The use of serial outpatient complete blood count (CBC) results to derive biologic variation: a new tool to gauge the acceptability of hematology testing.

INTRODUCTION: Most estimates of biologic variation (sb ) are based on periodically acquiring and storing specimens from reference subjects, followed by analysis within a tightly controlled analytic run. We demonstrate that reliable estimates of sb can be derived for virtually all constituents of the CBC from previously obtained paired patient results and summary QC data. METHODS: A laboratory data repository provided all of the outpatient CBC results measured over 20.5 months at a large Canadian referral laboratory. These CBC measurements were taken on one of four Beckman Coulter LH analyzers. A total of 1852 different patients had CBCs repeated at least twice within 84 h. We tabulated the pairs of intrapatient constituents that were separated by 0-6, 6-12, 12-18,… 72-78, and 78-84 h. The standard deviations of duplicates (SDD) of the paired data were then regressed against time. The y-intercept represents the sum of sb and short-term analytic variation (sa ): y0 =(s(2) a +s(2) b )(1/2) . The short-term imprecision was determined from normal range Coulter quality control specimens. RESULTS: Patient sb for hematocrit, MCH, absolute monocytes, and absolute neutrophils are extremely close to those determined by biologic variation experiments using healthy volunteers. Most of the other estimates of sb tended to be slightly lower than literature estimates. CONCLUSIONS: We describe a novel approach to deriving sb . The ratio of the sb to sa (a measure of sigma) indicates that the Beckman Coulter LH is extremely suitable for CBC monitoring of outpatients as well as for inpatients, whose sb is generally higher.

Head and neck tumour immunology: basic concepts and new clinical implications.

An understanding of the immune system and its modes of action is fundamental to understanding the causes, natural history, management and treatment of many diseases. As such, a grasp of the principles of immunology is essential for every physician.This paper represents a succinct overview of the immune system, discussing the major components in turn, in respect of structure, function and integrated organisation, in relation to head and neck cancer.

Collaborative philosophical enquiry for school children: cognitive effects at 10-12 years.

BACKGROUND: Debates about the modifiability of cognitive ability have been largely resolved by reports of successful 'thinking skills' interventions. However, such interventions are very diverse and generalization of effects relatively little explored. AIMS: This study investigated whether a thinking skills intervention involving collaborative interactive dialogue could lead not only to gains in measured verbal cognitive ability but also generalization to non-verbal and quantitative reasoning ability. SAMPLE: Randomly selected intervention children were aged 10 at pre-test (N=105, four classes/schools). Controls followed a normal curriculum (N=72, three classes/two schools). METHOD: Intervention children engaged in collaborative enquiry for 1 hour per week over 16 months. The control group received normal classroom experiences. The Cognitive Abilities Test was administered before and after the intervention. RESULTS: Intervention pupils showed significant standardized gains in verbal and also in non-verbal and quantitative aspects of reasoning, consistent across intervention schools. Boys and girls made significant gains. The highest quartile of pre-test ability showed the smallest gains. Controls did not gain in any aspect. CONCLUSIONS: Philosophical enquiry involving interactive dialogue led not only to significant gains in measured verbal cognitive ability but also generalization to non-verbal and quantitative reasoning ability, consistent across schools and largely irrespective of pupil gender and ability. The effect sizes from this large-scale field trial in one local authority exceeded those reported in the literature. Implications for theory building, replicability and sustainability are addressed.

Collaborative philosophical inquiry for schoolchildren: cognitive gains at 2-year follow-up.

BACKGROUND: Evidence of maintained gains from thinking skills interventions are rare in the literature even within sectors of education, let alone across sectors. AIMS: This study investigated the cognitive effects of collaborative philosophical inquiry at long-term 2-year follow-up, after the participants had transferred to secondary (high) school without experiencing further philosophical inquiry in the interim. SAMPLE: Sample attrition was greater in the control than in the experimental group, but 96 experimental and 52 control subjects were available. METHOD: Intervened children who engaged in collaborative inquiry for 1 hour per week over 16 months and whose pre-post cognitive gains were reported in Topping and Trickey (2007) were followed up 2 years later, some time after they had transferred from primary to secondary school, again using the Cognitive Abilities Test. RESULTS: The significant pre-post cognitive ability gains in the experimental group in primary school were maintained towards the end of their second year of secondary school. Higher achieving pupils were somewhat advantaged in sustaining these gains. The control group showed an insignificant but persistent deterioration in scores from pre- to post-test to follow-up. CONCLUSIONS: Given the pattern of sample attrition, the group difference seems likely to be underestimated. The study provides evidence of maintained cognitive gains from collaborative philosophical inquiry, transferred across contexts. Implications for future research, policy and practice are discussed.

