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Biomedical subjects

F Alonso

Publications and source records attributed to F Alonso.

At least 19 recordsLinked to original sources
PubMed2000

Induction of aggregation in porcine lymphoid cells by antibodies to CD46.

CD46 is a major transmembrane glycoprotein that belongs to the regulator of complement activation (RCA) family. Recently, mAbs to human CD46 were shown to suppress IL-12 production. Here, we describe that mAbs against different porcine CD46 epitopes induced a marked adhesion of normal lymphocytes. Addition of low amounts of antibody to freshly isolated lymphocytes or thymocytes resulted in the clustering of the cells. Cross-linking of CD46 molecules seems essential since Fab fragments failed to induce aggregation. This aggregation was dependent on active cell metabolism and on the presence of divalent cations and required a functional cytoskeleton. It was not inhibited by antibodies to CD18, CD29, CD2, CD11a and CD11b. Staurosporine, an inhibitor of protein kinases, partially blocked the aggregation. This finding is indicative of a role of protein kinases in the transduction of the signal generated by CD46 engagement.

Animals↗
PubMed2000

Porcine reproductive and respiratory syndrome (PRRS) virus down-modulates TNF-alpha production in infected macrophages.

The effect of porcine reproductive and respiratory syndrome (PRRS) virus infection on the synthesis and secretion of TNF-alpha and other pro-inflammatory cytokines by porcine alveolar macrophages (PAM) was investigated as well as the effect that TNF-alpha has on the replication of this virus. A clear reduction of phorbol myristate acetate (PMA)-induced expression of TNF-alpha mRNA was observed in cells incubated with PRRS virus. Moreover, the presence of PRRS virus also induced a decrease in IL-1 alpha and MIP-1 beta mRNAs expression with respect to PMA-stimulated uninfected cells. According to these results, exposure to the PRRS virus led to a reduction of the TNF-alpha protein in supernatants of PMA-stimulated PAM. On the other hand, addition of recombinant porcine TNF-alpha to cultures clearly reduced virus replication; however the addition of TNF-alpha to cultures containing IFN-alpha did not result in a further reduction of the produced by IFN-alpha alone. This indicates the lack of synergy in the effect of these cytokines on viral replication.

Animals↗
PubMed2000

A porcine cell surface receptor identified by monoclonal antibodies to SWC3 is a member of the signal regulatory protein family and associates with protein-tyrosine phosphatase SHP-1.

SWC3 was defined at the First International Swine CD Workshop as a specific myelomonocytic antigen of 230 kDa with mAbs 74-22-15, 6F3 and DH59B. In this report, we describe two new mAbs (BL1H7 and BA1C11) that react selectively with granulocytes, monocytes and macrophages. These monoclonal antibodies (mAbs) recognize a molecule in the range of 90-115 kDa in immunoprecipitation and/or Western blotting analyses. Two-colour FACS analyses showed that the distribution of BL1H7 and BA1C11 antigens was identical to that of SWC3. Moreover, in this assay, mAb 74-22-15 appeared to partially block the binding of mAbs BL1H7 and BA1C11, suggesting that all these mAbs reacted with the same or spatially close epitopes. Cross-blocking analyses indicated that it was the case with mAbs 74-22-15 and BL1H7. Immunoprecipitation experiments with mAbs 74-22-15, BL1H7 and BA1C11, followed by immunoblotting with mAb BL1H7 confirmed that all three mAbs recognize the same molecule. Analysis of the N-terminal sequence carried out on the affinity purified protein revealed homology with members of signal regulatory protein (SIRP) family. Like other members of this family, after treatment with sodium pervanadate, SWC3 became phosphorylated in tyrosines, and associated with the protein-tyrosine phosphatase SHP-1.

Animals↗
PubMed1999

The porcine 2A10 antigen is homologous to human CD163 and related to macrophage differentiation.

The mAb 2A10 recognizes a 120-kDa protein with sequence homology to the human CD163 and whose expression is restricted to the cells of the porcine monocyte/macrophage lineage. While most of tissue macrophages express high levels of 2A10 Ag, bone marrow cells and a subset of blood monocytes are negative for this marker. The percentage of 2A10+ blood monocytes ranges between 5-50% depending on the donor. The phenotypic analysis indicates that these cells are more similar to mature macrophages than 2A10- monocytes. 2A10+ monocytes express higher levels of swine histocompatibility leukocyte Ag II, CD16, and the adhesion molecules very late Ag-4 (CD49d) and LFA-1 (CD11a) than 2A10- monocytes, while CD14 and SWC1 expression is lower. Both monocyte subsets also differ in their functional capabilities. 2A10+ monocytes induce a greater allogeneic response on T lymphocytes than 2A10- cells. LPS-stimulated 2A10+ and 2A10- monocytes both produce proinflammatory cytokines (TNF-alpha and IL-1alpha), but antiinflammatory IL-10 is only detected on the latter population. When 2A10- monocytes were cultured in medium containing pig serum, they acquired some phenotypic features of 2A10+ cells, expressing the 2A10 Ag. In contrast, when they were cultured in the presence of L929 supernatant as a source of GM-CSF, the 2A10 Ag expression remained low, scarcely increasing over basal levels. 2A10+ cells cultured with pig serum developed features that resemble monocyte-derived dendritic cells. These results indicate that 2A10+ monocytes could constitute a cell population in a more advanced maturation stage than 2A10- circulating monocytes.

