Disruption of IKAROS activity in primitive chronic-phase CML cells mimics myeloid disease progression.

Without effective therapy, chronic-phase chronic myeloid leukemia (CP-CML) evolves into an acute leukemia (blast crisis [BC]) that displays either myeloid or B-lymphoid characteristics. This transition is often preceded by a clinically recognized, but biologically poorly characterized, accelerated phase (AP). Here, we report that IKAROS protein is absent or reduced in bone marrow blasts from most CML patients with advanced myeloid disease (AP or BC). This contrasts with primitive CP-CML cells and BCR-ABL1-negative acute myeloid leukemia blasts, which express readily detectable IKAROS. To investigate whether loss of IKAROS contributes to myeloid disease progression in CP-CML, we examined the effects of forced expression of a dominant-negative isoform of IKAROS (IK6) in CP-CML patients' CD34(+) cells. We confirmed that IK6 disrupts IKAROS activity in transduced CP-CML cells and showed that it confers on them features of AP-CML, including a prolonged increased output in vitro and in xenografted mice of primitive cells with an enhanced ability to differentiate into basophils. Expression of IK6 in CD34(+) CP-CML cells also led to activation of signal transducer and activator of transcription 5 and transcriptional repression of its negative regulators. These findings implicate loss of IKAROS as a frequent step and potential diagnostic harbinger of progressive myeloid disease in CML patients.

Chemotherapy enhances cross-presentation of nuclear tumor antigens.

Cross-presentation of tumor antigen is essential for efficient priming of naïve CD8⁺ T lymphocytes and induction of effective anti-tumor immunity. We hypothesized that the subcellular location of a tumor antigen could affect the efficiency of cross-presentation, and hence the outcome of anti-tumor responses to that antigen. We compared cross-presentation of a nominal antigen expressed in the nuclear, secretory, or cytoplasmic compartments of B16 melanoma tumors. All tumors expressed similar levels of the antigen. The antigen was cross-presented from all compartments but when the concentration was low, nuclear antigen was less efficiently cross-presented than antigen from other cellular locations. The efficiency of cross-presentation of the nuclear antigen was improved following chemotherapy-induced tumor cell apoptosis and this correlated with an increase in the proportion of effector CTL. These data demonstrate that chemotherapy improves nuclear tumor antigen cross-presentation and could be important for anti-cancer immunotherapies that target nuclear antigens.

Flow cytometry as a rapid and reliable method to quantify sperm viability in the honeybee Apis mellifera.

An important measure of male quality is sperm viability; i.e., the percentage of live sperm within an ejaculate, as this provides an accurate measure of the number of sperm potentially available for egg fertilization. Sperm viability is often determined by fluorescence microscopy using dyes that differentially stain viable and nonviable sperm, but the technique has a number of limitations. Here, a flow cytometry (FCM) method was developed, which allows the rapid determination of honeybee sperm viability, facilitating high throughput analyses. Using samples with known sperm viabilities, it was found that data obtained from FCM were more accurate and less variable compared with data obtained for the same samples using fluorescence microscopy. It was also found that a previously reported additional population of honeybee sperm found in datasets using FCM is caused by freeze-thawing samples. In conclusion, the method described here allows to quantify sperm viability of honeybees quickly and with high accuracy. This will be of great value for future scientific research and could also be of value to guide future bee breeding programs, given the agricultural importance of honeybees as pollinators.

Quantifying spore viability of the honey bee pathogen Nosema apis using flow cytometry.

