Potential use of ascorbic acid-based surfactants as skin penetration enhancers.

6-O-Ascorbic acid alkanoates (ASCn) are amphiphilic molecules having physical-chemical properties that depend on the alkyl chain length. The derivatives of low molecular weight (n < 11) have enough aqueous solubility to produce self-assemblies at room temperature ( approximately 25 degrees C), while those with longer alkyl chains possess a critical micellar temperature (CMT) higher than 30 degrees C. At higher temperatures (T degrees > CMT), ASCn aqueous suspensions turn into either micellar solutions or gel phases, depending on the length of the hydrophobic chain. On cooling, coagels are produced, which possess a lamellar structure that exhibit sharp X-ray diffraction patterns and optical birefringence. The semisolid consistency of such coagels is an interesting property to formulate dermatological pharmaceutical dosage forms able to solubilize and stabilize different drugs. The objective of the present study was the evaluation of the enhancing permeation effect of ASCn with different chain lengths and to correlate permeability changes with histological effects. With this purpose, ASCn coagels containing anthralin (antipsoriasic drug) or fluorescein isothiocyanate (FITC, hydrophobic fluorescent marker) were assayed on rat skin (ex vivo) and mice skin (in vivo), respectively. Also, histological studies were performed aimed at detecting some possible side effects of ASCn. No inflammatory cellular response was observed in the skin when ASCn coagels were applied, suggesting non-irritating properties. Light microscopy indicated slight disruption and fragmentation of stratum corneum. The penetration of ASCn through rat skin epidermis was very fast and quantitatively significant. The permeation of anthralin was significantly increased when the drug was vehiculized in ASCn coagels, compared to other pharmaceutical systems. The results indicated that ASC12 seems to have the highest enhancing effect on FITC permeation. ASC12 appears to be the compound that possesses the highest capacity to enhance the penetration of the drugs. Furthermore, it has the highest permeation of the serie.

B cells from aged mice exhibit reduced apoptosis upon B-cell antigen receptor stimulation and differential ability to up-regulate survival signals.

During ageing, autoimmune disorders and the higher susceptibility to infectious have been associated with alterations in the humoral immune response. We report that splenic B lymphocytes from aged mice exhibit lower level of apoptosis induced by B-cell antigen receptor (BCR) ligation in vitro. Respect to B cells from young mice the anti-mu stimulated aged B cells show similar Bcl-2 and Bcl-xL expression but differential kinetic of A1 degradation and a higher level of cFLIP and FAIM. Even though B cells from aged mice show minor Fas expression they exhibit the same susceptibility to anti-Fas induced apoptosis. Aged B cells also present upon BCR stimulation, a higher proliferative response and similar level of activation markers expression than B cells from young mice. These data agree with the observation that aged mice exhibit an increment of T2 and mature B cell subset which rapidly enters cell cycle upon BCR engagement. The diminished apoptosis after activation in aged mice could compromise homeostatic mechanism allowing the persistence of self and non-self antigen specific B cells.

Diminished percentage of antigen bearing cells in the lymph nodes of immune aged rats.

We have demonstrated previously that during experimental autoimmune prostatitis (EAP), aged rats show a diminished humoral autoimmune response. In the present paper we have studied the transport of the autoantigen from the site of injection toward lymphatic organs in rats of different ages with or without EAP. We used as autoantigen prostatic components (rat accessory glands (RAG)) conjugated with fluorescein isothiocyanate (FITC). Studies of flow cytometry, fluorescent microscopy and confocal microscopy show no differences in the percentage of RAG-FITC positive cells or in the localization of the cells in the popliteal lymph nodes of not-immunized young and aged rats. On the other hand, in 18-month-old rats immunized with either RAG or Ovalbumin there were lower levels of specific IgG antibodies and fewer antigen containing cells in the draining lymph nodes than those of 3- or 12-month-old rats. In all groups fluorescent cells were MHC class II positive and some were IgM positive. Our results demonstrate that in immunized 18-month-old rats there is a diminished percentage of cells bearing the antigen in the draining lymph nodes after antigen injection in the skin, related to the levels of specific antibodies able to form antigen-antibody complexes in the periphery.

Age-related alterations in inflammatory response during experimental autoimmune prostatitis.

