A novel somatostatin receptor ligand for human ACTH - and GH -secreting pituitary adenomas.

OBJECTIVE: Somatostatin receptor ligands have come to play a pivotal role in the treatment of both ACTH- and GH-secreting pituitary adenomas. Clinical efficacy averages 30-50%, thus a considerable number of patients with Cushing's disease or acromegaly remain unresponsive to this therapeutic approach. HTL0030310 is a new somatostatin receptor ligand selective for subtype 5 over subtype 2, thus with a different receptor profile compared to clinical somatostatin receptor ligands. DESIGN: Assessment of the effect of HTL0030310 on hormone secretion in human ACTH- and GH-secreting pituitary adenomas in vitro. METHODS: Primary cultures from 3 ACTH-secreting and 5 GH-secreting pituitary adenomas were treated with 1, 10 and 100 nM HTL0030310 alone or with 10 nM CRH or GHRH, respectively. Parallel incubations with 10 nM pasireotide were also carried out. ACTH and GH secretion were assessed after 4 and 24 hour incubation; SSTR2, SSTR3, SSTR5, GH and POMC expression were evaluated after 24 hours. RESULTS: HTL0030310 reduced unchallenged ACTH and POMC levels up to 50% in 2 ACTH-secreting adenomas and blunted CRH-stimulated ACTH/POMC by 20-70% in all 3 specimens. A reduction in spontaneous GH secretion was observed in 4 GH-secreting adenomas and in 2 specimens during GHRH co-incubation. SSTRs expression was detected in all specimens. CONCLUSIONS: This first study on a novel somatostatin receptor 5-preferring ligand indicates that HTL0030310 can inhibit hormonal secretion in human ACTH- and GH-secreting pituitary adenomas. These findings suggest a potential new avenue for somatostatin ligands in the treatment of Cushing's disease and acromegaly.

Can ultrasensitive thyroglobulin immunoassays avoid the need for ultrasound in thyroid cancer follow-up?

PURPOSE: Differentiated thyroid cancer (DTC) is the most common endocrine neoplasm, with a rising incidence and a long life expectancy. It has recently been suggested that patients with low- and intermediate-risk DTC with a good response to treatment at one year could be followed up using only highly sensitive immunoassays for thyroglobulin (Tg). The aim of this study was to examine the serum Tg levels in a series of DTC patients with histologically proven persistent or recurrent diseases. METHODS: The study involved 50 consecutive patients being routinely followed up at our center, whose clinical, histological, and biochemical data were retrospectively collected. RESULTS: The false-negative rate of ultrasensitive serum Tg assay was 14.3% (5/35) overall, and limited to anti-thyroglobulin autoantibodies (TgAb)-negative patients. Among them, only one patient had an excellent response to treatment at one-year follow-up and was diagnosed with a 4 mm recurrence, after more than seven years of periodic ultrasounds. The size of the neck lesion documented in the histological report was slightly larger in patients with detectable as opposed to negative Tg values (P < 0.05). CONCLUSIONS: Serum highly sensitive Tg is undetectable in a proportion of patients with a proven persistent or recurrent DTC. The reasons behind this phenomenon are still unknown. However, in low/intermediate-risk patients cured at one-year follow-up, highly sensitive Tg without neck US seems an appropriate strategy for patients' management.

Basal and Calcium-Stimulated Procalcitonin for the Diagnosis of Medullary Thyroid Cancers: Lights and Shadows.

