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Obesity and dyslipidaemia are features of the metabolic syndrome and risk factors for chronic kidney disease. The cellular mechanisms connecting metabolic syndrome with chronic kidney disease onset and progression remain largely unclear. We show that proximal tubular epithelium is a target site for lipid deposition upon overnutrition with a cholesterol-rich Western-type diet. Affected proximal tubule epithelial cells displayed giant vacuoles of lysosomal or autophagosomal origin, harbouring oxidised lipoproteins and concentric membrane layer structures (multilamellar bodies), reminiscent of lysosomal storage diseases. Additionally, lipidomic analysis revealed renal deposition of cholesterol and phospholipids, including lysosomal phospholipids. Proteomic profiles of renal multilamellar bodies were distinct from those of epidermis or lung multilamellar bodies and of cytoplasmic lipid droplets. Tubular multilamellar bodies were observed in kidney biopsies of obese hypercholesterolaemic patients, and the concentration of the phospholipidosis marker di-docosahexaenoyl (22:6)-bis(monoacylglycerol) phosphate was doubled in urine from individuals with metabolic syndrome and chronic kidney disease. The enrichment of proximal tubule epithelial cells with phospholipids and multilamellar bodies was accompanied by enhanced inflammation, fibrosis, tubular damage markers, and higher urinary electrolyte content. Concomitantly to the intralysosomal lipid storage, a renal transcriptional response was initiated to enhance lysosomal degradation and lipid synthesis. In cultured proximal tubule epithelial cells, inhibition of cholesterol efflux transport or oxysterol treatment induced effects very similar to the in vivo situation, such as multilamellar body and phospholipid amassing, and induction of damage, inflammatory, fibrotic, and lipogenic molecules. The onset of phospholipidosis in proximal tubule epithelial cells is a novel pathological trait in metabolic syndrome-related chronic kidney disease, and emphasises the importance of healthy lysosomes and nutrition for kidney well-being. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Background Susceptibility to pepsin digestion of candidate transgene products is regarded an important parameter in the weight-of-evidence approach for allergenicity risk assessment of genetically modified crops. It has been argued that protocols used for this assessment should better reflect physiological conditions encountered in representative food consumption scenarios. Aim To evaluate whether inclusion of more physiological conditions, such as sub-optimal and lower pepsin concentrations, in combination with pancreatin digestion, improved the performance of digestibility protocols used in characterization of protein stability. Methods Four pairs of established allergens and their related non/weakly-allergenic counterparts (seed albumins, muscle tropomyosins, plant lipid transfer proteins [LTP] and collagens) plus fish parvalbumin, were subjected to nine combinations of pH (1.2–2.5–4.0) and pepsin-to-protein ratio (PPR: 10–1–0.1 U/µg) for pepsin digestion, followed by pancreatin digestion in the presence of bile salts. Digestion was monitored by SDS-PAGE in conjunction with Coomassie staining and immunoblotting using rabbit antisera and human IgE. Results At pH 4.0 and at PPR 0.1 most proteins, both allergen and non-allergen, were highly resistant to pepsin. Under conditions known to favor pepsin proteolysis, the established major allergens Ara h 2, Pru p 3 and Pen a 1 were highly resistant to proteolysis, while the allergen Cyp c 1 was not. However, this resistance to pepsin digestion only made Ara h 2 and to a lesser extent Pen a 1 and Pru p 3 stand out compared to their non-allergenic counterparts. Largely irrespective of preceding pepsin digestion conditions, pancreatin digestion was very effective for all tested proteins, allergens and non-allergens, except for Cyp c 1 and bovine collagen. Conclusions Sub-optimal pH, low pepsin-to protein ratio, and sequential pepsin and pancreatin digestion protocols do not improve the predictive value in distinguish allergens from non-allergens. Digestion conditions facilitating such distinction differ per protein pair. Electronic supplementary material The online version of this article (10.1186/s13601-018-0216-9) contains supplementary material, which is available to authorized users.

Most patients with acute myeloid leukemia (AML) can only be cured when allogeneic hematopoietic stem-cell transplantation induces a graft-versus-leukemia immune response (GVL). Although the role of T cells and natural killer cells in tumor immunology has been established, less is known about the contribution of B cells. From B cells of high-risk patients with AML with potent and lasting GVL responses, we isolated monoclonal antibodies directed against antigens expressed on the cell surface of AML cells but not on normal hematopoietic and nonhematopoietic cells. A number of these donor-derived antibodies recognized the U5 snRNP200 complex, a component of the spliceosome that in normal cells is found in the cell. In AML however, the U5 snRNP200 complex is exposed on the cell membrane of leukemic blasts. U5 snRNP200 complex-specific antibodies induced death of AML cells in an Fc receptor-dependent way in the absence of cytotoxic leukocytes or complement. In an AML mouse model, treatment with U5 snRNP200 complex-specific antibodies led to significant tumor growth inhibition. Thus, donor-derived U5 snRNP200 complex-recognizing AML-specific antibodies may contribute to antitumor responses.

The number of individuals partaking in veganism has increased sharply in the last decade. Therefore, it is critical to look at the implications of vegan diets for public health. Although there are multiple health benefits of a vegan diet, studies have also linked the diet with deficiencies in various micronutrients. This study focuses on vitamin B12, because of its critical role in DNA synthesis and methylation. In light of these connections, a critical review of recent scientific literature is conducted to understand the effects of a B12 deficient diet on the genome and epigenome, and whether it can give rise to cancer. It is observed that a B12 deficiency leads to increased uracil misincorporation, leading to impaired DNA synthesis and genomic instability. The deficiency also leads to global hypomethylation of DNA, a hallmark of early carcinogenesis. The findings of this study highlight the need for increased awareness among vegans to ensure adequate B12 intake through supplementation or consumption of fortified products as a preventative measure. Additionally, the biofortification of staple crops and an improved version of fermented products with increased B12 content can be developed when inadequate intake seems otherwise inevitable.

