Further in vivo evidence implying DNA apurinic/apyrimidinic endonuclease activity in Trypanosoma cruzi oxidative stress survival.

Trypanosoma cruzi is under the attack of reactive species produced by its mammalian and insect hosts. To survive, it must repair its damaged DNA. We have shown that a base excision DNA repair (BER)-specific parasite TcAP1 endonuclease is involved in the resistance to H2 O2 . However, a putative TcAP1 negative dominant form impairing TcAP1 activity in vitro did not show any in vivo effect. Here, we show that a negative dominant form of the human APE1 apurinic/apyrimidinic (AP) endonuclease (hAPE1DN) induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to H2 O2 . Those results confirm that TcAP1 AP endonuclease activity plays an important role in epimastigote and in infective metacyclic trypomastigote oxidative DNA damage resistance leading to parasite persistence in the insect and mammalian hosts. All along its biological cycle and in its different cellular forms, T. cruzi, the etiological parasite agent of Chagas' disease, is under the attack of reactive species produced by its mammalian and insect hosts. To survive, T. cruzi must repair their oxidative damaged DNA. We have previously shown that a specific parasite TcAP1 AP endonuclease of the BER is involved in the T. cruzi resistance to oxidative DNA damage. We have also demonstrated that epimastigotes and cell-derived trypomastigotes parasite forms expressing a putative TcAP1 negative dominant form (that impairs the TcAP1 activity in vitro), did not show any in vivo effect in parasite viability when exposed to oxidative stress. In this work, we show the expression of a negative dominant form of the human APE1 AP endonuclease fused to a green fluorescent protein (GFP; hAPE1DN-GFP) in T. cruzi epimastigotes. The fusion protein is found both in the nucleus and cytoplasm of noninfective epimastigotes but only in the nucleus in metacyclic and cell-derived trypomastigote infective forms. Contrarily to the TcAP1 negative dominant form, the ectopic expression of hAPE1DN-GFP induces a decrease in epimastigote and metacyclic trypomastigote viability when parasites were exposed to increasing H2 O2 concentrations. No such effect was evident in expressing hAPE1DN-GFP cell-derived trypomastigotes. Although the viability of both wild-type infective trypomastigote forms diminishes when parasites are submitted to acute oxidative stress, the metacyclic forms are more resistant to H2 O2 exposure than cell-derived trypomastigotes.Those results confirm that the BER pathway and particularly the AP endonuclease activity play an important role in epimastigote and metacyclic trypomastigote oxidative DNA damage resistance leading to parasite survival and persistence inside the mammalian and insect host cells.

A Flap Endonuclease (TcFEN1) Is Involved in Trypanosoma cruzi Cell Proliferation, DNA Repair, and Parasite Survival.

FLAP endonucleases (FEN) are involved both in DNA replication and repair by processing DNA intermediaries presenting a nucleotide flap using its phosphodiesterase activity. In spite of these important functions in DNA metabolism, this enzyme was not yet studied in Trypanosomatids. Trypanosoma cruzi, the ethiological agent of Chagas disease, presents two dividing cellular forms (epimastigote and amastigote) and one non-proliferative, infective form (trypomastigote). The parasite survives DNA damage produced by reactive species generated in its hosts. The activity of a T. cruzi FLAP endonuclease (TcFEN1) was determined in the three cellular forms of the parasite using a DNA substrate generated by annealing three different oligonucleotides to form a double-stranded DNA with a 5' flap in the middle. This activity showed optimal pH and temperature similar to other known FENs. The substrate cut by the flap endonuclease activity could be ligated by the parasite generating a repaired DNA product. A DNA flap endonuclease coding sequence found in the T. cruzi genome (TcFEN1) was cloned, inserted in parasite expression vectors and transfected to epimastigotes. The purified native recombinant protein showed DNA flap endonuclease activity. This endonuclease was found located in the parasite nucleus of transfected epimastigotes and its over-expression increased both parasite proliferation and survival to H2 O2 . The presence of a flap endonuclease activity in T. cruzi and its nuclear location are indicative of the participation of this enzyme in DNA processing of flap fragments during DNA replication and repair in this parasite of ancient evolutive origin. J. Cell. Biochem. 118: 1722-1732, 2017. © 2016 Wiley Periodicals, Inc.

Lewy Body-like Pathology and Loss of Dopaminergic Neurons in Midbrain Organoids Derived from Familial Parkinson's Disease Patient.

