Receptor Density Research Articles

Characterization of oxytocin and vasopressin receptors in the Southern giant pouched rat and comparison to other rodents.

Vasopressin and oxytocin are well known and evolutionarily ancient modulators of social behavior. The distribution and relative densities of vasopressin and oxytocin receptors are known to modulate the sensitivity to these signaling molecules. Comparative work is needed to determine which neural networks have been conserved and modified over evolutionary time, and which social behaviors are commonly modulated by nonapeptide signaling. To this end, we used receptor autoradiography to determine the distribution of vasopressin 1a and oxytocin receptors in the Southern giant pouched rat (Cricetomys ansorgei) brain, and to assess the relative densities of these receptors in specific brain regions. We then compared the relative receptor pattern to 23 other species of rodents using a multivariate ANOVA. Pouched rat receptor patterns were strikingly similar to hamsters and voles overall, despite the variation in social organization among species. Uniquely, the pouched rat had dense vasopressin 1a receptor binding in the caudate-putamen (i.e., striatum), an area that might impact affiliative behavior in this species. In contrast, the pouched rat had relatively little oxytocin receptor binding in much of the anterior forebrain. Notably, however, oxytocin receptor binding demonstrated extremely dense binding in the bed nucleus of the stria terminalis, which is associated with the modulation of several social behaviors and a central hub of the social decision-making network. Examination of the nonapeptide system has the potential to reveal insights into species-specific behaviors and general themes in the modulation of social behavior.

Predation cues induce predator specific changes in olfactory neurons encoding defensive responses in agile frog tadpoles.

Although behavioural defensive responses have been recorded several times in both laboratory and natural habitats, their neural mechanisms have seldom been investigated. To explore how chemical, water-borne cues are conveyed to the forebrain and instruct behavioural responses in anuran larvae, we conditioned newly hatched agile frog tadpoles using predator olfactory cues, specifically either native odonate larvae or alien crayfish kairomones. We expected chronic treatments to influence the basal neuronal activity of the tadpoles' mitral cells and alter their sensory neuronal connections, thereby impacting information processing. Subsequently, these neurons were acutely perfused, and their responses were compared with the defensive behaviour of tadpoles previously conditioned and exposed to the same cues. Tadpoles conditioned with odonate cues differed in both passive and active cell properties compared to those exposed to water (controls) or crayfish cues. The observed upregulation of membrane conductance and increase in both the number of active synapses and receptor density at the postsynaptic site are believed to have enhanced their responsiveness to external stimuli. Odonate cues also affected the resting membrane potential and firing rate of mitral cells during electrophysiological patch-clamp recordings, suggesting a rearrangement of the repertoire of voltage-dependent conductances expressed in cell membranes. These recorded neural changes may modulate the induction of an action potential and transmission of information. Furthermore, the recording of neural activity indicated that the lack of defensive responses towards non-native predators is due to the non-recognition of their olfactory cues.

Preoperative assessment of lymph nodal metastases with [68Ga]Ga-DOTATOC PET radiomics for improved surgical planning in well-differentiated pancreatic neuroendocrine tumours.

Accurate identification of lymph node (LN) metastases is pivotal for surgical planning of pancreatic neuroendocrine tumours (PanNETs); however, current imaging techniques have sub-optimal diagnostic sensitivity. Aim of this study is to investigate whether [68Ga]Ga-DOTATOC PET radiomics might improve the identification of LN metastases in patients with non-functioning PanNET (NF-PanNET) referred to surgical intervention. Seventy-two patients who performed preoperative [68Ga]Ga-DOTATOC PET between December 2017 and March 2022 for NF-PanNET. [68Ga]Ga-DOTATOC PET qualitative assessment of LN metastases was measured using diagnostic balanced accuracy (bACC), sensitivity (SN), specificity (SP), positive and negative predictive values (PPV, NPV). SUVmax, SUVmean, Somatostatin receptor density (SRD), total lesion SRD (TLSRD) and IBSI-compliant radiomic features (RFs) were obtained from the primary tumours. To predict LN involvement, these parameters were engineered, selected and used to train different machine learning models. Models were validated using tenfold repeated cross-validation and control models were developed. Models' bACC, SN, SP, PPV and NPV were collected and compared (Kruskal-Wallis, Mann-Whitney). LN metastases were detected in 29/72 patients at histology. [68Ga]Ga-DOTATOC PET qualitative examination of LN involvement provided bACC = 60%, SN = 24%, SP = 95%, PPV = 78% and NPV = 65%. The best-performing radiomic model provided a bACC = 70%, SN = 77%, SP = 61%, PPV = 60% and NPV = 83% (outperforming the control model, p < 0.05*). In this study, [68Ga]Ga-DOTATOC PET radiomics allowed to increase diagnostic sensitivity in detecting LN metastases from 24 to 77% in NF-PanNET patients candidate to surgery. Especially in case of micrometastatic involvement, this approach might assist clinicians in a better patients' stratification.

