Diagnostic accuracy of a rapid E1-antigen test for chikungunya virus infection in a reference setting.

OBJECTIVES: Rapid diagnostic tests targeting virus-specific antigen could significantly enhance the diagnostic capacity for chikungunya virus infections. We evaluated the accuracy of an immunochromatographic antigen test for diagnosis of chikungunya in a reference laboratory for arboviruses. METHODS: An immunochromatographic rapid test that uses mouse monoclonal antibodies as a tracer against the E1-envelope protein of chikungunya (ARKRAY, Inc. Kyoto, Japan) was evaluated. Sensitivity was tested in sera from travellers with RT-PCR confirmed chikungunya virus infection (Eastern/Central/Southern African (ECSA) genotype) (n=9) and from patients diagnosed during the 2014-2015 chikungunya outbreak on Aruba (Asian genotype, n=30). Samples from patients with other febrile and non-febrile illnesses (n=26), sera spiked with Flavivirus and Alphavirus reference strains (n=13, including non-spiked serum), and samples containing other selected pathogens (n=20) were used to test specificity of the E1-antigen test. RESULTS: Sensitivity of the E1-antigen test was 8/9 (88.9%, 95% CI 56.5-98.0) for the ECSA genotype, but only 10/30 (33.3%, 95% CI 19.2-51.2) for the Asian genotype. Overall diagnostic specificity was 49/59 (83.1%, 95% CI 71.5-90.5). CONCLUSIONS: The E1-antigen test we evaluated had fair diagnostic sensitivity for ECSA genotype chikungunya, but low sensitivity for Asian genotype, and poor overall specificity. Antibodies that react across genotypes will be required for further development of a rapid test for chikungunya. Performance of new tests should be evaluated against different chikungunya genotypes.

Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis.

Although RNA interference (RNAi) knockdown screening of cancer cell cultures is an effective approach to predict drug targets or therapeutic/prognostic biomarkers, interactions among identified targets often remain obscure. Here, we introduce the nodes-and-connections RNAi knockdown screening that generates a map of target interactions through systematic iterations of in silico prediction of targets and their experimental validation. An initial RNAi knockdown screening of MCF-7 human breast cancer cells targeting 6560 proteins identified four signaling molecules required for their fulvestrant-induced apoptosis. Signaling molecules physically or functionally interacting with these four primary node targets were computationally predicted and experimentally validated, resulting in identification of four second-generation nodes. Three rounds of further iterations of the prediction-validation cycle generated third, fourth and fifth generation of nodes, completing a 19-node interaction map that contained three predicted nodes but without experimental validation because of technical limitations. The interaction map involved all three members of the death-associated protein kinases (DAPKs) as well as their upstream and downstream signaling molecules (calmodulins and myosin light chain kinases), suggesting that DAPKs play critical roles in the cytocidal action of fulvestrant. The in silico Kaplan-Meier analysis of previously reported human breast cancer cohorts demonstrated significant prognostic predictive power for five of the experimentally validated nodes and for three of the prediction-only nodes. Immunohistochemical studies on the expression of 10 nodal proteins in human breast cancer tissues not only supported their prognostic prediction power but also provided statistically significant evidence of their synchronized expression, implying functional interactions among these nodal proteins. Thus, the Nodes-and-Connections approach to RNAi knockdown screening yields biologically meaningful outcomes by taking advantage of the existing knowledge of the physical and functional interactions between the predicted target genes. The resulting interaction maps provide useful information on signaling pathways cooperatively involved in clinically important features of the malignant cells, such as drug resistance.

High-throughput applicable genomic sex typing of chicken by TaqMan real-time quantitative polymerase chain reaction.

