Anti-tau intrabodies: From anti-tau immunoglobulins to the development of functional scFv intrabodies.

Over the last decade, there has been a growing interest in intrabodies and their therapeutic potential. Intrabodies are antibody fragments that are expressed inside a cell to target intracellular antigens. In the context of intracellular protein misfolding and aggregation, such as tau pathology in Alzheimer's disease, intrabodies have become an interesting approach as there is the possibility to target early stages of aggregation. As such, we engineered three anti-tau monoclonal antibodies into single-chain variable fragments for cytoplasmic expression and activity: PT51, PT77, and hTau21. Due to the reducing environment of the cytoplasm, single-chain variable fragment (scFv) aggregation is commonly observed. Therefore, we also performed complementarity-determining region (CDR) grafting into three different stable frameworks to rescue solubility and intracellular binding. All three scFvs retained binding to tau after cytoplasmic expression in HEK293 cells, in at least one of the frameworks. Subsequently, we show their capacity to interfere with either mouse or mutant human tau aggregation in two different primary mouse neuron models and organotypic hippocampal slice cultures. Collectively, our work extends the current knowledge on intracellular tau targeting with intrabodies, providing three scFv intrabodies that can be used as immunological tools to target tau inside cells.

Development and Characterization of Mouse-Specific Anti-Tau Monoclonal Antibodies: Relevance for Analysis of Murine Tau in Cerebrospinal Fluid.

BACKGROUND: Clearance of tau seeds by immunization with tau antibodies is currently evaluated as therapeutic strategy to block the spreading of tau pathology in Alzheimer's disease and other tauopathies. Preclinical evaluation of passive immunotherapy is performed in different cellular culture systems and in wild-type and human tau transgenic mouse models. Depending on the preclinical model used, tau seeds or induced aggregates can either be of mouse, human or mixed origin. OBJECTIVE: We aimed to develop human and mouse tau-specific antibodies to discriminate between the endogenous tau and the introduced form in preclinical models. METHODS: Using hybridoma technology, we developed human and mouse tau-specific antibodies that were then used to develop several assays to specifically detect mouse tau. RESULTS: Four antibodies, mTau3, mTau5, mTau8, and mTau9, with a high degree of specificity for mouse tau were identified. Additionally, their potential application in highly sensitive immunoassays to measure tau in mouse brain homogenate and cerebrospinal fluid is illustrated, as well as their application for specific endogenous mouse tau aggregation detection. CONCLUSION: The antibodies reported here can be very important tools to better interpret the results obtained from different model systems as well as to study the role of endogenous tau in tau aggregation and pathology observed in the diverse mouse models available.

IP-LC-MSMS Enables Identification of Three Tau O-GlcNAcylation Sites as O-GlcNAcase Inhibition Pharmacodynamic Readout in Transgenic Mice Overexpressing Human Tau.

O-ß-linked N-acetylglucosaminylation (O-GlcNAcylation) modulates tau phosphorylation and aggregation: the pharmacological increase of tau O-GlcNAcylation upon treatment with inhibitors of O-GlcNAc hydrolase (OGA) constitutes a potential strategy to tackle neurodegenerative diseases. Analysis of tau O-GlcNAcylation could potentially be used as a pharmacodynamic biomarker both in preclinical and clinical studies. The goal of the current study was to confirm tau O-GlcNAcylation at S400 as a pharmacodynamic readout of OGA inhibition in P301S transgenic mice overexpressing human tau and treated with the OGA inhibitor Thiamet G and to explore if additional O-GlcNAcylation sites on tau could be identified. As a first step, an immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS) methodology was developed to monitor changes in O-GlcNAcylation around S400 of tau in mouse brain homogenate (BH) extracts. Second, additional O-GlcNAc sites were identified in in-house produced recombinant O-GlcNAcylated human tau at relatively high concentrations, thereby facilitating collection of informative LC-MS data for identification of low-concentration O-GlcNAc-tryptic tau peptides in human transgenic mouse BH extracts. This strategy enabled, for the first time, identification of three low abundant N-terminal and mid-domain O-GlcNAc sites of tau (at S208, S191, and S184 or S185) in human transgenic mouse BH. Data are openly available at data.mendeley.com (doi: 10.17632/jp57yk9469.1; doi: 10.17632/8n5j45dnd8.1; doi: 10.17632/h5vdrx4n3d.1).

