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Bacterial infections are a common and deadly threat to vulnerable patients. Alternative strategies to fight infection are needed. β-Glucan, an immunomodulator derived from the fungal cell wall, provokes resistance to infection by inducing trained immunity, a phenomenon that persists for weeks to months. Given the durability of trained immunity, it is unclear which leukocyte populations sustain this effect. Macrophages have a life span that surpasses the duration of trained immunity. Thus, we sought to define the contribution of differentiated macrophages to trained immunity. Our results show that β-glucan protects mice from Pseudomonas aeruginosa infection by augmenting recruitment of innate leukocytes to the site of infection and facilitating local clearance of bacteria, an effect that persists for more than 7 d. Adoptive transfer of macrophages, trained using β-glucan, into naive mice conferred a comparable level of protection. Trained mouse bone marrow–derived macrophages assumed an antimicrobial phenotype characterized by enhanced phagocytosis and reactive oxygen species production in parallel with sustained enhancements in glycolytic and oxidative metabolism, increased mitochondrial mass, and membrane potential. β-Glucan induced broad transcriptomic changes in macrophages consistent with early activation of the inflammatory response, followed by sustained alterations in transcripts associated with metabolism, cellular differentiation, and antimicrobial function. Trained macrophages constitutively secreted CCL chemokines and robustly produced proinflammatory cytokines and chemokines in response to LPS challenge. Induction of the trained phenotype was independent of the classic β-glucan receptors Dectin-1 and TLR-2. These findings provide evidence that β-glucan induces enhanced protection from infection by driving trained immunity in macrophages.

BACKGROUND: Monophosphoryl lipid A (MPLA) is a TLR4 agonist that has potent immunomodulatory properties and modulates innate immune function to improve host resistance to infection with common nosocomial pathogens in mice. The goal of this study was to assess the safety and efficacy of MPLA in a sheep model of burn injury and Pseudomonas aeruginosa pneumonia. The sheep provides a favorable model for pre-clinical testing as their response to TLR4 agonists closely mimics that of humans. METHODS: Twelve chronically instrumented adult female Merino sheep received 20% total body surface area, third-degree cutaneous burn under anesthesia and analgesia. At 24 hours after burn, sheep were randomly allocated to receive; 1) MPLA (2.5 μg/kg IV, n=6), or 2) vehicle (IV, n=6). At 24 hours after MPLA or vehicle treatment, Pseudomonas aeruginosa pneumonia was induced. Sheep were mechanically ventilated, fluid resuscitated and cardiopulmonary variables were monitored for 24 hours after induction of pneumonia. Cytokine production, vascular barrier function, and lung bacterial burden were also measured. RESULTS: MPLA infusion induced small and transient alterations in core body temperature, heart rate, pulmonary artery pressure, and pulmonary vascular resistance. Pulmonary mechanics were not altered. Vehicle-treated sheep developed severe acute lung injury during Pseudomonas aeruginosa pneumonia, which was attenuated by MPLA as indicated by improved PaO(2)/FiO(2) ratio, oxygenation index, and shunt fraction. Sheep treated with MPLA also exhibited less vascular leak, lower blood lactate levels, and lower modified organ injury score. MPLA treatment attenuated systemic cytokine production and decreased lung bacterial burden. CONCLUSIONS: MPLA was well tolerated in burned sheep and attenuated development of acute lung injury, lactatemia, cytokinemia, vascular leak, and hemodynamic changes caused by Pseudomonas aeruginosa pneumonia.

Study objective Postoperative delirium (POD) is a common neurological system disorder in surgical patients. Anesthesia providers have a wide choice of sedative agents involving different mechanisms in clinical practice, and the incidence of POD varies regarding which sedative agent administered. This network meta-analysis aimed to comprehensively analyze the safety and efficacy of each choice for patients. Design A network meta-analysis. Setting Vanderbilt University Medical Center. Measurements We searched PubMed, EMBASE, Ovid Medline and Cochrane Central Register of Controlled Trials (CENTRAL) through the end of September 2018 with the registration number CRD42018110585. The randomized controlled trials were identified and extracted by two reviewers independently. Commonly used sedative agents such as placebo, sevoflurane, desflurane, isoflurane, dexmedetomidine, propofol, midazolam, and ketamine were assessed in this network meta-analysis and the primary outcome was the incidence of POD. The data were synthesized by network meta-analysis. Pair-wise meta-analyses were conducted using the random-effects model. Each intervention was ranked according to its corresponding surface under the cumulative ranking curve (SUCRA) values. The GRADE framework was undertaken to evaluate the risk of bias. Main results We identified 39 RCTs and 5991 patients in this meta-analysis. Dexmedetomidine was found to be the most effective option in reducing POD, compared to midazolam, propofol, desflurane, and sevoflurane. The results revealed that dexmedetomidine was associated with a lower incidence of POD, whereas midazolam was associated with a significantly higher number of patients with delirium. Midazolam and propofol were also associated with a higher incidence of perioperative hypotension and bradycardia. Conclusion Our study provided meta-analytic evidence and suggested dexmedetomidine could be considered as the most effective sedative agent to reduce POD. However, clinical practitioners still need to weigh the pros and cons before choosing a sedative agent for individual patient.

Objectives Cardiopulmonary bypass-induced endothelial dysfunction has been inferred by changes in pulmonary vascular resistance, alterations in circulating biomarkers, and postoperative capillary leak. Endothelial-dependent vasomotor dysfunction of the systemic vasculature has never been quantified in this setting. The objective of the present study was to quantify acute effects of cardiopulmonary bypass on endothelial vasomotor control and attempt to correlate these effects with postoperative cytokines, tissue edema, and clinical outcomes in infants. Design Single-center prospective observational cohort pilot study. Setting Pediatric cardiac ICU at a tertiary children's hospital. Patients Children less than 1 year old requiring cardiopulmonary bypass for repair of a congenital heart lesion. Intervention None. Measurements and main results Laser Doppler perfusion monitoring was coupled with local iontophoresis of acetylcholine (endothelium-dependent vasodilator) or sodium nitroprusside (endothelium-independent vasodilator) to quantify endothelial-dependent vasomotor function in the cutaneous microcirculation. Measurements were obtained preoperatively, 2-4 hours, and 24 hours after separation from cardiopulmonary bypass. Fifteen patients completed all laser Doppler perfusion monitor (Perimed, Jarfalla, Sweden) measurements. Comparing prebypass with 2-4 hours postbypass responses, there was a decrease in both peak perfusion (p = 0.0006) and area under the dose-response curve (p = 0.005) following acetylcholine, but no change in responses to sodium nitroprusside. Twenty-four hours after bypass responsiveness to acetylcholine improved, but typically remained depressed from baseline. Conserved endothelial function was associated with higher urine output during the first 48 postoperative hours (R = 0.43; p = 0.008). Conclusions Cutaneous endothelial dysfunction is present in infants immediately following cardiopulmonary bypass and recovers significantly in some patients within 24 hours postoperatively. Confirmation of an association between persistent endothelial-dependent vasomotor dysfunction and decreased urine output could have important clinical implications. Ongoing research will explore the pattern of endothelial-dependent vasomotor dysfunction after cardiopulmonary bypass and its relationship with biochemical markers of inflammation and clinical outcomes.

