Fluorescent Antibody Staining Research Articles

Effects of eczema calming lotion on the stratum corneum in atopic dermatitis: Corneodesmosin and intercellular lipid lamellae.

Atopic dermatitis (AD) is characterized by compositional and structural changes to the skin at lesional sites. Alteration to the levels and organization of both protein and lipid components are associated with disease status and lead to impaired barrier and hydration. Corneodesmosin (CDSN) and the arrangement and length of the intercellular lipid lamellae (ICLL) are altered in disrupted skin states. The aim of this research was to profile the distribution of CDSN and the ICLL in the stratum corneum (SC) at lesional and non-lesional sites in AD-prone skin and to investigate the impact of an eczema calming lotion containing petroleum jelly, fatty acids, and colloidal oatmeal. An IRB-approved study was conducted with participants with active AD. From a small subset of participants, tape strips were collected from lesional and non-lesional sites on the arm, prior to and after twice daily application, over 4 weeks of an eczema calming lotion containing petroleum jelly, fatty acids, and colloidal oatmeal. Fluorescent antibody staining was used to investigate the distribution of CDSN. Transmission electron microscopy (TEM) was used to characterize the ICLL. The distribution/coverage of CDSN was similar between lesional and non-lesional sites at baseline; application of the lotion resulted in a more defined honeycomb/peripheral distribution. Normalized ICLL (nICLL) was lower in baseline samples from lesional sites relative to non-lesional sites. Application of the lotion increased this parameter by the end of the study at all sites. The eczema calming lotion containing petroleum jelly, fatty acids and colloidal oatmeal provided changes in corneodesmosomal proteins distribution and ICLL, consistent with improvements in corneocyte maturation and improved barrier function in the skin of individuals with atopic dermatitis.

Method for the growth of Herpes Simplex Virus (HSV-1 and HSV-2) in an oxygen-free environment

Vaginal herpes infections occur under low oxygen conditions, including anoxia. Using an anaerobic (0% O2) cell system, the infection rate, early protein expression, and progeny viability were determined. Anaerobic HeLa 229 cells (0% O2; anaerobic DMEM; 24h) were infected with MOI 1 of HSV1/2, then incubated under aerobic (control) and anaerobic conditions. Infectivity (6h post-infection (pi)) was assessed by ONPG activity and enumerating X-gal-stained cells. HSV1/2 viral immediate-early entry, early entry, and late entry protein expression were determined by fluorescent antibody staining followed by HSV1/2 glycoprotein B mouse monoclonal antibody and DAPI. Statistical analysis was performed using t-test and data were considered significant when p ≤ 0.05 (GraphPad Prism). HSV1/2 infection under standard aerobic conditions was approximately twice that measured for anaerobically infected cells. In addition, the level of HSV-2 infectivity was significantly (p < 0.05) higher (~8-fold) than that of HSV-1, regardless of oxygen level. HSV entry stage protein expression (ICP8, ICP27, VP5) was detected for anaerobic HSV1/2. A similar pattern was observed for anaerobically grown progeny of HSV1/2, i.e., the number of progeny was higher when anaerobic progenies were grown under aerobic conditions (anaerobic to aerobic) as compared to those grown under anaerobic conditions (anaerobic to anaerobic). In addition, the progeny levels grown solely under anaerobic conditions trended higher for HSV-1 than HSV-2 progeny but were not significantly different. These findings show that HSV1/2 can infect and replicate in anaerobically growing cells and that oxygen is not essential for productive replication.

Diagnosis of Legionnaires' Disease Assisted by Next-Generation Sequencing in a Patient with COVID-19.

Coinfection in COVID-19 patients is associated with worsening outcome. Among patients with COVID-19, Legionella pneumophila, a common cause of pneumonia, has been reported as a co-occurring respiratory infection. A nonspecific clinical presentation, however, makes an early diagnosis difficult. Bronchoalveolar lavage fluid was collected from a patient suffering from COVID-19 and presenting with pneumonia and sent for metagenomic analysis. Differential abundance analysis was carried out by comparing each taxon reads per million between the bronchoalveolar lavage fluid sample and the negative control. Two replicates of metagenomic sequencing were conducted on bronchoalveolar lavage fluid samples. Within each replicated sequencing, one negative control was sequenced for comparison of taxon abundance in the BALF sample. In both replicates, Legionella pneumophila was the only taxon with significantly higher abundance when compared with the negative control. PCR of the bronchoalveolar further confirmed the presence of L. pneumophila. Several studies have estimated that the incidence of Legionnaires' disease co-infection in patients with COVID-19 infection is approximately 0% to 1.5%. There are some common characteristics of COVID-19 and co-infection with Legionnaires' disease, making it difficult to diagnose bacterial infection early. The diagnosis of these cases is important due to the different treatments used. Current diagnostic tests for Legionnaires' disease include conventional culture, urinary antigen for L. pneumophila serogroup 1, polymerase chain reaction, direct fluorescent antibody stain, and paired serology. The current study demonstrated that metagenomics is a promising approach that facilitated the diagnosis of Legionnaires' disease.

