Virion polypeptide composition of the human papovavirus BK: comparison with simian virus 40 and polyoma virus.

The polypeptide composition of labeled BK virus was compared with that of simian virus 40 (SV40) and polyoma virus by co-electrophoresis of disrupted virions in polyacrylamide gels containing approximately 73% of the capsid protein and had a molecular weight of 39,000. It was smaller than VP1 of SV40 and polyoma virus. The other polypeptides of BK virus were similar in molecular weight to those of SV40. A comparison of the proteins of BK virus and SV40 iodinated with chloramine T before and after disruption in alkaline buffer at pH 10.5 revealed differences between the two viruses in the number and distribution of tyrosines available for iodination. The tryptic peptides of VP1, VP3, VP4, and VP5 combined of SV40 were compared with those of the same polypeptides of BK virus. Among the 19 peptides of VP1 resolved, only two were common to both viruses. The analyses of VP4 and VP5, the histone-like proteins, however, showed more similarity between the viruses, with 6 of 15 resolved peptides in common. The tryptic digests of VP3 were completely different.     

Genomic structure of human polyoma virus JC: nucleotide sequence of the region containing replication origin and small-T-antigen gene

The nucleotide sequence of the region of human polyoma virus JC DNA between 0.5 and 0.7 map units from a unique EcoRI cleavage site was determined and compared with those of the corresponding regions of another human polyoma virus, BK, and simian virus 40 DNAs. Within this region consisting of 945 base pairs, we located the origin of DNA replication near 0.7 map units, the entire coding region for small T antigen, and the splice junctions for large-T-antigen mRNA. The deduced amino acid sequences for small T antigen and the part of large T antigen markedly resembled those of polyoma virus BK and simian virus 40. The results strongly suggest that polyoma virus JC has the same organization of early genome as polyoma virus BK and simian virus 40 on the physical map, with the EcoRI site as a reference point.     

An SopB-mediated immune escape mechanism of Salmonella enterica can be subverted to optimize the performance of live attenuated vaccine carrier strains

Salmonellae have evolved several mechanisms to evade host clearance. Here, we describe the influence on bacterial immune escape of the effector protein SopB, which is translocated into the cytosol through a type III secretion system. Wild-type bacteria, as well as the sseC and aroA attenuated mutants exerted a stronger cytotoxic effect on dendritic cells (DC) than their SopB-deficient derivatives. Cells infected with the double sseC sopB, phoP sopB and aroA sopB mutants also exhibited higher expression of MHC, CD80, CD86 and CD54 molecules, and showed a stronger capacity to process and present an I-E(d)-restricted epitope from the influenza hemagglutinin (HA) to CD4+ cells from TCR-HA transgenic mice in vitro. The incorporation of an additional mutation into the sopB locus of the attenuated sseC, phoP and aroA mutants resulted in the stimulation of improved humoral and cellular immune responses following oral vaccination. The obtained results define a new potential immune escape strategy of this important pathogen, and also demonstrate that this mechanism can be subverted to optimize the immune responses elicited using Salmonella as a live vaccine carrier.     

LncRNA SNHG1 regulates immune escape of renal cell carcinoma by targeting miR-129-3p to activate STAT3 and PD-L1

Immune escape of renal cell carcinoma (RCC) impacts patient survival. However, the molecular mechanism of long noncoding RNA (lncRNA) small nucleolar RNA host gene 1 (SNHG1) in RCC immune escape remains unclear. Quantitative real-time PCR and western blotting results revealed that the expression of lncRNA SNHG1 and STAT3 were upregulated in RCC tissues and cells and that the expression of miR-129-3p was downregulated. Enzyme-linked immunosorbent assay results revealed the increased levels of immune-related factors (interferon-γ, tumour necrosis factor α, and interleukin-2) in RCC tissues. SNHG1 knockdown or miR-129-3p overexpression inhibited the proliferation and invasion of A498 and 786-O cells, while the proliferation and cytotoxicity of CD8⁺ T cells increased, which promoted the secretion of immune-related factors. STAT3 overexpression decreased the protective effect of miR-129-3p overexpression on RCC cell immune escape. In addition, miR-129-3p knockdown and STAT3 overexpression decreased the protective effect of lncRNA SNHG1 knockdown on RCC cell immune escape. In addition, PD-L1 expression was downregulated after lncRNA SNHG1 knockdown but upregulated after miR-129-3p knockdown and STAT3 overexpression. Dual-luciferase assays showed that lncRNA SNHG1 targets miR-129-3p, and miR-129-3p targets STAT3. RNA pull-down and RNA immunoprecipitation assays verified the regulatory relationship between SNHG1 and STAT3. In vivo, shSNHG1 prolonged the overall survival of RCC tumour model mice and inhibited RCC tumour growth and immune escape but increased CD8⁺ T cell infiltration in mice. Our findings provide an experimental basis for elucidating the molecular mechanisms of immune escape by RCC and reveal a novel target to treat this disease.     

