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1.
PLoS One ; 18(11): e0291937, 2023.
Article in English | MEDLINE | ID: mdl-38011121

ABSTRACT

Single-domain antibodies (sdAbs) derived from Camelidae heavy-chain-only antibodies (also called nanobodies or VHHs) have advantages over conventional antibodies in terms of their small size and stability to pH and temperature extremes, their ability to express well in microbial hosts, and to be functionally multimerized for enhanced properties. For these reasons, VHHs are showing promise as enteric disease therapeutics, yet little is known as to their pharmacokinetics (PK) within the digestive tract. To improve understanding of enteric VHH PK, we investigated the functional and structural stability of monomeric and multimeric camelid VHH-agents following in vitro incubation with intestinal extracts (chyme) from rabbits and pigs or fecal extracts from human sources, and in vivo in rabbits. The results showed that unstructured domains such as epitopic tags and flexible spacers composed of different amino acid sequences were rapidly degraded by enteric proteases while the functional core VHHs were much more stable to these treatments. Individual VHHs were widely variable in their functional stability to GI tract proteases. Some VHH-based agents which neutralize enteric Shiga toxin Stx2 displayed a functional stability to chyme incubations comparable to that of Stx2-neutralizing IgG and IgA mAbs, thus indicating that selected nanobodies can approach the functional stability of conventional immunoglobulins. Enteric PK data obtained from in vitro incubation studies were consistent with similar incubations performed in vivo in rabbit surgical gut loops. These findings have broad implications for enteric use of VHH-based agents, particularly VHH fusion proteins.


Subject(s)
Camelids, New World , Single-Domain Antibodies , Animals , Humans , Rabbits , Swine , Immunoglobulin Heavy Chains , Antibodies, Monoclonal , Amino Acid Sequence , Peptide Hydrolases
2.
Sci Rep ; 12(1): 11664, 2022 07 08.
Article in English | MEDLINE | ID: mdl-35803998

ABSTRACT

Single domain antibodies (sdAbs), also called nanobodies, have substantial biophysical advantages over conventional antibodies and are increasingly being employed as components of immunotherapeutic agents. One particularly favorable property is the ability to link different sdAbs into heteromultimers. This feature allows production of single molecules capable of simultaneously targeting more than one antigen. In addition, cooperative binding of multiple linked sdAbs to non-overlapping epitopes on the same target can produce synergistic improvements in target affinity, variant specificity, and in vivo potencies. Here we seek to test the option of increased component sdAbs in these heteromultimers by testing different sdAb heterohexamers in which each of the six camelid sdAb components (VHHs) can neutralize one of three different Botulinum neurotoxin (BoNT) serotypes, A, B or E. Each heterohexamer bound all three targeted BoNT serotypes and protected mice from at least 100 MIPLD50 of each serotype. To test the potential of mRNA therapeutics encoding long sdAb heteromultimers, one heterohexamer was encoded as replicating RNA (repRNA), formulated with a cationic nanocarrier, and delivered to mice via intramuscular injection. Heterohexamer antitoxin serum expression levels were easily detected by 8 h post-treatment, peaked at 5-10 nM around two days, and persisted for more than three days. Mice treated with the formulated repRNA one day post-treatment survived challenge with 100 MIPLD50 of each toxin serotype, demonstrating the function of all six component VHHs. Use of long sdAb multimers, administered as proteins or repRNA, offer the potential for substantially improved versatility in the development of antibody-based therapeutics.


Subject(s)
Antitoxins , Botulinum Toxins , Single-Domain Antibodies , Animals , Botulinum Toxins/genetics , Mice , RNA , Serogroup , Single-Domain Antibodies/genetics
3.
In Vitro Cell Dev Biol Anim ; 57(2): 160-173, 2021 Feb.
Article in English | MEDLINE | ID: mdl-33237403

