Increased Natural Killer (NK)-cell cytotoxicity and Trypanosoma cruzi-specific memory B cells in subjects with discordant serology for Chagas disease.

The presence of memory T cell specific for Trypanosoma cruzi in subjects with discordant serology for Chagas disease supports a cleared infection in these subjects. Using high-dimensional flow cytometry, ELISPOT assays and quantitative PCR, antibody-secreting cells and memory B cells specific for T. cruzi, total B-cell phenotypes, innate immune responses and parasite DNA were evaluated in serodiscordant, seropositive and seronegative subjects for T. cruzi infection. T. cruzi-specific memory B cells but no antibody-secreting cells specific for T. cruzi, increased proportion of nonclassical monocytes and increased levels of polyfunctional NK cells were found in serodiscordant compared with seropositive subjects. None of the serodiscordant subjects evaluated showed detectable parasite DNA, most of them did not show cardiac abnormalities and a group of them had had confirmed positive serology for Chagas disease. The unique immune profiles in serodiscordant subjects support that T. cruzi infection was cleared or profoundly controlled in these subjects.

Photoinactivation of Planktonic Cells, Pseudohyphae, and Biofilms of Candida albicans Sensitized by a Free-Base Chlorin and Its Metal Complexes with Zn(II) and Pd(II).

Invasive candidiasis is an important cause of morbidity and mortality, and its occurrence is increasing due to the growing complexity of patients. In particular, Candida albicans exhibits several virulence factors that facilitate yeast colonization in humans. In this sense, the photodynamic inactivation of yeasts is a promising new alternative to eliminate fungal infections. Herein, the photodynamic activity sensitized by a free-base chlorin (TPCF16) and its complexes with Zn(II) (ZnTPCF16) and Pd(II) (PdTPCF16) was investigated in order to eliminate C. albicans under different forms of cell cultures. A decrease in cell survival of more than 5 log was found in planktonic cells incubated with 5 µM TPCF16 or ZnTPCF16 upon 15 min of white-light irradiation. The mechanism of action mainly involved a type II pathway in the inactivation of C. albicans cells. In addition, the photodynamic action induced by these chlorins was able to suppress the growth of C. albicans in a culture medium. These photosensitizers were also effective to photoinactivate C. albicans pseudohyphae suspended in PBS. Furthermore, the biofilms of C. albicans that incorporated the chlorins during the proliferation stage were completely eradicated using 5 µM TPCF16 or ZnTPCF16 after 60 min of light irradiation. The studies indicated that these chlorins are effective photosensitizing agents to eliminate C. albicans as planktonic cells, pseudohyphae, and biofilms.

Mixed T Helper1/T Helper2/T Cytotoxic Profile in Subjects with Chronic Chagas Disease with Hypersensitivity Reactions to Benznidazole.

Dermatitis is the most common adverse event during treatment with benznidazole in chronic Chagas disease and is probably mediated by T cells. A set of molecules representative of the different type IV hypersensitivity reactions was evaluated in the circulation and skin biopsies of Trypanosoma cruzi-infected subjects presenting dermatitis during benznidazole administration. Through cytometric bead assays and enzyme-linked immunosorbent assay capture techniques, the serum levels of cytokines, chemokines, proapoptotic molecules, and mediators of the activation and migration of eosinophils and T cells were measured in subjects infected with Trypanosoma cruzi who exhibited skin adverse events (n = 22) and compared with those without adverse events (n = 37) during benznidazole therapy. Serum levels of interleukin- 5 (IL-5), soluble Fas cell surface death receptor ligand (FAS-L), and interferon γ-induced protein (IP-10) significantly increased at 7 to 30 days posttreatment with benznidazole and decreased thereafter in subjects with dermatitis but not in those without dermatitis. Circulating eotaxin levels were lower in subjects with dermatitis than in those without. Two patterns emerged in the skin biopsies: a T helper 1/T cytotoxic profile and a T helper 2/T cytotoxic profile with the presence of CD4+ and CD8+ T cells. Increased low-density lipoprotein (LDL), glutamic-oxaloacetic transaminase (GOT), uremia, and T cell activation emerged as risk factors for the development of dermatitis during benznidazole administration. These results support a delayed-type hypersensitivity reaction to benznidazole, involving CD4+ and CD8+ T cells and eosinophils, and a mixed cytokine profile. This study provides new insights for better management of adverse drug reactions to benznidazole. IMPORTANCE This study identified the risk factors for the development of adverse reactions to benznidazole and identified a set molecule to monitor the appearance of these reactions. This knowledge might improve the safety of benznidazole administration.

