VEGF-C attenuates renal damage in salt-sensitive hypertension

Salt-sensitive hypertension is a major risk factor for renal impairment leading to chronic kidney disease. High-salt diet leads to hypertonic skin interstitial volume retention enhancing the activation of the tonicity-responsive enhancer-binding protein (TonEBP) within macrophages leading to vascular endothelial growth factor C (VEGF-C) secretion and NOS3 modulation. This promotes skin lymphangiogenesis and blood pressure regulation. Whether VEGF-C administration enhances renal and skin lymphangiogenesis and attenuates renal damage in salt-sensitive hypertension remains to be elucidated. Hypertension was induced in BALB/c mice by a high-salt diet. VEGF-C was administered subcutaneously to high-salt-treated mice as well as control animals. Analyses of kidney injury, inflammation, fibrosis, and biochemical markers were performed in vivo. VEGF-C reduced plasma inflammatory markers in salt-treated mice. In addition, VEGF-C exhibited a renal anti-inflammatory effect with the induction of macrophage M2 phenotype, followed by reductions in interstitial fibrosis. Antioxidant enzymes within the kidney as well as urinary RNA/DNA damage markers were all revelatory of abolished oxidative stress under VEGF-C. Furthermore, VEGF-C decreased the urinary albumin/creatinine ratio and blood pressure as well as glomerular and tubular damages. These improvements were associated with enhanced TonEBP, NOS3, and lymphangiogenesis within the kidney and skin. Our data show that VEGF-C administration plays a major role in preserving renal histology and reducing blood pressure. VEGF-C might constitute an interesting potential therapeutic target for improving renal remodeling in salt-sensitive hypertension.     

Perpetuation of the Lyme Disease Spirochete Borrelia lusitaniae by Lizards

To determine whether the Lyme disease spirochete Borrelia lusitaniae is associated with lizards, we compared the prevalence and genospecies of spirochetes present in rodent- and lizard-associated ticks at a site where this spirochete frequently infects questing ticks. Whereas questing nymphal Ixodes ricinus ticks were infected mainly by Borrelia afzelii, one-half of the infected adult ticks harbored B. lusitaniae at our study site. Lyme disease spirochetes were more prevalent in sand lizards (Lacerta agilis) and common wall lizards (Podarcis muralis) than in small rodents. Although subadult ticks feeding on rodents acquired mainly B. afzelii, subadult ticks feeding on lizards became infected by B. lusitaniae. Genetic analysis confirmed that the spirochetes isolated from ticks feeding on lizards are members of the B. lusitaniae genospecies and resemble type strain PotiB2. At our central European study site, lizards, which were previously considered zooprophylactic for the agent of Lyme disease, appear to perpetuate B. lusitaniae.     

Engineering production of antihypertensive peptides in plants

To date, a number of antihypertensive peptides (AHPs) have been identified. Most of these are derived from proteins present in common edible consumables, including milk, egg, and plant foods. Consumption of these foods serves as means of AHP delivery and thus contributing favorable health benefits. It is hypothesized that food crops, either over-expressing AHP precursor proteins or producing particular peptides as heterologous components, may serve as viable vehicles for production and delivery of functional foods as alternative hypertension therapies. In recent years, genetic engineering efforts have been undertaken to add value to functional foods. Pioneering approaches have been pursued in several crop plants, such as rice and soybean. In this review, a summary of current tools used for discovery of new AHPs, as well as strategies and perspectives of capitalizing on these AHPs in genetic engineering efforts will be presented and discussed. The implications of these efforts on the development of functional foods for preventing and treating hypertension are also presented.     

Immunogenic properties of a lettuce-derived C4(V3)6 multiepitopic HIV protein

Elicitation of broad humoral immune responses is a critical factor in the development of effective HIV vaccines. In an effort to develop low-cost candidate vaccines based on multiepitopic recombinant proteins, this study has been undertaken to assess and characterize the immunogenic properties of a lettuce-derived C4(V3)6 multiepitopic protein. This protein consists of V3 loops corresponding to five different HIV isolates, including MN, IIIB, RF, CC, and RU. In this study, both Escherichia coli and lettuce-derived C4(V3)6 have elicited local and systemic immune responses when orally administered to BALB/c mice. More importantly, lettuce-derived C4(V3)6 has shown a higher immunogenic potential than that of E. coli-derived C4(V3)6. Moreover, when reactivity of sera from mice immunized with C4(V3)6 are compared with those elicited by a chimeric protein carrying a single V3 sequence, broader responses have been observed. The lettuce-derived C4(V3)6 has elicited antibodies with positive reactivity against V3 loops from isolates MN, RF, and CC. In addition, splenocyte proliferation assays indicate that significant T-helper responses are induced by the C4(V3)6 immunogen. Taken together, these findings account for the observed elicitation of broader humoral responses by the C4(V3)6 multiepitopic protein. Moreover, they provide further validation for the production of multiepitopic vaccines in plant cells as this serves not only as a low-cost expression system, but also as an effective delivery vehicle for orally administered immunogens.     