Background: Procalcitonin (proCt) was recently proposed as an alternative or in addition to calcitonin (Ct) in medullary thyroid cancer (MTC) diagnostics. Methods: Serum basal Ct (bCt) and proCt (bproCt) levels were measured before surgery from a consecutive series of patients with (n=43) and without (n=75) MTC, retrospectively collected in Padua. Serum bproCt, bCt and stimulated proCt and Ct (sproCt and sCt) were measured in another consecutive series of 33 patients seen at three tertiary-level institutions undergoing a calcium stimulation test prior to surgery, 20 of them with a final diagnosis of MTC, and 13 with non-MTC nodular disease. Results: Median bproCt levels were higher in MTC than in non-MTC. A positive correlation was found for bproCt with bCt (P<0.01, R2 = 0.75), and with tumor size (P<0.01, R2 = 0.39). The cut-off for bproCt differentiating between MTC and non-MTC patients was >0.07 ng/ml (sensitivity: 85.7%, specificity: 98.9%, positive predictive value [PPV]: 98.2%, negative predictive value [NPV]: 90.6%, P<0.01). While bproCt was >0.07 ng/ml in 38/39 (97.4%) patients with MTC >10 mm, it was above said cut-off only in 15/23 (65.2%) patients with tumors ≤10 mm. A sproCt >0.19 ng/ml was able to identify MTC [sensitivity: 90.0%, specificity:100.0%, PPV: 100.0%, NPV: 86.7% (P<0.01)]. Conclusions: Our data suggest that bproCt can be a good adjunct to Ct for MTC diagnostic purposes. In consideration of its high specificity, it can be used in combination with Ct in MTC diagnostics, particularly in the case of mildly elevated basal Ct levels.

Serum miR-375 for Diagnostic and Prognostic Purposes in Medullary Thyroid Carcinoma.

Purpose: Having previously demonstrated that tissue miR-375 expression in medullary thyroid carcinoma (MTC) tissues is linked to prognosis, the aim of this study was to assess the diagnostic and prognostic value of circulating miR-375 levels in MTC patients. Methods: A series of 68 patients with MTC was retrospectively retrieved and assessed in terms of their clinicopathological characteristics. MiR-375 levels were measured in all patients' presurgical blood samples. Both serum and tissue levels were tested prior to surgery in a subgroup of 57 patients. Serum miR-375 levels were also measured in serum from 49 patients with non-C-cell thyroid nodular diseases (non-CTN), 14 patients with pheochromocytoma, and 19 healthy controls. Results: Circulating miR-375 levels were 101 times higher in the serum of patients with MTC than in all other patients and controls, with no overlap (P < 0.01). No correlation emerged between serum and tissue miR-375 levels. Serum miR-375 levels were higher in MTC patients with N0 than in those with N1 disease (P = 0.01), and also in patients who were biochemically cured than in those who were not (P = 0.02). In the whole series of patients and controls, calcitonin (CT) and serum miR-375 levels were correlated at diagnosis (R2 = 0.40, P < 0.01), but in a U-shaped manner: a positive correlation was found with low CT levels, then the correlation turns negative as CT rises (in MTC patients). A negative correlation was indeed found in MTC patients between serum miR-375 and CT (R2 = -0.10, P = 0.01). On ROC curve analysis, a cut-off of 2.1 for serum miR-375 proved capable of distinguishing between MTC patients and the other patients and controls with a 92.6% sensitivity and a 97.6% specificity (AUC: 0.978, P < 0.01). Conclusions: Serum miR-375 levels can serve as a marker in the diagnosis of MTC, with a remarkable specificity. Serum miR-375 also proved a novel marker of prognosis in this disease. Further in vitro experiments to corroborate our results are currently underway.

Analytical performances of a chemiluminescence immunoassay for SARS-CoV-2 IgM/IgG and antibody kinetics.

