Vitamin D receptor (VDR) deficiency contributes to the development of experimental inflammatory bowel disease (IBD) in several different models. T cells have been shown to express the VDR, and T cells are targets of vitamin D. In this article we determined the effects of VDR expression on CD8+ T cells.
VDR KO CD8+ T cells, but not WT CD8+ T cells, induced colitis in Rag KO recipients. In addition, co-transfer of VDR KO CD8+ T cells with naïve CD4+ T cells accelerated colitis development. The more severe colitis was associated with rapidly proliferating naïve VDR KO CD8+ T cells and increased IFN-γ and IL-17 in the gut. VDR KO CD8+ T cells proliferated in vitro without antigen stimulation and did not downregulate CD62L and upregulate CD44 markers following proliferation that normally occurred in WT CD8+ T cells. The increased proliferation of VDR KO CD8+ cells was due in part to the higher production and response of the VDR KO cells to IL-2.
Our data indicate that expression of the VDR is required to prevent replication of quiescent CD8+ T cells. The inability to signal through the VDR resulted in the generation of pathogenic CD8+ T cells from rapidly proliferating cells that contributed to the development of IBD.
Vitamin D receptor; CD8+ T cells; Proliferation; Inflammatory bowel disease
The aim of this study was to assess the level of salivary immunoglobulins and periodontal status in smokers and non-smokers.
Materials and methods
Unstimulated saliva of 30 subjects (mean age 24.2 ± 3.5 years) who were smokers (test group) and of 30 subjects (mean age 25.3 ± 3.8 years) who were non-smokers (control group) was collected and centrifugated; IgA, IgG, and IgM were measured with the colorimetric immunoenzymatic method. Moreover, the following periodontal clinical parameters were recorded for each subject: plaque index (PI), gingival index (GI), probing depth (PD), and clinical attachment level (CAL).
A significantly (p< 0.05) lower Ig level was observed in smoking patients (IgA: 20.0 ± 1.2 mg/dl; IgM: 19.5 ± 1.6 mg/dl; IgG: 8.1 ± 1.4 mg/dl) compared to levels in the non-smoking control group (IgA: 234.1 ± 65.2 mg/dl; IgM: 121.0 ± 31.7 mg/dl; IgG: 1049.4 ± 102 mg/dl). In the test group, PI (2.2 ± 0.3), GI (2.4 ±0.5), PD (49.3 ± 9.2%), and CAL (49.3 ± 4.6%) were higher (p< 0.05) than those observed in the control group (PI: 0.8 ± 0.4; GI: 0.7 ± 0.3; PD: 10.6 ± 2.4%; CAL: 3.1 ± 0.8%).
Smoking subjects showed lower levels of salivary IgA, IgG, and IgM and a worse periodontal condition than non-smoking subjects. On the base of our study, as smoking subjects also had lower levels of IgA, IgG, and IgM in their saliva than non-smoking subjects, despite the fact that there is little evidence that the salivary Igs have a protective action against periodontitis and that the whole saliva does not result in whole from the salivary glands, it can be concluded that the deteriorated periodontal health conditions of these patients can be attributed in part to a lowering of the host’s defense due to a decrease in the quantity of Igs in salivary fluid.
Monocytes represent a heterogeneous population of cells subdivided according to the expression level of membrane antigens. A pro-inflammatory (intermediate/nonclassical) subpopulation of monocytes is defined by expression of CD16. CD163 seems to be characteristically preferentially expressed by immunosuppressive monocytes. The aim of our study was to evaluate the distribution of monocyte subpopulations in 71 patients with kidney allograft transplantation.
The phenotype was evaluated by flow cytometry in defined time points. The proportions of peripheral CD14+CD16+ monocytes were downregulated immediately after the kidney transplantation and basiliximab treatment partially attenuated this trend. The transient downregulation of the CD14+CD16+ subpopulation was adjusted to basal values in two months. The proportions of CD14+CD163+ monocytes were transiently upregulated early after the kidney transplantation and remained higher during the first month in most patients. In ATG treated patients, the expansion of CD14+CD163+ monocytes was delayed but their upregulation lasted longer. In vitro data showed the direct effect of ATG and methylprednisolone on expression of CD16 and CD163 molecules while basiliximab did not affect the phenotype of cultured monocytes.
We assume from our data that kidney allograft transplantation is associated with modulation of monocyte subpopulations (CD14+CD16+ and CD14+CD163+) partially affected by an immunosuppressive regime used.
CD14+CD16+; CD14+CD163+; Kidney; Monocytes; Subpopulations; Transplantation
Up to 40% of HIV-infected individuals receiving Highly Active Antiretroviral Therapy (HAART) have poor CD4+ T-cell recovery. The role of natural killer (NK) cells in immune recovery during HAART is not well understood. We described the profiles of NK cell subsets and their expression of activating receptor, NKG2D and cytotoxicity receptor NKp46 among suboptimal immune responders to despite four years of suppressive HAART.
