Role of chromogranin A-derived fragments after resection of nonfunctioning pancreatic neuroendocrine tumors.

PURPOSE: No single reliable biomarker is available for nonfunctioning pancreatic neuroendocrine tumors (NF-PanNETs). Vasostatin-1 (VS-1), the N-terminal fragment of chromogranin A (CgA), seems to be a more accurate biomarker compared to its precursor. Primary aim was to investigate the ability of VS-1, compared to total-CgA, to assess the effectiveness of surgical resection performed for NF-PanNETs. Secondary aim was to evaluate two additional CgA-derived fragments, pancreastatin (PST) and vasostatin-2 (VS-2), as possible biomarkers for NF-PanNETs. METHODS: Consecutive patients who underwent surgery for NF-PanNETs at San Raffaele Scientific Institute were included (n = 35). Plasma levels of CgA and CgA-derived fragments were measured by Enzyme-Linked ImmunoSorbent Assay (ELISA), preoperatively and postoperatively. RESULTS: Preoperative VS-1 was significantly higher compared to VS-1 measured on postoperative day 5 (POD5) (pre: 0.338 nM versus POD5: 0.147 nM, P < 0.001), whereas total-CgA significantly increased after surgery (pre: 1.123 nM versus POD5: 1.949 nM, P = 0.006). Overall, 24 patients showed ≥ 1 feature of tumor aggressiveness (T3-T4, nodal/distant metastases, Ki67 > 5%, microvascular/perineural invasion, necrosis). The median percentage decrease in VS-1 plasma levels was 63% (IQR 28-88%) among patients with aggressive tumors, compared to 13% (IQR 0-57%) in the remaining population (P = 0.033). No significant differences in terms of PST (P = 0.870) and VS-2 (P = 0.909) were observed between preoperative and postoperative time. CONCLUSION: VS-1 provides an early assessment of surgical efficacy in patients who undergo resection for NF-PanNETs, especially in those with aggressive neoplasms. Total-CgA, PST and VS-2 have no clinical utility in this setting.

Optimization of photon storage fidelity in ordered atomic arrays.

A major application for atomic ensembles consists of a quantum memory for light, in which an optical state can be reversibly converted to a collective atomic excitation on demand. There exists a well-known fundamental bound on the storage error, when the ensemble is describable by a continuous medium governed by the Maxwell-Bloch equations. However, these equations are semi-phenomenological, as they treat emission of the atoms into other directions other than the mode of interest as being independent. On the other hand, in systems such as dense, ordered atomic arrays, atoms interact with each other strongly and spatial interference of the emitted light might be exploited to suppress emission into unwanted directions, thereby enabling improved error bounds. Here, we develop a general formalism that fully accounts for spatial interference, and which finds the maximum storage efficiency for a single photon with known spatial input mode into a collection of atoms with discrete, known positions. As an example, we apply this technique to study a finite two-dimensional square array of atoms. We show that such a system enables a storage error that scales with atom number N a like ∼ ( log N a ) 2 ∕ N a 2 , and that, remarkably, an array of just 4 × 4 atoms in principle allows for an error of less than 1%, which is comparable to a disordered ensemble with an optical depth of around 600.

The impact of the non-invasive follicular thyroid neoplasm with papillary-like nuclear feature terminology in the routine diagnosis of thyroid tumours.

BACKGROUND: Due to the recent proposal of the non-invasive follicular thyroid neoplasm with papillary-like nuclear feature (NIFTP) category, the authors analyse the state of the art in the challenging diagnosis of follicular thyroid neoplasms in routine practice. METHODS AND RESULTS: A consecutive series of 200 histological diagnoses, with complete cytological correlation, was analysed following the introduction of the NIFTP definition. The study was conducted in a general hospital with a high prevalence of thyroid benign nodules that accounted for approximately 60% of surgically-treated nodules. The significant incidence of the new NIFTP category was 7%. Concurrently, a gradual decrease of the follicular variant of papillary thyroid carcinoma (fvPTC) was observed (3.5%). When evaluating the FNA biopsies within the NIFTP group, despite the systematic evaluation of nuclear crowding, enlargement, irregularities and clearing, the final cytological class was often indeterminate for malignancy (Thy3/III-IV, 71%). At histology, the application of the semiquantitative NIFTP score for the evaluation of the PTC-like nuclear features was able to discriminate benign lesions (score 0/1) from fvPTC (score 2/3). A certain degree of overlapping still persisted between NIFTP and fvPTC (score 2) or between NIFTP and benign lesions (score 1). CONCLUSIONS: In the routine evaluation of FNA biopsies, the presence of subtle and questionable PTC-like nuclear features still remains a controversial aspect of the diagnostic workflow. Given that the NIFTP category was introduced to stratify the low-risk group of thyroid tumours more precisely, pathologists should force themselves to apply the nuclear score rigorously and to classify cases assigned a score of 1 as benign proliferations.

