Alzheimer-like behavior and synaptic dysfunction in 3 × Tg-AD mice are reversed with calcineurin inhibition.

Alzheimer's disease is a progressive neurodegenerative disorder characterized by impairments in synaptic plasticity and cognitive performance. Current treatments are unable to achieve satisfactory therapeutic effects or reverse the progression of the disease. Calcineurin has been implicated as part of a critical signaling pathway for learning and memory, and neuronal calcineurin may be hyperactivated in AD. To investigate the effects and underlying mechanisms of FK506, a calcineurin inhibitor, on Alzheimer-like behavior and synaptic dysfunction in the 3 × Tg-AD transgenic mouse model of Alzheimer's disease, we investigated the effect of FK506 on cognitive function and synaptic plasticity in the 3 × Tg-AD transgenic mouse model of Alzheimer's disease. The results showed that FK506 treatment ameliorated cognitive deficits, as indicated by the decreased latency in the water maze, and attenuated tau hyperphosphorylation in 3 × Tg-AD mice. Treatment with FK506 also reduced the levels of certain markers of postsynaptic deficits, including PSD-95 and NR2B, and reversed the long-term potentiation deficiency and dendritic spine impairments in 3 × Tg-AD mice. These findings suggest that treatment with calcineurin inhibitors such as FK506 can be an effective therapeutic strategy to rescue synaptic deficit and cognitive impairment in familial Alzheimer's disease and related tauopathies.

Linagliptin ameliorates tacrolimus-induced renal injury: role of Nrf2/HO-1 and HIF-1α/CTGF/PAI-1.

BACKGROUND: Tacrolimus (TAC) is a frequently used immunosuppressive medication in organ transplantation. However, its nephrotoxic impact limits its long-term usage. This study aims to investigate the effect of linagliptin (Lina) on TAC-induced renal injury and its underlying mechanisms. METHODS AND RESULTS: Thirty-two Sprague Dawley rats were treated with TAC (1.5 mg/kg/day, subcutaneously) and/or Lina (5 mg/kg/day, orally) for 4 weeks. Histological examination was conducted, and serum and urinary biomarkers were measured to assess kidney function and integrity. Furthermore, ELISA, Western blot analysis and immunohistochemical assay were employed to determine signaling molecules of oxidative stress, profibrogenic, hypoxic, and apoptotic proteins. Tacrolimus caused renal dysfunction and histological deterioration evidenced by increased serum creatinine, blood urea nitrogen (BUN), urinary cystatin C, and decreased serum albumin as well as elevated tubular injury and interstitial fibrosis scores. Additionally, TAC significantly increased the expression of collagen type-1, alpha-smooth muscle actin (α-SMA), plasminogen activator inhibitor-1 (PAI-1), and transforming growth factor-beta1 (TGF-ß1) renal content. Moreover, TAC decreased the expression of nuclear factor erythroid-2-related factor2 (Nrf2), heme oxygenase 1 (HO-1), and mitochondrial superoxide dismutase (SOD2). In addition, TAC increased protein expression of hypoxia-inducible factor1-alpha (HIF-1α), connective tissue growth factor (CTGF), inducible nitric oxide synthase (iNOS), 8-hydroxy-2-deoxyguanosine (8-OHdG), as well as nitric oxide (NO), 4-hydroxynonenal, caspase-3 and Bax renal contents. Furthermore, TAC decreased Bcl-2 renal contents. The Lina administration markedly attenuated these alterations. CONCLUSION: Lina ameliorated TAC-induced kidney injury through modulation of oxidative stress, hypoxia, and apoptosis related proteins.

Induction of diabetes by Tacrolimus in a phenotypic model of obesity and metabolic syndrome.

