Fatal demyelinating disease is induced by monocyte-derived macrophages in the absence of TGF-ß signaling.

The cytokine transforming growth factor-ß (TGF-ß) regulates the development and homeostasis of several tissue-resident macrophage populations, including microglia. TGF-ß is not critical for microglia survival but is required for the maintenance of the microglia-specific homeostatic gene signature1,2. Under defined host conditions, circulating monocytes can compete for the microglial niche and give rise to long-lived monocyte-derived macrophages residing in the central nervous system (CNS)3-5. Whether monocytes require TGF-ß for colonization of the microglial niche and maintenance of CNS integrity is unknown. We found that abrogation of TGF-ß signaling in CX3CR1+ monocyte-derived macrophages led to rapid onset of a progressive and fatal demyelinating motor disease characterized by myelin-laden giant macrophages throughout the spinal cord. Tgfbr2-deficient macrophages were characterized by high expression of genes encoding proteins involved in antigen presentation, inflammation and phagocytosis. TGF-ß is thus crucial for the functional integration of monocytes into the CNS microenvironment.

Myeloid cell-specific topoisomerase 1 inhibition using DNA origami mitigates neuroinflammation.

Targeting myeloid cells, especially microglia, for the treatment of neuroinflammatory diseases such as multiple sclerosis (MS), is underappreciated. Our in silico drug screening reveals topoisomerase 1 (TOP1) inhibitors as promising drug candidates for microglial modulation. We show that TOP1 is highly expressed in neuroinflammatory conditions, and TOP1 inhibition using camptothecin (CPT) and its FDA-approved analog topotecan (TPT) reduces inflammatory responses in microglia/macrophages and ameliorates neuroinflammation in vivo. Transcriptomic analyses of sorted microglia from LPS-challenged mice reveal an altered transcriptional phenotype following TPT treatment. To target myeloid cells, we design a nanosystem using ß-glucan-coated DNA origami (MyloGami) loaded with TPT (TopoGami). MyloGami shows enhanced specificity to myeloid cells while preventing the degradation of the DNA origami scaffold. Myeloid-specific TOP1 inhibition using TopoGami significantly suppresses the inflammatory response in microglia and mitigates MS-like disease progression. Our findings suggest that TOP1 inhibition in myeloid cells represents a therapeutic strategy for neuroinflammatory diseases and that the myeloid-specific nanosystems we designed may also benefit the treatment of other diseases with dysfunctional myeloid cells.

An overlooked subset of Cx3cr1wt/wt microglia in the Cx3cr1CreER-Eyfp/wt mouse has a repopulation advantage over Cx3cr1CreER-Eyfp/wt microglia following microglial depletion.

BACKGROUND: Fluorescent reporter labeling and promoter-driven Cre-recombinant technologies have facilitated cellular investigations of physiological and pathological processes, including the widespread use of the Cx3cr1CreER-Eyfp/wt mouse strain for studies of microglia. METHODS: Immunohistochemistry, Flow Cytometry, RNA sequencing and whole-genome sequencing were used to identify the subpopulation of microglia in Cx3cr1CreER-Eyfp/wt mouse brains. Genetically mediated microglia depletion using Cx3cr1CreER-Eyfp/wtRosa26DTA/wt mice and CSF1 receptor inhibitor PLX3397 were used to deplete microglia. Primary microglia proliferation and migration assay were used for in vitro studies. RESULTS: We unexpectedly identified a subpopulation of microglia devoid of genetic modification, exhibiting higher Cx3cr1 and CX3CR1 expression than Cx3cr1CreER-Eyfp/wtCre+Eyfp+ microglia in Cx3cr1CreER-Eyfp/wt mouse brains, thus termed Cx3cr1highCre-Eyfp- microglia. This subpopulation constituted less than 1% of all microglia under homeostatic conditions, but after Cre-driven DTA-mediated microglial depletion, Cx3cr1highCre-Eyfp- microglia escaped depletion and proliferated extensively, eventually occupying one-third of the total microglial pool. We further demonstrated that the Cx3cr1highCre-Eyfp- microglia had lost their genetic heterozygosity and become homozygous for wild-type Cx3cr1. Therefore, Cx3cr1highCre-Eyfp- microglia are Cx3cr1wt/wtCre-Eyfp-. Finally, we demonstrated that CX3CL1-CX3CR1 signaling regulates microglial repopulation both in vivo and in vitro. CONCLUSIONS: Our results raise a cautionary note regarding the use of Cx3cr1CreER-Eyfp/wt mouse strains, particularly when interpreting the results of fate mapping, and microglial depletion and repopulation studies.

