Individual Physiological Adaptations Enable Selected Bacterial Taxa To Prevail during Long-Term Incubations.

Enclosure experiments are frequently used to investigate the impact of changing environmental conditions on microbial assemblages. Yet, how the incubation itself challenges complex bacterial communities is thus far unknown. In this study, metaproteomic profiling, 16S rRNA gene analyses, and cell counts were combined to evaluate bacterial communities derived from marine, mesohaline, and oligohaline conditions after long-term batch incubations. Early in the experiment, the three bacterial communities were highly diverse and differed significantly in their compositions. Manipulation of the enclosures with terrigenous dissolved organic carbon resulted in notable differences compared to the control enclosures at this early phase of the experiment. However, after 55 days, bacterial communities in the manipulated and the control enclosures under marine and mesohaline conditions were all dominated by gammaproteobacterium Spongiibacter In the oligohaline enclosures, actinobacterial cluster I of the hgc group (hgc-I) remained abundant in the late phase of the incubation. Metaproteome analyses suggested that the ability to use outer membrane-based internal energy stores, in addition to the previously described grazing resistance, may enable the gammaproteobacterium Spongiibacter to prevail in long-time incubations. Under oligohaline conditions, the utilization of external recalcitrant carbon appeared to be more important (hgc-I). Enclosure experiments with complex natural microbial communities are important tools to investigate the effects of manipulations. However, species-specific properties, such as individual carbon storage strategies, can cause manipulation-independent effects and need to be considered when interpreting results from enclosures.IMPORTANCE In microbial ecology, enclosure studies are often used to investigate the effect of single environmental factors on complex bacterial communities. However, in addition to the manipulation, unintended effects ("bottle effect") may occur due to the enclosure itself. In this study, we analyzed the bacterial communities that originated from three different salinities of the Baltic Sea, comparing their compositions and physiological activities both at the early stage and after 55 days of incubation. Our results suggested that internal carbon storage strategies impact the success of certain bacterial species, independent of the experimental manipulation. Thus, while enclosure experiments remain valid tools in environmental research, microbial community composition shifts must be critically followed. This investigation of the metaproteome during long-term batch enclosures expanded our current understanding of the so-called "bottle effect," which is well known to occur during enclosure experiments.

M-CSF and IL-34 expression as indicators for growth in sporadic vestibular schwannoma.

Macrophage colony stimulating factor and IL-34 are associated with clinical vestibular schwannoma progression. Investigating the biology behind vestibular schwannoma progression helps understanding tumor growth. Inflammation is important in the microenvironment of neoplasms. Macrophages are major players in the intratumoral infiltrate. These tumor-associated macrophages are known to stimulate angiogenesis and cell growth. M-CSF and IL-34 are cytokines that can regulate tumor-infiltrating macrophages. They are expressed by tumors and form potential targets for therapy. The goal of this study was to investigate these cytokines in vestibular schwannomas and to see if their expression is related to angiogenesis, macrophage numbers, cystic degeneration, and volumetric tumor progression. Immunohistochemical expression of M-CSF and IL-34 was analyzed in ten fast-growing vestibular schwannomas and in ten slow-growing vestibular schwannomas. Expression M-CSF and IL-34 were compared between fast- versus slow-growing and cystic versus non-cystic tumors. Data on macrophage numbers and microvessel density, known from earlier research, was also included. All tumors expressed M-CSF and its expression was higher in fast-growing tumors (p = 0.003) and in cystic tumors (p = 0.035). CD163 expression was higher in tumors with strong M-CSF expression (p = 0.003). All tumors expressed IL-34 as well, but no significant differences were found in relation to clinicopathological characteristics. This study demonstrated the expression of M-CSF and IL-34 in vestibular schwannomas. The results suggest that M-CSF is related to macrophage activity and tumor progression, making it a potential target for therapy. If a similar assumption can be made for IL-34 remains unclear.

Impact of Pseudomonas H6 surfactant on all external life cycle stages of the fish parasitic ciliate Ichthyophthirius multifiliis.