Porphyrin-Schiff Base Conjugates Bearing Basic Amino Groups as Antimicrobial Phototherapeutic Agents.

New porphyrin-Schiff base conjugates bearing one (6) and two (7) basic amino groups were synthesized by condensation between tetrapyrrolic macrocycle-containing amine functions and 4-(3-(N,N-dimethylamino)propoxy)benzaldehyde. This approach allowed us to easily obtain porphyrins substituted by positive charge precursor groups in aqueous media. These compounds showed the typical Soret and four Q absorption bands with red fluorescence emission (ΦF ~ 0.12) in N,N-dimethylformamide. Porphyrins 6 and 7 photosensitized the generation of O2(1Δg) (ΦΔ ~ 0.44) and the photo-oxidation of L-tryptophan. The decomposition of this amino acid was mainly mediated by a type II photoprocess. Moreover, the addition of KI strongly quenched the photodynamic action through a reaction with O2(1Δg) to produce iodine. The photodynamic inactivation capacity induced by porphyrins 6 and 7 was evaluated in Staphylococcus aureus, Escherichia coli, and Candida albicans. Furthermore, the photoinactivation of these microorganisms was improved using potentiation with iodide anions. These porphyrins containing basic aliphatic amino groups can be protonated in biological systems, which provides an amphiphilic character to the tetrapyrrolic macrocycle. This effect allows one to increase the interaction with the cell wall, thus improving photocytotoxic activity against microorganisms.

Reduced Trypanosoma cruzi-specific humoral response and enhanced T cell immunity after treatment interruption with benznidazole in chronic Chagas disease.

BACKGROUND: Interruption of benznidazole therapy due to the appearance of adverse effects, which is presumed to lead to treatment failure, is a major drawback in the treatment of chronic Chagas disease. METHODS: Trypanosoma cruzi-specific humoral and T cell responses, T cell phenotype and parasite load were measured to compare the outcome in 33 subjects with chronic Chagas disease treated with an incomplete benznidazole regimen and 58 subjects treated with the complete regimen, during a median follow-up period of 48 months. RESULTS: Both treatment regimens induced a reduction in the T. cruzi-specific antibody levels and similar rates of treatment failure when evaluated using quantitative PCR. Regardless of the regimen, polyfunctional CD4+ T cells increased in the subjects, with successful treatment outcome defined as a decrease of T. cruzi-specific antibodies. Regardless of the serological outcome, naive and central memory T cells increased after both regimens. A decrease in CD4+ HLA-DR+ T cells was associated with successful treatment in both regimens. The cytokine profiles of subjects with successful treatment showed fewer inflammatory mediators than those of the untreated T. cruzi-infected subjects. High levels of T cells expressing IL-7 receptor and low levels of CD8+ T cells expressing the programmed cell death protein 1 at baseline were associated with successful treatment following benznidazole interruption. CONCLUSIONS: These findings challenge the notion that treatment failure is the sole potential outcome of an incomplete benznidazole regimen and support the need for further assessment of the treatment protocols for chronic Chagas disease.

Magnetic Nanoplatforms for in Situ Modification of Macromolecules: Synthesis, Characterization, and Photoinactivating Power of Cationic Nanoiman-Porphyrin Conjugates.

