Multichannel apparatus for parallel monitoring of light scattering in Dictyostelium discoideum cell suspensions

Suspensions of Dictyostelium discoideum amoebae display free-running light scattering oscillations at the onset of development. We describe a device to monitor these oscillations in several samples in parallel. The apparatus consists of a thermostated cuvette holder where up to eight cuvettes containing cell suspension are inserted. Cells are aerated and kept in suspension via an airlift. Infrared light emitted from a five-diode array passes through the suspension and is detected by an array of five light detecting diodes. The resulting signal is digitized and recorded with a sampling rate of two measuring points/second. The parallel analysis approach allows determination of the effects of adding of agents or of variations in the external conditions in the same batch of amoebae at the same developmental time point. This represents an advantage over the conventional single cuvette approach, as oscillation characteristics themselves are developmentally regulated. Moreover, as the new experimental setup enables simultaneous analyses of up to eight samples, the behavior of wild-type and several mutant strains can be compared under identical experimental conditions.     

Human papillomavirus type 16 E7 peptide-directed CD8+ T cells from patients with cervical cancer are cross-reactive with the coronavirus NS2 protein

Human papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are required for cellular transformation and represent candidate targets for HPV-specific and major histocompatibility complex class I-restricted CD8(+)-T-cell responses in patients with cervical cancer. Recent evidence suggests that cross-reactivity represents the inherent nature of the T-cell repertoire. We identified HLA-A2 binding HPV16 E7 variant peptides from human, bacterial, or viral origin which are able to drive CD8(+)-T-cell responses directed against wild-type HPV16 E7 amino acid 11 to 19/20 (E7(11-19/20)) epitope YMLDLQPET(T) in vitro. CD8(+) T cells reacting to the HLA-A2-presented peptide from HPV16 E7(11-19(20)) recognized also the HLA-A2 binding peptide TMLDIQPED (amino acids 52 to 60) from the human coronavirus OC43 NS2 gene product. Establishment of coronavirus NS2-specific, HLA-A2-restricted CD8(+)-T-cell clones and ex vivo analysis of HPV16 E7 specific T cells obtained by HLA-A2 tetramer-guided sorting from PBL or tumor-infiltrating lymphocytes obtained from patients with cervical cancer showed that cross-reactivity with HPV16 E7(11-19(20)) and coronavirus NS2(52-60) represents a common feature of this antiviral immune response defined by cytokine production. Zero of 10 patients with carcinoma in situ neoplasia and 3 of 18 patients with cervical cancer showed > or =0.1% HPV16 E7-reactive T cells in CD8(+) peripheral blood lymphocytes. In vivo priming with HPV16 was confirmed in patients with cervical cancer or preinvasive HPV16-positive lesions using HLA-A2 tetramer complexes loaded with the E6-derived epitope KLPQLCTEL. In contrast, we could not detect E6-reactive T cells in healthy individuals. These data imply that the measurement of the HPV16 E7(11-19(20)) CD8(+)-T-cell response may reflect cross-reactivity with a common pathogen and that variant peptides may be employed to drive an effective cellular immune response against HPV.     

Impact of antigen presentation on TCR modulation and cytokine release: implications for detection and sorting of antigen-specific CD8+ T cells using HLA-A2 wild-type or HLA-A2 mutant tetrameric complexes

Soluble MHC class I molecules loaded with antigenic peptides are available either to detect and to enumerate or, alternatively, to sort and expand MHC class I-restricted and peptide-reactive T cells. A defined number of MHC class I/peptide complexes can now be implemented to measure T cell responses induced upon Ag-specific stimulation, including CD3/CD8/zeta-chain down-regulation, pattern, and quantity of cytokine secretion. As a paradigm, we analyzed the reactivity of a Melan-A/MART-1-specific and HLA-A2-restricted CD8(+) T cell clone to either soluble or solid-phase presented peptides, including the naturally processed and presented Melan-A/MART-1 peptide AAGIGILTV or the peptide analog ELAGIGILTV presented either by the HLA-A2 wild-type (wt) or mutant (alanineright arrowvaline aa 245) MHC class I molecule, which reduces engagement of the CD8 molecule with the HLA-A2 heavy chain. Soluble MHC class I complexes were used as either monomeric or tetrameric complexes. Soluble monomeric MHC class I complexes, loaded with the Melan-A/MART-1 peptide, resulted in CD3/CD8 and TCR zeta-chain down-regulation, but did not induce measurable cytokine release. In general, differences pertaining to CD3/CD8/zeta-chain regulation and cytokine release, including IL-2, IFN-gamma, and GM-CSF, were associated with 1) the format of Ag presentation (monomeric vs tetrameric MHC class I complexes), 2) wt vs mutant HLA-A2 molecules, and 3) the target Ag (wt vs analog peptide). These differences are to be considered if T cells are exposed to recombinant MHC class I Ags loaded with peptides implemented for detection, activation, or sorting of Ag-specific T cells.     

