Functional expression of chemokine receptor 2 by normal human eosinophils.

BACKGROUND: Within the granulocytes, the CC chemokines preferentially activate basophils and eosinophils on binding to chemokine receptors (CCRs). In vivo administration of neutralizing anti-monocyte chemoattractant protein 1 (MCP-1) antibodies can block accumulation of eosinophils in the lungs of antigen-challenged animals. OBJECTIVE: We studied a panel of chemokines for chemotactic activity in normal human eosinophils from healthy donors with a special focus on MCP-1, identified the respective receptor required for the biological response of eosinophils, and investigated mediators used for signal transduction. METHODS: Cells were enriched by magnetic cell sorting. Receptor expression in eosinophils was shown by RT-PCR and fluorescence-activated cell sorting. The biological response was tested in chemotaxis and calcium mobilization assays. RESULTS: Eosinophils have detectable mRNA for CCR2, and the receptor protein is expressed on cell surfaces. MCP-1 induces chemotaxis and calcium mobilization in eosinophils. The chemotactic activity of MCP-1 revealed a double-peaked dose-response curve; one of the peaks is abolished by addition of a blocking antibody to CCR2, but it is insensitive to blocking of CCR1 or CCR3. Specific enzyme inhibitors ruled out signaling characteristics of CCR2 in eosinophils. CONCLUSION: Normal human eosinophils express functional CCR2 on cell surfaces.

Characterization of monoclonal antibodies specific for the human growth hormone 22K and 20K isoforms.

We have derived and characterized a set of monoclonal antibodies (mAb) specific for the different human GH (hGH) isoforms. The binding characteristics of each antibody to the hGH isoforms (22K and 20K) were analyzed in direct and competitive immunoassays as well as by Western blot. We studied the effects of these mAb on the biological activity of hGH and showed that they specifically block their respective activities. Using these mAb, we developed several immunoassays that have been applied for the quantitation of the different hGH isoforms in body fluids. Therefore, these mAb may help to unravel the biological function of these variants.

Human monoclonal Fab fragments specific for viral antigens from combinatorial IgA libraries.

BACKGROUND: IgA constitutes the first line of immune defense, interacting with a variety of environmental antigens. Following infection with respiratory syncytial virus (RSV) individuals frequently exhibit elevated serum IgA titers specific for the virus. Previously combinatorial IgG libraries have successfully been used to clone such human antibody responses. OBJECTIVES: Here we evaluate the possibility of constructing combinatorial IgA libraries on the surface of filamentous phage to retrieve human viral-specific IgA Fab fragments. STUDY DESIGN: Bone marrow from an HIV-1 seropositive donor was used as RNA source to construct combinatorial IgA kappa and lambda libraries of approximately 10(7) clones. RESULTS: By affinity selection using an immobilized recombinant RSV FG protein, two unique IgA Fab fragments producing clones (AD5 and AD23) reactive with the selecting antigen were isolated. One of the Fab fragments was found to be specific for RSV F glycoprotein and bind with high apparent affinity (2 x 10(8) M-1). The other binds with lower affinity and exhibits cross-reactivity with other antigens. CONCLUSION: The strategy described, involving construction of combinatorial IgA libraries on the surface of filamentous phage, should be generally applicable to the investigation of both mucosal and systemic human IgA immune responses, and may become an important tool for evaluation of mucosal vaccine regimes.

Swine-reconstituted SCID mice as a model for African swine fever virus infection.

Injection of swine peripheral blood mononuclear cells into mice with severe combined immunodeficiency (SCID), resulted in the stable long-term establishment of a functional swine immune system (SCID-sw). Swine immunoglobulins were present in the serum of SCID-sw mice and swine cells were detected in the blood as well as in lymph nodes and spleen using monoclonal antibodies raised against cell subpopulations. Swine lymphocytes from reconstituted SCID mice responded in vitro to specific antigens or mitogens. When SCID-sw mice were challenged with African swine fever (ASF) virus, ASF virus-infected cells were detected in blood and spleen, and antiviral antibodies and virus-specific T cells were generated.

T cell receptor-associated alpha-phosphatidylinositol 3-kinase becomes activated by T cell receptor cross-linking and requires pp56lck.

