Cytotoxic T cell responses in HLA-A2.1 transgenic mice. Recognition of HLA alloantigens and utilization of HLA-A2.1 as a restriction element

Previous studies have indicated that the frequency of murine CTL precursors (CTLp) for human class I molecules is one to two orders of magnitude lower than that for murine class I alloantigens, and that this is due to species-specific structural differences between these molecules. Transgenic mice expressing the human class I MHC Ag HLA-A2.1 were used to examine changes in the frequency of class I HLA-specific precursors after T cell differentiation in an HLA-A2.1 positive environment. The HLA-A2.1 gene product was expressed at levels comparable to those of the endogenous H-2Db molecule in thymus, bone marrow, and spleen. By limiting dilution analysis, it was observed that the frequencies of CTLp in transgenic mice responding to the human alloantigens HLA-B7 or HLA-A2.2 were comparable to or lower than those in normal C57BL/6 mice, regardless of whether the Ag was presented on human or murine cells. Thus, expression of a human class I molecule in these animals did not result in an expansion of the number of CTLp specific for other human class I Ag. In addition, the frequency of HLA-A2.1-restricted, influenza specific CTLp was substantially lower than the frequency of H-2b restricted CTLp, indicating a poor utilization of HLA-A2.1 as a restricting element. Finally, the frequencies of CTLp for HLA-A2.1 expressed on syngeneic murine tumor cells were decreased significantly. Thus, expression of HLA-A2.1 in these animals appeared to induced tolerance to this Ag. Interestingly, however, these mice were not tolerant to the HLA-A2.1 molecule expressed on human cells. This indicates that the HLA-A2.1 associated epitopes expressed on murine and human cells differ and suggests that, under these circumstances, HLA-A2.1 acts as a restricting element for human nominal Ag. These results are discussed in the context of current models of T cell repertoire development.     

IL-12 administration leads to a transient depletion of T cells,B cells,and APCs and concomitant abrogation of the HLA-A2.1-restricted CTL response in transgenic mice

The injection of a mixture of bona fide T cell epitopes can lead to the occurrence of immunodominance, meaning that the immune response is focused on the recognition of a single epitope or a small portion of the epitopes injected. We have previously demonstrated that the administration of rIL-12 can counteract immunodominance in BALB/c mice. In this study, we show that the administration of rIL-12 to HLA-A2.1 transgenic mice (A2k(b) mice) abrogates specifically the immune response against HLA-A2.1-restricted HIV epitopes in the spleen. This lack of immune response is most probably due to a transient depletion of B cells, T cells, macrophages, and dendritic cells in this organ. Therefore, our study explains the mechanism of immunosuppression by rIL-12 in vivo.     

IgE isotype switch and IgE production are enhanced in IL-21-deficient but not IFN-gamma-deficient mice in a Th2-biased response

IgE plays a critical role in the pathogenesis of allergy and asthma. Therefore, suppression of IgE production would provide therapeutic benefits to patients suffering from these diseases. We have reported that the production of IgE is regulated differently in the spleen vs. the draining lymph nodes (LN). IgE isotype switch and IgE producing B cell expansion occur in the draining LN after antigen (Ag) immunization, but do not happen in the spleen. In addition, a population of pre-existing IgE+ cells is observed in the spleen of normal or sham immunized mice, but is not present in the draining LN. To further understand the regulation of IgE production in different lymphoid organs, and the potential inhibitory factors of IgE isotype switch in the spleen, the involvement of IL-21 and IFN-gamma in regulating IgE production was investigated by using the IL-21 and the IFN-gamma deficient mice. We found that in the absence of IL-21 IgE isotype switch and IgE+ cell clonal expansion were dramatically enhanced in the spleen and IgE isotype switch was partially increased in the draining LN. In addition, IgE production of the pre-existing CD19-CD5+B220(low) IgE+ cells in the spleen was also increased in the absence of IL-21 under physiological conditions. In contrast, using the IFN-gamma deficient mice, we did not observe a negative impact of IFN-gamma on either IgE isotype switch or IgE production. Our data suggest that IL-21 appears to be a critical cytokine to keep low IgE levels under physiological and pathological conditions.     