Skin in the Game: An Assay to Monitor Leukocyte Infiltration in Dermal Lesions of a Guinea Pig Model for Tick-Borne Rickettsiosis.

Intact, the skin typically serves as an effective barrier to the external world; however, once pathogens have breached this barrier via a wound, such as a tick bite, the surrounding tissues must recruit immune cells from the blood to neutralize the pathogen. With innate and adaptive immune systems being similar between the guinea pig and human systems, the ability of guinea pigs to show clinical signs of many infectious diseases, and the large size of guinea pigs relative to a murine model, the guinea pig is a valuable model for studying tick-borne and other pathogens that invade the skin. Here, we report a novel assay for assessing guinea pig leukocyte infiltration in the skin. Briefly, we developed an optimized six-color/eight-parameter polychromatic flow cytometric panel that combines enzymatic and mechanical dissociation of skin tissue with fluorescent antibody staining to allow for the immunophenotyping of guinea pig leukocytes that have migrated into the skin, resulting in inflammation. We designed this assay using a guinea pig model for tick-borne rickettsiosis to further investigate host–pathogen interactions in the skin, with preliminary data demonstrating immunophenotyping at skin lesions from infected ticks. We anticipate that future applications will include hypothesis testing to define the primary immune cell infiltrates responding to exposure to virulent, avirulent tick-borne rickettsiae, and tick-borne rickettsiae of unknown virulence. Other relevant applications include skin lesions resulting from other vector-borne pathogens, Staphylococcus aureus infection, and Buruli ulcer caused by Mycobacterium ulcerans.

A comparison of assays for the detection of Cryptosporidium parvum in the feces of scouring calves.

Cryptosporidium parvum is a zoonotic, protozoan parasite that causes potentially life-threatening diarrhea in the host and can be transmitted via the fecal-oral route. C. parvum can infect cattle and may be detected in their feces using a variety of tests. We compared the level of agreement, ease of procedure, and cost among PCR, lateral flow immunoassay, fluorescent antibody, and Kinyoun acid-fast stain direct smear tests. Over the course of 9 mo, 74 calf fecal samples were submitted and tested for C. parvum using all 4 tests. A Fleiss kappa value of 0.813 was obtained, indicating an excellent level of agreement among tests. Overall, the best test based on cost and ease of procedure was the Kinyoun acid-fast stain direct smear.

New Functions of Low-Molecular-Weight Hyaluronic Acid on Epidermis Filaggrin Production and Degradation

Hyaluronic acid (HA) is a high-molecular-weight polysaccharide with high moisturizing power. It is composed of repeating disaccharides of N-acetyl-D-glucosamine and D-glucuronic acid. Low-molecular-weight hyaluronan (LMHA) is obtained by changing the molecular weight or modifying the functional groups of HA and is commonly used together with HA in cosmetics. The objective of this study was to determine whether LMHA promotes the synthesis of filaggrin (FLG). We also investigated whether LMHA activates FLG-degrading enzymes. Three-dimensional (3D) models of the human epidermis were cultured with LMHA. Real-time PCR was used to quantify the mRNA levels of profilaggrin (proFLG), involucrin (IVL), and FLG-degrading enzymes. FLG protein levels were measured by fluorescent antibody staining and Western blotting. The mRNA was quantified using a 3D epidermis model, and it was observed that the mRNA levels of proFLG, IVL, caspase-14 (CASP14), and bleomycin hydrolase were increased by the application of LMHA. Immunofluorescence results showed an increase in FLG proteins, and results from experiments using 3D epidermis models showed that LMHA increased the activity of CASP14. This suggests that the topical application of LMHA would result in an increase in natural moisturizing factor and promote moisturization of the stratum corneum.

The Therapeutic Effect of Traditional LiuJunZi Decoction on Ovalbumin-Induced Asthma in Balb/C Mice

Aim To investigate the therapeutic effect of LiuJunZi decoction (LJZD) in an experimental model of asthma and uncover its potential mechanism. Materials and Methods The ovalbumin (OVA) was applied to induce asthma in Balb/C mice, and LJZD was orally administrated to asthmatic mice. The lung function and histological lesion were evaluated by airway hyperresponsiveness assay, lung edema assay, and hematoxylin and eosin staining. The amounts of CD4+CD25+Foxp3+ TReg cells were analyzed through combining fluorescent antibody staining with flow cytometry assay. The levels of inflammatory factors and immunoglobulins were detected by enzyme-linked immuno sorbent assay (ELISA). The expression of miR-21 and miR-146a was investigated by real-time PCR. The protein expression of activating protein-1 (AP-1), nuclear factor kappa-B (NF-κB), and NF-κB inhibitor alpha (IκBα) was determined by western blotting. Results LJZD improves OVA-induced asthma in Balb/C mice, which is manifested by decreasing lung edema, Penh levels, lung histological lesion, and inflammatory cell infiltration. LJZD increased the number of CD4+CD25+Foxp3+ TReg cells in blood mononuclear cells from asthmatic mice. Furthermore, LJZD reduced the levels of tumor necrosis factor-α (TNF-α), interleukin- (IL-) 4, IL-6, IgG1, and IgE, but increased interferon gamma (IFN-γ) expression, in serum of asthmatic mice, and also decreased the expression of IL-17a, IL-23, IL-25, and thymic stromal lymphopoietin (Tslp) in lung tissues. In addition, miR-21 and miR-146a expression and phospho (p)-NF-κB, p-IκBα, and AP-1 protein expression were inhibited by LJZD in lung tissues from asthmatic mice. Conclusion LJZD improved OVA-induced asthma in Balb/C mice by inhibiting allergic inflammation and Th2 immunoreaction, which might be associated with the inactivation of the NF-κB signaling pathway.