Summary: Metabolic reprogramming has emerged as a crucial regulator of immune cell activation, but how systemic metabolism influences immune cell metabolism and function remains to be investigated. To investigate the effect of dyslipidemia on immune cell metabolism, we performed in-depth transcriptional, metabolic, and functional characterization of macrophages isolated from hypercholesterolemic mice. Systemic metabolic changes in such mice alter cellular macrophage metabolism and attenuate inflammatory macrophage responses. In addition to diminished maximal mitochondrial respiration, hypercholesterolemia reduces the LPS-mediated induction of the pentose phosphate pathway (PPP) and the Nrf2-mediated oxidative stress response. Our observation that suppression of the PPP diminishes LPS-induced cytokine secretion supports the notion that this pathway contributes to inflammatory macrophage responses. Overall, this study reveals that systemic and cellular metabolism are strongly interconnected, together dictating macrophage phenotype and function. : The link between systemic and cellular metabolism is a neglected aspect in immunometabolism. Baardman et al. show that hypercholesterolemia alters macrophage metabolism and phenotype. The suppressed pentose phosphate pathway (PPP) in those “foam cell” macrophages attenuates inflammatory responses, signifying that systemic and cellular metabolism together regulate macrophage function. Keywords: immunometabolism, inflammation, macrophages, hypercholesterolemia, pentose phosphate pathway, Nrf2, meta-inflammation, foam cells, atherosclerosis, cardiovascular disease, metabolic disease

Summary Activation of macrophages by inflammatory stimuli induces reprogramming of mitochondrial metabolism to support the production of pro-inflammatory cytokines and nitric oxide. Hallmarks of this metabolic rewiring are downregulation of α-ketoglutarate formation by isocitrate dehydrogenase (IDH) and accumulation of glutamine-derived succinate, which enhances the inflammatory response via the activity of succinate dehydrogenase (SDH). Here, we identify the nuclear receptor Nur77 (Nr4a1) as a key upstream transcriptional regulator of this pro-inflammatory metabolic switch in macrophages. Nur77-deficient macrophages fail to downregulate IDH expression and accumulate higher levels of succinate and other TCA cycle-derived metabolites in response to inflammatory stimulation in a glutamine-independent manner. Consequently, these macrophages produce more nitric oxide and pro-inflammatory cytokines in an SDH-dependent manner. In vivo, bone marrow Nur77 deficiency exacerbates atherosclerosis development and leads to increased circulating succinate levels. In summary, Nur77 induces an anti-inflammatory metabolic state in macrophages that protects against chronic inflammatory diseases such as atherosclerosis., Graphical Abstract, Highlights • Genome-wide profiling indicates that Nur77 regulates macrophage mitochondrial metabolism • Nur77 inhibits IDH expression and TCA cycle activity in inflammatory macrophages • Nur77-deficient macrophages produce more nitric oxide and cytokines via SDH • Nur77 deficiency increases circulating succinate levels and atherosclerosis in vivo, Koenis et al. show that nuclear receptor Nur77 regulates the reprogramming of mitochondrial metabolism in inflammatory macrophages. Nur77-deficient macrophages accumulate higher levels of succinate and produce more pro-inflammatory cytokines and nitric oxide in a succinate dehydrogenase-dependent manner. In vivo, bone marrow Nur77 deficiency exacerbates atherosclerosis and increases circulating succinate levels.

Reactive oxygen species (ROS) formation by mitochondria is an incompletely understood eukaryotic process. I proposed a kinetic model [BioEssays (2011) 33, 88–94] in which the ratio between electrons entering the respiratory chain via FADH2 or NADH (the F/N ratio) is a crucial determinant of ROS formation. During glucose breakdown, the ratio is low, while during fatty acid breakdown, the ratio is high (the longer the fatty acid, the higher is the ratio), leading to higher ROS levels. Thus, breakdown of (very-long-chain) fatty acids should occur without generating extra FADH2 in mitochondria. This explains peroxisome evolution. A potential ROS increase could also explain the absence of fatty acid oxidation in long-lived cells (neurons) as well as other eukaryotic adaptations, such as dynamic supercomplex formation. Effective combinations of metabolic pathways from the host and the endosymbiont (mitochondrion) allowed larger varieties of substrates (with different F/N ratios) to be oxidized, but high F/N ratios increase ROS formation. This might have led to carnitine shuttles, uncoupling proteins, and multiple antioxidant mechanisms, especially linked to fatty acid oxidation [BioEssays (2014) 36, 634–643]. Recent data regarding peroxisome evolution and their relationships with mitochondria, ROS formation by Complex I during ischaemia/reperfusion injury, and supercomplex formation adjustment to F/N ratios strongly support the model. I will further discuss the model in the light of experimental findings regarding mitochondrial ROS formation.

Organotypic cultures from normal neocortical tissue obtained at epilepsy surgery show a severe injury response. This response involves both neuronal degeneration and the proliferation of reactive cells. A salient feature of the reactive cells is the co-expression of microglial and astrocytic markers. Surprisingly, the reactive cells also began to express neuronal markers Tubulin βIII and MAP2 adding to the confusion about their origin. Concomitant with their appearance in reactive cells MAP2 and Tubulin βIII expression disappeared from neurons. While NeuN expression decreased significantly, it did not entirely disappear from many neurons. Moreover, it was not observed in reactive cells, showing that NeuN is a reliable marker of neurons.

The latent HIV-1 reservoir in treated patients primarily consists of resting memory CD4+ T cells. Stimulating the T-cell receptor (TCR), which facilitates transition of resting into effector T cells, is the most effective strategy to purge these latently infected cells. Here we demonstrate that TCR-stimulated effector T cells still frequently harbor latent HIV-1. Renewed TCR-stimulation or subsequent activation with latency reversing agents (LRAs) did not overcome latency. However, interaction of infected effector cells with dendritic cells (DCs) triggered further activation of latent HIV-1. When compared to TCR-stimulation only, CD4+ T cells from aviremic patients receiving TCR+DC-stimulation reversed latency more frequently. Such a “one-two punch” strategy seems ideal for purging the reservoir. We determined that DC contact activates the PI3K-Akt-mTOR pathway in CD4+ T cells. This insight could facilitate the development of a novel class of potent LRAs that purge latent HIV beyond levels reached by T-cell activation.

Brain injury affects a significant number of people each year. Organotypic cultures from resected normal neocortical tissue provide unique opportunities to study the cellular and neuropathological consequences of severe injury of adult human brain tissue in vitro. The in vitro injuries caused by resection (interruption of the circulation) and aggravated by the preparation of slices (severed neuronal and glial processes and blood vessels) reflect the reaction of human brain tissue to severe injury. We investigated this process using immunocytochemical markers, reverse transcriptase quantitative polymerase chain reaction and Western blot analysis. Essential features were rapid shrinkage of neurons, loss of neuronal marker expression and proliferation of reactive cells that expressed Nestin and Vimentin. Also, microglia generally responded strongly, whereas the response of glial fibrillary acidic protein-positive astrocytes appeared to be more variable. Importantly, some reactive cells also expressed both microglia and astrocytic markers, thus confounding their origin. Comparison with post-mortem human brain tissue obtained at rapid autopsies suggested that the reactive process is not a consequence of epilepsy.