Introduction: The pathogenesis of Post-Transplant Diabetes Mellitus (PTDM) is complex and multifactorial and it resembles that of Type-2 Diabetes Mellitus (T2DM). One risk factor specific to PTDM differentiates both entities: the use of immunosuppressive therapy. Specifically, Tacrolimus interacts with obesity and insulin resistance (IR) in accelerating the onset of PTDM. In a genotypic model of IR, the obese Zucker rats, Tacrolimus is highly diabetogenic by promoting the same changes in beta-cell already modified by IR. Nevertheless, genotypic animal models have their limitations and may not resemble the real pathophysiology of diabetes. In this study, we have evaluated the interaction between beta-cell damage and Tacrolimus in a non-genotypic animal model of obesity and metabolic syndrome. Methods: Sprague Dawley rats were fed a high-fat enriched diet during 45 days to induce obesity and metabolic dysregulation. On top of this established obesity, the administration of Tacrolimus (1mg/kg/day) during 15 days induced severe hyperglycaemia and changes in morphological and structural characteristics of the pancreas. Results: Obese animals administered with Tacrolimus showed increased size of islets of Langerhans and reduced beta-cell proliferation without changes in apoptosis. There were also changes in beta-cell nuclear factors such as a decrease in nuclear expression of MafA and a nuclear overexpression of FoxO1A, PDX-1 and NeuroD1. These animals also showed increased levels of pancreatic insulin and glucagon. Discussion: This model could be evidence of the relationship between the T2DM and PTDM physiopathology and, eventually, the model may be instrumental to study the pathogenesis of T2DM.

Antascomicin B stabilizes FKBP51-Akt1 complexes as a molecular glue.

Antascomicin B is a natural product that similarly to the macrolides FK506 and Rapamycin binds to the FK506-binding protein 12 (FKBP12). FK506 and Rapamycin act as molecular glues by inducing ternary complexes between FKBPs and additional target proteins. Whether Antascomicin B can induce ternary complexes is unknown. Here we show that Antascomicin B binds tightly to larger human FKBP homologs. The cocrystal structure of FKBP51 in complex with Antascomicin B revealed that large parts of Antascomicin B are solvent-exposed and available to engage additional proteins. Cellular studies demonstrated that Antascomicin B enhances the interaction between human FKBP51 and human Akt. Our studies show that molecules with molecular glue-like properties are more prominent in nature than previously thought. We predict the existence of additional 'orphan' molecular glues that evolved to induce ternary protein complexes but where the relevant ternary complex partners are unknown.

Concomitant use of interleukin-2 and tacrolimus suppresses follicular helper T cell proportion and exerts therapeutic effect against lupus nephritis in systemic lupus erythematosus-like chronic graft versus host disease.

Introduction: Defective interleukin-2 (IL-2) production contributes to immune system imbalance in patients with systemic erythematosus lupus (SLE). Recent clinical studies suggested that low-dose IL-2 treatment is beneficial for SLE and the therapeutic effect is associated with regulatory T cell (Treg) expansion. Pharmacological calcineurin inhibition induces a reduction in the number of Tregs because they require stimulation of T cell receptor signaling and IL-2 for optimal proliferation. However, the activation of T cell receptor signaling is partially dispensable for the expansion of Tregs, but not for that of conventional T cells if IL-2 is present. Aim: We examined whether addition of IL-2 restores the Treg proportion even with concurrent use of a calcineurin inhibitor and if the follicular helper T cell (Tfh) proportion is reduced in an SLE-like murine chronic graft versus host disease model. Methods: Using a parent-into-F1 model, we investigated the effect of IL-2 plus tacrolimus on Treg and Tfh proportions and the therapeutic effect. Results: Treatment with a combination of IL-2 and tacrolimus significantly delayed the initiation of proteinuria and decreased the urinary protein concentration, whereas tacrolimus or IL-2 monotherapy did not significantly attenuate proteinuria. Phosphorylation of signal transducer and activator of transcription 3, a positive regulator of Tfh differentiation, was reduced by combination treatment, whereas phosphorylation of signal transducer and activator of transcription 5, a negative regulator, was not reduced. Conclusion: Addition of calcineurin inhibitors as adjunct agents may be beneficial for IL-2-based treatment of lupus nephritis.

Tacrolimus alleviates pulmonary fibrosis progression through inhibiting the activation and interaction of ILC2 and monocytes.