Genetic sex typing of vertebrate animals is an essential technique for research on reproductive phenomena such as sex determination of embryonic tissues. Polymerase chain reaction amplification of genomic DNA segments in the Z and W sex chromosomes has been widely used as a standard laboratory method to determine genetic sex of the chicken (Gallus gallus domesticus). However, conventional protocols for PCR determination of avian sex typically involve tedious steps of genomic DNA isolation, which often require relatively large amounts of tissue samples, and the purity of genomic DNA specimens significantly affects PCR efficiency. Moreover, detection of sex chromosome-specific PCR products by gel electrophoresis is prone to misjudgment caused by amplification of contaminating genomic DNA segments derived from tissue or DNA samples as well as previously generated PCR products. Thus, the credibility of genetic sex typing by conventional PCR-based methods that measure the relative amounts of the end product DNA amplicons critically depends on several experimental steps that are potentially vulnerable to errors. Here, we describe an optimized protocol of chicken genetic sex typing by TaqMan real-time quantitative PCR amplification of markers on the sex chromosomes. This TaqMan sex typing method accurately quantifies relative amounts of the Z and W sex chromosome markers directly from only 0.5 to 2 microL of total blood lysate without nucleic acid purification. The real-time amplification curves of the quantitative PCR reaction readily distinguished truly homozygous (ZZ) and heterozygous (ZW) sex chromosomes from contamination of the sex chromosomal DNA, ensuring highly credible sex determination. Thus, the TaqMan typing of chicken genetic sex has several advantageous features for high-throughput operation compared with conventional methods.

Polymorphisms in CCR5 chemokine receptor gene in Japan.

Mutations in the human CC chemokine receptor 5 (CCR5) gene may alter the expression or function of the protein product, thereby altering chemokine binding/signalling or human immunodeficiency virus type 1 (HIV-1) infection of the cells that normally express CCR5 protein. We performed a systematic survey of natural sequence variations in an 8.1-kb region of the entire CCR5 gene as well as CCR2V64I in 50 Japanese subjects and evaluated the effects of those variations on CCR5 promoter activity. We also analysed CCR5 promoters and CCR2V64I in 80 more Japanese and 186 Thais. There was no 32-bp deletion observed in Caucasians, but two types of non-synonymous substitutions were found in CCR5 genes of Japanese. Our results showed several novel characteristics of the CCR2-CCR5 haplotype structure that were not reported from studies on Caucasians and African-Americans. Specifically, we were able to show that the G allele at position -2852 from the CCR5 open reading frame in Japanese and Thais is the representative of the CCR5 promoter haplotype that was reported to be associated with rapid progression to acquired immune deficiency syndrome (AIDS) in HIV-1-infected individuals. Furthermore, nearly all non-synonymous polymorphisms in Japanese CCR5 occurred in haplotypes with elevated promoter activity. We thus hypothesized that there was a certain selective pressure favouring low levels of CCR5 expression during human evolution.

ERalpha-CITED1 co-regulated genes expressed during pubertal mammary gland development: implications for breast cancer prognosis.

Expression microarray analysis identified over 930 genes regulated during puberty in the mouse mammary gland. Most prominent were genes whose expression increased in parallel with pubertal development and remained high thereafter. Members of the Wnt, transforming growth factor-beta and oestrogen-signalling pathways were significantly overrepresented. Comparison to expression data from CITED1 knockout mice identified a subset of oestrogen-responsive genes displaying altered expression in the absence of CITED1. Included in this subset are stanniocalcin2 (Stc2) and amphiregulin (Areg). Chromatin immunoprecipitation revealed that ERalpha binds to oestrogen response elements in both the Stc2 and Areg genes in the mammary gland during puberty. Additionally, CITED1 and ERalpha localize to the same epithelial cells of the pubertal mammary gland, supporting a role for interaction of these two proteins during normal development. In a human breast cancer data set, expression of Stc2, Areg and CITED1 parallel that of ERalpha. Similar to ERalpha, CITED1 expression correlates with good outcome in breast cancer, implying that potential maintenance of the ERalpha-CITED1 co-regulated signalling pathway in breast tumours can indicate good prognosis.

A three-base-deletion polymorphism in the upstream non-coding region of human interleukin 7 (IL-7) gene could enhance levels of IL-7 expression.

