PAS-1, an Ascaris suum Protein, Modulates Allergic Airway Inflammation via CD8(+) gamma delta TCR(+) and CD4(+) CD25(+) FoxP3(+) T Cells

We have previously demonstrated that PAS-1, a 200 kDa protein from Ascaris suum, has a potent immunomodulatory effect on humoral and cell-mediated responses induced by APAS-3 (an allergenic protein from A. suum) or unrelated antigens. In this study, we investigated the mechanisms by which PAS-1 is able to induce this effect on an allergic airway inflammation induced by OVA in mice. C57BL/6 mice were adoptively transferred on day 0 with seven different PAS-1-primed cell populations: PAS-1-primed CD19(+) or B220(+) or CD3(+) or CD4(+) or CD8(+) or CD4(+) CD25) or CD4(+) CD25(+) lymphocytes. These mice were immunized twice with OVA and alum by intraperitoneal route (days 0 and 7) and challenged twice by intranasal route (days 14 and 21). Two days after the last challenge, the airway inflammation was evaluated by antibody levels, cellular migration, eosinophil peroxidase levels, cytokine and eotaxin production, and pulmonary mechanical parameters. Among the adoptively transferred primed lymphocytes, only CD4(+) CD25(+), CD8(+) or the combination of both T cells impaired the production of total IgE and OVA-specific IgE and IgG1 antibodies, eosinophilic airway inflammation, Th2-type cytokines (IL-4, IL-5 and IL-13), eotaxin release and airway hyperreactivity. Moreover, airway recruited cells from CD4(+) CD25(+) and CD8(+) T-cell recipient secreted more IL-10/TGF-beta and IFN-gamma, respectively. Moreover, we found that PAS-1 expands significantly the number of CD4(+) CD25(+) FoxP3(+) and CD8(+) gamma delta TCR(+) cells. In conclusion, these findings demonstrate that the immunomodulatory effect of PAS-1 is mediated by these T-cell subsets.

Endothelin A receptor antagonist modulates lymphocyte and eosinophil infiltration, hyperreactivity and mucus in murine asthma

Levels of endothelins are particularly high in the lung, and there is evidence that these peptides are involved in asthma. Asthma is a chronic inflammatory disease associated with lymphocyte infiltration. In the present study, we used a murine model of asthma to investigate the role of endothelins in lymphocyte and eosinophil infiltration into the airway hyperreactivity and mucus secretion. Sensitized C57B1/6 mice were treated with endothelin ET(A) receptor antagonist (BQ123) or endothelin ET(B) receptor antagonist (BQ788) 30 min before an antigen aerosol challenge. After 24 h, dose response curves to methacholine were performed in isolated lungs, FACS analysis of lymphocytes and eosinophil counts were performed in bronchoalveolar lavage fluid and mucus index was determined by histopathology. In sensitized and antigen-challenged mice there is a marked increase in the T CD(4)(+), T CD(8)(+), B220(+), T gamma delta(+) and NK1.1(+) lymphocyte subsets. Treatment with BQ123 further increased these cell populations. The number of eosinophils, airway hyperreactivity and mucus were all reduced by BQ123 treatment. The BQ788 had no significant effect on the parameters analyzed. Treatment with BQ123 reduced the endothelin concentration in lung homogenates, suggesting that endothelins exert a positive feedback on their synthesis. We show here that in murine asthma the ET(A) receptor antagonist up-regulates lymphocyte infiltration and reduces eosinophils, hyperreactivity and mucus. (C) 2008 Elsevier B.V. All rights reserved.

Inhibitory Signals Mediated by Programmed Death-1 Are Involved With T-Cell Function in Chronic Periodontitis

