Randomised controlled trial of an online cognitive training program in school-aged children with cerebral palsy.

BACKGROUND: Children with cerebral palsy (CP) experience deficits in nonverbal reasoning. The SMART online cognitive intervention has been associated with gains in IQ and nonverbal IQ in previous studies in typically developing school-aged children and children experiencing learning difficulties. AIM: To assess the efficacy of an online cognitive intervention in school-aged children with CP. METHODS AND PROCEDURES: 21 children with CP (male n = 17; 76.2%), mean age 9 y 8 m, SD 1 y 1 month (range 8 y 3 m to 12 y 6 m) were randomised into the intervention group (n = 9) or a waitlist control group. A mixed-methods approach with an explanatory sequential design was used, with a randomised controlled trial followed by qualitative interviews. Participants were assessed on measures of intelligence, academic ability, attention and executive functioning, and social-emotional functioning at baseline, then after completing the training, or the waitlist period. Analyses included ANCOVAs and paired samples t tests. Semi-structured interviews explored participants' experiences with the training. RESULTS AND OUTCOMES: Training completion was low with a mean of 16.9 modules completed out of 55 available. No significant effect of training was found for the primary outcome of intelligence, or for any secondary outcomes. Participants reported barriers and facilitators for accessing the program. IMPLICATIONS: Cognitive training programs addressing relational framing ability may require significant modifications before they can be effectively tested with children with CP.

Cognition and learning difficulties in a representative sample of school-aged children with cerebral palsy.

BACKGROUND: Nearly half of all children with CP experience intellectual impairment, with impacts on academic achievement. AIMS: To assess cognitive and academic functioning for primary-school aged children with CP METHODS AND PROCEDURES: This population-based cohort study assessed 93 participants (male n = 62; mean = 9 years 9 months, SD 1 y 1.8 months) on measures of fluid and crystallised intelligence (Raven's Coloured Progressive Matrices, Peabody Picture Vocabulary Test) and academic achievement (Wechsler Individual Achievement Test). Analyses included t-tests, Pearson's chi-square and regression. OUTCOMES AND RESULTS: 41 (44.1%) children met criteria consistent with intellectual developmental disorder. Academic skills were significantly below population means on word reading (M= 85.4, SD = 19.3), t(66) = -6.2, p < .001; spelling (M=83.3, SD=19.7) t(65) = -6.87, p < .001; and numerical operations (M=72.9, SD=21.7) Z = 66.0, p < .001. Cognitive ability was associated with GMFCS level (χ² (1, N = 93) = 16.15, p < .001) and diagnosis of epilepsy (χ² (2, N = 93) = 11.51 p = .003). Crystallised and fluid intelligence together accounted for 65% of the variance in word reading, 56% in spelling and 52% in numerical operations. IMPLICATIONS: Many children with CP experience academic challenges. Screening is recommended for all children with CP and full psychoeducational assessment undertaken when children with CP experience academic difficulties.

Preventable deaths among major trauma patients in Mersey Region, North Wales and the Isle of Man.

Preventable and unexpected deaths following injury were identified from among 1088 victims of major injuries arising in a defined population and area during a 12-month period. In hospital, 44 (16 per cent) deaths from blunt injury, one death from penetrating injury and one death from drowning were preventable. In patients sustaining blunt injuries, 22 per cent of non-head-injury deaths and 13 per cent of head-injury deaths were preventable. In all preventable head-injury deaths either a delay in operation (35 per cent) or no operation for mass lesions (65 per cent) occurred, often because of misdiagnosis as alcohol intoxication (22 per cent) or CVA (22 per cent). Multiple preventable factors were more likely in non-head-injury deaths and included missed injuries (67 per cent), poor airway care (57 per cent), delayed or no operation (52 per cent), undertransfusion (38 per cent) and inadequate surgery (19 per cent). By TRISS methodology the outcome was unexpected, in 53 per cent blunt injury deaths in hospital and 2.8 per cent of survivors. Three preventable blunt injury deaths (6.8 per cent) had probabilities of survival < 50 per cent and were not, therefore, identified as unexpected by TRISS. A preventable death rate of 16 per cent for blunt injuries equates to 638 preventable blunt injury deaths each year in England and Wales.

The epidemiology of major injuries in Mersey Region and North Wales.

