Allen-Masters syndrome as a cause of status ileus - case report.

INTRODUCTION: Internal hernias are rare and are encountered in a small percentage of cases. The hernia in the broad ligament of uterus (Allen-Masters syndrome) is a unique type of internal hernia which represents only approximately 4% of all internal hernias. CASE REPORT: We present the case of a 39-year-old woman admitted for clinical signs of mechanical bowel obstruction. CT examination revealed a dilated loop of small intestine in the left lower abdomen. The patient underwent laparoscopic surgery with the finding of an incarcerated small bowel loop in the ligamentum latum uteri. Small bowel deliberation and ligament defect suture were performed. CONCLUSION: A defect in the ligamentum latum uteri (Allen-Masters syndrome) is a rare diagnosis, usually discovered as an incidental finding in female patients with ileus. This syndrome may explain the vague problems of many patients whose symptoms include dyspareunia, dysmenorrhea, acute and chronic pelvic pain. Allen-Masters syndrome can be diagnosed and successfully managed by laparoscopic approach.

Preoperative non-selective administration of nutritional supplements to patients undergoing elective colorectal resection - standard of perioperative care?

INTRODUCTION: Preoperative nutritional support for oncosurgical patients is recommended to minimize the negative effects of potential malnutrition. Nutritional support is intended to adjust the pathophysiological reactions to surgery, reduce postoperative complications, shorten the length of hospital stay and speed up convalescence. The aim of the present study was to evaluate the effect of preoperative nutritional supplements (ONS oral nutritional supplements) on the physical and nutritional status of patients undergoing elective colorectal resection for cancer and to assess patients self-sufficiency after surgery. METHODS: This was a prospective, randomized, single-center clinical trial designed to assess self-sufficiency and return to normal activities in relation to preoperative ONS in patients undergoing elective colorectal surgery. Patients enrolled in the study were randomized to receive ONS twice daily for 7 days prior to surgery or no ONS. RESULTS: One hundred patients were included in the study. The rate of postoperative complications was comparable; no differences were found in postoperative values of laboratory nutritional parameters (albumin, prealbumin). The length of hospital stay was comparable; the stay in the ICU was shorter in patients taking ONS but the difference was not statistically significant. Differences between the study subgroups regarding muscle weight were not statistically significant. Patient self-sufficiency (assessed using the Barthel index) was comparable in both groups before and after surgery (p=0.717 and p=0.327). CONCLUSION: Non-selective preoperative administration of ONS to all patients undergoing elective colorectal resection does not reduce postoperative morbidity or speed up recovery. Patients self-sufficiency and their physical and nutritional status are not affected by preoperative nutritional support.

Latex and silicone drains in surgery − is the ban on rubber drains really a step forward or rather a step back?

The use of silicone and latex drains is an integral part of surgical practice. Experience and the review of the world literature show that silicone drain is characterized by a much lower rate of fibrotic reaction of the tissue around the drain. The ability of a latex, or rubber, or popularly called rubber drain, to induce the formation of ligaments in its surroundings is advantageously used in situations where the targeted formation of scar tissue is desired. This feature is absent in silicone drains. However, nowadays the rule in most surgical departments is to use almost exclusively silicone drains, which is based on prevention of latex allergy. This article is devoted to the description of the different and mutually irreplaceable use of silicone and latex drains. Subsequently, he also discusses the question of whether the twilight of the use of latex drains in modern medicine is really progress, or rather retrogression.

Technical modifications of appendiceal transection during laparoscopic appendectomy.

INTRODUCTION: Appendiceal transection is the most critical part of laparoscopic appendectomy (LAPPE). The aim of our study was to evaluate post-operative and economic outcomes of laparoscopic appendectomy with different technical modifications of transection of the appendix. METHODS: This was a prospective, randomized, unicenter clinical study comparing different techniques of appendiceal transection in patients with acute appendicitis during the study period (18 months). The patients were randomized to one of three arms - endoloop, hem-o-lok clips and the stapler. RESULTS: In total, 120 patients were enrolled in the study. The shortest operative time was noted in the hem-o-lok arm (37.3 minutes); mean length of hospital stay (3.7 days) was comparable in all study arms. Postoperative morbidity was 6.6%; all recorded complications were SSIs (Surgical Site Infections). The number of postoperative complications was comparable in all study arms. Mean direct costs of laparoscopic appendectomy were lowest in the hem-o-lok arm. According to our findings, LAPPE is not a profit making surgery irrespective of the type of appendiceal transection (mean profit in the study patients was CZK -4019). CONCLUSION: The rate of postoperative complications was similar for all the technical modifications of appendiceal stump closure. As indicated by the study outcomes, hem-o-lok clips have the potential of becoming the method of choice in securing the appendix base during LAPPE.

