Mário Arala-Chaves

A B-cell mitogen from a pathogenic trypanosome is a eukaryotic proline racemase

Lymphocyte polyclonal activation is a generalized mechanism of immune evasion among pathogens. In a mouse model of Trypanosoma cruzi infection (American trypanosomiasis), reduced levels of polyclonal lymphocyte responses correlate with resistance to infection and cardiopathy. We report here the characterization of a parasite protein with B-cell mitogenic properties in culture supernatants of infective forms, the cloning of the corresponding gene and the analysis of the biological properties of its product. We characterized the protein as a co-factor-independent proline racemase, and show that its expression as a cytoplasmic and/or membrane-associated protein is life-stage specific. Inhibition studies indicate that availability of the racemase active site is necessary for mitogenic activity. This is the first report to our knowledge of a eukaryotic amino acid racemase gene. Our findings have potential consequences for the development of new immune therapies and drug design against pathogens.

Evidence for circulating hemocyte proliferation in the shrimp Penaeus japonicus

We have observed by flow cytometric analysis that a small but consistent percentage of circulating hemocytes in phases S, G2 and M of the cell cycle can be detected in Penaeus japonicus. Significantly increased percentages of proliferating hemocytes (approximately three-fold increase) were observed after stimulation by lipopolysaccharide (LPS), p43 (an immunosuppressive lymphocyte mitogenic protein produced by Candida albicans) and a combination of LPS and p43. Moreover, an approximately six-fold increase in the percentage of proliferating hemocytes was also observed in animals infected with Fusarlum opp., as compared to non-infected shrimps. Furthermore, [3H] thymidine uptake in circulating hemocytes was 26 times greater in LPS stimulated than in non-stimulated shrimps. The present study suggests that circulating hemocytes of the shrimp P. japonicus can divide in vivo and that proliferation can be increased significantly after mitogenic or infectious stimulation.

Is there any kind of adaptive immunity in invertebrates

Abstract The possible existence of a peculiar form of adaptive immunity in invertebrates is important for a better understanding of immunological evolution and for the development of vaccination strategies. These may be relevant in the control of infectious diseases, common under intensive farming of economically important crustaceans. Adaptive immunity has been assumed to be absent from invertebrates because they lack the immunoglobulin (Ig), T cell receptor (TCR) and Major histocompatibility complex (Mhc) high diversity molecules. Since adhesion Ig super family (SF) molecules, which in mammals are known to be involved in adaptive immune response, are present in invertebrates, it can be postulated that they may also be responsible for invertebrate adaptive immunity. However, because invertebrate IgSF molecules are not phylogenetically homologous to those of vertebrates, the existence of an anticipatory immunity has not been accepted in invertebrates. It has also been postulated that the antigen receptors in invertebrates have a low range of diversity leading to similar responses to disparate immunostimulants. We have observed that the hemocyte proliferation rate (HPR) of Penaeus japonicus was increased by a similar extent after stimulation with different mitogens, although at a lower magnitude than after fungal infections. Besides, Drosophila responses discriminate between fungus and bacteria. Furthermore, upon comparison of the HPR after a single and a second challenge with fungal antigens, we observed that after a second challenge there was an increased HPR that correlated with cell activation. This increase was, however, much smaller than that observed in lymphocyte proliferation between a vertebrate primary and secondary immune response. This observation is suggestive of a peculiar form of adaptive immunity in invertebrates that can constitute, nevertheless, the basic tool for vaccination strategies.

Flow Cytometric Analysis of Molt-Related Changes in Hemocyte Type in Male and Female Penaeus japonicus

Hemocyte cell suspensions obtained from male and female Penaeus japonicus were individually analyzed by flow cytometry through forward and side light-scatter parameters. The hemocyte cell suspensions were further characterized after cell sorting. This type of cell analysis has several advantages over microscopy techniques. After staining with phenoloxidase and peroxidase, the hemocytes were classified into the three classic categories of hyaline, semigranular, and granular cells. Significant cyclic differences were detected among the molting stages in both sexes. The hyaline cell population was predominant before and soon after the molt, decreasing over the intermolt. This decrease was, however, more prolonged in females. Thus, the hyaline cell population was dominant in stages B, D0, and D1 in males and only in stages B and D1 in females. Semigranular cells became predominant in females during the D0 stage.