Background: Inhibitory signals mediated via molecules such as programmed death-1 (PD-1) play a critical role in downmodulating immune responses and maintaining peripheral tolerance. We investigated the involvement of cytokines and PD-1 engagement in mediating the T-cell unresponsiveness to bacterial and ubiquitous antigens in periodontal diseases. Methods: Gingival and peripheral blood samples from healthy individuals and patients with chronic periodontitis were collected and used for the subsequent assays. Leukocytes in the lesion site and blood were evaluated using flow cytometry. The production of interferon-gamma, interleukin-10, and transforming growth factor-P proteins was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of PD-1+cells in the inflamed gingiva was confirmed by immunofluorescence confocal microscopy for CD4 and PD-1 colocalization. Results: T cells from patients with chronic periodontitis proliferated poorly in response to Aggregatibacter actinomycetem comitans (previously Actinobacillus actinomycetemcomitans) antigen. T-cell unresponsiveness was not associated with imbalanced cytokine production. However, T cells from patients with chronic periodontitis expressed significantly higher levels of PD-1 either upon isolation or after culture with antigens. Moreover, PD-1 blocking did not result in significant T-cell proliferation in cells cultured with phytohemagglutinin or bacterial antigens. The blockade of PD-1 resulted in the increased production of IFN-gamma. In addition, CD4+ and CD8+ T cells expressing PD-1 accumulated in lesions with chronic periodontitis. Conclusion: These data show that PD-1 engagement could be involved in the modulation of IFN-gamma production by T cells in patients with chronic periodontitis. J Periodontol 2009,80:1833-1844.

CD8(+) T cell adjuvant effects of Salmonella FliCd flagellin in live vaccine vectors or as purified protein

Salmonella flagellin, the flagellum structural subunit, has received particular interest as a vaccine adjuvant conferring enhanced immunogenity to soluble proteins or peptides, both for activation of antibody and cellular immune responses. In the present study, we evaluated the Salmonella enterica FliCd flagellin as a T cell vaccine adjuvant using as model the 9-mer (SYVPSAEQI) synthetic H2(d)-restricted CD8(+) T cell-specific epitope (CS(280-288)) derived from the Plasmodium yoelii circumsporozoite (G) protein. The FliCd adjuvant effects were determined under two different conditions: (i) as recombinant flagella, expressed by orally delivered live S. Dublin vaccine strains expressing the target CS(280-288) peptide fused at the central hypervariable domain, and (ii) as purified protein in acellular vaccines in which flagellin was administered to mice either as a recombinant protein fused or admixed with the target CS(280-288) peptide. The results showed that CS(280-288)-specific cytotoxic CD8(+) T cells were primed when BALB/c mice were orally inoculated with the expressing the CS280-288 epitope S. Dublin vaccine strain. In contrast, mice immunized with purified FliCd admixed with the CS280-288 peptide and, to a lesser extent, fused with the target peptide developed specific cytotoxic CD8(+) T cell responses without the need of a heterologous booster immunization. The CD8(+) T cell adjuvant effects of flagellin, either fused or not with the target peptide, correlated with the in vivo activation of CD11c(+) dendritic cells. Taken together, the present results demonstrate that Salmonella flagellins are flexible adjuvant and induce adaptative immune responses when administered by different routes or vaccine formulations. (C) 2009 Elsevier Ltd. All rights reserved.

Estudo da distribui����o e quantifica����o dos linf��citos CD8 e CD20 nas les��es inflamat��rias periapicais cr��nicas

A les��o inflamat��ria periapical �� uma patologia bastante frequente, sendo na maioria dos casos, consequ��ncia da c��rie dental. O objetivo deste trabalho foi quantificar as popula����es linfocit��rias CD8+ e CD20+ em les��es inflamat��rias periapicais cr��nicas. Foram utilizadas 90 les��es inflamat��rias periapicais. Atrav��s da t��cnica de imunohistoqu��mica pelo m��todo da estreptoavidina-biotina, utilizou-se os marcadores CD8 e CD20 para identifica����o dos linf��citos T citot��xicos/supressores e linf��citos B, respectivamente. A contagem das c��lulas foi feita em 3 campos microsc��picos da l��mina, mantendo-se um aumento de 400 vezes. A m��dia da contagem das c��lulas CD8+ foi de 7,72 c��lulas para os cistos inflamat��rios, enquanto que nos grupos cisto abscedado e abscesso cr��nico foi 11,25 e 11,62, respectivamente, com diferen��a estatisticamente significante. A m��dia da contagem das c��lulas CD20+ foi 12,19; 11,06 e 12,91 c��lulas nos cistos abscedados, cistos inflamat��rios e abscessos cr��nicos, respectivamente, sem diferen��a estatisticamente significante. As les��es inflamat��rias periapicais supuradas apresentaram n��mero maior de linf��citos CD8+ do que as les��es n��o supuradas e; a presen��a de supura����o e prolifera����o epitelial nas les��es n��o interferiu na quantidade de linf��citos CD20+ presentes.

