The pertussis epidemic: informing strategies for prevention of severe disease.

To assess the impact of Bordetella pertussis infections in South Australia during an epidemic and determine vulnerable populations, data from notification reports for pertussis cases occurring between July 2008 and December 2009 were reviewed to determine the distribution of disease according to specific risk factors and examine associations with hospitalizations. Although the majority (66%) of the 6230 notifications for pertussis occurred in adults aged >24 years, the highest notification and hospitalization rate occurred in infants aged <1 year. For these infants, factors associated with hospitalization included being aged <2 months [relative risk (RR) 2·3, 95% confidence interval (CI) 1·60-3·32], Indigenous ethnicity (RR 1·7, 95% CI 1·03-2·83) and receiving fewer than two doses of pertussis vaccine (RR 4·1, 95% CI 1·37-12·11). A combination of strategies aimed at improving direct protection for newborns, vaccination for the elderly, and reducing transmission from close contacts of infants are required for prevention of severe pertussis disease.

Fire mosaics and reptile conservation in a fire-prone region.

Fire influences the distribution of fauna in terrestrial biomes throughout the world. Use of fire to achieve a mosaic of vegetation in different stages of succession after burning (i.e., patch-mosaic burning) is a dominant conservation practice in many regions. Despite this, knowledge of how the spatial attributes of vegetation mosaics created by fire affect fauna is extremely scarce, and it is unclear what kind of mosaic land managers should aim to achieve. We selected 28 landscapes (each 12.6 km(2) ) that varied in the spatial extent and diversity of vegetation succession after fire in a 104,000 km(2) area in the semiarid region of southeastern Australia. We surveyed for reptiles at 280 sites nested within the 28 landscapes. The landscape-level occurrence of 9 of the 22 species modeled was associated with the spatial extent of vegetation age classes created by fire. Biogeographic context and the extent of a vegetation type influenced 7 and 4 species, respectively. No species were associated with the diversity of vegetation ages within a landscape. Negative relations between reptile occurrence and both extent of recently burned vegetation (≤10 years postfire, n = 6) and long unburned vegetation (>35 years postfire, n = 4) suggested that a coarse-grained mosaic of areas (e.g. >1000 ha) of midsuccessional vegetation (11-35 years postfire) may support the fire-sensitive reptile species we modeled. This age class coincides with a peak in spinifex cover, a keystone structure for reptiles in semiarid and arid Australia. Maintaining over the long term a coarse-grained mosaic of large areas of midsuccessional vegetation in mallee ecosystems will need to be balanced against the short-term negative effects of large fires on many reptile species and a documented preference by species from other taxonomic groups, particularly birds, for older vegetation.

Analysis of the measurement precision of an amorphous silicon EPID used for MLC leaf position quality control and the long-term calibration stability of an optically controlled MLC.

Electronic portal imaging devices (EPIDs) have been shown to be suitable for multileaf collimator (MLC) leaf positioning quality control (QC). In our centre, a continuous dataset is available of 2 years of film measurements followed by 3 years of EPID measurements on five MLC-equipped linear accelerators of identical head design. The aim of this work was to analyse this unique dataset in order to determine the relative precision of film and EPID for MLC leaf positioning measurements and to determine the long-term stability of the MLC calibration. The QC dataset was examined and periods without MLC adjustments that contained at least four successive collimator position measurements (a minimum of 6 months) were identified. By calculating the standard deviations (SD) of these results, the reproducibility of the measurements can be determined. Comparison of the film and EPID results enables their relative measurement precision to be assessed; on average film gave an SD of 0.52 mm compared to 0.13 mm for EPIDs. The MLC and conventional collimator results were compared to assess MLC calibration stability; on average, for EPID measurements, the MLC gave an SD of 0.12 mm compared to 0.14 mm for a conventional collimator. The long-term relative individual leaf positions were compared and found to vary between 0.07 and 0.15 mm implying that they are stable over long time periods. These results suggest that the calibration of an optically controlled MLC is inherently very stable between disturbances to the optical system which normally occur on service days.

Use of an amorphous silicon EPID for measuring MLC calibration at varying gantry angle.

