Prognosis analysis of radical or modified radical hysterectomy and simple hysterectomy in patients with stage Ⅱ endometrial cancer / 中华妇产科杂志

Objective: To compare the prognosis and perioperative situation of patients with stage Ⅱ endometrial cancer (EC) between radical hysterectomy/modified radical hysterectomy (RH/mRH) and simple hysterectomy (SH). Methods: A total of 47 patients diagnosed EC with stage Ⅱ [International Federation of Gynecology and Obstetrics (FIGO) 2009] by postoperative pathology, from January 2006 to January 2021 in Peking University People's Hospital, were analyzed retrospectively. The patients were (54.4±10.7) years old, and the median follow-up time was 65 months (ranged 9-138 months). They were divided into RH/mRH group (n=14) and SH group (n=33) according to the scope of operation. Then the prognosis of patients between the groups were compared, and the independent prognostic factors of stage Ⅱ EC were explored. Results: (1) The proportions of patients with hypertension in RH/mRH group and SH group were 2/14 and 45% (15/33), the amounts of intraoperative blood loss were (702±392) and (438±298) ml, and the incidence of postoperative complications were 7/14 and 15% (5/33), respectively. There were significant differences (all P<0.05). (2) The median follow-up time of RH/mRH group and SH group were 72 vs 62 months, respectively (P=0.515). According to Kaplan-Meier analysis and log-rank method, the results showed that there were no significant difference in 5-year progression-free survival (PFS) rate (94.3% vs 84.0%; P=0.501), and 5-year overall survival rate (92.3% vs 92.9%; P=0.957) between the two groups. Cox survival analysis indicated that age, pathological type, serum cancer antigen 125 (CA125), and estrogen receptor (ER) status were associated with 5-year PFS rate (all P<0.05). But the scope of hysterectomy (RH/mRH and SH) did not affect the 5-year PFS rate of stage Ⅱ EC patients (P=0.508). And level of serum CA125 and ER status were independent prognostic factors for 5-year PFS rate (all P<0.05). Conclusions: This study could not find any survival benefit from RH/mRH for stage Ⅱ EC, but increases the incidence of postoperative complications. Therefore, the necessity of extending the scope of hysterectomy is questionable.

Peking University Third Hospital score: a comprehensive system to predict intra-operative blood loss in radical nephrectomy and thrombectomy / 中华医学杂志(英文版)

BACKGROUND@#Radical nephrectomy and thrombectomy is the standard surgical procedure for the treatment of renal cell carcinoma (RCC) with tumor thrombus (TT). But the estimation of intra-operative blood loss is only based on the surgeon's experience. Therefore, our study aimed to develop Peking University Third Hospital score (PKUTH score) for the prediction of intra-operative blood loss volume in radical nephrectomy and thrombectomy.@*METHODS@#The clinical data of 153 cases of renal mass with renal vein (RV) or inferior vena cava tumor thrombus admitted to Department of Urology, Peking University Third Hospital from January 2015 to May 2018 were retrospectively analyzed. The total amount of blood loss during operation is equal to the amount of blood sucked out by the aspirator plus the amount of blood in the blood-soaked gauze. Univariate linear analysis was used to analyze risk factors for intra-operative blood loss, then significant factors were included in subsequent multivariable linear regression analysis.@*RESULTS@#The final multivariable model included the following three factors: open operative approach (P < 0.001), Neves classification IV (P < 0.001), inferior vena cava resection (P = 0.001). The PKUTH score (0-3) was calculated according to the number of aforementioned risk factors. A significant increase of blood loss was noticed along with higher risk score. The estimated median blood loss from PKUTH score 0 to 3 was 280 mL (interquartile range [IQR] 100-600 mL), 1250 mL (IQR 575-2700 mL), 2000 mL (IQR 1250-2900 mL), and 5000 mL (IQR 4250-8000 mL), respectively. Meanwhile, the higher PKUTH score was, the more chance of post-operative complications (P = 0.004) occurred. A tendency but not significant overall survival difference was found between PKUTH risk score 0 vs. 1 to 3 (P = 0.098).@*CONCLUSION@#We present a structured and quantitative scoring system, PKUTH score, to predict intra-operative blood loss volume in radical nephrectomy and thrombectomy.

Surgical complexity and prognostic outcome of small volume renal cell carcinoma with high-level venous tumor thrombus and large volume renal cell carcinoma with low-level thrombus / 中华医学杂志(英文版)

