Properties of Pacific white shrimp (Litopenaeus vannamei) collagen and its degradation by endogenous proteinases during cold storage.

Many factors are responsible for the diminished quality of shrimp during cold storage, while the role of collagen has rarely been studied. This study therefore investigated the relationship between collagen degradation and changes of textural properties of Pacific white shrimp, and its hydrolysis by endogenous proteinases. The textural properties of shrimp decreased gradually along with disruption of shrimp muscle tissues, and the chewiness property of shrimp muscle showed a linear relationship with collagen contents in muscle during 6-day-storage at 4 °C. Pepsin-solubilized collagen in shrimp muscle consisted of one α1 chain and two α2 chains, revealing a typical tripeptide sequence (i.e., Gly-X-Y) in their molecules. In addition, collagen could be hydrolyzed by crude endogenous proteinases extracted from shrimp hepatopancreas, and serine proteinase plays a critical role in the process. These findings strongly suggested that the quality reduction of shrimp during cold storage is closely associated with collagen degradation.

Epidemiological characteristics of respiratory viruses in patients with acute respiratory infections during 2009-2018 in southern China.

BACKGROUND: Acute respiratory infections (ARIs) remain a significant public threat with high morbidity and mortality worldwide; viruses are significant pathogens that cause ARIs. This study was conducted to better understand the epidemiological characteristics of respiratory viruses circulating in southern China. METHODS: We collected 22,680 respiratory samples from ARI patients in 18 hospitals in southern China during 2009-2018; seven common respiratory viruses including Flu, RSV, PIV, hMPV, ADV, HCoV, and HBoV were screened using in-house real-time PCR. RESULTS: Of all samples, 9760 ARI cases (9760/22680, 43.03%) tested positive for the seven common respiratory viruses. The most detected virus was Flu (14.15%), followed by RSV (10.33%) and PIV (5.43%); Flu-A, PIV3, and HCoV-OC43 were the predominant subtypes. Although most of the viruses were detected in male inpatients, Flu was more likely detected in female outpatients. Flu infection was more likely to cause URTI (upper respiratory tract infection), whereas RSV infection was more likely to cause pneumonia and bronchitis. The prevalence of Flu was particularly high in 2009. The epidemic level was found notably high in 2014-2018 for RSV, in 2016-2018 for PIV, in the summer of 2018 for ADV, in the summer of 2016 and winter of 2018 for HCoV, and in the summer of 2011 and autumn of 2018 for HBoV. The co-detection rate of the seven viruses was 4.70%; RSV, PIV, and Flu were the most commonly co-detected viruses. CONCLUSIONS: This work demonstrates the epidemiological characteristics of seven common respiratory viruses in ARI patients in southern China.

Epidemiological characteristics and phylogenic analysis of human respiratory syncytial virus in patients with respiratory infections during 2011-2016 in southern China.

BACKGROUND: Human respiratory syncytial virus (RSV) is one of the most important pathogens that cause acute respiratory infections in children and immunocompromised adults. This work was conducted to understand the epidemiological and phylogenetic features of RSV in southern China during 2011-2016. METHODS: A total of 16 024 nasopharyngeal swabs were collected from patients with respiratory infections in 14 hospitals, and screened for RSV and seven other respiratory viruses using real-time PCR. Six hundred and twenty-three RSV-positive samples from 13 hospitals were further analyzed for subtypes. G gene sequencing and phylogenetic analysis were performed based on 46 RSV-A and 15 RSV-B strains. RESULTS: RSV was detected in 9.5% of the 16 024 specimens, the highest among the eight respiratory viruses screened. Most of these specimens came from inpatients and children under 3 years of age. The incidence of RSV-A (9.4%) was higher than that of RSV-B (4.4%) in children (<15 years), but not in adults (0.64% vs. 0.58%). A 2-year RSV-A dominance followed by a 1-year RSV-B dominance pattern was found. The co-detection rate of RSV was 25.1%. The main prevalent genotypes were NA1, ON1, and BA9. The prevalent RSV-A genotype in 2011-2012 was NA1, close to Chongqing and Brazil, but a new Hong Kong ON1 genotype was introduced and became the prevalent genotype in Guangzhou in 2014-2015. Deduced amino acid sequence analysis confirmed the ongoing evolution and a high selection pressure of RSV-A and B strains, especially in RSV-A ON1 and NA1 genotypes. CONCLUSIONS: This study demonstrated the molecular epidemiological characteristics of RSV in patients with respiratory infections in southern China.

