LIM domain only 7 negatively controls nonalcoholic steatohepatitis in the setting of hyperlipidemia.

BACKGROUND AND AIMS: Hyperlipidemia has been extensively recognized as a high-risk factor for NASH; however, clinical susceptibility to NASH is highly heterogeneous. The key controller(s) of NASH susceptibility in patients with hyperlipidemia has not yet been elucidated. Here, we aimed to reveal the key regulators of NASH in patients with hyperlipidemia and to explore its role and underlying mechanisms. APPROACH AND RESULTS: To identify the predominant suppressors of NASH in the setting of hyperlipidemia, we collected liver biopsy samples from patients with hyperlipidemia, with or without NASH, and performed RNA-sequencing analysis. Notably, decreased Lineage specific Interacting Motif domain only 7 (LMO7) expression robustly correlated with the occurrence and severity of NASH. Although overexpression of LMO7 effectively blocked hepatic lipid accumulation and inflammation, LMO7 deficiency in hepatocytes greatly exacerbated diet-induced NASH progression. Mechanistically, lysine 48 (K48)-linked ubiquitin-mediated proteasomal degradation of tripartite motif-containing 47 (TRIM47) and subsequent inactivation of the c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) cascade are required for the protective function of LMO7 in NASH. CONCLUSIONS: These findings provide proof-of-concept evidence supporting LMO7 as a robust suppressor of NASH in the context of hyperlipidemia, indicating that targeting the LMO7-TRIM47 axis is a promising therapeutic strategy for NASH.

Cavin1 activates the Wnt/ß-catenin pathway to influence the proliferation and migration of hepatocellular carcinoma.

INTRODUCTION AND OBJECTIVES: Cavin1 is a cell membrane caveolin, with controversial function in different tumors. Meanwhile, the role of Cavin1 in hepatocellular carcinoma (HCC) progression remains unclear. In this study, we attempted to elucidate the significance of Cavin1 in HCC occurrence and progression. MATERIALS AND METHODS: Cavin1 content was examined in HCC tissues and paired adjacent normal liver tissues by qRT-PCR and IHC among 81 HCC patients. The Cavin1-mediated regulation of HCC proliferation and metastasis was assessed through in vitro and in vivo experiments. Finally, using GSEA, we found out Cavin1 could be a potential regulator of the Wnt pathway. The alterations of the Wnt pathway-related proteins were identified by Western Blot analysis. RESULTS: Cavin1 was lower expressed in HCC, which implied poor survival outcomes in HCC patients. Phenotypic experiments revealed that Cavin1 strongly suppressed HCC proliferation and migration in vitro and in vivo. Besides, altered epithelial-mesenchymal transition (EMT)-related protein expressions were detected. Based on our GSEA analysis, Cavin1 activated the Wnt pathway, and Western Blot analysis revealed diminished ß-catenin, c-Myc, and MMP9 contents upon Cavin1 overexpression. CONCLUSIONS: Cavin1 suppresses HCC progression by modulating HCC proliferation and migration via inhibiting the Wnt/ß-catenin axis activation.

An Explainable Student Fatigue Monitoring Module with Joint Facial Representation.

Online fatigue estimation is, inevitably, in demand as fatigue can impair the health of college students and lower the quality of higher education. Therefore, it is essential to monitor college students' fatigue to diminish its adverse effects on the health and academic performance of college students. However, former studies on student fatigue monitoring are mainly survey-based with offline analysis, instead of using constant fatigue monitoring. Hence, we proposed an explainable student fatigue estimation model based on joint facial representation. This model includes two modules: a spacial-temporal symptom classification module and a data-experience joint status inferring module. The first module tracks a student's face and generates spatial-temporal features using a deep convolutional neural network (CNN) for the relevant drivers of abnormal symptom classification; the second module infers a student's status with symptom classification results with maximum a posteriori (MAP) under the data-experience joint constraints. The model was trained on the benchmark NTHU Driver Drowsiness Detection (NTHU-DDD) dataset and tested on an Online Student Fatigue Monitoring (OSFM) dataset. Our method outperformed the other methods with an accuracy rate of 94.47% under the same training-testing setting. The results were significant for real-time monitoring of students' fatigue states during online classes and could also provide practical strategies for in-person education.