Background@#Radical nephrectomy with thrombectomy is one of the most difficult and complicated urological operations. But the roles of renal tumor volume and thrombus level in surgical complexity and prognostic outcome are not clear. This study aimed to evaluate the surgical complexity and prognostic outcome between the volume of renal cell carcinoma (RCC) and the level of venous tumor thrombus.@*Methods@#The clinical data of 67 RCC cases with renal vein or inferior vena cava (IVC) tumor thrombus from January 2015 to May 2018 were retrospectively analyzed. Among these 67 cases, 21 (31.3%) were small tumors with high-level thrombus (tumor ≤7 cm in diameter and thrombus Neves Level II–IV), while 46 (68.7%) were large tumors with low-level thrombus group (tumor >7 cm in diameter and thrombus Level 0–I). Clinical features, operation details, and pathology data were collected. Univariable and multivariable logistic regression analyses were applied to evaluate the risk factors for small tumor with high-level thrombus.@*Results@#Patients with small tumors and high-level thrombus were more likely to have longer operative time (421.9 ± 135.1 min vs. 282.2 ± 101.9 min, t = 4.685, P < 0.001), more surgical bleeding volume (1200 [325, 2900] mL vs. 500 [180, 1000] mL, U = 270.000, P = 0.004), more surgical blood transfusion volume (800 [0, 1400] mL vs. 0 [0, 800] mL, U = 287.500, P = 0.004), more plasma transfusion volume (0 [0, 800] mL vs. 0 [0, 0] mL, U = 319.000, P = 0.004), higher percentage of open operative approach (76.2% vs. 32.6%, χ2 = 11.015, P = 0.001), higher percentage of IVC resection (33.3% vs. 0%, χ2 = 17.122, P < 0.001), and higher percentage of post-operative complications (52.4% vs. 19.6%, χ2 = 7.415, P = 0.010) than patients with large tumors and low-level thrombus. In multivariate analysis, decreased hemoglobin (Hb) (odds ratio [OR]: 0.956, 95% confidence interval [CI]: 0.926–0.986, P = 0.005) and non-sarcomatoid differentiation (OR: 0.050, 95% CI: 0.004–0.664, P = 0.023) were more likely to form small tumors with high-level tumor thrombus rather than large tumor with small tumor thrombus. The estimated mean cancerspecific survival times of small tumor with high-level thrombus and large tumor with low-level thrombus were 31.6 ± 3.8 months and 32.5 ± 2.9 months, without statistical significance (P = 0.955). After univariate and multivariate Cox proportional hazard survival regression analyses, only distant metastasis (hazard ratio [HR]: 3.839, P = 0.002), sarcomatoid differentiation (HR: 7.923, P < 0.001), alkaline phosphatase (HR: 2.661, P = 0.025), and severe post-operative complications (HR: 10.326, P = 0.001) were independent predictors of prognosis.@*Conclusions@#The level of the tumor thrombus was more important than the diameter of the primary kidney tumor in affecting the complexity of surgery. In the same T3 stage, neither the renal tumor diameter nor the tumor thrombus level was an independent risk factor for prognosis.

Surgical complexity and prognostic outcome of small volume renal cell carcinoma with high-level venous tumor thrombus and large volume renal cell carcinoma with low-level thrombus / 中华医学杂志(英文版)

BACKGROUND@#Radical nephrectomy with thrombectomy is one of the most difficult and complicated urological operations. But the roles of renal tumor volume and thrombus level in surgical complexity and prognostic outcome are not clear. This study aimed to evaluate the surgical complexity and prognostic outcome between the volume of renal cell carcinoma (RCC) and the level of venous tumor thrombus.@*METHODS@#The clinical data of 67 RCC cases with renal vein or inferior vena cava (IVC) tumor thrombus from January 2015 to May 2018 were retrospectively analyzed. Among these 67 cases, 21 (31.3%) were small tumors with high-level thrombus (tumor ≤7 cm in diameter and thrombus Neves Level II-IV), while 46 (68.7%) were large tumors with low-level thrombus group (tumor >7 cm in diameter and thrombus Level 0-I). Clinical features, operation details, and pathology data were collected. Univariable and multivariable logistic regression analyses were applied to evaluate the risk factors for small tumor with high-level thrombus.@*RESULTS@#Patients with small tumors and high-level thrombus were more likely to have longer operative time (421.9 ± 135.1 min vs. 282.2 ± 101.9 min, t = 4.685, P < 0.001), more surgical bleeding volume (1200 [325, 2900] mL vs. 500 [180, 1000] mL, U = 270.000, P = 0.004), more surgical blood transfusion volume (800 [0, 1400] mL vs. 0 [0, 800] mL, U = 287.500, P = 0.004), more plasma transfusion volume (0 [0, 800] mL vs. 0 [0, 0] mL, U = 319.000, P = 0.004), higher percentage of open operative approach (76.2% vs. 32.6%, χ = 11.015, P = 0.001), higher percentage of IVC resection (33.3% vs. 0%, χ = 17.122, P < 0.001), and higher percentage of post-operative complications (52.4% vs. 19.6%, χ = 7.415, P = 0.010) than patients with large tumors and low-level thrombus. In multivariate analysis, decreased hemoglobin (Hb) (odds ratio [OR]: 0.956, 95% confidence interval [CI]: 0.926-0.986, P = 0.005) and non-sarcomatoid differentiation (OR: 0.050, 95% CI: 0.004-0.664, P = 0.023) were more likely to form small tumors with high-level tumor thrombus rather than large tumor with small tumor thrombus. The estimated mean cancer-specific survival times of small tumor with high-level thrombus and large tumor with low-level thrombus were 31.6 ± 3.8 months and 32.5 ± 2.9 months, without statistical significance (P = 0.955). After univariate and multivariate Cox proportional hazard survival regression analyses, only distant metastasis (hazard ratio [HR]: 3.839, P = 0.002), sarcomatoid differentiation (HR: 7.923, P < 0.001), alkaline phosphatase (HR: 2.661, P = 0.025), and severe post-operative complications (HR: 10.326, P = 0.001) were independent predictors of prognosis.@*CONCLUSIONS@#The level of the tumor thrombus was more important than the diameter of the primary kidney tumor in affecting the complexity of surgery. In the same T3 stage, neither the renal tumor diameter nor the tumor thrombus level was an independent risk factor for prognosis.

