Focused ultrasound neuromodulation of the spleen activates an anti-inflammatory response in humans.

Focused ultrasound stimulation (FUS) activates mechanosensitive ion channels and is emerging as a method of noninvasive neuromodulation. In preclinical studies, FUS of the spleen (sFUS) activates an anti-inflammatory neural pathway which suppresses acute and chronic inflammation. However, the relevance of sFUS for regulating inflammatory responses in humans is unknown. Here, we used a modified diagnostic ultrasound imaging system to target the spleen of healthy human subjects with 3 min of continuously swept or stationary focused pulsed ultrasound, delivered at three different energy levels within allowable safety exposure limits. Potential anti-inflammatory effects of sFUS were assessed by measuring sFUS-elicited changes in endotoxin-induced tumor necrosis factor (TNF) production in whole blood samples from insonified subjects. We found that stimulation with either continuously swept or focused pulsed ultrasound has an anti-inflammatory effect: sFUS lowers TNF production for >2 h, with TNF returning to baseline by 24 h following sFUS. This response is independent of anatomical target (i.e., spleen hilum or parenchyma) or ultrasound energy level. No clinical, biochemical, or hematological parameters are adversely impacted. This is the first demonstration that sFUS suppresses the normal inflammatory response in humans, with potential implications for noninvasive bioelectronic therapy of inflammatory disorders.

Thiocarbazate building blocks enable the construction of azapeptides for rapid development of therapeutic candidates.

Peptides, polymers of amino acids, comprise a vital and expanding therapeutic approach. Their rapid degradation by proteases, however, represents a major limitation to their therapeutic utility and chemical modifications to native peptides have been employed to mitigate this weakness. Herein, we describe functionalized thiocarbazate scaffolds as precursors of aza-amino acids, that, upon activation, can be integrated in a peptide sequence to generate azapeptides using conventional peptide synthetic methods. This methodology facilitates peptide editing-replacing targeted amino acid(s) with aza-amino acid(s) within a peptide-to form azapeptides with preferred therapeutic characteristics (extending half-life/bioavailability, while at the same time typically preserving structural features and biological activities). We demonstrate the convenience of this azapeptide synthesis platform in two well-studied peptides with short half-lives: FSSE/P5779, a tetrapeptide inhibitor of HMGB1/MD-2/TLR4 complex formation, and bradykinin, a nine-residue vasoactive peptide. This bench-stable thiocarbazate platform offers a robust and universal approach to optimize peptide-based therapeutics.

Stimulation of the hepatoportal nerve plexus with focused ultrasound restores glucose homoeostasis in diabetic mice, rats and swine.

Peripheral neurons that sense glucose relay signals of glucose availability to integrative clusters of neurons in the brain. However, the roles of such signalling pathways in the maintenance of glucose homoeostasis and their contribution to disease are unknown. Here we show that the selective activation of the nerve plexus of the hepatic portal system via peripheral focused ultrasound stimulation (pFUS) improves glucose homoeostasis in mice and rats with insulin-resistant diabetes and in swine subject to hyperinsulinemic-euglycaemic clamps. pFUS modulated the activity of sensory projections to the hypothalamus, altered the concentrations of metabolism-regulating neurotransmitters, and enhanced glucose tolerance and utilization in the three species, whereas physical transection or chemical blocking of the liver-brain nerve pathway abolished the effect of pFUS on glucose tolerance. Longitudinal multi-omic profiling of metabolic tissues from the treated animals confirmed pFUS-induced modifications of key metabolic functions in liver, pancreas, muscle, adipose, kidney and intestinal tissues. Non-invasive ultrasound activation of afferent autonomic nerves may represent a non-pharmacologic therapy for the restoration of glucose homoeostasis in type-2 diabetes and other metabolic diseases.

Targeted peripheral focused ultrasound stimulation attenuates obesity-induced metabolic and inflammatory dysfunctions.

Obesity, a growing health concern, is associated with an increased risk of morbidity and mortality. Chronic low-grade inflammation is implicated in obesity-driven metabolic complications. Peripheral focused ultrasound stimulation (pFUS) is an emerging non-invasive technology that modulates inflammation. Here, we reasoned that focused ultrasound stimulation of the liver may alleviate obesity-related inflammation and other comorbidities. After 8 weeks on a high-fat high-carbohydrate "Western" diet, C57BL/6J mice were subjected to either sham stimulation or focused ultrasound stimulation at the porta hepatis. Daily liver-focused ultrasound stimulation for 8 weeks significantly decreased body weight, circulating lipids and mitigated dysregulation of adipokines. In addition, liver-focused ultrasound stimulation significantly reduced hepatic cytokine levels and leukocyte infiltration. Our findings demonstrate the efficacy of hepatic focused ultrasound for alleviating obesity and obesity-associated complications in mice. These findings suggest a previously unrecognized potential of hepatic focused ultrasound as a possible novel noninvasive approach in the context of obesity.

