Prospective evaluation of functional outcomes after laparoscopic sigmoidectomy with high tie of the inferior mesenteric artery for diverticular disease in consecutive male patients.

BACKGROUND: To date, there has been no consensus concerning the vascular approach during sigmoid colectomy for diverticular disease. The aim of this study was to determine the functional impact of elective laparoscopic sigmoidectomy performed with high ligation of the inferior mesenteric artery for diverticulitis in consecutive male patients. METHODS: Twenty-five consecutive patients of median age 53 years were enrolled in a prospective single-centre pilot study at a tertiary teaching hospital. Main outcome measures were functional results. Patients were asked to complete standardized, validated questionnaires to evaluate preoperative and 6 months postoperative bowel symptomatology (Jorge-Wexner Incontinence Score and KESS score), urinary function (IPSS), and sexual function (IIEF). Secondary outcomes were surgical data, morbidity, and quality of life (SF-36). RESULTS: There were no significant differences between preoperative and 6 months postoperative total scores for bowel symptomatology, urinary function, and sexual function. There were no perioperative deaths. The morbidity rate was 12% including three minor and no major events. Quality of life demonstrated statistically better general health (p < 0.01) and better medical status over the prior 4 weeks at 6 months after surgery, compared to baseline. This single-centre prospective study has a limited number of patients, relatively short follow-up time, and includes only male patients. CONCLUSION: Laparoscopic sigmoidectomy with high tie of the inferior mesenteric artery for diverticular disease does not induce functional disorders at 6 months after surgery. The benefit of the operation for quality of life is even greater for general health and medical status.

Opsonic antibodies evoked by hybrid peptide copies of types 5 and 24 streptococcal M proteins synthesized in tandem.

The protective immunogenicity of a hybrid peptide containing tandem copies of types 5 and 24 epitopes was investigated. Carboxy-terminal peptides of the cyanogen bromide-derived fragment 7 (CB7) of type 24 M protein were chemically synthesized, and then extended to include the first 20 residues of the amino-terminus of type 5 M protein. When emulsified in CFA and injected into rabbits without conjugation to a carrier, each of the synthetic hybrid peptides, designated S-M5(1-20)-S-CB7(23-35)C and S-M5(1-20)-S-CB(19-34), evoked opsonic antibodies against both types 5 and 24 streptococci without raising heart tissue-crossreactive immunity. These results suggest that tandem hybrid peptides may provide a new approach to the development of multivalent vaccines, not only to different serotypes of group A streptococci but perhaps also to a variety of other infectious agents.

Strategies for improving production and purification of a recombinant protein: rP30 of Toxoplasma gondii expressed in the yeast Schizosaccharomyces pombe.

Many problems concerned with the production and the purification of recombinant proteins must be addressed prior to launching an industrial production process. Among these problems, attention is focused on low-level expression that complicates the purification step and can jeopardise the process. The expression of a membrane protein, rP30, of Toxoplasma gondii in the yeast Schizosaccharomyces pombe led to a secretion of only 0.5 microg ml(-1). In order to obtain a sufficient quantity for biochemical characterization and evaluation in vitro diagnostic test development, strategies for both production and purification had to be optimized. First, the influence of four nitrogen sources (three peptones and yeast extract) on the growth rate, but also on the separation between the protein and the components of the fermentation broth was assessed. Second, batch and fed-batch fermentations were compared in terms of final biomass and rP30 concentrations. Third, three different protocols that included fixed and expanded bed ion exchange chromatography were compared for processing a large volume of feedstock. By using the most appropriate strategies, i.e. fed-batch fermentation, capture on EBA cation exchanger and affinity chromatography polishing, a purification factor of 1778 and a yield of 49% were achieved. These performances allowed a 12.5-fold increase for the overall rP30 process productivity.

Highly specific anti-estradiol antibodies: structural characterisation and binding diversity.

