ABSTRACT
BACKGROUND: We recently described a murine model of atopic dermatitis (AD) elicited by epicutaneous sensitization with ovalbumin (OVA). The skin lesions in these mice were characterized by a dermal infiltrate consisting of eosinophils and T cells and by increased expression of the TH2 cytokines IL-4 and IL-5. Epicutaneous sensitization induces a rise in the levels of serum total IgE and OVA-specific antibodies, further indicating that it elicits a predominantly TH2 response. OBJECTIVE: This study was undertaken to assess the roles of T cells, B cells, and CD40L-CD40 interactions in AD. METHODS: Mice with targeted gene deletions were sensitized with OVA. Histologic and immunohistochemical examinations, as well as measurements of IL-4 mRNA, were performed on OVA-sensitized skin. Total and antigen-specific serum IgE levels were determined. RESULTS: RAG2(-/-) mice, which lack both T and B cells, did not exhibit cellular infiltration, induction of dermal IL-4 mRNA, or elevation of serum IgE after OVA sensitization; all of these features were present in B-cell-deficient IgH(-/-) mice. T-cell receptor alpha(-/-) mice did not display cellular infiltration, IL-4 mRNA expression, or increased IgE levels after OVA sensitization, but these responses were elicited in T-cell receptor delta(-/-) mice after sensitization. Absence of CD40 had no effect on these responses. CONCLUSION: These results suggest that alphabeta T cells, but not gammadelta T cells, B cells, or CD40L-CD40 interactions, are critical for skin inflammation and the TH2 response in AD.
Subject(s)
B-Lymphocytes/physiology , CD40 Antigens/physiology , CD40 Ligand/physiology , Dermatitis, Atopic/metabolism , Receptors, Antigen, T-Cell, alpha-beta/physiology , Receptors, Antigen, T-Cell, gamma-delta/physiology , Animals , Disease Models, Animal , Eosinophils/immunology , Immunization , Interleukin-4/biosynthesis , Leukocytes, Mononuclear/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Ovalbumin/immunology , Skin/immunology , Skin/metabolismABSTRACT
IL-4 and CD40 ligation are essential for IgE synthesis by B cells. We have shown previously that hydrocortisone (HC) induces IgE synthesis in IL-4-stimulated human B cells. In this study we demonstrate that HC fails to induce IgE synthesis in B cells from CD40 ligand-deficient (CD40L-deficient) patients. Disruption of CD40L-CD40 interactions by soluble CD40-Ig fusion protein or anti-CD40L mAb blocked the capacity of HC to induce IgE synthesis in normal B cells. HC upregulated CD40L mRNA expression in PBMCs and surface expression of CD40L in PBMCs as well as in purified populations of T and B cells. Upregulation of CD40L mRNA in PBMCs occurred 3 hours after stimulation with HC and was inhibited by actinomycin D. Upregulation of CD40L mRNA and induction of IgE synthesis by HC were inhibited by the steroid hormone receptor antagonist RU-486. These results indicate that ligand-mediated activation of the glucocorticoid receptor upregulates CD40L expression in human lymphocytes.
Subject(s)
B-Lymphocytes/drug effects , CD40 Ligand/biosynthesis , Glucocorticoids/pharmacology , Immunoglobulin Class Switching , Immunoglobulin E/biosynthesis , B-Lymphocytes/immunology , CD40 Ligand/genetics , CD40 Ligand/immunology , Cells, Cultured , Humans , Hydrocortisone/pharmacology , Interleukin-4/pharmacology , Monocytes/drug effects , Monocytes/immunology , RNA, Messenger/analysis , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Up-RegulationABSTRACT
Advances in recombinant DNA manufacturing technology have now made possible the production of a highly purified and active recombinant factor IX (rFIX) product. Recombinant factor IX was developed by (1) stable insertion of the genes for both factor IX and PACE-SOL (a truncated, soluble serine protease needed to enhance the capacity of cells to remove the amino-terminal propeptide from rFIX) into Chinese hamster ovary cells; (2) selection of a cell line that was capable of expressing high amounts of active rFIX while growing in bioreactors containing a completely defined culture medium that does not contain blood or plasma products; and (3) inclusion of four independent chromatography steps, none of which require monoclonal antibodies. Furthermore, rFIX has been extensively tested to demonstrate similarity to plasma-derived factor IX and has been shown to be a consistent, high-purity product. For example, a high-specific-activity product (276+/-23 IU/mg) has been consistently produced throughout 65 consecutive batches from five consecutive manufacturing campaigns. Thus, rFIX offers a consistent and high-purity source of factor IX treatment for patients with hemophilia B.
