ABSTRACT
While increased DNA damage is a well-described feature of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), it is unclear whether all lineages and all regions of the marrow are homogeneously affected. In this study, we performed immunohistochemistry on formalin-fixed, paraffin-embedded whole-section bone marrow biopsies using a well-established antibody to detect pH2A.X (phosphorylated histone variant H2A.X) that recognizes DNA double-strand breaks. Focusing on TP53-mutated and complex karyotype MDS/AML, we find a greater pH2A.X+ DNA damage burden compared to TP53 wild-type neoplastic cases and non-neoplastic controls. To understand how double-strand breaks vary between lineages and spatially in TP53-mutated specimens, we applied a low-multiplex immunofluorescence staining and spatial analysis protocol to visualize pH2A.X+ cells with p53 protein staining and lineage markers. pH2A.X marked predominantly mid- to late-stage erythroids, whereas early erythroids and CD34+ blasts were relatively spared. In a prototypical example, these pH2A.X+ erythroids were organized locally as distinct colonies, and each colony displayed pH2A.X+ puncta at a synchronous level. This highly coordinated immunophenotypic expression was also seen for p53 protein staining and among presumed early myeloid colonies. Neighborhood clustering analysis showed distinct marrow regions differentially enriched in pH2A.X+/p53+ erythroid or myeloid colonies, indicating spatial heterogeneity of DNA-damage response and p53 protein expression. The lineage and architectural context within which DNA damage phenotype and oncogenic protein are expressed is relevant to current therapeutic developments that leverage macrophage phagocytosis to remove leukemic cells in part due to irreparable DNA damage. © 2024 The Pathological Society of Great Britain and Ireland.
Subject(s)
Mutation , Myelodysplastic Syndromes , Tumor Suppressor Protein p53 , Humans , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/pathology , Myelodysplastic Syndromes/metabolism , Middle Aged , DNA Damage , Male , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/pathology , Leukemia, Myeloid, Acute/metabolism , Aged , Female , DNA Breaks, Double-Stranded , Histones/metabolism , Histones/genetics , Bone Marrow/pathology , Bone Marrow/metabolism , Aged, 80 and over , ImmunohistochemistryABSTRACT
BACKGROUND: Prognosticating outcomes for traumatic brain injury (TBI) patients is challenging due to the required specialized skills and variability among clinicians. Recent attempts to standardize TBI prognosis have leveraged machine learning (ML) methodologies. This study evaluates the necessity and influence of ML-assisted TBI prognostication through healthcare professionals' perspectives via focus group discussions. METHODS: Two virtual focus groups included ten key TBI care stakeholders (one neurosurgeon, two emergency clinicians, one internist, two radiologists, one registered nurse, two researchers in ML and healthcare and one patient representative). They answered six open-ended questions about their perceptions and potential ML use in TBI prognostication. Transcribed focus group discussions were thematically analyzed using qualitative data analysis software. RESULTS: The study captured diverse perceptions and interests in TBI prognostication across clinical specialties. Notably, certain clinicians who currently do not prognosticate expressed an interest in doing so independently provided they had access to ML support. Concerns included ML's accuracy and the need for proficient ML researchers in clinical settings. The consensus suggested using ML as a secondary consultation tool and promoting collaboration with internal or external research resources. Participants believed ML prognostication could enhance disposition planning and standardize care regardless of clinician expertise or injury severity. There was no evidence of perceived bias or interference during the discussions. CONCLUSION: Our findings revealed an overall positive attitude toward ML-based prognostication. Despite raising multiple concerns, the focus group discussions were particularly valuable in underscoring the potential of ML in democratizing and standardizing TBI prognosis practices.
ABSTRACT
Digital histopathological images, high-resolution images of stained tissue samples, are a vital tool for clinicians to diagnose and stage cancers. The visual analysis of patient state based on these images are an important part of oncology workflow. Although pathology workflows have historically been conducted in laboratories under a microscope, the increasing digitization of histopathological images has led to their analysis on computers in the clinic. The last decade has seen the emergence of machine learning, and deep learning in particular, a powerful set of tools for the analysis of histopathological images. Machine learning models trained on large datasets of digitized histopathology slides have resulted in automated models for prediction and stratification of patient risk. In this review, we provide context for the rise of such models in computational histopathology, highlight the clinical tasks they have found success in automating, discuss the various machine learning techniques that have been applied to this domain, and underscore open problems and opportunities.