Indocyanine green fluorescence imaging-guided laparoscopic right posterior hepatectomy.

BACKGROUND: Laparoscopic right posterior hepatectomy is considered difficult on the basis of the surgery difficulty scoring system. In this study, we evaluated the safety and effectiveness of the technical application of indocyanine green (ICG) fluorescence imaging-guided laparoscopic right posterior hepatectomy. METHODS: Twenty-six patients who underwent ICG fluorescence imaging-guided laparoscopic right posterior hepatectomy at Hepatobiliary and Pancreatic Surgery Department of Zhongnan Hospital, Wuhan University, from June 2018 to December 2019, were included. The influence of patient position, trocar placement, hepatic inflow occlusion, central venous pressure (CVP), and the ICG fluorescence imaging-guided method were analyzed. RESULTS: In 17 patients, the left lateral position was maintained when the main tumor was in the S7, and in the remaining nine patients, the supine position was maintained with the right side of the body raised when the main tumor was in the S6. Ten patients who underwent preoperative injection of ICG were successfully developed for nonanatomical hepatectomy. Sixteen patients received intraoperative ICG injection for anatomical hepatectomy (2 cases had positive imaging findings, 14 cases had negative imaging findings, and 2 cases had failed imaging findings). All patients underwent the Pringle maneuver during the procedure. Four patients were preset with subhepatic vena cava blocking and one patient with suprahepatic inferior vena cava blocking. CVP was controlled at 3.00 ± 0.63 (mean ± SD) cmH2O. The operative time was 216.14 ± 52.05 min, and the bleeding volume was 128.57 ± 75.55 ml. Four patients had Clavien-Dindo level I complications, and one had level III complications. Postoperative hospitalization duration was 6.19 ± 1.40 days. There were 14 patients with hepatocellular carcinoma, 9 with metastatic liver malignancies, 2 with hepatic hemangioma, 1 with focal nodular hyperplasia of the liver, and 10 with hepatitis B liver cirrhosis. CONCLUSIONS: ICG fluorescence imaging guidance could be helpful for the safe implementation of laparoscopic right posterior hepatectomy.

TRIM45 facilitates NASH-progressed HCC by promoting fatty acid synthesis via catalyzing FABP5 ubiquitylation.

Non-alcoholic steatohepatitis (NASH) is rapidly surpassing viral hepatitis as the primary cause of hepatocellular carcinoma (HCC). However, understanding of NASH-progressed HCC remains poor, which might impede HCC diagnosis and therapy. In this study, we aim to identify shared transcriptional changes between NASH and HCC, of which we focused on E3 ligase TRIM45. We found TRIM45 exacerbates HCC cells proliferation and metastasis in vitro and in vivo. Further transcriptome analysis revealed TRIM45 predominantly affects fatty acid metabolism and oleic acid restored impaired proliferation and metastasis of TRIM45-deficient HCC cells. IP-tandem mass spectrum and FABP5 depriving experiment indicated that TRIM45 enhance fatty acid synthesis depending on FABP5 presence. Interestingly, we found TRIM45 directly added K33-type and K63-type poly-ubiquitin chains to FABP5 NLS domain, which ultimately promoted FABP5 nuclear translocation. Nuclear FABP5 interacted with PPARγ to facilitate downstream lipid synthesis gene expression. We observed TRIM45 accelerated NASH-to-HCC transition and exacerbated both NASH and NASH-HCC with the enhanced fatty acid production in vivo. Moreover, high concentration of fatty acid increased TRIM45 expression. The established mechanism was substantiated by gene expression correlation in TCGA-LIHC. Collectively, our research revealed a common lipid reprograming process in NASH and HCC and identified the cyclical amplification of the TRIM45-FABP5-PPARγ-fatty acid axis. This signaling pathway offers potential therapeutic targets for therapeutic intervention in NASH and NASH-progressed HCC.

