Infectious agents such as bacteria or viruses can grow rapidly. If a microorganism invades a host, it must be recognized rapidly and destroyed before it overwhelms the immune system. Limiting infection to a minimum in the early stage is critical for the outcome and the recovery from infection. The innate immune system has evolved to recognize a few highly conserved, constitutive structures present only in microorganisms, such as bacterial lipopolysaccharide (LPS), called pathogen-associated molecular patterns (PAMP). Toll-like receptors are the host receptors that recognize PAMP, ultimately activating a variety of transcription factors to induce expression of a wide spectrum of immune related genes, e.g. defensins. Defensins are antimicrobial peptides that play an important role in innate defense against microorganisms in plants and animals. Beta-defensins are the largest family of antimicrobial peptides, which can directly kill microorganisms and have regulatory effects on the immune system. Thirteen beta-defensins have been identified; however, the regulation of these genes has not been well-investigated in the chicken. The objective of this research was to understand constitutive and inducible gene expression of beta-defensins in chicken white blood cells. Real-time RT-PCR was used to quantify gene expression level before and after LPS stimulation. Transcription factor binding sites in the genes were identified to understand the gene expression regulation. From the expression profile results, most chicken beta-defensins had induced gene expression by LPS stimulation in the early phase (0- to 3-hour) and reduced gene expression in the late phase (3- to 8-hour). As for the level of gene expression, the results show that the induced gene expression in the early phase corresponded to the higher levels of expression at 3-hours after LPS stimulation, and the reduced gene expression in the late phase corresponded to the lower levels of gene expression at 8-hours after LPS stimulation.

Bovine herpesvirus-1 (BoHV-1) causes recurrent respiratory and genital infections in cattle; and predisposes them to lethal secondary bacterial infections. Vaccination is a primary strategy to prevent and reduce the severity of disease associated with BoHV-1, and to reduce virus transmission. While modified live (MLV) or killed (KV) BoHV-1 vaccines exist, these are expensive to produce, can cause disease (MLV) or may be ineffective (KV). Development of a DNA vaccine for BoHV-1 has the potential to address these shortcomings, but the very small amount of antigen expressed after DNA immunization presents a barrier to successful immunization of large animals. Engineering the vaccine to target this limited quantity of antigen to dendritic cells (DCs), the cells that prime immune responses, by attracting immature DCs (iDCs) to the vaccination site, is one way that DNA vaccine efficacy might be improved. Beta (β)-defensins are chemotactic peptides that, in studies with mice, improve induction of immune responses to DNA vaccines and this is due, at least in part, to their ability to attract iDCs to the site of vaccination. Accordingly, the objective of the studies described in this thesis was to determine whether using a bovine β-defensin in a DNA vaccine would enhance immune responses to the vaccine and subsequently protect cattle upon challenge with BoHV-1. First I characterized the bovine iDC and then used these cells to screen a panel of synthesized bovine β-defensins for chemotactic activity. The results showed that bovine neutrophil β-defensin (BNBD) 3, BNBD9 and enteric β-defensin (EBD) were equally the most chemotactic of the fourteen synthesized peptides for bovine iDCs. Because BNBD3 is the most abundant of the thirteen BNBDs and was able to attract CD1+ DCs when injected into the skin, I chose BNBD3 as the peptide I would use for the rest of the project. Next I constructed plasmids that expressed BNBD3; either alone or as a fusion construct with the BoHV-1 antigen truncated glycoprotein D (tgD), and then tested the effects of the plasmids as vaccines in both mice and cattle. In cattle, the addition of BNBD3 as a fusion strengthened the Th1 bias and increased cell-mediated immune responses to the DNA vaccine but not antibody response or protection from BoHV-1 infection. Given that inefficient humoral immune responses have been implicated in a lack of protection from BoHV-1 challenge, these results suggested that the successful BoHV-1 DNA vaccine would need to induce a much stronger humoral response. Lastly I assessed the ability of BNBD3 to improve humoral responses to pMASIA-tgD when complexed with the DNA vaccine and found that the vaccine complexed at a nanomolar peptide to DNA ratio of 125:1 increased humoral responses of mice. In vitro, treatment of mouse bone-marrow DCs with BNBD3 induced phenotypic and functional maturation/activation. This is an important aspect for vaccination in the skin, since after uptake, the DC must “mature” in order to traffic from the site of vaccination to the draining lymph node where induction of antigen-specific responses, by activated DCs, takes place. The findings in this thesis show that bovine β-defensins are chemotactic for bovine iDCs. I also show that using a bovine β-defensin as a fusion construct in a DNA vaccine enhances cell mediated but not humoral responses of cattle and yet this vaccine is protective against BoHV-1 challenge. I demonstrate that a bovine β-defensin, when used as a peptide to complex an antigen-encoding plasmid, can increase humoral responses. My work shows a multifunctional ability of bovine β-defensins to modulate and increase immune responses and suggests that bovine β-defensins likely have further untapped potential to enhance efficacy of DNA vaccines for large animals.

