Clearance Mechanisms Research Articles

The Biodistribution of the Novel Heme and Hemoglobin Scavenger Apohemoglobin-Haptoglobin Complex in Healthy C57BL/6 Mice

Background: Apohemoglobin-haptoglobin (apoHb-Hp) complex represents a bioengineered complex designed to scavenge hemoglobin and heme. These substances are released into the intravascular space during hemolysis, leading to vasoconstriction, inflammation, and disease progression. The use of apoHb-Hp complex has already demonstrated promising results in sickle cell and beta-thalassemia mouse models, highlighting its potential as a therapeutic agent. However, there are still crucial aspects that remain poorly understood, particularly concerning the biodistribution and pharmacokinetics associated during chronic administration. Understanding how the drug is distributed throughout the body is essential for optimizing its therapeutic potential, ensuring patient safety, and guiding both drug development and clinical use. Methods: To address these critical questions of apoHb-Hp biodistribution and pharmacokinetics, we conducted a study using twenty C57BL/6 mice from Jackson Laboratory. The mice were treated with the apoHb-Hp complex via daily injection every other day for different periods: 24 hours, one week, three weeks, and six weeks, with five mice in each subgroup. The dosage used was 80 mg/kg, and the concentration was 33.6 mg/mL. To evaluate the half-life and biodistribution of the complex, the last dose in each subgroup was radiolabeled with technetium-99m (99mTc-ApoHb-Hp).The intravascular 99mTc-ApoHb-Hp levels were measured after 30 minutes, 1 hour, 4 hours, and 24 hours to establish the drug's half-life. After the final tail vein sample collection, the mice were sacrificed, and their hearts, livers, spleens, lungs, and kidneys were weighed and collected to determine the amount of 99mTc-ApoHb-Hp present in each organ. Results: The results indicate that a significant portion of the 99mTc-ApoHb-Hp was accumulated in the liver, and regardless of the duration of dosing, the half-life for the 24 hour group was 10 hours, for the 1 week it was 6.5hrs, and for the 3 weeks it was 9hrs, showing no significant difference between the groups. hours. Liver accumulation favors the hepatocytes metabolism degradation of the apoHb-Hp complex from the body. There was no significant differences in half life based on dosing chronicity, likely suggesting that there was no additional degradation of ApoHb-Hp by immune mediated mechanisms such as antibody formation. In conclusion, Biodistribution of ApoHb-Hp was predominantly in the liver in both acute setting ( single dose) and chronic setting ( multiple doses). The average half life of the infusion was 5 hours and the predominant clearance mechanism was likely via hepatic elimination. Further research and development may help improve our understanding of the biodistribution of the apo Hb- Hp complex, therapy opening new doors for the treatment of hemolytic anemias using the ApoHb-Hp complex.

2529. Population pharmacokinetic models for cefepime and enmetazobactam derived from pooled Phase 1 to Phase 3 clinical studies

Abstract Background The investigational β-lactam/β-lactamase inhibitor combination of cefepime (FEP)/enmetazobactam (ENM) met criteria for non-inferiority and superiority compared to piperacillin/tazobactam in a phase 3 clinical trial of adult patients with complicated urinary tract infections (cUTI)/acute pyelonephritis (AP). In this study, a population pharmacokinetic (PK) model is presented that describes FEP and ENM plasma PK in adult cUTI/AP patients. Methods Plasma PK samples were analyzed from participants receiving FEP (n=588) and ENM (n=649) in three phase 1 (n=132), and single phase 2 (n=43) and phase 3 (n=488) clinical trials. Population PK parameters were assessed and the relationship between different covariates and model parameters were investigated. The effect of infection on disposition was assessed by testing disease covariates on fixed and random effects for all PK parameters. Results A two-compartment model with linear clearance of FEP (5.95 L/h) and ENM (7.68 L/h) from the central compartment best described the PK of both compounds. Common covariates for both compounds were de-indexed estimated glomerular filtration rate (eGFR), age, and body weight while ENM PK covariates also included cUTI infection and sex. Simulations showed that the only covariate requiring dose adjustment was renal function. The final model simultaneously described FEP and ENM plasma PK, which enabled estimates of the correlation of the random effects between PK parameters. Clearances and volumes of distribution (Vd) were strongly correlated within individuals between the two compounds (correlation coefficients >0.9). Variability was higher in cUTI patients although differences in mean ENM or FEP exposures between healthy subjects and cUTI patients were negligible. The total Vd was 16.9 L for FEP and 20.6 L for ENM. Clearance of both agents was found to depend on de-indexed eGFR (covariate coefficients ≥0.83), suggesting that renal filtration is the predominant clearance mechanism. The terminal elimination half-life was 2.2 h for FEP and 2.0 h for ENM. Final population PK model population parameter estimates for cefepime and enmetazobactam determined from the pooled phase 1 to phase 3 clinical studies Conclusion The population PK models for FEP and ENM predict exposures in cUTI patients, including subjects with renal impairment. These models support Monte-Carlo simulations and target attainment estimations in adult cUTI/AP patients. Disclosures Adam Belley, PhD, Allecra Therapeutics SAS: Advisor/Consultant Patrick Velicitat, MD, Allecra Therapeutics SAS: Salary

