Biology subjects

Kiupel, M.

Publications and source records attributed to Kiupel, M..

7 recordsLinked to original sources

bioRxiv2024

Material Composition and Implantation Site Affect in vivo Device Degradation Rate

Successful tissue engineering requires biomedical devices that initially stabilize wounds, then degrade as tissue is regenerated. However, the material degradation rates reported in literature are often conflicting. Incorporation of in situ monitoring functionality into implanted devices would allow real time assessment of degradation and potential failure. This necessitates introduction of contrast agent as most biomedical devices are composed of polymeric materials with no inherent contrast in medical imaging modalities. In the present study, computed tomography (CT)-visible radiopaque composites were created by adding 5-20wt% tantalum oxide (TaOx) nanoparticles into polymers with distinct degradation profiles: polycaprolactone (PCL), poly(lactide-co-glycolide) (PLGA) 85:15 and PLGA 50:50, representing slow, medium and fast degrading materials respectively. Radiopaque phantoms, mimicking porous tissue engineering devices, were implanted into mice intramuscularly or intraperitoneally, and monitored via CT over 20 weeks. Changes in phantom volume, including collapse and swelling, were visualized over time. Phantom degradation profile was determined by polymer matrix, regardless of nanoparticle addition and foreign body response was dictated by the implant site. In addition, degradation kinetics were significantly affected in mid-degrading materials, transitioning from linear degradation intramuscularly to exponential degradation intraperitoneally, due to differences in inflammatory responses and fluid flow. Nanoparticle excretion from degraded phantoms lagged behind polymer, and future studies will modulate nanoparticle clearance. Utilizing in situ monitoring, this study seeks to unify literature and facilitate better tissue engineering devices, by highlighting the relative effect of composition and implant site on important materials properties.

bioengineering↗

bioRxiv2024

Effect of Genotype and Age on a Defined Microbiota in Gnotobiotic SCID Piglets

Severe combined immunodeficient (SCID) individuals lack functional T and B lymphocytes, leading to a deficient adaptive immune system. SCID pigs are a unique large animal biomedical model as they possess many similarities to humans, allowing for the collection of translatable data in regenerative medicine, cancer, and other biomedical research topics. While many studies suggest early gut microbiota development is necessary for developing the intestinal barrier and immune system, these animals are often cesarian section derived, leaving them uncolonized for normal intestinal microflora. The hypothesis was that an increase in complexity of microbiota inoculum will allow for more stability in the composition of the gut microbiota of SCID piglets. This was tested across multiple litters of SCID piglets with three different defined microbiota consortium (2-strain, 6-strain, 7-strain). All piglets received their designated defined microbiota by oral gavage immediately after birth and again 24 hours later. There was no effect of SCID genotype on the composition of the gut microbiota, but there was a significant effect due to piglet age. Additionally, all three defined microbiota consortia were deemed safe to use in SCID piglets, and the 7-strain microbiota was the most stable over time. Based on these results, the 7-strain defined microbiota will be added to the SCID pig husbandry protocol, allowing for a more reproducible model.

genetics↗

bioRxiv2024

Engineered endosymbionts that modulate primary macrophage function and attenuate tumor growth by shifting the tumor microenvironment

Modulating gene expression in macrophages can be used to improve tissue regeneration and to redirect tumor microenvironments (TME) toward positive therapeutic outcomes. We have developed Bacillus subtilis as an engineered endosymbiont (EES) capable of residing inside the eukaryotic host cell cytoplasm and controlling the fate of macrophages. Secretion of mammalian transcription factors (TFs) from B. subtilis that expresses listeriolysin O (LLO; allowing the EES to escape destruction by the macrophage) modulated expression of surface markers, cytokines and chemokines, indicating functional changes in a macrophage/monocyte cell line. The engineered B. subtilis LLO TF strains were evaluated in murine bone marrow-derived macrophages (BMDMs) by flow cytometry, chemokine/cytokine profiling, metabolic assays and RNA-Seq. Delivery of TFs by the EES shifted BMDM gene expression, production of cytokine and chemokines and metabolic patterns, indicating that the TF strains could guide primary macrophage function. Thereafter, the ability of the TF strains to alter the TME was characterized in vivo, in an orthotopic murine model of triple-negative breast cancer to assess therapeutic effects. The TF strains altered the TME by shifting immune cell composition and attenuating tumor growth. Additionally, multiple doses of the TF strains were well-tolerated by the mice. The use of B. subtilis LLO TF strains as EES showed promise as a unique cancer immunotherapy by directing immune function intracellularly. The uses of EES could be expanded to modulate other mammalian cells over a range of biomedical applications.

synthetic biology↗

bioRxiv2023

In vivo Biomedical Imaging of Immune Tolerant, Radiopaque Nanoparticle-Embedded Polymeric Device Degradation

Biomedical implants remain an important clinical tool for restoring patient mobility and quality of life after trauma. While polymers are often used for devices, their degradation profile remains difficult to determine post-implantation. CT monitoring could be a powerful tool for in situ monitoring of devices, but polymers require the introduction of radiopaque contrast agents, like nanoparticles, to be distinguishable from native tissue. As device function is mediated by the immune system, use of radiopaque nanoparticles for serial monitoring therefore requires a minimal impact on inflammatory response. Radiopaque polymer composites were produced by incorporating 0-20wt% TaOx nanoparticles into synthetic polymers: polycaprolactone (PCL) and poly(lactide-co-glycolide) (PLGA). In vitro inflammatory response to TaOx was determined by monitoring mouse bone marrow derived macrophages on composite films. Nanoparticle addition stimulated only a slight inflammatory reaction, namely increased TNF secretion, mediated by changes to the polymer matrix properties. When devices (PLGA 50:50 + 20wt% TaOx) were implanted subcutaneously in a mouse model of chronic inflammation, no changes to device degradation were noted although macrophage number was increased over 12 weeks. Serial CT monitoring of devices post-implantation provided a detailed timeline of device structural collapse, with no burst release of the nanoparticles from the implant. Changes to the device were not significantly altered with monitoring, nor was the immune system ablated when checked via blood cell count and histology. Thus, polymer devices incorporating radiopaque TaOx NPs can be used for in situ CT monitoring, and can be readily combined with multiple medical imaging techniques, for a truly dynamic view biomaterials interaction with tissues throughout regeneration, paving the way for a more structured approach to biomedical device design.