A case control study utilized frozen peripheral blood mononuclear cells (PBMC) from a cohort of HIV-infected adults that initiated HAART in 2004/5, at CD4 < 200 cells/μl. Cases were ‘suboptimal’ responders; patients within the lowest quartile of CD4+ T-cell reconstitution, with a median CD4 count increase of 129 (-43-199) cells/μl (difference between CD4 count at baseline and after 4 years of HAART) and controls were ‘super-optimal’ responders; patients within the highest quartile of CD4 T-cell reconstitution with a median CD4 count increase of 528 (416-878) cells/μl). Expression of NK cell lineage markers (CD56+/-CD16+/-) and receptors NKG2D and NKp46, was measured among PBMC from 29 cases of ‘suboptimal’ responders’ and 23 controls of ‘super-optimal responders’, and compared among ‘suboptimal’ and ‘super-optimal’ responders. NK cell populations were compared using the Holm Sidak multiple comparison test and p values < 0.05 were considered statistically significant. Data was analyzed using FLOWJO and GraphPad Prism 6.
‘Suboptimal responders’ had a higher proportion of cytokine producing CD56++CD16+/- (CD56bri) NK cells than the ‘super-optimal responders’ p = 0.017, and CD56neg NK cells were lower among suboptimal than super-optimal responders (p = 0.007). The largest NK cell subset, CD56dim, was comparable among suboptimal responders and ‘super-optimal immune responders’. Expression of NKG2D and NKp46 receptors on NK cell subsets (CD56bri, CD56neg and CD56dim), was comparable among ‘suboptimal’ and ‘super-optimal’ immune responders.
The pro-inflammatory CD56++CD16-- NK cells were higher among ‘suboptimal’ responders relative to ‘super-optimal’ responders, despite four years of suppressive HAART. Alteration of NK cell populations could inhibit host immune responses to infections among suboptimal responders. We recommend further analysis of NK cell function among suboptimal immune responders in order to inform targeted interventions to optimize immune recovery among HAART-treated adults.
Natural killer cells; Suppressive antiretroviral therapy; HAART; Suboptimal immune recovery; HAART; Sub-saharan Africa
Toxic shock syndrome (TSS) is caused by an overwhelming host-mediated response to bacterial superantigens produced mainly by Staphylococcus aureus and Streptococcus pyogenes. TSS is characterized by aberrant activation of T cells and excessive release of pro-inflammatory cytokines ultimately resulting in capillary leak, septic shock, multiple organ dysfunction and high mortality rates. No therapeutic or vaccine has been approved by the U.S. Food and Drug Administration for TSS, and novel therapeutic strategies to improve clinical outcome are needed. Mesenchymal stromal (stem) cells (MSCs) are stromal cells capable of self-renewal and differentiation. Moreover, MSCs have immunomodulatory properties, including profound effects on activities of T cells and macrophages in specific contexts. Based on the critical role of host-derived immune mediators in TSS, we hypothesized that MSCs could modulate the host-derived proinflammatory response triggered by Staphylococcal enterotoxin B (SEB) and improve survival in experimental TSS.
Effects of MSCs on proinflammatory cytokines in peripheral blood were measured in wild-type C57BL/6 mice injected with 50 μg of SEB. Effects of MSCs on survival were monitored in fatal experimental TSS induced by consecutive doses of D-galactosamine (10 mg) and SEB (10 μg) in HLA-DR4 transgenic mice.
Despite significantly decreasing serum levels of IL-2, IL-6 and TNF induced by SEB in wild-type mice, human MSCs failed to improve survival in experimental TSS in HLA-DR4 transgenic mice. Similarly, a previously described downstream mediator of human MSCs, TNF-stimulated gene 6 (TSG-6), did not significantly improve survival in experimental TSS. Furthermore, murine MSCs, whether unstimulated or pre-treated with IFNγ, failed to improve survival in experimental TSS.
Our results suggest that the immunomodulatory effects of MSCs are insufficient to rescue mice from experimental TSS, and that mediators other than IL-2, IL-6 and TNF are likely to play critical mechanistic roles in the pathogenesis of experimental TSS.
Allergen recognition by dendritic cells (DCs) is a key event in the allergic cascade leading to production of IgE antibodies. C-type lectins, such as the mannose receptor and DC-SIGN, were recently shown to play an important role in the uptake of the house dust mite glycoallergen Der p 1 by DCs. In addition to mannose receptor (MR) and DC-SIGN the high and low affinity IgE receptors, namely FcϵRI and FcϵRII (CD23), respectively, have been shown to be involved in allergen uptake and presentation by DCs.
This study aims at understanding the extent to which IgE- and IgG-facilitated Der p 1 uptake by DCs influence T cell polarisation and in particular potential bias in favour of Th2. We have addressed this issue by using two chimaeric monoclonal antibodies produced in our laboratory and directed against a previously defined epitope on Der p 1, namely human IgE 2C7 and IgG1 2C7.
Flow cytometry was used to establish the expression patterns of IgE (FcϵRI and FcϵRII) and IgG (FcγRI) receptors in relation to MR on DCs. The impact of FcϵRI, FcϵRII, FcγRI and mannose receptor mediated allergen uptake on Th1/Th2 cell differentiation was investigated using DC/T cell co-culture experiments. Myeloid DCs showed high levels of FcϵRI and FcγRI expression, but low levels of CD23 and MR, and this has therefore enabled us to assess the role of IgE and IgG-facilitated allergen presentation in T cell polarisation with minimal interference by CD23 and MR. Our data demonstrate that DCs that have taken up Der p 1 via surface IgE support a Th2 response. However, no such effect was demonstrable via surface IgG.
