Generation of human TRIM5alpha mutants with high HIV-1 restriction activity.

Rhesus macaque tripartite motif (TRIM)5alpha potently inhibits early stages of human immunodeficiency virus (HIV)-1 replication, while the human orthologue has little effect on this virus. We used PCR-based random mutagenesis to construct a large library of human TRIM5alpha variants containing mutations in the PRYSPRY domain. We then applied a functional screen to isolate human cells made resistant to HIV-1 infection by the expression of a mutated TRIM5alpha. This protocol led to the characterization of a human TRIM5alpha variant containing a mutation at arginine 335 as conferring resistance to HIV-1 infection. The level of protection stemming from expression of this mutant was comparable to that of previously described mutations at position 332. R332/R335 double mutants decreased permissiveness to HIV-1 and to other lentiviruses by 20- to 50-fold in TE671 fibroblasts and in the T-cell line SUP-T1, and prevented HIV-1 spreading infection as efficiently as the rhesus macaque TRIM5alpha orthologue did. The finding that only two substitutions in human TRIM5alpha can confer resistance to HIV-1 at levels as high as one of the most potent natural orthologues of TRIM5alpha removes a roadblock toward the use of this restriction factor in human gene therapy applications.

Vascular injury induces posttranscriptional regulation of the Id3 gene: cloning of a novel Id3 isoform expressed during vascular lesion formation in rat and human atherosclerosis.

The molecular mechanisms that regulate the proliferation of smooth muscle cells (SMCs) of the vasculature in response to injury are poorly understood. Members of the inhibitor of DNA binding (Id) class of helix-loop-helix transcription factors are known to regulate the growth of a variety of cell types; however, the expression of the various Id genes in SMCs and in vascular lesions has not been examined. In the present study, the yeast 2-hybrid system was used to clone Id genes from a cultured rat aortic SMC library. By use of ubiquitous E proteins as bait, Id3 and a novel isoform of Id3 (Id3a) were cloned. Id3a is the product of alternative splicing of the Id3 gene, resulting in inclusion of a 115-bp "coding intron," which encodes a unique 29-amino acid carboxyl terminus for the Id3a protein. Unlike Id3, Id3a mRNA was not detected in the normal rat carotid artery. However, after balloon injury, Id3a was abundantly expressed throughout the neointimal layer. In addition, mRNA of the human homologue of Id3a (Id3L) was detected in human carotid atherosclerotic plaques. Adenovirus-mediated overexpression of these Id3 isoforms in cultured rat aortic SMCs revealed that infection of SMCs with an adenovirus overexpressing Id3a (in contrast to Id3) resulted in a significant decrease in cell number versus AdLacZ-infected cells. DNA fragmentation analysis suggested that this decrease in SMC viability was due to increased apoptotic activity in cells infected with adenovirus overexpressing Id3a. These results provide evidence that alternative splicing of the Id3 gene may represent an important mechanism by which neointimal SMC growth is attenuated during vascular lesion formation.

Multiple effects of an anti-human immunodeficiency virus nucleocapsid inhibitor on virus morphology and replication.

Human immunodeficiency virus type 1 nucleocapsid protein is a major structural component of the virion core and a key factor involved in proviral DNA synthesis and virus formation. 2,2'-Dithiobenzamides (DIBA-1) and related compounds that are inhibitors of NCp7 are thought to eject zinc ions from NCp7 zinc fingers, inhibiting the maturation of virion proteins. Here, we show that the presence of DIBA-1 at the time of virus formation causes morphological malformations of the virus and reduces proviral DNA synthesis. Thus, it seems that DIBA-1 is responsible for a "core-freezing effect," as shown by electron microscopy analyses. DIBA-1 can also directly interfere with the fate of the newly made proviral DNA in a manner independent of its effects on virion core formation. These data strongly suggest that nucleocapsid protein is a prime target for new compounds aimed at inhibiting human immunodeficiency virus and other retroviruses.

[Palliative treatment of adenocarcinoma of the cardia: is there a role for surgery?]. / Traitement palliatif de l'adénocarcinome du cardia: y a-t-il une place pour la chirurgie?

OBJECTIVES: The value of palliative surgery for adenocarcinoma of the cardia (AC) is controversial, and specific studies are lacking. The aim of this study was to report the results of a palliative resection for AC in 69 patients. METHODS: From 1980 to 1993, 69 patients (mean age 59 +/- 10 years) underwent a palliative resection for AC. Palliative resection was defined by macroscopically incomplete resection, tumoral involvement of resection margins, visceral or serosal metastasis, or N3 metastatic nodes. Patients were classified according to the diagnosis of palliation established preoperatively (group A, n = 26), peroperatively (group B, n = 35), or postoperatively (group C, n = 8) respectively. RESULTS: Six patients (8.7%) died postoperatively. Mortality rates were 3.8%, 8.6% and 25% in groups A, B and C, respectively. Twenty one patients (30%) had postoperative non-fatal complications. Median global survival was 9 months (mean 11 +/- 7 months) without significant difference between groups A, B and C. Forty-four out of 51 patients (86%) followed until death did not have dysphagia. The other patients were free of dysphagia during an average of 70% of the follow-up duration. Among the 14 patients surviving postoperatively with a tumoral esophageal margin, none experienced dysphagia from anastomotic recurrence during follow-up. CONCLUSIONS: In selected patients with AC, a palliative resection can be achieved with an acceptable mortality and a very good functional result. This result can justify a prospective comparison between palliative surgery and alternative treatments.

Role of the N-terminal zinc finger of human immunodeficiency virus type 1 nucleocapsid protein in virus structure and replication.

Nucleocapsid protein (NCp7) of human immunodeficiency virus type 1 is found covering the genomic RNA in the interior of the viral particle. It is a highly basic protein with two zinc fingers of the form CX2CX4HX4C which exhibit strong affinity for a zinc cation. To study the structure-function relationship of the N-terminal zinc finger of NCp7, this domain was either deleted or changed to CX2CX4CX4C. We examined virus formation and structure as well as proviral DNA synthesis. Our data show that these two NC mutations result in the formation of particles with an abnormal core morphology and impair the end of proviral DNA synthesis, leading to noninfectious viruses.

Mutations in the N-terminal domain of human immunodeficiency virus type 1 nucleocapsid protein affect virion core structure and proviral DNA synthesis.

Nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1) is a small basic nucleic acid binding protein containing two zinc fingers of the form (CX2CX4HX4C) and is present at about 2,000 copies inside the viral core. NCp7 molecules are tightly associated with the genomic RNA dimer to form the nucleocapsid, which also includes reverse transcriptase and integrase proteins. In vitro, NCp7 has been shown to bind specifically to HIV-1 RNA, inducing NCp7-NCp7 interactions. In the viral context, mutagenesis of amino acid residues in the zinc finger domains showed that NCp7 is responsible for the specific incorporation of genomic RNA into virions and is necessary for correct virion assembly and maturation. In this work, we investigated the consequences of mutating conserved basic residues in the N-terminal region that precedes the first zinc finger. Two of the mutants were poorly infectious and showed only limited, though significant, defects in RNA encapsidation and viral protein maturation. Electron microscopy, together with sucrose gradient analysis, revealed defects in particle core structure and heterogeneity among mutant virions. These defects were associated with strong reduction of proviral DNA synthesis and stability in newly infected cells. Taken together, these data show multiple and probably interdependent implications for the NCp7 protein in both early and late phases of the HIV-1 replicative cycle and emphasize it as a target for antiviral drug development.

[Results of the surgical treatment of cancer of the cardia]. / Résultats du traitement chirurgical du cancer du cardia.

OBJECTIVES: In the curative treatment of the adenocarcinoma of the cardia (AC), the extent of the esogastrectomy and the need for lymph node dissection are still debated. The palliative treatment of AC is now currently non-surgical. The aim of this study was (a): to assess early results of palliative surgery; and (b) to evaluate the results of curative resection with reference to the influence of the extent of gastrectomy and lymph node dissection on early results and long-term survival. Methods. From 1979 to 1989, 179 patients (mean age = 60 +/- 12 years) with AC had 45 palliative resections (mean age = 56 +/- 15) and 134 curative resections (mean age = 61 +/- 12). Thirty-eight proximal subtotal esogastrectomy (PSOG) and 7 total esogastrectomy (TOG) were palliative; 72 PSOG and 62 TOG extended to the spleen were curative and associated with lymphadenectomy. RESULTS: The operative mortality rate was 8.9% regardless of the palliative or curative intent of resection. After palliative resection, the mortality rate was 2.6% (1/38) after PSOG and 42.9% (3/7) after OGT = (p = 0.01); the median survival was 8 months. After curative resection, the mortality rate was 12.5% (9/72) after PSOG and 4.8% (3/62) after extended TOG (p = 0.2); actuarial 5-year survival rate was 42% after PSOG and 39% after extended TOG. CONCLUSIONS: These results suggests that: (a) palliative PSOG for AC can be performed with a low mortality; and (b) resection with extensive lymphadenectomy allows substantial survival regardless of the extent of gastrectomy.

