In vitro evolution of antibody affinity via insertional scanning mutagenesis of an entire antibody variable region.

We report a systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD (transposition-based random insertion and deletion mutagenesis) was used to generate large libraries with random in-frame InDels across the entire single-chain variable fragment gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (InScaM). An overall 256-fold affinity improvement of an anti-IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach, and suggests that the results of this InDel mutagenesis and conventional exploration of point mutations can synergize to generate antibodies with higher affinity.

Physiological behavior during stress anticipation across different chronic stress exposure adaptive models.

Anticipation of stress induces physiological, behavioral and cognitive adjustments that are required for an appropriate response to the upcoming situation. Additional research examining the response of cardiopulmonary parameters and stress hormones during anticipation of stress in different chronic stress adaptive models is needed. As an addition to our previous research, a total of 57 subjects (16 elite male wrestlers, 21 water polo player and 20 sedentary subjects matched for age) were analyzed. Cardiopulmonary exercise testing (CPET) on a treadmill was used as the laboratory stress model; peak oxygen consumption (VO2) was obtained during CPET. Plasma levels of adrenocorticotropic hormone (ACTH), cortisol, alpha-melanocyte stimulating hormone (alpha-MSH) and N-terminal-pro-B type natriuretic peptide (NT-pro-BNP) were measured by radioimmunometric, radioimmunoassay and immunoassay sandwich technique, respectively, together with cardiopulmonary measurements, 10 minutes pre-CPET and at the initiation of CPET. The response of diastolic blood pressure and heart rate was different between groups during stress anticipation (p = 0.019, 0.049, respectively), while systolic blood pressure, peak VO2 and carbon-dioxide production responses were similar. ACTH and cortisol increased during the experimental condition, NT-pro-BNP decreased and alpha-MSH remained unchanged. All groups had similar hormonal responses during stress anticipation with the exception of the ACTH/cortisol ratio. In all three groups, ΔNT-pro-BNP during stress anticipation was the best independent predictor of peak VO2 (B = 36.01, r = 0.37, p = 0.001). In conclusion, the type of chronic stress exposure influences the hemodynamic response during anticipation of physical stress and the path of hormonal stress axis activation. Stress hormones released during stress anticipation may hold predictive value for overall cardiopulmonary performance during the stress condition.

LAY SUMMARYThe study revealed differences in hormonal and hemodynamic responses during anticipation of stress between athletes and sedentary participants. Stress hormones released during stress anticipation may hold predictive value for overall cardiopulmonary performance during the stress condition.

Understanding the experience of rumination and worry: A descriptive qualitative survey study.

OBJECTIVES: Rumination and worry have been implicated in the onset, severity, maintenance and relapse risk of depression and anxiety disorders. Despite this, little research has examined individuals' personal experiences of these processes. This study investigates how individuals experience these processes, which will provide insight into these common features of mental disorders and inform the development of an online intervention specifically targeting rumination and worry. DESIGN: An online qualitative survey was conducted to gain insight into people's personal definitions, experiences with and understandings of rumination and worry. METHODS: Participants answered open- and close-ended questions about their personal understanding of rumination and worry, typical thought content, triggers, frequency, duration and coping strategies. Participant responses were coded into themes. Participants also completed self-report questionnaires of depression, anxiety and stress and repetitive negative thinking. RESULTS: Two hundred and seven adults completed the online survey (76% female; mean age = 28.2 years, range = 17-71), 51% of whom reported previously experiencing depression and anxiety. All participants were familiar with the concept of worry, whereas 28% of participants indicated they had never heard of rumination. Participants reported most commonly ruminating and/or worrying about personal relationships, past mistakes, negative experiences and conversations/social interactions. The most commonly reported triggers for rumination and/or worry were social situations/interpersonal interactions (25%) and negative events/experiences (24%). Distraction was the most common coping strategy (48%); however, 21% reported being unable to stop themselves from ruminating and/or worrying. CONCLUSIONS: The results provide a unique insight into the personal experiences and understandings of rumination and worry of potential end users of treatment programs targeting these processes.

