Memory B-cell persistence is independent of persisting immunizing antigen

Immunological memory in the antibody system is generated in T-cell-dependent responses and carried by long-lived memory B cells that recognize antigen by high-affinity antibodies. But it remains controversial whether these B cells represent true 'memory' cells (that is, their maintenance is independent of the immunizing antigen), or whether they are a product of a chronic immune response driven by the immunizing antigen, which can be retained in the organism for extended time periods on the surface of specialized antigen-presenting cells (follicular dendritic cells). Cell transfer experiments provided evidence in favour of a role of the immunizing antigen; however, analysis of memory cells in intact animals, which showed that these cells are mostly resting and can persist in the absence of detectable T-cell help or follicular dendritic cells, argued against it. Here we show, by using a genetic switch mediated by Cre recombinase, that memory B cells switching their antibody specificity away from the immunizing antigen are indeed maintained in the animal over long periods of time, similar to cells retaining their original antigen-binding specificity.     

Research advances in genetic engineering of plantibody

The combination of engineering antibody and plant biotechnology creates the plantibody. It has been reported that the engineering antibodies expressed in plant, no matter whether they are intact or small-molecular antibodies, keep their antigen-binding specificity, which means they are functional. This trait makes plantibody notable. Now the researches are focused mainly on the following three aspects: (i) Therapeutic antibody in clinic. It is expected to offer the opportunity of large-scale antibody procluction in agriculture systems, for the purpose of decreasing the cost greatly. Moreover, the property of multiple sexual hybridization between plants can be used to produce bior multi-functional antibodies and create new antibody medicines. (ii) Plant physiology. Engineering antibodies against plant hormones or some active materials can block their activities in plant, so the trait of plantibody could be used to study the growth and development of plant. (iii) Plant disease-resistance. The gene of antibody against plant virus can be transferred into plant to defend the intrusion of virus in order to cure the disease.     

Hybrid hybridomas and their use in immunohistochemistry

A normal antibody-producing cell only expresses one antibody, resulting in the well-known phenomenon of allelic exclusion. When two myeloma cells are fused, the derived hybrids are capable of co-dominantly expressing the antibody genes of both parents. Although the respective variable (V) and constant (C) region genes remain expressed in the same cis configuration, heavy and light chains of both parents are scrambled, and hybrid molecules are formed. The same is true when a myeloma and an antibody-producing cell are fused to produce a hybrid myeloma (hybridoma). Fusion therefore allows the production of hybrid immunoglobulin molecules containing two different combining sites. Hybrid molecules of this type retain antigen-binding activity and specificity. Bispecific monoclonal antibodies secreted by hybridomas may have a variety of uses in biology and in medicine. Here we have focused on their application in histochemistry. As an example, we have prepared and tested an anti-somatostatin-anti-peroxidase bispecific antibody. This way of producing hybrid molecules is superior to the production of hybrid antibodies by chemical reconstitution methods because the drastic treatment required for chain separation in the latter is likely to lead to some protein denaturation and loss of antibody activity. Intracellularly synthesized and assembled hybrids do not suffer from this disadvantage. In addition, the recombination of heavy and light chains from different antibody molecules is likely to lead to considerable waste.     

Replacing the complementarity-determining regions in a human antibody with those from a mouse

The variable domains of an antibody consist of a beta-sheet framework with hypervariable regions (or complementarity-determining regions--CDRs) which fashion the antigen-binding site. Here we attempted to determine whether the antigen-binding site could be transplanted from one framework to another by grafting the CDRs. We substituted the CDRs from the heavy-chain variable region of mouse antibody B1-8, which binds the hapten NP-cap (4-hydroxy-3-nitrophenacetyl caproic acid; KNP-cap = 1.2 microM), for the corresponding CDRs of a human myeloma protein. We report that in combination with the B1-8 mouse light chain, the new antibody has acquired the hapten affinity of the B1-8 antibody (KNP-cap = 1.9 microM). Such 'CDR replacement' may offer a means of constructing human monoclonal antibodies from the corresponding mouse monoclonal antibodies.     