Toll-like receptor, RIG-I-like receptors and the NLRP3 inflammasome: key modulators of innate immune responses to double-stranded RNA viruses

Double-stranded RNA (dsRNA), the genetic material for many RNA viruses, induces robust host immune responses via pattern recognition receptors, which include Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I-like receptors (RLRs) and the multi-protein NLRP3 inflammasome complex. The engagement of dsRNA receptors or inflammasome activation by viral dsRNA initiates complex intracellular signaling cascades that play essential roles in inflammation and innate immune responses, as well as the resultant development of adaptive immunity. This review focuses on signaling pathways mediated by TLR3, RLRs and the NLRP3 inflammasome, as well as the potential use of agonists and antagonists that target these pathways to treat disease.     

Characterization of the type and group specificities of the immune response in mice to murine leukemia viruses.

Normal sera from a variety of strains of inbred mice have precipitating antibodies to murine type C viruses that are detected by radioimmune precipitation assays. The results demonostrate that this humoral immune response is primarily directed against the AKR strain of murine leukemia virus (MuLV) proteins gp71, gp43, and p15(E). These sera also react with Friend- or Rauscher-MuLV in radioimmune precipitation assays. This reaction is not due to a separate immune response, but rather is primarily a consequence of the cross-reactivity of antibodies to the AKR strain of MuLV p15(E) with the p15(E) of these viruses. These data, using autogenous immune sera, emphasize the serological differences of the virion glycoproteins and the serological similarity of the p15(E) virion component of the viruses. Furthermore, based on the serological reactivities to the glycoproteins, the results suggest that the AKR strain of MuLV is endogenous to and expressed in mice, but that the Friend-Moloney-Rauscher virus group is not.     

Flt3配体胞外域的原核表达纯化及活性鉴定

目的:构建pET32a(+)-hFLext原核表达载体,诱导hFLext蛋白表达、纯化及活性鉴定.方法:以人淋巴细胞cDNA文库为模板,克隆hFlext,构建pET32a(+)-hFLext重组表达载体.转化大肠杆菌BL21,IPTG诱导蛋白表达,镍珠亲合层析纯化蛋白,SDS-PAGE及Western blot鉴定.细胞增殖实验检测其生物学活性.结果:成功克隆获得hFLext,并构建了pET32a(+)-hFLext重组表达载体.在大肠杆菌BL21,经1 mM IPTG 30℃诱导12 h,成功表达Trx-hFLext融合蛋白,主要以包涵体形式存在.经8M尿素变性包涵体蛋白,逐步透析复性,镍珠亲合层析纯化蛋白,SDS-PAGE及Western blot鉴定,成功获得高纯度的Trx-hFLext融合蛋白.细胞增殖实验证实其具有生物学活性,能够有效刺激脐血细胞增殖.结论:成功构建了pET32a(+)-hFLext重组表达载体,表达、纯化了具有生物学活性的Trx-hFLext融合蛋白,为造血干/祖细胞的体外扩增研究奠定了基础.     

Characterization of the local immune response to cyst antigens during the acute and elimination phases of primary murine giardiasis