Subject(s)
Machine Learning , Neoplasms , Humans , Neoplasms/genetics , Neoplasms/diagnosisABSTRACT
Streptococcus agalactiae is one of the main aetiological agents in large-scale mortalities of tilapia, having caused major economic losses to the aquaculture industry in recent years. This study describes the isolation and identification of the bacteria from cage-cultured Etroplus suratensis that experienced moderate to severe mortalities in Kerala, India. Gram-positive, catalase-negative S. agalactiae was identified from brain, eye and liver of the fish by antigen grouping and 16S rDNA sequencing. Multiplex PCR confirmed that the isolate belonged to capsular serotype Ia. Antibiotic susceptibility tests showed that the isolate was resistant to methicillin, vancomycin, tetracycline, kanamycin, streptomycin, ampicillin, oxacillin and amikacin. Histological sections of the infected E. suratensis brain revealed infiltration of inflammatory cells, vacuolation and meningitis. This report is the first description of S. agalactiae as a primary pathogen causing mortalities in E. suratensis culture in Kerala.
Subject(s)
Cichlids , Tilapia , Animals , Streptococcus agalactiae/genetics , India , Anti-Bacterial Agents/pharmacologyABSTRACT
In the present study, we studied the effect of ß-glucan on the activation of antiviral immune responses against nervous necrosis virus (NNV) taking into consideration the role of innate immune training. Sevenband grouper primary macrophages showed an attenuated proinflammatory response and elevated antiviral response to NNV infection. In vitro, priming of ß-glucan enhanced macrophage viability against NNV infection which is associated with the activation of sustained inflammatory cytokines gene expression. Observations were clear to understand that NLR Family CARD Domain Containing 3 (NLRC3) and caspase-1 activation and subsequent IL-1ß production were reduced in ß-glucan-primed macrophages. Subsequent markers for training including Lactate and abundance of HIF-1α were elevated in the cells following training. However, the lactate dehydrogenase (LDH) concentrations remained stable among the ß-glucan stimulated infected and uninfected groups suggesting similar macrophage health in both groups. In vivo, the NNV-infected fish primed with ß-glucan had a higher survival rate (60%) than the control NNV-infected group (40%). Our findings demonstrate that ß-glucan induced protective responses against NNV infection and studies are underway to harness its potential applicability for prime and boost vaccination strategies.
Subject(s)
Bass , Fish Diseases , Nodaviridae , RNA Virus Infections , beta-Glucans , Animals , Antiviral Agents/therapeutic use , Bass/immunology , Bass/virology , Fish Diseases/prevention & control , Fish Diseases/virology , RNA Virus Infections/prevention & control , RNA Virus Infections/veterinary , beta-Glucans/pharmacologyABSTRACT
Changes in the thermal optima of fish impacts changes in the physiology and immune response associated with infections. The present study showed that at suboptimal temperatures (17 °C), the host tries to evade viral infection by downregulating the inflammatory response through enhanced neuronal protection. There was significantly less abundance of IgM + B cells in the 17 °C group compared to that in the 25 °C group. An increased macrophage population (Iba1+) during the survival phase in fish challenged at 25 °C demonstrated inflammation. Optimal temperature challenge activated virus-induced senescence in brain cells, demonstrated with a heterochromatin-associated H3K9me3 histone mark. There was an abundant expression of anti-inflammatory cytokines in the brain of fish at the suboptimal challenge. Besides the cytokines, the expression of BDNF was significantly higher in the suboptimally challenged group, suggesting that its neuronal protection activity following NNV infection is mediated through TGFß. The suboptimal challenge resulted in H3k9ac displaying transcriptional competency, activation of trained immunity H3K4me3, and enrichment of H3 histone-lysine-4 monomethylation (H3K4me1), resulting in a robust re-stimulatory immune response. The observations from the H4 modifications showed that besides H4K12ac and H4K20m3, all the assayed modifications were significantly higher in suboptimal convalescent fishes. The suboptimally challenged fish acquired more methylation along cytosine residues than the optimally infected fish. Together, these observations suggest that optimal temperature results in an immune priming effect, whereas the protection enabled in suboptimal convalescent fishes is operated through epigenetically controlled trained immune functions.
