Macrophage SAMSN1 protects against sepsis-induced acute lung injury in mice.

OBJECTIVE: Inflammation and oxidative stress contribute to the progression of sepsis-induced acute lung injury (ALI). SAM domain, SH3 domain and nuclear localization signals 1 (SAMSN1) is a signaling adaptor protein, and mainly regulates inflammatory response of various immune cells. The present study generates macrophage-specific SAMSN1-knockout (Samsn1MKO) and SAMSN1-transgenic (Samsn1MTG) mice to investigate its role and mechanism in sepsis-induced ALI. METHODS: Samsn1MKO and Samsn1MTG mice were exposed to lipopolysaccharide (LPS) instillation or cecal ligation and puncture (CLP) surgery to induce sepsis-induced ALI. Bone marrow transplantation, cellular depletion and non-invasive adoptive transfer of bone marrow-derived macrophages (BMDMs) were performed to validate the role of macrophage SAMSN1 in sepsis-induced ALI in vivo. Meanwhile, BMDMs were isolated from Samsn1MKO or Samsn1MTG mice to further clarify the role of SAMSN1 in vitro. RESULTS: Macrophage SAMSN1 expression was increased in response to LPS stimulation, and negatively correlated with LPS-induced ALI in mice. Macrophage SAMSN1 deficiency exacerbated, while macrophage SAMSN1 overexpression ameliorated LPS-induced inflammation, oxidative stress and ALI in mice and in BMDMs. Mechanistically, we found that macrophage SAMSN1 overexpression prevented LPS-induced ALI though activating AMP-activated protein kinase α2 (AMPKα2) in vivo and in vitro. Further studies revealed that SAMSN1 directly bound to growth factor receptor bound protein 2-associated protein 1 (GAB1) to prevent its protein degradation, and subsequently enhanced protein kinase A (PKA)/AMPKα2 activation in a protein tyrosine phosphatase, non-receptor type 11 (PTPN11, also known as SHP2)-dependent manner. Moreover, we observed that macrophage SAMSN1 overexpression diminished CLP-induced ALI in mice. CONCLUSION: Our study documents the protective role of macrophage SAMSN1 against sepsis-induced inflammation, oxidative stress and ALI through activating AMPKα2 in a GAB1/SHP2/PKA pathway, and defines it as a promising biomarker and therapeutic target to treat sepsis-induced ALI.

Cefoperazone sodium/sulbactam sodium vs piperacillin sodium/tazobactam sodium for treatment of respiratory tract infection in elderly patients.

BACKGROUND: Respiratory tract infections in the elderly are difficult to cure and can easily recur, thereby posing a great threat to patient prognosis and quality of life. AIM: To investigate the therapeutic effects of different antibiotics in elderly patients with respiratory tract infection. METHODS: Seventy-four elderly patients with respiratory tract infection were randomly allocated to a study (n = 37; treated with cefoperazone sodium/sulbactam sodium) or control (n = 37; treated with piperacillin sodium/tazobactam sodium on the basis of routine symptomatic support) group. Both groups were treated for 7 d. Time to symptom relief (leukocyte recovery; body temperature recovery; cough and sputum disappearance; and rale disappearance time), treatment effect, and laboratory indexes [procalcitonin (PCT), C-reactive protein (CRP), white blood cell count (WBC), and neutrophil percentage (NE)] before and 7 d after treatment and the incidence of adverse reactions were assessed. RESULTS: In the study group, the time to WBC normalization (6.79 ± 2.09 d), time to body temperature normalization (4.15 ± 1.08 d), time to disappearance of cough and sputum (6.19 ± 1.56 d), and time to disappearance of rales (6.68 ± 1.43 d) were shorter than those of the control group (8.89 ± 2.32 d, 5.81 ± 1.33 d, 8.77 ± 2.11 d, and 8.69 ± 2.12 d, respectively; P = 0.000). Total effective rate was higher in the study group (94.59% vs 75.68%, P = 0.022). Serum PCT (12.89 ± 3.96 µg/L), CRP (19.62 ± 6.44 mg/L), WBC (20.61 ± 6.38 × 109/L), and NE (86.14 ± 7.21%) levels of the study group before treatment were similar to those of the control group (14.05 ± 4.11 µg/L, 18.79 ± 5.96 mg/L, 21.21 ± 5.59 × 109/L, and 84.39 ± 6.95%, respectively) with no significant differences (P = 0.220, 0.567, 0.668, and 0.291, respectively). After 7 d of treatment, serum PCT, CRP, WBC, and NE levels in the two groups were lower than those before treatment. Serum PCT (2.01 ± 0.56 µg/L), CRP (3.11 ± 1.02 mg/L), WBC (5.10 ± 1.83 × 109/L), and NE (56.35 ± 7.17%) levels were lower in the study group than in the control group (3.29 ± 0.64 µg/L, 5.67 ± 1.23 mg/L, 8.13 ± 3.01 × 109/L, and 64.22 ± 8.08%, respectively; P = 0.000). There was no significant difference in the incidence of adverse reactions between the groups (7.50% vs 12.50%, P = 0.708). CONCLUSION: Piperacillin sodium/tazobactam sodium is superior to cefoperazone sodium/ sulbactam sodium in the treatment of elderly patients with respiratory tract infection with a similar safety profile.

