A highly efficient tumor-infiltrating MDSC differentiation system for discovery of anti-neoplastic targets,which circumvents the need for tumor establishment in mice

Myeloid-derived suppressor cells (MDSCs) exhibit potent immunosuppressive activities in cancer. MDSCs infiltrate tumors and strongly inhibit cancer-specific cytotoxic T cells. Their mechanism of differentiation and identification of MDSC-specific therapeutic targets are major areas of interest. We have devised a highly efficient and rapid method to produce very large numbers of melanoma-infiltrating MDSCs ex vivo without inducing tumors in mice. These MDSCs were used to study their differentiation, immunosuppressive activities and were compared to non-neoplastic counterparts and conventional dendritic cells using unbiased systems biology approaches. Differentially activated/deactivated pathways caused by cell type differences and by the melanoma tumor environment were identified. MDSCs increased the expression of trafficking receptors to sites of inflammation, endocytosis, changed lipid metabolism, and up-regulated detoxification pathways such as the expression of P450 reductase. These studies uncovered more than 60 potential novel therapeutic targets. As a proof of principle, we demonstrate that P450 reductase is the target of pro-drugs such as Paclitaxel, which depletes MDSCs following chemotherapy in animal models of melanoma and in human patients. Conversely, P450 reductase protects MDSCs against the cytotoxic actions of other chemotherapy drugs such as Irinotecan, which is ineffective for the treatment of melanoma.Myeloid-derived suppressor cells (MDSCs) have been recognized as major contributors to tumor-induced immunosuppression. Tumor-infiltrating MDSCs strongly inhibit cytotoxic T cells, and their expansion favors tumor progression and metastasis [1, 2]. Counteracting MDSC activities strongly enhances anti-cancer treatments and prolongs survival. Specific MDSC elimination by chemotherapy significantly contributes to anti-tumor efficacy [3-5]. Interestingly, conventional dendritic cells (DCs) remain unaffected by some of these chemotherapy treatments and the mechanisms underlying selective MDSC susceptibility to these drugs are currently unknown. The availability of large numbers of tumor-infiltrating MDSCs would significantly improve research in their biology and functions, and facilitate anti-MDSC drug discovery.MDSCs in mice comprise a heterogeneous population of immature CD11b^high Gr-1⁺ myeloid cells [6]. However, their discrimination from other myeloid cells such as immature DCs, M2 macrophages, monocytes and neutrophils remains somewhat ambiguous. Nevertheless, mouse MDSCs are classified into monocytic (M) and granulocytic (G) subsets, which differ in Ly6C-Ly6G expression profiles. M-MDSCs are Ly6C^high Ly6G^−/low while G-MDSCs are Ly6C^int/low Ly6G^high. Both subsets suppress immune responses through several pathways, including L-arginine depletion through arginase-1 (arg-1) and inducible nitric oxide synthase (iNOS) activity, increased generation of reactive oxygen species (ROS) and production of immunosuppressive cytokines such as TGF-β [7, 8].For their study, MDSCs are isolated from the spleen or directly of tumors from a large number of tumor-bearing mice [9-11]. However, spleen MDSCs are phenotypically and functionally different from tumor-infiltrating MDSCs [11, 12]. Moreover, isolated intra-tumor MDSCs are usually contaminated with other myeloid cells, do not proliferate well ex vivo, lack plasticity of differentiation and are prone to apoptosis [9, 13, 14]. In addition, low MDSC numbers are obtained from within tumors [12, 15]. Ex vivo MDSC production systems have been developed, which rely on incubation of bone marrow (BM) cells with high concentrations of recombinant GM-CSF, alone or in combination with other cytokines, and sometimes supplemented with cancer cell-derived conditioning medium. Nevertheless, these methods achieve MDSC differentiation efficiencies of around 30%-40% of total cells [13, 14]. In practical terms, none of these methods have yet replaced the purification of MDSCs directly from tumors of cancer-bearing mice. Therefore, high-throughput and drug discovery studies with isolated intra-tumor MDSCs are certainly a challenge.     

