Local FK506 delivery induces osteogenesis in rat bone defect and rabbit spine fusion models.

Bone grafting procedures are commonly used for the repair, regeneration, and fusion of bones in a wide range of orthopaedic surgeries, including large bone defects and spine fusion procedures. Autografts are the clinical gold standard, though recombinant human bone morphogenetic proteins (rhBMPs) are often used, particularly in difficult clinical situations. However, treatment with rhBMPs can have off-target effects and increase surgical costs, adding to patients' already high economic and mental burden. Recent studies have identified that FDA-approved immunosuppressant drug, FK506 (Tacrolimus), can also activate the BMP pathway by binding to its inhibitors. This study tested the hypothesis that FK506, as a standalone treatment, could induce osteogenic differentiation of human mesenchymal stromal cells (hMSCs), as well as functional bone formation in a rat segmental bone defect model and rabbit spinal fusion model. FK506 enhanced osteogenic differentiation and mineralization of hMSCs in vitro. Standalone treatment with FK506 delivered on a collagen sponge produced consistent bone bridging of a critically sized rat femoral defect with functional mechanical properties comparable to naïve bone. In a rabbit single level posterolateral spine fusion model, treatment with FK506 delivered on a collagen sponge successfully fused the L5-L6 vertebrae at rates comparable to rhBMP-2 treatment. These data demonstrate the ability of FK506 to induce bone formation in human cells and two challenging in vivo models, and indicate FK506 can be utilized to treat a variety of spine disorders.

Local FK506 delivery induces osteogenesis in in vivo rat bone defect and rabbit spine fusion models.

Bone grafting procedures are commonly used for the repair, regeneration, and fusion of bones in in a wide range of orthopaedic surgeries, including large bone defects and spine fusion procedures. Autografts are the clinical gold standard, though recombinant human bone morphogenetic proteins (rhBMPs) are often used, particularly in difficult clinical situations. However, treatment with rhBMPs can have off-target effects and significantly increase surgical costs, adding to patients' already high economic and mental burden. Recent studies have identified that FDA-approved immunosuppressant drug, FK506 (Tacrolimus), can also activate the BMP pathway by binding to its inhibitors. This study tested the hypothesis that FK506, as a standalone treatment, could induce osteogenic differentiation of human mesenchymal stromal cells (hMSCs), as well as functional bone formation in a rat segmental bone defect model and rabbit spinal fusion model. FK506 potentiated the effect of low dose BMP-2 to enhance osteogenic differentiation and mineralization of hMSCs in vitro. Standalone treatment with FK506 delivered on a collagen sponge, produced consistent bone bridging of a rat critically-sized femoral defect with functional mechanical properties comparable to naïve bone. In a rabbit single level posterolateral spine fusion model, treatment with FK506 delivered on a collagen sponge successfully fused the L5-L6 vertebrae at rates comparable to rhBMP-2 treatment. These data demonstrate the ability of FK506 to induce bone formation in human cells and two challenging in vivo models, and indicate FK506 can be utilized either as a standalone treatment or in conjunction with rhBMP to treat a variety of spine disorders.

Gene-dosage- and sex-dependent differences in the prodromal-Like phase of the F344tgHD rat model for Huntington disease.

