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.

Outcomes of Extended Pedicle Technique vs Free Nipple Graft Reduction Mammoplasty for Patients With Gigantomastia.

BACKGROUND: Optimal reduction mammoplasty techniques to treat patients with gigantomastia have been debated and can involve extended pedicles (EP) or free nipple grafts (FNG). OBJECTIVES: The authors compared clinical, patient-reported, and aesthetic outcomes associated with reduction mammoplasty employing EP vs FNG. METHODS: A multi-institutional, retrospective study of adult patients with gigantomastia who underwent reduction mammoplasty at 2 tertiary care centers from 2017 to 2020 was performed. Gigantomastia was defined as reduction weight >1500 g per breast or sternal notch-to-nipple distance ≥40 cm. Surgeons at 1 institution employed the EP technique, whereas those at the other utilized FNG. Baseline characteristics, preoperative and postoperative BREAST-Q, and clinical outcomes were collected. Aesthetic outcomes were assessed in 1:1 propensity score-matched cases across techniques. Preoperative and postoperative photographs were provided to reviewers across the academic plastic surgery continuum (students to faculty) and non-medical individuals to evaluate aesthetic outcomes. RESULTS: Fifty-two patients met the inclusion criteria (21 FNG, 31 EP). FNG patients had a higher incidence of postoperative cellulitis (23% vs 0%, P < 0.05) but no other differences in surgical or medical complications. Baseline BREAST-Q scores did not differ between groups. Postoperative BREAST-Q scores revealed greater satisfaction with the EP technique (P < 0.01). The aesthetic assessment of outcomes in 14 matched pairs of patients found significantly better aesthetic outcomes in all domains with the EP procedure (P < 0.05), independent of institution or surgical experience. CONCLUSIONS: This multi-institutional study suggests that, compared with FNG, the EP technique for reduction mammoplasty provides superior clinical, patient-reported, and aesthetic outcomes for patients with gigantomastia.

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.

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.

Site-Specific Modification of Single-Chain Antibody Fragments for Bioconjugation and Vascular Immunotargeting.

The conjugation of antibodies to drugs and drug carriers improves delivery to target tissues. Widespread implementation and effective translation of this pharmacologic strategy awaits the development of affinity ligands capable of a defined degree of modification and highly efficient bioconjugation without loss of affinity. To date, such ligands are lacking for the targeting of therapeutics to vascular endothelial cells. To enable site-specific, click-chemistry conjugation to therapeutic cargo, we used the bacterial transpeptidase, sortase A, to attach short azidolysine containing peptides to three endothelial-specific single chain antibody fragments (scFv). While direct fusion of a recognition motif (sortag) to the scFv C-terminus generally resulted in low levels of sortase-mediated modification, improved reaction efficiency was observed for one protein, in which two amino acids had been introduced during cloning. This prompted insertion of a short, semi-rigid linker between scFv and sortag. The linker significantly enhanced modification of all three proteins, to the extent that unmodified scFv could no longer be detected. As proof of principle, purified, azide-modified scFv was conjugated to the antioxidant enzyme, catalase, resulting in robust endothelial targeting of functional cargo in vitro and in vivo.

Presentation and Referral Patterns in Pediatric Closed Hand Fractures.

BACKGROUND: Although pediatric hand fractures are common and generally have good outcomes, they remain a considerable source of anxiety for non-hand surgeons, who are less familiar with these injuries. We hypothesized that this anxiety may manifest as inefficiency in referral patterns. METHODS: The records of pediatric patients with isolated, closed hand fractures without concurrent trauma seen at our institution by a hand surgeon between January 2017 and December 2018 were retrospectively reviewed. RESULTS: There were 454 patients included; 62.1% were men, and the mean age was 9.6 years at initial encounter. Most patients (89.6%) were treated nonoperatively and incurred few complications (0.5%). Roughly half of all cases (n = 262) initially presented to an outside provider. Of these, 24.0% (n = 64 of 262) were evaluated by 2+ providers before a hand surgeon. Most commonly, these patients were referred from an outside emergency department (ED) to our ED before hand surgeon evaluation (n = 45 of 64). Forty-seven patients required surgery; however, none were performed urgently. Although a greater proportion of 7- to 11-year-old patients saw 2+ providers prior to a hand surgeon (P = .007), fewer required surgery (P < .001). CONCLUSIONS: Pediatric closed hand fractures are mainly treated nonoperatively and nonemergently with generally excellent outcomes. Our data suggest that many patients continue to be referred through the ED or multiple EDs/providers for treatment. These inefficient referral patterns demonstrate the need for better education for ED and primary care providers, as well as better communication between these providers and local pediatric hand surgeons. Advancements in these areas are likely to improve efficiency of care and decrease costs.

