Genotype and diet affect resistance, survival, and fecundity but not fecundity tolerance.

Insects are exposed to a variety of potential pathogens in their environment, many of which can severely impact fitness and health. Consequently, hosts have evolved resistance and tolerance strategies to suppress or cope with infections. Hosts utilizing resistance improve fitness by clearing or reducing pathogen loads, and hosts utilizing tolerance reduce harmful fitness effects per pathogen load. To understand variation in, and selective pressures on, resistance and tolerance, we asked to what degree they are shaped by host genetic background, whether plasticity in these responses depends upon dietary environment, and whether there are interactions between these two factors. Females from ten wild-type Drosophila melanogaster genotypes were kept on high- or low-protein (yeast) diets and infected with one of two opportunistic bacterial pathogens, Lactococcus lactis or Pseudomonas entomophila. We measured host resistance as the inverse of bacterial load in the early infection phase. The relationship (slope) between fly fecundity and individual-level bacteria load provided our fecundity tolerance measure. Genotype and dietary yeast determined host fecundity and strongly affected survival after infection with pathogenic P. entomophila. There was considerable genetic variation in host resistance, a commonly found phenomenon resulting from for example varying resistance costs or frequency-dependent selection. Despite this variation and the reproductive cost of higher P. entomophila loads, fecundity tolerance did not vary across genotypes. The absence of genetic variation in tolerance may suggest that at this early infection stage, fecundity tolerance is fixed or that any evolved tolerance mechanisms are not expressed under these infection conditions.

Absolute Differential Positronium-Formation Cross Sections.

The first absolute experimental determinations of the differential cross sections for the formation of ground-state positronium are presented for He, Ar, H2, and CO2 near 0°. Results are compared with available theories. The ratio of the differential and integrated cross sections for the targets exposes the higher propensity for forward emission of positronium formed from He and H2.

Identification and frequency of CCR5Δ32/Δ32 HIV-resistant cord blood units from Houston area hospitals.

OBJECTIVES: The use of umbilical cord blood (CB) that is genetically resistant to HIV infection has been proposed as a novel stem cell therapy for the treatment of patients with AIDS. These genetically unique CB units (CBUs) should be present in public CB banks at a predicted frequency. METHODS: The chemokine (C-C motif) receptor 5 (CCR5) genotypes of CBUs donated to the M. D. Anderson CB Bank by four Houston area hospitals were determined by polymerase chain reaction (PCR) and DNA sequencing. RESULTS: The frequency of CCR5Δ32/Δ32 CBUs was consistent with the frequency of the CCR5Δ32 allele in human populations, and was apparently dependent on the ethnic population of the parents of the newborns from whom the CBUs were collected. CONCLUSIONS: Routine genotyping to identify HIV-resistant CBUs could create a bank of CB-derived stem/progenitor cells with which to treat HIV infection.

Electron-like scattering of positronium.

Positronium (Ps), a hydrogen-like atom composed of an electron and its antimatter partner, the positron, is formed in considerable quantities whenever positrons interact with matter. It has unexpectedly been found to scatter from a wide variety of atoms and molecules in a way very similar to that of a bare electron moving at the same velocity, despite Ps being neutral and twice the mass.

Multi-laboratory evaluation of procedures for reducing the volume of cord blood: influence on cell recoveries.

