Distinct immune responses associated with vaccination status and protection outcomes after malaria challenge.

Understanding immune mechanisms that mediate malaria protection is critical for improving vaccine development. Vaccination with radiation-attenuated Plasmodium falciparum sporozoites (PfRAS) induces high level of sterilizing immunity against malaria and serves as a valuable tool for the study of protective mechanisms. To identify vaccine-induced and protection-associated responses during malarial infection, we performed transcriptome profiling of whole blood and in-depth cellular profiling of PBMCs from volunteers who received either PfRAS or noninfectious mosquito bites, followed by controlled human malaria infection (CHMI) challenge. In-depth single-cell profiling of cell subsets that respond to CHMI in mock-vaccinated individuals showed a predominantly inflammatory transcriptome response. Whole blood transcriptome analysis revealed that gene sets associated with type I and II interferon and NK cell responses were increased in prior to CHMI while T and B cell signatures were decreased as early as one day following CHMI in protected vaccinees. In contrast, non-protected vaccinees and mock-vaccinated individuals exhibited shared transcriptome changes after CHMI characterized by decreased innate cell signatures and inflammatory responses. Additionally, immunophenotyping data showed different induction profiles of vδ2+ γδ T cells, CD56+ CD8+ T effector memory (Tem) cells, and non-classical monocytes between protected vaccinees and individuals developing blood-stage parasitemia, following treatment and resolution of infection. Our data provide key insights in understanding immune mechanistic pathways of PfRAS-induced protection and infective CHMI. We demonstrate that vaccine-induced immune response is heterogenous between protected and non-protected vaccinees and that inducted-malaria protection by PfRAS is associated with early and rapid changes in interferon, NK cell and adaptive immune responses. Trial Registration: ClinicalTrials.gov NCT01994525.

Systems analysis of immune responses to attenuated P. falciparum malaria sporozoite vaccination reveals excessive inflammatory signatures correlating with impaired immunity.

Immunization with radiation-attenuated sporozoites (RAS) can confer sterilizing protection against malaria, although the mechanisms behind this protection are incompletely understood. We performed a systems biology analysis of samples from the Immunization by Mosquito with Radiation Attenuated Sporozoites (IMRAS) trial, which comprised P. falciparum RAS-immunized (PfRAS), malaria-naive participants whose protection from malaria infection was subsequently assessed by controlled human malaria infection (CHMI). Blood samples collected after initial PfRAS immunization were analyzed to compare immune responses between protected and non-protected volunteers leveraging integrative analysis of whole blood RNA-seq, high parameter flow cytometry, and single cell CITEseq of PBMCs. This analysis revealed differences in early innate immune responses indicating divergent paths associated with protection. In particular, elevated levels of inflammatory responses early after the initial immunization were detrimental for the development of protective adaptive immunity. Specifically, non-classical monocytes and early type I interferon responses induced within 1 day of PfRAS vaccination correlated with impaired immunity. Non-protected individuals also showed an increase in Th2 polarized T cell responses whereas we observed a trend towards increased Th1 and T-bet+ CD8 T cell responses in protected individuals. Temporal differences in genes associated with natural killer cells suggest an important role in immune regulation by these cells. These findings give insight into the immune responses that confer protection against malaria and may guide further malaria vaccine development. Trial registration: ClinicalTrials.gov NCT01994525.

Retrospective analysis of radiological investigation of surgically excised head and neck lipomas.