Absence of microglia or presence of peripherally-derived macrophages does not affect tau pathology in young or old hTau mice.

Microglia are implicated in the pathophysiology of several neurodegenerative disorders, including Alzheimer's disease. While the role of microglia and peripheral macrophages in regulating amyloid beta pathology has been well characterized, the impact of these distinct cell subsets on tau pathology remains poorly understood. We and others have recently demonstrated that monocytes can engraft the brain and give rise to long-lived parenchymal macrophages, even under nonpathological conditions. We undertook the current study to investigate the regulation of tau pathology by microglia and peripheral macrophages using hTau transgenic mice, which do not exhibit microglial activation/pathology or macrophage engraftment. To assess the direct impact of microglia on tau pathology we developed a protocol for long-term microglial depletion in Cx3cr1CreER R26DTA mice and crossed them with hTau mice. We then depleted microglia up to 3 months in both young and old mice, but no net change in forebrain soluble oligomeric tau or total or phosphorylated levels of aggregated tau was recorded. To investigate the consequence of peripherally-derived parenchymal macrophages on tau aggregation we partially repopulated the hTau microglial pool with peripheral macrophages, but this also did not affect levels of tau oligomers or insoluble aggregates. Our study questions the direct involvement of microglia or peripheral macrophages in the development of tau pathology in the hTau model.

Features of repeated muscle biopsies and phenotypes of monocytes in paired blood samples and clinical long-term response to treatment in patients with idiopathic inflammatory myopathy: a pilot study.

OBJECTIVES: In a pilot study we aimed to identify biomarkers in repeated muscle biopsies and paired blood samples, taken before and after conventional immunosuppressive therapy, in order to predict long-term therapeutic response in patients with idiopathic inflammatory myopathies (IIM). METHODS: Muscle biopsies were selected from 13 new onset patients, six responders and seven non-responders. Repeated muscle biopsies after a median of 11 months follow-up were available from 9 patients and paired peripheral blood mononuclear cells (PBMCs) from 5 patients. Treatment response after 3 years was defined by MMT-8 measuring muscle strength and the ACR/EULAR 2016 improvement criteria. Frozen biopsy sections were immunohistochemically stained for expression of CD3, CD66b, IL-15, CD68, CD163 and myosin heavy chain neonatal (MHCn). PBMCs were analysed by flow cytometry for monocyte phenotypes (CD14, CD16, CD68, CX3CR1, and CCR2). RESULTS: Before treatment there were no significant differences in any clinical or muscle biopsy variables or monocyte subsets between responders and non-responders. MMT-8 was significantly higher compared to baseline in the responders at 3-year follow-up. In responders the expression of CD68 in the repeated biopsies was significantly lower compared to non-responders (p<0.05). CONCLUSIONS: Baseline biopsy, monocyte profile or clinical data did not predict long-term treatment response, but in the repeated biopsy within 1 year of immunosuppressive treatment, the lower number of macrophages (CD68+) seemed to predict a more favourable long-term clinical response with regard to improved muscle strength.

Sex-Specific Effects of Microglia-Like Cell Engraftment during Experimental Autoimmune Encephalomyelitis.

Multiple sclerosis (MS) is a chronic neuroinflammatory disorder of the central nervous system (CNS) that usually presents in young adults and predominantly in females. Microglia, a major resident immune cell in the CNS, are critical players in both CNS homeostasis and disease. We have previously demonstrated that microglia can be efficiently depleted by the administration of tamoxifen in Cx3cr1CreER/+Rosa26DTA/+ mice, with ensuing repopulation deriving from both the proliferation of residual CNS resident microglia and the engraftment of peripheral monocyte-derived microglia-like cells. In this study, tamoxifen was administered to Cx3cr1CreER/+Rosa26DTA/+ and Cx3cr1CreER/+ female and male mice. Experimental autoimmune encephalomyelitis (EAE), a widely used animal model of MS, was induced by active immunization with myelin oligodendrocyte glycoprotein (MOG) one month after tamoxifen injections in Cx3cr1CreER/+Rosa26DTA/+ mice and Cx3cr1CreER/+ mice, a time point when the CNS niche was colonized by microglia derived from both CNS microglia and peripherally-derived macrophages. We demonstrate that engraftment of microglia-like cells following microglial depletion exacerbated EAE in Cx3cr1CreER/+Rosa26DTA/+ female mice as assessed by clinical symptoms and the expression of CNS inflammatory factors, but these findings were not evident in male mice. Higher major histocompatibility complex class II expression and cytokine production in the female CNS contributed to the sex-dependent EAE severity in mice following engraftment of microglia-like cells. An underestimated yet marked sex-dependent microglial activation pattern may exist in the injured CNS during EAE.

