Automated ELISA for potency measurements of therapeutic antibodies and antibody fragments.

Potency assays are essential for the development and quality control of biopharmaceutical drugs, but they are often a time limiting factor due to manual handling steps and consequently low analytical throughput. On the other hand, automation of potency assays can be challenging due to their complexity and the use of biological materials. ELISA (enzyme-linked immunosorbent assay) is widely used for potency determination and is a good candidate for automation as all ELISA types depend on the same basic steps: coating, blocking, sample incubation, detection, and signal measurement. Nevertheless, ELISA for relative potency measurements still require drug-specific development and assay validation thereby complicating automation efforts. To simplify potency testing by ELISA, we first developed a manual protocol generally applicable to different drugs and then adapted this protocol for automated measurements. We identified unexpected critical parameters which had to be adapted to transfer the manual ELISA to an automated liquid handling system and we demonstrated that gravimetric sample dilution is unnecessary with the automated protocol. Both manual and automated protocols were validated and compared using multiple biotherapeutics. The automated protocol showed similar or higher precision and accuracy when compared to the manual method.

Agenesis of maxillary lateral incisors: diagnosis and treatment options.

INTRODUCTION: There are different possibilities of orthodontic planning for cases with congenital absence of maxillary lateral incisors. This subject divides the opinion of orthodontists and oral rehabilitation clinicians, due to the advantages and disadvantages of each treatment option, which may involve opening spaces for future implants and/or prosthetic restorations, or closing the spaces by positioning the maxillary canines in the place of lateral incisors. The correct diagnosis and careful evaluation of each patient allow to determine the best therapeutic approach. This paper discusses the main topics to be considered when planning these cases. OBJECTIVES: To evaluate the main aspects related to orthodontic treatment planning in cases of congenital absence of maxillary lateral incisors, to aid the decision-making, with clinical and scientific basis.

Agenesis of maxillary lateral incisors: diagnosis and treatment options

ABSTRACT Introduction: There are different possibilities of orthodontic planning for cases with congenital absence of maxillary lateral incisors. This subject divides the opinion of orthodontists and oral rehabilitation clinicians, due to the advantages and disadvantages of each treatment option, which may involve opening spaces for future implants and/or prosthetic restorations, or closing the spaces by positioning the maxillary canines in the place of lateral incisors. The correct diagnosis and careful evaluation of each patient allow to determine the best therapeutic approach. This paper discusses the main topics to be considered when planning these cases. Objectives: To evaluate the main aspects related to orthodontic treatment planning in cases of congenital absence of maxillary lateral incisors, to aid the decision-making, with clinical and scientific basis.

RESUMO Introdução: Existem diferentes possibilidades de planejamento ortodôntico para os casos que apresentam ausência congênita de incisivos laterais superiores. Esse é um assunto que divide a opinião de ortodontistas e reabilitadores orais, devido às vantagens e desvantagens de cada uma das opções de tratamento, as quais podem envolver a abertura de espaços para futuros implantes e/ou restaurações protéticas ou o fechamento dos espaços, com posicionamento dos caninos superiores no lugar dos incisivos laterais. O correto diagnóstico e uma criteriosa avaliação de cada paciente permitem determinar a melhor abordagem terapêutica. Nesse artigo, serão discutidos os principais tópicos a serem considerados no planejamento desses casos. Objetivos: Avaliar os principais aspectos relacionados ao planejamento do tratamento ortodôntico nos casos de ausência congênita de incisivos laterais superiores, de maneira a auxiliar nas tomadas de decisão, com embasamento clínico e científico.

Orthodontic traction of impacted maxillary canines using segmented arch mechanics.

The principles of orthodontic mechanics strongly influence the success of impacted canine traction. The present study discusses the main imaging exams used for diagnosis and localization of impacted canines, the possible associated etiological factors and the most indicated mechanical solutions.

