Investigation of Two Precipitation Methods for Extracting Immunoglobulin Y (IgY) from Egg Yolks

Two groups of hens (control and immunization group) were arranged in an experimental design with an immunization schedule of 3 injections of BSA antigen. IgY antibodies were extracted from egg yolks by two precipitation processes (chloroform and polyethylene glycol precipitates) and quantified using a standard curve of protein concentration. The purification of IgYwas confirmed by SDS-PAGE. Total protein extracted from egg yoks were less contaminated with yellow pigments (lutein and zeaxanthin) by using chloroform precipitate. The 2nd week post-immunization, IgY concentration increased respectively to 3903 ± 726 μg.ml-1 (chloroform extraction process) and 2937 ± 294 μg.ml-1 (PEG extraction process) (P < 0.01). After 3rdimmunization, IgY level obtaining from in immunization group extracted by chloroform process (6633 ± 1166 μg.ml-1) increased 2.7 times higher than that in control group (2482 ± 414 μg.ml-1). Whereas IgY concentrations obtained from PEG extraction process were not significantly different between the experimental group and control group. Chloroform and PEG precipitation methods had the same protein profile on the SDSPAGE. IgY antibody was identified by the presence of bands corresponding with IgY heavy chain (67-70 kDa) and IgY light chain (25 kDa) for both precipitation processes.

To compare the impact of sitting and supine position on behavioral distress during immunization among children (1-3 years) in selected immunization clinics

Background of the study: Immunizations cause distress in children due to its acute pain. Younger children are particularly in need of intervention because they report more pain and display more behavioral distress during painful procedures. One of the effective non-pharmacological interventions of acute management is comfort position given by the parent. Comfort position provided by the parent during immunizations has been demonstrated to be useful in infants, toddlers and pre-school children. Yet, this simple intervention is not used on a routine basis. Aim: The aim of the study was to compare the impact of sitting and supine position on behavioral distress during immunization among children (1-3 years) in selected immunization clinics. Objectives of the study: To determine the impact of sitting position on behavioral distress of children receiving immunization (Group I - experimental group). 1. To determine the impact of supine position on behavioral distress of children receiving immunization (Group II - control group). 2. To compare the impact of sitting and supine position on behavioral distress of children during immunization. Methods: The research design adopted for the study was post test only control group design. Immunization clinic selected based on the convenience of the investigator. Purposive sampling technique used to select the sample and the sample were assigned randomly in to Group I(experimental group) and Group II(control group).To assess the impact of position: PBRS-R was used to assess the behavioral distress of children during immunization injection. Results: The collected data was analyzed by descriptive and inferential statistics. 1. Assessment of behavioral distress scores during immunization injection showed significant difference in mean scores in Group I (16.4±2.30) and in Group II (28.45±2.59). 2. Comparison of behavioral distress scores in Group I and Group II showed that there is a significant difference (p<0.05) in behavioral distress between experimental (Group I) and control (Group II) group. 3. Item wise comparison of behavioral distress scores in Group I and Group II showed that there is no significant difference(p<0.05) in behavioral distress between experimental (Group I) and control (Group II) group. Interpretation and conclusion: Findings of the study revealed that the comfort position, i.e., sitting position was effective in reducing behavioral distress during immunisation. Hence, paediatric nurses ought to promote the use of comfort position to reduce behavioral distress associated with painful procedure.

Evolution of anti-modified protein antibody responses can be driven by consecutive exposure to different post-translational modifications

