Detection and quantification of cytosolic DNA.

Cytosolic DNA sensing is emerging to be a critical component of the antitumor immune response by jumpstarting innate immune responses subsequent to the stimulation of the cGAS and STING pathway. Investigating the accumulation of DNA species in the cytosol is therefore an essential readout for promising anticancer strategies. In this chapter, we present different techniques that can be utilized to detect and quantify cytosolic DNA accumulation.

Quantification of eIF2alpha phosphorylation during immunogenic cell death.

Immunogenic cell death (ICD) is a particular modality of cell death that can be triggered by selected anticancer chemotherapeutics. Tumor cells undergoing ICD can induce an adaptive anticancer immune response that targets residual cancer cells with the same antigenic profile. The activation of a full-blown immune response against the tumor antigen is preceded by the release or exposure of danger associated molecular patterns (DAMPs) by tumor cells that stimulate the attraction, activation and maturation of dendritic cells and eventually the antigen-specific priming of cytotoxic T lymphocytes (CTLs). The phosphorylation of the eukaryotic translation initiation factor (EIF2A) is a pathognomonic characteristic of ICD, which governs the release/exposure of DAMPs such as ATP and calreticulin and thus the immunogenicity of cell death. Here we describe techniques to detect eIF2alpha phosphorylation for the assessment of ICD.

Flow Cytometry Analysis to Identify Human CD8+ T Cells.

Flow cytometry is a powerful technique allowing multiparameter detection and quantification of single cells or particles including cell size, granularity, cell components (DNA, mRNA), surface receptors, intracellular proteins, and signaling events. The flow cytometer operates via three main systems: the fluidics, optics, and electronics, which work together to analyze the physical and chemical properties of your sample. The first system, the fluidics, transports your sample in a single stream through the instrument, from the sample tube, pass the lasers, and is either sorted for further experiments or discarded into the waste vessel. The second system, the optical system, is composed of a series of lasers; for excitation of your sample and attached fluorescence antibodies as it passes, a series of lenses; and a detector system. The third system is the electronic component, which enables the photocurrent from the detector system to be changed into electronic pulses to be processed by a computer and analyzed by flow cytometry software. Flow cytometry is thus a powerful technique, which is commonly used to determine the expression of cell surface markers and intracellular molecules to define cells into different populations by fluorescently labeled antibodies.The staining procedure outlined below creates a single-cell suspension for staining with a panel of flow cytometry antibodies, which target different surface markers, to identify an array of cell types. After staining the sample is loaded into the flow cytometer, where the fluorescently labeled cells are excited as they pass by the laser emitting light at various wavelengths which are detected by the flow cytometer. Each fluorescent antibody has its own excitation and emission spectrum allowing the use of multiple antibodies. However, the emission spectrums of different fluorochromes can overlap each other, called spectral overlap. Thus, it is important to have good compensation controls to eliminate any antibody spillover.The staining methods from this technique can be used for different cell types by changing the surface marker targeted by the flow antibody. It is also important to use knowledge of the density of surface molecule for detection and brightness of fluorochrome to guide antibody selection and also to titrate all antibodies prior to use.This chapter's protocol has been designed specifically for detection of human CD8+ T cells defining the activation status of the cells by surface marker phenotyping.

Flow Cytometry Analysis to Identify Human CD4+ T Cell Subsets.

Flow cytometry is a powerful tool, which uses lasers to analyze a wide range of different characteristics of cells. It is commonly used to determine the expression of cell surface markers and intracellular molecules to define cells into different populations using cell size, granularity, and fluorescently labeled antibodies. Thus, flow cytometry enables simultaneous and mutliparameter analysis of single cells.During the staining procedure, a single cell suspension is created for staining with flow cytometry antibodies for analysis on the flow cytometer. The staining methods from this technique can be used for different cell types by changing the surface marker targeted by the flow antibody, provided all antibodies are titrated prior to use, and are chosen with knowledge of the density of surface molecule for detection and brightness of fluorochrome to guide antibody selection.This chapter's protocol has been designed specifically for detection of human CD4+ T cell subsets defining naïve and memory subpopulations by surface marker phenotyping.

Gene Modification and Immunological Analyses for the Development of Immunotherapy Utilizing T Cells Redirected with Antigen-Specific Receptors.