PURPOSE: Differentiating benign lipomas from malignant causes is challenging and preoperative investigative guidelines are not well-defined. The purpose of this study was to retrospectively identify cases of head and neck lipomas that were surgically resected over a 5-year period and to identify the radiological modality chosen and features discussed in the final report. Multidisciplinary outcomes and pathology reports were examined with a view to identifying high risk features of a lipoma to aid in future risk stratification. METHODS: Retrospective chart review of pathology characteristics, radiological features (modality, size, calcifications, septations, globular/nodular foci), multidisciplinary discussion and history of presenting complaint was performed. RESULTS: Two liposarcomas and 138 lipomas were identified. Twenty-two percent of all lipomas received radiological investigation. Twenty-two percent of imaging referrals were possibly inappropriate. Furthermore, radiological features suggestive of malignancy were not present in the final radiology report, X2 = 28.8, p < 0.0001. CONCLUSION: As expected, the incidence of liposarcoma is low. There is limited awareness of radiology referral guidelines superimposed with a tendency to over-investigate lipomas. Furthermore, radiological features suggestive of malignancy were inconsistently reported on and not documented in multidisciplinary discussions. Therefore, we propose a multidisciplinary checklist for referring physicians and radiologists to aid in diagnostic work-up.

T-SPOT.TB Reactivity in Southern African Children With and Without in Utero Human Immunodeficiency Virus Exposure.

Infants who are human immunodeficiency virus (HIV)-exposed uninfected (iHEU) experience higher risk of infectious morbidity than infants HIV-unexposed uninfected (iHUU). We compared tuberculosis (TB) infection prevalence in 418 Bacillus Calmette-Guérin vaccinated sub-Saharan African iHEU and iHUU aged 9-18 months using T-SPOT.TB. Prevalence of TB infection was low and did not differ by HIV exposure status.

Parathyroid carcinoma: Current management and outcomes - A systematic review.

OBJECTIVE: Parathyroid carcinoma is rarely encountered in clinical practice. When faced with this clinical challenge, there is currently a paucity of evidence available for the optimal management of patients with parathyroid carcinoma. This systematic review synthesizes the available literature to evaluate the optimal management approach, thus providing guidance for future management. METHODS: A systematic review was conducted according to PRISMA guidelines using Ovid MEDLINE, EMBASE, and PubMed databases for studies, published in English, reporting on parathyroid carcinoma. Full text of potentially eligible articles were reviewed by two authors independently and eligible studies selected. Treatment options and associated outcomes were evaluated. Descriptive statistics were used to describe pooled patient cohorts. RESULTS: 3203 articles were initially identified using the search criteria with 59 full-text articles then screened for eligibility. Seven articles, all retrospective studies, concerning 2307 patients (median 224/study). Parathyroidectomy alone was the most frequently utilised surgical approach across all studies, followed by en-bloc resection (with adjacent thyroid and/or nodal tissue). There was no difference in post-operative morbidity, mortality or survival between surgical approaches (p < 0.005). Patients who underwent either form of surgery had longer overall survival than those managed non-operatively (p < 0.005). CONCLUSION: Surgical resection is the optimal treatment of parathyroid carcinoma. However there remains no consensus on the optimal extent of surgery, and as such future randomised prospective studies are necessary to evaluate the effects of different surgical approaches on morbidity, mortality and oncologic outcomes. Following resection, long-term surveillance with PTH is advised.

Use of a Contained Mycobacterium tuberculosis Mouse Infection Model to Predict Active Disease and Containment in Humans.

Previous studies have identified whole-blood transcriptional risk and disease signatures for tuberculosis; however, several lines of evidence suggest that these signatures primarily reflect bacterial burden, which increases before symptomatic disease. We found that the peripheral blood transcriptome of mice with contained Mycobacterium tuberculosis infection (CMTI) has striking similarities to that of humans with active tuberculosis and that a signature derived from these mice predicts human disease with accuracy comparable to that of signatures derived directly from humans. A set of genes associated with immune defense are up-regulated in mice with CMTI but not in humans with active tuberculosis, suggesting that their up-regulation is associated with bacterial containment. A signature comprising these genes predicts both protection from tuberculosis disease and successful treatment at early time points where current signatures are not predictive. These results suggest that detailed study of the CMTI model may enable identification of biomarkers for human tuberculosis.

Contained Mycobacterium tuberculosis infection induces concomitant and heterologous protection.