Transient reversion of ras oncogene-induced cell transformation by antibodies specific for amino acid 12 of ras protein

The proteins encoded by the ras oncogene are thought to trigger expression of the transformed phenotype in some types of cancer cells. In human cells, the ras protein family consists of several members including normal (proto-oncogene) and mutant (oncogene) forms. In general, the proto-oncogene forms are thought to be involved in the normal growth control of cells, while the mutant forms (which apparently result from somatic mutation of the normal ras genes) appear to be responsible, in part, for the loss of normal growth control. On microinjection into living normal cells, the purified ras oncogene protein (p21) induces a characteristic loss of growth control in cells within several hours. The mutant forms of the different ras proteins typically contain a single amino-acid change, usually at position 12 or less frequently at position 61. Here we report that microinjection of antibodies specific for amino acid 12 of the oncogenic v-Ki-ras protein into cells transformed by this protein causes a transient reversion of the cells to a normal phenotype. The fact that this antibody inhibits binding of GTP to the v-Ki-ras protein supports the notion that GTP binding is essential to the transforming function of this oncogene product.     

Three-dimensional structure of an antigen-antibody complex at 6 A resolution

Present understanding of the three-dimensional structure of antibody combining sites is based on X-ray diffraction studies of myeloma immunoglobulins. The structures of the antigen-binding fragment (Fab) complexes of two of these immunoglobulins with small ligands have also been determined. However, there is no crystallographic information concerning the interactions of an antibody with an antigen, nor do we know the precise structure of antigenic determinants on protein molecules. We now report the first structure determination of an antigen-antibody complex at 6 A resolution. The structure of the complex between hen egg-white lysozyme and the Fab of a monoclonal anti-lysozyme antibody (D1.3) shows that the combining site of antibodies is not merely a cleft delineated by the complementarity-determining regions of the variable regions of the light and heavy chains, but is a larger area extending beyond it. A correspondingly large area of the antigen makes close contacts with the antibody, in agreement with the notion of a 'topographical' rather than 'sequential' antigenic determinant. The structural basis of cross-reactivities of an antibody with heterologous antigens and the effect of a single amino acid substitution on antigenic specificity can thus be visualized in the structural model presented here.     

Biological properties of human c-Ha-ras1 genes mutated at codon 12

Vertebrate genomes contain proto-oncogenes whose enhanced expression or alteration by mutation seems to be involved in the development of naturally occurring tumours. These activated genes, usually assayed by their ability to induce the malignant transformation of NIH 3T3 cells, are frequently related to the ras oncogene of Harvey (Ha-ras) or Kirsten (Ki-ras) murine sarcoma viruses, or a third member of this family (N-ras). Activation involves point mutation which often affect codon 12 (refs 16-26) of the encoded 21,000-molecular weight polypeptide (p21). To provide insight into structural requirements involved in p21 activation, we have now constructed 20 mutant c-Ha-ras1 genes by in vitro mutagenesis, each encoding a different amino acid at codon 12. Analysis of rat fibroblasts transfected with these altered genes demonstrates that all amino acids except glycine (which is encoded by normal cellular ras genes) and proline at position 12 activate p21, suggesting a requirement for an alpha-helical structure in this region of the polypeptide. The morphological phenotype of cells transformed by the activated genes can, however, depend on the particular amino acid at this position.     

Molecular machinery for non-vesicular trafficking of ceramide

Synthesis and sorting of lipids are essential for membrane biogenesis; however, the mechanisms underlying the transport of membrane lipids remain little understood. Ceramide is synthesized at the endoplasmic reticulum and translocated to the Golgi compartment for conversion to sphingomyelin. The main pathway of ceramide transport to the Golgi is genetically impaired in a mammalian mutant cell line, LY-A. Here we identify CERT as the factor defective in LY-A cells. CERT, which is identical to a splicing variant of Goodpasture antigen-binding protein, is a cytoplasmic protein with a phosphatidylinositol-4-monophosphate-binding (PtdIns4P) domain and a putative domain for catalysing lipid transfer. In vitro assays show that this lipid-transfer-catalysing domain specifically extracts ceramide from phospholipid bilayers. CERT expressed in LY-A cells has an amino acid substitution that destroys its PtdIns4P-binding activity, thereby impairing its Golgi-targeting function. We conclude that CERT mediates the intracellular trafficking of ceramide in a non-vesicular manner.     

