Impedance Flow Cytometry for Selection of Pollen Traits Under High Temperature Stress in Pepper

High temperature stress is a major limiting factor for pepper productivity, which will continue to be a problem under climate change scenarios. Developing heat tolerant cultivars is critical for sustained pepper production, especially in tropical and subtropical regions. In fruiting crops, like pepper, reproductive tissues, especially pollen, are the most sensitive to high temperature stress. Typically, pollen viability and germination are assessed through staining and microscopy, which is tedious and potentially inaccurate. To increase efficiency in assessing pollen traits of pepper, the use of impedance flow cytometry (IFC) has been proposed. We conducted three independent experiments to determine the most effective methodology to use IFC for evaluating pollen traits for heat tolerance in pepper. Seven floral developmental stages were evaluated, and stages 3, 4, and 5 were found to best combine high pollen concentration and activity. Flowers in development stages 3, 4, or 5 were then heat treated at 41, 44, 47, 50, and 55 °C or not heat treated (control). The critical temperature to assess heat tolerance using IFC was found to be 50 °C, with a reduction in pollen activity and concentration occurring at temperatures greater than 47 °C. Twenty-one entries of pepper were then accessed for pollen traits using the staining and IFC methods over 2 months, April (cooler) and June (hotter). Growing environment was found to be the greatest contributor to variability for nearly all pollen traits assessed, with performance during June nearly always being lower. PBC 507 and PBC 831 were identified as being new sources of heat tolerance, based on using IFC for assessing pollen. Pollen viability determined by staining and pollen activity determined using IFC were significantly positively correlated, indicating that IFC is an efficient and accurate method to assess pollen traits in pepper. This work provides a basis for further research in this area and supports more efficient breeding of heat-tolerant cultivars.

Case Report: Long-Term Survival of a Dog With Chronic Lymphocytic Leukemia Treated With Chlorambucil, Prednisolone, and Imatinib

A 7-year-old castrated male Poodle dog presented with chronic progressive lymphocytosis. Hematologic and peripheral blood smear findings included remarkable lymphocytosis with well-differentiated small lymphocytes. Cytology of bone marrow aspirate showed hypercellular integrity with infiltration of small mature lymphocytes, accounting for 45% of all nucleated cells. Flow cytometry of blood and marrow samples revealed neoplastic lymphocytes predominantly expressing the CD21 molecule. B-cell chronic lymphocytic leukemia (CLL) was diagnosed on an immunophenotypic analysis. Administrations of prednisolone and chlorambucil were initiated and the response was unremarkable. Therefore, additional treatment with imatinib was provided, which resolved the hematologic abnormalities associated with CLL. Flow cytometry after ~1 year of treatment showed normalization of the count of lymphocytes positive for CD21 and resolved hematologic lymphocytosis. The dog was followed-up for 2 years, and there were no severe adverse effects. This case indicates that imatinib may be a good option as an adjunctive therapy with prednisolone and chlorambucil treatment for CLL in dogs without treatment response.

Involvement of Peripheral Monocytes with IL-1β in the Pathogenesis of West Syndrome

Neuroinflammation has been implicated in the pathogenesis of West syndrome (WS). Inflammatory cytokines, including interleukin-1β(IL-1β), have been reported to be associated with epilepsy. However, the assessment of cytokine changes in humans is not always simple or deterministic. This study aimed to elucidate the immunological mechanism of WS. We examined the intracellular cytokine profiles of peripheral blood cells collected from 13 patients with WS, using flow cytometry, and measured their serum cytokine levels. These were compared with those of 10 age-matched controls. We found that the WS group had significantly higher percentages of inter IL-1β, interleukin-1 receptor antagonist (IL-1RA)-positive monocytes, and interferon gamma (IFN-γ) in their CD8+ T cells than the control group. Interestingly, the group with sequelae revealed significantly lower levels of intracellular IFN-γ and IL-6 in their CD8+ T and CD4+ T cells, respectively, than the group without sequelae. There was no correlation between the ratios of positive cells and the serum levels of a particular cytokine in the WS patients. These cytokines in the peripheral immune cells might be involved in the neuroinflammation of WS, even in the absence of infectious or immune disease. Overall, an immunological approach using flow cytometry analysis might be useful for immunological studies of epilepsy.

