Assessment of susceptible chemical modification sites of trastuzumab and endogenous human immunoglobulins at physiological conditions.

The quality control testing of chemical degradations in the bio-pharmaceutical industry is currently under controversial debate. Here we have systematically applied in vitro and in vivo stress conditions to investigate the influence of protein degradation on structure-function. Extensive purification and characterization enabled identification and functional assessment of the physiological degradation of chemical modification sites in the variable complementarity-determining regions (CDRs) and conserved region of trastuzumab. We demonstrate that the degradation of the solvent-accessible residues located in the CDR and the conserved fragment crystallizable region (Fc) occurs faster in vivo (within days) compared to the levels observed for bio-process and real-time storage conditions. These results hence question the rationality of extreme monitoring of low level alterations in such chemical modifications as critical patient safety parameters in product quality control testing, given that these modifications merely mirror the natural/physiological aging process of endogenous antibodies.

Assessment of Telomere Length, Phenotype, and DNA Content.

Telomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence in situ hybridization of fluorescently-labeled probes (Flow-FISH) without prior cell separation. After cells are stained for surface immunofluorescence, antigen-antibody complexes are covalently cross-linked onto cell membranes before FISH with a telomere-specific probe. Cells with long telomeres are included as internal standards. Addition of a DNA dye permits exclusion of proliferating cells during data analysis. DNA ploidy measurements of cells of interest and internal standard are performed on separate aliquots in parallel to Flow-FISH. Telomere fluorescence of G0/1 cells of subpopulations and internal standards obtained from Flow-FISH are normalized for DNA ploidy, and telomere length in subsets of interest is expressed as a fraction of the internal standard telomere length. © 2017 by John Wiley & Sons, Inc.

A scalable software framework for data integration in bioprocess development.

Effectiveness in lab workflows-despite progresses made in automation and lab informatics-is often hindered by insufficient integration of devices and data. The iLAB software framework, a middleware connecting and integrating devices and data, provides a plugin architecture that can be adapted to individual lab environments. Integration of devices is preferably based on standardized data and communication protocols. In addition to device integration, process data and result data from different sources (e.g. readers) are converted to a standard format and administered by a powerful database for further processing. In this paper, the use of iLAB in a bioprocess development application is described. Process parameters and measured values of two high-throughput bioreactor systems (96-well plates and 10 mL reactor vessels) are collected in the iLAB database. Data from screening experiments and offline data are visualized, analyzed, and compared. A filter algorithm allows searching for matching parameters in experiments as well as the comparison of correlated datasets, independently from the used bioreactor system. The database model enables the consolidation of data, the transition from data to information, and a solid base for management decisions on enterprise level.

International Society for the Advancement of Cytometry cell sorter biosafety standards.

Flow cytometric cell sorting of biological specimens has become prevalent in basic and clinical research laboratories. These specimens may contain known or unknown infectious agents, necessitating precautions to protect instrument operators and the environment from biohazards arising from the use of sorters. To this end the International Society of Analytical Cytology (ISAC) was proactive in establishing biosafety guidelines in 1997 (Schmid et al., Cytometry 1997;28:99-117) and subsequently published revised biosafety standards for cell sorting of unfixed samples in 2007 (Schmid et al., Cytometry Part A J Int Soc Anal Cytol 2007;71A:414-437). Since their publication, these documents have become recognized worldwide as the standard of practice and safety precautions for laboratories performing cell sorting experiments. However, the field of cytometry has progressed since 2007, and the document requires an update. The new Standards provides guidance: (1) for laboratory design for cell sorter laboratories; (2) for the creation of laboratory or instrument specific Standard Operating Procedures (SOP); and (3) on procedures for the safe operation of cell sorters, including personal protective equipment (PPE) and validation of aerosol containment.

Natural killer T cells in advanced melanoma patients treated with tremelimumab.

A significant barrier to effective immune clearance of cancer is loss of antitumor cytotoxic T cell activity. Antibodies to block pro-apoptotic/downmodulatory signals to T cells are currently being tested. Because invariant natural killer T cells (iNKT) can regulate the balance of Th1/Th2 cellular immune responses, we characterized the frequencies of circulating iNKT cell subsets in 21 patients with melanoma who received the anti-CTLA4 monoclonal antibody tremelimumab alone and 8 patients who received the antibody in combination with MART-126-35 peptide-pulsed dendritic cells (MART-1/DC). Blood T cell phenotypes and functionality were characterized by flow cytometry before and after treatment. iNKT cells exhibited the central memory phenotype and showed polyfunctional cytokine production. In the combination treatment group, high frequencies of pro-inflammatory Th1 iNKT CD8(+) cells correlated with positive clinical responses. These results indicate that iNKT cells play a critical role in regulating effective antitumor T cell activity.