Experimentally guided computational antibody affinity maturation with de novo docking, modelling and rational design.

Antibodies are an important class of therapeutics that have significant clinical impact for the treatment of severe diseases. Computational tools to support antibody drug discovery have been developing at an increasing rate over the last decade and typically rely upon a predetermined co-crystal structure of the antibody bound to the antigen for structural predictions. Here, we show an example of successful in silico affinity maturation of a hybridoma derived antibody, AB1, using just a homology model of the antibody fragment variable region and a protein-protein docking model of the AB1 antibody bound to the antigen, murine CCL20 (muCCL20). In silico affinity maturation, together with alanine scanning, has allowed us to fine-tune the protein-protein docking model to subsequently enable the identification of two single-point mutations that increase the affinity of AB1 for muCCL20. To our knowledge, this is one of the first examples of the use of homology modelling and protein docking for affinity maturation and represents an approach that can be widely deployed.

SAbPred: a structure-based antibody prediction server.

SAbPred is a server that makes predictions of the properties of antibodies focusing on their structures. Antibody informatics tools can help improve our understanding of immune responses to disease and aid in the design and engineering of therapeutic molecules. SAbPred is a single platform containing multiple applications which can: number and align sequences; automatically generate antibody variable fragment homology models; annotate such models with estimated accuracy alongside sequence and structural properties including potential developability issues; predict paratope residues; and predict epitope patches on protein antigens. The server is available at http://opig.stats.ox.ac.uk/webapps/sabpred.

The H3 loop of antibodies shows unique structural characteristics.

The H3 loop in the Complementarity Determining Region of antibodies plays a key role in their ability to bind the diverse space of potential antigens. It is also exceptionally difficult to model computationally causing a significant hurdle for in silico development of antibody biotherapeutics. In this article, we show that most H3s have unique structural characteristics which may explain why they are so challenging to model. We found that over 75% of H3 loops do not have a sub-Angstrom structural neighbor in the non-antibody world. Also, in a comparison with a nonredundant set of all protein fragments over 30% of H3 loops have a unique structure, with the average for all of other loops being less than 3%. We further observed that this structural difference can be seen at the level of four residue fragments where H3 loops present numerous novel conformations, and also at the level of individual residues with Tyrosine and Glycine often found in energetically unfavorable conformations. Proteins 2017; 85:1311-1318. © 2017 Wiley Periodicals, Inc.

Antibody informatics for drug discovery.

More and more antibody therapeutics are being approved every year, mainly due to their high efficacy and antigen selectivity. However, it is still difficult to identify the antigen, and thereby the function, of an antibody if no other information is available. There are obstacles inherent to the antibody science in every project in antibody drug discovery. Recent experimental technologies allow for the rapid generation of large-scale data on antibody sequences, affinity, potency, structures, and biological functions; this should accelerate drug discovery research. Therefore, a robust bioinformatic infrastructure for these large data sets has become necessary. In this article, we first identify and discuss the typical obstacles faced during the antibody drug discovery process. We then summarize the current status of three sub-fields of antibody informatics as follows: (i) recent progress in technologies for antibody rational design using computational approaches to affinity and stability improvement, as well as ab-initio and homology-based antibody modeling; (ii) resources for antibody sequences, structures, and immune epitopes and open drug discovery resources for development of antibody drugs; and (iii) antibody numbering and IMGT. Here, we review "antibody informatics," which may integrate the above three fields so that bridging the gaps between industrial needs and academic solutions can be accelerated. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

Recurrent spontaneous abortions, Hashimoto thyroiditis and alopecia totalis: response to anticoagulation and intravenous immunoglobulin therapy.

