W. Tamashiro - Academia.edu (original) (raw)

Papers by W. Tamashiro

Brazilian Journal of Medical and Biological Research, 2007

Aging is accompanied by a decrease in several physiological functions that make older individuals... more Aging is accompanied by a decrease in several physiological functions that make older individuals less responsive to environmental challenges. In the present study, we analyzed the immune response of female BALB/c mice (N = 6) of different ages (from 2 to 96 weeks) and identified significant age-related alterations. Immunization with hapten-protein (trinitrophenyl-bovine serum albumin) conjugates resulted in lower antibody levels in the primary and secondary responses of old mice (72 weeks old). Moreover, young mice (2, 16, and 32 weeks old) maintained specific antibodies in their sera for longer periods after primary immunization than did old mice. However, a secondary challenge efficiently induced memory in old mice, as shown by the increased antibody levels in their sera. The number of CD4 + and CD8 + T cells in the spleen increased until 8 weeks of age but there was no change in the CD4 + /CD8 + ratio with aging. Splenic T cells from old mice that had or had not been immunized were less responsive to concanavalin-A and showed reduced cytokine production compared to young mice (IL-2: 57-127 vs 367-1104 pg/mL, IFNγ: 2344-12,836 vs 752-23,106 pg/mL and IL-10: 393-2172 vs 105-2869 pg/mL in old and young mice, respectively). These data suggest that there are significant changes in the organization of the immune system throughout life. However, the relevance of these alterations for the functioning of the immune system is unknown.

Toxicon, 2003

A novel lectin from Talisia esculenta seeds (TEL) has recently been purified and characterized. I... more A novel lectin from Talisia esculenta seeds (TEL) has recently been purified and characterized. In this study we investigated the proinflammatory activity of TEL in mice using both the air-pouch and peritoneal cavity as well as paw oedema models. TEL (10-40 mg) induced significant neutrophil and mononuclear cell recruitment when injected into either mouse air-pouch or peritoneal cavity. The neutrophil accumulation into the air-pouch was dose-and time-dependent with a maximal response at 16 h, returning to control levels at 72 h whereas maximal mononuclear cell accumulation was observed at 24 h after TEL injection. The same profile of neutrophil accumulation was observed when this lectin was injected into mouse peritoneal cavity, although the maximal mononuclear cell recruitment was observed 48 h after TEL injection. Additionally, TEL (12.5-200 mg/paw) caused a dose-dependent mice paw, as evaluated at 4 h after the lectin injection. D-mannose, better than Dglucose, significantly inhibited TEL-induced neutrophil migration into the peritoneal cavity or air-pouch. D-galactose had no effect on TEL-induced neutrophil migration in either cavity studied. On the other hand, D-mannose slightly inhibited the TELinduced paw oedema, whereas neither D-glucose nor D-galactose affected this phenomenon. In conclusion, our data show that TEL induces neutrophil and mononuclear cell accumulation by a mechanism related to their specific sugar-binding properties.

Cancer cell international, Jan 10, 2005

A human malignant continuous cell line, named NG97, was recently established in our laboratory. T... more A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells present...

Research in Immunology, 1992

To verify whether anti-Trypanosoma-cruzi-proteinase antibodies are able to play a role in the dev... more To verify whether anti-Trypanosoma-cruzi-proteinase antibodies are able to play a role in the development of Chagas' disease, CBA/J and C57BL/6 mice were immunized with purified proteinase fractions: antibody production was studied and passive immunization experiments were carried out. No significant differences were observed in the titre, isotype composition and specificity of the antibodies produced by the mouse strains. Immune sera produced in one strain was able to protect mice of both strains, provided that the challenge did not exceed the number of parasites corresponding to 30-fold the LD50. The data presented suggest that anti-proteinase antibodies may play a role in immunoprotection.

Hybridoma, 2000

The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugat... more The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG 1 , and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r 2 (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h 2 1 and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays.

Scandinavian Journal of Immunology, 2004

The ingestion of most dietary protein can cause systemic tolerance, and such tolerance is easier ... more The ingestion of most dietary protein can cause systemic tolerance, and such tolerance is easier to induce in younger than in older mice. In this study, we examined whether oral tolerance to ovalbumin (OVA) could be induced in OVA‐T‐cell receptor (OVA‐TCR)‐specific transgenic mice. Continuous feeding or gavage with OVA induced tolerance, measured as reduced antibody production, in young and aged BALB/c mice, in a dose‐dependent manner, but this effect was not observed in transgenic mice. Once BALB/c mice became tolerant, this state was maintained for over 44 weeks, although the tolerant state could be reversed by adoptive cell transfer. DO11.10 mice did not become tolerant upon continuous feeding with OVA, and the adoptive transfer of naïve cells increased the levels of specific antibodies in their sera after antigenic challenge. The immunization schedule used here leads to a Th2‐dependent antibody response in normal BALB/c mice. However, the same schedule induced both Th1‐ and Th2‐...