Exome sequencing identifies MXRA5 as a novel cancer gene frequently mutated in non-small cell lung carcinoma from Chinese patients.

Lung cancer has become the top killer among malignant tumors in China and is significantly associated with somatic genetic alterations. We performed exome sequencing of 14 non-small cell lung carcinomas (NSCLCs) with matched adjacent normal lung tissues extracted from Chinese patients. In addition to the lung cancer-related genes (TP53, EGFR, KRAS, PIK3CA, and ROS1), this study revealed "novel" genes not previously implicated in NSCLC. Especially, matrix-remodeling associated 5 was the second most frequently mutated gene in NSCLC (first is TP53). Subsequent Sanger sequencing of matrix-remodeling associated 5 in an additional sample set consisting of 52 paired tumor-normal DNA samples revealed that 15% of Chinese NSCLCs contained somatic mutations in matrix-remodeling associated 5. These findings, together with the results from pathway analysis, strongly indicate that altered extracellular matrix-remodeling may be involved in the etiology of NSCLC.

Epidemiological characteristics of adult SCIWORA in Tianjin, China: a preliminary study.

INTRODUCTION: The epidemiology of spinal cord injury without radiographic abnormality (SCIWORA) is less frequently reported in adults as compared with children. The annual incidence of SCIWORA was approximately 5.74% per million in Tianjin from 2004 to 2008. Importantly, the epidemiological characteristics of adult SCIWORA may be different from that in children. The aim of this study was to evaluate the radiological-clinical data of patients with adult SCIWORA, and to relatively analyze the epidemiological features. MATERIALS AND METHODS: Inpatients with cervical SCIWORA who were 16 and above in Tianjin were admitted in municipal hospitals in Tianjin from 2004 to 2008; all the patients received MRI scanning in sagittal and axial views. Epidemiological characteristics, such as injury origin, injury level or severity, neurological scale and MRI feature were acquired. RESULTS: In total, 203 patients were enrolled. The average age among the adult groups was 55.9 years (men 55.8 years, women 53.6 years). SCIWORA occurred more commonly in adults in the 46-60 age group, and falls were the leading cause of injury (52.2%), followed by vehicular injury (28.6%). The most predominantly affected level was C4/5 (48.7%), followed by C5/6 (30.5%) and C3/4 (12.8%), respectively. The occurrence of central cord syndrome (50.2%) with posterior longitudinal ligament tear (43.8%) was relatively higher than other injury patterns. CONCLUSION: It is clear that adult cervical SCIWORA is different from that in the pediatric group. Our study highlights the epidemiological properties of adult SCIWORA in Tianjin, China. Differing from other reports, particularly epidemiology study, we represent the first report regarding adult SCIWORA from China. As the geriatric population increases, it is very important to set up an individualized evaluation system based on a nationally scaled epidemiological database. The results from our study will be useful in assisting in the creation of such a database.

A Novel Immune-Related Prognostic Signature Predicting Survival in Patients with Pancreatic Adenocarcinoma.

