Role of hepatitis B virus infection in pathogenesis of IgA nephropathy

AIM: To investigate the role of hepatitis B virus (HBV) in the pathogenesis of IgA nephropathy (IgAN).METHODS: HBV antigens (HBAg, or HBsAg, HBcAg, and HBeAg) in renal tissues with IgAN were detected by immunohistochemical technique. The distribution and localization of HBV DNA were observed by using in situ hybridization. Southern blot analysis was performed to reveal the state of renal HBV DNA.RESULTS: Among 100 patients with IgAN, HBs antigenemia was detected in 18 patients (18.00 %). HBAg in renal tissues was detected in 31 patients (31.00 %), the positive rate of HBAg, HBsAg and HBcAg was 64.52 % (20/31), 32.26 %(10/31), 32.26 % (10/31), respectively in glomeruli. HBcAg was also found in tubular epithelia and interstitia, which was 45.16 % (14/31) and 6.45 % (2/31), respectively. Five out of six cases with positive HBV DNA by in situ hybridization were proved to be HBV DNA positive by Southern blot analysis, and all were of the integrated form. Eight specimens were demonstrated to be HBV DNA positive by in situ hybridization, which was localized in the nuclei of tubular epithelial cells and glomerular mesangial cells as well as in infiltrated interstitial lymphocytes.CONCLUSION: There is a relationship between HBV infection and IgAN. In addition to the humoral immune damage mediated by HBAg-HBAb immune complex, the cellular mechanism mediated by HBV originating from renal cells in situ may be also involved in the pathogenesis of IgAN.

Existence and significance of hepatitis B virus DNA in kidneys of IgA nephropathy

AIM: To investigate the existence and significance of hepatitis B virus (HBV) DNA in the pathogenesis of IgA nephropathy(IgAN).METHODS: Fifty cases of IgAN with HBV antigenaemia and/or hepatitis B virus antigens (HBAg, or HBsAg, HBcAg)detected by immunohistochemistry in renal tissues were enrolled in our study. The distribution and localization of HBV DNA were observed using in situ hybridization.Southern blot analysis was performed to reveal the state of renal HBV DNA.RESULTS: Among the 50 patients with IgAN, HBs antigenemia was detected in 17 patients (34%). HBAg in renal tissues was detected in 48 patients (96%), the positive rate of HBAg, HBsAg, and HBcAg was 82% (41/50), 58% (29/50),and 42% (21/50) in glomeruli, respectively; and was 94%(47/50), 56% (28/50) and 78% (39/50) in tubular epithelia,respectively. Positive HBV DNA was detected in 72% (36/50)and 82% (41/50) cases in tubular epithelia and glomeruli respectively by in Situ hybridization, and the positive signals were localized in the nuclei of tubular epithelial cells and glomerular mesangial cells as well as infiltrated interstitial lymphocytes. Moreover, 68% (34/50) cases were proved to be HBV DNA positive by Southern blot analysis, and all were the integrated form.CONCLUSION: HBV infection might play an important role in occurrence and progress of IgAN. In addition to humoral immune damages mediated by HBAg-HBAb immune complex,renal tissues of some IgAN are directly infected with HBV and express HBAg in situ, and the cellular mechanism mediated by HBV originating from renal cells in situ may also be involved in the pathogenesis of IgAN.

Role of adhesion molecules and dendritic cells in rat hepatic/renal ischemia-reperfusion injury and anti-adhesive intervention with anti-P-selectin lectin-EGF domain monoclonal antibody

