Rômulo Tadeu Dias de Oliveira

Involvement of Regulatory T Cells in the Immunosuppression Characteristic of Patients with Paracoccidioidomycosis

ABSTRACT Patients with paracoccidioidomycosis (PCM) exhibit a suppression of the cellular immune response characterized by negative delayed-type hypersensitivity (DTH) to Paracoccidioides brasiliensis antigens, the apoptosis of lymphocytes, and high levels of expression of cytotoxic-T-lymphocyte-associated antigen 4 (CTLA-4), interleukin-10 (IL-10), and transforming growth factor β (TGF-β). The aim of this study was to investigate whether and how regulatory T cells (Treg cells) are involved in this immunosuppression by analyzing the number, phenotype, and activity of these cells in patients with active disease (AD group) and patients who had received treatment (TD group). Our results showed that the AD patients had more Treg cells than the TD patients or controls (C group) and also had elevated levels of expression of regulatory markers (glucocorticoid-induced tumor necrosis factor [TNF] receptor-related protein [GITR], CTLA-4, CD95L, LAP-1, and CD38). An analysis of regulatory activity showed that Treg cells from the AD group had greater activity than did cells from the other groups and that cell-cell contact is mandatory for this activity in the C group but was only partially involved in the regulatory activity of cells from AD patients. The addition of anti-IL-10 and anti-TGF-β neutralizing antibodies to the cultures showed that the production of cytokines may be another mechanism used by Treg cells. In conclusion, the elevated numbers of these cells with an increased regulatory phenotype and strong suppressive activity suggest a potential role for them in the immunosuppression characteristic of paracoccidioidomycosis. In addition, our results indicate that while Treg cells act by cell-cell contact, cytokine production also plays an important role.

Characterization of CD4+CD28null T cells in patients with coronary artery disease and individuals with risk factors for atherosclerosis

Risk factors for atherosclerosis may contribute to chronic low-grade inflammation. A highly cytotoxic and inflammatory CD4(+) cell subset (CD4(+)CD28(null) cells) has been associated with inflammatory diseases, including acute coronary syndromes (ACS). The aim of this study was to quantify and characterize CD4(+)CD28(null) cells in individuals with risk factors for atherosclerosis and patients with coronary artery disease (CAD). In order to achieve this goal, peripheral blood mononuclear cells (PBMCs) from individuals with risk factors for atherosclerosis and patients with CAD were analyzed using flow cytometry to detect cytotoxic molecules and evaluate the expression of homing receptors and inflammatory cytokines in CD4(+) cell subsets. The cells were evaluated ex vivo and after stimulation in culture. We found no differences in the proportions of CD4(+)CD28(null) cells among the groups. Compared with the CD4(+)CD28(+) population, the ex vivo CD4(+)CD28(null) subset from all groups expressed higher levels of granzymes A and B, perforin, granulysin and interferon-γ (IFN-γ). Individuals with risk factors and patients with ACS showed the highest levels of cytotoxic molecules. After stimulation, tumor necrosis factor-α (TNF-α) expression in the CD4(+)CD28(null) subset from these groups increased more than in the other groups. Stimulation with LPS decreased the expression of cytotoxic molecules by CD4(+)CD28(null) cells in all groups. In conclusion, our results show that risk factors for atherosclerosis may alter the CD4(+)CD28(null) cells phenotype, increasing their cytotoxic potential. Our findings also suggest that CD4(+)CD28(null) cells may participate in the early phases of atherosclerosis.

Níveis séricos de interleucina-6 (IL-6), interleucina-18 (IL-18) e proteína C reativa (PCR) na síndrome coronariana aguda sem supradesnivelamento do ST em pacientes com diabete tipo 2

BACKGROUND Atherosclerosis is an inflammatory disease, and serum levels of inflammatory markers such as interleukin 6 (IL-6), interleukin 18 (IL-18) and C-reactive protein (CRP) are used to evaluate patients with coronary artery disease. In patients with type-2 diabetes, atherosclerosis is related to a larger number of events such as myocardial infarction and death, when compared with patients without diabetes. OBJECTIVE To evaluate the inflammatory response in patients with diabetes and acute events of coronary instability. METHODS Two groups of patients were primarily selected. The first group was comprised of diabetic outpatients with stable angina (D-CCS) and presence of coronary artery disease on coronary angiography (n=36). The second group was comprised of diabetic patients seen in the emergency room with acute coronary syndrome (D-ACS) without ST-segment elevation (n=38). Non-diabetic patients with ACS (n=22) and CCS (n=16) comprised the control group. Serum levels of CRP, IL-6 and IL-18 were determined using nephelometry (CRP) and ELISA (IL-6 and IL-18) techniques. RESULTS Higher serum IL-6 levels were found in diabetic or non-diabetic patients with ACS than in the group with CCS. On the other hand, diabetic patients with ACS had higher CRP levels in comparison with the other groups. Serum IL-18 levels were not significantly different among the patients studied. CONCLUSION our findings suggest a more intense inflammatory activity in patients with coronary instability. This inflammatory activity, as measured by CRP, seems to be even more intense in diabetic patients.

