Rapid Site-Directed Mutagenesis Using Two-PCR-Generated DNA Fragments Reproducing the Plasmid Template.

We describe a new rapid and efficient polymerase chain reaction (PCR)-based site-directed mutagenesis method. This procedure is effective with any plasmid and it employs four oligonucleotide primers. One primer contains the desired mutation, the second is oriented in the opposite direction (one of these two primers should be phosphorylated), and the third and fourth should be coding in complementary fashion for a unique restriction site to be introduced in a nonessential region. The method consists of two simultaneous PCR reactions; the PCR products are digested with the enzyme that recognizes the newly introduced unique restriction site and then ligased and used to transform competent bacteria. Additionally, the use of Dpn I facilitates the elimination of template DNA. The newly introduced restriction site is essential for ligation in the correct orientation of the two-PCR products and is further used for mutant screening. Resulting plasmids carry both the new restriction site and the desired mutation. Using this method, more than 20 mutants have already been generated (using two different kinds of templates); all these mutants were sequenced for the desired mutation and transfected into AtT-20 cells and the expressed mutant proteins encoded by the vector were assayed.

In-depth analysis of hyaline fibromatosis syndrome frameshift mutations at the same site reveal the necessity of personalized therapy.

Hyaline fibromatosis syndrome is an autosomal recessive disease caused by mutations in ANTXR2, a gene involved in extracellular matrix homeostasis. Sixty percent of patients carry frameshift mutations at a mutational hotspot in exon 13. We show in patient cells that these mutations lead to low ANTXR2 mRNA and undetectable protein levels. Ectopic expression of the proteins encoded by the mutated genes reveals that a two base insertion leads to the synthesis of a protein that is rapidly targeted to the ER-associated degradation pathway due to the modified structure of the cytosolic tail, which instead of being hydrophilic and highly disordered as in wild type ANTXR2, is folded and exposes hydrophobic patches. In contrast, one base insertion leads to a truncated protein that properly localizes to the plasma membrane and retains partial function. We next show that targeting the nonsense mediated mRNA decay pathway in patient cells leads to a rescue of ANTXR2 protein in patients carrying one base insertion but not in those carrying two base insertions. This study highlights the importance of in-depth analysis of the molecular consequences of specific patient mutations, which even when they occur at the same site can have drastically different consequences.

Retroviral Integration Site Selection

The stable insertion of a copy of their genome into the host cell genome is an essential step of the life cycle of retroviruses. The site of viral DNA integration, mediated by the viral-encoded integrase enzyme, has important consequences for both the virus and the host cell. The analysis of retroviral integration site distribution was facilitated by the availability of the human genome sequence, revealing the non-random feature of integration site selection and identifying different favored and disfavored genomic locations for individual retroviruses. This review will summarize the current knowledge about retroviral differences in their integration site preferences as well as the mechanisms involved in this process.

The use of proteomics to identify markers associated with metastasis in "Leishmania Viannia" species the role of tryparedoxin peroxidase

