Extending the Coding Potential of Viral Genomes with Overlapping Antisense ORFs: A Case for the De Novo Creation of the Gene Encoding the Antisense Protein ASP of HIV-1

Gene overprinting occurs when point mutations within a genomic region with an existing coding sequence create a new one in another reading frame. This process is quite frequent in viral genomes either to maximize the amount of information that they encode or in response to strong selective pressure. The most frequent scenario involves two different reading frames in the same DNA strand (sense overlap). Much less frequent are cases of overlapping genes that are encoded on opposite DNA strands (antisense overlap). One such example is the antisense ORF, asp in the minus strand of the HIV-1 genome overlapping the env gene. The asp gene is highly conserved in pandemic HIV-1 strains of group M, and it is absent in non-pandemic HIV-1 groups, HIV-2, and lentiviruses infecting non-human primates, suggesting that the ~190-amino acid protein that is expressed from this gene (ASP) may play a role in virus spread. While the function of ASP in the virus life cycle remains to be elucidated, mounting evidence from several research groups indicates that ASP is expressed in vivo. There are two alternative hypotheses that could be envisioned to explain the origin of the asp ORF. On one hand, asp may have originally been present in the ancestor of contemporary lentiviruses, and subsequently lost in all descendants except for most HIV-1 strains of group M due to selective advantage. Alternatively, the asp ORF may have originated very recently with the emergence of group M HIV-1 strains from SIVcpz. Here, we used a combination of computational and statistical approaches to study the genomic region of env in primate lentiviruses to shed light on the origin, structure, and sequence evolution of the asp ORF. The results emerging from our studies support the hypothesis of a recent de novo addition of the antisense ORF to the HIV-1 genome through a process that entailed progressive removal of existing internal stop codons from SIV strains to HIV-1 strains of group M, and fine tuning of the codon sequence in env that reduced the chances of new stop codons occurring in asp. Altogether, the study supports the notion that the HIV-1 asp gene encodes an accessory protein, providing a selective advantage to the virus.

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Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5679-5687.1990 ◽

1990 ◽

Vol 10 (11) ◽

pp. 5679-5687

Author(s):

C K Barlowe ◽

D R Appling

Keyword(s):

Saccharomyces Cerevisiae ◽

De Novo ◽

Point Mutations ◽

Gene Replacement ◽

Molecular Genetic Analysis ◽

Yeast Cells ◽

Yeast Saccharomyces Cerevisiae ◽

Purine Synthesis

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

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IDH1 Splicing Alterations in Patients with AML and Their Relationship to Blood 2HG Levels

Blood ◽

10.1182/blood.v124.21.1060.1060 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1060-1060

Author(s):

Sophia Adamia ◽

Patricia Toniolo ◽

Amir T. Fathi ◽

Ilene Galinsky ◽

Suiyang Liu ◽

...

Keyword(s):