Rapidly Progressive Cognitive Decline and Varicella Zoster Blisters Associated With Teprotumumab in 2 Patients With Graves’ Orbitopathy

Introduction: Teprotumumab, an IGF-1R antagonist, was approved for treatment of Graves’ orbitopathy (GO) in 2020. Common side effects include nausea, diarrhea, muscle spasms, hearing impairment, dysgeusia, headache, dry skin, infusion reactions, and hyperglycemia. We report here 2 cases of unexpected adverse effects. Patient #1 is a 76-year-old man with Graves’ disease diagnosed in 2018, treated with methimazole 2.5mg every other day. His GO manifestations included diplopia, proptosis, and exposure keratopathy. Past history was significant for hypertension, hyperlipidemia, and prostate hyperplasia. He lived independently and walked several miles daily. Teprotumumab was initiated. Between the 4th and 5th doses, his family noted 6-weeks of rapidly progressive cognitive decline characterized by behavioral changes, confabulation, memory deficit, delirium/delusions/mania. He was admitted to neurology service and subsequent doses held. Lumbar puncture was unremarkable. TFT was normal before, during and after stopping teprotumumab. Thyroid antibodies were notable for elevated TSI but normal thyrotropin receptor Ab and TPO/TgAb. Other labs: IL2, IL6, ESR, CRP, heavy metals, IgG, IgM, SARS-CoV-2 IgM/IgGs, vitamins B1, B6, and B12 were all normal. His most recent repeat TPO Ab was 14 IU/mL (increased from <6), TgAb 18.4 IU/mL (increased from <1.0). EEG showed no epileptiform activity. Head MRI/MRA showed cerebral amyloid angiopathy without stenosis or aneurysms in the intracranial arterial vasculature. Symptoms progressed with IV glucocorticoids and IV Ig therapy. He subsequently underwent plasmapheresis with resolution of symptoms. Patient #2 is a 48-year-old man with Graves’ disease and GO diagnosed in 2018, initially treated with methimazole and subsequently underwent total thyroidectomy in 2019. Patient is euthyroid on levothyroxine. Ophthalmic signs and symptoms included significant proptosis and retrobulbar pain. He was started on teprotumumab infusions. After his 4th dose, he noted painful vesicles in a dermatomal distribution on his right posterior thigh. Direct fluorescent antibody stain was positive for varicella zoster, and rashes resolved with valacyclovir therapy. Discussion: Paraneoplastic/autoimmune encephalitis syndromes can be associated with antibodies to neuronal cell surfaces/synaptic proteins and may occur in the presence or absence of cancer. The incidence of varicella-zoster virus infection is associated with the immune status of the patient, disease-related immunocompromised, or iatrogenic immunosuppression, with age being the major risk factor for 90% of adult cases. This could be incidental, or related to other disorders, or perhaps teprotumumab may have activated auto-immunity. The exact mechanisms for rapidly progressive cognitive decline and shingles remains unclear.

High fat/high salt diet intake promotes Th17 activation in the small intestine in males but not females