Subtle modulation of antibody-binding properties by protein engineering often lies with an accurate structural and energetic description of how an antigen is recognised. Thus, with the intent to increase the affinity and add a bias in favour of natural estradiol compared with its chemically modified immunogen, we have determined the crystal structure of two anti-estradiol monoclonal antibodies, 10G6D6 and 17E12E5. Although generated against the same estradiol derivative, these antibodies share little sequence identity, which is reflected in dissimilar binding pockets and in different positioning of the steroid. In both antibodies the characteristic 17-hydroxyl group is buried deeply at the bottom of hydrophobic pockets and stabilised by hydrogen bonds. Apart from this similarity, the steroid is oriented differently in the respective binding pockets. The high specificity of both antibodies has been mapped out, and even closely related steroids show low cross-reactivity. The structural studies of the complex formed between 10G6D6 and 6-CMO-estradiol have identified contacts between the 6-CMO coupling linker and an arginine residue from the heavy chain CDR2 segment. This segment is now being targeted by random mutagenesis to select mutants with a preference for natural estradiol compared to the branched hapten.

[Topical anesthesia for out-patient adult cataract surgery without an anesthesiologist]. / L'anesthésie topique en chirurgie de la cataracte ambulatoire de l'adulte sans anesthésiste présent.

To review the local anesthesia environment in France in adult out-patient cataract surgery. The author considers the presence or absence of an anesthesiologist in the operating room. The report shows that in many circumstances there is no need for an anesthesiologist if the following criteria are respected: the adult is fully informed, in rather good health, with no acute risk factors, and surgery is performed by a senior surgeon in a certified operating room. In France, a move toward more flexible regulations is expected, with a new cooperation between ophthalmologists and anesthesiologists that will lead to a new true risk/benefit/obligation ratio. Respect of the individual and safety remain crucial requirements making systematic generalizations impossible.

Covalent linkage of the synthetic adjuvant MDP to the synthetic polypeptide (T,G)-A-L changes the specificity of the immune response at the T and B cell level.

It is now well established that the synthetic molecule MDP (N-acetylmuramyl-L-alanyl-D-isoglutamine) can be a good adjuvant of immunity when covalently linked to antigen. The question raised in this work is whether conjugation of antigen to the immunomodulatory molecule MDP can modify the specificity of the antibodies and T cells induced following immunization. Using the well characterized synthetic polypeptide antigen, poly(L-Tyr,L-Glu)-poly(DL-Ala)--poly(L-Lys) [(T,G)-A--L], we show that immunization of C57B1/6 (H-2b) mice with MDP-(T,G)-A--L conjugate elicits at least two types of antibody directed against the poly(DL-Ala)--poly(L-Lys) (A--L) part of the antigen, and against new determinant(s) formed by MDP and a portion of the (T,G)-A--L molecule. Interestingly, the poly-(L-Try L-Glu) side chains thought to constitute the major antigenic determinants of the (T,G)-A--L molecule were not recognized. Lymph node cells from (T,G)-A--L immunized mice can be equally well stimulated in vitro by (T,G)-A--L or by MDP-(T,G)-A--L, whereas lymph node cells from MDP-(T,G)-A--L primed animals can be stimulated only when challenged by the conjugate used for immunization, and not by the free synthetic polypeptide (T,G)-A--L. The data presented here show that the coupling of a low mol. wt molecule such as MDP (mol. wt approx. 500) to an antigen can greatly modify the immune response directed against this antigen. Furthermore, (1) different antibody specificities are elicited depending upon whether the priming is done with free MDP and antigen or with MDP covalently linked to the antigen; (2) although still accessible on the conjugate, an epitope which represents the major antigenic determinant on the free polypeptide appears to be silent when presented on the conjugate; and (3) new determinant(s) formed by the chemical linkage of the polypeptide to the synthetic adjuvant are involved in the priming of T cells.

Influence of CONH2 or COOH as C-terminus groups on the antigenic characters of immunogenic peptides.

The influence of the presence of a terminal COOH or CONH2 on the antigenic characters of synthetic immunogenic peptides has been studied on a streptococcal synthetic vaccine model. The obtained results show that when a peptide amide is used, the antibodies raised specifically against the amide group recognize neither free COOH nor the parent protein. The carboxamide group is thus unsuitable as was postulated for raising antibodies which recognize the peptide bond.

Influence of helical organization on immunogenicity and antigenicity of synthetic peptides.

