Gender potentially affects early postoperative hyponatremia in pituitary adenoma: XGBoost-based predictive modeling.

Purpose: The occurrence of hyponatremia is a prevalent complication following transnasal transsphenoidal surgery for pituitary adenoma surgery, which adversely affects patient prognosis, hospitalization duration, and rehospitalization risk. The primary objective of this study is to strengthen the correlation between clinical factors associated with pituitary adenoma and postoperative hyponatremia. Additionally, the study aims to develop a predictive model for postoperative hyponatremia in patients with pituitary adenoma, with the ultimate goal of establishing a basis for reducing the occurrence of postoperative hyponatremia following surgical interventions. Methods: The chi-square test or Fisher test was employed for nominal data, while the t-test or Mann-Whitney test was utilized for continuous data analysis. In cases where the data exhibited statistical differences, binary logistic analysis was conducted to examine the risk and protective factors associated with postoperative hyponatremia. XGBoost was employed to construct predictive models for hyponatremia in this study. The patients were partitioned into training and test sets, and the most suitable parameters were determined through five-fold cross-validation and subsequently utilized for training on the training set. The discriminatory capability was assessed on the internal validation set. Results and conclusions: Out of the total 280 patients included in this investigation, 82 patients experienced early postoperative hyponatremia. Among these individuals, male gender (P = 0.02, odds ratio = 1.98) was identified as a risk factor for early postoperative hyponatremia, while preoperative chloride levels (P = 0.021, odds ratio = 0.866) and surgery time (P = 0.039, odds ratio = 0.990) were identified as protective factors against postoperative hyponatremia. The XGBoost model exhibited a sensitivity of 94.2%, a specificity of 61.5%, a positive predictive value of 51.6%, a negative predictive value of 96%, and identified male gender, preoperative sodium, and preoperative cortisol as the most significant predictors. Our findings indicate that gender may have influence in the development of early postoperative hyponatremia in patients with pituitary adenomas.

The power and the promise of CAR-mediated cell immunotherapy for clinical application in pancreatic cancer.

BACKGROUND: Pancreatic cancer, referred to as the "monarch of malignancies," is a neoplastic growth mostly arising from the epithelial cells of the pancreatic duct and acinar cells. This particular neoplasm has a highly unfavorable prognosis due to its marked malignancy, inconspicuous initial manifestation, challenging early detection, rapid advancement, and limited survival duration. Cellular immunotherapy is the ex vivo culture and expansion of immune effector cells, granting them the capacity to selectively target malignant cells using specialized techniques. Subsequently, these modified cells are reintroduced into the patient's organism with the purpose of eradicating tumor cells and providing therapeutic intervention for cancer. PRESENT SITUATION: Presently, the primary cellular therapeutic modalities employed in the treatment of pancreatic cancer encompass CAR T-cell therapy, TCR T-cell therapy, NK-cell therapy, and CAR NK-cell therapy. AIM OF REVIEW: This review provides a concise overview of the mechanisms and primary targets associated with various cell therapies. Additionally, we will explore the prospective outlook of cell therapy in the context of treating pancreatic cancer.

The Effective Analysis for Blue Honeysuckle Extract in the Treatment of Hepatocellular Carcinoma.

To further determine how BHE affected the growth of HCC cells, the proportion of each cell cycle phase was explored in HCC cells by flow cytometry. Blue honeysuckle (Lonicera caerulea L.) is a species of bush that grows in eastern Russia. Blue honeysuckle extract (BHE) is rich in bioactive phytochemicals which can inhibit the proliferation of tumor cells. The mechanism underlying the anticancer activity of BHE in primary liver cancer is poorly understood. The purpose of this study was to evaluate the growth inhibition mechanism of bioactive substances from blue honeysuckle on hepatocellular carcinoma (HCC) cells and to explore its protein and gene targets. The compounds in BHE were determined by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS). Cell counting kit-8 (CCK8) assay was used to evaluate the effects of BHE on HCC cell proliferation, and flow cytometry assay (FCA) was used to determine how BHE arrested the proportion of each cell cycle phase in HCC cells. Western blot (WB) was performed to determine the expression of cell cycle-related proteins in HCC cells treated with different concentrations of BHE. The xenograft tumor animal models were established by HCC cell implantation. The results showed that cyanidin-3-o-glucoside and cyanidin-3-o-sophoroside which are the main biologically active components were detected in BHE. BHE is highly effective in inhibiting the proliferation of HCC cells by arresting the HCC cell cycle in the G2/M phase. BHE also downregulated the expression of conventional or classical dendritic cells-2 (cDC2) and cyclin B1 by promoting the expression of myelin transcription factor 1 (MyT1) in HCC cells. The weight and volume of xenografts were significantly decreased in the BHE treated groups when compared to the control group. BHE increased the expression of MyT1 in xenograft tissues. These findings showed that blue honeysuckle extract inhibits proliferation in vivo and in vitro by downregulating the expression of cDC2 and cyclin B1 and upregulating the expression of MyT1 in HCC cells.

