Modulation of NF-kappaB activation in Theileria annulata-infected cloned cell lines is associated with detection of parasite-dependent IKK signalosomes and disruption of the actin cytoskeleton

Summary Apicomplexan parasites within the genus Theileria have the ability to induce continuous proliferation and prevent apoptosis of the infected bovine leukocyte. Protection against apoptosis involves constitutive activation of the bovine transcription factor NF-kappaB in a parasite-dependent manner. Activation of NF-kappaB is thought to involve recruitment of IKK signalosomes at the surface of the macroschizont stage of the parasite, and it has been postulated that additional host proteins with adaptor or scaffolding function may be involved in signalosome formation. In this study two clonal cell lines were identified that show marked differences in the level of activated NF-kappaB. Further characterization of these lines demonstrated that elevated levels of activated NF-kappaB correlated with increased resistance to cell death and detection of parasite-associated IKK signalosomes, supporting results of our previous studies. Evidence was also provided for the existence of host- and parasite-dependent NF-kappaB activation pathways that are influenced by the architecture of the actin cytoskeleton. Despite this influence, it appears that the primary event required for formation of the parasite-dependent IKK signalosome is likely to be an interaction between a signalosome component and a parasite-encoded surface ligand.

The transforming parasite Theileria co-opts host cell mitotic and central spindles to persist in continuously dividing cells

The protozoan parasite Theileria inhabits the host cell cytoplasm and possesses the unique capacity to transform the cells it infects, inducing continuous proliferation and protection against apoptosis. The transforming schizont is a multinucleated syncytium that resides free in the host cell cytoplasm and is strictly intracellular. To maintain transformation, it is crucial that this syncytium is divided over the two daughter cells at each host cell cytokinesis. This process was dissected using different cell cycle synchronization methods in combination with the targeted application of specific inhibitors. We found that Theileria schizonts associate with newly formed host cell microtubules that emanate from the spindle poles, positioning the parasite at the equatorial region of the mitotic cell where host cell chromosomes assemble during metaphase. During anaphase, the schizont interacts closely with host cell central spindle. As part of this process, the schizont recruits a host cell mitotic kinase, Polo-like kinase 1, and we established that parasite association with host cell central spindles requires Polo-like kinase 1 catalytic activity. Blocking the interaction between the schizont and astral as well as central spindle microtubules prevented parasite segregation between the daughter cells during cytokinesis. Our findings provide a striking example of how an intracellular eukaryotic pathogen that evolved ways to induce the uncontrolled proliferation of the cells it infects usurps the host cell mitotic machinery, including Polo-like kinase 1, one of the pivotal mitotic kinases, to ensure its own persistence and survival.

Training-related modulations of the autonomic nervous system in endurance athletes: is female gender cardioprotective?

The risk of sudden death is increased in athletes with a male predominance. Regular physical activity increases vagal tone, and may protect against exercise-induced ventricular arrhythmias. We investigated training-related modulations of the autonomic nervous system in female and male endurance athletes. Runners of a 10-mile race were invited. Of 873 applicants, 68 female and 70 male athletes were randomly selected and stratified according to their average weekly training hours in a low (≤4 h) and high (>4 h) volume training group. Analysis of heart rate variability was performed over 24 h. Spectral components (high frequency [HF] and low frequency [LF] power in normalized units) were analyzed for hourly 5 min segments and averaged for day- and nighttime. One hundred and fourteen athletes (50 % female, mean age 42 ± 7 years) were included. No significant gender difference was observed for training volume and 10-mile race time. Over the 24-h period, female athletes exhibited a higher HF and lower LF power for each hourly time-point. Female gender and endurance training hours were independent predictors of a higher HF and lower LF power. In female athletes, higher training hours were associated with a higher HF and lower LF power during nighttime. In male athletes, the same was true during daytime. In conclusion, female and male athletes showed a different circadian pattern of the training-related increase in markers of vagal tone. For a comparable amount of training volume, female athletes maintained their higher markers of vagal tone, possibly indicating a superior protection against exercise-induced ventricular arrhythmias.

