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The replication of viruses in secondary lymphoid organs guarantees sufficient amounts of pattern-recognition receptor ligands and antigens to activate the innate and adaptive immune system. Viruses with broad cell tropism usually replicate in lymphoid organs; however, whether a virus with a narrow tropism relies on replication in the secondary lymphoid organs to activate the immune system remains not well studied. In this study, we used the artificial intravenous route of infection to determine whether Influenza A virus (IAV) replication can occur in secondary lymphatic organs (SLO) and whether such replication correlates with innate immune activation. Indeed, we found that IAV replicates in secondary lymphatic tissue. IAV replication was dependent on the expression of Sialic acid residues in antigen-presenting cells and on the expression of the interferon-inhibitor UBP43 ( Usp18 ). The replication of IAV correlated with innate immune activation, resulting in IAV eradication. The genetic deletion of Usp18 curbed IAV replication and limited innate immune activation. In conclusion, we found that IAV replicates in SLO, a mechanism which allows innate immune activation.

Immune evasion of pathogens can modify the course of infection and impact viral persistence and pathology. Here, using different strains of the lymphocytic choriomeningitis virus (LCMV) model system, we show that slower propagation results in limited type I interferon (IFN-I) production and viral persistence. Specifically, cells infected with LCMV-Docile exhibited reduced viral replication when compared to LCMV-WE and as a consequence, infection with LCMV-Docile resulted in reduced activation of bone marrow derived dendritic cells (BMDCs) and IFN-I production in vitro in comparison with LCMV-WE. In vivo, we observed a reduction of IFN-I, T cell exhaustion and viral persistence following infection of LCMV-Docile but not LCMV-WE. Mechanistically, block of intracellular protein transport uncovered reduced propagation of LCMV-Docile when compared to LCMV-WE. This reduced propagation was critical in blunting the activation of the innate and adaptive immune system. When mice were simultaneously infected with LCMV-Docile and LCMV-WE, immune function was restored and IFN-I production, T cell effector functions as well as viral loads were similar to that of mice infected with LCMV-WE alone. Taken together, this study suggests that reduced viral propagation can result in immune evasion and viral persistence.

Early and strong production of IFN-I by dendritic cells is important to control vesicular stomatitis virus (VSV), however mechanisms which explain this cell-type specific innate immune activation remain to be defined. Here, using a genome wide association study (GWAS), we identified Integrin alpha-E ( Itgae , CD103) as a new regulator of antiviral IFN-I production in a mouse model of vesicular stomatitis virus (VSV) infection. CD103 was specifically expressed by splenic conventional dendritic cells (cDCs) and limited IFN-I production in these cells during VSV infection. Mechanistically, CD103 suppressed AKT phosphorylation and mTOR activation in DCs. Deficiency in CD103 accelerated early IFN-I in cDCs and prevented death in VSV infected animals. In conclusion, CD103 participates in regulation of cDC specific IFN-I induction and thereby influences immune activation after VSV infection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Duhan, Khairnar, Kitanovski, Hamdan, Klein, Lang, Ali, Adomati, Bhat, Friedrich, Li, Krebs, Futerman, Addo, Hardt, Hoffmann, Lang and Lang.)

Ebola virus epidemics can be effectively limited by the VSV-EBOV vaccine (Ervebo) due to its rapid protection abilities; however, side effects prevent the broad use of VSV-EBOV as vaccine. Mechanisms explaining the efficient immune activation after single injection with the VSV-EBOV vaccine remain mainly unknown. Here, using the clinically available VSV-EBOV vaccine (Ervebo), we show that the cell-intrinsic expression of the interferon-inhibitor Usp18 in CD169 + macrophages is one important factor modulating the anti-Ebola virus immune response. The absence of Usp18 in CD169 + macrophages led to the reduced local replication of VSV-EBOV followed by a diminished innate as well as adaptive immune response. In line, CD169 -Cre +/ki x Usp18 fl/fl mice showed reduced innate and adaptive immune responses against the VSV wildtype strain and died quickly after infection, suggesting that a lack of Usp18 makes mice more susceptible to the side effects of the VSV vector. In conclusion, our study shows that Usp18 expression in CD169 + macrophages is one important surrogate marker for effective vaccination against VSV-EBOV, and probably other VSV-based vaccines also.