A monoclonal antibody against antigen-specific helper factor augments T-cell help

Antigen-specific molecules, commonly termed 'factors', have been shown to be released from helper and suppressor T cells. These factors mimic the activity of the cells that secrete them and there is much speculation about the relationship of antigen-specific factors to T-cell receptors for antigen. We have raised a variety of antisera in rabbits which were shown to react against conserved 'constant' determinants on either helper or suppressor factors independently of antigenic specificity or mouse strain of origin of the factor. In contrast, syngeneic mouse antisera were found to react with 'variable' factor determinants in an antigen-specific and mouse strain-dependent manner. These antisera thus define two regions on factor molecules, one 'variable' (related to antigen specificity) and the other 'constant' (related to function). However, potential contaminants in these antisera have limited their usefulness. Thus, we are now generating monoclonal antibodies against T-cell factors and report here the properties of a monoclonal antibody (AF3.44.4) which reacts with antigen-specific helper factors. This antibody also binds to helper T cells and, in the presence of antigen, augments helper cell induction in vitro, which, in turn, leads to enhanced antibody production in vitro. These characteristics suggest that AF3.44.4 recognizes a determinant shared by helper factor and the antigen receptor on helper T cells.     

Specific targeting of cytotoxic T cells by anti-T3 linked to anti-target cell antibody

The specificity of cytotoxic T lymphocytes (Tc) cells is conferred by an antigen-specific receptor, Ti, which in humans is physically associated with an invariant cell-surface glycoprotein, T3. Monoclonal antibodies specific for either T3 and Ti are able to elicit a variety of T-cell responses such as lymphokine production, mitogenesis and cytotoxicity. For example, human Tc cells lyse anti-T3-expressing hybridoma cells, but not cells of other specificity, presumably because anti-T3 on the hybridoma cells binds to T3 on the Tc cells and triggers lysis. Here, we have adapted approaches used in a different cytotoxic effector system, antibody-dependent cellular cytotoxicity (ADCC), to alter the specificity of Tc cell. Studies of ADCC showed that heteroaggregates containing anti-Fc receptor (Fc gamma R) antibody cross-linked to a second antibody bind to Fc gamma R on ADCC effectors and cause them to kill target cells bearing antigen recognized by the second antibody. The present studies use anti-T3-containing heteroaggregates to re-target human Tc cells to cells for which we have appropriate antibodies, including xenogeneic tumour cells and chicken erythrocytes. These results extend previous observations on the role of T3 in triggering cytotoxicity and suggest that effector cell re-targeting could be used for in vivo treatment of neoplasms and other pathogens that express distinctive surface antigens.     

Molecular characterization of single memory B cells

Primary antigenic exposure results in an initial antibody response and the T cell-dependent induction of B-cell memory. Memory B-cell differentiation is characterized by somatic hypermutation in antibody variable region genes (V) and selection of B cells expressing high-affinity variants of this antigen receptor. Despite our current understanding of B-cell memory, the origin of memory B cells and the regulation of their differentiation remain elusive. This is largely due to the difficulties in observing and purifying this minor component of the immunized spleen. Further, molecular characterization of memory B cells requires hybridoma formation which restricts analyses to only those clones capable of fusion and does not allow isolation of cells in a normal physiological state. We have therefore developed a unique system which allows isolation and unambiguous enumeration of IgG1+ memory B cells, based on six-parameter flow cytometry, secretion of antibody in clonal cultures and analysis of clonally expressed V genes using the polymerase chain reaction. Here we report that single IgG1+ antigen-binding B cells from an early secondary immune response proliferate in lipopolysaccharide-driven microcultures and produce antigen-specific IgG1 antibodies. Individual B-cell clones in these cultures express somatically mutated heavy chain V genes, confirming their designation as memory B cells. Although isolated memory B cells undergo extensive proliferation in vitro, V gene sequence analysis of their individual progeny shows that further hypermutation does not occur.     

Y-152和K-156在果蝇ADH催化中的作用

果蝇醇脱氢酶酪氨酸－１５２（Ｙ－１５２）和赖氨酸－１５６（Ｋ－１５６）处于同类脱氢酶的保守位点。经人工定点突变和酶动力学分析,前者的苯丙氨酸（Ｙ１５２Ｆ）、组氨酸（Ｙ１５２Ｈ）和谷氨酸突变体（Ｙ１５２Ｅ）及后者的异亮氨酸突变体（Ｋ１５６Ｉ）均丧失催化活性。而半胱氨酸－１５２（Ｙ１５２Ｃ）及精氨酸－１５６（Ｋ１５６Ｒ）突变体活性分别是对应野生型的０．２５％和２．２％。此外,Ｙ１５２Ｃ和Ｋ１５６Ｒ的Ｋ     

