Rational Design of an Epstein-Barr Virus Vaccine Targeting the Receptor-Binding Site.

Epstein-Barr virus (EBV) represents a major global health problem. Though it is associated with infectious mononucleosis and ∼200,000 cancers annually worldwide, a vaccine is not available. The major target of immunity is EBV glycoprotein 350/220 (gp350) that mediates attachment to B cells through complement receptor 2 (CR2/CD21). Here, we created self-assembling nanoparticles that displayed different domains of gp350 in a symmetric array. By focusing presentation of the CR2-binding domain on nanoparticles, potent neutralizing antibodies were elicited in mice and non-human primates. The structurally designed nanoparticle vaccine increased neutralization 10- to 100-fold compared to soluble gp350 by targeting a functionally conserved site of vulnerability, improving vaccine-induced protection in a mouse model. This rational approach to EBV vaccine design elicited potent neutralizing antibody responses by arrayed presentation of a conserved viral entry domain, a strategy that can be applied to other viruses.

Focal adhesions are controlled by microtubules through local contractility regulation.

Microtubules regulate cell polarity and migration via local activation of focal adhesion turnover, but the mechanism of this process is insufficiently understood. Molecular complexes containing KANK family proteins connect microtubules with talin, the major component of focal adhesions. Here, local optogenetic activation of KANK1-mediated microtubule/talin linkage promoted microtubule targeting to an individual focal adhesion and subsequent withdrawal, resulting in focal adhesion centripetal sliding and rapid disassembly. This sliding is preceded by a local increase of traction force due to accumulation of myosin-II and actin in the proximity of the focal adhesion. Knockdown of the Rho activator GEF-H1 prevented development of traction force and abolished sliding and disassembly of focal adhesions upon KANK1 activation. Other players participating in microtubule-driven, KANK-dependent focal adhesion disassembly include kinases ROCK, PAK, and FAK, as well as microtubules/focal adhesion-associated proteins kinesin-1, APC, and αTAT. Based on these data, we develop a mathematical model for a microtubule-driven focal adhesion disruption involving local GEF-H1/RhoA/ROCK-dependent activation of contractility, which is consistent with experimental data.

CTC service heterogeneity and design of a workforce calculator.

AIM: To determine the level of heterogeneity in delivery of computed tomography (CT) colonography services and develop a workforce calculator that accommodates the variation identified. MATERIALS AND METHODS: A national survey, based on the "WHO workforce indicators of staffing need", established activity standards for essential tasks in delivery of the service. From these data a workforce calculator was designed to guide the required staffing and equipment resource by service size. RESULTS: Activity standards were established as mode responses >70%. Service homogeneity was greater in areas where professional standards and guidance were available. The mean service size was 1,101. Did not attend (DNA) rates were lower where direct booking was available (p<0.0001). Service sizes were larger where radiographer reporting was embedded in reporting paradigms (p<0.024). CONCLUSION: The survey identified benefits of radiographer-led direct booking and reporting. The workforce calculator derived from the survey provides a framework to guide the resourcing of expansion while maintaining standards.

Recognition of single-stranded nucleic acids by small-molecule splicing modulators.

Risdiplam is the first approved small-molecule splicing modulator for the treatment of spinal muscular atrophy (SMA). Previous studies demonstrated that risdiplam analogues have two separate binding sites in exon 7 of the SMN2 pre-mRNA: (i) the 5'-splice site and (ii) an upstream purine (GA)-rich binding site. Importantly, the sequence of this GA-rich binding site significantly enhanced the potency of risdiplam analogues. In this report, we unambiguously determined that a known risdiplam analogue, SMN-C2, binds to single-stranded GA-rich RNA in a sequence-specific manner. The minimum required binding sequence for SMN-C2 was identified as GAAGGAAGG. We performed all-atom simulations using a robust Gaussian accelerated molecular dynamics (GaMD) method, which captured spontaneous binding of a risdiplam analogue to the target nucleic acids. We uncovered, for the first time, a ligand-binding pocket formed by two sequential GAAG loop-like structures. The simulation findings were highly consistent with experimental data obtained from saturation transfer difference (STD) NMR and structure-affinity-relationship studies of the risdiplam analogues. Together, these studies illuminate us to understand the molecular basis of single-stranded purine-rich RNA recognition by small-molecule splicing modulators with an unprecedented binding mode.

