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1.
PLoS One ; 19(3): e0299645, 2024.
Article in English | MEDLINE | ID: mdl-38478564

ABSTRACT

Monodominant tree communities can have phenotypic trait variation (intraspecific variation) as extreme as the trait variation across a forest with higher species diversity. An example of such forests is those composed of Quercus, an important genus of woody angiosperms in the montane neotropical forest. The Andean oak, or Quercus humboldtii Bonpl., is the sole member of this genus in South America and a characteristic component of montane ecosystems. Although there are several studies on the ecology and genetic structure of this species, there are few studies on the functional trait diversity among populations. Understanding functional traits can improve our comprehension of how organisms respond to various environmental conditions. In this study, we aimed to evaluate differences in six functional traits in individuals of the Andean oak, in two ontogenetic stages (juveniles and adults) from three populations with contrasting environmental conditions. Additionally, using T-statistics, we assessed the impact of external filters (e.g., climate, resource availability, large-scale biotic interactions) on population assembly. We found a remarkable level of functional differentiation among Andean oak forests, with all traits differing between populations and five traits differing between ontogenetic stages. External filters had a stronger influence in populations with more extreme environmental conditions. These findings emphasize the dynamic and context-dependent nature of functional traits in this species. However, given the limited exploration of functional diversity in Andean oak populations, further studies are needed to inform conservation efforts.


Subject(s)
Ecosystem , Quercus , Humans , Quercus/genetics , Forests , Trees , Ecology
2.
Case Rep Oncol ; 16(1): 613-620, 2023.
Article in English | MEDLINE | ID: mdl-37900810

ABSTRACT

We present the case of a 64-year-old female with stage IV gastric adenocarcinoma, pulmonary, and abdominal wall metastases, and no history of cardiovascular disease. In palliative care, she received systemic cytotoxic treatment with fluorouracil, leucovorin, oxaliplatin, and docetaxel protocol, which was well tolerated over five cycles. During cycle 6, she presented with cardiovascular symptoms with hemodynamic consequences while receiving oxaliplatin injection without docetaxel or 5-fluorouracil. She was transferred to the emergency department and then to the intensive care unit. She developed no complications during the hospital stay and was discharged after 10 days with preserved systolic function and no structural changes at the myocardial level. The electrocardiogram, echocardiogram, cardiac catheterization, and magnetic resonance imaging findings indicated an oxaliplatin-associated Takotsubo syndrome. The immunochemistry analysis showed PD-L1 expression level TPS: 40% and the foundation one genomic profiling revealed high mutation load, microsatellite instability, and HER2 not found. The patient is currently asymptomatic and on pembrolizumab monotherapy with good tolerance and partial treatment response.

3.
Chin Clin Oncol ; 12(4): 45, 2023 Aug.
Article in English | MEDLINE | ID: mdl-37599513

ABSTRACT

BACKGROUND: Tumor lysis syndrome (TLS) is recognized as an oncologic disorder with a variable incidence. TLS can cause the rapid destruction of tumor cells in response to oncologic therapy and is characterized by multiple electrolyte disturbances as well as its secondary complications, including death. This disease is common among patients with hematologic neoplasms, but very rare among those with solid tumors, as is the case with sarcomas. Such patients have a poor prognosis and increased risk of mortality. In the patient's particular case, this occurred after initiating third-line systemic therapy with gemcitabine associated with pazopanib, an event not previously described in the literature. CASE DESCRIPTION: We report the case of a patient with a history of high-grade sarcoma of the left lower limb T4N1M0 stage IIIB undergoing surgical management and exhibiting tumor progression with the need for third-line systemic therapy with pazopanib and gemcitabine. The patient presented with pain at the amputation site, inflammatory changes, and a tumor mass of large components on admission. They later developed electrolyte imbalance and acute renal injury compatible with TLS after systemic therapy was initiated. Pharmacological therapy, including rasburicase, was initiated based on the clinical and laboratory findings. Due to the progression of renal involvement, it was necessary to initiate haemodialysis, and during her hospital stay, the patient presented febrile syndrome associated with pancytopenia. The patient showed a favourable clinical response to the proposed antibiotic therapy and recovery of renal function, for which reason therapy was restarted with pazopanib and gemcitabine, the latter with a 20% reduction for the following cycles. Outpatient follow-up continued, completing eight cycles of treatment with good tolerance and partial clinical response; the patient died of respiratory complications eight months after discharge. CONCLUSIONS: There is limited evidence for TLS in patients with high-grade sarcoma in the literature related to the oncologic therapy used; this indicates that early risk evaluation along with prompt initiation of effective therapies is required to prevent the appearance of this type of complications in the short and long term.


