Immunofluorescent Antibody Staining Research Articles

Evaluation of the antibacterial activity of some plant essential oils against Ralstonia solanacearum and the molecular diagnosis of the bacterium

Background Evaluation of the antibacterial activity of some essential oils for controlling potato bacterial wilt. Objective In vitro and planta trials, five essential oils, lemongrass, eucalyptus, thyme, ginger, and peppermint, were evaluated for their aptitude to impede the progress of Ralstonia solanacearum. Materials and methods Five essential oils were assessed for their activity to inhibit R. solanacearum growth using the disc diffusion method. Certain volumes of each tested plant essential oil were added on filter paper discs using tetrazolium chloride agar medium, which contained the tested bacterium to obtain the proposed concentrations used to evaluate the most efficient oil for controlling potato bacterial wilt. Serological and molecular identification was also conducted. Results and conclusion R. solanacearum was characterized using cultural characteristics on the selective medium South Africa, “which is the most effective in suppressing the growth of contaminating microorganisms, and the highest recovery rate of the tested bacterium,” immunofluorescence antibody staining tomato bioassay test and molecular identification via real-time PCR (Taq-man) assay. In-vitro studies showed that all tested plant essential oils (lemongrass, eucalyptus, thyme, ginger, and peppermint) significantly reduced R. solanacearum progress. Eucalyptus (Eucalyptus globulus) and peppermint (Mentha piperita) essential oils were the most efficient in suppressing R. solanacearum growth, where it showed the highest mean inhibition zone, followed by thyme oil (Thymus vulgaris), lemongrass oil (Comybogen citratus), and ginger oil (Zingiber officinale), respectively. Phenotypic variances in bacterial cell construction have been noticed via a transmission electron microscope. Eucalyptus oil led to cell wall lysis, cell laceration, bubble-like structures, and degraded cellular components in bacterial cells.

TMIC-86. SPATIAL ORGANIZATION OF THE PEDIATRIC HIGH-GRADE GLIOMA TUMOR-IMMUNE LANDSCAPE

Abstract BACKGROUND Advances in cellular immunotherapies have shown promising results for brain tumors, including pediatric-type diffuse high-grade gliomas (pHGG). A comprehensive understanding of the tumor-immune microenvironment (TME) is essential for efficient target identification and novel therapeutic strategies. The interplay between cells and their spatial localization within the TME may affect immune cell phenotype and function. We characterized these interactions and phenotypes by single-cell (SC) spatial proteomic analysis. METHODS Thirty-two tissue biopsies of pHGGs were collected from the Princess Máxima Center biobank consisting of 14 diffuse midline gliomas, H3K27-altered (DMG) and 18 other pHGGs. Three cores per biopsy were assembled in a tissue micro-array (TMA). A cyclical multiplex immunofluorescence antibody staining technique by means of chemical antibody-removal was applied to the TMA sections. This resulted in hyperplexed images enabling detailed mapping of the spatial organization. Survival data of patients was collected retrospectively. Clusters of interacting cells were detected using cellular neighborhood (CN) analysis and SC distance analysis. RESULTS SC analysis revealed a large fraction of myeloid cells in the TME, up to 21% of total cells within a tumor, characterized by expression of IBA1 and CD68. We also identified some significant differences in the composition and spatial organization of DMGs compared to other pHGGs. More specifically, DMG biopsies showed lower abundance of CD163+/CD206+ immunosuppressive myeloid cells compared to other pHGGs (p< 0.001). Additionally, the presence of T cells was significantly lower in DMGs compared to other pHGGs (p< 0.001). CN analysis revealed a higher occurrence of myeloid/astrocyte-like neighborhoods in pHGGs other than DMG. The presence of this cluster in these tumors is associated with higher risk of dying (p< 0.05). Thus, the combination of our spatial proteomic SC technology framework and unique patient sample collection will offer essential insights into the cellular interplay in the TME, and its influence on patient outcomes.

Oocyst Shedding Dynamics in Children with Cryptosporidiosis: a Prospective Clinical Case Series in Ethiopia.

