Identification and HLA-Tetramer-Validation of Human CD4+ and CD8+ T Cell Responses against HCMV Proteins IE1 and IE2

Human cytomegalovirus (HCMV) is an important human pathogen. It is a leading cause of congenital infection and a leading infectious threat to recipients of solid organ transplants as well as of allogeneic hematopoietic cell transplants. Moreover, it has recently been suggested that HCMV may promote tumor development. Both CD4⁺ and CD8⁺ T cell responses are important for long-term control of the virus, and adoptive transfer of HCMV-specific T cells has led to protection from reactivation and HCMV disease. Identification of HCMV-specific T cell epitopes has primarily focused on CD8⁺ T cell responses against the pp65 phosphoprotein. In this study, we have focused on CD4⁺ and CD8⁺ T cell responses against the immediate early 1 and 2 proteins (IE1 and IE2). Using overlapping peptides spanning the entire IE1 and IE2 sequences, peripheral blood mononuclear cells from 16 healthy, HLA-typed, donors were screened by ex vivo IFN-γ ELISpot and in vitro intracellular cytokine secretion assays. The specificities of CD4⁺ and CD8⁺ T cell responses were identified and validated by HLA class II and I tetramers, respectively. Eighty-one CD4⁺ and 44 CD8⁺ T cell responses were identified representing at least seven different CD4 epitopes and 14 CD8 epitopes restricted by seven and 11 different HLA class II and I molecules, respectively, in total covering 91 and 98% of the Caucasian population, respectively. Presented in the context of several different HLA class II molecules, two epitope areas in IE1 and IE2 were recognized in about half of the analyzed donors. These data may be used to design a versatile anti-HCMV vaccine and/or immunotherapy strategy.     

Structural requirements of (2'-5') oligoadenylate for protein synthesis inhibition in human fibroblasts.

The structural requirements of (2'-5')-oligoadenylic acid (pppA(2'p5'A)x, X greater than or equal to 1 or (2'-5'An) for inhibition of protein synthesis in cells were examined with a modified calcium-coprecipitation technique, using a series of trinucleotide analogs (pppA2'p5'A2'p5'N, N=rC, rG, rU, T, dC, dG, dA). In this system both the degree and the duration of the inhibition of protein synthesis were dependent on the added concentration of (2'-5')A3. Of all the heterotrimers, only the deoxy A derivative was active as an inhibitor of protein synthesis, while the other members of the analog series were found to have no inhibitory effects. In competition experiments between (2'-5')A3 and the non-active analogs, three heterotrimers were shown to reduce the activity of (2'-5')A3 in protein inhibition. In contrast, the dephosphorylated (2'-5')A3 had no inhibitory effect and was not effective in blocking (2'-5')A3. These results indicate that the 5'-terminal triphosphate is important for binding of (2'-5')A3 to the site of (2'-5')An action and the adenine base at the 2'-terminus is important for activating the machinery responsible for protein synthesis inhibition in the cells, most likely the (2'-5')An-activated nuclease.     

Sponge OAS has a distinct genomic structure within the 2-5A synthetase family

2′,5′-Oligoadenylate synthetases (2-5A synthetases, OAS) are enzymes that play an important role in the interferon-induced antiviral defense mechanisms in mammals. Sponges, the evolutionarily lowest multicellular animals, also possess OAS; however, their function is presently unclear. Low homology between primary structures of 2-5A synthetases from vertebrates and sponges renders their evolutionary relationship obscure. The genomic structure of vertebrate OASs has been thoroughly examined, making it possible to elucidate molecular evolution and expansion of this gene family. Until now, no OAS gene structure was available from sponges to compare it with the corresponding genes from higher organisms. In the present work, we determined the exon/intron structure of the OAS gene from the marine sponge Geodia cydonium and found it to be completely different from the strictly conserved exon/intron pattern of the OAS genes from vertebrates. This finding was corroborated by the analysis of OAS genes from another sponge, Amphimedon queenslandica, whose genome was recently sequenced. Our data suggest that vertebrate and sponge OAS genes have no direct common intron-containing ancestor and two (sub)types of OAS may be discriminated. This study opens new perspectives for understanding the phylogenesis and evolution of 2-5A synthetases as well as functional aspects of this multigene family. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Abbreviations  The nomenclature of particular OASs in the present paper is based on the level of their sequence similarities. OAS1 refers to the OAS consisting of a single OAS domain. Capital letters (OAS1X) are used to differentiate between OAS1 types with sequence homologies of less than 50%; their variants are additionally marked in small letters (OASXx, sequence homology ~70 to ~95%). Labels prime and double prime denote sponge OAS1Xx gene haplotypes (sequence homology close to 100%, a few amino acid substitutions).     

