HGMB1 and RAGE as Essential Components of Ti Osseointegration Process in Mice.

The release of the prototypic DAMP High Mobility Group Box 1 (HMGB1) into extracellular environment and its binding to the Receptor for Advanced Glycation End Products (RAGE) has been described to trigger sterile inflammation and regulate healing outcome. However, their role on host response to Ti-based biomaterials and in the subsequent osseointegration remains unexplored. In this study, HMGB1 and RAGE inhibition in the Ti-mediated osseointegration were investigated in C57Bl/6 mice. C57Bl/6 mice received a Ti-device implantation (Ti-screw in the edentulous alveolar crest and a Ti-disc in the subcutaneous tissue) and were evaluated by microscopic (microCT [bone] and histology [bone and subcutaneous]) and molecular methods (ELISA, PCR array) during 3, 7, 14, and 21 days. Mice were divided into 4 groups: Control (no treatment); GZA (IP injection of Glycyrrhizic Acid for HMGB1 inhibition, 4 mg/Kg/day); RAP (IP injection of RAGE Antagonistic Peptide, 4 mg/Kg/day), and vehicle controls (1.5% DMSO solution for GZA and 0.9% saline solution for RAP); treatments were given at all experimental time points, starting 1 day before surgeries. HMGB1 was detected in the Ti-implantation sites, adsorbed to the screws/discs. In Control and vehicle groups, osseointegration was characterized by a slight inflammatory response at early time points, followed by a gradual bone apposition and matrix maturation at late time points. The inhibition of HMGB1 or RAGE impaired the osseointegration, affecting the dynamics of mineralized and organic bone matrix, and resulting in a foreign body reaction, with persistence of macrophages, necrotic bone, and foreign body giant cells until later time points. While Control samples were characterized by a balance between M1 and M2-type response in bone and subcutaneous sites of implantation, and also MSC markers, the inhibition of HMGB1 or RAGE caused a higher expression M1 markers and pro-inflammatory cytokines, as well chemokines and receptors for macrophage migration until later time points. In conclusion, HMGB1 and RAGE have a marked role in the osseointegration, evidenced by their influence on host inflammatory immune response, which includes macrophages migration and M1/M2 response, MSC markers expression, which collectively modulate bone matrix deposition and osseointegration outcome.

Overexpression of Trypanosoma cruzi High Mobility Group B protein (TcHMGB) alters the nuclear structure, impairs cytokinesis and reduces the parasite infectivity.

Kinetoplastid parasites, included Trypanosoma cruzi, the causal agent of Chagas disease, present a unique genome organization and gene expression. Although they control gene expression mainly post-transcriptionally, chromatin accessibility plays a fundamental role in transcription initiation control. We have previously shown that High Mobility Group B protein from Trypanosoma cruzi (TcHMGB) can bind DNA in vitro. Here, we show that TcHMGB also acts as an architectural protein in vivo, since the overexpression of this protein induces changes in the nuclear structure, mainly the reduction of the nucleolus and a decrease in the heterochromatin:euchromatin ratio. Epimastigote replication rate was markedly reduced presumably due to a delayed cell cycle progression with accumulation of parasites in G2/M phase and impaired cytokinesis. Some functions involved in pathogenesis were also altered in TcHMGB-overexpressing parasites, like the decreased efficiency of trypomastigotes to infect cells in vitro, the reduction of intracellular amastigotes replication and the number of released trypomastigotes. Taken together, our results suggest that the TcHMGB protein is a pleiotropic player that controls cell phenotype and it is involved in key cellular processes.

Molecular and functional characterization of single-box high-mobility group B (HMGB) chromosomal protein from Aedes aegypti.

High-mobility group B (HMGB) proteins have highly conserved, unique DNA-binding domains, HMG boxes, that can bind non-B-type DNA structures, such as bent, kinked and unwound structures, with high affinity. HMGB proteins also promote DNA bending, looping and unwinding. In this study, we determined the role of the Aedes aegypti single HMG-box domain protein AaHMGB; characterized its structure, spatiotemporal expression levels, subcellular localization, and nucleic acid binding activities; and compared these properties with those of its double-HMG-box counterpart protein, AaHMGB1. Via qRT-PCR, we showed that AaHMGB is expressed at much higher levels than AaHMGB1 throughout mosquito development. In situ hybridization results suggested a role for AaHMGB and AaHMGB1 during embryogenesis. Immunolocalization in the midgut revealed that AaHMGB is exclusively nuclear. Circular dichroism and fluorescence spectroscopy analyses showed that AaHMGB exhibits common features of α-helical structures and is more stably folded than AaHMGB1, likely due to the presence of one or two HMG boxes. Using several DNA substrates or single-stranded RNAs as probes, we observed significant differences between AaHMGB and AaHMGB1 in terms of their binding patterns, activity and/or specificity. Importantly, we showed that the phosphorylation of AaHMGB plays a critical role in its DNA-binding activity. Our study provides additional insight into the roles of single- versus double-HMG-box-containing proteins in nucleic acid interactions for better understanding of mosquito development, physiology and homeostasis.

Trypanosoma cruzi High Mobility Group B (TcHMGB) can act as an inflammatory mediator on mammalian cells.

BACKGROUND: High Mobility Group B (HMGB) proteins are nuclear architectural factors involved in chromatin remodeling and important nuclear events. HMGBs also play key roles outside the cell acting as alarmins or Damage-associated Molecular Patterns (DAMPs). In response to a danger signal these proteins act as immune mediators in the extracellular milieu. Moreover, these molecules play a central role in the pathogenesis of many autoimmune and both infectious and sterile inflammatory chronic diseases. PRINCIPAL FINDINGS: We have previously identified a High mobility group B protein from Trypanosoma cruzi (TcHMGB) and showed that it has architectural properties interacting with DNA like HMGBs from other eukaryotes. Here we show that TcHMGB can be translocated to the cytoplasm and secreted out of the parasite, a process that seems to be stimulated by acetylation. We report that recombinant TcHMGB is able to induce an inflammatory response in vitro and in vivo, evidenced by the production of Nitric Oxide and induction of inflammatory cytokines like TNF-α, IL-1ß and IFN-γ gene expression. Also, TGF-ß and IL-10, which are not inflammatory cytokines but do play key roles in Chagas disease, were induced by rTcHMGB. CONCLUSIONS: These preliminary results suggest that TcHMGB can act as an exogenous immune mediator that may be important for both the control of parasite replication as the pathogenesis of Chagas disease and can be envisioned as a pathogen associated molecular pattern (PAMP) partially overlapping in function with the host DAMPs.

The toxin biliatresone causes mouse extrahepatic cholangiocyte damage and fibrosis through decreased glutathione and SOX17.

UNLABELLED: Biliary atresia, the most common indication for pediatric liver transplantation, is a fibrotic disease of unknown etiology affecting the extrahepatic bile ducts of newborns. The recently described toxin biliatresone causes lumen obstruction in mouse cholangiocyte spheroids and represents a new model of biliary atresia. The goal of this study was to determine the cellular changes caused by biliatresone in mammalian cells that ultimately lead to biliary atresia and extrahepatic fibrosis. We treated mouse cholangiocytes in three-dimensional (3D) spheroid culture and neonatal extrahepatic duct explants with biliatresone and compounds that regulate glutathione (GSH). We examined the effects of biliatresone on SOX17 levels and determined the effects of Sox17 knockdown on cholangiocytes in 3D culture. We found that biliatresone caused disruption of cholangiocyte apical polarity and loss of monolayer integrity. Spheroids treated with biliatresone had increased permeability as shown by rhodamine efflux within 5 hours compared with untreated spheroids, which retained rhodamine for longer than 12 hours. Neonatal bile duct explants treated with the toxin showed lumen obstruction with increased subepithelial staining for α-smooth muscle actin and collagen, consistent with fibrosis. Biliatresone caused a rapid and transient decrease in GSH, which was both necessary and sufficient to mediate its effects in cholangiocyte spheroid and bile duct explant systems. It also caused a significant decrease in cholangiocyte levels of SOX17, and Sox17 knockdown in cholangiocyte spheroids mimicked the effects of biliatresone. CONCLUSION: Biliatresone decreases GSH and SOX17 in mouse cholangiocytes. In 3D cell systems, this leads to cholangiocyte monolayer damage and increased permeability; in extrahepatic bile duct explants, it leads to disruption of the extrahepatic biliary tree and subepithelial fibrosis. This mechanism may be important in understanding human biliary atresia. (Hepatology 2016;64:880-893).

The distinct C-terminal acidic domains of HMGB proteins are functionally relevant in Schistosoma mansoni.