Effect of Electrode Insertion Angle on Cochlear Implantation Outcomes in Adult and Children Patients with Sensorineural Hearing Loss.

Purpose: To determine the role played by electrode insertion angle in cochlear implantation (CI) outcomes in adult and children patients with sensorineural hearing loss (SNHL). Methods: Adults (n = 10) and children (n = 19) with SNHL undergoing CI in a tertiary specialized hospital were retrospectively enrolled. The measurements were evaluated before and after CI surgery using sound field audiometry and speech recognition tests. Questionnaires were used to assess subjective benefits. Electrode insertion angles were determined using postoperative X-rays. Results: Both adult and children patients showed significant improvements in hearing, speech performance, and audiology and speech-related quality of life after CI. The angular insertion depths of adult and children group were 323.70 ± 43.57° and 341.53 ± 57.07°, respectively, showing no significant difference. In the adult group, deeper insertion depths were found to be strongly linked to lower postoperative pure tone thresholds at 12 months and higher postoperative disyllabic Word Recognition and Sentence Recognition Scores at 6 months (all P < 0.05). In the children group, deeper insertion depth had a positive correlation with postoperative monosyllabic Word Recognition Scores 6 and 12 months after CI surgery (both P < 0.05). Multiple linear regression models were constructed to predict disyllabic Word Recognition Scores at 6 and 12 months postoperatively in the children group, in which insertion angle, duration of hearing loss, and preoperative questionnaire result were identified as dependent variables. Conclusions: Greater angular insertion depths resulted in improved hearing and speech performances after CI. The benefits of greater angular insertion depths can be found in both adult and children patients and last for at least 12 months. Clinicians are expected to determine the optimal implantation direction during CI and ensure the insertion depth to improve the speech rehabilitation of patients.

Clinical Value of Pepsinogen in the Screening, Prevention, and Diagnosis of Gastric Cancer.

OBJECTIVES: To compare the levels of serum pepsinogen (PG) in patients with gastric cancer (GC), patients with atrophic gastritis (AG), and healthy donors. Also, we explored the clinical value of PG detection for the diagnosis and treatment of GC. METHODS: The PG level in peripheral blood from patients and heathy donors was determined using an Abbott automatic chemiluminescence instrument. The study included 117 patients with GC confirmed by gastroscopy and histopathology, of whom 13 patients had cancer at stage I, 47 at stage II, 41 at stage III, and 16 at stage IV. The AG group included 122 patients, and the control group had 120 healthy donors. The relationship between serum PG levels and the occurrence and development of GC, as well as the evaluation of the clinical value of diagnostic tests based on serum PG detection, were investigated by receiver operating characteristic (ROC) curve analyses. RESULTS: Pepsinogen I (PGI) levels gradually decreased from the control group, the AG group, and the GC group. PGI exhibited high diagnostic value for GC (area under the curve [AUC], 0.834; cutoff, 51.2 ng/mL, sensitivity, 81.7%; specificity, 68.4%), PGII (AUC, 0.587; cutoff value, 13.05 ng/mL; sensitivity, 65.8%; specificity, 53.8%), and PGR (AUC, 0.752; cutoff, 5.65; sensitivity, 54.2%; specificity, 87.2%). The occurrence of GC was negatively correlated with serum levels of PGI (B = -0.054; OR = 0.947; 95% confidence interval [CI], 0.925-0.970; P <.001) and PGR (B = -0.420; OR = 0.657; 95% CI, 0.499-0.864; P = .003). CONCLUSIONS: The combined detection of PGI, PGII, and PGR has important clinical value for the screening, prevention, and diagnosis of GC and could allow for earlier detection, diagnosis, and treatment of GC.

