The role of interleukins in vitiligo: a systematic review.

Vitiligo is a disorder of the skin that causes depigmentation and asymptomatic macules whose exact cause is still unclear. Although its aetiology is not fully elucidated, the main theory of its pathomechanism is that it is associated with the autoimmune process. There is few summarized information about the role of inflammatory mediators, as interleukins, in vitiligo, so our aim was to present a systematic review of the role of interleukins in vitiligo, focusing on interleukins. In this review, we included all studies assessing interleukin levels in vitiligo patients conducted up to June 2017. Quality assessment of these studies was performed using the Newcastle-Ottawa Scale (NOS). The interleukins mainly involved were IL-2, IL-4, IL-6, IL-10 and IL-17. The studies highlight the crucial role of IL-17 in the onset and progression of the disease, and its synergistic action with IL-2, IL-6 and IL-33. Dysregulated levels of the interleukins were also correlated with the stage of disease, the affected skin surface area, and indicated as the main factor for lymphocyte infiltration found in depigmented regions. These findings illustrate the growing need for new therapies targeting vitiligo and further research into the role of interleukins as an area of particular interest.

Antinociceptive and anti-inflammatory activities of Hymenaea martiana Hayne (Fabaceae) in mice.

Hymenaea martiana is a species popularly known in Northeastern Brazil as "jatobá" and used in folk medicine to treat pain and inflammation. The aim of this work was to evaluate the antinociceptive and anti-inflammatory activity of H. martiana. In the present study, we carried out an investigation about the effects of the crude ethanolic extract (Hm-EtOH) and the ethyl acetate fraction (Hm-AcOEt) in models of nociception and inflammation in mice. Chemical (acetic acid-induced writhing and formalin) and thermal stimuli (hot plate) were used for the evaluation of antinociceptive activity, while for the anti-inflammatory profile paw edema induced by carrageenan was used, along with leukocyte migration to the peritoneal cavity. The presence of the flavonoid astilbin in the samples was characterized through HPLC-DAD-MS analysis. Hm-EtOH and Hm-AcOEt (100, 200 and 400 mg.kg-1, i.p.) significantly reduced the number of abdominal contortions and decreased the paw licking time in the formalin test. In the hot plate, the extract increased the latency time of animals. Hm-EtOH and Hm-AcOEt inhibited significantly the increase in the edema after the administration of carrageenan. Hm-EtOH and Hm-AcOEt inhibited leukocyte migration in the peritonitis test. HPLC-DAD-MS analysis of Hm-EtOH and Hm-AcOEt revealed the presence of the flavonoid astilbin in the samples. According to the results of this study, both Hm-EtOH and Hm-AcOEt have antinociceptive and anti-inflammatory activities, which could be related with the presence of flavonoid in the extracts. The results reinforce the popular use of this plant.

Alteration of clonal profile. III. T15 ontogenetic advantages are not sufficient for establishing idiotypic dominance in adoptive transfer.

We have examined the ontogeny of BALB/c plaque-forming cell (PFC) responses to phosphorylcholine (PC) from fetal and neonatal liver by using the (CBA/N x BALB/c)F1 transplantation model. In this system, thymus-dependent (PC-keyhole limpet hemocyanin) and thymus-independent class 1 (PC-Brucella abortus, PC-lipopolysaccharide) PC antigens stimulate B cell subpopulations, which functionally emerge early after transfer. Responsiveness to a thymus-independent class 2 antigen, C-polysaccharide extract of a Streptococcus pneumoniae mutant, is acquired later. The response to PC antigens tested initially exhibited T15 dominance. Non-T15 clones, which are not expressed to a great degree in normal BALB/c mice, are inherently slow in their rate of maturation; in adoptive transfer, however, they eventually comprise much of the transplanted anti-PC PFC response. Obviously, the advantages the T15 subset has in ontogeny do not result in idiotypic dominance once the immature cells are removed from the intact BALB/c environment. We discuss possible regulatory mechanisms involved in the alteration of the T15+:T15- ratio.

