The Immunological Imprinting Hypothesis proposes that juvenile anadromous fish respond to the pathogen fingerprint specific to their natal site by producing protective long lived plasma cells (LLPCs) ...that constitutively produce antibodies against those pathogens. Hence, fish returning to their natal streams have immunological protection from pathogens at that specific location. Here, we tested the hypothesis through analysis of antibody composition and usage in sockeye salmon populations in Alaska. Spleen and anterior kidney were sampled from salmon from six sites, and relative usage levels of six different Immunoglobulin VH gene families determined using RT-qPCR. Additionally, prevalence and pathogen loads were measured in each fish for Renibacterium salmoninarum, Flavobacterium psychrophilum, and Infectious Hematopoietic Necrosis Virus. Results revealed differences in VH usage, pathogen loads, and infection rates between spawning sites, while probability of infection was dependent on location for each pathogen analyzed. Further, several negative correlations between specific VH usage patterns and pathogen loads were uncovered. Greater understanding of site-dependent VH usage in spawning fish potentially suggests a method of natural immunization against common fish pathogens and thus protection of both farmed and wild populations.
•Spawning sockeye salmon displayed differences in VH usage, pathogen loads, and infection rates between spawning sites.•Probability of infection was dependent on location for each pathogen analyzed.•Negative correlations between specific VH usage patterns and pathogen loads were uncovered.
The Immunological Imprinting Hypothesis proposes that juvenile anadromous fish respond to the pathogen fingerprint specific to their natal site by producing protective long lived plasma cells (LLPCs) ...that constitutively produce antibodies against those pathogens. Hence, fish returning to their natal streams have immunological protection from pathogens at that specific location. Here, we tested the hypothesis through analysis of antibody composition and usage in sockeye salmon populations in Alaska. Spleen and anterior kidney were sampled from salmon from six sites, and relative usage levels of six different Immunoglobulin V
gene families determined using RT-qPCR. Additionally, prevalence and pathogen loads were measured in each fish for Renibacterium salmoninarum, Flavobacterium psychrophilum, and Infectious Hematopoietic Necrosis Virus. Results revealed differences in V
usage, pathogen loads, and infection rates between spawning sites, while probability of infection was dependent on location for each pathogen analyzed. Further, several negative correlations between specific V
usage patterns and pathogen loads were uncovered. Greater understanding of site-dependent V
usage in spawning fish potentially suggests a method of natural immunization against common fish pathogens and thus protection of both farmed and wild populations.
The development of hematopoietic stem cell (HSCs) gene therapy for DNA repair disorders, such as Fanconi anemia and Bloom syndrome, is challenging because of the induction of HSCs apoptosis by ...cytokine stimulation. Although the Baboon envelope pseudotyped lentiviral vector (BaEV-Rless-LV) has been reported as a non-stimulatory gene transfer tool, the virus titer of BaEV-Rless-LV is too low for use in clinical applications. Transfected 293 T cells with helper plasmids, including the BaEV-Rless plasmid, showed morphological changes, such as syncytium formation and detachment. To establish a novel protocol for producing a high titer of BaEV-Rless-LV, we optimized three aspects of a basic virus production protocol by focusing on modifying culture conditions and the use of reagents: the virus titer increased 3-fold when the amount of BaEV-Rless plasmid was increased 1.2-fold; the highest titer was obtained when the viral supernatant was harvested at 48-h post-transfection, despite complete syncytium formation and detachment of the 293 T cells; and the use of poly-L-lysine-coated culture plates to enhance the adhesion and proliferation of 293 T cells and prevent detachment doubled the titer. Collectively, our novel protocol resulted in a 10-fold titer increase compared to the basic protocol and may be useful in clinical applications for treating DNA repair disorders.
Display omitted
•Hematopoietic stem cell gene therapy for DNA repair disorders is challenging.•BaEV-Rless pseudotyped lentiviral vector could solve the issue by cytokine depletion.•Although the titer is too low, novel virus production protocol is needed.•We modified protocol focusing on syncytium and detachment of transfected 293 T cells.•Our novel protocol resulted in a 10-fold titer increase compared to basic protocol.
-Thalassemia (AT) is one of the most commonly occurring inherited hematological diseases. However, few treatments are available, and allogeneic bone marrow transplantation (BMT) is the only ...available therapeutic option for patients with severe AT. Research into AT has remained limited due to a lack of adult mouse models, with severe AT typically resulting in in utero lethality. By using a lipid nanoparticle (LNP) targeting the receptor CD117 and delivering a Cre mRNA (mRNACreLNPCD117), we were able to delete floxed -globin genes at high efficiency in hematopoietic stem cells (HSC) ex vivo. These cells were then engrafted in the absence or presence of a novel α-globin expressing lentiviral vector (ALS20I). Myeloablated mice transplanted with mRNACreLNPCD117-treated HSC showed a complete knockout of -globin genes. They demonstrated a phenotype characterized by the synthesis of hemoglobin H (-tetramers, or HbH), aberrant erythropoiesis, and abnormal organ morphology, culminating in lethality approximately eight weeks following engraftment. Mice receiving mRNACreLNPCD117-treated HSC with at least one copy of ALS20I survived long-term with normalization of erythropoiesis, decreased the production of HbH, and ameliorated the abnormal organ morphology. Furthermore, we tested ALS20I in erythroid progenitors derived from -globin-KO CD34+ and cells isolated from patients with both deletional and non-deletional HbH disease, demonstrating improvement in -globin/-globin mRNA ratio and reduction in the formation of HbH by HPLC. Our results demonstrate the broad applicability of LNP for disease modeling, characterization of a novel severe mouse model of AT, and the efficacy of ALS20I for treating AT.
