Identification of CUX1 as the recurrent chromosomal band 7q22 target gene in human uterine leiomyoma.

Uterine leiomyomas are benign solid tumors of mesenchymal origin which occur with an estimated incidence of up to 77% of all women of reproductive age. The majority of these tumors remains symptomless, but in about a quarter of cases they cause leiomyoma-associated symptoms including chronic pelvic pain, menorrhagia-induced anemia, and impaired fertility. As a consequence, they are the most common indication for pre-menopausal hysterectomy in the USA and Japan and annually translate into a multibillion dollar healthcare problem. Approximately 40% of these neoplasms present with recurring structural cytogenetic anomalies, including del(7)(q22), t(12;14)(q15;q24), t(1;2)(p36;p24), and anomalies affecting 6p21 and/or 10q22. Using positional cloning strategies, we and others previously identified HMGA1, HMGA2, RAD51L1, MORF, and, more recently, NCOA1 as primary target (fusion) genes associated with tumor initiation in four of these distinct cytogenetic subgroups. Despite the fact that the del(7)(q22) subgroup is the largest among leiomyomas, and was first described more than twenty years ago, the 7q22 leiomyoma target gene still awaits unequivocal identification. We here describe a positional cloning effort from two independent uterine leiomyomas, containing respectively a pericentric and a paracentric chromosomal inversion, both affecting band 7q22. We found that both chromosomal inversions target the cut-like homeobox 1 (CUX1) gene on chromosomal band 7q22.1 in a way which is functionally equivalent to the more frequently observed del(7q) cases, and which is compatible with a mono-allelic knock-out scenario, similar as was previously described for the cytogenetic subgroup showing chromosome 14q involvement.

Improved detection of circulating Aspergillus antigen by use of a modified pretreatment procedure.

Detection of circulating galactofuranose (galf) antigens, including galactomannan (GM), by the Platelia Aspergillus (PA) enzyme-linked immunosorbent assay (ELISA) is an important tool in the early diagnosis of invasive aspergillosis (IA). We used a modified pretreatment technique (MT) on consecutive negative PA ELISA plasma samples from IA patients in order to improve the detection of the fungal components present. Plasma samples (52) were collected from healthy donors, and 174 plasma samples with a galactomannan index (GMI) below 0.5 were collected from 25 unclassifiable and 23 IA patients. The PA ELISA reactivity of pretreated samples was determined before (conventional technique [CT]) and after (MT) filtration using a Microcon filter with a 50-kDa cutoff (Millipore). For the MT, the sensitivity of the PA ELISA increased from 42.9% (CT) to 78.6% (MT) using a cutoff for the GMI of 1.5 in the probable and proven group, whereas specificity slightly decreased from 98.7% to 96.1% in the control group. The 10-fold concentration step increased the GMI as high as 121-fold. The MT resulted not only in positive reactivity in samples that tested negative with the CT but also in the earlier detection of antigen by 2 to 17 days.

Development of a PCL-silica nanoparticles composite membrane for Guided Bone Regeneration.

The pivotal step in Guided Bone Regeneration (GBR) therapy is the insertion of a membrane for support and barrier functions. Here, we studied the effect of the addition of silica nanoparticles (Si-NPs) in electrospun poly(ε-caprolactone) (PCL) membranes to improve the mechanical and osteoconductive properties of the membranes. To this end, Si-NPs were firstly synthesized and then suspended in PCL solutions containing a polar solvent (2,2,2-trifluroethanol) and water with the addition of an anionic surfactant. Nanocomposite membranes were fabricated from the solutions through an electrospinning technique. Morphology, structure and chemical composition, and tensile properties of the membranes were analyzed. Membrane stability was determined by visual examination of the membranes after immersion in phosphate buffered saline. The effect of the materials on osteoblastic differentiation was evaluated by in vitro culture of the membranes with MC3T3-E1 osteoblastic cells. The results indicated that Si-NPs were successfully incorporated in the interior of the PCL electrospun fibers during the electrospinning process. Tensile modulus was significantly increased for composition S50 and tensile strength significantly increased for compositions S25 and S50. Membranes containing Si-NPs have shown to be cytocompatible. The results obtained demonstrate that the Si-NPs were homogeneously incorporated in the electrospun fibers, resulting in an improvement of the tensile properties of the prepared materials.

Bacterial SBP56 identified as a Cu-dependent methanethiol oxidase widely distributed in the biosphere.

Oxidation of methanethiol (MT) is a significant step in the sulfur cycle. MT is an intermediate of metabolism of globally significant organosulfur compounds including dimethylsulfoniopropionate (DMSP) and dimethylsulfide (DMS), which have key roles in marine carbon and sulfur cycling. In aerobic bacteria, MT is degraded by a MT oxidase (MTO). The enzymatic and genetic basis of MT oxidation have remained poorly characterized. Here, we identify for the first time the MTO enzyme and its encoding gene (mtoX) in the DMS-degrading bacterium Hyphomicrobium sp. VS. We show that MTO is a homotetrameric metalloenzyme that requires Cu for enzyme activity. MTO is predicted to be a soluble periplasmic enzyme and a member of a distinct clade of the Selenium-binding protein (SBP56) family for which no function has been reported. Genes orthologous to mtoX exist in many bacteria able to degrade DMS, other one-carbon compounds or DMSP, notably in the marine model organism Ruegeria pomeroyi DSS-3, a member of the Rhodobacteraceae family that is abundant in marine environments. Marker exchange mutagenesis of mtoX disrupted the ability of R. pomeroyi to metabolize MT confirming its function in this DMSP-degrading bacterium. In R. pomeroyi, transcription of mtoX was enhanced by DMSP, methylmercaptopropionate and MT. Rates of MT degradation increased after pre-incubation of the wild-type strain with MT. The detection of mtoX orthologs in diverse bacteria, environmental samples and its abundance in a range of metagenomic data sets point to this enzyme being widely distributed in the environment and having a key role in global sulfur cycling.

Paradoxical increase in circulating Aspergillus antigen during treatment with caspofungin in a patient with pulmonary aspergillosis.

A paradoxical increase in circulating Aspergillus antigen was observed during treatment with caspofungin in a patient with proven invasive aspergillosis. With the exception of treatment with the echinocandin, no other factors were found that might explain this clinical observation, which was supported by experiments done in vitro.

Non-culture-based diagnostics for opportunistic fungi.

The value of the diagnostic markers galactomannan and 1,3-beta-D-glucan for the diagnosis of opportunistic fungal infections is reviewed in this article. Both markers have undergone clinical evaluation, and increasing insight is emerging with respect to the causes of false-negative or false-positive reactivity. These data will help design protocols in which single or multiple markers are used to identify patients who require antifungal therapy.

Issues with galactomannan testing.

Within the past decade detection of the aspergillus antigen galactomannan has become an important and reliable tool for the early diagnosis of invasive aspergillosis. The galactomannan molecule, that is detected by the commercial sandwich ELISA (Platelia Aspergillus, Biorad), was found not to be a single molecule, but a family of molecules that have the epitope that reacts with the monoclonal antibody. Also the cut off level is now world-wide lowered to 0.5 which will help to further standardize and compare this diagnostic tool. Despite the advantages of galactomannan detection, there are several issues that have impact on its use in clinical practice. Both false negative and false positive reactivity is encountered and although the causes of false reactivity are not fully understood, new insights have become available which help us to optimize the use of the assay. This review discusses present issues with galactomannan testing with a view to future research and management.

Bifidobacterium lipoteichoic acid and false ELISA reactivity in aspergillus antigen detection.