Idiopathic pulmonary fibrosis (IPF) is a heterogeneous group of lung diseases with different etiologies and characterized by progressive fibrosis. This disease usually causes pulmonary structural remodeling and decreased pulmonary function. The median survival of IPF patients is 2-5 years. Predominantly accumulation of type II innate immune cells accelerates fibrosis progression by secreting multiple pro-fibrotic cytokines. Group 2 innate lymphoid cells (ILC2) and monocytes/macrophages play key roles in innate immunity and aggravate the formation of pro-fibrotic environment. As a potent immunosuppressant, tacrolimus has shown efficacy in alleviating the progression of pulmonary fibrosis. In this study, we found that tacrolimus is capable of suppressing ILC2 activation, monocyte differentiation and the interaction of these two cells. This effect further reduced activation of monocyte-derived macrophages (Mo-M), thus resulting in a decline of myofibroblast activation and collagen deposition. The combination of tacrolimus and nintedanib was more effective than either drug alone. This study will reveal the specific process of tacrolimus alleviating pulmonary fibrosis by regulating type II immunity, and explore the potential feasibility of tacrolimus combined with nintedanib in the treatment of pulmonary fibrosis. This project will provide new ideas for clinical optimization of anti-pulmonary fibrosis drug strategies.

From the Clinical to the Bench: Exploring the Insulin Modulation Effects of Tacrolimus and Belatacept.

Calcineurin inhibitors (CNIs) are critical in preventing rejection posttransplantation but pose an increased risk of post-transplant diabetes (PTD). Recent studies show that late conversion from CNIs to belatacept, a costimulation blocker, improves HbA1c in kidney transplant recipients with PTD or de novo diabetes. This study investigates whether the observed effects on PTD stem solely from CNI withdrawal or if belatacept influences PTD independently. The study assessed the impact of tacrolimus and belatacept on insulin secretion in MIN6 cells (a beta cell line) and rat islets. Tacrolimus and belatacept were administered to the cells and islets, followed by assessments of cell viability and insulin secretion. Tacrolimus impaired insulin secretion without affecting cell viability, while belatacept showed no detrimental effects on either parameter. These findings support clinical observations of improved HbA1c upon switching from tacrolimus to belatacept. Belatacept holds promise in islet or pancreas transplantation, particularly in patients with unstable diabetes. Successful cases of islet transplantation treated with belatacept without severe hypoglycemia highlight its potential in managing PTD. Further research is needed to fully understand the metabolic changes accompanying the transition from CNIs to belatacept. Preserving insulin secretion emerges as a promising avenue for investigation in this context.

Age-Related Brain Atrophy and the Positive Effects of Behavioral Enrichment in Middle-Aged Beagles.

Aging dogs serve as a valuable preclinical model for Alzheimer's disease (AD) due to their natural age-related development of ß-amyloid (Aß) plaques, human-like metabolism, and large brains that are ideal for studying structural brain aging trajectories from serial neuroimaging. Here we examined the effects of chronic treatment with the calcineurin inhibitor (CNI) tacrolimus or the nuclear factor of activated T cells (NFAT)-inhibiting compound Q134R on age-related canine brain atrophy from a longitudinal study in middle-aged beagles (36 females, 7 males) undergoing behavioral enrichment. Annual MRI was analyzed using modern, automated techniques for region-of-interest-based and voxel-based volumetric assessments. We found that the frontal lobe showed accelerated atrophy with age, while the caudate nucleus remained relatively stable. Remarkably, the hippocampus increased in volume in all dogs. None of these changes were influenced by tacrolimus or Q134R treatment. Our results suggest that behavioral enrichment can prevent atrophy and increase the volume of the hippocampus but does not prevent aging-associated prefrontal cortex atrophy.

Calcineurin regulates synaptic Ca2+-permeable AMPA receptors in hypothalamic presympathetic neurons via α2δ-1-mediated GluA1/GluA2 assembly.

