44 results on '"Gonzalez VD"'
Search Results
2. Multiparameter single-cell proteomic technologies give new insights into the biology of ovarian tumors.
- Author
-
Funingana IG, Bedia JS, Huang YW, Delgado Gonzalez A, Donoso K, Gonzalez VD, Brenton JD, Ashworth A, and Fantl WJ
- Subjects
- Female, Humans, Carboplatin therapeutic use, Tumor Microenvironment, Proteomics, Ovarian Neoplasms diagnosis, Ovarian Neoplasms etiology, Ovarian Neoplasms therapy
- 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., (© 2023. The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
3. Elevated CD47 is a hallmark of dysfunctional aged muscle stem cells that can be targeted to augment regeneration.
- Author
-
Porpiglia E, Mai T, Kraft P, Holbrook CA, de Morree A, Gonzalez VD, Hilgendorf KI, Frésard L, Trejo A, Bhimaraju S, Jackson PK, Fantl WJ, and Blau HM
- Subjects
- Animals, Mice, Muscle, Skeletal, Aging, Disease Progression, CD47 Antigen, Myoblasts
- 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 (CD47
hi ) 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., Competing Interests: Declaration of interests H.M.B. is cofounder of Rejuvenation Technologies Inc. and Epirium Bio. H.M.B. and E.P. are named inventors on patent application no. PCT/US2021/038549 held by Stanford University., (Copyright © 2022 Elsevier Inc. All rights reserved.)- Published
- 2022
- Full Text
- View/download PDF
4. Hybrid Fluorescent Mass-Tag Nanotrackers as Universal Reagents for Long-Term Live-Cell Barcoding.
- Author
-
Delgado-Gonzalez A, Laz-Ruiz JA, Cano-Cortes MV, Huang YW, Gonzalez VD, Diaz-Mochon JJ, Fantl WJ, and Sanchez-Martin RM
- Subjects
- Cell Line, Flow Cytometry methods, Proteomics, Electronic Data Processing, Fluorescent Dyes chemistry
- 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.
- Published
- 2022
- Full Text
- View/download PDF
5. Measuring trogocytosis between ovarian tumor and natural killer cells.
- Author
-
Delgado-Gonzalez A, Huang YW, Porpiglia E, Donoso K, Gonzalez VD, and Fantl WJ
- Subjects
- Cell Membrane metabolism, Female, Humans, Killer Cells, Natural metabolism, Ovarian Neoplasms metabolism, Trogocytosis
- 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)., Competing Interests: The authors declare no competing interests. We have a published patent related to this study: WO2021/050200, PCT/US2020/046195., (© 2022 The Author(s).)
- Published
- 2022
- Full Text
- View/download PDF
6. Correction to: Mass Cytometry for the Characterization of Individual Cell Types in Ovarian Solid Tumors.
- Author
-
Gonzalez VD, Huang YW, and Fantl WJ
- Published
- 2022
- Full Text
- View/download PDF
7. Mass Cytometry for the Characterization of Individual Cell Types in Ovarian Solid Tumors.
- Author
-
Gonzalez VD, Huang YW, and Fantl WJ
- Subjects
- Antibodies, Female, Flow Cytometry, Humans, Isotopes, Mass Spectrometry, Single-Cell Analysis, Ovarian Neoplasms
- 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., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
- Published
- 2022
- Full Text
- View/download PDF
8. High-grade serous ovarian tumor cells modulate NK cell function to create an immune-tolerant microenvironment.
- Author
-
Gonzalez VD, Huang YW, Delgado-Gonzalez A, Chen SY, Donoso K, Sachs K, Gentles AJ, Allard GM, Kolahi KS, Howitt BE, Porpiglia E, and Fantl WJ
- Subjects
- Antineoplastic Agents pharmacology, Carboplatin pharmacology, Cell Line, Tumor, Coculture Techniques, Cytokines metabolism, Cytotoxicity, Immunologic, Female, Humans, Killer Cells, Natural drug effects, Killer Cells, Natural metabolism, Lymphocytes, Tumor-Infiltrating drug effects, Lymphocytes, Tumor-Infiltrating metabolism, Neoplasms, Cystic, Mucinous, and Serous drug therapy, Neoplasms, Cystic, Mucinous, and Serous metabolism, Neoplasms, Cystic, Mucinous, and Serous pathology, Ovarian Neoplasms drug therapy, Ovarian Neoplasms metabolism, Ovarian Neoplasms pathology, Phenotype, Receptors, Natural Killer Cell metabolism, Tetraspanin 29 metabolism, Trogocytosis, Immune Tolerance drug effects, Killer Cells, Natural immunology, Lymphocytes, Tumor-Infiltrating immunology, Neoplasms, Cystic, Mucinous, and Serous immunology, Ovarian Neoplasms immunology, Tumor Escape drug effects, Tumor Microenvironment immunology
- Abstract
Tubo-ovarian high-grade serous carcinoma (HGSC) is unresponsive to immune checkpoint blockade despite significant frequencies of exhausted T cells. Here we apply mass cytometry and uncover decidual-like natural killer (dl-NK) cell subpopulations (CD56+CD9+CXCR3+KIR+CD3-CD16-) in newly diagnosed HGSC samples that correlate with both tumor and transitioning epithelial-mesenchymal cell abundance. We show different combinatorial expression patterns of ligands for activating and inhibitory NK receptors within three HGSC tumor compartments: epithelial (E), transitioning epithelial-mesenchymal (EV), and mesenchymal (vimentin expressing [V]), with a more inhibitory ligand phenotype in V cells. In cocultures, NK-92 natural killer cells acquire CD9 from HGSC tumor cells by trogocytosis, resulting in reduced anti-tumor cytokine production and cytotoxicity. Cytotoxicity in these cocultures is restored with a CD9-blocking antibody or CD9 CRISPR knockout, thereby identifying mechanisms of immune suppression in HGSC. CD9 is widely expressed in HGSC tumors and so represents an important new therapeutic target with immediate relevance for NK immunotherapy., Competing Interests: Declaration of interests The authors declare no competing interests. We have a published patent related to this study: WO2021/050200, PCT/US2020/046195., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
9. Terminal Effector CD8 T Cells Defined by an IKZF2 + IL-7R - Transcriptional Signature Express FcγRIIIA, Expand in HIV Infection, and Mediate Potent HIV-Specific Antibody-Dependent Cellular Cytotoxicity.
- Author
-
Naluyima P, Lal KG, Costanzo MC, Kijak GH, Gonzalez VD, Blom K, Eller LA, Creegan M, Hong T, Kim D, Quinn TC, Björkström NK, Ljunggren HG, Serwadda D, Katabira ET, Sewankambo NK, Gray RH, Baeten JM, Michael NL, Wabwire-Mangen F, Robb ML, Bolton DL, Sandberg JK, and Eller MA
- Subjects
- 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, 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
- 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., (Copyright © 2019 The Authors.)- Published
- 2019
- Full Text
- View/download PDF
10. Identification of NK Cell Subpopulations That Differentiate HIV-Infected Subject Cohorts with Diverse Levels of Virus Control.