Subject(s)
Bass , Fish Diseases , Nodaviridae , RNA Virus Infections , Virus Diseases , Animals , Bass/metabolism , Temperature , Antiviral Agents , Nodaviridae/physiology , Epigenesis, Genetic , Cytokines/metabolism , Necrosis , Fish Proteins/genetics , Fish Proteins/metabolismABSTRACT
Trained immunity refers to the memory acquired by innate immune cells, leading to cross-protection and non-specific responses to subsequent infection, thereby improving host survival. Trained immunity induction is a combined effect of immune signaling, metabolic changes, and epigenetic modifications. The present study evaluated the induction of markers of the phenomenon of trained immunity in common carp, which is trained using ß-glucan. The mammalian target of rapamycin (mtor) and hypoxia-inducible factor (hif1α), the metabolic basis of trained immunity; the histone deacetylase (hdac7), one of the markers of epigenetic modifications, metabolic activity of activated cells and expression profiles of proinflammatory cytokines viz. il6a, tnfαa2, and ifnγ were targeted in the study and analyzed in vivo. Besides in vivo analysis, in vitro analysis of mtorc2, hif1α, hdac7, and ifnγ were analyzed. In vitro analyses were performed on head kidney macrophages isolated and maintained in L-15 media and double trained with ß-glucan at 100µg/mL. The culture supernatant was collected at different time intervals and processed for expression studies. Healthy common carp were injected with ß-glucan at 20 mg/kg body weight for training followed by a resting phase for 6 days and were restimulated with the same dose. Head kidney was collected from the fish post-induction as well as post-restimulation. The expression profile of mtorc2, hdac7, and hif1α were found elevated post-stimulation of ß-glucan. Further, a significantly upregulated expression profile of proinflammatory cytokines (ifnγ, il6a and tnfαa2) was observed. Increased glycolysis in the cells post-ß-glucan stimulation was confirmed by the high lactate and LDH production detected in the cell culture supernatant. Overall, the study revealed the expression profile of the trained immunity markers and the increased metabolic activity in cells induced with ß-glucan, which further validates that the action of trained immunity is indispensable in fish on encounter with a potential ligand. The study supports the existing reports on trained immunity in teleost fish with evidence at the genomic level. However, further studies are required to understand the responses and actions of trained immune cells during infection in detail.
Subject(s)
Carps , beta-Glucans , Animals , Carps/genetics , Glucans/pharmacology , Trained Immunity , beta-Glucans/pharmacology , Cytokines/genetics , Cytokines/pharmacology , Mechanistic Target of Rapamycin Complex 2 , Immunity, Innate/genetics , MammalsABSTRACT
NLRC3 is identified as a unique regulatory NLR involved in the modulation of cellular processes and inflammatory responses. In this study, a novel Nod like receptor C3 (NLRC3) was functionally characterized from seven band grouper in the context of nervous necrosis virus infection. The grouper NLRC3 is highly conserved and homologous with other vertebrate proteins with a NACHT domain and a C-terminal leucine-rich repeat (LRR) domain and an N-terminal CARD domain. Quantitative gene expression analysis revealed the highest mRNA levels of NLRC3 were in the brain and gill followed by the spleen and kidney following NNV infection. Overexpression of NLRC3 augmented the NNV replication kinetics in primary grouper brain cells. NLRC3 attenuated the interferon responses in the cells following NNV infection by impacting the TRAF6/NF-κB activity and exhibited reduced IFN sensitivity, ISRE promoter activity, and IFN pathway gene expression. In contrast, NLRC3 expression positively regulated the inflammasome response and pro-inflammatory gene expression during NNV infection. NLRC3 negatively regulates the PI3K-mTOR axis and activated the cellular autophagic response. Delineating the complexity of NLRC3 regulation of immune response in the primary grouper brain cells following NNV infection suggests that the protein acts as a virally manipulated host factor that negatively regulated the antiviral immune response to augment the NNV replication.