Progress in tuberculosis vaccine development is hampered by an incomplete understanding of the immune mechanisms that protect against infection with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. Although the M72/ASOE1 trial yielded encouraging results (54% efficacy in subjects with prior exposure to Mtb), a highly effective vaccine against adult tuberculosis remains elusive. We show that in a mouse model, establishment of a contained and persistent yet non-pathogenic infection with Mtb ("contained Mtb infection", CMTB) rapidly and durably reduces tuberculosis disease burden after re-exposure through aerosol challenge. Protection is associated with elevated activation of alveolar macrophages, the first cells that respond to inhaled Mtb, and accelerated recruitment of Mtb-specific T cells to the lung parenchyma. Systems approaches, as well as ex vivo functional assays and in vivo infection experiments, demonstrate that CMTB reconfigures tissue resident alveolar macrophages via low grade interferon-γ exposure. These studies demonstrate that under certain circumstances, the continuous interaction of the immune system with Mtb is beneficial to the host by maintaining elevated innate immune responses.

Early whole blood transcriptional responses to radiation-attenuated Plasmodium falciparum sporozoite vaccination in malaria naïve and malaria pre-exposed adult volunteers.

BACKGROUND: Vaccination with radiation-attenuated Plasmodium falciparum sporozoites is known to induce protective immunity. However, the mechanisms underlying this protection remain unclear. In this work, two recent radiation-attenuated sporozoite vaccination studies were used to identify potential transcriptional correlates of vaccination-induced protection. METHODS: Longitudinal whole blood RNAseq transcriptome responses to immunization with radiation-attenuated P. falciparum sporozoites were analysed and compared across malaria-naïve adult participants (IMRAS) and malaria-experienced adult participants (BSPZV1). Parasite dose and method of delivery differed between trials, and immunization regimens were designed to achieve incomplete protective efficacy. Observed protective efficacy was 55% in IMRAS and 20% in BSPZV1. Study vaccine dosings were chosen to elicit both protected and non-protected subjects, so that protection-associated responses could be identified. RESULTS: Analysis of comparable time points up to 1 week after the first vaccination revealed a shared cross-study transcriptional response programme, despite large differences in number and magnitude of differentially expressed genes between trials. A time-dependent regulatory programme of coherent blood transcriptional modular responses was observed, involving induction of inflammatory responses 1-3 days post-vaccination, with cell cycle responses apparent by day 7 in protected individuals from both trials. Additionally, strongly increased induction of inflammation and interferon-associated responses was seen in non-protected IMRAS participants. All individuals, except for non-protected BSPZV1 participants, showed robust upregulation of cell-cycle associated transcriptional responses post vaccination. CONCLUSIONS: In summary, despite stark differences between the two studies, including route of vaccination and status of malaria exposure, responses were identified that were associated with protection after PfRAS vaccination. These comprised a moderate early interferon response peaking 2 days post vaccination, followed by a later proliferative cell cycle response steadily increasing over the first 7 days post vaccination. Non-protection is associated with deviations from this model, observed in this study with over-induction of early interferon responses in IMRAS and failure to mount a cell cycle response in BSPZV1.

Four-Gene Pan-African Blood Signature Predicts Progression to Tuberculosis.

Rationale: Contacts of patients with tuberculosis (TB) constitute an important target population for preventive measures because they are at high risk of infection with Mycobacterium tuberculosis and progression to disease.Objectives: We investigated biosignatures with predictive ability for incident TB.Methods: In a case-control study nested within the Grand Challenges 6-74 longitudinal HIV-negative African cohort of exposed household contacts, we employed RNA sequencing, PCR, and the pair ratio algorithm in a training/test set approach. Overall, 79 progressors who developed TB between 3 and 24 months after diagnosis of index case and 328 matched nonprogressors who remained healthy during 24 months of follow-up were investigated.Measurements and Main Results: A four-transcript signature derived from samples in a South African and Gambian training set predicted progression up to two years before onset of disease in blinded test set samples from South Africa, the Gambia, and Ethiopia with little population-associated variability, and it was also validated in an external cohort of South African adolescents with latent M. tuberculosis infection. By contrast, published diagnostic or prognostic TB signatures were predicted in samples from some but not all three countries, indicating site-specific variability. Post hoc meta-analysis identified a single gene pair, C1QC/TRAV27 (complement C1q C-chain / T-cell receptor-α variable gene 27) that would consistently predict TB progression in household contacts from multiple African sites but not in infected adolescents without known recent exposure events.Conclusions: Collectively, we developed a simple whole blood-based PCR test to predict TB in recently exposed household contacts from diverse African populations. This test has potential for implementation in national TB contact investigation programs.

