Avaliação proteica do humor aquoso de equinos / Evaluation of equine aqueous humor protein

A avaliação proteica do humor aquoso (HA) pode ser utilizada como método diagnóstico nas uveítes. Entretanto, estudos sobre as proteínas nesse fluido, em equinos hígidos, são escassos e apresentam variações conforme a metodologia empregada. Dessa forma, objetivou-se realizar a análise proteica e citológica do HA nessa espécie, bem como verificar sua correlação com as proteínas plasmáticas. Foram avaliados 13 equinos adultos (26 olhos), sem raça definida, machos ou fêmeas. Mediante aqueocentese, foi coletado 0,5 mL de humor aquoso de cada olho. Cada amostra foi encaminhada para quantificação proteica pelo método de Bradford modificado e pela eletroforese em gel de poliacrilamida - dodecil sulfato de sódio (SDS-PAGE), bem como para avaliação citológica. Por meio de venopunção, coletou-se sangue para determinação da concentração de proteínas séricas. Treze olhos (50% das amostras) apresentaram valor proteico médio de 40,3 mg/dL±6,45 e a eletroforese demonstrou presença de proteínas de massas mais elevadas que 43 KDa. Houve ausência de células em 96,15% das amostras (25 olhos). Equinos hígidos apresentaram baixa concentração de proteínas no HA. Já a correlação entre proteína no humor aquoso/proteína plasmática total foi de 0,56%.(AU)

Evaluation of equine aqueous humor (AH) proteins can help the diagnosis of uveitis. However, studies on proteins in this fluid in healthy horses are scarce and present variations according to the methodology employed. This study aimed to perform protein analysis and cytology of equine aqueous humor of healthy horses and verify its correlation with plasmatic proteins. Thirteen adult horses (26 eyes), mixed breed, male or female were evaluated. A volume of 0.5 mL of aqueous humor was collected through aqueocentesis from both eyes. The samples were submitted to protein quantification by modified Bradford method and to sodium dodecyl sulphate - polyacrylamide gel electrophoresis (SDS-PAGE), and to cytological evaluation. Blood was collected for determination of plasmatic protein concentration. Thirteen eyes (50% of the samples) had values larger than zero by the Bradford method, with an average of 40.3 mg/dl±6.45. Electrophoresis showed presence of higher masses of proteins (43 KDa). There were no cells in 96.15% of the samples (25 eyes). Healthy equines presented low protein concentration in the HA. The ratio between protein concentration in the aqueous humor / total plasma protein of 0.56%.(AU)

Estudo anatômico do trajeto do canal mandibular em felinos (Felis catus domesticus) / Anatomical study of the mandibular canal course in felines (Felis catus domesticus)

Objetivou-se descrever, por meio de tomografia computadorizada, o trajeto do canal mandibular (CM) em 20 gatos sem raça definida, com ausência de alterações na cavidade oral, provenientes do Centro de Controle de Zoonoses do Distrito Federal. Foram realizados cortes tomográficos com 2mm de espessura, acompanhando todo o trajeto do CM, tendo como referência a região do forame mandibular, as raízes distais e mesiais dos dentes pré-molares e molares e o forame mentoniano, obtendo-se medidas desde o CM até as faces vestibular, lingual, ventral e alveolar (profundidade) do corpo da mandíbula, bem como seu diâmetro. Pôde constatar que o CM manteve-se no aspecto lingual do corpo da mandíbula desde o forame mandibular até a raiz mesial do 1º pré-molar, onde se deslocou para a face vestibular, emergindo no forame mentoniano. Com relação à profundidade, seu trajeto sofreu declive a partir do forame mandibular até a região da raiz mesial do 1º molar, onde alcançou seu ponto mais profundo para prosseguir em suave ascensão até o forame mentoniano. Os dados apresentados contribuem para o estudo anatômico da mandíbula de gatos, bem como auxiliam no melhor planejamento e execução de procedimentos cirúrgicos na mandíbula dessa espécie.

This study aimed to describe the path of the mandibular canal (MC), using computerized tomography, in twenty mongrel cats, with no changes in the oral cavity, from the Zoonosis Control Center of the Federal District. 2mm thick tomographic sections were taken following the entire path of the mandibular canal, considering as reference the region of the mandibular foramen, the distal and mesial roots of premolar and molar teeth, and mental foramen, obtaining measurements from the MC until the buccal, lingual, ventral and alveolar (depth) surfaces of the mandibular body as well as its diameter. MC remained on the lingual aspect of the mandibular body from the mandibular foramen to the mesial root of the first premolar, where it displaced to the buccal surface, emerging from the mental foramen. Regarding the depth, we observed a downward path from the mandibular foramen to the mesial root of the first premolar, where it reached its deepest point, the path continued in gentle ascent until to the mental foramen. Our data contribute to the anatomical study of the feline jaw and allow a better planning and execution of surgical procedures in the mandible of this species.

