Counter-regulatory responses to postprandial hypoglycaemia in patients with post-bariatric hypoglycaemia vs surgical and non-surgical control individuals.

AIMS/HYPOTHESIS: Post-bariatric hypoglycaemia is an increasingly recognised complication of bariatric surgery, manifesting particularly after Roux-en-Y gastric bypass. While hyperinsulinaemia is an established pathophysiological feature, the role of counter-regulation remains unclear. We aimed to assess counter-regulatory hormones and glucose fluxes during insulin-induced postprandial hypoglycaemia in patients with post-bariatric hypoglycaemia after Roux-en-Y gastric bypass vs surgical and non-surgical control individuals. METHODS: In this case-control study, 32 adults belonging to four groups with comparable age, sex and BMI (patients with post-bariatric hypoglycaemia, Roux-en-Y gastric bypass, sleeve gastrectomy and non-surgical control individuals) underwent a postprandial hypoglycaemic clamp in our clinical research unit to reach the glycaemic target of 2.5 mmol/l 150-170 min after ingesting 15 g of glucose. Glucose fluxes were assessed during the postprandial and hypoglycaemic period using a dual-tracer approach. The primary outcome was the incremental AUC of glucagon during hypoglycaemia. Catecholamines, cortisol, growth hormone, pancreatic polypeptide and endogenous glucose production were also analysed during hypoglycaemia. RESULTS: The rate of glucose appearance after oral administration, as well as the rates of total glucose appearance and glucose disappearance, were higher in both Roux-en-Y gastric bypass groups vs the non-surgical control group in the early postprandial period (all p<0.05). During hypoglycaemia, glucagon exposure was significantly lower in all surgical groups vs the non-surgical control group (all p<0.01). Pancreatic polypeptide levels were significantly lower in patients with post-bariatric hypoglycaemia vs the non-surgical control group (median [IQR]: 24.7 [10.9, 38.7] pmol/l vs 238.7 [186.3, 288.9] pmol/l) (p=0.005). Other hormonal responses to hypoglycaemia and endogenous glucose production did not significantly differ between the groups. CONCLUSIONS/INTERPRETATION: The glucagon response to insulin-induced postprandial hypoglycaemia is lower in post-bariatric surgery individuals compared with non-surgical control individuals, irrespective of the surgical modality. No significant differences were found between patients with post-bariatric hypoglycaemia and surgical control individuals, suggesting that impaired counter-regulation is not a root cause of post-bariatric hypoglycaemia. TRIAL REGISTRATION: ClinicalTrials.gov NCT04334161.

Multiplexed Assay to Quantify the PP-Fold Family of Peptides in Human Plasma Using Microflow Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Peptide Tyr-Tyr (PYY1-36), pancreatic polypeptide (PP1-36) and neuropeptide Y (NPY1-36) constitute the PP-fold family of peptides that is involved in metabolic regulation. Very low plasma concentrations and cleavage into active 3-36 fragments challenge bioanalytical assays used for the quantification of these peptides. METHODS: We developed a multiplexed isotopic dilution assay to quantify PYY1-36, PP1-36, and NPY1-36 and their dipeptidyl peptidase-4 (DPP4)-derived metabolites PYY3-36, PP3-36 and NPY3-36. All peptides were immunocaptured from plasma using a monoclonal antibody and quantified by micro-ultra-HPLC-MS/MS. Blood samples from healthy volunteers were collected fasting and 30 min after nutrient stimulation. Method comparison was performed with commercial immunoassays. RESULTS: Linearity was shown in the measured intervals (r2 > 0.99). The lower limit of quantification (LLOQ) with a CV at 20% was 1.5 pM for PYY1-36 and PYY3-36, 3.0 pM for PP1-36 and PP3-36, 0.8 pM for NPY1-36 and 0.5 pM for NPY3-36. In all cases, intra- and inter-assay bias and imprecision were <21%. Pre-analytical stability required addition of a protease inhibitor cocktail. Physiological concentrations of PYY3-36, NPY3-36, PP1-36 and PP3-36 were above the LLOQ in 43% to 100% of the samples. PYY1-36 and NPY1-36 were above the LLOQ in 9% and 0% of the samples, respectively. Immunoassays showed higher concentrations of measurands and poor agreement when compared with micro-UHPLC-MS/MS. CONCLUSIONS: The assay allowed for specific multiplexed analysis of the PP-fold family of peptides and their DPP4-cleaved fragments in a single sample, thereby offering new perspectives to study the role and therapeutic potential of these essential peptide hormones in health and metabolic disease.