Isolation and characterisation of vascular endothelial growth factor-165 specific scFv fragments by phage display.

Vascular endothelial growth factor (VEGF) is a multifunctional cytokine which plays a major role in angiogenesis. Alternative splicing causes the production of several different isoforms (VEGF-A 121, 145, 165, 189, 206). VEGF is essential for tumor angiogenesis and several studies have correlated elevated VEGF levels with tumor stage, metastases and progression. Antibody phage display was employed to isolate two scFv antibody fragments, D8 and F10, with specificity for the VEGF165 isoform. It was shown by ELISA and competitive immunohistochemistry that each clone bound to VEGF165 but not VEGF121. Immunohistochemistry with D8 and F10 on colorectal carcinoma and adenoma sections revealed positive staining similar to that shown by a polyclonal VEGF antibody. The scFv antibody fragments, D8 and F10, will be useful in specifically detecting circulating VEGF165 in cancer patients as most studies to date have quantified the total level of circulating VEGF (121 and 165). These reagents will allow further elucidation of the role of VEGF in tumor angiogenesis.

Collaborative writing: the effects of metacognitive prompting and structured peer interaction.

BACKGROUND: The structured system for peer assisted learning in writing named Paired Writing (Topping, 1995) incorporates both metacognitive prompting and scaffolding for the interactive process. AIM: This study sought to evaluate the relative contribution of these two components to student gain in quality of writing and attitudes to writing, while controlling for amount of writing practice and teacher effects. SAMPLE: Participants were 28 ten- and eleven-year-old students forming a problematic mixed ability class. METHODS: All received training in Paired Writing and its inherent metacognitive prompting. Students matched by gender and pre-test writing scores were assigned randomly to Interaction or No Interaction conditions. In the Interaction condition, the more able writers became 'tutors' for the less able. In the No Interaction condition, the more able writers acted as controls for the tutors and the less able as controls for the tutees. Over six weeks, the paired writers produced five pieces of personal writing collaboratively, while children in the No Interaction condition did so alone. RESULTS: On pre- and post-project analyses of the quality of individual writing, all groups showed statistically significant improvements in writing. However, the pre-post gains of the children who wrote interactively were significantly greater than those of the lone writers. There was some evidence that the paired writers also had more positive self-esteem as writers. CONCLUSION: The operation and durability of the Paired Writing system are discussed.

Pre-operative serum vascular endothelial growth factor can select patients for adjuvant treatment after curative resection in colorectal cancer.

We aim to determine the clinical usefulness of pre-operative serum vascular endothelial growth factor (VEGF) as a predictor of outcome in patients undergoing curative resection for colorectal cancer. Serum VEGF was assayed by quantitative ELISA in 81 patients prior to curative resection for node-negative (n = 53) and node-positive (n = 28) disease. Median follow-up for patients without cancer death was 27 months (range 21-37). Pre-operative serum VEGF was significantly higher in patients who went on to develop metastases than those who did not (median, 713 pg ml-1 vs. 314 pg ml-1, P < 0.0001). Using multivariate Cox regression analysis, pre-operative serum VEGF was the most important prognostic factor independent of nodal status and adjuvant chemotherapy, and was superior to nodal status in predicting outcome (P < 0.00001). At 575 pg ml-1, pre-operative serum VEGF was 64% sensitive and 89% specific in predicting the development of metastases in curative resections, with a positive predictive value of 73% and a negative predictive value of 85%. Pre-operative serum VEGF is a powerful predictor of outcome following curative surgery for colorectal cancer. These data support the measurement of pre-operative serum VEGF as a method for selecting patients who require adjuvant therapy.