Author Correction: Noninvasive sub-organ ultrasound stimulation for targeted neuromodulation.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Noninvasive sub-organ ultrasound stimulation for targeted neuromodulation.

Tools for noninvasively modulating neural signaling in peripheral organs will advance the study of nerves and their effect on homeostasis and disease. Herein, we demonstrate a noninvasive method to modulate specific signaling pathways within organs using ultrasound (U/S). U/S is first applied to spleen to modulate the cholinergic anti-inflammatory pathway (CAP), and US stimulation is shown to reduce cytokine response to endotoxin to the same levels as implant-based vagus nerve stimulation (VNS). Next, hepatic U/S stimulation is shown to modulate pathways that regulate blood glucose and is as effective as VNS in suppressing the hyperglycemic effect of endotoxin exposure. This response to hepatic U/S is only found when targeting specific sub-organ locations known to contain glucose sensory neurons, and both molecular (i.e. neurotransmitter concentration and cFOS expression) and neuroimaging results indicate US induced signaling to metabolism-related hypothalamic sub-nuclei. These data demonstrate that U/S stimulation within organs provides a new method for site-selective neuromodulation to regulate specific physiological functions.

Immunization Elicits Antigen-Specific Antibody Sequestration in Dorsal Root Ganglia Sensory Neurons.

The immune and nervous systems are two major organ systems responsible for host defense and memory. Both systems achieve memory and learning that can be retained, retrieved, and utilized for decades. Here, we report the surprising discovery that peripheral sensory neurons of the dorsal root ganglia (DRGs) of immunized mice contain antigen-specific antibodies. Using a combination of rigorous molecular genetic analyses, transgenic mice, and adoptive transfer experiments, we demonstrate that DRGs do not synthesize these antigen-specific antibodies, but rather sequester primarily IgG1 subtype antibodies. As revealed by RNA-seq and targeted quantitative PCR (qPCR), dorsal root ganglion (DRG) sensory neurons harvested from either naïve or immunized mice lack enzymes (i.e., RAG1, RAG2, AID, or UNG) required for generating antibody diversity and, therefore, cannot make antibodies. Additionally, transgenic mice that express a reporter fluorescent protein under the control of Igγ1 constant region fail to express Ighg1 transcripts in DRG sensory neurons. Furthermore, neural sequestration of antibodies occurs in mice rendered deficient in neuronal Rag2, but antibody sequestration is not observed in DRG sensory neurons isolated from mice that lack mature B cells [e.g., Rag1 knock out (KO) or µMT mice]. Finally, adoptive transfer of Rag1-deficient bone marrow (BM) into wild-type (WT) mice or WT BM into Rag1 KO mice revealed that antibody sequestration was observed in DRG sensory neurons of chimeric mice with WT BM but not with Rag1-deficient BM. Together, these results indicate that DRG sensory neurons sequester and retain antigen-specific antibodies released by antibody-secreting plasma cells. Coupling this work with previous studies implicating DRG sensory neurons in regulating antigen trafficking during immunization raises the interesting possibility that the nervous system collaborates with the immune system to regulate antigen-mediated responses.

C1q and HMGB1 reciprocally regulate human macrophage polarization.

A healthy immune system results from a balance of stimulatory and inhibitory pathways that allow effective responses to acute insults, without descending into chronic inflammation. Failed homeostasis is characteristic of autoimmune diseases such as systemic lupus erythematosus. Although HMGB1 induces proinflammatory M1-like macrophage differentiation, we describe a mechanism by which C1q modulates this activity and collaborates with HMGB1 to induce the differentiation of monocytes to anti-inflammatory M2-like macrophages. These anti-inflammatory macrophages are unresponsive to dendritic cell induction factors, effectively removing them from participation in an adaptive immune response. This pathway is mediated through a complex with RAGE and LAIR-1 and depends on relative levels of C1q and HMGB1. Importantly, these data provide insight into a homeostatic mechanism in which C1q and HMGB1 can cooperate to terminate inflammation, and which may be impaired in C1q-deficient patients with autoimmune disease.