Role of Monocytes in the Up‐Regulation of the Early Activation Marker CD69 on B and T Murine Lymphocytes Induced by Microbial Mitogens

CD69 is an early marker of lymphoid cell activation. The authors report on an up‐regulation of CD69 in splenic B and T cells of C57Bl/6 mice after administration of lipopolysaccharide (LPS) or microbial immunosuppressive/mitogenic (ISM) proteins produced by C. albicans (p43) and African Swine Fever Virus (p36). This up‐regulation of CD69 was observed 6 and 24 h after mitogenic treatments. The same pattern of increased CD69 expression was observed in the lymph nodes of mice treated with p43 or LPS, whereas p36 treatment failed to induce increased CD69 expression in this organ. Intracellular calcium mobilization was induced in splenic B and T lymphocytes after incubation of total spleen cells with LPS, p43 or p36. This increase was higher in B than in T cells. Increased calcium mobilization was also seen in lymph node B cells after incubation with p43 or p36 and in lymph node T cells after p43 stimulation. Up‐regulation of CD69 expression on B and T cells was also observed after in vitro stimulation of spleen cells with the three mitogens used. Similar results were obtained with culture supernatants of macrophage/monocyte (Mφ) cells activated with LPS (LPS/MφCS). Stimulation of Mφ cells with LPS or with the ISM proteins is demonstrated by the increased production of nitrites by these cells. The increased in vitro expression of CD69 was, however, not abolished by monoclonal antibodies to Mφ cytokines such as IL‐6, IL‐10 or TNFα. No increased expression of CD69 was found in vitro on purified B or T cells, even when mixed upon stimulation with p43, p36, LPS or with LPS/MφCS. However, an increase in the expression of CD69 was observed on B cells co‐cultured with Mφ cells after treatment with LPS or p36. All three mitogens failed to induce increased CD69 expression on cultured T cells mixed with Mφ cells.

Increased Resistance to Systemic Candidiasis in Athymic or Interleukin-10-Depleted Mice

Candida albicans produces a virulence-associated immunomodulatory protein (p43), which activates lymphocytes polyclonally, suppresses specific antibody responses to Candida antigens, and potentiates systemic growth of C. albicans. In this study, athymic, unlike euthymic, C57BL/6 mice were resistant to systemic candidiasis and produced lower serum levels of interleukin (IL)-4 and IL-10. Pretreatment with p43 stimulated the production of both cytokines. Depletion of IL-10, but not of IL-4, in euthymic animals reestablished resistance and abrogated p43-dependent suppression of the specific antibody response and facilitated C. albicans growth. In agreement with these results, both immunosuppression and p43-mediated facilitation of the fungus growth were abolished in IL-10 knockout mice. These observations demonstrate the relevance of IL-10 in facilitating systemic candidiasis and suggest a critical role for the immunosuppressive virulence factor p43 in the process.

Semi‐Purification of an Immunosuppressor Substance Secreted by Streptococcus mutans that Plays a Role in the Protection of the Bacteria in the Host

Crude extracellular products of Streptococcus mutans (CEP‐Sm) suppress the proliferative response to phytohaemagglutinin of human peripheral blood mononuclear cells and the primary immune response of C57BL/6 mice to sheep erythrocytes. This immunosuppressive effect favours the survival of the microorganism, and the bacteria lose the ability to secrete immunosuppressor substance if previously subcultured several times. Cells incubated with CEP‐Sm and subsequently washed recover the ability to proliferate. Traces of CEP‐Sm or too short a time of contact between CEP‐Sm and the target immune system induced higher proliferative ratios or higher in vivo immune responses than controls, respectively. The proliferative values of cultures supplemented with CEP‐Sm were parallel to the control values up to a certain time, after which they dropped abruptly. This drop is followed by a prolileration, and the higher the amount of CEP‐Sm added to the cultures, the shorter the time until the proliferation. CEP‐Sm was fractionated by means of ion exchange chromatography followed by double preparative isoelectrofocusing, ending in a subfraction of isoelectric point between 3.9 and 4.2, containing a heat‐unstable material stainable by Coomassie blue bui unstainable by periodic acid Schiff or methylene blue, and having a maximum optical density of 280 nm.

