Timothy R. McGuire

Reduction in mortality associated with statin therapy in patients with severe sepsis.

Study Objective. To evaluate the effect of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors (statins) on mortality in patients with severe sepsis.

Changes in human bone marrow fat content associated with changes in hematopoietic stem cell numbers and cytokine levels with aging

Hematological deficiencies increase with aging, including anemias, reduced responses to hematopoietic stress and myelodysplasias. This investigation tested the hypothesis that increased bone marrow (BM) fat content in humans with age was associated with decreased numbers of side population (SP) hematopoietic stem cells, and this decrease correlated with changes in cytokine levels. BM was obtained from the femoral head and trochanteric region of the femur removed at surgery for total hip replacement (N = 100 subjects). In addition, BM from cadavers (N = 36), with no evidence of hip disease, was evaluated for fat content. Whole trabecular marrow samples were ground in a sterile mortar and pestle, and cellularity and lipid content determined. Marrow cells were stained with Hoechst dye and SP profiles were acquired. Plasma levels of insulin‐like growth factor (IGF)‐1, stromal‐derived factor (SDF)‐1 and interleukin (IL)‐6 were measured using ELISA. Fat content in the BM of human subjects and cadavers increased with age. The numbers of SP stem cells in BM as well as plasma IGF‐1 and SDF‐1 levels decreased in correlation with increased BM fat. IL‐6 had no relationship to changes in marrow fat. These data suggest that increased BM fat may be associated with a decreased number of SP stem cells and IGF‐1 and SDF‐1 levels with aging. These data further raise a more general question as to the role of adipose cells in the regulation of tissue stem cells.

An in vitro model for endothelial permeability: Assessment of monolayer integrity

SummaryAn essential component of anyin vitro model for endothelial permeability is a confluent cell monolayer. The model reported here utilizes primary human umbilical vein endothelial cells (HUVEC) cultured on recently developed polyethylene terephthalate micropore membranes. Using a modification of the Wright-Giemsa stain, confluent HUVEC monolayers grown on micropore membranes were routinely assessed using light microscopy. Determination of confluence using this method was confirmed by scanning electron microscopy. Transendothelial electrical resistance of HUVEC monolayers averaged 27.9±11.4 Ω · cm2, 10 to 21% higher than literature values. Studies characterizing the permeability of the endothelial cell monolayer to3H-inulin demonstrated a linear relationship between the luminal concentration of3H-inulin and its flux across HUVEC monolayers. The slope of the flux versus concentration plot, which represents endothelial clearance of3H-inulin, was 2.01±0.076 × 10−4 ml/min (r2=.9957). The permeability coefficient for the HUVEC monolayer-micropore membrane barrier was 3.17±0.427×10−6 cm/s with a calculated permeability coefficient of the HUVEC monolayer alone of 4.07±0.617×10−6 cm/s. The HUVEC monolayer reduced the permeability of the micropore membrane alone to3H-inulin (1.43±0.445×10−5 cm/s) by 78%. Evans blue dye-labeled bovine serum albumin could not be detected on the abluminal side without disruption of the HUVEC monolayer. These results demonstrate a model for endothelial permeability that can be extensively assessed for monolayer integrity by direct visualization, transendothelial electrical resistance, and the permeability of indicator macromolecules.

Human blood and marrow side population stem cell and Stro-1 positive bone marrow stromal cell numbers decline with age, with an increase in quality of surviving stem cells: Correlation with cytokines