GlycoProfileAssigner: automated structural assignment with error estimation for glycan LC data.

MOTIVATION: Sequencing glycan structures is a difficult problem that requires the use of multiple experimental approaches. One powerful approach to glycan sequencing is the combination of liquid chromatography with sequential exoglycosidase digestions; however, interpreting this can be difficult and time-consuming. To aid this process, we introduce GlycoProfileAssigner, software for automated structural assignment of glycan profile data from liquid chromatography experiments. RESULTS: GlycoProfileAssigner has been tested on human IgG data, and can retrieve the correct structure in 14 out of 16 peaks tested. AVAILABILITY AND IMPLEMENTATION: The programme and its source code is available at https://bitbucket.org/fergaljd/glycoprofileassigner CONTACT: : pauline.rudd@nibrt.ie SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

Computational survey of peptides derived from disulphide-bonded protein loops that may serve as mediators of protein-protein interactions.

BACKGROUND: Bioactive cyclic peptides derived from natural sources are well studied, particularly those derived from non-ribosomal synthetases in fungi or bacteria. Ribosomally synthesised bioactive disulphide-bonded loops represent a large, naturally enriched library of potential bioactive compounds, worthy of systematic investigation. RESULTS: We examined the distribution of short cyclic loops on the surface of a large number of proteins, especially membrane or extracellular proteins. Available three-dimensional structures highlighted a number of disulphide-bonded loops responsible for the majority of the likely binding interactions in a variety of protein complexes, due to their location at protein-protein interfaces. We find that disulphide-bonded loops at protein-protein interfaces may, but do not necessarily, show biological activity independent of their parent protein. Examining the conservation of short disulphide bonded loops in proteins, we find a small but significant increase in conservation inside these loops compared to surrounding residues. We identify a subset of these loops that exhibit a high relative conservation, particularly among peptide hormones. CONCLUSIONS: We conclude that short disulphide-bonded loops are found in a wide variety of biological interactions. They may retain biological activity outside their parent proteins. Such structurally independent peptides may be useful as biologically active templates for the development of novel modulators of protein-protein interactions.

Ocular lymphoma: overcoming a diagnostic challenge with an endoscopic sinus approach.

BACKGROUND: Ocular lymphomas are rare, indolent tumours that pose a diagnostic challenge. Patients may be asymptomatic or present with symptoms of increased orbital mass. An index of suspicion is required alongside an understanding of the diagnostics and staging required to facilitate expedited multi-disciplinary work-up and management. METHODS: A comprehensive literature review was performed. We present a series of three cases, each presenting their own diagnostic challenge. RESULTS: Although ocular lymphomas are notoriously difficult to diagnose histologically, our case series show procurement of a diagnostic histological sample using an image-guided endoscopic sinus approach. CONCLUSIONS: Although not typically involved in the diagnosis or management, the otolaryngologist may encounter ocular lymphoma masquerading as sinus disease or with disseminated disease at other sites in the head and neck. Endoscopic sinus surgery is considered a safe, effective approach for achieving a histological diagnosis in these cases.

CD4-mediated immunity shapes neutrophil-driven tuberculous pathology.

