Quantification of cellular action fibers alignment from confocal microscopic images.

The cellular rheology has recently undergone a rapid development with particular attention to the cytoskeleton mechanical properties and its main components - actin filaments, intermediate filaments, microtubules and crosslinked proteins. However it is not clear what are the cellular structural changes that directly affect the cell mechanical properties. Thus, in this work, we aimed to quantify the structural rearrangement of these fibers that may emerge in changes in the cell mechanics. We created an image analysis platform to study smooth muscle cells from different arteries: aorta, mammary, renal, carotid and coronary and processed respectively 31, 29, 31, 30 and 35 cell image obtained by confocal microscopy. The platform was developed in Matlab (MathWorks) and it uses the Sobel operator to determine the actin fiber image orientation of the cell, labeled with phalloidin. The Sobel operator is used as a filter capable of calculating the pixel brightness gradient, point to point, in the image. The operator uses vertical and horizontal convolution kernels to calculate the magnitude and the angle of the pixel intensity gradient. The image analysis followed the sequence: (1) opens a given cells image set to be processed; (2) sets a fix threshold to eliminate noise, based on Otsu's method; (3) detect the fiber edges in the image using the Sobel operator; and (4) quantify the actin fiber orientation. Our first result is the probability distribution II(Δθ) to find a given fiber angle deviation (Δθ) from the main cell fiber orientation θ0. The II(Δθ) follows an exponential decay II(Δθ) = Aexp(-αΔθ) regarding to its θ0. We defined and determined a misalignment index α of the fibers of each artery kind: coronary αCo = (1.72 ‘+ or =’ 0.36)rad POT -1; renal αRe = (1.43 + or - 0.64)rad POT -1; aorta αAo = (1.42 + or - 0.43)rad POT -1; mammary αMa = (1.12 + or - 0.50)rad POT -1; and carotid αCa = (1.01 + or - 0.39)rad POT -1. The α of coronary and carotid are statistically different (p < 0.05) among all analyzed cells. We discussed our results correlating the misalignment index data with the experimental cell mechanical properties obtained by using Optical Magnetic Twisting Cytometry with the same group of cells

Thalidomide as a treatment of intestinal angiodisplastic lesions in a patient with CREST syndrome – case report and literature review

A anemia ferropriva grave secundária à hemorragia digestiva por angiodisplasiasintestinais representa um grande desafio terapêutico. Comumente, as ectasias vasculares sãomúltiplas e dispersas ao longo do intestino, limitando a eficácia do tratamento hemostáticolocal. Nos últimos anos houve significativo avanço no tratamento anti-angiogênico sistêmico dasangiodisplasias intestinais, sendo talidomida a droga mais empregada para tal fim. Relatamoso caso de uma paciente de 49 anos com angiodisplasias intestinais secundárias a síndromeCREST (Calcinose, Raynaud, Dismotilidade Esofágica, Esclerodactilia e Telangiectasias). Apaciente apresentava quadro de melena recorrente e alta necessidade transfusional, e nãoobteve resposta clínica após realização de enteroscopia e eletro-coagulação das lesões complasma de argônio. Após a introdução de talidomida 100mg ao dia, a paciente evoluiu deforma bastante satisfatória. O caso apresentado neste texto, além de demonstrar sucesso datalidomida no tratamento de angiodisplasias intestinais refratárias à eletro-coagulação complasma de argônio, também revela eficácia da droga na situação específica da síndromeCREST. Tal fato pode ser de grande valia quando da abordagem de hemorragia intestinal porangiodisplasias nesses pacientes, representando nova opção terapêuticaThe severe ferropenic anemia secondary to digestive bleeding due to intestinalangiodisplastic lesions represents a great challenge. Commonly, angiodisplastic lesions aremultiples and disperse through the intestine and that fact limits local treatments. Over the lastyears, there was a great advance in the antiangiogenic treatment of intestinal angiodisplasticlesions and thalidomide was the most employed drug for this purpose. We report a case of a49 year-old patient with intestinal angiodisplastic lesions due to CREST syndrome (Calcinosis,Raynaud phenomenon, Esophageal dysmotility, Sclerodactyly, and Telangiectasia). The patientpresented repeated episodes of digestive bleeding and did not achieve clinical improvementafter enteroscopy and argon plasma coagulation. The treatment consisting of the introductionof thalidomide 100mg per day demonstrated success. The case presented in this text revealssuccess in the use of thalidomide in the treatment of intestinal angiodisplastic lesions, probablyrepresenting a new therapeutic optio

