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Objective To construct four types of glucagon-like peptide-1 (GLP-1) and human serum albumin (HSA) fusion proteins that can be realeased at different rate in vivo by introducing protease cleavage sites between these two moieties.The therapeutic effect and release rate are studied to achieve balanced pharmacokinetics ( PK) and pharmacody-namics ( PD) of GLP-1 and HSA fusion proteins.Methods The gene with different polypeptide joint of GLP-1 and HSA fusion proteins were synthesized by overlap extension PCR amplification, cloned into expression vector pPIC9 and transformed into Pichia pastoris GS115.Then, fusion proteins were obtained by protein purification after being induced by methanol.The preliminary PK and PD of the fusion proteins were studied after purification.Results The fusion protein Gly2-GLP-1-GGGGG-HSA showed no release while Gly2-GLP-1-VTR-HSA, Gly2-GLP-1-SARSVRA-HSA, and Gly2-GLP-1-GRSRVTRSV-HSA showed a slow, medium and fast release rate, respectively, after incubation with furin.In vitro biological activity test results dispalyed that each type of fusion protein promoted insulin secretion of MIN6 cells.In vivo PK test indicated the half-life size of fusion proteins was the largest in Gly2-GLP-1-GGGGG-HSA, followed by Gly2-GLP-1-VTR-HSA, Gly2-GLP-1-SARSVRA-HSA, and Gly2-GLP-1-GRSRVTRSV-HSA.In vivo PD test exhibited hypoglycemic activity that was the highest in Gly2-GLP-1-VTR-HSA, followed by Gly2-GLP-1-SARSVRA-HSA, Gly2-GLP-1-GRSRVTRSV-HSA, and Gly2-GLP-1-GGGGG-HSA.Conclusion GLP-1 can be released from fusion proteins with full activity after the introduction of protease cleavage sites.Releasable fusion proteins at an appropriate release rate have the most balanced PK and PD.
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<p><b>BACKGROUND</b>An important characteristic of renal cell carcinomas and adrenal tumors is that these tumors may expand into the renal vein and inferior vena cava, and transform into tumor thrombi. This study was to evaluate the use of piggyback liver transplant techniques for surgical management of urological tumors with inferior vena cava tumor thrombus.</p><p><b>METHODS</b>Nineteen patients with renal cell carcinomas or adrenal tumors with inferior vena cava tumor thrombus were treated from November 1995 to April 2008. Their ages ranged from 29 years to 76 years (mean 54 years). The extent of tumor thrombus was infrahepatic (level I) in 2, retrohepatic (level II) in 7, suprahepatic (level III) in 6, and intra-atrial (level IV) in 4 patients. We used cardiopulmonary bypass with deep hypothermic circulatory arrest to remove the thrombi in 3 cases of level IV and in 2 cases of level III. In all level II, 4 level III, and 2 level IV cases, we used piggyback liver transplant techniques to mobilize the liver off of the inferior vena cava and to separate the inferior vena cava from the posterior abdominal wall.</p><p><b>RESULTS</b>Mean operative time was 5.1 hours, mean estimated blood loss was 2289 ml and mean blood transfusion was 12.84 U. One patient with adrenal cortical carcinoma and level IV thrombus died in the immediate postoperative period. Three patients were lost to follow up, and the other 15 survivors were followed from 5 months to 56 months. Eight of these 15 patients died due to metastasis; however 7 were still alive at the last follow-up.