Diagnosis of Tuberculosis in HIV Co-infected Individuals: Current Status, Challenges and Opportunities for the Future.

Tuberculosis (TB) remains one of the most important causes of death among people co-infected with human immunodeficiency virus (HIV). The diagnosis of TB remains challenging in HIV co-infected individuals, due to a high frequency of smear-negative disease and high rates of extrapulmonary TB. Accurate, ease of use and rapid diagnosis of active TB are critical to the World Health Organization (WHO) End TB Strategy by 2050. Traditional laboratory techniques do not provide rapid and accurate results to effectively manage HIV co-infected patients. Over the last decade, molecular methods have provided significant steps in the fight against TB. However, many HIV co-infected patients do not have access to these molecular diagnostic tests. Given the costs closely related with confirming a TB diagnosis in HIV patients, an overtreatment for TB is used in this patient population. Nowadays, an estimated US $8 billion a year is required to provide TB treatment, which is very high compared with making an important strategy to improve the current diagnostic tests. This review focuses on current advances in diagnosing active TB with an emphasis on the diagnosis of HIV-associated TB. Also discussed are the main challenges that need to be overcome for improving an adequate initial diagnosis of active TB in HIV-positive patients.

Global Efforts in the Development of Vaccines for Tuberculosis: Requirements for Improved Vaccines Against Mycobacterium tuberculosis.

Currently, more than 9.0 million people develop acute pulmonary tuberculosis (TB) each year and about 1.5 million people worldwide die from this infection. Thus, developing vaccines to prevent active TB disease remains a priority. This article discusses recent progress in the development of new vaccines against TB and focusses on the main requirements for development of improved vaccines against Mycobacterium tuberculosis (M. tb). Over the last two decades, significant progress has been made in TB vaccine development, and some TB vaccine candidates have currently completed a phase III clinical trial. The potential public health benefits of these vaccines are possible, but it will need much more effort, including new global governance investment on this research. This investment would certainly be less than the annual global financial toll of TB treatment.

Modulation of tuberculosis-related immune responses by helminths.

Tuberculosis (TB) continues to be a major worldwide health problem, with 9.4 million newly emerging active tuberculosis cases and causes nearly 2 million deaths annually. Currently, experimental evidence for an strong association between helminths and diminished T helper (Th)1 immunity against Mycobacterium tuberculosis infection is based on studies which show that helminth-induced Th2, T regulatory (Treg) responses and alternatively activated macrophages contribute to enhanced susceptibility to TB. In this context, it has been shown that Th1 response is reduced in helminth coinfected TB patients. This article discusses what is presently known about the types of immune responses modulated by helminths to diminish the protective immune response to TB.

Antimicrobial peptides as parasiticidal against human trypanosomatids: mechanisms of action and current status in development.

Trypanosomes cause a variety of tropical diseases that affect the livelihood of individuals worldwide. The currently used pharmaceutical treatments rely on chemotherapy. However, many of these drugs are very expensive, and highly toxic. In addition, parasite resistance to several of the therapeutic drugs used is increasing. Therefore, there is a growing need for new control measures for many of these diseases. One new approach is the use of antimicrobial peptides (AMPs) to disease control, since these peptides can be used as potential anti-parasite effector molecules. This review summarizes and discusses the parasiticidal properties of AMPs for treating trypanosome infections, highlighting their mechanisms of action and current status in development.

A study on parasites in Mexican rheumatic disease patients.

Blastocystis hominis is a common human parasite with infection rates up to 50% in developing countries, and giardiasis is the commonest intestinal one in Mexico. No doubt, various parasites as Giardia lamblia and Entamoeba histolytica can cause rheumatic diseases. This study coproparasitoscopic analysis evaluated the cysts by B. hominis, G. lamblia, E. hartmani, E. coli and E. histolytica in Mexican rheumatic disease patients. Also, ELISA was used to detect E. histolytica, Ascaris lumbricoides, Toxocara canis, and Trichinella spiralis in Mexican patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). Thirty-six patients (24 with AS and 12 with RA) and 77 healthy control individuals were enrolled in this study. The frequencies of protozoan cysts were comparable in rheumatic disease patients (AS and RA) and healthy control donors (33 and 25 vs. 26%, respectively; p > 0.05). The frequency of antibodies to T. canis was significantly higher in AS patients than in healthy control donors (16 vs. 2.6%, respectively; p = 0.027), whereas no differences were observed for the prevalence of antibodies for the other parasites (E. histolytica, A. lumbricoides and T. spiralis) (p > 0.05). This information indicates the need to intensify educational efforts for the prevention of parasite infections associated with AS disease that cannot be controlled only by drugs.

