Ligand Valency Affects Transcytosis, Recycling and Intracellular Trafficking Mediated by the Neonatal Fc Receptor

The neonatal Fc receptor (FcRn) transports IgG across epithelial cell barriers to provide maternal antibodies to offspring and serves as a protection receptor by rescuing endocytosed IgG and albumin from lysosomal degradation. Here we describe the generation of polarized Madin–Darby canine kidney (MDCK) cells expressing rat FcRn (rFcRn) to investigate the potential requirement for ligand bivalency in FcRn-mediated transport. The rFcRn-MDCK cells bind, internalize and bidirectionally transcytose the bivalent ligands IgG and Fc across polarized cell monolayers. However, they cannot be used to study FcRn-mediated transport of the monovalent ligand albumin, as we observe no specific binding, internalization or transcytosis of rat albumin. To address whether ligand bivalency is required for transport, the ability of rFcRn to transcytose and recycle wild-type Fc homodimers (wtFc; two FcRn-binding sites) and a heterodimeric Fc (hdFc; one FcRn-binding site) was compared. We show that ligand bivalency is not required for transcytosis or recycling, but that wtFc is transported more efficiently than hdFc, particularly at lower concentrations. We also demonstrate that hdFc and wtFc have different intracellular fates, with more hdFc than wtFc being trafficked to lysosomes and degraded, suggesting a role for avidity effects in FcRn-mediated IgG transport

A freeze substitution fixation-based gold enlarging technique for EM studies of endocytosed nanogold-labeled molecules

We have developed methods to locate individual ligands that can be used for electron microscopy studies of dynamic events during endocytosis and subsequent intracellular trafficking. The methods are based on enlargement of 1.4 nm Nanogold attached to an endocytosed ligand. Nanogold, a small label that does not induce misdirection of ligand–receptor complexes, is ideal for labeling ligands endocytosed by live cells, but is too small to be routinely located in cells by electron microscopy. Traditional pre-embedding enhancement protocols to enlarge Nanogold are not compatible with high pressure freezing/freeze substitution fixation (HPF/FSF), the most accurate method to preserve ultrastructure and dynamic events during trafficking. We have developed an improved enhancement procedure for chemically fixed samples that reduced auto-nucleation, and a new pre-embedding gold enlarging technique for HPF/FSF samples that preserved contrast and ultrastructure and can be used for high-resolution tomography. We evaluated our methods using labeled Fc as a ligand for the neonatal Fc receptor. Attachment of Nanogold to Fc did not interfere with receptor binding or uptake, and gold-labeled Fc could be specifically enlarged to allow identification in 2D projections and in tomograms. These methods should be broadly applicable to many endocytosis and transcytosis studies

Long-term efficacy and safety of CT-P6 versus trastuzumab in patients with HER2-positive early breast cancer: final results from a randomized phase III trial

Purpose Equivalent efficacy was demonstrated for the biosimilar CT-P6 and trastuzumab following neoadjuvant therapy for patients with human epidermal growth factor receptor-2 (HER2)-positive early breast cancer. Following adjuvant treatment, efficacy and safety were comparable between treatments. We report updated safety and efficacy data after up to 3 years’ follow-up. Methods Following neoadjuvant chemotherapy with CT-P6/trastuzumab, patients underwent surgery and continued receiving adjuvant CT-P6/trastuzumab. The primary endpoint (previously reported) was pathological complete response. Time-to-event analyses (disease-free survival [DFS], progression-free survival [PFS], and overall survival [OS]), study drug-related and cardiac adverse events, and immunogenicity were assessed during post-treatment follow-up. Results Most patients entered the follow-up period (CT-P6: 259 [95.6%]; trastuzumab: 269 [96.8%]). After a median follow-up of 38.7 (CT-P6) and 39.6 (trastuzumab) months, medians were not reached for time-to-event parameters; estimated hazard ratios (HRs) and 3-year survival rates were similar between groups. Estimated HRs (95% confidence intervals) for CT-P6 versus trastuzumab were 1.23 (0.78–1.93) for DFS, 1.31 (0.86–2.01) for PFS, and 1.10 (0.57–2.13) for OS (intention-to-treat population). Safety findings were comparable between groups for the overall study and follow-up period, including study drug-related cardiac disorders (CT-P6: 22 [8.1%] patients; trastuzumab: 24 [8.6%] patients [overall]) and decreases in left ventricular ejection fraction. Immunogenicity was similar between groups. Conclusion The similarity of the time-to-event analyses between CT-P6 and trastuzumab supports the equivalence in terms of efficacy established for the primary endpoint. CT-P6 was well tolerated, with comparable safety and immunogenicity to trastuzumab. ClinicalTrials.gov: NCT02162667 (registered June 13, 2014

Telerehabilitation as a Method for Achieving Competencies in Physical and Rehabilitation Medicine Residency Training in a Developing Country: A Protocol for a Pilot Mixed-Methods Study