Pancreatic adenocarcinoma (PAAD) carries the lowest survival rate of all major organ cancers, which is of dismal prognosis and high mortality rate. Thus, the present study attempted to identify a few novel prognostic biomarkers and establish an immune-related prognostic signature which could predict the prognosis of PAAD. Four prognostic immune-related genes (IRGs) including S100A6, S100A10, S100A16, and SDC1 were screened by differentially expressed gene (DEG) identification and weighted gene coexpression network analysis (WGCNA). Subsequent analysis proved the high expression of these IRGs in PAAD tissues, suggested by TCGA-PAAD data, merged microarray-acquired dataset (MMD), GEPIA, and Oncomine webtool. By using MMD and TCGA-PAAD data, S100A6 (MMD: AUC = 0.897; TCGA: AUC = 0.843), S100A10 (MMD: AUC = 0.880; TCGA: AUC = 0.780), S100A16 (MMD: AUC = 0.878; TCGA: AUC = 0.838), and SDC1 (MMD: AUC = 0.885; TCGA: AUC = 0.812) exhibited excellent diagnostic efficiency for PAAD. By conducting connectivity map (CMap) analysis, we concluded that three molecule drugs (sulpiride, famotidine, and nalidixic acid) might have worked in the treatment of PAAD. Then, an immune-related prognostic index was constructed, which was validated as an independent prognostic factor for PAAD patients (P=0.004). We further constructed a nomogram by using this immune-related signature and age, the prognostic value of which was validated by using concordance index (C-index = 0.780) and area under curve (AUC = 0.909). Moreover, the immune-related prognostic signature was associated with response to anti-PD-1/L1 immunotherapy. To sum up, four IRGs were screened out and verified to be novel immune-related prognostic biomarkers in PAAD. Besides, sulpiride, famotidine, and nalidixic acid might be potential choices in the treatment of PAAD. An immune-related signature was established to show great potential for prognosis prediction for PAAD, independently, which might guide more effective immunotherapy strategies. A nomogram is further established by using this immune-related prognostic index, which might contribute to more effective prognosis prediction in PAAD patients.

USP53 plays an antitumor role in hepatocellular carcinoma through deubiquitination of cytochrome c.

Despite of advances in treatment options, hepatocellular carcinoma (HCC) remains nearly incurable and has been recognized as the third leading cause of cancer-related deaths worldwide. As a deubiquitinating enzyme, the antitumor effect of ubiquitin-specific peptidase 53 (USP53) has been demonstrated on few malignancies. In this study, we investigated the potential antitumor role of USP53 in HCC. The results showed that USP53 was downregulated in HCC tissues as well as in HCC cell lines using both in silico data as well as patient samples. Furthermore, the ectopic expression of USP53 inhibited the proliferation, migration and invasion, and induced the apoptosis of HCC cells. Co-immunoprecipitation (CO-IP) assay and mass spectrometry (MS) combined with the gene set enrichment analysis (GSEA) identified cytochrome c (CYCS) as an interacting partner of USP53. USP53 overexpression increased the stability of CYCS in HCC cells following cycloheximide treatment. Finally, the overexpression of CYCS compensated for the decreased apoptotic rates in cells with USP53 knocked down, suggesting that USP53 induced the apoptosis in HCC cells through the deubiquitination of CYCS. To summarize, we identified USP53 as a tumor suppressor as well as a therapeutic target in HCC, providing novel insights into its pivotal role in cell apoptosis.

Effects of combined treatment with sansanmycin and macrolides on Pseudomonas aeruginosa and formation of biofilm.

OBJECTIVE: To observe the effects of combined treatment with sansanmycin and macrolides on Pseudomonas aeruginosa and formation of biofilm. METHODS: Micro-dilution method was used to determine the minimal inhibitory concentrations (MICs) of sansanmycin, gentamycin, carbenicillin, polymyxin B, roxithromycin, piperacillin, and tazobactam. PA1 and PA27853 biofilms were observed under optical microscope after staining and under SEM after treatment with sansanmycin at different dosages and combined treatment with sansanmycin and roxithromycin. Viable bacteria in PA1 and PA27853 biofilms were counted after treatment with sansanmycin at different dosages or combined treatment with sansanmycin and roxithromycin. RESULTS: The MIC of sansanmycin was lower than that of gentamycin and polymyxin B, but was higher than that of carbenicillin. Roxithromycin enhanced the penetration of sansanmycin to PA1 and PA27853 strains through biofilms. PA1 and PA27853 biofilms were gradually cleared with the increased dosages of sansanmycin or with the combined sansanmycin and roxithromycin. CONCLUSION: Sub-MIC levels of roxithromycin and sansanmycin substantially inhibit the generation of biofilms and proliferation of bacteria. Therefore, combined antibiotics can be used in treatment of intractable bacterial infection.

