Cataract progression after primary pars plana vitrectomy for uncomplicated rhegmatogenous retinal detachments in young adults.

BACKGROUND: Scleral buckling is typically implemented to repair rhegmatogenous retinal detachments (RRD) in young patients. Therefore, there is limited data on post-pars plana vitrectomy (PPV) cataract formation in this cohort. We report the rates and risk factors of cataract progression after PPV for RRD repair in young eyes. METHODS: Retrospective single-center cohort study. Medical records of patients between the ages of 15 to 45 undergoing PPV for uncomplicated RRD between 2014 and 2020 were reviewed. RESULTS: Twenty-eight eyes from 26 patients met inclusion criteria. Cataracts developed in 20/28 (71%) eyes after PPV. After PPV, nuclear sclerotic cataract (NSC) rates were higher in patients above 35 (65%) compared to below 35 years (18%) (p = 0.024). Cataracts developed more frequently after macula-off RRDs (88%) compared to macula-on RRDs (50%) (p = 0.044) with NSC more common in macula-off detachments (p = 0.020). At postoperative month 2, all eyes with C3F8 gas developed cataracts compared to 59% of eyes with no gas (p = 0.040). CONCLUSIONS: Cataract formation was common and frequent after PPV. After PPV, young eyes and macula-on detachments developed cataracts less frequently than older eyes and macula-off detachments. If appropriate, a shorter acting gas tamponade should be considered in young eyes to minimize cataract formation.

Outcome of perioperative 24-hour infusion of intravenous lignocaine on pain and QoR-15 scores after breast cancer surgery-A randomised controlled trial.

Background and Aims: Despite many analgesic modalities available, postoperative pain management after breast cancer surgery remains a challenge, which translates into poor quality of recovery, if untreated. Intravenous lignocaine with its anti-inflammatory, antihyperalgesic, and analgesic properties could provide a good option for these patients. The aim of this study was to evaluate the effect of intravenous lignocaine on postoperative pain relief and quality of recovery in patients undergoing surgery for breast cancer. Methods: In this prospective double-blind placebo-controlled randomised study, sixty-six patients undergoing breast cancer surgery were assigned 1:1 to placebo or intravenous lignocaine (Group L). Group L received an intravenous 1.5 mg/kg of lignocaine bolus at induction, followed by an intravenous infusion of 1 mg/kg/h for 24 hours intravenously, while the control group was given equal volume of normal saline. Pain scores, opioid utilisation, and quality of recovery (QoR-15) at 24 hours and on the day of suture removal were compared. Results: Statistically significant reduction was observed in both static (P = 0.01, 6 hours) and dynamic postoperative pain (P = 0.030, 24 hours), with consequential delay in the need for the first dose of opioid (P = 0.014) as well as decreased 24-hour postoperative opioid consumption (P < 0.001) and decreased post-operative nausea and vomiting (PONV) (P < 0.05) in the lignocaine group. Global QoR-15 was significantly better at 24 -hours in group L on postoperative day 1 (P < 0.001), albeit there was no significant difference at suture removal. No lignocaine related side effects were observed. Conclusion: Intravenous lignocaine can be safely used as an alternative perioperative non-opioid analgesic for early postoperative pain and recovery.

Irregular bladder shapes identified in women with overactive bladder: an ultrasound nomogram.

In this study, an ultrasound-based bladder shape nomogram was developed using data from women without overactive bladder (OAB) and tested in women with OAB to identify irregular bladder shapes. The goal was development of a nomogram that can ultimately be used for non-invasive identification of a bladder shape-associated OAB phenotype. Transabdominal 3-dimensional (3D) bladder ultrasound images were collected at 1-minute intervals during urodynamics studies and at 5-10-minute intervals during oral hydration studies. These prospective studies enrolled women with and without OAB based on International Consultation on Incontinence questionnaire on OAB (ICIq-OAB) question 5a (OAB 5a≥2, without OAB 5a<2). Bladder perimeters were manually traced and refined using GE 4D-View software. Nomograms for the transverse, sagittal and coronal perimeter-volume relationships were developed for women without OAB. A power model was used to approximate upper and lower nomogram bounds with 95% confidence intervals. Nomograms were tested using data from women with OAB, and each participant was classified as having an irregular bladder shape based on the number of perimeter values outside the nomogram bounds. Nomograms were developed using 533 images from 27 women without OAB (14 from urodynamics and 13 from hydration studies) and were tested using 264 images from 24 women with OAB (16 urodynamics and 8 hydration). The sagittal perimeter nomogram provided the best results, with irregular sagittal perimeters identified in 6/24 (25%) women with OAB and 0/27 (0%) without OAB. An irregular sagittal perimeter was significantly associated with OAB (P<0.05). Ultrasound-based nomograms may enable feasible, non-invasive identification of a subgroup of women with bladder shape-associated OAB.