- Author
-
Pohlmeyer CW, Gonzalez VD, Irrinki A, Ramirez RN, Li L, Mulato A, Murry JP, Arvey A, Hoh R, Deeks SG, Kukolj G, Cihlar T, Pflanz S, Nolan GP, and Min-Oo G
- Subjects
- 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, HIV Infections immunology, Killer Cells, Natural immunology, Killer Cells, Natural virology
- 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., (Copyright © 2019 American Society for Microbiology.)- Published
- 2019
- Full Text
- View/download PDF
11. Commonly Occurring Cell Subsets in High-Grade Serous Ovarian Tumors Identified by Single-Cell Mass Cytometry.
- Author
-
Gonzalez VD, Samusik N, Chen TJ, Savig ES, Aghaeepour N, Quigley DA, Huang YW, Giangarrà V, Borowsky AD, Hubbard NE, Chen SY, Han G, Ashworth A, Kipps TJ, Berek JS, Nolan GP, and Fantl WJ
- Subjects
- Aged, Aged, 80 and over, Antibodies, Neoplasm metabolism, Carboplatin pharmacology, Cell Line, Tumor, Cluster Analysis, Cystadenocarcinoma, Serous metabolism, Cystadenocarcinoma, Serous pathology, Drug Resistance, Neoplasm drug effects, Female, Humans, Middle Aged, Neoplasm Proteins metabolism, Neoplasm Recurrence, Local pathology, Ovarian Neoplasms metabolism, Ovarian Neoplasms pathology, Phenotype, Prognosis, Flow Cytometry methods
- Abstract
We have performed an in-depth single-cell phenotypic characterization of high-grade serous ovarian cancer (HGSOC) by multiparametric mass cytometry (CyTOF). Using a CyTOF antibody panel to interrogate features of HGSOC biology, combined with unsupervised computational analysis, we identified noteworthy cell types co-occurring across the tumors. In addition to a dominant cell subset, each tumor harbored rarer cell phenotypes. One such group co-expressed E-cadherin and vimentin (EV), suggesting their potential role in epithelial mesenchymal transition, which was substantiated by pairwise correlation analyses. Furthermore, tumors from patients with poorer outcome had an increased frequency of another rare cell type that co-expressed vimentin, HE4, and cMyc. These poorer-outcome tumors also populated more cell phenotypes, as quantified by Simpson's diversity index. Thus, despite the recognized genomic complexity of the disease, the specific cell phenotypes uncovered here offer a focus for therapeutic intervention and disease monitoring., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2018
- Full Text
- View/download PDF
12. Atomic mass tag of bismuth-209 for increasing the immunoassay multiplexing capacity of mass cytometry.
- Author
-
Han G, Chen SY, Gonzalez VD, Zunder ER, Fantl WJ, and Nolan GP
- Subjects
- Antibodies, Monoclonal chemistry, Humans, Immunoassay, Immunoconjugates chemistry, Bismuth chemistry, Flow Cytometry methods, Killer Cells, Natural, Mass Spectrometry methods, Single-Cell Analysis methods
- 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 of209 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 of209 Bi3+ -labeling DTPA complexes was developed to evaluate the stoichiometric ratio of209 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., (© 2017 International Society for Advancement of Cytometry.)- Published
- 2017
- Full Text
- View/download PDF
13. High-throughput precision measurement of subcellular localization in single cells.
- Author
-
Burns TJ, Frei AP, Gherardini PF, Bava FA, Batchelder JE, Yoshiyasu Y, Yu JM, Groziak AR, Kimmey SC, Gonzalez VD, Fantl WJ, and Nolan GP
- Subjects
- Cell Nucleus metabolism, Cytoplasm metabolism, Cytoplasm ultrastructure, DNA Damage genetics, Humans, Protein Transport genetics, Subcellular Fractions ultrastructure, Flow Cytometry methods, High-Throughput Screening Assays methods, Single-Cell Analysis methods
- 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., (© 2017 International Society for Advancement of Cytometry.)
- Published
- 2017
- Full Text
- View/download PDF
14. The Human NK Cell Response to Yellow Fever Virus 17D Is Primarily Governed by NK Cell Differentiation Independently of NK Cell Education.
- Author
-
Marquardt N, Ivarsson MA, Blom K, Gonzalez VD, Braun M, Falconer K, Gustafsson R, Fogdell-Hahn A, Sandberg JK, and Michaëlsson J
- Subjects
- 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, Interferon Type I immunology, Killer Cells, Natural immunology, Receptors, KIR immunology, Yellow Fever Vaccine immunology, Yellow fever virus immunology
- 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., (Copyright © 2015 by The American Association of Immunologists, Inc.)
- Published
- 2015
- Full Text
- View/download PDF
15. Palladium-based mass tag cell barcoding with a doublet-filtering scheme and single-cell deconvolution algorithm.
- Author
-
Zunder ER, Finck R, Behbehani GK, Amir el-AD, Krishnaswamy S, Gonzalez VD, Lorang CG, Bjornson Z, Spitzer MH, Bodenmiller B, Fantl WJ, Pe'er D, and Nolan GP
- Subjects
- Software, Staining and Labeling methods, Algorithms, Flow Cytometry methods, Palladium, Single-Cell Analysis methods
- Abstract
Mass-tag cell barcoding (MCB) labels individual cell samples with unique combinatorial barcodes, after which they are pooled for processing and measurement as a single multiplexed sample. The MCB method eliminates variability between samples in antibody staining and instrument sensitivity, reduces antibody consumption and shortens instrument measurement time. Here we present an optimized MCB protocol. The use of palladium-based labeling reagents expands the number of measurement channels available for mass cytometry and reduces interference with lanthanide-based antibody measurement. An error-detecting combinatorial barcoding scheme allows cell doublets to be identified and removed from the analysis. A debarcoding algorithm that is single cell-based rather than population-based improves the accuracy and efficiency of sample deconvolution. This debarcoding algorithm has been packaged into software that allows rapid and unbiased sample deconvolution. The MCB procedure takes 3-4 h, not including sample acquisition time of ∼1 h per million cells.
- Published
- 2015
- Full Text
- View/download PDF
16. Immunoagglutination test to diagnose Chagas disease: comparison of different latex-antigen complexes.
- Author
-
Garcia VS, Gonzalez VD, Marcipar IS, and Gugliotta LM
- Subjects
- Chagas Disease immunology, Cross Reactions immunology, Humans, Latex Fixation Tests, Leishmania immunology, ROC Curve, Recombinant Proteins blood, Recombinant Proteins immunology, Sensitivity and Specificity, Antibodies, Protozoan blood, Antigens, Protozoan blood, Chagas Disease diagnosis, Trypanosoma cruzi immunology
- 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., (© 2014 John Wiley & Sons Ltd.)
- Published
- 2014
- Full Text
- View/download PDF
17. Latex-protein complexes from an acute phase recombinant antigen of Toxoplasma gondii for the diagnosis of recently acquired toxoplasmosis.
- Author
-
Peretti LE, Gonzalez VD, Marcipar IS, and Gugliotta LM
- Subjects
- Adsorption, Electrophoresis, Enzyme-Linked Immunosorbent Assay, Hydrogen-Ion Concentration, Latex chemistry, Polystyrenes chemical synthesis, Polystyrenes chemistry, Toxoplasmosis immunology, Toxoplasmosis parasitology, Acute-Phase Reaction immunology, Antigens, Protozoan immunology, Latex immunology, Polystyrenes immunology, Recombinant Proteins immunology, Toxoplasma immunology, Toxoplasmosis diagnosis
- Abstract
The synthesis and characterization of latex-protein complexes (LPC), from the acute phase recombinant antigen P35 (P35Ag) of Toxoplasma gondii and "core-shell" carboxylated or polystyrene (PS) latexes (of different sizes and charge densities) are considered, with the aim of producing immunoagglutination reagents able to detect recently acquired toxoplasmosis. Physical adsorption (PA) and chemical coupling (CC) of P35Ag onto latex particles at different pH were investigated. Greater amounts of adsorbed protein were obtained on PS latexes than on carboxylated latexes, indicating that hydrophobic forces govern the interactions between the protein and the particle surface. In the CC experiments, the highest amount of bound protein was obtained at pH 6, near the isoelectric point of the protein (IP=6.27). At this pH, it decreased both the repulsion between particle surface and protein, and the repulsion between neighboring molecules. The LPC were characterized and the antigenicity of the P35Ag protein coupled on the particles surface was evaluated by Enzyme-Linked ImmunoSorbent Assay (ELISA). Results from ELISA showed that the P35Ag coupled to the latex particles surface was not affected during the particles sensitization by PA and CC and the produced LPC were able to recognize specific anti-P35Ag antibodies present in the acute phase of the disease., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
18. Optimisation and standardisation of an immunoagglutination assay for the diagnosis of Trypanosoma cruzi infection based on latex-(recombinant antigen) complexes.