[Comparison of prognosis of small hepatocellular carcinoma patients with hepatitis B virus infection versus hepatitis C virus infection].

OBJECTIVE: To compare the prognosis of small hepatocellular carcinoma patients with hepatitis B virus infection versus hepatitis C virus infection. METHODS: 413 patients receiving curative resections at Tianjin Cancer Hospital for small HCC (< or = 3 cm) from January 1997 to December 2003 were divided into four groups: HCV only (n = 75), HBV only (n = 251), HBV and HCV (n = 33), and neither HBV nor HCV (NBNC, n = 54). The preoperative status and postoperative recurrence were recorded. Survival analysis were used to assess the impact of HBV/HCV status on HCC recurrence. RESULTS: Patients with HCV were associated with older age, lower mean preoperative platelet counts and albumin levels, higher mean prothrombin time, alanine aminotransferase and total bilirubin levels. Tumors in patients with HCV are multinodular and less differentiated, and were associated with a higher incidence of vascular invasion and cirrhosis. During the follow-up, the HCV group showed a higher incidence of intrahepatic recurrence and multiple recurrent lesions than the other patients. CONCLUSIONS: HCC patients with HCV infection tended to be older, and were characterized by more severe cirrhosis and higher incidence of tumor multinodular. The statistically significant determinants of reoccurrence in patients with small HCC were HCV infection, presence of vascular invasion and multiple tumors.

[Effects of Qiulirunfei extracta on immunological function and influenza virus in mice].

OBJECTIVE: To investigate the effect of Qiulirunfei extracta on immunological function and Influenza virus type A (FM1 strain) in ICR mice. METHODS: After given Qiulirunfei extracta, mice's body weight, spleen weight and lymphocyte stimulation index (SI) were tested; its antiviral effect was also observed in mice and chick embryo. RESULTS: Qiulirunfei extracta inhibited effectively the growth of influenza virus type A (FM1 strain) in chick embryo, and reduced the mortality rate of mice infected by influenza virus type A (FM1 strain). Compared with control group, body weight, spleen index in experiment group increased greatly, and SI of lymphocyte increased significantly too. CONCLUSION: Qiulirunfei extracta shows antiviral effects, and it is a good immune system enhancer.

Reconstruction of radial bone defects using the reinforced tissue-engineered periosteum: an experimental study on rabbit weightbearing segment.

BACKGROUND: The objective of this study was to compare the osteogenic potential of reinforced and conventional tissue-engineered periosteum. METHODS: Adipose-derived stromal cells of rabbits were induced into osteoblasts. Osteoinduced cells were seeded onto chitosan-tricalcium-phosphate-gelatin (Cs-TCP-Gel) and chitosan (Cs) scaffold, thus constructing the reinforced and conventional tissue-engineered periostea, respectively. Alkaline phosphatase (ALP) and von Kossa staining protocols were used to assess osteoblast phenotype.We surgically created a 15-mm-long bone defect in the right radii of New Zealand rabbits. The defects were treated with reinforced biomimetic periosteum in group A (n = 30) and treated with conventional tissue-engineered periosteum in group B (n = 30).Group C (n = 30) received CS-TCP-Gel scaffold alone, and group D (n = 30) served as untreated side (sham group). Radiologic,histologic, immunohistochemical, and histomorphometric studies were used to analyze healing pattern. RESULTS: ALP was remarkably expressed in the osteoinduced cells, indicating that osteoblastic differentiation was stable. Extracellular matrix calcification with dark nodule was detected by von Kossa staining. Compared with groups B and C, histologic results demonstrated that de novo osteogenesis proliferated in group A at 4 weeks. This was further confirmed by radiographic findings, which displayed the segmental gap completely healed by mature bone at 12 weeks. Robust expression of bone morphogenetic protein-2 in group A was also evident, whereas group D displayed poor osteogenic performance. Furthermore, histomorphometric and biomechanical results in group A demonstrated statistical significance over those in other groups (p 0.05). CONCLUSIONS: Our findings show that the reinforced tissue-engineered periosteum is superior to conventional one as a better biomimetic tissue,further indicating that it can repair the weight-bearing defects.

