Muscle metaboreflex stimulates the cardiac sympathetic afferent reflex causing positive feedback amplification of sympathetic activity: effect of heart failure.

Exercise intolerance is a hallmark symptom of heart failure and to a large extent stems from reductions in cardiac output that occur due to the inherent ventricular dysfunction coupled with enhanced muscle metaboreflex-induced functional coronary vasoconstriction, which limits increases in coronary blood flow. This creates a further mismatch between O2 delivery and O2 demand, which may activate the cardiac sympathetic afferent reflex (CSAR), causing amplification of the already increased sympathetic activity in a positive-feedback fashion. We used our chronically instrumented conscious canine model to evaluate if chronic ablation of afferents responsible for the CSAR would attenuate the gain of muscle metaboreflex before and after induction of heart failure. After afferent ablation, the gain of the muscle metaboreflex control of mean arterial pressure was significantly reduced before (-239.5 ± 16 to -95.2 ± 8 mmHg/L/min) and after the induction of heart failure (-185.6 ± 14 to -95.7 ± 12 mmHg/L/min). Similar results were observed for the strength (gain) of muscle metaboreflex control of heart rate, cardiac output, and ventricular contractility. Thus, we conclude that the CSAR contributes significantly to the strength of the muscle metaboreflex in normal animals with heart failure serving as an effective positive-feedback amplifier thereby further increasing sympathetic activity.NEW & NOTEWORTHY The powerful pressor responses from the CSAR arise via O2 delivery versus O2 demand imbalance. Muscle metaboreflex activation (MMA) simultaneously elicits coronary vasoconstriction (which is augmented in heart failure) and profound increases in cardiac work thereby upsetting oxygen balance. Whether MMA activates the CSAR thereby amplifying MMA responses is unknown. We observed that removal of the CSAR afferents attenuated the strength of the muscle metaboreflex in normal and subjects with heart failure.

The orphan nuclear receptor NR4A1 specifies a distinct subpopulation of quiescent myeloid-biased long-term HSCs.

Hematopoiesis is maintained throughout life by self-renewing hematopoietic stem cells (HSCs) that differentiate to produce both myeloid and lymphoid cells. The NR4A family of orphan nuclear receptors, which regulates cell fate in many tissues, appears to play a key role in HSC proliferation and differentiation. Using a NR4A1(GFP) BAC transgenic reporter mouse we have investigated NR4A1 expression and its regulation in early hematopoiesis. We show that NR4A1 is most highly expressed in a subset of Lin(-) Sca-1(+) c-Kit(+) CD48(-) CD150(+) long-term (LT) HSCs, and its expression is tightly associated with HSC quiescence. We also show that NR4A1 expression in HSCs is induced by PGE2, a known enhancer of stem cell engraftment potential. Finally, we find that both NR4A1(GFP+) and NR4A1(GFP-) HSCs successfully engraft primary and secondary irradiated hosts; however, NR4A1(GFP+) HSCs are distinctly myeloid-biased. These results show that NR4A1 expression identifies a highly quiescent and distinct population of myeloid-biased LT-HSCs.

Optical Imaging Demonstrates Tissue-Specific Metabolic Perturbations in Mblac1 Knockout Mice.

OBJECTIVE: Metabolic changes have been extensively documented in neurodegenerative brain disorders, including Parkinson's disease and Alzheimer's disease (AD). Mutations in the C. elegans swip-10 gene result in dopamine (DA) dependent motor dysfunction accompanied by DA neuron degeneration. Recently, the putative human ortholog of swip-10 (MBLAC1) was implicated as a risk factor in AD, a disorder that, like PD, has been associated with mitochondrial dysfunction. Interestingly, the AD risk associated with MBLAC1 arises in subjects with cardiovascular morbidity, suggesting a broader functional insult arising from reduced MBLAC1 protein expression and one possibly linked to metabolic alterations. METHODS: Our current studies, utilizing Mblac1 knockout (KO) mice, seek to determine whether mitochondrial respiration is affected in the peripheral tissues of these mice. We quantified the levels of mitochondrial coenzymes, NADH, FAD, and their redox ratio (NADH/FAD, RR) in livers and kidneys of wild-type (WT) mice and their homozygous KO littermates of males and females, using 3D optical cryo-imaging. RESULTS: Compared to WT, the RR of livers from KO mice was significantly reduced, without an apparent sex effect, driven predominantly by significantly lower NADH levels. In contrast, no genotype and sex differences were observed in kidney samples. Serum analyses of WT and KO mice revealed significantly elevated glucose levels in young and aged KO adults and diminished cholesterol levels in the aged KOs, consistent with liver dysfunction. DISCUSSION/CONCLUSION: As seen with C. elegans swip-10 mutants, loss of MBLAC1 protein results in metabolic changes that are not restricted to neural cells and are consistent with the presence of peripheral comorbidities accompanying neurodegenerative disease in cases where MBLAC1 expression changes impact risk.

