Affinity targeting of therapeutic proteins to the bone surface - local delivery of sclerostin neutralizing antibody enhances efficacy.

Currently available biotherapeutics for the treatment of osteoporosis lack explicit mechanisms for bone localization, potentially limiting efficacy and inducing unintended off-target toxicities. While various strategies have been explored for targeting the bone surface, critical aspects remain poorly understood, including the optimal affinity ligand, the role of binding avidity and circulation time, and, perhaps most importantly, whether or not this strategy can enhance the functional activity of clinically relevant protein therapeutics. To investigate, we generated fluorescent proteins (e.g., mCherry) with site-specifically attached small molecule (bisphosphonate, BP) or peptide (deca-aspartate, D10) affinity ligands. While both affinity ligands successfully anchored fluorescent protein to the bone surface, quantitative radiotracing revealed only modest femoral and vertebral accumulation and suggested a need for enhanced circulation time. To achieve this, we fused mCherry to the Fc fragment of human IgG1 and attached D10 peptides to each C-terminus. mCherry-Fc-D10 demonstrated ~80-fold increase in plasma exposure and marked increases in femoral and vertebral accumulation (13.6 ± 1.4% and 11.4 ± 1.3% of the injected dose/gram [%ID/g] at 24 hours, respectively). To determine if bone surface targeting could enhance the efficacy of a clinically relevant therapeutic, we generated a bone-targeted sclerostin neutralizing antibody, anti-sclerostin-D10. The targeted antibody demonstrated marked increases in bone accumulation and retention (20.9 ± 2.5% and 19.5 ± 2.5% ID/g in femur and vertebrae at 7 days) and enhanced effects in a murine model of ovariectomy-induced bone loss (BV/TV, connectivity density, and structure model index all increased [p < 0.001] vs. untargeted anti-sclerostin). Collectively, our results indicate the importance of both bone affinity and circulation time in achieving robust targeting of therapeutic proteins to the bone surface and suggest that this approach may enable lower doses and/or longer dosing intervals without reduction in biotherapeutic efficacy. Future studies will be needed to determine the translational potential of this strategy and its potential impact on off-site toxicities.

Several biologic therapies have been approved for osteoporosis, but they lack means of localization to bone tissue, potentially limiting their efficacy and leading to off-target toxicities. This manuscript investigates strategies for targeting biotherapeutics to the bone surface and asks the question of whether or not this approach can enhance functional activity and allow for lower or less frequent dosing. To define the key determinants of bone surface targeting, we begin by synthesizing fluorescent model proteins with different bone targeting tags. We show that even one tag is enough to make the surface of the femur and vertebrae fluorescent following systemic administration. The results are relatively modest at first, but when we combine the bone targeting tag with a second modification that makes the protein circulate in the body for a longer period of time, we observe a huge increase in bone surface delivery. We then synthesize a bone surface targeted version of a sclerostin-inhibiting antibody and show that it is more effective than the untargeted antibody and provides near complete protection of bone density despite relatively low dose. Our findings could have translational implications for existing bone therapies and help guide design of future strategies for optimized bone surface targeting.

Bispecific antibody shuttles targeting CD98hc mediate efficient and long-lived brain delivery of IgGs.

The inability of antibodies to penetrate the blood-brain barrier (BBB) is a key limitation to their use in diverse applications. One promising strategy is to deliver IgGs using a bispecific BBB shuttle, which involves fusing an IgG to a second affinity ligand that engages a cerebrovascular endothelial target and facilitates transport across the BBB. Nearly all prior efforts have focused on shuttles that target transferrin receptor (TfR-1) despite inherent delivery and safety challenges. Here, we report bispecific antibody shuttles that engage CD98hc, the heavy chain of the large neutral amino acid transporter (LAT1), and efficiently transport IgGs into the brain. Notably, CD98hc shuttles lead to much longer-lived brain retention of IgGs than TfR-1 shuttles while enabling more specific targeting due to limited CD98hc engagement in the brain parenchyma, which we demonstrate for IgGs that either agonize a neuronal receptor (TrkB) or target other endogenous cell-surface proteins on neurons and astrocytes.

Coagulofibrinolytic effects of recombinant soluble thrombomodulin in prolonged porcine cardiac arrest.

