Enriched transcriptome analysis of laser capture microdissected populations of single cells to investigate intracellular heterogeneity in immunostained FFPE sections

To investigate intracellular heterogeneity, cell capture of particular cell populations followed by transcriptome analysis has been highly effective in freshly isolated tissues. However, this approach has been quite challenging in immunostained formalin-fixed paraffin-embedded (FFPE) sections. This study aimed at combining the standard pathology techniques, immunostaining and laser capture microdissection, with whole RNA-sequencing and bioinformatics analysis to characterize FFPE breast cancer cell populations with heterogeneous expression of progesterone receptor (PR). Immunocytochemical analysis revealed that 60% of MCF-7 cells admixture highly express PR. Immunocytochemistry-based targeted RNA-seq (ICC-RNAseq) and in silico functional analysis revealed that the PR-high cell population is associated with upregulation in transcripts implicated in immunomodulatory and inflammatory pathways (e.g. NF-κB and interferon signaling). In contrast, the PR-low cell population is associated with upregulation of genes involved in metabolism and mitochondrial processes as well as EGFR and MAPK signaling. These findings were cross-validated and confirmed in FACS-sorted PR high and PR-low MCF-7 cells and in MDA-MB-231 cells ectopically overexpressing PR. Significantly, ICC-RNAseq could be extended to analyze samples captured at specific spatio-temporal states to investigate gene expression profiles using diverse biomarkers. This would also facilitate our understanding of cell population-specific molecular events driving cancer and potentially other diseases

The omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) reverses corticosterone-induced changes in cortical neurons

Background: Chronic exposure to the glucocorticoid hormone corticosterone exerts cellular stress-induced toxic effects that have been associated with neurodegenerative and psychiatric disorders. Docosahexaenoic acid is a polyunsaturated fatty acid that has been shown to be of benefit in stress-related disorders, putatively through protective action in neurons. Methods: We investigated the protective effect of docosahexaenoic acid against glucocorticoid hormone corticosterone-induced cellular changes in cortical cell cultures containing both astrocytes and neurons. Results: We found that glucocorticoid hormone corticosterone (100, 150, 200 μM) at different time points (48 and 72 hours) induced a dose- and time-dependent reduction in cellular viability as assessed by methyl thiazolyl tetrazolium. Moreover, glucocorticoid hormone corticosterone (200 μM, 72 hours) decreased the percentage composition of neurons while increasing the percentage of astrocytes as assessed by βIII-tubulin and glial fibrillary acidic protein immunostaining, respectively. In contrast, docosahexaenoic acid treatment (6 μM) increased docosahexaenoic acid content and attenuated glucocorticoid hormone corticosterone (200 μM)-induced cell death (72 hours) in cortical cultures. This translates into a capacity for docosahexaenoic acid to prevent neuronal death as well as astrocyte overgrowth following chronic exposure to glucocorticoid hormone corticosterone. Furthermore, docosahexaenoic acid (6 μM) reversed glucocorticoid hormone corticosterone-induced neuronal apoptosis as assessed by terminal deoxynucleotidyl transferase–mediated nick-end labeling and attenuated glucocorticoid hormone corticosterone-induced reductions in brain derived neurotrophic factor mRNA expression in these cultures. Finally, docosahexaenoic acid inhibited glucocorticoid hormone corticosterone-induced downregulation of glucocorticoid receptor expression on βIII- tubulin-positive neurons. Conclusions: This work supports the view that docosahexaenoic acid may be beneficial in ameliorating stress-related cellular changes in the brain and may be of value in psychiatric disorders

Recommendations for the standardization of bone marrow disease assessment and reporting in children with neuroblastoma; on behalf of the International Neuroblastoma Response Criteria Bone Marrow Working Group

