Targeting TNF-α to neoangiogenic vessels enhances lymphocyte infiltration in tumors and increases the therapeutic potential of immunotherapy

Abnormal tumor vasculature impairs T lymphocyte adhesion to endothelial cells and lymphocyte extravasation into neoplastic tissues, limiting the therapeutic potential of both active and adoptive immunotherapies. We have found that treatment of tumor-bearing mice with NGR-TNF, a Cys-Asn-Gly-Arg-Cys peptide-TNF fusion product capable of altering the endothelial barrier function and improving drug penetration in tumors, associated with the intratumor upregulation of leukocyte-endothelial cell adhesion molecules, the release of proinflammatory cytokines and chemokines, and the infiltration of tumor-specific effector CD8(+) T cells. As a result, NGR-TNF enhanced the therapeutic activity of adoptive and active immunotherapy, delaying tumor growth and prolonging survival. Furthermore, we have found that therapeutic effects of these combinations can be further increased by the addition of chemotherapy. Thus, these findings might be relevant for the design of novel immunotherapeutic approaches for cancer patients.     

miR-29ab1 Deficiency Identifies a Negative Feedback Loop Controlling Th1 Bias That Is Dysregulated in Multiple Sclerosis

Th cell programming and function is tightly regulated by complex biological networks to prevent excessive inflammatory responses and autoimmune disease. The importance of microRNAs (miRNAs) in this process is highlighted by the preferential Th1 polarization of Dicer-deficient T cells that lack miRNAs. Using genetic knockouts, we demonstrate that loss of endogenous miR-29, derived from the miR-29ab1 genomic cluster, results in unrestrained T-bet expression and IFN-γ production. miR-29b regulates T-bet and IFN-γ via a direct interaction with the 3' untranslated regions, and IFN-γ itself enhances miR-29b expression, establishing a novel regulatory feedback loop. miR-29b is increased in memory CD4(+) T cells from multiple sclerosis (MS) patients, which may reflect chronic Th1 inflammation. However, miR-29b levels decrease significantly upon T cell activation in MS patients, suggesting that this feedback loop is dysregulated in MS patients and may contribute to chronic inflammation. miR-29 thus serves as a novel regulator of Th1 differentiation, adding to the understanding of T cell-intrinsic regulatory mechanisms that maintain a balance between protective immunity and autoimmunity.     

Proteomic analysis of mouse brain cortex identifies metabolic down-regulation as a general feature of ischemic pre-conditioning

J. Neurochem. (2012) 122, 1219-1229. ABSTRACT: The molecular mechanisms that lead to ischemic pre-conditioning are not completely understood, and proteins are important players. We compared the mouse brain cortex proteome from different ischemia sets: transient (7?min) middle cerebral artery occlusion (7'MCAo, pre-conditioning stimulus), permanent MCAo (pMCAo, severe ischemia), and pMCAo 4?days after 7'MCAo (7'MCAo/pMCAo, pre-conditioned model). Proteins were analyzed by two-dimensional electrophoresis coupled to liquid chromatography-tandem mass spectrometry. Overall, 28 proteins were expressed differentially from sham controls, and identified. The ischemic pre-conditioning stimulus alone up-regulated the stress protein heat-shock protein 70 (HSP70), possibly activated by the androgen receptor. Western blotting confirmed the increased expression of HSP70 and showed that androgen receptor expression paralleled that of HSP70. In the ischemic-tolerant group (7'MCAo/pMCAo), a number of proteins over-expressed after pMCAo returned to sham levels, seven proteins remained up-regulated as in pMCAo, and five proteins mainly involved in energy metabolism and mitochondrial electron transport and unchanged in pMCAo were down-regulated only in ischemic tolerance, suggesting a role in brain pre-conditioning. Astrocytes participated in ischemic-tolerance induction, as shown by the down-regulation of glutamine synthetase in the 7'MCAo/pMCAo group. The results suggest that metabolic down-regulation was a general feature of ischemic pre-conditioning, playing a pivotal role in neuroprotection.     

