scholarly journals Effect of Coadministration of Vancomycin and BMP-2 on Cocultured Staphylococcus aureus and W-20-17 Mouse Bone Marrow Stromal CellsIn Vitro

2012 ◽  
Vol 56 (7) ◽  
pp. 3776-3784 ◽  
Author(s):  
A. H. Nguyen ◽  
S. Kim ◽  
W. J. Maloney ◽  
J. C. Wenke ◽  
Y. Yang
Keyword(s):  
Staphylococcus Aureus ◽  
Bone Marrow ◽  
Growth Factors ◽  
Bone Cell ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mouse Bone Marrow ◽  
Content Type ◽  
Proliferation And Differentiation ◽  
Dual Administration

ABSTRACTIn this study, we aimed to establish anin vitrobacterium/bone cell coculture model system and to use this model for dose dependence studies of dual administration of antibiotics and growth factorsin vitro. We examined the effect of single or dual administration of the antibiotic vancomycin (VAN) at 0 to 16 μg/ml and bone morphogenetic protein-2 (BMP-2) at 0 or 100 ng/ml on both methicillin-sensitiveStaphylococcus aureusand mouse bone marrow stromal cells (W-20-17) under both mono- and coculture conditions. Cell metabolic activity, Live/Dead staining, double-stranded DNA (dsDNA) amounts, and alkaline phosphatase activity were measured to assess cell viability, proliferation, and differentiation. An interleukin-6 (IL-6) enzyme-linked immunosorbent assay (ELISA) kit was used to test the bone cell inflammation response in the presence of bacteria. Our results suggest that, when delivered together in coculture, VAN and BMP-2 maintain their primary functions as an antibiotic and a growth factor, respectively. Most interestingly, this dual-delivery type of approach has shown itself to be effective at lower concentrations of VAN than those required for an approach relying strictly on the antibiotic. It may be that BMP-2 enhances cell proliferation and differentiation before the cells become infected. In coculture, a dosage of VAN higher than that used for treatment in monoculture may be necessary to effectively inhibit growth ofStaphylococcus aureus. This could mean that the coculture environment may be limiting the efficacy of VAN, possibly by way of bacterial invasion of the bone cells. This report of a coculture study demonstrates a potential beneficial effect of the coadministration of antibiotics and growth factors compared to treatment with antibiotic alone.

scholarly journals In vitro myelopoiesis stimulated by rapid medium exchange and supplementation with hematopoietic growth factors

Blood ◽  
1991 ◽  
Vol 78 (12) ◽  
pp. 3155-3161 ◽  
Author(s):  
RM Schwartz ◽  
SG Emerson ◽  
MF Clarke ◽  
BO Palsson
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Culture Medium ◽  
Culture Conditions ◽  
Hematopoietic Growth Factors ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Medium Exchange ◽  
Proliferation And Differentiation

Abstract We studied the effect of the combination of rapid culture medium exchange with the addition of the human hematopoietic growth factors interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (Epo) on the proliferation and differentiation of human long-term bone marrow cultures (LTBMCs). Individually and in combinations, IL-3, GM-CSF, and Epo were added to the culture medium of LTBMCs that were maintained with 50% medium volume exchange per day. The combination of IL-3 + GM-CSF + Epo generated the most prolific cultures with an order of magnitude increase in nonadherent cell production from weeks 2 through 8 in culture as compared with unsupplemented controls. Under these conditions, the cultures produced as many cells as were inoculated every 2 weeks and led to a greater than 2.5-fold expansion in terms of the number of nonadherent cells produced over a 6- to 8-week period. Furthermore, the LTBMCs produced nonadherent colony-forming unit-GM (CFU-GM) for more than 20 weeks. The rapid medium exchange combined with the addition of human hematopoietic CSFs significantly enhances the proliferation and differentiation of LTBMCs. These results indicate that addition of combinations of hematopoietic CSFs, together with a rapid medium exchange rate, can provide culture conditions that are suitable for the expansion of the progenitor cell pool and perhaps for the increased survival of hematopoietic stem cells in culture. Although these culture conditions still fall short of full reconstitution of functional human bone marrow, they provide an improved approach to hematopoietic cell culture that may permit the expansion and manipulation of progenitor cells in vitro.