TNFA Haplotype Genetic Testing Improves HLA in Estimating the Risk of Celiac Disease in Children

Background

TNF-α and IFN-γ play a role in the development of mucosal damage in celiac disease (CD). Polymorphisms of TNFA and IFNG genes, as well as of the TNFRSF1A gene, encoding the TNF-α receptor 1, might underlie different inter-individual disease susceptibility over a common HLA risk background. The aims of this study were to ascertain whether five SNPs in the TNFA promoter (-1031T>C,-857C>T,-376G>A,-308G>A,-238G>A), sequence variants of the TNFRSF1A gene and IFNG +874A>T polymorphism are associated with CD in a HLA independent manner.

Methods

511 children (244 CD, 267 controls) were genotyped for HLA, TNFA and INFG (Real Time PCR). TNFRSF1A variants were studied (DHPLC and sequence).

Results

Only the rare TNFA-1031C (OR=0.65, 95% CI:0.44-0.95), -857T (OR=0.42, 95% CI:0.27-0.65), -376A (OR=2.25, 95% CI:1.12-4.51) and -308A (OR=4.76, 95% CI:3.12-7.26) alleles were significantly associated with CD. One TNFRSF1A variant was identified (c.625+10A>G, rs1800693), but not associated with CD. The CD-correlated TNFA SNPs resulted in six haplotypes. Two haplotypes were control-associated (CCGG and TTGG) and three were CD-associated (CCAG, TCGA and CCGA). The seventeen inferred haplotype combinations were grouped (A to E) based on their frequencies among CD. Binary logistic regression analysis documented a strong association between CD and HLA (OR for intermediate risk haplotypes=178; 95% CI:24-1317; OR for high risk haplotypes=2752; 95% CI:287-26387), but also an HLA-independent correlation between CD and TNFA haplotype combination groups. The CD risk for patients carrying an intermediate risk HLA haplotype could be sub-stratified by TNFA haplotype combinations.

Conclusion

TNFA promoter haplotypes associate with CD independently from HLA. We suggest that their evaluation might enhance the accuracy in estimating the CD genetic risk.     

Effects of Inhibiting Dipeptidyl Peptidase-4 (DPP4) in Cows with Subclinical Ketosis

The inhibition of dipeptidyl peptidase-4 (DPP4) via specific inhibitors is known to result in improved glucose tolerance and insulin sensitivity and decreased accumulation of hepatic fat in type II diabetic human patients. The metabolic situation of dairy cows can easily be compared to the status of human diabetes and non-alcoholic fatty liver. For both, insulin sensitivity is reduced, while hepatic fat accumulation increases, characterized by high levels of non-esterified fatty acids (NEFA) and ketone bodies.Therefore, in the present study, a DPP4 inhibitor was employed (BI 14332) for the first time in cows. In a first investigation BI 14332 treatment (intravenous injection at dosages of up to 3 mg/kg body weight) was well tolerated in healthy lactating pluriparous cows (n = 6) with a significant inhibition of DPP4 in plasma and liver. Further testing included primi- and pluriparous lactating cows suffering from subclinical ketosis (β-hydroxybutyrate concentrations in serum > 1.2 mM; n = 12). The intension was to offer effects of DPP4 inhibition during comprehensive lipomobilisation and hepatosteatosis. The cows of subclinical ketosis were evenly allocated to either the treatment group (daily injections, 0.3 mg BI 14332/kg body weight, 7 days) or the control group. Under condition of subclinical ketosis, the impact of DPP4 inhibition via BI 14332 was less, as in particular β-hydroxybutyrate and the hepatic lipid content remained unaffected, but NEFA and triglyceride concentrations were decreased after treatment. Owing to lower NEFA, the revised quantitative insulin sensitivity check index (surrogate marker for insulin sensitivity) increased. Therefore, a positive influence on energy metabolism might be quite possible. Minor impacts on immune-modulating variables were limited to the lymphocyte CD4⁺/CD8⁺ ratio for which a trend to decreased values in treated versus control animals was noted. In sum, the DPP4 inhibition in cows did not affect glycaemic control like it is shown in humans, but was able to impact hyperlipemia, as NEFA and TG decreased.     

Development of an Indicator to Monitor Mediterranean Wetlands

Wetlands are sensitive ecosystems that are increasingly subjected to threats from anthropogenic factors. In the last decades, coastal Mediterranean wetlands have been suffering considerable pressures from land use change, intensification of urban growth, increasing tourism infrastructure and intensification of agricultural practices. Remote sensing (RS) and Geographic Information Systems (GIS) techniques are efficient tools that can support monitoring Mediterranean coastal wetlands on large scales and over long periods of time. The study aims at developing a wetland indicator to support monitoring Mediterranean coastal wetlands using these techniques. The indicator makes use of multi-temporal Landsat images, land use reference layers, a 50m numerical model of the territory (NMT) and Corine Land Cover (CLC) for the identification and mapping of wetlands. The approach combines supervised image classification techniques making use of vegetation indices and decision tree analysis to identify the surface covered by wetlands at a given date. A validation process is put in place to compare outcomes with existing local wetland inventories to check the results reliability. The indicator´s results demonstrate an improvement in the level of precision of change detection methods achieved by traditional tools providing reliability up to 95% in main wetland areas. The results confirm that the use of RS techniques improves the precision of wetland detection compared to the use of CLC for wetland monitoring and stress the strong relation between the level of wetland detection and the nature of the wetland areas and the monitoring scale considered.     