Relationship among α‑synuclein, aging and inflammation in Parkinson's disease (Review).

Parkinson's disease (PD) is a common neurodegenerative pathology whose major clinical symptoms are movement disorders. The main pathological characteristics of PD are the selective death of dopaminergic (DA) neurons in the pars compacta of the substantia nigra and the presence of Lewy bodies containing α-synuclein (α-Syn) within these neurons. PD is associated with numerous risk factors, including environmental factors, genetic mutations and aging. In many cases, the complex interplay of numerous risk factors leads to the onset of PD. The mutated α-Syn gene, which expresses pathologicalα-Syn protein, can cause PD. Another important feature of PD is neuroinflammation, which is conducive to neuronal death. α-Syn is able to interact with certain cell types in the brain, including through phagocytosis and degradation of α-Syn by glial cells, activation of inflammatory pathways by α-Syn in glial cells, transmission of α-Syn between glial cells and neurons, and interactions between peripheral immune cells and α-Syn. In addition to the aforementioned risk factors, PD may also be associated with aging, as the prevalence of PD increases with advancing age. The aging process impairs the cellular clearance mechanism, which leads to chronic inflammation and the accumulation of intracellular α-Syn, which results in DA neuronal death. In the present review, the age-associated α-Syn pathogenicity and the interactions between α-Syn and certain types of cells within the brain are discussed to facilitate understanding of the mechanisms of PD pathogenesis, which may potentially provide insight for the future clinical treatment of PD.

Exploring and Analyzing the Systemic Delivery Barriers for Nanoparticles.

Most nanomedicines require efficient in vivo delivery to elicit diagnostic and therapeutic effects. However, en route to their intended tissues, systemically administered nanoparticles often encounter delivery barriers. To describe these barriers, we propose the term "nanoparticle blood removal pathways" (NBRP), which summarizes the interactions between nanoparticles and the body's various cell-dependent and cell-independent blood clearance mechanisms. We reviewed nanoparticle design and biological modulation strategies to mitigate nanoparticle-NBRP interactions. As these interactions affect nanoparticle delivery, we studied the preclinical literature from 2011-2021 and analyzed nanoparticle blood circulation and organ biodistribution data. Our findings revealed that nanoparticle surface chemistry affected the in vivo behavior more than other nanoparticle design parameters. Combinatory biological-PEG surface modification improved the blood area under the curve by ~418%, with a decrease in liver accumulation of up to 47%. A greater understanding of nanoparticle-NBRP interactions and associated delivery trends will provide new nanoparticle design and biological modulation strategies for safer, more effective, and more efficient nanomedicines.

Characteristics of esophageal refluxate and symptoms in infants compared between pre-treatment and on treatment with proton pump inhibitors.