ABSTRACTKnowledge on the duration of Cryptosporidium oocyst shedding, and how shedding may be affected by subtypes and clinical parameters, is limited. Reduced transmission may be a secondary benefit of cryptosporidiosis treatment in high-prevalence areas. We conducted a prospective clinical case series in children of <5 years presenting with diarrhea to a health center and a hospital in Ethiopia over an 18-month period. Stool samples were collected repeatedly from children diagnosed with cryptosporidiosis for up to 60 days. Samples were examined, and Cryptosporidium shedding was quantified, using auramine phenol, immunofluorescent antibody staining, and quantitative PCR (qPCR). In addition, species determination and subtyping were used to attempt to distinguish between new infections and ongoing shedding. Duration and quantity of shedding over time were estimated by time-to-event and quantitative models (sex- and age-adjusted). We also explored how diarrheal severity, acute malnutrition, and Cryptosporidium subtypes correlated with temporal shedding patterns. From 53 confirmed cryptosporidiosis cases, a median of 4 (range 1 to 5) follow-up stool samples were collected and tested for Cryptosporidium. The median duration of oocyst shedding was 31 days (95% confidence interval [CI], 26 to 36 days) after onset of diarrhea, with similar estimates from the quantitative models (31 days, 95% CI 27 to 37 days). Genotype shift occurred in 5 cases (9%). A 10-fold drop in quantity occurred per week for the first 4 weeks. Prolonged oocyst shedding is common in a pediatric clinical population with cryptosporidiosis. We suggest that future intervention trials should evaluate both clinical efficacy and total parasite shedding duration as trial endpoints.IMPORTANCE Cryptosporidiosis is an important cause of diarrhea, malnutrition, and deaths in young children in low-income countries. The infection spreads from person to person. After infection, prolonged release of the Cryptosporidium parasite in stool (shedding) may contribute to further spread of the disease. If diagnosis and treatment are made available, diarrhea will be treated and deaths will be reduced. An added benefit may be to reduce transmission to others. However, shedding duration and its characteristics in children is not well known. We therefore investigated the duration of shedding in a group of young children who sought health care for diarrhea in a hospital and health center in Ethiopia. The study followed 53 children with cryptosporidiosis for 2 months. We found that, on average, children released the parasite for 31 days after the diarrhea episode started. Point-of-care treatment of cryptosporidiosis may therefore reduce onward spread of the Cryptosporidium parasite within communities and households.

Distinguishing COVID-19 From Influenza Pneumonia in the Early Stage Through CT Imaging and Clinical Features

BackgroundBoth coronavirus disease 2019 (COVID-19) and influenza pneumonia are highly contagious and present with similar symptoms. We aimed to identify differences in CT imaging and clinical features between COVID-19 and influenza pneumonia in the early stage and to identify the most valuable features in the differential diagnosis.MethodsSeventy-three patients with COVID-19 confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and 48 patients with influenza pneumonia confirmed by direct/indirect immunofluorescence antibody staining or RT-PCR were retrospectively reviewed. Clinical data including course of disease, age, sex, body temperature, clinical symptoms, total white blood cell (WBC) count, lymphocyte count, lymphocyte ratio, neutrophil count, neutrophil ratio, and C-reactive protein, as well as 22 qualitative and 25 numerical imaging features from non-contrast-enhanced chest CT images were obtained and compared between the COVID-19 and influenza pneumonia groups. Correlation tests between feature metrics and diagnosis outcomes were assessed. The diagnostic performance of each feature in differentiating COVID-19 from influenza pneumonia was also evaluated.ResultsSeventy-three COVID-19 patients including 41 male and 32 female with mean age of 41.9 ± 14.1 and 48 influenza pneumonia patients including 30 male and 18 female with mean age of 40.4 ± 27.3 were reviewed. Temperature, WBC count, crazy paving pattern, pure GGO in peripheral area, pure GGO, lesion sizes (1–3 cm), emphysema, and pleural traction were significantly independent associated with COVID-19. The AUC of clinical-based model on the combination of temperature and WBC count is 0.880 (95% CI: 0.819–0.940). The AUC of radiological-based model on the combination of crazy paving pattern, pure GGO in peripheral area, pure GGO, lesion sizes (1–3 cm), emphysema, and pleural traction is 0.957 (95% CI: 0.924–0.989). The AUC of combined model based on the combination of clinical and radiological is 0.991 (95% CI: 0.980–0.999).ConclusionCOVID-19 can be distinguished from influenza pneumonia based on CT imaging and clinical features, with the highest AUC of 0.991, of which crazy-paving pattern and WBC count play most important role in the differential diagnosis.

Cryptosporidium and Giardia in Livestock in Tigray, Northern Ethiopia and Associated Risk Factors for Infection: A Cross-Sectional Study.