Hematological deficiencies increase with aging leading to anemias, reduced hematopoietic stress responses and myelodysplasias. This study tested the hypothesis that side population hematopoietic stem cells (SP-HSC) would decrease with aging, correlating with IGF-1 and IL-6 levels and increases in bone marrow fat. Marrow was obtained from the femoral head and trochanteric region of the femur at surgery for total hip replacement (N=100). Whole trabecular marrow samples were ground in a sterile mortar and pestle and cellularity and fat content determined. Marrow and blood mononuclear cells were stained with Hoechst dye and the SP-HSC profiles acquired. Marrow stromal cells (MSC) were enumerated flow cytometrically employing the Stro-1 antibody, and clonally in the colony forming unit fibroblast (CFU-F) assay. Plasma levels of IGF-1 (ng/ml) and IL-6 (pg/ml) were measured by ELISA. SP-HSC in blood and bone marrow decreased with age but the quality of the surviving stem cells increased. MSC decreased non-significantly. IGF-1 levels (mean=30.7, SEM=2) decreased and IL-6 levels (mean=4.4, SEM=1) increased with age as did marrow fat (mean=1.2mmfat/g, SEM=0.04). There were no significant correlations between cytokine levels or fat and SP-HSC numbers. Stem cells appear to be progressively lost with aging and only the highest quality stem cells survive.

Inflammation Associated With Obesity: Relationship With Blood and Bone Marrow Endothelial Cells

The purpose of this study was to assess the inflammatory nature of obesity and its effect on blood and bone marrow endothelial cell populations. Obese patients (BMI ≥30) had significantly higher concentrations of the inflammatory marker C‐reactive protein (CRP) (P = 0.03) and lower concentrations of the anti‐inflammatory cytokine interleukin‐10 (IL‐10) (P = 0.05). This cytokine profile is consistent with obesity being an inflammatory condition and is further supported by the significant correlation between total white blood cell count and BMI (r = 0.15; P = 0.035). High BMI was associated with significantly lower numbers of early endothelial cells (CD45−/CD34+) in the bone marrow (r = −0.20; P = 0.0068). There was also a significant inverse correlation between BMI and a more mature endothelial cell phenotype (CD45−/31+) in the blood (r = −0.17; P = 0.02). In addition, there was a significant correlation between BMI‐ and endothelial‐related cells of hematopoietic origin (CD133+/VEGFR‐2+) in the bone marrow (r = −0.26; P = 0.0007). Patients with higher plasma IL‐10 and insulin‐like growth factor (IGF) concentrations had higher numbers of endothelial phenotypes in the bone marrow suggesting a protective effect of these anti‐inflammatory cytokines. In conclusion, this work confirms the inflammatory nature of obesity and is the first to report that obesity is associated with reduced endothelial cell numbers in the bone marrow of humans. These effects of obesity may be a potential mechanism for impaired tissue repair in obese patients.

Assay for etoposide in human serum using solid-phase extraction and high-performance liquid chromatography with fluorescence detection

An HPLC assay for etoposide in human serum was developed. Serum, spiked with podophyllotoxin (internal standard), was treated with sodium dodecyl sulphate prior to solid phase extraction. Analysis was performed on a 300x3.9 mm Bondclone 10 C18 column coupled with a fluorometric detector (lambda(ex) 230 nm, lambda(em) 330 nm). The retention times for etoposide and podophyllotoxin were 14 and 28 min respectively. The range of assay was 0.5 to 20 microg/ml with a detection limit of 0.2 microg/ml. This assay is suitable for use in clinical studies with etoposide.

Colorimetric determination of hydroxyurea in human serum using high-performance liquid chromatography

A high-performance liquid chromatography assay for hydroxyurea in human serum was developed based on a commercial colorimetric assay kit for urea (Sigma Diagnostics). Serum (0.5 ml), spiked with methylurea as an internal standard, was treated with 70% perchloric acid. Supernatant (0.2 ml) was combined with 0.7 ml of BUN acid reagent and 0.6 ml of BUN color reagent. The resulting colored reactant (100 microl) was analyzed on a 300 x 3.9 mm Bondclone 10 C18 column coupled with a UV-Vis detector, at 449 nm. The mobile phase was 13% acetonitrile in water. Retention times of colored derivatives of hydroxyurea and methylurea were 6.5 and 12.2 min, respectively. The log-log calibration curve was linear from 0.0065 to 1.31 mM. Average accuracy was 99.9+/-4.0% and the intra- and inter-day error of assay did not exceed 11%.

