Cellaca® PLX image cytometer as an alternative for immunophenotyping, GFP/RFP transfection efficiencies, and apoptosis analysis.

Cell and gene therapy is a fast-growing field for cancer therapeutics requiring reliable instrumentation and technologies. Key parameters essential for satisfying Chemistry Manufacturing and Controls criteria standards are routinely performed using flow cytometry. Recently, image cytometry was developed for cell characterization and cell-based assays but had not yet demonstrated sufficient sensitivity for surface marker detection. We developed the Cellaca® PLX image cytometry system and the respective methodologies required for immunophenotyping, GFP and RFP transfection/transduction efficiencies, and cell health analyses for routine cell characterization. All samples tested were compared directly to results from the CytoFLEX flow cytometer. PBMCs were stained with T-cell surface markers for immunophenotyping, and results show highly comparable CD3, CD4, and CD8 populations (within 5 %). GFP- or RFP-expressing cell lines were analyzed for transfection/transduction efficiencies, and the percentage positive cells and respective viabilities were equivalent on both systems. Staurosporine-treated Jurkat cells were stained for apoptotic markers, where annexin V and caspase-3 positive cells were within 5 % comparing both instruments. The proposed system may provide a complementary tool for performing routine cell-based experiments with improved efficiency and sensitivity compared to prior image cytometers, which may be significantly valuable to the cell and gene therapy field.

Preclinical Characterization and Phase I Study of an Anti-HER2-TLR7 Immune-Stimulator Antibody Conjugate in Patients with HER2+ Malignancies.

Immune-stimulator antibody conjugates (ISAC) combining tumor-targeting monoclonal antibodies with immunostimulatory agents allow targeted delivery of immune activators into tumors. NJH395 is a novel, first-in-class ISAC comprising a Toll-like receptor 7 (TLR7) agonist conjugated to an anti-HER2 antibody via a noncleavable linker payload. Preclinical characterization showed ISAC-mediated activation of myeloid cells in the presence of antigen-expressing cancer cells, with antigen targeting and TLR7 agonism contributing to antitumor activity. Safety, efficacy, immunogenicity, pharmacokinetics, and pharmacodynamics were investigated in a phase I, multicenter, open-label study in patients with HER2+ non-breast advanced malignancies (NCT03696771). Data from 18 patients enrolled in single ascending dose escalation demonstrated delivery of the TLR7-agonist payload in HER2+ tumor cells and induction of type I IFN responses, which correlated with immune modulation in the tumor microenvironment. Cytokine release syndrome was a common, but manageable, drug-related adverse event. Antidrug antibodies and neuroinflammation at high doses represented significant clinical challenges. Data provide proof-of-mechanism and critical insights for novel immunotherapies.

The volume-outcomes relationship in donation after circulatory death liver transplantation.

BACKGROUND: Donation after circulatory death (DCD) liver transplantation (LT) has become an effective mechanism for expanding the donor pool and decreasing waitlist mortality. However, it is unclear if low-volume DCD centers can achieve comparable outcomes to high-volume centers. METHODS: From 2011 to 2019 utilizing the United Network for Organ Sharing (UNOS) database, liver transplant centers were categorized into tertiles based on their annual volume of DCD LTs. Donor selection, recipient selection, and survival outcomes were compared between very-low volume (VLV, n = 1-2 DCD LTs per year), low-volume (LV, n = 3-5), and high-volume (HV, n > 5) centers. RESULTS: One hundred and ten centers performed 3273 DCD LTs. VLV-centers performed 339 (10.4%), LV-centers performed 627 (19.2%), and HV-centers performed 2307 (70.4%) LTs. 30-day, 90-day, and 1-year patient and graft survival were significantly increased at HV-centers (all P < .05). Recipients at HV-centers had shorter waitlist durations (P < .01) and shorter hospital lengths of stay (P < .01). On multivariable regression, undergoing DCD LT at a VLV-center or LV-center was associated with increased 1-year patient mortality (VLV-OR:1.73, 1.12-2.69) (LV-OR: 1.42, 1.01-2.00) and 1-year graft failure (VLV-OR: 1.79, 1.24-2.58) (LV-OR: 1.28, .95-1.72). DISCUSSION: Increased annual DCD liver transplant volume is associated with improved patient and graft survival.