A prospective epidemiological study was undertaken to determine the workload and patient characteristics for a putative trauma centre in a large defined area. One thousand and eighty-eight patients were included: 430 brought in dead, 309 hospital deaths and 349 survivors. Types of injury were: blunt 76 per cent, penetrating 3.6 per cent, burns 5.8 per cent, other 14 per cent. The incidence of blunt injury was 19/100,000 for patients arriving alive at hospital and accounted for 0.08 per cent of new A & E attendances. Eight per cent of blunt injury patients were children, 68 per cent were adults and 24 per cent elderly. Major causes of injury were: road accidents 67 per cent and falls 26 per cent. In patients arriving alive after blunt injuries, those who subsequently died were significantly older, more severely injured and more physiologically impaired. Hospital mortality was 45 per cent for blunt, 43 per cent for penetrating injuries, and 67 per cent for burns. TRISS methodology indicated 53 per cent of hospital deaths from blunt injuries were unexpected. Practically, it is questionable whether the incidence of major injuries is sufficient to provide the volume of patients necessary to sustain a Level I Trauma Centre. Nevertheless, concentration of injury service is essential, since no hospital receives sufficient patients to develop and maintain expertise.

Lymphocyte subset analysis by Boolean algebra: a phenotypic approach using a cocktail of 5 antibodies and 3 color immunofluorescence.

Commercial reagent kits for the evaluation of leukocyte subsets involve the staining of a panel of up to six tubes using combinations of pre-mixed fluorescein isothiocyanate (FITC) and R-phycoerythrin (PE) conjugated monoclonal antibodies. We describe a rapid method whereby total CD3+ T-cells, CD4+ T-cells (CD3+ CD4+), CD8+ T-cells (CD3+ CD8+), putative gamma delta-receptor-T-cells (CD3+ CD4- CD8-), and T-cells that are CD3+ CD4+ CD8+ as well as B-lymphocytes and NK-cells can be enumerated after staining in a single tube. Whole blood specimens are labelled with a mixture of antibodies: FITC-conjugated antibodies to CD4 and CD19, PE-conjugated antibodies to CD8 and CD16, and either peridinin chlorophyll protein (PerCP) or allophycocyanin (APC) labelling for antibodies to CD3. After recording 20,000 events the data were analysed on the Consort 32 computer system and LYSYS-II (Becton Dickinson, San Jose, CA) and all of the lymphocyte subset values were determined by Boolean algebra using a technique we refer to as Boolean gate analysis (BGA). Our study has shown that BGA is statistically equivalent to SimulSET lymphocyte subset analysis. Furthermore, the procedure reduces the number of tubes required to two with consequential saving in reagents, consumables, and time.

Clonal analysis of a bladder cancer cell line: an experimental model of tumour heterogeneity.

The continuous cell line UCRU BL 17CL was derived from a human invasive bladder cancer and expresses elements of transitional, squamous and glandular differentiation. Nine clones of this line were established by limit dilution and have been extensively characterised. Only six of these clones grew subcutaneously in nude mice. Of these, three have exhibited local invasion, each in one of five implanted mice. Although all xenografts expressed transitional, squamous and glandular elements, different histological subtypes predominated within each clone. Only clones which grew in nude mice formed colonies in semi-solid medium, and each responded differently to the influence of medium that had been conditioned by the growth of UCRU BL 17CL, suggesting the possible secretion of a growth factor by these cells. The DNA content and lectin binding profiles of the clones also reflected the heterogeneity of the line. UCRU BL 17CL and the nine clones provide a unique model for the study of tumour heterogeneity, progression and differentiation, and the potential autocrine regulation of growth of bladder cancer.

Graft-versus-host resistance induced by tolerant cell populations. Evidence against clonal deletion as a mechanism of transplantation tolerance.

Characterization of the effect of immunization of F1 hybrid hosts with low doses of parental cells has shown that the F1 hybrid response to the receptor for the unshared MHC antigen on the immunizing cell induces specific resistance to a GVH challenge from cells of the same parental strain. We have shown that cells from parental rats tolerant to the unshared MHC antigens are capable of inducing GVH resistance in F1 hybrids. Unlike cells from normal parental rats that induce GVH resistance only when given in low immunizing doses of 10(6) cells, 10(6)-10(8) cells from tolerant donors effectively immunize F1 hybrids. This effect does not appear to be the result of passive transfer of suppressor cells from the tolerant donor. An alternative explanation is that tolerant populations contain cells that express the receptor for the tolerated alloantigen. The finding that normal parental populations that have been deleted of receptor-bearing cells by passage through semiallogeneic intermediate hosts do not induce GVH resistance, whereas tolerant cell populations do, confirms that clonal deletion does not adequately account for the functional characteristics of the tolerant cells. Attempts to delete putative receptor-bearing cells from the tolerant population however produced equivocal results.