Hypertension is a major adverse effect of calcineurin inhibitors, such as tacrolimus (FK506) and cyclosporine, used clinically as immunosuppressants. Calcineurin inhibitor-induced hypertension (CIH) is linked to augmented sympathetic output from the hypothalamic paraventricular nucleus (PVN). GluA2-lacking, Ca2+-permeable AMPA receptors (CP-AMPARs) are a key feature of glutamatergic synaptic plasticity, yet their role in CIH remains elusive. Here, we found that systemic administration of FK506 in rats significantly increased serine phosphorylation of GluA1 and GluA2 in PVN synaptosomes. Strikingly, FK506 treatment reduced GluA1/GluA2 heteromers in both synaptosomes and endoplasmic reticulum-enriched fractions from the PVN. Blocking CP-AMPARs with IEM-1460 induced a larger reduction of AMPAR-mediated excitatory postsynaptic current (AMPAR-EPSC) amplitudes in retrogradely labelled, spinally projecting PVN neurons in FK506-treated rats than in vehicle-treated rats. Furthermore, FK506 treatment shifted the current-voltage relationship of AMPAR-EPSCs from linear to inward rectification in labelled PVN neurons. FK506 treatment profoundly enhanced physical interactions of α2δ-1 with GluA1 and GluA2 in the PVN. Inhibiting α2δ-1 with gabapentin, α2δ-1 genetic knockout, or disrupting α2δ-1-AMPAR interactions with an α2δ-1 C terminus peptide restored GluA1/GluA2 heteromers in the PVN and diminished inward rectification of AMPAR-EPSCs in labelled PVN neurons induced by FK506 treatment. Additionally, microinjection of IEM-1460 or α2δ-1 C terminus peptide into the PVN reduced renal sympathetic nerve discharges and arterial blood pressure elevated in FK506-treated rats but not in vehicle-treated rats. Thus, calcineurin in the hypothalamus constitutively regulates AMPAR subunit composition and phenotypes by controlling GluA1/GluA2 interactions with α2δ-1. Synaptic CP-AMPARs in PVN presympathetic neurons contribute to augmented sympathetic outflow in CIH. KEY POINTS: Systemic treatment with the calcineurin inhibitor increases serine phosphorylation of synaptic GluA1 and GluA2 in the PVN. Calcineurin inhibition enhances the prevalence of postsynaptic Ca2+-permeable AMPARs in PVN presympathetic neurons. Calcineurin inhibition potentiates α2δ-1 interactions with GluA1 and GluA2, disrupting intracellular assembly of GluA1/GluA2 heterotetramers in the PVN. Blocking Ca2+-permeable AMPARs or α2δ-1-AMPAR interactions in the PVN attenuates sympathetic outflow augmented by the calcineurin inhibitor.

Anti-inflammatory and antioxidative actions of tacrolimus (FK506) on human microglial HMC3 cell line.

Microglial cells are indispensable for the normal development and functioning of neurons in the central nervous system, where they play a crucial role in maintaining brain homeostasis by surveilling the microenvironment for signs of injury or stress and responding accordingly. However, in neurodegenerative diseases, the density and phenotypes of microglial cells undergo changes, leading to chronic activation and inflammation. Shifting the focus from neurons to microglia in drug discovery for neurodegenerative diseases has become an important therapeutic target. This study was aimed to investigate the potential of Tacrolimus (FK506) an FDA-approved calcineurin inhibitor, to modulate the pathology of neurodegenerative diseases through anti-inflammatory and antioxidative effects on microglial activation. The human microglia clone 3 (HMC3) cells were exposed to 1 µg/mL LPS in the presence and absence of doses of FK506. Survival rates of cells were determined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) method. Morphological evaluation of cells showed that FK506 restored the normal morphology of activated microglia. Furthermore, FK506 treatment increases the total antioxidant capacity and reduces the total oxidative capacity, indicating its potential antioxidant effects. Data from ELISA and RT-PCR analyses showed that LPS abolished its promoting effects on the release of proinflammatory IL-1ß and IL-6 cytokines in HMC3 cells, reflecting the anti-inflammatory effect of FK506. These findings support the idea that FK506 could be a promising therapeutic agent for neurodegenerative diseases by modulating microglial activation and reducing inflammation and oxidative stress.

Hepatitis E virus infects human testicular tissue and Sertoli cells.

Globally, hepatitis E virus (HEV) infections are prevalent. The finding of high viral loads and persistent viral shedding in ejaculate suggests that HEV replicates within the human male genital tract, but its target organ is unknown and appropriate models are lacking. We aimed to determine the HEV tropism in the human testis and its potential influence on male reproductive health. We conducted an ex vivo culture of human testis explants and in vitro culture of primary human Sertoli cells. Clinically derived HEV genotype 1 (HEV1) and HEV3 virions, as well as rat-derived HEV-C1, were used for inoculation. Transcriptomic analysis was performed on testis tissues collected from tacrolimus-treated rabbits with chronic HEV3 infection. Our findings reveal that HEV3, but not HEV1 or HEV-C1, can replicate in human testis explants and primary human Sertoli cells. Tacrolimus treatment significantly enhanced the replication efficiency of HEV3 in testis explants and enabled successful HEV1 infection in Sertoli cells. HEV3 infection disrupted the secretion of several soluble factors and altered the cytokine microenvironment within primary human Sertoli cells. Finally, intratesticular transcriptomic analysis of immunocompromised rabbits with chronic HEV infection indicated downregulation of genes associated with spermatogenesis. HEV can infect the human testicular tissues and Sertoli cells, with increased replication efficiency when exposed to tacrolimus treatment. These findings shed light on how HEV may persist in the ejaculate of patients with chronic hepatitis E and provide valuable ex vivo tools for studying countermeasures.

