From bench to bedside: Biosensing strategies to evaluate endocrine disrupting compounds based on epigenetic events and their potential use in medicine.

The relationship between endocrine system disorders and health risks due to chemical environmental compounds has become a growing concern in recent years. Involuntary exposure to endocrine disruptors (EDCs) is associated with the worldwide increase of diseases such as cancer, obesity, diabetes, and neurocortical disorders. EDCs are compounds that target the nuclear hormonereceptors (NHR) leading to epigenetic changes. Consequently, the use of biosensing strategies based on epigenetic events have a great potential to provide outstanding information about the exposition of EDCs and their evaluation in human health. This review addresses the novel trends in biosensing EDCs evaluation based on DNA methylation assays associated with different human diseases.

Suppression of RAC1-driven malignant melanoma by group A PAK inhibitors.

Activating mutations in the RAC1 gene have recently been discovered as driver events in malignant melanoma. Expression of this gene is associated with melanocyte proliferation, and melanoma cells bearing this mutation are insensitive to BRAF inhibitors such as vemurafenib and dabrafenib, and also may evade immune surveillance due to enhanced expression of PD-L1. Activating mutations in RAC1 are of special interest, as small-molecule inhibitors for the RAC effector p21-activated kinase (PAK) are in late-stage clinical development and might impede oncogenic signaling from mutant RAC1. In this work, we explore the effects of PAK inhibition on RAC1P29S signaling in zebrafish embryonic development, in the proliferation, survival and motility of RAC1P29S-mutant human melanoma cells, and on tumor formation and progression from such cells in mice. We report that RAC1P29S evokes a Rasopathy-like phenotype on zebrafish development that can be blocked by inhibitors of PAK or MEK. We also found and that RAC1-mutant human melanoma cells are resistant to clinical inhibitors of BRAF but are uniquely sensitive to PAK inhibitors. These data suggest that suppressing the PAK pathway might be of therapeutic benefit in this type of melanoma.

Evaluation of the nutritive value of sugarcane residues inoculated with fungus Fomes sp / Evaluación del valor nutritivo de residuos de caña de azúcar inoculado con hongo Fomes sp

Objective. Improve the nutritional value of mechanized sugarcane residues inoculating the fungus Fomes sp. EUM1. Materials and methods. The fungus Fomes was inoculated according to a 0, 0.1, 0.2, and 0.3% (w/v) treatment and incubated at a temperature of 35°C for 7, 10 and 13 days. It was obtained DM, OM, CP, ash, NDF and ADF and the effective degradation of DM, NDF and ADF, with an experimental factorial design of 3X3 and a completely randomized design. The factors were growing days in an Erlenmeyer flask (7, 10, and 13) and inoculum percentage (0.1, 0.2 and 0.3). The data were analyzed with the SAS statistical package. Results. Statistical significance was found in the interaction of the fungus growing days by percentage of inoculum, in the variables: DM, CP and pH. The NDF and ADF factor differed in the percentage of inoculum. Effective degradation showed significant for the same type of interaction in all the variables studied. Conclusions. The inoculation of the fungus increased ADF degradation by only 0.2% of the inoculum percentage, without any effect on effective degradation due to the use of soluble fractions at the beginning of the incubation. It is considered that the degradation occurs in stages that are important to consider for determining treatments to maximize the beneficial effects of the fungus in terms of ruminant nutrition.