Infections can result in a temporarily restricted unresponsiveness of the innate immune response, thereby limiting pathogen control. Mechanisms of such unresponsiveness are well studied in lipopolysaccharide tolerance; however, whether mechanisms of tolerance limit innate immunity during virus infection remains unknown. Here, we find that infection with the highly cytopathic vesicular stomatitis virus (VSV) leads to innate anergy for several days. Innate anergy is associated with induction of apoptotic cells, which activates the Tyro3, Axl, and Mertk (TAM) receptor Mertk and induces high levels of interleukin-10 (IL-10) and transforming growth factor β (TGF-β). Lack of Mertk in Mertk -/- mice prevents induction of IL-10 and TGF-β, resulting in abrogation of innate anergy. Innate anergy is associated with enhanced VSV replication and poor survival after infection. Mechanistically, Mertk signaling upregulates suppressor of cytokine signaling 1 (SOCS1) and SOCS3. Dexamethasone treatment upregulates Mertk and enhances innate anergy in a Mertk-dependent manner. In conclusion, we identify Mertk as one major regulator of innate tolerance during infection with VSV., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

Macrophages have important protective functions during infection with herpes simplex virus type 1 (HSV-1). However, molecular mechanisms that restrict viral propagation and protect from severe disease are unclear. Here we show that macrophages take up HSV-1 via endocytosis and transport the virions into multivesicular bodies (MVBs). In MVBs, acid ceramidase (aCDase) converts ceramide into sphingosine and increases the formation of sphingosine-rich intraluminal vesicles (ILVs). Once HSV-1 particles reach MVBs, sphingosine-rich ILVs bind to HSV-1 particles, which restricts fusion with the limiting endosomal membrane and prevents cellular infection. Lack of aCDase in macrophage cultures or in Asah1 -/- mice results in replication of HSV-1 and Asah1 -/- mice die soon after systemic or intravaginal inoculation. The treatment of macrophages with sphingosine enhancing compounds blocks HSV-1 propagation, suggesting a therapeutic potential of this pathway. In conclusion, aCDase loads ILVs with sphingosine, which prevents HSV-1 capsids from penetrating into the cytosol.

The replication of virus in secondary lymphoid organs is crucial for the activation of antigen-presenting cells. Balanced viral replication ensures the sufficient availability of antigens and production of cytokines, and both of which are needed for virus-specific immune activation and viral elimination. Host factors that regulate coordinated viral replication are not fully understood. In the study reported here, we identified Map3k14 as an important regulator of enforced viral replication in the spleen while performing genome-wide association studies of various inbred mouse lines in a model of lymphocytic choriomeningitis virus (LCMV) infection. When alymphoplasia mice ( aly/aly, Map3k14 aly/aly , or Nik aly/aly ) , which carry a mutation in Map3k14, were infected with LCMV or vesicular stomatitis virus (VSV), they display early reductions in early viral replication in the spleen, reduced innate and adaptive immune activation, and lack of viral control. Histologically, scant B cells and the lack of CD169 + macrophages correlated with reduced immune activation in Map3k14 aly/aly mice. The transfer of wildtype B cells into Map3k14 aly/aly mice repopulated CD169 + macrophages, restored enforced viral replication, and resulted in enhanced immune activation and faster viral control.

Background: Dysregulation of the B-cell activating factor (BAFF) system is involved in the pathogenesis of systemic lupus erythematosus (SLE). Increased serum concentrations of BAFF are related to lupus nephritis and disease activity among SLE patients. Recently, a variant of the BAFF-encoding gene, BAFF-var, was identified to be associated with autoimmune diseases, in particular SLE, and to promote the production of soluble BAFF. The present study aimed to assess the prevalence of BAFF-var in a cohort of 195 SLE patients and to analyze the association of the BAFF-var genotype (TNSF13B) with various manifestations of SLE., Methods: A cohort of 195 SLE patients from Central Europe, including 153 patients from the Swiss SLE Cohort Study and 42 patients from the University Hospital Essen, Germany, underwent genotyping for detection of BAFF-var allele., Results: Of the 195 patients, 18 (9.2%) tested positive for BAFF-var variant according to the minor allele frequency of 4.6%. The presence of BAFF-var was associated with the occurrence of lupus nephritis (p = 0.038) (p = 0.03 and p = 0.003). Among various organ manifestations of SLE, the presence of BAFF-var was associated with the occurrence of lupus nephritis (p = 0.038; odds ratio [OR], 2.4; 95% confidence interval [CI], 0.89-6.34) and renal activity markers such as proteinuria and hematuria (p = 0.03; OR, 2.4; 95% CI, 0.9-6.4 for proteinuria; p = 0.003; OR, 3.9; 95% CI, 1.43-10.76 for hematuria). SLE patients carrying the BAFF-var allele exhibited increased disease activity at study entry, as determined by the physician's global assessment (PGA: p = 0.002; OR, 4.8; 95% CI, 1.54-14.93) and the SLE Disease Activity Index (p = 0.012; OR, 3.5; 95% CI, 1.12-11.18). Consistent with that, the percentage of patients treated with immunosuppressive agents at study entry was higher among those carrying the BAFF-var allele than among those tested negative for BAFF-var (p = 0.006; OR, 3.7; 95% CI, 1.27-10.84)., Conclusions: Our results indicate an association between the BAFF-var genotype and increased severity of SLE. Determining the BAFF-var status of SLE patients may improve the risk stratification of patients for whom the development of lupus nephritis is more likely and thus may be helpful in the follow-up care and treatment of SLE patients.