RESUME En Amérique Centrale et en Amérique du Sud, la leishmaniose cutanéo-muqueuse (LCM) est provoquée par le protozoaire Leishmania du sous-genre Viannia dont font partie L. (V.) braziliensis, L. (V.) panamensis et L. (V.) guyanensis. Dans la LCM, après guérison apparente de la lésion primitive, des lésions secondaires peuvent apparaître dues à la migration de l'infection à partir du site d'inoculation vers les muqueuses de l'ororhino-pharynx. Ce type de dissémination, communément appelé métastase, peut se produire plusieurs années après la guérison de la lésion cutanée initiale, et est un facteur majeur contribuant à la morbidité associée à la LCM. L'expression reproductible de l'activité métastatique au sein de populations discrètes de leishmanies chez le hamster fournit un modèle expérimental permettant d'étudier le degré de virulence du parasite. Nous avons utilisé des clones de L. (V.) guyanensis présentant des phénotypes stables allant d'un caractère hautement métastatique (M+) à non-métastatique (M-) comme outils pour mettre en évidence des facteurs spécifiques liés à la métastase chez les leishmanies du Nouveau Monde. Des analyses protéomiques comparatives utilisant l'électrophorèse bidimensionnelle sur gel de polyacrylamide couplée à de la spectrométrie de masse ont permis l'identification de plusieurs formes de la tryparedoxine peroxidase (TXNPx) en tant que polypeptides associés au phénotype métastatique. TXNPx, une enzyme de la famille des peroxiredoxines (Prxs), protéines antioxydantes, fonctionne comme la dernière peroxydase d'une cascade d'oxydoréductases qui réduit le peroxyde d'hydrogène aux dépens de NADPH. Toutes les Prxs sont caractérisées par un (1-Cys Prx) ou par deux résidus cystéines (2-Cys Prx), respectivement placés dans un environnement structurel conservé de la protéine et sont centrales dans la réaction catalytique. Des immuno-empreintes (« immunoblotting ») ont révélé que TXNPx est présente sous forme dimérique dans les promastigotes (M+) alors que dans les promastigotes, (M-) TXNPx est présente sous forme monomérique et dimérique. Cette caractéristique spécifique de dimérisation pourrait expliquer les différentes activités enzymatiques observées entre les deux promastigotes (M+) et (M-) en présence de peroxyde d'hydrogène ainsi que leur différence de survie et de charge parasitaire à l'intérieur des macrophages. Par conséquent, le processus métastatique pourrait être lié à la capacité du parasite à échapper efficacement aux défenses microbicides de la cellule hôte. ABSTRACT In South and Central America, protozoan parasites of the Leishmania Viannia subgenus including L. (V.) braziliensis, L. (V.) guyanensis and L. (V). panamensis cause mucocutaneous leishmaniasis (MCL). In MCL, after apparent cure of the primary lesion, secondary lesions may appear in the nasopharyngeal tissues of the infected host due to dissemination of the infection from the inoculation site. This type of dissemination, known as metastasis, can occur several years after healing of the original cutaneous lesion, and is a major contributory factor to the morbidity associated with MCL. The reproducible expression of metastasis by discrete populations of Leishmania parasites in hamsters provides an experimental model to examine the expression of parasite virulence. We used laboratory clones of L. (V.) guyanensis with stable phenotypes ranging from highly metastatic (M+) to non-metastatic (M-) as tools for the discovery of specific factors associated with metastasis in New World Leishmania species. Comparative proteome analyses via 2D-electrophoresis (2-DE) coupled with mass spectrometry (MS) enabled the identification of various isoforms of tryparedoxin peroxidase (TXNPx) as polypeptides associated with the metastatic phenotype. TXNPx, an enzyme related to the antioxidant peroxiredoxin family (Prx) functions as the terminal peroxidase of a redox cascade that reduces hydroperoxides by NADPH. All Prxs are characterized by one (1-Cys Prx) or two cysteine residue(s) (2-Cys Prx), respectively, located in a conserved structural environment of the protein which are central for the catalytic reaction. Immunoblotting analysis revealed that, under non-reducing denaturing conditions, TXNPx is present in dimeric forms in (M+) promastigotes, whereas in (M-) promastigotes, both monomeric and dimeric forms are found. This specific dimerization feature may explain the different enzymatic activities of both (M+) and (M-) promastigote parasites in the presence of H2O2 and their difference in survival and parasite load inside macrophages. Therefore, the metastatic process could be related to the ability of the parasite to efficiently evade the microbicidal effect of the host cell.

Molecular basis of interaction between Na,K-ATPase and FXYD proteins.