During the course of a giardial infection, the host's immune system is presented with a variety of Giardia antigens as trophozoites differentiate, through encysting cells, to form the infective cysts. Previous studies examining the host's immune response during giardial infections have focused on trophozoite-derived antigens (Ags). In this study, we were interested to determine if the host's immune system reacts to cyst Ags during the acute and elimination phases, when there is cyst shedding. For this purpose, we used antigenic extracts from trophozoites (Troph), encysting cells (ENC), and purified giardial cyst walls (PCW), as well as purified recombinant cyst wall protein 2 (rCWP2). Comparative analysis of the parasite extracts using SDS-PAGE analysis and surface-enhanced laser desorption/ionization time of flight mass spectrometry resulted in the detection of 175 protein entities, of which 26 were Troph-specific proteins, 17 ENC-specific proteins, and 31 were PCW-specific proteins. On the other hand, we detected 34 proteins shared between Troph and ENC, 19 proteins that were shared between ENC and PCW, and 29 proteins that were common to Troph and PCW. Finally, we detected 19 proteins that were shared by all three extract samples. BALB/c mice were infected with 10(5)Giardia muris cysts and sacrificed either at the acute or elimination phases of infection (days 12 and 40, respectively), and lymphocytes were isolated from the Peyer's patches (PP). Using flow cytometry, we detected significant increases in the number of PP-derived CD4(+) and CD19(+), but not CD8(+) lymphocytes. Quantification of the number of mucosal IL-4 and IFN-gamma secreting T-lymphocytes by enzyme-linked immunosorbent spot assay showed that these cells reacted by secreting similar levels of IL-4 and IFN-gamma, regardless of the Ag or the phase of infection. Analysis of intestinal humoral immune responses by ELISA resulted in the detection of Ag-specific IgA and IgG intestinal antibodies. Regardless of the Ag tested, a trend was consistently observed where the concentration of local antibodies was found to be slightly increased by the acute phase, where we detected approximately 200microg/mg of specific IgA and approximately 300ng/ml of specific IgG in intestinal lavage of infected mice. By the elimination phase, the amount of specific antibodies was found to increase to approximately 600microg/mg of specific IgA and approximately 1300ng/ml of specific IgG antibodies. Finally, we tested the biological activity of these antibodies and found that they were able to reduce the ability of trophozoites to differentiate into cysts in vitro. Collectively, we believe these results demonstrate for the first time the existence of significant cellular and humoral immune responses against Giardia cyst Ags that may contribute to the reduction of cyst shedding in infected animals.     

An Agent-Based Model to study the epidemiological and evolutionary dynamics of Influenza viruses

Background  

Influenza A viruses exhibit complex epidemiological patterns in a number of mammalian and avian hosts. Understanding transmission of these viruses necessitates taking into account their evolution, which represents a challenge for developing mathematical models. This is because the phrasing of multi-strain systems in terms of traditional compartmental ODE models either requires simplifying assumptions to be made that overlook important evolutionary processes, or leads to complex dynamical systems that are too cumbersome to analyse.     

An antigenic determinant common to human chorionic somatomammotropin and human growth hormone revealed by limited proteolysis of immune complexes.

An epitope of human chorionic somatomammotropin for one of the monoclonal antibodies raised against the whole antigen has been identified. We compared the release of peptides from limited proteolysis of the antigen in the presence and absence of the related antibody. Using enzymes of different specificity, we could determine the amino acid sequence that can be considered at least inclusive of the epitope. The monoclonal antibody selected is 100% cross-reactive with human growth hormone, so the antigenic determinant identified is shared by the two protein hormones.     

Genetic 'budget' of viruses and the cost to the infected host: a theory on the relationship between the genetic capacity of viruses, immune evasion, persistence and disease

The nature of the pathogen-host relationship is recognized as being a dynamic coevolutionary process where the immune system has required ongoing adaptation and improvement to combat infection. Under survival pressure from sophisticated immune responses, adaptive processes for microbes, including viruses, have manifested as immune evasion strategies. This paper proposes a theory that virus immune evasion can be broadly classified into 'acquisition' or 'erroneous replication' strategies. Acquisition strategies are characteristic of large genome dsDNA viruses, which (i) replicate in the cell nucleus; (ii) have acquired host genes that can be used to directly manipulate responses to infection; (iii) are often latent for the lifetime of the host; and (iv) have little or no serious impact on health. Alternatively, erroneous replication strategies are characteristic of small genome RNA viruses, which are recognized as being the cause of many serious diseases in humans. It is proposed that this propensity for disease is due to the cytoplasmic site of replication and truncated temporal relationship with the host, which has limited or removed the evolutionary opportunity for RNA viruses to have acquired host genes. This has resulted in RNA viruses relying on error-prone replication strategies which, while allowing survival and persistence, are more likely to lead to disease due to the lack of direct viral control over potentially host-deleterious inflammatory and immune responses to infection.     

MTSEA prevents ligand binding to the human melanocortin-4 receptor by modification of cysteine 130 in transmembrane helix 3

We have investigated the effect of the sulfhydryl-reactive reagent, methyl thiosulfonate ethylammonium (MTSEA), on ligand binding to the human melanocortin-4 (MC4) receptor stably expressed in HEK-293 cells. MTSEA inhibited binding of the agonist, 125I-NDPalpha-MSH, and the antagonist, 125I-SHU9119, in a concentration-dependent manner. Pre-incubation of cells with either the agonist or antagonist protected from subsequent MTSEA inhibition of radioligand binding. Mutation of Cys130 in transmembrane helix 3 to alanine, whilst not affecting ligand binding, led to a complete loss of the inhibitory effect of MTSEA. Since other types of sulfhydryl-reactive reagents had no effect on ligand binding, we conclude that covalent modification of Cys130 by MTSEA disrupts ligand binding by neutralising a close-by negative charge, most likely on Asp126.     