In Huntington disease (HD) the prodromal phase has been increasingly investigated and is currently in focus for early interventional treatments. Also, the influence of sex on disease progression and severity in patients is under discussion, as a sex-specific impact has been reported in transgenic rodent models for HD. To this end, we have been studying these aspects in Sprague Dawley rats transgenic for HD. Here, we took up on the congenic F344tgHD rat model, expressing a fragmented Htt construct with 51 CAG repeats on an inbred F344 rat background and characterized potential sexual dimorphism and gene-dosage effects in rats during the pre-symptomatic phase (1-8 months of age). Our study comprises a longitudinal phenotyping of motor function, emotion and sensorimotor gating, as well as screening of metabolic parameters with classical and automated assays in combination with investigation of molecular HD hallmarks (striatal cell number and volume estimation, appearance of HTT aggregates). Differences between sexes became apparent during middle age, particularly in the motor and sensorimotor domains. Female individuals were generally more active, demonstrated different gait characteristics than males and less anxiolytic-like behavior. Alterations in both the time course and affected behavioral domains varied between male and female F344tgHD rats. First subtle behavioral anomalies were detected in transgenic F344tgHD rats prior to striatal MSN cell loss, revealing a prodromal-like phase in this model. Our findings demonstrate that the congenic F344tgHD rat model shows high face-validity, closely resembling the human disease's temporal progression, while having a relatively low number of CAG repeats, a slowly progressing pathology with a prodromal-like phase and a comparatively subtle phenotype. By differentiating the sexes regarding HD-related changes and characterizing the prodromal-like phase in this model, these findings provide a foundation for future treatment studies.

Young human alpha synuclein transgenic (BAC-SNCA) mice display sex- and gene-dose-dependent phenotypic disturbances.

Parkinson's disease (PD) is a common neurodegenerative movement disorder, characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of aggregated alpha synuclein (aSyn). The disease often presents with early prodromal non-motor symptoms and later motor symptoms. Diagnosing PD based purely on motor symptoms is often too late for successful intervention, as a significant neuronal loss has already occurred. Furthermore, the lower prevalence of PD in females is not well understood, highlighting the need for a better understanding of the interaction between sex and aSyn, the crucial protein for PD pathogenesis. Here, we conducted a comprehensive phenotyping study in 1- to 5-month-old mice overexpressing human aSyn gene (SNCA) in a bacterial artificial chromosome (BAC-SNCA). We demonstrate a SNCA gene-dose-dependent increase of human aSyn and phosphorylated aSyn, as well as a decrease in tyrosine hydroxylase expression in BAC-SNCA mice, with more pronounced effects in male mice. Phosphorylated aSyn was already found in the dorsal motor nucleus of the vagus nerve of 2-month-old mice. This was time-wise associated with significant gait altrations in BAC-SNCA mice as early as 1 and 3 months of age using CatWalk gait analysis. Furthermore, anxiety-related behavioral tests revealed an increase in anxiety levels in male BAC-SNCA mice. Finally, 5-month-old male BAC-SNCA mice exhibited a SNCA gene-dose-dependent elevation in energy expenditure in automated home-cage monitoring. For the first time, these findings describe early-onset, sex- and gene-dose-dependent, aSyn-mediated disturbances in BAC-SNCA mice, providing a model for sex-differences, early-onset neuropathology, and prodromal symptoms of PD.

Early Resistance Rehabilitation Improves Functional Regeneration Following Segmental Bone Defect Injury.

Mechanical loading is integral to bone development and repair. The application of mechanical loads through rehabilitation are regularly prescribed as a clinical aide following severe bone injuries. However, current rehabilitation regimens typically involve long periods of non-loading and rely on subjective patient feedback, leading to muscle atrophy and soft tissue fibrosis. While many pre-clinical studies have focused on unloading, ambulatory loading, or direct mechanical compression, rehabilitation intensity and its impact on the local strain environment and subsequent bone healing have largely not been investigated. This study combines implantable strain sensors and subject-specific finite element models in a pre-clinical rodent model with a defect size on the cusp of critically-sized. Animals were enrolled in either high or low intensity rehabilitation one week post injury to investigate how rehabilitation intensity affects the local mechanical environment and subsequent functional bone regeneration. The high intensity rehabilitation animals were given free access to running wheels with resistance, which increased local strains within the regenerative niche by an average of 44% compared to the low intensity (no-resistance) group. Finite element modeling demonstrated that resistance rehabilitation significantly increased compressive strain by a factor of 2.0 at week 1 and 4.45 after 4 weeks of rehabilitation. The resistance rehabilitation group had significantly increased regenerated bone volume and higher bone bridging rates than its sedentary counterpart (bone volume: 22.00 mm3 ± 4.26 resistance rehabilitation vs 8.00 mm3 ± 2.27 sedentary; bridging rates: 90% resistance rehabilitation vs 50% sedentary). In addition, animals that underwent resistance running had femurs with improved mechanical properties compared to those left in sedentary conditions, with failure torque and torsional stiffness values matching their contralateral, intact femurs (stiffness: 0.036 Nm/deg ± 0.006 resistance rehabilitation vs 0.008 Nm/deg ± 0.006 sedentary). Running on a wheel with no resistance rehabilitation also increased bridging rates (100% no resistance rehabilitation vs 50% sedentary). Analysis of bone volume and von Frey suggest no-resistance rehabilitation may improve bone regeneration and hindlimb functionality. These results demonstrate the potential for early resistance rehabilitation as a rehabilitation regimen to improve bone regeneration and functional recovery.