Establishment of Antibody-induced Immune Hemolytic Disease Model in SD Rats and Their Pathotogical Changes / 中国实验血液学杂志

OBJECTIVE@#To establish the model of antibody-induced immune hemolytic disease in SD rats so as to provide an experimental platform for the exploration of its pathogenesis, course of disease and evaluation of drug efficacy.@*METHODS@#The red blood cells(RBC) of SD rats were isolated and intraperitoneally injected into BALB/c mice to induce production of the antiserum to SD rat RBC. Twenty SD rats were randomly divided into 2 groups. The rats in the model group were injected with 0.1 ml antiserum via tail vein; the rats in the control group were injected with 0.1 ml saline via tail vein.The symptoms of rats, hemolysis-related indexes and histopathological changes of the main organs were observed in both groups after injection.@*RESULTS@#After the injection of antiserum, the SD rats in the model group displayed nasal flaring, laziness, decrease of ingestion and water intake, skin and mucosal jaundice, and gross hemoglobinuria. At the 4th day after the injection, the body weight of SD rats in the model group was significantly lower than that in the control group (P＜0.01), and the coefficiens of liver and spleen increased significantly (P＜0.01); The levels of WBC, MCV, MCH, DBIL, DBIL/TBIL and FHb all increased statistically significantly, and RBC, Hb, HCT, MCHC and Plt levels decreased significantly in comparison with the control group (P＜0.01). In the SD rats of model group, the hemolytic pathological changes were observed in liver, spleen, kidney, lung and small intestine, and erythroid proliferation was observed in bone marrow smears.@*CONCLUSION@#The immune hemolytic disease model of SD rats can be successfully established by injecting the serum aginst SD rat red blood cells into the tail vein of SD rats, showing the high success rate, good reproducibility and low cost.

Comparison of various imaging in the diagnosis of renal cell carcinoma with inferior vena cava tumor thrombus combined with bland thrombus / 北京大学学报(医学版)

OBJECTIVE@#To analyse the clinical and imaging data of patients with renal cell carcinoma (RCC) with inferior vena cava tumor thrombus (IVCTT), and to assess the diagnostic efficacy of ultrasound, enhanced computed tomography (CT) and enhanced magnetic resonance imaging (MRI) in the diagnosis of RCC with IVCTT combined with bland thrombus was assessed.@*METHODS@#We retrospectively analyzed the clinical and imaging data of 56 RCC patients with IVCTT who underwent radical nephrectomy and IVC thrombectomy between January 2014 and July 2018 in Department of Urology, Peking University Third Hospital. All the patients underwent US, enhanced CT and enhanced MRI preoperatively, and all the cases were confirmed with RCC with IVCTT by histological evaluation.@*RESULTS@#The criteria of RCC with IVCTT combined with bland thrombus was confirmed by intraoperative observation and postoperative pathology. The 56 patients were divided into bland thrombus group (n=18) and non bland thrombus group (n=38). Compared the two groups, it was found that the length of IVCTT was longer [(10.50 ± 5.55) cm vs.(6.66 ± 3.73) cm, P=0.014]; the ratio of diameter of IVCTT to maximum coronal diameter of IVC was closer to 1 [1.0 (0.7, 1.0) vs. 0.9 (0.2, 1.0), P=0.004]; the proportion of lower limb edema was higher [66.7(12/8)% vs.5.3%(2/36), P=0.005]；the proportion of segmental resection or interrupt of IVC was higher [66.7%(12/18) vs.15.8%(6/38), P<0.001], with statistical significance. Compared with the three imaging methods of US, enhanced CT and MRI, the highest sensitivity was MRI (77.8%), the highest specificity was enhanced MRI and enhanced CT (97.4%), the highest accuracy was enhanced CT and enhanced MRI (83.9%), the highest positive predictive value was enhanced CT (90.9%) and the highest negative predictive value was enhanced MRI (89.2%).@*CONCLUSION@#For the patients that RCC with IVCTT combined with bland thrombus, the length of IVCTT is longer, and the ratio of the diameter of IVCTT to the maximum corona diameter of IVC is closer to 1, and more likely to cause lower limb edema. Preoperative comprehensive evaluation of multiple images is needed to improve the accuracy of diagnosis.