ABSTRACT

The gastrointestinal tract (GIT), in particular, the small intestine, plays a significant role in food digestion, fluid and electrolyte transport, drug absorption and metabolism, and nutrient uptake. As the longest portion of the GIT, the small intestine also plays a vital role in protecting the host against pathogenic or opportunistic microbial invasion. However, establishing polarized intestinal tissue models in vitro that reflect the architecture and physiology of the gut has been a challenge for decades and the lack of translational models that predict human responses has impeded research in the drug absorption, metabolism, and drug-induced gastrointestinal toxicity space. Often, animals fail to recapitulate human physiology and do not predict human outcomes. Also, certain human pathogens are species specific and do not infect other hosts. Concerns such as variability of results, a low throughput format, and ethical considerations further complicate the use of animals for predicting the safety and efficacy xenobiotics in humans. These limitations necessitate the development of in vitro 3D human intestinal tissue models that recapitulate in vivo-like microenvironment and provide more physiologically relevant cellular responses so that they can better predict the safety and efficacy of pharmaceuticals and toxicants. Over the past decade, much progress has been made in the development of in vitro intestinal models (organoids and 3D-organotypic tissues) using either inducible pluripotent or adult stem cells. Among the models, the MatTek's intestinal tissue model (EpiIntestinal™ Ashland, MA) has been used extensively by the pharmaceutical industry to study drug permeation, metabolism, drug-induced GI toxicity, pathogen infections, inflammation, wound healing, and as a predictive model for a clinical adverse outcome (diarrhea) to pharmaceutical drugs. In this paper, our review will focus on the potential of in vitro small intestinal tissues as preclinical research tool and as alternative to the use of animals.


Subject(s)
Cell Culture Techniques , Inflammation/pathology , Intestine, Small/pathology , Models, Biological , Pharmaceutical Preparations/metabolism , Toxicity Tests , Animals , Humans , Intestine, Small/ultrastructure , Permeability
4.
PLoS Negl Trop Dis ; 11(1): e0005306, 2017 01.
Article in English | MEDLINE | ID: mdl-28095417

ABSTRACT

Schistosomiasis is a major disease of the developing world for which no vaccine has been successfully commercialized. While numerous Schistosoma mansoni worm antigens have been identified that elicit antibody responses during natural infections, little is known as to the identities of the schistosome antigens that are most prominently recognized by antibodies generated through natural infection. Non-reducing western blots probed with serum from schistosome-infected mice, rats and humans on total extracts of larval or adult schistosomes revealed that a small number of antigen bands predominate in all cases. Recognition of each of these major bands was lost when the blots were run under reducing condition. We expressed a rationally selected group of schistosome tegumental membrane antigens in insect host cells, and used the membrane extracts of these cells to unambiguously identify the major antigens recognized by S. mansoni infected mouse, rat and human serum. These results revealed that a limited number of dominant, reduction-sensitive conformational epitopes on five major tegumental surface membrane proteins: SmTsp2, Sm23, Sm29, SmLy6B and SmLy6F, are primary targets of mouse, rat and human S. mansoni infection sera antibodies. We conclude that, Schistosoma mansoni infection of both permissive (mouse) and non-permissive (rat) rodent models, as well as humans, elicit a dominant antibody response recognizing a limited number of conformational epitopes on the same five tegumental membrane proteins. Thus it appears that neither infecting schistosomula nor mature adult schistosomes are substantively impacted by the robust circulating anti-tegumental antibody response they elicit to these antigens. Importantly, our data suggest a need to re-evaluate host immune responses to many schistosome antigens and has important implications regarding schistosome immune evasion mechanisms and schistosomiasis vaccine development.


Subject(s)
Antibodies, Helminth/immunology , Epitopes/immunology , Membrane Proteins/immunology , Schistosoma mansoni/immunology , Schistosomiasis mansoni/immunology , Animals , Antibody Formation , Epitopes/chemistry , Epitopes/genetics , Female , Helminth Proteins/chemistry , Helminth Proteins/genetics , Helminth Proteins/immunology , Humans , Male , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Rats , Schistosoma mansoni/chemistry , Schistosoma mansoni/genetics , Schistosomiasis mansoni/parasitology
5.
Clin Vaccine Immunol ; 23(3): 213-8, 2016 Jan 06.
Article in English | MEDLINE | ID: mdl-26740390