Cancer immunotherapy has been developed and established as a new treatment modality. Recently, adoptive transfer therapy using T cells redirected with antigen-specific antitumor receptors, such as T-cell receptor (TCR) and chimeric antigen receptor (CAR), has demonstrated clinical benefits even in patients with refractory malignancies. To advance this treatment modality, both generation of gene-modified T cells and evaluation of their reactivity with high quality in vitro are required. To achieve this, it is important to establish the ways (1) to generate optimal viral particle for T-cell transduction, (2) to transduce antitumor receptors into T cells and expand redirected T cells efficiently, and (3) to assess the functionality of antigen-specific gene-modified T cells precisely. Here, we summarize established protocols to generate and analyze antitumor receptor-transduced T cells. These procedures help to further assess characteristics of gene-modified T cells, resulting in promotion of translational research for cancer immunotherapy.

In Vitro Differentiation of T Cells: From Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.

In this chapter, we describe a protocol for hematopoietic differentiation of human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived from non-T cells, followed by the differentiation of the T-cell lineage. Derivation of T cells from PSCs involves three steps: induction of PSCs to hematopoietic progenitor cells (HPCs), differentiation of HPCs into progenitor T cells, and maturation of progenitor T cells into mature T cells (CD8 single-positive (SP) or CD4 SP).

In Vitro Differentiation of T Cells: From Nonhuman Primate-Induced Pluripotent Stem Cells.

In this chapter, we describe a protocol for hematopoietic differentiation of nonhuman primate (NHP)-induced pluripotent stem cells (iPSCs) derived from T cells and generation of T cells. Derivation of T cells from PSCs involves three steps: induction of PSCs to hematopoietic progenitor cells (HPCs), differentiation of HPCs into progenitor T cells, and maturation of progenitor T cells into mature T cells, in particular CD8 single-positive (SP) T cells.

Clonogenic Culture of Mouse Thymic Epithelial Cells.

The thymus plays an essential role in the development and selection of T cells by providing a unique microenvironment that is mainly composed of thymic epithelial cells (TECs). We previously identified stem cells of medullary TECs (mTECs) that are crucial for central tolerance induction using a novel clonogenic culture system. We also found that medullary thymic epithelial stem cells (mTESCs) maintain life-long mTECs regeneration and central T cell self-tolerance in mouse models. The clonogenic efficiency of TECs in vitro is highly correlated to the TEC reconstitution activity in vivo. Here, we describe the clonogenic culture system to evaluate the self-renewing activity of TESCs. The colonies are derived from TESCs, are visualized and quantified by rhodamine-B staining on a feeder layer, and can be passaged in vitro. Thus, our system enables quantitative evaluation of TESC activity and is useful for dissecting the mechanisms that regulate TESC activity in physiological aging as well as in various clinical settings.

Detection of u-serrated patterns in direct immunofluorescence images of autoimmune bullous diseases by inhibition-augmented COSFIRE filters.

Direct immunofluorescence (DIF) microscopy of a skin biopsy is used by physicians and pathologists to diagnose autoimmune bullous dermatoses (AIBD). This technique is the reference standard for diagnosis of AIBD, which is used worldwide in medical laboratories. For diagnosis of subepidermal AIBD (sAIBD), two different types of serrated pattern of immunodepositions can be recognized from DIF images, namely n- and u-serrated patterns. The n-serrated pattern is typically found in the most common sAIBD bullous pemphigoid. Presence of the u-serrated pattern indicates the sAIBD subtype epidermolysis bullosa acquisita (EBA), which has a different prognosis and requires a different treatment. The manual identification of these serrated patterns is learnable but challenging. We propose an automatic technique that is able to localize u-serrated patterns for automated computer-assisted diagnosis of EBA. The distinctive feature of u-serrated patterns as compared to n-serrated patterns is the presence of ridge-endings. We introduce a novel ridge-ending detector which uses inhibition-augmented trainable COSFIRE filters. Then, we apply a hierarchical clustering approach to detect the suspicious u-serrated patterns from the detected ridge-endings. For each detected u-serrated pattern we provide a score that indicates the reliability of its detection. In order to evaluate the proposed approach, we created a data set with 180 DIF images for serration pattern analysis. This data set consists of seven subsets which were obtained from various biopsy samples under different conditions. We achieve an average recognition rate of 82.2% of the u-serrated pattern on these 180 DIF images, which is comparable to the recognition rate achieved by experienced medical doctors and pathologists.

Performance evaluation of direct fluorescent antibody, Focus Diagnostics Simplexa™ Flu A/B & RSV and multi-parameter customized respiratory Taqman® array card in immunocompromised patients.

