Specific activation of CD4- CD8- double-negative T cells by Trypanosoma cruzi-derived glycolipids induces a proinflammatory profile associated with cardiomyopathy in Chagas patients.

Cardiomyopathy is the most severe outcome of Chagas disease, causing more than 12 000 deaths/year. Immune cells participate in cardiomyopathy development either by direct tissue destruction, or by driving inflammation. We have shown that CD4- CD8- [double-negative (DN)] T cells are major sources of inflammatory and anti-inflammatory cytokines, associated with the cardiac (CARD) and indeterminate (IND) forms of Chagas disease, respectively. Here, we sought to identify Trypanosoma cruzi-derived components that lead to activation of DN T cells in Chagas patients. Glycolipid (GCL), lipid (LIP) and protein-enriched (PRO) fractions derived from trypomastigote forms of T. cruzi were utilized to stimulate cells from IND and CARD patients to determine DN T cell activation by evaluating CD69 and cytokine expression. We observed that GCL, but not LIP or PRO fractions, induced higher activation of DN T cells, especially T cell receptor (TCR)-γδ DN T, from IND and CARD. GCL led to an increase in tumour necrosis factor (TNF) and interleukin (IL)-10 expression by TCR-γδ DN T cells from IND, while inducing IFN-γ expression by TCR-γδ DN T cells from CARD. This led to an increase in the ratio IFN-γ/IL-10 in TCR-γδ DN T cells from CARD, favouring an inflammatory profile. These results identify GCL as the major T. cruzi component responsible for activation of DN T cells in chronic Chagas disease, associated predominantly with an inflammatory profile in CARD, but not IND. These findings may have implications for designing new strategies of control or prevention of Chagas disease cardiomyopathy by modulating the response to GCL.

Immunoregulatory networks in human Chagas disease.

Chagas disease, caused by the infection with Trypanosoma cruzi, is endemic in all Latin America. Due to the increase in population migration, Chagas disease has spread worldwide and is now considered a health issue not only in endemic countries. While most chronically infected individuals remain asymptomatic, approximately 30% of the patients develop a potentially deadly cardiomyopathy. The exact mechanisms that underlie the establishment and maintenance of the cardiac pathology are not clear. However, there is consistent evidence that immunoregulatory cytokines are critical for orchestrating the immune response and thus influence disease development or control. While the asymptomatic (indeterminate) form represents a state of balance between the host and the parasite, the establishment of the cardiac form represents the loss of this balance. Analysis of data obtained from several studies has led to the hypothesis that the indeterminate form is associated with an anti-inflammatory cytokine profile, represented by high expression of IL-10, while cardiac form is associated with a high production of IFN-gamma and TNF-alpha in relation to IL-10, leading to an inflammatory profile. Here, we discuss the immunoregulatory events that might influence disease outcome, as well as the mechanisms that influence the establishment of these complex immunoregulatory networks.

Immunoregulation in human American leishmaniasis: balancing pathology and protection.

Leishmaniasis covers a broad spectrum of diseases with distinct, and sometimes overlapping, characteristics. The common thread in all forms of leishmaniasis is the infection by the parasite Leishmania belonging to the genus Leishmania. Upon infection of humans, there can be at least three outcomes, (i) control of Leishmania by the host immune response resulting in asymptomatic disease, (ii) patent infection and development of a relatively mild form of leishmaniasis and (iii) patent infection and development of severe clinical forms. The factors that determine the outcome of an initial inoculation with Leishmania are many, with the species of Leishmania representing one of the strongest predictive factors for the development of a given clinical form of disease. This is seen with L. braziliensis and L. amazonensis, infection leading mostly to tegumentary forms of disease, and L. infantum with the potential to induce visceral disease. However, it is also clear that the host immune response is a key factor in disease progression, not only responsible for control of Leishmania, but also playing an important role in disease progression and pathology. This duality between protective and pathogenic immune responses in individuals infected with Leishmania in the Americas is the focus of this review.

Immunoregulatory profile of monocytes from cutaneous leishmaniasis patients and association with lesion size.

