Tumor necrosis factor(TNF-alpha) plays an improtant role in the process of anti-infection and anti-cancer. It can both protect and make damage to the host. In order to find new way of inhibiting its host-damaging activity, An E. coli flagella displayed random peptide library was constructed and TNF-alpha antagonist peptides were screened using the peptide library. After 5 rounds of panning and DNA sequencing, six peptide sequences were obtained. Two of them(TBP2, TBP3) have the same sequence frame of V--N-WG. After primary comparation of TNF-alpha binding ability, four peptides were synthesised and purified with RP-HPLC. The activity of inhibiting TNF-alpha was detected with L929 cell and MTT method. The data show that TBP2 and TBP3 can inhibit 90% of TNF-alpha activity when TNF-alpha gives about 30% cell toxicity on L929. The two sequences have not been reported.
Escherichia coli ; genetics ; Peptide Library ; Peptides ; isolation & purification ; pharmacology ; Tumor Necrosis Factor-alpha ; antagonists & inhibitors

OBJECTIVETo find out clinical characteristics and natural history of post transfusion HCV infection.

METHODS83 subjects who have received the blood from a same blood donor from January 1998 to July 2002 were investigated by the method combining cross-sectional study with retrospective study. HCV-antibody, HCV RNA, liver function, abdomen B-ultrasound and Fibroscan were detected.

RESULTSThe HCV-antibody were all positive. The HCV RNA of 56 out of the 83 cases were positive. The chronicity rates of hepatis C were 76.3% (29/38) in male patients and 60.00% (27/45) in female patients respectively, without significant difference (X² = 2.99, P = 0.11). The average age of the HCV RNA positive patients was (36.54 ± 14.37) years old. The average age of the HCV RNA negative patients was (27.43 ± 12.51) years old. A significant difference (T = -2.41, P = 0.018) existed between. The HCV genotype was type1b. Among the HCV RNA positive patients,10 cases were with mild asthenia, anorexia and abdominal distention, 9 cases with increased serum ALT, 12 cases.with chronic hepatitis and 1 case was diagnosed with decompensated liver cirrhosis.

CONCLUSIONThe clinical manifestations of HCV infection are occult and chronic. The chronicity rate is related to gender and the age when infection was caught.

Adolescent ; Adult ; Age Factors ; Aged ; Blood Donors ; Child ; Cross-Sectional Studies ; Female ; Follow-Up Studies ; Hepacivirus ; Hepatitis C ; etiology ; transmission ; virology ; Hepatitis C, Chronic ; etiology ; transmission ; virology ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Sex Factors ; Transfusion Reaction ; Young Adult

AIMTo determine the protective effect of recombinant human interleukin-2 (rhIL-2) in inhalant form on experimental respiratory tract infection with Klebsiella pneumoniae in mice.

METHODSMice were infected with the method of nasal intubation drip. During infection, mice were given rhIL-2 by sc injection and the method of nasal intubation drip. There were normal group, vehicle group, model group, rhIL-2 groups and gentamicin group. In the end, the pathological changes in the lung were observed. The survival time and the mortality within a week of each group were recorded. The total protein content, the albumin content, the activity of alkaline phosphatase and the activity of lactic dehydrogenase of broncho-alveolar lavage fluid (BALF) were dertermined and compared.

RESULTSSymptoms of Klebsiella pneumoniae were remarkably relieved because of rhIL-2 administration. The total protein content, the albumin content, the activity of alkaline phosphatase and the activity of lactic dehydrogenase of BALF were less than those in the vehicle group and the model group.

CONCLUSIONInhalation of rhIL-2 can alleviate the pathological changes in the lung after infection. At the same dose, it could be seen that the effect of rhIL-2 in inhalant form was better than that of the injection.

