Recent studies have shown that epidermal growth factor receptor (EGFR) is an important target for cancer therapy. The present study prepared single chain Fv (scFv) directed against EGFR. Balb/c mice were immunized by human carcinoma A431 cells, and total RNA of the splenic cells was extracted. VH and VL gene fragments were amplified by RT-PCR and further joined into scFv gene with a linker, then scFv gene fragments were ligated into the phagemid vector pCANTAB 5E. The phagemid containing scFv were transformed into electro-competent E. coli TG1 cells. The recombinant phage antibody library was constructed through rescuing the transformed cells with help phage M13K07. The specified recombinant phages were enriched through 5 rounds of affinity panning and the anti-EGFR phage scFv clones were screened and identified with ELISA. A total of 48 clones from the library were selected randomly and 45 clones were identified positive. After infecting E. coli HB2151 cells with one positive clone, soluble recombinant antibodies about 27 kD were produced and located in the periplasm and the supernatant. The result of sequencing showed that the scFv gene was 768 bp, which encoded 256 amino acid residues. VH and VL including 3 CDRs and 4 FRs, respectively, were all homologous to mouse Ig. The soluble scFv showed the specific binding activity to purified EGFR and EGFR located in carcinoma cell membrane. The successful preparation of anti-EGFR scFv will provide an EGFR targeted molecule for the development of antibody-based drugs and biological therapy of cancer.
Animals ; Antibodies, Monoclonal ; Cell Line, Tumor ; Humans ; Immunoglobulin Light Chains ; genetics ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Peptide Library ; Receptor, Epidermal Growth Factor ; genetics ; immunology ; Single-Chain Antibodies ; genetics ; immunology

The article investigates the feasibility of delivering drugs to brain via inner ear, and provides a novel route for delivering drugs to the brain tissues. Dexamethasone acetate (DA)-loaded solid lipid nanoparticles (SLN) was prepared by using Compritol 888 ATO as material. HPLC assays for the determination of DA, dexamethasone sodium phosphate (DSP) and dexamethasone (Dex) were developed, separately. DA-loaded SLN and DSP solution were administered after intratympanic injection (IT) or intravenous injection (IV). Perilymph ( PL) and cerebrospinal fluid (CSF) were collected periodically. The concentrations in PL and CSF were measured by HPLC, and used to estimate pharmacokinetic parameters of Dex in CSF. The AUC of Dex in CSF following IT DA-loaded SLN or DSP solution were respectively 2.5 and 4.3-fold higher than those following IV. After IT, DA-loaded SLN increased the AUC by 13 times and extended the MRT by 19 times, compared with the solution. Moreover, the AUC of Dex in PL following IT the SLN was 76% lower than that following IT the solution. Intra-cochlear administration shows great potential and offers a promising alternative to brain-targeted drug delivery.
Animals ; Anti-Inflammatory Agents ; administration & dosage ; pharmacokinetics ; Brain ; metabolism ; Cerebrospinal Fluid ; metabolism ; Dexamethasone ; administration & dosage ; analogs & derivatives ; metabolism ; pharmacokinetics ; Drug Delivery Systems ; Ear, Inner ; metabolism ; Fatty Acids ; chemistry ; Female ; Guinea Pigs ; Lecithins ; Male ; Nanoparticles ; Particle Size ; Perilymph ; metabolism ; Random Allocation ; Surface-Active Agents ; chemistry ; Tissue Distribution

OBJECTIVETo observe the effect of hypertonic sodium chloride hydroxyethyl starch 40 injection (HSH) in treatment of acute intracranial hypertension complicated by hemorrhagic shock in dogs, and explore the mechanism of the effects of HSH.

METHODSTwenty dogs were randomized into 4 equal groups, namely the 7.5% NaCl (HS) group, Ringer-Lactates solution (RL) group, hydroxyethyl strarch (HES) group, and HSH group. Canine models of acute intracranial hypertension complicated by hemorrhagic shock were established by epidural balloon inflation with saline and rapid discharge of the arterial blood. One hour after the induced shock, the dogs were given HS (6 ml/kg), RL of 3-fold volume of blood loss, HES of equivalent volume of blood loss, and HSH 8 ml/kg in the 4 groups, respectively. During the shock and resuscitationperiod, the intracranial pressure (ICP), mean arterial pressure (MAP) and cerebral perfusion pressure (CPP) of the dogs were monitored, and the serum sodium level and plasma osmolality were measured at 30 min, 1 h and 4 h after the resuscitation.