Amorphous silicon electronic portal imaging devices (EPIDs) are used to perform routine quality control (QC) checks on the multileaf collimators (MLCs) at this centre. Presently, these checks are performed at gantry angle 0 degrees and are considered to be valid for all other angles. Since therapeutic procedures regularly require the delivery of MLC-defined fields to the patient at a wide range of gantry angles, the accuracy of the QC checks at other gantry angles has been investigated. When the gantry is rotated to angles other than 0 degrees it was found that the apparent pixel size measured using the EPID varies up to a maximum value of 0.0015 mm per pixel due to a sag in the EPID of up to 9.2 mm. A correction factor was determined using two independent methods at a range of gantry angles between 0 degrees and 360 degrees . The EPID was used to measure field sizes (defined by both x-jaws and MLC) at a range of gantry angles and, after this correction had been applied, any residual gravitational sag was studied. It was found that, when fields are defined by the x-jaws and y-back-up jaws, no errors of greater than 0.5 mm were measured and that these errors were no worse when the MLC was used. It was therefore concluded that, provided the correction is applied, measurements of the field size are, in practical terms, unaffected by gantry angle. Experiments were also performed to study how the reproducibility of individual leaves is affected by gantry angle. Measurements of the relative position of each individual leaf (minor offsets) were performed at a range of gantry angles and repeated three times. The position reproducibility was defined by the RMS error in the position of each leaf and this was found to be 0.24 mm and 0.21 mm for the two leaf banks at a gantry angle of 0 degrees . When measurements were performed at a range of gantry angles, these reproducibility values remained within 0.09 mm and 0.11 mm. It was therefore concluded that the calibration of the Elekta MLC is stable at all gantry angles.

Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma.

Like many epithelial tumors, head and neck squamous cell carcinoma (HNSCC) contains a heterogeneous population of cancer cells. We developed an immunodeficient mouse model to test the tumorigenic potential of different populations of cancer cells derived from primary, unmanipulated human HNSCC samples. We show that a minority population of CD44(+) cancer cells, which typically comprise <10% of the cells in a HNSCC tumor, but not the CD44(-) cancer cells, gave rise to new tumors in vivo. Immunohistochemistry revealed that the CD44(+) cancer cells have a primitive cellular morphology and costain with the basal cell marker Cytokeratin 5/14, whereas the CD44(-) cancer cells resemble differentiated squamous epithelium and express the differentiation marker Involucrin. The tumors that arose from purified CD44(+) cells reproduced the original tumor heterogeneity and could be serially passaged, thus demonstrating the two defining properties of stem cells: ability to self-renew and to differentiate. Furthermore, the tumorigenic CD44(+) cells differentially express the BMI1 gene, at both the RNA and protein levels. By immunohistochemical analysis, the CD44(+) cells in the tumor express high levels of nuclear BMI1, and are arrayed in characteristic tumor microdomains. BMI1 has been demonstrated to play a role in self-renewal in other stem cell types and to be involved in tumorigenesis. Taken together, these data demonstrate that cells within the CD44(+) population of human HNSCC possess the unique properties of cancer stem cells in functional assays for cancer stem cell self-renewal and differentiation and form unique histological microdomains that may aid in cancer diagnosis.

Stem cells, cancer, and cancer stem cells.

Stem cell biology has come of age. Unequivocal proof that stem cells exist in the haematopoietic system has given way to the prospective isolation of several tissue-specific stem and progenitor cells, the initial delineation of their properties and expressed genetic programmes, and the beginnings of their utility in regenerative medicine. Perhaps the most important and useful property of stem cells is that of self-renewal. Through this property, striking parallels can be found between stem cells and cancer cells: tumours may often originate from the transformation of normal stem cells, similar signalling pathways may regulate self-renewal in stem cells and cancer cells, and cancer cells may include 'cancer stem cells' - rare cells with indefinite potential for self-renewal that drive tumorigenesis.

Evaluation of a new dual-specificity promoter for selective induction of apoptosis in breast cancer cells.

The conditional expression of lethal genes in tumor cells is a promising gene therapy approach for the treatment of cancer. The identification of promoters that are preferentially active in cancer cells is the starting point for this strategy. The combination of tissue-specific and tumor-specific elements offers the possibility to artificially develop such promoters. We describe the construction and characterization of a hybrid promoter for transcriptional targeting of breast cancer. In many cases, breast cancer cells retain the expression of estrogen receptors, and most solid tumors suffer from hypoxia as a consequence of their aberrant vascularization. Estrogen response elements and hypoxia-responsive elements were combined to activate transcription in cells that present at least one of these characteristics. When a promoter containing these elements is used to control the expression of the pro-apoptotic gene harakiri, the induction of cell death can be activated by estrogens and hypoxia, and inhibited by antiestrogens such as tamoxifen. Finally, we show evidence that these properties are maintained in the context of an adenoviral vector (AdEHhrk). Therefore, infection with this virus preferentially kills estrogen receptor-positive breast cancer cells, or cells growing under hypoxic conditions. We propose the use of this promoter for transcriptional targeting of breast cancer.

