Induced Lymphocyte Proliferation Research Articles

Immunopotentiality of Ayurvedic polyherbal formulations “Saribadi” and “Anantamul Salsa” with augmentation of IgM production and lymphocytes proliferation: A preliminary study

ObjectiveTo assess the immunopotentiality of Ayurvedic polyherbal preparations, “Saribadi” and “Anantamul Salsa”. MethodsFreshly prepared BALB/c mice splenocytes were cultured with “Saribadi” or “Anantamul Salsa” treatment [doses of 0.25%, 0.50%, 0.75%, 1.00%, 1.50%, 2.00%, 3.00% and 4.00% (v/v)] at 37 °C for 5 days. The immunoglobulin M (IgM) production and lymphocytes proliferation were determined by ELISA and MTT methods, respectively. Endotoxin contamination was assessed by treating the preparations with polymyxin B. ResultsThe doses of “Saribadi” [0.25%, 0.50%, 0.75% and 1.00% (v/v)] significantly increased IgM productions (0.966, 0.728, 0.695 and 0.615 μg/mL vs. control 0.265 μg/mL) and lymphocytes proliferation [absorbance 0.311, 0.394, 0.372 and 0.334 optical density (OD) vs. control 0.162 OD]. Similarly, the doses of “Anantamul Salsa” [0.50%, 0.75%, 1.00% and 1.50% (v/v)] promoted IgM productions (0.933, 0.919, 0.917 and 0.892 μg/mL vs. control 0.502 μg/mL) and the doses of “Anantamul Salsa” [0.50%, 0.75%, 1.00%, 1.50%, 2.00%, and 3.00% (v/v)] stimulated lymphocytes proliferation (absorbance 0.395, 0.326, 0.440, 0.398, 0.452 and 0.355 OD vs. control 0.199 OD). The activity of “Saribadi” and “Anantamul Salsa” was not retarded by the treatment of preparations with polymyxin B. ConclusionsImmunomodulatory activity of “Saribadi” and “Anantamul Salsa” was unveiled for the first time. “Saribadi” and “Anantamul Salsa” possess immunostimulating potential acting through the induction of lymphocyte proliferation and IgM production. These preparations may be useful in strengthening immune responses. However, further cellular and in vivo studies are required.

A novel rabies virus lipopeptide provides a better protection by improving the magnitude of DCs activation and T cell responses

Besides rabies virus neutralizing antibody, non-neutralizing antibody to internal vital proteins, interferon, and possibly cell-mediated immunity also have a critical role in preventing the infection by rabies virus (RV). We identified novel CTL and Th epitopes which could induce lymphocyte proliferation and IFN-γ, IL-4 production, and designed linear and branched lipopeptides with these selected CTL and Th epitopes. Compared to linear construct, branched lipopeptides, especially Lipo C, stimulate stronger phenotypic and functional maturation of DCs, as well as more efficient CD8+ T cell responses, evaluating by using FACS, G333–341 tetramer staining and specific CTL assay. Lipo C could also assist rabies vaccine to induce an instant rabies virus neutralizing antibody production, and better protection against rabies virus challenge at early stage. These data reveal that Lipo C could be a promising component for developing novel rabies vaccines.

Glycerol Monolaurate (GML) inhibits human T cell signaling, metabolism, and function by disrupting lipid dynamics

Abstract Glycerol Monolaurate (GML) is a naturally occurring fatty acid widely utilized in food, cosmetics, and homeopathic supplements. GML is a potent antimicrobial agent that targets a range of bacteria, fungi, and enveloped viruses. Interestingly, GML suppresses mitogen induced lymphocyte proliferation and inositol triphosphate production, suggesting that GML has immunomodulatory functions. In this study, we have mechanistically examined if GML affects the signaling, metabolism, and functional output of human primary T cells. We found that GML potently altered lipid order and disorder dynamics in the plasma membrane that resulted in reduced membrane localized clustering of the proteins LAT, PLC-γ, and AKT, events integral for proper T cell receptor (TCR) signal propagation. Altered membrane signaling events induced selective inhibition of TCR-induced phosphorylation of SLP-76, regulatory P85 subunit of PI3K, and AKT as well as abrogated calcium influx. In addition to signaling defects, GML treated cells have profoundly altered metabolism profiles characterized by suppressed oxidative phosphorylation and increased glycolysis. Functionally, GML treatment potently reduced TCR-induced production of the cytokines IL-2, IFN-γ, TNF-α, and IL-10. Our data reveal that the widely used anti-microbial agent GML alters the lipid dynamics of human T cells, leading to their defective signaling, metabolism, and function.

