Elucidation of the Reversible Self-Association Interface of a Diabody-Interleukin Fusion Protein Using Hydrogen-Exchange Mass Spectrometry and In Silico Modeling.

Reversible self-association (RSA) of therapeutic proteins presents major challenges in the development of high-concentration formulations, especially those intended for subcutaneous administration. Understanding self-association mechanisms is therefore critical to the design and selection of candidates with acceptable developability to advance to clinical trials. The combination of experiments and in silico modeling presents a powerful tool to elucidate the interface of self-association. RSA of monoclonal antibodies has been studied extensively under different solution conditions and have been shown to involve interactions for both the antigen-binding fragment and the crystallizable fragment. Novel modalities such as bispecific antibodies, antigen-binding fragments, single-chain-variable fragments, and diabodies constitute a fast-growing class of antibody-based therapeutics that have unique physiochemical properties compared to monoclonal antibodies. In this study, the RSA interface of a diabody-interleukin 22 fusion protein (FP-1) was studied using hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) in combination with in silico modeling. Taken together, the results show that a complex solution behavior underlies the self-association of FP-1 and that the interface thereof can be attributed to a specific segment in the variable light chain of the diabody. These findings also demonstrate that the combination of HDX-MS with in silico modeling is a powerful tool to guide the design and candidate selection of novel biotherapeutic modalities.

Reanalysis of clinical exome identifies the second variant in two individuals with recessive disorders.

Clinical exome/genome sequencing is increasingly being utilized by clinicians to diagnose various likely genetic conditions, but many cases remain undiagnosed. In a subset of those undiagnosed cases, a single heterozygous variant in an autosomal recessive (AR) condition with consistent phenotype may be identified, raising the question if a second variant is missing. Here, we report two cases of recessive conditions in which only one heterozygous variant was initially reported by clinical exome sequencing, and on research reanalysis a second heterozygous variant in trans was identified. We performed a review of the existing exome reanalysis literature and found that this aspect is often not emphasized. These findings highlight the importance of data reanalysis in undiagnosed cases where only a single disease-associated variant is identified in an AR condition with a strong link to presenting phenotype.

Primary Anti-Phospholipid Antibody Syndrome: Real-World Defining Features of Rethrombosis in the Course of Disease.

Objective: We aimed to identify features that allow differentiation of primary antiphospholipid syndrome (PAPS) patients that suffer recurrent thrombotic events (RTE) despite anticoagulation, from the other diagnosed PAPS patients. Methods: This was an exploratory study of anticoagulated PAPS patients attending an Autoimmune Diseases Unit (1998-2018). From 2016, anti-phospholipid antibodies and lupus anticoagulant were determined for each patient at consecutive visits, collected together with retrospective clinical characteristics, laboratory, and therapeutic markers and compared according to the occurrence of thrombotic events during follow-up. Results: Overall, two thirds of the patients were female, 93% were Caucasian, with a median age of 40 years at diagnosis, for a median time of 11.5 years in follow-up. Out of 54 patients, 10 were identified with RTE. There were no significant differences among the RTE and non-RTE patients as far as classical risk factors for clotting disorders. The RTE group was characterized by a higher proportion of younger patients, male sex and positivity for all laboratory markers, and initially and over follow-up as well as a sustained high-risk profile based on APS laboratory markers. Anticardiolipin IgG at onset was the only statistically significant marker of the RTE group. At the end of follow-up, consistent reversion to negative status was a rare event, observed in 20% of RTE vs. 25% of non-RTE patients. Conclusions: Despite therapy, we were able to identify features associated to thrombotic events in patients with PAPS. Prospectively regular clinical and laboratory monitoring might be warranted in order to treat APS more assertively.

Biological Therapy in Patients with Rheumatoid Arthritis in a Tertiary Center in Portugal: A Cross-Sectional Study.