Changes in Tryptophan Hydroxylase 2 and 5HT1A Receptor Density May Contribute to Developmental Variation in Response to Serotonin in the in vitro Bullfrog Brainstem

Bullfrog, Lithobates catesbeiana, is a vertebrate animal model that can be used to study toxicology and developmental physiology. Our research is focused on better understanding vulnerability to brainstem respiratory centers in the pathology of sudden infant death syndrome (SIDS). We propose that a link between nicotine exposure during development leads to changes in the serotonergic signaling cascades of respiratory coordinating neurons. Caudal raphe neurons send serotonergic fibers that arborize and terminate directly on or near respiratory neurons in the brainstem. In bullfrogs, serotonin elicits a developmentally dependent effect on respiratory motor output. Late metamorphic tadpoles demonstrate an increase in lung burst activity with exposure to low concentrations of serotonin (≤ 0.5μM 5-hydroxytryptamine; 5-HT) mediated in part by 5-HT1a receptor (5HT1aR) activation. This developmental change in activity may be due to changes in the number of serotonergic fibers and/or 5-HT1a receptor density. Using the NIH Basic Local Alignment Search Tool (BLAST) we compared human and bullfrog 5HT1aR and tryptophan hydroxylase 2 (TH2) protein sequences and found a 34 and 65 percent identity match respectively. Quantitative western blot analysis of both proteins indicates developmental shifts in the serotonergic network during metamorphosis. These results were supported by immunohistochemical staining of 5HT1aR and TH2 in caudal raphe containing brainstem sections from pre-metamorphic tadpoles and post-metamorphic frogs. Impairments in the maturation of this serotonergic network may underlie the toxidrome associated with developmental nicotine exposure and further support the increased risk of SIDS following that exposure. This research was supported by the University of Wisconsin- La Crosse (UWL) through support from the UWL College of Science and Health, two UWL Undergraduate Research Creative Grants and a UWL FY23/24 Faculty Research Grant. This is the full abstract presented at the American Physiology Summit 2024 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.

Exploring the pathogenetic mechanisms of Mycoplasmapneumoniae (Review).

Mycoplasmas, the smallest self-replicating prokaryotes without a cell wall, are the most prevalent and extensively studied species in humans. They significantly contribute to chronic respiratory tract illnesses and pneumonia, with children and adolescents being particularly vulnerable. Mycoplasma pneumoniae (M. pneumoniae) infections typically tend to be self-limiting and mild but can progress to severe or even life-threatening conditions in certain individuals. Extrapulmonary effects often occur without pneumonia, and both intrapulmonary and extrapulmonary complications operate through separate pathological mechanisms. The indirect immune-mediated damage of the immune system, vascular blockages brought on by vasculitis or thrombosis and direct harm from invasion or locally induced inflammatory cytokines are potential causes of extrapulmonary manifestations due to M. pneumoniae. Proteins associated with adhesion serve as the primary factor crucial for the pathogenicity of M. pneumoniae, relying on a specialized polarized terminal attachment organelle. The type and density of these host receptors significantly impact the adhesion and movement of M. pneumoniae, subsequently influencing the pathogenic mechanism and infection outcomes. Adjacent proteins are crucial for the proper assembly of the attachment organelle, with variations in the genetic domains of P1, P40 and P90 surfaces contributing to the variability of clinical symptoms and offering new avenues for developing vaccines against M. pneumoniae infections. M. pneumoniae causes oxidative stress within respiratory tract epithelial cells by adhering to host cells and releasing hydrogen peroxide and superoxide radicals. This oxidative stress enhances the vulnerability of host cells to harm induced by oxygen molecules. The lack of superoxide dismutase and catalase of bacteria allows it to hinder the catalase activity of the host cell, leading to the reduced breakdown of peroxides. Lung macrophages play a significant role in managing M. pneumoniae infection, identifying it via Toll-like receptor 2 and initiating the myeloid differentiation primary response gene 88-nuclear factor κΒ signaling cascade. However, the precise mechanisms enabling M. pneumoniae to evade intracellular host defenses remain unknown, necessitating further exploration of the pathways involved in intracellular survival. The present comprehensive review delves into the pathogenesis of M. pneumoniae infection within the pulmonary system and into extrapulmonary areas, outlining its impact.

Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Loss of tumor suppressor TMEM127 drives RET-mediated transformation through disrupted membrane dynamics.