AIM: To investigate the role of P-selectin, intercellular adhesion molecule-1 (ICAM-1) and dendritic cells (DCs)in liver/kidney of rats with hepatic/renal ischemiareperfusion injury and the preventive effect of anti-Pselectin lectin-EGF domain monoclonal antibody (anti-PsLEGFmAb) on the injury.METHODS: Rat models of hepatic and renal ischemiareperfusion were established. The rats were then divided into two groups, one group treated with anti-PsL-EGFmAb(n = 20) and control treated with saline (n = 20). Both groups were subdivided into four groups according to reperfusion time (1, 3, 6 and 24 h). The sham-operated group (n = 5) served as a control group. DCs were observed by the microscopic image method, while P-selectin and ICAM-1 were analyzed by immunohistochemistry.RESULTS: P-selectin increased significantly in hepatic sinusoidal endothelial cells and renal tubular epithelial cells 1 h after ischemia-reperfusion, and the expression of ICAM-1 was up-regulated in hepatic sinusoid and renal vessels after 6 h. CD1a+CD80+DCs gradually increased in hepatic sinusoidal endothelium and renal tubules and interstitium 1 h after ischemia-reperfusion, and there was the most number of DCs in 24-h group. The localization of DCs was associated with rat hepatic/renal function.These changes became less significant in rats treated with anti-PsL-EGFmAb.CONCLUSION: DCs play an important role in immune pathogenesis of hepatic/renal ischemia-reperfusion injury.Anti-PsL-EGFmAb may regulate and inhibit local DC immigration and accumulation in liver/kidney.

Construction and expression of a humanized M_2 autoantigen trimer and its application in the diagnosis of primary biliary cirrhosis

AIM: To construct and express a humanized M2 autoantigen trimer designated as BPO and to apply it in the diagnosis of primary biliary cirrhosis (PBC). METHODS: cDNA fragments encoding M2-reactive epitopes of pyruvate dehydrogenase complex Ez (PDCE2), branched chain 2-oxo-acid dehydrogenase complex E2 (BCOADC-E2) and 2-oxo-glutarate dehydrogenase complex E2 (OGDC-E2) were amplified with PCR using total RNA extracted from human peripheral mononuclear blood cells. The fragments were cloned into the plasmid vector pQE-30 and then transferred into E. coliM15 (pREP4) for expression, which was induced by isopropylthio-β-Dgalactoside. The expressed recombinant BPO protein was demonstrated by SDS-PAGE, Western-blotting and Immunoabsorption test, its antigenic reactivity and specificity were identified with seven M2-positive sera confirmed at Euroimmun Research Center (Germany).Using the purified BPO, M2 antibodies in sera from patients with PBC and other liver related diseases were detected with ELISA. RESULTS: The expressed BPO was observed with both antigenic reactivity and specificity of M2 autoantigens. The determination of M2 antibodies by BPO with ELISA was more sensitive than using the Euroimmun's kit with the coefficients of variation less than 10 % in both interassay and intraassay.With the newly established method, M2 antibodies were found in 100 % (20/20) of patients with PBC. Six cases of liver disease with unknown etiology and 1 patient with drug induced liver injury had detectable levels of serum M2antibodies. There were also 2 patients with autoimmune cholangitis and 1 with autoimmune hepatitis showing M2-antibody positive. CONCLUSION: Compared with the routine immunofluorescenoe assay and commercially available assay kit using porcine heart mitochondrial protein as the antigen, the detection system established in the present study shows higher sensitivity and specificity and may be used as a powerful tool for the diagnosis of PBC.

Effect of tetramethylpyrazine on P-selectin and hepatic/renal ischemia and reperfusion injury in rats

AIM: To investigate the effect of tetramethylpyrazine on hepatic/renal ischemia and reperfusion injury in rats. METHODS: Hepatic/renal funclJon, histopathotogical changes, and hepatic/renal P-selectin expression were studied with biochemical measurement and immunohistochemistry in hepatic/renal ischemia and reperfusion injury in rat models. RESULTS: Hepatic/renal insufficiency and histopathological damage were much less in the tetramethylpyrazine-treated group than those in the saline-treated groups. Hepatic/renal P-selectin expression was down regulated in the tetramethylpyrazine-treated group. CONCLUSION: P-selectin might mediate neutrophil infiltration and contribute to hepatic/renal ischemia and reperfusion injury. Tetramethylpyrazine might prevent hepatic/renal damage induced by ischemia and reperfusion iniury throuclh inhibition of P-selectin.

