Differential activation of innate and adaptive lymphocytes during latent or active infection with Mycobacterium tuberculosis.

Most individuals infected with Mycobacterium tuberculosis (Mtb) have latent tuberculosis (TB), which can be diagnosed with tests (such as the QuantiFERON-TB Gold test [QFT]) that detect the production of IFN-γ by memory T cells in response to the Mtb-specific antigens 6 kDa early secretory antigenic target EsxA (Rv3875) (ESAT-6), 10 kDa culture filtrate antigen EsxB (Rv3874) (CFP-10), and Mtb antigen of 7.7 kDa (Rv2654c) (TB7.7). However, the immunological mechanisms that determine if an individual will develop latent or active TB remain incompletely understood. Here we compared the response of innate and adaptive peripheral blood lymphocytes from healthy individuals without Mtb infection (QFT negative) and from individuals with latent (QFT positive) or active TB infection, to determine the characteristics of these cells that correlate with each condition. In active TB patients, the levels of IFN-γ that were produced in response to Mtb-specific antigens had high positive correlations with IL-1ß, TNF-α, MCP-1, IL-6, IL-12p70, and IL-23, while the proinflammatory cytokines had high positive correlations between themselves and with IL-12p70 and IL-23. These correlations were not observed in QFT-negative or QFT-positive healthy volunteers. Activation with Mtb-soluble extract (a mixture of Mtb antigens and pathogen-associated molecular patterns [PAMPs]) increased the percentage of IFN-γ-/IL-17-producing NK cells and of IL-17-producing innate lymphoid cell 3 (ILC3) in the peripheral blood of active TB patients, but not of QFT-negative or QFT-positive healthy volunteers. Thus, active TB patients have both adaptive and innate lymphocyte subsets that produce characteristic cytokine profiles in response to Mtb-specific antigens or PAMPs. These profiles are not observed in uninfected individuals or in individuals with latent TB, suggesting that they are a response to active TB infection.

Isolation of Mycobacterium lepromatosis and Development of Molecular Diagnostic Assays to Distinguish Mycobacterium leprae and M. lepromatosis.

BACKGROUND: Mycobacterium leprae was thought to be the exclusive causative agent of leprosy until Mycobacterium lepromatosis was identified in a rare form of leprosy known as diffuse lepromatous leprosy (DLL). METHODS: We isolated M. lepromatosis from a patient with DLL and propagated it in athymic nude mouse footpads. Genomic analysis of this strain (NHDP-385) identified a unique repetitive element, RLPM, on which a specific real-time quantitative polymerase chain reaction assay was developed. The RLPM assay, and a previously developed RLEP quantitative polymerase chain reaction assay for M. leprae, were validated as clinical diagnostic assays according to Clinical Laboratory Improvement Amendments guidelines. We tested DNA from archived histological sections, patient specimens from the United States, Philippines, and Mexico, and US wild armadillos. RESULTS: The limit of detection for the RLEP and RLPM assays is 30 M. leprae per specimen (0.76 bacilli per reaction; coefficient of variation, 0.65%-2.44%) and 122 M. lepromatosis per specimen (3.05 bacilli per reaction; 0.84%-2.9%), respectively. In histological sections (n = 10), 1 lepromatous leprosy (LL), 1 DLL, and 3 Lucio reactions contained M. lepromatosis; 2 LL and 2 Lucio reactions contained M. leprae; and 1 LL reaction contained both species. M. lepromatosis was detected in 3 of 218 US biopsy specimens (1.38%). All Philippines specimens (n = 180) were M. lepromatosis negative and M. leprae positive. Conversely, 15 of 47 Mexican specimens (31.91%) were positive for M. lepromatosis, 19 of 47 (40.43%) were positive for M. leprae, and 2 of 47 (4.26%) contained both organisms. All armadillos were M. lepromatosis negative. CONCLUSIONS: The RLPM and RLEP assays will aid healthcare providers in the clinical diagnosis and surveillance of leprosy.

Innate Lymphoid Cells Have Decreased HLA-DR Expression but Retain Their Responsiveness to TLR Ligands during Sepsis.