Leishmaniasis is an important tropical disease composed of several clinical forms that adversely affect millions of people globally. Critical cells involved in the host-Leishmania interaction are monocytes and macrophages, which act to protect against infections due to their ability to both control intracellular infections and regulate the subsequent adaptive immune response. Both soluble factors and cell surface receptors are keys in directing the immune response following interaction with pathogens such as Leishmania. Toll-like receptors (TLRs) have an essential role in immune responses against infections, but little is known about their role in human infection with Leishmania braziliensis. In this work, we evaluated peripheral blood CD14+ monocytes for the expression of immunoregulatory cytokines, co-stimulatory molecules and TLR9 from cutaneous leishmaniasis patients infected with L. braziliensis and noninfected individuals. Our results showed that patients present decreased expression of co-stimulatory molecules such as CD80 and CD86 following culture with media alone or after stimulus with soluble Leishmania antigen. Interestingly, TLR9 expression was higher after culture with soluble Leishmania antigen (SLA), suggesting a role of this molecule in immunoregulation of active disease. Lastly, higher frequencies of TLR9+ monocytes were correlated with greater lesion size. These findings demonstrate a peripheral monocytes profile compatible with important immunoregulatory potential.

Association of CD28 and CTLA-4 gene polymorphisms with aggressive periodontitis in Brazilians.

OBJECTIVE: Susceptibility to and severity of periodontal disease is influenced by gene polymorphisms related to the immune response. Co-stimulatory molecules, such as CD28 and CTLA-4, are critical in the development of such responses. Our hypothesis is that polymorphisms in genes that code for these molecules may be associated with periodontitis. The aim of the study was to investigate the association between +17 (T/C) CD28 and +49 (A/G) CTLA-4 gene polymorphisms and periodontitis in Brazilians. MATERIALS AND METHODS: Genomic DNA was obtained from oral swabs of 424 individuals categorized into three groups (control group, aggressive, and chronic periodontitis) considering clinical parameters such as probing depth and clinical attachment loss. The genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism. RESULTS: There was an association between the T(-) genotype of the CD28 polymorphism and aggressive periodontitis (P = 0.04). Moreover, the A(+) genotype for CTLA-4 was associated with greater clinical attachment loss in non-smokers with aggressive periodontitis (P = 0.006, OR = 16.25, CI = 2.25-117.11). CONCLUSIONS: These findings show that T(-) in CD28 + 17 (T/C) and the A(+) in CTLA-4 +49 (A/G) genotypes are associated with susceptibility to aggressive periodontal disease. Thus, our study highlights these polymorphisms as potential genetic susceptibility markers of periodontitis in Brazilians.

Highly conserved CDR3 region in circulating CD4(+)Vß5(+) T cells may be associated with cytotoxic activity in Chagas disease.

Human infection with Trypanosoma cruzi leads to Chagas disease, which presents as several different clinical conditions ranging from an asymptomatic form to a severe dilated cardiomyopathy. Several studies have demonstrated that T cells play a critical role in the development of cardiac pathology, as well as in immunoregulation during chronic disease. However, the mechanisms that drive protective or pathogenic T cell response are not known. We have shown that CD4(+) T cells from chagasic patients preferentially express T cell receptor (TCR) ß-chain variable region (Vß) 5. The aim of this work was to determine whether T cells expressing this particular Vß region displayed variable or restricted CDR3 sequences, as an indicator of the nature of the stimulus leading to the activation of these T cells in vivo. Additionally, we aimed to evaluate phenotypic characteristics of these cells that might be associated with pathology. CDR3 junctional region sequencing of Vß5·1 expressing CD4(+) T cells revealed the occurrence of a highly homologous CDR3 region with conserved TCR Jß region usage among patients with cardiac, but not indeterminate, Chagas disease. Moreover, correlation analysis indicated that the frequency of CD4(+)Vß5·1(+) cells is associated with granzyme A expression, suggesting that these cells might display cytotoxic function. Together these results provide new insight into T cell recognition of antigens involved in Chagas disease and suggest that these cells may be implicated in the pathogenesis of chagasic cardiomyopathy.

CD4(+) T cells defined by their Vß T cell receptor expression are associated with immunoregulatory profiles and lesion size in human leishmaniasis.