Background Coronavirus disease 2019, abbreviated to COVID-19, represents an emerging health threat worldwide as, after initial reports in China, it has continued to spread rapidly. The clinical spectrum of the disease varies from mild to severe acute respiratory distress syndrome (ARDS). Moreover, many patients can be asymptomatic, thus increasing the uncertainty of the diagnostic work-up. Laboratory tests play a pivotal role in the diagnosis and management of COVID-19, the current gold standard being real-time reverse transcription polymerase chain reaction (rRT-PCR) on respiratory tract specimens. However, the diagnostic accuracy of rRT-PCR depends on many pre-analytical and analytical variables. The measurement of specific COVID-19 antibodies (both IgG and IgM) should serve as an additional, non-invasive tool for disease detection and management. Methods The imprecision of the MAGLUMI™ 2000 Plus 2019-nCov IgM and IgG assays (Snibe, Shenzhen, China) was assessed by adopting the Clinical and Laboratory Standards Institute (CLSI) EP15-A3 protocol. Linearity of dilution and recovery was evaluated by means of mixes of high-level pools and low-level pools of serum samples. Immunoglobulin time kinetics were evaluated using a series of serum samples, repeatedly collected from COVID-19-positive patients at different times, from <5 days up to 26-30 days. Results Findings at the analytical validation of the assay carried out according to the CLSI EP15-A3 guideline demonstrated that imprecision and repeatability were acceptable (repeatability was <4% and <6% for IgM and IgG, respectively, whilst intermediate imprecision was <6%). In addition, results of dilution and recovery studies were satisfactory. The kinetics of COVID-19 antibodies confirmed previously reported findings, showing a rapid increase of both IgM and IgG after 6-7 days from the symptom onset. IgG had 100% sensitivity on day 12, whilst 88% was the higher positive rate achieved for IgM after the same time interval. Conclusions The findings of this study demonstrate the validity of the MAGLUMI 2000 Plus CLIA assay for the measurement of specific IgM and IgG in sera of COVID-19 patients, and for obtaining valuable data on the kinetics of both (IgM and IgG) COVID-19 antibodies. These data represent a pre-requisite for the appropriate utilization of specific antibodies for the diagnosis and management of COVID-19 patients.

Two-center comparison of 10 fully-automated commercial procalcitonin (PCT) immunoassays.

Background This two-center study was designed to verify comparability of procalcitonin (PCT) values among 10 different commercial immunoassays. Methods A total number of 176 routine lithium-heparin plasma samples were divided in identical aliquots and simultaneously analyzed with 10 different PCT immunoassays, including Kryptor BRAHMS PCT sensitive, Abbott Architect BRAHMS PCT, Beckman Coulter Access PCT (on Access and DXI), BioMérieux Vidas BRAHMS PCT, Diasorin Liaison BRAHMS PCT, Fujirebio Lumipulse G BRAHMS PCT, Roche BRAHMS PCT (on Cobas E801), Diazyme PCT (on Roche Cobas C702) and SNIBE Maglumi PCT. Results Highly significant correlation was always found across multiple comparisons, with correlation coefficients comprised between 0.918 and 0.997 (all p < 0.001). Bland and Altman plots analysis revealed highly variable bias among immunoassays, ranging between ±0.2% and ±38.6%. Diazyme PCT on Roche Cobas C702 and SNIBE Maglumi PCT displayed the larger overestimation, whilst PCT values were underestimated by Cobas BRAHAMS PCT. The agreement was always >80% (all p < 0.001), but varied largely across multiple comparisons, ranging between 90%-99% at 0.1 µg/L, 81%-99% at 0.25 µg/L, 83%-100% at 0.5 µg/L, 94%-100% at 2.0 µg/L and 90%-99% at 10 µg/L, respectively. The larger disagreement was observed comparing Diazyme PCT and Maglumi PCT with the other methods. Conclusions Although we found acceptable correlation among 10 commercial PCT immunoassays, the limited agreement at clinical decision thresholds remains a major issue, especially at lower end of PCT concentration, thus potentially contributing to jeopardize the clinical value of this biomarker.

Calcitonin measurement and immunoassay interference: a case report and literature review.

Calcitonin (CT) is currently the most sensitive serological marker of C-cell disease [medullary thyroid carcinoma (MTC) and C-cell hyperplasia]. Starting with a report on a case that occurred at our institution, this review focuses on trying to explain the reasons behind the poor specificity and sensitivity of the various CT immunoassays. A 15-year-old patient was referred to our institution in May 2014 for moderately elevated CT levels. Thyroid ultrasonography (US) documented a colloidal goiter. Secondary causes of the hypercalcitoninemia (hyperCT) were ruled out. The mismatch between the clinical picture and the laboratory results prompted us to search for other reasons for the patient's high CT levels, so we applied the heterophilic blocking tube (HBT) procedure to the patient's sera before the CT assay. Using this pretreatment step, his serum CT concentration dropped to <1 ng/L, as measured at the same laboratory. Measuring plasma CT has an important role in screening for C-cell disease, but moderately elevated serum CT levels need to be placed in their clinical context, bearing in mind all the secondary causes of C-cell hyperplasia and the possibility of laboratory interference, before exposing patients to the risks and costs of further tests.