Objetivo. Mejorar el valor nutritivo de los residuos de cosecha mecanizada de la caña de azúcar inoculando el hongo Fomes sp. EUM1. Materiales y métodos. El hongo Formes se inoculó de acuerdo al tratamiento 0, 0.1, 0.2 y 0.3% (p/v), incubándose a una temperatura de 35°C durante 7, 10 y 13 días. Se obtuvo la MS, MO, PC, cenizas, FDN y FDA y la degradación efectiva de la MS, FDN y FDA; con un diseño experimental de tipo factorial 3X3, con un diseño experimental completamente al azar. Los factores fueron días de crecimiento en matraz Erlenmeyer (7, 10, 13) y porcentajes de inclusión (0.1, 0.2 y 0.3). Los datos se analizaron con el paquete estadístico SAS. Resultados. Se encontró significancia estadística para la interacción días de crecimiento del hongo por porcentaje de inóculo, en las variables MS, PC y pH. La FDN y la FDA presentaron diferencias para el factor porcentaje de inóculo. La degradación efectiva mostró significancia para el mismo tipo de interacción, en todas las variables estudiadas. Conclusiones. La inoculación del hongo aumentó la degradación de la FDA, únicamente al 0.2% de porcentaje de inclusión, sin un efecto sobre la degradación efectiva, debido a la utilización de fracciones solubles al inicio de la incubación. Se considera que la degradación se produce por etapas que son importante considerar para la determinación de tratamientos que maximicen los efectos benéficos del hongo en términos de la nutrición de rumiantes.

[The snapping scapula as a sympton of a tumor in the scapulothoracic region]. / El chasquido escapular como síntoma de un tumor de la región escapulotorácica.

INTRODUCTION: The snapping scapula syndrome is a grating sensation located in the scapulothoracic region that appears with movement.This sign is occasionally related to tumors. OBJECTIVE: To show the high incidence of this relationship (clinical sign-tumor), and to be aware of it when performing a differential diagnosis. MATERIAL AND METHOD: Retrospective study of the elastofibromas dorsi (ED) and scapular osteochondromas (SO), which may have presented with the sign under study in our center over the last 17 years (1993-2009). Thirty-seven ED and 6 SO were identified. The series was divided into group A (ED) and group B (SO). Mean follow-up was 7 years. The cohorts are made up of 23 women and 4 men with a mean age of 57 years (42-78) in group A, there were 2 women and 4 men with a mean age of 20 years (11-28) in group B. Action was taken to identify the initial medical sign at diagnosis, the treatment carried out, and the outcome. RESULTS: Around 21% of these tumors are reported to be associated with physical activity. The initial symptom was a painful mass in 81% of the patients, followed by a scapular snapping or clicking in 30 out of the 43 patients (70%). The treatment of choice was resection in both groups. A noticeable improvement in terms of pain was seen (VAS 7.5 preoperatively, 2.8 postoperatively). CONCLUSION: The presence of snapping scapula has a strong relationship to tumors of the scapulothoracic region. Therefore it is important to be aware of this.

Human trabecular bone cells are able to express both osteoblastic and adipocytic phenotype: implications for osteopenic disorders.

The decrease in bone volume associated with osteoporosis and age-related osteopenia is accompanied by increased marrow adipose tissue formation. Reversal of this process may provide a novel therapeutic approach for osteopenic disorders. We have shown that cells cultured from human trabecular bone are not only osteogenic, but are able also to undergo adipocyte differentiation under defined culture conditions. Osteoblast differentiation was induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and adipocyte differentiation by dexamethasone (dex) plus 3-isobutyl-1-methylxanthine (IBMX) treatment. Adipogenesis was characterized by lineage-specific enzyme and gene activities, alpha-glycerophosphate-3-dehydrogenase activity, fatty acid binding protein, aP2 and lipoprotein lipase expression. Osteoblastogenesis was assessed by osteoblast characteristic 1,25(OH)2D3 induction of alkaline phosphatase activity and osteoblast-specific 1,25(OH)2D3-induced osteocalcin synthesis and release. We provide evidence for a common pluripotent mesenchymal stem cell that is able either to undergo adipogenesis or osteoblastogenesis, using clonal cell lines derived from human trabecular bone cell cultures. Adipogenesis can be induced also by long chain fatty acids and the thiazolidinedione troglitazone. Dex plus IBMX-induced adipogenesis can be inhibited by interleukin-1beta, tumor necrosis factor-alpha, and transforming growth factor-beta. Interestingly, and in contrast to extramedullary adipocyte differentiation as shown by mouse 3T3L-1 and a human liposarcoma SW872 cell line, trabecular bone adipogenesis was unaffected by insulin. Also, the formation of fully differentiated adipocytes from trabecular bone cells after troglitazone treatment and long chain fatty acids was dependent on increased expression of the nuclear hormone receptor peroxisome proliferator-activated receptor gamma2 caused by dex plus IBMX. Specific inhibition of marrow adipogenesis and promotion of osteoblastogenesis of a common precursor cell may provide a novel therapeutic approach to the treatment of osteopenic disorders.