Functional and physiological role of vitamin C transporters

Vitamin C (ascorbic acid) is required for the synthesis of collagen, carnitine, catecholamine and the neurotransmitter norepinephrine. Vitamin C also plays an important role in protection against oxidative stress. Transporters for vitamin C and its oxidized form dehydroascorbate (DHA) are crucial to keep vitamin concentrations optimal in the body. The human SLC23 family consists of the Na(+)-dependent vitamin C transporters SVCT1 (SLC23A1) and SVCT2 (SLC23A2) and the orphan transporter SVCT3 (SLC23A3). Phylogenetically, the SLC23 family belongs to the nucleobase-ascorbate transporter family although no specificity for nucleobases has yet been demonstrated for the human members of this family. In fact, the SVCT1 and SVCT2 transporters are rather specific for ascorbic acid. SVCT1 is expressed in epithelial tissues such as intestine, where it contributes to the maintenance of whole-body ascorbic acid levels, whereas the expression of SVCT2 is relatively widespread either to protect metabolically active cells and specialized tissues from oxidative stress or to deliver ascorbic acid to tissues that are in high demand of the vitamin for enzymatic reactions. DHA, the oxidized form of ascorbic acid is taken up and distributed in the body by facilitated transport via members of the SLC2/GLUT family (GLUT1, GLUT3, and GLUT4). Although, the main focus of this review is on the SLC23 family of ascorbic acid transporters, transporters of DHA and nucleobases are also briefly discussed for completeness.

Vaccination with the recombinant chimeric antigen recNcMIC3-1-R induces a non-protective Th2-type immune response in the pregnant mouse model for N. caninum infection

The major route of transmission of Neospora caninum in cattle is transplacentally from an infected cow to its progeny. Therefore, a vaccine should be able to prevent both the horizontal transmission from contaminated food or water and the vertical transmission. We have previously shown that a chimeric vaccine composed of predicted immunogenic epitopes of NcMIC3, NcMIC1 and NcROP2 (recNcMIC3-1-R) significantly reduced the cerebral infection in BALB/c mice. In this study, mice were first vaccinated, then mated and pregnant mice were challenged with 2×10(6)N. caninum tachyzoites at day 7-9 of pregnancy. Partial protection was only observed in the mice vaccinated with a tachyzoite crude protein extract but no protection against vertical transmission or cerebral infection in the dams was observed in the group vaccinated with recNcMIC3-1-R. Serological and cytokine analysis showed an overall lower cytokine level in sera associated with a dominant IL-4 expression and high IgG1 titers. Thus, the Th2-type immune response observed in the pregnant mice was not protective against experimental neosporosis, in contrary to the mixed Th1-/Th2-type immune response observed in the non-pregnant mouse model. These results demonstrate that the immunomodulation that occurs during pregnancy was not favorable for the protection against N. caninum infection conferred by vaccination with recNcMIC3-1-R.

Depicting community perspectives: repeat photography and participatory research as tools for assessing environmental services in Sagarmatha National Park, Nepal

Efforts have been made to provide a scientific basis for using environmental services as a conceptual tool to enhance conservation and improve livelihoods in protected mountain areas (MtPAS). Little attention has been paid to participatory research or locals’ concerns as environmental service (ES) users and providers. Such perspectives can illuminate the complex interplay between mountain ecosystems, environmental services and the determinants of human well-being. Repeat photography, long used in geographical fieldwork, is new as a qualitative research tool. This study uses a novel application of repeat photography as a diachronic photo-diary to examine local perceptions of change in ES in Sagarmatha National Park. Results show a consensus among locals on adverse changes to ES, particularly protection against natural hazards, such as landslides and floods, in the UNESCO World Heritage Site. We argue that our methodology could complement biophysical ecosystem assessments in MtPAS, especially since assessing ES, and acting on that, requires integrating diverse stakeholders’ knowledge, recognizing power imbalances and grappling with complex social-ecological systems.

The strategies of the Theileria parasite: a new twist in host-pathogen interactions

Theileria parasites infect and transform cells of the ruminant immune system. Continuous proliferation and survival of Theileria-transformed cells involves the well-orchestrated activation of several host-cell signalling pathways. Constitutive NF-kappa B (nuclear factor kappa B) activation is accomplished by recruiting the IKK (I kappa B kinase) complex, a central regulator of NF-kappa B pathways, to the surface of the transforming schizont, where it becomes permanently activated. Constitutive activation of the PI-3K-PKB [phosphoinositide 3-kinase-(Akt) protein kinase B] pathway is likely to be indirect and is essential for continuous proliferation. Theileria-transformed T cells express a range of anti-apoptotic proteins that can be expected to provide protection against apoptosis induced by death receptors, as well as cellular control mechanisms that are mobilised to eliminate cells that entered a cycle of uncontrolled proliferation.