[Tacrolimus Induces Pain by Upregulating the Synaptic Expression of AMPA Receptors in the Spinal Cord Dorsal Horn]. / ä»–å ‹èŽ«å¸é€šè¿‡ä¸Šè°ƒè„Šé«“èƒŒè§’AMPA受体的突触表达诱发疼痛.

Objective: To investigate the effects of long-term administration of tacrolimus (also known as FK506) on the pain-related behaviors in mice and to study the underlying mechanism of pain induced by FK506 via measuring the effect of FK506 on the synaptic expression and phosphorylation of alpha-amino-3-hyroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor in the spinal cord dorsal horn of mice. Methods: 1) A total of 24 mice were evenly and randomly assigned to two groups, a FK506 group and a Saline group. The FK506 group was given daily intraperitoneal injection of FK506 and the Saline group received normal saline. Both groups received injection once a day for 7 days in a row. Some of the mice ( n=6 in each group) were monitored for the changes in the paw withdrawal threshold (PWT), the paw withdrawal latency (PWL), and the spontaneous pain behaviors to establish the pain model. The other mice ( n=6 in each group) of each group underwent isolation of the dorsal horn when obvious pain symptoms were induced on day 7 of injection. Then, immunoblotting was performed to determine the synaptic expression and phosphorylation levels of GluA1 and GluA2 subunits of AMPA receptors. 2) The mice were randomly divided into two groups, FK506+calcineurin (CaN) group and FK506+Saline group ( n=6 in each group). After the pain model was constructed, the mice were given intrathecal injection of recombinant CaN (also know as 33 U) or normal saline. Then, 60 minutes later, the PWT and the PWL of the mice were measured to investigate the role of CaN in FK506-induced pain. 3) Another18 mice were selected. The mice were randomly and evenly assigned to three groups, a control group (receiving intraperitoneal injection of normal saline followed by intrathecal injection of normal saline), FK506+Saline group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of normal saline) and FK506+CaN group (receiving intraperitoneal injection of FK506 followed by intrathecal injection of CaN). Then, 60 minutes later, the spinal cords were isolated and subjected to immunoblotting assay to determine the role of CaN in FK506-induced AMPA receptor modification. Results: 1) After 7 consecutive days of intraperitoneal injection of FK506, the PWT and PWL of mice dropped significantly, reaching on day 7 as low as 22.3%±0.05% and 66.6%±0.05% of the control group, respectively ( P<0.01). The FK506-treated mice displayed evident spontaneous pain behavior, presenting significantly increased licking activities ( P<0.01). These results indicated that FK506-induced pain model was successfully established. Immunoblotting assay showed that the total expressions of GluA1 and GluA2 subunits in the spinal dorsal horn of the FK506 group remained unchanged in comparison with those of the Saline group. However, FK506 specifically induced an increase in the synaptic expression of GluA1. In addition, the phosphorylation levels of GluA1 at Ser845 and Ser831 in FK506-treated mice were significantly increased in comparison with those of the control group ( P<0.05). 2) Compared with those of the mice in the FK506+Saline group, the PWT and the PWL of mice in the FK506+CaN group were significantly increased ( P<0.05). 3) Compared with those of the FK506+Saline group, the synaptic expression of GluA1 were decreased in FK506+CaN group ( P<0.01) and the phosphorylation levels of GluA1 at Ser845 and Ser831 were significantly downregulated ( P<0.001). Conclusion: The hyper-expression and hyperphosphorylation of GluA1 subunit in the spinal cord dorsal horn resulting from CaN inhibition contributes to the FK506-induced pain syndrome. FK506 induces the synaptic hyper-expression and hyperphosphorylation of GluA1 in the dorsal horn of the spinal cord through CaN inhibition, thereby inducing pain.