INTRODUCTION: Although numerous studies indicated a link between antithyroid antibodies and recurrent spontaneous abortions (RSA), consensus on the treatment of this condition is still lacking. CASE REPORT: We present a case of a 35-year-old pregnant woman (gestation week 4) with primary hypothyroidism, total alopecia, high level of positive antithyroid antibodies, and history of two recurrent spontaneous abortions in early pregnancy. Along with L-thyroxin substitution, intravenous human immunoglobulin (IVIg) combined with anticoagulation and antiaggregation therapy was introduced. During pregnancy her scalp hair completely re-grew, and following gestation week 39 she delivered healthy female child. CONCLUSION: Thyroid antibodies could contribute to previous recurrent abortions in our patient. It is suggested that in older primiparas with Hashimoto thyroiditis and history of RSA, a combined treatment with IVIg, anticoagulation and antiaggregation therapy should be considered.

Glucocorticoid receptor and molecular chaperones in the pathogenesis of adrenal incidentalomas: potential role of reduced sensitivity to glucocorticoids.

Glucocorticoid (GC) sensitivity depends on glucocorticoid receptor (GR) and heat shock proteins (Hsps). We investigated whether common GR genes (ER22/23EK, N363S, Bcl I, and 9ß) and adrenocorticotropin receptor promoter polymorphisms influence susceptibility for unilateral adrenal incidentaloma (AI), plus GR and Hsp expression in tumorous (n = 19), peritumorous (n = 13) and normal adrenocortical (n = 11) tissues. Patients (n = 112), population-matched controls (n = 100) and tumor tissues (n = 32) were genotyped for these polymorphisms. Postdexamethasone serum cortisol was higher in patients (p < 0.001). GR gene variants, larger allele of Bcl I (odds ratio [OR] 2.9; 95% confidence interval [CI] 1.7-5.1; p < 0.001] and minor allele of 9ß (OR 3.0; 95% CI 1.6-5.7; p < 0.001) were independent predictors of AI. In patients, the first allele is linked with larger tumors (p = 0.002) and the latter with higher postdexamethasone cortisol levels (p = 0.025). Both allele carriers had lesser waist circumference (p = 0.02), similar adrenocorticotropin and higher basal (p = 0.024) and postdexamethasone cortisol concentrations (p < 0.001). Tumorous and constitutional genotypes were similar. GR-D is the major receptor isoform in normal adrenal cortex by Western blotting. Loss of other receptor isoforms, decrease in immunostaining for GR (p < 0.0001), underexpression of chaperones (p ≤ 0.01) and the presence of inducible Hsp70 were found in adenomas. In conclusion, GR gene variants, C allele of Bcl I and minor allele of 9ß, are associated with AIs. Their concurrent presence in patients reduces GC sensitivity. Normal adrenal cortex preferentially expresses GR-D. In adenomas, the lack of other GR isoforms and underexpression of heat shock proteins perhaps permanently impair GC signaling, which could promote dysregulated cortisol production and tumor growth. The innate GC sensitivity probably modifies these effects.

Diagnostic and prognostic value of 99mTc-Tektrotyd scintigraphy and 18F-FDG PET/CT in a single-center cohort of neuroendocrine tumors.

Introduction: The aim was to assess the diagnostic value of 99mTc-Tektrotyd scintigraphy (TCT) and positron emission tomography/computed tomography using F-18 fluorodeoxyglucose (18F-FDG PET/CT) in the detection and follow-up of neuroendocrine tumors (NETs), and their predictive value for disease progression. Material and methods: In this retrospective cohort, TCT and 18F-FDG PET/CT were performed in 90 patients (37 men, 53 women, mean age 52.7 ±15.1), with NET. Correlation of Ki67 and tumor grade versus Krenning score and SUVmax was assessed, Kaplan-Meier analysis was used for progression-free survival (PFS), and Cox regression analysis was performed to identify the association between progression-related factors and PFS. Results: Out of 90, true positive TCT was detected in 56 (62.2%) patients, true negative in 19 (21.1%), false positive in 4 (4.4%), false negative in 11 (12.2%), while 18F-FDG PET/CT was true positive in 69 (76.7%) patients, true negative in 10 (11.1%), false positive in 5 (5.5%), false negative in 6 (6.7%). Mean 18F-FDG PET/CT SUVmax was 6.8 ±6.2. Diagnostic sensitivity of TCT was 83.6%, specificity 82.6%, accuracy 83.3% vs. 18F-FDG PET/CT sensitivity was 92.0%, specificity 66.7%, accuracy 87.8%. A significant correlation between Ki67 and SUVmax was found in positive 18F-FDG PET/CT findings, unlike the correlation between Ki67 and Krenning score. Median PFS was 25 months (95% CI: 18.2-31.8), in 18F-FDG PET/CT positive patients 23 months (95% CI: 16.3-29.7) and 18F-FDG PET/CT negative 26 months (p = 0.279). Progression-free survival predictors were SUVmax and Krenning score. Conclusions: In our study, TCT and 18F-FDG PET/CT have high diagnostic accuracy in detection of NET. Higher Krenning score on TCT and SUVmax in positive 18F-FDG PET/CT findings are predictors of disease progression. 99mTc-Tektrotyd scintigraphy and 18F-FDG PET/CT can be useful complementary tools in management of patients with NETs and in predicting patients' outcome.