</p><p><b>CONCLUSIONS</b>An aggressive surgical approach is the only hope for curing patients diagnosed with urological tumors combined with inferior vena cava tumor thrombus. The use of piggyback liver transplant techniques to mobilize the liver off of the inferior vena cava provides excellent exposure of the inferior vena cava. Patients with a level II or level III inferior vena cava thrombus may be treated without using cardiopulmonary bypass.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Adrenal Gland Neoplasms , Pathology , General Surgery , Carcinoma, Renal Cell , Pathology , General Surgery , Liver Transplantation , Methods , Neoplastic Cells, Circulating , Vena Cava, Inferior , PathologyABSTRACT
Objective To discuss the treatment of renal or adrenal tumor with cancer thrombus in the inferior vena cava. Methods From Jan 1984 to Apr 2008,29 cases of renal or adrenal malignancy with thrombosis involving the inferior vena cava underwent treatment.The diagnoses were confirmed by Doppler uhrasonography,CT and MRI.In the 29 surgical patiens the tumor thrombus was level I in 7,level Ⅱ in 10,level Ⅲ in 8 and levelⅣin 4.According to TNM classification,23 cases were classified to T2N.M.,1 case was T2Nl Mo,1 case was TzNlMl,1 case was T3NoMo,2 case were T3NlMl and 1 case was T3N2Mo.The mean tumor size was 8.7(4.O-16.O)cm in diameter.The mean tumor thrombosis length was 3.2(2.5-4.0)cm in level I,5.3(4.5-6.0)cm in level Ⅱ,8.2(6.5-9.O)cm in levelⅢand 15.1(12.0-18.5)cm in level IV. Results The operation was performed succesgfullv in 29 patients Patholocieal examination showed that 18 cases of clear cellcarcinoma,3 cases sarcomatoid carcinoma,2 cases renal papillary adenocarcinoma,1 case renal cell carcinoma (undifferentiated),1 case granule carcinoma,3 cases adrenocortical carcinoma and 1 case metastatic malignant melanoma of adrenal gland.Of 29 patients,3 were out of contact.Twenty-six patients were followed up for 35(0-62)months after treatment,3-and 5-year survival rates were 15/26 and 11/26.Three-year survival rates for stage T2 and T3 were 14/22 and 1/4.Five-year survival rates for stage T2 and T3 were 10/22 and 1/4.Three-year survival rates for level I、Ⅱ、Ⅲ andⅣ were 4/6,5/8,5/8 and 1/4.Five-year survival rates for level I,Ⅱ、Ⅲ andⅣ were 3/6,4/8,3/8 and 1/4.Three-year survival rates for a tumor thrombus in the below or above diaphragm were 14/22 versus 1/4,5-year survival rates were 10/22 versus 1/4.Three-year and 5-year survival rates for the patients without distant metastases and lymph node involvement were 12/18 and 9/18.Three-year and 5-year surviral rates for the patients with distant metastases and lymph node involvement were 3/8 and 2/8.The 3 surgical patients with metastatic disease died at 6,10,22 months. Conclusions Surgical treatment could be the preferred approach for the patients of renal or adrenal tumor with cancer thrombus in the inferior vena cava without distant metastases and lymph node involvement.It could improve the quality of life and may prolong survival.
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Calcineurin inhibitors (CNIs) are playing an important role in preventing acute rejection in renal transplantation; however, their nephrotoxicity may impact long-term renal allograft survival. Several CNI-sparing regimens (such as CNI-avoidance or CNI-minimization) have shown at least comparable efficacy with standard-dose CNI regimens. Research continues to achieve the "best" balance between efficacy and toxicity of available immunosuppressive regimens.