Comparison of Sensation Event Descriptors in Participants with Overactive and Normal Bladders during Non-Invasive Hydration Studies.

PURPOSE: Despite the importance of alterations in bladder sensation, objective metrics to characterize sensation outside of urodynamics remain limited. A real-time sensation meter enables recording of sensation event descriptors throughout filling. The purpose of this study was to evaluate the differences in sensation event descriptor patterns between normal participants and those with OAB. METHODS: Normal and OAB participants were enrolled from responses to the ICIq-OAB survey question on urgency (Q5a: 0 vs. ≥ 3). Real-time bladder sensation on a 0%-100% scale was recorded on a validated tablet sensation meter throughout two fill-void cycles. The first and second fills were considered "slow" and "fast" respectively. After each sensation meter change (sensation event), a pop-up screen asked participants to characterize sensation with one or more of these descriptors: "tense," "pressure," "tingling," "painful," and/or "other." Oral hydration was achieved by rapid consumption of 2L G2® Gatorade. RESULTS: Data from 29 participants (12 normal/17 OAB) were analyzed. The rate of filling from bladder volume and fill duration, was greater for the fast fill in both groups. In the slow fill, "tingling" (64 ± 3% OAB vs. 77 ± 3% normal, p=0.008) and "tense" (78 ± 3% OAB vs. 94 ± 1% normal, p<0.001) occurred at lower sensations in OAB participants. CONCLUSION: During only the slow fill, OAB individuals experience the sensation descriptors of "tingling" and "tense" at earlier sensations than normal individuals. Therefore, this non-invasive method to evaluate real-time sensation descriptors during filling may identify important sensation patterns and improve understanding and phenotyping of OAB.

Deconstructing the peptide-MHC specificity of T cell recognition.

In order to survey a universe of major histocompatibility complex (MHC)-presented peptide antigens whose numbers greatly exceed the diversity of the T cell repertoire, T cell receptors (TCRs) are thought to be cross-reactive. However, the nature and extent of TCR cross-reactivity has not been conclusively measured experimentally. We developed a system to identify MHC-presented peptide ligands by combining TCR selection of highly diverse yeast-displayed peptide-MHC libraries with deep sequencing. Although we identified hundreds of peptides reactive with each of five different mouse and human TCRs, the selected peptides possessed TCR recognition motifs that bore a close resemblance to their known antigens. This structural conservation of the TCR interaction surface allowed us to exploit deep-sequencing information to computationally identify activating microbial and self-ligands for human autoimmune TCRs. The mechanistic basis of TCR cross-reactivity described here enables effective surveillance of diverse self and foreign antigens without necessitating degenerate recognition of nonhomologous peptides.

Crossreactivity of a human autoimmune TCR is dominated by a single TCR loop.

Self-reactive CD4 T cells are thought to have a central role in the pathogenesis of many chronic inflammatory human diseases. Microbial peptides can activate self-reactive T cells, but the structural basis for such crossreactivity is not well understood. The Hy.1B11 T cell receptor (TCR) originates from a patient with multiple sclerosis and recognizes the self-antigen myelin basic protein. Here we report the structural mechanism of TCR crossreactivity with two distinct peptides from human pathogens. The structures show that a single TCR residue (CDR3α F95) makes the majority of contacts with the self-peptide and both microbial peptides (66.7-80.6%) due to a highly tilted TCR-binding topology on the peptide-MHC surface. Further, a neighbouring residue located on the same TCR loop (CDR3α E98) forms an energetically critical interaction with the MHC molecule. These data show how binding by a self-reactive TCR favors crossreactivity between self and microbial antigens.

Disruption of hydrogen bonds between major histocompatibility complex class II and the peptide N-terminus is not sufficient to form a human leukocyte antigen-DM receptive state of major histocompatibility complex class II.

