Marie van der Merwe

p47phox is required for atherosclerotic lesion progression in ApoE–/– mice

NADPH oxidase is upregulated in smooth muscle cells (SMCs) in response to growth factor stimulation, concomitant with increased reactive oxygen species (ROS) production. We investigated the role of ROS production by NADPH oxidase in SMC responses to growth factors and in atherosclerotic lesion formation in ApoE(-/-) mice. SMCs from wild-type, p47phox(-/-), and gp91phox(-/-) mice differed markedly with respect to growth factor responsiveness and ROS generation. p47phox(-/-) SMCs had diminished superoxide production and a decreased proliferative response to growth factors compared with wild-type cells, whereas the response of gp91phox(-/-) SMCs was indistinguishable from that of wild-type SMCs. The relevance of these in vitro observations was tested by measuring atherosclerotic lesion formation in genetically modified (wild-type, p47phox(-/-), ApoE(-/-), and ApoE(-/-)/p47phox(-/-)) mice. ApoE(-/-)/p47phox(-/-) mice had less total lesion area than ApoE(-/-) mice, regardless of whether mice were fed standard chow or a high-fat diet. Together, these studies provide convincing support for the hypothesis that superoxide generation in general, and NADPH oxidase in particular, have a requisite role in atherosclerotic lesion formation, and they provide a rationale for further studies to dissect the contributions of ROS to vascular lesion formation.

Bacterial Inhibition of Phosphatidylcholine Synthesis Triggers Apoptosis in the Brain

Streptococcus pneumoniae is the most common cause of bacterial meningitis of high mortality and morbidity. Neurological sequelae include paralysis, mental retardation, and learning disorders. In humans, neurons of the hippocampus undergo apoptosis as a result of meningitis. Phosphatidylcholine (PtdCho) is an essential component of mammalian cell membranes and PtdCho deficiency, either due to chemicals or altered nutrition, leads to apoptosis, especially in hippocampal neurons. We show that apoptosis of a variety of brain cells after pneumococcal infection arises from inhibition of PtdCho biosynthesis, the first such activity described for a bacterium. Apoptosis inhibitors did not prevent the bacterial-dependent inhibition of PtdCho biosynthesis. Supplementation with exogenous lyso-phosphatidylcholine prevents cell death and treatment of mice with cytidine diphosphocholine attenuates hippocampal damage during meningitis, even after the onset of infection. We conclude that bacterial inhibition of PtdCho biosynthesis activates an apoptotic cascade that is a causative event in pathogenesis and amenable to therapeutic intervention.

A preterm pig model of lung immaturity and spontaneous infant respiratory distress syndrome

Respiratory distress syndrome (RDS) and bronchopulmonary dysplasia remain the leading causes of preterm infant morbidity, mortality, and lifelong disability. Research to improve outcomes requires translational large animal models for RDS. Preterm pigs delivered by caesarian section at gestation days (GD) 98, 100, 102, and 104 were provided 24 h of neonatal intensive care, monitoring (pulse oximetry, blood gases, serum biomarkers, radiography), and nutritional support, with or without intubation and mechanical ventilation (MV; pressure control ventilation with volume guarantee). Spontaneous development of RDS and mortality without MV are inversely related with GD at delivery and correspond with inadequacy of tidal volume and gas exchange. GD 98 and 100 pigs have consolidated lungs, immature alveolar architecture, and minimal surfactant protein-B expression, and MV is essential at GD 98. Although GD 102 pigs had some alveoli lined by pneumocytes and surfactant was released in response to MV, blood gases and radiography revealed limited recruitment 1-2 h after delivery, and mortality at 24 h was 66% (35/53) with supplemental oxygen provided by a mask and 69% (9/13) with bubble continuous positive airway pressure (8-9 cmH2O). The lungs at GD 104 had higher densities of thin-walled alveoli that secreted surfactant, and MV was not essential. Between GD 98 and 102, preterm pigs have ventilation inadequacies and risks of RDS that mimic those of preterm infants born during the saccular phase of lung development, are compatible with standards of neonatal intensive care, and are alternative to fetal nonhuman primates and lambs.