Postirradiation recovery of lymphoid cells in the rat.

Whole-body irradiation has been extensively used to ablate immune responsiveness in rodent recipients in adoptive allograft assays. This study was undertaken to determine the relative radioresistance and the tempo of regeneration, following whole-body irradiation, of cells involved in the allograft response. Six distinct cell populations have been identified in the lymphoid tissues of rats subjected to sublethal whole-body irradiation. The relative representation of these subpopulations was significantly different from that in nonirradiated controls. NK cells, macrophages, and plasma cells, which are present in very low numbers in cell suspensions prepared from normal lymphoid tissues, made up a significant proportion of the residual/regenerating population in the tissues of rats recovering from whole-body irradiation. More significantly perhaps, the mature T cell populations showed a significant increase in the T cytotoxic/suppressor to T helper cell ratio. These observations support the suggestion that a number of the cell types within the mixed cell population observed in the rejecting indicator grafts of irradiated recipients in adoptive allograft assays are host derived. The finding that the T cytotoxic/suppressor population is apparently more radioresistant than the T helper population supports a conclusion that graft rejection in irradiated recipients, restored with pure populations of T helper cells, may not be directly mediated by the injected cells but may be the result of collaboration between these and host-derived cytotoxic cell populations.

Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice.

Immunological tolerance has been demonstrated in double-transgenic mice expressing the genes for a neo-self antigen, hen egg lysozyme, and a high affinity anti-lysozyme antibody. The majority of anti-lysozyme B-cells did not undergo clonal deletion, but were no longer able to secrete anti-lysozyme antibody and displayed markedly reduced levels of surface IgM while continuing to express high levels of surface IgD. These findings indicate that self tolerance may result from mechanisms other than clonal deletion, and are consistent with the hypothesis that IgD may have a unique role in B-cell tolerance.

A new microsphere-based immunofluorescence assay using flow cytometry.

The quantitative and qualitative capacities of flow cytometric analysis that have made it such a powerful tool in studies of cellular antigens have not previously been exploited when dealing with non-cellular antigens. A new immunofluorescence assay technique was developed, using an indirect staining procedure with monoclonal anti-kappa antibodies, to detect human free kappa light chains covalently bound to microspheres of a size suitable for flow cytometry. The strength of the fluorescent signal produced on the microspheres was related to the amount of antigen bound and the size of the beads. At the time of this work large microspheres (i.e., greater than 3 micron in diameter) suitable for this application were only available as suspensions of polysized beads. The fluorescent signal detected on labelled beads was optimized by selecting for analysis, on the basis of the forward angle laser scatter, only those beads of largest diameter. There are many potential applications for this technique - microspheres can be used for the presentation of virtually any antigen or antibody. The analytical benefits inherent in flow cytometry would be a significant advantage in the development of quantitative assays using this method.

Stability of lectin binding properties expressed by human bladder carcinoma cell lines passaged in vitro or in nude mice.

Binding of a panel of lectins by sublines of two human bladder carcinoma cell lines, UCRU-BL-17 CL and UCRU-BL-13 CL, assessed flow cytometrically following passage of the cells in vitro and in nude mice, was compared with that of human leukaemic cell lines, K562 and HL60, and found to be different. Marked glycosylation of sublines of both bladder cancer cell lines was found compared with normal human bladder transitional epithelium (assessed cytochemically). Neuraminidase pretreatment increased the binding of some lectins indicating that some galactose and N-acetylgalactosamine residues were sialylated. Lectin binding by UCRU-BL-17 CL sublines was remarkably constant on prolonged passage in vitro even though the lines underwent changes in physical characteristics and ploidy when grown in nude mice. This suggests that glycosylation of the tumour cell surface may represent an intrinsic feature of this bladder tumour.

Simultaneous cytometric analysis for the expression of cytoplasmic and surface antigens in activated T cells.

