Antigen Labeling Research Articles

Red Blood Cell (RBC) Antigen Confirmation Workflow – A Positive Impact at an Academic Medical Center

Abstract Introduction/Objective Introduction: Blood group antigens, present on the surface of red blood cells (RBCs), play a crucial role in transfusion medicine. These antigens are recognized serologically and form the basis of RBC phenotyping. Understanding the significance of antigen-negative RBCs is essential for mitigating the risk of hemolytic transfusion reactions and hemolytic disease of the fetus and newborn (HDFN). This study aims to assess the impact of implementing a new RBC antigen confirmation workflow on transfusion service efficiency and patient outcomes. Methods/Case Report Method: The study involved collaboration between our Transfusion Services Department (TS), the Blood Center supplier, and the Apheresis department. A comprehensive review of historical practices and FDA recommendations regarding RBC antigen labeling was conducted. Changes were implemented to streamline antigen confirmation testing and labeling processes. The impact on TS workload, turnaround time, and patient care metrics was evaluated. Results (if a Case Study enter NA) Results: Implementation of the new RBC antigen confirmation workflow led to a significant reduction in antigen confirmation tests performed by TS, with a 607% decrease observed from 2020 to 2023. Concurrently, the workload for reference lab workups increased by 337% over the same period. This shift in workload distribution allowed TS staff to focus more on patient reference workup cases and case review. The improved workflow efficiency resulted in enhanced turnaround times for providing patients with antigen-negative RBCs, ultimately improving the patient experience. Conclusion Conclusion: The adoption of the new RBC antigen confirmation workflow has resulted in compliance with FDA labeling requirements, efficient utilization of TS staff resources, and decreased turnaround times for blood delivery. These improvements have positively impacted patient care by ensuring timely access to compatible blood products. Future steps include developing reports to track turnaround time for patients with special blood product needs and continuing collaboration with the blood supplier and the Apheresis department to optimize inventory management and transfusion processes.

Multicolour and lineage-specific interphase chromosome Flow-FISH: method development and clinical validation

Flow cytometry can be applied in the detection of fluorescence in situ hybridisation (FISH) signals to efficiently analyse chromosomal aberrations. However, such interphase chromosome (IC) Flow-FISH protocols are currently limited to detecting a single colour. Furthermore, combining IC Flow-FISH with conventional multicolour flow cytometry is difficult because the DNA-denaturation step in FISH assay also disrupts cellular integrity and protein structures, precluding subsequent antigen-antibody binding and hindering concurrent labeling of surface antigens and FISH signals. We developed a working protocol for concurrent multicolour flow cytometry detection of nuclear IC FISH signals and cell surface markers. The protocol was validated by assaying sex chromosome content of blood cells, which was indicative of chimerism status in patients who had received sex-mismatched allogeneic haematopoietic stem cell transplants (allo-HSCT). The method was also adapted to detect trisomy 12 in chronic lymphocytic leukaemia (CLL) subjects. We first demonstrated the feasibility of this protocol in detecting multiple colours and concurrent nuclear and surface signals with high agreement. In clinical validation experiments, chimerism status was identified in clinical samples (n=56) using the optimised IC Flow-FISH method; the results tightly corresponded to those of conventional slide-based FISH (R2=0.9649 for XX cells and 0.9786 for XY cells). In samples from patients who received sex-mismatched allo-HSCT, individual chimeric statuses in different lineages could be clearly distinguished with high flexibility in gating strategies. Furthermore, in CLL samples with trisomy 12, this method could demonstrate that enriched trisomy 12 FISH signal was present in B cells rather than in T cells. Finally, by performing combined labelling of chromosome 12, X chromosome, and surface markers, we could detect rare residual recipient CLL cells with trisomy 12 after allo-HSCT. This adaptable protocol for multicolour and lineage-specific IC Flow-FISH advances the technique to allow for its potential application in various clinical contexts where conventional FISH assays are currently being utilised.

