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Knowledge of virus and replication kinetic is one of the most important issues in the vaccine production. The present study aimed to evaluate the best harvesting time of H9N2 avian influenza virus (AIV) vaccine strain inoculated in specific pathogen free (SPF) embryonated chicken eggs (ECE)s. For this purpose, 10-5 dilution of AIV (A/Chicken/Iran/99/H9N2) was inoculated into 336, 11-day old ECEs at the rate of 0.1 ml/ECE via intera-allantoic. Amnio-allantoic fluid (AAF) and chorioalantoic membranes were collected at 2 hours intervals up to 96 post inoculations from 7 eggs in each trial. The presence of virus in the harvested suspensions was evaluated by Hemagglutination assay (HA) and Egg infective dose 50 (EID50) assays. Our results showed that, the best virus harvesting time for vaccine production is between 50 – 60 hours post inoculation (hpi) in ECEs. This finding may provide possibility of the sufficient and increasing rate of production, immunogenicity and economic factors in H9N2 influenza vaccine production.

Newcastle disease (ND) is one of the most important diseases that affect birds; the epizootic nature of the disease has caused severe economic losses in the poultry industry worldwide. In this experiment ND virus (NDV) was inactivated by two different chemicals binary ethylenimine (BEI) and formaldehyde. Formaldehyde was used at 0.1%, while BEI was used at concentrations of 1 to 4 mM. NDV inactivation with BEI was done in various incubation temperatures and periods and the best result (30 °C, 4 mM BEI and 21 hrs treatment) used as an experimental vaccine. Prepared inactivated NDV vaccines and a commercial vaccine were tested for their efficiency in generating humoral immune response in different groups of specific pathogen free (SPF) chicks. Test groups received 0.2 ml formaldehyde inactivated NDV (NDVF), BEI inactivated NDV (NDVEI) and Razi institute produced NDV vaccine (NDVR) subcutaneously respectively. HI Log 2 total mean titer of NDVEI group (8.42 ± 0.12) were significantly higher than NDVF (7.64 ± 0.16) and NDVR (7.86 ± 0.11) groups (p

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Background: The gliding surface of total knee endoprostheses is exposed to high loads due to patient weight and activity. These implant components are typically manufactured from ultra-high molecular weight polyethylene (UHMWPE). Crosslinking of UHMWPE by ionizing radiation results in higher wear resistance but induces the formation of free radicals which impair mechanical properties after contact with oxygen. Medium-crosslinked UHMWPE enriched with vitamin E (MXE) provides a balance between the parameters for a sustainable gliding surface, i.e., mechanical strength, wear resistance, particle size, and oxidation stability. Therefore, a gliding surface for knee endoprostheses made up from this material was developed, certified, and launched. The aim of this study is to compare this new gliding surface to the established predecessor in a non-inferiority design., Methods: This multicenter, binational randomized controlled trial will enroll patients with knee osteoarthritis eligible for knee arthroplasty with the index device. Patients will be treated with a knee endoprosthesis with either MXE or a standard gliding surface. Patients will be blinded regarding their treatment. After implantation of the devices, patients will be followed up for 10 years. Besides clinical and patient-related outcomes, radiological data will be collected. In case of revision, the gliding surface will be analyzed biomechanically and regarding the oxidative profile., Discussion: The comparison between MXE and the standard gliding surface in this study will provide clinical data to confirm preceding biomechanical results in vivo. It is assumed that material-related differences will be identified, i.e., that the new material will be less sensitive to wear and creep. This may become obvious in biomechanical analyses of retrieved implants from revised patients and in radiologic analyses., Trial Registration: ClinicalTrials.gov, NCT04618016. Registered 27 October 2020, https://clinicaltrials.gov/study/NCT04618016?term=vikep&checkSpell=false&rank=1 . All items from the World Health Organization Trial Registration Data Set can be found in Additional file 1., (© 2023. The Author(s).)