BACKGROUND: The current study was conducted to expedite international standardized reporting of bone marrow disease in children with neuroblastoma and to improve equivalence of care. METHODS: A multidisciplinary International Neuroblastoma Response Criteria Bone Marrow Working Group was convened by the US National Cancer Institute in January 2012 with representation from Europe, North America, and Australia. Practical transferable recommendations to standardize the reporting of bone marrow disease were developed. RESULTS: To the authors' knowledge, the current study is the first to comprehensively present consensus criteria for the collection, analysis, and reporting of the percentage area of bone marrow parenchyma occupied by tumor cells in trephine-biopsies. The quantitative analysis of neuroblastoma content in bone marrow aspirates by immunocytology and reverse transcriptase-quantitative polymerase chain reaction are revised. The inclusion of paired-like homeobox 2b (PHOX2B) for immunohistochemistry and reverse transcriptase-quantitative polymerase chain reaction is recommended. Recommendations for recording bone marrow response are provided. The authors endorse the quantitative assessment of neuroblastoma cell content in bilateral core needle biopsies-trephines and aspirates in all children with neuroblastoma, with the exception of infants, in whom the evaluation of aspirates alone is advised. It is interesting to note that 5% disease is accepted as an internationally achievable level for disease assessment. CONCLUSIONS: The quantitative assessment of neuroblastoma cells is recommended to provide data from which evidence-based numerical criteria for the reporting of bone marrow response can be realized. This is particularly important in the minimal disease setting and when neuroblastoma detection in bone marrow is intermittent, where clinical impact has yet to be validated. The wide adoption of these harmonized criteria will enhance the ability to compare outcomes from different trials and facilitate collaborative trial design

Characterization of lymphocyte subsets over a 24-hour period in Pineal-Associated Lymphoid Tissue (PALT) in the chicken

BACKGROUND: Homeostatic trafficking of lymphocytes in the brain has important relevance to the understanding of CNS disease processes. The pineal gland of the chicken contains large accumulations of lymphocytes that suggest an important role related to homeostatic circadian neuro-immune interactions. The purpose of this initial study was to characterize the lymphocyte subsets in the pineal gland and quantitate the distribution and frequency of lymphocyte phenotypes at two time points over the 24-hour light:dark cycle. RESULTS: PALT comprised approximately 10% of the total pineal area. Image analysis of immunocytochemically stained sections showed that the majority of lymphocytes were CD3(+ )(80%) with the remaining 20% comprising B-cells and monocytes (Bu-1(+)), which tended to distribute along the periphery of the PALT. T-cell subsets in PALT included CD4(+ )(75–80%), CD8(+ )(20–25%), TCRαβ/Vβ(1)(+ )(60%), and TCRγδ(+ )(15%). All of the T-cell phenotypes were commonly found within the interfollicular septa and follicles of the pineal gland. However, the ratios of CD8(+)/CD4(+ )and TCRγδ(+)/TCRαβ/Vβ(1)(+ )within the pineal tissue were each 1:1, in contrast to the PALT where the ratios of CD8(+)/CD4(+ )and TCRγδ(+)/TCRαβ/Vβ(1)(+ )each approximated 1:4. Bu-1(+ )cells were only rarely seen in the pineal interstitial spaces, but ramified Bu-1(+ )microglia/macrophages were common in the pineal follicles. Effects of the 24-h light:dark cycle on these lymphocyte-pineal interactions were suggested by an increase in the area of PALT, a decline in the density of TCRαβ/Vβ(1)(+ )cells, and a decline in the area density of Bu-1(+ )microglia at the light:dark interphase (1900 h) compared to the dark:light interphase (0700 h). CONCLUSION: The degree of lymphocyte infiltration in the pineal suggests novel mechanisms of neuro-immune interactions in this part of the brain. Our results further suggest that these interactions have a temporal component related to the 24-hour light:dark cycle and that CD8(+ )and TCRγδ(+ )T-cells are preferentially recruited to the pineal follicles. Pineal microglia/macrophages were common and represent an important candidate for mediating these lymphocyte-pineal interactions via secretion of cytokines and chemokines

Immunohistochemistry as an Important Tool in Biomarkers Detection and Clinical Practice

The immunohistochemistry technique is used in the search for cell or tissue antigens that range from amino acids and proteins to infectious agents and specific cellular populations. The technique comprises two phases: (1) slides preparation and stages involved for the reaction; (2) interpretation and quantification of the obtained expression. Immunohistochemistry is an important tool for scientific research and also a complementary technique for the elucidation of differential diagnoses which are not determinable by conventional analysis with hematoxylin and eosin. In the last couple of decades there has been an exponential increase in publications on immunohistochemistry and immunocytochemistry techniques. This review covers the immunohistochemistry technique; its history, applications, importance, limitations, difficulties, problems and some aspects related to results interpretation and quantification. Future developments on the immunohistochemistry technique and its expression quantification should not be disseminated in two languages—that of the pathologist and another of clinician or surgeon. The scientific, diagnostic and prognostic applications of this methodology must be explored in a bid to benefit of patient. In order to achieve this goal a collaboration and pooling of knowledge from both of these valuable medical areas is vita