The Role of Gut Microbiota in Tumor Immunotherapy.

Tumor immunotherapy is the fourth therapy after surgery, chemotherapy, and radiotherapy. It has made great breakthroughs in the treatment of some epithelial tumors and hematological tumors. However, its adverse reactions are common or even more serious, and the response rate in some solid tumors is not satisfactory. With the maturity of genomics and metabolomics technologies, the effect of intestinal microbiota in tumor development and treatment has gradually been recognized. The microbiota may affect tumor immunity by regulating the host immune system and tumor microenvironment. Some bacteria help fight tumors by activating immunity, while some bacteria mediate immunosuppression to help cancer cells escape from the immune system. More and more studies have revealed that the effects and complications of tumor immunotherapy are related to the composition of the gut microbiota. The composition of the intestinal microbiota that is sensitive to treatment or prone to adverse reactions has certain characteristics. These characteristics may be used as biomarkers to predict the prognosis of immunotherapy and may also be developed as "immune potentiators" to assist immunotherapy. Some clinical and preclinical studies have proved that microbial intervention, including microbial transplantation, can improve the sensitivity of immunotherapy or reduce adverse reactions to a certain extent. With the development of gene editing technology and nanotechnology, the design and development of engineered bacteria that contribute to immunotherapy has become a new research hotspot. Based on the relationship between the intestinal microbiota and immunotherapy, the correct mining of microbial information and the development of reasonable and feasible microbial intervention methods are expected to optimize tumor immunotherapy to a large extent and bring new breakthroughs in tumor treatment.

Clinicopathologic features of the ureteral neuroendocrine tumors.

BACKGROUND: Neuroendocrine tumors of the ureter are extremely rare. There are only a few case reports in the past decades. Their clinicopathologic features, therapy and prognosis are not that clear. METHODS: 5 cases of ureteral neuroendocrine tumors were collected and reviewed of the literature. Histomorphology, immunophenotype and ultrastructural features were observed by HE, immunohistochemistry, special staining and electron microscopy. The clinical pathological data were retrospectively analyzed and followed up. RESULTS: Among the 5 patients, 1 was female and 4 were male, aged 62-82 years. 2 cases manifested intermittent hematuria, 1 had lower abdominal pain with frequent urination and dysuria, 1 with hydronephrosis, and 1 had no manifestations. All the 5 patients were treated with nephroureterectomy, 3 of which were also treated with excision of bladder cuff, 1 also had lymphadenectomy. On presentation, 2 cases in T2N0M0 (stage II), 2 cases in T3N0M0 (stage III), and 1case in T3N2M0 (stage IV). 2 cases were small cell neuroendocrine carcinoma, 1 was large cell neuroendocrine carcinoma and 2 were atypical carcinoid. The tumor cells were positive for neuroendocrine markers (CD56, CgA, Syn). 1 case of vimentin-positive small cell neuroendocrine carcinoma has a very good prognosis. Grimelius stain and electron microscopy observation showed numerous neuroendocrine granules in the cytoplasm. CONCLUSION: Ureteral neuroendocrine tumors are extremely rare. Neuroendocrine markers (CD56, CgA, Syn) and epithelial markers (CKpan, CK7) are usually helpful. Grimelius special staining and electron microscopy observation can help to make a final diagnosis. Radical surgery together with postoperative adjuvant chemotherapy can improve the survival of patients. Vimentin may play a role in predicting the prognosis.