Previous studies have identified Th17 to be highly pathogenic during immune dysregulation. We have previously shown that chronic high fat diet (HFD) led to significant kidney medullary interstitial fibrosis and aortic hypertrophy compared to normal diet intake. Numerous studies have identified a connection between high fat and high salt diets and an increase in pathogenic Th17 cells. Although little is understood about Th17 activation in the gut and kidney when high fat and high salt diets are combined (HF/HS). Furthermore, little is known about whether there are sex differences in dietary immune responses. We hypothesized that chronic HF/HS diet compared to normal diet (ND) intake can exaggerate Th17 activation and reduce regulatory T cells (Treg) in the gut and kidney compared to ND in a sex dependent manner. The gut comprises the largest site of immune cells in the body and are ideally located for mediating immune responses to dietary challenges, thus we focused on the gut as well as the kidney. We immunophenotyped T cells with flow cytometry through cytokine production and transcriptional markers from 10-week-old C57Bl6/J mice provided ND (10% fat, 0.4% salt) or HF/HS (45% fat, 4% salt) diet for 10 weeks. Small intestines, colons, and kidneys were collected and isolated to single cell suspensions and stimulated for two hours before fluorescent antibody staining for flow cytometric analysis. Results are reported as percentage of CD4+ TCRb+. We first examined baseline immune cell (CD45+) and T cell populations (CD4+) and found no statistical differences between ND and HF/HS. After gating on CD4+ TCRb+ T cells, we gated on IL-17A+ IFNg- cells as our Th17 population. In the small intestine of male mice, we found a significant increase of Th17 cells in the HF/HS group compared to ND mice (ND: 7.05 1.12, HF/HS: 18.32 2.13, p=0.0009). However, in the colon and kidney, we found no significant difference in Th17 cells in the HF/HS and ND groups (Kidney: ND: 1.060.08, HF/HS: 1.140.13, p=0.5967; Colon: ND: 2.87 0.48, HF/HS: 4.83 0.83, p=0.0610). In the colon, we found significantly decreased frequencies of effector Tregs (FoxP3+ RORgt+) with HF/HS compared to ND (ND: 12.95 1.30, HF/HS: 6.400.72, p=0.0023). There were no significant differences in effector Tregs in the small intestine and kidney (Small Intestine: ND: 8.941.26, HF/HS: 7.080.78, p=0.2378; Kidney: ND:0.47 0.19, HF/HS: 0.43 0.20, p=0.9057). We found no significant differences between ND and HF/HS groups in all tissue sources and T cell phenotypes in female mice (Th17: Small Intestine: ND: 18.20 3.86, HF/HS: 16.361.90, p=0.6983; Colon: ND: 3.611.23, HF/HS: 3.020.71, p= 0.6886; Kidney: ND: 27.484.44, HF/HS: 18.963.11, p=0.1666; Treg: Small Intestine: ND: 9.581.20, HF/HS: 8.63 1.07, p=0.5762; Colon: ND: 5.701.14, HF/HS: 4.63 0.61, p=0.4237; Kidney: ND:0.84 0.31, HF/HS: 0.31 0.04, p=0.1505). In conclusion, HF/HS diet induced increased small intestinal Th17 cells and decreased colonic effector Tregs in male mice and not in female mice. These results indicate that a dietary challenge promotes a pathogenic environment in the gut in a sex-specific manner.

Multiscale Assay of Unlabeled Neurite Dynamics Using Phase Imaging with Computational Specificity.

Primary neuronal cultures have been widely used to study neuronal morphology, neurophysiology, neurodegenerative processes, and molecular mechanism of synaptic plasticity underlying learning and memory. However, the unique behavioral properties of neurons make them challenging to study, with phenotypic differences expressed as subtle changes in neuronal arborization rather than easy-to-assay features such as cell count. The need to analyze morphology, growth, and intracellular transport has motivated the development of increasingly sophisticated microscopes and image analysis techniques. Due to its high-contrast, high-specificity output, many assays rely on confocal fluorescence microscopy, genetic methods, or antibody staining techniques. These approaches often limit the ability to measure quantitatively dynamic activity such as intracellular transport and growth. In this work, we describe a method for label-free live-cell cell imaging with antibody staining specificity by estimating the associated fluorescence signals via quantitative phase imaging and deep convolutional neural networks. This computationally inferred fluorescence image is then used to generate a semantic segmentation map, annotating subcellular compartments of live unlabeled neural cultures. These synthetic fluorescence maps were further applied to study the time-lapse development of hippocampal neurons, highlighting the relationships between the cellular dry mass production and the dynamic transport activity within the nucleus and neurites. Our implementation provides a high-throughput strategy to analyze neural network arborization dynamically, with high specificity and without the typical phototoxicity and photobleaching limitations associated with fluorescent markers.

Effects of mannan-oligosaccharide and Bacillus subtilis supplementation to preweaning Holstein dairy heifers on body weight gain, diarrhea, and shedding of fecal pathogens

