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1.
Front Immunol ; 10: 2324, 2019.
Article in English | MEDLINE | ID: mdl-31632403

ABSTRACT

The recent discovery of long-lived plasma cells (LLPCs) in mammals, which provide a constant expression of specific high-affinity antibodies that mediate humoral memory, has caused a dramatic paradigm shift in the study of immunity and vaccine development. In teleost fish, there are few studies regarding the association between LLPCs and antibody production, and the affinity of the antibodies secreted by the LLPCs is poorly understood. In the present study, channel catfish (Ictalurus punctatus) were immunized with trinitrophenylated-keyhole limpet hemocyanin (TNP-KLH) to examine TNP-specific antibody titers, affinities, antibody-secreting cell (ASC) dynamic changes, and especially the affinity of secreted antibodies by LLPCs post-immunization. We demonstrated that TNP-specific LLPCs were generated starting at week 4 post-immunization, achieved a maximal number at week 8, and maintained a comparable level throughout the 18-week post-immunization period, which was correlated with the dynamics of serum antibody titers and affinity maturation in the response. The LLPCs appeared to mostly reside within, or migrate to, the anterior kidney (bone marrow-like tissue in mammals), but a small portion was also located in the spleen and peripheral blood. The antibodies produced by the LLPCs possessed high affinities, indicating that the generation and development of LLPCs were driven by affinity selection in teleosts. Collectively, the results of this study provide insights toward the evolutionary understanding of the affinity-dependent mechanism of LLPC generation and development.


Subject(s)
Antibodies/immunology , Antibody Affinity , Fish Proteins/immunology , Ictaluridae/immunology , Immunization , Plasma Cells/immunology , T-Lymphocytes/immunology , Animals , Antibodies/blood , Fish Proteins/blood , Ictaluridae/blood , Picrates/immunology , Picrates/pharmacology , Plasma Cells/metabolism , T-Lymphocytes/metabolism
2.
Int Immunopharmacol ; 52: 110-118, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28888779

ABSTRACT

BACKGROUND: Inflammation activated by oxidative stress can cause various diseases, such as asthma, rheumatoid arthritis, cancer, diabetes, etc. Plant constituents with sesquiterpene lactones possess antioxidant and anti-inflammatory properties. AIM: To determine the antioxidant and anti-inflammatory potential of isolated phytoconstituent from Cyathocline purpurea Buch-Ham ex D (CP). Don in laboratory animals. Furthermore, to understand the interactions involved in the binding of this compound to cyclooxygenase-2 (COX-2) via computational docking. METHODS: Phytoconstituent was isolated, purified and well characterized (using IR, NMR, and MS) from ethyl acetate fraction of CP methanolic extract. It was then evaluated for its in-vitro antioxidant activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydrogen peroxide (H2O2) and hydroxyl (OH) radical assays as well as in-vivo anti-inflammatory potential against carrageenan-induced paw edema model in rats. The molecular docking study was performed against the crystal structure of COX-2 to evaluate the binding potential of phytoconstituent towards this enzyme. RESULTS: The isolated compound 6α-hydroxy-4 [14], 10 [15]-guainadien-8α, 12-olide (HGN) showed significant (p<0.001) antioxidant activity with IC50 values of 76µg/mL. Administration of HGN (10 and 20mg/kg) significantly (p<0.001) reduced the increased paw volume after subplantar administration of carrageenan. It also exhibits good binding affinity towards with COX-2 with a docking score of -8.98 and Glide binding energy of -36.488kcal/mol shedding light on the potential mechanism of anti-inflammatory action. CONCLUSIONS: The presence of hydroxyl group in HGN provides a credential to its in-vivo anti-inflammatory and in-vitro antioxidant activities. Furthermore, the good binding affinity of HGN for the active site of COX-2 may open novel vistas in therapeutic option with natural antioxidants like Cyathocline purpurea to treat various inflammatory disorders.


Subject(s)
Anti-Inflammatory Agents/therapeutic use , Antioxidants/therapeutic use , Cyclooxygenase 2/metabolism , Edema/drug therapy , Inflammation/drug therapy , Phytotherapy/methods , Sesquiterpenes, Guaiane/therapeutic use , Animals , Asteraceae/immunology , Biphenyl Compounds/immunology , Carrageenan/toxicity , Cells, Cultured , Edema/chemically induced , Female , Humans , Hydrogen Peroxide/metabolism , Male , Mice , Oxidative Stress/drug effects , Picrates/immunology , Rats , Rats, Wistar
3.
Sci Rep ; 7(1): 5400, 2017 07 14.
Article in English | MEDLINE | ID: mdl-28710441