During viral infection, tight regulation of CD8+ T-cell functions determines the outcome of the disease. Recently, others and we determined that the natural killer (NK) cells kill hyperproliferative CD8+ T cells in the context of viral infection, but molecules that are involved in shaping the regulatory capability of NK cells remain virtually unknown. Here we used mice lacking the Fc-receptor common gamma chain (FcRγ, FcεRIγ, Fcer1g-/- mice) to determine the role of Fc-receptor and NK-receptor signaling in the process of CD8+ T-cell regulation. We found that the lack of FcRγ on NK cells limits their ability to restrain virus-specific CD8+ T cells and that the lack of FcRγ in Fcer1g-/- mice leads to enhanced CD8+ T-cell responses and rapid control of the chronic docile strain of the lymphocytic choriomeningitis virus (LCMV). Mechanistically, FcRγ stabilized the expression of NKp46 but not that of other killer cell-activating receptors on NK cells. Although FcRγ did not influence the development or activation of NK cell during LCMV infection, it specifically limited their ability to modulate CD8+ T-cell functions. In conclusion, we determined that FcRγ plays an important role in regulating CD8+ T-cell functions during chronic LCMV infection., Competing Interests: The authors have declared that no competing interests exist.

While the role of interferon during systemic disease is well known and its immune modulating functions and its role in antiviral activity were extensively studied, the role of IFN-I in the brain is less clear. Here we summarize the most important literature on IFN in homeostasis of the CNS and induction of an IFN response during viral infection in the brain. Furthermore, we present work on the roles of IFN in the developing brain as well as during inflammation in the brain. Lastly, we aim to enlighten the functions of IFN on the blood-brain barrier as well as circulation and in cognitive and psychological functions and degeneration. In short, CNS astrocytes produce IFN-β, which is of high relevance for homeostasis in the brain. IFN-β regulates phagocytic removal of myelin debris by microglia. IFN-I limits the permeability of the blood-brain barrier. Disruption of the blood-brain barrier facilitates entrance of peripheral lymphocytes and inflammation. Viral infections during vulnerable phases of embryonic development cause severe fetal pathology and debilitating impairments to human infants. The roles of IFN in these scenarios are diverse and include deficits due to overproduction of IFN during the developmental stage of the brain as seems to be the case in pseudo-TORCH2., Competing Interests: The authors declare no competing interests., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Dysfunction of CD8 + T cells can lead to the development of chronic viral infection. Identifying mechanisms responsible for such T cell dysfunction is therefore of great importance to understand how to prevent persistent viral infection. Here we show using lymphocytic choriomeningitis virus (LCMV) infection that carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is fundamental for recruiting lymphocyte-specific protein kinase (Lck) into the T cell receptor complex to form an efficient immunological synapse. CEACAM1 is essential for activation of CD8 + T cells, and the absence of CEACAM1 on virus-specific CD8 + T cells limits the antiviral CD8 + T cell response. Treatment with anti-CEACAM1 antibody stabilizes Lck in the immunological synapse, prevents CD8 + T cell exhaustion, and improves control of virus infection in vivo. Treatment of human virus-specific CD8 + T cells with anti-CEACAM1 antibody similarly enhances their proliferation. We conclude that CEACAM1 is an important regulator of virus-specific CD8 + T cell functions in mice and humans and represents a promising therapeutic target for modulating CD8 + T cells.

