Objective To assess the effect of variable durations of warm ischemia on renal function early after laparoscopic partial nephrectomy ( LPN ) and make the definite safety duration of renal warm ischemia.Methods The clinical data of 76 patients treated with LPN from October 2012 to June 2014 were retrospectively analyzed.The patients were divided into 3 groups based on warm ischemic time,namely group A (28 cases) with warm ischemia time less than 20 min,group B (34 cases) with warm ischemia time more than 20 min and less than 30 min, group C ( 14 cases ) with warm ischemia time more than 30 min.LPN was performed with renal artery clamping alone in all the patients.Preoperative and postoperative renal scintigraphic scan was performed to access glomerular filtration rate ( GFR) in all patients.The GFR values were compared among before, 1 week, 1 month and 3 months after operation.The factors predicting the early renal injury were assessed by multivariate regression analysis.Results The renal GFR of the kidney underwent LPN decreased 19.43(17.70,22.06) ml/min at 1 week,17.04(13.94,20.70) ml/min at 1 month,13.82(10.72,18.73) ml/min at 3 months after the surgery in group C,respectively.In group A, the renal GFR of the operated-side decreased 12.07(10.91,13.42) ml/min,10.04(9.16,11.75) ml/min, 8.44(7.07,9.72) ml/min,respectively.In group B, the renal GFR of the operated-side decreased 13.64 (12.48,16.72) ml/min,10.29(9.17,14.27)ml/min,9.63(7.85,12.59) ml/min,respectively.The GFR decreased greater in group C than that in group A and B(P<0.05).The total renal GFR decreased (10.70 ± 4.93)ml/min at three months in group C,compared with (5.64 ±4.12)ml/min in group A and (6.37 ± 4.32)ml/min in group B,respectively.The decreased value in group C was greater than that in group A and B(P<0.05).However,the differences of the total renal GFR among the 3 groups were not significant at 1 week and 1 month(P>0.05).The multivariate regression analysis revealed that warm ischemia duration was the independent risk factor of the early renal injury.Conclusions Warm ischemia duration is the major factor regarding the early renal recovery after LPN.Warm ischemia time more than 30 min may not only greatly affect the renal function but also the renal function recovery rate.

Objective To investigate the clinical value of serum VEGF levels in the diagnosis and treatment of colon cancer.Methods Serum VEGF was detected by ELISA,and CEA and CA199 concentration were detected by CLEIA in 66 patients with colon cancer,55 patients with colon benign diseases and 50 health persons.The value of individual and joint detection for VEGF in colon cancer was evaluated.Analysis had been done on relationships between serum VEGF and pathology,treatment effects and prognosis.Results The levels of serum VEGF in colon cancer group [(318.5±148.6) ng/L] were significantly higher than those in control group [(125.7±49.4) ng/L] and benign colon diseases [(136.9±52.6) ng/L] (t =8.830,8.805,all P ＜ 0.01).There was a positive correlation between serum levels of VEGF and depth of tumor size,tumor invasion,lymph node metastasis and TNM stage (P ＜ 0.01).The susceptibilities of VEGF,CEA,CA199 were 61％,45 ％,53 ％.The sensitivity of detection was improved to 86 ％ when the combined detection of VEGF,CEA and CA199 (x2 =11.237,P ＜ 0.01).The serum levels of VEGF in patients with colon cancer was significantly decreased after treatment in the 3,7,10 day compared with that before operation [(272.3±88.1),(236.8±77.4),(173.1±59.9) vs (318.5±148.6) ng/L,t=2.173,P ＜ 0.05; t =3.961,P ＜ 0.01; t=7.464,P ＜ 0.01],respectively.Conclusion The VEGF was related to the onset and progression and metastasis of colon cancer.It has clinical significancy for diagnosis of colon cancer and judgment of curative effect and prognosis.