Résumé au large public Notre corps est constitué de différents types de cellules. La condition minimale ou primordiale pour la survie des cellules est d'avoir de l'énergie. Cette tâche est assumée en partie par une protéine qui se situe dans la membrane de chaque cellule. Nommé Na, K¬ATPase ou pompe à sodium, c'est une protéine pressente dans toutes les cellules chez les mammifères est composée de deux sous-unités, α et β. En transportant 3 ions de sodium hors de la cellule et 2 ions de potassium à l'intérieur de la cellule, elle transforme l'énergie chimique sous forme de l'ATP en énergie motrice, qui permet aux cellules par la suite d'échanger des matériaux entre l'espace intracellulaire et extracellulaire ainsi que d'ingérer des nutriments provenant de son environnement. Le manque de cette protéine chez la souris entraîne la mort de l'embryon. Des défauts fonctionnels de cette protéine sont responsables de plusieurs maladies humaines comme par exemple, un type de migraine. En dehors de sa fonction vitale, cette protéine est également engagée dans diverses activités physiologiques comme la contractilité musculaire, l'activité nerveuse et la régulation du volume sanguin. Vue l'importance de cette protéine, sa découverte par Jens C. Skou en 1957 a été honorée d'un Prix Noble de chimie quarante ans plus tard. Depuis lors, nous connaissons de mieux en mieux les mécanismes de fonctionnement de la Na, K-ATPase. Entre autre, sa régulation par une famille de protéines appelées protéines FXYD. Cette famille contient 7 membres (FXYD 1-7). L'un d'entre eux nommé FXYD 2 est lié à une maladie héréditaire connue sous le nom de hypomagnesemia. Nous disposons actuellement d'informations concernant les conséquences de la régulation par les protéines FXYD sur activité de la Na, K-ATPase, mais nous savons très peu sur le mode d'interaction entre les protéines FXYD et la Na, K-ATPase. Dans ce travail de thèse, nous avons réussi à localiser des zones d'interaction dans la sous- unité a de la Na, K-ATPase et dans FXYD 7. En même temps, nous avons déterminé un 3ème site de liaison spécifique au sodium de la Na, K-ATPase. Une partie de ce site se situe à l'intérieur d'un domaine protéique qui interagit avec les protéines FXYD. De plus, ce site a été démontré comme responsable d'un mécanisme de transport de la Na, K-ATPase caractérisé par un influx ionique. En conclusion, les résultats de ce travail de thèse fournissent de nouvelles preuves sur les régions d'interaction entre la Na, K-ATPase et les protéines FXYD. La détermination d'un 3ème site spécifique au sodium et sa relation avec un influx ionique offrent la possibilité 1) d'explorer les mécanismes avec lesquels les protéines FXYD régulent l'activité de la Na, ATPase et 2) de localiser un site à sodium qui est essentielle pour mieux comprendre l'organisation et le fonctionnement de la Na, K-ATPase. Résumé Les gradients de concentration de Na+ et de K+ à travers la membrane plasmatique des cellules animales sont cruciaux pour la survie et l'homéostasie de cellules. De plus, des fonctions cellulaires spécifiques telles que la reabsorption de Na dans le rein et le côlon, la contraction musculaire et l'excitabilité nerveuse dépendent de ces gradients. La Na, K¬ATPase ou pompe à sodium est une protéine membranaire ubiquitaire. Elle crée et maintient ces gradients en utilisant l'énergie obtenu par l'hydrolyse de l'adénosine triphosphate. L'unité fonctionnelle minimale de cette protéine se compose d'une sous-unité catalytique α et d'une sous-unité régulatrice β. Récemment, il a été montré que des membres de la famille FXYD, sont des régulateurs tissu-spécifiques de la Na, K-ATPase qui influencent