Impaired Systemic Tetrahydrobiopterin Bioavailability and Increased Oxidized Biopterins in Pediatric Falciparum Malaria: Association with Disease Severity

Decreased bioavailability of nitric oxide (NO) is a major contributor to the pathophysiology of severe falciparum malaria. Tetrahydrobiopterin (BH₄) is an enzyme cofactor required for NO synthesis from L-arginine. We hypothesized that systemic levels of BH4 would be decreased in children with cerebral malaria, contributing to low NO bioavailability. In an observational study in Tanzania, we measured urine levels of biopterin in its various redox states (fully reduced [BH₄] and the oxidized metabolites, dihydrobiopterin [BH₂] and biopterin [B₀]) in children with uncomplicated malaria (UM, n = 55), cerebral malaria (CM, n = 45), non-malaria central nervous system conditions (NMC, n = 48), and in 111 healthy controls (HC). Median urine BH4 concentration in CM (1.10 [IQR:0.55–2.18] μmol/mmol creatinine) was significantly lower compared to each of the other three groups — UM (2.10 [IQR:1.32–3.14];p<0.001), NMC (1.52 [IQR:1.01–2.71];p = 0.002), and HC (1.60 [IQR:1.15–2.23];p = 0.005). Oxidized biopterins were increased, and the BH4:BH2 ratio markedly decreased in CM. In a multivariate logistic regression model, each Log10-unit decrease in urine BH4 was independently associated with a 3.85-fold (95% CI:1.89–7.61) increase in odds of CM (p<0.001). Low systemic BH4 levels and increased oxidized biopterins contribute to the low NO bioavailability observed in CM. Adjunctive therapy to regenerate BH4 may have a role in improving NO bioavailability and microvascular perfusion in severe falciparum malaria.     

The NLRP3 inflammasome is activated by nanoparticles through ATP,ADP and adenosine