Progressive accumulation of α-Synuclein (αSyn) in Lewy bodies (LBs) and loss of dopaminergic (DA) neurons are the hallmark pathological features of Parkinson's disease (PD). Although currently available in vitro and in vivo models have provided crucial information about PD pathogenesis, the mechanistic link between the progressive accumulation of αSyn into LBs and the loss of DA neurons is still unclear. To address this, it is critical to model LB formation and DA neuron loss, the two key neuropathological aspects of PD, in a relevant in vitro system. In this study, we developed a human midbrain-like organoid (hMBO) model of PD. We demonstrated that hMBOs generated from induced pluripotent stem cells (hiPSCs), derived from a familial PD (fPD) patient carrying αSyn gene (SNCA) triplication accumulate pathological αSyn over time. These cytoplasmic inclusions spatially and morphologically resembled diverse stages of LB formation and were composed of key markers of LBs. Importantly, the progressive accumulation of pathological αSyn was paralleled by the loss of DA neurons and elevated apoptosis. The model developed in this study will complement the existing in vitro models of PD and will provide a unique platform to study the spatiotemporal events governing LB formation and their relation with neurodegeneration. Furthermore, this model will also be beneficial for in vitro screening and the development of therapeutic compounds.

Protocol for controlled cortical impact in human cerebral organoids to model traumatic brain injury.

Modeling traumatic brain injury (TBI) has been a challenge. Rodent and cellular models have provided relevant contributions despite their limitations. Here, we present a protocol for a TBI model based on the controlled cortical impact (CCI) performed on human cerebral organoids (COs), self-assembled 3D cultures that recapitulate features of the human brain. Here, we generate COs from iPSCs obtained from reprogrammed fibroblasts. For complete details on the use and execution of this protocol, please refer to Ramirez et al. (2021).

Modeling Traumatic Brain Injury in Human Cerebral Organoids.

Traumatic brain injury (TBI) is a head injury that disrupts the normal brain structure and function. TBI has been extensively studied using various in vitro and in vivo models. Most of the studies have been done with rodent models, which may respond differently to TBI than human nerve cells. Taking advantage of the recent development of cerebral organoids (COs) derived from human induced pluripotent stem cells (iPSCs), which resemble the architecture of specific human brain regions, here, we adapted the controlled cortical impact (CCI) model to induce TBI in human COs as a novel in vitro platform. To adapt the CCI procedure into COs, we have developed a phantom brain matrix, matching the mechanical characteristics of the brain, altogether with an empty mouse skull as a platform to allow the use of the stereotactic CCI equipment on COs. After the CCI procedure, COs were histologically prepared to evaluate neurons and astrocyte populations using the microtubule-associated protein 2 (MAP2) and the glial fibrillary acidic protein (GFAP). Moreover, a marker of metabolic response, the neuron-specific enolase (NSE), and cellular death using cleaved caspase 3 were also analyzed. Our results show that human COs recapitulate the primary pathological changes of TBI, including metabolic alterations related to neuronal damage, neuronal loss, and astrogliosis. This novel approach using human COs to model TBI in vitro holds great potential and opens new alternatives for understanding brain abnormalities produced by TBI, and for the development and testing of new therapeutic approaches.

Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase.

Trypanosoma cruzi, the etiological agent of Chagas' disease, presents three cellular forms (trypomastigotes, epimastigotes and amastigotes), all of which are submitted to oxidative species in its hosts. However, T. cruzi is able to resist oxidative stress suggesting a high efficiency of its DNA repair machinery.The Base Excision Repair (BER) pathway is one of the main DNA repair mechanisms in other eukaryotes and in T. cruzi as well. DNA glycosylases are enzymes involved in the recognition of oxidative DNA damage and in the removal of oxidized bases, constituting the first step of the BER pathway. Here, we describe the presence and activity of TcNTH1, a nuclear T. cruzi DNA glycosylase. Surprisingly, purified recombinant TcNTH1 does not remove the thymine glycol base, but catalyzes the cleavage of a probe showing an AP site. The same activity was found in epimastigote and trypomastigote homogenates suggesting that the BER pathway is not involved in thymine glycol DNA repair. TcNTH1 DNA-binding properties assayed in silico are in agreement with the absence of a thymine glycol removing function of that parasite enzyme. Over expression of TcNTH1 decrease parasite viability when transfected epimastigotes are submitted to a sustained production of H2O2.Therefore, TcNTH1 is the only known NTH1 orthologous unable to eliminate thymine glycol derivatives but that recognizes and cuts an AP site, most probably by a beta-elimination mechanism. We cannot discard that TcNTH1 presents DNA glycosylase activity on other DNA base lesions. Accordingly, a different DNA repair mechanism should be expected leading to eliminate thymine glycol from oxidized parasite DNA. Furthermore, TcNTH1 may play a role in the AP site recognition and processing.