Autoimmune/inflammatory syndrome induced by adjuvants in a woman with Hashimoto thyroiditis and familial autoimmunity-a case report and literature review.

Introduction: Autoimmune/inflammatory syndrome induced by adjuvants (ASIA) consists of a wide spectrum of symptoms and immunological features that are believed to develop in predisposed individuals after exposure to an adjuvant, including a silicone breast implant (SBI). Different autoimmune diseases (AIDs) have been associated with ASIA, but ASIA development after SBI in women with Hashimoto thyroiditis (HT) and familial autoimmunity has rarely been described. Case report: A 37-year-old woman presented in 2019 with arthralgia, sicca symptoms, fatigue, + antinuclear antibody (ANA), + anti SSA, and + anticardiolipin Immunoglobulin G (IgG) antibodies. She was diagnosed with HT and vitamin D deficiency in 2012. The familial autoimmunity was present: the patient's mother had been diagnosed with systemic lupus erythematosus and secondary Sjogren's syndrome and her grandmother with cutaneous lupus and pernicious anemia. In 2017, the patient had a cosmetic SBI procedure that was complicated by repeated right breast capsulitis. After 2 years of irregular visits due to COVID-19, she presented with + ANA, + anticentromere antibodies both in sera and seroma, sicca syndrome, arthralgias, twinkling in extremities, abnormal capillaroscopic findings, and reduced diffusing capacity of the lungs for carbon monoxide. She was diagnosed with ASIA, and antimalarial and corticosteroid therapy were introduced. Conclusion: In patients with HT and familial autoimmunity, SBI should be carefully considered due to the possibility of ASIA development. Hashimoto thyroiditis, familial autoimmunity, and ASIA seem to be interconnected in the complex mosaic of autoimmunity in predisposed individuals.

Case report: Nocardial brain abscess in a persistently SARS-CoV-2 PCR positive patient with systemic lupus erythematosus.

Coronavirus disease (COVID-19) in immunocompromised patients represents a major challenge for diagnostics, surveillance, and treatment. Some individuals remain SARS-CoV-2 PCR-positive for a prolonged period. The clinical and epidemiological significance of this phenomenon is not well understood. We report a case of a patient with a history of systemic lupus erythematosus (SLE) who has been persistently SARS-CoV-2 PCR positive for 9 months, with multiple thromboembolic complications, and development of nocardial brain abscess successfully treated with surgery and antibiotics.

Successful Immunomodulatory Treatment of COVID-19 in a Patient With Severe ACTH-Dependent Cushing's Syndrome: A Case Report and Review of Literature.