IgE bound to its high affinity receptor plays an important role in Der p 1 uptake and processing by peripheral blood DCs and in Th2 polarisation of T cells.
Allergen; Dendritic cells; Der p 1; IgG; IgE
Currently used mouse models fail to fully reflect human immunity to tuberculosis (TB), which hampers progress in research and vaccine development. Bone marrow-liver-thymus (BLT) mice, generated by engrafting human fetal liver, thymus, and hematopoietic stem cells in severely immunodeficient NOD/SCID/IL-2Rγ-/- (NSG) mice, have shown potential to model human immunity to infection. We engrafted HLA-A2-positive fetal tissues into NSG mice transgenically expressing human leukocyte antigen (HLA)-A2.1 (NSG-A2) to generate NSG-A2-BLT mice and characterized their human immune response to Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection to assess the utility of this model for investigating human TB.
NSG-A2-BLT mice were infected intravenously with BCG and the immune response of engrafted human immune cells was characterized. After ex vivo antigenic stimulation of splenocytes, interferon (IFN)-γ-producing cells were detected by ELISPOT from infected, but not uninfected NSG-A2-BLT mice. However, the levels of secreted IFN-γ, determined by ELISA, were not significantly elevated by antigenic stimulation. NSG-A2-BLT mice were susceptible to BCG infection as determined by higher lung bacillary load than the non-engrafted control NSG-A2 mice. BCG-infected NSG-A2-BLT mice developed lung lesions composed mostly of human macrophages and few human CD4+ or CD8+ T cells. The lesions did not resemble granulomas typical of human TB.
Engrafted human immune cells in NSG-A2-BLT mice showed partial function of innate and adaptive immune systems culminating in antigen-specific T cell responses to mycobacterial infection. The lack of protection was associated with low IFN-γ levels and limited numbers of T cells recruited to the lesions. The NSG-A2-BLT mouse is capable of mounting a human immune response to M. tuberculosis in vivo but a quantitatively and possibly qualitatively enhanced effector response will be needed to improve the utility of this model for TB research.
Animal model; BCG; Tuberculosis; BLT mice; NSG mice
The interaction between the Leishmania parasite and the host cell involves complex, multifaceted processes. The disease severity in cutaneous leishmaniasis (CL) is largely dependent on the causative species. Most of the information on immune responses in human CL is available with respect to L. major infection and is lacking for L. tropica species. In this study, we employed cytokine/chemokine/receptor membrane cDNA array to capture comprehensive picture of immuno-determinants in localized human tissue during L. tropica infection. Expression of selected molecules was evaluated by real time PCR in dermal lesion tissues at pre- and post treatment stages. Plasma IL-17 level was estimated by sandwich ELISA.
The cDNA array analysis identified several immuno-determinants in tissue lesions of Indian CL including cytokines (IFN-γ, TNF-α, IL-1β, IL-10, IL-13), chemokines (IL-8, CCL2, CCL3, CCL4) and apoptotic molecules (Fas, TRAIL, IRF-1). Elevated mRNA levels of Th17 (IL-17, IL-23 and RORγt) and Treg (CD25, CTLA-4 and Foxp3) markers were observed in lesion tissues of CL patients compared to the control group, which subsided post treatment. Plasma IL-17 levels were found to be significantly higher in CL samples compared to controls.
In addition to defining comprehensive immunological responses inside lesion tissues of CL patients, our study demonstrated the presence of Th17 and Treg cells in CL caused by L. tropica.
cDNA array; Cutaneous Leishmaniasis; Cytokines; Th17 cell; Treg cell
Short dimeric or mulitmeric peptides derived from a highly conserved stretch of amino acids from gammaretroviral envelope proteins has been found to have immunosuppressive properties in vitro. Here we test the hypothesis that such immunosuppressive peptides may serve as immunomodulatory reagents for treatment of inflammatory disorders.
The anti-inflammatory effect of a synthetic retrovirus-derived immunosuppressive peptide of 17 amino acids was tested in two murine skin inflammation models, a TPA-induced acute toxic contact eczema model and an oxazolone-induced allergic contact dermatitis. Overall, mice (n = 24) treated with a topically applied cream containing the dimeric immunosuppressive peptide exhibited a reduction of 28.8% in ear thickness (range 20.1-42.5), whereas the application of a scrambled peptide dimer or a monomer of the immunosuppressive peptide remained without effect (p = 0.028). Furthermore, ear biopsies from mice treated with the dimeric immunosuppressive peptide showed a significant reduction in mRNA of the pro-inflammatory cytokines TNF-α, IL-17C, and IL-6 as well as the chemokine CXCL2 compared to mice treated with control peptides.
Using two murine skin inflammation models, we show that an immunosuppressive retroviral peptide is capable of reducing inflammatory disorders. The results indicate that virus-derived immunosuppressive peptides capable of down-regulating several proinflammatory cytokines may represent a novel class of drugs for the treatment of excess inflammation.
Administration of mercury at nontoxic doses induces systemic autoimmune disease in Brown Norway (BN) rats. The pathogenesis of lupus-like oral mucosal lesion by mercury-induced autoimmunity is still unclear, even though the oral mucosa is observed to be commonly affected in mercury-treated BN rats. In this study, we investigated the immunopathology of lupus-like oral mucosal lesions in a model of mercury-induced systemic autoimmunity.
