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Interaction Between Regulatory T Cells and Antibody-Producing B Cells for Immune Responses at the Upper Respiratory Mucosa Against Nontypeable Haemophilus influenzae: In Vitro Assay Model

Hirano, T;Kadowaki, Y;Matsunaga, T;Yoshinaga, K;Kawano, T;Moriyama, M;Suzuki, M;
Product: Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

The aim of this study was to investigate the effect of regulatory T cells (Tregs) on B-cell immune responses against outer membrane protein (OMP) from nontypeable Haemophilus influenzae (NTHi) in vitro, to clarify its exact mechanism from an immunologic standpoint. Mice were vaccinated intranasally with OMP to induce OMP-specific immune responses in the nasal mucosa. Mononuclear cells (MNCs) were collected from the nasal mucosa, and Tregs and helper T (Th) cells were isolated separately from the spleens of those mice. Three different cell culture groups were allocated: MNCs cocultured with Tregs, MNCs cocultured with Th cells, and MNCs cultured alone. At 24 and 72 hours after cell culture, the concentrations of various cytokines and antibodies in culture supernatants were measured to assess the effects of Tregs and Th cells on B-cell responses. Cytokine levels and specific anti-OMP antibody levels in culture media were determined using enzyme-linked immunosorbent assay. CD69 or CD80 expression on B220-positive cells was detected using flow cytometric analysis. Th1 and Th2 cytokine concentrations were significantly elevated in the 3 groups incubated with OMP from 24 to 72 hours. Additionally, interleukin-10 levels were significantly higher in the Treg and Th groups than in the control group. Levels of OMP-specific immunoglobulin A did not differ significantly among the groups. The ratios of CD69+B220+ B2 cells were nearly the same in the 3 groups; however, the ratio of CD80+B220+ B2 cells was higher in the control group than in the Treg and Th groups during incubation. Tregs and Th cells did not affect OMP-specific immunoglobulin A production in this study. However, these cells may partially inhibit B-cell functions, such as T-cell activation. These inhibitory effects may be related to interleukin-10.

PubMed ID: 31092026
359335932019-06-172019-06-1714:32:1914:32:192019-07-052019-07-0515:16:3115:16:31Hirano, T;Kadowaki, Y;Matsunaga, T;Yoshinaga, K;Kawano, T;Moriyama, M;Suzuki, M;Hirano, T;Kadowaki, Y;Matsunaga, T;Yoshinaga, K;Kawano, T;Moriyama, M;Suzuki, M;20192019Interaction Between Regulatory T Cells and Antibody-Producing B Cells for Immune Responses at the Upper Respiratory Mucosa Against Nontypeable Haemophilus influenzae: In Vitro Assay ModelInteraction Between Regulatory T Cells and Antibody-Producing B Cells for Immune Responses at the Upper Respiratory Mucosa Against Nontypeable Haemophilus influenzae: In Vitro Assay ModelThe Annals Of Otology, Rhinology, And LaryngologyThe Annals Of Otology, Rhinology, And Laryngology45S-51S45S-51S1281286_suppl6_suppl3109202631092026

The aim of this study was to investigate the effect of regulatory T cells (Tregs) on B-cell immune responses against outer membrane protein (OMP) from nontypeable Haemophilus influenzae (NTHi) in vitro, to clarify its exact mechanism from an immunologic standpoint. Mice were vaccinated intranasally with OMP to induce OMP-specific immune responses in the nasal mucosa. Mononuclear cells (MNCs) were collected from the nasal mucosa, and Tregs and helper T (Th) cells were isolated separately from the spleens of those mice. Three different cell culture groups were allocated: MNCs cocultured with Tregs, MNCs cocultured with Th cells, and MNCs cultured alone. At 24 and 72 hours after cell culture, the concentrations of various cytokines and antibodies in culture supernatants were measured to assess the effects of Tregs and Th cells on B-cell responses. Cytokine levels and specific anti-OMP antibody levels in culture media were determined using enzyme-linked immunosorbent assay. CD69 or CD80 expression on B220-positive cells was detected using flow cytometric analysis. Th1 and Th2 cytokine concentrations were significantly elevated in the 3 groups incubated with OMP from 24 to 72 hours. Additionally, interleukin-10 levels were significantly higher in the Treg and Th groups than in the control group. Levels of OMP-specific immunoglobulin A did not differ significantly among the groups. The ratios of CD69+B220+ B2 cells were nearly the same in the 3 groups; however, the ratio of CD80+B220+ B2 cells was higher in the control group than in the Treg and Th groups during incubation. Tregs and Th cells did not affect OMP-specific immunoglobulin A production in this study. However, these cells may partially inhibit B-cell functions, such as T-cell activation. These inhibitory effects may be related to interleukin-10.