Pregnant lady with eosinophilia: common cause, uncommon association.

Peripheral eosinophilia can have a myriad of causes and presents a diagnostic challenge in everyday practice. Tropical pulmonary eosinophilia (TPE), seen commonly in tropics, is an immunological reaction to filarial parasites. This disease can present with clinical features that closely mimic asthma, eosinophilic pneumonia, and Loffler's syndrome. Differentiating between these diseases is essential owing to marked differences in treatment. This can be further challenging in pregnancy as any wrong treatment is likely to affect both the mother and the child. We report a case of a pregnant lady who presented with eosinophilia and of how she was worked up to the correct diagnosis. There are only few reported cases of TPE in pregnancy, and there are no reported cases from India. The case also underlines the approach required in these patients to reach the correct diagnosis.

Immediate flapless implant placement in a smoker patient: A challenge for optimum aesthetics and secondary stability: A case report.

The objective of this case report was to restore the young patient with missing teeth and extruded tooth using an immediate implant and synthetic bone graft material for the esthetic and comfort purpose. A 21-year young man reported extrusion of a tooth as well as missing teeth. Clinical examination revealed missing teeth in relation to (irt) 11 and 21, extrusion of tooth in relation to (irt) 12, patient had generalized fluorosis, and localized marginal gingivitis with melanin pigmentation. The patient is a known smoker, and he was advised the cessation of smoking before the treatment. This paper describes a step-by-step approach to different treatment phases, starting with surgical guide fabrication, immediate implant surgical procedures, bone grafting procedure, and later prosthesis fabrication. Follow-up resulted in a satisfactory outcome.

Efficacy and safety of rucaparib in previously treated, locally advanced or metastatic urothelial carcinoma from a phase 2, open-label trial (ATLAS).

BACKGROUND: ATLAS evaluated the efficacy and safety of the PARP inhibitor rucaparib in patients with previously treated locally advanced/unresectable or metastatic urothelial carcinoma (UC). METHODS: Patients with UC were enrolled independent of tumor homologous recombination deficiency (HRD) status and received rucaparib 600 mg BID. The primary endpoint was investigator-assessed objective response rate (RECIST v1.1) in the intent-to-treat and HRD-positive (loss of genome-wide heterozygosity ≥10%) populations. Key secondary endpoints were progression-free survival (PFS) and safety. Disease control rate (DCR) was defined post-hoc as the proportion of patients with a confirmed complete or partial response (PR), or stable disease lasting ≥16 weeks. RESULTS: Of 97 enrolled patients, 20 (20.6%) were HRD-positive, 30 (30.9%) HRD-negative, and 47 (48.5%) HRD-indeterminate. Among 95 evaluable patients, there were no confirmed responses. However, reductions in the sum of target lesions were observed, including 6 (6.3%) patients with unconfirmed PR. DCR was 11.6%; median PFS was 1.8 months (95% CI, 1.6-1.9). No relationship was observed between HRD status and efficacy endpoints. Median treatment duration was 1.8 months (range, 0.1-10.1). Most frequent any-grade treatment-emergent adverse events were asthenia/fatigue (57.7%), nausea (42.3%), and anemia (36.1%). Of 64 patients with data from tumor tissue samples, 10 (15.6%) had a deleterious alteration in a DNA damage repair pathway gene, including four with a deleterious BRCA1 or BRCA2 alteration. CONCLUSIONS: Rucaparib did not show significant activity in unselected patients with advanced UC regardless of HRD status. The safety profile was consistent with that observed in patients with ovarian or prostate cancer. TRIAL REGISTRATION: This trial was registered in ClinicalTrials.gov (NCT03397394). Date of registration: 12 January 2018. This trial was registered in EudraCT (2017-004166-10).

Hereditary Palpebro-Gingival Fibrosis Syndrome.

This is a case report involving 3 siblings, ages 22, 21, and 3 years old, of consanguineous parents and Syrian descent with a particularly unique clinical phenotype and eyelid/gingival findings that have not been previously characterized. Full-thickness eyelid biopsies for 2 of the siblings were evaluated by ophthalmic pathology showed generalized fibrosis without any active destructive process or amyloid. Three of the 9 siblings were affected by this order with no familial history. Ophthalmic plastic surgeons should be aware of this new entity as genetic localization and further identification may help families affected by this disorder.