Aim: To evaluate coagulofibrinolytic abnormalities and the effects of ART-123 (recombinant human thrombomodulin alpha) in a porcine model of cardiac arrest and prolonged cardiopulmonary resuscitation (CA/CPR). Methods: Fifteen pigs (n = 5 per group) underwent 8 minutes of no-flow CA followed by 50 minutes of mechanical CPR, while 2 pigs underwent sham arrest. CA/CPR animals were randomized to receive saline or 1 mg/kg ART-123 pre-arrest (5 minutes prior to ventricular fibrillation) or post-arrest (2 minutes after initiation of CPR). Arterial and venous blood samples were drawn at multiple time points for blood gas analysis and measurement of plasma and whole blood markers of coagulation and fibrinolysis. Results: In saline-treated CA/CPR, but not sham animals, robust and persistent activation of coagulation and fibrinolysis was observed throughout resuscitation. After 50 minutes of CPR, plasma tests and thromboelastography indicated a mix of hypercoagulability and consumptive coagulopathy. ART-123 had a robust anticoagulant effect, reducing both thrombin-antithrombin (TAT) complexes and d-dimer (p < 0.05 for each). The duration of anticoagulant effect varied depending on the timing of ART-123 administration. Similarly, ART-123 when given prior to cardiac arrest was found to have pro-fibrinolytic effects, increasing free tissue plasminogen activator (tPA, p = 0.02) and decreasing free plasminogen activator inhibitor-1 (PAI-1, p = 0.04). Conclusion: A porcine model of prolonged CA/CPR reproduces many of the coagulofibrinolytic abnormalities observed in human cardiac arrest patients. ART-123 demonstrates a combination of anticoagulant and profibrinolytic effects, depending on the timing of its administration relative to cardiac arrest.

Prediction of in-hospital deterioration in normotensive pulmonary embolism remains elusive: external validation of the calgary acute pulmonary embolism score.

Acute pulmonary embolism (PE) is a frequently diagnosed condition. Prediction of in-hospital deterioration is challenging with current risk models. The Calgary Acute Pulmonary Embolism (CAPE) score was recently derived to predict in-hospital adverse PE outcomes but has not yet been externally validated. Retrospective cohort study of normotensive acute pulmonary embolism cases diagnosed in our emergency department between 2017 and 2019. An external validation of the CAPE score was performed in this population for prediction of in-hospital adverse outcomes and a secondary outcome of 30-day all-cause mortality. Performance of the simplified Pulmonary Embolism Severity Index (sPESI) and Bova score was also evaluated. 712 patients met inclusion and exclusion criteria, with 536 patients having a sPESI score of 1 or more. Among this population, the CAPE score had a weak discriminative power to predict in-hospital adverse outcomes, with a calculated c-statistic of 0.57. In this study population, an external validation study found weak discriminative power of the CAPE score to predict in-hospital adverse outcomes among normotensive PE patients. Further efforts are needed to define risk assessment models that can identify normotensive PE patients at risk for in hospital deterioration. Identification of such patients will better guide intensive care utilization and invasive procedural management of PE.

Bispecific antibody shuttles targeting CD98hc mediate efficient and long-lived brain delivery of IgGs.

The inability of antibodies and other biologics to penetrate the blood-brain barrier (BBB) is a key limitation to their use in diagnostic, imaging, and therapeutic applications. One promising strategy is to deliver IgGs using a bispecific BBB shuttle, which involves fusing an IgG with a second affinity ligand that engages a cerebrovascular endothelial target and facilitates transport across the BBB. Nearly all prior efforts have focused on the transferrin receptor (TfR-1) as the prototypical endothelial target despite inherent delivery and safety challenges. Here we report bispecific antibody shuttles that engage CD98hc (also known as 4F2 and SLC3A2), the heavy chain of the large neutral amino acid transporter (LAT1), and efficiently transport IgGs into the brain parenchyma. Notably, CD98hc shuttles lead to much longer-lived brain retention of IgGs than TfR-1 shuttles while enabling more specific brain targeting due to limited CD98hc engagement in the brain parenchyma. We demonstrate the broad utility of the CD98hc shuttles by reformatting three existing IgGs as CD98hc bispecific shuttles and delivering them to the mouse brain parenchyma that either agonize a neuronal receptor (TrkB) or target other endogenous antigens on specific types of brain cells (neurons and astrocytes).

Adverse Clinical Outcomes Among Patients With Acute Low-risk Pulmonary Embolism and Concerning Computed Tomography Imaging Findings.