Lymphodepleting effects and safety of pentostatin for nonmyeloablative allogeneic stem-cell transplantation

Nonmyeloablative allogeneic stem-cell transplantation (alloNST) is the focus of investigations searching for less-toxic transplantation regimens. We report studies on the kinetics of lymphodepletion and safety of pentostatin (PT) conditioning in alloNST. Patients with hematologic malignancy received mobilized blood from human leukocyte antigen-matched related (n=4) or unrelated (n=8) donors. PT 4 mg/m2 was administered on days -21, -20, and -19 and 200 cGy of total-body irradiation was administered on day -1, followed by cyclosporine A and mycophenolate mofetil. Mononuclear cell adenosine deaminase after PT was inhibited 84%. The absolute CD3+ cells decreased significantly by day -7 (49%) and CD19+ cells declined 92% by day -1. CD4+ cells were depressed more than CD8+ cells. Neutrophils and monocytes were minimally affected by PT. Median posttransplant peripheral blood chimerism on day 70 showed 95% donor leukocytes and 82.5% donor CD3 lymphocytes. PT demonstrated lymphodepleting effects and promising safety, supporting alloNST as early as 7 days after initiation of PT.

Clinically important interaction between statin drugs and Clostridium difficile toxin

Clostridium difficile associated disease (CDAD), a common type of antibiotic associated diarrhea, is increasing in frequency and affecting patients outside of traditional populations. At one time CDAD exclusively occurred in hospitalized patients or frail elderly patients receiving antibiotic therapy. It is now occurring more commonly in younger patients who are relatively healthy and may not be receiving antibiotics. Co-factors that might explain this increase incidence and changing demographic are of great public health interest. Recent investigations have identified gastric acid suppression, particularly via proton pump inhibitors, as a risk factor for the development of CDAD by mechanisms which are not entirely clear. C. difficile toxin comes as two major forms that are closely related, toxin A and toxin B and both are able to produce CDAD. These toxins have a glucosyltransferase domain that glucosylates actived Rho, a small GTP binding protein involved in multiple cellular signaling pathways. Glucosylation inactivates Rho and modifies cell cycle, cytoskeletal and inflammatory pathways. The lipid lowering drugs called statins also inhibit Rho but at an earlier step in the Rho pathway. Statins inhibit the isoprenylation of Rho and therefore inhibits membrane anchoring a key step in Rho signaling. We propose that statins potentiate C. difficile toxin effects on colonic epithelium which leads to an increased risk of CDAD. We present preliminary data from a retrospective cohort which demonstrated an increased rate of CDAD in patients receiving statins compared to non-statin controls. The weight of the evidence leads to our hypothesis that statins interact with C difficile toxin A and B causing an increase in the rate and severity of CDAD.

High-dose cyclophosphamide in multiple sclerosis patients undergoing autologous stem cell transplantation

High-dose cyclophosphamide (CTX) is commonly used in preparation for autologous and allogeneic stem cell transplantation. CTX is a pro-drug, which undergoes complex oxidative metabolism with the metabolites being eliminated both renally and hepatically. In the following study, we evaluated the pharmacokinetic characteristics of high-dose CTX in three patients undergoing autologous stem cell transplantation for multiple sclerosis. The plasma concentration-time profiles for CTX and its hydroxy-metabolite were similar in multiple sclerosis patients to those reported in cancer patients undergoing stem cell transplantation. There was an increase in drug clearance after the second CTX dose indicating that the drug induced its own metabolism consistent with reports in other populations receiving high-dose CTX. One of the three patients cleared the drug slowly but this was not associated with greater toxicity. The patient with the slow clearance value and therefore highest drug exposure had stable disability scores at 2 years posttransplant compared with baseline values taken prior to transplantation. In conclusion, in this small case series, there was no indication that CTX metabolism was different than that in other populations undergoing transplantation.