Using cooperative effects of different peptide structures synthesized in tandem, we have induced an alpha-helical structure in water solution on a peptide which, alone, is unorganized. This structure is particularly relevant in this case as the selected model protein (type 24 M protein of Streptococcus pyogenes) is an extended coiled-coil system. We were thus able to assess the importance of organization or unorganization of a unique amino acid sequence with regards to its immunogenicity and antigenicity. Although in a classical manner, antibodies cross-reacting with the protein can be obtained with the short, unorganized peptide, we demonstrate that conformation-specific antibodies are raised when longer, organized peptides are used as immunogens.

A monoclonal antibody against a synthetic peptide is specific for the free native human chorionic gonadotropin beta-subunit.

A totally synthetic molecule (109-145 peptide) analogous to the beta-subunit carboxyl terminus was used as an antigen in the development of antibodies by the hybridoma technique. A monoclonal antibody (702 D7) specifically recognized the free native beta-human CG (beta hCG). 702 D7 was of the immunoglobulin G1 subclass and was directed against an antigenic site localized in a 10-amino acid sequence (109-118) or less. The recognition of an epitope located in the 109-118 region could explain the specific recognition of beta hCG observed with 702 D7, in contrast to monoclonal antibodies directed against a 118-145 region with a recognition of both beta hCG and whole hCG, as observed with a second monoclonal antibody (1032) to synthetic peptide. Immunohistochemical results and preliminary data obtained from the immunoradiometric assay show that 702 D7 provides a clinical tool for the detection of free beta-subunit secretion even at low concentrations, and could allow the study of this subunit or its metabolites produced by normal and tumoral cells.

Cloning and characterization of a gene encoding a novel immunodominant antigen of Trypanosoma cruzi.

A Trypanosoma cruzi genomic expression library was screened with a pool of sera obtained from chronic chagasic patients. The recombinant antigen (Tc40) isolated from this library reacted with a large number of serum samples of chronic chagasic patients, suggesting that the presence of anti-Tc40 antibodies may be specifically associated to Chagas' disease. The full-length sequence of the Tc40 gene was determined after isolation of genomic and cDNA clones. The Tc40 cDNA includes a large open reading frame (2745 bp-long) that encodes a polypeptide of 100 kDa without any homology with previously described T. cruzi sequences. In contrast with other T. cruzi antigens whose immunodominant B-cell epitopes are composed by amino acid repetitive motifs, Tc40 does not show any amino acid repetition. Antibodies against the Tc40 recombinant protein reacted with three native polypeptides of 100, 41 and 38 kDa which are tightly associated with membranes or cytoskeleton and expressed in all developmental stages of the parasite life cycle. A transcript of 3.9-kb was detected in Northern blot analysis which is large enough to encode a 100 kDa polypeptide. Tc40 genes were mapped on a chromosomal band of 1.1 Mbp and in a few copies per haploid genome in the G strain.

Organization and expression of the gene encoding an immunodominant repetitive antigen associated to the cytoskeleton of Trypanosoma cruzi.

We have studied the genomic organization and expression of the gene encoding a high molecular mass (300 kDa) repetitive antigen associated with the cytoskeleton of Trypanosoma cruzi. Protease digestion of the native protein, restriction analysis of genomic DNA and sequencing of genomic and cDNA clones indicated that most of the protein is built up by tandemly arranged, nearly identical repeats of 68 amino acids. The gene size was estimated to be approx. 9.4 kb based on the sizes of the transcript and the native protein. The nucleotide sequence conservation among the repeats indicates that selective sequence homogenization, presumably through gene conversion, maintained the amino-acid sequence conservation. Two duplicated allelic forms of this gene were mapped in fragments of about 20 kb. In some strains an additional allele was located in a fragment of 9.4 kb. Our results suggest that this repetitive antigen is a structural protein which could be involved in the attachment of the flagellum to the cell body.

Synthetic peptide ELISAs for detection of and discrimination between group M and group O HIV type 1 infection.