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTKs) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumor pheochromocytoma (PCC) can be caused by activating mutations of the rearranged during transfection (RET) receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumor suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin-coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

Разработка и реализация протокола поведенческого эксперимента для оценки когнитивных способностей и социального поведения мышей после стресса в раннем возрасте

&lt;p style="text-align: justify;"&gt;This manuscript presents a protocol designed for the comprehensive investigation of early life stress (ELS) outcomes and a feasibility study conducted with this protocol. ELS alters normal development by interfering at various levels: hormonal changes, brain cellular architecture, epigenome, and chromosomal structural elements. The protocol combines classic behavioral tests with advanced molecular techniques to obtain comprehensive data and thus uncover the underlying mechanisms of ELS. In this protocol, the main source of stress is maternal separation. Briefly, a group of C57Bl/6 mice undergoes maternal separation; then, mice perform the radial maze test and the resident-intruder test. As a control, another group of mice stays undisturbed and performs the same behavioral tests in the same timeframe. After the behavioral tests, biosamples are collected, including urine for corticosterone measurements, peripheral blood, hippocampus, amygdala, and prefrontal cortex tissues for DNA isolation and its downstream analyses (DNA methylation profiling and telomere length measuring), and whole brains for immunohistochemistry analysis of the glucocorticoid receptor density. This protocol was successfully tested as a feasibility study for a large-scale investigation that addresses potential flaws to establish a robust methodology. This paper reports on a comprehensive approach to examining multiple aspects of development that interrogates a holistic analysis of multilayer and multidimensional data and may contribute valuable insights for both animal and human studies.&lt;/p&gt;

Multiscale Textile‐Based Haptic Interactions

Wearable haptic devices transmit information via touch receptors in the skin, yet devices located on parts of the body with high densities of receptors, such as fingertips and hands, impede interactions. Other locations that are well‐suited for wearables, such as the wrists and arms, suffer from lower perceptual sensitivity. The emergence of textile‐based wearable devices introduces new techniques of fabrication that can be leveraged to address these constraints and enable new modes of haptic interactions. This article formalizes the concept of “multiscale” interaction, an untapped paradigm for haptic wearables, enabling enhanced delivery of information via textile‐based haptic modules. In this approach, users choose the depth and detail of their haptic experiences by varying their interaction mode. Flexible prototyping methods enable multiscale haptic bands that provide both body‐scale interactions (on the forearm) and hand‐scale interactions (on the fingers and palm). A series of experiments assess participants’ ability to identify pressure states and spatial locations delivered by these bands across these interaction scales. A final experiment demonstrates the encoding of three‐bit information into prototypical multiscale interactions, showcasing the paradigm's efficacy. This research lays the groundwork for versatile haptic communication and wearable design, offering users the ability to select interaction modes for receiving information.

Smoking Progression and Nicotine-Enhanced Reward Sensitivity Predicted by Resting-State Functional Connectivity in Salience and Executive Control Networks.

The neural underpinnings underlying individual differences in nicotine-enhanced reward sensitivity (NERS) and smoking progression are poorly understood. Thus, we investigated whether brain resting-state functional connectivity (rsFC.) during smoking abstinence predicts NERS and smoking progression in young light smokers. We hypothesized that high rsFC between brain areas with high densities of nicotinic receptors (insula, anterior cingulate cortex [ACC], hippocampus, thalamus) and areas involved in reward-seeking (nucleus accumbens [NAcc], prefrontal cortex [PFC]) would predict NERS and smoking progression. Young light smokers (N = 64, age 18-24, M = 1.89 cigarettes/day) participated in the study. These individuals smoked between 5 and 35 cigarettes per week and lifetime use never exceeded 35 cigarettes per week. Their rsFC was assessed using functional magnetic resonance imaging after 14 hours of nicotine deprivation. Subjects also completed a probabilistic reward task after smoking a placebo on 1 day and a regular cigarette on another day. The probabilistic-reward-task assessed greater NERS was associated with greater rsFC between the right anterior PFC and right NAcc, but with reduced rsFC between the ACC and left inferior prefrontal gyrus and the insula and ACC. Decreased rsFC within the salience network (ACC and insula) predicted increased smoking progression across 18 months and greater NERS. These findings provide the first evidence that differences in rsFCs in young light smokers are associated with nicotine-enhanced reward sensitivity and smoking progression. NCT02129387 (preregistered hypothesis: www.clinicaltrials.gov). Weaker rsFC within the salience network predicted greater NERS and smoking progression. These findings suggest that salience network rsFC and drug-enhanced reward sensitivity may be useful tools and potential endophenotypes for reward sensitivity and drug-dependence research.