Live Case Demonstrations are Essential for the Success of Pediatric Urology Meetings in India.

PURPOSE: The purpose of the study was to survey the opinion of delegates attending national pediatric urology meeting regarding live case demonstrations (LCDs) to see whether these can be replaced with taped case demonstrations (TCDs) in future. METHODS: A questionnaire-based survey was conducted at the end of the 3-day annual conference and live operative workshop in pediatric urology. Apart from general data such as age of the respondent and type of practice setting, four key questions on LCDs with yes/no responses included: would you have attended this meeting if there were no LCDs? Are unedited videos (TCDs) as effective as LCDs for teaching? Do you think LCDs should be continued? Would you allow your child to be operated in LCD by an expert? For question 3, the outcomes were compared between junior surgeons (<45-year-old) and senior surgeons (>45-year-old). RESULTS: On analysis, 88/140 (62%) respondents (95% confidence interval [CI]: 54%-70%) said that they would not have attended the meeting if there were no LCDs; 70/139 (50%) respondents (95% CI: 42%-58%) felt that TCDs may be an effective alternative to LCDs; 129/144 (90%) respondents (95% CI: 83%-94%) felt that LCDs should be continued. For question 4, 101/129 (79%) said they would allow their child to be operated in LCD by an expert, while 28 (21%) did not agree for their child to be operated in LCD. There was no significant difference between junior and senior surgeons regarding support for LCDs (P = 0.15). CONCLUSIONS: In the Indian scenario, LCDs were favored as an essential part of pediatric urology meeting, attract more participation, and are likely to be important for the success of the meeting.

Three-Year Efficacy and Safety of the Misago Peripheral Stent for Superficial Femoral Artery Disease: Final Results from the OSPREY Trial.

PURPOSE: This study evaluated the long-term outcomes of the Misago peripheral stent trial (Terumo) for atherosclerotic lesions in the superficial femoral artery (SFA) in patients with claudication. MATERIALS AND METHODS: This was a prospective multicenter, single-arm, clinical trial of primary stent placement for de novo cases of SFA disease conducted in the United States and Asia. The primary endpoint was freedom from clinically driven target lesion revascularization (CD-TLR) at 36 months. Secondary outcomes were ankle-brachial index (ABI), Rutherford score, Walking Impairment Questionnaire (WIQ), a quality of life survey, and rate of device fracture. RESULTS: A total of 276 patients (64.4% male; mean age, 69.3 ± 10.1 years) were enrolled. Freedom from CD-TLR was 78.5% (95% confidence interval [CI], 73.0%-83.0%) at 24 months and 75.4% (95% CI, 69.6%-80.2%) at 36 months. Baseline ABI was 0.7 ± 0.1 and 0.98 ± 0.20 (P < .001) at 30 days after the procedure. Baseline Rutherford score was 3.6 ± 0.6 and 1.6 ± 1.0 30 at 30 days after the procedure (P < .001). Mean (and changed) ABI and Rutherford score at 36 months compared to day 30 after the procedure were, respectively, 0.91 (-0.1 ± 0.2) and 1.5 (-0.2 ± 1.1). WIQ score at baseline was 21.49 ± 26.30 and 50.51 ± 38.49 at 30 days after the procedure ( P < .001). The mean WIQ score at 2 years was 46.65 ± 37.31 (P = .12). Stent fracture rate at 36 months was 2.0% (4 of 202 patients). CONCLUSIONS: OSPREY (Occlusive-Stenotic Peripheral Artery Revascularization Study) 36-month data demonstrated persistent freedom from CD-TLR and sustained improvement in ABI and Rutherford score with primary stent placement for SFA lesions.

Enhanced neonatal Fc receptor function improves protection against primate SHIV infection.