The objective of this clinical trial was to evaluate the effectiveness of probiotic, prebiotic, and synbiotic supplementation on average daily weight gain (ADG), duration of diarrhea, age at incidence of diarrhea, fecal shedding of Cryptosporidium oocysts, enteric pathogens, and the odds of pneumonia in preweaning dairy heifer calves on a commercial dairy. Feeding prebiotics and probiotics may improve health and production of calves. Hence, healthy Holstein heifer calves (n = 1,801) from a large California dairy were enrolled at 4 to 12 h of age and remained in this study until weaning at 60 d of age. Calves were block randomized to 1 of 4 treatments: (1) control, (2) yeast culture enriched with mannan-oligosaccharide (prebiotic), (3) Bacillus subtilis (probiotic), and (4) combination of both products (synbiotic), which were fed in milk twice daily from enrollment until weaning. Serum total protein at enrollment and body weight at 7, 42, and 56 d of age were measured. Fecal consistency was assessed daily for the entire preweaning period. A subgroup of 200 calves had fecal samples collected at 7, 14, 21, and 42 d for microbial culture and enumeration of Cryptosporidium oocysts by direct fluorescent antibody staining. Synbiotic-treated calves had 19 g increased ADG compared with control calves for overall ADG, from 7 to 56 d. From 42 to 56 d, prebiotic-treated calves had 85 g greater ADG and synbiotic-treated calves had 78 g greater ADG than control calves. There was no difference in duration of the first diarrhea episode, hazard of diarrhea, or odds of pneumonia per calf with treatment. Probiotic-treated calves had 100 times lower fecal shedding of Cryptosporidium oocysts at 14 d and prebiotic-treated calves had fewer Escherichia coli and pathogenic E. coli at 42 d compared with control calves. Although there were no effects on duration of diarrhea or pneumonia incidence, greater ADG in the late preweaning period may reflect treatment effects on enteric pathogens during the rearing process. The decreased shedding of Cryptosporidium should reduce infectious pressure, environmental contamination, and public health risks from Cryptosporidium. Our findings suggest ADG and potential health benefits for calves fed prebiotics, probiotics, and synbiotics and can help the dairy industry make informed decisions on the use of these products in dairy production.

Analysis of Molecular Mechanism of YiqiChutan Formula Regulating DLL4-Notch Signaling to Inhibit Angiogenesis in Lung Cancer.

In order to explore the specific mechanism of YiqiChutan formula (YQCTF) in inhibiting the angiogenesis of lung cancer and its relationship with delta-like ligand 4- (DLL4-) Notch signaling, 30 healthy BALB/c-nu/nu rats were selected and divided into three groups: A549 group (implanted with lung adenocarcinoma cell line A549), NCI-H460 group (implanted with human lung large-cell carcinoma cell line NCI-H460), and NCI-H446 group (implanted with human lung small cell carcinoma cell line NCI-H446) for constructing lung cancer transplanted tumor models. After modeling, the group treated with normal saline was taken as control group, 200 mg/kg of YQCTF was adopted for intervention, and the tumor volume and growth inhibition rate were compared with the vascular targeted inhibitor Sorafenib. HE staining, CD31 fluorescent antibody staining, and microelectron microscopy were adopted to observe the neovascular endothelial cells of the transplanted tumor. The expression of VEGF, HIF-1α, DLL4, and Notch-1 in the transplanted tumors in each group was detected by Western blot and RT-PCR at the protein level or mRNA level. Compared with the control group, the YQCTF-treated group had obvious inhibitory effect on lung cancer transplanted tumor and lung cancer angiogenesis. In the YQCTF-treated group, the density of angiogenesis decreased significantly and the vascular lumen structure also decreased, and the expression levels of VEGF, HIF-1α, DLL4, and Notch-1 in the YQCTF-treated group were all lower than those in the control group. YQCTF could inhibit the growth of lung cancer transplanted tumor through antiangiogenesis, and it could also reduce the amount of angiogenesis in lung cancer transplanted tumor. In addition, the generation of lumen structure was also hindered, which was realized through the VEGF signaling pathway and DLL4-Notch signaling pathway.

Laboratory Diagnostic Tools for Syphilis: Current Status and Future Prospects.

With the increasing number of patients infected with syphilis in the past 20 years, early diagnosis and early treatment are essential to decline syphilis prevalence. Owing to its diverse manifestations, which may occur in other infections, the disease often makes clinicians confused. Therefore, a sensitive method for detecting T. pallidum is fundamental for the prompt diagnosis of syphilis. Morphological observation, immunohistochemical assay, rabbit infectivity test, serologic tests, and nucleic acid amplification assays have been applied to the diagnosis of syphilis. Morphological observation, including dark-field microscopy, silver-staining, and direct fluorescent antibody staining for T. pallidum, can be used as a direct detection method for chancre specimens in primary syphilis. Immunohistochemistry is a highly sensitive and specific assay, especially in the lesion biopsies from secondary syphilis. Rabbit infectivity test is considered as a sensitive and reliable method for detecting T. pallidum in clinical samples and used as a historical standard for the diagnosis of syphilis. Serologic tests for syphilis are widely adopted using non-treponemal or treponemal tests by either the traditional or reverse algorithm and remain the gold standard in the diagnosis of syphilis patients. In addition, nucleic acid amplification assay is capable of detecting T. pallidum DNA in the samples from patients with syphilis. Notably, PCR is probably a promising method but remains to be further improved. All of the methods mentioned above play important roles in various stages of syphilis. This review aims to provide a summary of the performance characteristics of detection methods for syphilis.