Objectively measured physical activity and sedentary time in European adolescents: the HELENA study

The authors' aim in this cross-sectional study was to characterize levels of objectively measured physical activity and sedentary time in adolescents from 9 European countries. The study comprised 2,200 European adolescents (1,184 girls) participating in the HELENA cross-sectional study (2006-2008). Physical activity was measured by accelerometry and was expressed as average intensity (counts/minute) and amount of time (minutes/day) spent engaging in moderate- to vigorous-intensity physical activity (MVPA). Time spent in sedentary behaviors was also objectively measured. Cardiorespiratory fitness was measured by means of the 20-m shuttle run test. Level of maternal education was reported by the adolescents. A higher proportion of boys (56.8% of boys vs. 27.5% of girls) met the physical activity recommendations of at least 60 minutes/day of MVPA. Adolescents spent most of the registered time in sedentary behaviors (9 hours/day, or 71% of the registered time). Both average intensity and MVPA were higher in adolescents with high cardiorespiratory fitness, and sedentary time was lower in the high-fitness group. There were no physical activity or sedentary time differences between maternal education categories. These data provide an objective measure of physical activity and amount of time spent in sedentary behaviors in a relatively large number of European adolescents.     

Intergenerational Cardiovascular Disease Risk Factors Involve Both Maternal and Paternal BMI

OBJECTIVE

To examine the association between parental BMI and offspring cardiovascular disease (CVD) risk factors.

RESEARCH DESIGN AND METHODS

The study comprised 940 children (9.5 ± 0.4 years) and 873 adolescents (15.5 ± 0.5 years). Parental weight and height were reported by the mother and the father, and BMI was calculated. CVD risk factors included total (sum of five skinfolds) and central (waist circumference) body fat, blood pressure, cardiorespiratory fitness, insulin sensitivity, total cholesterol, HDL cholesterol, triglycerides, and fibrinogen.

RESULTS

Maternal and paternal BMI were positively associated with total and central fatness in offspring (P < 0.001). BMIs of both parents were significantly related to fibrinogen levels (P < 0.02), but these associations disappeared when controlling for fatness. There was a positive relationship between maternal and paternal BMI and waist circumference in the offspring regardless of total adiposity and height (P < 0.001). Maternal BMI was negatively associated with offspring cardiorespiratory fitness independently of fatness (P < 0.02). These relationships persisted when overweight descendants were excluded from the analysis. There were no significant associations between parental BMI and the other CVD risk factors.

CONCLUSIONS

Both maternal and paternal BMI increase CVD risk factors of their offspring, characterized by total and central body fat, and higher maternal BMI was associated with poorer cardiorespiratory fitness. Our findings give further support to the concept that adiposity in parents transmits susceptibility to CVD risk to descendants, which is detectable even in the absence of overweight in offspring.Parental obesity substantially increases the risk of obesity in offspring through genetic, biological, or environmental influences (1). The fetal overnutrition hypothesis suggests that maternal obesity and/or gestational diabetes may predispose offspring to increased adiposity in adulthood (2). Human studies showed a greater influence of maternal than paternal BMI on offspring adiposity (3,4). In contrast, others suggested that the contribution of the mother and the father on both prenatal and postnatal programming of intergenerational obesity may be similar according to the genomic imprinting (5).Most of the studies focused on the relationships of maternal and paternal BMI with their offspring BMI provided contradictory results (3,6,7), and only one study compared the association of maternal and paternal BMI with total body fat in the offspring (8). Whether the parental BMI-offspring body fat relationship applies to other established cardiovascular disease (CVD) risk factors remains to be elucidated.Excess adiposity leads to increased CVD risk factors and biological pathway alterations as insulin resistance, dyslipemia, hypertension, systemic inflammation, and low cardiorespiratory fitness (9). Therefore, the parental BMI-offspring CVD risk factor relationship may be influenced by the offspring body composition.The European Youth Heart Study (EYHS) provides an opportunity to better understand the parental-descendant aggregation of CVD factors by controlling for other potential confounding factors that could mediate in this relationship. Therefore, the aim of this study was to determine the association between both maternal and paternal BMI and the offspring CVD risk factors including total and central body fat, cardiorespiratory fitness, insulin sensitivity, blood pressure, blood lipids, and fibrinogen. We also examined the role of offspring adiposity in this relationship.     

