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1.
Anal Chem ; 94(30): 10626-10635, 2022 08 02.
Article in English | MEDLINE | ID: mdl-35866879

ABSTRACT

Barcoding and pooling cells for processing as a composite sample are critical to minimize technical variability in multiplex technologies. Fluorescent cell barcoding has been established as a standard method for multiplexing in flow cytometry analysis. In parallel, mass-tag barcoding is routinely used to label cells for mass cytometry. Barcode reagents currently used label intracellular proteins in fixed and permeabilized cells and, therefore, are not suitable for studies with live cells in long-term culture prior to analysis. In this study, we report the development of fluorescent palladium-based hybrid-tag nanotrackers to barcode live cells for flow and mass cytometry dual-modal readout. We describe the preparation, physicochemical characterization, efficiency of cell internalization, and durability of these nanotrackers in live cells cultured over time. In addition, we demonstrate their compatibility with standardized cytometry reagents and protocols. Finally, we validated these nanotrackers for drug response assays during a long-term coculture experiment with two barcoded cell lines. This method represents a new and widely applicable advance for fluorescent and mass-tag barcoding that is independent of protein expression levels and can be used to label cells before long-term drug studies.


Subject(s)
Electronic Data Processing , Fluorescent Dyes , Cell Line , Flow Cytometry/methods , Fluorescent Dyes/chemistry , Proteomics
2.
J Immunol ; 203(8): 2210-2221, 2019 10 15.
Article in English | MEDLINE | ID: mdl-31519862

ABSTRACT

HIV-1 infection expands large populations of late-stage differentiated CD8 T cells that may persist long after viral escape from TCR recognition. In this study, we investigated whether such CD8 T cell populations can perform unconventional innate-like antiviral effector functions. Chronic untreated HIV-1 infection was associated with elevated numbers of CD45RA+CD57+ terminal effector CD8 T cells expressing FcγRIIIA (CD16). The FcγRIIIA+ CD8 T cells displayed a distinctive transcriptional profile between conventional CD8 T cells and NK cells, characterized by high levels of IKZF2 and low expression of IL7R This transcriptional profile translated into a distinct NKp80+ IL-7Rα- surface phenotype with high expression of the Helios transcription factor. Interestingly, the FcγRIIIA+ CD8 T cells mediated HIV-specific Ab-dependent cellular cytotoxicity (ADCC) activity at levels comparable with NK cells on a per cell basis. The FcγRIIIA+ CD8 T cells were highly activated in a manner that correlated positively with expansion of the CD8 T cell compartment and with plasma levels of soluble mediators of antiviral immunity and inflammation such as IP-10, TNF, IL-6, and TNFRII. The frequency of FcγRIIIA+ CD8 T cells persisted as patients initiated suppressive antiretroviral therapy, although their activation levels declined. These data indicate that terminally differentiated effector CD8 T cells acquire enhanced innate cell-like characteristics during chronic viral infection and suggest that HIV-specific ADCC is a function CD8 T cells use to target HIV-infected cells. Furthermore, as the FcγRIIIA+ CD8 T cells persist in treatment, they contribute significantly to the ADCC-capable effector cell pool in patients on antiretroviral therapy.


Subject(s)
Antibody-Dependent Cell Cytotoxicity/immunology , CD8-Positive T-Lymphocytes/immunology , HIV Infections/immunology , HIV-1/immunology , Ikaros Transcription Factor/immunology , Receptors, IgG/genetics , Receptors, Interleukin-7/immunology , Adolescent , Adult , Antibody-Dependent Cell Cytotoxicity/genetics , CD8-Positive T-Lymphocytes/pathology , Cell Differentiation/immunology , Cohort Studies , Female , Humans , Male , Middle Aged , Prospective Studies , Receptors, IgG/immunology , Young Adult
3.
J Virol ; 93(7)2019 04 01.
Article in English | MEDLINE | ID: mdl-30700608