ZEB1-mediated vasculogenic mimicry formation associates with epithelial-mesenchymal transition and cancer stem cell phenotypes in prostate cancer.

The zinc finger E-box-binding homeobox 1 (ZEB1) induced the epithelial-mesenchymal transition (EMT) and altered ZEB1 expression could lead to aggressive and cancer stem cell (CSC) phenotypes in various cancers. Tissue specimens from 96 prostate cancer patients were collected for immunohistochemistry and CD34/periodic acid-Schiff double staining. Prostate cancer cells were subjected to ZEB1 knockdown or overexpression and assessment of the effects on vasculogenic mimicry formation in vitro and in vivo. The underlying molecular events of ZEB1-induced vasculogenic mimicry formation in prostate cancer were then explored. The data showed that the presence of VM and high ZEB1 expression was associated with higher Gleason score, TNM stage, and lymph node and distant metastases as well as with the expression of vimentin and CD133 in prostate cancer tissues. Furthermore, ZEB1 was required for VM formation and altered expression of EMT-related and CSC-associated proteins in prostate cancer cells in vitro and in vivo. ZEB1 also facilitated tumour cell migration, invasion and clonogenicity. In addition, the effects of ZEB1 in prostate cancer cells were mediated by Src signalling; that is PP2, a specific inhibitor of the Src signalling, dose dependently reduced the p-Src527 level but not p-Src416 level, while ZEB1 knockdown also down-regulated the level of p-Src527 in PC3 and DU-145 cells. PP2 treatment also significantly reduced the expression of VE-cadherin, vimentin and CD133 in these prostate cancer cells. Src signalling mediated the effects of ZEB1 on VM formation and gene expression.

Epidemiology characteristics of human coronaviruses in patients with respiratory infection symptoms and phylogenetic analysis of HCoV-OC43 during 2010-2015 in Guangzhou.

Human coronavirus (HCoV) is one of the most common causes of respiratory tract infection throughout the world. To investigate the epidemiological and genetic variation of HCoV in Guangzhou, south China, we collected totally 13048 throat and nasal swab specimens from adults and children with fever and acute upper respiratory infection symptoms in Gunazhou, south China between July 2010 and June 2015, and the epidemiological features of HCoV and its species were studied. Specimens were screened for HCoV by real-time RT-PCR, and 7 other common respiratory viruses were tested simultaneously by PCR or real-time PCR. HCoV was detected in 294 cases (2.25%) of the 13048 samples, with most of them inpatients (251 cases, 85.4% of HCoV positive cases) and young children not in nursery (53.06%, 156 out of 294 HCoV positive cases). Four HCoVs, as OC43, 229E, NL63 and HKU1 were detected prevalent during 2010-2015 in Guangzhou, and among the HCoV positive cases, 60.20% were OC43, 16.67% were 229E, 14.97% were NL63 and 7.82% were HKU1. The month distribution showed that totally HCoV was prevalent in winter, but differences existed in different species. The 5 year distribution of HCoV showed a peak-valley distribution trend, with the detection rate higher in 2011 and 2013 whereas lower in 2010, 2012 and 2014. The age distribution revealed that children (especially those <3 years old) and old people (>50 years) were both high risk groups to be infected by HCoV. Of the 294 HCoV positive patients, 34.69% (101 cases) were co-infected by other common respiratory viruses, and influenza virus was the most common co-infecting virus (30/101, 29.70%). Fifteen HCoV-OC43 positive samples of 2013-2014 were selected for S gene sequencing and phylogenetic analysis, and the results showed that the 15 strains could be divided into 2 clusters in the phylogenetic tree, 12 strains of which formed a separate cluster that was closer to genotype G found in Malaysia. It was revealed for the first time that genotype B and genotype G of HCoV-OC43 co-circulated and the newly defined genotype G was epidemic as a dominant genotype during 2013-2014 in Guanzhou, south China.