Recipient Myeloid-Derived Immunomodulatory Cells Induce PD-1 Ligand–Dependent Donor CD4+Foxp3+ Regulatory T Cell Proliferation and Donor–Recipient Immune Tolerance after Murine Nonmyeloablative Bone Marrow Transplantation

We showed previously that nonmyeloablative total lymphoid irradiation/rabbit anti-thymocyte serum (TLI/ATS) conditioning facilitates potent donor–recipient immune tolerance following bone marrow transplantation (BMT) across MHC barriers via recipient invariant NKT (iNKT) cell-derived IL-4–dependent expansion of donor Foxp3+ naturally occurring regulatory T cells (nTregs). In this study, we report a more specific mechanism. Wild-type (WT) BALB/c (H-2d) hosts were administered TLI/ATS and BMT from WT or STAT6−/− C57BL/6 (H-2b) donors. Following STAT6−/− BMT, donor nTregs demonstrated no loss of proliferation in vivo, indicating that an IL-4–responsive population in the recipient, rather than the donor, drives donor nTreg proliferation. In graft-versus-host disease (GVHD) target organs, three recipient CD11b+ cell subsets (Gr-1highCD11c−, Gr-1intCD11c−, and Gr-1lowCD11c+) were enriched early after TLI/ATS + BMT versus total body irradiation/ATS + BMT. Gr-1lowCD11c+ cells induced potent H-2Kb+CD4+Foxp3+ nTreg proliferation in vitro in 72-h MLRs. Gr-1lowCD11c+ cells were reduced significantly in STAT6−/− and iNKT cell–deficient Jα18−/− BALB/c recipients after TLI/ATS + BMT. Depletion of CD11b+ cells resulted in severe acute GVHD, and adoptive transfer of WT Gr-1lowCD11c+ cells to Jα18−/− BALB/c recipients of TLI/ATS + BMT restored day-6 donor Foxp3+ nTreg proliferation and protection from CD8 effector T cell–mediated GVHD. Blockade of programmed death ligand 1 and 2, but not CD40, TGF-β signaling, arginase 1, or iNOS, inhibited nTreg proliferation in cocultures of recipient-derived Gr-1lowCD11c+ cells with donor nTregs. Through iNKT-dependent Th2 polarization, myeloid-derived immunomodulatory dendritic cells are expanded after nonmyeloablative TLI/ATS conditioning and allogeneic BMT, induce PD-1 ligand–dependent donor nTreg proliferation, and maintain potent graft-versus-host immune tolerance.

Mutation of Gly721 Alters DNA Topoisomerase I Active Site Architecture and Sensitivity to Camptothecin

DNA topoisomerase I (Top1p) catalyzes the relaxation of supercoiled DNA via a concerted mechanism of DNA strand cleavage and religation. Top1p is the cellular target of the anti-cancer drug camptothecin (CPT), which reversibly stabilizes a covalent enzyme-DNA intermediate. Top1p clamps around duplex DNA, wherein the core and C-terminal domains are connected by extended α-helices (linker domain), which position the active site Tyr of the C-terminal domain within the catalytic pocket. The physical connection of the linker with the Top1p clamp as well as linker flexibility affect enzyme sensitivity to CPT. Crystallographic data reveal that a conserved Gly residue (located at the juncture between the linker and C-terminal domains) is at one end of a short α-helix, which extends to the active site Tyr covalently linked to the DNA. In the presence of drug, the linker is rigid and this α-helix extends to include Gly and the preceding Leu. We report that mutation of this conserved Gly in yeast Top1p alters enzyme sensitivity to CPT. Mutating Gly to Asp, Glu, Asn, Gln, Leu, or Ala enhanced enzyme CPT sensitivity, with the acidic residues inducing the greatest increase in drug sensitivity in vivo and in vitro. By contrast, Val or Phe substituents rendered the enzyme CPT-resistant. Mutation-induced alterations in enzyme architecture preceding the active site Tyr suggest these structural transitions modulate enzyme sensitivity to CPT, while enhancing the rate of DNA cleavage. We postulate that this conserved Gly residue provides a flexible hinge within the Top1p catalytic pocket to facilitate linker dynamics and the structural alterations that accompany drug binding of the covalent enzyme-DNA intermediate.

DNA Topoisomerase I Domain Interactions Impact Enzyme Activity and Sensitivity to Camptothecin