Scandinavian Journal of Immunology, 2002

Natural killer (NK) cells may provide the basis for resistance to Trypanosoma cruzi infection, be... more Natural killer (NK) cells may provide the basis for resistance to Trypanosoma cruzi infection, because the depletion of NK1.1 cells causes high levels of parasitemia in young C57Bl/6 mice infected with T. cruzi. Indeed, NK1.1 cells have been implicated in the early production of large amounts of interferon (IFN)-g, an important cytokine in host resistance. The NK1.1 marker is also expressed on special subpopulations of T cells. Most NK1.1 T cells are of thymic origin, and their constant generation may be prevented by thymectomy. This procedure, by itself, decreased parasitemia and increased resistance in young mice. However, the depletion of NK1.1 cells by the chronic administration of a monoclonal antibody (MoAb) (PK-136) did not increase the parasitemia or mortality in thymectomized C57Bl/6 mice infected with T. cruzi (Tulahuen strain). To study the cross-talk between NK1.1 cells and conventional T cells in this model, we examined the expression of activation/memory markers (CD45RB) on splenic CD4 and CD8 T cells from young euthymic or thymectomized mice with or without depletion of NK1.1 cells and also in aged mice during acute infection. Resistance to infection correlated with the amount of CD4 T cells that are already activated at the moment of infection, as judged by the number of splenic CD4 T cells expressing CD45RB ±. In addition, the specific antibody response to T. cruzi antigens was precocious and an accumulation of immunoglobulin (Ig)M with little isotype switch occurred in euthymic mice depleted of NK1.1 cells. The data presented here suggest that NK1.1 cells have important regulatory functions in euthymic, but not in thymectomized mice infected with T. cruzi. These regulatory functions include a helper activity in the generation of effector or activated/memory T cells.

Research in Microbiology, 1989

Man~" s6~ologLoal r~ct[ens using red bl-3od cel;s ~R~C; ~,eh ~: r~aj~j ~mune haemolysis (RIH) and... more Man~" s6~ologLoal r~ct[ens using red bl-3od cel;s ~R~C; ~,eh ~: r~aj~j ~mune haemolysis (RIH) and indirect haemagglutination (IH) tests have often been used for the detection of cholera toxin (CT) and heat-labile (LT) enterotoxin produced by porcine and human Escherichia coli strains. In these tests, the enterotoxins bind to sheep, bovine and guinea-pig RBC without any ligand. We studied several factors which might interfere with such binding, as well as the nature of the receptors involved. Treatment of erythrocytes with different enzymes revealed that proteolytic enzymes had no effect on the adsorption of enterotoxins to RBC. Conversely, treatment with neuraminidase increased the adsorption. Experiments c~'ried out with delipidized RBC revealed that none of the enterotoxins under study bound to the cells thus treated. Pre-incubation of ganglioside fractions with the enterotoxins blocked RIH and IH reactions and the biological effect of them on Vero cells. Assaying RBC gangiioside fractions by thin-layer chromatography revealed the presence of GM1. Our results suggest that the receptors for GT and LT enterotoxins in sheep, bovine and guinea pig RBC are gangliosides: mainly GMI.

Zeitschrift f�r Parasitenkunde Parasitology Research, 1983

A fraction (FAd) capable of inhibiting specific agglutination reactions of anti-epimastigote sera... more A fraction (FAd) capable of inhibiting specific agglutination reactions of anti-epimastigote sera (anti-LE) was obtained by extracting the sediment of lyophilized epimastigote lysates (LE) with 0.05 M phosphate buffered saline, at 37 degrees C for 1 h. These conditions favored the action of parasite proteinase whose presence was detected by tandem-crossed immunoelectrophoresis experiments. As expected from the proteinase properties, the addition of 2-mercaptoethanol or sodium iodoacetate to the extracting solution resulted, respectively, in either increased or decreased amounts of protein in the resulting FAd. FAd components could be precipitated by the addition of Concanavalin A, methylated albumins or 0.1 N HCl. This fraction presented a single component when subjected to electrophoresis in 1% agarose gel with an electrophoretic mobility 1.2 times higher than that of human albumin. FAd component(s) were unable to penetrate 15% polycrylamide gel matrix unless 1% SDS was used. Under this condition four glycopeptide components, with Rm of 0.5, 0.55, 0.6 and 0.86, were detected. The antigenic determinants present in FAd resisted heating at 100 degrees C for 30 min and the prolonged action of pronase. However, these determinants were completely destroyed by the action of 25 mM sodium periodate, thus suggesting polysaccharide characteristics. Immunization of rabbits with FAd induced the production of antibodies that were unable to precipitate with either FAd or with parasite proteinase. These antibodies exhibited positive agglutination reactions with epimastigote forms and positive immunofluorescence and immunoperoxidase reactions with trypomastigote and amastigote forms of the different strains tested. FAd was able to inhibit these reactions as well as those obtained with anti-LE and anti-FA immune sera, whereas purified proteinase was unable to inhibit any of these reactions.

Mediators of Inflammation, 1992

Pretreatment of human neutrophils with recombinant tumour necrosis factor-alpha (rTNF-α) and/or i... more Pretreatment of human neutrophils with recombinant tumour necrosis factor-alpha (rTNF-α) and/or interleukin-8 (rIL-8), but not with either transforming growth factor-beta, interleukin-6 or interferon-gamma, rendered these cells less responsive to FMLP, in microchemotaxis assays. This inhibitory effect was dose dependent and more powerful when neutrophils were pretreated with a mixture of both cytokines. Intravenous injection of human rIL-8 (hrIL-8) and/or murine rTNF-α (mrTNF-α) also significantly reduced in vivo neutrophil migration into peritoneal cavities of rats stimulated with carrageenan. These data suggest that the defect in neutrophil migration during septicaemia or endotoxaemia may be the result of the continuous release of IL-8 and TNF-α into the circulation. Thus, either the selective control or blockade of releasing of these cytokines as well as of its effects on neutrophils may be clinically useful in reestablishing the cell defence mechanisms.

