Immunomodulatory activity in tumor-bearing mice treated with Withania somnifera extract

We investigated some actions of Withania somnifera on the growth and differentiation of hematopoietic precursors [granulocyte/macrophage colony cell formation (CFU-GM)] of normal animals and EAT bearers, which were treated with different doses (20, 50, or 100 mg/kg/day). We also evaluated the presence of colony stimulatory factors in the animal's serum, as well as its survival. Furthermore, we analyzed lymphocyte proliferation, IFN-ɤ, and TNF-α concentrations in treated bearing mice. Our results demonstrated Withania somnifera effectiveness on hematopoietic precursors growth and differentiation in marrow and spleen TAE-bearing mice. As it was already expected, EAT produced myelosuppression and increased CFU-GM spleen number concomitantly. The treatment of EAT-bearing animals with W.S. (20, 50, and 100 mg/Kg) produced a dose-dependent increase in myelopoiesis, an increase in a lifetime, and a reduction in spleen colony number. All this happened parallel to survival. As to lymphocyte proliferation, they were also dose-dependent in treated bearing animals. Concerning IFN-γ levels, we observed a significant reduction in non-treated bearing mice. Levels of TNF-α of treated bearing mice significantly increased when compared to the non-treated bearing group. These results are encouraging since they favor the use of W.S. extract in therapeutic combinations with other chemotherapeutic agents to reduce myelotoxicity and supplement the tumoricidal efficacy of this plant.

PHENOS: a high-throughput and flexible tool for microorganism growth phenotyping on solid media

ABSTRACTBACKGROUNDMicrobial arrays, with a large number of different strains on a single plate printed with robotic precision, underpin an increasing number of genetic and genomic approaches. These include Synthetic Genetic Array analysis, high-throughput Quantitative Trait Loci (QTL) analysis and 2-hybrid techniques. Measuring the growth of individual colonies within these arrays is an essential part of many of these techniques but is useful for any work with arrays. Measurement is typically done using intermittent imagery fed into complex image analysis software, which is not especially accurate and is challenging to use effectively. We have developed a simple and fast alternative technique that uses a pinning robot and a commonplace microplate reader to continuously measure the thickness of colonies growing on solid agar, complemented by a technique for normalizing the amount of cells initially printed to each spot of the array in the first place. We have developed software to automate the process of combining multiple sets of readings, subtracting agar absorbance, and visualizing colony thickness changes in a number of informative ways.RESULTSThe “PHENOS” pipeline (PHENotyping On Solid media), optimized for Saccharomyces yeasts, produces highly reproducible growth curves and is particularly sensitive to low-level growth. We have empirically determined a formula to estimate colony cell count from an absorbance measurement, and shown this to be comparable with estimates from measurements in liquid. We have also validated the technique by reproducing the results of an earlier QTL study done with conventional liquid phenotyping, and found PHENOS to be considerably more sensitive.CONCLUSIONS“PHENOS” is a cost effective and reliable high-throughput technique for quantifying growth of yeast arrays, and is likely to be equally very useful for a range of other types of microbial arrays. A detailed guide to the pipeline and software is provided with the installation files at https://github.com/gact/phenos.

Yeast Colonies: A Model for Studies of Aging, Environmental Adaptation, and Longevity

When growing on solid surfaces, yeast, like other microorganisms, develops organized multicellular populations (colonies and biofilms) that are composed of differentiated cells with specialized functions. Life within these populations is a prevalent form of microbial existence in natural settings that provides the cells with capabilities to effectively defend against environmental attacks as well as efficiently adapt and survive long periods of starvation and other stresses. Under such circumstances, the fate of an individual yeast cell is subordinated to the profit of the whole population. In the past decade, yeast colonies, with their complicated structure and high complexity that are also developed under laboratory conditions, have become an excellent model for studies of various basic cellular processes such as cell interaction, signaling, and differentiation. In this paper, we summarize current knowledge on the processes related to chronological aging, adaptation, and longevity of a colony cell population and of its differentiated cell constituents. These processes contribute to the colony ability to survive long periods of starvation and mostly differ from the survival strategies of individual yeast cells.

Augmentation of neutrophilic granulocyte progenitors in the bone marrow of mice with tumor-induced neutrophilia: cytochemical study of in vitro colonies

Transplantation of CE mammary carcinoma into mice has been shown to produce marked neutrophilia. Previous studies in vivo indicated a significant increase in marrow neutrophil production in these mice, but regulatory mechanisms of this neutrophilia have not been well understood. In order to obtain information about neutrophil production mechanisms at the progenitor cell level, the profile of marrow granulocyte-macrophage progenitors in mice with neutrophilia induced by this tumor was quantitatively analyzed by cytochemical staining of in vitro colonies to distinguish colonies of neutrophils (N-colony), macrophages (M-colony), and mixed cells (NM-colony). Cell cycle kinetics of progenitors were studied by in vivo administration of cytocidal drugs. The absolute number of N-colonies in a femur increased significantly and reached three times normal three to four weeks after tumor implantation. The number of NM-colonies also increased significantly by the fourth week, but the number of M-colonies was unchanged. The number of N-colonies in a femur related directly to the degree of neutrophilia. The increased number of N-colonies from the marrow of tumor-bearing mice was not attributed to a different time course of colony growth nor to a different sensitivity to CSA; instead, a significantly larger fraction of neutrophilic progenitors from the tumor-bearing mice were in active cell cycle than were those of normal mice. The day 14 tumor-bearing mouse serum demonstrated N-colony stimulating activity while the sera of normal mice and day 7 tumor- bearing mice were inhibitory for in vitro colony growth. These studies demonstrated an increase in the numbers and turnover rate of marrow neutrophilic progenitors in CE tumor-induced neutrophilia, suggesting that this tumor stimulates proliferation of these progenitors in vivo.