Purification is an important step in the production of viral vaccines that strongly affects product recovery and subsequent immune responses. The present study was carried out with the aim of improving the purification of infectious bursal disease virus (IBDV) by the tangential flow filtration (TFF) method. Then, the effect of the purified virus on the induction of immune responses against IBDV in specific pathogen free (SPF) chickens was investigated. The IBD07IR strain was propagated in embryonated SPF eggs. The virus was purified using a 100 kDa cassette. The quality of the recovered viruses was evaluated by titration. A total number of 60 SPF chickens were randomly divided into three groups (n = 20) and received the concentrated viral antigen, commercial live IBDV vaccine and phosphate-buffered saline at the age of 3 weeks by eye drop method. The bursa of Fabricius was examined histopathologically for possible changes. Sera were collected at 1-week intervals from day 0 until the end of 6 weeks after vaccination. The IBDV-specific antibody levels, induction of cell-mediated immunity and mRNA expression levels of cytokines were evaluated. The results showed that despite a relative raise in virus titer from 7.66 to 8.17 embryo infectious dose (EID) 50 mL -1 following purification, both the purified IBDV and commercial vaccine are able to induce strong immune responses against the virus. Within a context of egg-based IBDV vaccine production, a single-step TFF can be applied for the relatively purification. This platform requires a further study in the selection of multiple membranes to optimize the operating conditions and final product., Competing Interests: No conflict of interest is declared., (© 2024 Urmia University. All rights reserved.)

Gamma (γ)-radiation can target viral genome replication and preserve viral structural proteins compared to formalin inactivation. Thus, a stronger immunity could be induced after the inoculation of the irradiated virus. In this study, γ-irradiated low-pathogenic avian influenza virus-H9N2 (LPAIV-H9N2) was used to immunize the broiler chicken in two formulations, including γ-irradiated LPAIV-H9N2 with 20% Trehalose intranasally (IVT.IN) or γ-irradiated LPAIV-H9N2 plus Montanide oil adjuvant ISA70 subcutaneously (IV+ISA.SC) in comparison with formalin-inactivated LPAIV-H9N2 vaccine intranasally (FV.IN) or formalin-inactivated LPAIV-H9N2 plus ISA70 subcutaneously (FV+ISA.SC). Two vaccination regimes were employed; the first one was primed on day 1 and boosted on day 15 (early regime), and the second one was primed on day 11 and boosted on day 25 (late regime). A challenge test was performed with a live homologous subtype virus. Virus shedding was monitored by quantifying the viral load via RT-qPCR on tracheal and cloacal swabs. Hemagglutination inhibition (HI) antibody titration and stimulation index (SI) of the splenic lymphocyte proliferation were measured, respectively, by HI test and Cell Proliferation assay. Cytokine assay was conducted by the RT-qPCR on antigen-stimulated spleen cells. The results of the HI test showed significant increases in antibody titer in all vaccinated groups, but it was more evident in the IVT late vaccination regime, reaching 5.33 log 2 . The proliferation of stimulated spleen lymphocytes was upregulated more in the IVT.IN vaccine compared to other vaccines. The mRNA transcription levels of T-helper type 1 cytokines such as interferon-gamma (IFN-γ) and interleukin 2 (IL-2) were upregulated in all vaccinated groups at the late regime. Moreover, IL-6, a pro-inflammatory cytokine was upregulated as well. However, upregulation was more noticeable in the early vaccination than the late vaccination (p < 0.05) . After the challenge, the monitoring of virus shedding for the H9 gene represented an extremely low viral load. The body weight loss was not significant ( p > 0.05 ) among the vaccinated groups. In addition, the viral load of <10 0.5 TCID 50 /ml in the vaccinated chicken indicated the protective response for all the vaccines. Accordingly, the IVT vaccine is a good candidate for the immunization of broiler chicken via the intranasal route at late regime., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Motamedi Sedeh, Khalili, Wijewardana, Unger, Shawrang, Behgar, Moosavi, Arbabi and Hosseini.)