To examine longitudinal pH-impedance characteristics from those infants who remained on proton pump inhibitors therapy for gastroesophageal reflux disease (GERD) as parents/providers refused to discontinue therapy after 4 weeks. Eighteen infants with acid reflux index >3% underwent treatment, and pH-impedance data were compared prior to and on proton pump inhibitors at 42 ± 1 and 46 ± 1 weeks' postmenstrual age, respectively. Esophageal acid and bolus exposure, symptoms and swallowing characteristics were examined. Proton pump inhibitors reduces the acid-mediated effects of reflux but modifies impedance and clearance mechanisms (P < 0.05). Prolonged therapy did not reduce symptoms (P > 0.05). Infants evaluated while on proton pump inhibitors were 1.8 times more likely to have swallows before and after reflux. Prescription of proton pump inhibitors for objectively determined GERD should have time limits, as prolonged treatment can result in prolonged esophageal bolus clearance time without relieving symptoms.

K6-linked ubiquitylation marks formaldehyde-induced RNA-protein crosslinks for resolution

Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.

Abstract 11755: Efferocytic Regulator Rab11FIP1 Alleviates Inflammation and Cardiac Remodeling After Myocardial Infarction

Introduction: Defective efferocytosis drives cardiac dysfunction after myocardial infarction (MI); however, the underlying molecular mechanisms and regulators of macrophage efferocytosis remain unknown. Hypothesis: This study aimed to explore the modulator and its mechanisms of the continued clearance and degradation of phagolysosomal cargo during MI. Methods: Leveraging macrophage RAB11FIP1 knockout model, flow cytometry and PET-CT technologies, we examined the functional significance of RAB11FIP1 after MI. Moreover, a nanoliposome-linked ssDNA aptamer contained modified RAB11FIP1 mRNA was designed. Results: By comparing the transcriptomic signatures of our scRNA-seq data, we identified RAB11FIP1 was the most sensitively efferocytic gene among all phagosome-related signatures. RAB11FIP1 deficiency resulted in a considerable exacerbation in cardiac function, the accumulation of apoptotic cardiomyocytes and a reduced efferocytosis. RAB11FIP1-loss also led to increased infiltration of CCR2+ macrophages and neutrophils with upregulation of proinflammatory mediators, IL-1β, TNF-α, Ccl2 after MI. In mechanistic studies, RAB11FIP1 defect disabled the recycling of efferocytic receptor, Mertk, further impaired continued macrophages efferocytosis. Moreover, the scRNA-seq data revealed that the regulon activity of XBP1, which acted as the key ER-stress executor, was significantly activated in RAB11FIP1-null hearts, then, the activated XBP1 governed the transcriptional expression of pro-inflammatory mediators after MI. Finally, we constructed a nanoliposome-linked ssDNA aptamer to specifically target macrophages. RAB11FIP1-expressing phagocytes display a striking increase in efferocytosis and decline in inflammation, cardiac remodeling after MI. Conclusions: Collectively, these findings advance the relationship of RAB11FIP1 between macrophage efferocytosis, ER-stress response and the cardiac inflammation.

Advances in Therapeutic Contact Lenses for the Management of Different Ocular Conditions.

In the advent of an increasingly aging population and due to the popularity of electronic devices, ocular conditions have become more prevalent. In the world of medicine, accomplishing eye medication administration has always been a difficult task. Despite the fact that there are many commercial eye drops, most of them have important limitations, due to quick clearance mechanisms and ocular barrers. One solution with tremendous potential is the contact lens used as a medication delivery vehicle to bypass this constraint. Therapeutic contact lenses for ocular medication delivery have attracted a lot of attention because they have the potential to improve ocular bioavailability and patient compliance, both with minimal side effects. However, it is essential not to compromise essential features such as water content, optical transparency, and modulus to attain positive in vitro and in vivo outcomes with respect to a sustained drug delivery profile from impregnated contact lenses. Aside from difficulties like drug stability and burst release, the changing of lens physico-chemical features caused by therapeutic or non-therapeutic components can limit the commercialization potential of pharmaceutical-loaded lenses. Research has progressed towards bioinspired techniques and smart materials, to improve the efficacy of drug-eluting contact lenses. The bioinspired method uses polymeric materials, and a specialized molecule-recognition technique called molecular imprinting or a stimuli-responsive system to improve biocompatibility and support the drug delivery efficacy of drug-eluting contact lenses. This review encompasses strategies of material design, lens manufacturing and drug impregnation under the current auspices of ophthalmic therapies and projects an outlook onto future opportunities in the field of eye condition management by means of an active principle-eluting contact lens.