Systemic Immune Modulation Alters Local Bone Regeneration in a Delayed Treatment Composite Model of Non-Union Extremity Trauma.

Bone non-unions resulting from severe traumatic injuries pose significant clinical challenges, and the biological factors that drive progression towards and healing from these injuries are still not well understood. Recently, a dysregulated systemic immune response following musculoskeletal trauma has been identified as a contributing factor for poor outcomes and complications such as infections. In particular, myeloid-derived suppressor cells (MDSCs), immunosuppressive myeloid-lineage cells that expand in response to traumatic injury, have been highlighted as a potential therapeutic target to restore systemic immune homeostasis and ultimately improve functional bone regeneration. Previously, we have developed a novel immunomodulatory therapeutic strategy to deplete MDSCs using Janus gold nanoparticles that mimic the structure and function of antibodies. Here, in a preclinical delayed treatment composite injury model of bone and muscle trauma, we investigate the effects of these nanoparticles on circulating MDSCs, systemic immune profiles, and functional bone regeneration. Unexpectedly, treatment with the nanoparticles resulted in depletion of the high side scatter subset of MDSCs and an increase in the low side scatter subset of MDSCs, resulting in an overall increase in total MDSCs. This overall increase correlated with a decrease in bone volume (P = 0.057) at 6 weeks post-treatment and a significant decrease in mechanical strength at 12 weeks post-treatment compared to untreated rats. Furthermore, MDSCs correlated negatively with endpoint bone healing at multiple timepoints. Single cell RNA sequencing of circulating immune cells revealed differing gene expression of the SNAb target molecule S100A8/A9 in MDSC sub-populations, highlighting a potential need for more targeted approaches to MDSC immunomodulatory treatment following trauma. These results provide further insights on the role of systemic immune dysregulation for severe trauma outcomes in the case of non-unions and composite injuries and suggest the need for additional studies on targeted immunomodulatory interventions to enhance healing.

Bacterial urinary tract infection and subclinical bacteriuria in dogs receiving antineoplastic chemotherapy.

BACKGROUND: Immunosuppressive treatment with glucocorticoids and cyclosporine increases the risk for positive urine cultures (PUCs) in dogs. OBJECTIVE: To investigate the prevalence and incidence of PUC in dogs diagnosed with cancer and treated with antineoplastic chemotherapy while distinguishing between subclinical bacteriuria (SB) and urinary tract infection (UTI). ANIMALS: Forty-six client-owned dogs with nonurogenital cancer treated with antineoplastic chemotherapy. METHODS: Prospective observational longitudinal clinical study. Dogs in which a urine culture was performed before the start of and at least once during antineoplastic chemotherapy were included. A McNemar's test was used to investigate if the prevalence of PUC increased during antineoplastic chemotherapy. Positive urine cultures were categorized into SB and UTI and multiple PUCs from the same dog and category were grouped together as 1 episode of PUC. RESULTS: Urine culture was positive in 21/185 urine samples in 8/46 dogs. Antineoplastic chemotherapy did not influence the prevalence of PUC (P = 1.00), which was 11% (5/46 dogs; 95% confidence interval: 5-23%) before the start of and 13% (6/46 dogs; 95% confidence interval: 6-26%) during antineoplastic chemotherapy. Eight dogs had 10 episodes of PUC; 7/10 episodes were classified as SB, and in 3/10 episodes UTI (chronic prostatitis, prostatic abscess, and emphysematous cystitis) was diagnosed. Escherichia coli was the most common pathogen, isolated in 9/10 episodes. CONCLUSIONS AND CLINICAL IMPORTANCE: We did not find evidence that antineoplastic chemotherapy is a major predisposing factor for the development of PUC. Most dogs with PUC had SB.