Analgesic Effect of Dizocine Combined with Ropivacaine on Recurrent Neuropathic Pain in Peripheral Nerve Compression Rats.

To explore the analgesic effect of dizocine combined with ropivacaine on recurrent neuropathic pain in rat model of peripheral nerve compression. Rats were randomly divided into 5 groups: sham control group (S), peripheral nerve compression model group (M), dizocine group (D), ropivacaine group (R), and combined drug group (DR). Rat peripheral nerve compression model was constructed to observe the symptoms of the rats before and after surgery. Mechanical withdrawal threshold was measured on the 21st day after surgery. The electrophysiological changes of rat peripheral nerve were measured by biopotential recording system, including proximal latency, distal latency, and compound muscle action potential. The incubation period and nerve conduction velocity were further obtained. Histological changes were observed by HE staining and toluidine blue staining. Axon number and myelin damage grade were performed, and the ultrastructure was observed by transmission electron microscopy (TEM). The mechanical withdrawal threshold, nerve conduction velocity, and compound muscle action potential were effectively increased in combination group. However, the proximal latency, distal latency, and incubation period were significantly reduced. Furthermore, dizocine combined with ropivacaine can effectively reduce the degree of myelination. TEM shown that the DR group had the best therapeutic effect, and the histological appearance of the cross section was quite similar to that of the S group. Dizocine combined with ropivacaine has a significant analgesic effect in rat model of peripheral nerve compression.

Effect of microRNA-133a-3p/matrix metalloproteinase-9 axis on the growth of atherosclerotic vascular smooth muscle cells.

Atherosclerosis (AS) is the leading cause of cardiovascular disease and poses a threat to human health. MicroRNAs (miRNAs/miRs) are a group of endogenous small non-coding RNAs that have been identified to serve important roles in AS. However, the expression and role of miR-133a-3p in AS remains unclear. The aim of the present study was to investigate miR-133a-3p in AS and to determine its underlying mechanism. The level of miR-133a-3p expression in the blood and vascular plaque tissue of patients with AS was detected via reverse transcription-quantitative PCR (RT-qPCR). The role of miR-133a-3p in human vascular smooth muscle cells (hVSMCs) was investigated, following upregulation and downregulation of this miR in hVSMCs. Cell proliferation and apoptosis were determined using a Cell Counting kit-8 assay and flow cytometry, respectively. The results demonstrated the downregulation of miR-133a-3p in the blood and vascular plaque tissue of patients with AS. Matrix metallopeptidase-9 (MMP-9) was revealed to be a direct target gene of miR-133a-3p, which was upregulated in the blood and vascular plaque tissue of patients with AS. Furthermore, MMP-9 was determined to be negatively regulated by miR-133a-3p in hVSMCs. In addition, significant inhibition of hVSMC proliferation and induction of cell apoptosis were observed following MMP-9 downregulation and following transfection with the miR-133a-3p mimic. The effects of the miR-133a-3p mimic on hVSMC proliferation and apoptosis were reversed by MMP-9 over-expression. Overall, the results indicated that miR-133a-3p was downregulated in AS, which results in the inhibition of hVSMC proliferation and the induction of cell apoptosis via MMP-9. miR-133a-3p may therefore be a promising therapeutic target for the treatment of AS.

