MicroRNA are small noncoding RNA molecules that are involved in the control of gene expression. To investigate the role of microRNA in multiple sclerosis (MS), we performed genome-wide expression analyses of mRNA and microRNA in T-cells from MS patients and controls.
Heparin-anticoagulated peripheral blood was collected from MS-patients and healthy controls followed by isolation of T-cells. MicroRNA and RNA from T-cells was prepared and hybridized to Affymetrix miR 2.0 array and Affymetrix U133Plus 2.0 Human Genome array (Santa Clara, CA), respectively. Verifications were performed with real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).
We identified 2,452 differentially expressed genes and 21 differentially expressed microRNA between MS patients and controls. By Kolmogorov-Smirnov test, 20 of 21 differentially expressed microRNA were shown to affect the expression of their target genes, many of which were involved in the immune system. Tumor necrosis factor ligand superfamily member 14 (TNFSF14) was a microRNA target gene significantly decreased in MS. The differential expression of mir-494, mir-197 and the predicted microRNA target gene TNFSF14 was verified by real-time PCR and ELISA.
These findings indicate that microRNA may be important regulatory molecules in T-cells in MS.
Autoimmunity; T-cell; Microarray; MicroRNA
Impaired diabetic wound healing occurs as a consequence of excessive reactive oxygen species (ROS) and inflammatory cytokine production. We previously found that whey protein (WP) was able to normally regulate the ROS and inflammatory cytokines during the inflammatory phase (first day) in streptozotocin (STZ)-diabetic wound healing. This study was designed to assess the effect of WP on metabolic status, the inflammation and anti-inflammation response, oxidative stress and the antioxidant defense system during different phases of the wound healing process in diabetic rats. WP at a dosage of 100 mg/kg of body weight, dissolved in 1% CMC, was orally administered daily to wounded normal (non-diabetic) and STZ-induced diabetic rats for 8 days starting from the 1st day after wounding.
The data revealed that WP enhanced wound closure and was associated with an increase in serum insulin levels in diabetic rats and an alleviation of hyperglycemic and hyperlipidemic states in diabetic animals. The increase in insulin levels as a result of WP administration is associated with a marked multiplication of β-cells in the core of islets of Langerhans. WP induced a reduction in serum TNF-α, IL-1β and IL-6 levels and an increase in IL-10 levels, especially on the 4th day after wounding and treatment. WP also suppressed hepatic lipid peroxidation and stimulated the antioxidant defense system by increasing the level of glutathione and the activity of glutathione-S-transferase, glutathione peroxidase and superoxide dismutase (SOD) in wounded diabetic rats.
WP was observed to enhance wound closure by improving the diabetic condition, limiting prolonged inflammation, suppressing oxidative stress and elevating the antioxidant defense system in diabetic rats.
Whey proteins; Prolonged inflammation; Cytokines; Wound healing; Diabetic rats
Transforming growth factor (TGF)-β is a potent immunosuppressive cytokine necessary for cancer growth. Animal and human studies have shown that pharmacologic inhibition of TGF-β slows the growth rate of established tumors and occasionally eradicates them altogether. We observed, paradoxically, that inhibiting TGF-β before exposing animals to tumor cells increases tumor growth kinetics. We hypothesized that TGF-β is necessary for the anti-tumor effects of cytotoxic CD8+ T lymphocytes (CTLs) during the early stages of tumor initiation.
BALB/c mice were pretreated with a blocking soluble TGF-β receptor (sTGF-βR, TGF-β-blockade group, n=20) or IgG2a (Control group, n=20) before tumor inoculation. Tumor size was followed for 6 weeks. In vivo lymphocyte assays and depletion experiments were then performed to investigate the immunological basis of our results. Lastly, animals were pretreated with either sTGF-βR (n=6) or IgG2a (n=6) prior to immunization with an adenoviral vector encoding the human papillomavirus E7 gene (Ad.E7). One week later, flow cytometry was utilized to measure the number of splenic E7-specific CD8+ T cells.
