RESUMO
Tic disorders (TD) are developmental neuropsychiatric conditions often accompanied by comorbid conditions, and psychosocial hardships for child and family. The etiology of tics is unknown, and is complex and multifactorial. Stress is known to aggravate tic expression as well as associated comorbidities. Consequently, this study focused on possible connections between stress, emotion regulation, tic expression, and related psychopathology. Sixty consecutive admissions were assessed for perceived stress, emotional dysregulation, severity of obsessions and compulsions, anxiety, depression, attention deficit disorder, and tic expression at a TD clinic, in a university affiliated pediatric hospital. The results indicated that stress and emotion dysregulation were significantly related to both tic expression and severity of comorbidities. We discuss the role of emotion regulation dimensions regarding TD and related psychopathology as well as the mediating role of emotion regulation, and how they may contribute to the development of improved therapies for children with TD.
Assuntos
Transtorno do Deficit de Atenção com Hiperatividade , Regulação Emocional , Transtornos de Tique , Tiques , Síndrome de Tourette , Criança , Humanos , Tiques/complicações , Síndrome de Tourette/psicologia , Transtornos de Tique/psicologia , Transtorno do Deficit de Atenção com Hiperatividade/psicologia , Comorbidade , Índice de Gravidade de DoençaRESUMO
AIM: To evaluate the effects of various mixing solutions on the biocompatibility of mineral trioxide aggregate (MTA). METHODOLOGY: Human alveolar osteoblasts (hOAs) were incubated with eluates of 24 h-set cement discs of MTA mixed with sterile H2 O, 3% sodium hypochlorite (NaOCl), 4% articaine (Ultracain(®) D-S), 0.9% NaCl, Ringer's solution or citrated blood, respectively. The cell proliferation in the presence of eluates was assessed by real-time cell analysis, and the expression of genes associated with proliferation (histone H3, HistH3), inflammation (interleukin-6, IL-6, matrix metalloproteinases 1 and 3, MMP1, MMP3) or apoptosis (caspase 3, Casp3) was analysed by qPCR after 24 and 72 h. The ultrastructure of cells grown on cement discs was visualized by scanning electron microscopy (SEM), whilst actin cytoskeleton was monitored by fluorescence staining in the presence of eluates after 7 and 14 days. A repeated-measure analysis was performed, and P-values were adjusted by Tukey. RESULTS: Whilst articaine-MTA sustained hOA proliferation patterns similar to H2 O-MTA, NaOCl-MTA reduced hOA proliferation and significantly increased the expression of MMP1 and MMP3. The addition of H2 O and articaine modulated the gene expression of Casp3 or Hist3H3. The use of NaCl, Ringer and blood induced mRNA levels comparable to matched controls. With the exception of NaOCl-MTA, SEM and FM revealed regular hOA morphology for all mixing solutions. CONCLUSIONS: NaOCl was highly cytotoxic for hOAs whilst all other mixing solutions can be considered as convenient biocompatible mixing solutions as alternatives to H2 O for clinical use.
Assuntos
Compostos de Alumínio/uso terapêutico , Materiais Biocompatíveis/uso terapêutico , Compostos de Cálcio/uso terapêutico , Materiais Dentários/uso terapêutico , Óxidos/uso terapêutico , Silicatos/uso terapêutico , Apoptose/efeitos dos fármacos , Carticaína , Proliferação de Células/efeitos dos fármacos , Combinação de Medicamentos , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Soluções Isotônicas , Teste de Materiais , Microscopia Eletrônica de Varredura , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Reação em Cadeia da Polimerase em Tempo Real , Solução de Ringer , Hipoclorito de Sódio , Soluções , TranscriptomaRESUMO
Ultraviolet (UV) light treatment of implant surfaces has been demonstrated to enhance their bioactivity significantly. This study examined the effect of UV treatment of different zirconia surfaces on the response of primary human alveolar bone-derived osteoblasts (PhABO). Disks of two zirconia-based materials with two different surface topographies (smooth, roughened) were exposed to UV light. Qualitative and quantitative assessment of PhABO on zirconia surfaces, by means of immunofluorescence, scanning electron microscopy and DNA quantification at 4 and 24 h revealed a higher number of initially attached osteoblasts on UV-treated surfaces. Cell area and perimeter were significantly larger on all UV-treated surfaces (p<0.05). The proliferation activity was significantly higher on both roughened UV-treated surfaces than on untreated samples at day 3 of culture (p<0.05). The expression levels of collagen I, osteopontin and osteocalcin at day 14 and alkaline phosphatase activity at day 7 and 14 of culture period were similar among UV-treated and untreated surfaces. Alizarin-Red-Staining at day 21 demonstrated significantly more mineralised nodules on UV-treated samples than on untreated samples. Contact angle measurements and X-ray photoelectron spectroscopy showed that UV light transformed zirconia surfaces from hydrophobic to (super-) hydrophilic (p<0.05) and significantly reduced the atomic percentage of surface carbon. The results showed that UV light pre-treatment of zirconia surfaces changes their physicochemical properties and improves their attractiveness against PhABO, primarily demonstrated by an augmented cell attachment and spreading. This may result in faster healing and better bone-to-implant contact of zirconia implants in vivo following such a pre-treatment.
Assuntos
Materiais Biocompatíveis/química , Implantes Dentários , Osteoblastos/citologia , Raios Ultravioleta , Zircônio/química , Fosfatase Alcalina/metabolismo , Processo Alveolar/citologia , Materiais Biocompatíveis/farmacologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fenômenos Químicos/efeitos da radiação , Colágeno Tipo I/genética , Cadeia alfa 1 do Colágeno Tipo I , Expressão Gênica/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas/efeitos da radiação , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Osteocalcina/genética , Osteopontina/genética , Espectroscopia Fotoeletrônica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Propriedades de Superfície/efeitos da radiação , Fatores de TempoRESUMO
BACKGROUND: The goal of this study was to explore the notion that anomalies of self-experience (ASE) are a core, 'not-yet-psychotic' clinical phenotype of emerging schizophrenia and its spectrum. Method To accomplish this goal, we examined the relationship between ASE and commonly accepted risk markers in a sample of 87 help-seeking, non-psychotic adolescents (aged 14-18 years). ASE were assessed with the Examination of Anomalous Self-Experience (EASE), subclinical psychotic symptoms were assessed with the Prodromal Questionnaire and the Structured Interview for Prodromal Syndromes, deterioration in psychosocial functioning was assessed with the Social and Role Functioning Scales, and level of distress with the Mood and Anxiety Symptoms Questionnaire. RESULTS: About 82 participants completed the entire EASE interview. The number of participants who reported ASE at a clinically meaningful level (n = 18, 22%) was smaller than that who met diagnostic criteria for a prodromal syndrome (n = 28, 34%). The degree of overlap between the two conditions was moderate but statistically significant (χ2 (1) = 7.01, p = 0.008). An exploratory factor analysis revealed that ASE load on a different factor than prodromal symptoms and deterioration in functioning, but that there is a moderate correlation between the three factors. CONCLUSIONS: These results suggest that ASE are prevalent among non-psychotic help-seeking adolescents, yet at a considerably lower rate than prodromal symptoms. In addition, they suggest that ASE and prodromal symptoms constitute distinct but moderately related dimensions of potential risk. Taken together, they provide preliminary support for the clinical usefulness of supplementing and refining the methods of early detection of risk with assessment of ASE.
Assuntos
Sintomas Prodrômicos , Transtornos Psicóticos/psicologia , Esquizofrenia/diagnóstico , Psicologia do Esquizofrênico , Autoimagem , Adolescente , Diagnóstico Precoce , Análise Fatorial , Feminino , Humanos , Masculino , Aceitação pelo Paciente de Cuidados de Saúde , Transtornos Psicóticos/diagnóstico , Fatores de Risco , Inquéritos e QuestionáriosRESUMO
Novel findings broaden the concept of mechanotransduction (MT) in biophysically stimulated tissues such as the periodontium by considering nuclear MT, convergence of intracellular MT pathways, and mechanoresponsive cotranscription factors such as Yes-associated protein 1 (YAP1). Regarding periodontal disease, recent studies have elucidated the role of bacterial gingipain proteases in disturbing the barrier function of cadherins, thereby promoting periodontal inflammation. This leads to dysregulation of extracellular matrix homeostasis via proteases and changes the cell's biophysical environment, which leads to alterations in MT-induced cell behavior and loss of periodontal integrity. Newest experimental evidence from periodontal ligament cells suggests that the Hippo signaling protein YAP1, in addition to integrin-FAK (focal adhesion kinase) mechanosignaling, also regulates cell stemness. By addressing mechanosignaling-dependent transcription factors, YAP1 is involved in osteogenic and myofibroblast differentiation and influences core steps of autophagy. Recent in vivo evidence elucidates the decisive role of YAP1 in epithelial homeostasis and underlines its impact on oral pathologies, such as periodontitis-linked oral squamous cell carcinogenesis. Here, new insights reveal that YAP1 contributes to carcinogenesis via overexpression rather than mutation; promotes processes such as apoptosis resistance, epithelial-mesenchymal transition, or metastasis; and correlates with poor prognosis in oral squamous cell carcinoma. Furthermore, YAP1 has been shown to contribute to periodontitis-induced bone loss. Mechanistically, molecules identified to regulate YAP1-related periodontal homeostasis and disease include cellular key players such as MAPK (mitogen-activated protein kinase), JNK (c-Jun N-terminal kinase), Rho (Ras homologue) and ROCK (Rho kinase), Bcl-2 (B-cell lymphoma 2), AP-1 (activator protein 1), and c-myc (cellular myelocytomatosis). These findings qualify YAP1 as a master regulator of mechanobiology and cell behavior in human periodontal tissues. This review summarizes the most recent developments in MT-related periodontal research, thereby offering insights into outstanding research questions and potential applications of molecular or biophysical strategies aiming at periodontal disease mitigation or prevention.
Assuntos
Carcinoma de Células Escamosas , Neoplasias Bucais , Homeostase , Humanos , Mecanotransdução Celular , Fator de Transcrição AP-1RESUMO
We previously demonstrated that altered peptide ligands (APL) can partially activate T cells, resulting in multiple distinct functional phenotypes, including the induction of anergy. Such APL stimulate a unique pattern of T cell receptor (TCR) phospho-zeta species, and lack associated ZAP-70 kinase activity. While these data suggested that selective signaling pathways downstream of the TCR/CD3 molecules are activated upon APL stimulation, they did not directly demonstrate this. Thus, we pursued intracellular signaling events successfully stimulated by APL. Because our previous studies showed that cyclosporin A (CsA) completely inhibited anergy induction, we assessed whether TCR ligation by APL cause a rise in cytosolic calcium (Ca+2). Our results show that these ligands can induce Ca+2 transients, in contrast to data generated using analogue peptides in other antigen systems. These opposing results may reflect differences in the intracellular signaling pathways utilized by different APL, or may be due to the exquisite sensitivity of the assay used here. Importantly, the APL-stimulated Ca+2 induction is both initiated and sustained at lower levels than that stimulated by a strong agonist signal, but resembles that stimulated by a weaker agonist stimulus. Alone, the less than optimal Ca+2 induction does not cause anergy, because ionomycin treatment together with the APL does not result in a proliferative signal. Instead, we propose that a combination of this and other signaling pathways induces T cell anergy. Overall, these data support the concept of differential signaling in T cells, as a direct consequence of the phosphotyrosine status of the TCR/CD3 molecules.
Assuntos
Cálcio/metabolismo , Hemoglobinas/farmacologia , Ativação Linfocitária , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/farmacologia , Linfócitos T/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Feminino , Hemoglobinas/química , Camundongos , Camundongos Endogâmicos CBA , Oligopeptídeos/química , Fragmentos de Peptídeos/química , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
OBJECTIVES: The purpose of this study was to describe the type of toxin ingested, clinical presentation and outcome of dogs with status epilepticus (SE) due to acute poisoning presented to a large referral veterinary hospital. MATERIALS AND METHODS: Retrospective case series. Medical records of all dogs suffering from SE were reviewed (Jan 1, 2002 to April 30, 2009). RESULTS: Fourteen dogs with SE due to acute intoxication were identified. Toxicological analyses (qualitative analysis with gas chromatography/mass spectrometry; n=11) detected poisonings with carbofuran, crimidine, paraoxone, metaldehyde, strychnine and diazinon. In the other three cases the uptake of a known poison was observed (zink phosphide, metaldehyde). None of the dogs showed evidence of neurological disease up to the day of presentation. The dogs were hospitalised for 2-10 days (median 5 days). The survival rate was 85.7%. None of the dogs experienced any more seizures after discharge (median observation period 2.6 years). CONCLUSION AND CLINICAL RELEVANCE: Ancillary to the acute clinical presentation, preliminary reports (possible uptake of poisonous material) and an inconspicuous medical history may suggest a tentative diagnosis. Managed adequately, these patients can have a high survival rate. Clinicians should also keep uncommon intoxications in mind.
RESUMO
Airway epithelia are positioned at the interface between the body and the environment, and generate complex signaling responses to inhaled toxins and other stresses. Luminal mechanical stimulation of airway epithelial cells produces a propagating wave of elevated intracellular Ca(2+) that coordinates components of the integrated epithelial stress response. In polarized airway epithelia, this response has been attributed to IP(3) permeation through gap junctions. Using a combination of approaches, including enzymes that destroy extracellular nucleotides, purinergic receptor desensitization, and airway cells deficient in purinoceptors, we demonstrated that Ca(2+) waves induced by luminal mechanical stimulation in polarized airway epithelia were initiated by the release of the 5' nucleotides, ATP and UTP, across both apical and basolateral membranes. The nucleotides released into the extracellular compartment interacted with purinoceptors at both membranes to trigger Ca(2+) mobilization. Physiologically, apical membrane nucleotide-release coordinates airway mucociliary clearance responses (mucin and salt, water secretion, increased ciliary beat frequency), whereas basolateral release constitutes a paracrine mechanism by which mechanical stresses signal adjacent cells not only within the epithelium, but other cell types (nerves, inflammatory cells) in the submucosa. Nucleotide-release ipsilateral and contralateral to the surface stimulated constitutes a unique mechanism by which epithelia coordinate local and distant airway defense responses to mechanical stimuli.
Assuntos
Trifosfato de Adenosina/metabolismo , Comunicação Celular/fisiologia , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Uridina Trifosfato/metabolismo , Animais , Apirase/farmacologia , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Brônquios/citologia , Cálcio/metabolismo , Polaridade Celular/fisiologia , Células Cultivadas , Humanos , Processamento de Imagem Assistida por Computador , Camundongos , Camundongos Knockout , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2Y2 , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismo , Estresse MecânicoRESUMO
After the membrane impermeant dye Lucifer Yellow is introduced into the cytoplasmic matrix of J774 cells, the dye is sequestered within cytoplasmic vacuoles and secreted into the extracellular medium. In the present work we studied the intracellular transport of Lucifer Yellow in J774 macrophages and the nature of the cytoplasmic vacuoles into which this dye is sequestered. When the lysosomal system of J774 cells was prelabeled with a Texas red ovalbumin conjugate and Lucifer Yellow was then loaded into the cytoplasm of the cells by ATP-mediated permeabilization of the plasma membrane, the vacuoles that sequestered Lucifer Yellow 30 min later were distinct from the Texas red-stained lysosomes. After an additional 30 min Lucifer Yellow and Texas red colocalized in the same membrane bound compartments, indicating that the Lucifer Yellow had been delivered to lysosomes. We next prelabeled the plasma membrane of J774 cells with anti-macrophage antibody and Texas red protein A before Lucifer Yellow was loaded into the cells. The phase-lucent vacuoles that subsequently sequestered Lucifer Yellow also stained with Texas red, showing that they were part of the endocytic pathway. J774 cells were fractionated on percoll density gradients either 15 or 60 min after Lucifer Yellow was introduced into the cytoplasmic matrix of the cells. In cells fractionated after 15 min, Lucifer Yellow was contained within the fractions of light buoyant density that contain plasma membrane and endosomes; the dye later appeared in vesicles of higher density which contained lysosomes. Secretion of Lucifer Yellow from the cytoplasmic matrix of J774 cells is inhibited by the organic anion transport blocker probenecid. We found that probenecid also reversibly inhibited sequestration of dye, indicating that sequestration of dye within cytoplasmic vacuoles was also mediated by organic anion transporters. These studies show that the vacuoles that sequester Lucifer Yellow from the cytoplasmic matrix of J774 cells possess the attributes of endosomes. Thus, in addition to their role in sorting of membrane bound and soluble substances, macrophage endosomes may play a role in the accumulation and transport of molecules resident in the soluble cytoplasm.
Assuntos
Citoplasma/metabolismo , Corantes Fluorescentes/metabolismo , Isoquinolinas/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Transporte Biológico Ativo , Fracionamento Celular , Linhagem Celular , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Centrifugação com Gradiente de Concentração , Macrófagos/ultraestrutura , Microscopia Eletrônica , Microscopia de Fluorescência , Pinocitose , Probenecid/farmacologia , Temperatura , Vacúolos/metabolismoRESUMO
Connexins are gap junction proteins that form aqueous channels to interconnect adjacent cells. Rat osteoblasts express connexin43 (Cx43), which forms functional gap junctions at the cell surface. We have found that ROS 17/2.8 osteosarcoma cells, UMR 106-01 osteosarcoma cells, and primary rat calvarial osteoblastic cells also express another gap junction protein, Cx46. Cx46 is a major component of plasma membrane gap junctions in lens. In contrast, Cx46 expressed by osteoblastic cells was predominantly localized to an intracellular perinuclear compartment, which appeared to be an aspect of the TGN as determined by immunofluorescence colocalization. Hela cells transfected with rat Cx46 cDNA (Hela/Cx46) assembled Cx46 into functional gap junction channels at the cell surface. Both rat lens and Hela/Cx46 cells expressed 53-kD (nonphosphorylated) and 68-kD (phosphorylated) forms of Cx46; however, only the 53-kD form was produced by osteoblasts. To examine connexin assembly, monomers were resolved from oligomers by sucrose gradient velocity sedimentation analysis of 1% Triton X-100-solubilized extracts. While Cx43 was assembled into multimeric complexes, ROS cells contained only the monomer form of Cx46. In contrast, Cx46 expressed by rat lens and Hela/Cx46 cells was assembled into multimers. These studies suggest that assembly and cell surface expression of two closely related connexins were differentially regulated in the same cell. Furthermore, oligomerization may be required for connexin transport from the TGN to the cell surface.
Assuntos
Conexinas/química , Complexo de Golgi/ultraestrutura , Cristalino/ultraestrutura , Osteoblastos/ultraestrutura , Animais , Western Blotting , Compartimento Celular/efeitos dos fármacos , Células Cultivadas , Conexinas/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Cristalino/metabolismo , Peso Molecular , Monensin/farmacologia , Osteoblastos/metabolismo , Ratos , TransfecçãoRESUMO
We introduced several membrane-impermeant fluorescent dyes, including Lucifer Yellow, carboxyfluorescein, and fura-2, into the cytoplasmic matrix of J774 cells and thioglycollate-elicited mouse peritoneal macrophages by ATP permeabilization of the plasma membrane and observed the subsequent fate of these dyes. The dyes did not remain within the cytoplasmic matrix; instead they were sequestered within phase-lucent cytoplasmic vacuoles and released into the extracellular medium. We used Lucifer Yellow to study these processes further. In cells incubated at 37 degrees C, 87% of Lucifer Yellow was released from the cells within 30 min after dye loading. The dye that remained within the cells at this time was predominantly within cytoplasmic vacuoles. Lucifer yellow transport was temperature dependent and occurred against a concentration gradient; therefore it appeared to be an energy-requiring process. The fluorescent dyes used in these studies are all organic anions. We therefore examined the ability of probenecid (p-[dipropylsulfamoyl]benzoic acid), which blocks organic anion transport across many epithelia, to inhibit efflux of Lucifer Yellow, and found that this drug inhibited this process in a dose-dependent and reversible manner. Efflux of Lucifer Yellow from the cells did not require Na+ co-transport or Cl- antiport; however, it was inhibited by lowering of the extracellular pH. These experiments indicate that macrophages possess probenecid-inhibitable transporters which are similar in their functional properties to organic anion transporters of epithelial cells. Such organic anion transporters have not been described previously in macrophages; they may mediate the release of naturally occurring organic anions such as prostaglandins, leukotrienes, glutathione, bilirubin, or lactate from macrophages.
Assuntos
Proteínas de Transporte/metabolismo , Macrófagos/metabolismo , Probenecid/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Proteínas de Transporte de Ânions , Linhagem Celular , Permeabilidade da Membrana Celular , Citoplasma/metabolismo , Corantes Fluorescentes , Isoquinolinas , Cinética , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Camundongos , Microscopia de FluorescênciaRESUMO
Connexin(Cx)43 is the major gap junction protein present in osteoblasts. We have shown that overexpression of Cx45 in osteoblasts expressing endogenous Cx43 leads to decreased cell-cell communication (Koval, M., S.T. Geist, E.M. Westphale, A.E. Kemendy, R. Civitelli, E.C. Beyer, and T.H. Steinberg. 1995. J. Cell Biol. 130:987-995) and transcriptional downregulation of several osteoblastic differentiation markers (Lecanda, F., D.A. Towler, K. Ziambaras, S.-L. Cheng, M. Koval, T.H. Steinberg, and R. Civitelli. 1998. Mol. Biol. Cell 9:2249-2258). Here, using the Cx43-null mouse model, we determined whether genetic deficiency of Cx43 affects skeletal development in vivo. Both intramembranous and endochondral ossification of the cranial vault were delayed in the mutant embryos, and cranial bones originating from migratory neural crest cells were also hypoplastic, leaving an open foramen at birth. Cx43-deficient animals also exhibited retarded ossification of the clavicles, ribs, vertebrae, and limbs, demonstrating that skeletal abnormalities are not restricted to a neural crest defect. However, the axial and appendicular skeleton of Cx43-null animals were essentially normal at birth. Cell to cell diffusion of calcein was poor among Cx43-deficient osteoblasts, whose differentiated phenotypic profile and mineralization potential were greatly impaired, compared with wild-type cells. Therefore, in addition to the reported neural crest cell defect, lack of Cx43 also causes a generalized osteoblast dysfunction, leading to delayed mineralization and skull abnormalities. Cell to cell signaling, mediated by Cx43 gap junctions, was critical for normal osteogenesis, craniofacial development, and osteoblastic function.
Assuntos
Conexina 43/deficiência , Conexina 43/genética , Anormalidades Craniofaciais/genética , Osteoblastos/citologia , Osteoblastos/fisiologia , Osteogênese/genética , Animais , Osso e Ossos/citologia , Osso e Ossos/embriologia , Osso e Ossos/patologia , Divisão Celular , Desenvolvimento Embrionário e Fetal/genética , Genótipo , Idade Gestacional , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Crista Neural/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Crânio/embriologiaRESUMO
Many cells coordinate their activities by transmitting rises in intracellular calcium from cell to cell. In nonexcitable cells, there are currently two models for intercellular calcium wave propagation, both of which involve release of inositol trisphosphate (IP3)- sensitive intracellular calcium stores. In one model, IP3 traverses gap junctions and initiates the release of intracellular calcium stores in neighboring cells. Alternatively, calcium waves may be mediated not by gap junctional communication, but rather by autocrine activity of secreted ATP on P2 purinergic receptors. We studied mechanically induced calcium waves in two rat osteosarcoma cell lines that differ in the gap junction proteins they express, in their ability to pass microinjected dye from cell to cell, and in their expression of P2Y2 (P2U) purinergic receptors. ROS 17/2.8 cells, which express the gap junction protein connexin43 (Cx43), are well dye coupled, and lack P2U receptors, transmitted slow gap junction-dependent calcium waves that did not require release of intracellular calcium stores. UMR 106-01 cells predominantly express the gap junction protein connexin 45 (Cx45), are poorly dye coupled, and express P2U receptors; they propagated fast calcium waves that required release of intracellular calcium stores and activation of P2U purinergic receptors, but not gap junctional communication. ROS/P2U transfectants and UMR/Cx43 transfectants expressed both types of calcium waves. Gap junction-independent, ATP-dependent intercellular calcium waves were also seen in hamster tracheal epithelia cells. These studies demonstrate that activation of P2U purinergic receptors can propagate intercellular calcium, and describe a novel Cx43-dependent mechanism for calcium wave propagation that does not require release of intracellular calcium stores by IP3. These studies suggest that gap junction communication mediated by either Cx43 or Cx45 does not allow passage of IP3 well enough to elicit release of intracellular calcium stores in neighboring cells.
Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Conexina 43/fisiologia , Conexinas/fisiologia , Junções Comunicantes/fisiologia , Osteoblastos/fisiologia , Receptores Purinérgicos P2/fisiologia , Transdução de Sinais/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Linhagem Celular , Conexina 43/biossíntese , Conexinas/biossíntese , Cricetinae , Junções Comunicantes/efeitos dos fármacos , Heptanol/farmacologia , Humanos , Cinética , Osteoblastos/efeitos dos fármacos , Osteossarcoma , RNA Mensageiro/biossíntese , Ratos , Receptores Purinérgicos P2/biossíntese , Proteínas Recombinantes/biossíntese , Transdução de Sinais/efeitos dos fármacos , Suramina/farmacologia , Tapsigargina/farmacologia , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
Many cells express multiple connexins, the gap junction proteins that interconnect the cytosol of adjacent cells. Connexin43 (Cx43) channels allow intercellular transfer of Lucifer Yellow (LY, MW = 443 D), while connexin45 (Cx45) channels do not. We transfected full-length or truncated chicken Cx45 into a rat osteosarcoma cell line ROS-17/2.8, which expresses endogenous Cx43. Both forms of Cx45 were expressed at high levels and colocalized with Cx43 at plasma membrane junctions. Cells transfected with full-length Cx45 (ROS/Cx45) and cells transfected with Cx45 missing the 37 carboxyl-terminal amino acids (ROS/Cx45tr) showed 30-60% of the gap junctional conductance exhibited by ROS cells. Intercellular transfer of three negatively charged fluorescent reporter molecules was examined. In ROS cells, microinjected LY was transferred to an average of 11.2 cells/injected cell, while dye transfer between ROS/Cx45 cells was reduced to 3.9 transfer between ROS/Cx45 cells was reduced to 3.9 cells. In contrast, ROS/Cx45tr cells transferred LY to > 20 cells. Transfer of calcein (MW = 623 D) was also reduced by approximately 50% in ROS/Cx45 cells, but passage of hydroxycoumarin carboxylic acid (HCCA; MW = 206 D) was only reduced by 35% as compared to ROS cells. Thus, introduction of Cx45 altered intercellular coupling between cells expressing Cx43, most likely the result of direct interaction between Cx43 and Cx45. Transfection of Cx45tr and Cx45 had different effects in ROS cells, consistent with a role of the carboxyl-terminal domain of Cx45 in determining gap junction permeability or interactions between connexins. These data suggest that coexpression of multiple connexins may enable cells to achieve forms of intercellular communication that cannot be attained by expression of a single connexin.
Assuntos
Comunicação Celular/fisiologia , Permeabilidade da Membrana Celular , Conexina 43/biossíntese , Conexinas/biossíntese , Junções Comunicantes/fisiologia , Animais , Sequência de Bases , Galinhas , Cromonas/metabolismo , Conexinas/genética , Condutividade Elétrica , Eletrofisiologia , Citometria de Fluxo , Fluoresceínas/metabolismo , Imunofluorescência , Immunoblotting , Isoquinolinas/metabolismo , Microinjeções , Dados de Sequência Molecular , Osteoblastos/fisiologia , Ratos , Proteínas Recombinantes/biossíntese , Transfecção , Células Tumorais CultivadasRESUMO
BACKGROUND AND OBJECTIVE: Collagen type I elevation in cyclosporin A-induced gingival overgrowth supports evidence that gingival fibroblasts play a decisive role in the manifestation of the phenotype. To analyze the role of gingival fibroblasts under more in vivo-like conditions, we evaluated the effect of cyclosporin A on collagen type I gene and protein expression in gingival overgrowth-derived gingival fibroblasts established as cocultures with gingival keratinocytes as well as in matched gingival fibroblast monolayers. MATERIAL AND METHODS: Monolayers and cocultures of primary gingival fibroblasts were treated with cyclosporin A for 6 and 72 h. The expression of collagen type I mRNA was analyzed by quantitative real time polymerase chain reaction, while expression and secretion of collagen type I protein was analyzed by indirect immunofluorescence and western blotting. RESULTS: Compared with controls, significant elevation of collagen type I mRNA was restricted to cocultures after 6 and 72 h of treatment with cyclosporin A. In keratinocytes, collagen type I remained undetectable. In monolayers and cocultures, indirect immunofluorescence showed a slightly higher level of collagen type I protein in gingival fibroblasts in response to stimulation with cyclosporin A. Semiquantitative detection of collagen type I by western blotting demonstrated a nonsignificant increase for cell extracts in monolayers and cocultures. For secreted collagen type I, western blot analysis of the supernatants revealed elevated protein levels in cultures stimulated with cyclosporin A. Compared with the corresponding monolayers, the stimulatory effect of cyclosporin A on protein secretion was significant only in coculture. CONCLUSION: Our results indicate that collagen type I is a target of cyclosporin A and that gingival fibroblasts are decisive for the manifestation of the gingival overgrowth-phenotype. Furthermore, the results suggest that cocultures of gingival overgrowth-derived gingival fibroblasts and gingival keratinocytes permit analysis of cyclosporin A-induced effects under more in vivo-like conditions.
Assuntos
Colágeno Tipo I/análise , Ciclosporina/efeitos adversos , Fibroblastos/patologia , Gengiva/patologia , Crescimento Excessivo da Gengiva/induzido quimicamente , Queratinócitos/patologia , Adulto , Western Blotting , Linhagem Celular Transformada , Células Cultivadas , Técnicas de Cocultura , Colágeno Tipo I/efeitos dos fármacos , Colágeno Tipo I/genética , Células do Tecido Conjuntivo/efeitos dos fármacos , Células do Tecido Conjuntivo/patologia , Feminino , Fibroblastos/efeitos dos fármacos , Técnica Indireta de Fluorescência para Anticorpo , Gengiva/efeitos dos fármacos , Crescimento Excessivo da Gengiva/patologia , Humanos , Queratinócitos/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Fenótipo , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , Fatores de TempoRESUMO
Canine angiostrongylosis is a nematode infection in domestic dogs and wild carnivores. Few single case reports describing the occurrence of this disease in Germany exist and until recently angiostrongylosis has not been considered endemic in this country. The present report focuses on clinical, pathological and parasitological findings in two cases of fatal disseminated canine angiostrongylosis associated with multifocal haemorrhages in the central nervous system. Both animals, which lived in Germany, presented with rapidly progressive neurological signs including depression, ataxia, unilateral central blindness and epileptic seizures. Blood work revealed grossly elevated D-dimers and mild thrombocytopenia. Both animals were subsequently euthanised due to progressive clinical aggravation. Necropsy showed cerebral and lung haemorrhages in both animals. Multiple sections of nematode larvae consistent with Angiostrongylus vasorum were identified on histopathological sections of the brain, heart, kidney and lung in both animals and a predominantly granulomatous inflammation with the occurrence of multinucleated giant cells was observed. Adult nematodes were found in the larger lung arteries of one dog and Angiostrongylus infection was subsequently confirmed by PCR-analysis and sequencing in both dogs. A. vasorum larvae were not detected by faecal Baermann examination performed in one of the dogs. It was concluded that canine angiostrongylosis should be considered as differential diagnosis in dogs in Germany, even if faecal examination is negative. There is currently still a lack of studies investigating the occurrence of angiostrongylosis in dogs and intermediate hosts in Germany which would be necessary to survey the endemic realities of this disease.
Assuntos
Angiostrongylus , Hemorragia Cerebral/veterinária , Doenças do Cão/parasitologia , Infecções por Strongylida/veterinária , Animais , Encéfalo/patologia , Hemorragia Cerebral/complicações , Hemorragia Cerebral/epidemiologia , Hemorragia Cerebral/parasitologia , Doenças do Cão/epidemiologia , Cães , Evolução Fatal , Feminino , Alemanha/epidemiologia , Pulmão/patologia , Masculino , Infecções por Strongylida/complicações , Infecções por Strongylida/epidemiologia , Infecções por Strongylida/parasitologiaRESUMO
BACKGROUND: Congenital sensorineural deafness has been reported frequently in experimental mixed-breed white cats but there is a paucity of data on occurrence of deafness in client-owned pure-breed white cats. OBJECTIVE: To describe hearing status in client-owned pure-breed white cats. ANIMALS: Eighty-four pure-breed client-owned cats with white coat color of 10 registered breeds presented for routine hearing evaluation before breeding (1995-2008). METHODS: Hearing was assessed by click-evoked brainstem auditory evoked response. RESULTS: Overall deafness prevalence was 20.2%; 9 cats (10.7%) were bilaterally deaf and 8 cats (9.5%) were unilaterally deaf. There was no association between sex and deafness status (P= .85). Deafness status was associated with iris color (P= .04). CONCLUSIONS AND CLINICAL IMPORTANCE: Congenital sensorineural deafness frequently occurs in pure-breed cats with white coat color. Unilateral sensorineural deafness was as common as bilateral deafness.
Assuntos
Doenças do Gato/epidemiologia , Perda Auditiva Neurossensorial/veterinária , Estimulação Acústica/veterinária , Animais , Doenças do Gato/congênito , Gatos , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Feminino , Perda Auditiva Neurossensorial/congênito , Perda Auditiva Neurossensorial/epidemiologia , Masculino , PrevalênciaRESUMO
UNLABELLED: Pneumocystis jiroveci (formerly known as Pneumocystis carinii) is a fungal pathogen that causes pneumonia (PCP) in liver transplant recipients. Consequently, prophylaxis with trimethoprim-sulfamethoxazole (TMP/SMZ) is typically administered for at least 1 year at most liver transplant programs. At our center we have utilized a short-term (3-month) prophylactic regimen with TMP/SMZ for the past decade and report our experience and speculate on the potential widespread application of this approach. METHODS: For patients transplanted at our center between January 1997 and January 2007, we recorded all documented PCP infections by review of our liver transplant database and hospital-based electronic medical records system, both of which record all infections and culture results. RESULTS: We recorded no cases of PCP in any of the liver transplant recipients at our center during the study period. CONCLUSIONS: We report the absence of PCP in a large cohort of liver transplant recipients receiving a short-term (3-month) prophylaxis with TMP/SMZ. These findings provide a rational basis to consider short-term (3-month) PCP prophylaxis or avoidance of prophylaxis altogether in selected low-risk patients.
Assuntos
Antifúngicos/administração & dosagem , Transplante de Fígado/efeitos adversos , Pneumocystis carinii , Pneumonia por Pneumocystis/prevenção & controle , Sulfametoxazol/administração & dosagem , Trimetoprima/administração & dosagem , Estudos de Coortes , Colorado/epidemiologia , Centros Comunitários de Saúde , Esquema de Medicação , Quimioterapia Combinada , Humanos , Prontuários Médicos , Pneumonia por Pneumocystis/epidemiologia , Prevalência , Estudos RetrospectivosRESUMO
OBJECTIVE: Fractures involving the proximal one-third of the splint bone are relatively rare and are challenging to treat. A variety of management techniques have been reported in the literature. The aim of this retrospective case series was to describe the clinical presentation and evaluate the efficacy of bioabsorbable polylactic acid screws in internal fixation of proximal fractures of the 2nd and 4th metacarpal and metatarsal bones in horses. METHODS: The medical records, diagnostic images and outcome of all horses diagnosed with a proximal fracture of the splint bones and treated with partial resection and internal fixation of the proximal stump using bioabsorbable polylactic acid screws between 2014 and 2015 were reviewed. RESULTS: Eight horses met the inclusion criteria. The results showed that there were no complications encountered during screw placement or postoperatively. Six horses returned to full work 3 months after the operation and two horses remained mildly lame. On follow-up radiographs 12 months postoperatively (n = 2) the screws were not completely absorbed. The screws resulted in a cone-shaped radiolucency, which was progressively replaced from the outer margins by bone sclerosis. CONCLUSION: The use of bioabsorbable screws for fixation of proximal fractures of the splint bone appears to be a safe and feasible technique and may offer several advantages over the use of traditional metallic implants.
Assuntos
Parafusos Ósseos , Fixação Interna de Fraturas/veterinária , Fraturas Ósseas/veterinária , Cavalos/cirurgia , Ossos Metacarpais/cirurgia , Ossos do Metatarso/cirurgia , Implantes Absorvíveis , Animais , Feminino , Fixação Interna de Fraturas/métodos , Fraturas Ósseas/diagnóstico por imagem , Fraturas Ósseas/cirurgia , Alemanha , Cavalos/lesões , Masculino , Ossos Metacarpais/lesões , Ossos do Metatarso/lesões , Estudos Retrospectivos , Resultado do TratamentoRESUMO
We have examined cell coupling and expression of gap junction proteins in monolayer cultures of cells derived from human bone marrow stromal cells (BMC) and trabecular bone osteoblasts (HOB), and in the human osteogenic sarcoma cell line, SaOS-2. Both HOB and BMC cells were functionally coupled, since microinjection of Lucifer yellow resulted in dye transfer to neighboring cells, with averages of 3.4 +/- 2.8 (n = 131) and 8.1 +/- 9.3 (n = 51) coupled cells per injection, respectively. In contrast, little diffusion of Lucifer yellow was observed in SaOS-2 monolayers (1.4 +/- 1.8 coupled cells per injection, n = 100). Dye diffusion was inhibited by octanol (3.8 mM), an inhibitor of gap junctional communication. All of the osteoblastic cells expressed mRNA for connexin43 and connexin45, but not for connexins 26, 32, 37, 40, or 46. Whereas all of the osteoblastic cells expressed similar quantities of mRNA for connexin43, the poorly coupled SaOS-2 cells produced significantly less Cx43 protein than either HOB or BMC, as assessed by immunofluorescence and immunoprecipitation. Conversely, more Cx45 mRNA was expressed by SaOS-2 cells than by HOB or BMC. Thus, intercellular coupling in normal and transformed human osteoblastic cells correlates with the level of expression of Cx43, which appears to mediate intercellular communication in these cells. Gap junctional communication may serve as a means by which osteoblasts can work in synchrony and propagate locally generated signals throughout the skeletal tissue.