Therapeutic potential of LINS01 histamine H3 receptor antagonists as antineoplastic agents for triple negative breast cancer.

The aims of this work were to evaluate the expression of histamine H3 receptor (H3R) in triple negative breast cancer (TNBC) samples and to investigate the antitumoral efficacy and safety of the LINS01 series of H3R antagonists, through in silico, in vitro, and in vivo approaches. Antitumor activity of LINS01009, LINS01010, LINS01022, LINS01023 was assayed in vitro in 4T1 and MDA-MB-231 TNBC cells (0.01-100 µM), and in vivo in 4T1 tumors orthotopically established in BALB/c mice (1 or 20 mg/kg). Additionally, H3R expression was assessed in 50 human TNBC samples. We have described a higher H3R mRNA expression in basal-like/TNBC tumors vs. matched normal tissue using TCGA Pan-Cancer Atlas data, and a higher H3R expression in human tumor samples vs. peritumoral tissue evidenced by immunohistochemistry associated with poorer survival. Furthermore, while all the essayed compounds showed antitumoral properties, LINS01022 and LINS01023 exhibited the most potent antiproliferative effects by: i) inducing cell apoptosis and suppressing cell migration in 4T1 and MDA-MB-231 TNBC cells, and ii) inhibiting cell growth in paclitaxel-resistant 4T1 cells (potentiating the paclitaxel antiproliferative effect). Moreover, 20 mg/kg LINS01022 reduced tumor size in 4T1 tumor-bearing mice, exhibiting a safe toxicological profile and potential for druggability estimated by ADME calculations. We conclude that the H3R is involved in the regulation of TNBC progression, offering promising therapeutic potential for the novel LINS01 series of H3R antagonists.

Letter to the Editor Ross procedure valve function, clinical outcomes and predictors after 25 years follow up.

The following letter presents an answer of a comment of our work titled "Ross procedure: valve function, clinical outcomes and predictors after 25 years' follow-up," recently published in your journal by Rangwala et al.1 As our colleagues point out, the Ross procedure has excellent survival rates but a significant risk of valve dysfunction and therefore reintervention at follow-up. Although the survival advantage with the Ross procedure appears to be consistent compared with mechanical valve substitutes, this benefit is not as clear compared with biological valve substitutes. However, biological valve substitutes also have significant reintervention rates during follow-up. The different surgical modifications of the Ross procedure have not clearly demonstrated better results in follow-up in terms of autograft reintervention. This procedure can be performed in a medium-volume center with good results as long as adequate patient selection and adequate surgical training are carried out.

Ross procedure: valve function, clinical outcomes and predictors after 25 years' follow-up.

OBJECTIVE: To describe long-term outcomes of the Ross procedure in a single center and retrospective series after 25 years follow-up. METHODS: From 1997-2019 we included all consecutive patients who underwent Ross procedure at our center. Clinical and echocardiographic evaluations were performed at least yearly. Echocardiographic valvular impairment was defined as at least moderate autograft or homograft dysfunction. Reintervention outcomes included surgical and percutaneous approach. RESULTS: 151 Ross procedures were performed (mean age 28±12years, 21 %<16years, 70 %male). After 25 years follow-up (median 18 years, interquartile range 9-21, only 3 patients lost) 12 patients died (8 %); Autograft, homograft or any valve dysfunction were present in 38(26 %), 48(32 %) and 75(51 %), respectively; and reintervention in 22(15%), 17(11%) and 38(26 %) respectively. At 20 years of follow-up, probabilities of survival free from autograft, homograft or any valve dysfunction were 63 %, 60 % and 35 %; and from reintervention, 80 %, 85 % and 67 %, respectively. The learning curve period (first 12 cases) was independently associated to autograft dysfunction (HR 2.78, 95 %CI:1.18-6.53, p = 0.02) and reintervention (HR 3.76, 95 %CI: 1.46-9.70, p = 0.006). Larger native pulmonary diameter was also an independent predictor of autograft reintervention (HR 1.22, 95 %CI:1.03-1.45, p = 0.03). Homograft dysfunction was associated with younger age (HR 5.35, 95 %CI: 2.13-13.47, p<0.001) and homograft reintervention, with higher left ventricle ejection fraction (HR 1,10, 95 %CI:1.02-1.19, p<0.02). CONCLUSIONS: In this 25 years' experience after the Ross procedure, global survival was high, although autograft and homograft dysfunction and reintervention rates were not negligible. Clinical and echocardiographic variables can identify patients with higher risk of events in follow up.

