivity (ir). The reserpine-induced rise in pancreastatin-ir was due predominantly to an increase in pancreastatin 1-47, whereas the rise in YLSKEWEDA-ir was due to increases in three polypeptides: a 51-kDa YLSKEWEDA-ir polypeptide, CgA297-313, and CgA248-313. The latter predominated. The action of reserpine on both pancreastatin- and YLSKEWEDA-ir was found to be largely inhibited by the protein synthesis inhibitor cycloheximide. The results show that treatment of isolated chromaffin cells with reserpine induces both the selective proteolytic processing and peptidyl-glycine amidation of CgA and its derived fragments. As reserpine has a similar effect on proenkephalin in chromaffin cells, the results suggest that reserpine induces a general increase in the activity of the processing enzymes, partially by an increase in protein synthesis.     

Chromogranin A as a Crucial Factor in the Sorting of Peptide Hormones to Secretory Granules

Chromogranin A (CgA) is a soluble glycoprotein stored along with hormones and neuropeptides in secretory granules of endocrine cells. In the last four decades, intense efforts have been concentrated to characterize the structure and the biological function of CgA. Besides, CgA has been widely used as a diagnostic marker for tumors of endocrine origin, essential hypertension, various inflammatory diseases, and neurodegenerative disorders such as amyotrophic lateral sclerosis and Alzheimer’s disease. CgA displays peculiar structural features, including numerous multibasic cleavage sites for prohormone convertases as well as a high proportion of acidic residues. Thus, it has been proposed that CgA represents a precursor of biologically active peptides, and a “granulogenic protein” that plays an important role as a chaperone for catecholamine storage in adrenal chromaffin cells. The widespread distribution of CgA throughout the neuroendocrine system prompted several groups to investigate the role of CgA in peptide hormone sorting to the regulated secretory pathway. This review summarizes the findings and theoretical concepts around the molecular machinery used by CgA to exert this putative intracellular function. Since CgA terminal regions exhibited strong sequence conservation through evolution, our work focused on the implication of these domains as potential functional determinants of CgA. Characterization of the molecular signals implicating CgA in the intracellular traffic of hormones represents a major biological issue that may contribute to unraveling the mechanisms defining the secretory competence of neuroendocrine cells.     

Proteolytic cleavage of chromogranin A (CgA) by plasmin. Selective liberation of a specific bioactive CgA fragment that regulates catecholamine release

Chromogranin A (CgA), the major soluble protein in catecholamine storage vesicles, serves as a prohormone that is cleaved into bioactive peptides that inhibit catecholamine release, providing an autocrine, negative feedback mechanism for regulating catecholamine responses during stress. However, the proteases responsible for the processing of CgA and release of bioactive peptides have not been established. Recently, we found that chromaffin cells express components of the plasmin(ogen) system, including tissue plasminogen activator, which is targeted to catecholamine storage vesicles and released with CgA and catecholamines in response to sympathoadrenal stimulation, and high affinity cell surface receptors for plasminogen, to promote plasminogen activation at the cell surface. In the present study, we investigated processing of CgA by plasmin and sought to identify specific bioactive CgA peptides produced by plasmin proteolysis. Highly purified human CgA (hCgA) was produced by expression in Escherichia coli and purification using metal affinity chromatography. hCgA was digested with plasmin. Matrix-assisted laser desorption/ionization mass spectrometry identified a major peptide produced with a mass/charge ratio (m/z) of 1546, corresponding uniquely to hCgA-(360-373), the identity of which was confirmed by reverse phase high pressure liquid chromatography and amino-terminal microsequencing. hCgA-(360-373) was selectively liberated by plasmin from hCgA at early time points and was stable even after prolonged exposure to plasmin. The corresponding synthetic peptide markedly inhibited nicotine-induced catecholamine release from pheochromocytoma cells. These results identify plasmin as a protease, present in the local environment of the chromaffin cell, that selectively cleaves CgA to generate a bioactive fragment, hCgA-(360-373), that inhibits nicotinic-mediated catecholamine release. These results suggest that the plasminogen/plasmin system through its interaction with CgA may play a major role in catecholaminergic function and suggest a specific mechanism as well as a discrete CgA peptide through which this effect is mediated.     

Transforming growth factor beta1 and soluble Fas serum levels in hepatocellular carcinoma

In this study we assessed the usefulness of serum Transforming Growth Factor-beta1 (TGF-beta1) and soluble Fas (sFas) in distinguishing liver cirrhosis (LC) with and without hepatocellular carcinoma (HCC) as compared with alpha-fetoprotein (AFP). Serum TGF-beta1 and sFas levels were measured by ELISA in 51 LC patients, 54 patients with HCC and 30 healthy donors. Considering as a cut-off limit (mean+1SD of controls) 74 pg/ml and 637 pg/ml for TGF-beta1 and sFas, respectively, we computed serum concentrations of TGF-beta1 and sFas as a score (mean+/-SD). The positive frequency of serum TGF-beta1 levels in HCC patients (54%) was greater than in LC patients (26%) and healthy donors (3%). TGF-beta1 levels were higher in HCC (1.6+/-0.5) than in LC (1.1+/-0.2) (P<0.0001) and healthy donors (0.6+/-0.2). Using a cut-off limit of 82 pg/ml (mean+2SD), the positive frequency of TGF-beta1 was 20% in HCC patients. None of the controls and LC patients had TGF-beta1 levels higher than 82 pg/ml. The positive frequency of serum sFas levels was 100% in HCC patients, 98% in LC patients and 3% in healthy controls. Serum sFas levels were higher in HCC (2.5+/-0.7) than in LC (1.9+/-0.5) (P<0. 001) and healthy donors (0.6+/-0.3). No significant change of positive frequency was obtained by setting sFas cut-off at higher levels. sFas values did not correlate with TGF-beta1 levels. No relationship was found between TGF-beta1 amounts and AFP levels. However, in the 23% of HCC patients, with normal AFP values TGF-beta1 levels were higher than the cut off. These findings suggest the potential usefulness for TGF-beta1 assay in AFP-negative HCC.     

Limitations of Chromogranin A in clinical practice

Context: Usefulness of circulating Chromogranin A (CgA) for the diagnosis of neuroendocrine tumors (NEN) is controversial. The aim of the present study was to assess the actual role of this marker as diagnostic tool. Methods: Serum blood samples were obtained from 42 subjects affected with NEN, 120 subjects affected with non-endocrine neoplasias (non-NEN) and 100 non-neoplastic subjects affected with benign nodular goitre (NNG). Determination of CgA was performed by means of immunoradiometric assay. Results: The CgA levels among NEN-patients were not significantly different from NNG and non-NEN subjects. The Receiver operating characteristic (ROC) curves analysis failed to identify a feasible cut-off value for the differential diagnosis between NEN and the other conditions. Conclusion: Serum CgA is not helpful for the first-line diagnosis of NEN.