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IMPORTANCE: Risk factors for breast cancer–related lymphedema (BCRL) after axillary lymph node dissection (ALND) are poorly understood. OBJECTIVE: To evaluate rates of and risk factors associated with BCRL in a prospective cohort of women treated with ALND. DESIGN, SETTING, AND PARTICIPANTS: This prospective BCRL screening study performed at a tertiary cancer center enrolled women with breast cancer 18 years and older undergoing breast surgery and unilateral ALND in the primary setting or after sentinel lymph node biopsy. EXPOSURES: Risk of BCRL during the first 2 years after ALND and radiotherapy. MAIN OUTCOMES AND MEASURES: Patients were prospectively evaluated with arm volume (perometer) measurements, and BCRL was defined as a relative volume change of 10% or greater from baseline. Cumulative incidence of BCRL was assessed using competing risk analysis. Risk factors for BCRL were assessed on univariate and multivariable analyses. RESULTS: From November 2016 to March 2020, 304 patients were enrolled; 276 had at least 1 longitudinal measurement. Median (IQR) age was 48 (40-57) years; median (IQR) body mass index, calculated as weight in kilograms divided by height in meters squared, was 26.4 (22.5-31.2). Of the 276 patients included in the analysis, 29 (11%) self-identified as Asian, 55 (20%) as Black, 16 (6%) as Hispanic, 166 (60%) as White, and 10 (3%) as unknown race and ethnicity; 70% received neoadjuvant chemotherapy (NAC); 93% received nodal irradiation. The 24-month BCRL rate was 23.8% (95% CI, 17.9%-29.8%), with significant variation by race and ethnicity (24-month rate: 37.2% [Black], 27.7% [Hispanic], 22.5% [Asian], and 19.8% [White]; P = .004). The BCRL rates were also higher among patients receiving NAC vs up-front surgery (24-month rate: 29.3% vs 11.1%; P = .01). On multivariable analysis, Black race and Hispanic ethnicity (compared with White race) (odds ratio [OR], 3.88; 95% CI, 2.14-7.08 and OR, 3.01; 95% CI, 1.10-7.62, respectively; P

The Sigma 2 receptor (σ2R) was described pharmacologically more than three decades ago, but its molecular identity remained obscure until recently when it was identified as transmembrane protein 97 (TMEM97). We and others have shown that σ2R/TMEM97 ligands produce analgesia in mouse neuropathic pain models with a time course wherein analgesic onset is 24 hours following dosing. We sought to understand this unique anti-neuropathic pain effect by addressing two key questions: do these σ2R/TMEM97 compounds act selectively via the receptor, and what is their downstream mechanism on nociceptive neurons. Using male and female conventional knockout (KO) mice forTmem97,we find that a novel σ2R/TMEM97 binding compound, FEM-1689, requires the presence of the gene to produce analgesia in the spared nerve injury model in mice. Using primary mouse dorsal root ganglion (DRG) neurons, we demonstrate that FEM-1689 inhibits the integrated stress response and promotes neurite outgrowth via a σ2R/TMEM97-specific action. We extend the clinical translational value of these findings by showing that FEM-1689 reduces ISR and p-eIF2α levels in human sensory neurons and that it alleviates the pathogenic engagement of ISR by methylglyoxal. We also demonstrate that σ2R/TMEM97 is expressed in human nociceptors and satellite glial cells. These results validate σ2R/TMEM97 as a promising target for further development for the treatment of neuropathic pain.Significance StatementNeuropathic pain is a major medical problem that is poorly treated with existing therapeutics. Our findings demonstrate that σ2R/TMEM97 targeting with modulators creates analgesia in a mouse model via a specific action on the receptor. We also identify a potential mechanism of action, ISR inhibition, that links the receptor to cellular signaling events that have preclinical and clinical validation for pain relief. Our work suggests that σ2R/TMEM97 can be selectively engaged by specific small molecules to produce ISR inhibition in a subset of cells that are critical for neuropathic pain. σ2R/TMEM97-targeted therapeutics thus have the potential to offer effective pain relief without the side effects associated with currently available neuropathic pain medicines.

The goal of this paper is to investigate the effects of intonational differences across Basque varieties. We have chosen three Markina Basque contours as stimuli in a perception experiment: those that are used in declaratives, yes-no questions and verum focus sentences. Stimuli include both natural and manipulated contours. Three participant groups (with Markina Basque, another Basque variety or Spanish as L1) classified stimuli as questions or statements. The results show that Markina Basque speakers differ from the other two groups in the interpretation of the contours. Markina speakers attend mostly to the tonal contour of the last syllable to identify questions.

This study investigates the types of humor embedded in funny scientific posts on social media and their effects on engagement. We mapped the landscape of such posts on Twitter and Instagram through content analysis of their message attributes. Regression analyses were then conducted to examine how different humor types, communicative functions, and visual attributes were associated with liking, retweeting, and commenting. On Twitter, wordplay and satire were found to be positively related to posts’ engagement levels, while anthropomorphic humor was negatively associated with the presence of comments. On Instagram, humor had no relation to engagement.

