Search

Lymph node culture-positive tuberculosis (LNTB+) is associated with increased mycobacterial antigen-induced pro-inflammatory cytokine production compared to LN culture-negative tuberculosis (LNTB-). However, the frequencies of CD4+, CD8+ T cells and NK cells expressing Th1/Tc1/Type 1 (IFNγ, TNFα, IL-2), Th17/Tc17/Type 17 (IL-17A, IL-17F, IL-22) cytokines and cytotoxic (perforin [PFN], granzyme [GZE] B, CD107a) markers in LNTB+ and LNTB- individuals are not known. Thus, we have studied the unstimulated (UNS) and mycobacterial antigen-induced frequencies of CD4+, CD8+ T and NK cells expressing Th1, Th17 cytokines and cytotoxic markers using flow cytometry. The frequencies of CD4+, CD8+ T and NK cells expressing cytokines and cytotoxic markers were not significantly different between LNTB+ and LNTB- individuals in UNS condition. In contrast, upon Mtb antigen stimulation, LNTB+ individuals are associated with significantly increased frequencies of CD4+ T cells (PPD [IFNγ, TNFα], ESAT-6 PP [IFNγ, TNFα], CFP-10 PP [IFNγ, TNFα, IL-2]), CD8+ T cells (PPD [IFNγ], ESAT-6 PP [IFNγ], CFP-10 PP [TNFα]) and NK cells (PPD [IFNγ, TNFα], ESAT-6 PP [IFNγ, TNFα], CFP-10 PP [TNFα]) expressing Th1/Tc1/Type 1, but not Th17/Tc17/Type 17 cytokines and cytotoxic markers compared to LNTB- individuals. LNTB+ individuals did not show any significant alterations in the frequencies of CD4+, CD8+ T cells and NK cells expressing cytokines and cytotoxic markers compared to LNTB- individuals upon HIV Gag PP and P/I antigen stimulation. Increased frequencies of CD4+, CD8+ T and NK cells expressing Th1/Tc1/Type 1 cytokines among the LNTB+ group indicates that the presence of mycobacteria plays a dominant role in the activation of key correlates of immune protection or induces higher immunopathology., Competing Interests: The authors have declared that no competing interests exist.

Background: Neutrophils are important for host innate immune defense and mediate inflammatory responses. Pulmonary tuberculosis (PTB) is associated with increased neutrophil granular protein (NGP) levels in the circulation. However, the systemic levels of neutrophil granular proteins were not examined in tuberculous lymphadenitis (TBL) disease., Methods: We measured the systemic levels of NGP (myeloperoxidase [MPO], elastase and proteinase 3 [PRTN3]) in TBL and compared them to latent tuberculosis (LTB) and healthy control (HC) individuals. We also measured the pre-treatment (Pre-T) and post-treatment (Post-T) systemic levels of neutrophil granular proteins in TBL individuals upon anti-tuberculosis treatment (ATT) completion. In addition, we studied the correlation and discriminatory ability of NGPs using receiver operating characteristic (ROC) analysis., Results: Our data suggests that systemic levels of NGPs (MPO, PRTN3, elastase) were significantly reduced in TBL individuals compared to LTB and HC individuals. Similarly, after ATT, the plasma levels of MPO and elastase but not PRTN3 were significantly elevated compared to pre-treatment levels. NGPs (except PRTN3) were positively correlated with absolute neutrophil count of TBL, LTB and HC individuals. Further, NGPs were able to significantly discriminate TBL from LTB and HC individuals., Conclusion: Hence, we conclude reduced neutrophil granular protein levels might be associated with disease pathogenesis in TBL., Competing Interests: The authors have declared that no competing interests exist.