Glutathione-enriched baker's yeast: production, bioaccessibility and intestinal transport assays.

AIMS: A glutathione (GSH) yeast-based biomass (Saccharomyces cerevisiae) was used to investigate GSH stability, solubilization during gastrointestinal digestion and GSH intestinal transport. METHODS AND RESULTS: A postgrowing procedure was applied to improve intracellular GSH yeast content. The presence of adenine (ADE) in the biotransformation solution (CYS-GLY-GLU mixture) and alternatively, a glucose shot after 4-h incubation, allowed to obtain cells containing about GSH 1.6-1.7% dcw (dry cell weight) (control 0.5%). Yeast samples were subjected to in vitro gastrointestinal digestion and absorption assays employing Caco-2 and HT29-MTX cell lines in different proportions (100/0, 70/30 and 50/50). Trials were also performed to verify intestinal cell viability. CONCLUSIONS: At least 87% of ingested GSH is available in reduced form for intestinal absorption. In vitro GSH transport assays indicated that GSH is poorly absorbed (<20%). Nevertheless, studies in response to oxidative stress induced by H2 O2 demonstrated a protective role of the GSH-enriched biomass towards intestinal cell viability. SIGNIFICANCE AND IMPACT OF STUDY: An enriched GSH yeast-based biomass has been obtained using a postgrowing procedure. Although GSH present in enriched yeasts is poorly absorbed by intestinal cells, this biomass showed an intestinal local protective effect, improving cells viability when a simulated oxidative stress was applied.

SLC7A7, encoding a putative permease-related protein, is mutated in patients with lysinuric protein intolerance.

Lysinuric protein intolerance (LPI, MIM 222700) is an autosomal recessive multisystem disorder found mainly in Finland and Italy. On a normal diet, LPI patients present poor feeding, vomiting, diarrhoea, episodes of hyperammoniaemic coma and failure to thrive. Hepatosplenomegaly, osteoporosis and a life-threatening pulmonary involvement (alveolar proteinosis) are also seen. LPI is caused by defective cationic amino acid (CAA) transport at the basolateral membrane of epithelial cells in kidney and intestine. Metabolic derangement is characterized by increased renal excretion of CAA, reduced CAA absorption from intestine and orotic aciduria. The gene causing LPI was assigned using linkage analysis to chromosome 14q11.2 near the T-cell receptor alpha/delta chains locus, and a critical region has been defined. We have identified two new transcripts (SLC7A8 and SLC7A7) homologous to amino acid transporters, highly expressed in kidney and mapping in the LPI critical region. Mutational analysis of both transcripts revealed that SLC7A7 (for solute carrier family 7, member 7) is mutated in LPI. In five Italian patients, we found either an insertion or deletion in the coding sequence, which provides evidence of a causative role of SLC7A7 in LPI. Furthermore, we detected a splice acceptor change resulting in a frameshift and premature translation termination in four unrelated Finnish patients. This mutation may represent the founder LPI allele in Finland.

Non-type I cystinuria caused by mutations in SLC7A9, encoding a subunit (bo,+AT) of rBAT.

