Amino-Functionalized Nitrogen-Doped Graphene Quantum Dots for Efficient Enhancement of Two-Photon-Excitation Photodynamic Therapy: Functionalized Nitrogen as a Bactericidal and Contrast Agent.

BACKGROUND: Although graphene quantum dots (GQDs) have received considerable research attention for their applications in various fields, the use of GQDs, such as nitrogen-doped GQDs (N-GQDs) and amino-functionalized N-GQDs (amino-N-GQDs), as photosensitizers to facilitate photodynamic therapy (PDT) has received limited research intention. To address this research gap, this study prepared novel amino-N-GQDs and investigated their properties. METHODS: The amino-N-GQDs subjected to two-photon excitation (TPE) exhibited remarkable bactericidal capability in PDT. The bonding compositions of nitrogen and the amino-functionalized group played a critical role in their antimicrobial effects. RESULTS: Compared with amino-group-free N-GQDs and amino-N-free GQDs, the amino-N-GQDs generated a higher amount of reactive oxygen species, demonstrating their superior efficacy for two-photon PDT. Additionally, the intrinsic luminescence properties and high photostability of the amino-N-GQDs demonstrate their suitability as an effective two-photon contrast agent for tracking bacteria during two-photon biomedical imaging. CONCLUSION: The amino-N-GQD and their remarkable properties may provide an efficient alternative approach for observing and easily eliminating malignant microbes in the future.

Significant mucosal sIgA production after a single oral or parenteral administration using in vivo CD40 targeting in the chicken.

Many pathogens enter the host through mucosal surfaces and spread rapidly via the circulation. The most effective way to prevent disease is to establish mucosal and systemic immunity against the pathogen. However, current vaccination programs in poultry industry require repeated administrations of live-attenuated virus or large amounts (10 to 100µg) of antigen together with adjuvant to induce specific secretory IgA immune responses at the mucosal effector sites. In the present study, we show that a single administration of 0.4µg of oligopeptide complexed with an agonistic anti-chicken CD40 (chCD40) monoclonal antibody (Mab) effectively targets antigen-presenting cells of the bird's mucosa-associated lymphoid tissue in vivo, and induces peptide-specific secretory IgA (sIgA) in the trachea 7days post administration. Anti-chCD40 Mab-peptide complex was administered once to four-week old male Leghorns via various mucosal routes (orally, via cloacal drinking, or oculo-nasally) or via subcutaneous (s.c.) immunization. Immunization through any of the three mucosal induction routes induced significant peptide-specific mucosal sIgA responses 7 and 14days after immunization. Interestingly, s.c. injection of the complex also induced mucosal sIgA. Our data suggest in vivo targeting of CD40 as a potential adjuvant platform, particularly for the purpose of enhancing and speeding up mucosal vaccine responses in chickens, and potentially other food animals. This is the first study able to elicit specific sIgA immune responses in remote mucosal sites with a single administration of only 0.4µg of antigen.

Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens.

The African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (CTLs) could be the key to complete protection. Hence, generation of an efficacious subunit vaccine depends on identification of CTL targets along with a suitable delivery method that will elicit effector CTLs capable of eliminating ASFV-infected host cells and confer long-term protection. To this end, we evaluated the safety and immunogenicity of an adenovirus-vectored ASFV (Ad-ASFV) multiantigen cocktail formulated in two different adjuvants and at two immunizing doses in swine. Immunization with the cocktail rapidly induced unprecedented ASFV antigen-specific antibody and cellular immune responses against all of the antigens. The robust antibody responses underwent rapid isotype switching within 1 week postpriming, steadily increased over a 2-month period, and underwent rapid recall upon boost. Importantly, the primed antibodies strongly recognized the parental ASFV (Georgia 2007/1) by indirect fluorescence antibody (IFA) assay and Western blotting. Significant antigen-specific gamma interferon-positive (IFN-γ+) responses were detected postpriming and postboosting. Furthermore, this study is the first to demonstrate induction of ASFV antigen-specific CTL responses in commercial swine using Ad-ASFV multiantigens. The relevance of the induced immune responses in regard to protection needs to be evaluated in a challenge study.

Effect of Size-Dependent Photodestructive Efficacy by Gold Nanomaterials with Multiphoton Laser.

The photostability, photodestructive efficacy, two-photon excitation cross section, and two-photon fluorescence of gold nanoparticles conjugated with a hydrophilic photosensitizer, indocyanine green, via multiphoton laser exhibited an increased size effect in methicillin-resistant Staphylococcus aureus and A549 cancer cells that was dependent on the size of multifunctional gold nanomaterials, but the effect only occurred when nanomaterials within 100 nm in diameter were used. Besides, the enhanced effectiveness of photodestruction, photostability, and contrast probe indicated an additive effect in the therapeutic and imaging efficiency of multifunctional gold nanomaterials. Consequently, the preparation of the multifunctional gold nanomaterials and their use in biomedical applications via multiphoton laser is an alternative and potential therapeutic approach for killing bacteria and for ablating cancer cells.

Immunization of chickens with an agonistic monoclonal anti-chicken CD40 antibody-hapten complex: rapid and robust IgG response induced by a single subcutaneous injection.

Producing diagnostic antibodies in chicken egg yolk represents an alternate animal system that offers many advantages including high productivity at low cost. Despite being an excellent counterpart to mammalian antibodies, chicken IgG from yolk still represents an underused resource. The potential of agonistic monoclonal anti-CD40 antibodies (mAb) as a powerful immunological adjuvant has been demonstrated in mammals, but not in chickens. We recently reported an agonistic anti-chicken CD40 mAb (designated mAb 2C5) and showed that it may have potential as an immunological adjuvant. In this study, we examined the efficacy of targeting a short peptide to chicken CD40 [expressed by the antigen-presenting cells (APCs)] in enhancing an effective IgG response in chickens. For this purpose, an immune complex consisting of one streptavidin molecule, two directionally biotinylated mAb 2C5 molecules, and two biotinylated peptide molecules was produced. Chickens were immunized subcutaneously with doses of this complex ranging from 10 to 90 µg per injection once, and relative quantification of the peptide-specific IgG response showed that the mAb 2C5-based complex was able to elicit a strong IgG response as early as four days post-immunization. This demonstrates that CD40-targeting antigen to chicken APCs can significantly enhance antibody responses and induce immunoglobulin isotype-switching. This immunization strategy holds promise for rapid production of hapten-specific IgG in chickens.

Production and characterization of agonistic monoclonal antibodies against chicken CD40.

CD40 is mainly expressed by professional antigen-presenting cells (APCs). Its ligand, CD40L, is transiently expressed on activated CD4(+) T-cells. CD40-CD40L interactions mediate T-cell help to APCs and provide crucial signals for affinity maturation and B-cell class switching. In mammals, agonistic monoclonal anti-CD40 antibodies (mAbs) mimic the effects of CD40L on APCs, leading to enhanced T-cell priming and expansion, increased antibody production and class switching. In this study, we describe agonistic anti-chicken CD40 mAb 2C5. This mAb detected CD40 on primary chicken B-cells and macrophages, DT40 B-cells, and HD11 macrophages, induced NO synthesis in HD11 macrophages, and stimulated DT40 B-cell proliferation. These observations demonstrated at least partial functional equivalence of 2C5 to chicken CD154. This mAb may therefore constitute a new tool to study the role of CD40 in the chicken immune system, and its agonistic effects suggest that it could also be used as an adjuvant.