How to develop a standard operating procedure for sorting unfixed cells.

Written standard operating procedures (SOPs) are an important tool to assure that recurring tasks in a laboratory are performed in a consistent manner. When the procedure covered in the SOP involves a high-risk activity such as sorting unfixed cells using a jet-in-air sorter, safety elements are critical components of the document. The details on sort sample handling, sorter set-up, validation, operation, troubleshooting, and maintenance, personal protective equipment (PPE), and operator training, outlined in the SOP are to be based on careful risk assessment of the procedure. This review provides background information on the hazards associated with sorting of unfixed cells and the process used to arrive at the appropriate combination of facility design, instrument placement, safety equipment, and practices to be followed.

Isolation of synaptic terminals from Alzheimer's disease cortex.

Amyloid beta (Aß) oligomers and phosphorylated tau (p-tau) aggregates are increasingly identified as potential toxic intermediates in Alzheimer's disease (AD). In cortical AD synapses, p-tau co-localizes with Aß, but the Aß and p-tau peptide species responsible for synaptic dysfunction and demise remains unclear. The present experiments were designed to use high-speed cell sorting techniques to purify synaptosome population based on size, and then extend the method to physically isolate Aß-positive synaptosomes with the goal of understanding the nature of Aß and tau pathology in AD synapses. To examine the purity of size-gated synaptosomes, samples were first gated on size; particles with sizes between 0.5 and 1.5 microns were collected. Electron microscopy documented a homogenous population of spherical particles with internal vesicles and synaptic densities. Next, size-gated synaptosomes positive for Aß were collected by fluorescence activated sorting and then analyzed by immunoblotting techniques. Sorted Aß-positive synaptosomes were enriched for amyloid precursor protein (APP) and for Aß oligomers and aggregates; immunolabeling for p-tau showed a striking accumulation of p-tau aggregates compared to the original homogenate and purified synaptosomes. These results confirm co-localization of Aß and p-tau within individual synaptic terminals and provide proof of concept for the utility of flow sorting synaptosomes.

Autocrine endothelin-3/endothelin receptor B signaling maintains cellular and molecular properties of glioblastoma stem cells.

Glioblastoma stem cells (GSC) express both radial glial cell and neural crest cell (NCC)-associated genes. We report that endothelin 3 (EDN3), an essential mitogen for NCC development and migration, is highly produced by GSCs. Serum-induced proliferative differentiation rapidly decreased EDN3 production and downregulated the expression of stemness-associated genes, and reciprocally, two glioblastoma markers, EDN1 and YKL-40 transcripts, were induced. Correspondingly, patient glioblastoma tissues express low levels of EDN3 mRNA and high levels of EDN1 and YKL-40 mRNA. Blocking EDN3/EDN receptor B (EDNRB) signaling by an EDNRB antagonist (BQ788), or EDN3 RNA interference (siRNA), leads to cell apoptosis and functional impairment of tumor sphere formation and cell spreading/migration in culture and loss of tumorigenic capacity in animals. Using exogenous EDN3 as the sole mitogen in culture does not support GSC propagation, but it can rescue GSCs from undergoing cell apoptosis. Molecular analysis by gene expression profiling revealed that most genes downregulated by EDN3/EDNRB blockade were those involved in cytoskeleton organization, pause of growth and differentiation, and DNA damage response, implicating the involvement of EDN3/EDNRB signaling in maintaining GSC migration, undifferentiation, and survival. These data suggest that autocrine EDN3/EDNRB signaling is essential for maintaining GSCs. Incorporating END3/EDNRB-targeted therapies into conventional cancer treatments may have clinical implication for the prevention of tumor recurrence.

Baseline immune phenotypes and CD4+ T lymphocyte responses to antiretroviral therapy in younger versus older HIV-infected individuals.

