Macrophage Migration Inhibitory Factor Inhibition Is Deleterious for High-Fat Diet-Induced Cardiac Dysfunction

Aims

Development of metabolic syndrome is associated with impaired cardiac performance, mitochondrial dysfunction and pro-inflammatory cytokine increase, such as the macrophage migration inhibitory factor MIF. Depending on conditions, MIF may exert both beneficial and deleterious effects on the myocardium. Therefore, we tested whether pharmacological inhibition of MIF prevented or worsened metabolic syndrome-induced myocardial dysfunction.

Methods and Results

C57BL/6J mice were fed for ten weeks with 60% fat-enriched diet (HFD) or normal diet (ND). MIF inhibition was obtained by injecting mice twice a week with ISO-1, for three consecutive weeks. Then, triglycerides, cholesterol, fat mass, glucose intolerance, insulin resistance, ex vivo cardiac contractility, animal energetic substrate utilization assessed by indirect calorimetry and mitochondrial respiration and biogenesis were evaluated. HFD led to fat mass increase, dyslipidemia, glucose intolerance and insulin resistance. ISO-1 did not alter these parameters. However, MIF inhibition was responsible for HFD-induced cardiac dysfunction worsening. Mouse capacity to increase oxygen consumption in response to exercise was reduced in HFD compared to ND, and further diminished in ISO-1-treated HFD group. Mitochondrial respiration was reduced in HFD mice, treated or not with ISO-1. Compared to ND, mitochondrial biogenesis signaling was upregulated in the HFD as demonstrated by mitochondrial DNA amount and PGC-1α expression. However, this increase in biogenesis was blocked by ISO-1 treatment.

Conclusion

MIF inhibition achieved by ISO-1 was responsible for a reduction in HFD-induced mitochondrial biogenesis signaling that could explain majored cardiac dysfunction observed in HFD mice treated with MIF inhibitor.     

Profiling of Androgen Response in Rainbow Trout Pubertal Testis: Relevance to Male Gonad Development and Spermatogenesis

The capacity of testicular somatic cells to promote and sustain germ cell differentiation is largely regulated by sexual steroids and notably androgens. In fish species the importance of androgens is emphasized by their ability to induce sex reversal of the developing fries and to trigger spermatogenesis. Here we studied the influence of androgens on testicular gene expression in trout testis using microarrays. Following treatment of immature males with physiological doses of testosterone or 11-ketotestosterone, 418 genes that exhibit changes in expression were identified. Interestingly, the activity of testosterone appeared stronger than that of 11-ketotestosterone. Expression profiles of responsive genes throughout testis development and in isolated germ cells confirmed androgens to mainly affect gene expression in somatic cells. Furthermore, specific clusters of genes that exhibit regulation coincidently with changes in the natural circulating levels of androgens during the reproductive cycle were highlighted, reinforcing the physiological significance of these data. Among somatic genes, a phylogenetic footprinting study identified putative androgen response elements within the proximal promoter regions of 42 potential direct androgen target genes. Finally, androgens were also found to alter the germ line towards meiotic expression profiles, supporting the hypothesis of a role for the somatic responsive genes in driving germ cell fate. This study significantly increases our understanding of molecular pathways regulated by androgens in vertebrates. The highly cyclic testicular development in trout together with functions associated with regulated genes reveal potential mechanisms for androgen actions in tubule formation, steroid production, germ cell development and sperm secretion.     

Enzymatic Assays for the Diagnosis of Bradykinin-Dependent Angioedema

Background

The kinins (primarily bradykinin, BK) represent the mediators responsible for local increase of vascular permeability in hereditary angioedema (HAE), HAE I-II associated with alterations of the SERPING1 gene and HAE with normal C1-Inhibitor function (HAE-nC1INH). Besides C1-Inhibitor function and concentration, no biological assay of kinin metabolism is actually available to help physicians for the diagnosis of angioedema (AE). We describe enzymatic tests on the plasma for diagnosis of BK-dependent AE.

Methods

The plasma amidase assays are performed using the Pro-Phe-Arg-p-nitroanilide peptide substrate to evaluate the spontaneous amidase activity and the proenzyme activation. We analyzed data of 872 patients presenting with BK-dependent AE or BK-unrelated diseases, compared to 303 controls. Anti-high MW kininogen (HK) immunoblot was achieved to confirm HK cleavage in exemplary samples. Reproducibility, repeatability, limit of blank, limit of detection, precision, linearity and receiver operating characteristics (ROC) were used to calculate the diagnostic performance of the assays.