Anus perinei ventralis in adulthood - case report.

Anorectal malformations present a type of the most serious congenital malformations, either in terms of treatment or treatment outcomes. Anorectal atresia can be subdivided into three categories: the supralevator form, the intermediate type of atresia and the low translevator type. One of the clinical forms of low translevator type in girls is a perineal fistula opening just behind the vaginal entrance on the perineum, with a fully developed sphincter complex dorsally from the fistula (so called anus perinei ventralis). The golden standard of surgical treatment of anus perinei ventralis in children is Peñas procedure, which was used as a guideline for anorectal reconstruction in our adult patient, as well.

Changes in carbon and nitrogen pool during in-situ aeration of old landfills under varying conditions.

Emissions from old landfills via leachate and the gas phase are influenced by state and stability of the organic matter in the solid waste and by environmental conditions within the landfill. Remediation of landfills by means of in-situ aeration is one possibility to reduce these emissions. By establishing aerobic conditions, biological processes in the landfill are accelerated. To investigate the effects of this remediation technology, lab-scale experiments with column tests have been carried out. The main goal of the present work is to characterize the changes of the carbon and nitrogen compounds in the aerated solid waste, the leachate and the gas phase under varying conditions. The results demonstrate a clear reduction of emissions and a stabilization of the organic matter. Furthermore, it is shown that both the intensity of aeration and the amount of water affect biological processes to a certain extent. Even when columns were operated under anaerobic conditions after a long running period of aeration, the emissions remained low.

Multifunctional g3p-peptide tag for current phage display systems.

We have previously described a monoclonal antibody (mAb), 10C3, directed against the gene-3 protein (g3p) of filamentous phage M13, which was produced to study g3p fusion protein expression in Escherichia coli and its incorporation in the phage capsid [Tesar, M., Beckmann, C., Röttgen, P., Haase, B., Faude, U., Timmis, K., 1995. Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems. Immunology 1, 53-54]. In this study we report mapping of the antigenic epitope of the mAb 10C3, by means of short overlapping peptide-sequences [Frank, R., Overwin, H., 1996. Spot synthesis. In: Morris, G.E. (Ed.), Methods in Molecular Biology, Vol. 66: Epitope Mapping Protocols. Humana Press, Totowa, NJ, pp. 149-169.] comprising the C-terminal half of the g3-protein. A minimal recognizable peptide was found which is represented in the 11 amino acid sequence from positions 292 to 302 of g3p [Wezenbeek van, P.M.G.P., Hulsebos, T.J.M., Schoenmakers, J.G.G., 1980. Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd. Gene 11, 129-148]. In order to use the antibody also for detection and purification of recombinant proteins, such as single chain antibodies, the epitope was introduced as a tag sequence into the phagemid pHEN1 [Hoogenboom, H.R., Griffith, A.D., Johnson, K., Chiswell, D.J., Hudson, P., Winter, G., 1991. Multi-subunit proteins on the surface of the filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acid Res. 19, 4133-4137; Nissim, A., Hoogenboom, H.R., Tomlinson, I.M., Flynn, G., Midgley, C., Lane, D., Winter, G., 1994. Antibody fragments from a single pot phage display library as immunochemical reagents. EMBO J. 13 (3) 692-698]. Purified single chain antibodies containing this tag were detectable down to a concentration of 2 ng ml(-1) under non-denaturing conditions (ELISA) or 4 ng per lane on immunoblots. The high sensitivity of the antibody for the peptide tag was reflected in the antibody affinity constant K(D) of 6.80 x 10(-10) M, which was determined by real time biomolecular interaction analysis (BIA) based on surface plasmon resonance (SPR) [Karlsson, R., Fält, A., 1997. Experimental design for kinetic analysis of protein-protein interactions with surface plasmon resonance biosensors. J. Immunol. Methods 200, 121-133]. Finally, recombinant proteins in E. coli periplasmic extracts could be purified in a single step by affinity purification using immobilized mAb 10C3. These studies demonstrated that the new peptide-tag and its corresponding mAb represents a versatile tool for the detection of recombinant proteins selected by phage display technology.

Monitoring of scFv selected by phage display using detection of scFv-pIII fusion proteins in a microtiter scale assay.

