The carboxyl terminus of the hamster beta-adrenergic receptor expressed in mouse L cells is not required for receptor sequestration

The structural basis for agonist-mediated sequestration and desensitization of the beta-adrenergic receptor (beta AR) was examined by oligonucleotide-directed mutagenesis of the hamster beta AR gene and expression of the mutant genes in mouse L cells. Treatment of these cells with the agonist isoproterenol corresponded to a desensitization of beta AR activity. A mutant receptor that bound agonist but did not couple to adenylate cyclase showed a dramatically reduced sequestration response to agonist stimulation. In contrast, beta AR mutants in which the C-terminus was truncated and/or in which two regions that have been proposed as phosphorylation substrates for cAMP-dependent protein kinase were removed showed normal sequestration responses. These results demonstrate that agonist-mediated sequestration of the beta AR can occur in the absence of the C-terminus of the protein and reveal a strong correlation between effective coupling to Gs and sequestration.     

Random screening of proteins for HLA-A*0201-binding nine-amino acid peptides is not sufficient for identifying CD8 T cell epitopes recognized in the context of HLA-A*0201

HLA-A2 is the most frequent HLA molecule in Caucasians with HLA-A*0201 representing the most frequent allele; it was also the first human HLA allele for which peptide binding prediction was developed. The Bioinformatics and Molecular Analysis Section of the National Institutes of Health (BIMAS) and the University of Tübingen (Syfpeithi) provide the most popular prediction algorithms of peptide/MHC interaction on the World Wide Web. To test these predictions, HLA-A*0201-binding nine-amino acid peptides were searched by both algorithms in 19 structural CMV proteins. According to Syfpeithi, the top 2% of predicted peptides should contain the naturally presented epitopes in 80% of predictions (www.syfpeithi.de). Because of the high number of predicted peptides, the analysis was limited to 10 randomly chosen proteins. The top 2% of peptides predicted by both algorithms were synthesized corresponding to 261 peptides in total. PBMC from 10 HLA-A*0201-positive and CMV-seropositive healthy blood donors were tested by ex vivo stimulation with all 261 peptides using crossover peptide pools. IFN-gamma production in T cells measured by CFC was used as readout. However, only one peptide was found to be stimulating in one single donor. As a result of this work, we report a potential new T cell target protein, one previously unknown CD8-T cell-stimulating peptide, and an extensive list of CMV-derived potentially strong HLA-A*0201-binding peptides that are not recognized by T cells of HLA-A*0201-positive CMV-seropositive donors. We conclude that MHC/peptide binding predictions are helpful for locating epitopes in known target proteins but not necessarily for screening epitopes in proteins not known to be T cell targets.     

Radioimmunoassay of human serum antibody specific for adenovirus type 5-purified fiber.

A radioimmunoassay (RIA), utilizing a second antibody to separate immune complexes, was developed to provide a sensitive and specific measure of serum antibody to adenovirus type 5 (Ad 5) fiber. Purity of fiber antigen was ascertained by sodium dodecyl sulfate urea-polyacrylamide gel electrophoresis and isoelectric focusing in ampholyte pH gradients. After labeling with 125I to high specific activity, the iodinated fiber did not exhibit loss of antigenic reactivity and remained stable for 3 weeks when stored at minus 20 degrees C with supplemental protein. Rabbit anti-Ad 5 serum with a neutralization titer of 1:320 precipitated 50% of the labeled fiber at a serum dilution of 1:50,000 when tested by the RIA. In competition assays as little as 0.5 ng of unlabeled fiber per millimeter was sufficient to inhibit the 125I fiber-antibody reaction. Serum specimens from 20 volunteers, obtained before and after vaccination with purified Ad 5 fiber or hexon subunit vaccine, were tested by RIA, hemagglutination-inhibition (HI), and neutralization tests. A comparison of mean antibody titers of post-inoculation sera showed that the RIA was 300 and 1000 times more sensitive than the HI and neutralization tests, respectively. Moreover, 19 of the men who were negative by the standard serologic tests before vaccination were shown to have anti-fiber antibody, with a mean RIA titer of 1:1028. Specificity of the RIA was demonstrated by the lack of an increase in antibody to Ad 5 fiber among those individuals vaccinated with the hexon subunit. Thus, the development of a highly sensitive and reproducible RIA allows for the detection of antibody specific for the Ad 5 fiber in serum which contains antibodies to the different virion antigenic determinants associated with Ad 5.     