Inhibition of TGF-β before the injection of tumor cells resulted in significantly larger average tumor volumes on days 11, 17, 22, 26 and 32 post tumor-inoculation (p < 0.05). This effect was due to the inhibition of CTLs, as it was not present in mice with severe combined immunodeficiency (SCID) or those depleted of CD8+ T cells. Furthermore, pretreatment with sTGF-βR inhibited tumor-specific CTL activity in a Winn Assay. Tumors grew to a much larger size when mixed with CD8+ T cells from mice pretreated with sTGF-βR than when mixed with CD8+ T cells from mice in the control group: 96 mm3 vs. 22.5 mm3, respectively (p < 0.05). In addition, fewer CD8+ T cells were generated in Ad.E7-immunized mice pretreated with sTGF-βR than in mice from the control group: 0.6% total CD8+ T cells vs. 1.9%, respectively (p < 0.05).
These studies provide the first in vivo evidence that TGF-β may be necessary for anti-tumor immune responses in certain cancers. This finding has important implications for our understanding of anti-tumor immune responses, the role of TGF-β in the immune system, and the future development of TGF-β inhibiting drugs.
Malignant mesothelioma; Tumor immunology; Immune suppression; TGF-β; CD8+ Cytotoxic T cell
Though potentially linked to the basic physiology of stress response we still have no clear understanding of Gulf War Illness (GWI), a debilitating condition presenting complex immune, endocrine and neurological symptoms. Here we compared male (n = 20) and female (n = 10) veterans with GWI separately against their healthy counterparts (n = 21 male, n = 9 female) as well as subjects with chronic fatigue syndrome/ myalgic encephalomyelitis (CFS/ME) (n = 12 male, n = 10 female).
Subjects were assessed using a Graded eXercise Test (GXT) with blood drawn prior to exercise, at peak effort (VO2 max) and 4-hours post exercise. Using chemiluminescent imaging we measured the concentrations of IL-1a, 1b, 2, 4, 5, 6, 8, 10, 12 (p70), 13, 15, 17 and 23, IFNγ, TNFα and TNFβ in plasma samples from each phase of exercise. Linear classification models were constructed using stepwise variable selection to identify cytokine co-expression patterns characteristic of each subject group.
Classification accuracies in excess of 80% were obtained using between 2 and 5 cytokine markers. Common to both GWI and CFS, IL-10 and IL-23 expression contributed in an illness and time-dependent manner, accompanied in male subjects by NK and Th1 markers IL-12, IL-15, IL-2 and IFNγ. In female GWI and CFS subjects IL-10 was again identified as a delineator but this time in the context of IL-17 and Th2 markers IL-4 and IL-5. Exercise response also differed between sexes: male GWI subjects presented characteristic cytokine signatures at rest but not at peak effort whereas the opposite was true for female subjects.
Though individual markers varied, results collectively supported involvement of the IL-23/Th17/IL-17 axis in the delineation of GWI and CFS in a sex-specific way.
Cytokines; Chronic fatigue; Gulf war illness; Exercise challenge; Immune signaling; Classification model
Airway inflammation is mainly mediated by T helper 2 cells (Th2) that characteristically produce interleukin (IL)-4, IL-5, and IL-13. Epidemiological studies have revealed an inverse association between the dietary intake of vitamin A and the occurrence of asthma. Serum vitamin A concentrations are significantly lower in asthmatic subjects than in healthy control subjects. It has been reported that all-trans retinoic acid (ATRA), a potent derivative of vitamin A, regulates immune responses. However, its role in Th2-mediated airway inflammation remains unclear. We investigated the effects of ATRA in a mouse model of allergic airway inflammation.
We found that ATRA treatment attenuated airway inflammation and decreased mRNA levels of Th2- and Th17-related transcription factors. The data showed that airway inflammation coincided with levels of Th2- and Th17-related cytokines. We also showed that ATRA inhibited Th17 and promoted inducible regulatory T-cell differentiation, whereas it did not induce an obvious effect on Th2 differentiation in vitro. Our data suggest that ATRA may interfere with the in vivo Th2 responses via T-cell extrinsic mechanisms.
Administration of ATRA dramatically attenuated airway inflammation by inhibiting Th2 and Th17 differentiation and/or functions. ATRA may have potential therapeutic effects for airway inflammation in asthmatic patients.
Asthma; All-trans retinoic acid; Th2; Th17; Regulatory T cells
Most HIV-infected subjects exhibit a progressive rise in CD4 T-cell counts after initiation of highly active antiretroviral therapy (HAART). However, a subset of individuals exhibit very poor CD4 T-cell recovery despite effective control of HIV-RNA viraemia. We evaluated CD4 T-cell proliferation among suboptimal responders and its correlation with CD4 T-cell activation.