Discovery of a Series of Potent, Selective, and Orally Bioavailable Nucleoside Inhibitors of CD73 That Demonstrates In Vivo Antitumor Activity.

CD73 (ecto-5'-nucleotidase) has emerged as an attractive target for cancer immunotherapy of many cancers. CD73 catalyzes the hydrolysis of adenosine monophosphate (AMP) into highly immunosuppressive adenosine that plays a critical role in tumor progression. Herein, we report our efforts in developing orally bioavailable and highly potent small-molecule CD73 inhibitors from the reported hit molecule 2 to lead molecule 20 and then finally to compound 49. Compound 49 was able to reverse AMP-mediated suppression of CD8+ T cells and completely inhibited CD73 activity in serum samples from various cancer patients. In preclinical in vivo studies, orally administered 49 showed a robust dose-dependent pharmacokinetic/pharmacodynamic (PK/PD) relationship that correlated with efficacy. Compound 49 also demonstrated the expected immune-mediated antitumor mechanism of action and was efficacious upon oral administration not only as a single agent but also in combination with either chemotherapeutics or checkpoint inhibitor in the mouse tumor model.

Three-percent saline administration during pediatric critical care transport.

OBJECTIVES: The purpose of this study was to describe the administration of 3% saline (3%S) during pediatric critical care transport. METHODS: A retrospective study was performed on pediatric patients who underwent critical transport to Loma Linda University Children's Hospital from January 1, 2003, to June 30, 2007, and were given 3%S. Patients' demographics, admission diagnosis, route and amount of 3%S administration, serum electrolytes, vital signs, radiographic data, and Glasgow Coma Scale scores were collected and analyzed. RESULTS: A total of 101 children who received 3%S infusions during pediatric critical care transport were identified. Mean patient age was 5.9 years, and mean patient weight was 27.6 kg. The main indications for infusing 3%S were suspected cerebral edema (41%), intracranial bleed with edema (51%), and symptomatic hyponatremia (6%). The amount of 3%S bolus ranged from 1.2 to 24 mL/kg, with a mean of 5.4 mL/kg. Serum electrolytes before and after 3%S infusion demonstrated significant increases in sodium, chloride, and bicarbonate levels (P < 0.05). A significant reduction was also seen in serum urea nitrogen levels and anion gap. Radiographic imaging performed before 3%S infusion demonstrated findings consistent with concerns of increased intracranial pressure such as intracranial bleed and cerebral edema. The route of initial 3%S infusions was mainly through peripheral intravenous lines (96%). No complications related to the 3%S delivery such as local reactions, renal abnormalities, or central pontine myelinolysis were observed. CONCLUSIONS: It seems 3%S may be administered safely during pediatric critical transport and administration routes can include peripheral lines. With the importance of initiating therapy early to improve patient outcomes, the use of 3%S may benefit transported children with brain injury and suspected intracranial hypertension.

Prednisone-induced sustained remission in a patient with familial fibronectin glomerulopathy (GFND).