Subject(s)
Sarcoma , Tumor Lysis Syndrome , Female , Humans , Gemcitabine , Tumor Lysis Syndrome/etiology , Sarcoma/complications , Sarcoma/drug therapy , Electrolytes
4.
Int J Gen Med ; 16: 697-705, 2023.
Article in English | MEDLINE | ID: mdl-36866168

ABSTRACT

Purpose: This study aims to determine the seroprevalence of coronavirus disease (COVID-19) among health care workers and describe the associated sociodemographic and labor features. Patients and Methods: An observational study with an analytical component was conducted at a clinic in Cali, Colombia. The sample size was 708 health workers and they were selected by stratified random sampling. A Bayesian analysis was developed to determine the raw and adjusted prevalence. A Poisson regression model was used to estimate the prevalence ratios. Results: Overall seroprevalence of COVID-19 among healthcare workers was 29%. Miscellaneous services workers, healthcare, and administrative workers, was 38%, 33%, and 32%, respectively. Factors related to seropositivity were having a contact with a COVID-19 patient for >120 minutes and being diagnosed with COVID-19 by laboratory tests. Conclusion: The present study shows a adjusted seroprevalence of 29% in health workers, indicating a high level of disease transmission and an increased risk of infection in this population group.

5.
Adv Mater Technol ; 7(5)2022 May.
Article in English | MEDLINE | ID: mdl-35754760

ABSTRACT

The design and manufacture of an origami-based liver-on-a-chip device are presented, together with demonstrations of the chip's effectiveness at recapitulating some of the liver's key in vivo architecture, physical microenvironment, and functions. Laser-cut layers of polyimide tape are folded together with polycarbonate nanoporous membranes to create a stack of three adjacent flow chambers separated by the membranes. Endothelial cells are seeded in the upper and lower flow chambers to simulate sinusoids, and hepatocytes are seeded in the middle flow chamber. Nutrients and metabolites flow through the simulated sinusoids and diffuse between the vascular pathways and the hepatocyte layers, mimicking physiological microcirculation. Studies of cell viability, metabolic functions, and hepatotoxicity of pharmaceutical compounds show that the endothelialized liver-on-a-chip model is conducive to maintaining hepatocyte functions and evaluation of the hepatotoxicity of drugs. Our unique origami approach speeds chip development and optimization, effectively simplifying the laboratory-scale fabrication of on-chip models of human tissues without necessarily reducing their structural and functional sophistication.

6.
Front Microbiol ; 12: 656979, 2021.
Article in English | MEDLINE | ID: mdl-33767688

ABSTRACT

Investigating the dynamics of virus-host interactions in vivo remains an important challenge, often limited by the ability to directly identify virally infected cells. Here, we utilize a beta-lactamase activated fluorescent substrate to identify primary targets of murine gammaherpesvirus 68 (MHV68) infection in the peritoneal cavity. By optimizing substrate and detection conditions, we were able to achieve multiparameter characterization of infected cells and the ensuing host response. MHV68 infection leads to a pronounced increase in immune cells, with CD8+ T cells increasing by 3 days, and total infiltrate peaking around 8 days post-infection. MHV68 infection results in near elimination of large peritoneal macrophages (LPMs) by 8 days post-infection, and a concordant increase in small peritoneal macrophages (SPMs) and monocytes. Infection is associated with prolonged changes to myeloid cells, with a distinct population of MHC IIhigh LPMs emerging by 14 days. Targets of MHV68 infection could be readily detected. Between 1 and 3 days post-infection, MHV68 infects ∼5-10% of peritoneal cells, with >75% being LPMs. By 8 days post-infection, the frequency of MHV68 infection is reduced at least 10-fold, with infection primarily in SPMs, with few infected dendritic cells and B cells. Importantly, limiting dilution analysis indicates that at 3 days post-infection, the majority of MHV68-infected cells harbor latent rather than lytic virus at frequencies consistent with those identified based on reporter gene expression. Our findings demonstrate the utility of the beta-lactamase MHV68 reporter system for high throughput single-cell analysis and identify dynamic changes during primary gammaherpesvirus infection.