Estudo dos linf��citos CD20, CD8 e CD4 nas hiperplasias inflamat��rias e sua rela����o com a infec����o por candida sp

O objetivo do presente estudo �� mapear e quantificar as popula����es de c��lulas CD 20, CD 8 e CD 4+ em Hiperplasias inflamat��rias (HI) e estabelecer rela����o com a infec����o por Candida sp. Foram utilizados 41 casos de HI do Laborat��rio de Patologia Bucal da UFRGS. Novos cortes de todos os casos foram submetidos �� t��cnica de colora����o do PAS, criando ��� se 2 grupos: com e sem infec����o por Candida sp. Seguiu ��� se a marca����o imunohistoqu��mica com os anticorpos monoclonais anti CD 20, anti CD 8 e anti CD 4, para se avaliar a localiza����o, a distribui����o e quantifica����o das c��lulas positivamente marcadas em 3 campos consecutivos (400x), escolhidos sobre a ��rea de maior concentra����o do infiltrado inflamat��rio. Os resultados da recontagem dos campos mostraram que o examinador estava calibrado pelo teste ���t���de Student. As c��lulas CD 8+ apresentaram localiza����o pr��xima ��s hifas de Candida sp. e foram mais numerosas no grupo com infec����o (diferen��a estatisticamente significante p= 732 x 10-20). As c��lulas CD 20 e CD 4 positivas n��o apresentaram rela����o com a infec����o por Candida sp. Concluiu ��� se que as c��lulas CD 8+ apresentaram localiza����o relacionada ��s hifas de Candida sp., al��m de uma raz��o c��lulas positivamente marcada/ linf��citos totais estatisticamente mais alta no grupo com infec����o por Candida sp.

Old beagle dogs have lower faecal concentrations of some fermentation products and lower peripheral lymphocyte counts than young adult beagles

The effects of age on microbiota composition, gut fermentation end-product formation and peripheral lymphocyte numbers were compared between old and young adult Beagle dogs fed four kibble diets differing in yeast cell wall contents. The experiment had a double 4 x 4 Latin square design, one with four mature dogs (4 years old) and the other with four old dogs (10 years old), with four replicates (diets) per dog. In each period a 15d adaptation period preceded a 5d total collection of faeces for the digestibility trial. on day 21, fresh faecal samples were collected for the determination of bacterial enumeration, pH, biogenic amine and short-chain fatty acid. Flow cytometry was used for immunophenotypic evaluation. Dogs were fed four kibble diets with similar composition with 0, 0.15, 0.30 and 0.45% of yeast cell wall (as-fed), respectively. Data were evaluated using general linear models of Statistical Analysis Systems statistical software (P<0.05). No evidence of a difference in faecal bacteria counts between ages was found (total aerobes, total anaerobes, Bifidobacterium, Lactobacillus, Clostridium and Escherichia coli: P. 0.15). Faecal concentrations of butyrate, histamine, agmatine and spermine were lower (P <= 0.05) and faecal pH was higher (P=0.03) in older dogs than in mature adult dogs, suggesting an alteration in bacterial metabolic activity, or in the rate of intestinal absorption of these compounds. Concentrations of T-lymphocytes, T-cytotoxic lymphocytes and B-lymphocytes were also lower (P <= 0.01) in older dogs than in mature adult dogs. The study confirmed alterations in peripheral lymphocytes and revealed a reduced concentration of some fermentation end products in the colon of old dogs.

Express��o imuno-histoqu��mica do cd8, foxp3, Tgf &#946;, tnf &#945; e nf-&#1082;b em displasias epiteliais e Carcinomas epiderm��ides orais

