CNS myelin induces regulatory functions of DC-SIGN-expressing, antigen-presenting cells via cognate interaction with MOG.

Myelin oligodendrocyte glycoprotein (MOG), a constituent of central nervous system myelin, is an important autoantigen in the neuroinflammatory disease multiple sclerosis (MS). However, its function remains unknown. Here, we show that, in healthy human myelin, MOG is decorated with fucosylated N-glycans that support recognition by the C-type lectin receptor (CLR) DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) on microglia and DCs. The interaction of MOG with DC-SIGN in the context of simultaneous TLR4 activation resulted in enhanced IL-10 secretion and decreased T cell proliferation in a DC-SIGN-, glycosylation-, and Raf1-dependent manner. Exposure of oligodendrocytes to proinflammatory factors resulted in the down-regulation of fucosyltransferase expression, reflected by altered glycosylation at the MS lesion site. Indeed, removal of fucose on myelin reduced DC-SIGN-dependent homeostatic control, and resulted in inflammasome activation, increased T cell proliferation, and differentiation toward a Th17-prone phenotype. These data demonstrate a new role for myelin glycosylation in the control of immune homeostasis in the healthy human brain through the MOG-DC-SIGN homeostatic regulatory axis, which is comprised by inflammatory insults that affect glycosylation. This phenomenon should be considered as a basis to restore immune tolerance in MS.

The release of cytokines by macrophages is not affected by myelin ingestion.

Macrophages play an important role in demyelination in multiple sclerosis (MS). Activated macrophages ingest myelin particles, thereby acquiring a foamy appearance. Foamy macrophages in MS lesions were described as being anti-inflammatory. Therefore, these cells might play a role in modulating the inflammatory state of an active lesion. Here, we investigated the mechanism by which myelin uptake leads to skewing of macrophages toward an anti-inflammatory phenotype. Macrophages were incubated with myelin, leading to the development of foamy macrophages. Afterwards, the cells were stimulated with the TLR-4 ligand lipopolysaccharide (LPS), and cytokine production was determined. Interestingly, foamy macrophages appeared to have a reduced cytokine secretion and were LPS insensitive only when generated with one of the myelin preparations. The factor responsible for the different outcomes between different myelin batches turned out to be LPS. We demonstrated that LPS contamination induced insensitivity to LPS in foamy macrophages. On the contrary, foamy macrophages generated in the presence of LPS-free myelin were able to secrete cytokines upon activation. To conclude, myelin-laden macrophages were not LPS insensitive, indicating that they had not acquired an anti-inflammatory phenotype.

Expression of aberrantly glycosylated tumor mucin-1 on human DC after transduction with a fiber-modified adenoviral vector.

BACKGROUND: DC-presenting tumor Ag are currently being developed to be used as a vaccine in human cancer immunotherapy. To increase chances for successful therapy it is important to deliver full-length tumor Ag instead of loading single peptides. METHODS: In this study we used a fiber-modified adenoviral vector (rAd5F35) containing full-length tumor Ag cDNA to transduce human monocyte (Mo)-derived DC in vitro. Cells were efficiently transduced and survived for at least 3 days after adenoviral transduction. Phenotype and function after maturation of Mo-DC were not impaired by infection with adenovirus particles. Expression of the tumor-associated Ag mucin-1 (MUC1) was detected using MAb defining different MUC1 glycoforms. RESULTS: Non-transduced mature Mo-DC express endogenous MUC1 with normal glycosylation. After transduction with the rAd5F35-MUC1 adenoviral vector, Mo-DC also expressed MUC1 with tumor-associated glycosylation (Tn and T glycoforms), although no changes in mRNA levels of relevant glycosyltransferases could be demonstrated. DISCUSSION: The presence of aberrantly glycosylated MUC1 may influence Ag presentation of the tumor glycoforms of MUC1 to immune cells, affecting tumor cell killing. These findings could be highly relevant to developing strategies for cancer immunotherapy based on DC vaccines using MUC1 as tumor Ag.