The magnitude of CD4 increase (difference between absolute CD4 counts at baseline and absolute CD4 counts at 4 years of ART) was grouped into 4 quartiles for the 211 patients with sustained HIV-RNA viral suppression. Cases of ‘Suboptimal immune responders’ included patients within the lowest quartile [Median CD4 increase 165 (Range −43-298) cells/μl; n=52] and a comparison group of ‘Optimal immune responders’ was defined as patients within the highest quartile of CD4 increase [Median CD4 increase 528 (Range 417–878) cells/μl; n=52]. Frozen PBMC were thawed and analysed from a convenient sample of 39 suboptimal responders and 48 optimal responders after 4 years of suppressive antiretroviral therapy. T-cell activation was measured by proportions of T-cells expressing surface marker CD38 and HLADR (CD4+CD38+HLA-DR+ and CD8+CD38+HLA-DR+ cells). T-cell proliferation was determined by the extent of carboxyfluorescein diacetate succinimidyl ester (CFSE) dye dilution on culture day 5 of PBMCs in the presence of antigen (SEB, PPD, CMVpp65, GagA and GagD). Samples were analyzed on a FACS Calibur flow cytometer and flow data was analyzed using FlowJo and GraphPad.
Overall, CD4 T-cell proliferation on stimulation with SEB, PPD, CMVpp65, Gag A and Gag D.antigens, was lower among suboptimal than optimal responders; this was significant for SEB (CD4+ p=0.003; CD8+ p=0.048) and PPD antigens (CD8+ p=0.038). Among suboptimal responders, T-cell proliferation decreased with increasing immune activation (Negative correlation; slope = −0.13±−0.11) but not among optimal responders.
T-cell immune activation and exhaustion were associated with poor proliferation among suboptimal responders to HAART despite sustained viral suppression. We recommend studies to further understand the mechanisms leading to impaired T-cell function among suboptimal responders as well as the potential role of immune modulation in optimizing CD4 count and functional recovery after HAART.
T-cell proliferation; Immune activation; Suboptimal immune recovery; HAART immune responses; HIV/AIDS
Surgical intervention-related trauma contributes largely to the development of postoperative immunosuppression, with reduced resistance to secondary bacterial infection. This study compared the impact of laparotomy versus laparoscopy on macrophage-associated bactericidal ability and examined whether laparotomy renders the host more susceptible to microbial infection.
BALB/c mice were randomized into control, laparotomy, and laparoscopy groups. Laparotomy, but not laparoscopy, significantly downregulated CR3 expression on macrophages, diminished macrophage-induced uptake and phagocytosis of E. coli and S. aureus, and impaired macrophage-mediated intracellular bacterial killing. Consistent with this, mice that underwent laparotomy displayed substantially higher bacterial counts in the blood and visceral organs as well as a significantly enhanced mortality rate following bacterial infection, whereas mice subjected to laparoscopy did not show any defects in their bacterial clearance.
Laparotomy has an adverse effect on host innate immunity against microbial infection by impairing macrophage-mediated phagocytosis and killing of the invaded bacteria. By contrast, laparoscopy appears to preserve macrophage-associated bactericidal ability, thus alleviating the development of postoperative immunosuppression.
Laparotomy; Laparoscopy; Phagocytosis; Bactericidal activity; Innate immunity; Macrophages
Despite decades of extensive studies, the morbidity and mortality for acute lung injury/acute respiratory distress syndrome (ALI/ARDS) remained high. Particularly, biomarkers essential for its early diagnosis and prognosis are lacking.
Recent studies suggest that alveolar macrophages (AMs) at the exudative phase of ALI/ARDS initiate, amplify and perpetuate inflammatory responses, while they resolve inflammation in the recovery phase to prevent further tissue injury and perpetuated inflammation in the lung. Therefore, proteins relevant to this functional switch could be valuable biomarkers for ALI/ARDS diagnosis and prognosis. We thus conducted comparative analysis of the AM proteome to assess its dynamic proteomic changes during ALI/ARDS progression and recovery.
