Delfina Tavares

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.

Streptococcus agalactiae GAPDH Is a Virulence-Associated Immunomodulatory Protein

Certain extracellular proteins produced by several pathogenic microorganisms interfere with the host immune system facilitating microbial colonization and were thus designated virulence-associated immunomodulatory proteins. In this study, a protein with B lymphocyte stimulatory activity was isolated from culture supernatants of Streptococcus agalactiae strain NEM316. This protein, with an apparent molecular mass of 45 kDa, was identified as GAPDH by N-terminal amino acid sequencing. The gapC gene was cloned and expressed in Escherichia coli for the production of a recombinant histidyl-tagged protein. The recombinant GAPDH (rGAPDH), purified in an enzymatically active form, induced in vitro an up-regulation of CD69 expression on B cells from normal and BCR transgenic mice. In addition, rGAPDH induced an increase in the numbers of total, but not of rGAPDH-specific, splenic Ig-secreting cells in C57BL/6 mice treated i.p. with this protein. These in vitro- and in vivo-elicited B cell responses suggest that the B cell stimulatory effect of rGAPDH is independent of BCR specificity. A S. agalactiae strain overexpressing GAPDH showed increased virulence as compared with the wild-type strain in C57BL/6 mice. This virulence was markedly reduced in IL-10-deficient and anti-rGAPDH antiserum-treated mice. These results suggest that IL-10 production, which was detected at higher concentrations in the serum of rGAPDH-treated mice, is important in determining the successfulness of the host colonization by S. agalactiae and they highlight the direct role of GAPDH in this process. Taken together, our data demonstrate that S. agalactiae GAPDH is a virulence-associated immunomodulatory protein.

Enolase from Streptococcus sobrinus is an immunosuppressive protein

A strategy of Streptococcus sobrinus, a major agent of dental caries, to survive and colonize the host consists of the production of a protein that suppresses the specific antibody responses. We have cloned the gene coding for a protein with immunosuppressive activity. It contains an open reading frame of 1302 base pairs encoding a polypeptide with 434 amino acid residues and a molecular mass of 46910 Da. The gene product is homologous to enolases from several organisms. The polypeptide was expressed in Escherichia coli as a hexahistidine‐tagged protein and purified in a fluoride‐sensitive enzymatically active form. Pretreatment of mice with the S. sobrinus recombinant enolase suppresses a primary immune response against T‐cell dependent antigens. This immunosuppressive effect is specific to the antigen used in the immunization, as it is not observed when the immune response against other antigens is analysed. Furthermore, the S. sobrinus recombinant enolase stimulates an early production of interleukin‐10, an anti‐inflammatory cytokine, and not the pro‐inflammatory cytokine IFN‐γ. These observations indicate that enolase acts in the suppression of the specific host immune response against S. sobrinus infection.

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.

Oral Therapeutic Vaccination with Streptococcus sobrinus Recombinant Enolase Confers Protection against Dental Caries in Rats

Dental caries is among the more prevalent chronic human infections for which an effective human vaccine has not yet been achieved. Enolase from Streptococcus sobrinus has been identified as an immunomodulatory protein. In the present study, we used S. sobrinus recombinant enolase (rEnolase) as a target antigen and assessed its therapeutic effect in a rat model of dental caries. Wistar rats that were fed a cariogenic solid diet on day 18 after birth were orally infected with S. sobrinus on day 19 after birth and for 5 consecutive days thereafter. Five days after infection and, again, 3 weeks later, rEnolase plus alum adjuvant was delivered into the oral cavity of the rats. A sham-immunized group of rats was contemporarily treated with adjuvant alone. In the rEnolase-immunized rats, increased levels of salivary IgA and IgG antibodies specific for this recombinant protein were detected. A significant decrease in sulcal, proximal enamel, and dentin caries scores was observed in these animals, compared with sham-immunized control animals. No detectable histopathologic alterations were observed in all immunized animals. Furthermore, the antibodies produced against bacterial enolase did not react with human enolase. Overall, these results indicate that rEnolase could be a promising and safe candidate for testing in trials of vaccines against dental caries in humans.

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.

Leukocyte populations and cytokine expression in the mammary gland in a mouse model of Streptococcus agalactiae mastitis

Streptococcus agalactiae is a contagious, mastitis-causing pathogen that is highly adapted to survive in the bovine mammary gland. This study used a BALB/c mouse model of Streptococcus agalactiae mastitis to evaluate leukocyte populations in regional lymph nodes and cytokine expression in the mammary gland involved in the immune response against Streptococcus agalactiae. It was found that the bacteria replicated efficiently in the mammary gland, peaking after 24 h and increasing by 100-fold. Dissemination of bacteria to systemic organs was observed 6 h after infection. At the same time, a massive infiltration of polymorphonuclear cells and an increase in the inflammatory cytokines interleukin (IL)-1beta, IL-6 and tumour necrosis factor-alpha were detected in mammary glands, indicating an early inflammatory response. A decrease in the levels of inflammatory cytokines in mammary glands was observed 72 h after infection, accompanied by an increase in the levels of IL-12 and IL-10, which were related to a gradual decrease in bacterial load. An increase in the number of macrophages and B220(+) lymphocytes and similar increases in both CD4(+) and CD8(+) T cells in regional lymph nodes were observed, being most pronounced 5 days after infection. Moreover, increased levels of anti-Streptococcus agalactiae antibodies in the mammary gland were observed 10 days after infection. Overall, these data suggest that the host exhibits both innate and acquired immune responses in response to Streptococcus agalactiae mastitis.

Therapeutic Vaccine against Streptococcus sobrinus-induced Caries

Streptococcus sobrinus produces a virulence-associated immunomodulatory protein (VIP) which suppresses the host-specific immune response and induces the early production of IL-10. In this study, we evaluated the effects of therapeutic immunization with this VIP on the incidence of caries in S. sobrinus-infected rats. Groups of Wistar rats were orally infected with S. sobrinus and fed with sucrose-sweetened drinking water ad libitum. Five days later, rats were immunized intranasally with active or heat-inactivated VIP plus alum as adjuvant or PBS plus adjuvant (sham-immunized). After 3 wks, all rats were re-immunized as above. Evaluation of dental caries showed that VIP-immunized animals had significantly fewer enamel sulcal and proximal caries lesions than did the sham-immunized animals (p < 0.001). The protective effects following therapeutic VIP immunization were attributed to the induced salivary immunoglobulin A specific to the VIP. These results offer a promising and safe strategy for the development of a vaccine against dental caries.

rEnolase Maternal Immunization Confers Caries Protection on Offspring

Therapeutic vaccination with Streptococcus sobrinus recombinant enolase (rEnolase) protects rats from dental caries. Here, we investigated the effect that maternal rEnolase vaccination before pregnancy had on the offspring’s immune response to S. sobrinus oral infection and dental caries progression. Female Wistar rats were immunized by intranasal and subcutaneous routes with rEnolase adsorbed onto aluminum hydroxide as adjuvant or similarly treated with the adjuvant alone (sham-immunized). Ten days after the last administration, the immunized females were paired with a male rat. The oral immune responses to S. sobrinus infection and dental caries in the offspring were evaluated. The results showed that pups born from rEnolase-immunized mothers had higher levels of rEnolase-specific salivary IgA and IgG antibodies (indicating a placental antibody transfer) and lower sulcal and proximal enamel caries scores than rats born from sham-immunized mothers. In conclusion, rEnolase maternal immunization before pregnancy provides offspring with protection against S. sobrinus-induced dental caries.