Productive replication of human adenovirus type 5 in canine cells

Development of immunocompetent patient-like models that allow direct analysis of human adenovirus-based conditionally replicative adenoviruses (CRAds) would be beneficial for the advancement of these oncolytic agents. To this end, we explored the possibility of cross-species replication of human adenovirus type 5 (Ad5) in canine cells. With a panel of canine tumor cell lines of both epithelial and mesenchymal derivations, we demonstrate that human Ad5 can productively infect canine cells. Since the biological behavior and clinical presentation of certain dog tumors closely resemble those of their human counterparts, our results raise the possibility of exploiting canine models for preclinical analysis of candidate CRAd agents designed for human virotherapy.     

HLA-A*02:07 is a protective allele for EBV negative and a susceptibility allele for EBV positive classical Hodgkin lymphoma in China

HLA-A2 protects from EBV+ classical Hodgkin lymphoma (cHL) in Western Europe, but it is unknown whether this protective effect also exists in the Chinese population. We investigated the association of HLA-A2 and specific common and well documented HLA-A2 subtypes with EBV stratified cHL patients (n = 161) from the northern part of China. Quantitative-PCR and sequence-based subtyping was performed to identify HLA-A2 positive samples and their subtypes. 67 (42%) of the cHL patients were EBV+. There were no significant differences in percentages of HLA-A2 positivity between cHL and controls (65% vs 66%) and between EBV+ and EBV− cHL patients (70% vs 61%). The frequency distribution of HLA-A2 subtypes was significantly different between EBV stratified cHL subgroups and controls. This difference was most striking for the HLA-A*02:07 type with a frequency of 38% in EBV+ cHL, 8% in EBV− cHL and 20% in controls. Significant differences were also observed for the HLA-A*02:07, HLA-A2 (non-02:07) and the A2-negative typings between EBV+ cHL vs controls (p = 0.028), EBV− cHL vs controls (p = 0.045) and EBV+ vs EBV− cHL cases (p = 2×10⁻⁵). In conclusion, HLA-A*02:07 is a predisposing allele for EBV+ cHL and a protective allele for EBV− cHL in the northern Chinese population.     

Synthesis of human adenovirus type 2 fiber protein in Escherichia coli cells

We have cloned and expressed in Escherichia coli the gene encoding the trimeric fiber protein of human adenovirus type 2. A gene expression system based on bacteriophage T7 RNA polymerase was used. Optimal gene expression was obtained with 1-h induction, at a temperature of 30 degrees C. The synthesized protein constituted about 1% of total host-cell protein. During induction, the growth of bacteria carrying the plasmid containing the fiber gene, was retarded compared with that of bacteria carrying the plasmid without the fiber gene. This toxic effect of fiber protein on bacterial hosts could be diminished by addition of glucose to the medium and by maintaining the pH above 7, thus improving the yield of recombinant fiber protein. The fiber protein produced in E. coli is stable during the course of induction. It is insoluble in buffers at physiological pH, in various salt solutions, and in the presence of nonionic detergents. It can be solubilized in 1% sodium dodecyl sulfate or in urea solutions above 2 M. There are indications that recombinant fiber trimerizes spontaneously, since after the removal of urea by dialysis at pH 8, recombinant fibers runs similarly to native trimeric fiber, on nondenaturing polyacrylamide gels. This trimer has, however, a less compact structure than native Ad2 fiber, since during gel filtration recombinant protein is excluded before native protein. It is also more sensitive to chymotrypsin digestion than native fiber.     