Human osteoclastoma-derived stromal cells: correlation of the ability to form mineralized nodules in vitro with formation of bone in vivo.

It has been suggested that the stromal element of human osteoclastomas contains osteoblastic cells. In this study, we demonstrate that osteoclast-depleted, passaged stromal cells express alkaline phosphatase and osteocalcin in vitro and form mineralized nodules under appropriate culture conditions. In addition, we describe a model in which severe combined immunodeficient (SCID) mice were used to support the differentiation of these putative human osteoblast progenitors in vivo. Lesions formed from human stromal cells were identified using the OKa blood group antigen and human procollagen type I antibodies. By 21 days, the lesion was a complete bone unit: a fully mineralized cortex, remodeling trabeculae, and a highly cellular marrow space. Stromal cells derived from six out of seven osteoclastomas produced identical lesions. Further studies have demonstrated that the capacity of the osteoclastoma-derived stromal cells to form bone in vivo and in vitro is passage dependent; early passages were osteogenic in both model systems, while later passages were not. In conclusion, we have developed a model in which the osteogenic nature of cells can be confirmed in vivo. Furthermore, human osteoclastoma-derived stromal cells provide a source of these osteogenic cells to study human osteoblast differentiation, both in vivo and in vitro.

Beneficial effects of the phosphodiesterase inhibitors BRL 61063, pentoxifylline, and rolipram in a murine model of endotoxin shock.

Three inhibitors of calcium-dependent cyclic adenosine 3'5'-monophosphate (cAMP) dependent phosphodiesterase IV (PDE IV) were evaluated for their effects on lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF) production in vitro and in vivo and for their ability to protect mice from LPS-induced lethality in D-galactosamine (D-gal) sensitized mice. In vitro, on LPS-stimulated murine peritoneal macrophages (PEM), BRL 61063 (1,3-di(cyclopropylmethyl)-8-aminoxanthine) and rolipram (4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidone) had similar TNF inhibitory activity with an IC50 ranging from 0.1 to 0.5 microM. Pentoxifylline (PTX), (3,7-dimethyl-1-(5-oxohexyl)xanthine) was less potent with an IC50 = 100 microM. In vivo, there was a rank order potency on serum TNF levels in LPS challenged D-gal sensitized mice. BRL 61063 inhibited TNF production with an ID50 of 0.1 mg/kg, rolipram at 1 mg/kg, and PTX at 200 mg/kg. Thus, BRL 61063 is 2,000 times more potent than PTX in reducing TNF serum levels in this model. Interestingly, TNF is implicated as having a central pathogenic role in the LPS/D-gal model, since survival of animals correlated directly with reduction of serum TNF levels for all three compounds tested. It is proposed that potent inhibitors of TNF may have therapeutic activity in disease states where TNF appears to play a role in the pathogenesis of the disease.

Effects of pyridinyl imidazole compounds on murine TNF-alpha production.

The effects of SK&F 86002 and other pyridinyl imidazole compounds on murine cytokine production were investigated. In vitro, SK&F 86002 inhibited LPS stimulated TNF-alpha production by the RAW 264.7 cell line and by oil elicited peritoneal macrophages with an IC50 of 5 microM. In general, the activity was reflective of previous results obtained with human monocytes as SK&F 86002 and its analogs demonstrated identical rank order potency for TNF-alpha inhibition in both species. These compounds also inhibited TNF-alpha in vivo in a murine model of endotoxin shock. Following oral administration, SK&F 86002 and its analogs reduced serum TNF-alpha levels by > 80% and afforded 100% protection from lethality. In contrast, tenidap, a novel anti-inflammatory drug, had minimal to no effect on murine TNF-alpha production in the same assays. These data further extend the pharmacological profile of the pyridinyl imidazoles by demonstrating that these compounds potently inhibit murine TNF-alpha production both in vitro and in vivo.