Bax-interacting factor-1 inhibits cell proliferation and promotes apoptosis in prostate cancer cells.

Prostate cancer (PCa) is one of the most common malignant tumors and the second leading cause of cancer-related death among males. Bax-interacting factor-1 (Bif-1) is a member of Endophilin family, which binds to and activates the BAX protein in response to the apoptosis signaling pathway. Loss of Bif-1 may suppress the intrinsic pathway of apoptosis and promote tumorigenesis, but there is also converse evidence that Bif-1 could in part be responsible for the tumorigenesis and the role of Bif-1 in PCa development is not clear. In the present study, we aimed to understand the relationships between Bif-1 expression and PCa development. The mRNA and protein expression levels of Bif-1 in PCa cell lines, benign prostatic hyperplasia (BPH) (n=100) and PCa tissues (n=100, including low Gleason-scored PCa n=43 and high Gleason-scored PCa n=57) were detected and the effects of Bif-1 overexpression on the apoptosis, proliferation and migration in LNCaP cells were explored. Bif-1 mRNA levels of PCa cell lines were analyzed by real-time PCR and the protein levels were detected by western blotting. Bif-1 expression in BPH and PCa samples was detected by immunohistochemistry. To build Bif-1 overexpression PCa cells, Bif-1 gene was transfected into LNCaP cells by pcDNA3.1(+)­Bif-1 vector. Cell apoptosis was detected by flow cytometric analysis, cell proliferation measured by 3­(4,5­dimethylthiazol­2­yl)­2,5­diphenyltetrazolium bromide (MTT) assay and cell migration was analyzed by wound­healing assay. The results proved that Bif-1 is downregulated in both PCa cell lines (P<0.01) and clinical samples (P<0.05), and Bif-1 expression is suppressed with the cancer progression (BPH vs. PCa P<0.01, and low Gleason-scored PCa vs. high Gleason-scored PCa P<0.05). Overexpression of Bif-1 could significantly inhibit cell proliferation (P<0.05) and enhancing PCa cell apoptosis (P<0.05), but it did not affect the migration ability (P>0.05). Our findings give strong evidence that Bif-1 is involved in PCa tumorigenesis and acts as a suppressor in PCa progression, and may have significance in understanding the process of PCa development.

Outbreak of PER-1 and diversity of ß-lactamases among ceftazidime-resistant Pseudomonas aeruginosa clinical isolates.

A growing number of ß-lactamases have been reported in Pseudomonas aeruginosa clinical isolates. The aim of this study was to investigate the diversity of ß-lactamases in the collection of 51 ceftazidime-resistant P. aeruginosa clinical isolates in four hospitals of southern China. Among these isolates, variable degrees of resistance to other ß-lactam and non-ß-lactam agents were observed. Pulsed-field gel electrophoresis (PFGE) revealed a high degree of clonality with five main genotypes. Of the 51 isolates tested, 35 (68.6%) were identified as extended-spectrum ß-lactamase (ESBL) producers, with 35 producing PER-1, 1 CTX-M-3, 7 CTX-M-15 and 1 CTX-M-14. Most (82.9%, 29/35) PER-1-producing isolates were collected from two hospitals between January and April in 2008 and belonged to the same PFGE pattern (pattern B) with similar antibiogram and ß-lactamase profiles, which suggested an outbreak of this clone at the time. The prevalence of CTX-M-type ESBL (17.6%, 9/51) was unexpectedly high. One isolate was identified as producing VIM-2. Furthermore, we also reported an occurrence of a novel OXA-10 variant, OXA-246, in 14 P. aeruginosa isolates. In addition, AmpC overproduction was found to be the ß-lactamase-mediated mechanism responsible for ceftazidime resistance in 6 isolates (11.8%). Our results revealed an overall diversity of ß-lactamases and outbreak of a PER-1-producing clone among ceftazidime-resistant P. aeruginosa in southern China.

OprD mutations and inactivation in imipenem-resistant Pseudomonas aeruginosa isolates from China.