HIV-1 transcription depends on cellular transcription factors that bind to sequences in the long-terminal repeat (LTR) promoter. Each HIV-1 subtype has a specific LTR promoter configuration, and minor sequence changes in transcription factor binding sites (TFBSs) or their arrangement can influence transcriptional activity, virus replication and latency properties. Previously, we investigated the proviral latency properties of different HIV-1 subtypes in the SupT1 T cell line. Here, subtype-specific latency and replication properties were studied in primary PHA-activated T lymphocytes. No major differences in latency and replication capacity were measured among the HIV-1 subtypes. Subtype B and AE LTRs were studied in more detail with regard to a putative AP-1 binding site using luciferase reporter constructs. c-Jun, a member of the AP-1 transcription factor family, can activate both subtype B and AE LTRs, but the latter showed a stronger response, reflecting a closer match with the consensus AP-1 binding site. c-Jun overexpression enhanced Tat-mediated transcription of the viral LTR, but in the absence of Tat inhibited basal promoter activity. Thus, c-Jun can exert a positive or negative effect via the AP-1 binding site in the HIV-1 LTR promoter, depending on the presence or absence of Tat.

Chemokines (chemotactic cytokines) can have direct antimicrobial activity, which is apparently related to the presence of a distinct positively charged patch on the surface. However, chemokines can retain antimicrobial activity upon linearization despite the loss of their positive patch, thus questioning the importance of this patch for activity. Thrombocidin-1 (TC-1) is a microbicidal protein isolated from human blood platelets. TC-1 only differs from the chemokine NAP-2/CXCL7 by a two-amino acid C-terminal deletion, but this truncation is crucial for antimicrobial activity. We assessed the structure-activity relationship for antimicrobial activity of TC-1. Reduction of the charge of the TC-1-positive patch by replacing lysine 17 with alanine reduced the activity against bacteria and almost abolished activity against the yeast Candida albicans. Conversely, augmentation of the positive patch by increasing charge density or size resulted in a 2-3-fold increased activity against Staphylococcus aureus, Escherichia coli, and Bacillus subtilis but did not substantially affect activity against C. albicans. Reduction of TC-1 resulted in loss of the folded conformation, but this disruption of the positive patch did not affect antimicrobial activity. Using overlapping 15-mer synthetic peptides, we demonstrate peptides corresponding to the N-terminal part of TC-1 to have similar antimicrobial activity as intact TC-1. Although we demonstrate that the positive patch is essential for activity of folded TC-1, unfolded TC-1 retained antimicrobial activity despite the absence of a positive patch. This activity is probably exerted by a linear peptide stretch in the N-terminal part of the molecule. We conclude that intact TC-1 and unfolded TC-1 exert antimicrobial activity via distinct structural elements.

Enzyme reprofiling in bacteria during adaptation from one environmental condition to another may be regulated by both transcription and translation. However, little is known about the contribution of translational regulation. Recently, we have developed a pulse labeling method using the methionine analog azidohomoalanine to determine the relative amounts of proteins synthesized by Escherichia coli in a brief time frame upon a change in environmental conditions. Here we present an extension of our analytical strategy, which entails measuring changes in total protein levels on the same time scale as new protein synthesis. This allows identification of stable and labile proteins and demonstrates that altered levels of most newly synthesized proteins are the result of a change in translation rate rather than degradation rate. With this extended strategy, average relative translation rates for 10 min immediately after a switch from aerobiosis to anaerobiosis were determined. The majority of proteins with increased synthesis rates upon an anaerobic switch are involved in glycolysis and pathways aimed at preventing glycolysis grinding to a halt by a cellular redox imbalance. Our method can be used to compare relative translation rates with relative mRNA levels at the same time. Discrepancies between these parameters may reveal genes whose expression is regulated by translation rather than by transcription. This may help unravel molecular mechanism underlying changes in translation rates, e. g. mediated by small regulatory RNAs. Molecular & Cellular Proteomics 9:2508-2516, 2010

With the rise in prevalence of antibiotic- resistant bacteria, honey is increasingly valued for its antibacterial activity. To characterize all bactericidal factors in a medical-grade honey, we used a novel approach of successive neutralization of individual honey bactericidal factors. All bacteria tested, including Bacillus subtilis, methicillin-resistant Staphylococcus au- reus, extended-spectrum -lactamase producing Esche- richia coli, ciprofloxacin-resistant Pseudomonas aerugi- nosa, and vancomycin-resistant Enterococcus faecium, were killed by 10 -20% (v/v) honey, whereas >40% (v/v) of a honey-equivalent sugar solution was required for similar activity. Honey accumulated up to 5.62 0.54 mM H2O2 and contained 0.25 0.01 mM methyl- glyoxal (MGO). After enzymatic neutralization of these two compounds, honey retained substantial activity. Using B. subtilis for activity-guided isolation of the additional antimicrobial factors, we discovered bee defensin-1 in honey. After combined neutralization of H2O2, MGO, and bee defensin-1, 20% honey had only minimal activity left, and subsequent adjustment of the pH of this honey from 3.3 to 7.0 reduced the activity to that of sugar alone. Activity against all other bacteria tested depended on sugar, H2O2, MGO, and bee defensin-1. Thus, we fully characterized the antibacterial activity of medical-grade honey.—Kwak- man, P. H. S., te Velde, A. A., de Boer, L., Speijer, D., Vandenbroucke-Grauls, C. M. J. E., Zaat, S. A. J. How honey kills bacteria. FASEB J. 24, 000 - 000 (2010). www.fasebj.org

Hfq is a highly conserved pleiotropically acting prokaryotic RNA-binding protein involved in the post-transcriptional regulation of many stress-responsive genes by small RNAs. In this study, we show that Hfq of the strictly human pathogen Neisseria meningitidis is involved in the regulation of expression of components involved in general metabolic pathways, iron metabolism and virulence. A meningococcal hfq deletion strain (H44/76Deltahfq) is impaired in growth in nutrient-rich media and does not grow at all in nutrient-limiting medium. The growth defect was complemented by expression of hfq in trans. Using proteomics, the expression of 28 proteins was found to be significantly affected upon deletion of hfq. Of these, 20 proteins are involved in general metabolism, among them seven iron-responsive genes. Two proteins (PilE, TspA) are involved in adherence to human cells, a step crucial for the onset of disease. One of the differentially expressed proteins, GdhA, was identified as an essential virulence factor for establishment of sepsis in an animal model, studied earlier. These results show that in N. meningitidis Hfq is involved in the regulation of a variety of components contributing to the survival and establishment of meningococcal disease.