Nanomicellar Formulations Loaded with Histamine and Paclitaxel as a New Strategy to Improve Chemotherapy for Breast Cancer.

Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Currently, paclitaxel (PTX) represents the first-line therapy for TNBC; however it presents a hydrophobic behavior and produces severe adverse effects. The aim of this work is to improve the therapeutic index of PTX through the design and characterization of novel nanomicellar polymeric formulations composed of a biocompatible copolymer Soluplus® (S), surface-decorated with glucose (GS), and co-loaded either with histamine (HA, 5 mg/mL) and/or PTX (4 mg/mL). Their micellar size, evaluated by dynamic light scattering, showed a hydrodynamic diameter between 70 and 90 nm for loaded nanoformulations with a unimodal size distribution. Cytotoxicity and apoptosis assays were performed to assess their efficacy in vitro in human MDA-MB-231 and murine 4T1 TNBC cells rendering optimal antitumor efficacy in both cell lines for the nanoformulations with both drugs. In a model of TNBC developed in BALB/c mice with 4T1 cells, we found that all loaded micellar systems reduced tumor volume and that both HA and HA-PTX-loaded SG micelles reduced tumor weight and neovascularization compared with the empty micelles. We conclude that HA-PTX co-loaded micelles in addition to HA-loaded formulations present promising potential as nano-drug delivery systems for cancer chemotherapy.

The importance of particularising the model to estimate landfill GHG emissions.

Methane generation in landfills can be estimated using mathematical models. One of the most widespread estimation models is that developed by the Intergovernmental Panel on Climate Change (IPCC). Despite its popularity, the simplicity that characterises this model markedly limits the possibility of representing operation alternatives, which can strongly impact surface emissions and hinder the introduction of local data that are sometimes available. In this study, the IPCC model was applied to a case study from which field data on gas emissions were available. To fit the model to the studied landfill conditions, a series of modifications were made, including changes in Degradable Organic Carbon (DOC) and methane generation rate constant (k) values, and degradation times for some waste fractions, and by considering leachate carbon and the inclusion of gas lateral migration phenomena or changes in the methane oxidation factor. The model's Final Version improved the fit of its Initial Version to the experimentally estimated values in the case study by more than 65%. Some modifications, such as considering the carbon dragged by leachate or the contour migration of gas, have a minor impact on the model's fit. However, changes in the degradation time of some fractions according to their particular pretreatment or the modification of parameter k in accordance with the moisture conditions in each landfill phase, strongly influence the model's results. This highlights the importance of particularising estimation models to achieve more accurate results, which allow better estimates of the efficiency of mitigation measures for landfill gas emissions in each facility.

Genomic and proteomic characterization of two strains of Shigella flexneri 2 isolated from infants' stool samples in Argentina.

BACKGROUND: Shigella specie is a globally important intestinal pathogen disseminated all over the world. In this study we analyzed the genome and the proteomic component of two Shigella flexneri 2a clinical isolates, collected from pediatric patients with gastroenteritis of the Northwest region of Argentina (NWA) in two periods of time, with four years of difference. Our goal was to determine putative changes at molecular levels occurred during these four years, that could explain the presence of this Shigella`s serovar as the prevalent pathogen in the population under study. RESULTS: As previously reported, our findings support the idea of Shigella has a conserved "core" genome, since comparative studies of CI133 and CI172 genomes performed against 80 genomes obtained from the NCBI database, showed that there is a large number of genes shared among all of them. However, we observed that CI133 and CI172 harbors a small number of strain-specific genes, several of them present in mobile genetic elements, supporting the hypothesis that these isolates were established in the population by horizontal acquisition of genes. These differences were also observed at proteomic level, where it was possible to detect the presence of certain secreted proteins in a culture medium that simulates the host environment. CONCLUSION: Great similarities were observed between the CI133 and CI172 strains, confirming the high percentage of genes constituting the "core" genome of S. flexneri 2. However, numerous strain specific genes were also determined. The presence of the here identified molecular elements into other strain of our culture collation, is currently used to develop characteristic markers of local pathogens. In addition, the most outstanding result of this study was the first description of a S. flexneri 2 producing Colicin E, as one of the characteristics that allows S. flexneri 2 to persist in the microbial community. These findings could also contribute to clarify the mechanism and the evolution strategy used by this pathogen to specifically colonize, survive, and cause infection within the NWA population.

