Chaperone-mediated MHC-I peptide exchange in antigen presentation.

This work focuses on molecules that are encoded by the major histocompatibility complex (MHC) and that bind self-, foreign- or tumor-derived peptides and display these at the cell surface for recognition by receptors on T lymphocytes (T cell receptors, TCR) and natural killer (NK) cells. The past few decades have accumulated a vast knowledge base of the structures of MHC molecules and the complexes of MHC/TCR with specificity for many different peptides. In recent years, the structures of MHC-I molecules complexed with chaperones that assist in peptide loading have been revealed by X-ray crystallography and cryogenic electron microscopy. These structures have been further studied using mutagenesis, molecular dynamics and NMR approaches. This review summarizes the current structures and dynamic principles that govern peptide exchange as these relate to the process of antigen presentation.

Experimental Structures of Antibody/MHC-I Complexes Reveal Details of Epitopes Overlooked by Computational Prediction.

mAbs to MHC class I (MHC-I) molecules have proved to be crucial reagents for tissue typing and fundamental studies of immune recognition. To augment our understanding of epitopic sites seen by a set of anti-MHC-I mAb, we determined X-ray crystal structures of four complexes of anti-MHC-I Fabs bound to peptide/MHC-I/ß2-microglobulin (pMHC-I). An anti-H2-Dd mAb, two anti-MHC-I α3 domain mAbs, and an anti-ß2-microglobulin mAb bind pMHC-I at sites consistent with earlier mutational and functional experiments, and the structures explain allelomorph specificity. Comparison of the experimentally determined structures with computationally derived models using AlphaFold Multimer showed that although predictions of the individual pMHC-I heterodimers were quite acceptable, the computational models failed to properly identify the docking sites of the mAb on pMHC-I. The experimental and predicted structures provide insight into strengths and weaknesses of purely computational approaches and suggest areas that merit additional attention.

Experimental structures of antibody/MHC-I complexes reveal details of epitopes overlooked by computational prediction.

Monoclonal antibodies (mAb) to major histocompatibility complex class I (MHC-I) molecules have proved to be crucial reagents for tissue typing and fundamental studies of immune recognition. To augment our understanding of epitopic sites seen by a set of anti-MHC-I mAb, we determined X-ray crystal structures of four complexes of anti-MHC-I antigen-binding fragments (Fab) bound to peptide/MHC-I/ß2m (pMHC-I). An anti-H2-Dd mAb, two anti-MHC-I α3 domain mAb, and an anti-ß2-microglobulin (ß2m) mAb bind pMHC-I at sites consistent with earlier mutational and functional experiments, and the structures explain allelomorph specificity. Comparison of the experimentally determined structures with computationally derived models using AlphaFold Multimer (AF-M) showed that although predictions of the individual pMHC-I heterodimers were quite acceptable, the computational models failed to properly identify the docking sites of the mAb on pMHC-I. The experimental and predicted structures provide insight into strengths and weaknesses of purely computational approaches and suggest areas that merit additional attention.

Visible Light Mediated Co-Catalyzed Isocyanide Insertion with Sulfonyl Azide: Synthesis of Sulfonyl Carbamimidic Azide and Sulfonyl Aminotetrazole via Carbodiimide Intermediate.

Herein, we report an operationally simple and efficient protocol to prepare sulfonyl carbamimidic azide and N-sulfonyl aminotetrazole via Co-catalyzed three component coupling of sulfonyl azide (acts as nitrene source), isocyanide, and TMS-azide at room temperature under visible light. Initially, the carbamimidic azide is formed, which cyclizes only in the presence of base to deliver N-sulfonyl aminotetrazole in very good yields. The sulfonyl aminotetrazole can also be synthesized directly without isolating the carbamimidic azide in the presence of base. The sulfonyl azide is anticipated to generate nitrene and reacts with isocyanide to produce carbodiimide. Subsequent addition of azide (TMS-N3 ) to carbodiimide results in the formation of carbamimidic azide.

