Cerebral Vasocontraction Induced by Thrombin-Stimulated Washed Human Platelets - A New In Vitro Model to Study Cerebral Vasospasm

We compared the pharmacodynamic activities of levofloxacin versus vancomycin, with or without rifampin, in an in vitro model with infected platelet-fibrin clots simulating vegetations. Infected platelet-fibrin clots were prepared with human cryoprecipitate, human platelets, calcium, thrombin, and approximately 10(9) CFU of organisms (MSSA 1199 and MRSA 494) per g and then were suspended via monofilament line into the in vitro model containing Mueller-Hinton growth medium. Antibiotics were administered by bolus injection into the model to simulate human pharmacokinetics; the regimens simulated included levofloxacin at dosages of 800 mg every 24 h (q24h) and 400 mg q12h, vancomycin at 1 g q12h, and rifampin at 600 mg q24h. Each model was run in duplicate over a 72-h period. Infected platelet-fibrin clots were removed in duplicate from each model, weighed, homogenized, serially diluted with sterile 0.9% saline, and plated on tryptic soy agar plates and plates containing antibiotics at 3, 6, and 12 times the MIC to evaluate the emergence of resistance. Time-kill curves were constructed by plotting the inoculum size versus time. Residual inoculum at 72 h was used to compare regimens. All levofloxacin regimens were significantly better than vancomycin monotherapy against both isolates (P < 0.002). Against MSSA 1199, levofloxacin q24h was significantly better than all other regimens, including levofloxacin q12h (P < 0.002); however, no difference between the levofloxacin monotherapy and combination therapy (with rifampin) regimens against MRSA 494 was seen. Killing activity for levofloxacin appeared to correlate better with the peak/MIC ratio than with the area under the curve/MIC ratio. The addition of rifampin significantly enhanced the activity of vancomycin but had little effect upon the activity of levofloxacin. For MRSA 494, vancomycin plus rifampin resulted in the greatest killing (P < 0.05). Development of resistance was not detected with any regimen. Levofloxacin may be a useful therapeutic alternative in the treatment of staphylococcal endocarditis, and further study with animal models of endocarditis or clinical trials are warranted.

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Beneficial Influence of Platelets on Antibiotic Efficacy in an In Vitro Model of Staphylococcus aureus-Induced Endocarditis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.7.2551-2557.2004 ◽

2004 ◽

Vol 48 (7) ◽

pp. 2551-2557 ◽

Cited By ~ 19

Author(s):

Renee-Claude Mercier ◽

Robert M. Dietz ◽

Jory L. Mazzola ◽

Arnold S. Bayer ◽

Michael R. Yeaman

Keyword(s):

Staphylococcus Aureus ◽

In Vitro Model ◽

Antimicrobial Effect ◽

Human Platelets ◽

Inhibitory Effects ◽

Activated Platelets ◽

Vitro Model ◽

Antibiotic Efficacy ◽

Chamber Fluid

ABSTRACT Platelets contribute to antimicrobial host defense against infective endocarditis (IE) by releasing platelet microbicidal proteins (PMPs). We investigated the influence of thrombin-stimulated human platelets on the evolution of simulated IE in the presence and absence of vancomycin or nafcillin. Staphylococcus aureus strains differing in intrinsic susceptibility to PMPs or antibiotics were studied: ISP479C (thrombin-induced PMP-1 [tPMP-1] susceptible; nafcillin and vancomycin susceptible), ISP479R (tPMP-1 resistant; nafcillin and vancomycin susceptible), and GISA-NJ (tPMP-1 intermediate-susceptible; vancomycin intermediate-susceptible). Platelets were introduced and thrombin activated within the in vitro IE model 30 min prior to inoculation with S. aureus. At 0 to 24 h postinoculation, bacterial densities in chamber fluid and simulated endocardial vegetations (SEVs) were quantified and compared among groups. Activated platelets alone, or in combination with antibiotics, inhibited the proliferation of ISP479C in chamber fluid or SEVs over the initial 4-h period (P < 0.05 versus controls). Moreover, nafcillin-containing regimens exerted inhibitory effects beyond 4 h against ISP479C in both model phases. By comparison, activated platelets inhibited GISA-NJ proliferation in SEVs but not in chamber fluid. The combination of platelets plus nafcillin or vancomycin significantly inhibited proliferation of the GISA-NJ strain in SEVs compared to the effect of platelets or antibiotics alone (P < 0.05). In contrast, platelets did not significantly alter the antistaphylococcal efficacies of nafcillin or vancomycin against ISP479R. These data support our hypothesis that a beneficial antimicrobial effect may result from the interaction among platelets, PMPs, and anti-infective agents against antibiotic-susceptible or -resistant staphylococci that exhibit a tPMP-1-susceptible or -intermediate-susceptible phenotype.

