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Leprosy, a human chronic granulomatous disease caused by Mycobacterium leprae (M. leprae), remains endemic in certain countries despite the use of multidrug therapy. Recently, several host genes modulating the immune responses to M. leprae infection have been suggested to influence the acquisition and clinical course of leprosy. Lymphoid protein tyrosine phosphatase, encoded by the protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene, serves a negative regulatory role in T cell activation. The non-synonymous single-nucleotide polymorphism (SNP) rs2476601 (1858C>T) has been associated with autoimmune diseases. Here, the present study investigated if rs2476601 polymorphism was associated with leprosy in a Mexican mestizo population. Genotyping was performed in patients with leprosy (n=189) and control subjects (n=231) from regions with higher incidence of leprosy. Genotypic (P=0.44) and allelic frequencies (P=0.45) of the rs2476601 polymorphism were similar between patients and controls; genotypic frequencies were 91 vs. 94% for CC and 9 vs. 6% for CT, and the TT genotype was absent in both groups. Allelic frequencies were 96 vs. 97% for C, and 4 vs. 3% for T. In the same way, the genotypic (P=0.46) and allelic frequencies (P=0.47) from MB patients and controls were similar. In conclusion, there was a lack of association of the PTPN22 rs2476601 polymorphism with the development of leprosy, which suggests that this SNP was not a genetic risk factor for leprosy in the Mexican mestizo population studied.

Mexico is a country with sporadic leprosy cases, and the reemergence of drug resistance is a concern. In this study, molecular analysis of Mycobacterium leprae was employed to clarify the spread of drug-resistant leprosy. Thus, drug resistance-determining regions in the folP1, rpoB, and gyrA genes, which are associated with resistance to dapsone, rifampicin, and ofloxacin, respectively, were analyzed by direct sequencing of the PCR product. No mutations in the folP1 gene were observed in any of the 72 slit skin samples obtained from 38 patients, although two samples carrying a mutation at codon 425 in the rpoB gene, which confers resistance to rifampicin, a key component of multidrug therapy, were identified. In addition, a mutation at codon 91 in the gyrA gene, which correlates with ofloxacin resistance, was found in one sample. These results demonstrate the existence of rifampicin- and ofloxacin-resistant leprosy. Interestingly, wild-type and mutant sequences in the gyrA gene were found to coexist in one clinical sample. In addition, all three drug resistance-related mutations were found in only one of the two earlobes of the patients concerned, suggesting a possible pathway for the spread of drug-resistant M. leprae.

Tuberculosis (TB), an infection caused by Mycobacterium tuberculosis, is one of the ten main causes of death by infectious disease. When the mycobacteria are inhaled, they interact with immune cells in the respiratory tract in a specialized structure known as granuloma. Interleukin 17 (IL-17) contributes to formation and size of the granuloma, and it correlates with the recruitment of neutrophils. It has been demonstrated that other innate lymphocytes, such as Tγδ cells, contribute to the production of IL-17 during TB. Innate lymphoid cells (ILCs) are other innate lymphocytes that are classified into groups (ILC1, ILC2 and ILC3) according to their cytokine patterns. ILC3 produce IL-17, but it is not known if these cells participate in the response to TB infection. We evaluated, by flow cytometry, the frequency and IL-17 production of Th, Tgd, NK and ILCs (ILC1, ILC2 and ILC3) in the peripheral blood of healthy individuals (negative and positive for the Quantiferon [QTF] test, which detects latent TB), and of patients with active pulmonary TB. The numbers of Th cells, NK cells and ILCs (ILC1, ILC2 and ILC3) were decreased in TB patients, compared to healthy individuals. Peripheral blood was stimulated with M. tuberculosis soluble extract (MTSE) for 5 h in the presence of brefeldin A. After stimulation, Th cells from QTF-positive healthy individuals and TB patients produced IL-17. Tγδ cells also produced IL-17, although the percentage of these cells did not change after activation with MTSE. In some cases, NK, ILC2 and ILC3 from TB patients produced IL-17, but this production did not change after MTSE activation. These results indicate that Th and Tγδ cells contribute to IL-17 production during TB. This project was funded by SIP-IPN-México (20180254).