Residual host cell proteins (HCPs) in the drug product can affect product quality, stability, and/or safety. In particular, highly active hydrolytic enzymes at sub-ppm levels can negatively impact the shelf life of drug products but are challenging to identify by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) due to their high dynamic range between HCPs and biotherapeutic proteins. We employed new strategies to address the challenge: (1) native digest at a high protein concentration; (2) sodium deoxycholate added during the reduction step to minimize the inadvertent omission of HCPs observed with native digestion; and (3) solid phase extraction with 50% MeCN elution prior to LC-MS/MS analysis to ensure effective mAb removal. A 50 cm long nanoflow charged surface hybrid column was also packed to allow for higher sample load for increased sensitivity. Our workflow has increased the sensitivity for HCP identification by 10- to 100-fold over previous reports and showed the robustness as low as 0.1 ppm for identifying HCPs (34.5 to 66.2 kDa MW). The method capability was further confirmed by consistently identifying85% of 48 UPS-1 proteins (0.10 to 1.34 ppm, 6.3 to 82.9 kDa MW) in a monoclonal antibody (mAb) and the largest number (746) of mouse proteins from NIST mAb reported to date by a single analysis. Our work has filled a significant gap in HCP analysis for detecting and demonstrating HCP clearance, in particular, extremely low-level hydrolases in drug process development.

Background\ud A wide range of reviews have demonstrated the effectiveness and tolerability of Virtual Reality (VR) in a range of clinical areas and subpopulations. However, no previous review has explored the current maturity, acceptability, tolerability, and effectiveness of VR with intensive care patients.\ud \ud Aims\ud To identify the range of uses of VR for intensive care patients, classify their current phase of development, effectiveness, acceptability, and tolerability.\ud \ud Methods\ud A scoping review was conducted. A multi-database search was undertaken (inception to January 2021). Any type of study which examined the use of VR with the target application population of intensive care patients were included. Screening, data extraction, and assessment of quality were undertaken by a single reviewer. A meta-analysis and a descriptive synthesis were undertaken.\ud \ud Results\ud Six hundred and forty-seven records were identified, after duplicate removal and screening 21 studies were included (weak quality). The majority of studies for relaxation, delirium, and Post Traumatic Stress Disorder (PTSD) were at the early stages of assessing acceptability, tolerability, and initial clinical efficacy. Virtual Reality for relaxation and delirium were well-tolerated with completion rates of target treatment of 73.6%, (95% CI:51.1%-96%, I2 = 98.52%) 52.7% (95% CI:52.7%-100%, I2 = 96.8%). The majority of reasons for non-completion were due to external clinical factors. There were some potential benefits demonstrated for the use of VR for relaxation, delirium, and sleep.\ud \ud Conclusion\ud Virtual Reality for intensive care is a new domain of research with the majority of areas of application being in the early stages of development. There is great potential for the use of VR in this clinical environment. Further robust assessment of effectiveness is required before any clinical recommendations can be made.

Virtual reality (VR) as a digital technology has developed rapidly, becoming more realistic, portable, sensory and easier to navigate. Although studies have found VR to be effective for many clinical applications, patients and clinicians have described several barriers to the successful implementation of this technology. To remove barriers for implementation of VR in health care, a greater understanding is needed of how VR can integrate into clinical environments, particularly complex settings such as an intensive care unit.This study aimed to explore the perceived barriers and facilitators for the implementation of VR exposure therapy for intensive care patients and clinical staff.A qualitative study using an Interpretative Description approach was undertaken. Semi-structured focus groups were conducted with 13 participants: nine patients and four health care professionals. Focus groups explored barriers and facilitators of using virtual reality (VR) exposure therapy in intensive care. Thematic analysis was employed to produce codes and themes.In total, eight themes describing the perceived barriers and facilitators to implementing VR exposure therapy were identified. Four themes related to the perceived barriers of implementing VR exposure therapy in intensive care were identified: psychological, sensory, environmental and staff competency and confidence. There were a further four themes related to the perceived facilitators to the implementation of VR exposure therapy: staff training, patient capacity, orientation to technology and support during the intervention.This study identified novel barriers and facilitators that could be expected when implementing VR exposure therapy for patients' post-intensive care unit stay. The findings suggest that psychological barriers of fear and apprehension were expected to provoke patient avoidance of exposure therapy. Perceived barriers for staff focused on preparedness to deliver the VR exposure therapy and a lack of technological competence. Both patients and staff stated that a comprehensive induction, orientation and training could facilitate VR exposure therapy, improving engagement.This study has identified that with appropriate staff training, resources, and integration into current patient care pathways, VR exposure therapy may be a valuable intervention to support patient recovery following critical illness. Prior to undertaking VR exposure therapy, patients often need reassurance that side-effects can be managed, and that they can easily control their virtual exposure experience.

PurposeThis study aims to investigate practice changes among Southern and Northern California's radiation oncology centers during the COVID-19 pandemic.MethodsOn the online survey platform SurveyMonkey, we designed 10 survey questions to measure changes in various aspects of medical physics practice. The questions covered patient load and travel rules; scopes to work from home; new protocols to reduce corona virus disease-2019 (COVID-19) infection risk; availability of telemedicine; and changes in fractionation schedules and/or type of treatment plans. We emailed the survey to radiation oncology centers throughout Northern and Southern California, requesting one completed survey per center. All responses were anonymized, and data were analyzed using both qualitative and quantitative research methods.ResultsAt the end of a 4-month collection period (July 2, 2021 to October 11, 2021), we received a total of 61 responses throughout Southern and Northern California. On average, 4111 patients were treated per day across the 61 centers. New COVID-19-related department and hospital policies, along with hybrid workflow changes, infectious control policies, and changes in patient load have been reported. Results also showed changes in treatment methods during the pandemic, such as increased use of telemedicine, hypofractionation for palliative, breast cancer, and prostate cancer cases; and simultaneous boosts, compared to sequential boosts.ConclusionOur California radiation oncology center population study shows changes in various aspects of radiation oncology practices during the COVID-19 pandemic. This study serves as a pilot study to identify possible correlations and new strategies that allow radiation oncology centers to continue providing quality patient care while ensuring the safety of both staff and patients.

