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Bone healing commences with an inflammatory reaction which initiates the regenerative healing process leading in the end to reconstitution of bone. An unbalanced immune reaction during this early bone healing phase is hypothesized to disturb the healing cascade in a way that delays bone healing and jeopardizes the successful healing outcome. The immune cell composition and expression pattern of angiogenic factors were investigated in a sheep bone osteotomy model and compared to a mechanically-induced impaired/delayed bone healing group. In the impaired/delayed healing group, significantly higher T cell percentages were present in the bone hematoma and the bone marrow adjacent to the osteotomy gap when compared to the normal healing group. This was mirrored in the higher cytotoxic T cell percentage detected under delayed bone healing conditions indicating longer pro-inflammatory processes. The highly activated periosteum adjourning the osteotomy gap showed lower expression of hematopoietic stem cell markers and angiogenic factors such as heme oxygenase and vascular endothelial growth factor. This indicates a deferred revascularization of the injured area due to ongoing pro-inflammatory processes in the delayed healing group. Results from this study suggest that there are unfavorable immune cells and factors participating in the initial healing phase. In conclusion, identifying beneficial aspects may lead to promising therapeutical approaches that might benefit further by eliminating the unfavorable factors.

The pathology associated with malaria infection is largely due to the ability of infected human RBCs to adhere to a number of receptors on endothelial cells within tissues and organs. This phenomenon is driven by the export of parasite-encoded proteins to the host cell, the exact function of many of which is still unknown. Here we inactivate the function of one of these exported proteins, PFA66, a member of the J-domain protein family. Although parasites lacking this protein were still able to grow in cell culture, we observed severe defects in normal host cell modification, including aberrant morphology of surface knobs, disrupted presentation of the cytoadherence molecule PfEMP1, and a total lack of cytoadherence, despite the presence of the knob associated protein KAHRP. Complementation assays demonstrate that an intact J-domain is required for recovery to a wild-type phenotype and suggest that PFA66 functions in concert with a HSP70 to carry out host cell modification. Strikingly, this HSP70 is likely to be of host origin. ATPase assays on recombinant protein verify a functional interaction between PFA66 and residual host cell HSP70. Taken together, our data reveal a role for PFA66 in host cell modification, strongly implicate human HSP70s as being essential in this process and uncover a new KAHRP-independent molecular factor required for correct knob biogenesis.

Background Since malaria parasites highly depend on ribose 5-phosphate for DNA and RNA synthesis and on NADPH as a source of reducing equivalents, the pentose phosphate pathway (PPP) is considered an excellent anti-malarial drug target. In Plasmodium , a bifunctional enzyme named glucose 6-phosphate dehydrogenase 6-phosphogluconolactonase (GluPho) catalyzes the first two steps of the PPP. Pf GluPho has been shown to be essential for the growth of blood stage Plasmodium falciparum parasites. Methods Plasmodium vivax glucose 6-phosphate dehydrogenase ( Pv G6PD) was cloned, recombinantly produced in Escherichia coli , purified, and characterized via enzyme kinetics and inhibitor studies. The effects of post-translational cysteine modifications were assessed via western blotting and enzyme activity assays. Genetically encoded probes were employed to study the effects of G6PD inhibitors on the cytosolic redox potential of Plasmodium . Results Here the recombinant production and characterization of Pv G6PD, the C-terminal and NADPH-producing part of Pv GluPho, is described. A comparison with Pf G6PD (the NADPH-producing part of Pf GluPho) indicates that the P. vivax enzyme has higher K M values for the substrate and cofactor. Like the P. falciparum enzyme, Pv G6PD is hardly affected by S -glutathionylation and moderately by S -nitrosation. Since there are several naturally occurring variants of Pf GluPho, the impact of these mutations on the kinetic properties of the enzyme was analysed. Notably, in contrast to many human G6PD variants, the mutations resulted in only minor changes in enzyme activity. Moreover, nanomolar IC 50 values of several compounds were determined on P. vivax G6PD (including ellagic acid, flavellagic acid, and coruleoellagic acid), inhibitors that had been previously characterized on Pf GluPho. ML304, a recently developed Pf GluPho inhibitor, was verified to also be active on Pv G6PD. Using genetically encoded probes, ML304 was confirmed to disturb the cytosolic glutathione-dependent redox potential of P. falciparum blood stage parasites. Finally, a new series of novel small molecules with the potential to inhibit the falciparum and vivax enzymes were synthesized, resulting in two compounds with nanomolar activity. Conclusion The characterization of Pv G6PD makes this enzyme accessible to further drug discovery activities. In contrast to naturally occurring G6PD variants in the human host that can alter the kinetic properties of the enzyme and thus the redox homeostasis of the cells, the naturally occurring Pf GluPho variants studied here are unlikely to have a major impact on the parasites’ redox homeostasis. Several classes of inhibitors have been successfully tested and are presently being followed up.