Sepsis, one of the leading causes of death in intensive care units, is caused by a dysregulated host response to infection that leads to life-threatening organ dysfunction. The proinflammatory and anti-inflammatory responses activated by the infecting microorganism become systemic, and the sustained anti-inflammatory response induces a state of immunosuppression that is characterized by decreased expression of HLA-DR on monocytes, T cell apoptosis, and reduced production of TNF-α by monocytes and macrophages in response to TLR ligands. Innate lymphoid cells (ILCs) are lymphocytes that lack Ag-specific receptors and lineage-specific markers; they express HLA-DR and are activated by cytokines and by direct recognition of microbial molecules. In this study, we evaluated if ILCs are affected by the anti-inflammatory response during sepsis. We found that the number of peripheral blood ILCs was decreased in septic patients compared with healthy volunteers; this decrease was caused by a reduction in ILC1 and ILC3 and is associated with apoptosis, because ILCs from septic patients expressed active caspase 3. ILCs from septic patients had decreased HLA-DR expression but increased expression of the activating receptors NKp46 and NKp44; they also showed a sustained expression of CD127 (IL-7R α-chain) and retained their capacity to produce TNF-α in response to TLR ligands. These results indicate that during sepsis, ILCs have decreased HLA-DR expression and die via apoptosis, similar to monocytes and T cells, respectively. However, other effector functions of ILCs (activation through NKp46 and NKp44, TNF-α production) may remain unaffected by the immunosuppressive environment prevailing in septic patients.

Prediction of Structure and Molecular Interaction with DNA of BvrR, a Virulence-Associated Regulatory Protein of Brucella.

Brucellosis, also known as "undulant fever" is a zoonotic disease caused by Brucella, which is a facultative intracellular bacterium. Despite efforts to eradicate this disease, infection in uncontrolled domestic animals persists in several countries and therefore transmission to humans is common. Brucella evasion of the innate immune system depends on its ability to evade the mechanisms of intracellular death in phagocytic cells. The BvrR-BvrS two-component system allows the bacterium to detect adverse conditions in the environment. The BvrS protein has been associated with genes of virulence factors, metabolism, and membrane transport. In this study, we predicted the DNA sequence recognized by BvrR with Gibbs Recursive Sampling and identified the three-dimensional structure of BvrR using I-TASSER suite, and the interaction mechanism between BvrR and DNA with Protein-DNA docking and molecular dynamics (MD) simulation. Based on the Gibbs recursive Sampling analysis, we found the motif AAHTGC (H represents A, C, and T nucleotides) as a possible sequence recognized by BvrR. The docking and EMD simulation results showed that C-terminal effector domain of BvrR protein is likely to interact with AAHTGC sequence. In conclusion, we predicted the structure, recognition motif, and interaction of BvrR with DNA.

Ursolic and Oleanolic Acids Induce Mitophagy in A549 Human Lung Cancer Cells.

Ursolic and oleanolic acids are natural isomeric triterpenes known for their anticancer activity. Here, we investigated the effect of triterpenes on the viability of A549 human lung cancer cells and the role of autophagy in their activity. The induction of autophagy, the mitochondrial changes and signaling pathway stimulated by triterpenes were systematically explored by confocal microscopy and western blotting. Ursolic and oleanolic acids induce autophagy in A549 cells. Ursolic acid activates AKT/mTOR pathways and oleanolic acid triggers a pathway independent on AKT. Both acids promote many mitochondrial changes, suggesting that mitochondria are targets of autophagy in a process known as mitophagy. The PINK1/Parkin axis is a pathway usually associated with mitophagy, however, the mitophagy induced by ursolic or oleanolic acid is just dependent on PINK1. Moreover, both acids induce an ROS production. The blockage of autophagy with wortmannin is responsible for a decrease of mitochondrial membrane potential (Δψ) and cell death. The wortmannin treatment causes an over-increase of p62 and Nrf2 proteins promote a detoxifying effect to rescue cells from the death conducted by ROS. In conclusion, the mitophagy and p62 protein play an important function as a survival mechanism in A549 cells and could be target to therapeutic control.

MIF promotes a differential Th1/Th2/Th17 inflammatory response in human primary cell cultures: Predominance of Th17 cytokine profile in PBMC from healthy subjects and increase of IL-6 and TNF-α in PBMC from active SLE patients.