Abstract PR10: Analytical validation and preliminary clinical utility of PD-L1 and HLA I expression profiling of circulating tumor cells using automated exclusion-based sample preparation technology

Abstract Only a subset of patients with non-small cell lung cancer (NSCLC) respond to PD-L1 targeted therapy, even after solid tumor biopsy screening for PD-L1 expression. Downregulation of HLA I may contribute to this high prevalence of resistance, warranting its inclusion in attempts to increase response rates. While it is unclear if loss of HLA I occurs as a de novo or acquired resistance mechanism to immunotherapy, liquid biopsies could detect dynamic changes in expression levels, enable early detection of resistance, and accelerate the shift to alternate, more effective treatments. To quantify the expression of both PD-L1 and HLA I on circulating tumor cells (CTCs), we have developed automated exclusion-based sample preparation technology (ESP), which leverages the forces of surface tension and magnetism at the microscale for high-efficiency, high-throughput cell capture and manipulation. On-chip fluorescent antibody staining is performed for CTC identification, enumeration, and analysis of protein expression. A quantitative readout of protein expression is performed using a refined fluorescence microscopy approach wherein images of cells are acquired and analyzed with automated algorithms for uniformity and objectivity. To evaluate precision, accuracy, and analytical sensitivity of both ESP processing and quantitative microscopy, we used standardized cell lines, synthetic beads, and patient sample replicates. Calibration beads demonstrate high precision between multiple users for the quantitative microscopy (0.9% CV max of all wavelengths between 3 different analysts). ELISA beads with serial dilutions of protein standards demonstrate the accuracy of the quantitative microscopy (R2 values of 0.99 for both proteins). Cell lines demonstrate the precision of ESP for protein staining (3.4% CV max of two different cell lines tested on three different days). Patient sample replicates demonstrate precision for patients with as few as 3 CTCs per sample (6% CV max between triplicates). Early clinical utility of this assay was demonstrated on a preliminary patient cohort of 21 patient time points from 8 unique patients with NSCLC treated with immunotherapies. Phenotypic analysis for CTC expression of PD-L1 and HLA I with this assay demonstrated a sensitivity of 94% and specificity of 75% for detecting either resistant or stable disease during treatment with immunotherapies. Preliminary evaluation of patients with clear cell renal cell carcinoma demonstrates the feasibility of applying this approach to other cancer types. By confirming high performance, we ensure compatibility for clinical laboratory implementation and eventual application to better predict and detect resistance to PD-L1 targeted therapy in patients with NSCLC. The preliminary patient cohort suggests this diagnostic approach can detect fluctuations in expression of PD-L1 and HLA I in CTCs that also correspond to patient response to immunotherapy and warrants its pursuance in larger cohorts of patients with NSCLC and other tumor types. This abstract is also being presented as Poster B38. Citation Format: Jennifer L. Schehr, Zachery D. Schultz, Camila I. Hernandez, Matthew C. Mannino, Jay W. Warrick, Ticiana A. Leal, David J. Beebe, Joshua M. Lang. Analytical validation and preliminary clinical utility of PD-L1 and HLA I expression profiling of circulating tumor cells using automated exclusion-based sample preparation technology [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR10.

Dietary salt and endothelin A receptor activation regulates T cell responses in a tissue‐dependent manner

Numerous studies have investigated the role of high salt diet (HSD) by utilizing salt‐sensitive animal models or co‐administration with pro‐hypertensive insult. HSD is implicated in altering tissue‐dependent immunologic responses. Endothelin (ET‐1) is a major vasoconstrictor peptide synthesized in response to HSD. The ET‐1/endothelin A (ETA) receptor pathway has been implicated in regulating inflammatory responses, and to blunt the immunologic response with pro‐hypertensive conditions. However, the singular role of HSD on renal, colonic and splenic T cells is not fully understood. Therefore, we hypothesized that ETA receptor activation blunts T cell responses with HSD. Utilizing flow cytometry, we immunophenotyped renal, colonic, and splenic T cells in 12‐wk old male C57BL6/J mice provided normal salt diet (NSD, 0.4% NaCl food), ETA blockade (atrasentan, 10mg/kg/day) with NSD, HSD (4% NaCl food plus 1% NaCl drinking water), or co‐administration of atrasentan and HSD for 5 weeks. Kidneys, colons and spleens were collected for single cell suspensions. Isolated cells were stimulated followed by fluorescent antibody staining for flow cytometric analysis. Data are expressed as cell number means SEM (n=4/group) with statistical differences calculated by two‐way ANOVA with Tukey’s correction for multiple comparisons. HSD significantly blunted the total number of renal CD4+T cells mice compared to NSD as well as atrasentan treatment without further suppression of CD4+T cells under co‐administration of both HSD and atrasentan (NSD: 5217 619; Atrasentan: 1.8 0.15*; HSD: 2.4 0.32*; Atrasentan/HSD: 1.8 0.15*, x103, *p&lt;0.001). Colonic CD4+T cells were similarly blunted under Atrasentan‐only and HSD‐only administration, without further reduction under co‐administration. However, splenic CD4+T cells were significantly expanded under chronic atrasentan administration (NSD: 2.66 1.45; Atrasentan: 7.63 0.61#; HSD: 6.63 0.69#; Atrasentan/HSD: 6.46 0.96#, x106, #p&lt;0.05). We next examined IL‐17A production by CD4+T cells, which allows us to determine TH17 cells. Compared to NSD, HSD or atrasentan significantly blunted the total number of renal TH17 cells (NSD: 65 9; Atrasentan: 22 5*; HSD: 26 4*; Atrasentan/HSD: 20 3*, *p&lt;0.001). Colonic TH17cells were similarly blunted. However, splenicTH17cells were seemingly unaffected under chronic atrasentan administration with similar results under HSD and combination of HSD/Atrasentan (NSD: 2.9 1.4; Atrasentan: 8.6 1.6; HSD: 7.6 2.8; Atrasentan/HSD: 5.5 1.6, x104, p&gt;0.05). These findings suggest that HSD or ETA receptor activation may regulate T cell responsiveness in a tissue‐dependent manner.Support or Funding InformationU01HL117684, P01HL69999, AHA SFRN 24450002, P01HL136267, 1 F31 HL151264‐01