BMI and BMI Changes to All-cause Mortality among the Elderly in Beijing: a 20-year Cohort Study.

OBJECTIVE: To explore the association between body mass index (BMI) and all-cause mortality among the elderly in Beijing. METHODS: This analysis was based on the Beijing multidimensional longitudinal study of aging (BLSA), which included 2,090 subjects over 55 years old and was followed-up from 1992 to 2012. BMI-mortality curves were drawn to find the optimal BMI range with the lowest mortality. Cox proportional hazard models were used to obtain the hazard ratios (HRs) for BMI and BMI changes in the overall population and in specific stratified populations. RESULTS: During follow-up, 1,164 deaths were recorded; BMI-mortality curve was U-shaped, with the lowest mortality at a BMI of approximately 25 kg/m2. After adjusting for gender, age, smoking, drinking and some pre-existing diseases, HRs for underweight, overweight and obesity compared with normal weight were 1.372 (95% CI: 1.154-1.631), 0.767 (95% CI: 0.666-0.884) and 0.871 (95% CI: 0.830-1.246), respectively. HR for BMI drop was 3.245 (95% CI: 0.824-12.772) in the underweight group and 1.892 (95% CI: 0.830-1.246) in the normal weight group, HR for BMI rise was 1.795 (95% CI: 1.243-2.591) in normal weight group and 1.962 (95% CI: 1.202-3.203) in the overweight group. CONCLUSION: Keeping BMI in an overweight status and stable is related to a reduced mortality.

Constipation, fiber intake and non-compliance contribute to inadequate colonoscopy bowel preparation: a prospective cohort study.

OBJECTIVE: Adequate bowel preparation is important for colonoscopy. Currently available evidence on the determinants of poor bowel preparation is largely derived from studies in Western countries. We aimed to identify the risk factors for inadequate bowel preparation for colonoscopy in the Chinese population. METHODS: In this single-center study, patients admitted to the Outpatient Department between March 2013 and December 2015 and had indications for colonoscopy were prospectively enrolled. Questionnaires were administered to the patients. Their characteristics and procedure-related parameters such as procedure time were recorded. Bowel preparation was assessed using Boston bowel preparation scale score. RESULTS: A total of 409 patients with a mean age of 48.8 ± 12.9 years were enrolled in the study, 60.9% of whom were men. On univariate analysis, poor educational level (P = 0.020), chronic constipation (P = 0.001), taking no physical exercise after medication (P < 0.001), a high-fiber diet during the 24-h period immediately preceding the colonoscopy (P < 0.001), incomplete intake of medication (P < 0.001), the passage of yellow or dark stools before colonoscopy (P < 0.001), waiting time (P = 0.001) and stool frequency after medication (P = 0.048) were significantly associated with inadequate bowel preparation. On multivariate analysis, chronic constipation [odds ratio (OR) 2.05, 95% confidence interval (CI) 1.31-3.23, P = 0.002], incomplete intake of the medication (OR 2.77, 95% CI 1.47-5.21, P = 0.002) and a high-fiber diet within 24 h before colonoscopy (OR 2.15, 95% CI 1.40-3.28, P < 0.001) were independent risk factors for inadequate bowel preparation. CONCLUSIONS: Chronic constipation, poor compliance with treatment and high-fiber diet were predictors of poor bowel preparation. Patients with these risk factors require more effective strategies for bowel preparation.

Interplay between menin and Dnmt1 reversibly regulates pancreatic cancer cell growth downstream of the Hedgehog signaling pathway.