To protect against human immunodeficiency virus (HIV-1) infection, broadly neutralizing antibodies (bnAbs) must be active at the portals of viral entry in the gastrointestinal or cervicovaginal tracts. The localization and persistence of antibodies at these sites is influenced by the neonatal Fc receptor (FcRn), whose role in protecting against infection in vivo has not been defined. Here, we show that a bnAb with enhanced FcRn binding has increased gut mucosal tissue localization, which improves protection against lentiviral infection in non-human primates. A bnAb directed to the CD4-binding site of the HIV-1 envelope (Env) protein (denoted VRC01) was modified by site-directed mutagenesis to increase its binding affinity for FcRn. This enhanced FcRn-binding mutant bnAb, denoted VRC01-LS, displayed increased transcytosis across human FcRn-expressing cellular monolayers in vitro while retaining FcγRIIIa binding and function, including antibody-dependent cell-mediated cytotoxicity (ADCC) activity, at levels similar to VRC01 (the wild type). VRC01-LS had a threefold longer serum half-life than VRC01 in non-human primates and persisted in the rectal mucosa even when it was no longer detectable in the serum. Notably, VRC01-LS mediated protection superior to that afforded by VRC01 against intrarectal infection with simian-human immunodeficiency virus (SHIV). These findings suggest that modification of FcRn binding provides a mechanism not only to increase serum half-life but also to enhance mucosal localization that confers immune protection. Mutations that enhance FcRn function could therefore increase the potency and durability of passive immunization strategies to prevent HIV-1 infection.

Immunological and virological mechanisms of vaccine-mediated protection against SIV and HIV.

A major challenge for the development of a highly effective AIDS vaccine is the identification of mechanisms of protective immunity. To address this question, we used a nonhuman primate challenge model with simian immunodeficiency virus (SIV). We show that antibodies to the SIV envelope are necessary and sufficient to prevent infection. Moreover, sequencing of viruses from breakthrough infections revealed selective pressure against neutralization-sensitive viruses; we identified a two-amino-acid signature that alters antigenicity and confers neutralization resistance. A similar signature confers resistance of human immunodeficiency virus (HIV)-1 to neutralization by monoclonal antibodies against variable regions 1 and 2 (V1V2), suggesting that SIV and HIV share a fundamental mechanism of immune escape from vaccine-elicited or naturally elicited antibodies. These analyses provide insight into the limited efficacy seen in HIV vaccine trials.

Using queue to signal quality in primary care: The need for further empirical investigation.

In recent times, signalling quality via queues for generic services has received significant attention. However, the literature till date on primary care services has focussed on the optimal speed-quality trade-off to ensure higher service times and lower waiting times for the patients. Borrowing from the queue management literature on generic services, we aim to understand whether the hypothesis that queue is a signal of physician's quality is reasonable. Based on theoretical arguments, we justify the need to investigate this hypothesis on queue acting as a signal of quality in primary care. Although we conjecture that the queue length on arrival may act as a signal of quality, the waiting experienced after joining the queue is still considered expensive.

Multidrug Resistance Regulators MarA, SoxS, Rob, and RamA Repress Flagellar Gene Expression and Motility in Salmonella enterica Serovar Typhimurium.

Production of flagella is costly and subject to global multilayered regulation, which is reflected in the hierarchical control of flagellar production in many bacterial species. For Salmonella enterica serovar Typhimurium and its relatives, global regulation of flagellar production primarily occurs through the control of flhDC transcription and mRNA translation. In this study, the roles of the homologous multidrug resistance regulators MarA, SoxS, Rob, and RamA (constituting the mar-sox-rob regulon in S Typhimurium) in regulating flagellar gene expression were explored. Each of these regulators was found to inhibit flagellar gene expression, production of flagella, and motility. To different degrees, repression via these transcription factors occurred through direct interactions with the flhDC promoter, particularly for MarA and Rob. Additionally, SoxS repressed flagellar gene expression via a posttranscriptional pathway, reducing flhDC translation. The roles of these transcription factors in reducing motility in the presence of salicylic acid were also elucidated, adding a genetic regulatory element to the response of S Typhimurium to this well-characterized chemorepellent. Integration of flagellar gene expression into the mar-sox-rob regulon in S Typhimurium contrasts with findings for closely related species such as Escherichia coli, providing an example of plasticity in the mar-sox-rob regulon throughout the Enterobacteriaceae family.IMPORTANCE The mar-sox-rob regulon is a large and highly conserved stress response network in the Enterobacteriaceae family. Although it is well characterized in E. coli, the extent of this regulon in related species is unclear. Here, the control of costly flagellar gene expression is connected to the mar-sox-rob regulon of S Typhimurium, contrasting with the E. coli regulon model. These findings demonstrate the flexibility of the mar-sox-rob regulon to accommodate novel regulatory targets, and they provide evidence for its broader regulatory role within this family of diverse bacteria.