4-Hexyl-1,3-phenylenediol, a nuclear factor-κB inhibitor, improves photodamaged skin and clinical signs of ageing in a double-blinded, randomized controlled trial.

BACKGROUND: The nuclear factor-κB (NF-κB) pathway is a key mediator of inflammation; however, few studies have examined the direct effects of NF-κB inhibition on the skin. OBJECTIVES: To investigate NF-κB activity in cultured human fibroblasts and to investigate the effects of 4-hexyl-1,3-phenylenediol (an NF-κB inhibitor) on elastin and collagen gene expression in vitro and on the clinical appearance of photodamaged skin. METHODS: The amount and activity of NF-κB in human fibroblasts obtained from donors (17-78 years old) was measured after transfection with a NF-κB reporter and a luciferase promoter system. The expression of extracellular matrix (ECM) genes was determined using quantitative polymerase chain reaction. Women with moderate skin photodamage were randomized to daily treatment with a topical lotion containing 4-hexyl-1,3-phenylenediol (n = 30) or vehicle (n = 29) for 8 weeks, with clinical assessments at baseline and weeks 2, 4 and 8. RESULTS: Fibroblasts obtained from donors older than 50 years had higher NF-κB activity compared with cells from younger donors; inhibition of the NF-κB pathway with 4-hexyl-1,3-phenylenediol enhanced the expression of ECM genes. In women, treatment for 8 weeks with 4-hexyl-1,3-phenylenediol significantly improved crow's feet fine lines, cheek wrinkles, age spots, mottled pigmentation and radiance compared with both the vehicle and baseline. Furthermore, treatment with 4-hexyl-1,3-phenylenediol resulted in a twofold greater clinical improvement in overall photodamage compared with the vehicle group. CONCLUSIONS: Inhibition of the proinflammatory NF-κB pathway resulted in increased expression of ECM proteins in vitro and significant clinical improvement in photodamaged skin.

Involvement and alteration of the Sonic Hedgehog pathway is associated with decreased cholesterol level in trisomy 18 and SLO amniocytes.

BACKGROUND: Trisomy 18 and Smith-Lemli-Opitz syndrome are two polymalformative conditions in which a cholesterol defect has been noted. When they occur prenatally, they are associated with a decreased maternal unconjugated estriol (uE(3)) level. Cholesterol plays an essential role in the Sonic Hedgehog pathway, allowing Shh protein maturation leading to its maximal activity. Many malformations in these two syndromes occur in Shh dependent tissues. We thus sought to assess whether a cholesterol defect could affect the Shh pathway and explain some of the observed malformations. MATERIALS AND METHODS: We selected 14 cases of trisomy 18 and 3 cases of SLO in which the maternal uE(3) level was decreased and reported malformations were observed after fetopathological examination. We correlated the number of malformations with maternal uE(3) level. We then carried out cholesterol concentrations in separate culture media consisting of trisomy 18, SLO and control amniocytes. Finally, we analyzed the Shh pathway by testing the gene expression of several Shh components: GLI transcription factors, BMP2, BMP4, TGFß1, COL1A1 and COL1A2. RESULTS AND DISCUSSION: There was an inverse correlation between phenotypic severity and maternal uE(3) levels in SLO and trisomy 18. The cholesterol levels in the amniocyte culture media were correlated with maternal uE3 levels and were significantly lower in T18 and SLO amniocytes, reflecting cholesterol defects. There was an alteration in the Shh pathway since expression of several genes was decreased in T18 and SLO amniocytes. However, these cholesterol defects were not solely responsible for the altered Shh pathway and the malformations observed.