Objectives To determine whether synthetic phosphorylated hexa-acyl disaccharides provide antimicrobial protection in clinically relevant models of bacterial infection. Design Laboratory study. Setting University laboratory. Subjects BALB/c, C57BL/10J, and C57BL/10ScNJ mice. Interventions Mice were treated with lactated Ringer's (vehicle) solution, monophosphoryl lipid A, or phosphorylated hexa-acyl disaccharides at 48 and 24 hours prior to intraperitoneal Pseudomonas aeruginosa or IV Staphylococcus aureus infection. Leukocyte recruitment, cytokine production, and bacterial clearance were measured 6 hours after P. aeruginosa infection. In the systemic S. aureus infection model, one group of mice was monitored for 14-day survival and another for S. aureus tissue burden at 3 days postinfection. Duration of action for 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide was determined at 3, 10, and 14 days using a model of intraperitoneal P. aeruginosa infection. Effect of 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide on in vivo leukocyte phagocytosis and respiratory burst was examined. Leukocyte recruitment, cytokine production, and bacterial clearance were measured after P. aeruginosa infection in wild-type and toll-like receptor 4 knockout mice treated with 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide or vehicle to assess receptor specificity. Measurements and main results During intraperitoneal P. aeruginosa infection, phosphorylated hexa-acyl disaccharides significantly attenuated infection-induced hypothermia, augmented leukocyte recruitment and bacterial clearance, and decreased cytokine production. At 3 days post S. aureus infection, bacterial burden in lungs, spleen, and kidneys was significantly decreased in mice treated with monophosphoryl lipid A or phosphorylated hexa-acyl disaccharides, which was associated with improved survival. Leukocyte phagocytosis and respiratory burst functions were enhanced after treatment with monophosphoryl lipid A or phosphorylated hexa-acyl disaccharides. A time course study showed that monophosphoryl lipid A- and 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide-mediated protection against P. aeruginosa lasts for up to 10 days. Partial loss of augmented innate antimicrobial responses was observed in toll-like receptor 4 knockout mice treated with 3-deacyl 6-Acyl phosphorylated hexa-acyl disaccharide. Conclusions Phosphorylated hexa-acyl disaccharides significantly augment resistance against clinically relevant Gram-negative and Gram-positive infections via enhanced leukocyte recruitment, phagocytosis, and respiratory burst functions of innate leukocytes. Improved antimicrobial protection persists for up to 10 days and is partially mediated through toll-like receptor 4.

Background: Delirium, contributing to subsequent increased morbidity and mortality, is common in hospitalized patients, especially in patients undergoing major surgery. Published systematic reviews suggest that certain medications for delirium management may decrease pain scores and analgesic requirements, at the expense of troublesome side effects. People are urgently seeking non-medication strategies for delirium. Acupuncture and related techniques (ART) are increasingly used to provide medical care and have potential to prevent delirium, however, the efficacy of ART on delirium remains controversial.Methods: In accordance with the PRISMA-P guidelines, we will search PubMed, Embase, the Cochrane Library (Cochrane Central Register of Controlled Trials (CENTRAL)), Web of Science, Medline and the Cochrane Library for clinical trials on ART use in people, without language restrictions. The incidence of delirium will be defined our primary outcome. Additional outcomes will be the incidence of adverse events, pain score, consumption of analgesics, number of cumulative delirious days, hemodynamics, length of hospital stay. Two researchers independently complete further review work and data abstraction based on pre-specified inclusion and exclusion criteria. Any discrepancy will be solved by negotiation or a third investigators. Cochrane Risk of Bias tool will be used to assess the methodological quality of all the studies that are finally included. Statistical heterogeneity was assessed by forest plots, confidence intervals (CI) and I² statistic. If feasible, a meta-analysis of included results will be performed.Conclusions: This protocol will be favorable to bridge the gap between non-medication strategies and clinical diseases, and potential to help future guideline development in management of preventing delirium.Systematic review registration: The protocol was prospectively registered on the homepage of the International Prospective Register of Systematic Reviews (PROSPERO): https://www.crd.york.ac.uk/PROSPERO.

MicroRNA-mediated post-transcriptional regulation plays key roles in stem cell self-renewal and tumorigenesis. However, the in vivo functions of specific microRNAs in controlling mammary stem cell (MaSC) activity and breast cancer formation remain poorly understood. Here we show that miR-31 is highly expressed in MaSC-enriched mammary basal cell population and in mammary tumors, and is regulated by NF-κB signaling. We demonstrate that miR-31 promotes mammary epithelial proliferation and MaSC expansion at the expense of differentiation in vivo. Loss of miR-31 compromises mammary tumor growth, reduces the number of cancer stem cells, as well as decreases tumor-initiating ability and metastasis to the lung, supporting its pro-oncogenic function. MiR-31 modulates multiple signaling pathways, including Prlr/Stat5, TGFβ and Wnt/β-catenin. Particularly, it activates Wnt/β-catenin signaling by directly targeting Wnt antagonists, including Dkk1. Importantly, Dkk1 overexpression partially rescues miR31-induced mammary defects. Together, these findings identify miR-31 as the key regulator of MaSC activity and breast tumorigenesis.

Methyl CpG binding protein 2 (MeCP2) is involved in nerve regeneration following ischemic stroke, but the related mechanism remains unclear. Here, we found low MeCP2 expression in hippocampal tissues. Using functional analysis, we demonstrated that MeCP2 accelerated FOXO3a methylation and subsequently inhibited its expression, thus repressing the apoptosis of neuronal cells. Mechanistically, FOXO3a could bind to the promoter region of SPRY2, consequently inducing its transcription and promoting the expression of the downstream target gene ZEB1. Altogether, our study revealed that overexpression of MeCP2 can protect mice against ischemic brain injury via disruption of the FOXO3a/SPRY2/ZEB1 signaling axis. Our results identify ectopic expression of MeCP2 as a therapeutic target in ischemic stroke.