Boron Induces Lymphocyte Proliferation and Modulates the Priming Effects of Lipopolysaccharide on Macrophages.

Chemical mediators of inflammation (CMI) are important in host defense against infection. The reduced capacity of host to induce the secretion of these mediators following infection is one of the factors in host susceptibility to infection. Boron, which has been suggested for its role in infection, is reported in this study to increase lymphocyte proliferation and the secretion of CMI by the lipopolysaccharide (LPS)-stimulated peritoneal macrophages in BALB/c mice. Boron was administered to mice orally as borax at different doses for 10 consecutive days, followed by the stimulation of animals with ovalbumin and isolation of splenocytes for proliferation assay. The lymphocyte subsets were determined by flow cytometry in spleen cell suspension. The mediators of inflammation, TNF-α, IL-6, IL-1β and nitric oxide (NO), were measured in culture supernatant of LPS-primed macrophages isolated from borax treated mice. TNF and ILs were measured by ELISA. NO was determined by Griess test. The expression of inducible nitric oxide synthase (iNOS) in macrophages was studied by confocal microscopy. Results showed a significant increase in T and B cell populations, as indicated by an increase in CD4 and CD19, but not CD8, cells. Boron further stimulated the secretion of TNF-α, IL-6, IL-1β, NO and the expression of iNOS by the LPS-primed macrophages. The effect was dose dependent and most significant at a dose level of 4.6 mg/kg b. wt. Taken together, the study concludes that boron at physiological concentration induces lymphocyte proliferation and increases the synthesis and secretion of pro-inflammatory mediators by the LPS-primed macrophages, more specifically the M1 macrophages, possibly acting through Toll-like receptor. The study implicates boron as a regulator of the immune and inflammatory reactions and macrophage polarization, thus playing an important role in augmenting host defense against infection, with possible role in cancer and other diseases.

Decidual soluble factors, through modulation of dendritic cells functions, determine the immune response patterns at the feto-maternal interface

Dendritic cells (DCs) can acquire immunogenic or tolerogenic properties depending on intrinsic and tissue environmental factors. We aimed to determine the immunomodulatory effects of decidual soluble factors from abortion- and non-abortion-prone mice on DC functions. The decidual cell supernatants (DS) were obtained from abortion-prone and non-abortion-prone mice. Splenic DCs were treated with DS and conalbumin (as an antigen) and injected into the palms of the mice. After five days, regional lymph node cells were collected and cultured in the presence and absence of conalbumin. The proliferation of lymphocyte cells, the frequency of regulatory T cells (Tregs), and the production of IL-4 and IFN-γ were measured by [3H]thymidine incorporation, flow cytometry, and ELISA respectively. Our results indicated that DS from both abortion- and non-abortion-prone mice decreased the ability of DCs to induce lymphocyte proliferation and IFN-γ production, while enhanced their capacity to induce Tregs compared with non-treated DCs. Another important finding was that the immunosuppressive effects of DS from abortion-prone mice on DCs for inducing proliferative responses, developing Tregs, and producing IFN-γ by primed lymphocytes was less than DS from non-abortion-prone mice. We also found that only DS from non-abortion-prone mice could enhance the capacity of DCs to induce IL-4 production by primed lymphocytes. It can be concluded that decidua-secreted factors, by altering DC functions, can determine the pattern of immune responses at the fetomaternal interface and, subsequently, pregnancy outcome.

