Mycobacterium bovis Wild-Type BCG or Recombinant BCG Secreting Murine IL-18 (rBCG/IL-18) Strains in Driving Immune Responses in Immunocompetent or Immunosuppressed Mice.

Mycobacterium tuberculosis infections remain a global health problem in immunosuppressed patients. The effectiveness of BCG (Bacillus Calmette−Guérin), an anti-tuberculosis vaccine, is unsatisfactory. Finding a new vaccine candidate is a priority. We compared numerous immune markers in BCG-susceptible C57BL/6 and BCG-resistant C3H mice who had been injected with 0.9% NaCl (control) or with wild-type BCG or recombinant BCG secreting interleukin (IL)-18 (rBCG/IL-18) and in immunized mice who were immunocompromised with cyclophosphamide (CTX). The inoculation of rBCG/IL-18 in immunocompetent mice increased the percentage of bone marrow myeloblasts and promyelocytes, which were further elevated in the rBCG/IL-18/CTX-treated mice: C57BL/6 mice­3.0% and 11.4% (control) vs. 18.6% and 42.4%, respectively; C3H mice­1.1% and 7.7% (control) vs. 18.4% and 44.9%, respectively, p < 0.05. The bone marrow cells showed an increased mean fluorescence index (MFI) in the CD34 adhesion molecules: C57BL/6 mice­4.0 × 103 (control) vs. 6.2 × 103; C3H mice­4.0 × 103 (control) vs. 8.0 × 103, p < 0.05. Even in the CTX-treated mice, the rBCG/IL-18 mobilized macrophages for phagocytosis, C57BL/6 mice­4% (control) vs. 8%; C3H mice­2% (control) vs. 6%, and in immunocompetent mice, C57BL/6 induced the spleen homing of effector memory CD4+ and CD8+ T cells (TEM), 15% (control) vs. 28% and 8% (control) vs. 22%, respectively, p < 0.05. In conclusion, rBCG/IL-18 effectively induced selected immune determinants that were maintained even in immunocompromised mice.

Cytokine Biosignature of Active and Latent Mycobacterium Tuberculosis Infection in Children.

None of the currently used diagnostic tools are efficient enough in diagnosing Mycobacterium tuberculosis (M.tb) infection in children. The study was aimed to identify cytokine biosignatures characterizing active and latent tuberculosis (TB) in children. Using a multiplex bead-based technology, we analyzed the levels of 53 Th17-related cytokines and inflammatory mediators in sera from 216 BCG-vaccinated children diagnosed with active TB (TB) or latent TB (LTBI) as well as uninfected controls (HC). Children with active TB, compared to HC children, showed reduced serum levels of IL-17A, MMP-2, OPN, PTX-3, and markedly elevated concentrations of APRIL/TNFSF13. IL-21, sCD40L, MMP-2, and IL-8 were significantly differentially expressed in the comparisons between groups: (1) HC versus TB and LTBI (jointly), and (2) TB versus LTBI. The panel consisting of APRIL/TNFSF13, sCD30/TNFRSF8, IFN-α2, IFN-γ, IL-2, sIL-6Rα, IL-8, IL-11, IL-29/IFN-λ1, LIGHT/TNFSF14, MMP-1, MMP-2, MMP-3, osteocalcin, osteopontin, TSLP, and TWEAK/TNFSF12 possessed a discriminatory potential for the differentiation between TB and LTBI children. Serum-based host biosignatures carry the potential to aid the diagnosis of childhood M.tb infections. The proposed panels of markers allow distinguishing not only children infected with M.tb from uninfected individuals but also children with active TB from those with latent TB.

Decrease of IL-5 Production by Naive T Cells Cocultured with IL-18-Producing BCG-Pulsed Dendritic Cells from Patients Allergic to House Dust Mite.