Synergistic antitumour activity of RAF265 and ZSTK474 on human TT medullary thyroid cancer cells.

Medullary thyroid cancer (MTC) is an aggressive malignancy responsible for up to 14% of all thyroid cancer-related deaths. It is characterized by point mutations in the rearranged during transfection (RET) proto-oncogene. The activated RET kinase is known to signal via extracellular signal regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K), leading to enhanced proliferation and resistance to apoptosis. In the present work, we have investigated the effect of two serine/threonine-protein kinase B-Raf (BRAF) inhibitors (RAF265 and SB590885), and a PI3K inhibitor (ZSTK474), on RET-mediated signalling and proliferation in a MTC cell line (TT cells) harbouring the RETC634W activating mutation. The effects of the inhibitors on VEGFR2, PI3K/Akt and mitogen-activated protein kinases signalling pathways, cell cycle, apoptosis and calcitonin production were also investigated. Only the RAF265+ ZSTK474 combination synergistically reduced the viability of treated cells. We observed a strong decrease in phosphorylated VEGFR2 for RAF265+ ZSTK474 and a signal reduction in activated Akt for ZSTK474. The activated ERK signal also decreased after RAF265 and RAF265+ ZSTK474 treatments. Alone and in combination with ZSTK474, RAF265 induced a sustained increase in necrosis. Only RAF265, alone and combined with ZSTK474, prompted a significant drop in calcitonin production. Combination therapy using RAF265 and ZSTK47 proved effective in MTC, demonstrating a cytotoxic effect. As the two inhibitors have been successfully tested individually in clinical trials on other human cancers, our preclinical data support the feasibility of their combined use in aggressive MTC.

A contribution to detection of anticardiolipin and anti-ß2glycoprotein I antibodies: Comparison between a home-made ELISA and a fluorescence enzyme immunoassay.

BACKGROUND: Currently, ELISA for detection of anticardiolipin (aCL) and anti-ß2glycoprotein I (anti-ß2GPI) antibodies is not standardized. Recently, few studies have compared the performance of ELISA with that of fluorescence enzyme immunoassay (FEIA), but they have produced debatable results. The aim of this investigation was to compare ELISA with FEIA results in detecting aCL and anti-ß2GPI antibodies. METHODS: The study cohort included 94 primary antiphospholipid syndrome (PAPS) patients, 65 subjects with the clinical criteria for PAPS classification but ELISA negative for the laboratory criteria and 165 control subjects. Serum IgG/IgM aCL/anti-ß2GPI antibodies were determined using FEIA-EliA™ and a home-made ELISA. RESULTS: The sensitivities of the two methods were similar with the exception of IgM aCL which was found to be significantly higher in the PAPS patients using the ELISA method, even if IgM aCL was detected at a low level by both techniques. The two assays had a comparable specificity, a high/significant agreement and a significant correlation between the antibody levels. FEIA testing uncovered no significant prevalence of any antiphospholipid (aPL) antibody in the ELISA negative patients. CONCLUSION: Our results suggest that FEIA is comparable to a home-made ELISA.

Protein supplementation increases postexercise plasma myostatin concentration after 8 weeks of resistance training in young physically active subjects.

