Adaptive optics in spinning disk microscopy: improved contrast and brightness by a simple and fast method.

Multiconfocal microscopy gives a good compromise between fast imaging and reasonable resolution. However, the low intensity of live fluorescent emitters is a major limitation to this technique. Aberrations induced by the optical setup, especially the mismatch of the refractive index and the biological sample itself, distort the point spread function and further reduce the amount of detected photons. Altogether, this leads to impaired image quality, preventing accurate analysis of molecular processes in biological samples and imaging deep in the sample. The amount of detected fluorescence can be improved with adaptive optics. Here, we used a compact adaptive optics module (adaptive optics box for sectioning optical microscopy), which was specifically designed for spinning disk confocal microscopy. The module overcomes undesired anomalies by correcting for most of the aberrations in confocal imaging. Existing aberration detection methods require prior illumination, which bleaches the sample. To avoid multiple exposures of the sample, we established an experimental model describing the depth dependence of major aberrations. This model allows us to correct for those aberrations when performing a z-stack, gradually increasing the amplitude of the correction with depth. It does not require illumination of the sample for aberration detection, thus minimizing photobleaching and phototoxicity. With this model, we improved both signal-to-background ratio and image contrast. Here, we present comparative studies on a variety of biological samples.

Impact of endoscopic ultrasonography on the management of a prospective cohort of 700 consecutive patients referred for diagnostic endoscopic ultrasonography.

BACKGROUND: endoscopic ultrasonography (EUS) is a high accuracy technique for the study of many digestive diseases. The degree of knowledge about the impact of EUS on the management of these patients is inadequate. AIM: to determine the therapeutic impact of endoscopic ultrasonography (EUS) on a prospective cohort of patients. METHODS: all patients referred for EUS over a period of 2 years were prospectively evaluated in order to asses: 1. EUS provides new information not previously known; 2. theoretic impact of EUS on patient management; 3. real impact of EUS on final therapy; 4. changes in the aggressiveness of the therapeutic decision after EUS. RESULTS: 700 patients were included. Preoperative assessment of digestive tumors was the commonest indication. EUS provided "new information" in the 89% of the patients. With regard to endoscopist opinion, these findings should alter the management in 79% of patients ("theoretic impact"). However, EUS prompted a change in the management in 67% of patients ("real impact"). Final therapy post-EUS was less aggressive in 34% of patients. Changes in therapeutic decision were associated with EUS findings, alcohol intake and age ≥ 57 years old. CONCLUSIONS: 1) EUS findings, advanced age, and alcohol intake are associated with a change in the management in 2 out of every 3 patients referred for EUS. 2) Therapeutic decision (post-EUS) is less aggressive in a third of these patients, what should represent a significant economic saving.

Impacto de la ultrasonografía endoscópica en el manejo terapéutico de una cohorte prospectiva de 700 pacientes consecutivos referidos para USE diagnóstica / No disponible

Antecedentes: la ultrasonografía endoscópica (USE) es una técnica de gran precisión para el estudio de diferentes patologías digestivas. El grado de conocimiento sobre el impacto de la USE en el tratamiento de estos pacientes es escaso. Objetivo: determinar el impacto terapéutico de la USE en una cohorte prospectiva de pacientes. Material y métodos: estudio que evalúa de forma prospectiva a todos los pacientes remitidos para USE en un periodo de 2 años. Se analiza: a) si la USE aporta información no conocida previamente; b) el impacto teórico de la USE en el tratamiento; c) el impacto final/real de USE en el tratamiento; d) la variación en la decisión terapéutica tras realizar la USE. Resultados: se incluyeron 700 pacientes. La indicación más frecuente de USE era el estudio preoperatorio de tumores digestivos. La USE aportó “información nueva” en el 89% de los casos. En opinión del ecoendoscopista estos hallazgos deberían suponer un cambio terapéutico en el 79% de los pacientes (“impacto teórico”). Sin embargo, el tratamiento sólo se modificó en el 67% de los casos (“impacto real”). La estrategia terapéutica post-USE fue menos agresiva en el 34% de los casos. Los cambios en la actitud terapéutica se asociaban con los hallazgos de USE, ingesta de alcohol y edad > 57 años. Conclusiones: – Los hallazgos de la USE, la edad avanzada y el consumo de alcohol, se asocian con un cambio terapéutico en 2 de cada 3 pacientes remitidos a una unidad de USE. – La decisión terapéutica final (post-USE) es menos agresiva en un tercio de estos pacientes, lo que podría representar un ahorro económico significativo(AU)