OBJECTIVE: The purpose of the study was to determine associations between pre-antiretroviral therapy (ART) senescent CD8+ T lymphocytes and naïve versus non-naive CD8+ and CD4+ T lymphocyte subpopulations and CD4+ responses after initiation of ART in younger versus older individuals. METHODS: Retrospective analysis of 100 subjects with pre-ART cryopreserved peripheral blood mononuclear cells samples was performed with flow cytometry. Subjects were divided into four groups by age (30-50 years or > 50 years) and 96-week CD4+ response (<100 or >200 cells/mm(3)). All subjects had 96-week viral suppression to <50 copies/mm(3). Regression was utilized to investigate associations between pre-ART CD8+ and CD4+ T cell phenotypes with age and CD4+ response categories. RESULTS: Individuals <50 years had a lower frequency of senescent CD8+ T lymphocytes of the CD56 + 57+, CD56+, and CD28- phenotypes (95%CI -3.6 to -0.02; 95%CI -4.2 to -0.03; 95%CI -12.5 to -1.4, respectively) and a higher frequency of naïve (CD45RA + CD28+) CD8+ T lymphocytes (95%CI 2.6 to 10.9). Younger age and good CD4+ response were associated with a higher frequency of pre-ART naïve CD4+ T cells (95%CI 2.0 to 16.4 and 95%CI 1.5 to 15.6, respectively). CONCLUSIONS: Prior to ART, younger HIV-infected individuals have a higher frequency of naïve CD4+ and CD8+ T cells and lower frequency of senescent CD8+ T cell phenotypes.

Short communication: enhanced CD8+ T cell apoptosis in HIV-infected adolescents with virologic failure on protease inhibitor-based therapy.

In this study, we investigated the possibility of differential effects of protease inhibitor (PI)-containing (PI(+)) and PI-sparing (PI(-)) antiretroviral therapies (ART) on CD8(+) T cell apoptosis. We retrospectively analyzed both PD-1 expression and CD8(+) T cell apoptosis in a cross-sectional study of HIV-positive adolescents and young adults (mean age = 17.4 years), with perinatally or behaviorally acquired HIV infection. Fifty-one specimens of cryopreserved peripheral blood mononuclear cells (PBMCs) were analyzed using 7-color flow cytometry: 20 from patients receiving PI(+) ART, 14 from PI(-) ART, and 17 from the untreated. The results showed that percentages of PD-1(+) CD8(+) T cells were strongly correlated with plasma viral loads regardless of treatment (p = 0.0001). The percentage of PD-1(+) CD8(+) T cells was also positively associated with percentages of Annexin V(+) CD8(+) T cells (p = 0.04) in the PI(+)-treated group. The fraction of apoptotic (Annexin V(+)) CD8(+) T cells was associated with viral load in the patients receiving ART that contained one or more protease inhibitors (p = 0.029), but not in the PI(-) or untreated groups. In summary, we found a direct correlation between PD-1 expression on CD8(+) T cells and HIV levels that was not affected by types of medications used in the ART of those adolescents, suggesting that virological success is necessary for PD-1 downregulation. CD8(+) T cell apoptosis was linked to high levels of PD-1 expression and HIV viremia. However, there was a higher degree of apoptosis among viremic patients receiving PI therapy, suggesting an immunologically adverse effect of continuing PI(+) therapy after virological failure.

Prostate-specific membrane antigen associates with anaphase-promoting complex and induces chromosomal instability.

Prostate-specific membrane antigen (PSMA) is a transmembrane protein highly expressed in advanced and metastatic prostate cancers. The pathologic consequence of elevated PSMA expression in not known. Here, we report that PSMA is localized to a membrane compartment in the vicinity of mitotic spindle poles and associates with the anaphase-promoting complex (APC). PSMA-expressing cells prematurely degrade cyclin B and exit mitosis due to increased APC activity and incomplete inactivation of APC by the spindle assembly checkpoint. Further, expression of PSMA in a karyotypically stable cell line induces aneuploidy. Thus, these findings provide the first evidence that PSMA has a causal role in the induction of aneuploidy and might play an etiologic role in the progression of prostate cancer.

CREB is a critical regulator of normal hematopoiesis and leukemogenesis.

The cAMP-responsive element binding protein (CREB) is a 43-kDa nuclear transcription factor that regulates cell growth, memory, and glucose homeostasis. We showed previously that CREB is amplified in myeloid leukemia blasts and expressed at higher levels in leukemia stem cells from patients with myeloid leukemia. CREB transgenic mice develop myeloproliferative disease after 1 year, but not leukemia, suggesting that CREB contributes to but is not sufficient for leukemogenesis. Here, we show that CREB is most highly expressed in lineage negative hematopoietic stem cells (HSCs). To understand the role of CREB in hematopoietic progenitors and leukemia cells, we examined the effects of RNA interference (RNAi) to knock down CREB expression in vitro and in vivo. Transduction of primary HSCs or myeloid leukemia cells with lentiviral CREB shRNAs resulted in decreased proliferation of stem cells, cell- cycle abnormalities, and inhibition of CREB transcription. Mice that received transplants of bone marrow transduced with CREB shRNA had decreased committed progenitors compared with control mice. Mice injected with Ba/F3 cells expressing either Bcr-Abl wild-type or T315I mutation with CREB shRNA had delayed leukemic infiltration by bioluminescence imaging and prolonged median survival. Our results suggest that CREB is critical for normal myelopoiesis and leukemia cell proliferation.

Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-amino-actinomycin D.

This protocol describes a rapid and simple method for the identification of apoptotic cells. Owing to changes in membrane permeability, early apoptotic cells show an increased uptake of the vital DNA dye Hoechst 33342 (HO342) compared with live cells. The nonvital DNA dye 7-amino-actinomycin D (7-AAD) is added to distinguish late apoptotic or necrotic cells that have lost membrane integrity from early apoptotic cells that still have intact membranes as assayed by dye exclusion. The method is suitable to be combined with cell surface staining using Abs of interest labeled with fluorochromes that are compatible with HO342 and 7-AAD emissions. Surface antigen staining is carried out according to standard methods before staining for apoptosis. The basic assay can be completed in 30 min, and extra time is needed for cell surface antigen staining.

International Society for Analytical Cytology biosafety standard for sorting of unfixed cells.

BACKGROUND: Cell sorting of viable biological specimens has become very prevalent in laboratories involved in basic and clinical research. As these samples can contain infectious agents, precautions to protect instrument operators and the environment from hazards arising from the use of sorters are paramount. To this end the International Society of Analytical Cytology (ISAC) took a lead in establishing biosafety guidelines for sorting of unfixed cells (Schmid et al., Cytometry 1997;28:99-117). During the time period these recommendations have been available, they have become recognized worldwide as the standard practices and safety precautions for laboratories performing viable cell sorting experiments. However, the field of cytometry has progressed since 1997, and the document requires an update. METHODS: Initially, suggestions about the document format and content were discussed among members of the ISAC Biosafety Committee and were incorporated into a draft version that was sent to all committee members for review. Comments were collected, carefully considered, and incorporated as appropriate into a draft document that was posted on the ISAC web site to invite comments from the flow cytometry community at large. The revised document was then submitted to ISAC Council for review. Simultaneously, further comments were sought from newly-appointed ISAC Biosafety committee members. RESULTS: This safety standard for performing viable cell sorting experiments was recently generated. The document contains background information on the biohazard potential of sorting and the hazard classification of infectious agents as well as recommendations on (1) sample handling, (2) operator training and personal protection, (3) laboratory design, (4) cell sorter set-up, maintenance, and decontamination, and (5) testing the instrument for the efficiency of aerosol containment. CONCLUSIONS: This standard constitutes an updated and expanded revision of the 1997 biosafety guideline document. It is intended to provide laboratories involved in cell sorting with safety practices that take into account the enhanced hazard potential of high-speed sorting. Most importantly, it states that droplet-based sorting of infectious or hazardous biological material requires a higher level of containment than the one recommended for the risk group classification of the pathogen. The document also provides information on safety features of novel instrumentation, new options for personal protective equipment, and recently developed methods for testing the efficiency of aerosol containment.

Standard safety practices for sorting of unfixed cells.

Cell sorting of viable biological specimens has become widespread in laboratories involved in basic and clinical research. As these samples can contain infectious agents, precautions to protect instrument operators and the environment from hazards arising from the use of sorters are paramount. This unit presents a revised and updated version of the biosafety guidelines for sorting of unfixed cells established in 1977 by the International Society of Analytical Cytology (ISAC), whose recommendations have become recognized worldwide as the standard practices and safety precautions for laboratories performing viable cell-sorting experiments. The unit contains background information on the biohazard potential of sorting and the hazard classification of infectious agents as well as recommendations on (1) sample handling, (2) operator training and personal protection, (3) laboratory design, (4) cell sorter setup, maintenance, and decontamination, and (5) testing the instrument for the efficiency of aerosol containment.

Combined Flow Cytometric Measurement of Two Cell-Surface Antigens and DNA-RNA Content.

INTRODUCTIONFlow cytometry is frequently used to assess nucleic acid content in individual cells. Based on DNA content alone, however, cells in the quiescent G(0) phase cannot be discriminated from cells in the proliferative G(1) phase, as DNA content remains constant until S-phase entry. In contrast, by measuring RNA content in addition to DNA content, cells can be assigned to G(0) and cell-cycle subcompartments of G(1). Assessing phenotype at the same time as nucleic acid content allows determination of the cell-cycle status of subpopulations in mixed-cell preparations. This protocol describes an optimized method for combining dual-color cell-surface immunofluorescent staining with staining for DNA-RNA, adapted for a basic dual-laser flow cytometer with blue (488-nm) and red (633- or 647-nm) excitation. DNA is stained at low pH in the presence of saponin with 7-aminoactinomycin D (7-AAD), and RNA is stained with pyronin Y (PY). Both dyes are used at low concentration, and 7-AAD is exchanged with nonfluorescent actinomycin D to minimize fluorochrome-fluorochrome interactions, which can negatively affect detection of cell-surface antigen staining.