The Schistosoma mansoni High Mobility Group Box (HMGB) proteins SmHMGB1, SmHMGB2 and SmHMGB3 share highly conserved HMG box DNA binding domains but have significantly different C-terminal acidic tails. Here, we used three full-length and tailless forms of the S. mansoni HMGB proteins to examine the functional roles of their acidic tails. DNA binding assays revealed that the different lengths of the acidic tails among the three SmHMGB proteins significantly and distinctively influenced their DNA transactions. Spectroscopic analyses indicated that the longest acidic tail of SmHMGB3 contributes to the structural stabilisation of this protein. Using immunohistochemical analysis, we showed distinct patterns of SmHMGB1, SmHMGB2 and SmHMGB3 expression in different tissues of adult worms. RNA interference approaches indicated a role for SmHMGB2 and SmHMGB3 in the reproductive system of female worms, whereas for SmHMGB1 no clear phenotype was observed. Schistosome HMGB proteins can be phosphorylated, acetylated and methylated. Importantly, the acetylation and methylation of schistosome HMGBs were greatly enhanced upon removal of the acidic tail. These data support the notion that the C-terminal acidic tails dictate the differences in the structure, expression and function of schistosome HMGB proteins.

Nucleosome remodeling by the SWI/SNF complex is enhanced by yeast high mobility group box (HMGB) proteins.

The regulation of gene expression at the level of transcription involves the concerted action of several proteins and protein complexes committed to dynamically alter the surrounding chromatin environment of a gene being activated or repressed. ATP-dependent chromatin remodeling complexes are key factors in chromatin remodeling, and the SWI/SNF complex is the founding member. While many studies have linked the action of these complexes to specific transcriptional regulation of a large number of genes and much is known about their catalytic activity, less is known about the nuclear elements that can enhance or modulate their activity. A number of studies have found that certain High Mobility Group (HMG) proteins are able to stimulate ATP-dependent chromatin remodeling activity, but their influence on the different biochemical outcomes of this activity is still unknown. In this work we studied the influence of the yeast Nhp6A, Nhp6B and Hmo1 proteins (HMGB family members) on different biochemical outcomes of yeast SWI/SNF remodeling activity. We found that all these HMG proteins stimulate the sliding activity of ySWI/SNF, while transient exposure of nucleosomal DNA and octamer transfer catalyzed by this complex are only stimulated by Hmo1. Consistently, only Hmo1 stimulates SWI/SNF binding to the nucleosome. Additionally, the sliding activity of another chromatin remodeling complex, ISW1a, is only stimulated by Hmo1. Further analyses show that these differential stimulatory effects of Hmo1 are dependent on the presence of its C-terminal tail, which contains a stretch of acidic and basic residues.

Sexual variability in Histoplasma capsulatum and its possible distribution: what is going on?

Histoplasma capsulatum is a dimorphic fungal pathogen naturally found in the soil. Inhalation of conidia can result in pulmonary histoplasmosis and, in some cases, causes severe disseminated disease and death. This fungus is an ascomycete that has an anamorphic or asexual stage and a teleomorphic or sexual stage, known as Ajellomyces capsulatus, which results from (+) and (-) mating types. Sexual reproduction is regulated by a specialized genomic region known as the mating-type (MAT1) locus. The mating process in this heterothallic species is represented by isolates that contain only one of the two different MAT1 locus idiomorphs (MAT1-1 or MAT1-2) that have unrelated sequences encoding different transcription factors. In medically important dimorphic pathogens and in most ascomycete molds, one MAT locus idiomorph encodes a high-mobility-group (HMG) box-domain transcription factor, and the other idiomorph encodes an alpha-box domain transcription factor. There is scarce molecular information about H. capsulatum mating type although recombinant population structures have been reported that could occur in nature and this process has been documented in distinct models such as parasites and other fungi. In this review, we shall focus on published studies on H. capsulatum sexuality, and outline the distribution of the two H. capsulatum mating types in Latin America. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012).

