The Effect of the Food Matrix on the In Vitro Bio-Accessibility and IgE Reactivity of Peanut Allergens.

SCOPE: Factors such as food processing, the food matrix, and antacid medication may affect the bio-accessibility of proteins in the gastrointestinal tract and hence their allergenic activity. However, at present they are poorly understood. METHODS AND RESULTS: Roasted peanut flour was incorporated into either a chocolate dessert or cookie matrix and bio-accessibility were assessed using an in vitro digestion system comprising a model chew and simulated gastric and duodenal digestion. Protein digestion was monitored by SDS-PAGE and immunoreactivity analyzed by immunoblotting and immunoassay. IgE reactivity was assessed by immunoassay using serum panels from peanut-allergic subjects. Roasted peanut flour proteins proved highly digestible following gastro-duodenal digestion even when incurred into a food matrix, with only low molecular weight polypeptides of Mr < 8 kDa remaining. When gastric digestion was performed at pH 6.5 (simulating the effect of antacid medication), peanut proteins are not digested; subsequent duodenal digestion is also limited. IgE reactivity of the major peanut allergens Ara h 1, Ara h 2, and Ara h 6, although reduced, was retained after oral-gastro-duodenal digestion irrespective of digestion conditions employed. CONCLUSION: Peanut allergen bio-accessibility is unaffected by the dessert or cookie matrices whilst high intra-gastric pH conditions render allergens more resistant to digestion.

A framework for the in vitro evaluation of cancer-relevant molecular characteristics and mitogenic potency of insulin analogues.

Epidemiological and laboratory studies raise the possibility of a link between clinically prescribed insulin analogues and increased cancer risk. Accordingly, there is a regulatory mandate for cancer-related pre-clinical safety evaluation during insulin analogue development, but currently, there is no standardized framework for such in vitro evaluation. We tested human insulin; the super-mitogenic insulin, X10 and insulin-like growth factor I, in four cancer cell lines with a range of insulin-like growth factor-I receptor (IGF-IR)/IR (insulin receptor) ratios (HCT 116, HT-29, COLO 205 and MCF7) and related these to IGF-IR and IR expression in 17 human adenocarcinomas. All cell types were IR-A isoform dominant. We determined IGF-IR/IR signalling pathway endpoints in dose- and time-varying experiments, and performed mitogenic dose-response equivalent assays to derive EC50 values, and correlated these with IGF-IR/IR ratios. We superimposed relative EC50 values onto data from the literature in a meta-analysis. The IGF-IR/IR ratios varied from <1 to 12 in the selected cell lines; similar pattern ranges were observed in human adenocarcinomas. The three ligands demonstrated differential IR/IGF-IR and Akt phosphorylation, which correlated with cell-specific IGF-IR/IR ratios. Mitogenic profiles of X10 mimicked those for insulin-like growth factor I (IGF-I) and correlated with IGF-IR/IR ratios. The meta-analysis, adding data from five additional studies, supported the hypothesis that ligand mitogenic potency, relative to human insulin, increases with increasing cell-specific IGF-IR/IR ratio. This study established a framework for the in vitro evaluation of cancer-relevant bioassays for comparisons of insulin analogues, and specifically consolidated earlier studies that determination of the cell-specific IGF-IR/IR ratio is crucial for the interpretation of ranking relative biological activities.

High-sensitivity human papilloma virus genotyping reveals near universal positivity in anal squamous cell carcinoma: different implications for vaccine prevention and prognosis.