Brown Norway male rats were injected subcutaneously with either phosphate-buffered saline (control) or mercury at a dose of 1.0 mg per kilogram of body weight on days 0, 3, 5, and 7. Blood, kidney, and tongue samples were taken at various timepoints for evaluation by immunohistochemistry, RT-PCR, and lupus band test (LBT).
Oral mucosal lesions were classified according to three consecutive temporal phases on the basis of infiltration of immunocompetent cells as follows: (phase I) infiltration of MHC class II+ dendritic cells (DC) and macrophages; (phase II) addition of ED1+ macrophage infiltrates; and (phase III) focal infiltration of pan T cells following increased infiltration of DC and macrophages. Dense infiltration of DC and macrophages was observed in the basement membrane (BM) zone of the oral epithelium. Tissue expression of IL-4 mRNA was detected in early lesions (phase I), suggesting that locally produced IL-4 may be responsible for Th2-mediated immune response. A linear and continuous smooth pattern of fluorescence was observed in the oral epithelial BM in addition to renal glomeruli, indicating immune complex deposits.
Local autoimmune responses are involved in the pathogenesis of mercury-induced lupus-like lesions of the oral mucosa.
Mercury-induced autoimmunity; Oral mucosa; Lupus-like lesions; Brown Norway rats
Mycobacterium tuberculosis (Mtb) infections are still a major cause of death among all infectious diseases. Although 99% of individuals infected with Mtb develop a CD4+ Th1 and CD8+ T cell mediated immunity as measured by tuberculin skin test, this results only in partial protection and Mtb vaccines are not effective. Deviation of immune responses by pathogens towards a Th2 profile is a common mechanism of immune evasion, typically leading to the persistence of the microbes.
Here we tested the stimulatory capacity of selective Mtb antigens on human monocyte-derived dendritic cell (DC) maturation and cytokine production. DC maturation markers CD80, CD86 and CD83 were readily upregulated by H37Ra- and H37Rv-associated antigens, the 30-kDa (from Ag85 B complex) and 38-KDa Mtb antigens only partially induced these markers. All Mtb antigens induced variable levels of IL-6 and low levels of IL-10, there was no release of IL-12p70 detectable. Substantial IL-12p40 production was restricted to LPS or H37Ra and H37Rv preparations. Although the proliferation levels of primary T cell responses were comparable using all the differentially stimulated DC, the 30-kDa and 38-kDa antigens showed a bias towards IL-4 secretion of polarized CD4+ T cells after secondary stimulation as compared to H37Ra and H37Rv preparations.
Together our data indicate that 30-kDa and 38-kDa Mtb antigens induced only partial DC maturation shifting immune responses towards a Th2 profile.
Dendritic cells; Mycobacterium tuberculosis; T helper cell responses
The milk-derived protein human Casein alpha s1 (CSN1S1) has recently been detected in blood cells and was shown to possess proinflammatory properties. In the present study, we investigated the effect of CSN1S1 on the differentiation of monocytes.
Primary human monocytes were stimulated with recombinant CSN1S1 and compared to cells stimulated with GM-CSF/IL-4 or M-CSF/IFNγ. Morphological changes were assessed by microscopy and quantification of surface markers of differentiation by FACS analysis. Phagocytic activity of CSN1S1 stimulated cells was measured by quantification of zymosan labeled particle uptake. The role of mitogen activated protein kinases for CSN1S1-induced differentiation of monocytes and proinflammatory cytokine expression was assessed by supplementation of specific inhibitors.
CSN1S1 at a concentration of 10 μg/ml resulted in morphological changes (irregular shape, pseudopodia) and aggregation of cells, comparable to changes observed in M-CSF/IFNγ differentiated macrophages. Surface marker expression was altered after 24 h with an upregulation of CD14 (mean 2.5 fold) and CD64 (1.9 fold) in CSN1S1 stimulated cells. CSN1S1 treated cells showed a characteristic surface marker pattern for macrophages after 120 h of incubation (CD14high, CD64high, CD83low, CD1alow) comparable to changes observed in M-CSF/IFNγ treated monocytes. Furthermore, phagocytic activity was increased 1.4 and 1.9 fold following stimulation with 10 μg/ml CSN1S1 after 24 and 48 h, respectively. Early GM-CSF, but not GM-CSF/IL-4 induced differentiation of monocytes towards dendritic cells (DC) was inhibited by addition of CSN1S1. Finally, CSN1S1 induced upregulation of CD14 was impeded by inhibition of ERK1/2, while inhibition of the mitogen activated protein kinases JNK and p38 did not influence cellular differentiation. However, JNK and p38 inhibitors impeded CSN1S1 induced secretion of the proinflammatory cytokines IL-1b or IL-6.
CSN1S1 skews in vitro differentiation of monocytes towards a macrophage-like phenotype. Data is accumulating that functions of CSN1S1 are beyond nutritional properties and include immunomodulatory effects.
Macrophage; Inflammation; Interleukin-1; Interleukin-6; Milk; Differentiation
The immune system consists of multiple preformed and more specific adaptive immune responses, which are all subject to both positive and negative regulation. Programmed cell death protein 1 (PD-1) is a cell surface ligand implicated in the induction of anergy, Inducible T-cell Costimulator (ICOS) plays a stimulatory role in the development of both CD4+ and CD8+ T-cells, Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) plays a role in inhibitory regulation of T-cell activity, and T cell immunoglobulin and mucin protein 3 (Tim-3) has been described as a negative regulatory molecule in CD4+ helper type 1 cells and CD8+ cytotoxic type 1 cells. Each of these ligands is induced with T-cell activation allowing greater opportunity to have a regulatory role.