The NLR pyrin domain containing 3 (NLRP3) inflammasome is a major component of the innate immune system, but its mechanism of activation by a wide range of molecules remains largely unknown. Widely used nano-sized inorganic metal oxides such as silica dioxide (nano-SiO₂) and titanium dioxide (nano-TiO₂) activate the NLRP3 inflammasome in macrophages similarly to silica or asbestos micro-sized particles. By investigating towards the molecular mechanisms of inflammasome activation in response to nanoparticles, we show here that active adenosine triphosphate (ATP) release and subsequent ATP, adenosine diphosphate (ADP) and adenosine receptor signalling are required for inflammasome activation. Nano-SiO₂ or nano-TiO₂ caused a significant increase in P2Y1, P2Y2, A2_A and/or A2_B receptor expression, whereas the P2X7 receptor was downregulated. Interestingly, IL-1β secretion in response to nanoparticles is increased by enhanced ATP and ADP hydrolysis, whereas it is decreased by adenosine degradation or selective A2_A or A2_B receptor inhibition. Downstream of these receptors, our results show that nanoparticles activate the NLRP3 inflammasome via activation of PLC-InsP3 and/or inhibition of adenylate cyclase (ADCY)-cAMP pathways. Finally, a high dose of adenosine triggers inflammasome activation and IL-1β secretion through adenosine cellular uptake by nucleotide transporters and by its subsequent transformation in ATP by adenosine kinase. In summary, we show for the first time that extracellular adenosine activates the NLRP3 inflammasome by two ways: by interacting with adenosine receptors at nanomolar/micromolar concentrations and through cellular uptake by equilibrative nucleoside transporters at millimolar concentrations. These findings provide new molecular insights on the mechanisms of NLRP3 inflammasome activation and new therapeutic strategies to control inflammation.The inflammasome is a major factor of the innate immune system acting as a multiprotein platform to activate caspase-1. We showed recently that nanoparticles of TiO₂ (nano-TiO₂) and SiO₂ (nano-SiO₂) are sensed by the NLRP3 inflammasome to induce the release of mature IL-1β,¹ as observed previously with the environmental irritants asbestos or silica.² Despite the identification and characterisation of numerous sterile or microbial activators, the precise mechanisms mediating NLRP3 inflammasome activation remain to be determined. Here, we investigated whether ATP release and purinergic signalling through ATP, ADP and adenosine may be involved in inflammasome activation by nanoparticles. Intracellular ATP is released after cellular stress and/or activation, and purinergic signalling has been shown to modulate inflammation and immunity.^{3, 4} In the extracellular space, ATP is rapidly hydrolysed in a stepwise manner to ADP, AMP (adenosine monophosphate) and adenosine by ectoenzymes.⁴ Adenosine is then irreversibly hydrolysed to inosine by adenosine deaminase (ADA). Extracellular ATP (eATP) signals through both ATP-gated ion channels P2X and G protein-coupled receptor (GPCR) P2Y membrane receptors, whereas ADP signals through P2Y receptors and adenosine through P1 receptors (or A receptors).⁵ P2Y receptors and A receptors may be coupled to the G_q protein, which activates phospholipase C-beta (PLC-β), to the stimulatory G (G_s) protein, which stimulates adenylate cyclase inducing an increase in cyclic AMP (cAMP) levels, or to the G inhibitory (G_i) protein, which inhibits adenylate cyclase. Extracellular adenosine level is the result of adenosine production from extracellular ATP and ADP, its degradation into inosine and its reuptake by cells. Both ATP and adenosine can be transported outside of the cell via diffusion or active transport, whereas only adenosine can enter the cells through adenosine transporters.⁶ Most cells possess equilibrative and concentrative adenosine transporters (respectively, ENTs and CNTs), which allow adenosine to quickly cross the plasma membrane.⁷ Intracellular adenosine is converted to ATP via phosphorylation steps mediated by adenosine kinase (AK) and AMP kinase (AMPK). The basal physiological level of extracellular adenosine has been estimated to be in the range of 30–200 nM.⁸ ATP-derived adenosine and its subsequent signalling through P1 receptors have beneficial roles in acute disease states.^{4, 9} However, during tissue injury, elevated adenosine levels participate in the progression to chronic diseases by promoting aberrant wound healing leading to fibrosis in different organs including the lungs, liver, skin and kidney. In these conditions the blockade of adenosine signalling is beneficial.^{10, 11, 12, 13, 14, 15, 16} In murine models, ADA-knockout mice present high persistent adenosine levels, which lead to airspace enlargement and fibrosis, cardinal signs of COPD and IPF.^{14, 17, 18}Here we investigate in more detail the critical contribution of purinergic signalling in driving NLRP3 inflammasome activation in response to nanoparticles pointing out the effect of ATP, ADP, as well as adenosine and its receptors. We also identify ATP-derived adenosine as a potential activator of the inflammasome.     

Change in HbA1c Levels between the Age of 8 Years and the Age of 12 Years in Dutch Children without Diabetes: The PIAMA Birth Cohort Study

Objective

HbA1c is associated with cardiovascular risk in persons without diabetes and cardiovascular risk accumulates over the life course. Therefore, insight in factors determining HbA1c from childhood onwards is important. We investigated (lifestyle) determinants of HbA1c at age 12 years and the effects of growth on change in HbA1c and the tracking of HbA1c between the age of 8 and 12 years.

Study Design and Methods

Anthropometric measurements were taken and HbA1c levels were assessed in 955 children without diabetes aged around 12 years participating in the PIAMA birth cohort study. In 363 of these children HbA1c was also measured at age 8 years. Data on parents and children were collected prospectively by questionnaires.

Results

We found no significant association between known risk factors for diabetes and HbA1c at age 12 years. Mean(SD) change in HbA1c between ages 8 and 12 years was 0.6(0.7) mmol/mol per year (or 0.1(0.1) %/yr). Anthropometric measures at age 8 and their change between age 8 and 12 years were not associated with the change in HbA1c. 68.9% of the children remained in the same quintile or had an HbA1c one quintile higher or lower at age 8 years compared to age 12 years.

Conclusion

The lack of association between known risk factors for diabetes and HbA1c suggest that HbA1c in children without diabetes is relatively unaffected by factors associated with glycaemia. HbA1c at age 8 years is by far the most important predictor of HbA1c at age 12. Therefore, the ranking of HbA1c levels appear to be fairly stable over time.     