Clinical pathology and assessment of pathogen exposure in southern and Alaskan sea otters

The southern sea otter (Enhydra lutris nereis) population in California (USA) and the Alaskan sea otter (E. lutris kenyoni) population in the Aleutian Islands (USA) chain have recently declined. In order to evaluate disease as a contributing factor to the declines, health assessments of these two sea otter populations were conducted by evaluating hematologic and/or serum biochemical values and exposure to six marine and terrestrial pathogens using blood collected during ongoing studies from 1995 through 2000. Samples from 72 free-ranging Alaskan, 78 free-ranging southern, and (for pathogen exposure only) 41 debilitated southern sea otters in rehabilitation facilities were evaluated and compared to investigate regional differences. Serum chemistry and hematology values did not indicate a specific disease process as a cause for the declines. Statistically significant differences were found between free-ranging adult southern and Alaskan population mean serum levels of creatinine kinase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, calcium, cholesterol, creatinine, glucose, phosphorous, total bilirubin, blood urea nitrogen, and sodium. These were likely due to varying parasite loads, contaminant exposures, and physiologic or nutrition statuses. No free-ranging sea otters had signs of disease at capture, and prevalences of exposure to calicivirus, Brucella spp., and Leptospira spp. were low. The high prevalence (35%) of antibodies to Toxoplasma gondii in free-ranging southern sea otters, lack of antibodies to this parasite in Alaskan sea otters, and the pathogen's propensity to cause mortality in southern sea otters suggests that this parasite may be important to sea otter population dynamics in California but not in Alaska. The evidence for exposure to pathogens of public health importance (e.g., Leptospira spp., T. gondii) in the southern sea otter population, and the na?veté of both populations to other pathogens (e.g., morbillivirus and Coccidiodes immitis) may have important implications for their management and recovery.     

Back to basics for Bayesian model building in genomic selection

Numerous Bayesian methods of phenotype prediction and genomic breeding value estimation based on multilocus association models have been proposed. Computationally the methods have been based either on Markov chain Monte Carlo or on faster maximum a posteriori estimation. The demand for more accurate and more efficient estimation has led to the rapid emergence of workable methods, unfortunately at the expense of well-defined principles for Bayesian model building. In this article we go back to the basics and build a Bayesian multilocus association model for quantitative and binary traits with carefully defined hierarchical parameterization of Student's t and Laplace priors. In this treatment we consider alternative model structures, using indicator variables and polygenic terms. We make the most of the conjugate analysis, enabled by the hierarchical formulation of the prior densities, by deriving the fully conditional posterior densities of the parameters and using the acquired known distributions in building fast generalized expectation-maximization estimation algorithms.     

Modulation of DNA precursor pools, DNA synthesis, and ultraviolet sensitivity of a repair-dificient CHO cell line by deoxycytidine

The presence of 2 mM deoxycytidine (CdR) in growth medium substantially increased the deoxycytidine triphosphate (dCTP) and deoxuthymidine triphosphate (dTTP) pools in a Chinese hamster ovary cell line, CHO-K1, and in a radiation-sensitive mutant, xrs-5, derived from it (Jeggo et al., 1982). We observed significant differences in alkaline-sucrose gradient profiles of pulse-labeled DNA from unirradiated CHO-K1 and xrs-5 cells. For the latter cell line, a sizable fraction of the DNA synthesized during 5 or 10 min of growth subsequent to a 5-min radiolabeling period was found to co-sediment with large-chromosome DNA. This characteristics of xrs-5 was dramatically reduced by the presence of 2 mM CdR in the culture medium, and the UV resistance of the mutant increased to nearly that of the parent cell line under these culture conditions. These results show that the locus conferring UV-radiation sensitivity to xrs-5 affects DNA replication and that replicative activity and UV-radiation sensivity are jointly modulated by CdR, possibly through its impact on the size of deoxynucleoside triphosphate pools.     

Challenges in the Care of Clients with Established Cardiovascular Disease: Lessons Learned from Australian Community Pharmacists

Background

As primary healthcare professionals, community pharmacists have both opportunity and potential to contribute to the prevention and progression of chronic diseases. Using cardiovascular disease (CVD) as a case study, we explored factors that influence community pharmacists’ everyday practice in this area. We also propose a model to best illustrate relationships between influencing factors and the scope of community pharmacy practice in the care of clients with established CVD.

Methods

In-depth, semi-structured interviews were conducted with 21 community pharmacists in New South Wales, Australia. All interviews were audio-recorded, transcribed ad verbatim, and analysed using a “grounded-theory” approach.

Results

Our model shows that community pharmacists work within a complex system and their practice is influenced by interactions between three main domains: the “people” factors, including their own attitudes and beliefs as well as those of clients and doctors; the “environment” within and beyond the control of community pharmacy; and outcomes of their professional care. Despite the complexity of factors and interactions, our findings shed some light on the interrelationships between these various influences. The overarching obstacle to maximizing the community pharmacists’ contribution is the lack of integration within health systems. However, achieving better integration of community pharmacists in primary care is a challenge since the systems of remuneration for healthcare professional services do not currently support this integration.

Conclusion

Tackling chronic diseases such as CVD requires mobilization of all sources of support in the community through innovative policies which facilitate inter-professional collaboration and team care to achieve the best possible healthcare outcomes for society.