Beata Małachowska

Compensatory metabolic networks in pancreatic cancers upon perturbation of glutamine metabolism

Pancreatic ductal adenocarcinoma is a notoriously difficult-to-treat cancer and patients are in need of novel therapies. We have shown previously that these tumours have altered metabolic requirements, making them highly reliant on a number of adaptations including a non-canonical glutamine (Gln) metabolic pathway and that inhibition of downstream components of Gln metabolism leads to a decrease in tumour growth. Here we test whether recently developed inhibitors of glutaminase (GLS), which mediates an early step in Gln metabolism, represent a viable therapeutic strategy. We show that despite marked early effects on in vitro proliferation caused by GLS inhibition, pancreatic cancer cells have adaptive metabolic networks that sustain proliferation in vitro and in vivo. We use an integrated metabolomic and proteomic platform to understand this adaptive response and thereby design rational combinatorial approaches. We demonstrate that pancreatic cancer metabolism is adaptive and that targeting Gln metabolism in combination with these adaptive responses may yield clinical benefits for patients.

Evolutionarily conserved serum microRNAs predict radiation-induced fatality in nonhuman primates

Evolutionarily conserved serum microRNAs predict radiation-induced fatality in nonhuman primates. Radiation alert When a nuclear accident or attack occurs and people are exposed to radiation, a rapid and accurate response is vital for saving lives. In particular, it is important to identify those who had been exposed to radiation at all and to determine which victims are at risk of death or severe injury and require urgent treatment. Fendler et al. studied nonhuman primates who had been exposed to radiation and given a radioprotective drug or placebo, and then analyzed the animals’ blood and developed an miRNA-based classifier. By measuring the expression of five miRNAs (adjusted for gender), the authors were able to identify which primates had been exposed to radiation as well as their chances of survival. Effective planning for the medical response to a radiological or nuclear accident is complex. Because of limited resources for medical countermeasures, the key would be to accurately triage and identify victims most likely to benefit from treatment. We used a mouse model system to provide evidence that serum microRNAs (miRNAs) may effectively predict the impact of radiation on the long-term viability of animals. We had previously used nonhuman primates (NHPs) to demonstrate that this concept is conserved and serum miRNA signatures have the potential to serve as prediction biomarkers for radiation-induced fatality in a human population. We identified a signature of seven miRNAs that are altered by irradiation in both mice and NHPs. Genomic analysis of these conserved miRNAs revealed that there is a combination of seven transcription factors that are predicted to regulate these miRNAs in human, mice, and NHPs. Moreover, a combination of three miRNAs (miR-133b, miR-215, and miR-375) can identify, with nearly complete accuracy, NHPs exposed to radiation versus unexposed NHPs. Consistent with historical data, female macaques appeared to be more sensitive to radiation, but the difference was not significant. Sex-based stratification allowed us to identify an interaction between gender and miR-16-2 expression, which affected the outcome of radiation exposure. Moreover, we developed a classifier based on two miRNAs (miR-30a and miR-126) that can reproducibly predict radiation-induced mortality. Together, we have obtained a five-miRNA composite signature that can identify irradiated macaques and predict their probability of survival.

Prevalence and clinical impact of IgE-mediated food allergy in school children with asthma: a double-blind placebo-controlled food challenge study.

