RESUMO
The most advantageous approach to pathogen destruction in a urine-diversion toilet vault is to maximize the effects of various environmental factors (i.e., pH, temperature, moisture content, type of bulking agent, and storage time). To quantify these effects, a field experiment was set up, consisting of 6 urine-diversion toilet vaults, each with a different combination of feces and bulking agent (soil, ash, wood shavings, sodium hydroxide, or straw) and ventilation (ventpipe/no ventpipe). The pH of the mixes varied from 6.37 to 10.09. Temperature probes, which were connected to a data logger, were inserted to the heaps, and the logger monitored over a period of nearly 10 months. Mean heap temperatures ranged from 16.8 degrees C in winter to 27.6 degrees C in summer. In addition, samples were taken at intervals from the various heaps in the vaults and also from an open heap exposed to the elements. The samples were subjected to microbiological testing to quantify the pathogen dieoff over time. In the vaults, there was a 3log10 (99.9%) reduction of total coliform between 130 and 250 days, fecal coliform between 100 and 250 days, and fecal streptococci from 125 days and longer. In the open heap, these times varied, from 115 days for both total and fecal coliform, to 140 days for fecal streptococci. Viable Ascaris ova were reduced to zero between 44 and 174 days in the vaults and by 44 days in the open heap. The results of this research showed that ventilation of the vault by means of a ventpipe does not result in any meaningful difference in the vault temperature or the rate of pathogen dieoff. While the type of bulking agent used does not significantly affect the temperature of the heap, it does have an effect on the rate of pathogen dieoff. The ordinary soil mix was seen to give the best results, and this was ascribed to the effect of competing microorganisms in the soil itself. It is concluded that, for safety, vaults of urine-diversion toilets should be sized for a storage period of 9 to 12 months from the last use.
Assuntos
Fezes/microbiologia , Segurança , Banheiros , Dessecação , Desenho de Equipamento , Fezes/parasitologia , Fertilizantes/microbiologia , Fertilizantes/parasitologia , Concentração de Íons de Hidrogênio , Temperatura , Fatores de Tempo , Raios Ultravioleta , UrinaRESUMO
Many lymphocyte-activation-associated molecules are observed by immunohistochemistry in psoriasis vulgaris lesional skin. Non-T cells in lesional skin also express these molecules. We quantitatively measured the number of T cells expressing cell surface activation-associated molecules (CD69, CD25, CD122, HLA-DR) and co-stimulatory molecules (CD28, CTLA-4, CD80, CD86), including a Type 2 T cell marker (CD30) and CD11b, by flow cytometry of skin and peripheral blood. T cells in single cell suspensions of psoriatic lesional-epidermis-expressed HLA-DR (86%), CD69 (59%), CD25 (55%), CD122 (44%), and CD28 (91%). Dermal T cells showed similar percentages except for CD69 (17%). CD69 was found directly in lesional skin biopsies by immunohistochemistry. Both CD4 and CD8 subsets from lesional skin contained large populations of CD25+ cells with a bias towards CD8 activation in the epidermis and towards CD4 activation in the dermis. CD86, CD80, CTLA-4, CD30 and CD11b were expressed by less than 23% of the T cell populations from both the epidermis and dermis. CD30+CD4+ cells were found two-fold over CD8+ T cells. These results show that the majority of lesional lymphocytes are persistently activated. We also found the majority of Type 2 associated markers primarily on the CD4+ epidermal T cell population. Psoriatic blood contained elevated levels of T cells expressing CD25, primarily within the CD8+ subset. Thus the majority of lesional T cells expressed the three primary activation markers, while psoriatic blood T cells were distinguished by an increase in CD25, specifically within the CTL population.