Subject(s)
Factor IX/genetics , Factor IX/isolation & purification , Factor IX/standards , Animals , CHO Cells , Cricetinae , Factor IX/therapeutic use , Hemophilia B/drug therapy , Humans , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/standards , Recombinant Proteins/therapeutic useABSTRACT
The TRAF3 molecule interacts with the cytoplasmic carboxyl terminus (COOH terminus) of the Epstein-Barr virus-encoded oncogene LMP-1. NF-kappaB activation is a downstream signaling event of tumor necrosis factor receptor-associated factor (TRAF) molecules in other signaling systems (CD40 for example) and is an event caused by LMP-1 expression. One region capable of TRAF3 interaction in LMP-1 is the membrane-proximal 45 amino acids (188-242) of the COOH terminus. We show that this region contains the only site for binding of TRAF3 in the 200-amino acid COOH terminus of LMP-1. The site also binds TRAF2 and TRAF5, but not TRAF6. TRAF3 binds to critical residues localized between amino acids 196 and 212 (HHDDSLPHPQQATDDSG), including the PXQX(T/S) motif, that share limited identity to the CD40 receptor TRAF binding site (TAAPVQETL). Mutation of critical residues in the TRAF3 binding site of LMP-1 that prevents binding of TRAF2, TRAF3, and TRAF5 does not affect NF-kappaB-activating potential. Deletion mapping localized the major NF-kappaB activating region of LMP-1 to critical residues in the distal 4 amino acids of the COOH terminus (383-386). Therefore, TRAF3 binding and NF-kappaB activation occur through two separate motifs at opposite ends of the LMP-1 COOH-terminal sequence.
Subject(s)
Antigens, Viral/metabolism , Herpesvirus 4, Human/metabolism , NF-kappa B/metabolism , Oncogene Proteins, Viral/metabolism , Proteins/metabolism , Viral Matrix Proteins/metabolism , Zinc Fingers , Amino Acid Sequence , Binding Sites/genetics , Capsid , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed , NF-kappa B/genetics , TNF Receptor-Associated Factor 3 , Tumor Cells, CulturedABSTRACT
Using an adaptive strategy, Chinese hamster ovary (CHO) cell lines were developed that are capable of robust growth in serum-free suspension culture. These preadapted derivatives of the commonly used strain of CHO cells (CHO DUKX), termed PA-DUKX, were used for the introduction and stable expression of several heterologous human genes. A significant advantage of recombinant PA-DUKX cells was their ability to readily resume growth in serum-free suspension culture after transfection and amplification of heterologous genes. Expression of recombinant human proteins in PA-DUKX cells was quantitatively similar to that of lineages generated using conventional CHO DUKX cells. In addition, recombinant human proteins expressed by transfected PA-DUKX lineages were shown to be biochemically and structurally similar to those expressed in CHO DUKX cells, PA-DUKX host cell technology provides an opportunity for reducing the time and resources required to develop large-scale, suspension culture-based manufacturing processes employing serum-free medium. (c) 1996 John Wiley & Sons, Inc.
ABSTRACT
The current and potential impact of the human immunodeficiency virus (HIV) disease on the financial and human resources of institutions requires strategic planning by health care administrators and providers. The HIV disease devastates the lives of many individuals, and it continues to impact the health care industry with accelerating force. Fairfax Hospital has developed a comprehensive HIV program to address the current needs of people afflicted with this disease and to manage for the future. Goals and strategies are discussed.