Administration, Inhalation ; Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; Female ; Interleukin-2 ; administration & dosage ; pharmacology ; Klebsiella Infections ; drug therapy ; metabolism ; pathology ; Klebsiella pneumoniae ; Lung ; pathology ; Mice ; Mice, Inbred ICR ; Recombinant Proteins ; administration & dosage ; pharmacology

Objective　To observe the changes of glucocorticoi d receptor (GR) in hepatic cytoplasm in rats after scalding-induced pathologic al stress and its regulation. Methods　The receptor binding capa city (R0) and the apparent dissociation constant (Kd) of GR in hepatic cytopla sm of normal, low-degree and heavy-degree scalded rats were measured with rad io-ligand binding assay, with ［3H］ dexamethasone as ligand. The changes of R0 and Kd of GR were regulated by injections of anti-rat TNFα, IL-1β a ntibodies, α-melanocyte-stimulating hormone (α-MSH), and KPV peptide( Ac- D-Lys-L-Pro-D-Val) respectively in vivo. Results　The R 0 of GR in hepatic cytoplasm in rats 12 h after heavy-degree scalding ［Mass action robust: （205.52±30.14） fmol/mg; Scatchard: （208.45±30.78） fmol/mg ］were significantly lower than that of control group ［Mass action robust:（307 .86±24.22） fmol/mg;Scatchard:（306.71±27.96） fmol/mg］(P<0.01), but no s ignificant difference was found in the R0 of GR between the control and the ra ts 12 h after low-degree scalding ［Mass action robust: （285.19±16.62） fmol/ mg ; Scatchard: （296.64±16.06） fmol/mg］. The injection of anti-rat TNFα, IL-1β antibodies, α-MSH and KVP all prevented the decline of R0 of GR in h epatic cytoplasm in rats with severe scalding. Conclusion　The injections of anti-rat TNFα, IL-1β antibodies, α-MSH or KPV can attenuate the reduction of GR in rat hepatic cytoplasm caused by severe scalding-induced pathological stress to some extent.

miR-214 plays a major role in the self-renewal of skin tissue. However, whether miR-214 regulates the proliferation and differentiation of human hair follicle stem cells (HFSCs) is unknown. Primary HFSCs were isolated from human scalp skin tissue, cultured, and identified using flow cytometry. An miR-214 mimic and inhibitor were constructed for transfection into HFSCs. The MTS and colony formation assays examined cell proliferation. Immunofluorescence detected the localization and expression levels of TCF4, β-catenin, and differentiation markers. Luciferase reporter and TOP/FOP Flash assays investigated whether miR-214 targeted EZH2 and regulated the Wnt/β-catenin signaling pathway. Western blot determined the expression levels of enhancer of zeste homolog 2 (EZH2), Wnt/β-catenin signaling-related proteins, and HFSC differentiation markers in cells subjected to miR-214 transfection. miR-214 expression was remarkably decreased during the proliferation and differentiation of HFSCs into transit-amplifying (TA) cells. Downregulation of miR-214 promotes the proliferation and differentiation of HFSCs. Overexpression of miR-214 led to decreased expression of EZH2, β-catenin, and TCF-4, whereas downregulation of miR-214 resulted in increased expression of EZH2, β-catenin, and TCF-4 as well as TA differentiation markers. Immunofluorescence assay revealed that inhibiting miR-214 triggered the entry of β-catenin and TCF-4 into the nucleus. The luciferase reporter and TOP/FOP Flash assays demonstrated that miR-214 directly targets EZH2 and affects Wnt/β-catenin signaling. The miR-214/EZH2/β-catenin axis could be considered a candidate target in tissue engineering and regenerative medicine for HFSCs.
Antigens, Differentiation ; Blotting, Western ; Cell Proliferation ; Down-Regulation ; Flow Cytometry ; Fluorescent Antibody Technique ; Hair Follicle* ; Hair* ; Humans* ; In Vitro Techniques* ; Luciferases ; Regenerative Medicine ; Scalp ; Skin ; Stem Cells* ; Tissue Engineering ; Transfection

Age Factors ; Aged ; Anticoagulants ; adverse effects ; therapeutic use ; Atrial Fibrillation ; drug therapy ; Female ; Humans ; International Normalized Ratio ; Male ; Middle Aged