Association Between Gut Bacterial Diversity and Mortality in Septic Shock Patients: A Cohort Study.

BACKGROUND Gut bacterial diversity is decreased in a proportion of patients with septic shock. We attempted to validate the hypothesis that low bacterial diversity increases the risk of mortality. MATERIAL AND METHODS All patients with septic shock seen at 2 medical center from 2016 through 2019 were included in this cohort study. Total DNA was isolated from stool, and high-throughput sequencing was performed. Clinical data were extracted from patient medical records and hospital databases. Patients were grouped by gut microbiota bacterial diversity (measured by Shannon diversity index) on presentation. We used logistic regression analysis to evaluate the risk of 28-day mortality in septic patients with low Shannon diversity index. RESULTS Of the 150 patients enrolled in this study, low bacterial diversity (Shannon index <3.0) was found in 80 patients and normal diversity (Shannon index ≥3.0) was found in 70 patients. Low diversity was associated with a higher unadjusted mortality risk, compared to those with normal diversity (odds ratio [OR] 2.04, 95% confidence interval [CI] 1.35-2.83). However, this result became non-significant after adjusting the confounding factors such as age, sex, severity of disease, comorbid status, usage of probiotics, enteral nutrition, and antimicrobial drugs (OR 1.93, 95% CI 0.55-2.69). CONCLUSIONS Our study does not support that low gut bacterial diversity is an independent risk factor for mortality in intensive care unit patients with septic shock.

MicroRNA-199a-5p aggravates primary hypertension by damaging vascular endothelial cells through inhibition of autophagy and promotion of apoptosis.

The present study investigated the expression of microRNA (miRNA or miR)-199a-5p in the peripheral blood of patients with primary hypertension, and examined its mechanism of action in vascular endothelial cell injury induced by hypertension. A total of 57 patients with primary hypertension, who were treated at the Affiliated Hospital of Qingdao University (Qingdao, China) between December 2014 and November 2015 were included in the present study. Peripheral blood was collected from all patients. The expression of miR-199a-5p was measured using reverse-transcription quantitative polymerase chain reaction analysis. Human umbilical vein endothelial cells (HUVECs) were divided into negative control, miR-199a-5p mimics and rescue (co-transfected with miR-199a-5p mimics and inhibitor) groups. After transfection, the proliferation and apoptosis of HUVECs were evaluated by a Cell Counting Kit-8 assay, a bromodeoxyuridine incorporation assay and flow cytometry. Western blot analysis was used to determine the expression of proteins involved in autophagy-associated and adenosine monophosphate kinase (AMPK)/unc-51 like autophagy activating kinase 1 (ULK1) signaling pathways. Laser scanning confocal microscopy and electron microscopy were used to observe the autophagy of HUVECs. The expression of miR-199a-5p was elevated in peripheral blood of patients with hypertension, and was correlated with the progression of hypertension. Overexpression of miR-199a-5p inhibited the proliferation and promoted the apoptosis of HUVECs. Upon expression of miR-199a-5p, the transition between microtubule-associated proteins 1A/1B light chain 3B (LC3B)I and LC3BII proteins was inhibited, the expression of p62 protein was upregulated. In addition, miR-199a-5p decreased the numbers of autophagosomes and autolysosomes in HUVECs. The present study demonstrated that expression of miR-199a-5p is positively correlated with the severity of hypertension. Expression of miR-199a-5p aggravated vascular endothelial injury by inhibiting autophagy and promoting the apoptosis of HUVECs via downregulation of the AMPK/ULK1 signaling pathway.

Isoquercetin ameliorates myocardial infarction through anti-inflammation and anti-apoptosis factor and regulating TLR4-NF-κB signal pathway.

