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Biomedical subjects

Daniel G Remick

Publications and source records attributed to Daniel G Remick.

At least 37 records · Page 2Linked to original sources
PubMed2005

Sequential ELISA to profile multiple cytokines from small volumes.

Due to the low cost and relative effectiveness the enzyme-linked immunosorbent assay (ELISA) is widely used to measure the concentration of inflammatory cytokines in plasma and other sources. Blood volume represents a limiting factor in those mouse models requiring repeated sample collection at multiple time intervals to monitor the trajectory of inflammatory processes. The small blood volumes in such scenarios restrict the array of cytokines that can be measured using the traditional ELISA. The implementation of the sequential ELISA protocol presented here can dramatically increase the number of measured cytokines, since the plasma samples are not discarded after the initial assay but re-used to measure additional selected inflammatory proteins in consecutive tests. From the original 20 mul of blood volume collected, up to fifteen cytokines can be successfully assayed in five consecutive cycles. With more unstable cytokines analyzed in the initial cycles, no inter-assay interference and/or deterioration of samples occurs. The sequential ELISA technique based on commercially-available antibody pairs can be an attractive alternative to more advanced, costly methods. Given the simplistic validation procedure, the proposed sequential ELISA protocol has a wide potential for further modifications to include other inflammation-related targets.

Animals↗
PubMed2005

Altered Kupffer cell function in biliary obstruction.

BACKGROUND: An altered Kupffer cell (KC) response is thought to be responsible for the characteristic phenotype observed after biliary obstruction: a phenotype marked by a defect in the hepatic reticuloendothelial system and a hypersensitivity to endotoxin. Few studies, however, have directly examined KC function. We have sought to define the specific alterations in function and phenotype that occur in the KC after biliary obstruction. METHODS: KCs were isolated from female C57BL/6 mice 4 days after a sham or common bile duct ligation (CBDL) operation. Phagocytosis, oxidative burst potential, and intracellular bacterial killing were measured as markers of reticuloendothelial system function. The KC response to endotoxin was assessed by measuring tumor necrosis factor alpha and interleukin 6 levels in the media after stimulation with lipopolysaccharide (LPS) or with LPS plus LPS-binding protein (LBP). RESULTS: CBDL KCs demonstrated a significant increase in phagocytic ability and significantly decreased baseline oxidative stress, compared with Shams. The oxidative burst potential, however, was equivalent or higher for CBDL KCs. CBDL KCs also demonstrated increased numbers of viable intracellular bacteria after infection; however, it is unclear if this finding represents impaired intracellular bacterial killing or increased phagocytosis of bacteria. With respect to the KC response to endotoxin, CBDL KCs were found to be less sensitive to the stimulatory effects of LPS alone but were exquisitely sensitive to the effects of LBP. LBP levels were found to be significantly elevated in CBDL animals, and CBDL KCs demonstrated a dose-dependent, exaggerated tumor necrosis factor alpha and interleukin 6 response to LPS administered with LBP. CONCLUSIONS: KC function is clearly altered after biliary obstruction. Phagocytic ability is actually increased, although the ability of CBDL KCs to kill bacteria within the phagosome remains ill defined. CBDL KCs are exquisitely sensitive to the effects of LBP, and LBP levels are elevated after biliary obstruction. LBP may be responsible for the increased proinflammatory response observed after endotoxin challenge in animals with biliary obstruction.

Acute-Phase Proteins↗
PubMed2005

Correction of perioperative hypothermia decreases experimental sepsis mortality by modulating the inflammatory response.

OBJECTIVE: The objective of this study was to investigate if the correction of perioperative hypothermia improves sepsis survival. DESIGN: Mice with anesthesia-induced perioperative hypothermia had sepsis induced by cecal ligation and puncture and were treated with fluid resuscitation and antibiotics. The mice were either warmed (35 degrees C) or kept at room temperature for 1 hr in the immediate postoperative period. SETTING: This study was conducted at a university research laboratory. SUBJECTS: This study included adult, female outbred mice. INTERVENTIONS: Immediately after surgery, mice were randomized to 1 hr of warming or maintained at room temperature. Warming was accomplished by placing the mice in a cage preheated to 35 degrees C. MEASUREMENT AND MAIN RESULTS: The anesthesia-induced hypothermia resolved within 10 hrs, and perioperative warming reestablished normothermia within 1 hr. Restoring normothermia improved sepsis survival from 42% to 60% (p < .02). Warming also significantly corrected the changes in body weight, reflecting improved overall physiological status. To examine the mechanism of this beneficial response, plasma levels of interleukin-6 were assessed. Warming was associated with a decrease in interleukin-6 levels in both those mice that died as well as survivors, reflecting a blunting but not complete inhibition of the inflammatory response. Among surviving mice, warming also significantly increased the peripheral blood cell count, including the neutrophils, an indication that warming augmented innate immunity. CONCLUSIONS: Correction of perioperative hypothermia improves survival after sepsis by appropriately modulating the early inflammatory response.