ATM activation accompanies histone H2AX phosphorylation in A549 cells upon exposure to tobacco smoke

<p>Abstract</p> <p>Background</p> <p>In response to DNA damage or structural alterations of chromatin, histone H2AX may be phosphorylated on <it>Ser</it>139 by phosphoinositide 3-kinase related protein kinases (PIKKs) such as <it>ataxia telangiectasia </it>mutated (ATM), ATM-and Rad-3 related (ATR) kinase, or by DNA dependent protein kinase (DNA-PKcs). When DNA damage primarily involves formation of DNA double-strand breaks (DSBs), H2AX is preferentially phosphorylated by ATM rather than by the other PIKKs. We have recently reported that brief exposure of human pulmonary adenocarcinoma A549 cells or normal human bronchial epithelial cells (NHBE) to cigarette smoke (CS) induced phosphorylation of H2AX.</p> <p>Results</p> <p>We report here that H2AX phosphorylation in A549 cells induced by CS was accompanied by activation of ATM, as revealed by ATM phosphorylation on <it>Ser</it>1981 (ATM-S1981^P) detected immunocytochemically and by Western blotting. No cell cycle-phase specific differences in kinetics of ATM activation and H2AX phosphorylation were observed. When cells were exposed to CS from cigarettes with different tobacco and filter combinations, the expression levels of ATM-S1981^Pcorrelated well with the increase in expression of phosphorylated H2AX (γH2AX) (R = 0.89). In addition, we note that while CS-induced γH2AX expression was localized within discrete foci, the activated ATM was distributed throughout the nucleoplasm.</p> <p>Conclusion</p> <p>These data implicate ATM as the PIKK that phosphorylates H2AX in response to DNA damage caused by CS. Based on current understanding of ATM activation, expression and localization, these data would suggest that, in addition to inducing potentially carcinogenic DSB lesions, CS may also trigger other types of DNA lesions and cause chromatin alterations. As checkpoint kinase (Chk) 1, Chk2 and the p53 tumor suppressor gene are known to be phosphorylated by ATM, the present data indicate that exposure to CS may lead to their phosphorylation, with the downstream consequences related to the halt in cell cycle progression and increased propensity to undergo apoptosis. Defining the nature and temporal sequence of molecular events that are disrupted by CS through activation and eventual dysregulation of normal defense mechanisms such as ATM and its downstream effectors may allow a more precise understanding of how CS promotes cancer development.</p

Assignment of potential neurotransmitters and neuromodulators to physiologically and morphologically identified olfactory interneurons

Behavioral and physiological studies show that processing of odor information involves neuronal interactions among the glomeruli in the insect antennal lobe (AL). These interactions are mediated by a complex network of inhibitory and excitatory local interneurons (LNs), that structures the olfactory representation and ultimately determines the tuning profile of projection neurons. LNs have distinct morphological and intrinsic electrophysiological properties and in addi- tion to GABA and acetylcholine LNs may contain and release various peptides, that can potentially act as neurotransmitters or neuromodulators. In Periplan- eta americana two main LN types are known: 1) Spiking type I LNs (LN I), that generate Na+ driven action potentials upon odor stimulation and exhibit GABA- like immunoreactivity (GABA-LIR) and 2) nonspiking type II LNs, subdivided into type IIa and type IIb, with unknown transmitter, that do not generate Na+ driven action potentials. This LN diversity implies that these neurons serve dis- tinct functions in the olfactory system. Currently, the morphologically and phys- iologically distinct LN subtypes are not very well matched with the variety of neurotransmitters and -modulators. This, however, is an important prerequisite for a detailed understanding of the role of LNs in the olfactory circuit. While previous studies have investigated the neurochemical organization of the AL in various insect species, I unequivocally assigned the inventory of potentially neu- roactive substances to the functionally distinct LN types. For this purpose, I used a combination of whole-cell patch-clamp recording, sin- gle cell labeling and immunocytochemical methods, to analyze the inventory of neuroactive substances in the AL of P. americana. Using an antibody against the biosynthetic enzyme choline acetyltransferase (ChAT), a marker for cholinergic neurons, a subset of the nonspiking LN IIa with distinct physiological and morphological properties was identified as cholinergic. In these type IIa1 LNs (LN IIa1), odor stimulation evoked depolarizations that generated Ca2+ driven ’spikelets’, but not Na+ driven action potentials. In an effort to form a complete messenger profiling of identified LN types, the neuropeptide allatotropin (AT) was assigned to spiking LN I and tachykinin- related peptides (TKRPs) were assigned to both LN I and nonspiking LN IIa1. This suggests, that AT is coreleased with GABA in LN I, while TKRPs are core- leased with GABA in LN I as well as with acetylcholine in LN IIa1. Finally, a method is introduced, that offers the possibility to perform mass spec- trometry in electrophysiologically and morphologically identified interneurons, which allows a comprehensive data set of individual neurons to be built up