Background: Pulmonary tuberculosis (PTB) is characterized by elevated levels of acute phase proteins (APPs), but their association with tuberculous lymphadenitis (TBL) is poorly studied., Methods: We examined the systemic levels of APPs (alpha-2-macroglobulin [⍺-2MG], serum amyloid A [SAA], C-reactive protein [CRP] and haptoglobin [Hp]) in TBL, PTB, latent tuberculosis (LTB) and healthy controls (HC) at baseline and in TBL after the completion of anti-tuberculosis treatment (ATT). We have also examined the association of these proteins with lymph node (LN) size, culture grade and multiple versus single LN involvement., Results: TBL individuals exhibited increased systemic levels of ⍺-2MG, SAA, CRP and Hp in comparison to HCs and increased CRP levels in comparison to LTB individuals. TBL individuals also exhibited decreased systemic levels of Hp compared to PTB individuals. APPs were not significantly associated with LN size, LN involvement and culture grade, indicating a lack of association with disease severity. Following ATT, post-treatment levels of ⍺-2MG, CRP and Hp were significantly diminished compared to pre-treatment levels., Conclusion: TBL disease is characterized by altered levels of APPs at baseline and modulated following treatment, indicating the presence of systemic inflammation., Competing Interests: The authors have declared that no competing interests exist.

Background: Helminth infections are known to regulate cytokine responses in both pulmonary and latent tuberculosis infection. Whether helminth infections also modulate cytokine responses in extra-pulmonary tuberculosis, specifically tuberculous lymphadenitis (TBL), has not been examined thus far., Methodology: Hence, to determine the cytokine profile in helminth-TBL coinfection, we measured the systemic and mycobacterial (TB)-antigen stimulated levels of Type 1, Type 2, Type 17, regulatory and pro-inflammatory cytokines in TBL individuals coinfected with or without Strongyloides stercoralis (Ss) infection., Significant Findings: TBL-Ss+ individuals have significantly higher bacterial burdens in the affected lymph nodes in comparison to TBL-Ss- individuals. TBL-Ss+ individuals exhibit significantly enhanced plasma levels of Type 2 (IL-5 and IL-13), Type 17 (IL-17 and IL-22) and regulatory (IL-10) cytokines in comparison to TBL-Ss- individuals. In contrast, TBL-Ss+ individuals exhibit significantly diminished plasma levels of pro-inflammatory cytokines (IL-1α and GM-CSF) in comparison to TBL-Ss- individuals. TBL-Ss+ individuals also exhibit significantly diminished unstimulated or mycobacterial-antigen stimulated levels of Type 1, Type 17 or IL-1 family cytokines in comparison to TBL-Ss- individuals but no differences in mitogen stimulated cytokine levels., Conclusion: Therefore, our data reveal a profound influence of Ss infection on the bacteriological profile of TBL and suggesting that the underlying modulation of cytokine responses might be a mechanism by which this helminth infection could impart a detrimental effect on the pathogenesis of TBL disease., Competing Interests: The authors have declared that no competing interests exist.

Non-protein coding RNA (npcRNA) is a functional RNA molecule that is not translated into a protein. Bacterial npcRNAs are structurally diversified molecules, typically 50-200 nucleotides in length. They play a crucial physiological role in cellular networking, including stress responses, replication and bacterial virulence. In this study, by using an identified npcRNA gene (Sau-02) in Methicillin-resistant Staphylococcus aureus (MRSA), we identified the Gram-positive bacteria S. aureus. A Sau-02-mediated monoplex Polymerase Chain Reaction (PCR) assay was designed that displayed high sensitivity and specificity. Fourteen different bacteria and 18 S. aureus strains were tested, and the results showed that the Sau-02 gene is specific to S. aureus. The detection limit was tested against genomic DNA from MRSA and was found to be ~10 genome copies. Further, the detection was extended to whole-cell MRSA detection, and we reached the detection limit with two bacteria. The monoplex PCR assay demonstrated in this study is a novel detection method that can replicate other npcRNA-mediated detection assays.