Identification of hepatitis B virus-specific CTL epitopes presented by HLA-A33:03 in peripheral blood mononuclear cells from patients and transgenic mice

Cytotoxic T lymphocyte (CTL) epitopes in the HBV protein of hepatitis B virus (HBV) may play a key role in viral control and liver damage. The aim of this study was to identify and study the function of HLA-A^∗33:03-restricted CTL epitopes in HBV protein of the HBV genotypes B and C, which are epidemic in China. Sixteen HBV peptides were predicated by computational analysis, and synthesized peptides were examined for their affinity to HLA-A^∗33:03 using a stable cell line. After being analyzed by enzyme-linked immunospot and cytolytic activity assays, as well as the tetramers staining method using peripheral blood mononuclear cells isolated from HBV-infected patients, five peptides (Hbs245–253, HBs335–343, HBc119–127, HBc104–112, and HBp391–399) were chosen to further confirm their HLA_A^∗33:03 restriction in transgenic mice.     

The novel beta-secretase inhibitor KMI-429 reduces amyloid beta peptide production in amyloid precursor protein transgenic and wild-type mice

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of amyloid plaques and neurofibrillary tangles in the brain. The major component of the plaques, amyloid beta peptide (Abeta), is generated from amyloid precursor protein (APP) by beta- and gamma-secretase-mediated cleavage. Because beta-secretase/beta-site APP cleaving enzyme 1 (BACE1) knockout mice produce much less Abeta and grow normally, a beta-secretase inhibitor is thought to be one of the most attractive targets for the development of therapeutic interventions for AD without apparent side-effects. Here, we report the in vivo inhibitory effects of a novel beta-secretase inhibitor, KMI-429, a transition-state mimic, which effectively inhibits beta-secretase activity in cultured cells in a dose-dependent manner. We injected KMI-429 into the hippocampus of APP transgenic mice. KMI-429 significantly reduced Abeta production in vivo in the soluble fraction compared with vehicle, but the level of Abeta in the insoluble fraction was unaffected. In contrast, an intrahippocampal injection of KMI-429 in wild-type mice remarkably reduced Abeta production in both the soluble and insoluble fractions. Our results indicate that the beta-secretase inhibitor KMI-429 is a promising candidate for the treatment of AD.     

利用转基因小鼠及转染色体小鼠产生人抗体的研究进展

自单克隆抗体（mAb)技术问世以来,解决了生命科学的许多重要问题,但其鼠源生导致的HAMA反应在大大限制了它在人体治疗中的应用,因此,制备人抗体成了亟待解决的难题,转入Ig基因组小鼠与转梁色体小鼠 的构建成功为解决这一难题提供了可行途径,本文就这两条小鼠的构建及其在制备人抗体中的应用进展作一综述。     

Human parathyroid hormone as a secretory peptide in milk of transgenic mice

In a transgenic mouse model we have targeted the expression of recombinant human parathyroid hormone (hPTH) to the mammary gland yielding hPTH as a secretory, soluble peptide in milk. A 2.5 kb upstream regulatory sequence of the murine whey acidic protein (WAP) directed the expression of the hPTH cDNA in a fusion gene construct (WAPPTHSV2) containing the SV40 small t-antigen intron and polyadenylation site in the 3′ end. Established lines of transgenic mice secreted hPTH to milk in concentrations up to 415 ng/ml. Recombinant hPTH recovered from the milk was purified by HPLC and shown to be identical to hPTH standard as analyzed by SDS-PAGE followed by immunoblotting. Expression of the WAPPTHSV2 was limited to the mammary gland as analyzed by polymerase chain reaction (PCR) and Southern blot of reversed transcribed mRNA from different tissues. hPTH is an important bone anabolic hormone and may be a potentially important pharmaceutical for treatment of demineralization disorders such as osteoporosis. We present the transgenic animal as a possible production system for hPTH. © 1995 Wiley-Liss, Inc.     

