Diabetes Mellitus and Reprogrammed Glucose Metabolism in Pancreatic Cancer: Features for Clinical Translation

The reprogrammed metabolism of cancer cells underlies the shift of glucose energetics from the highly efficient oxidative phosphorylation to the less efficient aerobic glycolysis, the Warburg effect. This phenomenon, with the activation of the glutamine pathway, advantage survival and proliferation of pancreatic ductal adenocarcinoma (PDAC) cells, which live in an adverse hypoxic and nutrient restricted microenvironment. In PDAC, glucose metabolic alterations occur also at the whole organism, diabetes mellitus (DM) being diagnosed in approximately 60% to 80% of patients. The association beteen PDAC and DM is a dual face phenomenon, DM being both a risk factor for and a consequence of this tumor type. Data from epidemiology indicate that longstanding DM increases PDAC risk 1.5 to 2.0 fold, probably because of the pro-proliferative effects of hyperinsulinemia. By contrast early onset DM, i.e. diabetes diagnosed no more than two years prior to cancer diagnosis, is considered a consequence of PDAC. Secondary DM is due to complex interactions between tumor cells, tumor microenvironment and pancreatic endocrine cells. In this scenario the role of the inflammatory S100A8 calcium binding protein, matrix metalloproteinases, Vanin1 or amylin has been experimentally demonstrated. However, the efforts made to translate in the clinical practice any individual new poteantial biomarker failed, because none reached enough sensitivity and specificity to be considered a reliable biomarker to diagnose PDAC even in high risk subjects as those with new onset DM. Therefore the identification and clinical validation of new biomarkers remains a challenge for future studies

Monocyte distribution width (MDW) parameter as a sepsis indicator in intensive care units.

Abstract Objectives Patients in Intensive Care Units (ICU) are a high-risk population for sepsis, recognized as a major cause of admission and death. The aim of the current study was to evaluate the diagnostic accuracy and prognostication of monocyte distribution width (MDW) in sepsis for patients admitted to ICU. Methods Between January and June 2020, we conducted a prospective observational study during the hospitalization of 506 adult patients admitted to the ICU. MDW was evaluated in 2,367 consecutive samples received for routine complete blood counts (CBC) performed once a day and every day during the study. Sepsis was diagnosed according to Sepsis-3 criteria and patients enrolled were classified in the following groups: no sepsis, sepsis and septic shock. Results MDW values were significantly higher in patients with sepsis or septic shock in comparison to those within the no sepsis group [median 26.23 (IQR: 23.48–29.83); 28.97 (IQR: 21.27–37.21); 21.99 (IQR: 19.86–24.36) respectively]. ROC analysis demonstrated that AUC is 0.785 with a sensitivity of 66.88% and specificity of 77.79% at a cut-off point of 24.63. In patients that developed an ICU-acquired sepsis MDW showed an increase from 21.33 [median (IQR: 19.47–21.72)] to 29.19 [median (IQR: 27.46–31.47)]. MDW increase is not affected by the aetiology of sepsis, even in patients with COVID-19. In sepsis survivors a decrease of MDW values were found from the first time to the end of their stay [median from 29.14 (IQR: 26.22–32.52) to 25.67 (IQR: 22.93–30.28)]. Conclusions In ICU, MDW enhances the sepsis detection and is related to disease severity

Polyclonal and monoclonal B lymphocytes response in HCV-infected patients treated with direct-acting antiviral agents

Hepatitis C virus (HCV) chronic infection can be associated with extrahepatic manifestations such as mixed cryoglobulinaemia and lymphoproliferative disorders that are endowed with increased rates of morbidity and all-cause mortality. In this study, we used flow cytometry to evaluate the effect of interferon-free antiviral treatment on peripheral blood lymphocytes in HCV-infected patients with or without associated lymphoproliferative disorders. Flow cytometry analysis of peripheral blood lymphocytes was performed at baseline and at the end of treatment. In HCV-infected patients with lymphoproliferative disorders, we evaluated immunoglobulin (Ig) light chain \u3ba/\u3bb ratio variations as a measure of monoclonal B-cell response to antiviral therapy. Healthy volunteers were enrolled as controls. A total of 29 patients were included, nine with and 20 without lymphoproliferative disorders. Sustained virological response was achieved in 29 of 29 patients. We observed a significant reduction in the B-cell compartment (39% global reduction) in eight of nine HCV-infected patients with lymphoproliferative disorders after viral clearance. We recognized the same trend, even if less pronounced, in HCV-infected patients without lymphoproliferative disorders (9% global reduction). Among HCV-infected patients with lymphoproliferative disorders, three showed an improvement/normalization of the immunoglobulin light chain ratio, whereas in the remaining six patients monoclonal B cells persisted to be clonally restricted even 1\ua0year after the end of treatment. Our data show that DAAs treatment can be effective in reducing the frequency of pathological B cells in the peripheral blood of HCV-infected patients affected by HCV-associated lymphoproliferative disorders; however, monoclonal populations can persist after viral eradication