Subject(s)
Bass , Fish Diseases , Nodaviridae , RNA Virus Infections , Virus Diseases , Animals , Antiviral Agents , Brain/metabolism , Fish Proteins , Immunity, Innate/genetics , Inflammasomes/metabolism , Necrosis , Nodaviridae/physiology , RNA Virus Infections/veterinaryABSTRACT
The nervous necrosis virus (NNV) infection is generally observed in aquafarms when the seawater temperature is higher than 24 °C and the fishes seem to be refractory to disease at suboptimal temperatures below 20 °C suggesting a role of thermoregulation in NNV pathogenesis. The present study profiled the temperature-dependent regulation of cytokines (TNF-α, IL-1ß and IFN-γ), innate antiviral factors (IFN-1, Mx, ISG-15), adaptive immune factors (CD-4, CD-8, IgM), signaling regulators (SOCS-1, SOCS-3), transcription factors (STAT-1, STAT-3) and microglial and NCC/NK specific cell markers (TMEM-119 and NCCRP-1) during NNV challenge in seven-band grouper, Hyporthodus septemfasciatus. The co-habitation challenge at 17 °C with showed a sustained expression of proinflammatory cytokines and following rechallenge with a dose of 104 TCID50/100µL/fish at optimal temperature, the survivors also exhibited a stable expression of immune factors. The 100% survival following the challenge at sub-optimal (17 °C) and rechallenge at optimal (25 °C) was due to the stable and sustained activation of the immune response. However, at 25 °C, the rechallenge displayed a priming effect with hyperactivation of the immune system evident from the immune gene expression profile. The mortality pattern observed is co-related with the cytokine storm as is evident from the gene expression profile. Whereas, neither of the adaptive immune markers was suggestive of humoral immune response in the 17 °C groups. Also, the data suggest a possible role of NK cell and microglia in mediating antiviral immune response following infection in the brain at different temperatures, where, former is beneficial in restricting viral infection with higher host tolerance.
Subject(s)
Bass , Fish Diseases , Nodaviridae , RNA Virus Infections , Animals , Antiviral Agents/therapeutic use , Cytokines , Fish Diseases/drug therapy , Immunologic Factors , Necrosis , RNA Virus Infections/veterinary , TemperatureABSTRACT
During viral infection, proper regulation of immune signaling is essential to ensure successful clearance of virus. Immunoproteasome is constitutively expressed and gets induced during viral infection by interferon signaling and contributes to regulate proinflammatory cytokine production and activation of the NF-κB pathway. In this study, we identified Hs-PSMB8, a member of the proteasome ß-subunits (PSMB) family, as a negative regulator of NF-κB responses during NNV infection. The transient expression of Hs-PSMB8 delayed the appearance of cytopathic effect (CPE) and showed a higher viral load. The Hs-PSMB8 interacted with NNV which was confirmed using immunocolocalization and co-IP. Overexpression of Hs-PSMB8 diminished virus induced activation of the NF-κB promoters and downregulated the activation of IL-1ß, TNFα, IL6, IL8, IFNγ expression upon NNV infection. Collectively, our results demonstrate that PSMB8 is an important regulator of NF-κB signaling during NNV infection in sevenband grouper.
Subject(s)
Bass/genetics , Bass/immunology , Fish Diseases/immunology , Gene Expression Regulation/immunology , Immunity/genetics , Proteasome Endopeptidase Complex/genetics , Proteasome Endopeptidase Complex/immunology , Amino Acid Sequence , Animals , Fish Diseases/virology , Fish Proteins/chemistry , Fish Proteins/genetics , Fish Proteins/immunology , Gene Expression Profiling/veterinary , NF-kappa B/immunology , Nodaviridae/physiology , Phylogeny , Proteasome Endopeptidase Complex/chemistry , RNA Virus Infections/immunology , RNA Virus Infections/veterinary , RNA Virus Infections/virology , Sequence Alignment/veterinary , Signal Transduction/immunologyABSTRACT
Co-infection with parasites and bacteria is of frequent occurrence in aquaculture, leads to growth impedance otherwise mortality in fish depending on the varying degree of a load of primary pathogen either parasite or bacteria. The mechanistic regulation of immune response during co-infection in fish has merely documented. The aim of this study was to determine the impact of co-infection with Aeromonas hydrophila at three exposure doses of Argulus sp. on the innate immune responses and antioxidative stress enzymes of goldfish (Carassius auratus). The experimental fish were randomly distributed into eight treatment groups viz. T1 (control group without Argulus and A. hydrophila infection), T2 (fish exposed to a sub-lethal dose of A. hydrophila), T3 (low Argulus-infested fish), T4 (T3 + sub-lethal dose of A. hydrophila), T5 (moderate Argulus-infested fish), T6 (T5 + sub-lethal dose of A. hydrophila), T7 (high Argulus-infested fish) and T8 (T7+ sub-lethal dose of A. hydrophila) in duplicates. After distributing experimental fish into their respective treatment group, A. hydrophila was injected to T2, T4, T6 and T8. After the bacterial challenge, four fish from each experimental group were randomly sampled on 24, 72, and 168 h and subjected to the hematological, innate immune parameters and enzymatic analysis. In the co-infection group T8, a high degree of enhanced pathogenicity of A. hydrophila was noticed with increased mortalities (84.2%) in comparison to other groups. The current study shows a declining pattern in RBC, PCV and Hb values with the degree of parasite infestation without co-infection groups. Moreover, in the T8 group, exposure of a sub-lethal dose of bacteria resulted in a drastic reduction of the recorded parameters. Furthermore, a decreased value for WBC, monocyte and neutrophil was found in higher parasite group co-infected with a sub-lethal dose of bacteria relative to other co-infected groups during the experimental period. Also, a decrease in innate immune parameters and antioxidative stress enzymes were observed in the T8 group compared to T7 and T2 groups throughout the trial period. These findings indicate that a rise in the dose of Argulus infection improves A. hydrophila colonization in goldfish and contributes to suppression of the innate immune system and increased mortality.