BACKGROUND: Various procedures can be used to isolate stem and progenitor cells from cord blood. This study evaluated the hydroxyethyl starch sedimentation (HES) with two centrifugation steps, and the top and bottom (T&B) isolation of buffy coat following a single centrifugation, and two filter systems for processing cord blood, one developed by Asahi Kasei Medical (filter A) and the second by Terumo (filter B). METHODS: Each of seven laboratories was randomly assigned the evaluation of either the HES or T&B method and one of the filter methods (n=8 cord blood units, per laboratory, for each method). The leukocyte-containing fraction with the stem/progenitor cells was recovered from the filters by reverse flushing. Utilizing the routine traditional processing and testing procedures of each laboratory, in vitro parameters were determined, with samples obtained after collection, after processing and after freezing/thawing. The results were expressed as the percentage recovery of viable cells in processed vs. collected samples (performance 1; PF1) and in thawed vs. processed samples (performance 2; PF2). The composite results obtained by the seven laboratories were summarized. RESULTS: The median PF1 percentage recovery of total nucleated cells (TNC) was comparable with both traditional methods (HES 79%, T&B 86%) and statistically reduced with both filtration procedures (filter A 58%, filter B 61%). Mononuclear cell (MNC) PF1 recovery was highest statistically with the T&B method (91%) and reduced on using filter A (77%) and filter B (70%) and the HES method (72%). CD34+ cell recovery was judged to be essentially comparable with the four methods, although the range of unit recoveries differed. The percentage recovery of TNC and MNC in PF1 was influenced by the volume of the collected cord blood, especially with use of the filtration procedures. This correlated with TNC content. A greater percentage of red cells and platelets was removed during processing with both filter methods. The time to process cord blood preparations with filter A was significantly shorter than the other methods. Processing with the HES method took the longest time. The recoveries for TNC, MNC and CD34+ cells in PF2 did not appear to be influenced by the specific processing procedure. DISCUSSION: These data indicate that filters that capture stem and progenitor cells may be an appropriate methodology for processing cord blood collected for banking.

Immune function responds to selection for cuticular colour in Tenebrio molitor.

Cuticular colour in the mealworm beetle (Tenebrio molitor) is a quantitative trait, varying from tan to black. Population level variation in cuticular colour has been linked to pathogen resistance in this species and in several other insects: darker individuals are more resistant to pathogens. Given that cuticular colour has a heritable component, we have taken an experimental evolution approach: we selected 10 lines for black and 10 lines for tan adult cuticular phenotypes over at least six generations and measured the correlated responses to selection in a range of immune effector systems. Our results show that two immune parameters related to resistance (haemocyte density and pre-immune challenge activity of phenoloxidase (PO)) were significantly higher in selection lines of black beetles compared to tan lines. This may help to explain increased resistance to pathogens in darker individuals. Cuticular colour is dependent upon melanin production, which requires the enzyme PO that is present in its inactive form inside haemocytes. Thus, the observed correlated response to selection upon cuticular colour and immune variables probably results from these traits' shared dependence on melanin production.

Examining costs of induced and constitutive immune investment in Tenebrio molitor.

Central to the conceptual basis of ecological immunity is the notion that immune effector systems are costly to produce, run, and/or maintain. Using the mealworm beetle, Tenebrio molitor, as a model we investigated two aspects of the costs of innate immunity. We conducted an experiment designed to identify the cost of an induced immune response, and the cost of constitutive investment in immunity, as well as potential interactions. The immune traits under consideration were the encapsulation response and prophylactic cuticular melanization, which are mechanistically linked by the melanin-producing phenoloxidase cascade. If immunity is costly, we predicted reduced longevity and/or fecundity as a consequence of investment in either immune trait. We found a measurable longevity cost associated with producing an inducible immune response (encapsulation). In contrast to other studies, this cost was expressed under ad libitum feeding conditions. We found no measurable costs for constitutive investment in immunity (prophylactic investment in cuticular colour).

Fragmentation of positronium in collision with He atoms.

The absolute cross section for the fragmentation of positronium in collision with He atoms has been measured. The results are compared with available theories. The longitudinal energy distributions of positrons resulting from fragmentation have also been determined and are found to display a peak situated just below half the residual energy. This is suggestive of the occurrence of "electron loss to the continuum" in which the two residual charged particles lie in a low relative-velocity Coulomb-continuum state.

The analysis of forensic samples using laser micro-pyrolysis gas chromatography mass spectrometry.

