Selective Impairment of TH17-Differentiation and Protection against Autoimmune Arthritis after Overexpression of BCL2A1 in T Lymphocytes.

The inhibition of apoptotic cell death in T cells through the dysregulated expression of BCL2 family members has been associated with the protection against the development of different autoimmune diseases. However, multiple mechanisms were proposed to be responsible for such protective effect. The purpose of this study was to explore the effect of the T-cell overexpression of BCL2A1, an anti-apoptotic BCL2 family member without an effect on cell cycle progression, in the development of collagen-induced arthritis. Our results demonstrated an attenuated development of arthritis in these transgenic mice. The protective effect was unrelated to the suppressive activity of regulatory T cells but it was associated with a defective activation of p38 mitogen-activated protein kinase in CD4+ cells after in vitro TCR stimulation. In addition, the in vitro and in vivo TH17 differentiation were impaired in BCL2A1 transgenic mice. Taken together, we demonstrated here a previously unknown role for BCL2A1 controlling the activation of CD4+ cells and their differentiation into pathogenic proinflammatory TH17 cells and identified BCL2A1 as a potential target in the control of autoimmune/inflammatory diseases.

Bone Morphogenetic Protein and Activin Membrane-Bound Inhibitor, a Transforming Growth Factor ß Rheostat That Controls Murine Treg Cell/Th17 Cell Differentiation and the Development of Autoimmune Arthritis by Reducing Interleukin-2 Signaling.

OBJECTIVE: Transforming growth factor ß (TGFß) plays a prominent role in the establishment of immunologic tolerance, and mice lacking TGFß1 die of multiorgan inflammation early in life. TGFß controls the differentiation of CD4+ lymphocytes into Treg cells or proinflammatory Th17 cells. Although this dual capacity is modulated by the presence of additional cytokines around the activated cells, TGFß also dissociates Th17/Treg cell differentiation in a dose-dependent manner by mechanisms still unknown. The purpose of this study was to explore the contribution of bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) to the modulation of TGFß activity during the differentiation of CD4+ cells and in the control of immunologic tolerance in mice with collagen-induced arthritis (CIA). METHODS: The in vitro and in vivo Treg cell and Th17 cell differentiation and the development of CIA were compared in wild-type mice and BAMBI-deficient mice. RESULTS: BAMBI was induced after activation by TGFß and fixed the appropriate intensity level of TGFß signaling in CD4+ cells. Its deficiency protected mice against the development of CIA by a Treg cell- and TGFß-dependent mechanism. Mechanistically, BAMBI was found to regulate CD25 expression and interleukin-2 (IL-2) signaling in Treg cells and in IL-2- and/or TGFß-activated CD4+ cells and modulated Treg cell and Th17 cell differentiation both in vitro and in vivo. CONCLUSION: Taken together, the results indicate that BAMBI is a component of a rheostat-like mechanism that, through the control of TGFß and IL-2 signaling strength, regulates the differentiation of CD4+ lymphocytes and the development of autoimmune arthritis.

Distinct serum proteome profiles associated with collagen-induced arthritis and complete Freund's adjuvant-induced inflammation in CD38⁻/⁻ mice: The discriminative power of protein species or proteoforms.

Collagen-type-II-induced arthritis (CIA) is an autoimmune disease, which involves a complex host systemic response including inflammatory and autoimmune reactions. CIA is milder in CD38(-/-) than in wild-type (WT) mice. ProteoMiner-equalized serum samples were subjected to 2D-DiGE and MS-MALDI-TOF/TOF analyses to identify proteins that changed in their relative abundances in CD38(-/-) versus WT mice either with arthritis (CIA(+) ), with no arthritis (CIA(-) ), or with inflammation (complete Freund's adjuvant (CFA)-treated mice). Multivariate analyses revealed that a multiprotein signature (n = 28) was able to discriminate CIA(+) from CIA(-) mice, and WT from CD38(-/-) mice within each condition. Likewise, a distinct multiprotein signature (n = 16) was identified which differentiated CIA(+) CD38(-/-) mice from CIA(+) WT mice, and lastly, a third multiprotein signature (n = 18) indicated that CD38(-/-) and WT mice could be segregated in response to CFA treatment. Further analyses showed that the discriminative power to distinguish these groups was reached at protein species level and not at the protein level. Hence, the need to identify and quantify proteins at protein species level to better correlate proteome changes with disease processes. It is crucial for plasma proteomics at the low-abundance protein species level to apply the ProteoMiner enrichment. All MS data have been deposited in the ProteomeXchange with identifiers PXD001788, PXD001799 and PXD002071 (http://proteomecentral.proteomexchange.org/dataset/PXD001788, http://proteomecentral.proteomexchange.org/dataset/PXD001799 and http://proteomecentral.proteomexchange.org/dataset/PXD002071).