Infection with the Trypanosoma cruzi parasite is endemic in parts of Central and South America. Approximately 30% of those infected develop Chagas cardiomyopathy, the most common cause of heart failure in this region. No suitable biomarker is available that reflects the evolution of the disease. Although there is substantial evidence of a strong inflammatory reaction following infection that could activate matrix metalloproteinases (MMPs), their role in the development of Chagas cardiomyopathy is unknown. A cross-sectional study was conducted in Bucaramanga, Colombia, from 2002 to 2006, including 144 patients at different stages of Chagas disease and 44 control patients. The potential enzyme activities of MMP-2 and MMP-9 in plasma samples were determined by gelatin zymography. Clinical data including T cruzi serology, electrocardiograms, and echocardiograms were recorded for all patients. Densitometric analysis of potential enzyme activities in plasma samples showed a significant increase of 72-kd MMP-2 (P < .001) and 92-kd MMP-9 (P < .001) in T cruzi seropositive patients compared with control subjects. Matrix metalloproteinase 9 showed significantly increased activity in patients with abnormal electrocardiogram (P < .004) and with dilated cardiomyopathy compared (P < .001) with controls. Analysis of the MMP-2 and MMP-9 results in relation to clinical data revealed that abnormal heart relaxation correlated positively with high MMP-2 levels in patients with dilated cardiomyopathy (r = 0.75, P < .01). Plasma MMP-2 and MMP-9 both appear to be useful biomarkers for detecting the advent and progression of cardiomyopathy in T cruzi–infected individuals.

Hydrogen peroxide (H2O2) causes cardiac dysfunction through multiple mechanisms. As oxidative stress can activate matrix metalloproteinases (MMPs) and, in particular, MMP-2 activity is associated with oxidative stress injury in the heart, we hypothesized that MMP-2 activation by H2O2 in isolated rat hearts contributes to cardiac dysfunction in this model. Isolated working rat hearts were perfused at 37 degrees C with a recirculating Krebs-Henseleit buffer+/-5 mmol/L pyruvate, known to protect hearts from oxidative stress. H2O2 (300 micromol/L) was added as a single bolus after 20 min of equilibration, and cardiac function was monitored for 60 min. MMPs activities in both the heart and perfusate samples were assessed by gelatin zymography. Tissue high energy phosphates were analysed by HPLC. The actions of 2 MMP inhibitors, doxycycline (75 micromol/L) or Ro 31-9790 (3 micromol/L), were also assessed. H2O2 at 300 micromol/L produced a rapid decline in cardiac mechanical function, which was maximal at 5 min. A peak in perfusate MMP-2 activity was also observed at 5 min. The deleterious effect of H2O2 on cardiac function was abolished by pyruvate but not by the MMPs inhibitors. This study suggests that in intact hearts, H2O2 induces contractile dysfunction independent of MMPs activation.

Hydrogen peroxide (H 2 O 2 ) causes cardiac dysfunction through multiple mechanisms. As oxidative stress can activate matrix metalloproteinases (MMPs) and, in particular, MMP-2 activity is associated with oxidative stress injury in the heart, we hypothesized that MMP-2 activation by H 2 O 2 in isolated rat hearts contributes to cardiac dysfunction in this model. Isolated working rat hearts were perfused at 37 °C with a recirculating Krebs-Henseleit buffer ± 5 mmol/L pyruvate, known to protect hearts from oxidative stress. H 2 O 2 (300 µmol/L) was added as a single bolus after 20 min of equilibration, and cardiac function was monitored for 60 min. MMPs activities in both the heart and perfusate samples were assessed by gelatin zymography. Tissue high energy phosphates were analysed by HPLC. The actions of 2 MMP inhibitors, doxycycline (75 µmol/L) or Ro 31-9790 (3 µmol/L), were also assessed. H 2 O 2 at 300 µmol/L produced a rapid decline in cardiac mechanical function, which was maximal at 5 min. A peak in perfusate MMP-2 activity was also observed at 5 min. The deleterious effect of H 2 O 2 on cardiac function was abolished by pyruvate but not by the MMPs inhibitors. This study suggests that in intact hearts, H 2 O 2 induces contractile dysfunction independent of MMPs activation.

Hydrogen peroxide (H2O2) causes cardiac dysfunction through multiple mechanisms. As oxidative stress can activate matrix metalloproteinases (MMPs) and, in particular, MMP-2 activity is associated with oxidative stress injury in the heart, we hypothesized that MMP-2 activation by H2O2 in isolated rat hearts contributes to cardiac dysfunction in this model. Isolated working rat hearts were perfused at 37 degrees C with a recirculating Krebs-Henseleit buffer+/-5 mmol/L pyruvate, known to protect hearts from oxidative stress. H2O2 (300 micromol/L) was added as a single bolus after 20 min of equilibration, and cardiac function was monitored for 60 min. MMPs activities in both the heart and perfusate samples were assessed by gelatin zymography. Tissue high energy phosphates were analysed by HPLC. The actions of 2 MMP inhibitors, doxycycline (75 micromol/L) or Ro 31-9790 (3 micromol/L), were also assessed. H2O2 at 300 micromol/L produced a rapid decline in cardiac mechanical function, which was maximal at 5 min. A peak in perfusate MMP-2 activity was also observed at 5 min. The deleterious effect of H2O2 on cardiac function was abolished by pyruvate but not by the MMPs inhibitors. This study suggests that in intact hearts, H2O2 induces contractile dysfunction independent of MMPs activation.

The addition of Grignard reagents to aldehydes in the presence of chiral aminoalcohols shows a moderate enantioselectivity. The study carried out with a series of ligands allows the correlation between the structural characteristics and their reactivity.