Background Besides anti-citrullinated protein antibodies (ACPA), rheumatoid arthritis patients (RA) often display autoantibody reactivities against other post-translationally modified (PTM) proteins, more specifically carbamylated and acetylated proteins. Immunizing mice with one particular PTM results in an anti-modified protein antibody (AMPA) response recognizing different PTM-antigens. Furthermore, human AMPA, isolated based on their reactivity to one PTM, cross-react with other PTMs. However, it is unclear whether the AMPA-reactivity profile is “fixed” in time or whether consecutive exposure to different PTMs can shape the evolving AMPA response towards a particular PTM. Methods Longitudinally collected serum samples of 8 human individuals at risk of RA and 5 with early RA were tested with ELISA, and titers were analyzed to investigate the evolution of the AMPA responses over time. Mice (13 per immunization group in total) were immunized with acetylated (or carbamylated) protein (ovalbumin) twice or cross-immunized with an acetylated and then a carbamylated protein (or vice versa) and their serum was analyzed for AMPA responses. Results Human data illustrated dynamic changes in AMPA-reactivity profiles in both individuals at risk of RA and in early RA patients. Mice immunized with either solely acetylated or carbamylated ovalbumin (AcOVA or CaOVA) developed reactivity against both acetylated and carbamylated antigens. Irrespective of the PTM-antigen used for the first immunization, a booster immunization with an antigen bearing the other PTM resulted in increased titers to the second/booster PTM. Furthermore, cross-immunization skewed the overall AMPA-response profile towards a relatively higher reactivity against the “booster” PTM. Conclusions The relationship between different reactivities within the AMPA response is dynamic. The initial exposure to a PTM-antigen induces cross-reactive responses that can be boosted by an antigen bearing this or other PTMs, indicating the formation of cross-reactive immunological memory. Upon subsequent exposure to an antigen bearing another type of PTM, the overall reactivity pattern can be skewed towards better recognition of the later encountered PTM. These data might explain temporal differences in the AMPA-response profile and point to the possibility that the PTM responsible for the initiation of the AMPA response may differ from the PTM predominantly recognized later in time.

Supplemental Dietary Selenohomolanthionine Improve Antioxidant Activity and Immune Function in Weaned Beagle Puppies

The purpose of this study was to investigate the effects of dietary Selenohomolanthionine (SeHLan) on antioxidant status and immune response in canine parvovirus (CPV) vaccinated puppies. In this study, 30 weaned puppies were randomly divided into six groups: control group (–Se/–Vacc), immunization group (–Se/+Vacc), supplementation of sodium selenite group (SS/+Vacc, 0.35 mg/kg DM), low-dose SeHLan group (SeHLan-L/+Vacc, 0.35 mg/kg DM), mid-dose SeHLan group (SeHLan-M/+Vacc, 1.0 mg/kg DM), and high-dose SeHLan group (SeHLan-H/+Vacc, 2.0 mg/kg DM). The puppies were fed for 42 days and vaccinated with Vanguard Plus 5 on day 0 and day 21. Blood samples were collected on 7, 14, 21, 28, 35, 42 days post-immunization (PI) for determination of antioxidant indicators, lymphocyte proliferation index, serum cytokine concentration (IL-2, IL-4), canine polymorphonuclear neutrophils (PMN) phagocytic function, and the level of CPV antibody titers. The results showed that SeHLan supplementation raised the serum Se concentration and glutathione peroxidase (GSH-Px) activity in a dose-dependent manner (P &lt; 0.05). It also increased the activity of serum superoxide dismutase (SOD) and decreased serum malondialdehyde (MDA) content, especially in SeHLan-M/+Vacc group (1.0 mg/kg DM) (P &lt; 0.01). SeHLan supplementation significantly increased lymphocyte proliferation, IL-2, and IL-4 levels in canine serum, and enhanced phagocytosis of PMN in vaccinated puppies (P &lt; 0.05). Moreover, SeHLan supplementation shortened the CPV antibody production time and increased the CPV antibody titers (P &lt; 0.05). Of note, the beneficial effects of SeHLan were superior to those of SS. In conclusion, dietary SeHLan supplementation improved antioxidant activity, increased CPV antibody titers, and enhanced immune function in puppies after weaning. An appropriate dosage of SeHLan (1~2 mg/kg DM) may confer nutritional benefits in puppies.

Characterizing the innate and adaptive responses of immunized mice toBordetella pertussisinfection usingin vivoimaging and transcriptomic analysis

Bordetella pertussis(B. pertussis) is the causative agent of pertussis (whooping cough). Since the 1990s, pertussis has re-emerged in the United States despite an estimated 95% vaccine coverage. Our goal was to characterize neutrophil responses and gene expression profiles of murine lungs in the context of vaccination andB. pertussischallenge. We utilized a bioluminescent neutrophil mouse model (NECre luc) to track neutrophil recruitment. NECre luc mice were immunized with whole cell vaccine (WCV), acellular vaccine (ACV), or a truncated adenylate cyclase toxoid (RTX) vaccine. Neutrophil recruitment was measured in live mice across time and corroborated by flow cytometry and other data. WCV immunized mice showed signs of neutrophilia in response toB. pertussischallenge. Mice immunized with either ACV or WCV cleared the challenge infection; however immunization with RTX alone was not protective. RNA sequencing revealed distinctive gene expression profiles for each immunization group. We observed an increase in expression of genes associated with responses to infection, and changes in expression of distinct genes in each vaccine group, providing a complex view of the immune response toB. pertussisinfection in mice. This study suggests that combination of immunological analysis with transcriptomic profiling can facilitate discovery of pre-clinical correlates of protection for vaccine development.