Appraisal of snakebite incidence and mortality in Bolivia.

No information has been yet published on snakebite in Bolivia. The country includes very different ecological situations leading to various epidemiological risks. A study has been carried out to evaluate the incidence and location of snakebite, particularly in relation with altitude, in order to improve management. Investigations on snakebite epidemiology were based on a) cases treated in health facilities as reported by health authorities and b) household surveys carried out in areas with high variations of altitude, in various regions of Bolivia. An average of 700 bites was treated each year in Bolivia (national annual incidence = 8 bites per 100,000 people) with a great disparity between districts. Household surveys showed annual incidences ranged from 30 to 110 bites per 100,000 inhabitants depending on location. Annual mortality ranged 0.1-3.9 per 100,000 people. A significant and constant inverse correlation was shown between snakebite incidence and altitude, which may be explained by both snake and human distributions and activities. Notification of snakebite is useful for improving patient management and informing antivenom distribution. It should also involve the report of deaths and clinical details of envenomation.

Transgenic expression of soluble human CD5 enhances experimentally-induced autoimmune and anti-tumoral immune responses.

CD5 is a lymphoid-specific transmembrane glycoprotein constitutively expressed on thymocytes and mature T and B1a lymphocytes. Current data support the view that CD5 is a negative regulator of antigen-specific receptor-mediated signaling in these cells, and that this would likely be achieved through interaction with CD5 ligand/s (CD5L) of still undefined nature expressed on immune or accessory cells. To determine the functional consequence of loss of CD5/CD5L interaction in vivo, a new transgenic mouse line was generated (shCD5EµTg), expressing a circulating soluble form of human CD5 (shCD5) as a decoy to impair membrane-bound CD5 function. These shCD5EµTg mice showed an enhanced response to autologous antigens, as deduced from the presentation of more severe forms of experimentally inducible autoimmune disease (collagen-induced arthritis, CIA; and experimental autoimmune encephalitis, EAE), as well as an increased anti-tumoral response in non-orthotopic cancer models (B16 melanoma). This enhancement of the immune response was in agreement with the finding of significantly reduced proportions of spleen and lymph node Treg cells (CD4+CD25+FoxP3+), and of peritoneal IL-10-producing and CD5+ B cells, as well as an increased proportion of spleen NKT cells in shCD5EµTg mice. Similar changes in lymphocyte subpopulations were observed in wild-type mice following repeated administration of exogenous recombinant shCD5 protein. These data reveal the relevant role played by CD5/CD5L interactions on the homeostasis of some functionally relevant lymphocyte subpopulations and the modulation of immune responses to autologous antigens.

Evaluation of compliance to congenital Chagas disease treatment: results of a randomised trial in Bolivia.

BACKGROUND: A randomised, unblinded, clinical trial comparing two benznidazole regimens for congenital Chagas disease was carried out to determine whether simplification and reduction in the length of treatment could lead to better treatment compliance. METHODS: This study was conducted in Santa Cruz, Bolivia. Serological screening was carried out in pregnant women, and parasites were sought in the blood of newborns from seropositive mothers. Infected infants were randomly assigned to two treatment groups. Recovery was assessed by parasite seeking at 1 month and 2 months as well as serological tests at 9 months. Assessment of treatment adherence was based on weekly home visits and use of electronic monitors. RESULTS: Benznidazole was given to 63 newborns in group A (5 mg/kg in two daily doses for 60 days) and 61 newborns in group B (7.5 mg/kg in a single daily dose for 30 days). There was no difference in compliance between the two groups. The study confirmed the efficacy and good tolerance of both benznidazole regimens in the treatment of congenital Chagas disease. CONCLUSIONS: The short treatment should be preferred as it allows reducing the dose of benznidazole as well as the cost of treatment.

p27(Kip1) inhibits systemic autoimmunity through the control of Treg cell activity and differentiation.

