Local Blockade Research Articles

Botox: Beauty and Brains

Botulinum toxins (BTX), predominately BotulinumtoxinA, have been used for medical and aesthetic purposes since 1997. With nearly eight billion treatments performed worldwide for aesthetic purposes alone, there is extensive post-market experience with this drug. The known mechanism of action (MOA) of the local neurotransmitter blockade of acetylcholine lasts approximately 90 days. However, patients frequently report symptom and wrinkle relief beyond the 90-day period. There is extensive evidence that peripherally injected BTX, even at low aesthetic doses, induces structural and functional changes in the human brain. The MOA in the central nervous system (CNS) is presently unclear, with several proposed hypotheses to include the Social Feedback Hypothesis (SFH), Facial Feedback Hypothesis (FFH) and Emotional Proprioception (EP), Monoamine Theory and Brain-Derived Neurotrophic Peptide (BDFP), and Neuronal Transport Hypothesis. With the ability to modify the CNS, BTX for aesthetic use may become a novel treatment for CNS disorders, such as major depressive disorder (MDD). Over 30% of persons diagnosed with MDD had inadequate response to first-line treatments. Over 30% of persons diagnosed with treatment resistant depression (TRD) attempt suicide. BotulinumtoxinA may offer an effective adjunctive treatment of MDD/TRD.

PEG-Sheddable Nanodrug Remodels Tumor Microenvironment to Promote Effector T Cell Infiltration and Revise Their Exhaustion for Breast Cancer Immunotherapy.

Low infiltration of cytotoxic T lymphocytes and their exhaustion manifest the two concurrent main hurdles for achieving effective tumor immunotherapy of triple-negative breast cancer. It is found that Galectin-9 blockage can revise the exhaustion of effector T cells, meanwhile the repolarization of protumoral M2 tumor-associated macrophages (TAMs) into tumoricidal M1-like ones can recruit effector T cells infiltrating into tumor to boost immune responses. Herein, a sheddable PEG-decorated and M2-TAMs targeted nanodrug incorporating Signal Transducer and Activator of Transcription 6 inhibitor (AS) and anti-Galectin-9 antibody (aG-9) is prepared. The nanodrug responds to acidic tumor microenvironment (TME) with the shedding of PEG corona and the release of aG-9, exerting local blockade of PD-1/Galectin-9/TIM-3 interaction to augment effector T cells via exhaustion reversing. Synchronously, targeted repolarization of M2-TAMs into M1 phenotype by AS-loaded nanodrug is achieved, which promotes tumor infiltration of effector T cells and thus synergizes with aG-9 blockade to boost the therapeutic efficacy. Besides, the PEG-sheddable approach endows nanodrug with stealth ability to reduce immune-related adverse effects caused by AS and aG-9. This PEG sheddable nanodrug holds the potential to reverse the immunosuppressive TME and increase effector T cell infiltration, which dramatically enhances immunotherapy in highly malignant breast cancer.

The Antinociceptive Effect of Sympathetic Block is Mediated by Transforming Growth Factor β in a Mouse Model of Radiculopathy.

Although sympathetic blockade is clinically used to treat pain, the underlying mechanisms remain unclear. We developed a localized microsympathectomy (mSYMPX), by cutting the grey rami entering the spinal nerves near the rodent lumbar dorsal root ganglia (DRG). In a chemotherapy-induced peripheral neuropathy model, mSYMPX attenuated pain behaviors via DRG macrophages and the anti-inflammatory actions of transforming growth factor-β (TGF-β) and its receptor TGF-βR1. Here, we examined the role of TGF-β in sympathetic-mediated radiculopathy produced by local inflammation of the DRG (LID). Mice showed mechanical hypersensitivity and transcriptional and protein upregulation of TGF-β1 and TGF-βR1 three days after LID. Microsympathectomy prevented mechanical hypersensitivity and further upregulated Tgfb1 and Tgfbr1. Intrathecal delivery of TGF-β1 rapidly relieved the LID-induced mechanical hypersensitivity, and TGF-βR1 antagonists rapidly unmasked the mechanical hypersensitivity after LID+mSYMPX. In situ hybridization showed that Tgfb1 was largely expressed in DRG macrophages, and Tgfbr1 in neurons. We suggest that TGF-β signaling is a general underlying mechanism of local sympathetic blockade.