Background: Avian influenza virus (AIV) subtype H9N2 is a low pathogenic avian influenza virus (LPAIV)., Objective: This study aims to evaluate the humoral and cellular immunity in vaccinated mice and broiler chicken by irradiated AIV antigen plus carboxymethyl chitosan bounded iron oxide nanoparticles (CMC-IO NPs) as an adjuvant., Methods: AIV subtype H9N2 with 10 8.5 EID 50 /ml and haemagglutinin antigen assay about 10 log 2 was irradiated by 30 kGy gamma radiation dose. Then, the gamma-irradiated AIV was used as an inactivated vaccine and conjugated with CMC-IO NPs to improve immune responses on mice. IO NPs must be applied in all activated tests using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysulfosuccinimide sodium salt (sulfo-NHS), and then functionalized by CMC as IO-CMC. Fourier transform infrared (FTIR) spectra on functionalized IO-CMC showed a peak of 638 cm -1 which is a band between metal and O (Fe-O)., Results: Based on the comparison between the two X-ray diffraction (XRD) patterns on Fe 2 O 3 -NPs and IO-CMC, the characteristics of IO-NPs did not change after carboxymethylation. A CHN Analyzer was applied to measure the molecular weight of IO-CMC that was calculated as 1045 g. IO-CMC, irradiated AIV-IO-CMC and formalin AIV-IO-CMC were injected into 42 BALB/c mice in six groups. The fourth group was the negative control, and the fifth and sixth groups were inoculated by irradiated AIV-ISA70 and formalin AIV-ISA70 vaccines. An increase in haemagglutination inhibition (HI) antibody titration was observed in the irradiated AIV-IO-CMC and formalin AIV-IO-CMC groups (p < 0.05). In addition, increases in the lymphoproliferative activity of re-stimulated splenic lymphocytes, interfron-γ (IFN-γ) and interleukin-2 (IL-2) concentration in the irradiated AIV-IO-CMC group demonstrated the activation of Type 1 helper cells. The concentration of IL-4 was without any significant increases in non-group., Conclusions: Accordingly, Th2 activation represented no increase. Finally, the finding showed that AIV-IO-CMC was effective on enhancing immunogenicity as irradiated AIV antigen administered with a clinically acceptable adjuvant (i.e. IO-CMC)., (© 2021 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Avian influenza (AI) A subtype H9N2 virus belongs to Orthomyxoviridae family and causes low-pathogenic disease AI. The use of gamma-irradiated viral antigens has been developed in the production of effective vaccines. In this research, LPAIV H9N2 strain, A/Chicken/IRN/Ghazvin/2001, was multiplied on SPF eggs and irradiated by a Nordian gamma cell instrument. Irradiated and non-irradiated AI virus (AIV) samples were titrated by EID50 method and hemagglutinin (HA) antigen was analyzed by HA test as the WHO pattern method. Infectivity of irradiated virus was determined by egg inoculation method during four blind cultures. The results showed that after increasing the dose of gamma radiation, virus titer gradually decreased. D 10 value and optimum dose for complete virus inactivation were calculated by dose/response curve, 3.36 and 29.52 kGy, respectively. In addition, HA antigenicity of gamma-irradiated virus samples from 0 to 30 kGy was not changed. The results of safety test for gamma-irradiated AIV samples showed complete inactivation with gamma ray doses 30 and 35 kGy, without any multiplication on eggs after four blind cultures. According to the results of HA antigen assay and safety test, the gamma-irradiated and complete inactivated AIV subtype H9N2 is a good candidate as an inactivated immunogenic agent for poultry vaccination.

Background: Although the determination of the ABO antibody titers is necessary for the decision-making in ABOincompatible (ABOi) kidney transplantations, various methods for the determination of the ABO antibody titers are being used. However, the absence of uniform standards makes their comparability far more difficult. Two of the most commonly used methods are the tube method and the gel card method. In this study, we systematically investigate to what extent these two methods affect the result of ABO antibody titers., Methods: ABO antibodies were determined from plasmas of 90 donors (30 individuals each with blood group A, B, and O). Seven further donors with blood group A, B, and AB provided erythrocytes for the testing. A total of 360 ABO antibody titers were determined; 180 tests for each method, each with 90 determinations of immunoglobulin G (IgG) and immunoglobulin M (IgM) antibody titers. In addition, we also made a differentiation by blood groups to find out if and to what extent the blood groups have an impact on the results., Results: Our analysis shows that the choice of method has a highly significant (p < 0.0001) impact on the titer level of the ABO antibodies. The median values of ABO antibody titers determined by using the gel card method are two titer steps lower than the titers, which are determined when using the tube method. Moreover, our data shows that there are major differences in the ABO antibody titer level among the blood groups, regardless of the choice of methods., Conclusions: We consider changing to the gel card method for determining the ABO antibody titers as a simple and effective way to achieve a standardized and uniform method. Here, too, the clinicians should be provided with sufficient information by the laboratories, in order to draw the right consequence from this change, while considering all the relevant data. As a consequence of this study, the transplant center of the University of Hamburg-Eppendorf paired a change from tube to gel card regarding the ABO antibody titer determination of ABOi kidney transplantations with an intensification of the preoperative target titer from ≤ 1:8 to ≤ 1:4.

