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R L Simmons

Publications and source records attributed to R L Simmons.

At least 55 records · Page 3Linked to original sources
PubMed1996

Nitric oxide production by mouse bone marrow-derived dendritic cells: implications for the regulation of allogeneic T cell responses.

Dendritic cells (DC) are the most potent known antigen presenting cells, and play important roles both in immunity and tolerance induction. Nitric oxide (NO) is an important effector molecule that is involved in numerous aspects of the immune response. There have been no accounts to date of efforts to determine NO generation by well-characterized DC. In this report we describe the production of NO by highly purified DEC 205+ DC propagated from mouse bone marrow in response to granulocyte/macrophage-colony stimulating factor (GM-CSF) + interleukin-4 (IL-4). NO synthesis was induced in DC by interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS), and was blocked by the inhibitor of nitric oxide synthase (NOS), NG-monomethyl-L-arginine (NMMA). Both "mature" B7-2+ (CD86+) DC and B7-2- (CD86-) DC progenitors could be induced to release NO. NO was also recovered from the supernatants of primary mixed leukocyte cultures containing comparatively high concentrations of B7-2+ DC in relation to purified allogeneic T cells. Furthermore, inhibition of NO release in these cultures by NMMA resulted in an increase in T cell proliferation. These observations suggest that NO may be an important soluble mediator of the interaction between DC and activated T cells. In addition to its ability to inhibit T cell proliferation, NO was also shown to induce programmed cell death in DC. This was visualized by the detection of DNA strand breaks with in situ nick translation. The percentage of DC apoptosis correlated with the level of NO in the cultures. Apoptosis was inhibited by the addition of NMMA. These results indicate that DC have the capacity both to stimulate and potentially limit the same allogeneic T cell response, in accordance with their production of NO.

Animals↗
PubMed1996

"Suboptimal" kidney donors: the experience with tacrolimus-based immunosuppression.

Female, pediatric, and older donors have been associated with inferior graft survival after renal transplantation. We analyzed these three subgroups in 397 patients receiving tacrolimus-based immunosuppression. There were no differences in recipient age, incidence of retransplantation, or percentage of sensitized patients. Female donors, compared with male donors, were associated with comparable 1- and 3-year patient survival rates (96% and 93% vs. 95% and 92%, respectively) and comparable 1- and 3-year graft survival rates (90% and 80% vs. 88% and 81%, respectively). Renal function was also similar. Recipients of pediatric en bloc kidneys, when compared with recipients of other cadaveric kidneys, also had comparable 1- and 3-year patient survival rates (94% and 94% vs. 95% and 91%, respectively) and comparable 1- and 3-year graft survival rates (84% and 84% vs. 89% and 79%, respectively). Renal function was better in recipients of en bloc kidneys, with a mean serum creatinine level of 1.4+/-1.8 mg/dl vs. 2.0+/-1.5 mg/dl (P=0.01). In contrast to the first two subgroups, donors over 60 years of age, when compared with donors under 60 years of age, were associated with worse 1- and 3-year patient survival rates (88% and 80% vs. 96% and 94%, respectively; P<0.03) and worse 1- and 3-year graft survival rates (74% and 62% vs. 91% and 83%, respectively; P<0.0001). Renal function was worse in the older donor group, with a serum creatinine level of 2.7+/-1.2 mg/ml vs. 1.9+/-1.5 mg/dl (P=0.01). We conclude that, under tacrolimus-based immunosuppression, kidneys from female or very young pediatric donors are not associated with adverse outcomes, whereas kidneys from donors over 60 years of age are associated with inferior outcomes.

Adult↗
PubMed1996

Stable expression of human inducible nitric oxide synthase in V79 Chinese hamster cells.

A recombinant expression vector containing the full-length cDNA for human inducible nitric oxide (NO) synthase was constructed for constitutive expression in V79 Chinese hamster cells. Expression was followed by Western analyses using three different NO synthase antisera. Activity remained stable during 4 months of continued cultivation. Activities were 25 pmol min-1 mg-1 cytosolic protein with L-arginine and 47 pmol min-1 mg-1 cytosolic protein with NG-hydroxy-L-arginine as substrates. Activity was concentration-dependently inhibited by inhibitors such as NG-methyl-L-arginine, NG-nitro-L-arginine, NG-nitro-L-arginine methyl ester, aminoguanidine, and S-methyl-isothiourea. The rank order of inhibitor potencies was different from published results obtained with rodent inducible NOS. Parental V79 cells do not express and cannot be induced for NO synthase activity. Therefore, the genetically engineered V79 cell line is defined for the cDNA-encoded human inducible NO synthase. The new cell line may serve as a useful tool to study human inducible NO synthase.

