QBC for the diagnosis of human and canine american visceral leishmaniasis: preliminary data.

"Quantitative Buffy Coat" (QBC) is a direct and fast fluorescent method used for the identification of blood parasites. Since Leishmania chagasi circulates in blood, we decided to test it in American visceral leishmaniasis (AVL). Bone marrow (BM) and peripheral blood (PB) of 49 persons and PB of 31 dogs were analyzed. QBC was positive in BM of 11/11 patients with AVL and in 1/6 patients with other diseases. Amastigotes were identified in PB of 18/22 patients with AVL and in none without AVL. The test was positive in 30 out of the 31 seropositive dogs and in 28/28 dogs with Leishmania identified in other tissues. QBC is a promising method for diagnosis of human AVL, and possibly for the exam of PB of patients with AVL/AIDS, for the control of the cure and for the identification of asymptomatic carriers. Because it is fast and easy to collect and execute, QBC should be evaluated for programs of reservoir control.

Induced p21WAF1 expression acts to reverse myc myelomonocytic cell transformation.

Two murine myelomonocytic cells lines were used to examine p21WAF1 expression in myc-induced cell transformation. tEMmyc4 and FDLV are two v-myc-transformed immortalised myeloid cell lines exhibiting different transformed phenotypes. FDLV cells were derived from the transduction of v-myc into FDC-P1 cells and retain growth factor (IL-3) dependence, whereas tEMmyc4 cells were derived from the transduction of embryonal monocytes with v-myc and are growth factor-independent, constitutively express endogenous CSF-1, and are highly tumorigenic in syngeneic mice. Both cell lines were found to exhibit low p21WAF1 expression. When examined in tEMmyc4 cells, neither the p53-dependent pathway (mitomycin C or exogenous p53) nor p53-independent pathway (TPA or growth factor, CSF-1, stimulation) acted to increase p21WAF1 levels. Growth factor (IL-3) withdrawal, shown to reduce p21WAF1 levels in parental FDC-P1 cells, failed to do this in FDLV cells. The dependence of p21WAF1 expression on v-myc was further demonstrated by showing that a v-myc-targeted ribozyme, which acts to decrease v-myc RNA, increased p21WAF1 levels in tEMmyc4 cells. Enforced expression of exogenous p21WAF1 in tEMmyc4 cells with dysfunctional growth cycle (including growth arrest and increased susceptibility to apoptosis) was examined. p21WAF1 partially restored cell cycle regulation and apoptosis as well as inhibited the delayed cell cycle progression and apoptosis induced by mitomycin C or serum withdrawal. These results show p21WAF1 expression to be affected by v-myc and a restoration of p21WAF1 expression to partially reverse myc-mediated transformation.

The beta-galactosidase gene as a marker for hematopoietic reconstitution: individual cell analysis in a murine bone marrow transplantation model.

We have used a simple, single-gene retrovirus carrying the Escherichia coli beta-galactosidase reporter gene (lacZ), termed LlacZ. This virus was found to infect immortalized myeloid and lymphoid precursor/leukemic cell lines efficiently as well as primary murine bone marrow clonogenic progenitors, without apparent modulation of growth or phenotype. Following infection of bone marrow cells, a significant proportion of progenitors--36% of lineage-negative cells with low levels of c-kit expression (lin-/c-kit(lo)) known to be enriched with pluripotent hemopoietic stem cells, and 19% of Sca1-positive cells known to be enriched with transplantable cells with lymphomyeloid-reconstituting ability--were shown to express lacZ. Use of an LlacZ-infected population of post 5-fluorouracil bone marrow cells to reconstitute lethally irradiated mice demonstrated the presence of lacZ-expressing cells in the spleen at day 12 post-transplantation with provirus detected in individual spleen colonies (CFU-S). In the long term (3-6 months following transplantation), lacZ expression was detected in hematopoietic tissues of all recipient mice. The use of two-color in situ and flow cytometry analysis combined with lineage-specific antibodies showed lacZ expression in both myeloid and lymphoid cells in spleen and bone marrow. In addition, lacZ-expressing cells were detected in secondary recipient mice injected with bone marrow cells derived from primary LlacZ recipients. Overall, these data show the efficacy of a single gene vector for stem cell transduction, the utility of beta-galactosidase as a single cell marker for stem cell transduction and reconstitution ability, and the need for protocol optimization to see high-level multilineage gene expression.

Mutant N-ras induces myeloproliferative disorders and apoptosis in bone marrow repopulated mice.

Mutations that activate the N-ras oncogene are among the most frequently detected genetic alterations in human acute myeloid leukemias (AMLs), Philadelphia chromosome-negative myeloproliferative disorders (MPDs), and myelodysplastic syndromes (MDSs). However, because N-ras has not been shown to induce these disorders in an in vivo model, the role of N-ras in the evolution of myeloid leukemia is unclear. To investigate the potential of N-ras to induce myeloid leukemia, lethally irradiated mice were reconstituted with bone marrow (BM) cells infected with a retroviral vector carrying activated N-ras. Approximately 60% of these mice developed hematopoietic disorders, including severe MPDs resembling human chronic myelogenous leukemia (CML) or AML with differentiation (French-American-British [FAB] classification M2). Other reconstituted mice succumbed to hematopoietic defects that were pathologically similar to human MDSs. The latter disorders appeared to be due to a myeloid impairment that was demonstrated by enumeration of day-12 colony-forming units-spleen (CFU-S) and by in vitro colony assays. A high level of apoptosis associated with thymic atrophy and peripheral blood (PB) lymphopenia was also evident in N-ras reconstituted mice. Our results are consistent with a model in which antiproliferative effects are a primary consequence of N-ras mutations and secondary transforming events are necessary for the development of myeloid leukemia. This is the first report of an in vivo model for N-ras induced MPD and leukemia.

