Late transplant-associated thrombotic microangiopathy verified in bone marrow biopsy specimens is associated with chronic GVHD and viral infections.

OBJECTIVES: To describe late transplant-associated thrombotic microangiopathy (TA-TMA) as chronic endothelial complication in bone marrow (BM) after allogeneic hematopoietic stem cell transplantation (HSCT). METHODS: BM specimens along with conventional diagnostic parameters were assessed in 14 single-institutional patients with late TA-TMA (more than 100 days after HCST), including 11 late with history of early TA-TMA, 10 with early TA-TMA (within 100 days), and 12 non TA-TMA patients. Three non-HSCT patients served as control. The time points of BM biopsy were +1086, +798, +396, and +363 days after HSCT, respectively. RESULTS: Late TA-TMA patients showed an increase of CD34+ and von Willebrand Factor (VWF)+ microvascular endothelial cells with atypical VWF+ conglomerates forming thickened VWF+ plaque sinus in the BM compared to patients without late TA-TMA and non-HSCT. Severe chronic (p = .002), steroid-refractory GVHD (p = .007) and reactivation of HHV6 (p = .002), EBV (p = .003), and adenovirus (p = .005) were pronounced in late TA-TMA. Overall and relapse-free survival were shorter in late TA-TMA than in patients without late TA-TMA (5-year OS and RFS: 78.6% vs. 90.2%, 71.4% vs. 86.4%, respectively). CONCLUSION: Chronic allo-immune microangiopathy in BM associated with chronic, steroid-refractory GVHD and/or viral infections are key findings of late, high-risk TA-TMA, which deserves clinical attention.

Antioxidant and Anti-Inflammatory Activities of Zingiber montanum Oil in HepG2 Cells and Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

Improvement of antioxidant and anti-inflammatory functions is believed to be an effective strategy for protection against various diseases such as cancer, aging, and neurodegenerative disease. This study focused on investigating antioxidant and anti-inflammatory abilities of Zingiber montanum oil (ZMO) extracted by the supercritical CO2 fluid system in HepG2 cells and lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. Ten predominant constituents of ZMO were identified, in which triquinacene, 1,4-bis (methoxy), terpinen-4-ol, triquinacene, 1,4,7-tris (methoxy), α-terpinene, sabinene hydrate, and (E and Z)-1-(3,4-dimethoxyphenyl)butadiene account for 86.47%. ZMO exhibited anti-inflammatory capacity by inhibiting the formation of pro-inflammatory markers such as nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, interleukin (IL)-1ß, IL-6, and monocyte chemoattractant protein-1 in LPS-treated macrophages. The LPS-induced stimulation of nuclear factor-kappa B, signal transducer and activator of transcription 3 (Stat3) and mitogen-activated protein kinase (MAPK) pathways as evident from increased phosphorylation of IKKα/ß, IκBα, p65, Stat3, ERK, JNK, and p38 MAPK was also suppressed by ZMO pretreatment. Further, ZMO enhanced the expression of nuclear factor erythroid 2-related factor (Nrf2) and heme oxygenase-1 (HO-1), and concurrently, reduced intracellular reactive oxygen species accumulation in LPS-treated RAW 264.7 cells. In addition, ZMO treatment markedly upregulated the expression of Nrf2 as well as its target genes, HO-1 and NAD(P)H:quinone oxidoreductase 1 in HepG2 cells. These data propose that ZMO may be a potent candidate for prevention and/or treatment of inflammatory and oxidative conditions.

Pro-survival role for Parkinson's associated gene DJ-1 revealed in trophically impaired dopaminergic neurons.

The mechanisms underlying the selective death of substantia nigra (SN) neurons in Parkinson disease (PD) remain elusive. While inactivation of DJ-1, an oxidative stress suppressor, causes PD, animal models lacking DJ-1 show no overt dopaminergic (DA) neuron degeneration in the SN. Here, we show that aging mice lacking DJ-1 and the GDNF-receptor Ret in the DA system display an accelerated loss of SN cell bodies, but not axons, compared to mice that only lack Ret signaling. The survival requirement for DJ-1 is specific for the GIRK2-positive subpopulation in the SN which projects exclusively to the striatum and is more vulnerable in PD. Using Drosophila genetics, we show that constitutively active Ret and associated Ras/ERK, but not PI3K/Akt, signaling components interact genetically with DJ-1. Double loss-of-function experiments indicate that DJ-1 interacts with ERK signaling to control eye and wing development. Our study uncovers a conserved interaction between DJ-1 and Ret-mediated signaling and a novel cell survival role for DJ-1 in the mouse. A better understanding of the molecular connections between trophic signaling, cellular stress and aging could uncover new targets for drug development in PD.