Direct comparison of different therapeutic cell types susceptibility to inflammatory cytokines associated with COVID-19 acute lung injury

Background Although 90% of infections with the novel coronavirus 2 (COVID-19) are mild, many patients progress to acute respiratory distress syndrome (ARDS) which carries a high risk of mortality. Given that this dysregulated immune response plays a key role in the pathology of COVID-19, several clinical trials are underway to evaluate the effect of immunomodulatory cell therapy on disease progression. However, little is known about the effect of ARDS associated pro-inflammatory mediators on transplanted stem cell function and survival, and any deleterious effects could undermine therapeutic efficacy. As such, we assessed the impact of inflammatory cytokines on the viability, and paracrine profile (extracellular vesicles) of bone marrow-derived mesenchymal stromal cells, heart-derived cells, and umbilical cord-derived mesenchymal stromal cells. Methods All cell products were manufactured and characterized to established clinical release standards by an accredited clinical cell manufacturing facility. Cytokines and Extracellular vesicles in the cell conditioned media were profiled using proteomic array and nanoparticle tracking analysis. Using a survey of the clinical literature, 6 cytotoxic cytokines implicated in the progression of COVID-19 ARDS. Flow cytometry was employed to determine receptor expression of these 6 cytokines in three cell products. Based on clinical survey and flow cytometry data, a cytokine cocktail that mimics cytokine storm seen in COVID-19 ARDS patients was designed and the impact on cytokine cocktail on viability and paracrine secretory ability of cell products were assessed using cell viability and nanoparticle tracking analysis. Results Flow cytometry revealed the presence of receptors for all cytokines but IL-6, which was subsequently excluded from further experimentation. Despite this widespread expression, exposure of each cell type to individual cytokines at doses tenfold greater than observed clinically or in combination at doses associated with severe ARDS did not alter cell viability or extracellular vesicle character/production in any of the 3 cell products. Conclusions The paracrine production and viability of the three leading cell products under clinical evaluation for the treatment of severe COVID-19 ARDS are not altered by inflammatory mediators implicated in disease progression.

Ovariectomy increases paclitaxel-induced mechanical hypersensitivity and reduces anti-inflammatory CD4+ T cells in the dorsal root ganglion of female mice

Chemotherapy is often dose limiting due to the emergence of a debilitating neuropathy. IL-10 and IL-4 are protective against peripheral neuropathy, yet the cell source is unknown. Using flow cytometry, we found that naïve females had a greater frequency of anti-inflammatory CD4+ T cells in the dorsal root ganglion (DRG) than males. In response to paclitaxel, females had reduced hypersensitivity and a greater frequency of anti-inflammatory CD4+ T cells (FoxP3, IL-10, IL-4) in the DRG than ovariectomized and male mice. These findings support a model in which estrogen promotes anti- inflammatory CD4+ T cells in female DRG to suppress peripheral neuropathy.

Comparison of FACS and PCR for Detection of BCMA-CAR-T Cells

Chimeric-antigen-receptor (CAR)-T-cell therapy is already widely used to treat patients who are relapsed or refractory to chemotherapy, antibodies, or stem-cell transplantation. Multiple myeloma still constitutes an incurable disease. CAR-T-cell therapy that targets BCMA (B-cell maturation antigen) is currently revolutionizing the treatment of those patients. To monitor and improve treatment outcomes, methods to detect CAR-T cells in human peripheral blood are highly desirable. In this study, three different detection reagents for staining BCMA-CAR-T cells by flow cytometry were compared. Moreover, a quantitative polymerase chain reaction (qPCR) to detect BCMA-CAR-T cells was established. By applying a cell-titration experiment of BCMA-CAR-T cells, both methods were compared head-to-head. In flow-cytometric analysis, the detection reagents used in this study could all detect BCMA-CAR-T cells at a similar level. The results of false-positive background staining differed as follows (standard deviation): the BCMA-detection reagent used on the control revealed a background staining of 0.04% (±0.02%), for the PE-labeled human BCMA peptide it was 0.25% (±0.06%) and for the polyclonal anti-human IgG antibody it was 7.2% (±9.2%). The ability to detect BCMA-CAR-T cells down to a concentration of 0.4% was similar for qPCR and flow cytometry. The qPCR could detect even lower concentrations (0.02–0.01%). In summary, BCMA-CAR-T-cell monitoring can be reliably performed by both flow cytometry and qPCR. In flow cytometry, reagents with low background staining should be preferred.