A nanoplatform concept was developed to synthesize accessible photoactive magnetic nanoparticles (MNPs) of Fe3O4 coated with silica. This approach was based on the covalent binding of 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (TPPF20) to aminopropyl-grafted MNPs by nucleophilic aromatic substitution reaction (SNAr) to obtain conjugate MNP-P1. After in situ modification, the remaining pentafluorophenyl groups of TPPF20 attached to MNPs were substituted by dimethylaminoethoxy groups to form MNP-P2. The basic amine group of these conjugates can be protonated in aqueous media. In addition, MNP-P1 and MNP-P2 were intrinsically charged to produce cationic conjugates MNP+-P1 and MNP+-P2+ by methylation. All of them were easily purified by magnetic decantation in high yields. The average size of the MNPs was ∼15 nm, and the main difference between these conjugates was the greater coating with positive charges of MNP+-P2+, as shown by the zeta potential values. Absorption spectra exhibited the Soret and Q bands characteristic of TPPF20 linked to MNPs. Furthermore, these conjugates showed red fluorescence emission of porphyrin with quantum yields of 0.011-0.036. The photodynamic effect sensitized by the conjugates indicated the efficient formation of singlet molecular oxygen in different media, reaching quantum yield values of 0.17-0.34 in N,N-dimethylformamide. The photodynamic activity of the conjugates was evaluated to inactivate the Gram-positive bacteria Staphylococcus aureus, the Gram-negative bacteria Escherichia coli, and the yeast Candida albicans. The modified cationic MNP+-P2+ was the most effective conjugate for photodynamic inactivation (PDI) of microorganisms. Binding of this conjugate to bacteria and photoinactivation capability was checked by means of fluorescence microscopy. Also, sustainable use by recycling was determined after three PDI treatments. Therefore, this methodology is a suitable scaffold for the in situ modification of conjugates, and in particular, MNP+-P2+ represents a useful photodynamic active material to eradicate microorganisms.

Correction: Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis.

[This corrects the article DOI: 10.1371/journal.pntd.0006998.].

Trypanosoma cruzi-specific IFN-γ-producing cells in chronic Chagas disease associate with a functional IL-7/IL-7R axis.

BACKGROUND: The severity of cardiac disease in chronic Chagas disease patients is associated with different features of T-cell exhaustion. Here, we assessed whether the ability of T cells to secrete IFN-γ in response to T. cruzi was linked to disruption in immune homeostasis and inflammation in patients with chronic Chagas disease. METHODOLOGY/PRINCIPAL FINDINGS: PBMCs from chronic Chagas disease patients and uninfected controls were examined for frequencies of T. cruzi-responsive IFN-γ-producing cells by ELISPOT and cellular expression and function of IL-7R using flow cytometry. Serum levels of IL-7, IL-21, IL-27, soluble IL-7R, and inflammatory cytokines were also evaluated by ELISA or CBA techniques. Patients possessing T. cruzi-specific IFN-γ-producing cells (i.e. IFN-γ producers) had higher levels of memory T cells capable of modulating the alpha chain of IL-7R and an efficient response to IL-7 compared to that in patients lacking (i.e. IFN-γ nonproducers) parasite-specific T-cell responses. IFN-γ producers also showed low levels of soluble IL-7R, high basal expression of Bcl-2 in T cells and low basal frequencies of activated CD25+ T cells. Modulation of IL-7R was inversely associated with serum IL-6 levels and positively associated with serum IL-8 levels. Circulating IL-21 and IL-27 levels were not associated with the frequency of IFN-γ producing cells but were reduced in less severe clinical forms of the disease. In vitro stimulation of PBMCs with IL-7 or IL-27 enhanced IFN-γ production in IFN-γ producers but not in IFN-γ nonproducers. CONCLUSIONS/SIGNIFICANCE: Alterations of the IL-7/IL-7R axis and in the levels of inflammatory cytokines were linked to impaired T. cruzi-specific IFN-γ production. These alterations might be responsible of the process of immune exhaustion observed in chronic Chagas disease.

