Distribution Pattern Of Cells Research Articles

Developmental mechanisms underlying webbed foot morphological diversity in waterbirds

The webbed feet of waterbirds are morphologically diverse and classified into four types: the palmate foot, semipalmate foot, totipalmate foot, and lobate foot. To understand the developmental mechanisms underlying this morphological diversity, we conducted a series of comparative analyses. Ancestral state reconstruction based on phylogeny assumed that the lobate feet possessed by the common coot and little grebe arose independently, perhaps through distinct developmental mechanisms. Gremlin1, which encodes a bone morphogenetic protein (BMP) antagonist and inhibits interdigital cell death (ICD) in the foot plate of avian embryos, remained expressed in the interdigital tissues of webbed feet in the duck, common coot, little grebe, and great cormorant. Differences in Gremlin1 expression pattern and proliferating cell distribution pattern in the toe tissues of the common coot and little grebe support the convergent evolution of lobate feet. In the totipalmate-footed great cormorant, Gremlin1 was expressed in all interdigital tissues at St. 31, but its expression disappeared except along the toes by St. 33. The webbing of the cormorant’s totipalmate foot and duck’s palmate foot may have risen from distinct developmental mechanisms.

Distribution Patterns of Mast Cells on Skipjack (Katsuwonus Pelamis) Infested with Endoparasitic Worms as Triggers for Anaphylactic Reactions

This study aims to examine the pattern of mast cell distribution in the intestinal tract of skipjack fish (Katsuwono pelamis) infested with endoparasitic worms and prove the correlation between endoparasitic worms and mast cells which are always involved in hypersensitivity responses. The study was conducted in May to July 2018 at the Paotere Fish Auction Place and at the Animal Climatology Histology Laboratory of Hasanuddin University. A total of 30 skipjack fish originating from the Paotere TPI were used as research samples, the fish were then dissected to be taken inside the organs infested with endoparasitic worms and then fixed and made histopatalogis slide preparations with microtechnic procedures and hematoxylin-eosin staining to be observed under a microscope. The results showed that infestation of endoparasitic worms in the body of skipjack fish could grasp the active mast cells. Mast cells are distributed only to the gonads, gills and heart of skipjack fish (Katsuwonus pelamis).

The Photoreceptor Cells in Retina and Pineal Gland of Amphibian Hoplobatrachus rugulosus

Hoplobatrachus rugulosus, commonly referred to as tiger‐skinned frog, is to be chosen the animal model in this study. It has been found that both chicken α‐internexin and zebrafish inaa (an ortholog of mammalian α‐internexin) were developmentally distributed not only in photoreceptors of retina but also in the photoreceptor‐like cells of the pineal gland from our previous studies. However, from an evolutionary point of view, there is still lack of evidence to illustrate the temporal and spatial distribution of frog α‐internexin protein in the developing pineal gland. According to other studies about Xenopus laevis, the animal model that is usually used for amphibian developmental research, the mRNA sequence which encodes Xenopus α‐internexin had been identified as “xefiltin”. Although H. rugulosus is not the same species with X. laevis, both of them are classified in the same order. The aims of this study is to characterize the expression pattern of α‐internexin‐like proteins, internexin neuronal intermediate filament protein alpha S homeolog (ina.S) and low molecular weight neuronal intermediate filament S homeolog (nif.S) which might be expressed in the developing retina and the pineal gland of the tiger‐skinned frog. Thus, the specific antibodies were generated to detect the frog α‐internexin‐like proteins and to confirm the distribution of these proteins in the developing frog retina as well as the pineal gland by western blot and immunohistochemistry. Furthermore, the markers applied to detect the photoreceptor cell in the retina and the pineal gland are recoverin and XAP1, both of them are reported to be specific to photoreceptors. Based on the distribution patterns in frog retina, immunopositive staining of anti‐nif.S could be observed in the outer segment of photoreceptors, but anti‐ina.S could only be found in retinal neurons. In the pineal gland, both photoreceptor markers, recoverin and XAP1, could be detected. The α‐internexin‐like protein, ina.S and nif.S, could be detected only in a few photoreceptor‐like cells at the early stage of the frog pineal gland. In summary, the distribution patterns of photoreceptor cells in retina and in pineal gland of developing tiger‐skinned frogs has been characterized in this study. It can be concluded that XAP1 could be a good marker to identify the photoreceptor cells in the developing pineal gland of frogs.Support or Funding InformationGrant SponsorMinistry of Science and Technology, Taiwan; Grant Number: MOST 108‐2312‐B‐002‐030‐MY2.