ABSTRACT

Bacillus anthracis, the causative agent of anthrax, secretes three polypeptides, which form the bipartite lethal and edema toxins (LT and ET, respectively). The common component in these toxins, protective antigen (PA), is responsible for binding to cellular receptors and translocating the lethal factor (LF) and edema factor (EF) enzymatic moieties to the cytosol. Antibodies against PA protect against anthrax. We previously isolated toxin-neutralizing variable domains of camelid heavy-chain-only antibodies (VHHs) and demonstrated their in vivo efficacy. In this work, gene therapy with an adenoviral (Ad) vector (Ad/VNA2-PA) (VNA, VHH-based neutralizing agents) promoting the expression of a bispecific VHH-based neutralizing agent (VNA2-PA), consisting of two linked VHHs targeting different PA-neutralizing epitopes, was tested in two inbred mouse strains, BALB/cJ and C57BL/6J, and found to protect mice against anthrax toxin challenge and anthrax spore infection. Two weeks after a single treatment with Ad/VNA2-PA, serum VNA2-PA levels remained above 1 µg/ml, with some as high as 10 mg/ml. The levels were 10- to 100-fold higher and persisted longer in C57BL/6J than in BALB/cJ mice. Mice were challenged with a lethal dose of LT or spores at various times after Ad/VNA2-PA administration. The majority of BALB/cJ mice having serum VNA2-PA levels of >0.1 µg/ml survived LT challenge, and 9 of 10 C57BL/6J mice with serum levels of >1 µg/ml survived spore challenge. Our findings demonstrate the potential for genetic delivery of VNAs as an effective method for providing prophylactic protection from anthrax. We also extend prior findings of mouse strain-based differences in transgene expression and persistence by adenoviral vectors.


Subject(s)
Adenoviridae/genetics , Anthrax/prevention & control , Antigens, Bacterial/immunology , Bacterial Toxins/immunology , Immunization, Passive/methods , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Single-Chain Antibodies/genetics , Single-Chain Antibodies/immunology , Animals , Anthrax/immunology , Antibodies, Bacterial/genetics , Antibodies, Bacterial/immunology , Bacillus anthracis/immunology , Bacillus anthracis/pathogenicity , Female , Immune Sera/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Spores, Bacterial/immunology
6.
Plant Biotechnol J ; 13(1): 117-24, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25229405

ABSTRACT

We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VH H) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen-binding proteins and the heterodimer fusion protein containing two VH H domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin-producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.


Subject(s)
Antitoxins/immunology , Botulinum Toxins/immunology , Camelids, New World/immunology , Chlamydomonas reinhardtii/immunology , Chloroplasts/metabolism , Immunoglobulin Heavy Chains/immunology , Immunoglobulin Variable Region/immunology , Animals , Antibodies, Neutralizing/immunology , Antigens/immunology , Cell Survival , Chlamydomonas reinhardtii/genetics , Genetic Vectors/metabolism , Mice , Polymerase Chain Reaction , Recombinant Proteins/metabolism , Single-Domain Antibodies/immunology , Transformation, Genetic , Transgenes
7.
Infect Immun ; 83(1): 286-91, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25368111

ABSTRACT

Hemolytic-uremic syndrome (HUS), caused by Shiga toxin (Stx)-producing Escherichia coli (STEC), remains untreatable. Production of human monoclonal antibodies against Stx, which are highly effective in preventing Stx sequelae in animal models, is languishing due to cost and logistics. We reported previously that the production and evaluation of a camelid heavy-chain-only VH domain (VHH)-based neutralizing agent (VNA) targeting Stx1 and Stx2 (VNA-Stx) protected mice from Stx1 and Stx2 intoxication. Here we report that a single intramuscular (i.m.) injection of a nonreplicating adenovirus (Ad) vector carrying a secretory transgene of VNA-Stx (Ad/VNA-Stx) protected mice challenged with Stx2 and protected gnotobiotic piglets infected with STEC from fatal systemic intoxication. One i.m. dose of Ad/VNA-Stx prevented fatal central nervous system (CNS) symptoms in 9 of 10 animals when it was given to piglets 24 h after bacterial challenge and in 5 of 9 animals when it was given 48 h after bacterial challenge, just prior to the onset of CNS symptoms. All 6 placebo animals died or were euthanized with severe CNS symptoms. Ad/VNA-Stx treatment had no impact on diarrhea. In conclusion, Ad/VNA-Stx treatment is effective in protecting piglets from fatal Stx2-mediated CNS complications following STEC challenge. With a low production cost and further development, this could presumably be an effective treatment for patients with HUS and/or individuals at high risk of developing HUS due to exposure to STEC.