Importance: Most patients presenting to US emergency departments (EDs) with acute pulmonary embolism (PE) are hospitalized, despite evidence from multiple society-based guidelines recommending consideration of outpatient treatment for those with low risk stratification scores. One barrier to outpatient treatment may be clinician concern regarding findings on PE-protocol computed tomography (CTPE), which are perceived as high risk but not incorporated into commonly used risk stratification tools. Objective: To evaluate the association of concerning CTPE findings with outcomes and treatment of patients in the ED with acute, low-risk PE. Design, Setting, and Participants: This cohort study used a registry of all acute PEs diagnosed in the adult ED of an academic medical center from October 10, 2016, to December 31, 2019. Acute PE cases were divided into high- and low-risk groups based on PE Severity Index (PESI) class alone or using a combination of PESI class and biomarker results. The low-risk group was further divided based on the presence of concerning CTPE findings: (1) bilateral central embolus, (2) right ventricle-to-left ventricle ratio greater than 1.0, (3) right ventricle enlargement, (4) septal abnormality, or (5) pulmonary infarction. Data analysis was conducted from June to October 2022. Main Outcomes and measures: The primary outcome was all-cause mortality at 7 and 30 days. Secondary outcomes included hospitalization, length of stay, need for intensive care, use of echocardiography and/or bedside ultrasonography, and activation of the PE response team (PERT) . Results: Of 817 patients (median [IQR] age, 58 [47-71] years; 417 (51.0%) female patients; 129 [15.8%] Black and 645 [78.9%] White patients) with acute PEs, 331 (40.5%) were low risk and 486 (59.5%) were high risk by PESI score. Clinical outcomes were similar for all low-risk patients, with no 30-day deaths in the low-risk group with concerning CTPE findings (0 of 151 patients) vs 4 of 180 (2.2%) in the low-risk group without concerning CTPE findings and 88 (18.1%) in the high-risk group (P < .001). Low-risk patients with concerning CTPE findings were less frequently discharged from the ED than those without concerning CTPE findings (3 [2.0%] vs 14 [7.8%]; P = .01) and had more frequent echocardiography (87 [57.6%] vs 49 [27.2%]; P < .001) and PERT activation for consideration of advanced therapies (34 [22.5%] vs 11 [6.1%]; P < .001). Conclusions and Relevance: In this single-center study, CTPE findings widely believed to confer high risk were associated with increased hospitalization and resource utilization in patients with low-risk PE but not short-term adverse clinical outcomes.

Effect of pulmonary embolism response team on advanced therapies administered: The University of Michigan experience.

BACKGROUND: Pulmonary Embolism Response Teams (PERT) were employed at multiple institutions to bridge the gap between varied treatment options for acute PE and unclear evidence for optimal management. There is limited data regarding the impact of PERT on the use of advanced therapies and clinical outcomes. METHODS: We performed a retrospective single-center cohort study comparing patients that presented to the ED with an acute PE before and after the creation of PERT in June 2017 at our institution. We assessed utilization of advanced therapies, LOS, and mortality. RESULTS: A total of 817 patients (168 pre-PERT, 649 post-PERT) were evaluated in the ED with an acute PE between October 2016 and December 2019. Both groups were similar in demographics, comorbidities, and PESI score. There was a decrease in advanced therapy use (16 % vs. 7.5 %, p = 0.006) after PERT creation. Most notable decreases were in catheter-based therapies (8.5 % vs. 2.2 %, p = 0.008) and IVC filter placement (5.3 % vs. 3.2 %, p < 0.001). Median ICU LOS (2.5 days vs. 2.3 days, p = 0.55) and hospital LOS (3.1 vs. 3.0, p = 0.92) did not vary pre-PERT vs. post-PERT. In-hospital mortality (8.5 % vs. 5.0 %, p = 0.29) and 30-day all-cause mortality (1.2 % vs. 0.5 %, p = 0.28) were not different between the two groups as well. CONCLUSION: At our institution, PERT was associated with a decrease in advanced therapies administered to acute PE patients without affecting mortality or LOS. Additional studies to assess impact of this multi-disciplinary care team model on interventional therapies and clinical outcomes for PE at a broader level are necessary.

The MedConnect Program: Symptomatology, Return Visits, and Hospitalization of COVID-19 Outpatients Following Discharge From the Emergency Department.