Targeted Nanocarriers Co-Opting Pulmonary Intravascular Leukocytes for Drug Delivery to the Injured Brain.

Ex vivo-loaded white blood cells (WBC) can transfer cargo to pathological foci in the central nervous system (CNS). Here we tested affinity ligand driven in vivo loading of WBC in order to bypass the need for ex vivo WBC manipulation. We used a mouse model of acute brain inflammation caused by local injection of tumor necrosis factor alpha (TNF-α). We intravenously injected nanoparticles targeted to intercellular adhesion molecule 1 (anti-ICAM/NP). We found that (A) at 2 h, >20% of anti-ICAM/NP were localized to the lungs; (B) of the anti-ICAM/NP in the lungs >90% were associated with leukocytes; (C) at 6 and 22 h, anti-ICAM/NP pulmonary uptake decreased; (D) anti-ICAM/NP uptake in brain increased up to 5-fold in this time interval, concomitantly with migration of WBCs into the injured brain. Intravital microscopy confirmed transport of anti-ICAM/NP beyond the blood-brain barrier and flow cytometry demonstrated complete association of NP with WBC in the brain (98%). Dexamethasone-loaded anti-ICAM/liposomes abrogated brain edema in this model and promoted anti-inflammatory M2 polarization of macrophages in the brain. In vivo targeted loading of WBC in the intravascular pool may provide advantages of coopting WBC predisposed to natural rapid mobilization from the lungs to the brain, connected directly via conduit vessels.

The Impact of Combined Oral Contraceptives on Adolescents with Macromastia.

BACKGROUND: The combined oral contraceptive pill, containing both estrogen and progestin, is commonly prescribed to adolescents for numerous health benefits. However, there is concern among patients and providers that its use may exacerbate breast growth. This retrospective, case-control study examined the association between combined oral contraceptive pill use and macromastia-related breast hypertrophy and symptoms in adolescents. METHODS: A total of 378 patients undergoing reduction mammaplasty between the ages of 12 and 21 years were assessed for baseline and postoperative breast symptoms and combined oral contraceptive pill use. In addition, the medical records of 378 female controls of the same age range were retrospectively reviewed. RESULTS: Although a lower proportion of the macromastia cohort used any hormonal contraception compared to controls (37.8 percent versus 64.8 percent; OR, 0.33; 95 percent CI, 0.24 to 0.44; p < 0.001), they were more often prescribed combined oral contraceptive pills (82.5 percent versus 52.7 percent; OR, 1.93; 95 percent CI, 1.29 to 2.68; p < 0.001). Participants with macromastia who used combined oral contraceptive pills had a smaller median normalized amount of breast tissue resected during reduction mammaplasty than those who never used hormonal contraception (639.5 g/m 2 versus 735.9 g/m 2 ; p = 0.003). Combined oral contraceptive pills were not associated with breast-related symptoms or clinical impairment, or postoperative breast growth ( p > 0.05 for all). CONCLUSIONS: Combined oral contraceptive pill use during adolescence may be associated with developing less severe breast hypertrophy. Combined oral contraceptive pills do not appear to exacerbate macromastia-related symptoms or impact postoperative growth in young women following reduction mammaplasty. Although additional research is needed, providers are encouraged to consider combined oral contraceptive pills for their patients with macromastia when indicated and appropriate. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Complications and Quality of Life following Gynecomastia Correction in Adolescents and Young Men.

