[Functional results of bipolar hemiarthroplasty in patients over 65 years of age with intracapsular hip fractures]. / Resultados funcionales de hemiartroplastía bipolar en pacientes mayores de 65 años con fracturas intracapsulares de cadera.

INTRODUCTION: Bipolar hemiarthroplasty is an alternative for the treatment of displaced femoral neck fractures in elderly patients with low functional demand and associated comorbidities. The goal was to describe functionality in patients over 65 years of age with intracapsular fractures of the hip. MATERIAL AND METHODS: Retrospective review of patients over 65 years of age between January 2012 and May 2017. It was evaluated with the Harris Hip Score (HHS) and Oxford scale at six months and the year after surgery. Complications and mortality were documented during the first post-surgical year. RESULTS: 48 cases (12 men; 36 women), average age of 80.8 ± 7.7 years. Most common diseases: high blood pressure (77.1%), osteoporosis (37.5%), diabetes (33.3%) hypothyroidism (29.2%). The percentage of associated postoperative complications was 8.3% (four cases). The median HHS at six and 12 months was: 90.5 (DE: 77.5-96.0) and 96 (DE: 92-98), respectively. The Oxford scale was 45.5 (DE: 38.5-48.0) at six months and 47.0 (DE: 43.5-48.0) per postoperative year. 4.2% (two cases) died during the first post-surgical year and none were associated with the procedure. DISCUSSION: HA provides good functional outcomes in patients over 65 years of age, with a low rate of complications. No mortality associated with the surgical procedure was observed in our series and in the short term.

INTRODUCCIÓN: La hemiartroplastía bipolar (HA) es una alternativa para el tratamiento de las fracturas desplazadas de cuello femoral en pacientes de edad avanzada con baja demanda funcional y comorbilidades asociadas. El objetivo fue describir la funcionalidad en pacientes mayores de 65 años con fracturas intracapsulares de cadera tratados con HA. MATERIAL Y MÉTODOS: Revisión retrospectiva de pacientes mayores de 65 años entre Enero de 2012 y Mayo de 2017. Se evaluó con la escala de Harris (HHS) y Oxford a los seis meses y al año posterior a la cirugía. Se documentaron las complicaciones y la mortalidad durante el primer año postquirúrgico. RESULTADOS: 48 casos (12 hombres; 36 mujeres), edad promedio de 80.8 ± 7.7 años. Comorbilidades más frecuentes: hipertensión arterial (77.1%), osteoporosis (37.5%), diabetes (33.3%) e hipotiroidismo (29.2%). El porcentaje de complicaciones postoperatorias asociadas fue de 8.3% (cuatro casos). La mediana del HHS a los seis y 12 meses fue de: 90.5 (DE: 77.5-96.0) y 96 (DE: 92-98), respectivamente. La escala de Oxford fue de 45.5 (DE: 38.5-48.0) a los seis meses y de 47.0 (DE: 43.5-48) al año postoperatorio. El 4.2% (dos casos) fallecieron durante el primer año postquirúrgico y ninguno estuvo asociado al procedimiento. DISCUSIÓN: La HA ofrece buenos resultados funcionales en pacientes mayores de 65 años, con una tasa baja de complicaciones. En nuestra serie y en el corto plazo no se observó mortalidad asociada al procedimiento quirúrgico.

Biodegradable multi-walled carbon nanotubes trigger anti-tumoral effects.

Carbon nanotubes are of huge biotechnological interest because they can penetrate most biological barriers and, inside cells, can biomimetically interact with the cytoskeletal filaments, triggering anti-proliferative and cytotoxic effects in highly dividing cells. Unfortunately, their intrinsic properties and bio-persistence represent a putative hazard that relapses their application as therapies against cancer. Here we investigate mild oxidation treatments to improve the intracellular enzymatic digestion of MWCNTs, but preserving their morphology, responsible for their intrinsic cytotoxic properties. Cell imaging techniques and confocal Raman spectroscopic signature analysis revealed that cultured macrophages can degrade bundles of oxidized MWCNTs (o-MWCNTs) in a few days. The isolation of nanotubes from these phagocytes 96 hours after exposure confirmed a significant reduction of approximately 30% in the total length of these filaments compared to the control o-MWCNTs extracted from the cell culture medium, or the intracellular pristine MWCNTs. More interestingly, in vivo single intratumoral injections of o-MWCNTs triggered ca. 30% solid melanoma tumour growth-inhibitory effects while displaying significant signs of biodegradation at the tumoral/peri-tumoral tissues a week after the therapy has had the effect. These results support the potential use of o-MWCNTs as antitumoral agents and reveal interesting clues of how to enhance the efficient clearance of in vivo carbon nanotubes.

