Papers by Cristian Cortez
International Journal of Molecular Sciences
The topography and composition of dental implant surfaces directly impact mesenchymal cell adhesi... more The topography and composition of dental implant surfaces directly impact mesenchymal cell adhesion, proliferation, and differentiation, crucial aspects of achieving osseointegration. However, cell adhesion to biomaterials is considered a key step that drives cell proliferation and differentiation. The aim of this study was to characterize characterize the topography and composition of commercial titanium dental implants manufactured with different surface treatments (two sandblasted/acid-etched (SLA) (INNO Implants, Busan, Republic of Korea; BioHorizonsTM, Oceanside, CA, USA) and two calcium phosphate (CaP) treated (Biounite®, Berazategui, Argentina; Zimmer Biomet, Inc., Warsaw, IN, USA)) and to investigate their influence on the process of cell adhesion in vitro. A smooth surface implant (Zimmer Biomet, Inc.) was used as a control. For that, high-resolution methodologies such as scanning electron microscopy (SEM), X-ray dispersive spectroscopy (EDX), laser scanning confocal micros...
International Journal of Molecular Sciences, Feb 25, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
PLOS ONE, Jul 31, 2012
Host cell invasion and dissemination within the host are hallmarks of virulence for many pathogen... more Host cell invasion and dissemination within the host are hallmarks of virulence for many pathogenic microorganisms. As concerns Trypanosoma cruzi, which causes Chagas disease, the insect vector-derived metacyclic trypomastigotes (MT) initiate infection by invading host cells, and later blood trypomastigotes disseminate to diverse organs and tissues. Studies with MT generated in vitro and tissue culture-derived trypomastigotes (TCT), as counterparts of insect-borne and bloodstream parasites, have implicated members of the gp85/trans-sialidase superfamily, MT gp82 and TCT Tc85-11, in cell invasion and interaction with host factors. Here we analyzed the gp82 structure/function characteristics and compared them with those previously reported for Tc85-11. One of the gp82 sequences identified as a cell binding site consisted of an a-helix, which connects the N-terminal b-propeller domain to the C-terminal b-sandwich domain where the second binding site is nested. In the gp82 structure model, both sites were exposed at the surface. Unlike gp82, the Tc85-11 cell adhesion sites are located in the N-terminal b-propeller region. The gp82 sequence corresponding to the epitope for a monoclonal antibody that inhibits MT entry into target cells was exposed on the surface, upstream and contiguous to the a-helix. Located downstream and close to the a-helix was the gp82 gastric mucin binding site, which plays a central role in oral T. cruzi infection. The sequences equivalent to Tc85-11 laminin-binding sites, which have been associated with the parasite ability to overcome extracellular matrices and basal laminae, was poorly conserved in gp82, compatible with its reduced capacity to bind laminin. Our study indicates that gp82 is structurally suited for MT to initiate infection by the oral route, whereas Tc85-11, with its affinity for laminin, would facilitate the parasite dissemination through diverse organs and tissues.
PLOS ONE, Dec 31, 2013
Background: To invade target cells, Trypanosoma cruzi metacyclic forms engage distinct sets of su... more Background: To invade target cells, Trypanosoma cruzi metacyclic forms engage distinct sets of surface and secreted molecules that interact with host components. Serine-, alanine-, and proline-rich proteins (SAP) comprise a multigene family constituted of molecules with a high serine, alanine and proline residue content. SAP proteins have a central domain (SAP-CD) responsible for interaction with and invasion of mammalian cells by metacyclic forms. Methods and Findings: Using a 513 bp sequence from SAP-CD in blastn analysis, we identified 39 full-length SAP genes in the genome of T. cruzi. Although most of these genes were mapped in the T. cruzi in silico chromosome TcChr41, several SAP sequences were spread out across the genome. The level of SAP transcripts was twice as high in metacyclic forms as in epimastigotes. Monoclonal (MAb-SAP) and polyclonal (anti-SAP) antibodies produced against the recombinant protein SAP-CD were used to investigate the expression and localization of SAP proteins. MAb-SAP reacted with a 55 kDa SAP protein released by epimastigotes and metacyclic forms and with distinct sets of SAP variants expressed in amastigotes and tissue culture-derived trypomastigotes (TCTs). Anti-SAP antibodies reacted with components located in the anterior region of epimastigotes and between the nucleus and the kinetoplast in metacyclic trypomastigotes. In contrast, anti-SAP recognized surface components of amastigotes and TCTs, suggesting that SAP proteins are directed to different cellular compartments. Ten SAP peptides were identified by mass spectrometry in vesicle and soluble-protein fractions obtained from parasite conditioned medium. Using overlapping sequences from SAP-CD, we identified a 54-aa peptide (SAP-CE) that was able to induce host-cell lysosome exocytosis and inhibit parasite internalization by 52%. Conclusions: This study provides novel information about the genomic organization, expression and cellular localization of SAP proteins and proposes a triggering role for extracellular SAP proteins in host-cell lysosome exocytosis during metacyclic internalization.