Radiopharmaceutical transport in solid tumors via a 3-dimensional image-based spatiotemporal model

Lutetium-177 prostate-specific membrane antigen (177Lu-PSMA)-targeted radiopharmaceutical therapy is a clinically approved treatment for patients with metastatic castration-resistant prostate cancer (mCRPC). Even though common practice reluctantly follows “one size fits all” approach, medical community believes there is significant room for deeper understanding and personalization of radiopharmaceutical therapies. To pursue this aim, we present a 3-dimensional spatiotemporal radiopharmaceutical delivery model based on clinical imaging data to simulate pharmacokinetic of 177Lu-PSMA within the prostate tumors. The model includes interstitial flow, radiopharmaceutical transport in tissues, receptor cycles, association/dissociation with ligands, synthesis of PSMA receptors, receptor recycling, internalization of radiopharmaceuticals, and degradation of receptors and drugs. The model was studied for a range of values for injection amount (100–1000 nmol), receptor density (10–500 nmol•l–1), and recycling rate of receptors (10–4 to 10–1 min–1). Furthermore, injection type, different convection-diffusion-reaction mechanisms, characteristic time scales, and length scales are discussed. The study found that increasing receptor density, ligand amount, and labeled ligands improved radiopharmaceutical uptake in the tumor. A high receptor recycling rate (0.1 min–1) increased radiopharmaceutical concentration by promoting repeated binding to tumor cell receptors. Continuous infusion results in higher radiopharmaceutical concentrations within tumors compared to bolus administration. These insights are crucial for advancing targeted therapy for prostate cancer by understanding the mechanism of radiopharmaceutical distribution in tumors. Furthermore, measures of characteristic length and advection time scale were computed. The presented spatiotemporal tumor transport model can analyze different physiological parameters affecting 177Lu-PSMA delivery.

Functional connectivity subtypes of MDD and their associations with gene expression profile, neurotransmitter, and cognition

IntroductionThere’s large heterogeneity present in major depressive disorder (MDD) and controversial evidence on alterations of brain functional connectivity (FC), making it hard to elucidate the neurobiological basis of MDD. Subtyping is one promising solution to characterize this heterogeneity.ObjectivesTo identify neurophysiological subtypes of MDD based on FC derived from resting-state functional magnetic resonance imaging using large multisite data and investigate the differences in genetic mechanisms and neurotransmitter basis of FC alterations, and the differences of FC-related cognition between each subtype.MethodsConsensus clustering of FC patterns was applied to a population of 829 MDD patients from REST-Meta-MDD database after data cleaning and image quality control. Gene transcriptomic data derived from Allen Human Brain Atlas and neurotransmitter receptor/transporter density data acquired by using neuromap toolbox were used to characterize the molecular mechanism underlying each FC-based subtype by identifying the gene set and neurotransmitters/transporters showing high spatial similarity with the profiles of FC alterations between each subtype and 770 healthy controls. The FC-related cognition in each subtype was also selected by lasso regression.ResultsTwo stable neurophysiological MDD subtypes were found and labeled as hypoconnectivity (n=527) and hyperconnectivity (n=299) characterized by the FC differences in each subtype relative to controls, respectively. The two subtypes did not differ in age, sex, and scores of Hamilton Depression/Anxiety Scale.The genes related to FC alterations were enriched in ion transmembrane transport, synaptic transmission/organization, axon development, and regulation of neurotransmitter level for both subtypes, but specifically enriched in glial cell differentiation for hypoconnectivity subtype, while enriched in regulation of presynaptic membrane and regulation of neuron differentiation for hyperconnectivity subtype.FC alterations were associated with the density of 5-HT2a receptor in both subtypes. For hyperconnectivity subtype, FC alterations were also correlated with the density of norepinephrine transporter, glutamate receptor, GABA receptor, 5-HT1b receptor, and cannabinoid receptor.Both subtypes showed correlations between FC and categorization, motor inhibition, and localization. The FC in hypoconnectivity subtype correlated with response inhibition, selective attention, face recognition, sleep, empathy, expertise, uncertainty, and anticipation, while that was related to inference, speech perception, and reward anticipation in hyperconnectivity subtype.ConclusionsOur findings suggested the presence of two neuroimaging subtypes of MDD characterized by hypo or hyper-connectivity. The two subtypes had both shared and distinct genetic mechanisms, neurotransmitter receptor/transporter profiles, and cognition types.Disclosure of InterestNone Declared

Abstract 2610: Discovery of AM E3: A novel α-TLSPR monoclonal antibody for the ADC treatment of CRLF2-rearranged Ph-like B-ALL