To investigate the mechanisms involved in imipenem resistance of Pseudomonas aeruginosa in southern China, 61 imipenem-resistant P. aeruginosa clinical isolates were collected from 4 hospitals between October 2011 and June 2012. All isolates were resistant to imipenem, whereas 21.3% were susceptible or intermediate to meropenem. Variable degrees of resistance to other ß-lactam and non-ß-lactam antimicrobials were observed. PFGE revealed high-level of clonal diversity. Among the 61 isolates, 50 isolates had OprD loss by disrupted oprD mutations, including 43 with frameshift mutations of oprD and 7 with a premature stop codon by single point mutation. Six isolates were oprD-negative by PCR, suggestive of a major disruption of oprD genes. Five isolates had intact oprD but had reduced expression of oprD genes. In addition, only one isolate with disrupted oprD mutation by a premature stop codon was confirmed to be a metallo-ß-lactamase producer (IMP-9). Our results show that the loss of OprD, as well as reduced expression of oprD and MBL production, were the predominant mechanisms of imipenem resistance in P. aeruginosa in southern China.

Screening and identification of significant genes related to tumor metastasis and PSMA in prostate cancer using microarray analysis.

Tumor metastasis is one of the causes for the high mortality rate of prostate cancer (PCa) patients, yet the molecular mechanisms of PCa metastasis are not fully understood. In our previous studies, we found that PSMA suppresses the metastasis of PCa, yet the underlying mechanism remains unknown. To identify the genes related to tumor metastasis possibly regulated by PSMA, we performed tumor metastasis PCR array assay to analyze the differentially expressed tumor metastasis-related genes. Eighty-four tumor metastasis related genes were screened in si-PSMA LNCap cells (PSMA silenced by siRNA)/LNCap cells and in PC-3/LNcap cells, respectively. Expression levels of possible related genes were verified by real-time PCR in 4 prostate cancer cell lines (LNCap, 22RV1, PC-3 and DU145) and in 85 clinical samples (12 normal, 26 benign prostatic hypertrophy and 47 prostate cancer tissues). The results showed that 10 genes (including CDH6 and CXCL12) were upregulated and 4 genes (CCL7, ITGB3, MDM2 and MMP2) were downregulated in the si-PSMA LNCap cells. There were 41 genes significantly upregulated and 15 genes downregulated in PC-3 cells when compared with LNCap cells. Eight common genes were found in both the si-PSMA and PSMA(-) groups. CDH6, MMP3, MTSS1 were further identified as PSMA-related genes in the prostate cancer cell lines and clinical samples, and their expression showed a negative correlation with the stage of prostate cancer (P<0.0001) and PSMA level (P<0.05) in clinical samples, indicating their possible involvement in PSMA-related PCa metastasis regulation. These findings may provide insights into the mechanism involved in the suppression of PCa metastasis by PSMA and its possible interacting proteins, and may provide clues for further exploration of the molecular mechanism of PCa metastasis.

[Recombination analysis of enterovirus 71 strain isolated in Guangzhou, 2009].

To describe the recombination features of human enterovirus 71 strain Guangzhou09 isolated in Guangzhou in 2009, the complete nucleotide sequences of Guangzhou09 were analyzed by various of bioinformatics software. Phylogenetic analysis based on P1, P2 and P3 regions indicated that recombination occurred between EV71 and CVA4. Phylogenetic, similarity plot and bootscan analysis further revealed the recombination between EV71 genotype C strain Shanghai-FJ713317 and CVA4 strain HQ728260 at region 2B was close to the nucleotide position 4 027. This represents the first evidence for intertypic recombination between EV71 subtype C4 and CVA4 in Guangzhou.

Knockdown of clusterin inhibits the growth and migration of renal carcinoma cells and leads to differential gene expression.

Clusterin (CLU) is a glycoprotein involved in tumor progression, whose expression level correlates with the metastasis of renal cell carcinoma (RCC). However, the mechanism by which CLU plays an oncogenic role in RCC remains unclear. In this study, we used the human renal cancer cell 786-O as an experimental model. We knocked down CLU expression in the 786-O cells using lentiviral vector-mediated delivery of RNAi, and then compared the gene expression profiles between the knocked down CLU 786-O cells and control cells. We observed that CLU knockdown induced apoptosis and inhibited the proliferation and migration of 786-O cells. Microassay analysis revealed changes in the expression of 588 genes between the 786-O cells infected by a si-CLU lentivirus and the control cells, where 356 genes were upregulated and 232 were downregulated. Pathway analysis classified the differentially expressed genes into 17 upregulated and 12 downregulated pathways, including the PI3K/Akt, MAPK and VEGF pathways. In this study, we demonstrated that CLU acts as an oncogene in RCC by promoting cell proliferation and migration and inhibiting apoptosis. Microassay analysis may provide a platform for further characterization of the individual genes implicated in the development of RCC, providing new insights into the oncogenic role of CLU.

