Transcriptional profiling of the LPS induced NF-kappaB response in macrophages

BACKGROUND: Exposure of macrophages to bacterial products such as lipopolysaccharide (LPS) results in activation of the NF-kappaB transcription factor, which orchestrates a gene expression programme that underpins the macrophage-dependent immune response. These changes include the induction or repression of a wide range of genes that regulate inflammation, cell proliferation, migration and cell survival. This process is tightly regulated and loss of control is associated with conditions such as septic shock, inflammatory diseases and cancer. To study this response, it is important to have in vitro model systems that reflect the behaviour of cells in vivo. In addition, it is necessary to understand the natural differences that can occur between individuals. In this report, we have investigated and compared the LPS response in macrophage derived cell lines and peripheral blood mononuclear cell (PBMC) derived macrophages. RESULTS: Gene expression profiles were determined following LPS treatment of THP-1 cells for 1 and 4 hours. LPS significantly induced or repressed 72 out of 465 genes selected as being known or putative NF-kappaB target genes, which exhibited 4 temporal patterns of expression. Results for 34 of these genes, including several genes not previously identified as LPS target genes, were validated using real time PCR. A high correlation between microarray and real time PCR data was found. Significantly, the LPS induced expression profile of THP-1 cells, as determined using real time PCR, was found to be very similar to that of human PBMC derived macrophages. Interestingly, some differences were observed in the LPS response between the two donor PBMC macrophage populations. Surprisingly, we found that the LPS response in U937 cells was dramatically different to both THP-1 and PBMC derived macrophages. CONCLUSION: This study revealed a dynamic and diverse transcriptional response to LPS in macrophages, involving both the induction and repression of gene expression in a time dependent manner. Moreover, we demonstrated that the LPS induced transcriptional response in the THP-1 cell line is very similar to primary PBMC derived macrophages. Therefore, THP-1 cells represent a good model system for studying the mechanisms of LPS and NF-kappaB dependent gene expression.

Lack of effect of foods enriched with plant- or marine-derived n-3 fatty acids on human immune function

Background: Greatly increasing dietary flaxseed oil [rich in the n-3 polyunsaturated fatty acid (PUFA) alpha-linolenic acid (ALA)] or fish oil [rich in the long-chain n-3 PUFAs eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids] can reduce markers of immune cell function. The effects of more modest doses are unclear, and it is not known whether ALA has the same effects as its long-chain derivatives. Objective: The objective was to determine the effects of enriching the diet with ALA or EPA+DHA on immune outcomes representing key functions of human neutrophils, monocytes, and lymphocytes. Design: In a placebo-controlled, double-blind, parallel study, 150 healthy men and women aged 25-72 y were randomly assigned to I of 5 interventions: placebo (no additional n-3 PUFAs), 4.5 or 9.5 g ALA/d, and 0.77 or 1.7 g EPA+DHA/d for 6 mo. The n-3 PUFAs were provided in 25 g fat spread plus 3 oil capsules. Blood samples were taken at 0, 3, and 6 mo. Results: The fatty acid composition of peripheral blood mononuclear cell phospholipids was significantly different in the groups with higher intakes of ALA or EPA+DHA. The interventions did not alter the percentages of neutrophils or monocytes engaged in phagocytosis of Escherichia coli or in phagocytic activity, the percentages of neutrophils or monocytes undergoing oxidative burst in response to E. coli or phorbol ester, the proliferation of lymphocytes in response to a T cell mitogen, the production of numerous cytokines by monocytes and lymphocytes, or the in vivo delayed-type hypersensitivity response. Conclusion: An intake of f less than or equal to9.5 g ALA/d or less than or equal to1.7 g EPA+DHA/d does not alter the functional activity of neutrophils, monocytes, or lymphocytes, but it changes the fatty acid composition of mononuclear cells.