Alteration of clonal profile. I. Effect of sublethal irradiation on the responses to phosphorylcholine in BALB/c mice.

BALB/c mice exhibit greater than 90% H8 clonal dominance in the immune response to phosphorylcholine. Adult mice exposed to 500 rads were initally unable to produce a humoral immune response to both phosphorylcholine and trinitrophenol antigens, and the direct plaque-forming cell response was slowly regained over several weeks. Clonotypic analysis wity antisera directed against the H8 idiotype showed that the H8 clone initially dominated the recovery of the response to phosphorycholine but that 60 days after the irradiation significant numbers of non-H8 clones could be detected. This same pattern could be seen in mice irradiated with 100 rads, a dose that does not completely abrogate the H8 response to phosphorylcholine. Sublethal irradiation of neonates before they had acquired responsiveness to phosphorylcholine could also eventually lead to the emergence of non-H8 idiotypes. Thus, a radiosensitive element regulates the expression of clonal dominance in anti-phosphorylcholine responses of BALB/c mice.

Phosphorylcholine-specific helper T cells in mice with an X-linked defect of antibody production to the same hapten.

F1 male mice with the CBA/N X-linked defect that are unable to produce plaque-forming cell responses to phosphorylcholine (PC) provide normal PC-specific helper T-cell activity when compared to F1 female littermates. Inhibition of helper activity with anti-idiotypic antiserum indicates that PC-specific T cells from both NBF1 female and male mice possess predominantly BALB/c myeloma protein HOPC-8 idiotypic determinants. Therefore, the CBA/N defect cannot be explained as a deletion of genes coding for V-region anti-PC specificities. The demonstration of helper activity in NBF1 male mice, which occurs in the absence of anti-PC antibody synthesis, also demonstrates the endogenous origin of the T-cell receptor.

Mice with the xid defect have helper cells for T15 idiotype-dominant anti-phosphorylcholine primary and secondary plaque-forming cells responses.

We have examined the abilities of helper T cells from commercially available (CBA/N X BALB/c)F1 (NBF1) xid male and phenotypically normal female mice to help T15+ and T15- B cells to produce thymus-dependent phosphorylcholine (PC)-specific direct plaque-forming cell responses. Carrier-primed T cells from both male and female mice were found (a) to restore T15+ TD responses in congenitally athymic BALB/c mice, (b) to help PC-primed BALB/c splenic B cells produce predominantly T15+ responses, and (c) to provide help for T15+ and T15- PFC responses generated by PC-primed normal F1 splenic B cells. Furthermore, carrier-primed irradiated xid and normal recipients contributed adequate helper activity for T15 dominant responses. We therefore conclude that male and female NBF1 mice are equally capable of helping T15+ responses.

Antibody response of rabbit blood lymphocytes in vitro. Kinetics, clone size, and clonotype analysis in response to streptococcal group polysaccharide antigens.

Peripheral blood lymphocytes (PBL) of rabbits previously hyperimmunized against streptococcal groups A and A-variant antigens were stimulated in vitro by the corresponding vaccines to produce group-specific antibody. This response was dependent on an optimal cell density (2 X 10(6) cells/ml), on the presence of antigen, it was specific and cross-reactive due to a shared rhamnose backbone of the two polysaccharide antigens, and it was highly selective, such that in a 42-55-day culture 1 out of 20 viable cells was a specific PFC. During the exponential increase of the antibody concentration at a constant number of PFC, antibodies were secreted at a rate of 2.4 X 10(4) molecules/s per cell until a plateau level of antibody (40 mug/culture) was reached. The microculture system was used to determine the minimal frequency of group polysaccharide-specific precursor cells in the blood. Independent of the time elapsed since the last immunization this frequency was 1-3 X 10(-5), i.e., in the range of 1-2.8 X 10(2) precursor cells per ml blood. This number was further used together with the clonotype analysis of the culture supernates to calculate the frequencies of precursors of major and minor clonotypes. A hierachy of persisting clonal memory precursor cells was found indicating that clonal dominance is determined by locked-in frequency patterns and therefore it is a phenomenon based on numbers of cells that respond to the antigen.