- Author
-
Garcia VS, Gonzalez VD, Marcipar IS, Vega JR, and Gugliotta LM
- Subjects
- Chagas Disease blood, Chagas Disease immunology, Enzyme-Linked Immunosorbent Assay methods, Humans, Latex Fixation Tests methods, Recombinant Proteins blood, Recombinant Proteins immunology, Antigens, Protozoan blood, Chagas Disease diagnosis, Trypanosoma cruzi immunology
- 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., (© 2013 John Wiley & Sons Ltd.)
- Published
- 2014
- Full Text
- View/download PDF
19. Real-time study of protein adsorption kinetics in porous silicon.
- Author
-
Lasave LC, Urteaga R, Koropecki RR, Gonzalez VD, and Arce RD
- Subjects
- Adsorption, Glucose Oxidase chemistry, Horseradish Peroxidase chemistry, Kinetics, Models, Molecular, Porosity, Computer Systems, Proteins chemistry, Silicon chemistry
- Abstract
This paper presents an optical method for real-time monitoring of protein adsorption using porous silicon self-supported microcavities as a label-free detection platform. The study combines an experimental approach with a physical model for the adsorption process. The proposed model agrees well with experimental observations, and provides information about the kinetics of diffusion and adsorption of proteins within the pores, which will be useful for future experimental designs., (Copyright © 2013 Elsevier B.V. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
20. Differential loss of invariant natural killer T cells and FoxP3⁺ regulatory T cells in HIV-1 subtype A and subtype D infections.
- Author
-
Flach B, Naluyima P, Blom K, Gonzalez VD, Eller LA, Laeyendecker O, Quinn TC, Serwadda D, Sewankambo NK, Wawer MJ, Gray RH, Michael NL, Wabwire-Mangen F, Robb ML, Eller MA, and Sandberg JK
- Subjects
- Adaptive Immunity, Adolescent, Adult, Antigens, CD1d biosynthesis, CD4 Antigens metabolism, Female, Forkhead Transcription Factors metabolism, Humans, Interleukin-2 biosynthesis, Lymphocyte Activation, Male, Middle Aged, Young Adult, HIV Infections immunology, HIV Infections virology, HIV-1 classification, HIV-1 immunology, Natural Killer T-Cells immunology, T-Lymphocytes, Regulatory immunology
- Abstract
HIV-1 subtype D is associated with faster disease progression compared with subtype A. Immunological correlates of this difference remain undefined. We investigated invariant natural killer T (iNKT) cells and FoxP3⁺ regulatory T cells (Tregs) in Ugandans infected with either subtype. Loss of iNKT cells was pronounced in subtype D, whereas Tregs displayed more profound loss in subtype A infection. The iNKT cell levels were associated with CD4 T-cell interleukin-2 production in subtype A, but not in D, infection. Thus, these viral subtypes are associated with differential loss of iNKT cells and Tregs that may influence the quality of the adaptive immune response.
- Published
- 2013
- Full Text
- View/download PDF
21. Temporal dynamics of the primary human T cell response to yellow fever virus 17D as it matures from an effector- to a memory-type response.
- Author
-
Blom K, Braun M, Ivarsson MA, Gonzalez VD, Falconer K, Moll M, Ljunggren HG, Michaëlsson J, and Sandberg JK
- Subjects
- 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, CD4-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes immunology, Yellow Fever prevention & control, Yellow Fever Vaccine immunology, Yellow fever virus immunology
- 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.
- Published
- 2013
- Full Text
- View/download PDF
22. Synthesis of latex-antigen complexes from single and multiepitope recombinant proteins. Application in immunoagglutination assays for the diagnosis of Trypanosoma cruzi infection.
- Author
-
Garcia VS, Gonzalez VD, Caudana PC, Vega JR, Marcipar IS, and Gugliotta LM
- Subjects
- Adsorption, Electrophoresis, Polyacrylamide Gel, Epitopes, Immunologic Tests, Isoelectric Focusing, Light, Microscopy, Electron, Scanning, Particle Size, Scattering, Radiation, Styrene, Agglutination Tests methods, Antigens, Protozoan chemistry, Chagas Disease diagnosis, Chagas Disease immunology, Latex immunology, Recombinant Proteins biosynthesis, Trypanosoma cruzi
- Abstract
The physical adsorption and the chemical coupling of recombinant proteins of Trypanosoma cruzi onto polystyrene and core-shell carboxylated particles were respectively investigated with the ultimate aim of producing latex-protein complexes to be used in an immunoagglutination assay able to detect the Chagas disease. To this effect, two single proteins (RP1 and RP5) and a multiepitope protein derived from three antigenic peptides (CP2) were evaluated, and sensitizations were carried out at different pHs. The maximum physical adsorption was produced at pHs close to the protein isoelectric point (i.e., pH 6 for RP5 and pH 5 for RP1 and CP2). High fractions of antigens were chemically bound to the carboxyl groups, and the highest surface density of linked protein was also observed at pHs close to the protein isoelectric point. The three latex-protein complexes obtained by covalent coupling at such pHs were tested with sera from a panel of 16 infected and 16 non-infected patients. In the immunoagglutination assays, the latex-CP2 complex produced the best discrimination between positive and negative sera., (Copyright © 2012 Elsevier B.V. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
23. HCV infection induces a unique hepatic innate immune response associated with robust production of type III interferons.
- Author
-
Thomas E, Gonzalez VD, Li Q, Modi AA, Chen W, Noureddin M, Rotman Y, and Liang TJ
- Subjects
- 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, Hepacivirus immunology, Hepatitis C, Chronic immunology, Hepatocytes immunology, Immunity, Innate, Interferons metabolism, Liver immunology
- 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., (Copyright © 2012 AGA Institute. Published by Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
24. Innate and adaptive immune responses both contribute to pathological CD4 T cell activation in HIV-1 infected Ugandans.