[Human bocavirus and its current epidemic status in China].

Human bocavirus (HBoV) is classified in the family of parvovirdae, genus bocavirus. Besides parvovirus B19 and human parvovirus 4 (PARV4), HBoV isone of the parvoviruses currently known to infect and cause illness in human. So far, four different HBoVs (HBoV1-4) have been successively reported. The incidence of HBoVs infection varies widely, the clinical presentations of patients are different, and HBoVs are often co-detected with other pathogens. There are already quite a few report of HBoVs infection, and this article reviews and discusses the biological characters, epidemic characters, pathogenic mechanism, phylogenetic analyses of HBoVs and the epidemiological situation in China.

[Variation and evolution analysis of the PB1-F2 genes of novel influenza A viruses isolated from Guangzhou China].

To compare and analyze the variation of PB1-F2 genes of Influenza A Viruses from Guangzhou during 2009 to 2011 with the Influenza A Viruses from all over the world, to lay the foundation of functional research and interaction mechanism of the PB1-F2 protein. 17 Novel H1N1 influenza viruses and 1 seasonal H1N1 influenza virus have been isolated from human in Guangzhou during 2009 to 2011 that were cloned into pMD 18-T Vector for sequencing. Then, 68 PB1-F2 genes of IAVs from human around the world were downloaded from GenBank database and analyzed using molecular biological software. The phylogenetic tree result shows that the PB1-F2 genes of IAV from the world separated into two main groups. There is high homology of PB1-F2 genes of one Seasonal H1N1 virus and Novel H1N1 viruses which were isolated in Guangzhou compared with the global Novel H1N1 viruses. And all of them got the 11 amino acids truncated protein by mutation included one seasonal H1N1 strain isolated by our laboratory. There is no variation of PB1-F2 genes of Novel H1N1 virus in Guangzhou compared with the worldwide strains. However, one seasonal H1N1 virus which isolated by our laboratory shows analogous truncated mutation of PB1-F2 of Novel H1N1 virus, it reveals that the PB1-F2 gene might has done the early reassortment between the Novel H1N1 virus and seasonal H1N1 virus.

Surveillance and genome analysis of human bocavirus in patients with respiratory infection in Guangzhou, China.

Human bocavirus (HBoV) is a novel parvovirus associated with respiratory tract diseases and gastrointestinal illness in adult and pediatric patients throughout the world. To investigate the epidemiological and genetic variation of HBoV in Guangzhou, South China, we screened 3460 throat swab samples from 1686 children and 1774 adults with acute respiratory infection symptoms for HBoV between March 2010 and February 2011, and analyzed the complete genome sequence of 2 HBoV strains. Specimens were screened for HBoV by real-time PCR and other 6 common respiratory viruses by RT-PCR or PCR. HBoV was detected in 58 (1.68%) out of 3460 samples, mostly from pediatric patients (52/58) and inpatient children (47/58). Six adult patients were detected as HBoV positive and 5 were emergency cases. Of these HBoV positive cases, 19 (32.76%) had co-pathogens including influenza virus (n = 5), RSV (n = 5), parainfluenza (n = 4), adenovirus (n = 1), coronavirus (n = 7). The complete genome sequences of 2 HBoVs strains (Genbank no. JN794565 and JN794566) were analyzed. Phylogenetic analysis showed that the 2 HBoV strains were HBoV1, and were most genetically close to ST2 (GenBank accession number DQ0000496). Recombination analysis confirmed that HBoV strain GZ9081 was an intra-genotype recombinant strain among HBoV1 variants.

New alternatively spliced variant of prostate-specific membrane antigen PSM-E suppresses the proliferation, migration and invasiveness of prostate cancer cells.

