Effects of Regulatory T Cell Depletion on NK Cell Responses against Listeria monocytogenes in Feline Immunodeficiency Virus Infected Cats

Regulatory T cells (Treg) are key players in the maintenance of peripheral tolerance, preventing autoimmune diseases and restraining chronic inflammatory diseases. Evidence suggests Treg cells and NK cells have important roles in feline immunodeficiency virus (FIV) pathogenesis; however, in vivo studies investigating the interplay between these two cell populations are lacking. We previously described innate immune defects in FIV-infected cats characterized by cytokine deficits and impaired natural killer cell (NK) and NK T cell (NKT) functions. In this study, we investigated whether in vivo Treg depletion by treatment with an anti-feline CD25 monoclonal antibody would improve the innate immune response against subcutaneous challenge with Listeria monocytogenes (Lm). Treg depletion resulted in an increased overall number of cells in Lm-draining lymph nodes and increased proliferation of NK and NKT cells in FIV-infected cats. Treg depletion did not normalize expression of perforin or granzyme A by NK and NKT cells, nor did Treg depletion result in improved clearance of Lm. Thus, despite the quantitative improvements in the NK and NKT cell responses to Lm, there was no functional improvement in the early control of Lm. CD1a+ dendritic cell percentages in the lymph nodes of FIV-infected cats were lower than in specific-pathogen-free control cats and failed to upregulate CD80 even when Treg were depleted. Taken together, Treg depletion failed to improve the innate immune response of FIV-infected cats against Lm and this may be due to dendritic cell dysfunction

Associations of behaviour with secretory immunoglobulin A and cortisol in domestic cats during their first week in an animal shelter

We tested the hypothesis that during their first week in an animal shelter, cats exhibit groups of behaviours that are connected to mucosal immune and adrenal responses. The behaviour of 34 cats was observed from admission to day 5 and immunoglobulin A (S-IgA) and cortisol were quantified from faeces. A multidimensional model constructed by Principal Component Analysis indicated the presence of three distinct behavioural dimensions. Behaviours forming dimension 1 were hiding, flat postures, freeze, startle, crawl and retreat from humans. These were significantly contrasted (R -0.6 to -0.4) to dimension 3 behaviours which included normal patterns of feeding, grooming, sleeping and locomotion, sitting at the front of the cage while calmly observing activities, sleeping or resting while lying on their side, rubbing on cage items and friendly behaviour towards humans. Dimension 2 behaviours included persistent meowing, scanning, pacing and pushing, together with bouts of destructive behaviour, attempts to escape and redirected aggression. Dimension 2 was not significantly contrasted to dimension 3 (

Impact of oral probiotic Lactobacillus acidophilus vaccine strains on the immune response and gut microbiome of mice.

The potential role of probiotic bacteria as adjuvants in vaccine trials led to their use as nonparenteral live mucosal vaccine vectors. Yet, interactions between these vectors, the host and the microbiome are poorly understood. This study evaluates impact of three probiotic, Lactobacillus acidophilus, vector strains, and their interactions with the host's immune response, on the gut microbiome. One strain expressed the membrane proximal external region from HIV-1 (MPER). The other two expressed MPER and either secreted interleukin-1ß (IL-1ß) or expressed the surface flagellin subunit C (FliC) as adjuvants. We also used MPER with rice bran as prebiotic supplement. We observed a strain dependent, differential effect suggesting that MPER and IL-1β induced a shift of the microbiome while FliC had minimal impact. Joint probiotic and prebiotic use resulted in a compound effect, highlighting a potential synbiotic approach to impact efficacy of vaccination. Careful consideration of constitutive adjuvants and use of prebiotics is needed depending on whether or not to target microbiome modulation to improve vaccine efficacy. No clear associations were observed between total or MPER-specific IgA and the microbiome suggesting a role for other immune mechanisms or a need to focus on IgA-bound, resident microbiota, most affected by an immune response

Mucosal Immunogenicity of Genetically Modified <i>Lactobacillus acidophilus</i> Expressing an HIV-1 Epitope within the Surface Layer Protein

<div><p>Surface layer proteins of probiotic lactobacilli are theoretically efficient epitope-displaying scaffolds for oral vaccine delivery due to their high expression levels and surface localization. In this study, we constructed genetically modified <i>Lactobacillus acidophilus</i> strains expressing the membrane proximal external region (MPER) from human immunodeficiency virus type 1 (HIV-1) within the context of the major S-layer protein, SlpA. Intragastric immunization of mice with the recombinants induced MPER-specific and S-layer protein-specific antibodies in serum and mucosal secretions. Moreover, analysis of systemic SlpA-specific cytokines revealed that the responses appeared to be Th1 and Th17 dominant. These findings demonstrated the potential use of the <i>Lactobacillus</i> S-layer protein for development of oral vaccines targeting specific peptides.</p></div

Induction of MPER-specific antibody production by long-term immunization.