Morphometric analysis of nodules in human onchocerciasis collected in communities of the southern Chiapas focus, México.

Human onchocerciasis is a disease that remains as an important public health problem. The morphometric and physical characteristics of 363 Onchocerca volvulus nodules collected in the major endemic focus of onchocerciasis in Southern Chiapas (Soconusco), was assessed. In the present work we found that treatment the morphometry of 363 onchocercal nodules preserved in a 67% glycerol solution was determined by measuring the length, width and thick of each nodule with a Vernier caliper. The mass was determined with an analytical balance and the volume by measuring the water displacement, while the specific gravity was calculated by dividing mass over the volume. Statistical analysis was calculated for each parameter. The results showed that the nodules were rather longer than wider or thicker. Morphometric characteristics were 9.87 +/-3.70 (mean +/- standard deviation), 7.52 +/- 2.81, and 4.62 +/-+/- 2.06 mm for length, width and thick respectively. In regard to the shape, 62.81% of the nodules showed a lenticular shape, while 18.18% were spherical and 19.01% were ovoid. Based on the distribution of frequencies of the length, the nodules were classified in three groups: the "small" (5.77 +/- 0.73 mm; n = 104, 28.65%), the "medium" group (9.86 +/- 2.05 mm; n = 203 nodules, 55.92%), and the group of the "big" ones (16.03 +/- 1.91 mm; n = 56, 15.43%). Moreover, the physical characteristics were: for the mass 0.33 +/- 0.24 g, the volume of displaced water was 0.28 +/- 0.26 ml, and the specific gravity was 1.10 +/- 0.55 g/ml. The results indicated that most of the Mexican Onchocerca nodules have a lenticular shape with average size of 10x7x5 mm, which is useful in the knowledge of the genus biodiversity and can be taken as a parameter in clinical or epidemiological trials, where onchocerciasis remains as a public health problem.

Activation of ERK1/2 and TNF-alpha production are mediated by calcium/calmodulin, and PKA signaling pathways during Mycobacterium bovis infection.

Mycobacterium bovis bacillus Calmette-Guérin (BCG)-induced tumor necrosis factor (TNF)-alpha secretion via an extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase-dependent mechanism is an important host defence mechanism against Mycobacterium tuberculosis in human monocytes. We now define distinct signaling pathways that regulate induction of TNF-alpha and activation of ERK1/2 by intracellular signaling mechanisms during M. bovis infection. We determined that M. bovis BCG-induced ERK 1/2 activation occurs through a mechanism that requires intracellular calcium and likely involves a calmodulin-sensitive step. In contrast, M. bovis BCG can induce p38 mapk activation by a calcium (Ca2+)/calmodulin-independent mechanism. Interestingly, we present evidence that M. bovis BCG activates protein kinase A (PKA), since pretreatment of monocytes with H-89, a inhibitor of PKA activity, blocked the ability of M. bovis BCG to induce ERK1/2 activation. These results were further supported by the fact that treatment of cells with KT5720, another well-described inhibitor of PKA activity, significantly diminished the effect of M. bovis BCG on ERK1/2 activation. Furthermore, secretion of TNF-alpha in M. bovis-infected human monocytes was also dependent on Ca2+/calmodulin, and PKA pathways. Finally, addition of H-89 significantly diminished TNF-alpha mRNA expression in M. bovis-infected human monocytes. These results indicate that the Ca2+/calmodulin, and PKA pathways play important regulatory roles in monocyte signaling upon M. bovis infection.

