Héctor Romero-Ramírez

Enforced and prolonged CD40 ligand expression triggers autoantibody production in vivo

CD40, a glycoprotein expressed on B lymphocytes plays an important role in B cell development, growth and differentiation. The ligand for the CD40 is a 39‐kDa glycoprotein (CD154) expressed onthe surface of activated T lymphocytes and is essential for thymus‐dependent humoral immunity. The expression of CD154 is tightly regulated and its transient expression reduces the chances of potentially deleterious bystander activation of B cells. Stimulation through CD40 has been studied in vitro by using antibodies against CD40, by membranes of activated T cells or lately, by CD154 transfected cells. In this work we have evaluated the outcome of CD40‐CD40 ligand interaction in vitro and in vivo by using CD154‐transfected L929 cells. In vitro assaysshowed that CD154‐L929 cells can induce on B cells: IL‐4‐dependent proliferation, up‐regulation of CD23, CD54 and class II molecules and can also rescue WEHI‐231 B cell lymphoma from anti‐IgM‐induced apoptosis. Interestingly, in vivo assays revealed that when CD154‐L929 cells were inoculated into the spleen, mice developed a strong but transient production of anti‐erythrocyte autoantibodies. Through B lymphocyte activation with CD154‐transfected L929 cells both in vitro and in vivo, our data reveal that enforced and prolonged expression of CD40 ligand overcomes the tightly regulated mechanisms of B cell activation, triggering the production of autoantibodies. This system might be used to evaluate the early steps of an autoimmune response and the role of CD40‐CD154 in the induction of primary responses in vivo.

Generation and characterization of a rat monoclonal antibody against the RNA polymerase protein from Dengue Virus-2

Dengue virus (DENV) RNA replication requires 2 viral proteins, non-structural protein 3 (NS3) and NS5. NS5 consists of 2 functional domains: a methyltransferase (MTase) domain involved in RNA cap formation and located in the amino terminal region and a RNA-dependent RNA polymerase domain essential for virus replication and located in the carboxyl terminal region. To gain additional insight into the structural interactions between viral proteins and cellular factors involved in DENV RNA replication, we generated a panel of rat monoclonal antibodies (mAbs) against the NS5 MTase domain. Six rat mAbs were selected from 41 clones, of which clone 13G7 was further characterized. The specificity of this antibody for NS5 was demonstrated by western blot of DENV-infected cells, which revealed that this antibody recognizes all 4 DENV serotypes. Furthermore, Western blotting analysis suggested that this antibody recognizes a sequential epitope of the NS5 protein. Positive and specific staining with 13G7 was detected predominantly in nuclei of DENV-infected cells, similarly a pattern was observed in both in human and monkey cells. Furthermore, the NS5 staining co-localized with a Lamin A protein (Pierson index: 0.7). In summary, this monoclonal antibody could be used to identify and evaluate different cellular factors that may interact with NS5 during DENV replication.

Generation and characterization of a monoclonal antibody that cross-reacts with the envelope protein from the four dengue virus serotypes

Dengue viruses (DENVs; serotypes 1–4) are members of the flavivirus family. The envelope protein (E) of DENV has been defined as the principal antigenic target in terms of protection and diagnosis. Antibodies that can reliably detect the E surface glycoprotein are necessary for describing and mapping new DENV epitopes as well as for developing more reliable and inexpensive diagnostic assays. In this study, we describe the production and characterization of a monoclonal antibody (mAb) against a recombinant DENV‐2 E protein that recognizes a sequential antigen in both native and recombinant form located in domain II of the E protein of all four DENV serotypes. We confirmed that this mAb, C21, recognizes a sequence located in the fusion peptide. In addition, C21 does not have neutralizing activity against DENV‐2 in an in vitro system. Furthermore, the C21 mAb is an ideal candidate for the development of research reagents for studying DENV biology because it cross‐reacts with the four dengue serotypes.

CD38 expression in early B-cell precursors contributes to extracellular signal-regulated kinase-mediated apoptosis

CD38 is a 45 000 molecular weight transmembrane protein that is expressed in immature and mature lymphocytes. However, the expression and function of CD38 during B‐cell differentiation in mice is poorly understood. Here, we report that CD38 is expressed from the earliest stages of B‐cell development. Pre‐pro‐B, pro‐B, pre‐B and immature B cells from murine bone marrow all stained positive for CD38. Interestingly, CD38 expression increases with B‐cell maturation. To assess the role of CD38 during B‐cell maturation, CD38‐deficient mice were analysed. CD38−/− mice showed a significant increase in both the frequency of B‐lineage cells and the absolute numbers of pre‐pro‐B cells in bone marrow; however, no other differences were observed at later stages. CD38 cross‐linking in Ba/F3 cells promoted apoptosis and marked extracellular signal‐regulated kinase (ERK) phosphorylation, and these effects were reduced by treatment with the mitogen‐activated protein kinase/ERK kinase inhibitor PD98059, and similar effects were observed in B‐cell precursors from bone marrow. These data demonstrate that B‐cell precursors in mouse bone marrow express functional CD38 and implicate the early ligation of CD38 in the ERK‐associated regulation of the B‐lineage differentiation pathway.