Assuntos
Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/metabolismo , Antígenos HLA-DR/metabolismo , Imunoconjugados , Ativação Linfocitária , Psoríase/imunologia , Receptores de Interleucina-2/metabolismo , Subpopulações de Linfócitos T/imunologia , Abatacepte , Adolescente , Adulto , Idoso , Antígenos de Diferenciação/metabolismo , Autoimunidade , Antígeno B7-1/metabolismo , Antígeno B7-2 , Biomarcadores , Antígenos CD28/metabolismo , Relação CD4-CD8 , Antígeno CTLA-4 , Feminino , Humanos , Antígeno Ki-1/metabolismo , Lectinas Tipo C , Contagem de Linfócitos , Antígeno de Macrófago 1/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Psoríase/patologia , Psoríase/terapia , Subpopulações de Linfócitos T/patologiaRESUMO
Patients with paraneoplastic cerebellar degeneration (PCD) offer the opportunity to explore the mechanisms underlying tumor immunity and immune-mediated neuronal degeneration. Cytotoxic T lymphocytes (CTLs) specific for the PCD onconeural antigen cdr2 found in the blood of patients with PCD are likely to be effectors of PCD tumor immunity. Here, we suggest a role for CTLs in the autoimmune destruction of Purkinje neurons. More than 75% of the cells obtained from the cerebrospinal fluid (CSF) of PCD patients were CD3+ alphabeta T cells. In patients with active/progressive disease, 20% to 40% of CSF cells were activated T cells, and the CD4+ helper cells were Th1-type cells. Three PCD patients were given tacrolimus, a specific inhibitor of activated T cells, which markedly reduced these cells in the CSF. Tacrolimus also reduced the number of activated cdr2-specific CTLs in the peripheral blood, but did not lead to tumor recurrence. We suggest that activated cdr2-specific CTLs in the CSF contribute to Purkinje degeneration in PCD, and that tacrolimus therapy may benefit patients with paraneoplastic neurological disease and other T cell-mediated autoimmune neurological disorders.
Assuntos
Degeneração Paraneoplásica Cerebelar/líquido cefalorraquidiano , Linfócitos T/metabolismo , Feminino , Humanos , Ativação Linfocitária/imunologia , Pessoa de Meia-Idade , Degeneração Paraneoplásica Cerebelar/imunologia , Linfócitos T/imunologiaRESUMO
Psoriasis vulgaris is a skin disease potentially mediated by pro-inflammatory cytokines produced by type 1 lesional T cells. The capability of individual T cells to produce these cytokines in lesional skin is not known. In this study we measured the ability of lesional and peripheral blood T cells to produce intracellular interferon-gamma, tumor necrosis factor-alpha, interleukin-2, interleukin-4, and interleukin-10 proteins as detected by flow cytometric analysis. Cytokine synthesis was induced by activation with ionomycin/phorbol myristate acetate (in the presence of Brefeldin A, which inhibits the exocytosis of these cytokines). After stimulation, we found relatively high percentages of epidermal CD8 and CD4 T cells capable of producing interferon-gamma, tumor necrosis factor-alpha, and interleukin-2, whereas few T cells, < 11%, expressed interleukin-4 or interleukin-10. Hence both CD8+ and CD4+ T cells are capable of type 1 effector functions (TC1 and TH1, respectively). This activation scheme was repeated on peripheral blood T cells from psoriatic patients versus healthy controls, where we also found a type 1 bias. In order to evaluate quantitatively the type 1 cytokine bias, we compared the frequency of type 2 interleukin-4 producing versus type 1 interferon-gamma producing T cells in our assay and found a shift towards type 1 producing cells. This shift reveals a type 1 differentiation bias in both lesional areas and in the peripheral blood, which may indicate an imbalance within the T cell population, which is contributing to the chronic or sustained immunologic activation of T cells found in this disease.
Assuntos
Citocinas/biossíntese , Células Epidérmicas , Psoríase/patologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T/metabolismo , Células Th1/metabolismo , Adulto , Idoso , Relação CD4-CD8 , Diferenciação Celular/efeitos dos fármacos , Feminino , Humanos , Interferon gama/biossíntese , Interferon gama/sangue , Interleucina-2/biossíntese , Interleucina-2/sangue , Ionomicina/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Psoríase/sangue , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Células Th1/citologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Narrow-band (312 nm) ultraviolet B light (UVB) is a new form of therapy for psoriasis, but its mechanism of action is unknown. In a bilateral comparison clinical study, daily exposure of psoriatic plaques to broad-band UVB (290-320 nm) or 312-nm UVB depleted T cells from the epidermis and dermis of psoriatic lesions. However, 312-nm UVB was significantly more depleting in both tissue compartments. To characterize the mechanism of T cell depletion, assays for T cell apoptosis were performed on T cells derived from UVB-irradiated skin in vivo and on T cells irradiated in vitro with 312-nm UVB. Apoptosis was induced in T cells exposed to 50-100 mJ/cm2 of 312-nm UVB in vitro, as measured by increased binding of fluorescein isothiocyanate (FITC)-Annexin V to CD3(+) cells and by characteristic cell size/granularity changes measured by cytometry. In vivo exposure of psoriatic skin lesions to 312-nm UVB for 1-2 wk also induced apoptosis in T cells as assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reaction in tissue sections, by binding of FITC-Annexin V to CD3(+) T cells contained in epidermal cell suspensions, and by detection of apoptosis-related size shifts of CD3(+) cells. Induction of T cell apoptosis could be the main mechanism by which 312-nm UVB resolves psoriasis skin lesions.