Local Knowledge and Conservation of Seagrasses in the Tamil Nadu State of India

Local knowledge systems are not considered in the conservation of fragile seagrass marine ecosystems. In fact, little is known about the utility of seagrasses in local coastal communities. This is intriguing given that some local communities rely on seagrasses to sustain their livelihoods and have relocated their villages to areas with a rich diversity and abundance of seagrasses. The purpose of this study is to assist in conservation efforts regarding seagrasses through identifying Traditional Ecological Knowledge (TEK) from local knowledge systems of seagrasses from 40 coastal communities along the eastern coast of India. We explore the assemblage of scientific and local traditional knowledge concerning the 1. classification of seagrasses (comparing scientific and traditional classification systems), 2. utility of seagrasses, 3. Traditional Ecological Knowledge (TEK) of seagrasses, and 4. current conservation efforts for seagrass ecosystems. Our results indicate that local knowledge systems consist of a complex classification of seagrass diversity that considers the role of seagrasses in the marine ecosystem. This fine-scaled ethno-classification gives rise to five times the number of taxa (10 species = 50 local ethnotaxa), each with a unique role in the ecosystem and utility within coastal communities, including the use of seagrasses for medicine (e.g., treatment of heart conditions, seasickness, etc.), food (nutritious seeds), fertilizer (nutrient rich biomass) and livestock feed (goats and sheep). Local communities are concerned about the loss of seagrass diversity and have considerable local knowledge that is valuable for conservation and restoration plans. This study serves as a case study example of the depth and breadth of local knowledge systems for a particular ecosystem that is in peril.     

2''5'' oligoadenylate synthetase, an interferon induced enzyme: direct assay methods for the products, 2''5'' oligoadenylates and 2''5'' co-oligonucleotides.

The interferon induced enzyme 2'5' oligoadenylate synthetase produces 2'5' pppA(pA)n the first discovered natural nucleotide with a 2'5' linkage. We describe a direct assay of this enzyme based on separation by thin layer chromatography (TLC) of the substrate ATP and the products 2'5' pppA(pA)n (n larger than or equal to 1). This technique presents obvious advantages compared to the currently used methods. Moreover the enzyme uses other nucleotides as substrates forming co-oligonucleotides 2'5 pppA(pA)n pN (N = U,G,C,dA,dG,dT and dC). Additional procedures are described using different developing solvent systems for the separation of the core-2'5' oligonucleotides (2'5' A(pA)npN) containing AMP-residues entirely and those with another nucleotide at the 2' end.     

Full genome gene expression analysis of the heat stress response in Drosophila melanogaster

The availability of full genome sequences has allowed the construction of microarrays, with which screening of the full genome for changes in gene expression is possible. This method can provide a wealth of information about biology at the level of gene expression and is a powerful method to identify genes and pathways involved in various processes. In this study, we report a detailed analysis of the full heat stress response in Drosophila melanogaster females, using whole genome gene expression arrays (Affymetrix Inc, Santa Clara, CA, USA). The study focuses on up- as well as downregulation of genes from just before and at 8 time points after an application of short heat hardening (36 degrees C for 1 hour). The expression changes were followed up to 64 hours after the heat stress, using 4 biological replicates. This study describes in detail the dramatic change in gene expression over time induced by a short-term heat treatment. We found both known stress responding genes and new candidate genes, and processes to be involved in the stress response. We identified 3 main groups of stress responsive genes that were early-upregulated, early-downregulated, and late-upregulated, respectively, among 1222 differentially expressed genes in the data set. Comparisons with stress sensitive genes identified by studies of responses to other types of stress allow the discussion of heat-specific and general stress responses in Drosophila. Several unexpected features were revealed by this analysis, which suggests that novel pathways and mechanisms are involved in the responses to heat stress and to stress in general. The majority of stress responsive genes identified in this and other studies were downregulated, and the degree of overlap among downregulated genes was relatively high, whereas genes responding by upregulation to heat and other stress factors were more specific to the stress applied or to the conditions of the particular study. As an expected exception, heat shock genes were generally found to be upregulated by stress in general.     

The interferon alpha induced protein ISG12 is localized to the nuclear membrane.

Interferons exert their biological function mainly through the activation of interferon-stimulated genes (ISGs). ISG12 (originally designated p27) belongs to a family of small, interferon alpha inducible genes of unknown function. We have determined the 5' end sequence of ISG12 cDNA from the human cell lines HeLa and AMA by RACE. Comparing this sequence to ISG12 sequences in the expressed sequence tag (EST) database revealed the presence of two alternative splice variants of ISG12 in human cells exhibiting the same open reading frame. We have sequenced the promoter region of the ISG12 gene and found ISRE, IRF1/IRF2, and STAT elements correlating to the interferon alpha inducibility of the gene. Subsequently, we have expressed human ISG12, a 12-kDa hydrophobic protein in the baculovirus expression system and with a C-terminal FLAG-tag in the human cell line 293. Recombinant ISG12 sediments in the nuclear envelope in both cell types. Finally, we have been able to demonstrate the prevalence of the ISG12 gene product in the nuclear envelope of HeLa cells treated with interferon alpha by immunocytochemical analyses. ISG12 is the first interferon induced protein found localizing to the nuclear envelope.     

Mycoplasma hominis septicaemia.

Mycoplasma hominis septicaemia occurred in a patient with a malignant lymphoma of lymphoblastic type in leukaemic phase. M hominis was isolated several times from blood cultures with antibody titres against the micro-organism rising to a high level despite severe immunosuppression. M hominis was detected in the blood after subculture of the blood culture bottles despite their macroscopically normal appearance. The patient''s pyrexia resolved without treatment with antibiotics effective against M hominis.