Histamine H4 Receptor Expression in Triple-negative Breast Cancer: An Exploratory Study.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype. There are neither universally accepted prognostic markers nor molecular targets related to TNBC. The histamine H4 receptor (H4R) has been characterized in TNBC experimental models, demonstrating its critical role in tumor development and progression. In this study, H4R expression was compared in breast cancer subtypes and correlated with clinical features using The Cancer Genome Atlas data (Pan-Cancer Atlas). The H4R status was further evaluated by immunohistochemistry in 30 TNBC human samples in relation to clinicopathological parameters. Results indicate that H4R was downregulated in basal-like/TNBC compared with luminal A and normal breast-like tumors. The higher expression of H4R was associated with improved progression-free and overall survival outcomes in basal-like/TNBC. H4R immunoreactivity was detected in about 70% of tumors, and its expression was positively correlated with the levels in the histologically normal peritumoral tissue. High H4R expression in peritumoral tissue correlated with reduced number of lymph node involvement and unifocal TNBC, while it was associated with increased patient survival. In conclusion, the H4R might represent a potential prognostic biomarker in TNBC. Further studies in large cohorts are needed to better understand the significance of H4R in breast cancer biology.

Histamine H4 Receptor Agonism Induces Antitumor Effects in Human T-Cell Lymphoma.

The discovery of the human histamine H4 receptor (H4R) has contributed to our understanding of the role of histamine in numerous physiological and pathological conditions, including tumor development and progression. The lymph nodes of patients with malignant lymphomas have shown to contain high levels of histamine, however, less is known regarding the expression and function of the H4R in T-cell lymphoma (TCL). In this work we demonstrate the expression of H4R isoforms (mRNA and protein) in three human aggressive TCL (OCI-Ly12, Karpas 299, and HuT78). Histamine and specific H4R agonists (VUF8430 and JNJ28610244) significantly reduced cell viability in a dose-dependent manner (p < 0.05). The combined treatment with the H4R antagonist (JNJ7777120, 10 µM) reversed the effects of the H4R ligands. Importantly, we screened a drug repurposing library of 433 FDA-approved compounds (1 µM) in combination with histamine (10 µM) in Hut78 cells. Histamine produced a favorable antitumor effect with 18 of these compounds, including the histone deacetylase inhibitor panobinostat. Apoptosis, proliferation, and oxidative stress studies confirmed the antitumoral effects of the combination. We conclude that the H4R is expressed in TCL, and it is involved in histamine-mediated responses.

Tumor fibroso solitario gigante de pleura. Serie de casos / Giant Solitary Fibrous Tumor of the Pleura. Case Series

Resumen Introducción. El tumor fibroso solitario de pleura (TFSP) es una neoplasia poco frecuente, con aproximadamente 1.000 casos reportados en la literatura mundial. La aproximación diagnóstica inicial se realiza con estudios imagenológicos. Métodos. De forma retrospectiva, se recopilaron cuatro casos de pacientes con TFSP gigante operados en nuestra institución. Se describen las características sociodemográficas, clínicas, imagenológicas, macroscópicas y microscópicas de cada caso. Resultados. Todos los pacientes de la serie cursaron con manifestaciones clínicas, con un promedio de 23,75 meses de evolución. El 50% de los tumores se localizaron en la cavidad pleural derecha y el 50% en la izquierda. En tomografía computarizada (TC) de tórax, los cuatro casos se presentaron como una masa sólida, de densidad heterogénea, con diámetros mayores entre 17 y 22 cm y contornos variables (lisos en tres casos y lobulados en un paciente). Se observaron calcificaciones intratumorales en dos casos y derrame pleural en tres pacientes. En cirugía, todas las masas presentaron pedículos. El análisis histológico e inmuno-químico confirmó la naturaleza benigna de tres casos y malignidad en una de las neoplasias. Conclusiones. Los TFSP generalmente son benignos y de buen pronóstico. Sin embargo, entre 10 y 20% de esos tumores son malignos. Las imágenes diagnósticas pueden sugerir el diagnóstico de TFSP, pero la confirmación de la naturaleza de la lesión debe realizarse con el análisis histopatológico de toda la pieza quirúrgica.