Leishmaniasis is caused by infection with the protozoan parasite, Leishmania, that parasitizes human cells, and the cellular immune response is essential for controlling infection. In order to measure the host T cell response to Leishmania infection, we have measured the expansion, activation state and functional potential of specific T cells as identified by their T cell receptor Vß region expression. In a group of cutaneous leishmaniasis (CL) patients, we evaluated these characteristics in nine different T cell subpopulations as identified by their Vß region expression, before and after specific Leishmania antigen stimulation. Our results show: (1) an increase in CD4(+) T cells expressing Vß 5·2 and Vß 24 in CL compared to controls; (2) a Leishmania antigen-induced increase in CD4(+) T cells expressing Vß 5·2, 11, 12 and 17; (3) a profile of previous activation of CD4(+) Vß 5·2-, 11- and 24-positive T cells, with higher expression of CD45RO, HLA-DR, interferon-γ, tumour necrosis factor-α and interleukin-10 compared to other Vß-expressing subpopulations; (4) a positive correlation between higher frequencies of CD4(+) Vß5·2(+) T cells and larger lesions; and (5) biased homing of CD4(+) T cells expressing Vß 5·2 to the lesion site. Given that CL disease involves a level of pathology (ulcerated lesions) and is often followed by long-lived protection and cure, the identification of specific subpopulations active in this form of disease could allow for the discovery of immunodominant Leishmania antigens important for triggering efficient host responses against the parasite, or identify cell populations most involved in pathology.

Captopril increases the intensity of monocyte infection by Trypanosoma cruzi and induces human T helper type 17 cells.

The anti-hypertensive drug captopril is used commonly to reduce blood pressure of patients with severe forms of Chagas disease, a cardiomyopathy caused by chronic infection with the intracellular protozoan Trypanosoma cruzi. Captopril acts by inhibiting angiotensin-converting enzyme (ACE), the vasopressor metallopeptidase that generates angiotensin II and promotes the degradation of bradykinin (BK). Recent studies in mice models of Chagas disease indicated that captopril can potentiate the T helper type 1 (Th1)-directing natural adjuvant property of BK. Equipped with kinin-releasing cysteine proteases, T. cruzi trypomastigotes were shown previously to invade non-professional phagocytic cells, such as human endothelial cells and murine cardiomyocytes, through the signalling of G protein-coupled bradykinin receptors (B(2) KR). Monocytes are also parasitized by T. cruzi and these cells are known to be important for the host immune response during infection. Here we showed that captopril increases the intensity of T. cruzi infection of human monocytes in vitro. The increased parasitism was accompanied by up-regulated expression of ACE in human monocytes. While T. cruzi infection increased the expression of interleukin (IL)-10 by monocytes significantly, compared to uninfected cells, T. cruzi infection in association with captopril down-modulated IL-10 expression by the monocytes. Surprisingly, studies with peripheral blood mononuclear cells revealed that addition of the ACE inhibitor in association with T. cruzi increased expression of IL-17 by CD4(+) T cells in a B(2) KR-dependent manner. Collectively, our results suggest that captopril might interfere with host-parasite equilibrium by enhancing infection of monocytes, decreasing the expression of the modulatory cytokine IL-10, while guiding development of the proinflammatory Th17 subset.

TNFA and IL10 gene polymorphisms are not associated with periodontitis in Brazilians.

IL-10 and TNF-α are cytokines that have complex and opposing roles in the inflammatory responses. G/A polymorphisms at position -1082 of IL10 and -308 of TNFA genes have been reported to influence the expression of IL-10 and TNF-α, respectively. The aim of this study was to investigate the association between the IL10 (-1082) and TNFA (- 308) gene polymorphisms with different clinical forms or severity of periodontitis in a sample of Brazilian individuals. DNA was obtained from oral swabs of 165 Brazilian individuals, which were divided into three groups: individuals with chronic periodontitis, aggressive periodontitis and individuals without clinical evidence of periodontitis. Evaluation of IL10 and TNFA polymorphisms was performed by RFLP analysis. Statistical analysis of data was performed using the Χ² likelihood ratio and Fisher;s exact test. No significant differences in the genotype and allele distribution of either IL10 or TNFA were observed among individuals with different clinical forms or with different degrees of severity of periodontitis. Moreover, combined analysis of IL10 and TNFA polymorphisms did not show any association with periodontal status. As conclusion, the IL10 and TNFA gene promoter polymorphisms investigated are not associated with different clinical forms of periodontitis or with severity of the disease in the Brazilian population polymorphisms.