Magnetic resonance imaging of peripheral venous distension with cyclooxygenase inhibition in healthy humans

Background: Peripheral venous distension, induced by a volume infusion into the veins in an occluded limb, evokes a reflex pressor and sympathoexcitatory response in humans (venous distension reflex, VDR). However, to what extent the procedure alters the size of superficial and deep veins remains unclear. Our data showed that local cyclooxygenase (COX) inhibition could attenuate the muscle sympathetic nerve activity (MSNA) and blood pressure (BP) responses to venous distension. It is unclear if the COX blockade would alter the magnitudes of the venous distension. The purpose of this study was to evaluate the changes in superficial and deep vein sizes by the venous distension procedure in healthy subjects under local COX blockade and control conditions. This study is associated with a registered clinical trial (NCT03513770). Hypothesis: We hypothesized that the voxel volume of both superficial and deep veins would increase following peripheral venous distension. We further hypothesized that COX blockade would not alter the distribution of infused volume. Methods: The study had a randomized, double-blind, placebo-controlled, and crossover design with two separate visits. Thirteen healthy volunteers underwent peripheral venous distension by which a volume of solution (saline alone or 9 mg ketorolac tromethamine in saline) was infused into the median cubital fossa of an arterially occluded forearm. Each trial (saline or COX blockade) was separated by at least 24 hours. The total infused volume (i.e., 5% of the forearm volume) was held constant across both saline and COX blockade trails. The 3D proton density-weighted magnitude and phase images of the treated arm were obtained before and after the venous distension procedure via a 3T magnetic resonance imaging scanner (Siemens PrismaFit). The average vein voxel volume of 10 forearm veins (six superficial and four deep) was calculated from several adjacent slices of the images. Results: Vein voxel volume was significantly increased by venous distension in both superficial (both P < .001) and deep (both P < 0.01) veins in both trials. However, COX blockade did not influence this increase in voxel volume in either superficial (NSS: Δ2.34 ± 3.05 vs. COX blockade: Δ1.99 ± 2.27 mm3, P = 0.64) or deep (NSS: Δ1.02 ± 1.2 vs. COX blockade: Δ1.06 ± 1.42 mm3, P = 0.94) veins. Conclusion: These data suggest that both superficial and deep veins may contribute to the VDR. Importantly, the results suggest that any attenuation in the MSNA and BP responses to venous distension with COX blockade is not a result of altered magnitudes of the venous distension during the procedure. This study was supported by National Institutes of Health Grants R01 HL144781 (to J.C. and L.I.S). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Cyclooxygenase inhibition attenuates peripheral venous distension reflex in healthy humans

Background: Venous distension in limbs evokes a pressor reflex (venous distension reflex, VDR). Group III and IV nerves, innervating peripheral veins, are suggested as the afferent arm of the VDR. Prostaglandins stimulate/sensitize group III/IV nerves. The purpose of this study was to evaluate the effect of cyclooxygenase (COX) blockade on muscle sympathetic nerve activity (MSNA) and blood pressure (BP) responses to venous distension. Hypothesis: We hypothesized that inhibition of prostaglandin synthesis by local COX blockade would attenuate the MSNA and BP responses to peripheral venous distension. Methods: Nineteen healthy volunteers (age, 27 ± 5 years) participated in the study with a randomized, double-blind, placebo-controlled, and crossover study design with two separate visits. To induce venous distension, a volume of solution (saline alone or 9 mg ketorolac tromethamine in saline) was infused into the vein in the antecubital fossa of an arterially occluded forearm. During the procedure, beat-by-beat heart rate (HR), BP, and MSNA were recorded simultaneously. The vein size at the site with the catheter for the infusion was measured with ultrasound. This study is associated with a registered clinical trial (NCT03513770). Data and a summary of the results: In both visits, the venous distension procedure significantly increased the vein size, and the increase in vein size was not different between the visits (P > 0.05). A significant decrease (P < 0.001) in TXB2 level (an index for prostaglandin synthesis) by the procedure was observed only in the ketorolac visit. In both visits, the venous distension procedure significantly increased BP, HR, and MSNA (all, P < 0.05). The increase in mean arterial pressure (MAP) and MSNA in the ketorolac visit was significantly lower than in the control visit (ΔMAP, 7.0 ± 6.2 vs. 13.8 ± 7.7 mmHg; ΔMSNA, 6.0 ± 7.1 vs. 14.8 ± 7.7 bursts/minute; both, P < 0.05). Discussion and conclusion: The presented data show that COX blockade attenuates the responses in MSNA and BP to peripheral venous distension. The results suggest that COX products play a key role in evoking afferent activation responsible for the VDR. Vein size was measured with ultrasonography only at the catheter site. Some members of COX products have vasoactive effects. Future studies evaluating the effect of COX blockade on vein size in more detail using other approaches (e.g., magnetic resonance imaging) are warranted to verify the detailed mechanism of the attenuated VDR by the COX blockade. This study was supported by National Institutes of Health Grants R01 HL144781 (to J.C. and L.I.S). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Retinotectal plasticity induced by monocular enucleation during the critical period is dependent of A2a adenosine receptor: A possible role of astrocytes