Mediators of Inflammation, 1992

Inhibitory effect upon neutrophil migration to the inflammatory focus was previously detected in ... more Inhibitory effect upon neutrophil migration to the inflammatory focus was previously detected in the cell-free incubation fluid of lipopolysaccharide (LPS)-stimulated macrophage monolayers. In the present study we showed that the neutrophil recruitment inhibitory activity from this supernatant was mainly detected in a fraction (P2) obtained by gel filtration chromatography on Sephacryl S-300. P2 fraction was able to inhibit ‘in vivo’ neutrophil emigration induced by different inflammatory stimuli, but it did not affect ‘in vitro’ neutrophil chemotaxis induced by FMLP. When injected intravenously, P2 inhibited oedema induced by carrageenin or immunological stimulus but not the oedema induced by dextran, thus affecting cell-dependent inflammatory responses. It was observed that P2 also induced neutrophil migration when injected locally in peritoneal cavities. This activity was significantly reduced by pretreatment of the animals with dexamethasone. Cytokines, such as IL-8 and TNF-α th...

Mediators of Inflammation, 1997

Macrophages harvested from the peritoneal cavities of rats release a neutrophil chemotactic facto... more Macrophages harvested from the peritoneal cavities of rats release a neutrophil chemotactic factor (MNCF) in response to stimulation with Gram-negative bacterial lipopolysaccharide (LPS). MNCF has been shown to be active in rats treated with dexamethasone, a glucocorticoid that usually inhibits the neutrophil migration induced in this species by interleukin (IL)-1, tumour necrosis factor alpha (TNFα), IL-8, C5a and leukotriene B4(LTB4). Here we report that macrophages harvested from peritoneal cavities of mice, and stimulatedin vitrowith LPS, also release a factor that induces neutrophil migration in dexamethasone-treated animals. This chemotactic activity was neutralized by the incubation of the LPS-stimulated macrophage supernatants with a purified polyclonal IgG anti-mouse TNFα. In addition, significant amounts of TNF were detected in the supernatants. The neutrophil migration induced by intraperitoneal administration of recombinant murine TNFα was also unaffected by pretreatment...

Journal of Reproductive Immunology, 2008

Uterine natural killer (uNK) cells expand rapidly during endometrial decidualization and account ... more Uterine natural killer (uNK) cells expand rapidly during endometrial decidualization and account for 70% of leukocytes in early gestational uteri of humans and rodents. These cells make unique contributions to pregnancy, contributing to the success of embryo implantation and maintenance of decidual tissue that supports placental and fetal development. We postulated that uNK cells express molecules that are not shared by circulating NK (cNK) cells or other leukocytes and, therefore, would be immunogenic for male mice. We isolated viable uNK cells from gestation day 9 pregnant mice and inoculated them into syngeneic males. This induced antibodies reactive with mouse uNK cells but not with cNK cells or other lymphocytes. The antibodies reacted identically with uNK cells in tissue sections from five different mice strains from gestational day 7-12 and in pregnant rat uterus, suggesting that the recognized antigen should be a specific marker of uNK cell. Spleen cells from inoculated males were used subsequently to produce a monoclonal antibody reactive to a uNK cell surface antigen. These experiments confirm that uNK cells are a pregnancy-specific subset of NK cells expressing distinct surface antigen from those found in other tissues.

Food Research International, 2010

According to the Pan-American Health Organization (PAHO), there is a growing increase of degenera... more According to the Pan-American Health Organization (PAHO), there is a growing increase of degenerative diseases in countries of the Americas, bringing on disability and death of the population. As eating properly is the most effective way to reduce the risk of diseases, food scientists are seeking alternatives for more nutritional foods, mainly through the investigation of functional compounds in natural foods. Among the functional compounds, fructans stand out for their beneficial effects on the animal body, favoring the growth of bifidobacteria in the colon that improve gastrointestinal metabolism, as well as regulating the levels of serum cholesterol and the absorption of minerals. Besides, fructans seem to be involved in the positive modulation of the immune system, mainly in an increased resistance to infections and microbicidal activity as well as by the reduction of allergic reactions and cancer in experimental models. This paper aims to analyze literature data concerning the benefits of consuming the fructans contained in natural food, especially their immunomodulatory actions in both animals and humans.