Menin, the product of the Men1 gene, which is frequently mutated in pancreatic neuroendocrine tumors, acts as a chromatin-remodeling factor to modulate the transcription of cell cycle regulators by interacting with histone modification factors. However, the function of menin and its underlying mechanisms in pancreatic ductal adenocarcinoma remain unknown. Here, we found that menin inhibited pancreatic cancer cell growth in vitro and in vivo and that its expression was gradually lost during pancreatic carcinogenesis. Menin overexpression significantly activated the expression of the cyclin-dependent kinase (CDK) inhibitors p18 and p27, accompanied with a decrease in DNA methylation levels of p18 and p27 promoters. Mechanistically, we found that interaction of menin with DNA methyltransferase 1 (Dnmt1) competitively pulled down Dnmt1 from p18 and p27 promoters, leading to the downregulation of DNA methylation levels. Moreover, menin expression was suppressed by Dnmt1 downstream of the Hedgehog signaling pathway, and menin overexpression strongly antagonized the promotion effect of hedgehog signaling on pancreatic cancer cell proliferation. Taken together, the interaction between menin and Dnmt1 reversibly regulates pancreatic cancer cell growth downstream of Hedgehog pathways with complex mutual modulation networks, suggesting that the Hedgehog/Dnmt1/menin axis is a potential molecular target for pancreatic cancer therapy.

MicroRNA-10b is upregulated and has an invasive role in colorectal cancer through enhanced Rhoc expression.

We found that the difference in miR­10b expression between the tumor tissue and adjacent non­tumor tissue was significant. Outer periphery and portal vein serum miR­10b concentrations were significantly higher than those of the control. However, the outer periphery vein miR­10b concentrations were not significant when compared with the portal vein serum concentration in colorectal cancer. The expression levels of miR­10b were associated with higher­grade colorectal cancer. MiR­10b levels were markedly elevated in lymph node metastasis-positive tumor tissue compared with those in lymph node metastasis-free tumor tissue, and were correlated with a downregulation in Hoxd10 expression. Rhoc protein expression in tumor tissue was significantly amplified when compared to that of the control tissue group. An inverse correlation between Hoxd10 and Rhoc in immunohistochemistry and western blot analysis was observed (P<0.05). MiR-10b expression was also inversely correlated with Hoxd10 protein expression (P<0.05). Thus, miR­10b is potentially involved in the invasion of colorectal cancer.

[Effect of maternal high-fat diet before and during pregnancy on bone growth of neonatal offspring rats].

OBJECTIVE: To explore the mechanism and effect of maternal high-fat diet before and during pregnancy on bone growth of neonatal offspring rats. METHODS: Forty female Sprague-Dawley rats were divided into high-fat diet and control groups (n=20) that were fed with 35% high-fat diet and standard chow, respectively. After 8 weeks, 8 female rats from each group were sacrificed for liver pathological examinations and the other female rats were mated with male rats and fed continuously with 35% high-fat diet and standard chow throughout gestation, respectively. The body lengths (from apex nasi to end of tail) of the offspring rats from both groups were measured within 24 hours after birth. Enzyme-linked immunosorbent assay was used to detect serum insulin-like growth factor (IFG-I) levels. Liver pathological changes were observed under a light microscope. The expression of insulin receptor substrate 1 (IRS-1) and phosphorylation IRS-1 (Phospho-IRS-1) in tibia and femur samples were detected by immunohistochemistry. The expression of mitogen-activated protein kinase (MAPK) and phosphorylation MAPK (Phospho-MAPK), phosphatidylinositol 3-kinase (PI3K) and phosphorylation PI3K (Phospho-PI3K), protein kinase B (AKT1) and phosphorylation AKT1 (Phospho-AKT1) in tibia and femur samples were detected by Western blot. RESULTS: The offspring rats from the high-fat diet group showed a significant shorter body length compared with those from the control group (P<0.05). The level of serum IGF-I in offspring rats from the high-fat diet group decreased by 20.1% in comparison to those from the control group, but there was no significant difference between the two groups (P>0.05). Fatty degeneration was found in livers of both high-fat diet-fed maternal rats and their offspring rats under a light microscope. There were no significant differences in IRS-1 and Phospho-IRS-1 expression in chondrocytes of tibia and femur samples between the offspring rats of the two groups (P>0.05). The protein expression of MAPK in chondrocytes of tibia and femur samples of offspring rats from the high-fat diet group was higher than that from the control group (P<0.05). There were no significant differences of PI3K and AKT1/Phospho-AKT1 between the offspring rats of the two groups (P>0.05). CONCLUSIONS: A maternal high-fat diet before and during pregnancy may affect the bone growth of offspring rats in utero, which is possibly associated with the decreased IGF-I level. However, further study on the exact mechanism of IGF-I on the bone growth is needed.