Group B meningococcal outer membrane protein vaccine promote potent anti-viral effect

This report demonstrates a novel method to explore and evaluate the specific humoral/cellular immune response levels and immunoprotective effects of NMB0315 nucleic acid vaccine, recombinant protein vaccine and nucleic acid vaccine + recombinant protein vaccine in combination with mice, and to further explore the effective immunization method for NMB0315 vaccine. This route provides experimental basis. Nucleic acid vaccines [pcDNA3 1(+) / NMB0315] and recombinant protein vaccines (pET 30a / NMB0315) were prepared in large quantities, and immunologically or separately immunized female BALB/c mice were determined by nucleic acid priming protein boosting method. The specific humoral/cell immune response level, the in vitro bactericidal titer of immune serum, and the immunoprotective effect of the vaccine on mice infected with group B meningococcus were observed. Serum-specific IgG, IgG1, IgG2a and genital lavage fluids induced by NMB0315 nucleic acid vaccine group (pNMB0315 CpG), protein vaccine group (rNMB0315 FA) and combined immunization group (pNMB0315 CpG+rNMB0315 FA). The specific sIgA level reached the peak in the eighth week, and the A450 values were in vitro, and the in vitro bactericidal antibody titers of the nucleic acid vaccine group, the protein vaccine group and the combined immunization group were 1, 64, 1128, respectively. The immune protection rate of experimental mice were 70%, 95% and 80%, respectively. At 2, 4, 6, and 8 weeks, the ratio of IgG2a / IgG1 in the nucleic acid vaccine group, the recombinant protein vaccine group, and the combined immunization vaccine group was less than 1.

Simultaneous Administration of Recombinant Measles Viruses Expressing Respiratory Syncytial Virus Fusion (F) and Nucleo (N) Proteins Induced Humoral and Cellular Immune Responses in Cotton Rats

We previously reported that recombinant measles virus expressing the respiratory syncytial virus (RSV) fusion protein (F), MVAIK/RSV/F, induced neutralizing antibodies against RSV, and those expressing RSV-NP (MVAIK/RSV/NP) and M2-1 (MVAIK/RSV/M2-1) induced RSV-specific CD8+/IFN-γ+ cells, but not neutralizing antibodies. In the present study, MVAIK/RSV/F and MVAIK/RSV/NP were simultaneously administered to cotton rats and immune responses and protective effects were compared with MVAIK/RSV/F alone. Sufficient neutralizing antibodies against RSV and RSV-specific CD8+/IFN-γ+ cells were observed after re-immunization with simultaneous administration. After the RSV challenge, CD8+/IFN-γ+ increased in spleen cells obtained from the simultaneous immunization group in response to F and NP peptides. Higher numbers of CD8+/IFN-γ+ and CD4+/IFN-γ+ cells were detected in lung tissues from the simultaneous immunization group after the RSV challenge. No detectable RSV was recovered from lung homogenates in the immunized groups. Mild inflammatory reactions with the thickening of broncho-epithelial cells and the infiltration of inflammatory cells were observed in lung tissues obtained from cotton rats immunized with MVAIK/RSV/F alone after the RSV challenge. No inflammatory responses were observed after the RSV challenge in the simultaneous immunization groups. The present results indicate that combined administration with MVAIK/RSV/F and MVAIK/RSV/NP induces humoral and cellular immune responses and shows effective protection against RSV, suggesting the importance of cellular immunity.

Toxicity of 32.2 kDa MW Escherichia coli Pili Adhesin Isolated from Infertile Male Semen in Reproductive System