A new group B adenovirus receptor is expressed at high levels on human stem and tumor cells

CD46 is used by human group B adenoviruses (Ads) as a high-affinity attachment receptor. Here we show evidence that several group B Ads utilize an additional receptor for infection of human cells, which is different from CD46. We tentatively named this receptor receptor X. Competition studies with unlabeled and labeled Ads, recombinant Ad fiber knobs, and soluble CD46 and CD46 antibodies revealed three different subgroups of group B Ads, in terms of their receptor usage. Group I (Ad16, -21, -35, and -50) nearly exclusively uses CD46. Group II (Ad3, -7p, and -14) utilizes receptor X and not CD46. Group III (Ad11p) uses both CD46 and the alternative receptor X. Interaction of group II and III Ads with receptor X occurs via the fiber knob. Receptor X is an abundantly expressed glycoprotein that interacts with group II and III Ads at relatively low affinity in a Ca(2+)-dependent manner. This receptor is expressed at high levels on human mesenchymal and undifferentiated embryonic stem cells, as well as on human cancer cell lines. These findings have practical implications for stem cell and gene therapy.     

The type I insulin-like growth factor receptor is a motility receptor in human melanoma cells

Insulin-like growth factors I and II (IGF-I and II) and insulin are chemotactic agents for the human melanoma cell line A2058. As shown in this report, the motility receptor mediating this response is the heterodimeric type I IGF receptor. These three factors are able to compete with 125I-labeled IGF-I for binding to the cell surface with IC50 values equal to approximately 2 (IGF-I), approximately 150 (IGF-II), and approximately 300 nM (insulin). Cross-linking of 125I-IGF-I to the cell surface with disuccinimidyl suberate followed by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveals a 130-kDa protein (reduced) consistent with the alpha component of a type I receptor and a 38-kDa protein which does not bind insulin, and thus could be another IGF-I cell surface binding protein. The anti-IGF-I receptor monoclonal antibody (alpha IR-3) also competes with labeled IGF-I in binding experiments. In contrast, a control monoclonal antibody, matched to alpha IR-3 with respect to IgG subclass, has no significant effect on IGF-I binding. While alpha IR-3 inhibits the motility induced by IGF-I, IGF-II, and insulin, pertussis toxin (0.01-1.0 micrograms/ml) has no significant effect on the motility induced by the insulin-like growth factors or insulin on this cell line. Therefore, the type I IGF receptor appears to mediate a highly potent pertussis toxin-insensitive motility response to IGF-I, IGF-II, and insulin. In contrast, motility induced by the autocrine motility factor, a cytokine produced by the A2058 cells, is not affected by alpha IR-3 but is extremely sensitive to pertussis toxin. When mixtures of autocrine motility factor and IGF-I are employed to induce chemotaxis, the resulting motility is greater than that induced by either agent alone. These data indicate that motility in this melanoma cell line can be initiated through multiple receptors that stimulate the cells by separate transduction pathways. This capability to respond to multiple stimuli could enhance the metastatic potential.     

Carbohydrate structures of human interleukin 5 expressed in Chinese hamster ovary cells.

The asparagine-linked sugar chains of recombinant human interleukin 5 produced by Chinese hamster ovary cells were released quantitatively as oligosaccharides by hydrazinolysis. After N-acetylation followed by NaB3H4 reduction, each oligosaccharide was isolated by paper electrophoresis and serial lectin column chromatography. Study of their structures by sequential exoglycosidase digestion in combination with methylation analysis, revealed that they are bi-, tri-, and tetraantennary complex-type with fucosylated and non-fucosylated trimannosyl cores and high mannose type sugar chains. More than 80% of the sugar chains occur as biantennary complex-type sugar chains. Although acidic oligosaccharides amount to only 14% of the total oligosaccharides, their sialic acid residues occur exclusively as the Sia alpha 2----3Gal group. Removal of the sugar moiety from intact recombinant human interleukin 5 produced a 2.5-fold increase of its activity to induce IgM secretion.     