Macrophage migration Inhibitory Factor (MIF) is a cytokine associated with the pathogenesis of autoimmune inflammatory diseases. There is evidence that MIF functions in a positive-feedback-loop with proinflammatory cytokines and could perpetuate the inflammatory process in Systemic Lupus Erythematosus (SLE).The aim of this study was to assess the effect of recombinant-human-MIF (rhMIF) on the expression of Th1, Th2 and Th17 cytokines in Peripheral Blood Mononuclear Cells (PBMC) from Healthy Subjects (HS) and SLE patients. The PBMC were isolated from SLE patients classified according to the 1997 SLE ACR criteria and HS donors; all subjects included were women from an unrelated Mexican-Mestizo population. The PBMC isolated were stimulated with rhMIF, LPS and ISO-1 in different combinations; Th1, Th2 and Th17cytokine profiles levels were determined by MAGPIX Bio-plex assay in supernatants from cell cultures. We observed in supernatants of PBMCs from HS treated with rhMIF a predominance of Th17 cytokine profile with an increase of IL-17A, IL-17F and IL-21 versus PBMCs from SLE patients, which showed an inflammatory profile represented by increase of IL-6 cytokine. According to SLE remission/activity presented at enrollment in the study (Mex-SLEDAI index), the PBMC from active SLE patients showed higher levels of TNF-α and IL-6 versus PBMC from remission SLE patients. In conclusion, our results suggest that MIF can induce a differential inflammatory response in physiological and pathological conditions with a predominance of a Th17 cytokine profile in PBMC from HS and an increase in TNF-α and IL-6 expression in PBMC from active SLE patients.

HLA Alleles are Genetic Markers for Susceptibility and Resistance towards Leprosy in a Mexican Mestizo Population.

Despite the use of multidrug therapy, leprosy remains endemic in some countries. The association of several human leucocyte antigen (HLA) alleles and gene polymorphisms with leprosy has been demonstrated in many populations, but the major immune contributors associated to the spectrum of leprosy have not been defined yet. In this study, genotyping of HLA-A, -B, -DR, and -DQ alleles was performed in leprosy patients (n = 113) and control subjects (n = 117) from the region with the highest incidence for the disease in México. The odds of developing leprosy and lepromatous subtype were 2.12- and 2.74-fold higher in carriers of HLA-A*28, and 2.48- and 4.14-fold higher for leprosy and dimorphic subtype in carriers of DQB1*06. Interestingly, DQB1*07 was overrepresented in healthy individuals, compared to patients with leprosy (OR = 0.08) and the lepromatous subtype (OR = 0.06). These results suggest that HLA-A*28 is a marker for predisposition to leprosy and the lepromatous subtype and DQB1*06 to leprosy and the dimorphic subtype, while DQB1*07 might be a resistance marker in this Mestizo population.

Comparative proteomic profiles reveal characteristic Mycobacterium tuberculosis proteins induced by cholesterol during dormancy conditions.

Cholesterol has been reported to play an important role during Mycobacterium tuberculosis infection and during its dormant state inside the host. We present the determination of proteomic profiles of M. tuberculosis H37Rv in the presence of cholesterol as the sole carbon source under exponential growth and in two in vitro dormancy phases (NRP1 and NRP2). Using 2D-PAGE, we detected that M. tuberculosis expressed a high diversity of proteins in both exponential and non-replicative phases. We also found that cholesterol was involved in the overexpression of some proteins related to sulfur metabolism (CysA2), electron transport (FixB), cell wall synthesis (Ald), iron storage (BfrB), protein synthesis (Tig and EF-Tu) and dormancy maintenance (HspX and TB 31.7). According to our results we propose that proteins Ald, BfrB, FadA5 and TB31.7 are likely to play a fundamental role during in vitro dormancy of M. tuberculosis in the presence of cholesterol, helping to counteract its intracellular hostile microenvironment.

Differential activation of dendritic cells by Mycobacterium tuberculosis Beijing genotype.

Mycobacterium tuberculosis (Mtb) inhibits dendritric cells (DC) function in order to delay T cell response. Furthermore, there is increasing evidence that genetic diversity of Mtb strains can affect their interaction with the immune system. Beijing genotype has attracted attention because of its high prevalence and multi-drug resistance. Although it is known that this genotype is hypervirulent and differentially activates macrophages when compared to other genotypes, little is known about its interaction with DC. In order to address this issue, murine bone marrow derived DC (BMDC) were stimulated with soluble extracts (SE) from BCG, H37Rv, Canetti and Beijing genotypes. We observed that unlike other mycobacteria strains, SE-Beijing was unable to induce maturation of DC as assessed by cell surface MHC-II expression. DC stimulated with SE-Beijing failed to produce IL-12 and TNF-α, but did secrete IL-10. Interestingly, SE-Beijing induced CCR7 and PDL-1 on BMDC, but did not induce the expression of CD86. When BMDC stimulated with SE-Beijing were used to activate CD4+ cells they were unable to induce a Th1 response when compared with less virulent genotypes. These results indicate that Beijing is able to modulate DC activation and function, which may be related to the pathogenesis induced by this genotype.