Background: Cytokines play an important role in the pathogenesis of pulmonary tuberculosis (PTB)--Type 2 diabetes mellitus co-morbidity. However, the cytokine interactions that characterize PTB coincident with pre-diabetes (PDM) are not known., Methods: To identify the influence of coincident PDM on cytokine levels in PTB, we examined circulating levels of a panel of cytokines in the plasma of individuals with TB-PDM and compared them with those without PDM (TB-NDM)., Results: TB-PDM is characterized by elevated circulating levels of Type 1 (IFNγ, TNFα and IL-2), Type 17 (IL-17A and IL-17F) and other pro-inflammatory (IL-1β, IFNβ and GM-CSF) cytokines. TB-PDM is also characterized by increased systemic levels of Type 2 (IL-5) and regulatory (IL-10 and TGFβ) cytokines. Moreover, TB antigen stimulated whole blood also showed increased levels of pro-inflammatory (IFNγ, TNFα and IL-1β) cytokines as well. However, the cytokines did not exhibit any significant correlation with HbA1C levels or with bacterial burdens., Conclusion: Our data reveal that pre-diabetes in PTB individuals is characterized by heightened cytokine responsiveness, indicating that a balanced pro and anti - inflammatory cytokine milieu is a feature of pre-diabetes--TB co-morbidity.

Background: Helminth infections are known to modulate innate and adaptive immune responses in active and latent tuberculosis (TB). However, the role of helminth infections in modulating responses associated with inflammation and immune activation (reflecting disease activity and/or severity) in TB is not known., Methodology: We measured markers of inflammation and immune activation in active pulmonary TB individuals (ATB) with co-incidental Strongyloides stercoralis (Ss) infection. These included systemic levels of acute phase proteins, matrix metalloproteinases and their endogenous inhibitors and immune activation markers. As a control, we measured the systemic levels of the same molecules in TB-uninfected individuals (NTB) with or without Ss infection., Principal Findings: Our data confirm that ATB is associated with elevated levels of the various measured molecules when compared to those seen in NTB. Our data also reveal that co-incident Ss infection in ATB individuals is associated with significantly decreased circulating levels of acute phase proteins, matrix metalloproteinases, tissue inhibitors of matrix metalloproteinases as well as the systemic immune activation markers, sCD14 and sCD163. These changes are specific to ATB since they are absent in NTB individuals with Ss infection., Conclusions: Our data therefore reveal a profound effect of Ss infection on the markers associated with TB disease activity and severity and indicate that co-incidental helminth infections might dampen the severity of TB disease.

Background: Circulating T follicular helper (Tfh) cells represent a distinct subset of CD4+ T cells and are important in immunity to infections. Although they have been shown to play a role in experimental models of tuberculosis infection, their role in human tuberculosis remains unexplored., Aims/methodology: To determine the distribution of circulating Tfh cells in human TB, we measured the frequencies of Tfh cells ex vivo and following TB - antigen or polyclonal stimulation in pulmonary TB (PTB; n = 30) and latent TB (LTB; n = 20) individuals, using the markers CXCR5, PD-1 and ICOS., Results: We found that both ex vivo and TB - antigen induced frequencies of Tfh cell subsets was significantly lower in PTB compared to LTB individuals. Similarly, antigen induced frequencies of Tfh cells expressing IL-21 was also significantly lower in PTB individuals and this was reflected in diminished circulating levels of IL-21 and IFNγ. This was not accompanied by diminished frequencies of activated or memory B cell subsets. Finally, the diminution in frequency of Tfh cells in PTB individuals was dependent on IL-10, CTLA-4 and PD-L1 in vitro., Conclusions: Thus, PTB is characterized by adiminution in the frequency of Tfh cell subsets.