Background and objective Although hospitalization is required for only a minority of those infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the high rates of morbidity and mortality among these patients have led researchers to focus on the predictors of admission and adverse outcomes in the inpatient population. However, there is scarce data on the clinical trajectory of individuals symptomatic enough to present for emergency care, but not sick enough to be admitted. In light of this, we aimed to examine the symptomatology, emergency department (ED) revisits, and hospitalization of coronavirus disease 2019 (COVID-19) outpatients after discharge from the ED. Methods Adult patients with COVID-19 infection were prospectively enrolled after discharge from the ED between May and December 2020. Patients were followed up longitudinally for 14 days via phone interviews designed to provide support and information and to track symptomatology, ED revisits, and hospitalization. Results A volunteer, medical student-run program enrolled 199 COVID-19 patients discharged from the ED during the first nine months of the pandemic. Of the 176 patients (88.4%) who completed the 14-day protocol, 29 (16.5%) had a second ED visit and 17 (9.6%) were admitted, 16 (9%) for worsening COVID-19 symptoms. Age, male sex, comorbid illnesses, and self-reported dyspnea, diarrhea, chills, and fever were associated with hospital admission for patients with a subsequent ED visit. For those who did not require admission, symptoms generally improved following ED discharge. Age >65 years and a history of cardiovascular disease (CVD) were associated with a longer duration of cough, but generally, patient characteristics and comorbidities did not significantly affect the overall number or duration of symptoms. Conclusions Nearly one in five patients discharged from the ED with COVID-19 infection had a second ED evaluation during a 14-day follow-up period, despite regular phone interactions aimed at providing support and information. More than half of them required admission for worsening COVID-19 symptoms. Established risk factors for severe disease and self-reported persistence of certain symptoms were associated with hospital admission, while those who did not require hospitalization had a steady improvement in symptoms over the 14-day period.

A library of Rhodamine6G-based pH-sensitive fluorescent probes with versatile in vivo and in vitro applications.

Acidic pH is critical to the function of the gastrointestinal system, bone-resorbing osteoclasts, and the endolysosomal compartment of nearly every cell in the body. Non-invasive, real-time fluorescence imaging of acidic microenvironments represents a powerful tool for understanding normal cellular biology, defining mechanisms of disease, and monitoring for therapeutic response. While commercially available pH-sensitive fluorescent probes exist, several limitations hinder their widespread use and potential for biologic application. To address this need, we developed a novel library of pH-sensitive probes based on the highly photostable and water-soluble fluorescent molecule, Rhodamine 6G. We demonstrate versatility in terms of both pH sensitivity (i.e., pK a) and chemical functionality, allowing conjugation to small molecules, proteins, nanoparticles, and regenerative biomaterial scaffold matrices. Furthermore, we show preserved pH-sensitive fluorescence following a variety of forms of covalent functionalization and demonstrate three potential applications, both in vitro and in vivo, for intracellular and extracellular pH sensing. Finally, we develop a computation approach for predicting the pH sensitivity of R6G derivatives, which could be used to expand our library and generate probes with novel properties.

Anchoring IgG-degrading enzymes to the surface of platelets selectively neutralizes antiplatelet antibodies.

Immune thrombocytopenia (ITP) is an acquired bleeding disorder characterized by immunoglobulin G (IgG)-mediated platelet destruction. Current therapies primarily focus on reducing antiplatelet antibodies using immunosuppression or increasing platelet production with thrombopoietin mimetics. However, there are no universally safe and effective treatments for patients presenting with severe life-threatening bleeding. The IgG-degrading enzyme of Streptococcus pyogenes (IdeS), a protease with strict specificity for IgG, prevents IgG-driven immune disorders in murine models, including ITP. In clinical trials, IdeS prevented IgG-mediated kidney transplant rejection; however, the concentration of IdeS used to remove pathogenic antibodies causes profound hypogammaglobulinemia, and IdeS is immunogenic, which limits its use. Therefore, this study sought to determine whether targeting IdeS to FcγRIIA, a low-affinity IgG receptor on the surface of platelets, neutrophils, and monocytes, would be a viable strategy to decrease the pathogenesis of antiplatelet IgG and reduce treatment-related complications of nontargeted IdeS. We generated a recombinant protein conjugate by site-specifically linking the C-terminus of a single-chain variable fragment from an FcγRIIA antibody, clone IV.3, to the N-terminus of IdeS (scIV.3-IdeS). Platelets treated with scIV.3-IdeS had reduced binding of antiplatelet IgG from patients with ITP and decreased platelet phagocytosis in vitro, with no decrease in normal IgG. Treatment of mice expressing human FcγRIIA with scIV.3-IdeS reduced thrombocytopenia in a model of ITP and significantly improved the half-life of transfused platelets expressing human FcγRIIA. Together, these data suggest that scIV.3-IdeS can selectively remove pathogenic antiplatelet IgG and may be a potential treatment for patients with ITP and severe bleeding.