A dual role of the putative RNA dimerization initiation site of human immunodeficiency virus type 1 in genomic RNA packaging and proviral DNA synthesis.

In retroviruses, the genomic RNA is in the form of a 60S-70S complex composed of two identical genome-length RNA molecules tightly associated through numerous interactions. A major interaction, called the dimer linkage structure, has been found near the RNA 5' end and is probably involved in the control of translation, packaging, and recombination during proviral DNA synthesis. Recently, a small sequence corresponding to a stem-loop structure located in the 5' leader of human immunodeficiency virus type 1 (HIV-1) RNA was found to be required for the initiation of HIV-1 RNA dimerization in vitro and named the dimerization initiation site (E. Skripkin, J.-C. Paillart, R. Marquet, B. Ehresmann, and C. Ehresmann, Proc. Natl. Acad. Sci. USA 91: 4945-4949, 1994). To investigate the possible role of this 5' stem-loop in HIV-1 virion formation and infectivity, four mutant viruses were generated and analyzed in vivo. Results show that deletion of the stem-loop structure reduces infectivity by a factor of 10(3) whereas loop substitutions cause a decrease of 10- to 100-fold. The level of genomic RNA packaging was found to be decreased fivefold in mutants virions containing the stem-loop deletion and only twofold in the loop-substituted virions. Surprisingly, the second DNA strand transfer during reverse transcription was found to be severely impaired upon stem-loop deletion. Taken together, these results indicate that the stem-loop structure called the dimerization initiation site is a cis element acting on both genomic RNA packaging and synthesis of proviral DNA.

Laparoscopic mobilization of the stomach for oesophageal replacement.

Nine patients of mean(s.d.) age 61(8) years underwent oesophagogastrectomy with laparoscopic gastric mobilization and abdominal lymphadenectomy for oesophageal cancer. Moderate to severe airway obstruction was present in all patients, in whom the mean(s.d.) value of forced expiratory flow rate at 1 s was 65(17) (range 35-85) per cent of the predicted value. Six patients had an abdominal laparoscopic approach combined with a right open thoracotomy; the other three had a laparoscopic abdominal and transhiatal approach combined with a left cervicotomy. No patient required conversion to open laparotomy. All had an uneventful postoperative course with extubation occurring at the end of the surgical procedure (n = 2) or on day 1 after operation (n = 7). Mean(s.d.) duration of hospitalization was 10.3(3.1) (range 8-18) days. The laparoscopic approach for gastric mobilization and abdominal lymphadenectomy is safe and can be used in patients with impaired pulmonary function.

[Adenocarcinoma of the cardia: does the extent of gastric resection and lymph node excision influence survival?]. / Adénocarcinome du cardia: l'étendue de l'exérèse gastrique et du curage ganglionnaire influence-t-elle la survie?

OBJECTIVES: In the curative treatment of the adenocarcinoma of the cardia (AC), the extent of the esogastrectomy and the need for lymph node dissection are still debated. The palliative treatment of AC is now currently non-surgical. The aim of this study was: a) to assess early results of palliative surgery; b) to evaluate the results of curative resection with reference to the influence of the extent of gastrectomy and lymph node dissection on early results and long-term survival. METHODS: From 1979 to 1989, 179 patients (mean age = 60 +/- 12 years) with AC had 45 palliative resections (mean age = 56 +/- 15) and 134 curative resections (mean age = 61 +/- 12). Thirty-eight proximal subtotal esogastrectomies (PSOG) and 7 total esogastrectomies (TOG) were palliative; 72 PSOG and 62 TOG extended to the spleen were curative and associated with lymphadenectomy. RESULTS: The operative mortality rate was 8.9% regardless of the palliative or curative intent of resection. After palliative resection, the mortality rate was 2.6% (1 case out of 38) after PSOG and 42.9% (3 cases out of 7) after OGT (P = 0.01); the median survival was 8 months. After curative resection, the mortality rate was 12.5% (9 cases out of 72) after PSOG and 4.8% (3 cases out of 62) after extended TOG (P = 0.2); actuarial 5-year survival rate was 42% after PSOG and 39% after extended TOG. CONCLUSIONS: These results suggest that: a) palliative PSOG for AC can be performed with a low mortality; b) resection with extensive lymphadenectomy allows substantial survival regardless of the extent of gastrectomy.