OBJECTIVE: Despite the importance of Treg cells in the maintenance of immunologic tolerance, the mechanisms that control their generation and activity are unknown. Since the cell cycle inhibitor p27(Kip1) (p27) was involved in T cell anergy, we undertook this study to explore its role in both Treg cell processes. METHODS: The development of type II collagen-induced arthritis (CIA) and lupus-like abnormalities was compared between transgenic mice overexpressing human Bcl-2 in T cells (BCL2-TgT mice) and nontransgenic mice that were deficient or not deficient in p27. The contribution of Treg cells to disease evolution was also explored. Finally, the in vitro activity of Treg cells and their differentiation from naive CD4+ cells was compared between these strains of mice. RESULTS: BCL2-TgT mice were protected against CIA by a Treg cell-dependent mechanism. In association with this protection, the overexpression of Bcl-2 in T cells enhanced the differentiation and activity of Treg cells. Both Bcl-2 effects were independent of its antiapoptotic activity but dependent on its capacity to induce the expression of p27 that augmented the strength of transforming growth factor ß (TGFß) signaling in T cells. Accordingly, down-modulation of p27 expression in BCL2-TgT mice promoted CIA. In addition, p27 deficiency in aged C57BL/6 mice reduced the number and activity of Treg cells and induced the development of mild lupus-like abnormalities. CONCLUSION: Our results point to p27 as a critical regulator of Treg cell differentiation and function through the positive modulation of TGFß signaling strength in T cells.

Mice deficient in CD38 develop an attenuated form of collagen type II-induced arthritis.

CD38, a type II transmembrane glycoprotein expressed in many cells of the immune system, is involved in cell signaling, migration and differentiation. Studies in CD38 deficient mice (CD38 KO mice) indicate that this molecule controls inflammatory immune responses, although its involvement in these responses depends on the disease model analyzed. Here, we explored the role of CD38 in the control of autoimmune responses using chicken collagen type II (col II) immunized C57BL/6-CD38 KO mice as a model of collagen-induced arthritis (CIA). We demonstrate that CD38 KO mice develop an attenuated CIA that is accompanied by a limited joint induction of IL-1ß and IL-6 expression, by the lack of induction of IFNγ expression in the joints and by a reduction in the percentages of invariant NKT (iNKT) cells in the spleen. Immunized CD38 KO mice produce high levels of circulating IgG1 and low of IgG2a anti-col II antibodies in association with reduced percentages of Th1 cells in the draining lymph nodes. Altogether, our results show that CD38 participates in the pathogenesis of CIA controlling the number of iNKT cells and promoting Th1 inflammatory responses.

Influence of pregnancy on Trypanosoma cruzi parasitemia in chronically infected women in a rural Bolivian community.

To determine the role of pregnancy on Trypanosoma cruzi parasitemia, a matched cohort study was carried out in a rural Bolivian community comparing parasite rates in gravidae, puerperae, and non-pregnant infected women. A selection of 67 chronically infected women, who delivered between March 2004 and May 2005, were initially evaluated during the third trimester of pregnancy and again after delivery. They were matched for age, parity, and location with 104 seropositive non-pregnant women, who likewise had submitted blood for microscopic examination for T. cruzi parasites in June 2005. Seroreactive pregnant women had a higher rate of T. cruzi parasitemia (14.9%) than matched non-pregnant infected women (2.9%; P = 0.004). After delivery, parasitemia significantly decreased during puerperium (1.5%) compared with the period of pregnancy (14.9%; P = 0.03). This study showed an increase of parasite loads in maternal peripheral blood, during the third trimester, and a significant decline after delivery.

Door-to-door screening as a strategy for the detection of congenital Chagas disease in rural Bolivia.

OBJECTIVE: To demonstrate the feasibility of a house-to-house screening system used for congenital Chagas disease in rural areas based on an active search for pregnant women and newborns in their homes in addition to passive case detection in health facilities. METHODS: Exploratory phase conducted by the research team followed by an operational period coordinated by municipal health service. A blood sample was taken for serological and parasitological tests of Trypanosoma cruzi from pregnant women who were searching antenatal care or visited at home by field investigators. Infants born to T. cruzi-infected women were examined for infection at birth and again at 1 and 7 months of age. RESULTS: 64.5% of the pregnant women were infected. Congenital infection was diagnosed at birth in 4.0% (12/299) of the children born to seroreactive mothers. Twelve additional cases of infection (4%) were diagnosed in children between 1 and 7 months of age. Finally, 37% of the children were lost to follow-up in the exploratory phase and 53% during the operational phase (P=0.002), significantly fewer than in most passive case detection studies. CONCLUSION: Despite poorer outcomes after door-to-door screening activities have been transferred to the health system, a combined strategy based on active and passive case detection appeared to be efficient for identifying rural cases of congenital Chagas disease.

Exacerbation of type II collagen-induced arthritis in apolipoprotein E-deficient mice in association with the expansion of Th1 and Th17 cells.