Beneficial effects of SK&F 105809, a novel cytokine-suppressive agent, in murine models of endotoxin shock.

SK&F 105809 is a structurally novel dual inhibitor of lipoxygenase and cyclooxygenase-mediated arachidonic acid (AA) metabolism, which has demonstrated antiinflammatory activity in rodent models of inflammation. In addition, the active metabolite of this compound, SK&F 105561, has been shown in vitro to inhibit the production of the inflammatory cytokine interleukin-1 (IL-1) in human monocytes stimulated with lipopolysaccharide (LPS). We report here that in vitro SK&F 105561 also blocks the production of tumor necrosis factor (TNF) from human monocytes (IC50 0.8-3 microM). Furthermore, in a murine model of endotoxin shock in which animals are injected with LPS in combination with D-galactosamine (D-gal), SK&F 105809 (10, 30, and 100 mg/kg p.o.), delivered 30 min prior to LPS/D-gal, caused a dramatic reduction in serum TNF (40-90%) and protected the animals from the lethal effects of this treatment. Similar results were obtained in a second model of endotoxin shock in which mice were sensitized with Propionibacterium acnes 10 days prior to LPS injection. In this system 100-fold higher levels of serum TNF are elicited than with the LPS/D-gal model. Treatment with SK&F 105809 (30 and 100 mg/kg p.o.) delivered 30 min prior to LPS resulted in 90-100% inhibition of serum TNF. Protection from the lethal effects of LPS was observed at these doses in the P. acnes/LPS model.

Inhibition of lymphoproliferative responses by SK&F 105685, a novel anti-arthritic agent.

SK&F 105685 (N,N-Dimethyl-8,8-dipropyl-2-azaspiro[4.5]decane-2-propanamine+ ++ dihydrochloride) is a novel azaspirane with beneficial activity in animal models of autoimmune diseases such as adjuvant-induced arthritis and experimental autoimmune encephalomyelitis in the Lewis rat and lupus-like disease in the MRL mouse. The effect of SK&F 105685 on the proliferation of rat lymphoid cells was examined in vitro. The compound inhibited the proliferative response of spleen, thymus and lymph node cells to the mitogen concanavalin A (Con A) in a dose-dependent manner but had little or no effect on the mitogenic response of peripheral blood lymphocytes. Although less potent than cyclosporin A, SK&F 105685 was able to inhibit the proliferation of spleen cells stimulated with PMA and ionomycin or the mitogens phytohemagglutinin (PHA), Con A and pokeweed mitogen (PWM). Relatively early event(s) in cell proliferation were affected by SK&F 105685 since delaying addition of the drug by 24 to 48 hours after Con A stimulation of rat spleen cells resulted in reduced levels of suppression. The mode of action of SK&F 105685 appeared to differ from that of cyclosporin A or rapamycin. Unlike cyclosporin A, SK&F 105685 did not affect IL-2 production by Con A-stimulated spleen cells or the IL-2-producing Jurkat cell line, but, like rapamycin, the compound significantly reduced the IL-2-induced proliferation of rat ConA blasts. These results suggest that inhibition of lymphocyte proliferation by SK&F 105685 may require the activity of an intermediate effector cell(s) present in susceptible populations such as cells from the spleen, thymus, lymph nodes and Con A blast preparations but absent or present in low numbers in resistant populations such as peripheral blood cells. Indomethacin and NG-monomethyl-L-arginine (NGMMA), a competitive inhibitor of nitric oxide synthase, were both unable to relieve SK&F 105685-induced suppression of splenic Con A responses thereby ruling out a role for the production of prostaglandins or nitric oxide by macrophages as an intermediate in drug-mediated suppression. In summary, SK&F 105685 was unable to inhibit lymphoproliferative responses by a mechanism distinct from that of cyclosporin A or rapamycin and which appears to involve regulation of cellular interactions rather than a direct effect on responding lymphocytes.