Thesis (Ph. D.)--Case Western Reserve University, 2007. / [School of Medicine] Department of Molecular Biology and Microbiology. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Host defense peptides (HDPs) are a large group of small positively charged peptides that play an important role in innate immunity. Their role is more critical at early ages when other components of the immune system have not fully developed. There are three classes of avian HDPs: avian beta defensins (AvBDs), cathelicidins (Cath) and liver-expressed antimicrobial peptide 2 (LEAP-2). The objective was to compare expression of HDPs in male turkey poults at day of hatch (D0), D7, D14, D21 and D28 from the thymus, spleen, bursa, duodenum, jejunum and ileum. The expression of AvBD1, AvBD2, AvBD8, AvBD9, AvBD10, AvBD13, Cath2, Cath3 and LEAP-2 was measured using qPCR (n=6 birds/tissue/age). Data were analyzed by one-way ANOVA and Tukey's test, and significance considered at P ≤ 0.05. AvBDs and Caths exhibited greater expression in immune organs than intestinal tissues, with the greatest expression of AvBDs observed in the spleen. The intestinal tissues showed very low expression of AvBDs except for AvBD10 at D0. Similar to AvBDs, Caths expression in the immune organs was greater than the intestinal tissues with the spleen having the greatest expression among immune organs. Conversely, LEAP-2 showed greater expression in the intestinal tissues than in the immune tissues, which showed very low LEAP-2 expression unlike other HDPs. Understanding the differential expression of HDPs could reveal the innate immune status of poults, and may subsequently allow improvement of their health through appropriate mitigation strategies. / Master of Science

Orientador: Daniela Leal Zandim Barcelos / Resumo: O propósito deste estudo foi determinar a influência do tabagismo nos níveis de beta-defensina 3 (hBD 3) no fluido crevicular gengival (FCG) de indivíduos com periodontite crônica e avaliar sua relação com saúde e doença periodontal. Além disso, foi investigada a correlação deste peptídeo antimicrobiano com metaloproteinase da matriz (MMP-8) e citocinas pró (IL-10) e anti-inflamatórias (IL-1β, IFN-γ e TNF-α). Um total de 40 indivíduos com periodontite crônica, sendo 20 fumantes (PF) e 20 não fumantes (PNF), e 20 indivíduos sem doença periodontal (S) foram incluídos no estudo. Amostras de FCG de sítios sadios e doentes dos indivíduos com periodontite, e apenas de sítios sadios dos indivíduos periodontalmente saudáveis, foram coletadas com tiras de papel absorvente. A quantificação da hBD 3 foi feita pela técnica ELISA sanduíche e dos marcadores biológicos por ensaio Multiplex. Níveis significativamente menores de hBD 3 foram identificados nos sítios doentes de PF em comparação aos sítios doentes de PNF (p=0,02). Por outro lado, os níveis de hBD 3 nos sítios sadios de PF foram significativamente maiores que nos sítios sadios de PNF e S (p=0,006). Na comparação dos sítios dentro do grupo PF, foi verificado que os sítios doentes apresentavam níveis reduzidos de hBD 3 em comparação com sítios sadios (p=0,04). Já no grupo PNF, os níveis de hBD 3 foram mais elevados nos sítios doentes que nos sítios sadios (p=0,02). Uma correlação negativa foi observada entre os níveis de hBD 3 e MM... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The purpose of this study was to determine the influence of smoking on gingival crevicular fluid (GCF) levels of beta-defensin 3 (hBD 3) in individuals with chronic periodontitis and to evaluate its relationship with health and periodontal disease. In addition, the correlation of this antimicrobial peptide with matrix metalloproteinase (MMP-8) and pro (IL-10) and anti-inflammatory cytokines (IL-1β, IFN-γ and TNF-α) was investigated. A total of 40 individuals with chronic periodontitis, including 20 smokers (PS) and 20 non-smokers (PNS), and 20 subjects without periodontal disease (H) were included in the study. GCF samples from healthy and diseased sites of individuals with periodontitis, and only from healthy sites of periodontally healthy individuals, were collected with absorbent paper strips. Quantification of hBD 3 was performed by a sandwich ELISA assay and the biological markers levels were analyzed by a multiplex assay. Significantly lower levels of hBD 3 were identified in diseased sites of PS compared to diseased sites of PNS (p = 0.02). On the other hand, the levels of hBD 3 in healthy sites of PS were significantly higher than in healthy sites of PNS and H (p = 0.006). The comparison between the sites within the groups with periodontitis showed reduced levels of hBD 3 in diseased sites of PS compared to healthy sites (p = 0.04), while higher levels of this peptide was detected in diseased sites of PNS compared to healthy sites (p = 0.02). A negative correlation wa... (Complete abstract click electronic access below) / Mestre

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 121-133).