Abstract Introduction. Solitary fibrous tumor of the pleura (SFTP) is a rare neoplasm, with ~1.000 cases reported in the worldwide literature. The initial diagnostic approach is performed by imaging studies. Methods. Retrospectively, we collected four cases of patients with giant SFTP operated in our institution. The sociodemographic, clinical, imaging, macroscopic, and microscopic characteristics of each case are described. Results. All the patients in the series had clinical manifestations, with an average of 23.75 months of evolution. 50% of the tumors were located in the right pleural cavity and 50% in the left. In chest computed tomography (CT), the four cases presented as a solid mass, of heterogeneous density, with greater diameters between 17 and 22 cm, and variable contours (smooth in three cases and lobulated in one patient). Intratumoral calcifications were observed in two cases and pleural effusion in three patients. In surgery, all masses presented pedicles. The histological and immunochemical analysis confirmed the benign nature of three cases and malignancy in one of them. Conclusions. SFTPs are usually benign and have a good prognosis. However, between 10 and 20% of these tumors are malignant. Diagnostic images may suggest the diagnosis of SFTP, but confirmation of the nature of the lesion should be made with the histopathological analysis of the entire surgical specimen.

Histamine in cancer immunology and immunotherapy. Current status and new perspectives.

Cancer is the second leading cause of death globally and its incidence and mortality are rapidly increasing worldwide. The dynamic interaction of immune cells and tumor cells determines the clinical outcome of cancer. Immunotherapy comes to the forefront of cancer treatments, resulting in impressive and durable responses but only in a fraction of patients. Thus, understanding the characteristics and profiles of immune cells in the tumor microenvironment (TME) is a necessary step to move forward in the design of new immunomodulatory strategies that can boost the immune system to fight cancer. Histamine produces a complex and fine-tuned regulation of the phenotype and functions of the different immune cells, participating in multiple regulatory responses of the innate and adaptive immunity. Considering the important actions of histamine-producing immune cells in the TME, in this review we first address the most important immunomodulatory roles of histamine and histamine receptors in the context of cancer development and progression. In addition, this review highlights the current progress and foundational developments in the field of cancer immunotherapy in combination with histamine and pharmacological compounds targeting histamine receptors.

Free-living hard ticks (Ixodida: Ixodidae) from three different natural environments of Costa Rica.

This paper presents data on free-living ticks collected by flagging and using CO2 traps in three natural areas in Costa Rica: Carara National Park (CNP), Palo Verde National Park (PVNP), and a Private Forest Reserve in Sarapiquí (SPR). Data were analyzed calculating aspects of alpha diversity (species richness, entropy; dominance index, and evenness); and for beta diversity, compositional similarity between communities of ticks was also calculated. We collected 12,795 ticks belonging to 10 species: Amblyomma coelebs, Amblyomma dissimile, Amblyomma mixtum, Amblyomma naponense, Amblyomma cf. oblongoguttatum, Amblyomma cf. parvum, Amblyomma sabanerae, Amblyomma tapirellum, Haemaphysalis juxtakochi and Ixodes affinis. The number of species and individuals varied between sites: 5970 ticks were collected in CNP, 4443 in PVNP, and 2382 in SPR. Amblyomma cf. oblongoguttatum and A. cf. parvum were collected at all three sites, but A. mixtum was the most abundant species, even though it was not collected in SPR. Values of alpha diversity were calculated for CNP and SPR, while diversity in PVNP was the lowest of the three locations. Evenness was highest in SPR and lowest in CNP. The only community that presented dominance was PVNP. Beta diversity showed low similarity between the three locations with the lowest being CNP and SPR. For the three localities, estimates of the number of tick species based on presence/absence data was higher using flagging than CO2; and considering the stage of the ticks collected. More larvae were captured using CO2 traps than by flagging, while flagging was better for collecting adults. To our knowledge this is the first study in Costa Rica that compares these two sampling methods in three different environmental areas.

