Transcript imaging of the development of human T helper cells using oligonucleotide arrays.

Many pathological processes, including those causing allergies and autoimmune diseases, are associated with the presence of specialized subsets of T helper cells at the site of inflammation. Understanding the genetic program that controls the functional properties of T helper type 1 (Th1) versus T helper type 2 (Th2) cells may provide insight into the pathophysiology of inflammatory diseases. We compared the gene-expression profiles of human Th1 and Th2 cells using high-density oligonucleotide arrays with the capacity to display transcript levels of 6,000 human genes. Here we analyse the data sets derived from five independent experiments using statistical algorithms. This approach resulted in the identification of 215 differentially expressed genes, encoding proteins involved in transcriptional regulation, apoptosis, proteolysis, and cell adhesion and migration. A subset of these genes was further upregulated by exposure of differentiated Th1 cells to interleukin-12 (IL-12), as confirmed by kinetic PCR analysis, indicating that IL-12 modulates the effector functions of Th1 cells in the absence of antigenic stimulation. Functional assays and in vivo expression of selected genes have validated the biological relevance of our study. Our results provide new insight into the transcriptional program controlling the functional diversity of subsets of T helper cells.

Severe immunodeficiency associated with a human immunodeficiency virus 1 NEF/3'-long terminal repeat transgene.

We have generated several transgenic mouse strains carrying a human immunodeficiency virus 1 (HIV-1) NEF/3' long terminal repeat (LTR) transgene under control of a T cell-specific promoter-enhancer element, showing a depletion of CD4+ T cells in the thymus and periphery. The immunological functions of the line with the most dramatic changes in lymphocyte populations, B6/338L, were analyzed in greater detail. The presence of the transgene in the heterozygous animal is associated with a dominant severe immunodeficiency. Older animals develop lymph-adenopathy and splenomegaly. CD4+CD8+ and CD4+CD8- single positive thymocytes already are depleted in these mice at the earliest stages in ontogeny, and peripheral T cells are reduced in frequency and present cell surface marker expression, which is characteristic for memory and activated T cells. The immunological response of B6/338L mice to several viral infections is also greatly impaired. Thus, the HIV-1 NEF/3' LTR as transgene in T cells can cause immunodeficiency and disease with striking similarities to a known retrovirus-induced immunodeficiency called murine AIDS (H. C. Morse III, S. K. Chattopadhyay, M. Makino, T. N. Frederickson, A. W. Hügin, and J. W. Hartley. 1992. AIDS. 6:607).

Conjugation of a peptide autoantigen to gold nanoparticles for intradermally administered antigen specific immunotherapy.

Antigen specific immunotherapy aims to tolerise patients to specific autoantigens that are responsible for the pathology of an autoimmune disease. Immune tolerance is generated in conditions where the immune response is suppressed and thus gold nanoparticles (AuNPs) are an attractive drug delivery platform due to their anti-inflammatory effects and their potential to facilitate temporal and spatial delivery of a peptide autoantigen in conjunction with pro-tolerogenic elements. In this study we have covalently attached an autoantigen, currently under clinical evaluation for the treatment of type 1 diabetes (PIC19-A3 peptide), to AuNPs to create nanoscale (<5 nm), negatively charged (-40 to -60 mV) AuNP-peptide complexes for immunotherapy. We also employ a clinically approved microneedle delivery system, MicronJet600, to facilitate minimally-invasive intradermal delivery of the nanoparticle constructs to target skin-resident antigen presenting cells, which are known to be apposite target cells for immunotherapy. The AuNP-peptide complexes remain physically stable upon extrusion through microneedles and when delivered into ex vivo human skin they are able to diffuse rapidly and widely throughout the dermis (their site of deposition) and, perhaps more surprisingly, the overlying epidermal layer. Intracellular uptake was extensive, with Langerhans cells proving to be the most efficient cells at internalising the AuNP-peptide complex (94% of the local population within the treated region of skin). In vitro studies showed that uptake of the AuNP-peptide complexes by dendritic cells reduced the capacity of these cells to activate naïve T cells. This indicator of biological functionality encourages further development of the AuNP-peptide formulation, which is now being evaluated in clinical trials.

Stimulation of sea urchin H2B histone gene transcription by a chromatin-associated protein fraction depends on gene sequences downstream of the transcription start site.