Flow cytometry was used to quantitate the expression of PD-1, ICOS, CTLA-4 and Tim-3 in human T-cells from geriatric and younger subjects both at baseline and after in vitro induction by mitogen. The magnitude of expression of the molecules increased significantly on activated blasts after mitogen stimulation compared to their baseline levels in resting cells. The increase in CTLA-4 expressing CD8+ T-cells was significantly higher after in vitro induction in older persons, while the increase in cells expressing Tim-3 and PD-1 was significantly reduced. In CD4+ T-cells, a greater increase in CTLA-4 expressing cells in older persons was the only difference between the age groups.
We found several significant changes in the older individuals in regulatory elements of the adaptive immune system that occur particularly after immune activation. These differences could have ramifications to autoimmunity as well as immunology against infection and tumors.
Immunosenescence; CD4; CD8; Adaptive immune system; Anergy; Co-receptor; PD-1; CTLA-4; ICOS; Tim-3
Candida albicans water-soluble fraction (CAWS), a mannoprotein-β-glucan complex obtained from the culture supernatant of C. albicans NBRC1385, causes CAWS-mediated vasculitis (CAWS-vasculitis) in B6 and DBA/2 mice with mild and lethal symptoms, respectively. Why CAWS is lethal only in DBA/2 mice remains unknown.
We performed DNA microarray analyses using mRNA obtained from peripheral blood mononuclear cells (PBMCs) of B6 and DBA/2 mice and compared their respective transcriptomes. We found that the mRNA levels of interferon-γ (Ifng) and several genes that regulate the complement system, such as C3, C4, Cfb, Cfh, and Fcna, were increased dramatically only in DBA/2 mice at 4 and 8 weeks after CAWS administration. The dramatic increase was confirmed by quantitative real-time polymerase chain reactions (qRT-PCR). Moreover, mRNA levels of immune-related genes, such as Irf1, Irf7, Irf9, Cebpb, Ccl4, Itgam, Icam1, and IL-12rb1, whose expression levels are known to be increased by Ifng, were also increased, but only in DBA/2 mice. By contrast, the mRNA level of Dectin-2, the critical receptor for the α-mannans of CAWS, was increased slightly and similarly in both B6 and DBA/2 mice after CAWS administration.
Taken together, our results suggest that CAWS administration induces Dectin-2 mediated CAWS-vasculitis in both B6 and DBA/2 mice and the expression of Ifng, but only in DBA/2 mice, which led to increased expression of C3, C4, Cfb, Cfh, and Fcna and an associated increase in lethality in these mice. This model may contribute to our understanding of the pathogenesis of severe human vasculitis.
CAWS; DNA microarray; DBA/2; Kawasaki disease; Complement factors; Ifng
Active cancer immunotherapies are beginning to yield clinical benefit, especially those using peptide-pulsed dendritic cells (DCs). Different adjuvants, including Toll-like receptor (TLR) agonists, commonly co-administered to cancer patients as part of a DC-based vaccine, are being widely tested in the clinical setting. However, endogenous DCs in tumor-bearing individuals are often dysfunctional, suggesting that ex vivo educated DCs might be superior inducers of anti-tumor immune responses. We have previously shown that prothymosin alpha (proTα) and its immunoreactive decapeptide proTα(100–109) induce the maturation of human DCs in vitro. The aim of this study was to investigate whether proTα- or proTα(100–109)-matured DCs are functionally competent and to provide preliminary evidence for the mode of action of these agents.
Monocyte-derived DCs matured in vitro with proTα or proTα(100–109) express co-stimulatory molecules and secrete pro-inflammatory cytokines. ProTα- and proTα(100–109)-matured DCs pulsed with HER-2/neu peptides induce TH1-type immune responses, prime autologous naïve CD8-positive (+) T cells to lyse targets expressing the HER-2/neu epitopes and to express a polyfunctional profile, and stimulate CD4+ T cell proliferation in an HER-2/neu peptide-dependent manner. DC maturation induced by proTα and proTα(100–109) is likely mediated via TLR-4, as shown by assessing TLR-4 surface expression and the levels of the intracellular adaptor molecules TIRAP, MyD88 and TRIF.
Our results suggest that proTα and proTα(100–109) induce both the maturation and the T cell stimulatory capacity of DCs. Although further studies are needed, evidence for a possible proTα and proTα(100–109) interaction with TLR-4 is provided. The initial hypothesis that proTα and the proTα-derived immunoactive decapeptide act as “alarmins”, provides a rationale for their eventual use as adjuvants in DC-based anti-cancer immunotherapy.
Prothymosin alpha; Immunoreactive peptide; Dendritic cells; TH1 immune responses; TLR-4; Adjuvant; HER-2/neu peptides
Anti-glomerular basement membrane (GBM) disease is a well-known antibody-induced autoimmune disease. A few patients have glomerular C1q deposition, but it is usually absent on renal histopathology. The role of C1q deposition in kidney injury is unclear. Recently, anti-C1q antibodies are demonstrated to be pathogenic in the target organ damage of many autoimmune diseases, by facilitating C1q deposition and enhancing complement activation via classical pathway. In the current study, we investigated the associations between anti-C1q antibodies in sera and C1q deposition in kidney of patients with anti-GBM disease.