Cystinuria (MIM 220100) is a common recessive disorder of renal reabsorption of cystine and dibasic amino acids. Mutations in SLC3A1, encoding rBAT, cause cystinuria type I (ref. 1), but not other types of cystinuria (ref. 2). A gene whose mutation causes non-type I cystinuria has been mapped by linkage analysis to 19q12-13.1 (Refs 3,4). We have identified a new transcript, encoding a protein (bo, +AT, for bo,+ amino acid transporter) belonging to a family of light subunits of amino acid transporters, expressed in kidney, liver, small intestine and placenta, and localized its gene (SLC7A9) to the non-type I cystinuria 19q locus. Co-transfection of bo,+AT and rBAT brings the latter to the plasma membrane, and results in the uptake of L-arginine in COS cells. We have found SLC7A9 mutations in Libyan-Jews, North American, Italian and Spanish non-type I cystinuria patients. The Libyan Jewish patients are homozygous for a founder missense mutation (V170M) that abolishes b o,+AT amino-acid uptake activity when co-transfected with rBAT in COS cells. We identified four missense mutations (G105R, A182T, G195R and G295R) and two frameshift (520insT and 596delTG) mutations in other patients. Our data establish that mutations in SLC7A9 cause non-type I cystinuria, and suggest that bo,+AT is the light subunit of rBAT.

Natural history of gastro-entero-pancreatic and thoracic neuroendocrine tumors. Data from a large prospective and retrospective Italian epidemiological study: the NET management study.

BACKGROUND: The few epidemiological data available in literature on neuroendocrine tumors (NET) are mainly based on Registry databases, missing therefore details on their clinical and natural history. AIM: To investigate epidemiology, clinical presentation, and natural history of NET. DESIGN AND SETTING: A large national retrospective survey was conducted in 13 Italian referral centers. Among 1203 NET, 820 originating in the thorax (T-NET), in the gastro-enteropancreatic tract (GEP-NET) or metastatic NET of unknown primary origin (U-NET) were enrolled in the study. RESULTS: 93% had a sporadic and 7% a multiple endocrine neoplasia type 1 (MEN1)-associated tumor; 63% were GEP-NET, 33% T-NET, 4% U-NET. Pancreas and lung were the commonest primary sites. Poorly differentiated carcinomas were <10%, all sporadic. The incidence of NET had a linear increase from 1990 to 2007 in all the centers. The mean age at diagnosis was 60.0 ± 16.4 yr, significantly anticipated in MEN1 patients (47.7 ± 16.5 yr). Association with cigarette smoking and other non-NET cancer were more prevalent than in the general Italian population. The first symptoms of the disease were related to tumor burden in 46%, endocrine syndrome in 23%, while the diagnosis was fortuity in 29%. Insulin (37%) and serotonin (35%) were the most common hormonal hypersecretions. An advanced tumor stage was found in 42%, more frequently in the gut and thymus. No differences in the overall survival was observed between T-NET and GEP-NET and between sporadic and MEN1-associated tumors at 10 yr from diagnosis, while survival probability was dramatically reduced in U-NET. CONCLUSIONS: The data obtained from this study furnish relevant information on epidemiology, natural history, and clinico-pathological features of NET, not available from the few published Register studies.

Sodium hydroxide pretreatment of ensiled sorghum forage and wheat straw to increase methane production.

The aim of this study was to determine the effect of sodium hydroxide pretreatment on the chemical composition and the methane production of ensiled sorghum forage and wheat straw. NaOH pretreatment was conducted in closed bottles, at 40 °C for 24 h. Samples were soaked in a NaOH solution at different dosages (expressed in terms of total solids (TS) content) of 1 and 10% gNaOH/gTS, with a TS concentration of 160 gTS/L. At the highest NaOH dosage the reduction of cellulose, hemicelluloses and lignin was 31, 66 and 44%, and 13, 45 and 3% for sorghum and wheat straw, respectively. The concentration of soluble chemical oxygen demand (CODs) in the liquid phase after the pretreatment was also improved both for wheat straw and sorghum (up to 24 and 33%, respectively). Total sugars content increased up to five times at 10% gNaOH/gTS with respect to control samples, suggesting that NaOH pretreatment improves the hydrolysis of cellulose and hemicelluloses. The Biochemical Methane Potential (BMP) tests showed that the NaOH pretreatment favoured the anaerobic degradability of both substrates. At 1 and 10% NaOH dosages, the methane production increased from 14 to 31% for ensiled sorghum forage and from 17 to 47% for wheat straw. The first order kinetic constant increased up to 65% for sorghum and up to 163% for wheat straw.