We isolated a chromosomal protein fraction derived from chromatin of sea urchin embryos which specifically stimulated the expression of the histone H2B gene by a factor of 5- to 10-fold when the complete sea urchin histone gene repeat h22 was injected in Xenopus laevis oocyte nuclei. Gene manipulation experiments revealed the existence of two different target sites in the H2B gene which appear to mediate the response to injection of the stimulatory sea urchin chromatin-associated proteins; both are located downstream of the transcription initiation site. The first sequence element which is shown to be implicated is within, or at least includes, the H2B 5' untranslated leader sequence between nucleotides 11 and 76. The second element resides within an H2B DNA segment located near the 3' end of the gene, extending from 90 base pairs upstream of the mRNA 3' terminus to 140 base pairs in the spacer sequences downstream.

Bacterial transcript imaging by hybridization of total RNA to oligonucleotide arrays.

We have used high-density oligonucleotide probe arrays (chips) for bacterial transcript imaging. We designed a chip containing probes representing 106 Hemophilus influenzae genes and 100 Streptococcus pneumoniae genes. The apparent lack of polyadenylated transcripts excludes enrichment of mRNA by affinity purification and we thus used total, chemically biotinylated RNA as hybridization probe. We show that hybridization of Streptococcus RNA to a chip allows simultaneous quantification of the transcript levels. The sensitivity was found to be in the range of one to five transcripts per cell. The quantitative chip results were in good agreement with conventional Northern blot analysis of selected genes. This technology allows simultaneous and quantitative measurement of the transcriptional activity of entire bacterial genomes on a single oligonucleotide probe array.

The wheat germ cell-free system possesses processing activity for the precursor of human placental lactogen.

1. Total RNA was extracted from human term placenta and mRNA purified by chromatography on oligo(dT)-cellulose. The poly(A)-containing fraction stimulated amino acid incorporation 5- to 10-fold in the wheat germ cell-free system. Immunoprecipitation with an anti-lactogen serum indicated that 14-27% of the peptides synthesized in vitro contained antigenic determinants of this hormone. 2. Analysis of the [3H]leucine labelled product in the immunoprecipitate on sodium dodecyl sulfate-polyacrylamide gels revealed a complex mixture of polypeptides. Two heavily labelled bands (I and III) were seen corresponding in mobility with pre-lactogen (Mr = 25 000) and native lactogen (Mr = 22 200), each accounting for about 30% of the immunoprecipitable radioactivity. Two additional bands with an intermediate mobility were also observed. 3. Synthesis of the hormone was inhibited by 7-methylguanosine-5'-monophosphate suggesting the presence of a 7-methylguanosine 'cap' on the 5'-end of the mRNA for lactogen. 4. Peptide analysis of the cyanogen bromide cleavage products of band I, band III and authentic lactogen showed marked similarities in their primary structure. The precursor molecule, however, was lacking the N-terminal peptide present in authentic hormone indicating the presence of an extension of 25 amino acids at this side of the molecule. 5. The presence of one or several processing enzymes in the wheat germ cell-free system was indicated by the effect of Triton X-100. Low concentrations of this detergent (0.04%) while inhibiting the protein synthesizing activity for only 15%, completely abolished the precursor cleavage activity. Under these conditions only pre-lactogen was detected in the immunoprecipitate.

Genomic divergence of an HIV-2 from a German AIDS patient probably infected in Mali.

The complete nucleotide sequence of an HIV-2 isolate derived from a German AIDS patient with predominantly neurological symptoms is reported. The HIV-2BEN sequence is highly divergent from those of previously described HIV-2 and SIV strains. Evolutionary tree analysis of eight HIV-2 sequences reveals the existence of three HIV-2 groups. HIV-2BEN belongs to a group with two isolates from Ghana and The Gambia. Based on a comparison of HIV-2BEN with six HIV-2 isolates, SIVsmm and SIVmac, the variability of the structural env and gag proteins is similar within the HIV-2/SIVsmm/mac and HIV-1 groups. In contrast, the regulatory HIV-1 proteins are more highly conserved than those from HIV-2 strains. Multiple sequence alignments reveal that some domains of the envelope and regulatory proteins are well conserved among HIV-1, HIV-2/SIVsmm/mac, SIVagm and SIVmnd. The identification of conserved domains within the external glycoprotein could help to develop broadly active vaccines.

Expression of biologically active human T-cell lymphotropic virus type III reverse transcriptase in Bacillus subtilis.