The retinotectal topography of rats develops within the first three postnatal weeks during the critical period. Previous studies have shown that monocular enucleation results in plasticity of the intact retinotectal pathway in a time-dependent manner. Glial fibrillary acidic protein (GFAP), an astrocyte marker, is up-regulated after central nervous system injury. Adenosine is a neuromodulator involved in the development and plasticity of the visual system acting through the inhibitory A1 and excitatory A2a receptor activities. Herein, we examined whether adenosine receptors and astrocytes are crucial for monocular enucleation (ME)-induced plasticity. We also investigate whether A2a blockade alters retinotectal plasticity in an astrocyte-dependent manner. Lister Hooded rats were submitted to monocular enucleation at postnatal day 10 (PND10) or PND21 and, after different survival times, were processed for immunohistochemistry or western blotting assays. Another group underwent subpial implantation of ELVAX containing vehicle (DMSO) or SCH58261 (1 μM - an A2a receptor antagonist), simultaneously with ME at PND10. After a 72 h survival, GFAP content and the retinotectal plasticity were evaluated. Our data show that monocular enucleation leads to an upregulation in GFAP expression in the contralateral superior colliculus. At PND10, a slight increase in GFAP labeling was observed at 72 h post-enucleation, while at PND21 GFAP increase was detected in the deafferented superior colliculus after 1 to 3 weeks of survival. The content of adenosine receptors also varies in the contralateral target after ME. A transient increase in A1 receptors is observed in the early periods of plasticity, while A2a receptors are upregulated later. Interestingly, the local blockade of A2a receptors abolished the increase in GFAP and the retinotectal reorganization induced by monocular enucleation during the critical period. Taken together these results suggest a correlation between astrocytes and A2a adenosine receptors in the subcortical visual plasticity.

β-Catenin signaling in alveolar macrophages enhances lung metastasis through a TNF-dependent mechanism.

The main cause of malignancy-related mortality is metastasis. Although metastatic progression is driven by diverse tumor-intrinsic mechanisms, there is a growing appreciation for the contribution of tumor-extrinsic elements of the tumor microenvironment, especially macrophages, which correlate with poor clinical outcomes. Macrophages consist of bone marrow-derived and tissue-resident populations. In contrast to bone marrow-derived macrophages, the transcriptional pathways that govern the pro-metastatic activities of tissue-resident macrophages (TRMs) remain less clear. Alveolar macrophages (AMs) are a TRM population with critical roles in tissue homeostasis and metastasis. Wnt/β-catenin signaling is a hallmark of cancer and has been identified as a pathologic regulator of AMs in infection. We tested the hypothesis that β-catenin expression in AMs enhances metastasis in solid tumor models. Using a genetic β-catenin gain-of-function approach, we demonstrated that (a) enhanced β-catenin in AMs heightened lung metastasis; (b) β-catenin activity in AMs drove a dysregulated inflammatory program strongly associated with Tnf expression; and (c) localized TNF-α blockade abrogated this metastatic outcome. Last, β-catenin gene CTNNB1 and TNF expression levels were positively correlated in AMs of patients with lung cancer. Overall, our findings revealed a Wnt/β-catenin/TNF-α pro-metastatic axis in AMs with potential therapeutic implications against tumors refractory to the antineoplastic actions of TNF-α.