Netherton syndrome is a rare but severe autosomal recessive disorder with dominant impaired skin barrier function, caused by mutations in the SPINK5 (serine protease inhibitor Kazal-type 5) gene, which encodes LEKTI (lymphoepithelial Kazal-type-related inhibitor).To establish a murine model of Netherton syndrome based on CRISPR/Cas9 gene editing technology.Spink5-sgRNA was designed to target exon 3 of the mouse Spink5 gene. Cas9 mRNA and sgRNA were microinjected into the zygotes of C57BL/6J mice. Spink5 homozygous knockout mice were born from a heterozygous intercross, and the phenotype of these mice was compared with wild-type regarding gross morphology, histopathology and immunofluorescent detection of LEKTI.Following microinjection of zygotes using the CRISPR/Cas9 system, sequencing demonstrated a 22-bp deletion at exon 3 of the mouse Spink5 gene. Histological investigation revealed complete detachment of the stratum corneum from the underlying granular layer and an absence of LEKTI in skin from Spink5 homozygous knockout mice.The 22-bp deleted Spink5 transgenic mouse model demonstrates the clinical phenotype and genotype of human Netherton syndrome, representing a useful tool for future gene correction and skin barrier/inflammation studies.

Alloreactivity can drive autoimmune syndromes. After allogeneic hematopoietic stem cell transplantation (allo-HCT) chronic graft-versus-host disease (cGVHD), a B cell-mediated autoimmune-like syndrome, commonly occurs. Because donor-derived B cells continually develop under selective pressure from host alloantigens, aberrant B Cell Receptor (BCR)-activation and IgG production can emerge and contribute to cGVHD pathobiology. To better understand molecular programing of B cells under selective pressure of alloantigens, we performed scRNA-Seq analysis on high numbers of purified B cells from allo-HCT patients. An unsupervised analysis revealed 10 clusters, distinguishable by signature genes for maturation, activation and memory. We found striking transcriptional differences in the memory B cell compartment after allo-HCT compared to healthy or infected individuals. To identify intrinsic properties when B-cell tolerance is lost after allo-HCT, we then assessed clusters for differentially expressed genes (DEGs) between patients with vs. without autoimmune-like manifestations (Active cGVHD vs. No cGVHD, respectively). DEGs were found in Active cGVHD in both naive and BCR-activated clusters, suggesting functional diversity. Some DEGs were also differentially expressed across most clusters, suggesting common molecular programs that may promote B cell plasticity. Our study of human allo-HCT and cGVHD provides new understanding of B-cell memory in the face of chronic alloantigen stimulation.

Some G protein-coupled receptor (GPCR) ligands act as “biased agonists” that preferentially activate specific signaling transducers over others. Although GPCRs are primarily found at the plasma membrane, GPCRs can traffic to and signal from many subcellular compartments. Here, we determine that differential subcellular signaling contributes to the biased signaling generated by three endogenous ligands of the GPCR CXC chemokine receptor 3 (CXCR3). The signaling profile of CXCR3 changes as it traffics from the plasma membrane to endosomes in a ligand-specific manner. Endosomal signaling is critical for biased activation of G proteins, β-arrestins, and extracellular-signal-regulated kinase (ERK). In CD8 + T cells, the chemokines promote unique transcriptional responses predicted to regulate inflammatory pathways. In a mouse model of contact hypersensitivity, β-arrestin-biased CXCR3-mediated inflammation is dependent on receptor internalization. Our work demonstrates that differential subcellular signaling is critical to the overall biased response observed at CXCR3, which has important implications for drugs targeting chemokine receptors and other GPCRs.

Corneal nerve fibers provide sensation and maintain the epithelial renewal process. Insufficient corneal innervation can cause neurotrophic keratopathy. Here, topically delivered tacrolimus is evaluated for its therapeutic potential to promote corneal reinnervation in rats.A compartmentalized neuronal cell culture was used to determine the effect of locally delivered tacrolimus on sensory axon regeneration in vitro. The regenerating axons but not the cell bodies were exposed to tacrolimus (50 ng/mL), nerve growth factor (50 ng/mL), or a vehicle control. Axon area and length were measured after 48 hours. Then, a biodegradable nanofiber drug delivery system was fabricated via electrospinning of a tacrolimus-loaded polycarbonate-urethane polymer. Biocompatibility, degradation, drug biodistribution, and therapeutic effectiveness were tested in a rat model of neurotrophic keratopathy induced by stereotactic trigeminal nerve ablation.Sensory neurons whose axons were exposed to tacrolimus regenerated significantly more and longer axons compared to vehicle-treated cultures. Trigeminal nerve ablation in rats reliably induced corneal denervation. Four weeks after denervation, rats that had received tacrolimus topically showed similar limbal innervation but a significantly higher nerve fiber density in the center of the cornea compared to the non-treated control. Topically applied tacrolimus was detectable in the ipsilateral vitreal body, the plasma, and the ipsilateral trigeminal ganglion but not in their contralateral counterparts and vital organs after 4 weeks of topical release.Locally delivered tacrolimus promotes axonal regeneration in vitro and corneal reinnervation in vivo with minimal systemic drug exposure.Topically applied tacrolimus may provide a readily translatable approach to promote corneal reinnervation.