Cardioprotective Effect of Glycyrrhizin on Myocardial Remodeling in Diabetic Rats.

Myocardial fibrosis is one of the major complications of long-term diabetes. Hyperglycemia induced cardiomyocyte atrophy is a frequent pathophysiological indicator of diabetic heart. The objective of this study was to investigate the cardioprotective effect of glycyrrhizin (GLC) on myocardial damage in diabetic rats and assess the anti-inflammatory and anti-fibrotic effect of GLC. Our study demonstrates that hyperglycemia can elevate cardiac atrophy in diabetic animals. Type 2 diabetic fatty and the lean control rats were evaluated for cardiac damage and inflammation at 8-12 weeks after the development of diabetes. Western blot and immunohistochemical studies revealed that gap junction protein connexin-43 (CX43), cardiac injury marker troponin I, cardiac muscle specific voltage gated sodium channel NaV1.5 were significantly altered in the diabetic heart. Furthermore, oxidative stress mediator receptor for advanced glycation end-products (RAGE), as well as inflammatory mediator phospho-p38 MAPK and chemokine receptor CXCR4 were increased in the diabetic heart whereas the expression of nuclear factor erythroid-2-related factor 2 (Nrf2), the antioxidant proteins that protect against oxidative damage was reduced. We also observed an increase in the expression of the pleiotropic cytokine, transforming growth factor beta (TGF-ß) in the diabetic heart. GLC treatment exhibited a decrease in the expression of phospho-p38 MAPK, RAGE, NaV1.5 and TGF-ß and it also altered the expression of CX43, CXCR4, Nrf2 and troponin I. These observations suggest that GLC possesses cardioprotective effects in diabetic cardiac atrophy and that these effects could be mediated through activation of Nrf2 and inhibition of CXCR4/SDF1 as well as TGF-ß/p38MAPK signaling pathway.

A Model for Studying the Biomechanical Effects of Varying Ratios of Collagen Types I and III on Cardiomyocytes.

PURPOSE: To develop a novel model composed solely of Col I and Col III with the lower and upper limits set to include the ratios of Col I and Col III at 3:1 and 9:1 in which the structural and mechanical behavior of the resident CM can be studied. Further, the progression of fibrosis due to change in ratios of Col I:Col III was tested. METHODS: Collagen gels with varying Col I:Col III ratios to represent a healthy (3:1) and diseased myocardial tissue were prepared by manually casting them in wells. Absorbance assay was performed to confirm the gelation of the gels. Rheometric analysis was performed on each of the collagen gels prepared to determine the varying stiffnesses and rheological parameters of the gels made with varying ratios of Col I:Col III. Second Harmonic Generation (SHG) was performed to observe the 3D characterization of the collagen samples. Scanning Electron microscopy was used for acquiring cross sectional images of the lyophilized collagen gels. AC16 CM (human) cell lines were cultured in the prepared gels to study cell morphology and behavior as a result of the varying collagen ratios. Cellular proliferation was studied by performing a Cell Trace Violet Assay and the applied force on each cell was measured by means of Finite Element Analysis (FEA) on CM from each sample. RESULTS: Second harmonic generation microscopy used to image Col I, displayed a decrease in acquired image intensity with an increase in the non-second harmonic Col III in 3:1 gels. SEM showed a fiber-rich structure in the 3:1 gels with well-distributed pores unlike the 9:1 gels or the 1:0 controls. Rheological analysis showed a decrease in substrate stiffness with an increase of Col III, in comparison with other cases. CM cultured within 3:1 gels exhibited an elongated rod-like morphology with an average end-to-end length of 86 ± 28.8 µm characteristic of healthy CM, accompanied by higher cell growth in comparison with other cases. Finite element analysis used to estimate the forces exerted on CM cultured in the 3:1 gels, showed that the forces were well dispersed, and not concentrated within the center of cells, in comparison with other cases. CONCLUSION: This study model can be adopted to simulate various biomechanical environments in which cells crosstalk with the Collagen-matrix in diseased pathologies to generate insights on strategies for prevention of fibrosis.