BACKGROUND: Characterisation of human papilloma virus (HPV) infection in anal squamous cell carcinoma (ASCC) may have dual importance: first, aetiological; second, prognostic, informing outcome after chemo-radiotherapy (CRT). We undertook HPV genotyping, and allelic characterisations, to evaluate the aetiological role of HPV while simultaneously evaluating the impact of HPV genotyping on relapse-free (RFS) and overall survival (OS). METHOD: Dual-primer HPV genotyping (subtypes 6, 11, 16, 18, 31, 33, 45, 52, 58) and DNA sequencing of HPV 16 positive tumours were analysed in 151 consecutively referred ASCCs, previously characterised by immunohistochemistry for p16 expression. In 110 patients treated with CRT, factors influencing RFS and OS were evaluated using univariate and multivariate models. RESULTS: HPV positivity was observed in 95%. HPV 16 accounted for 89%; of these, 64% harboured the T350G E6 variant. HPV 16 positivity was significantly correlated with improved 5-year RFS (62% versus 40%; p = 0.027) and OS (59% versus 38%; p = 0.019). p16 expression was also significantly correlated with improved 5-year RFS (positive versus negative: 65% versus 16%; p < 0.0001) and OS (63% versus 13%; p < 0.0001). In multivariable models that included HPV 16 status, p16 status, sex, and age, p16 expression remained an independent prognostic factor for RFS (p < 0.0001) and OS (p = 0.002). CONCLUSION: In ASCC, near-universal HPV detection rates were demonstrated, higher than generally reported in the literature, and supporting the development of multivalent HPV vaccinations for prevention. By contrast, p16 negatively, but not HPV 16 genotype, is an independent adverse prognosticator after chemo-radiotherapy in patients with ASCC.

Alterations of insulin-like growth factor binding protein 3 (IGFBP-3) glycosylation in patients with breast tumours.

OBJECTIVES: Insulin-like growth factor binding protein-3 (IGFBP-3) is an important modulator of development and progression of breast cancer as it regulates the amount of free, physiologically active IGF-I and IGF-II. Changes in the glycosylation pattern within IGFBP-3 may affect its interaction with ligands. The aim of this study was to investigate whether such changes occur during disease progression. DESIGN AND METHODS: IGFBP-3 in serum samples from healthy women and from women with breast tumours was characterised in terms of its concentration (IRMA), glycosylation moiety (lectin-affinity chromatography) and distribution of molecular species (immunoblotting). RESULTS: In patients with benign tumours the concentration and carbohydrate content of IGFBP-3 was unaltered compared to healthy women. In patients with malignant tumours in most cases these two parameters were unchanged, but there were women whose concentration of IGFBP-3 was reduced and its structure was altered. In non-surviving cancer patients the concentration of IGFBP-3 was significantly reduced and these molecules contained a greater amount of biantennary complex type N-glycans having more mannose, fucose, bisecting GlcNAc and terminal sialic acid residues. CONCLUSION: Our results showed that breast cancer progression causes alterations of IGFBP-3 glycosylation. The extent of changes increases with breast cancer severity.

Characterisation of N-glycans bound to IGFBP-3 in sera from healthy adults.

Human IGFBP-3 contains three potential N-linked glycosylation sites. Published data concerning the type and saccharide composition of the N-glycans is scarce. The aim of this study was to characterise N-glycans covalently attached to IGFBP-3 from sera of healthy adults (men and women). In order to do that a panel of eight lectins covering broad saccharide specificity was used: agarose-immobilised SNA (Sambucus nigra agglutinin), Con A (lectin from Canavalia ensiformis), RCA I (Ricinus communis agglutinin I), PHA-E (Phaseolus vulgaris erythroagglutinin), PHA-L (P. vulgaris leukoagglutinin), succinylated WGA (wheat germ agglutinin), ECL (Erythrina cristagalli lectin) and UEA (Ulex europaeus agglutinin). IGFBP-3 interacted with SNA, Con A, RCA I, PHA-E and, to a much lesser extent, with PHA-L. These results indicate that human IGFBP-3 bears mostly biantennary complex type N-glycans with a very high content of alpha-2,6-linked Sia at their termini. Hybrid type and high-mannose type N-glycans are present, as well as a bisecting GlcNAc residue, which may be core fucosylated. N-glycosylation of IGFBP-3 follows the N-glycosylation pattern of major serum proteins. This study represents a ground for the future research of glycosylation pattern of IGFBP-3 from the circulation of men and women diagnosed with different illnesses.

