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Whole cell & culture filtrate proteins from prevalent genotypes of Mycobacterium tuberculosis provoke better antibody & T cell response than laboratory strain H 37 Rv.

Kumar G, Shankar H, Chahar M, Sharma P, Yadav VS, Chauhan DS, Katoch VM, Joshi B - Indian J. Med. Res. (2012)

Bottom Line: Of the WCE of 39 clinical isolates, 10 were found to be potent inducer of lymphoproliferation as well as cytokine secretion (P<0.05) in peripheral blood mononuclear cells from PPD+ healthy controls.Six culture filtrate proteins (CFPs) from these selected clinical isolates were also better inducers of antibody and T-cell response.Overall, our results revealed that the clinical isolates belonging to prevalent genotypes; CAS1_Del (ST-26), East African-Indian (ST-11) and Beijing family (ST-1) induced better antibody and T cell responses compared to H 37 Rv laboratory strain.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, National JALMA Institute for Leprosy & Other Mycobacterial Diseases (ICMR), Agra, India.

ABSTRACT

Background & objectives: The immune responses to different antigens of Mycobacterium tuberculosis H 37 Rv vary from patient to patient with tuberculosis (TB). Therefore, significant difference might be documented between the H 37 Rv with long histories of passages and recent clinical isolates of M. tuberculosis. In the present study, immune response of TB patients and healthy controls against 39 clinical M. tuberculosis isolates was correlated with laboratory strain H 37 Rv.

Methods: The antibody response was studied coating whole cell extracts and culture filtrate proteins of M. tuberculosis isolates and laboratory strain H 37Rv by enzyme linked immunosorbent assay (ELISA). Lymphoproliferation was studied by incorporation of tritiated thymidine and cytokines (IFN-γ and IL-4) by using commercially available kits.

Results: Sero-reactivity to whole cell extract (WCE) of 11 clinical isolates was higher with pooled serum and individual's serum from tuberculosis patients showed significant reactivity (P<0.05) to ten of these isolates using ELISA. Of the WCE of 39 clinical isolates, 10 were found to be potent inducer of lymphoproliferation as well as cytokine secretion (P<0.05) in peripheral blood mononuclear cells from PPD+ healthy controls. Six culture filtrate proteins (CFPs) from these selected clinical isolates were also better inducers of antibody and T-cell response.

Interpretation & conclusion: Overall, our results revealed that the clinical isolates belonging to prevalent genotypes; CAS1_Del (ST-26), East African-Indian (ST-11) and Beijing family (ST-1) induced better antibody and T cell responses compared to H 37 Rv laboratory strain. Further studies need to be done to purify and identify the dominant protein (s) using whole cell extract and culture filtrates from these immunologically relevant clinical M. tuberculosis isolates, which will be worthwhile to find out pathogenic factors, potential diagnostic markers and protective molecules for tuberculosis.

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Related in: MedlinePlus

(a): T-cell proliferation response and (b) IFN-γ levels secreted by PBMCs of healthy individuals induced by whole cell extracts from 39 clinical M. tuberculosis isolates (CI), H37Rv, PPD standard, and Mitogen phytohemagglutinin (PHA); Ctrl, unstimulated PBMC, Bar represents the mean value of stimulation index (SI) for each isolate. Statistical significance of T-cell proliferation response was calculated using paired t-test by comparing the stimulation index values of H37Rv and clinical isolates, P value for IFN-γ secretion was calculated by comparing unstimulated PBMCs (Ctrl) with WCE or CFP stimulated PBMCs, *P<0.05, **P<0.01, ***P<0.001.
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Figure 2: (a): T-cell proliferation response and (b) IFN-γ levels secreted by PBMCs of healthy individuals induced by whole cell extracts from 39 clinical M. tuberculosis isolates (CI), H37Rv, PPD standard, and Mitogen phytohemagglutinin (PHA); Ctrl, unstimulated PBMC, Bar represents the mean value of stimulation index (SI) for each isolate. Statistical significance of T-cell proliferation response was calculated using paired t-test by comparing the stimulation index values of H37Rv and clinical isolates, P value for IFN-γ secretion was calculated by comparing unstimulated PBMCs (Ctrl) with WCE or CFP stimulated PBMCs, *P<0.05, **P<0.01, ***P<0.001.