Animals↗
PubMed1996

Induction of nitric oxide synthase in mouse dendritic cells by IFN-gamma, endotoxin, and interaction with allogeneic T cells: nitric oxide production is associated with dendritic cell apoptosis.

Nitric oxide (NO) is an important effector molecule that is involved in immune regulation and host defense. In this study, highly purified NLDC 145+ (DEC-205+) MHC class II(bright) B7-2+ dendritic cells (DC) propagated from normal mouse bone marrow in response to granulocyte-macrophage CSF + IL-4 were induced to produce NO by IFN-gamma and LPS. NO production was inhibited by the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (NMMA). Nitrite also accumulated in mixed leukocyte culture supernatants as the result of coculture of DC with purified naive allogeneic T cells. Furthermore, NO production was induced by CD40 ligation. Suboptimal T cell proliferation observed at high relative concentrations of DC correlated with increased NO production and was mitigated by NMMA. Induction of mRNA for an inducible NOS (iNOS) in DC was confirmed by Northern blotting, whereas intracellular iNOS was visualized by two-color flow cytometry and by both immunofluorescent and immunogold labeling in a subpopulation of IFN-gamma + LPS-stimulated cells. Both endogenous NO production and exposure of unstimulated DC to the NO donor S-nitroso-N-acetyl-penicillamine (SNAP) resulted in DC apoptosis. Thus, although DC function initially as the most potent APCs for T cell activation, DC induced to synthesize NOS by IFN-gamma may inhibit (allogeneic) T cell proliferation: NO may suppress lymphocyte proliferation and also induce apoptosis of the most potent source of alloantigenic stimulation.

Animals↗
PubMed1996

Bystander injury of host lymphoid tissue during murine graft-verus-host disease is mediated by nitric oxide.

The suppressed lymphocyte proliferative responses characteristic of graft-versus-host disease (GVHD) are due, in part, to production of nitric oxide (NO). In order to more fully elucidate the role of NO during GVHD, an NO synthesis inhibitor, aminoguanidine (AG), was administered to unirradiated (C57BL/6J X DBA/2J)F1 mice injected with 5 X 10(7) C57BL/6J splenocytes. Administration of AG resulTed in abrogation of the elevation in serum NO2- + NO3- levels characteristic of GVHD. A significantly increased percentage of splenocytes of host phenotype (H2b/d, B220+, and THY1.2+) and a significantly higher hematocrit value were detected in GVHD animals receiving AG therapy. Additionally, the Con A-induced proliferative response of splenocytes obtained from GVHD mice receiving AG therapy was increased compared with the responses of splenocytes from animals that did not receive AG therapy. Parameters not affected by AG therapy included NO synthesis by recovered peritoneal macrophages, donor antihost cytolytic activity in splenocyte populations, serum GM-CSF levels and long-term engraftment of donor cells. These data indicate that NO may play a role in the destruction of both lymphoid and erythroid host tissue as well as the reduced lymphoproliferative responses associated with the acute phase of GVHD.

Animals↗
PubMed1996

Transcriptional regulation of human inducible nitric oxide synthase (NOS2) gene by cytokines: initial analysis of the human NOS2 promoter.