Distinct Treatment Outcomes of Antiparasitic Therapy in Trypanosoma cruzi-Infected Children Is Associated With Early Changes in Cytokines, Chemokines, and T-Cell Phenotypes.

Background: In contrast to adults, Trypanosoma cruzi-infected children have more broadly functional Trypanosoma cruzi-specific T cells, and the total T-cell compartment exhibits fewer signs of immune exhaustion. However, not much is known about the link between immunocompetence and the treatment efficacy for human Chagas disease. Methods: Using cytokine enzyme-linked immunosorbent spot (ELISPOT) polychromatic flow cytometry, cytometric bead assay, multiplex serological assays and quantitative PCR, we evaluated T. cruzi-specific T-cell and antibody immune responses, T-cell phenotypes and parasitemia in children in the early chronic phase of Chagas disease undergoing anti-Trypanosoma cruzi treatment. Results: Treatment with benznidazole or nifurtimox induced a decline in T. cruzi-specific IFN-γ- and IL-2-producing cells and proinflammatory cytokines and chemokines. T-cell responses became detectable after therapy in children bearing T-cell responses under background levels prior to treatment. The total frequencies of effector, activated and antigen-experienced T cells also decreased following anti-T. cruzi therapy, along with an increase in T cells expressing the receptor of the homeostatic cytokine IL-7. Posttreatment changes in several of these markers distinguished children with a declining serologic response suggestive of successful treatment from those with sustained serological responses in a 5-year follow-up study. A multivariate analysis demonstrated that lower frequency of CD4+CD45RA-CCR7-CD62L- T cells prior to drug therapy was an independent indicator of successful treatment. Conclusions: These findings further validate the usefulness of alternative metrics to monitor treatment outcomes. Distinct qualitative and quantitative characteristics of T cells prior to drug therapy may be linked to treatment efficacy.

The Significance of Discordant Serology in Chagas Disease: Enhanced T-Cell Immunity to Trypanosoma cruzi in Serodiscordant Subjects.

BACKGROUND: Subjects are considered infected with Trypanosoma cruzi when tested positive by at least two out of three serological tests, whereas a positive result in only one of up to three tests is termed "serodiscordant" (SD). Assessment of parasite-specific T-cell responses may help discriminate the uninfected from infected individuals among SD subjects. METHODS: Peripheral blood mononuclear cells from SD and seropositive (SP) subjects, who were born in areas endemic for T. cruzi infection but living in Buenos Aires city, Argentina, at the time of the study, and seronegative unexposed subjects were included for analysis. The function and phenotype of T cells were assessed by interferon-γ (IFN-γ) and interleukin (IL)-2 enzyme-linked immunospot assay and multiparameter flow cytometry. T. cruzi-specific antibodies were quantified by conventional serology and a multiplex assay format. RESULTS: SD subjects exhibited immunity cell responses to T. cruzi but in contrast to SP subjects, T cells in SD subjects more often display the simultaneous production of IFN-γ and IL-2 in response to T. cruzi antigens and have a resting phenotype. SD individuals also have higher IFN-γ spot counts, polyfunctional CD4+ T-cells enriched in IL-2 secreting cells and low levels of antibodies specific for a set of T. cruzi-derived recombinant proteins compared with the SP group. Long-term follow-up of SD individuals confirmed that humoral and T-cell responses fluctuate but are sustained over time in these subjects. T cells in SD subjects for T. cruzi infection did not recognize Leishmania antigens. CONCLUSION: Both T-cell and humoral responses in most subjects assessed by conventional tests as SD for T. cruzi infection indicate prior exposure to infection and the establishment of immunological memory suggestive of a resolved infection.

Prevention of congenital Chagas disease by Benznidazole treatment in reproductive-age women. An observational study.