Underestimated Peripheral Effects Following Pharmacological and Conditional Genetic Microglial Depletion.

Microglia, predominant parenchymal resident macrophages in the central nervous system (CNS), are crucial players in neurodevelopment and CNS homeostasis. In disease conditions, pro-inflammatory microglia predominate over their regulatory counterparts, and are thus a potential immunotherapeutic target. It has been well documented that microglia can be effectively depleted using both conditional genetic Cx3cr1Cre-diphtheria toxin receptor (DTR)/diphtheria toxin subunit A (DTA) animal models and pharmacological colony-stimulating factor 1 receptor (CSF1R) inhibitors. Recent advances using these approaches have expanded our knowledge of the multitude of tasks conducted by microglia in both homeostasis and diseases. Importantly, experimental microglial depletion has been proven to exert neuroprotective effects in an increasing number of disease models, mostly explained by reduced neuroinflammation. However, the comprehensive effects of additional targets such as circulating monocytes and peripheral tissue macrophages during microglial depletion periods have not been investigated widely, and for those studies addressing the issue the conclusions are mixed. In this study, we demonstrate that experimental microglial depletion using both Cx3cr1CreER/+Rosa26DTA/+ mice and different doses of CSF1R inhibitor PLX3397 exert crucial influences on circulating monocytes and peripheral tissue macrophages. Our results suggest that effects on peripheral immunity should be considered both in interpretation of microglial depletion studies, and especially in the potential translation of microglial depletion and replacement therapies.

Enforced microglial depletion and repopulation as a promising strategy for the treatment of neurological disorders.

Microglia are prominent immune cells in the central nervous system (CNS) and are critical players in both neurological development and homeostasis, and in neurological diseases when dysfunctional. Our previous understanding of the phenotypes and functions of microglia has been greatly extended by a dearth of recent investigations. Distinct genetically defined subsets of microglia are now recognized to perform their own independent functions in specific conditions. The molecular profiling of single microglial cells indicates extensively heterogeneous reactions in different neurological disorders, resulting in multiple potentials for crosstalk with other kinds of CNS cells such as astrocytes and neurons. In settings of neurological diseases it could thus be prudent to establish effective cell-based therapies by targeting entire microglial networks. Notably, activated microglial depletion through genetic targeting or pharmacological therapies within a suitable time window can stimulate replenishment of the CNS niche with new microglia. Additionally, enforced repopulation through provision of replacement cells also represents a potential means of exchanging dysfunctional with functional microglia. In each setting the newly repopulated microglia might have the potential to resolve ongoing neuroinflammation. In this review, we aim to summarize the most recent knowledge of microglia and to highlight microglial depletion and subsequent repopulation as a promising cell replacement therapy. Although glial cell replacement therapy is still in its infancy and future translational studies are still required, the approach is scientifically sound and provides new optimism for managing the neurotoxicity and neuroinflammation induced by activated microglia.

Establishing a Proteomics-Based Monocyte Assay To Assess Differential Innate Immune Activation Responses.

Innate immune cells are complex systems that can be simultaneously activated in a variety of ways. Common methods currently used to estimate the response of innate immune cells to stimuli are usually biased toward a single mode of activation. The aim of this study was to assess the possibility of designing an assay based on unbiased proteome analysis that would be capable of predicting the complex response of the innate immune system to various challenges. Monocytes were used as representative cells of the innate immune system. The underlying hypothesis was that their proteome response to different activating molecules would reflect the immunogenicity of these molecules. To identify the main modes of response, we treated the human monocytic THP-1 cell line with nine different stimuli. Differentiation and activation were determined to be the two major modes of monocyte response, with PMA causing the strongest differentiation and Pam3CSK4 causing the strongest proinflammatory activation. The established assay was applied to characterize the monocyte response to epidermal growth factor peptide containing isoaspartate, which induced differentiation but not proinflammatory activation. Because of its versatility, robustness, and specificity, this new assay is likely to find a niche among the more established immunological methods.