The expression of inducible nitric oxide synthase (NOS2) is complex and is regulated in part by gene transcription. In this investigation we studied the regulation of NOS2 in a human liver epithelial cell line (AKN-1) which expresses high levels of NOS2 mRNA and protein in response to tumor necrosis factor alpha, interleukin 1 beta, and interferon gamma (cytokine mix, CM). Nuclear run-on analysis revealed that CM transcriptionally activated the human NOS2 gene. To delineate the cytokine-responsive regions of the human NOS2 promoter, we stimulated AKN-1 cells with CM following transfection of NOS2 luciferase constructs. Analysis of the first 3.8 kb upstream of the NOS2 gene demonstrated basal promoter activity but failed to show any cytokine-inducible activity. However, 3- to 5-fold inductions of luciferase activity were seen in constructs extending up to -5.8 and -7.0 kg, and a 10-fold increase was seen upon transfection of a -16 kb construct. Further analysis of various NOS2 luciferase constructs ligated upstream of the thymidine kinase promoter identified three regions containing cytokine-responsive elements in the human NOS2 gene: -3.8 to -5.8, -5.8 to -7.0, and -7.0 to -16 kb. These results are in marked contrast with the murine macrophage NOS2 promoter in which only 1 kb of the proximal 5' flanking region is necessary to confer inducibility to lipopolysaccharide and interferon gamma. These data demonstrate that the human NOS2 gene is transcriptionally regulated by cytokines and identify multiple cytokine-responsive regions in the 5' flanking region of the human NOS2 gene.

Blotting, Northern↗
PubMed1996

Antibiotic-resistant enterococci and the changing face of surgical infections.

BACKGROUND: Enterococci have not been thought to play an important role in intra-abdominal infections because of their relatively low virulence. However, this notion is changing because of the recent emergence of these microbes as significant nosocomial pathogens. OBJECTIVES: To review the mechanisms of antibiotic resistance of enterococci and to discuss the significance of multidrug-resistant enterococci in surgical infections. DATA SOURCES: Medical and basic science literature relating to enterococci. DATA SYNTHESIS: In addition to having intrinsic resistance to a number of antibiotics, enterococci have the ability to acquire resistant genes through the exchange of plasmids or transposons from other bacterial species. Moreover, enterococci have been shown to transmit these genes to other bacterial species in turn. The extensive resistance of these microorganisms has led to their emergence as significant nosocomial pathogens, ranking second only to Escherichia coli in the number of pathogenic isolates recovered from patients in intensive care units. There has also been a marked increase in vancomycin-resistant enterococcal infections in surgical patients in the last 5 years. Some studies associate the prior use of vancomycin or third-generation cephalosporins with the emergence of these strains. Overall, enterococcal infections are associated with increased morbidity and mortality. CONCLUSIONS: In view of the marked resistance of enterococci to antibiotics and their ability to disseminate resistance genes, these microbes have become important pathogens. Enterococci pose a threat to surgical patients, often causing significant therapeutic dilemmas.

Drug Resistance, Microbial↗
PubMed1996

Biomaterial-neutrophil interactions: dysregulation of oxidative functions of fresh neutrophils induced by prior neutrophil-biomaterial interaction.

Biomaterial-associated infection results in increased morbidity and mortality, and may occur because of nonproductive premature activation of neutrophils resulting in impaired phagocyte function at the biomaterial surface in the event of bacterial challenge. To further explore the effects of this premature activation, we evaluated the supernatants of biomaterial associated neutrophils to determine whether soluble mediators were released, and the likely role of these mediators. We show that these supernatants contain a chemoattractant and thereby induce chemotaxis by fresh neutrophils. No evidence of enhanced oxidative free radical production by either unstimulated neutrophils or a primed response to other mediators occurs when neutrophils were incubated with these supernatants. We also examined the effect of adding fresh neutrophils to a biomaterial surface containing a previous inoculum of neutrophils, and observed that the fresh cells did not become stimulated to release reactive oxygen intermediates (ROI) and also exhibited impaired killing of staphylococci. These studies suggest that not only does the biomaterial surface activate the initial wave of neutrophils but that subsequent waves of neutrophils exhibit an impaired host-defense function. These results are consistent with the known impairment of host defense in the presence of biomaterials, and provide evidence for a long-term down-regulation of neutrophil function at biomaterial surfaces.

Biocompatible Materials↗
PubMed1996

Nitric oxide produced by cytokine-activated pulmonary artery smooth muscle cells is cytotoxic to cocultured endothelium.