The mammalian skeleton consists of bones that are formed in two different ways: long bones via endochondral ossification and flat bones via intramembranous ossification. These different formation modes may result in differences in the composition of the two bone types. Using the 2D-difference in gel electrophoresis technique and mass spectrometry, we analyzed the composition of murine mineral-associated proteins of calvaria and long bone. Considerable differences in protein composition were observed. Flat bones (calvariae) contained more soluble collagen (8x), pigment epithelium derived factor (3x) and osteoglycin (4x); whereas long bones expressed more chondrocalcin (3x), thrombospondin- 1 (4x), fetuin (4x), secreted phosphoprotein 24 (3x), and thrombin (7x). Although cystatin motifs containing proteins, such as secreted phosphoprotein 24 and fetuin are highly expressed in long bone, they did not inhibit the activity of the cysteine proteinases cathepsin B and K. The solubility of collagen differed which coincided with differences in collagen crosslinking, long bone containing 3x more (hydroxylysine)-pyridinoline. The degradation of long bone collagen by MMP2 (but not by cathepsin K) was impaired. These differences in collagen crosslinking may explain the differences in the proteolytic pathways osteoclasts use to degrade bone. Our data demonstrate considerable differences in protein composition of flat and long bones and strongly suggest functional differences in formation, resorption, and mechanical properties of these bone types.

Human chitotriosidase is specifically expressed by phagocytes, has anti-fungal activity towards chitin-containing fungi in vitro and in vivo, and is part of innate immunity. We studied the effect of toll-like receptor (TLR)- and nucleotide-binding oligomerization domain ( NOD)- 2 triggering on chitotriosidase expression and release by phagocytes. We find that TLR, but not NOD2 activation, regulates chitotriosidase release by neutrophils. Furthermore, both TLR and NOD2 activation resulted in diminished induction by monocytes. Lastly, NOD2 activation, but not TLR stimulation, induces chitinase expression in macrophages. We conclude that phagocyte-specific regulation is important for efficient eradication of chitin-containing pathogens. (c) 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved

Plasma lipoproteins, such as high-density lipoprotein (HDL), can serve as carriers for a wide range of proteins that are involved in processes such as lipid metabolism, thrombosis, inflammation and atherosclerosis. The identification of HDL-associated proteins is essential with regards to understanding these processes at the molecular level. In this study, a combination of proteomic approaches including 1-DE and 2-DE MALDI-TOF, isotope-coded affinity tag and Western blot analysis were employed to identify proteins associated with human HDL. To minimize potential losses of HDL-associated proteins during isolation, a one-step ultracentrifugation technique was applied and the quality of purified HDL was confirmed by nephelometry, high-performance gel chromatography, and Western blot analysis. MS analysis revealed the presence of 56 HDL-associated proteins including all known apolipoproteins and lipid transport proteins. Furthermore, proteins involved in hemostasis and thrombosis, the immune and complement system were found. In addition, growth factors, receptors, hormone-associated proteins and many other proteins were found to be associated with HDL. Our approach thus resulted in the identification of a large number of proteins associated with HDL. The combination of proteomic technologies proved to be a powerful and comprehensive tool for the identification of proteins on HDL.

The gene prmC , encoding the putative S -adenosyl- l -methionine (AdoMet)-dependent methyltransferase (MTase) of release factors (RFs) of the obligate intracellular pathogen Chlamydia trachomatis , was functionally analyzed. Chlamydial PrmC expression suppresses the growth defect of a prmC knockout strain of Escherichia coli K-12, suggesting an interaction of chlamydial PrmC with E. coli RFs in vivo. In vivo methylation assays carried out with recombinant PrmC and RFs of chlamydial origin demonstrated that PrmC methylates RFs within the tryptic fragment containing the universally conserved sequence motif Gly-Gly-Gln. This is consistent with the enzymatic properties of PrmC of E. coli origin. We conclude that C. trachomatis PrmC functions as an N 5 -glutamine AdoMet-dependent MTase, involved in methylation of RFs.

This review focuses on several aspects of humoral immunologic defence of the ocular surface and intraocular compartment. Secretory IgA (sIgA) is a major component of lacrimal fluid and contributes to the first line of defence against infection of the ocular surface. Recent findings show that part of the lacrimal gland (LG) IgA repertoire consists of so-called natural IgA antibodies. How B cells responsible for these natural IgA antibodies are distributed to effector mucosal sites like the LG is not known. Extensive data are now available on the regionalization of mucosal IgA responses in murine gut, involving peritoneal B cells, the prototypic natural antibody producing cells. By comparing elaborate experiments done in mice with the much less extensive data on the LG, it becomes clear that this gland is a unique, but poorly investigated effector organ of the mucosal immune system. In addition to the humoral immune response at the surface of the eye, the production of antibodies within the ocular compartment also has several fascinating features. The detection of pathogen-specific antibodies in intraocular fluid (IOF) of uveitis patients is accepted as a diagnostic tool, but the specificity of these intraocular antibodies was not investigated in much detail. Recent data however, demonstrate that even antibodies recognizing the same antigen in both serum and IOF still differ in the epitopes they recognize. This reveals that the intraocular compartment largely determines its own antibody profile in the defence against intra-ocular pathogens. Several models as to how an exclusive intra-ocular B cell repertoire may be generated are presented. (C) 2003 Elsevier Science Ltd. All rights reserved

Clinical non-relevant (CNR) IgM specific for Toxoplasma gondii is responsible for false-positive results in commercially available IgM assays. Using IgM immunoblotting, it is possible to distinguish between IgM in sera of acutely infected (AI) patients and CNR IgM. Especially the combination of staining of a 55 and 30 kD antigen in T.gondii lysate proved useful in this respect. The 55 kD antigen was identified as Rop1, while the 30 kD antigen was confirmed to be Sag1. However, the use of recombinant antigens instead of lysates for diagnostic assays would improve reproducibility. IgM recognized recombinant Rop1, but most CNR sera also had low anti-Rop1 titers. Although purified native Sag1 separated AI and CNR sera very well on immunoblot, IgM did not recognize recombinant Sag1 at all. Clearly, it is difficult to produce a recombinant Sag1 that can be recognized by IgM. Recombinant Rop1 might be suitable as one of the recombinant antigens in an IgM immunoblot assay, but has to be combined with at least one other immunogenic antigen.

Background: Latent HIV type I (HIV-1) infections can frequently occur in short-lived proliferating effector T lymphocytes. These latently infected cells could revert into resting T lymphocytes and thereby contribute to the establishment of the long-lived viral reservoir. Monocyte-derived dendritic cells can revert latency in effector T cells in vitro. Methods: Here we investigated the latency activation properties of tissue-specific immune cells, including a large panel of dendritic cell subsets, to explore in which body compartments effector T cells are most likely to maintain latent HIV-1 provirus and thus potentially contribute to the long-lived reservoir. Results: Our results demonstrate that blood or genital tract dendritic cells do not activate latent provirus in effector T cells, whereas gut or lymphoid dendritic cells induce virus production from latently infected effector T cells in our in-vitro model for latency. Toll-like receptor 3-induced interferon production by myeloid dendritic cells abolished the dendritic cells’ ability to induce viral gene expression. Conclusions: In this study, we show that HIV-1 provirus residing in effector T cells is activated from latency by tissue-specific dendritic cell subsets and other immune cells with remarkably different efficiencies. Conclusions: Our new assay system points to an important, neglected aspect of HIV-1 research: the ability of other immune cells, especially dendritic cells, to differentially affect latency establishment as well as virus reactivation.