Peptide presentation by MHC class II is of critical importance to the function of CD4+ T cells. HLA-DM resides in the endosomal pathway and edits the peptide repertoire of newly synthesized MHC class II molecules before they are exported to the cell surface. HLA-DM ensures MHC class II molecules bind high affinity peptides by targeting unstable MHC class II:peptide complexes for peptide exchange. Research over the past decade has implicated the peptide N-terminus in modulating the ability of HLA-DM to target a given MHC class II:peptide combination. In particular, attention has been focused on both the hydrogen bonds between MHC class II and peptide, and the occupancy of the P1 anchor pocket. We sought to solve the crystal structure of a HLA-DR1 molecule containing a truncated hemagglutinin peptide missing three N-terminal residues compared to the full-length sequence (residues 306-318) to determine the nature of the MHC class II:peptide species that binds HLA-DM. Here we present structural evidence that HLA-DR1 that is loaded with a peptide truncated to the P1 anchor residue such that it cannot make select hydrogen bonds with the peptide N-terminus, adopts the same conformation as molecules loaded with full-length peptide. HLA-DR1:peptide combinations that were unable to engage up to four key hydrogen bonds were also unable to bind HLA-DM, while those truncated to the P2 residue bound well. These results indicate that the conformational changes in MHC class II molecules that are recognized by HLA-DM occur after disengagement of the P1 anchor residue.

Mechanisms of peptide repertoire selection by HLA-DM.

Recently, crystal structures of key complexes in antigen presentation have been reported. HLA-DM functions in antigen presentation by catalyzing dissociation of an invariant chain remnant from the peptide binding groove and stabilizing empty MHC class II proteins in a peptide-receptive conformation. The crystal structure of a MHC class II-HLA-DM complex explains how HLA-DM stabilizes an otherwise short-lived transition state and promotes a rapid peptide exchange process that favors the highest-affinity ligands. HLA-DO has sequence similarity with MHC class II molecules yet inhibits antigen presentation. The structure of the HLA-DO-HLA-DM complex shows that it blocks HLA-DM activity as a substrate mimic. Alterations in the efficiency of DM-mediated peptide selection may contribute to autoimmune pathologies, which will be an exciting area for future investigation.

Crystal structure of the HLA-DM-HLA-DR1 complex defines mechanisms for rapid peptide selection.

HLA-DR molecules bind microbial peptides in an endosomal compartment and present them on the cell surface for CD4 T cell surveillance. HLA-DM plays a critical role in the endosomal peptide selection process. The structure of the HLA-DM-HLA-DR complex shows major rearrangements of the HLA-DR peptide-binding groove. Flipping of a tryptophan away from the HLA-DR1 P1 pocket enables major conformational changes that position hydrophobic HLA-DR residues into the P1 pocket. These conformational changes accelerate peptide dissociation and stabilize the empty HLA-DR peptide-binding groove. Initially, incoming peptides have access to only part of the HLA-DR groove and need to compete with HLA-DR residues for access to the P2 site and the hydrophobic P1 pocket. This energetic barrier creates a rapid and stringent selection process for the highest-affinity binders. Insertion of peptide residues into the P2 and P1 sites reverses the conformational changes, terminating selection through DM dissociation.

Self-reactive human CD4 T cell clones form unusual immunological synapses.

Recognition of self-peptide-MHC (pMHC) complexes by CD4 T cells plays an important role in the pathogenesis of many autoimmune diseases. We analyzed formation of immunological synapses (IS) in self-reactive T cell clones from patients with multiple sclerosis and type 1 diabetes. All self-reactive T cells contained a large number of phosphorylated T cell receptor (TCR) microclusters, indicative of active TCR signaling. However, they showed little or no visible pMHC accumulation or transport of TCR-pMHC complexes into a central supramolecular activation cluster (cSMAC). In contrast, influenza-specific T cells accumulated large quantities of pMHC complexes in microclusters and a cSMAC, even when presented with 100-fold lower pMHC densities. The self-reactive T cells also maintained a high degree of motility, again in sharp contrast to virus-specific T cells. 2D affinity measurements of three of these self-reactive T cell clones demonstrated a normal off-rate but a slow on-rate of TCR binding to pMHC. These unusual IS features may facilitate escape from negative selection by self-reactive T cells encountering very small amounts of self-antigen in the thymus. However, these same features may enable acquisition of effector functions by self-reactive T cells encountering large amounts of self-antigen in the target organ of the autoimmune disease.