INTRODUCTION: Clinical outcomes in rheumatoid arthritis have greatly improved with therapeutic advances. Despite the availability of substantial clinical trial evidence, there is a lack of real-life data. The aim of this study was to assess disease status and quality of life in an outpatient population treated with biological disease-modifying anti-rheumatic drugs. MATERIAL AND METHODS: Cross-sectional study recalling all patients ever treated in our unit with biological disease-modifying antirheumatic drugs. Clinical and demographic data, compliance, disease activity, functional status, joint deformities, and comorbidities were documented, and patients queried on occupational status, education, marital status and generic health related quality of life questionnaires. RESULTS: Recall was attended by 77 of the original 94 patients. At recall, median age was 63 years old, 82% of the patients were female and the median disease duration was 12 years. Biological therapy was started at a median of four years following disease onset. According to the disease activity score (DAS28), the percentage of patients with high, moderate, low disease activity or remission changed from 50, 45, 0 and 5 (pre-therapy) to 11, 37, 25 and 26 at recall, respectively; functional status was significantly improved. Seventy-five per cent of the patients retained the original treatment with good compliance. Lower Short Form-36 domain scores accompanied a low EQ-5D-3L score. Deceased patients (n = 6) had a lower estimated 10-year survival rate. In this group, biological therapy was discontinued at a higher frequency during follow-up. DISCUSSION: A high disease activity and a high HAQ disability index characterized most patients at pre-bDMARD onset. CONCLUSION: Despite therapy switches and regular follow-up, a significant percentage of patients still presented with moderate disease activity, functional impairment and a poor health-related quality of life.

Introdução: Avanços no tratamento da artrite reumatóide contribuiram para uma evolução favorável. Apesar de evidências substanciais provenientes de ensaios clínicos, são menos conhecidos os dados provenientes da vida real. O objetivo do estudo foi caracterizar a doença e a qualidade de vida em doentes sob fármacos biotecnológicos. Material e Métodos: Trata-se de um estudo transversal com recolha de dados clínicos relativos à adesão terapêutica, atividade da doença, capacidade funcional, deformidades articulares, comorbilidades e questionários de qualidade de vida relacionada com a saúde, estado civil, situação profissional e escolaridade. Resultados: Foram recrutados 77 doentes do grupo original de um total de 94. A mediana da idade foi 63 anos, 82% do sexo feminino e início de biológico cerca de quatro anos após o início da doença, com uma mediana de duração de 12 anos. De acordo com o disease activity score (DAS28), a percentagem de doentes com atividade alta, moderada, baixa ou em remissão mudou, respectivamente, de 50, 45, 0 e 5 pré- biológico para 11, 37, 25 e 26 na altura da re-avaliação, com melhoria funcional. Setenta e cinco por cento dos doentes mantiveram o tratamento original com boa adesão. Pontuações mais baixas do short form-36 associaram-se a uma baixa pontuação no EQ-5D-3L. No grupo de doentes que viriam a falecer (n = 6), foi observada uma menor esperança de vida aos 10 anos, assim como uma maior discontinuação da terapêutica biológica. Discussão: Pré-biológico, uma elevada percentagem dos doentes apresentava elevada atividade da doença e incapacidade funcional. Conclusão: Não obstante ajustes terapêuticos e seguimento regular, uma percentagem significativa de doentes mantinha atividade moderada e limitação funcional com baixa qualidade de vida relacionada com a saúde.

Impact of COVID-19 on Pediatric Gastroenterology Fellow Training in North America.

BACKGROUND: The COVID-19 pandemic has drastically changed healthcare systems and training around the world. The Training Committee of the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition sought to understand how COVID-19 has affected pediatric gastroenterology fellowship training. METHODS: A 21 question survey was distributed to all 77 pediatric gastroenterology fellowship program directors (PDs) in the North American Society for Pediatric Gastroenterology, Hepatology and Nutrition program director database via email on April 7. Responses collected through April 19, 2020 were analyzed using descriptive statistics. RESULTS: Fifty-one of 77 (66%) PDs from the United States, Canada, and Mexico responded to the survey. Forty-six of 51 (90%) PDs reported that they were under a "stay-at-home" order for a median of 4 weeks at the time of the survey. Two of the 51 (4%) programs had fellows participating in outpatient telehealth before COVID-19 and 39 of 51 (76%) at the time of the survey. Fellows stopped participating in outpatient clinics in 22 of 51 (43%) programs and endoscopy in 26 of 51 (52%) programs. Changes to inpatient care included reduced fellow staffing, limiting who entered patient rooms, and rounding remotely. Fellows in 3 New York programs were deployed to adult medicine units. Didactics were moved to virtual conferences in 47 of 51 (94%) programs, and fellows used various online resources. Clinical research and, disproportionately, bench research were restricted. CONCLUSIONS: This report provides early information of the impact of COVID-19 on pediatric fellowship training. Rapid adoption of telehealth and reduced clinical and research experiences were important changes. Survey information may spur communication and innovation to help educators adapt.

