Pituitary Adenylate Cyclase-Activating Polypeptide Ameliorates Experimental Acute Ileitis and Extra-Intestinal Sequelae

Background

The neuropeptide Pituitary adenylate cyclase-activating polypeptide (PACAP) plays pivotal roles in immunity and inflammation. So far, potential immune-modulatory properties of PACAP have not been investigated in experimental ileitis.

Methodology/Principal Findings

Mice were perorally infected with Toxoplasma (T.) gondii to induce acute ileitis (day 0) and treated daily with synthetic PACAP38 from day 1 to 6 post infection (p.i.; prophylaxis) or from day 4 to 6 p.i. (therapy). Whereas placebo-treated control mice suffered from acute ileitis at day 7 p.i. and succumbed to infection, intestinal immunopathology was ameliorated following PACAP prophylaxis. PACAP-treated mice exhibited increased abundance of small intestinal FOXP3+ cells, but lower numbers of ileal T lymphocytes, neutrophils, monocytes and macrophages, which was accompanied by less ileal expression of pro-inflammatory cytokines such as IL-23p19, IL-22, IFN-γ, and MCP-1. Furthermore, PACAP-treated mice displayed higher anti-inflammatory IL-4 concentrations in mesenteric lymph nodes and liver and higher systemic anti-inflammatory IL-10 levels in spleen and serum as compared to control animals at day 7 p.i. Remarkably, PACAP-mediated anti-inflammatory effects could also be observed in extra-intestinal compartments as indicated by reduced pro-inflammatory mediator levels in spleen (TNF-α, nitric oxide) and liver (TNF-α, IFN-γ, MCP-1, IL-6) and less severe histopathological sequelae in lungs and kidneys following prophylactic PACAP treatment. Strikingly, PACAP prolonged survival of T. gondii infected mice in a time-of-treatment dependent manner.

Conclusion/Significance

Synthetic PACAP ameliorates acute small intestinal inflammation and extra-intestinal sequelae by down-regulating Th1-type immunopathology, reducing oxidative stress and up-regulating anti-inflammatory cytokine responses. These findings provide novel potential treatment options of inflammatory bowel diseases.     

Reduction of Campylobacter jejuni in Broiler Chicken by Successive Application of Group II and Group III Phages

Background

Bacteriophage treatment is a promising tool to reduce Campylobacter in chickens. Several studies have been published where group II or group III phages were successfully applied. However, these two groups of phages are different regarding their host ranges and host cell receptors. Therefore, a concerted activity of group II and group III phages might enhance the efficacy of a treatment and decrease the number of resistant bacteria.

Results

In this study we have compared the lytic properties of some group II and group III phages and analysed the suitability of various phages for a reduction of C. jejuni in broiler chickens. We show that group II and group III phages exhibit different kinetics of infection. Two group III and one group II phage were selected for animal experiments and administered in different combinations to three groups of chickens, each containing ten birds. While group III phage CP14 alone reduced Campylobacter counts by more than 1 log₁₀ unit, the concomitant administration of a second group III phage (CP81) did not yield any reduction, probably due to the development of resistance induced by this phage. One group of chickens received phage CP14 and, 24 hours later, group II phage CP68. In this group of animals, Campylobacter counts were reduced by more than 3 log₁₀ units.

Conclusion

The experiments illustrated that Campylobacter phage cocktails have to be carefully composed to achieve the best results.     

ASIC2 Subunits Facilitate Expression at the Cell Surface and Confer Regulation by PSD-95

Acid-sensing ion channels (ASICs) are Na⁺ channels activated by changes in pH within the peripheral and central nervous systems. Several different isoforms of ASICs combine to form trimeric channels, and their properties are determined by their subunit composition. ASIC2 subunits are widely expressed throughout the brain, where they heteromultimerize with their partnering subunit, ASIC1a. However, ASIC2 contributes little to the pH sensitivity of the channels, and so its function is not well understood. We found that ASIC2 increased cell surface levels of the channel when it is coexpressed with ASIC1a, and genetic deletion of ASIC2 reduced acid-evoked current amplitude in mouse hippocampal neurons. Additionally, ASIC2a interacted with the neuronal synaptic scaffolding protein PSD-95, and PSD-95 reduced cell surface expression and current amplitude in ASICs that contain ASIC2a. Overexpression of PSD-95 also reduced acid-evoked current amplitude in hippocampal neurons. This result was dependent upon ASIC2 since the effect of PSD-95 was abolished in ASIC2−/− neurons. These results lend support to an emerging role of ASIC2 in the targeting of ASICs to surface membranes, and allows for interaction with PSD-95 to regulate these processes.     