V-myc in a simple, single gene retroviral vector causes rapid induction of leukemia and concomitant apoptosis following bone marrow transplantation.

We have previously developed an in vivo model of leukemogenesis utilizing mice reconstituted with genetically modified bone marrow cells. Based on those studies, a new single gene retroviral vector has been engineered which efficiently transfers v-myc into immature murine bone marrow cells. All reconstituted mice developed leukemia with a short latency period (5-11 weeks). In addition to hyperproliferation associated with elevated levels of PCNA, extensive apoptosis was also observed in all leukemic animals with p53 accumulating in the apoptotic cells. Whereas bax encoded protein, an effector of p53 apoptotic activity was detected in apoptotic cells, p21Waf1 protein, a potential mediator of p53 growth suppression was not detected in these cells suggesting that v-myc-induced apoptosis was independent of the ability of p53 to induce p21Waf1. These results indicate that apoptosis, a part of the cellular response to v-myc expression, does not prevent leukemia development and that hyperproliferation rather than abrogation of oncogene-induced apoptosis appears to be a critical event in v-myc-induced leukemia.

Planning strategies in disability management.

Successful disability management practice is a systemic approach to vocational rehabilitation that begins with planning at the organizational level. A strategic planning format is used to outline the basic tenets of planning and, through its description, describe the unique approach to workplace intervention disability management has to offer. A case study is used to illustrate a simple application of planning steps of needs assessment, establishing goals and objectives, and developing disability management strategies.

Increased frequency of the uncommon allele of a tumour necrosis factor alpha gene polymorphism in rheumatoid arthritis and systemic lupus erythematosus.

The frequency of the uncommon allele (TNF2) of a polymorphism in the promoter region of the tumour necrosis factor alpha (TNF alpha) gene in patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) was found to be 3 times that of the normal anglo-saxon population. In SLE patients, this allele was strongly associated with HLA-DR3 expression and was also more frequent in patients who did not have malar rash. Functional studies of normal monocyte cytokine production in vitro showed that this genotype was associated with increased IL-1 alpha protein production but there were no differences in the production of TNF alpha protein.

Lack of association between an interleukin-1 receptor antagonist gene polymorphism and systemic lupus erythematosus.

Non-MHC linked genes may contribute to genetic predisposition to the development of systemic lupus erythematosus. The possibility that cytokine genes may be involved was raised by the observation of increased frequency in expression of an uncommon allele of an interleukin-1 receptor antagonist gene polymorphism and SLE in a recent U.K. study. We have not been able to show any significant differences in expression of this allele in SLE patients as a whole or in any patient subgroups. Our results actually show a slight decrease in the expression of this allele in SLE patients compared with healthy controls and in SLE patients with malar rash compared with SLE patients without malar rash.

Cytokine production by normal human monocytes: inter-subject variation and relationship to an IL-1 receptor antagonist (IL-1Ra) gene polymorphism.

Monocytes from different individuals show variable cytokine production in response to a variety of stimuli. We wished to determine the sets of conditions (cytokine combinations) that would enable us to demonstrate stable inter-individual differences in the production of IL-1 alpha, IL-1 beta, IL-1Ra, on-6 and tumour necrosis factor-alpha (TNF-alpha) by monocytes. We assessed the ability of a number of recombinant human cytokines (granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-gamma (IFN-gamma), TNF-alpha, IL-4, IL-6, transforming growth factor-beta (TGF-beta), IL-10 and IL-1Ra)) to stimulate or inhibit the production of one or more of these monocyte products. GM-CSF was found to stimulate the production of all five of these cytokines in a highly reproducible manner. TNF-alpha also up-regulated production of IL-1 alpha, IL-1 beta, IL-1Ra and IL-6 by monocytes, but the variability in the results of cells cultured from the same individuals on different occasions was greater. Other cytokines either stimulated production of only some of the five cytokine products tested, or stimulated the production of some cytokine products while inhibiting production of others. This was especially evident when cytokines were used in combination with GM-CSF: IFN-gamma down-regulated production of IL-1Ra while up-regulating the production of IL-1 alpha/beta, IL-6 and TNF-alpha, while IL-4 had the exact opposite effect. Polymorphisms in regions of cytokine genes that affect transcription may account for some of the interindividual variation in cytokine production. We have shown that a stable estimate of cytokine production phenotype can be obtained when monocytes collected on at least two separate occasions are stimulated by GM-CSF in vitro. We have looked for a relationship between IL-1 production and an 86-bp variable repeat polymorphism in intron 2 of the IL-1Ra gene. A less common allele of this polymorphism (allele 2) was associated with increased production of IL-1Ra protein, and also reduced production of IL-1 alpha protein by monocytes.

The many stakeholders in return to work.

Return to work has become an increasingly important consideration in TBI rehabilitation. However, most of the efforts in return to work centers around client deficits and treatment with little attention paid to the larger context of the world of work. This article examines the agendas of other players in the world of work and the interplay of these agendas that influence successful outcomes in return to work.