RESULTSAll dogs had similar MAP, CPP, and ICP before resuscitation (P>0.05). After resuscitation, the MAP was significantly improved (P<0.01), but the dogs in HSH group exhibited the fastest response; with the exception of the dogs in HS group to have significantly decreased MAP 2 h after resuscitation (P<0.01), all the other dogs maintained the MAP for 4 h. The CPP was also significantly increased after resuscitation (P<0.01), and in HS group, CPP decreased significantly after 2 h (P<0.01), and HSH group maintained the high CPP after 4 h. The ICP was increased significantly in RL and HES groups after resuscitation (P<0.01), reaching the peak level at 1 and 3 h, respectively, but in HS and HSH groups, the ICP decreased significantly to the lowest level at 1 h (P<0.01) which was maintained for 4 h. After resuscitation, the plasma sodium and plasma osmolality were significantly increased in HSH and HS groups.

CONCLUSIONIn dogs with acute intracranial hypertension and hemorrhagic shock, HSH can effectively resuscitate hemorrhagic shock and decrease ICP, and the effect is longer-lasting than that of HS.

Acute Disease ; Animals ; Dogs ; Female ; Hydroxyethyl Starch Derivatives ; administration & dosage ; therapeutic use ; Intracranial Hypertension ; drug therapy ; etiology ; Male ; Plasma Substitutes ; administration & dosage ; therapeutic use ; Random Allocation ; Saline Solution, Hypertonic ; administration & dosage ; therapeutic use ; Shock, Hemorrhagic ; complications ; drug therapy ; Treatment Outcome

OBJECTIVETo investigate the expression pattern of human triggering receptor expressed on myeloid cells 1 (TREM-1) mRNA in peripheral blood mononuclear cells and its clinical significance in acute obstructive suppurative cholangitis (AOSC).

METHODSPeripheral blood mononuclear cells were collected from 36 patients with AOSC and 40 healthy adults. TREM-1 mRNA was determined by semi-quantitative RT-PCR, and TREM-1 protein by immunocytochemistry. Enzyme-linked immunosorbent assay (TNF-alpha) was used to detect the level of tumor necrosis factor-alpha (TNF-alpha), and immunoturbidimetry employed to detect C reactive protein.

RESULTSThe expression of TREM-1 mRNA relative to beta-actin was 1.007-/+0.252 in patients with AOSC, significantly higher than that in the healthy adults (0.457-/+0.053, P<0.05). The two groups also showed significantly different TREM protein expression (P<0.01). The AOSC patients exhibited significantly higher levels of TNF-alpha and C reactive protein than the healthy adults (P<0.01).

CONCLUSIONThe expression of human TREM-1 in peripheral blood mononuclear cells is up-regulated obviously in early stage of AOSC, probably suggesting an important role of TREM-1 in the development of AOSC.

Acute Disease ; Adult ; Aged ; Aged, 80 and over ; Biomarkers ; blood ; Case-Control Studies ; Cholangitis ; blood ; complications ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Leukocytes, Mononuclear ; metabolism ; Male ; Membrane Glycoproteins ; genetics ; metabolism ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Receptors, Immunologic ; genetics ; metabolism ; Sepsis ; blood ; etiology ; Triggering Receptor Expressed on Myeloid Cells-1

This study was aimed to analyze the survival status of patients with diffuse large B-cell lymphoma (DLBCL) and to investigate the influence of autologous hematopoietic stem cell transplantation (auto-HSCT), different pathological types, International Prognosis Idex (IPI) on prognosis. One hundred and sixteen cases of DLBCL were analyzed retrospectively. The treatment efficacy of R-CHOP alone and R-CHOP combined with auto-HSCT as well as the influence of different immunopathologic types, IPI, hypersensitive C-reactive protein (HSCRP), α-hydroxybutyric acid deaminase (HBDH) on the prognosis of DLBCL patients including overall survival (OS) rate, progression-free survival (PFS) rate were analyzed. The results indicated that the 5-year OS for all patients was 72.4%. in which 30 patients with Ann Arbor staging III-IV received auto-HSCT plus R-CHOP. The prognosis of the 30 patients was better than that of 86 cases received R-CHOP chemotherapy alone (5-year OS was 82.5% vs 69.0%, 5-year PFS was 77.1% vs 68.3%) (P < 0.05). The prognosis of patients in germinal center B-cell-like group (GCB group) was better than that of patients in activated B-cell-like group (ABC group). Some clinical features were associated with poor prognosis including OS and PFS, such as age, B symptoms, IPI scores, the level of LDH, HSCRP and HBDH (P < 0.05) in which the level of LDH, age ≥ 60 years and B symptoms were independent prognostic factors in DLBCL patients (P < 0.05). It is concluded that auto-HSCT combined with R-CHOP can improve the long-term survival of DLBCL patients. The prognosis of patients in GCB group is better than that of patients in the ABC group. The clinical features such as age, B symptoms, IPI scores and LDH are associated with prognosis.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antibodies, Monoclonal, Murine-Derived ; Antineoplastic Combined Chemotherapy Protocols ; Cyclophosphamide ; Doxorubicin ; Female ; Hematopoietic Stem Cell Transplantation ; Humans ; Lymphoma, Large B-Cell, Diffuse ; diagnosis ; therapy ; Male ; Middle Aged ; Prednisone ; Prognosis ; Retrospective Studies ; Vincristine ; Young Adult