Myostatin (MSTN) is a negative regulator of muscle growth even if some studies have shown a counterintuitive positive correlation between MSTN and muscle mass (MM). Our aim was to investigate the influence of 2 months of resistance training (RT) and diets with different protein contents on plasma MSTN, interleukin 1 beta (IL-1ß), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-α), and insulin-like growth factor 1 (IGF-1). Eighteen healthy volunteers were randomly divided in two groups: high protein (HP) and normal protein (NP) groups. Different protein diet contents were 1.8 and 0.85 g of protein·kg bw(-1)·day(-1) for HP and NP, respectively. Subjects underwent 8 weeks of standardized progressive RT. MSTN, IGF-1, IL-1ß, IL-6, and TNF-α were analyzed before and after the first and the last training sessions. Lean body mass, MM, upper-limb muscle area, and strength were measured. Plasma MSTN showed a significant increase (P<.001) after the last training in the HP group compared with NP group and with starting value. IGF-1 plasma concentration showed a positive correlation with MSTN in HP after the last training (r(2)=0.6456; P=.0295). No significant differences were found between NP and HP for IL-1ß, IL-6, TNF-α, and strength and MM or area. These findings suggest a "paradoxical" postexercise increase of plasma MSTN after 8 weeks of RT and HP diets. This MSTN elevation correlates positively with IGF-1 plasma level. This double increase of opposite (catabolic/anabolic) mediators could explain the substantial overlapping of MM increases in the two groups.

Adiponectin levels are reduced while markers of systemic inflammation and aortic remodelling are increased in intrauterine growth restricted mother-child couple.

AIM OF THE STUDY: To investigate the relationships between the adipocytokine levels, markers of inflammation, and vascular remodelling in pregnancies complicated by intrauterine growth restriction (IUGR). MATERIALS AND METHODS: This was a retrospective study. One hundred and forty pregnant patients were enrolled. Adiponectin, leptin, tumor necrosis factor α (TNFα), interleukin-6 (IL-6), and C reactive protein (CRP) were assessed in IUGR, small for gestational age (SGA), and appropriate for gestational age (AGA) mother-child couples at delivery. IUGR and SGA fetuses were defined as fetuses whose estimated fetal weight (EFW) was below 10th percentile for gestational age with and without umbilical artery (UA) Doppler abnormalities, respectively. Fetal aorta intima media thickness (aIMT) was evaluated by ultrasound in the same fetal groups. Data were analyzed by R (version 2.15.2). RESULTS: There were 37 IUGR mother-child couples, 33 SGA, and 70 AGA. Leptin, TNFα, IL-6, and CRP serum levels were higher in IUGR pregnant patients (P < 0.05). Adiponectin levels were significantly reduced in IUGR fetuses compared to SGA and AGA, while leptin, TNFα, and IL-6 levels were higher in IUGR group (P ≤ 0.05). Fetal aIMT was significantly higher in IUGR (P < 0.05) and in this group there was a negative correlation between aIMT and adiponectin/leptin ratio (A/L ratio) (P < 0.05) and between adiponectin and IL-6 levels (P < 0.05). CONCLUSIONS: In conclusion, compared to SGA and AGA, IUGR fetuses had reduced circulating levels of adiponectin and elevated measures of aIMT and several inflammatory markers. Moreover, adiponectin levels were negatively correlated with aIMT in IUGR fetuses suggesting a possible causal link between reduced adiponectin and vessel remodelling.

Insulin and body weight but not hyperandrogenism seem involved in seasonal serum 25-OH-vitamin D3 levels in subjects affected by PCOS.

PCOS patients were frequently characterized by lower plasma vitamin D levels. The mechanisms involved in this dysfunction remains still debated, therefore we evaluated the role of androgen, insulin and body weight on the serum VitD levels in women with or without PCOS. Eighty one patients 18-42 yrs old were studied into "SUMMER" and "WINTER" seasonal period: thirty seven PCOS, seventeen no-ovarian hyperandrogenic (noPCOS), twelve functional hypothalamic amenorrhea (FHA) and finally fifteen healthy (Con). Patients were further divided into: lean (L), obese (O), normo- (nINS) and hyperinsulinemic (hINS). All hormonal and metabolic parameters were measured at 1-7 days of the menstrual cycle. Our results show that VitD levels were lower in PCOS and in noPCOS than in FHA and Con, in particular in (O) and (hINS) PCOSs. Both in summer and in winter, PCOSs had basal VitD levels significantly lower than FHA and Con, whereas they were similar to noPCOS. Yet, LhINS and OPCOS had VitD levels lower than Con and noPCOS. VitD levels were comparable in LnINS PCOS and Con. In conclusion, PCOSs had levels of VitD lower than controls. Weight and hyperinsulinemia had a significant influence on these values. Finally, over 70% of our healthy patients had VitD deficiency.