Analysis of the Baroreceptor and Vestibular Receptor Inputs in the Rostral Ventrolateral Medulla following Hypotension in Conscious Rats

Input signals originating from baroreceptors and vestibular receptors are integrated in the rostral ventrolateral medulla (RVLM) to maintain blood pressure during postural movement. The contribution of baroreceptors and vestibular receptors in the maintenance of blood pressure following hypotension were quantitatively analyzed by measuring phosphorylated extracellular regulated protein kinase (pERK) expression and glutamate release in the RVLM. The expression of pERK and glutamate release in the RVLM were measured in conscious rats that had undergone bilateral labyrinthectomy (BL) and/or sinoaortic denervation (SAD) following hypotension induced by a sodium nitroprusside (SNP) infusion. The expression of pERK was significantly increased in the RVLM in the control group following SNP infusion, and expression peaked 10 min after SNP infusion. The number of pERK positive neurons increased following SNP infusion in BL, SAD, and BL+SAD groups, although the increase was smaller than seen in the control group. The SAD group showed a relatively higher reduction in pERK expression when compared with the BL group. The level of glutamate release was significantly increased in the RVLM in control, BL, SAD groups following SNP infusion, and this peaked 10 min after SNP infusion. The SAD group showed a relatively higher reduction in glutamate release when compared with the BL group. These results suggest that the baroreceptors are more powerful in pERK expression and glutamate release in the RVLM following hypotension than the vestibular receptors, but the vestibular receptors still have an important role in the RVLM.

Effect of Glutamate on the Vestibulo-Solitary Projection after Sodium Nitroprusside-Induced Hypotension in Conscious Rats

Orthostatic hypotension is most common in elderly people, and its prevalence increases with age. Attenuation of the vestibulo-sympathetic reflex (VSR) is commonly associated with orthostatic hypotension. In this study, we investigated the role of glutamate on the vestibulo-solitary projection of the VSR pathway to clarify the pathophysiology of orthostatic hypotension. Blood pressure and expression of both pERK and c-Fos protein were evaluated in the nucleus tractus solitarius (NTS) after microinjection of glutamate into the medial vestibular nucleus (MVN) in conscious rats with sodium nitroprusside (SNP)-induced hypotension that received baroreceptor unloading via sinoaortic denervation (SAD). SNP-induced hypotension increased the expression of both pERK and c-Fos protein in the NTS, which was abolished by pretreatment with glutamate receptor antagonists (MK801 or CNQX) in the MVN. Microinjection of glutamate receptor agonists (NMDA or AMPA) into the MVN increased the expression of both pERK and c-Fos protein in the NTS without causing changes in blood pressure. These results indicate that both NMDA and AMPA receptors play a significant role in the vestibulo-solitary projection of the VSR pathway for maintaining blood pressure, and that glutamatergic transmission in this projection might play a key role in the pathophysiology of orthostatic hypotension.

Additive Role of the Vestibular End Organ and Baroreceptors on the Regulation of Blood Pressure in Rats

Contribution of the vestibular end organ to regulation of arterial pressure was quantitatively compared with the role of baroreceptors in terms of baroreflex sensitivity and c-Fos protein expression in the rostral ventrolateral medulla (RVLM). Baroreflex sensitivity and c-Fos protein expression in the RVLM were measured in conscious rats that had undergone bilateral labyrinthectomy (BL) and/or baroreceptor unloading. BL attenuated baroreflex sensitivity during intravenous infusion of sodium nitroprusside (SNP), but did not significantly affect the sensitivity following infusion of phenylephrine (PE). Baroreflex sensitivity became positive following sinoaortic denervation (SAD) during infusion of PE and attenuated sensitivity during infusion of SNP. Baroreflex sensitivity also became positive following double ablation (BL+SAD) during infusion of PE, and attenuated sensitivity during infusion of SNP. c-Fos protein expression increased significantly in the RVLM in the sham group after SNP administration. However, the BL, SAD, and SAD+BL groups showed significant decreases in c-Fos protein expression compared with that in the sham group. The SAD group showed more reduced c-Fos protein expression than that in the BL group, and the SAD+BL group showed less expression than that in the SAD group. These results suggest that the vestibular system cooperates with baroreceptors to maintain arterial pressure during hypotension but that baroreceptors regulate arterial pressure during both hypotension and hypertension. Additionally, afferent signals for maintaining blood pressure from the vestibular end organs and the baroreceptors may be integrated in the RVLM.

