Biomarkers of inflammation and breast cancer risk: A case-control study nested in the EPIC-Varese cohort

Abstract Breast cancer (BC) is the leading cause of cancer death in women. Adipokines, and other inflammation molecules linked to adiposity, are suspected to be involved in breast carcinogenesis, however prospective findings are inconclusive. In a prospective nested case-control study within the EPIC-Varese cohort, we used conditional logistic regression to estimate rate ratios (RRs) for BC, with 95% confidence intervals (CI), in relation to plasma levels of C-reactive protein (CRP), tumor necrosis factor-alpha (TNF-α), interleukin-6, leptin, and adiponectin, controlling for BC risk factors. After a median 14.9 years, 351 BC cases were identified and matched to 351 controls. No marker was significantly associated with BC risk overall. Significant interactions between menopausal status and CRP, leptin, and adiponectin were found. Among postmenopausal women, high CRP was significantly associated with increased BC risk, and high adiponectin with significantly reduced risk. Among premenopausal women, high TNF-α was associated with significantly increased risk, and high leptin with reduced risk; interleukin-6 was associated with increased risk only in a continuous model. These findings constitute further evidence that inflammation plays a role in breast cancer. Interventions to lower CRP, TNF-α, and interleukin-6 and increase adiponectin levels may contribute to preventing BC

Adipocytokines, Hepatic and Inflammatory Biomarkers and Incidence of Type 2 Diabetes. The CoLaus Study.

CONTEXT: There is contradictory information regarding the prognostic importance of adipocytokines, hepatic and inflammatory biomarkers on the incidence of type 2 diabetes. The objective was to assess the prognostic relevance of adipocytokine and inflammatory markers (C-reactive protein - CRP; interleukin-1beta - IL-1β; interleukin-6- IL-6; tumour necrosis factor-α - TNF-α; leptin and adiponectin) and gamma-glutamyl transpeptidase (γGT) on the incidence of type 2 diabetes. METHODS: Prospective, population-based study including 3,842 non-diabetic participants (43.3% men, age range 35 to 75 years), followed for an average of 5.5 years (2003-2008). The endpoint was the occurrence of type 2 diabetes. RESULTS: 208 participants (5.4%, 66 women) developed type 2 diabetes during follow-up. On univariate analysis, participants who developed type 2 diabetes had significantly higher baseline levels of IL-6, CRP, leptin and γGT, and lower levels of adiponectin than participants who remained free of type 2 diabetes. After adjusting for a validated type 2 diabetes risk score, only the associations with adiponectin: Odds Ratio and (95% confidence interval): 0.97 (0.64-1.47), 0.84 (0.55-1.30) and 0.64 (0.40-1.03) for the second, third and forth gender-specific quartiles respectively, remained significant (P-value for trend = 0.05). Adding each marker to a validated type 2 diabetes risk score (including age, family history of type 2 diabetes, height, waist circumference, resting heart rate, presence of hypertension, HDL cholesterol, triglycerides, fasting glucose and serum uric acid) did not improve the area under the ROC or the net reclassification index; similar findings were obtained when the markers were combined, when the markers were used as continuous (log-transformed) variables or when gender-specific quartiles were used. CONCLUSION: Decreased adiponectin levels are associated with an increased risk for incident type 2 diabetes, but they seem to add little information regarding the risk of developing type 2 diabetes to a validated risk score

