Increased serum concentrations of estrogen-induced growth factors Midkine and FGF2 in NF1 patients with plexiform neurofibroma.

Neurofibromatosis type 1 (NF1) predisposes to the development of dermal and plexiform neurofibromas and serum of NF1 patients stimulates neurofibroma proliferation in vitro. This study aimed to determine whether, in NF1 patients, serum levels of midkine (MK) and fibroblast growth factor 2 (FGF2) were associated with the number and/or type of neurofibromas. In addition, their concentrations were correlated with serum levels of dehydroepiandrosterone sulfate (DHEAS), a neurosteroid secreted by the peripheral nervous system. We performed a case control-study and measured, by ELISA assay, serum concentrations of MK, FGF2, and DHEAS in 20 NF1 patients and 30 controls. We found increased serum levels of MK and FGF2 in NF1 patients between 30 and 50 years old. Their concentrations were significantly higher in NF1 patients with plexiform neurofibromas than in controls (P=0.003 for MK and P=0.008 for FGF2). As an underlying hormonal regulation was suspected, DHEAS serum levels were measured but no difference was observed between patients and controls. We also observed a strong association between MK and FGF2 levels (P=0.0001) in NF1 patients and controls. In conclusion, we point out MK and FGF2 as biomarkers for plexiform neurofibroma in NF1 patients. As both growth factors are estrogen-responsive genes and neurofibromin is a co-repressor of estrogen receptor alpha activity, we suggest that the increased serum levels of MK and FGF2 observed in NF1 patients might be due to estradiol hypersensitivity.

Oral Consumption of Bread from an RNAi Wheat Line with Strongly Silenced Gliadins Elicits No Immunogenic Response in a Pilot Study with Celiac Disease Patients.

Celiac disease (CD) is a genetically predisposed, T cell-mediated and autoimmune-like disorder caused by dietary exposure to the storage proteins of wheat and related cereals. A gluten-free diet (GFD) is the only treatment available for CD. The celiac immune response mediated by CD4+ T-cells can be assessed with a short-term oral gluten challenge. This study aimed to determine whether the consumption of bread made using flour from a low-gluten RNAi wheat line (named E82) can activate the immune response in DQ2.5-positive patients with CD after a blind crossover challenge. The experimental protocol included assessing IFN-γ production by peripheral blood mononuclear cells (PBMCs), evaluating gastrointestinal symptoms, and measuring gluten immunogenic peptides (GIP) in stool samples. The response of PBMCs was not significant to gliadin and the 33-mer peptide after E82 bread consumption. In contrast, PBMCs reacted significantly to Standard bread. This lack of immune response is correlated with the fact that, after E82 bread consumption, stool samples from patients with CD showed very low levels of GIP, and the symptoms were comparable to those of the GFD. This pilot study provides evidence that bread from RNAi E82 flour does not elicit an immune response after a short-term oral challenge and could help manage GFD in patients with CD.

Microarray analysis of Mycobacterium tuberculosis-infected monocytes reveals IL26 as a new candidate gene for tuberculosis susceptibility.

Differences in the activity of monocytes/macrophages, important target cells of Mycobacterium tuberculosis, might influence tuberculosis progression. With the purpose of identifying candidate genes for tuberculosis susceptibility we infected monocytes from both healthy elderly individuals (a tuberculosis susceptibility group) and elderly tuberculosis patients with M. tuberculosis, and performed a microarray experiment. We detected 78 differentially expressed transcripts and confirmed these results by quantitative PCR of selected genes. We found that monocytes from tuberculosis patients showed similar expression patterns for these genes, regardless of whether they were obtained from younger or older patients. Only one of the detected genes corresponded to a cytokine: IL26, a member of the interleukin-10 (IL-10) cytokine family which we found to be down-regulated in infected monocytes from tuberculosis patients. Non-infected monocytes secreted IL-26 constitutively but they reacted strongly to M. tuberculosis infection by decreasing IL-26 production. Furthermore, IL-26 serum concentrations appeared to be lower in the tuberculosis patients. When whole blood was infected, IL-26 inhibited the observed pathogen-killing capability. Although lymphocytes expressed IL26R, the receptor mRNA was not detected in either monocytes or neutrophils, suggesting that the inhibition of anti-mycobacterial activity may be mediated by lymphocytes. Additionally, IL-2 concentrations in infected blood were lower in the presence of IL-26. The negative influence of IL-26 on the anti-mycobacterial activity and its constitutive presence in both serum and monocyte supernatants prompt us to propose IL26 as a candidate gene for tuberculosis susceptibility.