Rapid detection of rpoB gene mutations in rif-resistant M. tuberculosis isolates by oligonucleotide microarray / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To detect the specific mutations in rpoB gene of Mycobacterium tuberculosis by oligonucleotide microarray.</p><p><b>METHODS</b>Four wild-type and 8 mutant probes were used to detect rifampin resistant strains. Target DNA of M. tuberculosis was amplified by PCR, hybridized and scanned. Direct sequencing was performed to verify the results of oligonucleotide microarray.</p><p><b>RESULTS</b>Of the 102 rifampin-resistant strains 98 (96.1%) had mutations in the rpoB genes.</p><p><b>CONCLUSION</b>Oligonucleotide microarray with mutation-specific probes is a reliable and useful tool for the rapid and accurate diagnosis of rifampin resistance in M. tuberculosis isolates.</p>

Upregulation of FasL/Fas expression and FasL/Fas-associated apoptosis in J774A.1 cells induced by Leptospira interrogans / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine the involvement of FasL/Fas pathway in apoptosis of J774A.1 cells induced by Leptospira interrogans.</p><p><b>METHODS</b>The cell infection model was established with mouse monocyte-macrophage J774A.1 cells infected by L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601. The morphological characteristics of apoptotic J774A.1 cells were observed by DAPI staining method, and the apoptosis rate was quantitatively determined by flow cytometry. FasL neutralizing antibody was applied to block the apoptosis. Expression of FasL or Fas in the L.interrogans strain 56601-infected J774A.1 cells was detected by flow cytometry using PE-conjugated monoclonal antibody.</p><p><b>RESULT</b>Chromatin condensation and marginalization were found in J774A.1 cells infected by L.interrogans strain 56601 for 4 h, which became more predominant for 24 h and karyorrhexis was present in some cells. When J774A.1 cells were infected for 4 h and 24 h, the apoptosis rates were 53.6% and 64.31%, respectively. However, the apoptosis rates were decreased to 10.27% and 15.9% after the cells were pre-treated with FasL neutralizing antibody. When J774A.1 cells were infected for 4 h and 24 h, FasL expression rates were increased to 21.69% and 65.70% from that of 4.19% before infection, and Fas expression rates were risen to 91.96% and 88.01% from that of 12.88% before infection.</p><p><b>CONCLUSION</b>Inducement of cell apoptosis is an important mechanism of L.interrogans strain 56601 injuring J774A.1 cells. The strain of L.interrogans is able to up-regulate FasL/Fas expression levels of host cells and induce apoptosis of the cells via FasL/Fas pathway.</p>

J774A.1 cell apoptosis induced by Leptospira interrogans and effects of caspase-3, -6 activation on apoptosis / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To investigate the apoptosis of J774A.1 cells induced by Leptospira interrogans and the effect of caspase-3, -6 activation on the apoptosis.</p><p><b>METHODS</b>Mouse monocyte-macrophage like cell line J774A.1 was infected by L.interrogans serogroup Icterohaemorrhagiae serovar icterohaemorrhagiae Lai strain 56601. The apoptosis or necrosis of infected cells was examined by flow cytometry using fluorescein labeling FITC-Annexin V/PI. The activity of caspase-3, -6, and their cleaved substrates PARP and Lamin A/C were measured by fluorometry and Western Blotting, respectively.</p><p><b>RESULT</b>L. interrogans strain Lai was able to induce apoptosis of J774A.1 cells and the maximal apoptotic rate was(48.81+/-5.95)% when microbe: cell ratio was 100: 1. The maximal activities of caspase-3 and -6 in the infected J774A.1 cells were (1453.41+/-36.07) and (618.65+/-39.82) FU, respectively, which were 16.38- and 9.98-fold of those uninfected cells. PARP and Lamin A/C in the infected cells were detected. Caspase-3 and -6 inhibitors remarkably blocked the L. interrogans-induced apoptosis in J774A.1 cells.</p><p><b>CONCLUSION</b>L. interrogans is able to induce the apoptosis of J774A.1 cells and intracellular caspase-3 and -6 are closely associated with the apoptosis.</p>

Sequences and expression pattern of mce gene in Leptospira interrogans of different serogroups / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine the frequency of mce gene in Leptospira interrogans, and to investigate the gene transcription levels of L. interrogans before and after infecting cells.</p><p><b>METHODS</b>The segments of entire mce genes from 13 L.interrogans strains and 1 L.biflexa strain were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of mce gene was constructed; the expression and output of the target recombinant protein rMce were examined by SDS-PAGE and Western Blot assay. Rabbits were intradermally immunized with rMce to prepare the antiserum, the titer of antiserum was measured by immunodiffusion test. The transcription levels of mce gene in L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 before and after infecting J774A.1 cells were monitored by real-time fluorescence quantitative RT-PCR.</p><p><b>RESULT</b>mce gene was carried in all tested L.interrogans strains, but not in L.biflexa serogroup Semaranga serovar patoc strain Patoc I. The similarities of nucleotide and putative amino acid sequences of the cloned mce genes to the reported sequences (GenBank accession No: NP712236) were 99.02%-100% and 97.91%-100%, respectively. The constructed prokaryotic expression system of mce gene expressed rMce and the output of rMce was about 5% of the total bacterial proteins. The antiserum against whole cell of L.interrogans strain 56601 efficiently recognized rMce. After infecting J774A.1 cells, transcription levels of the mce gene in L.interrogans strain 56601 were remarkably up-regulated.</p><p><b>CONCLUSION</b>The constructed prokaryotic expression system of mce gene and the prepared antiserum against rMce provide useful tools for further study of the gene function.</p>