Cell surface display of functional human MHC class II proteins: yeast display versus insect cell display.

Reliable and robust systems for engineering functional major histocompatibility complex class II (MHCII) proteins have proved elusive. Availability of such systems would enable the engineering of peptide-MHCII (pMHCII) complexes for therapeutic and diagnostic applications. In this paper, we have developed a system based on insect cell surface display that allows functional expression of heterodimeric DR2 molecules with or without a covalently bound human myelin basic protein (MBP) peptide, which is amenable to directed evolution of DR2-MBP variants with improved T cell receptor (TCR)-binding affinity. This study represents the first example of functional display of human pMHCII complexes on insect cell surface. In the process of developing this pMHCII engineering system, we have also explored the potential of using yeast surface display for the same application. Our data suggest that yeast display is a useful system for analysis and engineering of peptide binding of MHCII proteins, but not suitable for directed evolution of pMHC complexes that bind with low affinity to self-reactive TCRs.

T cell receptor recognition of self and foreign antigens in the induction of autoimmunity.

The major histocompatibility complex (MHC) on human chromosome 6 represents the most important genetic locus for a number of common human autoimmune diseases. Specific alleles that differ from closely related alleles by only one or a few amino acids in the peptide binding groove are frequently strongly associated with disease susceptibility, raising the important question of which peptide presentation events are critical in disease initiation and progression. This review will cover a number of topics pertinent to this fundamental question, including MHC linked disease susceptibility to autoimmune diseases, molecular mechanisms for the role of MHC molecules in autoimmune diseases as well as the recognition of self and microbial peptides by self-reactive T cell receptors (TCRs).

A highly tilted binding mode by a self-reactive T cell receptor results in altered engagement of peptide and MHC.

Self-reactive T cells that escape elimination in the thymus can cause autoimmune pathology, and it is therefore important to understand the structural mechanisms of self-antigen recognition. We report the crystal structure of a T cell receptor (TCR) from a patient with relapsing-remitting multiple sclerosis that engages its self-peptide-major histocompatibility complex (pMHC) ligand in an unusual manner. The TCR is bound in a highly tilted orientation that prevents interaction of the TCR-α chain with the MHC class II ß chain helix. In this structure, only a single germline-encoded TCR loop engages the MHC protein, whereas in most other TCR-pMHC structures all four germline-encoded TCR loops bind to the MHC helices. The tilted binding mode also prevents peptide contacts by the short complementarity-determining region (CDR) 3ß loop, and interactions that contribute to peptide side chain specificity are focused on the CDR3α loop. This structure is the first example in which only a single germline-encoded TCR loop contacts the MHC helices. Furthermore, the reduced interaction surface with the peptide may facilitate TCR cross-reactivity. The structural alterations in the trimolecular complex are distinct from previously characterized self-reactive TCRs, indicating that there are multiple unusual ways for self-reactive TCRs to bind their pMHC ligand.

Purification, identification and preliminary crystallographic studies of Pru du amandin, an allergenic protein from Prunus dulcis.

Food allergies appear to be one of the foremost causes of hypersensitivity reactions. Nut allergies account for most food allergies and are often permanent. The 360 kDa hexameric protein Pru du amandin, a known allergen, was purified from almonds (Prunus dulcis) by ammonium sulfate fractionation and ion-exchange chromatography. The protein was identified by a BLAST homology search against the nonredundant sequence database. Pru du amandin belongs to the 11S legumin family of seed storage proteins characterized by the presence of a cupin motif. Crystals were obtained by the hanging-drop vapour-diffusion method. The crystals belong to space group P4(1) (or P4(3)), with unit-cell parameters a = b = 150.7, c = 164.9 A.

Purification, identification and preliminary crystallographic studies of an allergenic protein from Lathyrus sativus.

A 24 kDa protein was purified from the seeds of Lathyrus sativus by ammonium sulfate fractionation and ion-exchange chromatography. The N-terminal amino-acid sequence showed significant homology with the 2S albumin class of seed storage proteins. The protein showed 85% sequence homology with the seed albumin of Pisum sativum within the 40 N-terminal residues. Crystals were obtained by the hanging-drop vapour-diffusion method. The crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 43.5, b = 82.7, c = 153.4 A.

Differential epitope positioning within the germline antibody paratope enhances promiscuity in the primary immune response.