Recruitment of CD8(+) T cells expressing granzyme A is associated with lesion progression in human cutaneous leishmaniasis.

Human infection with Leishmania braziliensis leads to the establishment of cutaneous leishmaniasis (CL), characterized by the appearance of skin lesions that progress from nonulcerated to ulcerated forms. Our goal was to characterize the immunological kinetics associated with this progression, comparing the cellular composition, cytokines and granzyme expression between lesions of patients with early (E-CL) and late stages (L-CL) of CL. Histopathological analysis showed that lesions from L-CL had more exuberant inflammatory infiltrate as compared to E-CL. Although E-CL and L-CL lesions were predominantly mononuclear, lesions from E-CL patients presented higher neutrophil and eosinophil counts than L-CL. While percentages of CD4(+) and of CD68(+) cells were slightly higher in L-CL, a fivefold increase of CD8(+) cells was observed in L-CL, as compared to E-CL. Moreover, CD8(+) T-cells from L-CL expressed significantly higher levels of granzyme A than E-CL. Interestingly, granzyme A expression was positively correlated with intensity of the inflammatory infiltrate in L-CL but not E-CL. Lastly, percentages of IFN-gamma(+) and IL-10(+) cells were higher in L-CL as compared to E-CL, with CD4(+) T-cells and CD68(+) monocytes as the main sources of these cytokines, respectively. These results suggest that recruitment of CD8(+) granzyme A(+) T cells is involved in lesion progression in human CL.

The leukocytes expressing DARPP-32 are reduced in patients with schizophrenia and bipolar disorder.

Bipolar disorder (BPD) and schizophrenia (SCZ) are severe disorders representing an enormous social, familiar and individual burden, being SCZ the most disabling psychiatric disorder characterized by psychosis and cognitive impairment. It is well known that SCZ and BPD are associated with abnormalities in dopamine signaling pathway. Recent data in the literature have demonstrated altered expression levels of some proteins involved in the modulation of this pathway in both brain and peripheral tissues. It was shown that protein and mRNA levels of dopamine and cAMP regulated phosphoprotein (DARPP-32) were downregulated in dorsolateral prefrontal cortex (DLPFC) of patients with SCZ or BPD when compared to controls. Due to the difficulty to access brain tissue and the absence of objective laboratory tests for bio-markers, we measured DARPP-32 expression in blood cell sub-populations (CD4+ T lymphocytes, CD56+ NK cells, CD19+ B lymphocytes and CD14+ monocytes) taking advantage of the close relation of nervous and immune systems. Using flow cytometry as the analytical method, our results have shown that the DARPP-32 expression was diminished in CD4+ T lymphocytes, CD19+ B lymphocytes and CD14+ monocytes of BPD patients and was also decreased in CD4+ T lymphocytes and CD56+ NK cells of SCZ patients. These results showed that DARPP-32 expression in immune cells agrees with reports of reduced DARPP-32 protein in the DLPFC of BPD or SCZ patients. Our data suggest that DARPP-32 expression in PBMC could be used as a source of bio-markers to help in the treatment response of neuropsychiatry disorders as a window to the changes in the brain of those patients.

Expression of neuronal calcium sensor-1 (NCS-1) is decreased in leukocytes of schizophrenia and bipolar disorder patients.

Schizophrenia (SCZ) and bipolar disorder (BPD) are severe illnesses representing an enormous social, familiar and individual burden that affect 1% of the population world-wide. Several evidences indicate abnormalities of the dopamine system in both SCZ and BPD. Neuronal calcium sensor-1 (NCS-1) is a protein that has many functions in neurotransmission such as inhibition of dopamine D(2) receptor desensitization, regulation of ionic channels and enhancement of exocytosis of neurotransmitters. In addition, NCS-1 protein expression and mRNA levels were found increased in pre-frontal cortex (PFC) of SCZ and BPD patients. NCS-1 expression in neural and neuroendocrine cells is well documented and, recently, it was shown that NCS-1 is also expressed in mast cells and neutrophils. NCS-1 has important functions in mast cells since it stimulates Fc epsilon RI-triggered exocytosis and the release of arachidonic acid metabolites. Then, due to the known close relation between the nervous and immune systems, we sought to investigate the NCS-1 expression in lymphocytes and monocytes (CD4+ T lymphocytes, CD56+ NK cells, CD19+ B lymphocytes and CD14+ monocytes) of SCZ and BPD patients. Using flow cytometry, our results have shown that NCS-1 expression was diminished in CD4+T lymphocytes, CD19+ B lymphocytes and CD14+ monocytes of BPD patients and also decreased in CD4+ T lymphocytes and CD56+ NK cells of SCZ patients. Results suggest that immune cells might be a cellular model for studies with SCZ and BPD patients considering NCS-1 functions. Efforts need to be done to investigate the motive of the decreased percentage of immune cells expressing NCS-1 in patients with SCZ and BPD.