Subject(s)
Humans , Calcineurin Inhibitors , Graft Rejection , Immunosuppressive Agents , Kidney TransplantationABSTRACT
<p><b>OBJECTIVE</b>To investigate the types and therapies of malignancies in renal allograft recipients.</p><p><b>METHODS</b>We retrospectively analyzed the occurrence, types, and therapies of malignancies in 498 renal allograft recipients who had received operations in Peking Union Medical College Hospital from May 1986 to October 2008.</p><p><b>RESULTS</b>Among 498 renal allograft recipients, 18 patients (3.6% ) were diagnosed with malignancies, which included bladder cancer (n = 5), renal pyloric cancer or ureteric cancer (n = 4), leukemia or lymphoma (n = 3), hepatic cancer (n = 2), skin cancer, rectum carcinoma, pulmonary carcinoma and thymoma (n = 1 each). Surgical operations were performed in 10 cases, 6 of whom survived with normal renal function and had no rejection of transplanted kidneys. Three patients with bladder cancer and one patient with ureteric cancer experienced recurrences 7 to 15 months after operations; among them one bladder cancer patient died. One hepatic carcinoma patient died of pulmonary metastasis 8 months after operation. One non-Hodgkin's lymphoma patient died 11 months after chemotherapy. Five cases with advanced unresectable malignancies died 8 to 17 months after the diagnosis.</p><p><b>CONCLUSIONS</b>The incidences of malignancies, especially urological epithelial carcinoma, are high in renal allograft recipients. Radical surgery of the solid malignancies is a preferred option.</p>
Subject(s)
Adult , Aged , Female , Humans , Male , Middle Aged , Kidney Transplantation , Neoplasms , Epidemiology , Therapeutics , Postoperative Complications , Epidemiology , Therapeutics , Retrospective StudiesABSTRACT
Objective To improve the diagnosis and treatment quality of juxtaglomerular cell tumor of the kidney. Methods Three patients(2 females, 1 male) were diagnosed with juxtaglomerular cell tumor of kidney and underwent nephron-sparing surgery. Case 1 was female, 15 years old. She presented with hypertention of 245/135 mm Hg. The serum kalium was 2.5--3.0 mmol/L. Thelaboratory examination suggested that in decubitus and standing position, the plasma renin activity (PRA) was 2.2 and 3.5 μg · L-1 · h-1 , angiotensin Ⅱ (ATⅡ) was 181.2 and 481.4 ng/L; aldosterone(ALD) was 332.4 and 747.9 pmol/L, respectively. Dynamic enhanced CT scanning demonstrated a tumor with the diameter of 1.3 cm in right kidney. Case 2 was male, 39 years old. He presented with hypertention of 180/120 mm Hg. The serum kalium was 2. 7--3.0 mmol/L. In decubitus and standing position, PRAwas8.1 and 9.2 μg·L-1 · h-1, ATⅡ was 198.3 and 279.1 ng/L, ALD was 285.3 and 761.7 pmol/L, respectively. Dynamic enhanced CT scanning showed a tumor with the diameter of 1.2 cm in right kidney. Case 3 was female, 26 years old. She presented with hypertention of 210/120 mm Hg. The serum kalium was 4. 1 mmol/L. In decubitus and standing position, PRA was 0.1 and 0.3 μg · L-1·h-1 , ATⅡ 56.2 and 71.5 ng/L, ALD 321.3 and 421.1 pmol/L, respec tively. On dynamic enhanced CT scanning, a tumor with a diameter of 3.0era was located in left kidney. Results Partial nephrectomy was successfully performed in 3 patients, including 1 (case 2) retroperitoneal laparoscopic surgery. Pathologic examination revealed encapsulated tumors in all cases. Light microscopically, the tumor consisted of clusters of polygonal cells, and the cell had centrally located nuclei and slightly eosinophilic cytoplasm. Thick walled vessels were usually present. The tumors showed positive immunostaining for actin and CD34. Three patients were followed up for 23,4, 26 months respectively and all remained normotensive without any treatment. No recurrence or metastasis occurred. Conclusions Hypertention, increased PRA, secondary aldosteronism, hypokalemia are characteristics for juxtaglomerular cell tumor of the kidney. Dynamic enhanced CT scanning has high sensitivity. Partial nephrectomy or enucleation of tumor are both effective surgical treatment. Retroperitoneal laparoscopic surgery is safe and effective as well.