Background: Despite similarities in mechanism and architecture, human DNA topoisomerase I (Top1) is ∼100-fold more sensitive to camptothecin (CPT) than yeast Top1. Results: Reciprocal swaps of conserved and divergent protein domains alter chimeric Top1 activity. Conclusion: Conserved core and C-terminal domains dictate Top1 biochemical behavior and intrinsic CPT sensitivity. Significance: Interactions between nonconserved structural domains of Top1 impair cell viability, independent of enzyme catalysis. During processes such as DNA replication and transcription, DNA topoisomerase I (Top1) catalyzes the relaxation of DNA supercoils. The nuclear enzyme is also the cellular target of camptothecin (CPT) chemotherapeutics. Top1 contains four domains: the highly conserved core and C-terminal domains involved in catalysis, a coiled-coil linker domain of variable length, and a poorly conserved N-terminal domain. Yeast and human Top1 share a common reaction mechanism and domain structure. However, the human Top1 is ∼100-fold more sensitive to CPT. Moreover, substitutions of a conserved Gly717 residue, which alter intrinsic enzyme sensitivity to CPT, induce distinct phenotypes in yeast. To address the structural basis for these differences, reciprocal swaps of yeast and human Top1 domains were engineered in chimeric enzymes. Here we report that intrinsic Top1 sensitivity to CPT is dictated by the composition of the conserved core and C-terminal domains. However, independent of CPT, biochemically similar chimeric enzymes produced strikingly distinct phenotypes in yeast. Expression of a human Top1 chimera containing the yeast linker domain proved toxic, even in the context of a catalytically inactive Y723F enzyme. Lethality was suppressed either by splicing the yeast N-terminal domain into the chimera, deleting the human N-terminal residues, or in enzymes reconstituted by polypeptide complementation. These data demonstrate a functional interaction between the N-terminal and linker domains, which, when mispaired between yeast and human enzymes, induces cell lethality. Because toxicity was independent of enzyme catalysis, the inappropriate coordination of N-terminal and linker domains may induce aberrant Top1-protein interactions to impair cell growth.

Disulfide Cross-links Reveal Conserved Features of DNA Topoisomerase I Architecture and a Role for the N Terminus in Clamp Closure

In eukaryotes, DNA topoisomerase I (Top1) catalyzes the relaxation of supercoiled DNA by a conserved mechanism of transient DNA strand breakage, rotation, and religation. The unusual architecture of the monomeric human enzyme comprises a conserved protein clamp, which is tightly wrapped about duplex DNA, and an extended coiled-coil linker domain that appropriately positions the C-terminal active site tyrosine domain against the Top1 core to form the catalytic pocket. A structurally undefined N-terminal domain, dispensable for enzyme activity, mediates protein-protein interactions. Previously, reversible disulfide bonds were designed to assess whether locking the Top1 clamp around duplex DNA would restrict DNA strand rotation within the covalent Top1-DNA intermediate. The active site proximal disulfide bond in full-length Top1-clamp534 restricted DNA rotation (Woo, M. H., Losasso, C., Guo, H., Pattarello, L., Benedetti, P., and Bjornsti, M. A. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 13767–13772), whereas the more distal disulfide bond of the N-terminally truncated Topo70-clamp499 did not (Carey, J. F., Schultz, S. J., Sisson, L., Fazzio, T. G., and Champoux, J. J. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 5640–5645). To assess the contribution of the N-terminal domain to the dynamics of Top1 clamping of DNA, the same disulfide bonds were engineered into full-length Top1 and truncated Topo70, and the activities of these proteins were assessed in vitro and in yeast. Here we report that the N terminus impacts the opening and closing of the Top1 protein clamp. We also show that the architecture of yeast and human Top1 is conserved in so far as cysteine substitutions of the corresponding residues suffice to lock the Top1-clamp. However, the composition of the divergent N-terminal/linker domains impacts Top1-clamp activity and stability in vivo.

Lung volume recruitment in a preterm pig model of lung immaturity.

A translational preterm pig model analogous to infants born at 28 wk of gestation revealed that continuous positive airway pressure results in limited lung recruitment but does not prevent respiratory distress syndrome, whereas assist-control + volume guarantee (AC+VG) ventilation improves recruitment but can cause injury, highlighting the need for improved ventilation strategies. We determined whether airway pressure release ventilation (APRV) can be used to recruit the immature lungs of preterm pigs without injury. Spontaneously breathing pigs delivered at 89% of term (model for 28-wk infants) were randomized to 24 h of APRV (n = 9) vs. AC+VG with a tidal volume of 5 ml/kg (n = 10). Control pigs (n = 36) were provided with supplemental oxygen by an open mask. Nutrition and fluid support was provided throughout the 24-h period. All pigs supported with APRV and AC+VG survived 24 h, compared with 62% of control pigs. APRV resulted in improved lung volume recruitment compared with AC+VG based on radiographs, lower Pco2 levels (44 ± 2.9 vs. 53 ± 2.7 mmHg, P = 0.009) and lower inspired oxygen fraction requirements (36 ± 6 vs. 44 ± 11%, P < 0.001), and higher oxygenation index (5.1 ± 1.5 vs. 2.9 ± 1.1, P = 0.001). There were no differences between APRV and AC+VG pigs for heart rate, ratio of wet to dry lung mass, proinflammatory cytokines, or histopathological markers of lung injury. Lung protective ventilation with APRV improved recruitment of alveoli of preterm lungs, enhanced development and maintenance of functional residual capacity without injury, and improved clinical outcomes relative to AC+VG. Long-term consequences of lung volume recruitment by using APRV should be evaluated.