Splice Variant ◽

Splice Variants ◽

Point Mutations ◽

Advisory Board ◽

Blood Levels ◽

Sequencing Analysis ◽

Isocitrate Dehydrogenase 1 ◽

Nested Polymerase Chain Reaction ◽

Stop Codons

Abstract Introduction: Point mutations in the human cytosolic isocitrate dehydrogenase 1 (IDH1) gene, or its mitochondrial homolog IDH2 gene, are frequent in patients with AML, particularly at residues R132 in IDH1 and, R140 or R172 in IDH2. These mutations cause loss of the enzymeÕs ability to catalyze the conversion of isocitrate to α-ketoglutarate and result in neomorphic production of R(-)-2-hydroxyglutarate (2HG). It has been reported that 2HG functions as an ÒoncometaboliteÓ. Interestingly, serum levels of 2HG are highly variable among patients with the same mutations, and occasionally are elevated even in patients without detectable mutations. Previously Chaturvedi et al (ASH 2011, Abstract) showed that an IDH1 splice variant promotes leukemogenesis in vivo in the absence of IDH1/IDH2 mutations. In this study, we examined the frequency of this splice variant and two other IDH1 novel splice variants in patients with AML, and looked for any relationships between splice variants, mutations, and 2HG levels. Methods: Cloning and sequencing analysis were used to identify IDH1 and IDH2 splice variants. DNA fragment analysis and capillary electrophoresis was used to evaluate expression of splice variants in 242 patients, of which 166 patients had 2HG blood levels measured. As controls we used two CD34+ bone marrow (BM) cells, three un-fractionated BM cells, and purified neutrophil and monocytes from three peripheral blood samples (PB) of normal donors. To detect point mutations, DNA fragments spanning exon 4 of IDH1 and IDH2 were amplified by nested polymerase chain reaction, and then PCR products were directly sequenced by Sanger sequencing. Results: We studied three splice variants of IDH1: IDH1Va (reported previously), and two variants discovered in the course of this study, IDH1Vb and IDH1Vc. Alignment analyses of these splice variants indicated that the IDH1Vb variant transcripts do not cause a frame shift, and retain native start and stop codons, while IDH1Va and IDH1Vc transcripts have frame shifts that create premature stop codons in the last exon. No splice variants of IDH2 were identified in these patient samples. Of the 242 AML samples (PB-166, BM-76) 51% expressed IDH1Va, 49% expressed IDH1Vb and 23% expressed IDHVc, almost always in combination with full length of IDH1 transcripts (IDH1FL). These IDH1 splice variants were not detected in the normal cells analyzed. We compared expression of IDH1 splice variants, presence of common point mutations, and levels of 2-HG in the 166 patients (Table 1). As expected, patients with point mutations in IDH1 or IDH2 generally had high levels of 2HG in their blood (> average 6104 ng/ml). Seven patients had detectable IDH1/IDH2 point mutations but low 2-HG (< average 222 ng/ml) blood levels. In these patients IDH1Va and IDH1Vb splice variants were detected frequently; Five of 7 patients expressed these variants together and/or in combination with the IDH1-FL mRNA. In contrast, 14 patients had no detectable IDH1/IDH2 mutations but elevated (> average 4430 ng/ml) 2-HG blood levels, twelve of these overexpressed the IDH1Vb splice variant transcripts (Table 1). Six of these 12 patients expressed only IDH1Vb transcripts together with IDH1FL; the average 2HG blood levels in these patients was 2875ng/ml. Conclusion: Though high 2-HG blood levels are correlated with the presence of IDH1/IDH2 mutations, the degree of 2-HG elevation may be altered by other events such as alternative splicing. The expression of IDH1 splice variants was commonly detected in AML samples. Studies to examine the effects of splice variants of IDH1 on 2HG production and transformation are underway. Figure 1 Figure 1. Disclosures Fathi: Millennium: Research Funding; Seattle Genetics: Advisory Board, Advisory Board Other; Agios: Advisory Board, Advisory Board Other. Yen:Agios: Employment. Kim:Agios: Employment. Zhu:Agios Pharmaceuticals: Employment, Stockholder Other. Kim:Yes, same as for being Author of Abstract #70656 Hide Full Conflict-of-Interest Disclosure I have relevant financial relationship(s) to disclose. Yes Name of Organization Type of relationship Agios Pharmaceuticals: Employment. Steensma:Novartis: Consultancy; Celgene: Consultancy; Ariad: Equity Ownership; Amgen: Consultancy. Stone:Agios: Consultancy.

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The likelihood of heterogeneity or additional mutation in KRAS or associated oncogenes to compromise targeting of oncogenic KRAS G12C

10.1101/149724 ◽

2017 ◽

Author(s):

Vincent L. Cannataro ◽

Stephen G. Gaffney ◽

Carly Stender ◽

Zi-Ming Zhao ◽

Mark Philips ◽

...