Contribution of ocular B-mode and triplex Doppler in the evaluation of 10 Poodle dogs with cataracts / Contribuição diagnóstica da ultrassonografia ocular e Doppler Triplex na avaliação de 10 cães Poodle com catarata

The findings of b-mode and especially triplex Doppler ocular ultrasound in the evaluation of 10 Poodle dogs with cataracts, which bring a contribution not yet reported in veterinary medicine, were reported. Ten Poodle dogs of varied ages and presenting cataracts were used. All animals were evaluated for ophthalmic and ultrasound examination. The ultrasound examination allowed the evaluation of the sonographic anatomy of the eye and measurement of the axial thickness of the lens (ATL). Using the Doppler mode, the blood flow of the ophthalmic artery and its vascular indexes, systolic velocity (SV), resistive index (RI) and pulsatility index (PI) were measured. Values found for ATL were 5.89±1.05 for the right eye (OD) and 6.07±1.32 for the left eye (OS). Values found using Doppler evaluation were SV OD: 26.54±7.05 and SV OS: 29.21±11.18; PI OD: 1.89±0.61 and PI OS: 1.7±0.35; RI OD: 0.76±0.1 and RI OS: 0.72±0.09 (OS). It was concluded that triplex Doppler was important for the determination of vascular indexes of the ophthalmic artery, which can be used for monitoring animals with hemodynamic alterations of the eyes and monitoring the therapy of ocular diseases.

Descreveram-se os achados da ultrassonografia ocular convencional e, principalmente, do modo Doppler Triplex na avaliação de 10 cães da raça Poodle com catarata, contribuindo com parâmetros ainda não relatados em medicina veterinária. Foram utilizados 10 cães de diferentes idades e da raça Poodle, apresentando graus variados de catarata. Os animais foram submetidos ao exame oftalmológico e ultrassonográfico. Por meio da ultrassonografia avaliaram-se a anatomia ultrassonográfica dos bulbos oculares e a espessura axial da lente (EAL). Por meio do modo Doppler verificaram-se o tipo de fluxo sanguíneo da artéria oftálmica e seus índices vasculares, a velocidade sistólica (VS), o índice de resistência (RI) e a pulsatividade (PI). Os valores de EAL para olho direito (OD) foi de 5,89±1,05 e para o olho esquerdo (OE) de 6,07±1,32. Por meio do Doppler, observaram-se VS para OD de 26,54±7,05 e VS para OE de 29,21±11,18; PI para OD de 1,89±0,61 e PI para OE de 1,7±0,35; RI para OD de 0,76±0,1 e PI para OE de 0,72±0,09. Concluiu-se que o modo Doppler Triplex mostrou-se importante para determinação das medidas dos índices vasculares da artéria oftálmica, podendo ser utilizada para o acompanhamento de alterações hemodinâmicas nos olhos dos animais acometidos e no acompanhamento da terapia de doenças oculares.

Estudo clínico, histopatológico e imunoistoquímico de neoplasias mamárias em cadelas / Clinical, histopathological and immunohistochemical study of mammary neoplasm in bitches

O objetivo foi realizar um estudo clínico e epidemiológico de neoplasias mamárias em cadelas, considerando-se histórico reprodutivo, exame físico, diagnóstico histopatológico e imunoistoquímico. Utilizaram-se 60 neoplasias mamárias, divididas em grupos (grupo 1 - benigno, e grupo 2 - maligno). Avaliaram-se dados do histórico reprodutivo, o exame físico e achados histopatológicos e imunoistoquímicos para fator de crescimento endotelial vascular. Ao estudo do histórico reprodutivo, encontraram-se 90% dos animais com irregularidade de cio, 86,63% das cadelas não foram medicadas com contraceptivos e 83,33% não eram castradas. Ao exame físico, não foi verificada diferença (p>0,05) entre grupos ao se avaliar consistência das massas, regularidade da superfície tumoral e localização anatômica dos tumores. Quanto ao tamanho das massas, verificou-se diferença entre os grupos (p=0,0077), com 0,78±1,13cm para o grupo 1 e 1,81±2,29cm para o grupo 2. Diagnosticaram-se 40% de massas benignas e 60% de malignas, de acordo com os tipos de neoplasias. Para VEGF, verificaram-se valores médios de 2,22±0,89 para tumores malignos e 1,66±0,91 para benignos, com diferença entre grupos (p=0,0315). As neoplasias mamárias em cadelas não apresentam características de histórico reprodutivo e de exame clínico que auxiliem o diagnóstico diferencial, sendo a histopatologia o único método para conclusão do diagnóstico e a imunoistoquímica podendo ser utilizada para prognóstico da lesão.