The Use of Imatinib Mesylate as a Lifesaving Treatment of Chronic Myeloid Leukemia Relapse after Bone Marrow Transplantation

We describe the response of imatinib as lifesaving treatment of chronic myeloid leukemia (CML) relapse in seven patients who underwent allogeneic bone marrow transplantation (alloBMT) at our institution over a period of 4 years. Retrospective analysis of their medical records revealed that a mean age at transplant was 45.2 years. The median time to diagnosis was 7.4 years after transplant. At relapse, four, two, and one patients were classified as having hematologic, major molecular, and cytogenetic relapse, respectively. At imatinib initiation, five had CML in a chronic phase, while one patient was diagnosed as having accelerated phase and blast crisis. All these patients could be evaluated for the therapeutic efficacy. At a mean of follow-up of 1.9 years of therapy, all evaluable patients achieved major molecular response without compromising safety. Consistent with available data, our results indicate that imatinib is safe and effective treatment option for patients with relapse after BMT

Porfirias: quadro clínico, diagnóstico e tratamento

As porfirias são doenças incomuns e de herança genética na maior parte dos casos. As porfirias são divididas em eritropoiéticas, hepáticas agudas e hepáticas crônicas. Os subtipos de maior relevância clínica são a porfiria cutânea tarda e a porfiria intermitente aguda. O diagnóstico das porfirias pode ser bastante difícil, dada a sobreposição de quadros clínicos e achados bioquímicos. A precisão do diagnóstico depende da dosagem de porfirinas urinárias e fecais, da análise da atividade enzimática de eritrócitos e, eventualmente, da pesquisa de mutações. O objetivo do presente artigo é realizar revisão literária das porfirias, com ênfase no diagnóstico e tratamento de seus diversos subtipos.Porphyrias are uncommon diseases that have genetic inheritance in the majority of the cases. Porphyrias are divided in: erythropoietic porphyria, acute hepatic porphyria and chronic hepatic porphyria. The subtypes with major clinical relevance are porphyria cutanea tarda and acute intermittent porphyria. Diagnosing porphyrias may be quite difficult, as there is significant overlapping between clinical and biochemical findings. The diagnosis depends on the measurement of urinary and fecal porphyrins, enzymatic analysis of erythrocytes and, eventually, analysis of mutations. The main purpose of this article is to make a review of porphyrias, with emphasis on diagnosis and treatment of its several subtypes

Non-HFE hemochromatosis

Hereditary hemochromatosis (HH) is an autosomal recessive disorder classically related to HFE mutations. However, since 1996, it is known that HFE mutations explain about 80% of HH cases, with the remaining around 20% denominated non-HFE hemochromatosis. Nowadays, four main genes are implicated in the pathophysiology of clinical syndromes classified as non-HFE hemochromatosis: hemojuvelin (HJV, type 2Ajuvenile HH), hepcidin (HAMP, type 2B juvenile HH), transferrin receptor 2 (TFR2, type 3 HH) and ferroportin (SLC40A1, type 4 HH). The aim of this review is to explore molecular, clinical and management aspects of non-HFE hemochromatosis

Quality of life scores differs between genotypic groups of patients with suspected hereditary hemochromatosis