Peripheral tolerance is an important mechanism by which the immune system can guarantee a second line of defense against autoreactive T and B cells. One autoimmune disease that is related to a break of peripheral tolerance is diabetes mellitus type 1. Using the RIP-GP mouse model, we analyzed the role of the spleen and lymph nodes (LNs) in priming CD8 + T cells and breaking peripheral tolerance. We found that diabetes developed in splenectomized mice infected with the lymphocytic choriomeningitis virus (LCMV), a finding showing that the spleen was not necessary in generating autoimmunity. By contrast, the absence of LNs prevented the priming of LCMV-specific CD8 + T cells, and diabetes did not develop in these mice. Additionally, we found that dendritic cells are responsible for the distribution of virus in secondary lymphoid organs, when LCMV was administered intravenously. Preventing this distribution with the sphingosine-1-phosphate receptor antagonist FTY720 inhibits the transport of antigen to peripheral LNs and consequently prevented the onset of diabetes. However, in case of subcutaneous infection, administration of FTY720 could not inhibit the onset of diabetes because the viral antigen is already presented in the peripheral LNs. These findings demonstrate the importance of preventing the presence of antigen in LNs for maintaining tolerance.

Background: Bile acids (BAs) are steroid molecules that are synthesized in the liver. In addition to their important role as a surfactant in solubilizing lipids and promoting the absorption of lipids in the gastrointestinal tract, they act as inflammagens. The role of BAs and their receptor farnesoid X receptor (FXR) during viral infection has not been studied in detail., Methods: By using FXR-deficient mice, we investigated the role of bile acid receptor FXR during infection with lymphocytic choriomeningitis virus (LCMV). The importance of FXR in inducing IFN-I and monocytes proliferation were investigated and viral titers and T cell exhaustion were analyzed at different time points., Results: This study shows that controlled levels of BAs activate FXR in hepatocytes and FXR in response upregulates the production of type I interferon. In turn, FXR maintains BAs within a balanced range to inhibit their toxic effects. The absence of FXR results in high levels of BAs, which inhibit the proliferation of monocytes and result in a defect in viral elimination, consequently leading to T cell exhaustion., Conclusion: We found that FXR contributes to IFN-I production in hepatocytes and balances BA levels to inhibit their toxic effects on monocytes., (© 2017 The Author(s) Published by S. Karger AG, Basel.)

Since the discovery of the ubiquitin system and the description of its important role in the degradation of proteins, many studies have shown the importance of ubiquitin-specific peptidases (USPs). One special member of this family is the USP18 protein (formerly UBP43). In the past two decades, several functions of USP18 have been discovered: this protein is not only an isopeptidase but also a potent inhibitor of interferon signaling. Therefore, USP18 functions as 'a' maestro of many biological pathways in various cell types. This review outlines multiple functions of USP18 in the regulation of various immunological processes, including pathogen control, cancer development, and autoimmune diseases.

Upon infection with persistence-prone virus, type I interferon (IFN-I) mediates antiviral activity and also upregulates the expression of programmed death ligand 1 (PD-L1), and this upregulation can lead to CD8 + T-cell exhaustion. How these very diverse functions are regulated remains unknown. This study, using the lymphocytic choriomeningitis virus, showed that a subset of CD169 + macrophages in murine spleen and lymph nodes produced high amounts of IFN-I upon infection. Absence of CD169 + macrophages led to insufficient production of IFN-I, lower antiviral activity and persistence of virus. Lack of CD169 + macrophages also limited the IFN-I-dependent expression of PD-L1. Enhanced viral replication in the absence of PD-L1 led to persistence of virus and prevented CD8 + T-cell exhaustion. As a consequence, mice exhibited severe immunopathology and died quickly after infection. Therefore, CD169 + macrophages are important contributors to the IFN-I response and thereby influence antiviral activity, CD8 + T-cell exhaustion and immunopathology.