In vitro infection of human cells with Mycobacterium tuberculosis.

It has been estimated that approximately 50% of individuals exposed to Mycobacterium tuberculosis never become tuberculin skin test positive, which may indicate that successful human immunological responses are able not only to inhibit mycobacterial growth, but also to kill the bacteria. Nevertheless, it has been extremely difficult to reproduce this effect in vitro and the ability of human phagocytes to eliminate the bacteria is controversial. This is one of the reasons why we do not fully understand either tuberculosis resistance or susceptibility. Nowadays there is a pressing need in tuberculosis vaccine research to find biomarkers of successful responses to tuberculosis and the use of in vitro models may allow the identification of the immunological mechanisms responsible for the mycobacterial killing that could be tested in clinical settings. Besides biosafety concerns, the manipulation of mycobacteria is technically very demanding, and the optimization of in vitro infection protocols have been difficult. As a result, there are a large number of variations in the methodology that make arduous the comparison of results obtained in different research groups. In this review an overview of the mycobacterial and human cellular models most frequently studied are presented, together with a description of several of the modifications tried in infection protocols, from the preparation of the inoculum to the quantification of surviving mycobacteria after infection. A comment about statistical methods that may improve the detection of relevant biological effects is also included.

Macrophages from elders are more permissive to intracellular multiplication of Mycobacterium tuberculosis.

The elderly account for a disproportionate share of all tuberculosis cases, and the population ageing may not fully explain this phenomenon. We have performed in vitro infection experiments to investigate whether there is an immunological basis for the apparent susceptibility of elders to tuberculosis. In our infection model, Mycobacterium tuberculosis induces a higher production of interleukin (IL)-6 and reactive oxygen species in macrophages from elders than from younger adults. This response did not prevent, however, an increased multiplication of M. tuberculosis in macrophages from elders as compared with the growth observed within cells from adults. By performing a factorial experiment, we have found that IFN-γ, but not IL-1ß, IL-6 or TNF-α, stimulate the macrophages to restrict the multiplication of the bacterium in macrophages from elders. Although monocytes from elders seem to be in a higher level of activation, we present evidences that protein tyrosine phosphorylation response induced by M. tuberculosis is stronger in monocytes from adults than from elders. Using a protein array that detects 71 tyrosine phosphorylated kinases, we identified Pyk2 as the only kinase that displayed a difference of intensity larger than 50 % in adults than in elders. Furthermore, monocytes from elders that were incubated in the presence of tyrosine kinase inhibitors (genistein and PP2) allowed a higher level of bacterial multiplication. These observations may help to explain the susceptibility of elders to tuberculosis. An unexpected result was that both genistein and its negative control, daidzein, abundant soy isoflavones, promoted intracellular mycobacterial growth.

Apoptosis and oxidative burst in neutrophils infected with Mycobacterium spp.