Different distribution patterns of plasmacytoid dendritic cells in discoid lupus erythematosus and lichen planopilaris demonstrated by CD123 immunostaining

BackgroundClinical and histological features may overlap between lichen planopilaris-associated and discoid lupus erythematosus-associated scarring alopecia. ObjectivesThe aim of this study was to demonstrate the cutaneous infiltration of plasmacytoid dendritic cells and to compare their distribution pattern in discoid lupus erythematosus and lichen planopilaris. MethodsTwenty-four cases of discoid lupus erythematosus and 30 cases of lichen planopilaris were examined for immunostaining of the CD123 marker. The percentage and distribution pattern of plasmacytoid dendritic cells and the presence of the plasmacytoid dendritic cells clusters were evaluted in the samples. ResultsThe number of plasmacytoid dendritic cells was higher in the discoid lupus erythematosus specimens. Aggregations of 10 cells or more (large cluster) were observed in half of the discoid lupus erythematosus specimens and only 2 lichen planopilaris, with 50% sensitivity and 93% specificity for differentiating discoid lupus erythematosus from lichen planopilaris. Study limitationsIncidence and prevalence of discoid lupus erythematosus-associated scarring alopecia in the scalp are low, so the samples size of our study was small. ConclusionsWe suggest that a plasmacytoid dendritic cells cluster of 10 cells or more is highly specific for distinguishing discoid lupus erythematosus from lichen planopilaris. It also appears that CD123 immunolabeling is valuable in both active and late stages of the disease.

Integrative Transcriptomic Analysis Reveals a Multiphasic Epithelial-Mesenchymal Spectrum in Cancer and Non-tumorigenic Cells.

Epithelial–mesenchymal transition (EMT), the conversion between rigid epithelial cells and motile mesenchymal cells, is a reversible cellular process involved in tumorigenesis, metastasis, and chemoresistance. Numerous studies have found that several types of tumor cells show a high degree of cell-to-cell heterogeneity in terms of their gene expression signatures and cellular phenotypes related to EMT. Recently, the prevalence and importance of partial or intermediate EMT states have been reported. It is unclear, however, whether there is a general pattern of cancer cell distribution in terms of the overall expression of epithelial-related genes and mesenchymal-related genes, and how this distribution is related to EMT process in normal cells. In this study, we performed integrative transcriptomic analysis that combines cancer cell transcriptomes, time course data of EMT in non-tumorigenic epithelial cells, and epithelial cells with perturbations of key EMT factors. Our statistical analysis shows that cancer cells are widely distributed in the EMT spectrum, and the majority of these cells can be described by an EMT path that connects the epithelial and the mesenchymal states via a hybrid expression region in which both epithelial genes and mesenchymal genes are highly expressed overall. We found that key patterns of this EMT path are observed in EMT progression in non-tumorigenic cells and that transcription factor ZEB1 plays a key role in defining this EMT path via diverse gene regulatory circuits connecting to epithelial genes. We performed Gene Set Variation Analysis to show that the cancer cells at hybrid EMT states also possess hybrid cellular phenotypes with both high migratory and high proliferative potentials. Our results reveal critical patterns of cancer cells in the EMT spectrum and their relationship to the EMT process in normal cells, and provide insights into the mechanistic basis of cancer cell heterogeneity and plasticity.