Isolation of Acanthamoeba-specific antibodies from a bacteriophage display library.

Acanthamoeba causes opportunistic eye infections in humans, which can lead to severe keratitis and may ultimately result in blindness. Current methods for identifying this organism rely on culture and microscopy. In this paper, we describe the isolation of antibody fragments that can be used for the unequivocal identification of Acanthamoeba. A bacteriophage antibody display library was used to isolate antibody fragments that bind specifically to Acanthamoeba. Individual clones were studied by enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence. Four antibody clones that specifically bind to Acanthamoeba spp. were identified.

Isolation of human colorectal tumour reactive antibodies using phage display technology.

Few highly specific anti-colorectal tumour antibodies exist. Here, we describe the isolation of a panel of colorectal tumour reactive antibodies, using bacteriophage-antibody display technology. A whole-cell based panning protocol has been used to isolate antibodies. panning against a cell type related to the colorectal tumour cells was used to remove antibodies reacting with common antigens. The 11 antibodies studied possess different levels of specificity, all bound to protein or glycoprotein molecules, and 2 of them recognised tunicamycin-sensitive epitopes. None of the antibodies recognise the commonly expressed colorectal-markers CEA or EP-CAM. Our work demonstrates the potential of this technology for the development of new anti-tumour reagents.

The role of monocytes and natural killer cells in mediating antibody-dependent lysis of colorectal tumour cells.

Monocytes and natural killer (NK) cells are known to be important effector cell populations in mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Purified monocyte and NK effector cell populations, from normal and colorectal cancer (CRC) patients, together with a number of murine (17-1A and 323/A3) and their chimaeric (c17-1A) or humanised (3622W94) equivalents, and chimaeric (c) SF25 were compared for their ability to mediate ADCC of colorectal tumour cells. The chimaeric and humanised antibodies were significantly better at mediating tumour lysis than their murine equivalents with all-effector populations. When effector cells from CRC patients were used the cSF25 antibody was significantly better than 3622W94 (P < 0.02) which, in turn, was significantly better than c17-1A (P < 0.03). Depletion of NK cells produced a decrease in specific tumour lysis with all antibodies. In addition a higher rate of NK cell death was observed in CRC patients during the assay than in normal controls. The chimaeric and humanised antibodies stained a similar percentage of the HT-29 target cells (>80%), but 3622W94 bound to significantly more cells from primary tumour biopsies than cSF-25 (P = 0.001). Together, the results suggest that NK cells are the most important effector cell type mediating ADCC in vitro, that there is some impairment of NK function in CRC patients, and that cSF25 is the most potent antibody. For use in vivo the anti-Ep-CAM antibody 3622W94 would appear to be the most suitable reagent for further study.

Advanced colorectal cancer is associated with impaired interleukin 12 and enhanced interleukin 10 production.

Interleukin 12 (IL-12) is a heterodimeric cytokine that has been demonstrated to have a major role in stimulating a cell-mediated antitumor response. IL-10, a product of T helper 2 lymphocytes, is its most potent inhibitor. The aim of this study was to investigate whether patients with colorectal cancer had an imbalance in production of IL-12 and IL-10 preoperatively, and whether this was associated with advanced disease at surgery. Blood was obtained before surgery from 60 patients with colorectal cancer and from 30 controls. Peripheral blood mononuclear cells were incubated with Staphylococcus aureus Cowan's strain 1 in vitro for 24 h to assess IL-12 expression after stimulation, and serum was used for IL-10 measurement. IL-12 and IL-10 levels were assessed by ELISA. A single pathologist staged the tumors according to the tumor-node-metastasis (TNM) and Dukes' classifications. Patients with colorectal cancer had significantly lower levels of IL-12 (P <0.001) and higher levels of IL-10(P = 0.004) compared to controls. In addition, lower levels of IL-12 were detected in those patients who were node positive (P<0.05), had Dukes' C lesions (P < or = 0.001), and T3 or T4 lesions (P<0.033) when compared to controls. Patients with Dukes' B and C lesions (P<0.01) and T3 and T4 lesions (P<0.05) also had higher levels of IL-10 compared to controls. This study is the first to demonstrate that patients with colorectal cancer have decreased IL-12 production and increased serum IL-10. This suggests an impaired T helper 1 cell-mediated antitumor response and provides some justification for exogenous IL-12 therapy or anti-IL-10 therapy in these patients.