- Author
-
Eller MA, Blom KG, Gonzalez VD, Eller LA, Naluyima P, Laeyendecker O, Quinn TC, Kiwanuka N, Serwadda D, Sewankambo NK, Tasseneetrithep B, Wawer MJ, Gray RH, Marovich MA, Michael NL, de Souza MS, Wabwire-Mangen F, Robb ML, Currier JR, and Sandberg JK
- Subjects
- ADP-ribosyl Cyclase 1 metabolism, Adolescent, Adult, Antigens, CD metabolism, Antigens, Viral immunology, Apoptosis Regulatory Proteins metabolism, CD4-Positive T-Lymphocytes immunology, Cell Proliferation, Chronic Disease, Disease Progression, Female, HIV Infections blood, HIV Infections immunology, HLA-DR Antigens metabolism, Humans, Immunologic Memory immunology, Interleukin-6 blood, Lipopolysaccharide Receptors blood, Male, Middle Aged, Programmed Cell Death 1 Receptor, Receptors, Antigen, T-Cell, alpha-beta metabolism, Uganda, Young Adult, Adaptive Immunity immunology, CD4-Positive T-Lymphocytes pathology, CD4-Positive T-Lymphocytes virology, HIV Infections virology, HIV-1 immunology, Immunity, Innate immunology, Lymphocyte Activation immunology
- Abstract
HIV-1 disease progression is associated with persistent immune activation. However, the nature of this association is incompletely understood. Here, we investigated immune activation in the CD4 T cell compartment of chronically HIV-1 infected individuals from Rakai, Uganda. Levels of CD4 T cell activation, assessed as co-expression of PD-1, CD38 and HLA-DR, correlated directly to viral load and inversely to CD4 count. Deeper characterization of these cells indicated an effector memory phenotype with relatively frequent expression of Ki67 despite their PD-1 expression, and levels of these cells were inversely associated with FoxP3+ regulatory T cells. We therefore use the term deregulated effector memory (DEM) cells to describe them. CD4 T cells with a DEM phenotype could be generated by antigen stimulation of recall responses in vitro. Responses against HIV-1 and CMV antigens were enriched among the DEM CD4 T cells in patients, and the diverse Vβ repertoire of DEM CD4 T cells suggested they include diverse antigen-specificities. Furthermore, the levels of DEM CD4 T cells correlated directly to soluble CD14 (sCD14) and IL-6, markers of innate immune activation, in plasma. The size of the activated DEM CD4 T cell subset was predictive of the rate of disease progression, whereas IL-6 was only weakly predictive and sCD14 was not predictive. Taken together, these results are consistent with a model where systemic innate immune activation and chronic antigen stimulation of adaptive T cell responses both play important roles in driving pathological CD4 T cell immune activation in HIV-1 disease.
- Published
- 2011
- Full Text
- View/download PDF
25. NK cells and CD1d-restricted NKT cells respond in different ways with divergent kinetics to IL-2 treatment in primary HIV-1 infection.
- Author
-
Kuylenstierna C, Snyder-Cappione JE, Loo CP, Long BR, Gonzalez VD, Michaëlsson J, Moll M, Spotts G, Hecht FM, Nixon DF, and Sandberg JK
- Subjects
- Dendritic Cells immunology, Humans, Interferon-gamma biosynthesis, Interferon-gamma immunology, Kinetics, Antigens, CD1d immunology, HIV Infections immunology, HIV-1 immunology, Interleukin-2 immunology, Killer Cells, Natural immunology, Natural Killer T-Cells immunology
- Abstract
Cytokine immunotherapy is being evaluated as adjunct treatment in infectious diseases. The effects on innate and adaptive immunity in vivo are insufficiently known. Here, we investigate whether combination treatment with antiretroviral therapy (ART) and Interleukin-2 (IL-2) of patients with primary HIV-1 infection induces sustained increases in circulating NKT cell and NK cell numbers and effector functions and investigate how changes are coordinated in the two compartments. Patients with primary HIV-1 infection starting ART were analyzed for numbers, phenotype and function of NKT cells, NK cells and dendritic cells (DC) in peripheral blood before, during and after IL-2 treatment. NKT cells expanded during IL-2 treatment as expected from previous studies. However, their response to α-galactosyl ceramide antigen were retained but not boosted. Myeloid DC did not change their numbers or CD1d-expression during treatment. In contrast, the NK cell compartment responded with rapid expansion of the CD56(dim) effector subset and enhanced IFNγ production. Expansions of NKT cells and NK cells retracted back towards baseline values at 12 months after IL-2 treatment ended. In summary, NKT cells and NK cells respond to IL-2 treatment with different kinetics. Effects on cellular function are distinct between the cell types and the effects appear not to be sustained after IL-2 treatment ends. These results improve our understanding of the effects of cytokine immunotherapy on innate cellular immunity in early HIV-1 infection., (© 2011 The Authors. Scandinavian Journal of Immunology © 2011 Blackwell Publishing Ltd.)
- Published
- 2011
- Full Text
- View/download PDF
26. Chronic immune activation in the T cell compartment of HCV/HIV-1 co-infected patients.
- Author
-
Sandberg JK, Falconer K, and Gonzalez VD
- Subjects
- ADP-ribosyl Cyclase 1 metabolism, Antiretroviral Therapy, Highly Active, Antiviral Agents therapeutic use, CD8-Positive T-Lymphocytes immunology, Disease Progression, Drug Therapy, Combination, HIV Infections drug therapy, HIV Infections immunology, HIV Infections virology, Hepatitis C, Chronic drug therapy, Hepatitis C, Chronic immunology, Hepatitis C, Chronic virology, Humans, Interferon-alpha therapeutic use, Membrane Glycoproteins metabolism, Polyethylene Glycols therapeutic use, Ribavirin therapeutic use, HIV Infections complications, HIV-1 immunology, Hepacivirus immunology, Hepatitis C, Chronic complications, Lymphocyte Activation immunology, T-Lymphocytes immunology
- Abstract
Activation of innate and adaptive immune mechanisms in response to infection is necessary to control and clear infections. However, chronic immune activation in human immunodeficiency virus 1 (HIV-1) infection has a series of detrimental effects and is a major driving force in HIV-1 disease progression. We recently found that patients with chronic hepatitis C virus (HCV)/HIV-1 co-infection display sharply elevated immune activation as determined by expression of CD38 in T cells. High immune activation was observed despite that these patients were on effective antiretroviral therapy (ART), which usually brings down activation levels in HIV-infected people. HCV treatment with pegylated interferon-α (IFNα) and ribavirin reduced activation, and this was at first glance unexpected as IFNα is believed to be involved in driving activation. Here, we briefly summarize these findings and discuss them in context of the emerging roles of the gut barrier and the liver in chronic immune activation and viral disease progression.
- Published
- 2010
- Full Text
- View/download PDF
27. Immunodiagnosis of Chagas disease: Synthesis of three latex-protein complexes containing different antigens of Trypanosoma cruzi.
- Author
-
Gonzalez VD, Garcia VS, Vega JR, Marcipar IS, Meira GR, and Gugliotta LM
- Subjects
- Humans, Hydrogen-Ion Concentration, Isoelectric Point, Recombinant Proteins chemical synthesis, Antigens, Protozoan analysis, Chagas Disease diagnosis, Latex chemistry, Trypanosoma cruzi immunology
- Abstract
This article describes the physical adsorption and the chemical coupling of 3 antigenic proteins of Trypanosoma cruzi onto polystyrene (PS) based latexes to be used as novel immunodiagnosis reagents for detecting the Chagas disease. The coupled proteins were a homogenate of T. cruzi, or a recombinant protein (either Ag36 or CP1). With the homogenate, between 30 and 60% of the total-linked protein was chemically coupled, showing a small dependence with the pH. For Ag36 and CP1, around 90% of the total-linked protein was chemically coupled, with a maximum coupling at pH 5 (i.e., close to the isoelectric point). The chemical coupling of CP1 was less affected by the pH than the coupling of Ag36., (2010 Elsevier B.V. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
