Platelet Rich Plasma and Culture Configuration Affect the Matrix Forming Phenotype of Bone Marrow Stromal Cells

We aim to examine the influence of platelet rich plasma (PRP) and spatial cues in cartilage/bone matrix forming proteins, and to evaluate the mitotic and chemotactic potential of PRP on human mesenchymal stem cells (hMSCs). Directed cell migration towards PRP gradients was assessed in chemotactic chambers, and recorded by time-lapse microscopy. hMSCs cultured in three-dimensional (3D) scaffolds were visualized by scanning electron microscopy; Hoechst dye was used to confirm cell confluence in 3D-constructs and monolayers before experimental treatment. MSCs were treated with 10% PRP lysate or 10% PRP lysate supplemented with TGF-β-based differentiation medium. The expression of cartilage (COL2A1, Sox9, ACAN, COMP), and bone (COL1A1, VEGF, COL10A1, Runx2) fundamental genes was assessed by real time PCR in monolayers and 3D-constructs. PRP had mitotic (p <.001), and chemotactic effect on hMSCs, Ralyleigh test p = 1.02E - 10. Two and three-week exposure of MSCs to PRP secretome in 3Dconstructs or monolayers decreased Sox9 expression (p <0.001 and p = 0.050) and COL2A1, (p = 0.011 and p = 0.019). MSCs in monolayers exposed to PRP showed increased ACAN (p = 0.050) and COMP (p <0.001). Adding TGF-β-based differentiation medium to PRP increased COMP, and COL2A1 expression at gene and protein level, but merely in 3D-constructs, p <0.001. TGF-β addition to monolayers reduced Sox9 (p <0.001), aggrecan (p = 0.004), and VEGF (p = 0.004). Cells exposed to PRP showed no changes in hypertrophy associated genes in either monolayers or 3Dconstructs. Our study suggests hMSCs have high-degree of plasticity having the potential to change their matrix-forming phenotype when exposed to PRP and according to spatial configuration.

Uso del colgajo pediculado miofascial temporal en cirugía reconstructiva posterior a maxilectomías con componente horizontal / Use of pediculated temporal myofascial flaps in reconstruction surgery after maxilectomies with horizontal components

Las resecciones oncológicas del tercio medio de la cara y órbita causan significativos defectos funcionales como el habla, deglución, masticación y estéticos, así como un alto nivel de trauma psicológico y físico para el paciente y sus familiares. Actualmente, los colgajos libres brindan una excelente opción reconstructiva, pero que requiere de instituciones especializadas en el área, con un recurso humano multidisciplinario así como equipos y materiales costosos que hacen difícil su ejecución. Nos proponemos evaluar la utilidad del colgajo pediculado miofascial temporal como alternativa reconstructiva en el cierre de fístulas oro-antrales y oro-nasales generadas en cirugías de senos paranasales que requieren de algún tipo de resección del componente horizontal. De un total de 43 maxilectomías realizadas en el servicio de cabeza y cuello del IOLR entre enero 2008 - diciembre 2011; 17 pacientes fueron sometidos al procedimiento de reconstrucción con colgajo miofascial temporal siguiendo los criterios de inclusión; se utilizaron dos tipos de análisis estadísticos, el primero descriptivo donde se calculan medidas de posición; el segundo un análisis de significación o validación estadística basados en la distribución t-Student; todos los contrastes de hipótesis se realizarón con un a=0,05 es decir una confianza del 95%. De los 17 pacientes evaluados, solo 4 (23,5%) de ellos presentaron dehiscencias. El colgajo pediculado miofascial temporal es una herramienta útil y eficaz para la reconstrucción del componente horizontal en maxilectomías independientemente de la extensión de la resección.

Oncological resections of the middle third of the face and orbit cause significant functional speech, swallowing, chewing and aesthetic defects, as well as a high level ofphysical and psychological trauma for the patient and their families. Currently, free flaps have provided an excellent reconstructive option, but it requires specialized institutions in the area, with a multidisciplinary human resource aswell as equipment and expensive materials that make it difficult for their implementation. We intend to evaluate the usefulness of the flap pediculated temporal myofascial as reconstructive alternative closure of fistulae oro-antrales and oro-nasales generated in surgery of par nasal sinuses that require some sort of resection of the horizontal component. Of a total of 43 maxillectomy made in the head and neck of the IOLR service between January 2008 - December 2011; 17 patients were subjected to the procedure of temporary myofascial flap reconstruction following the inclusion criteria; We used two types of statistical analysis, the first descriptive where are calculated measures of position; the second an analysis of significance or statistical validation based on the distribution of t-Student; all the contrasts of hypothesis will be done witha a= 0.05 is a 95% confidence. Of 17 patients evaluated, only 4 (23.5%) of them presented dehiscences. The pediculated temporal myofascial flap is a useful and effective tool for the reconstruction of the horizontal component in maxillectomy regardless of the extent of resection.

MHC-restricted and -unrestricted CD8 T cells: an evolutionary perspective.

BACKGROUND: In mammals, cytotoxic CD8 T cells are crucial effectors of a typical adaptive cellular immune response. They recognize and kill cells that express at their surface antigenic peptides complexed to major histocompatibility complex (MHC) class I molecules. Although T cells (undefined as to CD determinants) and associated in vitro cytotoxic activity have been described in a few amphibian and teleost species, their in vivo functions have yet to be characterized. METHODS AND RESULTS: CD8 function has been investigated in the frog Xenopus by antibody depletion, skin allografting, and tumor transplantation. Injection of adult frogs with anti-Xenopus CD8 monoclonal antibody effects transient CD8 T-cell depletion in vivo that correlates with delayed rejection of MHC-disparate skin allografts and an impaired immune response against transplanted syngeneic MHC class I-negative tumors. CONCLUSIONS: For the first time, CD8 T cells have been shown to be involved in acute skin allograft rejection in an ectothermic vertebrate. Our data also suggest that, at least in Xenopus, T cells that express a CD8 epitope may be effectors in MHC-unrestricted anti-tumor responses.

