Molecular epidemiology of apparent outbreak of invasive aspergillosis in a hematology ward.

During a 2-month period, five patients suffering from invasive infections caused by Aspergillus flavus or Aspergillus fumigatus were identified in the Hematology Department of the University Hospital Dijkzigt (Rotterdam, The Netherlands). To study the epidemiological aspects of invasive aspergillosis, strains from these patients and from the hospital environment, isolated during extensive microbiological screening, were subjected to genotyping. A novel DNA extraction technique, involving freezing, grinding, and direct lysis in guanidium isothiocyanate-containing buffers of mycelial material, was applied. DNA isolation was followed by typing by random amplification of polymorphic DNA (RAPD) analysis. This showed that strains isolated from all patients infected with the same fungal species were genotypically distinct, thus providing evidence against the possibility of an ongoing, single-source nosocomial outbreak. Strains could also be differentiated from strains of geographically diverse origins. However, an A. flavus strain from one of the patients was also frequently encountered in the hospital environment. As all environmental strains were collected after this patient had been diagnosed with invasive disease, the epidemiological value of this observation could not be ascertained. Intensive investigations showed no single source of A. flavus or other aspergilli. RAPD genotyping proved that the outbreak of invasive aspergillosis in the hematology ward consisted of a series of unrelated events and was not due to a common source within the hospital. RAPD fingerprinting of aspergilli may greatly facilitate future investigations of the epidemiology of invasive disease caused by these pathogens.     

Management considerations to implementing pharmaceutical care

Progressing towards the goal of PC requires a fundamental change to pharmacy practice. Strong leadership and management skills will be needed to facilitate this change. Even with enthusiastic and capable staff, implementation of the PC model will require considerable effort. Changes to the department's mission statement and organizational structure will be required. From this beginning, an action plan for the department can be developed. This plan includes the training of individuals and/or recruiting the necessary personnel. An ongoing education program, as well as determining the value of your service, is required. With successful implementation the PC model will lead to the acceptance of the pharmacist's role as the person responsible for identifying, preventing, and resolving drug-related problems.     

A continuous‐flow,high‐throughput,high‐pressure parahydrogen converter for hyperpolarization in a clinical setting

Pure parahydrogen (pH₂) is the prerequisite for optimal pH₂‐based hyperpolarization experiments, promising approaches to access the hidden orders of magnitude of MR signals. pH₂ production on‐site in medical research centers is vital for the proliferation of these technologies in the life sciences. However, previously suggested designs do not meet our requirements for safety or production performance (flow rate, pressure or enrichment). In this article, we present the safety concept, design and installation of a pH₂ converter, operated in a clinical setting. The apparatus produces a continuous flow of four standard liters per minute of ≈98% enriched pH₂ at a pressure maximum of 50 bar. The entire production cycle, including cleaning and cooling to 25 K, takes less than 5 h, only ≈45 min of which are required for actual pH₂ conversion. A fast and simple quantification procedure is described. The lifetimes of pH₂ in a glass vial and aluminum storage cylinder are measured to be T_1C(glass vial) = 822 ± 29 min and T_1C(Al cylinder) = 129 ± 36 days, thus providing sufficiently long storage intervals and allowing the application of pH₂ on demand. A dependence of line width on pH₂ enrichment is observed. As examples, ¹H hyperpolarization of pyridine and ¹³C hyperpolarization of hydroxyethylpropionate are presented. Copyright © 2012 John Wiley & Sons, Ltd.     

Tumor initiating but differentiated luminal-like breast cancer cells are highly invasive in the absence of basal-like activity

The majority of human breast cancers exhibit luminal epithelial differentiation. However, most aggressive behavior, including invasion and purported cancer stem cell activity, are considered characteristics of basal-like cells. We asked the following questions: Must luminal-like breast cancer cells become basal-like to initiate tumors or to invade? Could luminally differentiated cells within a basally initiated hierarchy also be tumorigenic? To answer these questions, we used rare and mutually exclusive lineage markers to isolate subsets of luminal-like and basal-like cells from human breast tumors. We enriched for populations with or without prominent basal-like traits from individual tumors or single cell cloning from cell lines and recovered cells with a luminal-like phenotype. Tumor cells with basal-like traits mimicked phenotypic and functional behavior associated with stem cells assessed by gene expression, mammosphere formation and lineage markers. Luminal-like cells without basal-like traits, surprisingly, were fully capable of initiating invasive tumors in NOD SCID gamma (NSG) mice. In fact, these phenotypically pure luminal-like cells generated larger and more invasive tumors than their basal-like counterparts. The tumorigenicity and invasive potential of the luminal-like cancer cells relied strongly on the expression of the gene GCNT1, which encodes a key glycosyltransferase controlling O-glycan branching. These findings demonstrate that basal-like cells, as defined currently, are not a requirement for breast tumor aggressiveness, and that within a single tumor there are multiple “stem-like” cells with tumorigenic potential casting some doubt on the hypothesis of hierarchical or differentiative loss of tumorigenicity.     