28. Innate immunity and chronic immune activation in HCV/HIV-1 co-infection.
- Author
-
Gonzalez VD, Landay AL, and Sandberg JK
- Subjects
- Dendritic Cells immunology, Disease Progression, HIV Infections virology, Hepatitis C, Chronic virology, Humans, Immunity, Innate immunology, Interferon-alpha immunology, Killer Cells, Natural immunology, Toll-Like Receptors immunology, HIV Infections immunology, HIV-1 immunology, Hepacivirus immunology, Hepatitis C, Chronic immunology
- Abstract
Innate immune responses are critical in the defense against viral infections. NK cells, myeloid and plasmacytoid dendritic cells, and invariant CD1d-restricted NKT cells mediate both effector and regulatory functions in this early immune response. In chronic uncontrolled viral infections such as HCV and HIV-1, these essential immune functions are compromised and can become a double edged sword contributing to the immunopathogenesis of viral disease. In particular, recent findings indicate that innate immune responses play a central role in the chronic immune activation which is a primary driver of HIV-1 disease progression. HCV/HIV-1 co-infection is affecting millions of people and is associated with faster viral disease progression. Here, we review the role of innate immunity and chronic immune activation in HCV and HIV-1 infection, and discuss how mechanisms of innate immunity may influence protection as well as immunopathogenesis in the HCV/HIV-1 co-infected human host., (Copyright 2009 Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
29. Expansion of functionally skewed CD56-negative NK cells in chronic hepatitis C virus infection: correlation with outcome of pegylated IFN-alpha and ribavirin treatment.
- Author
-
Gonzalez VD, Falconer K, Björkström NK, Blom KG, Weiland O, Ljunggren HG, Alaeus A, and Sandberg JK
- Subjects
- 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, CD56 Antigen immunology, Chemokine CCL4 immunology, Hepatitis C, Chronic immunology, Interferon-alpha therapeutic use, Killer Cells, Natural immunology, Ribavirin therapeutic use
- 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.
- Published
- 2009
- Full Text
- View/download PDF
30. High levels of chronic immune activation in the T-cell compartments of patients coinfected with hepatitis C virus and human immunodeficiency virus type 1 and on highly active antiretroviral therapy are reverted by alpha interferon and ribavirin treatment.
- Author
-
Gonzalez VD, Falconer K, Blom KG, Reichard O, Mørn B, Laursen AL, Weis N, Alaeus A, and Sandberg JK
- Subjects
- ADP-ribosyl Cyclase 1 immunology, Adult, Disease Progression, Female, Humans, Interferon-alpha immunology, Interferon-alpha therapeutic use, Lymphocyte Activation immunology, Male, Middle Aged, T-Lymphocyte Subsets immunology, T-Lymphocyte Subsets virology, Viral Load, Young Adult, Antiretroviral Therapy, Highly Active, Antiviral Agents therapeutic use, HIV Infections drug therapy, HIV Infections immunology, HIV Infections virology, HIV-1 immunology, Hepacivirus immunology, Hepatitis C, Chronic drug therapy, Hepatitis C, Chronic immunology, Hepatitis C, Chronic virology, Ribavirin therapeutic use, T-Lymphocytes immunology, T-Lymphocytes virology
- Abstract
Chronic immune activation is a driver of human immunodeficiency virus type 1 (HIV-1) disease progression. Here, we describe that subjects with chronic hepatitis C virus (HCV)/HIV-1 coinfection display sharply elevated immune activation as determined by CD38 expression in T cells. This occurs, despite effective antiretroviral therapy, in both CD8 and CD4 T cells and is more pronounced than in the appropriate monoinfected control groups. Interestingly, the suppression of HCV by pegylated alpha interferon and ribavirin treatment reduces activation. High HCV loads and elevated levels of chronic immune activation may contribute to the high rates of viral disease progression observed in HCV/HIV-1-coinfected patients.
- Published
- 2009
- Full Text
- View/download PDF
31. Elevated natural killer cell activity despite altered functional and phenotypic profile in Ugandans with HIV-1 clade A or clade D infection.
- Author
-
Eller MA, Eller LA, Ouma BJ, Thelian D, Gonzalez VD, Guwatudde D, McCutchan FE, Marovich MA, Michael NL, de Souza MS, Wabwire-Mangen F, Robb ML, Currier JR, and Sandberg JK
- Subjects
- Adult, CD4 Lymphocyte Count, CD56 Antigen blood, Female, Humans, Immunophenotyping, Interferon-gamma biosynthesis, Killer Cells, Natural immunology, Lysosomal-Associated Membrane Protein 1 blood, Male, Middle Aged, NK Cell Lectin-Like Receptor Subfamily C blood, Receptors, KIR2DL1 blood, Uganda, Acquired Immunodeficiency Syndrome immunology, HIV-1 classification
- Abstract
Background and Objective: Natural killer (NK) cells most likely contribute toward limiting HIV-1 replication, and investigation into their function throughout the course of infection is therefore important. We here aimed to determine the state of the NK cell compartment in Ugandans with untreated HIV-1 clade A or D infection in comparison with matched uninfected controls., Methods and Results: The function and phenotype of NK cells were investigated using 10-color flow cytometry. Surprisingly, NK cells displayed elevated production of interferon-gamma and macrophage inflammatory protein 1beta, as well as CD107a degranulation in infected subjects. This included unexpected levels of degranulation in the CD56bright subset of NK cells and high levels of macrophage inflammatory protein 1beta in CD56negative NK cells. HIV-1 infection was associated with reduced expression of KIR2DL1, NKG2A, CD161, and NKp30 in CD56dim and CD56negative NK cells, whereas lowered CD161 expression was the only alteration in the CD56bright subset. Interestingly, low CD4 counts were associated with increased levels of interferon-gamma and degranulation in CD56bright NK cells, as well as increased NKp44 expression in the CD56dim cells., Conclusions: NK cells in HIV-1-infected Ugandans display elevated activity, despite an altered functional and phenotypic profile. Furthermore, specific alterations in the CD56bright and CD56dim subsets occur in patients with severe CD4 loss.
- Published
- 2009
- Full Text
- View/download PDF
32. Reduction of the HIV-1 reservoir in resting CD4+ T-lymphocytes by high dosage intravenous immunoglobulin treatment: a proof-of-concept study.
- Author
-
Lindkvist A, Edén A, Norström MM, Gonzalez VD, Nilsson S, Svennerholm B, Karlsson AC, Sandberg JK, Sönnerborg A, and Gisslén M
- Abstract
Background: The latency of HIV-1 in resting CD4+ T-lymphocytes constitutes a major obstacle for the eradication of virus in patients on antiretroviral therapy (ART). As yet, no approach to reduce this viral reservoir has proven effective., Methods: Nine subjects on effective ART were included in the study and treated with high dosage intravenous immunoglobulin (IVIG) for five consecutive days. Seven of those had detectable levels of replication-competent virus in the latent reservoir and were thus possible to evaluate. Highly purified resting memory CD4+ T-cells were activated and cells containing replication-competent HIV-1 were quantified. HIV-1 from plasma and activated memory CD4+ T-cells were compared with single genome sequencing (SGS) of the gag region. T-lymphocyte activation markers and serum interleukins were measured., Results: The latent HIV-1 pool decreased with in median 68% after IVIG was added to effective ART. The reservoir decreased in five, whereas no decrease was found in two subjects with detectable virus. Plasma HIV-1 RNA >or= 2 copies/mL was detected in five of seven subjects at baseline, but in only one at follow-up after 8-12 weeks. The decrease of the latent HIV-1 pool and the residual plasma viremia was preceded by a transitory low-level increase in plasma HIV-1 RNA and serum interleukin 7 (IL-7) levels, and followed by an expansion of T regulatory cells. The magnitude of the viral increase in plasma correlated to the size of the latent HIV-1 pool and SGS of the gag region showed that viral clones from plasma clustered together with virus from activated memory T-cells, pointing to the latent reservoir as the source of HIV-1 RNA in plasma., Conclusion: The findings from this uncontrolled proof-of-concept study suggest that the reservoir became accessible by IVIG treatment through activation of HIV-1 gene expression in latently-infected resting CD4+ T-cells. We propose that IVIG should be further evaluated as an adjuvant to effective ART.