The aim of the present study was to investigate the protective mechanisms and identify the effects of isoquercetin on myocardial infarction in a rat model of acute myocardial infarction (AMI). Isoquercetin ameliorated myocardial infarct size, creatine kinase (CK), CK­MB and lactic dehydrogenase activity and inhibited inflammation, oxidative stress and heart cell apoptosis in a rat with AMI. Isoquercetin increased endothelial nitric oxide synthase, reduced inducible nitric oxide synthase levels and suppressed the Toll-like receptor 4­nuclear factor (TLR4­NF)­κB signaling pathway in a rat with AMI. Overall, isoquercetin ameliorated AMI through anti­inflammatory and anti­apoptotic factors, and regulation of the TLR4­NF­κB signaling pathway. Isoquercetin may therefore potentially exert a protective effect against AMI or other heart diseases.

MicroRNA-185 suppresses pancreatic cell proliferation by targeting transcriptional coactivator with PDZ-binding motif in pancreatic cancer.

The aim of the present study was to compare the expression of transcriptional coactivator with the PDZ-binding motif (TAZ) in pancreatic cancer (PC) patients, and to investigate the regulation mechanisms of TAZ in the proliferation of PC. PC tissues and matched peritumoral tissues, pancreatic juice and serum were collected from PC patients who underwent pancreatectomy between June 2012 and December 2015 at the Affiliated Hospital of Qingdao University (Qingdao, China). Pancreatic juice and serum were collected from patients with chronic pancreatitis as a control. The levels of taz mRNA expression in the samples were examined by reverse-transcription quantitative polymerase chain reaction, and the protein expression of TAZ was assessed by western blot analysis and ELISA. MicroRNAs (miRNAs) that regulate TAZ expression were also predicted by bioinformatics analysis and validated by dual luciferase reporter and rescue assays. In addition, the proliferation of PC cells was evaluated after transfection with TAZ small interfering RNA (siRNA) or its upstream miRNA agomir. Expression of TAZ was significantly increased in the PC tissues, pancreatic juice and serum of PC patients at the mRNA and protein levels compared with controls (P<0.05). Furthermore, TAZ was predicted and verified to be a target of miRNA (miR)-185, and miR-185 and TAZ were inversely expressed in samples from PC patients (P<0.05). In addition, TAZ siRNA or agomiR-185 transfection significantly inhibited human pancreatic adenocarcinoma cell proliferation (P<0.05). However, overexpression of TAZ in the agomiR-185 group rescued the inhibition (P<0.05). Finally, the expression of TAZ effector proteins, namely ankyrin repeat domain-containing protein and cysteine-rich 61, were upregulated in PC tissues (P<0.05), but repressed following transfection of PC cells with agomiR-185 (P<0.05). Thus, miR-185 may regulate the proliferation of PC by targeting TAZ, making it a promising diagnostic marker for PC.

Neuroglobin protects cardiomyocytes against apoptosis and cardiac hypertrophy induced by isoproterenol in rats.

Neuroglobin (Ngb) is well known as a physiological role in oxygen homeostasis of neurons and perhaps a protective role against hypoxia and oxidative stress. In this study, we found that Ngb is expressed in rat heart tissues and it is related to isoproterenol induced cardiac hypertrophy. Moreover, overexpression or knock-down of Ngb influences the expression of hypertrophic markers ANP and BNP and the ratio of hypertrophic cells in rat H9c2 myoblasts when isoproterenol treatment. The Annexin V-FITC/PI Staining, Western blot and qPCR analysis showed that the involvement in p53-mediated apoptosis of cardiomyocytes of Ngb is might be the mechanism. This protein could prevent the cells against ROS and POS-induced apoptosis not only in nervous systems but also in cardiomyocytes. From the results, it is concluded that Ngb is a promising protectant in the cardiac hypertrophy, it may be a candidate target to cardiac hypertrophy for clinic treatment.

Absolute quantification of a very virulent Marek's disease virus dynamic quantity and distributions in different tissues.

