Interleukin-10 determines viral clearance or persistence in vivo

Persistent viral infections are a major health concern. One obstacle inhibiting the clearance of persistent infections is functional inactivation of antiviral T cells. Although such immunosuppression occurs rapidly after infection, the mechanisms that induce the loss of T-cell activity and promote viral persistence are unknown. Herein we document that persistent viral infection in mice results in a significant upregulation of interleukin (IL)-10 by antigen-presenting cells, leading to impaired T-cell responses. Genetic removal of Il10 resulted in the maintenance of robust effector T-cell responses, the rapid elimination of virus and the development of antiviral memory T-cell responses. Therapeutic administration of an antibody that blocks the IL-10 receptor restored T-cell function and eliminated viral infection. Thus, we identify a single molecule that directly induces immunosuppression leading to viral persistence and demonstrate that a therapy to neutralize IL-10 results in T-cell recovery and the prevention of viral persistence.     

The impact of AUG start codon context on maize gene expression in vivo

Summary In mammals, the sequence context surrounding an AUG start codon can alter the efficiency at which translation is initiated. Less is known about the AUG context requirements for translation initiation in plants. Using a maize transient assay, we present evidence that the naturally occurring AUG start codon of the Alcohol dehydrogenase-1 is efficiently used in vivo. We have also tested the effects of upstream, out-of-frame AUGs on the translation of firefly luciferase reporter gene mRNAs. The presence of an upstream out-of-frame AUG, even when surrounded by a poor context, eliminated most luciferase expression, suggesting efficient translation initiation at the upstream AUG. The relaxed requirements for AUG context in maize suggest that plants and mammals may differ in their requirements for efficient translation initiation.     

Conversion of a mitochondrial gene for mammalian cytochrome c oxidase subunit II into its universal codon equivalent and expression in vivo and in vitro.

To begin to assess the independent structural and functional characteristics of the mitochondrially encoded subunits of mammalian cytochrome c oxidase, we have converted the cloned mitochondrial gene for rat subunit II (coxII) into its universal codon equivalent (ucoxII) by oligonucleotide-directed, site-specific mutagenesis. This involved synthesizing 12 oligodeoxynucleotides to achieve the 13 ATA to ATG and the 5 TGA to TGG changes needed. To express ucoxII in Escherichia coli, we used a number of different expression vectors in which the promoters and ribosome-binding sequences of the messenger RNA were varied. While ucoxII alone was expressed at a low level, a striking increase in the level of expression resulted when the ucoxII gene was fused to other E. coli genes. The COXII peptide was identified by proteolytic digestion, partial sequencing, and reaction with specific antisera. A cro-beta-galactosidase-COXII fusion protein has been purified, characterized, and used to produce polyclonal antibodies to the COXII peptide. The ucoxII gene was also expressed in a cell-free translation system and in Xenopus oocytes, yielding a nondenatured, membrane-associated peptide with the same apparent molecular weight as authentic subunit II. In oocytes and in a reticulocyte lysate in vitro system supplemented with microsomal membranes, the protein is glycosylated and coisolates with the washed membrane fraction. In both cases, the COXII peptide is soluble under mild conditions in a nonionic detergent and is precipitable by antibodies to subunit II. The production of subunit II in the in vitro translation system is stimulated as strongly by addition of soybean phospholipid vesicles as by microsomal membranes, providing further evidence of membrane insertion and stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lipopolysaccharide increases resistin gene expression in vivo and in vitro

Although resistin has been thought to be an important link between obesity and diabetes, recent results do not support this hypothesis. We speculated that resistin may be involved in inflammatory processes and be induced by inflammatory stimuli. In this study, we tested whether lipopolysaccharide (LPS) induced resistin expression in rats. The results show that resistin mRNA levels in white adipose tissue and white blood cells were increased by LPS treatment. LPS also increased resistin mRNA levels in 3T3-L1 adipocytes and human peripheral blood monocytes. The results suggest that resistin is involved in insulin resistance and probably in other inflammatory responses.     

Gene expression,nucleotide composition and codon usage bias of genes associated with human Y chromosome

Analysis of codon usage pattern is important to understand the genetic and evolutionary characteristics of genomes. We have used bioinformatic approaches to analyze the codon usage bias (CUB) of the genes located in human Y chromosome. Codon bias index (CBI) indicated that the overall extent of codon usage bias was low. The relative synonymous codon usage (RSCU) analysis suggested that approximately half of the codons out of 59 synonymous codons were most frequently used, and possessed a T or G at the third codon position. The codon usage pattern was different in different genes as revealed from correspondence analysis (COA). A significant correlation between effective number of codons (ENC) and various GC contents suggests that both mutation pressure and natural selection affect the codon usage pattern of genes located in human Y chromosome. In addition, Y-linked genes have significant difference in GC contents at the second and third codon positions, expression level, and codon usage pattern of some codons like the SPANX genes in X chromosome.     