Combined detection tumor markers for diagnosis and prognosis of gallbladder cancer.

AIM: To clarify the value of combined use of markers for the diagnosis of gallbladder cancer and prediction of its prognosis. METHODS: Serum cancer antigens (CA)199, CA242, carcinoembryonic antigen (CEA), and CA125 levels were measured in 78 patients with gallbladder cancer (GBC), 78 patients with benign gallbladder diseases, and 78 healthy controls using electrochemiluminescence. CA199, CA242, CEA, and CA125 levels and positive rates were analyzed and evaluated pre- and post-operatively. Receiver operator characteristic curves were used to determine diagnostic sensitivity and specificity of GBC. Survival time analysis, including survival curves, and multivariate survival analysis of a Cox proportional hazards model was performed to evaluate independent prognostic factors. RESULTS: Serum CA242, CA125, and CA199 levels in the GBC group were significantly higher when compared with those in the benign gallbladder disease and healthy control groups (P < 0.01). With a single tumor marker for GBC diagnosis, the sensitivity of CA199 was the highest (71.7%), with the highest specificity being in CA242 (98.7%). Diagnostic accuracy was highest with a combination of CA199, CA242, and CA125 (69.2%). CA242 could be regarded as a tumor marker of GBC infiltration in the early stage. The sensitivity of CA199 and CA242 increased with progression of GBC and advanced lymph node metastasis (P < 0.05). The 78 GBC patients were followed up for 6-12 mo (mean: 8 mo), during which time serum CA199, CA125, and CA242 levels in the recurrence group were significantly higher than in patients without recurrence (P < 0.01). The post-operative serum CA199, CA125, and CA242 levels in the non-recurrence group were significantly lower than those in the GBC group (P < 0.01). Multivariate survival analysis using a Cox proportional hazards model showed that cancer of the gallbladder neck and CA199 expression level were independent prognostic factors. CONCLUSION: CA242 is a marker of GBC infiltration in the early stage. CA199 and cancer of the gallbladder neck are therapeutic and prognostic markers.

[Effect of Roux-en-Y gastric bypass on quality of life in non-obese patients with type 2 diabetes mellitus].

OBJECTIVE: To observe the impact of Roux-en-Y gastric bypass on quality of life in non-obese patients with type 2 diabetes mellitus. METHODS: Thirty-seven non-obese patients with type 2 diabetes mellitus who underwent Roux-en-Y gastric bypass were prospectively studied. A 36-item short form healthy survey questionnaire(SF36), the diabetes treatment satisfaction questionnaire(DTSQ), and quality of life scale for patients with type 2 diabetes mellitus(DMQLS) were used to evaluate the quality of life for all the non-obese patients with type 2 diabetes mellitus preoperatively and 12 months postoperatively. RESULT: The blood glucose and lipid indexes were significantly decreased after operation(all P<0.05). SF36 showed the physical and mental synthesis scores at 12 month after operation were 74.6±18.3 and 79.8±14.9 respectively, higher than those at one week before operation(54.9±15.1 and 56.4±17.8, both P<0.01). DTSQ showed treatment satisfaction score was increaced significantly after operation(29.2±7.1 vs. 15.4±5.6, P<0.01). The quality of life evaluated by DMQLS, was also significantly improved(P<0.01). CONCLUSION: Roux-en-Y gastric bypass can significantly improve the quality of life for non-obese patients with type 2 diabetes mellitus.

Identification of a conserved B-cell epitope on reticuloendotheliosis virus envelope protein by screening a phage-displayed random peptide library.