Differential induction of TNF-alpha and NOS2 by mitogen-activated protein kinase signaling pathways during Mycobacterium bovis infection.

The role of mitogen-activated protein kinase (MAPK) signaling pathways in the regulation of TNF-alpha and NOS2 production by human monocytes infected with Mycobacterium bovis BCG was examined. Inhibition studies showed that ERK1/2 and p38 MAPK activation were necessary for the monocyte response to M. bovis infection. Analysis of MAPK activation showed rapid phosphorylation of ERK1/2 and p38 in response to M. bovis BCG. Phosphorylation was not due to an autocrine effect of TNF-alpha secretion, since an anti-TNF-alpha antibody had no significant effect on the levels of p38 phosphorylation. The inhibitor PD98059 significantly reduced M. bovis BCG-induced TNF-alpha production and almost completely abrogated phosphorylation of ERK1/2; in addition the potent MEK inhibitor U0126 also abrogated phosphorylation. In contrast, studies using inhibitors selective for ERK1/2 and p38 showed that p38 plays an essential role in the induction of NOS2, whereas the role of ERK1/2 was minor. These results suggest that ERK1/2 and p38 kinases differentially regulate the M. bovis BCG-mediated induction of TNF-alpha and NOS2 in human monocytes.

Infection of human monocytes with Mycobacterium bovis BCG induces production of CC-chemokines.

DESIGN: CC-chemokines are potent leukocyte activators and chemoattractants, which have an important role in granuloma formation, function critical for the immune responses to mycobacterial infection. This study investigated whether infection of human monocytes with Mycobacterium bovis bacillus Calmette-Guérin (BCG) elicits secretion of RANTES, macrophage inflammatory protein (MIP)-1alpha and MIP-1beta. METHODS: RANTES, MIP-1alpha and MIP-1beta synthesis was measured by the presence of protein secretion in the cell culture supernatant as determined by enzyme-linked immunosorbent assay. To investigate the mechanism of M. bovis BCG stimulation of RANTES, we carried out inhibition assays with antibodies to CD40 and we used an intracellular calcium chelator BAPTA-AM. RESULTS: Infection of human monocytes with M. bovis BCG induced RANTES, MIP-1alpha and MIP-1beta secretion in a dose-dependent manner. This stimulation of CC-chemokines production was not attributed to LPS contamination. M. bovis-induced RANTES secretion was dependent upon bacterial uptake and on tumor necrosis factor (TNF)-alpha. Interestingly, the production of RANTES by M. bovis BCG-infected monocytes occurs through a mechanism that requires intracellular calcium and was significantly inhibited (P<0.05) with antibodies to CD40. CONCLUSIONS: These results suggest that the ability of M. bovis BCG to produce CC-chemokines might lead to protection in the acquired immune response of mycobacterial infection and at the same time indicate that M. bovis BCG-induced RANTES secretion is mediated by CD40 and dependent on the intracellular calcium influx.

Signals involved in mycobacteria-induced CXCL-8 production by human monocytes.

CXC chemokine-interleukin-8 (IL-8) (CXCL-8) is a potent proinflammatory chemotactic factor that induces important immune responses for antimycobacterial defenses. However, little is known about the biochemical mechanisms by which the mycobacterial antigens upregulate the release of CXCL-8 from human monocytes. In this study, the mechanisms through which Mycobacterium bovis BCG induces CXCL-8 secretion in human monocytes were investigated. We found that M. bovis BCG induced the production of high levels of CXCL-8 by human monocytes. M. bovis-induced CXCL-8 secretion was unaffected by the protein kinase C (PKC) inhibitor bisindolylmaleimide. In contrast, preincubation of the monocytes with the protein tyrosine kinase (PTK) inhibitor genistein resulted in dose-dependent suppression of mycobacteria-induced CXCL-8 secretion. These results were further supported by the fact that treatment of monocytes with herbimycin-A, another well-described inhibitor of PTK activity with a different mechanism of action, significantly diminished the effect of M. bovis on CXCL-8 secretion. In addition, the specificity of this inhibition was demonstrated by the inability of herbimycin-A to block in a significant manner IL-1 beta induction of CXCL-8. Herbimycin-A significantly blocked tyrosine phosphorylation of p59(hck) in response to M. bovis. Finally, two specific NF-kappa B inhibitors, sulfasalazine and caffeic acid phenetyl ester (CAPE), strongly inhibited the production of CXCL-8 by human monocytes infected with M. bovis. These results show intracellular signaling pathways and a transcription factor involved in the M. bovis-mediated upregulation of CXCL-8 biosynthesis and release by human monocytes.