Differentiation-dependent expression of lectin binding sites on normal and neoplastic keratinocytes in vivo and in vitro.

We used lectins as probes to demonstrate the composition of membrane carbohydrates of canine keratinocytes in various functional stages and various degrees of differentiation. Keratinocytes during normal epidermal turnover were compared by lectin immunohistochemistry to cells of hyperplastic epidermis and neoplastic keratinocytes. Three types of epidermal tumors and oral squamous cell carcinomas were examined. In addition, two in vitro tissue culture systems for keratinocytes were studied and compared with in vivo epithelium. In normal skin, PNA reacted only weakly with basal cells, whereas in hyperplastic skin basal cells bound this lectin strongly, demonstrating increasing expression of PNA binding sites with increasing thickness of the stratified squamous epithelium. ConA bound to basal cell tumors only. In oral squamous cell carcinomas, the expression of distinct lectin binding sites correlated with certain histological growth patterns, e.g., UEA-I reacted with highly invasive tumors but not with tumors showing a solid growth pattern. Using cell surface iodination and polyacrylamide gel electrophoresis, distinct differences in cell membrane protein expression were demonstrated between normal and neoplastic keratinocytes. SDS-polyacrylamide gel electrophoresis of cultured normal and neoplastic keratinocytes revealed several cell surface proteins that are specific for either cell type. Neoplastic cells specifically express a 140 KD lectin binding cell surface glycoprotein. The results of this study show that lectin binding patterns of keratinocytes are dependent on the functional state and the degree of differentiation of the cells and demonstrate correlation of some histological growth patterns with distinct lectin binding phenotypes, suggesting association of expression of cell membrane carbohydrate moieties with growth patterns. In addition, close similarities between "lifted cultures" grown at the air-liquid interface and native tissue demonstrate the value of this culture system as a model for differentiated stratified squamous epithelium.     

High-level expression and immunogenicity of a porcine circovirus type 2 capsid protein through codon optimization in Pichia pastoris

The porcine circovirus type 2 (PCV2) capsid protein (Cap) is an important antigen for the development of vaccines. To achieve high-level expression of recombinant PCV2 Cap in Pichia pastoris, the wild-type Cap (wt-Cap) and optimized Cap (opti-Cap) gene fragments encoding the same amino acid sequence of PCV2 were amplified by PCR using DNA from lymph nodes of postweaning multisystemic wasting syndrome-suffered pigs and synthesized based on the codon bias of the methylotrophic yeast P. pastoris, respectively. The wt-Cap and opti-Cap gene fragments were inserted into the site between EcoRI and NotI sites in pPIC9K, which was under the control of the alcohol oxidase 1 (AOX1) promoter and α-mating factor signal sequence from Saccharomyces cerevisiae. The recombinant plasmids, designated as pPIC9K-wt-Cap and pPIC9K-opti-Cap, were linearized using SacI and transformed into P. pastoris GS115 by electroporation. The expressed intracellular soluble opti-Cap reached 174 μg/mL without concentration in a shake flask and kept good reactivity to PCV2-specific positive sera, whereas the wt-Cap could not be detectable throughout three times electroporation. Strong specific PCV2-Cap antibodies were elicited from piglets immunized with vaccine based on opti-Cap. To the best of our knowledge, the achieved opti-Cap yield is the highest ever reported. Our results demonstrated that codon optimization play an important role on the high-level expression of a codon-optimized PCV2-Cap gene in P. pastoris, and the vaccine based on opti-Cap may be a potential subunit vaccine candidate.     

Expression of the cauliflower mosaic virus capsid gene in vivo

Antisera against the N-terminal and C-terminal parts of the potential ORF IV product were used to analyse extracts from CaMV-infected turnip leaves by immunoblotting. Polypeptides of 87, 83, 82, 60 and 57 kDa were detected. The origin of these proteins is discussed.     