Generation and application of polyclonal antibody against replication and transcription activator of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma, the most common neoplasm in untreated HIV-1-infected individuals, and several B cell disorders. KSHV infection goes through lytic and latent phases, and the switch from latency to lytic replication is governed by viral replication and transcription activator (RTA). RTA consists of 691 amino acids, containing an N-terminal DNA-binding and a C-terminal activation domain. In the present study, polyclonal antibody against RTA was generated and evaluated. The C-terminal region of RTA (E482 approximately D691) was expressed in Escherichia coli, purified by affinity chromatography, and utilized to raise polyclonal antibody in BALB/c mice. High-affinity antisera were obtained, which successfully detected the antigen at a dilution of 1:13,500 for ELISA and 1:20,000 for Western blot analysis. The antibody can specifically recognize full-length RTA expressed in both E. coli and mammalian cells. Furthermore, endogenous RTA can be detected with the antibody in TPA-induced BCBL-1 cells under various conditions. These results suggested that the antibody is valuable for the investigation of biochemical properties and biological functions of RTA.

[Infection and latency of HSV-1 on rat bone marrow mesenchymal stem cells in vitro].

To culture bone marrow mesenchymal stem cells (BMSCs) of rat in vitro and observe HSV-1 infection on BMSCs, BMSCs were separated from the bone marrow and identified by alizarin red staining and detection of ALP. The morphology of HSV-1 infected BMSCs and the CPE were observed. The total DNA was extracted from HSV-1 infected BMSCs and the desired specific gene fragment of 477bp of HSV-1 was amplified by PCR. Results showed that after BMSCs were induced by mineral-fluid for 14 days, the ALP level was increased and the nodule calcification was formed. The induced BMSCs were manifested to have the characteristics of osteoblasts. CPE couldn't be found in HSV-1 latently infected BMSCs but the 477bp gene fragment was still detectable. HSV-1 could establish latent infection in BMSCs after 7 passages. This study indicated that rat BMSCs could be induced to differentiate into osteoblasts in vitro, therefore they can be used as the seed cells for the tissue engineering. HSV-1 can infect rat BMSCs and develop the latent infection in vitro.

[Complete genome sequence analysis of a newly isolated paramyxovirus Tianjin strain].

Paramyxovirus Tianjin strain is the high-pathogenic virus to primate and might also cause human lower respiratory tract infection. To determine the genome structure, variation features and phylogenetic position, the complete nucleotide sequence of paramyxovirus Tianjin strain was analyzed. The homology comparison and phylogenetic analysis of the nucleotide and the deduced amino acid sequences among paramyxovirus Tianjin strain and the 28 strains in seven genera and the 7 unclassified viruses of Paramyxoviridae were performed. The results suggested that Tianjin strain is a member of the Respirovirus genus in the Paramyxovirinae, Paramyxoviridae and has the closest relationship to Sendai virus. Its genome length and composition are similar to the previously published Sendai virus except one extra glutamic acid residue increasing at the C terminus of Large protein due to the genomic RNA mutation at position A15240C. 440 unique nucleotide variations of Tianjin strain lead to 110 amino acid residue changes, making it differed from any other Sendai viruses. The phylogenetic analysis reveals paramyxovirus Tianjin strain doesn't belong to any of the three known evolution lineages of Sendai viruses and locates at a separate evolution branch. The obvious distinctions of genome nucleotide sequence, host tropism and pathogenicity suggest that paramyxovirus Tianjin strain might represent a novel genotype of Sendai virus.

In vivo pharmacokinetics of hesperidin are affected by treatment with glucosidase-like BglA protein isolated from yeasts.