Hydrogen exchange in native and denatured states of hen egg-white lysozyme.

The hydrogen exchange kinetics of 68 individual amide protons in the native state of hen lysozyme have been measured at pH 7.5 and 30 degrees C by 2D NMR methods. These constitute the most protected subset of amides, with exchange half lives some 10(5)-10(7) times longer than anticipated from studies of small model peptides. The observed distribution of rates under these conditions can be rationalized to a large extent in terms of the hydrogen bonding of individual amides and their burial from bulk solvent. Exchange rates have also been measured in a reversibly denatured state of lysozyme; this was made possible under very mild conditions, pH 2.0 35 degrees C, by lowering the stability of the native state through selective cleavage of the Cys-6-Cys-127 disulfide cross-link (CM6-127 lysozyme). In this state the exchange rates for the majority of amides approach, within a factor of 5, the values anticipated from small model peptides. For a few amides, however, there is evidence for significant retardation (up to nearly 20-fold) relative to the predicted rates. The pattern of protection observed under these conditions does not reflect the behavior of the protein under strongly native conditions, suggesting that regions of native-like structure do not persist significantly in the denatured state of CM6-127 lysozyme. The pattern of exchange rates from the native protein at high temperature, pH 3.8 69 degrees C, resembles that of the acid-denatured state, suggesting that under these conditions the exchange kinetics are dominated by transient global unfolding. The rates of folding and unfolding under these conditions were determined independently by magnetization transfer NMR methods, enabling the intrinsic exchange rates from the denatured state to be deduced on the basis of this model, under conditions where the predominant equilibrium species is the native state. Again, in the case of most amides these rates showed only limited deviation from those predicted by a simple random coil model. This reinforces the view that these denatured states of lysozyme have little persistent residual order and contrasts with the behavior found for compact partially folded states of proteins, including an intermediate detected transiently during the refolding of hen lysozyme.

Hydrophobic clustering in nonnative states of a protein: interpretation of chemical shifts in NMR spectra of denatured states of lysozyme.

Chemical shifts of resonances of specific protons in the 1H NMR spectrum of thermally denatured hen lysozyme have been determined by exchange correlation with assigned native state resonances in 2D NOESY spectra obtained under conditions where the two states are interconverting. There are subtle but widespread deviations of the measured shifts from the values which would be anticipated for a random coil; in the case of side chain protons these are virtually all net upfield shifts and it is shown that this may be the averaged effect of interactions with aromatic rings in a partially collapsed denatured state. In a very few cases, notably that of two sequential tryptophan residues, it is possible to interpret these effects in terms of specific, local interresidue interactions. Generally, however, there is no correlation with either native state shift perturbations or with sequence proximity to aromatic groups. Diminution of most of the residual shift perturbations on reduction of the disulfide cross-links confirms that they are not simply effects of residues adjacent in the sequence. Similar effects of chemical denaturants, with the disulfides intact, demonstrate that the shift perturbations reflect an enhanced tendency to side chain clustering in the thermally denatured state. The temperature dependences of the shift perturbations suggest that this clustering is noncooperative and is driven by small, favorable enthalpy changes. While the extent of conformational averaging is clearly much greater than that observed for a homologous protein, alpha-lactalbumin, in its partially folded "molten globule" state, the results clearly show that thermally denatured lysozyme differs substantially from a random coil, principally in that it is partially hydrophobically collapsed.

Disruptive adolescents in the school system: comparative effectiveness of helping strategies.

Large sums of money are often spent on setting up different systems of helping or managing disruptive adolescents in the school system, without reference to objective data on effectiveness. This paper considers 24 alternative systems, scrutinizes the evaluation research on each, and notes that the most expensive systems are not necessarily the most effective. Furthermore, the task of resolving behavioural difficulties may be either under- or over-resourced in individual cases, building in either failure or waste. A guide is given to developing a sequence of intervention strategies of known cost-effectiveness, providing a fail-safe structure for helping disruptive adolescents in educational settings.