Systematic development of immunohistochemistry protocol for large cryosections-specific to non-perfused fetal brain

BackgroundImmunohistochemistry (IHC) is an important technique in understanding the expression of neurochemical molecules in the developing human brain. Despite its routine application in the research and clinical setup, the IHC protocol specific for soft fragile fetal brains that are fixed using the non-perfusion method is still limited in studying the whole brain. New methodThis study shows that the IHC protocols, using a chromogenic detection system, used in animals and adult humans are not optimal in the fetal brains. We have optimized key steps from Antigen retrieval (AR) to chromogen visualization for formalin-fixed whole-brain cryosections (20 µm) mounted on glass slides. ResultsWe show the results from six validated, commonly used antibodies to study the fetal brain. We achieved optimal antigen retrieval with 0.1 M Boric Acid, pH 9.0 at 70°C for 20 minutes. We also present the optimal incubation duration and temperature for protein blocking and the primary antibody that results in specific antigen labeling with minimal tissue damage. Comparison with existing methodsThe IHC protocol commonly used for adult human and animal brains results in significant tissue damage in the fetal brains with little or suboptimal antigen expression. Our new method with important modifications including the temperature, duration, and choice of the alkaline buffer for AR addresses these pitfalls and provides high-quality results. ConclusionThe optimized IHC protocol for the developing human brain (13–22 GW) provides a high-quality, repeatable, and reliable method for studying chemoarchitecture in neurotypical and pathological conditions across different gestational ages.

Melatonin Modulates Cell Cycle Dynamics and Promotes Hippocampal Cell Proliferation After Ischemic Injury in Neonatal Rats

Promoting neural cell proliferation may represent an important strategy for enhancing brain repair after developmental brain injury. The present study aimed to assess the effects of melatonin on cell proliferation after an ischemic injury in the developing hippocampus, focusing on cell cycle dynamics. After in vivo neonatal hypoxia–ischemia (HI), hippocampal cell cycle dynamics were assessed by flow cytometry, together with histological evaluation of dentate gyrus cellularity and proliferation. Melatonin significantly increased the number of proliferating cells in the G2/M phase as well as the proliferating cell nuclear antigen (PCNA) and doublecortin (DCX) labeling reduced by HI. In vivo BrdU labeling revealed a higher BrdU-positivity in the dentate gyrus of ischemic rats treated with melatonin, an effect followed by increased cellularity and preserved hippocampal tissue integrity. These results indicate that the protective effect of melatonin after ischemic injury in neonatal rats may rely on the modulation of cell cycle dynamics of newborn hippocampal cells and increased cell proliferation.

Research progress of colloidal gold technology

The colloidal gold technique is a technique that combines antigen-antibody immune reaction with colloidal gold labeling for antigen and antibody detection. This paper introduces the background, principle, application, advantages and disadvantages of this technique. The colloidal gold technique uses colloidal gold particles as markers to bind with antigens or antibodies and show red bands on the test strip. It has three methods: the double antibody sandwich method, competition method and indirect method. It can be used for things like environmental monitoring, food safety, and clinical diagnostics. It has the advantages of rapid, simple, low cost, and good stability, but also has the disadvantages of low sensitivity, poor specificity, difficult quantification, etc. In this paper, the background, principle, preparation method, application, advantages and disadvantages of colloidal gold are reviewed to provide readers with a comprehensive and systematic understanding. Although colloidal gold technology has made many achievements, there are still some challenges and difficulties in realizing the industrial application of colloidal gold, such as how to improve the stability, sensitivity, specificity and repeatability of colloidal gold.

Synplex: In silico modelling of the tumor microenvironment from multiplex images.

Multiplex immunofluorescence is a novel, high-content imaging technique that allows simultaneous in situ labeling of multiple tissue antigens. This technique is of growing relevance in the study of the tumor microenvironment, and the discovery of biomarkers of disease progression or response to immune-based therapies. Given the number of markers and the potential complexity of the spatial interactions involved, the analysis of these images requires the use of machine learning tools that rely for their training on the availability of large image datasets, extremely laborious to annotate. We present Synplex, a computer simulator of multiplexed immunofluorescence images from user-defined parameters: i. cell phenotypes, defined by the level of expression of markers and morphological parameters; ii. cellular neighborhoods based on the spatial association of cell phenotypes; and iii. interactions between cellular neighborhoods. We validate Synplex by generating synthetic tissues that accurately simulate real cancer cohorts with underlying differences in the composition of their tumor microenvironment and show proof-of-principle examples of how Synplex could be used for data augmentation when training machine learning models, and for the in silico selection of clinically relevant biomarkers. Synplex is publicly available at https://github.com/djimenezsanchez/Synplex.