BACKGROUND: Persistent adolescent gynecomastia negatively affects health-related quality of life. Surgery results in psychosocial improvements, but the effects of postoperative complications on health-related quality of life are unknown. The authors examined whether complications following adolescent gynecomastia surgery impact postoperative health-related quality of life. METHODS: Patients aged 12 to 21 years who underwent surgical correction of unilateral/bilateral gynecomastia between 2007 and 2019 were enrolled (n = 145). Relevant demographic and clinical data were obtained from medical records. Fifty-one patients completed the following surveys preoperatively, and at 6 months and 1, 3, 5, 7, 9, and 11 years postoperatively: 36-Item Short-Form Health Survey (Version 2), Rosenberg Self-Esteem Scale, and the 26-item Eating Attitudes Test. RESULTS: Within a median period of 8.6 months, 36 percent of breasts experienced at least one complication. The most common were residual tissue (12.6 percent), contour irregularities (9.2 percent), and hematomas (7.8 percent). Patients reported significant postoperative improvements in self-esteem and in seven health-related quality-of-life domains (Physical Functioning, Role-Physical, Bodily Pain, Vitality, Social Functioning, Role-Emotional, and Mental Health) at a median of 33.3 months. Postoperative survey scores did not vary by grade or procedure, or largely by body mass index category or complication status. However, patients aged younger than 17 years at surgery scored significantly higher than older patients in the Short-Form Health Survey Vitality and Mental Health domains postoperatively. CONCLUSIONS: Health-related quality-of-life improvements are achievable in adolescents through surgical correction of persistent gynecomastia. Postoperatively, patients largely experienced similar health-related quality-of-life gains irrespective of complication status, grade, surgical technique, or body mass index category. Minor postcorrection complications are but do not appear to limit postoperative health-related quality-of-life benefits.

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.

The Impact of Progestin-only Contraception on Adolescents with Macromastia.

Progestin-only contraception has become increasingly popular among adolescents. However, patients, parents, and providers share concerns regarding the potential impact that progestin-only contraception may have on breast growth. We sought to explore the impact of progestin-only contraception on breast hypertrophy and symptomatology in adolescents with macromastia. METHODS: Patients between the ages of 12 and 21 years undergoing reduction mammaplasty were prospectively assessed for baseline and postoperative breast symptomatology and medication use. The medical records of female controls within the same age range were retrospectively reviewed. RESULTS: A total of 378 participants with macromastia and 378 controls were included in analyses. A higher proportion of controls used progestin-only methods compared with participants with macromastia (28.0% versus 5.3%, P < 0.001). The most commonly prescribed methods were the depot medroxyprogesterone acetate injection (31.0%), levonorgestrel-containing intrauterine device (31.0%), and subdermal implant (26.2%). Patients with macromastia who used progestin-only contraception had a greater amount of breast tissue resected during reduction mammaplasty (P = 0.04), reported greater musculoskeletal pain (P = 0.008), and were roughly 500% more likely to experience breast pain (odds ratio, 4.94; 95% confidence interval, 1.58-15.47; P = 0.005) than those with macromastia who never used hormonal contraception. CONCLUSIONS: Adolescents with macromastia who use progestin-only contraception may have greater breast hypertrophy and worse breast and musculoskeletal pain. When appropriate, providers may wish to consider other contraception methods for patients who are at-risk for breast hypertrophy or those who suffer from macromastia-related symptoms.

Acute myeloid leukemia with hepatic infiltration presenting as obstructive jaundice.

We present the case of a 55-year-old woman who presented with laboratory studies concerning for acute myeloid leukemia (AML) as well as obstructive cholestasis. In similar previously reported cases, concerns of chemotherapy toxicity exacerbated by liver dysfunction or concerns of untreated, concurrent cholecystitis in a neutropenic patient often delay initiation of chemotherapy for full medical workup. At admission, our patient was started on the cytoreductive agent hydroxyurea. By day 10 of her medical workup, her liver function had improved with total bilirubin levels normalizing. At that time, full-dose 7 + 3 induction with cytarabine and daunorubicin was then initiated.

Management of Residual Spitz Nevus in Surgical Specimens following Biopsy and Excision.