Abstract 2737: A gene therapy with a novel PD-L1 inhibitory peptide secretory gene inhibits the growth of colon carcinoma in mice

Abstract In the United States, colorectal cancer (CRC) is the second leading cause of cancer-related death in both sexes. Immune checkpoint blockade therapy (ICBT) has emerged as a powerful new tool for cancer therapy. However, only CRCs associated with microsatellite instability (MSI) or DNA mismatch repair gene defects (dMMR, ~15% of all CRCs) are sensitive to this therapy. This poor sensitivity to ICBT is either due to poor tumor infiltration of functional T cells or T cell exhaustion. To overcome this, we investigated a combination treatment with an oncolytic virus and an immune checkpoint inhibitor (ICI). Because tumor cell oncolysis generates neoantigens and increases tumor immunogenicity, T cell infiltration into the tumor tissue will be increased and the efficacy of ICBT will be enhanced. Accordingly, local immune checkpoint inhibition coupled with oncolysis may be an ideal ICBT. In the present study, a novel peptide that interferes with the PD-1/PD-L1 immune checkpoint pathway, termed PD-L1 inhibitory peptide 3 (PD-L1ip3), was computationally designed, experimentally validated for its specific binding to PD-L1, and evaluated for its antitumor effects in cell culture and in a mouse colon carcinoma syngeneic murine model. Fourteen candidate peptide sequences were generated using the PinaColada algorithm and the x-ray structure of the PD-1:PD-L1 complex and screened in molecular dynamics simulations on the microsecond timescale. Two were chosen for experimental testing, wherein the peptide denoted PD-L1ip3 showed a binding affinity for PD-L1 in the micromolar range. In cell culture studies, treatment with PD-L1ip3, but not a similar peptide with a scrambled sequence, substantially increased death of CT26 colon carcinoma cells when co-cultured with murine CD8+ T cells primed by antigens from CT26 cells. In immunocompetent mice, growth of CT26 tumor cells transduced with the PD-L1ip3 gene by an adenovirus vector was significantly slower than that of un-transduced CT26 cells. This tumor growth attenuation was further enhanced by cotreatment with the peptide form of PD-L1ip3. The present study suggests that this peptide can stimulate host antitumor immunity via a blockade of the PD-1/PD-L1 pathway, thereby increasing CD8+ T cell-induced death of colon carcinoma cells. The tumor site-specific inhibition of PD-L1 by an adenovirus carrying the PD-L1ip3 gene, together with direct peptide treatment, may be used as a local immune checkpoint blockade therapy to inhibit colon carcinoma growth. This research was supported by Kansas State University Johnson Cancer Research center (MT), K-INBRE Scholar Award (MB), National Cancer Institute (MT, JC) and the National Science Foundation (JC). Citation Format: Susumu Ishiguro, Deepa Upreti, Molly Bassette, E. R. Azhagiya Singam, Ravindra Thakkar, Mayme Loyd, Makoto Inui, Jeffrey Comer, Masaaki Tamura. A gene therapy with a novel PD-L1 inhibitory peptide secretory gene inhibits the growth of colon carcinoma in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2737.

Abstract 5105: TIMP1 mediates astrocyte-dependent local immunosuppression in brain metastasis

Abstract Brain metastasis is an unmet clinical need, affecting between 10-30% of cancer patients with 200000 to 400000 newly diagnosis per annum in the US. Recently, several clinical trials have reported benefits using immunotherapy to treat brain metastasis. However, variability of the responses is broad and high benefit is found mainly in asymptomatic brain metastasis, while the benefit is dramatically reduced in the clinically relevant stage. Thus, it is currently unknown how to effectively target symptomatic brain metastases with immunotherapy. We previously reported a clinically relevant protumoral program driven by STAT3 activation in a subpopulation of reactive astrocytes in these advanced stages of the disease. Our current study further exploited the heterogeneity within the metastasis-associated microenvironment as a resource to identify novel therapeutic vulnerabilities to improve the benefits of immunotherapies based on immune checkpoint blocking antibodies (ICB) in symptomatic brain metastasis. Our results demonstrate that reactive astrocytes are strong immunomodulatory cells in brain tumors. We have identified the molecular profile of disease-associated glial cells and defined its connection to modulatory activities on specific lymphocyte populations in experimental brain metastasis as well as human-derived samples. scRNASeq and high content multiplex immunofluorescence allowed us to report a novel local immunomodulatory axis dependent on TIMP1 (astrocytes)/CD63 (CD8 T cells), which is present in brain metastasis patients with high immunoscore and would imply an additional immunosuppressive signal for potential ICB responders in brain metastasis. Genetic and pharmacologic approaches targeting this STAT3-dependent local immunomodulatory axis have allowed us to define the rationale to combine immune checkpoint blockade with a STAT3 inhibitor, which we previously used in patients. We proved that such combined immunotherapy boost the systemic activation of T cells while also preventing the local blockade. Additionally, our comprehensive strategy includes the possibility to stratify patients that are best qualified to benefit from this therapy by measuring TIMP1 in liquid biopsies from CSF. Even more, our data using Patient Derived Organotypic Cultures (PDOC) from fresh brain metastasis neurosurgeries confirms that our therapeutic strategy might benefit brain metastases generated from any primary source. In conclusion, we describe an immunosuppressive mechanism in the brain microenvironment that could explain the lack of response to ICB in patients with advanced brain metastasis. Our finding provides the rationale to implement complementary approaches targeting local immunosuppression to increase the benefit of immunotherapy in symptomatic brain metastasis. Citation Format: Neibla Priego, Pedro García-Gómez, Ana de Pablos-Aragoneses, María Perea-García, Laura Álvaro-Espinosa, Carolina Hernández-Oliver, Elena Martínez-Saez, Ángel Pérez-Núñez, Aurelio Hernández-Laín, Rebeca Sanz-Pamplona, Marc Schmitz, Stephen J. Crocker, Diego Serrano, Asís Palazón, RENACER Red Nacional de Metástasis Cerebral, Manuel Valiente. TIMP1 mediates astrocyte-dependent local immunosuppression in brain metastasis. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5105.