Escherichia coli (E. coli) is the leading cause of male genital tract infection with no symptoms of infertility. Protein E. coli pili hemagglutinin isolated from infertile male sperm with 32.2 kDa MW acts as adhesion in spermatozoa. This study aimed to prove whether E. coli pili adhesin 32.2 kDa MW is toxic to male reproductive system. Samples consisted of spermatozoa of 30 guinea pigs divided into three groups: control, immunized with E. coli pili adhesin 32.2 kDa MW protein, and transurethral infected E. coli. Observations of sperm motility, vitality and morphology were performed under a microscope. MDA levels and sperm DNA damage were measured by a spectrophotometer and comet assay method and observed using a fluorescent microscope. There was no difference between control and immunization group of E. coli pili adhesin in motility (p=0.499), vitality (p=0.817) and morphology (p=0.176); between control and transuretral infection groups in motility (p=0.000), vitality (p=0.000) and morphology (p=0.000); and between control and both treatment groups in motility (p=0.001), vitality (p=0,000) and morphology (p=0.000). Histologic analysis showed E. coli pili adhesin of 32.2 kDa MW immunization group did not suffer from testicular tissue damage, while the positive group showed a deterioration of seminiferous tubular cells. MDA levels differed between immunization group E. coli pili, transurethral infection group, and control (p=0.024) and between transurethral and control (p=0.007) groups. However, between control and immunized group with E. coli pili protein showed no difference (p=0.251). DNA damage differed (p=0.000) between immunized group with E. coli pili, transurethral infection and control group; between control and transurethral infected group (p=0.000); and between transurethral infection group and E. coli pili protein immunization group (p=0.000). However, between control and E. coli pili immunization group showed no difference (p=0.600). In conclusion, E. coli pili adhesin 32.2 kDa MW protein is not toxic for sperm quality and the quality of sperm molecules.

An Attenuated Cytomegalovirus Vaccine with a Deletion of a Viral Chemokine Gene Is Protective against Congenital CMV Transmission in a Guinea Pig Model

Development of a vaccine against congenital cytomegalovirus (CMV) infection is a public health priority, but CMVs encode immune evasion genes that complicate live virus vaccine design. To resolve this problem, this study employed guanosyl phosphoribosyl transferase (gpt) mutagenesis to generate a recombinant guinea pig CMV (GPCMV) with a knockout of a viral chemokine gene, GPCMV MIP (gp1). MIP deletion virus replicated with wild-type kinetics in cell culture but was attenuated in nonpregnant guinea pigs, demonstrating reduced viremia and reduced inflammation and histopathology (compared to a control virus with an intact GPCMV MIP gene) following footpad inoculation. In spite of attenuation, the vaccine was immunogenic, eliciting antibody responses comparable to those observed in natural infection. To assess its protective potential as a vaccine, either recombinant virus or placebo was used to immunize seronegative female guinea pigs. Dams were challenged in the early 3rd trimester with salivary gland-adapted GPCMV. Immunization protected against DNAemia (1/15 in vaccine group versus 12/13 in the control group,P<0.01). Mean birth weights were significantly higher in pups born to vaccinated dams compared to controls (98.7 g versus 71.2 g,P<0.01). Vaccination reduced pup mortality, from 35/50 (70%) in controls to 8/52 (15%) in the immunization group. Congenital GPCMV infection was also reduced, from 35/50 (70%) in controls to 9/52 (17%) in the vaccine group (P<0.0001). We conclude that deletion of an immune modulation gene can attenuate the pathogenicity of GPCMV while resulting in a viral vaccine that retains immunogenicity and demonstrates efficacy against congenital infection and disease.

Active immunization against the proregions of GDF9 or BMP15 alters ovulation rate and litter size in mice

The transforming growth factor β (TGFB) superfamily proteins bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9), are essential for mammalian fertility. Recent in vitro evidence suggests that the proregions of mouse BMP15 and GDF9 interact with their mature proteins after secretion. In this study, we have actively immunized mice against these proregions to test the potential in vivo roles on fertility. Mice were immunized with either N- or C-terminus proregion peptides of BMP15 or GDF9, or a full-length GDF9 proregion protein, each conjugated to keyhole limpet hemocyanin (KLH). For each immunization group, ovaries were collected from ten mice for histology after immunization, while a further 20 mice were allowed to breed and litter sizes were counted. To link the ovulation and fertility data of these two experimental end points, mice were joined during the time period identified by histology as being the ovulatory period resulting in to the corpora lutea (CL) counted. Antibody titers in sera increased throughout the study period, with no cross-reactivity observed between BMP15 and GDF9 sera and antigens. Compared with KLH controls, mice immunized with the N-terminus BMP15 proregion peptide had ovaries with fewer CL (P<0.05) and produced smaller litters (P<0.05). In contrast, mice immunized with the full-length GDF9 proregion not only had more CL (P<0.01) but also had significantly smaller litter sizes (P<0.01). None of the treatments affected the number of antral follicles per ovary. These findings are consistent with the hypothesis that the proregions of BMP15 and GDF9, after secretion by the oocyte, have physiologically important roles in regulating ovulation rate and litter size in mice.