Immunogenicity of nuclear-encoded LTB:ST fusion protein from Escherichia coli expressed in tobacco plants

Enterotoxigenic Escherichia coli (ETEC) is one of the main causative agents of diarrhea in infants and for travelers. Inclusion of a heat-stable (ST) toxin into vaccine formulations is mandatory as most ETEC strains can produce both heat-labile (LT) and ST enterotoxins. In this study, a genetic fusion gene encoding for an LTB:ST protein has been constructed and transferred into tobacco via Agrobacterium tumefaciens-mediated transformation. Transgenic tobacco plants carrying the LTB:ST gene are then subjected to GM1-ELISA revealing that the LTB:ST has assembled into pentamers and displays antigenic determinants from both LTB and ST. Protein accumulation of up to 0.05% total soluble protein is detected. Subsequently, mucosal and systemic humoral responses are elicited in mice orally dosed with transgenic tobacco leaves. This has suggested that the plant-derived LTB:ST is immunogenic via the oral route. These findings are critical for the development of a plant-based vaccine capable of eliciting broader protection against ETEC and targeting both LTB and ST. Features of this platform in comparison to transplastomic approaches are discussed.     

Altered intracellular calcium fluxes in pancreatic cancer induced diabetes mellitus: Relevance of the S100A8 N‐terminal peptide (NT‐S100A8)

After isolating NT‐S100A8 from pancreatic cancer (PC) tissue of diabetic patients, we verified whether this peptide alters PC cell growth and invasion and/or insulin release and [Ca²⁺]_i oscillations of insulin secreting cells and/or insulin signaling. BxPC3, Capan1, MiaPaCa2, Panc1 (PC cell lines) cell growth, and invasion were assessed in the absence or presence of 50, 200, and 500 nM NT‐S100A8. In NT‐S100A8 stimulated β‐TC6 (insulinoma cell line) culture medium, insulin and [Ca²⁺] were measured at 2, 3, 5, 10, 15, 30, and 60 min, and [Ca²⁺]_i oscillations were monitored (epifluorescence) for 3 min. Five hundred nanomolars NT‐S100A8 stimulated BxPC3 cell growth only and dose dependently reduced MiaPaCa2 and Panc1 invasion. Five hundred nanomolars NT‐S100A8 induced a rapid insulin release and enhanced β‐TC6 [Ca²⁺]_i oscillations after both one (F = 6.05, P < 0.01) and 2 min (F = 7.42, P < 0.01). In the presence of NT‐S100A8, [Ca²⁺] in β‐TC6 culture medium significantly decreased with respect to control cells (F = 6.3, P < 0.01). NT‐S100A8 did not counteract insulin induced phosphorylation of the insulin receptor, Akt and IκB‐α, but it independently activated Akt and NF‐κB signaling in PC cells. In conclusion, NT‐S100A8 exerts a mild effect on PC cell growth, while it reduces PC cell invasion, possibly by Akt and NF‐κB signaling, NT‐S100A8 enhances [Ca²⁺]_i oscillations and insulin release, probably by inducing Ca²⁺ influx from the extracellular space, but it does not interfere with insulin signaling. J. Cell. Physiol. 226: 456–468, 2011. © 2010 Wiley‐Liss, Inc.     

Proteomic profiling of the hypothalamus in two mouse models of narcolepsy

Narcolepsy is a disabling neurological disorder of sleepiness linked to the loss of neurons producing orexin neuropeptides in the hypothalamus. Two well‐characterized phenotypic mouse models of narcolepsy, loss‐of‐function (orexin‐knockout), and progressive loss of orexin (orexin/ataxin‐3) exist. The open question is whether the proteomics signatures of the hypothalamus would be different between the two models. To address this gap, we utilized a label‐free proteomics approach and conducted a hypothalamic proteome analysis by comparing each disease model to that of wild type. Following data processing and statistical analysis, 14 484 peptides mapping to 2282 nonredundant proteins were identified, of which 39 proteins showed significant differences in protein expression across groups. Altered proteins in both models showed commonalties in pathways for mitochondrial dysfunction and neuronal degeneration, as well as altered proteins related to inflammatory demyelination, insulin resistance, metabolic responses, and the dopaminergic and monoaminergic systems. Model‐specific alterations in insulin degraded enzyme (IDE) and synaptosomal‐associated protein‐25 were unique to orexin‐KO and orexin/ataxin‐3, respectively. For both models, proteomics not only identified clinically suspected consequences of orexin loss on energy homeostasis and neurotransmitter systems, but also identified commonalities in inflammation and degeneration despite the entirely different genetic basis of the two mouse models.