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Northern Initiative for Social Action (NISA) is a consumer-run, occupation-based, nonprofit organization located in northeastern Ontario, Canada. The NISA organization has grown in response to research revealing few opportunities for participation in personally meaningful and socially valued occupation for persons with mental illness living in the community of study. This article describes a mixed-design research study conducted by the ParNorth Research Unit of NISA and an occupational therapist. The study purposes were to (a) better understand the emerging characteristics of the NISA program and identify which the participants found helpful; (b) evaluate whether participation in NISA improved members' quality of life; and (c) ascertain whether participation reduced members' need for more traditional and costly methods of care (e.g., hospitalization, crisis services). Focus groups, daily participant observation, a quality of life interview, a consumer member survey and objective review of hospitalization data were used for data collection. Qualitative results indicated that NISA helped to meet participants' being, belonging, and becoming needs. Quantitative data indicated that overall, NISA members perceive an improvement in their subjective quality of life and sense of well-being. Their perceptions are supported by minimal use of crisis services and hospitalization, improved socioeconomic status, and several members' success in obtaining paid employment either within or outside NISA. Future challenges include the need to clearly describe the evolving NISA model and to ensure that the growth of this new organization does not exceed secured human or fiscal resources.

This unit presents an assay that has proven useful as an initial screening test is an HIV cytopathic effect (CPE) inhibition assay in which immortalized T cell lines (e.g., ATH8 or MT2) that are profoundly sensitive to the cytopathic effect of certain strains of HIV are utilized as target cells. Additional protocols assess the anti-HIV activity of certain candidate agents by measuring inhibition of syncytium formation or p24 gag protein production by ELISA. Calculation of the 50% inhibitory concentration (IC(50)) is also presented.

To compare ambulatory preceptors' and students' perceptions of the use of educational planning (setting goals, assessing needs, formulating objectives, choosing methods, and providing feedback and evaluation) in the office setting, we mailed a survey, which was returned by 127 longitudinal ambulatory preceptors and 168 first-year and second-year medical students. Faculty perceptions did not match student perceptions of what occurred in the longitudinal preceptor program teaching sessions in educational planning areas. Students perceived these activities were occurring with much less frequency than faculty perceived. Medical education needs to move beyond the usual faculty development workshop paradigm to a more comprehensive educational development model that includes training both faculty and students in core educational skills. This will enable the ambulatory setting to reach its full educational potential in training future physicians.

A method is described for the discrimination of Type III, late apoptotic, and necrotic cells, to improve the accuracy of proliferation and ploidy determinations of breast tumors. We selected an immunological probe, antitubulin antibody, and a DNA specific stain, propidium iodide (PI), both capable of crossing the permeable membranes of Type III, late apoptotic, and necrotic cells. This study utilized MDA-MB-175-VII breast carcinoma cells deprived of oxygen for up to 11 d to simulate intratumoral hypoxia, and 10 human breast tumors and mouse-human breast tumor xenografts disassociated by mechanical or enzymatic means. After 24 h under hypoxic conditions, the MDA cells displayed characteristics associated with both apoptosis and necrosis. Approximately 50% of day 1 cells showed membrane permeability by trypan blue and absence of DNA laddering; however, by day 3-4 characteristic apoptotic DNA laddering by gel electrophoresis was evident. Substantial DNA content loss, further evidenced by a reduction in PI staining and fluorescent microscopy, was obvious by day 5. By day 10, 98% of cells showed no propidium iodide staining by conventional PI live/dead cell gating, but were positive for antitubulin antibody staining. When the study was extended to the analysis of ten tumors, antitubulin antibody showed a range of 78%-96% staining with a median value of 87.5%, while PI staining showed a range of 8%-74% with a median value of 11.5%. This study demonstrates that a large percentage of cells in tumors and hypoxic cell populations have significantly reduced DNA content, such that conventional live/dead cell gating using PI may include many Type III cells as live cells, thus significantly altering data involving multicolor investigations.

The exclusion of non-tumor and dead cells from the analysis of live tumor cells can significantly improve the accuracy of prognostic indicators such as proliferative and DNA indexes. To target live breast tumor cells in a heterogeneous breast tumor model, we have designed a panel consisting of the DNA-specific dye DAPI and epithelial tissue-specific (cytokeratin), tumor-associated (MC5), proliferation-associated (proliferating cell nuclear antigen), and viability-associated (tubulin) markers. The breast tumor model consisted of a mixture of equal numbers of live and dead MDA-MB-175-VII (breast tumor) cells, live CEM (leukemic) cells, and live peripheral blood mononuclear cells. Targeting the live MDA cells in the mixture by gating on tubulin, cytokeratin, and MC5 resulted in a sevenfold increase in PCNA positivity (from 3% ungated to 22.3%), a 60% decrease in the %S-phase fraction (from 37.2% ungated to 15%), and elimination of extraneous hypodiploid and diploid components, enriching the tetraploid MDAs. These results are consistent with those obtained from unmixed live MDA cells. The combined utilization of this panel and "cumulative" electronic gating of the targeted population increases the number of relevant parameters that can be analyzed per sample and the accuracy of the resultant data.