Enhanced suppression of hormonal and metabolic responses to stress by application of combined spinal-epidural and general anaesthesia compared with combined spinal general anaesthesia during colorectal surgery.

The aim of the study was to determine the impact of combined spinal-epidural and general anesthesia (CSEGA) on the suppression of the hormonal and metabolic response to surgical stress and to compare it with the technique of combined spinal and general anesthesia (CSGA). Sixteen patients referred for elective colorectal surgery randomly divided into two groups on the basis of anesthesia (CSEGA and CSGA). Mean arterial blood pressure, heart rate, haemoglobin saturation, serum glucose, serum cortisol and urinary catecholamines (adrenaline, noradrenaline and dopamine) were determined at four distinct peri-operative time points. During the peri-operative period the overall haemodynamic and respiratory functions in patients that received CSEGA were superior compared to those in patients that received CSGA. Biochemical analysis revealed that in the sera of patients that received CSEGA the concentration of both glucose and cortisol was elevated to a lesser degree than that observed in the sera of patients that received CSGA. Furthermore, a significant reduction in post-operative urinary catecholamine (adrenaline and noradrenaline) excretion was apparent in the CSEGA group. We conclude that CSEGA is a more suitable form of combined anaesthesia for colorectal surgery with a clear advantage of a blunted surgery-mediated neuro-endocrine stress response.

Insulin-like growth factor I (IGF-I) as a sensitive biomarker of catabolism in patients with gastrointestinal diseases.

Gastrointestinal ailments evoke changes in the hypothalamic-pituitary-adrenal (HPA) axis and modulation of hepatic protein synthesis. We examined the catabolic effect of certain primary gastrointestinal diseases and surgery on the concentration of insulin-like growth factor I (IGF-I). Blood samples from patients with gastric cancer (GC), cholecystitis (CC), or inguinal hernia (IH) were taken before and after surgery. The concentrations of IGF-I, IGF binding protein-1 (IGFBP-1), insulin, cortisol, and glucose were determined. In GC patients the concentration of IGF-I was reduced and the concentrations of IGFBP-1 and cortisol were elevated preoperatively; after surgery, IGFBP-1 normalized. In CC patients the concentration of IGF-I was low and the concentration of IGFBP-1 was high before cholecystectomy; after surgery IGFBP-1 returned to normal and the concentration of cortisol increased. In IH patients the concentration of IGF-I was low and the concentrations of IGFBP-1 and cortisol were high before surgery; after laparotomy IGFBP-1 returned to normal. The metabolic changes were present in all analyzed patient groups, regardless of the severity of disease and nutrition. The concentration of IGF-I was reduced before surgery and remained reduced after, recommending IGF-I as a metabolic marker in both pre and postoperative examination of patients.

Differential influence of open surgery and sepsis on the circulating insulin-like growth factors and their binding proteins as representative metabolic markers.

OBJECTIVES: In critical illnesses and stress conditions many endocrine systems are disturbed. In the current study we determined the influence of open surgery, post-operative sepsis and its early therapy on the components of the insulin-like growth factor (IGF) system in patients with malignant gastric or pancreatic tumors. DESIGN AND METHODS: Twenty-one patients and eighty-one age- and sex-matched healthy subjects were included in this study. IGF-I, IGF-II, IGF binding proteins (IGFBPs), cortisol, insulin and protein concentrations (total, albumin and IgG) were determined pre-operatively, post-operatively, when sepsis was diagnosed and 48 h after initiating therapy. RESULTS: The concentrations of circulating IGF-I, IGF-II and IGFBP-3 were significantly lower in pre-operative patients compared to healthy subjects. Sepsis caused a further decrease in IGF-I and IGFBP-3 but an increase in IGFBP-1, IGFBP-2 and IGFBP-4 resulting in a redistribution of IGF molecules from ternary to binary complexes. CONCLUSIONS: The presence of malignant gastric or pancreatic tumors followed by post-operative sepsis caused a serious misbalance in components of the IGF system which failed to recover during the time of our longitudinal study.