We describe here a method for the efficient and rapid analysis of antigen binding characteristics of recombinant antibodies (ab) selected by phage display. This novel approach combines the bacterial production of soluble single chain ab (scFv)-pIII fusion proteins on a microtiter scale with the detection of these fusion proteins via a pIII-specific ab. It facilitates the parallel analysis of large numbers of clones and is more efficient than current analysis protocols. Applying this technique, we analysed phage display selection of tetanus toxoid (TTX) specific scFv with respect to: (i) the productive expression of fusion proteins; (ii) the enrichment of specific scFv in subsequent rounds of phage display selection on a polyclonal level; (iii) the antigen specificity of individual scFv clones; (iv) the antigen binding affinity of a selected scFv. A TTX-specific scFv (clone 4.3) was further examined in a mono- and bivalent form by surface plasmon resonance analysis. ScFv 4.3 possesses a subnanomolar affinity and a low off rate constant.

High-throughput generation and engineering of recombinant human antibodies.

The first version of the Human Combinatorial Antibody Library (HuCAL) is a single-chain Fv-based phage display library (HuCAL-scFv) with 2x10(9) members optimised for high-throughput generation and targeted engineering of human antibodies. 61% of the library genes code for functional scFv as judged by sequencing. We show here that since HuCAL-scFv antibodies are expressed in high levels in Escherichia coli, automated panning and screening in miniaturised settings (96- and 384-well format) have now become feasible. Additionally, the unique modular design of HuCAL-genes and -vectors allows the distinctly facilitated conversion of scFv into Fab, miniantibody and immunoglobulin formats, and the fusion with a variety of effector functions and tags not only convenient for therapeutic applications but also for high-throughput purification and detection. Thus, the HuCAL principle enables the rapid and high-throughput development of human antibodies by optimisation strategies proven useful in classical low molecular weight drug development. We demonstrate in this report that HuCAL is a very convenient source of human antibodies for various applications.

Application of FT-IR for assessment of the biological stability of landfilled municipal solid waste (MSW) during in situ aeration.

Disposal of untreated municipal solid waste leads to gaseous emissions as well as liquid degradation products. In situ aeration is an emerging means for remediation of abandoned landfills. It aims at an accelerated mineralization and stabilization of waste organic matter and thus reduces significantly the emission potential of the site. In order to prove the success of the technique, evaluation of the biological stability of the aerated material has been suggested. Fourier Transform Infrared Spectroscopy (FT-IR) provides comprehensive information on the chemical composition of solid waste samples. Different stages of organic matter degradation are reflected by changes in the infrared spectral pattern. In the present study the feasibility of applying FT-IR for assessment of the stability of waste material derived from abandoned landfills and for in situ aeration process control was investigated. Waste samples derived in the course of pilot-scale and lab-scale aeration experiments were characterized by FT-IR (4000-400 cm(-1), KBr-technique, transmission mode) and a set of conventional parameters describing biological stability. The occurrence of distinct indicator bands was correlated with chemical and biological waste properties using 206 solid waste samples. Visual spectra interpretation was found to be appropriate in proving a reduced emission potential of initially rather reactive waste (respiration activity over 4 days (RA(4)) > 7 mg O(2) g(-1) DM) during aeration. Furthermore, cluster analysis was applied successfully to differentiate between original and aerated waste samples, even for rather stable material (RA(4) < 7 mg O(2) g(-1) DM), when visual spectra interpretation was limited.

Foot-and-mouth disease virus protease 3C inhibits cellular transcription and mediates cleavage of histone H3.

Foot-and-mouth disease virus protease 3C is essential for the processing of the viral precursor polyprotein. It is shown here to also inhibit gene expression in baby hamster kidney cells after transient expression from transfected cDNA fragments. Protease 3C could not be detected by indirect immunofluorescence in contrast to other cDNA-encoded virus proteins, but protein synthesized de novo 16 hr after transfection could be detected by radioimmunoprecipitation. The cellular translation apparatus was, therefore, not inhibited. The enzyme, although produced as part of a fusion protein, was in size indistinguishable from that found in virus-infected cells. This suggested that the enzyme was released by autocatalysis from the recombinant fusion protein and from viral precursor protein in a similar manner. Transcription of protease 3C-encoding cDNA fragments as well as that of cotransfected fragments, which do not encode protease 3C, was inhibited as determined by hybridization assays. The shut off of transcription which was one of the cytopathic effects observed in virus-infected cells therefore correlates with the production of transactive protease 3C. The inhibitory molecular mechanism may involve truncation of the nuclear protein histone H3 at its N-terminus since this protein was found similarly truncated in virus-infected cells and after transfer of 3C-encoding cDNA fragments.