The induction of innate and adaptive immunity is essential for controlling viral infections. Limited or overwhelming innate immunity can negatively impair the adaptive immune response. Therefore, balancing innate immunity separately from activating the adaptive immune response would result in a better antiviral immune response. Recently, we demonstrated that Usp18-dependent replication of virus in secondary lymphatic organs contributes to activation of the innate and adaptive immune responses. Whether specific mechanisms can balance innate and adaptive immunity separately remains unknown. In this study, using lymphocytic choriomeningitis virus (LCMV) and replication-deficient single-cycle LCMV vectors, we found that viral replication of the initial inoculum is essential for activating virus-specific CD8(+) T cells. In contrast, extracellular distribution of virus along the splenic conduits is necessary for inducing systemic levels of type I interferon (IFN-I). Although enforced virus replication is driven primarily by Usp18, B cell-derived lymphotoxin beta contributes to the extracellular distribution of virus along the splenic conduits. Therefore, lymphotoxin beta regulates IFN-I induction independently of CD8(+) T-cell activity. We found that two separate mechanisms act together in the spleen to guarantee amplification of virus during infection, thereby balancing the activation of the innate and adaptive immune system., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Clinically used human vaccination aims to induce specific antibodies that can guarantee long-term protection against a pathogen. The reasons that other immune components often fail to induce protective immunity are still debated. Recently we found that enforced viral replication in secondary lymphoid organs is essential for immune activation. In this study we used the lymphocytic choriomeningitis virus (LCMV) to determine whether enforced virus replication occurs in the presence of virus-specific antibodies or virus-specific CD8(+) T cells. We found that after systemic recall infection with LCMV-WE the presence of virus-specific antibodies allowed intracellular replication of virus in the marginal zone of spleen. In contrast, specific antibodies limited viral replication in liver, lung, and kidney. Upon recall infection with the persistent virus strain LCMV-Docile, viral replication in spleen was essential for the priming of CD8(+) T cells and for viral control. In contrast to specific antibodies, memory CD8(+) T cells inhibited viral replication in marginal zone but failed to protect mice from persistent viral infection. We conclude that virus-specific antibodies limit viral infection in peripheral organs but still allow replication of LCMV in the marginal zone, a mechanism that allows immune boosting during recall infection and thereby guarantees control of persistent virus.

Background/aims: Unexpected transmissions of viral pathogens during solid organ transplantation (SOT) can result in severe, life-threatening diseases in transplant recipients. Immune activation contributes to disease onset. However mechanisms balancing the immune response against transmitted viral infection through organ transplantation remain unknown. Methods &, Results: Here we found, using lymphocytic choriomeningitis virus (LCMV), that transplantation of LCMV infected hearts led to exhaustion of virus specific CD8+ T cells, viral persistence in organs and survival of graft and recipient. Genetic depletion of Interleukin-10 (IL-10) resulted in strong immune activation, graft dysfunction and death of mice, suggesting that IL-10 was a major regulator of CD8+ T cell exhaustion during SOT. In the presence of memory CD8+ T cells, virus could be controlled. However sufficient antiviral immune response resulted in acute rejection of transplanted heart., Conclusion: We found that virus transmitted via SOT could not be controlled by naïve mice recipients due to IL-10 mediated CD8+ T cell exhaustion which thereby prevented immunopathology and graft failure whereas memory mice recipients were able to control the virus and induced graft failure., (© 2016 The Author(s) Published by S. Karger AG, Basel.)

Background: Graft versus host disease (GvHD) occurs in 20% of cases with patients having an MHC I matched bone marrow transplantation (BMT). Mechanisms causing this disease remain to be studied., Methods: Here we used a CD8+ T cell transgenic mouse line (P14/CD45.1+) and transgenic DEE mice bearing ubiquitously the glycoprotein 33-41 (GP33) antigen derived from the major lymphocytic choriomeningitis virus (LCMV) epitope to study mechanisms of tolerance in anti-host reactive CD8+ T cells after BMT., Results: We found that anti-host reactive CD8+ T cells (P14 T cells) were not negatively selected in the thymus and that they were present in wild type (WT) recipient mice as well as in DEE recipient mice. Anti-host reactive CD8+ T cells ignored the GP33 antigen expressed ubiquitously by host cells but they could be activated ex vivo via LCMV-infection. Lipopolysaccharides (LPS) induced transient cell damage in DEE mice bearing anti-host reactive CD8+ T cells after BMT, suggesting that induction of host inflammatory response could break antigen ignorance. Introducing the GP33 antigen into BM cells led to deletion of anti-host reactive CD8+ T cells., Conclusion: We found that after BMT anti-host reactive CD8+ T cells ignored host antigen in recipients and that they were only deleted when host antigen was present in hematopoietic cells. Moreover, LPS-induced immune activation contributed to induction of alloreactivity of anti-host reactive CD8+ T cells after BMT., (© 2016 S. Karger AG, Basel.)