Food Research International, 2011

The gradual increase of degenerative diseases observed in the last decades has been raising morbi... more The gradual increase of degenerative diseases observed in the last decades has been raising morbidity, incapacitation and mortality. The occurrence of these kinds of diseases is related to the aging of humanity as well as the unhealthy choices of individuals, particularly those dwelling in large urban centers, which are closely linked with poor nutrition, obesity, and tobacco and alcohol consumption. The introduction of functional compounds in the diet seems to be an attractive alternative to ameliorate the quality of life of all age groups. The prebiotics stand out because of their beneficial effects, favoring the growth of colonic microbiota, helping the gastrointestinal metabolism, and regulating the serum cholesterol and mineral absorption. Experimental data indicates that prebiotics could reduce the severity or incidence of degenerative diseases, such as neoplasias, diabetics, coronary diseases, and infectious diseases. They also seem to promote a positive modulation of the immune system. Their effects on the immune system could even be associated to increase of resistance to infection and microbicide capability, as well as to a decrease in allergic reactions. This article's goal is to analyze the immunomodulatory potential of prebiotics observed in experimental and trial studies.

FEBS Letters, 1999

A protein band of approximately 166 kDa was detected in the soluble fraction of root tips and you... more A protein band of approximately 166 kDa was detected in the soluble fraction of root tips and young leaves of maize seedlings, based on Western blot analysis using antibodies raised against mouse macrophage nitric oxide synthase (NOS) and rabbit brain NOS. NOS activity was present in these soluble fractions, as determined by L-[U-IR C]citrulline synthesis from L-[U-IR C]arginine. Immunofluorescence showed that the maize NOS protein is present in the cytosol of cells in the division zone and is translocated into the nucleus in cells in the elongation zone of maize root tips. These results indicate the existence of a NOS enzyme in maize tissues, with the localization of this protein depending on the phase of cell growth.

Clinical and Developmental Immunology, 2012

In this work, we evaluated the effects of administration of OVA on phenotype and function of intr... more In this work, we evaluated the effects of administration of OVA on phenotype and function of intraepithelial lymphocytes (IELs) from small intestine of transgenic (TGN) DO11.10 and wild-type BALB/c mice. While the small intestines from BALB/c presented a well preserved structure, those from TGN showed an inflamed aspect. The ingestion of OVA induced a reduction in the number of IELs in small intestines of TGN, but it did not change the frequencies of CD8+and CD4+T-cell subsets. Administration of OVA via oral + ip increased the frequency of CD103+cells in CD4+T-cell subset in IELs of both BALB/c and TGN mice and elevated its expression in CD8β+T-cell subset in IELs of TGN. The frequency of Foxp3+cells increased in all subsets in IELs of BALB/c treated with OVA; in IELs of TGN, it increased only in CD25+subset. IELs from BALB/c tolerant mice had lower expression of all cytokines studied, whereas those from TGN showed high expression of inflammatory cytokines, especially of IFN-γ, TGF-...

Brazilian Journal of Medical and Biological Research, 2010

Oral tolerance can be induced in some mouse strains by gavage or spontaneous ingestion of dietary... more Oral tolerance can be induced in some mouse strains by gavage or spontaneous ingestion of dietary antigens. In the present study, we determined the influence of aging and oral tolerance on the secretion of co-stimulatory molecules by dendritic cells (DC), and on the ability of DC to induce proliferation and cytokine secretion by naive T cells from BALB/c and OVA transgenic (DO11.10) mice. We observed that oral tolerance could be induced in BALB/c mice (N = 5 in each group) of all ages (8, 20, 40, 60, and 80 weeks old), although a decline in specific antibody levels was observed in the sera of both tolerized and immunized mice with advancing age (40 to 80 weeks old). DC obtained from young, adult and middle-aged (8, 20, and 40 weeks old) tolerized mice were less efficient (65, 17 and 20%, respectively) than DC from immunized mice (P < 0.05) in inducing antigen-specific proliferation of naive T cells from both BALB/c and DO11.10 young mice, or in stimulating IFN-γ, IL-4 and IL-10 production. However, TGF-β levels were significantly elevated in co-cultures carried out with DC from tolerant mice (P < 0.05). DC from both immunized and tolerized old and very old (60 and 80 weeks old) mice were equally ineffective in inducing T cell proliferation and cytokine production (P < 0.05). A marked reduction in CD86 + marker expression was observed in DC isolated from both old and tolerized mice (75 and 50%, respectively). The results indicate that the aging process does not interfere with the establishment of oral tolerance in BALB/c mice, but reduces DC functions, probably due to the decline of the expression of the CD86 surface marker.

Brazilian Journal of Medical and Biological Research, 2001

Brazilian Journal of Medical and Biological Research, 2007

Aging is accompanied by a decrease in several physiological functions that make older individuals... more Aging is accompanied by a decrease in several physiological functions that make older individuals less responsive to environmental challenges. In the present study, we analyzed the immune response of female BALB/c mice (N = 6) of different ages (from 2 to 96 weeks) and identified significant age-related alterations. Immunization with hapten-protein (trinitrophenyl-bovine serum albumin) conjugates resulted in lower antibody levels in the primary and secondary responses of old mice (72 weeks old). Moreover, young mice (2, 16, and 32 weeks old) maintained specific antibodies in their sera for longer periods after primary immunization than did old mice. However, a secondary challenge efficiently induced memory in old mice, as shown by the increased antibody levels in their sera. The number of CD4 + and CD8 + T cells in the spleen increased until 8 weeks of age but there was no change in the CD4 + /CD8 + ratio with aging. Splenic T cells from old mice that had or had not been immunized were less responsive to concanavalin-A and showed reduced cytokine production compared to young mice (IL-2: 57-127 vs 367-1104 pg/mL, IFNγ: 2344-12,836 vs 752-23,106 pg/mL and IL-10: 393-2172 vs 105-2869 pg/mL in old and young mice, respectively). These data suggest that there are significant changes in the organization of the immune system throughout life. However, the relevance of these alterations for the functioning of the immune system is unknown.