Incorporation of cis-9,trans-11 or trans-10,cis-12 conjugated linoleic acid into plasma and cellular lipids in healthy men

This study investigated the incorporation of cis-9,trans-11 conjugated linoleic acid (c9,t11 CLA) and trans-10,cis-12-CLA (t10,c12 CLA) into plasma and peripheral blood mononuclear cell (PBMC) lipids when consumed as supplements highly enriched in these isomers. Healthy men (n = 49, age 31 +/- 8 years) consumed one, two, and four capsules containing similar to600 mg of either c9,t11 CIA or t10,c12 CLA per capsule for sequential 8 week periods followed by a 6 week washout before consuming the alternative isomer. Both isomers were incorporated in a dosedependent manner into plasma phosphatidylcholine (PC) (c9,t11 CLA r = 0.779, t10,c12 CLA r = 0.738; P < 0.0001) and cholesteryl ester (CE) (c9,t11 CLA r = 0.706, t10,c12 CLA r = 0.788; P < 0.0001). Only t10,c12 CLA was enriched in plasma nonesterified fatty acids. Both c9,t11 CIA and t10,c12 CLA were incorporated linearly into PBMC total lipids (r = 0.285 and r = 0.273, respectively; P < 0.0005). The highest concentrations of c9,t11 CLA and t10,c12 CLA in PBMC lipids were 3- to 4-fold lower than those in plasma PC and CE. These data suggest that the level of intake is a major determinant of plasma and PBMC CLA content, although PBMCs appear to incorporate both CLA isomers less readily.

In vitro immunomodulatory activity of Lactobacillus fermentum CECT5716 and Lactobacillus salivarius CECT5713: two probiotic strains isolated from human breast milk

Commensal bacteria, including some species of lactobacilli commonly present in human breast milk, appear to colonize the neonatal gut and contribute to protection against infant infections, suggesting that lactobacilli could potentially modulate immunity. In this study, we evaluated the potential of two Lactobacillus strains isolated from human milk to modulate the activation and cytokine profile of peripheral blood mononuclear cell (PBMC) subsets in vitro. Moreover, these effects were compared to the same probiotic species of non-milk origin. Lactobacillus salivarius CECT5713 and Lactobacillus fermentum CECT5716 at 105, 106 and 107 bacteria/mL were co-cultured with PBMC (106/mL) from 8 healthy donors for 24 h. Activation status (CD69 and CD25 expressions) of natural killer (NK) cells (CD56+), total T cells (CD3+), cytotoxic T cells (CD8+) and CD4+ T cells was determined by flow cytometry. Regulatory T cells (Treg) were also quantified by intracellular Foxp3 evaluation. Regarding innate immunity, NK cells were activated by addition of both Lactobacillus strains, and in particular, the CD8+ NK subset was preferentially induced to highly express CD69 (90%, p<0.05). With respect to acquired immunity, approximately 9% of CD8+ T cells became activated after co-cultivation with L. fermentum or L salivarius. Although CD4+ T cells demonstrated a weaker response, there was a preferential activation of Treg cells (CD4+CD25+Foxp3+) after exposure to both milk probiotic bacteria (p<0.05). Both strains significantly induced the production of a number of cytokines and chemokines, including TNFα, IL-1β, IL-8, MIP-1α, MIP-1β, and GM-CSF, but some strain-specific effects were apparent. This work demonstrates that L salivarius CECT5713 and L. fermentum CECT5716 enhanced both natural and acquired immune responses, as evidenced by the activation of NK and T cell subsets and the expansion of Treg cells, as well as the induction of a broad array of cytokines.

Comparative effects of six probiotic strains on immune function in vitro

There is considerable interest in the strain specificity of immune modulation by probiotics. The present study compared the immunomodulatory properties of six probiotic strains of different species and two genera in a human peripheral blood mononuclear cell (PBMC) model in vitro. Live cells of lactobacilli (Lactobacillus casei Shirota, L. rhamnosus GG, L. plantarum NCIMB 8826 and L. reuteri NCIMB 11951) and bifidobacteria (Bifidobacterium longum SP 07/3 and B. bifidum MF 20/5) were individually incubated with PBMC from seven healthy subjects for 24 h. Probiotic strains increased the proportion of CD69+ on lymphocytes, T cells, T cell subsets and natural killer (NK) cells, and increased the proportion of CD25+, mainly on lymphocytes and NK cells. The effects on activation marker expression did not appear to be strain specific. NK cell activity was significantly increased by all six strains, without any significant difference between strains. Probiotic strains increased production of IL-1β, IL-6, IL-10, TNF-α, granulocyte-macrophage colony-stimulating factor and macrophage inflammatory protein 1α to different extents, but had no effect on the production of IL-2, IL-4, IL-5 or TNF-β. The cytokines that showed strain-specific modulation included IL-10, interferon-γ, TNF-α, IL-12p70, IL-6 and monocyte chemotactic protein-1. The Lactobacillus strains tended to promote T helper 1 cytokines, whereas bifidobacterial strains tended to produce a more anti-inflammatory profile. The results suggest that there was limited evidence of strain-specific effects of probiotics with respect to T cell and NK cell activation or NK cell activity, whereas production of some cytokines was differentially influenced by probiotic strains.