Autoantibodies are a hallmark of autoimmune diseases, such as rheumatoid arthritis, autoimmune hepatitis, and systemic lupus erythematosus (SLE). High titers of anti-nuclear antibodies are used as surrogate marker for SLE, however their contribution to pathogenesis remains unclear. Using murine model of SLE and human samples, we studied the effect of immune stimulation on relapsing of SLE. Although autoantibodies bound to target cells in vivo, only additional activation of CD8(+) T cells converted this silent autoimmunity into overt disease. In mice as well as in humans CD8(+) T cells derived IFN-γ enhanced expression of Fc-receptors on CD11b(+) cells. High expression of Fc-receptors allowed CD11b(+) cells to bind to antibody covered target cells and to destroy them in vivo. We found that autoantibodies induce clinically relevant disease when adaptive immunity, specific for disease non-related antigen, is activated., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

B cells are essential for antiviral immune defence because they produce neutralizing antibodies, present antigen and maintain the lymphoid architecture. Here we show that intrinsic signalling of CEACAM1 is essential for generating efficient B-cell responses. Although CEACAM1 exerts limited influence on the proliferation of B cells, expression of CEACAM1 induces survival of proliferating B cells via the BTK/Syk/NF-κB-axis. The absence of this signalling cascade in naive Ceacam1(-/-) mice limits the survival of B cells. During systemic infection with cytopathic vesicular stomatitis virus, Ceacam1(-/-) mice can barely induce neutralizing antibody responses and die early after infection. We find, therefore, that CEACAM1 is a crucial regulator of B-cell survival, influencing B-cell numbers and protective antiviral antibody responses.

Background: Type I interferon (IFN-I) predisposes to bacterial superinfections, an important problem during viral infection or treatment with interferon-alpha (IFN-α). IFN-I-induced neutropenia is one reason for the impaired bacterial control; however there is evidence that more frequent bacterial infections during IFN-α-treatment occur independently of neutropenia., Methods: We analyzed in a mouse model, whether Pseudomonas aeruginosa control is influenced by co-infection with the lymphocytic choriomeningitis virus (LCMV). Bacterial titers, numbers of neutrophils and the gene-expression of liver-lysozyme-2 were determined during a 24 hours systemic infection with P. aeruginosa in wild-type and Ifnar(-/-) mice under the influence of LCMV or poly(I:C)., Results: Virus-induced IFN-I impaired the control of Pseudomonas aeruginosa. This was associated with neutropenia and loss of lysozyme-2-expression in the liver, which had captured P. aeruginosa. A lower release of IFN-I by poly(I:C)-injection also impaired the bacterial control in the liver and reduced the expression of liver-lysozyme-2. Low concentration of IFN-I after infection with a virulent strain of P. aeruginosa alone impaired the bacterial control and reduced lysozyme-2-expression in the liver as well., Conclusion: We found that during systemic infection with P. aeruginosa Kupffer cells quickly controlled the bacteria in cooperation with neutrophils. Upon LCMV-infection this cooperation was disturbed., (© 2015 S. Karger AG, Basel.)

Background: Impairment of regulatory T cells (Tregs) in common autoimmune diseases seems likely. However, the extent of Treg deficiency (number, function) or differential susceptibility of T effector cells (Teffs) to suppression is not completely understood. We hypothesize that even in healthy individuals both cell populations are heterogeneous and differ in their suppressive capability and their susceptibility to suppression., Material and Methods: Lymphocytes were enriched by MACS for CD4(+)CD25(+) Tregs or CD4(+)CD25(-) Teffs. After multicolour staining (anti-CD25, anti-CD127, anti-CD49d or anti-CD45RA) highly purified Treg and Teff subpopulations were collected by FACS. Functional capacity of Tregs or suppressive susceptibility of Teffs was analyzed in an in vitro assay., Results: When CD4(+)CD25(high)CD127(-/low) CD49d(-) Tregs were tested on naive CD4(+)CD127(+)CD25(-)CD45RA(+) Teffs (93.8 %) suppression was almost complete, while the suppressive capacity of CD4(+)CD25(high)CD127(-/low) CD49d(+) Tregs was significantly less (71.8 %). Suppressive activity was low when CD4(+)CD25(high)CD127(-/low) CD49d(+) Tregs were analyzed on CD4(+)CD127(+)CD25(-)CD45RA(-) Teffs (48.7%)., Conclusion: Although CD49d(+) Tregs are functional, the suppressive capacity is significantly lower compared to CD49d(-) Tregs. CD45RA(+) Teffs can be completely suppressed, while CD45RA(-) Teffs display relative resistance. Phenotypic and functional heterogeneity of Tregs as well as Teffs has to be considered when analyzing deficiencies in immune regulation., (© 2014 S. Karger AG, Basel.)