Subject(s)
Aeromonas hydrophila , Arguloida , Goldfish , Gram-Negative Bacterial Infections/veterinary , Immunity, Innate/physiology , Parasitic Diseases, Animal/parasitology , Animals , Antioxidants , Catalase/genetics , Catalase/metabolism , Gene Expression Regulation, Enzymologic/immunology , Glutathione Peroxidase/genetics , Glutathione Peroxidase/metabolism , Gram-Negative Bacterial Infections/complications , Gram-Negative Bacterial Infections/immunology , Parasitic Diseases, Animal/complications , Parasitic Diseases, Animal/immunology , Stress, Physiological , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolismABSTRACT
OBJECTIVES: To evaluate the concordance between second-trimester anatomic ultrasound and fetal echocardiography in detecting minor and critical congenital heart disease in pregnancies meeting American Heart Association criteria. METHODS: We conducted a retrospective cohort study of pregnancies in which a second-trimester fetal anatomic ultrasound examination (18-26 weeks) and fetal echocardiography were performed between 2012 and 2018 at our institution based on American Heart Association recommendations. Anatomic ultrasound studies were interpreted by maternal-fetal medicine specialists and fetal echocardiographic studies by pediatric cardiologists. Our primary outcome was the proportion of critical congenital heart disease (CCHD) cases not detected by anatomic ultrasound but detected by fetal echocardiography. The secondary outcome was the proportion of total congenital heart disease cases missed by anatomic ultrasound but detected by fetal echocardiography. Neonatal medical records were reviewed for all pregnancies when obtained and available. RESULTS: Overall, 722 studies met inclusion criteria. Anatomic ultrasound and fetal echocardiography were in agreement in detecting cardiac abnormalities in 681(96.1%) studies (κ = 0.803; P < .001). The most common diagnosis not identified by anatomic ultrasound was a ventricular septal defect, accounting for 9 of 12 (75%) missed congenital heart defects. Of 664 studies with normal cardiac findings on the anatomic ultrasound examinations, no additional instances of CCHD were detected by fetal echocardiography. No unanticipated instances of CCHD were diagnosed postnatally. CONCLUSIONS: With current American Heart Association screening guidelines, automatic fetal echocardiography in the setting of normal detailed anatomic ultrasound findings provided limited benefit in detecting congenital heart defects that would warrant immediate postnatal interventions. More selective use of automatic fetal echocardiography in at-risk pregnancies should be explored.
Subject(s)
Heart Defects, Congenital , Pregnancy, High-Risk , Child , Echocardiography , Female , Fetal Heart/diagnostic imaging , Heart Defects, Congenital/diagnostic imaging , Humans , Infant, Newborn , Pregnancy , Retrospective Studies , Ultrasonography, PrenatalABSTRACT
In the present study, early uptake of nervous necrosis virus (NNV) in the tissues (gill, brain, skin, eye, heart) and immune response associated with the uptake in the gill and brain of seven-band grouper was investigated. The gill was found to act as a primary portal of entry for NNV during the initial phase of the water-borne infection. The presence of viral genome and infectious particles was demonstrated using quantitative (qPCR, viral titer) and qualitative (ISH) approach. Initially, an increased viral uptake was noticed, but the virus got cleared from the gills at the later phase of infection. Localization in the brain was evident at the blood-brain barrier followed by the brain parenchyma in the latter stage of infection. Nectin-4, an established NNV receptor, and GHSC70 showed an up-regulated expression throughout the challenge period initially in the gill and at latter phase in brain; however, it seems that the virus does not use gill as a primary replication site but brain as a permissive tissue. Combined activity as reflected by the up-regulation of cytokine, interferon, antigen-presenting cell, and immunoglobulin genes restricts early NNV replication in gill. Observations from the present study provide a better understanding of early NNV entry and also opens a window for further elucidating the modes of NNV neuro-invasion through systemic circulation.