As free-living organisms, alpha-proteobacteria produce reactive oxygen species (ROS) that diffuse into the surroundings; once constrained inside the archaeal ancestor of eukaryotes, however, ROS production presented evolutionary pressures – especially because the alpha-proteobacterial symbiont made more ROS, from a variety of substrates. I previously proposed that ratios of electrons coming from FADH2 and NADH (F/N ratios) correlate with ROS production levels during respiration, glucose breakdown having a much lower F/N ratio than longer fatty acid (FA) breakdown. Evidently, higher endogenous ROS formation did not hinder eukaryotic evolution, so how were its disadvantages mitigated? I propose that the resulting selection pressures favoured the evolution of a variety of eukaryotic ‘innovations’: peroxisomes for FA breakdown, carnitine shuttles, the linkage of beta-oxidation to antioxidant properties, uncoupling proteins (UCPs) and using mitochondrial uncoupling during beta-oxidation to reduce ROS. Recently observed relationships between peroxisomes and mitochondria further support the model.

Purpose Protein adsorption, cell adhesion and graft patency was compared in hydrophilic versus hydrophobic polymer-coated prosthetic vascular grafts. We hypothesize that in vivocompatibility of hydrophilic polymer-coated prosthetic vascular grafts is superior to in vivo compatibility of hydrophobic grafts. Methods A pairwise side-to-side common carotid artery interposition graft was placed eight female landrace goats (mean weight 55 kg). Protein adsorption was assessed using Western Blot in two hydrophilic and two hydrophobic grafts harvested after three days. Graft patency was monitored for 28 days in six goats with continuous wave Doppler ultrasonography. Adherence of endothelial cells, leukocytes and platelets was determined with ELISA and compared between the two graft types after 28 days. Results After three days, more ApoA-l, albumin and VEGF and less fibrin adsorbed to hydrophilic grafts. After 28 days, compared to hydrophobic grafts, higher numbers of endothelial cells were present on hydrophilic grafts (P=0.016), and less thrombocytes and leukocytes (P=0.012 and 0.024, respectively). Two out of eight hydrophobic grafts lost patency, while none of the hydrophilic grafts failed (P=0.157). Conclusions Hydrophilic polymer-coated vascular grafts have superior in vivo compatibility when compared to hydrophobic grafts as characterized by reduced platelet and leukocyte adherence as well as higher endothelialization.

Neisseria meningitidis (the meningococcus) is primarily a commensal of the human oropharynx that sporadically causes septicemia and meningitis. Meningococci adapt to diverse local host conditions differing in nutrient supply, like the nasopharynx, blood, and cerebrospinal fluid, by changing metabolism and protein repertoire. However, regulatory transcription factors and two-component systems in meningococci involved in adaptation to local nutrient variations are limited. We identified novel sibling small regulatory RNAs (Neisseria metabolic switch regulators [NmsRs]) regulating switches between cataplerotic and anaplerotic metabolism in this pathogen. Overexpression of NmsRs was tolerated in blood but not in cerebrospinal fluid. Expression of six tricarboxylic acid cycle enzymes was downregulated by direct action of NmsRs. Expression of the NmsRs themselves was under the control of the stringent response through the action of RelA. Small sibling regulatory RNAs of meningococci, controlling general metabolic switches, add an exciting twist to their versatile repertoire in bacterial pathogens., IMPORTANCE Regulatory small RNAs (sRNAs) of pathogens are coming to be recognized as highly important components of riboregulatory networks, involved in the control of essential cellular processes. They play a prominent role in adaptation to physiological changes as represented by different host environments. They can function as posttranscriptional regulators of gene expression to orchestrate metabolic adaptation to nutrient stresses. Here, we identified highly conserved sibling sRNAs in Neisseria meningitidis which are functionally involved in the regulation of gene expression of components of the tricarboxylic acid cycle. These novel sibling sRNAs that function by antisense mechanisms extend the so-called stringent response which connects metabolic status to colonization and possibly virulence as well as pathogenesis in meningococci.

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) can cure acute myeloid leukemia (AML) when the donor immune system generates a potent graft versus leukemia (GvL) response. While the role of T cells and NK cells in GvL immune responses has been established, the contribution of B cells to GvL responses is less clear. Using SEREX and other techniques, the presence of antibodies directed against established tumor antigens following allogeneic HSCT has been demonstrated, but because these antibodies were not obtained in monoclonal format, the function of these antibodies could not be analyzed. Aim: To investigate the role of antibodies produced by donor-derived B cells in GvL responses. Methods: We selected five patients with high-risk AML who remained disease-free for more than 5 years after allogeneic HSCT and thus have mounted a potent GvL response. From the peripheral blood of these patients we isolated memory B cells that we transduced with Bcl-6 and Bcl-xL, to establish antibody-producing clonal B cell lines. Blood was obtained 2 years after allogeneic HSCT. B cell lines were screened for the production of antibodies that specifically bound to surface antigens on AML cell lines and AML blasts isolated from patients in our clinic. Target identification was performed by immunoprecipitation and mass-spectometry. Results: We identified patient derived clonal B cell lines producing antibodies that recognized antigens expressed on the cell surface of AML cells, but not on normal hematopoietic and non-hematopoietic cells. Antibodies were donor-derived, and a number of these antibodies recognized the U5 snRNP200 complex. The U5 snRNP200 complex is a component of the spliceosome that in normal cells is located in the nucleus but that is exposed on the cell membrane of AML cells. U5 snRNP200 complex-specific antibodies were specific for allogeneic HSCT recipients with AML, as they were found in in 4 out of 5 AML patients screened, but were not found in multiple myeloma patients who received an allogeneic HSCT or in healthy individuals. Strikingly, U5 snRNP200 complex-specific antibodies induced death of AML cells in vitro, and, in a human AML mouse model, in vivo. Cell death was induced in the absence of cytotoxic leukocytes or of complement, through a non-apoptotic process that depended on destabilization of the cytoskeleton as cell death could be blocked by incubation of the target cells with cytochalasin D, an actin polymerization inhibitor. Cytotoxicity of the U5 snRNP200 antibodies was present at 4°C and 37°C, suggesting that cell death was induced by a passive process. Indeed, interaction of the antibodies with their target cells did not induce a calcium flux. Cytotoxicity of the antibodies depended on the Fc region of the antibody, since recombinant U5 snRNP200 complex-specific antibodies with a defective Fc region were not cytotoxic. Summary and Conclusions: Allogeneic HSCT recipients with robust donor anti-AML immunity generate antibodies against a component of the spliceosome, the U5 snRNP200 complex, that is expressed on the membrane of AML blasts. U5 snRNP200 antibodies are cytotoxic in vivo and in vitro, demonstrating the potency of the humoral immune system in tumor immunology. Citation Format: Marijn Gillissen, Martijn Kedde, Greta De Jong, Etsuko Yasuda, Sophie Levie, Arjen Bakker, Yvonne Claassen, Koen Wagner, Julien Villaudy, Martino Bohne, Dave Speijer, Paul Hensbergen, Pauline van Helden, Tim Beaumont, Hergen Spits, Mette D. Hazenberg. Acute myeloid leukemia patients cured after allogeneic hematopoietic stem cell transplantation generate tumor-specific cytotoxic antibodies that kill AML blasts [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A061.