The E1B 19-kilodalton protein is not essential for transformation of rodent cells in vitro by adenovirus type 5.

The newly constructed adenovirus type 5 mutant in1 carries a single AT base pair insertion immediately after nucleotide position 1715 in the E1B gene sequence which destroys the proximal AUG normally present in E1B messages and prevents production of intact E1B 19-kDa protein in infected cells. We have used in1, variants of in1 containing mutant alleles of viral genes known to enhance transformation frequency, and adenovirus type 5 mutant dl337 (S. Pilder, J. Logan, and T. Shenk, J. Virol. 52:664-671, 1984), in which the sequence between nucleotides 1770 and 1916 within the 19-kDa reading frame is deleted, to test the generally accepted hypothesis that this E1B protein is essential for the transformation of rodent cells and maintenance of the transformed phenotype. We find that these mutants transform rat embryo cells, rat kidney and mouse kidney primary cells, and cells of the 3Y1 rat line with decreased frequencies only when virus is added to these various cells at high input multiplicities of infection. In contrast, when lower doses of virus are used, the mutants transform with wild-type frequencies. Cells infected with higher doses of mutant virus show increased levels of DNA degradation and cell killing compared with those of cells infected with the same levels of wild-type virus, and these effects most likely contribute to the decreased transformation frequencies observed. On the basis of these results and the results of phenotypic analyses of numerous transformants, we propose that the E1B 19-kDa protein is not required for induction and/or maintenance of transformed-cell characteristics in rodent cells infected with adenovirus type 5.     

Characterization of the knob domain of the adenovirus type 5 fiber protein expressed in Escherichia coli.

The adenovirus fiber protein is used for attachment of the virus to a specific receptor on the cell surface. Structurally, the protein consists of a long, thin shaft that protrudes from the vertex of the virus capsid and terminates in a globular domain termed the knob. To verify that the knob is the domain which interacts with the cellular receptor, we have cloned and expressed the knob from adenovirus type 5 together with a single repeat of the shaft in Escherichia coli. The protein was purified by conventional chromatography and functionally characterized for its interaction with the adenovirus receptor. The recombinant knob domain bound about 4,700 sites per HeLa cell with an affinity of 3 x 10(9) M-1 and blocked adenovirus infection of human cells. Antibodies raised against the knob also blocked virus infection. By gel filtration and X-ray diffraction analysis of protein crystals, the knob was shown to consist of a homotrimer of 21-kDa subunits. The results confirm that the trimeric knob is the ligand for attachment to the adenovirus receptor.     

Cytotoxic T-lymphocyte responses to human papillomavirus type 16 E5 and E7 proteins and HLA-A*0201-restricted T-cell peptides in cervical cancer patients

Previously, we found that human papillomavirus type 16 (HPV-16) E5 protein is a tumor rejection antigen and can induce cytotoxic T-lymphocyte (CTL) activity. Therefore, in this study, human leukocyte antigen A*0201 (HLA-A*0201)-restricted human CTL epitopes of HPV-16 E5 protein were identified using a bioinformatics approach, and the abilities of these predicted peptides to induce an immune response in HLA-A*0201 transgenic mice were confirmed by assaying E5-specific CTLs and in vitro-generated CTLs from normal peripheral blood T lymphocytes of HLA-A2-positive human donors. Second, the CTL responses to HLA-A*0201 CTL epitopes (E5 63-71 and E7 11-20) were examined in HPV-16-infected patients with HLA-A2. Third, the effect of HLA-A-type alleles on CTL activities in response to the entire E5 and E7 proteins was examined in cervical cancer patients. E5 and E7 peptides (but not the whole proteins) stimulated E5- and E7-specific CTL recall responses in HPV-16- and HLA-A2-positive cervical cancer patients, and HPV-16 E5 and E7 proteins stimulated na?ve T cells in HPV-16-negative cervical cancer patients with HLA-A11 and -A24 haplotypes. In summary, this is the first demonstration that E5 63-71 is an HLA-A*0201-restricted T-cell epitope of HPV-16 E5.     