Experimental autoimmune prostatitis (EAP) is an experimental model of autoimmune disease, developed in Wistar rats against prostatic components. The 12-and 18-month-old rats with EAP show a higher cellular autoimmune response and lower humoral autoimmune response compared to 3-month-old rats. The analysis of NO(.) and O(2)(-) production by peritoneal exudate cells (PECs) resulted in a higher NO(.) and O(2)(-) production in EAP rats at all ages, compared to control animals. PECs from 12- and 18-month-old rats produced more NO(.) and less O(2)(-) than PECs from 3-month-old rats. However, lipopolysacharide (LPS) did not stimulate PECs from aged rats for NO(.) production as much as in 3-month-old rats and thus, turning out in a lower index of LPS-stimulation of PECs from aged rats, compared to 3-month-old rats. Furthermore, the mast cells number in prostates of EAP rats, especially the number of degranulated cells, was higher than in control animals, but no significant differences were found between 3- and 12-month-old control rats. In conclusion, these results show that aging affects differentially the inflammation mediators during EAP.

Changes in the development of experimental autoimmune prostatitis (EAP) by castration in aged rats.

During Experimental Autoimmune Prostatitis (EAP), 12-month-old rats show a higher cellular autoimmune response and lower humoral autoimmune response against prostatic components than 3-month-old rats subjected to the same antigen stimulus. We analyzed if thymus recovery by orchidectomy could affect the development of EAP in 12-month-old rats. Thirty days after gonadectomy, 12-month-old rats showed an increment in the thymic mass and in the thymocytes absolute number, with percentages of the four main cell subpopulations (defined by CD4-CD8 molecules expression) similar to the 3-month-old rats. The DTH response of castrated 12-month-old with EAP were diminished in comparison with sham-castrated 12-month-old rats with EAP, resembling the values observed in 3-month-old rats with EAP. The prostates of castrated 12-month-old rats with EAP did not show inflammatory mononuclear cell infiltration, as did control 3- and 12-month-old rats with EAP. Castration seems to modulate negatively EAP in 12-month-old rats, possibly through the regeneration of thymus after testosterone deprivation.

Effect of aging on experimental autoimmune prostatitis: differential kinetics of development.

We have studied the influence of aging on the kinetics of autoimmune response in Experimental Autoimmune Prostatis (EAP). EAP was induced in 3- and 12-month-old Wistar rats by i.d. immunization with a saline extract of rat male sex accessory glands (RAG), chemically modified, and emulsioned in CFA. After immunization, 12-month-old rats developed a faster and stronger specific DTH response against RAG and mononuclear infiltration in the prostate. The levels of total IgM and IgG against RAG were lower in 12-month-old rats than in 3-month-old rats, with a prevalence of IgG2a, IgG2b, and IgG2c subclasses in both ages. Immunization stimulated slightly the appearance of specific IgG1 to RAG only in 3-month-old rats but in 12-month-old rats there was no specific IgG1 to RAG. On the other hand, normal 12-month-old rats showed higher levels of some natural antibodies and their thymocytes and peripheral lymphocytes had a diminished proliferative capacity compared to 3-month-old rats. These data demonstrated that 12-month-old rats show parameters of an aged immune system and present an exacerbated autoimmune prostatitis compared with 3-month-old rats.

Age-associated changes in lymphoid and antigen-presenting cell functions in mice immunized with Trypanosoma cruzi antigens.

The purpose of these studies was to analyze the role of different immune cell populations in the immune response against Trypanosoma cruzi antigens in aged mice. Mice of different ages (3 and 12 months old) were immunized i.d. with S-105 plus Bordetella pertussis as adjuvant and we compared the activities of the lymph node cells taken from 3- and 12-month-old donor animals to transfer DTH or antibody production to 3-month-old recipients. This study revealed that adherent and non-adherent immune lymph node cells of aged donor animals did not transfer response against the foreign antigen (S-105) whereas 3-month-old non-adherent lymph node cells transferred a DTH response as well as helped the specific antibody production. When total lymph node cells from 3- and 12-month-old mice were mixed, we observed an inhibition of S-105 transferred response indicating a suppressive effect of aged cells on the 3-month-old mice cells. Furthermore, we analyzed the participation of antigen-presenting cells (APC) in the immune response changes related to the previously described aged mice. Peritoneal cavities cells (PC), pulsed in vivo with S-105, obtained from 3- and 12-month-old mice were transferred to normal recipients and a DTH response to S-105 was studied. We observed that the DTH response was lower in the recipients of aged PC with respect to recipients of young PC. The results suggest that APC from aged mice are involved in controlling the cellular immune response to S-105. Age-related changes in immune T cell and APC are discussed in the context of these observations.

