MGMT-inhibitor in combination with TGF-ßRI inhibitor or CDK 4/6 inhibitor increases temozolomide sensitivity in temozolomide-resistant glioblastoma cells.

BACKGROUND: Glioblastoma (GB) remains an incurable and deadly brain malignancy that often proves resistant to upfront treatment with temozolomide. Nevertheless, temozolomide remains the most commonly prescribed FDA-approved chemotherapy for GB. The DNA repair protein methylguanine-DNA methyl transferase (MGMT) confers resistance to temozolomide. Unsurprisingly temozolomide-resistant tumors tend to possess elevated MGMT protein levels or lack inhibitory MGMT promotor methylation. In this study, cultured human temozolomide resistance GB (43RG) cells were introduced to the MGMT inhibitor O6-benzylguanine combined with temozolomide and either LY2835219 (CDK 4/6 inhibitor) or LY2157299 (TGF-ßRI inhibitor) seeking to overcome GB treatment resistance. METHODS: Treatment effects were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, western blot, cell viability, and cell cycle progression. RESULTS: Our in vitro study demonstrated that sequential treatment of O6-Benzylguanine with either LY2385219 or LY2157299-enhanced temozolomide enhanced sensitivity in MGMT+ 43RG cells. Importantly, normal human neurons and astrocytes remained impervious to the drug therapies under these conditions. Furthermore, LY2835219 has additional anti-proliferative effects on cell cycling, including induction of an RB-associated G (1) arrest via suppression of cyclin D-CDK4/6-Rb pathway. LY2157299 enhances anti-tumor effect by disrupting TGF-ß-dependent HIF-1α signaling and by activating both Smad and PI3K-AKT pathways towards transcription of S/G2 checkpoints. CONCLUSION: This study establishes the groundwork for the development of a combinatorial pharmacologic approach by using either LY2385219 or LY2157299 inhibitor plus O6-Benzylguanine to augment temozolomide response in temozolomide-resistant GB cells.

Evaluating anti-tumor activity of palbociclib plus radiation in anaplastic and radiation-induced meningiomas: pre-clinical investigations.

PURPOSE: Meningiomas are common brain tumors, the majority of which are considered benign. Despite surgery and/or radiation therapy, recurrence rates are approximately 8-10%. One likely cause is the dysregulation of cyclin D-cyclin-dependent kinases 4 and 6 (CDK4/6)-retinoblastoma (Rb) pathway, which controls the cell cycle restriction point. This pathway is commonly dysregulated in anaplastic meningioma cell lines (AM) and radiation-induced meningioma cells (RIM), making it a rational target for anti-meningioma therapy. In this study, we investigate the effect of a CDK4/6 inhibitor, palbociclib, with radiation in relevant pre-clinical models. METHODS: In vitro cell culture, ex vivo slice culture and in vivo cell line-derived orthotopic xenograft animal models of AM/RIM were utilized to assess treatment efficacy with palbociclib plus radiation. Treatment effects were examined by immunoblot, cell viability, apoptosis, and cell cycle progression. RESULTS: The in vitro and ex vivo studies demonstrate that palbociclib plus radiation treatment reduced proliferation and has additional effects on cell cycling, including induction of an RB-associated G (1) arrest in Rb+ AM and RIM cells, but not in Rb- cells. Our results also demonstrated reduced CDK4 and CDK6 expression as well as reduced E2F target gene expression (CCNA2 and CCNE2) with the combination therapy. MRI results in vivo demonstrated reduced tumor size at 5 weeks when treated with 14 days palbociclib (10 mg/kg) plus 6 Gy radiation compared to saline-treated tumors. Finally, no hepatic toxicity was found after treatments. CONCLUSION: A pre-clinical murine model provides preclinical evidence for use of palbociclib plus radiation as a therapeutic agent for Rb+ meningiomas.

Infecciones bacterianas en pacientes con Pie Diabético. Hospital Regional de Ciudad del Este, Paraguay. Año 2015 / Bacterial infections in patients with Diabetic Foot. Regional Hospital of Ciudad del Este, Paraguay. Year 2015