Augmentation of neutrophilic granulocyte progenitors in the bone marrow of mice with tumor-induced neutrophilia: cytochemical study of in vitro colonies

Transplantation of CE mammary carcinoma into mice has been shown to produce marked neutrophilia. Previous studies in vivo indicated a significant increase in marrow neutrophil production in these mice, but regulatory mechanisms of this neutrophilia have not been well understood. In order to obtain information about neutrophil production mechanisms at the progenitor cell level, the profile of marrow granulocyte-macrophage progenitors in mice with neutrophilia induced by this tumor was quantitatively analyzed by cytochemical staining of in vitro colonies to distinguish colonies of neutrophils (N-colony), macrophages (M-colony), and mixed cells (NM-colony). Cell cycle kinetics of progenitors were studied by in vivo administration of cytocidal drugs. The absolute number of N-colonies in a femur increased significantly and reached three times normal three to four weeks after tumor implantation. The number of NM-colonies also increased significantly by the fourth week, but the number of M-colonies was unchanged. The number of N-colonies in a femur related directly to the degree of neutrophilia. The increased number of N-colonies from the marrow of tumor-bearing mice was not attributed to a different time course of colony growth nor to a different sensitivity to CSA; instead, a significantly larger fraction of neutrophilic progenitors from the tumor-bearing mice were in active cell cycle than were those of normal mice. The day 14 tumor-bearing mouse serum demonstrated N-colony stimulating activity while the sera of normal mice and day 7 tumor- bearing mice were inhibitory for in vitro colony growth. These studies demonstrated an increase in the numbers and turnover rate of marrow neutrophilic progenitors in CE tumor-induced neutrophilia, suggesting that this tumor stimulates proliferation of these progenitors in vivo.

A model of intramedullary hematopoietic microenvironments based on stereologic study of the distribution of endocloned marrow colonies

Hematopoietic colonies were studied in the marrow of alternate fraction- irradiated mice by light microscopic stereology to investigate the microenvironmental organization of marrow. Separate analyses of the relative colony cell density of undifferentiated, granulocytic, erythrocytic, and macrophage colonies in four marrow zones were carried out at 3, 4, and 5 days postirradiation (PI) for all colonies, all periarterial colonies, and all non-periarterial colonies. The results demonstrate a differential colony cell distribution that does not appear to be due to a preferential distribution of certain colony types around arteries. Undifferentiated colony cells showed a consistent predilection for endosteal and periarterial regions, with the majority of colony cells occurring along bone. Erythrocytic colony cells proliferated initially in intermediate and central marrow zones and along arteries. Granulocytic colony cells occurred in all areas at 3 days PI, but increased in density along bone thereafter. Macrophage colony cells occurred in all zones at 4 days PI, but at 5 days were concentrated in subosteal and central regions. Macrophage colonies also occurred periarterially. To explain these findings and the organization of normal bone marrow, we present a detailed model of the microenvironmental organization of intramedullary hematopoiesis. This model portrays the stroma as engendering distinct microenvironments for stem cell replication, stem cell commitment, and early progenitor cell proliferation.

A model of intramedullary hematopoietic microenvironments based on stereologic study of the distribution of endocloned marrow colonies

Hematopoietic colonies were studied in the marrow of alternate fraction- irradiated mice by light microscopic stereology to investigate the microenvironmental organization of marrow. Separate analyses of the relative colony cell density of undifferentiated, granulocytic, erythrocytic, and macrophage colonies in four marrow zones were carried out at 3, 4, and 5 days postirradiation (PI) for all colonies, all periarterial colonies, and all non-periarterial colonies. The results demonstrate a differential colony cell distribution that does not appear to be due to a preferential distribution of certain colony types around arteries. Undifferentiated colony cells showed a consistent predilection for endosteal and periarterial regions, with the majority of colony cells occurring along bone. Erythrocytic colony cells proliferated initially in intermediate and central marrow zones and along arteries. Granulocytic colony cells occurred in all areas at 3 days PI, but increased in density along bone thereafter. Macrophage colony cells occurred in all zones at 4 days PI, but at 5 days were concentrated in subosteal and central regions. Macrophage colonies also occurred periarterially. To explain these findings and the organization of normal bone marrow, we present a detailed model of the microenvironmental organization of intramedullary hematopoiesis. This model portrays the stroma as engendering distinct microenvironments for stem cell replication, stem cell commitment, and early progenitor cell proliferation.