Oxidation of melatonin and its catabolites, N-1-acetyl-N (2)-formyl-5-methoxykynuramine and N-1-acetyl-5-methoxykynuramine, by activated leukocytes

N-1-acetyl-N-2-formyl-5-methoxykynuramine (AFMK) and N-1-acetyl-5-methoxykynuramine (AMK), two melatonin catabolites, have been described as potent antioxidants. We aimed to follow the kinetics of AFMK and AMK formation when melatonin is oxidized by phorbol myristate acetate (PMA) and lipopolysaccharide (LPS)-activated leukocytes. An HPLC-based method was used for AFMK and AMK determination in neutrophil and peripheral blood mononuclear cell cultures supernatants. Samples were separated isocratically on a C18 reverse-phase column using acetonitrile/H2O (25:75) as the mobile phase. AFMK was detected by fluorescence (excitation 340 nm and emission 460 nm) and AMK by UV-VIS absorbance (254 nm). Activation of neutrophils and mononuclear cells with PMA produces larger amounts of AFMK than activation with LPS, probably due to the lower levels of reactive oxygen species formation and myeloperoxidase (MPO) degranulation that occurs when cells are stimulated with LPS. The concentration of AMK found in the supernatant was about 5-10% (from 18-hr cultures) compared with AFMK. This result may reflect its reactivity. Indeed AMK, but not AFMK, is easily oxidized by activated neutrophils in a MPO and hydrogen peroxide-dependent reaction. In conclusion, we defined a simple procedure for the determination of AFMK and AMK in biological samples and demonstrated the capacity of leukocytes to oxidize melatonin and AMK.

Cytokines and acute-phase serum proteins as markers of inflammatory regression during pulmonary tuberculosis treatment