Prediction of relapse-free survival according to adjuvant chemotherapy and regulator of chromosome condensation 2 (RCC2) expression in colorectal cancer

BackgroundThere is a need for improved selection of patients for adjuvant chemotherapy after resection of non-metastatic colorectal cancer (CRC). Regulator of chromosome condensation 2 (RCC2) is a potential prognostic biomarker. We report on the establishment of a robust protocol for RCC2 expression analysis and prognostic tumour biomarker evaluation in patients who did and did not receive adjuvant chemotherapy.Materials and methodsRCC2 was analysed in 2916 primary CRCs from the QUASAR2 randomised trial and two single-hospital Norwegian series. A new protocol using fluorescent antibody staining and digital image analysis was optimised. Biomarker value for 5-year relapse-free survival was analysed in relation to tumour stage, adjuvant chemotherapy and the molecular markers microsatellite instability, KRAS/BRAFV600E/TP53 mutations and CDX2 expression.ResultsLow RCC2 expression was scored in 41% of 2696 evaluable samples. Among patients with stage I–III CRC who had not received adjuvant chemotherapy, low RCC2 expression was an independent marker of inferior 5-year relapse-free survival in multivariable Cox models including clinicopathological factors and molecular markers (HR 1.45, 95% CI 1.09 to 1.94, p=0.012, N=521). RCC2 was not prognostic in patients who had received adjuvant chemotherapy, neither in QUASAR2 nor the pooled Norwegian series. The interaction between RCC2 and adjuvant chemotherapy for prediction of patient outcome was significant in stage III, and strongest among patients with microsatellite stable tumours (pinteraction=0.028).ConclusionsLow expression of RCC2 is a biomarker for poor prognosis in patients with stage I–III CRC and seems to be a predictive biomarker for effect of adjuvant chemotherapy.

Development of flow cytometry assays for measuring cell-membrane enzyme activity on individual cells

Background: Cell-membrane expressing enzymes such as ADAM (a disintegrin and metalloproteinase) superfamily members are thought to be key catalysts of vital cellular functions. To directly measure these enzymes and determine their association with particular cells and functions, individual-cell membrane-bound enzyme activity assays are required, but unavailable.Methods: We developed two such assays, using a fluorescence resonance energy transfer (FRET) peptide substrate (FPS) and flow cytometry. One assay measured live-cell natural processing of FPS and binding of its fluorescent product onto individual-cell membrane-bound enzymes. The other assay measured processing of specifically-bound and glutaraldehyde-crosslinked FPS, and consequent generation of its coupled fluorescent product onto individual-cell membrane-bound enzymes.Results: Confocal-microscopy imaging indicated that proteolytic processing of FPS selectively occurred on and labeled cell membrane of individual cells. The new assays measured specific increases of cell-associated FPS fluorescent product in substrate-concentration-, temperature- and time-dependent manners. A large proportion of processed FPS fluorescent products remained cell-associated after cell washing, indicating their binding to cell-membrane expressing enzymes. The assays measured higher levels of cell-associated FPS fluorescent product on wild-type than ADAM10-knockout mouse fibroblasts and on human monocytes than lymphocytes, which correlated with ADAM10 presence and expression levels on cell membrane, respectively. Furthermore, the enzyme activity assays could be combined with fluorescent anti-ADAM10 antibody staining to co-label and more directly associate enzyme activity and ADAM10 protein levels on cell membrane of individual cells.Conclusions: We report on two novel assays for measuring cell-membrane anchored enzyme activity on individual cells, and their potential use to directly study specific biology of cell-surface-expressing proteases.