Densidad de la masa ósea y marcadores bioquímicos del metabolismo óseo: estudio transversal en adultos residentes de Lima Metropolitana, Perú / Bone mass density and biochemical markers of bone metabolism: transversal study in adult residents of Metropolitan Lima, Peru

RESUMEN Objetivos. Determinar y correlacionar la densidad mineral ósea (DMO) y los marcadores bioquímicos del metabolismo óseo de adultos de 30 a 40 años de Lima, Perú, en el año 2018. Métodos. Estudio descriptivo, transversal, realizado a 84 mujeres y 53 varones, de 30 a 40 años de edad, residentes en Lima, Perú, sin manifestaciones ni tratamiento de enfermedades metabólicas óseas primarias ni secundarias. Se registró información del estado actual, antecedentes personales fisiológicos, patológicos, familiares y examen clínico. Se midió la DMO por absorciometría dual de rayos X en columna, cadera y antebrazo; en sangre, parathormona, osteocalcina, 25-hidroxi-vitamina-D, hormona luteinizante, estradiol en las mujeres, testosterona en los varones, calcio sérico, fósforo sérico, fosfatasa alcalina, proteínas totales y fracciones, creatinina; en orina N-telopéptido de enlaces de colágeno tipo I, calcio y fósforo, por métodos convencionales. Resultados. La DMO se efectuó a 62 mujeres y 40 varones; 2 mujeres y 1 varón tuvieron osteoporosis, y 3 mujeres y 2 varones osteopenia, retirados para la evaluación estadística; 58 mujeres y 35 varones tuvieron una DMO normal. Los varones tuvieron mayor DMO y concentraciones mayores de calcio, fosfatasa alcalina y creatinina; 14 mujeres y 2 varones tuvieron cifras bajas de 25-OH-vitaminaD. Hubo correlación positiva entre DMO y el IMC. Conclusiones. La DMO normal de personas de 30 a 40 años de Lima fue lineal y hubo una relación positiva con el IMC; hubo osteoporosis en 2 mujeres y 1 varón, osteopenia en 3 mujeres y 2 varones, cifras bajas de 25-hidroxi-vitamina-D en 16 personas.

ABSTRACT Objectives. To determine and correlate the bone mineral density (BMD) and the biochemical markers of bone metabolism in adults 30 to 40 years old from Lima, Perú, in year 2018. Methods. A total of 84 women and 53 men, 30 to 40 years old from Lima, Perú, were submmited to a descriptive, transversal study. They did not have symptoms nor treatment for bone metabolic diseases. Information about their present health state, personal and familiar physiological and pathological history and physical examinatión were registered. BMD was measured by dual energy-X-ray absorptiometry in spine, hip and forearm. Parathormone, osteocalcine, 25-hidroxi-vitamine-D, luteinizing hormone, estradiol in women, testosterone in men, calcium, phosphorus, alkaline fosfatase, total and fractionated proteins, creatinine were measured in blood; in urine N-telopéptide, calcium and phosphorus by conventional methods. Results. BMD was measured in 62 women and 40 males; 2 women and 1 man had osteoporosis and 3 women and 2 men osteopenia, who were taken out of statistical evaluation; 58 women and 35 men showed a normal BMD. Male subjects had a greater BMD, higher calcium, alkaline fosfatase and creatine blood concentrations than the female ones; 14 women y 2 men had low 25-OH-vitaminaD concentrations. A positive correlation between the BMD and BMI was found. Conclusions. The normal BMD of persons 30 to 40 years old showed a linear figure and there was a positive correlate with the BMI; premature osteoporosis in 2 women and 1 men as well as osteopenia in 3 women y 2 men was found, low concentrations of 25-hidroxi-vitamine-D was detected in 16 persons.

The Impact of a Comprehensive Resident Curriculum and Required Participation in "This Week in SCORE" on General Surgery ABSITE Performance and Well-Being.