Proper management of Spitz nevi continues to be debated, with treatment ranging from observation to surgery. To better characterize the outcome of surgical procedures performed for incomplete initial excision or biopsy, we sought to ascertain the histopathological presence of residual Spitz nevi in a set of surgical specimens. METHODS: We retrospectively reviewed 123 records with histologically-confirmed Spitz nevus. Data concerning treatment, clinical features, histopathological margin involvement, and presence of residual lesion on subsequent procedural specimens were collected. RESULTS: Fifty-three percent of lesions (n = 65) were initially sampled by shave or punch biopsy, and the remainder (n = 58) were formally excised without initial biopsy. The rates of re-excision for involved margins were: shave biopsy (92.2%), punch biopsy (78.6%), and formal excision (13.8%). In total, 61.0% of patients who underwent an initial procedure of any kind had involved margins, but only half of those re-excised for involved margins (57.6%) had histologically residual lesion on repeated excision. A significantly higher proportion of initial punch biopsies (90.9%) resulted in residual lesion (in secondary excision specimens) when compared with shave biopsy (48.9%) and formal excision (62.5%; P < 0.05). CONCLUSIONS: Findings suggest that clinicians may consider shave biopsy over punch biopsy for diagnosing suspected lesions, when indicated and appropriate. Given the rarity of malignant transformation and the frequency of residual nevus, observation may be reasonable for managing pediatric patients with histologically-confirmed Spitz nevi, who are post initial biopsy or excision despite known histopathological margin involvement.

Optimal Timing for Reduction Mammaplasty in Adolescents.

BACKGROUND: Reduction mammaplasty effectively alleviates symptoms and restores quality of life. However, operating on adolescents remains controversial, partly because of fear of potential postoperative breast growth. This cross-sectional study provides surgeons with a method to predict the optimal timing, or biological "sweet spot," for reduction mammaplasty to minimize the risk of breast regrowth in adolescents. METHODS: The authors reviewed the medical records of women aged 12 to 21 years who underwent reduction mammaplasty from 2007 to 2019. Collected data included symptomology, perioperative details, and postoperative outcomes. RESULTS: Four hundred eighty-one subjects were included in analyses and were, on average, 11.9 years old at first menses (menarche) and 17.9 years old at surgery. Six percent of subjects experienced postoperative breast growth. Breast size appears to stabilize considerably later in obese adolescents compared to healthy-weight and overweight patients, and breast growth in obese macromastia patients may not end until 9 years after menarche. Operating on obese women before this time point increased the likelihood of glandular breast regrowth by almost 120 percent (OR, 1.18; 95 percent CI, 1.11 to 1.26). Surgery performed less than 3 years after menarche, the commonly regarded end of puberty, increased the likelihood of glandular regrowth by over 700 percent in healthy-weight and overweight subjects (OR, 7.43; 95 percent CI, 1.37 to 40.41). CONCLUSIONS: Findings suggest that reduction mammaplasty age restrictions imposed by care providers and third-party payors may be arbitrary. Surgical readiness should be determined on an individual basis incorporating the patient's biological and psychological maturity, obesity status, potential for postoperative benefit, and risk tolerance for postoperative breast growth. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, III.

Biocompatible coupling of therapeutic fusion proteins to human erythrocytes.

Carriage of drugs by red blood cells (RBCs) modulates pharmacokinetics, pharmacodynamics, and immunogenicity. However, optimal targets for attaching therapeutics to human RBCs and adverse effects have not been studied. We engineered nonhuman-primate single-chain antibody fragments (scFvs) directed to human RBCs and fused scFvs with human thrombomodulin (hTM) as a representative biotherapeutic cargo (hTM-scFv). Binding fusions to RBCs on band 3/glycophorin A (GPA; Wright b [Wrb] epitope) and RhCE (Rh17/Hr0 epitope) similarly endowed RBCs with hTM activity, but differed in their effects on RBC physiology. scFv and hTM-scFv targeted to band 3/GPA increased membrane rigidity and sensitized RBCs to hemolysis induced by mechanical stress, while reducing sensitivity to hypo-osmotic hemolysis. Similar properties were seen for other ligands bound to GPA and band 3 on human and murine RBCs. In contrast, binding of scFv or hTM-scFv to RhCE did not alter deformability or sensitivity to mechanical and osmotic stress at similar copy numbers bound per RBCs. Contrasting responses were also seen for immunoglobulin G antibodies against band 3, GPA, and RhCE. RBC-bound hTM-scFv generated activated protein C (APC) in the presence of thrombin, but RhCE-targeted hTM-scFv demonstrated greater APC generation per bound copy. Both Wrb- and RhCE-targeted fusion proteins inhibited fibrin deposition induced by tumor necrosis factor-α in an endothelialized microfluidic model using human whole blood. RhCE-bound hTM-scFv more effectively reduced platelet and leukocyte adhesion, whereas anti-Wrb scFv appeared to promote platelet adhesion. These data provide a translational framework for the development of engineered affinity ligands to safely couple therapeutics to human RBCs.