Streamlined particle quantification (SParQ) plug-in is an automated fluorescent vesicle quantification plug-in for particle quantification in Fiji/ImageJ

ABSTRACT The endolysosomal system is critical for protein homeostasis in cells. A common way of studying protein transport and degradation (e.g. via autophagy) is by labeling vesicular structures such as endosomes, autophagosomes, lysosomes, or model substrates with fluorescent tags or by fluorescent antibody staining. Detailed analyses require quantification of hundreds of structures under various conditions. Typically, the images are analyzed individually with software such as the widely available Fiji/ImageJ (https://imagej.net/Fiji/Downloads), adjusting and thresholding each image and channel independently, which is a very labor intensive and fastidious task. To streamline the process, we developed a plug-in that, integrated into Fiji, enables the automated quantification of vesicular (i.e. punctate) structures. Importantly, the process still allows the operator to evaluate and have control over all the phases of quantification process. Abbreviations CMA: chaperone-mediated autophagy; CSV: comma separated values; eMI: endosomal microautophagy; Fiji: Fiji is just ImageJ; MA: macroautophagy; SParQ: Streamlined Particle Quantification

De novo generation of a functional human thymus from induced pluripotent stem cells

A proper functional thymus is required for generation of T cell–mediated immunity. This is dramatically illustrated by patients lacking a thymus, such as children with complete DiGeorge syndrome, which is fatal if left untreated.1-3 Therapeutic options for such patients are limited and confined to transplantation of small fragments from allogeneic neonatal thymi.

Central Nervous System Disease and Adenoviruses

Research Article| March 01 2019 Central Nervous System Disease and Adenoviruses AAP Grand Rounds (2019) 41 (3): 35. https://doi.org/10.1542/gr.41-3-35 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Central Nervous System Disease and Adenoviruses. AAP Grand Rounds March 2019; 41 (3): 35. https://doi.org/10.1542/gr.41-3-35 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search toolbar search search input Search input auto suggest filter your search All PublicationsAll JournalsAAP Grand RoundsPediatricsHospital PediatricsPediatrics In ReviewNeoReviewsAAP NewsAll AAP Sites Search Advanced Search Topics: adenoviruses, central nervous system dysfunction, adenovirus infections Source: Schwartz KL, Richardson SE, MacGregor D, et al. Adenovirus-associated central nervous system disease in children. J Pediatr. 2018 6 November [published online ahead of print]; doi: https://doi.org/10.1016/j.jpeds.2018.09.036Google Scholar Investigators from The Hospital for Sick Children (SickKids) in Toronto conducted a retrospective observational study to characterize adenovirus-associated central nervous system (CNS) disease in children. Three data sources were used: the SickKids microbiology and encephalitis databases (which include children who had been admitted to SickKids during 2000–2016 and 1996–2016, respectively) and a literature search. Children in the SickKids databases were eligible for the study if they were previously healthy; were 1 month to 18 years old; had neurologic symptoms; did not have a preexisting neurologic disorder; and had adenovirus detected by direct fluorescent antibody staining, culture, or polymerase chain reaction (PCR). Child cases identified from the literature search were eligible if they had a neurologic diagnosis other than febrile seizures and had microbiologic or serologic evidence of acute adenovirus infection. Clinical and demographic characteristics of identified children were abstracted from each data source. Using SickKids microbiology data, investigators determined the frequency and spectrum of neurologic complications of adenovirus disease. Using SickKids encephalitis data, investigators determined the frequency of adenovirus among children with encephalitis. Cases identified by literature search were then pooled with children identified in the SickKids databases (excluding those with febrile seizures) to characterize the adenovirus-associated CNS disease spectrum further. Multivariable logistic regression was used to identify risk factors associated with death or neurologic impairment. Of the 977 cases of adenovirus infection in the SickKids microbiology database, 32 (3.3%) had neurologic symptoms. Of these, 26 had febrile seizures, 4 had encephalitis, 1 had acute demyelinating encephalomyelitis, and 1 had aseptic meningitis. Of the 431 children in the SickKids encephalitis database, 8 (1.9%) had adenovirus (including the 4 with encephalitis identified in the SickKids microbiology database). There were 16 studies identified by the literature search involving 38 children, resulting in a pooled total of 48 cases. Of these, the median age was 2 years, and 40% were female. The most common prodromal symptoms were upper respiratory illness (48%), vomiting (22%), and diarrhea (19%). Hepatitis developed during hospitalization in 40%. There were 10 (21%) deaths, and 8 (17%) had permanent neurologic impairment. Children who developed seizures during their illness course had greater odds of death or permanent neurologic impairment (adjusted OR, 7.3; 95% CI, 1.69–31.45). The investigators conclude that adenovirus is a rare cause of CNS disease in children but can be fatal. Dr Brady has disclosed no financial relationship relevant to this commentary. This commentary does not contain a discussion of an unapproved/investigative use of a commercial product/device. Adenoviruses are common, accounting for at least 5%–10% of respiratory infections in children.1 Vomiting and diarrhea are often present concurrently.2 A specific viral etiology is usually not sought in children with these self-limited symptoms, so the incidence of adenovirus infections is difficult to ascertain. In the current study, the authors reviewed rare CNS manifestations of... You do not currently have access to this content.