Background: Tumor tissue resembles chronically inflamed tissue. Since chronic inflammatory conditions are a strong stimulus for bone marrow-derived cells (BMDCs) it can be assumed that recruitment of BMDCs into cancer tissue should be a common phenomenon. Several data have outlined that BMDC can influence tumor growth and metastasis, e.g., by inducing a paracrine acting feedback loop in tumor cells. Likewise, cell fusion and horizontal gene transfer are further mechanisms how BMDCs can trigger tumor progression., Results: Hygromycin resistant murine 67NR-Hyg mammary carcinoma cells were co-cultivated with puromycin resistant murine BMDCs from Tg(GFPU)5Nagy/J mice. Isolation of hygromycin/puromycin resistant mBMDC/67NR-Hyg cell clones was performed by a dual drug selection procedure. PCR analysis revealed an overlap of parental markers in mBMDC/67NR-Hyg cell clones, suggesting that dual resistant cells originated by cell fusion. By contrast, both STR and SNP data analysis indicated that only parental 67NR-Hyg alleles were found in mBMDC/67NR-Hyg cell clones favoring horizontal gene transfer as the mode of origin. RealTime-PCR-array analysis showed a marked up-regulation of Abcb1a and Abcb1b ABC multidrug transporters in mBMDC/67NR-Hyg clones, which was verified by Western Blot analysis. Moreover, the markedly increased Abcb1a/Abcb1b expression was correlated to an efficient Rhodamine 123 efflux, which was completely inhibited by verapamil, a well-known Abcb1a/Abcb1b inhibitor. Likewise, mBMDCs/67NR-Hyg clones revealed a marked resistance towards chemotherapeutic drugs including 17-DMAG, doxorubicin, etoposide and paclitaxel. In accordance to Rhodamine 123 efflux data, chemotherapeutic drug resistance of mBMDC/67NR-Hyg cells was impaired by verapamil mediated blockage of Abc1a/Abcb1b multidrug transporter function., Conclusion: Co-cultivation of mBMDCs and mouse 67NR-Hyg mammary carcinoma cells gave rise to highly drug resistant cells. Even though it remains unknown whether mBMDC/67NR-Hyg clones originated by cell fusion or horizontal gene transfer, our data indicate that the exchange of genetic information between two cellular entities is crucial for the origin of highly drug resistant cancer (hybrid) cells, which might be capable to survive chemotherapy.

Sinus node dysfunction and high-degree heart block are the major causes for electronic pacemaker implantation. Recently, genetically modified mesenchymal stromal cells (MSCs, also known as "mesenchymal stem cells") were demonstrated to generate pacemaker function in vivo. However, experimental approaches typically use open thoracotomy for direct cell injection into the myocardium. Future clinical implementation, however, essentially requires development of more gentle methods to precisely and efficiently apply specified stem cells at specific cardiac locations. In a "proof of concept" study, we performed selective power-controlled radiofrequency catheter ablation (RFCA) with eight ablation pulses (30 W, 60 seconds each) to induce heat-mediated lesions at the auricles of the cardiac right atrium of four healthy foxhounds. The next day, allogeneic MSCs (4.3 x 10(5) cells per kilogram of body weight) labeled with superparamagnetic iron oxide particles (SPIOs) were infused intravenously. Hearts were explanted 8 days later. High numbers of SPIO-labeled cells were identified in areas surrounding the RFCA-induced lesions by Prussian blue staining. Antibody staining revealed SPIO-labeled cells being positive for the typical MSC surface antigen CD44. In contrast, low levels of calprotectin, an antigen found on monocytes and macrophages, indicated negligible infiltration of monocytes in MSC-positive areas. Thus, RFCA allows targeting of MSCs to the cardiac right atrium, adjacent to the sinoatrial node, providing an opportunity to rescue or generate pacemaker function without open thoracotomy and direct injection of MSCs. This method presents a new strategy for cardiac stem cell application leading to an efficient guidance of MSCs into the myocardium. Disclosure of potential conflicts of interest is found at the end of this article.