Treatment options for antibody-mediated rejection (AMR) are limited. Recent studies have shown that inhibition of interleukin-6 (IL-6)/interleukin-6 receptor (IL-6R) signaling can reduce inflammation and slow AMR progression.We report our experience using monthly tocilizumab (anti-IL6R) in 25 pediatric renal transplant recipients with AMR, refractory to IVIg/Rituximab. From January 2013 to June 2019, a median (IQR) of 12 (6.019.0) doses of tocilizumab were given per patient. Serial assessments of renal function, biopsy findings, and HLA DSA (by immunodominant HLA DSA [iDSA] and relative intensity score [RIS]) were performed.Median (IQR) time from transplant to AMR was 41.4 (24.367.7) months, and time from AMR to first tocilizumab was 10.6 (8.317.6) months. At median (IQR) follow up of 15.8 (8.435.7) months post-tocilizumab initiation, renal function was stable except for 1 allograft loss. There was no significant decrease in iDSA or RIS. Follow up biopsies showed reduction in peritubular capillaritis (p = .015) and C4d scoring (p = .009). The most frequent adverse events were cytopenias.Tocilizumab in pediatric patients with refractory AMR was well tolerated and appeared to stabilize renal function. The utility of tocilizumab in the treatment of AMR in this population should be further explored.

Natural killer (NK) cells are an important defender against infections and tumors. Their function is regulated by the balance of inhibitory and activating receptors. Among all inhibitory NK receptors: killer immunoglobulin-like receptors (KIR) and CD94/NKG2A recognize human leukocyte antigen (HLA) Class I molecules, allowing NK cells to be 'licensed' to avoid autoreactivity, but be fully functional at the same time. Licensed NK cells can target malignant cells with altered or downregulated/missing 'self' antigens. NK cell attacking malignant cells is one of the mechanisms of graft-versus-leukemia (GVL) effect. Numerous studies have demonstrated that NK cells improve hematopoietic stem cell transplantation (HCT) survival by reducing relapse mortality through GVL effect. Therapeutic strategies, such as adoptive alloreactive NK cell transfer, CAR-NK cells, antibodies against NKG2A and KIR2DL1-3, have been utilized to treat hematological malignancies in HCT. In this review, NK cell functions, NK cell receptors and ligands, as well as common alloreactive NK donor selection algorithms for patients with hematological malignancies in the setting of HCT are discussed. The goal of this review is to provide insights on the controversial results and provide better understanding and resources on how to perform alloreactive donor NK cell selection in HCT.

Since the first allogeneic hematopoietic stem cell transplantation (HCT) was performed by Dr. E. Donnall Thomas in 1957, the field has advanced with new stem cell sources, immune suppressive regimens, and transplant protocols. Stem cells may be collected from bone marrow, peripheral or cord blood from an identical twin, a sibling, or a related or unrelated donor, which can be human leukocyte antigen (HLA) matched, mismatched, or haploidentical. Although HLA matching is one of the most important criteria for successful allogeneic HCT (allo-HCT) to minimize graft vs host disease (GVHD), prevent relapse, and improve overall survival, the novel immunosuppressive protocols for GVHD prophylaxis offered improved outcomes in haploidentical HCT (haplo-HCT), expanding donor availability for the majority of HCT candidates. These immunosuppressive protocols are currently being tested with the HLA-matched and mismatched donors to improve HCT outcomes further. In addition, fine-tuning the DPB1 mismatching and discovering the B leader genotype and mismatching may offer further optimization of donor selection and transplant outcomes. While the decision about a donor type largely depends on the patient's characteristics, disease status, and the transplant protocols utilized by an individual transplant center, there are general approaches to donor selection dictated by donor-recipient histocompatibility and the urgency for HCT. This review highlights recent advances in understanding critical factors in donor selection strategies for allo-HCT. It uses clinical vignettes to demonstrate the importance of making timely decisions for HCT candidates.

Diabetes is by far, the most common cause of neuropathy, inducing neurodegeneration of terminal sensory nerve fibers associated with loss of sensation, paresthesia, and persistent pain. Foretinib prevents die-back degeneration in cultured sensory and sympathetic neurons by rescuing mitochondrial activity and has been proven safe in prospective clinical trials. Here we aimed at investigating a potential neuroprotective effect of Foretinib in experimental diabetic neuropathy. A mouse model of streptozotocin induced diabetes was used that expresses yellow fluorescent protein (YFP) in peripheral nerve fibers under the thy-1 promoter. Streptozotocin-injected mice developed a stable diabetic state (blood glucose > 270 mg/dl), with a significant reduction of intraepidermal nerve fiber density by 25% at 5 weeks compared to the non-diabetic controls. When diabetic mice were treated with Foretinib, a significantly greater volume of the cutaneous nerve fibers (67.3%) in the plantar skin was preserved compared to vehicle treated (37.8%) and non-treated (44.9%) diabetic mice while proximal nerve fiber morphology was not affected. Our results indicate a neuroprotective effect of Foretinib on cutaneous nerve fibers in experimental diabetic neuropathy. As Foretinib treated mice showed greater weight loss compared to vehicle treated controls, future studies may define more sustainable treatment regimen and thereby may allow patients to take advantage of this neuroprotective drug in chronic neurodegenerative diseases like diabetic neuropathy.