Frontiers in Microbiology
Porphyromonas gingivalis is an oral human pathogen associated with the onset and progression of p... more Porphyromonas gingivalis is an oral human pathogen associated with the onset and progression of periodontitis, a chronic immune-inflammatory disease characterized by the destruction of the teeth-supporting tissue. P. gingivalis belongs to the genus Porphyromonas, which is characterized by being composed of Gram-negative, asaccharolytic, non-spore-forming, non-motile, obligatory anaerobic species, inhabiting niches such as the oral cavity, urogenital tract, gastrointestinal tract and infected wound from different mammals including humans. Among the Porphyromonas genus, P. gingivalis stands out for its specificity in colonizing the human oral cavity and its keystone pathogen role in periodontitis pathogenesis. To understand the evolutionary process behind P. gingivalis in the context of the Pophyoromonas genus, in this study, we performed a comparative genomics study with publicly available Porphyromonas genomes, focused on four main objectives: (A) to confirm the phylogenetic positio...
International Journal of Molecular Sciences
Periodontitis is a non-communicable chronic inflammatory disease characterized by the progressive... more Periodontitis is a non-communicable chronic inflammatory disease characterized by the progressive and irreversible breakdown of the soft periodontal tissues and resorption of teeth-supporting alveolar bone. The etiology of periodontitis involves dysbiotic shifts in the diversity of microbial communities inhabiting the subgingival crevice, which is dominated by anaerobic Gram-negative bacteria, including Porphyromonas gingivalis. Indeed, P. gingivalis is a keystone pathogen with a repertoire of attributes that allow it to colonize periodontal tissues and influence the metabolism, growth rate, and virulence of other periodontal bacteria. The pathogenic potential of P. gingivalis has been traditionally analyzed using classical biochemical and molecular approaches. However, the arrival of new techniques, such as whole-genome sequencing, metagenomics, metatranscriptomics, proteomics, and metabolomics, allowed the generation of high-throughput data, offering a suitable option for bacteria...
Clinical Oral Investigations
OBJECTIVES During periodontitis, chronic inflammation triggers soft tissue breakdown, and hyaluro... more OBJECTIVES During periodontitis, chronic inflammation triggers soft tissue breakdown, and hyaluronan is degraded into fragments of low molecular weight (LMW-HA). This investigation aimed to elucidate whether LMW-HA fragments with immunogenic potential on T lymphocytes remain in periodontal tissues after periodontal treatment. MATERIALS AND METHODS GCF samples were obtained from 15 periodontitis-affected patients and the LMW-HA, RANKL, and OPG levels were analyzed before and after 6 months of periodontal treatment by ELISA. Eight healthy individuals were analyzed as controls. Besides, human T lymphocytes were purified, exposed to infected dendritic cells, and pulsed with LMW-HA. Non-treated T lymphocytes were used as control. The expression levels of the transcription factors and cytokines that determine the Th1, Th17, and Th22 lymphocyte differentiation and function were analyzed by RT-qPCR. Similarly, the expression levels of RANKL and CD44 were analyzed. RESULTS In the GCF samples of periodontitis-affected patients, higher levels of LMW-HA were detected when compared with those of healthy individuals (52.1 ± 15.4 vs. 21.4 ± 12.2, p < 0.001), and these increased levels did not decrease after periodontal therapy (52.1 ± 15.4 vs. 45.7 ± 15.9, p = 0.158). Similarly, the RANKL levels and RANKL/OPG ratios did not change after periodontal therapy. Furthermore, in human T lymphocytes, LMW-HA induced higher expression levels of the Th1, Th17, and Th22-related transcription factors and cytokines, as well as CD44 and RANKL, as compared with non-treated cells. CONCLUSIONS In some patients, increased levels of LMW-HA persist in periodontal tissues after conventional periodontal therapy, and these remaining LMW-HA fragments with immunostimulatory potential could induce the polarization of a pathologic Th1/Th17/Th22-pattern of immune response on T lymphocytes. CLINICAL RELEVANCE The persistence of increased levels of LMW-HA in periodontal tissues after periodontal therapy could favor the recurrence of the disease and further breakdown of periodontal supporting tissues.