[How the bovine viral diarrhea virus outwits the immune system]

The interaction of bovine viral diarrhea virus (BVD virus) with its host has several unique features, most notably the capacity to infect its host either transiently or persistently. The transient infection stimulates an antiviral immune reaction similar to that seen in other transient viral infections. In contrast, being associated with immunotolerance specific for the infecting BVD viral strain, the persistent infection differs fundamentally from other persistent infections like those caused by lentiviruses. Whereas the latter are characterized by complex viral evasion of the host's adaptive immune response by mechanisms such as antigenic drift and interference with presentation of T cell epitopes, BVD virus avoids the immune response altogether by inducing both humoral and cellular immune tolerance. This is made possible by invasion of the fetus at an early stage of development. In addition to adaptive immunity, BVD virus also manipulates key elements of the host's innate immune response. The non-cytopathic biotype of BVD virus, which is capable of persistently infecting its host, fails to induce type I interferon. In addition, persistently infected cells are resistant to the induction of apoptosis by double-stranded RNA and do not produce interferon when treated with this pathogen-associated molecular pattern (PAMP) that signals viral infection. Moreover, when treated with interferon, cells persistently infected with non-cytopathic BVD virus do not clear the virus. Surprisingly, however, despite this lack of effect on persistent infection, interferon readily induces an antiviral state in these cells, as shown by the protection against infection by unrelated viruses. Overall, BVD virus manipulates the host's interferon defense in a manner that optimises its chances of maintaining the persistent infection as well as decreasing the risks that heterologous viral infections may carry for the host. Thus, since not all potential host cells are infected in animals persistently infected with BVD virus, heterologous viruses replicating in cells uninfected with BVD virus will still trigger production of interferon. Interferon produced by such cells will curtail the replication of heterologous viruses only, be that in cells already infected with BVD virus, or in cells in which the heterologous virus may replicate alone. From an evolutionary viewpoint, this strategy clearly enhances the chances of transmission of BVD virus to new hosts, as it attenuates the negative effects that a global immunosuppression would have on the survival of persistently infected animals.

Imatinib mesylate selectively impairs expansion of memory cytotoxic T cells without affecting the control of primary viral infections

Imatinib mesylate (imatinib) is a potent inhibitor of defined tyrosine kinases (TKs) and is effective in the treatment of malignancies characterized by constitutive activation of these TKs such as chronic myeloid leukemia and gastrointestinal stromal tumors. TKs also play an important role in T-cell receptor (TCR) signal transduction. Inhibitory as well as stimulating effects of imatinib on T cells and dendritic cells have been described. Here, we analyzed the effects of imatinib treatment on antiviral immune responses in vivo. Primary cytotoxic T-cell (CTL) responses were not impaired in imatinib-treated mice after infection with lymphocytic choriomeningitis virus (LCMV) or after immunization with a tumor cell line expressing LCMV glycoprotein (LCMV-GP). Similarly, neutralizing antibody responses to vesicular stomatitis virus (VSV) were not affected. In contrast, secondary expansion of LCMV-specific memory CTLs was reduced in vitro and in vivo, resulting in impaired protection against reinfection. In addition, imatinib treatment delayed the onset of diabetes in a CTL-induced diabetes model. In summary, imatinib treatment in vivo selectively inhibits the expansion of antigen-experienced memory CTLs without affecting primary T- or B-cell responses. Therefore, imatinib may be efficacious in the suppression of CTL-mediated immunopathology in autoimmune diseases without the risk of acquiring viral infections.