A major difficulty with the detection of circulating galactomannan, a cell-wall polysaccharide released by Aspergillus sp during growth, in the serodiagnosis of invasive aspergillosis is the occurrence of false-positive ELISA results, especially in neonates and infants. On the basis of molecule similarity, we postulate that a lipoteichoic acid of Bifidobacterium sp can act as epitope for the monoclonal antibody used in the ELISA. The neonatal gut is heavily colonised with Bifidobacterium sp and these bacteria or their lipoteichoic acid might cause ELISA reactivity with serum after translocation because of immaturity of the intestinal mucosa. If our hypothesis is correct, we might find a method to discriminate between false-positive and true-positive ELISA results and thereby prevent unnecessary pre-emptive treatment of patients.

Utility of Aspergillus antigen detection in specimens other than serum specimens.

The detection of circulating galactomannan in serum is an important tool for the early diagnosis of invasive aspergillosis. A commercial enzyme-linked immunosorbent assay (Platelia Aspergillus; BioRad) was shown to be both highly sensitive and specific for detection of galactomannan in serum samples. Despite the fact that this assay is validated for serum samples, specimens of other body fluids are increasingly used for detection of galactomannan, including urine, bronchoalveolar lavage fluid, and cerebrospinal fluid. Review of the literature shows that galactomannan can be detected in each of these samples from patients with invasive aspergillosis with higher sensitivity than is the case with culture, as well as early in the course of infection. However, the evidence thus far is based on case reports--predominantly retrospective studies--that often include heterogeneous patient populations and limited numbers of cases of proven infection. Clearly, well-designed prospective studies with systematic sampling and use of consensus case definitions are needed to compare the performance of antigen detection in samples other than serum specimens with that in serum specimens.

Detection of circulating galactomannan for the diagnosis and management of invasive aspergillosis.

The availability of the Platelia Aspergillus, a sandwich ELISA kit that detects circulating galactomannan, has been a major advance for managing patients at risk for invasive aspergillosis because of the early detection of the antigen. The assay is now widely used throughout the world, including the USA. Although initial studies that assessed the performance characteristics of this assay reported high sensitivity and specificity, more recent studies show significant variation in performance. The causes of this variability are multifactorial and, in large part, cannot be explained because there is insufficient understanding of the kinetics of galactomannan in vivo. We explored some of the factors that affect the release of the aspergillus antigen that bears the epitope that reacts with the monoclonal antibody used in the ELISA, its leakage from the site of infection into the blood, and its binding to substances present in the blood. Factors that affect the detection of antigen in blood are also discussed, most notably the pretreatment procedure aimed at liberating the antigen from immune complexes. Understanding the biology of galactomannan release by aspergillus will greatly enhance our understanding of the kinetics of this and other surrogate markers and allow their optimum use in the management of invasive aspergillosis.

Characterization of a recurrent t(1;2)(p36;p24) in human uterine leiomyoma.

Uterine leiomyomas are the most common neoplasms in women of reproductive age. Approximately 40% of these neoplasms show recurring structural cytogenetic anomalies, including del(7)(q22), t(12;14)(q15;q24), t(1;2)(p36;p24), and anomalies affecting 6p21 or 10q22. Using positional cloning strategies, we and others had previously identified HMGA1, HMGA2, RAD51L1, and MYST4 (previously referred to as MORF); as primary target (fusion) genes associated with tumor development in three of these distinct cytogenetic subgroups. Here, we report the positional cloning of a single, recurrent, leiomyoma-associated anomaly, t(1;2)(p36;p24). Molecular characterization of the reciprocal breakpoint intervals showed that that AJAP1 (alias SHREW1) and NPHP4 flank the breakpoint on chromosome 1 and that ITSN2 and NCOA1 flank the breakpoint on chromosome 2. Detailed analysis of the breakpoint regions revealed that in this particular case the translocation was associated with a 27-bp deletion on chromosome 1 and a 136-bp duplication on chromosome 2. No breakpoint-spanning (fusion) genes were identified. In silico prediction of transcription factor binding sites, however, indicated the presence of several such sites in the respective breakpoint regions, and major changes therein as a result of the t(1;2)(p36;p24) under investigation. We postulate that transcriptional deregulation of one or more of these breakpoint-flanking genes may contribute to the development of human uterine leiomyomas.

Value of Candida serum markers in patients with invasive candidiasis after myeloablative chemotherapy.