Results

Spontaneous amidase activity was significantly increased in all BK-dependent AE, associated with the acute phase of disease in HAE-nC1INH, but preserved in BK-unrelated disorders. The increase of the amidase activity was associated to HK proteolysis, indicating its relevance to identify kininogenase activity. The oestrogens, known for precipitating AE episodes, were found as triggers of enzymatic activity. Calculations from ROC curves gave the optimum diagnostic cut-off for women (9.3 nmol⋅min⁻¹⋅mL⁻¹, area under curve [AUC] 92.1%, sensitivity 80.0%, and specificity 90.1%) and for men (6.6 nmol·min⁻¹⋅mL⁻¹, AUC 91.0%, sensitivity 87.0% and specificity 81.2%).

Conclusion

The amidase assay represents a diagnostic tool to help physicians in the decision to distinguish between BK-related and –unrelated AE.     

NADPH Oxidase-Driven Phagocyte Recruitment Controls Candida albicans Filamentous Growth and Prevents Mortality

Candida albicans is a human commensal and clinically important fungal pathogen that grows as both yeast and hyphal forms during human, mouse and zebrafish infection. Reactive oxygen species (ROS) produced by NADPH oxidases play diverse roles in immunity, including their long-appreciated function as microbicidal oxidants. Here we demonstrate a non-traditional mechanistic role of NADPH oxidase in promoting phagocyte chemotaxis and intracellular containment of fungi to limit filamentous growth. We exploit the transparent zebrafish model to show that failed NADPH oxidase-dependent phagocyte recruitment to C. albicans in the first four hours post-infection permits fungi to germinate extracellularly and kill the host. We combine chemical and genetic tools with high-resolution time-lapse microscopy to implicate both phagocyte oxidase and dual-specific oxidase in recruitment, suggesting that both myeloid and non-myeloid cells promote chemotaxis. We show that early non-invasive imaging provides a robust tool for prognosis, strongly connecting effective early immune response with survival. Finally, we demonstrate a new role of a key regulator of the yeast-to-hyphal switching program in phagocyte-mediated containment, suggesting that there are species-specific methods for modulation of NADPH oxidase-independent immune responses. These novel links between ROS-driven chemotaxis and fungal dimorphism expand our view of a key host defense mechanism and have important implications for pathogenesis.     

Functional Characterization of Autoantibodies against Complement Component C3 in Patients with Lupus Nephritis

Lupus nephritis (LN) is a complication of the autoimmune disease systemic lupus erythematosus. Because the complement system plays a critical role in orchestrating inflammatory and immune responses as well as in the clearance of immune complexes, autoreactivity to complement components may have considerable pathological consequences. Autoantibodies against the central complement component C3 have been reported in systemic lupus erythematosus, but their molecular mechanism and functional relevance are not well understood. The objective of this study was to evaluate the frequency and the functional properties of the anti-C3 autoantibodies. Anti-C3 autoantibodies were measured in plasma of 39 LN patients, and identification of their epitopes on the C3 molecule was performed. By using surface plasmon resonance, we analyzed the influence of patient-derived IgG antibodies on the interaction of C3b with Factor B, Factor H, and complement receptor 1. The capacity of these antibodies to dysregulate the C3 convertase on the surface of endothelial cell was measured by flow cytometry. Here we report that the frequency of anti-C3 autoantibodies in LN is ∼30%. They inhibited interactions of the negative complement regulators Factor H and complement receptor 1 with C3b. An enhanced C3 deposition was also observed on human endothelial cells in the presence of C3 autoantibodies. In addition, anti-C3 autoantibody levels correlated with disease activity. In conclusion, the anti-C3 autoantibodies in LN may contribute to the autoimmune pathology by their capacity to overactivate the complement system.     