Innate immune environment in ileal pouch mucosa: α5 defensin up-regulation as predictor of chronic/relapsing pouchitis.

Defensins are small cationic peptides with antibacterial activity expressed in Paneth cells (α-defensins) or generally in intestinal epithelial cells (ß-defensins) that have a profound effect on gut microbiota. Chronic pouchitis, which occurs in 5% of patients after restorative proctocolectomy and can cause pouch failure, is associated to a significant increase of Clostridiaceae spp. The aim of this study was to gain further insight in the pathogenesis of pouch dysbiosis by exploring defensin expression. Thirty-two consecutive patients coming for follow-up endoscopy were recruited. On pouch biopsies, we cultured bacteria adherent to the mucosa and determined α- and ß-defensins and toll-like receptor-4 and -2 mRNA by quantitative real-time RT-PCR. Serum and mucosal levels of IL-1ß, IL-6 and TNF-α were measured with immunometric assays. Faecal lactoferrin was analysed by quantitative ELISA. After a median follow-up of 23 (IQR 20-24) months, the patients were contacted for a reassessment of current and past disease activity. During the follow-up, chronic/relapsing pouchitis was diagnosed in six patients. The mucosal level of α-5 and α-6 defensins correlated with chronic/relapsing pouchitis onset (τ = 0.30, p = 0.034 and τ = 0.28, p = 0.053, respectively). High levels of α-5 defensin resulted to be predictive of chronic/relapsing pouchitis [AUC = 74% (95% CI = 53-89%), p = 0.052]. Patients with high levels of α-5 and α-6 defensins had earlier pouchitis relapses (p = 0.009 and p = 0.034, respectively). High levels of α-5 defensin were associated to a significant risk of chronic/relapsing pouchitis [OR = 10.6 (95% CI = 1.2-97.6), p = 0.027]. At multivariate analysis, the mucosal levels of α-5 defensin and the number of CFU of mucosa-associated Clostridiaceae spp resulted to be independent predictors of chronic/relapsing pouchitis [ß = 0.46 (0.18), p = 0.024 and ß = 0.44 (0.18), p = 0.027, respectively]. In conclusion, chronic/relapsing pouchitis is associated to increased expression of mucosal HD-5 and to increased antimicrobial activity against Escherichia coli. In patients with chronic/relapsing pouchitis, HD-5 and TLR-4 over-expression is likely to create a hostile environment against Enterobacteriaceae, thus favouring Clostridiaceae spp by decreasing competing bacteria families.

TLR2 and TLR4 up-regulation and colonization of the ileal mucosa by Clostridiaceae spp. in chronic/relapsing pouchitis.

BACKGROUND: Chronic pouchitis, which can lead to pouch failure, occurs in approximately 5% of patients after restorative proctocolectomy for ulcerative colitis (UC). This work examined the interplay between the microbiota adherent to the ileal pouch mucosa and the mucosal immune system in chronic/relapsing pouchitis. PATIENTS AND METHODS: Thirty-two consecutive patients attending our surgical gastroenterological department following restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) for UC were considered eligible candidates for this study. Biopsy samples of bacteria adherent to the mucosa were collected. TLR4 and TLR2 mucosal expression was measured by Real Time RT-PCR. Serum and mucosal IL-1ß, IL-6, and TNF-α levels were assessed using immunometric assays. Fecal lactoferrin concentrations were determined by quantitative ELISA. After a median follow-up of 23 months (IQR 20-24 months) each patient underwent a global assessment of their clinical condition and disease activity status. RESULTS: Six patients were diagnosed with relapsing/chronic pouchitis during the follow-up period. Mucosal TLR2 and TLR4 expression was higher in the chronic/relapsing pouchitis group than in the no or only one episode of pouchitis group (P = 0.036 and P = 0.016, respectively). The number of colony forming units (CFU) of mucosa-associated Clostridiaceae spp. was higher in the former than in the latter group (P = 0.031). Clostridiaceae were associated to a significant risk of chronic/relapsing pouchitis [OR: 14 (95% CI 0.887-224.021), P = 0.045]. CONCLUSION: Chronic/relapsing pouchitis is associated to higher mucosal TLR2 and TLR4 expression. Mucosal colonization by Clostridiaceae spp seems to play a role in the pathogenesis of chronic/relapsing pouchitis.