In Vitro Expansion of Human Hematopoietic Cells with Delayed but Sustained Multi-Lineage Repopulating Activity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1270-1270
Author(s):  
Alice M.S. Cheung ◽  
Stefan Wohrer ◽  
Paul H. Miller ◽  
Suzan Imren ◽  
Shabnam Rostamirad ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Cord Blood ◽  
Stromal Cell ◽  
Human Cells ◽  
Mouse Bone Marrow ◽  
Self Renewal ◽  
Post Transplant ◽  
Nsg Mice

Abstract Abstract 1270 In vivo expansion of hematopoietic stem cells (HSCs) involves local interactions with stimuli generated from non-hematopoietic niche environments, but the full spectrum of molecular mechanisms responsible have remained elusive. Initial experiments in mice showed that highly purified HSCs from adult mouse bone marrow are consistently expanded 3–5-fold with full maintenance of their long term (≥6 months), serially transplantable, multi-lineage repopulating ability when cultured for 7 days in serum-free UG26 stromal-cell conditioned medium (CM) supplemented with 100 ng/ml mouse Steel Factor (SF) and 20 ng/ml mouse IL-11. To explore the potential effects of this CM on HSCs in human cord blood, we conducted an initial experiment in which CD34+CD38− cells were cultured for 7 days in UG26 CM supplemented with 100 ng/ml human Flt3-ligand, 100 ng/ml human SF, and 20 ng/ml each of human IL-3, IL-6 and G-CSF. The results of limiting dilution transplants of the cultured cells in intravenously injected NSG mice showed retention of input numbers of cells with equivalent robust 6-month lympho-myeloid repopulating activity. To characterize the initial target cells and determine whether their proliferative responses might be predictive of their self-renewal behavior, we set up single cell cultures with the CD49f+ subset of CD34+CD38−CD45RA−CD90+Rho−cells with the 5 growth factors in the presence or absence of CM. Under both conditions, 7/13 and 4/13 input cells, respectively, died within the first 72 hours in culture. The subsequent rate of proliferation of the survivors was similar with all completing a first division after 96 hours and a second division 24–48 hours later. By day 8, clones of variable sizes were noted (6–1100 and 4–200 cells/clone, respectively). Clones generated under the same conditions were pooled and injected intravenously into 2 NSG mice each. We then looked for the presence of human cells in the mice by analysis of serial bone marrow aspirates starting 3 weeks post-transplant. Human cells were detected in only one of each of the 2 pairs of mice and, interestingly, in both cases, no evidence of human cells was detectable until 3 months post-transplant. In the positive mouse injected with cells generated in the absence of CM, this repopulation was transient, peaking at ∼0.1% of the mouse bone marrow compartment at 4 months post-transplant and undetectable a month later. In contrast, in the positive recipient of cells from the cultures that contained CM, both lymphoid and myeloid human cells reached much higher levels (together making up ∼20% of the mouse bone marrow compartment) which were maintained for another 3 months when the mouse was sacrificed. Transplants of cells obtained at this time from the marrow gave positive repopulation of secondary mice. In a subsequent experiment, in which similar cultures were initiated with CD34+ cord blood cells, evidence of a late continuing effect of the CM was obtained with a net absolute expansion of CD34+CD45RA−CD90+ cell numbers during the interval between 12 and 21 days in vitro. These findings highlight the important potential of as yet unidentified secreted stromal cell factors to stabilize the stem cell state in HSCs stimulated to proliferate in vitro by growth factors that favor their self-renewal. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Abnormal in vitro proliferation and differentiation of T colony-forming cells in patients with lymphadenopathy syndrome

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1063-1069 ◽  
Author(s):  
Y Lunardi-Iskandar ◽  
V Georgoulias ◽  
W Rozenbaum ◽  
D Klatzmann ◽  
MC Coll ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Lymph Node ◽  
Growth Factors ◽  
Peripheral Blood ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Interleukin 2 ◽  
Proliferation And Differentiation