In order to find a new TPO-mimic peptide with similar activity to TPO while reducing the side effects, a TPO-mimic peptide (P1) screened from a random peptide library was restructured. The new structure of the TPO mimic peptide (P2) was synthesized. After coupling P2 with Dextran 10 and performing intermolecular oxidation, dextran-coupled and dimerized form of P2 were obtained, naming D-P2 and (P2)(2) respectively. The activities of the peptides in vitro were measured with MTT method. The results showed that the EC50 of P2 was 20 nmol/L, 700 times higher than P1. The EC50 of D-P2 and (P2)(2) were 0.35 nmol/L and 0.14 nmol/L, respectively. After administrating to the mouse, the peptides increased the number of platelets in the blood circulation obviously without influence on other blood cells. In conclusion, the TPO-mimic peptides have prospects in treating diseases related with thrombocytopenia.
Animals ; Female ; Male ; Mice ; Platelet Count ; Thrombopoietin ; analogs & derivatives ; chemical synthesis ; pharmacology

The aim of this study was to investigate the renal function in 149 patients receiving myeloablative allogeneic hematopoietic stem cell transplantation (allo-HSCT) from June 2005 to June 2010 in our hospital, and analyze the risk factors resulting in kidney insufficiency and experience in diagnose and therapy. The creatinine clearance (CrCL) and serial creatinine level were evaluated before and after allo-HSCT within 100 days and 1 year. Non-radiation conditioning regimens were used for any patients. The acute kidney insufficiency (AKI) was defined as at least a 1.5-fold rise in serum creatinine level after allo-HSCT within the first 100 days. The chronic kidney insufficiency (CKI) was defined as the creatinine clearance < basal level within 3 months to 1 year after allo-HSCT. The results showed that the kidney insufficiency was found in 41 patients, in which the incidence of AKI was 32/149 (21.5%). CsA, amphotericin B (P = 0.025) and ES (P = 0.022) were defined as risk factors for AKI. The incidence of CKI was 18/138 (13%). cGVHD (P = 0.013) and TA-TMA (P = 0.012) were associated with the development of CKI. The 2-year survival was lower in patients with kidney dysfunction than that in patients without kidney dysfunction (39% vs 74.1%, P < 0.001). The main factors resulting in kidney insufficiency were defined as infection (52%), GVHD (20%), TA-TMA (12%) and tumor relapse (12%). It is concluded that kidney insufficiency is an important complication of allo-HSCT. Careful monitoring kidney function, minimizing the use of amphotericin B, prophylaxis and effective treatment of fungal infection, GVHD and TA-TMA may be effective preventive measures to decrease the incidence of kidney insufficiency.
Acute Kidney Injury ; etiology ; Adolescent ; Adult ; Female ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Male ; Middle Aged ; Renal Insufficiency ; etiology ; Retrospective Studies ; Risk Factors ; Transplantation, Homologous ; Young Adult

OBJECTIVETo analyze the specificity, sensitivity and receiver operating characteristic (ROC) curve of plasma elafin for diagnosis of skin acute graft-versus-host disease (aGVHD), and to explore its clinical diagnostic value.

METHODSIncidence of skin aGVHD from fifty-three patients received allogeneic hematopoietic stem cell transplantation (allo-HSCT) were observed prospectively in Guangdong General Hospital from Apr 2010 to Aug 2011. The plasma concentrations of elafin were detected by enzyme-linked immunosorbent assay (ELISA). Skin biopsies were taken from 28 patients with skin rash, and elafin expression in the skin was detected by immunohistochemistry. Positive expression was defined as significant staining of at 50% of the depth of the epidermis, excluding the granular cell layer and the acrosyringium.