Antecedents: endoscopic ultrasonography (EUS) is a high accuracy technique for the study of many digestive diseases. The degree of knowledge about the impact of EUS on the management of these patients is inadequate. Aim: to determine the therapeutic impact of endoscopic ultrasonography (EUS) on a prospective cohort of patients. Methods: all patients referred for EUS over a period of 2 years were prospectively evaluated in order to asses: 1. EUS provides new information not previously known; 2. theoretic impact of EUS on patient management; 3. real impact of EUS on final therapy; 4. changes in the aggressiveness of the therapeutic decision after EUS. Results: 700 patients were included. Preoperative assessment of digestive tumors was the commonest indication. EUS provided “new information” in the 89% of the patients. With regard to endoscopist opinion, these findings should alter the management in 79% of patients (“theoretic impact”). However, EUS prompted a change in the management in 67% of patients (“real impact”). Final therapy post-EUS was less aggressive in 34% of patients. Changes in therapeutic decision were associated with EUS findings, alcohol intake and age >= 57 years old. Conclusions: 1) EUS findings, advanced age, and alcohol intake are associated with a change in the management in 2 out of every 3 patients referred for EUS. 2) Therapeutic decision (post- EUS) is less aggressive in a third of these patients, what should represent a significant economic saving(AU)

Hairy esophagus: una complicación de la cirugía reconstructiva faringoesofágica / No disponible

No disponible

“Enfermedad de las tres mentiras”: síndrome de la úlcera rectal solitaria / “The three-lies disease”: Solitary rectal ulcer syndrome

El síndrome de la úlcera rectal solitaria es una entidad benignapoco común cuyos síntomas más frecuentes son la rectorragia y eldolor anal. Los hallazgos anatomopatológicos son típicos. Se sueleevidenciar engrosamiento de la mucosa, elongación y distorsiónde las glándulas, una lámina propia edematosa con gran cantidadde colágena y engrosamiento de la muscularis mucosae. El diagnósticose realiza mediante endoscopia con toma de biopsias. Nosiempre se trata de lesiones ulceradas. Se suele localizar preferentementeen la cara rectal anterior y/o lateral, aunque hasta un30% de las lesiones son múltiples, existiendo casos de afectacióndel colon sigmoide y descendente. Por tanto, la presentación puedeser heterogénea y este es el motivo por el que esta entidad estambién conocida como la “enfermedad de las tres mentiras”.Presentamos un caso de síndrome de la ulcera rectal solitaria manifestadoendoscópicamente como una placa eritematosa localizadaen la cara lateral izquierda del recto

Solitary rectal ulcer syndrome is an uncommon benign conditioncharacterized by rectal bleeding, passage of mucus, and pain.Histological features are well established as obliteration of thelamina propria by fibrosis and smooth-muscle fibers extendingfrom a thickened muscularis mucosa to the lumen. Diagnosis canusually be made on sigmoidoscopy, and biopsies should always betaken. Ulceration is not universally present, and polypoid, non-ulceratedlesions and erythematous areas are also seen. The lesionor lesions are most often found on the anterior or anterolateralwall of the rectum, although they can also be located in the leftcolon and be more extensive or even circumferential. Lesions aremultiple in 30 percent of cases. These are the reasons why thisentity is also known as “the disease of three lies”. We report acase of solitary rectal ulcer syndrome presenting at endoscopywith an erythematous area on the left side wall of the rectum (AU)

[A prospective study about the usefulness of ultrasonographic monitoring after invasive liver procedures--liver biopsy and fine-needle aspiration (FNA)]. / Estudio prospectivo sobre la utilidad de la ecografía de control tras la realización de pruebas invasivas hepáticas: biopsia hepática y punción aspiración con aguja fina (PAAF).