Purpose Recent studies indirectly suggest a possible link between food allergy (FA) and asthma. Most of them have evaluated the occurrence of FA in asthmatic children, especially in the first year of life, using questionnaire-based studies or specific IgE (sIgE) assay. The aim of this study was to evaluate the prevalence and clinical impact of IgE-mediated FA in school children with asthma using a double-blind placebo-controlled food challenge (DBPCFC). Methods The study group consisted of school children with atopic asthma who were admitted to the Department of Pediatric Allergology, Gastroenterology and Nutrition, Medical University of Lodz, for the evaluation of food hypersensitivity. The diagnosis of FA was established using questionnaires, sIgE analysis, and the DBPCFC. Asthma severity and asthma control state were also assessed. Results A relationship between consumed food and complaints was reported in 180 children (49.7%). Seventy children (19.3%) were sensitized to food allergens. IgE-mediated FA was confirmed in 24 children (6.6%), while 11 children (3%) demonstrated respiratory symptoms. Food-induced asthma exacerbations were observed in 9 patients (2.5%). Statistically significant differences in the prevalence of atopic dermatitis (P<0.002), urticaria (P<0.03), digestive symptoms (P<0.03), rhinitis (P<0.02), sIgE level (P<0.001), positive family history of atopy (P<0.001) and FA in history (P<0.001) were found between asthmatic children with FA and those without. Children with food-induced asthma exacerbations demonstrated significantly greater severity, poorer controls, and worse morbidity compared to those without. Conclusions Although food-induced respiratory reactions in children with asthma were rare, they were classified as severe and associated with worse morbidity, greater severity, and poorer control. As the most commonly observed symptoms were coughing and rhinitis, which can be easily misdiagnosed, a proper diagnosis is essential for improving the management of both clinical conditions.

Association of microRNA-93, 190, 200b and Receptor Status in Core Biopsies from Stage III Breast Cancer Patients

Oncologists now favor more personalized treatment strategies in breast cancer patients. Gene expression analysis has been widely used, but less is known about epigenetic factors, for example, microRNAs (miRNAs). The aim of this study was to determine the relationship between selected miRNAs and receptor status in core biopsies sampled before preoperative chemotherapy in stage III locally advanced breast cancer (LABC) patients. In 37 LABC core biopsies, three miRNAs per sample were analyzed: hsa-miR-93-5p, hsa-miR-190a, and hsa-miR-200b-3p, and hsa-miR-103a-3p as an endogenous control (TaqMan(®) RT-PCR; Applied Biosystems). Receptor status was determined by a dedicated pathologist. The Mann-Whitney U, Shapiro-Wilk, and Levenes tests were used to compare related samples. Levels of miRNA-93 differed significantly in core biopsies of LABC patients with different expressions of ER (estrogen receptor) and PR (progesterone receptor). Higher levels of miRNA-93 were found in ER-negative (p=0.0027) and PR-negative patients (p=0.0185). Levels of miRNA-190 and 200b did not differ significantly in core biopsies of LABC patients who expressed ER and PR differently (p=0.7727, p=0.9434, p=0.6213, and p=0.1717). Levels of miRNA-93, 190, and 200b were not significantly different in core biopsies of LABC patients with different HER2 (human epidermal growth factor 2) expressions (p=0.8013, p=0.2609, and p=0.3222). The assessment of core biopsy miRNA profiles and receptor-based subtypes may identify new signaling pathways for improved breast cancer classification.

The NR3C1 Glucocorticoid Receptor Gene Polymorphisms May Modulate the TGF-beta mRNA Expression in Asthma Patients