Involvement of accessory cells in the Trypanosoma cruzi-induced inhibition of the polyclonal response of T lymphocytes.

Infection with Trypanosoma cruzi is characterized by hyporesponsiveness of the immune system during the acute phase of infection. To better understand the immunological mechanisms affected by T. cruzi, we studied if a reduced T cell proliferative response could originate from an inability of T cells to proliferate or a functional deficiency at the level of accessory cells (AC). The inhibitory effect exerted by T. cruzi was during the induction phase of the lymphoproliferative response, suggesting the participation of AC in the hyporesponse. Then we further investigated the potential of the parasite to interfere with accessory cell-dependent and -independent pathways of human T cell proliferation. Peripheral blood mononuclear cells and peripheral blood lymphocytes from healthy individuals, enriched for T cells, were analysed with regard to their proliferative capacity using: phytohaemagglutinin, immobilized anti-CD3 monoclonal antibody (MoAb) and MoAb to the CD28 antigen, anti-CD3 MoAb and recombinant IL-2 and anti-CD3 MoAb plus phorbol myristate acetate in the presence of parasites. Significant suppression of the proliferative response was caused by the parasite only when AC were present. The parasite markedly reduced the surface expression of HLA-DR and CD11b antigens, key molecules in PHA-induced proliferation. Addition of indomethacin to the culture failed to reverse the inhibitory effect of the parasites, suggesting that prostaglandin E2 was not involved. These data suggest that AC in contact with T. cruzi become incompetent as antigen presenting cell because they are unable to induce a normal proliferative response in T lymphocytes.

Trypanosoma cruzi: transfer of protection by lymph node cells obtained from mice immunized with exoantigens of pI 4.5.

Exoantigens of pI 4.5 (Ea 4.5) of T. cruzi released to the circulation of infected mice are able to induce partial protective immune response in mice (F. Cerbán et al. 1991, International Archives of Allergy and Applied Immunology 96, 35-40). In order to analyze the participation of cellular immunity in the parasitemia control, we i.d. immunized mice with Ea 4.5 plus Bordetella pertussis as adjuvant. The role of immune cells in protective immunity was examined by adoptive transfer experiments. The immune lymph node cells (LNC) transferred the capacity to control the parasitemia, since it was observed that the normal recipients of immune LNC, which were afterward infected, presented a significant decrease in parasite levels with respect to the animals receiving LNC from control mice. This capacity was absent in the spleen cells. In addition, polystyrene nonadherent cells from immune LNC transferred the capacity to control T. cruzi infection. It was observed that Ig+ cells and enriched T cells from immunized mice are able to control the parasitemia. To define epitopes of Ea 4.5 able to stimulate protective immunity, the levels of parasitemia were examined in mice immunized with Ea 4.5 untreated or treated with sodium metaperiodate. These animals presented similar levels of parasitemia and in both cases they are significantly lower than the parasitemias of the control animals, suggesting that the most relevant epitopes for the protective immune response that control the beginning of the infection are not carbohydrates. Later, on Day 30 postinfection only the animals immunized with untreated Ea 4.5 maintained a significant decrease in parasite levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of aging on autoimmune response to rat male accessory glands: young, but not aged, antigen-presenting cells efficiently induce suppression in aged rats.

The present report analyzes the suppressor cell system of aged rats in an experimental model of autoimmunity to rat male accessory glands (RAG). A state of specific suppression to RAG was induced when young rats are pretreated with peritoneal cells (PC) obtained from syngeneic young rats i.p. injected 2 h previously with chromatographic fraction I (Sephadex G-100) (FI) of RAG (yFI-PC). Although the yFI-PC injection diminished the DTH in aged rats the autoimmune response remained positive. Peritoneal cells obtained from aged rats injected with FI of RAG (oFI-PC) did not suppress the DTH response in either aged or young rats. In both young and aged, pretreatment with yFI-PC stimulates spleen cells capable of inducing suppression (inductor-phase suppressor cells) when they are transferred to young recipients. However, the spleen inductor-phase suppressor cells of 12-month-old rats are unable to suppress the autoimmune response in their own aged environment. To obtain effective suppression in 12-month-old rats, the injection of yFI-PC was necessary prior to and subsequent to immunization. In this work we observe that 12-month-old rats could efficiently induce inducer phase and effector-phase suppressor cells when the adequate young antigen-presenting cells were present to stimulate them.