bioengineering↗

bioRxiv2022

Bacterial Infection of the Placenta Induces Sex-Specific Responses in the Fetal Brain

BACKGROUNDEpidemiological data indicate that prenatal infection is associated with an increased risk of several neurodevelopmental disorders in the progeny. These disorders display sex differences in presentation. The role of the placenta, which is a target of prenatal infection, in the sex-specificity of neurodevelopmental abnormalities is unknown. We used an imaging-based animal model of the bacterial pathogen Listeria monocytogenes to identify sex-specific effects of placental infection on neurodevelopment of the fetus. METHODSPregnant CD1 mice were infected with a bioluminescent strain of Listeria on embryonic day 14.5 (E14.5). Excised fetuses were imaged on E18.5 to identify the infected placentas. The associated fetal brains were analyzed for gene expression and altered brain structure due to infection. The behavior of adult offspring affected by prenatal Listeria infection was analyzed. RESULTSPlacental infection induced sex-specific alteration of gene expression patterns in the fetal brain and resulted in abnormal cortical development correlated with placental infection levels. Furthermore, male offspring exhibited abnormal social interaction, whereas females exhibited elevated anxiety. CONCLUSIONPlacental infection by Listeria induced sex-specific abnormalities in neurodevelopment of the fetus. Prenatal infection also affected the behavior of the offspring in a sex-specific manner. ImpactO_LIPlacental infection with Listeria monocytogenes induces sexually dichotomous gene expression patterns in the fetal brain. C_LIO_LIAbnormal cortical lamination is correlated with placental infection levels. C_LIO_LIPlacental infection results in autism related behavior in male offspring and heightened anxiety level in female offspring. C_LI

microbiology↗

bioRxiv2021

Computer-Assisted Mitotic Count Using a Deep Learning-based Algorithm Improves Inter-Observer Reproducibility and Accuracy in canine cutaneous mast cell tumors

The mitotic count (MC) is an important histological parameter for prognostication of malignant neoplasms. However, it has inter- and intra-observer discrepancies due to difficulties in selecting the region of interest (MC-ROI) and in identifying/classifying mitotic figures (MFs). Recent progress in the field of artificial intelligence has allowed the development of high-performance algorithms that may improve standardization of the MC. As algorithmic predictions are not flawless, the computer-assisted review by pathologists may ensure reliability. In the present study we have compared partial (MC-ROI preselection) and full (additional visualization of MF candidate proposal and display of algorithmic confidence values) computer-assisted MC analysis to the routine (unaided) MC analysis by 23 pathologists for whole slide images of 50 canine cutaneous mast cell tumors (ccMCTs). Algorithmic predictions aimed to assist pathologists in detecting mitotic hotspot locations, reducing omission of MF and improving classification against imposters. The inter-observer consistency for the MC significantly increased with computer assistance (interobserver correlation coefficient, ICC = 0.92) compared to the unaided approach (ICC = 0.70). Classification into prognostic stratifications had a higher accuracy with computer assistance. The algorithmically preselected MC-ROIs had a consistently higher MCs than the manually selected MC-ROIs. Compared to a ground truth (developed with immunohistochemistry for phosphohistone H3), pathologist performance in detecting individual MF was augmented when using computer assistance (F1-score of 0.68 increased to 0.79) with a reduction in false negatives by 38%. The results of this study prove that computer assistance may lead to a more reproducible and accurate MCs in ccMCTs.

pathology↗

bioRxiv2019

Novel engraftment and T cell differentiation of human hematopoietic cells in Art-/- IL2RG-/ SCID pigs

Pigs with severe combined immunodeficiency (SCID) are an emerging biomedical animal model. Swine are anatomically and physiologically more similar to humans than mice, making them an invaluable tool for preclinical regenerative medicine and cancer research. One essential step in further developing this model is the immunological humanization of SCID pigs. In this work we have generated T- B- NK- SCID pigs through site directed CRISPR/Cas9 mutagenesis of IL2RG within a naturally occurring DCLRE1C (Artemis)-/- genetic background. We confirmed Art-/- IL2RG-/Y pigs lacked T, B, and NK cells in both peripheral blood and lymphoid tissues. Additionally, we and successfully performed a bone marrow transplant on one Art-/- IL2RG-/Y male SCID pig with a bone marrow from a complete swine leukocyte antigen (SLA) matched donor without conditioning to reconstitute porcine T and NK cells. Next, we performed in utero injections of cultured human CD34+ selected cord blood cells into the fetal Art-/- IL2RG-/Y SCID pigs. At birth, human CD45+ CD3{varepsilon}+ cells were detected in peripheral blood of in utero injected SCID piglets. Human leukocytes were also detected within the bone marrow, spleen, liver, thymus, and mesenteric lymph nodes of these animals. Taken together, we describe critical steps forwards the development of an immunologically humanized SCID pig model.\n\nOne sentence summaryWe have generated a T- B- NK- SCID pig model through site directed mutagenesis of IL2RG in a naturally occurring Artemis null background and show successful engraftment of human T and B cells in blood and lymphoid organs after in utero injection of human hematopoietic stem cells.

immunology↗