ABSTRACT

HIV infection is controlled immunologically in a small subset of infected individuals without antiretroviral therapy (ART), though the mechanism of control is unclear. CD8+ T cells are a critical component of HIV control in many immunological controllers. NK cells are also believed to have a role in controlling HIV infection, though their role is less well characterized. We used mass cytometry to simultaneously measure the levels of expression of 24 surface markers on peripheral NK cells from HIV-infected subjects with various degrees of HIV natural control; we then used machine learning to identify NK cell subpopulations that differentiate HIV controllers from noncontrollers. Using CITRUS (cluster identification, characterization, and regression), we identified 3 NK cell subpopulations that differentiated subjects with chronic HIV viremia (viremic noncontrollers [VNC]) from individuals with undetectable HIV viremia without ART (elite controllers [EC]). In a parallel approach, we identified 11 NK cell subpopulations that differentiated HIV-infected subject groups using k-means clustering after dimensionality reduction by t-neighbor stochastic neighbor embedding (tSNE) or linear discriminant analysis (LDA). Among these additional 11 subpopulations, the frequencies of 5 correlated with HIV DNA levels; importantly, significance was retained in 2 subpopulations in analyses that included only cohorts without detectable viremia. By comparing the surface marker expression patterns of all identified subpopulations, we revealed that the CD11b+ CD57- CD161+ Siglec-7+ subpopulation of CD56dim CD16+ NK cells are more abundant in EC and HIV-negative controls than in VNC and that the frequency of these cells correlated with HIV DNA levels. We hypothesize that this population may have a role in immunological control of HIV infection.IMPORTANCE HIV infection results in the establishment of a stable reservoir of latently infected cells; ART is usually required to keep viral replication under control and disease progression at bay, though a small subset of HIV-infected subjects can control HIV infection without ART through immunological mechanisms. In this study, we sought to identify subpopulations of NK cells that may be involved in the natural immunological control of HIV infection. We used mass cytometry to measure surface marker expression on peripheral NK cells. Using two distinct semisupervised machine learning approaches, we identified a CD11b+ CD57- CD161+ Siglec-7+ subpopulation of CD56dim CD16+ NK cells that differentiates HIV controllers from noncontrollers. These cells can be sorted out for future functional studies to assess their potential role in the immunological control of HIV infection.


Subject(s)
HIV Infections/immunology , Killer Cells, Natural/immunology , Killer Cells, Natural/virology , CD11b Antigen/immunology , CD56 Antigen/immunology , CD57 Antigens/immunology , CD8-Positive T-Lymphocytes/virology , Cell Line, Tumor , DNA, Viral/immunology , HIV Infections/virology , HIV-1/immunology , Humans , K562 Cells , NK Cell Lectin-Like Receptor Subfamily B/immunology , Receptors, IgG/immunology , Viremia/immunology , Viremia/virology
4.
Biomacromolecules ; 19(12): 4607-4616, 2018 12 10.
Article in English | MEDLINE | ID: mdl-30376297

ABSTRACT

Transdermal immunization is highly attractive because of the skin's accessibility and unique immunological characteristics. However, it remains a relatively unexplored route of administration because of the great difficulty of transporting antigens past the outermost layer of skin, the stratum corneum. In this article, the abilities of three poly( N-vinylcaprolactam) (PVCL)-based thermoresponsive assemblies-PVCL hydrogels and nanogels plus novel film forming PVCL/acrylic nanogels-to act as protein delivery systems were investigated. Similar thermal responses were observed in all systems, with transition temperatures close to 32 °C, close to that of the skin surface. The investigated dermal delivery systems showed no evidence of cytotoxicity in human fibroblasts and were able to load and release ovalbumin (OVA), a well-studied antigen, in a temperature-dependent manner in vitro. The penetration of OVA into ex vivo human skin following topical application was evaluated, where enhanced skin delivery was seen for the OVA-loaded PVCL systems relative to administration of the protein alone. The distinct protein release and skin penetration profiles observed for the different PVCL assemblies were here discussed on the basis of their structural differences.


Subject(s)
Antigens/chemistry , Drug Carriers , Hydrogels/chemistry , Nanoparticles/chemistry , Administration, Cutaneous , Antigens/administration & dosage , Caprolactam/chemistry , Dermis/drug effects , Dermis/pathology , Epidermis/drug effects , Epidermis/pathology , Humans , Hydrogels/administration & dosage , Nanoparticles/administration & dosage , Ovalbumin/administration & dosage , Ovalbumin/chemistry , Polyethylene Glycols/chemical synthesis , Polyethyleneimine/chemistry , Polymers/administration & dosage , Polymers/chemistry , Skin/metabolism , Skin Absorption/drug effects , Temperature , Vaccination
5.
Cytometry A ; 91(12): 1150-1163, 2017 12.
Article in English | MEDLINE | ID: mdl-29205767