Neutrophil and natural killer cell imbalances prevent muscle stem cell-mediated regeneration following murine volumetric muscle loss.

Volumetric muscle loss (VML) overwhelms the innate regenerative capacity of mammalian skeletal muscle (SkM), leading to numerous disabilities and reduced quality of life. Immune cells are critical responders to muscle injury and guide tissue resident stem cell­ and progenitor-mediated myogenic repair. However, how immune cell infiltration and intercellular communication networks with muscle stem cells are altered following VML and drive pathological outcomes remains underexplored. Herein, we contrast the cellular and molecular mechanisms of VML injuries that result in the fibrotic degeneration or regeneration of SkM. Following degenerative VML injuries, we observed the heightened infiltration of natural killer (NK) cells as well as the persistence of neutrophils beyond 2 wk postinjury. Functional validation of NK cells revealed an antagonistic role in neutrophil accumulation in part via inducing apoptosis and CCR1-mediated chemotaxis. The persistent infiltration of neutrophils in degenerative VML injuries was found to contribute to impairments in muscle stem cell regenerative function, which was also attenuated by transforming growth factor beta 1 (TGFß1). Blocking TGFß signaling reduced neutrophil accumulation and fibrosis and improved muscle-specific force. Collectively, these results enhance our understanding of immune cell­stem cell cross talk that drives regenerative dysfunction and provide further insight into possible avenues for fibrotic therapy exploration.

Transglutaminase 6 Is Colocalized and Interacts with Mutant Huntingtin in Huntington Disease Rodent Animal Models.

Mammalian transglutaminases (TGs) catalyze calcium-dependent irreversible posttranslational modifications of proteins and their enzymatic activities contribute to the pathogenesis of several human neurodegenerative diseases. Although different transglutaminases are found in many different tissues, the TG6 isoform is mostly expressed in the CNS. The present study was embarked on/undertaken to investigate expression, distribution and activity of transglutaminases in Huntington disease transgenic rodent models, with a focus on analyzing the involvement of TG6 in the age- and genotype-specific pathological features relating to disease progression in HD transgenic mice and a tgHD transgenic rat model using biochemical, histological and functional assays. Our results demonstrate the physical interaction between TG6 and (mutant) huntingtin by co-immunoprecipitation analysis and the contribution of its enzymatic activity for the total aggregate load in SH-SY5Y cells. In addition, we identify that TG6 expression and activity are especially abundant in the olfactory tubercle and piriform cortex, the regions displaying the highest amount of mHTT aggregates in transgenic rodent models of HD. Furthermore, mHTT aggregates were colocalized within TG6-positive cells. These findings point towards a role of TG6 in disease pathogenesis via mHTT aggregate formation.

Comprehensive phenotyping revealed transient startle response reduction and histopathological gadolinium localization to perineuronal nets after gadodiamide administration in rats.