Amending HIV Drugs: A Novel Small-Molecule Approach To Target Lupus Anti-DNA Antibodies.

Systemic lupus erythematosus is an autoimmune disease that can affect numerous tissues and is characterized by the production of nuclear antigen-directed autoantibodies (e.g., anti-dsDNA). Using a combination of virtual and ELISA-based screens, we made the intriguing discovery that several HIV-protease inhibitors can function as decoy antigens to specifically inhibit the binding of anti-dsDNA antibodies to target antigens such as dsDNA and pentapeptide DWEYS. Computational modeling revealed that HIV-protease inhibitors comprised structural features present in DWEYS and predicted that analogues containing more flexible backbones would possess preferred binding characteristics. To address this, we reduced the internal amide backbone to improve flexibility, producing new small-molecule decoy antigens, which neutralize anti-dsDNA antibodies in vitro, in situ, and in vivo. Pharmacokinetic and SLE model studies demonstrated that peptidomimetic FISLE-412,1 a reduced HIV protease inhibitor analogue, was well-tolerated, altered serum reactivity to DWEYS, reduced glomeruli IgG deposition, preserved kidney histology, and delayed SLE onset in NZB/W F1 mice.

Effects of novel muscarinic M3 receptor ligand C1213 in pulmonary arterial hypertension models.

Pulmonary hypertension (PH) is a complex disease comprising a pathologic remodeling and thickening of the pulmonary vessels causing an after load on the right heart ventricle that can result in ventricular failure. Triggered by oxidative stress, episodes of hypoxia, and other undetermined causes, PH is associated with poor outcomes and a high rate of morbidity. In the neonate, this disease has a similar etiology but is further complicated by the transition to breathing after birth, which requires a reduction in vascular resistance. Persistent pulmonary hypertension of the newborn (PPHN) is one form of PH that is frequently unresponsive to current therapies including inhaled nitric oxide (due to lack of proper absorption and diffusion), and other therapeutics targeting signaling mediators in vascular endothelium and smooth muscle. The need for novel agents, which target distinct pathways in pulmonary hypertension, remains. Herein, we investigated the therapeutic effects of novel muscarinic receptor ligand C1213 in models of PH We demonstrated that via M3 muscarinic receptors, C1213 induced activating- eNOS phosphorylation (serine-1177), which is known to lead to nitric oxide (NO) production in endothelial cells. Using signaling pathway inhibitors, we discovered that AKT and calcium signaling contributed to eNOS phosphorylation induced by C1213. As expected for an eNOS-stimulating agent, in ex vivo and in vivo models, C1213 triggered pulmonary vasodilation and induced both pulmonary artery and systemic blood pressure reductions demonstrating its potential value in PH and PPHN In brief, this proof-of-concept study provides evidence that an M3 muscarinic receptor functionally selective ligand stimulates downstream pathways leading to antihypertensive effects using in vitro, ex vivo, and in vivo models of PH.

Macrophage migration inhibitory factor promotes colorectal cancer.

A growing body of evidence implicates macrophage migration inhibitory factor (MIF) in tumorigenesis and metastasis. In this study, we investigated whether MIF expression was associated with clinicopathologic features of colorectal carcinoma (CRC), especially in tumors with hepatic metastasis, and whether neutralization of endogenous MIF using anti-MIF therapeutics would inhibit tumor growth and/or decrease the frequency of colorectal hepatic metastases in a mouse colon carcinoma model. The concentration of serum MIF was positively correlated with an increased risk of hepatic metastasis in human patients with CRC (R = 1.25, 95% confidence internal = 1.02-1.52, P = 0.03). MIF was also dramatically upregulated in human colorectal tissue, with 20-40 times as many MIF-positive cells found in the mucosa of patients with CRC than in normal tissue (P < 0.001 ANOVA). Moreover, in those patients with metastatic colorectal cancer in the liver, MIF-positive cells were similarly increased in the diseased hepatic tissue. This increased MIF expression was restricted to diseased tissue and not found in areas of the liver with normal morphology. In subsequent in vitro experiments, we found that addition of recombinant MIF to colonic cell lines significantly increased their invasive properties and the expression of several genes (for example, matrix metalloproteinase 9 and vascular endothelial growth factor) known to be upregulated in cancerous tissue. Finally, we treated mice that had been given CT26 colon carcinoma cell transplants with anti-MIF therapeutics--either the MIF-specific inhibitor ISO-1 or neutralizing anti-MIF antibodies--and observed a significant reduction in tumor burden relative to vehicle-treated animals. Taken together, these data demonstrate that MIF expression was not only correlated with the presence of colorectal cancer but also may play a direct role in cancer development.