Toxicon, 2003

A novel lectin from Talisia esculenta seeds (TEL) has recently been purified and characterized. I... more A novel lectin from Talisia esculenta seeds (TEL) has recently been purified and characterized. In this study we investigated the proinflammatory activity of TEL in mice using both the air-pouch and peritoneal cavity as well as paw oedema models. TEL (10-40 mg) induced significant neutrophil and mononuclear cell recruitment when injected into either mouse air-pouch or peritoneal cavity. The neutrophil accumulation into the air-pouch was dose-and time-dependent with a maximal response at 16 h, returning to control levels at 72 h whereas maximal mononuclear cell accumulation was observed at 24 h after TEL injection. The same profile of neutrophil accumulation was observed when this lectin was injected into mouse peritoneal cavity, although the maximal mononuclear cell recruitment was observed 48 h after TEL injection. Additionally, TEL (12.5-200 mg/paw) caused a dose-dependent mice paw, as evaluated at 4 h after the lectin injection. D-mannose, better than Dglucose, significantly inhibited TEL-induced neutrophil migration into the peritoneal cavity or air-pouch. D-galactose had no effect on TEL-induced neutrophil migration in either cavity studied. On the other hand, D-mannose slightly inhibited the TELinduced paw oedema, whereas neither D-glucose nor D-galactose affected this phenomenon. In conclusion, our data show that TEL induces neutrophil and mononuclear cell accumulation by a mechanism related to their specific sugar-binding properties.

Cancer cell international, Jan 10, 2005

A human malignant continuous cell line, named NG97, was recently established in our laboratory. T... more A human malignant continuous cell line, named NG97, was recently established in our laboratory. This cell line has been serially subcultured over 100 times in standard culture media presenting no sign of cell senescence. The NG97 cell line has a doubling time of about 24 h. Immunocytochemical analysis of glial markers demonstrated that cells are positive for glial fibrillary acidic protein (GFAP) and S-100 protein, and negative for vimentin. Under phase-contrast microscope, cultures of NG97 showed cells with variable morphological features, such as small rounded cells, fusiform cells (fibroblastic-like cells), and dendritic-like cells. However, at confluence just small rounded and fusiform cells can be observed. At scanning electron microscopy (SEM) small rounded cells showed heterogeneous microextentions, including blebs and filopodia. Dendritic-like cells were flat and presented extensive prolongations, making several contacts with small rounded cells, while fusiform cells present...

Research in Immunology, 1992

To verify whether anti-Trypanosoma-cruzi-proteinase antibodies are able to play a role in the dev... more To verify whether anti-Trypanosoma-cruzi-proteinase antibodies are able to play a role in the development of Chagas&#39; disease, CBA/J and C57BL/6 mice were immunized with purified proteinase fractions: antibody production was studied and passive immunization experiments were carried out. No significant differences were observed in the titre, isotype composition and specificity of the antibodies produced by the mouse strains. Immune sera produced in one strain was able to protect mice of both strains, provided that the challenge did not exceed the number of parasites corresponding to 30-fold the LD50. The data presented suggest that anti-proteinase antibodies may play a role in immunoprotection.

Hybridoma, 2000

The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugat... more The aim of this study was to produce anti-TNP monoclonal antibodies (MAbs) that could be conjugated and used for the detection of antigen-antibody reactions, in which the antigen specific-antibody had been previously bound to trinitrophenyl (TNP). For hybridoma production, SP2/0-Ag14 cells were fused with spleen cells from mice previously immunized with TNP-ovalbumin (TNP-OVA). After 10 days, enzyme-linked immunoadsorbent assay (ELISA) was used to detect anti-TNP antibodies in the supernatants, and five cultures were found to be strictly positive for TNP. Three of these were subsequently cloned by limiting dilution, and 15 clones were chosen for expansion based on the criterion of high reactivity against TNP. Anti-TNP MAbs produced by those clones were isotyped as IgG 1 , and purified by Sepharose-protein G affinity cromatography from ascites developed in BALB/c mice. Two purified MAbs (1B2.1B6 and 1B2.1E12) were coupled to horseradish peroxidase (HRPO). The resulting conjugates were evaluated in ELISA tests for interferon-gamma and interleukin-4 detection, in which the secondary anti-cytokine antibodies were coupled either to TNP or biotin. The performance of anti-TNP conjugates in these assays were compared with a biotin-streptavidin/peroxidase system. Both types of conjugates were similarly able to detect cytokines with r 2 (linear correlation coefficient) close to unity value. Growth studies of one of those hybridomas (1B2.1B6) yielded a specific growth rate of 0.042 h 2 1 and a doubling time of 16.5 h. Data discussed here show that at least two MAbs against TNP raised in this work can be used as a reagent for enzyme immunoassays.