Differential immune regulation of activated T cells between cutaneous and mucosal leishmaniasis as a model for pathogenesis.

Cutaneous (CL) and mucosal leishmaniasis (ML) are characterized by a predominant type 1 immune response (IFN-gamma and TNF-alpha production) and strong inflammatory response in the lesions with few parasites. This exacerbated type 1 response is more evident in ML as compared to CL. Our main hypothesis is that a differential immune regulation of T cell activation leads to over reactive T cells in ML. In the present study, we investigated immunological factors that could explain the mechanisms behind it by comparing some immune regulatory mechanisms between ML and CL patients: frequency of cells expressing co-stimulatory molecules, apoptotic markers, T cell activation markers; and ability of neutralizing antibodies to IL-2, IL-12 and IL-15 do down-regulate IFN-gamma production in leishmania antigen-stimulated peripheral blood mononuclear cells (PBMC). Interestingly, in CL anti-IL-2 and anti-IL-15 significantly suppressed antigen-specific IFN-gamma production, while in ML only anti-IL-2 suppressed IFN-gamma production. Finally, higher frequency of CD4+ T cells expressing CD28-, CD69+ and CD62L(low) were observed in ML as compared to CL. These data indicate that an exacerbated type 1 response in ML is differentially regulated and not appropriately down modulated, with increased frequencies of activated effectors T cells, maintaining the persistent inflammatory response and tissue damage observed in ML.

Interleukin-6 expression and gene polymorphism are associated with severity of periodontal disease in a sample of Brazilian individuals.

Interleukin (IL)-6 is an inflammatory mediator involved in bone resorption. G/C polymorphism at position -174 of the IL-6 gene has been reported to influence IL-6 expression, with the G allele associated with higher expression levels. The aims of this study were to investigate the expression of IL-6 as well as the incidence of IL-6 (-174) gene polymorphism and their correlation to the severity of periodontitis in Brazilians. Peripheral blood mononuclear cells were collected from 12 non-smoker individuals with periodontitis for evaluation of IL-6 expression using flow cytometry. We observed a positive correlation between the mean clinical attachment loss and intensity of expression of IL-6, in which the greater the attachment loss, the higher the expression of IL-6 (P=0 x 007, R2=0 x 52). Also, patients with severe periodontitis displayed a higher intensity of IL-6 expression compared to moderate periodontitis (P=0 x 04). To determine the occurrence of IL-6 gene polymorphism, DNA was obtained from oral swabs of 209 Brazilian individuals with and without periodontitis. Polymerase chain reaction, restriction endonuclease digestion and electrophoresis were performed, allowing for detection of the IL-6 (-174) polymorphism. We observed that non-smokers with moderate periodontitis (P=0 x 05) and control (P=0 x 04) groups displayed a higher incidence of the G genotype when compared to severe periodontitis. This suggests that the G genotype may represent a protective role in severity of periodontitis. Thus, the increased expression of IL-6 and IL-6 (-174) polymorphism are associated with periodontal disease severity in Brazilian individuals.

The IL1A (-889) gene polymorphism is associated with chronic periodontal disease in a sample of Brazilian individuals.