Targeted In Vivo Loading of Red Blood Cells Markedly Prolongs Nanocarrier Circulation.

Engineering drug delivery systems for prolonged pharmacokinetics (PK) has been an ongoing pursuit for nearly 50 years. The gold standard for PK enhancement is the coating of nanoparticles with polymers, namely polyethylene glycol (PEGylation), which has been applied in several clinically used products. In the present work, we utilize the longest circulating and most abundant component of blood─the erythrocyte─to improve the PK behavior of liposomes. Antibody-mediated coupling of liposomes to erythrocytes was tested in vitro to identify a loading dose that did not adversely impact the carrier cells. Injection of erythrocyte targeting liposomes into mice resulted in a ∼2-fold improvement in the area under the blood concentration versus time profile versus PEGylated liposomes and a redistribution from the plasma into the cellular fraction of blood. These results suggest that in vivo targeting of erythrocytes is a viable strategy to improve liposome PK relative to current, clinically viable strategies.

Supramolecular arrangement of protein in nanoparticle structures predicts nanoparticle tropism for neutrophils in acute lung inflammation.

This study shows that the supramolecular arrangement of proteins in nanoparticle structures predicts nanoparticle accumulation in neutrophils in acute lung inflammation (ALI). We observed homing to inflamed lungs for a variety of nanoparticles with agglutinated protein (NAPs), defined by arrangement of protein in or on the nanoparticles via hydrophobic interactions, crosslinking and electrostatic interactions. Nanoparticles with symmetric protein arrangement (for example, viral capsids) had no selectivity for inflamed lungs. Flow cytometry and immunohistochemistry showed NAPs have tropism for pulmonary neutrophils. Protein-conjugated liposomes were engineered to recapitulate NAP tropism for pulmonary neutrophils. NAP uptake in neutrophils was shown to depend on complement opsonization. We demonstrate diagnostic imaging of ALI with NAPs; show NAP tropism for inflamed human donor lungs; and show that NAPs can remediate pulmonary oedema in ALI. This work demonstrates that structure-dependent tropism for neutrophils drives NAPs to inflamed lungs and shows NAPs can detect and treat ALI.

A hybridoma-derived monoclonal antibody with high homology to the aberrant myeloma light chain.

The identification of antibody variable regions in the heavy (VH) and light (VL) chains from hybridomas is necessary for the production of recombinant, sequence-defined monoclonal antibodies (mAbs) and antibody derivatives. This process has received renewed attention in light of recent reports of hybridomas having unintended specificities due to the production of non-antigen specific heavy and/or light chains for the intended antigen. Here we report a surprising finding and potential pitfall in variable domain sequencing of an anti-human CD63 hybridoma. We amplified multiple VL genes from the hybridoma cDNA, including the well-known aberrant Sp2/0 myeloma VK and a unique, full-length VL. After finding that the unique VL failed to yield a functional antibody, we discovered an additional full-length sequence with surprising similarity (~95% sequence identify) to the non-translated myeloma kappa chain but with a correction of its key frameshift mutation. Expression of the recombinant mAb confirmed that this highly homologous sequence is the antigen-specific light chain. Our results highlight the complexity of PCR-based cloning of antibody genes and strategies useful for identification of correct sequences.

Site-Specific Modification of Single-Chain Affinity Ligands for Fluorescence Labeling, Radiolabeling, and Bioconjugation.