佐剂CpG-ODN与重组HBsAg疫苗联合免疫乙型肝炎病毒转基因小鼠的免疫应答研究

目的 以CpG-ODN为佐剂与重组HBsAg(rHBsAg)疫苗合用,研究其对乙型肝炎病毒转基因(HBV Tg)小鼠模型的免疫应答效果.方法 40只HBV Tg小鼠随机分为4组,每组小鼠分别注射rHBsAg疫苗(单用rHBsAg组)、rHBsAg疫苗+CpG-ODN(试验组)、rIFNα-2b(IFN组)、生理盐水(对照组).经多次免疫HBV转基因小鼠,于免疫前、后不同时间采血,动态观察各组小鼠血清中HBsAg量、抗-HBs阳性率和HBV DNA的变化,检测肝组织中HBsAg的表达.检测免疫小鼠的外周血T淋巴细胞亚群和白细胞介素2(IL-2)、IL-12(p70)以及γ干扰素(IFN-γ)的含量,分别检测免疫小鼠的脾细胞增殖和细胞毒性T淋巴细胞(CTL)杀伤功能并计算各组小鼠肝组织活性指数(HAI).结果 rHBsAg组和rHBsAg+CpG组在免疫小鼠后2周100%诱导抗-HBs;rHBsAg+CpG组能显著降低血清中的HBsAg量或使HBsAg转阴,rHBsAg+CpG组肝组织中HBsAg的表达量与血清中一样降低,并降低血清中HBV DNA的拷贝数.rHBsAg组的CD3+、CD4+、CD8+细胞在T细胞中所占百分比,IL-2、IL-12(p70)和IFN-γ的含量以及淋巴细胞特异性增殖和杀伤效应均明显高于对照组(P＜0.05).rHBsAg+CpG组与rHBsAg组比较,免疫小鼠产生更强的HBV特异性细胞应答(P＜0.05),且以Th1型细胞免疫应答为主.在rHBsAg+CpG组肝组织中出现大量淋巴细胞,肝脏的HAI在4个组中最高.结论 CpG-ODN作为佐剂可以增强重组HBsAg疫苗诱导HBV转基因小鼠产生抗病毒免疫应答,重组HBsAg疫苗辅以CpG ODN可作为免疫治疗慢性HBV感染的可行性途经.     

Plastid targeting and transient expression of rat liver ATP: citrate lyase in pea protoplasts

Protoplasts isolated from pea leaves (Pisum sativum L. cv. Hurst Greenshaft) were electroporated in the presence of plasmid pDR#1, which contains the rat liver ATP:citrate lyase gene fused to a duplex 35S cauliflower mosaic virus promoter with a transit peptide sequence of the Rubisco small subunit. The level of enzyme expression and viability of protoplasts were both influenced by polyethylene glycol treatment before electroporation. Under the optimised electroporation conditions, an average increase of ATP:citrate lyase activity of 14% was observed in the transfected cells after 24 h, with a similar magnitude of change in the abundance of the corresponding mRNA. Immunoblot analysis confirmed the correct expression and targeting of ATP:citrate lyase protein in the chloroplasts of pea protoplasts. These results provide a basis for the establishment of a procedure for targeting heterologous protein into pea plastids in the presence of a transit peptide. Received: 14 June 1996 / Revision received: 24 November 1996 / Accepted: 4 January 1997     

The 1st step initiation essential for allergen‐specific IgE antibody production upon the 2nd step: Induction of non‐specific IgE+ small B cells containing secondly‐sensitized allergen‐specific ones in mice firstly‐sensitized with an allergen