A bacterial biosurfactant isolated from Pseudomonas (strain H6) has previously been shown to have a lethal effect on the oomycete Saprolegnia diclina infecting fish eggs. The present work demonstrates that the same biosurfactant has a strong in vitro antiparasitic effect on the fish pathogenic ciliate Ichthyophthirius multifiliis. Three life cycle stages (the infective theront stage, the tomont and the tomocyst containing tomites) were all susceptible to the surfactant. Theronts were the most sensitive showing 100% mortality in as low concentrations as 10 and 13 µg/ml within 30 min. Tomonts were the most resistant but were killed in concentrations of 100 µg/ml. Tomocysts, which generally are considered resistant to chemical and medical treatment, due to the surrounding protective cyst wall, were also sensitive. The surfactant, in concentrations of 10 and 13 µg/ml, penetrated the cyst wall and killed the enclosed tomites within 60 min. Rainbow trout fingerlings exposed to the biosurfactant showed no adverse immediate or late signs following several hours incubation in concentrations effective for killing the parasite. This bacterial surfactant may be further developed for application as an antiparasitic control agent in aquaculture.

Agar composition affects in vitro screening of biocontrol activity of antagonistic microorganisms.

Agar-based screening assays are the method of choice when evaluating antagonistic potential of bacterial biocontrol-candidates against pathogens. We showed that when using the same medium, but different agar compositions, the activity of a bacterial antagonist against Agrobacterium was strongly affected. Consequently, results from in vitro screenings should be interpreted cautiously.

Targeting survivin as a potential new treatment for chondrosarcoma of bone.

Chondrosarcomas are malignant cartilage-forming bone tumors, which are intrinsically resistant to chemo- and radiotherapy, leaving surgical removal as the only curative treatment option. Therefore, our aim was to identify genes involved in chondrosarcoma cell survival that could serve as a target for therapy. siRNA screening for 51 apoptosis-related genes in JJ012 chondrosarcoma cells identified BIRC5, encoding survivin, as essential for chondrosarcoma survival. Using immunohistochemistry, nuclear as well as cytoplasmic survivin expression was analyzed in 207 chondrosarcomas of different subtypes. Nuclear survivin has been implicated in cell-cycle regulation while cytoplasmic localization is important for its anti-apoptotic function. RT-PCR was performed to determine expression of the most common survivin isoforms. Sensitivity to YM155, a survivin inhibitor currently in phase I/II clinical trial for other tumors, was examined in 10 chondrosarcoma cell lines using viability assay, apoptosis assay and cell-cycle analysis. Survivin expression was found in all chondrosarcoma patient samples. Higher expression of nuclear and cytoplasmic survivin was observed with increasing histological grade in central chondrosarcomas. Inhibition of survivin using YM155 showed that especially TP53 mutant cell lines were sensitive, but no caspase 3/7 or PARP cleavage was observed. Rather, YM155 treatment resulted in a block in S phase in two out of three chondrosarcoma cell lines, indicating that survivin is more involved in cell-cycle regulation than in apoptosis. Thus, survivin is important for chondrosarcoma survival and chondrosarcoma patients might benefit from survivin inhibition using YM155, for which TP53 mutational status can serve as a predictive biomarker.

Src kinases in chondrosarcoma chemoresistance and migration: dasatinib sensitises to doxorubicin in TP53 mutant cells.