Interactions of amyloidogenic proteins with mitochondrial protein import machinery in aging-related neurodegenerative diseases.

Most mitochondrial proteins are targeted to the organelle by N-terminal mitochondrial targeting sequences (MTSs, or "presequences") that are recognized by the import machinery and subsequently cleaved to yield the mature protein. MTSs do not have conserved amino acid compositions, but share common physicochemical properties, including the ability to form amphipathic α-helical structures enriched with basic and hydrophobic residues on alternating faces. The lack of strict sequence conservation implies that some polypeptides can be mistargeted to mitochondria, especially under cellular stress. The pathogenic accumulation of proteins within mitochondria is implicated in many aging-related neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases. Mechanistically, these diseases may originate in part from mitochondrial interactions with amyloid-β precursor protein (APP) or its cleavage product amyloid-β (Aβ), α-synuclein (α-syn), and mutant forms of huntingtin (mHtt), respectively, that are mediated in part through their associations with the mitochondrial protein import machinery. Emerging evidence suggests that these amyloidogenic proteins may present cryptic targeting signals that act as MTS mimetics and can be recognized by mitochondrial import receptors and transported into different mitochondrial compartments. Accumulation of these mistargeted proteins could overwhelm the import machinery and its associated quality control mechanisms, thereby contributing to neurological disease progression. Alternatively, the uptake of amyloidogenic proteins into mitochondria may be part of a protein quality control mechanism for clearance of cytotoxic proteins. Here we review the pathomechanisms of these diseases as they relate to mitochondrial protein import and effects on mitochondrial function, what features of APP/Aβ, α-syn and mHtt make them suitable substrates for the import machinery, and how this information can be leveraged for the development of therapeutic interventions.

Low Dose Rituximab Efficiently Clears Circulating CLL Cells and Maintains Sensitivity to Antibody Dependent Cellular Phagocytosis (ADCP)

Introduction: We have recently shown that high frequency (2 x week) low dose (50 mg)(HFLD) rituximab (RTX) and acalabrutinib is highly effective initial therapy for patients (n=38) with progressive CLL (ClinicalTrials.gov NCT03788291, PMID:36689726). The first dose of 50 mg of IV rituximab administered at 25 mg/h decreased the median circulating CLL cell count by 84% from pre-treatment baseline at 1h with no further decrease in the count during the remainder of the infusion (PMID:37003030). During the first hour of infusion, median CLL cell membrane CD20 levels decreased to 65% of baseline and were 41% of baseline at the end of the infusion. Median serum rituximab concentration was 2 mcg/ml at 1h and 10 mcg/ml at the end of the infusion. Median complement (CH50) concentration decreased to 91% of baseline at 1 h and to 81% of baseline at the end of the infusion. The cessation of clearance of CLL cells from the circulation within 1h of starting rituximab therapy is thus unlikely to be caused by low rituximab levels, decreased CLL cell CD20 expression or low serum complement levels. We and others have previously shown that ADCP is the primary mechanism of clearance of anti-CD20 mAb opsonized B cells in both humans and mice. We hypothesized that clearance of circulating CLL cells decreases because residual circulating CLL cells are resistant to mAb induced ADCP. To test this hypothesis, we measured in vitro ADCP sensitivity of CLL cells sampled from patients before and after rituximab treatment. Methods: Blood specimens were collected from 13 patients prior to starting therapy (C1D1_pre), 1h after start of IV rituximab infusion (C1D1_1h), at end of rituximab infusion (C1D1_post), and 48 h after initiation of therapy (C1D3_pre). Sensitivity of circulating CLL cells to in vitro ADCP was measured using live cell time-lapse high-content microscopy imaging of phagocytosis of CLL cells by human monocyte derived macrophages (hMDM) as previously described (PMID:32005699). Phagocytosis was quantified using a void index and sensitivity of samples was compared using the area under the curve of the 2h void index plot (AUC). Experiments were done in triplicate with co-cultures of hMDM and CLL cells without mAb as controls. Rituximab (10 mcg/ml) was used to test CD20 mAb mediated ADCP and alemtuzumab (10 mcg/ml) to test non-CD20 mediated ADCP. Results: Figure (median and 95% confidence intervals) shows that neither rituximab nor alemtuzumab induced ex-vivo ADCP as measured by median void index AUC was significantly altered by prior in vivo exposure of CLL cells to rituximab (Wilcoxon Match-Paired Sign test; all p&amp;gt;0.05). Discussion: The rapid decrease in circulating CLL cells induced by IV rituximab occurred only within the first hour after initiation of mAb infusion. Our measurements show that serum rituximab and complement concentrations and CLL cell membrane CD20 levels should be adequate to support additional CLL cell clearance after 1h. We now demonstrate that residual circulating CLL cells remain sensitive to in vitro ADCP by both rituximab and alemtuzumab. This suggests that despite reduced levels of surface antigen, circulating cells remain sensitive to mAb mediated clearance. Alternative explanations for the limited clearance of circulating CLL cells include: 1. Saturation of the limited cytotoxic capacity of the innate immune effector cells (Kupffer cells and splenic macrophages) compatible with our previously reported data on finite macrophage ADCP capacity (PMID:32556153); 2. Mobilization of CLL cells from lymphoid tissue; and 3. Temporary sequestration of rituximab opsonized CLL cells in tissue (e.g. spleen or lungs), which is unlikely given the observed decrease in lymph node size following initiation of therapy. Ongoing research is testing these hypotheses.