Antinociceptive and anti-inflammatory activities of Hymenaea martiana Hayne (Fabaceae) in mice / Atividade antinociceptiva e anti inflamatória de Hymenaea martiana Hayne (Fabaceae) em camundongos

Hymenaea martiana is a species popularly known in Northeastern Brazil as "jatobá" and used in folk medicine to treat pain and inflammation. The aim of this work was to evaluate the antinociceptive and anti-inflammatory activity of H. martiana. In the present study, we carried out an investigation about the effects of the crude ethanolic extract (Hm-EtOH) and the ethyl acetate fraction (Hm-AcOEt) in models of nociception and inflammation in mice. Chemical (acetic acid-induced writhing and formalin) and thermal stimuli (hot plate) were used for the evaluation of antinociceptive activity, while for the anti-inflammatory profile paw edema induced by carrageenan was used, along with leukocyte migration to the peritoneal cavity. The presence of the flavonoid astilbin in the samples was characterized through HPLC-DAD-MS analysis. Hm-EtOH and Hm-AcOEt (100, 200 and 400 mg.kg-¹, i.p.) significantly reduced the number of abdominal contortions and decreased the paw licking time in the formalin test. In the hot plate, the extract increased the latency time of animals. Hm-EtOH and Hm-AcOEt inhibited significantly the increase in the edema after the administration of carrageenan. Hm-EtOH and Hm-AcOEt inhibited leukocyte migration in the peritonitis test. HPLC-DAD-MS analysis of Hm-EtOH and Hm-AcOEt revealed the presence of the flavonoid astilbin in the samples. According to the results of this study, both Hm-EtOH and Hm-AcOEt have antinociceptive and anti-inflammatory activities, which could be related with the presence of flavonoid in the extracts. The results reinforce the popular use of this plant.

Hymenaea martiana é uma espécie popularmente conhecida no Nordeste do Brasil como “jatobᔠe usada na medicina popular para tratar a dor e a inflamação. O objetivo deste trabalho foi avaliar a atividade antinociceptiva e anti inflamatória de H. martiana. No presente estudo, foram avaliados os efeitos do extrato etanólico bruto (Hm-EtOH) e da fração acetato de etila (Hm-AcOEt) em modelos de nocicepção e inflamação em camundongos. Foram utilizados estímulos químicos (contorções abdominais induzidas por ácido acético e teste da formalina) e estímulo térmico (teste da placa quente) para avaliação da atividade antinociceptiva, enquanto no perfil anti-inflamatório foi utilizado o teste do edema de pata induzido por carragenina e migração de leucócitos para a cavidade peritoneal. A presença do flavonoide astilbina nas amostras foi caracterizada através de análise por CLAE-DAD-EM. Hm-EtOH e o Hm-AcOEt (100, 200 e 400 mg.kg-¹, i.p.) reduziram significativamente o número de contorções abdominais e diminuíram o tempo de lambida da pata no teste da formalina. No teste da placa quente, houve aumento do tempo de latência dos animais. Hm-EtOH e Hm-AcOEt inibiram significativamente o aumento do edema após a administração de carragenina, bem como inibiram a migração de leucócitos no teste de peritonite. A análise por CLAE-DAD-EM de Hm-EtOH e Hm-AcOEt revelou a presença do flavonoide astilbina nas amostras. De acordo com os resultados deste estudo, tanto Hm-EtOH quanto o Hm-AcOEt possuem atividades antinociceptiva e anti-inflamatória, o que pode estar relacionado à presença do flavonoide. Os resultados reforçam o uso popular desta planta.