SARS-CoV-2 antibodies recognize 23 distinct epitopic sites on the receptor binding domain.

The COVID-19 pandemic and SARS-CoV-2 variants have dramatically illustrated the need for a better understanding of antigen (epitope)-antibody (paratope) interactions. To gain insight into the immunogenic characteristics of epitopic sites (ES), we systematically investigated the structures of 340 Abs and 83 nanobodies (Nbs) complexed with the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein. We identified 23 distinct ES on the RBD surface and determined the frequencies of amino acid usage in the corresponding CDR paratopes. We describe a clustering method for analysis of ES similarities that reveals binding motifs of the paratopes and that provides insights for vaccine design and therapies for SARS-CoV-2, as well as a broader understanding of the structural basis of Ab-protein antigen (Ag) interactions.

Chaperone function in antigen presentation by MHC class I molecules-tapasin in the PLC and TAPBPR beyond.

Peptide loading of MHC-I molecules plays a critical role in the T cell response to infections and tumors as well as to interactions with inhibitory receptors on natural killer (NK) cells. To facilitate and optimize peptide acquisition, vertebrates have evolved specialized chaperones to stabilize MHC-I molecules during their biosynthesis and to catalyze peptide exchange favoring high affinity or optimal peptides to permit transport to the cell surface where stable peptide/MHC-I (pMHC-I) complexes are displayed and are available for interaction with T cell receptors and any of a host of inhibitory and activating receptors. Although components of the endoplasmic reticulum (ER) resident peptide loading complex (PLC) were identified some 30 years ago, the detailed biophysical parameters that govern peptide selection, binding, and surface display have recently been understood better with advances in structural methods including X-ray crystallography, cryogenic electron microscopy (cryo-EM), and computational modeling. These approaches have provided refined mechanistic illustration of the molecular events involved in the folding of the MHC-I heavy chain, its coordinate glycosylation, assembly with its light chain, ß2-microglobulin (ß2m), its association with the PLC, and its binding of peptides. Our current view of this important cellular process as it relates to antigen presentation to CD8+ T cells is based on many different approaches: biochemical, genetic, structural, computational, cell biological, and immunological. In this review, taking advantage of recent X-ray and cryo-EM structural evidence and molecular dynamics simulations, examined in the context of past experiments, we attempt a dispassionate evaluation of the details of peptide loading in the MHC-I pathway. By critical evaluation of several decades of investigation, we outline aspects of the peptide loading process that are well-understood and indicate those that demand further detailed investigation. Further studies should contribute not only to basic understanding, but also to applications for immunization and therapy of tumors and infections.

SARS-CoV-2 antibodies recognize 23 distinct epitopic sites on the receptor binding domain.

The COVID-19 pandemic and SARS-CoV-2 variants have dramatically illustrated the need for a better understanding of antigen (epitope)-antibody (paratope) interactions. To gain insight into the immunogenic characteristics of epitopic sites (ES), we systematically investigated the structures of 340 Abs and 83 nanobodies (Nbs) complexed with the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein. We identified 23 distinct ES on the RBD surface and determined the frequencies of amino acid usage in the corresponding CDR paratopes. We describe a clustering method for analysis of ES similarities that reveals binding motifs of the paratopes and that provides insights for vaccine design and therapies for SARS-CoV-2, as well as a broader understanding of the structural basis of Ab-protein antigen (Ag) interactions.

Structural mechanism of tapasin-mediated MHC-I peptide loading in antigen presentation.

Loading of MHC-I molecules with peptide by the catalytic chaperone tapasin in the peptide loading complex plays a critical role in antigen presentation and immune recognition. Mechanistic insight has been hampered by the lack of detailed structural information concerning tapasin-MHC-I. We present here crystal structures of human tapasin complexed with the MHC-I molecule HLA-B*44:05, and with each of two anti-tapasin antibodies. The tapasin-stabilized peptide-receptive state of HLA-B*44:05 is characterized by distortion of the peptide binding groove and destabilization of the ß2-microglobulin interaction, leading to release of peptide. Movements of the membrane proximal Ig-like domains of tapasin, HLA-B*44:05, and ß2-microglobulin accompany the transition to a peptide-receptive state. Together this ensemble of crystal structures provides insights into a distinct mechanism of tapasin-mediated peptide exchange.