The occurrence and species/genotypes of Cryptosporidium and Giardia duodenalis infecting young livestock in selected districts of Tigray, Ethiopia were investigated, along with risks associated with infection. A total of 757 faecal samples were collected from calves, lambs, and goat kids from four rural districts in Tigray, and also from calves in periurban Mekelle, Tigray's main city, and analysed for Cryptosporidium oocysts and Giardia cysts. Farmers answered questionnaires regarding potential risk factors at sample collection. Immunofluorescent antibody staining was used for parasite detection, and PCR at selected genes and sequencing of positive samples was used for molecular characterisation. The occurrence of Cryptosporidium infection was 10, 9, and 4% in calves, lambs, and goat kids, respectively; equivalent figures for Giardia infection were 39, 32, and 21%. Molecular characterisation of Cryptosporidium isolates revealed C. ubiquitum, subtype XIIa in all three host species; C. ryanae in calves and goat kids; C. andersoni and C. bovis were identified only in calves, and C. xiaoi was identified in lambs. For Giardia, Assemblage E predominated in all host species, but among calf isolates we also identified a few potentially zoonotic genotypes (assemblages A (AI) and Assemblage B). Periparturient care was shown to be a particularly relevant risk factor for infection, and infections were less likely to occur under extensive management systems. Our major findings were widespread occurrence of both parasites in livestock, and the apparent lack of the most common zoonotic species. Our results are discussed in relation to other relevant studies. As our study was conducted in Tigray, further investigation in different settings in Ethiopia could provide relevant information on transmission and zoonotic potential. In addition, given the dependency on healthy animals for the livelihoods of the population of Tigray, investigation of the effect of these common parasites on livestock productivity is important.

CD200S-positive granulated lymphoid cells in endometrium appear to be CD56-positive uterine NK cells

The eutopic secretory phase endometrium in endometriosis overproduces and releases a soluble immunosuppressive CD200 molecule (CD200L) and is populated by stromal cells that contain a truncated CD200 (CD200S) that promotes a proinflammatory environment. The CD200S+ cell population persists when pregnancy occurs and are abundant in the early pregnancy decidua of women with missed abortion. In the present study, CD200S+, CD56+, and CD68+ cells were enumerated in formalin-fixed paraffin-embedded tissue sections from women with endometriosis and non-endometriosis controls. CD200S+ cells were more numerous than CD68+ macrophages and were similar in number and location to CD56bright endometrial NK cells. In some endometria, there was an additional population of CD200S- CD56+ NK cells. In ectopic endometrial peritoneal deposits and in ectopic myometrial deposits (adenomyosis), CD200S+ cells were less frequent, consistent with the known paucity of CD56+ NK cells in sites of ectopic deposits. CD200S+ cell frequency was greater in stroma surrounding the smaller ectopic cystic deposits. Dual immunofluorescent antibody staining confirmed CD200S+ cells were CD56+ NK cells. CD200S+ NK cell frequency may be greater in endometriosis patients’ endometrium and may affect embryo survival in early pregnancy. In our opinion, regulation of alternative splicing that results in CD200S rather than CD200L may provide new diagnostic and therapeutic options for women with endometriosis.

Differentiation of immortalized human multi-lineage progenitor to alveolar type 2-like cells: angiotensin-converting enzyme 2 expression and binding of severe acute respiratory syndrome coronavirus 2 spike and spike 1 proteins

Along with the nasal epithelium, the lung epithelium is a portal of entry for sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and many other respiratory viruses. In the case of SARS-CoV-2, the virus surface spike proteins bind to the angiotensin-converting enzyme 2 (ACE-2) receptor to facilitate entry into the respiratory epithelium. Alveolar type 2 (AT2) cells are committed respiratory progenitor cells responsible for the integrity and regeneration of the respiratory epithelium and production of respiratory surfactant proteins. AT2 cells express high levels of surface ACE-2 and thus are a leading target for primary infection by SARS-CoV-2. This study describes a method for directly differentiating telomerase reverse transcriptase-immortalized human cord blood-derived multi-lineage progenitor cells (MLPCs) to AT2-like cells for the purpose of generating an in vitro cellular platform for viral studies. Differentiation was confirmed with the acquisition of AT2 and absence of alveolar type 1 (AT1) specific markers by confocal microscopy. Expression of the ACE-2 receptor was confirmed by immunofluorescence antibody staining, quantitative reverse transcription polymerase chain reaction and binding of biotinylated SARS-CoV-2 spike and spike 1 proteins. The binding of biotinylated spike proteins was specifically blocked by unlabeled spike proteins and neutralizing antibodies. Additionally, it was demonstrated that the spike protein was internalized after binding to the surface membrane of the cells. The authors defined the culture conditions that enabled AT2-like cells to be repeatedly passaged and cryopreserved without further differentiation to AT1. The authors’ method provides a stable and renewable source of AT2 cells for respiratory viral binding, blocking and uptake studies.

Use of EP3533-Enhanced Magnetic Resonance Imaging as a Measure of Disease Progression in Skeletal Muscle of mdx Mice.