New strategies for developing vaccines against the H5N1 avian influenza virus. Proceeding of The First International Avian Influenza Summit, University of Arkansas- October 16-17, 2023”

The H5N1 avian influenza virus poses a significant threat to poultry populations and human health, with sporadic outbreaks and the potential for pandemic emergence. Traditional vaccine development approaches have faced challenges in achieving broad-spectrum protection due to the high mutation rate of the virus. This abstract outlines novel strategies that have emerged to address these hurdles. Firstly, advances in reverse genetics technology have enabled the creation of recombinant vaccines, allowing for the precise manipulation of viral genes to enhance immunogenicity. These platforms facilitate the development of multi-valent vaccines capable of targeting multiple H5N1 strains simultaneously. Additionally, viral replicant or no-replicant vector vaccines employing non-pathogenic viruses as delivery systems show promise in eliciting robust immune responses against H5N1. Secondly, integrating computational modeling and artificial intelligence techniques in antigen design has accelerated the identification of conserved epitopes within H5N1, including MHC I, enabling the development of more effective and broadly protective vaccine candidates. Furthermore, using adjuvants, immune potentiators, antigen labeling for immune system cells, and novel delivery systems has enhanced the efficacy of H5N1 vaccines, providing more durable and cross-protective immunity. Moreover, implementing advanced manufacturing technologies of protein expression, such as cell-based (animal/plants/yeasts/bacteria) and virus-like particle (VLP) platforms, has streamlined production processes and increased vaccine accessibility. These platforms offer scalable and adaptable solutions to meet global demands during outbreaks and potential pandemics. Lastly, incorporating one health approach, emphasizing collaborative efforts between human, animal, and environmental health sectors, is crucial for comprehensive pandemic preparedness. Surveillance systems, early warning mechanisms, and rapid response strategies are integral to a proactive defense against H5N1 outbreaks. In conclusion, this abstract highlights the promising strides in developing vaccines against the H5N1 avian influenza virus through innovative approaches. These strategies, ranging from advanced genetic engineering techniques to cutting-edge computational modeling, offer a comprehensive toolkit for achieving broader and more durable protection against this formidable pathogen. By synergizing these advancements with a holistic health framework, we can significantly enhance our ability to mitigate the impact of H5N1 outbreaks on animal and human populations.

Chemoenzymatic Tagging of Tn/TF/STF Antigens in Living Systems.

Truncated mucin-type O-glycans, such as Tn-associated antigens, are aberrantly expressed biomarkers of cancer, but remain challenging to target. Reactive antibodies to these antigens either lack high-affinity or are prone to antigen escape. Here, we have developed a robust chemoenzymatic strategy for the global labeling of Tn-associated antigens, i.e. Tn (GalNAcα-O-Ser/Thr), Thomsen-Friedenreich (Galβ1-3GalNAcα-O-Ser/Thr, TF) and STF (Neu5Acα2-3Galβ1-3GalNAcα-O-Ser/Thr, STF) antigens, in human whole blood with high efficiency and selectivity. This method relies on the use of the O-glycan sialyltransferase ST6GalNAc1 to transfer a sialic acid-functionalized adaptor to the GalNAc residue of these antigens. By tagging, the adaptor functionalized antigens can be easily targeted by customized strategies such as, but not limited to, chimeric antigen receptor T-Cells (CAR-T). We expect this tagging system to find broad applications in cancer diagnostics and targeting in combination with established strategies.

Cell-Based Outer Vocal Fold Replacement Both Treats and Prevents Vocal Fold Scarring in Rabbits.

Numerous pharmacological and cell-based treatments have shown promise in preventing vocal fold (VF) scarring when applied at the time of injury. A common clinical scenario, however, is the finding of mature scar impeding voicing. Many treatments are less effective in remodeling existing scar tissue. This objective of this study is to determine if a cell-based outer vocal fold replacement (COVR) effectively restores VF function when applied to existing scar. Eighteen rabbits were allocated to three groups: unilateral COVR implant at the time of cordectomy (acute COVR); unilateral cordectomy followed by COVR implant 2 months later (chronic COVR); and unilateral cordectomy followed by sham implant surgery 2 months later (chronic scar). Larynges were harvested 2 months after implant or sham surgery. All larynges in the COVR groups demonstrated human leukocyte antigen labeling on immunohistochemistry (IHC). COVR groups had increased hyaluronic acid content compared with normal. VF stiffness as measured by elastic moduli in acute COVR and chronic COVR were similar to their contralateral unoperated VF. COVR implantation in both acutely injured and chronically scarred VF demonstrate persistence of implanted cells, restored tissue biomechanics, and increased hyaluronic acid content. NA Laryngoscope, 134:764-772, 2024.

