Programmed Cell Death in Procyclic Form Trypanosoma brucei rhodesiense - Identification of Differentially Expressed Genes during Con A Induced Death

Trypanosoma brucei rhodesiense can be induced to undergo apoptosis after stimulation with Con A. As cell death in these parasites is associated with de novo gene expression we have applied a differential display technique, Randomly Amplified Differential Expressed Sequence-Polymerase Chain Reaction (RADES-PCR) to the study of gene expression during Con A induced cell death in these organisms. Twenty-two differentially displayed products have been cloned and sequenced. These represent the first endogenous genes to be identified as implicated in cellular death in trypanosomatids (the most primitive eukaryote in which apoptosis has been described). Evidence for an ancestral death machinery, `proto-apoptosis' in single celled organisms is discussed.

Bcl9/Bcl9l are critical for Wnt-mediated regulation of stem cell traits in colon epithelium and adenocarcinomas.

Canonical Wnt signaling plays a critical role in stem cell maintenance in epithelial homeostasis and carcinogenesis. Here, we show that in the mouse this role is critically mediated by Bcl9/Bcl9l, the mammalian homologues of Legless, which in Drosophila is required for Armadillo/beta-catenin signaling. Conditional ablation of Bcl9/Bcl9l in the intestinal epithelium, where the essential role of Wnt signaling in epithelial homeostasis and stem cell maintenance is well documented, resulted in decreased expression of intestinal stem cell markers and impaired regeneration of ulcerated colon epithelium. Adenocarcinomas with aberrant Wnt signaling arose with similar incidence in wild-type and mutant mice. However, transcriptional profiles were vastly different: Whereas wild-type tumors displayed characteristics of epithelial-mesenchymal transition (EMT) and stem cell-like properties, these properties were largely abrogated in mutant tumors. These findings reveal an essential role for Bcl9/Bcl9l in regulating a subset of Wnt target genes involved in controlling EMT and stem cell-related features and suggest that targeting the Bcl9/Bcl9l arm of Wnt signaling in Wnt-activated cancers might attenuate these traits, which are associated with tumor invasion, metastasis, and resistance to therapy.

Cell populations in lesions of cutaneous leishmaniasis of leishmania (L.) amazonensis- infected rhesus macaques, Macaca mulatta

The cellular nature of the infiltrate in cutaneous lesion of rhesus monkeys experimentally infected with Leishmania (L.) amazonensis was characterized by immunohistochemistry. Skin biopsies from infected animals with active or healing lesions were compared to non-infected controls (three of each type) to quantitate inflammatory cell types. Inflammatory cells (composed of a mixture of T lymphocyte subpopulations, macrophages and a small number of natural killer cells and granulocytes) were more numerous in active lesions than in healing ones. T-cells accounted for 44.7 ± 13.1% of the infiltrate in active lesions (versus CD2+= 40.3 ± 5.7% in healing lesions) and T-cell ratios favor CD8+ cells in both lesion types. The percentage of cells expressing class II antigen (HLA-DR+) in active lesions (95 ± 7.1%) was significantly higher (P < 0.005) from the healing lesions (42.7 ± 12.7%). Moreover, the expression of the activation molecules CD25 (@ 16%), the receptor for interleukin-2, suggests that many T cells are primed and proliferating in active lesions. Distinct histopathological patterns were observed in lesions at biopsy, but healing lesions contained more organized epithelioid granulomas and activated macrophages, followed by fibrotic substitution. The progression and resolution of skin lesions appears to be very similar to that observed in humans, confirming the potential for this to be used as a viable model to study the immune response in human cutaneous leishmaniasis.