Potentiation of cytokine-induced proliferation of human Natural Killer cells by intravenous immunoglobulin G

Intravenous IgG (IVIG) therapy can be used for immunomodulation. IL-2 is an immunoregulatory cytokine. We evaluated IVIG modulation of human blood lymphocyte response to IL-2 and other cytokines. Neither IVIG nor low concentrations of IL-2 (3–30U/ml) induced lymphocyte proliferation, but in combination they synergistically enhanced proliferation of NK cells. The CD56bright cells expanded more than CD56dim NK cells, with 90% of NK cells dividing up to 8 generations by day 6, while <8% of T cells divided. IVIG also potentiated NK cell proliferation with IL-12, IL-15 and IL-18. The IVIG+cytokine-expanded NK cells were less cytotoxic for K562 cells, than NK cells with cytokine alone. IVIG also enhanced interferon-γ production with IL-2, IL-12 and IL-18. In conclusion, IVIG selectively potentiates NK cell proliferation and interferon-γ secretion with IL-2, IL-12, IL-15 and IL-18 in vitro. These findings warrant evaluation in vivo in relation to NK cells and the immunoregulatory actions of IVIG.

Survey of immuno-allergological ultra high dilution research

Experiments about basic research in Immuno-allergology reported by M. Bastide and B. Poitevin in Ultra High Dilution (1994) have been appraised from a 20 year perspective. The numerous experiments published mainly focus on immunological regulation, inflammatory process and basophil activation. They are analyzed according to one essential criterion: repeatability. The commentary reflects the research details made available in a recently published literature review, also published in French. The regulatory effect of high dilution of bursin on immune response has been observed in multiple experiments but not reproduced by independent teams. The immunomodulating effect of Thymulin has been confirmed in mice. Rhus toxicodendron has an anti-inflammatory activity on different models, from mother tincture (TM) to very high dilutions. The homeopathic complex Canova activates macrophages in vitro and in vivo, induces lymphocyte proliferation, and reduces the size of tumors and mortality of sarcoma-bearing mice. Some homeopathic medicines used in clinical inflammation modulate in vitro the neutrophil activation, with variability in the protocols used and in the medicines tested. In allergology, high dilution histamine has an inhibitory effect on basophil activation in multicenter trials and with independent teams, either with methods implying a change in basophil staining or with flow cytometry. However, high dilution histamine had no effect in some well-conducted experiments. The inhibitory effect of Apis mellifica has not been studied with the flow cytometry method, as well as the activation of basophil by anti-IgE high dilution, published in Nature. Despite considerable research activity in immuno-allergology and a great increase in the number of publications, there is still not in this domain a "gold standard" trial in basic research in homeopathy. The most studied system, the inhibitory effect of histamine high dilutions on basophil activation, requires clarifications of various factors, including individual sensitivity. For scientific and epistemological reasons, the same work should be carried out for independent reproduction of the experiments conducted with anti-IgE and Apis mel high dilution, in complement of the new axes of research in immunoallergology developed since 20 years.

Identification of Toxoplasma gondii protein fractions induce immune response against melanoma in mice.

Dendritic cells (DCs) play a crucial role in the initiation of adaptive immune responses against tumor cells. We recently found that protein components of Toxoplasmagondii (T.gondii) could mature DCs efficiently. Therefore, in this study, we aimed to find the most effective protein components of T.gondii which are able to mature DCs and consequently instruct immune responses in tumor-bearing mice. Soluble tachyzoite antigens (STAgs) were fractionated by ammonium sulfate precipitation and subsequently by anion-exchange HPLC. Immature DCs (iDCs) were treated by these protein fractions and were monitored for IL-12p70 and IL-10 production. Moreover, the capacity of mature DCs (mDCs) to induce lymphocyte proliferation was investigated. Ultimately, we analyzed the ability of mDCs in instructing immune responses in tumor-bearing mice. We found that ammonium sulfate fraction one (A1) matured-DCs produced higher IL-12 level and IL-12/IL-10 ratio; therefore, this fraction was selected for further fractionation by anion-exchange HPLC. The results showed that anion-exchange HPLC fraction 14 (C14) matured-DCs secrete higher levels of IL-12p70 and IL-12p70/IL-10 ratio. Survival of the mice matured by A1 fraction increased significantly compared to other groups. Moreover, SDS-PAGE electrophoresis showed that different obtained fractions have distinct proteins based on their size. These results demonstrate that two protein fractions of T.gondii are able to mature DCs more efficient.