The SH3 domain of HS1 protein recognizes lysine-rich polyproline motifs

HS1 is a protein involved in erythroid proliferation and apoptotic cell death, containing several structurally significant motifs including a C-terminal SH3 domain. HPK1 is a member of the Ste20-related kinase family, which contains four proline-rich sequences and is constitutively associated with HS1 in hematopoietic cells. Recombinant fusion protein GST-SH3_HS1 was expressed to assess the binding properties of 16 peptides derived from the HPK1 proline-rich regions. The binding affinities were determined by non-immobilized ligand interaction assay by circular dichroism. Our results revealed that the classical PxxPxK class II binding motif is not sufficient to induce the interaction with the GST-SH3_HS1 domain, an event dependent on the presence of additional basic residue(s) located at the C-terminus of the PxxPxK motif: Lys₋₅ in P2 peptide and Lys₋₈ in P4c peptide. Lys replacement by Arg residues decreases the ligand binding affinity. The finding that both SH3_HS1 domain and full-length HS1 protein bind to P2 peptide with similar affinity demonstrates that the whole protein sequence does not affect the interaction properties of the domain. In silico models of SH3_HS1 as a complex with P2 or P4c highlight the domain residues that interact with the recognition determinants of the peptide ligand and that cooperate in the complex stabilization.     

Ca2+ ion transport through channels formed by α-hemolysin analyzed using a microwell array on a Si substrate

For the functional analysis of ion channel activity, an artificial lipid bilayer suspended over microwells was formed that ruptured giant unilamellar vesicles on a Si substrate. Ca(2+) ion indicators (fluo-4) were confined in the microwells by sealing the microwells with a lipid bilayer. An overhang formed at the microwells prevented the lipid membrane from falling into them and allowed the stable confinement of the fluorescent probes. The transport of Ca(2+) ions through the channels formed by α-hemolysin inserted in a lipid membrane was analyzed by employing the fluorescence intensity change of fluo-4 in the microwells. The microwell volume was very small (1-100 fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/μm(2), and this is much smaller than the ion current in a standard electrophysiological measurement. Smaller microwells will make it possible to mimic a local ion concentration change in the cells, although the signal to noise ratio must be further improved for the functional analysis of a single channel. We demonstrated that a microwell array with confined fluorescent probes sealed by a lipid bilayer could constitute a basic component of a highly sensitive biosensor array that works with functional membrane proteins. This array will allow us to realize high throughput and parallel testing devices.     

QTL mapping under truncation selection in homozygous lines derived from biparental crosses

In plant breeding, a large number of progenies that will be discarded later in the breeding process must be phenotyped and marker genotyped for conducting QTL analysis. In many cases, phenotypic preselection of lines could be useful. However, in QTL analyses even moderate preselection can have a significant effect on the power of QTL detection and estimation of effects of the target traits. In this study, we provide exact formulas for quantifying the change of allele frequencies within marker classes, expectations of marker contrasts and the variance of the marker contrasts under truncation selection, for the general case of two QTL affecting the target trait and a correlated trait. We focused on homozygous lines derived at random from biparental crosses. The effects of linkage between the marker and the QTL under selection as well as the effect of selection on a correlated trait can be quantified with the given formulas. Theoretical results clearly show that depending on the magnitude of QTL effects, high selection intensities can lead to a dramatic reduction in power of QTL detection and that approximations based on the infinitesimal model deviate substantially from exact solutions. The presented formulas are valuable for choosing appropriate selection intensity when performing QTL mapping experiments on the data on phenotypically preselected traits and enable the calculation and bias correction of the effects of QTL under selection. Application of our theory to experimental data revealed that selection-induced bias of QTL effects can be successfully corrected.     

Genomic signature of natural and anthropogenic stress in wild populations of the waterflea Daphnia magna: validation in space, time and experimental evolution

Natural populations are confronted with multiple selection pressures resulting in a mosaic of environmental stressors at the landscape level. Identifying the genetic underpinning of adaptation to these complex selection environments and assigning causes of natural selection within multidimensional selection regimes in the wild is challenging. The water flea Daphnia is a renowned ecological model system with its well-documented ecology, the possibility to analyse subfossil dormant egg banks and the short generation time allowing an experimental evolution approach. Capitalizing on the strengths of this model system, we here link candidate genome regions to three selection pressures, known to induce micro-evolutionary responses in Daphnia magna: fish predation, parasitism and land use. Using a genome scan approach in space, time and experimental evolution trials, we provide solid evidence of selection at the genome level under well-characterized environmental gradients in the wild and identify candidate genes linked to the three environmental stressors. Our study reveals differential selection at the genome level in Daphnia populations and provides evidence for repeatable patterns of local adaptation in a geographic mosaic of environmental stressors fuelled by standing genetic variation. Our results imply high evolutionary potential of local populations, which is relevant to understand the dynamics of trait changes in natural populations and their impact on community and ecosystem responses through eco-evolutionary feedbacks.     