TGFß regulates persistent neuroinflammation by controlling Th1 polarization and ROS production via monocyte-derived dendritic cells.

Intracerebral levels of Transforming Growth Factor beta (TGFß) rise rapidly during the onset of experimental autoimmune encephalomyelitis (EAE), a mouse model of Multiple Sclerosis (MS). We addressed the role of TGFß responsiveness in EAE by targeting the TGFß receptor in myeloid cells, determining that Tgfbr2 was specifically targeted in monocyte-derived dendritic cells (moDCs) but not in CNS resident microglia by using bone-marrow chimeric mice. TGFß responsiveness in moDCs was necessary for the remission phase since LysM(Cre) Tgfbr2(fl/fl) mice developed a chronic form of EAE characterized by severe demyelination and extensive infiltration of activated moDCs in the CNS. Tgfbr2 deficiency resulted in increased moDC IL-12 secretion that skewed T cells to produce IFN-γ, which in turn enhanced the production of moDC-derived reactive oxygen species that promote oxidative damage and demyelination. We identified SNPs in the human NOX2 (CYBB) gene that associated with the severity of MS, and significantly increased CYBB expression was recorded in PBMCs from both MS patients and from MS severity risk allele rs72619425-A carrying individuals. We thus identify a novel myeloid cell-T cell activation loop active in the CNS during chronic disease that could be therapeutically targeted. GLIA 2016;64:1925-1937.

Human macrophages induce CD4(+)Foxp3(+) regulatory T cells via binding and re-release of TGF-ß.

While pro-inflammatory immune responses are a requirement to combat microbes, uncontrolled self-directed inflammatory immune responses are the hallmark of autoimmune diseases. Restoration of immunological tolerance involves both suppression of ongoing tissue-destructive immune responses and re-education of the host immune system. Both functionally immunosuppressive macrophages (M2) and regulatory T cells (Tregs) are implicated in these processes. Their mutual interaction is synergistic in this context and adoptive transfer of each cell type has been functioning as immunotherapy in experimental models, being particularly effective when using M2 macrophages generated with an optimized interleukin-4 (IL-4)/interleukin-10 (IL-10)/transforming growth factor-ß (TGF-ß) combination. As a prerequisite for eventual translation of M2 therapy into clinical settings we herein studied the induction, stability and mechanism of generation of human induced Tregs (iTregs) by M2 macrophages generated with IL-4/IL-10/TGF-ß. The supernatants of monocyte-derived human M2 macrophages robustly induced FOXP3 and other Treg signature molecules such as CTLA-4 and IKZF4 in human naïve CD4 T cells. M2-induced iTregs displayed enhanced FOXP3 stability and low expression of pro-inflammatory cytokines interferon-γ and IL-17, as well as functional immunosuppressive activity compared with control T cells. The FOXP3-inducing activity was dependent on TGF-ß, which was both expressed and captured with re-release by M2 macrophages into the soluble supernatant fraction, in which the TGF-ß was not confined to extracellular vesicles such as exosomes. We propose that adoptive transfer of human M2 macrophages may be exploited in the future to induce Tregs in situ by delivering TGF-ß, which could be developed as a therapeutic strategy to target autoimmune and other inflammatory diseases.

Unbiased expression mapping identifies a link between the complement and cholinergic systems in the rat central nervous system.

The complement system is activated in a wide spectrum of CNS diseases and is suggested to play a role in degenerative phenomena such as elimination of synaptic terminals. Still, little is known of mechanisms regulating complement activation in the CNS. Loss of synaptic terminals in the spinal cord after an experimental nerve injury is increased in the inbred DA strain compared with the PVG strain and is associated with expression of the upstream complement components C1q and C3, in the absence of membrane attack complex activation and neutrophil infiltration. To further dissect pathways regulating complement expression, we performed genome-wide expression profiling and linkage analysis in a large F2(DA × PVG) intercross, which identified quantitative trait loci regulating expression of C1qa, C1qb, C3, and C9. Unlike C1qa, C1qb, and C9, which all displayed distinct coregulation with different cis-regulated C-type lectins, C3 was regulated in a coexpression network immediately downstream of butyrylcholinesterase. Butyrylcholinesterase hydrolyses acetylcholine, which exerts immunoregulatory effects partly through TNF-α pathways. Accordingly, increased C3, but not C1q, expression was demonstrated in rat and mouse glia following TNF-α stimulation, which was abrogated in a dose-dependent manner by acetylcholine. These findings demonstrate new pathways regulating CNS complement expression using unbiased mapping in an experimental in vivo system. A direct link between cholinergic activity and complement activation is supported by in vitro experiments. The identification of distinct pathways subjected to regulation by naturally occurring genetic variability is of relevance for the understanding of disease mechanisms in neurologic conditions characterized by neuronal injury and complement activation.