Scandinavian Journal of Immunology, 2004

The ingestion of most dietary protein can cause systemic tolerance, and such tolerance is easier ... more The ingestion of most dietary protein can cause systemic tolerance, and such tolerance is easier to induce in younger than in older mice. In this study, we examined whether oral tolerance to ovalbumin (OVA) could be induced in OVA‐T‐cell receptor (OVA‐TCR)‐specific transgenic mice. Continuous feeding or gavage with OVA induced tolerance, measured as reduced antibody production, in young and aged BALB/c mice, in a dose‐dependent manner, but this effect was not observed in transgenic mice. Once BALB/c mice became tolerant, this state was maintained for over 44 weeks, although the tolerant state could be reversed by adoptive cell transfer. DO11.10 mice did not become tolerant upon continuous feeding with OVA, and the adoptive transfer of naïve cells increased the levels of specific antibodies in their sera after antigenic challenge. The immunization schedule used here leads to a Th2‐dependent antibody response in normal BALB/c mice. However, the same schedule induced both Th1‐ and Th2‐...

Scandinavian Journal of Immunology, 2002

Natural killer (NK) cells may provide the basis for resistance to Trypanosoma cruzi infection, be... more Natural killer (NK) cells may provide the basis for resistance to Trypanosoma cruzi infection, because the depletion of NK1.1 cells causes high levels of parasitemia in young C57Bl/6 mice infected with T. cruzi. Indeed, NK1.1 cells have been implicated in the early production of large amounts of interferon (IFN)-g, an important cytokine in host resistance. The NK1.1 marker is also expressed on special subpopulations of T cells. Most NK1.1 T cells are of thymic origin, and their constant generation may be prevented by thymectomy. This procedure, by itself, decreased parasitemia and increased resistance in young mice. However, the depletion of NK1.1 cells by the chronic administration of a monoclonal antibody (MoAb) (PK-136) did not increase the parasitemia or mortality in thymectomized C57Bl/6 mice infected with T. cruzi (Tulahuen strain). To study the cross-talk between NK1.1 cells and conventional T cells in this model, we examined the expression of activation/memory markers (CD45RB) on splenic CD4 and CD8 T cells from young euthymic or thymectomized mice with or without depletion of NK1.1 cells and also in aged mice during acute infection. Resistance to infection correlated with the amount of CD4 T cells that are already activated at the moment of infection, as judged by the number of splenic CD4 T cells expressing CD45RB ±. In addition, the specific antibody response to T. cruzi antigens was precocious and an accumulation of immunoglobulin (Ig)M with little isotype switch occurred in euthymic mice depleted of NK1.1 cells. The data presented here suggest that NK1.1 cells have important regulatory functions in euthymic, but not in thymectomized mice infected with T. cruzi. These regulatory functions include a helper activity in the generation of effector or activated/memory T cells.

Research in Microbiology, 1989

Man~" s6~ologLoal r~ct[ens using red bl-3od cel;s ~R~C; ~,eh ~: r~aj~j ~mune haemolysis (RIH) and... more Man~" s6~ologLoal r~ct[ens using red bl-3od cel;s ~R~C; ~,eh ~: r~aj~j ~mune haemolysis (RIH) and indirect haemagglutination (IH) tests have often been used for the detection of cholera toxin (CT) and heat-labile (LT) enterotoxin produced by porcine and human Escherichia coli strains. In these tests, the enterotoxins bind to sheep, bovine and guinea-pig RBC without any ligand. We studied several factors which might interfere with such binding, as well as the nature of the receptors involved. Treatment of erythrocytes with different enzymes revealed that proteolytic enzymes had no effect on the adsorption of enterotoxins to RBC. Conversely, treatment with neuraminidase increased the adsorption. Experiments c~'ried out with delipidized RBC revealed that none of the enterotoxins under study bound to the cells thus treated. Pre-incubation of ganglioside fractions with the enterotoxins blocked RIH and IH reactions and the biological effect of them on Vero cells. Assaying RBC gangiioside fractions by thin-layer chromatography revealed the presence of GM1. Our results suggest that the receptors for GT and LT enterotoxins in sheep, bovine and guinea pig RBC are gangliosides: mainly GMI.

Zeitschrift f�r Parasitenkunde Parasitology Research, 1983

A fraction (FAd) capable of inhibiting specific agglutination reactions of anti-epimastigote sera... more A fraction (FAd) capable of inhibiting specific agglutination reactions of anti-epimastigote sera (anti-LE) was obtained by extracting the sediment of lyophilized epimastigote lysates (LE) with 0.05 M phosphate buffered saline, at 37 degrees C for 1 h. These conditions favored the action of parasite proteinase whose presence was detected by tandem-crossed immunoelectrophoresis experiments. As expected from the proteinase properties, the addition of 2-mercaptoethanol or sodium iodoacetate to the extracting solution resulted, respectively, in either increased or decreased amounts of protein in the resulting FAd. FAd components could be precipitated by the addition of Concanavalin A, methylated albumins or 0.1 N HCl. This fraction presented a single component when subjected to electrophoresis in 1% agarose gel with an electrophoretic mobility 1.2 times higher than that of human albumin. FAd component(s) were unable to penetrate 15% polycrylamide gel matrix unless 1% SDS was used. Under this condition four glycopeptide components, with Rm of 0.5, 0.55, 0.6 and 0.86, were detected. The antigenic determinants present in FAd resisted heating at 100 degrees C for 30 min and the prolonged action of pronase. However, these determinants were completely destroyed by the action of 25 mM sodium periodate, thus suggesting polysaccharide characteristics. Immunization of rabbits with FAd induced the production of antibodies that were unable to precipitate with either FAd or with parasite proteinase. These antibodies exhibited positive agglutination reactions with epimastigote forms and positive immunofluorescence and immunoperoxidase reactions with trypomastigote and amastigote forms of the different strains tested. FAd was able to inhibit these reactions as well as those obtained with anti-LE and anti-FA immune sera, whereas purified proteinase was unable to inhibit any of these reactions.