Antinociceptive and anti-inflammatory activities of Hymenaea martiana Hayne (Fabaceae) in mice

Abstract Hymenaea martiana is a species popularly known in Northeastern Brazil as jatobá and used in folk medicine to treat pain and inflammation. The aim of this work was to evaluate the antinociceptive and anti-inflammatory activity of H. martiana. In the present study, we carried out an investigation about the effects of the crude ethanolic extract (Hm-EtOH) and the ethyl acetate fraction (Hm-AcOEt) in models of nociception and inflammation in mice. Chemical (acetic acid-induced writhing and formalin) and thermal stimuli (hot plate) were used for the evaluation of antinociceptive activity, while for the anti-inflammatory profile paw edema induced by carrageenan was used, along with leukocyte migration to the peritoneal cavity. The presence of the flavonoid astilbin in the samples was characterized through HPLC-DAD-MS analysis. Hm-EtOH and Hm-AcOEt (100, 200 and 400 mg.kg-1, i.p.) significantly reduced the number of abdominal contortions and decreased the paw licking time in the formalin test. In the hot plate, the extract increased the latency time of animals. Hm-EtOH and Hm-AcOEt inhibited significantly the increase in the edema after the administration of carrageenan. Hm-EtOH and Hm-AcOEt inhibited leukocyte migration in the peritonitis test. HPLC-DAD-MS analysis of Hm-EtOH and Hm-AcOEt revealed the presence of the flavonoid astilbin in the samples. According to the results of this study, both Hm-EtOH and Hm-AcOEt have antinociceptive and anti-inflammatory activities, which could be related with the presence of flavonoid in the extracts. The results reinforce the popular use of this plant.

Resumo Hymenaea martiana é uma espécie popularmente conhecida no Nordeste do Brasil como jatobá e usada na medicina popular para tratar a dor e a inflamação. O objetivo deste trabalho foi avaliar a atividade antinociceptiva e anti-inflamatória de H. martiana. No presente estudo, foram avaliados os efeitos do extrato etanólico bruto (Hm-EtOH) e da fração acetato de etila (Hm-AcOEt) em modelos de nocicepção e inflamação em camundongos. Foram utilizados estímulos químicos (contorções abdominais induzidas por ácido acético e teste da formalina) e estímulo térmico (teste da placa quente) para avaliação da atividade antinociceptiva, enquanto no perfil anti-inflamatório foi utilizado o teste do edema de pata induzido por carragenina e migração de leucócitos para a cavidade peritoneal. A presença do flavonoide astilbina nas amostras foi caracterizada através de análise por CLAE-DAD-EM. Hm-EtOH e o Hm-AcOEt (100, 200 e 400 mg.kg-1, i.p.) reduziram significativamente o número de contorções abdominais e diminuíram o tempo de lambida da pata no teste da formalina. No teste da placa quente, houve aumento do tempo de latência dos animais. Hm-EtOH e Hm-AcOEt inibiram significativamente o aumento do edema após a administração de carragenina, bem como inibiram a migração de leucócitos no teste de peritonite. A análise por CLAE-DAD-EM de Hm-EtOH e Hm-AcOEt revelou a presença do flavonoide astilbina nas amostras. De acordo com os resultados deste estudo, tanto Hm-EtOH quanto o Hm-AcOEt possuem atividades antinociceptiva e anti-inflamatória, o que pode estar relacionado à presença do flavonoide. Os resultados reforçam o uso popular desta planta.

Antinociceptive and anti-inflammatory activities of Hymenaea martiana Hayne (Fabaceae) in mice / Atividade antinociceptiva e anti-inflamatória de Hymenaea martiana Hayne (Fabaceae) em camundongos