Relationship between mucosa-associated microbiota and inflammatory parameters in the ileal pouch after restorative proctocolectomy for ulcerative colitis.

BACKGROUND: Our aim was to assess the relationship between the ileal-pouch microbiota and inflammatory parameters in patients operated on for ulcerative colitis. METHODS: In this cross-sectional study, 32 consecutive outpatients returning for follow-up endoscopy were recruited. Pouch biopsies were obtained during endoscopy for culture of bacteria adherent to the mucosa, histology, and analysis of local inflammation (IL-1ß, IL-6, and TNFα by immunometric assay; and toll-like receptor [TLR] 2 and 4 mRNA by quantitative real-time PCR). Fecal samples were collected for analysis of lactoferrin by ELISA. RESULTS: Granulocyte and monocyte mucosal infiltration correlated directly with mucosal Bacteriodiaceae spp. counts. Clostridiaceae spp. counts showed a direct correlation with mucosal ulceration and number of daily stools. In patients with pouchitis, Enterococcaceae spp. counts were less than in healthy patients. Enterobacteriaceae spp., Streptococcaceae spp. and Enterococcaceae spp. counts correlated inversely with immune cell infiltration. TLR-2 and TLR-4 mRNA, and mucosal levels of IL-1ß levels all correlated directly with Veilonella spp. counts. CONCLUSION: Bacteriodaceae spp. and, Clostridiaceae spp. may be associated with inflammation of the pouch mucosa. Conversely, Enterococcaceae spp., and possibly Enterobacteriaceae spp. and Streptococcaceae spp., may have an active role in maintaining immunologic homeostasis within the pouch mucosa.

In vitro model for IgE mediated food allergy.

BACKGROUND: In intestinal food allergy, the non-specificity of gastrointestinal symptoms and the limited access to the reacting organ are the reasons for the limited understanding of the pathophysiology of this disease and the difficulties in establishing an appropriate diagnosis in the individual patient. OBJECTIVE: To develop an in vitro model reproducing pathophysiological mechanisms of IgE mediated food allergy. METHODS: Distal duodenum biopsies of nine patients with food allergy and 10 control subjects were cultured for 3 h with medium alone and with 1 mg/ml of peptic-tryptic digest of wheat gliadin, wheat albumins, and apple proteins. Each biopsy was used for conventional histological examination and for immunohistochemical detection of IgE-positive cells. We have also analyzed the expression of tight junction proteins, occludin, claudin-1, and ZO-1 by immunoconfocal microscopy. Histamine and tryptase release were measured in the culture medium and collected at 0, 30 min, and 3 h of culture using an enzyme and radio immunoassay, respectively. RESULTS: Exposure of small intestinal biopsy specimens of patients with food allergy to food allergens led to a significative increase of IgE-positive cells with a significative increase of histamine and tryptase release and an altered expression of tight junction proteins. No differences were found in intestinal biopsies of controls, cultured with or without food antigens. CONCLUSIONS: Small intestinal organ culture is a functional model of food allergy and could be considered as an in vitro oral food challenge, with evident reduction of costs and risks for the patients.

Mucosal immune environment in colonic carcinogenesis: CD80 up-regulation in colonic dysplasia in ulcerative colitis.