Tuberculosis is still increasing and was declared a worldwide sanitary emergency by the World Health Organization (WHO) in 1995. Its control is difficult due to long treatment duration and lack of markers of treatment success or failure. Cytokines such as IFN-gamma and TNF-alpha, a central factor in immune response against Mycobacterium tuberculosis, are responsible for the interaction between T lymphocytes and the infected macrophage and are also produced during this interaction. As proinflammatory cytokines have a close relationship with mycobacteria clearance, in fact even preceding it, they could be used as markers for inflammatory activity and response to treatment. Proinflammatory cytokines act in the liver and stimulate a strong local and systemic acute-phase response as a result of homeostatic and physiological responses also induced by them. Acute-phase proteins produced by cytokine activity are useful diagnostic markers that could also be used to monitor treatment response as they can be serially quantified. The objective of this study was to evaluate IFN-gamma, TNF-alpha, IL-10 and TGF-beta production in supernatant of peripheral blood mononuclear cell (PBMC) and monocyte (MO) cultures, as well as serum acute-phase response through total protein, albumin, globulin, C-reactive protein (CRP), alpha-1-acid glycoprotein (AGP), and erythrocyte sedimentation rate (ESR) as regression markers of inflammatory response during pulmonary tuberculosis treatment. Twenty blood donors (G1) from the Blood Bank at Botucatu School of Medicine's University Hospital (BSM-UH) were evaluated once and 28 pulmonary tuberculosis patients (G2): 13 from BSM-UH and 15 from the Bauru State Health Secretariat. Patients were evaluated at three moments of treatment: before (M1), at three months (M2), and at the end (M3). Cytokines were determined in 20ml of peripheral blood (ELISA), with or without activation: lipopolysaccharide (LPS) for MO culture and phytohemagglutinin (PHA) for PBMC culture. Acute-phase protein behavior in G2 throughout treatment was: Globulins: M1> M2, M1> M3 (rho < 0.001); CRP: M1> M2> M3 (.< 0.001); AGP for men: M1> M2, M1> M3 (rho < 0.001); ESR for men: M1> M2, M1> M3 (rho < 0.0016) and for women: M1> M2 (.< 0.025). Comparison between cytokine levels found in supernatant of MO and PBMC cultures, with and without stimulus, in G1 and G2 during treatment showed: TNF-alpha (with/ without LPS) at M1: G2> G1; at M2: G2> G1 (rho < 0.001); (without LPS) at M3: G2> G1 (rho < 0.001), (with LPS) at M3: G2> G1 (rho < 0.028); IFN-. (with and without PHA) at M1: G2> G1; at M2: G2> G1 (rho < 0.001); IL-10 (with and without LPS) at M1: G2> G1; at M2: G2> G1; at M3: G2> G1 (rho < 0.001); TGF-beta (with and without LPS) at M1: G2> G1; at M2: G2> G1 (rho < 0.001), (without LPS) at M3: G2> G1 (rho < 0.001). In G2, all cytokines in supernatant of MO and PBMC cultures, with and without stimulus, showed: M1> M2> M3 (rho < 0.01). Levels of globulins, CRP, AGP, and ESR in patients with pulmonary tuberculosis before treatment (M1) were significantly higher than reference values, suggesting their use as diagnostic markers and indicators of treatment. The CRP decreasing values along treatment could be taken as a marker of the regression of inflammatory process and of response to treatment in patients with pulmonary tuberculosis.Regarding cytokines, there was significant increase in TNF-alpha, IFN-gamma, IL-10, and TGF-alpha levels before and at three months treatment, with and without stimulus; in TNF-a and IL-10 lvels, with and without stimulus, as well as in TGF-alpha levels without stimulus at six months. Patients had higher levels of all studied cytokines than controls before treatment, and these values decreased along treatment. In this study, pulmonary tuberculosis patients showed a Th0 cytokine profile before treatment, with the production of both Th1 (IFN-gamma) and Th2 (IL-10) cytokines, in addition to TNF-alpha inflammatory and TGF-alpha regulatory and fibrosis-inducer cytokines. At the end of treatment, all had evolved to Th2 profile, probably in an attempt to reduce the harmful effects of the proinflammatory activity of the Th1 cytokine profile and of the still above-normal levels of TNF-alpha. The high levels of TGF-alpha, also found in these patients, are related to its important role in the extracellular matrix deposition and fibrosis induction that characterize tuberculosis healing process. IFN-gamma was the only cytokine reaching normal levels at the end of treatment, which suggests its use as a marker of response to treatment.

Citocinas e proteínas de fase aguda do soro como marcadores de regressão da resposta inflamatória ao tratamento da tuberculose pulmonar

OBJETIVO: Analisar o padrão de citocinas pró- e antiinflamatórias e da resposta de fase aguda (RFA) como marcadores de resposta ao tratamento da tuberculose pulmonar. MÉTODOS: Determinação dos níveis de interferon-gama (IFN-γ), tumor necrosis factor-alpha (TNF-α, fator de necrose tumoral-alfa), interleucina-10 (IL-10) e transforming growth factor-beta (TGF-β, fator transformador de crescimento-beta), pelo método ELISA, em sobrenadante de cultura de células mononucleares do sangue periférico e monócitos, assim como dos níveis de proteínas totais, albumina, globulinas, alfa-1-glicoproteína ácida (AGA), proteína C reativa (PCR) e velocidade de hemossedimentação (VHS) em 28 doentes com tuberculose pulmonar, em três tempos: antes (T0), aos três meses (T3) e aos seis meses (T6) de tratamento, em relação aos controles saudáveis, em um único tempo. RESULTADOS: Os pacientes apresentaram valores maiores de citocinas e RFA que os controles em T0, com diminuição em T3 e diminuição (TNF-α, IL-10, TGF-β, AGA e VHS) ou normalização (IFN-γ e PCR) em T6. CONCLUSÕES: PCR, AGA e VHS são possíveis marcadores para auxiliar no diagnóstico de tuberculose pulmonar e na indicação de tratamento de indivíduos com baciloscopia negativa; PCR (T0 > T3 > T6 = referência) pode também ser marcador de resposta ao tratamento. Antes do tratamento, o perfil Th0 (IFN-γ, IL-10, TNF-α e TGF-β), indutor de e protetor contra inflamação, prevaleceu nos pacientes; em T6, prevaleceu o perfil Th2 (IL-10, TNF-α e TGF-β), protetor contra efeito nocivo pró-inflamatório do TNF-α ainda presente. O comportamento do IFN-γ (T0 > T3 > T6 = controle) sugere sua utilização como marcador de resposta ao tratamento.

