Enhanced lentiviral transduction of monocyte-derived dendritic cells in the presence of conditioned medium from dying monocytes.

Lentiviral vectors (LVs) are attractive vehicles for the transduction of human dendritic cells (DCs) in order to mobilize their endogenous antigen presentation pathways. We analyzed here how to improve the efficiency of LV transduction, which we performed at the initial stages of the differentiation of purified monocytes into dendritic cells (Mo-DCs). Using LVs pseudotyped with the vesicular stomatitis virus envelope G glycoprotein (VSV-G), we found that a conditioned medium derived from dying monocytes (MCM) improved by 2- to 10- fold the proportion of transduced Mo-DCs. This enhanced transduction efficiency requires the presence of MCM during the initial stage of LV transduction and does not affect the phenotype and antigen presentation function of terminally differentiated Mo-DCs. Importantly, we found that MCM derived from a human acute monocytic leukemia cell line, THP-1, was equally effective. The MCM activity was heat stable (56 degrees C) and was present in the soluble fraction after high-speed centrifugation. Altogether our results show that a soluble factor present in dying monocyte cultures can replace advantageously facilitating agents such as Polybrene, to achieve high LV transductions levels. This protocol can be performed with autologous monocytes and is therefore applicable in clinical settings.

Efficient transduction of monocyte- and CD34+-derived Langerhans cells with lentiviral vectors in the absence of phenotypic and functional maturation.

BACKGROUND: Gene delivery in dendritic cells (DC) has raised considerable interest to modulate DC functions and induce therapeutic immunity or tolerance in an antigen-specific fashion. Among immature DC, Langerhans cells (LC) are attractive candidates for antigen delivery using lentiviral vectors (LV). METHODS: LC derived from monocytes (Mo-LC), or derived from CD34+ cells (CD34-LC) in the presence of cytokine cocktail, were transduced with LV expressing enhanced green fluorescent protein (E-GFP) under the control of the ubiquitous phosphoglycerate kinase (PGK) promoter at a multiplicity of infection of 18, at days 0 to 3 for Mo-LC, or at days 0 to 12 for CD34-LC. We assessed gene transfer levels from the percentage of E-GFP+ cells in the final cultures, and examined the morphology, immunophenotype, state of differentiation and function of transduced LC. RESULTS: Day 0 transduction of monocytes or CD34+ progenitors before cytokine pre-activation and LC differentiation resulted in stable gene expression in 7.8% of Mo-LC and 24% of CD34-LC. Monocyte-derived DC (Mo-DC) differentiated in serum-free medium were also efficiently transduced up to 13.2%. Interestingly, Mo-LC cells committed towards LC phenotype were permissive for transduction up to day 3. Transduction levels of CD34-LC peaked at day 6 to 44% and decreased thereafter. LV transduction did not perturb viability, phenotype and function of E-GFP-expressing LC. CONCLUSIONS: LC generated ex vivo can serve as vaccine vehicles in humans through efficient transduction by LV. These LC will be helpful to assess in vitro the immunogenicity of gene therapy vectors, from the characterization of their phenotypic and functional maturation.

Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles.

Dendritic cells constitutively secrete a population of small (50-90 nm diameter) Ag-presenting vesicles called exosomes. When sensitized with tumor antigenic peptides, dendritic cells produce exosomes, which stimulate anti-tumor immune responses and the rejection of established tumors in mice. Using a systematic proteomic approach, we establish the first extensive protein map of a particular exosome population; 21 new exosomal proteins were thus identified. Most proteins present in exosomes are related to endocytic compartments. New exosomal residents include cytosolic proteins most likely involved in exosome biogenesis and function, mainly cytoskeleton-related (cofilin, profilin I, and elongation factor 1alpha) and intracellular membrane transport and signaling factors (such as several annexins, rab 7 and 11, rap1B, and syntenin). Importantly, we also identified a novel category of exosomal proteins related to apoptosis: thioredoxin peroxidase II, Alix, 14-3-3, and galectin-3. These findings led us to analyze possible structural relationships between exosomes and microvesicles released by apoptotic cells. We show that although they both represent secreted populations of membrane vesicles relevant to immune responses, exosomes and apoptotic vesicles are biochemically and morphologically distinct. Therefore, in addition to cytokines, dendritic cells produce a specific population of membrane vesicles, exosomes, with unique molecular composition and strong immunostimulating properties.

[Etiologic course of heart valve diseases surgically treated during 20 years in France]. / Evolution des étiologies des valvulopathies opérées en France sur une période de 20 ans.