Adipocyte-derived extracellular vesicles: characterisation and function

Extracellular vesicles (EVs) are submicron vesicles released from cells as intercellular communicators. EV research is hindered by a current lack of standardisation. However, mounting evidence suggests a role for EVs in both physiological and pathophysiological processes. Little is known about adipocyte-derived EVs despite the recognition of adipose tissue (AT) as an endocrine organ and the role of dysfunctional AT in disease. Obese AT develops regions of hypoxia and inflammation, leading to obesity-associated metabolic complications. The aim of this thesis was to explore EVs as novel adipocyte communicators, characterising their release under physiological and disease-like conditions. Nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) were assessed for their accuracy and usability for EV quantification. NTA and TRPS both accurately quantified EVs though NTA was more user-friendly. The choice of anticoagulant, filtering and storage of EVs all affected EV concentration. Physiological EV release from 3T3-L1 adipocytes was characterised pre- and post-adipogenesis. EV generation increased prior to adipogenesis and EVs were enriched in pro-signalling fatty acids and proteins characteristic of the original cell. Therefore, EVs may aid the initiation of adipogenesis. Hypoxia was then used to pathologically generate EVs to mimic adipocyte obesity. Adipocyte EV release increased in hypoxia; these EVs were enriched in pro-signalling fatty acids and monocyte chemoattractant protein-1. Hypoxic EVs were then analysed for their interaction with macrophages (Mϕ). Hypoxic EVs may increase Mϕ migration and promote an anti-inflammatory Mϕ phenotype; further repeats are needed to confirm this. Finally, adipocyte markers were detected in plasma EVs suggesting the presence of circulating adipocyte-derived EVs in vivo. Plasma EVs were also reduced in hypercholesterolaemia patients by routine apheresis treatment. In conclusion, adipocytes release EVs which may assist intercellular communication in both physiological and disease-like conditions. Adipocyte-derived EVs can be detected in vivo and may provide novel biomarkers of obesity-associated diseases

Effects of Transplanting Bone Marrow Stromal Cells Transfected with CXCL13 on Fracture Healing of Diabetic Rats

Background/Aims: Diabetic fracture have poor treatment and serious complications. Therefore, how to treat diabetic fracture is receiving increasing attention. This study aimed to investigate the effects of transplanting CXCL13-stimulated bone marrow stromal cells (BMSCs) on the fracture healing in diabetic rats. Methods: In vitro, RT-PCR was employed to examine the expression of CXCL13 in BMSCs in high glucose environment. MTT assay and apoptosis assay were utilized to determine the effects of CXCL13 overexpression on the proliferation and apoptosis of BMSCs respectively. ALP staining was applied to detect the ALP activity. In vivo, CXCL13-stimulated BMSCs were transplanted into the fracture sites of diabetic rats. At the 1st week, 2nd weeks, 4th week and 6th week after the operation, bone mineral density (BMD) and callus area measurement, ELISA detection, and HE staining were performed to evaluate the fracture healing. Results: Low BMD and less area of callus in diabetic rats showed that the recovery after fracture was worse in diabetic rats than in non-diabetic rats. Meanwhile, the expression of CXCL13 in serum was lower in diabetic rats than in non-diabetic rats. Overexpression of CXCL13 promoted the proliferation of BMSCs in vitro high glucose environment. After BMSCs transfected with CXCL13 being transplanted into the fracture sites of diabetic rats, it was found that the fracture healing was enhanced and ALP expression in serum became higher. HE staining results further verified the effects of transplantation of BMSCs transfected with CXCL13 on fracture healing in diabetic rats. Conclusion: These finding indicated that CXCL13 may play a critical role in the process of fracture healing, which could provide a deeper insight into molecular targets for the fracture healing in diabetic people

Acute intermittent porphyria. Inflammation, diet and biomarkers in acute intermittent porphyria