II. The Effects of Thalidomide Treatment on Autoimmune-Prone NZB and MRL Mice are Consistent with Stimulation of the Central Immune System

We describe here some immunomodulatory effects of thalidomide on autoimmune‐prone mice. The highly increased synthesis of splenic IgM in NZB mice, of splenic and lymph node IgG of different subclasses in MRL/n mice, and of splenic and lymph node IgGl in MRL/lpr mice was markedly inhibited by thalidomide treatment. After a single treatment with 3mg of thalidomide, the following changes were observed in NZB mice: (i) an initial decrease in the numbers of large CD5+μhigh, and in the numbers of total CD5+μ−, CD5−μhigh, CD5+μhigh lymphocyte populations of the pleural cavity followed by a late increase in the numbers of large cells of the three cell populations; (ii) a consistent increase in the numbers of a CD5lowμlow pleural lymphoid population; (iii) a consistent reduction in the numbers of splenic large CD5+ B cells and an oscillatory increase in the number of cells with CD5− phenotype; (iv) a late reduction in the numbers of splenic total CD5+ B cells. These results are consistent with the notion that thalidomide controls a disease‐associated expansion of B cells in autoimmune prone mouse strains through a stimulatory effect of the drug on the immune system.

I. Immunostimulatory Effect of Thalidomide in Normal C57BL/6 Mice is Compatible with Stimulation of a Highly Connected Central Immune System

Although thalidomide has been used with success in the treatment of increasing numbers of autoimmune diseases, the therapeutic effects have not been satisfactorily explained so far. We describe here some findings that may contribute to a better understanding of the immunomodulatory effects of this drug. Several immunological changes were observed after treating C57BL/6 mice with 3 mg of thalidomide. The numbers of natural IgM PFC against sheep red blood cells were increased in the spleen, and occasionally a dramatic oscillatory increase in the numbers of non‐specific splenic IgM and IgG PFC was observed in these mice. However, these oscillatory increases were progressively lower, after two and three treatments with thalidomide at 20‐day intervals. Furthermore, the absolute numbers of splenic CD5+ B and CD5 B lymphocytes were increased whereas depletion of CD4+ CD8+ cells in the thymus and of lymphoid cells in the bone marrow was seen after a single treatment with 3 mg of thalidomide. Taken together, these results suggest that thalidomide stimulates both peripheral and central immune systems and consequently enhances the connectivity of the central immune system.

The biological effects induced in mice by p36, a proteinaceous factor of virulence produced by African swine fever virus, are mediated by interleukin-4 and also to a lesser extent by interleukin-10.

We have previously presented indirect evidence that both specific immunosuppression and lymphocyte mitogenicity induced in mice by p36, a proteinaceous factor of virulence produced by porcine monocytes infected by African swine fever virus, were consistent with a Th2‐driven response. Here we show: (1) Interleukin‐4 (IL‐4) and interleukin‐10 (IL‐10) mRNA expression in the spleen and thymus of C57BL/6 mice were displayed early after p36 inoculation. The expression of thymic IL‐10 mRNA occurred, however, later than that of IL‐4 mRNA. (2) Increased serum levels of these two cytokines were also soon detected after the protein inoculation. (3) Both immunosuppressive and mitogenic effects of p36 were absent in IL‐4 gene‐targeted mice and partially abrogated in mice depleted of IL‐4 by neutralizing monoclonal antibodies. (4) IL‐10 depletion abrogated the immunosuppressive but not the p36 lymphocyte mitogenic biological effects. (5) The increase in the serum concentrations of both IL‐4 and IL‐10 were lower in thymectomized than in non‐thymectomized mice. (6) The expression of interferon‐γ (IFN‐γ) mRNA was weakly or not at all induced in p36‐treated mice. Taken together, these results are in agreement with the promotion of a Th2 immune response induced by p36.