Abstract Patients with acquired immunodeficiency syndrome (AIDS) present impaired colony growth and in vitro differentiation capacity of peripheral blood and bone marrow T colony-forming cells (T-CFC). We show that peripheral blood, bone marrow, and lymph node T-CFC from patients with persistent lymphadenopathy syndrome (LAS), a syndrome that can precede AIDS, displayed similar abnormalities. Indeed, peripheral blood T-CFC generated a low number of colonies in seven out of 12 patients, and almost no colonies were obtained from bone marrow cells of all patients. The simultaneous study of T-CFC from peripheral blood and lymph node mononuclear cells seems to provide a reliable indicator for the risk of developing AIDS. The six patients who developed AIDS displayed extremely low numbers of peripheral blood T- CFC (13 +/- 17 colonies per 5 X 10(4) cells), and in two of them, no colonies could be obtained from lymph node T-CFC. The remaining patients who had not developed AIDS displayed a higher number of peripheral blood T-CFC (141 +/- 113 per 5 X 10(4) cells) and lymph node T-CFC, which, in addition, preserved their clonogenic capacity. In some patients, peripheral blood and lymph node, but not bone marrow, T-CFC were capable of generating colonies in the absence of added growth factors or mitogens, whereas in others, colony formation was obtained with purified interleukin 2 (IL 2) alone. Both spontaneous and IL 2- induced colony formation was abrogated by a monoclonal antibody against the IL 2 receptor. Taken together, these findings suggest that at least some T-CFC expressed IL 2 receptors. Colonies generated either in the presence or in the absence of added growth factors were composed of T4+, T6+, and T8+ cells, indicating impaired in vitro T-CFC differentiation. These findings indicate that a dramatic quantitative and qualitative impairment of the proliferation and differentiation of peripheral blood and lymph node T-CFC precedes the clinical evolution from LAS to AIDS.

scholarly journals Mucosal Immunization with a Staphylococcus aureus IsdA-Cholera Toxin A2/B Chimera Induces Antigen-Specific Th2-Type Responses in Mice

2011 ◽  
Vol 18 (9) ◽  
pp. 1543-1551 ◽  
Author(s):  
Britni M. Arlian ◽  
Juliette K. Tinker
Keyword(s):  
Staphylococcus Aureus ◽  
Epithelial Cells ◽  
Cholera Toxin ◽  
Cellular Proliferation ◽  
Mucosal Vaccine ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mucosal Immunization ◽  
Content Type ◽  
Human Nasal Epithelial Cells

ABSTRACTStaphylococcus aureusis a leading cause of opportunistic infection worldwide and a significant public health threat. The iron-regulated surface determinant A (IsdA) adhesin is essential forS. aureuscolonization on human nasal epithelial cells and plays an important role in iron acquisition and resistance to human skin defenses. Here we investigated the murine immune response to intranasal administration of a cholera toxin A2/B (CTA2/B) chimera containing IsdA. Plasmids were constructed to express the IsdA-CTA2/B chimera and control proteins inEscherichia coli. Proper construction of the chimera was verified by SDS-PAGE, Western blotting, GM1 enzyme-linked immunosorbent assay (ELISA), and confocal microscopy. Groups of female BALB/c mice were mock immunized or immunized with IsdA-CTA2/B, IsdA mixed with CTA2/B, or IsdA alone, followed by one booster immunization at 10 days postpriming. Analysis of serum IgG and nasal, intestinal, and vaginal IgA suggested that mucosal immunization with IsdA-CTA2/B induces significant IsdA-specific humoral immunity. Functionalin vitroassays revealed that immune serum significantly blocks the adherence ofS. aureusto human epithelial cells. Splenocytes from mice immunized with IsdA-CTA2/B showed specific cellular proliferation and production of interleukin-4 (IL-4) afterin vitrostimulation. Immunization with IsdA-CTA2/B drove isotype switching to IgG1, indicative of a Th2-type response. Our results suggest that the immunogenicity of theS. aureusIsdA-CTA2/B chimera merits further investigation as a potential mucosal vaccine candidate.

scholarly journals In VivoEfficacy of Ceftaroline Fosamil in a Methicillin-Resistant Panton-Valentine Leukocidin-Producing Staphylococcus aureus Rabbit Pneumonia Model

2014 ◽  
Vol 58 (4) ◽  
pp. 1855-1861 ◽  
Author(s):  
Delphine Croisier-Bertin ◽  
Davy Hayez ◽  
Sonia Da Silva ◽  
Delphine Labrousse ◽  
Donald Biek ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Pulmonary Tissue ◽  
Ceftaroline Fosamil ◽  
Methicillin Resistant ◽  
Content Type ◽  
Panton Valentine Leukocidin ◽  
Valentine Leukocidin