Abstract Background: Patients affected by Ph-like B-cell acute lymphoblastic leukemia (B-ALL) have a high mortality rate and elevated levels of disease relapse upon chemotherapy treatment. More than 60% of Ph-like B-ALL is characterized by genomic rearrangements resulting in high expression level of the CRLF2/TLSPR receptor on the cell surface. While significant advances have been made in developing targeted agents for B-ALL treatment, the CRLF2-rearranged subpopulation is in complete absence of selective and effective therapies. Here, we report the identification of α-TLSPR monoclonal antibodies with therapeutic potential and the preclinical validation of AM E3 as an Antibody-Drug Conjugate (ADC) for the treatment of CRLF2-rearranged Ph-like B-ALL. Material and Methods: Leveraging a high-complex phage display platform we have first identified several fully human α-TLSPR monoclonal antibodies with single-digit nanomolar affinity for the target. Among them, AM E3 emerged as suitable for ADC development due to its rapid internalization by CRLF2-expressing cells. As a proof of concept, upon conjugation with the cytotoxic payload tesirine (SG3249) AM E3 was validated for anti-tumor response in both CRLF2-rearranged in vitro cell lines and in vivo xenograft models. Further, since human/mouse CRLF2 homology is low (˂35%), we have also developed an ADC surrogate against murine TLSPR to establish a therapeutic window for the ADC therapy in CRLF2-rearranged preclinical models. Results: ADC AM E3 demonstrated strong cytotoxicity (EC50=40pM) on cell lines derived from hCRLF2-rearranged B-ALL patients. In xenograft models a single injection of ADC AM E3 (0.7mg/Kg) showed antitumoral activity proportional to hCRLF2/TSLPR receptor density at the cell surface. Moreover, ADC AM E3 treatment reduces tumor growth and disease burden of hCRLF2-rearranged B-ALL orthotopic xenografts and extends survival over standard induction therapy. ADC surrogate mB4 AM23, which retains similar properties as the human ADC AM E3, reduces tumor growth and is well tolerated in xenograft models engineered to express mCRLF2/TSLPR. Conclusions: AM E3 is a novel α-TLSPR mAb with ideal features for the development of a first-in-class ADC therapy. α-TLSPR ADC demonstrated in vitro and in vivo efficacy as a monotherapy for the treatment of CRLF2/TLSPR rearranged Ph-like B-ALLs and proven to be well-tolerated in surrogate animal studies. Citation Format: Claudia Dall'Armi, Valentina Di Biasio, Chantal Paolini, Daniela Natale, Francesco Scalabrì, Antonino Missineo, Nadine Alaimo, Maurizio Nuzzo, Anna Demartis, Pamela Di Pasquale, Roberto Benoni, Fulvia Caretti, Elisa Beghetto, Fabrizio Colaceci, Romina Sasso, Vincenzo Pucci, Alessandro Carugo, Licia Tomei, Carlo Toniatti. Discovery of AM E3: A novel α-TLSPR monoclonal antibody for the ADC treatment of CRLF2-rearranged Ph-like B-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2610.

Abstract 4272: Disruption of hyaluronan metabolism alters glioma cell proliferation by ligand dependent and independent mechanisms

Abstract Hyaluronan (HA) is the main constituent of the brain extracellular matrix and is an essential component of the glioblastoma (GBM) tumor microenvironment. The biosynthesis and catabolism of hyaluronan has multiple roles in tissue architecture and cell signaling. Dysregulation of these mechanisms is crucial in pathological processes such as cancer, inflammation or tissue remodeling. The role of HA is believed to be strongly dependent on its size (molecular weight), location, and cell receptor density and activity. HA is produced intracellularly by HA synthases (HAS1-3) and can also be catabolized endogenously by hyaluronidases (HYAL1-3).The fabrication of controlled microenvironments provided by three dimensional (3D) models helps to elucidate the role of HA signaling in GBM tumors. We exploit here the advantages of these models to understand the role of hyaluronan in the local behavior of GBM after radiation therapy and selected targeted inhibitors. We have established engineered brain tumor biomaterials based on functionalized hydrogels to monitor the response of patient-derived xenograft cell populations with different molecular signatures in combination with microfluidic devices. We have analyzed shifts in metabolism, hyaluronan secretion, as well as hyaluronan synthetic enzymes (HAS) and hyaluronidases (HYAL) activity in an array of patient derived GBM cells. We reveal that endogenous HA plays a role in mitochondrial respiration and cell proliferation in a tumor subtype dependent manner. We also show that HA fragments can enhance tumor metabolism and growth through ligand dependent and independent mechanisms. We provide bioengineered platforms and strategies to predict and stratify tumors in order to achieve more efficient combinatorial treatments targeting endogenous hyaluronan production. This work emphasizes the potential of these preclinical models to predict and accelerate cancer treatments, more relevant in malignancies with limited treatment options. Citation Format: Edward R. Neves, Joseph Mueller, Achal Anand, Kimberly Selting, Hui Xu, Roddel Remy, Brendan Harley, Sara Pedron-Haba. Disruption of hyaluronan metabolism alters glioma cell proliferation by ligand dependent and independent mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4272.