As putative treatments are developed for Duchenne muscular dystrophy (DMD), sensitive, non-invasive measures are increasingly important to quantify disease progression. Fibrosis is one of the histological hallmarks of muscular dystrophy and has been directly linked to prognosis. EP3533 is a novel contrast agent with an affinity to collagen 1 that has demonstrated a significant and high correlation to ex vivo fibrosis quantification. Halofuginone is an established anti-fibrotic compound shown to reduce collagen skeletal muscle fibrosis in murine models of DMD. This experiment explored whether EP3533 could be used to detect signal change in skeletal muscle of mdx mice before and after a 12 week course of halofuginone compared to controls. Four age-matched groups of treated and untreated mice were evaluated: 2 groups of mdx (n = 8 and n = 13, respectively), and 2 groups of BL10 mice (n = 5 and n = 3, respectively). Treated mice received an intraperitoneal injection with halofuginone three times per week for 12 weeks, with the remaining mice being given vehicle. Both mdx groups and the untreated BL10 were scanned at baseline, then all groups were scanned on week 13. All subjects were scanned using a 7T Varian scanner before and after administration of EP3533 using a T1 mapping technique. Mice underwent grip testing in week 13 prior to dissection. Skeletal muscle was used for Masson's trichrome quantification, hydroxyproline assay, and immunofluorescent antibody staining. Untreated mdx mice demonstrated a significant increase in R1 signal from pre- to post-treatment scan in three out of four muscles (gastrocnemius p = 0.04, hamstrings p = 0.009, and tibialis anterior p = 0.01), which was not seen in either the treated mdx or the BL10 groups. Histological quantification of fibrosis also demonstrated significantly higher levels in the untreated mdx mice with significant correlation seen between histology and EP3533 signal change. Forelimb weight adjusted-grip strength was significantly lower in the untreated mdx group, compared to the treated group. EP3533 can be used over time as an outcome measure to quantify treatment effect of an established anti-fibrotic drug. Further studies are needed to evaluate the use of this contrast agent in humans.

The role of cytomegalovirus and interleukin-8 in destabilization of atherosclerotic lesions in humans

Relevance. Currently, the role of persistent infections in the atherogenesis development mechanism is not fully understood. Therefore, its important to analyze the role of viral infection against the background of the pro-inflammatory cytokines expression in atherosclerotic plaque destabilization.&#x0D; The aim of the work was a comparative immunohistochemical study of cytomegalovirus (CMV) and IL-8 in different types of human atherosclerotic lesions during their destabilization.&#x0D; Materials and methods. The study was carried out on 130 autopsy samples of human aorta. CMV was detected by direct immunofluorescent antibody staining. IL-8 was detected by two-stage streptavidin-biotin antibody staining.&#x0D; Results. It has been shown that active detection of both CMV and IL-8 is characteristic of atherogenesis foci of the arterial intima with the most intense immune-inflammatory changes. The obtained results indicate the synergism of the cellular response to CMV and IL-8 in the vascular wall during the destabilization atherosclerotic lesions process.&#x0D; Conclusion. According to the results of the work, it can be concluded that both the presence of CMV in atherosclerotic lesions and the high production of IL-8 play a significant role in the formation of unstable atherosclerotic plaques in the vascular wall.

Direct Oral Anticoagulants Reduce Hypercoagulopathy and Preserve Podocyte Function in an Experimental Model of Glomerular Disease

Proteinuric glomerular diseases are a leading cause of chronic kidney disease (CKD). Both pre-CKD glomerular disease and established CKD are major risk factors for thrombosis. Glomerular capillary podocyte injury is a key determinant of CKD progression and results in massive proteinuria accompanied by an acquired hypercoagulopathy that drives thrombotic risk. Unfortunately, the routine use of anticoagulant prophylaxis during glomerular proteinuria (GP) remains controversial due to both a lack of agreement regarding indications and no randomized controlled trial data demonstrating both safety and efficacy. We have recently used rat glomerular disease models to reveal that: (1) Proteinuria is directly correlated with hypercoagulopathy and in vivo thrombosis and (2) Thrombin, the key effector enzyme of the coagulation system, directly injures podocytes during proteinuria. What is not yet known is the ability of direct oral anticoagulant (DOAC) therapy to improve these important CKD and thrombosis outcomes. Thus, the aim of the present study is to determine if DOACs simultaneously reduce podocytopathy and enable effective thromboprophylaxis during GP. We hypothesized that DOACs would simultaneously preserve podocyte function and reduce hypercoagulopathy, in a podocyte-specific rodent model of glomerular disease. We utilized the podocin promotor-human diphtheria toxin receptor (pDTR) transgenic rat model to induce highly specific podocyte injury following a single I.P. injection of 50 ng/kg diphtheria toxin (DT). DT-induced proteinuria was subsequently treated daily by oral gavage with 1) Dabigatran (20 mg/kg; Dabi), 2) Rivaroxaban (3 mg/kg; Riva), or 3) Sham (saline) and compared to healthy controls (n=3-6/group). Morning spot urine and citrated plasma samples were collected from each group at day 10 post-DT. Endogenous Thrombin Potential (ETP) was measured by Technothrombin TGA assay, without and with DOAC-Stop reagent. Glomeruli were isolated from the kidney, dissociated into single-cell suspensions and analyzed by flow cytometry following immunofluorescent antibody and TUNEL staining. Both Dabi and Riva significantly reduced proteinuria (Fig A) and podocytopathy (TUNEL positive podocyte fraction; Fig B), while concomitantly correcting elevated ETP levels (Fig C open symbols). Addition of DOAC-Stop (Fig C closed symbols) revealed an insignificant (P=0.18) trend toward partial ETP reduction, consistent with DOAC-mediated reduction of the underlying GP-mediated hypercoagulopathy (via indirect, antiproteinuric effects). In conclusion, dabigatran and rivaroxaban reduce proteinuria and enhance podocyte health in concert with alleviation of the acquired hypercoagulopathy in a podocyte-specific rodent model of glomerular disease. Overall these data suggest DOAC treatment as a novel approach to simultaneously reduce both podocytopathy and thrombotic co-morbidities during glomerular disease. Additional experiments using this model to determine DOAC efficacy on in vivo thrombosis are in progress. Results from these preclinical studies should inform subsequent randomized controlled DOAC trials that may transform care for patients with glomerular disease by mitigating their risk of both CKD progression and thrombosis. Disclosures No relevant conflicts of interest to declare.