Anesthesia, General↗
PubMed2005

Interleukin-8.

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Animals↗
PubMed2005

Evaluation of endotoxin models for the study of sepsis.

Many strategies have been proposed for the treatment of sepsis, and most of the proposed treatment modalities have failed in clinical trials. Many of the previous treatment protocols called for blocking the activity of a single, clearly defined mediator. The underlying hypothesis was that sepsis induced a specific mediator that then caused organ injury and death. This simple, linear reasoning was frequently based on cytokines that were defined using endotoxin models of sepsis. The endotoxin models were widely used to study the pathophysiology of sepsis and were felt to adequately reproduce the full spectrum of inflammatory changes observed in patients with sepsis. Based on mortality and hematologic changes, these assumptions appeared justified. As the models were examined more closely, and directly compared with focus of infection models that more accurately portray the changes in sepsis, it became apparent that the endotoxin models did not accurately mimic the patient with sepsis. In the endotoxin models, the explosive release of cytokines into the circulating blood volume was reproducibly found regardless of the species studied (human, primate, pig, rat, or mouse). This lead to a series of anticytokine sepsis trials, all of which failed. The cytokine response in focus of infection models, such as that induced by cecal ligation and puncture, was examined and found to be more similar to that observed in patients with sepsis. When cytokine inhibitor strategies were used in the cecal ligation and puncture model, they were also generally found to lack efficacy. Compounds that have been shown to be effective at reducing mortality in endotoxin models should be re-evaluated in more clinically relevant models of sepsis.

Animals↗
PubMed2005

Improved survival in mice given systemic gene therapy in a gram negative pneumonia model.

BACKGROUND: We previously demonstrated an essential role for lipopolysaccharide binding protein (LBP) in the pulmonary immune response to Gram-negative bacterial infection. LBP knockout mice had significantly higher mortality, greater rates of bacteremia, and higher counts of viable bacteria in their lungs at sacrifice compared with wild-type controls. We postulate that systemic LBP gene therapy will reconstitute a protective innate immune response in LBP knockout mice and that overexpression of LBP in wild-type mice may offer a survival advantage. METHODS: 12-16 week old female C57BL/6 wild-type mice and age matched LBP knockout mice were given 5 x 10(9) PFU of recombinant adenovirus containing either the gene for LBP or the irrelevant control protein beta-galactosidase by tail vein injection. 72 hours later each mouse was administered 1 x 10(3) CFU of Klebsiella pneumoniae by intratracheal injection. RESULTS: Administration of LBP by systemic gene therapy to LBP knockout mice improved survival from Klebsiella pneumonia to a level equivalent or better than wild-type mice exposed to the same dose of bacteria (36 versus 25%). Wild-type mice given the LBP gene therapy demonstrated increased 7 day survival from Klebsiella pneumonia when compared with controls treated with beta-galactosidase (68 versus 30%, p = 0.03). CONCLUSIONS: Systemic gene therapy with intravenous adenoviral vector transfer of LBP significantly improves survival in LBP knockout mice. Overexpression of LBP in wild-type mice improves survival from Klebsiella pneumonia. Raising levels of LBP in the setting of Gram-negative pneumonia may be of therapeutic benefit.

Acute-Phase Proteins↗
PubMed2005

Signal pathways underlying homocysteine-induced production of MCP-1 and IL-8 in cultured human whole blood.

