Although cervical cancer is preventable with early detection, it remains the second most common malignancy among women. An understanding of how proteins change in their expression during a particular diseased state such as cervical cancer will contribute to an understanding of how the disease develops and progresses. Potentially, it may also lead to the ability to predict the occurrence of the disease. With this in mind, we aimed to identify differentially expressed proteins in the plasma of cervical cancer patients. Plasma from control, cervical intraepithelial neoplasia (CIN) grade 3 and squamous cell carcinoma (SCC) stage IV subjects was resolved by two-dimensional gel electrophoresis and the resulting proteome profiles compared. Differentially expressed protein spots were then identified by mass spectrometry. Eighteen proteins were found to be differentially expressed in the plasma of CIN 3 and SCC stage IV samples when compared with that of controls. Competitive ELISA further validated the expression of cytokeratin 19 and tetranectin. Functional analyses of these differentially expressed proteins will provide further insight into their potential role(s) in cervical cancer-specific monitoring and therapeutics.
Cryptocaryon irritans causes Cyptocaryonosis or white spot disease in a wide range of marine fish including Lates calcarifer (Asian seabass). However, the immune response of this fish to the parasite is still poorly understood. In this study, quantitative polymerase chain reaction (qPCR) was performed to assess the expression profile of immune-related genes in L. calcarifer infected by C. irritans. A total of 21 immune-related genes encoding various functions in the fish immune system were utilized for the qPCR analysis. The experiment was initiated with the infection of juvenile fish by exposure to theronts from 200 C. irritans cysts, and non-infected juvenile fish were used as controls. Spleen, liver, gills and kidney tissues were harvested at three days post-infection from control and infected fish. In addition, organs were also harvested on day-10 post-infection from fish that had been allowed to recover from day-4 up to day-10 post-infection. L. calcarifer exhibited pathological changes on day-3 post-infection with the characteristic presence of white spots on the entire fish body, excessive mucus production and formation of a flap over the fish eye. High quality total RNA was extracted from all tissues and qPCR was performed. The qPCR analysis on the cohort of 21 immune-related genes of the various organs harvested on day-3 post-infection demonstrated that most genes were induced significantly (p C. irritans infection (10 days post-infection), expression of the immune-related genes was down-regulated to levels similar to the control fish. These results provide insights into the interaction between C. irritans and L. calcarifer and suggest that the innate immune system plays an important role in early defence against parasite infection allowing the fish to eventually recover from the infection.
The development of oral vaccine formulation is crucial to facilitate an effective mass immunization program for various vaccine-preventable diseases. In this work, the efficacy of hepatitis B antigen delivered by bacterial nanocellulose/poly(acrylic acid) composite hydrogel microparticles (MPs) as oral vaccine carriers was assessed to induce both local and systemic immunity. Optimal pH-responsive swelling, mucoadhesiveness, protein drug loading, and drug permeability were characterized by MPs formulated with minimal irradiation doses and acrylic acid concentration. The composite hydrogel materials of bacterial nanocellulose and poly(acrylic acid) showed significantly greater antigen release in simulated intestinal fluid while ensuring the integrity of antigen. In in vivo study, mice orally vaccinated with antigen-loaded hydrogel MPs showed enhanced vaccine immunogenicity with significantly higher secretion of mucosal immunoglobulin A, compared to intramuscular vaccinated control. The splenocytes from the same group demonstrated lymphoproliferation and significant increased secretion of interleukin-2 cytokines upon stimulation with hepatitis B antigen. Expression of CD69 in CD4+ T lymphocytes and CD19+ B lymphocytes in splenocytes from mice orally vaccinated with antigen-loaded hydrogel MPs was comparable to that of the intramuscular vaccinated control, indicating early activation of lymphocytes elicited by our oral vaccine formulation in just two doses. These results demonstrated the potential of antigen-loaded hydrogel MPs as an oral vaccination method for hepatitis B.
Hepatitis B virus (HBV) infection is a major cause of chronic liver disease that may progress to liver cirrhosis and hepatocellular carcinoma. Host immune responses represent the key determinants of HBV clearance or persistence. Here, we investigated the role of the early activation marker, CD69 and effector cytokines, granzyme B (GrB) and IFN-γ in the exhaustion of innate-like TCR Vα7.2+CD4+T cells, in 15 individuals with chronic HBV (CHB) infection where six were HBV DNA+ and nine were HBV DNA-. The percentage of cytokine-producing T cells and MAIT cells were significantly perturbed in HBV patients relative to healthy controls (HCs). The intracellular expression of GrB and IFN-γ was significantly reduced in MAIT cells derived from HBV-infected patients as compared to HCs, and the levels correlated with the percentage and levels [mean fluorescence intensity (MFI)] of CD69 expression. The total expression of CD69 (iMFI) was lower in CHB patients as compared to HCs. The frequency of CD69+ cells correlated with the levels of cytokine expression (MFI), particularly in CHB patients as compared to HCs. In summary, the polyfunctionality of peripheral T cells was significantly reduced among CHB patients, especially in the TCR Vα7.2+CD4+T cells, and the levels of cytokine expression correlated with functional cytokine levels.
Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy.