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Introduction To predict postoperative myocardial infarction rates in patients who undergo noncardiac surgery, the Canadian Cardiovascular Society Guidelines on Perioperative Cardiac Risk Assessment and Management recommends assessment of brain natriuretic peptide (BNP) in certain patients. Serial troponins are measured if the BNP level is elevated. In certain cases, Revised Cardiac Risk Index (RCRI) alone does not perform well, for example, during vascular surgery. Cardiac events occur in 20% of all vascular surgery patients. The odds ratio for such events is 9.2 if ST segments were depressed by 1 mm intraoperatively (relative to the PR interval) within the first 48 hours postoperatively. Increasing the number of cables and pads from three to five for electrocardiogram (EKG) increases the sensitivity from around 30% to over 80% for ischemic events relative to a formal EKG stress test, and then the monitor continuously displays not only lead II but also lead V5. Methods Our hypothesis was that raising awareness about diagnostic and therapeutic options to reduce the risk of postoperative myocardial infarction would increase the use of five pads. We conducted open-ended surveys at six hospitals to assess the reasons for choosing three pads. In our university hospital practice, we measured a cross-sectional incidence of using three pads before and, once again, a month after an intervention during a single morning. Several resident conferences encouraged the use of five pads. Education included weekly lectures and informal discussions with other staff during surgery, demonstrating that using five pads allows interrogation of an entire 12-lead EKG. In comparison, three pads only allow viewing three leads. Results At baseline, only three pads were available in 96% of our 23 operating rooms. Five cables were available in eight of those surgeries, but two were taped off to the side. Surveys unveiled scarcity of equipment and, more importantly, disempowerment (i.e., knowing how to diagnose or when to treat ischemia). After several conferences, the prevalence of equipment availability of only three pads fell to 47%. Conclusions Education enumerated details of recognizing ischemic configurations of ST depression. Next, education revealed methods to interrupt the progression of ischemia to infarction such as elevated blood pressure and hematocrit, reducing heart rate, and calling a cardiology consultant if the anesthesiologist wishes to draw serial troponins. Barriers to implementing an enhanced recovery after surgery (ERAS) pathway began with a need for more access to manage stress tests or optimize blood pressure medications after a preoperative anesthesia evaluation. The intraoperative barrier was knowing what to do if ST depression occurs. Therefore, we began raising awareness by encouraging the addition of an element of a future ERAS pathway, adding a cost of only $1 to monitor lead V5. Future ERAS pathways can include preoperative stress tests and consults, as found in published guidelines.
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AIMS: While gas-fermenting acetogens have been engineered to secrete non-native metabolites such as butyrate, acetate remains the most thermodynamically favourable product. An alternative to metabolic engineering is to exploit native capabilities for CO-to-acetate conversion by coculturing an acetogen with a second bacterium that provides efficient acetate-butyrate conversion. METHODS AND RESULTS: We used dynamic metabolic modelling to computationally evaluate the CO-to-butyrate conversion capabilities of candidate coculture systems by exploiting the diversity of human gut bacteria for anaerobic synthesis of butyrate from acetate and ethanol. A preliminary screening procedure based on flux balance analysis was developed to identify 48 gut bacteria which satisfied minimal growth rate and acetate-to-butyrate conversion requirements when cultured on minimal medium containing acetate and a simple sugar not consumed by the paired acetogen. A total of 170 acetogen/gut bacterium/sugar combinations were dynamically simulated for continuous growth using a 70/30 CO/CO2 feed gas mixture and minimal medium computationally determined for each combination. CONCLUSIONS: While coculture systems involving the acetogens Eubacterium limosum or Blautia producta yielded low butyrate productivities and CO-to-ethanol conversion had minimal impact on system performance, dynamic simulations predicted a large number of promising coculture designs with Clostridium ljungdahlii or C. autoethanogenum as the CO-to-acetate converter. Pairings with the gut bacterium Clostridium hylemonae or Roseburia hominis were particularly promising due to their ability to generate high butyrate productivities over a range of dilution rates with a variety of sugars. The higher specific acetate secretion rate of C. ljungdahlii proved more beneficial than the elevated growth rate of C. autoethanogenum for coculture butyrate productivity. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study demonstrated that metabolic modelling could provide useful insights into coculture design that can guide future experimental studies. More specifically, our predictions generated several favourable designs, which could serve as the first coculture systems realized experimentally.
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Butiratos , Clostridium , Clostridiales , Técnicas de Cocultivo , Eubacterium , Fermentación , HumanosRESUMEN
AIMS: To identify putative mutualistic interactions driving community composition in polymicrobial chronic wound infections using metabolic modelling. METHODS AND RESULTS: We developed a 12 species metabolic model that covered 74% of 16S rDNA pyrosequencing reads of dominant genera from 2963 chronic wound patients. The community model was used to predict species abundances averaged across this large patient population. We found that substantially improved predictions were obtained when the model was constrained with genera prevalence data and predicted abundances were averaged over 5000 ensemble simulations with community participants randomly determined according to the experimentally determined prevalences. Staphylococcus and Pseudomonas were predicted to exhibit a strong mutualistic relationship that resulted in community growth rate and diversity simultaneously increasing, suggesting that these two common chronic wound pathogens establish dominance by cooperating with less harmful commensal species. In communities lacking one or both dominant pathogens, other mutualistic relationship including Staphylococcus/Acinetobacter, Pseudomonas/Serratia and Streptococcus/Enterococcus were predicted consistent with published experimental data. CONCLUSIONS: Mutualistic interactions were predicted to be driven by crossfeeding of organic acids, alcohols and amino acids that could potentially be disrupted to slow chronic wound disease progression. SIGNIFICANCE AND IMPACT OF THE STUDY: Approximately 2% of the US population suffers from nonhealing chronic wounds infected by a combination of commensal and pathogenic bacteria. These polymicrobial infections are often resilient to antibiotic treatment due to the nutrient-rich wound environment and species interactions that promote community stability and robustness. The simulation results from this study were used to identify putative mutualistic interactions between bacteria that could be targeted to enhance treatment efficacy.
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Bacterias/aislamiento & purificación , Microbiota , Simbiosis , Infección de Heridas/microbiología , Bacterias/clasificación , Bacterias/genética , Bacterias/metabolismo , Fenómenos Fisiológicos Bacterianos , Coinfección/microbiología , HumanosRESUMEN
OBJECTIVES: To describe a chemotherapy protocol combining lomustine and doxorubicin in canine histiocytic sarcoma, including outcomes and toxicity. MATERIALS AND METHODS: Retrospective review of case records for dogs with histiocytic sarcoma treated with lomustine and doxorubicin (± cyclophosphamide) alternating every 2 weeks. Data collected included signalment, clinical signs, clinicopathological abnormalities, extent of disease, response, toxicity, time to tumour progression and survival time. RESULTS: Of 17 dogs, 15 had disseminated or metastatic disease. The median number of chemotherapy cycles (one dose of each drug) received was three; most dogs discontinued therapy due to progressive disease. Dose reductions or delays occurred in 18% of cycles. The overall response rate was 58%, with a median time to tumour progression of 185 (range, 59 to 268) days for responders. The overall median survival time was 185 (18 to 402) days. No significant prognostic factors were identified. CLINICAL SIGNIFICANCE: The protocol appeared well-tolerated, had some efficacy against canine histiocytic sarcoma in the study population and could be considered as an alternative to single-agent protocols; prospective comparison may be warranted.
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Antineoplásicos/administración & dosificación , Enfermedades de los Perros/tratamiento farmacológico , Doxorrubicina/administración & dosificación , Sarcoma Histiocítico/veterinaria , Lomustina/administración & dosificación , Animales , Protocolos de Quimioterapia Combinada Antineoplásica , Enfermedades de los Perros/mortalidad , Enfermedades de los Perros/patología , Perros , Femenino , Sarcoma Histiocítico/tratamiento farmacológico , Masculino , Estudios Retrospectivos , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
BACKGROUND: The utility of whole body magnetic resonance imaging (MRI) in detecting bone marrow infiltration in dogs with cancer has not been investigated. OBJECTIVES: To assess the feasibility of 3T body MRI for bone marrow assessment in dogs with hematopoietic neoplasia. ANIMALS: Seven dogs with B-cell lymphoma, 3 dogs with myelodysplastic syndrome (MDS), and 2 clinically normal dogs. METHODS: A prospective study of dogs with hematopoetic cancer was conducted using T1W, T2W, In-Phase, Out-of-Phase and STIR pulse sequences of the body excluding the head prior to bone marrow sampling. The relative signal intensity of a midlumbar vertebral body and a midshaft femoral bone marrow was compared by visual and point region of interest analysis to regional skeletal muscle. RESULTS: Similarity of femoral diaphyseal and vertebral body marrow signal intensity to that of skeletal muscle on the Out-of-Phase sequence was useful in distinguishing the 3 dogs with hypercellular marrow because of MDS from the 7 dogs with B-cell lymphoma and from the 2 clinically normal dogs. 1/7 dogs with lymphoma had proven bone marrow involvement but normal cellularity and less than 5% abnormal cells. Unaffected midfemoral marrow had greater signal intensity than skeletal muscle and unaffected vertebral marrow had less signal intensity than skeletal muscle on the Out-of-Phase sequence. CONCLUSIONS AND CLINICAL IMPORTANCE: 3T, Out-of-Phase MR pulse sequence was useful in distinguishing diffuse bone marrow infiltrate (MDS) from minimally or unaffected marrow using skeletal muscle for signal intensity comparison on whole body MRI.
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Médula Ósea/patología , Enfermedades de los Perros/patología , Neoplasias Hematológicas/veterinaria , Linfoma de Células B/veterinaria , Imagen por Resonancia Magnética/veterinaria , Síndromes Mielodisplásicos/veterinaria , Estadificación de Neoplasias/veterinaria , Animales , Enfermedades de los Perros/diagnóstico , Perros , Neoplasias Hematológicas/diagnóstico , Neoplasias Hematológicas/patología , Linfoma de Células B/complicaciones , Síndromes Mielodisplásicos/diagnóstico , Síndromes Mielodisplásicos/patología , Estadificación de Neoplasias/métodosRESUMEN
We performed genomewide gene expression analysis of 35 samples representing 6 common histologic subtypes of canine lymphoma and bioinformatics analyses to define their molecular characteristics. Three major groups were defined on the basis of gene expression profiles: (1) low-grade T-cell lymphoma, composed entirely by T-zone lymphoma; (2) high-grade T-cell lymphoma, consisting of lymphoblastic T-cell lymphoma and peripheral T-cell lymphoma not otherwise specified; and (3) B-cell lymphoma, consisting of marginal B-cell lymphoma, diffuse large B-cell lymphoma, and Burkitt lymphoma. Interspecies comparative analyses of gene expression profiles also showed that marginal B-cell lymphoma and diffuse large B-cell lymphoma in dogs and humans might represent a continuum of disease with similar drivers. The classification of these diverse tumors into 3 subgroups was prognostically significant, as the groups were directly correlated with event-free survival. Finally, we developed a benchtop diagnostic test based on expression of 4 genes that can robustly classify canine lymphomas into one of these 3 subgroups, enabling a direct clinical application for our results.
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Biomarcadores de Tumor/metabolismo , Enfermedades de los Perros/clasificación , Linfoma de Células B/veterinaria , Linfoma de Células T/veterinaria , Animales , Estudios de Cohortes , Biología Computacional , Supervivencia sin Enfermedad , Enfermedades de los Perros/mortalidad , Enfermedades de los Perros/patología , Perros , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Estudio de Asociación del Genoma Completo/veterinaria , Inmunofenotipificación , Linfoma de Células B/clasificación , Linfoma de Células B/metabolismo , Linfoma de Células B/patología , Linfoma de Células T/clasificación , Linfoma de Células T/metabolismo , Linfoma de Células T/patología , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Pronóstico , ARN Neoplásico/genéticaRESUMEN
BACKGROUND: Tumors have heterogeneous properties, which could be explained by the existence of hierarchically and biologically distinct tumor cells such as tumor-initiating cells (TICs). This model is clinically important, as TICs are promising targets for cancer therapies. However, TICs in spontaneous B-cell lymphoma have not been conclusively identified. HYPOTHESIS/OBJECTIVES: Tumor cells with a progenitor phenotype exist in B-cell lymphoma, reflecting a hierarchical organization. ANIMALS: Twenty-eight client-owned dogs with previously untreated B-cell lymphoma and 6 healthy dogs. METHODS: This was a prospective study. Flow cytometry was used to identify lymphoid progenitor cells (LPCs) that coexpressed hematopoietic progenitor antigens CD34, CD117, and CD133, with lymphoid differentiation markers CD21 and/or CD22 in B-cell lymphoma. The polymerase chain reaction for antigen receptor rearrangements was used to analyze clonality and relatedness of tumor populations. A xenograft model with NOD/SCID/IL-2Rγ(-/-) mice was adapted to expand and serially transplant primary canine B-cell lymphoma. RESULTS: LPCs were expanded in lymph nodes from 28 dogs with B-cell lymphoma compared with 6 healthy dogs (P= .0022). LPCs contained a clonal antigen receptor gene rearrangement identical to that of the bulk of tumor cells. Canine B-cell lymphoma xenografts in recipient mice that maintained LPCs in the tumors were recurrently observed. CONCLUSIONS AND CLINICAL IMPORTANCE: These results suggest the presence of a hierarchy of tumor cells in B-cell lymphoma as has been demonstrated in other cancers. These findings have the potential to impact not only the understanding of lymphoma pathogenesis but also the development of lymphoma therapies by providing novel targets for therapy.
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Enfermedades de los Perros/patología , Tejido Linfoide/patología , Linfoma de Células B/patología , Células Madre Neoplásicas/patología , Antígeno AC133 , Animales , Antígenos CD/análisis , Antígenos CD/inmunología , Antígenos CD34/análisis , Antígenos CD34/inmunología , Estudios de Cohortes , Modelos Animales de Enfermedad , Enfermedades de los Perros/inmunología , Perros , Femenino , Citometría de Flujo/veterinaria , Glicoproteínas/análisis , Glicoproteínas/inmunología , Inmunofenotipificación/veterinaria , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Linfoma de Células B/inmunología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/inmunología , Péptidos/análisis , Péptidos/inmunología , Estudios Prospectivos , Proteínas Proto-Oncogénicas c-kit/análisis , Proteínas Proto-Oncogénicas c-kit/inmunología , Organismos Libres de Patógenos Específicos , Estadísticas no Paramétricas , Trasplante Heterólogo/veterinariaRESUMEN
Cytotoxic T-lymphocyte responses to subcellular antigens are enhanced when antigens are presented on cell-sized silica microbeads called large multivalent immunogens (LMIs). LMIs prepared with tumour cell membrane fragments have induced partial remissions in humans with melanoma and renal cell carcinoma. The purpose of this phase I study was to evaluate the safety of LMIs, prepared with autologous lymphoma cell membranes, along with subcutaneous interleukin 2 (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF) in dogs with untreated B-cell lymphoma. After lymph node excision and induction chemotherapy, five dogs were vaccinated with three weekly doses of LMI alone; five with LMI and subcutaneous IL-2 and five with LMI, IL-2 and GM-CSF. No significant toxicity was noted, treatment did not adversely affect disease-free interval and half of the dogs showed measurable delayed-type hypersensitivity reactions to intradermal challenge with LMI, suggesting specific cell-mediated immunity.
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Antígenos de Neoplasias/uso terapéutico , Autoantígenos/administración & dosificación , Enfermedades de los Perros/tratamiento farmacológico , Factor Estimulante de Colonias de Granulocitos y Macrófagos/administración & dosificación , Inmunoterapia/veterinaria , Interleucina-2/administración & dosificación , Linfoma de Células B/veterinaria , Animales , Antígenos de Neoplasias/administración & dosificación , Terapia Combinada/veterinaria , Supervivencia sin Enfermedad , Enfermedades de los Perros/inmunología , Enfermedades de los Perros/cirugía , Perros , Esquema de Medicación/veterinaria , Femenino , Hipersensibilidad Tardía/veterinaria , Inyecciones Subcutáneas/veterinaria , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/inmunología , Linfoma de Células B/cirugía , Masculino , Microesferas , Resultado del TratamientoRESUMEN
BACKGROUND: Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) contains crucial information about tumour heterogeneity and the transport limitations that reduce drug efficacy. Mathematical modelling of drug delivery and cellular responsiveness based on underutilised DCE-MRI data has the unique potential to predict therapeutic responsiveness for individual patients. METHODS: To interpret DCE-MRI data, we created a modelling framework that operates over multiple time and length scales and incorporates intracellular metabolism, nutrient and drug diffusion, trans-vascular permeability, and angiogenesis. The computational methodology was used to analyse DCE-MR images collected from eight breast cancer patients at Baystate Medical Center in Springfield, MA. RESULTS: Computer simulations showed that trans-vascular transport was correlated with tumour aggressiveness because increased vessel growth and permeability provided more nutrients for cell proliferation. Model simulations also indicate that vessel density minimally affects tissue growth and drug response, and nutrient availability promotes growth. Finally, the simulations indicate that increased transport heterogeneity is coupled with increased tumour growth and poor drug response. CONCLUSION: Mathematical modelling based on DCE-MRI has the potential to aid treatment decisions and improve overall cancer care. This model is the critical first step in the creation of a comprehensive and predictive computational method.
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Antineoplásicos/farmacocinética , Neoplasias de la Mama/diagnóstico , Neoplasias de la Mama/tratamiento farmacológico , Imagen por Resonancia Magnética , Adulto , Anciano , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/patología , Simulación por Computador , Medios de Contraste , Femenino , Humanos , Persona de Mediana Edad , Modelos Biológicos , Neovascularización Patológica/diagnóstico , Neovascularización Patológica/tratamiento farmacológico , Valor Predictivo de las PruebasRESUMEN
Steady-state and dynamic flux balance analysis (DFBA) was used to investigate the effects of metabolic model complexity and parameters on ethanol production predictions for wild-type and engineered Saccharomyces cerevisiae. Three metabolic network models ranging from a single compartment representation of metabolism to a genome-scale reconstruction with seven compartments and detailed charge balancing were studied. Steady-state analysis showed that the models generated similar wild-type predictions for the biomass and ethanol yields, but for ten engineered strains the seven compartment model produced smaller ethanol yield enhancements. Simplification of the seven compartment model to two intracellular compartments produced increased ethanol yields, suggesting that reaction localisation had an impact on mutant phenotype predictions. Further analysis with the seven compartment model demonstrated that steady-state predictions can be sensitive to intracellular model parameters, with the biomass yield exhibiting high sensitivity to ATP utilisation parameters and the biomass composition. The incorporation of gene expression data through the zeroing of metabolic reactions associated with unexpressed genes was shown to produce negligible changes in steady-state predictions when the oxygen uptake rate was suitably constrained. Dynamic extensions of the single and seven compartment models were developed through the addition of glucose and oxygen uptake expressions and transient extracellular balances. While the dynamic models produced similar predictions of the optimal batch ethanol productivity for the wild type, the single compartment model produced significantly different predictions for four implementable gene insertions. A combined deletion/overexpression/insertion mutant with improved ethanol productivity capabilities was computationally identified by dynamically screening multiple combinations of the ten metabolic engineering strategies. The authors concluded that extensive compartmentalisation and detailed charge balancing can be important for reliably screening metabolic engineering strategies that rely on modification of the global redox balance and that DFBA offers the potential to identify novel mutants for enhanced metabolite production in batch and fed-batch cultures.
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Etanol/metabolismo , Modelos Biológicos , Saccharomyces cerevisiae/metabolismo , Biología de Sistemas/métodos , Adenosina Trifosfato/metabolismo , Algoritmos , Biomasa , Reactores Biológicos , Simulación por Computador , Fermentación , Perfilación de la Expresión Génica , Glucosa/metabolismo , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxígeno/metabolismoRESUMEN
BACKGROUND CONTEXT: Achieving solid implant fixation to osteoporotic bone presents a clinical challenge. New techniques and devices are being designed to increase screw-bone purchase of pedicle screws in the lumbar spine via a novel cortical bone trajectory that may improve holding screw strength and minimize loosening. Preliminary clinical evidence suggests that this new trajectory provides screw interference that is equivalent to the more traditionally directed trajectory for lumbar pedicle screws. However, a biomechanical study has not been performed to substantiate the early clinical results. PURPOSE: Evaluate the mechanical competence of lumbar pedicle screws using a more medial-to-lateral path (ie, "cortical bone trajectory") than the traditionally used path. STUDY DESIGN: Human cadaveric biomechanical study. METHODS: Each vertebral level (L1-L5) was dual-energy X-ray absorptiometry (DXA) scanned and had two pedicle screws inserted. On one side, the traditional medially directed trajectory was drilled and tapped. On the contralateral side, the newly proposed cortical bone trajectory was drilled and tapped. After qCT scanning, screws were inserted into their respective trajectories and pullout and toggle testing ensued. In uniaxial pullout, the pedicle screw was withdrawn vertically from the constrained bone until failure occurred. The contralateral side was tested in the same manner. In screw toggle testing, the vertebral body was rigidly constrained and a longitudinal rod was attached to each screw head. The rod was grasped using a hydraulic grip and a quasi-static, upward displacement was implemented until construct failure. The contralateral pedicle screw was tested in the same manner. Yield pullout (N) and stiffness (N/mm) as well as failure moment (N-m) were compared and bone mineral content and bone density data were correlated with the yield pullout force. RESULTS: New cortical trajectory screws demonstrated a 30% increase in uniaxial yield pullout load relative to the traditional pedicle screws (p=0.080), although mixed loading demonstrated equivalency between the two trajectories. No significant difference in construct stiffness was noted between the two screw trajectories in either biomechanical test or were differences in failure moments (p=0.354). Pedicle screw fixation did not appear to depend on bone quality (DXA) yet positive correlations were demonstrated between trajectory and bone density scans (qCT) and pullout force for both pedicle screws. CONCLUSIONS: The current study demonstrated that the new cortical trajectory and screw design have equivalent pullout and toggle characteristics compared with the traditional trajectory pedicle screw, thus confirming preliminary clinical evidence. The 30% increase in failure load of the cortical trajectory screw in uniaxial pullout and its juxtaposition to higher quality bone justify its use in patients with poor trabecular bone quality.
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Tornillos Óseos , Ensayo de Materiales , Fusión Vertebral/instrumentación , Absorciometría de Fotón , Fenómenos Biomecánicos , Falla de Equipo , Humanos , Vértebras Lumbares/cirugía , Osteoporosis/cirugíaRESUMEN
Artificial microbial ecosystems have been increasingly used to understand principles of ecology. These systems offer unique capabilities to mimic a variety of ecological interactions that otherwise would be difficult to study experimentally in a reasonable period of time. However, the elucidation of the genetic bases for these interactions remains a daunting challenge. To address this issue, we have designed and analysed a synthetic symbiotic ecosystem in which the genetic nature of the microbial interactions is defined explicitly. A mathematical model of the gene regulatory network in each species and their interaction through quorum sensing mediated intercellular signalling was derived to investigate the effect of system components on cooperative behaviour. Dynamic simulation and bifurcation analysis showed that the designed system admits a stable coexistence steady state for sufficiently large initial cell concentrations of the two species. The steady-state fraction of each species could be altered by varying model parameters associated with gene transcription and signalling molecule synthesis rates. The design also admitted a stable steady state corresponding to extinction of the two species for low initial cell concentrations and stable periodic solutions over certain domains of parameter space. The mathematical analysis was shown to provide insights into natural microbial ecosystems and to allow identification of molecular targets for engineering system behaviour.
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Fenómenos Fisiológicos Bacterianos , Ecosistema , Regulación Bacteriana de la Expresión Génica/fisiología , Modelos Biológicos , Percepción de Quorum/fisiología , Simbiosis/fisiología , Proliferación Celular , Simulación por ComputadorRESUMEN
The mechanisms underlying insufficient insulin secretion and loss of beta-cell mass in feline and human type 2 diabetes mellitus are incompletely understood. However, islet amyloid polypeptide (IAPP)-derived islet amyloidosis (IA) has been linked to increased rates of beta-cell apoptosis and, therefore, our goal was to develop an in vitro model of IAPP fibrillogenesis using isolated pancreatic islets from mice transgenic for human IAPP (hIAPP Tg mice). Islets from hIAPP Tg mice, from mice transgenic for non-amyloidogenic murine IAPP (mIAPP Tg mice), and from the FVB background strain were exposed to normal (5.5 mM) or high (28 mM) glucose conditions in cell culture for 8 days. On days 0 and 8, islets were collected for electron microscopy (EM). EM showed no abnormalities in the mIAPP Tg or FVB islets at either time point. On day 8, hIAPP Tg islets cultured at high glucose concentration formed extracellular IAPP-derived flocculent deposits. No significant differences in rates of apoptosis were found between groups. Our findings, therefore, show that in vitro culture of hIAPP Tg mouse islets under high glucose conditions produces a readily available and rapidly inducible model of IAPP-derived fibrillogenesis and enables the study of early phases of the molecular pathogenesis of IA.
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Amiloide/metabolismo , Amiloidosis/metabolismo , Islotes Pancreáticos/metabolismo , Precursores de Proteínas/metabolismo , Amiloide/genética , Amiloidosis/patología , Animales , Apoptosis , Células Cultivadas , Femenino , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Polipéptido Amiloide de los Islotes Pancreáticos , Islotes Pancreáticos/patología , Islotes Pancreáticos/ultraestructura , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Precursores de Proteínas/genéticaRESUMEN
Inhibition of p38 kinase blocks the production of tumor-promoting factors in the multiple myeloma (MM) bone marrow microenvironment. Proteasome inhibitors MG132 and bortezomib have been shown to have direct cytotoxic effects on MM cells. We show that a selective inhibitor of p38alpha, SCIO-469, enhances the ability of MG132 and bortezomib to induce the apoptosis of MM cells. Previously, we showed that p38 inhibition with SCIO-469 enhances MM cytotoxicity of bortezomib by inhibiting the transient expression and phosphorylation of Hsp27, a downstream target of p38. Here we show that continued treatment of MM cells with bortezomib leads to a SCIO-469-enhanced downregulation of Hsp27 and to increased MM apoptosis. Furthermore, we show that p38 inhibition enhances the bortezomib-induced MM apoptosis by upregulation of p53 and downregulation of Bcl-X(L) and Mcl-1. In a mouse xenograft plasmacytoma model of MM, we found that inhibiting p38 augments the effects of bortezomib in decreasing MM tumor growth in vivo. Thus, in addition to its role in suppressing an activated MM microenvironment, co-treatment with a p38 inhibitor, such as SCIO-469, may enhance the cytotoxicity of bortezomib by modulating pro-apoptotic and anti-apoptotic factors in MM cells, suggesting great potential for co-therapy.
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Proteínas de Choque Térmico/metabolismo , Indoles/farmacología , Proteína Quinasa 14 Activada por Mitógenos/antagonistas & inhibidores , Mieloma Múltiple/metabolismo , Proteínas de Neoplasias/metabolismo , Inhibidores de Proteasas/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Proteína bcl-X/metabolismo , Administración Oral , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Apoptosis/efectos de los fármacos , Ácidos Borónicos/administración & dosificación , Ácidos Borónicos/farmacología , Bortezomib , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Activación Enzimática/efectos de los fármacos , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/efectos de los fármacos , Humanos , Técnicas In Vitro , Indoles/administración & dosificación , Inyecciones Intravenosas , Leupeptinas/farmacología , Ratones , Ratones Desnudos , Proteína Quinasa 14 Activada por Mitógenos/metabolismo , Chaperonas Moleculares , Mieloma Múltiple/enzimología , Proteínas de Neoplasias/efectos de los fármacos , Inhibidores de Proteasas/administración & dosificación , Pirazinas/administración & dosificación , Pirazinas/farmacología , Células Tumorales Cultivadas , Proteína p53 Supresora de Tumor/efectos de los fármacos , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína bcl-X/efectos de los fármacosRESUMEN
The annulus fibrosus and its attachment to the bony tympanic ring were studied in a series of mammals. In the pallid bat, Antrozous pallidus, there is an extensive plexus of large interconnected blood sinuses in the part of the annulus that borders the tympanic bone. The spaces between the sinuses are packed with smooth muscle cells. Most of the cells have a predominately radial orientation; they extend from the bony tympanic sulcus to a dense collagenous matrix (apical zone) where radially oriented fibers of the pars tensa are confluent with the annulus. The muscles and vessels constitute a myovascular zone. A structurally similar myovascular zone is also present in the European hedgehog. In rodents, the annulus lacks the large interconnected blood sinuses but many small vessels are present. Smooth muscle is concentrated in the broad area of attachment of the annulus to the tympanic bone. In the gerbil, smooth muscle seems to be concentrated in the central part of the width of the annulus where it is attached to bone and radiates toward the tympanic membrane. In humans collections of radially oriented smooth muscle cells were found in several locations. The smooth muscle in all species studied appears to form a rim of contractile elements for the pars tensa. This arrangement suggests a role in controlling blood flow and/or creating and maintaining tension on the tympanic membrane.
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Membrana Timpánica/anatomía & histología , Anciano , Animales , Quirópteros , Femenino , Gerbillinae , Erizos , Humanos , Lactante , Masculino , Ratones , Persona de Mediana Edad , Músculo Liso/anatomía & histología , Músculo Liso/irrigación sanguínea , Filogenia , Ratas , Especificidad de la Especie , Membrana Timpánica/irrigación sanguíneaRESUMEN
Leptin produced by both adipose tissue and the placental trophoblast, has been proposed to regulate numerous aspects of human conceptus development. Although recent animal studies have suggested an additional role for the polypeptide in fetal lung maturation, no evidence has been reported in primates. Therefore, we employed the baboon (Papio sp.), a well-characterized primate model for human pregnancy, to determine the presence and ontogeny of leptin receptor in fetal lung with advancing gestation. Lungs were collected from fetal baboons, early in gestation (days 58-62, n = 4), at mid gestation (days 98-102, n = 4), and late in gestation (days 158-165, n = 4) (term 184 days). mRNA transcripts for leptin (LEP) and both long and short intracellular domain isoforms of the leptin receptor (LEP-R(L) and LEP-R(S)) were assessed by RT-PCR. leptin receptor protein was evaluated by immunoblotting and cell types expressing leptin receptor were identified in late pregnancy by immunohistochemistry. Fetal serum leptin concentrations, determined by RIA, remained relatively unchanged at 5.7 +/- 1.1 ng/ml (mean +/- s.e.m.) in mid pregnancy and 8.4 +/- 3.0 ng/ml in late pregnancy (P > 0.05). Although leptin were detectable in fetal lung, no changes in transcript abundance were apparent with advancing gestation. However, transcripts for both LEP-R(L) and LEP-R(S) receptor isoforms increased several-fold (P < 0.05) in fetal lung between mid and late gestation, while leptin receptor protein was detectable only in late pregnancy. leptin receptor was localized in distal pulmonary epithelial cells, including type II pneumocytes. In conclusion, leptin is present in the fetal baboon and its receptor is enhanced during late gestation in cells responsible for the synthesis of pulmonary surfactant. Collectively, these and past findings may suggest a modulatory role for the polypeptide in pulmonary development and/or may identify leptin receptor as a physiological marker of primate fetal lung maturity.
Asunto(s)
Pulmón/embriología , Papio/fisiología , Receptores de Superficie Celular/metabolismo , Animales , Femenino , Sangre Fetal/química , Edad Gestacional , Humanos , Immunoblotting , Inmunohistoquímica/métodos , Leptina/análisis , Leptina/sangre , Leptina/genética , Pulmón/química , Modelos Animales , Embarazo , Isoformas de Proteínas/análisis , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/análisis , Receptores de Superficie Celular/análisis , Receptores de Superficie Celular/genética , Receptores de Leptina , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Previous studies demonstrating the presence of collagen II in the basilar membrane have used a biochemical approach or have used immunohistochemistry at the light microscopic level. In this investigation both the presence and arrangement of collagen II were demonstrated at the ultrastructural level using pre- and post-embedding immunoelectron microscopy. Labeling was dependent on the development of protocols to expose epitopes while maintaining identifiable ultrastructure. Both positive and negative controls indicate that the labeling was specific for collagen II. Collagen II was detected in the fibrous sheet of the pars tecta and in the two fibrous layers of the pars pectinata. It was detected in situ and on isolated individual 10-12 nm fibrils. The presence of collagen II in all the fibrous layers of the basilar membrane places constraints on the biomechanical properties of this important structure.
Asunto(s)
Membrana Basilar/metabolismo , Colágeno Tipo II/metabolismo , Animales , Membrana Basilar/ultraestructura , Colágeno Tipo II/inmunología , Colágeno Tipo II/ultraestructura , Reacciones Cruzadas , Microscopía Electrónica de Rastreo/métodos , Microscopía Inmunoelectrónica/métodos , Adhesión en Plástico , ConejosRESUMEN
This paper describes the development of a protocol that can be used to detect collagen II in the healthy adult basilar membrane (BM) at the electron microscopic level. This protocol required aggressive epitope exposure techniques to break the crosslinks that bind the collagen molecules tightly into fibrils and to remove a dense mat of ground substance that surrounds the fibrils. On the other hand, the steps had to be carefully controlled to preserve BM ultrastructure and the collagen II epitopes that are typically labile. These requirements were satisfied by introducing a targeted crosslink breakage method and by regulating the duration of epitope exposure based on changes in tissue appearance observed with differential interference contrast microscopy. High levels of immunolabeling were achieved by substituting tissue preservation techniques for most or all of fixation; this was important because fixation reduces antigenicity directly and impedes epitope exposure. When these techniques were combined with more traditional trypsin and pepsin treatments, the result was dense immunolabeling and preservation of ultrastructure that allowed accurate localization of the immunolabeling. This pre-embedding immunoelectron microscopic method is the first to be carried out on the BM and may be adaptable to future studies of the BM as well as other tissues with similar molecular composition.
Asunto(s)
Membrana Basilar/metabolismo , Colágeno Tipo II/metabolismo , Microscopía Inmunoelectrónica/métodos , Animales , Membrana Basilar/ultraestructura , Colágeno Tipo II/inmunología , Colágeno Tipo II/ultraestructura , Epítopos/aislamiento & purificación , Inmunohistoquímica/métodos , Microscopía de Interferencia , Adhesión en Plástico , ConejosRESUMEN
In order to analyze the entry of solutes through the round window membrane, a quantitative description of round window anatomy in relationship to scala tympani is required. High-resolution magnetic resonance microscopy was used to visualize the fluid spaces and tissues of the inner ear in three dimensions in isolated, fixed specimens from guinea pigs. Each specimen was represented as consecutive serial slices, with a voxel size of approximately 25 microm(3). The round window membrane, and its relationship to the terminal portion of scala tympani in the basal turn, was quantified in six specimens. In each image slice, the round window membrane and scala tympani were identified and segmented. The total surface area of the round window membrane averaged 1.18 mm(2) (S.D. 0.08, n=6). The length and variation of cross-sectional area as a function of distance for the cochlear aqueduct was determined in five specimens. The cochlear aqueduct was shown to enter scala tympani at the medial limit of the round window membrane, which corresponded to a distance of approximately 1 mm from the end of the scala when measured along its mid-point. These data are of value in simulating drug and other solute movements in the cochlear fluids and have been incorporated into a public-domain simulation program available at http://oto.wustl.edu/cochlea/.