ABSTRACT

Mass cytometry (or CyTOF) is an atomic mass spectrometry-based single-cell immunoassay technology, which has provided an increasingly systematic and sophisticated view in basic biological and clinical studies. Using elemental reporters composed of stable heavy metal isotopes, more than 50 cellular parameters are measured simultaneously. However, this current multiplexing does not meet the theoretical capability of CyTOF instrumentation with 135 detectable channels, primarily due to the limitation of available chemistries for conjugating elemental mass tags to affinity reagents. To address this issue, we develop herein additional metallic mass tag based on bismuth-209 (209 Bi) for efficient conjugation to monoclonal antibody. This enables the use of an addtional channel m/z = 209 of CyTOF for single-cell immunoassays. Bismuth has nearly the same charge-to-radius ratio as lanthanide elements; thus, bismuth(III) cations (209 Bi3+ ) could coordinate with DTPA chelators in the same geometry of O- and N-donor groups as that of lanthanide. In this report, the coordination chemistry of 209 Bi3+ with DTPA chelators and Maxpar® X8 polymers were investigated in details. Accordingly, the protocols of conjugating antibody with bismuth mass tag were provided. A method based on UV-Vis absorbance at 280 nm of 209 Bi3+ -labeling DTPA complexes was developed to evaluate the stoichiometric ratio of 209 Bi3+ cations to the conjugated antibody. Side-by-side single-cell analysis experiments with bismuth- and lanthanide-tagged antibodies were carried out to compare the analytical sensitivities. The measurement accuracy of bismuth-tagged antibody was validated within in vitro assay using primary human natural killer cells. Furthermore, bismuth-tagged antibodies were successfully employed in cell cycle measurements and high-dimensional phenotyping immunoassays. © 2017 International Society for Advancement of Cytometry.


Subject(s)
Bismuth/chemistry , Flow Cytometry/methods , Killer Cells, Natural , Mass Spectrometry/methods , Single-Cell Analysis/methods , Antibodies, Monoclonal/chemistry , Humans , Immunoassay , Immunoconjugates/chemistry
6.
Cytometry A ; 91(2): 180-189, 2017 02.
Article in English | MEDLINE | ID: mdl-28094900

ABSTRACT

To quantify visual and spatial information in single cells with a throughput of thousands of cells per second, we developed Subcellular Localization Assay (SLA). This adaptation of Proximity Ligation Assay expands the capabilities of flow cytometry to include data relating to localization of proteins to and within organelles. We used SLA to detect the nuclear import of transcription factors across cell subsets in complex samples. We further measured intranuclear re-localization of target proteins across the cell cycle and upon DNA damage induction. SLA combines multiple single-cell methods to bring about a new dimension of inquiry and analysis in complex cell populations. © 2017 International Society for Advancement of Cytometry.


Subject(s)
Flow Cytometry/methods , High-Throughput Screening Assays/methods , Single-Cell Analysis/methods , Cell Nucleus/metabolism , Cytoplasm/metabolism , Cytoplasm/ultrastructure , DNA Damage/genetics , Humans , Protein Transport/genetics , Subcellular Fractions/ultrastructure
7.
Clin Chem Lab Med ; 55(4): 595-604, 2017 Mar 01.
Article in English | MEDLINE | ID: mdl-27658149

ABSTRACT

BACKGROUND: P35 and P22 Toxoplasma gondii proteins are recognized by specific IgG at the early infection stage, making them ideal for acute toxoplasmosis pregnancy control. Both proteins have been studied to discriminate between acute and chronic toxoplasmosis. However, results were hardly comparable because different protein obtainment procedures led to different antigens, the reference panels used were not optimally typified, and avidity tests were either not performed or narrowly examined. METHODS: We bioinformatically predicted P35 and P22 regions with the highest density of epitopes, and expressed them in pET32/BL21DE3 alternative expression system, obtaining the soluble proteins rP35a and rP22a. We assessed their diagnostic performance using pregnant woman serum samples typified as: not infected, NI (IgG-, IgM-), typical-chronic, TC (IgM-, IgG+), presumably acute, A (IgG+, IgM+, low-avidity IgG), and recently chronic, RC (IgG+, IgM+, high-avidity IgG). RESULTS: rP35a performed better than rP22a to differentiate A from RC, the areas under the curve (AUC) being 0.911 and 0.818, respectively. They, however, performed similarly to differentiate A from TC+RC (AUC: 0.915 and 0.907, respectively). rP35a and rP22a evaluation by avidity ELISA to discriminate A from RC rendered AUC values of 0.974 and 0.921, respectively. The indirect ELISA and avidity ELISA results analyzed in tandem were consistent with those obtained using commercial kits. CONCLUSIONS: rP35a and rP22a features suggest that, with complementary use, they could replace parasite lysate for toxoplasmosis infection screening and for acute toxoplasmosis diagnosis. Our proposal should be validated by a longitudinal study and may lead to a reliable toxoplasmosis pregnancy control, performing tests in only one serum sample.