Amino Acid Sequence↗
PubMed1999

Monoclonal antibodies 2F6/8 and 2A10/8 recognize a porcine antigen (SWC7) expressed on B cells and activated T cells.

This report describes the production and characterization of two monoclonal antibodies (mAbs), 2F6/8 and 2A10/8, that recognize a porcine antigen (SWC7) expressed on B cells in lymphoid tissues. The antigen was not detectable on resting PBMC but its expression could be induced after treatment with phorbol esters (PMA) but not by ConA, PWM, LPS or Ca ionophore. Kinetic studies showed that the antigen was expressed 24 h after PMA treatment, peaked at day 2 or 3 and slightly declined by day 6. Interestingly, the antigen was also found on a subset of CD3 + T cells, with levels of expression similar to those of B cells. By immunohistochemistry, the antigen was detected on follicular dendritic cells of germinal centers in tonsils, spleen, lymph nodes and Peyer's patches. MAb 2F6/8 precipitates a molecule of approximately 40 kDa under non-reducing conditions, and 24 kDa under reducing conditions. The restricted and tightly regulated expression of this antigen may reflect an important role in B cell differentiation within the germinal center. These mAbs will be useful reagents for phenotypic analysis of porcine lymphoid cell populations by flow cytometry and immunohistochemistry.

Animals↗
PubMed1999

Analysis of cellular immune response in pigs recovered from porcine respiratory and reproductive syndrome infection.

The cellular immune response to a European isolate of porcine reproductive and respiratory syndrome (PRRS) virus in animals recovered from the experimental infection has been studied in vitro. Peripheral blood mononuclear cells (PBMC) from these pigs proliferated specifically when they were stimulated with PRRS virus. This response was not detectable until 4 weeks after inoculation and remained for more than 3 months. Addition of blocking monoclonal antibodies to the cultures showed that this proliferation was mainly dependent on CD4(+) cells with the participation of SLA-class II molecules. T-cell cultures established by stimulating responding cells with PRRS virus and maintained in culture for up to 3 weeks showed an increase of CD8(+) CD4(+) and CD4(-) CD8(+) subsets within activated cells, gated according to their light scatter parameters, whereas CD4(+) CD8(-) cells declined along the time in culture. Within the activated cells, those expressing the TcR gammadelta receptor also increased, being most of them also positive for the CD8 marker. By RT-PCR, T-cells responding to the virus showed a Th1 type cytokine production pattern. During the culture period the cytotoxic activity against K-562 cells increased from 15 to 35% of specific lysis. This cellular immune response may play a relevant role in the clearance of PRRS virus and the recovery of the infection.

Animals↗
PubMed1999

Circulating levels of immunoreactive beta-endorphin in polycystic ovary syndrome.

The plasma levels of beta-endorphin were studied in 64 women with polycystic ovarian disease (PCOD), from whom was selected a group of 23 women with normal weight and amenorrhea of < 36 days. On day 21, beta-endorphin levels were: mean 64.92 pg/ml; SD 37.32 pg/ml; 95% CI 48.38-81.47 pg/ml. It was also observed that their levels of opioid peptide were reduced, compared with women who had normal ovulatory cycles, both in the follicular phase (mean 70.93 pg/ml; SD 24.59 pg/ml; 95% CI 76.84-99.77 pg/ml) and luteal phase (mean 88.30 pg/ml; SD 31.80 pg/ml; 95% CI 76.84-99.77 pg/ml). The results were statistically significant (p < 0.05) for levels in PCOD patients compared with those of the luteal phase in women with normal ovulatory cycles. The decreased levels of beta-endorphin were negatively related to luteinizing hormone (LH) levels, which might explain the rise of LH levels in women with PCOD who control their weight and at the time of amenorrhea, although it is not clear if central opioid activity is reflected in the peripheral blood.

Adolescent↗
PubMed1999

African swine fever virus infection induces tumor necrosis factor alpha production: implications in pathogenesis.