OBJECTIVE: To determine the need to perform ultrasound scans to all patients after liver biopsy or fine-needle aspiration (FNA) in order to detect complications with or without symptoms. MATERIAL AND METHODS: After liver biopsy or FNA using a regular protocol the patient is observed for 24 hours at the hospital, and all patients undergo an abdominal sonography at that time even in the absence of evident complications. RESULTS: 298 liver biopsies and 98 FNAs were performed. There were complications in 37 patients (9.34%): 36 (9.09%) were minor complications such as pain, vasovagal episodes, or small bleeding, and 1 (0.25%) was a major complication with severe hemorrhage. Only 1 out of all 396 procedures had a complication detected by ultrasounds (intrahepatic hematoma) while the patient was asymptomatic. CONCLUSIONS: The low incidence of complications occurring without symptoms, and their favorable course suggest that routine ultrasonography is not necessary after these techniques, and that it should be only performed when a complication is suspected.

Pancreatitis aguda como diagnóstico debut de un hiperparatiroidismo primario / Acute pancreatitis as clinical presentation of a primary hyperparatiroidism

No disponible

Estudio prospectivo sobre la utilidad de la ecografía de control tras la realización de pruebas invasivas hepáticas: biopsia hepática y punción aspiración con aguja fina (PAAF) / A prospective study about the usefulness of ultrasonographic monitoring after invasive liver procedures –liver biopsy and fine-needle aspiration (FNA)

Objetivo: establecer si es necesario realizar una ecografía de control a todos los pacientes sometidos a una biopsia hepática o una punción aspiración con aguja fina, para detectar posibles complicaciones con o sin repercusión clínica. Material y métodos: tras la realización de una biopsia hepática o una punción aspiración con aguja fina según el protocolo habitual, se mantiene al paciente en observación durante 24 horas, realizándose en ese momento una ecografía a todos los pacientes aunque no presenten datos clínicos de complicación. Resultados: se llevaron a cabo 298 biopsias hepáticas y 98 punciones mediante aguja fina. Presentaron complicaciones un total de 37 pacientes (9,34%), de las cuales 36 (9,09%) fueron complicaciones menores en forma de dolor, síncope vasovagal o hemorragia leve y 1 (0,25%) complicación mayor en forma de hemorragia grave. De las 396 exploraciones tan sólo uno de los casos presentó una complicación detectada en la ecografía (hematoma intraparenquimatoso) encontrándose asintomático. Conclusiones: la baja incidencia de complicaciones, que cursan de forma asintomática, y la buena evolución de las mismas hacen poco rentable la realización de ecografía de control tras la realización de dichas técnicas diagnósticas, siendo necesaria tan sólo en el caso de sospecha clínica de complicación

Objective: to determine the need to perform ultrasound scans to all patients after liver biopsy or fine-needle aspiration (FNA) in order to detect complications with or without symptoms. Material and methods: after liver biopsy or FNA using a regular protocol the patient is observed for 24 hours at the hospital, and all patients undergo an abdominal sonography at that time even in the absence of evident complications. Results: 298 liver biopsies and 98 FNAs were performed. There were complications in 37 patients (9.34%): 36 (9.09%) were minor complications such as pain, vasovagal episodes, or small bleeding, and 1 (0.25%) was a major complication with severe hemorrhage. Only 1 out of all 396 procedures had a complication detected by ultrasounds (intrahepatic hematoma) while the patient was asymptomatic. Conclusions: the low incidence of complications occurring without symptoms, and their favorable course suggest that routine ultrasonography is not necessary after these techniques, and that it should be only performed when a complication is suspected

["The three-lies disease": solitary rectal ulcer syndrome]. / "Enfermedad de la tres mentiras": síndrome de la úlcera rectal solitaria.