Single-chain protein affinity ligands are recombinant polypeptides that recreate the antigen-binding site of parental, monoclonal antibodies (mAbs) or present unique binding surfaces derived from display technologies, computational design, or other approaches. These diverse ligands have several advantages over full-length mAbs as agents for delivery of small molecule, protein, and nanoparticle cargoes to desired sites in the body. However, they present unique challenges for modification and bioconjugation. Fusion of a LPXTGG motif, or "sortag," and a 5-amino acid, flexible linker to the C-terminus of these affinity ligands enables high-efficiency transpeptidation by the bacterial enzyme, Sortase A, and site-specific addition of fluorophores, radiolabels, or functional groups for oriented and stoichiometrically controlled bioconjugation. We describe in detail this method and address several challenges and pitfalls in the purification and characterization of modified single-chain affinity ligands.

Early Convalescent Plasma for High-Risk Outpatients with Covid-19.

BACKGROUND: Early administration of convalescent plasma obtained from blood donors who have recovered from coronavirus disease 2019 (Covid-19) may prevent disease progression in acutely ill, high-risk patients with Covid-19. METHODS: In this randomized, multicenter, single-blind trial, we assigned patients who were being treated in an emergency department for Covid-19 symptoms to receive either one unit of convalescent plasma with a high titer of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or placebo. All the patients were either 50 years of age or older or had one or more risk factors for disease progression. In addition, all the patients presented to the emergency department within 7 days after symptom onset and were in stable condition for outpatient management. The primary outcome was disease progression within 15 days after randomization, which was a composite of hospital admission for any reason, seeking emergency or urgent care, or death without hospitalization. Secondary outcomes included the worst severity of illness on an 8-category ordinal scale, hospital-free days within 30 days after randomization, and death from any cause. RESULTS: A total of 511 patients were enrolled in the trial (257 in the convalescent-plasma group and 254 in the placebo group). The median age of the patients was 54 years; the median symptom duration was 4 days. In the donor plasma samples, the median titer of SARS-CoV-2 neutralizing antibodies was 1:641. Disease progression occurred in 77 patients (30.0%) in the convalescent-plasma group and in 81 patients (31.9%) in the placebo group (risk difference, 1.9 percentage points; 95% credible interval, -6.0 to 9.8; posterior probability of superiority of convalescent plasma, 0.68). Five patients in the plasma group and 1 patient in the placebo group died. Outcomes regarding worst illness severity and hospital-free days were similar in the two groups. CONCLUSIONS: The administration of Covid-19 convalescent plasma to high-risk outpatients within 1 week after the onset of symptoms of Covid-19 did not prevent disease progression. (SIREN-C3PO ClinicalTrials.gov number, NCT04355767.).

Molecularly Engineered Nanobodies for Tunable Pharmacokinetics and Drug Delivery.

The use of single-domain antibody fragments, or nanobodies, has gained popularity in recent years as an alternative to traditional monoclonal antibody-based approaches. Relatively little is known, however, about the utility of nanobodies as targeting agents for delivery of therapeutic cargoes, particularly to vascular epitopes or in the setting of acute inflammatory conditions. We used a nanobody (VCAMelid) directed against mouse vascular cell adhesion molecule 1 (VCAM-1) and techniques for site-specific radiolabeling and bioconjugation to measure targeting to sites of constitutive and inducible antigen expression and investigate the impact of various characteristics (affinity, valence, circulation time) on nanobody biodistribution and pharmacokinetics. Engineering of VCAMelid for bivalent binding (BiVCAMelid) increased affinity by an order of magnitude and provided 2.8- and 3.6-fold enhancements in splenic and brain targeting in naive mice, with a further 2.6-fold increase in brain uptake in the setting of focal CNS inflammation. In contrast, introduction of an albumin-binding arm (VCAM/ALB8) did not affect binding affinity, but its prolonged circulation time resulted in 3.5-fold and 17.4-fold increases in splenic and brain uptake at 20 min post-dose and remarkable 40-, 25-, and 15-fold enhancements in overall exposure of blood, spleen, and brain, respectively, relative to both VCAMelid and BiVCAMelid. Both therapeutic protein (superoxide dismutase, SOD-1) and nanocarrier (liposome) delivery were enhanced by conjugation to VCAM-1 targeted nanobodies. The bispecific VCAM/ALB8 maintained its superiority over VCAMelid in enhancing both circulation time and organ targeting of SOD-1, but its advantages were largely blunted by conjugation to liposomes.

Selective targeting of nanomedicine to inflamed cerebral vasculature to enhance the blood-brain barrier.