Since the decline in new cases of infection by insect/vector, congenital Chagas disease has become more relevant in the transmission of Chagas disease. Treatment with benznidazole significantly reduces the parasitemia, which constitutes an important factor linked to vertical transmission. The objective of this study was to evaluate whether treatment with benznidazole previously administered to women of childbearing age can prevent or reduce the incidence of new cases of congenital Chagas disease. An historical cohort study that included all women in reproductive age (15-45 years) assisted in our center was designed. We included 67 mothers with chronic Chagas disease; 35 women had not been treated prior to pregnancy, 15 had been treated prior to pregnancy and 17 gave birth prior and after treatment with benznidazole. Eight mothers gave birth to 16 children with congenital Chagas disease (8/67, 12%). The prevalence of congenital Chagas was 16/114 (14%) children born to untreated mothers and 0/42 (0%) children born to benznidazole- treated mothers, p=0.01. No significant differences were observed in clinical, serologic, epidemiological or socioeconomic baseline variables between mothers with and without children born with congenital Chagas. A 32% conversion rate to negative serology was observed in benznidazole-treated women after long-term follow up. Antiparasitic treatment administered to women in reproductive age can prevent the occurrence of congenital Chagas disease.

Early evolution of endoparasitic group in powdery mildews: molecular phylogeny suggests missing link between Phyllactinia and Leveillula.

Of the 17 genera of the Erysiphaceae, only four genera (viz. Leveillula, Phyllactinia, Pleochaeta and Queirozia) exhibit (partly) endoparasitism. To investigate early evolution of this endoparasitic nature, we performed molecular phylogenetic analyses of powdery mildews belonging to the tribe Phyllactinieae collected in North and South America. The most ancestral taxa in the tribe Phyllactinieae belong to the Pleochaeta/Queirozia group, from which the genus Phyllactinia was derived. Finally, the truly endoparasitic genus Leveillula emerged from a part of Phyllactinia The present study showed clear evolutional polarity in the powdery mildews concerned (that is, partly endoparasitic group evolved from ectoparasitic group) and then a truly endoparasitic group emerged from a partly endoparasitic group. In addition, a group with distinctly dimorphic conidia proved to be basal in the Phyllactinieae, and a group without distinctly dimorphic conidia was derived from that group. The present analyses clearly showed that Leveillula derived from a part of the "Basal Phyllactinia group". However, all sister taxa to Leveillula were distributed in North and South America. Because the putative geographic origin of Leveillula is assumed to be Central and Western Asia or the Mediterranean region, we postulate a missing link during the evolution of Leveillula from Phyllactinia Based on the present phylogenetic studies and the new rules of the International Code of Nomenclature for algae, fungi, and plants (McNeill et al. 2012), the following new species and taxonomic re-allocations are proposed: Phyllactinia bougainvilleae sp. nov., Ph. caricae comb. nov., Ph. caricicola comb. nov., Ph. durantae comb. nov., Ph. leveilluloides sp. nov., Ph. obclavata comb. nov., and Ph. papayae comb. nov.

Treatment Success in Trypanosoma cruzi Infection Is Predicted by Early Changes in Serially Monitored Parasite-Specific T and B Cell Responses.

BACKGROUND: Chagas disease is the highest impact parasitic disease in Latin America. We have proposed that changes in Trypanosoma cruzi-specific immune responses might serve as surrogate indicators of treatment success. Herein, we addressed in a long-term follow-up study whether cure achieved after treatment can be predicted by changes in non-conventional indexes of anti-parasite serological and T cell activities. METHODOLOGY/PRINCIPAL FINDINGS: T. cruzi-specific T cell responses, as measured by interferon-γ ELISPOT and T. cruzi-specific antibodies assessed by ELISA, hemagglutination and immunofluorescence tests as well as by a multiplex assay incorporating 14 recombinant T. cruzi proteins were measured in 33 patients at 48-150 months post-benznidazole treatment. Cure - as assessed by conventional serological tests - was associated with an early decline in T. cruzi-specific IFN-γ-producing T cells and in antibody titers measured by the multiplex serological assay. Changes in the functional status and potential of T. cruzi-specific T cells, indicative of reduced antigen stimulation, provided further evidence of parasitological cure following benznidazole treatment. Patients showing a significant reduction in T. cruzi-specific antibodies had higher pre-therapy levels of T. cruzi-specific IFN-γ- producing T cells compared to those with unaltered humoral responses post-treatment. CONCLUSIONS/SIGNIFICANCE: Monitoring of appropriate immunological responses can provide earlier and robust measures of treatment success in T. cruzi infection.