Measurement of Suppressor Activity of T CD4+CD25+ T Reg Cells Using Bromodeoxyuridine Incorporation Assay

The suppressor effect of T regulatory lymphocytes in co-cultures with T effector cells obtained by magnetic columns from healthy donors and activated by CD3/CD28 was measured by a proliferation assay using BrdU incorporation and an ELISA test. Tritiated thymidine incorporation was used as a reference since it is the gold standard for proliferation assays. Both methods were used simultaneously in the same samples in order to compare them. Correlation between them was statistically significant (p < 0.001). The purification using magnetic columns was very efficient since CD4+CD25+ cells were also FOXP3+ therefore; they were identified as suppressor T cells. The use of BrdU incorporation in suppression assays is an excellent method that avoids the use of radioactive contaminating materials.

CD38 protein deficiency induces autoimmune characteristics and its activation enhances IL-10 production by regulatory B cells

CD38 is a transmembrane protein expressed in B lymphocytes, and is able to induce responses as proliferation, differentiation or apoptosis. Several reports propose that CD38 deficiency accelerates autoimmune processes in murine models of autoimmune diabetes, lymphoproliferation and rheumatoid arthritis. Other reports have shown elevated CD38 expression in B and T cells from patients with autoimmunity; however, the role of CD38 is still unclear in the development of autoimmunity. Recently, it has been characterized as CD1dhi CD5+ regulatory B cell subpopulation able to produce IL‐10, and the loss of these cells exacerbates the autoimmunity in murine models. Here, we report that CD38−/− mice exhibited elevated titres of ANAS, anti‐dsDNA autoantibodies from 12 months of age and were higher by 16 months of age and mice presented kidney damage. Interestingly, there is a reduction in the survival of CD38−/− mice compared to the WT. Furthermore, CD38 is highly expressed by CD1dhigh CD5+ regulatory B cells, and the agonistic anti‐CD38 stimulus plus LPS was able to increase the percentage of this cell subset and its ability to induce IL‐10 production. Together, these results suggest that CD38 could play a role in the control of autoimmune diseases through their expression on regulatory B cells.

Identification of Helicobacter pylori Strain cagPAI+ and cagPAI− Antigens by IgG Antibodies from Sera of Experimentally Colonized Meriones unguiculatus (Mongolian gerbils)

Background:  Mongolian gerbils that are experimentally infected with Helicobacter pylori develop a chronic inflammation that is similar to natural infections in humans. The aim of this study was to compare the antigens of H. pylori cagPAI+ and cagPAI− strains that are expressed during Meriones unguiculatus colonization.

Regulatory IFN-γ-producing killer dendritic cells are enhanced in B6.MLR-Faslpr/J lupus-prone mice

A novel cell population denominated IFN‐γ‐producing killer dendritic cells (IKDCs) have been recently described. These cells are lymphocytes lacking B‐ or T‐ receptors, but they can be identified by the presence of B220+CD38+CD49b+ and low CD11c, among other cell surface markers. The main characteristics of IKDCs are the production of IFN‐γ and the ability to spontaneously kill tumor cells. We found that this population increases in B6.MLR‐Faslpr/J mice. Interestingly, IKDCs increase with age and are more abundant in mice older than 6 months onward. To analyze whether these cells have any role in the induction of the lupus‐like phenotype in the B6.MLR‐Faslpr/J mice, IKDCs were purified and transferred into 6‐month‐old B6.MRL‐Faslpr/J mice, then the presence of anti‐nuclear antibodies (ANAS) and anti‐dsDNA antibodies were analyzed 2 and 4 months after the transfer. The results showed a reduction in the levels of these autoantibodies and increased survival of these mice, indicating that these cells may have a regulatory function. In vitro assays demonstrated that IKDCs reduced the proliferation of both autoreactive B and T cells, suggesting that these may be the mechanisms used by these cells to ameliorate the lupus‐like phenotype in the B6.MRL‐Faslpr/J mice.