Detection of erythropoiesis-stimulating agents in a single dried blood spot.

The use of dried blood spots (DBS) for anti-doping purposes would facilitate an increase in the number of blood samples because it eliminates the need for specialized personnel and involves minimal invasiveness, reduced costs, stability, and easy transportation and storage. Here, the electrophoretic methodology established by the World Anti-Doping Agency (WADA) to detect erythropoiesis-stimulating agents (ESAs) has been adapted to evaluate their applicability to DBS. A qualitative procedure to detect recombinant erythropoietin (rEPO), novel erythropoiesis-stimulating protein (NESP), and continuous erythropoietin receptor activator (CERA) in a single DBS was optimized and validated. For rEPO and NESP, confirmation was performed in finger-prick DBS from a pilot study and an administration patients study, respectively. For CERA, detection capabilities were evaluated in DBS prepared with modeled-blood spiked with known concentrations of the protein. Main validation parameters concerning DBS sampling such as stability, hematocrit influence, and blood type (capillary vs. venous) described minor variations. Onsite drying appeared not to be essential before transport. Intra- and inter-day variation range was 2.9%-23.5%. Linearity was maintained (r ≥ 0.9) and ESAs were robustly recovered (CV ≤ 20.2%). The validated method permitted the detection of treated subjects after 48 hours and 17 days of rEPO and NESP administration, respectively. The reproduction of a CERA pharmacokinetics showed good possibilities for the method with a detection window that could reach 16 days after its actual administration. Thus, results provided here reinforce the suitability of DBS blood sampling for the analysis of ESA misuse in sports drug testing.

Reversible glutathionylation of Sir2 by monothiol glutaredoxins Grx3/4 regulates stress resistance.

The regulatory mechanisms of yeast Sir2, the founding member of the sirtuin family involved in oxidative stress and aging, are unknown. Redox signaling controls many cellular functions, especially under stress situations, with dithiol glutaredoxins (Grxs) playing an important role. However, monothiol Grxs are not considered to have major oxidoreductase activity. The present study investigated the redox regulation of yeast Sir2, together with the role and physiological impact of monothiol Grx3/4 as Sir2 thiol-reductases upon stress. S-glutathionylation of Sir2 upon disulfide stress was demonstrated both in vitro and in vivo, and decreased Sir2 deacetylase activity. Physiological levels of nuclear Grx3/4 can reverse the observed post-translational modification. Grx3/4 interacted with Sir2 and reduced it after stress, thereby restoring telomeric silencing activity. Using site-directed mutagenesis, key cysteine residues at the catalytic domain of Sir2 were identified as a target of S-glutathionylation. Mutation of these residues resulted in cells with increased resistance to disulfide stress. We provide new mechanistic insights into Grx3/4 regulation of Sir2 by S-deglutathionylation to increase cell resistance to stress. This finding offers news perspectives on monothiol Grxs in redox signaling, describing Sir2 as a physiological substrate regulated by S-glutathionylation. These results might have a relevant role in understanding aging and age-related diseases.

Sir2 is induced by oxidative stress in a yeast model of Huntington disease and its activation reduces protein aggregation.

Huntington disease (HD) is a neurodegenerative disorder caused by expansion of CAG trinucleotide repeats, leading to an elongated polyglutamine sequence (polyQ) in the huntingtin protein. Misfolding of mutant polyQ proteins with expanded tracts results in aggregation, causing cytotoxicity. Oxidative stress in HD has been documented in humans as important to disease progression. Using yeast cells as a model of HD, we report that when grown at high glucose concentration, cells expressing mutant polyQ do not show apparent oxidative stress. At higher cell densities, when glucose becomes limiting and cells are metabolically shifting from fermentation to respiration, protein oxidation and catalase activity increases in relation to the length of the polyQ tract. Oxidative stress, either endogenous as a result of mutant polyQ expression or exogenously generated, increases Sir2 levels. Δ sir2 cells expressing expanded polyQ lengths show signs of oxidative stress even at the early exponential phase. In a wild-type background, isonicotinamide, a Sir2 activator, decreases mutant polyQ aggregation and the stress generated by expanded polyQ. Taken together, these results describe mutant polyQ proteins as being more toxic in respiring cells, causing oxidative stress and an increase in Sir2 levels. Activation of Sir2 would play a protective role against this toxicity.