Streptococcus mutans is a pathogen related to the occurrence of human dental caries. The determination of total amounts of mutans streptococci, as well as the proportion related to other oral bacteria, is of interest when assessing the risk of developing caries. In this context, it is better to use a sensitive, specific and non-time consuming method such as the polymerase chain reaction (PCR), than to use culture and biochemical identification methods. In this work we identified potentially cariogenic strains of S. mutans and assessed the relationship with the dmft, DMFT or dmft/DMFT index. Using DNA isolated from dental plaque, a 192 bp sequence was identified and amplified from the spaP gene and a 722 bp sequence from the dexA gene. The results suggest that it is important to evaluate the presence of cariogenic S. mutans strains in plaque content rather than the accumulation of plaque itself. However, other factors like diet, hygiene, genetic background, the flow rate of saliva and the presence of specific antibodies, also play a key role in the development of caries.

Sepsis is a systemic inflammatory response to infection, which leads to life-threatening organ dysfunction. During sepsis, many cells of the innate and adaptive immune systems have functional alterations; in particular, monocytes produce reduced levels of pro-inflammatory cytokines in response to microbial products (MAMPs). Innate lymphoid cells (ILCs) are cells with lymphocyte morphology that lack antigen-specific receptors and respond directly to cytokines and MAMPs; it is not known if ILCs are affected during sepsis. In this study, we used flow cytometry to evaluate the frequency of ILCs in the peripheral blood of septic patients and healthy controls, as well as the expression of CD56, CCR6 and NKp44 and the production of IL-17 and IFN-γ by ILCs. ILCs were defined as CD45+ lineage- CD127+ cells. The number of total lymphocytes and the number of ILCs were significantly lower in septic patients, compared to healthy controls, with ILCs representing around 0.5% of peripheral blood lymphocytes. In both healthy controls and septic patients, CD56 defines two populations of ILCs: a higher percentage of the CD56− cells express CCR6, compared to the CD56+ cells, while a higher percentage of the CD56+ cells express NKp44, compared to the CD56− cells. CD56− ILCs are the main producers of IL-17, while CD56+ cells are the main producers of IFN-γ, in both healthy controls and septic patients. The production of these two cytokines is increased in the ILCs from septic patients, compared to the ILCs from healthy controls. This is in contrast with the decreased cytokine production that is observed in monocytes and T lymphocytes from septic patients. This project was funded by CONACyT-México (219661).

We have previously shown that human neutrophils (NEU) stimulated with live Mycobacterium tuberculosis H37Rv (Mtb) release exovesicles, named EXO-Mtb. When autologous macrophages were incubated with EXO-Mtb, they increased the expression of CD86 and MHC II and produced inflammatory cytokines. Here we demonstrate that EXO-Mtb can also reduce the intracellular load of Mtb in autologous macrophages. Since autophagy has been shown to control mycobacterial growth, LC3II expression was measured on infected macrophages after incubation with EXO-Mtb (4, 6 and 24h). Control EXO preparations included those released spontaneously from NEU, or after stimulation with PMA or fMLP. Our results showed that of all EXO preparations used, EXO-Mtb induced the largest amounts of LC3II protein, which correlates with a significant reduction of colony forming units (CFU). This phenomenon could be reverted with the addition of Wortmannin, an autophagy inhibitor. It is important to remark that there was not a 100% control of intracellular mycobacterial growth. Further experiments should be done to better understand the role of ectosomes in NEU-macrophage communication through exovesicles.