Glomerulopathy with Fibronectin Deposits (GFND) is a rare, autosomal dominant disease characterized by proteinuria, hematuria and progressive renal failure associated with glomerular deposition of fibronectin, frequently resulting in end-stage renal disease (ESRD). There is no established treatment for this condition beyond conservative measures such as blood pressure control and the use of angiotensin-converting enzyme (ACE) inhibitors. We present a case of GFND associated with progressive chronic kidney disease (CKD) and nephrotic range proteinuria showing a sustained response to prednisone treatment. A 27-year-old G2P2 Caucasian female presented with 3 g/day of proteinuria, serum creatinine (Cr) 0.7 mg/dL, inactive urinary sediment and normotension without medication. She was part of a large family with glomerular disease, including three members who died of cerebral hemorrhage or stroke in their thirties. The patient's kidney biopsy showed mesangial deposition of fibronectin consistent with GFND. No interstitial fibrosis was seen. Genotyping revealed the Y973C FN1 gene mutation. Despite maximal tolerable ACE inhibition, proteinuria increased to 4-6 g/g Cr and serum Cr increased to 1.0 mg/dL. She was treated with prednisone 60 mg (~ 1 mg/Kg) daily for 2 mos and then tapered by ~ 0.2 mg/Kg every month for 6 mos of total therapy. Proteinuria decreased to ~ 1 g/g Cr for > 5 years and serum Cr stabilized in the 1.2 mg/dL range with treatment. No significant side effects were encountered. In conclusion, this protocol should be considered in GFND patients with nephrotic range proteinuria despite maximal angiotensin system inhibition who have relatively preserved renal function.

Ibudilast in healthy volunteers: safety, tolerability and pharmacokinetics with single and multiple doses.

AIMS: To investigate the safety, tolerability and pharmacokinetics (PK) of ibudilast after a single-dose and a multiple-dose regimen. METHODS: Healthy adult male (n = 9) and female (n = 9) volunteers were evaluated over a 17-day stay in a Phase 1 unit. Subjects were randomized 1 : 3 to either oral placebo or ibudilast at 30-mg single administration followed by 14 days of 30 mg b.i.d. Complete safety analyses were performed and, for PK, plasma and urine samples were analysed for ibudilast and its major metabolite. RESULTS: Ibudilast was generally well tolerated. No serious adverse events occurred. Treatment-related adverse events included hyperhidrosis, headache and nausea. Two subjects discontinued after a few days at 30 mg b.i.d. because of vomiting. Although samples sizes were too small to rule out a sex difference, PK were similar in men and women. The mean half-life for ibudilast was 19 h and median T(max) was 4-6 h. Mean (SD) steady-state plasma C(max) and AUC(0-24) were 60 (25) ng ml(-1) and 1004 (303) ng h ml(-1), respectively. Plasma levels of 6,7- dihydrodiol-ibudilast were approximately 30% of the parent. CONCLUSIONS: Ibudilast is generally well tolerated in healthy adults when given as a single oral dose of 30 mg followed by 30 mg b.i.d. (60 mg day(-1)) for 14 days. Plasma PK reached steady state within 2 days of starting the b.i.d. regimen. Exposure to ibudilast was achieved of a magnitude comparable to that associated with efficacy in rat chronic pain models.

Inhibition of arginase by CB-1158 blocks myeloid cell-mediated immune suppression in the tumor microenvironment.

BACKGROUND: Myeloid cells are an abundant leukocyte in many types of tumors and contribute to immune evasion. Expression of the enzyme arginase 1 (Arg1) is a defining feature of immunosuppressive myeloid cells and leads to depletion of L-arginine, a nutrient required for T cell and natural killer (NK) cell proliferation. Here we use CB-1158, a potent and orally-bioavailable small-molecule inhibitor of arginase, to investigate the role of Arg1 in regulating anti-tumor immunity. METHODS: CB-1158 was tested for the ability to block myeloid cell-mediated inhibition of T cell proliferation in vitro, and for tumor growth inhibition in syngeneic mouse models of cancer as a single agent and in combination with other therapies. Tumors from animals treated with CB-1158 were profiled for changes in immune cell subsets, expression of immune-related genes, and cytokines. Human tumor tissue microarrays were probed for Arg1 expression by immunohistochemistry and immunofluorescence. Cancer patient plasma samples were assessed for Arg1 protein and L-arginine by ELISA and mass spectrometry, respectively. RESULTS: CB-1158 blocked myeloid cell-mediated suppression of T cell proliferation in vitro and reduced tumor growth in multiple mouse models of cancer, as a single agent and in combination with checkpoint blockade, adoptive T cell therapy, adoptive NK cell therapy, and the chemotherapy agent gemcitabine. Profiling of the tumor microenvironment revealed that CB-1158 increased tumor-infiltrating CD8+ T cells and NK cells, inflammatory cytokines, and expression of interferon-inducible genes. Patient tumor samples from multiple histologies expressed an abundance of tumor-infiltrating Arg1+ myeloid cells. Plasma samples from cancer patients exhibited elevated Arg1 and reduced L-arginine compared to healthy volunteers. CONCLUSIONS: These results demonstrate that Arg1 is a key mediator of immune suppression and that inhibiting Arg1 with CB-1158 shifts the immune landscape toward a pro-inflammatory environment, blunting myeloid cell-mediated immune evasion and reducing tumor growth. Furthermore, our results suggest that arginase blockade by CB-1158 may be an effective therapy in multiple types of cancer and combining CB-1158 with standard-of-care chemotherapy or other immunotherapies may yield improved clinical responses.