Enhancement of natural antibodies in mice immunized with exoantigens of pI 4.5 from Trypanosoma cruzi.

This paper deals with the enhancement of natural antibodies in mice immunized with a previously purified exoantigen of Trypanosoma cruzi from infected mouse plasma by isoelectric focusing, called Ea 4.5. A simultaneous rinse of IgG antibodies recognizing acidic sciatic nerve antigen (SNA) and other conserved antigens such as myoglobin, actin, thyroglobulin, and tubulin was observed. The highest level of antibodies was revealed when myoglobin was used as antigen in the ELISA test. Good correlation was found between the level of antibodies reactive with SNA and with highly conserved antigens. Furthermore, absorption experiments showed that a fraction of antibodies binding SNA are polyreactive and also react with the highly conserved antigens. The histological studies of sciatic nerve, heart and skeletal muscle performed 1 month after the last immunization revealed no modifications with respect to the control animals. Based on these and a previous result [1], indicating that injection of Ea 4.5 induced in mice a partial protection against T. cruzi, the possibility exists that a percentage of antibodies induced by Ea 4.5 may correspond to the natural autoantibody type and take part in protective and/or pathogenic effects.

Aging: the cellular immune response against autologous and foreign antigens is affected before the humoral response.

In this study we examined the auto- and hetero-immune response in mice of different ages immunized with antigens of Trypanosoma cruzi (S-105). We observed that 20-day- and 12-month-old mice showed decreased response to foreign antigens and increased response to autoantigens, compared with 3-month-old immunized mice. The 6-month-old mice showed hetero- and auto-immune cellular responses similar to those of 12-month-old animals; however, the humoral response was similar to that of 3-month-old animals against either antigen, suggesting that the compartments of the immune response are altered at different moments in the same individual. Immune response against a foreign antigen is correlated with the presence of cellular infiltrate in skeletal and heart muscle whereas no modifications in the tissue are noticed in animals with an autoimmune response. Also, we observed from cell transfer experiments that lymph node cells are involved in the dysregulation that we noticed with aging.

Immune response in mice immunized with acidic antigenic fractions from Trypanosoma cruzi cytosol.

The humoral and cellular immune responses as well as the resistance to infection with bloodstream forms of T. cruzi were studied in mice immunized with acidic antigenic fractions from parasite cytosol, F III and F IV, plus Bordetella pertussis as adjuvant. The immunization with F III induced positive ITH and DTH responses to homologous antigens. In mice immunized with F IV, the ITH was negative and four out of six animals presented positive DTH reactions. In both groups of mice the analysis of IgG against T. cruzi showed that the major isotype elicited was IgG1. Specific IgE was also detected in sera from F III immunized mice, thus confirming the presence of homocytotropic antibodies. The parasitemias reached by F III and F IV immunized mice after challenge were lower than those of the controls showing in this way a partial protection against the acute infection. The histological studies of heart and skeletal muscle performed two months after the infection revealed variable mononuclear infiltration in all infected mice despite immunization.

Trypanosoma cruzi exoantigens: can those recognized by sera from chagasic patients trigger a protective immune response in mice?

Exoantigens (Ea) of Trypanosoma cruzi released in blood during the acute phase of experimental murine infection and recognized as antigens by sera from chagasic patients were grouped into two zones: one zone of pI 4-5 (Ea4-5), which had components of 35 kDa, 50 kDa and slightly higher than 100 kDa, MW, and another zone, of pI 6-7 (Ea 6-7), with Ea of 50 kDa, 66-80 kDa and higher than 100 kDa. Immunization of mice with Ea4-5 or Ea6-7 prior to infection induced a protective immune response, as judged by levels of parasitaemia which were significantly lower than those of controls. Analysis of the immune response by skin test revealed that both groups of Ea induced immediate type hypersensitivity, the values of which were higher in animals immunized with Ea4-5. These antigens also induced specific cellular immunity (delayed-type hypersensitivity). There was a direct correlation between intensity of reactivity and the drop in the number of blood forms of parasites in these animals. Antibodies able to fix the epimastigote surface were also detected by ELISA and the immunofluorescence test in mice immunized with Ea4-5 or Ea6-7. There were no qualitative or quantitative differences in the antibody induced by the two groups of Ea; the main isotypes of these antibodies which recognized Ea expressed on the parasite surface were IgG1 and IgG2.