Diverse biological effects of the essential oil from Iranian Trachyspermum ammi

Trachyspermum ammi (Apiaceae) is a plant with a good reputation in the traditional Persian and Ayurvedic medicine. The hydrodistilled essential oil from the fruits of T. ammi, known as ‘ajwain oil’, is used in countries such as Iraq, Iran, Afghanistan, Pakistan, and India in the preparation of curry, to flavour several foods, as preservative, and in perfumery. At therapeutic level, ajwain oil is employed in the treatment of gastrointestinal ailments, lack of appetite and bronchial problems. In the present work, the essential oil of T. ammi growing in Iran was analysed by GC–FID and GC–MS showing thymol (67.4%), p-cymene (17.9%) and γ-terpinene (11.3%) as the major constituents. Afterwards, we investigated the biological effects displayed by ajwain oil, namely the antimicrobial and antioxidant activity, the cytotoxicity on tumour cells, and the induction of lymphocyte proliferation. In addition, the inhibition on nicotinate mononucleotide adenylyltransferase (NadD), which is a promising new target for developing novel antibiotics, was evaluated. The antimicrobial effects of ajwain oil, measured by the agar disc diffusion method, were relevant, with inhibition zones higher than those of reference antibiotics, especially on Staphylococcus aureus and Candida albicans (34.7 and 54.3mm, respectively). This effect was not due to the enzymatic inhibition on NadD. The ajwain oil exhibited a considerable dose-dependent inhibition on the ABTS radical cation, with an IC50 value of 22.4μg/mL. MTT assay revealed that ajwain oil is particularly cytotoxic on colon carcinoma cells, with a IC50 value of 9.6μg/mL. Finally, PBMC proliferation assay revealed some role for the ajwain oil within the network of interactions of the cells of the immune system.

Purification and Identification of a Novel Antimicrobial Protein from the Rabbit Sacculus Rotundus and its Effect on Cellular Immune Function in Mice

The aim of this study was to identify the antimicrobial substances in the sacculus rotundus and evaluate their effects on mammalian immune function and molecular mechanisms. Chromatographic techniques were used to purify antimicrobial substances. Components with antimicrobial activity were analyzed by NanoLC-ESI-MS/MS. Immunosuppressed mice were given crude protein extract or purified antimicrobial protein by gavage for 4 weeks, and serum cytokine levels were measured. Splenic lymphocyte proliferation assays were also performed. We obtained histone cluster 1, H2bc-like, which had antimicrobial activity. We termed this protein rabbit sacculus rotundus antimicrobial histone, according to its primary source. Sequence analysis and alignment suggested that EKTPVKKKARKSAGAAKRKA was a core segment of the protein mediating its antimicrobial activity. Treatment with high doses of the crude protein extract for 14, 21, and 28 days significantly increased interferon-γ (IFN-γ) levels compared to the control. Interleukin (IL)-4 levels were also increased significantly on days 7, 14, 21, and 28 in the experimental group. Moreover, treatment with the crude protein extract stimulated a significant increase in lymphocyte proliferation on days 14 and 28. Administration of antimicrobial protein isolated from the rabbit sacculus rotundus to immunosuppressed mice increased serum IFN-γ and IL-4 levels and induced lymphocyte proliferation, thereby promoting cellular immune function in immunosuppressed mice.

Inflammatory environment and oxidized LDL convert circulating human proangiogenic cells into functional antigen-presenting cells.