135 proteins were characterized to be differentially expressed between AMs at the exudative and recovery phase. MALDI-TOF-MS and peptide mass fingerprint (PMF) analysis characterized 27 informative proteins, in which 17 proteins were found with a marked increase at the recovery phase, while the rest of 10 proteins were manifested by the significantly higher levels of expression at the exudative phase.
Given the role of above identified proteins played in the regulation of inflammatory responses, cell skeleton organization, oxidative stress, apoptosis and metabolism, they have the potential to serve as biomarkers for early diagnosis and prognosis in the setting of patients with ALI/ARDS.
ALI/ARDS; Alveolar macrophages; Biomarker; 2D PAGE; MALDI-TOF-MS
Proton currents are required for optimal respiratory burst in phagocytes. Recently, HVCN1 was identified as the molecule required for the voltage-gated proton channel activity associated with the respiratory burst in neutrophils. Although there are similarities between eosinophils and neutrophils regarding their mechanism for respiratory burst, the role of proton channels in eosinophil functions has not been fully understood.
In the present study, we first identified the expression of the proton channel HVCN1 in mouse eosinophils. Furthermore, using HVCN1-deficient eosinophils, we demonstrated important cell-specific effector functions for HVCN1. Similar to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils produced significantly less reactive oxygen species (ROS) upon phorbol myristate acetate (PMA) stimulation compared with WT eosinophils. In contrast to HVCN1-deficient neutrophils, HVCN1-deficient eosinophils did not show impaired calcium mobilization or migration ability compared with wild-type (WT) cells. Uniquely, HVCN1-deficient eosinophils underwent significantly increased cell death induced by PMA stimulation compared with WT eosinophils. The increased cell death was dependent on NADPH oxidase activation, and correlated with the failure of HVCN1-deficient cells to maintain membrane polarization and intracellular pH in the physiological range upon activation.
Eosinophils require proton channel HVCN1 for optimal ROS generation and prevention of activation-induced cell death.
Proton channel; HVCN1; Eosinophil
Hepatitis C virus (HCV) is an RNA virus which has been known to cause acute and chronic necro-inflammatory disease of the liver. It is the leading cause of end-stage liver disease and hepatocellular carcinoma. HIV is known to have a negative impact on the natural disease outcome and immune response of HCV infection, whereas the reverse remains unclear. We evaluated the impact of HCV co-infection on recovery of CD4+ and CD8+ T-cells and liver enzyme levels before and after initiation of highly active antiretroviral therapy (HAART) in HIV/HCV co-infected patients.
A hospital-based, observational, prospective cohort study design was used for this study. Pre-antiretroviral treatment (Pre-ART) and under HAART HIV mono-infected and HCV/HIV co-infected individuals who are under regular follow-up were recruited for this study. 387 blood samples were collected from volunteer, known HIV positive Ethiopian patients and screened for HCV. Twenty five HCV/HIV co-infected patients were prospectively followed for four years. CD4+ and CD8+ T-cells and liver enzyme levels were determined annually for each of the participant.
The prevalence of HCV/HIV co-infection in this study was 6.5%. Both HCV/HIV co-infected and HIV mono-infected under HAART groups showed CD4+ recovery (343 Vs 426; P < 0.004, OR = 4.97, 95% CI = 2.41 to 10.27) respectively; but, the recovery rate was higher in mono-infected (80 Vs 426) than co-infected group (148 Vs 343). The recovery and/or decline pattern of CD8+ T-cells was the same with that of CD4+. In 75% of co-infected groups, the mean alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were above the upper limit of normal reference range. Analyses restricted to individuals who initiated HAART and pre-ART showed similar results.
We found that CD4+ T-cell recovery was negatively affected by the presence of ongoing HCV replication in under HAART co-infected individuals and fast decline of CD4+ T-cells in pre-ART patients. It was also associated with increased ALT and AST enzyme levels in both HAART initiated and treatment naïve co-infected patients.
Immunological; HCV/HIV co-infection; Pre-ART; HAART; CD4+; CD8+; GOT; GPT; Alkaline phosphatase
Infection with parasite protozoa is a long-term health issue in tropical and subtropical regions throughout the world. The Toll-like receptor (TLR) signaling pathway is one of the first-responding defense systems against Leishmania. The aim of this study was to investigate the expression of TLR2 and TLR9 in jejunum and colon and its correlation with CD11c, CD11b, and CD14 receptors used as markers for dendritic cells and macrophages.