Background: Hereditary hemochromatosis (HH) encompasses a group of autosomal recessive disorders mainly characterized by enhanced intestinal absorption of iron and its accumulation in parenchymal organs. HH diagnosis is based on iron biochemical and magnetic resonance imaging (MRI) assessment, and genetic testing. Questionnaires, such as SF-36 (short form health survey), have been increasingly used to assess the impact of diseases on the patient's quality of life (QL). In addition, different genotypes are identified as results of genetic tests in patients with suspected primary iron overload. In the present study, our aim was to evaluate whether domains of QL are different according to genotypic groups in patients suspected of HH. Methods: Seventy-nine patients with primary iron overload were included and two genotypic groups were formed (group 1: homozygous genotype for the HFE p.Cys282Tyr mutationgroup 2: other genotypes). Results: Group 1 had higher means of plasma transferrin saturation (86 +/- 19%) and serum ferritin (1669 +/- 1209 ng/mL) compared to group 2 (71 +/- 12%, 1252 +/- 750 ng/mL, respectivelyp = 0.001). Four domains were significantly different among groups 1 and 2: physical functioning (p = 0.03), bodily pain (p = 0.03), vitality (p = 0.02) and social functioning (p = 0.01). Conclusions: Our main finding was that patients with p. Cys282Tyr homozygosity had a worse QL scenario assessed by SF-36, compared with patients with iron overload without the same genotype. Being aware of this relationship between genotypes and QL might be helpful in the overall management of patients suspected of hereditary hemochromatosis.Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES) [2013/09295-3]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), Brazil [2013/20614-3]Univ Sao Paulo, Heart Inst InCor, Lab Genet & Mol Cardiol, Med Sch, Av Doutor Eneas de Carvalho Aguiar 44, BR-05403900 Sao Paulo, SP, BrazilSanta Casa Med Sch, Hematol & Hemotherapy Sect, Sao Paulo, BrazilAcad Ciencia & Tecnol, Sao Jose Do Rio Preto, BrazilFundacao Pro Sangue, Hemoctr Sao Paulo, Sao Paulo, SP, BrazilUniv Sao Paulo, Sao Paulo, SP, BrazilUniv Sao Paulo, Med Sch, Hosp Clin, Hematol Serv, Sao Paulo, BrazilUniv Sao Paulo, Med Sch, Hosp Clin, Hematol & Hemotherapy Discipline, Sao Paulo, BrazilUniv Rennes, Pontchaillou Univ Hosp, Liver Dis Unit, Rennes, FranceNatl Reference Ctr Rare Iron Overload Dis Genet O, Rennes, FranceUniv Fed Sao Paulo UNIFESP, Dept Pharmacol, Sao Paulo, BrazilUniv Fed Sao Paulo UNIFESP, Dept Pharmacol, Sao Paulo, BrazilCAPES: 2013/09295-3FAPESP: 2013/20614-3Web of Scienc

Study of the mechanical properties of vascular smooth muscle cells under physiological and pathological situations