7.
Matter ; 4(1): 217-240, 2021 Jan 06.
Article in English | MEDLINE | ID: mdl-33718864

ABSTRACT

In this study, we present the photosynthetic oxygen (O2) supply to mammalian cells within a volumetric extracellular matrix-like construct, whereby a three-dimensional (3D)-bioprinted fugitive pattern encapsulating unicellular green algae, Chlamydomonas reinhardtii (C. reinhardtii), served as a natural photosynthetic O2-generator. The presence of bioprinted C. reinhardtii enhanced the viability and functionality of mammalian cells while reducing the hypoxic conditions within the tissues. We were able to subsequently endothelialize the hollow perfusable microchannels formed after enzymatic removal of the bioprinted C. reinhardtii-laden patterns from the matrices following the initial oxygenation period, to obtain biologically relevant vascularized mammalian tissue constructs. The feasibility of co-culture of C. reinhardtii with human cells, the printability and the enzymatic degradability of the fugitive bioink, as well as the exploration of C. reinhardtii as a natural, eco-friendly, cost-effective, and sustainable source of O2 would likely promote the development of engineered tissues, tissue models, and food for various applications.

8.
Gut ; 69(10): 1818-1831, 2020 10.
Article in English | MEDLINE | ID: mdl-31988194

ABSTRACT

OBJECTIVE: To investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target. DESIGN: FLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases. RESULTS: FLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis. CONCLUSIONS: Based on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients.


Subject(s)
Carcinogenesis , Cell Proliferation , Colorectal Neoplasms , Neovascularization, Pathologic , RNA, Long Noncoding , STAT3 Transcription Factor/metabolism , Animals , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinogenesis/drug effects , Carcinogenesis/genetics , Cell Proliferation/drug effects , Cell Proliferation/genetics , Colorectal Neoplasms/genetics , Colorectal Neoplasms/therapy , Drug Discovery , Gene Expression Regulation, Neoplastic , Genetic Markers , Genetic Therapy , Humans , Mice , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Pharmacogenomic Testing , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Vascular Endothelial Growth Factor A/metabolism
9.
Front Mol Biosci ; 7: 612801, 2020.
Article in English | MEDLINE | ID: mdl-33585561

ABSTRACT

The Nobel Prize-deserving concept of blocking inhibitory pathways in T cells, to unleash their anti-tumoral capacity, became one of the pillars of cancer treatment in the last decade and has resulted in durable clinical responses for multiple cancer types. Currently, two of the most important goals in cancer immunotherapy are to understand the mechanisms resulting in failure to checkpoint blockade and to identify predictive immunological biomarkers that correlate to treatment response, disease progression or adverse effects. The identification and validation of biomarkers for routine clinical use is not only critical to monitor disease or treatment progression, but also to personalize and develop new therapies. To achieve these goals, powerful research tools are needed. Flow cytometry stands as one of the most successful single-cell analytical tools used to characterize immune cell phenotypes to monitor solid tumors, hematological malignancies, minimal residual disease or metastatic progression. This technology has been fundamental in diagnosis, treatment and translational research in cancer clinical trials. Most recently, the need to evaluate simultaneously more features in each cell has pushed the field to implement more powerful adaptations beyond conventional flow cytometry, including Full Spectrum Flow Cytometry (FSFC). FSFC captures the full emission spectrum of fluorescent molecules using arrays of highly sensitive light detectors, and to date has enabled characterization of 40 parameters in a single sample. We will summarize the contributions of this technology to the advancement of research in immunotherapy studies and discuss best practices to obtain reliable, robust and reproducible FSFC results.