Chickens infected with Marek's disease virus (MDV) carry the virus consistently for a long time, which increases the incidence and rate of virus-induced multi-organ tumors and increases its potential for horizontal transmission. There is a positive correlation between very virulent (vv) MDV quantity and the pathology. The purpose of this study was to determine the vvMDV loads dynamics in different phases, and the correlation between the viral quantity and tumor development. We used a SYBR Green duplex real-time quantitative PCR (q-PCR) assay to detect and quantify MDV loads and distributions in different tissues, targeting the Eco-Q protein gene (meq) of the virus and the house-keeping ovotransferrin (ovo) gene of chickens. The q-PCR was performed using different tissue DNA preparations derived from chickens which were infected with 1,000 pfu of the SDWJ1302 strain and tissue samples were collected from control and MDV-infected birds on 7, 10, 15, 21, 28, 40, 60, and 90 d post-infection (DPI). The data indicated that the MDV genome was almost quantifiable in immune organs of infected chickens as early as 7 DPI, and the number of MDV genome copies in the blood and different organs peaked by 28 DPI, but then gradually decreased by 40 DPI. The levels of viral quantity in the lymphocytes, liver, and spleen were all higher than those in other organs, and that in the feather follicles was the highest among different phases of MDV infection. The vvMDV could still be detected in peripheral blood and tissues by 90 DPI, and the vast existence of virus will stimulate tissue destruction. The data provided further evidence of viral infection involving multi-organ distribution and mainly involving immune organ proliferation, resulting in immunosuppression.

Transcriptional activity comparison of different sites in recombinant Marek's disease virus for the expression of the H9N2 avian influenza virus hemagglutinin gene.

Over the last two decades, much attention has been paid to MDV-vectored recombinant vaccines. Many factors have influenced their protective efficacy, and insertion site has been among the main influential factors for the expression of foreign genes in recombinant Marek's disease virus (rMDV). To compare the transcriptional activity of different sites of rMDV, an H9N2 avian influenza virus hemagglutinin gene (AIV-H9N2-HA) expression cassette that used the bi-directional promoter of serotype 1 MDV (MDV1) in the 1.8kb RNA transcript direction (p1.8kb) as a promoter was inserted into 4 different regions of MDV using the bacterial artificial chromosome (BAC) vector and FLP/FRT recombination technique. The insertion regions included 3 of its own sites (US2, US10 and one of Meq genes) in the MDV genome and a foreign site (gpt gene) in the BAC vector. Quantitative PCR and enzyme-linked immunosorbent assay (ELISA) were used to analyze and compare the H9N2-HA expression levels of these different rMDVs both at the mRNA level and at the protein level. The results indicated that among the four tested insertion regions, the HA expression cassette in the US2 region demonstrated the highest activity, followed by that in the Meq region, which was almost equal to that of US10. Further, the expression cassette had the lowest activity in the foreign region gpt gene. The above data could be useful for choosing proper recombinant insertion regions in the construction of rMDV to express different foreign genes, and it is a prerequisite for developing effective MDV-vectored recombinant vaccines.

Comparative transcriptional activity of five promoters in BAC-cloned MDV for the expression of the hemagglutinin gene of H9N2 avian influenza virus.

On the basis of recent studies, much attention has been given to recombinant MDV (rMDV)-based vaccines. During the construction of rMDV, the activity of promoters to transcribe foreign genes is one of the major factors that can affect protective efficacy. To investigate the transcription activity and efficacy of five different promoters, the advantage of an existing rMDV BAC infectious clone that had been previously constructed was used to construct rMDVs. The expression cassette of the hemagglutinin gene (HA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain was inserted into the US2 region under five selected promoters. These five promoters included three MDV endogenous promoters (the promoter for the gB gene and a bi-directional promoter in both directions for pp38 (ppp38) and 1.8 kb RNA transcripts (p1.8 kb)), and two exogenous promoters (CMV and SV40). Among these five promoters, the CMV promoter demonstrated the highest activity, followed by p1.8 kb and SV40, which had a similar transcriptional activity level. Two of the MDV endogenous promoters showed much lower transcriptional activities, particularly the promoter ppp38, which had the lowest activity. The results of the in vivo experiment proved that none of the three recombinant viruses of rGX-CMV-HA, rGX-SV40-HA and rGX-p1.8kb-HA provided protection in SPF chickens. Chickens vaccinated with rGX-pPP38-HA induced 50% and rGX-gB-HA induced 25% protection against the challenge with H9N2, respectively.