CRP is related to higher leptin levels in minority peripubertal females regardless of adiposity levels

Overweight is related to higher levels of C-reactive protein (CRP) and leptin, which have been independently associated with increased risk for diabetes, cardiovascular disease, and the metabolic syndrome. Elevated CRP may trigger leptin resistance by inhibiting the binding of leptin to its receptors. We cross-sectionally examined the relationship between CRP, leptin, BMI z-score, percent body fat (%BF) assessed by air plethysmography (BodPod), and insulin sensitivity (SI) and acute insulin response (AIRg) measured by intravenous glucose tolerance test in 51 Latina and African-American females (77% Latina), mean age 9.2 (±0.9) years, at either Tanner Pubertal Stage (TPS) 1 (n = 25) or TPS 2 (n = 26). Females at TPS 2 had higher BMI z-scores, %BF (23% ± 10.1 vs. 30% ± 10.0, P = 0.02), AIRg (976.7 ± 735.2 vs. 1555.3 ± 1,223 μIU/ml, P = 0.05), fasting insulin (11.0 ± 10.8 vs. 17.2 ± 13.6 μlU/ml, P = 0.00) and leptin levels (11.0 ± 7.1 vs. 19.6 ± 10.9 ng/ml, P < 0.001) than those at TPS 1. There were no ethnic differences in any of the measured variables. CRP was positively correlated with BMI z-score (P = 0.001), %BF (P = 0.006), fasting insulin and AIRg (P = 0.02), and fasting leptin (P = 0.00), and negatively correlated with SI (P = 0.05). A linear regression model showed that CRP independently explained 10% (P = 0.00) of the variance in leptin after adjusting %BF, TPS, ethnicity, habitual physical activity and SI. Hence, low-grade inflammation may contribute to prolonged leptin exposure and leptin resistance, even in healthy children.     

The pressure/volume relationship during dobutamine stress echocardiography in transplanted heart: comparison with quality of life and coronary anatomy

Background

Cardiac allograft vasculopathy (CAV) is a major late complication in cardiac transplant recipients and has a relevant impact on outcome of these patients. Aims of this study: to compare, in cardiac transplant recipients patients, the diagnostic value of pressure/volume relationship (ESPVR) during dobutamine stress echocardiography (DSE) for coronary artery disease, assessed by Multislice Computed Tomography (MSCT), and by coronary angiography (CA). We also analyzed any possible relationship between ESPVR and the Health Related Quality of Life of the patients (HRQoL), evaluated by SF–36 questionnaire.

Methods

25 consecutive patients underwent DSE within 24 hours after MSCT coronary angiogram and then they underwent CA. The HRQoL questionnaire was administered to the patients in the settings of DSE. They were followed-up for 6 months.

Results

DSE has a sensitivity in detecting CAV of 67%, specificity of 95%, positive predictive value of 67% and negative predictive value of 95%; DSE with ESPVR has a sensitivity of 100%, specificity of 95%, positive predictive value of 75%, negative predictive value of 100%; MSCT has a sensitivity of 100%; specificity of 82%; positive predictive value of 43%; negative predictive value of 100%. Htx recipients with a flat-biphasic ESPVR, although asymptomatic, perceived a worst HRQoL compared with the up-sloping ESPVR population, and this is statistically significant for the general health (p 0.0004), the vitality (p 0.0013) and the mental health (p 0.021) SF-36 subscale.

Conclusions

Evaluation with DSE and ESPVR is accurate in the clinical control of heart transplant recipients reserving invasive evaluation only for patients with abnormal contractility indexes.