Assuntos
Apoptose/efeitos da radiação , Psoríase/patologia , Psoríase/radioterapia , Pele/efeitos da radiação , Terapia Ultravioleta , Adulto , Anexina A5/metabolismo , Complexo CD3/análise , Tamanho Celular , Epiderme/imunologia , Epiderme/patologia , Epiderme/efeitos da radiação , Eritema/etiologia , Feminino , Humanos , Marcação In Situ das Extremidades Cortadas , Masculino , Psoríase/imunologia , Pele/imunologia , Pele/patologia , Raios UltravioletaRESUMO
The onset and persistence of psoriatic lesions are linked to the presence of an inflammatory infiltrate of CD3+ lymphocytes that includes CD4+ and CD8+ subsets. Since a primary susceptibility factor for psoriasis is the Class I HLA-Cw6 molecule, we set out to learn more about the features of the epidermal CD8+ lymphocytes. The markers tested were GMP-17, a cytotoxic granule protein found in activated cytotoxic lymphocytes (CTLs), and the alpha chain of the IL-2 receptor (CD25), a plasma membrane molecule found on activated T cells. Lymphocytes in lesional skin expressed the GMP-17 protein, whereas lymphocytes in non-lesional skin, resolving lesional skin and normal skin had little or no GMP-17. By flow cytometry analysis, lesional epidermal GMP-17+ cells were CD8+CD3+, with a subpopulation expressing the activation marker CD25+. Due to the abundance of activated GMP-17+CD8+CD3+ lymphocytes (the phenotype of activated cytotoxic cells) in psoriatic lesions compared to non-lesional and normal skin, we hypothesize that they are contributing directly to the psoriatic phenotype.
Assuntos
Linfócitos/imunologia , Proteínas , Psoríase/imunologia , Linfócitos T Citotóxicos/imunologia , Complexo CD3/análise , Complexo CD3/imunologia , Antígenos CD8/análise , Antígenos CD8/imunologia , Epiderme/patologia , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos/análise , Fator Estimulador de Colônias de Granulócitos/imunologia , Humanos , Imuno-Histoquímica , Ativação Linfocitária , Linfócitos/química , Proteínas de Membrana/análise , Proteínas de Membrana/imunologia , Fenótipo , Proteínas de Ligação a Poli(A) , Psoríase/genética , Proteínas de Ligação a RNA/análise , Proteínas de Ligação a RNA/imunologia , Pele/química , Pele/citologia , Pele/patologia , Antígeno-1 Intracelular de Células T , Subpopulações de Linfócitos T/química , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Citotóxicos/química , Linfócitos T Citotóxicos/citologiaRESUMO
Most types of human oculocutaneous albinism (OCA) result from mutations in the gene for tyrosinase (OCA1) or the P protein (OCA2), although other types of OCA have been described but have not been mapped to specific loci. Melanocytes were cultured from an African-American with OCA, who exhibited the phenotype of Brown OCA, and his normal fraternal twin. Melanocytes cultured from the patient with OCA and the normal twin appeared brown versus black, respectively. Melanocytes from both the patient with OCA and the normal twin demonstrated equal amounts of NP-40-soluble melanin; however, melanocytes from the patient with OCA contained only 7% of the amount of insoluble melanin found from the normal twin. Tyrosinase- related protein-1 (TRP-1) was not detected in the OCA melanocytes by use of various anti-TRP-1 probes. Furthermore, transcripts for TRP-1 were absent in cultured OCA melanocytes. The affected twin was homozygous for a single-bp deletion in exon 6, removing an A in codon 368 and leading to a premature stop at codon 384. Tyrosine hydroxylase activity of the OCA melanocytes was comparable to controls when assayed in cell lysates but was only 30% of controls when assayed in intact cells. We conclude that this mutation of the human TRP-1 gene affects its interaction with tyrosinase, resulting in dysregulation of tyrosinase activity, promotes the synthesis of brown versus black melanin, and is responsible for a third genetic type of OCA in humans, which we classify as "OCA3."