Glucocorticosteroids (GCs) are basic drugs in therapy of a number of diseases, including chronic diseases of the respiratory system. They are the most important anti-inflammatory drugs in the treatment of asthma. GCs after binding to the glucocorticoid receptor (GR) form the complex (transcription factor), which acts on promoter and regulatory parts of genes enhancing the expression of anti-inflammatory proteins and decreasing the proinflammatory protein synthesis, including numerous cytokines mediating inflammation in the course of asthma. Non-sensitivity or resistance to GCs favours an increase in the TGF-β expression. This cytokine plays a central role in asthma inducing fibroblast differentiation and extracellular matrix synthesis. TGF-β isoforms, 1, 2 and 3, are located on chromosome 19q13, 1q41 and 14q24, respectively. GCs reduce TGF-β 1 and TGF-β 2 production and significantly decrease the expression of upregulated TGF-β 1 and TGF-β 2 mRNA induced by exogenous TGF-β. In asthma, TGF-β may play a role in the development of the peribronchiolar and subepithelial fibrosis, which contributes to a significant clinical exacerbation of asthma. Therefore, it is possible that NR3C1 glucocorticoid receptor gene polymorphisms could exert varied effects on the TGF-β mRNA expression and fibrotic process in lungs of asthmatic patients. The aim of the study was to evaluate the impact of polymorphic forms (Tth111I, BclI, ER22/23EK, N363S) of the NR3C1 gene on the level of the TGF-β 1 mRNA expression. A total of 173 patients with asthma and 163 healthy volunteers participated in the study. Genotyping of Tth111I, BclI, ER22/23EK, and N363S polymorphisms of the NR3C1 gene was performed by using PCR-HRM and PCR-RFLP techniques. TGF-β mRNA was assessed by real time RT-PCR. Tth111I SNP significantly (p = 0.0115) correlated with the TGF-β 1 mRNA expression level. The significance of AA and GG genotypes of Tth111I SNP in increasing and decreasing the level of the TGF-β 1 mRNA expression was demonstrated. Both BclI SNP and ER22/23EK SNP did not affect the expression level of the cytokine analysed. The N363S SNP AA genotype of NR3C1 gene statistically significantly influenced the increase in the level of the TGF-β 1 mRNA expression. Thus, SNPs of NR3C1 gene play an important regulatory function in the bronchi of patients suffering from asthma. In the case of the occurrence of Tth111I and N363S polymorphic forms of the gene studied, a reduced ability of GCs to inhibit the TGF-β 1 expression can be observed.

The impact of food allergens on airway responsiveness in schoolchildren with asthma: A DBPCFC study

Despite the growing evidence of a possible link between asthma and food allergy (FA), so far, the involvement of food in inducing respiratory symptoms has not been fully evaluated. The objective of this study was to evaluate the impact of food allergens on respiratory symptoms and bronchial reactivity (BHR) in schoolchildren with asthma.

Population-based estimates for double diabetes amongst people with glucokinase monogenic diabetes, GCK-MODY