Subject(s)
HIV Infections/nursing , HIV-1 , Health Resources/organization & administration , Patient Care Team/organization & administration , Hospital Bed Capacity, 500 and over , Hospitals, Proprietary/organization & administration , Humans , Patient Care Planning/organization & administration , Patient Education as Topic , VirginiaABSTRACT
The protein-tyrosine kinase activity of pp60c-src (c-Src) is inhibited by phosphorylation of tyr527, within the c-Src c-terminal tail. Genetic and biochemical data have suggested that this negative regulation requires an intact Src homology 2 (SH2) domain. Since SH2 domains recognize phosphotyrosine, it is possible that these two non-catalytic domains associate, and thereby repress c-Src kinase activity. Consistent with this model, an isolated Src SH2 domain expressed in bacteria as a GST fusion protein bound in vitro to a synthetic phosphotyrosine-containing peptide modeled on the C-terminal 13 residues of the c-Src tail. Binding was absolutely dependent on phosphorylation of tyr527 in the tail peptide, and was modified by both the length and sequence of the peptide. Competition experiments indicated only a moderate binding affinity between the Src SH2 domain and the phosphorylated tail. A distinct phosphotyrosine-containing peptide previously identified as binding the Src SH2 domain with high affinity stimulated c-Src tyrosine kinase activity in vitro, possibly by competing with the endogenous tail phosphorylation site for binding to the SH2 domain. Indeed, this activation was competitively inhibited by purified bacterial Src SH2 domain. These data provide direct evidence that the c-Src tail has an intrinsic affinity for the Src SH2 domain, and suggest that such an interaction in the intact molecule contributes to maintaining c-Src in an inactive form.
Subject(s)
Protein Sorting Signals/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Enzyme Activation , Mice , Molecular Sequence Data , Phosphorylation , Tyrosine/metabolismABSTRACT
Phosphorylation of the carboxyl terminus of pp60c-src, the product of the c-src protooncogene, at Tyr-527 suppresses its tyrosine kinase activity and transforming potential. It has been proposed that the phosphorylated carboxyl terminus of pp60c-src inhibits kinase activity by binding to the SH2 (src homology 2) domain. We have synthesized peptides corresponding to the carboxyl-terminal 13 residues of pp60c-src phosphorylated and nonphosphorylated at Tyr-527. A highly transforming mutant, pp60c-src(F527), in which Tyr-527 is mutated to Phe, bound to the phosphorylated peptide immobilized to Affi-Gel 10. Binding of the phosphorylated peptide was abolished by deletion of residues 144-175 in the SH2 domain but not by deletion of residues 93-143, which removes most of the SH3 domain. The phosphorylated peptide also bound to pp60v-src, the transforming protein of Rous sarcoma virus. Only traces of pp60v-src and pp60c-src(F527) bound to the corresponding nonphosphorylated c-src peptide. Normal pp60c-src bound much less efficiently to the phosphorylated peptide than did pp60c-src(F527). A phosphorylated peptide corresponding to the carboxyl terminus of the c-fgr protein also bound to pp60c-src(F527), but with weaker affinity. Furthermore, the phosphorylated synthetic carboxyl-terminal pp60c-src peptide markedly inhibited phosphorylation of pp60c-src(F527) during cytoskeletal kinase assays. These results provide direct evidence for models in which the phosphorylated carboxyl terminus of pp60c-src binds intramolecularly or intermolecularly to the SH2 domain of the c-src protein.
Subject(s)
Phosphopeptides/metabolism , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins pp60(c-src)/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Chickens , Chromatography, Affinity , Chromosome Deletion , Enzyme Activation , Genes, src , Mice , Models, Structural , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphopeptides/chemical synthesis , Phosphorylation , Phosphotyrosine , Protein Binding , Protein Conformation , Protein-Tyrosine Kinases/genetics , Proto-Oncogene Proteins pp60(c-src)/chemical synthesis , Proto-Oncogene Proteins pp60(c-src)/genetics , Substrate Specificity , Transfection , Tyrosine/analogs & derivativesABSTRACT
AIDS presents health care providers with complex medical management issues as well as the need to confront their own fears and prejudices. Hospice programs provide specialized care to persons who are in the final stages of the disease. As the epidemic grows, they increasingly also must respond to HIV-infected staff. A comprehensive workplace program prepares the hospice provider for these challenges and ensures an appropriate response. The four essential elements of such a program are policy development, staff education, supervisory training, and compliance monitoring. Policies to consider include infection control, occupational exposure, confidentiality, and the response to HIV-infected patients and employees. Comprehensive education ensures quality care for AIDS patients. Particularly crucial is the education of managers and supervisors. After policy implementation and training, procedures must be instituted to monitor compliance and develop corrective action. A bibliography of additional resource materials is provided.