Hematopoietic myeloblasts give rise to macrophages, dendritic cells, and neutrophils. Circulating myeloid cells detect invading microbes using pattern recognition receptors and subsequently orchestrate an innate immune response to contain and kill the pathogens. This innate immune response establishes an inflammatory niche characterized by hypoxia due to host and pathogen factors. Hypoxia-inducible factor (HIF) transcription factors are the primary regulators of the myeloid response to hypoxia. In particular, HIF-1α is a critical hub that integrates hypoxic and immunogenic signals during infection or inflammation. Hypoxia induces HIF-1α stabilization, which drives metabolic and phenotypic reprogramming of myeloid cells to maximize antimicrobial potential. HIF-1α activity in myeloid-derived cells enhances the host response to infection, but may also play a role in pathogenic inflammatory processes, such as atherosclerosis. In this review, we summarize recent advances that have elucidated the mechanism by which myeloid cells regulate HIF-1α activity and, in turn, how HIF-1α shapes myeloid cell function.

Interleukin (IL)-15 is essential for natural killer (NK), NKT and memory (m) CD8+ T cell development and function, and is currently under investigation as an immunotherapeutic agent for the treatment of cancer. Recently, the creation of IL-15 superagonist by complexing IL-15 and its high affinity receptor alpha (IL-15 Rα) in solution, inspired by the natural trans-presentation of IL-15, advances the potential of IL-15-based tumor immunotherapy. IL-15 superagonist shows promising advantages over monomeric IL-15 such as sustaining high circulating concentrations due to prolonged half-life and more potently stimulating NK and CD8+ T effector lymphocytes. So far, there are three different forms of recombinant IL-15 superagonist fusion protein based on configurational modifications. Gene therapy using engineered cells co-expressing IL-15/IL-15 Rα complex for cancer treatment is also emerging. All forms have demonstrated efficacy in causing tumor regression in animal studies, which provides strong rationale for advancing IL-15 superagonist through clinical trials. To date, there are fourteen phase I/II IL-15 superagonist trials in cancer patients and one phase I trial in HIV patients. Information generated by ongoing trials regarding the toxicity and efficacy of IL-15 superagonist is awaited. Finally, we elaborate on immunotoxicity caused by IL-15 superagonist in preclinical studies and discuss important safety considerations.

Natural killer (NK) cells are large granular lymphocytes largely recognized for their importance in tumour surveillance and the host response to viral infections. However, as the major innate lymphocyte population, NK cells also coordinate early responses to bacterial infections by amplifying the antimicrobial functions of myeloid cells, especially macrophages, by production of interferon-γ (IFN-γ). Alternatively, excessive NK cell activation and IFN-γ production can amplify the systemic inflammatory response during sepsis resulting in increased physiological dysfunction and organ injury. Our understanding of NK cell biology during bacterial infections and sepsis is mostly derived from studies performed in mice. Human studies have demonstrated a correlation between altered NK cell functions and outcomes during sepsis. However, mechanistic understanding of NK cell function during human sepsis is limited. In this review, we will review the current understanding of NK cell biology during sepsis and discuss the challenges associated with modulating NK cell function during sepsis for therapeutic benefit.

MicroRNA-mediated post-transcriptional regulation plays key roles in stem cell self-renewal and tumorigenesis. However, the in vivo functions of specific microRNAs in controlling mammary stem cell (MaSC) activity and breast cancer formation remain poorly understood. Here we show that miR-31 is highly expressed in MaSC-enriched mammary basal cell population and in mammary tumors, and is regulated by NF-κB signaling. We demonstrate that miR-31 promotes mammary epithelial proliferation and MaSC expansion at the expense of differentiation in vivo. Loss of miR-31 compromises mammary tumor growth, reduces the number of cancer stem cells, as well as decreases tumor-initiating ability and metastasis to the lung, supporting its pro-oncogenic function. MiR-31 modulates multiple signaling pathways, including Prlr/Stat5, TGFβ and Wnt/β-catenin. Particularly, it activates Wnt/β-catenin signaling by directly targeting Wnt antagonists, including Dkk1. Importantly, Dkk1 overexpression partially rescues miR31-induced mammary defects. Together, these findings identify miR-31 as the key regulator of MaSC activity and breast tumorigenesis.

Interleukin 15 is essential for the development and differentiation of NK and memory CD8+ (mCD8+) T cells. Our laboratory previously showed that NK and CD8+ T lymphocytes facilitate the pathobiology of septic shock. However, factors that regulate NK and CD8+ T lymphocyte functions during sepsis are not well characterized. We hypothesized that IL-15 promotes the pathogenesis of sepsis by maintaining NK and mCD8+ T cell integrity. To test our hypothesis, the pathogenesis of sepsis was assessed in IL-15–deficient (IL-15 knockout, KO) mice. IL-15 KO mice showed improved survival, attenuated hypothermia, and less proinflammatory cytokine production during septic shock caused by cecal ligation and puncture or endotoxin-induced shock. Treatment with IL-15 superagonist (IL-15 SA, IL-15/IL-15Rα complex) regenerated NK and mCD8+ T cells and re-established mortality of IL-15 KO mice during septic shock. Preventing NK cell regeneration attenuated the restoration of mortality caused by IL-15 SA. If given immediately prior to septic challenge, IL-15–neutralizing IgG M96 failed to protect against septic shock. However, M96 caused NK cell depletion if given 4 d prior to septic challenge and conferred protection. IL-15 SA treatment amplified endotoxin shock, which was prevented by NK cell or IFN-γ depletion. IL-15 SA treatment also exacerbated septic shock caused by cecal ligation and puncture when given after the onset of sepsis. In conclusion, endogenous IL-15 does not directly augment the pathogenesis of sepsis but enables the development of septic shock by maintaining NK cell numbers and integrity. Exogenous IL-15 exacerbates the severity of sepsis by activating NK cells and facilitating IFN-γ production.