Tissue invasive helminth infections and tuberculosis (TB) are co-endemic in many parts of the world and can trigger immune responses that might antagonize each other. We have previously shown that helminth infections modulate the Th1 and Th17 responses to mycobacterial-antigens in latent TB. To determine whether helminth infections modulate antigen-specific and non-specific immune responses in active pulmonary TB, we examined CD4(+) and CD8(+) T cell responses as well as the systemic (plasma) cytokine levels in individuals with pulmonary TB with or without two distinct helminth infections-Wuchereria bancrofti and Strongyloides stercoralis infection. By analyzing the frequencies of Th1 and Th17 CD4(+) and CD8(+) T cells and their component subsets (including multifunctional cells), we report a significant diminution in the mycobacterial-specific frequencies of mono- and multi-functional CD4(+) Th1 and (to a lesser extent) Th17 cells when concomitant filarial or Strongyloides infection occurs. The impairment in CD4(+) and CD8(+) T cell cytokine responses was antigen-specific as polyclonal activated T cell frequencies were equivalent irrespective of helminth infection status. This diminution in T cell responses was also reflected in diminished circulating levels of Th1 (IFN-γ, TNF-α and IL-2)- and Th17 (IL-17A and IL-17F)-associated cytokines. Finally, we demonstrate that for the filarial co-infections at least, this diminished frequency of multifunctional CD4(+) T cell responses was partially dependent on IL-10 as IL-10 blockade significantly increased the frequencies of CD4(+) Th1 cells. Thus, co-existent helminth infection is associated with an IL-10 mediated (for filarial infection) profound inhibition of antigen-specific CD4(+) T cell responses as well as protective systemic cytokine responses in active pulmonary TB.

Buprofezin is an insect growth regulator and widely used insecticide in Malaysia. The present study evaluated the toxic effects of buprofezin on the embryo and larvae of African catfish (Clarias gariepinus) as a model organism. The embryos and larvae were exposed to 7 different concentrations (0, 0.05, 0.5, 5, 25, 50 and 100 mg/L) of buprofezin. Each concentration was assessed in five replicates. Eggs were artificially fertilized and 200 eggs and larvae were subjected to a static bath treatment for all the concentrations. The mortality of embryos was significantly increased with increasing buprofezin concentrations from 5 to 100 mg/L (p< 0.05). However, the mortality was not significantly different (p<0.05) among the following concentrations: 0 (control), 0.05, 0.5 and 5 mg/L. Data obtained from the buprofezin acute toxicity tests were evaluated using probit analysis. The 24 h LC50 value (with 95% confidence limits) of buprofezin for embryos was estimated to be 6.725 (3.167-15.017) mg/L. The hatching of fish embryos was recorded as 68.8, 68.9, 66.9, 66.4, 26.9, 25.1 and 0.12% in response to 7 different concentrations of buprofezin, respectively. The mortality rate of larvae significantly (p<0.05) increased with increasing buprofezin concentrations exposed to 24-48 h. The 24 and 48 h LC50 values (with 95% confidence limits) of buprofezin for the larvae was estimated to be 5.702 (3.198-8.898) and 4.642 (3.264-6.287) mg/L respectively. There were no significant differences (p>0.05) in the LC50 values obtained at 24 and 48 h exposure times. Malformations were observed when the embryos and larvae exposed to more than 5 mg/L. The results emerged from the study suggest that even the low concentration (5 mg/L) of buprofezin in the aquatic environment may have adverse effect on the early embryonic and larval development of African catfish.