It was shown that the severity of kidney injury was comparable between patients with and without C1q deposition, including the prevalence of oliguria/auria, the median percentage of crescents in glomeruli and the mean concentration of serum creatinine. Serum anti-C1q antibodies were detected in 15/25 (60%) patients with a low titer. The prevalence of C1q deposition in kidney was comparable between patients with and without serum anti-C1q antibodies (26.7% vs. 30.0%, p > 0.05). No association was found between anti-C1q antibodies and the severity of kidney injury.
The classical pathway of complement may not play a pathogenic role in the kidney injury of human anti-GBM disease. Anti-C1q antibodies could be detected in more than half of patients, which need further investigations.
Anti-glomerular basement membrane disease; Complement; Classical pathway; C1q; Anti-C1q antibody
Activin A is a pleiotrophic regulatory cytokine, the ablation of which is neonatal lethal. Healthy human alveolar macrophages (AMs) constitutively express activin A, but AMs of patients with pulmonary alveolar proteinosis (PAP) are deficient in activin A. PAP is an autoimmune lung disease characterized by neutralizing autoantibodies to Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF). Activin A can be stimulated, however, by GM-CSF treatment of AMs in vitro. To further explore pulmonary activin A regulation, we examined AMs in bronchoalveolar lavage (BAL) from wild-type C57BL/6 compared to GM-CSF knockout mice which exhibit a PAP-like histopathology. Both human PAP and mouse GM-CSF knockout AMs are deficient in the transcription factor, peroxisome proliferator activated receptor gamma (PPARγ).
In sharp contrast to human PAP, activin A mRNA was elevated in mouse GM-CSF knockout AMs, and activin A protein was increased in BAL fluid. Investigation of potential causative factors for activin A upregulation revealed intrinsic overexpression of IFNγ, a potent inducer of the M1 macrophage phenotype, in GM-CSF knockout BAL cells. IFNγ mRNA was not elevated in PAP BAL cells. In vitro studies confirmed that IFNγ stimulated activin A in wild-type AMs while antibody to IFNγ reduced activin A in GM-CSF knockout AMs. Both IFNγ and Activin A were also reduced in GM-CSF knockout mice in vivo after intratracheal instillation of lentivirus-PPARγ compared to control lentivirus vector. Examination of other M1 markers in GM-CSF knockout mice indicated intrinsic elevation of the IFNγ-regulated gene, inducible Nitrogen Oxide Synthetase (iNOS), CCL5, and interleukin (IL)-6 compared to wild-type. The M2 markers, IL-10 and CCL2 were also intrinsically elevated.
Data point to IFNγ as the primary upregulator of activin A in GM-CSF knockout mice which in addition, exhibit a unique mix of M1-M2 macrophage phenotypes.
Interferon gamma; Activin A; Alveolar macrophages
Some studies have shown that nasally administered immunobiotics had the potential to improve the outcome of influenza virus infection. However, the capacity of immunobiotics to improve protection against respiratory syncytial virus (RSV) infection was not investigated before.
The aims of this study were: a) to evaluate whether the nasal administration of Lactobacillus rhamnosus CRL1505 (Lr05) and L. rhamnosus CRL1506 (Lr06) are able to improve respiratory antiviral defenses and beneficially modulate the immune response triggered by TLR3/RIG-I activation; b) to investigate whether viability of Lr05 or Lr06 is indispensable to modulate respiratory immunity and; c) to evaluate the capacity of Lr05 and Lr06 to improve the resistance of infant mice against RSV infection.
Nasally administered Lr05 and Lr06 differentially modulated the TLR3/RIG-I-triggered antiviral respiratory immune response. Lr06 administration significantly modulated the production of IFN-α, IFN-β and IL-6 in the response to poly(I:C) challenge, while nasal priming with Lr05 was more effective to improve levels of IFN-γ and IL-10. Both viable Lr05 and Lr06 strains increased the resistance of infant mice to RSV infection while only heat-killed Lr05 showed a protective effect similar to those observed with viable strains.
The present work demonstrated that nasal administration of immunobiotics is able to beneficially modulate the immune response triggered by TLR3/RIG-I activation in the respiratory tract and to increase the resistance of mice to the challenge with RSV. Comparative studies using two Lactobacillus rhamnosus strains of the same origin and with similar technological properties showed that each strain has an specific immunoregulatory effect in the respiratory tract and that they differentially modulate the immune response after poly(I:C) or RSV challenges, conferring different degree of protection and using distinct immune mechanisms. We also demonstrated in this work that it is possible to beneficially modulate the respiratory defenses against RSV by using heat-killed immunobiotics.
Lactobacillus rhamnosus; Nasal treatment; Poly(I:C); Sntiviral immunity; Respiratory tract; Respiratory syncytial virus
Although DNA vaccine holds a great potential for cancer immunotherapy, effective long-lasting antitumoral immunity sufficient to induce durable responses in cancer patients remains to be achieved. Considering the pivotal role of dendritic cells (DC) in the antigen processing and presentation, we prepared DC-targeting DNA vaccines by fusing tumor-associated antigen HER2/neu ectodomain to single chain antibody fragment (scFv) from NLDC-145 antibody specific for DC-restricted surface molecule DEC-205 (scFvNLDC-145), and explored its antitumoral efficacy and underlying mechanisms in mouse breast cancer models.