Circulating endothelial cells and endothelial progenitors as predictive markers of clinical response to bevacizumab-based first-line treatment in advanced colorectal cancer patients.

BACKGROUND: Despite the consistent clinical results demonstrated by studies on anti-angiogenic drugs targeted against the vascular endothelial growth factor in metastatic colorectal cancer (mCRC) patients, no specific direct/indirect biomarker of their efficacy has been validated. In this field, circulating endothelial cells (CECs) and endothelial progenitor cells (CEPs) have recently been proposed as noninvasive biomarkers. PATIENTS AND METHODS: The absolute numbers of CEPs, total CECs (tCECs) and their resting (rCECs) and activated subsets were evaluated by multiparameter flow cytometry in 40 mCRC patients at baseline and before the administration of the third and sixth course of a bevacizumab-based first-line treatment. Fifty healthy subjects were utilized as control. RESULTS: The overall response rate was 80%, overall clinical benefit was 90% and median progression-free survival (PFS) was 13.8 months. In our patients, tCECs and rCECs were significantly increased compared with healthy subjects. The patients who achieved a radiological response showed, at baseline, a significant decrease of rCECs and a trend in decrease of tCECs in comparison with patients not achieving response. Finally, a baseline absolute number of tCEC and rCEC <40 cells/ml was evidenced in patients with a longer PFS. No correlation was found regarding CEP. CONCLUSIONS: Our study suggests significant correlations between both tCEC and rCEC baseline levels and the antitumor efficacy of a bevacizumab-based combination therapy in mCRC patients, thus confirming that these biomarkers could be used in the clinical setting as an early predictor of tumor response.

Immunological effects of bevacizumab-based treatment in metastatic colorectal cancer.

OBJECTIVE: The efficacy of bevacizumab in metastatic colorectal cancer (mCRC) could be related not only to its well-known antiangiogenetic properties but also to a hypothetical effect on the immune system of the host. METHODS: We enrolled mCRC patients treated with a bevacizumab-based first-line therapy. Lymphocyte and dendritic cell subsets were evaluated at baseline, 3rd and 6th cycle. The clinical efficacy was estimated as response rate and progression-free survival. Forty healthy subjects were used as reference. RESULTS: Fifty-one patients were enrolled. In comparison with healthy subjects, they showed a decrease of T and B cell compartments. Bevacizumab ameliorated the impairment of lymphocyte subsets, especially for T cells. Responders showed a trend toward an increase of CD3 (p = 0.07) and CD4 (p = 0.05). Among patients with a progression-free survival >1 year, only CD19 (p = 0.033) and CD20 (p = 0.013) showed a significant increase. No baseline impairment and no significant modification of dendritic cells were found. CONCLUSION: Bevacizumab-based therapy is able to increase B and T cell compartments. The expansion of T lymphocytes could imply an amelioration of dendritic cell-presenting capacity. These effects correlate with a more favourable clinical outcome and could be taken into account in clinical protocols aimed at combining antiangiogenetic-therapy with immunotherapy in mCRC.

Cadmium elicits alterations in mitochondrial morphology and functionality in C3H10T1/2Cl8 mouse embryonic fibroblasts.