11.
J Surg Res ; 213: 90-99, 2017 06 01.
Article in English | MEDLINE | ID: mdl-28601338

ABSTRACT

BACKGROUND: Presence of circulating endothelial cells (CECs) in systemic circulation may be an indicator of endothelial damage and/or denudation, and the body's response to repair and revascularization. Thus, we hypothesized that aggregated platelets (AgPlts) can disrupt/denude the endothelium and contribute to the presence of CEC and EC-derived particles (ECDP). METHODS: Endothelial cells were grown in glass tubes and tagged with/without 0.5 µm fluorescent beads. These glass tubes were connected to a mini-pump variable-flow system to study the effect of circulating AgPlts on the endothelium. ECs in glass tube were exposed to medium alone, nonaggregated platelets (NAgPlts), AgPlts, and 90 micron polystyrene beads at a flow rate of 20 mL/min for various intervals. Collected effluents were cultured for 72 h to analyze the growth potential of dislodged but intact ECs. Endothelial damage was assessed by real time polymerase chain reaction (RT-PCR) for inflammatory genes and Western blot analysis for von Willebrand factor. RESULTS AND CONCLUSION: No ECs and ECDP were observed in effluents collected after injecting medium alone and NAgPlts, whereas AgPlts and Polybeads drastically dislodged ECs, releasing ECs and ECDP in effluents as the time increased. Effluents collected when endothelial cell damage was seen showed increased presence of von Willebrand factor as compared to control effluents. Furthermore, we analyzed the presence of ECs and ECDPs in heart failure subjects, as well as animal plasma samples. Our study demonstrates that circulating AgPlts denude the endothelium and release ECs and ECDP. Direct mechanical disruption and shear stress caused by circulating AgPlts could be the underlying mechanism of the observed endothelium damage.


Subject(s)
Blood Platelets/physiology , Endothelial Cells/physiology , Human Umbilical Vein Endothelial Cells/physiology , Platelet Aggregation/physiology , Animals , Biomarkers/metabolism , Blotting, Western , Cells, Cultured , Enzyme-Linked Immunosorbent Assay , Humans , Real-Time Polymerase Chain Reaction , Sheep
12.
PLoS One ; 12(6): e0179084, 2017.
Article in English | MEDLINE | ID: mdl-28591228

ABSTRACT

We evaluated the importance of neutrophils in the development of chronic lesions caused by L. Viannia spp. using the hamster as experimental model of American Cutaneous Leishmaniasis (ACL). Neutrophils infiltrated the lesion within the first six hours post-infection. Inhibition of this early infiltration using a polyclonal antibody or cyclophosphamide was associated with transient parasite control but the protective effect vanished when lesions became clinically apparent. At lesion onset (approximately 10 days p.i.), there was an increased proportion of both uninfected and infected macrophages, and subsequently a second wave of neutrophils infiltrated the lesion (after 19 days p.i.) This second neutrophil infiltration was associated with lesion necrosis and ulceration (R2 = 0.75) and maximum parasite burden. Intradermal delivery of N-formylmethionyl-leucyl-phenylalanine (fMLP), aimed to increase neutrophil infiltration, resulted in larger lesions with marked necrosis and higher parasite burden than in mock treated groups (p<0.001 each). In contrast, reduced neutrophil infiltration via cyclophosphamide-mediated depletion led to more benign lesions and lower parasite loads compared to controls (p<0.001 each). Neutrophils of the second wave expressed significantly lower GM-CSF, reactive oxygen species and nitric oxide than those of the first wave, suggesting that they had less efficient anti-leishmania activity. However, there was increased inflammatory cytokines and expression of neutrophil proteases (myeloperoxidase, cathepsin G and elastase) in lesions during the second wave of neutrophil infiltration compared with the levels reached during the first wave (6h p.i.). This suggests that augmented neutrophil proteases and inflammatory cytokines during the secondary wave of neutrophils could contribute to skin inflammation, ulceration and necrosis in ACL. The overall results indicate that neutrophils were unable to clear the infection in this model, and that the second wave of neutrophils played an important role in the severity of ACL.


Subject(s)
Inflammation/blood , Leishmaniasis, Cutaneous/blood , Necrosis/blood , Neutrophil Infiltration , Animals , Cricetinae , Disease Models, Animal , Female , Humans , Inflammation/parasitology , Inflammation/physiopathology , Leishmania/pathogenicity , Leishmaniasis, Cutaneous/parasitology , Leishmaniasis, Cutaneous/physiopathology , Macrophages/pathology , Necrosis/parasitology , Necrosis/physiopathology , Neutrophils/pathology , Nitric Oxide/metabolism , Parasite Load , Reactive Oxygen Species/metabolism , United States
13.
J Med Case Rep ; 11(1): 172, 2017 Jun 27.
Article in English | MEDLINE | ID: mdl-28651636

