生物可降解微球作为乙型肝炎基因免疫佐剂的研究

探讨生物可降解微球对基因免疫的增强作用。采用有机溶剂蒸发法制备聚乳酸聚乙醇酸共聚 物（ＰＬＧＡ）微球,构建含有乙型肝炎病毒表面抗原Ｓ基因的ｐＲＣ－ＣＭＶ真核表达载体,用微球与基因 载体共孵育法制备其混合物。肌肉注射免疫Ｂａｌｂ／ｃ小鼠。结果表明：微球注射组的血清抗体滴度达到 ｌ：１６００,其效果与乙型肝炎病毒表面抗原加铝佐剂注射组相近,而裸ＤＮＡ注射组没有反应。说明了 生物可降解微球可显著的提高基因免疫的免疫反应。     

Selective labeling and detection of specific RNAs in an RNA mixture

We report here a unique approach to selectively label and detect specific RNA in an RNA mixture (without separation or purification) using DNA polymerase, dNTP labels, and a short synthetic DNA template complementary to the 3(')-terminus of the RNA. The detection sensitivity is high, at attomole level (10-18 mole). The selective principle was demonstrated by individually labeling and detecting RNAs in a RNA mixture when different templates were provided. By taking advantage of the template-directed selectivity, poly(A) tail-containing mRNA in total RNA was detected and labeled at the 3(')-terminal on a poly(T) template. Nonradioactive labels, such as fluorophore and antigen labels, may also be used; this method can be applied in methodology for direct detection and quantification of viral RNAs.     

Role of Adenovirus Structural Proteins in the Cessation of Host-Cell Biosynthetic Functions

Two of the adenovirus capsid proteins, the fiber and the hexon, complexed with either KB cell or type 5 adenovirus deoxyribonucleic acid (DNA). Maximal binding occurred at 0.01 m NaCl; increasing the ionic strength of the reaction mixture to 0.2 m NaCl resulted in a decrease in the association of either antigen to DNA. Variations of pH between 6.3 and 8.4 did not affect the binding of fiber antigen to DNA. Below pH 7.5, however, there was a small decrease in the ability of the hexon to bind nucleic acid. The association between the adenovirus structural proteins and DNA was reversible and was independent of whether the DNA was native or denatured. The fiber or hexon protein inhibited the DNA-dependent ribonucleic acid (RNA) polymerase and the DNA polymerase from KB cells. On a weight basis, the fiber protein inhibited enzymatic activity to a greater extent than the hexon. Increasing the template DNA concentration decreased this inhibition. The inhibition of the DNA-dependent RNA polymerase activity by either antigen could be reversed by increasing the ionic strength of the reaction mixture. After infection of KB cells with type 5 adenovirus, the levels of DNA and RNA polymerases remained unchanged for 15 to 20 hr. Thereafter, the specific activity of both enzymes decreased. By 30 hr postinfection, the polymerase activities were only about 30% of the enzyme activities in uninfected cells.     

Radioimmunoassay of an antibody to phi X 174 DNA.RNA hybrid.

A fast and simple radioimmunoassay (RIA) technique was developed for an antibody to DNA·RNA hybrid using protein A-bearing Staphylococcus aureus cells as immuno-adsorbent and a glass microfiber filter for the deparation of free antigen and antibody-antigen complex. A simple method for preparing ³H-labeled DNA·RNA hybrid using single-stranded circular DNA of viruses and Escherichia coli RNA polymerase (nucleosidetri-phosphate: RNA nucleotidyltransferase, EC 2.7.7.6) is also described. In comparison to a hybrid made of natural sequences, a synthetic homopolymer hybrid poly (A)·poly(dT) was found to be a poor competitor for the antibody by this RIA technique.     

Catalytically inactive,purified RNase H1: A specific and sensitive probe for RNA–DNA hybrid imaging

R-loops are three-stranded nucleic acid structures with both physiological and pathological roles in cells. R-loop imaging generally relies on detection of the RNA–DNA hybrid component of these structures using the S9.6 antibody. We show that the use of this antibody for imaging can be problematic because it readily binds to double-stranded RNA (dsRNA) in vitro and in vivo, giving rise to nonspecific signal. In contrast, purified, catalytically inactive human RNase H1 tagged with GFP (GFP-dRNH1) is a more specific reagent for imaging RNA–DNA hybrids. GFP-dRNH1 binds strongly to RNA–DNA hybrids but not to dsRNA oligonucleotides in fixed human cells and is not susceptible to binding endogenous RNA. Furthermore, we demonstrate that purified GFP-dRNH1 can be applied to fixed cells to detect hybrids after their induction, thereby bypassing the need for cell line engineering. GFP-dRNH1 therefore promises to be a versatile tool for imaging and quantifying RNA–DNA hybrids under a wide range of conditions.     