O-glycosylation of NnTreg lymphocytes recognized by the Amaranthus leucocarpus lectin.

O-glycosidically-linked glycans have been involved in development, maturation, homing, and immune regulation in T cells. Previous reports indicate that Amaranthus leucocarpus lectin (ALL), specific for glycans containing galactose-N-acetylgalactosamine and N-acetylgalactosamine, recognizes human naïve CD27(+)CD25(+)CD4(+) T cells. Our aim was to evaluate the phenotype of CD4(+) T cells recognized by ALL in peripheral blood mononuclear cells obtained from healthy volunteers. CD4(+) T cells were isolated by negative selection using magnetic beads-labeled monoclonal antibodies; the expression of T regulatory cell phenotypic markers was assessed on ALL-recognized cells. In addition, IL-4, IL-10, IFN-γ, and TGF-ß intracellular production in ALL (+) cells was also evaluated. The analyses of phenotypic markers and intracellular cytokines were performed through flow cytometry. ALL-recognized CD4(+) T cells were mainly CD45RA(+), CCR7(+) cells. Although 52 ± 10% CD25(+)Foxp3(+) cells were positive to ALL, only 34 ± 4% of ALL (+) cells corresponded to CD25(+)Foxp3(-) cells. Intracellular cytokines in freshly obtained ALL (+)CD4(+) T cells exhibited 8% of IL-4, 15% of IL-10, 2% of IFN-γ, and 15% of TGF-ß, whereas ALL (-)CD4(+) T cells depicted 1% of IL-4, 2% of IL-10, <1% of IFN-γ, and 6% of TGF-ß. Our results show that galactose-N-acetylgalactosamine and N-galactosamine-bearing CD4(+) T cells expressed phenotypic markers of NnTreg cells.

Effect of selective serotonin reuptake inhibitors and immunomodulator on cytokines levels: an alternative therapy for patients with major depressive disorder.

Major depressive disorder (MDD) is a psychiatric illness that presents as a deficit of serotonergic neurotransmission in the central nervous system. MDD patients also experience alterations in cortisol and cytokines levels. Treatment with selective serotonin reuptake inhibitors (SSRIs) is the first-line antidepressant regimen for MDD. The aim of this study was to determine the effect of a combination of SSRIs and an immunomodulator-human dialyzable leukocyte extract (hDLE)-on cortisol and cytokines levels. Patients received SSRIs or SSRIs plus hDLE. The proinflammatory cytokines IL-1 ß , IL-2, and IFN- γ ; anti-inflammatory cytokines IL-13 and IL-10; and 24-h urine cortisol were measured at weeks (W) 0, 5, 20, 36, and 52 of treatment. The reduction in cortisol levels in the SSRI-treated group was 30% until W52, in contrast, the combined treatment induced a 54% decrease at W36. The decline in cortisol in patients who were treated with SSRI plus hDLE correlated with reduction of anti-inflammatory cytokines and increases levels of proinflammatory cytokines at the study conclusion. These results suggest that the immune-stimulating activity of hDLE, in combination with SSRIs, restored the pro- and anti-inflammatory cytokine balance and cortisol levels in depressed patients versus those who were given SSRIs alone.

Immunological evaluation of captive green sea turtle (Chelonia mydas) with ulcerative dermatitis.

Ulcerative dermatitis (UD) is common in captive sea turtles and manifests as skin erosions and ulcers associated with gram-negative bacteria. This study compared clinically healthy and UD-affected captive turtles by evaluating hematology, histopathology, immunoglobulin levels, and delayed-type hypersensitivity assay. Turtles with UD had significantly lower weight, reduced delayed-type hypersensitivity (DTH) responses, and higher heterophil:lymphocyte ratios. This study is the first to assay DTH in green turtles (Chelonia mydas) and suggests that UD is associated with immunosuppression.

[Efectividad de la suspensión bacteriana sublingual en la prevención de infecciones de vías aéreas de repetición en pediatría].