Interleukin 7 (IL-7) is a key factor in the survival, development and proliferation of B and T lymphocytes. Elevation of plasma IL-7 has been reported in several lymphopenia cases such as HIV-1 patients. After patients started to receive antiretroviral drugs and their CD4(+) cell counts had recovered, IL-7 in plasma decreased to normal levels. There are considerable variations in the levels of plasma IL-7 as well as the rate of CD4(+) T-cell restoration. Although pre-treatment plasma IL-7 levels have been shown to be prognostic for the rate of post-treatment CD4(+) T-cell restoration, the mechanisms responsible for the variations in plasma IL-7 and rate of CD4(+) T-cell restoration are still completely unknown. In the study here, we searched for genetic polymorphisms that might affect levels of IL-7 gene expression. For this purpose, we used 1658-bp PCR-amplified fragments of the IL-7 gene containing 1470 bp of the upstream non-coding region obtained from 151 Japanese and 234 Thai subjects. We found two novel human genetic polymorphisms in the upstream non-coding region of the IL-7 gene. The luciferase reporter assay demonstrated that one of those polymorphisms could increase the gene expression of IL-7. We speculate that this polymorphism, a three base ATC deletion just upstream of an out-of-frame ATG codon in the upstream non-coding region of the IL-7 gene, reduces the efficiency of translation from the upstream, out-of-frame ATG, resulting in increased translation efficiency from the authentic ATG of IL-7. Although the frequency of this allele is very low, it would be interesting to analyse this polymorphism in HIV-1-infected individuals with different rates of immune reconstitution after treatment with a highly active antiretroviral therapy.

CITED1 homozygous null mice display aberrant pubertal mammary ductal morphogenesis.

Expression microarray analysis identified CITED1 among a group of genes specifically upregulated in the pubertal mouse mammary gland. At puberty, CITED1 localizes to the luminal epithelial cell population of the mammary ducts and the body cells of the terminal end buds. Generation of CITED1 gene knockout mice showed that homozygous null mutants exhibit retarded mammary ductal growth at puberty and, in addition, dilated ductal structures with a lack of spatial restriction of the subtending branches. Analysis of CITED1 homozygous null and heterozygous null mammary gland gene expression using microarrays suggested that the mammary-specific phenotype seen in the homozygous null females is due to a disturbance in the transcription of a number of key mediators of pubertal ductal morphogenesis. These include estrogen and TGFbeta responsive genes, such as the EGFR/ErbB2 ligand, amphiregulin, whose transcription we suggest is directly or indirectly regulated by CITED1.

Selective coactivation of estrogen-dependent transcription by CITED1 CBP/p300-binding protein.

CITED1, a CBP/p300-binding nuclear protein that does not bind directly to DNA, is a transcriptional coregulator. Here, we show evidence that CITED1 functions as a selective coactivator for estrogen-dependent transcription. When transfected, CITED1 enhanced transcriptional activation by the ligand-binding/AF2 domain of both estrogen receptor-alpha (ERalpha) and ERbeta in an estrogen-dependent manner, but it affected transcriptional activities of other nuclear receptors only marginally. CITED1 bound directly to ERalpha in an estrogen-dependent manner through its transactivating domain, and this binding activity was separable from its p300-binding activity. CITED1 was strongly expressed in nulliparous mouse mammary epithelial cells and, when expressed in ER-positive MCF-7 breast cancer cells by transduction, exogenous CITED1 enhanced sensitivity of MCF-7 cells to estrogen, stabilizing the estrogen-dependent interaction between p300 and ERalpha. The estrogen-induced expression of the transforming growth factor-alpha (TGF-alpha) mRNA transcript was enhanced in the CITED1-expressing MCF-7 cells, whereas estrogen-induced expression of the mRNA transcripts for progesterone receptor or pS2 was not affected. Chromatin immunoprecipitation assay revealed that endogenous CITED1 is recruited to the chromosomal TGF-alpha promoter in MCF-7 cells in an estrogen-dependent manner but not to the pS2 promoter. These results suggest that CITED1 may play roles in regulation of estrogen sensitivity in a gene-specific manner.

Over-expression of MSG1 transcriptional co-activator increases melanin in B16 melanoma cells: a possible role for MSG1 in melanogenesis.