OBJECTIVE: This Week in SCORE (TWIS) is a biennial, general surgery curriculum comprised of weekly online modules, readings, and multiple-choice quizzes. In this study, we examined the impact of required TWIS on American Board of Surgery In-Training Examination (ABSITE) scores and well-being among categorical general surgery residents. DESIGN, SETTING, AND PARTICIPANTS: TWIS quiz completion became required in 2017. Residents attended weekly lectures, and ABSITE performance was incentivized with educational stipends. Surveys were distributed to assess study preferences, learning styles, burnout, and grit. Thirty-six categorical general surgery residents who took ABSITE in both 2017 and 2018 were evaluated in a paired-sample, retrospective analysis. RESULTS: After requiring TWIS, median ABSITE percentile increased by 12% (65%-77%, p = 0.001). Weekly TWIS completion (59% vs 89%, p < 0.001) and quiz results (62% vs 69%, p = 0.005) also improved. During this time, emotional exhaustion and depersonalization declined significantly, yet overall burnout scores did not change. Of 21 survey respondents, 66.7% (n = 14) increased weekly study time by a median of 2.5 hours. However, less than half used Surgical Council on Resident Education as their primary study tool. Only 23.8% (n = 5) reported that mandatory TWIS modified their study behavior, while 90.4% (n = 19) felt the culture of education had improved. CONCLUSIONS: After TWIS participation became required, ABSITE performances improved. Formalized curriculum with frequent assessment may foster accountability among residents, enhancing educational climate, well-being, and test performance.

Congenital Atrophic Plaque: Fibroblastic Connective Tissue Nevus.

Fibroblastic connective tissue nevus (FCTN) is a rare, benign, dermal mesenchymal hamartoma that affects children. We report a 15-year-old boy with a congenital FCTN and describe the clinical, dermatoscopic, and histopathologic features.

Incorporation of Phosphonate into Benzonaphthyridine Toll-like Receptor 7 Agonists for Adsorption to Aluminum Hydroxide.

Small molecule Toll-like receptor 7 (TLR7) agonists have been used as vaccine adjuvants by enhancing innate immune activation to afford better adaptive response. Localized TLR7 agonists without systemic exposure can afford good adjuvanticity, suggesting peripheral innate activation (non-antigen-specific) is not required for immune priming. To enhance colocalization of antigen and adjuvant, benzonaphthyridine (BZN) TLR7 agonists are chemically modified with phosphonates to allow adsorption onto aluminum hydroxide (alum), a formulation commonly used in vaccines for antigen stabilization and injection site deposition. The adsorption process is facilitated by enhancing aqueous solubility of BZN analogs to avoid physical mixture of two insoluble particulates. These BZN-phosphonates are highly adsorbed onto alum, which significantly reduced systemic exposure and increased local retention post injection. This report demonstrates a novel approach in vaccine adjuvant design using phosphonate modification to afford adsorption of small molecule immune potentiator (SMIP) onto alum, thereby enhancing co-delivery with antigen.

Rational design of small molecules as vaccine adjuvants.

Adjuvants increase vaccine potency largely by activating innate immunity and promoting inflammation. Limiting the side effects of this inflammation is a major hurdle for adjuvant use in vaccines for humans. It has been difficult to improve on adjuvant safety because of a poor understanding of adjuvant mechanism and the empirical nature of adjuvant discovery and development historically. We describe new principles for the rational optimization of small-molecule immune potentiators (SMIPs) targeting Toll-like receptor 7 as adjuvants with a predicted increase in their therapeutic indices. Unlike traditional drugs, SMIP-based adjuvants need to have limited bioavailability and remain localized for optimal efficacy. These features also lead to temporally and spatially restricted inflammation that should decrease side effects. Through medicinal and formulation chemistry and extensive immunopharmacology, we show that in vivo potency can be increased with little to no systemic exposure, localized innate immune activation and short in vivo residence times of SMIP-based adjuvants. This work provides a systematic and generalizable approach to engineering small molecules for use as vaccine adjuvants.