Acute intermittent porphyria (AIP) is a rare autosomal dominant inherited metabolic disease with lowered function of the enzyme hydroxymethylbilan synthase due to mutation in this gene of the heme synthesis. The resulting excess of aminolevulinic acid and porphobilinogen can result in acute abdominal pain, paresis, fatigue, increased risk for kidney failure and hepatocellular carcinoma. Some drugs, fasting, infections and stress can trigger AIP attacks. Metabolic diseases like diabetes mellitus are associated with inflammation. Therefore we investigated if there was inflammation in AIP measured as elevated levels of plasma cytokines. Handling of blood samples can have effect on the cytokine levels, so we compared the use of plasma using four different anticoagulants with serum. The effects of storage temperature, freezing and thawing were also examined. We concluded that one should analyse cytokines in EDTA plasma samples stored at 4ºC for up to 4 hours before centrifugation. We established reference ranges for cytokines from 162 healthy persons. By using a multiplex assay, we found elevated cytokines in the 50 AIP cases as a sign of low-grade inflammation, compared to the 50 matched controls. The inflammation may be one of the explanations for the higher risk for HCC in AIP. We found lower levels of prealbumin and lower kidney function in symptomatic vs. asymptomatic AIP cases. The fasting C-peptide and insulin levels were lower in the symptomatic AIP cases compared to matched controls. Persons with AIP are recommended a high carbohydrate intake because glucose inhibits the first step in the heme synthesis. The cases and controls filled out a 7-days diet logbook, had their anthropometric measures done, filled out a questionnaire on lifestyle-factors and was interviewed by a physician. Blood was analysed for cytokines, vitamins, glucose metabolism measures and fats etc. Persons with AIP had a lower carbohydrate intake than recommended by guidelines

Proteomic and clinical insights into polycystic ovary syndrome in adolescents

Despite its high prevalence, our understanding of the pathophysiology of polycystic ovary syndrome (PCOS) is lacking. Consequently, the way we diagnose and manage this common condition is inadequate, which is especially true for adolescents. This thesis aims to expand the body of knowledge regarding PCOS in adolescents. It will explore the clinical phenotype of PCOS, in addition to using proteomic techniques to better understand the biological mechanisms which underpin this condition. However, to do this, we must first comprehend ‘normal’ menstrual patterns in these pubertal years. As such, this thesis begins by seeking to define menstrual and ovulatory ‘normality’ in the first year following menarche, by systematically reviewing relevant literature. Following this, data are presented from a longitudinal study evaluating the clinical presentation and phenotype of adolescents with a suspected diagnosis of PCOS. The latter part of the thesis focuses on the use of proteomic techniques to broaden our understanding of PCOS. Discovery proteomic analysis of urine samples is employed firstly to explore the biological pathways associated with PCOS, and secondly to identify specific proteins which are differentially expressed in adolescents with PCOS, which may form a pool of non-invasive candidate biomarkers. Inflammation was identified as the most significant biological process associated with PCOS in discovery analysis, and these findings were validated in subsequent targeted proteomic panels. Validation studies were undertaken in a larger cohort of adolescents with PCOS, and then comparison was also made to adults with PCOS. Finally, all results from this thesis are summarised, the findings discussed, and their implications considered, alongside future work

