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.

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.

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-gamma (IFN-gamma) 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.

Purification, and biochemical and biological characterization of an immunosuppressive and lymphocyte mitogenic protein secreted by Streptococcus sobrinus.

An immunosuppressive/mitogenic (ISM) protein was purified from the supernatants of cultures of Streptococcus sobrinus with an isoelectric point of 4.75 and a relative molecular mass of 38 kDa (p38). Treatment of C57BL/6 mice with p38 induced an increase in the numbers of non-specific splenic Ig-secreting plaque-forming cells (PFC) with peak responses on day 3 for IgM-secreting PFC and on day 5 for IgG-secreting PFC, with an isotype pattern consisting predominantly of IgG2a and IgG2b. This increase was accompanied by a lymphocyte blastogenic response of both T and B lymphocytes. The in vitro effects of p38 on pure B, T and total splenic lymphocytes indicated that this ISM protein was primarily a B cell mitogen, being T cells activated subsequently by the generation of B blasts. Suppression of the murine primary immune response against sheep red blood cells was observed in C57BL/6 mice treated 4 days before with p38. The amino acid sequence of the N-terminus of p38 has a significant similarity with several enolases, particularly with rabbit enolase. However, the biological effects ascribed to p38 have not been detected after in vivo treatment with that enolase. The immunosuppressive effect of p38 was abrogated by depletion of IL-10 but not of IL-4. In agreement with this observation IL-10 was the only cytokine detected in serum of C57BL/6 mice after p38 treatment and the peak of serum levels was observed as soon as 2 h after treatment.

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.

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 (M phi) cells activated with LPS (LPS/M phi CS). Stimulation of M phi 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 M phi cytokines such as IL-6, IL-10 or TNF alpha. 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/M phi CS. However, an increase in the expression of CD69 was observed on B cells co-cultured with M phi cells after treatment with LPS or p36. All three mitogens failed to induce increased CD69 expression on cultured T cells mixed with M phi cells.

Immunoprotection against systemic candidiasis in mice.

We have previously described an immunosuppressive B cell mitogenic (ISM) protein, p43, produced by Candida albicans, which plays an important role in the survival of the microorganism in the host. The N-terminal amino acid sequence of p43 was found to be different from all amino acid sequences registered in updated protein databanks. Immunization of BALB/c mice with p43 partially neutralized the biological effects of this protein, namely depletion of bone marrow pre-B and B cells, the increased numbers of total and large B and CD4+ lymphocytes, and the non-specific polyclonal response of splenic IgG2a-, IgG2b- and IgM-secreting plaque forming cells. Immunization of BALB/c mice with p43 fully protected the mice against the fungal infection. In contrast, immunization with C. albicans sonicates (Cs) was not protective. Our data indicated that specific antibodies against p43 protected, whereas those against Cs facilitated C. albicans infection. Thus, the ratio between anti-p43 and anti-Cs antibody titres was much lower in the non-protected mice (Cs-immunized and control non-immunized) than in p43-immunized mice. Moreover, passive administration of specific anti-p43 antibodies significantly protected against fungal infection, whereas passive administration of specific anti-Cs antibodies markedly increased the susceptibility to C. albicans infection. These observations are discussed on the basis of alternative approaches of immunointervention.

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.

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 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 IgG1 in MRL/lpr mice was markedly inhibited by thalidomide treatment. After a single treatment with 3 mg of thalidomide, the following changes were observed in NZB mice: (i) an initial decrease in the numbers of large CD5+ microhigh, and in the numbers of total CD5+ micro-, CD5- microhigh, CD5+ microhigh 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 CD5low microlow 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.

Immunological activities of a Candida albicans protein which plays an important role in the survival of the microorganism in the host.