The activation of a phosphatidylinositol (PI) 3-kinase is one of the earliest intracellular responses to T lymphocyte stimulation. We have used a human T cell line, Jurkat, and an antibody that recognizes the clonotype of its T cell receptor (TcR) to characterize the association of PI 3-kinase with the TcR and its activation in response to TcR cross-linking. We show that the TcR is constitutively associated with the alpha isoform of the PI 3-kinase p85 regulatory subunit. Stimulation of Jurkat cells through the TcR results in the rapid and transient activation of the associated PI 3-kinase. In addition, our results indicate that the tyrosine kinase pp56lck is essential for PI 3-kinase activation via the TcR.

[Sociomedical syndrome. Intervention strategies to manage a new public health problem]. / El síndrome socio-sanitario. Estrategias de intervención ante un problema nuevo en salud pública.

Medical and social services are confronted with the increasing demands that our ageing society presents. Some patients (specially geriatric, chronically disabled and oncologic patients) pose multiple needs of social and medical care that very often are not met in a coordinated and comprehensive way. In this work authors present case management as a tool to achieve a better organization of social and medical resources in accordance with the demands of this growing part of the population.

Biological activity of bacterial surface components: bacterial extracts and defined bacterial cell wall components as immunomodulators.

Bacterial extracts obtained from pathogenic strains occurring in lung infections (Broncho Vaxom) or urogenital infections (Urovaxom) as well as defined surface components of Gram-negative bacteria purified from bacteria or obtained by chemical synthesis were tested for their immunomodulatory properties in a murine system. The bacterial extracts were able to act as immunogens inducing an antigen-specific response. Both the bacterial extracts and the purified bacterial cell wall components constituted polyclonal activators of murine splenic B cells, as demonstrated by proliferation assays measuring the incorporation of [3H]thymidine into DNA. They were also able to act as immunoadjuvants increasing the SRBC and the BSA-TNP specific immune response, and could induce tumor cytotoxicity in bone marrow-derived macrophages. The results show that bacterial extracts and defined bacterial surface components constitute immunogens as well as immunomodulators in vitro and in vivo.

Polyclonal B-cell activation by a synthetic analogue of bacterial lipoprotein is functionally different from activation by bacterial lipopolysaccharide.

The reactivity of 38 murine strains to a synthetic analogue of bacterial lipoprotein, tripalmitoyl-pentapeptide (TPP), was tested and compared with the reactivity to lipopolysaccharide (LPS). These strains include common laboratory mice and H-2 recombinant inbred lines, as well as some newly bred lines originating from animals recently captured in different regions of Europe. All animals analysed were reactive to TPP and polyclonally activated to proliferation and immunoglobulin synthesis. Large differences in mitogen reactivities of various H-2 recombinant inbred strains suggest that MHC or closely linked gene products influence the reactivity to the LPS and TPP mitogens. By analysing the frequencies of precursor cells reactive to TPP or LPS and the isotype patterns obtained after stimulation, we demonstrated that both mitogens activate individual B cells in different ways.

Alkaline phosphatase activity as a membrane marker for activated B cells.

Alkaline phosphatase activity was assayed by a microtiter assay (with p-nitrophenylphosphate used as substrate) in the plasma membrane of mouse spleen cells activated in vitro by the B cell mitogen LPS and the T cell-dependent B cell mitogen, PWM. No activity was detected in spleen cells cultured in the presence of the T cell mitogens PHA and Con A. This alkaline phosphatase activity was detected in the blast-enriched 30 to 40% fraction of discontinuous Percoll gradients in LPS-treated cultures, and the enzymatic activity assayed was susceptible to inhibition by the alkaline phosphatase inhibitors EDTA and L-phenylalanine. These data support the idea that the membrane alkaline phosphatase activity could be used as a marker for activated B cells.

Immunoglobulin C-gene expression. III. Possible induction of specific genetic events in activated B lymphocytes by the polyclonal stimuli driving clonal expansion.