We have investigated changes in gene expression in mouse peritoneal macrophages following infection with virulent Mycobacterium tuberculosis . Using differential-display reverse transcription-PCR (RT-PCR), we have identified a gene that was markedly down-regulated within 6 h of infection and remained so for the duration of the experiment (5 days). On sequencing, this gene was found to encode the murine cytochrome c oxidase subunit VIIc (COX VIIc). Down-regulation of COX VIIc during M. tuberculosis infection was confirmed by three independent techniques: limiting-dilution RT-PCR, RNase protection assay, and Northern analysis. Limiting-dilution RT-PCR and Northern analysis were also used to analyze the specificity of this regulation; heat-killed M. tuberculosis , Mycobacterium bovis BCG, and latex beads had no effect on expression of COX VIIc. Down-regulation of this enzyme was also confirmed by using adherent cells isolated from spleens of M. tuberculosis -infected mice. These ex vivo macrophages showed apoptotic features, suggesting a possible involvement of cytochrome c oxidase in the programmed cell death of the host cells.

Despite the introduction of multidrug therapy and the overall reduction of leprosy prevalence in Mexico, the disease remains endemic in certain regions of the country. A genetic basis for the immune susceptibility to Mycobacterium leprae has already been established in different populations worldwide. In this study, we investigated the possible association of the HLA-DRB1 alleles with leprosy in a Mexican Mestizo popula- tion. The results demonstrated that the HLA-DRB1*01 allele is associated with lepromatous and dimorphic leprosy (P

DNA was isolated from mycobacteria by a simplified procedure. Cells were suspended in 6 M guanidinium chloride, the suspension was cooled to -70 degrees C, then incubated at 65 degrees C for 10 min, cooled in ice, deproteinized by chloroform and DNA was recovered from the supernatant. The procedure was used to obtain DNA from several mycobacteria (1 x 10(9) or more cells) including Mycobacterium neoaurum, M. fortuitum, M. phlei and M. smegmatis. Each of the species was shown to have two ribosomal RNA operons per genome, and preliminary evidence was obtained which suggests that one of these operons is homologous with one of the operons of M. smegmatis.

Human Limbal Epithelial Cells (hLEC) are stem cells that give rise to corneal epithelium. After corneal damage, hLEC produce large amounts of IL-8 and IL-6, inducing inflammation in cornea and conjunctiva. Despite inflammation is necessary to repair the ocular surface since this process may be potentially harmful and could lead to corneal opacity. Ophthalmic infectious diseases have been treated with human dialyzable leukocyte extracts (hDLE). Clinical observations in hDLE-treated patients, have suggested an apparent control of ocular inflammatory injuries, without changes in the re-epithelialization process.To determine the inflammatory cytokine profile in supernatants (SN) of hLEC cultured with hDLE.hLEC were obtained from cadaver donors. hDLE were added to the hLEC cultures, and SN were collected at different times (1h, 3h, 6h, and 24h). IL-1?, IL 6, IL-8, IL-12p70 and TNF-? were measured in SN with cytometric bead arrays.The majority of isolated cells were CK19+/vimentin+/p63+, indicating that cultured-cells were limbal epithelial stem cells. Limbal cells responded to hDLE by diminishing the secretion of IL-8 and IL-6. Secretion of IL-8 and IL-6 was down-regulated significantly at 24h of culture with hDLE. Interestingly, hDLE did not induce secretion of IL-1 ?, TNF-?, and IL-12p70 in hLEC at any evaluated times.hDLE down-regulates secretion of IL-8 and IL-6 without induction of IL-1 ?, TNF-a, and IL-12p70 in hLEC. Our results provide a basis to understand some clinical effects, related to control ocular inflammation, that have been observed in patients treated with hDLE.