A protein with an isoelectric point of 4.3 and a relative molecular mass of 43 kDa (p43) was purified from the supernatants of the cultures of pathogenic Candida albicans but could not be detected in the supernatants of cultures of this fungus with pathogenicity previously attenuated after being repeatedly subcultured in vitro. Treatment of BALB/c and C57BL/6 mice with p43 resulted in (i) marked increases in the numbers of splenic immunoglobulin-secreting plaque-forming cells (PFC) with peak responses of immunoglobulin M (IgM) PFC preceding those of IgG PFC, with an isotype restriction pattern of IgG2a > IgG2b > IgG3 > IgG1 > IgM, and (ii) specific immunosuppression of the murine primary immune response against sheep erythrocytes. Immunosuppressive and B-cell mitogenic properties of p43 were quantitatively associated and inversely correlated with susceptibility to C. albicans infection. C57BL/6 mice treated with p43 2 days before inoculation with C. albicans were considerably more susceptible to the fungal infection than untreated mice. The immunobiological and chemical properties of p43 are compared with previously described immunosuppressive and B-cell mitogenic proteins produced by bacteria and viruses, and strategies for immunointervention are discussed.

Peripheral lymphoid hyperplasia and central lymphoid depletion in mice treated with a bacterial B-cell mitogen (F3'EP-Si/p90).

In order to further understand the mechanism mediating the mitogenic and immunosuppressor effects of p90, a protein produced by Streptococcus intermedius, flow cytometric studies were performed on peripheral and central lymphoid organs of mice treated with this protein. p90 induced a strong blastogenic B-cell response in the spleen and lymph nodes, followed by a slight but significant polyclonal T-cell activation. B-cell repertoire analysis indicated that polyclonal B-cell responses affected similarly both CD5+ and conventional (CD5-) B cells in the spleen. Repertoire analysis of T cells failed to reveal any preferential stimulation of the V beta T-cell receptor (V beta-TcR) families studied. Peripheral lymphoid hyperplasia was observed concomitantly with central lymphoid depletion. In the bone marrow, pre-B and B cells were profoundly depleted, with a more pronounced effect on small pre-B cells. In the thymus, double-positive (CD4+CD8+) thymocytes were preferentially eliminated, with a relative enrichment of single positive (either CD4+ or CD8+) and double-negative (CD4-CD8-) thymocytes.

V-region-related and -unrelated immunosuppression accompanying infections.

This paper discusses current evidence for the relationship between polyclonal lymphocyte activation, specific immunosuppression with decreased resistance, and autoimmune pathology, that are all often found associated with infections by a variety of virus, bacteria and parasites. The central question of class determination of immune effector activities is considered in the context of the cellular targets for nonspecific mitogenic activities associated with infection. A model is presented to integrate these findings: mitogens produced by the microorganism or the infected cells are preferentially active on CD5 B cells; the resulting over-production of IL-10 will tend to bias all immune activities into a Th2-mode of effector functions, with high titers of polyclonal antibodies and little or no production of gamma IFN and other "inflammatory" lymphokines that often mediate resistance. In turn, these conditions allow for parasite persistence and the corresponding long-term disregulation of self-directed immune reactivities, resulting in autoimmunity in the chronic phase. This model would predict that selective immunization with the mitogenic principles involved in deregulation, could stand better chances than strategies of vaccination based on immunopotentiation against other, functionally neutral antigenic epitopes. It is argued, however, that the complexity of immune responses and their regulation, together with our ignorance on the genetic controls of class-determination, offer poor prospects for a scientifically-based, rational development of vaccines in the near future. It is suggested that empirically-based and technologically developed vaccines might succeed, while basic scientific approaches are reinforced and given the time to provide a better understanding of those processes.

Recombinational biases in the rearranged C1-inhibitor genes of hereditary angioedema patients.