BACKGROUND: Molecular assays for diagnosis of Flu A, Flu B, and RSV with short turn-around-time (TAT) are of considerable clinical importance. In addition, rapid and accurate diagnosis of a large panel of viral and atypical pathogens can be crucial in immunocompromised patients. OBJECTIVES: First, to evaluate the performance of the Simplexa™ Direct assay system in comparison with direct fluorescent antibody (DFA) and customized Taqman® Array Card (TAC) testing for RSV, Flu A, and Flu B in immunocompromised patients. Second, to evaluate different algorithms for the detection of respiratory pathogens in terms of cost, turn-around-time (TAT) and diagnostic yield. STUDY DESIGN: We collected 125 nasopharyngeal swabs (NTS) and 25 BAL samples from symptomatic immunocompromised patients. Samples for which Simplexa™ and TAC results were discordant underwent verification testing. The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. RESULTS: The overall sensitivity was significantly lower for DFA testing than for the two molecular methods (p<0.05). Performance characteristics of Simplexa™ testing were not significantly different compared to TAC testing (p>0.1). For BAL samples only, the sensitivity and specificity of the Simplexa™ assay was 100%. In total, 6.7, 16 and 18% of samples were positive for Flu A, Flu B or RSV by DFA, Simplexa™ and TAC testing, respectively. When considering not only these pathogens but also all results for TAC, the method identified 93 samples with one or more respiratory pathogens (62%). A co-infection rate of 15.3% was found by TAC. The estimated costs and TAT were 8.2€ and 2h for DFA, 31.8€ and 1.5h for Simplexa™ and 55€ and 3h for TAC testing. CONCLUSIONS: Performing the Simplexa™ test 24h a day/7 days a week instead of DFA would considerably improve the overall sensitivity and time-to-result, albeit at a higher cost generated in the laboratory. Performing the TAC would increase the diagnostic yield and detection of co-infections significantly.

Pénfigo herpetiforme: revisión de un caso en edad pediátrica / Herpetiforme pemphigus: report of a pediatric case

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Mapeo por inmunofluorescencia para el diagnóstico de epidermólisis ampollosa congénita / Immunofluorescence Mapping for Diagnosis of Congenital Epidermolysis Bullosa

Las herramientas para el diagnóstico en las epidermólisis ampollosas (EA) han tenido un gran avance desde que Hintner et al, introdujeron el mapeo antigénico como prueba diagnóstica en este grupo de genodermatosis. La utilización de anticuerpos monoclonales/policlonales dirigidos contra algunas de las proteínas específicas que conforman la epidermis y la membrana basal epidérmica, han servido para clasificar los 4 tipos de epidermólisis ampollosa y subclasificar todas sus variantes. Ante la presencia de un recién nacido con ampollas surgen diagnósticos diferenciales múltiples, en donde la microscopia de luz orienta el diagnostico de epidermólisis ampollosa. Sin embargo, el mapeo por inmunofluorescencia y la microscopia electrónica permiten confirmar y clasificar a las epidermólisis ampollosas congénitas. En este artículo, se explica la importancia y metodología para desarrollar la técnica de mapeo antigénico por inmunofluorescencia, con el propósito de clasificar y subclasificar las epidermólisis ampollosas (AU)

The tools for diagnosis of epidermolysis bullosa have advanced greatly since Hintner's group introduced antigen mapping as a diagnostic test for this family of genodermatoses. Monoclonal or polyclonal antibodies raised against some of the specific proteins found in the epidermis and basement membrane of the epidermis have allowed 4 types of epidermolysis bullosa de be identified and all variants to be classified. When a newborn baby presents with blisters, many conditions are implicated in the differential diagnosis. Examination under an optical microscope can suggest epidermolysis bullosa, but immunofluorescence mapping and electron microscopy are required for confirmation of the diagnosis and further classification of congenital epidermolysis bullosa. This article explains the importance of immunofluorescence antigen mapping and describes the methods employed for classification and subclassification of epidermolysis bullosa (AU)

Monolithic silicon chip for immunofluorescence detection on single magnetic beads.

While fluorescence detection is widely used for bioassays owing to its high sensitivity, a complete fluorescent microscopy setup, comprised of a light source, optical filters, a microscope body, and a camera, still is bulky equipment, compromising its use in a point-of-care environment. Here we propose an integrated monolithic silicon chip for integrated magnetic manipulation and optical detection of fluorescently labeled magnetic beads. Our approach permits microscopeless measurement of the fluorescence of a single microparticle. We demonstrate the viability of this approach by the detection of cancer biomarker 5D10 monoclonal antibodies (mAbs) in a noncompetitive sandwich immunoassay performed on the surface of magnetic beads, in a phosphate buffered saline-bovine serum albumin (PBS-BSA) solution, with a detection limit of 1 ng mL(-1).