Two of the better characterized antimicrobial mechanisms displayed by human neutrophils are the reactive oxygen species (ROS) production and the induction of apoptosis. Their importance in mycobacterial infections is, however, controversial and we aimed to analyze them simultaneously in neutrophils infected with either Mycobacterium tuberculosis or the non-pathogenic M. gordonae. Neither species is eliminated by neutrophils but the pattern exhibited for both activities is completely different. M. tuberculosis induces ROS production and apoptosis but M. gordonae does not. Additional evidence was provided by an attenuated strain of M. gordonae that, although it has become susceptible to the antimicrobial activity of neutrophils, it still does not promote ROS production or apoptosis. Therefore no relationship could be established between any of these activities and the ability of neutrophils to kill mycobacteria. We have also observed that neutrophil concentration, a variable that is important in the antimicrobial activity against other pathogens, has no influence in the mycobacterial intracellular growth.

Cytokines as immunomodulators in tuberculosis therapy.

The use of cytokines for therapeutic purposes is limited by their high cost and toxicity. Nevertheless, the emergence of extensively drug-resistant tuberculosis (XDR TB), for which chemotherapy is ineffective, has again made cytokine-based therapy attractive as one of the last available options. The results of clinical trials treating pulmonary tuberculosis with cytokines have not been encouraging, making it clear that therapeutic strategies utilizing a single cytokine are inadequate. To develop effective cytokine-based XDR TB therapies, more basic research will be needed to achieve a better understanding of how cytokines promote a successful immune response. We not only have to investigate cytokines already known to participate in tuberculosis, but also the role of other cytokines and chemokines that may enhance both the mycobacterial killing activity of effector cells and the restriction of bacterial intracellular multiplication. There are already several patents involving cytokines for therapeutic use, in the hope of stimulating the immune system in a variety of infectious diseases, including tuberculosis. The validity of these patents needs to be reassessed from a clinical standpoint, and new applications of patents concerning cytokines potentially useful in XDR TB treatment should be encouraged.

Detection of inhibition of antimicrobial activity by mycobacterial lysates in human monocytes infected with Legionella pneumophila.

Antimicrobial activity in human monocytes infected with Mycobacterium tuberculosis has been difficult to demonstrate in vitro, and the molecular mechanisms allowing the bacteria to survive intracellularly are unknown. As a means to test the influence of bacterial products in the microbicidal activity of monocytes we have developed an infection model with Legionella pneumophila, which is killed by interferon gamma activated cells. We demonstrate that this model is useful because M. tuberculosis lysates inhibit one hundred fold the interferon gamma induced activity against L. pneumophila. Comparable degrees of inhibition are also detected when we use lysates from the less pathogenic Mycobacterium gordonae and the pathogenic Staphylococcus aureus, suggesting the participation of a common mechanism. This hypothesis is supported by the fact that the pattern of cytokine secretion is similar in all cases. A significant difference is, however, observed when we used lysates from the non-pathogenic Escherichia coli, which resulted in the recovery of low numbers of bacteria, probably because they induce the cell death of infected monocytes.

Human phagocytes lack the ability to kill Mycobacterium gordonae, a non-pathogenic mycobacteria.

Non-pathogenic mycobacteria, like Mycobacterium gordonae, are rarely associated to disease. The analysis of the mechanisms which are successful against them in the human host may provide useful information to understand why they fail against the pathogenic M. tuberculosis. We have developed an infection model to test the ability of human phagocytes to kill two strains of M. gordonae, HL184G and an attenuated variety, HL184Gat. As controls we included a strain of M. tuberculosis (HL186T) and another one of L. pneumophila (ATCC13151). We observed that human phagocytes lack the intrinsic ability to eliminate either M. gordonae or M. tuberculosis, but they can kill the attenuated strain. We found a relationship between pathogenicity and the pattern of cytokine production. Thus, both the pathogenic M. tuberculosis and Legionella pneumophila, but not the non-pathogenic M. gordonae, induced the production of significantly different levels of IL-1beta, IL-6 and TNF-alpha in monocytes and IL-8 in neutrophils. Although both monocytes and neutrophils killed HL184Gat, but not HL184G, the patterns of cytokine production induced by either strain were identical. Addition of INF-gamma and/or TNF-alpha did not enhance the antimycobacterial activity of phagocytes.