Prostaglandins (PG) are potent mediators of intercellular communication, and PGE2 at high concentration is immunosuppressive for T-cell-mediated immunity. We studied the kinetics of PGE2 production and the expression of the enzymes related to its synthesis during the course of experimental pulmonary tuberculosis. Secondly, we analysed the pathological and immunological changes produced by the pharmacological suppression of PG production. In BALB/c mice infected via the trachea with Mycobacterium tuberculosis H37Rv there is an initial phase of partial resistance, dominated by type 1 cytokines plus tumour necrosis factor-alpha (TNF-alpha) and expression of the inducible form of nitric oxide synthase (iNOS), followed by a phase of progressive disease. During the early phase of the infection some activated macrophages located in the alveolar-capillary interstitium and in granulomas showed strong PGE2 immunostaining. However, PGE2 concentrations were relatively low and stable. Animals in this early phase of infection were treated with niflumic acid, a potent and specific blocker of cyclo-oxygenase 2, the rate-limiting enzyme of PG production. In comparison with control animals, the suppression of PG synthesis produced higher inflammation and expression of TNF-alpha, interleukin-1alpha and interferon-gamma (IFN-gamma), but almost complete disappearance of iNOS expression, which coexisted with a significant increment of bacterial load. The late progressive phase in this experimental model is characterized by progressive pneumonia, small granulomas and diminished expression of IFN-gamma, TNF-alpha and iNOS in coexistence with high expression of IL-4. Strong PGE2 immunostaining was seen in foamy macrophages localized in the pneumonic areas, and the PGE2 concentration was four-fold higher in this late phase of infection than during the early phase. When PG production was suppressed in animals suffering advanced phase infection, a significant reduction of pneumonia and bacillus load with striking increment of granuloma size was seen, and the expression of IFN-gamma, TNF-alpha and iNOS was also improved. These findings demonstrate a significant participation of PGE2 in the pathogenesis of pulmonary tuberculosis, showing that during the early phase of the infection there are low PGE2 concentrations which contribute to iNOS expression permitting the temporal control of bacillus growth, while the high PGE2 concentrations during the late phase of the disease contribute to down-regulate cell-mediated immunity, permitting disease progression.

Periodontal Disease is bacterial infection of tooth-supporting tissues leading to chronic inflammation and loss of teeth, the immunopathologic mechanisms include inflammatory cells and proinflammatory cytokines; persistent local inflammation develops the expression of stress molecules as Hsp90, Hsp70 and P53 also autoantibodies. The rol of cellular stress and autoimmunity in periodontal disease is not understood. Objetives: Determine the expression of cell stress molecules and auto-antibodies in periodontal disease patients. Methods: 34 patient with severe chronic periodontitis and 14 healthy people were clinical evaluated. Periodontal tissue and sera were obtained. Electroforesis, Western blot and Immunodetection to identify Hsp70, Hsp90 and P53 was set up, Hsp 90 were evaluated by RT-PCR and IFI for ANCA, antiepithelial antibodies in mouse skin and ANA using Hep2 cells. Results: 57% were positives for ANCA, total of sera were positives for nuclear and/or cytoplasmic antibodies and 57% presented anti-epithelial antibodies, Hsp90, Hsp70 and P53 is expressed according with the severity of disease. Conclusions: The expression profile of Hsps in periodontal tissue is according with the severity, and could be associated with cellular stress because of the damage mechanism induced by periodontal pathogens and trigger necrotic or apoptotic phenomena. Destruction of extracellular matrix in periodontal tissue could be mediated by auto-antibodies that recognize self structures.

Major Depressive Disorder (MDD) is a psychiatric illness that commonly displays a deficit of serotonergic neurotransmission in the central nervous system. MDD patients also show alterations in alterations in cortisol and cytokines levels. Those patients are treated with selective serotonin reuptake inhibitors (SSRIs). The aim of this study was to assess the efficacy of the combined treatment with SSRIs plus an immunomodulator -dialyzable leukocyte extract (DLE) - in restoring the balance of both the serotoninergic system and the endocrinoimmune parameters. A total of 34 patients diagnosed with MDD received anti-depressant treatment with SSRIs plus DLE. As control 31 patients received only SSRIs. Proinflammatory cytokines (Th1) IL-1β, IL-2, IFN-γ, anti-inflammatory cytokines (Th2) IL-13, IL-10, and 24-h urine cortisol were determined at weeks (W) 0, 5, 20, 36 and 52 of treatment. The maximum reduction in cortisol levels in SSRIs group was only 30% (W52). By contrast, the treatment with SSRIs plus DLE decreased 53% the cortisol levels from W20. Reduced cortisol in patients treated with SSRI plus DLE correlated with reductions in the concentration of Th2-cytokine and increases in Th1-cytokines at the end of the study. These results suggest that the immune-stimulating action of DLE, in association with the effects of SSRIs, allowed the immune system to balance the ratio of Th1/Th2 cytokines associated and decreased levels of cortisol.