Proteomics Reveals a Role for Attachment in Monocyte Differentiation into Efficient Proinflammatory Macrophages.

Monocytes are blood-borne cells of the innate immune system. They can be differentiated and activated into proinflammatory macrophages that might be employed in tumor immune therapy. Monocyte exposure to lipopolysaccharide (LPS) is a standard method to induce a proinflammatory macrophage state, with the resultant population comprising both adherent and nonadherent cells. In the current study, we aimed to identify the differences in proteomes of these monocyte subpopulations, which addresses a more general question about the role of attachment in monocyte differentiation. Label-free proteomics of a model of human monocytes (THP-1 cell line) revealed that the cells remaining in suspension upon LPS treatment were activated by cytokines and primed for rapid responsiveness to pathogens. In terms of proteome change, the adhesion process was orthogonal to activation. Adherent cells exhibited signs of differentiation and enhanced innate immune responsivity, being closer to macrophages. These findings indicate that adherent, LPS-treated cells would be more appropriate for use in tumor therapeutic applications.

Abrogation of adenosine A1 receptor signalling improves metabolic regulation in mice by modulating oxidative stress and inflammatory responses.

AIMS/HYPOTHESIS: Adenosine is an important regulator of metabolism; however, the role of the A1 receptor during ageing and obesity is unclear. The aim of this study was to investigate the effects of A1 signalling in modulating metabolic function during ageing. METHODS: Age-matched young and aged A 1 (also known as Adora1)-knockout (A1(-/-)) and wild-type (A1(+/+)) mice were used. Metabolic regulation was evaluated by body composition, and glucose and insulin tolerance tests. Isolated islets and islet arterioles were used to detect islet endocrine and vascular function. Oxidative stress and inflammation status were measured in metabolic organs and systemically. RESULTS: Advanced age was associated with both reduced glucose clearance and insulin sensitivity, as well as increased visceral adipose tissue (VAT) in A1(+/+) compared with A1(-/-) mice. Islet morphology and insulin content were similar between genotypes, but relative changes in in vitro insulin release following glucose stimulation were reduced in aged A1(+/+) compared with A1(-/-) mice. Islet arteriolar responses to angiotensin II were stronger in aged A1(+/+) mice, this being associated with increased NADPH oxidase activity. Ageing resulted in multiple changes in A1(+/+) compared with A1(-/-) mice, including enhanced NADPH oxidase-derived O2(-) formation and NADPH oxidase isoform 2 (Nox2) protein expression in pancreas and VAT; elevated levels of circulating insulin, leptin and proinflammatory cytokines (TNF-α, IL-1ß, IL-6 and IL-12); and accumulation of CD4(+) T cells in VAT. This was associated with impaired insulin signalling in VAT from aged A1(+/+) mice. CONCLUSIONS/INTERPRETATION: These studies emphasise that A1 receptors regulate metabolism and islet endocrine and vascular functions during ageing, including via the modulation of oxidative stress and inflammatory responses, among other things.

Adoptive transfer of cytokine-induced immunomodulatory adult microglia attenuates experimental autoimmune encephalomyelitis in DBA/1 mice.