<p>Mice received GAD19 orally every 2 weeks for 14 weeks. (a) Diluted serum (1/100) was analyzed by ELISA at each time point. Arrows represent timing of the gavage. (b) Endpoint titers (or absorbance at 450 nm) of MPER-specific serum IgG, cecal IgA, vaginal IgA, and vaginal IgG. Each symbol represents an individual mouse.</p

Mucosal Immune Response to Feline Enteric Coronavirus Infection

Feline infectious peritonitis is a devastating, fatal disease of domestic cats caused by a pathogenic mutant virus derived from the ubiquitous feline enteric coronavirus (FECV). Infection by FECV is generally subclinical, and little is known about the mucosal immune response that controls and eliminates the virus. We investigated the mucosal immune response against FECV in an endemically infected breeding colony over a seven-month period. Thirty-three cats were grouped according to FECV seropositivity and fecal virus shedding into na&iuml;ve/immunologically quiescent, convalescent and actively infected groups. Blood, fecal samples and colon biopsies were collected to assess the mucosal and systemic immunologic and virologic profile. Results showed that cats with active FECV infections have strong systemic IgG and mucosal IgA responses that wane after virus clearance. Significant FECV-specific mucosal T cell IFN&gamma; responses were not detected in any of the three groups. A shift toward an inflammatory state in the mucosa was suggested by increased IL17:FoxP3 expression. However, no histologic abnormalities were observed, and no shifts in lymphocyte subpopulation phenotype or proliferation were noted. Together, the results suggest that control of FECV is mediated by humoral mucosal and systemic responses and that perturbations in the primary reservoir organ (colon) are minimal

Validation of genetically modified <i>L</i>. <i>acidophilus</i> producing MPER-displaying S-layer proteins.

<p>The <i>L</i>. <i>acidophilus slpA</i> gene in NCK1909 was replaced with the modified <i>slpA</i> gene including MPER-encoding sequences by homologous recombination in NCK2208. (a) The gene replacement of <i>slpA</i> with the modified <i>slpA</i> was confirmed by PCR. L, DNA ladder marker. Amplified DNA fragments using primers, AK_62 and AK_65 (lane 1 and 4), AK_62 and AK_57 (lane 2 and 5), or AK_56 and AK_65 (lane 3 and 6). (b) Detection of the MPER epitope in S-layer (SlpA) protein using 2F5 mAb. Total cell proteins and purified S-layer proteins of NCK1909 and NCK2208 were separated by SDS-PAGE. The gels were stained with CBB or blotted onto PVDF membrane followed by western blot analysis using 2F5 (anti-MPER monoclonal human IgG). (c) The exposed MPER epitope was detected by flow cytometry. The <i>L</i>. <i>acidophilus</i> strains labeled with 2F5 and Alexa Fluor 488-conjugated anti-human IgG were analyzed. Relative fluorescence intensity of each strain was shown as histogram plot.</p

Typing of classes and subclasses of MPER-specific antibodies.

<p>Sera from GAD19-immunized mice were analyzed by ELISA. Each value plus SD (standard deviation) was shown.</p

Desmoglein-1 is a minor autoantigen in dogs with pemphigus foliaceus

The majority of human patients with pemphigus foliaceus (PF) have circulating IgG autoantibodies that target conformational epitopes on the desmosomal cadherin desmoglein-1 (dsg1). Limited studies using immunoblot techniques suggested that the principal autoantigen in dogs with PF might also be dsg1. It was the objective of this study to test this hypothesis. A comprehensive survey of canine PF sera was conducted using a novel screening strategy that detects conformational epitopes. This method consists of the ectopic expression of canine dsg1 at the surface of human 293T epithelial kidney cells and their live screening, i.e. prior to fixation. Out of seven control human PF sera that bound to canine epidermis, three (57%) contained IgG autoantibodies that recognized ectopically expressed canine dsg1 with a membrane and punctate pattern. Out of 83 canine PF sera only five (6%) contained IgG that recognized canine dsg1. Consistent with findings for human PF sera obtained in this study, autoantibody binding was conformation- and glycosylation-dependent as demonstrated by calcium chelation with EDTA and tunicamycin or wheat germ agglutinin treatment, respectively. In conclusion, these studies establish canine dsg1 as a minor autoantigen for canine PF. Antigenic epitopes appear to be conformation- and glycosylation-dependent