Pulmonary Mycobacterium tuberculosis (Mtb) infection results in highly heterogeneous lesions ranging from granulomas with central necrosis to those primarily comprised of alveolitis. While alveolitis has been associated with prior immunity in human post-mortem studies, the drivers of these distinct pathologic outcomes are poorly understood. Here, we show that these divergent lesion structures can be modeled in C3HeB/FeJ mice and are regulated by prior immunity. Using quantitative imaging, scRNAseq, and flow cytometry, we demonstrate that Mtb infection in the absence of prior immunity elicits dysregulated neutrophil recruitment and necrotic granulomas. In contrast, prior immunity induces rapid recruitment and activation of T cells, local macrophage activation, and diminished late neutrophil responses. Depletion studies at distinct infection stages demonstrated that neutrophils are required for early necrosis initiation and necrosis propagation at chronic stages, whereas early CD4 T cell responses prevent neutrophil feedforward circuits and necrosis. Together, these studies reveal fundamental determinants of tuberculosis lesion structure and pathogenesis, which have important implications for new strategies to prevent or treat tuberculosis.

Mycobacterium tuberculosis infection associated immune perturbations correlate with antiretroviral immunity.

Infection with Mycobacterium tuberculosis (MTB) remains one of the most important opportunistic infections in people with HIV-1 (PWH). While active Tuberculosis (TB) leads to rapid progression of immunodeficiency in PWH, the interaction between MTB and HIV-1 during the asymptomatic phase of both infections remains poorly understood. In a cohort of individuals with HIV (PWH) with and without suppressed HIV-1 viral load, the transcriptomic profiles of peripheral blood mononuclear cells (PBMC) clustered in individuals infected with Mycobacterium tuberculosis (MTB) compared to carefully matched controls. Subsequent functional annotation analysis disclosed alterations in the IL-6, TNF, and KRAS pathways. Notably, MTB-associated genes demonstrated an inverse correlation with HIV-1 viremia, evident at both on individual gene level and when employed as a gene score. In sum, our data show that MTB infection in PWH is associated with a shift in the activation state of the immune system, displaying an inverse relationship with HIV-1 viral load. These results could provide an explanation for the observed increased antiretroviral control associated with MTB infection in PWH.

Preimmunization correlates of protection shared across malaria vaccine trials in adults.

Identifying preimmunization biological characteristics that promote an effective vaccine response offers opportunities for illuminating the critical immunological mechanisms that confer vaccine-induced protection, for developing adjuvant strategies, and for tailoring vaccination regimens to individuals or groups. In the context of malaria vaccine research, studying preimmunization correlates of protection can help address the need for a widely effective malaria vaccine, which remains elusive. In this study, common preimmunization correlates of protection were identified using transcriptomic data from four independent, heterogeneous malaria vaccine trials in adults. Systems-based analyses showed that a moderately elevated inflammatory state prior to immunization was associated with protection against malaria challenge. Functional profiling of protection-associated genes revealed the importance of several inflammatory pathways, including TLR signaling. These findings, which echo previous studies that associated enhanced preimmunization inflammation with protection, illuminate common baseline characteristics that set the stage for an effective vaccine response across diverse malaria vaccine strategies in adults.

Longitudinal immune profiling after radiation-attenuated sporozoite vaccination reveals coordinated immune processes correlated with malaria protection.

Background: Identifying immune processes required for liver-stage sterilizing immunity to malaria remains an open problem. The IMRAS trial comprised 5x immunizations with radiation-attenuated sporozoites resulting in 55% protection from subsequent challenge. Methods: To identify correlates of vaccination and protection, we performed detailed systems immunology longitudinal profiling of the entire trial time course including whole blood transcriptomics, detailed PBMC cell phenotyping and serum antigen array profiling of 11 IMRAS radiation-attenuated sporozoite (RAS) vaccinees at up to 21 timepoints each. Results: RAS vaccination induced serum antibody responses to CSP, TRAP, and AMA1 in all vaccinees. We observed large numbers of differentially expressed genes associated with vaccination response and protection, with distinctly differing transcriptome responses elicited after each immunization. These included inflammatory and proliferative responses, as well as increased abundance of monocyte and DC subsets after each immunization. Increases in Vδ2 γδ; T cells and MAIT cells were observed in response to immunization over the course of study, and CD1c+ CD40+ DC abundance was significantly associated with protection. Interferon responses strongly differed between protected and non-protected individuals with high interferon responses after the 1st immunization, but not the 2nd-5th. Blood transcriptional interferon responses were correlated with abundances of different circulating classical and non-classical monocyte populations. Conclusions: This study has revealed multiple coordinated immunological processes induced by vaccination and associated with protection. Our work represents the most detailed immunological profiling of a RAS vaccine trial performed to date and will guide the design and interpretation of future malaria vaccine trials.