Serum F-protein concentration following halothane or isoflurane anaesthesia.

We have investigated the prevalence of hepatic injury following uncomplicated anaesthesia using a sensitive and specific marker of hepatic damage, the serum F-protein concentration. The median variation in serum F-protein in fit adults over six days is 16 ng/ml, minimum 0 ng/ml, maximum 36 ng/ml. A significant rise in serum F-protein was demonstrated six days following anaesthesia and surgery, but not earlier after 3 or 24 h. There was no significant difference between patients who received halothane (n = 12) or isoflurane (n = 13). These changes were not related to duration of anaesthesia, quantity of delivered volatile agent or mode of ventilation. Hepatocellular damage may occur following anaesthesia for minor surgery.

Induction of non-specific suppressor cells and myeloregulatory effects of an immunomodulatory azaspirane, SK&F 105685.

Administration of the immunosuppressive agent, SK&F 105685, has demonstrated immunosuppressive activity in several animal models of autoimmunity such as adjuvant arthritis and experimental autoimmune encephalomyelitis. The mechanism of action of SK&F 105685 in these autoimmune disease models appears to be the induction of non-specific suppressor cells (SC) detected in the spleen and bone marrow of treated animals. In this study we have examined the kinetics of SC appearance in the spleen and bone marrow following treatment with 30 mg/kg/day, p.o., for 1-6 days. SC activity was apparent following a single dose and increased with successive treatments. Treatment with SK&F 105685 also resulted in significantly enhanced myelopoiesis as measured by a 128% increase in the frequency of bone marrow myeloid progenitors (CFU-GM). Mechanistic studies indicated that in vitro treatment of bone marrow stromal cell cultures with SK&F 105685 upregulated the production of colony stimulating activity (CSA) detectable in a rat CFU-GM assay. Further, in vitro studies revealed that the SC in the bone marrow or spleens of SK&F 105685-treated rats admixed with normal marrow cells inhibited CFU-GM formation at a six-fold less cell concentration than cells obtained from control rats. These in vitro results suggest that the SK&F 105685-induced myelopoiesis is regulated by the subsequent generation of SC.

Protective effect of SK&F 86002, a novel dual inhibitor of arachidonic acid metabolism, in murine models of endotoxin shock: inhibition of tumor necrosis factor as a possible mechanism of action.

The effect of a new, structurally novel, dual inhibitor of arachidonic acid (AA) metabolism, SK&F 86002, was studied in two murine models of endotoxin shock. The first model was the injection of C57BL/6 mice with lipopolysaccharide (LPS) in combination with D-galactosamine (D-gal), which resulted in death within 6-48 hr. Treatment of these mice with SK&F 86002 (100 mg/kg, p.o.) 30 min to 2 hr prior to the administration of D-gal and LPS protected the animals from mortality. Protection was also provided by treatment with the corticosteroid dexamethasone, whereas only partial protection was afforded by the dual inhibitor of AA metabolism phenidone and the cyclooxygenase inhibitors naproxen and indomethacin. In a similar dosing protocol, SK&F 86002 also protected mice in a second endotoxin shock model in which mice sensitized with Proprionibacterium acnes received LPS 10 days later. Moreover, partial protection was obtained when SK&F 86002 was administered therapeutically after LPS injection. The administration of SK&F 86002 to P. acnes/LPS-treated mice decreased serum levels of tumor necrosis factor (TNF), which was not observed following naproxen or indomethacin treatment. Consistent with the role of TNF in this model of endotoxin shock was the observation that treatment with antibodies against TNF also prevented or reduced mortality.