Carbamylated erythropoietin ameliorates the metabolic stress induced in vivo by severe chronic hypoxia.

Ischemia and chronic hypoxia (CH) trigger a variety of adverse effects arising from metabolic stress that injures cells. In response to reduced O2, hypoxia-inducible factor 1alpha (HIF-1alpha) activates erythropoietin (Epo) as well as many other target genes that counteract the effects of O2 deficiency. Epo produced by the kidney stimulates erythrocyte production, leading to decreased HIF-1alpha production by improved tissue O2 delivery. However, Epo is produced by many other tissues, and it is currently unclear to what extent, if any, locally produced Epo modulates HIF-1alpha expression. Derivatives of Epo that possess tissue-protective activities but do not stimulate erythropoiesis [e.g., carbamylated Epo (CEpo)] are useful tools with which to determine whether exogenous Epo modulates HIF-1alpha in the absence of changes in hemoglobin concentration. We compared the effects of CH (6.5% O2 for 10 days) with or without CEpo administered by daily s.c. injection (10 microg/kg of body weight). CEpo administration did not alter the survival rate, weight loss, or increased hemoglobin concentration associated with CH. Therefore, CEpo does not directly suppress HIF-mediated erythropoiesis. CEpo does, however, prevent CH-induced neuronal increases of HIF-1alpha and Epo receptor-associated immunoreactivity (a measure of stress) while reducing the apoptotic index. In contrast, the myocardium did not exhibit increased HIF-1alpha expression during CH, although CEpo did reduce the apoptotic index. These observations therefore demonstrate that CEpo administration reduces the metabolic stress caused by severe CH, resulting in improved cellular survival independent of erythrocyte production.

Carbamylated erythropoietin reduces radiosurgically-induced brain injury.

Gamma knife radiosurgery is an attractive noninvasive treatment of brain tumors and vascular malformations that minimizes collateral tissue damage. However, exposure of normal tissue to even low-dose radiation triggers a cascade of acute and chronic injury and potentially significant morbidity and mortality. Because many irradiated patients now survive for years, identifying methods to prevent radiotherapy-induced collateral tissue damage is a major focus of current research. Erythropoietin (EPO), a cytokine produced locally by many tissues in response to injury, antagonizes apoptosis, reduces inflammation, and promotes healing. Systemic administration of recombinant EPO, widely used for treatment of anemia, provides robust protection from numerous insults in a variety of tissues, including the brain. Although irradiation injury is likely sensitive to EPO, the hematopoietic activity of EPO is undesirable in this setting, increasing erythrocyte number and predisposing to thrombosis. To avoid these potential adverse effects, we developed carbamylated EPO (CEPO) which does not stimulate the bone marrow. In this study, we show that CEPO (50 microg kg(-1) intraperitoneally) improves functional outcome when administered to adult rats just before, and then once daily for 10 d after, a necrotizing dose of radiation (100 Gy) to the right striatum. Immediately following irradiation, use and reflex movements of the contralateral forelimb to vibrissae stimulation were abnormal but rapidly improved in animals receiving CEPO. Moreover, histological examination revealed that the extent of brain necrosis after 90 days was reduced by approximately 50%. These findings further extend the kinds of injury for which administration of a tissue-protective cytokine provides benefit.

Cytoprotective doses of erythropoietin or carbamylated erythropoietin have markedly different procoagulant and vasoactive activities.

Recombinant human erythropoietin (rhEPO) is receiving increasing attention as a potential therapy for prevention of injury and restoration of function in nonhematopoietic tissues. However, the minimum effective dose required to mimic and augment these normal paracrine functions of erythropoietin (EPO) in some organs (e.g., the brain) is higher than for treatment of anemia. Notably, a dose-dependent risk of adverse effects has been associated with rhEPO administration, especially in high-risk groups, including polycythemia-hyperviscosity syndrome, hypertension, and vascular thrombosis. Of note, several clinical trials employing relatively high dosages of rhEPO in oncology patients were recently halted after an increase in mortality and morbidity, primarily because of thrombotic events. We recently identified a heteromeric EPO receptor complex that mediates tissue protection and is distinct from the homodimeric receptor responsible for the support of erythropoiesis. Moreover, we developed receptor-selective ligands that provide tools to assess which receptor isoform mediates which biological consequence of rhEPO therapy. Here, we demonstrate that rhEPO administration in the rat increases systemic blood pressure, reduces regional renal blood flow, and increases platelet counts and procoagulant activities. In contrast, carbamylated rhEPO, a heteromeric receptor-specific ligand that is fully tissue protective, increases renal blood flow, promotes sodium excretion, reduces injury-induced elevation in procoagulant activity, and does not effect platelet production. These preclinical findings suggest that nonerythropoietic tissue-protective ligands, which appear to elicit fewer adverse effects, may be especially useful in clinical settings for tissue protection.