Abstract 2579: A nectin-4 PET radiotracer for pharmacodynamic evaluation of enfortumab vedotin (EV) therapy in urothelial carcinoma

Abstract Introduction: Enfortumab vedotin (EV), an antibody-drug conjugate targeting nectin-4, offers hope for urothelial carcinoma patients (UC) but prolonged use leads to treatment-related toxicities often necessitating dose adjustments. How EV engages nectin-4 at the tumor and its relevance to efficacy is unknown. To understand the pharmacodynamics (target engagement) of EV at the tumor across FDA approved dosing levels, we developed a nectin-4 specific peptide-based radiotracer, [68Ga]AJ647, for positron emission tomography (PET) imaging. Methods: Six UC cell lines of luminal (HT-1376, UC-14, RT-112) and basal (UC-9, SCaBER, T24) morphological type were selected for in vitro and in vivo testing of [68Ga]AJ647 and nectin-4 target engagement by EV. Cell uptake and competitive binding assays were conducted to test [68Ga]AJ647 specificity for nectin-4 in the presence or absence of EV. Flow cytometry derived receptor density was used to correlate [68Ga]AJ647 uptake in cells with nectin-4 expression. Pharmacokinetics, biodistribution and specificity of [68Ga]AJ647 for nectin-4 expression was evaluated in six xenograft models by PET imaging and biodistribution studies. Target engagement by EV was quantified by measuring unoccupied nectin-4 levels by [68Ga]AJ647-PET in HT-1376 xenografts at different doses (15, 9, and 6 mg/kg) and time (1, 3, and 6 days). Results: [68Ga]AJ647 was generated in 20-30% radiochemical yield with &amp;gt;95% purity. In vitro assays showed distinctive uptake in cell lines (HT-1376 &amp;gt; UC-14 &amp;gt; RT-112; UC-9 &amp;gt; SCaBER &amp;gt; T24) that correlated with nectin-4 density (R2=0.85). PET and biodistribution studies showed tractable pharmacokinetics of [68Ga]AJ647 enabling high contrast images in 60 min and exhibiting nectin-4 dependent uptake in all the six xenografts. Uptake of [68Ga]AJ647 in UC cells reduced in a dose dependent manner in the presence of EV, indicating that [68Ga]AJ647 measures unoccupied nectin-4, and paving the way for EV target engagement evaluation. For target engagement evaluation, [68Ga]AJ647 accumulation in HT-1376 tumors in saline treated mice was assumed as 100% unoccupied nectin-4 levels. In mice treated with EV for 24 hrs (n=3-5), unoccupied nectin-4 levels were 21.1±11.6, 23.2±14.5, and 24.3±18.8 for 15, 9, and 6 mg/kg doses, respectively. Whereas on day 6, unoccupied nectin-4 levels showed significant differences at 29.5±15.8, 64.2±21.0, and 89.5±18.9 for 15, 9, and 6 mg/kg dose, respectively. These data indicate that FDA approved doses of EV show higher target engagement at 24 h but a dramatic reduction at lower doses over time. Conclusion: [68Ga]AJ647-PET quantifies variable nectin-4 expression in tumors. Furthermore, [68Ga]AJ647-PET quantifies EV therapy induced pharmacodynamics at the tumor. Such real-time ADC target engagement assessment could inform patient selection and dosing strategies to enhance efficacy and minimize toxicities. Citation Format: Akhilesh Mishra, Ajay K. Sharma, Kuldeep Gupta, Burles A. Johnson, David J. McConkey, Jeannie Hoffman-Censits, Sridhar Nimmagadda. A nectin-4 PET radiotracer for pharmacodynamic evaluation of enfortumab vedotin (EV) therapy in urothelial carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2579.