Collective occupation as a means of overcoming Occupational Apartheid: the case of the struggle for the right to health of the Mama Cultiva Grouping / Ocupação coletiva como meio de superação do Apartheid Ocupacional: o caso da luta pelo direito à saúde da Associação Mama Cultiva / Ocupación colectiva como medio de superación del Apartheid Ocupacional: el caso de la lucha por el derecho a la salud de la Agrupación Mama Cultiva

Abstract The objective of this article is to turn the practices used by Occupação Coletiva da Mamá Cultiva visible to advocate to the right to access health, which would be limited by a context of occupational apartheid. A qualitative research was carried out, with a phenomenological approach, in which the information is produced by semi-structured interviews and discussion groups, with 6 groups participants. The information obtained is organized through a categorical matrix and analyzed through simple categories. The main conclusion is that the resistance practices are strongly rooted in a collective identity. This is related to the shared experiences of group individuals, especially those associated with the experience of psychosocial suffering caused by stigmatization, criminalization and prevalent medicalization.

Resumo O objetivo deste artigo é tornar visíveis as práticas utilizadas pela Ocupação Coletiva da Mama Cultiva para exercer o direito à saúde, o que seria limitado por um contexto de apartheid ocupacional. Neste estudo optou-se por pesquisa qualitativa, com abordagem fenomenológica, na qual a informação é produzida por meio de entrevistas semiestruturadas e grupos de discussão, com 6 participantes do grupo. A informação obtida é ordenada através de uma matriz categorial e é analisada através de categorias simples. A principal conclusão é que as práticas de resistência estão fortemente enraizadas na identidade coletiva. Isto está relacionado às experiências compartilhadas pelos sujeitos do grupo, especialmente aqueles associados à experiência de sofrimento psicossocial causado pela estigmatização, criminalização e a medicalização prevalente.

Resumén El objetivo de este artículo es hacer visibles las prácticas realizadas por la Ocupación Colectiva Mama Cultiva, para defender el derecho al acceso a la salud, que estaría limitado por un contexto de apartheid ocupacional. Se realizó una investigación cualitativa, con un enfoque fenomenológico, en la que la información se produce mediante entrevistas semiestructuradas y grupos de discusión, con 6 participantes. La información obtenida se organiza a través de una matriz categorial y se analiza a través de categorías simples. La conclusión principal es que las prácticas de resistencia están fuertemente arraigadas en una identidad colectiva. Esto se relaciona con las experiencias compartidas de los sujetos del grupo, especialmente aquellos asociados con la experiencia del malestar psicosocial causado por la estigmatización, la criminalización y la medicalización prevalente.

[Inflammatory bowel diseases: an immunological approach]. / Enfermedad inflamatoria intestinal: Una mirada inmunológica.

Inflammatory bowel diseases (IBD) are inflammatory diseases with a multifactorial component that involve the intestinal tract. The two relevant IBD syndromes are Crohn's disease (CD) and ulcerative colitis (UC). One factor involved in IBD development is a genetic predisposition, associated to NOD2/CARD15 and Toll-like receptor 4 (TLR4) polymorphisms that might favor infectious enterocolitis that is possibly associated to the development of IBD. The identification of specific immunologic alterations in IBD and their relationship to the etiology of the disease is a relevant research topic. The role of intra and extracellular molecules, such as transcription factors and cytokines that are involved in the inflammatory response, needs to be understood. The relevance of immunologic molecules that might drive the immune response to a T helper (Th) 1, Th 2 or the recently described Th 17 phenotype, has been demonstrated in animal models and clinical studies with IBD patients. CD and UC predominantly behave with a Th 1 and Th 2 immune phenotype, respectively. Recently, an association between CD and Th 17 has been reported. The knowledge acquired from immunologic and molecular research will help to develop accurate diagnostic methods and efficient therapies.