Subject(s)
Antigens, Protozoan/blood , Enzyme-Linked Immunosorbent Assay/methods , Pregnancy Complications, Parasitic/diagnosis , Protozoan Proteins/blood , Toxoplasmosis/diagnosis , Antigens, Protozoan/genetics , Antigens, Protozoan/immunology , Antigens, Protozoan/metabolism , Female , Humans , Immunoglobulin G/blood , Pregnancy , Protozoan Proteins/genetics , Protozoan Proteins/immunology , Protozoan Proteins/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Toxoplasma/pathogenicity
8.
J Immunol ; 195(7): 3262-72, 2015 Oct 01.
Article in English | MEDLINE | ID: mdl-26283480

ABSTRACT

NK cells play an important role in the defense against viral infections. However, little is known about the regulation of NK cell responses during the first days of acute viral infections in humans. In this study, we used the live attenuated yellow fever virus (YFV) vaccine 17D as a human in vivo model to study the temporal dynamics and regulation of NK cell responses in an acute viral infection. YFV induced a robust NK cell response in vivo, with an early activation and peak in NK cell function at day 6, followed by a delayed peak in Ki67 expression, which was indicative of proliferation, at day 10. The in vivo NK cell response correlated positively with plasma type I/III IFN levels at day 6, as well as with the viral load. YFV induced an increased functional responsiveness to IL-12 and IL-18, as well as to K562 cells, indicating that the NK cells were primed in vivo. The NK cell responses were associated primarily with the stage of differentiation, because the magnitude of induced Ki67 and CD69 expression was distinctly higher in CD57(-) NK cells. In contrast, NK cells expressing self- and nonself-HLA class I-binding inhibitory killer cell Ig-like receptors contributed, to a similar degree, to the response. Taken together, our results indicate that NK cells are primed by type I/III IFN in vivo early after YFV infection and that their response is governed primarily by the differentiation stage, independently of killer cell Ig-like receptor/HLA class I-mediated inhibition or education.


Subject(s)
Interferon Type I/immunology , Killer Cells, Natural/immunology , Receptors, KIR/immunology , Yellow Fever Vaccine/immunology , Yellow fever virus/immunology , Adult , Antibodies, Neutralizing/immunology , Antigens, CD/biosynthesis , Antigens, Differentiation, T-Lymphocyte/biosynthesis , B-Lymphocytes/immunology , CD57 Antigens/metabolism , Cell Differentiation/immunology , Cell Proliferation , Histocompatibility Antigens Class I/immunology , Humans , Interferon Type I/blood , Interleukin-12 Subunit p35/immunology , Interleukin-18/immunology , K562 Cells , Ki-67 Antigen/biosynthesis , Killer Cells, Natural/cytology , Lectins, C-Type/biosynthesis , Lymphocyte Activation/immunology , Middle Aged , T-Lymphocytes/immunology , Vaccines, Attenuated/immunology , Viral Load/immunology , Viral Vaccines/immunology
9.
Parasitology ; 144(8): 1073-1078, 2017 Jul.
Article in English | MEDLINE | ID: mdl-28290263

ABSTRACT

The aim of this work was to obtain a reagent based on latex particles for ruling out acute toxoplasmosis in pregnant women by immunoagglutination (IA). Latex-protein complexes (LPC) were previously synthesized coupling the recombinant protein of Toxoplasma gondii P22Ag and the homogenate of the parasite to latex particles with different size, chemical functionality and charge density. LPC were tested in IA assays against a panel of 72 pregnant women serum samples. Results were analysed through receiver operating characteristic curves, determining area under the curve (AUC), sensitivity, specificity positive and negative predictive values (PPV and NPV, respectively). It was observed that the antigenicity of proteins was not affected during sensitization by either physical adsorption or covalent coupling. The best results in the sense of maximizing discrimination of low avidity sera from chronic ones were observed for the IA test based on latex particles with carboxyl functionality and the recombinant P22Ag, obtaining an AUC of 0·94, a sensitivity of 100% and a NPV of 100%. In this way, the proposed test could be useful for the toxoplasmosis diagnosis in pregnant women, with the advantages of being cheap, rapid and easy to be implemented.


Subject(s)
Agglutination Tests , Antigens, Protozoan/chemistry , Latex/immunology , Reagent Kits, Diagnostic , Toxoplasma/immunology , Toxoplasmosis/diagnosis , Female , Humans , Latex/metabolism , Pregnancy , Recombinant Proteins/immunology , Sensitivity and Specificity
10.
Exp Parasitol ; 182: 9-15, 2017 Nov.
Article in English | MEDLINE | ID: mdl-28867354

ABSTRACT

Visceral leishmaniasis is a public health problem worldwide. The early diagnosis in dogs is crucial, since they are an epidemiologically relevant reservoir of the disease. The aim of a field study is to early identify the disease allowing rapid intervention to reduce its effects. We propose an immunoagglutination test as a visual in situ method for diagnosis of canine visceral leishmaniasis. Latex-protein complexes were sensitized by covalent coupling of a chimeric recombinant antigen of Leishmania spp. onto polystyrene latex with carboxyl functionality. The reaction time and the antigen concentration under which the immunoagglutination assay shows greater discrimination between the responses of a positive control serum and a negative control serum were determined. Then, the latex-protein complexes were evaluated as a visual diagnostic tool with a panel of 170 sera. The test may be read between 2 and 5 min and can be performed even using sera with elevated concentration of lipids, bilirubin or with variable percentage of hemolysis. The sensitivity, the specificity and the diagnostic accuracy were 78%; 100% and >80%, respectively. The visual immunoagglutination test is of potential application as a method for field studies because it shows results in less than 5 min, it is easy to implement and does not require sophisticated equipment.