Microglia are resident antigen-presenting cells in the central nervous system (CNS) that either suppress or promote disease depending on their activation phenotype and the microenvironment. Multiple sclerosis (MS) is a chronic inflammatory disease causing demyelination and nerve loss in the CNS, and experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate pathogenic mechanisms and therapeutic effects. We isolated and cultured microglia from adult mouse brains and exposed them to specific combinations of stimulatory molecules and cytokines, the combination of IL-4, IL-10, and TGF-ß yielding the optimal regime for induction of an immunosuppressive phenotype (M2). M2 microglia were characterized by decreased expression or production of CD86, PD-L1, nitric oxide, and IL-6, increased expression of PD-L2, and having a potent capacity to retain their phenotype on secondary proinflammatory stimulation. M2 microglia induced regulatory T cells, suppressed T-cell proliferation, and downmodulated M1-associated receptor expression in M1 macrophages. Myelin oligodendrocyte glycoprotein (MOG)-induced EAE was induced in DBA/1 mice and at different time points (0, 5, 12, or 15 days postimmunization) 3 × 105 M2 microglia were transferred intranasally. A single transfer of M2 microglia attenuated the severity of established EAE, which was particularly obvious when the cells were injected at 15 days postimmunization. M2 microglia-treated mice had reduced inflammatory responses and less demyelination in the CNS. Our findings demonstrate that adult M2 microglia therapy represents a novel intervention that alleviated established EAE and that this therapeutic principle may have relevance for treatment of MS patients.

Variability in C-type lectin receptors regulates neuropathic pain-like behavior after peripheral nerve injury.

INTRODUCTION: Neuropathic pain is believed to be influenced in part by inflammatory processes. In this study we examined the effect of variability in the C-type lectin gene cluster (Aplec) on the development of neuropathic pain-like behavior after ligation of the L5 spinal nerve in the inbred DA and the congenic Aplec strains, which carries seven C-type lectin genes originating from the PVG strain. RESULTS: While both strains displayed neuropathic pain behavior early after injury, the Aplec strain remained sensitive throughout the whole study period. Analyses of several mRNA transcripts revealed that the expression of Interleukin-1ß, Substance P and Cathepsin S were more up-regulated in the dorsal part of the spinal cord of Aplec rats compared to DA, indicating a stronger inflammatory response. This notion was supported by flow cytometric analysis revealing increased infiltration of activated macrophages into the spinal cord. In addition, macrophages from the Aplec strain stimulated in vitro displayed higher expression of inflammatory cytokines compared to DA cells. Finally, we bred a recombinant congenic strain (R11R6) comprising only four of the seven Aplec genes, which displayed similar clinical and immune phenotypes as the Aplec strain. CONCLUSION: We here for the first time demonstrate that C-type lectins, a family of innate immune receptors with largely unknown functions in the nervous system, are involved in regulation of inflammation and development of neuropathic pain behavior after nerve injury. Further experimental and clinical studies are needed to dissect the underlying mechanisms more in detail as well as any possible relevance for human conditions.

Clodronate liposomes reduce excessive scar formation in a mouse model of burn injury by reducing collagen deposition and TGF-ß1 expression.

Clodronate liposome injection is an effective approach to selectively and specifically depleting macrophages. Macrophages play a crucial role in cutaneous wound healing and are associated with excessive scar formation. Use of clodronate liposomes to enhance cutaneous wound healing and reduce scar formation could represent a major advance in wound therapy and hypertrophic scar treatment. This study aimed to investigate the effects of subcutaneous or intraperitoneal injection of clodronate liposomes on cutaneous wound healing and scar formation. A burn injury mouse model was used. Mice were treated with subcutaneous or intraperitoneal injection of clodronate liposomes. Wound healing time was analyzed and scar tissues were harvested for hematoxylin and eosin (HE) staining, reverse transcription polymerase chain reaction (RT-PCR) and Western blot analyses. Wound healing time in treated mice was extended. HE showed that the basal layer of the epidermis in treated scars was flattened, the dermis layer was not significantly thickened, and collagen fibers were well arranged, with few cells and micro vessels. RT-PCR and Western blot analyses showed that the levels of TGF-ß1 and collagen I-α2 were decreased in treated mice. Clodronate liposomes reduce excessive scar formation and delay cutaneous wound healing possibly by reducing collagen deposition and macrophage-derived TGF-ß1 expression.

Genetic variability in the rat Aplec C-type lectin gene cluster regulates lymphocyte trafficking and motor neuron survival after traumatic nerve root injury.