Regulation of p53 localization.

Despite intensive study of p53, the regulation of p53 cellular localization is still poorly understood. This is an overview of the elements and molecules involved in p53 nucleocytoplasmic transportation. These include the nuclear import and export signals of p53, inhibition of p53 nuclear import and export by oligomerization, MDM2-mediated p53 nuclear export, and possible roles of p53 phosphorylation in regulating p53 cellular localization. Finally, questions regarding p53 cellular trafficking will also be discussed.

Molecular cloning and characterization of a novel regulator of G-protein signaling from mouse hematopoietic stem cells.

A novel regulator of G-protein signaling (RGS) has been isolated from a highly purified population of mouse long-term hematopoietic stem cells, and designated RGS18. It has 234 amino acids consisting of a central RGS box and short divergent NH(2) and COOH termini. The calculated molecular weight of RGS18 is 27,610 and the isoelectric point is 8.63. Mouse RGS18 is expressed from a single gene and shows tissue specific distribution. It is most highly expressed in bone marrow followed by fetal liver, spleen, and then lung. In bone marrow, RGS18 level is highest in long-term and short-term hematopoietic stem cells, and is decreased as they differentiate into more committed multiple progenitors. The human RGS18 ortholog has a tissue-specific expression pattern similar to that of mouse RGS18. Purified RGS18 interacts with the alpha subunit of both G(i) and G(q) subfamilies. The results of in vitro GTPase single-turnover assays using Galpha(i) indicated that RGS18 accelerates the intrinsic GTPase activity of Galpha(i). Transient overexpression of RGS18 attenuated inositol phosphates production via angiotensin receptor and transcriptional activation through cAMP-responsive element via M1 muscarinic receptor. This suggests RGS18 can act on G(q)-mediated signaling pathways in vivo.

Cell wound assays.

Many mammalian and invertebrate cells that reside in mechanically active tissue environments normally and frequently experience disruptions in plasma membrane integrity which are rapidly repaired and do not lead to cellular death. This unit describes methods for identifying "wounded" cells, often present as a minority in culture or tissues. The use of these methods can provide information about the frequency and extent of cellular wounding.

A novel, conditionally replicative adenovirus for the treatment of breast cancer that allows controlled replication of E1a-deleted adenoviral vectors.

The efficiency of gene therapy strategies against cancer is limited by the poor distribution of the vectors in the malignant tissues. To solve this problem, a new generation of tumor-specific, conditionally replicative adenoviruses is being developed. To direct the replication of the virus to breast cancer, we have considered one characteristic present in a great proportion of these cancers, which is the expression of estrogen receptors (ERs). On the basis of the wild-type adenovirus type 5, we have constructed a conditionally replicative adenovirus (Ad5ERE2) in which the E1a and E4 promoters have been replaced by a portion of the pS2 promoter containing two estrogen-responsive elements (EREs). This promoter induces transcriptional activation of the E1a and E4 units in response to estrogens in cells that express the ERs. Ad5ERE2 is able to kill ER(+) human breast cancer cell lines as efficiently as the wild-type virus, but has decreased capacity to affect ER(-) cells. By complementation of the E1a protein in trans, Ad5ERE2 allows restricted replication of a conventional E1a-deleted adenoviral vector. When a virus expressing the proapoptotic gene Bc1-xs (Clarke et al., Proc. Natl. Acad. Sci. U.S.A. 1995;92:11024-11028) is used in combination with Ad5ERE2, the ability of both viruses to induce cell death is dramatically increased, and the effect can be modulated by addition of the antiestrogen tamoxifen.

Complex social organization reflects genetic structure and relatedness in the cooperatively breeding bell miner, Manorina melanophrys.

The cooperatively breeding bell miner, Manorina melanophrys, differs from most other cooperative breeding species in the complexity of its social system, where discrete social organization occurs on at least three levels. Microsatellite markers were used to investigate the degree of genetic structure underlying the social organization of M. melanophrys by comparing colonies, coteries and nest contingents. The genetic data confirmed behavioural observations of M. melanophrys living in male kin-based groups between which females disperse short distances to breed. Estimates of FST revealed restricted gene flow between eight colonies located within 30 km that was significantly associated with geographical distance when the two most distant colonies were included. Within a high density colony significant differences were found between coteries; analysis of the degree of relatedness between coterie members showed that this is due to related individuals associating preferentially with each other. Similarly, the contingent of individuals attending a nest were generally close relatives of the young they were aiding, supporting models invoking kin selection as the selective agency mediating helping.

A bipartite nuclear localization signal is required for p53 nuclear import regulated by a carboxyl-terminal domain.