Inhibition of nuclear factor-kappa B enhances the capacity of immature dendritic cells to induce antigen-specific tolerance in experimental autoimmune encephalomyelitis.

Autoimmune disorders develop as a result of deregulated immune responses that target self-antigens and cause destruction of healthy host tissues. Because dendritic cells (DCs) play an important role in the maintenance of peripheral immune tolerance, we are interested in identifying means of enhancing their therapeutic potential in autoimmune diseases. It is thought that during steady state, DCs are able to anergize potentially harmful T cells bearing T cell receptors that recognize self-peptide-major histocompatibility complexes. The tolerogenic capacity of DCs requires an immature phenotype, which is characterized by a reduced expression of costimulatory molecules. On the contrary, activation of antigen-specific naive T cells is enhanced by DC maturation, a process that involves expression of genes controlled by the transcription factor nuclear factor (NF)-kappaB. We evaluated the capacity of drugs that inhibit NF-kappaB to enhance the tolerogenic properties of immature DCs in the experimental autoimmune encephalomyelitis (EAE) model. We show that andrographolide, a bicyclic diterpenoid lactone, and rosiglitazone, a peroxisome proliferator-activated receptor gamma agonist, were able to interfere with NF-kappaB activation in murine DCs. As a result, treated DCs showed impaired maturation and a reduced capacity to activate antigen-specific T cells. Furthermore, NF-kappaB-blocked DCs had an enhanced tolerogenic capacity and were able to prevent EAE development in mice. The tolerogenic feature was specific for myelin antigens and involved the expansion of regulatory T cells. These data suggest that NF-kappaB blockade is a potential pharmacological approach that can be used to enhance the tolerogenic ability of immature DCs to prevent detrimental autoimmune responses.

Andrographolide interferes with T cell activation and reduces experimental autoimmune encephalomyelitis in the mouse.

Andrographolide is a bicyclic diterpenoid lactone derived from extracts of Andrographis paniculata, a plant indigenous to South Asian countries that shows anti-inflammatory properties. The molecular and cellular bases for this immunomodulatory capacity remain unknown. Here, we show that andrographolide is able to down-modulate both humoral and cellular adaptive immune responses. In vitro, this molecule was able to interfere with T cell proliferation and cytokine release in response to allogenic stimulation. These results were consistent with the observation that T cell activation by dendritic cells (DCs) was completely abolished by exposing DCs to andrographolide during antigen pulse. This molecule was able to interfere with maturation of DCs and with their ability to present antigens to T cells. Furthermore, in vivo immune responses such as antibody response to a thymus-dependent antigen and delayed-type hypersensitivity were drastically diminished in mice by andrographolide treatment. Finally, the ability of andrographolide to inhibit T cell activation was applied to interfere with the onset of experimental autoimmune encephalomyelitis (EAE), an inflammatory demyelinating disease of the central nervous system that is primarily mediated by CD4(+) T cells and serves as an animal model for human multiple sclerosis. Treatment with andrographolide was able to significantly reduce EAE symptoms in mice by inhibiting T cell and antibody responses directed to myelin antigens. Our data suggest that andrographolide is able to efficiently block T cell activation in vitro, as well as in vivo, a feature that could be useful for interfering with detrimental T cell responses.

Enfermedad inflamatoria intestinal: Una mirada inmunológica: [revisión] / Inflammatory bowel diseases: An immunological approach: [review]

Inflammatory bowel diseases (IBD) are inflammatory diseases with a multifactorial component that involve the intestinal tract. The two relevant IBD syndromes are Crohn's disease (CD) and ulcerative colitis (UC). One factor involved in IBD development is a genetic predisposition, associated to NOD2/CARD15 and Toll-like receptor 4 (TLR4) polymorphisms that might favor infectious enterocolitis that is possibly associated to the development of IBD. The identification of specific immunologic alterations in IBD and their relationship to the etiology of the disease is a relevant research topic. The role of intra and extracellular molecules, such as transcription factors and cytokines that are involved in the inflammatory response, needs to be understood. The relevance of immunologic molecules that might drive the immune response to a T helper (Th) 1, Th 2 or the recently described Th 17 phenotype, has been demonstrated in animal models and clinical studies with IBD patients. CD and UC predominantly behave with a Th 1 and Th 2 immune phenotype, respectively. Recently, an association between CD and Th 17 has been reported. The knowledge acquired from immunologic and molecular research will help to develop accurate diagnostic methods and efficient therapies.