Identification of a Dual-Specific T Cell Epitope of the Hemagglutinin Antigen of an H5 Avian Influenza Virus in Chickens

Avian influenza viruses (AIV) of the H5N1 subtype have caused morbidity and mortality in humans. Although some migratory birds constitute the natural reservoir for this virus, chickens may play a role in transmission of the virus to humans. Despite the importance of avian species in transmission of AIV H5N1 to humans, very little is known about host immune system interactions with this virus in these species. The objective of the present study was to identify putative T cell epitopes of the hemagglutinin (HA) antigen of an H5 AIV in chickens. Using an overlapping peptide library covering the HA protein, we identified a 15-mer peptide, H5246–260, within the HA1 domain which induced activation of T cells in chickens immunized against the HA antigen of an H5 virus. Furthermore, H5246–260 epitope was found to be presented by both major histocompatibility complex (MHC) class I and II molecules, leading to activation of CD4+ and CD8+ T cell subsets, marked by proliferation and expression of interferon (IFN)-γ by both of these cell subsets as well as the expression of granzyme A by CD8+ T cells. This is the first report of a T cell epitope of AIV recognized by chicken T cells. Furthermore, this study extends the previous finding of the existence of dual-specific epitopes in other species to chickens. Taken together, these results elucidate some of the mechanisms of immune response to AIV in chickens and provide a platform for creation of rational vaccines against AIV in this species

Expression of Antimicrobial Peptides in Cecal Tonsils of Chickens Treated with Probiotics and Infected with Salmonella enterica Serovar Typhimurium ▿

Several strategies currently exist for control of Salmonella enterica serovar Typhimurium colonization in the chicken intestine, among which the use of probiotics is of note. Little is known about the underlying mechanisms of probiotic-mediated reduction of Salmonella colonization. In this study, we asked whether the effect of probiotics is mediated by antimicrobial peptides, including avian beta-defensins (also called gallinacins) and cathelicidins. Four treatment groups were included in this study: a negative-control group, a probiotic-treated group, a Salmonella-infected group, and a probiotic-treated and Salmonella-infected group. On days 1, 3, and 5 postinfection (p.i.), the cecal tonsils were removed, and RNA was extracted and used for measurement of avian beta-defensin 1 (AvBD1), AvBD2, AvBD4, AvBD6, and cathelicidin gene expression by real-time PCR. The expressions of all avian beta-defensins and cathelicidin were detectable in all groups, irrespective of treatment and time point. Probiotic treatment and Salmonella infection did not affect the expression of any of the investigated genes on day 1 p.i. Furthermore, probiotic treatment had no significant effect on the expression of the genes at either 3 or 5 days p.i. However, the expression levels of all five genes were significantly increased (P < 0.05) in response to Salmonella infection at 3 and 5 days p.i. However, administration of probiotics eliminated the effect of Salmonella infection on the expression of antimicrobial genes. These findings indicate that the expression of antimicrobial peptides may be repressed by probiotics in combination with Salmonella infection or, alternatively, point to the possibility that, due to a reduction in Salmonella load in the intestine, these genes may not be induced

Probiotics Stimulate Production of Natural Antibodies in Chickens

Commensal bacteria in the intestine play an important role in the development of immune response. These bacteria interact with cells of the gut-associated lymphoid tissues (GALT). Among cells of the GALT, B-1 cells are of note. These cells are involved in the production of natural antibodies. In the present study, we determined whether manipulation of the intestinal microbiota by administration of probiotics, which we had previously shown to enhance specific systemic antibody response, could affect the development of natural antibodies in the intestines and sera of chickens. Our findings demonstrate that when 1-day-old chicks were treated with probiotics, serum and intestinal antibodies reactive to tetanus toxoid (TT) and Clostridium perfringens alpha-toxin in addition to intestinal immunoglobulin A (IgA) reactive to bovine serum albumin (BSA) were increased in unimmunized chickens. Moreover, IgG antibodies reactive to TT were increased in the intestines of probiotic-treated chickens compared to those of untreated controls. In serum, IgG and IgM reactive to TT and alpha-toxin were increased in probiotic-treated, unimmunized chickens compared to levels in untreated controls. However, no significant difference in serum levels of IgM or IgG response to BSA was observed. These results are suggestive of the induction of natural antibodies in probiotic-treated, unimmunized chickens. Elucidating the role of these antibodies in maintenance of the chicken immune system homeostasis and immune response to pathogens requires further investigation

Photophysical Properties and Kinetic Studies of 2-Vinylpyridine-Based Cycloplatinated(II) Complexes Containing Various Phosphine Ligands