Introdução: As células do músculo liso vascular (CMLV) são quiescentes nos vasos adultos, com baixa capacidade de migração e de secreção de matriz extracelular, caracterizando fenótipo contrátil. Evidências apontam para a heterogeneidade fenotípica das CMLV ao longo da árvore arterial: há distribuição heterogênea de doenças e de resposta a determinadas drogas nos diferentes vasos, além de variabilidade de expressão dos genes de proteínas contráteis de músculo liso entre eles. O papel das CMLV, em fase adulta, é classicamente descrito como restrito à regulação do tônus de pequenos vasos, sendo insignificante a contribuição da mecânica das CMLV para a complacência das artérias elásticas. Existe a hipótese de que a viscoelasticidade das CMLV contribua para a mecânica final das artérias, sendo o enrijecimento dessas células associado à rigidez arterial. Objetivo: Estudar a variabilidade das propriedades mecânicas e de expressão proteica das CMLV, ao longo da árvore arterial, buscando identificar moduladores regionais para esse fenótipo. Avaliar se situações clínicas sabidamente associadas à rigidez arterial (envelhecimento, sexo feminino pós-menopausa, ancestralidade genética africana, diabetes mellitus e tabagismo) cursam com enrijecimento de CMLV. Métodos: 1) Estudou-se a composição e a organização da camada média de diferentes artérias. As CMLV desses vasos foram avaliadas quanto à viscoelasticidade de citoplasma (G), por meio do ensaio de Citometria Magnético Ótica de Oscilação e, quanto à expressão proteica global, usando cromatografia multidimensional e espectrometria de massas em tandem de alta resolução (Proteômica Shotgun). Os dados mecânicos obtidos foram correlacionados com as características da matriz extracelular (MEC) dos vasos de origem (porcentagem de elastina e quantidade de MEC). Em paralelo, foi realizado experimento de estiramento cíclico (10%/1Hz) das CMLV das diferentes artérias por 24 e 48h, seguido pela mensuração de rigidez de citoplasma. 2) Foram isoladas as CMLV de fragmentos de artéria mamária de 80 pacientes submetidos à cirurgia de revascularização do miocárdio, células essas que foram avaliadas quanto à viscoelasticidade de citoplasma (G, G\' e G\'\'). Elaborou-se modelo estatístico para avaliar se as variáveis clínicas idade, sexo feminino, ancestralidade africana, tabagismo e diabetes mellitus estavam associadas a alterações de mecânica celular. Resultados: 1) A viscoelasticidade das CMLV variou significativamente entre as artérias. As CMLV provenientes de artérias distais (artérias femoral e renal) mostraram-se significativamente mais rígidas que as CMLV de aorta torácica (p < 0,001). Identificou-se correlação negativa entre rigidez de CMLV e quantidade de MEC / elastina na camada média vascular. O regime de estiramento cíclico por 48h reduziu globalmente a rigidez das CMLV. As CMLV provenientes da aorta torácica expressaram maior quantidade de proteínas relacionadas com a estrutura e a organização do citoesqueleto em relação às CMLV da artéria femoral. 2) Constatou-se variabilidade interindividual de viscoelasticidade de CMLV e associação entre tabagismo e sexo feminino com enrijecimento de CMLV. Conclusões: As CMLV são heterogêneas quanto às propriedades mecânicas, à organização do citoesqueleto e à expressão proteica ao longo da árvore arterial, reforçando o conceito de plasticidade fenotípica das CMLV. A mecânica das CMLV é modulada pelas características da MEC e pela tensão circunferencial cíclica aplicada às paredes vasculares pelo fluxo sanguíneo. Mulheres pós-menopausa e tabagistas exibem enrijecimento de CMLV, sendo esse fato um provável contribuinte para a rigidez arterial associada a essas condições e um possível alvo terapêutico a ser avaliado futuramenteRational: Vascular smooth muscle cells (VSMC) lose their ability to migrate and secrete extracellular matrix (ECM) with the end of vascular development, condition known as contractile phenotype and reversible in the presence of vascular injury. There is evidence of heterogeneity of VSMC phenotype along arterial tree, as the distribution of diseases (atherosclerosis) and the response to drugs vary between different vessels, as well as the expression of smooth muscle-contractile protein genes. The role played by VSMC mechanics on determining large arteries\' compliance was always considered irrelevant. It has been hypothesized that the VSMC mechanical properties are important for vascular mechanics, especially in the pathological scenario, where VSMC stiffening may be associated with arterial rigidity. Goals: Study the variation of VSMC mechanics and protein expression along arterial tree, identifying regional modulators of this phenotype. Evaluate if clinical situations associated with arterial rigidity (ageing, post-menopausal women, African ancestry, diabetes mellitus and smoking) concur with VSMC stiffening. Methods: 1) Different arteries were studied in terms of composition and organization of their media layer. VSMC isolated from these arteries were evaluated regarding cytoplasm viscoelasticity, measured using Optical Magnetic Twisting Cytometry Assay (OMTC), and protein expression, using two-dimensional liquid chromatography and tandem mass spectrometry (Shotgun Proteomics). Mechanical data were correlated with ECM characteristics (percentage of elastin and ECM amount) of the vessels of origin. In parallel, VSMC of different arteries were subjected to cyclic stretching (10%/1Hz) during 24 and 48h, followed by the measurement of their cytoplasm rigidity. 2) VSMC were isolated from fragments of mammary artery of 80 patients subjected to coronary bypass surgery and evaluated regarding their viscoelasticity (G, G\' e G\'\'). A statistic model was elaborated to address if the clinical variables age, female sex, African ancestry, smoking and diabetes mellitus were associated with changes of VSMC mechanics. Results: 1) VSMC viscoelasticity varied significantly amongst the studied arteries. VSMC from heart-distant arteries (femoral and renal arteries) were stiffer than VSMC from thoracic aorta (p < 0,001). There was a negative correlation between VSMC rigidity and the amount of ECM / percentage of elastin within the media layer. 48h-cyclic stretching was associated with a global reduction of VSMC rigidity. VSMC of thoracic aorta expressed significantly more proteins associated with cytoskeleton structure and organization than VSMC of femoral artery. 2) There was a significant inter-individual variation of VSMC viscoelasticity. Smoking and female sex were associated with VSMC stiffening. Conclusion: VSMC mechanics, cytoskeleton organization and protein expression are heterogeneous along arterial tree. VSMC mechanical properties are modulated by ECM characteristics and by regional mechanical forces. This reinforces the concept of phenotypic heterogeneity of VSMC. Post-menopausal women and smokers exhibit stiffer VSMC, representing an important factor for the understanding of the arterial rigidity associated with these conditions and also a possible future therapeutic targe