Orthodontic traction of impacted maxillary canines using segmented arch mechanics

ABSTRACT The principles of orthodontic mechanics strongly influence the success of impacted canine traction. The present study discusses the main imaging exams used for diagnosis and localization of impacted canines, the possible associated etiological factors and the most indicated mechanical solutions.

RESUMO Os princípios vetoriais da mecânica ortodôntica têm influência direta no sucesso do tracionamento dos caninos impactados. O objetivo desse artigo é discorrer sobre os possíveis fatores etiológicos associados à impacção dos caninos, os exames de imagem indicados no processo de diagnóstico e localização das unidades retidas, e as melhores soluções mecânicas para o tracionamento desses dentes.

LAPS Insulin115: A novel ultra-long-acting basal insulin with a unique action profile.

AIMS: To conduct a comprehensive pre-clinical study of the novel ultra-long acting insulin analogue LAPS Insulin115. METHODS: Pharmacokinetic/pharmacodynamic studies comparing LAPS Insulin115 with other basal insulins were conducted in genetically diabetic (db/db) mice. Insulin signalling in the major target organs was analysed using Western blot after single subcutaneous injection in wild-type male Wistar rats. Using in vitro assays we analysed transendothelial transport, insulin receptor (IR) interaction, and the mitogenic and metabolic properties of LAPS Insulin115. Furthermore, IR downregulation after long-term exposure to high concentrations of LAPS Insulin115 was analysed using an in vitro desensitization/resensitization model. RESULTS: The novel Fc-conjugated insulin derivative LAPS Insulin115 showed an extensively prolonged pharmacokinetic and pharmacodynamic profile in rodents. Despite its size of 59 kDa, LAPS Insulin115 passes the vascular endothelial barrier and induces insulin signalling in all major target tissues in rats. In vitro, LAPS Insulin115 showed a very slow onset of action because of its reduced IR affinity; however, after long-term stimulation it was equipotent in respect to its metabolic potency and showed no increased mitogenic action when compared with regular insulin. Remarkably, under conditions of chronic exposure, LAPS Insulin115 does not induce irreversible desensitization of target cells, which is probably attributable to much less prominent IR downregulation. CONCLUSION: Thus, LAPS Insulin115 exhibits a unique in vivo and in vitro profile and thereby represents an excellent candidate for a once-weekly insulin analogue.

Extrações de molares na Ortodontia / Molar extractions in orthodontics

O tratamento ortodôntico com extração de molares em pacientes adultos é tecnicamente mais complexo, devido a inúmeros fatores. Em geral, o espaço a ser fechado é maior do que o espaço dos pré-molares e, por isso, a ancoragem é crítica e o tempo de tratamento mais longo. É comum esses casos apresentarem algum grau de comprometimento periodontal por causa da idade dos pacientes e, portanto, necessitam de maior controle da mecânica ortodôntica para reduzir os efeitos colaterais do fechamento do espaço. Por isso, bons resultados de finalização são mais difíceis de ser alcançados. Sendo assim, este artigo tem como objetivo apresentar as razões para a indicação de extração de molares nos tratamentos ortodônticos, as contraindicações, as diferentes fases da mecânica ortodôntica, esclarecer os fatores envolvidos nesse tipo de planejamento e tratamento e apresentar casos clínicos tratados com extração de molares.

Orthodontic treatment with extraction of molars in adult patients is technically more complex due to a number of factors. In general, the space to be closed is greater than premolar spaces rendering critical anchorage and longer treatment time. Often such cases exhibit some degree of periodontal involvement due to patient age. Hence, the need for greater control over orthodontic mechanics to reduce the side effects of space closure. Therefore, good finishing results can be more difficult to achieve. Thus, the purpose of this article is to determine the reasons for molar extraction indications, describe different stages of orthodontic mechanics, and explain the issues involved in this kind of planning and treatment. Additionally, it aims at describing some treatments with molar extractions.