Middle East respiratory syndrome coronavirus (MERS-CoV) was identified as a novel human coronavirus and posed great threat to public health world wide,which calls for the development of effective and safe vaccine urgently. In the study, peptide epitopes tagrgeting spike antigen were predicted based on bioinformatics methods. Nine polypeptides with high scores were synthesized and linked to keyhole limpet hemocyanin (KLH). Female BALB/C mice were immunized with individual polypeptide-KLH, and the total IgG was detected by ELISA as well as the cellular mediated immunity (CMI) was analyzed using ELIs-pot assay. The results showed that an individual peptide of YVDVGPDSVKSACIEVDIQQTFFDKTWPRPIDVSKADGI could induce the highest level of total IgG as well as CMI (high frequency of IFN-γ secretion) against MERS-CoV antigen in mice. Our study identified a promising peptide vaccine candidate against MERS-CoV and provided an experimental support for bioinformatics-based design of peptide vaccine.
Animals ; Antibodies, Viral ; immunology ; Computational Biology ; Coronavirus Infections ; immunology ; prevention & control ; virology ; Female ; Humans ; Immunization ; Mice ; Mice, Inbred BALB C ; Middle East Respiratory Syndrome Coronavirus ; genetics ; immunology ; Peptides ; administration & dosage ; genetics ; immunology ; Spike Glycoprotein, Coronavirus ; administration & dosage ; genetics ; immunology ; Viral Vaccines ; administration & dosage ; genetics ; immunology

Objective To construct a prokaryotic plasmid to express the testis development related gene 1 (TDRG1) recombinant protein and obtain anti-TDRG1 mAb by immunizing mice, and to identify the biological properties of the mAb. Methods The coding sequence of TDRG1 was amplified by RT-PCR from normal human testis tissue and cloned into the vector pET21, and then was expressed in the E. coli BL 21(DE3) to get TDRG1 recombinant protein. The purified TDRG1 recombinant protein was injected to immunize the BALB/C mice to develop anti-TDRG1 mAb. Splenocytes of the immunized mice were collected and fused with the mouse myeloma cell line Sp2/0 cells. The hybridoma cells that secreted anti-TDRG1 mAb were subcloned with limited dilution. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate titers and subtypes of mAb. Western blot and immunohistochemistry were used to detect specificity of mAb.Results The prokaryotic plasmid expressing the recombinant protein was constructed, and the TDRG1 recombinant protein was expressed and purified. Two hybridoma cell lines that secreted anti-TDRG1 mAb were obtained. The titer of the mAb in ascites was 1∶1.6×10~6, and the subtype of the mAb was IgG_1. Westem blot and immunohistochemistry analysis indicated the mAb showed specific combination with TDRG1 protein in human testes.Conclusion The TDRG1 recombinant protein is highly purified and has strong antigenicity. The anti-TDRG1 mAb is produced successfully.

Objective To investigate the diagnostic value of capsule endoscopy (CE), CT enterography (CTE), ileocolonoscopy and small bowel follow through (SBFT) for small bowel Crohn's disease (CD). Methods Fifty-seven consecutive patients with CD underwent ileocolonoscopy, CTE, CE, and SBFT. It included the presence of the following symptoms and signs: abdominal pain, weight loss,diarrhea, fever and positive fecal occult blood test. The location and the characteristics of intestinal and extra-intestinal lesions detected by four techniquks were compared. The proportions of patients with positive findings using each examination were compared. Results Of the 57 patients, 50 underwent ileocolonoscopy, terminal ileum lesion was found in 33 patients (66. 00% ), the remaining 17 (34.0%)were normal; among 34 patients who had CTE, 29 of small bowel lesion were found (85. 29% ); CE were performed in 27 patients, due to prolonged gastric transit one time, the capsule did not reach the cecum in one patient during battery lifetime. CE showed small bowel lesion in 26 patients (96.30% ); SBF was performed in 39 patients and 26 of small bowel lesion were detected (66. 67% ). CE had the highest diagnostic yield for CD and ileocolonoscopy had the lowest, and there were statistically significant difference among the 4 examinations (P = 0. 006 ). The combinded positive rates of two methods were: CE + CTE 92. 86% (13/14), SBFT + CTE 90. 91% (20/22), CE + ileocolonoscopy 95. 65% (22/23), CE + SBFT100% (17/17), ileocolonoscopy + CTE 89. 66% ( 26/29 ), ileocolonoscopy + SBFT 77.78% ( 28/36 ), but there were no significant differences between each two examinations. Conclusion CE, CTE have a higher yield in depicting mild to moderate finding of CD than SBFT. CE is better for assessing early mucosal disease,whereas CTE is better for detecting transmural and extraluminal abnormalities. Most important, CE plus CTE may depict nonobstructive CD of the small bowel when conventional techniques such as ileocolonoscopy or SBFF have negative or inconclusive finding. CE provides us explanations for the symptoms of patients, decision to follow up or therapy.