Multiple sclerosis (MS) is an autoimmune disorder directed against self antigens of the central nervous system. CD4(+)CD25(+)FoxP3(+) regulatory T cell (T(reg)) mediated suppression is an essential mechanism of self-tolerance. We studied whether changes in the suppressive function of a mixture of CD25(high) and CD25(intemediate) expressing T(reg) cells in myelin basic protein (MBP)-induced proliferation occurred in untreated MS patients. Suppression of MBP-induced proliferation was observed in 13 out of 29 (45%) MS patients; this was significantly (p<0.05) less compared with 17 out of 19 (89%) healthy individuals. Relative T(reg) counts was significantly increased in MS patients (mean+/-S.D.; 20+/-8%) compared with healthy individuals (15+/-5%). These findings suggest that impaired T(reg) function may be involved in pathogenesis of MS.

New statistics are developed to gather the contribution of many alleles at different loci to common diseases. Both inferential and descriptive statistics are included in order to uncover epistatic effects as well as heterogeneity. The problem of multiple testing is circumvented by considering a global null hypothesis. Global testing is supplemented by descriptive methods that make use of measures like odds ratio or the P-value of individually tested allele combinations. Visualization helps to reflect complex data sets. The methods described here have been scrutinized by statistical simulations, and we show that power gains can be substantial as compared to single locus statistics. Typing data of multiple sclerosis patients and controls are investigated, representing an example of larger scale information in screening candidate genes for their impact on complex diseases. New insights emerge from this data set demonstrating genetic heterogeneity and evidence for epistasis.

In order to investigate the interrelation between immunogenetic host and bacterial factors the T-cell receptor (TCR) polymorphism TCRBV6S1 A/B, HLA-DRB1 alleles and cagA status was analyzed in 380 unrelated German individuals. H. pylori infection with cagA-positive bacterial strains was significantly associated with peptic ulcer disease in the German cohort. Patients homozygous for the non-functional TCRBV6S1B allele and presenting with peptic ulcer disease showed no CagA-specific antibodies in the majority of cases. There was no association between HLA-DRB1 alleles and the CagA status of infected individuals, although certain alleles show significant association to the infection status in different populations.

Objective: Genetic variability influences susceptibility to several diseases and depends on the specific ethnic background of individuals. HLA-class II genes have repeatedly been investigated as candidate genes for predisposition to Helicobacter pylori infection. Certain HLA-DQA1 alleles have been reported to be associated with gastric and duodenal ulcer disease in infected patients in the Japanese population. But conflicting results were reported on European and Japanese populations., Methods: HLA-DRB1 typing of 382 German individuals with well-defined H. pylori status and different clinical course of the disease was performed by polymerase chain reaction and allele-specific oligonucleotide hybridization., Results: No association with the infection status itself was observed in the German cohort. Similar results have been found in other European populations. In contrast, re-analysis of published data in a Japanese cohort revealed a highly significant association of DRB1*1501 with uninfected controls (P = 0.00035). In the German population, the carrier frequency of DRB1*15 was higher in H. pylori-positive individuals with gastric or duodenal ulcer but without statistical significance (gastric ulcer: odds ratio, 2.13; chi2 = 3.77; P = 0.05; Bonferroni correction, Pc = not significant; and duodenal ulcer: odds ratio, 2.15; chi2 = 3.4; P = 0.06; Pc = not significant). In infected individuals, autoantibodies to gastric mucosa were investigated, but no statistical significant difference in carrier frequencies of HLA-DRB1 alleles was evident., Conclusion: The DRB1*1501-DQA1*01021-DQB1*0602 haplotype seems to provide protection from H. pylori infection in the Japanese population, whereas genetic variability in HLA-class II genes has only a minor impact on H. pylori infection and its clinical course in the European population.

Because primate males usually invest very little in offspring, male reproductive success will mainly be determined by access to fertile females with differences in access leading to differential male reproductive success. To determine the outcome of alternative male reproductive routes, we investigated a wild population of Hanuman langurs at Ramnagar, South Nepal, where groups were either one-male or multimale. Paternity was established by DNA analysis from faeces for 42 infants in five groups. In one-male groups all infants were sired by the only resident male in the group. In multimale groups the alpha male fathered significantly more infants (57%) than all other resident males. Nonresident males sired at least 21% of all infants born into multimale groups. Because of the lower mean number of infants sired by alpha males during their whole tenure as alpha compared with males in one-male groups (2.3 versus 6.8) and the higher maximum value (8.9 versus 6.0), the alpha male route is considered the riskier option. Based on demographic data we suggest that the considerable variance in short-term reproductive success might not translate to the same degree into differences in long-term reproductive success. Copyright 2001 The Association for the Study of Animal Behaviour.