Development of immunoprecipitation - two-dimensional liquid chromatography - mass spectrometry methodology as biomarker read-out to quantify phosphorylated tau in cerebrospinal fluid from Alzheimer disease patients.

In Alzheimer's disease (AD) brain, one of the histopathological hallmarks is the neurofibrillary tangles consisting of aggregated and hyperphosphorylated tau. Currently many tau binding antibodies are under development to target the extracellular species responsible for the spreading of the disease in the brain. As such, an in-house developed antibody JNJ-63733657 with picomolar affinity towards tau phosphorylated at both T212 and T217 (further named p217+tau) was recently tested in phase I clinical trial NCT03375697. Following multiple dose administration in healthy subjects and subjects with AD, there were dose dependant reductions in free p217+tau fragments in cerebrospinal fluid (CSF) following antibody administration, as measured with a novel single molecule ELISA assay (Simoa PT3 x PT82 assay), demonstrating epitope engagement of the therapeutic antibody [Galpern, Haeverans, Janssens, Triana-Baltzer, Kolb, Li, Nandy, Mercken, Van Kolen, Sun, Van Nueten, 2020]. Total p217+tau levels also were reduced in CSF as measured with the Simoa PT3 x PT82 assay. In this study we developed an orthogonal immunoprecipitation - liquid chromatography - triple quadrupole mass spectrometry (IP-LC-TQMS) assay to verify the observed reductions in total p217+ tau levels. In this assay, an excess of JNJ-63733657 is added to the clinical CSF to ensure all p217+tau is bound by the antibody instead of having a pool of bound and unbound antigen and to immunoprecipitate all p217+tau, which is followed by on-bead digestion with trypsin to release surrogate peptides. Tryptic peptides with missed cleavages were monitored when phosphorylation occurred close to the cleavage site as this induced miscleavages. Compared with acidified mobile phases typically used for peptide analysis, reversed phase LC with mobile phase at basic pH resulted in sharper peaks and improved selectivity and sensitivity for the target peptides. With this setup a diphospho-tau tryptic peptide SRTPSLPTPPTREPK*2 could be measured with pT217 accounting for at least one of the phospho-sites. This is the first time that the presence of a diphopsho-tau peptide is reported to be present in human CSF. A two-dimensional LC-TQMS method was developed to remove matrix interferences. Selective trapping of diphospho-peptides via a metal oxide chromatography mechanism was achieved in a first dimension with a conventional reversed phase stationary phase and acidified mobile phase. Subsequent elution at basic pH enabled detection of low picomolar p217+tau levels in human CSF (lower limit of quantification: 2 pM), resulting in an approximate 5-fold increase in sensitivity. This enabled the quantification of total p217+tau in CSF leading to the confirmation that in addition to reductions in free p217+tau levels total p217+tau levels were also reduced following administration of the tau mAb JNJ-63733657, correlating with the previous measurement with the PT3 x PT82 Simoa assay. An orthogonal sample clean-up using offline TiO2/ZrO2 combined with 1DLC-TQMS was developed to confirm the presence of mono-ptau (pT217) tryptic peptides in CSF.

Ankle-brachial index according to pulse pressure and microalbuminuria in hypertensive patients: a prospective study in family medicine.