Potentiating antibody-dependent killing of cancers with CAR T cells secreting CD47-SIRPα checkpoint blocker

AbstractChimeric antigen receptor (CAR) T-cell therapy has shown success in the treatment of hematopoietic malignancies; however, relapse remains a significant issue. To overcome this, we engineered “Orexi” CAR T cells to locally secrete a high-affinity CD47 blocker, CV1, at the tumor and treated tumors in combination with an orthogonally targeted monoclonal antibody. Traditional CAR T cells plus the antibody had an additive effect in xenograft models, and this effect was potentiated by CAR T-cell local CV1 secretion. Furthermore, OrexiCAR-secreted CV1 reversed the immunosuppression of myelomonocytoid cells both in vitro and within the tumor microenvironment. Local secretion of the CD47 inhibitor bypasses the CD47 sink found on all cells in the body and may prevent systemic toxicities. This combination of CAR T-cell therapy, local CD47 blockade, and orthogonal antibody may be a combinatorial strategy to overcome the limitations of each monotherapy.

Exon junction complex shapes the m6A epitranscriptome

N6-methyladenosine (m6A), the most abundant modification of mRNA, is essential for normal development and dysregulation promotes cancer. m6A is highly enriched in the 3’ untranslated region (UTR) of a large subset of mRNAs to influence mRNA stability and/or translation. However, the mechanism responsible for the observed m6A distribution remains enigmatic. Here we find the exon junction complex shapes the m6A landscape by blocking METTL3-mediated m6A modification close to exon junctions within coding sequence (CDS). Depletion of EIF4A3, a core component of the EJC, causes increased METTL3 binding and m6A modification of short internal exons, and sites close to exon-exon junctions within mRNA. Reporter gene experiments further support the role of splicing and EIF4A3 deposition in controlling m6A modification via the local steric blockade of METTL3. Our results explain how characteristic patterns of m6A mRNA modification are established and uncover a role of the EJC in shaping the m6A epitranscriptome.

Targeting 5-HT2A receptors and Kv7 channels in PFC to attenuate chronic neuropathic pain in rats using a spared nerve injury model

Chronic pain remains a disabling disease with limited therapeutic options. Pyramidal neurons in the prefrontal cortex (PFC) express excitatory Gq-coupled 5-HT2A receptors (5-HT2AR) and their effector system, the inhibitory Kv7 ion channel. While recent publications show these cells innervate brainstem regions important for regulating pain, the cellular mechanisms underlying the transition to chronic pain are not well understood. The present study examined whether local blockade of 5-HT2AR or enhanced Kv7 ion channel activity in the PFC would attenuate mechanical allodynia associated with spared nerve injury (SNI) in rats. Following SNI, we show that inhibition of PFC 5-HT2ARs with M100907 or opening of PFC Kv7 channels with retigabine reduced mechanical allodynia. Parallel proteomic and RNAScope experiments evaluated 5-HT2AR/Kv7 channel protein and mRNA. Our results support the role of 5-HT2ARs and Kv7 channels in the PFC in the maintenance of chronic pain.