Atopic dermatitis(AD) is a T cell-mediated skin disease associated with a Th2 local response and skin inflammation is associated with CLA expression. In this work we evaluated expression of CLA on CD4 and CD8 T cells in 2 groups of patients one of them received metilprednisolone(mP-Advantan-) as treatment(Group 1) and second group received mP and dialyzable leukocyte extracts(DLE-Transferon-) as immune modulator(Group 2). Methods: Double blind randomized placebo controlled study, approved by hospital ethical committee. PBMC were obtained before, during (14 day) and at the end of treatment (28 day) and CLA expression on CD4 and CD8 was evaluated by flow cytometry. Results: Group 1:Frequency of CD4+CLA+T cells before treatment 1%±0.6 vs 1.9%±0.9 p=0.02 after treatment. CD8+CLA+T cells before treatment 4.5%±0.9 vs 8.1%±1.9 p=0.0002 after treatment. Group 2: CD8+CLA+T cells before treatment 5.3%±2.1 vs 8.8%±1.8 p=0.005 after treatment. Conclusion: We treated 58 AD-pediatric patients with mP or with mP+DLE, in both groups clinical outcome was improved after therapy; mP-treated patients induced CLA on CD4+ and CD8+ Tcells, while DLE-treated patients induced only CD8+CLA+T cells. Despite CLA expression is associated with local skin inflammation, some authors have suggested that CLA expression is also induced in Tregs; it is possible that DLE- induced CD8+CLA+ T could be enriched Tregs since it has been reported a diminished frequency of AD activation after DLE immune modulation.

Allergic conjunctivitis (AC) is a frequent disease that affects the ocular surface, The study of immune physiopathology of allergic conjunctivitis is focused on T helper cells. Recently it has been suggested that γδ T lymphocytes are necessary for the maintenance of the ocular allergic response, however there are not studies in humans about the role of γδ T lymphocytes in human AC. The aim of this study was to evaluate the frequency of γδ T cells and chemokines in tears of AC patients. Patients: 31 patients were included (16 men and 15 women), AC diagnosis was based on clinical history, physical and eye examination. PBMC were obtained from patients and healthy controls and frequency of γδ T cells was evaluated by flow cytometry; tear samples were also collected to determine chemokine concentrations by CBA. Results: γδ T lymphocytes were 11.9 times increased in AC-patients (22.63 ± 2.9%) compared to healthy donors (1.9 ± 1.9%) (p=0.002), chemokine determination in tears showed increased MIG/CXCL9 in AC patients (1604 ± 1404 pg/ml) when compared with healthy subjects (568 ±841pg/ml) p< 0.02. Conclusion: γδ T cells could be also involved in human allergic conjunctivitis as is has been demonstrated in animal models, it is possible that γδ T cells could migrate to conjunctiva due to several chemokines such as CXCL9, significant increased in AC patients, sustaining the local inflammation in conjunctiva

Aim: To describe clinical and immunological characteristics of human microbial keratitis. Methods. 28 patients with bacterial and fungal keratitis were included. Characteristics of ocular lesions such as size of corneal ulcer (epithelial defect), infiltration, hypopyon, were considered; immunological evaluation included determination of IL-1b, IL-6, IL-8, IL-10, IL-12 and TNF-a by cytometric bead arrays in samples obtained from infected and non-infected eyes; frequency of CD4, CD8, CD19 and CD56 lymphocytes were also determined on peripheral blood mononuclear cells (PMBC). Results. Non-significant differences were observed in area of epithelial defect, stromal infiltration, nor hypopyon, when compared; however, we observed a characteristic immunological profile apparently related to keratitis origin: IL-8 >IL-6 in bacterial keratitis; IL-8>IL-6 with increased frequency of circulating CD3-CD56+ cells in gram-negative keratitis; and IL-8=IL-6 >IL-1b in fungal keratitis. Conclusions. This study is the first in evaluate clinical and immunological characteristics of patients with corneal ulcers, despite we did not observe a clinical profile clearly related with the type of keratitis, characterization of immunological status could aid to understand immune pathophysiological mechanisms underlying each type of keratitis. IL-6 and IL-8 could be a target for therapy in severe microbial keratitis