Management of maternal antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is a systemic autoimmune disease which manifests as thrombotic and/or obstetric adverse events, mediated by persistent circulating antiphospholipid antibodies (aPL) detected by means of three tests: lupus anticoagulant, anticardiolipin and anti ß2-glycoprotein I antibodies. It can be isolated or associated with other autoimmune rheumatic diseases. During pregnancy, patients with APS have a higher risk of obstetric complications and a higher thrombotic risk due to the pregnancy itself. Therefore, a preconception counselling is crucial to assist the patient and her family in planning the pregnancy and to optimise the management by implementing preventive measures that can allow the best outcomes for both the mother and the baby. In clinical practice, we can distinguish between different subsets of patients that require alternative approaches: patients with obstetric APS, patients with thrombotic APS, patients with APS associated with other autoimmune diseases, and asymptomatic individuals (aPL carriers). Pregnancy and foetal outcomes have greatly improved in the past 2 decades as a result of a therapeutic implementation based on individual risk stratification and a combination of low dose aspirin and heparin. Additional strategies have been suggested for women with pregnancy failure despite this management.

Heterogeneous lupus-specific lesions and treatment outcome, in a single patient, over a period of time.

The report highlights the importance of strict clinico-histological correlations when skin biopsies are performed in diagnostic doubt in systemic lupus erythematosus. Furthermore, PUVA is never indicated in autoimmune conditions involving photosensitivity, due to high potential for internal and cutaneous aggravation of the disease, as the authors observed in this case.

Enteric Virome and Bacterial Microbiota in Children With Ulcerative Colitis and Crohn Disease.

OBJECTIVES: We examined the fecal virome and bacterial community composition of children with Crohn disease (CD), ulcerative colitis (UC), and healthy controls to test the hypothesis that unique patterns of viral organisms and/or presence of bacterial pathogens may be identified that could contribute to the pathogenesis of pediatric inflammatory bowel disease (IBD). METHODS: Fecal samples from 24 children (mean 12.2 years) with CD (n = 7) or UC (n = 5) and similar aged controls (n = 12) were processed to determine individual viromes. Viral sequences were identified through translated protein sequence similarity search. Bacterial microbiota were determined by sequencing of the V4 region of the 16S rRNA gene. RESULTS: Only a few human viruses were detected, so virome analyses focused on bacterial viruses. The relative abundance of Caudovirales was greater than that of Microviridae phages in both IBD and healthy controls. Caudovirales phages were more abundant in CD (mean 80.8%) than UC (48.8%) (P = 0.05) but not controls. The richness of viral strains in Microviridae but not Caudovirales was higher in controls than CD (P = 0.05) but not UC cases. No other measure of phage abundance, richness, or Shannon diversity showed significant difference between the 2 IBD and control groups. Bacterial microbiota analysis revealed that IBD diagnosis, albumin, hemoglobin, erythrocyte sedimentation rate, and probiotic supplementation correlated to the composition of gut bacterial microbiota. CONCLUSIONS: Minor patterns in gut virome and bacterial community composition distinguish pediatric IBD patients from healthy controls. Probiotics are associated with bacterial microbiota composition. These exploratory results need confirmation in larger studies.

Disease activity assessment of rheumatic diseases during pregnancy: a comprehensive review of indices used in clinical studies.