The only currently available anti-tuberculosis vaccine, Bacillus Calmette-Guérin (BCG), has been reported to also protect against unrelated diseases, including inflammatory diseases such as allergic asthma. Recombinant BCG strains that produce IL-18 have been shown to enhance Th1 responses over non-recombinant BCG and to reduce IL-5 production and bronchoalveolar eosinophilia in mice. However, their ability to decrease the immune polarization of human Th2 cells is not known. Here, we show that BCG and recombinant BCG producing human IL-18 (rBCG-hIL-18) induced the maturation of Der p 1-stimulated monocyte-derived dendritic cells (MD-DCs) from healthy controls and from patients allergic to house dust mites. After incubation with mycobacteria and Der p 1, MD-DCs produced significantly more IL-23 and IP-10 but had no effect on IL-12p70 or IL-10 production compared to Der p 1-pulsed MD-DCs in the absence of mycobacteria. In the presence of Der p 1, BCG- and rBCG-hIL-18-pulsed MD-DCs cocultured with naive, but not with memory T cells from allergic patients, resulted in a decrease in IL-5 production compared to non-pulsed MD-DCs cultured in the presence of Der p 1. BCG, and especially rBCG-hIL-18, may thus be potential therapeutic tools to reduce exacerbated Th2 responses in patients with allergic asthma.

IL-18 and related function proteins associated with tuberculosis severity and screening for active TB among patients with non-mycobacterial community-acquired pneumonia (CAP).

BACKGROUND: Differentiation of active pulmonary tuberculosis (TB) from non-mycobacterial community-acquired pneumonia (CAP) still remains a diagnostic challenge. OBJECTIVE: The study aimed to quantify the IL-18, IFN-γ, IL-18BP, IL-37, and IP-10 levels in serum and Mycobacterium tuberculosis (M.tb) antigens-stimulated blood cultures from TB or CAP patients and explore if the proteins can be a useful basis for discriminating these diseases. METHODS: In total, 124 Polish adults, including mild/moderate (M/MTB) or advanced (ATB) TB patients, and CAP patients, were enrolled in the study. The concentrations of IL-18, IL-18BP, IFN-γ, IL-37, and IP-10 in sera and M.tb-stimulated cultures were measured by ELISA. RESULTS: The most specific and sensitive serum proteins discriminating TB from CAP were IP-10 and IL-18BP; however, IP-10 had the highest AUC in the ROC curve for the diagnosis. Serum IP-10 and IL-18BP levels increased significantly in M/MTB or ATB groups. The IL-18BP elevation in ATB group was accompanied by an increase in IL-18. No single protein measured in M.tb-stimulated cultures differed TB from CAP patients. CONCLUSIONS: The combined analysis of serum IL-18BP and IP-10 might be considered as an auxiliary tool in the differentiation of TB from CAP.

Interleukin-18, Functional IL-18 Receptor and IL-18 Binding Protein Expression in Active and Latent Tuberculosis.

A thorough understanding of the processes modulating the innate and acquired immune response to Mycobacterium tuberculosis (M.tb) infection in the context of gene expression is still a scientific and diagnostic problem. The study was aimed to assess IL-18, IL-18 binding protein (IL-18BP), IL-18R, IFN-γ, and IL-37 mRNA expression in patients with active tuberculosis (ATB) and healthy volunteers with latent M.tb-infection (LTB) or M.tb-uninfected healthy controls (Control). The relative mRNA expression was assessed in the buffy coat blood fraction using the qPCR method. In total, 97 BCG-vaccinated Polish adults were enrolled in the study. The relative expression of IL-18 and IL-18BP mRNA was significantly elevated in the ATB and LTB groups. In ATB, but not LTB individuals, the overexpression of IL-18 and IL-18BP, as well as a significant increase in IFN-γ mRNA expression, might be considered as a manifestation of active tuberculosis disease. No statistically significant differences were observed in the IL-37 mRNA expression among the studied groups. Particularly noteworthy is the outstanding reduction in the relative expression of IL-18R mRNA in the LTB group as compared to the ATB and Control group. Reduced expression of IL-18R in LTB group may, at least partially, prevent the development of a pathological inflammatory reaction and promote the maintenance of homeostatic conditions between host immunity and M.tb.

IL-18/IL-37/IP-10 signalling complex as a potential biomarker for discriminating active and latent TB.