MP14-05 EXTRACELLULAR VESICLE BIOMARKER SIGNATURE DETECTION PLATFORM FOR GUIDING CANCER DETECTION AND THERAPY

You have accessJournal of UrologyCME1 Apr 2023MP14-05 EXTRACELLULAR VESICLE BIOMARKER SIGNATURE DETECTION PLATFORM FOR GUIDING CANCER DETECTION AND THERAPY Thomas Osinski, Samuel Walker, Karen Doersch, Jonathan Flax, and James McGrath Thomas OsinskiThomas Osinski More articles by this author , Samuel WalkerSamuel Walker More articles by this author , Karen DoerschKaren Doersch More articles by this author , Jonathan FlaxJonathan Flax More articles by this author , and James McGrathJames McGrath More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003234.05AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Extracellular Vesicles (EVs) have tremendous potential in cancer diagnostics and personalized cancer therapeutics. They are abundant in all biofluids, carry molecular markers of their cell-of-origin and can be readily obtained via noninvasive techniques. Current EV biomarker assessments have several limitiations. Pooled EVs from samples have low detection sensitivity for less abundant biomarkers and preclude colocalization of biomarkers on individual EVs. Currently, expensive specialized devices requiring large biofluid inputs are needed for more detailed EV analysis. Here, we develop an easy to use and inexpensive device that permits optical detection of multiple markers on individual EVs using only 10 µl of serum. This work investigates whether the microfluidic strategy we call ‘catch and display for liquid biopsy [CAD-LB]’ can detect biomarkers on single EVs. We also sought to determine the dynamic range of accurate quantitation using purified EVs and serum and validate detection of immune checkpoint proteins (PD-L1 and/or PD-1), which may help to distinguish immune checkpoint immunotherapy (ICI) responders from non-responders in bladder cancer. METHODS: For this study EVs were derived from: 1. ultracentrifuge purified urinary EVs; 2. EVs from media of 5637 bladder cancer and primary bladder epithelium cell lines and; 3. Serum from patients with advanced urothelial carcinoma or healthy controls. EVs were labeled with the pan-EV marker carboxyfluorescein succinimidyl ester and fluorescently labeled primary antibodies. Small volumes (10 µL) of solutions were captured on our membrane with nanopores in a transparent membrane and imaged using fluorescence microscopy. Detection range was assessed by serial dilutions of EVs. EV surface antigen labeling was assessed by CAD-LB. RESULTS: 1. Counting curves demonstrate a wide dynamic range of CAD-LB (102 – 106 EVs) for purified EVs while serum plateaus at lower inputs (∼104 EVs). 2. CAD-LB detects PD-1 and PD-L1 biomarkers in urothelial carcinoma patients’ serum. 3. CAD-LB detects T cell biomarker CD3 on approximately 1-2% of EVs in healthy control serum. CONCLUSIONS: CAD-LB can detect rare and clinically significant EV associated biomarkers in conditioned medium and patient serum samples. Furthermore, CAD-LB provides single EV characterization with small sample volumes. These qualities make CAD-LB a potentially useful tool to help discover novel markers that could aid in diagnosis and treatment decisions for cancer patients. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e183 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Thomas Osinski More articles by this author Samuel Walker More articles by this author Karen Doersch More articles by this author Jonathan Flax More articles by this author James McGrath More articles by this author Expand All Advertisement PDF downloadLoading ...

Dissecting the contributions of epitope accessibility in B cell activation.