BACKGROUND: The gp90 protein of avian reticuloendotheliosis-associated virus (REV-A) is an important envelope glycoprotein, which is responsible for inducing protective antibody immune responses in animals. B-cell epitopes on the gp90 protein of REV have not been well studied and reported. METHODS AND RESULTS: This study describes the identification of a linear B-cell epitope on the gp90 protein by screening a phage-displayed 12-mer random peptide library with the neutralizing monoclonal antibody (mAb) A9E8 directed against the gp90. The mAb A9E8 recognized phages displaying peptides with the consensus motif SVQYHPL. Amino acid sequence of the motif exactly matched (213)SVQYHPL(219) of the gp90. Further identification of the displayed B cell epitope was conducted using a set of truncated peptides expressed as GST fusion proteins and the Western blot results indicated that (213)SVQYHPL(219) was the minimal determinant of the linear B cell epitope recognized by the mAb A9E8. Moreover, an eight amino acid peptide SVQYHPLA was proven to be the minimal unit of the epitope with the maximal binding activity to mAb A9E8. The REV-A-positive chicken serum reacted with the minimal linear epitopes in Western blot, revealing the importance of the eight amino acids of the epitope in antibody-epitope binding activity. Furthermore, we found that the epitope is a common motif shared among REV-A and other members of REV group. CONCLUSIONS AND SIGNIFICANCE: We identified (213)SVQYHPL(219) as a gp90-specific linear B-cell epitope recognized by the neutralizing mAb A9E8. The results in this study may have potential applications in development of diagnostic techniques and epitope-based marker vaccines against REV-A and other viruses of the REV group.

Evaluation of recombinant fowlpox virus expressing infectious bronchitis virus S1 gene and chicken interferon-γ gene for immune protection against heterologous strains.

A recombinant fowlpox virus (rFPV-IFNγS1) that co-expressed the infectious bronchitis virus (IBV) S1 gene and the chicken interferon-γ gene has been constructed. To evaluate the efficacy of the recombinant fowlpox virus vaccine against heterotypic IBV strains, 60 4-week-old Specific-Pathogen-Free (SPF) chickens were inoculated with this vaccine and 3 weeks post inoculation challenged with the homotypic IBV strain LX4 and the heterotypic IBV strains LHB, LHLJ04XI, LTJ95I and LSC99I. Antibodies against IBV were detected in vaccinated chickens 1-week post inoculation. The number of CD4(+) and CD8(+) T-lymphocytes in the peripheral blood increased rapidly in the vaccinated groups challenged with strains LX4, LHB and LHLJ04XI. There were significant differences in the number of CD4(+) and CD8(+) T-lymphocytes between the vaccinated groups challenged with strains LTJ95I and LSC99I and all the control groups. The morbidity was below 30% in vaccinated groups challenge with strains LX4, LHB and LHLJ04XI, but was 40% greater than that in the other groups. In addition, the lesions and the amount of virus shedding were less severe in the vaccinated groups challenged by strains LX4, LHB and LHLJ04XI when compared with the other groups, but there was no significant difference in the average body weight of the chickens in all groups (all p>0.05). These results indicate that the rFPV-IFNγS1 protected chickens against challenge with homotypic IBV strain LX4 and heterotypic strains LHLJ04XI and LHB.

A novel polymer-lipid hybrid nanoparticle for efficient nonviral gene delivery.

AIM: To develop a novel non-viral vector with high transfection efficiency and low cytotoxicity. METHODS: Poly (ethylene glycol)-distearoylphosphatidylethanolamine (PEG-DSPE) was incorporated into polymer-lipid hybrid nanoparticles (PLN) to construct a PEG-DSPE modified long circulating PLN (L-PLN). The L-PLN was prepared by the emulsifying-solvent evaporation method, L-PLN and L-PLN/DNA complexes were characterized. Both HEK293 and MDA-MB-231 cells transfected by L-PLN/DNA complexes were observed under a fluorescence microscope. The transfection efficiency of the complexes to HEK293 cells was further evaluated by flow cytometry. RESULTS: The GFP fluorescence intensity in HEK293 cells transfected by the L-PLN/DNA complexes (N/P=10) was about 37.2%, which was higher than those transfected by PLN alone or commercial Lipofectamine 2000. The L-PLN exhibited minimal toxicity at a low N/P ratio compared with other vectors. CONCLUSION: L-PLN as a novel gene delivery system, has higher transfection efficiency and acceptable cytotoxicity compared to the corresponding PLN, which is beneficial for the development of non-viral gene transfer vectors and may offer an alternative strategy for the future gene therapy.

