Missouri College

Myogenic differentiation (MyoD), a class II basic helix-loop-helix transcription factor, regulates multiple cell functions, including fibrosis in many organs, but remains unknown in the cornea. This study characterized the expression of MyoD in non-fibrotic and fibrotic rabbit and human donor corneas and investigated the effects of MyoD gene silencing on corneal myofibroblast dedifferentiation in vitro and fibrosis in vivo. New Zealand White rabbits, human donor corneas, human corneal stromal fibroblasts (CSFs), and human corneal myofibroblasts (CMFs) were used. MyoD shRNA or scrambled shRNA was delivered into CMFs via Lipofectamine 3000 and rabbit cornea via 2-kDa polyethylenimine conjugated to gold nanoparticles (PEI2-GNPs). Quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting quantified the expression of profibrotic genes, intermediate filaments, fibroblast-specific protein 1 (FSP1), and proliferation marker Ki67 in CMF-/+ MyoD gene silencing. MyoD expression was detected in human and rabbit corneal epithelium, stroma, and endothelium. MyoD levels were significantly greater in fibrotic human corneas (P < 0.001) and rabbit corneas (P < 0.0001) than corresponding non-fibrotic corneas. Also, CMFs had significantly greater MyoD levels than CSFs (P < 0.0001). MyoD shRNA-silenced CMFs acquired a characteristic fibroblast phenotype and showed reduced fibrotic genes, alpha smooth muscle actin (αSMA; P < 0.0001), fibronectin (FN; P = 0.0015), collagen type I (ColI; P = 0.0011), and collagen type IV (ColIV; P = 0.0053) mRNA levels compared to the scrambled shRNA-delivered CMFs. Further, immunofluorescence demonstrated increased vimentin (P < 0.001), FSP1 (P < 0.01), and Ki67 expression (P = 0.0349) and decreased desmin expression (P < 0.001) in MyoD shRNA-silenced CMFs than the scrambled shRNA-delivered CMFs. MyoD shRNA-delivered rabbit corneas had less scarring in vivo (P = 0.368). MyoD is expressed in cornea and could amend CMF dedifferentiation and fibrosis in an injured cornea. Additional studies are warranted to define the potential of MyoD in corneal fibrosis management.

Immune imprinting, in which prior antigenic exposures biases recall toward dominant epitopes, constrains the breadth and durability of influenza vaccine protection. Here we show that targeted variation across multiple hemagglutinin (HA) head sites (A, B, and D) between sequential A(H3N2) vaccines reprograms epitope hierarchy-redirecting recall toward conserved, subdominant head and stem epitopes. In a controlled ferret model mimicking imprinting-like recall in humans, antigenically distant priming accelerates neutralizing antibody induction, broadens reactivity, enhances cross-protection, and reduces viral shedding after drifted virus challenge. Epitope mapping and structural analysis confirms redirection toward conserved epitopes; single-cell transcriptomics and ELISpot assays reveal amplified germinal center B cell and Th1 responses. This "epitope hierarchy reshaping" links targeted antigenic variation to enhanced B cell competition, amplified T cell help, and improved viral control. This principle is likely applicable to vaccines against other rapidly evolving viruses where strong imprinting effects similarly limit immune breadth.

Aging is associated with colon epithelial barrier integrity and upregulation of myelopoiesis in the bone marrow (BM). Alamandine (Ala) and MrgD are novel members of the renin angiotensin system (RAS). This study tested the hypothesis that Ala restores the colon epithelial barrier integrity in aging via modulating gut-BM axis. Mice of age 2-3 (Young) or 22-24 months (Old) were treated with saline or Ala by using Osmotic pumps. The intestinal permeability was evaluated by using FITC-dextran. Lgr5+Olfm4+ intestinal stem cells (ISCs), Wnt3a and β-catenin were evaluated by immunohistochemistry or western blotting. Fecal microbiome was analyzed by 16S rRNA sequencing. Monocyte-macrophages were characterized by flow cytometry. Cecal or serum bacterial metabolites were analyzed. The pro-myelopoietic potential of cecal supernatants (CS) was tested in the Young-BM cells. MrgD was expressed in ISCs, which was decreased in the Old. Increased intestinal permeability in aging was reversed by Ala. In the colon organoids, Ala increased Wnt3a levels that were antagonized by the NF449, SQ22536 or 666-15. Ala restored phospho-CREB and active β-catenin levels that were decreased in the Old colon-organoids. Ala increased the richness and β-diversity of the aging microbiome and decreased Bacillota/Bacteroidota. Ala decreased the CD80+ and increased CX3CR+ cells in the Old colons. Old-CS induced myelopoiesis in vitro in BM cells with higher number of monocytes and pro-inflammatory macrophages which was not observed in the CS derived from Ala-treated Old mice. Ala is a promising pharmacological agent for reversing the leaky gut of aging by restoring homeostasis in the gut-BM axis.