β-thalassemia is a disorder caused by altered hemoglobin protein synthesis and affects individuals worldwide. Severe forms of the disease, left untreated, can result in death before the age of 3 ...years (1). The standard of care consists of chronic and costly palliative treatment by blood transfusion combined with iron chelation. This dual approach suppresses anemia and reduces iron-related toxicities in patients. Allogeneic bone marrow transplant is an option, but limited by the availability of a highly compatible HSC donor. While gene therapy is been explored in several trials, its use is highly limited to developed regions with centers of excellence and well-established healthcare systems (2). Hence, there remains a tremendous unmet medical need to develop alternative treatment strategies for β-thalassemia (3). Occurrence of aberrant splicing is one of the processes that affects β-globin synthesis in β-thalassemia. The (C>G) IVS-2-745 is a splicing mutation within intron 2 of the β-globin gene. It leads to an aberrantly spliced mRNA that incorporates an intron fragment. This results in an in-frame premature termination codon that inhibits β-globin production. Here, we propose the use of uniform 2'-O-methoxyethyl (2'-MOE) splice switching oligos (SSOs) to reverse this aberrant splicing in the pre-mRNA. With these lead SSOs we show aberrant to wild type splice switching. This switching leads to an increase of adult hemoglobin (HbA) up to 80% in erythroid cells from patients with the IVS-2-745 mutation. Furthermore, we demonstrate a restoration of the balance between β-like- and α-globin chains, and up to an 87% reduction in toxic α-heme aggregates. While examining the potential benefit of 2'-MOE-SSOs in a mixed sickle-thalassemic phenotypic setting, we found reduced HbS synthesis and sickle cell formation due to HbA induction. In summary, 2'-MOE-SSOs are a promising therapy for forms of β-thalassemia caused by mutations leading to aberrant splicing.
Nursing notes in Electronic Health Records (EHR) contain critical health information, including fall risk factors. However, an exploration of fall risk prediction using nursing notes is not well ...examined. In this study, we explored deep learning architectures to predict fall risk in older adults using text in nursing notes and medications in the EHR. EHR predictor data and fall events outcome data were obtained from 162 older adults living at TigerPlace, a senior living facility located in Columbia, MO. We used pre-trained BioWordVec embeddings to represent the words in the clinical notes and medications and trained multiple recurrent neural network-based natural language processing models to predict future fall events. Our final model predicted falls with an accuracy of 0.81, a sensitivity of 0.75, a specificity of 0.83, and an F1 score of 0.82. This preliminary exploratory analysis provides supporting evidence that fall risk can be predicted from clinical notes and medications. Future studies will utilize additional data modalities available in the EHR to potentially improve fall risk prediction from EHR data.
The Immunological Imprinting Hypothesis proposes that juvenile anadromous fish respond to the pathogen fingerprint specific to their natal site by producing protective long lived plasma cells (LLPCs) ...that constitutively produce antibodies against those pathogens. Hence, fish returning to their natal streams have immunological protection from pathogens at that specific location. Here, we tested the hypothesis through analysis of antibody composition and usage in sockeye salmon populations in Alaska. Spleen and anterior kidney were sampled from salmon from six sites, and relative usage levels of six different Immunoglobulin VH gene families determined using RT-qPCR. Additionally, prevalence and pathogen loads were measured in each fish for Renibacterium salmoninarum, Flavobacterium psychro-philum, and Infectious Hematopoietic Necrosis Virus. Results revealed differences in VH usage, pathogen loads, and infection rates between spawning sites, while probability of infection was dependent on location for each pathogen analyzed. Further, several negative correlations between specific VH usage patterns and pathogen loads were uncovered. Greater understanding of site-dependent VH usage in spawning fish potentially suggests a method of natural immunization against common fish pathogens and thus protection of both farmed and wild populations.
Inherited monogenic disorders such as beta-hemoglobinopathies (BH) are fitting candidates for treatment via gene therapy by gene transfer or gene editing. The reported safety and efficacy of ...lentiviral vectors in preclinical studies have led to the development of several clinical trials for the addition of a functional beta-globin gene. Across trials, dozens of transfusion-dependent patients with sickle cell disease (SCD) and transfusion-dependent beta-thalassemia (TDT) have been treated via gene therapy and have achieved reduced transfusion requirements. While overall results are encouraging, the outcomes appear to be strongly influenced by the level of lentiviral integration in transduced cells after engraftment, as well as the underlying genotype resulting in thalassemia. In addition, the method of procurement of hematopoietic stem cells can affect their quality and thus the outcome of gene therapy both in SCD and TDT. This suggests that new studies aimed at maximizing the number of corrected cells with long-term self-renewal potential are crucial to ensure successful treatment for every patient. Recent advancements in gene transfer and bone marrow transplantation have improved the success of this approach, and the results obtained by using these strategies demonstrated significant improvement of gene transfer outcome in patients. The advent of new gene-editing technologies has suggested additional therapeutic options. These are primarily focused on correcting the defective beta-globin gene or editing the expression of genes or genomic segments that regulate fetal hemoglobin synthesis. In this review, we aim to establish the potential benefits of gene therapy for BH, to summarize the status of the ongoing trials, and to discuss the possible improvement or direction for future treatments.
PDF