Má oclusão Classe II, 2ª divisão de Angle com sobremordida exagerada e discrepância acentuada / Angle Class II, division 2 malocclusion with severe overbite and pronounced discrepancy

Este artigo relata o tratamento de uma jovem paciente, com 13,8 anos de idade, que apresentava uma má oclusão Classe II, segunda divisão de Angle, retenção prolongada de dentes decíduos, mordida cruzada dentária, sobremordida exagerada, dentre outros desvios da normalidade. Inicialmente, a abordagem ortodôntica envolveu uma expansão da maxila, seguida pelo uso do aparelho extrabucal de Kloehn e aparelhagem ortodôntica fixa. O resultado obtido demonstra a importância de um diagnóstico e planejamento criteriosos, bem como a necessidade de colaboração do paciente durante o tratamento ortodôntico. Este caso clínico foi apresentado à Diretoria do Board Brasileiro de Ortodontia e Ortopedia Facial (BBO), representando a categoria livre, como parte dos requisitos para a obtenção do título de diplomado pelo BBO.

This article reports the treatment of a young patient at 13.8 years of age who presented with an Angle Class II, division 2 malocclusion, prolonged retention of deciduous teeth, dental crossbite and severe overbite, among other abnormalities. At first, the approach involved rapid maxillary expansion followed by the use of Kloehn headgear and fixed orthodontic appliance. Treatment results demonstrate the importance of careful diagnosis and planning as well as the need for patient compliance during treatment. This case was presented to the Brazilian Board of Orthodontics and Facial Orthopedics (BBO). It is representative of the free category and fulfills part of the requirements for obtaining the BBO Diploma.

The adipocyte-myocyte axis in insulin resistance.

Insulin resistance in skeletal muscle is linked to an elevated adipose tissue mass, as is found in obesity, but can also be observed in lipodystrophy, in which adipose tissue is greatly reduced. Adipose tissue releases endocrine and metabolic mediators and is actively involved in crosstalk with skeletal muscle, a process that precedes and underlies the development of insulin resistance in muscles. Adipokines including tumor necrosis factor alpha, interleukin-6, leptin and adiponectin influence insulin signaling in skeletal muscle. Free fatty acids, their metabolites and ectopic fat in muscle also contribute to insulin resistance. Recent research indicates inflammation, endoplasmic reticulum stress and oxidative stress could be underlying mechanisms at the center of the development of insulin resistance. Insights into the role of macrophages in adipose tissue add to the complicated interplay between adipose tissue and skeletal muscle.

Pathways leading to muscle insulin resistance--the muscle--fat connection.

Type 2 diabetes is a heterogeneous disease characterized by hyperglycemia and insulin resistance in peripheral tissues such as adipose tissue and skeletal muscle. This review focuses on obesity as one of the major environmental factors contributing to the development of diabetes. It has become evident that adipose tissue represents an active secretory organ capable of releasing a variety of cytokines such as TNFalpha, IL-6, adiponectin and other still unknown factors that might constitute the missing link between adipose tissue and insulin resistance. In fact, adipocyte-derived factors are significantly increased in obesity and represent good predictors of the development of type 2 diabetes. The negative crosstalk between adipocytes and skeletal muscle cells leads to disturbances in muscle cell insulin signalling and insulin resistance involving major pathways in inflammation, cellular stress and mitogenesis. Positive regulators of insulin sensitivity include the adipocyte hormone adiponectin and inhibitors of inflammatory pathways such as JNK-, IKK- and ERK-inhibitors. In summary, a better knowledge of intracellular and intercellular mechanisms by which adipose tissue affects skeletal muscle cell physiology may help to develop new strategies for diabetes treatment.

Cytokine secretion by human adipocytes is differentially regulated by adiponectin, AICAR, and troglitazone.