Keyword(s):

Targeted Therapy ◽

De Novo ◽

Point Mutations ◽

Treatment Resistance ◽

Braf V600e ◽

Intratumor Heterogeneity ◽

Resistance Mutations ◽

Ras Genes ◽

Fitness Advantage

AbstractActivating mutations in RAS genes are associated with approximately 20% of all human cancers. New targeted therapies show preclinical promise in inhibiting the KRAS G12C variant, however, concerns exist regarding the effectiveness of such therapies in vivo given the possibilities of existing intratumor heterogeneity or de novo mutation leading to treatment resistance. We performed deep sequencing of 27 KRAS G12 positive lung tumors and found no evidence of other oncogenic mutations within KRAS or within commonly mutated downstream genes that could confer resistance at the time of treatment. Furthermore, we estimate the de novo mutation rate in KRAS position 12 and in genes downstream of KRAS. We find that mutations that confer resistance are about as likely to occur downstream of KRAS as within KRAS. Moreover, we present an approach for estimation of the selection intensity for these point mutations that explains their high prevalence in tumors. Our approach predicts that BRAF V600E would provide the highest fitness advantage for de novo resistant subclones. Overall, our findings suggest that resistance to targeted therapy of KRAS G12C positive tumors is unlikely to be present at the time of treatment and, among the de novo mutations likely to confer resistance, mutations in BRAF, a gene with targeted inhibitors presently available, result in subclones with the highest fitness advantage.One Sentence SummaryMutations conferring resistance to KRAS G12C targeted therapy are unlikely to be present at the time of resection, and the likely mechanisms of evolved resistance are predicted be ones that are responsive to therapies that are in development or that are already available.

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Convergent evolution as an indicator for selection during acute HIV-1 infection

10.1101/168260 ◽

2017 ◽

Cited By ~ 1

Author(s):

Frederic Bertels ◽

Karin J. Metzner ◽

Roland Regoes

Keyword(s):

Convergent Evolution ◽

Adaptive Immune System ◽

Purifying Selection ◽

Selective Advantage ◽

Single Individual ◽

Viral Genomes ◽

Acute Hiv ◽

Different Populations ◽

Neutral Models ◽

Hiv 1

AbstractConvergent evolution describes the process of different populations acquiring similar phenotypes or genotypes. Complex organisms with large genomes only rarely and only under very strong selection converge to the same genotype. In contrast, independent virus populations with very small genomes often acquire identical mutations. Here we test the hypothesis of whether convergence in early HIV-1 infection is common enough to serve as an indicator for selection. To this end, we measure the number of convergent mutations in a well-studied dataset of full-length HIV-1envgenes sampled from HIV-1 infected individuals during early infection. We compare this data to a neutral model and find an excess of convergent mutations. Convergent mutations are not evenly distributed across the env gene, but more likely to occur in gp41, which suggests that convergent mutations provide a selective advantage and hence are positively selected. In contrast, mutations that are only found in an HIV-1 population of a single individual are significantly affected by purifying selection. Our analysis suggests that comparisons between convergent and private mutations with neutral models allow us to identify positive and negative selection in small viral genomes. Our results also show that selection significantly shapes HIV-1 populations even before the onset of the adaptive immune system.

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Rodent Cells Support Key Functions of the Human Immunodeficiency Virus Type 1 Pathogenicity Factor Nef

Journal of Virology ◽

10.1128/jvi.79.3.1655-1665.2005 ◽

2005 ◽

Vol 79 (3) ◽

pp. 1655-1665 ◽

Cited By ~ 35

Author(s):