To test the hypothesis that patient-controlled analgesia (PCA) contributes to improvement of hemorheology in patients undergoing hip arthroplasty.120 patients, aged 60 - 75 years old, undergoing hip arthroplasty under spinal anesthesia, were randomly divided into group PCA (n = 60) and control group (n = 60). Patients in PCA group received PCA in postoperative 3 days. Blood samples from the median cubital vein were collected at five time points: before anesthesia (T1), after surgery (T2), 6 h after surgery (T3), 24 h after surgery (T4), 48 h after surgery (T5). Hemorheological parameters were measured, including whole blood viscosity at a high shear rate (Hηb), whole blood viscosity at a low shear rate (Lηb), reduced viscosity (ηr), plasma viscosity (ηp), hematocrit (Hct), erythrocyte aggregation index(EAI) and erythrocyte deformation index (EDI). Noninvasive blood pressure and heart rate at T1-5 and pain scoring of visual analogue scale (VAS) score at T2-5 were recorded.(1) Compared with T1, Hηb, Lηb, ηp, ηr decreased significantly at T3-5 with EAI decreased significantly at T5 in group PCA (p 0.05), EDI increased significantly at T5 in group C (p 0.05). (2) Compared with group C, Hηb, Lηb, ηp, ηr, EAI decreased significantly at T5 with Lηb concurrently decreased at T4 in group PCA (p 0.05).Postoperative pain may increase blood viscosity in patients undergoing hip arthroplasty, mainly via plasma viscosity, erythrocyte aggregation and rigidity, and which could be improved by postoperative PCA.ZIEL: Prüfung der Hypothese, dass die patientengesteuerte Analgesie (PCA) bei Patienten nach Hüftarthroplastik zur Verbesserung der Hämorheologie beiträgt. METHODEN: 120 Patienten im Alter von 60 – 75 Jahren, die sich einer Hüftarthroplastik unter Spinalanästhesie unterzogen, wurden per Randomisierung der PCA-Gruppe (n = 60) bzw. der Kontrollgruppe (n = 60) zugewiesen. Patienten der PCA-Gruppe wurden postoperativ über 3 Tage mittels PCA behandelt. Zu fünf Zeitpunkten wurden Blutproben aus der Ellenbeugenvene entnommen: vor der Anästhesie (T1), nach der Operation (T2), 6 h nach der Operation (T3), 24 h nach der Operation (T4) und 48 h nach der Operation (T5). Als hämorheologische Parameter wurden die Viskosität des Vollbluts bei hoher Schergeschwindigkeit (Hηb), die Viskosität des Vollbluts bei geringer Schergeschwindigkeit (Lηb), die reduzierte Viskosität (ηr), die Plasmaviskosität (ηp), der Hämatokrit (Hct) sowie der Erythrozytenaggregationsindex (EAI) und der Erythrozytendeformationsindex (EDI) erhoben. Zudem wurden nichtinvasive Blutdruck- und Herzfrequenzmessungen zu den Zeitpunkten T1 – 5 sowie Schmerzbeurteilungen anhand einer visuellen Analogskala (VAS) zu den Zeitpunkten T2 – 5 aufgezeichnet. ERGEBNISSE: (1) In der PCA-Gruppe hatten sich die Parameter Hηb, Lηb, ηp und ηr zu den Zeitpunkten T3 – 5 und der EAI zum Zeitpunkt T5 signifikant vermindert, jeweils gegenüber Zeitpunkt T1 (p 0,05); in der Kontrollgruppe hatte sich der EDI zum Zeitpunkt T5 signifikant erhöht (p 0,05). (2) Im Vergleich zur Kontrollgruppe wurde in der PCA-Gruppe zum Zeitpunkt T5 eine signifikante Verminderung der Parameter Hηb, Lηb, ηp, ηr und EAI bei gleichzeitiger Verminderung der Lηb zum Zeitpunkt T4 beobachtet (p 0,05). SCHLUSSFOLGERUNG: Bei Patienten, die sich einer Hüftarthroplastik unterziehen, können postoperative Schmerzen die Blutviskosität erhöhen, überwiegend über die Plasmaviskosität, Erythrozytenaggregation und -rigidität, was sich mittels postoperativer PCA verbessern ließ.

Treatment with the TLR4 agonist MPLA augments innate resistance to common bacterial pathogens. However, the cellular and molecular mechanisms by which MPLA augments innate immunocyte functions are not well characterized. This study examined the importance of MyD88- and TRIF-dependent signaling for leukocyte mobilization, recruitment, and activation following administration of MPLA. MPLA potently induced MyD88- and TRIF-dependent signaling. A single injection of MPLA caused rapid mobilization and recruitment of neutrophils, a response that was largely mediated by the chemokines CXCL1 and -2 and the hemopoietic factor G-CSF. Rapid neutrophil recruitment and chemokine production were regulated by both pathways although the MyD88-dependent pathway showed some predominance. In further studies, multiple injections of MPLA potently induced mobilization and recruitment of neutrophils and monocytes. Neutrophil recruitment after multiple injections of MPLA was reliant on MyD88-dependent signaling, but effective monocyte recruitment required activation of both pathways. MPLA treatment induced expansion of myeloid progenitors in bone marrow and upregulation of CD11b and shedding of L-selectin by neutrophils, all of which were attenuated in MyD88- and TRIF-deficient mice. These results show that MPLA-induced neutrophil and monocyte recruitment, expansion of bone marrow progenitors and augmentation of neutrophil adhesion molecule expression are regulated by both the MyD88- and TRIF-dependent pathways.

Infectious diseases remain a threat to critically ill patients, particularly with the rise of antibiotic-resistant bacteria. Septic shock carries a mortality of up to ∼40% with no compelling evidence of promising therapy to reduce morbidity or mortality. Septic shock survivors are also prone to nosocomial infections. Treatment with toll-like receptor 4 (TLR4) agonists have demonstrated significant protection against common nosocomial pathogens in various clinically relevant models of infection and septic shock. TLR4 agonists are derived from a bacteria cell wall or synthesized de novo, and more recently novel small molecule TLR4 agonists have also been developed. TLR4 agonists augment innate immune functions including expansion and recruitment of innate leukocytes to the site of infection. Recent studies demonstrate TLR4-induced leukocyte metabolic reprogramming of cellular metabolism to improve antimicrobial function. Metabolic changes include sustained augmentation of macrophage glycolysis, mitochondrial function, and tricarboxylic acid cycle flux. These findings set the stage for the use of TLR4 agonists as standalone therapeutic agents or antimicrobial adjuncts in patient populations vulnerable to nosocomial infections.

Monophosphoryl lipid A (MPLA) is a clinically used TLR4 agonist that has been found to drive nonspecific resistance to infection for up to 2 wk. However, the molecular mechanisms conferring protection are not well understood. In this study, we found that MPLA prompts resistance to infection, in part, by inducing a sustained and dynamic metabolic program in macrophages that supports improved pathogen clearance. Mice treated with MPLA had enhanced resistance to infection with Staphylococcus aureus and Candida albicans that was associated with augmented microbial clearance and organ protection. Tissue macrophages, which exhibited augmented phagocytosis and respiratory burst after MPLA treatment, were required for the beneficial effects of MPLA. Further analysis of the macrophage phenotype revealed that early TLR4-driven aerobic glycolysis was later coupled with mitochondrial biogenesis, enhanced malate shuttling, and increased mitochondrial ATP production. This metabolic program was initiated by overlapping and redundant contributions of MyD88- and TRIF-dependent signaling pathways as well as downstream mTOR activation. Blockade of mTOR signaling inhibited the development of the metabolic and functional macrophage phenotype and ablated MPLA-induced resistance to infection in vivo. Our findings reveal that MPLA drives macrophage metabolic reprogramming that evolves over a period of days to support a macrophage phenotype highly effective at mediating microbe clearance and that this results in nonspecific resistance to infection.