SFMAC: a novel method for analyzing multiple parameters on lymphocytes with a single fluorophore in cell-microarray system.

The analysis of single cells with multiple parameters in flow cytometry or microscopy requires suitable combinations of fluorophores and optical filters. The growing demands for the multiplex analysis of cells increase the requirements for developing new fluorophores and techniques. We have developed a novel method of analyzing a large number of cells with multiple parameters on a single-cell basis using a single fluorophore. Cells were arrayed onto a microwell array chip with an array of 45,000 microwells, which could capture single cells, stained with a phycoerythrin (PE)-conjugated antibody to a marker, and analyzed with a cell-scanner. After analysis, we photobleached the PE molecules by irradiating the sample with blue light. Because the fluorescence of PE was not recovered after the photobleaching and the analyzed cells remained in the same microwells on the chip, we could repeatedly stain and analyze the same cells with other markers using PE. We applied a method of analyzing lymphocytes from 100 microL of peripheral blood for cytokine secretion and expression of intracellular proteins as well as for multiple cell surface markers. This novel method enables us to analyze multiple markers with a single fluorophore using a simple apparatus. The method may expand the scope of cytometry.

Infecciones virales de vías respiratorias en los primeros seis meses de vida / Viral respiratory tract infections in the first six months of life

Antecedentes: El virus respiratorio sincitial (VRS) y otros virus son causas conocidas de hospitalización en lactantes. Menos conocido es el patrón de virus en infecciones extrahospitalarias en menores de 6 meses. Objetivo: El objetivo de este estudio es describir las características clínicas y los factores epidemiológicos asociados con las infecciones respiratorias virales de ámbito extrahospitalario en menores de 6 meses. Pacientes y métodos: Estudio prospectivo en cohorte de niños de las áreas 8 y 9 de Madrid controlados desde el nacimiento mediante llamadas telefónicas quincenales durante una temporada invernal. Se registraron datos clínicos y epidemiológicos en cuestionarios prediseñados. Se exploró y recogió el aspirado nasofaríngeo (ANF) cuando el paciente presentó sintomatología compatible con una infección respiratoria. El diagnóstico de los virus más comunes se realizó con inmunofluorescencia directa (IFD) y amplificación genómica (PCR). Resultados: Fueron seleccionados 316 recién nacidos. Se realizaron 1.865 llamadas telefónicas (mediana 4), y 106 visitas, en 89 de las cuales se confirmó la enfermedad. Los síntomas más frecuentes fueron rinitis (91 %) y tos (69 %). El diagnóstico clínico principal fue infección respiratoria de vías altas (82 %); 17 de 72 ANF realizados (23,2 %) fueron positivos. Se detectaron rinovirus (41,1 %) y VRS (35,2 %). Ingresaron un 16 % (17/106) de los niños atendidos por enfermedad (el 5,3 % de la cohorte), diagnosticados de síndrome febril y de bronquiolitis. No encontramos ningún factor epidemiológico asociado con la infección respiratoria viral en los casos positivos. Conclusiones: En nuestro medio las infecciones respiratorias de los lactantes son en su mayoría banales y no precisan atención hospitalaria. El rinovirus y el VRS son los principales agentes etiológicos. No se encontraron factores epidemiológicos relacionados con la infección respiratoria asociada a virus (AU)

Background: Respiratory syncytial virus and Influenza virus infections are known causes of hospital admission in infants. It is less well known the pattern of virus infections in infants under 6 months of age in the outpatient setting. Objective: To describe the clinical and epidemiological pattern of community-acquired viral respiratory infections in infants under 6 months. Patients and methods: A cohort of infants from the 8 and 9 Madrid Health Districts was followed by telephone calls every two weeks since birth during the epidemic winter season. Clinical and epidemiological data were collected in pre-designed questionnaires. Nasopharyngeal aspirate was obtained in every patient with symptoms compatible with respiratory infection. Diagnosis of the more common virus was made with direct immunofluorescence and nucleic acid amplification test (PCR). Results: Were recruited 316 newborns. The 1,865 phone calls made (median 4 for every child), produced 106 visits, and the illness confirmed in 89 illness. Rhinitis (91 %) and cough (69 %) were the most common symptoms. Upper respiratory infection was the principal clinical diagnosis (84.5 %), and 17 of the 72 samples (23.2 %) were positive. Most common viruses were RSV (41.1 %) and rhinovirus (35.2 %). Of the children visited, 17 out of 106 (16 %) (5.3 % of the cohort) were admitted to hospital. Diagnoses were febrile syndrome and bronchiolitis. We did not find any epidemiological factor associated with viral respiratory infection in positive cases. Conclusions: In our population most of the respiratory infections in infants are minor and do not need hospital assistance. Rhinovirus and RSV are the major pathogens. We did not find any epidemiological factor associated with viral respiratory infection (AU)

Evaluation of direct immunofluorescence test for diagnosis of upper respiratory tract infection by Chlamydia pneumoniae.