- Published
- 2009
- Full Text
- View/download PDF
33. Severe functional impairment and elevated PD-1 expression in CD1d-restricted NKT cells retained during chronic HIV-1 infection.
- Author
-
Moll M, Kuylenstierna C, Gonzalez VD, Andersson SK, Bosnjak L, Sönnerborg A, Quigley MF, and Sandberg JK
- Subjects
- Adult, Antigens, CD metabolism, Antigens, CD1d metabolism, Apoptosis Regulatory Proteins metabolism, Cell Proliferation, Chronic Disease, Female, HIV Infections virology, Humans, Interferon-gamma metabolism, Male, Middle Aged, Natural Killer T-Cells metabolism, Programmed Cell Death 1 Receptor, Antigens, CD immunology, Antigens, CD1d immunology, Apoptosis Regulatory Proteins immunology, HIV Infections immunology, HIV-1, Interferon-gamma immunology, Natural Killer T-Cells immunology
- Abstract
Invariant CD1d-restricted NKT cells play important roles in regulating both innate and adaptive immunity. They are targeted by HIV-1 infection and severely reduced in number or even lost in many infected subjects. Here, we have investigated the characteristics of NKT cells retained by some patients despite chronic HIV-1 infection. NKT cells preserved under these circumstances displayed an impaired ability to proliferate and produce IFN-gamma in response to CD1d-restricted lipid antigen as compared with cells from uninfected control subjects. HIV-1 infection was associated with an elevated expression of the inhibitory programmed death-1 (PD-1) receptor (CD279) on the CD4(-) subset of NKT cells. However, blocking experiments indicated that the functional defects in NKT cells were largely PD-1-independent. Furthermore, the elevated PD-1 expression and the functional defects were not restored by anti-retroviral treatment, and the NKT cell numbers in blood did not recover significantly in response to treatment. The functional phenotype of NKT cells in these patients suggests an irreversible immune exhaustion due to chronic activation in vivo. The data demonstrate a severe functional impairment in the remaining NKT-cell compartment in HIV-1-infected patients, which limits the prospects to mobilize these cells in immunotherapy approaches in patients.
- Published
- 2009
- Full Text
- View/download PDF
34. Elevated numbers of Fc gamma RIIIA+ (CD16+) effector CD8 T cells with NK cell-like function in chronic hepatitis C virus infection.
- Author
-
Björkström NK, Gonzalez VD, Malmberg KJ, Falconer K, Alaeus A, Nowak G, Jorns C, Ericzon BG, Weiland O, Sandberg JK, and Ljunggren HG
- Subjects
- CD8-Positive T-Lymphocytes metabolism, CD8-Positive T-Lymphocytes pathology, CD8-Positive T-Lymphocytes virology, Cell Differentiation genetics, Cell Differentiation immunology, Cells, Cultured, Hepatitis C, Chronic metabolism, Humans, Immunophenotyping, Killer Cells, Natural metabolism, Killer Cells, Natural pathology, Lymphocyte Count, Middle Aged, Receptors, Antigen, T-Cell, alpha-beta biosynthesis, Receptors, Antigen, T-Cell, alpha-beta genetics, Receptors, IgG physiology, Receptors, KIR biosynthesis, Receptors, KIR genetics, T-Lymphocyte Subsets metabolism, T-Lymphocyte Subsets pathology, T-Lymphocyte Subsets virology, CD8-Positive T-Lymphocytes immunology, Cytotoxicity, Immunologic genetics, Hepacivirus immunology, Hepatitis C, Chronic immunology, Hepatitis C, Chronic pathology, Killer Cells, Natural immunology, Receptors, IgG biosynthesis, T-Lymphocyte Subsets immunology
- Abstract
CTL are crucial in the defense against viral infections. In the course of investigating peripheral blood and intrahepatic CD8 T cells in patients with chronic hepatitis C virus (HCV) infection, we observed a significant population of CD8 T cells expressing the FcgammaRIIIA (CD16) receptor. This observation led us to characterize these cells with respect to their phenotype and function in a cohort of patients with chronic HCV infection as well as in healthy blood donors. On average, 10% of peripheral blood CD8 T cells from HCV-infected patients expressed CD16 compared with only a few percent in healthy donors. CD16(+) CD8 T cells displayed a late-stage effector phenotype with high levels of perforin. These cells exhibited a restricted TCR profile suggesting underlying clonal expansion. Stimulation of CD16 on CD8 T cells evoked a vigorous response similar to that of CD16 stimulation in NK cells. Our data suggest that CD8 T cells, during chronic HCV infection in humans, continue to differentiate beyond defined stages of terminal effector cells, acquiring CD16 and NK cell-like functional properties.
- Published
- 2008
- Full Text
- View/download PDF
35. Expansion of CD56- NK cells in chronic HCV/HIV-1 co-infection: reversion by antiviral treatment with pegylated IFNalpha and ribavirin.
- Author
-
Gonzalez VD, Falconer K, Michaëlsson J, Moll M, Reichard O, Alaeus A, and Sandberg JK
- Subjects
- Adult, Aged, CD56 Antigen metabolism, Female, Flow Cytometry, HIV Infections complications, HIV Infections immunology, HIV-1, Hepatitis C, Chronic complications, Hepatitis C, Chronic immunology, Humans, Interferon alpha-2, Interferon-alpha therapeutic use, Killer Cells, Natural immunology, Lymphocyte Subsets immunology, Male, Middle Aged, Polyethylene Glycols therapeutic use, Recombinant Proteins, Ribavirin therapeutic use, Viremia drug therapy, Antiviral Agents therapeutic use, HIV Infections drug therapy, Hepatitis C, Chronic drug therapy, Killer Cells, Natural drug effects, Lymphocyte Subsets drug effects
- Abstract
Co-infection with HCV and HIV-1 is a problem of increasing importance and the role of innate cellular immunity in this co-infection is incompletely understood. Here, we have observed sharply elevated numbers of CD56(-)CD16(+) perforin(low) NK cells in HCV/HIV-1 co-infected subjects on antiretroviral therapy. Interestingly, this expansion of unconventional CD56(-) NK cells rapidly reverted when HCV was suppressed by IFNalpha and ribavirin treatment, and was not seen in mono-infected control groups. In vitro experiments suggested that this effect of treatment was due to suppression of HCV viremia rather than a direct effect of IFNalpha on these cells. In contrast, the conventional CD56(+) NK cells were largely unchanged in subjects with high HCV loads, although they exhibited slightly decreased perforin expression. With delayed kinetics, the CD56(bright) immuno-regulatory NK cell subset temporarily increased to supranormal levels in response to HCV treatment. In contrast to the NK compartment, the CD1d-restricted NKT cells were severely reduced by the co-infection and not restored by treatment. Together, our data suggest that the high HCV loads in HCV/HIV-1 co-infection alter the NK cell compartment in a way not observed in HCV mono-infection.
- Published
- 2008
- Full Text
- View/download PDF
36. Spontaneous HCV clearance in HCV/HIV-1 coinfection associated with normalized CD4 counts, low level of chronic immune activation and high level of T cell function.