Neonatal lymphocyte subpopulations analysis and maternal preterm premature rupture of membranes: a pilot study

Abstract Objectives Preterm premature rupture of membranes (pPROM) causes preterm delivery, and increases maternal T-cell response against the fetus. Fetal inflammatory response prompts maturation of the newborn's immunocompetent cells, and could be associated with unfavorable neonatal outcome. The aims were (1) to examine the effects of pPROM on the newborn's and mother's immune system and (2) to assess the predictive value of immune system changes in neonatal morbidity. Methods Mother-newborn pairs (18 mothers and 23 newborns) who experienced pPROM and controls (11 mothers and 14 newborns), were enrolled. Maternal and neonatal whole blood samples underwent flow cytometry to measure lymphocyte subpopulations. Results pPROM-newborns had fewer naïve CD4 T-cells, and more memory CD4 T-cells than control newborns. The effect was the same for increasing pPROM latency times before delivery. Gestational age and birth weight influenced maturation of the newborns' lymphocyte subpopulations and white blood cells, notably cytotoxic T-cells, regulatory T-cells, T-helper cells (absolute count), and CD4/CD8 ratio. Among morbidities, fewer naïve CD8 T-cells were found in bronchopulmonary dysplasia (BPD) (p=0.0009), and more T-helper cells in early onset sepsis (p=0.04). Conclusions pPROM prompts maturation of the newborn's T-cell immune system secondary to antigenic stimulation, which correlates with pPROM latency. Maternal immunity to inflammatory conditions is associated with a decrease in non-major histocompatibility complex (MHC)-restricted cytotoxic cells

Inflammation and pancreatic cancer: molecular and functional interactions between S100A8, S100A9, NT-S100A8 and TGFβ1

BACKGROUND: In order to gain further insight on the crosstalk between pancreatic cancer (PDAC) and stromal cells, we investigated interactions occurring between TGF\u3b21 and the inflammatory proteins S100A8, S100A9 and NT-S100A8, a PDAC-associated S100A8 derived peptide, in cell signaling, intracellular calcium (Cai2+) and epithelial to mesenchymal transition (EMT). NF-\u3baB, Akt and mTOR pathways, Cai2+ and EMT were studied in well (Capan1 and BxPC3) and poorly differentiated (Panc1 and MiaPaCa2) cell lines. RESULTS: NT-S100A8, one of the low molecular weight N-terminal peptides from S100A8 to be released by PDAC-derived proteases, shared many effects on NF-\u3baB, Akt and mTOR signaling with S100A8, but mainly with TGF\u3b21. The chief effects of S100A8, S100A9 and NT-S100A8 were to inhibit NF-\u3baB and stimulate mTOR; the molecules inhibited Akt in Smad4-expressing, while stimulated Akt in Smad4 negative cells. By restoring Smad4 expression in BxPC3 and silencing it in MiaPaCa2, S100A8 and NT-S100A8 were shown to inhibit NF-\u3baB and Akt in the presence of an intact TGF\u3b21 canonical signaling pathway. TGF\u3b21 counteracted S100A8, S100A9 and NT-S100A8 effects in Smad4 expressing, not in Smad4 negative cells, while it synergized with NT-S100A8 in altering Cai2+ and stimulating PDAC cell growth. The effects of TGF\u3b21 on both EMT (increased Twist and decreased N-Cadherin expression) and Cai2+ were antagonized by S100A9, which formed heterodimers with TGF\u3b21 (MALDI-TOF/MS and co-immuno-precipitation). CONCLUSIONS: The effects of S100A8 and S100A9 on PDAC cell signaling appear to be cell-type and context dependent. NT-S100A8 mimics the effects of TGF\u3b21 on cell signaling, and the formation of complexes between TGF\u3b21 with S100A9 appears to be the molecular mechanism underlying the reciprocal antagonism of these molecules on cell signaling, Cai2+ and EMT