Roles of intracellular calcium and NF-kappaB in the Bacillus Calmette-Guérin-induced secretion of interleukin-8 from human monocytes.

Interleukin (IL)-8 secretion contributes to the early host response against mycobacterial infection by increasing local inflammation and recruiting professional phagocytes. Because the mechanisms through which Mycobacterium bovis Calmette-Guérin (BCG) induces IL-8 secretion are unknown, the aim of the present study was to characterize the nature of IL-8 production induced by BCG in human monocytes. In this study, we found that the induction of IL-8 synthesis was dose- and time-dependent after stimulation with BCG. This IL-8 secretion was not attributed to LPS contamination or the presence of TNF-alpha. We also determined that BCG-induced IL-8 secretion occurs through a mechanism that requires intracellular calcium and likely involves a calmodulin-sensitive step. Interestingly, BCG-induced secretion of IL-8 from human monocytes resulted from transcriptional up-regulation of the IL-8 gene. Moreover, we present evidence that BCG activates nuclear translocation of the transcription factor NF-kappaB, since pretreatment of monocytes with sulfasalazine, a inhibitor of NF-kappaB activity, blocked the ability of BCG to induce IL-8 secretion in a dose-dependent manner, producing 92.5% inhibition at a concentration of 2 mM. These results were further supported by the fact that treatment of cells with curcumin, another well-described inhibitor of NF-kappaB activity with a different mechanism of action, significantly diminished the effect of BCG on IL-8 secretion. Together, these studies are the first to demonstrate that BCG-induced IL-8 secretion by human monocytes appears to be mediated by intracellular Ca(2+) and is NF-kappaB-dependent and at the same time suggest that production of IL-8 in response to M. bovis BCG can contribute to the initial local and systemic inflammatory response in human tuberculosis.

Protein tyrosine kinase regulates FAS-mediated apoptosis in human BCG-infected monocytes.

Apoptosis of monocytes/macrophages has emerged as a central regulatory event in the defense against mycobacterial infections. The involvement of protein tyrosine kinases (PTK) in Fas-mediated apoptosis in T cells is well established, but the possible role of PTK in Fas-dependent death of human bacillus Calmette-Guerin (BCG)-infected monocytes remains unclear. Here, we first examined the expression and function of Fas on BCG-infected human monocytes by flow cytometry. The results demonstrated that BCG-infected monocytes expressed significant Fas protein levels. In addition, engagement of the Fas antigen with its agonistic antibody (Ab) resulted in apoptosis of monocytes, as monitored by DNA analysis and fluorescence-activated cell sorter (FACS) analysis. The apoptotic action of Fas was suppressed significantly by genistein, indicating a role for PTK in this death process. Consistent with this observation, herbimycin A and tyrphostin, two selective tyrosine kinase inhibitors with different mechanisms of action, effectively inhibited Fas-mediated apoptosis of BCG-infected monocytes, as demonstrated by DNA content analysis. Moreover, we confirmed the effect of genistein, herbimycin A, and tyrphostin by examining apoptosis with the terminal transferase dUTP nick endlabeling (TUNEL) assay. Collectively, these data demonstrate that Fas-induced apoptosis may represent an important mechanism for eliminating BCG-activated human monocytes and that this apoptosis is due, at least in part, to signaling via a PTK pathway.

CD40 ligand expression in mycobacterium bovis BCG infection and its regulation by cytokines: a direct role of interleukin 12.