Monogenic diabetes has become an increasingly active area of research in recent years, allowing for the establishment of several large registries and epidemiologic estimates [1–3]. In the paediatric population, mutation of the glucokinase gene (GCK) is most frequently responsible for the development of monogenic diabetes, typically characterized by low HbA1c levels and low risk of complications [2]. However, we have observed patients with poor metabolic control in the Polish GCK maturity-onset diabetes of the young (MODY) cohort, and hypothesized that such individuals may have a second cause of diabetes. To calculate the expected number of patients with different overlapping types of diabetes, we used data from available epidemiologic reports [1–3] and the Central Statistical Office of Poland (www.stat.gov.pl/bdlen/ app/strona.html?p_name-indeks). To estimate the frequency of this ‘double diabetes’, we assumed the frequency of monogenic diabetes to equal 108 per million individuals [1], out of whom approximately one third were expected to have GCK-MODY. The overall prevalence of diabetes in Poland was estimated using the International Diabetes Federation (IDF) Diabetes Atlas [4]. To cover the whole population from 0 to 79 years, we added prevalence data on paediatric patients described in earlier epidemiologic reports [2,5] to the IDF estimates (which covered ages 20–79 years). The distribution of major types of diabetes was assumed to be 90% Type 2 and 10% Type 1 diabetes [6]. As there was no evidence of monogenic diabetes being protective against either Type 1 or Type 2 diabetes, we multiplied the probability of having these types by the number of individuals with monogenic diabetes. Patient recruitment and data collection in the group of individuals with GCK-MODY was described in earlier reports [7–9] and in the Supporting Information (Appendix S1). The study was approved by the Bioethics Committee of the Medical University of Lodz. All study participants gave their informed consent. In Poland, 1.54 million (4.0%) individuals have diabetes [4]. Based on earlier estimates, the total number of patients with monogenic diabetes would equal 4162 (95% CI 4038–4290). The number of patients with Type 2 diabetes overlapping on monogenic diabetes in Poland should thus reach 150 (3.6%, 95% CI 3.1–4.3). Similarly, 17 individuals with concomitant Type 1diabetes and monogenic diabetes would be expected from such an estimate [0.4% (95% CI 0.3–0.6)]. To determine how many current patients with such overlapping phenotypes there are, we evaluated individuals in the Polish Nationwide Registry of Monogenic Diabetes with GCKMODY, as its distinctive phenotype marked out individuals with highly unusual phenotype characteristics. Of the 285 eligible patients, eight individuals (2.8%) had HbA1c levels > 59 mmol/mol (7.5%; see also Supporting Information, Fig. S1). Seven of them had shown features of Type 2 diabetes at the time of diagnosis (Table 1), were more likely to be obese [odds ratio (95% CI): 11.78 (1.90–79.27)], more likely to manifest polyuria/polydipsia [odds ratio (95% CI): 5.92 (0.96–6.7)] and required insulin treatment more frequently [odds ratio (95% CI): 20.64 (2.46–173.10)] than the rest of the group with GCK-MODY. BMI status was significantly associated with HbA1c level (Fd.f.=3 = 103.75; P < 0.0001); 31 (10.88%) patients were overweight and 11 (3.95%) were obese. However, obesity alone did not seem sufficient to explain high HbA1c. Out of 11 individuals with BMI > 30 kg/m, only two had HbA1c levels > 59 mmol/mol. This suggested that an additional factor, such as insulin resistance or impaired b-cell function, was present in the seven individuals with Type 2 diabetes-like phenotype and GCK-MODY. The eighth patient suspected for double diabetes showed features of Type 1 diabetes. The probability of monogenic diabetes computed with the MODY calculator [10] for the eight identified patients was well below the threshold recommended for the UK population. This confirmed that, had the patients not been identified as relatives of children previously screened for GCK-MODY, or not developed diabetes in childhood, they would not be considered eligible for genetic testing. Considering the circumstances in which patients with double diabetes were enrolled into the genetic screening programme, it seems well justified that a paediatric-centred genetic screening is a feasible solution that would not exclude them on the basis of metabolic control and traits uncommon to monogenic diabetes. The HbA1c threshold of 59 mmol/mol (7.5%) used in our search for atypical GCK-MODY was selected because of its importance in guidelines for MODY diagnosis and current screening programmes. The observed number of seven patients with Type 2 and GCK-MODY diabetes identified through our search was in line with a recent paper by Steele et al., who showed that six out of 235 individuals with GCK-MODY had high HbA1c levels [66–79 mmol/mol (8.2–9.4%)], accompanied by traits of Type 2 diabetes [11]. Both frequencies are lower than the expected 3.6% based on prevalence of Type 2 diabetes, but the difference may result from more aggressive treatment or relatively young age of the cohort. It is, however, evident that such a therapeutic challenge will arise as more and more patients with various types of monogenic diabetes are identified using novel molecular methods. As therapeutic trials on such patients are unlikely, it seems reasonable to focus on achievement of metabolic goals in patients with double diabetes, even if this mandates treatment intensification not routinely recommended in GCK-MODY. In conclusion, the coexistence of a second type of diabetes with GCK-MODY may hinder recruitment to genetic screening programmes and may necessitate a more aggressive therapeutic approach.

TP53 Promoter Methylation in Primary Glioblastoma: Relationship with TP53 mRNA and Protein Expression and Mutation Status

Reduced expression of TP53 by promoter methylation has been reported in several neoplasms. It remains unclear whether TP53 promoter methylation is associated with reduced transcriptional and protein expression in glioblastoma (GB). The aim of our work was to study the impact of TP53 methylation and mutations on TP53 mRNA level and protein expression in 42 molecularly characterized primary GB tumors. We also evaluate the impact of all molecular alterations on the overall patient survival. The frequency of TP53 promoter methylation was found in 21.4%. To the best of our knowledge, this is the first report showing such high frequency of TP53 promoter methylation in primary GB. There was no relation between TP53 promoter methylation and TP53 mRNA level (p=0.5722) and between TP53 promoter methylation and TP53 protein expression (p=0.2045). No significant associations were found between TP53 mRNA expression and mutation of TP53 gene (p=0.9076). However, significant association between TP53 mutation and TP53 protein expression was found (p=0.0016). Our data suggest that in primary GB TP53 promoter methylation does not play a role in silencing of TP53 transcriptional and protein expression and is probably regulated by other genetic and epigenetic mechanisms associated with genes involved in the TP53 pathway.