Hymenaea martiana is a species popularly known in Northeastern Brazil as "jatobá" and used in folk medicine to treat pain and inflammation. The aim of this work was to evaluate the antinociceptive and anti-inflammatory activity of H. martiana. In the present study, we carried out an investigation about the effects of the crude ethanolic extract (Hm-EtOH) and the ethyl acetate fraction (Hm-AcOEt) in models of nociception and inflammation in mice. Chemical (acetic acid-induced writhing and formalin) and thermal stimuli (hot plate) were used for the evaluation of antinociceptive activity, while for the anti-inflammatory profile paw edema induced by carrageenan was used, along with leukocyte migration to the peritoneal cavity. The presence of the flavonoid astilbin in the samples was characterized through HPLC-DAD-MS analysis. Hm-EtOH and Hm-AcOEt (100, 200 and 400 mg.kg-1, i.p.) significantly reduced the number of abdominal contortions and decreased the paw licking time in the formalin test. In the hot plate, the extract increased the latency time of animals. Hm-EtOH and Hm-AcOEt inhibited significantly the increase in the edema after the administration of carrageenan. Hm-EtOH and Hm-AcOEt inhibited leukocyte migration in the peritonitis test. HPLC-DAD-MS analysis of Hm-EtOH and Hm-AcOEt revealed the presence of the flavonoid astilbin in the samples. According to the results of this study, both Hm-EtOH and Hm-AcOEt have antinociceptive and anti-inflammatory activities, which could be related with the presence of flavonoid in the extracts. The results reinforce the popular use of this plant.

Hymenaea martiana é uma espécie popularmente conhecida no Nordeste do Brasil como "jatobá" e usada na medicina popular para tratar a dor e a inflamação. O objetivo deste trabalho foi avaliar a atividade antinociceptiva e antiinflamatória de H. martiana. No presente estudo, foram avaliados os efeitos do extrato etanólico bruto (Hm-EtOH) e da fração acetato de etila (Hm-AcOEt) em modelos de nocicepção e inflamação em camundongos. Foram utilizados estímulos químicos (contorções abdominais induzidas por ácido acético e teste da formalina) e estímulo térmico (teste da placa quente) para avaliação da atividade antinociceptiva, enquanto no perfil anti-inflamatório foi utilizado o teste do edema de pata induzido por carragenina e migração de leucócitos para a cavidade peritoneal. A presença do flavonoide astilbina nas amostras foi caracterizada através de análise por CLAE-DAD-EM. Hm-EtOH e o Hm-AcOEt (100, 200 e 400 mg.kg-1, i.p.) reduziram significativamente o número de contorções abdominais e diminuíram o tempo de lambida da pata no teste da formalina. No teste da placa quente, houve aumento do tempo de latência dos animais. Hm-EtOH e Hm-AcOEt inibiram significativamente o aumento do edema após a administração de carragenina, bem como inibiram a migração de leucócitos no teste de peritonite. A análise por CLAE-DAD-EM de Hm-EtOH e Hm-AcOEt revelou a presença do flavonoide astilbina nas amostras. De acordo com os resultados deste estudo, tanto Hm-EtOH quanto o Hm-AcOEt possuem atividades antinociceptiva e anti-inflamatória, o que pode estar relacionado à presença do flavonoide. Os resultados reforçam o uso popular desta planta.

Protein kinase C inhibition induces apoptosis and ceramide production through activation of a neutral sphingomyelinase.

We report that WEHI-231 undergo apoptosis following exposure to the protein kinase C inhibitors chelerythrine chloride and calphostin C. Following the addition of chelerythrine or calphostin C to WEHI-231 cells, ceramide production increased over baseline levels with a concurrent decrease in sphingomyelin. More detailed examinations determined that the ceramide accumulation resulted from activation of neutral, but not acidic, sphingomyelinase. These results suggest an antagonistic relationship between protein kinase C activity and ceramide in the signaling events preceding apoptosis.

Loss of ceramide production confers resistance to radiation-induced apoptosis.