We have analyzed the production of tumor necrosis factor alpha (TNF-alpha) induced by in vitro infection with African swine fever (ASF) virus (ASFV) and the systemic and local release of this inflammatory cytokine upon in vivo infection. An early increase in TNF-alpha mRNA expression was detected in ASFV-infected alveolar macrophages, and high levels of TNF-alpha protein were detected by ELISA in culture supernatants from these cells. When animals were experimentally infected with a virulent isolate (E-75), enhanced TNF-alpha expression in mainly affected organs correlated with viral protein expression. Finally, elevated levels of TNF-alpha were detected in serum, corresponding to the onset of clinical signs. TNF-alpha has been reported to be critically involved in the pathogenesis of major clinical events in ASF, such as intravascular coagulation, tissue injury, apoptosis, and shock. In the present study, TNF-alpha containing supernatants from ASFV-infected cultures induced apoptosis in uninfected lymphocytes; this effect was partially abrogated by preincubation with an anti-TNF-alpha specific antibody. These results suggest a relevant role for TNF-alpha in the pathogenesis of ASF.

African Swine Fever↗
PubMed1999

Sarcoptic mange in Spanish ibex from Spain.

The Spanish ibex (Capra pyrenaica hispanica) population of the "Sierras de Cazorla, Segura y Las Villas" Nature Park (Spain) was isolated as the result of a severe epidemic of sarcoptic mange. In this context, the dynamic characteristics of the disease were analyzed in a wild group consisting of 35 animals from the beginning of the epizootic (when the mating period started) to the extinction of the population due to mange. Monthly tracking permitted the sequential characterization of the pathology in each animal. The duration of the disease was 2 to 3 mo, evolving to severe disease and terminating in death. Incidence and prevalence rates in terms of morbidity and severity, and mortality and lethality were calculated. At the end of the mating season, 81% of the population were affected. There were no statistically significant differences in severity of the disease across sex or age categories of the animals. Most of the carcasses were found in caves used as refuge and/or near rivers or streams. Additionally, 46 of the 63 (73%) ibex captured in different areas of the nature park were naturally infected with the Sarcoptes scabiei. Infected ibex were examined for number of mites during the initial stage of the disease (n = 3), in the development stage (n = 12), in the consolidation stage (n = 17), and in the chronic stage (n = 14). The prevalence of mites in different anatomical regions was determined in each of these phases of the infection. A histological study of the skin lesions was conducted in 22 animals. Both the clinical and the pathological (macroscopic and microscopic) aspects of the sarcoptic mange in Spanish ibex corresponded to the classic description of sarcoptic mange in other wild and domestic small ruminants.

Age Factors↗
PubMed1998

Overview of the Second International Workshop to define swine cluster of differentiation (CD) antigens.

The aim of the Second International Swine Cluster of Differentiation (CD) Workshop, supported by the Veterinary Immunology Committee (VIC) of the International Union of Immunological Societies (IUIS), was to standardize the assignment of monoclonal antibodies (mAb) reactive with porcine leukocyte differentiation antigens and to define new antibody clusters. At the summary meeting of the workshop in July, 1995, revisions in the existing nomenclature for Swine CD were approved, so that the rules are now in accord with those for human and ruminant CD. Swine CD numbers will now be given to clusters of mAb to swine orthologues of human CD molecules when homology is proven by (1) suitable tissue distribution and lymphoid cell subset expression, (2) appropriate molecular mass of the antigen recognized by the mAbs, and (3) reactivity of mAbs with the cloned swine gene products, or cross-reactivity of the mAb on the human gene products. In some cases, this reactivity would not be fully proven, mainly due to the lack of cloned gene products; for these CD antigens, the respective clusters will be assigned by the prefix 'w' which will lead to 'wCD' antigens. As a result of the Second International Swine CD Workshop the assignment of 16 mAb to existing CD groups (CD2a, CD4a, CD5a, wCD6, wCD8, CD14, CD18a, wCD21, wCD25) was confirmed, and 2 mAb to existing swine workshop clusters (SWC). More importantly, for the work on the porcine immune system, was the definition of 5 new swine CD antigens, namely CD3 (recognized by 6 new mAb and 3 epitopes), CD16 (1 new mAb), wCD29 (2 mAb), CD45RA (3 mAb) and CD45RC (1 new mAb). Finally, the demarcation of two new SWC molecules in swine, SWC8 (2 mAb) and SWC9 (2 mAb) was confirmed.

Animals↗
PubMed1998

Porcine myelomonocytic markers: summary of the Second International Swine CD Workshop.