Frontiers in Cell and Developmental Biology, 2022
Niemann-Pick type A (NPA) disease is a fatal lysosomal neurodegenerative disorder caused by the d... more Niemann-Pick type A (NPA) disease is a fatal lysosomal neurodegenerative disorder caused by the deficiency in acid sphingomyelinase (ASM) activity. NPA patients present severe and progressive neurodegeneration starting at an early age. Currently, there is no effective treatment for this disease and NPA patients die between 2 and 3 years of age. NPA is characterized by an accumulation of sphingomyelin in lysosomes and dysfunction in the autophagy-lysosomal pathway. Recent studies show that c-Abl tyrosine kinase activity downregulates autophagy and the lysosomal pathway. Interestingly, this kinase is also activated in other lysosomal neurodegenerative disorders. Here, we describe that c-Abl activation contributes to the mechanisms of neuronal damage and death in NPA disease. Our data demonstrate that: 1) c-Abl is activated in-vitro as well as in-vivo NPA models; 2) imatinib, a clinical c-Abl inhibitor, reduces autophagy-lysosomal pathway alterations, restores autophagy flux, and lower...
International Journal of Molecular Sciences, 2022
Senescent cells express a senescence-associated secretory phenotype (SASP) with a pro-inflammator... more Senescent cells express a senescence-associated secretory phenotype (SASP) with a pro-inflammatory bias, which contributes to the chronicity of inflammation. During chronic inflammatory diseases, infiltrating CD4+ T lymphocytes can undergo cellular senescence and arrest the surface expression of CD28, have a response biased towards T-helper type-17 (Th17) of immunity, and show a remarkable ability to induce osteoclastogenesis. As a cellular counterpart, T regulatory lymphocytes (Tregs) can also undergo cellular senescence, and CD28− Tregs are able to express an SASP secretome, thus severely altering their immunosuppressive capacities. During periodontitis, the persistent microbial challenge and chronic inflammation favor the induction of cellular senescence. Therefore, senescence of Th17 and Treg lymphocytes could contribute to Th17/Treg imbalance and favor the tooth-supporting alveolar bone loss characteristic of the disease. In the present review, we describe the concept of cellul...
Journal of Periodontology, 2020
This is the author manuscript accepted for publication and has undergone full peer review but has... more This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as
Molecular Genetics and Metabolism, 2020
Oral Diseases, 2019
The serotype b of Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) induces higher... more The serotype b of Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) induces higher cytokine production in dendritic cells (DCs) compared with the other serotypes. However, this increased immuno-stimulatory potential was modified when DCs were co-infected with the other A. actinomycetemcomitans serotypes. This study aimed to analyze whether the production of interferon-gamma (IFN-γ), C-reactive protein (CRP), matrix metalloproteinase (MMP)-2, and MMP-9, as well as the activity of osteoclasts, also vary when DCs are co-infected with the A. actinomycetemcomitans serotypes. Materials and Methods: Human DCs were stimulated with the A. actinomycetemcomitans serotypes using the following stimulatory conditions: serotype a/b/c/a+b/a+c/b+c/a+b+c. The IFN-γ, CRP, and MMP-2 levels were quantified by ELISA. The active form of MMP-9 was quantified using fluorescent functional assays. The MMP-2 gelatinolytic activity was identified by zymogram. The osteoclast activity was determined by quantifying the TRAP expression and resorption-pit formation using cytochemistry and osteo-assays. Results: Higher levels of IFN-γ, CRP, MMP-2, MMP-9, and osteoclast activity were detected when DCs were stimulated with the serotype b of A. actinomycetemcomitans compared with the others. This increased immuno-stimulatory potential attributed to serotype b diminished when DCs were co-infected with the serotype a. Conclusions: This study provides new insights into the virulence of A. actinomycetemcomitans and reveals important differences in the immuno-stimulatory and pro-destructive potential among its serotypes.
PLOS Pathogens, 2019
V-ATPases are part of the membrane components of pathogen-containing vacuoles, although their fun... more V-ATPases are part of the membrane components of pathogen-containing vacuoles, although their function in intracellular infection remains elusive. In addition to organelle acidification, V-ATPases are alternatively implicated in membrane fusion and anti-inflammatory functions controlled by ATP6V 0 d2, the d subunit variant of the V-ATPase complex. Therefore, we evaluated the role of ATP6V 0 d2 in the biogenesis of pathogen-containing vacuoles using ATP6V 0 d2 knock-down macrophages infected with the protozoan parasite Leishmania amazonensis. These parasites survive within IFNγ/LPS-activated inflammatory macrophages, multiplying in large/fusogenic parasitophorous vacuoles (PVs) and inducing ATP6V 0 d2 upregulation. ATP6V 0 d2 knock-down decreased macrophage cholesterol levels and inhibited PV enlargement without interfering with parasite multiplication. However, parasites required ATP6V 0 d2 to resist the influx of oxidized low-density lipoprotein (ox-LDL)derived cholesterol, which restored PV enlargement in ATP6V 0 d2 knock-down macrophages by replenishing macrophage cholesterol pools. Thus, we reveal parasite-mediated subversion of host V-ATPase function toward cholesterol retention, which is required for establishing an inflammation-resistant intracellular parasite niche.