[Imaging of cancer of the esophagus: ultrasound-endoscopy or computed tomography?]. / Imagerie du cancer de l'oesophage: échoendoscopie ou tomodensitométrie?

The aim of this retrospective study, in 100 patients who had an esophagectomy from 1987 to 1992 for squamous cell carcinoma of the thoracic esophagus, was to determine the accuracy of endoscopic ultrasonography (EUS) and computed tomography (CT) in the preoperative staging of esophageal squamous cell carcinoma (SCC). The EUS report were compared to the pathological findings for the T stage. Both EUS and CT reports were compared with surgical and pathological findings for N stage (N0/N1) and mediastinal extension. In the 100 patients, EUS accuracy for T stage was 80%; EUS was more accurate for N stage than CT (71% vs 55% respectively; p < 0.02), and as accurate as CT for mediastinal extension (86% vs 85%). In the 26 SCC estimated superficial by EUS (all passed through), EUS accuracy was 92% for T stage and 77% for the distinction between mucosal and submucosal tumours; for N stage, EUS and CT accuracies were equivalent (77% vs 81%). In the 44 SCC passed and estimated advanced by EUS, EUS accuracy for T stage was 68%; EUS was more accurate than CT for N stage (77% vs 48%, p < 0.01) and as accurate as CT for mediastinal extension (86 vs 84%). In the 30 SCC not passed by EUS, accuracies of EUS and CT were equivalent for N stage (57% vs 47%) and for mediastinal extension (73% vs 73%). This study suggests that: a) for SCC estimated superficial by EUS, EUS does not differentiate reliably mucosal from submucosal tumors and CT is useless; b) routine EUS seems unnecessary for SCC not passed through by endoscopy; c) CT is still useful in patients with advanced SCC, mainly for detection of distant metastases.

Associated primary esophageal and lung carcinoma: a study of 39 patients.

From 1979 to 1992, of 1,294 patients with esophageal squamous cell carcinoma, 39 patients (3.2%) (38 male patients, 1 female patient; mean age, 58 years) had associated primary lung carcinoma. Criteria for the diagnosis of primary lung carcinoma were: (1) non-squamous cell carcinoma tumors, (2) tumors existing before the esophageal squamous cell carcinoma, and (3) solitary squamous cell carcinoma presenting with endobronchial involvement. The two tumors were observed synchronously in 22 patients (56%) and metachronously in 17, with a mean tumor-free interval of 46 months (range, 18 to 77 months). In patients with synchronous disease, 10 underwent nonoperative treatment or a palliative surgical procedure, and 12 (55%) underwent a curative operation. In patients with metachronous disease, a curative operation was performed in all for the first tumor and in 9 (53%) for the second tumor. The overall postoperative mortality rate was 15%. Two patients (10%) died after the curative operation. None of the patients died who underwent curative esophagectomy combined with lobectomy. For the patients with synchronous disease, the 5-year survival rate was 11% in those who underwent a curative operation, and the longest survival in those who received palliative treatment was 18 months. For the patients with metachronous disease, the 5-year survival rates from the date of the diagnosis of the second tumor were 17% for those who had a curative operation and 11% for those who received palliative treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

[Perineal hernia after abdominoperineal amputation. Role of epiploplasty and/or irradiation]. / Hernie périnéale après amputation abdomino-périnéal. Role de l'épiplooplastie et/ou de l'irradiation.

Healing of the perineal wound after abdominoperineal amputation is a difficult technical problem for surgeons to resolve. The majority of current therapeutic protocols include pre- or post-operative radiotherapy, and most study results show a reduction in perineal relapses when compared with a control group, but at the price of delayed healing of the perineal wound. This delayed healing reaction varies with the radiation dose. The use of epiploplasty shortens the healing period, but when performed after pre-operative radiotherapy it can predispose to the development of a perineal hernia. Four cases of this complication are described. No standard procedure for treatment of these hernias exists, and although it is possible to use the uterus to fill the perineal cavity, the most effective method appears to be a double perineal and abdominal pathway using a plate.

[Long-term results of mastectomy in surgically treatble cancers at the Institut Curie from 1960 to 1973]. / Résultats à long terme de l'amputation du sein dans les cancers opérables à l'institut Curie de 1960 à 1973.