- Author
-
Falconer K, Gonzalez VD, Reichard O, Sandberg JK, and Alaeus A
- Subjects
- CD4 Lymphocyte Count, Female, HIV-1, Humans, Middle Aged, HIV Infections complications, HIV Infections immunology, HIV Infections virology, Hepacivirus isolation & purification, Hepatitis C, Chronic complications, Hepatitis C, Chronic immunology, Hepatitis C, Chronic virology, Lymphocyte Activation immunology, T-Lymphocytes immunology
- Abstract
Background/objective: Spontaneous HCV clearance in HCV/HIV-1 coinfected patients is likely to be very rare given the damage to the immune system caused by HIV-1 infection. The search for immune correlates of spontaneous clearance is important for the understanding of pathogenesis as well as for the development of possible new treatment strategies., Study Design/results: A cohort of HCV/HIV-1 coinfected patients was followed. Plasma HCV viral load was measured and T cell immunity in peripheral blood samples was assessed using multi-color flow cytometry. One HCV/HIV-1 coinfected patient spontaneously became HCV-RNA negative after being positive for several years. This patient displayed a normalized CD4 counts on successful HAART, low level of T cell activation and a high level of T cell function as compared to HCV/HIV-1 coinfected control subjects., Conclusions: A beneficial immune status including a low level of chronic T cell activation and a high level of T cell function may be one factor that contributes to improved chances of clearance of chronic HCV infection in HIV-1 infected patients.
- Published
- 2008
- Full Text
- View/download PDF
37. Latex of immunodiagnosis for detecting the Chagas disease: II. Chemical coupling of antigen Ag36 onto carboxylated latexes.
- Author
-
Gonzalez VD, Gugliotta LM, Giacomelli CE, and Meira GR
- Subjects
- Animals, Antibodies, Protozoan chemistry, Antibodies, Protozoan immunology, Humans, Immunologic Tests, Materials Testing, Serum Albumin, Bovine chemistry, Trypanosoma cruzi immunology, Antigens, Protozoan chemistry, Chagas Disease diagnosis, Immunoassay instrumentation, Latex chemical synthesis
- Abstract
A novel immunodiagnosis reagent for detecting the Chagas Disease was developed, by chemical coupling of antigen Ag36 of Trypanosoma cruzi onto two (carboxylated and core-shell) latexes. The coupling reactions involved the use of a carbodiimide intermediate. Bovine serum albumin (BSA) was used as a model protein for determining the appropriate conditions for its physical and chemical coupling. BSA showed an increased adsorption onto the base carboxylated latexes, with respect to a PS latex without carboxyl groups. The chemical bonding experiments only involved the carboxylated latexes. With BSA, the final density of covalently bound protein was 2.30 mg/m(2). In addition, around 55% of the total linked protein was chemically coupled, and the reaction was little affected by the pH. With Ag36, the final density of covalently bound protein was 2.44 mg/m(2), around 80% of the total linked protein was chemically coupled, and the chemical coupling was maximum at pH = 5 (i.e., close to the isoelectric point).
- Published
- 2008
- Full Text
- View/download PDF
38. Latex of immunodiagnosis for detecting the Chagas disease. I. Synthesis of the base carboxylated latex.
- Author
-
Gonzalez VD, Gugliotta LM, and Meira GR
- Subjects
- Animals, Antibodies, Protozoan immunology, Humans, Immunologic Tests, Materials Testing, Models, Theoretical, Trypanosoma cruzi immunology, Chagas Disease diagnosis, Immunoassay instrumentation, Latex chemical synthesis
- Abstract
This article investigates the synthesis of two (monodisperse, carboxylated, and core-shell) latexes, through a batch and a semibatch emulsion copolymerizations of styrene (St) and methacrylic acid (MAA) onto polystyrene latex seeds. A mathematical model of the process was developed that predicts conversion, average particle size, and surface density of carboxyl groups. The model was adjusted to the batch reaction measurements, and then it was used in the design of the semibatch experiment. The semibatch reaction involved an initial homopolymerization of St followed by instantaneous addition of MAA-St-initiator. Compared with the batch reaction results, the semibatch policy more than doubled the surface density of carboxyl groups. The second part of this series describes the development of an immunodiagnosis latex-protein complex for detecting the Chagas disease, by coupling an antigen of Trypanosoma cruzi onto the produced carboxylated latexes.
- Published
- 2008
- Full Text
- View/download PDF
39. Application of nine-color flow cytometry for detailed studies of the phenotypic complexity and functional heterogeneity of human lymphocyte subsets.
- Author
-
Gonzalez VD, Björkström NK, Malmberg KJ, Moll M, Kuylenstierna C, Michaëlsson J, Ljunggren HG, and Sandberg JK
- Subjects
- Antigens, CD1 analysis, Antigens, CD1d, CD8-Positive T-Lymphocytes metabolism, CD8-Positive T-Lymphocytes virology, Cytokines metabolism, Fluorescent Dyes, HIV-1 genetics, HIV-1 immunology, Humans, Immunophenotyping instrumentation, K562 Cells, Orthomyxoviridae immunology, Phenotype, gag Gene Products, Human Immunodeficiency Virus immunology, Antigens, CD analysis, CD8-Positive T-Lymphocytes immunology, Flow Cytometry instrumentation, Immunity, Cellular, Immunophenotyping methods, Killer Cells, Natural immunology, Lymphocyte Activation, Lymphocyte Subsets immunology
- Abstract
Innate and adaptive cellular immunity is initiated, directed and regulated by a vast array of cell surface receptors. Attempts to harness the cellular immune system in translational settings such as immunotherapy and vaccine development require tools to accurately describe and isolate lymphocytes with specific characteristics. One such tool, flow cytometry, is undergoing a revolution in instrumentation and reagents, providing opportunities for high resolution phenotypic and functional analysis of lymphocytes. Here, we demonstrate how nine-color flow cytometry can be adapted, optimized and applied to investigate the phenotypic complexity and functional heterogeneity of human lymphocyte subsets. We provide examples of studies of adaptive T cell responses against viruses, as well as the assessment of CD1d-restricted NKT cells and NK cells. We discuss the importance of this technology for detailed investigations of lymphocyte subsets in studies of infectious diseases and cancer.
- Published
- 2008
- Full Text
- View/download PDF
40. CXCR5+ CCR7- CD8 T cells are early effector memory cells that infiltrate tonsil B cell follicles.
- Author
-
Quigley MF, Gonzalez VD, Granath A, Andersson J, and Sandberg JK
- Subjects
- Adult, Antibody Formation, Antigens, CD biosynthesis, Antigens, Differentiation, T-Lymphocyte biosynthesis, B-Lymphocytes cytology, B-Lymphocytes immunology, Cell Differentiation, Chemokine CXCL13 pharmacology, Chemotaxis, Leukocyte drug effects, Coculture Techniques, Humans, Inducible T-Cell Co-Stimulator Protein, Lymphocyte Activation, Lymphocyte Cooperation, Palatine Tonsil immunology, Palatine Tonsil ultrastructure, Receptors, CCR7 analysis, Receptors, OX40 biosynthesis, T-Lymphocyte Subsets cytology, T-Lymphocyte Subsets drug effects, T-Lymphocytes, Cytotoxic cytology, T-Lymphocytes, Cytotoxic drug effects, CD8 Antigens analysis, Cytokines metabolism, Germinal Center cytology, Immunologic Memory immunology, Palatine Tonsil cytology, Receptors, CXCR5 analysis, T-Lymphocyte Subsets immunology, T-Lymphocytes, Cytotoxic immunology
- Abstract
Naive and central memory CD8 T cells use CCR7 to recirculate through T cell zones of secondary lymphoid organs where they can encounter antigen. Here we describe a subset of human CD8 T cells expressing CXCR5 which enables homing in response to CXCL13 produced within B cell follicles. CXCR5+ CD8 T cells were found in tonsil B cell follicles, and isolated cells migrated towards CXCL13 in vitro. They expressed CD27, CD28, CD45RO, CD69, and were CD7low, and produced IFN-gamma and granzyme A but lacked perforin, a functional profile suggesting that these cells are early effector memory cells in the context of contemporary T cell differentiation models. Receptors important in the interaction with B cells, including CD70, OX40 and ICOS, were induced upon activation, and CXCR5+ CD8 T cells could to some extent support survival and IgG production in tonsil B cells. Furthermore, CXCR5+ CD8 T cells expressed CCR5 but no CCR7, suggesting a migration pattern distinct from that of follicular CD4 T cells. The finding that a subset of early effector memory CD8 T cells use CXCR5 to locate to B cell follicles indicates that MHC class I-restricted CD8 T cells are part of the follicular T cell population.