BACKGROUND: Currently, there are serious limitations in the direct diagnosis of active tuberculosis (ATB). We evaluated the levels of the IL-18/IL-37/IP-10 signalling complex proteins in Mycobacterium tuberculosis (M.tb)-specific antigen-stimulated QuantiFERON® Gold In-Tube (QFT) cultures and in serum samples from ATB patients, healthy individuals with latent M.tb infection (LTBI) and healthy controls (HC) to examine whether combined analyses of these proteins were useful in the differentiation of M.tb states. METHODS: The concentrations of IL-18, IL-18BP, IFN-γ, IL-37 and IP-10 in the serum and QFT supernatants were measured using specific enzyme-linked immunosorbent assay (ELISA) kits. Free IL-18 levels were calculated using the law of mass action. RESULTS: Increased concentrations of total and free IL-18, IL-18BP, IFN-γ and IP-10 in the sera of ATB patients were detected. These increases were not counterbalanced by the overproduction of IL-37. Complex co-expression of serum IL-18BP and IL-37, IP-10 and IFN-γ was identified as the highest discriminative biomarker set for the diagnosis of ATB. CONCLUSIONS: Our results suggest that the IL-18 signalling complex might be exploited by M. tuberculosis to expand the clinical manifestations of pulmonary TB. Therefore, direct analysis of the serum components of the IL-18/IL-37 signalling complex and IP-10 may be applicable in designing novel diagnostic tests for ATB.

Lack of significant effect of interleukin-18 gene variants on tuberculosis susceptibility in the Polish population.

BACKGROUND: Polymorphisms in genes encoding cytokines are known to determine susceptibility to Mycobacterium tuberculosis (M.tb) infection. In particular, interleukin-18 (IL-18), an inducer of interferon-gamma (IFN-γ), playing an important role in anti-mycobacterial immune responses, may influence the risk of developing active tuberculosis (TB). AIM: A case-control study was performed to investigate whether two promoter polymorphisms of the IL-18 gene at positions -137G/A (rs187238) and -607A/C (rs1946518) affect the serum level of IL-18 and might be associated with genetic susceptibility to tuberculosis (TB) in the Polish population. METHODS: Two IL-18 gene promoter SNPs were detected by an allele-specific polymerase chain reaction. Serum IL-18 levels were measured immunoenzymatically using Human Total IL-18 ELISA DuoSet. RESULTS: A single-gene analysis showed no differences either in allele or genotype frequencies of the studied SNPs between TB patients and healthy controls. No significant differences in the frequencies of any of the haplotypes between TB patients and healthy controls were found. None of the polymorphic variants of IL-18(-137G/A) or IL-18(-607A/C) SNP was associated with IL-18 producing capability. CONCLUSION: Our results suggest that IL-18(-137G/A) and IL-18(-607A/C) polymorphisms may not be risk factors for susceptibility to TB in the Polish population. Increased serum IL-18 level observed in TB patients has no genetic background, but is a consequence of M.tb infection. Further studies with a higher sample size are required to confirm these findings.

Effect of Cyclophosphamide Treatment on Central and Effector Memory T Cells in Mice.

Immunological memory is a key feature of adaptive immunity. It provides the organism with long-lived and robust protection against infection. The important question is whether cyclophosphamide (CP), as immunosuppressive agent used in cancer therapy and in some autoimmune diseases, may act on the memory T-cell population. We investigated the effect of CP on the percentage of central memory T cells (TCM) and effector memory T cells (TEM) in the mouse model of CP-induced immunosuppression (8-10-week-old male C57BL/6 mice CP treated for 7 days at the daily dose of 50 µg/g body weight [bw], manifested the best immunosuppression status, as compared to lower doses of CP: 10 or 20 µg/g bw). The CP induced a significant decrease in the percentage of CD8+ (TCM), compared to nonimmunosuppressed mice. This effect was not observed in the case of CD4+ TCM population. The percentage of gated TEM with CD4 and CD8 phenotype was significantly decreased in CP-treated mice, as compared to the control ones. Taken together, the above data indicate that CP-induced immunosuppression in mice leads to a reduction in the abundance of central memory cells possessing preferentially CD8+ phenotype as well as to a reduction in the percentage of effector memory cells (splenocytes both CD4+ and CD8+), compared to the cells from nonimmunosuppressed mice. These findings in mice described in this article may contribute to the understanding of the complexity of the immunological responses in humans and extend research on the impact of the CP model of immunosuppression in mice and memory T-cell populations.

Tuberculin skin test reaction is related to memory, but not naive CD4+ T cell responses to mycobacterial stimuli in BCG-vaccinated young adults.