We developed and evaluated two peptide-based immunoassays to confirm and discriminate between group M and group O HIV-1 infection. These assays are based on in vitro competition for antibody binding between M and O peptides. The first EIA is based on competition between group M and group O gp41 immunodominant domains and the second on competition between group O and group M V3 regions of gp120. Two panels of sera were used: the first consisted of 109 sera collected from 27 group O- and 92 group M-infected patients in whom the HIV isolates had been genotyped by sequencing or heteroduplex mobility assay. In this panel, the combination of the two assays correctly discriminated 106 samples (100% group O and 96.7% group M samples). The second panel, used for the field evaluation of the two assays, consisted of 157 samples from HIV-1-infected Cameroonian patients, 33 strains having been genotyped. The combination of the two techniques in a serogrouping algorithm discriminated 147 of these samples, 74 being HIV-1 group O and 73 group M. These results always correlated with genotyping results. The 10 sera that were not successfully classified by these assays were from early seroconverters. Altogether, the two assays clearly differentiated 263 of 276 (94.9%) samples in the two panels. On the basis of the genotyping results, the positive predictive value for group discrimination in the two panels was 100% for both GSEIA assays. Our peptide-blocking group-specific EIAs for differentiation and confirmation of HIV-1 group M and group O infection are complementary tools for epidemiological studies and surveillance of HIV-1 group O strain trafficking.

Determination of anti-Toxoplasma gondii immunoglobulin G avidity: adaptation to the Vidas system (bioMérieux).

The determination of specific anti-Toxoplasma gondii IgG avidity has been proposed to improve the determination of the date of toxoplasmic seroconversion in pregnant women. In this study, we adapted this serological technique to the Vidas system (bioMérieux) using 6 M urea as the dissociating agent. We studied 356 sera, including 42 sequential sera from sero-conversions in pregnant women. Our results show that the test is easy to use, and that an avidity index higher than 0.300 allows the exclusion of a recent infection acquired less than 4 months before serum sampling.

Subperiosteal subtemporal approach: technique and applications.

The authors describe a craniofacial surgical approach that they have used since 1987. Based on existing anatomical continuity between the subtemporal plane and the cranial periosteum, it consists of a direct subperiosteal subtemporal cleavage. Preservation of continuity between the muscle and the periosteum allows the operator to return the temporalis muscle to a normal position at the end of the operation when the scalp is sutured. This approach avoids superficial cleaving of the temporalis muscle or section and reinsertion to the fronto-orbital rim. It also allows access to the upper third of the face without jeopardizing the frontal ramus of the facial nerve.

A potential anti-pregnancy vaccine built by conjugation of the beta-subunit of human chorionic gonadotropin to adjuvant-active muramyl peptide.

The beta-subunit of human chorionic gonadotropin (hCG) conjugated to tetanus toxoid is being investigated as a vaccine for human fertility control. Initial clinical trials indicated that the level of antibody response induced by such an immunogen was not always sufficient to prevent pregnancy. Therefore, efforts are being made to evaluate new carriers for the beta-subunit and to select adjuvants to yield a more efficient vaccine. In the present report, we demonstrate that conjugates of the beta-subunit of hCG with muramyl dipeptide (MDP), or its nonpyrogenic derivative murabutide, may have potential as an effective antipregnancy vaccine. The copolymer of beta hCG and MDP administered with Al(OH)3 to mice induced a high anti-beta hCG response, better than that induced by the conjugate of beta hCG to tetanus toxoid given with Al(OH)3. Moreover, the antibodies induced by such an immunogen were competent for neutralizing the biological activity of hCG in vivo. Even more interesting, a copolymer of beta hCG and of murabutide induced high levels of biologically active antibodies. This immunogen may represent a promising candidate for the development of an efficient vaccine for human fertility control.

[Patient information and preoperative informed consent: comments on an experience]. / L'information du patient et le consentement écrit à l'opération: commentaires d'une expérience.

We gave consideration to the new legal obligation to a medical doctor to prove that he has given to a patient adequate information. 312 cases of cataract or glaucoma received written information about their disease and the risks of surgery. An informed consent regarding the operation and the post-opérative care were signed by all patients. Information cover surgical risks, sides effects, unpredictable events, care steps and all last minute protocol modifications. These documents were given when scheduling the surgery. No cancellation and no objection were noticed. The French Code Civil article 1315 was not dedicated to medical exercise but it concerns physicians. The way to give a proof is not clear. Nothing is asked in the by-laws and every medical doctor can choose the method he prefers. A signature is not an obligation and an informed consent is not an authorization. A discussion is opened to clear the best and safest recommendations to obtain a legal proof of information. Now, written texts seem to be the easiest procedure. Quality medical records and personal relation between patient and surgeon are of great importance to prevent patient unsatisfaction and disappointment.