Dilated intercellular spaces in subtypes of gastroesophagic reflux disease.

BACKGROUND: Dilatation of the intercellular spaces by electron microscopy has been considered as an early morphological marker of tissue injury in gastroesophageal reflux disease. The degree of dilatation in Barret's esophagus in currently unknown. OBJECTIVES: To determine the frequency of dilated intervellular spaces in Barrett's esophagus. MATERIAL AND METHODS: Cross-sectional and prospective analysis of consecutive patients with gastroesophageal reflux disease. We selected symptomatic patients > 18 years and both sexes. Patients with recent PPI use (< 14 days), H-2 antagonists, NSAID's or previous upper GI tract surgery were excluded. VARIABLES INCLUDED: Clinical-demographic data, Carlsson-Dent score, conventional endoscopy findings, pH-metry results (in non-erosive) and normal mucosal biopsies at 2 and 5 cm above the squamocolumnar junction. Dilation of intercellular spaces was measured by electron microscopy. STATISTICS: Chi square test with a significance level 0.05 was calculated. The following four groups were compared: a) non-erosive reflux disease (n = 14); b) erosive esophagitis (n = 5); c) Barrett's esophagus (n = 13); and d) healthy controls (n = 5). RESULTS: the dilation of intervellular spaces was increasingly greater form non-erosive revlux to Barrett's esophagus and higher in biopsies taken at 5 cm than at 2 cm of the squamous columnar junction (2.72 +/- 1.35 vs. 1.71 +/- 0.48 microg) (p = 0.001). There was no difference between biopsies at 2 and 5 cm in the order groups. CONCLUSION: dilation of intercellular spaces was greater in Barrett s esophagus than in the other groups and higher at 5 cm from the squamocolumnar junction.

Tubulin cofactor B regulates microtubule densities during microglia transition to the reactive states.

Microglia are highly dynamic cells of the CNS that continuously survey the welfare of the neural parenchyma and play key roles modulating neurogenesis and neuronal cell death. In response to injury or pathogen invasion parenchymal microglia transforms into a more active cell that proliferates, migrates and behaves as a macrophage. The acquisition of these extra skills implicates enormous modifications of the microtubule and actin cytoskeletons. Here we show that tubulin cofactor B (TBCB), which has been found to contribute to various aspects of microtubule dynamics in vivo, is also implicated in microglial cytoskeletal changes. We find that TBCB is upregulated in post-lesion reactive parenchymal microglia/macrophages, in interferon treated BV-2 microglial cells, and in neonate amoeboid microglia where the microtubule densities are remarkably low. Our data demonstrate that upon TBCB downregulation both, after microglia differentiation to the ramified phenotype in vivo and in vitro, or after TBCB gene silencing, microtubule densities are restored in these cells. Taken together these observations support the view that TBCB functions as a microtubule density regulator in microglia during activation, and provide an insight into the understanding of the complex mechanisms controlling microtubule reorganization during microglial transition between the amoeboid, ramified, and reactive phenotypes.

Tubulin cofactor B plays a role in the neuronal growth cone.

Tubulin cofactors, initially identified as alpha-, beta-tubulin folding proteins, are now believed to participate in the complex tubulin biogenesis and degradation routes, and thus to contribute to microtubule functional diversity and dynamics. However, a concrete role of tubulin cofactor B (TBCB) remains to be elucidated because this protein is not required for tubulin biogenesis, and it is apparently not essential for life in any of the organisms studied. In agreement with these data, here we show that TBCB localizes at the transition zone of the growth cones of growing neurites during neurogenesis where it plays a role in microtubule dynamics and plasticity. Gene silencing by means of small interfering RNA segments revealed that TBCB knockdown enhances axonal growth. In contrast, excess TBCB, a feature of giant axonal neuropathy, leads to microtubule depolymerization, growth cone retraction, and axonal damage followed by neuronal degeneration. These results provide an important insight into the understanding of the controlling mechanisms of growth cone microtubule dynamics.

