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RNA trans-splicing has become a versatile tool in the gene therapy of monogenetic diseases. This technique is especially valuable for the correction of mutations in large genes such as COL7A1, which underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa. Over 800 mutations spanning the entire length of the COL7A1 gene have been associated with defects in type VII collagen, leading to excessive fragility of epithelial tissues, the hallmark of dystrophic epidermolysis bullosa (DEB). In the present study, we designed an RNA trans-splicing molecule (RTM) that is capable of repairing any given mutation within a 4200 nucleotide region spanning the 3′ half of COL7A1. The selected RTM, RTM28, was able to induce accurate trans-splicing into endogenous COL7A1 pre-mRNA transcripts in a type VII collagen-deficient DEB patient-derived cell line. Correct trans-splicing was detected at the RNA level by semiquantitative RT-PCR and correction of full-length type VII collagen was confirmed at the protein level by immunofluorescence and western blot analyses. Our results demonstrate that RTM28, which covers >60% of all mutations reported in DEB and is thus the longest RTM described so far for the repair of COL7A1, represents a promising candidate for therapeutic applications.

In the cross-sectional Study of Health in Pomerania (SHIP-0), subjects with an adequate magnesium supply had a lower risk of periodontal disease and more teeth than those with low magnesium levels. The authors analyzed 5-y follow-up data (SHIP-1) to determine whether the baseline magnesium levels had a long-term effect on attachment level and number of teeth lost. Of the participants examined dentally in the baseline study, 3,300 (75%) were examined for progression or recession of periodontal attachment level after 5 y. For 2,432 subjects, the authors related the outcome variables of periodontal attachment level and tooth loss to baseline characteristics, especially serum magnesium and calcium concentrations, as well as systemic markers of inflammation. The progression of periodontitis was associated with the magnesium/calcium (Mg/Ca) ratio at baseline in a dose-dependent manner. Progression of mean attachment loss was prevented in the upper quartile of the Mg/Ca ratio (P< 0.001) with antagonistic effects of magnesium and calcium irrespective of inflammatory state. With respect to tooth loss, Mg/Ca exerted dimorphic effects. In inflammatory states as indicated by high C-reactive protein (>3 mg/L), tooth loss was prevented in subjects with high Mg/Ca ratio (incidence rate ratio = 0.60, 95% confidence interval: 0.45 to 0.80, P= 0.001), but the contrary was observed in subjects with low C-reactive protein levels (incidence rate ratio = 1.14, 95% confidence interval: 0.97 to 1.34, NS). Similar results were observed with stratifying the regression on tooth loss by interleukin 6 or fibrinogen threshold. An adequate magnesium serum level and Mg/Ca balance may prevent progression of attachment level and tooth loss, especially in inflammatory states.Knowledge Transfer Statement:The results of this study present evidence that an adequate magnesium supply may be important in the prevention of periodontal diseases and future tooth loss. A diet high in magnesium could improve periodontal health, notwithstanding its beneficial effects on systemic disease. In populations with a high prevalence of hypomagnesemia, additional intake of supplements is advisable.

Summary: Background. Reports on long-term outcomes after endoscopic axillary lymph node dissection (ALND) of breast cancer patients are still lacking in the medical literature. The objective of this prospective study was to assess the oncological and functional outcomes in breast cancer patients after endoscopic ALND. Methods. Fifty-five breast cancer patients were prospectively enrolled, of whom 52 were available for follow-up with a median of 71.9thinspmonths (range 11–96). The following oncological and functional endpoints were evaluated during follow-up at several time points: occurrence of local, axillary and distant metastases, seroma or infection, shoulder mobility (range of motion), numbness, pain, presence of lymphoedema as well as restriction in activities of daily living. Results. In 52 patients endoscopic ALND of level I and II was successfully performed. Two port-site metastases (2/52, 4%) occurred, one of which in a patient with negative axillary lymph nodes. The same patient suffered from the only axillary recurrence (1/52, 2%). Three patients (3/52, 6%) developed lymphoedema. No other functional adverse events (shoulder mobility, pain, numbness, hypertrophic scar) were noticed at the end of the observation period. Conclusion. The present investigation with long-term follow-up after endoscopic ALND – the first one in the literature – reveals minor morbidity, good functional and cosmetic results. In contrary to conventional surgery, the endoscopic procedure is associated with the occurrence of port-site metastases, not seen in the open approach. Axillary recurrences do not appear more frequently when compared with results after conventional ALND. In the meantime the less invasive sentinel lymph node (SLN) biopsy is the established standard technique in evaluating the axillary lymph node status.