Scientific Reports, 2019
Trypanosoma cruzi, the causative agent of Chagas disease, has a dense coat of GPI-anchored virule... more Trypanosoma cruzi, the causative agent of Chagas disease, has a dense coat of GPI-anchored virulence factors.T.cruziGPI-anchored adhesin GP82 is encoded by a repertoire of transcripts containing several in-frame initiation codons located up-stream from that adjacent to the predicted signal peptide (SP). Transfection ofT.cruziepimastigotes with constructs encoding GP82 starting at the SP or from the farthest up-stream methionine confirmed protein expression on the parasite cell surface, comparable to the native GP82. Proteins were fully functional, inducing parasite adhesion to HeLa cells and lysosome mobilization, events required for parasite invasion. Transgenic and native GP82 proteins showed indistinguishable electrophoretic mobility, suggesting similar processing of the SP. Deletion of SP generated a ~72 kDa protein devoid ofN-linked oligosaccharides allowing irrefutable identification of GP82 precursor. SP transposition to an internal region of GP82 rendered the signal unrecogn...
Molecular Neurodegeneration, 2019
Background: Amyotrophic lateral sclerosis (ALS) is a multifactorial fatal motoneuron disease with... more Background: Amyotrophic lateral sclerosis (ALS) is a multifactorial fatal motoneuron disease without a cure. Ten percent of ALS cases can be pointed to a clear genetic cause, while the remaining 90% is classified as sporadic. Our study was aimed to uncover new connections within the ALS network through a bioinformatic approach, by which we identified C13orf18, recently named Pacer, as a new component of the autophagic machinery and potentially involved in ALS pathogenesis. Methods: Initially, we identified Pacer using a network-based bioinformatic analysis. Expression of Pacer was then investigated in vivo using spinal cord tissue from two ALS mouse models (SOD1 G93A and TDP43 A315T) and sporadic ALS patients. Mechanistic studies were performed in cell culture using the mouse motoneuron cell line NSC34. Loss of function of Pacer was achieved by knockdown using short-hairpin constructs. The effect of Pacer repression was investigated in the context of autophagy, SOD1 aggregation, and neuronal death. Results: Using an unbiased network-based approach, we integrated all available ALS data to identify new functional interactions involved in ALS pathogenesis. We found that Pacer associates to an ALS-specific subnetwork composed of components of the autophagy pathway, one of the main cellular processes affected in the disease. Interestingly, we found that Pacer levels are significantly reduced in spinal cord tissue from sporadic ALS patients and in tissues from two ALS mouse models. In vitro, Pacer deficiency lead to impaired autophagy and accumulation of ALS-associated protein aggregates, which correlated with the induction of cell death. Conclusions: This study, therefore, identifies Pacer as a new regulator of proteostasis associated with ALS pathology.
International Journal of Molecular Sciences, 2018
Niemann–Pick type C (NPC) disease is a rare neurovisceral cholesterol storage disorder that arise... more Niemann–Pick type C (NPC) disease is a rare neurovisceral cholesterol storage disorder that arises from loss of function mutations in the NPC1 or NPC2 genes. Soon after birth, some patients present with an aggressive hepatosplenomegaly and cholestatic signs. Histopathologically, the liver presents with large numbers of foam cells; however, their role in disease pathogenesis has not been explored in depth. Here, we studied the consequences of gadolinium chloride (GdCl3) treatment, a well-known Kupffer/foam cell inhibitor, at late stages of NPC liver disease and compared it with NPC1 genetic rescue in hepatocytes in vivo. GdCl3 treatment successfully blocked the endocytic capacity of hepatic Kupffer/foam measured by India ink endocytosis, decreased the levels CD68—A marker of Kupffer cells in the liver—and normalized the transaminase levels in serum of NPC mice to a similar extent to those obtained by genetic Npc1 rescue of liver cells. Gadolinium salts are widely used as magnetic res...