Pregnancy following bariatric surgery: maternal considerations

Background The mechanism behind the perinatal complications associated with obesity in pregnancy is not fully understood. There is convincing evidence that pregnancies following bariatric surgery have a lower incidence of gestational diabetes (GDM), pre-eclampsia (PE), large for gestational age (LGA) neonates, higher incidence of small for gestational age (SGA) neonates and moderately preterm birth. The mechanism for this is also unknown, however, could be related to changes in maternal insulin resistance and other metabolic pathways involved in glucose and fat metabolism. Aims 1. To investigate the effects of bariatric surgery on maternal insulin resistance, waist to hip ratio (WHR), blood pressure and components of fat and glucose metabolism such as adipokines, pro-inflammatory hormones, incretins and metabolites. 2. To compare the lipoprotein profile of obese women and women with a normal BMI in the third trimester, without previous bariatric surgery. Method We conducted a prospective, longitudinal study comparing pregnant women with previous bariatric surgery to those without surgery. The following were assessed: 1. Insulin, glucose, glycosylated haemoglobin (HbA1c), Homeostasis Model Assessment of insulin resistance (HOMA-IR) and the Matsuda Index were measured using fasting blood samples collected at 28 weeks gestation. 2. Maternal weight, height, waist to hip ratio (WHR) and blood pressure were measured at all antenatal visits. 3. Fasting blood samples at 28+0-30+0 weeks’ gestation were used to measure peptide hormones, adipokines, pro-inflammatory hormones and incretins. 4. Untargeted metabolomics with proton Nuclear Magnetic Resonance (H1 NMR) was performed on samples obtained at six time points: 11+0-14+0 (T1), 20+0-24+0 (T2), 28+0-30+0 (T3), 30+0-33+0 (T4) and 35+0-37+6 (T5) weeks’ gestation, and within 72 hours of delivery (T6). H1 NMR lipoprotein profiling was performed for pregnant women recruited without previous bariatric surgery at 28+0-30+0 weeks’ gestation. Results were compared between women with normal BMI and women who were obese (BMI ≥ 30kg/m2). Results The no surgery group had higher median insulin resistance (IR), [2.20 (IQR 1.53-3.38)] compared to the post bariatric surgery group [1.15 (IQR 1.04 -2.07); p <0.05] and post malabsorptive bariatric surgery group, [1.08 (0.99 – 1.23; p <0.05]. Pregnant women with previous bariatric surgery had significantly lower leptin levels at 28-30 weeks [13.3ng/ml (IQR 9.71-15.36)] compared to women with no surgery [20.84ng/ml (IQR 18.12-24.1); p<0.05]. Maternal adiponectin levels at 28-30 weeks of gestation were higher in the post bariatric women [4.9µg/ml (IQR 2.9-6.7)] compared to no surgery women [2.43 µg/ml (IQR 1.8-3.2); p <0.05]. Pregnant women with previous malabsorptive bariatric surgery had an altered serum metabolome by T4 (30-33 weeks) and T5 (35-37 weeks) compared to those without bariatric surgery (p=0.027 and p=0.006, respectively). There is a lower serum level of unsaturated lipids, isobutyrate, leucine, isoleucine and N-acetyl glycoprotein and higher level of glutamine and D-ß-hydroxybutyrate. The lipoprotein profile of women at 28 weeks gestation without surgery showed that, compared to women with normal BMI, obese women have higher levels of HDL4 Triglyceride (p=0.02) VLDL1 Phospholipid (p=0.023) and VLDL1 Cholesterol (p=0.02) and lower levels of HDL, HDL1 cholesterol (p=0.02, 0.02), LDL2, LDL3 cholesterol (p=0.03, 0.02) and HDL1 phospholipid (p=0.03). Conclusion The study has demonstrated that women with previous bariatric surgery have a reduction in insulin resistance, especially post malabsorptive surgery. In the third trimester, they have a lower leptin and higher adiponectin level. These findings may explain the reduced incidence of GDM and LGA babies seen in this group.Open Acces

Peptidome and Proteome Peritoneal Dialysate Evolutionary Atlas (P3DEVOATLAS)

Peritoneal membrane (PM) failure in patients with end stage renal disease submitted to peritoneal dialysis (PD) cannot be predicted and does not occur in every patient in the same sequence and to the same extent. Moreover, long-term PD leads to morphological and functional alterations in the PM, reducing the lifespan of this dialysis up to five years, and forcing the replacement of PD by other renal replacement therapies. This represents a lower quality of life for the patients and extra cost of tens of million euros per year for the Portuguese National Health System. Peritoneal dialysis effluent (PDE) represents an underestimated biochemical window into the peritoneum and a useful reservoir of potential clinical biomarkers. Therefore, this work aims to develop longitudinal studies to unravel the evolution of the peptidome and proteome of the PDE with time, to identify specific molecular changes that can be particularly interesting for the understanding and early detection of long-term PM alterations. To achieve this goal, mass spectrometry (MS)-based methods are needed to improve PDE proteome and peptidome analysis and to overcome some drawbacks that can arise from such a complex biological sample that can hamper the proteome and peptidome coverage. For this reason, this thesis is focused also in the use of sample treatments and methodologies to reduce PDE sample complexity prior to MS analysis. Therefore, different methods of sample treatment were assessed with success as proteomics tools for getting insight into the PDE proteome and peptidome. Furthermore, this research constitutes the first proteome and peptidome-based longitudinal study of PD patient. In addition, the results represent the highest proteome and peptidome coverage ever achieved for this complex sample. Hence, this knowledge could be useful for the proteomic and clinical PD-devoted research community