[Opinion of patients on the functioning of an ophthalmologic surgical outpatient center within a hospital. Analysis and consequences]. / L'opinion des patients sur le fonctionnement d'une structure indépendante de chirurgie oculaire sans hospitalisation au sein d'un hôpital. Analyses et conséquences.

GOAL: The purpose of this analysis is to appreciate the patients perception of ambulatory cataract surgery performed in an out patient center localized inside a public hospital. MATERIAL AND METHODS: An anonymous postal questionnaire was designed to cover the aspects of ambulatory surgery which could influence the patients perception. 576 patients were included and the study collected 487 answers to 12 questions. Authors give attention to the pros and cons detected during the study time. RESULTS: The key question shows that most patients recommend this procedure as the treatment of choice. The percentage increases with surgical experience of surgeons: the response rate to the questionnaire was both rapid and impressive. Final visual acuity did not influence significatively the overall perceptions of patients. CONCLUSION: Answers to the 12 questions point of importance of information, coordination and preparation. A high level of surgical and anesthetic formation is required to obtain an easy out patient ambulatory procedure.

[Topical anesthesia for cataract surgery in adults]. / Anesthésie topique pour l'opération de la cataracte de l'adulte.

PURPOSE: This prospective study compares the analgesia obtained by topical anaesthesia versus a single peribulbar anaesthesia. The comparison is made during clearcornear phacoemulsification in adult patient. METHODS: Ninety-six eyes are operated with Lidocaine 2% (66) or Tetracaine 1% (30) topical anaesthesia, 38 eyes received an unique anterior periocular injection. The same surgeon performs the phacoemulsification in 2 different centers. Analgesia is evaluated in the per and post operative period by the surgeon and a nurse. RESULTS: Four cases (4.2%) in the topical anaesthesia group required a complementary subconjonctival anaesthetic injection. Seven cases (7.3%) had pain during surgery but without any need for a complementary injection. In the other group 1 injection was necessary (2.5%). A slight pain was felt by 5% of patients during surgery. CONCLUSION: Topical anaesthesia is reliable but needs a patients selection, a well trained staff and a non beginner surgeon. The advantages are obvious for out patient surgery if no premedication is associated. Many doctors will continue to ask for an akinesia during cataract surgery even is the analgesia is obtained.

[Monoclonal antibodies directed against the beta hCG subunit and against a synthetic peptide analog of the carboxyl-terminal end. Clinical use in vivo and in vitro]. / Anticorps monoclonaux dirigés contre la sous-unité beta hCG et contre un peptide synthétique analogue de l'extrémité carboxyl-terminale: utilisation clinique in vivo et in vitro.

In order to detect specifically the beta-hCG, we have produced monoclonal antibodies (Mabs), using as immunogens hCG, beta hCG or a totally synthetic molecule (109-145 peptide) analogous to the beta-subunit carboxyl terminus. 34 fusion experiments were performed and led to 8 Mabs which presented a high reactivity to 125I beta hCG in the screening test. Mab D1E8 was directed to the 1-115 region of beta hCG and was cross reactive with hLH (greater than 60%). Mabs 702, 1032, and 1211 were directed against three different epitopes located in the 109-145 region and were specific for the beta hCG. The in vivo localization of tumors containing beta hCG ty tomoscintigraphy (SPECT) was evaluated in 5 patients by injecting 131I-Mab D1E8, selected for imaging because of its high affinity to beta hCG (Ka = 1.9 X 10(9)). SPECT was performed 48 h and 96 h after injection: 2 patients with proven tumor sites had positive "immunoscintigraphy" results. One patient, simultaneously injected with 125I non-specific IgG, had a tumor resection: the count ratio between normal and tumoral tissues revealed a low specific uptake. Two and three-site immunoradiometric assays (IRMA) were performed with monoclonal antibodies purified from nude mice ascitic fluids. IRMA were based upon two or three Mabs, with D1E8 on a solid phase and 125I Mab 702 or a mixing of 125I-702 and 125I-1032 as tracer. The specificity of IRMA was demonstrated by the absence of binding with increasing amount of hLH, hFSH and hTSH up to 5000 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)