A 2.4-kb DNA fragment from the pol region of the human T-cell lymphotropic virus, encoding the protease, reverse transcriptase and a portion of the endonuclease N-terminus was stably introduced into a high-level Bacillus subtilis expression system under inducible control of the Escherichia coli lac regulatory elements. The major expression plasmid, pRTL11, contains a bacteriophage T5 promoter/lac operator element, which is controlled by lac repressor, supplied by the secondary plasmid, pBL1. Upon IPTG induction, a 90-kDa polyprotein is synthesised and subsequently proteolytically cleaved to reveal 64-kDa and 52-kDa polypeptides. Partial purification reveals that reverse transcriptase activity co-migrates with these two polypeptides.

CD4-affinity purification of recombinant and native HIV gp120 and comparison of the affinity constants for the receptor.

Soluble CD4-immunoglobulin chimeric proteins were covalently attached to CNBr-activated Sepharose. This affinity matrix was used to establish a powerful new method to isolate different species of the HIV external glycoprotein gp120 from cell-free culture supernatants. Recombinant gp120 was expressed in Baculovirus-infected insect cells and isolated from cell-free culture supernatants. The recombinant protein has an apparent molecular mass of 130 kDa. These two gp120 species were shown to be of identical molecular size after complete deglycosylation achieved by endoglycosidase treatment, and they bound to CD4-H gamma l with the same binding constant, that was reported for native forms of gp120 and CD4. Thus the different glycosylation of gp120 does not influence its affinity to CD4 and the gp120-CD4 complex can be reversibly dissociated.

Purification and in vitro-phospholabeling of secretory envelope proteins E1 and E2 of hepatitis C virus expressed in insect cells.

The putative envelope glycoproteins of hepatitis C virus (HCV), E1 and E2, were expressed as recombinant, secretory proteins in Sf9 insect cells through infection with recombinant baculoviruses. The influenza virus hemagglutinin signal sequence (HASS) was inserted upstream of the HCV-cDNAs in order to effect secretion. Furthermore, a hexa-histidine tag for purification on a Ni(2+)-nitrilotriacetic acid (Ni(2+)-NTA) column and a protein kinase A (PKA) recognition sequence for in vitro-phospholabeling were fused upstream of the HCV-cDNA. E1- and E2 proteins lacking their carboxy-terminal, hydrophobic sequence were produced by baculovirus-infected insect cells in bioreactors of 23 1. The medium was concentrated and proteins were purified under native conditions on Ni(2+)-NTA columns. Purified proteins could be phospholabeled in vitro using the catalytic subunit of protein kinase. A isolated from bovine heart and gamma-[32P]ATP. Labeled E1 and E2 proteins expressed in insect cells could be immunoprecipitated with sera from HCV-infected patients. Co-expression of these E1 and E2 proteins led to the formation of E1-E2 complexes within the insect cell and to secretion of these complexes into the medium.

Mutation of conserved N-glycosylation sites around the CD4-binding site of human immunodeficiency virus type 1 GP120 affects viral infectivity.

Infection by the human immunodeficiency virus type 1 (HIV-1) is initiated through interaction of its exterior envelope glycoprotein gp120 with the CD4 receptor on target cells. To address the possible role of N-glycosylation of HIV-1 gp120 in binding CD4, we mutated different conserved N-glycosylation site Asn-residues in the vicinity of the putative CD4 binding site, as single mutations or in combinations. Authentic and mutant gp120 proteins were produced using the baculovirus expression system. All mutant proteins were produced and secreted at similar levels and could be immunoprecipitated with an HIV(+)-serum. Furthermore, all glycosylation mutants retained the full capacity to bind CD4 except for a triple mutant which showed reduced binding. Different gp120 mutant genes were then introduced in an infectious proviral DNA clone. Upon transfection of MT-2 cells, the authentic HIV-1 clone induced maximal virus production after 6 days. In the case of the triple glycosylation mutant, comparable virus production was first reached after a delay of about 12 days. Moreover, in contrast to native HIV, the mutant virus induced no typical multinucleated giant cells. These results suggest that the attached carbohydrates around the CD4-binding site of gp120, may contribute to the generation of this protein domain required for high affinity receptor interaction.

Tumor necrosis factor receptor p55 mediates induction of HIV type 1 expression in chronically infected U1 cells.

