The Effect of Valproic Acid on Mesenchymal Pluripotent Cell Proliferation and Differentiation in Extracellular Matrices

Valproic acid (2-n-propylpentanoic acid, VPA) is a widely used antiepileptic and anticonvulsant drug. Previous studies have reported that VPA effects osteogenesis in vivo and in vitro, yet it remains unclear whether VPA promotes cell differentiation of osteoblasts derived from mesenchymal cells. The purpose of this study was to clarify the effect of VPA on undifferentiated pluripotent mesenchymal cell proliferation and differentiation into osteoblasts while analyzing the impact of the absence or presence of extracellular matrices (ECMs). Mouse mesenchymal cells were cultured on non-coated plastic, type I collagen-coated, and fibronectin-coated plates in the absence or presence of VPA. A cell proliferation assay was performed in which modified formazan dye content was analyzed and proliferation nuclear antigen (PCNA)-positive cells were counted at various concentrations of VPA. A high concentration of VPA did not clearly alter cell morphology, but large numbers of stress fibers were observed in these cells and the cell proliferation ratio was decreased with positive PCNA counts. In the presence of matrices, the cell proliferation ratio decreased at low VPA concentrations compared with the ratio obtained in the absence of these ECMs. On the other hand, VPA promoted osteoblastic differentiation in the presence of type I collagen. These findings indicate that for undifferentiated mesenchymal cells, VPA promotes a decrease in the cell proliferation rate in the presence of ECMs and promotes osteoblastic differentiation, both of which could provide insight into additional mechanisms of osteoblastic cell differentiation caused by VPA

Trypanosoma congolense: High erythropoietic potential in infected yearling cattle during the acute phase of the anemia

N'Dama (Bos taurus) cattle are known to tolerate trypanosome infections, developing less severe anemia and lower parasitemia, compared to Boran (Bos indicus) cattle. Young calves were also reported to be more resistant to trypanosomiasis than adult cattle. To expore the basis for this resistance, the erythropoietic response was evaluated in four naive yearling N'Dama calves and four age-matched Boran calves which developed anemia over a 140-day primary infection with Trypanosoma congolense clone IL 13E3. Similar levels of parasites were detected in the two breeds until 42 days postinfection (dpi). During the same period, a rapid and greater colony-forming units-erythroid response in the bone marrow of yearling Boran calves, compared with N'Dama calves, may explain the unusual absence of breed differences in mean packed cell volumes (PCV). However, this early erythropoietic response was transient and did not result in any rise in PCV in the Boran calves. Moreover, dispite the persistent severe anemia, the erythroid response subsided from 70 dpi onward. In contrast, in the N'Dama calves, following the erythroid response, the mean PCV was gradually compensated from 56 dpi onward and reached 30 percent by 126 dpi. This period of PCV recovery coincided with low and intermittent parasitemia and a return of the erythroid progenitor levels to near preinfection values. Elevated levels of erythroid progeniotrs in the N'Dama calves, occurring 1 week after trypanocidal treatment, returned the PCV to preinfection values. These results suggest that the age of the Bos indicus cattle has important impact on the early bone marrow response in primary T. congolense infection and confirmed previous suggestions of a high erythropoietic potential in trypanosome-infection and confirmed previous suggestions of a high erythropoietic potential in trypanosome-infected young calves

Rise in erythropoietin concentrations in experimental Trypanosoma congolense infection of calves