Transfusion of post-operative shed blood: laboratory characteristics and clinical utility.

Increased awareness of the potential hazards of allogenic blood transfusion, such as incompatibility reactions, metabolic and immunologic disorders, or transmission of viral diseases, has led to an emphasis on allogeneic blood alternatives. For orthopaedic surgery, several autologous transfusion modalities have emerged as alternatives to allogeneic blood transfusion, avoiding its immunomodulatory effects. Among them, transfusion or return of post-operative salvaged shed blood has become popular in major orthopaedic procedures. However, although the effectiveness of this blood-saving method is well documented, several authors have questioned its safety and recommended the use of washed blood. Therefore, this review analyses the haematologic characteristics of unwashed filtered shed blood, including metabolic status and survival of red blood cells, the components of the haemostatic system, the content of fat particles, bacterial and tumour cells and the possibility of their removal, the content of inflammatory mediators, and the effects on the patient's immune system. From data reviewed in this paper, it can be concluded that post-operative salvage of blood seems to be an excellent source of functional and viable red cells without many of the transfusion-related risks and with some immuno-stimulatory effects. In addition, from our experience, post-operative re-infusion of unwashed shed blood after major spine procedures has proved to reduce post-operative homologous transfusion requirements and to complement pre-operative autologous blood donation, without any clinically relevant complication.

Approach for defining endogenous reference genes in gene expression experiments.

The quantification of gene expression by real-time polymerase chain reaction (PCR) has revolutionized the field of gene expression analysis. Due to its sensitivity and flexibility it is becoming the method of choice for many investigators. However, good normalization protocols still have to be implemented to facilitate data exchange and comparison. We have designed primers for 10 unrelated genes and developed a simple protocol to detect genes with stable expression that are suitable for use as endogenous reference genes for further use in the normalization of gene expression data obtained by real-time PCR. Using this protocol, we were able to identify human proteosome subunit Y as a reliable endogenous reference gene for human umbilical vein endothelial cells treated for up to 18 h with TNFalpha, IL-4, or IFNgamma and for B cells isolated from healthy controls and patients suffering from IgA nephropathy. Other optional endogenous reference genes that can be considered are phosphomannomutase (PPMM) and actin for endothelial cells and glyceraldehyde-3-phosphate dehydrogenase and PPMM for B cells.

Acute phase response in patients undergoing lumbar spinal surgery: modulation by perioperative treatment with naproxen and famotidine.

In orthopaedic surgery, perioperative administration of non-steroidal anti-inflammatory drugs has been shown to reduce postoperative pain and analgesic consumption. In addition, preoperative administration of ibuprofen has proved to reduce interleukin-6 (IL-6) release, while that of ranitidine reduced postoperative IL-6-induced C-reactive protein synthesis in patients undergoing abdominal surgery. However, it has not been established whether the preoperative administration of both types of drugs may reduced the postoperative inflammatory reaction after instrumented spinal surgery. Accordingly, our objective was to investigate the effects of preoperative treatment with naproxen plus famotidine on the postoperative systemic inflammatory reaction in patients undergoing instrumented lumbar spinal surgery. Forty consecutive patients scheduled for elective instrumented spinal fusion were alternately assigned to receive either naproxen (500 mg/day, p.o.) plus famotidine (40 mg/day, p.o.) for 7 days before operation, or no adjuvant treatment. Haematological parameters, acute phase proteins, complement fractions, immunoglobulins and cytokines were determined 7 days and immediately before surgery, and on days 0, 1, 2 and 7 after surgery. Haematological parameters, clinical data, duration of surgery, blood loss, perioperative blood transfusion and postoperative complications were similar in the two groups, although pretreated patients showed lower increases in body temperature and required less analgesic medication. Compared with preoperative levels, IL-6 levels were significantly increased postoperatively in all patients with no differences between groups. C-reactive protein, alpha(1)-acid-glycoprotein and haptoglobin levels were also significantly increased postoperatively in all patients; however, they were significantly lower in pretreated patients. In conclusion, perioperative treatment with naproxen plus famotidine was well tolerated and reduced the acute phase response after instrumented spinal surgery. However, further research is needed to determine the best dose and timing of preoperative treatment administration, and to correlate these changes with long-term clinical results.