Effect of aging on the autoimmune response to rat male accessory glands: deficit of I-E-positive peritoneal cells capable of inducing suppression.

The present report analyzes the ability to induce suppression to rat male accessory gland (RAG) autoantigens and the characteristics of T suppressor (Ts)-inducer peritoneal cells (PC) in old rats which show increased autoimmune responses. The injection of young rats with a purified fraction (FI) of RAG 10 and 3 days prior to immunization with chemically modified RAG (MRAG) markedly reduced the immune response to RAG autoantigens when compared with young rats which had only been immunized (controls), while the pretreatment of old rats did not block the delayed-type hypersensitivity reaction to MRAG when compared with control old rats. The study of cell surface markers on PC from rats injected i.p. 2 h previously with FI of RAG (FI-PC) showed an increase of OX-6 (I-A) and a decrease of OX-17 (I-E) in FI-PC of old rats with respect to FI-PC of young animals, which showed a selective increase of I-E+ Ts-inducer PC. The i.p. injection of FI-PC from old rats into young recipients, 10 and 3 days prior to immunization with MRAG in complete Freund's adjuvant, did not modify the autoimmune response when compared with controls. By contrast, the injection of young and old rats with FI-PC from young animals induced a significant suppression of the autoimmune response. The reduced percentage of I-E+ suppressor-inducer PC provides an explanation for the diminished ability to induce suppression to RAG autoantigens in old rats.

Mouse IgE response against exoantigens of Trypanosoma cruzi.

This work describes the occurrence of anaphylactic antibodies against exoantigens of Trypanosoma cruzi in infected or immunized mice. The results obtained show that the exoantigens induce an IgE response when injected into mice with complete Freund's adjuvant or in animals infected with 10(2) trypomastigotes. Among the antigens recognized by anti-exoantigen IgE, two molecules of 27 and 45 kD were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by western blotting. Immunofluorescence analysis located some target antigens on the parasite surface.

IgE dependent autoimmune response. Effect of Trypanosoma cruzi infection.

The autoimmune response to mouse accessory glands (MAG) was investigated in male BALB/c mice immunized with different doses of chemically modified mouse accessory glands (MMAG) and complete Freund's adjuvant (CFA). This autoimmune response was studied at several time intervals using the skin test with MAG. It was found that 5 mg of MMAG induced on the day 15 an autoimmune response detected by specific skin test at 20 min., 3 h and 24 h. The results of the immediate type hypersensitivity (ITH) were higher than those with the other skin tests. In order to study the type of immunoglobulin involved, the ITH was also analyzed by passive cutaneous anaphylaxis (PCA) at different time intervals with treated and untreated sera at 56 degrees C. The findings suggest the presence of reaginic antibodies, IgE being the major antibody as detected by enzime linked immunosorbent assay (ELISA). The MAG was subsequently fractionated using Sephandex G-100 and the fractions thus obtained (FI,FII and FIII) were used to challenge mice immunized with MMAG. It was found that FI was the only fraction which revealed an ITH similar to that revealed by MAG. The effect of infection with Trypanosoma cruzi on the autoimmune response to MAG was analyzed with different mouse groups intraperitoneally treated with 2 x 10(3) blood trypomastigotes/animal at several time intervals: namely, on days -5, 0, +5 and +10 with respect to the immunization with MMAG. The autoimmune response to MAG showed suppression when the animals received the parasites on the same day as the autoantigen.

Participation of different cellular types in the enhancement of autoimmune response of old animals to sex accessory glands in male rats: importance of macrophages.

In a previous work, we showed that the immunization of male rats, 3 and 12 months old, with saline extract of rat male accessory glands chemically modified (MRAG) and human serum albumin (HSA) induced a higher humoral and cellular autoimmune response in old animals than in young ones. We have also demonstrated that the facilitation of the autoimmune response is transferred by spleen total cells of 12-month-old animals. The immune response to HSA was not modified. In this work, the cellular type involved in such facilitation was analyzed. For this transference experiment, cells enriched in T and B lymphocytes and macrophages were used. The results showed that the macrophage is the main cellular type involved. However, the transference was only total with the three cellular types together. The study, performed with macrophages pulsed in vivo with MRAG-HSA and then transferred to normal recipients, indicated that although the macrophages from young and old animals were capable of presenting the antigens, the latter did this with significantly greater efficiency for the autoantigen.