A Randomized Trial of Pharmacogenetic Warfarin Dosing in Naive Patients with Non-Valvular Atrial Fibrillation

Genotype-guided warfarin dosing have been proposed to improve patient's management. This study is aimed to determine whether a CYP2C9- VKORC1- CYP4F2-based pharmacogenetic algorithm is superior to a standard, clinically adopted, pharmacodynamic method. Two-hundred naive patients with non-valvular atrial fibrillation were randomized to trial arms and 180 completed the study. No significant differences were found in the number of out-of-range INRs (INR3.0) (p = 0.79) and in the mean percentage of time spent in the therapeutic range (TTR) after 19 days in the pharmacogenetic (51.9%) and in the control arm (53.2%, p = 0.71). The percentage of time spent at INR>4.0 was significantly lower in the pharmacogenetic (0.7%) than in the control arm (1.8%) (p = 0.02). Genotype-guided warfarin dosing is not superior in overall anticoagulation control when compared to accurate clinical standard of care

SLC22A3 polymorphisms do not modify pancreatic cancer risk, but may influence overall patient survival

Expression of the solute carrier (SLC) transporter SLC22A3 gene is associated with overall survival of pancreatic cancer patients. This study tested whether genetic variability in SLC22A3 associates with pancreatic cancer risk and prognosis. Twenty four single nucleotide polymorphisms (SNPs) tagging the SLC22A3 gene sequence and regulatory elements were selected for analysis. Of these, 22 were successfully evaluated in the discovery phase while six significant or suggestive variants entered the validation phase, comprising a total study number of 1,518 cases and 3,908 controls. In the discovery phase, rs2504938, rs9364554, and rs2457571 SNPs were significantly associated with pancreatic cancer risk. Moreover, rs7758229 associated with the presence of distant metastases, while rs512077 and rs2504956 correlated with overall survival of patients. Although replicated, the association for rs9364554 did not pass multiple testing corrections in the validation phase. Contrary to the discovery stage, rs2504938 associated with survival in the validation cohort, which was more pronounced in stage IV patients. In conclusion, common variation in the SLC22A3 gene is unlikely to significantly contribute to pancreatic cancer risk. The rs2504938 SNP in SLC22A3 significantly associates with an unfavorable prognosis of pancreatic cancer patients. Further investigation of this SNP effect on the molecular and clinical phenotype is warranted

Persistent Megalocystic Ovary Following in Vitro Fertilization in a Postpartum Patient with Polycystic Ovarian Syndrome

SummaryObjectiveOvarian hyperstimulation syndrome (OHSS) is more severe when pregnancy occurs, as the developing pregnancy produces human chorionic gonadotropin, which stimulates the ovary's persistent growth. If no pregnancy occurs, the syndrome will typically resolve within 1 week. In a maintained pregnancy, slow resolution of symptoms usually occurs over 1-2 months.Case ReportA 31-year-old woman, gravida 2, para 1, aborta 1, with polycystic ovary syndrome underwent in vitro fertilization (IVF) with clomiphene citrate and follicle-stimulating hormone/gonadotropin releasing hormone-antagonist stimulation. During transvaginal oocyte retrieval, enlarged bilateral ovaries were noted. She had an episode of OHSS after IVF/embryo transfer, for which paracentesis was performed three times. Pregnancy was achieved. Throughout antenatal examinations, bilateral ovaries were enlarged. She delivered a healthy baby by cesarean section at term. However, 1 month after delivery, the bilateral ovary had not shrunk, and levels of tumor markers CA125 and CA199 were 50.84 and 41.34 U/mL, respectively. At laparotomy for suspected malignancy, both adnexae formed “kissing ovaries”, which were multinodulated with yellow serous fluid. Specimens from wedge resection submitted for frozen section showed a benign ovarian cyst. The final pathology report showed bilateral follicle cysts.ConclusionWith the increasing use of gonadotropins in the management of infertility, ovarian enlargement secondary to hyperstimulation is common. Generally, symptoms appear between the 6th and 13th weeks of pregnancy and disappear thereafter. The hyperstimulated ovary often subsides after the first trimester. This case is unusual as the megalocystic ovary persisted after delivery. To the best of our knowledge, we report the first case of enlarged bilateral ovaries persisting 2 months after delivery

Common genetic variants associated with pancreatic adenocarcinoma may also modify risk of pancreatic neuroendocrine neoplasms (vol 39, pg 360, 2018)