Honey bees are hosts to more than 80 different parasites, some of them being highly virulent and responsible for substantial losses in managed honey bee populations. The study of honey bee pathogens and their interactions with the bees' immune system has therefore become a research area of major interest. Here we developed a fast, accurate and reliable method to quantify the viability of spores of the honey bee gut parasite Nosema apis. To verify this method, a dilution series with 0, 25, 50, 75, and 100% live N. apis was made and SYTO 16 and Propidium Iodide (n = 35) were used to distinguish dead from live spores. The viability of spores in each sample was determined by flow cytometry and compared with the current method based on fluorescence microscopy. Results show that N. apis viability counts using flow cytometry produced very similar results when compared with fluorescence microscopy. However, we found that fluorescence microscopy underestimates N. apis viability in samples with higher percentages of viable spores, the latter typically being what is found in biological samples. A series of experiments were conducted to confirm that flow cytometry allows the use of additional fluorescent dyes such as SYBR 14 and SYTOX Red (used in combination with SYTO 16 or Propidium Iodide) to distinguish dead from live spores. We also show that spore viability quantification with flow cytometry can be undertaken using substantially lower dye concentrations than fluorescence microscopy. In conclusion, our data show flow cytometry to be a fast, reliable method to quantify N. apis spore viabilities, which has a number of advantages compared with existing methods.

Developments in the immunophenotypic analysis of haematological malignancies.

Immunophenotyping is the method by which antibodies are used to detect cellular antigens in clinical samples. Although the major role is in the diagnosis and classification of haematological malignancies, applications have expanded over the past decade. Immunophenotyping is now used extensively for disease staging and monitoring, to detect surrogate markers of genetic aberrations, to identify potential immuno-therapeutic targets and to aid prognostic prediction. This expansion in applications has resulted from developments in antibodies, methodology, automation and data handling. In this review we describe recent advances in both the technology and applications for the analysis of haematological malignancies. We highlight the importance of the expanding repertoire of testing capability for diagnostic, prognostic and therapeutic applications. The impact and significance of immunophenotyping in the assessment of haematological neoplasms are evident.

Flow cytometric analysis of lymphocyte subset kinetics in Bali cattle experimentally infected with Jembrana disease virus.

Jembrana disease virus (JDV) is an unusual bovine lentivirus that causes an acute and sometimes fatal disease after a short incubation period in Bali cattle (Bos javanicus). The pathological changes occur primarily in lymphoid tissues, which feature proliferating lymphoblastoid-like cells predominantly throughout parafollicular (T-cell) areas, and atrophy of follicles (B-cell) areas. Five Bali cattle were experimentally infected with JDV and all developed typical clinical signs of Jembrana disease characterised by a transient febrile response, enlargement of superficial lymph nodes and a significant leukopenia. Flow cytometric analysis of PBMC during the acute (febrile) disease phase showed that the reduced number of lymphocytes was due to a significant decrease in both the proportion and absolute numbers of CD4(+) T cells, but not CD8(+) T-cells or CD21(+) B-cells. At the end of the febrile phase, total numbers of both CD8(+) T-cells and CD21(+) B-cells increased significantly, while CD4(+) T-cell numbers remained below normal values, resulting in a significantly reduced CD4(+):CD8(+) ratio. We speculate that the persistent depletion of CD4(+) T cells following JDV infection, through lack of CD4(+) T cell help to B cells, may explain the lack of production of JDV-specific antibodies for several weeks after recovery despite an increase in CD21(+) B cell numbers. Further, our previous data showing that IgG(+) plasma cells are targets for JDV infection, correlated with our current data demonstrating an increase in CD8(+) T cell numbers, supports the suggestion that anti-viral cytotoxic T cell or other cell-mediated immune responses may be critical in the recovery process, although this remains to be formally demonstrated for JDV.

Relationship between inflammatory cytokines and self-report measures of training overload.

It has been purported that inflammatory cytokines may be responsible for the aetiology of overtraining. The aim of the present study was to investigate the relationship between self-reported measures of overtraining and inflammatory cytokines. Eight elite male rowers were monitored in their natural training environment for 8 weeks prior to the 2007 Rowing World Championships. During this period of intense endurance training, self-report measures of overtraining and inflammatory cytokines (Interleukin (IL)-1beta, IL-6, IL-8, IL-10, IL-12p70, and Tumor Necrosis Factor (TNF)-alpha) were assessed fortnightly. Consistent with previous findings, proinflammatory cytokines IL-1beta and TNF-alpha were significantly associated (p