Laser micropyrolysis gas chromatography-mass spectrometry is used for the analysis of paint, photocopier toner, and synthetic fiber materials to test the forensic potential of this emerging technology. It uses a laser microprobe to selectively target very small parts of the materials for GC-MS analysis. Whereas the paint and the toner samples were amenable to direct laser pyrolysis, the synthetic fibers proved transparent to the 1064 nm laser radiation. The difficulty with the fibers demonstrates that a specific laser wavelength may not be appropriate for all types of materials. Nevertheless, the fibers were able to be indirectly pyrolyzed by impregnation in a strongly absorbing graphite matrix. A vast array of hydrocarbon pyrolysates was detected from the different materials studied. Unique product distributions were detected from each sample and in sufficient detail to facilitate individual molecular characterization (i.e., molecular fingerprinting). The integrity of the laser data were confirmed by comparison to data obtained from the same samples by the more conventional pyroprobe pyrolysis GC-MS method. The high spatial resolution and selectivity of the laser method may be advantageous for specific forensic applications, however, further work may be required to improve the reproducibility of the data.

An international survey of unrelated umbilical cord blood banking.

OBJECTIVES: To investigate operational and technical practices within the field of cord blood banking. MATERIALS AND METHODS: Cord blood banks world-wide were invited to participate in a survey of collection, processing and testing. The survey covered a 12-month period up to August 1998. RESULTS: Replies were received from 18 cord blood banks. Analysis of the survey responses demonstrated wide variations in many aspects of cord blood banking. CONCLUSION: There is a need for standardization to ensure adoption of best practice.

The single strand conformational analysis of cattle and human single nucleotide polymorphisms may be biased towards specific sequence motifs that minimize local secondary structure of single strand DNA.

Single strand conformational polymorphisms (SSCP) resulting from point mutations were found to be associated preferentially with two DNA sequence motifs. These motifs are (1) three or more of the same base but in which the polymorphism is not due to length variation and (2) a region of polypurine or polypyrimidine bases. These motifs were identified after SSCP alleles from cattle were sequenced. The sequence difference and flanking sequence for each single nucleotide polymorphism are shown. The motifs were also found in SSCP from humans chosen at random from the literature, in which the alleles had been sequenced. There is a low level of complementarity of adjacent bases in these motifs and they should represent regions of low secondary structure in the single stranded DNA. Regions of high secondary structure, such as palindromes, were found in the same sample to have allelic variation that was not detected by SSC analysis. These results give a rule of thumb for selecting the particular part of a DNA fragment to be selected for testing for polymorphisms, but this rule clashes with rules used to design primers to amplify sequences using the PCR, namely, minimise hydrogen bonding within and between primers and reduce self-complementarity.

Informal education: a hidden element of clinical nurse consultant practice.

A survey was conducted to investigate the amount of informal education provided by 16 community-based clinical nurse consultants (CNCs) in Sydney, Australia. The survey results indicated that the CNCs spent a substantial amount of time in informal teaching, of which only 3% was recorded as a legitimate educational activity. Findings from the survey support what has been reported in the literature, that skilled knowledge can be gained in the workplace when learners interact with experts. Through the avenue of informal teaching CNCs can positively contribute to quality nursing care by addressing on-the-job learning needs of community nurses.

Differences between the radiation hybrid and genetic linkage maps of bovine chromosome 5 resolved with a quasi-phylogenetic method of analysis.

Two major differences were detected in gene order between the radiation hybrid map and the genetic linkage map of bovine Chromosome (Chr) 5, and these were resolved by analyzing the raw radiation hybrid data by a quasi-phylogenetic method. Seventeen loci were typed on the new cattle whole genome radiation hybrid panel. Most of these loci are framework loci and include AGLA293, BM315, BM6026, BP1, BZRP, CD9, CSSM22, CSSM34, CYP2D@, ETH2, ETH10, ETH152, IGF1, LALBA, SLC2A3, SYT1, and TPI1. BP1 was found to be closer to the centromere than either BM6026 or SYT1 with two standard computer software packages for analyzing radiation hybrid panel data. This is inconsistent with any of the genetic linkage maps as well as their consensus. CYP2D@ was placed between ETH2 and BZRP, and this is also inconsistent with the genetic linkage maps, since CYP2D@ should be the most telomeric of the loci tested in this study. Resolution was reached by analyzing the raw radiation hybrid data for clones that bind some but not all of the loci, and the binding pattern was more consistent with the linkage maps and less consistent with the software-generated radiation hybrid map. The comparative mapping data confirm the relative inversion of gene order of SYT1 compared with humans and mice. A non-polymorphic fragment for CD9 indicates the conservation of gene order for three loci located on human Chr 12p. The genes of bovine Chr 5 conserved on human Chr 12p are located separately from the genes conserved on human Chr 12q. It is recommended that the raw data for radiation hybrid maps be made publicly available so that conflicts in gene order can be evaluated explicity.