Pregnancy requires a special management in women with inflammatory rheumatic diseases (RDs), with the aim of controlling maternal disease activity and avoiding fetal complications. Despite the heterogeneous course of RDs during pregnancy, their impact on pregnancy largely relates to the extent of active inflammation at the time of conception. Therefore, accurate evaluation of disease activity is crucial for the best management of pregnant patients. Nevertheless, there are limitations in using conventional measures of disease activity in pregnancy, as some items included in these instruments can be biased by symptoms or by physiological changes related to pregnancy and the pregnancy itself may influence laboratory parameters used to assess disease activity. This article aims to summarize the current literature about the available instruments to measure disease activity during pregnancy in RDs. Systemic lupus erythematosus is the only disease with instruments that have been modified to account for several adaptations which might interfere with the attribution of signs or symptoms to disease activity during pregnancy. No modified-pregnancy indices exist for women affected by other RDs, but standard indices have been applied to pregnant patients. The current body of knowledge shows that the physiologic changes that occur during pregnancy need to be either adapted from existing instruments or developed to improve the management of pregnant women with RDs. Standardized instruments to assess disease activity during pregnancy would be helpful not only for clinical practice but also for research purposes.

Obstetric Anti-phospholipid Syndrome: State of the Art.

PURPOSE OF REVIEW: This review focuses on new pathogenesis and clinical-therapeutic aspects of obstetric anti-phospholipid syndrome (ob-APS) in the last 5 years. RECENT FINDINGS: The pathogenesis of ob-APS is multifactorial, including placental infarctions, infiltration of inflammatory cells that cause acute and chronic inflammation, leading to uncontrolled inflammation and poor pregnancy outcomes. A preconception counseling and a patient-tailored treatment are fundamental to improve maternal and fetal outcomes. Thanks to conventional treatment, based on low-dose aspirin and heparin, 70% of women with ob-APS can have successful pregnancies. Women with positive anti-phospholipid antibodies (aPL) without clinical manifestations ("aPL carriers") or with obstetric manifestation not fulfilling ob-APS criteria need to be further investigated in order to assess their best management. Great interest has been given to drugs that could interact in the pathophysiological mechanisms, such as hydroxychloroquine, statins, and eculizumab. These drugs could be considered for patients refractory to conventional therapy.

Airway management in a patient with Crouzon syndrome proposed to orthognathic surgery.

Airway management is one of the most important anaesthetist's skills as major complications of airway management, although rare, can be among the most life threatening in medicine. Crouzon syndrome is a rare condition with physical characteristics that can result in difficult airway manipulation. A correct preanaesthetic evaluation and a planned preinduction strategy should be designed to facilitate intubation.

De novo inflammatory bowel disease after pediatric kidney or liver transplant.

A subset of children who receive a liver and/or kidney transplant develop de novo inflammatory bowel disease-like chronic intestinal inflammation, not explained by infection or medications, following transplant. We have conducted a single-center, retrospective case series describing the unique clinical and histologic features of this IBD-like chronic intestinal inflammation following solid organ transplant. At our center, nine of 327 kidney or liver recipients developed de novo IBD following transplant (six liver, two kidney, one liver-kidney). Most children presented with prolonged hematochezia and diarrhea and were treated with aminosalicylates. At time of diagnosis, five were not currently using mycophenolate mofetil for transplant immunosuppression. Histologic and endoscopic findings at IBD diagnosis included inflammation, ulcerations, granulomas, and chronic colitis. Since diagnosis, no patients have required surgical intervention, or escalation to biologic therapy, nor developed stricturing or perianal disease. In this case series, de novo post-transplant IBD developed in 4% of pediatric liver and/or kidney recipients; however, it often does not fit the classic patterns of Crohn's disease or ulcerative colitis.

Complications of Endoscopic Retrograde Cholangiopancreatography in Pediatric Patients; A Systematic Literature Review and Meta-Analysis.