Ionizing radiation mediates cell death, in part, through chromosomal damage following one or more cell divisions. X-rays also induce programmed cell death (apoptosis) in some cell types both in vitro and in vivo. Both neutral and acidic sphingomyelinases, which generate the lipid second messenger ceramide, are reported to induce apoptosis following ionizing radiation and other death signals such as tumor necrosis factor alpha and Fas ligand. Herein we report that a loss of ceramide production from a neutral sphingomyelinase generates a radioresistant phenotype as measured by a marked decrease in apoptosis. A WEHI-231 subline made deficient in ceramide production was found to be resistant to apoptosis compared with the parental subline following treatment with X-rays. The resistant subline underwent two to three subsequent cell divisions following X-irradiation, confirming that X-rays induce cell death through both mitotic and apoptotic mechanisms. These data suggest that loss of ceramide production following X-rays represents an extranuclear mechanism for the development of radioresistance. Modulation of extranuclear signals may increase tumor cell killing following radiation and represent new cellular targets for cancer therapy.

Suppression of Bcl-2 messenger RNA production may mediate apoptosis after ionizing radiation, tumor necrosis factor alpha, and ceramide.

Recent studies have proposed that tumor necrosis factor alpha (TNF-alpha) and ionizing radiation induce apoptosis by activating hydrolysis of sphingomyelin to ceramide. Bcl-2 and a related gene, Bcl-X, inhibit several forms of apoptosis. Herein, we report that internucleosomal DNA fragmentation, characteristic of apoptosis and induced by ionizing radiation, is accompanied by concomitant decreases in Bcl-2 and Bcl-X mRNA levels in HL-60 and U-937 human leukemia cells. Apoptotic DNA fragmentation after exposure to TNF-alpha and C2-ceramide was also associated with down-regulation of Bcl-2 mRNA in HL-60 and U-937 cells, while Bcl-X mRNA production was unaffected. These results suggest that modulation of Bcl-2 gene expression may be a target for ceramide-mediated apoptosis following exposure to ionizing radiation and TNF-alpha. Changes in Bcl-2 expression may be the basis for the interactive killing observed between radiation and TNF-alpha in some human and tumor cells.

Decreasing the apoptotic threshold of tumor cells through protein kinase C inhibition and sphingomyelinase activation increases tumor killing by ionizing radiation.

Approximately 30% of cancer deaths result from the failure to control local and regional tumors. The goal of radiotherapy is to maximize local and regional tumor cell killing while minimizing normal tissue destruction. Attempts to enhance radiation-mediated tumor cell killing using halogenated pyrimidines, antimetabolites, and other DNA-damaging agents or sensitizers of hypoxic tumor cells have met with only modest clinical success. In an unique strategy to modify tumor radiosensitivity, we used an inhibitor of the protein kinase C group A and B isoforms, chelerythrine chloride (chelerythrine), to enhance the killing effects of ionizing radiation (IR). Protein kinase C activity plays a central role in cellular proliferation, differentiation, and apoptosis. Chelerythrine increases sphingomyelinase activity and enhances IR-mediated cell killing through induction of apoptotic tumor cell death in a radioresistant tumor model both in vitro and in vivo. Although previous reports have suggested that IR-mediated apoptosis correlates with tumor volume reduction, we demonstrate for the first time that lowering the apoptotic threshold increases tumor cell killing in vivo.

Studies on the induction of apoptosis in WEHI 231 cells by pharmacological agents and lipid mediators. Sphingosine and ceramide induce apoptosis in WEHI 231.

We report the results of a systematic study of the effects of pharmacological agents known to cause or modify physiological cell death (PCD). Using WEHI 231 cells as a model, we investigated the effects of dexamethasone, cAMP, selected growth factors/ cytokines, DNA damaging agents, metabolic inhibitors and lipid mediators. We found that WEHI 231 cells are not affected by cAMP(1-90 microM) or TGFbeta (1-50 ng/ml), both of which are known to induce PCD in other systems. We also failed to detect protection from PCD in WEHI 231 cells cultured with Zn(++), E64 and leupeptin. In contrast, dexamethasone (400 microg/ml), etoposide (10(-4)M), emetine (10(-5)M), calyculin (10(-5)M), sphingosine (8-16 microM) and ceramide (20-40 microM) all cause PCD in WEHI 231 cells. The effects of ceramide can be blocked by LPS but not by overexpression of bcl2.The role of killer lipids in PCD is discussed.