Assuntos
Albinismo Oculocutâneo/genética , Melanócitos/metabolismo , Glicoproteínas de Membrana , Oxirredutases , Proteínas/genética , Deleção de Sequência , Albinismo Oculocutâneo/classificação , Albinismo Oculocutâneo/patologia , Sequência de Bases , Células Cultivadas , Primers do DNA , Di-Hidroxifenilalanina/análise , Doenças em Gêmeos/genética , Éxons , Técnica Indireta de Fluorescência para Anticorpo , Homozigoto , Humanos , Masculino , Melanócitos/citologia , Melanócitos/patologia , Dados de Sequência Molecular , Monofenol Mono-Oxigenase/genética , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Pele/metabolismo , Gêmeos Dizigóticos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Skin irradiation with ultraviolet B (UVB) is a common and often durable treatment for psoriasis and other inflammatory skin disorders. We studied the effects of UVB on keratinocytes and leukocytes in psoriatic tissue and in culture. In nine patients treated repetitively, most of the cellular and molecular changes that typify the psoriatic epidermis reverted to normal. Keratinocyte hyperplasia, assessed by expression of the Ki-67 cell cycle antigen, decreased by 70%, and residual cell proliferation was appropriately confined to the basal layer. Epidermal thickening was reduced by 60%, and a granular layer reformed. Expression of keratin 16, as well as suprabasal integrin alpha 3 and insulin-like growth factor-1 receptors, was eliminated, whereas filagrin increased markedly. UVB also depleted > 90% of the CD3+, CD8+, and CD25+ T cells from the psoriatic epidermis, whereas dermal T cells were only minimally depressed. The latter finding parallels the known inability of these doses of UVB to penetrate the dermis. In tissue culture, UVB was antiproliferative and cytotoxic toward T cells and keratinocytes, but the T cells were 10-fold more sensitive. Furthermore, low doses of UVB induced apoptosis in lymphocytes but not keratinocytes, as detected by the TUNEL (TdT-mediated dUTP-biotin nick end labeling) technique. The selective effects of UVB on intraepidermal T cells in situ and in culture support the hypothesis that epidermal alterations in psoriasis can be normalized by a depletion of activated intraepidermal T cells.
Assuntos
Queratinócitos/patologia , Psoríase/radioterapia , Subpopulações de Linfócitos T/efeitos da radiação , Células Cultivadas , Relação Dose-Resposta Imunológica , Epiderme/patologia , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Queratinócitos/efeitos da radiação , Psoríase/patologia , Raios UltravioletaRESUMO
The Smyth line (SL) chicken is an animal model for the human acquired depigmentary disorder vitiligo. Affected birds from this line express a postnatal loss of melanocytes in feather and ocular tissues. This vitiligo-like depigmentation is considered to be a disorder with two interacting components: melanocyte dysfunctions and autoimmune reactions. Previously, SL chicks were shown to express high levels of circulating autoantibodies that bind to chicken melanocyte proteins with molecular masses between 65 and 80 kd. Three mammalian melanocyte proteins known to have isoforms in this molecular mass range are tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-2. Of these, only tyrosinase is reported to be expressed in chicken melanocytes. The results presented in this study indicate that, of these three candidate proteins, TRP-1 is the primary antigen recognized by the SL autoantibodies. SL autoantibodies recognize a chicken melanocyte protein that is different from that of tyrosinase or the candidate chicken TRP-2. In addition, several types of experiments incriminate TRP-1 as the primary mammalian melanocyte antigen recognized by SL autoantibodies. We further verified that chicken melanocytes expressed messages for TRP-1 by finding positive signals on Northern blots of chicken melanocyte RNA probed with mammalian TRP-1 cDNA fragments. Therefore, we conclude from these results that the SL autoantibodies primarily recognize TRP-1 in mammalian melanocytes and suggest that chicken melanocytes express a homologue of TRP-1 (the human gp75 and the murine brown/b locus protein).
Assuntos
Autoanticorpos/imunologia , Glicoproteínas de Membrana , Oxirredutases , Proteínas/imunologia , Animais , Northern Blotting , Galinhas , Modelos Animais de Doenças , Immunoblotting , Terapia de Imunossupressão/métodos , Isomerismo , Melanócitos/imunologia , Peso Molecular , Testes de Precipitina , Proteínas/química , Vitiligo/imunologiaRESUMO
Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histaminergic H1 receptors on cultured bovine adrenal chromaffin cells stimulated a rapid increase in TOH phosphorylation (within 5 s) that was sustained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellular Ca2+. In contrast, the H1-mediated response was abolished by preloading the cells with BAPTA acetoxymethyl ester (50 microM) and significantly reduced by prior exposure to caffeine (10 mM for 10 min) to deplete intracellular Ca2+. Tryptic-phosphopeptide analysis by HPLC revealed that the H1 response in the presence or absence of extracellular Ca2+ resulted in a major increase in the phosphorylation of Ser19 with smaller increases in that of Ser40 and Ser31. In contrast, although a brief stimulation with nicotine (30 microM for 60 s) also resulted in a major increase in Ser19 phosphorylation, this response was abolished in the absence of extracellular Ca2+. These data indicate that the mobilization of intracellular Ca2+ plays a crucial role in supporting H1-mediated TOH phosphorylation and may thus have a potentially important role in regulating catecholamine synthesis.