Transoral robotic surgery in Ireland: the beginning.

BACKGROUND: Transoral robotic surgery (TORS) has shown promising results in the treatment of myriad head and neck pathologies but is now most commonly used in the investigation and management of oropharyngeal squamous cell carcinoma. AIMS: The aim of this study was to report our cases of the newly introduced TORS, particularly its role in identifying primary of unknown origin and the potential implications for patients. A literature review and our early experience should begin to debunk some of the criticisms of TORS including setup times and cost. METHODS: Prospective data was collected from all patients undergoing transoral robotic surgery including demographics, indication, histology results in primary of unknown origin and complications. RESULTS: We have performed 36 TORS procedures in total ranging from intermediate to major complex. Our complication rate is low, and this has improved with the passage of time. Haemorrhage rates remain at 5.6% (n = 2), and the average length of stay is 1 day. Successful identification of a primary tumour in cancer of unknown primary was 80% (n = 8). CONCLUSIONS: We anticipate the integration of TORS into routine practice in the investigation and management of a number of ENT pathologies following robust clinical trials.

Neutrophil degranulation, NETosis and platelet degranulation pathway genes are co-induced in whole blood up to six months before tuberculosis diagnosis.

Mycobacterium tuberculosis (M.tb) causes tuberculosis (TB) and remains one of the leading causes of mortality due to an infectious pathogen. Host immune responses have been implicated in driving the progression from infection to severe lung disease. We analyzed longitudinal RNA sequencing (RNAseq) data from the whole blood of 74 TB progressors whose samples were grouped into four six-month intervals preceding diagnosis (the GC6-74 study). We additionally analyzed RNAseq data from an independent cohort of 90 TB patients with positron emission tomography-computed tomography (PET-CT) scan results which were used to categorize them into groups with high and low levels of lung damage (the Catalysis TB Biomarker study). These groups were compared to non-TB controls to obtain a complete whole blood transcriptional profile for individuals spanning from early stages of M.tb infection to TB diagnosis. The results revealed a steady increase in the number of genes that were differentially expressed in progressors at time points closer to diagnosis with 278 genes at 13-18 months, 742 at 7-12 months and 5,131 detected 1-6 months before diagnosis and 9,205 detected in TB patients. A total of 2,144 differentially expressed genes were detected when comparing TB patients with high and low levels of lung damage. There was a large overlap in the genes upregulated in progressors 1-6 months before diagnosis (86%) with those in TB patients. A comprehensive pathway analysis revealed a potent activation of neutrophil and platelet mediated defenses including neutrophil and platelet degranulation, and NET formation at both time points. These pathways were also enriched in TB patients with high levels of lung damage compared to those with low. These findings suggest that neutrophils and platelets play a critical role in TB pathogenesis, and provide details of the timing of specific effector mechanisms that may contribute to TB lung pathology.

CycloPs: generating virtual libraries of cyclized and constrained peptides including nonnatural amino acids.

We introduce CycloPs, software for the generation of virtual libraries of constrained peptides including natural and nonnatural commercially available amino acids. The software is written in the cross-platform Python programming language, and features include generating virtual libraries in one-dimensional SMILES and three-dimensional SDF formats, suitable for virtual screening. The stand-alone software is capable of filtering the virtual libraries using empirical measurements, including peptide synthesizability by standard peptide synthesis techniques, stability, and the druglike properties of the peptide. The software and accompanying Web interface is designed to enable the rapid generation of large, structurally diverse, synthesizable virtual libraries of constrained peptides quickly and conveniently, for use in virtual screening experiments. The stand-alone software, and the Web interface for evaluating these empirical properties of a single peptide, are available at http://bioware.ucd.ie .