Improved radioimmunotherapy of colorectal cancer xenografts using antibody mixtures against carcinoembryonic antigen and colon-specific antigen-p

Summary Radioimmunotherapy of GW-39 human colonic tumor xenografts grown in the hamster cheek pouch with¹³¹I-labeled NP-4 anti-(carcinoembryonic antigen) (CEA) and¹³¹I-labeled Mu-9 anti-(color-specific antigen-p) (CSAp) murine monoclonal antibodies, administered in combination, was more effective than using either antibody alone for tumor masses less than 0.5 cm³ in size. The antibody mixture had no therapeutic advantage for larger tumors. Therapeutic efficacy was determined by measuring the change in tumor size over time, quantifying the absolute number of tumors responding to radioantibody therapy, and determining the percentage growth inhibition of each treatment at various times after radioantibody administration. Several mechanisms are discussed to explain the improved tumoricidal effect of the antibody mixture noted in this model system, such as (a) the possibility that an antibody mixture could target a greater number of tumor cells, (b) the potential for antibody mixtures to provide better tumor distribution and (c) the possibility that antibodies administered in combination can increase the magnitude of tumor uptake of individual radioantibodies, thereby resulting in a greater radiation dose delivered to the tumor.     

Identification of the RNA N-glycosidase activity of ricin in castor bean extracts by an electrochemiluminescence-based assay

A simple electrochemiluminescence-based assay for RNA N-glycosidase activity has been modified to permit its use with authentic extracts of Ricinus communis (castor beans) and Abrus precatorius (jequirity seeds)—the natural sources of ricin and abrin. Modifications include the addition of an RNase inactivator to the reaction mixture, elimination of a signal-enhancing monoclonal antibody, and optimization of the incubation temperature. Concurrent testing with two substrates provides a diagnostic tool enabling castor bean toxins to be differentiated from a larger selection of N-glycosidase toxins than was previously examined.     

Transfer Agent of Immunity

Antibody formation in vitro was studied using erythrocytes (RBC) as antigen and immunocytoadhesion as the technique for detection of antibody-forming cells. Spleen cells (SPC) of nonimmune mice gained the ability to produce antibody after treatment with ribonucleic acid (RNA) preparation extracted from allogeneic mice immunized with xenogeneic or allogeneic RBC. It was also found that a small proportion of SPC from individual mice of certain strains formed antibody against autologous RBC when the cells were treated in vitro with RNA preparation obtained from the spleen of an allogeneic mouse immunized with RBC of that individual. No converting ability was observed in the RNA preparation from spleen of nonimmune autologous or allogeneic mice. The converting activity of immune RNA preparation was shown to be sensitive to ribonuclease treatment. These evidences exclude the possible contribution of antigen or fragments thereof in the RNA preparation to the induction of antibody formation in RNA recipient cells.     

In vitro deamination of cytosine to uracil in single-stranded DNA by apolipoprotein B editing complex catalytic subunit 1 (APOBEC1)

Apolipoprotein B-editing complex catalytic subunit 1 (APOBEC1) is the catalytic component of an RNA-editing complex that deaminates C6666 --> U in apolipoprotein B RNA in gastrointestinal tissue, thereby generating a premature stop codon. Whereas RNA is the physiological substrate of APOBEC1, recent experiments have strongly indicated that, when expressed in bacteria, APOBEC1 and some of its homologues can deaminate cytosine in DNA. Indeed, genetic evidence demonstrates that the physiological function of activation-induced deaminase, a B lymphocyte-specific APOBEC1 homologue, is to perform targeted deamination of cytosine within the immunoglobulin locus, thereby triggering antibody gene diversification. However, biochemical evidence of in vitro DNA deamination by members of the APOBEC family is still needed. Here, we show that deamination of cytosine to uracil in DNA can be achieved in vitro using partially purified APOBEC1 from extracts of transformed Escherichia coli. Thus, APOBEC1 can deaminate cytosine in both RNA and DNA. Strikingly, its activity on DNA is specific for single-stranded DNA and exhibits dependence on local sequence context.     

Quantitative specificity‐based display library screening identifies determinants of antibody‐epitope binding specificity

Despite the critical importance of molecular specificity in bimolecular systems, in vitro display technologies have been applied extensively for affinity maturation of peptides and antibodies without explicitly measuring the specificity of the desired interaction. We devised a general strategy to measure, screen, and evolve specificity of protein ligand interactions analogous to widely used affinity maturation strategies. The specificity of binding to target and nontarget antibodies labeled with spectrally distinct fluorophores was measured simultaneously in protein mixtures via multiparameter flow cytometry, thereby enabling screening for high target antibody specificity. Isolated antibody specific ligands exhibited varying specificity, revealing critical amino acid determinants for target recognition and nontarget avoidance in complex mixtures. Molecular specificity in the mixture was further enhanced by quantitative directed evolution, yielding a family of epitopes exhibiting improved specificities equivalent, or superior to, the native peptide antigen to which the antibody was raised. Specificity screening simultaneously favored affinity, yielding ligands with three‐fold improved affinity relative to the parent epitope. Quantitative specificity screening will be useful to screen, evolve, and characterize the specificity of protein and peptide interactions for molecular recognition applications.     