Subject(s)
Adenoviruses, Human/genetics , Antibodies, Neutralizing/therapeutic use , Escherichia coli Infections/drug therapy , Escherichia coli O157/immunology , Hemolytic-Uremic Syndrome/drug therapy , Shiga Toxin 1/antagonists & inhibitors , Shiga Toxin 2/antagonists & inhibitors , Animals , Antibodies, Neutralizing/genetics , Disease Models, Animal , Drug Carriers/administration & dosage , Escherichia coli Infections/immunology , Escherichia coli Infections/microbiology , Escherichia coli O157/genetics , Female , Genetic Vectors , Hemolytic-Uremic Syndrome/immunology , Hemolytic-Uremic Syndrome/microbiology , Injections, Intramuscular , Mice , Shiga Toxin 1/immunology , Shiga Toxin 2/immunology , Survival Analysis , Swine , Time Factors
8.
PLoS One ; 9(8): e106422, 2014.
Article in English | MEDLINE | ID: mdl-25170904

ABSTRACT

Current therapies for most acute toxin exposures are limited to administration of polyclonal antitoxin serum. We have shown that VHH-based neutralizing agents (VNAs) consisting of two or more linked, toxin-neutralizing heavy-chain-only VH domains (VHHs), each binding distinct epitopes, can potently protect animals from lethality in several intoxication models including Botulinum neurotoxin serotype A1 (BoNT/A1). Appending a 14 amino acid albumin binding peptide (ABP) to an anti-BoNT/A1 heterodimeric VNA (H7/B5) substantially improved serum stability and resulted in an effective VNA serum half-life of 1 to 2 days. A recombinant, replication-incompetent, adenoviral vector (Ad/VNA-BoNTA) was engineered that induces secretion of biologically active VNA, H7/B5/ABP (VNA-BoNTA), from transduced cells. Mice administered a single dose of Ad/VNA-BoNTA, or a different Ad/VNA, via different administration routes led to a wide range of VNA serum levels measured four days later; generally intravenous > intraperitoneal > intramuscular > subcutaneous. Ad/VNA-BoNTA treated mice were 100% protected from 10 LD50 of BoNT/A1 for more than six weeks and protection positively correlated with serum levels of VNA-BoNTA exceeding about 5 ng/ml. Some mice developed antibodies that inhibited VNA binding to target but these mice displayed no evidence of kidney damage due to deposition of immune complexes. Mice were also successfully protected from 10 LD50 BoNT/A1 when Ad/VNA-BoNTA was administered up to 1.5 hours post-intoxication, demonstrating rapid appearance of the protective VNA in serum following treatment. Genetic delivery of VNAs promises to be an effective method of providing prophylactic protection and/or acute treatments for many toxin-mediated diseases.


Subject(s)
Antibodies, Neutralizing/blood , Botulinum Antitoxin/immunology , Botulism/prevention & control , Dependovirus/genetics , Animals , Botulinum Antitoxin/genetics , Botulinum Antitoxin/metabolism , Botulism/blood , Botulism/immunology , Dependovirus/metabolism , Disease Models, Animal , Genetic Vectors/administration & dosage , Mice , Vaccination
9.
Infect Immun ; 81(12): 4592-603, 2013 Dec.
Article in English | MEDLINE | ID: mdl-24082082