BACKGROUND: We recently demonstrated that rat pulmonary artery smooth muscle (RPASM) generates maximal nitric oxide (NO) when exposed to inflammatory cytokines, such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Our hypothesis is that NO produced by cytokine-stimulated RPASM has local cytotoxic effects on endothelium. Accordingly, we designed a pulmonary smooth muscle and endothelial coculture experiment in which the effects of NO on endothelium can be distinguished from the direct effects of cytokines. METHODS: RPASM cells were incubated with a mixture of TNF-alpha (500 units/ml) and IFN-gamma (100 units/ml) for 24 hours. This cytokine mixture was then removed and the NO-producing smooth muscle cells were incubated in a coculture transwell system with rat pulmonary artery endothelial (RPAE) cells. Subsequent NO production (as measured by nitrite concentration in cell supernatants), and the number of viable attached endothelial cells were then measured at 48 hours. RESULTS: RPASM continued to produce large amounts of NO, in the absence of further cytokine stimulation, after a 24-hour exposure to TNF-alpha and IFN-gamma. This RPASM-generated NO decreased the number of viable attached endothelial cells after 24 hour RPASM-RPAE coculture by 57%. The competitive stereospecific inhibitor of inducible NO synthase (iNOS), NG-monomethyl-L-arginine (NMA), returned the inducible NO production to basal levels and reversed the cytotoxic effects on endothelial cells. The number of viable attached endothelial cells returned to control levels. CONCLUSIONS: The NO produced by cytokine-activated RPASM has local cytotoxic effects on RPAE in coculture. Such NO produced in the vasculature may be a factor in the origin of acute lung injury under conditions of trauma and sepsis.

Animals↗
PubMed1996

Functional changes of intestinal intraepithelial lymphocytes during acute graft versus host disease: correlation with phenotype.

While the pathological characteristics of acute graft versus host disease (GvHD) have been defined for many target organs, little attention has been paid to the functional changes to lymphocytes in target organs such as the liver and small intestine. We have shown previously, utilizing a nonlethal parent (C57BL/6J) into the F1 (C57BL/6J x DBA2J F1) GvHD model, that the intestinal mucosa is infiltrated exclusively by donor T cells with a CD8+ phenotype during the first 3 weeks post-GvHD induction. The present study investigated the functional changes associated with the phenotypic changes of intestinal intraepithelial lymphocytes (IEL) during the acute phase of GvHD. IEL displayed specific anti-host cytolytic activity during the second and third week after GvHD induction. In addition, enhanced cytolytic activity, detected in the presence of anti-CD3 antibody, was apparent during the second through fourth week, with a peak in the third week after GvHD induction. The IEL also showed enhanced proliferative responses to immobilized anti-CD3 during the first 2 weeks of acute GvHD, while profound inhibition of proliferation was observed in the splenocyte population of the same animals. During weeks 2 and 3 post-GvHd induction, the V beta distribution of host IEL remained unchanged while the V beta distribution of infiltrating donor CD8+ cells resembled that of the donor IEL population. In the small intestine, the increased cytolytic and proliferative activity of IEL during the course of the disease may provide a rationale for the involvement of this organ in the pathology associated with GvHD.

Acute Disease↗
PubMed1996

Effect of nitric oxide on staphylococcal killing and interactive effect with superoxide.

The role of reactive nitrogen intermediates (RNI) such as nitric oxide (.NO) in host defense against pyogenic microorganisms is unclear, and the actual interactive effect of RNI and reactive oxidative intermediates (ROI) for microbial killing has not been determined. Since, in nature, ROI and RNI might be generated together within any local infection, we evaluated the separate and interactive effects of .NO and O2- on staphylococcal survival by using a simplified system devoid of eukaryotic cells. These studies showed that prolonged exposure of staphylococci to .NO does not result in early loss of viability but instead is associated with a dose-related delayed loss of viability. This effect is abrogated by the presence of hemoglobin, providing further evidence that the effect is RNI associated. Superoxide-mediated killing also is dose related, but in contrast to RNI-mediated killing, it is rapid and occurs within 2 h of exposure. We further show that the interaction of .NO and O(2)- results in decreased O(2)--mediated staphylococcal killing at early time points. .NO, however, appears to enhance or stabilize microbial killing over prolonged periods of incubation. This study did not produce evidence of early synergism of ROI and RNI, but it does suggest that .NO may contribute to host defense, especially when ROI-mediated killing is compromised.

Dose-Response Relationship, Drug↗
PubMed1996

Counterprotective effect of erythrocytes in experimental bacterial peritonitis is due to scavenging of nitric oxide and reactive oxygen intermediates.