Oliver T. Keppler ◽

Ina Allespach ◽

Lismarie Schüller ◽

David Fenard ◽

Warner C. Greene ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

De Novo ◽

Small Animal ◽

Transgenic Rat ◽

Host Determinants ◽

Immunodeficiency Virus ◽

Key Factor ◽

Hiv 1

ABSTRACT After infection with human immunodeficiency virus (HIV), progression toward immunodeficiency is governed by a complex interplay of viral and host determinants. The viral accessory protein Nef is a key factor for the development of AIDS. Strains of HIV and simian immunodeficiency virus that lack functional nef genes either do not induce AIDS or do so only after a significant delay. The validity of a transgenic-small-animal model for de novo infection by HIV will depend on its ability to recapitulate the actions of critical factors of viral pathogenicity, such as Nef. We assessed the ability of rat, mouse, and hamster cells to support key effector functions of Nef. In cell lines from rodents, the subcellular distribution of wild-type HIV type 1 strain SF2 Nef and mutants was comparable to that in human cells. Nef downregulated human CD4 from the cell surface, was associated with p21-activated kinase activity, and enhanced the infectivity of HIV-1 virions. Importantly, these Nef-induced effects, as well as the downregulation of rat CD4 and major histocompatibility complex class I molecules, could also be demonstrated in primary T lymphocytes and macrophages from human CD4-transgenic rats. Thus, HIV-1 Nef exerts key functions in rodent cells. In line with our ongoing efforts to establish a transgenic-rat model of HIV disease, these results indicate that important aspects of viral pathogenesis could be addressed in a transgenic-rodent model permissive for de novo infection and that such a model would be valuable for evaluating the function of Nef in vivo.

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Potential Impact of Fluopyram on the Frequency of the D123E Mutation in Alternaria solani

Plant Disease ◽

10.1094/pdis-06-17-0853-re ◽

2018 ◽

Vol 102 (3) ◽

pp. 656-665 ◽

Cited By ~ 4

Author(s):

Mitchell J. Bauske ◽

S. K. R. Yellareddygari ◽

Neil C. Gudmestad

Keyword(s):

Disease Control ◽

Solanum Tuberosum L ◽

Management Strategies ◽

Point Mutations ◽

Fungal Growth ◽

Alternaria Solani ◽

Selective Advantage ◽

Field Trials

Succinate dehydrogenase-inhibiting (SDHI) fungicides have been widely applied in commercial potato (Solanum tuberosum L.) fields for the control of early blight, caused by Alternaria solani Sorauer. Five-point mutations on three AsSdh genes in A. solani have been identified as conferring resistance to SDHI fungicides. Recent work in our laboratory determined that A. solani isolates possessing the D123E mutation, or the substitution of aspartic acid for glutamic acid at position 123 in the AsSdhD gene, were collected at successively higher frequencies throughout a 3-year survey. In total, 118 A. solani isolates previously characterized as possessing the D123E mutation were evaluated in vitro for boscalid and fluopyram sensitivity. Over 80% of A. solani isolates with the D123E mutation evaluated were determined to be highly resistant to boscalid in vitro. However, effective concentration at which the fungal growth is inhibited by 50% values of isolates with the D123E mutation to fluopyram, ranging from 0.2 to 3 µg/ml, were sensitive and only slightly higher than those of baseline isolates to fluopyram, which ranged from 0.1 to 0.6 µg/ml. Five A. solani isolates with the D123E mutation were further evaluated in vivo for percent disease control obtained from boscalid and fluopyram compared with two wild-type isolates, three isolates possessing the F129L mutation, two isolates possessing the H134R mutation, two isolates possessing the H133R mutation, and one isolate with the H278R mutation. Relative area under the dose response curve values for boscalid and fluopyram were significantly lower for all five D123E-mutant isolates, demonstrating reduced disease control in vivo. In field trials, the frequency of A. solani isolates with the D123E mutation recovered from treatments receiving an in-furrow application of fluopyram ranged from 5 to 37%, which was significantly higher compared with treatments receiving foliar applications of standard protectants, in which the frequency of the D123E mutation in isolates ranged from 0 to 2.5%. Results suggest that A. solani isolates possessing the D123E mutation have a selective advantage under the application of fluopyram compared with SDHI-sensitive isolates, as well as isolates possessing other mutations conferring SDHI resistance. These data illustrate the importance of implementing fungicide resistance management strategies and cautions the use of fluopyram for in-furrow applications that target other pathogens of potato.