The ex vivo experimentation with surgically discarded human skin represents a unique methodology amenable for mechanism and pharmacologic agent studies without the involvement of human subjects. Here, we describe a protocol that includes preparation, culture, and stimulation of human skin explants, and subsequent analyses by quantitative reverse transcription PCR and immunostaining. This protocol may also be applied for ex vivo studies of murine skin, reducing animal numbers and potentially harmful treatments. In our hands, this protocol has been used for wound healing, viral infection, and hair growth–related studies. Graphical abstract: Cartoon of explant skin culture. Skin explant sits on top of a gelatin surgical sponge saturated with culture medium at an air–liquid interface.

ObjectiveTo investigate serum neurofilament light protein (sNfL) levels in patients with spinocerebellar ataxia type 3 (SCA3) and to determine whether they are associated with disease severity.MethodsThis cross-sectional study enrolled 185 healthy controls and 235 ATXN3 mutation carriers (17 asymptomatic stage, 20 preclinical stage, and 198 ataxic stage). We measured sNfL levels with the single molecule array (Simoa) platform. Clinical disease severity was assessed using the Scale of Assessment and Rating of Ataxia (SARA) and the Inventory of Nonataxia Signs (INAS). In a subgroup of 50 ataxic stage patients, we further evaluated the gray matter volume and the integrity of white matter fibers by MRI.ResultssNfL concentrations were elevated in asymptomatic, preclinical, and ataxic ATXN3 mutation carriers compared to controls (12.18 [10.20–13.92], 21.84 [18.37–23.45], 36.06 [30.04–45.90], and 8.24 [5.92–10.84] pg/mL, median [interquartile range], respectively, p < 0.001). sNfL correlated with SARA (r = 0.406, 95% confidence interval [CI] 0.284–0.515, p < 0.0001) and INAS (r = 0.375, 95% CI 0.250–0.487, p < 0.0001), and remained significant after adjustment for age and CAG repeats. In addition, we observed negative correlations of the sNfL with gray matter volume in the left precentral gyrus and the left paracentral lobule as well as with the mean diffusivity in widespread white matter tracts.ConclusionOur results demonstrate that sNfL levels are increased in SCA3 and are associated with clinical disease severity, which supports sNfL as a biomarker for disease severity in SCA3.Classification of evidenceThis study provides Class II evidence that in patients with SCA3, sNfL elevations are associated with clinical disease severity.

Breast cancer is the second most common malignancy affecting women and accounts for 15% of cancer-related mortality globally. Over 70% of breast cancers are hormone receptor (HR) positive (estrogen receptor and/or progesterone receptor positive) and human epidermal growth factor receptor 2 negative (HER2-). HR+HER2- breast cancers are poorly responsive to chemotherapy and recurrence and mortality remain high for patients with locally advanced disease. Thus, more therapeutic strategies are needed. Dendritic cell (DC) activation is an important pathway for T cell priming and subsequent improvements in immunosurveillance. In this study, we determined the ability of DC-targeted therapy with agonistic CD40 antibody to restore anti-tumor immunologic function in a murine orthotopic HR+HER2- breast cancer model. The Brpkp110 cell line was injected orthotopically into the abdominal mammary glands of 8 to 10-week-old C57BL/6 mice. Tumor-bearing mice treated with agonistic CD40 antibody (FGK4.5, 100 ug intraperitoneal) demonstrated decreased tumor growth and increased intratumoral CD8 T cells at 2 weeks. Both intratumoral DCs and tumor-associated macrophages in mice treated with agonistic CD40 were found to upregulate PD-L1 and CD40. CD8 T cell depletion abrogated the anti-tumor effect of agonistic CD40. Our data suggest that agonistic CD40 therapy may be a viable strategy for tumor inhibition and activation of anti-tumor immunity in HR+HER2- breast cancer. Citation Format: Jennifer Zhang, Olivia Lanchoney, Nikhil Joshi, Nune Markosyan, Robert Vonderheide. CD40 agonism inhibits tumor growth in murine hormone receptor positive breast cancer via CD8 T cells [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy; 2022 Oct 21-24; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(12 Suppl):Abstract nr A67.

Fc galactosylation is a critical quality attribute for anti-tumor recombinant immunoglobulin G (IgG)-based monoclonal antibody (mAb) therapeutics with complement-dependent cytotoxicity (CDC) as the mechanism of action. Although the correlation between galactosylation and CDC has been known, the underlying structure–function relationship is unclear. Heterogeneity of the Fc N-glycosylation produced by Chinese hamster ovary (CHO) cell culture biomanufacturing process leads to variable CDC potency. Here, we derived a kinetic model of galactose transfer reaction in the Golgi apparatus and used this model to determine the correlation between differently galactosylated species from CHO cell culture process. The model was validated by a retrospective data analysis of more than 800 historical samples from small-scale and large-scale CHO cell cultures. Furthermore, using various analytical technologies, we discovered the molecular basis for Fc glycan terminal galactosylation changing the three-dimensional conformation of the Fc, which facilitates the IgG1 hexamerization, thus enhancing C1q avidity and subsequent complement activation. Our study offers insight into the formation of galactosylated species, as well as a novel three-dimensional understanding of the structure–function relationship of terminal galactose to complement activation in mAb therapeutics.