Protection of chickens against infectious bronchitis by a recombinant fowlpox virus co-expressing IBV-S1 and chicken IFNgamma.

A fowlpox virus expressing the chicken infectious bronchitis virus (IBV) S1 gene of the LX4 strain (rFPV-IBVS1) and a fowlpox virus co-expressing the S1 gene and the chicken type II interferon gene (rFPV-IBVS1-ChIFNgamma) were constructed. These viruses were assessed for their immunological efficacy on specific-pathogen-free (SPF) chickens challenged with a virulent IBV. Although the antibody levels in the rFPV-IBVS1-ChIFNgamma-vaccinated group were lower than those in the attenuated live IB vaccine H120 group and the rFPV-IBVS1 group, the rFPV-IBVS1-ChIFNgamma provided the strongest protection against an IBV LX4 virus challenge (15 out of 16 chickens immunized with rFPV-IBVS1-ChIFNgamma were protected), followed by the attenuated live IB vaccine (13/16 protected) and the rFPV-IBVS1 (12/16 protected). Compared to those of the rFPV-IBVS1 and the attenuated live IB vaccine groups, chickens in the rFPV-IBVS1-ChIFNgamma group eliminated virus more quickly and decreased the presence of viral antigen more significantly in renal tissue. Examination of affected tissues revealed abnormalities in the liver, spleen, kidney, lung and trachea of chickens vaccinated with the attenuated live IB vaccine and the rFPV-IBVS1 vaccine. In rFPV-IBVS1-ChIFNgamma-vaccinated chickens, pathological changes were also observed in those organs, but were milder and lasted shorter. The lesions in the mock control group were the most severe and lasted for at least 20 days. This study demonstrated that chicken type II interferon increased the immunoprotective efficacy of rFPV-IBVS1-ChIFNgamma and normal weight gain in vaccinated chickens although it inhibited serum antibody production.

Monoclonal antibodies and conserved antigenic epitopes in the C terminus of GP5 protein of the North American type porcine reproductive and respiratory syndrome virus.

Glycoprotein 5 (GP5) is the major glycoprotein of porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the gene encoding rtGP5, lacking signal peptide sequence, was expressed as GST-fusion protein in E. coli. Fifteen monoclonal antibodies (MAbs) against rtGP5 were developed and used to probe a series of GP5 peptides by ELISA, in which two MAbs specifically recognized the epitope GP5EP3 (146-156aa), four recognized GP5EP5 (164-180aa) and nine recognized GP5EP7 (192-200aa). After precise analysis by sequential deletion of the terminal amino acid residues, the three minimal epitopes (R(152)LYRWR(156), E(169)GHLIDLKRV(178) and Q(196)WGRL(200)) were determined, which were highly conserved among the North American type isolates, with the exception of one amino acid mutation (L(200) to P(200)). Mutational analysis showed that the mutant (Q(196)WGRP(200)) could be recognized by four of nine anti-GP5EP7 MAbs, indicating Q(196)WGRP(200) was also one minimal epitope. Western blot analysis showed that GP5EP5 and GP5EP7 (L(200) or P(200)) could be recognized by PRRSV-positive sera of CH-1a and/or BJ-4, suggesting GP5EP5 and GP5EP7 (L(200) or P(200)) were antigenic epitopes in the PRRSV-infected pigs. MAbs against GP5EP3, GP5EP5, and GP5EP7 could react with MARC-145 cells infected with the North American type isolates from China in IFA. However, very interestingly, when the highly pathogenic PRRSV, represented by HUN4, was passaged in MARC-145 cells, MAbs against GP5EP7 did not react with HUN4-F20-HUN4-F112 (20-112th passage virus), where Q(196)WGRL(200) had mutated to R(196)WGRL(200). Due to no mutations observed in GP5EP3 and GP5EP5, MAbs against GP5EP3 and GP5EP5 could recognize HUN4-F20-HUN4-F112. All the results herein might deepen the understanding of the antigen structure of in the C terminus of GP5 and facilitate the development of diagnostic antigens of the North American type PRRSV in China.