OBJECTIVE: To explore the bidirectional relationship between the development of rheumatoid arthritis (RA) and atherosclerosis using bovine type II collagen (CII)-immunized B10.RIII apoE(-/-) mice, a murine model of spontaneous atherosclerosis and collagen-induced arthritis (CIA). METHODS: Male B10.RIII apoE(-/-) mice and wild-type controls were immunized with 150 µg of CII emulsified in Freund's complete adjuvant (CFA). The clinical, radiologic, and histopathologic severity of CIA, the levels of circulating IgG1 and IgG2a anti-CII antibodies, the expression of proinflammatory and antiinflammatory cytokines in the joints, and the percentages of Th1, Th17, and Treg lymphocytes in the draining lymph nodes were evaluated during CIA induction. In addition, the size of atherosclerotic lesions was assessed in these mice 8 weeks after CIA induction. RESULTS: B10.RIII apoE(-/-) mice that were immunized with CII and CFA developed an exacerbated CIA that was accompanied by increased joint expression of multiple proinflammatory cytokines and by the expansion in the draining lymph nodes of Th1 and Th17 cells. In contrast, the size of vascular lesions in B10.RIII apoE(-/-) mice was not affected by the development of CIA. CONCLUSION: Our findings indicate that a deficiency in apolipoprotein E and/or its consequences in cholesterol metabolism act as accelerating factors in autoimmunity by promoting Th1 and Th17 inflammatory responses.

Antibody drop in newborns congenitally infected by Trypanosoma cruzi treated with benznidazole.

OBJECTIVE: To compare the drop of Chagas antibody titres between non-infected and congenitally infected newborns treated by two doses of benznidazole, aiming at evaluating the recovery time and giving recommendations regarding serological criteria of recovery. METHODS: During a clinical trial, the drop of Trypanosoma cruzi antibody titres measured by ELISA tests was followed during the first year of life in congenitally infected newborns treated with different doses of benznidazole and compared to T. cruzi antibody titres in non-parasitaemic newborns. Confirmation of recovery was given by two negative serological tests: Chagas Stat-Pak (CSP) (immunochromatography) and Chagatest v3.0 (ELISA). RESULTS: In non-parasitaemic infants of infected mothers, antibodies of maternal origin disappeared in <8 months while in infected infants, T. cruzi antibodies decreased more slowly and disappeared in 9-16 months allowing to confirm the recovery. All CSP tests were negative before the ninth month while about 10% of ELISA tests remained positive at the 12th month. CONCLUSIONS: Recovery may be confirmed in most cases at 10 months. The CSP test was compared to Chagatest v3.0 ELISA and appeared to give a reliable response. The decrease rate of antibodies does not depend on treatment modes.

GITR contributes to the systemic adjuvanticity of the Escherichia coli heat-labile enterotoxin.

The Escherichia coli heat-labile enterotoxin (LT) possesses a powerful mucosal and systemic adjuvant effect. However, little is known about the cellular and molecular basis of the immunostimulatory activity of LT at the mucosal level, and even less information is available on the mechanisms underlying its systemic adjuvant activity. In this study, we show that distinct mechanisms are responsible for the parenteral and mucosal adjuvanticity of LT. Indeed, the systemic administration of LT upregulates the expression of glucocorticoid-induced TNFR-related protein (GITR), but not other activation markers, in naive T cells. Using WT and GITR-deficient mice and LT and its enzymatically inactive mutant LTK63 as adjuvants, we show that the induction of GITR expression in T cells accounts for the systemic immunostimulatory capacity of LT, which requires an intact enzymatic activity. In contrast, the mucosal administration of LT does not induce GITR expression on Peyer's patche T cells and accordingly no differences are observed in the mucosal adjuvanticity of LT between WT and GITR-deficient mice. Altogether, our results demonstrate the distinct effect of LT after parenteral administration when compared with the mucosal delivery, and describe a new mechanism of LT adjuvanticity related to its ability to induce the expression of GITR in CD4(+) T cells.

Sensitivity and specificity of Chagas Stat-Pak test in Bolivia.

OBJECTIVE: To compare the results of an immunochromatographic test performed on whole blood, Chagas Stat-Pak, with those of an ELISA test using recombinant antigens. METHOD: We tested 995 subjects of a rural population of all ages in the south of Bolivia, 459 pregnant women of the same population and 1030 urban women giving birth from the east of Bolivia. RESULTS: The sensitivity of the CSP test for the entire studied population (n = 2484) was 94.73% [93.35-96.10]; the specificity was 97.33% [96.50-98.15]. However, the specificity differed significantly between rural pregnant and urban birthing women, which could be attributed either to differences of parasite strain or Chagas prevalence. CONCLUSION: The test is simple of use, reliable, relatively inexpensive (<2 US$ each test) and its performances are compatible with a field use for large-scale screenings.