Rural and remote mental health placements for nursing students.

Clinical placements can be instrumental in encouraging nursing students to consider a future career in rural Australia. Twenty nursing students from a metropolitan university were provided with the opportunity to undertake a clinical placement in mental health in a rural or remote setting. The majority of placements were between 2 and 4 weeks in length. They took place in community health centres and rehabilitation centres in New South Wales and in hospital inpatient facilities, remote areas and community health services in the Northern Territory. On return from the placement, students completed an open-question pro forma giving their views and impressions of their experiences and contrasting this with clinical experience gained in urban settings. The content of the students' responses was analysed and presented under the following themes: scale relating to urban and rural differences in population; geographical and health facility size; staffing matters; the environment; the students' perception of the clients; and professional interaction. Additional material relating to remote area placements is presented. Questions are raised about the maintenance of students' interest and the development of rural careers.

Follow up of workers previously exposed to silver solder containing cadmium.

OBJECTIVES: To study longitudinal biological monitoring data on urinary and blood cadmium collected in a small cohort of nine workers who had been brazing for several years with solders containing cadmium. METHODS: Cadmium was measured by neutron activation analysis in livers and kidneys, and estimates of renal function were carried out in 1983 and 1995. During the intervening period exposure to cadmium was dramatically reduced by local exhaust ventilation control and substitution of the solder containing cadmium. RESULTS: From urinary protein measurements there was evidence within the group of increasing renal tubular damage over the 12 year period, even though exposure to cadmium was dramatically reduced over this period and almost eliminated by 1995. There was no evidence from serum creatinine of decreasing glomerular filtration rate, and the renal tubular handling of calcium, phosphate, or urate had not worsened significantly. Blood and urinary cadmium concentrations reduced significantly over the 12 year period but were still substantial in 1995. Blood cadmium concentrations tended to reflect cadmium body burden in 1995 when exposure had been low for several years, and decreased most significantly during 1983-90. By contrast urinary cadmium concentrations only decreased significantly from about 1990 onwards. Urinary cadmium was not significantly correlated with liver or kidney cadmium concentration in either 1983 or 1995. This may be due to the level of tubular dysfunction in the cohort. Calculated cumulative excretion of cadmium over the 12 year period was substantially greater than the loss of cadmium measured in livers and kidneys and the derived loss in body burden. Reasons for this are discussed. It is possible that in cohorts, where renal damage is apparent, urinary concentrations reflect a substantial component of current exposure rather than stored body losses. CONCLUSIONS: The data reinforce the concept that blood cadmium concentrations may not always reflect recent exposure, but may reflect body burden derived from historical exposure depending on the degree of current exposure; and that the decline in urinary and blood cadmium measurements after removal from, or reduction in, exposure will be slow and depend on the historical body burden.

Cord blood banking in London: the first 1000 collections.