Burn patients are susceptible to infections due, in part, to immune dysfunction. Upregulation of programmed death-1 (PD-1) receptor on T cells and programmed cell death ligand-1 (PD-L1) on myeloid cells contribute to immune dysfunction in nonburn-related sepsis. We hypothesized that PD-1/PDL1 interactions contribute to immune dysfunction after burn injury. To determine the impact of burn injury and infection on PD-L1, PD-1 and costimulatory receptor expression by leukocytes and its relationship to T cell functions. The efficacy of anti-PD-L1 antibody was evaluated in a clinically relevant mouse model of burn injury and bacterial infection. Mice underwent 35% scald burn followed by Pseudomonas aeruginosa or Staphylococcus aureus infection on day 4 postburn. Anti-PD-L1 was administered on day 3 postburn. Numbers and phenotype of leukocytes, plasma cytokine concentrations, bacterial clearance, organ injury, and survival were assessed. Burn injury and infection with P. aeruginosa caused a significant upregulation of PD-L1 on myeloid cells, along with a decrease in T cell numbers and function, significant multiorgan injury, and decreased survival. Treatment with anti-PD-L1 antibody improved bacterial clearance, reduced organ injury, and enhanced survival during Pseudomonas burn wound infection. Furthermore, anti-PD-L1 effectively protected against multiorgan injury, and improved bacterial clearance and survival following systemic S. aureus infection after burn injury. Blockade of PD-1/PD-L1 interactions might represent a viable treatment to improve outcomes among critically ill burn-injured subjects and increased leukocyte PD-L1 expression could serve as a valuable biomarker to select appropriate patients for such treatment.

Emerging evidence indicates that even subtle changes in the expression of key genes of signalling pathways can have profound effects. MicroRNAs (miRNAs) are masters of subtlety and generally have only mild effects on their target genes. The microRNA miR-31 is one of the major microRNAs in many cutaneous conditions associated with activated keratinocytes, such as the hyperproliferative diseases psoriasis, non-melanoma skin cancer and hair follicle growth. miR-31 is a marker of the hair growth phase, and in our miR-31 transgenic mouse model it impairs the function of keratinocytes. This leads to aberrant proliferation, apoptosis, and differentiation that results in altered hair growth, while the loss of miR-31 leads to increased hair growth. Through in vitro and in vivo studies, we have defined a set of conserved miR-31 target genes, including LATS2 and STK40, which serve as new players in the regulation of keratinocyte growth and hair follicle biology. LATS2 can regulate growth of keratinocytes and we have identified a function of STK40 that can promote the expression of key hair follicle programme regulators such as HR, DLX3 and HOXC13.

Monophosphoryl lipid A (MPLA), a less toxic derivative of lipopolysaccharide (LPS), is employed as a vaccine adjuvant and is under investigation as a non-specific immunomodulator. However, the differential response of human leukocytes to MPLA and LPS has not been well characterized. The goal of this study was to compare the differential transcriptomic response of human blood to LPS and MPLA. Venous blood from human volunteers was stimulated with LPS, MPLA or vehicle. Gene expression was determined using microarray analysis. Among 21,103 probes profiled, 136 and 130 genes were differentially regulated by LPS or MPLA, respectively. Seventy four genes were up-regulated and 9 were down-regulated by both ligands. The remaining genes were differentially induced by either agent. Ingenuity Pathway Analysis predicted that LPS and MPLA share similar upstream regulators and have comparable effects on canonical pathways and cellular functions. However, some pro-inflammatory cytokine and inflammasome-associated transcripts were more strongly induced by LPS. In contrast, only the macrophage-regulating chemokine CCL7 was preferentially up-regulated by MPLA. In conclusion, LPS and MPLA induce similar transcriptional profiles. However, LPS more potently induces pro-inflammatory cytokine and inflammasome-linked transcripts. Thus, MPLA is a less potent activator of the pro-inflammatory response but retains effective immunomodulatory activity.

The HIV-1 accessory protein Nef is considered to play an important role in the development of a podocyte phenotype in HIV-1 associated nephropathy. We hypothesized that Nef may be altering the podocyte phenotype both structurally and functionally. To elucidate the involved mechanisms, podocyte proteins interacting with Nef were identified using GST pull down assay and yeast two hybrid assay. The GST pull down assay on protein extracts made from stable colonies of conditionally immortalized human podocytes expressing Nef (Nef/CIHP) displayed a band at 45 kD, which was identified as actin by mass spectrometry. Yeast two hybrid assay identified the following Nef-interacting proteins: syntrophin, filamin B, syntaxin, translational elongation factor 1, and zyxin. The Nef-actin and Nef-zyxin interactions were confirmed by co-localization studies on Nef/CIHP stable cell lines. The co-localization studies also showed that Nef/CIHP stable cell lines had a decreased number of actin filaments (stress fibers), displayed formation of lamellipodia, and increased number of podocyte projections (filopodia). Nef/CIHP displayed an enhanced cortical F-actin score index (P

Sepsis is a leading cause of death among severely burned patients. Burn injury disrupts the protective skin barrier and causes immunological dysfunction. In our previous studies, we found that burn injury and wound infection causes a significant decline in lymphocyte populations, implying adaptive immune system dysfunction. In the present study, we examined the effect of treatment with Fms-like tyrosine kinase-3 Ligand (Flt3L) on T cell phenotype and function in a model of burn wound sepsis. FLt3L is an essential cytokine required for hematopoietic progenitor cell development and expansion of both myeloid and lymphoid lineages. Flt3L has been shown to potentiate innate immune functions of dendritic cells and neutrophils during burn wound sepsis. However, the ability of Flt3L to improve T cell function during burn wound sepsis has not been previously evaluated.Mice underwent 35% total body surface area scald burn and were treated with Flt3L (10 μg) or vehicle daily via the intraperitoneal route starting 1 day after burn injury. On day 4 after burn injury, Pseudomonas aeruginosa was used to induce wound infection. Leukocytes in spleen and wound draining lymph nodes were characterized using flow cytometry. Bacterial clearance, organ injury, and survival were also assessed.Flt3L treatment prevented the decline in splenic CD4 and CD8 T cells caused by burn injury and infection. Flt3L treatment also attenuated the decline in CD28 expression on CD4 and CD8 T cells and IFNγ production by CD8 T cells in the spleen and wound draining lymph nodes. Furthermore, Flt3L decreased the levels of programmed death ligand 1 expression on splenic dendritic cells and macrophages. Flt3 treatment improved systemic bacterial clearance, decreased liver and kidney injury, and significantly improved survival in mice with burn wound sepsis.Burn injury and associated sepsis causes significant loss of T cells and evidence of T cell dysfunction. Flt3L attenuates T cell dysfunction and improves host resistance to burn wound sepsis in mice.