Background X-linked agammaglobulinemia (XLA) is characterized by impaired B-cell differentiation caused by mutations in Bruton's tyrosine kinase (Btk) gen. Btk is expressed in myeloid cells and recent evidence support that it participates in Toll like receptor signaling, but results regarding its rol in XLA patients are contradictories. Objective To evaluate lipopolysaccharide (LPS)-induced pro-inflammatory cytokine response in peripheral blood mononuclear cells (PBMC) from XLA patients. Methods Thirteen patients with XLA were included in the study. PBMC LPS-induced TNF-α, IL-1β, IL-6, and IL-10 production was determined by ELISA and compared with that obtained from matched healthy controls. Cytokine production was correlated with the severity of the mutation, affected domain and clinical characteristics. Results In response to LPS, PBMC from XLA patients produced significantly higher amounts of pro-inflammatory cytokines and IL-10 compared with controls and this production is not influenced by the neither severity of mutation or the affected domain. PBMC from patients with a history of more hospital admissions before diagnosis and patients with lower expression of Btk in monocytes produced higher levels of TNF-α and IL-1β, respectively. PBMC from patients with lower IgA levels showed a higher production of TNF-α and IL-1β. Less severe (punctual) mutations in Btk gene were associated with higher IgG levels at diagnosis. Conclusions Our results demonstrated a predominantly inflammatory response in XLA patients after LPS stimuli and suggest a TLR signaling dysregulation in absence of Btk. This response may be influenced by environmental factors.

Mycobacterium leprae , a non-cultivable bacteria, is the causative agent of leprosy. Despite huge effort from WHO leprosy remains a public health problems in some parts of the world. One of the principal goals of leprosy research is to develop serological tests that will allow identification and early treatment of leprosy patients. In this work, M. habana a cultivable non-pathogenic mycobacterium and candidate vaccine for leprosy, was used to obtain soluble antigenic extracts (MHSE). By Western blot (WB) analysis MHSE proteins were reacted with sera form lepromatous leprosy patients (LL), active tuberculosis patients (TB) and healthy donors (H) from an endemic leprosy area. LL sera diluted 1:2,000 showed reactivity with a 28-30 KDa doublet antigen in MHSE. No reactivity with these bands was observed at 1:2,000 or lower sera dilutions of TB or H donors. After performing two dimension PAGE and WB analysis with the same sera, the doublet band was shown to have several spots. Further characterization was done using Tandem Mass Spectrometry (LC/ESI-MS/MS) and two proteins were identified: enoyl- coenzyme A hydratase (lipid metabolism) and antigen 85B (Ag85B, mycolyltransferase). These proteins represent promising candidates for the design of a reliable tool for the serodiagnosis of the infectious form of leprosy.

Mendelian susceptibility to mycobacterial disease (MSMD) is a heterogenous syndrome. The clinical characteristics include repetitive local infection and in some cases dissemination. In this work we analyzed a 5 years old boy with an uncharacterized mycobacterial infection. His parents are first degree cousins. Blood samples were collected and cultured 48 hours with medium, BCG or BCG+IL-12. Supernatants were assayed for IFN-γ by ELISA. PBMC were cultured with PMA-Ionomicyn and IL12R was measured by flow cytometry. We found low production of IFN γ after stimulation with BCG+IL12 that seems to correlate with an IL-12R defect. Flow cytometry showed a decrease of IL-12RB1 expression. Sequencing revealed a point mutation (A182T), which generates E61G change in the extracellular domain. Furthermore, the uncommon clinical features in this case might suggest another primary immunodeficiency.