Infecciones bacterianas en pacientes con Pie Diabético. Hospital Regional de Ciudad del Este, Paraguay. Año 2015 RESUMEN La Organización Mundial de la Salud define el pié diabético como una "situación de infección, ulceración o también destrucción de los tejidos profundos de los pies, asociada a anormalidades neurológicas y varios grados de enfermedad vascular periférica en los miembros inferiores de pacientes con Diabetes Mellitus. El objetivo del trabajo fue identificar la prevalencia de microorganismos que causaron infecciones en pacientes diagnosticados con pie diabético atendidos en el Hospital Regional de Ciudad del Este, Paraguay, durante el año 2015. Estudio descriptivo, transversal y retrospectivo. El muestreo fue no probabilístico, por conveniencia, y se incluyeron 115 muestras correspondientes a 94 pacientes. Fue utilizada la base de datos del Laboratorio de Bacteriología del Hospital Regional de Ciudad del Este. De los 94 pacientes con pie diabético, 52% fueron hombres. El 25% de las infecciones se produjeron en pacientes con edades comprendidas entre 51 a 60 años. Fueron aislados 21 microorganismos diferentes en las 115 muestras. El 80% (75) fueron monomicrobianos, y 20% (40) polimicrobianos. Entre los Gram positivos, el microorganismo más frecuentemente aislado fue el Staphylococcus aureus 19% (22) y el Enterococcus spp. 6% (6) y entre los Gram negativos fueron la Klebsiella pneumoniae13% (16) y Acinetobacter spp. 12% (14). Los resultados de las pruebas de sensibilidad antimicrobiana demostraron que el 100% de las cepas de S. aureus fueron resistentes a la Oxacilina y alta resistencia de las cepas de K. pneumoniae a las Cefalosporinas. Las cepas de Acinetobacter spp. fueron 100% resistentes a las cefalosporinas y Piperacilina. Los microorganismos aislados y el perfil de resistencia antimicrobiana que los mismos presentan, coinciden con la bibliografía, y es muy importante implementar programas de prevención a esta patología a fin de evitar amputaciones en este tipo de pacientes. Palabras claves: bacterias; pie; diabético; hospital; Paraguay.

The World Health Organization defines the diabetic foot as a "situation of infection, ulceration or destruction of the deep tissues of the feet. It is associated with neurological abnormalities and various degrees of peripheral vascular disease in the lower limbs of patients with Diabetes Mellitus". The objective of the research was to identify the prevalence of microorganisms which caused infections in patients diagnosed with diabetic foot treated at the Regional Hospital of Ciudad del Este, Paraguay, during the year 2015. It is a descriptive, crosssectional and retrospective study. The sampling was nonprobabilistic, for convenience, and 115 samples were included corresponding to 94 patients. The database of the Department of Bacteriology of the Regional Hospital of Ciudad del Este was used. Of the 94 patients with diagnosed diabetic foot, 52% were men. 25% of the infections occurred in patients with ages ranging from 51 to 60 years. 21 microorganisms were isolated in the 115 different samples. 80% (75) were monomicrobial, and 20% (40) polymicrobial. The most isolated bacterium in this group of bacteria was the Staphylococcus aureus (19%). Among the Gram positive bacilli, the most frequently isolated microorganism was Staphylococcus aureus, 19% (22) and Enterococcus spp. 6% (6) and among Gram negatives: Klebsiella pneumoniae 13% (16) and Acinetobacter spp. 12% (14). The results of the sensitivity tests demonstrated that 100% of the strains of S. aureus were resistant to Oxacillin and 100% of the strains of K. pneumoniae showed resistance to cefhems. 100% of the strains of Acinetobacter spp were resistant to Cefotaxime and Piperacillin. The isolated microorganisms and the antimicrobial resistance profile they present coincide with the literature, and it is of most importance to implement prevention programs regarding this pathology in order to avoid amputations in this type of patients. Key words: bacteria; foot; diabetic; h Palabras claves: bacterias; pie; diabético; hospital; Paraguay.

Validation of a reaction volume reduction protocol for analysis of Y chromosome haplotypes targeting DNA databases.

The use of Y chromosome haplotypes, important for the detection of sexual crimes in forensics, has gained prominence with the use of databases that incorporate these genetic profiles in their system. Here, we optimized and validated an amplification protocol for Y chromosome profile retrieval in reference samples using lesser materials than those in commercial kits. FTA® cards (Flinders Technology Associates) were used to support the oral cells of male individuals, which were amplified directly using the SwabSolution reagent (Promega). First, we optimized and validated the process to define the volume and cycling conditions. Three reference samples and nineteen 1.2 mm-diameter perforated discs were used per sample. Amplification of one or two discs (samples) with the PowerPlex® Y23 kit (Promega) was performed using 25, 26, and 27 thermal cycles. Twenty percent, 32%, and 100% reagent volumes, one disc, and 26 cycles were used for the control per sample. Thereafter, all samples (N = 270) were amplified using 27 cycles, one disc, and 32% reagents (optimized conditions). Data was analyzed using a study of equilibrium values between fluorophore colors. In the samples analyzed with 20% volume, an imbalance was observed in peak heights, both inside and in-between each dye. In samples amplified with 32% reagents, the values obtained for the intra-color and inter-color standard balance calculations for verification of the quality of the analyzed peaks were similar to those of samples amplified with 100% of the recommended volume. The quality of the profiles obtained with 32% reagents was suitable for insertion into databases.

Burnout syndrome in oral and maxillofacial surgeons: a critical analysis.