The Oral Epithelial Dysplasia (OED) is the lesion that precedes or co-exists with the Oral Squamous Cell Carcinoma (OSCC), presenting molecular and/or histological similar alterations. The divergences about the malignization potential of OEDs and the role of inflammation in this process make hard the early diagnosis and evaluation of OSCCs aggressiveness. Thus, it became the goal of this study to evaluate the role of inflammation in oral carcinogenesis and tumoral aggressiveness. For this purpose a morphological study was performed in 20 OED cases and 40 OSCC cases to detect the malignization potential of OEDs and the histologic malignancy grading (HMG) of OSCCs, analyzing superficial masses for dismorphism evaluation and the invasive front for evaluation of tumoral growing; and immunohistochemical, using anti-CD8, anti-FOXP3, anti-TGF&#946;, anti-TNF&#945; and anti-NF-&#1082;B antibodies, comparing their with the types lesion, histological degree and intensity of the inflammatory infiltrate. The results were statistically significant for the parameters: cell maturity (p=0,0001), masses presence (p=0,038) and dismorphism (p=0,037), when associated to HMG. To compare the expression of the markers with the types lesion, a significantly higher expression of CD8 (p=0,001) and NF-&#1082;B (p=0,002) in the OED, and also a smaller expression of the epithelial TGF&#946; in the severe OEDs (p=0,011), without significant expression between OSCC degrees. By relating the expression of the studied markers with the inflammatory infiltrate intensity, a positive relation was observed with: inflammatory TNF&#945;(p=0,003), epithelial TNF&#945; and NF-&#1082;B (p=0,051 and p=0,004), in OEDs; and with CD8 (p=0,021) and TNF&#945; (p=0,015) in conjunctive OSCCs; and a negative relation with epithelial TNF&#945; (p=0,034) in OSCCs. No significant relation was found between FOXP3 with any of the studied variables. These findings lead to the conclusion that, the study of the invasive front is as important as the study of superficial masses for the evaluation of tumoral aggressiveness; the intensity of the inflammatory infiltrate has no use as a parameter for prognostic evaluation of OSCC in routine exams, but, the molecular events detected in this study may be necessary to give basis for determining the malignant potential in OEDs and aggressiveness in OSCCs

Lymphocyte transformation assay for C neoformans antigen is not reliable for detecting cellular impairment in patients with Neurocryptococcosis

Background: Cryptococcus neoformans causes meningitis and disseminated infection in healthy individuals, but more commonly in hosts with defective immune responses. Cell-mediated immunity is an important component of the immune response to a great variety of infections, including yeast infections. We aimed to evaluate a specific lymphocyte transformation assay to Cryptococcus neoformans in order to identify immunodeficiency associated to neurocryptococcosis (NCC) as primary cause of the mycosis.Methods: Healthy volunteers, poultry growers, and HIV-seronegative patients with neurocryptococcosis were tested for cellular immune response. Cryptococcal meningitis was diagnosed by India ink staining of cerebrospinal fluid and cryptococcal antigen test (Immunomycol-Inc, SP, Brazil). Isolated peripheral blood mononuclear cells were stimulated with C. neoformans antigen, C. albicans antigen, and pokeweed mitogen. The amount of H-3-thymidine incorporated was assessed, and the results were expressed as stimulation index (SI) and log SI, sensitivity, specificity, and cut-off value (receiver operating characteristics curve). We applied unpaired Student t tests to compare data and considered significant differences for p<0.05.Results: The lymphotoxin alpha showed a low capacity with all the stimuli for classifying patients as responders and non-responders. Lymphotoxin alpha stimulated by heated-killed antigen from patients with neurocryptococcosis was not affected by TCD4+ cell count, and the intensity of response did not correlate with the clinical evolution of neurocryptococcosis.Conclusion: Response to lymphocyte transformation assay should be analyzed based on a normal range and using more than one stimulator. The use of a cut-off value to classify patients with neurocryptococcosis is inadequate. Statistical analysis should be based on the log transformation of SI. A more purified antigen for evaluating specific response to C. neoformans is needed.

Cell and humoral immunity in endemic pemphigus foliaceus

Foram avaliados dezesseis doentes portadores de p��nfigo foli��ceo end��mico, dez com a forma localizada da doen��a (Grupo G1) e seis com a forma disseminada (Grupo G2), com os objetivos de correlacionar o quadro cl��nico e laboratorial desses pacientes com o perfil imunol��gico dos mesmos, e verificar a rela����o dos t��tulos dos anticorpos antiepiderme circulantes, identificados pela imunofluoresc��ncia indireta, com intensidade da les��o e com a evolu����o das les��es em tratamento. Foram realizados: hemograma completo, quantifica����o de subpopula����o de c��lulas mononucleares por anticorpos monoclonais e estudo da transforma����o bl��stica de linf��citos e quantifica����o de anticorpos circulantes por meio da rea����o de imunofluoresc��ncia indireta. Observou-se leucocitose principalmente no grupo G2, diminui����o dos valores relativos das subpopula����es de linf��citos CD3+ e CD4+ e tend��ncia �� diminui����o dos valores relativos da subpopula����o CD8+ nos doentes (Grupos G1 e G2). Os ��ndices de transforma����o bl��stica de linf��citos frente �� fitohemaglutinina revelaram n��veis mais elevados nos doentes (Grupos G1 + G2), que nos controles. A rea����o de imunofluoresc��ncia indireta foi positiva em 100% dos doentes do grupo G2 e em 80% do grupo G1 A mediana dos valores dos t��tulos foi maior no grupo G2, quando comparado com o grupo G1. A an��lise global dos resultados permite concluir que a imunidade celular est�� preservada, e que existe uma rela����o entre os t��tulos de anticorpos obtidos �� rea����o de imunofluoresc��ncia indireta e extens��o da les��o cut��nea.