In vivo targeting assay demonstrated that scFvNLDC-145 specifically delivered DNA vaccine-encoded antigen to DC. Compared with untargeted HER2/neu DNA vaccines, vaccination with scFvNLDC-145-HER2/neu markedly promoted the HER2/neu-specific cellular and humoral immune responses with long-lasting immune memory, resulting in effective protection against challenge of HER2/neu-positive D2F2/E2 breast tumor while ineffective in parental HER2/neu-negative D2F2 breast tumor. More importantly, in combination with temporary depletion of regulatory T cells (Treg) by low-dose cyclophosphamide, vaccination with scFvNLDC-145-HER2/neu induced the regression of established D2F2/E2 breast tumor and significantly retarded the development of spontaneous mammary carcinomas in transgenic BALB-neuT mice.
Our findings demonstrate that DC-targeted DNA vaccines for in vivo direct delivery of tumor antigens to DC could induce potent antigen-specific cellular and humoral immune responses and, if additional combination with systemic Treg depletion, was able to elicit an impressively therapeutic antitumoral activity, providing a rationale for further development of this approach for cancer treatment.
DNA vaccine; DC-targeted; HER2/neu; Breast cancer; Cyclophosphamide
C-reactive protein (CRP) is a nonspecific, acute-phase protein that rises in response to infectious and non-infectious inflammatory processes. Infections are the single largest cause of neonatal deaths globally.
The primary aim of this study is to examine the association between CRP gene polymorphism and serum levels of CRP in correlation with early onset sepsis (EOS) infection in newborns living in Taif city, Saudi Arabia. The second aim is to examine the relationship between specific IgG/IgG subclasses and early onset sepsis (EOS) infection among these newborns.
Staphylococcus aureus (S. aureus) is one of the most common organisms related to sepsis infection in the newborn at King Abdel Aziz Specialist Hospital (KAASH). This study was conducted in Taif city, at KAASH’s neonatal intensive care unit between March and August 2012. Neonates were consecutively enrolled onto the study having met our inclusion criteria (as per our research protocol).
The CRP concentration level was analysed using NycoCard® CRP Single Test. CRP -286 (C>T>A) A polymorphisms were analyzed using Pyrosequencing technology for CRP genotyping. IgG subclasses were analysed in the study population using ELISA.
Logistic regression analyses showed that the AA and AC genotypes were negatively associated amongst EOS neonates compared to suspected neonates. The frequency of CC and CT were significantly associated with the EOS neonates compared to the suspected group. The levels of specific IgG1, IgG2 and IgG3 antibodies were significantly lower amongst EOS compared to the suspected group.
Taken together, the CRP-286 (C>T>A) A genotype polymorphism and specific IgG antibodies isotype levels can contribute to a reduced risk of EOS. Furthermore, CRP has a potential use in detecting EOS in neonates, which may mean earlier detection and management of EOS and subsequently better clinical outcome.
CRP; CRP gene Polymorphism; IgG subclasses; Early onset sepsis (EOS); Saudi Arabia
Host genetic variations may contribute to disease susceptibility of influenza. IL-1A and IL-1B are important inflammatory cytokines that mediate the inflammation and initiate the immune response against virus infection. In this study, we investigated the relationship between single-nucleotide polymorphisms (SNPs) of Interleukin-1A (IL-1A) and Interleukin-1B (IL-1B) and the susceptibility to 2009 pandemic A/H1N1 influenza (A(H1N1)pdm09). 167 patients whom were confirmed with A(H1N1)pdm09 and 192 healthy controls were included in this study. Four SNPs (rs1304037, rs16347, rs17561, rs2071373) in IL1A gene and three SNPs (rs1143623, rs3917345, rs1143627) in IL1B gene were genotyped by using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry platform, and the associations of the genetic variants of IL-1 with susceptibility to A(H1N1)pdm09 were then assessed.
The polymorphisms of rs17561 in IL1A gene and rs1143627 in IL1B gene were found to be associated with susceptibility to A(H1N1)pdm09 with P values of 0.003 (OR 2.08, 95% CI 1.27-3.41) and 0.002 (OR 1.62 , 95% CI 1.20-2.18), respectively. However, no significant difference in allelic frequency was observed for other SNPs between cases and controls.
This study provides a new insight into pathogenesis of A(H1N1)pdm09, suggesting that genetic variants of IL-1A and IL-1B may exert a substantial impact on the susceptibility of A(H1N1)pdm09 virus infection.
Interleukin-1; H1N1; Influenza; Single-nucleotide polymorphisms (SNPs)
Recent investigations suggest that neutrophils play an important role in the immune response to lung cancer as well as chronic obstructive pulmonary disease (COPD). The aim of this study was to evaluate the amount of neutrophils and markers of their activity in lung cancer and COPD and in coexistence of these two diseases.
In total, 267 persons were included in the study: 139 patients with lung cancer, 55 patients with lung cancer and COPD, 40 patients with COPD, and 33 healthy subjects. Peripheral blood and BAL fluid samples were obtained for cell count analysis and determination of NE, MPO levels and ROS production. NE and MPO levels in the serum and BAL fluid were determined by ELISA. ROS production was analyzed by flow cytometer.