Carcinoma medular de mama. Caracterización de una serie de casos, 2010-2019

IntroductionMedullary carcinoma of the breast is a rare subtype of invasive carcinoma that accounts for about 3-5% of all cases of breast cancer. The aim of this study was to characterize a series of cases with anatomopathological diagnosis of medullary carcinoma of the mother, from Hospital Clínico Quirúrgico Docente Joaquín Albarrán, in the period 2010 to 2019. MethodA retrospective, descriptive and observational study was conducted, in which the biopsy records were reviewed, collecting variables such as age and macroscopic and histological characteristics. ResultsIn the period from 2010 to 2019, the histopathological diagnosis of 9 cases of medullary carcinoma of the breast was made, of which 77.8% were typical medullary carcinoma of the breast and 22.2%, atypical. Only 33.3% of the lesions presented intratumoral necrosis; lymphatic permeation was present in 100% of cases; lymph node metastasis was confirmed in 33.3% of cases; The immunohistochemical study revealed that 88.9% of the tumors did not express hormonal and Her-2 receptors, so they were classified as triple negative or basal molecular subtype. ConclusionFor the histopathological diagnosis of CMM, the presence of lymphoplasmocytic infiltrate is essential, always detecting the intensity and distribution pattern of lymphocytes and plasma cells in relation to the characteristics of tumor cells (individual disposition and characteristics). The triple negative molecular subtype was the most frequent.

Chemokine CXCL14-like immunoreactivity in the αMSH-producing cells and PRL-producing cells of the flat-tailed house gecko pituitary.

The distribution pattern of chemokine CXCL14-immunoreactive cells was examined by immunohistochemistry in the pituitary of the gecko Hemidactylus platyurus. Immunoreactive cells were observed in the pars intermedia and pars distalis of the pituitary, but not in the pars nervosa. All α-melanocyte-stimulating hormone (αMSH)-producing cells were immunoreactive for CXCL14 in the pars intermedia. The CXCL14-immunoreactive cells corresponded to prolactin (PRL)-producing cells but not to other adenohypophyseal-hormone-producing cells in the pars distalis. CXCL14 secreted from αMSH-producing cells and PRL-producing cells may regulate insulin release from β cells in the pancreatic islets as well as glucose uptake in the muscle cells together with αMSH and/or PRL. In addition, secreted CXCL14 with αMSH and/or PRL may act as a bioactive factor regulating hormone release in the adenohypophyseal cells of the reptilian pars distalis.

Distribution pattern and molecular signature of cholinergic tuft cells in human gastro-intestinal and pancreatic-biliary tract

Despite considerable recent insight into the molecular phenotypes and type 2 innate immune functions of tuft cells in rodents, there is sparse knowledge about the region-specific presence and molecular phenotypes of tuft cells in the human digestive tract. Here, we traced cholinergic tuft cells throughout the human alimentary tract with immunohistochemistry and deciphered their region-specific distribution and biomolecule coexistence patterns. While absent from the human stomach, cholinergic tuft cells localized to villi and crypts in the small and large intestines. In the biliary tract, they were present in the epithelium of extra-hepatic peribiliary glands, but not observed in the epithelia of the gall bladder and the common duct of the biliary tract. In the pancreas, solitary cholinergic tuft cells were frequently observed in the epithelia of small and medium-size intra- and inter-lobular ducts, while they were absent from acinar cells and from the main pancreatic duct. Double immunofluorescence revealed co-expression of choline acetyltransferase with structural (cytokeratin 18, villin, advillin) tuft cell markers and eicosanoid signaling (cyclooxygenase 1, hematopoietic prostaglandin D synthase, 5-lipoxygenase activating protein) biomolecules. Our results indicate that region-specific cholinergic signaling of tuft cells plays a role in mucosal immunity in health and disease, especially in infection and cancer.

ANGI-11. SEX DIFFERENCES IN IMAGING-BASED ASSESSMENT OF GLIOBLASTOMA INVASION

Abstract INTRODUCTION Neuroimaging dogma for glioblastoma asserts that hyperintensity on T1Gd MRI reveals the bulk of the tumor, while T2/FLAIR signal indicates edema. However, it is unclear whether this edema results from immune response or increased tumor cells. Further, one significant driver of the known sex differences in glioblastoma may be differences in immune response, due to the X-linkage of many immune genes. Based on this, we hypothesized that assumptions regarding tumor cellularity in T2/FLAIR images should be tailored to the biological sex of the patient. METHODS Using a retrospective cohort of 18 primary glioblastoma patients receiving multiple image-localized biopsies (82 total) and standard MRI, we assessed: distance of biopsy from T1Gd and T2 areas; a pathologist’s score of percent tumor cell density; and an imaging-based invasion metric, D/ρ. This metric is derived from the biomathematical Proliferation-Invasion model of glioma growth, which features two parameters, net growth rate (ρ) and net invasion rate (D). Their ratio D/ρ is related to degree of invasion, and is estimated from volumetric measurements of MRI abnormalities. Additionally, 25 patient-derived xenograft models implanted in females were grown until moribund, at which point brains were excised and stained for DAPI (to show all cells) and Lamin (to highlight tumor cells). Image processing of lamin-stained sections defines contours of intensity correlating with cell density. RESULTS Outside both the T1Gd and T2 region, male patient biopsies had higher tumor cell densities than females. Males also tended to have higher invasion metrics. Although each set derived from different patients, preclinical metrics of invasion were positively correlated with clinical invasion in females but negatively correlated in males. CONCLUSION Our preliminary finding that cell distribution patterns correlate with imaging metrics differently between the sexes supports the hypothesis that the degree of tumor cell density represented on certain MRI sequences may be sex-specific.