ABSTRACT

IgG3, passively administered together with small proteins, induces enhanced primary humoral responses against these proteins. We previously found that, within 2 h of immunization, marginal zone (MZ) B cells capture IgG3-antigen complexes and transport them into splenic follicles and that this requires the presence of complement receptors 1 and 2. We have here investigated the localization of IgG3 anti-2, 4, 6-trinitrophenyl (TNP)/biotin-ovalbumin-TNP immune complexes in the follicles and the involvement of classical versus total complement activation in this process. The majority (50-90%) of antigen inside the follicles of mice immunized with IgG3-antigen complexes co-localized with the follicular dendritic cell (FDC) network. Capture of antigen by MZ B cells as well as antigen deposition on FDC was severely impaired in mice lacking C1q or C3, and lack of either C1q or C3 also impaired the ability of IgG3 to enhance antibody responses. Finally, IgG3 efficiently primed for a memory response against small proteins as well as against the large protein keyhole limpet hemocyanine.


Subject(s)
Antigens/immunology , Complement C1q/genetics , Complement C3/genetics , Dendritic Cells, Follicular/immunology , Immunoglobulin G/metabolism , Ovalbumin/immunology , Adoptive Transfer , Animals , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/metabolism , Antigens/chemistry , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Biotin/chemistry , Biotin/immunology , Complement Activation , Complement C1q/deficiency , Complement C3/deficiency , Dendritic Cells, Follicular/cytology , Hemocyanins/chemistry , Hemocyanins/immunology , Hybridomas/immunology , Immunization, Passive , Immunoglobulin G/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Ovalbumin/chemistry , Picrates/chemistry , Picrates/immunology , Receptors, Complement/genetics , Receptors, Complement/immunology , Receptors, Complement 3d/genetics , Receptors, Complement 3d/immunology , Spleen/cytology , Spleen/immunology , Whole-Body Irradiation
4.
J Chromatogr A ; 1426: 102-9, 2015 Dec 24.
Article in English | MEDLINE | ID: mdl-26654254

ABSTRACT

This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants.


Subject(s)
Immunoglobulin Fab Fragments/chemistry , Peptide Library , Picrates/immunology , Animals , Binding Sites , Chromatography, Liquid , Complementarity Determining Regions/chemistry , Complementarity Determining Regions/genetics , Computer Simulation , Humans , Immunoglobulin Fab Fragments/genetics , Ligands , Mice , Mutagenesis, Site-Directed
5.
Nat Immunol ; 15(4): 354-364, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24562309

ABSTRACT

Innate lymphoid cells (ILCs) regulate stromal cells, epithelial cells and cells of the immune system, but their effect on B cells remains unclear. Here we identified RORγt(+) ILCs near the marginal zone (MZ), a splenic compartment that contains innate-like B cells highly responsive to circulating T cell-independent (TI) antigens. Splenic ILCs established bidirectional crosstalk with MAdCAM-1(+) marginal reticular cells by providing tumor-necrosis factor (TNF) and lymphotoxin, and they stimulated MZ B cells via B cell-activation factor (BAFF), the ligand of the costimulatory receptor CD40 (CD40L) and the Notch ligand Delta-like 1 (DLL1). Splenic ILCs further helped MZ B cells and their plasma-cell progeny by coopting neutrophils through release of the cytokine GM-CSF. Consequently, depletion of ILCs impaired both pre- and post-immune TI antibody responses. Thus, ILCs integrate stromal and myeloid signals to orchestrate innate-like antibody production at the interface between the immune system and circulatory system.


Subject(s)
Antibody Formation , B-Lymphocytes/immunology , Lymphocytes/immunology , Plasma Cells/immunology , Spleen/immunology , Animals , Antibodies/blood , Antigens, T-Independent/immunology , Blood Proteins/immunology , Cell Adhesion Molecules , Cell Communication/immunology , Cell Differentiation , Cells, Cultured , Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Humans , Immunity, Innate , Immunoglobulins/metabolism , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mucoproteins/metabolism , Neutrophils/immunology , Nuclear Receptor Subfamily 1, Group F, Member 3/metabolism , Picrates/immunology , Signal Transduction/immunology , Stromal Cells/immunology
6.
J Aquat Anim Health ; 24(4): 258-73, 2012 Dec.
Article in English | MEDLINE | ID: mdl-23134222

