Nine cases of primary localized cutaneous amyloidosis were studied by immunoperoxidase technique (ABC method) employing anti-keratin antibodies. All specimens were examined using consecutive paraffin sections to confirm the correspondence between amyloid existing area and reactive sites. Anti-keratin antibody 34pE which recognize 68, 58, 56.5, 56kd keratin peptides reacted with amyloid deposits in both lichen amyloidosus and macular amyloidosis. However, anti-keratin antibodies 34pB4 and 35pH did not react with amyloids. In general, Dylon staining positive material, keratin reacted with 34pE and amyloid P showed similar distribution in serial sections, but did not show the same one. Several keratin bodies reacted with 34pE, which were not stained with Dylon staining or antiamyloid P were found in the dermis of one specimen. These results suggest that immunohistochemical staining with antikeratin antibody 34pE using formalin-fixed, paraffin-embedded sections appeared to be a useful method in studying the histogenesis of primary localized cutaneous arnyloidosis.
Amyloid* ; Amyloidosis* ; Antibodies* ; Catalytic Domain ; Dermis ; Immunoenzyme Techniques ; Lichens ; Paraffin ; Peptides ; Plaque, Amyloid*

While indirect targeting strategies using reporter-genes are taking center stage in current molecular imaging research, another vital strategy has long involved direct imaging of specific receptors using radiolabeled ligands. Recently, there is renewal of immense interest in this area with particular attention to the epidermal growth factor receptor (EGFR), a transmembrane glycoprotein critically involved in the regulation of many cellular functions and malignancies. Recently, two novel classes of EGFR-targeting anticancer drugs have entered clinical trials with great expectations. These are monoclonal antibodies such as cetuximab that target the extracellular domain, and small molecule tyrosine kinase inhibitors such as gefitinib (Iressa) and erlotinib (Tarceva) that target the catalytic domain of the receptor. However, early results have showed disappointing survival benefits, disclosing a major challenge for this therapeutic strategy; namely, the need to identify tumors that are most likely to respond to the agents. To address this important clinical issue, several noninvasive imaging techniques are under investigation including radiolabeled probes based on small molecule tyrosine kinase inhibitors, anti-EGFR antibodies, and EGF peptides. This review describes the current status, limitations, and future prospects in the development of radiotracer methods for EGFR imaging.
Antibodies ; Antibodies, Monoclonal ; Antibodies, Monoclonal, Humanized ; Catalytic Domain ; Epidermal Growth Factor ; Glycoproteins ; Ligands ; Molecular Imaging ; Peptides ; Protein-Tyrosine Kinases ; Quinazolines ; Receptor, Epidermal Growth Factor ; Cetuximab ; Erlotinib Hydrochloride

Hyperacute rejection (HAR) is the major obstacle to xenotransplantation. In HAR complement (C') cascade is activated following the binding of xenoreactive antibodies to the donor tissue. Complement receptor type 1 (CR1), the most efficient protein in inhibiting activated C's, was modified with membrane cofactor protein (MCP) to make a more efficient C'-inhibiting hybrid molecule. Modification was done by swapping the four active site short consensus repeats (SCRs) of MCP for the SCRs 8-11 of CR1. The hybrid molecule (CR1/MCP) was expressed on the surface of mouse L cells. When the complement inhibitory activity of the CR1/MCP protein was compared with that of the wild CR1 (wCR 1) protein, CR1/MCP's inhibitory activity was weaker than wCR1's. CR1/MCP protein's L cell protecting activity from complement's attack was more prominent in adherent state than in suspension state. From these results it was suggested that the conformational direction of MCP's inhibitory action on C' is different from that of CR1 and most of the MCP expression seems to be confined to the apical side but not to the basal side of the L cell in adherent state. The wCR1's expression seems to be present on all sides of the L cell. Finally, the inverted direction of SCRS-11 of CR1 or variable length of the serine-threoninrich structure of MCP could be tried to make other CR1/MCP variants with more powerful C' inhibitory activities.
Animals ; Antibodies ; Antigens, CD46 ; Catalytic Domain ; Complement System Proteins* ; Consensus ; Humans ; Mice ; Receptors, Complement ; Tissue Donors ; Transplantation, Heterologous

OBJECTIVElo investigate the effects of anti-VEGF antibody and anti-VEGF hairpin ribozyme gene on the proliferation, apoptosis and related gene expression of the leukemia K562 cells and the possible molecular mechanisms.

METHODSK562 cells were cultured in different concentrations of anti-VEGF antibody. The recombinant eukaryotic expression plasmid (pcDNA3-RZ) containing anti-VEGF hairpin ribozyme gene and the vector-alone were introduced into K562 cells by lipofectamine mediation. Cell proliferative capacity was determined by MTT, colony formation assay and cells cycles analysis. Cell apoptosis was assayed by DNA ladder and Annexin V -FITC/PI flow cytometry.