The objective was to conduct a clinical and epidemiological study of mammary cancer in bitches, considering their reproductive history, physical examination, histopathological and immunohistochemical diagnosis. We used 60 breast tumors which were divided into groups (group 1 - group 2 and benign - malignant). We evaluated data from the reproductive history, physical examination and histopathology and immunohistochemistry for VEGF. The study of reproductive history had 90% of irregular estrus, 86.63% of the dogs were not tested with contraceptives and 83.33% were not castrated. On physical examination, there was no difference (p>0.05) between groups regarding the consistency of the masses, surface regularity of the tumor and anatomic location of tumors. As for the masses, there was a difference between groups (p=0.0077), with 0.78±1.13cm for group 1 and 1.81±2.29 cm for group 2. 40% of benign masses and 60% of malignant masses were diagnosed, according to the types of malignancies. For VEGF, the average values were 2.22±0.89 for malignant tumors and 1.66±0.91 for benign, with differences between groups (p=0.0315). The mammary tumors do not exhibit characteristics of reproductive history and clinical examination to help the differential diagnosis, and histopathology is the only method for completion of diagnosis and immunohistochemistry which can be used for injury prognosis.

Avaliação de 67 casos de protrusão da glândula da terceira pálpebra em cães (2005-2010) / Evaluation of 67 cases of prolapse of the third eyelid gland in dogs (2005-2010)

Avaliaram-se 67 casos de protrusão da glândula da terceira pálpebra (cherry eye) em cães entre 2005 e 2010. Foram analisados a incidência da doença por gênero, a raça, a idade, o acometimento uni ou bilateral e a eficácia da técnica de Morgan pocket no reposicionamento da glândula. A protrusão da glândula da terceira pálpebra foi mais frequente em fêmeas (62,6%), e a raça mais acometida foi Lhasa Apso, seguida de cães sem raça definida (SRD). A idade dos animais variou entre dois meses e 13 anos (média de três anos), sendo mais frequente em animais com idade abaixo de 24 meses. A afecção manifestou-se no olho direito de 30 (44,8%) cães, no olho esquerdo de 18 (26,8%), e, em 19 animais (28,4%), a condição foi bilateral. Verificou-se que a técnica de Morgan pocket foi eficiente na resolução da afecção e requer mais cuidados no pós-operatório das raças grandes e gigantes.

In this study, 67 cases of dogs with prolapse of the third eyelid gland (cherry eye) between 2005 and 2010 were evaluated. The incidence of the disease by gender, age and breed was checked, and also if involvement was uni or bilateral and the effectiveness of the Morgan pocket technique of repositioning the gland. Prolapse of the third eyelid was more incident in females (62.6%), and Lhasa Apso and mongrel dogs were the most affected breeds. The age of the animals ranged between two months and 13 years (average of three years), and cases were more frequent in animals younger than 24 months. The disease manifested itself in the right eye of 30 (44.8%) dogs, in the left eye of 18 (26.8%) dogs, and in 19 animals (28.4%) the condition was bilateral. Morgan pocket technique was effective in the resolution of the complaint and demands more intensive care in post-surgery of large and giant breeds.

Deciphering chondrocyte behaviour in matrix-induced autologous chondrocyte implantation to undergo accurate cartilage repair with hyaline matrix.

Since the emergence in the 1990s of the autologous chondrocytes transplantation (ACT) in the treatment of cartilage defects, the technique, corresponding initially to implantation of chondrocytes, previously isolated and amplified in vitro, under a periosteal membrane, has greatly evolved. Indeed, the first generations of ACT showed their limits, with in particular the dedifferentiation of chondrocytes during the monolayer culture, inducing the synthesis of fibroblastic collagens, notably type I collagen to the detriment of type II collagen. Beyond the clinical aspect with its encouraging results, new biological substitutes must be tested to obtain a hyaline neocartilage. Therefore, the use of differentiated chondrocytes phenotypically stabilized is essential for the success of ACT at medium and long-term. That is why researchers try now to develop more reliable culture techniques, using among others, new types of biomaterials and molecules known for their chondrogenic activity, giving rise to the 4th generation of ACT. Other sources of cells, being able to follow chondrogenesis program, are also studied. The success of the cartilage regenerative medicine is based on the phenotypic status of the chondrocyte and on one of its essential component of the cartilage, type II collagen, the expression of which should be supported without induction of type I collagen. The knowledge accumulated by the scientific community and the experience of the clinicians will certainly allow to relief this technological challenge, which influence besides, the validation of such biological substitutes by the sanitary authorities.

Mechanisms involved in the desflurane-induced post-conditioning of isolated human right atria from patients with type 2 diabetes.