AIM: To elucidate the mechanisms underlying homocysteine (Hcy)-induced chemokine production. METHODS: Human whole blood was pretreated with inhibitors of calmodulin (CaM), protein kinase C (PKC), protein tyrosine kinase (PTK), mitogen-activated protein kinase (MAPK), and NF-kappaB and activators of PPARgamma for 60 min followed by incubation with Hcy 100 micromol/L for 32 h. The levels of mitogen chemokine protein (MCP)-1 and interleukin-8 (IL-8) were determined by enzyme-linked immunosorbant assay (ELISA). RESULTS: Inhibitors of PKC (calphostin C, 50-500 nmol/L and RO-31-8220, 10-100 nmol/L), CaM (W7, 28-280 micromol/L), ERK1/2 MAPK (PD 98059, 2-20 micromol/L), p38 MAPK (SB 203580, 0.6-6 micromol/L), JNK MAPK (curcumin, 2-10 micromol/L), and NF-kappaB (PDTC, 10-100 nmol/L) markedly reduced Hcy 100 micromol/L-induced production of MCP-1 and IL-8 in human cultured whole blood, but the inhibitors of PTK (genistein, 2.6-26 micromol/L and tyrphostin, 0.5-5 micromol/L) had no obvious effect on MCP-1 and IL-8 production. PPARgamma activators (ciglitazone 30 micromol/L and troglitazone 10 micromol/L) depressed the Hcy-induced MCP-1 production but not IL-8 production in the cultured whole blood. CONCLUSION: Hcy-induced MCP-1 and IL-8 production is mediated by activated signaling pathways such as PKC, CaM, MAPK, and NF-kappaB. Our results not only provide clues for the signal transduction pathways mediating Hcy-induced chemokine production, but also offer a plausible explanation for a pathogenic role of hyperhomocysteinemia in these diseases.

Calmodulin↗
PubMed2005

Role of interleukin-6 in mortality from and physiologic response to sepsis.

Previous studies have suggested that interleukin-6 (IL-6) serves as both a marker and a mediator for the severity of sepsis. We tested whether interleukin 6 knockout (IL-6KO) mice were more susceptible to sepsis mortality induced by cecal ligation and puncture. IL-6KO and wild-type (WT) mice were subjected to increasing degrees of sepsis severity. Physiologic support was given with fluids and appropriate antibiotics. Plasma IL-6 levels were determined 6 h after the onset of sepsis, and a complete hematologic profile was performed on day 2. As expected, increasing sepsis severity resulted in greater and more rapid mortality. However, the mortality was nearly identical in the IL-6KO and WT mice. All WT septic mice had high plasma levels of IL-6 6 h after the onset of sepsis, while IL-6KO were near or below the lower limit of detection. Among the WT mice, mortality was significantly higher in mice with plasma IL-6 >3,000 pg/ml. Both IL-6KO and WT mice destined to die in the early stages of sepsis had substantial and nearly identical weight gain in the first 24 h. However, at later stages the WT mice had significantly greater weight loss than the KO mice. The KO mice failed to develop the characteristic hypothermia within the first 24 h of severe sepsis routinely observed in the WT mice. These data demonstrate that IL-6 serves as a marker of disease severity in sepsis and does modulate some physiologic responses, but complete lack of IL-6 does not does not alter mortality due to sepsis.

Animals↗
PubMed2004

Prevention and reversal of pulmonary inflammation and airway hyperresponsiveness by dexamethasone treatment in a murine model of asthma induced by house dust.

The morbidity and mortality from asthma in the Western world have increased 75% in the past 20 years. Recent studies have demonstrated that sensitization to cockroach allergens correlates strongly with the increased asthma morbidity for adults and children. We investigated whether dexamethasone administered before or after allergen challenge would inhibit the pulmonary inflammation and airway hyperresponsiveness in a mouse model of asthma induced by a house dust extract with high levels of cockroach allergens. For the prevention experiment, mice were treated with an intraperitoneal injection of dexamethasone 1 h before each pulmonary challenge, and airway hyperresponsiveness was measured 24 h after the last challenge. Mice were killed 48 h after the last challenge. For the reversal study, airway hyperresponsiveness was measured 24 h after the last challenge, and the mice were treated with dexamethasone. Dexamethasone treatment before allergen challenge significantly reduced the pulmonary recruitment of inflammatory cells, myeloperoxidase activity in the lung, airway hyperreactivity, and total serum IgE levels compared with PBS-treated mice. Additionally, dexamethasone treatment could significantly reduce the airway hyperreactivity of an established asthmatic response. These results demonstrate that dexamethasone not only prevents but also halts the asthmatic response induced by house dust containing cockroach allergens. This model exhibits several features of human asthma that may be exploited in the study of pathophysiological mechanisms and potential therapeutic interventions.

Acute Disease↗
PubMed2004

Effects of platelet inhibitors on propyl gallate-induced platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activation.