PSM-E is a newly discovered alternatively spliced variant of prostate-specific membrane antigen (PSMA). In the current study, its role on the proliferation, invasiveness and migration in prostate cancer cell lines was analyzed. PSM-E and PSMA (as a comparison) eukaryotic expression vectors pcDNA3.0/PSM-E and pcDNA3.0/PSMA were constructed, validated by RT-PCR and Western blotting, and PSMA/PSM-E overexpression PC-3 cell models were built. Gene interference was used to block PSMA and the expression of its splice variants in LNCap cells. Three shRNA fragments were synthesized against PSMA, cloned into the vector pSilencer 2.1-U6-neo, their interference effect was evaluated by RT-PCR and Western blotting, and pSilencer 2.1-U6-neo­shRNA3 (named p­shRNA3) was chosen in further analyses. Growth curves were drawn to observe the proliferation change, which showed that PSM-E had the potential to suppress proliferation (P<0.05), but no significant change was observed in PSMA/PC-3 cells and in PSMA/PSM-E interfering LNCap cells (P>0.05). Cross-river test showed that the migration speeds of PSM-E/PC-3 and PSMA/PC-3 were both significantly slower than the vector negative control, and faster in p-shRNA3 interfering LNCap cells compared with its vector negative control (P<0.05), and no significant difference existed between PSM-E/PC-3 and PSMA/PC-3 (P>0.05). Transwell assay showed that the invasive cells of both PSMA/PC-3 and PSM-E/PC-3 were fewer compared to the vector negative control (P<0.05), and the invasive suppression effect of PSM-E was weaker than PSMA (P<0.05), and accordingly, invasiveness of interfering LNCaP cells was enhanced compared with the vector negative control (P<0.05). These results showed that PSM-E could suppress proliferation, migration and invasiveness of prostate cancer cells. Its suppression effect on cell proliferation is stronger compared to PSMA and the suppression effect on invasiveness is weaker than that of PSMA.

A two-year surveillance of 2009 pandemic influenza A (H1N1) in Guangzhou, China: from pandemic to seasonal influenza?

In this two-years surveillance of 2009 pandemic influenza A (H1N1) (pH1N1) in Guangzhou, China, we reported here that the scale and duration of pH1N1 outbreaks, severe disease and fatality rates of pH1N1 patients were significantly lower or shorter in the second epidemic year (May 2010-April 2011) than those in the first epidemic year (May 2009-April 2010) (P<0.05), but similar to those of seasonal influenza (P>0.05). Similar to seasonal influenza, pre-existing chronic pulmonary diseases was a risk factor associated with fatal cases of pH1N1 influenza. Different from seasonal influenza, which occurred in spring/summer seasons annually, pH1N1 influenza mainly occurred in autumn/winter seasons in the first epidemic year, but prolonged to winter/spring season in the second epidemic year. The information suggests a tendency that the epidemics of pH1N1 influenza may probably further shift to spring/summer seasons and become a predominant subtype of seasonal influenza in coming years in Guangzhou, China.

Study on the effects of allitride and its mechanisms on renal cell carcinoma in vitro.

OBJECTIVE: To study the killing effect of allitride on human renal cell carcinoma cell line Ketr-3 and its possible mechanisms. METHODS: The Ketr-3 cells were treated with allitride and the morphological changes were observed with inverted microscope. The cytotoxicity was estimated through theamine blue tetrazolium bromide (MTT). Apoptotic cells were detected by in situ cell apoptosis detection kit, and confirmed by flow cytometry. Changes of apoptosis rate cell cycle were assessed by flow cytometry. Caspase-3 (cysteineaspartate specific proteinase) mRNA was detected by semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and Caspase-3 protease activity was estimated with colorimetry. RESULTS: MTT assay and morphological changes confirmed the killing effect of allitride on Ketr-3 cellline. FCM also showed that S-phase and G2/M-phase arrest was induced. RT-PCR and colorimetry confirmed that there was apparently a rise of Caspase-3 mRNA and Caspase-3 protease activity. CONCLUSION: Allitride could kill Ketr-3 effectively by inducing apoptosis. Cell cycle arrest and up-regulation of Caspase-3 may play an important role in the mechanisms of killing effect.