Autoimmune diseases: a leading cause of death among young and middle-aged women in the United States.

OBJECTIVES: This study assessed the effect of autoimmune diseases on mortality among women. METHODS: Counts of autoimmune disease deaths were compared with frequencies of the 10 "official" leading causes of death among women in the United States in 1995. RESULTS: Autoimmune disease deaths exceeded the frequency of the 10th leading cause in every age category of women younger than 65 years and exceeded that for the eighth leading cause in the 15 to 24, 25 to 44, and 45 to 64 years age groups. CONCLUSIONS: Autoimmune diseases constitute a leading cause of death among young and middle-aged women. This fact is obscured by current methods used to identify leading causes.

Melatonin suppression by light in euthymic bipolar and unipolar patients.

BACKGROUND: Previous studies have suggested that bipolar patients are supersensitive to light suppression of melatonin and that this may be a trait marker for genetic vulnerability. The present study was an attempt to replicate and extend this observation. Propranolol hydrochloride effects were compared with light effects because of the documented influence of beta-adrenergic receptors on melatonin production. Nighttime levels of corticotropin and cortisol were also examined as potential trait vulnerability markers. METHODS: Melatonin levels in euthymic bipolar patients (n= 29) were tested before and after 500-lux light was administered between 2 and 4 AM and on a separate night in the dark. Results were compared with those of a group of patients with unipolar depression (n= 24) and with those of a group of non-psychiatrically ill control subjects (n= 50). Lithium effects and propranolol effects were tested in subgroups. RESULTS: No group differences were seen in light suppression among bipolar patients, unipolar patients, and controls; an analysis of the whole group did not reveal differences in propranolol effect, differences in corticotropin or cortisol levels, or evidence for a lithium effect. However, patients with bipolar I affective disorder showed the following: (1) significantly lower melatonin levels on the light night, at baseline and following light exposure; and (2) a later peak time for melatonin on the dark night. CONCLUSIONS: The general hypothesis of increased light sensitivity in bipolar patients was not supported. However, melatonin secretion abnormalities were confirmed in the subgroup with bipolar I disorder. Further assessments of circadian rhythm disruption as a vulnerability marker in bipolar illness are indicated.

Mechanisms for the release and redistribution of 2,4,5,2',4',5'-hexachlorobiphenyl (6-CB) from hepatic tissues in the rat.

The translocation of 6-[14C]CB from rat hepatic tissues to various media was studied employing in situ hepatic perfusion and primary hepatocyte culture techniques. 6-[14C]CB release from the hepatic tissues of female rats pretreated with 2 microCI 6-CB was dependent on the relative proportion of perfusate buffer components. Approximately 10% of hepatic 6-CB was released into buffer containing either 4% BSA or 4% BSA and 100 mg/dl exogenous human very low density lipoproteins (VLDL). 6-CB release was significantly increased under simulated hyperlipidemic conditions (400 mg VLDL/dl). Release declined when BSA was eliminated or replaced with Dextran. The distribution of 6-CB between the triacylglycerol (TG)-rich VLDL and the protein buffer components was found to be dependent on the ratio of TG:protein. Under hyperlipidemic perfusate conditions, approximately 83% of the 6-CB associated with the BSA fraction. Under normolipidemic conditions, 99% of the 6-CB associated with BSA. The concentration of 6-CB in TG was greatly increased under hyperlipidemic conditions. Thus, 6-CB distribution under simulated normolipidemic conditions could not be explained by saturation of the VLDL fraction. Approximately 15% of 6-CB was released from hepatocytes prepared from late pregnant or age-matched control rats. Eighty percent of 6-CB was associated with VLDL secreted from hepatocytes. The TG:protein ratio in culture media was approximately 1: 6 while ratios of 1:20 or 1:600 occurred in the perfusion studies. These data suggest that 6-CB may be released from hepatic tissues in association with newly synthesized TG, but that once in the circulation, its distribution is dependent on the ratio of TG to protein present.

The effect of monooxygenase inducing agents on the incorporation of [35S]methionine into hepatic microsomal protein of rainbow trout (Salmo gairdneri).

Treatment of rainbow trout (Salmo gairdneri) with 150 mg/kg BNF resulted in an increase in hepatic microsomal monooxygenase activity as assessed by ECOD and EROD when compared to those activities in corn oil-pretreated animals. Administration of 100 mg/kg 2,4,5,2',4',5'-hexachlorobiphenyl (6-CB) to trout had no significant effect on these catalytic activities or on BeND. The amount of radioactivity in hepatic microsomes at 24, 48 or 72 hr following the administration of 75 muCi of [35S]methionine was consistently higher in animals pretreated with BNF than in those treated with corn oil or 6-CB. Autoradiography/fluorography of electrophoretograms demonstrated the appearance of at least three radiolabeled bands in the 50,000-60,000 mol. wt range in solubilized microsomes from BNF-treated fish which were not present in microsomes from control animals or fish treated with 6-CB. These data indicate that the stimulation of hepatic microsomal catalytic activities observed following the administration of 3-MC-type agents to rainbow trout is due, at least in part, to induction of enzyme(s) rather than activation of existing enzyme(s). These results further support the observation that fish appear to be non-responsive to phenobarbital-type inducing agents.