Mauricio Afonso Vericimo

Frequency of Natural Regulatory CD4+CD25+ T Lymphocytes Determines the Outcome of Tolerance across Fully Mismatched MHC Barrier through Linked Recognition of Self and Allogeneic Stimuli

We show in this study that long-term tolerance to allogeneic skin grafts can be established in the absence of immunosuppression by the combination of the following elements: 1) augmenting the frequency of regulatory CD4+CD25+ T cells (Treg) and 2) presentation of the allogeneic stimuli through linked recognition of allo- and self-epitopes on semiallogeneic F1 APCs. BALB/c spleen cells enriched for CD4+CD25+ T lymphocytes were transferred either to BALB/c nu/nu mice or to BALB/c nu/nu previously injected with F1(BALB/c × B6.Ba) spleen cells, or else grafted with F1(BALB/c × B6.Ba) skin (chimeric BALB/c nu/nu-F1). Chimeric BALB/c nu/nu-F1 reconstituted with syngeneic CD25+-enriched spleen cells were unable to reject the previously transferred F1(BALB/c × B6.Ba) spleen cells or F1(BALB/c × B6.Ba) skin grafts, and a specific tolerance to a secondary B6 graft was obtained, with rejection of third-party CBA grafts. BALB/c nu/nu mice reconstituted only with syngeneic CD25+-enriched spleen cells rejected both B6 and CBA skin grafts. In contrast, when chimeric BALB/c nu/nu-F1 were reconstituted with spleen populations comprising normal frequencies of Treg cells, the linked recognition of allo and self resulted in breaking of self tolerance and rejection of syngeneic grafts, strongly suggesting that linked recognition works in both directions, either to establish tolerance to allo, or to break tolerance to self, the critical parameter being the relative number of Treg cells.

A new 2S albumin from Jatropha curcas L. seeds and assessment of its allergenic properties

Significant effort has been made world-wide to boost biofuels with the expectation of a positive contribution to renewable fuel and greenhouse gas reduction. Jatropha curcas L. has proved to be an opportunistic crop in tropical areas, particularly in unfavorable environments. For this reason, analyses of toxicity and allergy caused by its seeds and pollen are necessary. A 12kDa, allergenic 2S albumin, denoted Jat c 1, was isolated from Physic nut (J. curcas) seeds. Jat c 1 binds IgE attached to rat mast cells, inducing histamine release. It also showed strong cross-reactivity with the major allergens from castor bean, Ric c 1 and Ric c 3.

Structural analysis and binding properties of isoforms of tarin, the GNA-related lectin from Colocasia esculenta

The lectins, a class of proteins that occur widely in animals, plants, fungi, lichens and microorganisms, are known for their ability to specifically bind to carbohydrates. Plant lectins can be classified into 12 families including the Galanthus nivalis agglutinin (GNA)-related lectin superfamily, which is widespread among monocotyledonous plants and binds specifically to mannose, a behavior that confers remarkable anti-tumor, anti-viral and insecticidal properties on these proteins. The present study characterized a mitogenic lectin from this family, called tarin, which was purified from the crude extract from taro (Colocasia esculenta). The results showed that tarin is a glycoprotein with 2-3% carbohydrate content, composed of least 10 isoforms with pIs ranging from 5.5 to 9.5. The intact protein is a heterotetramer of 47kDa composed of two non-identical and non-covalently associated polypeptides, with small subunits of 11.9kDa and large subunits of 12.6kDa. The tarin structure is stable and recovers or maintains its functional structure following treatments at different temperatures and pH. Tarin showed a complex carbohydrate specificity, binding with high affinity to high-mannose and complex N-glycans. Many of these ligands can be found in viruses, tumor cells and insects, as well as in hematopoietic progenitor cells. Chemical modifications confirmed that both conserved and non-conserved amino acids participate in this interaction. This study determined the structural and ligand binding characteristics of a GNA-related lectin that can be exploited for several different purposes, particularly as a proliferative therapeutic molecule that is able to enhance the immunological response.