Subject(s)
Bass , Fish Diseases/immunology , Immunity , Nodaviridae/physiology , RNA Virus Infections/veterinary , Animals , Brain/virology , Fish Diseases/virology , Gills/virology , RNA Virus Infections/immunology , RNA Virus Infections/virologyABSTRACT
INTRODUCTION: Predictors of non-response in mismatch repair deficiency cancers are poorly understood. Upregulation of the canonical Wnt pathway has been associated with decreased immune cell infiltration in many cancer types. The relationship between Wnt/ß-catenin pathway activation and the programmed death-ligand 1 axis in endometrial cancer remains poorly characterized. This study evaluates ß-catenin expression in a well characterized cohort of endometrial cancers by mismatch repair status and programmed death-ligand 1 expression. METHODS: Whole sections of formalin-fixed, paraffin embedded tissue from 23 Lynch syndrome-associated carcinomas, 20 mutL homolog-1 (MLH1) promoter hypermethylated carcinomas, and 19 mismatch repair intact carcinomas were evaluated. Immunohistochemistry staining for ß-catenin and programmed death-ligand 1 was performed on all cases. Programmed death-ligand 1 expression was scored in both the tumor and the peri-tumoral immune compartment. Tumor staining was classified as positive when membranous (programmed death-ligand 1) staining was present in ≥1% of tumor cells. Immune stromal staining was scored as positive when ≥5% of peritumoral and intratumoral immune cells (including lymphocytes and macrophages) showed reactivity. RESULTS: Six tumors (6/62, 9.7%) demonstrated nuclear expression of ß-catenin (4 were Lynch syndrome-associated, 1 was MLH1 methylated, 1 was mismatch repair intact). The majority of tumors with nuclear ß-catenin expression demonstrated concomitant tumoral programmed death-ligand 1 expression (5/6, 83.3%) and were more likely to demonstrate tumoral programmed death-ligand 1 expression compared to tumors without nuclear ß-catenin expression (83.3% vs 39.3%, p=0.04). Both tumoral and immune cell expression of programmed death-ligand 1 was statistically significantly associated with mismatch repair deficient tumors. DISCUSSION: Tumors demonstrating nuclear ß-catenin expression were more likely to express tumoral programmed death-ligand 1 staining than tumors without nuclear ß-catenin expression. Nuclear ß-catenin expression could be a potential predictive biomarker for non-response to immune checkpoint inhibition in mismatch repair deficient tumors. Nuclear ß-catenin expression status should be considered as a translational endpoint in future clinical trials of immune checkpoint inhibition in endometrial cancer.
Subject(s)
B7-H1 Antigen/biosynthesis , DNA Mismatch Repair , Endometrial Neoplasms/genetics , Endometrial Neoplasms/metabolism , beta Catenin/biosynthesis , Aged , B7-H1 Antigen/genetics , Endometrial Neoplasms/pathology , Female , Humans , Immunohistochemistry , Microsatellite Instability , Middle Aged , MutL Protein Homolog 1/genetics , MutL Protein Homolog 1/metabolism , Neoplasm Staging , Retrospective Studies , Wnt Signaling Pathway , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
The prevalence of carbapenem resistance among bacterial isolates from selected water bodies receiving hospital effluents and adjoining aquaculture farms in Kerala, India, was studied. Klebsiella pneumoniae followed by Escherichia coli, Klebsiella oxytoca, Enterobacter aerogenes and Acinetobacter baumannii were the predominant isolates. Antibiotic sensitivity of these isolates was determined by Kirby-Bauer disc diffusion method. Nearly 60% of the Enterobacteriaceae isolates screened were multidrug resistant of which 16.6% were carbapenem resistant. The carbapenem-resistant Enterobacteriaceae were further screened for the presence of New Delhi metallo ß-lactamase-1 and cephalosporin resistance encoding genes. All NDM-1 isolates were highly resistant to carbapenem, cephalosporin, aminoglycosides, quinolones, tetracycline, and sulphonamides. K. pneumoniae harboring blaNDM-1 gene and E. coli isolates with blaCTX-M-15 and blaSHV-11 genes were detected in hospital discharge points. In aquaculture farms too, carbapenem-resistant K. pneumoniae with blaNDM-1 gene and E. coli isolates with blaCTX-M-15 were observed, although there was no use of antibiotics in these farms. However, other carbapenemase genes such as blaTEM, blaVIM, blaIMP and blaGIM were not detected in any of these isolates. The results suggest the increased prevalence of carbapenem-resistant Enterobacteriaceae in the water bodies receiving hospital effluent and its dissemination to adjacent aquaculture farms, posing a serious threat to public health.