Mediators of Inflammation, 1992

Pretreatment of human neutrophils with recombinant tumour necrosis factor-alpha (rTNF-α) and/or i... more Pretreatment of human neutrophils with recombinant tumour necrosis factor-alpha (rTNF-α) and/or interleukin-8 (rIL-8), but not with either transforming growth factor-beta, interleukin-6 or interferon-gamma, rendered these cells less responsive to FMLP, in microchemotaxis assays. This inhibitory effect was dose dependent and more powerful when neutrophils were pretreated with a mixture of both cytokines. Intravenous injection of human rIL-8 (hrIL-8) and/or murine rTNF-α (mrTNF-α) also significantly reduced in vivo neutrophil migration into peritoneal cavities of rats stimulated with carrageenan. These data suggest that the defect in neutrophil migration during septicaemia or endotoxaemia may be the result of the continuous release of IL-8 and TNF-α into the circulation. Thus, either the selective control or blockade of releasing of these cytokines as well as of its effects on neutrophils may be clinically useful in reestablishing the cell defence mechanisms.

Mediators of Inflammation, 1992

Inhibitory effect upon neutrophil migration to the inflammatory focus was previously detected in ... more Inhibitory effect upon neutrophil migration to the inflammatory focus was previously detected in the cell-free incubation fluid of lipopolysaccharide (LPS)-stimulated macrophage monolayers. In the present study we showed that the neutrophil recruitment inhibitory activity from this supernatant was mainly detected in a fraction (P2) obtained by gel filtration chromatography on Sephacryl S-300. P2 fraction was able to inhibit ‘in vivo’ neutrophil emigration induced by different inflammatory stimuli, but it did not affect ‘in vitro’ neutrophil chemotaxis induced by FMLP. When injected intravenously, P2 inhibited oedema induced by carrageenin or immunological stimulus but not the oedema induced by dextran, thus affecting cell-dependent inflammatory responses. It was observed that P2 also induced neutrophil migration when injected locally in peritoneal cavities. This activity was significantly reduced by pretreatment of the animals with dexamethasone. Cytokines, such as IL-8 and TNF-α th...

Mediators of Inflammation, 1997

Macrophages harvested from the peritoneal cavities of rats release a neutrophil chemotactic facto... more Macrophages harvested from the peritoneal cavities of rats release a neutrophil chemotactic factor (MNCF) in response to stimulation with Gram-negative bacterial lipopolysaccharide (LPS). MNCF has been shown to be active in rats treated with dexamethasone, a glucocorticoid that usually inhibits the neutrophil migration induced in this species by interleukin (IL)-1, tumour necrosis factor alpha (TNFα), IL-8, C5a and leukotriene B4(LTB4). Here we report that macrophages harvested from peritoneal cavities of mice, and stimulatedin vitrowith LPS, also release a factor that induces neutrophil migration in dexamethasone-treated animals. This chemotactic activity was neutralized by the incubation of the LPS-stimulated macrophage supernatants with a purified polyclonal IgG anti-mouse TNFα. In addition, significant amounts of TNF were detected in the supernatants. The neutrophil migration induced by intraperitoneal administration of recombinant murine TNFα was also unaffected by pretreatment...

Journal of Reproductive Immunology, 2008

Uterine natural killer (uNK) cells expand rapidly during endometrial decidualization and account ... more Uterine natural killer (uNK) cells expand rapidly during endometrial decidualization and account for 70% of leukocytes in early gestational uteri of humans and rodents. These cells make unique contributions to pregnancy, contributing to the success of embryo implantation and maintenance of decidual tissue that supports placental and fetal development. We postulated that uNK cells express molecules that are not shared by circulating NK (cNK) cells or other leukocytes and, therefore, would be immunogenic for male mice. We isolated viable uNK cells from gestation day 9 pregnant mice and inoculated them into syngeneic males. This induced antibodies reactive with mouse uNK cells but not with cNK cells or other lymphocytes. The antibodies reacted identically with uNK cells in tissue sections from five different mice strains from gestational day 7-12 and in pregnant rat uterus, suggesting that the recognized antigen should be a specific marker of uNK cell. Spleen cells from inoculated males were used subsequently to produce a monoclonal antibody reactive to a uNK cell surface antigen. These experiments confirm that uNK cells are a pregnancy-specific subset of NK cells expressing distinct surface antigen from those found in other tissues.