Background/Objectives: Brucella is a major global One Health threat, causing an estimated 2.1 million human infections and substantial livestock losses annually, with no vaccine currently available for humans, underscoring the urgent need for a safe and effective vaccine. Methods: Employing a reverse vaccinology approach, a novel 175-mer multiepitope vaccine (Mvax) targeting Brucella FrpB was computationally designed in this study, incorporating two B-cell, two MHC class I (MHC-I), and three MHC class II (MHC-II) epitopes selected for their high predicted antigenicity, safety, and IFN-γ-inducing potential. Human β-defensin-3 (hBD3) was fused to the N-terminus as an adjuvant, followed by comprehensive in silico evaluation of the construct. Results: Population coverage analysis predicted 99.59% global MHC class I/II coverage for selected epitopes. In silico analyses predicted that Mvax has high solubility (Protein-SOL score: 0.808), a high antigenicity score (VaxiJen: 1.06), and a negative GRAVY index (−0.881), indicating favorable predicted physicochemical characteristics. iMODS, CABS-Flex 3, and molecular dynamics simulations suggested theoretical stability trends for the modeled vaccine complexes. C-ImmSim immune simulations further predicted elevated Th1 cell populations and associated cytokines (IL-12, IFN-γ, IL-2) following both single and multiple simulated Mvax exposures. Conclusions: The computational analyses described here provide a theoretical modeling basis for an antivirulence multi-epitope vaccine design against human brucellosis, with predicted metrics and simulated immune responses requiring empirical validation.

Canine extramedullary plasmacytomas are typically benign tumors of the skin, oral cavity, and alimentary tract that are cured by surgical excision. This tumor is rarely metastatic and aggressive. We report an unusual plasmacytoma in a dog that had been presented because of dyspnea. Aside from evidence of pleural effusion, no cutaneous lesions or other abnormalities were detected on physical examination. Nearly 2 L of pleural fluid were removed by thoracocentesis, and a sample was submitted for cytologic examination. The pleural fluid had increased protein and cell concentrations, with a predominance of individualized, large, round, atypical cells. Those cells frequently had Russell body-like intracytoplasmic structures, as seen on microscopic examination of modified Wright-stained concentrated slide preparations. Together, these findings were strongly supportive of a neoplastic plasma cell exudate. Immunohistochemical (CD3, CD20, MUM1, IgG, λ light chain) staining and B-cell PCR for antigen receptor rearrangement analysis performed on formalin-fixed, paraffin-embedded pleural cell-pellet sections confirmed a novel, clonal, IgG lambda extramedullary plasmacytoma with Mott cell differentiation that was most likely metastatic from a non-cutaneous primary site. Metastatic plasma cell neoplasia with voluminous serous cavity effusion carries a grave prognosis in humans, but has not been reported previously in dogs, to our knowledge.

PurposeMitochondria regulate cellular activity in a tissue-selective manner. The role of mitochondria in corneal fibrosis is elusive. This study investigated changes in mitochondrial DNA (mtDNA) and mitochondrial transcription factor A (TFAM) in human corneal stromal fibroblasts (CSFs) and corneal myofibroblast (CMFs) and effects on corneal fibrosis in vitro and ex vivo.MethodsHealthy donor human corneas were used to generate CSFs and ex vivo culture. CMF formation was induced by transforming growth factor beta-1 (TGFβ1) in vitro and human cornea by nitrogen mustard (NM) ex vivo. mtTFA/TFAM CRISPR/Cas9 KO plasmid, Lipofectamine CRISPRMAX, and TrueCut Cas9 Protein v2 were used for gene editing. Long-range PCR and quantitative reverse-transcription PCR (qRT-PCR) measured mtDNA transcription, mtDNA quantity, and ratios of mtDNA to nuclear DNA (nDNA). Immunofluorescence and immunoblotting quantified protein expression. The MitoSOX assay was used to analyze mitochondrial reactive oxygen species (mtROS).ResultsHuman CMFs showed significantly reduced mtDNA copies (P < 0.01) and mtDNA-to-nDNA ratios (P < 0.05) compared to CSFs. Significant time-dependent increases in mRNA levels of α-smooth muscle actin (αSMA) and nDNA-transcribed genes and decreases in TFAM and mtDNA-transcribed genes were noted during CSF transdifferentiation to CMFs (P < 0.05, P < 0.001, or P < 0.0001). Correspondingly, time-dependent decreases in TFAM and increases in Rieske iron–sulfur (Fe-S) and αSMA protein (P < 0.0001) and mtROS and ROS levels (P < 0.0001) were observed. TFAM silencing arrested fibrotic events and exhibited reduced αSMA and enhanced mtDNA (P < 0.001). The NM-induced fibrotic human cornea showed decreased TFAM and increased αSMA compared to naïve corneas (P < 0.01).ConclusionsWe observed that mtDNA plays an important role in corneal fibroblast transdifferentiation to myofibroblast and that TFAM has the potential to modulate this process in an injured cornea. Additional studies are warranted.