ABSTRACT

BACKGROUND: Adult hemophagocytic lymphohistiocytosis is a secondary immunopathologic phenomenon, mainly secondary to malignancy, infection, or autoimmune disorders. The performance of diagnostic criteria, studied in the pediatric population, is yet to be validated in the adult population. Some of the criteria include cytopenias and organomegaly that are inherent features to malignant processes, thus making the diagnosis of hemophagocytic lymphohistiocytosis a challenge in patients with cancer. CASE PRESENTATION: We describe the case of a 54-year-old white man with history of metastatic maxillary sinus adenoid cystic carcinoma who had severe liver injury and cytopenias with progressive clinical deterioration. We performed an evaluation, by flow cytometry, of the expression of surface markers in his natural killer cells that revealed remarkable abnormalities. His syndrome eventually fulfilled criteria for hemophagocytic lymphohistiocytosis and he received therapy with steroids with interval clinical improvement. Unfortunately, he refused further cytotoxic treatment and died 2 weeks later. CONCLUSIONS: The conventional criteria for the diagnosis of hemophagocytic lymphohistiocytosis are suboptimal for adult patients with cancer resulting in delays in diagnosis and timely initiation of treatment. The diagnostic criteria have to be re-evaluated in patients with cancer; novel, easily available, and accurate diagnostic methods are needed.


Subject(s)
Carcinoma, Adenoid Cystic/complications , Lymphohistiocytosis, Hemophagocytic/complications , Lymphohistiocytosis, Hemophagocytic/diagnosis , Biomarkers, Tumor , Fatal Outcome , Flow Cytometry , Humans , Killer Cells, Natural/immunology , Lymphohistiocytosis, Hemophagocytic/immunology , Male , Middle Aged , Multiple Organ Failure
15.
Cell Rep ; 12(11): 1731-9, 2015 Sep 22.
Article in English | MEDLINE | ID: mdl-26344765

ABSTRACT

Autophagy, an intracellular degradation and energy recycling mechanism, is emerging as an important regulator of immune responses. However, the role of autophagy in regulating neutrophil functions is not known. We investigated neutrophil biology using myeloid-specific autophagy-deficient mice and found that autophagy deficiency reduced neutrophil degranulation in vitro and in vivo. Mice with autophagy deficiency showed reduced severity of several neutrophil-mediated inflammatory and autoimmune disease models, including PMA-induced ear inflammation, LPS-induced breakdown of blood-brain barrier, and experimental autoimmune encephalomyelitis. NADPH oxidase-mediated reactive oxygen species generation was also reduced in autophagy-deficient neutrophils, and inhibition of NADPH oxidase reduced neutrophil degranulation, suggesting NADPH oxidase to be a player at the intersection of autophagy and degranulation. Overall, this study establishes autophagy as an important regulator of neutrophil functions and neutrophil-mediated inflammation in vivo.


Subject(s)
Autophagy/physiology , Inflammation/metabolism , Inflammation/pathology , Neutrophils/metabolism , Neutrophils/pathology , Animals , Inflammation Mediators/metabolism , Mice , Mice, Inbred C57BL , NADPH Oxidases/metabolism , Reactive Oxygen Species/metabolism
16.
Biosens Bioelectron ; 74: 698-704, 2015 Dec 15.
Article in English | MEDLINE | ID: mdl-26210466

ABSTRACT

Microarrays are a powerful platform for rapid and multiplexed analysis in a wide range of research fields. Electrical readout systems have emerged as an alternative to conventional optical methods for microarray analysis thanks to its potential advantages like low-cost, low-power and easy miniaturization of the required instrumentation. In this work an automated electrical readout system for low-cost glass-slide microarrays is described. The system enables the simultaneous conductimetric detection of up to 36 biorecognition events by incorporating an array of interdigitated electrode transducers. A polydimethylsiloxane microfluidic structure has been designed that creates microwells over the transducers and incorporates the microfluidic channels required for filling and draining them with readout and cleaning solutions, thus making the readout process fully automated. Since the capture biomolecules are not immobilized on the transducer surface this readout system is reusable, in contrast to previously reported electrochemical microarrays. A low-density microarray based on a competitive enzymatic immunoassay for atrazine detection was used to test the performance of the readout system. The electrical assay shows a detection limit of 0.22±0.03 µg L(-1) similar to that obtained with fluorescent detection and allows the direct determination of the pesticide in polluted water samples. These results proved that an electrical readout system such as the one presented in this work is a reliable and cost-effective alternative to fluorescence scanners for the analysis of low-density microarrays.