Bacillus Calmette-Guérin (BCG) is the only vaccine available against tuberculosis and the tuberculin skin test (TST) is the most widely used method to detect BCG take. However, subjects may remain TST-negative, even after several BCG administrations. To investigate some of the potential reasons underlying this inability of developing tuberculin sensitivity in response to BCG we compared the effect of different mycobacterial stimuli in the groups differently responding to tuberculin. TST was performed on 71 healthy adults aged 25-30 years, who had received BCG in their childhood, and considered TST-positive at ≥10 mm. Dendritic cells (DCs) were incubated with PPD, live BCG or rBCGhIL-18, producing human IL-18. The latter strain was used to investigate whether the production of IL-18 could overcome some of the immune read-out limitations in the TST-negative subjects. CD86, CD80, CD40, and DC-specific intracellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) expression was analysed by flow cytometry and IL-10, IL-23 and IP-10 secretion in culture supernatants by ELISA. In DCs-T cell co-cultures with naive and memory CD4+ T cells, the IFN-γ and IL-10 levels were determined by ELISA. We found no difference in IL-10 and IFN-γ production by naive T cells between the TST-negative and TST-positive subjects. However, IFN-γ was produced in significantly higher amounts by memory T cells incubated with PPD, BCG or rBCGhIL-18-pulsed DCs in TST-positive than in TST-negative subjects, whereas the numbers of the IFN-γ-producing T cells were similar in both groups. This difference may be partially due to a decreased CD40 and enhanced reduction in DC-SIGN expression by DCs of TST-negative versus TST-positive subjects. A strong effect of IL-18 expression by rBCGhIL-18 on IL-23 production by the DC was seen in both groups, which likely was the reason for the increased IFN-γ production by naïve T cells upon incubation with mycobacteria-pulsed DC, regardless of the TST status.

CD14-159C/T polymorphism in the development of delayed skin hypersensitivity to tuberculin.

The skin tuberculin test (TST), an example of a delayed-type hypersensitivity (DTH) reaction, is based on measuring the extent of skin induration to mycobacterial tuberculin (PPD). Little is known about the genetic basis of TST reactivity, widely used for diagnosing TB infection. The study investigated the relationship of the single base change polymorphic variants in CD14 gene (CD14(-159C/T)) with the development of DTH to PPD in BCG-vaccinated Polish Caucasian individuals. We found persistent lack of TST reactivity in about 40% of healthy subjects despite receiving more than one dose of BCG. The TST size was negatively correlated with the number of BCG inoculations. The distribution of C/T genotype was significantly more frequent among TST-negative compared with TST-positive individuals. The concentration of serum sCD14 was positively associated with mCD14 expression, but not with the TST status or CD14(-159C/T) polymorphism. A significant increase in mCD14 expression and serum sCD14 levels was found in TB group. We hypothesize that CD14(-159C/T) polymorphic variants might be one of genetic components in the response to attenuated M. bovis BCG bacilli.

Mycobacteria-derived biomarkers for tuberculosis diagnosis.

Tuberculosis (TB) remains an escalating problem worldwide. The current diagnostic methods do not always guarantee reliable diagnosis. TB treatment is a time-consuming process that requires the use of several chemotherapeutics, to which mycobacteria are becoming increasingly resistant. This article focuses on the potential utility of biomarkers of mycobacterial origin with potential implications for TB diagnosis. Properly standardized indicators could become new diagnostic tools, improving and streamlining the identification of Mycobacterium tuberculosis infection and the implementation of appropriate therapy. These markers can also potentially provide a quick confirmation of effectiveness of new anti-mycobacterial drugs and TB vaccines, leading to a possible application in practice.

Two-Year Follow-up Study of Mycobacterium tuberculosis Antigen-Driven IFN-γ Responses and Macrophage sCD14 Levels After Tuberculosis Contact.