Construction of recombinant Marek's disease virus (MDV) lacking the meq oncogene and co-expressing AIV-H9N2 HA and NA genes under control of exogenous promoters.

To develop a recombinant Marek's disease virus (rMDV1) co-expressing the hemagglutinin gene (HA) and neuramidinase gene (NA) from a low pathogenic avian influenza virus (LPAIV) H9N2 strain and lacking the meq oncogene that shares homology with the Jun/Fos family of transcriptional factors, a wild strain of MDV GX0101 was used as parental virus, the HA and NA genes co-expression cassette under control of the CMV and SV40 early promoters was inserted at two meq sites of GX0101 to form a new meq knock-out mutant MDV (MZC12HA/NA) through homologous recombination. MZC12HA/NA was reconstituted by transfection of recombinant BAC-MDV DNA into the secondary chicken embryo fibroblast (CEF) cells. Highly purified MZC12HA/NA was obtained after four rounds of plaque purification and proliferation. In vitro growth properties of recombinant virus were also inspected and concluded that the MZC12HA/NA had the same growth kinetics in CEF cultures as its parental wild type virus GX0101. Southern blot indicated that co-expression cassette was successfully inserted at two copies sites of meq gene, so two meq genes were knocked-out completely. RT-qPCR showed transcription and expression levels of the HA and NA genes were both significantly higher than that of GX0101 own pp38 gene. Indirect fluorescence antibody (IFA) test, and Western blot analyses indicated that HA and NA genes were co-expressed simultaneously under control of the different promoters but meq genes were not. These results herald a new and effective recombinant meq-deleted MDV-based AIV-H9N2 vaccine may be useful in protecting chickens from very virulent MDV and H9N2 challenges.

Construction of recombinant Marek's disease virus (rMDV) co-expressing AIV-H9N2-NA and NDV-F genes under control of MDV's own bi-directional promoter.

To qualitatively analyze and evaluate a bi-directional promoter transcriptional function in both transient and transgenic systems, several different plasmids were constructed and recombinant MDV type 1 strain GX0101 was developed to co-express a Neuraminidase (NA) gene from Avian Influenza Virus H9N2 strain and a Fusion (F) gene from the Newcastle disease virus (NDV). The two foreign genes, NDV-F gene and AIV-NA gene, were inserted in the plasmid driven in each direction by the bi-directional promoter. To test whether the expression of pp38/pp24 heterodimers are the required activators for the expression of the foreign genes, the recombinant plasmid pPpp38-NA/1.8kb-F containing expression cassette for the two foreign genes was co-transfected with a pp38/pp24 expression plasmid, pBud-pp38-pp24, in chicken embryo fibroblast (CEF) cells. Alternatively, plasmid pPpp38-NA/1.8kb-F was transfected in GX0101-infected CEFs where the viral endogenous pp38/pp24 were expressed via virus infection. The expression of both foreign genes was activated by pp38/pp24 dimers either via virus infection, or co-expression. The CEFs transfected with pPpp38-NA/1.8kb-F alone had no expression. We chose to insert the expression cassette of Ppp38-NA/1.8kb-F in the non-essential region of GX0101ΔMeq US2 gene, and formed a new rMDV named MZC13NA/F through homologous recombination. Indirect fluorescence antibody (IFA) test, ELISA and Western blot analyses indicated that F and NA genes were expressed simultaneously under control of the bi-directional promoter, but in opposite directions. The data also indicated the activity of the promoter in the 1.8-kb mRNA transcript direction was higher than that in the direction for the pp38 gene. The expression of pp38/pp24 dimers either via co-tranfection of the pBud-pp38-pp24 plasmid, or by GX0101 virus infection were critical to activate the bi-directional promoter for expression of two foreign genes in both directions. Therefore, the confirmed function of the bi-directional promoter provides better feasibilities to insert multiple foreign genes in MDV genome based vectors.