Objective To investigate the development strategy of novel T cell based vaccine against HCV infection.Methods BALB/c mice were primed with pSCK-based DNA vaccine and boosted with type 5 adenoviral vector-based vaccine, which expressed the structural proteins ( Core, E1 and E2) de-rived from a Chinese HCV patient (genotype 1b, Hebei strain).Enzyme linked immunospot assay (ELIS-POT) and intracellular cytokine staining ( ICS) were used to analyze the elicited antigen-specific immune re-sponses and the efficacy of cross-protection.Results Immunization of mice with the prime-boost vaccination strategy elicited stronger T cell immune responses against multiple HCV antigens than using the DNA vac-cines alone, especially the IFN-γ-secreting T cell responses against E1 protein as indicated by ELISPOT as-say.ICS data indicated that the prime-boost regimen elicited more TNF-α-producing CD4+and IFN-γ-produ-cing CD8+T cells against E1 protein and high levels of IFN-γ-producing CD4+and CD8+T cells against E2 protein in comparison with immunization with DNA vaccines.Moreover, the prime-boost vaccination was ca-pable of eliciting effective cross-protection in a surrogate challenge model based on a recombinant heterolo-gous HCV (JFH1, 2a) vaccinia virus.Conclusion The prime-boost vaccination using DNA and rAd5-based vaccine expressing HCV structural antigens induced significant cellular immune response and cross-protection in mice, suggesting the possibility of using it as a promising T cell based vaccine against HCV in-fection.

Objective To construct recombinant adenovirus Ad/MDC-VP1 and investigate its im-muno-boosting effect of the mice primed with the experimental DNA vaccine against Coxsackievirus infection. Methods The recombinant adenovirus Ad/MDC-VP1 was constructed and packaged. The Western blot analysis was used to verify the target protein. BALB/c mice were divided into four groups: Ad/MDC-VP1 group, pcDNA3/MDC-VP1 group, pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost group and PBS group. The mice in each group were immunized intramuscularly. The titers of serum IgG and neutralizing antibody were tested by ELISA and trace neutralization assay, respectively. The lymphocytes proliferation activity and specific CTL cytotoxic activity were tested by CCK-8 assay. The mice in each group were challenged with le-thal dose of Coxsackievirus, and the assay of the serum virus titers and the observation of protection efficacy against Coxsackievirus infection were carried out. Results The recombinant adenovirus Ad/MDC-VP1 was successfully constructed and the target protein was expressed. It was observed that the titers of CVB3 VP1 specific antibody, lymphocyte stimulation index, CTL cytotoxicity activities and protection rate of the pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost group were much higher than those of the rest groups( P < 0.05), and the titer of serum virus was lower after CVB3 challenged ( P < 0.05 ). Conclusion Both the cellular and humoral immune responses in mice could been significantly enhanced by the pcDNA3/MDC-VP1 prime-Ad/MDC-VP1 boost strategy.

Objective To compare the immune effects of Coxsackievirus B3 (CVB3) capsid protein VP1 expressed bacterially, recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1which express VP1 protein in mice. Methods After expressed in prokaryotic cells, VP1 protein was purified. Recombinant adenovirus rAd/VP1 and recombinant plasmid pcDNA3/VP1 were amplified and extracted. Six to 8-week-old, male BALB/c mice were divided into four groups randomly. Each group contained 18 mice. The mice of pcDNA3/VP1 group or VP1 protein group were immunized intramuscularly with three injections at three weeks apart, of recombinant plasmid pcDNA3/VP1 at a dose of 100 μg/mouse or recombinant protein VP1 at a dose of 50 μg/mouse. The mice of rAd/VP1 group were immunized intramuscularly twice at two weeks interval with rAd/VP1 at a dose of 1.2 × 107 PFU. The control group was mock-immunized with 100 μl of PBS intramuscularly. Mice were bled from the retroorbital sinus plexus every two weeks after each immunization. ELISA and micro-neutralization test were used to detect levels of CVB3-specific IgG antibody and neutralizing antibody titers in the sera of immunized mice. Three weeks after the last immunization, the cytotoxic T lymphocyte(CTL) killing activity of spleen lymphocytes was detected with CCK-8 assay. Subsequently, virus titers in the sera of immunized mice were determined by the 50% cell culture infective dose( CCID50 ) assay on HeLa cell monolayers and percentage of animals surviving were observed after lethal CVB3 attack over a period of 21 days. Results The titers of specific IgG antibody and neutralizing antibody in sera of VP1 protein immunized mice were higher than other groups( P ＜0.05 ). While CTL killing activity of spleen lymphocytes of VP1 protein immunized mice was lower than mice in rAd/VP1 group( P ＜0. 05). Virus titers in sera of VP1 protein immunized mice were lower than the mice in pcDNA3/VP1 or rAd/VP1 groups ( P ＜ 0.05 ), while survival rate was significantly higher than these two groups ( P ＜ 0.05 ).Conclusion VP1 protein induced higher level of humoral immune response and acquired obvious immune protection effects in mice. The immunizing potency of VP1 protein vaccine surpassed plasmid pcDNA3/VP1or recombinant adenovirus rAd/VP1. It appeared to be a promising candidate among the three different vaccines.