A method of two-colour immunofluorescence staining has been developed to allow the simultaneous analysis of both surface and cytoplasmic antigens. This involves the use of direct fluorochrome antibody conjugates for cell-surface antigen staining, followed by cell permeabilization and the staining of cytoplasmic antigens with biotinylated antibodies and streptavidin-fluorochrome conjugates. Fluorochrome-antibody conjugates bound to cell-surface epitopes were found not to be affected by the subsequent permeabilisation and cytoplasmic staining. This method was used to examine the surface phenotype of T cells expressing a cytoplasmic antigen, STA. STA is a unique determinant detected in activated human T cells by the monoclonal antibody K-1-21, which also recognizes a cross-reactive conformation-dependent epitope on human free kappa light chains. Cytometric analysis showed that STA is found in both Leu 2a+ cytotoxic/suppressor T cells and Leu 3a+ helper/inducer T cells but is not induced in the Leu 15+ population which contains suppressor T cells. STA was also shown to be an activation antigen in murine T cells.

Establishment and characterization of a new human bladder cancer cell line showing features of squamous and glandular differentiation.

Tumour-cell heterogeneity has been studied in a continuous cell line, UCRU-BL-17CL, established from a xenografted human primary bladder carcinoma. The cell line, grown in vitro for more than 30 generations, reflects the pathology of both the xenograft from which it was derived and the original human tumour. It comprises mainly adenocarcinoma cells which secrete mucin in vitro, as well as squamous and transitional carcinoma cells. Features of both adenocarcinomatous and squamous differentiation have been observed within the same cell. The line expresses ABH blood group isoantigens, binds to peanut lectin and reacts with monoclonal antibodies (MAbs) raised against keratin and against normal and malignant epithelial cells. It also reacts with MAbs against ras p21 proteins and the epidermal growth factor receptor (EGFR). It shows high levels of lactic acid dehydrogenase isozyme 5, consistent with a high-grade tumour, forms colonies in methylcellulose and is tumorigenic in nude mice. The karyotype (human) shows many marker chromosomes, consistent with expression of EGF receptors and ras p21 proteins, and an 11:13 translocation. DNA content, as studied by flow cytometry, reveals a shift from tetraploid to near triploid. This line may provide a useful model for studies of the histogenesis of bladder cancer and the relationship between transitional-cell carcinoma and the other histological subtypes of this disease.

Cellular requirements for mixed leucocyte reactivity in the cyclostome, Eptatretus stoutii.

Unfractionated peripheral blood leucocytes from allogeneic hagfish proliferated vigorously in one-way mixed leucocyte reaction (MLR) at 20-22 degrees. Maximum responsiveness was observed after 5 days of co-culture of responder and irradiated stimulator cells. Separation of leucocytes on the basis of laser scatter parameters yielded a small leucocyte population which responded but did not stimulate in MLR and a large leucocyte population capable of stimulating but not responding. Adherent cell depletion of the total leucocyte population did not affect the ability to respond in MLR but did ablate the stimulatory capacity of hagfish leucocytes. Together with previous phenotypic studies, these results confirm the presence of lymphocyte and macrophage/accessory cell populations in hagfish peripheral leucocytes.

High dose suppression of human anti-influenza A virus responses using T cell clones.

Human T cells exposed to high concentrations of influenza A virus cause specific suppression of the in vitro antibody response to the virus, but the phenomenon does not require viable T cells. In order to investigate the mechanism of this form of suppression, IL-2-dependent T cell clones of helper phenotype (CD4+, HLA-DR+, IL-2R+) were prepared with specificity for influenza A (Mem/Bel) and B (B HK) viruses and the non-crossreacting antigen purified protein derivative (PPD). When pulsed with high dose Mem/Bel virus, all three clones transferred suppression equally well to effector cultures of syngeneic or allogeneic E+ and E- cells stimulated with an immunogenic dose of the same virus. Thus, although suppression was specific at the level of expression, the induction phase was non-specific and non-major histocompatibility complex (MHC) restricted and did not involve interaction of antigen with the T cell receptor. HLA-DR, CD3 and CD8 determinants were excluded as the binding site for the virus by two-colour immunofluorescent staining and flow cytometric analysis. The concentrations of antigen required for high dose suppression inhibited antigen-specific proliferation by the clones; on the other hand, they remained partially sensitive to IL-2 and could still release gamma-interferon. These findings suggest that this phenomenon is distinct from conventional antigen-specific suppression mediated by CD8 T cells, but may play a biologically important role in regulating immune responses at least to viral antigens.