Autotransfusión en cirugía de columna lumbar / Autologous transfusion in surgery of the lumbar spine

Objetivos. En este estudio se pretendía evaluar la efectividad de la autotransfusión, en sus modalidades preoperatoria (donación preoperatoria de sangre autóloga, DPSA) y postoperatoria (recuperación/reinfusión de la sangre de los drenajes postoperatorios, ATP), para reducir la exposición a transfusión de sangre homóloga (TSH), y las complicaciones derivadas de la misma, en pacientes de cirugía de columna lumbar. Pacientes y métodos: Se han incluido en el estudio 122 pacientes consecutivos sometidos a artrodesis de columna lumbar o lumbosacra instrumentada (Allospine, Sulzer, CH) que fueron divididos en 4 grupos de acuerdo con la estrategia transfusional empleada: Grupo A (Control, sólo sangre homóloga), Grupo B (ATP), Grupo C (DPSA) y Grupo D (DPSA+ATP). La recuperación y reinfusión de la sangre drenada durante las primeras 6 horas del postoperatorio se realizó con el sistema ConstaVac CBCII (Stryker, USA). Se ha valorado la efectividad ( por ciento de pacientes que evitan la TSH) y el rendimiento ( por ciento de unidades de sangre autóloga reinfundidas) del programa de autotransfusión y la incidencia de complicaciones postoperatorias. Resultados: Los pacientes del grupo A (control, n = 37) recibieron exclusivamente sangre homóloga (1,92 ñ 0,2 U/pte), mientras que en los pacientes del grupo B (ATP, n = 29) la reinfusión de 405 ñ 29 ml/pte (19 U, 74 por ciento del sangrado postoperatorio total) redujo en un 60 por ciento las necesidades de TSH postoperatorias (0,28 vs 0,67 U/pte) y en un 20 por ciento las necesidades de TSH totales (1,56 vs 1,92 U/pte), sin que hubiese diferencias con respecto al grupo control en tasa media de transfusión (2,25 ñ 0,19 U/pte). Cinco pacientes de Grupo A y 4 del Grupo B no recibieron TSH. En los 2 grupos siguientes, se realizó una DPSA a corto plazo de 2 U/pte (7 y 1 días antes de la intervención), que se utilizó sola (Grupo C, n = 24) o en combinación con ATP (Grupo D, n = 32).En el grupo C los requerimientos transfusionales fueron de 1,79 ñ 0,1 U/pte, utilizándose el 83 por ciento de la DPSA y sólo 2 pacientes recibieron TSH (Efectividad 93 por ciento).El grupo D presentó un mayor sangrado y una menor ratio varones/mujeres, por lo que, además de utilizarse 61 U de DPSA (Rendimiento 95 por ciento) y 24 U de ATP (58 por ciento del sangrado postoperatorio), fueron necesarias 9 U de TSH en 7 pacientes (Efectividad 78 por ciento), para una tasa media de transfusión de 3,03 ñ 0,15 U/pte (p < 0,01). La efectividad global del programa de donación preoperatoria (grupos C y D) fue del 84 por ciento y el porcentaje de unidades no utilizadas del 11 por ciento (Rendimiento 89 por ciento). No hubo diferencias significativas en los niveles de hemoglobina pre y postoperatorios o al alta, en la tasa de incidencia de complicaciones infecciosas ni en la duración de la estancia hospitalaria entre los distintos grupos. Tampoco se registraron efectos adversos clínicamente relevantes durante la realización de la DPSA o la ATP. Conclusiones: La DPSA a corto plazo se ha mostrado como un procedimiento efectivo y seguro para reducir las TSH en pacientes sometidos a cirugía de columna lumbar instrumentada, mientras que la asociación de ATP a la DPSA podría ser útil en aquellos casos en los que no pueden donarse el número de unidades requeridas o se espera un sangrado postoperatorio abundante (AU)

[Autotransfusion after cardiac surgery. Hematological, biochemical and immunological properties of shed mediastinal blood]. / Autotransfusión postoperatoria en cirugía cardíaca. Características hematológicas, bioquímicas e inmunológicas de la sangre recuperada del drenaje mediastínico.