BACKGROUND: C-type lectin (CLEC) receptors are important for initiating and shaping immune responses; however, their role in inflammatory reactions in the central nervous system after traumatic injuries is not known. The antigen-presenting lectin-like receptor gene complex (Aplec) contains a few CLEC genes, which differ genetically among inbred rat strains. It was originally thought to be a region that regulates susceptibility to autoimmune arthritis, autoimmune neuroinflammation and infection. METHODS: The inbred rat strains DA and PVG differ substantially in degree of spinal cord motor neuron death following ventral root avulsion (VRA), which is a reproducible model of localized nerve root injury. A large F2 (DAxPVG) intercross was bred and genotyped after which global expressional profiling was performed on spinal cords from F2 rats subjected to VRA. A congenic strain, Aplec, created by transferring a small PVG segment containing only seven genes, all C-type lectins, ontoDA background, was used for further experiments together with the parental strains. RESULTS: Global expressional profiling of F2 (DAxPVG) spinal cords after VRA and genome-wide eQTL mapping identified a strong cis-regulated difference in the expression of Clec4a3 (Dcir3), a C-type lectin gene that is a part of the Aplec cluster. Second, we demonstrate significantly improved motor neuron survival and also increased T-cell infiltration into the spinal cord of congenic rats carrying Aplec from PVG on DA background compared to the parental DA strain. In vitro studies demonstrate that the Aplec genes are expressed on microglia and upregulated upon inflammatory stimuli. However, there were no differences in expression of general microglial activation markers between Aplec and parental DA rats, suggesting that the Aplec genes are involved in the signaling events rather than the primary activation of microglia occurring upon nerve root injury. CONCLUSIONS: In summary, we demonstrate that a genetic variation in Aplec occurring among inbred strains regulates both survival of axotomized motor neurons and the degree of lymphocyte infiltration. These results demonstrate a hitherto unknown role for CLECs for intercellular communication that occurs after damage to the nervous system, which is relevant for neuronal survival.

DNA aptamers that target human glioblastoma multiforme cells overexpressing epidermal growth factor receptor variant III in vitro.

AIM: Aptamers are oligonucleic acid or peptide molecules that bind to a specific target molecule in cells, thus may act as effective vehicles for drug or siRNA delivery. In this study we investigated the DNA aptamers that target human glioblastoma multiforme (GBM) cells overexpressing epidermal growth factor receptor variant III (EGFRvIII), which was linked to radiation and chemotherapeutic resistance of this most aggressive brain tumor. METHODS: A 73-mer ssDNA library containing molecules with 30 nt of random sequence flanked by two primer hybridization sites was chosen as the initial library. Cell systematic evolution of ligands by exponential enrichment (Cell-SELEX) method was used to select the DNA aptamers that target EGFRvIII. The binding affinity of the aptamers was measured using a cell-based biotin-avidin ELISA. RESULTS: After 14 rounds of selection, four DNA aptamers (32, 41, 43, and 47) that specifically bound to the EGFRvIII-overexpressing human glioma U87Δ cells with Kd values of less than 100 nmol/L were discovered. These aptamers were able to distinguish the U87Δ cells from the negative control human glioma U87MG cells and HEK293 cells. Aptamer 32 specifically bound to the EGFRvIII protein with an affinity similar to the EGFR antibody (Kd values of aptamer 32 and the EGFR antibody were 0.62±0.04 and 0.32±0.01 nmol/L, respectively), and this aptamer was localized in the cell nucleus. CONCLUSION: The DNA aptamers are promising molecular probes for the diagnosis and treatment of GBM.

[Effect of protein on the detection of antipsychotic drug residues in human serum and its elimination].

Using pure human serum albumin (HSA) as the model protein, the effects of protein on the extraction of antipsychotic drugs (APDs: diazepam, chlorpromazine hydrochloride, and perphenazine) in human serum sample were studied. The present paper investigated the interaction between APDs and HSA by fluorescence spectrometry in detail The influences of different ethanol concentration solution on protein denaturation were studied by Rayleigh scattering. The results showed that APDs strongly bound with HSA. In the phi (ethanol) 80% extracting solution, a slow but full protein denaturation takes place, which causes the unfolding of protein and the dissociation of drugs. Then K2 HPO4 was added into the extracting solution to form aqueous two-phase system, and meanwhile the drug residues were extracted into upper phase with high extraction efficiencies. After filtration, the upper phase was ready for analysis of drug residues by HPLC system. The detection limits were in the range of 18.8-38.4 ng x mL(-1), and the spiked recovery was 94.2%-98.7% for determination of antipsychotic drugs in human serum. The method is efficient, solvent-saving, environment-friendly, and accurate.