Efficacy of PCR-bassed techniqes and remote sensing technology for detecting potato brown rot infection caused by R. solanacearum

Bacterial wilt of potatoes (Solanum tuberosum) caused by Ralstonia solanacearum phylotype II sequevar I (race 3 biovar 2), is one of the most serious diseases of potatoes. This study aimed to detect such deteriorations through the remotely sensed data. In this regard, naturally infested potato fields at Abo Alkhawi, Al Tayareya and Om Saber villages at Behera governorate, Egypt, were used for selective isolation of R. solanacearum. On Semi Selective South Africa (SMSA) medium, 30 isolates were randomly screened and tested by immunofluorescence antibody staining (IFAS) and confirmed biologically by tomato plantlets (cv. Pinto) reaction. All isolates showed positive results with Real-time PCR. Based on the phylotype specific multiplex (Pmx) – PCR data, all isolates in concern were R. solanacearum and affiliated to phylotype II as 372- bp amplicon. Spectral indices were applied on the Landsat 8 and RapidEye satellite images acquired in January and April 2019 including Normalized Difference Moisture Index (NDMI), Normalized Difference Vegetation Index (NDVI), Normalized Difference Salinity Index (NDSI) and Modified Chlorophyll Absorption Ratio Index (MCARI). Results from the remote sensing analyses showed medium to relatively high salinity and moisture content of soils concerened. Moreover, plants chlorophyll levels are in medium levels which is not typical to condition associated with usual circumstances. Further investigation, in a greater academic depth, are needed for pathological field surveys.

The metastasis suppressor CD82/KAI1 represses the TGF-β 1 and Wnt signalings inducing epithelial-to-mesenchymal transition linked to invasiveness of prostate cancer cells.

The epithelial-to-mesenchymal transition (EMT) is closely associated with cancer invasion and metastasis. Since the transforming growth factor β (TGF-β) and Wnt signals induce EMT in various epithelial cell types, we examined whether and how the CD82/KAI1 metastasis suppressor affects the TGF-β and Wnt signal-dependent EMT in human prostate cancer cells. The invasiveness of cancer cells was evaluated by examining their ability to pass through the basement membrane matrigel. The subcellular localizations of Smad4 and β-catenin proteins were respectively examined by confocal microscopy following immunofluorescence antibody staining and immunoblotting analysis following subcellular fractionation. The transcriptional activities of the TGF-β1 -responsive TRE and Wnt-responsive Tcf/Lef promoters were determined by a luciferase reporter assay following transfection of the recombinant reporter vector into the cell. TGF-β1 and Wnt3a treatments of human prostate cancer cells without CD82 expression resulted in not only increased invasiveness but also EMT involving the development of motile structures, downregulation of E-cadherin, and upregulation of the mesenchymal proteins. However, in the cells with high levels of CD82, the TGF-β1 and Wnt3a stimulations neither elevated invasiveness nor induced EMT. Furthermore, the TGF-β1 signaling events occurring in the CD82-deficient cells, such as phosphorylation of Smad2, nuclear translocation of Smad4, and transactivation of the TRE promoter, did not take place in the high CD82-expressing cells. Further, high CD82 expression interfered with the Wnt signal-dependent alterations in the phosphorylation pattern of glycogen synthase kinase 3β (GSK-3β) in prostate cancer cells, which allowed GSK-3β to continue phosphorylating β-catenin, thereby attenuating the Wnt signaling effects on the nuclear translocation of β-catenin and subsequent transactivation of the Tcf/Lef promoter. The results of the present study suggest that CD82/KAI1 functions in suppressing TGF-β1 - and Wnt-induced EMT in prostate cancer cells by inhibiting the TGF-β1 /Smad and Wnt/β-catenin pathways. Therefore, loss or decrease of CD82 expression is likely to render prostate cancer cells prone to respond to the TGF-β1 and Wnt signals with EMT, resulting in the development of a motile and invasive mesenchymal phenotype related to the initiation of the metastatic cascade.