The identification and characterization of new immunogenic egg components: implications for evaluation and control of the immunopathogenic T cell response in schistosomiasis

In schistosomiasis, granuloma formation to parasite eggs signals the beginning of a chronic and potentially life-threatening disease. Granulomas are strictly mediated by CD4+ T helper (Th) cells specific for egg antigens; however, the number and identity of these T cell-sensitizing molecules are largely unknown. We have used monoclonal T cell reagents derived from egg-sensitized individuals as probes to track down, isolate and positively identify several egg antigens; this approach implicitly assures that the molecules of interest are T cell immunogens and, hence, potentially pathogenic. The best studied and most abundant egg component is the Sm-p40 antigen. Sm-p40 and its peptide 234-246 elicit a strikingly immunodominant Th-1-polarized response in C3H and CBA mice, which are H-2k strains characterized by severe egg-induced immunopathology. Two additional recently described T cell-sensitizing egg antigens are Schistosoma mansoni phosphoenolpyruvate carboxykinase (Sm-PEPCK) and thioredoxin peroxidase-1 (Sm-TPx-1). In contrast to Sm-p40, both of these molecules induce a more balanced Th-1/Th-2 response, and are relatively stronger antigens in C57BL/6 mice, which develop smaller egg granulomas. Importantly, Sm-p40 and Sm-PEPCK have demonstrated immunogenicity in humans. The findings in the murine model introduce the important notion that egg antigens can vary significantly in immunogenicity according to the host's genetic background. A better knowledge of the principal immunogenic egg components is necessary to determine whether the immune responses to certain antigens can serve as indicators or predictors of the form and severity of clinical disease, and to ascertain whether such responses can be manipulated for the purpose of reducing pathology.

Destruction of conditional insulinoma cell lines in NOD mice: a role for autoimmunity.

AIMS/HYPOTHESIS: betaTC-tet (H2(k)) is a conditional insulinoma cell line derived from transgenic mice expressing a tetracycline-regulated oncogene. Transgenic expression of several proteins implicated in the apoptotic pathways increase the resistance of betaTC-tet cells in vitro. We tested in vivo the sensitivity of the cells to rejection and the protective effect of genetic alterations in NOD mice. METHODS: betaTC-tet cells and genetically engineered lines expressing Bcl-2 (CDM3D), a dominant negative mutant of MyD88 or SOCS-1 were transplanted in diabetic female NOD mice or in male NOD mice with diabetes induced by high-dose streptozotocin. Survival of functional cell grafts in NOD-scid mice was also analyzed after transfer of splenocytes from diabetic NOD mice. Autoreactive T-cell hybridomas and splenocytes from diabetic NOD mice were stimulated by betaTC-tet cells. RESULTS: betaTC-tet cells and genetically engineered cell lines were all similarly rejected in diabetic NOD mice and in NOD-scid mice after splenocyte transfer. In 3- to 6-week-old male NOD mice treated with high-dose streptozotocin, the cells temporarily survived, in contrast with C57BL/6 mice treated with high-dose streptozotocin (indefinite survival) and untreated 3- to 6-week-old male NOD mice (rejection). The protective effect of high-dose streptozotocin was lost in older male NOD mice. betaTC-tet cells did not stimulate autoreactive T-cell hybridomas, but induced IL-2 secretion by splenocytes from diabetic NOD mice. CONCLUSION/INTERPRETATION: The autoimmune process seems to play an important role in the destruction of betaTC-tet cells in NOD mice. Genetic manipulations intended at increasing the resistance of beta cells were inefficient. Similar approaches should be tested in vivo as well as in vitro. High dose streptozotocin influences immune rejection and should be used with caution.

Protease-activated receptor 2 signalling promotes dendritic cell antigen transport and T-cell activation in vivo.

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c(high) DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44(+) CD62(-)) CD4(+) and CD8(+) T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC(+) DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0.95% versus 0.47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.