Assessment of telomere length, phenotype, and DNA content.

Telomere sequences at the end of chromosomes control somatic cell division; therefore, telomere length in a given cell population provides information about its replication potential. This unit describes a method for flow cytometric measurement of telomere length in subpopulations using fluorescence in situ hybridization of fluorescently-labeled probes (Flow-FISH) without prior cell separation. After cells are stained for surface immunofluorescence, antigen-antibody complexes are covalently cross-linked onto cell membranes before FISH with a telomere-specific probe. Cells with long telomeres are included as internal standards. Addition of a DNA dye permits exclusion of proliferating cells during data analysis. DNA ploidy measurements of cells of interest and internal standard are performed on separate aliquots in parallel to Flow-FISH. Telomere fluorescence of G(0/1) cells of subpopulations and internal standards obtained from Flow-FISH are normalized for DNA ploidy and telomere length in subsets of interest is expressed as a fraction of the internal standard telomere length.

Biosafety concerns for shared flow cytometry core facilities.

Many researchers who need flow cytometry for their projects have neither sufficient funds nor the work volume to justify the purchase of an analytic cytometer or cell sorter. In shared flow cytometry facilities, costs for instrument purchases, cytometer maintenance, and personnel are pooled to provide economic services for a multitude of users when they are required. Owing to the diverse nature of the samples that are submitted to core facilities, the biohazard potential of the samples can vary dramatically. For the safety of facility personnel and users, it is critical that information about hazards contained in the samples be transmitted to instrument operators before flow cytometry experiments are started. During 1999 the former Biosafety Committee of the International Society for Analytical Cytology formulated a framework biosafety questionnaire for shared facilities designed to request information about the hazard potential of experimental samples from investigators who wish to use the facility. In this report we review safety issues that are pertinent to flow cytometry core facilities by discussing the individual components of this biosafety questionnaire.

Simultaneous flow cytometric analysis of two cell surface markers, telomere length, and DNA content.

BACKGROUND: Various protocols for estimation of telomere length in individual cells by flow cytometry using fluorescence in situ hybridization of fluorescently labeled peptide nucleic acid (PNA) probes (Flow-FISH) have been described. Combined analysis of telomere length and cell phenotype, however, remains difficult because few fluorochromes with suitable emission spectra tolerate the harsh conditions needed for DNA denaturation during hybridization of the telomere-specific PNA probe. We overcame these problems and developed a method for measuring telomere length in cell subsets characterized by the expression of two surface antigens. METHODS: Alexa Fluor 488 and Alexa Fluor 546 were used for cell surface staining. Antigen-antibody complexes were covalently cross-linked onto the cell membrane before Flow-FISH. Cells were hybridized with a PNA probe conjugated to cyanine 5 (Cy5). Hoechst 33342 (HO342) was added for determination of cellular DNA content. For assay standardization, we added an aliquot of a single batch of 1,301 cells to each sample as an internal control before hybridization with the PNA probe. Samples were prepared in duplicate and analyzed on a standard three-laser BD LSR flow cytometer. For assay validation, the same samples were analyzed in parallel to correlate the percentage of telomere length of the sample versus 1,301 control cells to the mean size of terminal restriction fragments (TRFs) of DNA as determined by Southern gel analysis. RESULTS: The method permitted clear identification of lymphocyte subsets in samples hybridized for Flow-FISH, with subset frequencies comparable to those of untreated samples. At a concentration of 10 nM, the Cy5-labeled telomere-specific PNA probe produced a bright fluorescence signal well separated from background. Addition of HO342 in low concentration did not interfere with Cy5 telomere fluorescence, produced adequate DNA histograms, and permitted clear identification of cell phenotype. The probe concentration of 10 nM also proved optimal for inclusion of 1,301 control cells for assay standardization. Telomere length estimations by the current method correlated highly with TRF calculations by Southern gel hybridization (r(2)= 0.9, P = 0.0003). Application of our protocol to the analysis of human CD8CD28 lymphocyte subsets showed that CD8(+bright)CD28(-) lymphocytes generally exhibit shorter telomeres than CD8(+bright)CD28(+) cells. These data concurred with previous results of telomere shortening in CD8(+)CD28(-) T cells that were obtained by using different techniques. CONCLUSIONS: The multiparameter Flow-FISH protocol permitted rapid determination of differences in telomere length in subpopulations characterized by two surface markers without prior cell separation.