Gadolinium based contrast agents (GBCAs) are widely used in clinical MRI since the mid-1980s. Recently, concerns have been raised that trace amounts of Gadolinium (Gd), detected in brains even long time after GBCA application, may cause yet unrecognized clinical consequences. We therefore assessed the behavioral phenotype, neuro-histopathology, and Gd localization after repeated administration of linear (gadodiamide) or macrocyclic (gadobutrol) GBCA in rats. While most behavioral tests revealed no difference between treatment groups, we observed a transient and reversible decrease of the startle reflex after gadodiamide application. Residual Gd in the lateral cerebellar nucleus was neither associated with a general gene expression pathway deregulation nor with neuronal cell loss, but in gadodiamide-treated rats Gd was associated with the perineuronal net protein aggrecan and segregated to high molecular weight fractions. Our behavioral finding together with Gd distribution and speciation support a substance class difference for Gd presence in the brain after GBCA application.

[Disseminated protothecosis with ulcerative granulomatous colitis in a Rhodesian Ridgeback from Germany]. / Ulzerative granulomatöse Kolitis durch Prototheca spp. bei einem Rhodesian Ridgeback in Deutschland.

A 10-month-old male Rhodesian Ridgeback was presented to the Clinic of Small Animal Medicine, LMU, Germany, with a 6-month history of chronic diarrhea and hematochezia. The dog lived in Germany and had never traveled abroad. Complete blood count and serum biochemistry performed by the referring veterinarian revealed neutrophilia, hyperkalemia, and hyponatremia, with a basal cortisol of 4.3 µg/dl, which excluded hypoadrenocorticism. Since antibiotic treatment had not resulted in any improvement, a 2 week course of prednisolone administration had been initiated, leading to a marked deterioration of intestinal signs and a significant weight loss of 6 kg. At the time of referral, the patient was markedly emaciated, dehydrated, hypovolemic and had a rectal temperature of 39.6 °C. Abdominal ultrasound showed a thickened and irregular colonic wall. On colonoscopy, an irregular colonic mucosa with ulcerations was observed. Histopathologic examination revealed an ulcerative granulomatous colitis, and on Periodic acid-Schiff reaction (PAS) numerous organisms consistent with Prototheca spp. were identified. Prototheca zopfii infection was confirmed by culture and MALDI-TOF MS. In order to test for an underlying immunodeficiency, immunoglobulin levels in serum were determined. IgM was decreased, while IgG and IgA levels were within the reference interval. Due to deterioration of general condition, grave prognosis and costs of a treatment trial, the patient was euthanized one week later, and necropsy was performed. Prototheca spp. were detected on histopathologic examination in the lymphnodes, however not in the eyes or the central nervous system. Protothecosis should be considered an differential diagnosis in dogs with chronic diarrhea and ulcerative granulomatous colitis even in dogs living in Germany. Histopathologic examination of colonic biopsies with special stains such as PAS is recommended in every dog with signs of chronic large bowel disease in order to avoid missing this rare infectious disease.

[Bacterial urinary tract infection and subclinical bacteriuria in the dog: a current review]. / Bakterielle Harnwegsinfektion und subklinische Bakteriurie des Hundes: eine aktuelle Übersicht.

Bacterial urinary tract infection (UTI) is a common clinical presentation in dogs and a frequent reason for the prescription of antimicrobial drugs. UTI refers to adherence, multiplication and persistence of an infectious agent within the urogenital system. This causes an associated inflammatory response as well as the pertaining clinical signs. Depending on the site of infection, UTI's may be classified as bacterial cystitis, prostatitis or pyelonephritis. In contrast, subclinical bacteriuria (SB) is defined as the presence of a significant number of bacteria in the urine of an individual not showing clinical signs referrable to UTI. UTI's typically occur as a consequence of ascending pathogen migration from the host's own fecal or distal urogenital microbial flora. The most commonly isolated pathogen in cases of UTI and SB is Escherichia coli. The diagnosis is based on clinical signs and the results of urine examination and culture. The recently revised guidelines of the International Society for Companion Animal Infectious Diseases provide detailed recommendations for diagnosis and management of different forms of bacterial UTI's in dogs. Adherence to treatment guidelines will improve treatment success and is imperative in avoiding further deterioration of the antimicrobial resistance situation.