Accuracy of white blood cell count, C-reactive protein, interleukin-6 and tumor necrosis factor alpha for diagnosing late neonatal sepsis

OBJECTIVE To evaluate the diagnostic value for late neonatal sepsis of white blood cell count (WBC) and assays for C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), in isolation and in conjunction. METHODS This was a diagnostic test validation study. Chemiluminescence was used to assay CRP, IL-6 and TNF-alpha at the time of clinical suspicion and again after 24 and 48 hours, whereas the WBC was performed only once, at the time of suspicion. Patients were classified into three groups based on clinical progress and culture results: confirmed sepsis (CS), probable sepsis (PS), and not infected (NI). Statistical analysis was performed using the Wilcoxon and chi-square tests and Friedman analysis of variance; cutoffs were defined by plotting receiver operator characteristic curves. RESULTS The total study sample comprised 82 children, 42 of whom were classed as CS, 16 as PS and 24 as NI. At all three test times, the medians for CRP and IL-6 were significantly more elevated in the CS and PS groups, while the medians for TNF-alpha were abnormal only in the CS group. The CRP test had elevated indices of diagnostic utility at all three test times, better accuracy than the WBC and similar accuracy to the first IL-6 and TNF-alpha assays. There was no statistical difference between the cytokines, nor between them and the WBC. Combining tests did not increase diagnostic power, with the exception of the combination of WBC with CRP2 and when the sequential CRP assays were combined. CONCLUSIONS Both CRP and WBC were useful for the diagnosis of late neonatal sepsis and comparable with IL-6 and TNF-alpha. Accuracy increased when CRP and WBC were combined and when sequential CRP assay results were used.

Differential expression of cytokines, chemokines and chemokine receptors in patients with coronary artery disease.

Monocytes/macrophages and lymphocytes have a key role in the pathogenesis of atherosclerosis through the production of inflammatory and anti-inflammatory cytokines. We evaluated mRNA expression and protein production of CCL2, CXCL8, CXCL9, CXCL10, IFN-gamma and IL-10 in vitro as well as the expression of the CCR2 and CXCR3 receptors in peripheral blood mononuclear cells (PBMCs) of patients with coronary artery disease (CAD) and healthy controls in the presence or absence of oxidized LDL (oxLDL). Patients with CAD showed higher constitutive expression of CCL2, CXCL8, CXCL9, CXCL10 and IFN-gamma mRNA and, after stimulation with oxLDL, higher expression of CCL2 and CXCL8 mRNA than the control group. We also detected higher levels of CCL2 and CXCL8 in supernatants of oxLDL-stimulated PBMCs from CAD patients than in corresponding supernatants from controls. Patients with CAD had a higher percentage of constitutive CCR2(+) and CXCR3(+) cells after stimulation with oxLDL. Among CAD patients, the main differences between the stable (SA) and unstable angina (UA) groups were lower IL-10 mRNA production in the latter group. Altogether, our data suggest that PBMCs from CAD patients are able to produce higher concentrations of chemokines and cytokines involved in the regulation of monocyte and lymphocyte migration and retention in atherosclerotic lesions.

Acurácia diagnóstica do leucograma, proteína C-reativa, interleucina-6 e fator de necrose tumoral-alfa na sepse neonatal tardia