The Effects of Apohemoglobin-Haptoglobin on Heme-Induced Coagulation

Introduction: In conditions with significant hemolysis, like sickle cell and thalassemia, red blood cells break down, leading to higher levels of cell-free hemoglobin (Hb) in the bloodstream. When this cell-free Hb is oxidized into methemoglobin, it releases heme, a crucial iron-protoporphyrin complex essential for oxygen transport. However, excessive free heme can become problematic, since plasma concentrations of the body protective proteins, haptoglobin and hemopexin, to scavenge and clear hemoglobin and heme is very limited. Thus, these protective mechanisms can get overwhelmed by high levels of free Hb or heme in the blood. Additionally, heme can interact with the coagulation cascade, potentially causing harmful pro-coagulant effects in the body. To address this, we introduce a new synthetic scavenger protein called apohemoglobin-haptoglobin (apoHb+Hp). It mimics hemopexin's function and binds to free heme with even greater affinity. Moreover, it utilizes haptoglobin's clearance mechanism for efficient removal from the plasma. Our aim is for apoHb+Hp to effectively clear free heme from the plasma, thereby alleviating pro-coagulatory markers induced by heme. This novel approach may enhance the body's natural defense against heme, leading to potential therapeutic benefits. Materials and Methods: This study used male Sprague-Dawley rats to investigate the effect of apoHb+Hp on coagulation markers during conditions of excess heme. To do so, 20% of the blood volume, estimated as 7% of body mass, was removed from the animal over a 10 minute period. After 10 minutes of rest, the removed blood volume was re-infused to the animal as either 20% lactated ringers, 20% apoHb+Hp, 10% heme bound to human serum albumin (HSA) and 10% apoHb+Hp, or 20% heme HSA. After a 30-minute rest period, 70 µm and 5 mL blood samples were drawn from the tail vein and arterial catheter, respectively, to evaluate measures of the coagulation cascade including prothrombin time, active partial thromboplastin time, fibrinogen, soluble glycoprotein V, Thrombin/ antithrombin complex (TAT), thromboelastography (TEG). Results: Re-injection of Heme HSA resulted in activation of the coagulation system as evidenced by an increase in TAT and soluble glycoprotein V. This effect was reversed with co administration of ApoHb-Hp. Heme HSA caused activation of the extrinsic pathway, as measured by TEG (EXTEM) and a reduction in PT, this effect was ameliorated upon treatment with apoHb-Hp. No notable changes were seen in the intrinsic pathway factors as evidenced by the PTT or TEG/ INTEM. Discussion: Previous literature has established that heme has significant effects in the extrinsic and common pathways of the coagulation cascade, and our study is consistent with these results. When combined with heme HSA, however, apoHb+Hp was able to improve some of the thrombotic effects induced by the addition of heme HSA to circulation. Conclusions: Excess free heme can contribute to a hypercoagulable state. As expected, our findings showed significantly notable pro-coagulatory state in the presence of excessive heme in the blood. ApoHb+Hp attenuates heme-induced prothrombotic states to some extent in the extrinsic and common pathways. ApoHb-Hp may be a potential therapeutic strategy in hemolytic anemias and further studies to evaluate its role in pathologic states associated with hemolysis is warranted.