A yeast-based assay identifies drugs that interfere with immune evasion of the Epstein-Barr virus

Epstein-Barr virus (EBV) is tightly associated with certain human cancers, but there is as yet no specific treatment against EBV-related diseases. The EBV-encoded EBNA1 protein is essential to maintain viral episomes and for viral persistence. As such, EBNA1 is expressed in all EBV-infected cells, and is highly antigenic. All infected individuals, including individuals with cancer, have CD8⁺ T cells directed towards EBNA1 epitopes, yet the immune system fails to detect and destroy cells harboring the virus. EBV immune evasion depends on the capacity of the Gly-Ala repeat (GAr) domain of EBNA1 to inhibit the translation of its own mRNA in cis, thereby limiting the production of EBNA1-derived antigenic peptides presented by the major histocompatibility complex (MHC) class I pathway. Here we establish a yeast-based assay for monitoring GAr-dependent inhibition of translation. Using this assay we identify doxorubicin (DXR) as a compound that specifically interferes with the GAr effect on translation in yeast. DXR targets the topoisomerase-II–DNA complexes and thereby causes genomic damage. We show, however, that the genotoxic effect of DXR and various analogs thereof is uncoupled from the effect on GAr-mediated translation control. This is further supported by the observation that etoposide and teniposide, representing another class of topoisomerase-II–DNA targeting drugs, have no effect on GAr-mediated translation control. DXR and active analogs stimulate, in a GAr-dependent manner, EBNA1 expression in mammalian cells and overcome GAr-dependent restriction of MHC class I antigen presentation. These results validate our approach as an effective high-throughput screening assay to identify drugs that interfere with EBV immune evasion and, thus, constitute candidates for treating EBV-related diseases, in particular EBV-associated cancers.KEY WORDS: EBV-associated cancers, Cell-based drug screening, EBNA1 GAr domain, Yeast-based models, Immune evasion, Doxorubicin, Daunorubicin, 5-fluorouracil     

Morphological, physiological and behavioural evaluation of a ‘Mice in Space’ housing system

Environmental conditions likely affect physiology and behaviour of mice used for life sciences research on Earth or in Space. Here, we analysed the effects of cage confinement on the weightbearing musculoskeletal system, behaviour and stress of wild-type mice (C57BL/6JRj, 30 g b.wt., total n = 24) housed for 25 days in a prototypical ground-based and fully automated life support habitat device called “Mice in Space” (MIS). Compared with control housing (individually ventilated cages) the MIS mice revealed no significant changes in soleus muscle size and myofiber distribution (type I vs. II) and quality of bone (3-D microarchitecture and mineralisation of calvaria, spine and femur) determined by confocal and micro-computed tomography. Corticosterone metabolism measured non-invasively (faeces) monitored elevated adrenocortical activity at only start of the MIS cage confinement (day 1). Behavioural tests (i.e., grip strength, rotarod, L/D box, elevated plus-maze, open field, aggressiveness) performed subsequently revealed only minor changes in motor performance (MIS vs. controls). The MIS habitat will not, on its own, produce major effects that could confound interpretation of data induced by microgravity exposure during spaceflight. Our results may be even more helpful in developing multidisciplinary protocols with adequate scenarios addressing molecular to systems levels using mice of various genetic phenotypes in many laboratories. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Orderly pattern of development of the autoantibody response in (New Zealand White x BXSB)F1 lupus mice: characterization of target antigens and antigen spreading by two-dimensional gel electrophoresis and mass spectrometry

Immunoblots of a two-dimensional PAGE-separated HL-60 cell proteomic map and mass spectrometry were combined to characterize proteins targeted by autoantibodies produced by male (New Zealand White x BXSB)F(1) (WB) mice that develop lupus and anti-phospholipid syndrome. Analysis of sera sequentially obtained from seven individual mice at different ages showed that six proteins, vimentin, heat shock protein 60, UV excision-repair protein RAD23, alpha-enolase, heterogeneous nuclear ribonucleoprotein L, and nucleophosmin, were the targets of the B cell autoimmune response, and that autoantibodies to them were synthesized sequentially in an orderly pattern that recurred in all the male WB mice analyzed: anti-vimentin first and anti-nucleophosmin last, with anti-RAD23 and anti-heat shock protein 60, then anti-alpha-enolase and anti-heterogeneous nuclear ribonucleoprotein L Abs occuring concomitantly. Anti-vimentin reactivity always appeared before anti-cardiolipin and anti-DNA Abs, suggesting that vimentin is the immunogen initiating the autoimmune process. The pattern of HL-60 proteins recognized by female WB sera differed from that of male sera, indicating that the Y chromosome-linked autoimmune acceleration gene is not an accelerator but a strong modifier of the autoimmune response. Thus, 1) combining two-dimensional PAGE and mass spectrometry constitutes a powerful tool to identify the set of Ags bound by autoantibodies present in a single serum and the whole autoantibody pattern of an autoimmune disease; 2) the diversification of the autoimmune response in male WB mice occurs in a predetermined pattern consistent with Ag spreading, and thus provides a useful model to further our understanding of the development of the autoantibody response in lupus.     