Abnormalities in type III collagen in the arterial walls cause certain familial intracranial aneurysms (IAs); however, it remains unknown whether COL3A1 variants contribute to the risk of sporadic IAs. To study whether COL3A1 variants are associated with sporadic IAs, the association of COL3A1 variants with sporadic IAs was tested in 298 cases and 488 controls, replicated in an independent population of 192 cases and 1,690 controls, and further verified in 633 patients with intra-cerebral hemorrhage, 1,074 hypertensives, and 1,883 controls. We found that allele A of SNP rs1800255 conferred a 1.71-fold increased risk for IAs (adjusted odds ratio: OR = 1.71, 95% confidence interval: CI 1.19-2.45, P = 0.004) and results in an amino acid change of Ala698Thr, which led to a lower thermal stability of the peptide. These results were confirmed in the independent study. The associations were independent of the presence of hemorrhagic stroke and hypertension. These results support the view that the functional variant of COL3A1 is genetic risk factors for IAs in the Chinese population.

An important issue raised in testing new neuroprotective/restorative treatments for Parkinson's disease (PD) is the optimal stage in the disease process to initiate therapy. Current palliative treatments are effective in the early disease stages raising ethical concerns about substituting an experimental treatment for a proven therapy. Thus, we have endeavored to create a stable 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP) nonhuman primate model of early PD. The new model was created by controlling for dose and route administration of MPTP (unilateral intracarotid infusion), and age of the animals (middleaged, 16–19 years old) in 27 female rhesus monkeys. All animals showed stable parkinsonian features lasting for up to 12-month as per behavioral evaluation. Compared with late-stage PD animals, postmortem analysis demonstrated that more dopaminergic neurons remained in the substantia nigra pars compacta, and more fibers were found in the striatum. In addition, tissue levels of striatal dopamine and its metabolites were also higher. Our results support that a milder but stable PD model can be produced in middle-aged rhesus monkeys.

Infection is the leading cause of death in severely burned patients that survive the acute phase of injury. Neutrophils are the first line of defense against infections, but hospitalized burn patients frequently cannot mount an appropriate innate response to infection. Thus, immune therapeutic approaches aimed at improving neutrophil functions after burn injury may be beneficial. Prophylactic treatment with the TLR4 agonist monophosphoryl lipid A is known to augment resistance to infection by enhancing neutrophil recruitment and facilitating bacterial clearance. This study aimed to define mechanisms by which monophosphoryl lipid A treatment improves bacterial clearance and survival in a model of burn-wound sepsis. Burn-injured mice were treated with monophosphoryl lipid A or vehicle, and neutrophil mobilization was evaluated in the presence or absence of Pseudomonas aeruginosa infection. Monophosphoryl lipid A treatment induced significant mobilization of neutrophils from the bone marrow into the blood and sites of infection. Neutrophil mobilization was associated with decreased bone marrow neutrophil CXCR4 expression and increased plasma G-CSF concentrations. Neutralization of G-CSF before monophosphoryl lipid A administration blocked monophosphoryl lipid A-induced expansion of bone marrow myeloid progenitors and mobilization of neutrophils into the blood and their recruitment to the site of infection. G-CSF neutralization ablated the enhanced bacterial clearance and survival benefit endowed by monophosphoryl lipid A in burn-wound-infected mice. Our findings provide convincing evidence that monophosphoryl lipid A-induced G-CSF facilitates early expansion, mobilization, and recruitment of neutrophils to the site of infection after burn injury, allowing for a robust immune response to infection.

IL-15 is currently undergoing clinical trials to assess its efficacy for treatment of advanced cancers. The combination of IL-15 with soluble IL-15Rα generates a complex termed IL-15 superagonist (IL-15 SA) that possesses greater biological activity than IL-15 alone. IL-15 SA is considered an attractive antitumor and antiviral agent because of its ability to selectively expand NK and memory CD8+ T (mCD8+ T) lymphocytes. However, the adverse consequences of IL-15 SA treatment have not been defined. In this study, the effect of IL-15 SA on physiologic and immunologic functions of mice was evaluated. IL-15 SA caused dose- and time-dependent hypothermia, weight loss, liver injury, and mortality. NK (especially the proinflammatory NK subset), NKT, and mCD8+ T cells were preferentially expanded in spleen and liver upon IL-15 SA treatment. IL-15 SA caused NK cell activation as indicated by increased CD69 expression and IFN-γ, perforin, and granzyme B production, whereas NKT and mCD8+ T cells showed minimal, if any, activation. Cell depletion and adoptive transfer studies showed that the systemic toxicity of IL-15 SA was mediated by hyperproliferation of activated NK cells. Production of the proinflammatory cytokine IFN-γ, but not TNF-α or perforin, was essential to IL-15 SA–induced immunotoxicity. The toxicity and immunological alterations shown in this study are comparable to those reported in recent clinical trials of IL-15 in patients with refractory cancers and advance current knowledge by providing mechanistic insights into IL-15 SA–mediated immunotoxicity.

Immunosuppression is increasingly being recognized as one of the causes of increased morbidity and mortality during sepsis. Both innate and adaptive immune system dysfunction have been shown to cause an impaired ability to eradicate the primary infection and also lead to frequent occurrence of secondary opportunistic infections. Pre-clinical and clinical studies have shown that inhibitory immune checkpoint molecules, including programmed death-1 (PD-1), programmed death ligand-1 (PD-L1), cytotoxic T lymphocyte antigen-4 (CTLA-4), T cell membrane protein-3 (TIM-3), Lymphocyte activation-gene-3 (LAG-3) and 2B4, are upregulated during the course of sepsis. Engagement of these inhibitory molecules on various immune cells has been consistently shown to inhibit innate immune cell functions (e.g., phagocytosis, cytokine production and pathogen clearance) and also lead to impaired T cell competence. In numerous pre-clinical models of sepsis, therapeutic agents aimed at blocking engagement of inhibitory immune checkpoints on immune cells have been shown to improve innate and adaptive immune cell functions, increase host resistance to infection and significantly improve survival. Therefore, immunotherapy with immune cell checkpoint inhibitors holds significant potential for the future of sepsis therapy and merits further investigation.