Pancreatic neuroendocrine neoplasms (pNEN) account for less than 5% of all pancreatic neoplasms and genetic association studies on susceptibility to the disease are limited. We sought to identify possible overlap of genetic susceptibility loci between pancreatic ductal adenocarcinoma (PDAC) and pNEN; therefore, PDAC susceptibility variants (n=23) from Caucasian genome-wide association studies (GWAS) were genotyped in 369 pNEN cases and 3,277 controls from the PANcreatic Disease ReseArch (PANDoRA) consortium to evaluate the odds associated with pNEN risk, disease onset and tumor characteristics. Main effect analyses showed four PDAC susceptibility variants – rs9854771, rs1561927, rs9543325 and rs10919791 to be associated with pNEN risk. Subsequently, only associations with rs9543325, rs10919791 and rs1561927 were noteworthy with false positive report probability (FPRP) tests. Stratified analyses considering age at onset (50 year threshold), showed rs2736098, rs16986825 and rs9854771 to be associated with risk of developing pNEN at a younger age. Stratified analyses also showed some SNPs to be associated with different degrees of tumor grade, metastatic potential and functionality. Our results identify known GWAS PDAC susceptibility loci, which may also be involved in sporadic pNEN etiology and suggest that some genetic mechanisms governing pathogenesis of these two entities may be similar, with few of these loci being more influential in younger cases or tumor subtypes

Modulazione termica dell'espressione della subunità catalitica della tossina difterica e delle sue varianti CRM176 e CRM197 nella terapia genica del carcinoma del pancreas

Despite surgery and/or chemotherapy more than 80% of patients affected by pancreatic cancer do not survive more than 5 years after diagnosis. Gene therapy with bacterial toxins might be an effective approach for pancreatic cancer treatment. The aims of the present study were: 1. to create expression vectors, encoding highly toxic compounds, which can be activated and modulated by heat; 2. to verify, in vitro, their possible application in pancreatic cancer gene therapy. First objective The catalytic domain of diphtheria toxin (DTA) and of its variants CRM176 and CRM197 have been employed in this study as potent inhibitors of protein synthesis. To confine the expression of these cytotoxic agents to neoplastic tissue we focused our attention on heat inducible promoters, which allow a selective spatial and time control of gene induction by external heat. We chose the promoter of HSPA6 (encoding Hsp70B' protein) because it is strictly inducible, its basal expression levels being barely detectable in most tissues. We engineered three expression vectors with different heat-inducible HSPA6-derived promoter sequences driving the reporter gene eGFP: V1 containing a 473bp commercial sequence with a 91% homology to HSPA6; V2 with a 104bp sequence designed by us containing five Heat Shock Elements in tandem and the minimal promoter of HSPA6; V3 containing the 104bp sequence located downstream the 473bp promoter. In order to define the optimal heat shock temperature and exposure time we used four pancreatic cancer cell lines stably transfected with V1: the highest levels of eGFP expression (measured by Q-RT-PCR and FACS analysis) were obtained with a heat shock of 42,5°C for 1,5 hours. After setting the experimental conditions we compared the eGFP protein and mRNA levels in V1, V2 and V3 transfected pancreatic cancer cell lines. At 37°C there were minimal eGFP expression levels in cells transfected with all three vectors. After heat shock, however, V1, V2 and V3 transfected cells behaved differently: V1 and V2 transfected lines showed low and similar levels of expression (fold increase in mRNA about 8) while V3 transfected cells were highly induced by heat reaching up to 30 times the basal levels. V3, therefore, was shown to offer the best combination of high transciption efficiency and low background levels and was selected to drive the expression of DTA and its less toxic variants. Second objective The growth of all cell lines, transfected with DTA or its twenty fold less active variant CRM176, was significantly delayed even at 37°C. In other words the basal transcription levels of these toxins are sufficient to cause cell death; therefore DTA and CRM176 cannot be considered suitable candidates for gene therapy protocols using V3 promoter. At 37°C the supposedly inactive toxin CRM197 caused mild distress in transfected cells. After heat shock this phenomenon was amplified: cell growth was reduced in all CRM197 transfected cell lines. These findings indicate that the lethal effects of CRM197 are probably dose correlated. A spatial and time controlled expression of this toxin variant might offer the opportunity of combining its cytotoxic effects with its immunogenic properties, which may help antitumor immune system reaction. Conclusions The expression vector with the V3 heat-inducible promoter driving the CRM197 variant can be considered a promising starting point for future in vivo applications of pancreatic cancer gene therapy