The function of human circulating PACs has been described extensively. However, little focus has been placed on understanding how these cells differ in their functions in the presence of microenvironments mimicking vascular inflammation. We hypothesized that exposure to proinflammatory cytokines or the oxLDL, an autoantigen abundant in advanced atherosclerotic plaques, converts PACs into immune-modulating/proinflammatory cells. Hence, we examined the effect of oxLDL and inflammatory stimuli on their phenotype by use of a functional genomics model based on secretome and whole genome transcriptome profiling. PACs obtained from culturing a PBMC fraction in angiogenic medium were primed with DC differentiation cytokines and then exposed to proinflammatory cytokines or oxLDL. Under these conditions, PACs converted into APCs, expressed maturation markers CD80 and CD83, and showed an increased up-regulation of CD86. APCcy and APCox induced a robust T cell BrdU incorporation. Despite a similar ability to induce lymphocyte proliferation, APCcy and APCox differed for the secretory pathway and mRNA expression. Analysis of the differentially expressed genes identified 4 gene "clusters," showing reciprocal modulation in APCcy vs. APCox, justifying, according to functional genomics analyses, a different putative function of the cells in antigen processing. Together, these data show that treatment with inflammatory cytokines or oxLDL converts human PAC phenotypes and functions into that of APCs with similar lymphocyte-activating ability but distinct maturation degree and paracrine functions.

TLR-4 cooperates with Dectin-1 and mannose receptor to expand Th17 and Tc17 cells induced by Paracoccidioides brasiliensis stimulated dendritic cells.

The concomitant use of diverse pattern recognition receptors (PRRs) by innate immune cells can result in synergistic or inhibitory activities that profoundly influence anti-microbial immunity. Dectin-1 and the mannose receptor (MR) are C-type lectin receptors (CLRs) previously reported to cooperate with toll-like receptors (TLRs) signaling in the initial inflammatory response and in the induction of adaptive Th17 and Tc17 immunity mediated by CD4+ and CD8+ T cells, respectively. The protective immunity against paracoccidioidomycosis, the most prevalent fungal infection of Latin America, was previously shown to be influenced by these T cell subsets motivating us to study the contribution of TLRs, Dectin-1, and MR to the development of Th17/Tc17 immunity. First, curdlan a specific Dectin-1 agonist was used to characterize the influence of this receptor in the proliferative response and Th17/Tc17 differentiation of naïve lymphocytes induced by Paracoccidioides brasiliensis activated dendritic cells (DCs) from C57BL/6 mice. Then, wild type (WT), Dectin-1–/–, TLR-2–/–, and TLR-4–/– DCs treated or untreated with anti-Dectin-1 and anti-MR antibodies were used to investigate the contribution of these receptors in lymphocyte activation and differentiation. We verified that curdlan induces an enhanced lymphocyte proliferation and development of IL-17 producing CD4+ and CD8+ T cells. In addition, treatment of WT, TLR-2–/–, and TLR-4–/– DCs by anti-Dectin-1 antibodies or antigen presentation by Dectin-1–/– DCs led to decreased lymphoproliferation and impaired Th17 and Tc17 expansion. These responses were also inhibited by anti-MR treatment of DCs, but a synergistic action on Th17/Tc17 differentiation was mediated by TLR-4 and MR. Taken together, our results indicate that diverse TLRs and CLRs are involved in the induction of lymphocyte proliferation and Th17/Tc17 differentiation mediated by P. brasiliensis activated DCs, but a synergist action was restricted to Dectin-1, TLR-4, and MR.

Toxicity of cobalt-chromium nanoparticles released from a resurfacing hip implant and cobalt ions on primary human lymphocytes in vitro.