The London Cord Blood Bank was established with the aim of collecting, processing and storing 10000 unrelated stem cell donations for the significant number of children in the UK requiring transplantation, for whom a matched unrelated bone marrow donor cannot be found. Collection is performed at two hospitals by dedicated cord blood bank staff after delivery of the placenta. Mothers are interviewed regarding medical, ethnic and behavioural history by nurse counsellors and sign a detailed consent form. Donations are returned to the bank for processing. Volume reduction is undertaken by a simple, closed, semi-automated blood processing system, with excellent recovery of progenitor cells. Units are cryopreserved and stored in the vapour phase of liquid nitrogen. Blood samples from mothers and cord blood donations are tested for the UK mandatory red cell and microbiology markers for blood donors. Donations are typed for HLA-A, B and DR at medium resolution (antigen split) level using sequence-specific oligonucleotide probing and sequence-specific priming techniques. The selection of collection hospitals on the basis of ethnic mix has proven effective, with 41.5% of donations derived from non-European caucasoid donors. Bacterial contamination of collections has been dramatically reduced by implementation of improved umbilical cord decontamination protocols.

Cord blood banking: volume reduction of cord blood units using a semi-automated closed system.

Clinical evidence indicates that placental/umbilical cord blood (CB) is an alternative source of haematopoietic stem cells for bone marrow reconstitution. To establish a CB bank large panels of frozen, HLA-typed CB units need to be stored. Cryopreserved, unprocessed CB units require vast storage space. This study describes a method, using the Optipress II Automated Blood Component Extractor (Opti II) from Baxter Healthcare Corporation, to reduce the volume of the CB collection, preserving the quantity and quality of the progenitor cells, in a closed system. The CB collection was transferred to a triple bag system, centrifuged to produce a buffy coat layer and processed using a standard Opti II protocol to separate the whole blood into three components: plasma, buffy coat and buffy coat-depleted red cell concentrate. The buffy coat volume was standardised to 25 ml; mean reduced volume of 24.5 ml (s.d. 1.5 ml) with 53% red cell depletion. Good recovery of cells was observed: 92%, 98%, 96% and 106% recovery of nucleated, mononuclear, CD34+ and total colony-forming cells, respectively. Using this method for processing CB units reduces storage requirement by two-thirds but preserves the quantity and quality of the progenitor cells.

CD34+-selected peripheral blood progenitor cell transplantation in patients with multiple myeloma: tumour cell contamination and outcome.

Thirty-six patients with multiple myeloma (23 PR1, nine PR2, four stable disease) were entered into a pilot study evaluating the use of CD34+-selected peripheral blood progenitor cell transplantation (PBPCT) following high-dose melphalan alone or high-dose melphalan and total body irradiation. Peripheral blood progenitor cells (PBPCs) were mobilized with cyclophosphamide and granulocyte colony stimulating factor (G-CSF). CD34+ selection using the Cellpro Ceprate-SC system was performed in 22 cases with an adequate yield in 20. 10 patients failed to mobilize sufficient cells to permit selection and in four cases selection was not performed for other reasons. 16 patients therefore received unselected PBPC. Tumour cell contamination was evaluated by IgH gene fingerprinting (fpPCR). Harvested PBPC were fpPCR positive in 13/20 CD34+-selected cases and remained positive after selection in seven. Harvested PBPC were studied in 9/16 patients receiving unselected cells; fpPCR was positive in five and negative in four. There was no difference in event-free survival (EFS) between the CD34+-selected group and the unselected group (median 21 and 26 months, respectively, P=ns). The CD34+-selection process therefore reduced contamination but did not eliminate it completely, and in this small non-randomized study there was no apparent clinical benefit of CD34+ selection.

Activation of stress-activated protein kinases by hepatocyte isolation induces gene 33 expression.

Gene 33 is a putative immediate early gene and we have shown that mRNA encoding for gene 33 exhibits a transient increase as a result of the procedures used for hepatocyte isolation. The stress-activated protein kinases p46 JNK, p54 JNK, and p38 SAPK are activated by hepatocyte isolation and precede changes in gene 33 mRNA content. Although each SAPK isoform shows a distinctive profile of activity during isolation and subsequent hepatocyte culture, in each case the activation is transient and is largely reversed within 3 h of hepatocyte isolation. SB 203580, a p38 SAPK inhibitor, prevents the change to gene 33 expression in response to hepatocyte isolation. Given the possible role of gene 33 as an immediate early gene, the data presented here have general implications for control of hepatocyte proliferation and differentiation.