Mentions: Cell mediated immune response of whole cell extracted and culture filtrate proteins: The WCE of CI1, 5, 6, 11, 20, 23, 27, 31, 32 and 39 induced significantly higher levels of T-cell proliferation as compared to H37Rv and IFN-γ secretion as compared to unstimulated PBMCs (fig. 2a, 2b and Table III) but IL-4 levels was not significantly higher for any WCE of clinical isolate as well as for H37Rv as compared to unstimulated PBMCs (data not shown). The relative efficacies of CFP preparations from 10 selected clinical isolates of M. tuberculosis were also analyzed by stimulating PBMCs from healthy donors. A comparison of the different CFP preparations revealed that the CFP of CI5, 11, 20, 27, 31, 32 and 39 clinical isolates were statistically more potent in inducing cell proliferation than those of H37Rv and PPD (P<0.05 for CI5, 11, 20, 27, 39 and <0.001 for CI31) (Fig. 3a). CFP preparations of clinical isolates which were significant inducers of T-cell proliferation were also the potent stimulator of IFN-γ secretion by PBMCs (Fig. 3b and 3c). Responses to different CFP preparations indicated that the CFPs derived from CI39 induced maximum IFN-γ, which was significantly higher (P <0.001) than the response to CFPs of H37Rv and PPD (Fig. 3b). IL-4 concentration was also determined in culture supernatants of PBMCs stimulated with or without CFPs (data not shown). A moderate increase in IL-4 secretion was induced only by some CFPs (CI5, CI32 and CI39). The relative efficacy of different CFP preparations to induce T-cell proliferation and IFN-γ secretion were not similar for all PBMC preparations. Efficacy of different CFP preparations from clinical isolates in proliferation and IFN-γ assays were correlated and a positive correlation between the two parameters of T-cell activation was observed for clinical isolates (r, 0.697; Fig. 3c).


Whole cell & culture filtrate proteins from prevalent genotypes of Mycobacterium tuberculosis provoke better antibody & T cell response than laboratory strain H 37 Rv.

Kumar G, Shankar H, Chahar M, Sharma P, Yadav VS, Chauhan DS, Katoch VM, Joshi B - Indian J. Med. Res. (2012)

(a): T-cell proliferation response and (b) IFN-γ levels secreted by PBMCs of healthy individuals induced by whole cell extracts from 39 clinical M. tuberculosis isolates (CI), H37Rv, PPD standard, and Mitogen phytohemagglutinin (PHA); Ctrl, unstimulated PBMC, Bar represents the mean value of stimulation index (SI) for each isolate. Statistical significance of T-cell proliferation response was calculated using paired t-test by comparing the stimulation index values of H37Rv and clinical isolates, P value for IFN-γ secretion was calculated by comparing unstimulated PBMCs (Ctrl) with WCE or CFP stimulated PBMCs, *P<0.05, **P<0.01, ***P<0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3401709&req=5