ABSTRACT

Lake trout Salvelinus namaycush on thiamine-replete and thiamine-depleted diets were evaluated for the effects of thiamine status on in vivo responses to the T-dependent antigen trinitophenol (TNP)-keyhole limpet hemocyanin (TNP-KLH), the T-independent antigen trinitrophenol-lipolysaccaharide (TNP-LPS), or Dulbecco's phosphate-buffered saline (DPBS; negative control fish). Plasma antibody concentrations were evaluated for possible differences in total anti-TNP activity as well as differences in response kinetics. Associations between anti-TNP activity and muscle and liver thiamine concentrations as well as ratios of muscle-to-liver thiamine to anti-TNP activity were also examined. Thiamine-depleted lake trout that were injected with TNP-LPS exhibited significantly more anti-TNP activity than thiamine-replete fish. The depleted fish injected with TNP-LPS also exhibited significantly different response kinetics relative to thiamine-replete lake trout. No differences in activity or kinetics were observed between the thiamine-replete and -depleted fish injected with TNP-KLH or in the DPBS negative controls. Anti-TNP activity in thiamine-depleted lake trout injected with TNP-KLH was positively associated with muscle thiamine pyrophosphate (thiamine diphosphate; TPP) concentration. A negative association was observed between the ratio of muscle-to-liver TPP and T-independent responses. No significant associations between anti-TNP activity and tissue thiamine concentration were observed in the thiamine-replete fish. We demonstrated that thiamine deficiency leads to alterations in both T-dependent and T-independent immune responses in lake trout.


Subject(s)
Antibodies/metabolism , Fish Diseases/immunology , T-Lymphocytes/physiology , Thiamine Deficiency/veterinary , Thiamine/pharmacology , Trout/immunology , Animal Feed/analysis , Animal Nutritional Physiological Phenomena , Animals , Diet/veterinary , Hemocyanins/chemistry , Hemocyanins/immunology , Picrates/chemistry , Picrates/immunology , Thiamine/administration & dosage , Thiamine Deficiency/immunology
7.
Immunogenetics ; 63(9): 599-609, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21660592

ABSTRACT

The programmed, stepwise acquisition of immunocompetence that marks the development of the fetal immune response proceeds during a period when both T cell receptor and immunoglobulin (Ig) repertoires exhibit reduced junctional diversity due to physiologic terminal deoxynucleotidyl transferase (TdT) insufficiency. To test the effect of N addition on humoral responses, we transplanted bone marrow from TdT-deficient (TdT(-/-)) and wild-type (TdT(+/+)) BALB/c mice into recombination activation gene 1-deficient BALB/c hosts. Mice transplanted with TdT(-/-) cells exhibited diminished humoral responses to the T-independent antigens α-1-dextran and (2,4,6-trinitrophenyl) hapten conjugated to AminoEthylCarboxymethyl-FICOLL, to the T-dependent antigens NP(19)CGG and hen egg lysozyme, and to Enterobacter cloacae, a commensal bacteria that can become an opportunistic pathogen in immature and immunocompromised hosts. An exception to this pattern of reduction was the T-independent anti-phosphorylcholine response to Streptococcus pneumoniae, which is normally dominated by the N-deficient T15 idiotype. Most of the humoral immune responses in the recipients of TdT(-/-) bone marrow were impaired, yet population of the blood with B and T cells occurred more rapidly. To further test the effect of N-deficiency on B cell and T cell population growth, transplanted TdT-sufficient and -deficient BALB/c IgM(a) and congenic TdT-sufficient CB17 IgM(b) bone marrow were placed in competition. TdT(-/-) cells demonstrated an advantage in populating the bone marrow, the spleen, and the peritoneal cavity. TdT deficiency, which characterizes fetal lymphocytes, thus appears to facilitate filling both central and peripheral lymphoid compartments, but at the cost of altered responses to a broad set of antigens.


Subject(s)
Lymphocyte Activation/immunology , Animals , Antigens, CD19/immunology , B-Lymphocytes/immunology , Bone Marrow/immunology , Bone Marrow Transplantation/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , DNA Nucleotidylexotransferase/genetics , DNA Nucleotidylexotransferase/immunology , Dextrans/immunology , Enterobacter cloacae/immunology , Ficoll/immunology , Haptens/immunology , Immunity, Humoral , Immunoglobulin M/immunology , Mice , Mice, Inbred BALB C , Muramidase/immunology , Phosphorylcholine/immunology , Picrates/immunology , Spleen/immunology , Streptococcus pneumoniae/immunology
8.
Eur J Pharmacol ; 605(1-3): 153-7, 2009 Mar 01.
Article in English | MEDLINE | ID: mdl-19248227

ABSTRACT

Previously, we indicated that athymic BALB/c-nu/nu (nude) mice that had been repeatedly treated with 2,4,6-trinitrochlorobenzene (TNCB) failed to exhibit chronic scratching behavior in spite of the accumulation of dermal mast cells in the lesion. The mice also failed to produce specific IgE or potent dermatitis. In the present study, therefore, we aimed to examine the role of IgE and repeated hapten treatment in the induction of scratching behavior and dermatitis using nude mice and trinitrophenol (TNP)-specific IgE-transgenic mice. The ears of nude mice were treated with TNCB 6 times at intervals of 48 h, and TNP-specific IgE was administered to the mice intravenously before the sixth TNCB treatment. The nude mice that had been supplemented with IgE exhibited a persistent increase in scratching behavior and continuous degranulation of mast cells. Furthermore, a potent immediate ear swelling was induced, although no biphasic dermatitis pattern was observed. On the other hand, the IgE-transgenic mice failed to exhibit persistent increases in scratching behavior after a single TNCB treatment, although biphasic ear swelling was observed. These results indicate that specific IgE plays an essential role in the induction of persistent increases in scratching behavior and continuous degranulation of mast cells. Furthermore, repeated challenge with the hapten also plays an important role in persistent increases in scratching behavior through accumulation and continuous activation of mast cells.