Role of cofactors B (TBCB) and E (TBCE) in tubulin heterodimer dissociation.

Tubulin folding cofactors B (TBCB) and E (TBCE) are alpha-tubulin binding proteins that, together with Arl2 and cofactors D (TBCD), A (TBCA or p14) and C (TBCC), participate in tubulin biogenesis. TBCD and TBCE have also been implicated in microtubule dynamics through regulation of tubulin heterodimer dissociation. Understanding the in vivo function of these proteins will shed light on the Kenny-Caffey/Sanjad-Sakati syndrome, an important human disorder associated with TBCE. Here we show that, when overexpressed, TBCB depolymerizes microtubules. We found that this function is based on the ability of TBCB to form a binary complex with TBCE that greatly enhances the efficiency of this cofactor to dissociate tubulin in vivo and in vitro. We also show that TBCE, TBCB and alpha-tubulin form a ternary complex after heterodimer dissociation, whereas the free beta-tubulin subunit is recovered by TBCA. These complexes might serve to escort alpha-tubulin towards degradation or recycling, depending on the cell requirements.

Native tubulin-folding cofactor E purified from baculovirus-infected Sf9 cells dissociates tubulin dimers.

Tubulin-folding cofactor E (TBCE) is an alpha-tubulin-binding protein involved in the formation of the tubulin dimer and in microtubule dynamics, through the regulation of tubulin heterodimer dissociation. TBCE has also been implicated in two important related human disorders, the Kenny-Caffey and Sanjad-Sakati syndromes. The expression of TBCE as a recombinant protein in bacteria results in the formation of insoluble inclusion bodies in the absence of denaturing agents. Although the active protein can be obtained from mammalian tissues, biochemical studies of TBCE present a special challenge. To express and purify native TBCE, a recombinant baculovirus expression system was used. Native wild-type TBCE purified from Sf9 extracts was sequentially purified chromatographically through cation exchange, hydrophobic interaction, and high-resolution gel-filtration columns. Mass spectrometric analysis identified 30% of the sequence of human TBCE. A stoichiometric excess of purified TBCE dissociated tubulin heterodimers. This reaction produced a highly unstable TBCE-alpha-tubulin complex, which formed aggregates. To distinguish between the aggregation of tubulin dimers induced by TBCE and tubulin dissociation, TBCE and tubulin were incubated with tubulin-folding cofactor A (TBCA). This cofactor captures the beta-tubulin released from the heterodimer with a stoichiometry of 1:1, as previously demonstrated. The beta-tubulin polypeptide was recovered as TBCA-beta-tubulin complexes, as demonstrated by non-denaturing gel electrophoresis and specific antibodies directed against beta-tubulin and TBCA.

Intracanal temperature rise evaluation during the usage of the System B: replication of intracanal anatomy.