OBJECTIVES: To systematically review risks and summarize reported complication rates associated with the performance of endoscopic retrograde cholangiopancreatography (ERCP) in children during the past 2 decades. STUDY DESIGN: A systematic literature search of MEDLINE, Embase, and Web of Science from January 1995 to January 2016 was conducted for observational studies published in English. Studies reporting ERCP complications in patients <21 years without history of liver transplant or cholecystectomy were included. A summary estimate of the proportion of children who experienced complications following ERCP was derived via a random effects meta-analysis. RESULTS: Thirty-two studies involving 2612 children and 3566 procedures were included. Subjects' ages ranged from 3 days to 21 years. Procedures were performed for biliary (54%), pancreatic (38%), and other (8%) indications; 56% of ERCPs were interventional. The pooled complication rate was 6% (95% CI 4%- 8%). Procedural complications included post-ERCP pancreatitis (166, 4.7%), bleeding (22, 0.6%), and infections (27, 0.8%). The pooled estimate of post-ERCP pancreatitis was 3% (95% CI 0.02-0.05), and other complications were 1% (95% CI 0.02-0.05). In the subgroup with neonatal cholestasis, the pooled complication rate was 3% (95% CI 0.01-0.07). Adult and pediatric gastroenterologists and surgeons performed the ERCPs. Available data limited the ability to report differences between pediatric-trained and other endoscopists. CONCLUSIONS: Complications associated with pediatric ERCP range widely in severity and are reported inconsistently. Our review suggests 6% of pediatric ERCPs have complications. Further studies that use systematic and standardized methodologies are needed to determine the frequency and risk factors for ERCP-related complications.

Improved enteral tolerance following step procedure: systematic literature review and meta-analysis.

Surgical management of children with short bowel syndrome (SBS) changed with the introduction of the serial transverse enteroplasty procedure (STEP). We conducted a systematic review and meta-analysis using MEDLINE and SCOPUS to determine if children with SBS had improved enteral tolerance following STEP. Studies were included if information about a child's pre- and post-STEP enteral tolerance was provided. A random effects meta-analysis provided a summary estimate of the proportion of children with enteral tolerance increase following STEP. From 766 abstracts, seven case series involving 86 children were included. Mean percent tolerance of enteral nutrition improved from 35.1 to 69.5. Sixteen children had no enteral improvement following STEP. A summary estimate showed that 87 % (95 % CI 77-95 %) of children who underwent STEP had an increase in enteral tolerance. Compilation of the literature supports the belief that SBS subjects' enteral tolerance improves following STEP. Enteral nutritional tolerance is a measure of efficacy of STEP and should be presented as a primary or secondary outcome. By standardizing data collection on children undergoing STEP procedure, better determination of nutritional benefit from STEP can be ascertained.

Addition of Histology to the Paris Classification of Pediatric Crohn Disease Alters Classification of Disease Location.

OBJECTIVES: The aim of the study was to investigate the value of microscopic findings in the classification of pediatric Crohn disease (CD) by determining whether classification of disease changes significantly with inclusion of histologic findings. METHODS: Sixty patients were randomly selected from a cohort of patients studied at the Pediatric Inflammatory Bowel Disease Clinic at the University of California, San Francisco Benioff Children's Hospital. Two physicians independently reviewed the electronic health records of the included patients to determine the Paris classification for each patient by adhering to present guidelines and then by including microscopic findings. RESULTS: Macroscopic and combined disease location classifications were discordant in 34 (56.6%), with no statistically significant differences between groups. Interobserver agreement was higher in the combined classification (κ = 0.73, 95% confidence interval 0.65-0.82) as opposed to when classification was limited to macroscopic findings (κ = 0.53, 95% confidence interval 0.40-0.58). When evaluating the proximal upper gastrointestinal tract (Paris L4a), the interobserver agreement was better in macroscopic compared with the combined classification. CONCLUSIONS: Disease extent classifications differed significantly when comparing isolated macroscopic findings (Paris classification) with the combined scheme that included microscopy. Further studies are needed to determine which scheme provides more accurate representation of disease extent.

Idosos praticantes e não praticantes de exercícios físicos: uma comparação do estado de equilíbrio / Elderly practitioners and non physical exercise practitioners: a comparison of the balance of state

O objetivo deste estudo foi avaliar e comparar o equilíbrio corporal estático edinâmico entre um grupo de idosos praticantes de exercícios físicos (hidroginástica) e um grupo de idosos não praticantes de exercícios físicos. Através dos resultados, utilizando ametodologia proposta, verificou-se que, ao analisar individualmente os grupos do pré- para o pós-teste, ambos não obtiveram melhoras significativas. Ao comparar, porém, o equilíbrio entre os dois grupos no pré- e pós-teste pode-se afirmar que houve melhoras significativas nogrupo de praticantes de hidroginástica, com p=0,046 (pré-teste), e p=0,038 (pós-teste).(AU)