Molecular cloning of yeast cytochrome C-like polypeptide expressed in human lung carcinoma: An antigen recogizable by lung cancer-specific human monoclonal antibody

Summary We previously determined the amino acid sequence to the epitope (ATLFKTR) of cytochrome c fromCandida krusei, which is cross-reactive to the lung cancer-specific human monoclonal antibody HB4C5. Here we report that an antigen messenger RNA, which codes for a structure similar to the cytochrome c epitope, is expressed in the human lung adenocarcinoma A549. Sequencing analysis has revealed that this messenger RNA encodes a novel 190 amino acid polypeptide of 21-kDa containing an amino acid sequence (ALLFFT) similar to the cytochrome c epitope, although the total messenger RNA sequence is apparently different from the cytochrome c messenger RNA. Western analysis indicated that an antibody-recognizable 21-kDa antigen which has the same molecular weight as the predicted polypeptide is expressed in the A549 adenocarcinoma. Thein vitro translated product of the antigen messenger RNA and synthesized ALLFFT peptide were both shown to be reactive with the monoclonal antibody, indicating that this protein contains the epitope which enables A549 cells to specifically react with the antibody. The antigen mRNA was not expressed in non-transformed fibroblasts, suggesting that the antigen mRNA expression was associated with cellular transformation. Also in part of the antigen nucleotide sequence, there was a segment that had about 90% homology to the long terminal repeat sequence (no. 297–475) of the human endogenous retrovirus HERV-K10, which was related to the mouse mammary tumor virus.     

Antigen structural requirements for recognition by a cyclobutane thymine dimer-specific monoclonal antibody.

A monoclonal antibody (TDM-2) specific to a UV-induced cyclobutane pyrimidine dimer (T[cis-syn]T) has previously been established; however,the immunization had used UV-irradiated calf-thymus DNA containing a heterogeneous mixture of photoproduct sites. We investigated here the structural requirements of antigen recognition by the antibody using chemically synthesized antigen analogs. TDM-2 bound with cis-syn,but not trans-syn thymine dimer,and could bind strongly with four nucleotide analogs in which the cis-syn pyrimidine dimer was located in the center. Antigen analogs containing abasic linkers at the 5'- or 3'-side of the cis-syn cyclobutane pyrimidine dimer were synthesized and tested for binding to TDM-2. The results indicated that TDM-2 recognizes not only the cyclobutane ring but also both the 5'- and 3'-side nucleosides of the cyclobutane dimer. Furthermore,it was proved that either the 5'- or 3'-side phosphate group at a cyclobutane dimer site was absolutely required for the affinity to TDM-2. The antibody showed a strong binding to single stranded DNA but indicated little binding to double stranded DNA.     

Persistent Sin Nombre virus infection in the deer mouse (Peromyscus maniculatus) model: sites of replication and strand-specific expression

To address Sin Nombre (SN) virus persistence in deer mice, we sacrificed experimentally infected deer mice at eight time points from day 21 to day 217 postinoculation (p.i.) and examined their tissues for viral nucleocapsid (N) antigen expression and both negative-strand (genomic) and positive-strand (replicative/mRNA) viral S segment RNA titers. All the animals that we inoculated developed persistent infections, and SN virus could be isolated from tissues throughout the course of infection. The transition from an acute to a persistent pattern of infection appeared to occur between days 60 and 90 p.i. Beginning on day 60 p.i., the heart, brown adipose tissue (BAT), and lung retained antigen expression and genomic viral RNA the most frequently. We found a statistically significant association among the presence of replicative RNA in the heart, lung, and BAT, widespread antigen expression (in > or =5 tissues), and RNA viremia. Of these three tissues, the heart retained negative-strand RNA and viral N antigen the most consistently (in 25 of 26 animals). During persistence, there were two distinct patterns of infection: restricted versus disseminated tissue involvement. Mice with the restricted pattern exhibited N antigen expression in < or =3 tissues, an absence of viral RNA in the blood, neutralizing antibody titers of < or =1:1,280 (P = 0.01), and no replicative RNA in the heart, lung, or BAT. Those with the "disseminated" pattern showed N antigen expression in > or =5 tissues, neutralizing antibody titers of 1:160 to 1:20,480, replicative RNA in the heart, lung, and BAT at a high frequency, and RNA viremia. Virus could be isolated consistently only from mice that demonstrated the disseminated pattern. The heart, lung, and BAT are important sites for the replication and maintenance of SN virus during persistent infection.     

Enhanced chemiluminescence ELISA for Listeria specific antigen in cerebrospinal fluid using an FITC-anti-FITC system

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of a soluble Listeria monocytogenes serogroup 4 antigen in cerebrospinal fluid samples (CSFs). In the ELISA an anti-Listeria monoclonal antibody, immobilized onto assay wells, was used to capture antigen from CSFs. the captured antigen was then reacted with a fluorescein isothiocyanate (FITC) conjugate of the same anti-Listeria antibody, which was detected with a horseradish peroxidase conjugate of a monoclonal antibody to FITC. The presence of antigen was detected by an enhanced chemiluminescence assay using a camera luminometer. Antigen was detected in the CSFs taken from five out of seven patients with culture proven L. monocytogenes serogroup 4 central nervous system infections, and in none of the CSFs taken from 25 other patients.