Expression of tissue inhibitor of matrix metalloproteinases-1 during aging in rat liver

AIM: To investigate the expression and role of tissueinhibitor of matrix metalloproteinases-1 (TIMP-1) during natural aging in rat liver and to detect the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9.METHODS: The rats were divided into 3-mo-old group (n = 5), 10-mo-old group (n = 5) and 24-mo-old group(n = 5). Histopathologic changes of liver were observed with HE and Masson stain. The location and protein expressions of TIMP-1 were determined by immunohistochemistry and Westem blot; message RNA (mRNA) levels were measured in livers from rats of various ages by semi-quantitative reverse transcriptional polymerase chain reaction (RT-PCR). In addition, the expression of MMP-2 and MMP-9was assessed by RT-PCR and Western blot.RESULTS: Histologic examination showed that the aging liver had excessive fatty degeneration and collagen deposition. Immunohistochemical staining showed that TIMP-1 related antigen in livers was located in cytoplasm. The proteinexpression of TIMP-1 was significantly higher in the oldestanimals and the mRNA expression was increased significantlyin the 24-mo-old rats (t= 4.61, P= 0.002＜0.05, 24-vs 10-mo-old rats; t= 4.31, P= 0.003＜0.05, 24- vs 3-mo-oldrats). The expression of MMP-2 and MMP-9 had no change during aging; the ratios TIMP-1/MMP-2 and TIMP-1/MMP-9 in aging liver were significantly higher than those in maturation and young livers.CONCLUSION: TIMP-1 may play an important role in the process of liver aging.

Association of two polymorphisms of tumor necrosis factor gene with acute biliary pancreatitis

AIM: To investigate TNF-α-308 and TNFB polymorphisms in acute biliary pancreatitis (ABP) and to related them to the plasma TNF-α levels.METHODS: Genomic DNA was prepared from peripheral blood leukocytes. Genotypes and allele frequencies were determined in patients (n=127) and healthy controls (n=-102)using restriction fragment length polymorphism analysis of polymerase chain reaction (PCR) products. Reading the size of digested bands from polyacrylamide gel demonstrated the two alleles TNF1 and TNF2, or the two alleles TNFB1and TNFB2.RESULTS: The frequencies of TNF2 polymorphism and TNFB2 polymorphism were both similar in patients with mild or severe pancreatitis, so were in pancreatitis patients and in controls. Patients with septic shock showed a significantly higher prevalence of the TNF2 than those without. No significant differences were found in the genotype distribution of TNF-α-308 and TNFB among different groups. Plasma TNF-α levels did not differ significantly in ASBP patients displaying different alleles of the TNF gene studied.CONCLUSION: Results indicate that TNF gene polymorphisms studied play no part in determination of disease severity or susceptibility to acute biliary pancreatitis; however, TNF2polymorphism is associated with septic shock from ASBP.Genetic factors are not important in determining plasma TNF-α levels in ASBP.

Synergistic antitumor effect of TRAIL and doxorubicin on colon cancer cell line SW480

AIM: TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) has been reported to specifically induce apoptosis of cancer cells although only a small percentage of cell lines were sensitive to it. Cell lines not responding to TRAIL in vitro were said to be more prone to apoptosis when TRAIL was combined with another anticancer agent.Generally, factors affecting drug-sensitivity involve many apoptosis-related proteins, including p53. The expression of wild-type p53 gene was proposed as an important premise for tumor cells responding to chemotherapy. The present study was to investigate the cell killing action of TRAIL on colon cancer cell line SW480, its synergistic effect with doxorubicin, and the possible mechanisms.METHODS: SW480 cells were cultured in the regular condition and incubated with different levels of agents.Morphologic changes in these cells after treatment were observed under phase-contrast microscope and cytotoxicity by TRAIL alone and in combination with doxorubicin was quantified by a 1-day microculture tetrazolium dye (MTT) assay. In addition, flow cytometry assay (FCM) and transmission electron microscopy were used to detect apoptosis among these cells. Variation of p53 protein level among different groups according to concentrations of agents was measured by Western blot assay.RESULTS: (1) SW480 cells were not sensitive to TRAIL,with IC50>l mg·L^1 and dose-independent cytotoxicity. (2)SW480 cells were sensitive to doxorubicin at a certain degree,with dose-dependent cytotoxicity and IC50=65.25±3.48μmol·L^-1. (3) TRAIL could synergize with doxorubicin to kill SW480 cells effectively, which was represented by the boosted killing effect of doxorubicin on theses cells. IC50 of doxorubicin against SW480 cells sharply reduced when it was combined with TRAIL. (4) Subtoxic TRAIL (100 μg·L^-1),combined with subtoxic doxorubicin (0.86 μmol·L^-1), could kill SW480 cells sufficiently. Cytotoxicity by MTT assay arrived at 80.12±2.67 %, which was significantly higher than that by TRAIL or doxorubicin alone, with P=0.006 and 0.003 respectively. This killing effect was partly due to apoptosis. It was proved by large amounts of apoptotic cells under phase-contrast microscopy, cell apoptosis rate of 76.82±1.93 % by FCM assay and typical apoptotic morphology observed through transmission electron microscopy. Increase of apoptosis after combined treatment had no relation with protein level of p53 (p>0.05).CONCLUSION: SW480 cells are not sensitive to TRAIL, but TRAIL can synergize with lower concentra^on of doxorubidn to induce apoptosis effectively. The status of p53 protein is not involved in the mechanism of synergistic apoptosis. It suggests the potential therapeutic applicability of the combination of TRAIL with doxorubidn against colon cancers.