Tumor necrosis factor alpha (TNF-alpha) is a potent inducer of human immunodeficiency virus type 1 (HIV-1) expression in chronically infected cells. The aim of this study was to investigate the role played by the two known TNF-alpha receptors, TNFR-p55 and TNFR-p75, in the activation of HIV-1 expression. As a model system the latently infected human promonocytic cell line U1 was stimulated with wild-type TNF-alpha, with TNF-alpha muteins that specifically bind to one or the other receptor or with receptor-specific monoclonal antibodies. Induction of HIV-1 expression, measured by p24 core antigen capture enzyme-linked immunosorbent assay (ELISA), was found to be exclusively triggered by TNFR-p55 stimulation. However, our results also showed that the addition of TNFR-p75-specific ligands negatively modulated the HIV-1 expression induced via TNFR-p55.

Molecular cloning and characterization of a German HIV-1 isolate.

The genetic diversity of HIV-1 is well documented. Except for the HIV-1 isolate LAV-1BRU, no nucleic acid sequence of a European isolate of HIV-1 has been published to date. To further investigate the extent of the genetic variability and the evolution of HIV-1, we have isolated, cloned, and subsequently sequenced HIV-1 from a German patient with AIDS-related complex. Comparative studies of the nucleic acid sequence revealed that this isolate, designated HAN2, is highly divergent from the North American and African subtypes of HIV-1 and may represent a European subtype of HIV-1. Furthermore, a full-length molecular clone was derived from this isolate which was infectious in human T-cell lines. Therefore this new isolate will be particularly useful for studies on the genetic evolution and biology of HIV-1 as well as for testing antiviral substances and for developing vaccines.

Reduced sensitivity to saquinavir: an update on genotyping from phase I/II trials.

A genotypic analysis of the HIV-1 proteinase was performed on clinical specimen obtained from patients after different periods of Saquinavir (SQV) treatment. Proteinase genes of integrated proviral DNA from PBMC were isolated by PCR, cloned and individual sequences were obtained. Genotypic resistance was defined by the Gly48-->Val and Leu90-->Met exchanges. Frequencies and kinetics of resistance development will be reported for phase I/II trials V13330. V13329, O13328 and ACTG229 in patients on monotherapy or combination therapy with RT inhibitors. Data from V13330 have been analysed in more detail for correspondence of genotypic and phenotypic resistance and any correlation between resistance and changes in plasma viral RNA load. Furthermore, we will discuss the data from our extensive proteinase gene sequence collection with respect to mutational changes which would be indicative of resistance to other inhibitors of HIV-1 proteinase.

Competitive polymerase chain reaction assay for quantitation of HIV-1 DNA and RNA.

A quantitative PCR assay for the detection of HIV-1 nucleic acids is described. The assay is based on a competitive internal standard nucleic acid which can be discriminated from target sequences by the presence of a new restriction enzyme site. The method was used to quantitate plasmid molecules containing HIV-1 sequences, HIV-1 DNA and HIV-1 RNA purified from HIV-1-infected tissue culture cells as well as HIV-1 DNA present in the peripheral blood mononuclear cells of an AIDS patient. The assay will be valuable for assessing viral load in AIDS patients and for the study of viral gene expression.

Identification of an amino acid substitution involved in the reduction of sensitivity of HIV-1 to an inhibitor of viral proteinase.

Clones derived from HIV variants previously characterized as resistant to Ro31-8959, an inhibitor of viral proteinase (PR), were sequenced. Substitution of glycine by valine at position 48 of the PR protein was found. None of the 20 clones derived from wild type HTLV-IIIB contain this mutation. Since such a position is located in a conserved region of PR, it is possible that the substitution can affect the interaction of the enzyme with the inhibitor.

Conserved residues Pro-109 and Asp-116 are required for interaction of the human immunodeficiency virus type 1 integrase protein with its viral DNA substrate.

The human immunodeficiency virus type 1 integrase protein can be specifically cross-linked to viral long terminal repeat substrate oligonucleotides in vitro by using UV light. Site-directed mutagenesis and deletion analyses were used to define the domains involved in the interaction of integrase with the viral DNA substrate. Our results showed that mutation of conserved residues Pro-109 and Asp-116, which are found to be critical for the endonuclease and integration activities of IN protein, abolished the ability of the protein to cross-link to its DNA substrate. Furthermore, deletion analysis experiments showed that removal of 39 amino acids from the amino terminus and deletion of 15 amino acids from the carboxyl terminus abolished DNA cross-linking.