Vascular morphogenesis by adult bone marrow progenitor cells in three-dimensional fibrin matrices

The neovascularization of tissues is accomplished by two distinct processes: de novo formation of blood vessels through the assembly of progenitor cells during early prenatal development (vasculogenesis), and expansion of a pre-existing vascular network by endothelial cell sprouting (angiogenesis), the main mechanism of blood vessel growth in postnatal life. Evidence exists that adult bone marrow (BM)-derived progenitor cells can contribute to the formation of new vessels by their incorporation into sites of active angiogenesis. Aim of this study was to investigate the in vitro self-organizing capacity of human BM mononuclear cells (BMMNC) to induce vascular morphogenesis in a three-dimensional (3D) matrix environment in the absence of pre-existing vessels. Whole BMMNC as well as the adherent and non-adherent fractions of BMMNC were embedded in fibrin gels and cultured for 3-4 weeks without additional growth factors. The expression of hematopoietic-, endothelial-, smooth muscle lineage, and stem cell markers was analyzed by immunohistochemistry and confocal laser-scanning microscopy. The culture of unselected BMMNC in 3D fibrin matrices led to the formation of cell clusters expressing the endothelial progenitor cell (EPC) markers CD133, CD34, vascular endothelial growth factor receptor (VEGFR)-2, and c-kit, with stellar shaped spreading of peripheral elongated cells forming tube-like structures with increasing complexity over time. Cluster formation was dependent on the presence of both adherent and non-adherent BMMNC without the requirement of external growth factors. Developed vascular structures expressed the endothelial markers CD34, VEGFR-2, CD31, von Willebrand Factor (vWF), and podocalyxin, showed basement-membrane-lined lumina containing CD45+ cells and were surrounded by alpha-smooth muscle actin (SMA) expressing mural cells. Our data demonstrate that adult human BM progenitor cells can induce a dynamic self organization process to create vascular structures within avascular 3D fibrin matrices suggesting a possible alternative mechanism of adult vascular development without involvement of pre-existing vascular structures.     

Is IP-10 a Better Biomarker for Active and Latent Tuberculosis in Children than IFNγ?

Background

The blood based interferon-gamma release assays (IGRA) for the diagnosis of tuberculosis do not discriminate between active TB disease and latent TB infection (LTBI). The search for distinguishing biomarkers therefore continues, as the accurate diagnosis of tuberculosis is particularly challenging in children. IFN-γ-inducible protein 10 (IP-10/CXCL10) has recently been evaluated as a marker for active TB in adults with promising results.

Aim

To investigate this new biomarker for active TB and LTBI in paediatrics.

Method

We measured IP-10 levels using ELISA in supernatants of whole blood samples stimulated with TB-specific-antigens and negative control antigen.

Results

IP-10 is produced in high levels following mycobacterial antigen stimulation in active TB (n = 17) and LTBI (n = 16) compared to controls (n = 16) and to IFN-γ. The baseline levels of IP-10 are increased in active TB and in LTBI, but there is no significant difference of stimulated levels of IP-10 between active TB and LTBI.

Conclusions

IP-10 is a biomarker for tuberculosis in children. However like IFNγ, IP-10 also does not distinguish between active TB and LTBI.     

Effects of voltage‐gated calcium channel subunit genes on calcium influx in cultured C. elegans mechanosensory neurons

Voltage‐gated calcium channels (VGCCs) serve as a critical link between electrical signaling and diverse cellular processes in neurons. We have exploited recent advances in genetically encoded calcium sensors and in culture techniques to investigate how the VGCC α₁ subunit EGL‐19 and α₂/δ subunit UNC‐36 affect the functional properties of C. elegans mechanosensory neurons. Using the protein‐based optical indicator cameleon, we recorded calcium transients from cultured mechanosensory neurons in response to transient depolarization. We observed that in these cultured cells, calcium transients induced by extracellular potassium were significantly reduced by a reduction‐of‐function mutation in egl‐19 and significantly reduced by L‐type calcium channel inhibitors; thus, a main source of touch neuron calcium transients appeared to be influx of extracellular calcium through L‐type channels. Transients did not depend directly on intracellular calcium stores, although a store‐independent 2‐APB and gadolinium‐sensitive calcium flux was detected. The transients were also significantly reduced by mutations in unc‐36, which encodes the main neuronal α₂/δ subunit in C. elegans. Interestingly, while egl‐19 mutations resulted in similar reductions in calcium influx at all stimulus strengths, unc‐36 mutations preferentially affected responses to smaller depolarizations. These experiments suggest a central role for EGL‐19 and UNC‐36 in excitability and functional activity of the mechanosensory neurons. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Microbial nitrate respiration--genes, enzymes and environmental distribution