Apoptosis and ultrastructural lesions in Vero and J774A.1 cells induced by Leptospira interrogans / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine the ability of adhering and invading to host cells and the related pathologic changes of Leptospira spp. with different virulence.</p><p><b>METHODS</b>A special Fontana silver staining method was developed to observe the ability of L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 and serogroup Pomona serovar pomona strain 56608, and L.biflexa serogroup Samaranga serovar patoc strain Patoc I to adhere Vero and J774A.1 cells. Ultrastructural lesions of the infected cells were examined by electron microscopy. By using flow cytometry with fluorescein labeling of FITC-Annexin V/PI,apoptosis and necrosis of the Vero and J774A.1 cells induced by the leptospiral strains before and after inactivation with ultraviolet treatment were detected, respectively.</p><p><b>RESULTS</b>The adhering rates of L.interrogans strains 56601 and 56608 to the Vero cells were 24.2% and 22.9% (P>0.05), while to the J774A.1 cells were 49.0% and 46.9% (P>0.05), respectively. L.biflexa strain Patoc I did not adhere to these two host cells. After the two strains of L.interrogans invaded two different cell lines, the special phagosomes containing the Leptospira were formed and similar cell ultrastructural lesions, such as chromatilysis and chromatin condensation, vacuolar degeneration, mitochondrion swelling and mitochondrial crista disappearance, and endoplasmic reticulum swelling and paramembranous ribosome disappearance, were observed. The apoptosis rates in the Vero cells caused by the L.interrogans strains 56601 and 56608 before and after ultraviolet inactivation were 84.4%, 82.8% and 77.9%, 86.1%, respectively. The L.interrogans strain 56601 mainly induced the terminal apoptosis in Vero cells with the rates of 68.0% and 52.9% before or after inactivation, while the L.interrogans strain 56608 mainly induced the early apoptosis in Vero cells with apoptosis rates of 64.1% and 50.1% before or after inactivation. In the J774A.1 cells, the L.interrogans strain 56608 caused cell necrosis (before and after inactivation) and apoptosis that was dominated by terminal apoptosis.</p><p><b>CONCLUSION</b>The established Fontana silver staining method can be used to observe adhesion of L.interrogans.L.interrogans can invade host cells through endocytosis which causes untrastructural lesions. The necrosis or apoptosis induced by L.interrogans are affected by different host cell lines but not the virulence of L.interrogans strains.</p>

Phospholipase C activity and alteration of intracellular free Ca2+ levels during internalization of Leptospira interrogans / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To determine the effects of leptospiral strains with different virulence on intracellular free Ca(2+)level and its relation with phospholipase C (PLC) activity of L.interrogans.</p><p><b>METHODS</b>L.interrogans-j infection cell modals were established with Vero and J774A.1 cell lines. Vero and J774A.1 cells were co-incubated with L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 (strong virulence) and serogroup Pomona serovar pomona strain 56608 (weak virulence) and L.biflexa serogroup Samaranga serovar patoc strain Patoc I (non-virulence). Intracellular free Ca(2+)levels were detected by laser scanning confocal microscopy with specific fluorescence labeling of fluoj3/AM. Using [(3)H] PIP2 as the substrate, the PLC activities in the culture supernatant, and cytoplasma and cytomembrane of the three strains of Leptospira were measured by isotope assay.</p><p><b>RESULTS</b>The baseline intracellular free Ca(2+)levels in the normal Vero and J774A.1 cells were (102.3+/-8.2)% and (105.9+/-7.3)%,respectively. The fluorescence intensity in the two cell lines incubated with L.biflexa strain Patoc I were fluctuated in range of (102.3+/-8.2)%approximate, equals(102.2+/-8.3)% during the observation period. The intracellular free Ca(2+)levels in the two cell lines infected with L.interrogans strain 56601 showed elevation with double peak patterns, with first peaks of (430.5+/-35.7)%, (747.5+/-18.5)% and the second peak of (380.6+/-17.4)%, (804.6+/-22.4)%, respectively. When the cells were infected with L.interrogans strain 56608, the intracellular free Ca(2+)levels were rising slowly with a single slope-like pattern, with the maximal of (235.0+/-19.3)% in Vero cells and (402.4+/-17.4)% in J774A.1 cells, which were significantly lower than those in the cells infected with L.interrogans strain 56601 (P<0.01). The culture supernatants, and cytoplasma and cytomembrane proteins of all three strains displayed PLC activities (P<0.05).</p><p><b>CONCLUSION</b>The cells infected with L.interrogans of different virulence show distinct intracellular free Ca(2+)levels and peak patterns. The different host cell lines can affect the intracellular free Ca(2+)levels, which is not related to the PLC activity in the leptospiral strains.</p>