Subject(s)
Antibodies, Protozoan/blood , Dog Diseases/diagnosis , Latex Fixation Tests/veterinary , Leishmania infantum/immunology , Leishmaniasis, Visceral/veterinary , Animals , Antigens, Protozoan/immunology , Blotting, Western/veterinary , Disease Reservoirs , Dog Diseases/parasitology , Dogs , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/parasitology , Recombinant Proteins/immunology , Sensitivity and Specificity
11.
J Immunol ; 190(5): 2150-8, 2013 Mar 01.
Article in English | MEDLINE | ID: mdl-23338234

ABSTRACT

The live attenuated yellow fever virus (YFV) 17D vaccine provides a good model to study immune responses to an acute viral infection in humans. We studied the temporal dynamics, composition, and character of the primary human T cell response to YFV. The acute YFV-specific effector CD8 T cell response was broad and complex; it was composed of dominant responses that persisted into the memory population, as well as of transient subdominant responses that were not detected at the memory stage. Furthermore, HLA-A2- and HLA-B7-restricted YFV epitope-specific effector cells predominantly displayed a CD45RA(-)CCR7(-)PD-1(+)CD27(high) phenotype, which transitioned into a CD45RA(+)CCR7(-)PD-1(-)CD27(low) memory population phenotype. The functional profile of the YFV-specific CD8 T cell response changed in composition as it matured from an effector- to a memory-type response, and it tended to become less polyfunctional during the course of this transition. Interestingly, activation of CD4 T cells, as well as FOXP3(+) T regulatory cells, in response to YFV vaccination preceded the kinetics of the CD8 T cell response. The present results contribute to our understanding of how immunodominance patterns develop, as well as the phenotypic and functional characteristics of the primary human T cell response to a viral infection as it evolves and matures into memory.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Yellow Fever Vaccine/immunology , Yellow Fever/prevention & control , Yellow fever virus/immunology , Adolescent , Adult , CD4-Positive T-Lymphocytes/pathology , CD4-Positive T-Lymphocytes/virology , CD8-Positive T-Lymphocytes/pathology , CD8-Positive T-Lymphocytes/virology , Cytokines/biosynthesis , Cytokines/immunology , HLA Antigens/genetics , HLA Antigens/immunology , Histocompatibility Testing , Humans , Immunologic Memory , Immunophenotyping , Middle Aged , Time Factors , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/immunology , Yellow Fever/immunology , Yellow Fever/pathology , Yellow Fever/virology , Yellow Fever Vaccine/administration & dosage
12.
Trop Med Int Health ; 19(1): 37-46, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24219561

ABSTRACT

OBJECTIVE: To determine the conditions under which the immunoagglutination assay to detect Chagas disease, obtained from a novel latex-(chimeric recombinant antigen) complex, shows greater discrimination between the responses of a positive control serum and a negative control serum. METHODS: The following variables were determined: (i) the sensitisation mechanism, (ii) the emulsifier employed for protein desorption, (iii) the reaction time, (iv) the ionic strength of the reaction medium, (v) the particle concentration, (vi) the presence of blocking agents, (vii) the presence of polyethyleneglycol as potentiator of reaction and (viii) the antigen and antibody concentrations. The search of optimal conditions was investigated by varying one variable at a time. To this effect, monodisperse latex particles sensitised with a recombinant chimeric protein (CP1) were subjected to different conditions. The agglutination reaction was followed by measuring the changes in the optical absorbance by turbidimetry. RESULTS: The maximum discrimination between negative and positive sera was obtained at a reaction time of 5 min, when latex complexes with a concentration of covalently coupled protein of 2.90 mg/m(2) were put in contact with undiluted sera in buffer borate pH 8-20 mm containing glycine (0.1 m) and polyethyleneglycol 8000 (3% w/v). Finally, the latex-protein complex was tested under the obtained optimal conditions, with a panel of Trypanosoma cruzi-positive sera, leishmaniasis-positive sera and -negative sera for both parasites. CONCLUSION: The immunoagglutination test based on the latex-CP1 complex could be used as a screening method for detecting Chagas disease. This test is rapid, easy to implement and could be used under field conditions; but its results should be confirmed by reference techniques like ELISA, HAI, and IFI.