Nitrate is a key node in the network of the assimilatory and respiratory nitrogen pathways. As one of the ‘fixed’ forms of nitrogen, nitrate plays an essential role in both nature and industry. For bacteria, it is both a nitrogen source and an electron acceptor. In agriculture and wastewater treatment, nitrate respiration by microorganisms is an important issue with respect to economics, greenhouse gas emission and public health. Several microbial processes compete for nitrate: denitrification, dissimilatory nitrate reduction to ammonium and anaerobic ammonium oxidation. In this review we provide an up to date overview of the organisms, genes and enzymes involved in nitrate respiration. We also address the molecular detection of these processes in nature. We show that despite rapid progress in the experimental and genomic analyses of pure cultures, knowledge on the mechanism of nitrate reduction in natural ecosystems is still largely lacking.     

Analysis of omega-3 and omega-6 fatty acid-derived lipid metabolite formation in human and mouse blood samples

Mass spectrometry techniques have enabled the identification of different lipid metabolites and mediators derived from omega-6 and omega-3 polyunsaturated fatty acids (n-6 and n-3 PUFA) that are implicated in various biological processes. However, the broad-spectrum assessment of physiologically formed lipid metabolites and mediators in blood samples has not been presented so far. Here lipid mediators and metabolites of the n-6 PUFA arachidonic acid as well as the long-chain n-3 PUFA eicosapentaenoic acids (EPA) and docosahexaenoic acid (DHA) were measured in human blood samples as well as in mouse blood. There were detectable but mostly very low amounts of the assayed compounds in human native plasma samples, whereas in vitro activation of whole blood with the calcium ionophore A23187 led to highly significant increases of metabolite formation, with a predominance of the 12-lipoxygenase (12-LOX) products 12-hydroxyeicosatetraenoic acid (12-HETE), 12-hydroxyeicosapentaenoic acid (12-HEPE) and 14-hydroxydocosahexaenoic acid (14-HDHA). A23187 activation also led to significant increases in the formation of 5-LOX products including leukotriene B(4) (LTB(4)), leukotriene B(5) (LTB(5)) as well as of 15-LOX products and prostaglandin E(2) (PGE(2)) and thromboxane B(2) (TXB(2)). Levels were similar or even higher in A23187-activated mouse blood. The approach presented here thus provides a protocol for the comprehensive and concomitant assessment of the generation capacity of n-3 and n-6 PUFA-derived lipid metabolites as well as thromboxanes and prostaglandins in human and murine blood samples. Further studies will now have to evaluate lipid metabolite generation capacity in different physiological and pathophysiological contexts.     

Association between TAS2R38 gene polymorphisms and colorectal cancer risk: a case-control study in two independent populations of Caucasian origin

Molecular sensing in the lingual mucosa and in the gastro-intestinal tract play a role in the detection of ingested harmful drugs and toxins. Therefore, genetic polymorphisms affecting the capability of initiating these responses may be critical for the subsequent efficiency of avoiding and/or eliminating possible threats to the organism. By using a tagging approach in the region of Taste Receptor 2R38 (TAS2R38) gene, we investigated all the common genetic variation of this gene region in relation to colorectal cancer risk with a case-control study in a German population (709 controls and 602 cases) and in a Czech population (623 controls and 601 cases). We found that there were no significant associations between individual SNPs of the TAS2R38 gene and colorectal cancer in the Czech or in the German population, nor in the joint analysis. However, when we analyzed the diplotypes and the phenotypes we found that the non-taster group had an increased risk of colorectal cancer in comparison to the taster group. This association was borderline significant in the Czech population, (OR = 1.28, 95% CI 0.99–1.67; P_value = 0.058) and statistically significant in the German population (OR = 1.36, 95% CI 1.06–1.75; P_value = 0.016) and in the joint analysis (OR = 1.34, 95% CI 1.12–1.61; P_value = 0.001). In conclusion, we found a suggestive association between the human bitter tasting phenotype and the risk of CRC in two different populations of Caucasian origin.     