BackgroundIn the second year of the COVID-19 pandemic, Physical and Rehabilitation Medicine (PRM) residents in a developing country continue to face a lack of in-person clinical exposure and learning opportunities. With the unprecedented shift to virtual care, it remains uncertain whether residents can achieve PRM competencies using telerehabilitation as a method of instruction.ObjectiveTo determine the PRM residents' ability to achieve competencies through telerehabilitation, as perceived by different stakeholders (residents, chief residents, training officers, and department heads).MethodsThis will be a pilot mixed-methods study, employing concurrent triangulation, in the Department of Rehabilitation Medicine in one large private medical center and one large government hospital in Manila, Philippines. There will be two phases of online data collection upon approval by their respective research ethics board. The first phase will involve an online Likert-scale questionnaire to obtain the residents' self-perceived attainment of competencies and learning of PRM topics and skills specified by the International Society of Physical and Rehabilitation Medicine and the Philippine Board of Rehabilitation Medicine. The results of the survey will then be summarized and presented in a focus group discussion (FGD) with the department heads, training officers, and chief residents of the two institutions in an attempt to explain the residents' perceptions on their competencies achieved through virtual care. Afterwards, the qualitative data obtained from the FGD will then be thematically analyzed, and mixed methods integration will be employed to generate knowledge and recommendations.DiscussionIt is hypothesized that the majority of the residents had little to no experience with telerehabilitation pre-pandemic. Suddenly telerehabilitation was used to augment clinical training during the pandemic. It is uncertain whether telerehabilitation can help residents achieve competencies in the different domains of training, namely: patient safety and quality patient care; medical knowledge and procedural skills; interpersonal and communication skills; practice- and systems-based learning and improvement; reintegration of people with disabilities into the society; medical ethics and public health; quality assurance; policies of care and prevention for disabled people; and professionalism. The study results can provide insights on the aspects of a PRM curriculum that may have to be modified to ensure the training program is sensitive and appropriate to the changing training needs of the residents amid the pandemic and similar crises that may disrupt in-person clinical encounters in the future

Chromosomal aberrations among Filipino health workers at the chemotherapy oncology wards/ clinics of a tertiary government hospital

Introduction. Chromosomal mutations are causal events in neoplasia development. Biomarker cytogenetic assays can determine exposure to mutagenic agents in occupational settings. This study assessed early biological marker chromosomal aberrations among health workers in the chemotherapy oncology wards/ clinics, exploring its association to the subjects\u27 occupational, environmental and baseline profile. Methods. This was an IRB approved cross-sectional exploratory study among hospital personnel working in the chemotherapy oncology facility of a tertiary government hospital, who underwent structured interview and blood extraction for cytogenetic assay after informed consent. Study funds only permitted assay of 44 specimens of 144 planned sample size, hence, Stata 6.0 only analyzed data from 44 subjects. Results. All 44 subjects had varying exposure to chemotherapy drug infusions. Of these, 79% had 1.0 breaks per cell (hypersensitive). Predominantly chromatid breaks (CTB), chromatid gaps (CTG), sister chromatid exchanges (SCE) were seen. No significant association was shown between mutagenic sensitivity and baseline characteristics, but with small sample size. Conclusion. 21% borderline to hypersensitive mutagenic sensitivity among oncology workers at the tertiary government hospital is relatively significant, despite small sample size, connoting a must preventive promotive practice of chemotherapy administration in the workplace

Efficacy and safety of CT-P6 versus reference trastuzumab in HER2-positive early breast cancer: updated results of a randomised phase 3 trial

Purpose Neoadjuvant CT-P6, a trastuzumab biosimilar, demonstrated equivalent efficacy to reference trastuzumab in a phase 3 trial of HER2-positive early-stage breast cancer (EBC) (NCT02162667). We report post hoc analyses evaluating pathological complete response (pCR) and breast pCR alongside additional efficacy and safety measures. Methods Following neoadjuvant treatment and surgery, patients received adjuvant CT-P6 or trastuzumab (6 mg/kg) every 3 weeks for ≤ 1 year. Results In total, 271 and 278 patients received CT-P6 and trastuzumab, respectively. pCR and breast pCR rates were comparable between treatment groups regardless of age, region, or clinical stage. Overall, 47.6% (CT-P6) and 52.2% (trastuzumab) of patients experienced study drug-related treatment-emergent adverse events (TEAEs), including 17 patients reporting heart failure (CT-P6: 10; trastuzumab: 7). Two CT-P6 and three trastuzumab patients discontinued adjuvant treatment due to TEAEs. Conclusion Adjuvant CT-P6 demonstrated comparable efficacy and safety to trastuzumab at 1 year in patients with HER2-positive EBC, supporting CT-P6 and trastuzumab comparability