ses propriétés de transport. Cependant, on connaît peu de chose au sujet de la nature moléculaire de l'interaction entre les protéines FXYD et la Na, K-ATPase. Dans cette étude, nous fournissons, pour la première fois, l'évidence directe que des résidus du domaine transmembranaire (TM) 9 de la sous-unité α de la Na, K-ATPase sont impliqués dans l'interaction fonctionnelle et structurale avec les protéines FXYD. De plus nous avons identifié des régions dans le domaine transmembranaire de FXYD 7 qui sont importantes pour l'association stable avec la Na, K-ATPase et une série de résidus responsables des régulations fonctionnelles. Nous avons aussi montré les contributions fonctionnelles du TM 9 de la Na, K-ATPase à la translocation de Na + en déterminant un 3ème site spécifique au Na+. Ce site se situe probablement dans un espace entre TM 9, TM 6 et TM 5 de la sous-unité α de la pompe à sodium. De plus, nous avons constaté que le 3ème site de Na + est fonctionnellement lié à un courant entrant de la pompe sensible à l'ouabaïne et activé par le pH acide. En conclusion, ce travail donne de nouvelles perspectives de l'interaction structurale et fonctionnelle entre les protéines FXYD et la Na, K-ATPase. En outre, les contributions fonctionnelles de TM 9 offrent de nouvelles possibilités pour explorer le mécanisme par lequel les protéines FXYD régulent les propriétés fonctionnelles de la Na, K-ATPase. La détermination du 3ème site au Na + fournit une compréhension avancée du site spécifique au Na + de la Na, K-ATPase et du mécanisme de transport de la Na, K-ATPase. Summary The Na+ and K+ gradients across the plasma membrane of animal cells are crucial for cell survival and homeostasis. Moreover, specific tissue functions such as Na+ reabsorption in kidney and colon, muscle contraction and nerve excitability depend on the maintenance of these gradients. Na, K-ATPase or sodium pump, an ubiquitous membrane protein, creates and maintains these gradients by using the energy from the hydrolysis of ATP. The minimal functional unit of this protein is composed of a catalytic α subunit and a regulatory β subunit. Recently, members of the FXYD family, have been reported to be tissue-specific regulators of Na, K-ATPase by influencing its transport properties. However, little is known about the molecular nature of the interaction between FXYD proteins and Na, K-ATPase. In this study, we provide, for the first time, direct evidence that residues from the transmembrane (TM) domain 9 of the α subunit of Na, K-ATPase are implicated in the functional and structural interaction with FXYD proteins. Moreover, we have identified regions in the TM domain of FXYD 7 important for the stable association with Na, K-ATPase and a stretch of residues responsible for the functional regulations. We have further revealed the functional contributions of TM 9 of the Na, K-ATPase α subunit to the Na+ translocation by determining a 3rd Na+-specific cation binding site. This site is likely in a space between TM 9, TM 6 and TM 5 of the a subunit of the sodium pump. Moreover, we have found that the 3rd Na+ binding site is functionally linked to an acidic pH- activated ouabain-sensitive inward pump current. In conclusion, this work gives new insights into the structural and functional interaction between FXYD proteins and Na, K-ATPase. Functional contributions of TM 9 offer new possibilities to explore the mechanism by which FXYD proteins regulate functional properties of Na, K-ATPase. The determination of the 3rd Na+ binding site provides an advanced understanding concerning the Na+ -specific binding site of Na, K-ATPase and the 3rd Na+ site related transport mechanism.