Clostridioides difficile infection (CDI) is a toxin-mediated gastro-intestinal disease. Yet, C. difficile is a phylogenetically diverse species that includes many non-toxigenic strains. In general, these are understudied, despite having significant potential impact for our understanding of the colonization process and as therapeutic modalities. Here, we present an in-depth characterization-including the complete genome sequence-of the non-toxigenic C. difficile strain L-NTCD03. This strain belongs to PCR ribotype 416, clade 4 and multilocus sequence type 39. It is resistant to multiple antimicrobials, but not those used for treatment of CDI. We validated the relevance of the cfr(B) gene from this strain in antimicrobial resistance to clindamycin, linezolid, retapamulin, and streptogramin A. We found the L-NTCD03 strain to be non-toxic in various assays. Altogether, L-NTCD03 is a promising candidate for developing into a live biotherapeutic product.

Serotonin reuptake inhibitors (SSRIs) are commonly prescribed to pregnant women experiencing depression. Such drugs, however, might adversely affect placenta and fetal brain development. Parietal trophoblast giant cells (pTGCs) in the mouse placenta are postulated to internalize maternal serotonin (5-HT) via transport through SERT, encoded by Slc6a4, and to provide the initial source of 5-HT to the emerging brain via the placental-brain axis. Genetic deletion of Slc6a4 in pTGCs has been hypothesized to impact placental and fetal brain development. A transgenic mouse line with high-affinity SERT, encoded by Slc6a4, was selectively deleted by pairing mice with Cre recombinase linked to Prl2c2, with LoxP sites flanking the Slc6a4 gene. PRL2C2 is solely expressed by pTGCs and other giant cells of the placenta. To compare placental and fetal brain development in selective Slc6a4 KO and WT mice, 5-HT content in the placenta and fetal brains of conceptuses was measured. No significant differences in 5-HT content were evident between knockout (KO) and wild-type (WT) placentas or fetal brains. However, there were significantly fewer pTGCs in KO placentas compared to WT (p ≤ 0.05). Sexually dimorphic differences in gene expression were evident in the placenta and fetal brain between KO and WT counterparts, with female conceptuses showing the most dramatic responses, including decrease in Prl7a2, Prl5a1, Prl3a1, Slc28a3, and Ceacam 15 in female placental samples. These findings suggest that ablation of Slc6a4 in pTGC disrupts the placenta-brain axis in a sex-dependent manner. The results might have important clinical ramifications for pregnant women being treated with SSRIs.

The management of diabetes mellitus (DM) in cats can benefit from an integration of medical and nutritional strategies, based on an understanding of the dynamic nature of the disease, together with an appreciation of the interrelationships between nutritional status and clinical status. In this context, a new classification system for feline DM is proposed, comprising three clinical status categories: those at risk of developing DM, those with clinical DM and those in diabetic remission. The influence of individual dietary components (carbohydrate, protein, fat and water fractions) on clinical outcomes is discussed, followed by overarching principles for the dietary management of diabetic cats, based on both the limited scientific evidence and the clinical experience of the authors. Key aspects of nutritional assessment, the principles of therapeutic weight reduction and feeding practices are covered. Using the classification framework, recommendations for nutritional management are proposed for cats at risk of development of DM, cats with clinical DM and those in clinical remission. Successful implementation of these recommendations can be achieved by using a relationship-centred approach, where owner concerns are addressed and goals for management are agreed in partnership. It is hoped that these perspectives will help guide veterinary professionals in their clinical management decisions, thereby improving health outcomes in cats in all three categories of DM.