Twenty four dogs infected with Leishmania infantum were used in this study. Cytometry was carried out in lamina propria cells from jejunum and colon using markers for TLR2, TLR9, CD11b, CD11c and CD14.
Cellular inflammatory exudate was diffuse in the mucosa and submucosa, predominately comprising mononuclear cells: plasma cells, macrophages, and lymphocytes. Despite the parasite load, microscopy showed no erosion was evident in the epithelial mucosa layers. The colon harbored more parasites than the jejunum. Flow cytometry revealed higher frequency of TLR2+ and CD11c+ dendritic cells in the colon than in the jejunum. Conversely, TLR9-expressing cells were more frequent in jejunum. Moreover, frequency of macrophages (CD11b+ and CD14+) expressing simultaneity TLR9 were lower in the colon than in jejunum, while CD11c+ cells predominated in the colon. Despite of the negative ELISA serum results, IL-10 and TNF-α were higher in jejunum than colon of infected animals. However, IL-4 was higher in colon than jejunum of infected animals. A higher expression these cytokines were demonstrated in infected dogs compared to uninfected dogs.
There was no correlation between clinical signs and pathological changes and immunological and parasitological findings in the gastrointestinal tract in canine visceral leishmaniasis. However, jejunum showed a lower parasite load with increased frequency and expression of CD11b, TLR9, CD14/CD11b/TLR9 receptors and IL-10 and TNF-α cytokines. Conversely, the colon showed a higher parasite load along with increased frequency and expression of TLR2, CD11c receptors, and IL-4 cytokine. Thus, Leishmania infantum is able to interfere in jejunum increased expression of TLR2, TLR9, CD11b, CD14, CD14/CD11b/TLR9 receptors, IL-10, and TNF-α; and in colon increased expression of CD11c, TLR2, TLR9, CD11b, CD14 e, CD14/CD11b/TLR9 receptors, IL-10, and TNF-α.
Canine visceral leishmaniasis; Jejunum and colon; Toll-like receptors 2 and 9; Parasite burden; Leishmania infantum
Genetic polymorphisms observed in various disease states associated with sensitivity or resistance to specific treatments have been a robust area of investigation for decades, with the potential to allow clinicians to make evidence-based decisions on the appropriate course of treatment. This study aimed to evaluate whether genetic polymorphisms of the signal transducer and activator of transcription 6 gene (STAT6) could be associated with a sustained virological response (SVR) among patients infected with hepatitis C virus genotypes 1 and 2 (HCV-1 and HCV-2) who were treated with peginterferon plus ribavirin (PEG-IFNα-RBV). We analyzed the associations between SVR to PEG-IFNα-RBV therapy and 4 single nucleotide polymorphisms (SNPs) in STAT6. This study included Taiwanese Chinese patients infected with either HCV-1 (n = 265) or HCV-2 (n = 195) in the presence or absence of an SVR. Among the STAT6 SNPs examined, the dosage effect of the A allele and allele frequency in rs1059513 were inversely correlated with SVR in patients infected with HCV-1 (P = 0.0179 and P = 0.0235, respectively). This effect was not observed in patients infected with HCV-2. The GG, GGG, and GGGC STAT6 haplotypes comprising 2, 3, and 4 SNPs (rs1059513, rs703817, rs324015, and rs3024974) were found to be associated with SVR, and their presence may increase the probability of a successful treatment outcome in patients infected with HCV-1 (P = 0.0273, 0.0352, and 0.0368, respectively). Moreover, a multivariate logistic regression model for predicting an SVR revealed that the presence of the GGGC haplotype carriers mutually affected the outcome of PEG-IFNα-RBV treatment. The presence of STAT6 SNPs and the association with SVR demonstrated that STAT6 polymorphisms might influence the therapeutic outcomes of patients infected with HCV-1 under standard-of-care (SOC) treatment.
Hepatitis C virus; Standard of care treatment; Sustained virological response; Signal transducer and activator of transcription 6
The editors of BMC Immunology would like to thank all of our reviewers who have contributed to the journal in Volume 13 (2012).