Lack of Correlation between Seminal and Plasma HIV-1 Viral Loads is Associated with CD4T Cell Depletion in Therapy-naïve HIV-1+ Patients

The present study was conducted to investigate a possible correlation between plasma (PVL) and seminal viral load (SVL) on treatment-naïve HIV-1-infected patients in Vitória, ES, Brazil. We also evaluated whether the progressive immunosuppression associated with HIV disease (as evidenced by declining CD4 T cell counts) has any impact on the correlation between PVL and SVL HIV-1. Viral load on paired blood and semen samples from 56 consecutive treatment-naïve patients were evaluated and compared to CD4 cell counts. Viral load and T cell counts (cells/µl) were determined by NASBA and by flow cytometry, respectively. Overall, a strong positive correlation between PVL and SVL (rho = 0.438, p = 0.001) was observed. However, when patients were grouped according to their CD4 counts, this correlation was only significant among patients with CD4 counts > 200 cells/µl. Results presented here demonstrate the existence of a strong correlation between PVL and SVL on patients with CD4 cell counts > 200 cells/µl, suggesting that this association may correlate with disease progression.

The C76R transmembrane activator and calcium modulator cyclophilin ligand interactor mutation disrupts antibody production and B-cell homeostasis in heterozygous and homozygous mice.

BackgroundMutations in TNFRSF13B, the gene encoding transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI), are found in 10% of patients with common variable immunodeficiency. However, the most commonly detected mutation is the heterozygous change C104R, which is also found in 0.5% to 1% of healthy subjects. The contribution of the C104R mutation to the B-cell defects observed in patients with common variable immunodeficiency therefore remains unclear.ObjectiveWe sought to define the functional consequences of the C104R mutation on B-cell function.MethodsWe performed in vitro studies of TACI C104R expression and signaling. A knock-in mouse with the equivalent mutation murine TACI (mTACI) C76R was generated as a physiologically relevant model of human disease. We examined homozygous and heterozygous C76R mutant mice alongside wild-type littermates and studied specific B-cell lineages and antibody responses to T cell-independent and T cell-dependent challenge.ResultsC104R expression and ligand binding are significantly diminished when the mutant protein is expressed in 293T cells or in patients' cell lines. This leads to defective nuclear factor κB activation, which is proportionally restored by reintroduction of wild-type TACI. Mice heterozygous and homozygous for mTACI C76R exhibit significant B-cell dysfunction with splenomegaly, marginal zone B-cell expansion, diminished immunoglobulin production and serological responses to T cell-independent antigen, and abnormal immunoglobulin synthesis.ConclusionsThese data show that the C104R mutation and its murine equivalent, C76R, can significantly disrupt TACI function, probably through haploinsufficiency. Furthermore, the heterozygous C76R mutation alone is sufficient to disturb B-cell function with lymphoproliferation and immunoglobulin production defects.

Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 µg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.

Mast cell changes in experimental diabetes: focus on attenuation of allergic events

The prevalence of atopic diseases and diabetes is increasing worldwide though the concurrence of these pathologies in individual patients is found less frequent than it would be predicted. Moreover, co-existence of diabetes and allergy is generally marked by attenuation of their respective symptoms, and effective treatment of one disease exacerbates the other. This review gives an update of the state-of-the-art concerning the intercurrence of allergy and diabetes, particularly focusing on the consequences to the allergen-evoked vascular and cellular changes. It is proposed that the reduction in mast cell numbers and reactivity may be a pivotal mechanism behind the mutual exclusion phenomenon.

Innate signaling promotes formation of regulatory nitric oxide-producing dendritic cells limiting T-cell expansion in experimental autoimmune myocarditis.