Pan-cancer analysis of the CASP gene family in relation to survival,tumor-infiltrating immune cells and therapeutic targets

The cysteinyl aspartate protease (caspase, or CASP) gene family plays a significant role in programmed cell death, inflammation and immunity. However, the correlation between CASP family members and prognosis and tumor-infiltrating lymphocytes in different tumors has not been determined. We investigated the role of CASP genes in cancer prognosis and their relationship with clinicopathological parameters. We also evaluated the correlation between the expression of CASP family members and cancer immune infiltration and evaluated whether these molecules can be used as targets for immunotherapy. The CASP1/2/4/5/7/9 genes may represent prognostic factors and therapeutic targets for breast cancer, hepatocellular carcinoma and pancreatic cancer. Another finding is that the CASP1/4/5 genes help to regulate innate immunity and T cell immunity and may also have an important effect on tumor checkpoint inhibition. These findings may elucidate the roles played by CASP family members in cancer progression and identify strategies to promote collaborative activities in the context of immunotherapy.     

Contribution of coupling between human myometrial beta2-adrenoreceptor and the BK(Ca) channel to uterine quiescence

The ₂-adrenergic receptor (₂-AR) and the large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel have been shown, separately, to be involved in mediating uterine relaxation. Our recent studies reveal that the levels of both ₂-AR and BK_Ca channel proteins in pregnant human myometrium decrease by 50% after the onset of labor. We present direct evidence in support of a structural and functional association between the ₂-AR and the BK_Ca channel in pregnant human myometrium. Localization of both proteins is predominantly plasmalemmal, with 60% of ₂-AR colocalizing with the BK_Ca channel. Coimmunoprecipitation studies indicate that BK_Ca and ₂-AR are structurally linked by direct protein-protein interactions. Functional correlation was confirmed by experiments of human myometrial contractility in which the BK_Ca channel blocker, paxilline, significantly antagonized the relaxant effect of the ₂-AR agonist ritodrine. These novel findings provide an insight into the coupling between the ₂-AR and BK_Ca channel and may have utility in the application of this signaling cascade for therapeutic potential in the management of preterm labor. ₂-adrenergic receptor; myometrium; potassium channel; preterm labor; uterine contraction     

Activation of human complement by IgG antisperm antibody and the demonstration of C3 and C5b-9-mediated immune injury to human sperm

To investigate the role of C in the pathogenesis of antisperm antibody (ASA)-mediated infertility, we evaluated the binding and biologic effects of antisperm IgG and autologous C on human sperm. A flow cytometric assay using motile sperm as a target for IgG ASA+ (n = 30) and ASA- (n = 5) sera was developed for the concomitant detection of sperm-bound IgG and the initial (C3d) and terminal (C5b-9) C components on the surface of human sperm. Of the 30 IgG ASA+ sera evaluated by flow cytometry, 15 (50%) and 22 (73.3%) sera were also positive for sperm-bound C3d and C5b-9, respectively. Monomeric IgG purified from C-fixing ASA+ serum was able to bind to sperm and induced deposition of C3 on the sperm surface in the presence of human C. Incubation of motile sperm with C-fixing immune sera resulted in a significant loss (43 to 87%) of motility associated with characteristic C5b-9-induced alterations in sperm morphology leading ultimately to sperm lysis. When motile sperm were cocultured with purified polymorphonuclear leukocytes (PMN) in the presence of C-fixing immune sera, the binding of sperm heads to the PMN resulted in the formation of sperm rosettes, whereas non C-fixing or control sera had no such effect. Transmission electron microscopy of thin sections of the rosettes revealed ingestion of the sperm by the human PMN. These data suggested that 1) antibody bound to sperm is capable of activating autologous C by the classical pathway; 2) binding of both IgG and C proteins initiates C3-mediated sperm binding to PMN and sperm inactivation by deposition of membrane attack complex (MC5b-9) of C; and 3) concomitant detection of sperm-bound IgG, C3d, and C5b-9 may serve as an indicator of C-fixing cytotoxic ASA in the sera of infertile couples.     

An introduction to parasitic wasps of Drosophila and the antiparasite immune response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts'' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts¹. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response². Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited¹. Strains of L. boulardi also produce virus-like particles³ although they differ significantly in their ability to succeed on D. melanogaster¹. Some of these L. boulardi strains are difficult to grow on D. melanogaster¹ as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms^1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.Wasp infection activates immune signaling (Fig. 4)⁴. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)^5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).We present representative data with Toll signal transduction pathway components Dorsal and Spätzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).