2007P - Spatio-temporal separation of tumor infiltrating CD8+ T-cells and HER2/neu+ tumor cells in tumor-immune milieu of infiltrating ductal carcinoma of the breast

BackgroundDesignated tumor infiltrating lymphocytes (TIL) within tumor microenvironment (TME) comprised of both regulatory cells and effector cells, and their interaction with tumor cells account for the immune escape and immunosurveillance mechanisms respectively. The number and location of CD8+ tumor infiltrating lymphocytes (TILs) are the measures of immune response and carries prognostic significance and predictive potential in the era of immuno-oncology. The present study was an attempt to find the relation between the treatment response and the spatio-temporal separation of tumor infiltrating CD8+ T-cells and HER2/neu+ tumor cells in breast cancer TME. MethodsIn this study, a total of 7 breast cancer patients were enrolled and surgically removed fresh tumor tissues were evaluated for the baseline HER2/neu and CD8 expression by immunohistochemistry (IHC) using dual stain (duplex IHC). The tumor tissues were also tested for the response to HER2 blocker in a novel humanized ex vivo platform that preserves tumour microenvironment using tumour explants, maintained in defined tumour grade-matched matrix support and autologous patient serum. ResultsTumors responded to HER2 blockade had a majority of the CD8+ T cells (60-80%) concentrated around the HER2/neu+ tumor area (within 50-100µm). While, the non-responding tumors had significantly lesser number of the CD8+T cells (20-40%) proximal to HER2/neu+ zone compared to the responders. The present study indeed delineated a preferential spatial relationship of tumor infiltrating CD8+ cells in HER2/neu+ tumor region especially for the recognition of tumor antigen, cross presentation and tumor cell killing. ConclusionsAssessment of distribution pattern of effector cells surrounding tumor area had a correlation on the efficacy of HER2 inhibition. It warrants validation in the larger cohort of patient tumors to underpin its clinical relevance for possible immunotherapies like combination of check point inhibitors with HER2/neu blocker and others. Identification of other co-stimulatory and pathway molecules on the tumors not responded to HER2 blocker is being studied to understand the mechanism of resistance. Legal entity responsible for the studyThe authors. FundingMitra RxDx Inc. DisclosureAll authors have declared no conflicts of interest.

Abstract 4579: A comparative immune-landscape within tumor-microenvironment: Pattern of distribution of immune cells in tumor versus tumor-adjacent normal tissues