This review assesses the effect of electronic nicotine delivery systems (ENDS) on cigarette smoking cessation as compared other types of nicotine replacement therapies.

Inherited cardiomyopathies are among the major causes of heart failure and associated with significant mortality and morbidity. Currently, over 70 genes have been linked to the etiology of various forms of cardiomyopathy, some of which are X-linked. Due to the lack of appropriate cell and animal models, it has been difficult to model these X-linked cardiomyopathies. With the advancement of induced pluripotent stem cell (iPSC) technology, the ability to generate iPSC lines from patients with X-linked cardiomyopathy has facilitated in vitro modelling and drug testing for the condition. Nonetheless, due to the mosaicism of the X-chromosome inactivation, disease phenotypes of X-linked cardiomyopathy in heterozygous females are also usually more heterogeneous, with a broad spectrum of presentation. Recent advancements in iPSC procedures have enabled the isolation of cells with different lyonisation to generate isogenic disease and control cell lines. In this review, we will summarise the current strategies and examples of using an iPSC-based model to study different types of X-linked cardiomyopathy. The potential application of isogenic iPSC lines derived from a female patient with heterozygous Danon disease and drug screening will be demonstrated by our preliminary data. The limitations of an iPSC-derived cardiomyocyte-based platform will also be addressed.

Protein A chromatography is currently the industry gold-standard for monoclonal antibody and Fc-fusion protein purification. The high cost of Protein A, however, makes resin lifetime and resin reuse an important factor for process economics. Typical resin lifetime studies performed in the industry usually examine the effect of resin re-use on binding capacity, yield, and product quality without answering the fundamental question of what is causing the decrease in performance. A two part mechanistic study was conducted in an attempt to decouple the effect of the two possible factors (resin hydrolysis and/or degradation vs. resin fouling) on column performance over lifetime of the most commonly used alkali-stable Protein A resins (MabSelect SuRe and MabSelect SuRe LX). The change in binding capacity as a function of sodium hydroxide concentration (rate of hydrolysis), temperature, and stabilizing additives was examined. Additionally, resin extraction studies and product cycling studies were conducted to determine cleaning effectiveness (resin fouling) of various cleaning strategies. Sodium hydroxide-based cleaning solutions were shown to be more effective at preventing resin fouling. Conversely, cold temperature and the use of stabilizing additives in conjunction with sodium hydroxide were found to be beneficial in minimizing the rate of Protein A ligand hydrolysis. An effective and robust cleaning strategy is presented here to maximize resin lifetime and thereby the number of column cycles for future manufacturing processes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:708-715, 2017.

The spreadsheet file reported herein provides centroid data, descriptive of deuterium uptake, for the FabFragment of NISTmAb (PDB: 5K8A) reference material, as measured by the bottom-up hydrogen-deuterium exchange mass spectrometry (HDX-MS) method. The protein sample was incubated in deuterium-rich solutions under uniform pH and salt concentrations between 3.6 oC and 25.4 oC for seven intervals ranging over (0 to 14,400) s plus a ∞pseudo s control. The deuterium content of peptic peptide fragments were measured by mass spectrometry. These data were reported by fifteen laboratories, which conducted the measurements using orbitrap and Q-TOF mass spectrometers. The cohort reported ≈ 78,900 centroids for 430 proteolytic peptide sequences of the heavy and light chains of NISTmAb, providing nearly 100 % coverage. In addition, some groups reported ≈ 10,900 centroid measurements for 77 peptide sequences of the Fc fragment. The instrumentation and physical and chemical conditions under which these data were acquired are documented.

Purpose Multimodal analgesia with acetaminophen and/or nonsteroidal anti-inflammatory drugs is recommended for the treatment of postoperative pain. Although oral fixed-dose combinations (FDCs) are available, parenteral administration may be clinically justified. The goal of this study was to investigate the clinical efficacy and safety of an intravenous FDC of ibuprofen and acetaminophen after bunionectomy. Methods This study was a prospective, randomized, double-blind, multicenter, placebo-controlled factorial clinical trial conducted at 2 clinical research centers in the United States between November 2016 and June 2017. Eligible patients (male and female subjects, aged 18–65 years, reporting pain intensity levels ≥40 mm on a 100-mm visual analog scale (VAS) after distal, first metatarsal bunionectomy) were randomized (3:3:3:2) to receive the FDC (ibuprofen 300 mg + acetaminophen 1000 mg), ibuprofen 300 mg, acetaminophen 1000 mg, or placebo (vehicle), administered as 15-minute intravenous infusions every 6 hours for 48 hours. The primary efficacy end point was the time-adjusted sum of pain intensity differences from baseline over 48 hours (SPID48). In addition to VAS pain intensity scores, pain relief scores, time to perceptible and meaningful pain relief, the use of rescue medication, and participant's global evaluations of the study drug were recorded. Adverse events occurring during the 48-hour treatment period were included in the safety analysis. Findings A total of 276 participants were enrolled; most were female (82%), the mean age was 42.4 years, and the median baseline VAS was 67 mm, indicating moderate to severe pain. SPID48 was significantly higher for the FDC (23.4 [2.5] mm) than for ibuprofen (9.5 [2.5] mm), acetaminophen (10.4 [2.5] mm), and placebo (−1.3 [3.1] mm; all, P Implications The study found that repeated administration of an intravenous FDC of ibuprofen and acetaminophen provided statistically significant improvement in SPID48 over comparable doses of either monotherapy without an increase in adverse events. ClinicalTrials.gov identifier: NCT02689063 .