Development of a new automated method for the quantification of nuclear immunohistochemical markers

Antecedentes: La evaluación de marcadores inmunohistoquímicos se realiza con fines diagnósticos, terapéuticos e investigadores de forma manual. La utilización del análisis informatizado de imágenes digitales para evaluar estos marcadores aún no es suficientemente eficaz. Objetivos: Diseñar un nuevo procedimiento informatizado para cuantificar marcadores inmunohistoquímicos nucleares y evaluar los efectos de la compresión de imágenes. Métodos: El procedimiento desarrollado consta de diferentes etapas, donde se evalúan diferentes marcadores immunohistoquímicos utilizados en cáncer de mama y en linfoma. Resultados: El análisis estadístico demostró una gran validez del método automatizado. La redondez fue el único parámetro morfológico afectado por la compresión. Unos factores correctores fueron desarrollados para corregir esta afectación y la variabilidad en la cuantificación producida por esta afectación. Conclusiones: Este nuevo procedimiento automatizado es un método objetivo, más rápido y reproducible que tiene un excelente nivel de precisión, incluso con imágenes digitales de elevada complejidad y también en imágenes comprimidas. Background: The evaluation of immunohistochemical markers is carried out manually for diagnostic, therapeutic and research purposes. The use of a computerized digital image analysis to evaluate these markers is not sufficiently effective yet.Objectives: To design a new computerized procedure to quantify nuclear immunohistochemical markers and evaluate the effects of image compression.Methods: The procedure developed consists of several stages which evaluate different immunohistochemical markers used in breast cancer and lymphoma.Results: Statistical analysis demonstrated a high validity of the automated method. The roundness was the only morphological parameter affected by compression. Some correction factors were developed to correct this disorder and the variability in the measurement caused by this disorder.Conclusions: This new automated process is objective, faster and it has also an excellent level of accuracy, even with highly complex digital images and compressed images

The Isotropic Fractionator as a Tool for Quantitative Analysis in Central Nervous System Diseases

One major aim in quantitative and translational neuroscience is to achieve a precise and fast neuronal counting method to work on high throughput scale to obtain reliable results.Here we tested the Isotropic Fractionator (IF) method for evaluating neuronal and non-neuronal cell loss in different models of central nervous system (CNS) pathologies.Sprague-Dawley rats underwent: (i) ischemic brain damage; (ii) intraperitoneal injection with kainic acid (KA) to induce epileptic seizures; and (iii) monolateral striatal injection with quinolinic acid (QA) mimicking human Hungtington’s disease.All specimens were processed for IF method and cell loss assessed.Hippocampus from KA-treated rats and striatum from QA-treated rats were carefully dissected using a dissection microscope and a rat brain matrix. Ischemic rat brains slices were first processed for TTC staining and then for IF.In the ischemic group the cell loss corresponded to the neuronal loss suggesting that hypoxia primarily affects neurons. Combining IF with TTC staining we could correlate the volume of lesion to the neuronal loss; by IF, we could assess that neuronal loss also occurs contralaterally to the ischemic side.In the epileptic group we observed a reduction of neuronal cells in treated rats, but also evaluated the changes in the number of non-neuronal cells in response to the hippocampal damage