Subject(s)
Carbapenem-Resistant Enterobacteriaceae , Cephalosporin Resistance , Water Microbiology , beta-Lactamases , Anti-Bacterial Agents/pharmacology , Carbapenem-Resistant Enterobacteriaceae/drug effects , Carbapenem-Resistant Enterobacteriaceae/genetics , Cephalosporin Resistance/genetics , Escherichia coli/drug effects , Escherichia coli/genetics , Hospitals , India , Klebsiella pneumoniae/drug effects , Microbial Sensitivity Tests , beta-Lactamases/geneticsABSTRACT
Nectin-4/PVRL4 belonging to the family of immunoglobulin-like cell adhesion molecules was identified as a potential cellular receptor for several animal viruses. Here we show that nervous necrosis virus that causes viral nervous necrosis in teleosts uses the same receptor in its life cycle. Transfection of SSN-1â¯cell lines with an expression vector encoding Nectin-4 rendered them to be more susceptible to NNV. Immunofluorescence microscopy on Nectin-4 expressing cells revealed that the protein interacted with NNV specifically. A virus binding assay indicated that Nectin-4 was a bonafide receptor that supported virus attachment to the host cell whereas siRNA directed against Nectin-4 blocked NNV infections in grouper primary brain cells. Results of the present study will improve our understanding of the pathogenesis of NNV infection and provide a target for the development of novel antiviral interventions in marine finfish aquaculture.
Subject(s)
Bass/genetics , Bass/immunology , Fish Diseases/immunology , Nectins/genetics , Nectins/immunology , Animals , Fish Proteins/genetics , Fish Proteins/immunology , Nodaviridae/physiology , RNA Virus Infections/immunology , RNA Virus Infections/veterinaryABSTRACT
Interferon regulatory factor (IRF) 3 and IRF7 are key regulators of type I interferon (IFN) gene expression for the antiviral immune response. In the present study, interferon regulatory factor 3 and 7 from Asian seabass, namely AsIRF3 and AsIRF7 were cloned and characterized. The full-length cDNA sequence of IRF3 and IRF7 consisted of 2965 and 2343 bp respectively. AsIRF3 and AsIRF7 were true orthologes of vertebrate IRF3/7 and showed similar domain organization, with an N-terminal DBD which consisted five tryptophan residues in IRF3 and four in IRF7, a C-terminal IRF3 domain and a serine rich region. Both IRF3 and 7 constitutively expressed during the ontogenesis and in all tissues of healthy fish. The expression of both genes was up-regulated following NNV challenge with obvious transcript abundance in brain heart and kidney. Ectopic expression of AsIRF3 and AsIRF7 displayed activation of ISRE/NF-κB promoters and modulation of interferon, ISGs and pro-inflammatory cytokine gene expression. These observations indicated that IRF3 and IRF7 play an important role in Asian seabass's antiviral defense and the RIG-IRF-IFN axis is conserved in the species.