The stem cell growth factor receptor KIT is not expressed on interstitial cells in bladder

The mast/stem cell growth factor receptor KIT has long been assumed to be a specific marker for interstitial cells of Cajal (ICC) in the bladder, with possible druggable perspectives. However, several authors have challenged the presence of KIT⁺ ICC in recent years. The aim of this study was therefore to attempt to clarify the conflicting reports on KIT expression in the bladder of human beings, rat, mouse and guinea pig and to elucidate the possible role of antibody‐related issues and interspecies differences in this matter. Fresh samples were obtained from human, rat, mouse and guinea pig cystectomies and processed for single/double immunohistochemistry/immunofluorescence. Specific antibodies against KIT, mast cell tryptase (MCT), anoctamin‐1 (ANO1) and vimentin were used to characterize the cell types expressing KIT. Gut (jejunum) tissue was used as an external antibody control. Our results revealed KIT expression on mast cells but not on ICC in human, rat, mouse and guinea pig bladder. Parallel immunohistochemistry showed KIT expression on ICC in human, rat, mouse and guinea pig gut, which confirmed the selectivity of the KIT antibody clones. In conclusion, we have shown that KIT⁺ cells in human, rat, mouse and guinea pig bladder are mast cells and not ICC. The present report is important as it opposes the idea that KIT⁺ ICC are present in bladder. In this perspective, functional concepts of KIT⁺ ICC being involved in sensory and/or motor aspects of bladder physiology should be revised.     

Purification and characterization of recombinant human interleukin 5 expressed in Chinese hamster ovary cells

Recombinant human interleukin 5 (rhIL-5) expressed in Chinese hamster ovary cells was purified and characterized. Molecular heterogeneity was observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two major components of Mr around 40,000 were detected under non-reducing conditions. However, under reducing conditions, the Mr of rhIL-5 was determined to be 22,000 and 20,000. After treatment with endoglycosidase F, a band with an apparent Mr of 18,000 was observed. Treatment of rhIL-5 with 2-mercaptoethanol followed by N-ethylmaleimide resulted in its dissociation into a monomeric form. This alkylated rhIL-5 was biologically less active than intact rhIL-5. These results suggest that rhIL-5 exists as a dimer, and that the heterogeneity of rhIL-5 is mainly due to the difference in the content of carbohydrate. Moreover, the formation of disulfide bond(s) might be important for the biological activity of rhIL-5.     

Toll-like receptor 2 is expressed by alveolar epithelial cells type II and macrophages in the human lung

The ability of the host to recognize pulmonary invasion by pathogenic organisms and establish an appropriate host response to infection requires innate immune defense mechanisms. Early bacterial clearance in the lung is mediated by alveolar macrophages (AM) and polymorphonuclear neutrophils. Additionally alveolar epithelial cells type II (AEC-II) may act as immunoregulatory cells. The toll-like receptors (TLR) are part of this innate immune defense, recognizing conserved patterns on microorganisms. Toll-like receptor 2 (TLR2) is crucial in detecting components of gram-positive bacteria and mycobacteria. Signals initiated by the interaction of TLR2 with bacterial components direct the subsequent inflammatory response. The detection of TLR2 mRNA in human lung tissue prompted us to localize the expression of mRNA and protein at the cellular level using a novel method for tissue fixation. We utilized HOPE-fixed lung specimen sections for targeting mRNA by in situ hybridization and protein by immunohistochemistry using the monoclonal antibody TL2.1. In normal lung areas the expression of TLR2 mRNA and protein was found to be located in cells resembling AEC-II and AM. Expression of mRNA was verified by RT-PCR and DNA sequencing. These results indicate a potential mechanism of increased immunosurveillance at the alveolar level controlling the localized infection.