Clinical data regarding the prediction of active tuberculosis (TB) development in close TB contacts are scarce. To address this problem, we performed a 2-year follow-up study of Mycobacterium tuberculosis (M.tb) antigen-driven IFN-gamma responses and serum levels of soluble macrophage CD14 receptor in individuals with recent or prolonged M.tb exposure. Between June 2011 and June 2013, we studied 60 healthy Polish adults with recent household or long-term work TB contact and individuals without known M.tb exposure. All of them underwent baseline and repeated testing with IGRA (IFN-gamma release assay) and serum sCD14 ELISA quantification. Frequencies of IGRA results differed at the baseline and follow-up testing. IGRA reversions were noticed in almost one-third of Work TB Contacts and no participants from the Household TB Contact group. IGRA conversions were found in 40 % of Household TB Contacts. No correlation between the IGRA result and the sCD14 level was observed. IFN-γ variability has important implications for clinical practice and requires caution in interpreting the results to distinguish new infections from nonspecific inter-individual variations in cytokine responses. The impairment of IFN-γ response in some individuals with prolonged M.tb exposure representing a resistant immune status does not allow considering IGRA results as reliable and credible. Monitoring the serum sCD14 level can reduce the likelihood of a false prediction of active TB development in close TB contacts showing an M.tb-specific increase in the IFN-gamma production in repeated IGRA testing.

Interleukin 18 (IL-18) as a target for immune intervention.

Interleukin 18 (IL-18) is a pleiotropic cytokine involved in the regulation of innate and acquired immune response. In the milieu of IL-12 or IL-15, IL-18 is a potent inducer of IFN-gamma in natural killer (NK) cells and CD4 T helper (Th) 1 lymphocytes. However, IL-18 also modulates Th2 and Th17 cell responses, as well as the activity of CD8 cytotoxic cells and neutrophils, in a host microenvironment-dependent manner. It is produced by various hematopoietic and nonhematopoietic cells, including dendritic cells and macrophages. In an organism, bioactivity of the cytokine depends on the intensity of IL-18 production, the level of its natural inhibitory protein - IL-18BP (IL-18 binding protein) and the surface expression of IL-18 receptors (IL-18R) on the responding cells. This review summarizes the biology of the IL-18/IL-18BP/IL-18R system and its role in the host defense against infections. The prospects for IL-18 application in immunotherapeutic or prophylactic interventions in infectious and non-infectious diseases are discussed.

Immune response gene polymorphisms in tuberculosis.

Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (M.tb), remains a leading public health problem in most parts of the world. Despite the discovery of the bacilli over 100 years ago, there are still many unanswered questions about the host resistance to TB. Although one third of the world's population is infected with virulent M.tb, no more than 5-10% develop active disease within their lifetime. A lot of studies suggest that host genetic factors determine the outcome of M.tb-host interactions, however, specific genes and polymorphisms that govern the development of TB are not completely understood. Strong evidence exists for genes encoding pattern recognition receptors (TLR, CD14), C-type lectins, cytokines/chemokines and their receptors (IFN-γ, TNF-α, IL-12, IL-10, MCP-1, MMP-1), major histocompatibility complex (MHC) molecules, vitamin D receptor (VDR), and proton-coupled divalent metal ion transporters (SLC11A1). Polymorphisms in these genes have a diverse influence on the susceptibility to or protection against TB among particular families, ethnicities and races. In this paper, we review recent discoveries in genetic studies and correlate these findings with their influence on TB susceptibility.

The balance between pro- and anti-inflammatory cytokines in the immune responses to BCG and DTwP vaccines.

Bacillus Calmette-Guérin (BCG) and pertussis vaccines have been found to be insufficient and their further improvement is required. In order to develop improved vaccines, a better understanding of the main pathways involved in the host's protective immunity to the pathogens is crucial. We address the question as to whether the balance between pro- and anti-inflammatory cytokine production might affect the host responses to BCG and diphtheria-tetanus toxoids-whole cell pertussis (DTwP) vaccines. The study population consisted of 118 healthy people, age range 18-30 years, who had been subjected to BCG and DTwP vaccination according to the state policy. Tuberculin skin testing (TST) revealed a delayed type hypersensitivity (DTH) to PPD (purified protein derivative) in 53% volunteers. The variability in development of the BCG-driven DTH to tuberculin prompted us to address a question as to whether Th1/Th2 polarization is involved in the lack of skin responsiveness to PPD. PPD-stimulated blood lymphocytes from TST(+) participants produced significantly more IFN-γ and less IL-10 than lymphocytes from TST(-) volunteers. However, TST(-) volunteers' sera contained more anti-pertussis IgG but not anti-diphtheria toxin IgG. Mycobacterial antigens and particularly PPD induced a higher expression of HLA-DR and co-stimulatory CD80 receptors on DCs from TST(+) than TST(-) participants. BCG but not PPD pulsed DCs from TST(-) volunteers produced significantly more IL-10. Mycobacterial antigen stimulated DCs from TST(+) volunteers induced a more intense IFN-γ production in co-cultures with autologous lymphocytes than the cells from TST(-) participants. Differences among the types of dendritic cell activities contribute to development of tuberculin reactivity in BCG vaccinated volunteers.

Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults.

Tuberculosis remains an enormous global burden, despite wide vaccination coverage with the Bacillus Calmette-Guérin (BCG), the only vaccine available against this disease, indicating that BCG-driven immunity is insufficient to protect the human population against tuberculosis. In this study we constructed recombinant BCG producing human IL-18 (rBCGhIL-18) and investigated whether human IL-18 produced by rBCGhIL-18 modulates DC functions and enhances Th1 responses to mycobacterial antigens in humans. We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective. In contrast, both BCG strains decreased the DC-SIGN expression on human DCs. The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG. In a coculture system of CD4(+) T cells and loaded DCs, rBCGhIL-18 favoured strong IFN-γ but also IL-10 production by naive T cells but not by memory T cells. This was much less the case for nonrecombinant BCG. Thus the expression of IL-18 by recombinant BCG increases IL-23, IP-10, and IL-10 expression by human DCs and enhances their ability to induce IFN-γ and IL-10 expression by naive T cells, without affecting the maturation phenotype of the DCs.

Helicobacter pylori-driven modulation of NK cell expansion, intracellular cytokine expression and cytotoxic activity.

During Helicobacter pylori (Hp) infections, innate immune cells may be positively or negatively modulated by Hp compounds or by Hp-induced cytokines. We have shown previously that the natural cytotoxic activity of PBMC was lower in Hp-infected [Hp(+)] than Hp-uninfected individuals [Hp(-)]. Here, we asked whether the Hp-modulated cytotoxic amplitude is associated with changes in the number of NK cells, their activation or intracellular cytokine expression. Flow cytometry immunophenotyping of PBMC was performed with regard to the surface receptors CD3, CD56 and CD25, and intracellular cytokine expression of IL-2, IFN-γ and IL-10 after in vitro stimulation with Hp glycine acid extract (GE), Hp LPS or standard Escherichia coli LPS. Hp GE-driven enhancement of lymphocyte cytotoxic activity was associated with the expansion of CD3(-)CD56(+)CD25(+) NK cells and the up-regulation of IFN-γ and/or IL-2 synthesis, up to the higher level in Hp(-) than in Hp(+), while Hp LPS-mediated decrease in lymphocyte cytotoxicity was accompanied by the lack of CD3(-)CD56(+)CD25(+) NK propagation, the inhibition of pro-inflammatory cytokine expression and intense expansion of IL-10-producing NK cells. Thus, the cytotoxic and cytokine activities of NK cells were dependent on the type of antigenic challenge and the Hp status, that is, NK cells could be modulated positively by Hp GE Ags and negatively by Hp LPS.

Interferon-gamma assay in combination with tuberculin skin test are insufficient for the diagnosis of culture-negative pulmonary tuberculosis.