Targeted Inhibition of EGFR and Glutaminase Induces Metabolic Crisis in EGFR Mutant Lung Cancer.

Cancer cells exhibit increased use of nutrients, including glucose and glutamine, to support the bioenergetic and biosynthetic demands of proliferation. We tested the small-molecule inhibitor of glutaminase CB-839 in combination with erlotinib on epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) as a therapeutic strategy to simultaneously impair cancer glucose and glutamine utilization and thereby suppress tumor growth. Here, we show that CB-839 cooperates with erlotinib to drive energetic stress and activate the AMP-activated protein kinase (AMPK) pathway in EGFR (del19) lung tumors. Tumor cells undergo metabolic crisis and cell death, resulting in rapid tumor regression in vivo in mouse NSCLC xenografts. Consistently, positron emission tomography (PET) imaging with 18F-fluoro-2-deoxyglucose (18F-FDG) and 11C-glutamine (11C-Gln) of xenografts indicated reduced glucose and glutamine uptake in tumors following treatment with CB-839 + erlotinib. Therefore, PET imaging with 18F-FDG and 11C-Gln tracers can be used to non-invasively measure metabolic response to CB-839 and erlotinib combination therapy.

DNA binding ligands with improved in vitro and in vivo potency against drug-resistant Staphylococcus aureus.

Potent in vivo activity against methicillin-resistant Staphylococcus aureus (MRSA) has been difficult to achieve with previously reported DNA binding antibacterials. Herein, we describe an efficient access to a focused library of new analogues yielding compounds with improved activity in a mouse peritonitis model. The most potent molecules (14 and 19) exhibit efficacy against MRSA at ED50 values of approximately 1 and approximately 5 mg/kg, respectively, and display excellent in vitro activity against vancomycin-resistant S. aureus.

DNA binding ligands targeting drug-resistant bacteria: structure, activity, and pharmacology.

We describe the lead optimization and structure-activity relationship of DNA minor-groove binding ligands, a novel class of antibacterial molecules. These compounds have been shown to target A/T-rich sites within the bacterial genome and, as a result, inhibit DNA replication and RNA transcription. The optimization was focused on N-terminal aromatic heterocycles and C-terminal amines and resulted in compounds with improved in vivo tolerability and excellent in vitro antibacterial potency (MIC >/= 0.031 microg/mL) against a broad range of Gram-positive pathogens, including drug-resistant strains such as methicillin-resistant Stapylococcus aureus (MRSA), penicillin-resistant Streptococcus pneumoniae (PRSP), and vancomycin-resistant Enterococcus faecalis (VRE). In a first proof-of-concept study, a selected compound (35) showed in vivo efficacy in a mouse peritonitis model against methicillin-sensitive S. aureus infection with an ED(50) value of 30 mg/kg.

Helium-oxygen therapy for pediatric acute severe asthma requiring mechanical ventilation.