Erythrocytes (RBC) in the peritoneal cavity significantly increase the lethality of bacterial peritonitis. The lethality is known to be associated with, and perhaps due to, increased bacterial counts in the peritoneal cavity. The mechanism is unknown. In this study, we investigated the hypothesis that RBC scavenge reactive oxygen intermediates (ROI) and nitric oxide (NO), so that the counterprotective effect is due to a loss of the microbiostatic activity of both ROI and NO. To study this effect, rats were subjected to a peritoneal inoculation of live Escherichia coli without RBC (nonlethal dose) or with RBC (lethal dose). The adjuvant effect of RBC was not modified by NG-monomethyl-L-arginine (NMA, an NO synthase inhibitor), superoxide dismutase, catalase, mannitol, or a combination of these agents. Furthermore, the increased number of bacteria in the peritoneal cavity in the presence of RBC was unaffected by these treatments. The administration of NMA with bacteria alone (no RBC) converted a nonlethal model into a lethal one associated with higher intraperitoneal bacterial counts. A similar effect was seen with superoxide dismutase and catalase but not with mannitol. During bacterial peritonitis in the absence of RBC, superoxide and NO formation (determined by the total nitrite plus nitrate formed) was detected in the ascites and inducible NO synthase mRNA expression was present in the peritoneal cells. In the absence of RBC, superoxide was detected and oxidation of dihydrorhodamine to rhodamine was observed, indicating that peroxynitrite was produced. Both were blocked by the inclusion of RBC. Preinjection with a low inoculum of killed bacteria protected the rats from a subsequent lethal peritoneal bacterial challenge; this effect was reversed by scavenging ROI and NO. The protective effect of killed bacterial pretreatment was lost when RBC were placed in the peritoneal cavity. In vitro bactericidal activity of NO- and ROI-generating macrophages was also inhibited by RBC or by inhibiting ROI and NO formation. Taken together, these data are consistent with the hypothesis that RBC can impair bacterial clearance by removing both NO and ROI, suggesting that NO in combination with superoxide may be important to the antimicrobial defenses of the peritoneal cavity.

Animals↗
PubMed1996

Protein kinase A activation is required for IL-1-induced nitric oxide production by cardiac myocytes.

We have previously reported that interleukin-1 beta (IL-1) alone induced the transcription of inducible nitric oxide synthase (iNOS) mRNA and nitric oxide (NO) production by isolated neonatal rat cardiac myocytes (CM). The present studies were undertaken to explore the signal transduction pathways involved in IL-1-induced NO production by CM. The addition of IL-1 to CM resulted in a peak rise in both adenosine 3',5'-cyclic monophosphate (cAMP) and protein kinase A (PKA) activities by 10 min followed by rapid declines and return to basal levels within 60 min. The PKA inhibitor KT-5720 completely blocked NO-2 production by IL-1-stimulated CM (P < 0.01; n = 12). The protein kinase C (PKC) inhibitor, calphostin C, had no effect on NO2- production by IL-1 stimulated CM [P = not significant (NS); n = 12]. The addition of PKA+cAMP to cytosols derived from IL-1-treated CM did not directly enhance iNOS enzyme activity (P = NS; n = 3). CM treated with IL-1 alone stained positively for iNOS protein by immunohistochemistry. iNOS staining was absent in CM treated with IL-1+KT-5720. KT-5720 resulted in an earlier disappearance of iNOS mRNA from IL-1-treated CM, as detected by semiquantitative reverse transcriptase-polymerase chain reaction. We report for the first time that PKA (but not PKC) activation is required for IL-1-induced NO production by CM.

Animals↗
PubMed1996

Vascular gene transfer of the human inducible nitric oxide synthase: characterization of activity and effects on myointimal hyperplasia.