Background: Since the Intergroup 0116 study was published in 2000, adjuvant postoperative chemoradiotherapy using CT-planned and 3D conformal/intensity-modulated radiotherapy has been offered routinely to fit patients with resected gastric cancer at Princess Margaret Hospital .The objective of this study was to analyze patterns of disease recurrence with respect to the radiotherapy volumes., Methods: For the date and site (local, locoregional, or distant) of the first recurrence, medical records were reviewed for all patients treated at Princess Margaret Hospital with adjuvant chemoradiotherapy for resected gastric adenocarcinoma (January 1, 2000 to November 30, 2009). Patients whose recurrences were limited to local and/or regional sites were selected for further analysis. Available diagnostic imaging of the recurrence site was registered to the original planning radiotherapy dataset for contouring. If necessary to respect changes in anatomy, the contour was translocated on the basis of anatomic descriptors. The center of mass for each recurrence was identified as a point and its location was categorized according to the isodose encompassing it; in field (90 % or more), marginal (50-89 %), or out of field (less than 50 %)., Results: Of all 197 patients, 14 (7 %) had isolated locoregional failure, constituting 20 % of all 71 patients with a recurrence. Successful fusions were feasible in five cases. Of these recurrences, four were in field and one was marginal. In a further four cases, visual inspection was used, showing one in-field recurrence, one marginal recurrence, and two out-of-field recurrences. In five patients, either a useable original dataset or diagnostic imaging of the recurrence was not available., Conclusions: The rates of isolated local/locoregional tumor recurrence in this study were low. Of the small number of recurrences available for analysis, most (five of nine) were in field. Further studies involving a larger cohort of patients might allow a more meaningful analysis of trends in the recurrence site with evolving radiotherapy techniques.

Background: Influenza A is a virus that affects a wide range of animals and also human beings. Avian influenza virus (AIV) subtype H9N2 has the potential to create influenza pandemic and vaccination is a common solution for this problem. The vaccine, used for rapid intervention, should be safe to use and highly effective, after a single administration. Chitosan nanoparticles (CNP) have already been recommended as a new adjuvant for inactivated AIV H9N2 vaccine immunization., Objectives: This study aimed at the evaluation and better understanding of optimum concentration of CNP preparations and also, assessment of loading capacity of AIV into CNP, as an adjuvant in specific pathogen-free (SPF) chickens., Materials and Methods: For measurement of vaccine-antibody response, different types of CNP were injected intramuscularly, in a single dose, to 21-day-old specific pathogen-free chickens. Chickens were monitored for the efficacy of the nanoparticles and, also, their immune response, during a follow up of 7 weeks, by using hemagglutination-inhibition (HI) test. The CNP were prepared according to modified ionic gelation method and inactivated antigen was loaded in four hemagglutinin units (HAU) concentrations. Loading capacity of nanoparticles was determined by hemagglutination (HA) method. Inactivated A/H9N2 AIV was mixed with chitosan of low molecular weight., Results: The CNP did not cause any mortality or side effects, when chickens were administered the prepared vaccine. The results strongly showed that this novel vaccine significantly enhances the immunogenicity of inactivated AIV, comparing with ISA70 (SEPPIC, Puteaux, France) adjuvant that is used routinely in the Razi Serum and Vaccine Research and Production Institute, Karaj, Iran, to reduce ISA70's side effects., Conclusions: The AIV loaded into CNP vaccines induce appropriate antibody titers, after a single immunization, while requiring a low dose of antigen. The CNP also represent an interesting new platform for antigen delivery and a promising adjuvant candidate for H9N2 inactivated influenza vaccine.