PLoS neglected tropical diseases, 2016
The question whether metacylic trypomastigote (MT) forms of different T. cruzi strains differenti... more The question whether metacylic trypomastigote (MT) forms of different T. cruzi strains differentially release surface molecules, and how they affect host cell invasion, remains to be fully clarified. We addressed that question using T. cruzi strains that differ widely in the ability to invade cells. Metacyclic forms were incubated at 37°C for 1 h in complete D10 medium or in nutrient-deprived PBS containing Ca2+ and Mg2+ (PBS++). The conditioned medium (CM), collected after parasite centrifugation, was used for cell invasion assays and Western blot analysis, using monoclonal antibodies directed to gp82 and gp90, the MT surface molecules that promote and negatively regulate invasion, respectively. CM of poorly invasive G strain (G-CM) contained high amounts of gp90 and gp82, either in vesicles or as soluble molecules. CM of highly invasive CL strain (CL-CM) contained gp90 and gp82 at very low levels. HeLa cells were incubated for 1 h with CL strain MT in D10, in absence or in the pre...
Parasites & vectors, 2016
Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last t... more Outbreaks of acute Chagas disease by oral infection have been reported frequently over the last ten years, with higher incidence in northern South America, where Trypanosoma cruzi lineage TcI predominates, being responsible for the major cause of resurgent human disease, and a small percentage is identified as TcIV. Mechanisms of oral infection and host-cell invasion by these parasites are poorly understood. To address that question, we analyzed T. cruzi strains isolated from chagasic patients in Venezuela, Guatemala and Brazil. Trypanosoma cruzi metacyclic trypomastigotes were orally inoculated into mice. The mouse stomach collected four days later, as well as the stomach and the heart collected 30 days post-infection, were processed for histological analysis. Assays to mimic parasite migration through the gastric mucus layer were performed by counting the parasites that traversed gastric mucin-coated transwell filters. For cell invasion assays, human epithelial HeLa cells were inc...
Scientific Reports, 2016
Mevalonate kinase (MVK) is an essential enzyme acting in early steps of sterol isoprenoids biosyn... more Mevalonate kinase (MVK) is an essential enzyme acting in early steps of sterol isoprenoids biosynthesis, such as cholesterol in humans or ergosterol in trypanosomatids. MVK is conserved from bacteria to mammals and localizes to glycosomes in trypanosomatids. During the course of T. cruzi MVK characterization, we found that, in addition to glycosomes, this enzyme may be secreted and modulate cell invasion. To evaluate the role of TcMVK in parasite-host cell interactions, TcMVK recombinant protein was produced and anti-TcMVK antibodies were raised in mice. TcMVK protein was detected in the supernatant of cultures of metacyclic trypomastigotes (MTs) and extracellular amastigotes (EAs) by Western blot analysis, confirming its secretion into extracellular medium. Recombinant TcMVK bound in a non-saturable dose-dependent manner to HeLa cells and positively modulated internalization of T. cruzi EAs but inhibited invasion by MTs. In HeLa cells, TcMVK induced phosphorylation of MAPK pathway ...
Cellular Microbiology, 2015
A fundamental question to be clarified concerning the host cell invasion by Trypanosoma cruzi is ... more A fundamental question to be clarified concerning the host cell invasion by Trypanosoma cruzi is whether the insect-borne and mammalian-stage parasites use similar mechanisms for invasion. To address that question, we analysed the cell invasion capacity of metacyclic trypomastigotes (MT) and tissue culture trypomastigotes (TCT) under diverse conditions. Incubation of parasites for 1 h with HeLa cells in nutrient-deprived medium, a condition that triggered lysosome biogenesis and scattering, increased MT invasion and reduced TCT entry into cells. Sucrose-induced lysosome biogenesis increased HeLa cell susceptibility to MT and resistance to TCT. Treatment of cells with rapamycin, which inhibits mammalian target of rapamycin (mTOR), induced perinuclear lysosome accumulation and reduced MT invasion while augmenting TCT invasion. Metacylic trypomastigotes, but not TCT, induced mTOR dephosphorylation and the nuclear translocation of transcription factor EB (TFEB), a mTOR-associated lysosome biogenesis regulator. Lysosome biogenesis/scattering was stimulated upon HeLa cell interaction with MT but not with TCT. Recently, internalized MT, but not TCT, were surrounded by colocalized lysosome marker LAMP2 and mTOR. The recombinant gp82 protein, the MT-specific surface molecule that mediates invasion, induced mTOR dephosphorylation, nuclear TFEB translocation and lysosome biogenesis/ scattering. Taken together, our data clearly indicate that MT invasion is mainly lysosome-dependent, whereas TCT entry is predominantly lysosomeindependent.
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Papers by Cristian Cortez