This mini-review summarizes techniques applied in, and results obtained with, proteomic studies of human immunodeficiency virus type 1 (HIV-1)-T cell interaction. Our group previously reported on the use of two-dimensional differential gel electrophoresis (2D-DIGE) coupled to matrix assisted laser-desorption time of flight peptide mass fingerprint analysis, to study T cell responses upon HIV-1 infection. Only one in three differentially expressed proteins could be identified using this experimental setup. Here we report on our latest efforts to test models generated by this data set and extend its analysis by using novel bioinformatic algorithms. The 2D-DIGE results are compared with other studies including a pilot study using one-dimensional peptide separation coupled to MS(E), a novel mass spectrometric approach. It can be concluded that although the latter method detects fewer proteins, it is much faster and less labor intensive. Last but not least, recent developments and remaining challenges in the field of proteomic studies of HIV-1 infection and proteomics in general are discussed.

GBA3, also known as cytosolic beta-glucosidase, is thought to hydrolyze xenobiotic glycosides in man. Deficiency of glucocerebrosidase (GBA), a beta-glucosidase degrading glucosylteramide, underlies Gaucher disease. We examined GBA3, which recently was proposed to degrade glucosylceramide and influence the clinical manifestation of Gaucher disease. Recombinant GBA3 was found to hydrolyze artificial substrates such as 4-methylumbelliferyl-beta-D-glucoside and C6-NBD-glucosylceramide, but hydrolysis of naturally occurring lipids like glucosylceramide and glucosylsphingosine was hardly detected. Consistent with this, inhibition of GBA3 in cultured cells using a novel inhibitor (alpha-1-C-nonyl-DIX) did not result in an additional increase in glucosylceramide as compared to GBA inhibition alone. Examination of the GBA3 gene led to the identification of a common substitution in its open reading frame (1368T -> A), resulting in a truncated GBA3 protein missing the last a-helix of its (beta/alpha)(8) barrel. Both recombinant 1368A GBA3 and 1368A enzyme from spleen of a homozygous individual were found to be inactive. Amongst non-neuronopathic (type 1) Gaucher disease patients, we subsequently identified individuals being wild-type, heterozygous, or homozygous for the GBA3 1368T -> A mutation. No correlation was observed between GBA3 1368A/T haplotypes and severity of type 1 Gaucher disease manifestation. In conclusion, GBA3 does not seem to modify type 1 Gaucher disease manifestation. (C) 2010 Elsevier Inc. All rights reserved

The aim of this study was to compare plasma and serum protein profiles in elderly acute hip fracture patients with and without delirium. The spectra from surface-enhanced laser desorption ionization (SELDI) using time-of-flight (TOF) mass spectrometry of 16 patients without and 16 patients with delirium (two groups of eight and eight) scored using the Confusion Assessment Method were compared. The most discriminating peak of 15.9 kDa in plasma in a testing group of eight patients with delirium to eight patients without delirium was confirmed in an independent validation group. Taking both groups together, three discriminating peaks of 7.97, 15.9, and 16.0 kDa were found in delirious patients. These peaks presumably correspond to hemoglobin-beta, its doubly charged ion, and its glycosylated form. (The Journal of Neuropsychiatry and Clinical Neurosciences 2009; 21: 284-291)

Udgivelsesdato: 2009-Aug Gaucher disease is an inherited lysosomal storage disorder, characterized by massive accumulation of glucosylceramide-laden macrophages in the spleen, liver and bone marrow as a consequence of deficient activity of glucocerebrosidase. Gaucher disease has been the playground to develop new therapeutic interventions such as enzyme-replacement therapy and substrate-reduction therapy. The availability of these costly therapies has stimulated research regarding suitable biomarkers to monitor onset and progression of disease, as well as the efficacy of therapeutic intervention. Given the important role of storage cells in the pathology, various attempts have been made to identify proteins in plasma or serum reflecting the body burden of these pathological cells. In this review, the existing data regarding biomarkers for Gaucher disease, as well as the current application of biomarkers in clinical management of Gaucher patients are discussed. Moreover, the use of several modern proteomic technologies for the identification of Gaucher biomarkers is reviewed.

The plasma proteome of type I Gaucher disease patients was investigated by 2D gel electrophoresis (2DGE). Using the classical procedure with 8 M urea treated plasma, several high molecular weight proteins were absent from Gaucher plasma specimens, while additional low molecular weight proteins were visible. The latter were identified as proteolytic degradation products. Adding small amounts of patient plasma to control plasma gave extensive protein breakdown. The presence of 2.2 M thiourea/7.7 M urea in the rehydration solution totally prevented breakdown. In the 'urea only' solution, protease(s) uniquely present in Gaucher plasma, appear to be still active towards other denatured plasma proteins at low pH. Therapy of patients results in gradual disappearance of proteolytic capacity from plasma specimens, indicating it to be related to the presence of Gaucher storage cells. The proteolytic activity could be partly removed from Gaucher plasma samples by Concanavalin A, suggesting that glycoproteins are involved. Reduction of proteolysis by Pepstatin A and Leupeptin implies that cathepsins. proteases known to be overproduced by Gaucher storage cells, are involved. In conclusion, 2DGE Gaucher plasma proteomes should be interpreted cautiously given the abnormal high levels of proteases associated with this disorder. (c) 2008 Elsevier B.V. All rights reserved

A biomarker is an analyte that indicates the presence of a biological process linked to the clinical manifestations and outcome of a particular disease. An ideal biomarker provides indirect but ongoing determinations of disease activity. In the case of lysosomal storage disorders (LSDs), metabolites or proteins specifically secreted by storage cells are good candidates for biomarkers. Potential clinical applications of biomarkers are found in improved diagnosis, monitoring of disease progression and assessment of therapeutic correction. These applications are illustrated by reviewing the use of plasma chitotriosidase in the clinical management of patients with Gaucher disease, the most common LSD. The ongoing debate on the value of biomarkers in patient management is addressed. Novel analytical methods have revolutionized the identification and measurement of biomarkers at the protein and metabolite level. Recent developments in biomarker discovery by proteomics are described and the future for biomarkers of LSDs is discussed.Conclusion: Besides direct applications for biomarkers in patient management, biomarker searches are likely to render new insights into pathophysiological mechanisms and metabolic adaptations, and may provide new targets for therapeutic intervention.