Manganese is the link between frataxin and iron-sulfur deficiency in the yeast model of Friedreich ataxia.

Friedreich ataxia is a human neurodegenerative and myocardial disease caused by decreased expression of the mitochondrial protein frataxin. Proteomic analysis of the mutant yeast model of Friedreich ataxia presented in this paper reveals that these cells display increased amounts of proteins involved in antioxidant defenses, including manganese-superoxide dismutase. This enzyme shows, however, lower activity than that found in wild type cells. Our results indicate that this lack of activity is a consequence of cellular manganese deficiency, because in manganese-supplemented cultures, cell manganese content, and manganese-superoxide dismutase activity were restored. One of the hallmarks of Friedreich ataxia is the decreased activity of iron/sulfur-containing enzymes. The activities of four enzymes of this group (aconitase, glutamate synthase, succinate dehydrogenase, and isopropylmalate dehydratase) have been analyzed for the effects of manganese supplementation. Enzyme activities were recovered by manganese treatment, except for aconitase, for which, a specific interaction with frataxin has been demonstrated previously. Similar results were obtained when cells were grown in iron-limited media suggesting that manganese-superoxide dismutase deficiency is a consequence of iron overload. In conclusion, these data indicate that generalized deficiency of iron-sulfur protein activity could be a consequence of manganese-superoxide dismutase deficiency, and consequently, it opens new strategies for Friedreich ataxia treatment.

Tumor-infiltrating cells as a prognostic factor in Hodgkin's lymphoma: a quantitative tissue microarray study in a large retrospective cohort of 267 patients.

The present study aimed to describe the general tissular composition of the immune infiltrate observed in Hodgkin's lymphoma (HL) and its possible relationship with clinical and survival prognostic factors. In this retrospective study of 267 HL patients, the relative proportions of infiltrating T lymphocytes (CD4+, CD8+), natural killer cells (CD 56+, CD 57+), cytotoxic cells (Granzyme B+, TIA-1+) and dendritic cells (CD 21+, S-100+) were quantified immunohistochemically with tissue microarray technology. Our results confirm the predominance of CD4 + T lymphocytes in the background of tumoral cells, in addition to a high number of cytotoxic lymphocytes (CD8, CD 57 and TIA-1). Patients with low numbers of infiltrating CD8, CD 56, CD 57+cells and high numbers of Granzyme B and TIA-1+cells presented a significantly unfavourable clinical course (presence of leukocytosis, B symptoms, advanced clinical stage (III/IV), non-responding patients). A reduced infiltration of CD4+T lymphocytes was related with the presence of Epstein - Barr virus. Significantly longer survival times were observed in patients with a high level of infiltrating CD 57, as well as a low level of Granzyme B and TIA-1+cells (log-rank test). When evaluated in a multivariate model, high levels of infiltrating TIA-1 and Granzyme B+cells were shown to be independent prognostic factors that negatively influenced overall survival. The presence of TIA-1+cells was found to be the only unfavorable prognostic factor of event-free survival and disease-free survival. The overall detection of tumor-infiltrating cells in HL confirms the importance of cytotoxic T lymphocyte infiltration (Granzyme B and TIA-1+cells) in these patients. Independently of the classical clinical and pathological features, these cells appear to be an unfavourable prognostic factor in HL and, more particularly, the presence of cytotoxic TIA-1+cells.

Genomic imbalances associated with methotrexate resistance in human osteosarcoma cell lines detected by comparative genomic hybridization-based techniques.

Methotrexate (MTX) is one of the most important drugs for osteosarcoma (OS) treatment. To identify genetic aberrations associated with the development of MTX resistance in OS cells, in addition to the previously reported expression changes of dihydrofolate reductase (DHFR) and reduced folate carrier (RFC) genes, comparative genomic hybridization (CGH)-based techniques were used. The direct comparison between MTX-resistant variants of U-2OS or Saos-2 human OS cell lines with their respective parental cell lines by CGH on chromosomes revealed that development of MTX resistance was associated with gain of the chromosomal regions 5q12-q15 and 11q14-qter in U-2OS variants, and with gain of 8q22-qter in Saos-2 variants. Further analyses by CGH on microarrays demonstrated a progressively increasing gain of mixed lineage leukemia (MLL) gene (11q23) in U-2OS MTX-resistant variants, which was also confirmed by fluorescence in situ hybridization (FISH), in addition to gain of FGR (1p36), amplification/overexpression of DHFR, and slight decrease of RFC expression. In Saos-2 MTX-resistant variants, gain of MYC (8q24.12-q24.13) was detected, together with a remarkable decrease of RFC expression. Further analyses of DHFR, MLL, MYC, and RFC gene status in four additional human OS cell lines revealed that only gain of DHFR and MLL were associated with an inherent lower sensitivity to MTX. These data demonstrate that genetic analyses with complementary techniques are helpful for the identification of new candidate genes, which might be considered for an early identification of MTX unresponsive tumors.