MSG1 is a 27 kDa nuclear protein that is expressed strongly in melanotic B16 melanoma cells but very weakly in amelanotic B16 cells. Transient expression of B16 cells with an expression vector for MSG1 resulted in an increase in levels of the enzyme dopachrome tautomerase but not tyrosinase, as detected by western blotting. Stable transfection of B16 melanoma cells with plasmids containing the full length MSG1 or its deletion mutants, however, generated cell lines that showed an increase in levels of tyrosinase, dopachrome tautomerase and cellular melanin when compared with control transfected cells. Our results suggest that MSG1 plays an important role in melanogenesis, by regulating the levels of the enzymes of the pigmentary system via tyrosinase and dopachrome tautomerase.

Sendai virus C protein physically associates with Stat1.

BACKGROUND: The P/C gene of the Sendai virus (SeV), a member of the family Paramyxoviridae, encodes C protein, which plays a crucial role in counteracting the antiviral effect of interferon (IFN). The C protein blocks IFN signalling to prevent the activation of IFN stimulated genes. However, its underlying molecular mechanism remains to be defined. RESULTS: Signal transducer and activator of transcription 1 (Stat1) is a critical component of IFN-alpha/beta and IFN-gamma signalling. We found that both unphosphorylated Stat1 and tyrosine-phosphorylated (pY) Stat1 were present in a form of aberrant high molecular weight complexes (HMWCs) of over 2 MDa in infected cell extracts under low-salt conditions. Of recombinant vaccinia viruses carrying each SeV gene, only those expressing the C gene induced Stat1-HMWC. SeV infected cell extracts further displayed an in vitro ability to convert the pY-Stat1 homodimer to pY-Stat1-HMWC. This cell extract activity was not seen after removal of the C protein from the extracts. C protein was therefore involved in the formation of HMWCs. The HMWCs decomposed into smaller complexes in a high-salt buffer, and under this stringent (high-salt) condition, as well as a physiological (isotonic) condition, both unphosphorylated Stat1 and pY-Stat1 were co-precipitated with anti-C antibody. CONCLUSION: The C protein physically associates with Stat1. This suggests that SeV C protein directly targets Stat1 for inhibitory control on the transcriptional activation of IFN stimulated genes.

Novel polymorphisms in human macrophage inflammatory protein-1 alpha (MIP-1alpha) gene.

Human macrophage inflammatory protein-1 alpha (MIP-1alpha) is a chemotactic cytokine, which binds to macrophages, T cells, and B cells affecting their activation. We found novel polymorphisms at four sites within MIP-1alpha gene in Japanese population: C to T in exon 2; A to G in intron 2; C to G and A to G in exon 3. They occurred on the same allele. Although MIP-1alpha effectively suppresses the replication of HIV-1 in vitro, we observed no statistically significant difference in the allele frequency of this polymorphism between HIV-1-infected and uninfected individuals in Japanese population. Since an increased transcription level of MIP-1alpha has been reported to be associated with inflammatory diseases such as atopic dermatitis, we also investigated the frequency of these polymorphisms among patients with atopic dermatitis, HIV-1-infected individuals (with a normal IgE level), and healthy donors. A small increase in ratio of homozygotes to other genotypes was observed in patients with atopic dermatitis (P = 0.04).

Quintuple deglycosylation mutant of simian immunodeficiency virus SIVmac239 in rhesus macaques: robust primary replication, tightly contained chronic infection, and elicitation of potent immunity against the parental wild-type strain.

We previously generated a mutant of simian immunodeficiency virus (SIV) lacking 5 of a total of 22 N-glycans in its external envelope protein gp120 with no impairment in viral replication capability and infectivity in tissue culture cells. Here, we infected rhesus macaques with this mutant and found that it also replicated robustly in the acute phase but was tightly, though not completely, contained in the chronic phase. Thus, a critical requirement for the N-glycans for the full extent of chronic infection was demonstrated. No evidence indicating reversion to a wild type was obtained during the observation period of more than 40 weeks. Monkeys infected with the mutant were found to tolerate a challenge infection with wild-type SIV very well. Analyses of host responses following challenge revealed no neutralizing antibodies against the challenge virus but strong secondary responses of cytotoxic T lymphocytes against multiple antigens, including Gag-Pol, Nef, and Env. Thus, the quintuple deglycosylation mutant appeared to represent a novel class of SIV live attenuated vaccine.