Abstract Introduction: Tumor and immune cells can determine each other’s fate. Immunotherapy targets either the PD1-PD-L1 axis which modulates immune-checkpoints or the cytotoxic CTLA4-mediated immune invasion of tumor cells. Thus the success of cellular-dynamics based immunotherapy depends on the makeup of the immune landscape within a given tumor as compared to its adjacent normal tissue. Aim: We hypothesize that as the immune-environment directly or indirectly shapes and determines the fate, drug response, and progression of an established tumor, the immune-landscape of the tumor-microenvironment will be characteristically different from that of the tumor-adjacent normal tissue. We studied the distribution pattern of selected immune cells in the tumor and tumor-adjacent normal tissues from patients with cancers of lung and ovary. Methods: The study was approved by Avera IRB. FFPE tumor and tumor-adjacent normal tissue samples in pairs from surgically resected tissues were used for IHC to identify CD3 (Anti-CD3 [SP7] ab16669), CD4 ([EPR6855] ab133616), CD8 (ab85792), CD68 (Dako. #M0876), CD163 (Cell Signaling #93498), FOXP3 ([236A/E7] ab20034) in immune cells and PD-L1 (Clone 22C3; Dako. #M3653) in tumor cells & tumor-associated macrophages. Tonsil tissue was used for the IHC validation. Tumor and normal cells from paired specimens were compared using H&E stain & Ki-67, pS6RP, cl-caspase 3, CIP2A, and CD31 IHC-stains based on criteria of evaluation for individual protein(s). Anatomic pathological evaluation for the diagnosis of the disease was carried out by Pathologist. Results: IHC expression of CD3, CD4, CD8, CD68, CD163, and FOXP3 in immune cells and PD-L1 in tumor cells was found heterogeneous. Although each tumor presented an individual pattern of distribution of immune cells, in most tumor samples, the identified immune cells were of tumor-cooperating phenotypes. Tumoricidal cells were rarely observed within the tumor compartment, and barely present in spaces, while tumor-friendly immune cells were frequent. In lung tumors, alveolar macrophages were predominantly of tumor-cooperating M2 types (CD68+/CD163+), whereas tumoricidal M1 macrophages (CD68+) were either absent or rare. The expression of PD-L1 in tumors did not correlate participation of any other immune cells in the tumor-microenviroment. Lung macrophages were highly positive for PD-L1 in which the intensity ranged from punctate to high. Conclusion: We present for the first time, a tissue-based analysis of distribution pattern of different immune cells in histological subtypes of the lung and ovarian cancers, in the tumor and tumor-adjacent normal tissues. The relationship between the immune-landscape of the tumor and tumor-adjacent normal tissue is being worked out and will be presented in the meeting. Citation Format: Xiaoqian Lin, Read Sulaiman, Jennifer Aske, Paul Mayer, Luis Rojas-Espaillat, David Starks, Pradip De, Brian Leyland-Jones, Nandini Dey. A comparative immune-landscape within tumor-microenvironment: Pattern of distribution of immune cells in tumor versus tumor-adjacent normal tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4579.

Temporal analysis of distribution pattern of islet cells and antioxidant enzymes for diabetes onset in postnatal critical development window in rats

AimAt performing a temporal analysis of the distribution pattern of islet endocrine cells and antioxidant enzymes in diabetic rats during the post-natal critical development window. Main methodsThe newborns received streptozotocin (STZ) at birth for diabetes induction, and control females received the vehicle. The animals were euthanized at different lifetimes: D5, D10, D15, and D30. Morphological analysis of pancreas and biochemical assays was performed. Key findingsThe STZ-induced rats presented irregular shape of islet on D5 and there was an attempt to restore of this shape in other life moment studied. There was an increase progressive in islet area, however they maintained smaller than those of control rats, with lower labeling intensity for insulin, higher for glucagon and somatostatin, lower for SOD-1 was lower in the islets of the STZ-induced animals at all times studied and for GSH-Px in D10 and D30. SignificanceAlthough STZ-induced diabetic rats presented compensatory mechanisms to restore the mass of endocrine cells, this was not sufficient since these rats developed the diabetic state. This was confirmed by the oral glucose tolerance test from D30. In addition, the delta (δ)-cells presented ectopic location in islets, indicating a possible relationship for beta (β)-cell mass restoration. There was a response of the pancreas to reduce the hyperglycemia in the first month of life. Furthermore, the cells from the endocrine pancreas of diabetic animals show a decline of antioxidant enzymatic, contributing to the increased susceptibility of cells to hyperglycemia-induced ROS in this postnatal critical development window.

Genetic Elimination of Connective Tissue Growth Factor in the Forebrain Affects Subplate Neurons in the Cortex and Oligodendrocytes in the Underlying White Matter.