A bioassay was used to measure erythropoietin (EPO) concentrations in calves with haemorrhagic anaemia due to blood loss and in calves with anaemia due to Trypanosoma congolense infection. The bioactivity of EPO was measured in the assay by its stimulatory effect on 125I-deoxyuridine incorporation in spleen cells from phenylhydrazine-treated mice. Erythropoietin concentrations in blood-volume-depleted calves were elevated 6 h after blood loss, maximal (1225 mU/ml) at 33 h and below detection limits at 72 h. Reticulocytes (0.05 ± 0.1%) appeared in blood by 72 h, peaked at 120 h and disappeared from the circulation by 7 days after bleeding. The packed cell volume (PCV) started increasing at 120 h and reached near pre-bleeding values by 14 days. In T. congolense-infected calves, parasites were first detected in the peripheral blood 12 days post-infection (dpi). Parasitaemia peaked (5 x 105 trypanosomes/ml of blood) at 15-18 dpi and, thereafter, several waves of parasitaemia were observed, but the peaks gradually diminished. Undiluted plasma from T. congolense-infected calves suppressed 125I-deoxyuridine incorporation into spleen cells from 13 dpi onwards. The suppressive effect of plasma was partly negated by five-fold dilution, which made possible the detection of increased EPO concentrations during the acute and chronic stages of the anaemia. The highest EPO peaks, reaching 2300 mU/ml in one calf, were detected during the chronic stage of the infection. At 15-39 dpi, there was a transient bone-marrow erythropoietic response characterized by an increase in mean corpuscular volume and a decrease in mean corpuscular haemoglobin concentration but with few reticulocytes (0.4%). However, from 76 dpi onwards, this response waned despite low PCV and elevated EPO concentrations. These results suggest that there is an ineffective erythroid response in the face of elevated EPO concentrations during bovine trypanosomiasis. The negative effect of plasma and serum from trypanosome-infected calves on the in-vitro bioactivity of EPO suggests the presence of inhibitory factors

Comparative bone marrow responses during Trypanosoma congolense infection in N'Dama and Boran cattle

Anaemia is a major cause of morbidity and mortality in bovine trypanosomiasis and has served as the main parameter to monitor the severity of the disease. In fact, trypanosome-infected cattle experience intermittent episodes of pancytopaenia including anaemia, leucopaenia and trombocytopaenia. Considering the multiple blood cell lineages affected by the disease, it was hypothesized that a defect in the bone marrow, site of origin of those cells, might be a cause. Study of the bone marrow response was intitiated in the laboratory using a Trypanosoma congolense rechallenge infection in five Boran cattle. The cattle developed a non-responsive, normocytic, normocytic, normochromic anaemia during the first ten weeks of the infection, characterized by low levles of erythroid progenitors in the bone marrow. Subsequently, between week 10 and 14, a responsive macrocytic hypochromic anaemia followed, characterized by peaks of CFU-E, slight increase of BFU-E levels and appearance of BFU-E in the peripheral blood. During trypanosome infection the trypanotolerant N'Dama cattle usually develop a moderate anaemia associated with lower parasitaemia when compared to the susceptible Zebu cattle such as the Boran. The present study was undertaken to determine if the ability of the N'Dama cattle to better control anaemia resides in a superior bone marrow response

Trypanosoma congolense: Comparative effects of a primary infection on bone marrow progenitor cells from N'Dama and Boran cattle