Impact of histamine H4 receptor deficiency on the modulation of T cells in a murine breast cancer model.

BACKGROUND: The histamine H4 receptor (H4R) is preferentially expressed in immune cells and is a potential therapeutic target for inflammatory and autoimmune diseases. This study aimed at further exploring the role of H4R in the immunobiology of breast cancer. METHODS: We used wild type (WT) and H4R deficient mice (KO) to evaluate whether H4R genotypes show a different distribution of T cell subsets in spleens, tumours and tumour draining lymph nodes (TDLN) in a syngeneic ErbB2-positive breast cancer model developed orthotopically with LM3 cells and its impact on tumour growth. RESULTS: The presence of tumours had a differential impact on the distribution of T cells in TDLN from KO mice compared to WT ones. At day 21 post-inoculation (p.i.) of cells, despite no significant changes in the tumour weight, TDLN from KO mice showed a significantly increased proportion of CD8+ T cells compared to WT mice. At day 38 p.i. of cells a reduced tumour weight was evident in KO mice. This was accompanied by a decreased proportion of CD4+CD25+FoxP3+ regulatory T cells in TDLN of KO compared to WT mice. Tumour-bearing KO mice showed a better survival compared to WT mice. CONCLUSIONS: H4R-mediated mechanisms may modulate the immune tumour microenvironment, promoting an immunosuppressive milieu. Results suggest that H4R could be explored as an immunotherapeutic target with potential benefit in combination with immunotherapy. Further preclinical and clinical studies are necessary to confirm this hypothesis.

Epidemiological study of prevalent pathogens in the Northwest region of Argentina (NWA).

Northwest Argentina (NWA) is a poor economic-geographical region, with the highest rate of diarrhea diseases. At the moment, there are no reports showing the epidemiological status of this region that would allow to establish methods for prevention and control of these infections and to indicate of the prevalent pathogen that produces them. Therefore we carried out an epidemiological study of the gastroenteritis etiological agents and their incidence in the pediatric population. A total of 17 823 fecal samples were collected, 14 242 from HNJ-Tuc, 2,257 from CePSI-Stgo and 1,324 from HINEP-Cat. In 2,595 samples a bacterial agent was identified, the 93.64% corresponded to Shigella/Salmonella clinical isolates. Shigella genus was the prevalent pathogen, being Shigella flexneri 2 the most frequent serotype. Most of the Shigella clinical isolates presented themselves as multidrug-resistant (MDR), harboring 2 to 3 genetic resistance determinants. 50% of the affected patients were children under 4 years old. Here, we demonstrate that bacterial gastrointestinal diseases strongly affect the health of NWA population. The appearance of epidemic outbreaks, as happened during 2014, suggest that they may be related to the socio-economic poverty of NWA. Recently, Shigella flexneri 2 has become the highest NWA´s incidence infectious agent. The acquisition of new antibiotic resistance determinants may play an important role in their adaptation and persistence.

Methods for histological characterization of cryo-induced myocardial infarction in a rat model.