BACKGROUND AND OBJECTIVE: It has been proposed that genotypes reflective of polymorphisms in cytokine genes can predispose individuals to disease by enhancing inflammatory processes. The C/T polymorphism at position -889 of the IL1A gene influences interleukin-1alpha expression, with the T allele inducing higher expression. The aim of this study was to evaluate the association of the IL1A (-889) gene polymorphism in Brazilian individuals with different clinical forms of periodontitis and severity of disease. MATERIAL AND METHODS: DNA was obtained from oral swabs of 163 Brazilian individuals and was amplified using the polymerase chain reaction (PCR). Products were submitted to digestion and were analyzed by electrophoresis to distinguish the C and T alleles. RESULTS: A significant difference in the genotype distribution was observed when comparing the chronic periodontitis group with the control group, evaluating only nonsmokers (chi-squared analysis = 9.91; p = 0.007), as well as when smokers were included (chi-squared analysis = 6.36; p = 0.04). Moreover, we observed a higher incidence of the T allele in the chronic periodontitis group (37.8%) when compared with the control group (18.4%) in nonsmokers (p = 0.006, odds ratio = 2.69, confidence interval = 1.27-5.68) and also when smokers were included (p = 0.03, odds ratio = 1.87, confidence interval = 0.98-3.56). No statistical difference was observed when the aggressive periodontitis group was compared with the control group. With regard to severity of disease, no statistical difference was observed. CONCLUSION: These data show an association of the IL1A (-889) polymorphism with chronic periodontitis in Brazilian individuals.

Mucosal leishmaniasis patients display an activated inflammatory T-cell phenotype associated with a nonbalanced monocyte population.

Leishmania braziliensis is a parasite that can induce at least two clinical forms of leishmaniasis in humans: cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML). In humans, the specific mechanisms that determine which form will develop following infection are not well established. In this study, peripheral blood mononuclear cells from 17 CL and 9 ml patients were compared both ex vivo and after culture with soluble leishmania antigen (SLA). Patients with ML presented a higher frequency of activated T cells as measured by ex vivo frequencies of (CD4+)(CD69+), (CD4+)(CD28-), (CD4+)(CD62L-) and (CD8+)(CD69+) than those with CL. Moreover, after stimulation with SLA, patients with ML presented a higher frequency of TNF-alpha-producing CD4+ and CD14+ cells than CL individuals. While CL patients displayed a positive correlation between the frequency of IL-10 and TNF-alpha-producing monocytes, the ML patients did not. This lack of a positive correlation between IL-10-producing and TNF-alpha-producing monocytes in ML patients could lead to a less controlled inflammatory response in vivo. These results corroborate with a model of an exacerbated, unregulated, immune response in ML patients and point to key immunomodulatory leucocyte populations and cytokine networks that may be involved in the development of immunopathology in ML patients.

Effect of LACK and KMP11 on IFN-gamma production by peripheral blood mononuclear cells from cutaneous and mucosal leishmaniasis patients.

The immune modulatory properties of recombinant antigens Kinetoplasmid membrane protein-11 (KMP11) and Leishmania homologue of receptors for activated C kinase (LACK) in cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML) patients were evaluated. The mean levels of interferon-gamma (IFN-gamma) in soluble leishmania antigen (SLA) stimulated peripheral blood mononuclear cells (PBMC) of ML and CL patients were 5625 +/- 2333 pg/ml and 4422 +/- 3665 pg/ml, respectively. IFN-gamma was not detected in cultures stimulated with KMP11 or LACK. Interleukin-10 (IL-10) concentration in SLA, KMP11 and LACK-stimulated PBMC of ML patients was 13 +/- 12 pg/ml, 285 +/- 388 pg/ml and 802 +/- 483 pg/ml, respectively. Addition of KMP11 or LACK to SLA-stimulated PBMC of CL and ML patients enhanced IL-10 production (P < 0.05). Addition of KMP11 decreased IFN-gamma levels by 52% in CL patients and by 19% in ML patients. Addition of LACK to SLA-stimulated cultures decreased IFN-gamma levels by 58% in CL patients and by 30% in ML patients. Neutralization of IL-10 abrogated the downregulatory effect of LACK and KMP11. The modulatory properties of LACK and KMP11 are due to induction of IL-10 production and may be helpful for attenuating chronic inflammatory diseases. However, in some clinical conditions, as demonstrated for ML, these molecules are not able to suppress the IFN-gamma response, even inducing IL-10 production.

Phenotypic and functional characteristics of CD28+ and CD28- cells from chagasic patients: distinct repertoire and cytokine expression.