Subject(s)
Dermatitis, Allergic Contact/immunology , Immunoglobulin E/immunology , Pruritus/immunology , Animals , Dermatitis, Allergic Contact/physiopathology , Disease Models, Animal , Female , Haptens/immunology , Immunoglobulin E/administration & dosage , Mast Cells/immunology , Mice , Mice, Inbred BALB C , Mice, Nude , Mice, Transgenic , Picrates/immunology , Picryl Chloride/administration & dosage , Picryl Chloride/immunology , Pruritus/etiology
9.
Immunol Cell Biol ; 87(3): 241-8, 2009.
Article in English | MEDLINE | ID: mdl-19139762

ABSTRACT

The injection of antigen into the anterior chamber (AC, intracameral injection) of a murine eye induces the generation of splenic CD8(+) regulatory T cells (AC-SPL cells) that effect the antigen-specific suppression of a delayed-type hypersensitivity (DTH) reaction. Here we show (i) for the first time that the local antigen-specific suppression of DTH-induced swelling in immunized mice either by an intracameral injection of antigen or by the direct injection of CD8(+) AC-SPL cells into an antigen-challenged site is associated with an absence of infiltrated mononuclear cells, (ii) that the local antigen-specific suppression of the DTH reaction by CD8(+) AC-SPL cells requires compatibility between the Qa-1 but not H2 antigen haplotype of the immunized recipient and the injected AC-SPL regulatory T cells, (iii) that the suppression of the DTH reaction by CD8(+) AC-SPL cells requires the expression of Qa-1 but not H2 antigens and is not due to bystander suppression.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Histocompatibility Antigens Class I/immunology , Hypersensitivity, Delayed/therapy , Immunosuppression Therapy , Animals , Anterior Chamber , CD8-Positive T-Lymphocytes/transplantation , H-2 Antigens/genetics , H-2 Antigens/immunology , Haplotypes , Histocompatibility Antigens Class I/genetics , Hypersensitivity, Delayed/pathology , Immune Tolerance/genetics , Mice , Ovalbumin/immunology , Picrates/immunology , Picrates/pharmacology , Spleen/immunology , T-Lymphocytes, Regulatory/immunology
10.
Biotechnol Appl Biochem ; 52(Pt 1): 79-87, 2009 Jan.
Article in English | MEDLINE | ID: mdl-18627355

ABSTRACT

Nowadays, recombinant antibody and phage display technology enable the efficient generation of immunotools and a subsequent manipulation for optimized affinity, specificity or overall performance. Such advantages are of particular interest for haptenic target structures, such as TNT (2,4,6-trinitrotoluene). The toxicity of TNT and its breakdown products makes a reliable and fast detection of low levels in aqueous samples highly important. In the present study, we aimed for the generation of scFvs (single-chain antibody fragments) specific for the TNT-surrogate TNP (2,4,6-trinitrophenyl) and their subsequent production as monoclonal avian IgY immunoglobulins providing improved assay performance. Therefore we subjected a human synthetic scFv library to selection following different strategies. TNP-specific human antibody fragments could be identified, characterized for their primary structure and evaluated for production as soluble scFv in Escherichia coli. Additionally, a murine TNP-specific antibody fragment was obtained from the hybridoma 11B3; however, the prokaryotic expression level was found to be limited. To generate and evaluate immunoglobulin formats with superior characteristics, all recombinant antibody fragments then were converted into two different chimaeric bivalent IgY antibody formats. After expression in mammalian cells, the IgY antibodies were assessed for their reactivity towards TNT. The IgY antibodies generated on the basis of the combinatorial library proved to be useful for detection of TNT, thereby emphasizing the high potential of this approach for the development of detection devices for immunoassay-based techniques.