Propyl gallate (PG) is a platelet agonist characterized by inducing platelet aggregation, protein tyrosine phosphorylation, and platelet factor 3 activity. The mechanisms of platelet activation following PG stimulation were examined by pre-incubating platelets with well-defined platelet inhibitors using platelet aggregation, protein tyrosine phosphorylation, activated plasma clotting time, and annexin V binding by flow cytometry. PG-induced platelet aggregation and tyrosine phosphorylation of multiple proteins were substantially abolished by aspirin, apyrase, and abciximab (c7E3), suggesting that PG is associated with activation of platelet cyclooxygenase 1, adenosine phosphate receptors, and glycoprotein IIb/IIIa, respectively. The phosphorylation of the cytoskeletal enzyme pp60(c-src) increased following PG stimulation, but was blunted by pre-incubation of platelets with aspirin, apyrase, and c7E3, suggesting that tyrosine kinase is important for the signal transduction of platelet aggregation. Propyl gallate also activates platelet factor 3 by decreasing the platelet coagulation time and increasing platelet annexin V binding. Platelet incubation with aspirin, apyrase, and c7E3 did not alter PG-induced platelet coagulation and annexin V binding. The results suggest that platelet factor 3 activation and membrane phosphotidylserine expression were not involved with activation of platelet cyclooxygenase, adenosine phosphate receptors, and glycoprotein IIb/IIIa. PG is unique in its ability to stimulate platelet aggregation and coagulation simultaneously, and platelet inhibitors in this study affect only platelet aggregation but not platelet coagulation.

Annexin A5↗
PubMed2004

Humane endpoints in shock research.

In biomedical research using animal models, the phrase "humane endpoints" refers to predetermined criteria used to judge when the research animals should be humanely euthanized. The intended goal of humane endpoints is to minimize the distress or suffering of research animals; however, if applied incorrectly, this well-intended concept could lead to premature decisions and inaccurate data, resulting in a waste of animal life. A concensus on specific endpoints for shock and inflammation research is not available but several biochemical, physical and behavioral parameters have been suggested for other research models. In addition, the authors have found, in the studies presented here, that increasing body weight, decreased body temperature, and inability to ambulate are important parameters in a model of cecal ligation and puncture. However, it is clear that the applicability of these endpoints may change with the model of disease, intensity of insults, experimental treatments and other factors. Consequently, humane endpoints should be assigned cautiously and preferably after preliminary studies to prevent aberrant research results. In order to accomplish this, investigators must become aware of certain concepts including: when to implement endpoints, what endpoints to consider, and how to establish the endpoints for their studies. Equipped with the basic principles of humane endpoints, investigators can make informed decisions that meet current standards of animal care while still achieving the scientific goals of their research studies.

Animal Welfare↗
PubMed2004

Development of a sensitive microarray immunoassay and comparison with standard enzyme-linked immunoassay for cytokine analysis.

Cytokine and cytokine inhibitors represent important components of the inflammatory response in patients with trauma, shock, and sepsis. Many investigators wish to quantify cytokines and it would be advantageous to measure multiple cytokines in a multiplex manner to obtain an inflammatory profile rather than a single value. Using the well-accepted standard enzyme-linked immunoassay (ELISA) as a basis, a microarray immunoassay (MI) was designed to measure 16 different human cytokines simultaneously. The MI was performed by spotting antibodies on nitrocellulose pads affixed to glass slides. Detection of the mediators was performed with biotin-conjugated antibodies followed by fluorescently labeled streptavidin. All antibodies and other reagents were purchased commercially. The MI achieved a lower limit of detection that was generally similar to traditional ELISAs (approximately 4-12 pg/mL) and also had a similar coefficient of variation. In the multiplexed MI, there was no cross reactivity between mediators. To verify the utility of the MI, cytokines and cytokine inhibitors were measured in endotoxin stimulated human blood by both ELISA and MI. Virtually identical cytokine concentrations were measured by both methods. These results describe the development of a sensitive, specific and cost-effective multiplexed microarray immunoassay that produces values similar to traditional ELISAs.

Antibodies↗
PubMed2004

Model of angiogenesis in mice with severe combined immunodeficiency (SCID) and xenoengrafted with Epstein-Barr virus-transformed B cells.

Xenoengraftment of human cells in mice with severe combined immunodeficiency (SCID) has been used as a model system to study the mechanisms of B-cell lymphomagenesis. In the study reported here, we determined that SCID mice can also be used as a model to study angiogenesis in B-cell lymphomas. The C.B-17 scid/scid mice were xenotransplanted with Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCL), and we determined whether CD31, a marker found on endothelial cells, was detected in the human B-cell lymphomas that developed in these mice. Microvessel formation was identified by use of immunohistochemical staining for CD31. To assess possible mechanisms of angiogenic stimulus, we analyzed the expression of interleukin 8 (IL-8), a chemokine documented to promote angiogenesis, in non-small-cell lung cancer and bronchogenic carcinomas. We observed that a panel of LCL and LCL-lymphomas expressed IL-8 mRNA and protein. Neutralization of IL-8, however, did not inhibit lymphomagenesis, suggesting that IL-8 is not essential for angiogenesis in this model. To examine other parameters of angiogenesis, we identified expression of vascular endothelial growth factor in the lymphomas. These data suggest that angiogenesis accompanies EBV-associated B-cell lymphoma development, but IL-8 is not essential for this process. Thus, the SCID mouse model is amenable to testing of anti-angiogenic factors.