Dead cells represent a significant source of interference in the flow cytometric analysis of viable cells primarily due to nonspecific uptake of probes, increased autofluorescence, and altered antigen expression and DNA content. Traditional methods of dead cell exclusion, based on light scatter or uptake of dyes such as propidium iodide (PI) or fluorescein diacetate (FDA), are appropriate for the analysis of fresh, relatively homogeneous samples. However, they are incompatible with the development in this laboratory of a solid tumor monoclonal antibody panel incorporating combined surface and intracellular staining: Light scatter is unreliable in heterogeneous samples such as solid tumors, and most of the widely used viability probes are incompatible, due to weak or reversible binding, with the use of permeabilizing agents for intracellular staining. To determine the best viability marker for inclusion in the solid tumor panel, we compared cultured cells held under hypoxic conditions for up to 15 days after harvest, stained with eight viability probes, and processed according to the solid tumor panel procedure (unprocessed cells from each day, stained with PI, were used as standards). The viability probes included PI (in processed and unprocessed samples); 7-aminoactinomycin D (7-AAD); TO-PRO-3; laser dye styryl (LDS)-751; ethidium monoazide (EMA); and actin, cytokeratin, and tubulin indirectly labelled with sheep-alpha-mouse-FITC (SAM-FITC). The selection criteria for the best viability probe included broad cell type specificity: low nonspecific staining of live cells, specific staining of dead cells strong enough to withstand the permeabilization procedure, high signal-to-noise ratio throughout the time course, and compatibility with the four other fluorescent probes making up the tumor antibody panel. TO-PRO-3, LDS-751, and PI (in processed cells) stained both live and dead cells indiscriminately. Actin-SAM-FITC, EMA, and 7-AAD did not display sufficiently high signal-to-noise ratios over the entire time course. Cytokeratin-SAM-FITC was acceptable in every respect other than its specificity only for cells of epithelial origin. Tubulin-SAM-FITC alone satisfied all the criteria and was selected for inclusion in the monoclonal antibody panel as a viability probe.

The expression of human immunodeficiency virus type 1 (HIV-1) in infected cells is induced (or enhanced) by a number of agents including phorbol myristate acetate (PMA), phytohemagglutinin (PHA), certain infectious agents, certain cytokines, and ultraviolet light. ACH2 cells represent latently HIV-1-infected T-cells, which produce only a low level of HIV-1 in vitro. We found that various anti-cancer agents including 5-azacytidine (5-AZC), 5-fluorouracil (5-FU), methotrexate, cytosine arabinoside, and vinblastine potentiated the expression of HIV-1 in ACH2 cells. There was no evidence of altered DNA methylation patterns in ACH2 cells cultured with 5-FU unlike with 5-AZC. The NF-kappa B binding activity was found to be enhanced in ACH2 cells exposed to 5-FU (but not in those exposed to 5-AZC) as assessed by the mobility shift assay using an oligonucleotide containing two NF-kappa B binding sites. These data suggest that the use of certain anti-cancer agents may induce (or enhance) the expression of HIV-1.

The chaperone protein Hsc70 is an ATPase of unknown mechanism, although the crystal structure of the 44-kDa ATPase domain has been solved. This structure shows that the hydroxyl of threonine 204 is located close to the gamma-phosphate of ATP, in a position where it might be an intermediate phosphate acceptor in the hydrolysis reaction. We made two point mutations at residue 204 of Hsc70, threonine to valine (T204V) and threonine to glutamic acid (T204E). The wild-type ATPase domain had a Km for ATP of approximately 1 microM; the mutants had Km values of approximately 90 microM. The kcat values for the mutant proteins were also increased. After crystallization, the structures of the T204V and T204E proteins were solved and refined with data to 2.3- and 2.4-A resolution, respectively. The overall tertiary structure of the mutants showed little change from the wild type; however, significant changes were observed in the active site. Analysis of the structures suggested possible reasons for the changes in kinetic constants. Threonine 204 does not seem to be an obligatory intermediate phosphate acceptor in the hydrolysis reaction since the mutants retained appreciable ATPase activity.