[Isolation of enteropathogenic Escherichia coli O157:H16 identified in a diarrhea case in a child and his household contacts in La Pampa Province, Argentina]. / Aislamiento de Escherichia coli enteropatógeno O157:H16 de un caso de diarrea infantil y sus contactos familiares en La Pampa, Argentina.

Enteropathogenic Escherichia coli (EPEC) is a major causative agent of acute diarrhea in children in developing countries. This pathotype is divided into typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence of the bfp virulence factor associated with adhesion, encoded in the pEAF plasmid. In the present study, the isolation of aEPEC O157:H16 from a bloody diarrhea case in a child and his household contacts (mother, father and sister) is described. The strain was characterized as E. coli O157:H16 eae-ɛ-positive, sorbitol fermenter with ß-glucuronidase activity, susceptible to all antimicrobials tested, and negative for virulence factors stx1, stx2, ehxA and bfp. XbaI-PFGE performed on all isolates showed the AREXHX01.1040 macrorestriction pattern, with 100% similarity. These results highlight the importance of epidemiological surveillance of E. coli O157-associated diarrhea cases identified in children and their family contacts, as well as the incorporation of molecular techniques that allow the detection of the different E. coli pathotypes.

Aislamiento de Escherichia coli enteropatógeno 0157: H16 de un caso de diarrea infantil y sus contactos familiares en La Pampa, Argentina / Isolation of enteropathogenic Eschjerichia coli0157:H16 identified in a diarrhea case in a chil and his household contacts in La Pampa Province, Argentina

Escherichia coli enteropatógeno (EPEC) es uno de los principales agentes de diarrea infantil aguda en los países en desarrollo. Se clasifica en típico (tEPEC) y atípico (aEPEC) sobre la base de la presencia del factor bfp, asociado a la adherencia y codificado en el plásmido pEAF. Se describe el aislamiento de E. coli O157:H16, de la categoría aEPEC, en un caso de diarrea sanguinolenta infantil y en sus contactos familiares. De las muestras de materia fecal del niño, de la madre, del padre y de la hermana se aisló E. coli O157:H16 eae-??-positivo, sorbitol-positivo, ß-glucuronidasa-positivo, sensible a los antimicrobianos ensayados, y negativo para los factores stx1, stx2, ehxA y bfp. Por XbaI-PFGE, todos los aislamientos presentaron el patrón de macrorrestricción AREXHX01.1040, con 100% de similitud. Es importante la vigilancia epidemiológica de los casos de diarrea asociados a E. coli O157 y sus contactos familiares, y la incorporación de técnicas para detectar los distintos patotipos de E. coli

Enteropathogenic Escherichia coli (EPEC) is a major causative agent of acute diarrhea in children in developing countries. This pathotype is divided into typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence of the bfp virulence factor associated with adhesion, encoded in the pEAF plasmid. In the present study, the isolation of aEPEC O157:H16 from a bloody diarrhea case in a child and his household contacts (mother, father and sister) is described. The strain was characterized as E. coli O157:H16 eae-??-positive, sorbitol fermenter with ß-glucuronidase activity, susceptible to all antimicrobials tested, and negative for virulence factors stx1, stx2, ehxA and bfp. XbaI-PFGE performed on all isolates showed the AREXHX01.1040 macrorestriction pattern, with 100% similarity. These results highlight the importance of epidemiological surveillance of E. coli O157-associated diarrhea cases identified in children and their family contacts, as well as the incorporation of molecular techniques that allow the detection of the different E. coli pathotypes

Perturbed T cell IL-7 receptor signaling in chronic Chagas disease.