Status of heavy metals and metalloid concentrations in water resources and associated health risks in parts of Indo-Gangetic plain, India

Groundwater is a major source for the supply of water for drinking as well as supporting the industrial and irrigation demands in the urban and peri-urban regions of Shahjahanpur, Uttar Pradesh, India. However, the groundwater quality of the area is deteriorating due to rapid urbanization, industrialization and intensive agriculture. The present study investigates the heavy metal(loid)s pollution in surface and groundwater and the associated health risks to the inhabitants. The results revealed that most of the analyzed heavy metal(loid)s remained within the acceptable limits set by the Bureau of Indian Standards (BIS, 2012) except for As, Fe, Mn and Ni. The collective impact of heavy metals on water quality was assessed using heavy metal pollution index (HPI) which revealed low pollution in November 2018 and low to medium pollution in October 2021. Ficklin-Caboi plot indicates that metals fall in ‘low’ to ‘high’ classes and their mobility in 30% and 45% of groundwater samples in November 2018 and October 2021 respectively was comparatively higher in the aqueous phase due to slightly lower pH conditions. The evaluated Non-Carcinogenic Hazard Index (HI) was significantly higher than the threshold limit (>1) for children as compared to adults. Relatively higher hazard quotient of As over Fe, Mn and Ni revealed its maximum contribution to the hazard index. The total carcinogenic risk values also exceeded the prescribed acceptable limit (1 × 10−4) for children compared to adults. The greater vulnerability of children can be attributed to several factors such as higher water consumption per unit of body weight of children compared to adults; toxicokinetic differences, immature metabolic enzyme systems and clearance mechanisms that result in longer half-lives of contaminants. Overall, the study reveals an escalated concern regarding heavy metal(loid)s induced health risk and recommends the adoption of conventional techniques for the removal of these metals from water resources.

Pharmacological Inhibition of the AQP4 Water Channel Activity Causes an Aggravation of Alpha-Synuclein Pathology in the Substantia Nigra in a Rat Model of Parkinson’s Disease

The misfolding of the protein α-synuclein, which leads to the formation of neurototoxic oligomers and aggregates, is one of the main causes of loss of dopaminergic (DA) neurons within the substantia nigra pars compacta (SNpc) in Parkinson’s disease (PD). We previously found that pharmacological inhibition of the water channel aquaporin-4 (AQP4), participating in the mechanisms of brain clearance of amyloidogenic proteins, caused the aggravation of neurodegeneration in the nigrostriatal system and the development of motor disturbances in a lactacystin model of PD. It was hypothesized that the progression of neurodegeneration can be a result of the excessive accumulation of pathologic forms of α-synuclein due to the AQP4 inhibition. The aim of this study is to determine whether pharmacological inhibition of AQP4 activity in a rat model of preclinical PD leads to an aggravation in α-synuclein pathology. The experiments were performed on male Wistar rats. AQP4 activity was suppressed using the intracerebroventricular injection of inhibitor TGN-020. To reproduce the model of the preclinical stage of PD, a specific proteasome inhibitor lactacystin (LC) was used. It was injected bilaterally into the SNpc. Immunoblotting methods and confocal microscopy were applied. The LC model of PD was characterized by a pathologic accumulation of total water-soluble and Ser129-phosphorylated forms of α-synuclein, as well as by formation of insoluble α-synuclein aggregates in the DA-neurons of SNpc. TGN-020 caused a significant aggravation of α-synuclein pathology in the LC model of PD. It was manifested by a marked increase in the level of water-soluble and modified forms of α-synuclein and by the 1.9-fold rise in the amount of α-synuclein aggregates in SN. We suppose that the disfunction of AQP4 which is involved in glymphatic system functioning, can be one of the mechanisms leading to the neurodegeneration and accumulation of amyloidogenic proteins in brain parenchyma during PD. The water channel AQP4 might be a target for the development of new therapeutic approaches aimed at attenuation of the cytotoxicity, accumulation and distribution of α-synuclein during the development of PD-like pathology.