With cell culture titers and productivity increasing in the last few years, pressure has been placed on downstream purification to look at alternative strategies to meet the demand of biotech products with high dose requirements. Even when the upstream process is not continuous (perfusion based), adopting a more productive and/or continuous downstream process can be of significant advantage. Due to the recent trend in exploring continuous processing options for biomolecules, several enabling technologies have been assessed at Biogen. In this paper, we evaluate the capability of one of these technologies to streamline and improve our downstream mAb purification platform. Current conventional downstream polishing steps at Biogen are operated in flow-through mode to achieve higher loadings while maintaining good selectivity. As titers increase, this would result in larger columns and larger intermediate product pool holding tanks. A semicontinuous downstream process linking the second and third chromatography steps in tandem can reduce/eliminate intermediate holding tanks, reduce overall processing time, and combine unit operations to reduce validation burdens. A pool-less processing technology utilizing inline adjustment functionality was evaluated to address facility fit challenges for three high titer mAbs. Two different configurations of polishing steps were examined: (i) anion exchange and hydrophobic interaction and (ii) anion exchange and mixed mode chromatography. Initial laboratory scale proof of concept studies showed comparable performance between the batch purification process and the pool-less process configuration.

Long noncoding RNAs (lncRNAs) control diverse biological processes. Here the lncRNA SPRY4-IT1 is linked with TJ expression and subsequent gut permeability., Epithelial cells line the intestinal mucosa and form an important barrier to a wide array of noxious substances in the lumen. Disruption of the barrier integrity occurs commonly in various pathologies. Long noncoding RNAs (lncRNAs) control diverse biological processes, but little is known about the role of lncRNAs in regulation of the gut permeability. Here we show that the lncRNA SPRY4-IT1 regulates the intestinal epithelial barrier function by altering expression of tight junction (TJ) proteins. SPRY4-IT1 silencing led to dysfunction of the epithelial barrier in cultured cells by decreasing the stability of mRNAs encoding TJ proteins claudin-1, claudin-3, occludin, and JAM-1 and repressing their translation. In contrast, increasing the levels of SPRY4-IT1 in the intestinal mucosa protected the gut barrier in mice exposed to septic stress by increasing the abundance of TJ proteins. SPRY4-IT1 directly interacted with TJ mRNAs, and this process was enhanced through the association with the RNA-binding protein HuR. Of interest, the intestinal mucosa from patients with increased gut permeability exhibited a decrease in the levels of SPRY4-IT1. These findings highlight a novel role for SPRY4-IT1 in controlling the intestinal epithelial barrier and define a mechanism by which SPRY4-IT1 modulates TJ expression by altering the stability and translation of TJ mRNAs.

© 2017 American Chemical Society. Bispecific antibodies have great potential to be the next-generation biotherapeutics due to their ability to simultaneously recognize two different targets. Compared to conventional monoclonal antibodies, knob-into-hole bispecific antibodies face unique challenges in production and characterization due to the increase in variant possibilities, such as homodimerization in covalent and noncovalent forms. In this study, a storage- and pH-sensitive hydrophobic interaction chromatography (HIC) profile change was observed for the hole-hole homodimer, and the multiple HIC peaks were explored and shown to be conformational isomers. We combined traditional analytical methods with hydrogen/deuterium exchange mass spectrometry (HDX MS), native mass spectrometry, and negative-staining electron microscopy to comprehensively characterize the hole-hole homodimer. HDX MS revealed conformational changes at the resolution of a few amino acids overlapping the CH2-CH3 domain interface. Conformational heterogeneity was also assessed by HDX MS isotopic distribution. The hole-hole homodimer was demonstrated to adopt a more homogeneous conformational distribution during storage. This conformational change is likely caused by a lack of CH3 domain dimerization (due to the three "hole" point mutations), resulting in a unique storage- and pH-dependent conformational destabilization and refolding of the hole-hole homodimer Fc. Compared with the hole-hole homodimer under different storage conditions, the bispecific heterodimer, guided by the knob-into-hole assembly, proved to be a stable conformation with homogeneous distribution, confirming its high quality as a desired therapeutic. Functional studies by antigen binding and neonatal Fc receptor (FcRn) binding correlated very well with the structural characterization. Comprehensive interpretation of the results has provided a better understanding of both the homodimer variant and the bispecific molecule.