Prevention of postmenopausal bone loss with long-cycle hormone replacement therapy

OBJECTIVE: Postmenopausal bone loss and osteoporotic fractures can be prevented by hormone replacement therapy (HRT). However, opposed HRT may increase the risk of breast cancer above that associated with estrogen alone and in non-hysterectomized women estrogen substitution alone increases the risk of uterine cancer, which triggered renewed interest in long-cycle HRT regimens (estrogen replacement therapy with progesterone-free intervals up to 6 months). The effects on bone of such long-cycle HRT regimens are unknown. The objective of the present study was to compare the effects on bone and the endometrium of long-cycle HRT and conventional HRT. METHODS: Seventy-three healthy non-hysterectomized postmenopausal women were randomized to either conventional HRT (estradiol (E2) 2 mg/d during 12 days, E2 2 mg/d plus 1 mg/d of norethisterone acetate (NETA) during 10 days, E2 1 mg/d for 6 days) or long-cycle HRT treatment (two cycles with E2 2 mg/d during 28 days, followed by one cycle of conventional HRT and repeated every 3 months). Primary endpoint was the change in bone mineral density (BMD) at the lumbar spine (LS) over 24 months. RESULTS: BMD at LS increased significantly versus baseline in both treatment groups (conventional HRT +3.8 +/- 0.6%, long-cycle HRT +3.3 +/- 0.5%, p < 0.0001 for both) with no significant difference between treatment groups over 24 months. Similar significant BMD increases versus baseline were observed at the femoral neck, while biochemical markers of bone turnover (osteocalcin and deoxypyridinoline) were significantly decreased over 24 months. There were no endometrial or breast related adverse events reported. CONCLUSION: Long-cycle HRT may be a valid alternative to conventional HRT with regard to protection against postmenopausal bone loss.

CD4+ T cell help improves CD8+ T cell memory by retained CD27 expression

CD4+ T cell help during the priming of CD8+ T lymphocytes imprints the capacity for optimal secondary expansion upon re-encounter with antigen. Helped memory CD8+ T cells rapidly expand in response to a secondary antigen exposure, even in the absence of T cell help and, are most efficient in protection against a re-infection. In contrast, helpless memory CTL can mediate effector function, but secondary expansion is reduced. How CD4+ T cells instruct CD8+ memory T cells during priming to undergo efficient secondary expansion has not been resolved in detail. Here, we show that memory CTL after infection with lymphocytic choriomeningitis virus are CD27(high) whereas memory CTL primed in the absence of CD4+ T cell have a reduced expression of CD27. Helpless memory CTL produced low amounts of IL-2 and did not efficiently expand after restimulation with peptide in vitro. Blocking experiments with monoclonal antibodies and the use of CD27(-/-) memory CTL revealed that CD27 ligation during restimulation increased autocrine IL-2 production and secondary expansion. Therefore, regulating CD27 expression on memory CTL is a novel mechanism how CD4+ T cells control CTL memory.

Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense

Although eosinophils are considered useful in defense mechanisms against parasites, their exact function in innate immunity remains unclear. The aim of this study is to better understand the role of eosinophils within the gastrointestinal immune system. We show here that lipopolysaccharide from Gram-negative bacteria activates interleukin-5 (IL-5)- or interferon-gamma-primed eosinophils to release mitochondrial DNA in a reactive oxygen species-dependent manner, but independent of eosinophil death. Notably, the process of DNA release occurs rapidly in a catapult-like manner--in less than one second. In the extracellular space, the mitochondrial DNA and the granule proteins form extracellular structures able to bind and kill bacteria both in vitro and under inflammatory conditions in vivo. Moreover, after cecal ligation and puncture, Il5-transgenic but not wild-type mice show intestinal eosinophil infiltration and extracellular DNA deposition in association with protection against microbial sepsis. These data suggest a previously undescribed mechanism of eosinophil-mediated innate immune responses that might be crucial for maintaining the intestinal barrier function after inflammation-associated epithelial cell damage, preventing the host from uncontrolled invasion of bacteria.

Interferon-gamma regulates idiopathic pneumonia syndrome, a Th17+CD4+ T-cell-mediated graft-versus-host disease