Antibody-mediated immunity is highly specific and is activated by B cell exposure to pathogens or antigens. Antigen bound to the B cell receptor (BCR) triggers signaling cascades that can lead to B cell activation, antigen phagocytosis, and the eventual secretion of antibodies. Although our understanding of the biochemical and biophysical events involved in B cell activation have advanced considerably in recent years, the specific features of antigens that contribute to BCR engagement, B cell activation, and ultimately the production of antibodies are not well-defined. Among the factors that may contribute, the accessibility of particular epitopes on the antigenic surface is thought to play an important role. In this line of thinking, accessible epitopes are more easily bound by the BCR, directing the adaptive immune response to these sites. However, because different epitopes also differ in their sequence and structure, decoupling the contributions of accessibility from other contributing factors has been challenging. To address this challenge, we have developed an experimental system to specifically dissect the contributions of epitope accessibility in BCR engagement and B cell activation. Using engineered Ramos B cells that express BCRs specific to different epitopes on the fusion protein of respiratory syncytial virus (RSV) that can be presented in either membrane-proximal or membrane-distal conformations, we are able to compare the efficiency of BCR engagement and B cell activation to a single epitope while varying its accessibility. We track these processes using fluorescence microscopy combined with site-specific labeling of viral antigens and the BCR. These experiments will help to identify physical constraints imposed on BCR binding and B cell activation which may contribute to the immunodominance of particular epitopes on RSV and other viral pathogens.

Gene expression analysis of antioxidant and DNA methylation on the rat liver after 4-week wood preservative chromated copper arsenate exposure.

Our previous 4-week repeated dose toxicity study showed that wood preservative chromated copper arsenate (CCA) induced hepatocellular hypertrophy accompanied by biochemical hepatic dysfunction and an increase in oxidative stress marker, 8-hydroxydeoxyguanosine, in female rats. To further explore the molecular mechanisms of CCA hepatotoxicity, we analyzed 10%-buffered formalin-fixed liver samples from female rats for cell proliferation, apoptosis, and protein glutathionylation and conducted microarray analysis on frozen liver samples from female rats treated with 0 or 80 mg/kg/day of CCA. Chemical analysis revealed that dimethylated arsenical was the major metabolite in liver tissues of male and female rats. CCA increase labeling indices of proliferating cell nuclear antigen and decrease terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling accompanied with increased expression of protein glutathionylation, indicating a decrease in glutathione (GSH) in hepatocytes of female rats. Microarray analysis revealed that CCA altered gene expression of antioxidants, glutathione-S-transferase (GST), heat shock proteins and ubiquitin-proteasome pathway, cell proliferation, apoptosis, DNA methylation, cytochrome P450, and glucose and lipid metabolism in female rats. Increased expression of GSTs, including Gsta2, Gsta3, Mgst1, and Cdkn1b (p27), and decreased expression of the antioxidant Mt1, and DNA methylation Dnmt1, Dnmt3a, and Ctcf were confirmed in the liver of female rats in a dose-dependent manner. Methylation status of the promoter region of the Mt1 was not evidently changed between control and treatment groups. The results suggested that CCA decreased GSH and altered the expression of several genes, including antioxidants, GST, and DNA methylation, followed by impaired cell proliferation in the liver of female rats.

Dietary Capsaicin Reduces Chemically Induced Rat Urinary Bladder Carcinogenesis.

Capsaicin (CAP) is the compound responsible for pungency in chili peppers, presenting several biological properties. But its general safety and effectiveness in the context of carcinogenesis has not been fully clarified. Thus, the present study evaluated whether dietary CAP modifies the development of urothelial lesions induced by the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in male Sprague-Dawley rats. Animals were randomly allocated into 6 groups: G1 - treated with 0.05% BBN in drinking water (weeks 1-12) and received a balanced diet (weeks 1-20); G2 and G3-treated with BBN (weeks 1-12) and received a balanced diet with 0.01 or 0.02% CAP (weeks 1-20), respectively; G4 and G5- only received a balanced diet with 0.01 or 0.02% CAP (weeks 1-20), respectively; G6 - only received a balanced diet (weeks 1-20). At the end of week 20, the incidence and types of urothelial lesions, proliferating cell nuclear antigen (PCNA) labeling index, and matrix metalloproteinases (MMP) 2 and 9 activities were analyzed. A significant reduction was observed in the incidence and multiplicity of simple (p = 0.020 and p = 0.011) and nodular/papillary (p = 0.030 and p = 0.003) hyperplasias and papillomas/carcinomas (p = 0.023 and p = 0.020), epithelial proliferation (p = 0.007) and in the activity of the intermediate form of MMP-2 (p < 0.001) and pro-MMP-9 activities (p < 0.002), in BBN + 0.02% CAP (G3) group in comparison to BBN (G1) group. Capsaicin intake per se did not alter body weight, liver and kidney weights, urothelial histology or serum biochemical parameters. Thus, dietary CAP was safe and showed a protective effect against rat BBN-induced urothelial carcinogenesis.