Ligation of the left anterior descending (LAD) coronary artery has been commonly employed to induce myocardial infarction (MI) in animals; however, it is known to pose setbacks in the form of cardiac arrhythmias and unpredictable areas of necrotic damage. Cryo-infarction is an alternate method that has been adopted to create a reproducible model of a myocardial injury. In this study, Sprague-Dawley rats were subjected to thoracotomy followed by cryo-induced infarction of the heart, while the control-sham group was only subjected to thoracotomy following which the heart was collected from all animals. Tissue sections were stained with hematoxylin and eosin and analyzed to determine cardiac muscle density, fiber length, and fiber curvature. Observations revealed reduced muscle density, cardiac fiber length, and distorted fibers in infarcted tissue sections. Gomori's Trichrome staining was performed on tissue sections to study the effects of post MI on collagen, which showed enhanced intensity of collagen staining indicating fibrosis for the experimental models as compared to the sham models, an established consequence to myocardial injury. Immunohistochemical staining of the tissue sections with DAPI and connexin-43 (Cx-43) revealed that there was reduced DAPI staining and a less pronounced expression of Cx-43 in the experimental samples as compared to the sham samples. Results implied significant cell damage resulting from the cryo-infarction, subsequently disrupting and disaggregating the functional Cx-43 junction in cardiac myocytes, which is essential for normal and healthy cardiac physiology and function. This quantitative histological study of cryo-induced MI in a rat model can aid others attempting to optimize MI models in rats via cryo-injury, to study cardiac disease progression, and to aid in the construction of engineered cardiac tissues.

Hydrogel scaffolds with elasticity-mimicking embryonic substrates promote cardiac cellular network formation.

Hydrogels are a class of biomaterials used for a wide range of biomedical applications, including as a three-dimensional (3D) scaffold for cell culture that mimics the extracellular matrix (ECM) of native tissues. To understand the role of the ECM in the modulation of cardiac cell function, alginate was used to fabricate crosslinked gels with stiffness values that resembled embryonic (2.66 ± 0.84 kPa), physiologic (8.98 ± 1.29 kPa) and fibrotic (18.27 ± 3.17 kPa) cardiac tissues. The average pore diameter and hydrogel swelling were seen to decrease with increasing substrate stiffness. Cardiomyocytes cultured within soft embryonic gels demonstrated enhanced cell spreading, elongation, and network formation, while a progressive increase in gel stiffness diminished these behaviors. Cell viability decreased with increasing hydrogel stiffness. Furthermore, cells in fibrotic gels showed enhanced protein expression of the characteristic cardiac stress biomarker, Troponin-I, while reduced protein expression of the cardiac gap junction protein, Connexin-43, in comparison to cells within embryonic gels. The results from this study demonstrate the role that 3D substrate stiffness has on cardiac tissue formation and its implications in the development of complex matrix remodeling-based conditions, such as myocardial fibrosis.

Histamine H4 receptor agonists induce epithelial-mesenchymal transition events and enhance mammosphere formation via Src and TGF-ß signaling in breast cancer cells.

Epithelial-mesenchymal transition (EMT) contributes to cell invasion and metastasis during the progression of epithelial cancers. Though preclinical evidence suggests a role for histamine H4 receptor (H4R) in breast cancer growth, its function in the EMT is less known. In this study we proposed to investigate the effects of H4R ligands on EMT and mammosphere formation as a surrogate assay for cancer stem cells in breast cancer cells with different invasive phenotype. We also investigated the participation of Src and TGF-ß signaling in these events. Breast cancer cells were treated with the H4R agonists Clobenpropit, VUF8430 and JNJ28610244 and the H4R antagonist JNJ7777120. Immunodetection studies showed cytoplasmic E-cadherin, cytoplasmic and nuclear beta-catenin, nuclear Slug and an increase in vimentin and α-smooth muscle actin expression. There was also an enhancement in cell migration and invasion assessed by transwell units. All these effects were prevented by JNJ7777120. Moreover, H4R agonists induced an increase in phospho-Src levels detected by Western blot. Results revealed the involvement of phospho-Src in EMT events. Upon treatment with H4R agonists there was an increase in phospho-ERK1/2 and TGF-ß1 levels by Western blot, in Smad2/3 positive nuclei by indirect immunofluorescence, and in tumor spheres formation by the mammosphere assay. Notably, the selective TGF-ß1 kinase/activin receptor-like kinase inhibitor A83-01 blocked these effects. Moreover, cells derived from mammospheres exhibited higher Slug expression and enhanced migratory behavior. Collectively, findings support the interaction between H4R and TGF-ß receptor signaling in the enhancement of EMT features and mammosphere formation and point out intracellular TGF-ß1 as a potential mediator of these events.