Biomedical subjects

Carol Miller-Graziano

Publications and source records attributed to Carol Miller-Graziano.

7 recordsLinked to original sources

PubMed2006

Cell-specific expression and pathway analyses reveal alterations in trauma-related human T cell and monocyte pathways.

Monitoring genome-wide, cell-specific responses to human disease, although challenging, holds great promise for the future of medicine. Patients with injuries severe enough to develop multiple organ dysfunction syndrome have multiple immune derangements, including T cell apoptosis and anergy combined with depressed monocyte antigen presentation. Genome-wide expression analysis of highly enriched circulating leukocyte subpopulations, combined with cell-specific pathway analyses, offers an opportunity to discover leukocyte regulatory networks in critically injured patients. Severe injury induced significant changes in T cell (5,693 genes), monocyte (2,801 genes), and total leukocyte (3,437 genes) transcriptomes, with only 911 of these genes common to all three cell populations (12%). T cell-specific pathway analyses identified increased gene expression of several inhibitory receptors (PD-1, CD152, NRP-1, and Lag3) and concomitant decreases in stimulatory receptors (CD28, CD4, and IL-2Ralpha). Functional analysis of T cells and monocytes confirmed reduced T cell proliferation and increased cell surface expression of negative signaling receptors paired with decreased monocyte costimulation ligands. Thus, genome-wide expression from highly enriched cell populations combined with knowledge-based pathway analyses leads to the identification of regulatory networks differentially expressed in injured patients. Importantly, application of cell separation, genome-wide expression, and cell-specific pathway analyses can be used to discover pathway alterations in human disease.

Gene Expression Profiling↗

PubMed2005

Simultaneous aberrations in Mphi and T cell function adversely affect trauma patients' clinical outcome: a possible faulty IL-13 feedback loop.

Possible defective trauma patients' Mphi-T-cell feedback interactions between T cell IL-13 production and IL-1beta and IL-18 Mphi secretion were assessed. Mphi produced IL-1 and IL-18 augment T cell IL-13, which in turn limits excessive macrophage activation. Immunodepressed patients' T cells (depressed proliferation to alphaCD3 + alphaCD4) had decreased IL-13 production concomitant to aberrant Mphi activities ( upward arrow mTNFalpha, downward arrow IL-10) and consequent multiple organ failure (MOF). Decreased IL-13 levels in patients' T cell and diminished Mphi supernatant augmentation of healthy controls' T cell IL-13 production appeared concomitantly, suggesting patients' aberrant monokine levels might intensify in vivo T cell dysfunction severity. Patients' Mphi supernatants, which failed to augment controls' T cell IL-13 production, had depressed IL-1beta and lower induction of IL-18 than immunocompetent patients' Mphi, but combined addition of IL-1beta and IL-18 restored these Mphis' IL-13 enhancing activity. These data suggest that immunodepressed patients' aberrant monokine and depressed T cell IL-13 production are independent but synergistic contributors to emergence of MOF.

Adolescent↗

PubMed2005

A network-based analysis of systemic inflammation in humans.

Oligonucleotide and complementary DNA microarrays are being used to subclassify histologically similar tumours, monitor disease progress, and individualize treatment regimens. However, extracting new biological insight from high-throughput genomic studies of human diseases is a challenge, limited by difficulties in recognizing and evaluating relevant biological processes from huge quantities of experimental data. Here we present a structured network knowledge-base approach to analyse genome-wide transcriptional responses in the context of known functional interrelationships among proteins, small molecules and phenotypes. This approach was used to analyse changes in blood leukocyte gene expression patterns in human subjects receiving an inflammatory stimulus (bacterial endotoxin). We explore the known genome-wide interaction network to identify significant functional modules perturbed in response to this stimulus. Our analysis reveals that the human blood leukocyte response to acute systemic inflammation includes the transient dysregulation of leukocyte bioenergetics and modulation of translational machinery. These findings provide insight into the regulation of global leukocyte activities as they relate to innate immune system tolerance and increased susceptibility to infection in humans.

Acute Disease↗

PubMed2005

Application of genome-wide expression analysis to human health and disease.

The application of genome-wide expression analysis to a large-scale, multicentered program in critically ill patients poses a number of theoretical and technical challenges. We describe here an analytical and organizational approach to a systematic evaluation of the variance associated with genome-wide expression analysis specifically tailored to study human disease. We analyzed sources of variance in genome-wide expression analyses performed with commercial oligonucleotide arrays. In addition, variance in gene expression in human blood leukocytes caused by repeated sampling in the same subject, among different healthy subjects, among different leukocyte subpopulations, and the effect of traumatic injury, were also explored. We report that analytical variance caused by sample processing was acceptably small. Blood leukocyte gene expression in the same individual over a 24-h period was remarkably constant. In contrast, genome-wide expression varied significantly among different subjects and leukocyte subpopulations. Expectedly, traumatic injury induced dramatic changes in apparent gene expression that were greater in magnitude than the analytical noise and interindividual variance. We demonstrate that the development of a nation-wide program for gene expression analysis with careful attention to analytical details can reduce the variance in the clinical setting to a level where patterns of gene expression are informative among different healthy human subjects, and can be studied with confidence in human disease.

Cluster Analysis↗

PubMed2004

Two third-year medical student-level laboratory shock exercises without large animals.