Characterization of nCD64 expression in neutrophils and levels of s-TREM-1 and HMGB-1 in patients with suspected infection admitted in an emergency department / Caracterización de la expresión de nCD64 en neutrófilos y de los niveles de s-TREM-1 y HMGB-1 en pacientes con sospecha de infección admitidos en el departamento de emergencias

Introduction: The nCD64 receptor, the soluble triggering receptor expressed in myeloid cells (s-TREM-1), and the high mobility group-box 1 protein (HMGB-1) have been proposed as significant mediators in sepsis. Objective: To evaluate the prognostic value of these markers in patients with suspected infection recently admitted in an emergency department (ED). Materials and methods: All patients who presented to the ED with suspected infection were eligible for enrollment in this study. Baseline clinical data, Sequential Organ Failure Assessment score (SOFA) score, APACHE II score, HMGB-1 levels, s-TREM-1 levels, and nCD64 levels were analyzed. The HMGB-1 and sTREM-1 serum concentrations were determined using commercially available ELISA kits, and CD64 on the surface of neutrophils was measured by flow cytometry. Results:. A total of 579 patients with suspected infection as their admission diagnosis were enrolled in this study. The median patient age was 50 years (IQR = 35-68). Morbidity during the 28-day followup period was 11.1% (n=64). The most frequent diagnosis at the time of admission was communityacquired pneumonia (CAP) in 23% (n=133) patients, followed by soft tissue infection in 16.6% (n=96), and urinary tract infection in 15% (n=87). After multivariable analysis, no significant association was identified between any biomarker and 28-day mortality. Conclusion: In the context of a tertiary care hospital emergency department in a Latin-American city, the nCD64 receptor, s-TREM-1, and HMGB-1 biomarkers do not demonstrate prognostic utility in the management of patients with infection. The search continues for more reliable prognostic markers in the early stages of infection.

Introducción. El receptor CD64, receptor soluble ´desencadenador´ expresado en células mieloides (sTREM-1) y la proteína del grupo Box-1 de alta movilidad (HMGB-1), se han propuesto como mediadores en la sepsis. Objetivo. Evaluar el valor pronóstico de estos marcadores en pacientes con sospecha de infección, recientemente admitidos en un departamento de emergencias. Materiales y métodos. Se incluyeron en el estudio pacientes que consultaron al hospital con sospecha de infección. Se analizó la base de datos clínica, el puntaje SOFA, el puntaje APACHE II, los niveles de HMGB-1, los niveles de sTREM-1 y los niveles de nCD64. Se determinaron las concentraciones en suero de HMGB-1 y sTREM-1, usando kits de ELISA disponibles comercialmente, y la de CD64 se midió por citometría de flujo. Resultados. Se analizaron 579 pacientes con sospecha de infección al ingreso. La edad media fue de 50 años (rango intercuartílico=35-68), y 11,1 % (n=64) murieron durante el seguimiento de 28 días. El diagnóstico más frecuente en el momento del ingreso fue neumonía adquirida en la comunidad, en 23 % (n=133) de los pacientes, seguida de infección de tejidos blandos, en 16,6 % (n=96), e infección urinaria, en 15 % (n=87). Después de un análisis multivariado, no hubo asociación significativa entre ningún biomarcador y la mortalidad a los 28 días. Conclusión. Los resultados sugieren que en el contexto de un departamento de emergencias de tercer nivel de una ciudad latinoamericana típica, los tres marcadores evaluados no ofrecieron ninguna ventaja en el pronóstico de infección. La búsqueda de marcadores pronósticos más confiables en estadios tempranos de la infección aún continúa abierta.

Characterization of nCD64 expression in neutrophils and levels of s-TREM-1 and HMGB-1 in patients with suspected infection admitted in an emergency department.

INTRODUCTION: The nCD64 receptor, the soluble triggering receptor expressed in myeloid cells (s-TREM-1), and the high mobility group-box 1 protein (HMGB-1) have been proposed as significant mediators in sepsis. OBJECTIVE: To evaluate the prognostic value of these markers in patients with suspected infection recently admitted in an emergency department (ED). MATERIALS AND METHODS: All patients who presented to the ED with suspected infection were eligible for enrollment in this study. Baseline clinical data, Sequential Organ Failure Assessment score (SOFA) score, APACHE II score, HMGB-1 levels, s-TREM-1 levels, and nCD64 levels were analyzed. The HMGB-1 and sTREM-1 serum concentrations were determined using commercially available ELISA kits, and CD64 on the surface of neutrophils was measured by flow cytometry. RESULTS: . A total of 579 patients with suspected infection as their admission diagnosis were enrolled in this study. The median patient age was 50 years (IQR = 35-68). Morbidity during the 28-day followup period was 11.1% (n=64). The most frequent diagnosis at the time of admission was communityacquired pneumonia (CAP) in 23% (n=133) patients, followed by soft tissue infection in 16.6% (n=96), and urinary tract infection in 15% (n=87). After multivariable analysis, no significant association was identified between any biomarker and 28-day mortality. CONCLUSION: In the context of a tertiary care hospital emergency department in a Latin-American city, the nCD64 receptor, s-TREM-1, and HMGB-1 biomarkers do not demonstrate prognostic utility in the management of patients with infection. The search continues for more reliable prognostic markers in the early stages of infection.