Unleashing the tumor-specific immune response by immunotherapies such as checkpoint inhibitors or allogeneic stem cell transplantation can result in long lasting tumor regressions. It is thought that T cell responses are responsible for tumor rejection. Here we tested the hypothesis that B cells contribute to tumor regression following immunotherapy by analysing the tumor-specific antibody repertoire in patients suffering from acute myeloid leukemia (AML), a high-risk malignancy with a poor prognosis. These patients receive an allogeneic hematopoietic stem cell transplantation (HSCT) which resets the immune system and can lead to potent graft versus leukemia (GvL) responses. We selected three patients with high-risk AML who mounted potent GvL responses after allogeneic HSCT. Of these patients we established antibody-producing clonal B cell lines following transduction of memory B cells from peripheral blood with BCL-6 and Bcl-xL and screened those for producing antibodies specifically binding to surface antigens on AML cell lines and AML blasts. A number of antibodies were identified that recognized primary AML blasts isolated from newly diagnosed AML patients, but did not bind to healthy bone marrow, peripheral blood mononuclear cells or tissues such as liver, skin and colon. Strikingly, 40% of these AML-specific antibodies induced direct cell death of cultured AML cell lines and of primary AML blasts. Cytotoxicity of the antibodies was rapid, occurred both at 37°C and 4°C and could be prevented by Cytochalasin D, an actin polymerization inhibitor that stabilizes the cytoskeleton. This, in combination with the observation that cell death could not be prevented by apoptosis inhibitors indicated that the tumor cells were killed through a necrotic pathway like oncosis. Interestingly, most cytotoxic antibodies recognized an antigen, which in normal cells is expressed in the nucleus but in AML cells is present on the membrane. Together these data indicate that tumor selective antibodies can be elicited following allogeneic HSCT in AML patients with a strong GvL response. The direct cytotoxic activities against tumor cells of a proportion of these antibodies suggest that they contribute to the GvL response. Citation Format: Marijn Gillissen, Martijn Kedde, Etsuko Yasuda, Sophie Levie, Arjen Bakker, Yvonne Claassen, Martino Bohne, Dave Speijer, Daisy Picavet, Jan Stap, Pauline van Helden, Tim Beaumont, Hergen Spits, Mette Hazenberg. Donor derived cytotoxic antibodies from transplanted AML patients targeting a cell surface expressed nuclear antigen. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B052.

Unleashing the tumor-specific immune response by immunotherapies such as checkpoint inhibitors or allogeneic stem cell transplantation can result in long lasting tumor regression. While the role of T cells in such graft versus leukemia (GvL) immune responses has been established, the contribution of B cells to GvL responses is less clear. Using SEREX and other techniques, the presence of antibodies directed against established tumor antigens following allogeneic hematopoietic stem cell transplantation (HSCT) has been demonstrated. The function of these antibodies remains to be established though. Here we tested the hypothesis that B cells contribute to GvL. We selected three patients with high-risk AML who mounted potent GvL responses after allogeneic HSCT. Of these patients we established antibody-producing clonal B cell lines following transduction of memory B cells from the peripheral blood of these patients with Bcl-6 and Bcl-xL and screened those for producing antibodies specifically binding to surface antigens on AML cell lines and AML blasts. A number of antibodies were identified that recognized primary AML blasts isolated from newly diagnosed AML patients, but did not bind to healthy bone marrow, peripheral blood mononuclear cells or tissues such as liver, skin and colon. Target identification analyses revealed two novel, AML-specific surface proteins as primary targets of these antibodies. These newly identified AML-specific antigens are widely expressed on different types of AML, including blasts of the last 17 AML patients that entered our clinic of whom diagnosis material was made available for this project. Strikingly, antibody binding to one of these targets induced direct cell death of cultured AML cell lines and of primary AML blasts. Cytotoxicity of the antibodies was rapid, occurred both at 37o C and 4o C and could be prevented by Cytochalasin D, an actin polymerization inhibitor that stabilizes the cytoskeleton. This, in combination with the observation that cell death could not be prevented by apoptosis inhibitors indicated that the tumor cells were killed through a necrotic pathway like oncosis. Other antibodies induced antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent-cytotoxicity (CDC). Together these data indicate that tumor selective antibodies are elicited following allogeneic HSCT in AML patients with a strong GvL response. The direct and indirect cytotoxic activities against tumor cells of these antibodies suggest that they contribute to the GvL response. Disclosures No relevant conflicts of interest to declare.

A wide range of pathogens, including human immunodeficiency virus type 1 (HIV-1), hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, Mycobacterium , Leishmania , and Helicobacter pylori , can interact with dendritic cell (DC)-specific ICAM3-grabbing nonintegrin (DC-SIGN), expressed on DCs and a subset of B cells. More specifically, the interaction of the gp120 envelope protein of HIV-1 with DC-SIGN can facilitate the transfer of virus to CD4 + T lymphocytes in trans and enhance infection. We have previously demonstrated that a multimeric Le X component in human milk binds to DC-SIGN, preventing HIV-1 from interacting with this receptor. Biochemical analysis reveals that the compound is heat resistant, trypsin sensitive, and larger than 100 kDa, indicating a specific glycoprotein as the inhibitory compound. By testing human milk from three different mothers, we found the levels of DC-SIGN binding and viral inhibition to vary between samples. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and matrix-assisted laser desorption ionization analysis, we identified bile salt-stimulated lipase (BSSL), a Lewis X (Le X )-containing glycoprotein found in human milk, to be the major variant protein between the samples. BSSL isolated from human milk bound to DC-SIGN and inhibited the transfer of HIV-1 to CD4 + T lymphocytes. Two BSSL isoforms isolated from the same human milk sample showed differences in DC-SIGN binding, illustrating that alterations in the BSSL forms explain the differences observed. These results indicate that variations in BSSL lead to alterations in Le X expression by the protein, which subsequently alters the DC-SIGN binding capacity and the inhibitory effect on HIV-1 transfer. Identifying the specific molecular interaction between the different forms may aid in the future design of antimicrobial agents.