Effect of age on the gastrointestinal-associated mucosal immune response of humans

Age-related changes in gastrointestinal-associated mucosal immune response have not been well studied. Thus, we investigated the effect of age on this response and compared these responses to those of peripheral immune cells. Saliva, blood, and intestinal biopsies were collected from young and old healthy subjects to determine immunoglobulin (Ig) levels and to isolate peripheral blood mononuclear cells, intraepithelial lymphocytes (IELs), and lamina propria lymphocytes (LPLs). Although subject age did not influence the level of total IgA found in saliva, IgA levels in serum increased (p < .05) with age. Older subjects' peripheral blood mononuclear cell proliferation and IL-2 production were significantly lower than those of young subjects. LPLs from older subjects produced significantly less IL-2 in response to all stimuli than did that from the young. IEL's ability to proliferate and produce IL-2 was not affected by subject age. Thus, LPL but not IEL demonstrated an age-related decline in immune function similar to that seen in peripheral lymphocytes.

IL-12 and neutralization of endogenous IL-10 revert the in vitro antigen-specific cellular immunosuppression of paracoccidioidomycosis patients

Treatment of patients with paracoccidioidomycosis is still a challenge. Patients present defective lymphoproliferation and IFN-γ responses to the main Paracoccidioides brasiliensis antigen (gp43), which correlates with disease severity. Here, we demonstrated that the patients show also a defective synthesis of interleukin (IL)-12. Therefore, we attempted to revert this immune disfunction by adding IL-12 and neutralizing anti-IL-10 antibody to gp-43-stimulated peripheral blood mononuclear cell cultures. Both treatments increased IFN-γ secretion to levels observed with healthy sensitized individuals, but affected proliferation only modestly. When combined, the treatments further increased IFN-γ synthesis and cell proliferation. The addition of suboptimal concentrations of IL-2 also further increased the IL-12-mediated secretion of IFN-γ. Interestingly, the immune modulation was mostly antigen-specific, since the responses to Candida albicans' antigen were not affected. These results suggest that appropriate immune intervention with cytokines and/or anti-cytokines may help in the treatment of PCM. © 2002 Elsevier Science Ltd. All rights reserved.

Proinflammatory profile of in vitro monocytes in the ageing is affected by lymphocytes presence

Background: Aging is associated with complex and constant remodeling of the immune function, resulting in an increasing susceptibility to infection and others diseases. The infections caused by Gram-negative microorganisms, present in nursing homes and hospitals, constitute one of the most common infections in the elderly, and are mainly combated by innate immune cells. Although the functions of innate immunity seem more preserved during aging than of adaptive immune mechanisms, two systems operate in an integrated way in the body, so that injury in one part of the immune system inevitably affects the other as they are part of a defensive network. The aim of this study was to investigate the in vitro production of proinflammatory (TNF-α, IL-6, IL-1β, CXCL-8 and MCP-1) and anti-inflammatory (TGF-β and IL-10) cytokines by monocytes, stimulated or not (basal) with lipopolysaccharide, from healthy young and elderly subjects. By means of PBMCs, we also studied if cytokine profile is altered in these different patient groups, in the presence of lymphocytes, under the same experimental conditions.Results: The monocytes from elderly presented higher basal production of TNF-α, MCP-1 and lower of TGF-β than young monocytes. PBMC showed similar cytokines production, irrespective age or stimulation presence. In the presence of lymphocytes, the spontaneous production of IL-10 was higher and of TGF-β was lower than monocytes, regardless of age. After LPS-stimulation, the presence of lymphocytes resulted in increased IL-6, IL-1β, MCP-1 and IL-10 and decreased CXCL-8 and TGF-β in comparison to pure culture of monocytes from young patients. With age, the same differences were observed, except for CXCL-8 and TGF-β which production was the same between monocytes and PBMC stimulated with LPS.Conclusion: These findings reinforce the systemic state of inflamm-aging frequently reported in elderly and considered a factor of susceptibility to numerous diseases. Still, the cytokine production from just monocytes of the elderly showed alterations, while in the lymphocyte presence not, suggesting an immunomodulator role of lymphocytes on monocytes. In addition, the differences between the production patterns by LPS-stimulated PBMC between young and elderly volunteers can be related with an imbalance in response against Gram-negative bacteria in throughout life. © 2013 Pinke et al.; licensee BioMed Central Ltd.