BACKGROUND: Desflurane triggers post-conditioning in the diabetic human myocardium. We determined whether protein kinase C (PKC), mitochondrial adenosine triphosphate-sensitive potassium (mitoK(ATP)) channels, Akt, and glycogen synthase kinase-3ß (GSK-3ß) were involved in the in vitro desflurane-induced post-conditioning of human myocardium from patients with type 2 diabetes. METHODS: The isometric force of contraction (FoC) of human right atrial trabeculae obtained from patients with type 2 diabetes was recorded during 30 min of hypoxia followed by 60 min of reoxygenation. Desflurane (6%) was administered during the first 5 min of reoxygenation either alone or in the presence of calphostin C (PKC inhibitor) or 5-hydroxydecanoate (5-HD) (mitoK(ATP) channel antagonist). Phorbol 12-myristate 13-acetate (PKC activator) and diazoxide (a mitoK(ATP) channel opener) were superfused during early reoxygenation. The FoC at the end of the 60 min reoxygenation period was compared among treatment groups (FoC(60); mean and sd). The phosphorylation of Akt and GSK-3ß was studied using western blotting. RESULTS: Desflurane enhanced the recovery of force [FoC(60): 79 (3)% of baseline] after 60 min of reoxygenation when compared with the control group (P>0.0001). Calphostin C and 5-HD abolished the beneficial effect of desflurane-induced post-conditioning (both P<0.0001). Phorbol 12-myristate 13-acetate and diazoxide enhanced the FoC(60) when compared with the control group (both P<0.0001). Desflurane increased the level of phosphorylation of Akt and GSK-3ß (P<0.0001). CONCLUSIONS: Desflurane-induced post-conditioning in human myocardium from patients with type 2 diabetes was mediated by the activation of PKC, the opening of the mitoK(ATP) channels, and the phosphorylation of Akt and GSK-3ß.

Chronic exposure of bone morphogenetic protein-2 favors chondrogenic expression in human articular chondrocytes amplified in monolayer cultures.

Articular cartilage is a specialized connective tissue containing chondrocytes embedded in a network of extracellular macromolecules such as type II collagen and presents poor capacity to self-repair. Autologous chondrocyte transplantation (ACT) is worldwide used for treatment of focal damage to articular cartilage. However, dedifferentiation of chondrocytes occurs during the long term culture necessary for mass cell production. The aim of this study was to investigate if addition of bone morphogenetic protein (BMP)-2, a strong inducer of chondrogenic expression, to human chondrocytes immediately after their isolation from cartilage, could help to maintain their chondrogenic phenotype in long-term culture conditions. Human articular chondrocytes were cultured according to the procedure used for ACT. Real-time PCR and Western blotting were performed to evaluate the cellular phenotype. Exogenous BMP-2 dramatically improves the chondrogenic character of knee articular chondrocytes amplified over two passages, as assessed by the BMP-2 stimulation on type II procollagen expression and synthesis. This study reveals that BMP-2 could potentially serve as a therapeutic agent for supporting the chondrogenic phenotype of human articular chondrocytes expanded in the conditions generally used for ACT.

Rhein, the metabolite of diacerhein, reduces the proliferation of osteoarthritic chondrocytes and synoviocytes without inducing apoptosis.

OBJECTIVE: The aim of this study was to determine the effects of pharmacologically relevant concentrations of rhein (1,8-dihydroxy-3-carboxyanthraquinone) on the cell proliferation rate of human chondrocytes and synoviocytes. METHODS: Cultures of human osteoarthritic synoviocytes and chondrocytes were incubated with 10(-6), 10(-5), and 10(-4) M rhein. [3H]thymidine incorporation was used to determine rhein proliferative effects after incubation periods of 24 h, 48 h, and 1 week. The cytotoxicity of the drug was assayed with a nonradioactive assay kit. Nuclear extracts were used to detect variations in cell-cycle proteins (p21, p27, and cyclin D1) by Western blotting. The effect of rhein on apoptosis was investigated by measurement of caspase-3/7 activity and DNA fragmentation. RESULTS: Rhein was found to downregulate the proliferation rate of both chondrocytes and synoviocytes, two-fold for 10(-5) M rhein and five- to six-fold for 10(-4) M rhein. No cytotoxicity of the drug was observed. Rhein (10(-4) M) decreased caspase-3/7 activity and did not induce DNA fragmentation. Western blots showed that 10(-4) M rhein increased the expression of p21 and/or p27, but not that of cyclin D1. CONCLUSIONS: Rhein has previously been shown to reduce the interleukin (IL)-1beta deleterious effects on osteoarthritis (OA) cartilage through inhibition of the expression of degrading enzymes. Here, rhein was also found to inhibit proliferation of both synoviocytes and chondrocytes, suggesting that the drug may decrease the development of the inflammatory synovial tissue that accompanies joint pathologies. Both its anti-catabolic and anti-proliferative effects may explain its beneficial effect in the treatment of joint diseases.