Allelic effects on uromodulin aggregates drive autosomal dominant tubulointerstitial kidney disease

Missense mutations in the uromodulin (UMOD) gene cause autosomal dominant tubulointerstitial kidney disease (ADTKD), one of the most common monogenic kidney diseases. The unknown impact of the allelic and gene dosage effects and fate of mutant uromodulin leaves open the gap between postulated gain‐of‐function mutations, end‐organ damage and disease progression in ADTKD. Based on two prevalent missense UMOD mutations with divergent disease progression, we generated UmodC171Y and UmodR186S knock‐in mice that showed strong allelic and gene dosage effects on uromodulin aggregates and activation of ER stress and unfolded protein and immune responses, leading to variable kidney damage. Deletion of the wild‐type Umod allele in heterozygous UmodR186S mice increased the formation of uromodulin aggregates and ER stress. Studies in kidney tubular cells confirmed differences in uromodulin aggregates, with activation of mutation‐specific quality control and clearance mechanisms. Enhancement of autophagy by starvation and mTORC1 inhibition decreased uromodulin aggregates. These studies substantiate the role of toxic aggregates as driving progression of ADTKD‐UMOD, relevant for therapeutic strategies to improve clearance of mutant uromodulin.

Nanoparticle-Mediated Strategies for Enhanced Drug Penetration and Retention in the Airway Mucosa.

Airway mucus is a complex viscoelastic gel composed mainly of water, glycoproteins, lipids, enzymes, minerals, etc. Among them, glycoproteins are the main factors determining mucus's gel-like rheology. Airway mucus forms a protective barrier by secreting mucin, which represents a barrier for absorption, especially for more lipophilic drugs. It rapidly removes drugs from the airway through the physiological mucus clearance mechanism so drugs cannot remain in the lungs or reach the airway epithelial tissue for a long time. Significant progress has been made in enhancing drug lung deposition recently, but strategies are still needed to help drugs break through the lung mucosal barrier. Based on the physiopathological mechanisms of airway mucus, this paper reviews and summarizes strategies to enhance drug penetration and retention in the airway mucosa mediated by nano-delivery systems, including mucosal permeation systems, mucosal adhesion systems, and enzyme-modified delivery systems. On this basis, the potential and challenges of nano-delivery systems for improving airway mucus clearance are revealed. New ideas and approaches are provided for designing novel nano-delivery systems that effectively improve drug retention and penetration in the airway mucus layer.

The neuraminidase activity of influenza A virus determines the strain-specific sensitivity to neutralization by respiratory mucus.

The respiratory tract of humans is constantly exposed to potentially harmful agents, such as small particles or pathogens, and thus requires protective measures. Respiratory mucus that lines the airway epithelia plays a major role in the prevention of viral infections by limiting the mobility of viruses, allowing subsequent mucociliary clearance. Understanding the interplay between respiratory mucus and viruses can help elucidate host and virus characteristics that enable the initiation of infection. Here, we tested a panel of primary influenza A viruses of avian or human origin for their sensitivity to mucus derived from primary human airway cultures and found that differences between virus strains can be mapped to viral neuraminidase activity. We also show that binding of influenza A viruses to decoy receptors on highly glycosylated mucus components constitutes the major inhibitory function of mucus against influenza A viruses.