Abstract 4180: Evaluation of CD38 pharmacodynamics in multiple myeloma using PET with a Ga-68 labelled peptide

Abstract Introduction: Multiple myeloma (MM) is a disease of plasma cells that primarily arises in the bone marrow. CD38 is a receptor expressed uniformly and with high density in MM. Several CD38 targeted therapeutics are in clinic for MM. However, how those therapeutics engage or modulate CD38 is poorly understood particularly in lesions that cannot be assessed with routine blood testing. CD38 specific radiotracers for positron emission tomography (PET) could be helpful for detection of MM and for assessing therapy response early by measuring CD38 pharmacodynamics non-invasively. Here, we report the development of a first-in-class peptide-based PET radiotracer, [68Ga]AJ206, evaluation of its pharmacokinetics (PK), specificity for non-invasive CD38 detection, and its potential to evaluate therapy induced CD38 pharmacodynamics in mouse models of MM. Methods: A bicyclic peptide, AJ206, was synthesized and its binding affinity for CD38 was determined by surface plasmon resonance. AJ206 was labelled with Gallium-68 in high radiochemical yields and purity. In vitro uptake of [68Ga]AJ206 was carried out in four MM cell lines with variable CD38 expression (MOLP8, MM1S, RPMI8226, and U266). PK and biodistribution of [68Ga]AJ206 were evaluated by PET in cell line derived xenografts, in patient derived xenografts (PDXs) and in a disseminated disease model. PET data was corroborated by ex vivo biodistribution studies. [68Ga]AJ206 in vivo specificity for CD38 was confirmed by co-injection of non-radioactive AJ206 (1 mg/kg) and cross-correlative immunohistochemistry (IHC) of matched xenografts. Furthermore, ability of [68Ga]AJ206 to quantify CD38 pharmacodynamics was established by treating PDX models with all-trans retinoic acid (ATRA) and cross validating PET imaging observations with IHC. Results: AJ206 exhibits high affinity (KD,19 nM) for human CD38. In vitro binding assays showed variable [68Ga]AJ206 uptake in MM cells that correlated with receptor density measured by flow cytometry. Dynamic PET-MR imaging revealed high and specific uptake of [68Ga]AJ206 in MOLP8 tumor within 5 min that retained for at least 90 min. In contrast, [68Ga]AJ206 exhibited rapid clearance from normal tissues providing high contrast images at 60 minutes and a high tumor-to-muscle ratio of 27.1±2.7. Tumor uptake was reduced by &amp;gt; 80 % in mice receiving blocking dose, confirming the specificity of the radiotracer. Also, [68Ga]AJ206 could detect CD38 expression in different MM xenografts, PDXs and disseminated disease models which was corroborated by flow cytometry and IHC findings. Moreover, [68Ga]AJ206 successfully quantified the increased CD38 expression in PDXs following ATRA treatment. Conclusion: [68Ga]AJ206 is a CD38 specific high affinity peptide-based PET imaging agent that can be used to non-invasively quantify CD38 pharmacodynamics in MM, with potential applications in other diseases with CD38 involvement. Citation Format: Ajay Kumar Sharma, Kuldeep Gupta, Akhilesh Mishra, Gabriela Lofland, Ian Marsh, Dhiraj Kumar, Gabriel Ghiaur, Philip Imus, Steven P. Rowe, Robert Hobbs, Christian B. Gocke, Sridhar Nimmagadda. Evaluation of CD38 pharmacodynamics in multiple myeloma using PET with a Ga-68 labelled peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4180.

Influence of sex hormones on the aggressive behavior during peck order establishment and stabilization in meat and egg type chickens

In the poultry industry, broiler and layer strains are genetically selected for different purposes (e.g., high meat-yield and high egg-production). Genetic selection for productivity can have unintended consequences on the behavioral repertoire of the birds, including aggression. Alongside the increasing societal concern regarding the welfare of animal in agriculture, the number of countries that are advocating the prohibition of using battery cages for laying hens has resulted in the transition and adoption of cage-free or free-range systems. Thus, both broiler and layer chickens are housed in large flocks rather than housed individually in cages. Housing birds in groups increases the opportunity for birds to engage in social behaviors, including aggression, that are used to establish social status. Aggressive interactions are associated with the risk of injury and the potential for a subordinate animal to have unmet needs (e.g., access to feed). The aim of this study was to characterize the relationships among aggressive behavior, neurobiology, and hormones during peck order establishment and social hierarchy stabilization of 2 divergently selected strains (meat- and egg-type chicken). Meat-type strains performed more male on male (P < 0.001), male on female (P < 0.0001), and female on female (P < 0.0001) non-reciprocal aggression behavior (NRA) than egg-type strains. Greater serum testosterone and estradiol concentrations in the weeks after the peck order establishment were observed in meat-type birds compared those in egg-type birds for both males and females (all P < 0.05). Greater (P < 0.05) cellular densities of androgen receptors, but not estrogen receptors, were observed in the hypothalamus of meat-type birds compared to egg-type birds. These findings suggest that greater sex hormone concentrations in the meat-type birds may be a consequence of genetic selection for rapid growth resulting in more sex hormones-induced aggressive behavior.