Pathogenicity and Viral Distribution of Wild Type Porcine Epidemic Diarrhea Virus and Its Cell Culture Adapted Porcine Epidemic Diarrhea Virus

Swine enteritis in all ages is caused by porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGE), rotavirus, Eimeria spp. etc., and is often fatal among neonatal piglets. This study aimed to compare the pathogenicity and nucleotide sequence of ORF3 between wild-type porcine epidemic diarrhea virus (wt-PEDV) and cell culture-adapted PEDV (ca-PEDV). A total of 30 colostrum- deprived piglets that were 1 day old were inoculated with either wt-PEDV or ca-PEDV to compare the villus height and crypt depth (VH/CD) ratio, amount of viral nucleic acid in jejunum, and open reading frame (ORF) 3 nucleotide sequence via immunofluorescence labeling of viral antigen and western blot analysis. Mean jejunal villus height and crypt depth (VH/CD) ratio in piglets infected orally with Korean strain of PEDV were 6.8 ± 0.8, 7.0 ± 0.8, 8.2 ± 1.0, 8.0 ± 1.3, and 7.9 ± 1.1 in uninfected control piglets; 7.2 ± 1.2, 5.2 ± 1.4, 2.7 ± 0.3, 1.4 ± 0.2, and 1.5 ± 0.1 in wt-PEDV-infected piglets; and 7.6 ± 1.8, 3.2 ± 0.4, 2.2 ± 0.3, 1.3 ± 0.3, and 1.1 ± 0.2 in ca-PEDV-infected piglets at 12, 24, 36, 48, and 60 h post inoculation. Conclusively, the mean VH/CD ratio for wt-PEDV piglets was significantly different from that of ca-PEDV piglets at 24, 36, and 60 h post inoculation. The in situ hybridization (ISH) positive results in piglets orally infected with the Korean strain of PEDV in jejunal villus were 4.0 ± 0.8, 10.3 ± 0.5, 7.3 ± 0.5, 7.0 ± 0.8, and 5.3 ± 0.5 in ca-PEDV-infected piglets; and 4.3 ± 0.9, 16.7 ± 1.2, 9.3 ± 0.5, 9.0 ± 0.8, and 5.7 ± 0.9 in wt-PEDV-infected piglets. Conclusively, a significant difference was observed in each group. A greater amount of PEDV nucleic acid was detected in the jejunal tissues (P < 0.05) of the ca-PEDV-inoculated piglets than in those of the wt-PEDV-infected piglets at 24, 36, and 60 h post-inoculation. The nucleotide sequences of wt-PEDV and ca-PEDV were nearly identical (98.7% homology); nucleotide substitutions were noted in ORF3 that caused some amino acid changes. Statistically significant differences were observed in the pathogenicity of ca-PEDV compared with its parental wt-PEDV; ORF3 nucleotide changes were identified in ca-PEDV that possibly influenced PEDV pathogenicity.

Immunohistochemical expression of FAP and PCNA in neoplastic epithelial colonic lesions

Background Colorectal carcinoma is one of the leading causes of morbidity and mortality worldwide. Fibroblast-activation protein is expressed in cancer-associated fibroblasts and its expression is upregulated during the transformation of benign colorectal tissue into cancer. Proliferating-cell nuclear antigen expression was used as a marker of cell proliferation and is considered as a marker able to identify aggressive adenomas that may in turn become malignant. Aim To study the expression of fibroblast-activation protein and proliferating-cell nuclear antigen in neoplastic epithelial colonic lesions and their role in adenoma–carcinoma sequence. Patients and methods This retrospective study aimed to evaluate the expression of fibroblast-activation protein and proliferating-cell nuclear antigen in 80 cases of neoplastic epithelial colonic lesions. Results In malignant colorectal epithelial tumors, high fibroblast-activation protein expression was detected with an increase in histological grade, depth of invasion, vascular invasion, and lymph-node metastasis. No fibroblast-activation protein expression was detected in all benign cases. In included benign cases, there was a significant relation between proliferating-cell nuclear antigen labeling index and both histological type and grade of dysplasia. In the included malignant cases, there was a significant relation between proliferating-cell nuclear antigen labeling index with grade of tumor and the depth of invasion. Conclusions Both fibroblast-activation protein and proliferating-cell nuclear antigen labeling index suggested to have a role in early detection of malignant transformation and had a prognostic role in malignant cases.