Efflux of cholesterol and phospholipids derived from the haemoglobin-lipid adduct in human red blood cells into plasma.

OBJECTIVE: The interior of red blood cells (RBCs) contains a variable amount of cholesterol and phospholipids bound to haemoglobin (Hb). This current study was devised to determine if this pool of lipids (termed Hb-Ch) was available for exchange with plasma lipoproteins. DESIGN AND METHODS: We studied the in vitro efflux of lipids from human RBCs into fasting plasma in men with either low (control group) or high Hb-Ch (study group). RESULTS: When plasma was incubated with a two-fold excess of autologous RBCs the plasma cholesterol level increased due to a decrease in the level of cholesterol from the RBC membrane (in the control group) and due to a decrease in the level of cholesterol both from the RBC membrane and the Hb-Ch fraction (in the study group). The loss of Hb-Ch-derived phospholipids during lipid efflux was roughly equal to that of Hb-Ch-derived cholesterol. The loss of RBC cholesterol into plasma high-density lipoproteins (HDL) was more pronounced in our study group and correlated with the loss of cholesterol from Hb-Ch. CONCLUSION: The Hb-Ch adduct significantly contributes to the lipid efflux from RBCs into plasma. The majority of cholesterol released from Hb-Ch appears in the plasma HDL fraction suggesting that Hb-Ch may play a role in reverse cholesterol transport in vivo.

A comparison of sevoflurane-fentanyl and neuroleptic anaesthesia for laparoscopic cholecystectomy of mildly obese patients.

The current study was formulated to evaluate the advantages and disadvantages of sevoflurane-fentanyl and neuroleptic anaesthesia for laparoscopic cholecystectomy surgery of mildly obese patients. Laparoscopic cholecystectomy was performed using either sevoflurane-fentanyl or neuroleptic anaesthesia. Intra-operative haemodynamic stability, surgical duration, awakening time, severity of post-operative pain and hormonal stress parameters (serum and urinary cortisol, serum insulinlike growth factor and serum insulin) were determined. The duration of both surgery and patient hospitalisation were similar in both study groups. Patients that received sevoflurane-fentanyl exhibited a significantly shorter awakening time compared to neuroleptic anaesthesia. In addition, the same group of patients had less post-operative complications (nausea, vomiting, urinary retention and respiratory insufficiency) despite more abdominal pain. The severity of postoperative pain and the requirement of additional analgesia (up to 12 hours post-surgery) were greater in patients that received sevoflurane-fentanyl. The serum and urinary cortisol concentrations were significantly increased in post-operative patients that received neuroleptic anaesthesia whereas the insulin-like growth factor-I concentration in both study groups significantly decreased post-operatively. Our results clearly show that neuroleptic anaesthesia attenuates the stress response to laparoscopic cholecystectomy to a lesser extent than sevoflurane-fentanyl. The latter anaesthesia is recommended for laparoscopic cholecystectomy of mildly obese patients.

The effect of laparoscopy on the IGF system in patients diagnosed with acute cholecystitis.