Connective tissue growth factor (CTGF) is a secreted extracellular matrix-associated protein, which play a role in regulating various cellular functions. Although the expression of CTGF has been reported in the cortical subplate, its function is still not clear. Thus, to explore the significance of CTGF in the brain, we created a forebrain-specific Ctgf knockout (FbCtgf KO) mouse model. By crossing Ctgffl/fl mice with Emx1-Cre transgenic mice, in which the expression of Cre is prenatally initiated, the full length Ctgf is removed in the forebrain structures. In young adult (2–3 months old) FbCtgf KO mice, subplate markers such as Nurr1 and Cplx3 are still expressed in the cortical layer VIb; however, the density of the subplate neurons is increased. Interestingly, in these mutants, we found a reduced structural complexity in the subplate neurons. The distribution patterns of neurons and glial cells, examined by immunohistochemistry, are comparable between genotypes in the somatosensory cortex. However, increased densities of mature oligodendrocytes, but not immature ones, were noticed in the external capsule underneath the cortical layer VIb in young adult FbCtgf KO mice. The features of myelinated axons in the external capsule were then examined using electron microscopy. Unexpectedly, the thickness of the myelin sheath was reduced in middle-aged (>12 months old), but not young adult FbCtgf KO mice. Our results suggest a secretory function of the subplate neurons, through the release of CTGF, which regulates the density and dendritic branching of subplate neurons as well as the maturation and function of nearby oligodendrocytes in the white matter.

Architecture and Life History of Female Germ-Line Cysts in Clitellate Annelids.

Animal female and male germ-line cells often form syncytial units termed cysts, clusters, or clones. Within these cysts, the cells remain interconnected by specific cell junctions known as intercellular bridges or ring canals, which enable cytoplasm to be shared and macromolecules and organelles to be exchanged between cells. Numerous analyses have shown that the spatial organization of cysts and their functioning may differ between the sexes and taxa. The vast majority of our knowledge about the formation and functioning of germ-line cysts comes from studies of model species (mainly Drosophila melanogaster); the other systems of the cyst organization and functioning are much less known and are sometimes overlooked. Here, we present the current state of the knowledge of female germ-line cysts in clitellate annelids (Clitellata), which is a monophyletic taxon of segmented worms (Annelida). The organization of germ-line cysts in clitellates differs markedly from that of the fruit fly and vertebrates. In Clitellata, germ cells are not directly connected one to another, but, as a rule, each cell has one ring canal that connects it to an anuclear central cytoplasmic core, a cytophore. Thus, this pattern of cell distribution is similar to the germ-line cysts of Caenorhabditis elegans. The last decade of studies has revealed that although clitellate female germ-line cysts have a strong morphological plasticity, e.g., cysts may contain from 16 to as many as 2500 cells, the oogenesis always shows a meroistic mode, i.e., the interconnected cells take on different fates; a few (sometimes only one) become oocytes, whereas the rest play the role of supporting (nurse) cells and do not continue oogenesis.This is the first comprehensive summary of the current knowledge on the organization and functioning of female germ-line cysts in clitellate annelids.

Quantifying Compartment-Specific Protein Translocation in Astrocytes by Object-Oriented Image Analysis: Mitochondrial Translocation of PKCδ.

Assessing the amount and subcellular distribution of protein expression is a key component in modern cell biological and medical research. We studied protein kinase Cδ (PKCδ) as a potential regulator of mitochondrial metabolism in astrocytes, and sought to evaluate mitochondrial translocation of PKCδ since this is an important determinant of its function. Apart from visualizing compartment specific localization of mobile proteins such as PKCδ, we also wanted to determine what amount of a cell's total content of a particular protein is located to a specific compartment, or translocated comparing control and experimental condition.We develop a semiquantitative parameter that indicates the relative protein distribution to two subcellular compartments, starting from standard two-channel fluorescence microscopy images. We studied the mitochondrial translocation of PKCδ in astrocytes using double immunofluorescence microscopy and object-oriented image analysis. In one channel, the protein of interest (PKCδ) is labeled, in the other the compartment or organelle of interest (mitochondria, using cytochrome oxidase IV). Both channels were binarized, turned into object populations, and the channel specific values for total area and integrated intensity extracted. From these values, the "intensity density ratio" (IDR) is calculated, a standardized parameter to easily compare distribution patterns in different cells or ROIs. IDR is highly sensitive to changes in localization pattern, and thus easily detects protein translocation in comparison between control and experimental condition. In our study, medium application of glutamate was found to result in partial PKCδ translocation to mitochondria, a statistically highly significant result based only on a limited number of acquired images.