BACKGROUND: Chondrosarcomas are malignant cartilage-forming tumours of bone. Because of their resistance to conventional chemotherapy and radiotherapy, currently no treatment strategies exist for unresectable and metastatic chondrosarcoma. Previously, PI3K/AKT/GSK3ß and Src kinase pathways were shown to be activated in chondrosarcoma cell lines. Our aim was to investigate the role of these kinases in chemoresistance and migration in chondrosarcoma in relation to TP53 mutation status. METHODS: We used five conventional and three dedifferentiated chondrosarcoma cell lines and investigated the effect of PI3K/AKT/GSK3ß pathway inhibition (enzastaurin) and Src pathway inhibition (dasatinib) in chemoresistance using WST assay and live cell imaging with AnnexinV staining. Immunohistochemistry on tissue microarrays (TMAs) containing 157 cartilaginous tumours was performed for Src family members. Migration assays were performed with the RTCA xCelligence System. RESULTS: Src inhibition was found to overcome chemoresistance, to induce apoptosis and to inhibit migration. Cell lines with TP53 mutations responded better to combination therapy than wild-type cell lines (P=0.002). Tissue microarray immunohistochemistry confirmed active Src (pSrc) signalling, with Fyn being most abundantly expressed (76.1%). CONCLUSION: These results strongly indicate Src family kinases, in particular Fyn, as a potential target for the treatment of inoperable and metastatic chondrosarcomas, and to sensitise for doxorubicin especially in the presence of TP53 mutations.

Restoration of chemosensitivity for doxorubicin and cisplatin in chondrosarcoma in vitro: BCL-2 family members cause chemoresistance.

BACKGROUND: Chondrosarcomas are malignant cartilage-forming tumors notorious for their resistance to conventional chemo- and radiotherapy. Postulated explanations describe the inaccessibility due to abundant hyaline cartilaginous matrix, presence of multidrug resistance (MDR) pumps, and expression of anti-apoptotic BCL-2 family members. MATERIALS AND METHODS: We studied the sensitivity of chondrosarcoma cell lines (SW1353, CH2879, JJ012, OUMS27) and two primary cultures for doxorubicin and cisplatin. We examined the role of extracellular matrix using three-dimensional (3D) pellet models and MDR pump activity using fluorescence-activated cell sorter analysis. The role of BCL-2 family members was investigated using the BH3 mimetic ABT-737. RESULTS: Chondrosarcoma cells showed highest resistance to cisplatin. 3D cell pellets, morphologically strongly resembling chondrosarcoma in vivo, confirmed nuclear incorporation of doxorubicin. MDR pump activity was heterogeneous among cultures. Chondrosarcoma cells responded to ABT-737 and combination with doxorubicin led to complete loss of cell viability and apoptosis with cytochrome C release. CONCLUSIONS: Despite MDR pump activity and abundance of hyaline cartilaginous matrix, doxorubicin is able to accumulate in the cell nuclei. By repairing the apoptotic machinery, we were able to sensitize chondrosarcoma cells to doxorubicin and cisplatin, indicating an important role for BCL-2 family members in chemoresistance and a promising new treatment strategy for inoperable chondrosarcoma.

COX-2 expression in chondrosarcoma: a role for celecoxib treatment?

Chondrosarcomas are resistant to conventional chemo- and radiotherapy. A subset of chondrosarcomas arises secondarily in the benign tumour syndromes enchondromatosis (EC) and multiple osteochondromas (MO), and prevention of tumour development would greatly improve prognosis. We therefore investigated the effect of selective COX-2 inhibition on chondrosarcoma growth. COX-2 expression was studied in central- and peripheral cartilaginous tumours. The effect of COX-2 inhibition was assessed in four high-grade chondrosarcoma cell lines using celecoxib and NS-398 treatment. COX-2 activity (prostaglandin E(2) (PGE(2)) ELISA) and cell viability were measured. The (prophylactic) effect of celecoxib on chondrosarcoma growth in vivo was studied for 8 weeks using a xenograft model of cell line CH2879 in immunoincompetent nude mice. High COX-2 protein expression was mainly found in solitary peripheral chondrosarcoma and in enchondromatosis-related central chondrosarcoma, which was confirmed by qPCR. After 72h of celecoxib treatment, a significant decrease in cell viability was observed in three chondrosarcoma cell lines. In vivo, celecoxib initially slowed tumour growth in chondrosarcoma xenografts; however, after prolonged treatment relapsed tumour growth was observed. Tumour volume was negatively associated with celecoxib serum levels, and seemed smaller in the high-dose prophylactic treatment group. We confirmed the expression of COX-2 in 65% of chondrosarcomas, and COX-2 inhibition by celecoxib diminished cell viability in vitro. The initial response and the decrease in tumour volume with increased celecoxib serum levels in vivo supported a role for celecoxib, although relapsed tumour growth after 6 weeks was worrisome. Also the role of high-dose prophylactic celecoxib in preventing the development of benign and malignant cartilage tumours in EC and MO patients deserves further investigation.