OBJECTIVE: Early diagnosis of infectious cases and treatment of tuberculosis (TB) are important strategies for reducing the incidence of this disease. Unfortunately, traditional TB diagnostic methods are time-consuming and often unreliable. This study compared the accuracy and reliability of the tuberculin skin test (TST) and interferon (IFN)-γ-based assay (IGRA) for the diagnosis of active pulmonary TB Polish cases that could or could not be confirmed by M. tuberculosis (M.tb) culture. METHODS: In total, 126 adult patients with clinically active TB or non-mycobacterial, community-acquired lung diseases (NMLD) hospitalised at the Regional Specialised Hospital of Tuberculosis, Lung Diseases and Rehabilitation in Tuszyn, Poland were enrolled in the present study. Sensitivity, specificity, positive predicted value (PPV), negative predicted value (NPV), and analytic accuracy (Acc) of TST and IGRA testing for the diagnosis of culture-positive and culture-negative TB patients were calculated. The quantities of IFN-γ produced in the response to M.tb specific antigens (TB Ag - Nil) in the cultures of blood from patients with active TB and NMLD patients were also analysed. RESULTS: The IGRA sensitivity in culture-positive and culture-negative TB patients was similar, measuring 65.1% and 55.6%, respectively. The sensitivity of TST did not differ from the parameters designated for IGRA, measuring 55.8% in culture-positive and 64.9% in culture-negative TB. The sensitivity of TST and IGRA was age-dependent and decreased significantly with the age of the patients. No differences in the frequency or intensity of M.tb-stimulated IFN-γ production, as assessed by IGRA testing between culture-positive and culture-negative TB were noticed. Significantly lower concentrations of IFN-γ were observed in patients with advanced TB forms compared with those with mild or moderate TB pathologies. CONCLUSIONS: Our results do not show that a combination of IGRA and TST might be a step forward in the diagnosis of culture-negative TB cases. However, M. tuberculosis-stimulated IFN-γ levels might help to assess the extent of pulmonary TB lesions.

Antigen-specific lymphocyte proliferation as a marker of immune response in guinea pigs with sustained Helicobacter pylori infection.

Helicobacter pylori (H. pylori) bacteria are human pathogens causing symptomatic gastritis, peptic ulcer or gastric cancer. Little is known about the kinetics of immune responses in H. pylori infected patients because the initial moment of infection has not been identified. Various animal models are used to investigate the immune processes related to H. pylori infection. In this study we checked whether H. pylori infection in guinea pigs, mimicking natural H. pylori infection in humans, resulted in the development of specific immune responses to H. pylori antigens by measuring the proliferation of lymphocytes localized in mesenteric lymph nodes, spleen and peripheral blood. The maturity of macrophages and cytokines, delivered by monocyte-macrophage lineage or lymphocytes, were considered as mediators, which might influence the lymphocyte blastogenic response. The obtained results showed the activation of T cells localized in mesenteric lymph nodes by H. pylori antigens in H. pylori infected guinea pigs four weeks postinfection. The blastogenic activity of lymphocytes was shaped by their interaction with antigen presenting cells, which were present in the cell cultures during the whole culture period. Moreover, the balance between cytokines derived from adherent leukocytes including interleukin 8--IL-8 as well as interferon gamma--IFN-γ, and transforming growth factor beta--TGF-ß delivered by lymphocytes, was probably important for the successful proliferation of lymphocytes. The H. pylori specific lymphocytes were not propagated in peripheral blood and spleen of H. pylori infected animals. The modulation of immunocompetent cells by H. pylori antigens or their different distribution cannot be excluded.

The lack of L-PG production and the repercussions of it in regards to M. Tuberculosis interactions with mononuclear phagocytes.

The lysine connection with phosphatidylglycerol (PG) alters the M. tuberculosis(Mtb) surface charge, and consequently it may decrease the bacterial vulnerability to antimicrobial action of the immune cells. The aim of the study was to assess the significance of PG lysinylation in the Mtb interactions with mononuclear phagocytes. Both the Mtb strain with deletion of lysX gene (Mtb-lysX) which is responsible for PG lysinylation as well as the complemented strain (Mtb-compl) was used to infect human blood monocytes or THP-1 cells. The monocytes were obtained by MACS technique, or THP-1 cells. The Mtb-lysX strain has exhibited the enhanced sensitivity to HNP 1-3. However, it was not susceptible to bactericidal action of cathepsin G. The LysX deletion did not influence the Mtb ability of monocyte induction to IL-10 secretion. The intra- and extracellular expression of MHC-II was similarly reduced after the Mtb-lysX or Mtb-Rv infections. Noticeably significant is that the Mtb strain with deleted lysX has not affected the intensity of the gene expression of cathepsin G compared to the uninfected monocytes. That is the clear contrast to what the Mtb-Rv strain has proved. The obtained results suggest that the Mtb ability to lysinylate PG is a participatory element in mycobacterial strategy of survival inside phagocytic cells. However, the extended studies are needed to determine its influence on the other immune cells and define its role in the developing of Mtb infection.