Correction to: Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

There was a typo in author Andrew Wahba's name in the initial online publication. The original article has been corrected.

Monitoring of intracerebellarly-administered natural killer cells with fluorine-19 MRI.

PURPOSE: Medulloblastoma (MB) is the most common malignant brain tumor in children. Recent studies have shown the ability of natural killer (NK) cells to lyse MB cell lines in vitro, but in vivo successes remain elusive and the efficacy and fate of NK cells in vivo remain unknown. METHODS: To address these questions, we injected MB cells into the cerebellum of immunodeficient mice and examined tumor growth at various days after tumor establishment via bioluminescence imaging. NK cells were labeled with a fluorine-19 (19F) MRI probe and subsequently injected either intratumorally or contralaterally to the tumor in the cerebellum and effect on tumor growth was monitored. RESULTS: The 19F probe efficiently labeled the NK cells and exhibited little cytotoxicity. Fluorine-19 MRI confirmed the successful and accurate delivery of the labeled NK cells to the cerebellum of the mice. Administration of 19F-labeled NK cells suppressed MB growth, with the same efficacy as unlabeled cells. Immunohistochemistry confirmed the presence of NK cells within the tumor, which was associated with induction of apoptosis in tumor cells. NK cell migration to the tumor from a distal location as well as activation of apoptosis was also demonstrated by immunohstochemistry. CONCLUSIONS: Our results show that NK cells present a novel opportunity for new strategies in MB treatment. Further, 19F-labeled NK cells can suppress MB growth while enabling 19F MRI to provide imaging feedback that can facilitate study and optimization of therapeutic paradigms.

Expression of carcinoma, apoptosis, and cell-death-related genes are determinants for sensitivity of pediatric cancer cell lines to lysis by natural killer cells.

Natural killer (NK) cells have potential utility in pediatric cancer immunotherapy for their ability to lyse diverse tumor targets, lack of dependence on mutation-associated tumor antigens, and for their relative safety demonstrated so far in clinical trials. Here, we evaluate the cytotoxic potential of expanded NK cells against a well-characterized panel of pediatric cancer cell lines representing Ewing sarcoma, rhabdomyosarcoma, neuroblastoma, lymphoma, leukemia, and brain tumors. We correlate their sensitivity NK cell lysis with tumor phenotypic, transcriptomic, and genetic determinants, and correlate known immunogenetic determinants with donor NK cell potency. Although ligand expression on cell lines stratified according to hematologic versus nonhematologic cancer types, the sensitivity to NK cell lysis varied widely and did not correlate with cancer type, expression of individual activating or inhibitory ligands, gene-expression clusters of NK cell ligands, disease status (newly diagnosed or relapsed), or MYCN amplification. Rather, sensitivity to NK cell-mediated lysis was associated with a novel 96-gene cluster of predominantly carcinoma-, apoptosis-, and cell death-related pathways, and with functional p53 status. NK cell potency was strongly associated with activating KIR gene content, but not with KIR/KIR-ligand mismatch. This study suggests that adoptive immunotherapy with expanded NK cells has the potential for a wide range of pediatric cancers, identifies potential biomarkers of efficacy and response, and establishes a foundation for using this cell line panel for the preclinical evaluation of immunotherapies.

Self-assembling influenza nanoparticle vaccines elicit broadly neutralizing H1N1 antibodies.

Influenza viruses pose a significant threat to the public and are a burden on global health systems. Each year, influenza vaccines must be rapidly produced to match circulating viruses, a process constrained by dated technology and vulnerable to unexpected strains emerging from humans and animal reservoirs. Here we use knowledge of protein structure to design self-assembling nanoparticles that elicit broader and more potent immunity than traditional influenza vaccines. The viral haemagglutinin was genetically fused to ferritin, a protein that naturally forms nanoparticles composed of 24 identical polypeptides. Haemagglutinin was inserted at the interface of adjacent subunits so that it spontaneously assembled and generated eight trimeric viral spikes on its surface. Immunization with this influenza nanoparticle vaccine elicited haemagglutination inhibition antibody titres more than tenfold higher than those from the licensed inactivated vaccine. Furthermore, it elicited neutralizing antibodies to two highly conserved vulnerable haemagglutinin structures that are targets of universal vaccines: the stem and the receptor binding site on the head. Antibodies elicited by a 1999 haemagglutinin-nanoparticle vaccine neutralized H1N1 viruses from 1934 to 2007 and protected ferrets from an unmatched 2007 H1N1 virus challenge. This structure-based, self-assembling synthetic nanoparticle vaccine improves the potency and breadth of influenza virus immunity, and it provides a foundation for building broader vaccine protection against emerging influenza viruses and other pathogens.