Abstract 3635: CTC and ctDNA profiling to detect 6 NCCN-guideline recommended classes of alterations for immunotherapy and targeted therapy selection using sample from a single blood draw

Abstract Introduction The availability of targeted and immunotherapies has provided NSCLC patients with more effective treatment options. However, this has resulted in an increase in the number and modality of tests required for treatment selection. Given 30-50% of advanced lung cancer patients have insufficient or unavailable tissue for comprehensive genomic profiling, there is a need for a non-invasive assay that can accurately detect all guideline-recommended markers for NSCLC treatment selection. To meet this need, we have developed a blood test that detects six classes of alterations (SNV, Indels, Rearrangements, CNA, Microsatellite Instability and PD-L1 expression) for therapy selection. Methods &amp; Results Three tubes of blood from a routine blood draw were sent to our CLIA-certified and/or CAP accredited lab for analysis. PD-L1 expression was evaluated in circulating tumor cells (CTCs) utilizing two different assays; (i) Immunofluorescence (IF) antibody staining, (ii) mRNA qPCR. CTCs were captured on the CMxTM CTC Platform coated with lipid bilayer and antibodies to EpCAM. PD-L1 expression results were highly correlated between IF and qPCR assays in ten solid tumor cell lines (lung, breast, prostate and colorectal cancer) spiked into whole blood to mimic the actual patient CTC capture process. In an ongoing study on clinical samples from NSCLC patients (N=20), we observed greater than 90% concordance between tissue (IHC by 22C3 PD-L1 clone) and blood (CTC IF and mRNA assays). A proprietary Single Molecule Sequencing (SMSEQTM) NGS assay was performed on plasma in order to detect 5 classes of genomic alterations (SNV, Indels, Rearrangements, CNA, MSI) from ctDNA. This assay was validated in accordance with the latest ACMG and AMP guidelines to accurately detect variants at low mutant allele fraction (.1% for SNVs and Indels, 1% for rearrangements and 5 copies for CNA) with high sensitivity and specificity. MSI status was determined by assessing nucleotide repeat sequences in five standard markers (BAT-25, BAT-26, MONO-27, NR-21, NR-24), and was detectable down to a MAF of 1%. In an ongoing study on clinical samples from NSCLC patients (N=20), we observed high concordance of MSI status between tissue (immunohistochemistry for dMMR/MSI status) and blood (ctDNA SMSEQ assay). Conclusion Tissue insufficiency and procurement challenges are the primary reasons why ~90% of patients diagnosed with advanced NSCLC are not comprehensively tested per NCCN-guidelines in the community setting where most cancer is treated, leading to suboptimal treatment selection. An accurate blood test that detects all 6 NCCN-recommended markers for immunotherapy and targeted therapy selection has the potential to significantly improve adherence to NCCN testing guidelines and enable optimal treatment selection. Citation Format: Huangpin B. Hsieh, Jen-chia Wu, Feng-Ming Lin, Julian Lucas, Alex Atkins, Pratyush Gupta, Hung-Jen Shao, Yen-Lin Chen, Wen-Jie Huang, Chia-Hsun Hsieh, Ruey Kuen Hsieh, Kuo-Wei Chen, Ming-Hong Yen, Mana Javey, Shih-En Chang, Twinkal Marfatia, Drew Watson, Mahul Amin, Ashish Nimgaonkar, Oscar Segurado, Rui Mei. CTC and ctDNA profiling to detect 6 NCCN-guideline recommended classes of alterations for immunotherapy and targeted therapy selection using sample from a single blood draw [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3635.