The aim of this study was to investigate the effect of regulatory T cells (Tregs) on B-cell immune responses against outer membrane protein (OMP) from nontypeable Haemophilus influenzae (NTHi) in vitro, to clarify its exact mechanism from an immunologic standpoint. Mice were vaccinated intranasally with OMP to induce OMP-specific immune responses in the nasal mucosa. Mononuclear cells (MNCs) were collected from the nasal mucosa, and Tregs and helper T (Th) cells were isolated separately from the spleens of those mice. Three different cell culture groups were allocated: MNCs cocultured with Tregs, MNCs cocultured with Th cells, and MNCs cultured alone. At 24 and 72 hours after cell culture, the concentrations of various cytokines and antibodies in culture supernatants were measured to assess the effects of Tregs and Th cells on B-cell responses. Cytokine levels and specific anti-OMP antibody levels in culture media were determined using enzyme-linked immunosorbent assay. CD69 or CD80 expression on B220-positive cells was detected using flow cytometric analysis. Th1 and Th2 cytokine concentrations were significantly elevated in the 3 groups incubated with OMP from 24 to 72 hours. Additionally, interleukin-10 levels were significantly higher in the Treg and Th groups than in the control group. Levels of OMP-specific immunoglobulin A did not differ significantly among the groups. The ratios of CD69+B220+ B2 cells were nearly the same in the 3 groups; however, the ratio of CD80+B220+ B2 cells was higher in the control group than in the Treg and Th groups during incubation. Tregs and Th cells did not affect OMP-specific immunoglobulin A production in this study. However, these cells may partially inhibit B-cell functions, such as T-cell activation. These inhibitory effects may be related to interleukin-10.

1.41.4

... The vaccine contained a mixture of 10 µg OMP and 1 µg cholera toxin as an adjuvant (List Biological Laboratories, Campbell, California, USA) suspended in phosphate-buffered saline. ...

 

Author did not specify which Cholera toxin was utilized.  List Labs Product #101B/C - Cholera Toxin from Vibrio Cholerae has been discontinued; however, List Labs still provides Product #100B - Cholera Toxin (AZIDE-FREE) from Vibrio cholerae.  

To follow, are our recommendations for transitioning from Product #101B/C to Product #100B:

Product #101B, Cholera Toxin from Vibrio cholerae (1 mg), has been discontinued.  Product #100B, Cholera Toxin (AZIDE-FREE) from Vibrio cholerae, may be substituted for Product #101B, if the user accounts for the formulation differences.  Both products contain the same quantity of toxin per vial (1.0 mg).  For Product #100B, reconstitution with 1.0 mL of water results in a 1 mg/mL toxin solution in a buffer with the following component concentrations:  0.05 M Tris, 0.2 M NaCl, 0.001 M Na2EDTA at pH 7.5.  For Product #101B, reconstitution with half that amount of water (0.5 mL) resulted in a 2 mg/mL toxin solution with the same concentrations of the buffer components, and in addition, Product #101B contained 0.003 M NaN3.  The user may add the sodium azide preservative to product #100B, if desired.

Product #101C, Cholera Toxin from Vibrio cholerae (2 mg), has been discontinued.  Product #100B, Cholera Toxin (AZIDE-FREE) from Vibrio cholerae, may be substituted for #101C, if the researcher accounts for the formulation differences.  While Product #101C contained 2.0 mg per vial, Product #100 contains 1.0 mg per vial.  When Product #100B is reconstituted with 1.0 mL of water, the resulting 1 mg/mL toxin solution will have buffer components with the following concentrations:  0.05 M Tris, 0.2 M NaCl, 0.001 M Na2EDTA at pH 7.5.  For Product #101C, reconstitution with less than half that amount of water (0.4 mL) resulted in a 5 mg/mL toxin solution in the same buffer, and in addition, Product #101C contained 0.003 M NaN3.  The user may add the sodium azide preservative to product #100B, if desired. 