Figure 2: (a): T-cell proliferation response and (b) IFN-γ levels secreted by PBMCs of healthy individuals induced by whole cell extracts from 39 clinical M. tuberculosis isolates (CI), H37Rv, PPD standard, and Mitogen phytohemagglutinin (PHA); Ctrl, unstimulated PBMC, Bar represents the mean value of stimulation index (SI) for each isolate. Statistical significance of T-cell proliferation response was calculated using paired t-test by comparing the stimulation index values of H37Rv and clinical isolates, P value for IFN-γ secretion was calculated by comparing unstimulated PBMCs (Ctrl) with WCE or CFP stimulated PBMCs, *P<0.05, **P<0.01, ***P<0.001.
Mentions: Cell mediated immune response of whole cell extracted and culture filtrate proteins: The WCE of CI1, 5, 6, 11, 20, 23, 27, 31, 32 and 39 induced significantly higher levels of T-cell proliferation as compared to H37Rv and IFN-γ secretion as compared to unstimulated PBMCs (fig. 2a, 2b and Table III) but IL-4 levels was not significantly higher for any WCE of clinical isolate as well as for H37Rv as compared to unstimulated PBMCs (data not shown). The relative efficacies of CFP preparations from 10 selected clinical isolates of M. tuberculosis were also analyzed by stimulating PBMCs from healthy donors. A comparison of the different CFP preparations revealed that the CFP of CI5, 11, 20, 27, 31, 32 and 39 clinical isolates were statistically more potent in inducing cell proliferation than those of H37Rv and PPD (P<0.05 for CI5, 11, 20, 27, 39 and <0.001 for CI31) (Fig. 3a). CFP preparations of clinical isolates which were significant inducers of T-cell proliferation were also the potent stimulator of IFN-γ secretion by PBMCs (Fig. 3b and 3c). Responses to different CFP preparations indicated that the CFPs derived from CI39 induced maximum IFN-γ, which was significantly higher (P <0.001) than the response to CFPs of H37Rv and PPD (Fig. 3b). IL-4 concentration was also determined in culture supernatants of PBMCs stimulated with or without CFPs (data not shown). A moderate increase in IL-4 secretion was induced only by some CFPs (CI5, CI32 and CI39). The relative efficacy of different CFP preparations to induce T-cell proliferation and IFN-γ secretion were not similar for all PBMC preparations. Efficacy of different CFP preparations from clinical isolates in proliferation and IFN-γ assays were correlated and a positive correlation between the two parameters of T-cell activation was observed for clinical isolates (r, 0.697; Fig. 3c).

Bottom Line: Of the WCE of 39 clinical isolates, 10 were found to be potent inducer of lymphoproliferation as well as cytokine secretion (P<0.05) in peripheral blood mononuclear cells from PPD+ healthy controls.Six culture filtrate proteins (CFPs) from these selected clinical isolates were also better inducers of antibody and T-cell response.Overall, our results revealed that the clinical isolates belonging to prevalent genotypes; CAS1_Del (ST-26), East African-Indian (ST-11) and Beijing family (ST-1) induced better antibody and T cell responses compared to H 37 Rv laboratory strain.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, National JALMA Institute for Leprosy & Other Mycobacterial Diseases (ICMR), Agra, India.

ABSTRACT

Background & objectives: The immune responses to different antigens of Mycobacterium tuberculosis H 37 Rv vary from patient to patient with tuberculosis (TB). Therefore, significant difference might be documented between the H 37 Rv with long histories of passages and recent clinical isolates of M. tuberculosis. In the present study, immune response of TB patients and healthy controls against 39 clinical M. tuberculosis isolates was correlated with laboratory strain H 37 Rv.

Methods: The antibody response was studied coating whole cell extracts and culture filtrate proteins of M. tuberculosis isolates and laboratory strain H 37Rv by enzyme linked immunosorbent assay (ELISA). Lymphoproliferation was studied by incorporation of tritiated thymidine and cytokines (IFN-γ and IL-4) by using commercially available kits.

Results: Sero-reactivity to whole cell extract (WCE) of 11 clinical isolates was higher with pooled serum and individual's serum from tuberculosis patients showed significant reactivity (P<0.05) to ten of these isolates using ELISA. Of the WCE of 39 clinical isolates, 10 were found to be potent inducer of lymphoproliferation as well as cytokine secretion (P<0.05) in peripheral blood mononuclear cells from PPD+ healthy controls. Six culture filtrate proteins (CFPs) from these selected clinical isolates were also better inducers of antibody and T-cell response.

Interpretation & conclusion: Overall, our results revealed that the clinical isolates belonging to prevalent genotypes; CAS1_Del (ST-26), East African-Indian (ST-11) and Beijing family (ST-1) induced better antibody and T cell responses compared to H 37 Rv laboratory strain. Further studies need to be done to purify and identify the dominant protein (s) using whole cell extract and culture filtrates from these immunologically relevant clinical M. tuberculosis isolates, which will be worthwhile to find out pathogenic factors, potential diagnostic markers and protective molecules for tuberculosis.

Show MeSH
Related in: MedlinePlus