Background:Periodontitis is a bacterial inflammatory disease leading to attachment loss with the consequence of tooth loss. There exists a multifactorial risk pattern including bacterial challenge, smoking, age, gender, diabetes, and socioeconomic and genetic factors. Smoking has the highest impact on the course of the disease modulated by all the other factors. Here, we report the relationship between smoking and the genetic polymorphism of interleukin‐1 (IL‐1). Methods:In a randomly selected population‐based study, we genotyped 1,085 test persons for the IL‐1 genotype, examined their periodontal status, and assessed their smoking behavior including present and past quality and quantity of smoking. Results:There was a significant dose‐effect relationship between the exposure to tobacco smoke and the extent of periodontal disease assessed as attachment loss and tooth loss. Moreover, there was a gene‐environmental interaction. Subjects bearing at least one copy of the variant allele 2 at positions IL‐1A –889 and IL‐1B +3954 (genotype positive) had an enhanced smoking‐associated periodontitis risk as compared to their IL‐1 genotype‐negative counterparts. With genotype‐negative non‐smokers as a reference, logistic regression resulted in odds ratios of 0.98 (95% confi‐ dence interval: 0.83 to 1.14), 2.37 (1.96 to 2.87), and 4.50 (2.30 to 8.82) for genotype‐positive non‐smokers, genotype‐negative smokers, and genotype‐positive smokers, respectively. Conclusions:There is a gene‐environmental interaction between smoking and the IL‐1 genetic polymorphism. Smokers bearing the genotype‐positive IL‐1 allele combination have an increased risk of periodontitis. The IL‐1 genotype has no influence in non‐smokers. J Periodontol 2004;75:236‐242.

Background: The aim of this study was to identify predisposing factors for complications after gallstone spillage during laparoscopic cholecystectomy (LC). Methods: Papers derived from Medline search and papers from reference lists within these papers were studied. Ninety-one reports on complications caused by lost gallstones published between 1991 and 1998 were analyzed. These patients were compared with cases in published series on LC in general. Results: Gallbladder perforation (20%) and stone spillage (9%) were the two most common complications of LC which occurred during the dissection (75%) and removal (25%) of the gallbladder. Predisposing factors for developing complications after stone spillage were: older age, male sex, acute cholecystitis, spillage of pigment stones, number of stones (>15) or size of the stone (Ø > 1.5 cm), and perihepatic localization of lost stones. CT-scan and ultrasound examination proved best for the recognition of complications caused by lost stones. Explorative laparotomy and surgical removal of the stones was the most frequently used therapy. Conclusions: Gallbladder perforation and stone spillage might cause hazardous complications. In cases with loss of numerous or large pigment stones which cannot be retrieved by laparoscopy, intraoperative conversion to open surgery can be justified.

Biologists have long argued that runs of alternating purines and pyrimidines could form alternative DNA structures, which might regulate transcription. Here, we report that simple sequence repeat polymorphisms in the tilapia prolactin 1(prl 1) promoter are associated with differences in prl 1gene expression and the growth response of salt-challenged fishes. Individuals homozygous for long microsatellite alleles express less prl 1in fresh water but more prl 1in half-seawater than fishes with other genotypes. Our work provides the first in vivo evidence that differences in microsatellite length among individuals may indeed affect gene expression and that variance in expression has concomitant physiological consequences. These results suggest that dinucleotide microsatellites represent an under-appreciated source of genetic variation for regulatory evolution.