Mapping IgE-binding epitopes of Ric c 1 and Ric c 3, allergens from Ricinus communis, by mast cell degranulation assay.

Ric c 1 and Ric c 3 are the major castor bean allergens. In order to identify continuous IgE-epitopes in Ric c 1 and Ric c 3, pools of sera from rats immunized with a pool of 2S albumin from these seeds, Ric c 1 and Ric c 3 overlapping synthetic peptides, were used to screen for IgE-binding epitopes. The allergenic properties were monitored by mast cell degranulation assays, histamine quantification and human-IgE binding. Large and small chains isolated from these proteins present allergenic properties. Four continuous epitopes were identified in Ric c 3 and two in Ric c 1. This knowledge may allow the induction of protective antibody responses to antagonize the IgE recognition.

Cross-sectional study of serum reactivity to Anisakis simplex in healthy adults in Niterói, Brazil.

Although the incidence of anisakiasis is rising worldwide, its frequency is still unknown in Brazil. The aim of this study was to verify immunoreactivity to Anisakis simplex antigens in healthy adults and determine its possible relationship with frequency of fish consumption and allergy symptoms. A prospective cross-sectional study was carried out with 67 volunteers recruited from a military facility in Niterói, Brazil. The subjects completed a structured questionnaire and serum titers of specific anti-Anisakis IgE and IgG antibodies were measured. The association between frequency of fish intake and IgE reactivity was evaluated by Fisher’s exact test. Almost all subjects (97.0%, 65/67) that consumed seafood; 64.6% (42/65) ate fish at least once weekly. Of all seafood consumers, 56.9% (37/65) reported allergy symptoms, being gut allergies most often cited (35.5%). IgE seroreactivity to Anisakis simplex was found in 20.9% of subjects (14/67), with 13.4% (9/67) reacting exclusively to somatic antigen, 3.0% (2/67) exclusively to excretory/secretory antigens and 4.5% (3/67) to both antigens. There was a significant association between frequency of fish consumption and positive serology (p = 0.019). An immunoblot assay for Anisakis antigens showed different positive bands for IgG. The direct relationship between ELISA reactivity and frequency of fish intake and absence of association with allergy symptoms suggests previous contact with Anisakis simplex antigens.

A technique for the intra-gastric administration of live larvae of Anisakis simplex in mice

To understand the mechanisms of infection and to attempt to simulate human infection by the Anisakidae family, many in vivo experimental approaches have been developed. The aim was to develop and present a technique for the induction of an oral infection through the use of an intra-gastric gavage of live Anisakis simplex in mice. A commercial pediatric gastric tube (No. 4) was cut longitudinally to produce a 3-cm slit at the distal end where the larva was placed to then be administered to the stomach of the mouse. There were no abnormal clinical complications before, during or after the procedure. In conclusion oral infection through the direct delivery of larvae in the stomach is simple and effective.

Experimental anisakid infections in mice.

Anisakidosis is a human parasitic disease caused by infections with members of the Anisakidae family. Accidental infection after fish intake affects the gastrointestinal tract as a consequence of mechanical damage caused by migrating larvae. Infections can also trigger allergies, hives, severe asthma or anaphylaxis with angioedema. Although mouse models of intraperitoneal antigenic stimulation exist, enabling immunological studies, few models using gastric introduction of live larvae are available for the study of immunological and gastrointestinal damage in mice. This study was designed to characterize serum reactivity against Anisakis spp. and Contracaecum spp. in Balb/c mice following orogastric inoculation and to assess gastrointestinal damage. These anisakid species were classified at the Universidade Federal Fluminense (UFF) School of Veterinary Medicine and materials for live larval inoculation were developed at the UFF Immunobiology laboratory. Live larvae were inoculated following injection with a NaCl solution. Blood samples were collected and sera screened for immunoglobulin (Ig)E and IgG anti-larva responses to both nematodes, specific for somatic and excretory/secretory antigens, by enzyme-linked immunosorbent assay (ELISA). The means of the optical densities were analysed using analysis of variance (ANOVA) with Tukeys post-hoc test and the general linear model. This analysis identified the presence of anti-IgG seroreactivity to both somatic and excretory/secretory Anisakis antigens in inoculated animals compared with controls (P< 0.001), and no gastric or intestinal damage was observed. These experiments demonstrated that introduction of live Contracaecum spp. into the gastrointestinal tract did not elicit serum sensitization in animals.