The ability of Bcl-x(L) and Bcl-2 to prevent apoptosis can be differentially regulated.

Although expression of Bcl-2 has been shown to prevent apoptosis under many circumstances, there are several systems in which Bcl-2 fails to promote cell survival. We have previously reported that Bcl-2 and Bcl-x(L) display differential ability to protect WEHI-231 cells from multiple inducers of apoptosis. A possible explanation for this paradox was provided by the discovery of Bax. Bax is a Bcl-2-related protein which can inhibit the ability of Bcl-2 to enhance the survival of growth factor-dependent cell lines in the absence of growth factor. Consistent with the possibility that Bcl-2 function in WEHI-231 cells is inhibited by Bax, WEHI-231 cells were found to express a high level of Bax. To directly test the effects of Bax expression on Bcl-x(L) function, FL5.12 cells were transfected with both genes. Although Bax overexpression can inhibit Bcl-2 from prolonging cell survival upon growth factor withdrawal, Bax overexpression did not inhibit Bcl-x(L) from preventing apoptosis in this cell line. Although Bcl-2 and Bcl-x(L) were both found to be able to form heterodimers with Bax, the majority of Bax in both cases was not complexed to a partner. Our data suggest that Bcl-x(L) does not function by simply preventing the formation of Bax homodimers which promote cell death. Instead Bax appears to display selectivity in its ability to inhibit Bcl-2 but not Bcl-x(L) from prolonging survival. Furthermore, our data suggest that the abilities of Bcl-2 and Bcl-x(L) to promote cell survival are not identical and can be independently regulated within a cell. Regulation of a cell's apoptotic threshold is likely to result from a complex set of interactions among Bcl-2 family members and other, as yet uncharacterised, regulators of apoptosis.

Bcl-2 and Bcl-2-related proteins in apoptosis regulation.

In this review we have discussed the importance of Bcl-2 and related proteins in the regulation of apoptotic cell death in mammalian systems. It is clear that Bcl-2 plays a critical role in controlling many forms of PCD. Bcl-2 seems to have particular significance in lymphocyte development and the function of the immune system. We have also discussed the increasing size of the newly identified Bcl-2 family. There are a number of Bcl-2 homologues in human, murine, avian, nematode, and viral systems. The evolutionary conservation of the function of the Bcl-2 homologues, reinforces the importance of PCD in all complex organisms. Some of these bcl-2-like genes function as agonists and others as antagonists. Despite the seemingly universal importance of Bcl-2, it is unable to prevent PCD in all systems. In addition, we have described a role for other Bcl-2 family members in systems in which Bcl-2 is ineffective and supplied a potential rationale for the large number of genes involved in the regulation of PCD. Identification and functional analysis of the Bcl-2 family members reveals the complex nature of cell death regulation. As we begin to appreciate the significance of PCD in the control of development and homeostasis, its regulation at the molecular level is becoming better understood. Bcl-2 has long been the only known intracellular regulator of the PCD pathway(s), although its ability to prevent apoptosis is not universal. We now know that bcl-2 is only one member of an evolutionary conserved family of genes which display different patterns of expression as well as function. At least two family members, Bcl-xs and Bax, act in opposition to Bcl-2. The discovery of these new family members, including those with Bcl-2-like function and antagonists, should help clear up the discrepancies seen in Bcl-2's ability to protect cells from PCD. In doing so, we will be able to further define the pathways associated with cell death signaling. The study of these family members, as well as the non-related genes of the PCD pathways (ced-3, ced-4, ice) should lead us to understanding of how cells of multicellular organisms make decisions to die.

Delayed antigen presentation by epidermal Langerhans cells to cloned T h1 and T h2 cells.