Hintergrund.Der perianale, durch humane Papillomaviren verursachte Buschke-Löwenstein-Tumor (Riesenkondylom) ist sehr selten. Obwohl es sich histologisch um eine benigne Neoplasie handelt, ist sein klinisches Wachstumsmuster maligne mit Infiltration und Destruktion der Umgebung. Charakteristisch sind die hohe Rezidivrate und die häufige Transformation in ein malignes Plattenepithelkarzinom. Therapie der Wahl ist die radikale chirurgische Resektion. Fallbericht.Eine 56-jährige Frau stellte sich wegen eines perianalen Abszesses vor, der in einem ausgedehnten Buschke-Löwenstein-Tumor mit Infiltration von Rektum und Vagina entstanden war. Nach Ausheilung des Abszesses erfolgte die abdominoperineale Rektumresektion en bloc mit Hinterwand der Vagina und perinealem Weichteilgewebe. Die Histologie ergab ein aus dem Riesenkondylom entstandenes, im Gesunden entferntes Plattenepithelkarzinom. Mittels eines transpelvin eingebrachten myokutanen M.-rectus-abdominis-Insellappens konnten die Vaginalhinterwand rekonstruiert und der Defekt im kleinen Becken gefüllt werden. Nach primärer Wundheilung folgte die pelvine Bestrahlung. Schlussfolgerung.Die simultane plastisch-chirurgische Rekonstruktion eines großen pelvinoperinealen Weichteildefektes mit dem transpelvinen M.-rectus-abdominis-Lappen erlaubt eine primäre Heilung mit beschleunigter Rehabilitation und risikoarmer Nachbestrahlung, da der Dünndarm nicht ins kleine Becken prolabiert und damit nicht ins Bestrahlungsfeld gerät. Introduction.The Buschke Löwenstein tumor (giant condyloma) in its perianal variant is an extremely rare disease caused by human papilloma virus. Although of histologically benign appearance, it infiltrates and destroys the surrounding tissue. There is a high risk of local recurrence and malignant transformation. The treatment of choice is wide surgical resection. Case.A 56-year-old woman presented with perianal giant condyloma infiltrating the rectum and vagina. The extensive soft tissue defect resulting from wide resection was filled with a transpelvic myocutaneous rectus abdominis flap. Histology showed a squamous cell carcinoma arising in the Buschke Löwenstein tumor with clear resection margins. Therefore, the patient was irradiated locally after uneventful primary wound healing. Conclusion.A simultaneous reconstruction of a large pelvinoperineal soft tissue defect with the transpelvic myocutaneous rectus abdominis flap allows primary healing, accelerated rehabilitation, and safe adjuvant radiotherapy without risk of serious radiation damage to the small bowel by preventing it from protruding into the pelvic defect.

Diagnosis and Treatment of Acute Left-Sided Colonic Diverticulitis – Swiss ExperiencesLeft-sided colonic diverticulitis is quite common in Western societies. Until recently, the initial imaging was contrast enema with water-soluble contrast media. This test has now been replaced by computed tomography (CT: oral, rectal and intravenous contrast administration). In addition to high sensitivity of CT for the diagnosis of diverticulitis (97%), the severity of the disease can be assessed. Furthermore, CT has its role in the diagnosis and treatment of associated abscess formation, in predicting the likelihood of medical treatment failure and, finally, in predicting the risk of secondary complications after initial conservative treatment. CT is the safest and most cost-effective diagnostic test. If surgery is needed, the diseased bowel segment should be removed. The question is whether one should carry out a primary anastomosis or a Hartmann''s procedure. In our experience of over 30 years, resection with primary anastomosis can safely be performed even in advanced stages of the disease (peritonitis). Overall morbidity is high (20–30%). Anastomotic leakage occurs in 1–3%. In all operated patients the mortality rate is below 10% and in patients with mild disease below 1%. Morbidity and mortality rates are higher in Hartmann''s procedure. Therefore, resection with primary anastomosis can be recommended as the procedure of choice.

The MAGE-1 gene encodes a protein encompassing a HLA-A1-restricted target epitope for cytolytic T lymphocytes. Monoclonal antibodies directed against the MAGE-1 protein were tested for usage in immunohistology of routine pathology material. Seven formalinfixed, paraffin-embedded malignant melanomas were studied by the Avidin-Biotin complex (ABC) method with or without different antigen retrieval methods. Native, frozen tissues from the same tumours were used to validate the results by immunohistochemistry on frozen sections, by PCR for mRNA and by protein demonstration in tissue extracts using western blotting. Of 4 monoclonal antibodies tested, mAB 34B and mAB 77B were highly efficient in detecting MAGE-1 protein in deparaffinised sections with the regular ABC method after microwave pretreatment. In a series of an additional 28 patients 75% expressed MAGE-1, 50% in a substantial proportion. Follow-up studies in 6 patients indicate that the expression pattern remains stable but may change substantially within a short range. Immunohistology is thus a rapid and well-established method that might be used to select and monitor HLA-A1 positive patients with malignant melanoma and other candidate tumours for MAGE-1-directed immuno-therapy.

Summary.: With a response rate of 60.6 % to a questionnaire sent out in 1994 it is possible to give an overview of the situation in hernia surgery at the 142 surgical clinics of Switzerland. In general, regional anesthesia is the preferred method (67.4 %). Together, Shouldice repair and fascia transversalis repair cover over 90 % of the repair techniques in primary hernias. In recurrent hernias the same techniques dominate (70.2 %) a large spectrum of different operation methods; implantation of foreign materials is rare (5.9 %). Only 4.5 % of all Swiss surgical clinics have some experience with endoscopic hernia surgery.