Profiling of high-grade central osteosarcoma and its putative progenitor cells identifies tumourigenic pathways.

BACKGROUND: Osteosarcoma is the most prevalent primary malignant bone tumour in children and young adults, with poor survival in 40% of patients. To identify the signalling pathways involved in tumourigenesis, we compared gene expression in osteosarcoma with that in its presumed normal counterparts. METHODS: Genome-wide expression profiles were generated from 25 high-grade central osteosarcoma prechemotherapy biopsies, 5 osteoblastomas, 5 mesenchymal stem cell (MSC) populations and these same MSCs differentiated into osteoblasts. Genes that were differentially expressed were analysed in the context of the pathways in which they function using the GenMAPP programme. RESULTS: MSCs, osteoblasts, osteoblastomas and osteosarcomas clustered separately and thousands of differentially expressed genes were identified. The most significantly altered pathways are involved in cell cycle regulation and DNA replication. Several upstream components of the Wnt signalling pathway are downregulated in osteosarcoma. Two genes involved in degradation of beta-catenin protein, the key effectors of Wnt signalling, Axin and GSK3-beta, show decreased expression, suggesting that Wnt signalling is no longer under the control of regular signals. Comparing benign osteoblastomas with osteosarcomas identified cell cycle regulation as the most prominently changed pathway. CONCLUSION: These results show that upregulation of the cell cycle and downregulation of Wnt signalling have an important role in osteosarcoma genesis. Gene expression differences between highly malignant osteosarcoma and benign osteoblastoma involve cell cycle regulation.

Diversity and functional analysis of LuxR-type transcriptional regulators of cyclic lipopeptide biosynthesis in Pseudomonas fluorescens.

Cyclic lipopeptides (CLPs) are produced by many Pseudomonas species and have several biological functions, including a role in surface motility, biofilm formation, virulence, and antimicrobial activity. This study focused on the diversity and role of LuxR-type transcriptional regulators in CLP biosynthesis in Pseudomonas species and, specifically, viscosin production by Pseudomonas fluorescens strain SBW25. Phylogenetic analyses showed that CLP biosynthesis genes in Pseudomonas strains are flanked by LuxR-type regulators that contain a DNA-binding helix-turn-helix domain but lack N-acylhomoserine lactone-binding or response regulator domains. For SBW25, site-directed mutagenesis of the genes coding for either of the two identified LuxR-type regulators, designated ViscAR and ViscBCR, strongly reduced transcript levels of the viscABC biosynthesis genes and resulted in a loss of viscosin production. Expression analyses further showed that a mutation in either viscAR or viscBCR did not substantially (change of <2.5-fold) affect transcription of the other regulator. Transformation of the DeltaviscAR mutant of SBW25 with a LuxR-type regulatory gene from P. fluorescens strain SS101 that produces massetolide, a CLP structurally related to viscosin, restored transcription of the viscABC genes and viscosin production. The results further showed that a functional viscAR gene was required for heterologous expression of the massetolide biosynthesis genes of strain SS101 in strain SBW25, leading to the production of both viscosin and massetolide. Collectively, these results indicate that the regulators flanking the CLP biosynthesis genes in Pseudomonas species represent a unique LuxR subfamily of proteins and that viscosin biosynthesis in P. fluorescens SBW25 is controlled by two LuxR-type transcriptional regulators.

Trivalent inactivated subunit influenza vaccine Influvac: 25-Year experience of safety and immunogenicity.