A series of cycloplatinated(II) complexes with general formula of [PtMe(Vpy)(PR3)], Vpy = 2-vinylpyridine and PR3 = PPh3 (1a); PPh2Me (1b); PPhMe2 (1c), were synthesized and characterized by means of spectroscopic methods. These cycloplatinated(II) complexes were luminescent at room temperature in the yellow–orange region’s structured bands. The PPhMe2 derivative was the strongest emissive among the complexes, and the complex with PPh3 was the weakest one. Similar to many luminescent cycloplatinated(II) complexes, the emission was mainly localized on the Vpy cyclometalated ligand as the main chromophoric moiety. The present cycloplatinated(II) complexes were oxidatively reacted with MeI to yield the corresponding cycloplatinated(IV) complexes. The kinetic studies of the reaction point out to an SN2 mechanism. The complex with PPhMe2 ligand exhibited the fastest oxidative addition reaction due to the most electron-rich Pt(II) center in its structure, whereas the PPh3 derivative showed the slowest one. Interestingly, for the PPhMe2 analog, the trans isomer was stable and could be isolated as both kinetic and thermodynamic product, while the other two underwent trans to cis isomerization

Highly Emissive Cycloplatinated(II) Complexes Obtained by the Chloride Abstraction from the Complex [Pt(ppy)(PPh3)(Cl)]:Employing Various Silver Salts

In the present investigation, the precursor complex [Pt(ppy)(PPh3)(Cl)], 3, ppy = 2-phenylpyridinyl, undergoes the chloride abstraction reaction using various AgX (X = PF6, BF4, NO3 and CH3COO) salts. Depending on the nature of anions in AgX salts (coordinating or noncoordinating), the products can be neutral or ionic. In the cases of NO3 and CH3COO, they can be coordinated to the Pt center so that the neutral complexes [Pt(ppy)(PPh3)(NO3)], 4a, and [Pt(ppy)(PPh3)(CH3COO)], 4b, are formed. In contrast, the ionic complexes [{Pt(ppy)(PPh3)(CH3CN)}PF6], 5a, and [{Pt(ppy)(PPh3)(CH3CN)}BF4], 5b, can be generated when the AgPF6 ([{Ag(CH3CN)4}PF6]) or AgBF4 ([{Ag(CH3CN)4}BF4]) salts are used in which the PF6 and BF4 stand as counteranions. In these two complexes, CH3CN fills the empty ligand position which can be present as solvent or ligand in the initial silver salts. The structures of the new complexes were accurately deduced from the multinuclear (1H, 31P{1H}, 195Pt{1H}) NMR spectroscopy and further authenticated by X-ray crystallography. Interestingly, the complexes are green emitters under various states and temperature conditions for which nonchelating L/X (PPh3/NO3 or PPh3/CH3COO) and L/L (PPh3/CH3CN) ancillary ligands exist in the structure of cycloplatinated(II) complexes. The photophysical properties of these new complexes, supported by density functional theory (DFT) and time-dependent DFT (TD-DFT) calculations, were investigated by photoluminescence and UV–vis spectroscopies

Cytokine mRNA expression in splenocytes in response to H5_246–260.

<p>In three replicate experiments, spleen mononuclear cells from vaccinated birds were incubated with peptide or peptide solvent for 24 hours before RNA extraction and cytokine analysis using real-time PCR. Relative expression of IFN-γ (A), IL-10 (B) mRNA normalized to β-actin expression in stimulated and unstimulated spleen mononuclear cells are presented. Comparisons were made using the REST program and considered significant at P≤0.05 (*).</p

Proliferative response to H5_246–260 in sorted CD4+ and CD8+ spleen cells.

<p>Spleen mononuclear cells from vaccinated birds, in 6 replicate experiments, were sorted into CD4+ and CD8+ T cell subsets followed by co-culture with APC and peptide, H5_246–260 (10 µM) at an APC to responder ratio of either 2∶1 or 10∶1. T cell subsets were also cultured with or without the peptide in the absence APC (no APC). In another control group (APC alone), APCs were cultured in the absence or presence of the peptide. Proliferative response was measured at 72 hours. Results for CD4+ (A) and CD8+ (B) are presented as an average stimulation index, (mean cpm of treatment/mean of control for all independent experimental replicates).</p

Sequence Alignment of B7 peptide from HA of A/turkey/Ireland/1378/83 (H5N8) subtype with the corresponding peptides derived from HA of other influenza virus subtypes.

<p>Bold-faced residues represent differences in the sequence of corresponding peptides compared to H5_246–260.</p

Granzyme A and perforin mRNA expression in CD8+ T cell subsets.

<p>Spleen cells from vaccinated birds in three replicate experiments, were co-cultured with peptide solvent or peptide, H5_246–260 (10 µM) for 24 hours followed by magnetic cell sorting into CD4+ and CD8+ T cell subsets. Sorted cells were used for RNA extraction and cytokine analysis using real-time PCR. Relative expression of granzyme A (A), perforin (B) mRNA expression normalized to β-actin in CD4+ and CD8+ T cells treated with the peptide or peptide solvent are presented. Comparisons between peptide-treated and control cells were made using the REST program and considered significant at P≤0.05 (*).</p