CHARACTERISATION OF NF‐KB ACTIVATION IN REGENERATING FIBRES OF OLD MICE

Sarcopenia is the age‐related loss of muscle mass and function. In human studies a decrease of up to 50% in muscle mass and function is observed from the ages of 25 to 70 years and this contributes significantly to frailty and loss of independence in the ageing population. An increase in pro‐inflammatory cytokines is associated with many age‐related conditions including sarcopenia (particularly TNF‐alpha and IL‐6). Many inflammatory processes are under the direct influence of the NF‐κB pathway and a chronic activation of NF‐κB has been shown in muscles from old mice and in accelerated models of muscle ageing (eg. Vasilaki et al, 2006). Additional studies using immunofluorescent antibody staining demonstrated the presence of p65 (a subunit of NF‐κB) only in muscle fibres containing centrally located nuclei, suggesting that NF‐kB was only active in regenerating muscle fibres.The aim of this study was to determine the extent and nature of activation of NF‐kB during regeneration in a well established mouse model of muscle degeneration and regeneration using the myotoxin, barium chloride (BaCl‐2‐). BaCl‐2‐ was injected into the extensor digitorum longus (EDL) muscles of adult (8 months) and old (22 month) wild‐type (WT) C57BL/6 mice. At defined time points the muscles were quickly excised, weighed and frozen in OCT for transverse sectioning. Changes in muscle morphology were observed through haemotoxylin and eosin staining while fluorescently labelled antibodies against p65 targeted the cellular localization of NF‐κB.Centrally located nuclei were present in the majority of the fibres at 14, 28 and 60 days post BaCl2 injection. Nuclear translocation of p65 was seen 14 and 28 days post BaCl2 injection indicating activation of the canonical NF‐κB pathway in regenerating fibres.The results of these experiments reveal an association between muscle fibre regeneration and activation of the canonical NF‐κB pathway. Further studies will determine the effect of activation of NF‐kB during regeneration on inflammatory gene expression and compare with activation in ageing models.Support or Funding InformationFunded by the National Institutes of Health (NIA).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Linear epitopes in African swine fever virus p72 recognized by monoclonal antibodies prepared against baculovirus-expressed antigen.

Protein p72 is the major capsid protein of African swine fever virus (ASFV) and is an important target for test and vaccine development. Monoclonal antibodies (mAbs) were prepared against a recombinant antigenic fragment, from amino acid (aa) 20-303, expressed in baculovirus. A total of 29 mAbs were recovered and tested by immunofluorescent antibody (IFA) staining on ASFV Lisbon-infected Vero cells. Six antibodies were IFA-positive and selected for further characterization. Epitope mapping was performed against overlapping polypeptides expressed in E. coli and oligopeptides. Based on oligopeptide recognition, the mAbs were divided into 4 groups: mAb 85 (aa 165-171); mAbs 65-3 and 6H9-1 (aa 265-280); mAbs 8F7-3 and 23 (aa 280-294); and mAb 4A4 (aa 290-303). All mAbs were located within a highly conserved region in p72. This panel of antibodies provides the opportunity to develop new assays for the detection of ASFV antibody and antigen.

Pneumocystis jirovecii immunostain versus Gomori/Grocott methenamine silver stain of bronchoalveolar lavage in cell blocks: an institutional experience

Current approaches to Pneumocystis jirovecii (PCJ) screening on bronchioalveolar lavage samples (BAL) include Gomori/Grocott methenamine silver stain (GMS), toluidine blue O stain, Wright-Giemsa stain, immunofluorescent antibody stain, and polymerase chain reaction. Another method available is PCJ immunohistochemistry stain (PCJ IHC). There are no published series evaluating the efficacy of PCJ IHC in cell block preparation of BAL, we sought to compare GMS versus PCJ IHC at our institution. We performed a retrospective analysis at our institution of all BAL with cell blocks where PCJ IHC and GMS were done simultaneously since March 2015. 982 BAL samples were identified from 640 patients (median age: 54 years; range: 1-84 years). For 895 cases, GMS and PCJ IHC were performed simultaneously. PCJ was identified in 14 samples, from 13 patients (2.2% of patients) using PCJ IHC. GMS stains were read as positive in only 6 of these 14 cases (42.8%); in two of those cases, PCJ was easily identified on routine Papanicolaou stains. We repeated GMS staining on those 14 cases following before-schedule maintenance in our Ventana Benchmark Autostainer, identifying 12 cases positive. In addition, a significantly higher number of organisms was seen on repeat GMS (median: 58) than the original GMS (median: 8.7). Nevertheless, a statistically significant higher number of organisms was detected by PCJ IHC (median: 474). PCJ IHC performed in cell block is more sensitive and specific than GMS and is a reliable marker when a low number of PCJ organisms are present.

Role of Protein Quality Control Failure in Alcoholic Hepatitis Pathogenesis.