Cyclosporin a modulation of tumor necrosis factor gene expression and effects in vitro and in vivo

We investigated, in vitro and in vivo, the cyclosporin A (CsA) regulation of LPS-induced TNF gene expression and subsequent pathophysiologic changes. In vitro dose-response kinetics data showed that CsA inhibited TNF bioactivity in the supernatant without delaying its production, whereas Northern blot and in situ hybridization analysis demonstrated that CsA did not inhibit TNF mRNA expression. We then sought to examine the in vivo effects of CsA (75 mg/kg) in CBA/J mice that were primed with CFA, and injected 2 wk later with LPS. CsA demonstrated suppression of local levels (ascites) of TNF as measured by either bioactivity or an anti-murine TNF ELISA. However, CsA did not decrease mRNA for TNF, or cell-associated TNF. In vivo kinetics studies were performed to show that CsA blocked both local (ascites) and systemic (plasma) LPS-induced TNF production without delaying these effects. CsA inhibited the neutrophilia and lymphopenia that developed after the LPS challenge, but did not block the lung neutrophilic infiltrate. These observations are helpful in understanding the role of the macrophage in CsA immunosuppression, particularly with regard to the ability of CsA to block LPS-induced TNF secretion.     

In vivo and in vitro inhibition of JE gene expression by glucocorticoids

Glucocorticoids are potent anti-inflammatory agents which affect cell growth and migration in a wide variety of systems and have profound effects on monocytes, decreasing their circulating number as well as inhibiting their accumulation at sites of inflammation and injury. Although the mechanisms by which glucocorticoids regulate gene induction have been established, the mechanisms by which they inhibit inflammation or cell growth and migration have yet to been determined. JE is one of the most abundant genes induced by platelet-derived growth factor (PDGF) in vitro and is also induced in vivo in response to ischemia or injury. JE encodes a low molecular weight glycoprotein that functions in part as a monocyte chemotactic factor and thus may be important in recruiting monocytes to sites of tissue injury and/or inflammation. We report that glucocorticoids block the induction of JE mRNA by serum or PDGF in cultured vascular smooth muscle cells. The effect of glucocorticoids appears largely due to destabilization of JE mRNA and has specificity for JE, in that other "early" PDGF-inducible genes are not inhibited by glucocorticoids. The effect of glucocorticoids also occurs in vivo: methyl prednisolone blocks the constitutive expression and inhibits the ischemia-induced elevation of JE mRNA levels in rat kidneys. The inhibition of JE mRNA accumulation by glucocorticoids may be related to the anti-inflammatory effects of these agents and defines JE as a member of what may be a group of PDGF-inducible genes that are responsive to corticosteroids.     

A型流感病毒NS1基因密码子去优化改造引起病毒毒力减弱

根据A型流感病毒密码子使用偏嗜性,选取稀有密码子对A/Puerto Rico/8/34(H1N1)病毒NS1基因内部110个氨基酸区域进行密码子同义突变改造,并全基因合成NS基因,利用反向遗传操作技术拯救出含有密码子去优化NS1基因的重组病毒(deoNS)。体外细胞噬斑形成实验和病毒生长曲线证明该病毒在MDCK细胞内的感染和复制能力比野生型病毒低约1000倍;BALB/c小鼠体内致病力实验证明deoNS病毒不能引起小鼠发病和死亡,该病毒在小鼠肺内的复制滴度比野生型病毒低100～1000倍。本研究探索了通过基因组密码子去优化改造途径降低A型流感病毒毒力的可行性,首次证明流感病毒NS1基因密码子去优化同义突变可以降低病毒毒力,为流感减毒活疫苗的研究提供了新的思路。     

Enhanced expression of PCV2 capsid protein in Escherichia coli and Lactococcus lactis by codon optimization

Capsid protein (Cap) of porcine circovirus type 2 (PCV2) encoded by orf2 is a main structural protein with strong immunoreactivity. However, capsid protein is expressed poorly in prokaryotic organisms because of differences in codon usage. In this study, we introduce 24 synonymous mutations into orf2 by mutagenic primers and overlap extension polymerase chain reaction (OE-PCR) technique. Fourteen rare codons of orf2 were replaced with preferable codons used in Escherichia coli cells. Moreover, the nuclear localization signal (NLS) region rich in rare codon clusters at the 5′ end was deleted. The codon-optimized genes demonstrated higher levels of expression compared with wild-type genes. The influence of rare codons on the gene expression was eliminated by mutation. Western blot analysis confirmed the immunoreactivity of the proteins expressed by mutated genes. Further testing demonstrated that the mutated genes were also expressed successfully in Lactococcus lactis NZ9000. The immunologically active Cap proteins produced by recombinant strains have the potential applications for serological diagnostic assays and vaccine development against PCV2-associated diseases.     