Enhancing Antibody Exposure in the Central Nervous System: Mechanisms of Uptake, Clearance, and Strategies for Improved Brain Delivery

Antibodies (mAbs) are attractive molecules for their application as a diagnostic and therapeutic agent for diseases of the central nervous system (CNS). mAbs can be generated to have high affinity and specificity to target molecules in the CNS. Unfortunately, only a very small number of mAbs have been specifically developed and approved for neurological indications. This is primarily attributed to their low exposure within the CNS, hindering their ability to reach and effectively engage their potential targets in the brain. This review discusses aspects of various barriers such as the blood–brain barrier (BBB) and blood–cerebrospinal fluid (CSF) barrier (BCSFB) that regulate the entry and clearance of mAbs into and from the brain. The roles of the glymphatic system on brain exposure and clearance are being described. We also discuss the proposed mechanisms of the uptake of mAbs into the brain and for clearance. Finally, several methods of enhancing the exposure of mAbs in the CNS were discussed, including receptor-mediated transcytosis, osmotic BBB opening, focused ultrasound (FUS), BBB-modulating peptides, and enhancement of mAb brain retention.

An autonomic mode of brain activity.

The relevance of interactions between autonomic and central nervous systems remains unclear for human brain function and health, particularly when both systems are challenged under sleep deprivation (SD). We measured brain activity (with fMRI), pulse and respiratory signals, and baseline brain amyloid beta burden (with PET) in healthy participants. We found that SD relative to rested wakefulness (RW) resulted in a significant increase in synchronized low frequency (LF, < 0.1Hz) activity in an autonomically-related network (AN), including dorsal attention, visual, and sensorimotor regions, which we previously found to have consistent temporal coupling with LF pulse signal changes (regulated by sympathetic tone). SD resulted in a significant phase coherence between the LF component of the pulse signal and a medial network with peak effects in the midbrain reticular formation, and between LF component of the respiratory variations (regulated by respiratory motor output) and a cerebellar network. The LF power of AN during SD was significantly and independently correlated with pulse-medial network and respiratory-cerebellar network phase coherences (total adjusted R2 = 0.78). Higher LF power of AN during SD (but not RW) was associated with lower amyloid beta burden (Cohen's d = 0.8). In sum, SD triggered an autonomic mode of synchronized brain activity that was associated with distinct autonomic-central interactions. Findings highlight the direct relevance of global cortical synchronization to brain clearance mechanisms.

In Silico Investigation of the Clinical Translatability of Competitive Clearance Glucose-Responsive Insulins.

The glucose-responsive insulin (GRI) MK-2640 from Merck was a pioneer in its class to enter the clinical stage, having demonstrated promising responsiveness in in vitro and preclinical studies via a novel competitive clearance mechanism (CCM). The smaller pharmacokinetic response in humans motivates the development of new predictive, computational tools that can improve the design of therapeutics such as GRIs. Herein, we develop and use a new computational model, IM3PACT, based on the intersection of human and animal model glucoregulatory systems, to investigate the clinical translatability of CCM GRIs based on existing preclinical and clinical data of MK-2640 and regular human insulin (RHI). Simulated multi-glycemic clamps not only validated the earlier hypothesis of insufficient glucose-responsive clearance capacity in humans but also uncovered an equally important mismatch between the in vivo competitiveness profile and the physiological glycemic range, which was not observed in animals. Removing the inter-species gap increases the glucose-dependent GRI clearance from 13.0% to beyond 20% for humans and up to 33.3% when both factors were corrected. The intrinsic clearance rate, potency, and distribution volume did not apparently compromise the translation. The analysis also confirms a responsive pharmacokinetics local to the liver. By scanning a large design space for CCM GRIs, we found that the mannose receptor physiology in humans remains limiting even for the most optimally designed candidate. Overall, we show that this computational approach is able to extract quantitative and mechanistic information of value from a posteriori analysis of preclinical and clinical data to assist future therapeutic discovery and development.

The Diagnostic, Prognostic, and Therapeutic Potential of Cell-Free DNA with a Special Focus on COVID-19 and Other Viral Infections.

Cell-free DNA (cfDNA) in human blood serum, urine, and other body fluids recently became a commonly used diagnostic marker associated with various pathologies. This is because cfDNA enables a much higher sensitivity than standard biochemical parameters. The presence of and/or increased level of cfDNA has been reported for various diseases, including viral infections, including COVID-19. Here, we review cfDNA in general, how it has been identified, where it can derive from, its molecular features, and mechanisms of release and clearance. General suitability of cfDNA for diagnostic questions, possible shortcomings and future directions are discussed, with a special focus on coronavirus infection.