ABSTRACT

Shiga toxin-producing Escherichia coli (STEC) is a major cause of severe food-borne disease worldwide, and two Shiga toxins, Stx1 and Stx2, are primarily responsible for the serious disease consequence, hemolytic-uremic syndrome (HUS). Here we report identification of a panel of heavy-chain-only antibody (Ab) V(H) (VHH) domains that neutralize Stx1 and/or Stx2 in cell-based assays. VHH heterodimer toxin-neutralizing agents containing two linked Stx1-neutralizing VHHs or two Stx2-neutralizing VHHs were generally much more potent at Stx neutralization than a pool of the two-component monomers tested in cell-based assays and in vivo mouse models. We recently reported that clearance of toxins can be promoted by coadministering a VHH-based toxin-neutralizing agent with an antitag monoclonal antibody (MAb), called the "effector Ab," that indirectly decorates each toxin molecule with four Ab molecules. Decoration occurs because the Ab binds to a common epitopic tag present at two sites on each of the two VHH heterodimer molecules that bind to each toxin molecule. Here we show that coadministration of effector Ab substantially improved the efficacy of Stx toxin-neutralizing agents to prevent death or kidney damage in mice following challenge with Stx1 or Stx2. A single toxin-neutralizing agent consisting of a double-tagged VHH heterotrimer--one Stx1-specific VHH, one Stx2-specific VHH, and one Stx1/Stx2 cross-specific VHH--was effective in preventing all symptoms of intoxication from Stx1 and Stx2 when coadministered with effector Ab. Overall, the availability of simple, defined, recombinant proteins that provide cost-effective protection against HUS opens up new therapeutic approaches to managing disease.


Subject(s)
Escherichia coli Infections/immunology , Hemolytic-Uremic Syndrome/immunology , Shiga Toxin 1/immunology , Shiga Toxin 2/immunology , Single-Domain Antibodies/immunology , Animals , Antibodies, Monoclonal/immunology , Enteropathogenic Escherichia coli/immunology , Enteropathogenic Escherichia coli/metabolism , Female , Mice , Molecular Sequence Data , Shiga Toxin 1/metabolism , Shiga Toxin 2/metabolism , Shiga-Toxigenic Escherichia coli/immunology , Shiga-Toxigenic Escherichia coli/metabolism
10.
J Leukoc Biol ; 94(5): 865-75, 2013 Nov.
Article in English | MEDLINE | ID: mdl-23804807

ABSTRACT

The protein kinase Btk has been implicated in the development, differentiation, and activation of B cells through its role in the BCR and TLR signaling cascades. These receptors and in particular, the BCR and either TLR7 or TLR9 also play a critical role in the activation of autoreactive B cells by RNA- or DNA-associated autoantigens. To explore the role of Btk in the development of autoreactive B cells, as well as their responses to nucleic acid-associated autoantigens, we have now compared Btk-sufficient and Btk-deficient mice that express a prototypic RF BCR encoded by H- and L-chain sdTgs. These B cells bind autologous IgG2a with low affinity and only proliferate in response to IgG2a ICs that incorporate DNA or RNA. We found that Btk-sufficient RF(+) B cells mature into naïve FO B cells, all of which express the Tg BCR, despite circulating levels of IgG2a. By contrast, a significant proportion of Btk-deficient RF(+) B cells acquires a MZ or MZ precursor phenotype. Remarkably, despite the complete inability of RF(+) Xid/y B cells to respond to F(ab')2 anti-IgM, RF(+) Xid/y B cells could respond well to autoantigen-associated ICs. These data reveal unique features of the signaling cascades responsible for the activation of autoreactive B cells.


Subject(s)
B-Lymphocytes/immunology , Lymphocyte Activation , Protein-Tyrosine Kinases/physiology , Receptors, Antigen, B-Cell/physiology , Rheumatoid Factor/analysis , Toll-Like Receptors/physiology , Agammaglobulinaemia Tyrosine Kinase , Animals , Female , Male , Mice , Mice, Inbred BALB C , Mice, Inbred CBA , Mice, Transgenic
11.
J Biol Chem ; 287(47): 39789-99, 2012 Nov 16.
Article in English | MEDLINE | ID: mdl-23019335