Differential expression of perforin in muscle-infiltrating T cells in polymyositis and dermatomyositis.

Polymyositis (PM) and dermatomyositis (DM) are the prototypical inflammatory diseases of skeletal muscle. In PM, CD8+ T cells invade and destroy muscle fibers, whereas humoral effector mechanisms prevail in DM. We studied the expression of the cytotoxic mediator perforin in inflammatory cells in PM and DM muscle by semiquantitative PCR, immunohistochemistry and confocal laser microscopy. Similar levels of perforin mRNA were expressed in PM and DM, and abundant perforin-expressing CD3+CD8+ and CD3+ CD4+ T cells were observed in both diseases. However, there was a striking difference in the intracellular localization of perforin. In DM, perforin was distributed randomly in the cytoplasm of the inflammatory T cells. In contrast, 43% of the CD8+ T cells that contacted a muscle fiber in PM showed perforin located vectorially towards the target muscle fiber. The results suggest (a) that the random distribution of perforin in the cytoplasm of muscle-infiltrating T cells observed in DM reflects nonspecific activation, and (b) that the vectorial orientation observed only in PM reflects the specific recognition via the T cell receptor of an antigen on the muscle fiber surface, pointing to a perforin- and secretion-dependent mechanism of muscle fiber injury.     

CNN-Gd(3+) enables cell nucleus molecular imaging of prostate cancer cells: the last 600 nm

Molecular imaging is defined as the characterization and measurement of biological processes at the cellular and molecular level. Molecular imaging, therefore, necessitates a sufficient amount of contrast agent within the cell. Consequently, we realized that the intracellular uptake and cell compartment specificity of the commonly used interstitial contrast agent gadolinium (Gd(3+)) with a cell-nucleus directed peptide module could be helpful. This modular molecule is characterized by a Gd(3+)-complex module that is bound to a transmembrane transport unit (TPU) of human origin and further to a nucleus-directed address module (nuclear localization sequence) resulting in a specific cell nucleus-directed nuclear localization sequence-conjugated Gd(3+)-complex (CNN-Gd(3+)-complex). By use of magnetic resonance imaging, Gd(3+) was detected within DU-145 prostate cancer cells after only 10 min. The nuclear localization was confirmed with confocal laser scanning microscopy. The resulting MRI signal enhancement only slightly decreased over the next 48 h compared with an absolute loss of signal enhancement after only 8 h when a random target sequence was used. Therefore, our method seems promising for in vivo application in molecular imaging.     

Antigen loading of dendritic cells with whole tumor cell preparations

Dendritic cells (DC) based vaccinations have been widely used for the induction of anti-tumoral immunity in clinical studies. Antigen loading of DC with whole tumor cell preparations is an attractive method whenever tumor cell material is available. In order to determine parameters for the loading procedure, we performed dose finding and timing experiments. We found that apoptotic and necrotic melanoma cells up to a ratio of one-to-one, equivalent to 1mg/ml protein per 1 x 10(6) DC, can be added to monocyte derived DC without effecting DC recovery extensively. Using the isolated protein content of tumor cells (lysate) as a parameter, up to 5 mg/ml protein per 1 x 10(6) DC can be added. To achieve significant protein uptake at least 1 mg/ml of protein have to be added for more than 24 h as tested with FITC-labelled ovalbumin. Maturation inducing cytokines can be added simultaneously with the tumor cell preparations to immature DC without affecting the uptake. Furthermore, we tested the feasibility of cryopreservation of loaded and matured DC to facilitate the generation of ready to use aliquots. DC were cryopreserved in a mix of human serum albumin, DMSO and 5% glucose. After thawing, surface expression of molecules indicating the mature status (CD83, costimulatory and MHC molecules), was found to be unaltered. Furthermore, cryopreserved DC kept the capability to stimulate allogenic T-cell proliferation in mixed leukocyte reactions at full level. Loaded and matured DC pulsed with influenza matrix peptide (IMP) retained the capacity to induce the generation of IMP-specific cytotoxic T-lymphocytes after cryopreservation as measured by ELISPOT and tetramer staining. The expression of the chemokine receptor CXCR-4 and CCR-7 remained unaltered during cryopreservation and the migratory responsiveness towards MIP-3beta was unaltered as measured in a migration assay. Thus we conclude that the large scale loading and maturation of DC with whole tumor cell preparations can be performed in a single session. These data will facilitate the clinical application of DC loaded with whole tumor cell preparations.