AIM: To evaluate and determine intracanal temperature rises at 2 and 4 mm from the working length (WL) necessary to obtain proper replication of intracanal anatomy with gutta-percha (GP) using the System B heat source during vertical condensation. METHODOLOGY: A split-tooth model was prepared and artificial shallow depressions were cut in the buccal canal wall 2 and 4 mm from the WL. At the same level on the palatal wall holes were drilled to adapt two thermocouples. The canal was filled using GP in a vertical condensation technique by placing the System B plugger at 2 and 4 mm from the WL in groups A and B, respectively. Two control groups in which no GP was used were carried out placing the plugger 2 and 4 mm from the WL (groups A.c and B.c, respectively) and activating the heat source. Recording of temperature rise was carried out during the filling procedure for groups A and B and during activation for control groups A.c and B.c; the highest temperatures were recorded. After each filling was completed, 3 min were allowed for the GP to cool and the model divided to reveal the filling. Images of the GP were taken with a CCD camera to evaluate the presence of replication of artificial round depressions. In control groups, the temperature was recorded for 20 s after a 3 s activation of the heat source. The rise in temperature was compared between the groups individually at each level (2 or 4 mm) and statistically analysed using one-way anova and Fisher PLSD tests at 5% of significance level (P < 0.05). RESULTS: Mean temperature rises of 14 +/- 3 and 12 +/- 2 degrees C at 2 and 4 mm from the WL, respectively, were observed in group A fillings, and 4 +/- 1 and 6 +/- 1 degrees C at 2 and 4 mm, respectively, in group B fillings. Recordings at 2 mm showed significantly (P < 0.05) higher temperature rises with group A.c when compared with groups B and B.c. Replication of intracanal anatomy with GP was always found in group A fillings at both levels but only 4 mm from the WL in group B fillings. CONCLUSIONS: Positioning the plugger close to WL and a temperature rise of 6 degrees C were necessary to obtain replication of intracanal anatomy. A mean temperature rise of 4 degrees C at 2 mm from WL (group B) resulted in no replication of intracanal anatomy. Further studies simulating clinical conditions are necessary.

Transcytosis of protein through the mammalian cerebral epithelium and endothelium. III. Receptor-mediated transcytosis through the blood-brain barrier of blood-borne transferrin and antibody against the transferrin receptor.

Diferric-transferrin (Tf; 80K mol. wt.) and the OX26 antibody (150K mol. wt.) against the transferrin receptor (TfR) were evaluated in the rat at light and ultrastructural levels as potential vehicles for the blood to brain transcellular transfer (transcytosis) of native horseradish peroxidase (40K mol. wt.), which by itself does not cross the blood-brain barrier (BBB). OX26, the Fab fragment of OX26 (50K mol. wt.), and Tf complexed to two ferric ions were conjugated to HRP irreversibly in a 1:1 molar ratio. The indirect immunoperoxidase technique with OX26 as the monoclonal primary antibody applied to the surface of cryostat sections or delivered intravenously to the live rat revealed TfRs on BBB capillaries, arterioles, and venules; TfRs were absent on non-BBB vessels supplying the circumventricular organs (i.e., median eminence, choroid plexus). OX26-HRP and OX26(Fab)-HRP delivered intravenously and diferric-Tf-HRP administered into the carotid artery labeled BBB vessels throughout the CNS without discernible disruption of the BBB or extravasation of the blood-borne probes into the brain parenchyma. No reaction product for the probes was observed in sites deficient in a BBB. Each of the macromolecular conjugates was endocytosed by BBB endothelia and labeled presumptive endocytic vesicles, endosomes, and dense bodies. OX26-HRP and Tf-HRP, but not OX26(Fab)-HRP, appeared to undergo transcytosis through BBB endothelia for subsequent labeling of perivascular cells. Distinct differences in the intracellular and extracellular distributions between OX26-HRP and Tf-HRP were identified: (1) endocytosis and sequestration of blood-borne OX26-HRP within BBB endothelia were more prominent than those for diferric-Tf-HRP; (2) only OX26-HRP labeled the Golgi complex in BBB endothelia; (3) peroxidase labeling of CNS perivascular clefts and perivascular cells in rats receiving diferric-Tf-HRP was conspicuous at less than 1 h postinjection but not so in rats with blood-borne OX26-HRP at 5 min through 6 h postinjection; and (4) peroxidase-labeled CNS neurons and glial cells were identified readily in rats receiving diferric-Tf-HRP. The results suggest that the receptor-mediated, transendothelial transfer of Tf-HRP from blood to brain is more efficient and direct than that of OX26-HRP. Labeling of the Golgi complex in BBB endothelia with blood-borne OX26-HRP implies that the transendothelial transfer of OX26-HRP follows intraendothelial pathways associated with the process of adsorptive transcytosis. A diagram is provided depicting the possible intracellular and transcellular pathways within BBB endothelia available to blood-borne diferric-Tf and OX26 as vectors for delivery into the CNS of non-lipid-soluble macromolecules that otherwise are denied entry by the blood-brain fluid barriers.

Transcytosis of protein through the mammalian cerebral epithelium and endothelium. II. Adsorptive transcytosis of WGA-HRP and the blood-brain and brain-blood barriers.