Subject(s)
Immunoglobulin Variable Region/immunology , Immunoglobulins/immunology , Picrates/immunology , Trinitrotoluene/immunology , Amino Acid Sequence , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Chickens , Environmental Monitoring/methods , Escherichia coli/metabolism , Gene Library , Haptens , Humans , Hybridomas/metabolism , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/genetics , Immunoglobulins/biosynthesis , Immunoglobulins/genetics , Mice , Models, Molecular , Molecular Sequence Data , Peptide Library , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology
11.
J Immunol ; 181(12): 8745-52, 2008 Dec 15.
Article in English | MEDLINE | ID: mdl-19050295

ABSTRACT

The relative ability of IgG subclasses to cause acute inflammation and the roles of specific effector mechanisms in this process are not clear. We explored this in an in vivo model of glomerular inflammation in the mouse. Trinitrophenol was planted on the glomerular basement membrane after conjugation to nephrotoxic Ab. The relative nephritogenicity of anti-trinitrophenol switch variant mAbs was then explored and shown to be IgG2a > IgG2b, with no disease caused by IgG1. Using knockout mice, we showed that FcgammaRIII was necessary for both neutrophil influx and glomerular damage induced by IgG2a and IgG2b. Surprisingly, IgG1 did not cause disease although it binds to FcgammaRIII. Using blocking Abs, we showed that this was explained by an additional requirement for FcgammaRIV, which does not bind to IgG1. IgG2a- or IgG2b-induced neutrophil influx was not affected by deficiency of either FcgammaRI or C3. Bone marrow chimeras were constructed to test the effect of combined deficiency of FcgammaRI and C3, and there was no effect on IgG2a- or IgG2b-mediated neutrophil influx. However, IgG2b-induced albuminuria and thrombosis were reduced in C3-deficient mice, showing an additional role for complement in IgG2b-mediated glomerular damage. The results show that IgG2a and IgG2b are the pathogenic subclasses in acute neutrophil-mediated glomerular inflammation, with an indispensable role for both FcgammaRIII and FcgammaRIV. Additionally, complement contributes to IgG2b-induced glomerular injury.


Subject(s)
Antibodies, Monoclonal/toxicity , Glomerulonephritis/immunology , Glomerulonephritis/pathology , Immunoglobulin Class Switching/genetics , Immunoglobulin G/classification , Immunoglobulin G/toxicity , Receptors, IgG/physiology , Acute Disease , Animals , Antibodies, Monoclonal/genetics , Complement Activation/genetics , Complement Activation/immunology , Complement C3/deficiency , Complement C3/genetics , Glomerulonephritis/metabolism , Immunoglobulin G/biosynthesis , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Picrates/immunology , Picrates/toxicity , Proteinuria/immunology , Proteinuria/pathology , Receptors, IgG/deficiency , Receptors, IgG/genetics , Thrombosis/immunology , Thrombosis/pathology
12.
Proteomics ; 8(14): 2840-8, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18655052

ABSTRACT

Antigen arrays are becoming widely used tools for the characterization of the complexity of humoral immune responses. Current antibody profiling techniques provide modest and indirect information about the effector functions of the antibodies that bind to particular antigens. Here we introduce an antigen array-based approach for obtaining immune profiles reflecting antibody functionality. This technology relies on the parallel measurement of antibody binding and complement activation by features of the array. By comparing sera from animals immunized against the same antigen under different conditions, we show that identifying the position of an antigen in a 2-D space, derived from antibody binding and complement deposition, permits distinction between immune profiles characterized by diverse antibody isotype distributions. Additionally, the technology provides a biologically interpretable graphical representation of the relationship between antigen and host. Our data suggest that 2-D immune profiling could enrich the data obtained from proteomic scale serum profiling studies.


Subject(s)
Antibody Formation , Antigens/immunology , Protein Array Analysis , Animals , Antibodies/metabolism , Antibodies/physiology , Antibody Affinity , Antibody Specificity/genetics , Antigens/metabolism , Binding Sites, Antibody , Complement System Proteins/deficiency , Complement System Proteins/metabolism , Complement System Proteins/physiology , Epitopes/genetics , Epitopes/immunology , Immunoglobulin G/biosynthesis , Immunoglobulin Isotypes/biosynthesis , Immunoglobulin Isotypes/metabolism , Immunoglobulin M/biosynthesis , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Picrates/administration & dosage , Picrates/immunology , Picrates/metabolism , Signal Transduction/immunology
14.
Gastroenterology ; 134(7): 2014-24, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18424268