OBJETIVO: Avaliar o valor do leucograma, proteina C-reativa (PCR), interleucina-6 (IL-6) e do fator de necrose tumoral-alfa (TNF-α), isoladamente e em conjunto, na deteccao da sepse neonatal tardia. METODOS: Estudo de validacao diagnostica. A PCR, IL-6 e TNF-α foram dosados por quimioluminescencia a suspeita clinica, 24 e 48 horas depois, e o leucograma unicamente a suspeita. De acordo com evolucao clinica e resultados de culturas, tres grupos foram definidos: sepse comprovada (SC), sepse provavel (SP) e nao infectados (NI). Os testes estatisticos utilizados foram os de Wilcoxon, qui-quadrado e analise de variância de Friedman e os limites de corte foram obtidos pela construcao da curva ROC. RESULTADOS: Estudaram-se 82 criancas, sendo 42 no grupo SC, 16 no SP e 24 NI. Nos tres momentos, as medianas da PCR e da IL-6 mostraram-se significativamente mais elevadas nos grupos SC e SP, e as do TNF-α alteraram-se apenas no grupo SC. Os indices diagnosticos da PCR foram elevados nos tres momentos e com acuracia superior a do leucograma e semelhante a da IL-6 e a do TNF-α em suas primeiras medidas. Entre as citocinas, nao houve diferenca estatistica entre elas, nem em relacao ao leucograma. A associacao dos testes nao aumentou a capacidade diagnostica, exceto na combinacao entre leucograma e PCR2 e na dosagem seriada de PCR. CONCLUSOES: A PCR e o leucograma mostram-se uteis no diagnostico de sepse neonatal tardia e comparaveis a IL-6 e ao TNF-α. A acuracia aumentou com a associacao PCR-leucograma e a dosagem seriada da PCR.

Phenotypic and Functional Characterization of NK Cells in Human Immune Response against the Dimorphic Fungus Paracoccidioides brasiliensis

Besides their role in fighting viral infection and tumor resistance, recent studies have shown that NK cells also participate in the immune response against other infectious diseases. The aim of this study was to characterize the possible role of NK cells in the immune response against Paracoccidioides brasiliensis. Purified NK cells from paracoccidioidomycosis patients and healthy individuals were incubated with P. brasiliensis yeast cells or P. brasiliensis-infected monocytes, with or without the addition of recombinant IL-15. We found that NK cells from paracoccidioidomycosis patients exhibit a lower cytotoxic response compared with healthy individuals. NK cells are able directly to recognize and kill P. brasiliensis yeast cells, and this activity seems to be granule-dependent but perforin-independent, whereas the cytotoxicity against P. brasiliensis-infected monocytes is perforin-dependent. These results indicate that NK cells participate actively in the immune response against the P. brasiliensis infection either by directly destroying yeast cells or by recognizing and killing infected cells. Granulysin is the possible mediator of the cytotoxic effect, as the reduced cytotoxic activity against the yeast cells detected in patients with paracoccidioidomycosis is accompanied by a significantly lower frequency of CD56+granulysin+ cells compared with that in healthy controls. Furthermore, we show that NK cells released granulysin in cultures after being stimulated by P. brasiliensis, and this molecule is able to kill the yeast cells in a dose-dependent manner. Another important finding is that stimulated NK cells are able to produce proinflammatory cytokines (IFN-γ and TNF-α) supporting their immunomodulatory role in the infection.

Detection of TCD4+ subsets in human carotid atheroma

Activated TCD4(+) cells are detected in human atherosclerotic plaques which indicate their participation in disease progression and destabilization. Among these cells, IFN-γ-producing T cells (TH1) are recognized as having a pro-atherogenic role. Recently, the IL-17-producing T helper lineage of cells (TH17) has been identified in atherosclerotic lesions. They have been linked to atheroma development through the production of pro-inflammatory mediators present in these lesions. Furthermore, IL-22 producing TCD4(+) cells (TH22) have been identified in the atheromatous environment, but their presence and function has not been investigated. The aim of this study was to analyze the immune response mediated by pro-inflammatory subtypes of TCD4(+) cells in atheromatous lesions. Atherosclerotic plaques of 57 patients with critical stenosis of carotid submitted to endarterectomy were evaluated. Three carotid fragments from organ donors were used as control. mRNA analysis showed expression of TH1 (IFN-γ, T-bet, IL-2, IL-12p35, TNF-α and IL-18); TH2 (GATA-3); TH17 (IL-17A, IL-17RA, Rorγt, TGF-β, IL-6, IL-1β, IL-23p19, CCL20, CCR4 and CCR6) and TH22 (IL-22 and Ahr) related markers. Asymptomatic patients showed higher expression of mRNA of IL-10, TGF-β, CCR4 and GATA-3 when compared to symptomatic ones. Immunohistochemistry analysis showed higher levels of IL-23, TGF-β, IL-1β and IL-18 in macrophages and foam cells in unstable lesions compared to stable and control ones. In vitro stimulation of atheroma cells induced IL-17 and IFN-γ production. Finally we were able to detect, the following subpopulations of TCD3(+) cells: TCD4(+) IFN-γ(+), TCD4(+)IL-17(+), TCD4(+)IL-4(+), TCD4(+)IL-22(+) and double positive cells (IFN-γ/IL-17(+), IFN-γ/IL-22(+) or IL-17/IL-22(+)). Our results showed the presence of distinct TCD4(+) cells subsets in human carotid lesions and suggest that interactions among them may contribute to the atheroma progression and destabilization.