Subject(s)
Antigens, Protozoan/blood , Chagas Disease/diagnosis , Trypanosoma cruzi/immunology , Chagas Disease/blood , Chagas Disease/immunology , Enzyme-Linked Immunosorbent Assay/methods , Humans , Latex Fixation Tests/methods , Recombinant Proteins/blood , Recombinant Proteins/immunology
13.
Trop Med Int Health ; 19(11): 1346-54, 2014 Nov.
Article in English | MEDLINE | ID: mdl-25175083

ABSTRACT

OBJECTIVE: To evaluate the diagnostic performance of novel latex-protein complexes obtained from different antigens of Trypanosoma cruzi through immunoagglutination test using a panel of T. cruzi-positive sera, leishmaniasis-positive sera and negative sera for both parasites. METHODS: Complexes' behaviour using total parasite homogenate (TPH), two simple recombinant proteins (RP1 and RP5) and two chimeric recombinant proteins (CP1 and CP2) was comparatively evaluated. The area under ROC curves was used as an index of accuracy. Sensitivity, specificity and discrimination efficiency were assessed. RESULTS: All recombinant antigens showed higher specificity than TPH. The lower specificity of TPH was mainly due to cross-reacting peptides between T. cruzi and Leishmania spp. In turn, all performance indicators were higher for CP1 and CP2 than for RP1 and RP5. The carboxylated latex-CP2 (C2-CP2) complex was able to detect antibodies against T. cruzi. The values of area under ROC curve (0.96), sensitivity (92.3%, 95% CI: 79.4-100.0%) and specificity (84.0%, 95% CI: 67.6-100.0%) indicate that the assay could be used as a screening test. CONCLUSION: The C2-CP2 complex could be an important tool to carry out sero-epidemiological studies.


Subject(s)
Antibodies, Protozoan/blood , Antigens, Protozoan/blood , Chagas Disease/diagnosis , Trypanosoma cruzi/immunology , Chagas Disease/immunology , Cross Reactions/immunology , Humans , Latex Fixation Tests , Leishmania/immunology , ROC Curve , Recombinant Proteins/blood , Recombinant Proteins/immunology , Sensitivity and Specificity
14.
Gastroenterology ; 142(4): 978-88, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22248663

ABSTRACT

BACKGROUND & AIMS: Polymorphisms in the IL28B gene have been associated with clearance of hepatitis C virus (HCV), indicating a role for type III interferons (IFNs) in HCV infection. Little is known about the function of type III IFNs in intrinsic antiviral innate immunity. METHODS: We used in vivo and in vitro models to characterize the role of the type III IFNs in HCV infection and analyzed gene expression in liver biopsy samples from HCV-infected chimpanzees and patients. Messenger RNA and protein expression were studied in HCV-infected hepatoma cell lines and primary human hepatocytes. RESULTS: HCV infection of primary human hepatocytes induced production of chemokines and type III IFNs, including interleukin (IL)-28, and led to expression of IFN-stimulated genes (ISGs). Chimpanzees infected with HCV showed rapid induction of hepatic type III IFN, associated with up-regulation of ISGs and minimal induction of type I IFNs. In liver biopsy specimens from HCV-infected patients, hepatic expression of IL-28 correlated with levels of ISGs but not of type I IFNs. HCV infection produced extensive changes with gene expression in addition to ISGs in primary human hepatocytes. The induction of type III IFNs is regulated by IFN regulatory factor 3 and nuclear factor κB. Type III IFNs up-regulate ISGs with a different kinetic profile than type 1 IFNs and induce a distinct set of genes, which might account for their functional differences. CONCLUSIONS: HCV infection results predominantly in induction of type III IFNs in livers of humans and chimpanzees; the level of induction correlates with hepatic levels of ISGs. These findings might account for the association among IL-28, level of ISGs, and recovery from HCV infection and provide a therapeutic strategy for patients who do not respond to IFN therapy.


Subject(s)
Hepacivirus/immunology , Hepatitis C, Chronic/immunology , Hepatocytes/immunology , Immunity, Innate , Interferons/metabolism , Liver/immunology , Animals , Antiviral Agents/therapeutic use , Biopsy , Cell Line, Tumor , Chemokine CXCL10/metabolism , Gene Expression Regulation , Hepacivirus/genetics , Hepatitis C, Chronic/drug therapy , Hepatitis C, Chronic/genetics , Hepatitis C, Chronic/pathology , Hepatocytes/pathology , Hepatocytes/virology , Humans , Interferon Regulatory Factor-3/metabolism , Interferon Type I/metabolism , Interferons/genetics , Interleukins/metabolism , Liver/pathology , Liver/virology , NF-kappa B/metabolism , Pan troglodytes , RNA, Messenger/metabolism , Signal Transduction , Time Factors , Up-Regulation
15.
Res Vet Sci ; 155: 69-75, 2023 Feb.
Article in English | MEDLINE | ID: mdl-36641975