Human keratinocytes maintain reversible anti-apoptotic defenses in vivo and in vitro

Human keratinocytes proliferate and differentiate in an epidermal environment where induction of apoptosis can be triggered by ultraviolet radiation (UVR), activated lymphocytes and cytokines. The purpose of this study was to determine whether keratinocytes were susceptible to apoptosis induced by ionophore, ultra-violet radiation, cytokines or crosslinking of CD95 (Fas/APO-1). In normal human skin exposed to two minimal erythema doses of ultraviolet radiation, suprabasal cells were the first keratinocytes to demonstrate apoptotic nuclei, and by 48 h apoptotic cells were identified throughout the mid to upper epidermis. However, most keratinocytes resisted apoptosis and UVR-induced apoptosis was not observed in basal cells, or in the most differentiated epidermis. Human keratinocytes and keratinocyte cell lines cultured in vitro developed maximal apoptosis 48 h after radiation. Human keratinocytes cultured in full growth factor supplements were resistant to UVR-induced apoptosis compared to keratinocyte cell lines or to a lymphoid cell line (HL60) susceptible to apoptosis. Keratinocyte cell lines were completely resistant to apoptosis induced by interferon-, interferon-, IL-2, IL-6, TNF-, IL-1Ra, and GM-CSF. A subset of the cells in cultures of keratinocytes and transformed keratinocyte cell lines died by apoptosis in response to anti-Fas, IL-1 and TNF- plus IFN- and ionophore. Second passage freshly isolated human keratinocytes were much more resistant to apoptosis induced by ionophore, anti-Fas and cytokines than were transformed keratinocyte cell lines. Calcium shift to induce differentiation in second-passage keratinocyte cultures made keratino-cytes even more resistant to UVR-induced apoptosis. This parallels the lack of UVR-induced apoptosis observed in the most differentiated keratinocytes in irradiated human skin. Both keratinocytes and kerati-nocyte cell lines express rather low levels of the anti-apoptotic proteins bcl-2 and bcl-x compared to other apoptosis-resistant cell types. The differences between keratinocytes and keratinocyte cell lines in suscepti-bility to apoptosis are not explained by difference in expression of bcl-2 or bcl-x. Finally, withdrawal of growth factors from keratinocytes decreased cell survival following UVR and increased the induction of apoptosis. Inhibition of protein synthesis with cyclo-heximide also made keratinocytes more susceptible to UVR-induced apoptosis, indicating that anti-apop-totic defences in cultured keratinocytes are dependent on active protein synthesis. These experiments show that the strong keratinocyte defences against apoptosis are stratified within the epidermis, and can be altered by differentiation and growth factor withdrawal.     

Comparison of gene expression during in vivo and in vitro postnatal retina development

Retina explants are widely used as a model of neural development. To define the molecular basis of differences between the development of retina in vivo and in vitro during the early postnatal period, we carried out a series of microarray comparisons using mouse retinas. About 75% of 8,880 expressed genes from retina explants kept the same expression volume and pattern as the retina in vivo. Fewer than 6% of the total gene population was changed at two consecutive time points, and only about 1% genes showed more than a threefold change at any time point studied. Functional Gene Ontology (GO) mapping for both changed and unchanged genes showed similar distribution patterns, except that more genes were changed in the GO clusters of response to stimuli and carbohydrate metabolism. Three distinct expression patterns of genes preferentially expressed in rod photoreceptors were observed in the retina explants. Some genes showed a lag in increased expression, some showed no change, and some continued to have a reduced level of expression. An early downregulation of cyclin D1 in the explanted retina might explain the reduction in numbers of precursors in explanted retina and suggests that external factors are required for maintenance of cyclin D1. The global view of gene profiles presented in this study will help define the molecular changes in retina explants over time and will provide criteria to define future changes that improve this model system.     

Erythropoietin gene expression in vitro and in vivo detected by in situ hybridization

Macrophages derived from unstimulated and unseparated mouse bone marrow cells have been shown to release erythropoietin into the extracellular fluid. Additional proof that macrophages can produce the hormone would be a demonstration that the gene is expressed and the mature protein released. In situ hybridization using a 1.2 kb biotinylated erythropoietin DNA probe demonstrates that both cultured macrophages and those present in normal mouse bone marrow express the gene. These results are discussed in terms of the role played by the macrophage in the hemopoietic cellular microenvironment and indicate that a subpopulation is responsible for this function and that cell interactions play an important role in hemopoietic differentiation.