Identification of a novel splice-site mutation in the CYP1A2 gene.

AIMS: To identify the molecular basis for a low CYP1A2 metabolic status, as determined by a caffeine phenotyping test, in a 71-year-old, nonsmoking, Caucasian woman who presented with very high clozapine concentrations despite being administered a standard dose of the drug. METHODS: The nucleotide sequence of the 7 exons, exon-intron boundaries and 5'-flanking region of the CYP1A2 gene was analysed by direct sequencing. RESULTS: Only one heterozygous point mutation was identified in the donor splice site of intron 6 (3534G > A) of CYP1A2. This mutation could cause abnormal RNA splicing and therefore lead to a truncated nonfunctional enzyme. No other carrier of this mutation was identified in a population of 100 unrelated healthy Caucasians. CONCLUSIONS: This is the first report of a splice-site mutation affecting the CYP1A2 gene. This polymorphism is a likely explanation for the low CYP1A2 activity associated with high clozapine concentrations in this patient.

Antiviral activity of a small-molecule inhibitor of arenavirus glycoprotein processing by the cellular site 1 protease.

Arenaviruses merit interest as clinically important human pathogens and include several causative agents, chiefly Lassa virus (LASV), of hemorrhagic fever disease in humans. There are no licensed LASV vaccines, and current antiarenavirus therapy is limited to the use of ribavirin, which is only partially effective and is associated with significant side effects. The arenavirus glycoprotein (GP) precursor GPC is processed by the cellular site 1 protease (S1P) to generate the peripheral virion attachment protein GP1 and the fusion-active transmembrane protein GP2, which is critical for production of infectious progeny and virus propagation. Therefore, S1P-mediated processing of arenavirus GPC is a promising target for therapeutic intervention. To this end, we have evaluated the antiarenaviral activity of PF-429242, a recently described small-molecule inhibitor of S1P. PF-429242 efficiently prevented the processing of GPC from the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) and LASV, which correlated with the compound's potent antiviral activity against LCMV and LASV in cultured cells. In contrast, a recombinant LCMV expressing a GPC whose processing into GP1 and GP2 was mediated by furin, instead of S1P, was highly resistant to PF-429242 treatment. PF-429242 did not affect virus RNA replication or budding but had a modest effect on virus cell entry, indicating that the antiarenaviral activity of PF-429242 was mostly related to its ability to inhibit S1P-mediated processing of arenavirus GPC. Our findings support the feasibility of using small-molecule inhibitors of S1P-mediated processing of arenavirus GPC as a novel antiviral strategy.

An autoregulatory loop controlling CYP1A1 gene expression: role of H(2)O(2) and NFI.

Cytochrome P450 1A1 (CYP1A1), like many monooxygenases, can produce reactive oxygen species during its catalytic cycle. Apart from the well-characterized xenobiotic-elicited induction, the regulatory mechanisms involved in the control of the steady-state activity of CYP1A1 have not been elucidated. We show here that reactive oxygen species generated from the activity of CYP1A1 limit the levels of induced CYP1A1 mRNAs. The mechanism involves the repression of the CYP1A1 gene promoter activity in a negative-feedback autoregulatory loop. Indeed, increasing the CYP1A1 activity by transfecting CYP1A1 expression vectors into hepatoma cells elicited an oxidative stress and led to the repression of a reporter gene driven by the CYP1A1 gene promoter. This negative autoregulation is abolished by ellipticine (an inhibitor of CYP1A1) and by catalase (which catalyzes H(2)O(2) catabolism), thus implying that H(2)O(2) is an intermediate. Down-regulation is also abolished by the mutation of the proximal nuclear factor I (NFI) site in the promoter. The transactivating domain of NFI/CTF was found to act in synergy with the arylhydrocarbon receptor pathway during the induction of CYP1A1 by 2,3,7,8-tetrachloro-p-dibenzodioxin. Using an NFI/CTF-Gal4 fusion, we show that NFI/CTF transactivating function is decreased by a high activity of CYP1A1. This regulation is also abolished by catalase or ellipticine. Consistently, the transactivating function of NFI/CTF is repressed in cells treated with H(2)O(2), a novel finding indicating that the transactivating domain of a transcription factor can be targeted by oxidative stress. In conclusion, an autoregulatory loop leads to the fine tuning of the CYP1A1 gene expression through the down-regulation of NFI activity by CYP1A1-based H(2)O(2) production. This mechanism allows a limitation of the potentially toxic CYP1A1 activity within the cell.

A functional and structural study of the major metalloprotease secreted by the pathogenic fungus Aspergillus fumigatus.