OBJECTIVE: IGFs and IGF binding proteins in the circulation are subject to modulation by a number of catabolic states including inflammation, trauma and surgery. We sought to determine the impact of laparoscopy on the IGF system in patients diagnosed with acute cholecystitis. DESIGN: The components of the IGF system, cortisol, glucose and insulin concentration in both the serum and urine of patients were compared to those from a healthy group of subjects. An additional comparison was made between pre- and post-laparoscopically assisted patients. PATIENTS: Thirty patients diagnosed with acute cholecystitis and 81 matched controls were included in the study. MEASUREMENTS: Radioimmunoassays were used to determine the IGF-I, IGF-II, insulin and cortisol concentrations. The concentration of IGFBP-3 was measured using an immunoradiometric assay. The GOD-PAP method was used to determine the glucose concentration. Gel filtration chromatography was performed to calculate the IGF binary/ternary complex ratio. The amount of sialic acid in IGFBP-3 was determined by affinity chromatography. The presence of IGFBPs in serum was determined by both immunoblotting and ligandblotting. RESULTS: The concentrations of circulating IGF-I, IGF-II and IGFBP-3 were significantly reduced in pre-operative patients compared to healthy subjects. No further reductions were observed post-laparoscopy. Immunoblotting and ligandblotting demonstrated a decreased amount of IGFBP-3 in both pre- and post-operative patients compared to healthy subjects. Increased levels of IGFBP-2 and IGFBP-1 were observed in pre-operative patients compared to healthy subjects, but laparoscopy did not cause further elevation. No alteration in the IGF binary/ternary complex ratio was witnessed between any of the study groups. A significant increase in the sialic acid content of IGFBP-3 was seen in both patient groups when compared to healthy subjects. The level of urinary cortisol was significantly increased in post-operative patients, whereas the urinary IGF-I concentration was decreased compared to healthy subjects. CONCLUSIONS: Our results indicate that acute cholecystitis causes several significant changes in the circulating IGF system. Laparoscopy, however, does not aggravate such changes, but elevates urinary cortisol.

Characterisation of insulin-like growth factor receptors and insulin receptors in the human placenta using lectin affinity methods.

Insulin and insulin-like growth factor receptors (IR, IGF-IR, IGF-IIR) from human placental cell membranes were solubilised and their glycoprotein properties were studied in terms of their interaction with five lectins: wheat germ agglutinin (WGA), banana lectin (BanLec), phytohaemagglutinin (PHA), concanavalin A (Con A), and Sambucus nigra agglutinin (SNA). The pattern of binding to the immobilised lectins indicated that the glycosylation of the IGF-IR, IGF-IIR and IR differed. We found several populations of receptors in placental cell membranes, differing with respect to their oligosaccharide moieties. IGF-IIR populations bore highly branched complex type N-glycans with a very high content of oligosaccharides terminating with Sia, high-mannose type N-glycans and hybrid type N-glycans. All these glycans seemed to be attached to the same IGF-II receptor molecules. Two major glycoforms of IR were detected, one having multiple highly branched N-glycans with a low content of terminal Sia and the other, having high-mannose type glycans attached to multiple N-glycosylation sites. As for the IGF-IR, multiple glycoforms were detected, bearing complex type N-glycans with various content of Sia-terminating branches, hybrid type N-glycans or high-mannose type N-glycans. The specific binding of (125)I-IGF-II to its receptor increased in the presence of immobilised WGA and SNA, which might imply the existence of a mammalian lectin counterpart whose potential physiological significance may lie in different targeting to various membrane compartments, thereby potentially modifying their cell signalling pathways.

The insulin-like growth factor system in the circulation of patients with viral infections.

The insulin-like growth factor (IGF) system was examined in the circulation of patients with viral infections (herpes simplex virus, HSV; cytomegalovirus, CMV; rotavirus, RV and adenovirus, AV). The serum concentrations of IGF-I, IGF-II and cortisol were measured by radioimmunoassay, while IGF-binding proteins (IGFBPs) were characterised by ligand-affinity blotting. Although both IGF-I and IGF-II concentrations were significantly lower in patients with viral infections (p<0.05) than in healthy persons, the IGF-II/IGF-I ratio was increased (p<0.05). No correlation between the concentration of IGF-I and IGF-II and the intensity of the antibody response to infection was observed. Ligand-affinity blotting demonstrated decreased amounts of IGFBP-3 (patients with HSV, CMV, AV and some patients with RV), increased IGFBP-2 (some patients with HSV and RV) and IGFBP-1 (patients with RV). Serum cortisol was significantly elevated (p<0.05) in patients infected with HSV, CMV and RV. The alterations observed can be interpreted as induction of the hypothalamic-pituitary-adrenal axis and suppression of the growth hormone (GH)/IGF axis under the influence of viral infection.

Insulin-like growth factors in patients with liver cysts.