BACKGROUND: Historically, medical schools have taught principles of hemodynamic shock using large animal models. Such exercises are infrequent today due to the increasing aversion of students and the wider community to the use of large animals in teaching. Herein, we describe two alternative exercises that communicated basic science and clinical principles of shock effectively. METHODS: We developed two complementary, distinct single-afternoon laboratory exercises for third-year medical students. The first exercise (lab) demonstrated three principles: (1) in vitro cytokine-induced apoptosis (illustrating mechanisms and consequences of sepsis), (2) the hemodynamic manifestations of hypovolemia and septic shock in rats, and (3) the effects of fluid resuscitation or vasopressor administration in these same rat models. In the second exercise, students managed the diagnosis, initial resuscitation, surgical treatment, and ICU care of a "patient" with abdominal sepsis, using a manikin-based patient simulator and actual patient test data. Current basic science and clinical literature were incorporated. RESULTS: Efficacy was evaluated by polling students in one of four rotations (n = 25). Educational value of the lab exercise was rated 3.70 (1, worst rating; 5, best rating), whereas its applicability to clinical care was rated 4.35. Educational value and clinical applicability of the patient simulator were rated 4.52 and 4.76, respectively. CONCLUSIONS: These exercises combining laboratory demonstrations of the pathophysiologic mechanisms and manifestations of shock with simulation were judged effective and clinically relevant while fulfilling the National Institutes of Health (NIH) mandate to reduce use of experimental animals.

Animals↗

PubMed2004

Abnormal PGE(2) regulation of monocyte TNF-alpha levels in trauma patients parallels development of a more macrophage-like phenotype.

Some trauma patients' monocytes (MO) increase TNF-alpha levels concomitant to augmenting production of the TNF-alpha inhibitor prostaglandin E2 (PGE2), suggesting posttrauma MO insensitivity to PGE2 effects. This study assesses additional posttrauma MO PGE2 insensitivity effects on altering TNF-alpha form (membrane versus secreted), down-regulating MO receptor expression, and depressing MO APC function. Posttrauma MO TNF-alpha insensitivity to exogenous and autocrine PGE2 correlated to accumulation of TNF-alpha primarily as a membrane-bound cytokine (mTNF-alpha). MO retention of mTNF-alpha correlated with unfavorable clinical outcomes and loss of antigen-presenting cell (APC) function as assessed by depressed MLR and dendritic cell (DC) differentiation. MO TNF-alpha sensitivity to down-regulation by IL-10 was retained, suggesting that PGE2-related functions are specifically altered in these patients' MO. Freshly isolated MO from all trauma patients had decreased expression of Toll-like receptor 4 (TLR4) for gram-negative bacteria. Exogenous PGE2 at high (10 (-6) M) or low (10 (-8) M) concentrations decreased normals' and further decreased APC-competent patients' MO TLR4 expression but had no effect on TLR2. Patients' APC-dysfunctional MO failed to further down-regulate their TLR4 expression in response to additional PGE2, demonstrating another form of PGE2 insensitivity. One of the primary MO prostaglandin receptors, eicosanoid receptor 4 (EP4), was decreased on patients' APC dysfunctional MO, suggesting that depressed EP4 expression could contribute to PGE2 insensitivity in patients' MO. The APC dysfunctional MO's dysregulation of TLR4 expression paralleled increased macrophage-like characteristics such as increased CD64 expression density, elevated mTNF-alpha production, and increased PGE2 levels. Increased PGE2 levels still decreased patients' MO APC functions but failed to depress either MO TLR4 expression or mTNF-alpha levels, suggesting differential involvement of EP receptors in postinjury PGE2-mediated effects.

Antigen-Presenting Cells↗

PubMed2002

Down-regulated circulating PMN function after injury despite enhanced p38 MAPK activity.

BACKGROUND: Neutrophils (PMN) are initially primed by injury. However, remaining PMN found in the circulation postinjury demonstrate a down-regulated phenotype with inhibited apoptosis, CXCR2 expression, and endotoxin (LPS) responsiveness that may contribute to infectious complications and organ dysfunction. The p38 mitogen activated protein kinase (MAPK) signal transduction pathway has been implicated in regulating each of these PMN functions. We, therefore, hypothesize that p38 signaling is similarly down-regulated in postinjury circulating PMN. MATERIALS AND METHODS: PMN were isolated from trauma patients (ISS > 20, postinjury day 3) and concurrently from healthy volunteers (control). PMN were cultured with LPS (100 ng/mL) and p38 activity assessed by Western blotting of cell lysates using a dual phosphospecific antibody for phosphorylated, active p38. In separate experiments, PMN from healthy volunteers were cultured in plasma from either healthy or injured subjects +/- LPS and p38 activity assessed by a cell-free in vitro kinase assay using the transcription factor, ATF-2, as a substrate. Apoptosis and IL-1beta secretion of the cultured PMN from each experimental condition were also quantified. RESULTS: Circulating PMN from trauma patients have an upregulated LPS signaled p38 MAPK response (threefold) compared to PMN from healthy volunteers (p < 0.05). Furthermore, circulating factors present in trauma plasma can transfer this response to normal PMN. There was no significant alteration in apoptosis following LPS treatment between control and trauma patient's PMN (control: 62 +/- 3% vs. trauma: 55 +/- 5%), but there was decreased secretion of IL-1beta (control: 100 +/- 28 pg/mL vs. trauma: 12 +/- 5 pg/mL, p < 0.05). CONCLUSION: We conclude that injury down-regulates selective PMN function despite enhanced p38 activity. These results suggest a shift in the role of PMN p38 signal transduction following injury with additional critical regulation of LPS responses downstream to p38 MAPK activity.

Apoptosis↗