The mechanisms of protein quality control in hepatocytes in cases of alcoholic hepatitis (AH) including ufmylation, FAT10ylation, metacaspase 1 (Mca1), ERAD (endoplasmic reticulum-associated degradation), JUNQ (juxta nuclear quality control), IPOD (insoluble protein deposit) autophagocytosis, and ER stress are reviewed. The Mallory–Denk body (MDB) formation develops in the hepatocytes in alcoholic hepatitis as a consequence of the failure of these protein quality control mechanisms to remove misfolded and damaged proteins and to prevent MDB aggresome formation within the cytoplasm of hepatocytes. The proteins involved in the quality control pathways are identified, quantitated, and visualized by immunofluorescent antibody staining of liver biopsies from patients with AH. Quantification of the proteins are achieved by measuring the fluorescent intensity using a morphometric system. Ufmylation and FAT10ylation pathways were downregulated, Mca1 pathways were upregulated, autophagocytosis was upregulated, and ER stress PERK (protein kinase RNA-like endoplasmic reticulum kinase) and CHOP (CCAAT/enhancer-binding protein homologous protein) mechanisms were upregulated. In conclusion: Despite the upregulation of several pathways of protein quality control, aggresomes (MDBs) still formed in the hepatocytes in AH. The pathogenesis of AH is due to the failure of protein quality control, which causes balloon-cell change with MDB formation and ER stress.

Characterization of arteriovenous identity in the developing neonate mouse retina

The murine retina has become an ideal model to study blood vessel formation. Blood vessels in the retina undergo various processes, including remodeling and differentiation, to form a stereotypical network that consists of precisely patterned arteries and veins. This model presents a powerful tool for understanding many different aspects of angiogenesis including artery and vein (AV) cell fate acquisition and differentiation. However, characterization of AV differentiation has been largely unexplored in the mouse retinal model. In this study, we describe the expression of previously established AV markers and assess arteriovenous acquisition and identity in the murine neonatal retina. Using in situ hybridization and immunofluorescent antibody staining techniques, we analyzed numerous AV differentiation markers such as EphB4-EphrinB2 and members of the Notch pathway. We find that at postnatal day 3 (P3), when blood vessels are beginning to populate the retina, AV identity is not immediately established. However, by P5 expression of many molecular identifiers of arteries and veins become restricted to their respective vessel types. This molecular distinction is more obvious at P7 and remains unchanged through P9. Overall, these studies indicate that, similar to the embryo, acquisition of AV identity occurs in a step-wise process and is largely established by P7 during retina development.

Immunofluorescent antibody staining of intact Drosophila larvae.

Antibody staining is a vital technique for studying the development of many model organisms, including Drosophila. Reliable protocols have long been available for antibody staining of Drosophila whole-mount embryos and dissected larvae. By contrast, methods for staining whole larvae have rarely been reported, are unreliable, and fail to work on large third-instar larvae. This has become a major limitation to understanding the role of multitissue interactions such as neural circuit formation and cell metastasis. We have modified existing embryo protocols to develop a reliable method for antibody staining of whole Drosophila larvae of any developmental stage. The procedure consists of a bleach wash, enzymatic digestion, first fixation, 'cracking', second fixation, (optional) Proteinase K (Pro-K) or sonication treatment, antibody staining, clearing, and mounting. The method takes longer than typical antibody stains of dissected larval tissues-12 or 16 d, depending on the size of the larvae, compared with 2-3 d for embryos or dissected tissue stains-but time is saved by eliminating the need for larval dissections and by allowing hundreds of larvae to be batch-processed. The method also works well for staining embryos, even late-stage embryos with cuticles, allowing characterization from early embryogenesis to the end of larval development.

Chikungunya virus transmission between Aedes albopictus and laboratory mice

BackgroundChikungunya virus (CHIKV) is a mosquito-borne alphavirus associated with epidemics of acute and chronic arthritic disease in humans. Aedes albopictus has emerged as an important new natural vector for CHIKV transmission; however, mouse models for studying transmission have not been developed.Methods Aedes albopictus mosquitoes were infected with CHIKV via membrane feeding and by using infected adult wild-type C57BL/6 mice. Paraffin sections of infected mosquitoes were analysed by immunofluorescent antibody staining using an anti-CHIKV antibody. CHIKV-infected mosquitoes were used to infect adult C57BL/6 and interferon response factor 3 and 7 deficient (IRF3/7-/-) mice.ResultsFeeding mosquitoes on blood meals with CHIKV titres > 5 log10CCID50/ml, either by membrane feeding or feeding on infected mice, resulted in ≥ 50 % of mosquitoes becoming infected. However, CHIKV titres in blood meals ≥ 7 log10CCID50/ml were required before salivary glands showed significant levels of immunofluorescent staining with an anti-CHIKV antibody. Mosquitoes fed on blood meals of 7.5 (but not 5.9) log10CCID50/ml were able efficiently to transmit virus to adult C57BL/6 and IRF3/7-/- mice, with the latter mice showing overt signs of arthritis post-infection.ConclusionsThe results provide a simple in vivo model for studying transmission of CHIKV from mosquitoes to mammals and also argue against a resistance barrier to CHIKV infection in adult mice.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1838-1) contains supplementary material, which is available to authorized users.