RESULTSAmong 28 patients with skin rash, twenty-five were considered as skin aGVHD by clinical diagnosis, seventeen were confirmed as skin aGVHD by pathological biopsy. 11 cases were elafin positive by immunohistochemical staining. Elafin protein was overexpressed in aGVHD skin tissue (P = 0.001). Plasma concentrations of elafin were significantly higher in patients with skin aGVHD (positive) group than in those without skin aGVHD (negative) group (P = 0.005), among which there being no statistically significant difference in plasma elafin level between patients with grade I skin aGVHD group and negative group(P = 0.971), but being statistically significant difference compared patients with grade II-IV skin aGVHD group with those with grade I skin aGVHD group (P = 0.02) and with negative group (P = 0.008). Using the pathological diagnosis as the gold standard, the estimated specificity and the sensitivity of clinical diagnosis criteria were 27.3% and 100%, respectively, and those of tissue elafin protein level were 100% and 64.7%, respectively. The area under the ROC curve was 0.909 (0.797 - 1.021) when plasma concentrations of elafin was used in diagnosis of skin aGVHD. The sensitivity was 82.4% and the specificity was 81.8 % when the critical value was set at 1456.043 µg/L.

CONCLUSIONPlasma concentration of elafin is significantly higher at the onset of skin aGVHD. It can be used as biochemical marker of skin aGVHD and has higher value in diagnosis of skin aGVHD.

Adolescent ; Adult ; Elafin ; blood ; Female ; Graft vs Host Disease ; blood ; diagnosis ; etiology ; Hematopoietic Stem Cell Transplantation ; adverse effects ; Humans ; Male ; Middle Aged ; ROC Curve ; Sensitivity and Specificity ; Skin Diseases ; blood ; diagnosis ; etiology ; Young Adult

Many antineoplastic agents can cause myelosuppression and thrombocytopenia. Thrombopoietin (TPO) is believed to be the major cytokine affecting the proliferation and maturation of megakaryocytes and increasing circulating platelet levels. We have designed and synthesized a TPO mimetic peptide, it can increase circulating platelet levels in vivo. For increasing half-life and forming dimer, the peptide was expressed as chimeric proteins with human IgG1 Fc fragments. The cDNA of TPO mimetic peptide was synthesized chemically and linked respectively to 5' terminus of human IgG1 Fc cDNA fragments in various length (Fc1: Fc 5' 648 bp; Fc2: Fc 5' 270 bp; Fc3: Fc 5' 267 bp; Fc4: Fc 5' 90 bp), and cloned into expression plasmid pET28a (+) for constructing four recombinant plasmids. By transforming the four recombinant plasmids into E. coli. BL21 (DE3) respectively, we got 3 kinds of engineered E. coli which express TPO + Fc chimeric proteins(28 kD TPO + Fc1, 12 kD TPO + Fc2 and 12 kD TPO + Fc3) at high level respectively, the expressed proteins were purified with DEAE-Sepharose FF and S-Sepharose FF column. The bioactivities of the expressed chimeric proteins(TPO + Fc1, TPO + Fc2 and TPO + Fc3), TPO mimetic peptide, and PEG4000 coupled TPO mimetic peptide were evaluated with Ba/F3-mp1 in vitro and with carboplatin-induced thrombocytopenia mice in vivo, the expressed chimeric proteins have higher activity than TPO mimetic peptide both in vitro and in vivo, the EC50 on Ba/F3-mp1 cells were 13, 10, 10, 50, and 25 nmol/L respectively, all of them can increase circulating platelet counts. Their imol/Lunogenicity were valuated in mice, none of them can elicit mice to produce antibodies to TPO mimetic peptide, meanwhile three TPO + Fc chimeric proteins can elicit mice to produce antibodies to human IgG1 Fc. These studies have laid basis for production of TPO mimetic peptide by genetic engineering.
Animals ; Base Sequence ; Blood Platelets ; drug effects ; Cell Division ; drug effects ; Female ; Humans ; Immunoglobulin Fc Fragments ; genetics ; immunology ; pharmacology ; Immunoglobulin G ; genetics ; Male ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Oligopeptides ; chemical synthesis ; genetics ; pharmacology ; Recombinant Fusion Proteins ; genetics ; immunology ; pharmacology ; Thrombocytopenia ; blood ; immunology ; Thrombopoietin ; genetics ; immunology ; pharmacology