OBJECTIVE: To illustrate the use of helium-oxygen gas mixtures as therapy for pediatric patients with acute severe asthma requiring conventional mechanical ventilation. DESIGN: Retrospective review. SETTING: Tertiary care children's teaching hospital. PATIENTS: All mechanically ventilated patients with severe asthma admitted to the pediatric intensive care unit from August 1994 to October 2000. INTERVENTIONS: Within 24 hrs of intubation or admission, patients were stabilized on volume ventilation, bronchodilator therapy, corticosteroids, and antibiotics when indicated. Hypercapnia was permitted while maintaining arterial blood gas pH > or =7.25. A helium-oxygen gas mixture then was begun with helium flow set at 5-7 L/min, and oxygen flow was titrated to maintain desired oxygen saturation. Only sedated, chemically paralyzed patients with adequate pre-helium-oxygen and post-helium-oxygen measurements were statistically analyzed. MEASUREMENTS AND MAIN RESULTS: Twenty-eight mechanically ventilated patients with severe asthma placed on helium-oxygen gas mixtures were identified who met study entry criteria. Mean patient age was 8.8 yrs (range, 1.1-14.6). Before helium-oxygen therapy began, mean peak inspiratory pressure was 40.5 +/- 4.2 cm H(2)O, mean arterial blood gas pH was 7.26 +/- 0.05, and mean CO(2) partial pressure was 58.2 +/- 8.5 torr. After patients were placed on helium-oxygen therapy, there was a significant decrease in mean peak inspiratory pressure to 35.3 +/- 3.0 cm H(2)O. Mean pH increased significantly to 7.32 +/- 0.06, and mean partial pressure CO(2) decreased significantly to 50.5 +/- 7.4 torr. Initial mean inspired helium was 57 +/- 4% (range, 32-74). Mechanical ventilation days ranged from 1 to 23 days (mean, 5.0). Hospital stay ranged from 4 to 29 days (mean, 10.1), with an average pediatric intensive care unit stay of 6.9 days (range, 2-24). There were two incidences of pneumothorax. CONCLUSIONS: In the pediatric patient with severe asthma requiring conventional mechanical ventilation, helium-oxygen administration appears to be a safe therapy and may assist in lowering peak inspiratory pressure and improving blood gas pH and partial pressure CO(2).

Treatment of oseltamivir-resistant influenza A (H1N1) virus infections in mice with antiviral agents.

Influenza A/Mississippi/03/2001 (H1N1) and A/Hong Kong/2369/2009 (H1N1) viruses containing the neuraminidase gene mutation H275Y (conferring resistance to oseltamivir) were adapted to mice and evaluated for suitability as models for lethal infection and antiviral treatment. The viral neuraminidases were resistant to peramivir and oseltamivir carboxylate but sensitive to zanamivir. Similar pattern of antiviral activity were seen in MDCK cell assays. Lethal infections were achieved in mice with the two viruses. Oral oseltamivir at 100 and 300mg/kg/day bid for 5day starting at -2h gave 30% and 60% protection from death, respectively, due to the A/Mississippi/03/2001 infection. Intraperitoneal treatments with zanamivir at 30 and 100mg/kg/day starting at -2h gave 60% and 90% protection, respectively. Neither compound at <300mg/kg/day protected mice when treatments began at +24h. Amantadine was effective at 10, 30, and 100mg/kg/day, rimantadine was protective at 10 and 30mg/kg/day (highest dose tested), and ribavirin was active at 30 and 75mg/kg/day, with survival ranging from 60-100% for oral treatments initiated at -2h. For treatments begun at +24h, amantadine was protective at 30 and 100mg/kg/day, rimantadine showed efficacy at 10 and 30mg/kg/day, and ribavirin was active at 75mg/kg/day, with 60-100% survival per group. In the A/Hong Kong/2369/2009 infection, oral oseltamivir at 100 and 300mg/kg/day starting at -2h gave 50% and 70% protection from death, respectively. These infection models will be useful to study newly discovered anti-influenza virus agents and to evaluate compounds in combination.

Efficacy of combined therapy with amantadine, oseltamivir, and ribavirin in vivo against susceptible and amantadine-resistant influenza A viruses.