Adverse tissue responses to prostheses wear particles and released ions are important contributors to hip implant failure. In implant-related adverse reactions T-lymphocytes play a prominent role in sustaining the chronic inflammatory response. To further understand the involvement of lymphocytes in metal-on-metal (MoM) implant failure, primary human lymphocytes were isolated and treated with cobalt-chromium (Co-Cr) wear debris and Co ions, individually, and in combination, for 24, 48 and 120 h. There was a significant increase in cell number where debris was present, as measured by the Neutral Red assay. Interleukin-6 (IL-6), interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) secretion levels significantly decreased in the presence of metal particles, as measured by ELISA. Interleukin-2 (IL-2) secretion levels were significantly decreased by both debris and Co ions. Flow cytometry analysis showed that the metal nanoparticles induced a significant increase in apoptosis after 48-h exposure. This investigation showed that prolonged exposure (120 h) to metal debris induces lymphocyte proliferation, suggesting that activation of resting lymphocytes may have occurred. Although cytokine production was affected mainly by metal debris, cobalt toxicity may also modulate IL-2 secretion, and even Co ion concentrations below the MHRA guideline levels (7 ppb) may contribute to the impairment of immune regulation in vivo in patients with MoM implants.

Construction of recombinant human IFNα-2b BCG and its antitumor effects on bladder cancer cells in vitro.

We constructed recombinant Bacille Calmette-Guérin (rBCG) that secreted human interferon alpha 2b (hIFNα-2b), and investigated its antitumor effects on bladder cancer cells in vitro. The recombinant plasmid phIFN-α-2b was constructed using pMAO-4 and transformed into BCG. The supernatant was collected at various times and IFN-γ, interleukin (IL)-12, and tumor necrosis factor (TNF)-α were detected using an enzyme-linked immunosorbent assay. EJ cells were cultivated for 24, 48, and 72 h, together with rBCG, wild-type BCG (wBCG), or wBCG+IFN-α-2b. rBCG capable of secreting cytokine IFNα-2b was constructed. On the 4th day of culture, the IFNα-2b secreted by rBCG reached a maximum. wBCG and rBCG showed no significant difference on cell growth rate over 7 days of incubation in 7H9 medium. wBCG and rBCG were both positive for acid-fast staining, and showed mycobacterial characteristics of intercellular connection in clusters with no clear abnormalities. Higher levels of IFN-γ, TNF-α, and IL-12 were induced by rBCG compared with wBCG or MAO4-rBCG (P < 0.05). rBCG may induce lymphocyte proliferation; the proliferation ratio was higher than those induced by wBCG and wBCG+IFN. rBCG had direct anti-proliferative effects on EJ cells. An MTT assay showed that rBCG inhibited the proliferation of bladder cancer cells and had more activity compared with wBCG (P < 0.05). The highest anti-tumor activity of lymphocytes was stimulated by rBCG (20.31-51.22%). rBCG-IFNα-2b induces enhanced cytotoxicity against bladder cancer cells in vitro and may be used as an alternative to BCG for bladder cancer patients.

Setae from larvae of the northern processionary moth (Thaumetopoea pinivora, TP) stimulate proliferation of human blood lymphocytes in vitro.

Larvae of the Northern pine processionary moth (Thaumetopoea pinivora, TP) carry microscopic needles (setae), which by penetrating skin and mucous membranes, may cause inflammatory/immune derived symptoms in man. In the present study the stimulatory effects of setae on human blood lymphocytes in vitro was investigated. Blood mononuclear cells were separated from venous blood or buffy coat of ten healthy individuals, six previously exposed to setae and four with no known exposure. Lymphoproliferation was measured as uptake of 3H-thymidine. Setae were prepared from TP larvae. Setae and saline setae extracts stimulated proliferation of T-lymphocytes in the presence of monocytic cells. Stimulation was pronounced in cells from persons who had been exposed to setae, and weak in cells from non-exposed donors. Chitin also induced lymphocyte proliferation in most donors, but to a lesser extent and independently of donor's previous exposure to setae. In conclusion, setae contain molecules that in the presence of monocytes activate human T-lymphocytes to proliferation. The antigenic nature of stimulatory molecules was supported by the significantly stronger lymphocyte response in persons previously exposed to setae than in non-exposed donors. The nature of such molecules remains to be defined.