BACKGROUND: In patients with ulcerative colitis (UC) the inconsistency between the rate of dysplasia and actual cancer incidence suggests the presence of an immunosurveillance mechanism. The aim of our study was to analyse the expression of CD80 and CD86 during the different stages of UC-associated and in non-inflammatory carcinogenesis. PATIENTS AND METHODS: Sixty-two patients affected with UC, UC with colonic dysplasia, UC and cancer, colonic adenoma, or colonic cancer and 11 healthy subjects were enrolled in our study. Tissue samples were taken from surgical specimens during colonic resection or during colonoscopy. Mucosal mRNA expression of CD80 and CD86 was quantified with real time polymerase chain reaction (RT-PCR). CD80, CD86 and p53 expressions and lamina propria mononuclear cell populations (CD3, CD20 and CD68) were analysed by immunohistochemistry. Mucosal levels of IL-1ß, IL-2 and IFN-γ were measured with immunometric assays. RESULTS: Among UC patients, CD80 protein expression was higher in those with dysplasia (p=0.017). In non-inflammatory carcinogenesis pathway CD80 protein and mRNA expressions were lower compared to the corresponding steps in the UC pathway. CD80 expression was directly correlated with the lamina propria mononuclear cell populations (T and B lymphocytes and monocytes). CD80 protein, but not CD80 mRNA, expression was significantly and directly correlated with IL-2 expression. CONCLUSION: CD80 resulted to be up-regulated in UC with dysplasia, while it was down-regulated in cancer. CD80 mucosal levels correlate with lamina propria T-cell and with IL-2 expression suggesting that it may elicit an active role in the immunosurveillance mechanism.

Rapid intraoperative parathyroid hormone assay in fine needle aspiration for differential diagnosis in thyroid and parathyroid surgery.

BACKGROUND: The purpose of our study was to investigate the efficacy of rapid intraoperative parathyroid hormone (rIO-PTH) analysis performed using material collected by fine needle aspiration as a parameter to distinguish anatomical structures which the surgeon is not able to identify clearly as parathyroid or lymph node during thyroid/parathyroid surgery. Before surgery, all patients gave written, informed consent. METHODS: We measured the rIO-PTH on 50 anatomical structures, 32 presumed parathyroids and 18 presumed lymph nodes from 22 patients, who underwent thyroid/parathyroid surgery between May and September 2009. RESULTS: The rIO-PTH values obtained on parathyroids ranged from 85.4 pmol/L to more than 2106 pmol/L, with a rIO-PTH median of 263.25 pmol/L. Results obtained on lymph nodes ranged from <0.52 pmol/L up to 20.4 pmol/L, with a rIO-PTH median equal to 1.31 pmol/L. Results of the Mann-Whitney test showed a rIO-PTH median significantly different (p<0.0001) between samples obtained from parathyroid glands and lymph node tissue. CONCLUSIONS: We demonstrated that rapid intraoperative measurement of parathyroid hormone is a good parameter for the differential diagnosis of parathyroid vs. lymph node tissue. We suggest extending the use of this technique to the field of parathyroid preservation during thyroid/parathyroid surgery in those cases where the parathyroids prove difficult to recognize on the basis of topographic or morphologic criteria.

Evaluation of erythrocyte band 3 phosphotyrosine level, glutathione content, CA-125, and human epididymal secretory protein E4 as combined parameters in endometriosis.

OBJECTIVE: To investigate the biochemical parameters of the erythrocyte response to diamide-induced oxidative stress, alone or as adjuncts to serum values of CA-125 and human epididymal secretory protein E4 (HE4), in the diagnosis and study of endometriosis. SETTING: University of Padova. DESIGN: Prospective study. PATIENT(S): Forty-five patients of reproductive age undergoing laparoscopy. INTERVENTION(S): All women were studied for endometriotic foci during laparoscopic surgery. Forty-one had laparoscopically and histologically confirmed endometriosis, and four did not. Twenty women with confirmed endometriosis were reassessed 1-4 months later. MAIN OUTCOME MEASURE(S): CA-125 and HE4 and two new parameters evaluated in erythrocytes after diamide-induced stress, that is, band 3 tyrosine phosphorylation (Tyr-P) level and decrease in total glutathione content (ΔGSH), were assessed in all patients. RESULT(S): In association with serum CA-125 levels but not with HE4, diamide-related erythrocyte band 3 Tyr-P and ΔGSH were significantly higher in patients with endometriosis and were able to discriminate with high sensitivity and specificity between patients before and after surgery. CONCLUSION(S): Endometriosis is associated with an increase in systemic oxidative stress, affecting the antioxidative defenses of circulating erythrocytes. All related implications, including evaluation of other oxidative stress-related changes, warrant further study.