Forty five mAbs submitted to the Second International Swine CD workshop were analyzed by six different laboratories for their possible reactivity with porcine myelomonocytic cells using flow cytometry and immunohistochemistry. As a result of these analyses, a new swine workshop cluster, SWC9, composed of two mAbs that recognize an antigen selectively expressed on mature macrophages, was defined. In addition, several mAbs were identified, allowing the differentiation of granulocytes from monocytes/macrophages, or monocytes from macrophages. Further work is required to identify the antigen recognized by these mAbs. Nevertheless, they should already prove useful for the identification of different stages in the macrophage maturation/differentiation, and will certainly aid analyses on the complexity of the mononuclear phagocyte system in the pig. Finally, the cross-reactivity of three anti-human CD14 mAbs with porcine myelomonocytic cells was established in this workshop.

Animals↗
PubMed1998

Workshop studies with monoclonal antibodies identifying a novel porcine differentiation antigen, SWC9.

Two monoclonal antibodies (mAb) within cluster M4 of the myeloid section of the Second International Swine CD Workshop, C4 (No. 144) and PM18-7 (No. 192), showed reactivity with thymocytes and among cells of myelomonocytic origin with mature macrophages but not with monocytes and granulocytes. Both mAb recognize a protein showing two bands of 205 kDa and 130 kDa under both reducing and non-reducing conditions. Although epitope mapping with these mAb could not be performed, this cluster received the SWC9 designation.

Animals↗
PubMed1998

A reliability study of the Spanish version of the social behaviour schedule (SBS) in a population of adults with learning disabilities.

The reliability of the Spanish version of the Social Behaviour Schedule (SBS) was tested in a vocational setting on a sample of 64 subjects with learning disabilities. Test-retest assessment showed a good percentage of agreement (80%) and adequate kappa values for most SBS items. The overall percentage of agreement of inter-rater reliability was 85% and kappa values were moderate to nearly perfect for 52% of items. Inter-informant analyses produced poorer results, with an average agreement of 43% and inadequate kappa values on 42% of items. The intraclass correlation coefficient (ICC) was 0.64 for test-retest, 0.76 for inter-rater assessment and 0.94 for inter-informant assessment. The Spearman correlation coefficient was adequate on the test-retest and inter-rater analyses, but not on inter-informant analysis. This low inter-informant agreement could be attributed to environmental factors which alter the reliability of reports from different informants in community settings with high levels of normalization. In such environments, an interview with a key informant may not suffice, and both a careful review of the clinical record and a direct interview with subjects may enhance the reliability of the information attained.

Adult↗
PubMed1997

Monoclonal antibodies to a high molecular weight isoform of porcine CD45: biochemical and tissue distribution analyses.

This report describes the obtention and characterization of two monoclonal antibodies (mAbs), 6E3/7 [mAb 6E3/7 was submitted to the Second International Swine CD Workshop, where it has been assigned to CD45R] and 3C3/9, which recognize the isoform of highest molecular weight of porcine CD45. This conclusion is based on their cell reactivity and tissue distribution, identical to that reported for the human high molecular weight isoform of CD45, and on data from immunoprecipitation and immunoblotting analyses which show that these mAbs react with the largest polypeptide of those precipitated by mAb 2A5, that recognizes an epitope shared by all CD45 isoforms. These mAbs react with 60% of peripheral blood mononuclear cells (PBMC) but not with alveolar macrophages, granulocytes, platelets or erythrocytes. Antigen expression on PBMC is heterogeneous and is reduced after in vitro activation with mitogens. B cells and CD8+ T cells express more antigen than CD4+ T cells. Using immunoperoxidase techniques, the antigen was detected on B cell areas of lymph nodes and Peyer's patches, and on a subpopulation of medullary thymocytes. These mAbs will be useful reagents for functional and phenotypic analysis of porcine lymphoid cell populations by flow cytometry and immunohistochemistry.

Animals↗
PubMed1997

African swine fever virus-specific cytotoxic T lymphocytes recognize the 32 kDa immediate early protein (vp32).

African swine fever (ASF) virus-specific cytotoxic T lymphocyte (CTL) activity has been studied in a model in which SLA inbred minipigs were experimentally infected with an attenuated isolate of the virus. The CTL assays were performed using alveolar macrophages as target cells. The specific lysis is mediated by purified CD8+ lymphocytes but not by CD4+ cells and can be blocked by incubation with anti-SLA class I monoclonal antibodies. The purified CD8+ population produced high levels of interferon-gamma after ASF virus stimulation. In an attempt to define the viral proteins recognized by CTL, target cells infected with a recombinant vaccinia virus (VV) expressing the ASF virus p32, an immediate early protein during ASF virus replication, were recognized and lysed by CTL. This assay may be useful for VV recombinant screening in order to identify other potential target ASF virus proteins.

African Swine Fever Virus↗