Octavio M. Rivero-Lezcano

Wiskott-Aldrich syndrome protein physically associates with Nck through Src homology 3 domains.

In the second of a series of experiments designed to identify p47nck-Src homology 3 (SH3)-binding molecules, we report the cloning of SAKAP II (Src A box Nck-associated protein II) from an HL60 cDNA expression library. This molecule has been identified as a cDNA encoding the protein product of WASP, which is mutated in Wiskott-Aldrich syndrome patients. Studies in vivo and in vitro demonstrated a highly specific interaction between the SH3 domains of p47nck and Wiskott-Aldrich syndrome protein. Furthermore, anti-Wiskott-Aldrich syndrome protein antibodies recognized a protein of 66 kDa by Western blot (immunoblot) analysis. In vitro translation studies identified the 66-kDa protein as the protein product of WASP, and subcellular fractionation experiments showed that p66WASP is mainly present in the cytosol fraction, although significant amounts are also present in membrane and nuclear fractions. The main p47nck region implicated in the association with p66WASP was found to be the carboxy-terminal SH3 domain.

In Vivo Monitoring of Intracellular ATP Levels in Leishmania donovani Promastigotes as a Rapid Method To Screen Drugs Targeting Bioenergetic Metabolism

ABSTRACT A method for the rapid screening of drugs targeting the bioenergetic metabolism of Leishmania spp. was developed. The system is based on the monitoring of changes in the intracellular ATP levels of Leishmania donovani promastigotes that occur in vivo, as assessed by the luminescence produced by parasites transfected with a cytoplasmic form of Phothinus pyralisluciferase and incubated with free-membrane permeabled-luciferin analogued-luciferin–[1-(4,5-dimethoxy-2-nitrophenyl) ethyl ester]. A significant correlation was obtained between the rapid inhibition of luminescence with parasite proliferation and the dissipation of changes in mitochondrial membrane potential (ΔΨm) produced by buparvaquone or plumbagin, two leishmanicidal inhibitors of oxidative phosphorylation. To further validate this test, a screen of 14 standard leishmanicidal drugs, using a 50 μM cutoff, was carried out. Despite its semiquantitative properties and restriction to the promastigote stage, this test compares favorably with other bioenergetic parameters with respect to time and cell number requirements for the screening of drugs that affect mitochondrial activity.

CCL20 is overexpressed in Mycobacterium tuberculosis-infected monocytes and inhibits the production of reactive oxygen species (ROS)

CCL20 is a chemokine that attracts immature dendritic cells. We show that monocytes, cells characteristic of the innate immune response, infected with Mycobacterium tuberculosis express the CCL20 gene at a much higher level than the same cells infected with non‐tuberculous mycobacteria. Interferon (IFN)‐γ, a fundamental cytokine in the immune response to tuberculosis, strongly inhibits both the transcription and the translation of CCL20. We have also confirmed that dendritic cells are a suitable host for mycobacteria proliferation, although CCL20 does not seem to influence their intracellular multiplication rate. The chemokine, however, down‐regulates the characteristic production of reactive oxygen species (ROS) induced by M. tuberculosis in monocytes, which may affect the activity of the cells. Apoptosis mediated by the mycobacteria, possibly ROS‐dependent, was also inhibited by CCL20.

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.

Non-chemotactic influence of CXCL7 on human phagocytes. Modulation of antimicrobial activity against L. pneumophila.