Optimal in vitro culture conditions for murine predominant immature CD8a+ dendritic cells.

BACKGROUND: The prospects of using immature CD8a(+) dendritic cells (DC2) to establish transplant immunologic tolerance and treatments for autoimmune diseases in the future are promising. However, the methods for inducing DC2 are still being explored. The present study was aimed to investigate the optimal in vitro conditions for preparing large numbers of predominant DC2 from murine bone marrow cells. METHODS: Three groups of bone marrow cells cultured under different conditions were examined, namely a cytokine-induced experimental group (cytokine group), a control group with a low concentration of granulocyte-macrophage colony stimulating factor (GM-CSF, low GM-CSF group) and a control group without endogenous cytokines. The cytokine group was cultured with 5 ng/ml GM-CSF, 25 ng/ml Flt3 ligand (Flt3L), 20 ng/ml interleukin 4 (IL-4) and 100 ng/ml stem cell factor (SCF). The low GM-CSF control group was cultured with 0.4 ng/ml GM-CSF, 25 ng/ml Flt3L and 100 ng/ml SCF, without IL-4. The control group without exogenous cytokines was cultured without additional cytokines. All cells were cultured at 37 degrees C under 5% CO2. On days 3, 7 and 16, 4-color flow cytometry was carried out to analyze the cell phenotypes, and the total cell numbers were counted to analyze the cell yields. Phase-contrast microscopy was used to observe the cell morphologies. RESULTS: The cytokine group exhibited higher proportions of typical immature CD8a(+) DC, especially on day 3, but the total cell number and DC2 proportion decreased during prolonged culture. The low GM-CSF control group showed the same tendencies as the cytokine group on days 16 and 22, but produced higher total cell numbers (P<0.05) with lower DC2 proportions and cell numbers. The control group without exogenous cytokines spontaneously generated a certain proportion of DC2, but with low total cell and DC2 numbers that decreased rapidly, especially during prolonged culture (days 7 and 16, P<0.05). CONCLUSIONS: Culture in the presence of 5 ng/ml GM-CSF, 25 ng/ml Flt3L, 20 ng/ml IL-4 and 100 ng/ml SCF can rapidly induce large quantities of predominant immature CD8a(+) DC from murine bone marrow cells. Therefore, these represent optimal culture conditions for preparing murine immature DC2 in vitro.

High expression of PSM-E correlated with tumor grade in prostate cancer: a new alternatively spliced variant of prostate-specific membrane antigen.

BACKGROUND: Prostate-specific membrane antigen (PSMA) overexpressed in prostate cancer (PCa) has been targeted for therapy and diagnosis of PCa. In the current study, PSMA cDNA was cloned from PCa tissue by RT-PCR. After sequencing, a new spliced variant of PSMA (PSM-E) was discovered and its specificity in PCa was evaluated. METHODS: PSM-E and PSMA mRNA were measured in LNCaP, PC-3 and prostate or nonprostatic malignancies. Following transfection of PC-3 with PSM-E cDNA in the pcDNA3.0 vector, PSM-E expression was measured by immunofluorescence and Western-blot. PSM-E and PSMA mRNA levels were quantified by a real-time PCR assay in normal prostate (n = 7), benign prostatic hyperplasia (BPH) (n = 22) and PCa (n = 41). The correlation between their levels and tumor grade was analyzed. RESULTS: PSM-E cDNA is identical to PSMA except for a 97-nucleotide region and a 93-nucleotide region. PSM-E and PSMA mRNA were detected in PCa and LNCaP, not in PC-3; PSMA could be detected in some nonprostatic tumors whereas PSM-E not. The expression of PSM-E protein was detected in transfected cells. Significant difference of PSM-E mRNA levels was observed among normal prostate, BPH and PCa (P < 0.001), and PSM-E levels increased with increasing Gleason score (r = 0.514, P < 0.001). PSMA mRNA levels were higher in BPH and PCa than in normal prostate (P < 0.001), but no difference between BPH and PCa, no significant correlation was observed between PSMA levels and Gleason score (r = 0.229, P = 0.057). CONCLUSIONS: PSM-E may be a potential prognostic indicator for PCa progression and may be a new target antigen for therapy of PCa.