Between 1982 and 2006, 76 clinical studies (including the annual update studies required for licensing in Europe) were performed with the trivalent inactivated subunit influenza vaccine Influvac. In all, 6415 subjects were vaccinated, of whom 5034 were eligible for safety evaluation and 4534 for efficacy evaluation. Treatment-emergent adverse events occurred in 13.7% of subjects. Transient mild-to-moderate local and systemic reactions occurred in up to half of subjects. Post-marketing surveillance confirmed the well-established safety profile reported for inactivated influenza vaccines. All three serological criteria for immunogenicity of the Committee for Medicinal Products for Human Use (CHMP) were met for all three virus strain (sub)types in healthy adults, elderly (over 60 years), nursing home residents, and those at high risk of influenza-related complications. In an additional trial in children aged 3 -- 12 years, all three CHMP criteria for adults were met for all three virus strains. Influvac is thus immunogenic and safe, and is a suitable vaccine to combat the annually recurring medical and economic burden of influenza epidemics.

Regulation of cyclic lipopeptide biosynthesis in Pseudomonas fluorescens by the ClpP protease.

Cyclic lipopeptides produced by Pseudomonas species exhibit potent surfactant and broad-spectrum antibiotic properties. Their biosynthesis is governed by large multimodular nonribosomal peptide synthetases, but little is known about the genetic regulatory network. This study provides, for the first time, evidence that the serine protease ClpP regulates the biosynthesis of massetolides, cyclic lipopeptides involved in swarming motility, biofilm formation, and antimicrobial activity of Pseudomonas fluorescens SS101. The results show that ClpP affects the expression of luxR(mA), the transcriptional regulator of the massetolide biosynthesis genes massABC, thereby regulating biofilm formation and swarming motility of P. fluorescens SS101. Transcription of luxR(mA) was significantly repressed in the clpP mutant, and introduction of luxR(mA) restored, in part, massetolide biosynthesis and swarming motility of the clpP mutant. Site-directed mutagenesis and expression analyses indicated that the chaperone subunit ClpX and the Lon protease are not involved in regulation of massetolide biosynthesis and are transcribed independently of clpP. Addition of Casamino Acids enhanced the transcription of luxR(mA) and massABC in the clpP mutant, leading to a partial rescue of massetolide production and swarming motility. The results further suggested that, at the transcriptional level, ClpP-mediated regulation of massetolide biosynthesis operates independently of regulation by the GacA/GacS two-component system. The role of amino acid metabolism and the putative mechanisms underlying ClpP-mediated regulation of cyclic lipopeptide biosynthesis, swarming motility, and growth in P. fluorescens are discussed.

Myxoid tumours of soft tissue: the so-called myxoid extracellular matrix is heterogeneous in composition.

AIM: Myxoid tumours of soft tissue are characterized by their so-called 'myxoid' extracellular matrix. The aim was to investigate the composition and possible function of this matrix which is poorly understood. METHODS AND RESULTS: Using Alcian Blue staining with and without pretreatment with hyaluronidase and application of the critical electrolyte concentration method followed by densitometry, the glycosaminoglycan composition of three different myxoid tumours was studied. The composition of glycosaminoglycans varied with tumour type and grade, despite their general characterization as myxoid tumours. Intramuscular myxoma contained similar amounts of the various glycosaminoglycans as grade I myxofibrosarcoma; grade III myxofibrosarcoma contained less hyaluronic acid and more heparan sulphate, whereas extraskeletal myxoid chondrosarcoma contained predominantly chondroitin-4 and -6 sulphates. Western blot identified albumin as a major protein in tumour lysates, and its presence in the extracellular matrix and cytoplasm of the majority of tumours was demonstrated by immunohistochemistry; production of albumin by the tumour cells was confirmed by quantitative polymerase chain reaction. CONCLUSIONS: The extracellular matrix of myxoid tumours of soft tissue has a heterogeneous composition consisting of, amongst others, glycosaminoglycans and albumin, which appear to play an active role in their morphogenesis.

Massetolide A biosynthesis in Pseudomonas fluorescens.

Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various Pseudomonas strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in P. fluorescens SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated massA, massB, and massC, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the mass genes contained several genes found in other Pseudomonas CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most Pseudomonas CLP gene clusters known to date, the mass genes are not physically linked but are organized in two separate clusters, with massA disconnected from massB and massC. Quantitative real-time PCR analysis indicated that transcription of massC is strongly reduced when massB is mutated, suggesting that these two genes function in an operon, whereas transcription of massA is independent of massBC and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by N-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of P. fluorescens SS101 and plays an important role in biofilm formation.