- Published
- 2007
- Full Text
- View/download PDF
41. CD8 T cell effector maturation in HIV-1-infected children.
- Author
-
Jordan KA, Furlan SN, Gonzalez VD, Karlsson AC, Quigley MF, Deeks SG, Rosenberg MG, Nixon DF, and Sandberg JK
- Subjects
- Adolescent, CD8-Positive T-Lymphocytes pathology, CD8-Positive T-Lymphocytes physiology, Cell Differentiation, Child, Child, Preschool, Gene Products, gag immunology, Granzymes, HIV Infections pathology, HIV Infections virology, Humans, Immunologic Memory, In Vitro Techniques, Interferon-gamma metabolism, Leukocyte Common Antigens metabolism, Membrane Glycoproteins metabolism, Peptide Fragments immunology, Perforin, Pore Forming Cytotoxic Proteins, Receptors, CCR7, Receptors, Chemokine metabolism, Serine Endopeptidases metabolism, env Gene Products, Human Immunodeficiency Virus, CD8-Positive T-Lymphocytes immunology, HIV Infections immunology, HIV-1
- Abstract
HIV-1 infection generates maturational responses in overall CD4 and CD8 T cell populations in adults, with elevated expression of lytic effector molecules perforin and granzyme B, and reduced expression of CCR7 and CD45RA. Here, we have found that these marked effects were significantly less pronounced in children, both in terms of the skewed CCR7/CD45RA expression profile as well as the increased perforin expression. Similar to adults, HIV-specific CD8 cells in children were largely CD27+ CD45RA- and lacked perforin. However, one pediatric subject with late-stage infection displayed robust expansion of Gag 77-85-specific CD8 T cells which were perforin+ and lytic, but lacked expression of CD27 and IFNgamma. Our data indicate that the T cell effector maturation induced by HIV-1 infection is markedly weaker in children as compared to adults. The data also suggest, however, that the perforin-deficient state of HIV-specific CD8 T cells in children may be reversible.
- Published
- 2006
- Full Text
- View/download PDF
42. Contamination by larger particles of two almost-uniform latices: analysis by combined dynamic light scattering and turbidimetry.
- Author
-
Gonzalez VD, Gugliotta LM, Vega JR, and Meira GR
- Abstract
Multiangle dynamic light scattering (MDLS) and turbidimetry (T) were applied (both individually and combined) for determining the contamination by larger particles of two almost-uniform polystyrene (PS) latices. Latex 1 was synthesized in our laboratories, and it contained a main population diameter of 340 nm together with a small fraction of larger particles. This latex was used as the base material for producing an immunoassay kit. Latex 2 was obtained by a simple blend of two uniform PS standards. The proposed data treatment calculates the diameter and number fraction of the large particles contamination assuming that the PSDs are bimodal. The calculation involves minimizing the errors between the measurements and their theoretical predictions. When analyzed by combined MDLS-T, the contamination of Latex 1 involved number fraction 0.6% and particle diameter 865 nm. The T average diameter is a function of the measurement wavelength, and the highest deviations of this average to an increasing contamination by large particles were always observed at the higher wavelengths. The DLS average diameter is a function of the measurement angle, but in this case it is impossible to determine a priori the angle of observation that provides the largest deviation of this average diameter to an increasing contamination.
- Published
- 2005
- Full Text
- View/download PDF
43. Expansion of CD7(low) and CD7(negative) CD8 T-cell effector subsets in HIV-1 infection: correlation with antigenic load and reversion by antiretroviral treatment.
- Author
-
Aandahl EM, Quigley MF, Moretto WJ, Moll M, Gonzalez VD, Sönnerborg A, Lindbäck S, Hecht FM, Deeks SG, Rosenberg MG, Nixon DF, and Sandberg JK
- Subjects
- Adult, Anti-Retroviral Agents therapeutic use, Antigens, Viral immunology, CD4 Lymphocyte Count, CD8-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes virology, Case-Control Studies, Cell Proliferation, Disease Progression, HIV Infections drug therapy, Humans, Middle Aged, T-Lymphocyte Subsets cytology, T-Lymphocyte Subsets immunology, T-Lymphocyte Subsets virology, Viral Load, Anti-Retroviral Agents pharmacology, Antigens, CD7 analysis, CD8-Positive T-Lymphocytes cytology, HIV Infections immunology
- Abstract
The antiviral response of CD8 T cells involves the differentiation of naive T cells into distinct types of effector and memory cells, which may be distinguished by the level of CD7 expression. We have investigated CD8 T cells in adults and children infected with HIV-1 to determine the disease relevance of cell subsets defined by CD7. CD8 T cells from patients infected with HIV-1 displayed profound down-modulation of CD7 expression as compared with healthy subjects, with expansion of both CD7(low) and CD7(negative) effector subsets. Loss of CD7(high) cells correlated directly with HIV-1 load and was particularly pronounced in patients with rapid disease progression. CD8 T cells specific for HIV-1, as well as Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were predominantly found in the CD7(low) effector cell subset. Furthermore, recovery of CD4 counts on antiretroviral therapy was associated with reversion of the skewed CD7 profile in CD8 T cells. Thus, effector CD8 T-cell subsets distinguished by lowered CD7 expression expand in a manner that correlates with the magnitude of HIV-1, EBV, and CMV antigenic challenge and contract in response to successful antiretroviral treatment. The results are discussed in relation to the dual roles of CD7 as a receptor of both costimulation and cell death.
- Published
- 2004
- Full Text
- View/download PDF
44. Latex particle size distribution by dynamic light scattering: novel data processing for multiangle measurements.
- Author
-
Vega JR, Gugliotta LM, Gonzalez VD, and Meira GR
- Abstract
Multiangle dynamic light scattering (DLS) provides a better estimate of particle size distributions (PSD) than single-angle DLS. However, multiangle data treatment requires appropriate weighting of each autocorrelation measurement prior to calculation of the PSD. The weighting coefficients may be directly obtained from (i). the autocorrelation baselines or (ii). independent measurement of the average light intensity by elastic light scattering. However, the propagation of errors associated with such procedures may intolerably corrupt the PSD estimate. In this work, an alternative recursive least-squares calculation is proposed that estimates the weighting coefficients on the basis of the complete autocorrelation measurement. The method was validated through a numerical example that simulates the analysis of a polystyrene latex with a bimodal PSD and with "measurements" taken at 10 detection angles. The ill-conditioned nature of the problem determines that the "true" PSD cannot be recovered, even in the absence of errors. A sensitivity analysis was carried out to determine the effect of errors in the weighting coefficients on the PSD recoveries.
- Published
- 2003
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.