There was a significant amount of non‐specific, but not of allergen (e.g., papain, mite feces and four kinds of pollen)‐specific, IgE antibodies (Abs) in the sera of normal mice. An i.n. injection of each allergen without adjuvant into mice caused an increase in total IgE Ab titers with a similar time course in the serum. However, the stage of initiation of allergy varied from allergen to allergen. Submandibular lymph node cells from normal mice contained papain‐, but not mite feces‐ or pollen‐specific IgE⁺ cells and an i.n. injection of papain induced papain‐specific IgE Abs in the serum. In contrast, one (i.n.) or two (i.n. and s.c) injections of mite feces induced neither mite feces‐specific IgE⁺ cells in the lymph nodes nor mite feces‐specific IgE Abs in the serum. I.n. sensitization with cedar pollen induced cedar pollen‐specific IgE⁺ small B cells in the lymph nodes on Day 10, when non‐specific IgE Ab titers reached a peak in the serum, implying induction of related allergen‐specific IgE⁺ small cells as well. In fact, a second (s.c.) injection of ragweed (or cedar) pollen into mice sensitized i.n. once with cedar (or ragweed) pollen, but not with mite feces, induced a large amount of ragweed (or cedar) pollen‐specific IgE Abs in the serum. These results indicate that when firstly‐sensitized non‐specific IgE⁺ small B cells in mouse lymph nodes include some secondly‐sensitized allergen‐specific ones, mice produce IgE Abs specific for the secondly‐injected allergen.

     

Production of stable isotope enriched antimicrobial peptides in Escherichia coli: An application to the production of a 15N-enriched fragment of lactoferrin

A method is described for the production of recombinant isotopically enriched peptides in E. coli. Peptides are produced in high yield as fusion proteins with ketosteroid isomerase which form insoluble inclusion bodies. This insoluble form allows easy purification, stabilizes the peptide against degradation and prevents bactericidal activity of the peptide. Cyanogen bromide cleavage released peptide which was conjugated with alkylamines to form lipopeptide. An important advantage of this system is that it allows production of peptides that are toxic to bacteria, which we have demonstrated on a dodecapeptide based on residues 21–31 of human bactericidal protein lactoferrin.     

Two routes for production and purification of Fab fragments in biopharmaceutical discovery research: Papain digestion of mAb and transient expression in mammalian cells

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody–antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1–20 mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS–PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.     

Recombinant protein production by large-scale transient gene expression in mammalian cells: state of the art and future perspectives

The expansion of the biologics pipeline depends on the identification of candidate proteins for clinical trials. Speed is one of the critical issues, and the rapid production of high quality, research-grade material for preclinical studies by transient gene expression (TGE) is addressing this factor in an impressive way: following DNA transfection, the production phase for TGE is usually 2-10 days. Recombinant proteins (r-proteins) produced by TGE can therefore enter the drug development and screening process in a very short time--weeks. With "classical" approaches to protein expression from mammalian cells, it takes months to establish a productive host cell line. This article summarizes efforts in industry and academia to use TGE to produce tens to hundreds of milligrams of r-proteins for either fundamental research or preclinical studies.     

Tie2-Cre transgenic mice: a new model for endothelial cell-lineage analysis in vivo

Endocardial cells are thought to contribute at least in part to the formation of the endocardial cushion mesenchyme. Here, we created Tie2-Cre transgenic mice, in which expression of Cre recombinase is driven by an endothelial-specific promoter/enhancer. To analyze the lineage of Cre expressing cells, we used CAG-CAT-Z transgenic mice, in which expression of lacZ is activated only after Cre-mediated recombination. We detected pan-endothelial expression of the Cre transgene in Tie2-Cre;CAG-CAT-Z double-transgenic mice. This expression pattern is almost identical to Tie2-lacZ transgenic mice. However, interestingly, we observed strong and uniform lacZ expression in mesenchymal cells of the atrioventricular canal of Tie2-Cre;CAG-CAT-Z double-transgenic mice. We also detected lacZ expression in the mesenchymal cells in part of the proximal cardiac outflow tract, but not in the mesenchymal cells of the distal outflow tract and branchial arch arteries. LacZ staining in Tie2-Cre;CAG-CAT-Z embryos is consistent with endocardial-mesenchymal transformation in the atrioventricular canal and outflow tract regions. Our observations are consistent with previously reported results from Cx43-lacZ, Wnt1-Cre;R26R, and Pax3-Cre;R26R transgenic mice, in which lacZ expression in the cardiac outflow tract identified contributions in part from the cardiac neural crest. Tie2-Cre transgenic mice are a new genetic tool for the analyses of endothelial cell-lineage and endothelial cell-specific gene targeting.     