BACKGROUND: Cadmium is a widespread carcinogen. We previously showed that the administration of low CdCl2 doses for 24 h to healthy C3H10T1/2Cl8 mouse embryonic fibroblast cell line at the beginning of Cell Transformation Assay (CTA), up regulates genes involved in metal scavenging and antioxidant defense, like metallothioneines, glutathione S-transferases and heat shock proteins. Still, although most cells thrive normally in the following weeks, malignancy is triggered by CdCl2 and leads to the appearance of foci of transformed cells at the end of the CTA. In this work we aim at elucidating the early metabolic deregulation induced by cadmium, underlying healthy cell transformation into malignant cells. METHODS: Respiratory metabolism was investigated through Seahorse Agilent assays, while oxidative stress level was assessed through fluorescent probes; DNA damage was evaluated by Comet assay, and mitochondrial morphology was analyzed in confocal microscopy. RESULTS: Results show that the initial response to CdCl2 involves mitochondria rearrangement into a perinuclear network. However, SOD1 and SOD2 activities are inhibited, leading to increased superoxide anion level, which in turn causes DNA strand breaks. From the metabolic point of view, cells increase their glycolytic flux, while all extra NADH produced is still efficiently reoxidized by mitochondria. CONCLUSIONS: Our results confirm previously shown response against cadmium toxicity; new data about glycolytic increase and mitochondrial rearrangements suggest pathways leading to cell transformation. GENERAL SIGNIFICANCE: In this work we exploit the widely used, well known CTA, which allows following healthy cells transformation into a malignant phenotype, to understand early events in cadmium-induced carcinogenesis.

A six-colour flow cytometric method for simultaneous detection of cell phenotype and apoptosis of circulating endothelial cells.

Blood circulating endothelial cells (CECs), with their resting and activated subsets, (rCECs and aCECs) and circulating progenitors cells (CEPs) are two extremely rare cell populations that are important in tissue vascularization. Their number and function are modulated in diseases involving vascular injury, such as human tumours. Although a consensus on the phenotypic definition of endothelial cells, as well as on the optimal enumeration technique, is still lacking, the number of clinical studies based on assessment of these cells is rapidly expanding, as well as the analytical methods employed. The present study aimed to develop a rapid and sensitive flow cytometric method of quantifying and characterizing CECs (with both their subsets and the apoptotic fraction) and CEPs. We analysed peripheral blood samples from 21 subjects with a six-colour flow cytometric approach allowing detection of the cell phenotype of CECs and CEPs using a monoclonal antibodies panel and a dedicated gating strategy. Apoptotic CECs were detected with Annexin V and dead cells with 7-amino-actinomycin D staining. The described technique proved to be a new, reliable, tool increasing our knowledge of the biology of CECs and CEPs and can readily be applied in the study of many pathological conditions characterized by endothelial damage.

Flow cytometric detection of circulating dendritic cells in healthy subjects.

Dendritic cells (DCs) are the key antigen-presenting cells controlling the initiation of the T cell- dependent immune response. Currently, two peripheral blood DC subsets have been identified on the basis of their CD11c expression. The CD11c-negative (CD11c-) DCs (expressing high levels of CD123) are designated as lymphoid-derived DCs (DC2), whereas the CD11c+/CD123- cells, do identify the myeloid-derived DCs (DC1). A growing number of studies have been conducted in recent years on both the quantitative and functional alterations of DCs and their subsets in different pathological conditions. In the present study we assessed, using two different flow cytometric (FCM) techniques, the normal profile of blood DCs in 50 italian adult healthy subjects (M/F: 25/25, median age 42.5 years, range 20-65). The percentage and the absolute number of DCs and their subsets, were obtained starting from whole blood samples in two ways: 1) by calculating the number of DCs when gated as lineage-negative/ HLA-DR+ and identifing the two subsets as CD11c+ (DC1) and CD123+ (DC2) and 2) by using three specific markers: BDCA.1 (CD11c+ high/CD123+ low, myeloid DCs); BDCA.2 (CD11c-/ CD123+high, lymphoid DCs); BDCA.3 (CD11c+low /CD123-, myeloid DCs). Six parameters, 4-color FCM analysis were perfomed with a BD FACSCanto equipment. The mean values of the percentage and of the absolute number were: 0.5+/-0.2% and 30+/-11 cells/microL for DCs; 0.2+/-0.1% and 15+/-6 cells/microL for DC1; 0.2+/-0.1% and 15+/-7 cells/microL for DC2. The same values were: 0.2+/-0.1% and 16+/-7 cells/microL for BDCA.1; 0.2+/-0.1% and 12+/-7 cells/microL for BDCA.2; 0.02+/-0.01% and 2+/-1 cells/microL for BDCA.3, respectively. Our study confirmes that the two types of FCM analysis are able to identify the DC population. We also provides the first reference values on normal rates and counts of blood DCs in italian adult healthy subjects.