ABSTRACT

The key step in the activation of autoreactive B cells is the internalization of nucleic acid containing ligands and delivery of these ligands to the Toll-like Receptor (TLR) containing endolysosomal compartment. Ribonucleoproteins represent a large fraction of autoantigens in systemic autoimmune diseases. Here we demonstrate that many uridine-rich mammalian RNA sequences associated with common autoantigens effectively activate autoreactive B cells. Priming with type I IFN increased the magnitude of activation, and the range of which RNAs were stimulatory. A subset of RNAs that contain a high degree of self-complementarity also activated B cells through TLR3. For the RNA sequences that activated predominantly through TLR7, the activation is proportional to uridine-content, and more precisely defined by the frequency of specific uridine-containing motifs. These results identify parameters that define specific mammalian RNAs as ligands for TLRs.


Subject(s)
Autoimmunity , Lymphocyte Activation , Membrane Glycoproteins/immunology , RNA/immunology , Toll-Like Receptor 3/immunology , Toll-Like Receptor 7/immunology , Animals , Ligands , Membrane Glycoproteins/genetics , Mice , Mice, Inbred BALB C , Mice, Knockout , RNA/genetics , Toll-Like Receptor 3/genetics , Toll-Like Receptor 7/genetics , Uridine/genetics , Uridine/immunology
12.
J Virol ; 86(10): 5660-73, 2012 May.
Article in English | MEDLINE | ID: mdl-22438545

ABSTRACT

Viral drug toxicity, resistance, and an increasing immunosuppressed population warrant continued research into new avenues for limiting diseases associated with human cytomegalovirus (HCMV). In this study, a small interfering RNA (siRNA), siX3, was designed to target coding sequences within shared exon 3 of UL123 and UL122 transcripts encoding IE1 and IE2 immediate-early proteins of HCMV. Pretreatment of cells with siX3 reduced the levels of viral protein expression, DNA replication, and progeny virus production compared to control siRNA. Two siRNAs against UL54 and overlapping transcripts (UL55-57) were compared to siX3 in HCMV infection and were also found to be effective at inhibiting HCMV replication. Further investigation into the effects of the siRNAs on viral replication showed that pretreatment with each of the siRNAs resulted in an inhibition in the formation of mature replication compartments. The ability of these siRNAs to prevent or reduce certain cytopathic effects associated with HCMV infection was also examined. Infected cells pretreated with siX3, but not siUL54, retained promyelocytic leukemia (PML) protein in cellular PML bodies, an essential component of this host intrinsic antiviral defense. DNA damage response proteins, which are localized in nuclear viral replication compartments, were reduced in the siX3- and siUL54-treated cells. siX3, but not siUL54, prevented DNA damage response signaling early after infection. Therapeutic efficacy was demonstrated by treating cells with siRNAs after HCMV replication had commenced. Together, these findings suggest that siRNAs targeting exon 3 of the major IE genes or the UL54-57 transcripts be further studied for their potential development into anti-HCMV therapeutics.


Subject(s)
Cytomegalovirus Infections/virology , Cytomegalovirus/genetics , DNA-Directed DNA Polymerase/genetics , Immediate-Early Proteins/genetics , RNA Interference , Trans-Activators/genetics , Viral Proteins/genetics , Virus Replication , Cell Line , Cytomegalovirus/physiology , Cytomegalovirus Infections/genetics , Cytomegalovirus Infections/metabolism , DNA Damage , DNA-Directed DNA Polymerase/metabolism , Down-Regulation , Gene Expression Regulation, Viral , Humans , Immediate-Early Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Promyelocytic Leukemia Protein , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Trans-Activators/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Tumor Suppressor Proteins/genetics , Tumor Suppressor Proteins/metabolism , Viral Proteins/metabolism
13.
Immunol Lett ; 143(1): 85-91, 2012 Mar 30.
Article in English | MEDLINE | ID: mdl-22285306