Vancomycin relieves tacrolimus-induced hyperglycemia by eliminating gut bacterial beta-glucuronidase enzyme activity.

Up to 40% of transplant recipients treated long-term with tacrolimus (TAC) develop post-transplant diabetes mellitus (PTDM). TAC is an important risk factor for PTDM, but is also essential for immunosuppression after transplantation. Long-term TAC treatment alters the gut microbiome, but the mechanisms of TAC-induced gut microbiota in the pathogenesis of PTDM are poorly characterized. Here, we showed that vancomycin, an inhibitor of bacterial beta-glucuronidase (GUS), prevents TAC-induced glucose disorder and insulin resistance in mice. Metagenomics shows that GUS-producing bacteria are predominant and flourish in the TAC-induced hyperglycemia mouse model, with upregulation of intestinal GUS activity. Targeted metabolomics analysis revealed that in the presence of high GUS activity, the hydrolysis of bile acid (BAs)-glucuronic conjugates is increased and most BAs are overproduced in the serum and liver, which, in turn, activates the ileal farnesoid X receptor (FXR) and suppresses GLP-1 secretion by L-cells. The GUS inhibitor vancomycin significantly eliminated GUS-producing bacteria and inhibited bacterial GUS activity and BAs levels, thereby enhancing L-cell GLP-1 secretion and preventing hyperglycemia. Our results propose a novel clinical strategy for inhibiting the bacterial GUS enzyme to prevent hyperglycemia without requiring withdrawal of TAC treatment. This strategy exerted its effect through the ileal bile acid-FXR-GLP-1 pathway.

Antiviral activity of immunosuppressors alone and in combination against human adenovirus and cytomegalovirus.

Human adenovirus (HAdV) and cytomegalovirus (HCMV) cause high morbidity and mortality in patients undergoing solid organ transplantation (SOT) and haematopoietic stem cell transplantation (HSCT). Immunosuppressors are used universally to prevent graft-vs-host disease in HSCT and graft rejection in SOT. The long-term use of these drugs is associated with a high risk of infection, but there is also evidence of their specific interference with viral infection. This study evaluated the antiviral activity of immunosuppressors commonly used in clinical practice in SOT and HSCT recipients in vitro to determine whether their use could be associated with reduced risk of HAdV and HCMV infection. Cyclophosphamide, tacrolimus, cyclosporine, mycophenolic acid, methotrexate, everolimus and sirolimus presented antiviral activity, with 50% inhibitory concentration (IC50) values at low micromolar and sub-micromolar concentrations. Mycophenolic acid and methotrexate showed the greatest antiviral effects against HAdV (IC50=0.05 µM and 0.3 µM, respectively) and HCMV (IC50=10.8 µM and 0.02 µM, respectively). The combination of tacrolimus and mycophenolic acid showed strong synergistic antiviral activity against both viruses, with combinatory indexes (CI50) of 0.02 and 0.25, respectively. Additionally, mycophenolic acid plus cyclosporine, and mycophenolic acid plus everolimus/sirolimus showed synergistic antiviral activity against HAdV (CI50=0.05 and 0.09, respectively), while methotrexate plus cyclosporine showed synergistic antiviral activity against HCMV (CI50=0.29). These results, showing antiviral activity in vitro against both HAdV and HCMV, at concentrations below the human Cmax values, may be relevant for the selection of specific immunosuppressant therapies in patients at risk of HAdV and HCMV infections.

Nerve Wrap for Local Delivery of FK506/Tacrolimus Accelerates Nerve Regeneration.