Assuntos
Medula Suprarrenal/enzimologia , Fosfosserina/metabolismo , Receptores Histamínicos H1/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Cafeína/farmacologia , Cálcio/fisiologia , Bovinos , Colforsina/farmacologia , Ionomicina/farmacologia , Mapeamento de Peptídeos , Dibutirato de 12,13-Forbol/farmacologia , Fosforilação , Receptores Nicotínicos/efeitos dos fármacos , Transdução de SinaisRESUMO
Smyth line (SL) chickens, which develop a depigmenting disorder similar to human vitiligo, produce circulating anti-melanocyte antibodies (Austin, L.M. et al., (1992) The detection of melanocyte autoantibodies in the Smyth chicken model for vitiligo. Clin. Immunol. Immunopathol., 64:112-120). In order to characterize these autoantibodies, we studied the reactivity of cultured chicken, mouse, and human melanocytes, as well as frozen sections of chicken feather follicles and embryonic eyes, against SL serum, employing indirect immunofluorescence. Light Brown Leghorn (LBL) serum was used as a negative control. Chicken (SL and LBL), mouse, and human melanocytes exhibited greater fluorescence with SL serum than with LBL serum (up to a 1:60,000 dilution). The fluorescent pattern was predominant along the perimeter of the cells, suggesting plasma membrane staining. Fluorescence-activated flow cytometry analysis and immunocytochemical localization at the ultrastructural level using intact chicken cells supported this hypothesis. Melanocytes were readily stained in cryosections of regenerating feather follicles and embryonic eyes incubated with SL, but not LBL, serum. In addition, amelanotic melanocytes in albino chicken feathers reacted with SL serum. SL serum also preferentially stained cells emigrating from cultured avian neural tubes and within the dermis of the proliferative germ of regenerating feather follicles suggesting that melanoblasts express the antigens. We conclude that Smyth line serum contains melanocyte autoantibodies that cross-react with mouse and human melanocytes, are able to bind to pigment cells within tissues, and recognize antigens expressed in the cytoplasm and on the surface of melanocytes and melanoblasts.
Assuntos
Autoanticorpos/imunologia , Doenças Autoimunes/imunologia , Membrana Celular/imunologia , Galinhas/imunologia , Modelos Animais de Doenças , Melanócitos/imunologia , Vitiligo/imunologia , Animais , Autoantígenos/biossíntese , Autoantígenos/imunologia , Doenças Autoimunes/sangue , Doenças Autoimunes/genética , Células Cultivadas , Embrião de Galinha , Galinhas/genética , Citoplasma/imunologia , Células Dendríticas/imunologia , Olho/embriologia , Olho/imunologia , Plumas/imunologia , Imunofluorescência , Humanos , Melanócitos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL/imunologia , Crista Neural/imunologia , Especificidade de Órgãos , Especificidade da Espécie , Vitiligo/sangue , Vitiligo/genéticaRESUMO
Smyth line (SL) chickens are phenotypically characterized by a posthatch depigmentation (vitiligo) of the feathers. The destruction of melanocytes in the feather follicle as well as in other tissues such as the choroid is genetically determined. Previous studies have shown that bursectomy or treatment with immunosuppressive agents decreases the incidence and severity of SL depigmentation (1). These observations implicate a role for the immune system, specifically the humoral component, in melanocyte destruction. In this study we show that there are circulating melanocyte-specific autoantibodies in the sera of depigmented SL chicks which are not present in sera from Light Brown Leghorn (LBL) control chicks. By immunoblots and by immunoprecipitation of radiolabeled melanocyte proteins, SL autoantibodies were shown to bind to multiple melanocyte proteins between 65 and 80 kDa. These proteins are not detected in SL fibroblasts. By immunoblotting, the incidence of autoantibodies for these 65- to 80-kDa proteins was determined to be 95% in depigmented SL chicks (n = 20), 0% in normally pigmented SL chicks (n = 8), and 5% in LBL chicks (n = 20). Melanocyte autoantibodies are detectable in the sera of affected chicks at or several weeks prior to the expression of depigmentation. This information, plus previously published data, implicate melanocyte autoantibodies in the depigmentary phenomenon of vitiligo observed in Smyth line chickens.