Pharmacomechanical Catheter-Directed Thrombolysis With the Bashir Endovascular Catheter for Acute Pulmonary Embolism: The RESCUE Study.

BACKGROUND: Catheter-directed thrombolysis (CDT) has been associated with rapid recovery of right ventricular (RV) function. The Bashir catheter was developed for enhanced thrombolysis in large vessels such as the pulmonary arteries (PAs) with lower doses of tissue plasminogen activator (tPA). OBJECTIVES: The aim of this study was to evaluate the efficacy and safety of tPA infused using a pharmacomechanical (PM) CDT device called the Bashir endovascular catheter in patients with intermediate-risk acute pulmonary embolism (PE). METHODS: Patients with symptoms of acute PE with computed tomographic evidence of RV dilatation were enrolled. The Bashir catheter was used to deliver 7 mg tPA into each PA over 5 hours. The primary efficacy endpoint was the core laboratory-assessed change in computed tomographic angiography-derived RV/left ventricular (LV) diameter ratio at 48 hours, and the primary safety endpoint was serious adverse events (SAEs) including major bleeding at 72 hours. RESULTS: At 18 U.S. sites, 109 patients were enrolled. The median device placement time was 15 minutes. At 48 hours after PM-CDT, the RV/LV diameter ratio decreased by 0.56 (33.3%; P < 0.0001). PA obstruction as measured by the refined modified Miller index was reduced by 35.9% (P < 0.0001). One patient (0.92%) had 2 SAEs: a retroperitoneal bleed (procedure related) and iliac vein thrombosis (device related). Two other procedure-related SAEs were epistaxis and non-access site hematoma with anemia. CONCLUSIONS: PM-CDT with the Bashir endovascular catheter is associated with a significant reduction in RV/LV diameter ratio and a very low rate of adverse events or major bleeding in patients with intermediate-risk acute PE. The notable finding was a significant reduction in PA obstruction with low-dose tPA. (Recombinant tPA by Endovascular Administration for the Treatment of Submassive PE Using CDT for the Reduction of Thrombus Burden [RESCUE]; NCT04248868).

Chaperones and Catalysts: How Antigen Presentation Pathways Cope With Biological Necessity.

Immune recognition by T lymphocytes and natural killer (NK) cells is in large part dependent on the identification of cell surface MHC molecules bearing peptides generated from either endogenous (MHC I) or exogenous (MHC II) dependent pathways. This review focuses on MHC I molecules that coordinately fold to bind self or foreign peptides for such surface display. Peptide loading occurs in an antigen presentation pathway that includes either the multimolecular peptide loading complex (PLC) or a single chain chaperone/catalyst, TAP binding protein, related, TAPBPR, that mimics a key component of the PLC, tapasin. Recent structural and dynamic studies of TAPBPR reveal details of its function and reflect on mechanisms common to tapasin. Regions of structural conservation among species suggest that TAPBPR and tapasin have evolved to satisfy functional complexities demanded by the enormous polymorphism of MHC I molecules. Recent studies suggest that these two chaperone/catalysts exploit structural flexibility and dynamics to stabilize MHC molecules and facilitate peptide loading.

The Predictive Individual Effect for Survival Data.