Clinical Significance of Peripheral Blood Lymphocyte Sensitivity to Glucocorticoids for the Differentiation of High-risk Patients With Decreased Allograft Function After Glucocorticoid Withdrawal in Renal Transplantation

PurposeA reliable biomarker to differentiate high-risk recipients who will experience a decrease in allograft function after glucocorticoid withdrawal has not been established in renal transplantation. We examined the clinical significance of peripheral blood lymphocyte sensitivity to glucocorticoids in vitro for the differentiation of the high-risk patients after glucocorticoid reduction/withdrawal in renal transplant recipients. MethodsThe study included 44 renal transplant recipients with stable allograft function. Peripheral lymphocyte responses to suppressive effects of cortisol, methylprednisolone, cyclosporine, and tacrolimus in mitogen assay procedures in vitro were examined. Clinical outcome after glucocorticoid reduction/withdrawal was retrospectively compared between recipients with lymphocytes normally sensitive to the drugs and those with hyposensitivity. The receiver-operating characteristic (ROC) curve analysis was undertaken for setting the cutoff IC50 values of the drugs against the T cell mitogen–induced lymphocyte proliferation to differentiate the high-risk recipients with decreased allograft function after glucocorticoid withdrawal. FindingsThe median (range) IC50 value for cortisol in the recipients who showed decreased renal function due to glucocorticoid withdrawal was 10,000 (570.9–72,279.3) ng/mL (n = 9), which was significantly higher than the value of 351.6 (2.0–10,000) ng/mL in the recipients who had not experienced glucocorticoid withdrawal symptoms (n = 35) (P < 0.001). Similarly, the median (range) IC50 value for methylprednisolone in the recipients who showed decreased renal function after glucocorticoid withdrawal was 69.1 (21.5–1442.7) ng/mL (n = 9), which was significantly higher than the value of 13.8 (0.7–1000) ng/mL in the recipients who had not experienced glucocorticoid withdrawal symptoms (n = 30) (P < 0.003). In contrast, there was no significant difference in the median IC50 values of cyclosporine and tacrolimus between the 2 recipient subgroups. The ROC curve analyses for the IC50 values of the immunosuppressive drugs estimated the cutoff value of cortisol and methylprednisolone to be 3580.0 and 21.5 ng/mL, respectively. The ROC AUCs for cortisol and methylprednisolone were 0.83 and 0.84, respectively. According to the cutoff IC50 value, the incidence of decreased allograft function in the low cortisol sensitivity (IC50 >3580.0 ng/mL) subgroup was 7 of 13 patients, which was significantly higher than that of the higher sensitivity subgroup of 2 of 31 (P = 0.0012). A similar case was observed using the cutoff IC50 value of methylprednisolone (P = 0.0012), whereas recipient grouping according to the cutoff IC50 values of cyclosporine and tacrolimus failed to differentiate the high-risk recipients with decreased allograft function after glucocorticoid withdrawal. ImplicationsGlucocorticoid pharmacodynamics in lymphocytes of individual patient origin is a reliable biomarker for differentiation of renal transplant recipients who will experience a safe reduction/withdrawal of glucocorticoid.

Intracellular Bacteria Interfere with Dendritic Cell Functions: Role of the Type I Interferon Pathway

Dendritic cells (DCs) orchestrate host defenses against microorganisms. In infectious diseases due to intracellular bacteria, the inefficiency of the immune system to eradicate microorganisms has been attributed to the hijacking of DC functions. In this study, we selected intracellular bacterial pathogens with distinct lifestyles and explored the responses of monocyte-derived DCs (moDCs). Using lipopolysaccharide as a control, we found that Orientia tsutsugamushi, the causative agent of scrub typhus that survives in the cytosol of target cells, induced moDC maturation, as assessed by decreased endocytosis activity, the ability to induce lymphocyte proliferation and the membrane expression of phenotypic markers. In contrast, Coxiella burnetii, the agent of Q fever, and Brucella abortus, the agent of brucellosis, both of which reside in vacuolar compartments, only partly induced the maturation of moDCs, as demonstrated by a phenotypic analysis. To analyze the mechanisms used by C. burnetii and B. abortus to alter moDC activation, we performed microarray and found that C. burnetii and B. abortus induced a specific signature consisting of TLR4, TLR3, STAT1 and interferon response genes. These genes were down-modulated in response to C. burnetii and B. abortus but up-modulated in moDCs activated by lipopolysaccharide and O. tsutsugamushi. This transcriptional alteration was associated with the defective interferon-β production. This study demonstrates that intracellular bacteria specifically affect moDC responses and emphasizes how C. burnetii and B. abortus interfere with moDC activation and the antimicrobial immune response. We believe that comparing infection by several bacterial species may be useful for defining new pathways and biomarkers and for developing new treatment strategies.