The virosomal influenza vaccine Invivac: immunogenicity and tolerability compared to an adjuvanted influenza vaccine (Fluad in elderly subjects.

Several approaches are currently being pursued in order to improve the efficacy of influenza vaccines in elderly individuals and others who have impaired immune responses to conventional influenza vaccines. There are two influenza vaccines available for elderly subjects: Fluad (Chiron) and Invivac (Solvay Pharmaceuticals). The present clinical study was a randomized, endpoint-blind, parallel group study in elderly subjects aged 61 years and older to investigate the safety and immunogenicity of these vaccines as compared to a standard influenza vaccine Invivac (Solvay Pharmaceuticals). The three vaccines had similar immunogenicity results, whereas the tolerability profile of Invivac was better as compared to Fluad.

Clinical experience with inactivated, virosomal influenza vaccine.

Current available influenza vaccines are safe and effective in preventing influenza. Nevertheless, there is a need for influenza vaccines with improved efficacy in the elderly. This need is underscored by both the observation that influenza has a major clinical and economic impact in the elderly and the fact that currently available vaccines are generally less effective in elderly than in younger subjects. Several approaches are currently being pursued in order to improve the efficacy of influenza vaccines in elderly individuals and others who have impaired immune responses to conventional influenza vaccines. A novel antigen-presenting strategy to overcome impaired immune responses is the use of virosomes. Previously, data on safety and reactogenicity have been published regarding the use of virosomal influenza vaccines. Data from three recent clinical trials are presented here. The first of these was a comparative study of a virosomal vaccine and a conventional subunit vaccine in "at-risk" adults with underlying chronic illness. The virosomal vaccine demonstrated comparable tolerability to the subunit vaccine, with about 98% of patients reporting tolerability to be good or very good. The vast majority of adverse events reported were mild to moderate in severity. With both vaccine types, mean HI titres decreased with age for both the A-H1N1 and B influenza virus strains, but for the A-H3N2 strain (the most virulent of the three strains), mean HI titres did not decrease with age, suggesting a better response with the virosomal vaccine when compared to the subunit vaccine. All three studies explored the long-term persistence of antibodies after vaccination with virosomal influenza vaccines. Immunogenicity declined over time but remained high at 4, 6 and 12 months post-vaccination compared to baseline, indicating that adequate seroprotection is achievable for the duration of the influenza season. Virosomal vaccines may induce better immunity in elderly subjects and may be more effective in reducing morbidity and mortality in this age group.

Aggressive angiomyxoma: a clinicopathological and immunohistochemical study of 11 cases with long-term follow-up.