Serological probes for some foot-and-mouth disease virus nonstructural proteins.

Foot-and-mouth disease virus (FMDV) O1 Kaufbeuren-specific cDNA fragments were subcloned into the E. coli expression vector pRIT.2T. Fusion proteins thus produced in bacteria were purified by affinity chromatography and inoculated into rabbits. Three sera thus obtained were found to be monospecific for FMDV proteins 3A, 3C, and 3D, respectively. Two others were prevalently directed against protein 2C, but in addition, either to protein 2B or to protein 3A. Five out of six mature nonstructural virus proteins can therefore be separately investigated in FMDV-infected cells, either by indirect immunofluorescence or by radioimmunoprecipitation. Immunofluorescence shows all investigated proteins to be located exclusively in the cytoplasm. One of them, protein 2C, transiently forms aggregates at the periphery of cells. Radioimmunoprecipitation confirmed current knowledge on maturation of FMDV proteins. It was further used to characterize postinfectional sera with regard to FMDV-specific antibodies. Cattle and guinea pig were found to have responded differently to FMDV nonstructural antigens. Furthermore, antigenicity of yet to be described FMDV polypeptides was observed in the guinea pig.

Genetic relationships among Pseudomonas stutzeri strains based on molecular typing methods.

Detailed characterization of the genetic variability among strains belonging to Pseudomonas stutzeri was achieved using different rapid molecular typing methods based on polymerase chain reaction (PCR), Southern blot and Western blot. Consensus motifs complementary to fragments of repetitive elements dispersed throughout the genomes of bacteria were used as primers and allowed differentiation at subspecies levels. Further and simple differentiation was also achieved based on the direct amplification of spacer regions between 16S and 23S rRNA, combined with single-strand conformation polymorphism (SSCP) analysis of the generated fragments. These methods are fast, sensitive, reliable for determining relationships, and have demonstrated a great genetic diversity among the strains of Ps. stutzeri studied in agreement with the heterogeneous phenotypic traits of the species.

Detection of hepatitis A virus proteins in infected BS-C-1 cells.

Hepatitis A virus (HAV) is distinguished from other picornaviruses by its tropism for the liver in infected hosts, a nonlytic infection in hepatocytes, and a slow and nonlytic growth cycle in cultured cells. Although the genome structure and organization of HAV appear to be similar to those of the other picornaviruses, the viral proteins synthesized in infected cells have not been previously characterized. We have utilized specific antisera raised in rabbits to recombinant HAV proteins expressed in Escherichia coli in an effort to identify both structural and nonstructural proteins in BS-C-1 cells throughout the course of a viral replication cycle. Replication was monitored by dot blot hybridization of viral genomes. Structural proteins VP0, VP1, VP2, and VP3 were found to accumulate during the infection cycle as did viral RNA. Nonstructural proteins 2C and 3D were not detected on immunoblots, although a minor amount of 2C could be detected by immunoprecipitation of lysates of radiolabeled, infected cells. The relative sensitivities of the various antisera were determined, and the failure to observe nonstructural proteins was shown not to be due to decreased sensitivity of the detection reagents. Thus, it appears that HAV nonstructural proteins do not accumulate in infected cells to levels comparable to those of capsid proteins.

Replication of hepatitis A viruses with chimeric 5' nontranslated regions.

The role of the 5' nontranslated region in the replication of hepatitis A virus (HAV) was studied by analyzing the translation and replication of chimeric RNAs containing the encephalomyocarditis virus (EMCV) internal ribosome entry segment (IRES) and various lengths (237, 151, or 98 nucleotides [nt]) of the 5'-terminal HAV sequence. Translation of all chimeric RNAs, truncated to encode only capsid protein sequences, occurred with equal efficiency in rabbit reticulocyte lysates and was much enhanced over that exhibited by the HAV IRES. Transfection of FRhK-4 cells with the parental HAV RNA and with chimeric RNA generated a viable virus which was stable over continuous passage; however, more than 151 nt from the 5' terminus of HAV were required to support virus replication. Single-step growth curves of the recovered viruses from the parental RNA transfection and from transfection of RNA containing the EMCV IRES downstream of the first 237 nt of HAV demonstrated replication with similar kinetics and similar yields. When FRhK-4 cells infected with recombinant vaccinia virus producing SP6 RNA polymerase to amplify HAV RNA were transfected with plasmids coding for these viral RNAs or with subclones containing only HAV capsid coding sequences downstream of the parental or chimeric 5' nontranslated region, viral capsid antigens were synthesized from the HAV IRES with an efficiency equal to or greater than that achieved with the EMCV IRES. These data suggest that the inherent translation efficiency of the HAV IRES may not be the major limiting determinant of the slow-growth phenotype of HAV.