Cholesterol accumulation and diabetes in pancreatic beta-cell-specific SREBP-2 transgenic mice: a new model for lipotoxicity

To determine the role of cholesterol synthesis in pancreatic beta-cells, a transgenic model of in vivo activation of sterol-regulatory element binding protein 2 (SREBP-2) specifically in beta-cells (TgRIP-SREBP-2) was developed and analyzed. Expression of nuclear human SREBP-2 in beta-cells resulted in severe diabetes as evidenced by greater than 5-fold elevations in glycohemoglobin compared with C57BL/6 controls. Diabetes in TgRIP-SREBP-2 mice was primarily due to defects in glucose- and potassium-stimulated insulin secretion as determined by glucose tolerance test. Isolated islets of TgSREBP-2 mice were fewer in number, smaller, deformed, and had decreased insulin content. SREBP-2-expressing islets also contained increased esterified cholesterol and unchanged triglycerides with reduced ATP levels. Consistently, these islets exhibited elevated expression of HMG-CoA synthase and reductase and LDL receptor, with suppression of endogenous SREBPs. Genes involved in beta-cell differentiation, such as PDX1 and BETA2, were suppressed, explaining loss of beta-cell mass, whereas IRS2 expression was not affected. These phenotypes were dependent on the transgene expression. Taken together, these results indicate that activation of SREBP-2 in beta-cells caused severe diabetes by loss of beta-cell mass with accumulation of cholesterol, providing a new lipotoxic model and a potential link of disturbed cholesterol metabolism to impairment of beta-cell function.     

Expression and assembly of largest foreign protein in chloroplasts: oral delivery of human FVIII made in lettuce chloroplasts robustly suppresses inhibitor formation in haemophilia A mice

Inhibitor formation is a serious complication of factor VIII (FVIII) replacement therapy for the X‐linked bleeding disorder haemophilia A and occurs in 20%–30% of patients. No prophylactic tolerance protocol currently exists. Although we reported oral tolerance induction using FVIII domains expressed in tobacco chloroplasts, significant challenges in clinical advancement include expression of the full‐length CTB‐FVIII sequence to cover the entire patient population, regardless of individual CD4⁺ T‐cell epitope responses. Codon optimization of FVIII heavy chain (HC) and light chain (LC) increased expression 15‐ to 42‐fold higher than the native human genes. Homoplasmic lettuce lines expressed CTB fusion proteins of FVIII‐HC (99.3 kDa), LC (91.8 kDa), C2 (31 kDa) or single chain (SC, 178.2 kDa) up to 3622, 263, 3321 and 852 μg/g in lyophilized plant cells, when grown in a cGMP hydroponic facility (Fraunhofer). CTB‐FVIII‐SC is the largest foreign protein expressed in chloroplasts; despite a large pentamer size (891 kDa), assembly, folding and disulphide bonds were maintained upon lyophilization and long‐term storage as revealed by GM1‐ganglioside receptor binding assays. Repeated oral gavages (twice/week for 2 months) of CTB‐FVIII‐HC/CTB‐FVIII‐LC reduced inhibitor titres ~10‐fold (average 44 BU/mL to 4.7 BU/mL) in haemophilia A mice. Most importantly, increase in the frequency of circulating LAP‐expressing CD4⁺CD25⁺FoxP3⁺ Treg in tolerized mice could be used as an important cellular biomarker in human clinical trials for plant‐based oral tolerance induction. In conclusion, this study reports the first clinical candidate for oral tolerance induction that is urgently needed to protect haemophilia A patients receiving FVIII injections.     