We have investigated the role of CXCL7 in the immune response of human phagocytes against the intracellular bacteria Mycobacterium tuberculosis and Legionella pneumophila. We have observed that polymorphonuclear neutrophil (PMN) chemotaxis induced by the supernatants of infected monocyte derived macrophages (MDM) may be attributed to CXCL8 rather than CXCL7, although both chemokines are present in large quantities. We have also found that CXCL7 is present not only in the supernatants of MDM, but also in the supernatants of PMN of some, but not all, individuals. Western blot analysis revealed that, in both MDM and PMN supernatants appeared two bands with molecular weights consistent with the platelet basic protein (PBP) and the neutrophil activating protein-2 (NAP-2) sizes. Regarding the influence on infected cells, recombinant NAP-2 enhanced the antimicrobial activity of IFNγ activated MDM against L. pneumophila, but not against M. tuberculosis. In addition, U937 cells transfected with a NAP-2 construct inhibited the intracellular multiplication of L. pneumophila, supporting its role in the modulation of the antimicrobial activity. Finally, U937 cells transfected with the NAP-2 construct showed an adherence that was dramatically enhanced when the substrate was fibronectin. We conclude that human phagocytes produce CXCL7 variants that may have a significant influence on the immune response against bacterial pathogens.

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.

Specificity of protein interactions with highly related SRC homology (SH) domains of FGR and FYN protein-tyrosine kinases

As an approach toward identification and isolation of cellular proteins that may act as substrates or effectors of the SRC‐family of protein‐tyrosine kinases, fusion proteins containing noncatalytic elements of two highly related SRC‐family members were tested for their ability to recognize distinct molecules present in lysates of cells known to normally express both enzymes. Our results demonstrate differences of protein binding between the SH2 elements of FYN and FGR kinases, but do not discriminate proteins binding to their SH3 domains.

Aproximación a la incidencia real de tuberculosis en el Área de Salud de León: aplicación del método captura-recaptura para comparar 2 fuentes de información

INTRODUCTION The actual incidence of tuberculosis is probably higher than that previously published in national and international records. Under-reporting is estimated to fluctuate between 7% and 27%, according to studies. OBJECTIVE To estimate the incidence rate of tuberculosis in the area of León for 2008 and 2009 using the capture-recapture method in order to compare two sources of information: prescribed tuberculostatic drugs (combination of rifampicin-isoniazid) and the regional epidemiological surveillance system register (SIVE). METHOD Retrospective descriptive study in an area of 351,086 inhabitants of tuberculosis cases using as sources: (i), information on prescribed tuberculostatic drugs, and (ii), the SIVE register. We calculated incidence rates for each source by the capture-recapture method. We analyzed epidemiological and demographic data, symptoms, diagnosis, treatment and follow-up. RESULTS The incidence based on the SIVE data for 2008 was 18.80/100,000 inhabitants and according to the pharmacy register, the rate was 26.77. The estimated value for 2009 based on the SIVE data was 18.23/100,000 inhabitants, and according to the pharmacy register, it was 22.50. After applying the capture-recapture method, the annual incidence for 2008 was 44.14/100,000 (95% CI; 37.88-50.41) and for 2009, it was 34.17/100,000 (95% CI; 30.19-38.17). In the study of all these years we have found that the number of cases were higher in the pharmacy register than the SIVE one. CONCLUSIONS The SIVE data on the incidence of tuberculosis in our study area underestimates the actual incidence rate. The source of information that involves case record of tuberculosis in the community is under-used. The capture-recapture method is a good alternative to measure the incidence of tuberculosis, and to check the surveillance systems.

Immunological response to Mycobacterium tuberculosis infection in blood from type 2 diabetes patients

The convergence of tuberculosis and diabetes represents a co-epidemic that threatens progress against tuberculosis. We have investigated type 2 diabetes as a risk factor for tuberculosis susceptibility, and have used as experimental model whole blood infected in vitro with Mycobacterium tuberculosis. Blood samples from diabetic patients were found to have a higher absolute neutrophil count that non-diabetic controls, but their immune functionality seemed impaired because they displayed a lower capacity to phagocytose M. tuberculosis, a finding that had been previously reported only for monocytes. In contrast, an increased production of TNFα was detected in infected blood from diabetic patients. Despite the altered phagocytic capacity showed by cells from these patients, the antimicrobial activity measured in both whole blood and monocyte derived macrophages was similar to that of controls. This unexpected result prompts further improvements in the whole blood model to analyze the immune response of diabetes patients to tuberculosis.