Implementation of an HIV-1 Triple-Target NAT Assay in the Routine Screening at Three German Red Cross Blood Centres.

BACKGROUND: Blood product safety was significantly improved by the introduction of NAT testing in the late 1990s, resulting in a strong decrease of transfusion-transmitted infections (TTIs). Due to the occurrence of HIV-1 NAT test failures as a consequence of mismatch mutations in the amplicon regions of mono-target NAT assays, the Paul Ehrlich Institute mandated the implementation of multi-target NAT assays for HIV-1 in 2014. Commercial suppliers mostly developed dual-target NAT assays, with only one implementing a triple-target NAT assay. METHODS: The HIV-1 triple-target NAT assay v3 (GFE Blut) was tested on mutated specimens and synthetic DNA bearing mutations that resulted in sample underquantification or false-negative test results. In addition, data from 2 years routine testing at three German Red Cross Blood centres were analysed. RESULTS: The HIV-1 triple-target PCR could compensate for all mutations tested and could compensate the loss of one amplicon without a significant loss of sensitivity. Data from 2 years routine testing showed a solid performance. CONCLUSION: The HIV-1 triple-target v3 assay (GFE Blut) can compensate mutations in target sequences better than a dual-target assay and is applicable to high-throughput screening, thus increasing blood product safety.

Sestrin-2, a repressor of PDGFRß signalling, promotes cigarette-smoke-induced pulmonary emphysema in mice and is upregulated in individuals with COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. COPD is caused by chronic exposure to cigarette smoke and/or other environmental pollutants that are believed to induce reactive oxygen species (ROS) that gradually disrupt signalling pathways responsible for maintaining lung integrity. Here we identify the antioxidant protein sestrin-2 (SESN2) as a repressor of PDGFRß signalling, and PDGFRß signalling as an upstream regulator of alveolar maintenance programmes. In mice, the mutational inactivation of Sesn2 prevents the development of cigarette-smoke-induced pulmonary emphysema by upregulating PDGFRß expression via a selective accumulation of intracellular superoxide anions (O2(-)). We also show that SESN2 is overexpressed and PDGFRß downregulated in the emphysematous lungs of individuals with COPD and to a lesser extent in human lungs of habitual smokers without COPD, implicating a negative SESN2-PDGFRß interrelationship in the pathogenesis of COPD. Taken together, our results imply that SESN2 could serve as both a biomarker and as a drug target in the clinical management of COPD.

Antioxidant activity of sestrin 2 controls neuropathic pain after peripheral nerve injury.

AIMS: Neuropathic pain is a chronic debilitating disease that is often unresponsive to currently available treatments. Emerging lines of evidence indicate that reactive oxygen species (ROS) are required for the development and maintenance of neuropathic pain. However, little is known about endogenous mechanisms that neutralize the pain-relevant effects of ROS. In the present study, we tested whether the stress-responsive antioxidant protein Sestrin 2 (Sesn2) blocks the ROS-induced neuropathic pain processing in vivo. RESULTS: We observed that Sesn2 mRNA and protein expression was up-regulated in peripheral nerves after spared nerve injury, a well-characterized model of neuropathic pain. Sesn2 knockout (Sesn2(-/-)) mice exhibited considerably increased late-phase neuropathic pain behavior, while their behavior in acute nociceptive and in inflammatory pain models remained unaffected. The exacerbated neuropathic pain behavior of Sesn2(-/-) mice was associated with elevated ROS levels and an enhanced activating transcription factor 3 up-regulation in sensory neurons, and it was reversed by the ROS scavenger N-tert-Butyl-α-phenylnitrone. In contrast, administration of the ROS donor tert-butyl hydroperoxide induced a prolonged pain behavior in naive Sesn2(-/-) mice. INNOVATION: We show that the antioxidant function of Sesn2 limits neuropathic pain processing in vivo. CONCLUSION: Sesn2 controls ROS-dependent neuropathic pain signaling after peripheral nerve injury and may, thus, provide a potential new target for the clinical management of chronic neuropathic pain conditions.