BACKGROUND: Activation and clonal expansion of T cells require not only the recognition of processed antigen on the surface of the antigen-presenting cell (APC) by T-cell receptor (TCR), but also involve co-stimulatory signals that are provided by the simultaneous engagement of cell surface molecules expressed by both the APC and the T cell. Interaction between CD40 and its ligand (CD40L) is known to mediate host immune response and T-cell-mediated effector functions in mycobacterial infections in mice. In this work, we investigated the capacity of Mycobacterium bovis (M. bovis) BCG to induce the expression of CD40L on human T cells. METHODS: Human cells were obtained from healthy adults by centrifugation using Ficoll/Hypaque. Cells (1 x 10(6)) were incubated in RPMI medium with BCG. After incubation at 37 degrees C in 5% CO(2) atmosphere for 40 h, cells were collected and double-stained with anti-CD40L-PE and anti-CD4-FITC or anti-CD8-FITC mAb. The quantification of positively stained population was based on samples stained with isotype control antibodies analyzed on a FACScan. RESULTS: M. bovis BCG stimulation induced a significant amount of CD40L expression on CD4+ T cells, while CD40L was not significantly detected on human CD8+ T cells. The highest CD40L expression on BCG-activated T cells in synergism with interleukin-12 endogenous occurred after a 40-h cell culture with a dose of 10 microg/mL of BCG (84.86 +/- 11.77; mean +/- standard deviation [SD]). This CD40L expression on BCG-activated human T cells was significantly inhibited by anti-IL-12 mAb (5.08 +/- 1.7; mean +/- SD). In contrast, anti-IFN-gamma and anti-IL-2 mAb did not have an important role in this expression. CONCLUSIONS: These results indicate that the capacity of BCG to induce CD40L expression on human cells represents a novel mechanism underlying the regulation of cellular responses against tuberculosis. Furthermore, the results showed a direct role of IL-12 to enhance the expression of CD40L on BCG-activated human cells.

Induction of apoptosis in bacillus Calmette-Guérin-activated T cells by transforming growth factor-beta.

In view of the critical role played by bacillus Calmette-Guérin (BCG) in the development and functional activation of protective T cells against tuberculosis, it has become important to understand the mechanisms by which cytokines regulate BCG-mediated immune responses. There is evidence that cytokine-mediated suppression of T cell function by mechanisms, including apoptosis, may reduce host resistance in tuberculosis. However, it is unclear whether cytokine-mediated suppression of antigen-responsive T cells through apoptotic mechanisms may be operating during human cellular activation induced by BCG. Here we present evidence, for the first time, that treatment of BCG-activated T cells with transforming growth factor-beta (TGF-beta) induces cellular apoptosis. These results were further supported by the fact that treatment of cells with a blocking mAb directed to TGF-beta significantly inhibited the percentage of apoptosis induced by TGF-beta. Interestingly, TGF-beta-mediated death of BCG-activated T cells in cultures containing interleukin (IL)-12 was observed. Moreover, our results demonstrated the induction of apoptosis by TGF-beta in BCG-activated T cells cultured in the presence of exogenous IL-12. In addition, our data indicated that TGF-beta significantly inhibited both BCG-induced cell growth determined by thymidine uptake and BCG-induced IFN-gamma secretion. Finally, TGF-beta-induced apoptosis in BCG-activated T cells correlated inversely with BCG-induced IFN-gamma secretion. Taken together, these findings indicate that TGF-beta induces apoptosis in human T cells activated with BCG and at the same time suggest that loss of BCG-reactive T cells through apoptotic mechanisms could contribute to an increased susceptibility to Mycobacterium tuberculosis infection.

Induction of transforming growth factor-beta 1 production in human cells by herpes simplex virus.