Improvement in clinical signs and cellular immunity of dogs with visceral leishmaniasis using the immunomodulator P-MAPA

This study investigated the immunotherapeutic potential of the protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride immuno-modulator (P-MAPA) on canine visceral leishmaniasis. Twenty mongrel dogs presenting clinical symptoms compatible with leishmaniasis and diagnosis confirmed by the detection of anti-leishmania antibodies were studied. Ten dogs received 15 doses of the immunomodulator (2.0mg/kg) intramuscularly, and 10 received saline as a placebo. Skin and peripheral blood samples were collected following administration of the immunomodulator. The groups were followed to observe for clinical signals of remission; parasite load in the skin biopsies using real-time PCR, the cytokines IL-2, IL-10 and IFN-γ in the supernatant of peripheral blood mononuclear cells stimulated in vitro with either total promastigote antigen or phytohemagglutinin measured by capture ELISA, and changes in CD4+ and CD8+ T cell subpopulations evaluated by flow cytometry. Comparison between the groups showed that treatment with the immunomodulator promoted improvement in clinical signs and a significant reduction in parasite load in the skin. In peripheral blood mononuclear cell cultures, supernatants showed a decrease in IL-10 levels and an increase in IL-2 and IFN-γ. An increase in CD8+ T cells was observed in peripheral blood. In addition, the in vitro leishmanicidal action of P-MAPA was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and no leishmanicidal activity was detected. These findings suggest that P-MAPA has potential as an immunotherapeutic drug in canine visceral leishmaniasis, since it assists in reestablishing partial immunocompetence of infected dogs. © 2013 Elsevier B.V.

Leukotrienes Are Upregulated and Associated with Human T-Lymphotropic Virus Type 1 (HTLV-1)-Associated Neuroinflammatory Disease

Leukotrienes (LTs) are lipid mediators involved in several inflammatory disorders. We investigated the LT pathway in human T-lymphotropic virus type 1 (HTLV-1) infection by evaluating LT levels in HTLV-1-infected patients classified according to the clinical status as asymptomatic carriers (HACs) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) patients. Bioactive LTB4 and CysLTs were both increased in the plasma and in the supernatant of peripheral blood mononuclear cell cultures of HTLV-1-infected when compared to non-infected. Interestingly, CysLT concentrations were increased in HAM/TSP patients. Also, the concentration of plasma LTB4 and LTC4 positively correlated with the HTLV-1 proviral load in HTLV-1-infected individuals. The gene expression levels of LT receptors were differentially modulated in CD4(+) and CD8(+) T cells of HTLV-1-infected patients. Analysis of the overall plasma signature of immune mediators demonstrated that LT and chemokine amounts were elevated during HTLV-1 infection. Importantly, in addition to CysLTs, IP-10 was also identified as a biomarker for HAM/TSP activity. These data suggest that LTs are likely to be associated with HTLV-1 infection and HAM/TSP development, suggesting their putative use for clinical monitoring.

HIV-1 Proviral DNA Loads (as Determined by Quantitative PCR) in Patients Subjected to Structured Treatment Interruption after Antiretroviral Therapy Failure

The impact of Structured Treatment Interruption (STI) in peripheral blood mononuclear cell (PBMC) proviral reservoirs in 41 highly active antiretroviral therapy (HAART)-treated viremic individuals at baseline and 12 weeks after STI was determined using quantitative PCR (qPCR). Viral load increased 0.7 log(10) and CD4 decreased 97.5 cells/mm(3) after 12 weeks. A total of 28 of the 41 individuals showed an increased proviral load, 19 with a statistically significant increase above 10%. An increase in active viral replication is an important factor in the replenishment of the proviral reservoir even for short time periods.