Fungalysins are secreted fungal peptidases with the ability to degrade the extracellular matrix proteins elastin and collagen and are thought to act as virulence factors in diseases caused by fungi. Fungalysins constitute a unique family among zinc-dependent peptidases that bears low sequence similarity to known bacterial peptidases of the thermolysin family. The crystal structure of the archetype of the fungalysin family, Aspergillus fumigatus metalloprotease (AfuMep), has been obtained for the first time. The 1.8 Å resolution structure of AfuMep corresponds to that of an autoproteolyzed proenzyme with separate polypeptide chains corresponding to the N-terminal prodomain in a binary complex with the C-terminal zinc-bound catalytic domain. The prodomain consists of a tandem of cystatin-like folds whose C-terminal end is buried into the active-site cleft of the catalytic domain. The catalytic domain harbouring the key catalytic zinc ion and its ligands, two histidines and one glutamic acid, undergoes a conspicuous rearrangement of its N-terminal end during maturation. One key positively charged amino-acid residue and the C-terminal disulfide bridge appear to contribute to its structural-functional properties. Thus, structural, biophysical and biochemical analysis were combined to provide a deeper comprehension of the underlying properties of A. fumigatus fungalysin, serving as a framework for the as yet poorly known metallopeptidases from pathogenic fungi.

Nucleotide sequence of the 5' noncoding region and part of the gag gene of mouse mammary tumor virus; identification of the 5' splicing site for subgenomic mRNAs.

We have determined the sequence of the first 1371 nucleotides at the 5' end of the genome of mouse mammary tumor virus using molecularly cloned proviral DNA of the GR virus strain. The most likely initiation codon used for the gag gene of mouse mammary tumor virus is the first one, located 312 nucleotides from the 5' end of the viral RNA. The 5' splicing site for the subgenomic mRNA's is located approximately 288 nucleotides downstream from the 5' end of the viral RNA. From the DNA sequence the amino acid sequence of the N-terminal half of the gag precursor protein, including p10 and p21, was deduced (353 amino acids).

Ectopic human telomerase catalytic subunit expression maintains telomere length but is not sufficient for CD8+ T lymphocyte immortalization.

Like most somatic human cells, T lymphocytes have a limited replicative life span. This phenomenon, called senescence, presents a serious barrier to clinical applications that require large numbers of Ag-specific T cells such as adoptive transfer therapy. Ectopic expression of hTERT, the human catalytic subunit of the enzyme telomerase, permits fibroblasts and endothelial cells to avoid senescence and to become immortal. In an attempt to immortalize normal human CD8(+) T lymphocytes, we infected bulk cultures or clones of these cells with a retrovirus transducing an hTERT cDNA clone. More than 90% of transduced cells expressed the transgene, and the cell populations contained high levels of telomerase activity. Measuring the content of total telomere repeats in individual cells (by flowFISH) we found that ectopic hTERT expression reversed the gradual loss of telomeric DNA observed in control populations during long term culture. Telomere length in transduced cells reached the levels observed in freshly isolated normal CD8(+) lymphocytes. Nevertheless, all hTERT-transduced populations stopped to divide at the same time as nontransduced or vector-transduced control cells. When kept in IL-2 the arrested cells remained alive. Our results indicate that hTERT may be required but is not sufficient to immortalize human T lymphocytes.

GroEL and CCT are catalytic unfoldases mediating out-of-cage polypeptide refolding without ATP.

Chaperonins are cage-like complexes in which nonnative polypeptides prone to aggregation are thought to reach their native state optimally. However, they also may use ATP to unfold stably bound misfolded polypeptides and mediate the out-of-cage native refolding of large proteins. Here, we show that even without ATP and GroES, both GroEL and the eukaryotic chaperonin containing t-complex polypeptide 1 (CCT/TRiC) can unfold stable misfolded polypeptide conformers and readily release them from the access ways to the cage. Reconciling earlier disparate experimental observations to ours, we present a comprehensive model whereby following unfolding on the upper cavity, in-cage confinement is not needed for the released intermediates to slowly reach their native state in solution. As over-sticky intermediates occasionally stall the catalytic unfoldase sites, GroES mobile loops and ATP are necessary to dissociate the inhibitory species and regenerate the unfolding activity. Thus, chaperonin rings are not obligate confining antiaggregation cages. They are polypeptide unfoldases that can iteratively convert stable off-pathway conformers into functional proteins.

On the cellular site of two-pore channel TPC1 action in the Poaceae.