ABSTRACT

BACKGROUND & AIMS: Treatment with ex vivo expanded regulatory T cells (Tregs) is regarded as a promising therapeutic approach in inflammatory bowel disease but is hampered by impaired Treg accumulation and function at inflammatory sites. We aim to study whether antigen-specific redirected Tregs can overcome these limitations. METHODS: We developed transgenic mice whose T cells, including Tregs, express chimeric receptor (CR) made of antibody variable region as recognition unit and T-cell stimulatory and costimulatory domains to activate specifically in response to the predetermined model antigen 2,4,6-trinitrophenol (TNP). RESULTS: TNP-specific CR-bearing Tregs were potently and specifically activated by exogenous TNP and suppressed effector T cells in the absence of costimulatory B7-CD28 interaction. TNP-specific transgenic (Tg) mice were resistant to 2,4,6-trinitrobenzene sulphonic acid (TNBS) colitis but not to other hapten-mediated colitis. Adoptive transfer of CR-bearing Tregs to wild-type mice with TNBS colitis was associated with significant amelioration of colitis and improved survival. Although TNP-specific CR-bearing Tregs did not suppress oxazolone colitis, they cured it after addition of traces of TNBS to oxazolone-inflamed colons, demonstrating a "bystander" effect. In vivo imaging of adoptively transferred CR-bearing Tregs demonstrated that they preferentially migrate to TNBS-induced colonic mucosal lesions within hours of induction of colitis. CONCLUSIONS: Tregs can be redirected with specificity distinct from that of pathogenic lymphocytes, accumulate at colonic inflammatory lesions, and suppress effector T cells in a specific, nonmajor histocompatibility complex-restricted, and noncostimulatory-dependent manner, resulting in significant amelioration of colitis. Hopefully, this approach will lead to a novel therapy for inflammatory bowel disease, as well as other inflammatory diseases.


Subject(s)
Colitis/therapy , Colon/immunology , Immunotherapy, Adoptive , Lymphocyte Activation , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes, Regulatory/transplantation , Animals , Bystander Effect , CD28 Antigens/immunology , Cell Line , Cell Movement , Colitis/chemically induced , Colitis/immunology , Colitis/pathology , Colitis/prevention & control , Colon/pathology , Disease Models, Animal , Forkhead Transcription Factors/metabolism , Histocompatibility Antigens/immunology , Immunity, Mucosal , Immunoglobulin Variable Region/immunology , Intestinal Mucosa/immunology , Mice , Mice, Inbred BALB C , Mice, Transgenic , Oxazolone , Picrates/immunology , Receptors, Antigen, T-Cell/genetics , Receptors, IgG/immunology , Recombinant Fusion Proteins/immunology , T-Lymphocytes, Regulatory/immunology , Time Factors , Trinitrobenzenesulfonic Acid
15.
Science ; 314(5807): 1936-8, 2006 Dec 22.
Article in English | MEDLINE | ID: mdl-17185603

ABSTRACT

Innate immune signals mediated by Toll-like receptors (TLRs) have been thought to contribute considerably to the antibody-enhancing effects of vaccine adjuvants. However, we report here that mice deficient in the critical signaling components for TLR mount robust antibody responses to T cell-dependent antigen given in four typical adjuvants: alum, Freund's complete adjuvant, Freund's incomplete adjuvant, and monophosphoryl-lipid A/trehalose dicorynomycolate adjuvant. We conclude that TLR signaling does not account for the action of classical adjuvants and does not fully explain the action of a strong adjuvant containing a TLR ligand. This may have important implications in the use and development of vaccine adjuvants.


Subject(s)
Adjuvants, Immunologic , Antibody Formation , B-Lymphocytes/immunology , Signal Transduction , Toll-Like Receptors/immunology , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/metabolism , Animals , Antigens, T-Independent/immunology , Cell Wall Skeleton/immunology , Cord Factors/immunology , Freund's Adjuvant/immunology , Haptens , Hemocyanins/immunology , Immunity, Innate , Immunization , Immunization, Secondary , Immunoglobulins/blood , Ligands , Lipid A/analogs & derivatives , Lipid A/immunology , Lipopolysaccharides/immunology , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/metabolism , Picrates/immunology , T-Lymphocytes/immunology , Toll-Like Receptors/metabolism , Vaccines/immunology
16.
Int Arch Allergy Immunol ; 141(2): 168-71, 2006.
Article in English | MEDLINE | ID: mdl-16899984

ABSTRACT

BACKGROUND: Homologous passive cutaneous anaphylaxis (PCA) is an established technique that provides a useful tool to study local inflammation due to mast cell activation. Since mast cells are responsive to repeated challenge by IgE receptor crosslinking in vitro, we investigated the responsiveness of mice to repeated PCA activation. METHODS: Female BALB/c mice were sensitized with intradermal injection of IgE into the ear and provocated through intravenous injection of trinitrophenol-BSA. Repeated provocation was administered, and the outcome was determined by colorimetrical measurement of Evans blue dye leakage. RESULTS: After local IgE injection, mice could be challenged to demonstrate a PCA reaction at least up to 13 days after IgE sensitization. In contrast, passively sensitized mice did not respond to repeated antigen challenge, i.e. a second provocation administered between 1 and 12 days after the first PCA reaction. However, if these mice were intradermally resensitized with IgE at the same site after the first challenge, they became responsive to the repeated challenge. CONCLUSIONS: Mast cells become desensitized upon PCA reaction, and resensitization with IgE is critical for mice repeatedly stimulated by PCA activation.