Background: Upper respiratory tract infections (URIs) are very common in the pediatric population. Most of these infections are mild, but due to their chronicity they affect quality of life (QoL), in addition to high costs for medical care. The use of bacterial extracts (BE) that stimulate general immunity can reduce its frequency and improve the QoL of the patient. Objective: Evaluate the effectiveness of a BE in the prevention of ARVI in children from 1 to 6 years of age. Methods: Children between the ages of 1 and 6 years, with a diagnosis of RAVI, were randomized into 3 different groups, with medical follow-up at 6 and 12 weeks after the start. The EB was administered with different doses to each group. An ANOVA test with a Tukey post hoc is used for multiple comparisons (maximum type I error of 0.05). Results: 33 children (12 girls) with a mean age of 3.11 years were included. The average frequency of RAVI prior to treatment was 2.2 events/month and 0.9 and 0.4 events/month at 6 and 12 weeks, respectively. The IVARS were reduced by 76.9% at 3 months of treatment. (Graph). No adverse effects were reported. Conclusions: BE is safe and effective in reducing the frequency of RAVI in children, in agreement with the literature. There is not enough published scientific evidence, but the BE seems to have an application in the prevention and treatment of RAVI. Sublingual administration is comfortable in this age group.

Antecedentes: Las infecciones de vías aéreas superiores (IVASR) son muy frecuentes en la población pediátrica. La mayoría de estas infecciones son leves, pero por la cronicidad afectan la calidad de vida (CdV), además de elevados costos por la atención médica. El uso de extractos bacterianos (EB) que estimulen la inmunidad general pueden reducir su frecuencia y mejorar la CdV del paciente. Objetivo: Evaluar la efectividad de un EB en la prevención de IVASR en niños de 1 a 6 años. Métodos: Se aleatorizaron niños entre 1 y 6 años, con diagnóstico IVASR en 3 grupos distintos, seguimiento médico a las 6 y 12 semanas tras el inicio. El EB se administró con dosis distintas a cada grupo. Se utiliza una prueba de ANOVA con un post hoc Tukey para comparaciones múltiples (error tipo I máximo de 0.05). Resultados: Se incluyeron 33 niños (12 niñas) con una media de edad de 3.11 años. La frecuencia de IVASR previo al tratamiento en promedio fue de 2.2 eventos/mes y de 0.9 y de 0.4 eventos/mes a las 6 y 12 semanas respectivamente. La IVARS se redujeron un 76.9% a los 3 meses de tratamiento. (Gráfica). No se reportaron efectos adversos. Conclusiones: El EB es seguro y efectivo en disminuir la frecuencia de IVASR en niños en concordancia con la literatura. No hay suficiente evidencia científica publicada pero el EB parece tener aplicación en la prevención y tratamiento de las IVASR. La administración sublingual es cómoda en este grupo etario.

Extracellular vesicles from Mycobacterium tuberculosis-infected neutrophils induce maturation of monocyte-derived dendritic cells and activation of antigen-specific Th1 cells.

Tuberculosis remains one of the leading public health problems in the world. The mechanisms that lead to the activation of the immune response against Mycobacterium tuberculosis have been extensively studied, with a focus on the role of cytokines as the main signals for immune cell communication. However, less is known about the role of other signals, such as extracellular vesicles, in the communication between immune cells, particularly during the activation of the adaptive immune response. In this study, we determined that extracellular vesicles released by human neutrophils infected with M. tuberculosis contained several host proteins that are ectosome markers. In addition, we demonstrated that extracellular vesicles released by human neutrophils infected with M. tuberculosis released after only 30 min of infection carried mycobacterial antigens and pathogen-associated molecular patterns, and we identified 15 mycobacterial proteins that were consistently found in high concentrations in extracellular vesicles released by human neutrophils infected with M. tuberculosis; these proteins contain epitopes for CD4 T-cell activation. We found that extracellular vesicles released by human neutrophils infected with M. tuberculosis increased the expression of the costimulatory molecule CD80 and of the coinhibitory molecule PD-L1 on immature monocyte-derived dendritic cells. We also found that immature and mature dendritic cells treated with extracellular vesicles released by human neutrophils infected with M. tuberculosis were able to induce IFN-γ production by autologous M. tuberculosis antigen-specific CD4 T cells, indicating that these extracellular vesicles acted as antigen carriers and transferred mycobacterial proteins to the antigen-presenting cells. Our results provide evidence that extracellular vesicles released by human neutrophils infected with M. tuberculosis participate in the activation of the adaptive immune response against M. tuberculosis.