INTRODUCTION: Microalbuminuria, as determined by the urinary albumin to creatinine (AC) ratio, is a marker of target organ damage (TOD) in hypertensive patients. Pulse pressure (PP) predicts arterial elasticity and the ankle-brachial index (ABI) is a marker of cardiovascular morbidity. TOD reduction should be achieved through improvements in these indices. OBJECTIVE: To determine whether ABI, calculated as the ratio between mean PP in the upper and lower limbs, is associated with a reduction in renal damage, as measured by the AC ratio. METHODS: This was a prospective interventional study based on an intention-to-treat analysis in an opportunity sample of patients treated by three specialists in family medicine, with three-monthly follow-up over a total of six months. Blood pressure was measured in arms and ankles, and PP was calculated and used to determine right and left ABI and mean overall ABI. The AC ratio was determined by urine dipstick test. Descriptive and inferential statistical analysis was performed. RESULTS: A sample of 75 patients were studied (42.4% women), of whom(42.4% women), of whom 27.6% were diabetic, 46.7% overweight/obese and 49.3% dyslipidemic. overweight/obese and 49 dyslipidemic. There were statistical differences for right ABI (as determined by PP) and for mean overall ABI (as determined by mean PP in lower and upper limbs). Bivariate correlation analysis showed that in the group with improved PP between the first and the third observations, n=23 (40%), there was a statistically significant reduction in AC ratio (r = -0.924, two-tailed p < 0.001); the opposite was observed in the group with reduced PP, in which the AC ratio increased. DISCUSSION: ABI determined by systolic blood pressure is an excellent predictor of hemodynamic alterations. Increased ABI, based on PP, was accompanied by improved urinary AC ratio. These results are in line with the international literature. CONCLUSIONS: An improvement in urinary AC ratio--a predictor of TOD--is observed when an improvement in the ankle PP/brachial PP ratio is achieved.

[Non-steroidal anti-inflammatory drugs prescription in General Practice setting: 435 does informatics have any influence?]. / Prescrição de anti-inflamatórios não esteróides no ambiente de Medicina Geral e Familiar: a informática influencia?

AIMS: In the ambulatory setting of General Practice/Family Medicine, a retrospective, observational study was carried out to ascertain variation on non-steroidal anti-inflammatory drugs prescription, in an informatics environment where name (brand or chemical), size of package, price and the existence of generic medicines is available. METHODS: Observational transversal study conducted in February 2008 with no prescription study warning given to the doctors in the study period. The differences in volume prescription in Daily Defined Dose (DDD) per registered patient in the middle of 2006 and 2007 two semesters, as well as the value of prescription measured by the price per registered patient were calculated and medicines were studied by the third level of the Portuguese medicines classification very similar to the ATC. The price per DDD was calculated as well. RESULTS: DDD volume and value declined throughout the study comparing entire years or semesters being "Oxicans" the only exception. Even though, the net volume and value decreased between 2006 and 2007 comparing both semesters of 2006 and of 2007. Price per DDD decreased for all medicines except for oxicans with an increase from the first to the second semesters of 2007. CONCLUSIONS: Informatics environment influences the prescription of non-steroidal anti-inflammatory drugs, reducing the number of DDD and the price per registered patient.

Qualidade da consulta de Medicina Geral e Familiar: conseguem os médicos julgar corretamente a qualidade sentida pelos doentes? / The Quality of General Prectice and Family Consultation: are doctors prepared to correctly evaluate how the patients feel about?