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 111-134).

Porphyromonas gingivalis produces proteases capable of degrading cytokines, host heme proteins, and some antimicrobial peptides. In this work, I show that P. gingivalis culture supernatants fully or partially degrade human neutrophil peptide alpha-defensins and human beta-defensins after 30 minutes. This observation suggests that proteases from P. gingivalis degrade defensins and this activity could abrogate defensin-related innate immune functions.

Diss. (sammanfattning) Umeå : Univ., 2007. / Härtill 4 uppsatser.

Made available in DSpace on 2015-03-26T13:47:13Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1442239 bytes, checksum: 5f0041bc266aa2b219384708dc4dd876 (MD5) Previous issue date: 2012-11-20 / In response to an infection, blood cells such as neutrophils produce defensins within minutes to two hours to aid pathogens lysis and stimulate chemotaxis, reaching peak levels in the body in 24 hours. These peptides can also be produced without any stimulus by epithelial cells of the integumentary system. The aim of this work was to study the expression of β-defensins (cBD102 and cBD103) of canine skin diagnosed with canine visceral leishmaniasis (CVL). There were selected 43 skin samples from dogs positive to CVL and healthy, they were included in paraffin blocks and divided into three groups: I desquamative dermatitis (DD) with 16 blocks, II ulcerative dermatitis (UD) 21 blocks and the control group (6 blocks without canine skin problems). It was used the indirect immunoperoxidase technique (IIP) to confirm the LVC diagnosis and the identification of β-defensins, using human primary antibodies (hBD-2 and hBD-3, SIGMA®). The experimental delineation was completely casualized, with a confidence level of 0.95. Note that there was difference (p = 0.03) in the immunostaining intensity between groups, showing higher marcation intensity with cBD102 and cBD103 in the epidermis of the control group compared with DD and DU groups. In the dermis there was difference (p = 0.001) of the two β-defensins studied, being the cBD103 expression higher than the cBD102. This study is the first one relating to the immunological interaction of the extension and intensity expression between the β-defensin cBD102 and cBD103 and canine visceral leishmaniasis in the DD and DU dermatological standards, assessed by immunohistochemistry. / Em resposta a uma infeção as defensinas são produzidas em questão de minutos ou até 2 horas por células sanguíneas como neutrófilos, para auxiliar a lise de patógenos e estimular a quimiotaxia, alcançando níveis máximos no organismo em 24 horas. Também estes peptídeos podem ser produzidos sem estimulo algum pelas células epiteliais do sistema tegumentário. Com base no exposto anteriormente, o objetivo deste trabalho foi estudar a expressão de β-defensinas (cBD102 e cBD103) de pele de cães diagnosticados com leishmaniose visceral canina (LVC). Para tal, foram selecionados 43 amostras de pele entre cães acometidos por LVC e sadios incluídas em blocos de parafina, distribuídos em 3 grupos: I com dermatite descamativa (DD) por 16 blocos, II dermatite ulcerativa (DU) 21 blocos e o grupo controle (6 blocos de cães sem problemas dermatológicos). Foi empregada a técnica de imunoperoxidase indireta (IPI) para a confirmação do diagnóstico de LVC e identificação de β-defensinas, usando os anticorpos primários humanos (hBD102 e hBD103, SIGMA®). O delineamento foi inteiramente casualizado, adotando 0,05% de probabilidade para o erro Tipo I. Nota-se que houve diferença (p=0,003) na intensidade da imunomarcação entre os grupos, observando-se maior intensidade de marcação da cBD102 e cBD103 no grupo controle quando comparados com os grupos DD e DU. Na derme houve diferença (p=0,001) entre a expressão das duas β-defensinas estudadas, sendo a expressão de cBD103 superior a de cBD102. O presente estudo é o primeiro referente à interação imunológica da expressão de extensão e intensidade entre a β-defensina cBD102 e cBD103 e a leishmaniose visceral canina, nos padrões dermatológicos DD e DU, avaliados pela técnica de imunoistoquímica.