Solitary rectal ulcer syndrome is an uncommon benign condition characterized by rectal bleeding, passage of mucus, and pain. Histological features are well established as obliteration of the lamina propria by fibrosis and smooth-muscle fibers extending from a thickened muscularis mucosa to the lumen. Diagnosis can usually be made on sigmoidoscopy, and biopsies should always be taken. Ulceration is not universally present, and polypoid, non-ulcerated lesions and erythematous areas are also seen. The lesion or lesions are most often found on the anterior or anterolateral wall of the rectum, although they can also be located in the left colon and be more extensive or even circumferential. Lesions are multiple in 30 percent of cases. These are the reasons why this entity is also known as "the disease of three lies". We report a case of solitary rectal ulcer syndrome presenting at endoscopy with an erythematous area on the left side wall of the rectum.

Intracellular retention of the two isoforms of the D(2) dopamine receptor promotes endoplasmic reticulum disruption.

The dopamine D(2) receptor exists as a long (D(2a)) and a short (D(2b)) isoform generated by alternative splicing of the corresponding transcript, which modifies the length of the third cytoplasmic loop implicated in heterotrimeric G-protein-coupling. Anatomical data suggested that this segment regulates the intracellular traffic and localization of the receptor. To directly address this question we used a combination of tagging procedures and immunocytochemical techniques to detect each of the two D(2) receptor isoforms. Surprisingly, most of the newly synthesized receptors accumulate in large intracellular compartments, the plasma membrane being only weakly labeled, without significant difference between the two receptor isoforms. Double labeling experiments showed that this localization corresponded neither to endosomal compartments nor to the Golgi apparatus. The D(2) receptor is mostly retained in the endoplasmic reticulum (ER), the long isoform more efficiently than the short one. It is accompanied by a striking vacuolization of the ER, roughly proportional to the expression levels of the two receptor isoforms. This phenomenon is partly overcome by treatment with pertussis toxin. In addition, an intrinsic activity of the D(2) receptor isoforms is revealed by [(35)S]-GTP gamma S binding and cAMP assay, which suggested that expression of weakly but constitutively active D(2) receptors promotes activation of heterotrimeric G protein inside the secretory pathway. This mechanism may participate in the regulation of the cellular traffic of the D(2) receptors isoforms.

Biosynthesis and intracellular post-translational processing of normal and mutant platelet glycoprotein GPIb-IX.

The multisubunit leucine-rich glycoprotein (GP) Ib-IX-V complex mediates von Willebrand factor-dependent platelet adhesion at sites of blood-vessel injury. Molecular defects of this receptor are reported to cause the Bernard-Soulier haemorrhagic disorder. To gain insight into the mechanisms controlling expression of normal and defective receptors, we performed pulse-chase metabolic studies and detailed analysis of intracellular processing in GPIb-IX-transfected Chinese-hamster ovary cells. In the native complex, after early subunit association, sugars N-linked to the three subunits are trimmed and sialylated in the Golgi compartment and GPIbalpha undergoes extensive O-glycosylation. Surface biotinylation during chase demonstrated that only fully processed complexes reach the cell surface. Tunicamycin treatment revealed that early N-glycosylation is not required for O-glycosylation of GPIbalpha and surface expression of the complex. Biosynthetic studies were then performed on a Bernard-Soulier variant based on previous description of abnormal GPIbalpha size and decreased surface expression. The mutant complex associated normally, but displayed defective processing of its N-linked sugars and abnormal O-glycosylation of GPIbalpha. Confocal immunofluorescence microscopy revealed that the mutant complexes could reach the cell surface but also accumulated intracellularly, while use of compartment specific markers showed strong co-localization in the endoplasmic reticulum (ER) and ER-to-Golgi intermediate compartments ('ERGIC') and only slight labelling of the cis-Golgi. Blockade before the Golgi was confirmed by brefeldin A treatment, which restored O-glycosylation and processing of N-linked sugars. The present study has shown that transfer from the ER to the Golgi represents an important step for controlling post-translational processing and surface expression of normal GPIb-IX-V complex.

Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes.

In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to the trans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network.

In vitro production of dendritic cells from human blood monocytes for therapeutic use.

Dendritic cells (DC) are professional antigen-presenting cells that are promising adjuvants for clinical immunotherapy. Methods to generate in vitro large numbers of functional human DC using either peripheral blood monocytes or CD34(+) pluripotent hematopoietic progenitor cells have been now developed. For this purpose, their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. In the present review, we give our experience of such a procedure: it includes collection of mononuclear cells by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + human serum (autologous patient's serum or AB serum) or in a serum-free medium (AIM V). The characteristics of monocyte-derived DC grown in these various conditions varied mainly regarding their phenotype and their morphology in confocal microscopy, whereas no significant differences were found in their capacity to phagocytize latex particles and to stimulate allogeneic (MLR) or autologous lymphocytes (antigen-presentation tests). The DC were also cryopreserved in bags (either by putting the bags directly in a -80 degrees C mechanical freezer or using a classical liquid nitrogen controlled-rate freezer at -1 degrees C/min) in a solution containing 10% dimethyl sulfoxide (Me(2)SO) and 2% human albumin in doses of DC available for several infusions. The mean recoveries after freezing and thawing were not statistically different (around 70%). The immunophenotype of DC, as well as the T lymphocyte-stimulating capacity, were not modified by the freezing--thawing procedure. The results obtained demonstrate that the experimental conditions we set up are easily applicable in clinical trials and lead to large numbers of well-defined DC. Clinical trials using DC already published will be discussed.

Unstimulated human CD4 lymphocytes express a cytoplasmic immature form of the common cytokine receptor gamma-chain.

As a component of various cytokine receptors, common cytokine receptor gamma-chain (gamma(c)) is essential in the development of the immune system and plays an important role in different stages of inflammatory and immune responses. Here we establish that resting CD4 T cells and the Jurkat CD4 T cell line do not express the mature form of gamma(c) (64 kDa) recognized by mAb Tugh4. However, these cells constitutively transcribe the corresponding gamma(c) gene. This apparent paradox was solved by the demonstration that polyclonal anti-gamma(c) Abs detected endoglycosidase-H-sensitive immature forms of gamma(c) (54-58 kDa) expressed by quiescent CD4 T lymphocytes and Jurkat cells. Immature gamma(c) is characterized as an intracellular component localized in the endoplasmic reticulum. Pulse-chase analysis shows that the immature gamma(c) is rapidly degraded after synthesis. After activation of CD4 T lymphocytes, and as seen in the CD4 T cell line Kit 225, the endoglycosidase-H-resistant mature form of gamma(c) is detectable at the cell surface and in the endosomal compartment. For the first time, our results demonstrate that a cytokine receptor chain may be constitutively produced as an immature form. Furthermore, this supports the notion that expression of the functional form of gamma(c) may require intracellular interactions with lineage- or subset-specific molecular partners.

CD1a molecules traffic through the early recycling endosomal pathway in human Langerhans cells.

In this work, we studied the localization and traffic of CD1a molecules in human epidermal Langerhans cells and the ability of these cells to stimulate CD1a-restricted T cell clones. We found that CD1a was spontaneously internalized into freshly isolated Langerhans cells, where it was rapidly distributed to the early/sorting endosomes and then to the early/recycling endosomes. In the latter compartments, CD1a colocalized with Rab11, a small GTPase known to be involved in the recycling of transmembrane proteins from early endosomes to the cell surface. In the steady state, intracellular CD1a was mainly located in Rab11+ recycling endosomal compartments. When endocytosis was blocked, intracellular CD1a moved rapidly from the early/recycling endosomes to the cell surface where it accumulated. The resultant increase in the cell surface expression of CD1a enhanced the capacity of Langerhans cells to stimulate a CD1a-restricted T cell clone. These findings are consistent with a dynamic exchange of CD1a between recycling compartments and the plasma membrane and suggest that the antigen-presenting function of CD1a depends on its traffic through the early/recycling endosomal pathway.