Members of the ISWI family of chromatin remodelers mobilize nucleosomes to control DNA accessibility and, in some cases, are required for recovery from DNA damage. However, it remains poorly understood how the non-catalytic ISWI subunits BAZ1A and BAZ1B might contact chromatin to direct the ATPase SMARCA5. Here, we find that the plant homeodomain of BAZ1A, but not that of BAZ1B, has the unusual function of binding DNA. Furthermore, the BAZ1A bromodomain has a non-canonical gatekeeper residue and binds relatively weakly to acetylated histone peptides. Using CRISPR-Cas9-mediated genome editing we find that BAZ1A and BAZ1B each recruit SMARCA5 to sites of damaged chromatin and promote survival. Genetic engineering of structure-designed bromodomain and plant homeodomain mutants reveals that reader modules of BAZ1A and BAZ1B, even when non-standard, are critical for DNA damage recovery in part by regulating ISWI factors loading at DNA lesions and supporting transcriptional programs required for survival., ISWI chromatin remodelers regulate DNA accessibility and have been implicated in DNA damage repair. Here, the authors uncover functions, in response to DNA damage, for the bromodomain of the ISWI subunit BAZ1B and for the non-canonical PHD and bromodomain modules of the paralog BAZ1A.

Sex differences and circadian variation are two major factors that affect the expression of drug-processing genes. This study aimed to examine sex differences in the circadian variation of hepatic cytochrome P450 (Cyp) genes and corresponding nuclear receptors. Adult mice were acclimated to environmentally controlled facilities for 2 wks, and livers were collected every 4 h during a 24-h period. Total RNA and protein were isolated and subjected to real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis. The mRNA expression of the aryl hydrocarbon receptor (AhR) and AhR-regulated Cyp1a1 and Cyp1a2 were higher in females and higher during the light phase. The mRNA expression of constitutive and rostane receptor (CAR) and CYP2B10 protein was female-predominant and higher in the dark phase. Pregnane X receptor (PXR) peaked around 18:00 h, but PXR-regulated Cyp3a11 and Cyp3a25 were higher at 10:00 h, without apparent sex dimorphism at protein levels. Peroxisome proliferator-activated receptor-α (PPARα), Cyp4a10, and Cyp4a14 were higher in females and peaked between 14:00 and 18:00 h. The mRNA levels of farnesoid X receptor (FXR), Cyp7a1, and Cyp27a1 peaked around 18:00 h and CYP7A1 protein was higher during the dark phase and higher in females. Cyp7b1(male-predominant) and Cyp2a4 (female-predominant) both showed circadian variation. Circadian variation of hepatic clock genes such as nuclear receptor Rev-erbα, cryptochrome 1 (Cry1), and brain muscle ARNT-like protein 1 (Bmal1) showed distinct patterns. Sex differences and circadian rhythmicity of Cyp genes and corresponding nuclear receptors exist in mouse liver that could impact xenobiotic metabolism and toxicity at different times of the day.

Simtuzumab is a monoclonal antibody against the lysl-oxidase like-2 (LOXL2) enzyme. 544 subjects with idiopathic pulmonary fibrosis (IPF) were randomized, 1:1, to either simtuzumab 125 mg subcutaneously once per week or placebo. The study was prematurely stopped because of lack of efficacy for the primary endpoints of progression free survival (PFS) in both the intent-to-treat (ITT) group and in a pre-specified subgroup with elevated serum LOXL2 levels. PFS was defined as time to death or a 10% decrease in forced vital capacity (FVC) percent predicted. Table 1 compares the primary and key secondary efficacy endpoints. For PFS in the ITT group, the hazard ratio (HR) (95% CI) for treatment benefit was 1.13 (0.88 to 1.45). For PFS in the high LOXL2 group, the HR was 1.03 (0.74-1.43). There were no significant differences in patterns of adverse events between simtuzumab and placebo treated subjects. Simtuzumab was not shown to be effective in treating IPF.

The heterogeneity in therapeutic antibodies arising from buried unpaired cysteines has not been well studied. This paper describes the characterization of two unpaired cysteines in a recombinant humanized IgG1 monoclonal antibody (referred to as mAb A). The reversed-phase high-performance liquid chromatography (RP-HPLC) analysis of mAb A samples showed three distinct peaks, indicating the presence of three species. The heterogeneities observed in the RP-HPLC have been determined to arise from unpaired cysteines (Cys-22 and Cys-96) that are buried in the V(H) domain. The Fab containing free thiols (referred to as "free-thiol Fab") and the Fab containing the disulfide (referred to as "intact Fab") of mAb A were generated through limited Lys-C digestion and purified with an ion exchange chromatography method. The binding of free-thiol Fab and intact Fab to its antigen was measured in a cell-based binding assay and an enzyme linked immunosorbent assay. The unpaired cysteines in the Fab of mAb A were found to have no significant impact on the binding to its target. Consistent with these Fab binding data, the enriched intact mAb A containing free thiols was determined to be fully active in a potency assay. The data reported here demonstrate that the redox status of cysteines is potentially a major source of heterogeneity for an antibody.

The conductive polymer polyaniline is blended with conventional industrial thermoplastics in order to obtain an electrically conductive polymer blend with adequate mechanical properties. Processing these polyblends into foams yields a porous conductive material that exhibits immense application potential such as dynamic separation media and low-density electrostatic discharge protection. In the current study, the morphology of a thermally processable blend consisting of an electrically conductive polyaniline-dodecylbenzene sulfonic acid complex and poly(methyl methacrylate) is explored using a two-phase batch foaming setup. The effect of blend composition and processing parameters on the resulting porous morphology is investigated. The impact of the underlying microstructure and blend composition on the frequency dependent electrical conductivity is elucidated using multiple linear regression and a model is proposed. Finally, dielectric analysis is utilized to experimentally identify the critical dispersion frequency of an unfoamed blend composition near the percolation threshold.