Regeneration in zebrafish lateral line neuromasts: expression of the neural progenitor cell marker sox2 and proliferation-dependent and-independent mechanisms of hair cell renewal.

Mechanosensory hair cells are essential for audition in vertebrates, and in many species, have the capacity for regeneration when damaged. Regeneration is robust in the fish lateral line system as new hair cells can reappear after damage induced by waterborne aminoglycoside antibiotics, platinum-based drugs, and heavy metals. Here, we characterize the loss and reappearance of lateral line hair cells induced in zebrafish larvae treated with copper sulfate using diverse molecular markers. Transgenic fish that express green fluorescent protein in different cell types in the lateral line system have allowed us to follow the regeneration of hair cells after different damage protocols. We show that conditions that damage only differentiated hair cells lead to reappearance of new hair cells within 24 h from nondividing precursors, whereas harsher conditions are followed by a longer recovery period that is accompanied by extensive cell division. In order to characterize the cell population that gives rise to new hair cells, we describe the expression of a neural stem cell marker in neuromasts. The zebrafish sox2 gene is strongly expressed in neuromast progenitor cells, including those of the migrating lateral line primordium, the accessory cells that underlie the hair cells in neuromasts, and in interneuromastic cells that give rise to new neuromasts. Moreover, we find that most of the cells that proliferate within the neuromast during regeneration express this marker. Thus, our results describe the dynamics of hair cell regeneration in zebrafish and suggest the existence of at least two mechanisms for recovery of these cells in neuromasts.

Anophthalmia-esophageal atresia syndrome caused by an SOX2 gene deletion in monozygotic twin brothers with markedly discordant phenotypes.

The clinical combination of anophthalmia/microphthalmia and esophageal atresia was first recognized in 1988 as a distinct variable multi-system malformation syndrome and since then at least 17 cases of the disease have been described, all of them sporadic in occurrence. We report a heterozygous SOX2 gene mutation underlying the syndrome of anophthalmia/microphthalmia-esophageal atresia and demonstrate that this entity can be associated to considerable clinical variability as shown by the discordant ocular phenotype observed in monozygotic twin brothers carrying an SOX2 deletion. This is the first report describing a strikingly discordant eye phenotype in monozygotic twins with the condition, with one of our patients being the first reported individual carrying an SOX2 lesion associated with unilateral eye defect. We discuss the probable sources for this remarkable phenotypic heterogeneity of the anophthalmia/microphthalmia syndrome in individuals with an identical genetic constitution.

Identification of neural crest competence territory: role of Wnt signaling.

In recent years, research on neural crest induction has allowed the identification of several molecules as candidates for neural crest inducers. Although many of these molecules have the ability to induce neural crest in different assays, a general mechanism of neural crest induction that includes a description of the tissues that produce the inductive signals and the time and steps in which this process takes place remains elusive. To better understand the mechanism of neural crest induction, we developed an assay that has been used previously by Nieuwkoop to study anterior-posterior pattern of the neural plate. Folds of competent ectoderm were implanted in different positions of a young neurula embryo, and the induction of neural crest was analyzed using the expression of the neural crest marker Xslug. We identified a very localized region of the early neurula where it is possible to get neural crest induction, whereas all of the regions tested showed a clear induction of the neural plate marker Xsox2. These results indicate that there is a region in the embryo with the appropriate combination of signals needed to induce neural crest cells; we called this region the neural crest competence territory. In addition, our results show that neural crest induction is always accompanied by neural plate induction, but there are many cases where neural plate was induced without neural crest. These results support the model in which the neural crest is induced by an interaction between neural plate and epidermis, but they also suggest that additional signals are required. By making grafts of different sizes and implanting them in the epidermis or the neural plate, we concluded that one of the inductive signals is produced in the dorsal region of the embryo and travels into the ectoderm. Finally, by performing gain- and loss-of-function of Wnt signaling experiments, we show that this pathway plays an important role not only in neural crest induction but also in the specification of the neural crest competence territory. Developmental Dynamics 229:109-117, 2004.