Insulin-like growth factors (IGFs) play an important role in cell growth and differentiation, and the liver is the main source of IGFs and IGF-binding proteins (IGFBPs) that appear in the circulation. The effect of liver cysts on the circulating IGF system was studied in this work. Serum concentrations of IGF-I and -II were measured by radioimmunoassay, IGFBP patterns were characterised by ligand-affinity and immunoblotting, and a lectin-binding assay was used to investigate the glyco component of IGFBP-3 complexes. IGF-I and -II concentrations in patients with cysts were significantly lower compared to those in healthy individuals (P<0.0001 and P<0.01, respectively), and the decrease was related to age but not sex. The overall mean concentrations of IGF-I and -II were not significantly different whether the cysts were caused by Echinococcus granulosus, cross-reactive pathologies, or some other factor. IGFBP profiles correlated with the amount of IGF present: patients with lower IGF-I concentrations expressed decreased IGFBP-3 and elevated IGFBP-2 levels. Increased IGFBP-3 proteolytic activity in the patients' blood was not detected by immunoblotting. In the lectin-binding assay, IGFBP-3 complexes in the circulation of patients demonstrated reactivity similar to that in healthy persons, suggesting that the overall structure of the saccharide moieties of the IGFBP-3 complexes was not significantly altered due to liver cyst formation.

Circulating insulin-like growth factors in patients infected with Helicobacter pylori.

OBJECTIVES: The level of insulin-like growth factors (IGFs) and their binding proteins may change in acutely ill humans. The aim of this work was to examine the changes in the IGF system in patients suffering from infection induced by Helicobacter pylori (H. pylori). DESIGN AND METHODS: The serum concentrations of IGF-I, IGF-II and cortisol were measured by radioimmunoassay. IGFBP patterns were characterized by ligand-affinity blotting, and a lectin-binding assay was used to investigate the possible changes in the glycocomponent of IGFBP-3. RESULTS: Both IGF-I and IGF-II concentrations were significantly lower in patients with H. pylori infection (P < 0.001 for IGF-I and P = 0.016 for IGF-II) compared to healthy individuals, whereas the level of cortisol was significantly elevated in analyzed patients (P < 0.001). Autoradiography demonstrated the increased presence of IGFBP-2 and IGFBP-1, together with a decreased level of IGFBP-3. CONCLUSIONS: The circulating IGF/IGFBP system is altered in patients infected with H. pylori. The increased level of cortisol suggests the involvement of the hypothalamic/pituitary/adrenal axis that stimulates the elevation of blood glucose, probably in coordination with decreased IGF activity to minimize anabolic metabolism.

Insulin-like growth factors and their binding proteins in the circulation of patients with echinococcosis, trichinellosis and toxoplasmosis.

BACKGROUND: Insulin-like growth factors (IGFs) are polypeptide hormones that play anabolic roles in cellular growth and metabolism. Their activity is regulated by binding proteins (IGFBPs) and degradation mechanisms. The liver is regarded as the main source of circulating forms and the levels change in various disease states. The aim of the study was to explore the effects of parasitic infections on the circulating IGFs and IGFBPs. METHODS: Peptide concentrations in sera of patients with echinococcosis, trichinellosis and toxoplasmosis were measured by radioimmunoassay, while IGFBP patterns were characterised by ligand-affinity blotting and gel chromatography. RESULTS: IGF-I levels were reduced in trichinellosis (p=0.016), with the increased relative amounts of IGFBP-1, -2 and -4. The significantly reduced levels of IGF-I (p<0.001), IGF-II (p=0.017) and IGFBP-3 and the increased presence of IGFBP-2 were found in patients with echinococcosis. CONCLUSIONS: In subjects with trichinellosis probably the combined effects of inadequate nutrition and the immunological response occurred. A possible explanation for the changes found in patients with echinococcosis is that the liver is a frequent target organ for Echinococcus granulosus. Since liver cysts were present in these patients, it can be expected that hepatic injury may affect liver metabolism leading to altered IGF/IGFBP profiles.