Chronic human Chagas' disease ranges from an asymptomatic to a severe cardiac clinical form. The involvement of the host's immune response in the development and maintenance of chagasic pathology has been demonstrated by several groups. We have shown that activated T-cells lacking CD28 expression are increased in the peripheral blood of chagasic patients (CP), suggesting a relationship between these cells and disease. In order to better characterize this cell population, determining their possible role in immunoregulation of human Chagas' disease, we evaluated the expression of TCR-Vbeta regions 2, 3.1, 5, 8 and 17, as well as the expression of IFN-gamma, TNF-alpha, IL-4 and IL-10 by CD28+ and CD28- cells from polarized indeterminate and cardiac CP. Flow cytometric analysis demonstrated equivalent TCR-Vbeta usage between CD4+CD28+ and CD4+CD28- cells from all groups (chagasic and healthy controls). However, there was a predominance of Vbeta5 expression in the CD28+ and CD28- populations in the CP groups (indeterminate and cardiac). Interestingly, CD8+CD28- cells from CP, but not from nonchagasic individuals, displayed a reduced frequency of most analysed Vbetas when compared with the CD8+CD28+ subpopulation. Comparison of V-beta expression in CD28+ or CD28- cell populations among individuals from different groups also showed several interesting differences. Functionally, cardiac CP displayed a higher frequency of IFN-gamma, TNF-alpha and IL-4 producing lymphocytes than indeterminate CP. Correlation analysis between the frequency of cytokine expressing cells, and the frequency of CD4+ T-cells with differential expression of CD28 demonstrated that CD4+CD28- T-cells were positively correlated with TNF-alpha in cardiac and with IL-10 in indeterminate CP, suggesting that these cells might have an important regulatory role in human Chagas' disease.

Antigen specific correlations of cellular immune responses in human leishmaniasis suggests mechanisms for immunoregulation.

Regulation of the immune response directed against Leishmania is critical for the establishment of effective control of the disease. It is likely that some types of immune responses directed against Leishmania can lead to more severe clinical forms of leishmaniasis causing a poor control of the pathogen and/or pathology, while others lead to resolution of the infection with little pathology as in cutaneous leishmaniasis. To gain a better understanding of the possible role that subpopulations of T cells, and their associated cytokines have on disease progression and/or protective immune responses to L. braziliensis infection, a detailed study of the frequency of activated and memory T cells, as well as antigen specific, cytokine producing T cells was carried out. Following the determination of cytokine producing mononuclear cell populations in response to total Leishmania antigen (SLA), and to the recombinant antigen LACK, correlation analysis were performed between specific cytokine producing populations to identify models for cellular mechanisms of immunoregulation in human cutaneous leishmaniasis. These studies have shown: (1) a positive correlation between ex vivo CD45RO frequencies and antigen specific cytokine (IFN-gamma or IL-10) producing cells; (2) a negative correlation between ex vivo CD69 expression and the frequency of IFN-gamma producing cells; (3) a positive correlation amongst SLA specific, IFN-gamma or TNF-alpha and IL-10 producing lymphocytes with one another; and (4) a higher frequency of IL-10 producing, parasite specific (anti-SLA or anti-LACK), lymphocytes are correlated with a lower frequency of TNF-alpha producing monocytes, demonstrating an antigen specific delivery of IL-10 inducing negative regulation of monocyte activity.

Evaluation of alternative reporter genes for the yeast two-hybrid system

The yeast two-hybrid system is a powerful tool for screening protein-protein interactions and has also been used for large-scale studies. We evaluated two protein-coding sequences as reporter genes for the yeast two-hybrid system, to determine if it was suitable as an alternative screening strategy. Aspergillus awamori glucoamylase activity results in clear haloes around colonies producing this enzyme after growth on starch plates and staining with iodine vapors. However, transcription activation by Gal4 on Gal-regulated promoters was insufficient for this type of phenotypic visualization. A modified gene of Aequoria victoria enhanced green fluorescent protein (EGFP) was tested to determine its suitability for interaction screenings with flow cytometry. When the EGFP reporter gene system was incorporated into the cells, Gal4 transcriptional activation produced sufficient fluorescence for detection with the flow cytometer, especially when there were strong interactions