Correlation between the promiscuity of the primary antibody response and conformational flexibility in a germline antibody was addressed by using germline antibody 36-65. Crystallographic analyses of the 36-65 Fab with three independent dodecapeptides provided mechanistic insights into the generation of antibody diversity. While four antigen-free Fab molecules provided a quantitative description of the conformational repertoire of the antibody CDRs, three Fab molecules bound to structurally diverse peptide epitopes exhibited a common paratope conformation. Each peptide revealed spatially different footprints within the antigen-combining site. However, a conformation-specific lock involving two shared residues, which were also associated with hapten binding, was discernible. Unlike the hapten, the peptides interacted with residues that undergo somatic mutations, suggesting a possible mechanism for excluding "nonspecific" antigens during affinity maturation. The observed multiple binding modes of diverse epitopes within a common paratope conformation of a germline antibody reveal a simple, yet elegant, mechanism for expanding the primary antibody repertoire.

Crystal structures of the PNA-porphyrin complex in the presence and absence of lactose: mapping the conformational changes on lactose binding, interacting surfaces, and supramolecular aggregations.

The extraordinary recognition specificity of lectins for carbohydrate ligands appears to be violated as they also bind to porphyrins and other noncarbohydrate ligands. In this study, crystal structures of meso-tetrasulfonatophenylporphyrin (H(2)TPPS) bound to peanut agglutinin (PNA) in the presence and absence of lactose were determined. The binding of H(2)TPPS with PNA involved 11 molecules of H(2)TPPS in different supramolecular stacking arrangements associated with a tetramer of PNA in the crystals of the PNA-H(2)TPPS binary complex as well as the PNA-H(2)TPPS-lactose ternary complex. The ternary complex involved lactose binding only to two subunits of the PNA tetramer, which did not have porphyrin interacting in the vicinity of the carbohydrate-binding site. Comparison of the two structures highlighted the plasticity of the carbohydrate-binding site expressed in terms of the conformational change in lactose binding. The unusual quaternary structure of PNA, which results in exposed protein-protein interaction sites, might be responsible for the porphyrin binding. The association of porphyrin in diverse oligomeric stacking arrangements observed in the PNA-H(2)TPPS complex suggested the possibility of protein-porphyrin aggregation under abnormal physiological conditions. The structures described here provide a possible native conformation of the carbohydrate-binding site of PNA in the absence of the ligand, highlight mapping of the unsaturated binding surfaces of PNA using porphyrin interactions, indicate new leads toward possible application of this lectin in photodynamic therapy, and exhibit diverse modes of porphyrin-lectin interactions with implications to porphyria, a disease that results from abnormal accumulation of porphyrins.

Identification and characterization of EhCaBP2. A second member of the calcium-binding protein family of the protozoan parasite Entamoeba histolytica.

Entamoeba histolytica, an early branching eukaryote, is the etiologic agent of amebiasis. Calcium plays a pivotal role in the pathogenesis of amebiasis by modulating the cytopathic properties of the parasite. However, the mechanistic role of Ca(2+) and calcium-binding proteins in the pathogenesis of E. histolytica remains poorly understood. We had previously characterized a novel calcium-binding protein (EhCaBP1) from E. histolytica. Here, we report the identification and partial characterization of an isoform of this protein, EhCaBP2. Both EhCaBPs have four canonical EF-hand Ca(2+) binding domains. The two isoforms are encoded by genes of the same size (402 bp). Comparison between the two genes showed an overall identity of 79% at the nucleotide sequence level. This identity dropped to 40% in the 75-nucleotide central linker region between the second and third Ca(2+) binding domains. Both of these genes are single copy, as revealed by Southern hybridization. Analysis of the available E. histolytica genome sequence data suggested that the two genes are non-allelic. Homology-based structural modeling showed that the major differences between the two EhCaBPs lie in the central linker region, normally involved in binding target molecules. A number of studies indicated that EhCaBP1 and EhCaBP2 are functionally different. They bind different sets of E. histolytica proteins in a Ca(2+)-dependent manner. Activation of endogenous kinase was also found to be unique for the two proteins and the Ca(2+) concentration required for their optimal functionality was also different. In addition, a 12-mer peptide was identified from a random peptide library that could differentially bind the two proteins. Our data suggest that EhCaBP2 is a new member of a class of E. histolytica calcium-binding proteins involved in a novel calcium signal transduction pathway.