Subject(s)
Biological Assay/instrumentation , Biosensing Techniques/instrumentation , Conductometry/instrumentation , Glass/chemistry , Lab-On-A-Chip Devices , Microarray Analysis/instrumentation , Equipment Design , Equipment Failure Analysis , Immunoassay/instrumentation , Microelectrodes , Reproducibility of Results , Sensitivity and Specificity
17.
Blood ; 124(19): 3007-15, 2014 Nov 06.
Article in English | MEDLINE | ID: mdl-25202140

ABSTRACT

Langerhans cell histiocytosis (LCH) is a myeloproliferative disorder characterized by lesions composed of pathological CD207(+) dendritic cells with an inflammatory infiltrate. BRAFV600E remains the only recurrent mutation reported in LCH. In order to evaluate the spectrum of somatic mutations in LCH, whole exome sequencing was performed on matched LCH and normal tissue samples obtained from 41 patients. Lesions from other histiocytic disorders, juvenile xanthogranuloma, Erdheim-Chester disease, and Rosai-Dorfman disease were also evaluated. All of the lesions from histiocytic disorders were characterized by an extremely low overall rate of somatic mutations. Notably, 33% (7/21) of LCH cases with wild-type BRAF and none (0/20) with BRAFV600E harbored somatic mutations in MAP2K1 (6 in-frame deletions and 1 missense mutation) that induced extracellular signal-regulated kinase (ERK) phosphorylation in vitro. Single cases of somatic mutations of the mitogen-activated protein kinase (MAPK) pathway genes ARAF and ERBB3 were also detected. The ability of MAPK pathway inhibitors to suppress MAPK kinase and ERK phosphorylation in cell culture and primary tumor models was dependent on the specific LCH mutation. The findings of this study support a model in which ERK activation is a universal end point in LCH arising from pathological activation of upstream signaling proteins.


Subject(s)
Histiocytosis, Langerhans-Cell/genetics , Histiocytosis, Langerhans-Cell/metabolism , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 1/metabolism , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Dendritic Cells/metabolism , Disease Progression , Erdheim-Chester Disease/genetics , Erdheim-Chester Disease/metabolism , HEK293 Cells , Histiocytosis, Sinus/genetics , Histiocytosis, Sinus/metabolism , Humans , MAP Kinase Signaling System/genetics , Mutation, Missense , Xanthogranuloma, Juvenile/genetics , Xanthogranuloma, Juvenile/metabolism
18.
Anal Chim Acta ; 832: 44-50, 2014 Jun 17.
Article in English | MEDLINE | ID: mdl-24890693

ABSTRACT

Low-density protein microarrays are emerging tools in diagnostics whose deployment could be primarily limited by the cost of fluorescence detection schemes. This paper describes an electrical readout system of microarrays comprising an array of gold interdigitated microelectrodes and an array of polydimethylsiloxane microwells, which enabled multiplexed detection of up to thirty six biological events on the same substrate. Similarly to fluorescent readout counterparts, the microarray can be developed on disposable glass slide substrates. However, unlike them, the presented approach is compact and requires a simple and inexpensive instrumentation. The system makes use of urease labeled affinity reagents for developing the microarrays and is based on detection of conductivity changes taking place when ionic species are generated in solution due to the catalytic hydrolysis of urea. The use of a polydimethylsiloxane microwell array facilitates the positioning of the measurement solution on every spot of the microarray. Also, it ensures the liquid tightness and isolation from the surrounding ones during the microarray readout process, thereby avoiding evaporation and chemical cross-talk effects that were shown to affect the sensitivity and reliability of the system. The performance of the system is demonstrated by carrying out the readout of a microarray for boldenone anabolic androgenic steroid hormone. Analytical results are comparable to those obtained by fluorescent scanner detection approaches. The estimated detection limit is 4.0 ng mL(-1), this being below the threshold value set by the World Anti-Doping Agency and the European Community.


Subject(s)
Electric Conductivity , Microelectrodes , Protein Array Analysis/instrumentation , Immunoassay/instrumentation , Immunoassay/methods , Protein Array Analysis/methods , Sensitivity and Specificity , Testosterone/analogs & derivatives , Testosterone/chemistry
19.
Am J Respir Crit Care Med ; 189(1): 16-29, 2014 Jan 01.
Article in English | MEDLINE | ID: mdl-24251647