A lethal mutation in mice eliminates the slow calcium current in skeletal muscle cells

Contraction of a vertebrate skeletal muscle fibre is triggered by electrical depolarization of sarcolemmal infoldings termed transverse-tubules (t-tubules), which in turn causes the release of calcium from an internal store, the sarcoplasmic reticulum (SR). The mechanism that links t-tubular depolarization to SR calcium release remains poorly understood. In principle, this link might be provided by the prominent slow calcium current that has been described in skeletal muscle cells of adult frogs and rats. However, blocking this current does not abolish the depolarization-induced contractile responses of frog muscle, and the function of this slow calcium current is unknown. Here we describe measurements of calcium currents in developing skeletal muscle cells of normal rats and mice, and of mice with muscular dysgenesis, a mutation that causes excitation-contraction (E-C) coupling to fail. We find that a slow calcium current is present in skeletal muscle cells of normal animals but absent from skeletal muscle cells of mutant animals. The effect of the mutation is specific to the slow calcium current of skeletal muscle; a fast calcium current is present in developing skeletal muscle cells of both normal and mutant animals, and slow calcium currents are present in cardiac and sensory neurones of mutant animals. We believe this to be the first report of a mutation affecting calcium currents in a multicellular organism. The effects of the mutation raise important questions about the relationship between the slow calcium current and skeletal muscle E-C coupling.     

Hybrid antibodies can target sites for attack by T cells

It would be advantageous in the case of certain diseases to be able to focus a strong T-cell response at a chosen target, for example, in treating cancer or infections that have escaped the normal host response. At present, it seems inconceivable that we could use antigen-specific lines or clones of effector T cells for this purpose because of complications due to the major histocompatibility restriction of T-cell specificity and the problem of rejection of transplanted effector cells. Here we describe a novel technology which combines the power of T lymphocytes in eliminating unwanted cells and causing beneficial inflammatory reactions with the great advantages of monoclonal antibodies (their specificity and availability). We show that heteroconjugates of monoclonal antibodies (referred to hereafter as hybrid antibodies), in which one of the component binding sites is anti-T-cell receptor and the other component binding site is directed against any chosen target antigen, can focus T cells to act at the targeted site. Monoclonal antibodies directed against the T-cell receptor, such as the anti-allotype used here, are mitogenic for resting T cells and can be used to induce effector T cells carrying the T-cell receptor determinant which can then be directed against the target by a hybrid antibody.     

基于生物免疫系统克隆选择机理和免疫网络理论的免疫算法

提出一种基于生物免疫系统克隆选择机理和免疫网络理论的免疫算法.该算法通过抗体的克隆选择和变异过程,完成对入侵抗原的清除,实现免疫防御的功能;利用免疫网络调节的思想选择抗体记忆细胞,完成知识的学习和积累,实现免疫自稳的功能;利用所建立的抗体记忆矩阵实现对类似入侵抗原的快速应答,行使免疫监视识别功能.该算法利用生物变异机制实现抗体的自适应调节,使系统具有自适应、自学习能力.在加热炉状态识别的应用研究表明,本文所提出的算法在解决数据识别方面具有较好的效果.     

Effects of interlinker sequences on the biological properties of bispecific single-chain antibodies

Single-chain bispecific antibody (scBsAb) is one of the promising genetic engineering antibody formats for clinical application. But the effects of interlinker sequenees on the biological properties of bispecific single-chain antibodies have not been studied in detail. Three interlinker sequences were designed and synthesized, and denominatedas Fc, HSA, 205C‘, respectively. Universal vectors with these different interlinker sequences for scBsAb expression in E.coli were constructed. A model scBsAb based on a reshaped single-chain antibody (scFv) against human CD3 and a scFv directed against human ovarian carcinoma were generated and expressed in E. coli. The results of SDS-PAGE and Western blot showed that the different interlinker sequences did not affect the expression level of scBsAb. However, as demonstrated by ELISA and pharmacokinetics studies performed in mice, scBsAbs with different interlinker sequenees had difference in the antigen-binding activities and terminal haw-life time (T1/2β) in vivo, the interlinker HSA could remarkably prolong the retention time of scBsAb in blood. These results indicated that the peptide sequence of intertinker could affect important biological properties of scBsAb, such as antigen-binding properties and stability in vivo. So, selection of an appropriate interlinker sequence is very important for scBsAb construction. Optimal interlinker can bring scBsAb biological properties more suitable for clinical application.