Untersuchungen zur selektiven Deletion alloreaktiver Spender-T- Lymphozyten durch CD178-modifizierte dendritische Zellen im Rahmen der allogenen Blutstammzelltransplantation

Eine der häufigsten Komplikationen bei der allogenen Blutstammzelltransplantation stellt die Transplantat-gegen-Wirt-Erkrankung (Graft versus Host Disease, GvHD) dar. Sie wird durch allogene Spender-T-Lymphozyten verursacht, die Gewebe des Transplantatempfängers erkennen und inflammatorische Entzündungsprozesse auslösen. Neben dieser Alloreaktivität induzieren Spender-T-Lymphozyten jedoch auch immuntherapeutisch erwünschte Transplantat-gegen-Leukämie-Reaktionen (Graft versus Leukemia, GvL-Reaktion), bei denen residuelle Tumor- bzw. Leukämiezellen im Patienten durch Spender-T-Zellen spezifisch erkannt und eliminiert werden. Im Rahmen einer verbesserten Immmuntherapie wird daher versucht, GvHD-reaktive und GvL-reaktive Spender-T-Lymphozyten effizient voneinander zu separieren und so eine wirkungsvolle GvHD-Prophylaxe bzw. optimierte GvL-Induktion zu erreichen. In diesem Kontext war es Ziel dieser Arbeit, murine dendritische Zellen (DZ) so zu modifizieren, daß sie für die spezifische Deletion alloreaktiver T-Zellen in murinen GvHD/GvL-Tiermodellen eingesetzt werden können. Die Modifikation der DZ sollte dazu führen, daß über das CD95/CD178-System Aktivierungs-induzierter Zelltod (activation induced cell death, AICD) in alloreaktiven T-Zellen ausgelöst wird. Hierzu wurden für die Modifikation der DZ zwei verschiedene Mechanismen angewandt: a) die Transfektion der DZ mit CD178-mRNA sowie b) die zielgerichtete Immobilisierung von hCD178-X-Fusionsproteinen auf Oberflächenmolekülen von DZ bzw. T-Zellen. Als Positivkontrolle für die Induktion CD95-vermittelter Apoptose diente der agonistische anti-CD95-Antikörper Jo2. Bei der Transfektion muriner DZ mit mRNA zeigte sich anhand des Reportergens EGFP, daß aus dem Knochenmark generierte DZ mit hoher Effizienz mit EGFP-mRNA transfizierbar waren. Im Falle von hCD178-mRNA führte die Transfektion jedoch zu einer insuffizienten CD178-Expression, die mit den regulatorischen Eigenschaften der zytoplasmatischen CD178-Region in Verbindung gebracht werden konnte. So führte die Verwendung einer zytoplasmatisch trunkierten Form der CD178-mRNA (CD178Dzyt) zu einer durchflußzytometrisch nachweisbaren CD178-Expression in DZ. Mit diesen CD178Dzyt-exprimierenden DZ konnte in einem Proliferationstest die Proliferation alloreaktiver T-Zellen inhibiert werden. Die Beladung von DZ bzw. von T-Zellen mit hCD178-X-Fusionsproteinen führte in vitro ebenfalls zu einer deutlichen Reduktion von Alloreaktivität. Dabei konnte eine spezifische Deletion/Inhibition alloreaktiver T-Zellen nachgewiesen werden. Die Elimination alloreaktiver T-Zellen erfolgte in beiden Verfahren über AICD. Darüber hinaus wurde eine Bifunktionalität der Fusionsproteine festgestellt, da sie neben der Induktion CD95-vermittelter Apoptose auch in der Lage waren, die Kostimulation allogener T-Zellen effizient zu inhibieren. Mit Hilfe adoptiver T-Zell-Transferexperimente konnten abschließend die in vitro gewonnenen Ergebnisse in vivo in zwei verschiedenen GvHD-Mausmodellen bestätigt werden.