Aberrant expression of MSG1 transcriptional activator in human malignant melanoma in vivo.

MSG1 was originally isolated as a candidate pigmentation-related gene. MSG1 mRNA transcripts were expressed strongly in cultured human and mouse normal epidermal melanocytes, and in highly pigmented mouse melanoma cells, while its expression was very weak in cultured non-pigmented human melanoma cells. Thus, MSG1 was initially proposed to be a melanocyte-specific gene, and its possible role in pigmentation has been speculated. It was found recently that the MSG1 protein interacts functionally with Smad4, which plays a pivotal role in signal transduction of transforming growth factor-beta. In this study, we analyzed MSG1 protein expression by immunohistochemistry using human tumor samples from nevus and malignant melanoma to reveal its role in pigmentation and melanoma development in vivo. A relatively strong but heterogeneous expression of MSG1 protein was seen in melanomas compared with weak expression in nevi. In nevi, MSG1 expression was mostly confined to the pigmented region, while it was expressed in both pigmented and non-pigmented regions in melanoma. Intracellularly, MSG1 protein was localized in the cytoplasm of nevus cells, but was seen in both nuclei and cytoplasm of melanoma cells. These results support a hypothesis that MSG1 plays a role in pigmentation. It is also suggested that MSG1 may be involved in malignant transformation of pigment cells. Alternatively, the aberrant expression of MSG1 in melanoma cells might be due to the abnormal environment, including aberrant cytokine or growth factor expression, associated with melanoma formation.

Naturally occurring deletional mutation in the C-terminal cytoplasmic tail of CCR5 affects surface trafficking of CCR5.

CCR5 is an essential coreceptor for the cellular entry of R5 strains of human immunodeficiency virus type 1 (HIV-1). CCR5-893(-) is a single-nucleotide deletion mutation which is observed exclusively in Asians (M. A. Ansari-Lari, et al., Nat. Genet. 16:221-222, 1997). This mutant gene produces a CCR5 which lacks the entire C-terminal cytoplasmic tail. To assess the effect of CCR5-893(-) on HIV-1 infection, we generated a recombinant Sendai virus expressing the mutant CCR5 and compared its HIV-1 coreceptor activity with that of wild-type CCR5. Although the mutant CCR5 has intact extracellular domains, its coreceptor activity was much less than that of wild-type CCR5. Flow cytometric analyses and confocal microscopic observation of cells expressing the mutant CCR5 revealed that surface CCR5 levels were greatly reduced in these cells, while cytoplasmic CCR5 levels of the mutant CCR5 were comparable to that of the wild type. Peripheral blood CD4(+) T cells obtained from individuals heterozygous for this allele expressed very low levels of CCR5. These data suggest that the CCR5-893(-) mutation affects intracellular transport of CCR5 and raise the possibility that this mutation also affects HIV-1 transmission and disease progression.

Duplication of the primary encapsidation and dimer linkage region of human immunodeficiency virus type 1 RNA results in the appearance of monomeric RNA in virions.

The dimerization initiation site (DIS) and the dimer linkage sequences (DLS) of human immunodeficiency virus type 1 have been shown to mediate in vitro dimerization of genomic RNA. However, the precise role of the DIS-DLS region in virion assembly and RNA dimerization in virus particles has not been fully elucidated, since deletion or mutation of the DIS-DLS region also abolishes the packaging ability of genomic RNA. To characterize the DIS-DLS region without altering packaging ability, we generated mutant constructs carrying a duplication of approximately 1,000 bases including the encapsidation signal and DIS-DLS (E/DLS) region. We found that duplication of the E/DLS region resulted in the appearance of monomeric RNA in virus particles. No monomers were observed in virions of mutants carrying the E/DLS region only at ectopic positions. Monomers were not observed when pol or env regions were duplicated, indicating an absolute need for two intact E/DLS regions on the same RNA for generating particles with monomeric RNA. These monomeric RNAs were most likely generated by intramolecular interaction between two E/DLS regions on one genome. Moreover, incomplete genome dimerization did not affect RNA packaging and virion formation. Examination of intramolecular interaction between E/DLS regions could be a convenient tool for characterizing the E/DLS region in virion assembly and RNA dimerization within virus particles.