Abnormal p53 cellular localization has been considered to be one of the mechanisms that could inactivate p53 function. To understand the regulation of p53 cellular trafficking, we have previously identified two p53 domains involved in its localization. A basic domain, Lys(305)-Arg(306), is required for p53 nuclear import, and a carboxyl-terminal domain, namely the cytoplasmic sequestration domain (CSD) from residues 326-355, could block the nuclear import of Lys(305) or Arg(306) mutated p53. To characterize further the function of these two domains, we demonstrate in this report that the previously described major nuclear localization signal works together with Lys(305)-Arg(306) to form a bipartite and functional nuclear localization sequence (NLS) for p53 nuclear import. The CSD could block the binding of p53 to the NLS receptor, importin alpha, and reduce the efficiency of p53 nuclear import in MCF-7, H1299, and Saos-2 cells. The blocking effect of the CSD is not due to the enhancement of nuclear export or oligomerization of the p53. These results indicate that the CSD can regulate p53 nuclear import by controlling access of the NLS to importin alpha binding.

The nuclear import of p53 is determined by the presence of a basic domain and its relative position to the nuclear localization signal.

It has been reported that Lysine-305 is needed for the nuclear import of the p53 protein (Liang et al., 1998). In the present study, further mutagenesis analyses were carried out between Lys-305 and the major nuclear localization signal (NLS I) of p53. It was found that a single mutation of Arg-306 resulted in the defect of p53 nuclear import. This effect is the same as that of Lys-305 mutation. Other mutations between Arg-306 and NLS I have no effect on the nuclear import of p53. However, deletions of more than two amino acids between this region abolished the transport of p53 into the nucleus. These results indicate that a basic domain other than the well defined NLS is required for the nuclear import of p53. A spacer between this basic domain and NLS I is necessary for the entrance of p53 into the cell nucleus.

Germ cell tumor: differentiation of viable tumor, mature teratoma, and necrotic tissue with FDG PET and kinetic modeling.

PURPOSE: To evaluate the feasibility of positron emission tomography (PET) with 2-[fluorine-18]-fluoro-2-deoxy-D-glucose (FDG) in patients with germ cell tumor (GCT) to monitor treatment and differentiate residual masses after chemotherapy. MATERIALS AND METHODS: Twenty-six FDG PET studies were performed in 21 patients with GCT, FDG uptake of tumors was interpreted visually, and the lean standardized uptake value (SUVlean) was determined. Tumor kinetic rate constants (K1, k2, k3) and net rate of FDG phosphorylation (K = [K1.k3]/[k2 + k3]) in tumors were calculated from the dynamic data by means of a three-compartment model, assuming k4 = 0. RESULTS: Viable tumors (n = 10) showed intense FDG uptake and could easily be differentiated visually from mature teratoma (n = 6) and necrosis or scar (n = 10). The SUVlean of residual viable tumors (4.51 +/- 1.34 [mean +/- SD]) was higher than that of mature teratoma (1.38 +/- 0.71) and necrosis or scar (1.05 +/- 0.29) (P < .05). Although neither the visual interpretation nor SUVlean differentiated mature teratoma from necrosis or scar, there were statistically significant differences in the kinetic rate constants K1 and K between mature teratoma and necrosis or scar as follows: K1, 0.113 mL/min/g +/- 0.026 versus 0.036 mL/min/g +/- 0.005 (P < .05); K, 0.005 mL/min/g +/- 0.003 versus 0.0008 mL/min/g +/- 0.0001 (P < .05). CONCLUSION: FDG PET with kinetic analysis appears to be a promising method for management of disease in patients with GCT after treatment.

Role of p53 in the regulation of irradiation-induced apoptosis in neuroblastoma cells.

Wild-type p53 plays a crucial role in the control of apoptosis following ionizing radiation (IR); conversely, mutant p53 is associated with IR resistance. Although wild-type p53 is expressed in virtually all neuroblastoma tumors, treatment failures secondary to inadequate local control with radiotherapy are a problem in patients with advanced stage disease. This apparent paradox is the focus of our interest. The Shep-1 neuroblastoma cell line is highly resistant to IR. This cell line contains a wild-type p53 gene and is an ideal model for studying the mechanism of IR resistance in this disease. Following high-dose IR, cell fractionation demonstrated that p53 is induced and targeted to the nucleus. The induced p53 is functional as p53-responsive genes (Waf-1 and MDM-2) are appropriately induced following IR. Intriguingly, overexpression of p53 could reverse the inherent IR resistance of Shep-1 cells. Multiple cell lines expressing variable levels of exogenous temperature-sensitive p53 were generated. Pulse induction of p53 alone did not affect Shep-1 cell viability, while induction of p53, followed by IR, resulted in cell death and DNA fragmentation proportional to the dose of IR and the level of p53 expression. These findings demonstrate that p53 overexpression renders Shep-1 cells IR-sensitive and suggest that large quantities of exogenous p53 can overcome the factors inhibiting p53-mediated, IR-induced apoptosis.