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In-vitro model for the ultrastructural study of the formation of thrombi in human platelets

Blood Coagulation & Fibrinolysis ◽

10.1097/01.mbc.0000214712.96244.47 ◽

2006 ◽

Vol 17 (2) ◽

pp. 161-164 ◽

Cited By ~ 4

Author(s):

Doris Cerecedo ◽

Sirenia González ◽

Mónica Mondragón ◽

Elba Reyes ◽

Ricardo Mondragón

Keyword(s):

Ultrastructural Study ◽

In Vitro Model ◽

Human Platelets ◽

Vitro Model

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Cytosolic Ca2+ Oscillations in Human Cerebrovascular Endothelial Cells after Subarachnoid Hemorrhage

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/jcbfm.2008.87 ◽

2008 ◽

Vol 29 (1) ◽

pp. 57-65 ◽

Cited By ~ 17

Author(s):

Wolfram Scharbrodt ◽

Yaser Abdallah ◽

Sascha A Kasseckert ◽

Dragan Gligorievski ◽

Hans M Piper ◽

...

Keyword(s):

Endothelial Cells ◽

Subarachnoid Hemorrhage ◽

Cerebral Vasospasm ◽

In Vitro Model ◽

Molecular Mechanisms ◽

Vascular Inflammation ◽

Cell Contraction ◽

Vitro Model ◽

Cerebrovascular Endothelial Cells

Molecular mechanisms of cerebral vasospasm after subarachnoid hemorrhage (SAH) include specific modes of cell signaling like activation of nuclear factor (NF)-kB and vascular cell adhesion molecules (VCAM)-1 expression. The study's hypothesis is that cisternal cerebral spinal fluid (CSF) from patients after SAH may cause Ca2+ oscillations which induce these modes of vascular inflammation in an in vitro model of human cerebral endothelial cells (HCECs). HCECs were incubated with cisternal CSF from 10 SAH patients with confirmed cerebral vasospasm. The CSF was collected on days 5 and 6 after hemorrhage. Cytosolic Ca2+ concentrations and cell contraction as an indicator of endothelial barrier function were examined by fura-2 microflurometry. Activation of NF-κB and VCAM-1 expression were measured by immunocytochemistry. Incubation of HCEC with SAH-CSF provoked cytosolic Ca2+ oscillations (0.31 ± 0.09 per min), cell contraction, NF-κB activation, and VCAM-1 expression, whereas exposure to native CSF had no significant effect. When endoplasmic reticulum (ER) Ca2+-ATPase and ER inositol trisphosphate (IP3)-sensitive Ca2+ channels were blocked by thapsigargin or xestospongin, the frequency of the Ca2+ oscillations was reduced significantly. In analogy to the reduction of Ca2+ oscillation frequency, the blockers impaired HCEC contraction, NF-κB activation, and VCAM-1 expression. Cisternal SAH-CSF induces cytosolic Ca2+ oscillations in HCEC that results in cellular constriction, NF-κB activation, and VCAM-1 expression. The Ca2+ oscillations depend on the function of ER Ca2+-ATPase and IP3-sensitive Ca2+ channels.

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An in vitro model for investigation of vitamin A effects on wound healing

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000692 ◽

2021 ◽

pp. 1-6

Author(s):

Hoda Keshmiri Neghab ◽

Mohammad Hasan Soheilifar ◽

Gholamreza Esmaeeli Djavid

Keyword(s):

Wound Healing ◽

Endothelial Cell ◽

Vitamin A ◽

In Vitro Model ◽

Cellular Proliferation ◽

Endothelial Cell Migration ◽

Normal Fibroblast ◽

Anti Inflammatory ◽

Vitro Model

Abstract. Wound healing consists of a series of highly orderly overlapping processes characterized by hemostasis, inflammation, proliferation, and remodeling. Prolongation or interruption in each phase can lead to delayed wound healing or a non-healing chronic wound. Vitamin A is a crucial nutrient that is most beneficial for the health of the skin. The present study was undertaken to determine the effect of vitamin A on regeneration, angiogenesis, and inflammation characteristics in an in vitro model system during wound healing. For this purpose, mouse skin normal fibroblast (L929), human umbilical vein endothelial cell (HUVEC), and monocyte/macrophage-like cell line (RAW 264.7) were considered to evaluate proliferation, angiogenesis, and anti-inﬂammatory responses, respectively. Vitamin A (0.1–5 μM) increased cellular proliferation of L929 and HUVEC (p < 0.05). Similarly, it stimulated angiogenesis by promoting endothelial cell migration up to approximately 4 fold and interestingly tube formation up to 8.5 fold (p < 0.01). Furthermore, vitamin A treatment was shown to decrease the level of nitric oxide production in a dose-dependent effect (p < 0.05), exhibiting the anti-inflammatory property of vitamin A in accelerating wound healing. These results may reveal the therapeutic potential of vitamin A in diabetic wound healing by stimulating regeneration, angiogenesis, and anti-inﬂammation responses.

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