Septic shock remains one of the leading causes of death in the USA. However, the immunological mechanisms of septic shock are still not well understood. Interleukin (IL)-15 is an essential mediator for natural killer (NK) and memory (m) CD8+T cell survival, as indicated by the deficits of these cells in IL-15 knockout (KO) mice. As NK and CD8+T cells are implicated in the pathogenesis of septic shock, we hypothesized that IL-15KO mice will be resistant to septic shock due to their lack of NK and mCD8+T cells. IL-15KO mice showed a significant survival advantage over wild type (WT) control mice during septic shock caused by cecal ligation and puncture (CLP) or LPS challenge. IL-15KO mice displayed less sepsis-induced hypothermia and attenuated plasma IL-6 production in our sepsis models. Treatment with low-dose IL-15 superagonist (SA) regenerated NK and mCD8+T cells in IL-15KO mice and reestablished mortality in these mice during CLP- and LPS-induced septic shock. However, if NK and CD8+T cell regeneration was prevented by anti-asialoGM1 and anti-CD8α prior to IL-15 SA treatment, IL-15 SA failed to reestablish sepsis-induced mortality in IL-15 KO mice. When given to WT mice 4 days prior to CLP or LPS challenge, M96, an IL-15 neutralizing IgG, caused 80.8% depletion of splenic NK cells and conferred protection against septic shock. However, when given shortly prior to CLP or LPS challenge, M96 did not deplete splenic NK cells and failed to confer protection against septic shock. In conclusion, IL-15 contributes to the lethality of septic mice by maintaining NK and mCD8+ T cell populations, but does not play a direct role in septic shock. Thus, NK and mCD8+ T cells may represent effective therapeutic targets for septic shock.

Infection is the leading cause of death in severely burned patients. Prophylactic treatment with the TLR4 agonist monophosphoryl lipid A (MPLA) induces resistance to subsequent bacterial challenge. Treatment of mice with MPLA enhances bacterial clearance, leading to improved survival in a model of P. aeruginosa burn wound infection. The current study was aimed to define the mechanisms responsible for improved bacterial clearance and survival in MPLA-treated burn-infected mice, and to determine if MPLA could protect against clinically relevant Gram positive and fungal pathogens. Mice underwent severe burn injury, followed by systemic treatment with MPLA or vehicle control for 2 days. Mice were then inoculated with P. aeruginosa topically or intraperitoneally, and responding neutrophils were measured in bone marrow, blood, burn wound and peritoneal cavity. In later experiments, burned mice were challenged systemically with S. aureus or C. albicans. MPLA treatment induced G-CSF production, decreased bone marrow neutrophil numbers and increased neutrophil numbers in the blood, peritoneal cavity and burn wound site. G-CSF was essential for MPLA-mediated survival, bacterial clearance and neutrophil trafficking. MPLA also improved survival against S. aureus and C. albicans systemic infections after burn. This suggests that G-CSF facilitates MPLA-induced trafficking of neutrophils, allowing for a more rapid response to sites of infection. The ability of MPLA to enhance antimicrobial responses and its ability to protect against a variety of clinically relevant pathogens make it an attractive therapeutic candidate for use in burn patients for the prevention of post-burn infections.

Interleukin (IL)-15 is currently undergoing clinical trials to assess its anti-tumor efficacy. IL-15 is essential for the generation, function and survival of natural killer (NK), natural killer T (NKT) and memory phenotype (MP) CD8+T cells. The combination of IL-15 with its soluble IL-15 receptor α (IL-15Rα) generates a complex termed IL-15 superagonist (IL-15 SA) that exhibits more profound biological activities on NK, NKT and MP CD8+T cells than IL-15 alone. Thus, IL-15 SA is a potentially more attractive therapeutic agent for use against advanced tumors. However, the safety of IL-15 SA administration in vivo is not clearly elucidated. This study was designed to evaluate toxic consequences of IL-15 SA in mice. Administration of 2 μg IL-15 SA for 4 days caused a time- and dose-dependent immune-toxicity, characterized by hypothermia, weight loss, liver damage and death. At 2ug, IL-15 SA expanded NK cells in spleens and livers compared to vehicle and significantly increased NK cell expression of CD69, NKG2D and NKp46 as well as production of IFNγ, granzyme B and perforin. IL-15 SA also prompted NK cell maturity with a preferential expansion of the pro-inflammatory (CD27+ CD11b+) NK subset. Mice depleted of NK cells by anti-asialoGM1or anti-NK1.1 were resistant to IL-15 SA-induced toxicity, whereas adoptive transfer of wild type NK cells to Rag2−/−γc−/− mice re-established IL-15 SA toxicity. While IL-15 SA expanded NKT and mCD8+ T cells, mice depleted of NKT or CD8+ T cells remained sensitive to IL-15 SA-mediated toxicity. In addition, IFNγ KO, but not TNF-α KO or perforin KO, mice were resistant to IL-15 SA-induced toxicity. In conclusion, high-dose IL-15 SA causes systemic immunotoxicity via induction of NK cell proliferation and activation.

Background Animal models that mimic human biology are important for successful translation of basic science discoveries into the clinical practice. Recent studies in rodents have demonstrated the efficacy of TLR4 agonists as immunomodulators in models of infection. However, rodent models have been criticized for not mimicking important characteristics of the human immune response to microbial products. The goal of this study was to compare genomic responses of human and sheep blood to the TLR4 agonists lipopolysaccharide (LPS) and monophosphoryl lipid A (MPLA). Methods Venous blood, withdrawn from six healthy human adult volunteers (~ 28 years old) and six healthy adult female sheep (~3 years old), was mixed with 30 μL of PBS, LPS (1μg/mL) or MPLA (10μg/mL) and incubated at room temperature for 90 minutes on a rolling rocker. After incubation, 2.5 mL of blood was transferred to Paxgene Blood RNA tubes. Gene expression analysis was performed using an Agilent Bioanalyzer with the RNA6000 Nano Lab Chip. Agilent gene expression microarrays were scanned with a G2565 Microarray Scanner. Differentially expressed genes were identified. Results 11,431 human and 4,992 sheep probes were detected above background. Among them 1,029 human and 175 sheep genes were differentially expressed at a stringency of 1.5-fold change (p 1.5-fold changes in human samples. Genes of major inflammatory mediators, such as IL-1, IL-6 and IL-8, TNF alpha, NF-kappaB, ETS2, PTGS2, PTX3, CXCL16, KYNU, and CLEC4E were similarly (>2-fold) upregulated by LPS and MPLA in both species. Conclusion The genomic responses of peripheral blood to LPS and MPLA in sheep are quite similar to those observed in humans, supporting the use of the ovine model for translational studies that mimic human inflammatory diseases and the study of TLR-based immunomodulators.