The limited efficacy of existing antiviral therapies for influenza--coupled with widespread baseline antiviral resistance--highlights the urgent need for more effective therapy. We describe a triple combination antiviral drug (TCAD) regimen composed of amantadine, oseltamivir, and ribavirin that is highly efficacious at reducing mortality and weight loss in mouse models of influenza infection. TCAD therapy was superior to dual and single drug regimens in mice infected with drug-susceptible, low pathogenic A/H5N1 (A/Duck/MN/1525/81) and amantadine-resistant 2009 A/H1N1 influenza (A/California/04/09). Treatment with TCAD afforded >90% survival in mice infected with both viruses, whereas treatment with dual and single drug regimens resulted in 0% to 60% survival. Importantly, amantadine had no activity as monotherapy against the amantadine-resistant virus, but demonstrated dose-dependent protection in combination with oseltamivir and ribavirin, indicative that amantadine's activity had been restored in the context of TCAD therapy. Furthermore, TCAD therapy provided survival benefit when treatment was delayed until 72 hours post-infection, whereas oseltamivir monotherapy was not protective after 24 hours post-infection. These findings demonstrate in vivo efficacy of TCAD therapy and confirm previous reports of the synergy and broad spectrum activity of TCAD therapy against susceptible and resistant influenza strains in vitro.

The glial modulatory drug AV411 attenuates mechanical allodynia in rat models of neuropathic pain.

Controlling neuropathic pain is an unmet medical need and we set out to identify new therapeutic candidates. AV411 (ibudilast) is a relatively nonselective phosphodiesterase inhibitor that also suppresses glial-cell activation and can partition into the CNS. Recent data strongly implicate activated glial cells in the spinal cord in the development and maintenance of neuropathic pain. We hypothesized that AV411 might be effective in the treatment of neuropathic pain and, hence, tested whether it attenuates the mechanical allodynia induced in rats by chronic constriction injury (CCI) of the sciatic nerve, spinal nerve ligation (SNL) and the chemotherapeutic paclitaxel (Taxol). Twice-daily systemic administration of AV411 for multiple days resulted in a sustained attenuation of CCI-induced allodynia. Reversal of allodynia was of similar magnitude to that observed with gabapentin and enhanced efficacy was observed in combination. We further show that multi-day AV411 reduces SNL-induced allodynia, and reverses and prevents paclitaxel-induced allodynia. Also, AV411 cotreatment attenuates tolerance to morphine in nerve-injured rats. Safety pharmacology, pharmacokinetic and initial mechanistic analyses were also performed. Overall, the results indicate that AV411 is effective in diverse models of neuropathic pain and support further exploration of its potential as a therapeutic agent for the treatment of neuropathic pain.

Proton spectroscopy detected myoinositol in children with traumatic brain injury.

Previous studies have shown that proton magnetic resonance spectroscopy (MRS) is useful in predicting neurologic prognosis in children with traumatic brain injury (TBI). Reductions in N-acetyl derived metabolites and presence of lactate have been predictive of poor outcomes. We examined another spectroscopy metabolite, myoinositol (mI), to determine whether it is altered after TBI. Found primarily in astrocytes, mI functions as an osmolyte and is involved in hormone response pathways and protein-kinase C activation. Myoinositol is elevated in the newborn brain and is increased in a variety of diseases. We studied 38 children (mean age 11 y; range 1.6-17 y) with TBI using quantitative short echo time occipital gray and parietal white matter proton MRS at a mean of 7 d (range 1-17 d) after injury. We found that occipital gray matter mI levels were increased in children with TBI (4.30 +/- 0.73) compared with controls (3.53 +/- 0.48; p = 0.003). We also found that patients with poor outcomes 6-12 mo after injury had higher mI levels (4.78 +/- 0.68) than patients with good outcomes (4.15 +/- 0.69; p < 0.05). Myoinositol is elevated after pediatric TBI and is associated with a poor neurologic outcome. The reasons for its elevation remain unclear but may be due to astrogliosis or to a disturbance in osmotic function.

DNA binding ligands targeting drug-resistant Gram-positive bacteria. Part 1: Internal benzimidazole derivatives.

Novel DNA minor-groove binding ligands with a promising antibacterial profile are described. Apart from excellent in vitro potency against multiple Gram-positive bacterial strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VRE), and penicillin-intermediate Streptococcus pneumoniae (PISP), a small subset of compounds was active against Gram-negative bacteria such as Escherichia coli (E. coli).

DNA binding ligands with in vivo efficacy in murine models of bacterial infection: optimization of internal aromatic amino acids.

DNA binding ligands with potent antimicrobial activity against Gram-positive bacteria were further optimized by variation of the internal aromatic amino acids. This modification led to compounds with improved in vivo efficacy in lethal murine models of peritonitis (methicillin-resistant S. aureus, MRSA) and lung infection (S. pneumoniae).