Transforming growth factor-beta (TGF-beta) is a cytokine of particular interest in human retrovirus infections because it can abrogate antigen-specific cellular activation. Although TGF-beta production has been observed in HIV infections, there is no evidence that herpes simplex virus (HSV)-stimulated human cells produce this cytokine. Here we present evidence, for the first time, that in vitro infection of human mononuclear cells with HSV type 1 (HSV-1) induced the release of TGF-beta1 protein. The production of this cytokine was time dependent and was found highly significant (p < 0.001) after 48 h. In addition, we observed that the secretion of TGF-beta1 was dependent on the concentration of human cells. It was found that virus needs to replicate in human cells for the production of TGF-beta1, as UV-inactivated virus did not induce significant production of cytokine protein. Interestingly, increased HSV-1-induced TGF-beta1 production in cultures containing antiinterleukin (IL)-12 or antiinterferon (IFN)-gamma antibodies was observed, whereas an irrelevant antibody had no effect on the production of this cytokine. Taken together, these findings indicate that human cells synthetize TGF-beta1 in response to HSV-1 and at the same time suggest that HSV-1-induced TGF-beta1 production may be one of the mechanisms by which HSV can at least partly evade activation of the host immune system.

Interleukin-12 regulates the production of Bacille Calmette-Guérin-induced interferon-gamma from human cells in a CD40-dependent manner.

Interferon (IFN)-gamma is a cytokine which plays a critical role in the host defence against human tuberculosis infection. There is evidence that interleukin (IL)-12 can exert a potent effect in stimulating the production of IFN-gamma, and it is well known that a costimulatory signal provided by CD40 may enhance IL-12 production by monocytes/macrophages. However, it is unclear whether CD40-CD40L stimulation is able to regulate the production of mycobacterial-induced IFN-gamma through an IL-12-dependent pathway. In this study, we investigated the capacity of the Bacille Calmette-Guérin (BCG) strain of Mycobacterium bovis to induce the production of interferon-gamma through IL-12 and/or CD40 costimulation from human cells. Our data demonstrate that anti-IL-12 antibodies markedly reduced the levels of IFN-gamma produced by the BCG-stimulated human cells, while exogenous recombinant (r)IL-12 up-regulated the production of IFN-gamma. In addition, the stimulatory effect of IL-12 on BCG-induced IFN-gamma secretion was specific, as it was significantly abolished in the presence of anti-IL-12 antibodies. We also observed that the presence of an anti-CD40L monoclonal antibody significantly inhibited the production of IL-12 and IFN-gamma by human cells activated with BCG. In contrast, an isotype control antibody showed no effect on cytokine production. Furthermore, the presence of a trimeric soluble CD40L agonist (CD40T) in cultures increased the production of IL-12 and IFN-gamma. Importantly, the stimulatory capacity of CD40T on BCG-induced IFN-gamma secretion was blocked by a monoclonal antibody against IL-12, indicating that the effect of CD40T on T cells was mediated through IL-12. Together, these studies are the first to demonstrate that BCG-induced IFN-gamma production by human cells appears to be mediated by IL-12 in a CD40-dependent manner and suggest that CD40-CD40L activation may be a critical mediator in regulating the immune response to stimulation with BCG.

Regulation of interleukin-12 production in human cells stimulated with Mycobacterium bovis BCG.

Interleukin 12 (IL-12) is a monokine which plays a critical role in resistance to Mycobacterium tuberculosis infection. However, little is known about the regulation of IL-12 production by human cells stimulated with BCG. Here we report that in vitro infection of human mononuclear cells with M. bovis BCG induces the release of IL-12 protein. We also observed that the ability of BCG to stimulate release of IL-12 from human cells was significantly inhibited by IL-10. The inhibitory effect of IL-10 on the secretion of IL-12 was specific, as it was significantly abolished in the presence of anti-IL-10 neutralizing monoclonal antibody. These results were further confirmed as anti-IL-10 antibodies markedly increased the levels of IL-12, suggesting that BCG-induced IL-10, as well as exogenous IL-10, can regulate IL-12 production by human cells stimulated with M. bovis BCG. Interestingly, IFN-gamma production in response to BCG had no significant increase by the addition of neutralizing antibodies to IL-10. Moreover, anti-IL-12 antibodies markedly reduced the levels of IFN-gamma produced by BCG-stimulated human cells and abrogated the capacity of anti-IL-10 to increase BCG-induced IFN-gamma. These studies are the first to demonstrate a regulatory effect on IL-12 production by human cells infected with M. bovis BCG and at the same time suggest that IL-12 may play an essential role during the human immune response to M. bovis BCG stimulation.