AIMS: To report the clinicopathological and immunohistochemical features and longer term biological behaviour of aggressive angiomyxoma, an uncommon mesenchymal neoplasm occurring predominantly in the pelvi-perineal region of adults. Using immunohistochemistry, possible overexpression of CDK4 and MDM2 was analysed, which might point to (cyto)genetic alteration(s) in chromosome region 12q13-15, an area reported to be altered in this tumour entity. METHODS AND RESULTS: Cases (n=11) of aggressive angiomyxoma were retrieved from the consultation files of the Comprehensive Cancer Centre of the Middle Netherlands (IKMN) panel for soft tissue tumours. Clinical and follow-up information were obtained, and immunohistochemical analysis was performed using antibodies directed against vimentin, cytokeratin AE1/AE3, desmin, alpha-smooth-muscle actin, CD34, S-100 protein, oestrogen receptors, CDK4 and MDM2. Five patients were female (age range 24-47 years; median 39 years), and six patients were male (age range 36-69 years; median 44.5 years). Of 11 cases, 10 arose in the pelvi-perineal area and 1 arose in the abdominal cavity in close relation to the bladder. Morphology was consistent with previous reports of this entity. Immunohistochemically, 8 of 11 cases were desmin positive (5 of 5 positive in females; 3 of 6 positive in males), 6 of 11 cases were positive for alpha-smooth-muscle actin, 5 of 11 cases were CD34 positive, 11 of 11 cases, irrespective of gender, were positive for oestrogen receptors and 3 of 11 cases were positive for cytokeratin AE1/AE3. Strong, diffuse nuclear positivity for CDK4 expression was present in all 6 cases tested, while only 1 of 11 cases tested for MDM2 showed weak focal positivity. Clinical follow-up in all cases (range 1-216 months; median 72 months) showed one local recurrence (9%) after 36 months. No metastases or tumour-related deaths were noted. CONCLUSIONS: The sex distribution of cases reported in this study was roughly equal, in contrast to previous reports emphasising the predominance of this tumour in females. Our study confirms the local aggressive nature of aggressive angiomyxoma, although our local recurrence rate is lower than previous reports (9% versus 36-72%); no metastases and/or disease-related patient deaths were documented. All cases arising in females were positive for desmin, while three of the six cases arising in males were negative for desmin, supporting previous findings and indicating that the lesion may be somewhat different in males. The strong diffuse positivity for CDK4 in all six cases tested goes some way in implicating CDK4, either directly or indirectly, in tumourigenesis. The negative immunostaining for MDM2 would argue against functional amplification of this gene.

Virosomal influenza vaccine: a safe and effective influenza vaccine with high efficacy in elderly and subjects with low pre-vaccination antibody titers.

In 14 clinical studies, various efficacy and safety aspects of a new virosomal influenza vaccine (Invivac) were assessed in 2865 subjects. The virosomal influenza vaccine fully complies with the Committee for Proprietary Medicinal Products (CPMP) requirement for immunogenicity of influenza vaccines. In particular, in a subset of subjects with low pre-vaccination titers (thus those persons who actually need protection by a vaccine), between 76 and 99% of subjects (dependent on age, health status and vaccine components) achieved protective hemagglutination inhibiting (HI) antibody titers after vaccination with the virosomal influenza vaccine. Acceptable frequencies of well-known local and systemic reactions were observed in healthy adults and risk subjects in clinical studies and in a post-marketing study population. These reactions were transient and generally not severe, and did not cause major inconvenience. In conclusion, Invivac is an efficacious and safe vaccine for the protection against influenza in healthy and chronically ill adult subjects. The vaccine is especially efficacious in subjects with low pre-vaccination immunity.

Epidermal growth factor-like repeats mediate lateral and reciprocal interactions of Ep-CAM molecules in homophilic adhesions.

Ep-CAM is a new type of cell adhesion molecule (CAM) which does not structurally resemble the members of the four major families (cadherins, integrins, selectins, and CAMs of the immunoglobulin superfamily) and mediates Ca(2+)-independent, homophilic adhesions. The extracellular domain of Ep-CAM consists of a cysteine-rich region, containing two type II epidermal growth factor (EGF)-like repeats, followed by a cysteine-poor region. We generated mutated Ep-CAM forms with various deletions in the extracellular domain. These deletion mutants, together with monoclonal antibodies recognizing different epitopes in the extracellular domain, were used to investigate the role of the EGF-like repeats in the formation of intercellular contacts mediated by Ep-CAM molecules. We established that both EGF-like repeats are required for the formation of Ep-CAM-mediated homophilic adhesions, including the accumulation of Ep-CAM molecules at the cell-cell boundaries, and the anchorage of the Ep-CAM adhesion complex to F-actin via alpha-actinin. Deletion of either EGF-like repeat was sufficient to inhibit the adhesion properties of the molecule. The first EGF-like repeat of Ep-CAM is required for reciprocal interactions between Ep-CAM molecules on adjacent cells, as was demonstrated with blocking antibodies. The second EGF-like repeat was mainly required for lateral interactions between Ep-CAM molecules. Lateral interactions between Ep-CAM molecules result in the formation of tetramers, which might be the first and necessary step in the formation of Ep-CAM-mediated intercellular contacts.