ABSTRACTCeftaroline, the active metabolite of the prodrug ceftaroline fosamil, is a cephalosporin with broad-spectrumin vitroactivity against Gram-positive organisms, including methicillin-resistantStaphylococcus aureus(MRSA) and multidrug-resistantStreptococcus pneumoniae(MDRSP), and common Gram-negative pathogens. This study investigated thein vivoactivity of ceftaroline fosamil compared with clindamycin, linezolid, and vancomycin in a severe pneumonia model due to MRSA-producing Panton-Valentine leukocidin (PVL). A USA300 PVL-positive clone was used to induce pneumonia in rabbits. Infected rabbits were randomly assigned to no treatment or simulated human-equivalent dosing with ceftaroline fosamil, clindamycin, linezolid, or vancomycin. Residual bacterial concentrations in the lungs and spleen were assessed after 48 h of treatment. PVL expression was measured using a specific enzyme-linked immunosorbent assay (ELISA). Ceftaroline, clindamycin, and linezolid considerably reduced mortality rates compared with the control, whereas vancomycin did not. Pulmonary and splenic bacterial titers and PVL concentrations were greatly reduced by ceftaroline, clindamycin, and linezolid. Ceftaroline, clindamycin, and linezolid were associated with reduced pulmonary tissue damage based on significantly lower macroscopic scores. Ceftaroline fosamil, clindamycin, and, to a lesser extent, linezolid were efficient in reducing bacterial titers in both the lungs and spleen and decreasing macroscopic scores and PVL production compared with the control.

Surface Electric Fields of Apatite Electret Promote Osteoblastic Responses

2012 ◽  
Vol 529-530 ◽  
pp. 357-360 ◽  
Author(s):  
Miho Nakamura ◽  
Akiko Nagai ◽  
Kimihiro Yamashita
Keyword(s):  
Bone Marrow ◽  
Quantitative Analysis ◽  
Electric Fields ◽  
Mtt Assay ◽  
Precursor Cells ◽  
Mouse Bone Marrow ◽  
Proliferation And Differentiation

The osteoblast behaviors on the biomaterial substrates are recognized to play a fundamental role in osteoconduction process. The purpose of this study was to evaluate the in vitro behaviors of osteoblasts cultured on electrically polarized hydroxyapatite (HA), having the enhanced osteobonding abilities. Osteoblasts derived from mouse bone marrow were seeded onto the polarized HA and investigated the proliferation and differentiation. The polarization had effects on the proliferation of osteoblast precursor cells based on the MTT assay. The acceleration was emerged as the early achievement to the confluence on the N-HA and P-HA. The quantitative analysis of the results of ALP and AR-S staining, the charges induced on the HA surface accelerated the differentiation from the osteoblast precursor cells to mature osteoblasts.

scholarly journals Characterization of Alpha-ToxinhlaGene Variants, Alpha-Toxin Expression Levels, and Levels of Antibody to Alpha-Toxin in Hemodialysis and Postsurgical Patients with Staphylococcus aureus Bacteremia

2014 ◽  
Vol 53 (1) ◽  
pp. 227-236 ◽  
Author(s):  
Batu K. Sharma-Kuinkel ◽  
Yuling Wu ◽  
David E. Tabor ◽  
Hoyin Mok ◽  
Bret R. Sellman ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Monoclonal Antibody ◽  
Clinical Outcome ◽  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Enzyme Linked Immunosorbent Assay ◽  
Alpha Toxin ◽  
Content Type ◽  
Antibody Levels

Alpha-toxin is a majorStaphylococcus aureusvirulence factor. This study evaluated potential relationships betweenin vitroalpha-toxin expression ofS. aureusbloodstream isolates, anti-alpha-toxin antibody in serum of patients withS. aureusbacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwentspatyping andhlasequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encodedhla(197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%).In vitroalpha-toxin levels were inversely associated with survival (cure, 2.19 μg/ml, versus failure, 1.09 μg/ml;P< 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95;P< 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27;P< 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressingS. aureusisolates (P< 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis ofhlarevealed 12 distincthlagenotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinicalS. aureusisolates. Higherin vitroalpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producingS. aureusexhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study.

scholarly journals Abnormal in vitro proliferation and differentiation of T colony-forming cells in patients with lymphadenopathy syndrome