FGFR-ERK signaling is an essential component of tissue separation

Formation of a constriction and tissue separation between parent and young polyp is a hallmark of the Hydra budding process and controlled by fibroblast growth factor receptor (FGFR) signaling. Appearance of a cluster of cells positive for double phosphorylated ERK (dpERK) at the late separation site indicated that the RAS/MEK/ERK pathway might be a downstream target of the Hydra Kringelchen FGFR. In fact, inhibition of ERK phosphorylation by the MEK inhibitor U0126 reversibly delayed bud detachment and prevented formation of the dpERK-positive cell cluster indicating de novo-phosphorylation of ERK at the late bud base. In functional studies, a dominant-negative Kringelchen FGFR prevented bud detachment as well as appearance of the dpERK-positive cell cluster. Ectopic expression of full length Kringelchen, on the other hand, induced a localized rearrangement of the actin cytoskeleton at sites of constriction, localized ERK-phosphorylation and autotomy of the body column. Our data suggest a model in which (i) the Hydra FGFR targets, via an unknown pathway, the actin cytoskeleton to induce a constriction and (ii) FGFR activates MEK/ERK signaling at the late separation site to allow tissue separation.     

Autophagy is defective in collagen VI muscular dystrophies, and its reactivation rescues myofiber degeneration

Autophagy is crucial in the turnover of cell components, and clearance of damaged organelles by the autophagic-lysosomal pathway is essential for tissue homeostasis. Defects of this degradative system have a role in various diseases, but little is known about autophagy in muscular dystrophies. We have previously found that muscular dystrophies linked to collagen VI deficiency show dysfunctional mitochondria and spontaneous apoptosis, leading to myofiber degeneration. Here we demonstrate that this persistence of abnormal organelles and apoptosis are caused by defective autophagy. Skeletal muscles of collagen VI-knockout (Col6a1(-/-)) mice had impaired autophagic flux, which matched the lower induction of beclin-1 and BCL-2/adenovirus E1B-interacting protein-3 (Bnip3) and the lack of autophagosomes after starvation. Forced activation of autophagy by genetic, dietary and pharmacological approaches restored myofiber survival and ameliorated the dystrophic phenotype of Col6a1(-/-) mice. Furthermore, muscle biopsies from subjects with Bethlem myopathy or Ullrich congenital muscular dystrophy had reduced protein amounts of beclin-1 and Bnip3. These findings indicate that defective activation of the autophagic machinery is pathogenic in some congenital muscular dystrophies.     

Virgin forests in Romania and Bulgaria: results of two national inventory projects and their implications for protection

Despite extensive forest destruction in the Middle Ages and later intensive commercial forest management, remnants of virgin forests remained spared in some Central, Eastern and South-Eastern European countries. These virgin forests are the last examples of original forests in this part of Europe. That is why their protection becomes an important issue of current European forestry and nature protection policy. But the knowledge about the location and the area of virgin forests in these countries is incomplete up till now. This article has the prime goal to present a conceptual framework what virgin forests might be (“A conceptual framework for defining of virgin forests” section). Based on this framework, a working methodology has been tested in Bulgaria and Romania (“Results of the two national projects in Romania and in Bulgaria” section and further). For this reason two projects have been carried out by the Royal Dutch Society of Nature Conservation (KNNV) in close co-operation with the Forestry Institutes in Romania and in Bulgaria. The results of these projects are described in general terms and further analysis in the future is necessary to describe specific features like forest structure and spatial heterogeneity of these forests. Based on the results of the inventory, principles of sustainable protection and management of the mapped virgin forests were defined and described in the research reports. The usefulness of the inventory became evident already during the EU pre-accession period of both countries while preparing the NATURA 2000 network. The remaining virgin forests of temperate Europe are an inexhaustible source of ecological information about biodiversity, structure, natural processes and overall functioning of undisturbed forest ecosystems. Their research will reveal information which can be used for ecological restoration of man-made forests which are degraded through intensive forestry practices over the last centuries. The last virgin forests of temperate Europe represent an irreplaceable part of the natural capital of Europe and are worth to be protected by law. Their last remnants in South-Eastern and Eastern Europe are endangered by commercial activities. A full inventory of remaining virgin forests in all countries of temperate Europe is a matter of highest urgency. A representative selection of virgin forest sites should be declared by UNESCO as World Heritage Sites.