Peripheral nerve injuries (PNIs) occur frequently and can lead to devastating and permanent sensory and motor function disabilities. Systemic tacrolimus (FK506) administration has been shown to hasten recovery and improve functional outcomes after PNI repair. Unfortunately, high systemic levels of FK506 can result in adverse side effects. The localized administration of FK506 could provide the neuroregenerative benefits of FK506 while avoiding systemic, off-target side effects. This study investigates the utility of a novel FK506-impregnated polyester urethane urea (PEUU) nerve wrap to treat PNI in a previously validated rat infraorbital nerve (ION) transection and repair model. ION function was assessed by microelectrode recordings of trigeminal ganglion cells responding to controlled vibrissae deflections in ION-transected and -repaired animals, with and without the nerve wrap. Peristimulus time histograms (PSTHs) having 1 ms bins were constructed from spike times of individual single units. Responses to stimulus onsets (ON responses) were calculated during a 20 ms period beginning 1 ms after deflection onset; this epoch captures the initial, transient phase of the whisker-evoked response. Compared to no-wrap controls, rats with PEUU-FK506 wraps functionally recovered earlier, displaying larger response magnitudes. With nerve wrap treatment, FK506 blood levels up to six weeks were measured nearly at the limit of quantification (LOQ ≥ 2.0 ng/mL); whereas the drug concentrations within the ION and muscle were much higher, demonstrating the local delivery of FK506 to treat PNI. An immunohistological assessment of ION showed increased myelin expression for animals assigned to neurorrhaphy with PEUU-FK506 treatment compared to untreated or systemic-FK506-treated animals, suggesting that improved PNI outcomes using PEUU-FK506 is mediated by the modulation of Schwann cell activity.

Calcineurin-dependent contributions to fitness in the opportunistic pathogen Candida glabrata.

The protein phosphatase calcineurin is vital for the virulence of the opportunistic fungal pathogen Candida glabrata. The host-induced stresses that activate calcineurin signaling are unknown, as are the targets of calcineurin relevant to virulence. To potentially shed light on these processes, millions of transposon insertion mutants throughout the genome of C. glabrata were profiled en masse for fitness defects in the presence of FK506, a specific inhibitor of calcineurin. Eighty-seven specific gene deficiencies depended on calcineurin signaling for full viability in vitro both in wild-type and pdr1∆ null strains lacking pleiotropic drug resistance. Three genes involved in cell wall biosynthesis (FKS1, DCW1, FLC1) possess co-essential paralogs whose expression depended on calcineurin and Crz1 in response to micafungin, a clinical antifungal that interferes with cell wall biogenesis. Interestingly, 80% of the FK506-sensitive mutants were deficient in different aspects of vesicular trafficking, such as endocytosis, exocytosis, sorting, and biogenesis of secretory proteins in the endoplasmic reticulum (ER). In response to the experimental antifungal manogepix that blocks GPI-anchor biosynthesis in the ER, calcineurin signaling increased and strongly prevented cell death independent of Crz1, one of its major targets. Comparisons between manogepix, micafungin, and the ER-stressing tunicamycin reveal a correlation between the degree of calcineurin signaling and the degree of cell survival. These findings suggest that calcineurin plays major roles in mitigating stresses of vesicular trafficking. Such stresses may arise during host infection and in response to antifungal therapies.IMPORTANCECalcineurin plays critical roles in the virulence of most pathogenic fungi. This study sheds light on those roles in the opportunistic pathogen Candida glabrata using a genome-wide analysis in vitro. The findings could lead to antifungal developments that also avoid immunosuppression.

The Effect of Local Purified Exosome Product, Stem Cells, and Tacrolimus on Neurite Extension.

PURPOSE: The combination of cellular and noncellular treatments has been postulated to improve nerve regeneration through a processed nerve allograft. This study aimed to evaluate the isolated effect of treatment with purified exosome product (PEP), mesenchymal stem cells (MSCs), and tacrolimus (FK506) alone and in combination when applied in decellularized allografts. METHODS: A three-dimensional in vitro-compartmented cell culture system was used to evaluate the length of regenerating neurites from the neonatal dorsal root ganglion into the adjacent peripheral nerve graft. Decellularized nerve allografts were treated with undifferentiated MSCs, 5% PEP, 100 ng/mL FK506, PEP and FK506 combined, or MSCs and FK506 combined (N = 9/group) and compared with untreated nerve autografts (positive control) and nerve allografts (negative control). Neurite extension was measured to quantify nerve regeneration after 48 hours, and stem cell viability was evaluated. RESULTS: Stem cell viability was confirmed in all MSC-treated nerve grafts. Treatments with PEP, PEP + FK506, and MSCs + FK506 combined were found to be superior to untreated allografts and not significantly different from autografts. Combined PEP and FK506 treatment resulted in the greatest neurite extension. Treatment with FK506 and MSCs was significantly superior to MSC alone. The combined treatment groups were not found to be statistically different. CONCLUSIONS: Although all treatments improved neurite outgrowth, treatments with PEP, PEP + FK506, and MSCs + FK506 combined had superior neurite growth compared with untreated allografts and were not found to be significantly different from autografts, the current gold standard. CLINICAL RELEVANCE: Purified exosome product, a cell-free exosome product, is a promising adjunct to enhance nerve allograft regeneration, with possible future avenues for clinical translation.