Immunophenotypes for 272 patients with acute myelogenous leukemia (AML) were analyzed using a panel of 22 antibodies. Numerical evidence for unusual coexpressions (present in normal marrow at < or = 0.1%) of surface markers on > or = 10% of the blast cells was found in 85% of all cases. Asynchronous expression of myeloid differentiation antigens occurred in 70% of the cases. Unusual coexpression of T-lymphoid, B-lymphoid, or natural killer (NK) markers with myeloid markers occurred in 38%, 13%, and 21%, respectively, of all AML cases. Two- and three-color analyses confirmed coexpression in 15 of 15 cases, and indicated that these percentages are an underestimate, because coexpression can be demonstrated in cases without numerical overlap. These data indicate that the unusual coexpression of normal differentiation antigens is a common occurrence in AML. Markers in 12 of 13 patients were similar between presentation and relapse, and in two patients, unusual phenotypes detected at first relapse were shown at second relapse, indicating these immunophenotypes are stable in the majority of AML patients. Significant correlations were found between t(8;21) cytogenetics and coexpression of CD19 with CD15 or CD34, t(9;22) and coexpression of CD19 and CD34, and t(15;17) and coexpression of CD2 and myeloid antigens. Multiparameter fluorescence analysis allows detection of unusual phenotypes when the blast counts are < 5% (classical remission). Analysis of 16 patients in remission indicated the presence of presentation phenotypes in 0.2% to 7.9% of the lymphocyte + blast light scatter region, representing 0.03% to 1.4% of the total nucleated marrow cells. Of the 16 patients with > or = 4 months follow-up after detection of these cells, 6 of 6 patients with > or = 0.2% unusual presentation phenotypic marrow cells have relapsed, while 9 of 10 patients with < 0.2% remain in remission. The detection of cells with the unusual presentation phenotype may reflect residual AML cells, and their increase may predict relapse.

Reported is the case of a 24-year-old female who presented to the Emergency Department complaining of lower abdominal pain and vaginal bleeding, whose initial transvaginal ultrasound was interpreted as showing a viable intrauterine pregnancy (IUP) of 8 weeks gestation. Repeat transvaginal ultrasound during a subsequent Emergency Department (ED) visit 3 days later revealed bilateral ectopic pregnancies of 6.5 weeks gestation. ED physicians should be familiar with the limitations of transvaginal sonography, and should be wary of early "intrauterine" pregnancies that are diagnosed ultrasonographically by inexperienced operators.

An experimental method of orchidopexy for high inguinal or abdominal testes, in the dog, using microsurgical anastomosis of the testicular vessels to vessels of the thigh or inferior epigastric vessels, is described. Experimental results suggest that this is a feasible method of treatment of such testes. A clinical trial of the method has commenced.

Non-neuronal accessory cells of mouse dorsal root ganglion cultures secrete a complex that stimulates neurite outgrowth in neonatal sympathetic ganglion neurons. A monoclonal antibody that binds to these two cell types also immunoprecipitates neurite outgrowth complex from mouse conditioned medium and binds to mouse brain tissue. Genetic analysis of the component recognized by the antibody revealed that it maps to the Thy-1 locus on mouse chromosome 9. Further studies of cell-type specificity, sedimentation analysis and antibody competition, confirmed that it is indistinguishable from the product of the Thy-1 locus. The finding of an association between Thy-1 and neurite outgrowth complex in the mouse argues for a role of the Thy-1 locus in the interactions of neurons with their surroundings.

Three hundred fifty-one alert emergency department patients with post-traumatic neck pain were evaluated prospectively. Seven (2%) had proven fractures or ligament disruptions. The immediate onset of neck pain and the presence of posterior midline cervical tenderness each had 100% sensitivity, with specificity of 65% and 48%, respectively. Discharged patients were followed up by telephone or letter at a mean of 25 +/- 20 weeks. Of this group, 63% saw another physician, and 43% had persistent moderate-to-severe neck pain or neurologic symptoms at a mean follow-up time of 24 weeks after injury. Of those who had not had a cervical radiograph while in the ED, 52% later obtained one. In addition, 66% of the discharged patients were pursuing litigation. The results suggest that it may be possible to identify alert patients with cervical spine injury by means of history and examination only; however, a larger study confirming these results is required. The high frequency of post-ED radiography, the high prevalence of persistent symptoms after injury, and frequent involvement in litigation are factors to be considered when evaluating ED patients with post-traumatic neck pain. These factors may support the validity of obtaining cervical spine radiographs on many more of these patients than high-yield criteria would dictate.