DNA structural changes responsible for hereditary angioedema were sought in the C1-inhibitor gene, which contains unusually dense clusters of Alu repeats in various orientations. Among patients belonging to 45 unrelated families, eight partial C1-inhibitor gene deletions and a partial duplication were found. Four deletions had one of the boundaries within the gene and the other in extragenic regions--in three cases 5' of the gene and in one case 3' of the gene. The boundaries of the partial duplication and of the remaining four deletions mapped instead within a few kilobases of exon 4. The same element--Alu 1--the first of three tandem Alu repeats preceding exon 4, contained one of the breakpoints of each of these five rearrangements. Moreover, these recombination breakpoints spread over the entire length of Alu 1, in contrast with the tight clustering observed near the 5' end of Alu sequences rearranged in other human genes. Thus, two uncommon recombinational biases are observed in the Alu rearrangements of hereditary angioedema patients; one promotes the occurrence of intragenic breakpoints in a single Alu repeat, and the other allows the breaks to be distributed over the entire Alu structure rather than within the hot spot of the left Alu monomer. A region of potential Z-DNA structure, located 1.7 kb upstream of Alu 1, may contribute to both peculiarities.

Susceptibility to infection with Mycobacterium avium is paradoxically correlated with increased synthesis of specific anti-bacterial antibodies.

A comparison was made between the levels of splenic and intestinal (Peyer's patches and thin intestinal epithelium) Ig production of C57BL/6 germ free and conventional C57BL/6, BALB/c, DBA/2 and C3H/He mice and the susceptibility to Mycobacterium avium infection, evaluated by the number of bacterial colony-forming units (CFU) found in the liver and in the spleen of the animals. Mice received an i.p. injection of either 5 x 10(6), 10(7) or 10(8) bacteria, or were given the larger inoculum intragastrically. Alternatively, mice were treated with an i.p. injection of M. avium bacterial sonicates. A marked increase of splenic IgA production, quantitatively associated with the size of the inoculum and thus with the degree of infection, was observed in susceptible compared to relatively resistant mice. This increase was observed at an earlier time following infection with the larger rather than with the smaller inocula. Consistent significant increases in splenic production of IgG isotypes were only observed in the susceptible mice after infection with the intermediate and larger inocula whereas a comparative increase of IgM was only clearly observed after infection with the larger inoculum. Intestinal Ig production remained unchanged, however, in both susceptible and relatively resistant mice after i.p. infection. Also, all mice were resistant to M. avium infection by the intragastric route and with this site of entry splenic and intestinal Ig production remained unchanged. Susceptibility to M. avium infection was also quantitatively associated with increased levels of circulating specific anti-bacterial antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of B and T lymphocytes in the specific immunosuppression induced by a protein released by porcine monocytes infected with African swine fever virus.

Some immunobiological aspects of host responses to an immunosuppressive protein (p36) released by porcine monocytes upon infection with African swine fever virus were analysed in a murine system. Treatment of normal, adult C57BL/6 mice with p36 (i) significantly delayed allogenic skin graft rejection; (ii) suppressed the specific plaque-forming cell response to immunization with heterologous erythrocytes; but (iii) induced marked increases in the numbers of 'background' splenic Ig-secreting plaque-forming cells. Cytofluorometric analysis of spleen cells revealed that a considerable fraction of all B cells, as well as CD4 and CD8 T lymphocytes, undergo blast transformation after p36 treatment. The immunosuppressive effects do not seem to result from 'antigenic competition', for they cannot be induced by even higher doses of pig albumin or by culture products of non-infected pig monocytes. Suppression of specific antibody responses and stimulation of 'background' plaque-forming cells are both T cell-dependent, since they are markedly reduced in thymectomized mice and in animals treated with anti-CD4 or anti-CD8 antibodies. This suggests the relationship between non-specific stimulation and specific suppression of 'unrelated' immune responses and reinforces the notion that viral-associated immunosuppression may be due to overstimulation. The present murine experimental model may prove valuable in the study of immunosuppression associated with infection, even for microorganisms which do not infect mice.

Low T- and B-cell reactivity is an apparently paradoxical request for murine immunoprotection against Streptococcus mutans. Murine protection can be achieved by immunization against a B-cell mitogen produced by these bacteria.