Drug targeting to inflammatory brain pathologies such as stroke and traumatic brain injury remains an elusive goal. Using a mouse model of acute brain inflammation induced by local tumor necrosis factor alpha (TNFα), we found that uptake of intravenously injected antibody to vascular cell adhesion molecule 1 (anti-VCAM) in the inflamed brain is >10-fold greater than antibodies to transferrin receptor-1 and intercellular adhesion molecule 1 (TfR-1 and ICAM-1). Furthermore, uptake of anti-VCAM/liposomes exceeded that of anti-TfR and anti-ICAM counterparts by ∼27- and ∼8-fold, respectively, achieving brain/blood ratio >300-fold higher than that of immunoglobulin G/liposomes. Single-photon emission computed tomography imaging affirmed specific anti-VCAM/liposome targeting to inflamed brain in mice. Intravital microscopy via cranial window and flow cytometry showed that in the inflamed brain anti-VCAM/liposomes bind to endothelium, not to leukocytes. Anti-VCAM/LNP selectively accumulated in the inflamed brain, providing de novo expression of proteins encoded by cargo messenger RNA (mRNA). Anti-VCAM/LNP-mRNA mediated expression of thrombomodulin (a natural endothelial inhibitor of thrombosis, inflammation, and vascular leakage) and alleviated TNFα-induced brain edema. Thus VCAM-directed nanocarriers provide a platform for cerebrovascular targeting to inflamed brain, with the goal of normalizing the integrity of the blood-brain barrier, thus benefiting numerous brain pathologies.

Shoulder Pseudodislocation Associated with Calcific Tendinitis/Bursitis and Diagnosed by Point of Care Ultrasound.

BACKGROUND: Shoulder pseudodislocation, or "drooping shoulder," presents with acute pain and deformity of the joint, with radiographs demonstrating inferior subluxation of the humeral head relative to the glenoid fossa. The diagnosis must be made promptly and distinguished from true glenohumeral dislocation, both to avoid unnecessary attempts at closed reduction and to facilitate investigation of the underlying cause, which may include septic arthritis, hemarthrosis, or other emergent etiologies. Point-of-care ultrasound (POCUS) may be useful in the evaluation of emergency department (ED) patients with suspected pseudodislocation. CASE REPORT: A 50-year old female presented to the ED with an acutely painful and deformed shoulder but atypical history and physical examination. Initial radiography appeared to show a glenohumeral dislocation, but POCUS, done to guide intra-articular lidocaine injection, led to recognition of pseudodislocation and subsequent diagnosis of calcific tendinitis/bursitis, a condition not previously associated with inferior humeral subluxation. WHY SHOULD AN EMERGENCY PHYSICIAN BE AWARE OF THIS?: Shoulder pseudodislocation must be considered in the evaluation of patients with suspected glenohumeral dislocation, but atypical features on history, physical examination, or initial plain radiography. POCUS may facilitate prompt diagnosis and identification of the underlying etiology.

Combining vascular targeting and the local first pass provides 100-fold higher uptake of ICAM-1-targeted vs untargeted nanocarriers in the inflamed brain.

New advances in intra-arterial (IA) catheters offer clinically proven local interventions in the brain. Here we tested the effect of combining local IA delivery and vascular immunotargeting. Microinjection of tumor necrosis factor alpha (TNFα) in the brain parenchyma causes cerebral overexpression of Inter-Cellular Adhesion Molecule-1 (ICAM-1) in mice. Systemic intravenous injection of ICAM-1 antibody (anti-ICAM-1) and anti-ICAM-1/liposomes provided nearly an order of magnitude higher uptake in the inflamed vs normal brain (from ~0.1 to 0.8%ID/g for liposomes). Local injection of anti-ICAM-1 and anti-ICAM-1/liposomes via carotid artery catheter provided an additional respective 2-fold and 5-fold elevation of uptake in the inflamed brain vs levels attained by IV injection. The uptake in the inflamed brain of respective untargeted IgG counterparts was markedly lower (e.g., uptake of anti-ICAM-1/liposomes was 100-fold higher vs IgG/liposomes). These data affirm the specificity of the combined effect of the first pass and immunotargeting. Intravital real-time microscopy via cranial window revealed that anti-ICAM-1/liposomes, but not IgG/liposomes bind to the lumen of blood vessels in the inflamed brain within minutes after injection. This straightforward framework provides the basis for translational efforts towards local vascular drug targeting to the brain.