Inactivation of sestrin 2 induces TGF-beta signaling and partially rescues pulmonary emphysema in a mouse model of COPD.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. Cigarette smoking has been identified as one of the major risk factors and several predisposing genetic factors have been implicated in the pathogenesis of COPD, including a single nucleotide polymorphism (SNP) in the latent transforming growth factor (TGF)-beta binding protein 4 (Ltbp4)-encoding gene. Consistent with this finding, mice with a null mutation of the short splice variant of Ltbp4 (Ltbp4S) develop pulmonary emphysema that is reminiscent of COPD. Here, we report that the mutational inactivation of the antioxidant protein sestrin 2 (sesn2) partially rescues the emphysema phenotype of Ltbp4S mice and is associated with activation of the TGF-beta and mammalian target of rapamycin (mTOR) signal transduction pathways. The results suggest that sesn2 could be clinically relevant to patients with COPD who might benefit from antagonists of sestrin function.

Wnt/beta-catenin signaling controls development of the blood-brain barrier.

The blood-brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/beta-catenin (beta-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of beta-cat in vivo enhances barrier maturation, whereas inactivation of beta-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of beta-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of beta-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of beta-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown.

The epithelial membrane protein 1 is a novel tight junction protein of the blood-brain barrier.

In the central nervous system, a constant microenvironment required for neuronal cell activity is maintained by the blood-brain barrier (BBB). The BBB is formed by the brain microvascular endothelial cells (BMEC), which are sealed by tight junctions (TJ). To identify genes that are differentially expressed in BMEC compared with peripheral endothelial cells, we constructed a subtractive cDNA library from porcine BMEC (pBMEC) and aortic endothelial cells (AOEC). Screening the library for differentially expressed genes yielded 26 BMEC-specific transcripts, such as solute carrier family 35 member F2 (SLC35F2), ADP-ribosylation factor-like 5B (ARL5B), TSC22 domain family member 1 (TSC22D1), integral membrane protein 2A (ITM2A), and epithelial membrane protein 1 (EMP1). In this study, we show that EMP1 transcript is enriched in pBMEC compared with brain tissue and that EMP1 protein colocalizes with the TJ protein occludin in mouse BMEC by coimmunoprecipitation and in rat brain vessels by immunohistochemistry. Epithelial membrane protein 1 expression was transiently induced in laser-capture microdissected rat brain vessels after a 20-min global cerebral ischemia, in parallel with the loss of occludin immunoreactivity. The study identifies EMP1 as a novel TJ-associated protein of the BBB and suggests its potential role in the regulation of the BBB function in cerebral ischemia.

Disruption of the gene encoding the latent transforming growth factor-beta binding protein 4 (LTBP-4) causes abnormal lung development, cardiomyopathy, and colorectal cancer.

Transforming growth factor-betas (TGF-betas) are multifunctional growth factors that are secreted as inactive (latent) precursors in large protein complexes. These complexes include the latency-associated propeptide (LAP) and a latent transforming growth factor-beta binding protein (LTBP). Four isoforms of LTBPs (LTBP-1-LTBP-4) have been cloned and are believed to be structural components of connective tissue microfibrils and local regulators of TGF-beta tissue deposition and signaling. By using a gene trap strategy that selects for integrations into genes induced transiently during early mouse development, we have disrupted the mouse homolog of the human LTBP-4 gene. Mice homozygous for the disrupted allele develop severe pulmonary emphysema, cardiomyopathy, and colorectal cancer. These highly tissue-specific abnormalities are associated with profound defects in the elastic fiber structure and with a reduced deposition of TGF-beta in the extracellular space. As a consequence, epithelial cells have reduced levels of phosphorylated Smad2 proteins, overexpress c-myc, and undergo uncontrolled proliferation. This phenotype supports the predicted dual role of LTBP-4 as a structural component of the extracellular matrix and as a local regulator of TGF-beta tissue deposition and signaling.