Disruption of cell proliferation and apoptosis balance in the testes of crossbred cattle-yaks affects spermatogenic cell fate and sterility.

The balance between proliferation, differentiation and apoptosis is well-coordinated in spermatogenesis for the timely production of appropriate numbers of sperm in animals. Disruption or decrease in sperm production is due to many conditions, including changes in testicular cell fate balance. Interspecies hybridization of domestic yaks and cattle results in sterility in males because of spermatogenic arrest; however, the underlying mechanisms involved in sterility are still unclear. In the present study, we investigated the proliferation and apoptosis status during the development of yaks and crossbred cattle-yaks using immunohistochemistry of proliferating cell nuclear antigen and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assays. Testicular tissues from yaks (immature: 1year old, mature: 2-3 years old) and backcrossed hybrids (2year old) were collected and used to investigate the expression of each parameter in testicular cells. During the maturation of yak testes, proliferation and apoptosis became active only in spermatogenic cells, and not in other somatic cells, such as Sertoli cells, myoid cells and Leydig cells. Furthermore, hybrid cattle-yak testes maintained proliferation ability but less apoptotic ability in spermatogenic cells when compared to yaks of the same age, suggesting that normal spermatogenic cell fate control is disrupted by changes in the balance between proliferation and apoptosis. In addition, Leydig cell proliferation rate was higher than apoptosis rate in the cattle-yak testes, indicating an increased number of Leydig cells, which may affect spermatogenesis through changes in steroidogenesis. Although epigenetic changes may be involved in cattle-yak testes, further studies are needed to clarify the modulation of proliferation and apoptosis to elucidate the mechanisms of infertility in hybrid cattle-yak males.

Previous Usutu Virus Exposure Partially Protects Magpies (Pica pica) against West Nile Virus Disease But Does Not Prevent Horizontal Transmission.

The mosquito-borne flaviviruses USUV and WNV are known to co-circulate in large parts of Europe. Both are a public health concern, and USUV has been the cause of epizootics in both wild and domestic birds, and neurological cases in humans in Europe. Here, we explore the susceptibility of magpies to experimental USUV infection, and how previous exposure to USUV would affect infection with WNV. None of the magpies exposed to USUV showed clinical signs, viremia, or detectable neutralizing antibodies. After challenge with a neurovirulent WNV strain, neither viremia, viral titer of WNV in vascular feathers, nor neutralizing antibody titers of previously USUV-exposed magpies differed significantly with respect to magpies that had not previously been exposed to USUV. However, 75% (6/8) of the USUV-exposed birds survived, while only 22.2% (2/9) of those not previously exposed to USUV survived. WNV antigen labeling by immunohistochemistry in tissues was less evident and more restricted in magpies exposed to USUV prior to challenge with WNV. Our data indicate that previous exposure to USUV partially protects magpies against a lethal challenge with WNV, while it does not prevent viremia and direct transmission, although the mechanism is unclear. These results are relevant for flavivirus ecology and contention.

Two-Stage Portal Vein Ligation Facilitates Liver Regeneration Safely in Rats with Liver Cirrhosis

Background Portal vein (PV) embolization is performed prior to extended hepatectomy for the damaged liver to increase future remnant liver volume and prevent postoperative liver failure. This study examined whether two-stage PV ligation (PVL) increased regeneration and hypertrophy of the future remnant liver compared to conventional PVL, and whether two-stage PVL was safe for damaged liver. Method We produced a cirrhotic liver rat model with perioperatively maintained fibrosis. Rats were divided into: Group A (70%PVL), ligation of left branch of PV; Group B (90%PVL), ligation of right and left branches of PV; and Group C (two-stage 90%PVL), two-stage PVL with left branch ligation of PV followed by right branch ligation 7 days later. To evaluate liver regeneration, liver weight ratios, proliferating cell nuclear antigen (PCNA) labeling index (LI), mitotic index (MI), and TdT-mediated dUTP-biotin nick end labeling (TUNEL) LI in the non-ligated caudate lobe were measured. Results Fourteen-day survival rate was 20% in Group B but 100% in Group C. TUNEL LI differed significantly between Groups A and B at 2 and 7 days postoperatively. Weight ratios were significantly higher in Group C than in Groups A and B at 14 days postoperatively. PCNA LI and MI in the non-ligated caudate lobe decreased to preoperative levels by 7 days postoperatively in Groups A and B, but remained elevated until 14 days postoperatively in Group C. Conclusion In cirrhotic liver rats, two-stage PVL avoided the lethal liver failure seen with one-stage PVL, and significantly facilitated liver regeneration more than one-stage PVL.