We have previously demonstrated that immune responses in subjects with chronic Trypanosoma cruzi infection display features common to other persistent infections with signs of T cell exhaustion. Alterations in cytokine receptor signal transduction have emerged as one of the cell-intrinsic mechanisms of T cell exhaustion. In this study, we performed an analysis of the expression of IL-7R components (CD127 and CD132) on CD4(+) and CD8(+) T cells and evaluated IL-7-dependent signaling events in patients at different clinical stages of chronic chagasic heart disease. Subjects with no signs of cardiac disease showed a decrease in CD127(+)CD132(+) cells and a reciprocal gain of CD127(-)CD132(+) in CD8(+) and CD4(+) T cells compared with either patients exhibiting heart enlargement or uninfected controls. T. cruzi infection, in vitro, was able to stimulate the downregulation of CD127 and the upregulation of CD132 on T cells. IL-7-induced phosphorylation of STAT5 as well as Bcl-2 and CD25 expression were lower in T. cruzi-infected subjects compared with uninfected controls. The serum levels of IL-7 were also increased in chronic chagasic patients. The present study highlights perturbed IL-7/IL-7R T cell signaling through STAT5 as a potential mechanism of T cell exhaustion in chronic T. cruzi infection.

Polyfunctional T cell responses in children in early stages of chronic Trypanosoma cruzi infection contrast with monofunctional responses of long-term infected adults.

BACKGROUND: Adults with chronic Trypanosoma cruzi exhibit a poorly functional T cell compartment, characterized by monofunctional (IFN-γ-only secreting) parasite-specific T cells and increased levels of terminally differentiated T cells. It is possible that persistent infection and/or sustained exposure to parasites antigens may lead to a progressive loss of function of the immune T cells. METHODOLOGY/PRINCIPAL FINDINGS: To test this hypothesis, the quality and magnitude of T. cruzi-specific T cell responses were evaluated in T. cruzi-infected children and compared with long-term T. cruzi-infected adults with no evidence of heart failure. The phenotype of CD4(+) T cells was also assessed in T. cruzi-infected children and uninfected controls. Simultaneous secretion of IFN-γ and IL-2 measured by ELISPOT assays in response to T. cruzi antigens was prevalent among T. cruzi-infected children. Flow cytometric analysis of co-expression profiles of CD4(+) T cells with the ability to produce IFN-γ, TNF-α, or to express the co-stimulatory molecule CD154 in response to T. cruzi showed polyfunctional T cell responses in most T. cruzi-infected children. Monofunctional T cell responses and an absence of CD4(+)TNF-α(+)-secreting T cells were observed in T. cruzi-infected adults. A relatively high degree of activation and differentiation of CD4(+) T cells was evident in T. cruzi-infected children. CONCLUSIONS/SIGNIFICANCE: Our observations are compatible with our initial hypothesis that persistent T. cruzi infection promotes eventual exhaustion of immune system, which might contribute to disease progression in long-term infected subjects.

Optimization of cellular uptake of zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine for maximal photoinactivation of Candida albicans.