Introduction: Periodontitis is an inflammatory process in response to dental biofilm and leads to periodontal tissue destruction. The aim of this study was the comparison of outcomes using either an enamel matrix derivative (EMD) or a nanocrystalline hydroxyapatite (NHA) in regenerative periodontal therapy after 6 and 12 months., Methods: Using a parallel group, prospective randomized study design, we enrolled 19 patients in each group. The primary outcome was bone fill after 12 months. Attachment gain, probing pocket depth (PPD) reduction, and recession were secondary variables. Additionally, early wound healing and adverse events were assessed. Data analysis included test of noninferiority of NHA group (test) compared to EMD group (reference) in bone fill. Differences in means of secondary variables were compared by paired t-test, frequency data by exact χ(2) test., Results: Both groups showed significant bone fill, reduction of PPD, increase in recession, and gain of attachment after 6 and 12 months. No significant differences between groups were found at any time point. Adverse events were comparable between both groups with a tendency of more complaints in the NHA group., Conclusion: The clinical outcomes were similar in both groups. EMD could have some advantage compared to NHA regarding patients comfort and adverse events. The trial is registered with ClinicalTrials.gov NCT00757159.

There are little information about growth properties of low pathogenic (LP) avian influenza virus (AIV) in embryonated chicken eggs (ECEs) at different incubation temperatures. Knowledge of this information increases the quantity and quality of antigen in vaccine production process. For this purpose, 10(-5) dilution of AIV (A/Chicken/Iran/99/H9N2) was inoculated (Intra-allantoic) into 400, 11-day old specific pathogen free (SPF) ECEs in the 0.1 mL per ECE rate and incubated in 32, 33, 34, 35, 36, 37.5, 38, 39 ˚C for 72 hr in 65% humidity. Early death embryos in first 24 hr were removed. Amnio-allantoic fluid was withdrawn into the measuring cylinder, and tested for hemagglutination (HA) activity and egg infective dose 50 (EID50). The utilizable ECEs and amnio-allantoic fluid volume was significantly increased in 35 ˚C, (p < 0.05). Significant difference in HA and EID50 titers, were seen only in 39 ˚C group. Therefore, 35°C is an optimum temperature for incubation of inoculated ECEs.

The WNT signaling is deregulated in most human colorectal cancers (CRC). Promoter methylation has been proposed as an alternative mechanism to inactivate genes in tumors. To gain insight into the methylation silencing of the WNT pathway during colorectal carcinogenesis, we examined the aberrant methylation profile of four genes, APC, Axin1, Axin2, and GSK3β in an unselected series of 112 sporadic colorectal tumors by methylation specific PCR. It has been suggested that the Axin2 C148T SNP is associated with the risk of developing certain types of cancers. To assess the contribution of Axin2 SNP to CRC susceptibility, we examined the Axin2 C148T genotype in CRC patients and 170 healthy controls by PCR-RFLP. The frequency of CRCs with at least one gene methylated was 18.75%. Promoter methylation of Axin2 and APC genes was detected in 7.1 and 11.9% of tumors, respectively. No aberrant methylation was found in Gsk3β and Axin1 gene in these tumor series. The methylation status of APC had no significant association with clinical parameters. But, promoter methylation of Axin2 was sex-related, occurring more frequently in females (P = 0.002). The frequency of Axin2 C148T genotypes were similar in patients and controls. Moreover, we observed no association between the Axin2 SNP and risk of CRC in patients stratified by age, sex, and smoking status. However, the heterozygote CT genotype was associated with a reduced CRC risk in distal patients compared with proximal patients (OR = 0.3; 95% CI 0.1-0.9, P = 0.04). Our findings indicate that Axin1 and GSK3β methylation play a minor role in colorectal carcinogenesis.