Carob fruit extract-enriched meat, as preventive and curative treatments, improves gut microbiota and colonic barrier integrity in a late-stage T2DM model

Epidemiological and experimental studies have suggested that dietary fiber and proanthocyanidins play an important role on gut microbiota (GM), colonic integrity and body health. Type 2 Diabetes Mellitus (T2DM) is a prevalent disease in which the modifications in the GM and colonic markers stand out. This manuscript hypothesizes the consumption of functional meat enriched in carob fruit extract [CFE; CFE-restructured meat (RM)] ameliorates the dysbiosis and colonic barrier integrity loss in a late-stage T2DM rat model induced by the conjoint action of a high-saturated-fat/high-cholesterol diet (Chol-diet) and a low dose of streptozotocin (STZ) plus a nicotinamide (NAD) injection. Three groups of eight rats were used: (1) D group, a T2DM control group, fed the Chol-diet; (2) ED group, a T2DM preventive strategy group fed the CFE-Chol-diet since the beginning of the study; and (3) DE group, a T2DM curative treatment group, fed the CFE-Chol-diet once the diabetic state was confirmed. The study lasted 8 weeks. Amount and variety of GM, feces short-chain-fatty acids (SCFAs), colonic morphology [crypt depth and density, goblet cells, proliferating cell nuclear antigen (PCNA) and transferase dUTP nick end labelling (TUNEL) indexes] and tight junctions were evaluated. A global colonic index combining 17 markers (GCindex) was calculated. ED rats displayed higher levels of GM richness, SCFAs production, crypt depth, and goblet cells than the D group. DE group showed lower Enterobacteriaceae abundance and greater TUNEL index and occludin expression in the distal colon than D counterpart. GCindex differentiated the colonic health status of the experimental groups in the order (ED > DE > D; P < 0.001) as a 17–51 range-quotation, ED, DE, and D groups displayed the values 43, 32.5, and 27, respectively. Thus, CFE-RM used as a T2DM preventive therapy could induce higher GM richness, more adequate SCFAs production, and better colonic barrier integrity. Furthermore, CFE-RM used with curative purposes induced more modest changes and mainly at the distal colonic mucosa. Further studies are needed to confirm this study’s results, to ascertain the benefits of consuming proanthocyanidins-rich fiber during different T2DM stages.

Evidence of BK Polyomavirus Infection in Urothelial but not Renal Tumors from a Single Center Cohort of Kidney Transplant Recipients.

Emerging evidence indicates that reactivation of BK polyomavirus (BKPyV) in the kidney and urothelial tract of kidney transplant recipients (KTRs) may be associated with cancer in these sites. In this retrospective study of a single center cohort of KTRs (n = 1307), 10 clear cell renal cell carcinomas and 5 urinary bladder carcinomas were analyzed from 15 KTRs for the presence of BKPyV infection through immunohistochemistry and fluorescent in situ hybridization (FISH). Three of these patients had already exhibited biopsy-proven polyomavirus-associated nephropathies (PyVAN). Although the presence of BKPyV large-T antigen was evident in the urothelium from a kidney removed soon after PyVAN diagnosis, it was undetectable in all the formalin-fixed and paraffin-embedded (FFPE) blocks obtained from the 10 kidney tumors. By contrast, large-T antigen (LT) labeling of tumor cells was detected in two out of five bladder carcinomas. Lastly, the proportion of BKPyV DNA-FISH-positive bladder carcinoma nuclei was much lower than that of LT-positive cells. Taken together, our findings further strengthen the association between BKPyV reactivation and cancer development in KTRs, especially bladder carcinoma.