ABSTRACT

Visceral leishmaniasis is a zoonotic infectious disease with a severe impact on humans and animals. Infection is transmitted by phlebotomine sand flies. The dogs are main reservoir for human infection. A rapid and accurate diagnosis of canine visceral leishmaniasis is essential for an efficient surveillance program. The aim of this study was to assess the performance of a rapid immunochromatographic strip test based on functionalized colored particles and a new recombinant antigenic protein, as a visual "in situ" method for the diagnosis of canine visceral leishmaniasis. The results were evaluated using an in-house ELISA assay with the same antigen. Both tests produced concordant results and the immunochromatographic strip test showed good diagnostic sensitivity (98%) and specificity (95%). Finally, meta-analysis was used to compare the sensitivity and specificity of the here developed test with the results of commercial immunochromatographic strip tests obtained from literature.


Subject(s)
Dog Diseases , Leishmania infantum , Leishmaniasis, Visceral , Dogs , Animals , Humans , Leishmaniasis, Visceral/diagnosis , Leishmaniasis, Visceral/veterinary , Microspheres , Antigens, Protozoan , Immunoassay/veterinary , Immunoassay/methods , Enzyme-Linked Immunosorbent Assay/veterinary , Enzyme-Linked Immunosorbent Assay/methods , Sensitivity and Specificity , Dog Diseases/diagnosis , Dog Diseases/epidemiology
16.
Semin Immunopathol ; 45(1): 43-59, 2023 01.
Article in English | MEDLINE | ID: mdl-36635516

ABSTRACT

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy. Its diagnosis at advanced stage compounded with its excessive genomic and cellular heterogeneity make curative treatment challenging. Two critical therapeutic challenges to overcome are carboplatin resistance and lack of response to immunotherapy. Carboplatin resistance results from diverse cell autonomous mechanisms which operate in different combinations within and across tumors. The lack of response to immunotherapy is highly likely to be related to an immunosuppressive HGSOC tumor microenvironment which overrides any clinical benefit. Results from a number of studies, mainly using transcriptomics, indicate that the immune tumor microenvironment (iTME) plays a role in carboplatin response. However, in patients receiving treatment, the exact mechanistic details are unclear. During the past decade, multiplex single-cell proteomic technologies have come to the forefront of biomedical research. Mass cytometry or cytometry by time-of-flight, measures up to 60 parameters in single cells that are in suspension. Multiplex cellular imaging technologies allow simultaneous measurement of up to 60 proteins in single cells with spatial resolution and interrogation of cell-cell interactions. This review suggests that functional interplay between cell autonomous responses to carboplatin and the HGSOC immune tumor microenvironment could be clarified through the application of multiplex single-cell proteomic technologies. We conclude that for better clinical care, multiplex single-cell proteomic technologies could be an integral component of multimodal biomarker development that also includes genomics and radiomics. Collection of matched samples from patients before and on treatment will be critical to the success of these efforts.


Subject(s)
Ovarian Neoplasms , Proteomics , Female , Humans , Carboplatin/therapeutic use , Ovarian Neoplasms/diagnosis , Ovarian Neoplasms/etiology , Ovarian Neoplasms/therapy , Tumor Microenvironment
17.
Methods Mol Biol ; 2424: 59-94, 2022.
Article in English | MEDLINE | ID: mdl-34918287

ABSTRACT

Mass cytometry aka Cytometry by Time-Of-Flight (CyTOF) is one of several recently developed multiparametric single-cell technologies designed to address cellular heterogeneity within healthy and diseased tissue. Mass cytometry is an adaptation of flow cytometry in which antibodies are labeled with stable heavy metal isotopes and the readout is by time-of-flight mass spectrometry. With minimal spillover between channels, mass cytometry enables readouts of up to 60 parameters per single cell. Critically, mass cytometry can identify minority cell populations that are lost in bulk tissue analysis. Mass cytometry has been used to great effect for the study of immune cells. We have extended its use to examine single cells within disaggregated solid tissues, specifically freshly resected tubo-ovarian high-grade serous tumors. Here we detail our protocols designed to ensure the production of high-quality single-cell datasets. The methodology can be modified to accommodate the study of other solid tissues.


Subject(s)
Ovarian Neoplasms , Antibodies , Female , Flow Cytometry , Humans , Isotopes , Mass Spectrometry , Single-Cell Analysis
18.
STAR Protoc ; 3(2): 101425, 2022 06 17.
Article in English | MEDLINE | ID: mdl-35693208

ABSTRACT

Trogocytosis is an active transport mechanism by which one cell extracts a plasma membrane fragment with embedded molecules from an adjacent cell in a contact-dependent process leading to the acquisition of a new function. Our protocol, which has general applicability, consolidates and optimizes existing protocols while highlighting key experimental variables to demonstrate that natural killer (NK) cells acquire the tetraspanin CD9 by trogocytosis from ovarian tumor cells. For complete details on the use and execution of this protocol, please refer to Gonzalez et al. (2021).