Animals↗
PubMed2004

Homocysteine induces production of monocyte chemoattractant protein-1 and interleukin-8 in cultured human whole blood.

AIM: To investigate whether increased plasma L-homocysteine (Hcy) level could promote monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) in cultured whole blood. METHODS: Human whole blood or different type of peripheral blood cells from health volunteers were incubated with Hcy and/or the inhibitors. MCP-1 and IL-8 level were measured by ELISA assay. RESULTS: Hcy 10-1000 micromol/L induced production of MCP-1 and IL-8 in cultured human whole blood (P<0.05). The major cellular source of these chemokines comed from monocytes. Meanwhile,Hcy also promoted the upregulation of MPO level even at the 10 micromol/L in the cultured whole blood. The intracellular ROS, particular the OH. radicals, play extremely important role in the Hcy-induced MCP-1 and IL-8 production. CONCLUSION: Increased Hcy level in plasma (hyperhomocysteinemia) induced MCP-1 and IL-8 secretion in cultured human whole blood, especially in monocytes via oxidative stress mechanism.

Cells, Cultured↗
PubMed2003

Homocysteine mediated expression and secretion of monocyte chemoattractant protein-1 and interleukin-8 in human monocytes.

Homocysteine (Hcy) is an independent risk factor for cardiovascular disease. Monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) are major chemokines for leukocyte trafficking and have been identified in atheromatous plaques. MCP-1 and IL-8 have been found to express mainly by macrophages in human lesion. We undertook this study to determine whether Hcy could induce the secretion of chemokines from human monocytes and, if so, to explore the mediating mechanism. We found that clinically relevant levels of Hcy (10 to 1000 micromol/L) increased the protein secretion and mRNA expression as well as activity of MCP-1 and IL-8 in cultured primary human monocytes. These effects of Hcy were primarily mediated by reactive oxygen species (ROS) through NAD(P)H oxidase, because Hcy could upregulate the production of ROS and the inhibitors of protein kinase C, calmodulin, free radical scavengers, or NAD(P)H oxidase abolished Hcy-induced ROS production and MCP-1 and IL-8 secretion in these cells. Furthermore, the inhibitors of mitogen-activated protein kinase (p38 and extracellular signal-regulated kinase 1/2) and nuclear factor-kappaB or the activator of peroxisome proliferator-activated receptor gamma (PPARgamma) significantly decreased Hcy-induced MCP-1 and IL-8 secretion in these cells. These data indicate that pathophysiological levels of Hcy can alter human monocyte function by upregulating MCP-1 and IL-8 expression and secretion via enhanced formation of intracellular ROS originated from NAD(P)H oxidase source via calmodulin or protein kinase C signaling pathways and that Hcy-induced ROS subsequently activates mitogen-activated protein kinase (p38 and ERK1/2) and nuclear factor-kappaB in a PPARgamma activator-sensitive manner. Thus, activation of PPARgamma may become a therapeutic target for preventing Hcy-induced proatherogenic effects.

Antioxidants↗
PubMed2003

Improved sensitivity of colorimetric compared to chemiluminescence ELISAs for cytokine assays.

Cytokines are often measured using ELISAs and chemiluminescence (CMIL) is reported to exhibit increased sensitivity compared to colorimetric (COL) assays. CMIL also has a wider dynamic detection range. We sought to directly compare ELISAs for measuring human TNF and IL-8 using CMIL or COL. CMIL substrates with glow fluorescence were obtained from 4 different commercial sources while the COL substrate was TMB. ELISAs for TNF and IL-8 were run under identical conditions and the standard curve extended from 0.5 to 4000 pg/ML. The COL substrate demonstrated a sigmoid shaped curve when plotted on a log-linear scale while the CMIL continued to increase up to the highest concentration. Both substrates were modeled most accurately by a 4 parameter equation with R values > 0.99. The standard curves for both the IL-8 and TNF demonstrated a lower limit of detection (LLD) for the COL comparable to the CMIL detection system. To precisely define the LLD quadruplicate blanks were run and the mean plus 4 standard deviations were used. By these criteria, the COL assay routinely had a LLD of < 1.5 pg/ML which was better than any of the CMIL substrates. Our data demonstrate the COL assays have the same or better sensitivity than CMIL and are significantly less expensive.

Colorimetry↗