ABSTRACT

Systemic autoimmune diseases are characterized by the development of autoantibodies directed against a limited subset of nuclear antigens, including DNA. DNA-specific B cells take up mammalian DNA through their B cell receptor, and this DNA is subsequently transported to an endosomal compartment where it can potentially engage TLR9. We have previously shown that ssDNA-specific B cells preferentially bind to particular DNA sequences, and antibody specificity for short synthetic oligodeoxynucleotides (ODNs). Since CpG-rich DNA, the ligand for TLR9 is found in low abundance in mammalian DNA, we sought to determine whether antibodies derived from DNA-reactive B cells showed binding preference for CpG-rich native dsDNA, and thereby select immunostimulatory DNA for delivery to TLR9. We examined a panel of anti-DNA antibodies for binding to CpG-rich and CpG-poor DNA fragments. We show that a number of anti-DNA antibodies do show preference for binding to certain native dsDNA fragments of differing sequence, but this does not correlate directly with the presence of CpG dinucleotides. An antibody with preference for binding to a fragment containing optimal CpG motifs was able to promote B cell proliferation to this fragment at 10-fold lower antibody concentrations than an antibody that did not selectively bind to this fragment, indicating that antibody binding preference can influence autoreactive B cell responses.


Subject(s)
Antibodies, Antinuclear/immunology , CpG Islands , DNA/immunology , Animals , Antibody Specificity , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Cell Proliferation , Cells, Cultured , DNA/chemistry , Mice , Mice, Inbred BALB C
14.
PLoS Pathog ; 7(5): e1001342, 2011 May.
Article in English | MEDLINE | ID: mdl-21589897

ABSTRACT

DNA damage resulting from intrinsic or extrinsic sources activates DNA damage responses (DDRs) centered on protein kinase signaling cascades. The usual consequences of inducing DDRs include the activation of cell cycle checkpoints together with repair of the damaged DNA or induction of apoptosis. Many DNA viruses elicit host DDRs during infection and some viruses require the DDR for efficient replication. However, the mechanism by which DDRs are activated by viral infection is poorly understood. Human cytomegalovirus (HCMV) infection induces a DDR centered on the activation of ataxia telangiectasia mutated (ATM) protein kinase. Here we show that HCMV replication is compromised in cells with inactivated or depleted ATM and that ATM is essential for the host DDR early during infection. Likewise, a downstream target of ATM phosphorylation, H2AX, also contributes to viral replication. The ATM-dependent DDR is detected as discrete, nuclear γH2AX foci early in infection and can be activated by IE proteins. By 24 hpi, γH2AX is observed primarily in HCMV DNA replication compartments. We identified a role for the E2F1 transcription factor in mediating this DDR and viral replication. E2F1, but not E2F2 or E2F3, promotes the accumulation of γH2AX during HCMV infection or IE protein expression. Moreover, E2F1 expression, but not the expression of E2F2 or E2F3, is required for efficient HCMV replication. These results reveal a novel role for E2F1 in mediating an ATM-dependent DDR that contributes to viral replication. Given that E2F activity is often deregulated by infection with DNA viruses, these observations raise the possibility that an E2F1-mediated mechanism of DDR activation may be conserved among DNA viruses.


Subject(s)
Cell Cycle Proteins/genetics , Cytomegalovirus Infections/virology , Cytomegalovirus/physiology , DNA Damage , DNA-Binding Proteins/genetics , E2F1 Transcription Factor/metabolism , Protein Serine-Threonine Kinases/genetics , Tumor Suppressor Proteins/genetics , Virus Replication/physiology , Ataxia Telangiectasia Mutated Proteins , Caffeine/pharmacology , Cell Cycle Proteins/metabolism , Cell Line , Cytomegalovirus/genetics , Cytomegalovirus/growth & development , DNA Repair/physiology , DNA, Viral/biosynthesis , DNA, Viral/genetics , DNA-Binding Proteins/metabolism , E2F1 Transcription Factor/genetics , Fibroblasts/cytology , Fibroblasts/metabolism , Fibroblasts/virology , Fluorescent Antibody Technique , Histones/metabolism , Humans , Immediate-Early Proteins/metabolism , Immunoblotting , Phosphodiesterase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/metabolism , RNA, Small Interfering/metabolism , Signal Transduction , Trans-Activators/metabolism , Tumor Suppressor Proteins/metabolism
15.
Mol Cell Biol ; 24(7): 2968-77, 2004 Apr.
Article in English | MEDLINE | ID: mdl-15024084