Langerhans (LC) cells require incubation with protein antigen for several days before the cells effectively stimulate proliferation of cloned, H-2 restricted, antigen-specific T h cells. In contrast, splenic antigen-presenting cells are immediately effective. LC are immediately competent, however, if an immunogenic peptide rather than the intact protein is the immunogen, indicating that resident or unchallenged LC have the required class II MHC and can provide the signals necessary for T-cell proliferation but may lack the capacity to internalize or cleave protein antigens. We propose that delayed antigen presentation by LC may be intrinsic and advantageous for promoting early systemic immunity. LC stimulate cloned T h1 and T h2 cells equally well, suggesting that LC may not limit or bias the type of immunity that occurs with cutaneous antigenic challenge.

Production of thyroid-stimulating antibodies in mice by immunization with T-cell epitopes of human thyrotropin receptor.

We examined whether mice, immunized with TSH receptor (TSH-R) peptides, which are known to be T-cell epitopes in patients with Graves' disease, would show thyroid-stimulating antibody (TSAb). We immunized DBA/1J mice with TSH-R peptide amino acids 132-150, 145-163, 158-176, and 172-186 and with a pool of these four peptides. The antibodies produced in these mice were evaluated by measurement of TSAb activity using Chinese hamster ovary cells expressing human TSH-R. Seven of 20 mice showed TSAb activity that could be partially blocked with TSH-R peptides. To assess the role of T-cell epitope-specific T-cells in the production of TSAb, we transferred a T-cell line developed from a TSAb-positive mouse to other syngeneic DBA/1J mice. Two of 4 recipient mice showed TSAb activities. These findings suggest that specific T-cell epitopes of TSH-R play a crucial role in the production of TSAb.

Immunosuppression of thyroiditis.

Immunization of mice with 50 micrograms human thyroglobulin (TG) in complete Freund's adjuvant leads to histological thyroiditis; production of IgG, IgA, and IgM anti-TG antibodies; and in vitro proliferative responses after incubation of lymphocytes with TG. Oral administration of 500 micrograms TG at four intervals before Tg immunization and once afterward causes up to 80% suppression of these responses. The effect is antigen specific and dose dependent. Feeding TG after immunization produces a 40% reduction in responses. We wished to define the mechanism of this antigen-specific oral tolerization. Popliteal lymph nodes (PLN) of orally tolerized animals (T) are reduced in size compared to those in immunized (I) animals not fed TG. PLN and mesenteric lymph nodes (MLN) of I animals produce interleukin-2 (IL-2) and interferon-gamma (IFN gamma) after in vitro incubation with TG, typical of an inflammatory immune response. PLN and MLN of tolerized animals do not proliferate in response to antigen, do not produce IL-2 or IFN gamma, but do not produce the cytokines IL-4 and transforming growth factor-beta (TGF beta). Mixing in vitro of spleen cells from T and I animals causes a reduction in the immune response when incubated with TG, but no reduction in response to purified protein derivative (PPD) (the antigen in complete Freund's adjuvant). When T splenocytes are incubated with TG and PPD together, the response to TG and PPD is suppressed. Partially purified CD8+ cells from tolerized animals produce IL-4 and TGF beta after exposure to human TG and induce suppression, whereas partially purified CD4+ cells produce IL-2 and IFN gamma and do not cause suppression. MLN cells do not proliferate in response to antigen, but do produce inhibitory cytokines. T animals appear to shift the immune response from a Th-1 helper cell subset response to a Th-2 helper cell immunosuppressive response. In this model, oral tolerization produces a dramatic reduction in the immune response. Exposure of MLN to oral TG appears to cause the production of regulatory cells that migrate to spleen and PLN. In vitro studies demonstrate that on exposure to antigen, these regulatory cells produce IL-4 and TGF beta, which suppress all aspects of specific immune responsiveness and nonspecifically suppress other ongoing immune responses (bystander effect). Oral tolerization may include some element of T cell deletion or anergy. This model defines an experimental system with possible relevance to immunosuppression of human autoimmune thyroid disease.