New agents in medical oncology and the risk of venous thromboembolism.

Over the past several years the medical approach to cancer patients has made important steps forward both in the field of novel, selective, antiproliferative agents and more effective supportive therapies. A greater understanding of the molecular pathways regulating cell proliferation and metastasis has led to the identification of a range of targets specifically inhibited by these new drugs. The clinical development of these compounds (the so called "targeted therapies") has shown distinctive adverse effects with respect to standard chemotherapeutic agents but the potential increasing risk of venous thromboembolism remains unvaried. In fact, the incidence of this potentially life-threatening complication in patients receiving standard chemotherapy ranges from about 11% to 20% and even more depending on the type of drug administered and on the possible association with other anti-neoplastic and supportive therapies. In this paper we reviewed all the available evidences concerning the increasing risk of venous thromboembolism in cancer patients during treatment with new agents currently used in medical oncology together with data concerning the clinical value of a concomitant prophylactic anticoagulation. At present, additional information concerning safety in terms of thromboembolic risk of novel biological and molecular therapies should be collected from specifically designed original basic science studies and clinical trials in order to optimize their use in current oncology practice.

lncRNA profiling in early-stage chronic lymphocytic leukemia identifies transcriptional fingerprints with relevance in clinical outcome.

Long non-coding RNAs (lncRNAs) represent a novel class of functional RNA molecules with an important emerging role in cancer. To elucidate their potential pathogenetic role in chronic lymphocytic leukemia (CLL), a biologically and clinically heterogeneous neoplasia, we investigated lncRNAs expression in a prospective series of 217 early-stage Binet A CLL patients and 26 different subpopulations of normal B-cells, through a custom annotation pipeline of microarray data. Our study identified a 24-lncRNA-signature specifically deregulated in CLL compared with the normal B-cell counterpart. Importantly, this classifier was validated on an independent data set of CLL samples. Belonging to the lncRNA signature characterizing distinct molecular CLL subgroups, we identified lncRNAs recurrently associated with adverse prognostic markers, such as unmutated IGHV status, CD38 expression, 11q and 17p deletions, and NOTCH1 mutations. In addition, correlation analyses predicted a putative lncRNAs interplay with genes and miRNAs expression. Finally, we generated a 2-lncRNA independent risk model, based on lnc-IRF2-3 and lnc-KIAA1755-4 expression, able to distinguish three different prognostic groups in our series of early-stage patients. Overall, our study provides an important resource for future studies on the functions of lncRNAs in CLL, and contributes to the discovery of novel molecular markers with clinical relevance associated with the disease.

Restoration of growth hormone (GH) response to GH-releasing hormone in elderly and obese subjects by acute pharmacological reduction of plasma free fatty acids.