Impact of contemporary patterns of chemotherapy utilization on survival in patients with advanced cancer of the urinary tract: a Retrospective International Study of Invasive/Advanced Cancer of the Urothelium (RISC).

Background: Cisplatin-based combination chemotherapy is the standard treatment of advanced urinary tract cancer (aUTC), but 50% of patients are ineligible for cisplatin according to recently published criteria. We used a multinational database to study patterns of chemotherapy utilization in patients with aUTC and determine their impact on survival. Patients and methods: This was a retrospective study of patients with: UTC (bladder, renal pelvis, ureter or urethra); advanced disease (stages T4b and/or N+ and/or M+); urothelial, squamous or adenocarcinoma histology. Primary objective was overall survival (OS). Eligibility-for-cisplatin was defined by Eastern Cooperative Oncology Group performance status ≤ 1, creatinine clearance ≥ 60 ml/min, no hearing loss, no neuropathy and no heart failure. Cox regression multivariate analyses were used to establish independent associations of cisplatin versus noncisplatin-based chemotherapy on OS. Results: 1794 patients treated between 2000 and 2013 at 29 centers were analyzed. Median follow-up was 29.1 months. About 1333 patients (74%) received first-line chemotherapy: the use of first-line chemotherapy was associated with longer OS: [hazard ratio (HR): 1.91, 95% confidence interval (CI): 1.67-2.20]. Type of first-line chemotherapy received was: cisplatin-based 669 (50%), carboplatin-based 399 (30%) and other 265 (20%). Cisplatin use was an independent favorable prognostic factor (HR: 1.54, 95% CI: 1.35-1.77). This benefit was independent of baseline characteristics or comorbidities but was associated with eligibility-for-cisplatin: eligible patients treated with cisplatin lived longer than those who were not (HR: 1.74, 95% CI: 1.36-2.21), while such benefit was not observed among ineligible patients. About 26% of patients who did not receive cisplatin were eligible for this agent. Median OS of ineligible patients was poor irrespective of the chemotherapy used. Conclusions: The importance of applying published criteria of eligibility-for-cisplatin was confirmed in a multinational, real-world setting in aUTC. The reasons for deviations from these criteria set targets to improve adherence. Effective therapies for cisplatin-ineligible patients are needed.

A Numerically Subdominant CD8 T Cell Response to Matrix Protein of Respiratory Syncytial Virus Controls Infection with Limited Immunopathology.

CD8 T cells are involved in pathogen clearance and infection-induced pathology in respiratory syncytial virus (RSV) infection. Studying bulk responses masks the contribution of individual CD8 T cell subsets to protective immunity and immunopathology. In particular, the roles of subdominant responses that are potentially beneficial to the host are rarely appreciated when the focus is on magnitude instead of quality of response. Here, by evaluating CD8 T cell responses in CB6F1 hybrid mice, in which multiple epitopes are recognized, we found that a numerically subdominant CD8 T cell response against DbM187 epitope of the virus matrix protein expressed high avidity TCR and enhanced signaling pathways associated with CD8 T cell effector functions. Each DbM187 T effector cell lysed more infected targets on a per cell basis than the numerically dominant KdM282 T cells, and controlled virus replication more efficiently with less pulmonary inflammation and illness than the previously well-characterized KdM282 T cell response. Our data suggest that the clinical outcome of viral infections is determined by the integrated functional properties of a variety of responding CD8 T cells, and that the highest magnitude response may not necessarily be the best in terms of benefit to the host. Understanding how to induce highly efficient and functional T cells would inform strategies for designing vaccines intended to provide T cell-mediated immunity.