Cellular uptake and photodynamic action of zinc(II) 2,9,16,23-tetrakis[4-(N-methylpyridyloxy)]phthalocyanine (ZnPPc4⁺) was examined in Candida albicans. In vitro investigations showed that ZnPPc4⁺ was rapidly bound to C. albicans cells. The binding of phthalocyanine to cells was dependent on ZnPPc4⁺ concentrations (1-10 µM) and cells densities (106-108 cells mL⁻¹). A high amount of ZnPPc4⁺ retained in the cells after two washing steps, indicating a strong interaction between the photosensitizer and C. albicans. The uptake was temperature dependent, although the difference between 37 °C and 4 °C was about 10 %. Also, the amount of ZnPPc bound to C. albicans was affected when the cells were incubated for a longer time with azide and 2,4-dinitrophenol (DNP) prior to treatment with ZnPP4⁺. Cell survival after irradiation was dependent on the irradiation period, ZnPPc4⁺ concentration and cells density. Photoinactivation of C. albicans cells was elevated even after two washing steps. The strong dependence of uptake on cell density reveals the strength and avidity of the binding of ZnPPc4⁺ to C. albicans cells. The accumulation behaviour of ZnPPc4⁺ suggests that mainly an affinity-mediated binding mechanism can be involved. Therefore, ZnPPc4⁺ is an interesting phthalocyanine for photodynamic inactivation (PDI) of yeasts in liquid suspensions.

Allopurinol reduces antigen-specific and polyclonal activation of human T cells.

Allopurinol is the most popular commercially available xanthine oxidase inhibitor and it is widely used for treatment of symptomatic hyperuricaemia, or gout. Although, several anti-inflammatory actions of allopurinol have been demonstrated in vivo and in vitro, there have been few studies on the action of allopurinol on T cells. In the current study, we have assessed the effect of allopurinol on antigen-specific and mitogen-driven activation and cytokine production in human T cells. Allopurinol markedly decreased the frequency of IFN-γ and IL-2-producing T cells, either after polyclonal or antigen-specific stimulation with Herpes Simplex virus 1, Influenza (Flu) virus, tetanus toxoid and Trypanosoma cruzi-derived antigens. Allopurinol attenuated CD69 upregulation after CD3 and CD28 engagement and significantly reduced the levels of spontaneous and mitogen-induced intracellular reactive oxygen species in T cells. The diminished T cell activation and cytokine production in the presence of allopurinol support a direct action of allopurinol on human T cells, offering a potential pharmacological tool for the management of cell-mediated inflammatory diseases.

Inhibitory receptors are expressed by Trypanosoma cruzi-specific effector T cells and in hearts of subjects with chronic Chagas disease.

We had formerly demonstrated that subjects chronically infected with Trypanosoma cruzi show impaired T cell responses closely linked with a process of T cell exhaustion. Recently, the expression of several inhibitory receptors has been associated with T cell dysfunction and exhaustion. In this study, we have examined the expression of the cytotoxic T lymphocyte antigen 4 (CTLA-4) and the leukocyte immunoglobulin like receptor 1 (LIR-1) by peripheral T. cruzi antigen-responsive IFN-gamma (IFN-γ)-producing and total T cells from chronically T. cruzi-infected subjects with different clinical forms of the disease. CTAL-4 expression was also evaluated in heart tissue sections from subjects with severe myocarditis. The majority of IFN-γ-producing CD4(+) T cells responsive to a parasite lysate preparation were found to express CTLA-4 but considerably lower frequencies express LIR-1, irrespective of the clinical status of the donor. Conversely, few IFN-γ-producing T cells responsive to tetanus and diphtheria toxoids expressed CTLA-4 and LIR-1. Polyclonal stimulation with anti-CD3 antibodies induced higher frequencies of CD4(+)CTAL-4(+) T cells in patients with severe heart disease than in asymptomatic subjects. Ligation of CTLA-4 and LIR-1 with their agonistic antibodies, in vitro, reduces IFN-γ production. Conversely, CTLA-4 blockade did not improved IFN-γ production in response to T. cruzi antigens. Subjects with chronic T. cruzi infection had increased numbers of CD4(+)LIR-1(+) among total peripheral blood mononuclear cells, relative to uninfected individuals and these numbers decreased after treatment with benznidazole. CTLA-4 was also expressed by CD3(+) T lymphocytes infiltrating heart tissues from chronically infected subjects with severe myocarditis. These findings support the conclusion that persistent infection with T. cruzi leads to the upregulation of inhibitory receptors which could alter parasite specific T cell responses in the chronic phase of Chagas disease.