Introduction: Patients with Cushing's syndrome (CS) represent a highly sensitive group during corona virus disease 2019 (COVID-19) pandemic. The effect of multiple comorbidities and immune system supression make the clinical picture complicated and treatment challenging. Case report: A 70-year-old female was admitted to a covid hospital with a severe form of COVID-19 pneumonia that required oxygen supplementation. Prior to her admission to the hospital she was diagnosed with adrenocorticotropic hormone (ACTH)-dependent CS, and the treatment of hypercortisolism had not been started yet. Since the patient's condition was quickly deteriorating, and with presumend immmune system supression due to CS, we decided on treatement with intraveonus immunoglobulins (IVIg) that enabled quick onset of immunomodulatory effect. All comorbidities were treated with standard of care. The patient's condition quickly stabilized with no direct side effects of a given treatment. Conclusion: Treatment of COVID-19 in patients with CS faces many challenges due to the complexity of comorbidity effects, immunosupression and potential interactions of available medications both for treatment of COVID-19 and CS. So far, there are no guidelines for treatment of COVID-19 in patients with active CS. It is our opinion that immunomodulating therapies like IVIg might be an effective and safe treatment modality in this particularly fragile group of patients.

Assessment of the left ventricular chamber stiffness in athletes.

Since diastolic dysfunction is an early sign of the heart disease, detecting diastolic disturbances is predicted to be the way for early recognizing underlying heart disease in athletes. So-called chamber stiffness index (E/e')/LVDd was predicted to be useful in distinguishing physiological from pathological left ventricular hypertrophy, because it was shown to be reduced in athletes. It remains unknown whether it is reduced in all athletic population. Standard and tissue Doppler were used to assess cardiac parameters at rest in 16 elite male wrestlers, 21 water polo player, and 20 sedentary subjects of similar age. In addition to (E/e')/LVDd index, a novel (E/e')/LVV, (E/e')/RVe'lat indices were determined. Progressive continuous maximal test on treadmill was used to assess the functional capacity. VO(2) max was the highest in water polo players, and higher in wrestlers than in controls. LVDd, LVV, LVM/BH(2.7) were higher in athletes. Left ventricular early diastolic filling velocity, deceleration and isovolumetric relaxation time did not differ. End-systolic wall stress was significantly higher in water polo players. RV e' was lower in water polo athletes. Right atrial pressure (RVE/e') was the highest in water polo athletes. (E/e'lat)/LVDd was not reduced in athletes comparing to controls (water polo players 0.83 ± 0.39, wrestlers 0.73 ± 0.29, controls 0.70 ± 0.28; P = 0.52), but (E/e's)/RVe'lat better distinguished examined groups (water polo players 0.48 ± 0.37, wrestlers 0.28 ± 0.15, controls 0.25 ± 0.16, P = 0.015) and it was the only index which predicted VO(2) max. In conclusion, intensive training does not necessarily reduce (E/e'lat)/LVDd index. A novel index (E/e's)/RVe'lat should be investigated furthermore in detecting diastolic adaptive changes.

The association of glucocorticoid receptor polymorphism with metabolic outcomes in menopausal women with adrenal incidentalomas.

OBJECTIVES: To investigate whether BclI polymorphism in the glucocorticoid receptor gene influences hypothalamic-pituitary-adrenal (HPA) axis regulation, body composition and metabolic parameters in women with adrenal incidentalomas (AIs). STUDY DESIGN: A cross-sectional study. MAIN OUTCOME MEASURES: We analyzed 106 women with AIs. Insulin resistance was assessed using a homeostasis model while HPA activity was assessed using dexamethasone suppression tests (DST), basal ACTH, urinary free cortisol, and midnight serum cortisol level. Body composition was analyzed using dual-energy X-ray absorptiometry. DNA was obtained from peripheral blood leucocytes and BclI polymorphism was detected using PCR, RFLP and DNA sequencing. RESULTS: BclI carriers in comparison with those with wild-type BclI had less suppressed cortisol after DST-0.5 mg (126.4 ± 111.4 vs 80.9 ± 75.7 nmol/l, p = 0.026) and had a lower prevalence of impaired glucose tolerance and of type 2 diabetes mellitus (T2DM). BclI carriers had a higher percentage of leg fat mass (FM), lower left-sided limb muscle mass and a decline in total lean body mass. Duration of menopause remained a strong predictor of appendicular lean mass index (ALMI) (ß=-0.125, p = 0.034). BclI polymorphism was significantly associated with sum of legs FM percentage (ß=0.327, p = 0.048). T2DM was negatively associated with BclI polymorphism, after adjusting for age, truncal FM, ALMI, and sum of legs FM (OR=0.158, 95%CI 0.031-0.806, p = 0.027). CONCLUSIONS: BclI polymorphism is associated with tissue-specific glucocorticoid sensitivity, relative glucocorticoid resistance of the HPA axis and peripheral adipose tissue, and glucocorticoid hypersensitivity at the muscle level. By modulating glucocorticoid and insulin sensitivity, BclI polymorphism appears to reduce the risk of T2DM in women with AIs.