Background: Tuberculosis (TB) is associated with oxidative stress and the induction of host anti-oxidants to counteract this response. Heme oxygenase-1 (HO-1) is a critical promoter of cytoprotection in diverse disease models including mycobacterial infection. Nevertheless, the pattern of expression of HO-1 in human tuberculosis has not been studied. Here, we examine expression of HO-1 in M. tuberculosis-exposed and -infected individuals and test its ability to distinguish active from latent and successfully treated TB cases. In addition, we assess correlations between plasma levels of HO-1 and cytokines closely associated with the immunopathogenesis of TB., Methods: Cross-sectional and longitudinal analyses of levels of HO-1, acute phase proteins and pro-inflammatory cytokines were performed in plasma samples from individuals with active pulmonary, extra-pulmonary or latent TB infection and healthy controls as part of a prospective cohort study in South India., Results: Systemic levels of HO-1 were dramatically increased in individuals with active pulmonary and extra-pulmonary tuberculosis and particularly those with bilateral lung lesions and elevated bacillary loads in sputum. HO-1 levels effectively discriminated active from latent tuberculosis with higher predictive values than either C-reactive protein or serum amyloid protein. Moreover, there was a marked reduction in HO-1 levels in active TB cases following anti-tuberculous therapy but not in those who failed treatment. Pulmonary TB patients displaying the highest concentrations of HO-1 in plasma exhibited significantly elevated plasma levels of interleukin (IL)-10, interferon (IFN)-γ and IL-17 and diminished levels of tumor necrosis factor (TNF)-α., Conclusion: These findings establish HO-1 levels as a potentially useful parameter for distinguishing active from latent or treated pulmonary tuberculosis, that is superior in this respect to the measurement of other acute inflammatory proteins.

Background: Although Type 1 cytokine responses are considered protective in pulmonary tuberculosis (PTB), their role as well as those of Type 2, 17 and immunoregulatory cytokines in tuberculous lymphadenitis (TBL) and latent tuberculosis (LTB) have not been well studied., Aim and Methods: To identify cytokine responses associated with pulmonary tuberculosis (TB), TB lymphadenitits and latent TB, we examined mycobacterial antigen-specific immune responses of PTB, TBL and LTB individuals. More specifically, we examined ESAT-6 and CFP-10 induced Type 1, Type 2 and Type 17 cytokine production and their regulation using multiplex ELISA., Results: PTB individuals exhibited a significantly lower baseline as well as antigen-specific production of Type 1 (IFNγ, TNFα and IL-2); Type 2 (IL-4) and Type 17 (IL-17A and IL-17F) cytokines in comparison to both TBL and LTB individuals. TBL individuals exhibited significantly lower antigen-specific IFNγ responses alone in comparison to LTB individuals. Although, IL-10 levels were not significantly higher, neutralization of IL-10 during antigen stimulation resulted in significantly enhanced production of IFNγ, IL-4 and IL-17A in PTB individuals, indicating that IL-10 mediates (at least partially) the suppression of cytokine responses in PTB., Conclusion: Pulmonary TB is characterized by an IL-10 dependent antigen-specific suppression of Type 1, Type 2 and Type 17 cytokines, reflecting an important association of these cytokines in the pathogenesis of active TB.

Background: Th1 and Th17 responses are known to play an important role in immunity to pulmonary tuberculosis (PTB), although little is known about their role in extrapulmonary forms of tuberculosis (TB)., Methods: To identify the role of Th1, Th17, and Th22 cells in multi-focal TB lymphadenitis (TBL), we examined mycobacteria-specific immune responses in the whole blood of individuals with PTB (n = 20) and compared them with those with TBL (n = 25)., Results: Elevated frequencies of CD4(+) T cells expressing IFN- γ, TNF-α, and IL-2 were present in individuals with TBL compared with those with PTB at baseline and in response to ESAT-6 and CFP-10. Similarly, increased frequencies of CD4(+) T cells expressing IL-17A, IL-17F, and IFN-γ were also present in individuals with TBL at baseline and following ESAT-6 and CFP-10 stimulation although no significant difference in frequency of Th22 cells was observed. Finally, frequencies of Th1 (but not Th17) cells exhibited a significantly negative correlation with natural regulatory T cell frequencies at baseline., Conclusions: Multi-focal TB lymphadenitis is therefore characterized by elevated frequencies of Th1 and Th17 cells, indicating that Th1 and Th17 responses in TB disease are probably correlates of disease severity rather than of protective immunity.