ABSTRACT

The retinoblastoma protein (Rb)/E2F pathway links cellular proliferation control to apoptosis and is critical for normal development and cancer prevention. Here we define a transcription-mediated pathway in which deregulation of E2F1 by ectopic E2F expression or Rb inactivation by E7 of human papillomavirus type 16 signals apoptosis by inducing the expression of Chk2, a component of the DNA damage response. E2F1- and E7-mediated apoptosis are compromised in cells from patients with the related disorders ataxia telangiectasia and Nijmegen breakage syndrome lacking functional Atm and Nbs1 gene products, respectively. Both Atm and Nbs1 contribute to Chk2 activation and p53 phosphorylation following deregulation of normal Rb growth control. E2F2, a related E2F family member that does not induce apoptosis, also activates Atm, resulting in phosphorylation of p53. However, we found that the key commitment step in apoptosis induction is the ability of E2F1, and not E2F2, to upregulate Chk2 expression. Our results suggest that E2F1 plays a central role in signaling disturbances in the Rb growth control pathway and, by upregulation of Chk2, may sensitize cells to undergo apoptosis.


Subject(s)
Apoptosis/physiology , Cell Cycle Proteins/metabolism , DNA-Binding Proteins , Nuclear Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Transcription Factors/metabolism , Animals , Ataxia Telangiectasia Mutated Proteins , Cells, Cultured , Checkpoint Kinase 2 , E2F Transcription Factors , E2F1 Transcription Factor , E2F2 Transcription Factor , Fibroblasts/cytology , Fibroblasts/physiology , Gene Expression Regulation , Humans , Mice , Mice, Transgenic , Oncogene Proteins, Viral/metabolism , Papillomavirus E7 Proteins , Retinoblastoma Protein/metabolism , Signal Transduction/physiology , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins
16.
Mol Cell Biol ; 22(15): 5308-18, 2002 Aug.
Article in English | MEDLINE | ID: mdl-12101227

ABSTRACT

It has been proposed that the E2F1 transcription factor serves as a link between the Rb/E2F proliferation pathway and the p53 apoptosis pathway by inducing the expression of p19ARF, a protein that regulates p53 stability. We find that although p19ARF contributes to p53 accumulation in response to E2F expression, p19ARF is not required for E2F1-mediated apoptosis. E2F1 can signal p53 phosphorylation in the absence of p19ARF, similar to the observed modifications to p53 in response to DNA damage. These modifications are not observed in the absence of p19ARF following expression of E2F2, an E2F family member that does not induce apoptosis in mouse embryo fibroblasts but can induce p19ARF and p53 protein expression. p53 modification is found to be crucial for E2F1-mediated apoptosis, and this apoptosis is compromised when E2F1 is coexpressed with a p53 mutant lacking many N- and C-terminal phosphorylation sites. Additionally, E2F1-mediated apoptosis is abolished in the presence of caffeine, an inhibitor of phosphatidylinositol 3-kinase-related kinases that phosphorylate p53. These findings suggest that p53 phosphorylation is a key step in E2F1-mediated apoptosis and that this modification can occur in the absence of p19ARF.


Subject(s)
Apoptosis/physiology , Cell Cycle Proteins , DNA-Binding Proteins , Nuclear Proteins , Transcription Factors/metabolism , Tumor Suppressor Protein p53/metabolism , Animals , Cell Line , Cyclin-Dependent Kinase Inhibitor p16 , E2F Transcription Factors , E2F1 Transcription Factor , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression , Mice , Mutation , Phosphorylation , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , Transcription Factors/genetics , Transfection , Tumor Suppressor Protein p14ARF/deficiency , Tumor Suppressor Protein p14ARF/genetics , Tumor Suppressor Protein p14ARF/metabolism , Tumor Suppressor Protein p53/deficiency , Tumor Suppressor Protein p53/genetics
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