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The Per-1 Short Isoform Inhibits de novo HIV-1 Transcription in Resting CD4+ T-cells

Current HIV Research ◽

10.2174/1570162x17666190218145048 ◽

2019 ◽

Vol 16 (6) ◽

pp. 384-395 ◽

Cited By ~ 3

Author(s):

Li Zhao ◽

Mei Liu ◽

Jiayue Ouyang ◽

Zheming Zhu ◽

Wenqing Geng ◽

...

Keyword(s):

T Cells ◽

Cd4 T Cells ◽

De Novo ◽

Inhibitory Effect ◽

Negative Regulator ◽

Viral Loads ◽

Transcription Blockage ◽

Negative Factors ◽

Hiv 1

Background: Understanding of the restriction of HIV-1 transcription in resting CD4+ Tcells is critical to find a cure for AIDS. Although many negative factors causing HIV-1 transcription blockage in resting CD4+ T-cells have been found, there are still unknown mechanisms to explore. Objective: To explore the mechanism for the suppression of de novo HIV-1 transcription in resting CD4+ T-cells. Methods: In this study, a short isoform of Per-1 expression plasmid was transfected into 293T cells with or without Tat's presence to identify Per-1 as a negative regulator for HIV-1 transcription. Silencing of Per-1 was conducted in resting CD4+ T-cells or monocyte-derived macrophages (MDMs) to evaluate the antiviral activity of Per-1. Additionally, we analyzed the correlation between Per-1 expression and viral loads in vivo, and silenced Per-1 by siRNA technology to investigate the potential anti-HIV-1 roles of Per-1 in vivo in untreated HIV-1-infected individuals. Results: We found that short isoform Per-1 can restrict HIV-1 replication and Tat ameliorates this inhibitory effect. Silencing of Per-1 could upregulate HIV-1 transcription both in resting CD4+ Tcells and MDMs. Moreover, Per-1 expression is inversely correlated with viral loads in Rapid progressors (RPs) in vivo. Conclusion: These data together suggest that Per-1 is a novel negative regulator of HIV-1 transcription. This restrictive activity of Per-1 to HIV-1 replication may contribute to HIV-1 latency in resting CD4+ T-cells.

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Human Immunodeficiency Virus Viremia Induces Plasmacytoid Dendritic Cell Activation In Vivo and Diminished Alpha Interferon Production In Vitro

Journal of Virology ◽

10.1128/jvi.01545-07 ◽

2008 ◽

Vol 82 (8) ◽

pp. 3997-4006 ◽

Cited By ~ 63

Author(s):

John C. Tilton ◽

Maura M. Manion ◽

Marlise R. Luskin ◽

Alison J. Johnson ◽

Andy A. Patamawenu ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

De Novo ◽

Type I Interferons ◽

Type I ◽

Interferon Production ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) infection has been associated with perturbations of plasmacytoid dendritic cells (PDC), including diminished frequencies in the peripheral blood and reduced production of type I interferons (IFNs) in response to in vitro stimulation. However, recent data suggest a paradoxical increase in production of type 1 interferons in vivo in HIV-infected patients compared to uninfected controls. Using a flow cytometric assay to detect IFN-α-producing cells within unseparated peripheral blood mononuclear cells, we observed that short-term interruptions of antiretroviral therapy are sufficient to result in significantly reduced IFN-α production by PDC in vitro in response to CpG A ligands or inactivated HIV particles. The primary cause of diminished IFN-α production was reduced responsiveness of PDC to de novo stimulation, not diminished per cell IFN-α production or migration of cells to lymphoid organs. Real-time PCR analysis of purified PDC from patients prior to and during treatment interruptions revealed that active HIV-1 replication is associated with upregulation of type I IFN-stimulated gene expression. Treatment of hepatitis C virus-infected patients with IFN-α2b and ribavirin for hepatitis C virus infection resulted in a profound suppression of de novo IFN-α production in response to CpG A or inactivated HIV particles, similar to the response observed in HIV-infected patients. Together, these results suggest that diminished production of type I interferons in vitro by PDC from HIV-1-infected patients may not represent diminished interferon production in vivo. Rather, diminished function in vitro is likely a consequence of prior activation via type I interferons or HIV virions in vivo.