Subject(s)
Ovarian Neoplasms , Trogocytosis , Cell Membrane/metabolism , Female , Humans , Killer Cells, Natural/metabolism , Ovarian Neoplasms/metabolism
19.
Cell Stem Cell ; 29(12): 1653-1668.e8, 2022 12 01.
Article in English | MEDLINE | ID: mdl-36384141

ABSTRACT

In aging, skeletal muscle strength and regenerative capacity decline, due in part to functional impairment of muscle stem cells (MuSCs), yet the underlying mechanisms remain elusive. Here, we capitalize on mass cytometry to identify high CD47 expression as a hallmark of dysfunctional MuSCs (CD47hi) with impaired regenerative capacity that predominate with aging. The prevalent CD47hi MuSC subset suppresses the residual functional CD47lo MuSC subset through a paracrine signaling loop, leading to impaired proliferation. We uncover that elevated CD47 levels on aged MuSCs result from increased U1 snRNA expression, which disrupts alternative polyadenylation. The deficit in aged MuSC function in regeneration can be overcome either by morpholino-mediated blockade of CD47 alternative polyadenylation or antibody blockade of thrombospondin-1/CD47 signaling, leading to improved regeneration in aged mice, with therapeutic implications. Our findings highlight a previously unrecognized age-dependent alteration in CD47 levels and function in MuSCs, which underlies reduced muscle repair in aging.


Subject(s)
CD47 Antigen , Myoblasts , Animals , Mice , Muscle, Skeletal , Aging , Disease Progression
20.
J Immunol ; 183(10): 6612-8, 2009 Nov 15.
Article in English | MEDLINE | ID: mdl-19846870

ABSTRACT

NK cells are important innate immune effector cells, normally characterized as CD56(+)CD3(-) lymphocytes. In this study, we report that CD56(-)CD16(+) NK cells expand in many patients with chronic hepatitis C virus infection. These CD56(-) NK cells were functionally impaired with respect to cytokine production upon target cell recognition, in comparison to CD56(dim) and CD56(bright) NK cell subsets. In particular, CD56(-) NK cells were strikingly defective in their polyfunctional response as measured by the coexpression of MIP-1beta, IFN-gamma, TNF-alpha, and CD107a degranulation. The ability of these cells to mediate three or four of these functions was poor; expression of MIP-1beta alone dominated their response. CD56(-) NK cells retained expression of receptors such as the natural cytotoxicity receptors and NKG2D, whereas the expression of CD57 and perforin was lower when compared with CD56(dim) NK cells. Interestingly, pretreatment levels of CD56(-) NK cells correlated with the outcome of pegylated IFN-alpha and ribavirin treatment. In patients with CD56(-) NK cells in the range of healthy subjects, 80% reached a sustained virological response to treatment, whereas only 25% of patients with levels clearly above those in healthy subjects experienced a sustained virological response. Thus, chronic hepatitis C virus infection is associated with an expansion of CD56(-) NK cells functionally skewed toward MIP-1beta production only. Furthermore, high levels of these cells reveal a disturbance in innate cellular immunity that is associated with an impaired ability to respond to antiviral treatment with IFN-alpha and ribavirin.


Subject(s)
CD56 Antigen/immunology , Chemokine CCL4/immunology , Hepatitis C, Chronic/immunology , Interferon-alpha/therapeutic use , Killer Cells, Natural/immunology , Ribavirin/therapeutic use , Adult , Antiviral Agents/therapeutic use , CD56 Antigen/metabolism , CD57 Antigens/immunology , CD57 Antigens/metabolism , Cell Degranulation/drug effects , Cell Degranulation/immunology , Chemokine CCL4/metabolism , Female , Hepatitis C, Chronic/drug therapy , Humans , Interferon-gamma/immunology , Interferon-gamma/metabolism , Killer Cells, Natural/metabolism , Killer Cells, Natural/virology , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Lymphocyte Subsets/virology , Lysosomal-Associated Membrane Protein 1/immunology , Lysosomal-Associated Membrane Protein 1/metabolism , Male , Middle Aged , NK Cell Lectin-Like Receptor Subfamily K/immunology , NK Cell Lectin-Like Receptor Subfamily K/metabolism , Perforin/immunology , Perforin/metabolism , Receptors, Natural Killer Cell/immunology , Receptors, Natural Killer Cell/metabolism , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolism
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