A comparative immunohistochemical study of epidermal and dermal/perifollicular Langerhans cell concentration in discoid lupus erythematosus and lichen planopilaris: a cross-sectional study

There are times when differentiation between discoid lupus erythematosus (DLE) and lichen planopilaris (LPP) becomes quite challenging clinicopathologically. The aim of this study was to evaluate and compare the concentration, distribution pattern and role of Langerhans cells (LCs), identified by CD1a staining in DLE and LPP. Twenty-five specimens of skin biopsies from patients diagnosed with LPP and DLE were included. Immunohistochemistry (IHC) staining was performed against CD1a antigen to assess and compare the concentration and distribution pattern of LCs. Compared with LPP, the mean number of epidermal CD1a+ cells per three high power fields was significantly lower in DLE ( p = 0.003). On the other hand, DLE cases had a significantly higher mean number of dermal/perifollicular CD1a+ cells in three high power fields than LPP cases ( p = 0.01). A small sample size and limited IHC markers. There are differences in the density and distribution pattern of LCs in LPP and DLE in the epidermis and perifollicular regions. Our findings of a statistically significant decrease in LC concentration in the epidermis of DLE cases and also in the perifollicular region of LPP may serve as helpful clues in further characterization of these entities, especially in equivocal cases. However, more extensive studies are required to better understand the underlying immunopathogenesis of these diseases in providing further clues to a specific diagnosis.

Differential involvement of the three nuclear estrogen receptors during oogenesis in European sea bass (Dicentrarchus labrax)†.

Estrogens are involved in a wide range of processes in vertebrate reproduction through ligand activation of their specific cognate receptors. In most teleosts, three nuclear estrogen receptor subtypes have been identified (Esr1, Esr2a, and Esr2b). Differences in ligand binding affinity and seasonal expression patterns in reproductive tissues among these Esr subtypes suggest distinct roles during oogenesis, vitellogenesis, and spermatogenesis. This study focuses on the role of the Esr subtypes in European sea bass (Dicentrarchus labrax) oogenesis and their endocrine regulation. The coding genes of the three Esr subtypes are highly expressed in reproduction-related tissues such as pituitary, gonad, and liver. Quantification of esr1, esr2a, and esr2b expression in the ovary and liver during a whole reproductive cycle showed different patterns depending on stage and subtype, suggesting differential roles of the three receptors in the regulation of oogenesis and vitellogenesis. Esr2a and Esr2b also showed differences in transcriptional activity and ligand affinity when functionally characterized in HEK293 cells. Finally, for the first time in teleosts, the localization of the three Esr subtypes in ovarian follicles and their regulation by gonadotropins is described. Immunodetection of the receptors revealed different distribution patterns in follicular cells and various subcellular locations of the oocyte. Gonadotropin stimulation of ovarian follicles in different stages of vitellogenesis showed a consistent induction of esrb2b expression by Fsh. All together, these data reinforce the hypothesis that each estrogen receptor plays a specific role in oogenesis.

Does Calypogeia azurea (Calypogeiaceae, Marchantiophyta) occur outside Europe? Molecular and morphological evidence.

Oil bodies are the unique feature of most liverworts. Their shape, color and distribution pattern in leaf and underleaf cells are important taxonomic features of the genus Calypogeia. Most species of the genus Calypogeia have pellucid and colorless oil bodies, whereas colored, including gray to pale brown, purple-brown or blue oil bodies, are rare. To date, C. azurea was the only species with blue oil bodies to have been considered as a species of the Holarctic range. This species has been noted in various parts of the northern hemisphere–from North America, through Europe to the Far East. The aim of this study was to determine the genetic diversity of C. azurea from different parts of its distribution range and to ascertain whether blue oil bodies appeared once or several times in the evolution of the genus Calypogeia. The phylogenetic analyses based on four plastid regions (rbcL, trnG, trnL, trnH-psbA) and one nuclear region (ITS2) revealed that C. azurea is presently a paraphyletic taxon, with other Calypogeia species nested among C. azurea accessions that were clustered into four different clades. Based on the level of genetic divergence (1.03–2.17%) and the observed morphological, ecological and geographical differences, the evaluated clades could be regarded as previously unrecognized species. Four species were identified: C. azurea Stotler & Crotz (a European species corresponding to the holotype), two new species from Pacific Asia—C. orientalis Buczkowska & Bakalin and C. sinensis Bakalin & Buczkowska, and a North American species which, due to the lack of identifiable morphological features, must be regarded as the cryptic species of C. azurea with a provisional name of C. azurea species NA.