Borrelia burgdorferi is a spirochete pathogen transmitted among warm-blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.

Hypervariable parts of mitochondrial DNA (mtDNA) were amplified enzymatically and sequenced directly by using genomic DNA from single plucked human hairs. This method has been applied to study mtDNA sequence variation among 15 members of the !Kung population. A genealogical tree relating these aboriginal, Khoisan-speaking southern Africans to 68 other humans and to one chimpanzee has the deepest branches occurring amongst the !Kung, a result consistent with an African origin of human mtDNA. Fifteen cases of unrelated individuals having identical sequences in the most variable parts of the mtDNA control region were found within populations of !Kung, Western Pygmies, and Eastern Pygmies, but no cases of identity were evident among these populations. This and other evidence of geographic structuring of the mitochondrial diversity in Africa, together with knowledge of the rate of accumulation of base changes in human mtDNA, implies that the average rate at which female lineages have moved their home bases during hunter-gatherer times could be as low as 13 meters per year. The technique of enzymatic amplification and direct sequencing applied to readily collected, highly stable biological materials such as hairs makes it possible to examine with high resolution many representatives of virtually any population.

The activity of ketocyclazocine, a putative kappa opioid receptor agonist, was studied and compared with that of morphine, a mu opioid receptor agonist, and cyclazocine, a putative kappa and sigma opioid receptor agonist, vs. placebo in 10 drug abusers. The measures included vital signs and pupil measurements, established and new observer- and subject-completed psychopharmacologic questionnaires and several methods of drug discrimination. The results indicate that ketocyclazocine is different from morphine-like agonists in that it produces only minimal miosis and lacks euphoriant action. It causes a dysphoric state and was clearly discriminated from morphine. The dysphoria and pattern of responses was similar to cyclazocine though ketocyclazocine was discriminated from cyclazocine. This is consistent with the concept that morphine and ketocyclazocine have separate modes of primary activity. The similarity between ketocyclazocine and cyclazocine obscures the assignment of particular drug effects to activity at the kappa receptor.

We analyzed the geographic distribution of the Ixodes ricinus-like ticks in eastern North America by comparing the mitochondrial 16S rDNA sequences of specimens sampled directly from the field during the 1990s. Two distinct lineages are evident. The southern clade includes ticks from the southeastern and middle-eastern regions of the United States. The range of the northern clade, which appears to have been restricted to the northeastern region until the mid-1900s, now extends throughout the northeastern and middle-eastern regions. These phyletic units correspond to northern and southern taxa that have previously been assigned specific status as Ixodes dammini and Ixodes scapularis, respectively. The expanding range of I. dammini appears to drive the present outbreaks of zoonotic disease in eastern North America that include Lyme disease and human babesiosis.

With a standard set of primers directed toward conserved regions, we have used the polymerase chain reaction to amplify homologous segments of mtDNA from more than 100 animal species, including mammals, birds, amphibians, fishes, and some invertebrates. Amplification and direct sequencing were possible using unpurified mtDNA from nanogram samples of fresh specimens and microgram amounts of tissues preserved for months in alcohol or decades in the dry state. The bird and fish sequences evolve with the same strong bias toward transitions that holds for mammals. However, because the light strand of birds is deficient in thymine, thymine to cytosine transitions are less common than in other taxa. Amino acid replacement in a segment of the cytochrome b gene is faster in mammals and birds than in fishes and the pattern of replacements fits the structural hypothesis for cytochrome b. The unexpectedly wide taxonomic utility of these primers offers opportunities for phylogenetic and population research.

Periodontitis is a bacterial inflammatory disease leading to attachment loss with the consequence of tooth loss. There exists a multifactorial risk pattern including bacterial challenge, smoking, age, sex, diabetes, socio-economic and genetic factors. Smoking has the highest impact on the course of the disease modulated by all the other factors. Here, we report the relationship between smoking and the polymorphisms of genetic polymorphisms inflicted in the pathogenesis.

Periodontitis is a bacterial inflammatory disease leading to attachment loss with the consequence of tooth loss. There exists a multifactorial risk pattern including bacterial challenge, smoking, age, sex, diabetes, socio-economic and genetic factors. Smoking has the highest impact on the course of the disease modulated by all the other factors. Here, we report the relationship between smoking and the polymorphisms of genetic polymorphisms inflicted in the pathogenesis.