The aim of this study was to determine the prevalence of burnout syndrome among Brazilian oral and maxillofacial surgeons and its relationship with socio-demographic, clinical, and habit variables. The sample of this study comprised 116 surgeons. The syndrome was quantified using the Maslach Burnout Inventory (General Survey), which defines burnout as the triad of high emotional exhaustion, high depersonalization, and low personal accomplishment. The criteria of Grunfeld et al. were used to evaluate the presence of the syndrome (17.2%). No significant differences between the surgeons diagnosed with and without the syndrome were observed according to age (P=0.804), sex (P=0.197), marital status (P=0.238), number of children (P=0.336), years of professional experience (P=0.102), patients attended per day (P=0.735), hours worked per week (P=0.350), use of alcohol (P=0.148), sports practice (P=0.243), hobbies (P=0.161), or vacation period per year (P=0.215). Significant differences occurred in the variables sex in the emotional exhaustion subscale (P=0.002) and use or not of alcohol in the personal accomplishment subscale (P=0.035). Burnout syndrome among Brazilian surgeons is average, showing a low personal accomplishment.

Development of temporomandibular joint ankylosis in rats using stem cells and bone graft.

64 male Wistar rats were used: 24 for the removal of stem cells, 4 as a control group, and 36 for the experiment, in which either stem cells or bone graft was used. The rats were divided into groups according to the type of procedure and time span (15, 30 or 60 days). The joints were submitted to histological study in order to score the ankylosis. The mean differences between initial and final maximal mouth opening (MMO) were gradually increased from 15 to 60 days, for all times of evaluation for both groups, being statistically significant at 15 days (p=0.045) in the bone-graft group. When both groups were compared, the mean differences between initial and final MMO were statistically significant at 15 days (p=0.018) and 30 days (p=0.029). In relation to the histological scores, in the bone-graft group almost all animals had intra-articular fibrosis at all times of evaluation (n=17). In the stem-cell group, there was new bone at 15 days (n=4), 30 days (n=3) and 60 days (n=4). The study model permitted the development of fibrous ankylosis in the majority of animals for both groups and no bony bridge was observed.

Treatment of chronic mandibular dislocations using a new miniplate.

Temporomandibular joint (TMJ) dislocation is defined as an excessive forward movement of the condyle beyond the articular eminence with complete separation of the articular surfaces and fixation in that position. The aim of this study was to describe a modified miniplate designed for treating chronic mandibular dislocations and evaluate the results of its placement in one patient, who was followed for 18 months. The treatment of chronic mandibular dislocation using this modified miniplate was shown to be efficient in relation to the postoperative maximal mouth opening, recurrence and articular function.

Treatment of chronic mandibular dislocations using miniplates: follow-up of 8 cases and literature review.

Temporomandibular joint (TMJ) dislocation is an excessive forward movement of the condyle beyond the articular eminence with complete separation of the articular surfaces and fixation in that position. This study reports 8 cases using miniplates for chronic mandibular dislocations, evaluates the results and critically reviews the literature. The sample was obtained from the records of the Oswaldo Cruz University Hospital and comprises patients undergoing chronic mandibular dislocation treatment using 2.0mm titanium miniplates between August 2002 and March 2004. Pre- and postoperative assessment included a thorough history and physical examination to determine the maximal mouth opening, presence of pain and sounds, frequency of luxations, recurrence rate and presence of facial nerve paralysis. The mean maximal mouth opening preoperatively was 42.75+/-11.53 mm and was 45.62+/-8.52 mm postoperatively. There was no facial nerve paralysis. Miniplate fracture was observed in 2 cases and there was one recurrence. Treating chronic mandibular dislocation using miniplates was shown to be efficient in relation to postoperative maximal mouth opening, recurrence and articular function, however, the possibility of the miniplate fracturing must be considered.

Cellular autoreactivity against heat shock protein 60 in renal transplant patients: peripheral and graft-infiltrating responses.

Autoreactivity to heat shock protein 60 (Hsp60) has been implicated in the pathogenesis and regulation of chronic inflammation, especially in autoimmune diseases. In transplantation, there is a lack of information regarding the cytokine profile and specificity of cells that recognize self-Hsp60 as well as the kinetics of autoreactivity following transplantation. We studied the cellular reactivity of peripheral and graft-infiltrating lymphocytes against Hsp60 in renal transplant patients. Cytokine production induced by this protein in peripheral blood mononuclear cells indicated a predominance of interleukin (IL)-10 during the late post-transplantation period, mainly in response to intermediate and C-terminal peptides. Patients with chronic rejection presented reactivity to Hsp60 with a higher IL-10/interferon (IFN)-gamma ratio compared to long-term clinically stable patients. Graft-infiltrating T cell lines, cocultured with antigen-presenting cells, preferentially produced IL-10 after Hsp60 stimulation. These results suggest that, besides its proinflammatory activity, autoreactivity to Hsp60 in transplantation may also have a regulatory role.