Invasive Candida infections are associated with a significant morbidity and mortality. Detection of circulating biomarkers has been shown to precede conventional diagnostic methods, which is important in improving outcome. We investigated the performance of multiple biomarkers using Candida antigen and anti-Candida antibody detection systems of Platelia and Serion and beta-d-glucan detection in serial serum samples from patients, treated for leukemia, with invasive candidiasis. The performance of single assays and combined detection appeared different for patients with 1 or more episodes of neutropenia and is therefore related to the phase of therapy for the underlying leukemia of the patient. These new insights may help to optimize the diagnostic strategies for the diagnosis of invasive candidiasis.

In vitro release by Aspergillus fumigatus of galactofuranose antigens, 1,3-beta-D-glucan, and DNA, surrogate markers used for diagnosis of invasive aspergillosis.

Aspergillus markers are becoming increasingly important for the early diagnosis of invasive aspergillosis. The kinetics of release of these surrogate markers, however, is largely unknown. We investigated the release of beta-(1-5)-galactofuranosyl (galf) antigens (Platelia Aspergillus), 1,3-beta-D-glucan (BG) (Fungitell), and DNA (PCR) in an in vitro model of Aspergillus fumigatus. The results showed that release is correlated to the growth phase of the fungus, which depends on available nutrients. Whereas galf antigens and BG are released during logarithmic growth, DNA is released only after mycelium breakdown. During early logarithmic growth, galf antigens seem to be released somewhat earlier than BG. Furthermore, galf antigen concentrations of more than 120,000 times the serum cutoff value (0.5 ng/ml) can be measured, while BG concentrations reach a value only 978 times the serum cutoff value (60 pg/ml). During lytical growth, release of galf antigens further increased to a maximum level, which depended on pH. After that, the concentration of galf antigens stayed high (pH 7.4) or decreased to zero within 4 days (pH 5.0). In contrast to galf antigens, BG concentration decreased after 1 day of growth. The decrease of galf components seems to be due to the enzyme beta-galactofuranosidase, which is able to destroy galf epitopes and whose activity fluctuates in the culture filtrates in parallel with galf antigen concentration. Fungal DNA seems to be released only due to autolysis caused by nutrient limitation. In conclusion, several factors clearly influence the release of surrogate markers in vitro. These same factors might also play a role at the infection site of Aspergillus disease in humans.

Bifidobacterial lipoglycan as a new cause for false-positive platelia Aspergillus enzyme-linked immunosorbent assay reactivity.

We previously hypothesized that a lipoglycan of Bifidobacterium bifidum subsp. pennsylvanicum cross-reacts with the Platelia Aspergillus (PA) enzyme-linked immunosorbent assay (ELISA) based on the presence of galactofuranosyl epitopes in the cell wall (M. A. S. H. Mennink-Kersten, R. R. Klont, A. Warris, H. J. M. Op den Camp, and P. E. Verweij, Lancet 363:325-327, 2004). We tested this hypothesis by testing bacterial suspensions of different bifidobacterial species and other gram-positive and -negative bacteria with the PA ELISA, which is used to detect circulating galactomannan for the serodiagnosis of invasive aspergillosis. Furthermore, neonatal fecal samples were enumerated for bifidobacteria by fluorescence in situ hybridization (FISH) and tested for PA ELISA reactivity. All bifidobacteria, except B. infantis and B. adolescentis, showed reactivity 6- to 600-fold higher compared to the controls (i.e., Micrococcus luteus and Propionibacterium freudenreichii, which contain a cell wall lipomannan). Eggerthella lenta showed a 25-fold-higher reactivity. ELISA reactivity was clearly shown to be associated with bacterial lipoglycans containing a beta-1,5-galactofuranosyl chain. All neonatal feces showed PA ELISA reactivity and associated numbers of bifidobacteria. Since high concentrations of bifidobacteria are present in the human gut, these bacteria or excreted lipoglycan may cause false serum PA ELISA reactivity in selected patient groups, especially neonates.