Immunosuppressive enzyme-responsive nanoparticles for enhanced accumulation in liver allograft to overcome acute rejection.

Acute rejection is a life-threatening complication after liver transplantation. Immunosuppressants such as tacrolimus are used to inhibit acute rejection of liver grafts in clinic. However, inefficient intragraft accumulation may reduce the therapeutic outcomes of tacrolimus. Here, an enzyme-responsive nanoparticle is developed to selectively enhance the accumulation of tacrolimus in liver allograft through enzyme-induced aggregation to refine immunotherapeutic efficacy of tacrolimus. The nanoparticles are composed of amphiphilic tacrolimus prodrugs synthesized by covalently conjugating tacrolimus and matrix metalloproteinase 9 (MMP9)-cleavable peptide-containing methoxy poly (ethylene glycol) to poly (l-glutamic acid). Upon exposure to MMP9, which is overexpressed in rejected liver allografts, the nanoparticles undergo a morphological transition from spherical micellar nanoparticles to microscale aggregate-like scaffolds. Intravenous administration of MMP9-responsive nanoparticles into a rat model of acute liver graft rejection results in enhanced nanoparticle accumulation in allograft as compared to nonresponsive nanoparticles. Consequently, the MMP9-responsive nanoparticles significantly inhibit intragraft inflammatory cell infiltration and proliferation, maintain intragraft immunosuppressive environment, alleviate graft damage, improve liver allograft function, abate weight loss and prolong recipient survival. This work proves that morphology-switchable enzyme-responsive nanoparticles represent an innovative strategy for selectively enhancing intragraft accumulation of immunosuppressive agents to improve treatment of liver allograft rejection.

Generation of Bioactivity-Tailored FK506/FK520 Analogs by CRISPR Editing in Streptomyces tsukubaensis.

For a potential application of FK506 in the treatment of acute kidney failure only the FKBP12 binding capability of the compound is required, while the immunosuppressive activity via calcineurin binding is considered as a likely risk to the patients. The methoxy groups at C13 and C15 are thought to have significant influence on the immunosuppressive activity of the molecule. Consequently, FK506 analogs with different functionalities at C13 and C15 were generated by targeted CRISPR editing of the AT domains in module 7 and 8 of the biosynthetic assembly line in Streptomyces tsukubaensis. In addition, the corresponding FK520 (C21 ethyl derivative of FK506) analogs could be obtained by media adjustments. The compounds were tested for their bioactivity in regards to FKBP12 binding, BMP potentiation and calcineurin sparing. 15-desmethoxy FK506 was superior to the other tested analogs as it did not inhibit calcineurin but retained high potency towards FKBP12 binding and BMP potentiation.

Propagation of conformational instability in FK506-binding protein FKBP12.

FKBP12 is the archetype of the FK506 binding domains that define the family of FKBP proteins which participate in the regulation of various distinct physiological signaling processes. As the drugs FK506 and rapamycin inhibit many of these FKBP proteins, there is need to develop therapeutics which exhibit selectivity within this family. The long ß4-ß5 loop of the FKBP domain is known to regulate transcriptional activity for the steroid hormone receptors and appears to participate in regulating calcium channel activity for the cardiac and skeletal muscle ryanodine receptors. The ß4-ß5 loop of FKBP12 has been shown to undergo extensive conformational dynamics, and here we report hydrogen exchange measurements for a series of mutational variants in that loop which indicate deviations from a two-state kinetics for those dynamics. In addition to a previously characterized local transition near the tip of this loop, evidence is presented for a second site of conformational dynamics in the stem of this loop. These mutation-dependent hydrogen exchange effects extend beyond the ß4-ß5 loop, primarily by disrupting the hydrogen bond between the Gly 58 amide and the Tyr 80 carbonyl oxygen which links the two halves of the structural rim that surrounds the active site cleft. Mutationally-induced opening of the cleft between Gly 58 and Tyr 80 not only modulates the global stability of the protein, it promotes a conformational transition in the distant ß2-ß3a hairpin that modulates the binding affinity for a FKBP51-selective inhibitor previously designed to exploit a localized conformational transition at the homologous site.