The aim of this study was to analyse the evolution of the aetiologies of operated valvular heart disease over a 20 year period and to assess the consequences. Two thousand five hundred and ninety eight patients who underwent conservative surgery or valve replacement were included. This population was classified according to the date of surgery into 4 groups, each corresponding to a 5 year period. Analysis of these results, especially in the groups operated between 1970 and 1974 and between 1985 and 1989 shows: 1) a change in aetiology: decreased incidence of rheumatic valvular disease (36 versus 61%) and an increased incidence of degenerative lesions (43 versus 23%) (p less than 0.0001); 2) a change in the valvular lesions operated, aortic stenosis has become the commonest operated condition (43 versus 27%) (less than 0.001); 3) an increased age (59 +/- 14 versus 49 +/- 13 years, p less than 0.0001) with a higher proportion of patients over 70 years of age (22 versus 1%, p less than 0.001); 4) a higher incidence of mixed coronary and valvular surgery (11 versus 2%, p less than 0.001).

[Bacteriology of acute otitis, in current practice, in 1987. Therapeutic implications]. / Bactériologie de l'otite aiguë, en ville, en 1987. Implications thérapeutiques.

After presenting the methodology of their study and the structure of their chain of swabs, the authors report the bacteriological results and therapeutic consequences which they imply. In this study, which was conducted in children between the ages of 3 months and 3 years, Haemophilus influenzae was the bacterium most frequently isolated, regardless of the age-group. It was followed by Streptococcus pneumoniae and Staphylococcus aureus. Neither the recurrent nature of the otitis nor the child's life style influenced the predominance of Haemophilus influenzae. 23% of the strains were beta-lactamase producers. The production of beta-lactamase prevented the use of amoxicillin. The increasingly frequent resistance of Streptococcus pneumoniae and the poor sensitivity of Haemophilus influenzae discourage the use of macrolids; the combination of amoxicillin-clavulanic acid obtained a better score with an efficacy of 89%.

[Retrospective study of 33 cases of obstetric paralysis of the brachial plexus]. / Etude rétrospective de 33 paralysies obstétricales du plexus brachial.

Thirty-three cases of obstetrical paralysis of the brachial plexus have been seen in 15 years in the gynaecological and obstetrical department of the University Hospital Pitié Salpêtrière. The authors, from studying these cases, work out the factors that tend to bring about this pathological condition. They are: multiparity, excessive weight gain in pregnancy, fetal excessive growth, prolonged second stage of labour, instrumental delivery and shoulder dystocia in cephalic presentations. In breech presentations it is mainly faulty performance of the manoeuvres that are needed to deliver the shoulders. These lesions involve, in most cases, the roots of C5 and C6. Spontaneous regression occurs very often. The authors have found 25% of sequellae. It is difficult to prevent shoulder dystocia. It consists in realising well the size of the fetus and using instruments really sensibly when the presenting part is high in a primiparous woman. In a multiparous woman one has to be on the look-out for increasing macrosomia of the fetus and it is important to teach the manoeuvres that are necessary to deal with shoulder dystocia. As far as breech delivery is concerned the major risk is the delivery itself without there being any need for fetal or maternal criteria to alter matters. Therefore prevention in this presentation has to be the presence of a competent obstetrician at each delivery always.

[Obstetrical injuries of the newborn infant. Retrospective study apropos of 20,409 births]. / Traumatismes obstétricaux du nouveau-né. Enquête rétrospective à propos de 20 409 naissances.

We have been able to find 291 cases of obstetric trauma when we analysed the 20,409 deliveries which took place in the Pitié maternity between 1971 and 1980. It has been shown that bony lesions did well in the long and short term but nerve lesions had a far less good prognosis. This pathology should become far less when it is possible to recognize better when the fetus is large and when such traumatising manoeuvres as high forceps and assisted deliveries of large breech babies are stopped.

[Myocardial infarction following administration of oral contraceptive agents. 3 cases]. / Infarctus du myocarde après contraceptifs oraux. 3 observations

The authors report the cases of three women aged 23, 24 and 28 years, receiving oestro-progestational agents and suffering a myocardial infarction. 25 cases in the literature are reviewed. Infarction is in most cases the first symptom. The clinical picture has no special features other than the frequency of anterior localisations. Coronary arteriography carried out in 15 cases showed a lesion of one vessel in 10 patients. The most common finding was a thrombosis of the left anterior descending. The coronaries were almost of completely normal in 3 cases, diffuse lesions being found only in 2 patients. The long term prognosis, at first sight favourable since the problem is one of localised lesions, is worsened by the frequency of ventricular contraction abnomalities. Myocardial infarction in patients taking oestro-progestational agents remains rare. A logical hypothesis to explain it would be a synergistic effect between the hypercoagulability induced by the drug and the risk factors in the individual patient. They are present with a frequency similar to that seen in other series of young patients with coronary disease.

Auto-anti-Pra: a 'second' example in a newborn.

A second example of anti-Pra was recognized in a newborn infant which exhibited several signs of development disease. The antibody was complement-fixing 19S IgM and showed a high thermal range but no detectable haemolytic anaemia was associated. It gave negative reactions with a panel of animal cells as well as with two samples of human En(a-) cells. The latter, however, could absorb the antibody.