The Influence of Methylsulfonylmethane on Inflammation-Associated Cytokine Release before and following Strenuous Exercise.

Background. Inflammation is associated with strenuous exercise and methylsulfonylmethane (MSM) has been shown to have anti-inflammatory properties. Methods. Physically active men were supplemented with either placebo or MSM (3 grams per day) for 28 days before performing 100 repetitions of eccentric knee extension exercise. Ex vivo and in vitro testing consisted of evaluating cytokine production in blood (whole blood and isolated peripheral blood mononuclear cells (PBMCs)) exposed to lipopolysaccharide (LPS), before and through 72 hours after exercise, while in vivo testing included the evaluation of cytokines before and through 72 hours after exercise. Results. LPS stimulation of whole blood after MSM supplementation resulted in decreased induction of IL-1β, with no effect on IL-6, TNF-α, or IL-8. After exercise, there was a reduced response to LPS in the placebo, but MSM resulted in robust release of IL-6 and TNF-α. A small decrease in resting levels of proinflammatory cytokines was noted with MSM, while an acute postexercise increase in IL-10 was observed with MSM. Conclusion. Strenuous exercise causes a robust inflammatory reaction that precludes the cells from efficiently responding to additional stimuli. MSM appears to dampen the release of inflammatory molecules in response to exercise, resulting in a less incendiary environment, allowing cells to still have the capacity to mount an appropriate response to an additional stimulus after exercise.Background. Inflammation is associated with strenuous exercise and methylsulfonylmethane (MSM) has been shown to have anti-inflammatory properties. Methods. Physically active men were supplemented with either placebo or MSM (3 grams per day) for 28 days before performing 100 repetitions of eccentric knee extension exercise. Ex vivo and in vitro testing consisted of evaluating cytokine production in blood (whole blood and isolated peripheral blood mononuclear cells (PBMCs)) exposed to lipopolysaccharide (LPS), before and through 72 hours after exercise, while in vivo testing included the evaluation of cytokines before and through 72 hours after exercise. Results. LPS stimulation of whole blood after MSM supplementation resulted in decreased induction of IL-1β, with no effect on IL-6, TNF-α, or IL-8. After exercise, there was a reduced response to LPS in the placebo, but MSM resulted in robust release of IL-6 and TNF-α. A small decrease in resting levels of proinflammatory cytokines was noted with MSM, while an acute postexercise increase in IL-10 was observed with MSM. Conclusion. Strenuous exercise causes a robust inflammatory reaction that precludes the cells from efficiently responding to additional stimuli. MSM appears to dampen the release of inflammatory molecules in response to exercise, resulting in a less incendiary environment, allowing cells to still have the capacity to mount an appropriate response to an additional stimulus after exercise.

Time-restricted feeding of a high-fat diet in male C57BL/6 mice reduces adiposity but does not protect against increased systemic inflammation

Time-restricted feeding (TRF) limits the duration of food availability without altering diet composition and can combat obesity in humans and mice. For this study we evaluated the effect of timing of food access during a TRF protocol on weight gain, adiposity, and inflammation. Young male C57BL/6 mice were placed on a high-fat (HF) diet (45% fat) for 8 weeks. Food access was unrestricted (HF) or restricted to 6 h per day, either for the first half (HF-early) or the second half (HF-late) of the active phase to resemble a window of time for food consumption early or late in the day in a human population. Weight, obesity-associated parameters, and inflammation were measured. TRF reduced weight gain over the 8-week period in mice consuming the same high-fat diet. Consistent with decreased weight gain in the TRF groups, body fat percentage, liver triglycerides, and plasma leptin and cholesterol levels were reduced. Adipose tissue inflammation, measured by CD11b+F4/80+ macrophage infiltration, was reduced in both TRF groups, but systemic tumor necrosis factor-α was increased in all groups consuming the high-fat diet. The HF-late group gained more weight than the HF-early group and had increased insulin resistance, while the HF-early group was protected. Therefore, a TRF protocol is beneficial for weight management when a high-fat diet is consumed, with food consumption earlier in the day showing greater health benefits. However, increased inflammatory markers in the TRF groups suggest that diet components can still increase inflammation even in the absence of overt obesity.