Polyclonal activation of resting B lymphocytes by either lipopolysaccharide or specific helper cells recognizing antigens on B cell membranes results in selective patterns of IgG subclass expression among plaque-forming cells. We have studied the IgG subclasses of plaque-forming cells generated in cultures of purified B cell blasts selected for reactivity to either LPS or helper cells, and restimulated by either lipopolysaccharide, specific helper cells, or as "bystanders", by nonspecific B cell growth factors. Development of IgG1 plaque-forming cells is observed only when clonal expansion is maintained by specific helper cells, whereas IgG3 secretion specifically requires stimulation by lipopolysaccharide and the absence of helper cell activity. Furthermore, exposure of resting B lymphocytes to specific helper cell induces, in 48 h, an irreversible loss of the potential to produce IgG3. Other than showing that helper cell-dependent B cell growth and maturation is more complex than previously suspected, these results suggest that differentiation signals or factors induce specific DNA recombination and deletion events.

Hapten-specific helper T cells III. Fine specificity of the (4-hydroxy-3-nitro-phenyl)acetyl (NP)-specific response in Igh-1b mice.

Helper cells, with specificity for the haptens (4-hydroxy-3-nitro-phenyl)acetyl (NP) or (4-hydroxy-5-iodo-3-nitro-phenyl)acetyl (NIP), were raised in B10.BR mice by in vivo priming and in vitro long-term enrichment with hapten-derivatized syngeneic spleen cells. Upon co-culture with the homologous antigen (NP or NIP self), selected helper cells specifically responded by proliferation and by inducing large numbers of B cells to clonal expansion and immunoglobulin secretion. Criss-cross experiments demonstrated the nonheteroclitic nature of antigen recognition by helper cells, as the proliferative and helper cell activities were in every case one order of magnitude higher when confronted with the homologous hapten used for immunization.

Immunoglobulin C-gene expression. I. The commitment to IgG subclass of secretory cells is determined by the quality of the nonspecific stimuli.

Polyclonal stimulation of normal splenic B lymphocytes with either lipopolysaccharide (LPS) or helper T lymphocytes specific for B cell surface antigens results in the selective expression of IgG subclasses by the secretory cells: in addition to IgM-secreting plaque-forming cells (PFC), thymus-independent stimulation leads to the development of IgG2 and IgG3 PFC, while helper cell-dependent activation leads to IgG1 and IgG2 PFC. This cannot be solely explained by selective stimulation of distinct B cell subpopulations, because purified LPS-reactive blasts if restimulated by helper cells switch to IgG1 while if maintained with LPS switch to IgG3. The simultaneous stimulation of splenic B cells with LPS and helper cells results in additive IgM and IgG2 responses, but in the selective suppression of IgG3 PFC with a concomitant synergic enhancement of IgG1 responses. These results are interpreted to indicate that the expression of IgG C genes in proliferating B lymphocytes is directed by the quality of nonspecific stimuli.

Different macrophage requirement in the specific and polyclonal responses induced by TNP-LPS and LPS.

The in vitro anti-trinitrophenyl (TNP) response induced by TNP-lipopolysaccharide (TNP-LPs) in mouse spleen cells was eliminated by passage through Sephadex G10 columns. Conditioned medium obtained from peritoneal macrophages restored the response, indicating the supportive role of such cells in the response. Conversely, polyclonal B-cell activation mediated by LPS was not affected by Sephadex G10 treatment, as judged by incorporation of 3H-thymidine, generation of specific anti-TNP plaque-forming cells, and induction of polyclonal IgM-secreting cells. The failure of the LPS moiety of TNP-LPS to induce B-cell activation without macrophage help suggests a restriction in the number of available anti-LPS receptor molecules when the Ig anti-TNP receptor interacts with the haptenic (TNP) groups. This restriction can be due to the attachment of the TNP molecules to residues located in the vicinity of the lipid A mitogenic region of the carrier.

Wheat germ agglutinin activates macrophages for collaboration with B cells.

Wheat germ agglutinin (WGA), added to Mishell-Dutton-type cultures of nude spleen cells challenged with erythrocyte antigens, allows a primary specific plaque-forming cell (PFC) response. WGA, however, does not display direct mitogenicity to either T or B lymphocytes. The activity of WGA in the specific PFC response is macrophage-dependent and parallels a nonspecific PFC response in the same cultures.