[Human chondrocyte responsiveness to bone morphogenetic protein-2 after their in vitro dedifferentiation: potential use of bone morphogenetic protein-2 for cartilage cell therapy]. / Réponse des chondrocytes humains à la bone morphogenetic protein-2 après leur dédifférenciation in vitro : utilisation potentielle de la bone morphogenetic protein-2 pour la thérapie cellulaire du cartilage.

AIM OF THE STUDY: Cartilage has a limited capacity for healing after trauma. Autologous chondrocyte implantation is widely used for the treatment of patients with focal damage to articular cartilage. Chondrocytes are isolated from biopsy specimen, cultured in monolayers on plastic then transplanted over the cartilage defect. However, chondrocyte amplification on plastic triggers their dedifferentiation. This phenomenon is characterized by loss of expression of type II collagen, the most abundant cartilage protein. The challenge for autologous chondrocyte implantation is to provide patients with well-differentiated cells. The aim of the present study was to test the capability of bone morphogenetic protein (BMP)-2 to promote redifferentiation of human chondrocytes after their expansion on plastic. MATERIALS AND METHODS: Chondrocytes extracted from nasal cartilage obtained after septoplasty were serially cultured in monolayers. After one, two or three passages, BMP-2 was added to the culture medium. The cellular phenotype was characterized at the gene level by using RT-PCR. The expression of genes coding for type II procollagen with the ratio of IIB/IIA forms, aggrecan, Sox9, osteocalcin and type I procollagen was monitored. RESULTS: Our results show that BMP-2 can stimulate chondrogenic expression of the chondrocytes amplified on plastic, without inducing osteogenic expression. However, this stimulatory effect decreases with the number of passages. CONCLUSION: The efficiency of autologous chondrocyte implantation could be improved by using chondrocytes treated with BMP-2 during their in vitro preparation.

Differentiation potential of human muscle-derived cells towards chondrogenic phenotype in alginate beads culture.

OBJECTIVE: The aim of this study was to evaluate the differentiation potential of two populations of muscle-derived cells (CD56- and CD56+) towards chondrogenic phenotype in alginate beads culture and to compare the effect of transforming growth factor beta 1 (TGFbeta1) on the differentiation process in these populations. METHODS: Muscle CD56- and CD56+ cells were cultured in alginate beads, in a chondrogenic medium, containing or not TGFbeta1 (10 ng/ml). Cultures were maintained for 3, 7, 14 or 21 days in a humidified culture incubator. At harvest, one culture of each set was fixed for alcian blue staining and aggrecan detection. The steady-state level of matrix macromolecules mRNA was assessed by real-time polymerase chain reaction (PCR). Protein detection was performed by western-blot analysis. The binding activity of nuclear extracts to Cbfa1 DNA sequence was also evaluated by electrophoretic mobility shift assays (EMSA). RESULTS: Chondrogenic differentiation of both CD56+ and CD56- muscle-derived cells was improved in alginate scaffold, even without growth factor, as suggested by increased chondrogenesis markers expression during the culture. Furthermore, TGFbeta1 enhanced the differentiation process and allowed to maintain a high expression of markers of mature chondrocytes. Of importance, the combination of alginate and TGFbeta1 treatment resulted in a further down-regulation of collagen type I and type X, as well as Cbfa1 both expression and binding activity. CONCLUSIONS: Thus, alginate scaffold and chondrogenic medium are sufficient to lead both populations CD56+ and CD56- towards chondrogenic differentiation. Moreover, TGFbeta1 enhances this process and allows to maintain the chondrogenic phenotype by inhibiting terminal differentiation, particularly for CD56- cells.

Hematological profile of healthy Pantaneiro horses / Valores do hemograma em cavalos sadios da raça Pantaneiro

Determinaram-se os valores do hemograma de 187 eqüinos sadios da raça Pantaneira. Para a composição de seis grupos experimentais consideraram-se a faixa etária e o sexo. Verificaram-se 6,2± 1,2 a 8,9± 2,3 hemácias/µl (x10(6)), 32,0± 4,0 a 37,0± 2,8 por cento de volume globular (VG), 11,5± 1,0 a 12,8± 1,6g de teor de hemoglobina/dl, 11,0± 3,2 a 17,0± 2,8 leucócitos/µl (x 10³), 4,9± 1,8 a 7,5± 1,5 neutrófilos segmentados/µl (x 10³) e 5,2± 2,0 a 8,6± 2,0 linfócitos/µl (x 10³). Observaram-se valores mais baixos na contagem de hemácias, e VG e valores mais altos do volume globular médio (VGM) em cavalos castrados (acima de 25 meses de idade). As contagens de leucócitos e neutrófilos segmentados em potros com até oito meses de idade apresentaram valores mais elevados que os encontrados em animais acima dos 25 meses de idade. Constatou-se que não houve influência do sexo nos parâmetros analisados.