OBJECTIVE: Although it is well documented that the reinfusion of unwashed shed blood reduces postoperative homologous blood requirements after cardiac surgery, the efficacy and safety of the technique has been questioned on the basis of several possible adverse effects. We therefore aimed to evaluate the extent of mediastinal shed blood damage by cardiopulmonary bypass (CPB) and extravasation. PATIENTS AND METHOD: Five perioperative blood samples were obtained from each of the 20 patients undergoing cardiac surgery: by venipuncture, during the induction of anesthesia (sample 1), at the end of CPB (sample 2), and one hour after mediastinal shed blood reinfusion (sample 5), and from the cardiotomy reservoir at the first and sixth postoperative hours (samples 3 and 4). Hematological, biochemical and immunological parameters, and the metabolic and functional status of shed erythrocytes were studied in each sample. RESULTS: Hematological and biochemical values were lower at the end of CPB and in shed blood, but not after reinfusion. Inversely, plasma free hemoglobin levels and echinocyte formation were elevated after CPB and in shed blood, the latter correlating with the decrease in serum albumin levels due to hemodilution. No alterations were detected in erythrocyte median corpuscular fragility, D-glucose, L-phenylalanine and L-serine uptakes or 2,3-bisphosphoglycerate content. Plasma IL-10 levels were elevated at the end of CPB, whereas shed blood showed increased levels of IL-1beta, IL-6 and IL-10. Shed blood retrieval did not modify the pattern of circulating cytokines found at the end of CPB. CONCLUSIONS: Shed mediastinal blood salvaged after cardiac surgery seems to be an excellent source of red cells, which conserve functional and metabolic status; once reinfused, shed blood does not modify the inflammatory response of the patient to CPB.

[Autotransfusion after orthopedic surgery. Analysis of quality, safety and efficacy of salvaged shed blood]. / Autotransfusión postoperatoria en cirugía ortopédica. Un análisis de la calidad, seguridad y eficacia de la sangre recuperada de los drenajes postoperatorios.

A series of immunosuppressant mechanisms can manifest during surgical procedures, mediated by immune system cells or by humoral factors, to which the immunosuppressant effects of anesthesia or blood transfusion may be added, possibly further prejudicing the patient's immunological status, having important clinical repercussions such as increased incidence of postoperative infection or tumor reappearance. Autotransfusion of various types is an effective alternative to homologous transfusion as the former avoids immunodepressant effects. Preoperative autotransfusion [preoperative donation of autologous blood (PTAB)] has been shown to be one of the safest and most effective techniques and is the gold standard for autotransfusion. Problems of over collection, anemia and over transfusion that sometimes occur with PTAB can be solved with better screening procedures. Intraoperative autotransfusion (IAT) and postoperative autotransfusion (PAT) avoid such problems completely. However, IAT is only cost-effective in certain procedures (bleeding > 1,000-1,500 mL) and is not applicable in others, such as knee arthroplasty. PAT, on the other hand, in addition to being a good complement to other autotransfusion methods, may be the technique of choice in some procedures, such as knee arthroplasty, particularly if PTAB is contraindicated or if it is logistically difficult for a hospital to provide.However, in spite of its demonstrated efficacy, PAT of filtered blood has many critics, who warn of possible side effects and recommend the use of washed blood, which would make the procedure enormously more expensive unless it is performed with the same equipment used for IAT. Therefore, this review will analyze the hematologic characteristics of filtered blood, including metabolic status and survival of red blood cells, the components of the hemostatic system and inflammatory mediators, the content of fat particles and the possibility of their clearance, the incidence of infections and the dissemination of tumor cells. This analysis can reach the conclusion that salvaged filtered blood is a source of red blood cells of sufficient quality to be safely reinfused and that their reinfusion contributes significantly to reduce the need for homologous blood.