In sepsis, tumor necrosis factor (TNF) is the key factor triggering respiratory burst, tissue injury and disseminated coagulation. Anti-TNF strategies based on monoclonal antibodies or F(ab’)2 fragments have been used in sepsis with contradictory results. Immunoglobulin new antigen receptors (IgNAR) are a unique subset of antibodies consisting of five constant (CNAR) and one variable domains (VNAR). VNAR domains are the smallest, naturally occurring, antibody-based immune recognition units, having potential use as therapy.
Our aim was to explore the impact of an anti-TNF VNAR on survival in an experimental model of endotoxic shock. Also, mRNA expression and serum protein of several inflammatory molecules were measured.
Endotoxic shock was induced by lipopolysaccharide (LPS) in male Balb/c mice. Animals were treated with anti-TNF VNAR domains, F(ab’)2 antibody fragments, or saline solution 15 minutes before, 2 h and 24 h after lethal dose100 (LD100) LPS administration. TNF blockade with either VNAR domains or F(ab’)2 fragments were associated with lower mortality (60% and 75%, respectively) compared to LD100. Challenge with LPS induced significant production of serum TNF and interleukins -10 and -6 at 3 h. After that, significant reduction of IL-6 at 24 h (vs 3 h) was shown only in the VNAR group. Nitrites level also increased in response to LPS.
In liver, TNF and IL-10 mRNA expression showed a pro-inflammatory imbalance in response to LPS. Blocking TNF was associated with a shift towards an anti-inflammatory status; however, polarization was more pronounced in animals receiving F(ab’)2 fragments than in those with VNAR therapy. With regard to IL-6, gene expression was increased at 3 h in all groups. TNF blockade was associated with rapid and sustained suppression of IL-6 expression, even more evident in the VNAR group. Finally, expression of inducible-nitric oxide synthase (iNOS) increased in response to LPS at 3 h, but this was decreased at 24 h only in the anti-TNF VNAR group.
Anti-TNF VNAR single domains improved survival in a murine model of endotoxic shock. Protection was associated with regulation in the TNF/IL-10 balance, attenuation of IL-6 and iNOS gene expression in the liver as well as decreased serum IL-6 concentration.
Endotoxic shock; Sepsis; Anti-TNF; VNAR; Inflammation
BATF plays important roles in the function of the immune system. Batf null mice are deficient in both CD4+ Th17 cells and T follicular helper cells and possess an intrinsic B cell defect that leads to the complete absence of class switched Ig. In this study, Tg mice overexpressing BATF in T cells were used together with Batf null mice to investigate how altering levels of BATF expression in T cells impacts the development and function of a recently characterized population of iNKT cells expressing IL-17 (iNKT-17).
BATF has a direct impact on IL-17 expression by iNKT cells. However, in contrast to the Th17 lineage where BATF activates IL-17 expression and leads to the expansion of the lineage, BATF overexpression restricts overall iNKT cell numbers while skewing the compartment in vivo and in vitro toward an iNKT-17 phenotype.
This work is the first to demonstrate that BATF joins RORγt as the molecular signature for all IL-17 producing cells in vivo and identifies BATF as a component of the nuclear protein network that could be targeted to regulate IL-17-mediated disease. Interestingly, these studies also reveal that while the Il17a gene is a common target for BATF regulation in Th17 and iNKT-17 cells, this regulation is accompanied by opposite effects on the growth and expansion of these two cell lineages.
BATF; Activator-protein-1; iNKT cells; IL-17; Mouse models
In holoendemic Plasmodium falciparum transmission areas such as western Kenya, severe malarial anemia [SMA, hemoglobin (Hb) < 6.0 g/dL, with any density parasitemia] is the most common clinical manifestation of severe malaria resulting in high rates of pediatric morbidity and mortality in these regions. Previous studies associated interleukin (IL)-13 with pathogenesis of different infectious diseases, including P. falciparum malaria. However, the functional roles of polymorphic variants within the IL-13 promoter in conditioning susceptibility to SMA remain largely unexplored. As such, the association between the IL-13 variants -7402 T/G (rs7719175) and -4729G/A (rs3091307) and susceptibility to SMA was determined in children (n = 387) presenting with clinical symptoms of falciparum malaria and resident in a holoendemic transmission region in western Kenya. Our results indicated no difference in the proportions of individual genotypes among children presenting with non-SMA (n = 222) versus SMA (n = 165). Similarly, there was no associations between the individual genotypes (-7402 T/G and -4729G/A) and SMA. Additional analyses, however, revealed that proportions of individuals with -7402 T/-4729A (TA) haplotype was significantly higher in children presenting with SMA than non-SMA group (P = 0.043). A further multivariate logistic regression analyses, controlling for confounding factors, demonstrated that carriage of the TA haplotype was associated with increased susceptibility to SMA (OR; 1.564, 95% CI; 1.023-2.389, P = 0.039). In addition, circulating levels of IL-13 were comparable between the clinical groups as well as across genotypes and haplotypes. Collectively, findings presented here suggest that haplotypes within the IL-13 promoter at -7402 T/G and -4729G/A may modulate SMA pathogenesis, but do not affect circulating IL-13 levels.