C57BL/6 mice thymectomized as adults or depleted of CD4+ cells were much less susceptible than intact conventional mice to the B-cell mitogenic and specific immunosuppressive effects of a protein designated as F5'EP-Sm secreted by Streptococcus mutans. These mice were also considerably more resistant to infection by these bacteria than intact individuals. The immunosuppressor effect of F5'EP-Sm was also abrogated, however, in conventional intact mice when immunized intraperitoneally against heat-inactivated F5'EP-Sm. On the other hand, resistance to bacterial infection could be achieved by immunization of conventional intact C57BL/6 mice against heat-inactivated F5'EP-Sm by intraperitoneal or intradermal routes even when the animals were infected 3 months after immunization and even when the immunization procedure did not include Freund's adjuvant, which was the case with the intradermal route. Interestingly, the protection against the bacterial infection was accompanied by only a minor increase in specific serum antibodies against F5'EP-Sm. These results are discussed in the context of adequate strategies for immunoprotection against Streptococcus mutans and other micro-organisms which are secretors of substances that share both B-cell mitogenic and immunosuppressive properties and which are thus able to suppress the immune response by overstimulation of the immune system of the host.

Erythema multiforme associated with autoreactivity to 17 alpha-hydroxyprogesterone.

We report a case of a 23-year-old woman who was afflicted with disseminated skin erythema multiforme-like eruptions that started at the menarche, relapsed at the premenstrual periods, dramatically spread during two pregnancies and cleared after abortion; the skin lesions responded dramatically to thalidomide treatment. A high-affinity binding factor to 17 alpha-hydroxyprogesterone (17-OHP) was found in the serum of this patient. Her lymphocytes did not proliferate in vitro after exposure to exogenous 17-OHP but showed significant chromatin activation. There was a decreased expression of HLA antigens at the surface of the patient's blood lymphocytes. This is a unique well-documented case of erythema multiforme most possibly due to autoreactivity to 17-OHP; the precise mechanism(s) of this autoreactivity has not been established.

Correlation between B-cell mitogenicity and immunosuppressor effects of a protein released by porcine monocytes infected with African swine fever virus.

Virus-free supernatants of cultured swine monocytes infected by African swine fever virus (ASFV) suppressed in vitro proliferation of porcine and human blood mononuclear cells in response to phytohemagglutinin and the in vivo primary immune response of C57BL/6 mice against sheep RBC. The supernatants were fractionated by discontinuous ion-exchange chromatography and subfractionated by double-step preparative isoelectric focusing. The pool of the most purified active subfractions (F5'EP-ASFV) is made up of heat-unstable material, can be stained by silver nitrate, and has an isoelectric point of 3.88, a maximal optical density at 280 nm, and a mass of 36,000 daltons. In vivo kinetic studies in nonimmunized C57BL/6 mice were performed on days 1, 2, 3, 5, and 7 after injection with 50 micrograms of F5'EP-ASFV protein. Compared with the untreated mice, the treated mice had a noticeable increase in nonspecific immunoglobulin-secreting splenic plaque-forming cells (PFC) with the following isotype profile: IgG2a greater than IgG2b greater than IgG3 greater than IgG1 congruent to IgM. Three days after treatment with the active material, specific IgM PFC against sheep RBC increased up to 23-fold. In C57BL/6 mice immunized against sheep RBC 2 days after treatment with F5'EP-ASFV, the increase in nonspecific PFC was followed by a suppression of specific PFC response in the respective isotype. When C57BL/6 mice were treated after priming with sheep RBC, however, there was little or no suppression of specific PFC and the increase in nonspecific PFC was considerably lower than that in the other F5'EP-ASFV-treated mice. In this case, kinetic curves of specific vs nonspecific PFC of each isotype were mirror images. Mice treated with 200 micrograms of F5'EP-ASFV protein died with hemorrhagic diastasis.