Interrogating a Multifactorial Model of Breast Conserving Therapy with Clinical Data

Most women with early stage breast cancer do not require removal of the entire breast to treat their cancer; instead, up to 70% of women can be effectively and safely treated by breast conserving therapy (BCT) with surgical removal of the tumor only (lumpectomy) followed by radiation treatment of the remaining breast tissue. Unfortunately, the final contour and cosmesis of the treated breast is suboptimal in approximately 30% of patients. The ability to accurately predict breast contour after BCT for breast cancer could significantly improve patient decision-making regarding the choice of surgery for breast cancer. Our overall hypothesis is that the complex interplay among mechanical forces due to gravity, breast tissue constitutive law distribution, inflammation induced by radiotherapy and internal stress generated by the healing process play a dominant role in determining the success or failure of lumpectomy in preserving the breast contour and cosmesis. We have shown here from a first patient study that even in the idealistic situation of excellent cosmetic outcome this problem requires multiscale modeling. We propose a method to decide which component of the model works best for each phase of healing and what parameters should be considered dominant and patient specific. This patient study is part of a clinical trial registered on ClinicalTrial.gov, identifier NCT02310711.     

Out of the Black Sea: Phylogeography of the Invasive Killer Shrimp Dikerogammarus villosus across Europe

The amphipod Dikerogammarus villosus has colonized most of the European main inland water bodies in less than 20 years, having deteriorating effect on the local benthic communities. Our aim was to reveal the species phylogeography in the native Black Sea area, to define the source populations for the colonization routes in continental Europe and for the newly established UK populations. We tested for the loss of genetic diversity between source and invasive populations as well as along invasion route. We tested also for isolation by distance. Thirty three native and invasive populations were genotyped for mtDNA (COI, 16S) and seven polymorphic nuclear microsatellites to assess cryptic diversity (presence of deeply divergent lineages), historical demography, level of diversity within lineage (e.g., number of alleles), and population structure. A wide range of methods was used, including minimum spanning network, molecular clock, Bayesian clustering and Mantel test. Our results identified that sea level and salinity changes during Pleistocene impacted the species phylogeography in the Black Sea native region with four differentiated populations inhabiting, respectively, the Dnieper, Dniester, Danube deltas and Durungol liman. The invasion of continental Europe is associated with two sources, i.e., the Danube and Dnieper deltas, which gave origin to two independent invasion routes (Western and Eastern) for which no loss of diversity and no isolation by distance were observed. The UK population has originated in the Western Route and, despite very recent colonization, no drastic loss of diversity was observed. The results show that the invasion of the killer shrimp is not associated with the costs of loosing genetic diversity, which may contribute to the success of this invader in the newly colonized areas. Additionally, while it has not yet occurred, it might be expected that future interbreeding between the genetically diversified populations from two independent invasion routes will potentially even enhance this success.     

P-glycoprotein activity in human Caucasian male lymphocytes does not follow its increased expression during aging

P-glycoprotein (P-gp) is a transmembrane protein that mediates the efflux of innumerous structurally unrelated compounds. It was initially found over-expressed in tumor cells, associated to a multidrug resistance phenotype (MDR). Then, P-gp was found constitutively expressed in excretory cells/tissues and in circulating cells, such as lymphocytes. Considering the importance of this transporter in the establishment of therapeutic protocols and the existence of contradictory results, this study aimed at evaluating the influence of aging in the expression and function of P-gp in human lymphocytes, comparing two different methodologies to assess both parameters. P-gp activity and expression were evaluated in lymphocytes isolated from whole blood samples of 65 healthy caucasian male donors, divided into two groups according to age (group 1: under 30-years old; group 2: above 60-years old). P-gp expression was assessed using the anti-P-gp monoclonal antibody, UIC2, in the presence and in absence of vinblastine (Vbl). P-gp activity was evaluated measuring the efflux rate of the fluorescent P-gp substrate rhodamine 123 (Rho 123) and also using UIC2 shift assay. Flow cytometric analysis was performed to assess all the proceedings. Furthermore, P-gp expression and each of the P-gp activity determination methods were compared, through correlation analysis and linear regression models. We observed a significant age-dependent increase in mean P-gp expression (p = 0.029), which was not reflected in the transporter's activity (p > 0.050). Statistical analysis allowed selection of UIC2 shift assay over Rho 123 efflux assay as a more selective method to assess P-gp activity. Despite the significant correlation between P-gp expression and P-gp activity found in lymphocytes (Gp1(group 1)-r = 0.609, p < 0.001; Gp2-r = 0.461, p = 0.012), using UIC2 shift assay, these data reinforce the need for P-gp activity assessment, rather than P-gp expression determination alone, when starting new therapeutic regimens with P-gp substrates, especially in men older than 60 years of age.