Role of soluble Fas ligand in autoimmune diseases

AIM: To investigate the role of soluble Fas ligand in autoimmune diseases.METHODS: RT-PCR was performed to amplify sFasL cDNA from the total RNA extracted from activated human peripheral blood lymphocytes. DNA fragments were cloned into PCR vector. After sequenced, sFasL gene fragments were inserted into pQE-31 vector and expressed in E. Coli M15 respectively. Proteins were purified through affinity chromatography column with ligand of 6xHis tag and identified by SDS-PAGE and Western blot. Mice were immunized with sFasL protein and specific anti-serum was harvested 6 wk after immunization. Monoclonal anti-human FasL antibody was made from the immunized mice. Serum level of sFasL in different patients was detected using antiFasL antibodies from the immunized mice.RESULTS: The protein expressed was 24 ku by SDS-PAGE electrophrosis. The protein was specially bound to antihuman FasL antibody by Western blot analysis. The sFasL protein could induce Jurket cell apoptosis in vitro. The concentration of serum sFasL in patients with autoimmune diseases was higher than that in normal individuals, sFasL could reduce arthritis in collagen induced arthritis (CIA) mice model by subcutaneous injection.CONCLUSION: sFasL may be involved in either induction of apoptosis or autoimmune diseases. Furthermore, sFasL may have potential application in treatment of autoimmune diseases.

The Effectof Ligustrazine on Peritoneal Transportin Peritoneal Dialysis

In order to investigate the effectof ligustrazine (L ig) i.p. on peritoneal permeability in peritoneal dialysis and its side effects,creatinine was given intravenously and continuously to m aintain the high plasm a creatinine level.All the rabbits were divided into three groups:norm al control group(group A) ,group B treated with0 .12 % L ig and group C treated with0 .2 4 % L ig. The peritoneal dialysis of all rabbits lasted2 h.The plasma and dialysate levels of glucose,protein and creatinine were observed immediate,30 min,6 0 min,90 m in,12 0 min after dialysis.Creasti- nine dialysate/ plasm a ratio (D/ P) ,protein D/ P ratio,glucose D/ Do at different time points after dialysis and creatinine mass transfer area coefficient (MTAC) at 12 0 m in were calculated. The structures of peritoneum were observed under optical microscope and electron microscope after continuously intraperitoneal injection of L ig for14 days.The results showed that the90 - min and 12 0 - min creatinine D/ P ratios in the group C were higher than in the group A.The12 0 - m in creati- nine MATC in the group C was higher than in the group A.The rabbits treated with L ig did not show significant structure changes of peritoneum and signs of peritoneal irritation.Itwas suggest- ed that L ig could increase m ass transfer ability of peritoneum without significant side effects.