IL-13; Promoter polymorphisms; Haplotypes; Severe malaria anemia
Allergic sensitisation has been ascribed to a dysregulated relationship between allergen-specific Th1, Th2 and regulatory T cells. We hypothesised that the relationship between these T cell subsets could be better defined using a short-term allergen stimulation system followed by direct analysis of CD154-positive T cells. Using peripheral blood samples from birch pollinosis patients and healthy non-atopic controls, we sought to explore the frequencies and phenotype of birch-stimulated CD154-positive T helper cells following ex vivo birch allergen stimulation.
Activated CD154-positive Th1, Th2 and Tr1-like cells, that co-expressed IFNγ, IL-4 and IL-10 respectively, were identified in both birch-allergic and non-allergic participants. We observed a close correlation between Th1, Th2 and Tr1-like cell frequency in non-allergic volunteers, such that the three parameters increased together to maintain a low Th2: Th1 ratio. The relationship between Th1, Th2 and Tr1-like responses was dysregulated in birch-allergic patients, with abrogation of the IL-10 response and a higher Th2: Th1 ratio. A close correlation was observed between Th2 cell frequency and the absolute concentration of birch-specific IgE within the birch-allergic group, and we confirmed previous reports of a more differentiated T cell phenotype in allergic subjects.
The findings demonstrate an important balance between IFNγ, IL-4 and IL-10 T cell responses to birch allergen in health, where Th2 responses to allergens were frequently observed, but apparently balanced by Th1 and regulatory responses. The detection of CD154 positive T cells after short-term antigen stimulation may be a useful method for the detection of T cell responses to allergens when cost, speed and convenience are priorities.
Allergen-specific T cell; Birch pollen allergy; Ex vivo phenotyping; Flow cytometry
Bacterial DNA is well-known for its potent immunostimulatory properties which have been attributed to the abundance of CpG dinucleotides within the genomes of prokaryotes. Research has found that mammalian TLR9 is a receptor which mediates the immune response to CpG DNA; however, its functional properties in non-mammalian vertebrates are still poorly characterized. Leukocytes isolated from lower vertebrates, including teleosts, respond to CpG DNA and TLR9 has been identified in many fish species; however, the ligand-binding properties of fish TLR9 have, so far, not been studied. The fact that some vertebrates, like chicken, lack TLR9 and use an alternative molecule (TLR21) as a receptor for CpGs has questioned the functional conservation of TLR9 within vertebrates.
In the current study, TLR9 from Atlantic salmon (SsTLR9) has been found to interact with synthetic oligonucleotides via a CpG-independent but a pH-dependent mechanism. The endogenous receptor, expressed by primary mononuclear phagocytes colocalizes with CpG oligonucleotides (ODNs) in vesicles that appear to be endosomes. When overexpressed in salmonid cell lines, SsTLR9 spontaneously activates ISRE-containing promoters of genes involved in the IFN response; however, the transgenic receptor fails to translocate to CpG-containing endosomes. This indicates that only specific immune cell types have the ability to relocate the receptor to the appropriate cellular compartments where it may become activated by its ligand. In addition, through co-precipitation and mass spectrometry, two salmon proteins - hnRNPA0 and NCOA5, which both contain RNA-binding domains (RRM), were found to bind CpG ODNs, suggesting they may be involved in the CpG response in salmon leukocytes.
The presented data are the first to demonstrate that the DNA-binding properties of TLR9 are conserved between teleosts and mammals. The current study also identifies additional molecules which may function as mediators of the immunostimulatory properties of foreign DNA.