Blockade of sympathetic ganglia improves vascular dysfunction in septic shock.

Sepsis/septic shock activates the sympathetic nervous system (SNS) to deal with the infection stress. However, an imbalanced or maladaptive response due to excessive or uncontrolled activation characterizes autonomic dysfunction. Our hypothesis was that reducing this excessive activation of the autonomic nervous system would impact positively in sepsis. Using ganglionic blockers as a pharmacological approach, the main aim of the present report was to assess the role of ganglionic transmission in the vascular dysfunction associated with sepsis.Sepsis was induced in rats by cecal ligation and puncture (CLP). One hour after CLP surgery, rats were treated subcutaneously with hexamethonium (15 mg/kg; ganglionic blocker), pentolinium (5 mg/kg; a blocker with a higher selectivity for sympathetic ganglia compared to hexamethonium), or vehicle (PBS). Basal blood pressure and the response to adrenergic agonists were evaluated at 6 and 24 h after CLP surgery. Reactivity to vasoconstrictors, nitric oxide (NO) synthase 2 (NOS-2) expression, IL-1 and TNF plasma levels, and density of α1 adrenergic receptors were evaluated in the aorta 24 h after CLP.Septic shock resulted in hypotension and hyporesponsiveness to norepinephrine and phenylephrine, increased plasma cytokine levels and NOS-2 expression in the aorta, and decreased α1 receptor density in the same vessel. Pentolinium but not hexamethonium recovered responsiveness and α1 adrenergic receptor density in the aorta. Both blockers normalized the in vivo response to vasoconstrictors, and reduced plasma IL-1 and NOx levels and NOS-2 expression in the aorta.Blockade of ganglionic sympathetic transmission reduced the vascular dysfunction in experimental sepsis. This beneficial effect seems to be, at least in part, due to the preservation of α1 adrenergic receptor density and to reduced NOS-2 expression and may lead to adjuvant ways to treat human sepsis.

Functional reorganization of memory processing in the hippocampus is associated with neuroprotector GLP-1 levels in type 2 diabetes

Type 2 diabetes (T2D) often impairs memory functions, suggesting specific vulnerability of the hippocampus. In vivo neuroimaging studies relating encoding and retrieval of memory information with endogenous neuroprotection are lacking. The neuroprotector glucagon-like peptide (GLP-1) has a high receptor density in anterior/ventral hippocampus, as shown by animal models. Using an innovative event-related fMRI design in 34 participants we investigated patterns of hippocampal activity in T2D (n = 17) without mild cognitive impairment (MCI) versus healthy controls (n = 17) during an episodic memory task. We directly measured neurovascular coupling by estimating the hemodynamic response function using event-related analysis related to encoding and retrieval of episodic information in the hippocampus. We applied a mixed-effects general linear model analysis and a two-factor ANOVA to test for group differences. Significant between-group differences were found for memory encoding, showing evidence for functional reorganization: T2D patients showed an augmented activation in the posterior hippocampus while anterior activation was reduced. The latter was negatively correlated with both GLP-1 pre- and post-breakfast levels, in the absence of grey matter changes. These results suggest that patients with T2D without MCI have pre-symptomatic functional reorganization in brain regions underlying episodic memory, as a function of the concentration of the neuroprotective neuropeptide GLP-1.

Clemastine and metformin extend the window of NMDA receptor surface expression in ageing oligodendrocyte precursor cells

In the central nervous system, oligodendrocyte precursor cells (OPCs) proliferate and differentiate into myelinating oligodendrocytes throughout life, allowing for ongoing myelination and myelin repair. With age, differentiation efficacy decreases and myelin repair fails; therefore, recent therapeutic efforts have focused on enhancing differentiation. Many cues are thought to regulate OPC differentiation, including neuronal activity, which OPCs can sense and respond to via their voltage-gated ion channels and glutamate receptors. However, OPCs’ density of voltage-gated ion channels and glutamate receptors differs with age and brain region, and correlates with their proliferation and differentiation potential, suggesting that OPCs exist in different functional cell states, and that age-associated states might underlie remyelination failure. Here, we use whole-cell patch-clamp to investigate whether clemastine and metformin, two pro-remyelination compounds, alter OPC membrane properties and promote a specific OPC state. We find that clemastine and metformin extend the window of NMDAR surface expression, promoting an NMDAR-rich OPC state. Our findings highlight a possible mechanism for the pro-remyelinating action of clemastine and metformin, and suggest that OPC states can be modulated as a strategy to promote myelin repair.