BACKGROUND: Activation of innate pattern-recognition receptors promotes CD4+ T-cell-mediated autoimmune myocarditis and subsequent inflammatory cardiomyopathy. Mechanisms that counterregulate exaggerated heart-specific autoimmunity are poorly understood. METHODS AND RESULTS: Experimental autoimmune myocarditis was induced in BALB/c mice by immunization with α-myosin heavy chain peptide and complete Freund's adjuvant. Together with interferon-γ, heat-killed Mycobacterium tuberculosis, an essential component of complete Freund's adjuvant, converted CD11b(hi)CD11c(-) monocytes into tumor necrosis factor-α- and nitric oxide synthase 2-producing dendritic cells (TipDCs). Heat-killed M. tuberculosis stimulated production of nitric oxide synthase 2 via Toll-like receptor 2-mediated nuclear factor-κB activation. TipDCs limited antigen-specific T-cell expansion through nitric oxide synthase 2-dependent nitric oxide production. Moreover, they promoted nitric oxide synthase 2 production in hematopoietic and stromal cells in a paracrine manner. Consequently, nitric oxide synthase 2 production by both radiosensitive hematopoietic and radioresistant stromal cells prevented exacerbation of autoimmune myocarditis in vivo. CONCLUSIONS: Innate Toll-like receptor 2 stimulation promotes formation of regulatory TipDCs, which confine autoreactive T-cell responses in experimental autoimmune myocarditis via nitric oxide. Therefore, activation of innate pattern-recognition receptors is critical not only for disease induction but also for counterregulatory mechanisms, protecting the heart from exaggerated autoimmunity.

Giardia duodenalis: analysis of secreted proteases upon trophozoite-epithelial cell interaction in vitro

Protease secretion by Giardia duodenalis trophozoites upon interaction with epithelial cells and its association with the parasite adhesion was studied in co-cultures of parasites with IEC6 epithelial cell monolayers in the presence or absence of protease inhibitors. Proteolytic activity in supernatants from trophozoites was enhanced when they were co-cultured with IEC6 cells. This activity was strongly inhibited by pre-incubation of live trophozoites with E-64 and TPCK and a concomitant inhibition of parasite adhesion to IEC6 cells was observed. These data suggest that trophozoites secrete cysteine-type proteases that play a role in the adhesion of G. duodenalis to epithelial cells.

Squamous cell carcinoma in 6 patients with chronic discharging osteomyelitis: Is it really rare?

Introduction: Development of a squamous cell carcinoma (Marjolin's ulcer) is a rare but well-known complication of chronic discharging osteomyelitis. A high index of suspicion and highquality of histopathological examination are paramount in order to make the correct diagnosis. Methods: During a 15-year period (1993 and 2008), patients with long-standing chronic osteomyelitis with clinical symptoms of >1 year of duration, were retrospectively reviewed. Included were patients with histologically confirmed squamous-cell carcinoma associated with chronic wound overlying the site of chronic osteomyelitis. Clinical features and treatment approaches of these patients were analyzed. Results: During the study period, 6 patients were identified (2 women and 4 men) aged 52 to 67 years (mean 59 years). All patients had a long history of chronic discharging osteomyelitis (12, 19, 21, 30, 39 and 40 years), localized in the lower (5 patients) or upper extremitiy (1 patient). All tumors were histologically highly-differentiated squamous-cell carcinomas involving the deep soft tissues and the bone. 5 of 6 patients were initially misdiagnosed as chronic bone infection since bacteria were isolated in wound swabs, including Staphylococcus aureus (n = 3), Escherichia coli (n = 1) and Staphylococcus epidermidis (n = 1). Treatment consisted of major amputation in 4 patients and radical surgical excision in 2 patients refusing amputation. 4 patients were lost to follow-up due to return to their country of origin, the remaining 2 patients were without signs of tumor recurrence (both after major amputation). Conclusion: Malignant transformation of is a rare, but serious complication of long-standing discharging chronic osteomyelitis. All 6 patients were diagnosed after >10 years of persistent or recurrent wounds. It should be particularly suspected in case of a pathological fracture, development of exophytic mass and changes in local pattern of the ulcers itself. Multiple biopsies, including deep soft tissues and bone, are recommended to distinguish between chronic osteomyelitis, pseudoepitheliomatous hyperplasia and highly-differentiated squamous cell carcinoma. Early diagnosis and a team approach (orthopaedic and plastic surgeon) are crucial for optimal management of the patient