Construction and identification of the prokaryotic expression system of rLTB/rCTB-rOmpL1/1 fusion genes / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct prokaryotic expression systems of ltB/ctB-ompL1/1 fusion genes and to determine the L.interrogans carrying status in leptospirosis patients with the expression products.</p><p><b>METHODS</b>The fusion genes ltB-ompL1/1 and ctB-ompL1/1 were constructed using linking primer PCR method. SDS-PAGE was used to examine expression of the target recombinant proteins rLTB-rOmpL1/1 and rCTB-rOmpL1/1. Western blot and GM1-ELISA were used to measure the immunogenic and GM(1)-binding activities of rLTB-rOmpL1/1 and rCTB-rOmpL1/1, respectively. PCR and MAT were performed to detect the expression of ompL1 gene in 97 wild L.interrogans strains. Antibodies against ompL1 gene products in serum samples of 228 leptospirosis patients were detected with ELISA method.</p><p><b>RESULTS</b>The homogeneity of nucleotide and putative amino acid sequence of ltB-jompL1/1 and ctB-ompL1/1 fusion genes were 99.7 % - 99.9 % and 99.5 % - 100 %, in comparison with the reported corresponding sequences. Expression outputs of both rLTB-rOmpL1/1 and rCTB-rOmpL1/1, mainly present in inclusion body, accounted for 10% of the total bacterial protein. Both rLTB-rOmpL1/1 and rCTB-rOmpL1/1 could combine to rabbit anti-rOmpL1/1 serum and bovine GM(1). 89.7 % of L.interrogans wild strains had ompL1 gene. 87.6% of the wild L.interrogans strains presented positive results for MAT (titers :1:4-1:256) with the rabbit anti-rOmpL1/1 or anti-rOmpL1/2 sera. 86.8% and 88.6% of the patients' serum samples were positive for rOmpL1/1 and rOmpL1/2 antibodies, respectively.</p><p><b>CONCLUSION</b>The fusion proteins, rLTB-rOmpL1/1 and rCTB-rOmpL1/1, showed high immunogenic and GM(1)-binding activities. ompL1 gene is extensively distributed and frequently expressed in different serogroups of L.interrogans and its products expressed by different genotypes exhibit extensive cross-antigenicity.</p>

Construction and application of prokaryotic expression system of Leptospira interrogans lipL32/1-lipL41/1 fusion gene / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct lipL32/1-lipL41/1 fusion gene and its prokaryotic expression system and to determine frequencies of carrying and expression of lipL32 and lipL41 genes in L.interrogans wild strains and specific antibody levels in sera from leptospirosis patients.</p><p><b>METHODS</b>lipL32/1-lipL41/1 fusion gene was constructed using linking primer PCR method and the prokaryotic expression system of the fusion gene done with routine techniques. SDS-PAGE was used to examine expression of the target recombinant protein rLipL32/1-rLipL41/1. Immunogenicity of rLipL32/1-rLipL41/1 was identified by Western blot. PCR and MAT were performed to detect carrying and expression of lipL32 and lipL41 genes in 97 wild L.interrogans strains. Antibodies against products of lipL32 and lipL41 genes in serum samples from 228 leptospirosis patients were detected by ELISA method.</p><p><b>RESULTS</b>The homogeneity of nucleotide and putative amino acid sequence of lipL32/1-lipL41/1 fusion gene were 99.9 % and 99.8 % in comparison with the reported sequences. Expression output of the target recombinant protein rLipL32/1-rLipL41/1, mainly present in inclusion body, accounted for 10 % of the total bacterial proteins. Both the rabbit antisera against rLipL32/1 and rLipL41/1 could combine to rLipL32/1-rLipL41/1. 97.9 % and 87.6 % of the L.interrogans wild strains had lipL32 and lipL41 genes, respectively. 95.9 % and 84.5 % of the wild strains were positive for MAT with titers of 1:4 - 1:128 using rabbit anti-rLipL32s or anti-rLipL41s sera, respectively. 94.7 % - 97.4 % of the patients'serum samples were positive for rLipL32s antibodies, while 78.5 % - 84.6 % of them were rLipL41s antibodies detectable.</p><p><b>CONCLUSION</b>lipL32/1-jlipL41/1 fusion gene and its prokaryotic expression system were successfully constructed. The expressed fusion protein had qualified immunogenicity. Both the lipL32 and lipL41 genes are extensively carried and frequently expressed by different serogroups of L.interrogans, and their expression products exhibit cross-antigenicity.</p>