The mechanism of prolonged graft survival following removal of the regional lymph node.

The immune responsiveness of rats in which the rejection of RT-1-incompatible neonatal heart grafts had been prevented by removal of the draining lymph node was examined. It was found that within three days of node removal animals developed a state of specific unresponsiveness characterized by the failure to reject secondary grafts made into the contralateral leg with intact draining nodes. Studies of recipients with long-surviving grafts revealed that their serum contained significant levels of cytotoxic alloantibody although their cells behaved like those from naive donors in mixed lymphocyte interactions, graft-versus-host responses, and adoptive allograft assays. There was no evidence for the presence of sensitized T cells or suppressor T cells in the tissues of recipients with long-surviving grafts. The findings support the suggestion that prolonged graft survival following node removal is due to enhancement actively induced by the passage of graft antigen into the circulation at a time when the induction of the cell-mediated response has been aborted by removal of the node regional to the graft.

Evolutionary development of lymphocyte heterogeneity: leucocyte subpopulations in the Pacific hagfish.

Flow cytometric analysis of forward angle versus 90 degree scatter patterns of hagfish peripheral blood revealed two distinct leucocyte populations with size characteristics analogous to mammalian monocytes/granulocytes (hagfish large leucocytes) and small lymphocytes (hagfish small leucocytes). A cell population enhanced for the small leucocytes was obtained by density gradient centrifugation. Over 70% of the small leucocyte population consistently stained with a rabbit antiserum directed against polypeptide determinants on hagfish immunoglobulin, while staining of the large cell population was greatly reduced (less than 10%). A panel of monoclonal antibodies raised against a crude hagfish leucocyte preparation distinguished the two cell populations and revealed the existence of subpopulations of both small and large leucocytes.

The role of the regional lymph node in the response to secondarily vascularized grafts.

Studies using neonatal hearts grafted into the foot pads of adult rats have shown significant differences in the tempo of rejection in various RT1-incompatible combinations of donor and recipient rats. The model allows simultaneous study of events in the graft and in the regional node draining the graft. Removal of the regional node in the inductive stages of the immune response resulted in highly significant prolongation of graft survival. This effect was not due to lymphatic interruption per se or to clonal deletion. The effect was independent of the presence of the primary graft. Second grafts implanted in animals from which both the original graft and its regional node had been removed showed prolonged survival. Once survival of the original graft, from which the node was removed, was established, survival of second grafts bearing the same antigens was also prolonged, although third-party grafts were rejected in first set time. The data suggest that the microenvironment and anatomical connections of the lymph node that first receives antigen, or the cells that have contacted antigen in the graft, or both, play a vital role in an orderly sequence of cellular interaction and migration that culminates in graft rejection. Interruption of this sequence by node removal appears to divert the alloimmune response toward specific enhancement of the graft.

Hypogammaglobulinaemia associated with abnormalities of both B and T lymphocytes in patients with chronic lymphatic leukaemia.

The underlying basis for hypogammaglobulinaemia in patients with chronic lymphatic leukaemia (CLL) was investigated by measurement if immunoglobulin produced in vitro in cultures of pokeweek mitogen-stimulated B and T lymphocytes. B and T cells were separated by sheep red blood cell rosette techniques and, by culture of these cells from CLL patients in various combinations with B or T cells from normal subjects, it was possible to measure independently the function of B lymphocytes and the helper or suppressor function of T lymphocytes. By these methods it was found that the B lymphocytes of six of eight patients failed to produce immunoglobulins in vitro. B lymphocytes from two patients appeared to produce immunoglobulins in vitro. T lymphocytes from five of the eight patients had low or undetectable helper T cell function and in six patients their T lymphocytes had excessive suppressor activity in comparison to T lymphocyte populations from normal subjects. Whether the primary abnormality in the CLL T cell populations was a deficiency of helper T cells or excess of suppressor T cells was uncertain from these studies. These results suggest that immunoglobulin production by B lymphocytes from most patients with CLL was abnormal but also that T cells from CLL patients may be abnormal in respect to their role in immunoglobulin production at an early stage of the disease. These findings may assist in understanding the pathogenesis of this disease and lead to new approaches in treatment.