BACKGROUND: Nitric oxide (NO) has been shown to decrease myointimal hyperplasia in injured blood vessels. We hypothesize inducible No synthase (iNOS) gene transfer even at low efficiency will provide adequate local no production to achieve this goal. MATERIALS AND METHODS: A retroviral vector containing the human iNOS cDNA (DFGiNOS) was used to transfer the iNOS gene into vascular cells and isolated blood vessels to answer the following questions: can vascular endothelial and smooth muscle cells support iNOS activity and will low efficiency iNOS gene transfer suppress myointimal hyperplasia in injured porcine arteries? RESULTS: DFGiNOS-infected sheep pulmonary artery endothelial cells (SPAEC) expressed significant iNOS mRNA and protein, releasing nitrite levels of 155.0 +/- 10.7 nmol/mg protein/24 h vs. 5.5 +/- 1.1 by control cells. Transduced rat smooth muscle cells (RSMC) also expressed abundant iNOS mRNA and protein, but, in contrast to SPAEC, NO synthesis was dependent on exogenous tetrahydrobiopterin (BH4) (291.8 +/- 10.4 nmol nitrite/mg protein/24 hr with BH4, 37.7 +/- 2.6 without BH4). Only porcine arteries infected with DFGiNOS following balloon injury exhibited a 3-fold increase in total NO synthesis and a 15-fold increase in cGMP levels over control vessels in a BH4 dependent fashion, despite only a 1% gene transfer efficiency. Transfer of iNOS completely prevented the 53% increase in myointimal thickness induced by balloon catheter injury; the administration of a NOS inhibitor reversed this effect. CONCLUSIONS: These in vitro findings suggest that vascular iNOS gene transfer may be feasible. Furthermore, a low gene transfer efficiency may be sufficient to inhibit myointimal hyperplasia following arterial balloon injury, although a source of BH4 may be required.

Animals↗
PubMed1995

A central role for IL-1 beta in the in vitro and in vivo regulation of hepatic inducible nitric oxide synthase. IL-1 beta induces hepatic nitric oxide synthesis.

We have previously demonstrated that high levels of inducible nitric oxide synthase (iNOS) expression in hepatocytes required a combination of LPS and TNF-alpha, IL-1 beta, and IFN-gamma. The need for such a complex regulatory system seemed unwarranted based on the importance of NO in the liver. Therefore, we investigated whether individual cytokines could induce NO synthesis in hepatocytes and characterized some of the mechanisms involved. Rat hepatocytes were stimulated in vitro with escalating doses of TNF-alpha, IL-1 beta, or IFN-gamma. Only IL-1 beta induced high levels of iNOS mRNA and corresponding NO2- + NO3- production, and dexamethasone and cycloheximide blocked a majority of this response. Nuclear run-on experiments revealed that IL-1 beta upregulated iNOS gene transcription. IL-1 receptor antagonist protein (IL-1ra) competitively inhibited IL-1 beta-stimulated NO synthesis, implying activation through a cell-specific receptor. Rats injected with both LPS and IL-1ra showed decreased hepatic iNOS mRNA and plasma NO2- + NO3- compared with rats given LPS alone, indicating that IL-1 beta plays a role in regulating iNOS expression within the liver in vivo during endotoxemia. The soluble TNF receptor antagonist, PEG-(rsTNF-RI)2, also suppressed hepatic iNOS mRNA levels and plasma NO2- + NO3- increases, supporting a role for this cytokine in LPS-induced iNOS expression. Finally, IL-1 beta at high doses also induced iNOS mRNA and significant NO2- + NO3- production in cultures of primary human hepatocytes. These data indicate an important role for IL-1 beta in the regulation of hepatic NO synthesis.

Animals↗
PubMed1995

Regulation of constitutive nitric oxide synthase activity by the human heart.

We and others have provided indirect evidence for the presence of a constitutive nitric oxide synthase (cNOS) in the mammalian heart. We now provide more direct evidence for the regulation of a myocardial cNOS in the hearts of patients undergoing elective cardiopulmonary bypass (CPB). cNOS enzyme activity was demonstrable in both cytosolic (8.3 +/- 0.02 pmol/min/mg) and membrane (11.1 +/- 0.4 pmol/min/mg) preparations derived from human atrial pectinate muscles obtained at the time of CPB (n = 6). Plasma nitrite (NO2-) + plasma nitrate (NO3-) levels from the beating hearts of patients before bypass were reduced from 146 +/- 33 to 5.1 +/- 50 pmol/min/g after cardiac arrest during CPB (n = 23; p < 0.002 by Student's t test). Thus, the human myocardium constitutively produces nitric oxide that is regulated by the contractile state of the heart.

Adult↗