Milica Kovacevic-Filipovic

Interleukin-6 (IL-6) and low O2 concentration (1%) synergize to improve the maintenance of hematopoietic stem cells (pre-CFC)

Low O2 concentration (1%) favors the self‐renewal of hematopoietic stem cells and inhibits committed progenitors (CFC). Since IL‐6 influences both stem cells and committed progenitors at 20% O2, we studied its effects in cultures at 1% O2. The pre‐CFC activity in Lin− population of mouse bone marrow was analyzed following 10 days of serum‐free culture in medium (LC1) supplemented with IL‐3 with and without IL‐6, at 20 and 1% O2 and phenotypic differentiation and proliferative history monitored. The IL‐6 receptor expression and initiation of VEGF‐A synthesis were also investigated. At 20% O2, the effects of IL‐6 on pre‐CFC were negligible but effects on CFC were apparent; conversely, at 1% O2, the IL‐6 enhances activity of pre‐CFC but not of CFC. Unlike at 20% O2, at 1% O2 a subpopulation of cells remained Lin− in spite of extensive proliferation. However, the absolute number of Lin− cells, did not correlate with pre‐CFC activity. A relative increase in VEGF transcripts at 1% O2 in presence of IL‐3 alone was enhanced by the addition of IL‐6. IL‐6 enhanced pre‐CFC activity at 1% O2 and this was correlated to the induction of VEGF. These data reinforce the concept that physiologically low oxygenation of bone marrow is a regulator of stem cell maintenance. Since the 20% O2 does not exist in tissues in vivo, further studies in vitro at lower O2 concentrations should revise our knowledge relating to cytokine effects on stem and progenitor cells. J. Cell. Physiol. 212: 68–75, 2007.

CD34+ cells obtained from “good mobilizers” are more activated and exhibit lower ex vivo expansion efficiency than their counterparts from “poor mobilizers”

BACKGROUND: The classification of patients into “good” or “poor” mobilizers is based on CD34+ cell count in their peripheral blood (PB) after granulocyte–colony‐stimulating factor (G‐CSF) injection. We hypothesized that, apart from their mobilization from marrow to the blood, the response to G‐CSF of CD34+ cells also includes activation of proliferation, metabolic activity, and proliferative capacity.

Serum amyloid A isoforms in serum and milk from cows with Staphylococcus aureus subclinical mastitis

Serum amyloid A proteins (SAA) are very sensitive acute phase proteins, displaying multiple isoforms in plasma and different body fluids. They are currently under investigation as biomarkers of diseases. The aim of the present study was to compare the concentration and isoform expression of SAA in serum and milk of cows with bacteriologically negative milk (control group) and naturally occurring Staphylococcus aureus (S. aureus) subclinical mastitis (subclinical mastitis group). Somatic cell count (SCC) and bacteriological analyses were performed to establish the control and subclinical mastitis group. SAA concentration was evaluated using a commercial ELISA kit, while expression of different isoforms (serum A-SAA and milk M-SAA3 isoforms) was visualized by denaturing isoelectrical focusing and immunoblotting. The SAA concentrations in sera and milk of cows in the subclinical mastitis group were three and 100 times higher than in those from the control group of cows, respectively. Cows in the subclinical mastitis group had more acidic SAA isoforms in serum with the most prominent one at pI 5.5. This isoform was not detected in sera from the control group. Milk samples in the subclinical mastitis group contained abundant highly alkaline M-SAA3 isoforms and most of the serum isoforms, except for that at pI 5.5. In the subclinical mastitis group SAA isoforms with equivalent pI as serum isoforms accounted for 20% of the total SAA concentration in milk. There were significant differences in the concentrations and isoform patterns of SAA in serum and milk between the control and subclinical mastitis groups of cows. Also, we demonstrated that serum SAA isoforms were not transferred to milk proportion to their plasma content.

Low-oxygen and high-carbon-dioxide atmosphere improves the conservation of hematopoietic progenitors in hypothermia

BACKGROUND: During short‐term storage of hematopoietic cells (HCs) at 4°C a substantial decline in number and in functional capacity of progenitors occurs after 3 days. We hypothesized that physiologic O2 and CO2 concentrations of hematopoietic tissue microenvironment (approx. 3% O2 and approx. 6% CO2) could improve cell viability and functionality during storage at 4°C.

Hypoxia/Hypercapnia‐Induced Adaptation Maintains Functional Capacity of Cord Blood Stem and Progenitor Cells at 4°C

We analyzed the effect of exposure to hypoxic/hypercapnic (HH) gas mixture (5% O2/9% CO2) on the maintenance of functional cord blood CD34+ hematopoietic stem and progenitor cells in severe hypothermia (4°C) employing the physiological and proteomic approaches. Ten‐day exposure to HH maintained the Day 0 (D‐0) level of hematopoietic stem cells as detected in vivo on the basis of hematopoietic repopulation of immunodeficient mice—short‐term scid repopulating cells (SRC). Conversely, in the atmospheric air (20% O2/0.05% CO2), usual condition used for cell storage at 4°C, stem cell activity was significantly decreased. Also, HH doubled the survival of CD34+ cells and committed progenitors (CFCs) with respect to the atmospheric air (60% vs. 30%, respectively). Improved cell maintenance in HH was associated with higher proportion of aldehyde dehydrogenase (ALDH) positive cells. Cell‐protective effects are associated with an improved maintenance of the plasma and mitochondrial membrane potential and with a conversion to the glycolytic energetic state. We also showed that HH decreased apoptosis, despite a sustained ROS production and a drop of ATP amount per viable cell. The proteomic study revealed that the global protein content was better preserved in HH. This analysis identified: (i) proteins sensitive or insensitive to hypothermia irrespective of the gas phase, and (ii) proteins related to the HH cell‐protective effect. Among them are some protein families known to be implicated in the prolonged survival of hibernating animals in hypothermia. These findings suggest a way to optimize short‐term cell conservation without freezing. J. Cell. Physiol. 229: 2153–2165, 2014.