Increased pro-inflammatory cytokine production after lipopolysaccharide stimulation in patients with X-linked agammaglobulinemia.

PURPOSE: To evaluate the lipopolysaccharide (LPS)-induced pro-inflammatory cytokine response by peripheral blood mononuclear cells (PBMCs) from XLA patients. METHODS: Thirteen patients with XLA were included in the study. LPS-induced TNF-α, IL-1ß, IL-6, and IL-10 production was determined in PBMCs from patients and matched healthy controls by ELISA. Cytokine production was correlated with the severity of mutation, affected domain and clinical characteristics. RESULTS: In response to LPS, PBMCs from XLA patients produced significantly higher amounts of pro-inflammatory cytokines and IL-10 compared to controls, and this production was influenced neither by the severity of the mutation nor the affected domain. PBMCs from patients with a history of more hospital admissions before their diagnosis produced higher levels of TNF-α. PBMCs from patients with lower serum IgA levels showed a higher production of TNF-α and IL-1ß. Less severe (punctual) mutations in the Btk gene were associated with higher serum IgG levels at diagnosis. CONCLUSIONS: Our results demonstrate a predominantly inflammatory response in XLA patients after LPS stimulation and suggest a deregulation of TLR signaling in the absence of Btk. This response may be influenced by environmental factors.

Mycobacterium tuberculosis Beijing genotype induces differential cytokine production by peripheral blood mononuclear cells of healthy BCG vaccinated individuals.

Members of the Mycobacterium tuberculosis (Mtb) Beijing genotype are a major concern due to their high prevalence in tuberculosis patients and their high rate of multi-drug resistance. Although it has been shown that Beijing modifies macrophage behavior, little is known about how this genotype could affect the cellular immune response. In order to address this issue, peripheral blood mononuclear cells (PBMC) from healthy BCG vaccinated individuals were stimulated with protein extracts from three Mycobacterium tuberculosis genotypes: Canetti, H37Rv and Beijing evaluating T cell proliferation and cytokine production. In this system both CD4+ and CD8+ proliferated in a similar manner independently of the Mtb genotype used for stimulation. Regarding cytokines, all strains induced similar levels of IFN-γ, but were unable to induce IL-4 and TGF-ß. Contrasting, Canetti strain induced lower production of IL-10, TNF-α and IL-12 compared to H37Rv and Beijing. Interestingly, PBMC stimulated with the Beijing strain produced the highest levels of IL-12 and IL-10 than those stimulated with other strains. This differential cytokine expression could affect the pathogenesis induced by Beijing strain through the modulation of inflammatory process in the host, but the precise mechanisms by which this cytokine environment affects the Beijing strain pathogenesis needs further characterization.

Lepromatous leprosy patients produce antibodies that recognise non-bilayer lipid arrangements containing mycolic acids.

Non-bilayer phospholipid arrangements are three-dimensional structures that form when anionic phospholipids with an intermediate structure of the tubular hexagonal phase II are present in a bilayer of lipids. Antibodies that recognise these arrangements have been described in patients with antiphospholipid syndrome and/or systemic lupus erythematosus and in those with preeclampsia; these antibodies have also been documented in an experimental murine model of lupus, in which they are associated with immunopathology. Here, we demonstrate the presence of antibodies against non-bilayer phospholipid arrangements containing mycolic acids in the sera of lepromatous leprosy (LL) patients, but not those of healthy volunteers. The presence of antibodies that recognise these non-bilayer lipid arrangements may contribute to the hypergammaglobulinaemia observed in LL patients. We also found IgM and IgG anti-cardiolipin antibodies in 77% of the patients. This positive correlation between the anti-mycolic-non-bilayer arrangements and anti-cardiolipin antibodies suggests that both types of antibodies are produced by a common mechanism, as was demonstrated in the experimental murine model of lupus, in which there was a correlation between the anti-non-bilayer phospholipid arrangements and anti-cardiolipin antibodies. Antibodies to non-bilayer lipid arrangements may represent a previously unrecognised pathogenic mechanism in LL and the detection of these antibodies may be a tool for the early diagnosis of LL patients.