A valorização da atividade médica em Medicina Geral e Familiar (MGF), conhecida no Brasil como Medicina de Família e Comunidade, depende, entre outros fatores, do resultado de um encontro médico/doente. O especialista em MGF, ao ter de gerir a sua atividade com base nos pilares fundamentais do contexto, da ciência e da atitude, deve ponderar que a qualidade é algo que depende também da interface que ele contata. Objetivos: verificar a satisfação dos pacientes com a consulta médica de Clínica Geral. Averiguar a capacidade de os médicos avaliarem corretamente a satisfação dos pacientes na consulta. Foi feito um estudo observacional, transversal, com intenção analítica. O material utilizado consistiu em: três médicos de Medicina Geral e Familiar de um Centro de Saúde, questionário validado e usuários da consulta em dois dias distintos de trabalho. Métodos: em dois dias de atividade, apenas sabidos no início de período laboral, foram entregues pelos médicos o questionário com 11 afirmações. No final da cada consulta os médicos preenchiam questionário sobre os mesmos pontos, mas, na sua óptica e após a saída do paciente. Critérios de inclusão: maiores de 16 anos, capacidade de leitura e escrita e aceitação para participar. Análise estatística descritiva e inferencial ( 2 e t de student). Análise de diferenças na resposta às afirmações do questionário, por um modelo de correlação bi-variada com o coeficiente tau-b de Kendall com sensibilidade a 5%. Resultados: recebidos 43 (64,2%) dos questionários entregues. Idade média de 42,7±16,8 anos, sendo majoritariamente do sexo feminino (72,1%). Para 51,3% da amostra não terá havido possibilidade de falar sobre as preocupações de saúde e para 50% não parecem ter sido recebidas instruções acerca de estilos de vida saudáveis para a doença, e para 56,1% o tempo de duração da consulta não terá sido suficiente. Sem diferenças com significado as respostas por sexo, grupo etário e formação. Apenas na questão de o médico querer advogar a saúde do doente, as respostas são consonantes entre doentes e médicos. Conclusões: boa avaliação da consulta nos 11 fatores estudados. Os médicos revelam baixa capacidade de avaliação correta da satisfação dos pacientes na consulta, apenas estando médico e doente de acordo quanto à disponibilidade do médico para advogar a saúde do paciente.

One of the criteria for evaluating medical activity in the field of Family and General Practice, in Brazil known as Family and Community Practice, is the satisfaction obtained in the doctor-patient encounter. The general practitioner, while performing his activity based on the fundamental pillars context, science and attitude, has to consider that quality is something that has to be seen from the viewpoint of the patient as well. Objectives: verify the degree of satisfaction of patients with the general practice consultation and the capacity of the practitioners to correctly predict the satisfaction of their patients with the consultation. An observational transverse study was carried out with the intent to analyze this question. The subjects ans material used in the study consisted of: three general practitioners from a Primary Care Unit, a validated questionnaire and users of consultations on two distinct workdays. Method: during two specific days, only revealed in the beginning of each work shift, the doctors asked the patients to answer a questionnaire containing 11 statements. In the end of each consultation, after the patient had left, the doctors answered the same questionnaire from their own perspective. Criteria for inclusion in the study: over 16 years of age, ability to read and write and consensual participation. Data were submitted to descriptive and inferential statistical analysis (chi square and students t test). The differences in the answers to the statements contained in the questionnaire were analyzed with a bivariable correlation model using Kendalls tau-b coefficient with sensibility of 5%. Results: Forty three (64,2%) of the distributed questionnaires were returned. The age mean of responders was 42,7±16,8 years, most of them of feminine sex (72,1 %). 51,3% of the sample claimed that they were not given the chance to speak about their health concerns, 50% seem not to have received instructions about healthier life styles in relation to their disease and in the opinion of 56,1% the consultation was too short. The answers showed no significant differences in relation to sex, age group and educational level of the responders. The only question in which doctors and patients agreed was that the doctors were ready to care for the health of the patients. Conclusions: the consultations were considered good with regard to the 11 factors studied. The doctors reveal low capacity for correctly predicting the satisfaction of their patients with the consultation. Doctors and patients only agree with respect to the doctors readiness to care for the patients health.

Dente natal: relato de um caso clínico / Natal tooth: clinical case

É muito baixa a incidência de dentes natais e neonatais. Por esta razão, os autores relatam um caso de dente natal, em que o paciente foi encaminhado à Clínica de Odontopediatria do Departamento de Estomatologia da Universidade Federal de Santa Maria, para tratamento. A conduta clínica executada pelos autores, após exame clínico e radiográfico, foi a exodontia sob anestesia local, para evitar possíveis complicações para a criança e para a mãe

Estudo da prevalência de agenesias dentárias: estudo em um grupo de crianças de Santa Maria - RS / Prevalence of tooth agenesis: study on a group of children in Santa Maria

Os autores realizam uma pesquisa de observação do número de dentes ausentes congênitamente em um grupo de crianças