Murine dialyzable spleen extracts (DSE) are similar to Transfer Factors in that they can transfer cell mediated immunity (CMI) from an immunocompetent organism into a naïve one in an antigen-specific manner. Lymphoid organs from rats treated with transfer factor have shown histological changes and increased lymphoblasts and plasmocytes. In this work we investigated if treating mice with DSE obtained from OVA immunized mice (DSEova) could modify the humoral immune response against OVA in naïve mice. Methods. DSEova preparation: splenic cell suspensions from OVA immunized BALB/c mice were disrupted by freeze-thaw cycles, extracts were then dialyzed with a membrane pore of 10 kDa. BALB/c mice were treated with 20μg of DSEova alone. Serum samples were obtained between days 0-36, and IgM and IgG anti-OVA were assayed by ELISA. Mice were sacrified on days 9, 15 and 23 post-immunization, and germinal centers (GC) in lymph nodes were identified by immunohistochemistry using PNA and THY-1. Results. Compared to control saline-treated animals, DSEova treated mice produced IgM and IgG anti-OVA. Compared to saline-treated mice, DSEova-mice showed increased numbers of GC, but of smaller size than OVA-treated mice. Conclusion. In this in-vivo model, compared to control-mice, DSEova would appear to stimulate the Ag-specific antibody responses and the appearance of GC.

We have previously shown that M. tuberculosis induce neutrophil extracellular traps (NETs) formation. Since NETs could capture M. tuberculosis bacilli but were unable to kill them, we hypothesized that these structures might be involved in maintaining the infectious focus localized and at the same time allowing macrophages to arrive at the infection site where they probably would phagocyte mycobacteria trapped in NETs. In this study we tested if phagocytosis of M. tuberculosis trapped in NETs versus M. tuberculosis alone had differential effect on macrophage proinflammatory cytokine gene expression. Methods: human monocyte-derived macrophages were left unstimulated or infected either with NETs, M. tuberculosis H37Rv trapped in NETs or M. tuberculosis H37Rv alone for 24h and proinflammatory cytokine gene expression was analyzed using SABioscience nylon membrane macroarrays. Results: M. tuberculosis trapped in NETs induced a lower gene expression profile of proinflamatory cytokines in comparison with that induced by M. tuberculosis alone in 2 of 3 subjects analyzed. In the other subject, NETs acted as an adjuvant, increasing the proinflammatory response against M. tuberculosis.

The LPS is a structural component of the external membrane of the Gram negative bacteria and is the most powerful of the inflammation inductors. High doses of LPS have as consequence a high production of inflammatory cytokines, which causes the endotoxic shock. The Dializable Leukocyte Extracts (DLE) is the dialyzable fraction with lower molecular weight to12 KDa which come from leukocytes lysates. In this work a model of endotoxic shock LPS was induced established in C57BL6 male 6 weeks of age, the dose for endotoxic shock induction was 130mg/kg mouse weight. The doses of 0.001ug and 0.0001ug mDLE showed protection against endotoxic shock death in 10% of the mice and the 0.01ug mDLE dose protected against of endotoxic shock death from 10 to 30% of the mice. Levels of TNF-alpha, IL-6, IL-12, MCP-1, IFN-gamma, IL-10 and TGF-beta were measured in serum of mice endotoxic shock induced and treated with mDLE, the groups in which LPS was administrated and then treated with 0.01ug, 0.001ug or 0.0001ug of mDLE showed smaller levels of inflammatory cytokines and more higher levels of antiinflammatory cytokines than the LPS treated control group. The splenic index was measured in the endotoxic shock induced and mDLE treated groups, the group LPS and the 0.001ug of mDLE treated showed the higher splenic index in comparison with the LPS control group. So the DLE can be use as a potential therapeutic agent as adjuvant in the treatment of sepsis.

Dialyzable leukocyte extracts (DLE) are obtained from freezed-thawed buffy coats and subsequent dialysis (