863 Blocking the LFA-1 signaling pathway reverses alopecia areata in C3H/HeJ mice

Alopecia areata (AA) is caused by T cell-mediated autoimmune attack of the hair follicle. Therefore, inhibition of T lymphocyte migration and pathogenic activity represents an attractive therapeutic approach in the treatment of T lymphocyte-mediated autoimmune diseases, including AA. The lymphocyte function antigen-1 (LFA-1) receptor signaling pathway plays a crucial role in T cell activation, migration of T cells to target tissues and formation of the classical cytolytic immune synapse. We previously showed that the intercellular adhesion molecule 1(ICAM-1), one of the major ligands for LFA-1, is upregulated in lesional skin of both human patients and C3H/HeJ mice with AA. We observed that CD44+CD8+effector/memory T cell expressed high levels of LFA-1 within skin draining lymph nodes as well as within AA lesional skin. Here, we investigated the role of LFA-1 signaling pathway in AA using C3H/HeJ mice, and found that blockade of the LFA-1 signaling pathway using either anti-LFA-1 or anti-ICAM-1 mAbs prevented the development of AA in C3H/HeJ skin grafted mice. Immunohistochemical staining of skin biopsies show that anti-LFA-1- or anti-ICAM-1-treated mice have a striking decrease in skin T cell infiltration together with reduced expression of AA-associated markers (MHC-I and MHC-II). Furthermore, we observed that LFA-1 signaling blockade not only reduced CD44+CD8 +effector/memory T cell migration into the skin, but also led to enrichment of Tregs in skin draining lymph nodes. To limit systemic exposure, we topically treated C3H/HeJ AA mice with lifitegrast, a small molecule antagonist of LFA-1, and found that lifitegrast was effective in reversal of AA. Taken together, we demonstrated that LFA-1 plays a crucial role in T cell migration and activation in AA, and that reversal of AA was achieved by local LFA-1 signaling blockade using topical delivery. Our results invite further clinical investigation of LFA-1 pathway inhibition as a therapeutic target in AA treatment.

Estrogens rapidly shape synaptic and intrinsic properties to regulate the temporal precision of songbird auditory neurons.

Sensory neurons parse millisecond-variant sound streams like birdsong and speech with exquisite precision. The auditory pallial cortex of vocal learners like humans and songbirds contains an unconventional neuromodulatory system: neuronal expression of the estrogen synthesis enzyme aromatase. Local forebrain neuroestrogens fluctuate when songbirds hear a song, and subsequently modulate bursting, gain, and temporal coding properties of auditory neurons. However, the way neuroestrogens shape intrinsic and synaptic properties of sensory neurons remains unknown. Here, using a combination of whole-cell patch clamp electrophysiology and calcium imaging, we investigate estrogenic neuromodulation of auditory neurons in a region resembling mammalian auditory association cortex. We found that estradiol rapidly enhances the temporal precision of neuronal firing via a membrane-bound G-protein coupled receptor and that estradiol rapidly suppresses inhibitory synaptic currents while sparing excitation. Notably, the rapid suppression of intrinsic excitability by estradiol was predicted by membrane input resistance and was observed in both males and females. These findings were corroborated by analysis of in vivo electrophysiology recordings, in which local estrogen synthesis blockade caused acute disruption of the temporal correlation of song-evoked firing patterns. Therefore, on a modulatory timescale, neuroestrogens alter intrinsic cellular properties and inhibitory neurotransmitter release to regulate the temporal precision of higher-order sensory neurons.

Global REACH 2018: increased adrenergic restraint of blood flow preserves coupling of oxygen delivery and demand during exercise at high-altitude.