Mastectomy remains the treatment of reference for breast cancer. Pre or post-operative radiotherapy was the only adjuvant therapy possible for many years. This series of 2166 cases, treated between 1960 and 1973, analyses the survival, loco-regional recurrence rate, metastases and complications as a function of initial UICC staging and the type of treatment chosen. With follow up periods ranging from 15 to 25 years, we have shown that the clinical course is marked by recurrences for metastases occurring principally between the 2nd and 5th year, and that the number of events occurring beyond 10 years is still considerable. This study justifies the long term surveillance of patients. The results of current therapies need to be assessed in relation to historical data with a sufficiently long follow up, in order to confirm their efficacy.

Improved early results of elective hepatic resection for liver tumors. One hundred consecutive hepatectomies in cirrhotic and noncirrhotic patients.

Liver resection for a neoplasm was performed in 100 patients between 1979 and 1987. There were 43 hepatocellular carcinomas (70% of them arising in patients with cirrhosis), 28 metastases from colorectal cancers, 20 benign tumors, and 9 miscellaneous tumors. Forty-nine patients had a major liver resection, 36 a segmentectomy, and 15 a nonanatomic liver resection. Great care was taken to avoid intraoperative and postoperative bleeding, including late ligation of the hepatic vein, the use of Kelly fracture and resorbable clips for hemostasis of transection planes in 74 patients, and temporary clamping of the portal pedicle in 22. Drainage of the abdomen was avoided in 21 patients to prevent ascitic leakage to decrease the postoperative hospital stay. Operative mortality was 1%. There were eight major complications, including one bile leak, one subphrenic abscess, and three subphrenic hematomas. The use of resorbable clips significantly reduced operative time and transfusion requirements. In patients with cirrhosis, temporary clamping of the hepatic pedicle significantly decreased blood loss. Avoiding drainage significantly decreased the postoperative hospital stay.

Patterns of improvement in resection of hepatocellular carcinoma in cirrhotic patients: results of a non drainage policy.

A prolonged ascitic leak through abdominal drains is a source of postoperative complications and of prolonged postoperative hospital stay after liver resection for hepatocellular carcinoma (HCC) in cirrhotic patients. Therefore we elected to abstain from routine abdominal drainage in the last 14 resections in cirrhotic livers. A significantly smaller number of patients had postoperative complications following liver resections without drainage (7%) than historical controls with abdominal drainage (59%, p less than 0.01). The number of complications related to ascites was significantly greater in patients with abdominal drainage (76%) than without (0%, p less than 0.001). Postoperative hospital stay was also significantly longer following resections with abdominal drainage (19 +/- 4 days) than in patients without (12 +/- 1 days, p less than 0.01). The long postoperative hospital stay in patients with abdominal drainage was related to ascitic discharge for a mean period of 13 +/- 10 days. No clinically significant accumulation of ascites was noted in patients without drainage. A more frequent utilization of hepatic vascular inflow occlusion did not account for the better results in the group of patients without drainage. These results suggest that routine abdominal drainage should not be used following liver resection for HCC in cirrhotic patients. This appears to be another of the technical details improving postoperative results in these patients.

[Resections of hepatocellular carcinomas in cirrhosis: results of a prospective study of 28 resections]. / Résections des carcinomes hépatocellulaires sur cirrhose: résultats d'une étude prospective de 28 résections.

Twenty-eight liver resections were performed in 24 patients with cirrhosis and hepatocellular carcinoma: 6 major hepatectomies, 13 limited, and 9 atypical liver resections. Postoperative variceal rebleeding was precluded in 5 patients with previous bleeding by preoperative sclerotherapy. Intraoperative bleeding was minimized in 16 patients by clamping the hepatic pedicle. Ascites formation was prevented by reduction of intraoperative and postoperative fluid infusion. There were no operative deaths. Benign postoperative complications occurred in 5 patients (18 p. 100). In one patient, carcinoma was not found in the resected specimen. Nineteen of the 27 others tumors were less than 5 cm in diameter (70 p. 100). Twenty-two tumors were encapsulated (81 p. 100). One and two-year actuarial survival rates were 66 p. 100 and 48 p. 100 in the group of 23 patients after resection of one, or more than one tumor, respectively. In 17 patients with a tumor less than 5 cm in diameter, one and two-year survivals were 76 p 100 and 51 p. 100, respectively. In 16 patients with a free margin of healthy tissue of more than 10 mm, one and two-year survivals were 85 p. 100 and 61 p. 100 respectively. Our results suggest that: 1) the operative risk of liver resection in cirrhosis is low, provided preventive measures are taken to avoid intraoperative bleeding and postoperative variceal bleeding and ascites, and 2) late survival is good in selected groups of patients after resection of hepatocellular carcinoma and cirrhosis.