Innate immunity; Toll-like receptors; CpG oligonucleotides; Teleosts; RNA recognition motifs
Blomia tropicalis is a dust mite and an important source of allergens in tropical regions. Up to now, the assays to diagnose atopy to this mite use whole body extract as antigens. However, anti-B. tropicalis IgE antibodies cross-react with Ascaris lumbricoides antigens, hindering the diagnosis of allergy to this mite. In this study, B. tropicalis recombinant allergens were evaluated with the purpose of developing an immunodiagnostic assay for allergy to this mite with greater specificity than those commercially available.
Two B. tropicalis allergens (Blo t 5 and Blo t 21) were cloned into a plasmidial expression vector, expressed in Escherichia coli and purified by affinity chromatography. Sixty-three sera containing anti-B. tropicalis extract (BtE) IgE antibodies were used to investigate IgE reactivity to the recombinant Blot 5 and 21 allergens. Inhibition assays with 20 sera pre-adsorbed with A. lumbricoides extract were performed using rBlo t 5, rBlo t 21, and BtE as antigens. All the assays were carried using indirect ELISA.
Eighty-two point nine percent and 80.0% of the sera with anti-BtE antibodies from 35 children reacted with rBlo t 5 and rBlo t 21, respectively, whereas 92.8% and 89.3% of the 28 sera with anti-BtE antibodies from adult asthma patients reacted with the same allergens, and 96.4% of these sera reacted with a mixture of rBlo t 5 and rBlo t 21. In an inhibition ELISA, the absorption of sera by A. lumbricoides extract affected less the reaction with rBlo t 5 and rBlo t 21 than with BtE.
The rBlo t 5 and rBlo t 21 allergens contain important epitopes recognized by IgE antibodies of individuals allergic to B. tropicalis antigens. Moreover, the assays using the recombinant allergens had lower IgE cross-reactivity with A. lumbricoides antigens, a fact which would confers higher specificity to serodiagnostic assays than the crude mite extract. However, additional recombinant allergens should be evaluated in order to reach the same sensitivity of the commercially available assays based on mite extract.
Blomia tropicalis; Recombinant allergens; Immunodiagnosis; Cross-reactivity; Ascaris lumbricoides; Sensitivity; Specificity
An in silico study was carried out to identify antigens for their possible collective use as vaccine candidates against diseases caused by different classes of pathogenic mycobacteria with significant clinical relevance. The genome sequences of the relevant causative agents were used in order to search for orthologous genes among them. Bioinformatics tools permitted us to identify several conserved sequences with 100% identity with no possibility of cross-reactivity to the normal flora and human proteins. Nine different proteins were characterized using the strain H37Rv as reference and taking into account their functional category, their in vivo expression and subcellular location. T and B cell epitopes were identified in the selected sequences. Theoretical prediction of population coverage was calculated for individual epitopes as well as their combinations. Several identical sequences, belonging to six proteins containing T and B cell epitopes which are not present in selected microorganisms of the normal microbial flora or in human proteins were obtained.
Mycobacterium smegmatis (Ms) is a nonpathogenic mycobacteria of rapid growth, which shares many characteristics with Mycobacterium tuberculosis (MTB), the major causative agent of tuberculosis. MTB has several cell wall glycolipids in common with Ms, which play an important role in the pathogenesis of tuberculosis and the induction of a protective immune response against MTB infection in some animal models. In this study, the humoral immune response and cross reactivity against MTB, of liposomes containing a mixture of cell wall glycolipids of Ms and commercial lipids was evaluated, in order to study its possible use as a component of a vaccine candidate against tuberculosis. Liposomes containing total lipids extracted from Ms, distearoyl phosphatidyl choline and cholesterol were prepared by the dehydration-rehydration technique. Balb/c mice were immunized with the liposomes obtained and the antibody response and cross reactivity against MTB were tested by ELISA. Total lipids extract from Ms showed the presence of several polar glycolipids in common with MTB, such as phosphatidylinositol mannosides. Liposomes that contained glycolipids of Ms were capable of inducing a specific IgG antibody response that allowed the recognition of surface antigens of MTB. The results of this study demonstrated the presence of immunogenic glycolipids in Ms, which could be included to enhance the protective effects of subunit vaccine formulations against tuberculosis.