Using in vitro clonogenic assays, the changes in haemopoietic progenitor cell levels were compared in the bone marrow of three adult trypanotolerant N'Dama cattle and three age-matched trypanosusceptible Boran cattle over 17 weeks (119 days) of a primary Trypanosoma congolense (clone IL 1180) infection. As the infection progressed, a clear tendency of the parasitaemia to decrease was seen in the N'Damas, while it remained high throughout the infection in the Borans. The decline in the colony-forming units-granulocyte macrophage (CFU-GM) between 7 and 42 days positinfection (dpi) corresponded withthe decreased numbers of neutrophils and monocytes in the blood observed in both breeds. Thereafter, a further significant drop in the CFU-GM levels was observed in the Borans which may partially explain the continued decrease in the numbers of neutrophils and monocytes in blood. In contrast, a significant peak of CFU-GM above preinfection levels was observed in the N'Damas on 49 dpi, which could partially explain the subsequent recovery of the numbers of neutrophils and monocytes in blood. When compared to the N'Damas, the Borans had a more dramatic drop in the packed cell volume (PCV) from 25 dpi onwards, resulting in significantly lower PCFrom 46-49 dpi on wards, the mean PCV stabilised at significantly lower levels in the Borans than in the N'Damas. The mean corpuscular volume (MCV) levels increased in both breeds, but at a much faster rate in the Borans. The clonogenic assays demonstrated an erythropoietic response, characterised by peaks above preinfecton levels of both the early and late erythroid progenitor cells (respectively, burstforming units-erythroid, BFU-E, and colony-forming units-erythroid, CFU-E), occurring between 35 and 70 dpi in both breeds of cattle. However, despite a more severe anaemia in the Borans, the magnitude of their erythroid response was similar to that of the N'Damas, suggesting that the response of the Borans was insufficient to compensate for the greater degree of anaemia. Moreover, the mean PCV did not improve in the Borans, indicating the ineffectiveness of their erythropoietic response. An increased rate of erythrocyte destruction and/or a defective differentiation and maturation of erythroid precursors have also been shown to be partially responsible for this persistent anaemia. From 98 dpi onwards, despite the persistent low PCV, the MCV decreased to preinfection levels and low CFU-E numbers were observed in the Borans. Over the same period, in the N'Damas the N'Damas the mean PCV progressively increased to reach 25 percent, which fell within the low normal range for cattle. This may partially explain the return of the BFU-E to preinfection levels and the slight decrease in the CFU-E in the N'Damas. During the acute phase of a T. congolense infection, both the granulomonocytic and erythroid progenitors were increased more effectively in the N'Dama cattle than in the Boran cattle, when compared to the degree of cytopaenia in blood; this might correlate with the superior ability of the N'Damas to maintain gigher numbers of granulocytes, monocytes, and erythrocytes in blood

Kinetics of hematopoietic progenitor cells during a Trypanosoma congolense rechallenge infection in Boran cattle

In a preliminary study, using clonogenic assays, the in vitro kinetics of committed haemopoietic progenitors were monitored during a Trypanosoma congolense rechallenge infection in five trypanosusceptible Boran cattle. Early in the infection (week 2), in the absence of any detectable parasitaemia, a drop in the number of nucleated marrow cells was recorded. This was accompanied by a marked but transient decrease in the levels of the colony-forming units-erythroid (CFU-E) followed by a partial recovery by weeks 3-4 after infection. The burst-forming units-erythroid (BFU-E) and the colony-forming units-granulocyte macrophage (CFU-GM) also significantly decreased between weeks 2 and 4. After a transient rise at weeks 3-5 postinfection, the CFU-GM steadily declined and remained below preinfection levels throughout the infection. The BFU-E remained below preinfection levels until the end of the experiment. The drop in nucleated marrow cells associated with the decreased numbers of CFU-E, BFU-E and CFU-GM was suggestive of a defect at the pluripotential stem cell level early in the infection (week 2). The erythrocyte indices, i.e. mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC), were unchanged until week 10 postinfection. Two animals became severely anaemic; one was euthanised at week 8 and one treated at week 9. The three remaining animals developed chronic anaemia with mean packed cell volume (PCV) fluctuating around 18%-19% between weeks 11 and 14. Low parasitaemia levels were recorded during that period. A CFU-E peak above preinfection levels was noted at week 12 and BFU-E appeared in the peripheral blood culture of two animals between weeks 11 and 14. A progressive rise in MCV associated with a gradual decrease in MCHC also characterised that period. A return to near preinfection levels was recorded for the numbers of all three progenitors three weeks after trypanocidal treatment followed by a full recovery five months after treatment. Although ineffective haemopoiesis has been suggested to contribute to the anaemia of bovine trypanosomiasis, this is the first demonstration of a negative effect on erythroid development in cultures of bone marrow of trypanosome-infected cattle