Morphological evidence of the potential for adsorptive transcytosis of protein through the mammalian blood-brain fluid barriers, first reported from this laboratory in the mouse, has been confirmed and expanded upon in rats injected intravenously or into the lateral cerebral ventricle/subarachnoid space with with exogenous lectin wheatgerm agglutinin (WGA) conjugated to horseradish peroxidase (HRP). Blood-borne WGA-HRP rapidly enters cerebral endothelia by the process of adsorptive endocytosis and labels the vascular tree throughout the CNS. At 3 h post-injection and longer, WGA-HRP occupies the perivascular clefts and labels perivascular cells and basal lamina; this suspected transendothelial transfer of the lectin conjugate from blood to brain involves specific constituents of the endothelial endomembrane system of organelles (e.g., plasmalemma, vesicles, endosomes, Golgi complex). Within 6 h, reaction product is evident in extracellular clefts beyond the perivascular basal lamina and labels endocytic vesicles, endosomes, and dense bodies within cells and processes of the neuropil. Exposure of the abluminal surface of blood-brain barrier endothelia for 1-18 h to WGA-HRP delivered into the cerebral ventricles or subarachnoid space indicates blood-brain barrier endothelia do not engage in demonstrable adsorptive endocytosis at the abluminal surface. In this preparation, no endothelial organelles comparable to those sequestering blood-borne WGA-HRP are labelled with the lectin conjugate; hence, significant adsorptive transcytosis of WGA-HRP through cerebral endothelia from brain to blood is unlikely. The demonstrable difference in membrane internalization of the luminal versus abluminal plasmalemma of blood-brain barrier endothelia suggests the blood-brain barrier is polarized regarding adsorptive endocytosis of WGA-HRP. If adsorptive transcytosis of macromolecules through the blood-brain barrier does occur, the process appears unidirectional, from blood to brain but not from brain to blood. Absence of demonstrable endocytosis at the abluminal front is an enigma in the scheme of transcytosis through the blood-brain barrier from blood to brain insofar as exocytosis and endocytosis are complementary events in the cellular secretory process. This unconventional membrane behavior associated with the abluminal plasmalemma argues against a significant transcytosis of blood-borne protein through blood-brain barrier endothelia. The potential for transcytosis of macromolecules through the blood-cerebrospinal fluid barrier of choroid plexus epithelia is not as problemmatic as that through blood-brain barrier endothelia; additional evidence is provided to suggest choroid plexus epithelia participate in adsorptive endocytosis circumferentially and adsorptive transcytosis of WGA-HRP bidirectionally between the blood and cerebrospinal fluid.

Angiogenesis and the blood-brain barrier in solid and dissociated cell grafts within the CNS.

Available evidence suggests that blood vessels indigenous to solid CNS and peripheral tissues grafted to the brain are sustained and maintain the morphological and permeability characteristics they manifest in normal life. Furthermore, these vessels of graft origin anastomose (albeit not rapidly) with vessels of the surrounding host tissue predominantly at the host-graft interface and less so, or not at all, within the graft itself. For these reasons, blood-brain and brain-blood barriers, evident in the late fetal and neonatal CNS, can be expected to exist within CNS grafts placed intracerebrally or extracerebrally, providing the graft remains viable. Peripheral neural and non-neural tissues not possessing cellular barriers to circulating macromolecules do not acquire such barriers subsequent to their transplantation within the CNS. The absence of a blood-brain barrier in the adrenal gland grafted intracerebrally may be relevant for the treatment of Parkinson's disease with blood-borne therapeutics. Compared to solid tissue grafts, cell suspension grafts have the potential of becoming vascularized rapidly. That cell suspensions of neurons and of glia are supplied with BBB vessels of host origin and that the permeability characteristics of host BBB vessels are altered by a tumor cell suspension reaffirm the belief that the type of transplanted cell/tissue indeed determines the permeability characteristics of the blood vessels supplying it. The suspected immunologic privilege of the CNS is not absolute. Eventual host rejection of allografts placed within the third ventricle may be a dual consequence of the absence of a BBB at the level of the host median eminence and involvement of the minor histocompatibility complex.