BACKGROUND: The call for patient-focused drug development is loud and clear, as expressed in the twenty-first Century Cures Act and in recent guidelines and initiatives of regulatory agencies. Among the factors contributing to modernized drug development and improved health-care activities are easily interpretable measures of clinical benefit. In addition, special care is needed for cancer trials with time-to-event endpoints if the treatment effect is not constant over time. OBJECTIVE: To quantify the potential clinical survival benefit for a new patient, would he/she be treated with the test or control treatment. METHODS: We propose the predictive individual effect which is a patient-centric and tangible measure of clinical benefit under a wide variety of scenarios. It can be obtained by standard predictive calculations under a rank preservation assumption that has been used previously in trials with treatment switching. RESULTS: We discuss four recent Oncology trials that cover situations with proportional as well as non-proportional hazards (delayed treatment effect or crossing of survival curves). It is shown that the predictive individual effect offers valuable insights beyond p-values, estimates of hazard ratios or differences in median survival. CONCLUSION: Compared to standard statistical measures, the predictive individual effect is a direct, easily interpretable measure of clinical benefit. It facilitates communication among clinicians, patients, and other parties and should therefore be considered in addition to standard statistical results.

Perspectives on Virtual (Remote) Clinical Trials as the "New Normal" to Accelerate Drug Development.

Although the digital revolution has transformed many areas of human endeavor, pharmaceutical drug development has been relatively slow to embrace the emerging technologies to enhance efficiency and optimize value in clinical trials. The topic has garnered even greater attention in the face of the coronavirus disease 2019 (COVID-19) outbreak, which has caused unprecedented disruption in the conduct of clinical trials and presented considerable challenges and opportunities for clinical trialists and data analysts. In this paper, we highlight the potential opportunity with virtual or digital clinical trials as viable options to enhance efficiency in drug development and, more importantly, in offering diverse patients easier and attractive means to participate in clinical trials. Special reference is made to the implication of artificial intelligence and machine-learning tools in trial execution and data acquisition, processing, and analysis in a virtual trial setting. Issues of patient safety, measurement validity, and data integrity are reviewed, and considerations are put forth with reference to the mitigation of underlying regulatory and operational barriers.

Structures of synthetic nanobody-SARS-CoV-2 receptor-binding domain complexes reveal distinct sites of interaction.

Combating the worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the emergence of new variants demands understanding of the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here, we report five X-ray crystal structures of sybodies (synthetic nanobodies) including those of binary and ternary complexes of Sb16-RBD, Sb45-RBD, Sb14-RBD-Sb68, and Sb45-RBD-Sb68, as well as unliganded Sb16. These structures reveal that Sb14, Sb16, and Sb45 bind the RBD at the angiotensin-converting enzyme 2 interface and that the Sb16 interaction is accompanied by a large conformational adjustment of complementarity-determining region 2. In contrast, Sb68 interacts at the periphery of the SARS-CoV-2 RBD-angiotensin-converting enzyme 2 interface. We also determined cryo-EM structures of Sb45 bound to the SARS-CoV-2 spike protein. Superposition of the X-ray structures of sybodies onto the trimeric spike protein cryo-EM map indicates that some sybodies may bind in both "up" and "down" configurations, but others may not. Differences in sybody recognition of several recently identified RBD variants are explained by these structures.

A delicate balance: Psychotropic polypharmacy and anti-cholinergic use are correlated with fall incidence in Australian inpatients with dementia.

BACKGROUND: Persons with dementia commonly experience a range of behavioural and psychological symptoms, including agitation, aggression, perceptual disturbances, and depression. While psychotropic medications are regularly prescribed to mitigate these symptoms, these agents also carry a broad adverse effect profile. This study aimed to characterize psychotropic medication use in patients with dementia, as well as identify prescribing factors associated with falls in this cohort. METHODS: This retrospective study collected longitudinal demographic and medication data from all patients admitted to a neuro-cognitive unit at an Australian metropolitan hospital over a 2-year period. Psychotropic polypharmacy and psychotropic agent use per patient-fortnight were investigated for their association with inpatient falls. RESULTS: All patients (n = 147) were prescribed at least one psychotropic medication, with 96% receiving anti-psychotic medications and 90% receiving benzodiazepines. Patient fall rate was significantly associated with anticholinergic drug use (Incidence rate ratio: 2.2; P < .001), as well as concomitant use of ≥5 daily psychotropic agents (Incidence rate ratio: 3.1; P = .001). CONCLUSIONS: Patients with dementia are routinely prescribed a wide variety of psychotropic medications. Use of anticholinergic drugs and psychotropic polypharmacy are correlated with fall incidence in persons with dementia.