17Beta-oestradiol up-regulates the expression of a functional UDP-glucose dehydrogenase in articular chondrocytes: comparison with effects of cytokines and growth factors.

OBJECTIVES: To investigate the mechanisms by which cytokines and 17beta-oestradiol (17beta-E2) modulate gene expression and activity of uridine diphosphoglucose dehydrogenase (UGDH), a key enzyme of GAG synthesis in articular chondrocytes. METHODS: Rabbit articular chondrocytes (RAC) from 3-week-old animals were incubated for 24 h with TGF-beta, insulin like growth factor-I (IGF-I), IL-1beta, IL-6 and 17beta-E2. GAG synthesis was measured by [35S]-sulphate labelling and the expression of the UGDH gene was estimated by both real-time polymerase chain reaction and western blotting, whereas the enzyme activity was assayed by a spectrophotometric procedure. In addition, the transcriptional activity of several UGDH gene promoter constructs was determined in RAC transiently transfected with wild-type or deleted human oestrogen receptor-alpha gene (hER alpha66 or hER alpha46, respectively). RESULTS: 17Beta-E2 and its receptor hER alpha66 enhanced GAG neosynthesis in rabbit articular chondrocytes, as did TGF-beta1 whereas IL-1beta decreased this synthesis. 17Beta-E2 was found to exert positive regulatory effects at mRNA, protein and UGDH activity levels. In addition, the receptor hER alpha66, but not hER alpha46, increased the transcriptional activity of the UGDH gene. In contrast, no clear correlation between transcription, translation and activity of the UGDH was found under the effects of the cytokines studied. However, TGF-beta enhanced the enzyme activity, whereas IL-1beta, IL-6 and IGF-I were without significant effect. CONCLUSIONS: 17Beta-E2 enhanced GAG synthesis in chondrocytes via up-regulation of the UGDH gene expression and enzyme activity. These data provide insights into the molecular mechanisms involved in the regulation of the UGDH gene and offer new approaches to investigate its potential alteration in joint diseases.

Interleukin-1beta up-regulation of Smad7 via NF-kappaB activation in human chondrocytes.

OBJECTIVE: We have previously shown that interleukin-1beta (IL-1beta) impairs transforming growth factor beta (TGFbeta) signaling through TGFbeta receptor type II (TGFbetaRII) down-regulation and Smad7 up-regulation. This mechanism could account for the reduced responsiveness of osteoarthritic chondrocytes to TGFbeta and the cartilage breakdown linked to this disease. The aim of this study was to investigate the molecular mechanism underlying the IL-1beta-induced stimulation of Smad7 in human articular chondrocytes. METHODS: Human articular chondrocytes were treated with IL-1beta in the presence of TGFbeta1, pyrrolidine dithiocarbamate (a repressor of the NF-kappaB pathway), or cycloheximide. Then, steady-state messenger RNA and protein levels were estimated by real-time reverse transcription-polymerase chain reaction and immunocytology. In addition, transient transfections of p65 expression vector or p65-targeted short hairpin RNA were performed to define the effect of NF-kappaB on Smad7 expression. RESULTS: TGFbetaRII overexpression restored the TGFbeta response of human articular chondrocytes. However, this effect was transient, implying that a secondary mechanism was responsible for the alteration of the TGFbeta response with long-term exposure to IL-1beta. Moreover, IL-1beta caused a late induction of the inhibitory Smad7. This effect was direct, since it did not require de novo synthesis. In addition, we established, by experiments with gain/loss of function, that the up-regulation of Smad7 by IL-1beta is mediated through the NF-kappaB pathway, especially the p65 subunit. CONCLUSION: These findings clarify the regulatory process of IL-1beta on Smad7 expression. Understanding the molecular basis of IL-1beta induction of Smad7 and the reduction of chondrocyte responsiveness to TGFbeta provides new insights into the molecular mechanisms of osteoarthritis and may facilitate the identification of novel approaches for its treatment.

NFkappaB mediates IL-1beta-induced down-regulation of TbetaRII through the modulation of Sp3 expression.