The resistance of 220 coagulase-negative Staphylococci (CNS) (associated with animal disease) to 13 antibiotics were determined using the disk diffusion method. 35.9% of multidrug-resistant coagulase-negative Staphylococci (MR-CNS) exhibited resistance to five or more than five antibiotics; all of these bacteria were resistant to methicillin too. The new Streptomyces sp. ABRIINW111 was isolated from the Zagros Mountains Hamadan, Iran. The 16S rDNA sequence of the isolate indicated that it has 98% similarity to S. levis, but some mutations in the alpha and gamma regions of the 16S rDNA sequence emphasize the probability of the existence of a new species. Preliminary and secondary antibacterial screenings revealed that the isolate is active against gram negative and positive bacteria. The diethyl ether extracted metabolite of the Streptomyces sp. ABRIINW111 showed an effective antibacterial activity against MR-CNS. So the diethyl ether extract of the new Streptomyces sp. strain ABRIINW111 can inhibit the MR-CNS in vitro, and it can offer a new approach to treat MR-CNS infectious patients.

Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the folate metabolic pathway. We aimed to test the hypothesis that C677T and A1298C variants of MTHFR predispose to microsatellite instable (MSI) colorectal cancer. We determined MTHFR genotypes in 175 sporadic colorectal cancer patients and a total of 231 normal controls in Shiraz, Southern Iran. Among the genotypes found in our samples, MTHFR CT and CT+TT were associated with increased risk for CRC incidence [odds ratio (OR)=2.4, 95% confidence interval (95%CI)=1.8-4.4; OR=2.4, 95%CI=1.6-3.6, respectively]. Double heterozygotes 677CT/1298AC and double homozygote 677TT/1298AA and 677CC/1298CC genotypes also showed a significantly increased risk of developing CRC compared with the wild-type 677CC/1298AA genotypes of the controls. Among the 151 tumors tested, 36 (23.8%) were MSI+. MSI was more common in proximal tumors (OR=10.4; 95%CI=3.9-27.8) and in smokers (OR=2.9; 95%CI=1.3-6.7). In a case-control comparison, the MTHFR 677CT+TT genotype was strongly associated with MSI (OR=2.6; 95%CI=1.3-5.3). Hypermethylation of mismatch repair genes was positively related with MSI incidence in these tumor series (P=0.00). Our data suggest that the MTHFR 677CT+TT variant genotype may be a risk factor for MSI+ cancer.

Background: 5,10-Methylenetetrahydrofolate reductase (MTHFR) plays a central role in folate metabolism. Previous studies have suggested an association between the MTHFR 1298A/C polymorphism and several diseases, such as cardiovascular and psychiatric diseases, neural tube defects, diabetes, and cancer. Currently, either PCR-restriction fragment length polymorphism (RFLP) technique or real-time PCR using Taqman assay are used to determine the MTHFR 1298 genotype., Methods: We developed a simple and efficient approach that employs mutagenically separated PCR to genotype MTHFR 1298A/C polymorphism. Two forward mutagenic allele-specific primers of different lengths for MTHFR 1298A/C were paired with the same reverse primer in a one-tube assay to genotype 20 genomic DNA samples., Results: Electrophoresis on 2.5% agarose gel showed two allele-specific fragments, a 113-bp A allele-specific and a 93-bp C allele-specific PCR product. The results were confirmed by the conventional PCR-RFLP method., Conclusions: We conclude that mutagenically separated PCR could be used as an alternative simple, reliable, and cost-effective method for the genotyping of MTHFR 1298A/C polymorphism.

Endovaginal sonography in combination with HSG is an effective screening tool in evaluating patients with postmenopausal bleeding. Endovaginal sonography is highly sensitive for detecting endometrial carcinoma and can identify patients at low risk for endometrial disease obviating the need for endometrial sampling in this subgroup of patients. In patients with abnormal findings at sonography, a detailed morphologic analysis can be used to determine which patients can undergo blind endometrial sampling successfully versus those who would benefit from hysteroscopic guidance. In patients in whom endovaginal sonography and HSG are inadequate, MRI may provide additional information on the appearance of the endometrium, particularly in patients in whom endometrial sampling is difficult (eg, patients with cervical stenosis).