Value of P-glycoprotein and clinicopathologic factors as the basis for new treatment strategies in high-grade osteosarcoma of the extremities.

PURPOSE: To evaluate the prognostic value of P-glycoprotein and clinicopathologic parameters in a large series of high-grade osteosarcoma (OS) patients treated at the Rizzoli Institute. PATIENTS AND METHODS: With the use of immunohistochemistry, P-glycoprotein was assessed in 149 patients with primary, nonmetastatic, high-grade OS who were homogeneously treated with chemotherapy protocols based on doxorubicin, high-dose methotrexate, and cisplatin and the addition of ifosfamide in the postoperative phase. RESULTS: P-glycoprotein positivity was found in 47 of 149 cases (32%) and was significantly associated with a higher incidence of relapse and a worse outcome, as was age younger than 12 years and tumor volume greater then 150 mL at diagnosis. Multivariate analysis further confirmed the prognostic value of these parameters, which all were independent adverse prognostic factors. Event-free survival and proportional hazards regression analyses confirmed that overexpression of P-glycoprotein at clinical onset is the most important adverse prognostic factor for high-grade OS patients treated with these chemotherapy protocols. CONCLUSION: Increased P-glycoprotein levels, together with tumor volume and age, should be taken into consideration to identify, at time of diagnosis, subgroups of OS patients with a higher risk of recurrence. This subgroup identification will constitute the basis for drawing individualized treatment protocols on the basis of risk evaluation, with the aim of using more aggressive chemotherapy, or combination chemotherapy with other adjuvants, only in those patients for which more aggressive regimens are strictly necessary and warranted.

Effectiveness of Ecteinascidin-743 against drug-sensitive and -resistant bone tumor cells.

PURPOSE: The identification of new drugs is strongly needed for bone tumors.Ecteinascidin-743 (ET-743), a highly promising antitumor agent isolated from the marine tunicate Ecteinascidia turbinata, is currently under Phase II clinical investigation in Europe and the United States for treatment of soft tissue sarcoma. In this study, we analyzed the preclinical effectiveness of this drug in osteosarcoma and Ewing's sarcoma. EXPERIMENTAL DESIGN: The effects of ET-743 were evaluated against a panel of human osteosarcoma and Ewing's sarcoma cell lines characterized by different drug responsiveness and compared with the effects of standard anticancer agents. In addition, combination treatments with ET-743 and the other standard chemotherapy agents for sarcoma were analyzed to highlight the best drug-to-drug interaction RESULTS: A potent activity of ET-743 was clearly observed against both drug-sensitive and drug-resistant (multidrug-resistant, methotrexate- and cisplatin-resistant) bone tumor cells at concentrations that are easily achievable in patients (pM to nM range). Ewing's sarcoma cells appeared to be particularly sensitive to the effects of this drug. The analysis of the effects of ET-743 on cell cycle, apoptosis, and differentiation indicated that both osteosarcoma and Ewing's sarcoma cells had a slower progression through the different phases of the cell cycle after treatment with ET-743. However, the drug was able to induce a massive apoptosis in Ewing's sarcoma but not in osteosarcoma cells. In the latter neoplasm, ET-743 showed a differential effect, as indicated by the significant increase in the expression and activity of alkaline phosphatase, a marker of osteoblastic differentiation. Concurrent exposure of cells to ET-743 and other chemotherapeutic agents resulted in greater than additive interactions when doxorubicin and cisplatin were used, whereas subadditive effects were observed with methotrexate, vincristine, and actinomycin D. CONCLUSIONS: Overall, these results encourage the inclusion of this drug in the treatment of patients with bone tumors, although a careful design of new regimens is required to identify the best therapeutic conditions.