Chronic exposure to hypoxia (high-altitude, HA; >4000m) attenuates the vasodilatory response to exercise and is associated with a persistent increase in basal sympathetic nerve activity (SNA). The mechanism(s) responsible for the reduced vasodilatation and exercise hyperaemia at HA remains unknown. We hypothesized that heightened adrenergic signalling restrains skeletal muscle blood flow during handgrip exercise in lowlanders acclimatizing to HA. We tested nine adult males (n=9) at sea-level (SL; 344 m) and following 21-28days at HA (∼4300m). Forearm blood flow (FBF; duplex ultrasonography), mean arterial pressure (MAP; brachial artery catheter), forearm vascular conductance (FVC; FBF/MAP), and arterial and venous blood sampling (O2 delivery ( ) and uptake ( )) were measured at rest and during graded rhythmic handgrip exercise (5%, 15% and 25% of maximum voluntary isometric contraction; MVC) before and after local α- and β-adrenergic blockade (intra-arterial phentolamine and propranolol). HA reduced ΔFBF (25% MVC: SL: 138.3±47.6 vs. HA: 113.4±37.1mlmin-1 ; P=0.022) and Δ (25% MVC: SL: 20.3±7.5 vs. HA: 14.3±6.2mlmin-1 ; P=0.014) during exercise. Local adrenoreceptor blockade at HA restored FBF during exercise (25% MVC: SLα-β blockade : 164.1±71.7 vs. HAα-β blockade : 185.4±66.6mlmin-1 ; P=0.947) but resulted in an exaggerated relationship between and ( / slope: SL: 1.32; HA: slope: 1.86; P=0.037). These results indicate that tonic adrenergic signalling restrains exercise hyperaemia in lowlanders acclimatizing to HA. The increase in adrenergic restraint is necessary to match oxygen delivery to demand and prevent over perfusion of contracting muscle at HA. KEY POINTS: In exercising skeletal muscle, local vasodilatory signalling and sympathetic vasoconstriction integrate to match oxygen delivery to demand and maintain arterial blood pressure. Exposure to chronic hypoxia (altitude, >4000m) causes a persistent increase in sympathetic nervous system activity that is associated with impaired functional capacity and diminished vasodilatation during exercise. In healthy male lowlanders exposed to chronic hypoxia (21-28days; ∼4300m), local adrenoreceptor blockade (combined α- and β-adrenergic blockade) restored skeletal muscle blood flow during handgrip exercise. However, removal of tonic adrenergic restraint at high altitude caused an excessive rise in blood flow and subsequently oxygen delivery for any given metabolic demand. This investigation is the first to identify greater adrenergic restraint of blood flow during acclimatization to high altitude and provides evidence of a functional role for this adaptive response in regulating oxygen delivery and demand.

RETRACTED ARTICLE: Copper-based metal–organic framework impedes triple-negative breast cancer metastasis via local estrogen deprivation and platelets blockade

Metastasis is one of the main causes of failure in the treatment of triple-negative breast cancer (TNBC). Abnormally estrogen level and activated platelets are the key driving forces for TNBC metastasis. Herein, an “ion/gas” bioactive nanogenerator (termed as IGBN), comprising a copper-based MOF and loaded cisplatin-arginine (Pt-Arg) prodrug is developed for metastasis-promoting tumor microenvironment reprogramming and TNBC therapy. The copper-based MOF not only serves as a drug carrier, but also specifically produces Cu2+ in tumors, which catalytic oxidizing estrogen to reduce estrogen levels in situ. Meanwhile, the rationally designed Pt-Arg prodrug reduced into cisplatin to significantly promote the generation of H2O2 in the tumor, then permitting self-augmented cascade NO gas generation by oxidizing Arg through a H2O2 self-supplied way, thus blocking platelet activation in tumor. We clarified that IGBN inhibited TNBC metastasis through local estrogen deprivation and platelets blockade, affording 88.4% inhibition of pulmonary metastasis in a 4T1 mammary adenocarcinoma model. Notably, the locally copper ion interference, NO gas therapy and cisplatin chemotherapy together resulted in an enhanced therapeutic efficacy in primary tumor ablation without significant toxicity. This “ion/gas” bioactive nanogenerator offers a robust and safe strategy for TNBC therapy.Graphical

The arcuate nucleus: A site of synergism between Angiotensin II and leptin to increase sympathetic nerve activity and blood pressure in rats

The action of leptin in brain to increase sympathetic nerve activity (SNA) and blood pressure depends upon functional Angiotensin II (AngII) type 1a receptors (AT1aR); however, the sites and mechanism of interaction are unknown. Here we identify one site, the hypothalamic arcuate nucleus (ArcN), since prior local blockade of AT1aR in the ArcN with losartan or candesartan in anesthetized male rats essentially eliminated the sympathoexcitatory and pressor responses to ArcN leptin nanoinjections. Unlike mice, in male and female rats, AT1aR and LepR rarely co-localized, suggesting that this interdependence occurs indirectly, via a local interneuron or network of neurons. ArcN leptin increases SNA by activating pro-opiomelanocortin (POMC) inputs to the PVN, but this activation requires simultaneous suppression of tonic PVN Neuropeptide Y (NPY) sympathoinhibition. Because AngII-AT1aR inhibits ArcN NPY neurons, we propose that loss of AT1aR suppression of NPY blocks leptin-induced increases in SNA; in other words, ArcN-AngII-AT1aR is a gatekeeper for leptin-induced sympathoexcitation. With obesity, both leptin and AngII increase; therefore, the increased AT1aR activation could open the gate, allowing leptin (and insulin) to drive sympathoexcitation unabated, leading to hypertension.