Subject(s)
Desensitization, Immunologic , Immune Tolerance , Immunoglobulin E/administration & dosage , Passive Cutaneous Anaphylaxis/immunology , Animals , Dose-Response Relationship, Drug , Female , Indicators and Reagents , Mast Cells/immunology , Mice , Mice, Inbred BALB C , Picrates/immunology , Serum Albumin, Bovine/immunology
17.
Scand J Immunol ; 62(5): 453-61, 2005 Nov.
Article in English | MEDLINE | ID: mdl-16305642

ABSTRACT

Antibodies, administered together with their specific antigen, can feedback-regulate antibody responses to this antigen. IgG1, IgG2a and IgG2b enhance antibody responses to soluble protein antigens. This effect is primarily mediated by FcRs as enhancement is impaired in FcR gamma-/- mice, reported to lack Fc gammaRI and Fc gammaRIII because of deletion of the common FcR gamma chain. Also IgG3 can enhance antibody responses. However, this effect is unperturbed in FcR gamma-/- mice but severely impaired in complement-depleted animals and in animals lacking complement receptor 1 and 2. Although this argues against involvement of Fc gammaRs, FcR gamma-/- mice may express one-fifth of the normal levels of Fc gammaRI and, in addition, Fc gammaRI has been suggested to bind IgG3. We re-investigated the dependence of IgG3-mediated enhancement on Fc gammaRs using a mouse strain selectively lacking Fc gammaRI and found that IgG3-mediated enhancement is completely normal. Unlike IgE and IgG2a, which are both thought to enhance T-cell proliferation via FcR-mediated antigen presentation, IgG3 was a poor enhancer of T-cell proliferation both in vivo and in vitro. These findings argue against a significant involvement of Fc gammaRs in IgG3-mediated enhancement of antibody responses and support our previous conclusion that complement plays a major role.


Subject(s)
Antibody Formation/immunology , Immunoglobulin G/immunology , Receptors, IgG/genetics , Adoptive Transfer , Animals , Antibody Formation/drug effects , Antigen Presentation/drug effects , Antigen Presentation/immunology , CD4-Positive T-Lymphocytes/immunology , Hemocyanins/immunology , Immunoglobulin G/pharmacology , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, Transgenic , Ovalbumin/immunology , Picrates/immunology , Receptors, IgG/immunology , Vaccination
18.
Ann Allergy Asthma Immunol ; 94(5): 575-80, 2005 May.
Article in English | MEDLINE | ID: mdl-15945561

ABSTRACT

BACKGROUND: Rapid administration of suboptimal antigen induces transient unresponsiveness in patients with IgE antibodies to beneficial medications, but the molecular mechanisms of desensitization are poorly understood. Mast cells (MCs) have been implicated as the target cells. OBJECTIVE: To establish a physiologic model of IgE-antigen desensitization using mouse bone marrow-derived MCs (mBMMCs) from wild-type and signal transducer and activator of transcription 6 (STAT6)-deficient mice. METHODS: The mBMMCs were sensitized with dinitrophenyl (DNP) IgE or trinitrophenyl (TNP) IgE and activated with DNP/TNP-human serum albumin. For desensitization, suboptimal doses of DNP/TNP-human serum albumin were administered at fixed intervals. RESULTS: Desensitized mBMMCs failed to respond to an optimal dose of antigen, indicating successful desensitization. Desensitization was time dependent, with 5 minutes of antigen exposure being optimal. Resensitization with DNP-IgE did not reverse the process. The desensitized cells were responsive to calcium ionophore and phorbol myristate acetate. Thus, the desensitization reaction alters an early event in the high-affinity IgE receptor (FcepsilonRI)-dependent signaling pathway in a nontoxic manner. The mBMMCs from STAT6-null mice could not be desensitized by suboptimal doses of antigen. CONCLUSIONS: Mast cells can be rendered unresponsive by rapid administration of suboptimal doses of antigen in the presence of calcium, similar to in vivo desensitizations. The STAT6-null mBMMCs cannot be desensitized, providing the first molecular target in this inhibitory process.