OBJECTIVETo study the epidemiologic characteristics of virus-induced acute diarrhea in children under 5 years old in Taiyuan, Shanxi province.

METHODSStool specimens and clinical data were collected from 346 inpatients with acute diarrhea from children less than 5 years old. Rotavirus-positive specimens were identified by ELASA kit. Calicivirus and astrovirus were detected by reverse transcription-polymerase chain reaction (RT-PCR). Adenovirus was done by polymerase chain reaction (PCR).

RESULTSOf the 346 specimens, the percentage of samples with Rotavirus, Calicivirus, Astrovirus, and Adenovirus was 40.8%, 7.5%, 6.4% and 3.2%. Among 141 rotavirus positive samples, serotype G1 (42.6%) was the predominant strain. More than 95% of viral diarrhea patients under hospitalization occurred among children younger than 2 years.

CONCLUSIONRotavirus is the major pathogen contributing to the acute diarrhea. The disease generally peaks at autumn/winter. The predominant rotavirus strain circulated was G1P[8].

Age Distribution ; Child, Preschool ; China ; epidemiology ; Diarrhea ; epidemiology ; virology ; Female ; Humans ; Infant ; Male ; Virus Diseases ; epidemiology ; virology ; Viruses ; classification ; genetics ; isolation & purification

OBJECTIVETo investigate the association of Apa I polymorphism in vitamin D receptor (VDR) gene with bone mass in men.

METHODSThe VDR Apa I genotype was determined by PCR-restriction fragment length polymorphism (RFLP) in 388 unrelated healthy men aged 46-80 years of Han nationality in Shanghai city. Bone mineral density (BMD) and bone mineral content (BMC) at lumber spine 1-4 (L1-4) and at any sites of proximal femur including to femoral neck (Neck), trochanter (Troch) and Ward's striangle (Ward's) were measured by duel-energy X-ray absorptiometry.

RESULTSFrequencies distribution of VDR Apa I genotype were aa for 48.1%, Aa for 44.2% and AA 7.7%. The allele frequencies of Apa I polymorphism were in Hardy-Weinberg equilibrium. No significant association was found between Apa I genotype and BMD or BMC in group of all population or in subgroup of men below 60 years. In men above 60 years, the significant association was found between VDR Apa I genotype and BMD or BMC at L1-4, Neck and Ward's (P < 0.05, P < 0.01) and compared with Aa and aa genotype, AA genotype had significantly higher mean BMD and BMC at L1-4, Neck and Ward's (P < 0.05, P < 0.01). But Apa I genotype is not associated with BMD and BMC at Troch.

CONCLUSIONSApa I polymorphism is associated with bone mass in men above 60 years, and AA genotype has higher bone mass. Apa I polymorphism in VDR gene possibly influence loss of trabecular and cortical bone mass in old men.

Age Factors ; Aged ; Aged, 80 and over ; Alleles ; Bone Density ; Humans ; Male ; Middle Aged ; Phenotype ; Polymorphism, Restriction Fragment Length ; Receptors, Calcitriol ; genetics ; Transcription Factors ; genetics ; Zinc Fingers ; genetics

OBJECTIVETo investigate the effect of HLA-Cw on haploidentical hematopoietic stem cell transplantation (HHSCT) without T-cell depletion.

METHODSHLA-Cw were detected with PCR-SSP, the clinical data of 21 cases of haploidentical hematopoietic stem cell transplantation, including 8 standard risk and 13 high risk cases from July 2002 to March 2006 were summarized, and the effect of HLA-Cw in HHSCT was analyzed.