Autotransfusión postoperatoria en cirugía ortopédica. Un análisis de la calidad, seguridad y eficacia de la sangre recuperada de los drenajes postoperatorios / Autotransfusion after orthopedic surgery. Analysis of quality, safety and efficacy of salvaged shed blood

En los procesos quirúrgicos pueden evidenciarse una serie de mecanismos de inmunosupresión, mediados tanto por las células del sistema inmunitario como por factores humorales, a los que se les pueden sumar los efectos inmunosupresores de la anestesia y las transfusiones sanguíneas, que pueden deteriorar aún más el estado inmunológico del paciente, lo que puede tener importantes repercusiones clínicas como aumento de infecciones postoperatorias o de recidivas tumorales.La autotransfusión, en sus distintas modalidades, se presenta como una alternativa eficaz a las transfusiones de sangre homóloga al evitar los efectos inmunosupresores de las mismas. La autotransfusión preoperatoria (donación preoperatoria de sangre autóloga) ha demostrado ser una de las técnicas transfusionales más seguras y eficaces y constituye el "patrón oro" en autotransfusión. Los problemas de sobrecolección, anemización y sobretransfusión que a veces presenta la donación preoperatoria de sangre autóloga pueden solucionarse con una mejor selección de los pacientes.Mediante la autotransfusión intra y postoperatoria se obvian por completo estos problemas. No obstante, la autotransfusión intraoperatoria sólo es coste-efectiva en determinadas intervenciones (hemorragia > 1.000-1.500 ml) y no es aplicable en otras, como la cirugía protésica de rodilla. Por su parte, la autotransfusión postoperatoria, además de ser un buen complemento del resto de técnicas de autotransfusión, en determinadas intervenciones quirúrgicas, como la de prótesis de rodilla, puede ser la técnica de elección, máxime si la donación preoperatoria de sangre autóloga está contraindicada en el paciente o es logísticamente difícil de implantar en el centro.Sin embargo, a pesar de haber demostrado su eficacia, la autotransfusión postoperatoria en forma de sangre filtrada cuenta aún con un gran número de detractores, los cuales advierten sobre una serie de posibles efectos adversos y preconizan el uso de sangre lavada, lo que encarecería enormemente el procedimiento, salvo que se realice con el mismo equipo utilizado para la autotransfusión intraoperatoria durante la intervención. Por ello, en la presente revisión se lleva a cabo un análisis detallado de las características hematológicas de la sangre filtrada, el estado metabólico y la supervivencia de sus hematíes, el contenido en componentes del sistema hemostásico y de mediadores inflamatorios, el contenido de partículas grasas y las posibilidades de eliminación y la incidencia de complicaciones infecciosas y de diseminación de células tumorales. De este análisis se puede llegar a la conclusión de que la sangre total filtrada y no lavada es una fuente de eritrocitos de calidad suficiente para ser reinfundidos sin problemas, y que su reinfusión contribuye significativamente a una reducción de las necesidades de sangre homóloga (AU)

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Análisis de la relación coste-efectividad de un programa de autotransfusión en cirugía protésica primaria de rodilla y cadera / Analysis of the cost-effectiveness relation is an autotransfusion program in primary prosthetic surgery of the knee and hip

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Postoperative blood salvage and reinfusion in spinal surgery: blood quality, effectiveness and impact on patient blood parameters.