We previously showed that interleukin-1beta (IL-1beta) down-regulation of type II TGFbeta receptor (TbetaRII) involves NFkappaB pathway and requires de novo synthesis of a yet unknown protein. Here, we demonstrate that this effect is mediated through Sp1 site located at position -25 of human TbetaRII promoter. Inhibition of transcription factors binding (decoy oligonucleotides or mithramycin) abolished IL-1beta effect. EMSA and ChIP revealed that this treatment induced Sp3 binding to cis-sequence whereby IL-1beta exerts its transcriptional effects whereas it decreased that of Sp1. Moreover, although the cytokine did not modulate Sp1 expression, it increased that of Sp3 via NFkappaB pathway. Experiments of gain and loss of function clearly showed that Sp3 inhibited TbetaRII expression whereas its silencing abolished IL-1beta effect. In addition, both Sp1 and Sp3 were found to interact with NFkappaB, which therefore may indirectly interact with TbetaRII pro moter. Altogether, these data suggest that IL-1beta decreases TbetaRII expression by inducing Sp3 via NFkappaB and its binding on core promote at the expense of Sp1, which could explain the loss of cell responsiveness in certain conditions. These findings bring new insights in the knowledge of the interference between two antagonistic transduction pathways involved in multiple physiopathological processes.

Interleukin-1beta impairment of transforming growth factor beta1 signaling by down-regulation of transforming growth factor beta receptor type II and up-regulation of Smad7 in human articular chondrocytes.

OBJECTIVE: Extracellular matrix deposition is tightly controlled by a network of regulatory cytokines. Among them, interleukin-1beta (IL-1beta) and transforming growth factor beta1 (TGFbeta1) have been shown to play antagonistic roles in tissue homeostasis. The purpose of this study was to determine the influence of IL-1beta on TGFbeta receptor type II (TGFbetaRII) regulation and TGFbeta1 responsiveness in human articular chondrocytes. METHODS: TGFbeta1-induced gene expression was analyzed through plasminogen activator inhibitor 1 and p3TP-Lux induction. Receptor-activated Smad (R-Smad) phosphorylation, TGFbeta receptors, and Smad expression were determined by Western blotting and real-time reverse transcription-polymerase chain reaction techniques. Signaling pathways were investigated using specific inhibitors, messenger RNA (mRNA) silencing, and expression vectors. RESULTS: IL-1beta down-regulated TGFbetaRII expression at both the protein and mRNA levels and led to inhibition of the TGFbeta1-induced gene expression and Smad2/3 phosphorylation. Moreover, IL-1beta strongly stimulated the expression of inhibitory Smad7. TGFbetaRII overexpression abolished the loss of TGFbeta1 responsiveness induced by IL-1beta. The decrease in TGFbetaRII required de novo protein synthesis and involved both the NF-kappaB and JNK pathways. CONCLUSION: We demonstrate that IL-1beta impairs TGFbeta1 signaling through down-regulation of TGFbetaRII, which is mediated by the p65/NF-kappaB and activator protein 1/JNK pathways, and secondarily through the up-regulation of Smad7. These findings show that there is cross-talk in the signaling of 2 regulatory cytokines involved in inflammation.

A silanized hydroxypropyl methylcellulose hydrogel for the three-dimensional culture of chondrocytes.

Articular cartilage has limited intrinsic repair capacity. In order to promote cartilage repair, the amplification and transfer of autologous chondrocytes using three-dimensional scaffolds have been proposed. We have developed an injectable and self-setting hydrogel consisting of hydroxypropyl methylcellulose grafted with silanol groups (Si-HPMC). The aim of the present work is to assess both the in vitro cytocompatibility of this hydrogel and its ability to maintain a chondrocyte-specific phenotype. Primary chondrocytes isolated from rabbit articular cartilage (RAC) and two human chondrocytic cell lines (SW1353 and C28/I2) were cultured into the hydrogel. Methyl tetrazolium salt (MTS) assay and cell counting indicated that Si-HPMC hydrogel did not affect respectively chondrocyte viability and proliferation. Fluorescent microscopic observations of RAC and C28/I2 chondrocytes double-labeled with cell tracker green and ethidium homodimer-1 revealed that chondrocytes proliferated within Si-HPMC. Phenotypic analysis (RT-PCR and Alcian blue staining) indicates that chondrocytes, when three-dimensionnally cultured within Si-HPMC, expressed transcripts encoding type II collagen and aggrecan and produced sulfated glycosaminoglycans. These results show that Si-HPMC allows the growth of differentiated chondrocytes. Si-HPMC therefore appears as a potential scaffold for three-dimensional amplification and transfer of chondrocytes in cartilage tissue engineering.