Analysis of a potential myristoylation site in hepatitis A virus capsid protein VP4.

The VP4 capsid protein of several picornaviruses has been shown to be myristoylated at an N-terminal glycine residue. Myristoylation occurs after removal of an initial methionine residue or a leader peptide, resulting in the exposure of an N-terminal eight amino acid myristoylation signal including a consensus G-x-x-x-T/S motif. Analysis of the amino acid sequence of hepatitis A virus (HAV) capsid protein reveals a potential myristoylation site beginning at position 5 of the VP4 sequence. To assess the significance of this apparent myristoylation signal, mutations were engineered (G to A; T to N) to alter the consensus sequence as well as the potential cleavage site that would be required to remove the short leader. An additional mutant was constructed in which the proposed leader was deleted. Expression of these HAV sequences in BS-C-1 cells showed that leader cleavage did not occur in the wild-type or mutant proteins, although the threonine to asparagine mutation resulted in reduced translation and processing efficiency. Transfection of BS-C-1 or FRhK-4 cells with transcripts derived from the wild-type or mutagenized cDNA clones gave rise to infectious virus, with no detectable incorporation of myristate. The results indicate that HAV does not require leader cleavage and myristoylation of VP4 for growth in cultured cells.

Hepatitis A virus polyprotein synthesis initiates from two alternative AUG codons.

The genomic RNA of hepatitis A virus has two potential translation initiation sites for synthesis of a 251-kDa polyprotein. It is not known which of these AUG codons, located at positions 735-737 and 741-743, is used in vitro or in vivo. Site-directed mutagenesis was carried out to eliminate each start codon independently. Transcripts from the unmodified and modified cDNA clones were used either to program an in vitro translation system or for transfection of BS-C-1 cells. In vitro and in vivo translation data revealed preferential usage of the downstream AUG located at position 741 to 743, although either site could be utilized in the absence of the other. Both modified RNAs were able to induce productive infections in BS-C-1 cells. Deletion of almost all of the 5'-untranslated region (5'UTR) of the RNA, however, stimulated selection of AUG 735-737 in vitro resulting in equal utilization of both sites, suggesting a strong influence of the 5'UTR for directing the ribosome to a specific internal initiation site.

Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems.

UNLABELLED: A monoclonal antibody directed against the gene 3 product (pIII) of filamentous phage M13 was produced to study pIII-fusion protein expression in E. coli and its incorporation in the phage capsid. The protein was gel-purified from E. coli expression cultures harboring the genetic information of pIII under the control of an inducible lac promoter. To study pIII-fusion protein expression, phage display systems were applied in which either the whole pIII or the C-terminal half was used (McCafferty et al. (1990) Nature (London) 348, 552-554; Szardenings and Collins (1990) Gene 94, 1-7; Barbas and Lerner (1991) In: METHODS: Companion to METHODS in Enzymology, Combinatorial Immunoglobulin Libraries on the Surface of Phage (Phabs): Rapid Selection of Antigen-Specific Fabs, Vol. 2, Academic Press, Orlando, pp. 119-124). In all cases, the monoclonal antibody was able to detect the native and the recombinant protein in E. coli and on the phage tip using non-denaturing (ELISA) and denaturing (SDS-PAGE, immunoblot analysis) conditions. All selected pIII-specific monoclonal antibodies were found to be directed against epitopes within amino acids 198 to 406 of pIII, which is necessary for capsid incorporation and therefore included in all pIII-mediated phage display designs.

Identification of foot-and-mouth disease virus replication in vaccinated cattle by antibodies to non-structural virus proteins.

Antibodies raised in cattle against foot-and-mouth disease virus by vaccination or by experimental infection were distinguished. Vaccination elicited only antibodies to virus capsid proteins and the polymerase 3D. Virus replication in cattle elicited additional antibodies directed against the non-structural proteins 2B, 2C, 3AB1, and/or 3C irrespective of prior vaccination or whether the cattle exhibited symptoms of disease. Non-permissive mice inoculated with virus responded in the same way, indicating that antibodies raised due to the transient presence of antigen are safely recognized by the method applied which was radioimmunoprecipitation. All kinds of infections were thus detected and it was possible to differentiate between cattle exposed or not exposed to challenge in the field, and further between protected animals and possible virus carriers.