Although reinfusion of salvaged shed blood has become popular in major orthopaedic procedures, this blood saving technique is still controversial. In an effort to assess the functional and metabolic status of shed blood erythrocytes and the impact of postoperative shed blood reinfusion on allogenic blood requirements and patient's blood parameters, analyses of perioperative blood samples were performed in 28 consecutive orthopaedic patients undergoing spinal fusion, in which postoperative shed blood was collected and reinfused with the ConstaVac CBC II device. In comparison with a previous series of 31 patients, this procedure reduced allogenic blood requirements by almost 30% (P < 0.05), without any increase in postoperative complications. Postoperative shed blood presented lower haematological values and higher plasma-free haemoglobin (PFHB) levels than preoperative blood, without any disturbance in morphology, median corpuscular fragility (MCF) or erythrocyte adenosine triphosphate (ATP) and diphosphoglycerate (DPG) content. Serum concentrations of enzymes--glutamate-oxalacetate aminotransferase (GOT), glutamate-piruvate aminotransferase (GPT), creatine kinase (CK), lactate dehydrogenase (LDH)--and inflammatory cytokines (IL-1beta, IL-6) were elevated in shed blood. After reinfusion, there was no alteration in coagulation parameters or cytokine levels. Serum levels of some enzymes increased at the end of surgery and remained elevated at postoperative day 2 (CK) or 7 (GOT, LDH), with a higher increase if postoperative autotransfusion was used as a blood saving method. Therefore, caution should be taken when these serum enzyme levels are used for diagnosis. In conclusion, salvaged shed blood in orthopaedic procedures of the spine seems to be an excellent source of red cells which are not significantly damaged, keeping a normal functional and metabolic status, and reduces allogenic blood requirements without significant side effects.

[Pre and post-operative autotransfusion. A comparative study of hematology, biochemistry and red cell metabolism in pre-donated blood and blood from post-operative surgical drainage]. / Autotransfusión pre y postoperatoria. Estudio comparativo de la hematología, bioquímica y metabolismo eritrocitario en sangre predonada y sangre de drenaje postoperatorio.

PURPOSE: The widespread use of aggressive surgical procedures, along with the increasing incidence of traffic accidents, has raised the necessity of homologous blood beyond the supplies of blood banks. This fact, plus the risks of homologous transfusion and the costs of blood bank maintenance, has prompted the advance of blood saving procedures such as autotransfusion, both in the pre-deposit (preoperative autotransfusion, POA) and the surgical drainage reinfusion (postoperative autotransfusion, SDR) modalities. As there is some controversy about the use of one or the other of the above procedures, the purposes of this study were: 1) to analyse the haematological and biochemical characteristics of blood, both pre-donated and stored at 4 degrees C for 4 weeks, and that recovered from surgical drains, from patients subjected to programmed orthopaedic or heart surgery; 2) to assess the metabolic and functional state of red cells attained from that blood, and 3) to compare the results achieved. MATERIAL AND METHODS: The following data were examined: red cell count, haematocrit, haemoglobin, red cell indicates, white cell count, platelet count, free plasma haemoglobin, red cell morphology, glucose, cholesterol, triglycerides, phospholipids, serum proteins and their fractions, ions, histamine, red cell glucose and amino acid transport, and ATP and 2,3-DPG content. RESULTS: The red cells, haemoglobin and haematocrit concentrations in POA blood did not show significant variations during the storage for 4 weeks and their values were significantly higher than found in drained blood. The biochemical values showed heterogeneous variations. Glucose and amino acid uptake by red cells of POA blood slightly decreased in the first 2 weeks of storage but always less than measured in SDR red cells. In POA blood it was noticed a progressive decrease in intra-erythrocytic ATP and 2,3 DPG, those levels being normal in SDR. DISCUSSION: In spite of lower haematocrit and haemoglobin but higher free plasma haemoglobin content, drained blood had higher ATP and 2,3-DPG concentration than pre-deposit, stored blood. Drained blood showed also less ion alterations and probably a lesser immunosuppressor capability. Thus, postoperative blood recovery seems a good source of red cell, with high oxygen transport power, and so, alone or in combination with pre-donated blood, it may contribute to reduce the necessities for homologous blood and decrease its risks.