BACKGROUND: Chlamydia pneumoniae causes a variety of respiratory infections and is involved in cardiovascular diseases. Diagnosis of C. pneumoniae infection currently relies on antibody detection by microimmunofluorescence (MIF), which has limited use, and is the retrospective diagnosis for acute infection. OBJECTIVE: Find an effective early diagnosis of acute upper respiratory infection, or use in combination with MIF to accurately diagnose the infection by C. pneumoniae. MATERIAL AND METHOD: Direct immunofluorescence (DIF) was developed to detect C. pneumoniae in nasopharyngeal specimens obtained from patients with upper respiratory tract infection, and normal individuals. IgM and IgG antibodies against C. pneumoniae by MIF were determined for evaluation of the detected C. pneumoniae and seroconversion. RESULTS: DIF gave positive results in 29 of 37 (78.4%) samples from 31 patients. Fifteen samples positive by DIF illustrated antibody titers interpreted as acute C. pneumoniae infection, and eight DIF positive samples showed antibody titers of chronic infection. Negative results by both DIF and MIF were found in two patients and 23 of 25 by DIF but 20 of 25 by MIF in normal subjects. Five paired sera subsequently collected from three of the 31 patients illustrated seroconversion 2-4 months after the primary specimen collection, which gave positive results by DIF but negative for antibodies. Significant association was found between C. pneumoniae detection by DIF and antibodies by MIF when analysis was done in the group of patients and normal subjects (p < 0.001; Pearson chi-square test). CONCLUSION: DIF could be an alternative assay for early diagnosis of C. pneumoniae infection, and may be used in combination with MIF for accurate diagnosis of acute C. pneumoniae infection.

A novel technique to eliminate cross-contamination when making wells on slides for rabies diagnosis.

The direct fluorescent antibody (DFA) rabies diagnostic test requires demarcating desirable areas of brain tissue slip smear slides to be stained, traditionally achieved by applying paint from a tech pen or using a wax pencil to form a circle or dam-like ring or well into which rabies conjugate is expelled. Unfortunately, using these instruments poses a possibility of cross-contamination by transfer via the pen or pencil tip of rabies antigen from one slide to another. A new method was developed to avoid cross-contamination. The open end of a disposable glass test tube, dipped into a shallow reservoir of nail polish, was used to apply a dam-like ring about the slip smear area to be stained, after which the test tube was discarded, thereby precluding tissue transfer.

Methods in virus diagnosis: immunofluorescence revisited.

BACKGROUND: Immunofluorescence (IF) has been used in many laboratories for virus diagnosis but has begun to fall inappropriately out of favour as a diagnostic method as pressure on budgets and for objective quality control increases. OBJECTIVES: To review the status, value and benefits of IF. CONCLUSIONS: IF has, we believe, still a valuable role to play in routine virus diagnosis because it is rapid, accurate (with properly validated reagents), flexible and, by giving feedback on the quality of the specimens collected, promotes dialogue with the customer clinicians to their benefit and to that of the diagnostic laboratory. These benefits are not easily duplicated by other methods or techniques. While such rapid diagnosis primarily benefits the individual patient, providing results within a clinically relevant time has a secondary effect of increasing use of the service. It is our experience that the availability of rapid IF diagnosis (as opposed to culture or serology) for respiratory viral infections leads to a substantial increase in its use, thereby enhancing the amount and breadth of the resultant epidemiological data.

Report from a workshop on multianalyte microsphere assays.

Multiplexed assays using fluorescent microspheres is an exciting technique that has been gaining popularity among researchers, particularly those in the public health field. Part of its popularity is due to its flexibility, as both immunoassays and oligonucleotide hybridization assays can be developed on this platform. This report summarizes a workshop held by the Centers for Disease Control and Prevention that discussed issues surrounding these assays and the Luminex 100 xMAP instrument. Topics included instrumentation, assay design, sample matrix and volume, quality control, and development of commercial applications.