The slow vacuolar (SV) channel has been characterized in different dicots by patch-clamp recordings. This channel represents the major cation conductance of the largest organelle in most plant cells. Studies with the tpc1-2 mutant of the model dicot plant Arabidopsis thaliana identified the SV channel as the product of the TPC1 gene. By contrast, research on rice and wheat TPC1 suggested that the monocot gene encodes a plasma membrane calcium-permeable channel. To explore the site of action of grass TPC1 channels, we expressed OsTPC1 from rice (Oryza sativa) and TaTPC1 from wheat (Triticum aestivum) in the background of the Arabidopsis tpc1-2 mutant. Cross-species tpc1 complementation and patch-clamping of vacuoles using Arabidopsis and rice tpc1 null mutants documented that both monocot TPC1 genes were capable of rescuing the SV channel deficit. Vacuoles from wild-type rice but not the tpc1 loss-of-function mutant harbor SV channels exhibiting the hallmark properties of dicot TPC1/SV channels. When expressed in human embryonic kidney (HEK293) cells OsTPC1 was targeted to Lysotracker-Red-positive organelles. The finding that the rice TPC1, just like those from the model plant Arabidopsis and even animal cells, is localized and active in lyso-vacuolar membranes associates this cation channel species with endomembrane function.

Laparoscope use and surgical site infections in digestive surgery

Rapport de synthèse : But: comparer les taux d'infections du site chirurgical (ISC) en fonction de la voie d'abord, ouverte ou laparoscopique, pour 3 procédures : l'appendicectomie, la cholécystectomie et la colectomie. Evaluer l'effet de la laparoscopie sur l'ISC pour ces trois interventions. Contexte : la laparoscopie est associée à de nombreux avantages par rapport à la chirurgie ouverte. Parmi ceux-ci, des taux inférieurs d'ISC ont été rapportés lors de laparoscopie. Ceci a été décrit en particulier lors de cholécystectomie. Mais des biais tels que le manque de suivi après la sortie de l'hôpital, et certains facteurs confondants, auraient pu contribuer à l'observation de différences entre ces deux techniques. Méthode : étude descriptive basée sur des données collectées entre mars 1998 et décembre 2004 de manière prospective dans le cadre d'un programme de surveillance des ISC dans 8 hôpitaux suisses. Ce programme comportait un suivi standardisé après le départ de l'hôpital. Les taux d'ISC ont été comparés après interventions faites par laparoscopie et chirurgie ouverte. Différents paramètres pouvant influencer la survenue d'une infection ont été identifiés en utilisant des modèles de régression logistiques. Résultats : les taux d'ISC après interventions par laparoscopie et par voie ouverte ont été respectivement de 59/1051 (5.6%) versus 117/1417 (8.3%) après appendicectomie (p = 0.01), 46/2606 (1.7%) versus 35/144 (7.9%) après cholécystectomie (p < 0.0001), et 35/311 (11.3%) versus 400/1781 (22.5%) après colectomie (p < 0,0001). Après ajustement, les interventions par laparoscopie étaient associées à un taux inférieur d'ISC : odds ratio = 0.61 (IC 95% : 0.43 - 0.87) pour l'appendicectomie, 0.27 (0.16 - 0.43) pour la cholécystectomie et 0.43 (0.29 - 0.63) pour la colectomie. Discussion et conclusion : bien que les patients aient quitté plus tôt l'hôpital après une intervention laparoscopique, leur suivi à un mois a été identique, ce qui a permis d'éviter une sous-estimation des ISC après chirurgie laparoscopique. De plus, l'analyse multivariée a inclus de nombreux facteurs potentiellement confondants, et l'utilisation de la laparoscopie était indépendamment et significativement liée à un effet protecteur à l'égard de l'ISC. La laparoscopie lors d'appendicectomie, cholécystectomie et colectomie semble diminuer le taux d'ISC en comparaison à la même chirurgie pratiquée par voie ouverte. Lorsqu'elle est faisable, cette voie d'abord minimalement invasive devrait être préférée à la chirurgie ouverte.