For more than a decade, protein-replacement therapy has been employed successfully for the treatment of Gaucher disease. Recently, a comparable therapy has become available for the related lipid-storage disorder Fabry disease. Two differently produced recombinant alpha-galactosidase A (alpha-gal A) preparations are used independently for this purpose. Agalsidase alpha is obtained from human fibroblasts that have been modified by gene activation; agalsidase beta is obtained from Chinese hamster ovary cells that are transduced with human alpha-gal A cDNA. It has previously been claimed that alpha-gal A mRNA undergoes editing, which may result in coproduction of an edited protein (Phe 396 Tyr) that might have a relevant physiological function. We therefore analyzed the occurrence of alpha-gal A editing, as well as the precise nature, in this respect, of the therapeutic enzymes. No indications were obtained for the existence of editing at the protein or RNA level. Both recombinant enzymes used in therapy are unedited and are capable of functionally correcting cultured fibroblasts from Fabry patients in their excessive globotriaosylceramide accumulation. Although RNA editing is apparently not relevant in the case of alpha-gal A, a thorough analysis of the potential occurrence of editing of transcripts is nevertheless advisable in connection with newly developed protein-replacement therapies.

Secretory IgA (sIgA) antibodies in human tears and milk were found to recognize protein disulfide isomerase (PDI) on a Toxoplasma gondii lysate immunoblot (IB). These antibodies were already detectable in tears of infants. To determine the epitope containing-regions on PDI, we generated truncated versions of recombinant PDI that differ by 8‐10 amino acids in length. By IB, it was found that the sIgA epitopes were confined to conserved regions of PDI, including the functionally essential thioredoxin-like domain. This suggested the capacity of sIgA to react with PDI of other species, which was confirmed by recognition of human PDI by IgA in tears. In contrast, anti-T. gondii PDI antibodies generated by immunization were not able to cross-react. Binding to the thioredoxin-like domain on IB could be gradually abrogated by incubation with peptide constituting the same domain. By consecutive investigation of the function of the protein targeted by sIgA, the presence of antibody in relation to age and analysis of the epitope constituting regions on PDI we demonstrate that sIgA directed against PDI are self-reactive natural antibodies. Furthermore, analysis of antibody epitopes on an antigen is a useful method to distinguish conventional, affinity-matured antibodies from natural antibodies. The presence at early age and continuity of anti-PDI sIgA in relation to age suggests the existence of B cells secreting germline-encoded antibodies in human mucosa outside of the gut. Overall, the PDI-specific antibodies are clearly part of the natural antibody repertoire, suggesting an active role for these antibodies in the innate defense against pathogens.

The human gene POLG encodes the catalytic subunit of mitochondrial DNA polymerase, but its precise roles in mtDNA metabolism in vivo have not hitherto been documented. By expressing POLG fusion proteins in cultured human cells, we show that the enzyme is targeted to mitochondria, where the Myc epitope-tagged POLG is catalytically active as a DNA polymerase. Long-term culture of cells expressing wild-type POLG-myc revealed no alterations in mitochondrial function. Expression of POLG-myc mutants created dominant phenotypes demonstrating important roles for the protein in mtDNA maintenance and integrity. The D198A amino acid replacement abolished detectable 3'-5' (proofreading) exonuclease activity and led to the accumulation of a significant load (1:1700) of mtDNA point mutations during 3 months of continuous culture. Further culture resulted in the selection of cells with an inactivated mutator polymerase, and a reduced mutation load in mtDNA. Transient expression of POLG-myc variants D890N or D1135A inhibited endogenous mitochondrial DNA polymerase activity and caused mtDNA depletion. Deletion of the POLG CAG repeat did not affect enzymatic properties, but modestly up-regulated expression. These findings demonstrate that POLG exonuclease and polymerase functions are essential for faithful mtDNA maintenance in vivo, and indicate the importance of key residues for these activities.

Mitochondria constitute the major energy-producing compartment of the eukaryotic cell. These organelles contain many molecules of DNA that contribute only a handful of proteins required for energy production. Mutations in the DNA of mitochondria were identified as a cause of human disease a quarter of a century ago, and they have subsequently been implicated in ageing. The process whereby deleterious variants come to dominate a cell, tissue or human is the subject of debate. It is likely to involve multiple, often competing, factors, as selection pressures on mitochondrial DNA can be both indirect and intermittent, and are subjected to rapid change. Here, we assess the different models and the prospects for preventing the accumulation of deleterious mitochondrial DNA variants with time.

HIV-1 latency remains a formidable barrier towards virus eradication as therapeutic attempts to purge these reservoirs are so far unsuccessful. The pool of transcriptionally silent proviruses is established early in infection and persists for a lifetime, even when viral loads are suppressed below detection levels using anti-retroviral therapy. Upon therapy interruption the reservoir can re-establish systemic infection. Different cellular reservoirs that harbor latent provirus have been described. In this study we demonstrate that HIV-1 can also establish a silent integration in actively proliferating primary T lymphocytes. Co-culturing of these proliferating T lymphocytes with dendritic cells (DCs) activated the provirus from latency. Activation did not involve DC-mediated C-type lectin DC-SIGN signaling or TCR-stimulation but was mediated by DC-secreted component(s) and cell-cell interaction between DC and T lymphocyte that could be inhibited by blocking ICAM-1 dependent adhesion. These results imply that circulating DCs could purge HIV-1 from latency and re-initiate virus replication. Moreover, our data show that viral latency can be established early after infection and supports the idea that actively proliferating T lymphocytes with an effector phenotype contribute to the latent viral reservoir. Unraveling this physiologically relevant purging mechanism could provide useful information for the development of new therapeutic strategies that aim at the eradication of HIV-1 reservoirs., Author Summary Combination therapy can suppress the viral load in HIV-1 infected individuals to undetectable levels, but does not lead to complete virus eradication. Even after many years of successful therapy the virus is still present in long-lived cells as a latently integrated provirus. HIV-1 can re-establish systemic infection from this reservoir when therapy stops. Purging attempts in patients have been unsuccessful and HIV-1 latency remains a formidable barrier to virus eradication. Different cellular reservoirs that harbor latent HIV-1 proviruses have been described to consist mainly of resting memory T lymphocytes. Yet how this reservoir in memory T lymphocytes is established is still unclear as infection of these cells is very inefficient. In this paper we demonstrate that HIV-1 can establish a latent provirus in activated effector T lymphocytes. We observed that for every virus producing cell there is at least one other cell harboring a latent provirus, illustrating that latent infections occur frequently. Proliferating T lymphocytes are generally short-lived and their contribution to the total cellular reservoir thus seems limited. However, these activated T lymphocytes can revert into resting memory T lymphocytes and become part of the long-lived viral reservoir.

Methods We have studied the effects of human milk (HM) and seminal plasma (SP) on interfering with the capture of HIV-1 through binding to DC-SIGN on dendritic cells and prevent the subsequent transfer of HIV-1 to CD4 cells. HM and SP were tested in Raji-DC-SIGN and immature dendritic cell transfer assays to determine interference with HIV-1 capture and transfer. Fractionation and MALDI-TOF biochemical analysis was utilized to identify proteins with DC-SIGN binding activity.