Eukaryotic expression of Leptospira interrogans lipL32/1-ompL1/1 fusion gene encoding genus-specific protein antigens and the immunoreactivity of expression products / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To construct the eukaryotic expression system of L.interrogans lipL32/1-ompL1/1 fusion gene and to identify the immunoreactivity of expression products.</p><p><b>METHODS</b>PCR with linking primer was used to construct the fusion gene lipL32/1-ompL1/1. The P.pastoris eukaryotic expression system of the fusion gene, pPIC9K-lipL32/1-ompL1/1-P. pastorisGS115, was constructed after the fusion gene was cloned and sequenced. Colony with phenotype His(+)Mut(+) was isolated by using MD and MM plates and His(+) Mut(+) transformant with high resistance to G418 was screened out by using YPD plate. Using lysate of His(+) Mut(+) colony with high copies of the target gene digested with yeast lyase as the template and 5'AOX1 and 3'AOX1 as the primers, the target fusion gene in chromosome DNA of the constructed P. pastoris engineering strain was detected by PCR. Methanol in BMMY medium was used to induce the target recombinant protein rLipL32/1-rOmpL1/1 expression. rLipL32/1-rOmpL1/1 in the medium supernatant was extracted by using ammonium sulfate precipitation and Ni-NTA affinity chromatography. Output and immunoreactivity of rLipL32/1-rOmpL1/1 were measured by SDS-PAGE and Western blot methods, respectively.</p><p><b>RESULTS</b>Amplification fragments of the obtained fusion gene lipL32/1-ompL1/1 was 1794 bp in size. The homogeneity of nucleotide and putative amino acid sequences of the fusion gene were as high as 99.94 % and 100 %, respectively, compared with the sequences of original lipL32/1 and ompL1/1 genotypes. The constructed eukaryotic expression system was able to secrete rLipL32/1-rOmpL1/1 with an output of 10 % of the total proteins in the supernatant, which located the expected position after SDS-PAGE. The rabbit anti-rLipL32/1 and anti-rOmpL1/1 sera could combine the expressed rLipL32/1-rOmpL1/1.</p><p><b>CONCLUSION</b>An eukaryotic expression system with high efficiency in P.pastoris of L.interrogans lipL32/1-ompL1/1 fusion gene was successfully constructed in this study. The expressed fusion protein shows specific immunoreactivity, which can be used as a potential antigen for developing a novel vaccine of L.interrogans.</p>

Investigation on cagA/vacA dominant genotypes and the coinfection of Helicobacter pylori isolates from patients in Zhejiang / 中华流行病学杂志

<p><b>OBJECTIVE</b>To determine cagA/vacA dominant genotypes of Helicobacter pylori in patients suffering from chronic gastritis (CG) or peptic ulcer (PU), and to understand the correlation of different genotype H. pylori infection, coinfection and the gastroduodenal diseases.</p><p><b>METHODS</b>H. pylori strains were isolated from antrum and corpus samples on 42 patients with CG and 36 patients with PU. Polymerase chain reaction was used to detect cagA and the s and m regions of vacA in 156 H. pylori isolates from both antrum and corpus. The distribution of H. pylori genotypes and coinfection in CG and PU was analyzed.</p><p><b>RESULTS</b>Almost all of the isolated H. pylori strains were cagA positive. In region of vacA, only one genotype of signal region (s1a) and four genotypes of the middle region (m1, m2, m1b and m1b-m2) were found. The proportions of s1a/m1, s1a/m2, s1a/m1b, s1a/m1b-m2 and coinfection of multiple H. pylori strains in 78 isolates from antrum samples were 6.4%, 55.1%, 26.9%, 1.3% and 3.8%; and the related proportions of those from corpus samples were 6.4%, 53.8%, 25.6%, 3.8% and 5.1%, respectively. Sixteen (20.5%) patients had multiple H. pylori strains with different cagA and vacA genotypes, and multiple samples were better than single sample taken from one stomach to increase the positive proportion of coinfection.</p><p><b>CONCLUSION</b>cagA(+) s1a/m2 was the dominant genotype of H. pylori in the CG or PU patients followed by cagA(+) s1a/m1b in the Zhejiang area of China. Some of the patients were coinfected with multiple H. pylori strains of different cagA and vacA genotypes. However, there was no significant correlation between the genotypes or mixed infection with multiple strains, CG or PU.</p>

Cloning, expression and identification of hpaA gene from a clinical isolate of Helicobacter pylori / 浙江大学学报·医学版

<p><b>OBJECTIVE</b>To clone Helicobacter pylori adhesin (hpaA) gene,to construct the expression vector of the gene and to identify immunogenicity of the fusion protein.</p><p><b>METHODS</b>The hpaA gene from a clinical isolate Y06 of H.pylori was amplified by high fidelity PCR. The nucleotide sequence of the target DNA amplification fragment was sequenced after T-A cloning. The expression vector pET32a with inserted hpaA gene was constructed. hpaA fusion protein was expressed in E.coli strain BL21DE3 induced by IPTG at different dosages. Western blot using antibody against whole cell of H.pylori as well as immunodiffusion assay using antiserum of rabbit against the fusion protein was applied to determine immunogenicity of the fusion protein.</p><p><b>RESULTS</b>In comparison with the reported corresponding sequences, the homology of nucleotide sequence of the cloned hpaA gene was from 94.25% approximate, equals 97.32%, while the homology of its putative amino acid sequence was as high as 95.38% approximate, equals 98.46%. The expression output of HpaA fusion protein in pET32a-hpaA-BL21DE3 system was approximately 40% of the total bacterial proteins. HpaA fusion protein was able to combine with antibody against whole cell of H.pylori and induce rabbit to preduce high titer antibody after the animal was immunized with the protein.</p><p><b>CONCLUSION</b>An expression system with high efficiency of H.pylori hpaA gene has been established successfully. The expressed HpaA fusion protein with satisfactory immunogenicity and immunoreactivity can be used as antigen in H.pylori vaccine.</p>