Involvement of the intrinsic and extrinsic cell-death pathways in the induction of apoptosis of mature lymphocytes by the Escherichia coli heat-labile enterotoxin.

Escherichia coli heat-labile enterotoxin (LT) exhibits a broad range of immunomodulatory activities, including the induction of lymphocyte-programmed cell death. In previous studies, we have demonstrated that in vivo LT promotes apoptosis of immature T and B cells through the stimulation of endogenous glucocorticoids. In the present study, we show that the extrinsic cell-death pathway as well as the apoptosis-inducing factor do not participate in the LT-induced elimination of thymocytes. In contrast to developing lymphocytes, LT promotes the death of mature lymphocytes by both glucocorticoid- and Fas death receptor/Fas ligand-dependent mechanisms. However, the dependency of these mechanisms in the LT-induced cell-death activity seems to be different among CD4(+) and CD8(+) T cells. Altogether, our study shows that the same bacterial toxin can induce apoptosis of lymphoid cells through several mechanisms depending on the status of differentiation of these cells.

Th17 development and autoimmune arthritis in the absence of reactive oxygen species.

Dendritic cells (DC) express a functional NADPH oxidase and produce reactive oxygen species (ROS) upon interaction with microbes and T cells. Exposure to ROS leads to DC activation and maturation, as evidenced by phenotypic and functional changes. We have evaluated how endogenous ROS production affects the cytokine secretion pattern and T cell-activating capacity of bone marrow-derived murine DC. DC treated with ROS scavengers, as well as DC from mice that lack a functional NADPH oxidase (and thereby inherently deficient in ROS production) produced significantly increased levels of IL-1beta, IL-6, TNF-alpha and TGF-beta in response to microbial activation. DC deficient in ROS production induced high levels of IFN-gamma and IL-17 in responding T cells after Ag-specific or superantigen-induced activation. Finally, we show that ROS deficiency affected the induction of a T cell-dependent inflammatory condition, collagen-induced arthritis (CIA). C57BL/6 mice that lack a functional NADPH oxidase developed a severe and erosive CD4-dependent CIA, whereas the majority of the congenic wild-type animals remained healthy. These data suggest that ROS act as immunomodulators in DC-driven T cell activation and perhaps also in T cell-dependent immunopathology.

Epidemiological evaluation of Chagas disease in a rural area of southern Bolivia.

We evaluated the prevalence of Chagas disease using a rapid screening test (Chagas Stat-Pak), confirmed by ELISA, in Caraparí, a village of 9000 inhabitants in southern Bolivian Chaco. The prevalence of Trypanosoma cruzi was estimated in a sample of 995 people. The prevalence adjusted on age was 51.2% and was proportionally related to age. We also observed a very significant cline from the south to the north of the locality, where the prevalence ranged from 40 to 80%. In children younger than 11 years, the prevalence was 21.5%, which confirmed the importance of residual vector transmission despite several years of vector control. Among women of procreation age, the prevalence was 63.9%, resulting in a high risk of congenital transmission. The control of the disease requires an increase in vector control and improvement of dwellings before considering children's treatment with trypanocide.

Serodiagnóstico de la Trypanosomiasis Americana en una población infantil: análisis epidemiológico / Serodiagnostic of the trypanosiomasis american in a poblation childlike, epidemiological analysis

La enfermedad de Chagas, ha sido reconocida como un problema mayor de Salud Pública estos últimos años. En Bolivia, la población infantil presenta un alto riesgo de infección por Trypanosoma cruzi en zonas endémicas. En este estudio, 1479 sueros provenientes de niños de 4 a 10 años de una zona peri-urbana de la ciudad de Cochabamba fueron analizados. Dos pruebas serológicas (IFI) y (ELISA) fueron utilizadas para la detección de anticuerpos anti Trypanosoma cruzi y todos los individuos fueron evaluados por un cuestionario epidemiológico y un examen clínico. Un 5.6 por ciento de seropositividad fue encontrado en el total de individuos estudiados. Esta población comprende dos grupos de niños provenientes de dos estratos sociales diferentes. Esta situación esta correlacionada significativamente con el riesgo de infección por Trypanosoma cruzi. Por otra parte, el análisis estadístico realizado entre las variables clínicas, hematológicas y antropométricas en relación a la infección Trypanosoma cruzi, nos demuestra una dependencia neta entre estas variables, salvo algunos aspectos antoropométricos (talla, peso) y hemáticos (población de segmentados) que presentan una tendencia relativa