Food Research International, 2010

According to the Pan-American Health Organization (PAHO), there is a growing increase of degenera... more According to the Pan-American Health Organization (PAHO), there is a growing increase of degenerative diseases in countries of the Americas, bringing on disability and death of the population. As eating properly is the most effective way to reduce the risk of diseases, food scientists are seeking alternatives for more nutritional foods, mainly through the investigation of functional compounds in natural foods. Among the functional compounds, fructans stand out for their beneficial effects on the animal body, favoring the growth of bifidobacteria in the colon that improve gastrointestinal metabolism, as well as regulating the levels of serum cholesterol and the absorption of minerals. Besides, fructans seem to be involved in the positive modulation of the immune system, mainly in an increased resistance to infections and microbicidal activity as well as by the reduction of allergic reactions and cancer in experimental models. This paper aims to analyze literature data concerning the benefits of consuming the fructans contained in natural food, especially their immunomodulatory actions in both animals and humans.

Food Research International, 2011

The gradual increase of degenerative diseases observed in the last decades has been raising morbi... more The gradual increase of degenerative diseases observed in the last decades has been raising morbidity, incapacitation and mortality. The occurrence of these kinds of diseases is related to the aging of humanity as well as the unhealthy choices of individuals, particularly those dwelling in large urban centers, which are closely linked with poor nutrition, obesity, and tobacco and alcohol consumption. The introduction of functional compounds in the diet seems to be an attractive alternative to ameliorate the quality of life of all age groups. The prebiotics stand out because of their beneficial effects, favoring the growth of colonic microbiota, helping the gastrointestinal metabolism, and regulating the serum cholesterol and mineral absorption. Experimental data indicates that prebiotics could reduce the severity or incidence of degenerative diseases, such as neoplasias, diabetics, coronary diseases, and infectious diseases. They also seem to promote a positive modulation of the immune system. Their effects on the immune system could even be associated to increase of resistance to infection and microbicide capability, as well as to a decrease in allergic reactions. This article's goal is to analyze the immunomodulatory potential of prebiotics observed in experimental and trial studies.

FEBS Letters, 1999

A protein band of approximately 166 kDa was detected in the soluble fraction of root tips and you... more A protein band of approximately 166 kDa was detected in the soluble fraction of root tips and young leaves of maize seedlings, based on Western blot analysis using antibodies raised against mouse macrophage nitric oxide synthase (NOS) and rabbit brain NOS. NOS activity was present in these soluble fractions, as determined by L-[U-IR C]citrulline synthesis from L-[U-IR C]arginine. Immunofluorescence showed that the maize NOS protein is present in the cytosol of cells in the division zone and is translocated into the nucleus in cells in the elongation zone of maize root tips. These results indicate the existence of a NOS enzyme in maize tissues, with the localization of this protein depending on the phase of cell growth.

Clinical and Developmental Immunology, 2012

In this work, we evaluated the effects of administration of OVA on phenotype and function of intr... more In this work, we evaluated the effects of administration of OVA on phenotype and function of intraepithelial lymphocytes (IELs) from small intestine of transgenic (TGN) DO11.10 and wild-type BALB/c mice. While the small intestines from BALB/c presented a well preserved structure, those from TGN showed an inflamed aspect. The ingestion of OVA induced a reduction in the number of IELs in small intestines of TGN, but it did not change the frequencies of CD8+and CD4+T-cell subsets. Administration of OVA via oral + ip increased the frequency of CD103+cells in CD4+T-cell subset in IELs of both BALB/c and TGN mice and elevated its expression in CD8β+T-cell subset in IELs of TGN. The frequency of Foxp3+cells increased in all subsets in IELs of BALB/c treated with OVA; in IELs of TGN, it increased only in CD25+subset. IELs from BALB/c tolerant mice had lower expression of all cytokines studied, whereas those from TGN showed high expression of inflammatory cytokines, especially of IFN-γ, TGF-...

Brazilian Journal of Medical and Biological Research, 2010

Oral tolerance can be induced in some mouse strains by gavage or spontaneous ingestion of dietary... more Oral tolerance can be induced in some mouse strains by gavage or spontaneous ingestion of dietary antigens. In the present study, we determined the influence of aging and oral tolerance on the secretion of co-stimulatory molecules by dendritic cells (DC), and on the ability of DC to induce proliferation and cytokine secretion by naive T cells from BALB/c and OVA transgenic (DO11.10) mice. We observed that oral tolerance could be induced in BALB/c mice (N = 5 in each group) of all ages (8, 20, 40, 60, and 80 weeks old), although a decline in specific antibody levels was observed in the sera of both tolerized and immunized mice with advancing age (40 to 80 weeks old). DC obtained from young, adult and middle-aged (8, 20, and 40 weeks old) tolerized mice were less efficient (65, 17 and 20%, respectively) than DC from immunized mice (P < 0.05) in inducing antigen-specific proliferation of naive T cells from both BALB/c and DO11.10 young mice, or in stimulating IFN-γ, IL-4 and IL-10 production. However, TGF-β levels were significantly elevated in co-cultures carried out with DC from tolerant mice (P < 0.05). DC from both immunized and tolerized old and very old (60 and 80 weeks old) mice were equally ineffective in inducing T cell proliferation and cytokine production (P < 0.05). A marked reduction in CD86 + marker expression was observed in DC isolated from both old and tolerized mice (75 and 50%, respectively). The results indicate that the aging process does not interfere with the establishment of oral tolerance in BALB/c mice, but reduces DC functions, probably due to the decline of the expression of the CD86 surface marker.

Brazilian Journal of Medical and Biological Research, 2001