Changes in airway resistance induced by nasal inhalation of cold dry, dry, or moist air in normal individuals

Fontanari, Pierre, Henri Burnet, Marie CarolineZattara-Hartmann, and Yves Jammes. Changes in airway resistanceinduced by nasal inhalation of cold dry, dry, or moist air in normalindividuals. J. Appl. Physiol. 81(4):1739-1743, 1996.Nasopulmonary bronchomotor reflexes elicited bymechanical or irritant stimulation of the nose have been described inanimals and asthmatic patients. However, few studies were devoted tothe consequences of nasal breathing of cold and dry air or of only dryor only moist air on the bronchomotor control in normal individuals.The present study reported changes in interruption resistance (Rint)measured during eupneic breathing of moderately cold (4 or10°C) and dry [0.3% relative humidity (RH)] airor of room air at 23°C that is either dry (0.3% RH) or moist (97%RH). Nasal inhalation of cold (4°C) dry air or of only dryair significantly increased baseline Rint value (17 and 21%,respectively) throughout the 15-min test periods. The response to cold was significantly accentuated when the air temperature was lowered to 10°C (42%). After nasal anesthesia orinhalation of a cholinergic antagonist, cold air did not induce achange in Rint. Nasal inhalation of moist room air had no effect. No Rint changes were measured during oral breathing of the three testagents. It is concluded that the activation of cold receptors orosmoreceptors in the nasal mucosa induces protective bronchoconstrictor responses in normal individuals.

     

FDG PET scans as evaluation of clinical response to dendritic cell vaccination in patients with malignant melanoma

Background

Measurements of tumour metabolism by [18F]fluorodeoxyglucose positron emission tomography (FDG-PET) have been successfully applied to monitor tumour response after chemo- and chemo-radiotherapy and may not have the same limitations as other morphological imaging techniques. In this study it is investigated whether FDG-PET might add information on the efficacy of immune therapy.

Materials and methods

In a retrospective analysis data from patients with advanced progressive melanoma, treated with DC vaccinations and evaluated by PET/CT scans at baseline as well as after 6 vaccinations were analysed. If a patient achieved stable disease according to RECIST, additional vaccinations were given. The PET scans were evaluated according to EORTC guidelines.

Results

PET/CT scans from 13 patients were evaluated. According to RECIST 3 patients achieved stable disease and 10 patients progressed. Interestingly, when evaluated by PET scans 2 patients had partial metabolic response and 1 patient had complete metabolic response of the 2 index lesions even though a new lesion appeared simultaneously. Ten patients were seen to have stable or progressive metabolic disease.

Conclusion

By adding PET scans to the CT evaluation of patients treated with DC vaccines, a more detailed picture of the single lesions was found. This seems to improve the clinical evaluation of the treatment. The lack of correlation between the PET and CT scans suggests that some of the increases in target lesions seen in CT scans might be due to oedema or immune-infiltrates and not progression of the disease. Thus, further investigation into the contribution of PET scans to the evaluation of cancer immunotherapy is needed.     

The Relationship between Parental Depressive Symptoms,Family Type,and Adolescent Functioning

It is evident that parental depressive symptoms negatively influence adolescent behavior and various psychosocial outcomes. Certain family types like families with a chronically ill parent and single parent families are more vulnerable to parental depressive symptoms. However, the relationship between these symptoms, family type, and adolescent functioning remains largely unclear. This study examined relations between self-report of parental depressive symptoms and adolescent functioning in 86 two-parent families including a parent with a chronic medical condition, 94 families with healthy single parents, and 69 families with 2 healthy parents (comparison group). Parents completed the Beck Depression Inventory. Adolescents filled in the Youth Self-Report measuring problem behavior, and other instruments measuring psychosocial outcomes (stress, grade point average, school problems, and self-esteem). Multilevel analyses were used to examine the effects of family type, parental depressive symptoms, adolescents'' gender and age, and interaction effects on adolescent functioning. The results indicated that adolescents with chronically ill and single parents had a lower grade point average (p<.01) than the comparison group. Adolescents of single parents reported more internalizing problems (p<.01) and externalizing problems (p<.05) than children from the other family types. Parental depressive symptoms were strongly related to child report of stress (p<.001). Adolescents of depressed chronically ill parents were particularly vulnerable to internalizing problems (interaction effect, p<.05). Older children and girls, and especially older girls, displayed more internalizing problems and stress. It can be concluded that growing up with a chronically ill parent in a family with 2 parents may have less impact on adolescent problem behavior than growing up in a single parent family. Health practitioners are encouraged to be attentive to the unique and combined influence of family type and parental depressive symptoms on adolescent functioning. Older and female adolescents deserve particular attention.