Subject(s)
Fish Diseases/immunology , Fish Proteins/genetics , Fish Proteins/immunology , Gene Expression Regulation/immunology , Immunity, Innate/genetics , Perciformes/genetics , Perciformes/immunology , Amino Acid Sequence , Animals , Base Sequence , DNA Virus Infections/immunology , Fish Proteins/chemistry , Gene Expression Profiling/veterinary , Interferon Regulatory Factor-3/chemistry , Interferon Regulatory Factor-3/genetics , Interferon Regulatory Factor-3/immunology , Interferon Regulatory Factor-7/chemistry , Interferon Regulatory Factor-7/genetics , Interferon Regulatory Factor-7/immunology , Nodaviridae/physiology , Phylogeny , Sequence Alignment/veterinaryABSTRACT
Member of the dynamin family of large GTPases, dynamin-related protein 1 (Drp1) dependent mitochondrial fission is an intricate process regulating both cellular and organ dynamics. Present study shows that NNV perturbs mitochondrial dynamics by promoting Drp-1 dependent mitochondrial fission, which attenuates MAVS mediated downstream signaling. NNV infected SISS cells revealed induction in Drp1 expression and subsequent translocation into mitochondria. The level of MAVS expression was up-regulated over a period of 24 hpi and declined with the progression of NNV infection at 48 and 72 hpi confirmed by western blot and mRNA transcript analysis. Drp-1 displayed its association with fragmented mitochondria and the transcript abundance was significant post infection along with Mff. Expression levels of IRF-3 IFN-1 and Mx followed a similar pattern with abundant expression at 48 hpi and diminished expression during the further period. Importantly, silencing of Drp1 caused significant elevation in the RLR downstream molecules and reduction in viral RNA expression. These results suggest that NNV-induced mitochondrial fission serve to attenuate host RLR signaling. This provides an illustration of host-pathogen interaction in which the virus evades innate immunity by enhancing mitochondrial fission and perturbs MAVS, and the downstream molecules.
Subject(s)
DEAD Box Protein 58/immunology , Dynamins/immunology , Fish Diseases/immunology , Fish Proteins/immunology , Mitochondrial Dynamics/immunology , RNA Virus Infections/immunology , Animals , Bass , Cell Line , Nodaviridae , Reactive Oxygen Species/immunology , Signal Transduction , Spleen/cytologyABSTRACT
The innate immune signaling adapter, Mitochondrial antiviral signaling protein (MAVS) coordinates the signals received from two independent RLRs (RIG-1 and MDA5) to induce IFN & interferon stimulatory genes (ISGs). In the present study, we report identification of an orthologue of MAVS from Lates calcarifer (LcMAVS) and its functional role in piscine RLR signaling. The LcMAVS-cDNA was cloned into pcDNA and transfected into SISS cells. LcMAVS was detected to be a 61KDa protein in western blot. Confocal microscopy demonstrated the mitochondrial localization of LcMAVS. In addition, pcDNA-MAVS transfected cells were protected against Nervous Necrosis Virus (NNV) infection as manifested by the delayed appearance of cytopathic effect (CPE) and decreased viral transcript levels. Ectopic expression of LcMAVS resulted in activation of an ISRE-containing promoter (52 folds over control cells) as well as transcriptional expression of IRF-3, IFN-1 and IFN-inducible genes including Mx and ISG15 (p<0.05). These results suggest that LcMAVS is involved in the antiviral immunity as one of the adaptors in fish IFN-activation pathway.
Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Fish Diseases/immunology , Immunity, Innate/genetics , Nodaviridae/physiology , Perciformes/immunology , Animals , Bass/immunology , Fish Proteins/genetics , Fish Proteins/immunology , Host-Pathogen Interactions , RNA Virus Infections/immunologyABSTRACT
Galectin-9 is a b-galactoside-binding tandem repeat galectin that regulates many cellular functions, ranging from cell adhesion to pathogen recognition. In spite of extensive study of mammalian galectin importance in immune system, little is known about that of fish. To study the normal expression and immune response of Labeo rohita to pathogens, a tandem-repeat galectin-9 from Labeo rohita was identified and named LrGal-9. Its full-length cDNA was 1534 bp encoded 291 amino acids (35.12â¯KDa), shared the highest 81% identity with the galectin-9 of Danio rerio. LrGal-9 identified in this study lacked signal peptide and a transmembrane domain like galectin-9 members reported in other fishes. Quantitative PCR showed that LrGal-9 was lowly expressed in gill, muscle, heart, highly expressed in tested immune tissues (intestine, kidney, liver, spleen) in normal body. After Aeromonas hydrophila challenge, LrGal-9 was remarkably increased in all tested immune tissues in a time-dependent manner. These results suggest that LrGal-9 plays a role in innate immunity in Labeo rohita.