The alpha-melanocyte stimulating hormone is related to heart rate during exercise recovery.

Alpha-melanocyte-stimulating hormone (alpha-MSH) is a part of the hormonal stress system with proven cardiovascular effects. Heart rate recovery (HRR) following exercise is strongly correlated to overall fitness and future adverse cardiovascular events. The current study examined the predictive value of alpha-MSH for HRR following exercise testing.Cardiopulmonary exercise testing (CPET) on a treadmill was used to measure HR and oxygen consumption (V̇O2) in 16 elite male wrestlers (W), 21 water polo player (WP) and 20 sedentary subjects (C) matched for age. Plasma levels of alpha-MSH were measured by radioimmunoassay technique in four phases of CPET: 1) 10 min pre-CPET at rest; 2) at the initation of CPET; 3) at peak CPET; and 4) at the third minute of recovery. The WP group had significantly higher HRR compared to than W and C groups, who did not have significantly different values. Significant difference in alpha-MSH measurements and patterns during CPET between groups was not observed (p > 0.05). When combining all three groups, we observed a significant correlation between V̇O2 recovery and alpha-MSH recovery/peak (r = -0.3, p = 0.022). HRR and ΔHRR/peak significantly correlated with alpha-MSH at all four measurment points (r = -0.4; p < 0.01 for all). On multiple regression analysis, which included anthropometric and hormonal measures, the best independent predictor of HRR and ΔHRR/peak was alpha-MSH during recovery (B = -1.0, -0.5; SE = 0.3, 0.1; CI = -1.5 to -0.4, -0.7 to -0.2; p = 0.001 respectively). In conclusion, alpha-MSH measured during exercise recovery holds predictive value for HRR and ΔHRR/peak, suggesting a contributing role to integrative regulation of overall cardiopulmonary performance. CONDENSED ABSTRACT: Present study examined the predictive value of alpha-melanocyte stimulating hormone (alpha-MSH) for heart rate recovery (HRR) in elite male wrestlers, water polo players and sedentary subjects matched for age. Alpha-MSH measured during exercise recovery holds predictive value for HRR and ΔHRR/peak, suggesting a contributing role to integrative regulation of overall cardiopulmonary performance.

Acute Response to Endurance Exercise Stress: Focus on Catabolic/anabolic Interplay Between Cortisol, Testosterone, and Sex Hormone Binding Globulin in Professional Athletes.

BACKGROUND: Endocrine system plays a major role in both permissive and regulatory activities in order to adequately respond to physical stress of exercise. But level and direction of activation depend on many factors and are not easily interpreted. METHODS: We tested a group of male professional athletes (21 water polo players and 15 wrestlers), together with 20 sedentary controls matched by age. All participants took a continuous progressive exercise stress test on a treadmill until exhaustion and plateau of oxygen consumption (VO2). Blood samples for cortisol, sex hormone binding globulin (SHBG) and testosterone were drawn in four time points: baseline (B), start of the test (S), point of maximal strain (MAX) and in the 3rd minute of recovery period (R). RESULTS: Cortisol levels significantly increased in both groups, but the response between S and MAX was more pronounced in controls (p=0.036). The athletes had significantly higher levels of cortisol in all points in test, except during R (p=0.118), when their cortisol levels gradually started to decline. Significant increase in total testosterone was in great deal a consequence of increase in SHBG level (p<0.01 for both). Consequently, calculated free testosterone significantly decreased during test (p=0.008), and the drop was more pronounced in athletes. This was in concordance with significant correlation between SHBG and cortisol level demonstrated in athletes, but not in controls. CONCLUSIONS: It seems that high intensity endurance exercise favors catabolic response, but the level of response highly depends on a previous level of training.