ABSTRACT

RATIONALE: IL-4Rα, the common receptor component for IL-4 and IL-13, plays a critical role in IL-4- and IL-13-mediated signaling pathways that regulate airway inflammation and remodeling. However, the regulatory mechanisms underlying IL-4Rα turnover and its signal termination remain elusive. OBJECTIVES: To evaluate the role of STUB1 (STIP1 homology and U-Box containing protein 1) in regulating IL-4R signaling in airway inflammation. METHODS: The roles of STUB1 in IL-4Rα degradation and its signaling were investigated by immunoblot, immunoprecipitation, and flow cytometry. The involvement of STUB1 in airway inflammation was determined in vivo by measuring lung inflammatory cells infiltration, mucus production, serum lgE levels, and alveolar macrophage M2 activation in STUB1(-/-) mice. STUB1 expression was evaluated in airway epithelium of patients with asthma and lung tissues of subjects with chronic obstructive pulmonary disease. MEASUREMENTS AND MAIN RESULTS: STUB1 interacted with IL-4Rα and targeted it for ubiquitination-mediated proteasomal degradation, terminating IL-4 or IL-13 signaling. STUB1 knockout cells showed increased levels of IL-4Rα and sustained STAT6 activation, whereas STUB1 overexpression reduced IL-4Rα levels. Mice deficient in STUB1 had spontaneous airway inflammation, alternative M2 activation of alveolar macrophage, and increased serum IgE. STUB1 levels were increased in airways of subjects with asthma or chronic obstructive pulmonary disease, suggesting that up-regulation of STUB1 might be an important feedback mechanism to dampen IL-4R signaling in airway inflammation. CONCLUSIONS: Our study identified a previously uncharacterized role for STUB1 in regulating IL-4R signaling, which might provide a new strategy for attenuating airway inflammation.


Subject(s)
Pneumonia/physiopathology , Receptors, Interleukin-4/physiology , Signal Transduction/physiology , Ubiquitin-Protein Ligases/physiology , Adult , Animals , Asthma/physiopathology , Child , Down-Regulation/physiology , Female , Flow Cytometry , Humans , Immunoblotting , Immunoprecipitation , Macrophage Activation/physiology , Male , Mice , Mice, Knockout , Proteasome Endopeptidase Complex/physiology , Pulmonary Disease, Chronic Obstructive/physiopathology , Real-Time Polymerase Chain Reaction , Receptors, Cell Surface/physiology , STAT6 Transcription Factor/physiology , Ubiquitin/physiology
20.
Immunity ; 39(3): 537-47, 2013 Sep 19.
Article in English | MEDLINE | ID: mdl-24035364

ABSTRACT

Autophagy and phagocytosis are conserved cellular functions involved in innate immunity. However, the nature of their interactions remains unclear. We evaluated the role of autophagy in regulating phagocytosis in macrophages from myeloid-specific autophagy-related gene 7-deficient (Atg7⁻/⁻) mice. Atg7⁻/⁻ macrophages exhibited higher bacterial uptake when infected with Mycobacterium tuberculosis (Mtb) or with M. tuberculosis var. bovis BCG (BCG). In addition, BCG-infected Atg7⁻/⁻ mice showed increased bacterial loads and exacerbated lung inflammatory responses. Atg7⁻/⁻ macrophages had increased expression of two class A scavenger receptors: macrophage receptor with collagenous structure (MARCO) and macrophage scavenger receptor 1 (MSR1). The increase in scavenger receptors was caused by increased activity of the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) transcription factor resulting from accumulated sequestosome 1 (SQSTM1 or p62) in Atg7⁻/⁻ macrophages. These insights increase our understanding of the host-pathogen relationship and suggest that therapeutic strategies should be designed to include modulation of both phagocytosis and autophagy.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Autophagy/immunology , Heat-Shock Proteins/metabolism , Microtubule-Associated Proteins/metabolism , NF-E2-Related Factor 2/metabolism , Phagocytosis/immunology , Receptors, Immunologic/biosynthesis , Scavenger Receptors, Class A/biosynthesis , Animals , Autophagy-Related Protein 7 , Bacterial Load/immunology , Cells, Cultured , Macrophages/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Microtubule-Associated Proteins/genetics , Mycobacterium Infections/immunology , Mycobacterium bovis/growth & development , Mycobacterium bovis/immunology , Mycobacterium tuberculosis/growth & development , Mycobacterium tuberculosis/immunology , Pneumonia/immunology , Pneumonia/microbiology , Receptors, Scavenger/biosynthesis , Receptors, Scavenger/immunology , Sequestosome-1 Protein , Up-Regulation
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