Cooperation of a single lysine mutation and a C-terminal domain in the cytoplasmic sequestration of the p53 protein.

Cytoplasmic sequestration of the p53 tumor suppresser protein has been proposed as a mechanism involved in abolishing p53 function. However, the mechanisms regulating p53 subcellular localization remain unclear. In this report, we analyzed the possible existence of cis-acting sequences involved in intracellular trafficking of the p53 protein. To study p53 trafficking, the jellyfish green fluorescent protein (GFP) was fused to the wild-type or mutated p53 proteins for fast and sensitive analysis of protein localization in human MCF-7 breast cancer, RKO colon cancer, and SAOS-2 sarcoma cells. The wild-type p53/GFP fusion protein was localized in the cytoplasm, the nucleus, or both compartments in a subset of the cells. Mutagenesis analysis demonstrated that a single amino acid mutation of Lys-305 (mt p53) caused cytoplasmic sequestration of the p53 protein in the MCF-7 and RKO cells, whereas the fusion protein was distributed in both the cytoplasm and the nucleus of SAOS-2 cells. In SAOS-2 cells, the mutant p53 was a less efficient inducer of p21/CIP1/WAF1 expression. Cytoplasmic sequestration of the mt p53 was dependent upon the C-terminal region (residues 326-355) of the protein. These results indicated the involvement of cis-acting sequences in the regulation of p53 subcellular localization. Lys-305 is needed for nuclear import of p53 protein, and amino acid residues 326-355 can sequester mt p53 in the cytoplasm.

A method of limited replication for the efficient in vivo delivery of adenovirus to cancer cells.

Replication-deficient viral vectors are currently being used in gene transfer strategies to treat cancer cells. Unfortunately, viruses are limited in their ability to diffuse through tissue. This makes it virtually impossible to infect the majority of tumor cells in vivo and results in inadequate gene transfer. This problem can be addressed by allowing limited viral replication. Limited viral replication facilitates greater penetration of virions into tissue and can improve gene transfer. We have developed a strategy of limited viral replication using AdRSVlaclys, a chemically modified E1-deleted adenovirus, to codeliver an exogenous plasmid encoding the adenovirus E1 region. This system allows one round of viral replication. We examined the effect of this limited adenovirus replication in vitro and in vivo. In culture, codelivery of virus and pE1 resulted in a large increase in infected cells when compared with control cells exposed to virus and pUC19. In experiments on nude mice bearing HeLa ascites tumors, intraperitoneal injection of AdRSVlaclys/pE1 resulted in a significantly higher percentage of infected HeLa cells as compared with the PBS controls (p < 0.05) or the AdRSVlaclys/pUC19 controls (p < 0.01). These data demonstrate that the transcomplementation of replication-deficient adenovirus with exogenous E1 DNA leads to limited replication, and this controlled replication enhances gene transfer efficiency of adenovirus in vivo.

Targeting cancer cell death with a bcl-XS adenovirus.

Transformation is a complex cellular process that requires several genetic abnormalities. In many cases, one of these abnormalities is an inhibition of PCD, which provides a selective advantage for tumor cells. This has been recently shown in an in vivo model, where overexpression of Bcl-XL, is a crucial step in the progression from hyperplasia to neoplasia and is accompanied by a significant decrease in tumor apoptosis [56]. Frequently, overexpression of a member of the Bcl-2 family results in a block in cell death and appears to nullify many built-in cellular defense mechanisms against cancer. Such a block presents a problem because radiation and chemotherapy, standard cancer treatments, ultimately exert their effect by induction of apoptosis and would also be made less effective. Therefore, to better treat cancer it may be necessary to develop novel methods to overcome the effects of the Bcl-2 family. One way to approach this problem is to target the cause--the molecular machinery that allows a cancer cell to survive. Advances in our understanding of apoptosis has identified the Bcl-2 family as a mediator of most apoptosis pathways, including those initiated by oncogenes, tumor suppressor genes, growth factor withdrawal, and external damaging signals. Therefore, functional inhibition of Bcl-2 family members is lethal to many cancer cells. Using gene transfer technology, we can now deliver genes that accomplish this goal. Further investigation will reveal whether this translates to improved therapy in the future.