Assessment of cytokine values in serum by RT-PCR in HIV-1 infected individuals with and without highly active anti-retroviral therapy (HAART)

Funda����o de Amparo �� Pesquisa do Estado de S��o Paulo (FAPESP)

Intestinal parasitic infections in HIV/AIDS patients: epidemiological, nutritional and immunological aspects

This study applied a socioeconomic questionnaire designed to evaluate the frequency of intestinal parasites and characterize epidemiological, nutritional, and immunological variables in 105 HIV/AIDS patients - with and without parasitic infections, attending the Day Hospital in Botucatu, UNESP, from 2007 to 2008. Body mass index was calculated and the following tests performed: parasitological stool examinations; eosinophil, IgE, CD4(+) T and CD8(+) T lymphocyte cell counts; albumin test; viral load measure; and TNF-alpha, IFN-gamma, IL-2, IL-5 and IL-10 cytokine levels. Results were positive for parasitic intestinal infections in 12.4% of individuals. Most patients had good socioeconomic conditions with basic sanitation, urban dwellings, treated water supply and sewage, good nutritional and immunological status and were undergoing HAART. Parasites were found at the following frequencies: Entamoeba - five patients (38.5%), Giardia lamblia-four (30.7%), Blastocystis hominis-three (23.0%), Endolimax nana-two (15.4%), and Ascaris lumbricoides - one (7.7%). There were no significant differences between the two groups for eosinophils, albumin, IgE, CD4(+) T and CD8(+) T lymphocytes, INF-gamma, IL-2, or IL-10. Most patients also showed undetectable viral load levels. Significant differences were found for TNF-alpha and IL-5. These results show the importance of new studies on immunodeficient individuals to increase understanding of such variables.

Teste tubercul��nico em indiv��duos com infec����o pelo v��rus da imunodefici��ncia humana: rela����o com n��mero de linf��citos T perif��ricos e atividade tuberculosa

OBJETIVO: Avaliar os resultados do teste tubercul��nico e relacion��-los com a presen��a ou n��o de tuberculose em atividade e com a contagem de linf��citos T CD4+/CD8+. M��TODOS: Foram revisados 802 prontu��rios de pacientes com s��ndrome da imunodefici��ncia adquirida atendidos no per��odo de agosto de 1985 a mar��o de 2003. Cento e oitenta e cinco pacientes realizaram o teste tubercul��nico (23,1%) e, destes, 107 eram do sexo masculino (57,8%). A m��dia de idade no grupo de reatores ao teste tubercul��nico foi de 30,6 anos, com desvio-padr��o de 6,62 anos, e entre os n��o reatores de 34,45 anos com desvio-padr��o de 10,32 anos. Foram constitu��dos dois grupos de estudo: reatores ao teste tubercul��nico, com 28 pacientes, e n��o reatores ao teste tubercul��nico, com 157 pacientes. RESULTADOS: Grande parte dos indiv��duos foi pouco responsiva ao teste tubercul��nico. Constatou-se, no grupo de reatores, maior porcentagem de indiv��duos com tuberculose ativa �� ��poca da realiza����o do teste, quando se comparou com os n��o reatores. Dez pacientes entre os reatores e onze entre os n��o reatores apresentavam alguma forma cl��nica de tuberculose em atividade �� ��poca da realiza����o do teste, sendo que seis do primeiro grupo e oito do segundo tinham contagem de linf��citos T CD4+ menor que 200 c��lulas/mm��. CONCLUS��O: Indura����es maiores do que 5 mm n��o se relacionaram com contagens absolutas mais altas de c��lulas T CD4+.