B1 cells contribution to susceptibility in experimental paracoccidioidomycosis: immunoglobulin isotypes and repertoire determination

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America. The experimental murine model has been used to approach the disease, with susceptible and resistant mice strains that reproduce most of the main human immunological features. Since the hypergammaglobulinemia observed in susceptible mice and humans might have an influence on B1 cells, we investigated its role during the experimental infection with Paracoccidiodes brasiliensis. CBA/Nxid mice, deficient in B1 cells, and CBA/Nxid reconstituted with B1 cells isolated from the non-mutant CBA/J strain were infected with 106 yeast forms of P. brasiliensis. At the 8th and 22nd week post infection the DTH response of CBA/Nxid mice was significantly higher after 24 h of P. brasiliensis antigens inoculation and the specific humoral response was reduced, in comparison to CBA/J or recCBA/Nxid. Production of NAbs is a hallmark of the B1 subset. Higher Ig productions to auto antigens such as DNA, MBP and RBC were observed in CBA/J infected mice or recCBA/Nxid. Anti P. brasiliensis IgG2a was produced by CBA/Nxid mice early in infection, while CBA/J or recCBA/Nxid presented increased levels of this isotype only after the 8th week of infection. Furthermore, western blotting analysis showed that CBA/Nxid mice expanded less clones against P. brasiliensis antigens, with weakly detectable anti-gp43 antibodies while CBA/J mice produce IgM anti-gp43 at the 2nd week of infection and IgG anti-gp43 at the 2nd and 8th week. On the other hand, recognition of gp70, a fungal antigen that, as gp43, inhibits macrophage activation was not compromised in B1 deficient mice. These results suggest that B1 cells might have influence in the kinetic of production of protective isotypes of immunoglobulins and their repertoire that could contribute to an early drive towards a Th2 response, affecting the cellular response in susceptible mice during experimental paracoccidiodomycosis.

Resposta imunológica a antígenos de Hysterothylacium deardorffoverstreetorum de peixes teleósteos

Anisaquidosis is a disease caused by parasites of Anisakidae family and is characterized by gastrointestinal and allergic reactions. The Anisakis simplex is a more pathogenic Anisakidae to humans and is highly allergenic. However, other species of this family also have characteristics that are harmful to humans, but little is known about the immunogenicity this parasites. The objective of this study was to experimentally assess the immunogenic potential of the parasite Hysterothylacium deardorffoverestreetorum (H.D) in mice. Mice of inbred BALB/c strain were divided into three groups and received three immunizations of the following antigenic preparations obtained from L3 larvae H.D: Crude larval extract of H.D (CEH) Extract secreted / excreted larvae H.D. (ESH) and crude extract of larvae after excretion / secretion (EEH). Serum samples were obtained on different days after immunization to determine the levels of circulating specific antibodies by enzyme-linked immunosorbent assay (ELISA). The results show increased production of immunoglobulin (Ig) G after the second immunization with a gradual increase after the third immunization. Regarding IgE reactivity, this occurred later, demonstrating a progressive increase only after the third immunization. Cellular immunity was evaluated by intradermal, and showed statistically significant result compared to the control used. This experiment is the first description of the pathogenic potential of this parasite in mammals and represents a breakthrough in the diagnosis of human Anisakidosis.

Access to the tracheal pulmonary pathway in small rodents

As vias de acesso traqueopulmonar vem sendo utilizadas em diversos modelos experimentais que estudam a acao de farmacos e agentes infecciosos, alem de enfermidades. Tendo em vista a sua importância e as dificuldades associadas, o presente artigo de atualizacao propoe-se a dar ao pesquisador as informacoes necessarias para o emprego das tecnicas de acesso traqueopulmonar em pequenos roedores.