Antibodies to molluscum contagiosum virus in the general population and susceptible patients.

BACKGROUND: Since many attempts to cultivate molluscum contagiosum virus (MCV) in vitro have been unsuccessful, it is difficult to prepare a large quantity of antigens. To assess the seroprevalence of antibodies against MCV in 508 subjects with or without clinical MCV infection, a truncated recombinant protein from open-reading frame MC133L was synthesized using Sendai virus expression system and applied to enzyme-linked immunosorbent assay as an antigen. OBSERVATIONS: Antibodies to MCV were present in 7 (58%) of 12 patients with molluscum contagiosum, 7 (6%) of 108 healthy controls, 7 (9%) of 76 with atopic dermatitis, and 7 (18%) of 39 patients with systemic lupus erythematosus, although no clinical MCV infection was observed in the latter 3 groups. Of 7 human immunodeficiency virus (HIV)-positive patients with molluscum contagiosum, 1 (14%) was antibody positive, compared with 5 (2%) of 266 HIV-positive patients without molluscum contagiosum. Serum samples from patients with atopic dermatitis and systemic lupus erythematosus showed a higher reactivity (P<.001) than those from healthy controls, while serum samples from HIV-positive subjects showed a lower reactivity (P<. 001). CONCLUSION: The humoral immune response to MCV is usually confined to patients with molluscum contagiosum and may be affected by the immunological condition of the host.

X inactivation in the mouse embryo deficient for Dnmt1: distinct effect of hypomethylation on imprinted and random X inactivation.

It has been suggested that DNA methylation plays a crucial role in genomic imprinting and X inactivation. Using DNA methyltransferase 1 (Dnmt1)-deficient mouse embryos carrying X-linked lacZ transgenes, we studied the effects of genomic demethylation on X inactivation. Based on the expression pattern of lacZ, the imprinted X inactivation in the visceral endoderm, a derivative of the extraembryonic lineage, was unaffected in Dnmt1 mutant embryos at the time other imprinted genes showed aberrant expression. Random X inactivation in the embryonic lineage of Dnmt1 mutant embryos, however, was unstable as a result of hypomethylation, causing reactivation of, at least, one lacZ transgene that had initially been repressed. Our results suggest that maintenance of imprinted X inactivation in the extraembryonic lineage can tolerate extensive demethylation while normal levels of methylation are required for stable maintenance of X inactivation in the embryonic lineage.

Crystal structure of recombinant native SDF-1alpha with additional mutagenesis studies: an attempt at a more comprehensive interpretation of accumulated structure-activity relationship data.

Crystal structures, forms 1 and 2, of recombinant native stromal cell-derived factor-1alpha (SDF-1alpha), expressed using the Sendai virus expression vector system, have been determined by x-ray crystallography at 2.0 A resolution. The crystal of form 1 is almost isomorphous with that used in the previous crystal structure analysis of the synthetic [N33A] mutant of SDF-1alpha (Dealwis, C., et al. Proc. Natl. Acad. Sci. USA 1998;95, 6941-6946). However, the present structure analysis led to considerably better refinement statistics, revealing an error in the structural assignment of N-terminal residues in the previous report. Comparison of the solution structure, as previously determined by nuclear magnetic resonance (NMR) spectroscopy, and the present structure, with two monomers in the asymmetric unit, reveals several local conformational differences. Alanine scan mutagenesis studies for each residue in the so-called RFFESH motif revealed that only the first residue, Arg12, is effective in enhancing receptor binding (and successive activation). A new notion that steric restraint between Arg8 and Arg12 is favorable (if not vital) for retaining SDF activities appears to explain more consistently the structure-activity relationship data accumulated to date. Four guiding principles are presented that may be useful for designing potent therapeutic compounds interfering with HIV-1 infection through competition at the CXCR4 coreceptor.