Subject(s)
Antigens/administration & dosage , Desensitization, Immunologic , Mast Cells/immunology , Trans-Activators/deficiency , Animals , Antibodies, Monoclonal/immunology , Antigens/immunology , Bone Marrow/immunology , Dinitrobenzenes/immunology , Dose-Response Relationship, Immunologic , Immunization , Mice , Mice, Inbred BALB C/genetics , Mice, Inbred C57BL/genetics , Picrates/immunology , STAT6 Transcription Factor , Time Factors , Trans-Activators/genetics , Trans-Activators/immunology
19.
J Neuroimmunol ; 153(1-2): 40-9, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15265662

ABSTRACT

Antigen injection into the eye's anterior chamber (AC) induces the antigen-specific suppression of delayed-type hypersensitivity (DTH) that is mediated by NKT cells and splenic CD8+ suppressor T cells. Because the AC, uveal tissues, the thymus and spleen required to induce anterior chamber-associated immune deviation (ACAID) have dense sympathetic innervations, we examined the effects of chemical sympathectomy of mice by 6-hydroxydopamine (6-OHDA) on the induction of the suppression of contact sensitivity to trinitrophenol (TNP) induced by the injection of TNP-bovine serum albumin (BSA) into the anterior chamber. DTH measured as contact sensitivity to picrylchloride was not induced in mice that received 6-OHDA before immunization with TNP-BSA. Although spleen cells from 6-OHDA-treated TNP-BSA-immunized mice produced IFN-gamma when stimulated by TNP-BSA, the number of DTH-initiating hepatic NKT cells was reduced markedly in 6-OHDA-treated mice. Chemically denervated mice did not produce splenic suppressor T cells or thymic NKT cells that activate splenic suppressor T cells. We suggest that an intact sympathetic nervous system (SNS) is required to maintain cellular immunoregulation.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , Spleen/cytology , Sympathetic Nervous System/immunology , Animals , Anterior Chamber/drug effects , Anterior Chamber/immunology , Antibody Formation , Desipramine/pharmacology , Drug Interactions , Enzyme Inhibitors/pharmacology , Enzyme-Linked Immunosorbent Assay/methods , Female , Hypersensitivity, Delayed/etiology , Hypersensitivity, Delayed/immunology , Immune Tolerance/drug effects , Immunization/methods , Immunosuppression Therapy , Interferon-gamma/biosynthesis , Killer Cells, Natural/immunology , Leukocytes, Mononuclear/immunology , Liver/cytology , Liver/immunology , Lymphocyte Activation/drug effects , Mice , Mice, Inbred C57BL , Oxidopamine/toxicity , Picrates/immunology , Picrates/pharmacology , Rheology , Serum Albumin, Bovine/immunology , Spleen/immunology , Sympathectomy/methods , Sympathetic Nervous System/drug effects , Sympatholytics/toxicity , Thymus Gland/cytology , Thymus Gland/immunology , Trinitrobenzenesulfonic Acid/immunology
20.
Scand J Immunol ; 59(5): 469-77, 2004 May.
Article in English | MEDLINE | ID: mdl-15140057

ABSTRACT

In susceptible mice, mercuric chloride induces a systemic autoimmune disease characterized by increased serum levels of immunoglobulin (Ig) G1 and IgE, production of anti-nucleolar autoantibodies (ANolA) and formation of renal IgG deposits. We have previously hypothesized that mercury confers more adverse immunological effects on those mouse strains, which are genetically prone to develop spontaneous autoimmune diseases than on normal strains. In this study, we tested our hypothesis in tight skin 1 (Tsk1/+) mice, a murine model for human scleroderma. As a support for our hypothesis, we observed that in Tsk1/+ mice, B cells were spontaneously hyperactive and that treatment with mercury induced a strong immune/autoimmune response in these mice, but not in their non-Tsk (+/+) littermates. This response was characterized by the formation of high numbers of splenic IgG1, IgG2b and IgG3 antibody-secreting cells, increased serum levels of IgE, production of IgG1 antibodies against single-stranded DNA (ssDNA), trinitrophenol (TNP) as well as thyroglobulin and the development of renal IgG1 deposits. Neither Tsk1/+ mice nor F1 hybrid crosses between this strain, and mercury susceptible B10.S (H-2(s)) were able to produce IgG1-ANolA in response to mercury. Moreover, mercury-induced immune activation in Tsk1/+ was not able to potentiate the progression of skin fibrosis in this strain. Thus, exposure to mercury accelerates the immune dysregulation, but not the development of skin fibrosis in Tsk1/+ mice.


Subject(s)
Fibrosis/immunology , Mercuric Chloride/immunology , Mercuric Chloride/toxicity , Scleroderma, Systemic/immunology , Skin/drug effects , Animals , Autoantibodies/biosynthesis , Autoantibodies/drug effects , B-Lymphocytes/immunology , DNA, Single-Stranded/immunology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Female , Fibrosis/pathology , Immunoglobulin G/biosynthesis , Immunoglobulin G/drug effects , Immunoglobulin Isotypes , Kidney/pathology , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Male , Mice , Mice, Mutant Strains , Picrates/immunology , Skin/immunology , Skin/pathology , Thyroglobulin/immunology
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