Inhibition of Mycobacterium bovis BCG-induced tumor necrosis factor alpha secretion in human cells by transforming growth factor beta.

The effect of exogenous transforming growth factor beta (TGF-beta) on Mycobacterium bovis BCG-induced tumor necrosis factor alpha (TNF-alpha) production by human mononuclear cells was studied. It was found that TNF-alpha production by human cells stimulated with BCG was significantly inhibited by TGF-beta. The specificity of the observed inhibition was demonstrated, since the addition of an anti-TGF-beta neutralizing monoclonal antibody completely reversed the inhibitory effect. Furthermore, the suppressive effect of TGF-beta on TNF-alpha secretion in this system was not due to a direct cytotoxic effect, since cell viability was comparable in the presence or absence of TGF-beta. Interestingly, our results demonstrated comparative suppressive effects of TGF-beta and interleukin-10 on BCG-induced TNF-alpha secretion. Together, the data demonstrate, for the first time, that TGF-beta inhibits BCG-induced TNF-alpha secretion by human cells.

Depletion of endogenous interleukin-10 augments interleukin-1 beta secretion by Mycobacterium bovis BCG-reactive human cells.

In this study, we found evidence that the interleukin-10 (IL-10) protein is functionally relevant in Mycobacterium bovis BCG-induced cytokine synthesis, as neutralization of endogenously synthesized IL-10 in human cells activated with BCG resulted in a two- to threefold increase in the level of IL-1 beta. When exogenous recombinant human IL-10 was added to human mononuclear cells, a significant reduction of BCG-induced IL-1 beta secretion was observed. This inhibitory effect was not attributed to a cytotoxic effect, since trypan blue exclusion studies indicated no loss of cell viability in the presence of IL-10, and it was specific, as it was completely abolished in the presence of anti-IL-10 neutralizing monoclonal antibody while an irrelevant antibody used as a control had no effect. Taken together, these are the first studies that demonstrate that the depletion of endogenous IL-10 via anti-IL-10 antibody results in a very significantly enhanced BCG-induced IL-1 beta secretion and that the addition of exogenous IL-10 to human mononuclear cells stimulated with BCG inhibits IL-1 beta production. Further experimental work is needed to determine if the neutralization of IL-10 activity via anti-IL-10 antibody indeed enhances cytokine synthesis in vivo. However, the present results may be of importance, since the use of anti-IL-10 antibody could presumably contribute to the protective immunity induced by BCG against tuberculosis via an increase in cytokine synthesis that would amplify antimicrobial systems.

Interleukin-4 inhibits secretion of interleukin-1beta in the response of human cells to mycobacterial heat shock proteins.

Cellular activation induced by Mycobacterium bovis bacillus Calmette-Guérin (BCG) and heat shock proteins (HSP) leads to the production of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. In this study, we found that IL-4 significantly suppressed IL-1beta secretion induced by BCG and the 70- and 65-kDa HSP. When exogenous recombinant human IL-4 was added to human mononuclear cells, a dose- and time-related inhibition of the 70-kDa HSP- and BCG-induced IL-1beta secretion was observed. IL-1beta secretion was maximally inhibited at 24 h of culture, and this inhibitory effect was sustained at a later time point of culture (120 h). In addition, IL-2, another T-cell-derived cytokine acting on monocytes, had no effect on IL-1beta secretion induced by either BCG or the 70-kDa HSP, indicating that in these experiments not all cytokines could immunoregulate IL-1beta secretion. This inhibitory effect was not due to a cytotoxic effect of IL-4, since the viabilities of human mononuclear cells were comparable in the presence and absence of IL-4. IL-4 was also able to inhibit the secretion of IL-1beta by mycobacterium-stimulated cells from three rheumatoid arthritis patients. This inhibitory effect of IL-4 was reversed with a blocking anti-IL-4 antibody. Finally, IL-4 inhibited IL-6 secretion by mycobacterium-activated human cells. These results suggest that IL-4 may be important in the regulation of the immune response to mycobacterial antigens.