Relationship between arsenic and selenium in workers occupationally exposed to inorganic arsenic

The interaction between arsenic (As) and selenium (Se) has been one of the most extensively studied. The antagonism between As and Se suggests that low Se status plays an important role in aggravating arsenic toxicity in diseases development. The objective of this study was to assess the Se contents in biological samples of inorganic As exposed workers (n=61) and in non-exposed subjects (n=52). Median (Me) total arsenic concentration in urine of exposed workers was 21.83μg/g creat. (interquartile range (IQR) 15.49-39.77) and was significantly higher than in the control group - (Me 3.75μg/g creat. (IQR 2.52-9.26), p<0.0001). The median serum Se concentrations in the study group and the control were: 54.20μg/l (IQR 44.2-73.10μg/l) and 55.45μg/l (IQR 38.5-69.60μg/l) respectively and did not differ significantly between the groups. In the exposed group we observed significantly higher urine concentrations of selenosugar 1 (SeSug 1) and selenosugar 3 (SeSug3) than in the control group Me: 1.68μg/g creat. (IQR 1.25-2.97 vs Me: 1.07μg/g creat. (IQR 0.86-1.29μg/g), p<0.0001 for SeSug1; Me: 0.45μg/g creat. (IQR 0.26-0.69) vs Me: 0.28μg/g creat. (IQR 0.17-0.45μg/g), p=0.0021). In the multivariate model, after adjusting to cofounders (age, BMI, job seniority time, consumption of fish and seafood and smoking habits) the high rate of arsenic urine wash out (measured as a sum of iAs+MMA+DMA) was significantly associated with the high total selenium urine excretion (B=0.14 (95%CI (confidence interval) 0.05-0.23)). Combination of both arsenic and selenium status to assess the risk of arsenic-induced diseases requires more studies with regard to both the analysis of speciation, genetics and the influence of factors such as nutritional status.

Essential oils reduce autonomous response to pain sensation during self-monitoring of blood glucose among children with diabetes.

Abstract Background: Essential oils were proven to possess analgesic activity in adults. Children with diabetes are exposed to highly painful interventions such as self-monitoring of blood glucose (SMBG). Objective: An evaluation of the analgesic properties of two essential oils during SMBG in diabetic children. Subjects: We included 73 hospitalized children (age<18 years) with well-controlled type 1 diabetes. Methods: The study extended over a period of 1 month (2 weeks for control group and 1 week for orange and lavender oil application). The measurements were performed four times per day in a shared room during SMBG. Pain intensity was evaluated by visual analog scale (VAS) and change of baseline heart rate (ΔHR%). An aromatherapy device was used to disperse essential oils in the testing room. Results: We performed 647 individual measurements of pain intensity and ΔHR%. Girls reported higher VAS scores [median, Me 0.5 (interquartile range, IQR 0–1) vs. 0 (IQR 0–0.5), p=0.0036]. Both age and duration of diabetes correlated with ΔHR% [r=–0.14, p=0.0005; r=–0.12, p=0.0025]. Negative correlations were also noted for VAS/age [r=–0.12, p=0.0030] and VAS/duration of diabetes [r=–0.12, p=0.0034]. Aromatherapy did not alter the VAS score (p=0.40), while ΔHR% decreased with borderline significance (p=0.0639). After adjustment for patient’s age and sex lower ΔHR% was associated with essential oil application (p=0.0252). Aromatherapy did not have any influence on VAS scores in multivariate analysis (p=0.35). Conclusion: Aromatherapy decreased the autonomic response to a painful stimulus by lowering ΔHR%, but did not affect the perception of pain reported by VAS.