Review on the extraction of calcium supplements from eggshells to combat waste generation and chronic calcium deficiency.

When faced with a plethora of issues, the possibility of one problem becoming the solution of another is a rare, yet beneficial scenario. This report explores the prospect of viewing the accumulation of organic waste matter in India as a potential calcium reservoir to relieve the issue of calcium deficiency in the population. Waste generation has seen gradual growth, and it has created a problem of waste disposal. A large segment of the generated waste primarily consists of food waste which contains significant amounts of nutrients. Food waste such as eggshells, waste from shellfish, bones, and fish scales contain good amounts of bioavailable calcium, and large quantities of this discarded bioavailable calcium remain unused. Global studies show India to have significantly lower levels of calcium intake than the global average, thus increasing the risk of calcium deficiency-related diseases. Furthermore, research shows that for over the past half of the century, the intake of dietary calcium has declined drastically throughout India. This has led to chronic calcium deficiency-related diseases throughout most of the Indian population. Hence, development of calcium supplements from calcium-rich waste material has the potential to not only reduce the strain on waste management, but also to provide the calcium-deficient population with a cheaper alternative to traditional supplements. Owing to the abundance and ease of separation, eggshells have been chosen as the focus of the review. This review highlights and compares their extraction methods of providing cheap calcium supplements while reducing the amount of eggshell waste.

Structures of synthetic nanobody-SARS-CoV-2-RBD complexes reveal distinct sites of interaction and recognition of variants.

The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emergence of new variants demands understanding the structural basis of the interaction of antibodies with the SARS-CoV-2 receptor-binding domain (RBD). Here we report five X-ray crystal structures of sybodies (synthetic nanobodies) including binary and ternary complexes of Sb16-RBD, Sb45-RBD, Sb14-RBD-Sb68, and Sb45-RBD-Sb68; and Sb16 unliganded. These reveal that Sb14, Sb16, and Sb45 bind the RBD at the ACE2 interface and that the Sb16 interaction is accompanied by a large CDR2 shift. In contrast, Sb68 interacts at the periphery of the interface. We also determined cryo-EM structures of Sb45 bound to spike (S). Superposition of the X-ray structures of sybodies onto the trimeric S protein cryo-EM map indicates some may bind both "up" and "down" configurations, but others may not. Sensitivity of sybody binding to several recently identified RBD mutants is consistent with these structures.

Demographic diversity of participants in Pfizer sponsored clinical trials in the United States.

The approval of new medicinal agents requires robust efficacy and safety clinical trial data demonstrated to be applicable to population subgroups. Limited data have previously been reported by drug sponsors on the topic of clinical trial diversity. In order to establish a baseline of diversity in our clinical trials that can be used by us and other sponsors, an analysis of clinical trial diversity was conducted covering race, ethnicity, sex, and age. This analysis includes Pfizer interventional clinical trials that initiated enrollment between 2011 through 2020. The data set comprises 213 trials with 103,103 US participants. The analysis demonstrated that overall trial participation of Black or African American individuals was at the US census level (14.3% vs 13.4%), participation of Hispanic or Latino individuals was below US census (15.9% vs 18.5%), and female participation was at US census (51.1% vs 50.8%). The analysis also examined the percentage of trials that achieved racial and ethnic distribution levels at or above census levels. Participant levels above census were achieved in 56.1% of Pfizer trials for Black or African American participants, 51.4% of trials for White participants, 16.0% of trials for Asian participants, 14.2% of trials for Native Hawaiian and Pacific Islander participants, 8.5% of trials for American Indian and Alaska Native participants, and 52.3% of trials for Hispanic or Latino participants. The results presented here provide a baseline upon which we can quantify the impact of our ongoing efforts to improve racial and ethnic diversity in clinical trials.