Nutrient oxidation and metabolic rate as affected by meals containing different proportions of carbohydrate and fat, in healthy young women

Summary The amount and composition of food eaten influence body weight regulation, which requires that, in the long term, energy intake matches energy expenditure and that the oxidation rate to be equal to intake for each and separate nutrients. The aim of this study was to examine the influence of two liquid formulas with different macronutrient composition, a high carbohydrate (HC) meal as compared to a high fat (HF) meal, on substrate oxidation and on thermic effect of food (TEF). Eighteen lean and healthy women which were fed a HC diet during the 3 preceding d were studied for a further 4 h after meal intake. The test meals provided fixed energy intake and whose calculated FQ were 0.77 for HF meal, and 0.96 for HC meal. The mean NPRQs were higher (P<0.01) in the HC group than in the HF group, even with values greater than 1.00 indicating net lipid synthesis (NL), and which correlated with metabolic rate (MR) value (P<0.05), glucose (P<0.05), and heart rate (HR) values. Carbohydrate (CHO) oxidation was higher with the HC than with HF meal (P<0.01) and correlated with the MR (P<0.05). Protein oxidation rate rose above baseline (P<0.01); this increase was accompanied by with a negative CHO balance. It is concluded that the change in fuel selection and the increase of TEF is mainly due to CHO intake and metabolism, respectively, and that surplus of dietary CHO of preceding days together with a large load of CHO can exceed the glycogen storage capacity and trigger NL. Received: 29 September 1998, Accepted: 14 April 1999     

Interaction Effect of the Mediterranean Diet and an Obesity Genetic Risk Score on Adiposity and Metabolic Syndrome in Adolescents: The HELENA Study

Obesity and metabolic syndrome (MetS) are worldwide major health challenges. The Mediterranean diet (MD) is associated with a better cardiometabolic profile, but these beneficial effects may be influenced by genetic variations, modulating the predisposition to obesity or MetS. The aim was to assess whether interaction effects occur between an obesity genetic risk score (obesity-GRS) and the MD on adiposity and MetS in European adolescents. Multiple linear regression models were used to assess the interaction effects of an obesity-GRS and the MD on adiposity and MetS and its components. Interaction effects between the MD on adiposity and MetS were observed in both sex groups (p < 0.05). However, those interaction effects were only expressed in a certain number of adolescents, when a limited number of risk alleles were present. Regarding adiposity, a total of 51.1% males and 98.7% females had lower body mass index (BMI) as a result of higher MD adherence. Concerning MetS, only 9.9% of males with higher MD adherence had lower MetS scores. However, the same effect was observed in 95.2% of females. In conclusion, obesity-related genotypes could modulate the relationship between MD adherence and adiposity and MetS in European adolescents; the interaction effect was higher in females than in males.     

Activity of soluble aminopeptidase A and dipeptidyl peptidase IV and membrane-bound aminopeptidase B and pyroglutamyl peptidase I in adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis

Objective

To analyze soluble and membrane-bound peptidase activities in the tonsils and adenoids removed from patients with adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis.

Methods

A total of 48 tissue samples from patients undergoing adenoidectomy and tonsillectomy for adenoid hyperplasia, tonsillar hyperplasia or chronic tonsillitis were analyzed. The catalytic activity of a pool of peptidases in the soluble (dipeptidyl peptidase IV, aminopeptidase A, aminopeptidase N and cystinyl aminopeptidase) and membrane-bound (prolyl endopeptidase, aspartyl aminopeptidase, aminopeptidase B and pyroglutamyl peptidase I) fractions was measured fluorometrically.

Results

The activity of membrane-bound aminopeptidase B was higher in cases of chronic tonsillitis and adenoid hyperplasia than in tonsillar hyperplasia, p = 0.004. Soluble dipeptidyl peptidase IV and membrane-bound pyroglutamyl peptidase I were found to be more active in tissues from male chronic tonsillitis tissues, p < 0.05, while membrane-bound aminopeptidase B activity was higher in tissues of females with tonsillar hyperplasia, p < 0.001. In the case of chronic tonsillitis, soluble aminopeptidase A was found to have a higher level of activity in tissues from children than those from adults, p = 0.005.

Conclusions

Our results suggest a potential role of soluble aminopeptidase A, soluble dipeptidyl peptidase IV, membrane-bound aminopeptidase B and membrane-bound pyroglutamyl peptidase I in the pathobiology of adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis that is differently regulated as a function of gender. These finfings may modify in the future the clinical approach to these diseases.