Novel Mutations in Serbian MEN1 Patients: Genotype-phenotype Correlation.

BACKGROUND: Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant cancer syndrome characterized by the occurrence of primary hyperparathyroidism (PHPT), pituitary adenoma (PA) and pancreatic neuroendocrine tumor (pNET). Whether the underlying mutations in MEN1 gene predict clinical presentation of affected heterozygotes or not, is still a matter of a debate. METHODS: Clinical and genetic analysis of 90 consecutive MEN1 patients was performed in a retrospective, single - center study. RESULTS: MEN1 mutation was found in 67 (74.4%) patients belonging to 31 different families. Twenty nine different heteozygous mutations were found, including 6 novel point mutations (W220G, 941delG, 1088del7, 1184insA, 1473del10, 1602del17) and one large deletion of exon 8. Truncating mutations predicted development of pNETs (OR=5.8, 95% CI 1.7 - 19.7%) and PHPT (OR=4.3, 95% CI 1.5 - 12.4%). CONCLUSIONS: Large number of novel mutations among MEN1 patients confirmed previously reported data. PNETs and PHPT were more frequent in patients with truncating mutations.

Antibodies binding the head domain of P2X4 inhibit channel function and reverse neuropathic pain.

P2X4 is a ligand-gated ion channel implicated in neuropathic pain. Drug discovery efforts targeting P2X4 have been unsuccessful largely because of the difficulty in engineering specificity and selectivity. Here, we describe for the first time the generation of a panel of diverse monoclonal antibodies (mAbs) to human and mouse P2X4, capable of both positive and negative modulation of channel function. The affinity-optimised anti-P2X4 mAb IgG#151-LO showed exquisite selectivity for human P2X4 and induced potent and complete block of P2X4 currents. Site-directed mutagenesis of P2X4 revealed the head domain as a key interaction site for inhibitory mAbs. Inhibition of spinal P2X4 either by intrathecal delivery of an anti-P2X4 mAb or by systemic delivery of an anti-P2X4 bispecific mAb with enhanced blood-spinal cord barrier permeability produced long-lasting (>7 days) analgesia in a mouse model of neuropathic pain. We therefore propose that inhibitory mAbs binding the head domain of P2X4 have therapeutic potential for the treatment of neuropathic pain.

Discovery and characterisation of an antibody that selectively modulates the inhibitory activity of plasminogen activator inhibitor-1.

Plasminogen activator inhibitor-1 (PAI-1) is a serine protease inhibitor (serpin) that regulates fibrinolysis, cell adhesion and cell motility via its interactions with plasminogen activators and vitronectin. PAI-1 has been shown to play a role in a number of diverse pathologies including cardiovascular diseases, obesity and cancer and is therefore an attractive therapeutic target. However the multiple patho-physiological roles of PAI-1, and understanding the relative contributions of these in any one disease setting, make the development of therapeutically relevant molecules challenging. Here we describe the identification and characterisation of fully human antibody MEDI-579, which binds with high affinity and specificity to the active form of human PAI-1. MEDI-579 specifically inhibits serine protease interactions with PAI-1 while conserving vitronectin binding. Crystallographic analysis reveals that this specificity is achieved through direct binding of MEDI-579 Fab to the reactive centre loop (RCL) of PAI-1 and at the same exosite used by both tissue and urokinase plasminogen activators (tPA and uPA). We propose that MEDI-579 acts by directly competing with proteases for RCL binding and as such is able to modulate the interaction of PAI-1 with tPA and uPA in a way not previously described for a human PAI-1 inhibitor.