Synthetic nanobody-SARS-CoV-2 receptor-binding domain structures identify distinct epitopes.

The worldwide spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands unprecedented attention. We report four X-ray crystal structures of three synthetic nanobodies (sybodies) (Sb16, Sb45 and Sb68) bind to the receptor-binding domain (RBD) of SARS-CoV-2: binary complexes of Sb16-RBD and Sb45-RBD; a ternary complex of Sb45-RBD-Sb68; and Sb16 unliganded. Sb16 and Sb45 bind the RBD at the ACE2 interface, positioning their CDR2 and CDR3 loops diametrically. Sb16 reveals a large CDR2 shift when binding the RBD. Sb68 interacts peripherally at the ACE2 interface; steric clashes with glycans explain its mechanism of viral neutralization. Superposing these structures onto trimeric spike (S) protein models indicates these sybodies bind conformations of the mature S protein differently, which may aid therapeutic design. ONE SENTENCE SUMMARY: X-ray structures of synthetic nanobodies complexed with the receptor-binding domain of the spike protein of SARS-CoV-2 reveal details of CDR loop interactions in recognition of distinct epitopic sites.

First-in-Human Study to Assess the Safety and Feasibility of the Bashir Endovascular Catheter for the Treatment of Acute Intermediate-Risk Pulmonary Embolism.

BACKGROUND: The Bashir Endovascular Catheter (BEC) is a novel pharmaco-mechanical device designed to enhance thrombolysis by increasing the exposure of thrombus to endogenous and exogenous thrombolytics. The aim of this prospective, multicenter, single-arm study was to evaluate the feasibility and initial safety of the BEC in patients with acute intermediate-risk pulmonary embolism (PE). METHODS: Patients with symptomatic PE and right ventricular to left ventricular diameter ratio ≥0.9 as documented by computer tomography angiography were eligible for enrollment. The primary safety end points were device related death or adverse events, and major bleeding within 72 hours after BEC directed therapy. RESULTS: Nine patients were enrolled across 4 US sites. The total dose of r-tPA (recombinant tissue-type plasminogen activator) was 14 mgs in bilateral PE and 12 mgs in unilateral PE over 8 hours delivered via the expanded BEC. At 30-day follow-up, there were no deaths or device-related adverse events. At 48 hours post-BEC therapy, the right ventricular to left ventricular diameter ratio decreased from 1.52±0.26 to 0.97±0.06 (P=0.0009 [95% CI, 0.33-0.82]; 37.0% reduction). Thrombus burden as measured by the Modified Miller Index decreased from 25.4±5.3 to 16.0±4.0 (P=0.0005; [95% CI, 5.5-13.4]; 37.1% reduction). CONCLUSIONS: In this early feasibility study of the BEC for intermediate-risk PE, there were no deaths or device-related adverse events and a significant reduction in right ventricular to left ventricular diameter ratio and thrombus burden. Registration: URL: https://www.clinicaltrials.gov. Unique identifier: NCT03927508.

Congenital Portosystemic Shunts and Liver Hemangiomas in Children: Is There an Association?

Liver hemangiomas are benign vascular tumors of infancy. They can have vascular shunting mostly arteriovenous and sometimes arterioportal or portosystemic, which improves as hemangiomas involute. In contrast, congenital portosystemic shunts are developmental vascular anomalies that may go undetected for years, with significant sequelae. We describe a child with a history of multiple cutaneous and liver hemangiomas in infancy and later diagnosis of congenital portosystemic shunt. Past experience of a similar patient and a current baby followed for liver hemangiomas with portosystemic shunts, is also shared. Literature is reviewed for known association. We suggest longer-term follow-up for babies with liver hemangiomas.