Construction of an infectious Marek's disease virus bacterial artificial chromosome and characterization of protection induced in chickens.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphoma in poultry. The complete genome of the avirulent vaccine strain MDV-814 was cloned as an infectious bacterial artificial chromosome (BAC) using an 8.8-kb fragment containing the self-designed selective marker guanosine phosphoriboxyl transferase. The recombinant virus MDV-814-BAC was generated by co-transfection of a BAC transfer vector and MDV-814 total DNA, and was purified by eight rounds of selective passaging. The infectivity of the BAC DNA clone was validated by MDV reconstitution from chicken embryo fibroblasts transfected with MDV-BAC DNA, which was extracted from electroporated Escherichia coli DH10B cells. In vitro, the BAC-derived virus had similar biological characteristics and growth kinetics as the wild-type parental and recombinant viruses, and chickens immunized with BAC derivatives by various delivery mechanisms acquired protection against virulent MDV challenge. Construction of this MDV-BAC may aid the development of recombinant vaccines-containing multiple antigens.

Protection of chickens from Newcastle disease and infectious laryngotracheitis with a recombinant fowlpox virus co-expressing the F, HN genes of Newcastle disease virus and gB gene of infectious laryngotracheitis virus.

A recombinant fowlpox virus (rFPV) coexpressing the Newcastle disease virus (NDV) fusion and hemagglutinin-neuraminidase genes and infectious laryngothracheitis virus (ILTV) glycoprotein B gene was constructed. This virus was then evaluated for its ability to protect specific-pathogen-free (SPF) chickens against clinical symptoms and death after challenge by virulent NDV and ILTV. SPF chickens were grouped and vaccinated with the rFPV and commercial NDV (La Sota) and ILTV attenuated live vaccine (Nobilis ILT), respectively. After challenge with NDV 10 days postvaccination, 70% of chickens vaccinated with rFPV were protected from death, whereas 100% of the commercial NDV-vaccinated chickens were protected from death. In contrast, 100% of the unvaccinated chickens died after challenge. After challenge with ILTV, both the rFPV and commercial ILTV-vaccinated chickens were completely protected from death and 70% of chickens were protected from respiratory signs. In comparison, 100% of the unvaccinated chickens developed severe respiratory disease and 10% of chickens died. The protective efficacy was also measured by the antibody responses and isolation of challenge viruses. Results showed that this rFPV could be a potential vaccine for preventing NDV and ILTV by a single immunization.

[Construction and characterization of a recombinant fowlpox virus co-expressing F, HN genes of Newcastle disease virus and gB gene of infectious laryngnotracheitis virus].

The Fusion (F) and Haemagglutinin-Neuraminidase (HN) genes of Newcastle disease virus (NDV) and the glycoprotein B (gB) gene of infectious laryngothracheitis virus (ILTV) as well as a LacZ reporter gene were all inserted into a nonessential gene of fowlpox virus (FPV) 017 strain by homologous recombination. The NDV and ILTV genes were each under the control of a fowlpox virus immediate early/late promoter (LP2EP2) while the LacZ reporter gene expression cassette was regulated by a P11 late promoter. A recombinant FPV harboring the F, HN and gB genes as well as the LacZ gene, designated as rFPV-F/HN/gB/LacZ, was obtained after ten cycles of blue plaque purification. The presence of the NDV and ILTV genes was confirmed by PCR. The expression of the recombinant proteins in rFPV-F/HN/gB/LacZ were characterized by Western blot (F and gB proteins) and indirect immunofluorescence test (F, HN and gB proteins). The results demonstrated that all four foreign proteins, which were encoded within a 10 kb gene fragment, could be expressed authentically and efficiently. Compared to the parental virus, rFPV-F/HN/gB/LacZ showed no obvious difference with respect to virus replication and cytopathogenic effects in chicken embryo fibroblasts (CEF) cell culture. Overall, our work suggests that FPV can be a useful live virus vector for the expression of multi- foreign genes against multiple avian pathogens.