Abstract PR11: Hyperspectral imaging tools capture the spatial organization of cell subsets within the tumor microenvironment

Abstract Immune cells are a major component of the tumor microenvironment (TME). The spatial organization of immune cell subpopulations within the TME is recognized to have biologic significance and clinical relevance. For example, spatial organization of immune cell subsets within the TME is critical for the inhibition of cytotoxic T-cell activity through direct interaction of ligand (PD-L1) with receptor (PD-1)). However, precise spatial deconvolution is limited by the lack of imaging algorithms for in situ multiplex single cell analyses as flow cytometry does not preserve data in the spatial dimension. To this end, we have developed a hyperspectral imaging platform designed for analyzing multichannel immunohistochemical-stained tissue sections for generating cell density data and reconstructing spatial architecture for tumor biology as well as clinical association studies. Whole-tissue sections from 20 lung adenocarcinomas with at least 5 years’ follow-up were stained for CD3 (pan-T cell), CD8 (cytotoxic T cell), and CD79a (B cell and plasma cell) and counterstained with hematoxylin. Multispectral images were acquired for five fields of view and analyzed to quantify cell types. Regions of Interest (ROIs) were then identified and analyzed in order to quantify cell-cell spatial relationships. Nonrandom patterns of immune cell distributions were identified using the Monte Carlo resampling method (500 iterations). Cell counts, densities, spatial relationships, and significant immune cell distributions were associated with clinical features (Kruskal-Wallis p<0.001). Our analysis generated 234 image files for analysis, with an average of 16,400 cells per image. The densities of intratumoral CD8+ cytotoxic T cells were significantly higher in nonrecurrent cases, agreeing with literature reports. Similarly, cell sociology deductions identified relationships associated with metastasis: tumor cells in nonmetastatic cases had increased numbers of CD8+ cytotoxic T-cell neighbors. Following Monte Carlo analysis, nonrandom cell~cell spatial proximities emerged that were not identified at a cell density level. We have developed a hyperspectral imaging platform capable of quantifying cell-cell spatial relationships within tissue sections. This technology can be applied to larger clinical cohorts for the study of therapeutically targetable immune cell subsets with the goal of identifying patterns that correlate with clinical response and patient outcome. This abstract is also being presented as Poster B16. Citation Format: Katey S.S. Enfield, Spencer D. Martin, Sonia H.Y. Kung, Paul Gallagher, Calum E. MacAulay, Martial Guillaud, Wan L. Lam. Hyperspectral imaging tools capture the spatial organization of cell subsets within the tumor microenvironment [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr PR11.

Cytology smears for DNA extraction: Practical approach for selecting the best slide.

Next generation sequencing (NGS) to detect actionable genetic abnormalities is standard of care in advanced stage lung adenocarcinoma. Many studies have shown that the molecular results obtained from fine needle aspiration cytology material are comparable to those obtained from formalin-fixed tissue samples. We undertook this study to validate DNA extraction from cytology material for molecular studies and to find any correlation between DNA yield, pattern of tumour cells and tumour fraction. DNA was extracted from 34 cytology slides of pulmonary adenocarcinoma cases with predetermined EGFR mutation status. Cytology slides were reviewed for pattern of tumour distribution and tumour fraction. NGS was performed on five slides with variable DNA and compared with original results. There were 14 alcohol-fixed and 20 air-dried smears. The mean DNA yield was 1.74μg and median of 0.4μg (range, 0.02-21μg). Tumour fractions varied from 10% to 90%. No correlation was found between tumour fraction and DNA yield (P=0.14). The mean DNA yield was high in slides with tumour throughout the slide (sheets or scattered clusters) as compared to rare scattered clusters and/or single cells. EGFR mutation was found in four of the five cases sent for NGS lung panel while one case revealed BRAF mutation. DNA with good quantity and quality can be extracted from the cytology slides for NGS irrespective of type of fixation. DNA yield has better correlation with distribution pattern of tumour cells on the slides rather than tumour fraction.