Experiment and research on development and characterizations of anodic alumina membrane for use in hemodialysis

Objective: The correlation between various formative conditions and the pore characterizations of the anodic alumina membrane is investigated to seek the optimal conditions for the formation of anodic alumina membrane. Methods: High purity aluminum foils are used as the starting materials. The anodization is conducted in three types of electrolytes, 3% sulfuric acid, 5% sulfuric acid and 2.7% oxalic acid, respectively, with different voltages at 0℃ for 48h. The characterizations of the pore size, the effective porosity and the pore porosity are observed and determined by scanning electron microscopy. The hydraulic conductances of the membranes are measured to confirm that the pores are open and evaluate the permselectivity of the membranes. Results: The experimental result shows that the ordered pore arrays are obtained for oxidation under our experimental conditions. With the increasing of the voltage, the pore size and pore porosity increased significantly ( P < 0.05) , while the effective porosity decreased significantly ( P < 0.05 ) with the same electrolyte. The pore size formed in 3% sulfuric acid or 5% sulfuric acid is much smaller than in 2.7% oxalic acid as an electrolyte. The hydraulic conductance of anodic alumina membrane that formed under our experimental condition is higher than those of the membranes are available currently used in clinical. Conclusion: The results suggest that the optimal conditions for the formation of anodic alumina membrane that used in hemodialysis are in 3% or 5% sulfuric acid with 12.5V to 17.5V at 0℃ for 48h.

EFFECTS OF CORDYCEPS SINENSIS PREPARATION ON BODY PROTEIN AND AMINO ACID METABOLISM IN PATIENTS AND RATS WITH CHRONIC RENAL FAILURE

Objective To study the effects of Cordyceps sinensis (CS) on the metabolism of body pro-tein and intra-extracellular amino acids in patients with chronic renal failure( CRF ) , and on the rates of proteinsyn thesis in rats with CRF. Methods In patients with CRF, free amino acid concentrations in plasma and skeletal muscle before and after CS treatment were measured by the LKB-4400 amino acid automatic analytical instrument and the changes of body protein metabolism were observed by the method of ^15N-labeled glycine.Meanwhile, the rates of protein synthesis in liver (SL% /d) and muscle (SM% /d) of rats with CRF were de-terminedd by ^3H-phenylalanine radioactive tracer. Results After patients with CRF were treated by CS, the Leu, Ile, Thr , Lys, Cys, Tyr concentrations in plasma approached the normal levels. In one sample of skele-tal muscle the Thr and Lys concentrations approached the normal, whereas both the intracellular and extracellu-lar Val concentrations were still remarkably decreased as compared with the normal controls. Moreover, the ni-trogen flow rate (Q), rates of protein synthesis (S) and catabolism (C), and amino nitrogen utilization ratio(S/Q) in patients with CRF and the SL% /d and SM% /d in rats with CRF were significantly increased as com-pared with those before CS treatment. Conclusion CS can notably improve the amino acid metabolism, pro-mote the body protein synthesis in patients with CRF, and increase the rates of SL% /d and SM% /d in rats with CRF.

Effects of antisense oligonucleotides on theexpression of macrophage migration inhibitoryfactor on macrophages

To investigate the effects of antisense oligonucleotides on the expression of macrophage migration inhibitory factor (MIF) on macrophages, the mouse phosphorothioate oligonucleotides were designed and synthesized with the sequences of antisense, 5′-TACGGATACAAGTAGCAC-3′; Sense, 5′-ATGCCTATGTTCATCGTG-3′; Missense, 5′-CTCTCAGACTCGATCTGT-3′. These phosphorothioate oligonucleotides were then transfected into cultured macrophages ( RAW264.7 ) by luciferase vector, and the transfected macrophages were incubated with Lipopolysaccharide (LPS) (1?ng/ml) for various periods of times and collected afterwards. The content of MIF protein in the cultural supernatants was determined by ELISA, cellular RNA extracted and the expression of MIF mRNA was examined by RT-PCR analysis. The experimental results showed that LPS could induce a time-dependent specific expression of MIF on macrophages, in which the MIF mRNA in cells and the MIF protein in cultural supernatants appeared after 3 h and reached their highest concentration at 9-12?h after LPS stimulation. The levels of mRNA and proteins in the macrophages treated with antisense olignucleotides were decreased significantly after stimulation with LPS in comparison with that of stimulation with LPS alone or with that with LPS plus sense or missense oligonucleotides. There were no differences among those without LPS stimulation. It is concluded that macrophages stimulated with LPS express MIF, and the antisense olignucleotides of MIF inhibit the expression of MIF mRNA as well as the secretion of MIF proteins in macrophages.