The percentage, cell count of neutrophils and neutrophil to lymphocyte ratio in the peripheral blood were significantly higher in lung cancer patients with or without COPD compared to COPD patients or healthy individuals (P < 0.05). The percentage and cell count of neutrophils in BAL fluid were significantly lower in patients with lung cancer with or without COPD than in patients with COPD (P < 0.05). However, BAL fluid and serum levels of both NE and MPO were significantly higher in patients with lung cancer than COPD patients or healthy individuals (P < 0.05). Neutrophils produced higher amounts of ROS in patients with lung cancer with or without COPD compared with COPD patients or healthy individuals (P < 0.05).
The results from this study demonstrate higher degree of local and systemic neutrophilic inflammation in patients with lung cancer (with or without COPD) than in patients with COPD.
Lung cancer; Chronic obstructive pulmonary disease; Neutrophils; Reactive oxygen species
T-cell receptor diversity correlates with immune competency and is of particular interest in patients undergoing immune reconstitution. Spectratyping generates data about T-cell receptor CDR3 length distribution for each BV gene but is technically complex. Flow cytometry can also be used to generate data about T-cell receptor BV gene usage, but its utility has not been compared to or tested in combination with spectratyping.
Using flow cytometry and spectratype data, we have defined a divergence metric that quantifies the deviation from normal of T-cell receptor repertoire. We have shown that the sample size is a sensitive parameter in the predicted flow divergence values, but not in the spectratype divergence values. We have derived two ways to correct for the measurement bias using mathematical and statistical approaches and have predicted a lower bound in the number of lymphocytes needed when using the divergence as a substitute for diversity.
Using both flow cytometry and spectratyping of T-cells, we have defined the divergence measure as an indirect measure of T-cell receptor diversity. We have shown the dependence of the divergence measure on the sample size before it can be used to make predictions regarding the diversity of the T-cell receptor repertoire.
Rheumatoid arthritis is a chronic inflammatory disease with a strong MHC class II component and where many patients develop characteristic autoantibodies towards the noncoding amino acid citrulline. Such anti-citrullinated protein antibodies (ACPA) have recently been put forward as an independent predictive factor for treatment response by co-stimulation blockade by CTLA4-Ig (abatacept). We have performed a mechanism of action study to dissect T cell functionality in RA patients with long-standing disease undergoing abatacept treatment and the influence of ACPA status.
Peripheral blood samples were collected from RA patients as they started CTLA4-Ig treatment and 3 and 6 months later. A general decrease of regulatory T cell subsets was observed in the cohort. Additionally within the ACPA-positive group significant down-regulation of all key T cell effector subsets including Th1, Th2, and Th17 was observed by analyzing cytokines by intracellular flow cytometry and in cell culture supernatants.
RA synovial fluid samples were cultured in vitro in the presence or absence of CTLA4-Ig (abatacept). T cell cytokine production was diminished, but without increasing the functional capacity of CD4+CD25hi regulatory T cells as previously demonstrated in the context of TNF-blockade and anti-IL6R therapy.
Our immunological study of T cell functionality in RA patients, both ACPA-positive and ACPA-negative, starting biological therapy with the co-stimulation blockade abatacept (CTLA4-Ig) supports the recently published registry study implicating ACPA seropositivity as an independent predictive factor to treatment response as we observed the most striking effect on T cell subset modulation in ACPA-positive patients. These data further support the notion of RA as a disease with several sub-entities, where the ACPA-positive fraction represents a classical HLA-associated autoimmune disorder while ACPA-negative patients may have other driving forces apart from classical adaptive immune responses.
Rheumatoid arthritis; Autoimmunity; T lymphocyte; Cytokines; Regulatory T cells; Abatacept; ACPA
Data regarding the quantitative expression of TCR Vβ subpopulations in children with autoimmune diseases provided interesting and sometimes conflicting results. The aim of the present study was to assess by comparative flow cytometric analysis the peripheral blood CD4+ TCR Vβ repertoire of children with an organ-specific autoimmune disorder, such as type 1 diabetes mellitus (T1DM), in comparison to children with a systemic autoimmune disease, such as Systemic Lupus Erythematosus (SLE) in comparison to healthy age-matched controls of the same ethnic origin. The CD4+ TCR Vβ repertoire was analysed by flow cytometry in three groups of participants: a) fifteen newly diagnosed children with T1DM (mean age: 9.2 ± 4.78 years old), b) nine newly diagnosed children with SLE, positive for ANA and anti-dsDNA, prior to treatment (mean age: 12.8 ±1.76 years old) and c) 31 healthy age-matched controls (mean age: 6.58 ± 3.65 years old), all of Hellenic origin.
CD4 + TCR Vβ abnormalities (± 3SD of controls) were observed mainly in SLE patients. Statistical analysis revealed that the CD4 + Vβ4 chain was significantly increased in patients with T1DM (p < 0.001), whereas CD4 + Vβ16 one was significantly increased in SLE patients (p < 0.001) compared to controls.
CD4 + Vβ4 and CD4 + Vβ16 chains could be possibly involved in the cascade of events precipitating the pathogenesis of T1DM and SLE in children, respectively.
TCR Vβ repertoire; Flow cytometry; T1DM; SLE; Children