AB0008 Blys/April Gene Expression in Synovial Fluid and Blood in Rheumatoid Arthritis

BackgroundThe B Lymphocyte Stimulator (BLyS) and A PRoliferation-Inducing Ligand (APRIL) signaling pathway has an important role in the selection, maturation and survival of B cells.Dysregulation of BLyS/APRIL is involved in...

Giardia duodenalis stimulates partial maturation of bovine dendritic cells associated with altered cytokine secretion and induction of T‐cell proliferation

Giardia duodenalis is an important intestinal parasite in animals and humans. The role of dendritic cells (DC) in the initiation of the immune response against G.duodenalis is poorly documented. The aim of this study was to test the hypothesis that G.duodenalis interferes with bovine DC function. Therefore, the effect of trophozoites and excretion/secretion products on bovine monocyte-derived dendritic cells (MoDC) was investigated. We assessed MoDC maturation and cytokine production of G.duodenalis-stimulated MoDC and the ability of these MoDC to take up antigen and induce lymphocyte proliferation. Little or no upregulation of maturation markers CD40 and CD80 was measured, but MHCII expression was increased after stimulation with low parasite concentrations. A dose-dependent decrease in ovalbumin uptake was observed in G.duodenalis-stimulated MoDC. In addition, stimulated MoDC induced proliferation of CD3(-) , γδ-T-cells and TCRαβ(+) CD4(+) and CD8(+) T-cells. Increased transcription of TGF-β was shown in CD4(+) T cells, and increased TNF-α, TGF-β, IL-10 and IL-4 were seen in γδ-T-cells. We found no evidence that G.duodenalis has a regulatory or inhibitory effect on bovine MoDC. MoDC stimulated with G.duodenalis are functionally active and able to induce proliferation of T cells that produce both pro- and anti-inflammatory cytokines.

Dynamic identification of H2 epitopes from Leishmania (Leishmania) amazonensis cysteine proteinase B with potential immune activity during murine infection

Peptides from the COOH-terminal extension of cysteine proteinase B from Leishmania (Leishmania) amazonensis (cyspep) can modulate immune responses in vertebrate hosts. With this hypothesis as base, we used the online analysis tool SYFPEITHI to predict seven epitopes from this region with potential to bind H2 proteins. We performed proliferation tests and quantified reactive T lymphocytes applying a cytometry analysis, using samples from draining lymph node of lesions from L. (L.) amazonensis-infected mice. To define reactivity of T cells, we used complexes of DimerX (H2 D(b):Ig and H2 L(d):Ig) and the putative epitopes. Additionally, we applied surface plasmon resonance to verify real time interactions between the putative epitopes and DimerX proteins. Five peptides induced blastogenesis in BALB/c cells, while only two presented the same property in C57BL/6 mouse cells. In addition, our data indicate the existence of CD8+ T lymphocyte populations able to recognize each tested peptide in both murine strains. We observed an overlapping of results between the peptides that induced lymphocyte proliferation and those capable of binding to the DimerX in the surface plasmon resonance assays thus indicating that using these recombinant proteins in biosensing analyses is a promising tool to study real time molecular interactions in the context of major histocompatibility complex epitopes. The data gathered in this study reinforce the hypothesis that cyspep-derived peptides are important factors in the murine host infection by L. (L.) amazonensis.