Hesperidin is an abundant flavanone glycoside in citrus fruits and has been reported to possess a wide range of biological activities. However, hesperidin has poor bioavailability. Here, we tested the hypothesis that hesperetin found in chenpi will have a better bioavailability than hesperidin and that treatment of hesperidin with the glucosidase-like yeast Bg1A protein will increase its bioavailability. The results indicate that hesperidin in pure or extract form is hydrolyzed by BglA protein extracted from Sporobolomyces singularis or expressed in Escherichia coli BL21 (DE3). This biotransformation affected the plasma pharmacokinetics of total hesperetin in rats, in that the plasma T max was significantly shorter after administration of BglA protein-treated hesperidin than after administration of hesperidin extract. In addition, the area under the curve values for total hesperetin after administration of Bg1A-treated hesperidin were approximately 4-fold higher by oral administration and 3-fold higher by intravenous administration, respectively. In contrast, the plasma clearance value and volume of distribution after administration of Bg1A-treated hesperidin extract or pure hesperetin were significantly smaller than after administration of untreated hesperidin extract or pure hesperidin. This is the first study that systemically determines the absolute bioavailability of hesperidin and hesperetin simultaneously, shows clearly that hesperetin is more bioavailable than hesperidin regardless of the route of administration, and shows that prior transformation of hesperidin to hesperetin via fermentation should significantly increase its bioavailability because of the action of the yeast glycosidase-like protein BglA.

[Construction of recombinant eukaryotic expression plasmid of murine chemokine MIP-1alpha and coimmunization with HSV-II DNA vaccine].

AIM: To construct the recombinant eukaryotic expression plasmid of murine macrophage inflammatory protein-1alpha (MIP-1alpha) and investigate the effect of MIP-1alpha as an adjuvant on immune response induced by herpes simplex virus type II glycoprotein D (HSV-II gD) DNA vaccine. METHODS: Using total RNA from RAW264.7 cells stimulated with LPS, the whole code sequence of murine MIP-1alpha was amplified by RT-PCR and inserted into pcDNA3 at Hind III/Xba I restriction sites. The recombinant eukaryotic expression plasmid Pm was transiently expressed in COS-7 cells and its specificity was demonstrated by RT-PCR and Boyden chemotaxis chamber assay. BALB/c mice were immunized with gD DNA vaccine and/or MIP-1alpha, and the effect of MIP-1alpha on gD DNA vaccine was evaluated by detecting anti-HSV-II antibody, antigen-specific lymphoproliferative responses, and examining survival rates after mice were challenged intravaginally with HSV-II. RESULTS: The recombinant eukaryotic expression plasmid of murine MIP-1alpha was constructed, and it was revealed that immune responses of HSV-II gD DNA vaccine were enhanced by coimmunization with MIP-1alpha. CONCLUSION: The murine MIP-1alpha can be used as an adjuvant of HSV-II gD DNA vaccine.

Macrophage inflammatory protein-1alpha expression plasmid enhances DNA vaccine-induced immune responses against HSV-2.

In this study, we examined the effectiveness of macrophage inflammatory protein (MIP)-1alpha cDNA as a HSV-2 DNA vaccine adjuvant. pcDNA3-gD (pgD) and pcDNA3-MIP-1alpha (pMIP-1alpha) were co-injected to examine the modulatory effects of MIP-1alpha on immune phenotype and protection against lethal challenge with HSV-2. We found that Th-cell proliferative responses were dramatically enhanced by co-injection of pgD and pMIP-1alpha compared with injection of pgD alone. The secretion of IL-2 and IFN-gamma was also significantly increased by pgD and pMIP-1alpha co-injection; however, the production of cytokines IL-4 and IL-10 was not affected by co-injection. pgD and pMIP-1alpha co-injection resulted in a moderate enhancement of systemic gD-specific antibody level, but mucosal secretory IgA was markedly enhanced. When BALB/c mice were challenged intravaginally with 100 LD50 of HSV-2 strain Sav, pMIP-1alpha co-injection with pgD improved their survival rate and significantly reduced both the number of mice with lesions and the lesion severity. Therefore, MIP-1alpha cDNA as a HSV-2 DNA vaccine adjuvant drives antigen-specific Th1-type responses, reducing HSV-2-derived morbidity and mortality.