Recombinant human bile salt-stimulated lipase: an example of defectiveO-glycosylation of a protein produced in milk of transgenic mice

The expression of recombinant human bile salt-stimulated lipase (bssl) was targeted to the lactating mammary gland of transgenic mice. Expression of recombinant genes comprisingbssl cDNA, or alternatively genomicbssl DNA, under control of regulatory elements derived from the murine whey acidic protein (wap) gene was achieved and evaluated. Constructs containing genomicbssl sequences mediated high levels (0.5–1, mg ml^–1) of recombinant human BSSL in the milk. The recombinant BSSL produced was purified, biochemically characterized and compared to native BSSL and recombinant BSSL produced in mouse C127 and hamster CHO cells. Recombinant BSSL derived from transgenic mice showed a different migration and distribution after SDS-PAGE electrophoresis, lower apparent molecular mass on size-exclusion chromatography and no detectable interactions with a panel of lectins. These results indicate a significantly lower degree ofO-glycosylation of recombinant BSSL in milk from transgenic mice than was found for the native enzyme or recombinant CHO- or C127 cell-produced BSSL. Despite these differences, mouse-milk-derived recombinant BSSL exhibited similar lipase activity, the same, stability to low pH and similar sensitivity to elevated temperatures as the native enzyme. The observation that mouse-C127-cell-produced recombinant BSSL is heavilyO-glycosylated makes species-related restrictions less attractive as an explanation for the reducedO-glycosylation.     

Proteomic analysis of protein expression and oxidative modification in r6/2 transgenic mice: a model of Huntington disease

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by motor, psychiatric, and cognitive symptoms. The genetic defect responsible for the onset of the disease, expansion of CAG repeats in exon 1 of the gene that codes for huntingtin on chromosome 4, has been unambiguously identified. On the other hand, the mechanisms by which the mutation causes the disease are not completely understood yet. However, defects in energy metabolism of affected cells may cause oxidative damage, which has been proposed as one of the underlying molecular mechanisms that participate in the etiology of the disease. In our effort to investigate the extent of oxidative damage occurring at the protein level, we used a parallel proteomic approach to identify proteins potentially involved in processes upstream or downstream of the disease-causing huntingtin in a well established HD mouse model (R6/2 transgenic mice). We have demonstrated that the expression levels of dihydrolipoamide S-succinyltransferase and aspartate aminotransferase increase consistently over the course of disease (10-week-old mice). In contrast, pyruvate dehydrogenase expression levels were found to be decreased in 10-week-old HD transgenic mice compared with young (4-week-old) mice. Our experimental approach also led to the identification of oxidatively modified proteins. Six proteins were found to be significantly oxidized in old R6/2 transgenic mice compared with either young transgenic mice or non-transgenic mice. These proteins are alpha-enolase, gamma-enolase (neuron-specific enolase), aconitase, the voltage-dependent anion channel 1, heat shock protein 90, and creatine kinase. Because oxidative damage has proved to play an important role in the pathogenesis and the progression of Huntington disease, our results for the first time identify specific oxidatively modified proteins that potentially contribute to the pathogenesis of Huntington disease.     

Brain response to traumatic brain injury in wild-type and interleukin-6 knockout mice: a microarray analysis

Traumatic injury to the brain is one of the leading causes of injury-related death or disability. Brain response to injury is orchestrated by cytokines, such as interleukin (IL)-6, but the full repertoire of responses involved is not well known. We here report the results obtained with microarrays in wild-type and IL-6 knockout mice subjected to a cryolesion of the somatosensorial cortex and killed at 0, 1, 4, 8 and 16 days post-lesion. Overall gene expression was analyzed by using Affymetrix genechips/oligonucleotide arrays with approximately 12,400 probe sets corresponding to approximately 10,000 different murine genes (MG_U74Av2). A robust, conventional statistical method (two-way anova) was employed to select the genes significantly affected. An orderly pattern of gene responses was clearly detected, with genes being up- or down-regulated at specific timings consistent with the processes involved in the initial tissue injury and later regeneration of the parenchyma. IL-6 deficiency showed a dramatic effect in the expression of many genes, especially in the 1 day post-lesion timing, which presumably underlies the poor capacity of IL-6 knockout mice to cope with brain damage. The results highlight the importance of IL-6 controlling the response of the brain to injury as well as the suitability of microarrays for identifying specific targets worthy of further study.