GH induces lipolysis in vivo, increasing plasma free fatty acid (FFA) levels; in turn, FFA are able to reduce GH release, and acipimox, a nicotinic acid analog able to block lipolysis, enhances in normal subjects the GH response to GHRH. Obesity and old age are characterized by a blunted GH response to several stimuli, including GHRH; reports also indicate high plasma FFA levels in obesity and sometimes in the elderly. The aim of this study was to evaluate the possible role of FFA in GH release in obese and elderly subjects. According to a randomized, single blind, cross-over protocol, six healthy subjects, six obese subjects, and six elderly subjects received on 2 different days, with a 1-week interval, placebo or acipimox (250 mg, orally) at 0700 and 1100 h; GHRH [GHRH-(1-44)NH2; 50 micrograms in healthy subjects and in elderly subjects, 100 micrograms in obese subjects] was injected iv at 1300 h, and blood samples for evaluation of plasma FFA, blood glucose, serum insulin (IRI), and serum GH levels were taken from 1200 to 1500 h. Plasma FFA levels were always lower (P < 0.05) after acipimox than after placebo (0.03 +/- 0.01 vs. 0.13 +/- 0.02 g/L in healthy subjects, 0.09 +/- 0.01 vs. 0.27 +/- 0.02 g/L in obese, 0.02 +/- 0.005 vs. 0.17 +/- 0.01 g/L in elderly subjects); serum IRI levels were also lower (P < 0.05) after acipimox than after placebo in the three groups of subjects (16 +/- 3 vs. 30 +/- 5, 120 +/- 30 vs. 181 +/- 32, and 21 +/- 3 vs. 49 +/- 9 pmol/L); both FFA (P < 0.05) and IRI levels (P < 0.05) were higher in obese than in healthy or elderly subjects after placebo and acipimox. Blood glucose levels were not different in the three groups of subjects after either placebo or acipimox. The integrated GH response to GHRH-(GH delta area) was always greater (P < 0.05) after acipimox than after placebo (4677 +/- 633 vs. 1599 +/- 373 in healthy, 1469 +/- 230 vs. 343 +/- 114 in obese, 2304 +/- 759 vs. 325 +/- 133 micrograms/L.120 min in elderly subjects); after both placebo and acipimox, the GH delta area was greater (P < 0.05) in healthy subjects than in obese or elderly subjects. The GH delta area of elderly and obese subjects after acipimox was not different from the GH delta area of healthy subjects after placebo. Changes in GH delta areas were not significantly related to changes in FFA or IRI induced by acipimox; in contrast, absolute values of FFA and IRI as well as basal GH levels were all significantly related to the GH delta area. At multiple regression analysis, FFA was the only significant predictor of GH delta area. These data indicate that acute pharmacological reduction of plasma FFA levels restores the blunted GH response to GHRH commonly observed in obese and elderly subjects: however, when lipolysis is blocked to a similar extent, healthy subjects still show a higher GH delta area than obese or elderly subjects. As FFA are the best predictor of the GH delta area, we suggest that in obesity, the blunted GH release is due to high FFA levels, whereas in the elderly there might be an abnormal sensitivity to normal FFA levels.

Assessment of growth hormone (GH) plasma clearance rate, half-life, and volume of distribution in acromegalic patients: the combined GH-octreotide infusion.

In acromegaly, high GH levels are primarily attributable to GH hypersecretion, but the contribution of GH clearance is still under debate and is difficult to assess. In the present study, GH plasma clearance rate (PCR), half-life (t1/2), and volume of distribution (VD) were assessed in seven acromegalic patients and seven normal lean subjects, after suppression of endogenous GH release by octreotide, with use of a constant GH infusion. An octreotide sc infusion was started the night before the day of the test (2100 h) and maintained throughout the study. On the day of the test, exogenous GH was constantly infused iv for 6 h (0900-1500 h) in order to achieve a new steady state of GH levels. After the cessation of GH infusion, the decay curve of serum GH levels was monitored for 1 h. In both groups, GH PCR was calculated from the steady state serum GH levels, and GH t1/2 was estimated from the monoexponential analysis of the GH disappearance curve. Estimates of VD were derived from PCR and t1/2. In acromegalic patients, GH PCR was 2.5 +/- 0.2 mL/kg.min-1, and GH t1/2 and VD were 15.7 +/- 1.0 min and 54.9 +/- 5.5 mL/kg, respectively. GH PCR and GH t1/2 of acromegalic patients were higher and lower, respectively, than those of normal subjects (PCR, 1.7 +/- 0.2 mL/kg.min-1, P < 0.02; t1/2, 18.4 +/- 0.6 min, P < 0.05). VD was not significantly different in the two groups. In summary, in acromegalic patients GH kinetic parameters can be reliably assessed by using a constant GH infusion after suppression of endogenous GH release by octreotide. Our results also indicate that the increased circulating GH levels observed in acromegaly are attributable only to GH overproduction and do not depend on an alteration in the processes of GH distribution or disappearance.

Evidence for an inhibitory effect of physiological levels of insulin on the growth hormone (GH) response to GH-releasing hormone in healthy subjects.