Periphilin is strongly expressed in the murine nervous system and is indispensable for murine development.

Periphilin is involved in multiple processes in vivo. To explore its physiological role from an organismic perspective, we generated mice with a gene trap insertion in the periphilin-1 gene. Based on beta-gal reporter activity, a widespread periphilin expression was evident, especially in the developing somites and limbs, the embryonic nervous system, and the adult brain. In accordance with this broad expression, homozygous deficiency of periphilin was lethal in early embryogenesis. Mice with a heterozygous deficiency did not show any abnormalities of brain morphology and function, neither histologically nor regarding the transcriptome. Interestingly, the reduction of the periphilin-1 gene dosage was compensated by an increased expression of the remaining wild-type allele in the brain. These results point to an indispensable function of periphilin during murine development and an important role in the nervous system, reflected by a strong and tightly regulated expression in the murine brain.

Regulation of astrocyte inflammatory responses by the Parkinson's disease-associated gene DJ-1.

The Parkinson's disease (PD)-associated gene DJ-1 mediates direct neuroprotection. The up-regulation of DJ-1 in reactive astrocytes also suggests a role in glia. Here we show that DJ-1 regulates proinflammatory responses in mouse astrocyte-rich primary cultures. When treated with a Toll-like receptor 4 agonist, the bacterial endotoxin lipopolysaccharide (LPS), Dj-1-knockout astrocytes generated >10 times more nitric oxide (NO) than littermate controls. Lentiviral reintroduction of DJ-1 restored the NO response to LPS. The enhanced NO production in Dj-1(-/-) astrocytes was mediated by a signaling pathway involving reactive oxygen species leading to specific hyperinduction of type II NO synthase [inducible NO synthase (iNOS)]. These effects coincided with significantly increased phosphorylation of p38 mitogen-activated protein kinase (MAPK), and p38(MAPK) inhibition suppressed NO production and iNOS mRNA and protein induction. Dj-1(-/-) astrocytes also induced the proinflammatory mediators cyclooxygenase-2 and interleukin-6 significantly more strongly, but not nerve growth factor. Finally, primary neuron cultures grown on Dj-1(-/-) astrocytes became apoptotic in response to LPS in an iNOS-dependent manner, directly demonstrating the neurotoxic potential of astrocytic DJ-1 deficiency. These findings identify DJ-1 as a regulator of proinflammatory responses and suggest that loss of DJ-1 contributes to PD pathogenesis by deregulation of astrocytic neuroinflammatory damage.

Structural determinants of the C-terminal helix-kink-helix motif essential for protein stability and survival promoting activity of DJ-1.

Mutations in the PARK7 gene encoding DJ-1 cause autosomal recessive Parkinson disease. The most deleterious point mutation is the L166P substitution, which resides in a structure motif comprising two alpha-helices (G and H) separated by a kink. Here we subjected the C-terminal helix-kink-helix motif to systematic site-directed mutagenesis, introducing helix-incompatible proline residues as well as conservative substitutions into the helical interface. Furthermore, we generated deletion mutants lacking the H-helix, the kink, and the entire C terminus. When transfected into neural and nonneural cell lines, steady-state levels of G-helix breaking and kink deletion mutants were dramatically lower than wild-type DJ-1. The effects of H-helix breakers were comparably smaller, and the non-helix breaking mutants only slightly destabilized DJ-1. The decreased steady-state levels were due to accelerated protein degradation involving in part the proteasome. G-helix breaking DJ-1 mutations abolished dimer formation. These structural perturbations had functional consequences on the cytoprotective activities of DJ-1. The destabilizing mutations conferred reduced cytoprotection against H(2)O(2) in transiently retransfected DJ-1 knock-out mouse embryonic fibroblasts. The loss of survival promoting activity of the DJ-1 mutants with destabilizing C-terminal mutations correlated with impaired anti-apoptotic signaling. We found that wild-type, but not mutant DJ-1 facilitated the Akt pathway and simultaneously blocked the apoptosis signal-regulating kinase 1, with which DJ-1 interacted in a redox-dependent manner. Thus, the G-helix and kink are critical determinants of the C-terminal helix-kink-helix motif, which is absolutely required for stability and the regulation of survival-promoting redox signaling of the Parkinson disease-associated protein DJ-1.