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Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme.

Molecular and Cellular Biology ◽

10.1128/mcb.10.11.5679 ◽

1990 ◽

Vol 10 (11) ◽

pp. 5679-5687 ◽

Cited By ~ 33

Author(s):

C K Barlowe ◽

D R Appling

Keyword(s):

Saccharomyces Cerevisiae ◽

De Novo ◽

Point Mutations ◽

Gene Replacement ◽

Molecular Genetic Analysis ◽

Yeast Cells ◽

Yeast Saccharomyces Cerevisiae ◽

Purine Synthesis

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Dysregulated Microglial Cell Activation and Proliferation Following Repeated Antigen Stimulation

Frontiers in Cellular Neuroscience ◽

10.3389/fncel.2021.686340 ◽

2021 ◽

Vol 15 ◽

Author(s):

Sujata Prasad ◽

Wen S. Sheng ◽

Shuxian Hu ◽

Priyanka Chauhan ◽

James R. Lokensgard

Keyword(s):

Cell Activation ◽

Microglial Cell ◽

De Novo ◽

Cell Epitope ◽

Viral Reactivation ◽

Activation Markers ◽

Memory Cd8 T Cells ◽

Neurotropic Viruses ◽

Hiv 1

Upon reactivation of quiescent neurotropic viruses antigen (Ag)-specific brain resident-memory CD8+ T-cells (bTRM) may respond to de novo-produced viral Ag through the rapid release of IFN-γ, which drives subsequent interferon-stimulated gene expression in surrounding microglia. Through this mechanism, a small number of adaptive bTRM may amplify responses to viral reactivation leading to an organ-wide innate protective state. Over time, this brain-wide innate immune activation likely has cumulative neurotoxic and neurocognitive consequences. We have previously shown that HIV-1 p24 Ag-specific bTRM persist within the murine brain using a heterologous prime-CNS boost strategy. In response to Ag restimulation, these bTRM display rapid and robust recall responses, which subsequently activate glial cells. In this study, we hypothesized that repeated challenges to viral antigen (Ag) (modeling repeated episodes of viral reactivation) culminate in prolonged reactive gliosis and exacerbated neurotoxicity. To address this question, mice were first immunized with adenovirus vectors expressing the HIV p24 capsid protein, followed by a CNS-boost using Pr55Gag/Env virus-like particles (HIV-VLPs). Following the establishment of the bTRM population [>30 days (d)], prime-CNS boost animals were then subjected to in vivo challenge, as well as re-challenge (at 14 d post-challenge), using the immunodominant HIV-1 AI9 CD8+ T-cell epitope peptide. In these studies, Ag re-challenge resulted in prolonged expression of microglial activation markers and an increased proliferative response, longer than the challenge group. This continued expression of MHCII and PD-L1 (activation markers), as well as Ki67 (proliferative marker), was observed at 7, 14, and 30 days post-AI9 re-challenge. Additionally, in vivo re-challenge resulted in continued production of inducible nitric oxide synthase (iNOS) with elevated levels observed at 7, 14 and 30 days post re-challenge. Interestingly, iNOS expression was significantly lower among challenged animals when compared to re-challenged groups. Furthermore, in vivo specific Ag re-challenge produced lower levels of arginase (Arg)-1 when compared with the challenged group. Taken together, these results indicate that repeated Ag-specific stimulation of adaptive immune responses leads to cumulative dysregulated microglial cell activation.

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