Thoracic Rat Spinal Cord Contusion Injury Induces Remote Spinal Gliogenesis but Not Neurogenesis or Gliogenesis in the Brain

After spinal cord injury, transected axons fail to regenerate, yet significant, spontaneous functional improvement can be observed over time. Distinct central nervous system regions retain the capacity to generate new neurons and glia from an endogenous pool of progenitor cells and to compensate neural cell loss following certain lesions. The aim of the present study was to investigate whether endogenous cell replacement (neurogenesis or gliogenesis) in the brain (subventricular zone, SVZ; corpus callosum, CC; hippocampus, HC; and motor cortex, MC) or cervical spinal cord might represent a structural correlate for spontaneous locomotor recovery after a thoracic spinal cord injury. Adult Fischer 344 rats received severe contusion injuries (200 kDyn) of the mid-thoracic spinal cord using an Infinite Horizon Impactor. Uninjured rats served as controls. From 4 to 14 days post-injury, both groups received injections of bromodeoxyuridine (BrdU) to label dividing cells. Over the course of six weeks post-injury, spontaneous recovery of locomotor function occurred. Survival of newly generated cells was unaltered in the SVZ, HC, CC, and the MC. Neurogenesis, as determined by identification and quantification of doublecortin immunoreactive neuroblasts or BrdU/neuronal nuclear antigen double positive newly generated neurons, was not present in non-neurogenic regions (MC, CC, and cervical spinal cord) and unaltered in neurogenic regions (dentate gyrus and SVZ) of the brain. The lack of neuronal replacement in the brain and spinal cord after spinal cord injury precludes any relevance for spontaneous recovery of locomotor function. Gliogenesis was increased in the cervical spinal cord remote from the injury site, however, is unlikely to contribute to functional improvement.     

Catecholaminergic Gene Polymorphisms Are Associated with GI Symptoms and Morphological Brain Changes in Irritable Bowel Syndrome

Background

Genetic and environmental factors contribute to the pathophysiology of irritable bowel syndrome (IBS). In particular, early adverse life events (EALs) and the catecholaminergic system have been implicated.

Aims

To investigate whether catecholaminergic SNPs with or without interacting with EALs are associated with: 1) a diagnosis of IBS, 2) IBS symptoms and 3) morphological alterations in brain regions associated with somatosensory, viscerosensory, and interoceptive processes.

Methods

In 277 IBS and 382 healthy control subjects (HCs), 11 SNPs in genes of the catecholaminergic signaling pathway were genotyped. A subset (121 IBS, 209 HCs) underwent structural brain imaging (magnetic resonance imaging [MRI]). Logistic and linear regressions evaluated each SNP separately and their interactions with EALs in predicting IBS and GI symptom severity, respectively. General linear models determined grey matter (GM) alterations from the SNPs and EALs that were predictive of IBS.

Results

1) Diagnosis: There were no statistically significant associations between the SNPs and IBS status with or without the interaction with EAL after adjusting for multiple comparisons. 2) Symptoms: GI symptom severity was associated with ADRA1D rs1556832 (P = 0.010). 3) Brain morphometry: In IBS, the homozygous genotype of the major ADRA1D allele was associated with GM increases in somatosensory regions (FDR q = 0.022), left precentral gyrus (q = 0.045), and right hippocampus (q = 0.009). In individuals with increasing sexual abuse scores, the ADRAβ2 SNP was associated with GM changes in the left posterior insula (q = 0.004) and left putamen volume (q = 0.029).

Conclusion

In IBS, catecholaminergic SNPs are associated with symptom severity and morphological changes in brain regions concerned with sensory processing and modulation and affect regulation. Thus, certain adrenergic receptor genes may facilitate or worsen IBS symptoms.