Apoptosis of Parathyroid Cell in 5／6 Nephrectomized Rats and its Change Induced by Calcitrid

Objective To study the apoptosis of parathyroid cell in chronic renal failure(CRF) and observe its change induced by calcitriol.Methods In the studies,Sprague-Dawley rats were divided into three groups(8in each group).The five-sixth renal nephrectomy(NX) were performed in group A,5/6 NX treated with calciriol 50ng/d,subcutaneously administared for 8 weeks in group B and sham-operated control in group C.All parathyroid glands of rats were taken under anatomic microscope to evaluate.After 8 weeks apoptosis rate(APO) and cell proliferative cycle of all parathyroid gland of rats were determined by flowmetry,meanwhile the serum Ca^2+,P^3-,ALP,iPTH and urinary creatinine clearance(CCr) were also measured.Results Both the 5/6 NX rats and 5/6NX rats treated with calcitriol show remarkable decreasing in CCr compared to the sham-operated rats.Serum ALP and iPTH in group A were significantly higher than those of group B and group C.APO in group A was(0.23±0.19)%,it was significantly lower than that of group B(1．32±0.45)%,respectively(P<0.01).While both Speriod cell rate(SPR) and proliferation index(PI) in group A [(11.99±2.73)% and (16.93±3.42)%] were significantly higher than those in group B [(2.95±1.58)%and (5.24±2.29%）]and group Cu [(0.73±0.46)% and (1.76±0.70)%],P<0.01.But SPF and PI in group B were still higher than in group C,P<0.05.Conclusion The outstanding proliferation of parathyroid cells may be partly given off by the reduction of its apoptosis at least and calcitriol may induce apoptosis of parathyroid cells in 5/6 renal nephrectomy rats.

Clinical and Pathological Studies on Severe Parathyroid Hyperplasia in Uremic Patients with Secondary Hyperparathyroidism

Objective To evaluate clinical significance of parathyroid proliferation in secondary hyperpar athyroidism(SHPT).Methods The specimens of parathyroid were taken from 7 patients with SHPT and resistance to medical therapy.The histological sections were routinely stained with hematoxylin-eoxin(HE) stain,and according to macroscopical and microscopical examinations,divided into two types:diffuse type (D-type) with a normal lobular constitution and nodular type(N-type).The sum of parathyroid cells under HE stain were calculated by computer image analysis system.Results There was 16.1 times increase in the weight of parathyroid of CRF patients with SHPT and 1.86 times increase in sum of parathyroid cells.The proliferation of N-type parathyroid was especially noticeable.Conclusion We suggest that D-type hyperplasia parathryoid should be selected in autotransplantation,expecially quite small piece,in order to prevent symtom recurrence of SHPT.

Renoprotective effect of combining angiotensin Ⅱ receptor blockers and statins in diabetic rats

Recent studies suggest that treatment with angiotensin Ⅱ type 1 (AT1) receptor blockers and lipid lowering agents, namely the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, or statins may have beneficial effects on renal function independent of lowering actions on blood pressure and cholesterol.^(1,2) However, the renal effects of the combination of AT1 receptor blockers and statins in experimental diabetes are unknown. The aims of the present study were to determine whether valsartan and fluvastatin would lower the expression of nuclear factor kappa B ((NF-κB))and monocyte chemoattractant protein (MCP-1) in the tubulointerstitium and improve renal function and to see whether treatment with a combination of valsartan and fluvastatin would have any extra beneficial effect in streptozotocin (STZ)-induced unilaterally nephrectomized diabetic rats.