The objective of this study was to evaluate and compare the static and dynamic body balance among a group of elderly practitioners of physical exercise (aerobics) and a non-practicing group of elderly of exercise. From the results using the proposed method, it was found that by individually analyzing the groups for the pre-post test, both not achieved significant improvement. But when comparing the balance between the two groups in the pre and post-test can be stated that there was significant improvement in the group of water gymnastics classes, with p = 0,046 (pre-test), p = 0,038 (post-test).(AU)

Idosos praticantes e não praticantes de exercícios físicos: uma comparação do estado de equilíbrio / Elderly practitioners and non physical exercise practitioners: a comparison of the balance of state

O objetivo deste estudo foi avaliar e comparar o equilíbrio corporal estático e dinâmico entre um grupo de idosos praticantes de exercícios físicos (hidroginástica) e um grupo de idosos não praticantes de exercícios físicos. Através dos resultados, utilizando a metodologia proposta, verificou-se que, ao analisar individualmente os grupos do pré- para o pós-teste, ambos não obtiveram melhoras significativas. Ao comparar, porém, o equilíbrio entre os dois grupos no pré- e pós-teste pode-se afirmar que houve melhoras significativas no grupo de praticantes de hidroginástica, com p=0,046 (pré-teste), e p=0,038 (pós-teste).

The objective of this study was to evaluate and compare the static and dynamic body balance among a group of elderly practitioners of physical exercise (aerobics) and a non-practicing group of elderly of exercise. From the results using the proposed method, it was found that by individually analyzing the groups for the pre-post test, both not achieved significant improvement. But when comparing the balance between the two groups in the pre and post-test can be stated that there was significant improvement in the group of water gymnastics classes, with p = 0,046 (pre-test), p = 0,038 (post-test).

Characterization of anti-Listeria bacteriocins isolated from shellfish: potential antimicrobials to control non-fermented seafood.

This work had as main objectives to characterize two bacteriocins produced by lactic acid bacteria (LAB) previously isolated from non-fermented seafood, in order to evaluate their potential as new food protective agents. The two bacteriocinogenic isolates were identified by Polymerase Chain Reaction (PCR) using genus- and species-specific primers, and confirmed by 16S rDNA sequencing, as Enterococcus faecium and Pediococcus pentosaceus. The antimicrobial spectrum of each strain included several indicator microorganisms, some of them also isolated from seafood. Growth of Listeria innocua, L. monocytogenes, Staphylococcus aureus, Bacillus cereus and other LAB species were inhibited, although no inhibition of Gram-negative microorganisms was observed. Proteolytic, but not lipolytic or glycolytic enzymes, completely inactivated the antimicrobial effect of both cell-free supernatants confirming the proteinaceous nature of the inhibitors. The antimicrobial activity was maintained after treatment with NaCl, SDS, Triton X-100, Tween 20, Tween 80 and EDTA after 2 h or 5 h of exposure and both bacteriocins were stable over a wide range of pH and temperatures. Production of bacteriocin by E. faecium (bacALP7) was detected initially at exponential phase and reached a maximum activity of 25,600 AU/ml in the early stationary phase, whereas bacteriocin production by P. pentosaceus ALP57 (bacALP57) reached the maximum at exponential phase with 12,800 AU/ml. The bacteriocins did not kill L. monocytogenes ESB54 nor L. innocua 2030c however, cellular growth was reduced. The partially purified bacteriocins, bacALP7 and bacALP57, were below 6.5 kDa in size as determined by Tricine-SDS gel electrophoresis. E. faecium and P. pentosaceus contained DNA fragments corresponding in size to those recorded for enterocin B and pediocin PA-1, respectively. Sequencing of the fragments from both bacteriocins confirmed the homology. To our knowledge, for the first time two LAB producing bacteriocins similar to pediocin PA-1 and enterocin B, were isolated from non-fermented shellfish. The adaptation of the cultures to seafood matrices may be advantageous in terms of application as a biopreservation strategy for reduction of L. monocytogenes levels in seafood products.