RESULTSTwenty patients achieved sustained, full-donor-type engraftment. The HLA-Cw matched and mismatched groups attained neutrophil recovery at a median of 12 days and 13 days, and platelet recovery to more than 20 x 10(9)/L at a median of 20 days and 23 days respectively (P > 0.05). The cumulative incidences of grades II-IV acute GVHD were 76.9% in HLA-Cw matched group and 14.3% in the mismatched group(P < 0.05). The incidences of chronic GVHD were 85.7% in HLA-Cw matched group and 57.1% in the mismatched group(P > 0.05). The 28 months disease-free survival probabilities were 49.0% in HLA-Cw matched group, and 85.7% in the mismatched group (P > 0.05). The Karnofsky score of survival patients was over 90%.

CONCLUSIONHLA-Cw mismatched in donor and recipient of HHSCT is beneficial for reducing II-IV aGVHD, and being in favor of long term survival.

Adolescent ; Adult ; Child ; Female ; Follow-Up Studies ; Graft vs Host Disease ; immunology ; HLA-C Antigens ; immunology ; Hematopoietic Stem Cell Transplantation ; Humans ; Male ; Middle Aged ; Survival Rate ; Transplantation, Homologous ; immunology

OBJECTIVETo explore the effect of cardiopulmonary bypass (CPB) on the profile of protein expression in peripheral blood mononuclear cells (PBMCs).

METHODSEleven patients undergoing cardiac surgery under cardiopulmonary bypass were enrolled in the study. Peripheral blood samples were collected before CPB (T0), 1 h after CPB (T1) and at the end of operation (T2), and PBMCs were obtained by gradient centrifugation. The profile of protein expression was analyzed using 2-D gel electrophoresis (2-DE) and mass spectrometry. The candidate proteins were further identified by Western blotting.

RESULTCompared to protein profile at T0, 12 protein spots were identified to be up-regulated in PBMCs at T1 (P <0.05), among which S100A9 reached the peak level at T1 and decreased after operation,but not returned to its initial level.

CONCLUSIONResults indicate that 12 proteins are likely to be involved in CPB, however, their roles need to be elucidated.

Adult ; Cardiopulmonary Bypass ; Electrophoresis, Gel, Two-Dimensional ; Female ; Humans ; Leukocytes, Mononuclear ; metabolism ; Male ; Mass Spectrometry ; Middle Aged ; Proteome ; metabolism ; Proteomics ; Young Adult

BACKGROUNDCD4(+) T cells play a crucial role in the pathogenesis of aplastic anaemia. However, the mechanisms of over-proliferation, activation, infiltration of bone marrow and damage to haematopoietic cells of CD4(+) T cells in aplastic anaemia are unclear. Therefore, we screened differentially expressed genes of bone marrow CD4(+) T cells of aplastic anaemia patients and normal donors by suppressive subtractive hybridization to investigate the pathogenesis of aplastic anaemia.

METHODSThe bone marrow mononuclear cells of a first visit aplastic anaemia patient and a healthy donor of the same age and sex were isolated using lymphocyte separating medium by density gradient centrifugation. With the patients as "tester" and donor as "driver", their CD4(+) T cells were separated with magnetic bead sorting and a cDNA library established by suppressive subtractive hybridization. Then 15 of the resulting subtracted cDNA clones were randomly selected for DNA sequencing and homological analysis. With semiquantitative RT-PCR, bone marrow samples from 20 patients with aplastic anaemia and 20 healthy donors assessed the expression levels of differentially expressed genes from SSH library.

RESULTSPCR detected 89 clones in the library containing an inserted fragment of 100 bp to 700 bp. Among 15 sequenced clones, 12 were known genes including 3 repeated genes. Compared with normal donors, there were 9/12 genes over-expressed in bone marrow CD4(+) T cells of patients with aplastic anaemia. The effects of these genes included protein synthesis, biology oxidation, signal transduction, proliferative regulation and cell migration. Not all these genes had been reported in the mechanisms of haematopoietic damage mediated by CD4(+) T cells in aplastic anaemia.

CONCLUSIONSScreening and cloning genes, which regulate functions of CD4(+) T cells, are helpful in elucidating the mechanisms of over proliferation, activation, infiltrating bone marrow and damaging haematopoietic cells of CD4(+) T cells in aplastic anaemia.

Adult ; Anemia, Aplastic ; genetics ; Bone Marrow Cells ; metabolism ; CD4-Positive T-Lymphocytes ; metabolism ; CREB-Binding Protein ; genetics ; Gene Library ; Humans ; Male ; Nucleic Acid Hybridization ; methods ; Reverse Transcriptase Polymerase Chain Reaction ; T Cell Transcription Factor 1 ; genetics