Cell cycle-regulated phosphorylation of hamartin, the product of the tuberous sclerosis complex 1 gene, by cyclin-dependent kinase 1/cyclin B.

Tuberous sclerosis complex is a tumor suppressor gene syndrome whose manifestations can include seizures, mental retardation, and benign tumors of the brain, skin, heart, and kidneys. Hamartin and tuberin, the products of the TSC1 and TSC2 genes, respectively, form a complex and inhibit signaling by the mammalian target of rapamycin. Here, we demonstrate that endogenous hamartin is threonine-phosphorylated during nocodazole-induced G2/M arrest and during the G2/M phase of a normal cell cycle. In vitro assays showed that cyclin-dependent kinase 1 phosphorylates hamartin at three sites, one of which (Thr417) is in the hamartin-tuberin interaction domain. Tuberin interacts with phosphohamartin, and tuberin expression attenuates the phosphorylation of exogenous hamartin. Hamartin with alanine mutations in the three cyclin-dependent kinase 1 phosphorylation sites increased the inhibition of p70S6 kinase by the hamartin-tuberin complex. These findings support a model in which phosphorylation of hamartin regulates the function of the hamartin-tuberin complex during the G2/M phase of the cell cycle.

Erythropoietin selectively attenuates cytokine production and inflammation in cerebral ischemia by targeting neuronal apoptosis.

Ischemic brain injury resulting from stroke arises from primary neuronal losses and by inflammatory responses. Previous studies suggest that erythropoietin (EPO) attenuates both processes. Although EPO is clearly antiapoptotic for neurons after experimental stroke, it is unknown whether EPO also directly modulates EPO receptor (EPO-R)-expressing glia, microglia, and other inflammatory cells. In these experiments, we show that recombinant human EPO (rhEPO; 5,000 U/kg body weight, i.p.) markedly reduces astrocyte activation and the recruitment of leukocytes and microglia into an infarction produced by middle cerebral artery occlusion in rats. In addition, ischemia-induced production of the proinflammatory cytokines tumor necrosis factor, interleukin 6, and monocyte chemoattractant protein 1 concentration is reduced by >50% after rhEPO administration. Similar results were also observed in mixed neuronal-glial cocultures exposed to the neuronal-selective toxin trimethyl tin. In contrast, rhEPO did not inhibit cytokine production by astrocyte cultures exposed to neuronal homogenates or modulate the response of human peripheral blood mononuclear cells, rat glial cells, or the brain to lipopolysaccharide. These findings suggest that rhEPO attenuates ischemia-induced inflammation by reducing neuronal death rather than by direct effects upon EPO-R-expressing inflammatory cells.

Isoform switching of Cdc6 contributes to developmental cell cycle remodeling.

Xenopus laevis early development is characterized by rapid and synchronous cleavage cycles, which consist of alternating S and M phases. At midblastula transition, zygotic transcription begins and these cleavage cycles are replaced by longer cell division cycles that include gap phases and checkpoints. Herein, we demonstrate developmentally regulated Cdc6 isoform switching that contributes to this developmental cell cycle remodeling. Cdc6 is an essential component of the eukaryotic DNA replication machine that licenses each origin to one round of DNA replication each cell division cycle. The originally characterized Xenopus Cdc6 isoform (here termed Xcdc6A) and a novel isoform (Xcdc6B) have divergent N-terminal regulatory regions and different temporal patterns of expression. Although abundant in the early embryo, Xcdc6A becomes undetectable following midblastula transition. In contrast, while Xcdc6B is present in the early embryo, it is nonfunctional, as judged by lack of chromatin binding. In somatic tissue, however, Xcdc6B binds chromatin and its inhibition blocks entry into S phase. This is the first example of developmental regulation of Cdc6, raising intriguing implications for cell cycle remodeling during embryogenesis.

Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC.

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S.pombe.