Expression of Mycobacterium leprae HSP65 in tobacco and its effectiveness as an oral treatment in adjuvant-induced arthritis.

Transgenic plants are able to express molecules with antigenic properties. In recent years, this has led the pharmaceutical industry to use plants as alternative systems for the production of recombinant proteins. Plant-produced recombinant proteins can have important applications in therapeutics, such as in the treatment of rheumatoid arthritis (RA). In this study, the mycobacterial HSP65 protein expressed in tobacco plants was found to be effective as a treatment for adjuvant-induced arthritis (AIA). We cloned the hsp65 gene from Mycobacterium leprae into plasmid pCAMBIA 2301 under the control of the double 35S promoter from cauliflower mosaic virus. Agrobacterium tumefaciens bearing the pChsp65 plasmid was used to transform tobacco plants. Incorporation of the hsp65 gene was confirmed by PCR, reverse transcription-PCR, histochemistry, and western blot analyses in several transgenic lines of tobacco plants. Oral treatment of AIA rats with the HSP65 protein allowed them to recover body weight and joint inflammation was reduced. Our results suggest a synergistic effect between the HSP65 expressed protein and metabolites presents in tobacco plants.

Granulocyte colony-stimulating factor produces a decrease in IFNgamma and increase in IL-4 when administrated to healthy donors.

Hematopoietic stem cells transplantation (HSCT) is the leading curative therapy for a variety of hematological and hereditary diseases; however, graft versus host disease (GVHD), an immunologic phenomenon that is favored by Th1 cytokines and cytotoxic cells from donors, is present frequently and is one of the most important causes of transplant related mortality. Peripheral blood HSCT is the preferred source of stem cells in almost 100% of the cases of autologous HSCT and in 70% of allogeneic transplants. The best mobilizing agent to get the stem cells out from the bone marrow is the Granulocyte-Colony Stimulating Factor (G-CSF). In this work, our main objective was to study a possible correlation between the graft cell dose and the patient's clinical outcome. We evaluated the immunologic changes produced by G-CSF in the lymphocyte and cytokine profiles in allogeneic HSC donors. HSC from twelve donors were mobilized with G-CSF at 16 microg/kg/day, for 5 days. Basal Peripheral Blood (BPB), Mobilized Peripheral Blood (MPB), and aphaeresis mononuclear cells (G-MNC) samples were taken from all donors. Using flow cytometry, we quantified CD19(+), CD3(+), CD3(+)CD4(+), CD3(+)CD8(+), NK, NKT, DC1, and DC2 cells. Cytokines were determined by ELISA in culture supernatants. CD19(+) (p = 0.001), DC1 (p < 0.002) and DC2 (p < 0.001) cells were increased in MPB with respect to BPB. An increase in Th2 cytokines such as (IL-4) and a decrease in Th1 cytokines (IFNgamma, IL-2) were also found in MPB samples. In conclusion, Th1 and Th2 cytokines are relevant in predicting the clinical outcome after allogeneic peripheral blood HSCT.

Valproic acid inhibits interferon-γ production by NK cells and increases susceptibility to Listeria monocytogenes infection.

Valproic acid (VPA) is a drug commonly used for epileptic seizure control. Recently, it has been shown that VPA alters the activation of several immune cells, including Natural Killer (NK) cells, which play an important role in the containment of viruses and intracellular bacteria. Although VPA can increase susceptibility to extracellular pathogens, it is unknown whether the suppressor effect of VPA could affect the course of intracellular bacterial infection. This study aimed to evaluate the role of VPA during Listeria monocytogenes (L.m) infection, and whether NK cell activation was affected. We found that VPA significantly augmented mortality in L.m infected mice. This effect was associated with increased bacterial load in the spleen, liver, and blood. Concurrently, decreased levels of IFN-γ in serum and lower splenic indexes were observed. Moreover, in vitro analysis showed that VPA treatment decreased the frequency of IFN-γ-producing NK cells within L.m infected splenocytes. Similarly, VPA inhibited the production of IFN-γ by NK cells stimulated with IL-12 and IL-18, which is a crucial system for early IFN-γ production in listeriosis. Finally, VPA decreased the phosphorylation of STAT4, p65, and p38, without affecting the expression of IL-12 and IL-18 receptors. Altogether, our results indicate that VPA increases the susceptibility to Listeria monocytogenes infection and suggest that NK cell is one of the main targets of VPA, but further work is needed to ascertain this effect.