RATIONALE: Pulmonary complications of hematopoietic stem cell transplantation include infections and graft-versus-host diseases, such as idiopathic pneumonia syndrome (IPS). Conflicting data exist regarding the role of the interferon (IFN)-gamma-producing Th1 CD4(+) T-cell subset and IL-17A in IPS. OBJECTIVES: To determine the role of IFN-gamma and IL-17A in the establishment of pulmonary graft-versus-host disease. METHODS: A semiallogeneic murine model based on C57BL/6 x BALB/c as recipients with transplantation of BALB/c RAG2(-/-) bone marrow and transfer of different genetic knockout T cells (T-bet(-/-), IFN-gamma(-/-), IFN-gammaR(-/-)) on a BALB/c background. Lung tissue was examined for parenchymal changes and infiltrating cells by histology and fluorescence-activated cell sorter analysis. MEASUREMENTS AND MAIN RESULTS: After transfer of semiallogeneic bone marrow together with donor CD4(+) T cells lacking IFN-gamma or T-bet-a T-box transcription factor controlling Th1 commitment-we found severe inflammation in the lungs, but no enhancement in other organs. In contrast, wild-type donor CD4(+) T cells mediated minimal inflammation only, and donor CD8(+) T cells were not required for IPS development. Mechanistically, the absence of IFN-gamma or IFN-gamma signaling in pulmonary parenchymal cells promoted expansion of IL-17A-producing CD4(+) T cells and local IL-17A release. In vivo depletion of IL-17A reduced disease severity. CONCLUSIONS: One mechanism of IFN-gamma protection against IPS is negative regulation of the expansion of pathogenic IL-17A-producing CD4(+) T cells through interaction with the IFN-gamma receptor on the pulmonary parenchymal cell population.

6th International Immunoglobulin Symposium: poster presentations

The posters presented at the 6th International Immunoglobulin Symposium covered a wide range of fields and included both basic science and clinical research. From the abstracts accepted for poster presentation, 12 abstracts were selected for oral presentations in three parallel sessions on immunodeficiencies, autoimmunity and basic research. The immunodeficiency presentations dealt with novel, rare class-switch recombination (CSR) deficiencies, attenuation of adverse events following IVIg treatment, association of immunoglobulin (Ig)G trough levels and protection against acute infection in patients with X-linked agammaglobulinaemia (XLA) and common variable immunodeficiency (CVID), and the reduction of class-switched memory B cells in patients with specific antibody deficiency (SAD). The impact of intravenous immunoglobulin on fetal alloimmune thrombocytopenia, pregnancy and postpartum-related relapses in multiple sclerosis and refractory myositis, as well as experiences with subcutaneous immunoglobulin in patients with multi-focal motor neuropathy, were the topics presented in the autoimmunity session. The interaction of dendritic cell (DC)-SIGN and alpha2,6-sialylated IgG Fc and its impact on human DCs, the enrichment of sialylated IgG in plasma-derived IgG, as wells as prion surveillance and monitoring of anti-measles titres in immunoglobulin products, were covered in the basic science session. In summary, the presentations illustrated the breadth of immunoglobulin therapy usage and highlighted the progress that is being made in diverse areas of basic and clinical research, extending our understanding of the mechanisms of immunoglobulin action and contributing to improved patient care.

Gene therapy for immunodeficiency due to adenosine deaminase deficiency

BACKGROUND: We investigated the long-term outcome of gene therapy for severe combined immunodeficiency (SCID) due to the lack of adenosine deaminase (ADA), a fatal disorder of purine metabolism and immunodeficiency. METHODS: We infused autologous CD34+ bone marrow cells transduced with a retroviral vector containing the ADA gene into 10 children with SCID due to ADA deficiency who lacked an HLA-identical sibling donor, after nonmyeloablative conditioning with busulfan. Enzyme-replacement therapy was not given after infusion of the cells. RESULTS: All patients are alive after a median follow-up of 4.0 years (range, 1.8 to 8.0). Transduced hematopoietic stem cells have stably engrafted and differentiated into myeloid cells containing ADA (mean range at 1 year in bone marrow lineages, 3.5 to 8.9%) and lymphoid cells (mean range in peripheral blood, 52.4 to 88.0%). Eight patients do not require enzyme-replacement therapy, their blood cells continue to express ADA, and they have no signs of defective detoxification of purine metabolites. Nine patients had immune reconstitution with increases in T-cell counts (median count at 3 years, 1.07x10(9) per liter) and normalization of T-cell function. In the five patients in whom intravenous immune globulin replacement was discontinued, antigen-specific antibody responses were elicited after exposure to vaccines or viral antigens. Effective protection against infections and improvement in physical development made a normal lifestyle possible. Serious adverse events included prolonged neutropenia (in two patients), hypertension (in one), central-venous-catheter-related infections (in two), Epstein-Barr virus reactivation (in one), and autoimmune hepatitis (in one). CONCLUSIONS: Gene therapy, combined with reduced-intensity conditioning, is a safe and effective treatment for SCID in patients with ADA deficiency. (ClinicalTrials.gov numbers, NCT00598481 and NCT00599781.)