Modulation of MHC class II transport and lysosome distribution by macrophage-colony stimulating factor in human dendritic cells derived from monocytes.

The macrophage-colony stimulating factor (M-CSF) has been already shown to affect the function of dendritic cells (DC). Therefore, the differentiation of dendritic cells into macrophages (M(PHI)) might represent a pathway which could inhibit the immune response initiated by DC. Because Major Histocompatibility Complex class II molecules (MHC-II) are crucial for DC function, we asked whether M-CSF may influence the intracellular transport of MHC-II in monocyte derived DC. We found that, at early stages, M-CSF induced first a rapid redistribution of MHC-II from the MHC-II containing compartments (MIIC) to the plasma membrane and second an increase in MHC-II synthesis as observed with LPS or TNF-(alpha). These processes were associated with the sorting of MHC-II from lysosomal membranes which underwent a drastic structural reorganization. However, in contrast to tumor necrosis factor (TNF)-(alpha) or lipopolysaccharide (LPS), M-CSF neither potentiated the allostimulatory function of DC nor allowed the stabilization of MHC-II at the cell surface, but rather increased MHC-II turnover. We conclude that the rapid modulation of MHC-II transport and distribution may participate in the inhibitory effect of M-CSF on DC function and differentiation.

Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network.

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20 degrees C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.

Characterization of CD1e, a third type of CD1 molecule expressed in dendritic cells.

Dendritic cells express several alternatively spliced CD1e mRNAs. These molecules encode proteins characterized by the presence of either one, two, or three alpha domains and either a 51- or 63-amino acid cytoplasmic domain. Moreover, mRNAs encoding isoforms lacking the transmembrane domain are observed. Several of these CD1e isoforms were expressed in transfected cells, and two of them, with three alpha domains, displayed a particular processing pathway. These latter isoforms slowly leave the endoplasmic reticulum due to the presence of atypical dilysine motifs in the cytoplasmic tail. These molecules are associated with the beta(2)-microglobulin and accumulate in late Golgi and late endosomal compartments. In the latter compartments, they are cleaved into soluble forms that appear to be stable. In dendritic cells, these isoforms are mainly located in the Golgi apparatus, and upon maturation they are redistributed to late endosomal compartments. This work demonstrates the existence of CD1e molecules. As compared with other CD1 molecules, CD1e displays fundamentally different properties and therefore may represent a third type of CD1 molecules.

In vitro generation of dendritic cells from human blood monocytes in experimental conditions compatible for in vivo cell therapy.

DC are professional APC that are promising adjuvants for clinical immunotherapy. Methods to generate in vitro large numbers of functional human DC using either peripheral blood monocytes or CD34+ pluripotent HPC have been developed recently. However, the various steps of their in vitro production for further clinical use need to fit good manufacturing practice (GMP) conditions. Our study focused on setting up such a full procedure, including collection of mononuclear cells (MNC) by apheresis, separation of monocytes by elutriation, and culture of monocytes with GM-CSF + IL-13 + autologous serum (SAuto) in sterile Teflon bags. The procedure was first developed with apheresis products from 7 healthy donors. Its clinical feasibility was then tested on 7 patients with breast cancer. The characteristics of monocyte-derived DC grown with SAuto (or in some instances with a pooled AB serum) were compared with those obtained in the presence of FBS by evaluation of their phenotype, their morphology in confocal microscopy, and their capacity to phagocytize latex particles and to stimulate allogeneic (MLR) or autologous lymphocytes (antigen-presentation tests). The results obtained demonstrate that the experimental conditions we set up are easily applicable in clinical trials and lead to large numbers of well-defined SAuto-derived DC as efficient as those derived with FBS.