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Apolipoprotein A-I-Milano(AIM), a natural variant, not only inhibits the initiation and progression of atherosclerosis, but also makes the preexisting atherosclerotic lesions regress. AIM gene, at which N-terminal codens were optimized, was subcloned into the expression vector of pET22b. Recombiant plasmids were transformed into E. coli strain BL21 (DE3) and induced with IPTG. The expressed apoliprotein A-I-Milano was soluble in E. coli and was about 38% of total cell lysate. Purified by Butyl Sepharose 4F. F hydrophobic chromatography and Q Sepharose H.P. anion exchange chromatography, followed by ultrafiltration with Vivaspin 20 (30 000MW), AIM monomer was obtained in a purity of more than 95%. Activity assay of binding of AIM monomer to lipid indicates that association of AIM monomer with DMPC is slower than normal apoA-I but DMPC number associated by AIM monomer is more than by apoA-I. This results will be important for studying structure, function of AIM, specially clinical application.
Subject(s)
Humans , Apolipoprotein A-I , Genetics , Escherichia coli , Genetics , Metabolism , Mutant Proteins , Genetics , Recombinant Proteins , Genetics , SolubilityABSTRACT
To increase the in vivo half-life of human CNTF mutein AX15 (R13K), HSA-AX15 (R13K) fusion protein was constructed by the fusion of the C-terminus of HSA to the N-terminus of AX15 (R13K) via an 11 amino acids linker. HSA-AX15 (R13K) fusion protein was purified to homogeneity by cation exchange chromatography, reverse phase chromatography and gel filtration after expressed in pichia pastoris. TF-1 cell survival bioassay showed the biological activity of AX15 (R13K) was not affected by the fusion to HSA. It was demonstrated that tertian injection of 4.8 mg/kg HSA-AX15 (R13K) fusion protein could produce more potent anti-obesity effects on KM mice than daily injection of 1.6 mg/kg AX15 (R13K). The long-acting form of hCNTF variant has the potential to reduce discomfort by requiring fewer injections and to minimize the side-effects by decreasing the dosage and fluctuation of plasma concentration, and thus has superior clinical application.
Subject(s)
Animals , Humans , Mice , Ciliary Neurotrophic Factor , Genetics , Mutant Proteins , Genetics , Pichia , Genetics , Metabolism , Recombinant Fusion Proteins , Genetics , Serum Albumin , GeneticsABSTRACT
AX15 is a mutein of naturally occurring human ciliary neurophic factor (hCNTF), with improved biological activity, stability and solubility. AX15 is susceptible to protease degradation when expressed in Pichia pastoris. Amino acid sequencing revealed the degradation was occurred behind position 12 and 13 amino acid residues, which constitute a dibasic site, RR. Based on the substrate specificity of KEX2, a KEX2 resistant mutein of AX15-AX15 (R13K) was constructed, in which RR was replaced by RK. It was demonstrated that the stability of AX15 (R13K) improved significantly, as no degradation was detected even after 120 hours of induction. AX15 (R13K) was purified to homogeneity by ultrafiltration and gel filtration. TF-1 cell survival bioassay showed AX15 (R13K) had equivalent specific activity to AX15. The protease resistant mutein of AX15 may have greater in vivo stability and thus have superior therapeutic potential.
Subject(s)
Humans , Ciliary Neurotrophic Factor , Genetics , Genetic Vectors , Mutant Proteins , Genetics , Mutation , Peptide Hydrolases , Chemistry , Pichia , Genetics , Metabolism , Recombinant Proteins , GeneticsABSTRACT
Human parathyroid hormone (hPTH) was highly expressed in Escherichia coli by inserted the synthesized whole hPTH cDNA into the vectors pBV220 and pET22b. After expression and disruption, the purified product was acquired through cation exchange chromatography and reverse phase chromatography. From the results of N-terminal sequencing and MALDI-TOF-MS analysis the recombiant prtein was indentified as intact hPTH. In in vitro Bioassays the recombinant hPTH stimulated adenylate cyclase as the standard did. In ovariectomized rats the recombinant hPTH markedly increased the femoral bone mass and bone mineral density.