The adherens junction (AJ) is important for maintaining uterine structural integrity, composition of the luminal environment, and initiation of implantation by virtue of its properties of cell-cell recognition, adhesion, and establishment of cell polarity and permeability barriers. In this study, we investigated the uterine changes of AJ components E-cadherin, beta-catenin, and alpha-catenin at their mRNA and protein levels, together with the cellular distribution of meprinbeta, phospho-beta-catenin, and active beta-catenin proteins, in hamsters that show only ovarian progesterone-dependent uterine receptivity and implantation. By in situ hybridization and immunofluorescence, we have demonstrated that uterine epithelial cells expressed three of these AJ proteins and their mRNAs prior to and during the initial phase of implantation. Immunofluorescence study showed no change in epithelial expression patterns of uterine AJ proteins from Days 1 to 5 of pregnancy. With advancement of the implantation process, AJ components were primarily expressed in cells of the secondary decidual zone (SDZ), but not in the primary decidual zone (PDZ). In contrast, we noted strong expression of beta-catenin and alpha-catenin proteins in the PDZ, but not in the SDZ, of mice. Taken together, these results suggest that AJ proteins contribute to uterine barrier functions by cell-cell adhesion to ensure protection of the embryo. In addition, cleavage of E-cadherin by meprinbeta might contribute to weakening uterine epithelial cell-cell contact for blastocyst implantation. We also report that the nuclear localization of active beta-catenin from Day 4 onward in hamsters implies that beta-catenin/Wnt-signal transduction is activated in the uterus during implantation and decidualization.

The objective was to study the expression of zonula occludens-2, a tight junction protein, during preimplantation hamster embryonic development, to predict its possible localization, source, and roles in trophectoderm differentiation and blastocyst formation in this species. Comparison of zonula occludens-2 expression pattern between the hamster and mouse preimplantation embryos from the zygote up to the blastocyst stage was also an objective of this study. Zonula occludens-2 localization was noted in nuclei of blastomeres in all stages of hamster and mouse embryonic development. Compared to mice, where zonula occludens-2 was first localized in the interblastomere membrane at the morula stage, hamster embryos had membranous zonula occludens-2 localization from the 2-cell stage onwards. Based on combined results of immunolocalization study in parthenogenic embryos and ovarian and epididymal sections, and quantitative PCR done in oocytes and all developmental stages of preimplantation embryos, perhaps there was a carry-over of zonula occludens-2 proteins or mRNA from the dam to the embryo. Based on these findings, we inferred that maternally derived zonula occludens-2 was involved in nuclear functions, as well as differentiation of blastomeres and blastocoel formation during preimplantation embryonic development in the hamster.

Angiotensin II (Ang II) has been reported to play an important role in both the development and progression of renal injury. Many of the downstream effects of Ang II are mediated through the activation of nuclear factor-kappaB (NF-kappaB). In the present study, we evaluated the effect of Ang II on the activation of Ets-1 (a transcription factor) in tubular cells. In addition, we studied the expression of pro-inflammatory molecules transcribed by Ets-1 in response to Ang II. Mice receiving Ang II infusion showed enhanced renal cortical mRNA expression of Ets-1. Immunolabeling studies localized the expression of Ets-1 in distal tubular cells of mice receiving Ang II. However, this effect of Ang II was mitigated by telmisartan, an AT1-receptor blocker. Mice receiving Ang II infusion also showed increased tubular cell expression of macrophage chemoattractant protein-1 (MCP-1), plasminogen activator inhibitor-1 (PAI-1), and p21 when compared with control mice; nevertheless, this effect of Ang II was attenuated by telmisartan. In in vitro studies, Ang II enhanced mRNA expression of Ets-1 by MDCK cells. However, this effect of Ang II was inhibited by losartan, an AT1-receptor blocker. Losartan also inhibited Ang II-induced PAI-1 and p21 expression by MDCK cells. These findings indicate that Ang II-induced tubular cell expression Ets-1 and associated downstream signaling is mediated through AT1 receptors.

There is a great need for the development of noninvasive, highly sensitive, and widely available imaging methods that can potentially be used to longitudinally monitor treatment of Parkinson's disease (PD). Here we report the monitoring of GDNF-induced functional changes of the basal ganglia in hemiparkinsonian monkeys via pharmacological MRI measuring the blood oxygenation level-dependent (BOLD) response to a direct dopamine agonist (apomorphine, APO). After testing BOLD responsiveness to APO in their normal state, two additional scans were taken with the same dose of APO stimulation after induced parkinsonism. Then all animals were chronically treated with GDNF for 18 weeks by a programmable pump and catheter system. The catheter was surgically implanted into the right putamen and connected to the pump via flexible polyurethane tubing. phMRI scans were taken at both 6 and 18 weeks while they received 22.5 μg of GDNF per day. In addition, behavioral changes were monitored throughout the entire study. The primary finding of this study was that APO-evoked activations in the DA denervated putamen were attenuated by the chronic intraputamenal infusion of GDNF accompanied by improvements of parkinsonian features, movement speed, and APO-induced rotation compared to data collected before the chronic GDNF treatment. The results suggest that phMRI methods in combination with administration of a selective DA agonist may be useful for monitoring neurorestorative therapies in PD patients in the future.

Aldosterone has attracted significant consideration for its role in the progression of renal injury. Since apoptotic cell loss contributes to the deterioration of renal function, we examined the effect of aldosterone on tubular cell apoptosis. To determine dose and time course effect, human renal proximal tubular (HK2) cells were treated with aldosterone at different doses and for variable time periods followed by evaluation for apoptosis. To determine the role of mineralocorticoid receptors (MR) and oxidative stress, HK2 cells were treated with either vehicle or aldosterone in the presence or absence of spironolactone/antioxidants/free radical scavengers (FRS) followed by evaluation for apoptosis. The presence of MR was evaluated using RT-PCR. Reactive oxygen species (ROS) generation was evaluated using redox-sensitive dyes. Effect of aldosterone was evaluated on dephosphorylation of phospho-Bad and accumulation of cytosolic cytochrome c. Human tubular cells express MR. Aldosterone promotes tubular cell apoptosis in a dose- and time-dependent manner. This effect of aldosterone is mediated through MR and associated with generation of ROS. Antioxidants and FRS partially attenuated the proapoaptotic effect of aldosterone. Aldosterone enhanced dephosphorylation of phospho-Bad and accumulation of cytosolic cytochrome c. We conclude that aldosterone-induced tubular cell apoptosis is mediated through the activation of the mitochondrial pathway and generation of ROS.