Blood ◽  
1986 ◽  
Vol 67 (4) ◽  
pp. 1063-1069
Author(s):  
Y Lunardi-Iskandar ◽  
V Georgoulias ◽  
W Rozenbaum ◽  
D Klatzmann ◽  
MC Coll ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Lymph Node ◽  
Growth Factors ◽  
Peripheral Blood ◽  
Colony Formation ◽  
Mononuclear Cells ◽  
Bone Marrow Cells ◽  
Interleukin 2 ◽  
Proliferation And Differentiation

Patients with acquired immunodeficiency syndrome (AIDS) present impaired colony growth and in vitro differentiation capacity of peripheral blood and bone marrow T colony-forming cells (T-CFC). We show that peripheral blood, bone marrow, and lymph node T-CFC from patients with persistent lymphadenopathy syndrome (LAS), a syndrome that can precede AIDS, displayed similar abnormalities. Indeed, peripheral blood T-CFC generated a low number of colonies in seven out of 12 patients, and almost no colonies were obtained from bone marrow cells of all patients. The simultaneous study of T-CFC from peripheral blood and lymph node mononuclear cells seems to provide a reliable indicator for the risk of developing AIDS. The six patients who developed AIDS displayed extremely low numbers of peripheral blood T- CFC (13 +/- 17 colonies per 5 X 10(4) cells), and in two of them, no colonies could be obtained from lymph node T-CFC. The remaining patients who had not developed AIDS displayed a higher number of peripheral blood T-CFC (141 +/- 113 per 5 X 10(4) cells) and lymph node T-CFC, which, in addition, preserved their clonogenic capacity. In some patients, peripheral blood and lymph node, but not bone marrow, T-CFC were capable of generating colonies in the absence of added growth factors or mitogens, whereas in others, colony formation was obtained with purified interleukin 2 (IL 2) alone. Both spontaneous and IL 2- induced colony formation was abrogated by a monoclonal antibody against the IL 2 receptor. Taken together, these findings suggest that at least some T-CFC expressed IL 2 receptors. Colonies generated either in the presence or in the absence of added growth factors were composed of T4+, T6+, and T8+ cells, indicating impaired in vitro T-CFC differentiation. These findings indicate that a dramatic quantitative and qualitative impairment of the proliferation and differentiation of peripheral blood and lymph node T-CFC precedes the clinical evolution from LAS to AIDS.

scholarly journals In vitro myelopoiesis stimulated by rapid medium exchange and supplementation with hematopoietic growth factors

Blood ◽  
1991 ◽  
Vol 78 (12) ◽  
pp. 3155-3161 ◽  
Author(s):  
RM Schwartz ◽  
SG Emerson ◽  
MF Clarke ◽  
BO Palsson
Keyword(s):  
Bone Marrow ◽  
Growth Factors ◽  
Culture Medium ◽  
Culture Conditions ◽  
Hematopoietic Growth Factors ◽  
Hematopoietic Stem ◽  
Gm Csf ◽  
Medium Exchange ◽  
Proliferation And Differentiation

We studied the effect of the combination of rapid culture medium exchange with the addition of the human hematopoietic growth factors interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and erythropoietin (Epo) on the proliferation and differentiation of human long-term bone marrow cultures (LTBMCs). Individually and in combinations, IL-3, GM-CSF, and Epo were added to the culture medium of LTBMCs that were maintained with 50% medium volume exchange per day. The combination of IL-3 + GM-CSF + Epo generated the most prolific cultures with an order of magnitude increase in nonadherent cell production from weeks 2 through 8 in culture as compared with unsupplemented controls. Under these conditions, the cultures produced as many cells as were inoculated every 2 weeks and led to a greater than 2.5-fold expansion in terms of the number of nonadherent cells produced over a 6- to 8-week period. Furthermore, the LTBMCs produced nonadherent colony-forming unit-GM (CFU-GM) for more than 20 weeks. The rapid medium exchange combined with the addition of human hematopoietic CSFs significantly enhances the proliferation and differentiation of LTBMCs. These results indicate that addition of combinations of hematopoietic CSFs, together with a rapid medium exchange rate, can provide culture conditions that are suitable for the expansion of the progenitor cell pool and perhaps for the increased survival of hematopoietic stem cells in culture. Although these culture conditions still fall short of full reconstitution of functional human bone marrow, they provide an improved approach to hematopoietic cell culture that may permit the expansion and manipulation of progenitor cells in vitro.

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