COMPARISON OF INTERMITTENT BOLUS AND CONTINUOUS INFUSION OF 0.1% LEVOBUPIVACAINE WITH FENTANYL FOR EPIDURAL LABOR ANALGESIA

Objectives: This study was carried out to compare intermittent bolus and continuous infusion for epidural labor analgesia in terms of total dose requirement of local anesthetic and quality of analgesia as primary objective. Secondary objective was level of sensory block, motor block, hemodynamic variables, mode of delivery, duration of second stage, neonatal outcome, side effects, and postpartum complications. Methods: Eighty women of ASA physical status I or II, with single pregnancy, cephalic presentation and cervical dilatation 3–5 cm, that is, during active labor were included in the study. Patients were randomly divided into two groups. In Group A, a bolus of 8 ml of 0.1% levobupivacaine (plain) with fentanyl 2 mcg/ml was given every hour and in Group B, an infusion of 0.1% levobupivacaine (plain) with fentanyl 2 mcg/ml at 8 ml/h was given. Pain scores using visual analog scale and verbal rating score, additional bolus requirement and total dose of local anesthetic, motor blockade, fetal and neonatal outcome, mode of delivery, and duration of second stage were recorded and compared. Side effects and postpartum complications if any were documented. Results: Additional bolus requirement and total dose of local anesthetic were significantly high in Group B (45.60±6.67 mg) as compared to Group A (34.20±5.58 mg). There was no difference in the quality of analgesia, neonatal outcome, mode of delivery, duration of second stage, side effects, and complications. Conclusion: Intermittent epidural bolus is better in terms of less drug consumption and less number of additional bolus requirement.

POS-042 C3d-Directed Factor H Targeting Delivers Potent and Durable Complement Inhibition and Disease-Modifying Efficacy In Kidney and Skin Without Inhibiting Systemic Complement

POS-042 C3d-Directed Factor H Targeting Delivers Potent and Durable Complement Inhibition and Disease-Modifying Efficacy In Kidney and Skin Without Inhibiting Systemic Complement

Liposomal bupivacaine-No breakthrough in postoperative pain management

One of the main limitations concerning the use of local anesthetics is due to their restricted duration of action. In recent years, liposomal formulations with prolonged release kinetics have been developed to extend the pharmacological duration of action of the 1‑stage peripheral regional anesthesia (single-shot procedure) and thus bring about alonger duration of action. The focus here is particularly on achieving postoperative freedom from pain for at least 24 h (or even better 48 h) and thus early mobilization of patients using on-demand medication causing (at most) minor local sensory blockade without causing motor impairments (at least that is the ideal). Therefore, methods of utilizing slow-release drugs as seen in liposomal carrier systems have experienced increasing scientific attention in the last few years. Acommon modern pharmacological example with atheoretically significantly longer duration of action is liposomal bupivacaine, an amide local anesthetic. Due to amultivesicular liposome structure, the retarded release of the active component bupivacaine HCl leads to atheoretical pharmacological effectiveness of up to 72 h. Previous studies consistently showed asafety profile comparable to conventional bupivacaine HCl. Liposomal bupivacaine has been approved by the U.S. Food and Drug Administration (FDA) under the trade name Exparel© (Pacira Pharmaceuticals, Parsippany, NJ, USA) since 2011; however, its use is currently limited to local wound infiltration, transverse abdominis plane (TAP) blocks, and interscalene nerve blocks of the brachial plexus. In 2020, the European Medicines Agency (EMA) also approved the use of liposomal bupivacaine for blockade of the brachial plexus or the femoral nerve and as afield block or for wound infiltration to treat postoperative pain. So far, studies on the clinical effectiveness of liposomal bupivacaine have been very heterogeneous and there have been no conclusive meta-analyses with sufficient rigor or significance. Recent systematic reviews and meta-analyses, combining the results of clinical studies regarding the analgesic efficiency of liposomal bupivacaine in different fields of application, consistently refuted any benefit of clinical relevance provided by the liposomal formulation. There is currently sufficient evidence to now end the ongoing debate around liposomal bupivacaine. The aim of this work is to give the reader acurrent, evidence-based overview of this substance.