It has been previously reported that in healthy subjects, the acute reduction of free fatty acids (FFA) levels by acipimox enhances the GH response to GHRH. In the present study, the GH response to GHRH was evaluated during acute blockade of lipolysis obtained either by acipimox or by insulin at different infusion rates. Six healthy subjects (four men and two women, 25.8 +/- 1.9 yrs old, mean +/- SE) underwent three GHRH tests (50 micrograms iv, at 1300 h) during: 1) iv 0.9% NaCl infusion (1200-1500 h) after oral acipimox administration (250 mg) at 0700 h and at 1100 h; 2) 0.1 mU.kg-1.min-1 euglycemic insulin clamp (1200-1500 h) after oral acipimox administration (250 mg at 0700 h and at 1100 h); 3) 0.4 mU.kg-1.min-1 euglycemic insulin clamp (1200-1500 h) after oral placebo administration (at 0700 and 1100 h). Serum insulin (immunoreactive insulin) levels were significantly different in the three tests (12 +/- 2, 100 +/- 10, 194 +/- 19 pmol/L, P < 0.06), plasma FFA were low and similar (0.04 +/- 0.003, 0.02 +/- 0.005, 0.02 +/- 0.003, not significant), and the GH response to GHRH was progressively lower (4871 +/- 1286, 2414 +/- 626, 1076 +/- 207 micrograms/L 120 min), although only test 3 was significantly different from test 1 (P < 0.05). Pooling the three tests together, a significant negative regression was observed between mean serum immunoreactive insulin levels and the GH response to GHRH (r = -0.629, P < 0.01). Our results indicate that in healthy subjects, acipimox and hyperinsulinemia produce a similar decrease in FFA levels and that at similar low FFA, the GH response to GHRH is lower during insulin infusion than after acipimox. These data suggest that insulin exerts a negative effect on GH release. Because the insulin levels able to reduce the GH response to GHRH are commonly observed during the day, for instance during the postprandial period, we conclude that the insulin negative effect on GH release may have physiological relevance.

Overexpression of wild-type and mutant mucolipin proteins in mammalian cells: effects on the late endocytic compartment organization.

Mucolipin-1 is a 65-kDa membrane protein encoded by the MCOLN1 gene, which is mutated in patients with mucolipidosis type IV (MLIV), a rare neurodegenerative lysosomal storage disorder. We studied the subcellular localization of wild-type and three different mutant forms (T232P, F408del and F465L) of mucolipin by expressing Myc-tagged proteins in HeLa cells. The overexpressed wild-type mucolipin colocalizes to late endocytic structures and induces an aberrant distribution of these compartments. F408del and F465L MLIV mutant proteins show a distribution similar to the wild-type protein, whereas T232P is retained in the endoplasmic reticulum. Among the mutants, only F408del induces a redistribution of the late endocytic compartment. These findings suggest that the overexpression of the mucolipin cation channel influences the dynamic equilibrium of late endocytic compartments.

Laparoscopic bilateral adrenalectomy for persistent Cushing's disease after transsphenoidal surgery.

BACKGROUND: We performed bilateral laparoscopic adrenalectomies on four patients (three women and one man) with Cushing's disease (pituitary-dependent Cushing's syndrome) showing persistent hypercortisolism after transsphenoidal surgery. METHODS: The technique for bilateral transperitoneal laparoscopic adrenalectomy was derived from the one previously adopted by our group for unilateral adrenalectomy and previously described. Eight trocars were used, of which two were used for both left and right adrenalectomy. RESULTS: Bilateral laparoscopic adrenalectomy was performed in a one-stage procedure in the three women and, because of the abundant abdominal fat of the patient, in a two-stage procedure (after a 1-week interval) in the man. Operating times for the three women were 255 minutes, 230 minutes, and 220 minutes, and for the man 170 minutes for right adrenalectomy and 140 minutes for left adrenalectomy. No surgical or anesthesiologic complications were encountered. All patients were discharged from the hospital within 5 days after operation. At present, after follow-up periods of 23, 8, 6, and 18 months, all patients show remission of Cushing's disease and undetectable cortisol levels. CONCLUSIONS: Our experience suggests that bilateral laparoscopic adrenalectomy is a safe and effective procedure and a valid therapeutic option in patients with Cushing's disease showing persistent hypercortisolism after transsphenoidal surgery. However, the decision to remove both adrenal glands in such patients needs to be weighed against the risk of their having Nelson's syndrome or other long-term complications.