Adipose tissue is an active endocrine organ producing a variety of cytokines and chemokines, which may be involved in the deregulation of glucose and lipid homeostasis as well as in the inflammatory state observed in obesity. We have shown previously that differentiated human adipocytes secrete a variety of cytokines which are able to induce skeletal muscle insulin resistance. However, the regulation of these factors by anti-diabetic drugs has remained mainly undefined. Secretion of IL-6, IL-8, MIP-1alpha/beta, and MCP-1 by adipocytes was found to be downregulated by adiponectin. In parallel to adiponectin, the AMPK activator AICAR also decreased the secretion of most of the measured cytokines including IL-6 and MIP-1alpha/beta but not IL-8. In contrast, the thiazolidinedione troglitazone only slightly reduced cytokine secretion despite increasing the phosphorylation of AMPK. In conclusion, we show that adipocyte secretion is strongly inhibited by the anti-diabetic adipocyte hormone adiponectin, an effect that can also be mimicked by the AMPK activator AICAR. However, the PPARgamma agonist troglitazone is much less effective in reducing cytokine secretion.

Monocyte chemotactic protein-1 is a potential player in the negative cross-talk between adipose tissue and skeletal muscle.

Adipose tissue is a major secretory and endocrine active organ producing a variety of bioactive proteins that may regulate energy metabolism and insulin sensitivity. In several studies, we have already shown that adipocyte-secretory products induce skeletal muscle insulin resistance. However, the precise nature of these factors has remained elusive. Human adipocytes were found to secrete various cytokines including IL-6, IL-8, macrophage inflammatory protein-1alpha/beta, and monocyte chemotactic protein-1 (MCP-1). Among these candidates, MCP-1 alone impaired insulin signaling in skeletal muscle cells at doses similar to its physiological plasma concentrations (200 pg/ml), whereas IL-6, IL-8, and macrophage inflammatory protein-1beta were effective at very high concentrations only. In addition, MCP-1 significantly reduced insulin-stimulated glucose uptake in the myocytes. Expression analysis of chemokine receptors in skeletal muscle cells revealed the presence of chemokine CXC motif receptor 1/2 and chemokine CC motif receptor 1/2/4/5/10. The action of MCP-1 on insulin signaling in skeletal muscle cells occurs via ERK1/2 activation but does not involve activation of the nuclear factor kappaB pathway. In conclusion, our data show that adipocytes secrete various adipokines that may be involved in the negative cross-talk between adipose tissue and skeletal muscle. Human skeletal muscle cells are highly sensitive toward MCP-1, which impairs insulin signaling and glucose uptake at concentrations even below that found in the circulation. However, other cytokines that are released by adipocytes impair insulin action only at supraphysiological concentrations. Therefore, MCP-1 may represent a molecular link in the negative cross-talk between adipose tissue and skeletal muscle assigning a completely novel important role to MCP-1 besides inflammation.

Autocrine action of adiponectin on human fat cells prevents the release of insulin resistance-inducing factors.

The adipocyte hormone adiponectin is negatively correlated with obesity and insulin resistance and may exert an important antidiabetes function. In this study, primary human skeletal muscle cells were cocultured with human fat cells or incubated with adipocyte-conditioned medium in the presence or absence of the globular domain of adiponectin (gAcrp30) to analyze its capacity to restore normal insulin signaling in the muscle cells. Human skeletal muscle cells cocultured with adipocytes or treated with adipocyte-conditioned medium showed an impaired Akt and glycogen synthase kinase 3 serine phosphorylation in response to insulin. Furthermore, insulin-stimulated GLUT4 translocation was reduced by adipocyte-conditioned medium. Impaired insulin signaling was normalized upon addition of gAcrp30 to the coculture. Further, adipocyte-conditioned medium generated in the presence of gAcrp30 was unable to perturb insulin-stimulated Akt phosphorylation. Concomitant addition of gAcrp30 and adipocyte-conditioned medium to the myocytes failed to restore normal insulin action. Protein array analysis of adipocyte-conditioned medium indicated that the secretion of at least eight different cytokines was diminished in response to gAcrp30. We therefore suggest that adiponectin operates as a key regulator of adipocyte secretory function. This autocrine action may prevent the induction of skeletal muscle insulin resistance and may partly explain the antidiabetes action of this hormone.