Differences in the inflammatory response between patients with and those without diabetes mellitus after coronary stenting.

BACKGROUND Patients with diabetes mellitus who undergo coronary stenting are at increased risk of restenosis. It is known that inflammation plays a crucial role in restenosis. OBJECTIVE We assessed the inflammatory response to elective coronary stent implantation (CSI) in stable diabetic and nondiabetic patients. METHODS C-reactive protein (CRP), soluble (s) P-selectin, and soluble intercellular adhesion molecule (sICAM)-1 plasma levels were determined in diabetic (n = 51) and nondiabetic (n = 56) patients before and 48 hours and 4 weeks after bare metal stenting (BMS). RESULTS Diabetic patients presented significantly higher inflammatory marker levels before and after CSI. Nonetheless, diabetic and nondiabetic patients had postintervention peak of markers attained within 48 hours. At baseline, diabetic and nondiabetic patients presented CRP levels of 5.0 +/- 20.1 (P < or = 0.04) and 3.8 +/- 9.4 microg/ml and, at 48 hours postintervention, 22.0 +/- 20.2 (P = 0.001; P = 0.002) and 12.6 +/- 11.3 (P < or = 0.0001) microg/ml. Regarding sP-selectin, diabetic and nondiabetic patients obtained levels of, at baseline, 182 +/- 118 (P < or = 0.04) and 105 +/- 48 ng/ml and, at 48 hours, 455 +/- 290 (P = 0.001; P < or = 0.01) and 215 +/- 120 (P < or = 0.04) ng/ml. For diabetic and nondiabetic patients, sICAM-1 levels were, at baseline, 248 +/- 98 (P < or = 0.04) and 199 +/- 94 ng/ml and, at 48 hours, 601 +/- 201 (P = 0.001; P < or = 0.01) and 283 +/- 220 (P = 0.001) ng/ml. At 4 weeks, for all patients, markers returned to preprocedural levels: versus before PCI: *P = 0.001, (section sign)P < or = 0.0001; versus nondiabetic patients: (#)P < or = 0.04, (paragraph sign)P = 0.002, (Upsilon)P < or = 0.01. CONCLUSIONS Diabetic and nondiabetic patients exhibited a temporal inflammatory response after an elective BMS. However, diabetic patients present higher preprocedural levels of CRP, sP-selectin, and sICAM-1 and reveal a further exacerbated inflammatory response after intervention. The differences in inflammatory response may have implications in restenosis within these two sets of patients.

Circulating levels of chemokines in psoriasis

Abstract Chemokines may contribute to local and systemic inflammation in patients with psoriasis. Previous studies have demonstrated the importance of chemokine ligands and receptors in the recruitment of T cells into psoriatic lesional skin and synovial fluid. The aim of this study was to evaluate the levels of Th1-related chemokines in psoriasis and to investigate any association with disease severity. We quantified serum levels of CXCL9, CXCL10 and CXCL16 and the frequencies of CD4+CXCR3+ T lymphocytes through ELISA and flow cytometry, respectively. A total of 38 patients with psoriasis and 33 controls were included. There were no significant differences in chemokine levels between psoriasis and control groups. Patients with psoriatic arthritis had lower median level of CXCL10 when compared with controls (p = 0.03). There were no significant correlations between serum chemokines analyzed and disease severity. Frequencies of CD4+CXCR3+ T cells were lower in patients with psoriasis than in controls (p < 0.01). A sensitivity analysis excluding patients on systemic therapy yielded similar results. Serum concentrations of CXCL9, CXCL10 and CXCL16 were not increased in the psoriasis group or correlated with disease severity. Systemic levels of chemokine ligands do not seem to be sensitive biomarkers of disease activity or accurate parameters to predict response to therapy. Frequencies of CD4+CXCR3+ T cells were decreased in the peripheral blood of psoriasis patients, possibly due to recruitment to inflammatory lesions.