Immunoglobulin: production, mechanisms of action and formulations

Human immunoglobulin (Ig) began to be applied in the clinical practice with the treatment of primary immunodeficiencies. Quickly, applications of Ig increased, as its anti-inflammatory and immunomodulatory functions were elucidated. Currently, Ig is the most commonly used blood product. Ig is obtained by processing plasma; methods, in particular, techniques to reduce plasma viral loads have been evolving over the years and include: pasteurization, solvent/ detergent treatment, caprylic acid treatment and nanofiltration. These methods contribute to increased safety and quality of blood products. The mechanisms of action of Ig not only involve the blockade of Fc receptors of phagocytes, but also control complement pathways, idiotype-anti-idiotype dimer formation, blockage of superantigen binding to T cells, inhibition of dendritic cells and stimulation of regulatory T cells (Tregs). There are several formulations of Ig available, each one with its own peculiar characteristics. In Brazil, there is stringent legislation regulating the quality of Ig. Only Ig products that completely fulfill the quality control criteria are released for use. These standards involve different tests from visual inspection to determination of anti-complementary activity. This paper will further review the history and current status of Ig, including its production and mechanisms of action. The formulations available in Brazil and also the criteria of quality control currently applied will be presented

Study of possible clinical and laboratory predictors of alloimmunization against red blood cell antigens in cancer patients

Background: The inflammatory background of patients influences the process of alloimmunization against red blood cell antigens. Proof of this statement to clinical practice is still lacking. Objective: The aim of this study was to verify whether factors related to disease severity and inflammatory status of cancer patients can predict alloimmunization. Methods: This was a case-control study in which alloimmunized oncologic patients treated between 2009 and 2012 were compared with a non-alloimmunized control group regarding the severity of the disease (metastasis/performance status/body mass index) and C-reactive protein levels. Results: The groups did not differ significantly in terms of C-reactive protein, Eastern Cooperative Oncology Group (ECOG)/Karnofsky performance status, presence of metastasis and body mass index. Conclusion: It is not possible to predict alloimmunization in cancer patients based on severity of illness and inflammatory markers. Strategies of screening patients by phenotyping blood based on these criteria are not justified