Large quantities of Product #101B or Product #101C may be obtained through Bulk Requests and Customer Orders

... The vaccine contained a mixture of 10 µg OMP and 1 µg cholera toxin as an adjuvant (List Biological Laboratories, Campbell, California, USA) suspended in phosphate-buffered saline. ...

 

Author did not specify which Cholera toxin was utilized.  List Labs Product #101B/C - Cholera Toxin from Vibrio Cholerae has been discontinued; however, List Labs still provides Product #100B - Cholera Toxin (AZIDE-FREE) from Vibrio cholerae.  

To follow, are our recommendations for transitioning from Product #101B/C to Product #100B:

Product #101B, Cholera Toxin from Vibrio cholerae (1 mg), has been discontinued.  Product #100B, Cholera Toxin (AZIDE-FREE) from Vibrio cholerae, may be substituted for Product #101B, if the user accounts for the formulation differences.  Both products contain the same quantity of toxin per vial (1.0 mg).  For Product #100B, reconstitution with 1.0 mL of water results in a 1 mg/mL toxin solution in a buffer with the following component concentrations:  0.05 M Tris, 0.2 M NaCl, 0.001 M Na2EDTA at pH 7.5.  For Product #101B, reconstitution with half that amount of water (0.5 mL) resulted in a 2 mg/mL toxin solution with the same concentrations of the buffer components, and in addition, Product #101B contained 0.003 M NaN3.  The user may add the sodium azide preservative to product #100B, if desired.

Product #101C, Cholera Toxin from Vibrio cholerae (2 mg), has been discontinued.  Product #100B, Cholera Toxin (AZIDE-FREE) from Vibrio cholerae, may be substituted for #101C, if the researcher accounts for the formulation differences.  While Product #101C contained 2.0 mg per vial, Product #100 contains 1.0 mg per vial.  When Product #100B is reconstituted with 1.0 mL of water, the resulting 1 mg/mL toxin solution will have buffer components with the following concentrations:  0.05 M Tris, 0.2 M NaCl, 0.001 M Na2EDTA at pH 7.5.  For Product #101C, reconstitution with less than half that amount of water (0.4 mL) resulted in a 5 mg/mL toxin solution in the same buffer, and in addition, Product #101C contained 0.003 M NaN3.  The user may add the sodium azide preservative to product #100B, if desired. 

Large quantities of Product #101B or Product #101C may be obtained through Bulk Requests and Customer Orders

https://journals.sagepub.com/doi/abs/10.1177/0003489419837994https://journals.sagepub.com/doi/abs/10.1177/00034894198379942019-06-012019-06-0110.1177/000348941983799410.1177/0003489419837994Cholera Toxin (AZIDE-FREE) from Vibrio choleraeCholera Toxin (AZIDE-FREE) from Vibrio choleraethirano@oita-u.ac.jpthirano@oita-u.ac.jpActivation;Adjuvant;Analysis;Anti;Antibodies;Antibody;Assay;Assess;B-cell;Biological;Cell;Cell Culture;Cholera;Control;Culture;Cytokine;Enzyme;Expression;In vitro;Induce;Inhibit;Inhibitory;Interleukin;Isolated;Linked;List;List Biological;Mechanism;Membrane;Mucosa;Nasal;Positive;Production;Protein;Regulatory;Respiratory;Specific;Study;T-cell;Toxin;Vaccine;The Annals Of Otology, Rhinology, And LaryngologyActivation;Adjuvant;Analysis;Anti;Antibodies;Antibody;Assay;Assess;B-cell;Biological;Cell;Cell Culture;Cholera;Control;Culture;Cytokine;Enzyme;Expression;In vitro;Induce;Inhibit;Inhibitory;Interleukin;Isolated;Linked;List;List Biological;Mechanism;Membrane;Mucosa;Nasal;Positive;Production;Protein;Regulatory;Respiratory;Specific;Study;T-cell;Toxin;Vaccine;The Annals Of Otology, Rhinology, And Laryngology100B100Binteraction-between-regulatory-t-cellsinteraction-between-regulatory-t-cells