Acute phase protein response in cows with staphylococcus aureus subclinical mastitis

Inflammation at a local and systemic level is a complex process that involves the synthesis of acute phase proteins (APPs) with multiple functions in the regulation of the inflammatory process itself. The aim of this work was to define local and systemic APPs response induced by natural Staph. aureus subclinical infection of the mammary gland in dairy cows with a different number of quarters involved. Midlactation dairy cows (n=30) were devided into three groups. First group were cows with bacteriologically negative milk samples (BN group), second group were cows with one quarter infected with Staph. aureus (SaQ1) and third group were cows with two quarters infected (SaQ2). Milk samples were analyzed for inflammation indicators: serum amyloid A (SAA) and somatic cell count (SCC). Serum samples where analyzed for SAA, haptoglobin (Hp), ceruloplasmin (Cp) and albumin concentration. Also, complete blood count (CBC) was done. SCC and SAA increased in quarter milk samples, being lowest in the BN group and highest in the SaQ2 group. In serum samples, SAA, Hp, Cp and albumin concentrations were significantly higher only in the SaQ2 group comparing with BN group. The leukocyte number, as well as hemoglobin concentration were in the physiological range in all three groups of cows. These results confirm that the magnitude of tissue injury has an impact on APPs concentration. They also demonstrate that cows having Staph. aureus sublinical infections of two mammary quarters have a more pronounced systemic APP response than cows with only one quarter involved.

Ovarian surface epithelium of hypothyroid newborn and neonatal rats: From proliferating cell nuclear antigen and caspase-3 perspectives

Introduction. The ovarian surface epithelium (OSE) undergoes intensive regeneration and remodelling after each ovulation during the whole reproductive period. This process increases the risk of one of the most common ovarian tumors in women and the female dog. Considering the fact that maternal hypothyroidism highly impacts cell proliferation and cell death during folliculogenesis in the early neonatal period, we aimed to analyze its effect on OSE morphology and dynamics. Materials and Methods. The study was performed on newborn (24-h-old) and neonatal (4-day-old) female rats, a randomized trial between the control and hypothyroid groups, born under controlled circumstances and hypothyroid mothers, respectively. Their ovaries were analyzed histologically and processed to determine the OSE cell height as an average value of four measurement points. Also, the immunopositivity of the proliferating cell nuclear antigen (PCNA) and caspase-3 were assessed semiquantitatively. Results and Conclusions. No major structural differences of OSE were found between groups within the given ages except for a slight increment of OSE cell height and incompleteness of apical cell membrane with cytoplasmic projections in hypothyroid animals. PCNA immunopositivity of the OSE cells was higher in ovaries of hypothyroid animals of both ages in comparison to the controls. Moreover, only scarce OSE cells were caspase-3 positive in both groups and ages, with no difference in immunopositivity. Our study confirms the impact of hypothyroidism in the early postnatal period on morphology and proliferation rate of OSE cells, with no effect on caspase-3 dependent cell removal, which may serve as a premise for future investigation of potential carcinogenesis, in terms of prevention and treatment of ovarian cancer.

Peripartal leukogram in cows with and without retained placenta.

The aim of this study was to investigate whether prepartal leukogram in cows with retained placenta could indicate the presence of subclinical systemic inflammatory response before the onset of disease. After calving, sixteen highly pregnant Holstein cows, aged 3 to 9 years, without clinical signs of the disease prior to calving were divided into two groups: the first group (n=9) were animals without retained placenta, or any visible inflammation after birth; the second group (n=7) were cows with retained placenta. Blood was sampled three times before parturition, at intervals of one week, and once 24 hours after birth. The number of total leukocytes, segmented and non segmented neutrophilic granulocytes (NG), lymphocytes and monocytes were determined by standard laboratory techniques. The results have shown that in the group of cows with retained placenta the number of mature neutrophils was slightly elevated in the third, second and last week before calving, and equal number of non segmented neutrophils in regard to the group with no retention. The results have also shown that, in both groups of cows, 24 hours after calving, the number of total leukocytes and the number of segmented neutrophils decreased, but the number of the non segmented neutrophils increased. Based on this, we can conclude that cows with retained placenta had no systemic inflammatory response during three weeks prepartal period, but 24 hours after calving, systemic inflammatory response was documented in all the cows. Moreover, the intensity of inflammatory response in cows with retained placenta was not more pronounced in comparison to cows without retained placenta. [Projekat Ministarstva nauke Republike Srbije, br. 175061]