OBJECTIVETo explore new methods for the early diagnosis of pancreatic cancer through detection of K-ras and p53 mutations in pancreatic juice and stool.

METHODS201 patients in PUMC Hospital from 1994 - 2000 and 60 control individuals were enrolled in this study. K-ras point mutation was detected by PCR-RFLP while p53 mutation was detected by PCR-SSCP.

RESULTSK-ras mutation was found in pancreatic juice in 87.8% (36/41) of pancreatic cancer patients and 23.5% (4/17) of benign pancreatic disease patients. In 261 stool specimens, amplification found mutations successfully in 235 patients (90%). K-ras mutation was found in stool in 88% (66/75) of pancreatic cancer patients, 51.1% (24/47) of benign pancreatic disease patients and 19.6% (9/46) of normal individuals. p53 mutation was found in pancreatic juice in 47.4% (18/38) of pancreatic cancer patients and 12.5% (2/16) of benign pancreatic disease patients. p53 mutation was found in stool in 37.1% (23/62) and 19.1% (4/21) of chronic pancreatitis patients.

CONCLUSIONK-ras mutation in pancreatic juice has higher diagnosis sensitivity and specificity, and therefore may be used as a supplement in the diagnosis of pancreatic cancer. Detection of K-ras mutation combined with p53 mutation in stool can aid in the screening of pancreatic cancer.

Feces ; chemistry ; Genes, p53 ; Genes, ras ; Humans ; Mutation ; Pancreatic Juice ; metabolism ; Pancreatic Neoplasms ; diagnosis ; genetics ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Polymorphism, Single-Stranded Conformational

OBJECTIVE: To determine the diagnostic value of real-time Rigiscan monitoring and Doppler ultrasonography following intracavernous injection of prostaglandin E1in erectile dysfunction(ED). METHODS: From January 1998 to December 2007, 1 392 ED patients completed the test. PGE(1) was injected into corpus cavernosum penis near the radix penis with injection guns. After 1 or 2 minutes, the Rigiscan Plus device was used for real-time evaluation of penile rigidity. Optimal injected dosage of each patient with positive reaction was determined. Injected dosage of patients with negative reaction was gradually increased from to 60 microg. As the dosage was established, color Doppler ultrasonography was used to assess the morphodynamic features of cavernosal arteries. RESULTS: Altogether 1 039 patients were positive, while 353 patients were negative by Rigiscan. In the 1 039 positive patients,663 were injected with 10 microg PGE(1), 265 patients 20 microg, 97 patients 30 microg,and 14 patients 60 microg while 89(13.4%),39(14.7%),41(42.3%),and 10(71.4%) patients were diagnosed vascular ED. Of the 353 negative patients, 267(75.64%) patients were diagnosed vascular ED. During intracavernous pharmacological testing,the more PGE(1) was used, the more vascular ED patients would be found(P<0.001). CONCLUSION PGE(1) is safe and effective for the erectile responses. The dosage of PGE(1) following intracavernous injection is related to vascular ED. Real-time Rigiscan monitoring combined with color Doppler ultrasonograph can be helpful to diagnose penile vascular diseases.
Adult ; Alprostadil ; administration & dosage ; Erectile Dysfunction ; diagnosis ; diagnostic imaging ; physiopathology ; Humans ; Male ; Penile Erection ; Penis ; blood supply ; diagnostic imaging ; Ultrasonography, Doppler, Color