Plasma level and genetic variation of apolipoprotein E in patients with lipoprotein glomerulopathy

Background Lipoprotein glomerulopathy (LPG) is a renal disease characterized by thrombus-like lipoproteins in the glomerular capillaries and its abnormal lipoprotein profiles with marked elevation of apolipoprotein E (apoE). In this study, 15 Chinese patients with LPG were involed in exploring the association of the genetic variation and its plasma level in the pathogenesis of LPG.Methods A retrospective analysis of the clinical and pathological features was made in 15 patients with LPG. Plasma concentrations of apoE were measured with radial immunodiffusion assay. Genetic variations of apoE gene were detected using polymerase chain reaction and restriction fragment length polymorphism. Glomerular deposition of apoA, apoB and apoE in these patients were detected by immunofluorescence staining using monoclonal antibodies. Results Biochemical profiles of lipids and lipoproteins revealed markedly elevated levels of triglyceride, apoB and apoE, but approximately normal levels of total cholesterol, apoA1 and lipoprotein(a) [Lp(a)], which resembled familial hypertriglyceridemia. Genetic analysis demonstrated that the genotype distribution of apoE were 7 cases with (ε3/ε 4,)4 cases with ε3/ε 3 and 2 cases with ε2/ε 3. The other 2 cases (a mother and her son) showed a same distinct band. The band pattern of later 2 cases was quite similar to the apoE variant of Tokyo type. The calculated allele frequency of ε 4 was relatively high in cases with LPG in comparison with that in the normal controls. We further divided the 13 patients into three groups according to their genotypes of apoE. Patients with the genotype of apoE ε2/ε3 showed a lower level of plasma apoE as compared to those with apoE ε3/ε4 (P<0.05). The serum level of high-density lipoprotein (HDL) was the lowest in patients with the genotype of apoE ε3/ε4. No difference was found among the patients with different apoE genotype in the other clinical and pathological characteristics. Conclusions The genotype of apoE ε3/ε4 is the predominant one in Chinese patients with LPG. Patients with this genotype tend to have a higher plasma level of apoE and more severe lipid dysmetabolism. No correlation was found between the genotype of apoE and the clinical features in patients with LPG.

Generation of mouse anti-human urate anion exchanger antibody by genetic immunization and its identification

Background Human urate anion exchanger (hURAT1) as a major urate transporter expressed on renal tubular epithelial cells regulates blood urate level by reabsorbing uric acid. Antibody is an important tool to study hURAT1. This study aimed, by genetic immunization, to produce mouse anti-hURAT1 polyclonal antibody with high throughput and high specificity and to detect the location of hURAT1 in human kidney.Methods Human renal total RNA was isolated and the entire cDNA of hURAT1 was amplified by RT-PCR. The sequence of intracellular high antigenicity fragment (A280 to R349) was chosen by prediction software of protein antigenicity, and its cDNA was amplified from cDNA of hURAT1, and then cloned into pBQAP-TT vector to construct recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization. Mice were inoculated with this recombinant plasmid and two other adjuvant plasmids, pCMVi-GMCSF and pCMVi-Flt3L, which helped to enhance the antibody’s generation. After four weeks, the mice were sacrificed to obtain the anti-hURAT1 antibody from serum. The antibody was identified by western blot analysis and immunohistochemistry. At the same time, rabbit anti-hURAT1 antibody was produced by protein immunization. The specificity and efficiency between the rabbit and mouse anti-hURAT1 antibody were compared by western blot analysis and immunohistochemistry. Results The entire cDNA of hURAT1 and cDNA of its intracellular high immunogenic fragment were amplified successfully. Recombinant plasmid pBQAP-TT-hURAT1-210 for genetic immunization was confirmed by restriction digestion and sequencing. Both!the mouse anti-hURAT1 antibody and rabbit anti-hURAT1 antibody recognized 58kD hURAT1 and 64kD glycosylated hURAT1 protein bands in western blot. Immunohistochemically, hURAT1 was located at the brush border membrane of renal proximal tubular cells. In addition, the throughput and specificity of the mouse anti-hURAT1 antibody were higher than those of the rabbit anti-hURAT1 antibody.Conclusion Genetic immunization can generate anti-hURAT1 polyclonal antibody of high throughput and specificity.