RESULTSThe anti-VEGF antibody was able to inhibit growth and induce apoptosis of K562 cells in a dose-dependent manner. Exposure to anti-VEGF antibody at 0. 165 microg/ml for 72 hours, the cells exhibited typical DNA ladders. The apoptosis rate peaked at antibody concentration of 0. 825 microg/ml. RT-PCR showed a decrease of MRP and TOPO II expression but a relative constant expression of GST. The introduction of exogenous anti-VEGF ribozyme gene resulted in a decrease of the proliferative capacity and colony forming efficiency from (30.5 +/- 3.3) % in control group to (16.3 +/- 2.8) % in K562/RZ group, higher G1 and lower S ratio in cell cycle distribution in comparison with the control groups. Typical DNA fragmentation and higher Annexin V + ratio occurred in K562/RZ cells after treated with 0.5 micromot/L of As2O3 for 3 days, the apoptosis rate increased from 13.4% in control group to 31. 5% in As2O3 treated group.

CONCLUSIONAnti-VEGF antibody can inhibit growth, induce apoptosis and down-regulate the expression of MRP, TOPO II genes of K562 cells in vitro. Transfection with anti-VEGF ribozyme gene can inhibit the proliferation of the cells by delaying the progression G1 into S phase in cell cycles and induce cell apoptosis by down-regulating VEGF gene expression.

Antibodies ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Gene Expression Regulation ; Genetic Vectors ; Humans ; K562 Cells ; Liposomes ; RNA, Catalytic ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; immunology ; metabolism

The expression vectors of the gene encoding ScFv-2F3 were transformed into E. coli BL21(DE3). Clones of higher expression were first selected, then were grown in the presence of IPTG at 37 degrees C to induce its expression. The culture conditions were carefully optimized. It was found that optimal conditions were as follows: the induction was started as OD590 reached to 1.0-1.8; the concentration of IPTG was 0.3-0.5 mmol/L and induction time is 7 h. The yield of ScFv-2F3 expressed in the selected clones is about 20% of the total proteins. The optimal culture conditions were successfully applied to fermenter of 50 L. The conditions of washing the inclusion bodies were also optimized. A two-step method was used to renature the inclusion body. The expression product of interest and its biological activities were characterized with Western blotting and ELISA. A novel selenium-containing single-chain abzyme with GPX activity was prepared.
Antibodies, Catalytic ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Bioreactors ; microbiology ; Cloning, Molecular ; Escherichia coli ; Gene Expression ; Immunoglobulin Fragments ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Inclusion Bodies ; metabolism ; Protein Folding ; Protein Renaturation ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Selenium ; metabolism

OBJECTIVETo develop monoclonal antibodies against the catalytic subunit of human telomerase hTERT for its expression detection of human tumors.

METHODSA dominant epitope in hTERT (peptide hTERT(9))was automatically synthesized based on Fmoc method, and was used to immunize BALB/c mice. Hybridomas were generated and screened by ELISA for specific monoclonal antibodies, and the characterization of which were performed by Western blotting and immunohistochemical staining.

RESULTSAntigenic peptide hTERT(9) was synthesized and confirmed by MALDI-TOF-MS and HPLC analysis. Three hybridoma cell lines secreting anti-hTERT(9) antibodies designated as H4, G8 and A11 were established after primary screening and consequent three rounds of limited dilution. Both of H4 and G8 were IgM, while A11 was IgG1 in isotyping. The competitive assay showed that the antibodies were hTERT(9) specific, and the affinity of G8 was stronger than that of H4 and A11 assayed by affinity ranking. However, in Western blotting, both of H4 and G8 stained an about 123 000 protein band with HeLa and 293 cell extracts but not with normal 2BS cells. Besides, positive staining presented in the nucleus of HeLa, while 2BS was non-reactive immunohistochemically. The sections from paraffin-embedded blocks of 127 cases of human cancer, 40 of precancerous and 19 of benign tumors were in situ stained by G8 antibody, the results showed that the human cancer tissues were 80.31% (102/127) positive in specific nuclear reaction, on the contrary, only a minority of precancerous lesions present weak positive (17.5%, 7/40), and negative in benign tumors (0/19).

CONCLUSIONSThe monoclonal antibodies developed against synthetic peptide were hTERT-specific and could recognize both the native and the denatured form. Thus their use in immunoblotting or immunohistochemistry for detecting the telomerase hTERT expression of cancer cell and tissues was promising.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Binding, Competitive ; Blotting, Western ; methods ; Catalytic Domain ; DNA-Binding Proteins ; Female ; HeLa Cells ; Humans ; Immunohistochemistry ; methods ; Mice ; Mice, Inbred BALB C ; Neoplasms ; enzymology ; pathology ; Telomerase ; chemical synthesis ; immunology