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Biomedical subjects

M A West

Publications and source records attributed to M A West.

At least 37 records · Page 2Linked to original sources
PubMed1999

In vitro macrophage endotoxin tolerance: defective in vitro macrophage map kinase signal transduction after LPS pretreatment is not present in macrophages from C3H/HeJ endotoxin resistant mice.

Altered endotoxin (LPS) signal transduction in macrophages (Mphi) may mediate development of organ dysfunction in sepsis. C3H/HeJ Mphi have a specific genetic defect that renders them "tolerant" to in vitro LPS activation. LPS tolerance can be induced in normal C3H/HeN Mphi following in vitro LPS pretreatment. In these experiments, in vitro LPS-stimulated activation of Mphi mitogen-activated protein (MAP) kinases were compared in C3H/HeJ and C3H/HeN mice. C3H/HeJ and C3H/HeN Mphi were cultured+/-10 ng/mL LPS pretreatment for 24 h, then stimulated with 0-1,000 ng/mL LPS for 6 h. Western blots were performed on lysates with monoclonal antibody to active ERK1,2 (p42/44), stress-activated protein kinase (SAPK, p54/46), and p38 kinase. Supernatant TNF or IL-1 was determined by bioassay. High dose LPS stimulation activated ERK, SAPK, and p38 kinases in both C3H/HeN and C3H/HeJ Mphi. ERK activation, p46 SAPK, and p38 activation were inhibited in C3H/HeN Mphi after LPS pretreatment, whereas they were unchanged or increased in HeJ Mphi. TNF secretion was significantly decreased in C3H/HeN Mphi following LPS pretreatment, but absent in C3H/HeJ Mphi at all times. Mphi from normal C3H/HeN mice rendered endotoxin tolerant by in vitro, low dose LPS pretreatment have specific signal transduction defects that are not present in genetically LPS hyporesponsive C3H/HeJ mice.

Animals↗
PubMed1999

Inhibitory kappaBalpha control of nuclear factor-kappaB is dysregulated in endotoxin tolerant macrophages.

The transcription factor nuclear factor (NF)-kappaB is thought to be required for endotoxin-stimulated tumor necrosis factor (TNF) and interleukin (IL)-1 gene transcription. Nuclear translocation of NF-kappaB is regulated by the cytoplasmic inhibitory factor IkappaBalpha. Low-dose lipopolysaccharide (LPS) pretreatment modulates cytokine release by altering subsequent LPS-activated signal transduction pathways. In this study, we examined the effect of LPS pretreatment exposure on IkappaBalpha and NF-kappaB following activation with LPS. Murine macrophages (Mphi were exposed to a range of LPS concentrations +/-24 h PreRx with 10 ng/mL LPS pretreatment. Cytoplasmic IkappaBalpha (Western immunoblot) and NF-kappaB (gel-shift assay) were assayed 30 min after LPS activation. Gene transcription for TNF was measured 6 h after LPS activation using RT-PCR. In the absence of LPS pretreatment, IkappaBalpha disappeared from the cytoplasm coincident with nuclear translocation of NF-kappaB. Tolerant Mphi had markedly enhanced levels of IkappaBalpha and normal to increased levels of NF-kappaB translocation with a different electrophoretic shift. LPS activation enhanced cytokine gene transcription in a dose-dependent manner, and this was unaltered by LPS pretreatment. Endotoxin-tolerant Mphi also had increased cytoplasmic levels of the p65 subunit of NF-kappaB. LPS tolerance is associated with increases of cytoplasmic IkappaBalpha p65, as well as enhanced NF-kappaB. We conclude that control of NF-kappaB translocation by IkappaBalpha is dysregulated in endotoxin-tolerant Mphi.

Animals↗
PubMed1999

Wound management in perforated appendicitis.

Open wound management after perforated appendicitis was common practice but, recently, primary closure has been advocated to reduce costs and morbidity. Hospital records from 319 adults who underwent appendectomy from 1993 to 1996 were reviewed to identify surgical wound infections (SWIs) and examine risk factors. Information about age, length of stay (LOS), operative time, white blood cell count, and antibiotic administration were obtained. Perforation was either noted at operation or identified microscopically by the pathologist. If primary wound closure was performed, patients with acute appendicitis and perforation had a 4-fold higher readmission rate, a 5-fold increase in SWI, and twice the LOS compared with patients with acute appendicitis without perforation. Patients with grossly perforated acute appendicitis had no difference in LOS if the wound was treated open or closed primarily. No patient with microscopic perforation and primary wound closure developed SWI. Primary wound closure after acute appendicitis was safe in the absence of clinical perforation. In the presence of clinical appendiceal perforation the wound should be left open.

Acute Disease↗
PubMed1999

Civilian rectal trauma: a changing perspective.

BACKGROUND: Recently the Organ Injury Scaling Committee of the American Association for the Surgery of Trauma developed a Rectal Injury Scaling System (RISS). Little data exist regarding its clinical utility. METHODS: We retrospectively reviewed 45 patients with rectal injuries to assess the impact of the RISS on patient management and outcome. We compared RISS grade I patients (group I, partial-thickness injury) with patients with grades 2, 3, and 4 injuries (group II, full-thickness injury). RESULTS: Group II underwent distal rectal washout and repair of the injury twice as often and had a significantly higher rate of diversion of the fecal stream. This was associated with a 3-fold increase in complications. The only complications in group I were in patients managed with diversion of the fecal stream and distal rectal washout. CONCLUSIONS: Our data suggest that aggressive surgical management for RISS grade I injury may not be necessary. Implementation of therapy based on the RISS may improve outcomes of civilian rectal trauma.

Adolescent↗
PubMed1998

Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells.

The Arabidopsis LEAFY COTYLEDON1 (LEC1) gene is required for the specification of cotyledon identity and the completion of embryo maturation. We isolated the LEC1 gene and showed that it functions at an early developmental stage to maintain embryonic cell fate. The LEC1 gene encodes a transcription factor homolog, the CCAAT box-binding factor HAP3 subunit. LEC1 RNA accumulates only during seed development in embryo cell types and in endosperm tissue. Ectopic postembryonic expression of the LEC1 gene in vegetative cells induces the expression of embryo-specific genes and initiates formation of embryo-like structures. Our results suggest that LEC1 is an important regulator of embryo development that activates the transcription of genes required for both embryo morphogenesis and cellular differentiation.

Amino Acid Sequence↗
PubMed1998

Assessment of the relationship between timing of fixation of the fracture and secondary brain injury in patients with multiple trauma.

BACKGROUND: It has been suggested that early fixation of a fracture is deleterious to eventual neurologic outcome. We undertook this study to determine whether the timing of fracture fixation is correlated to neurologic outcome. METHODS: We retrospectively reviewed patients with severe head and orthopedic injuries requiring fracture fixation. Patients were divided into two groups: early fracture fixation (< 24 hours after injury) and late fracture fixation (> 24 hours after injury). RESULTS: One hundred twenty-three patients met entry criteria. During fracture fixation, the early group had a significant 2-, 3-, and 2-fold increase in crystalloid, blood infusion, and blood loss, respectively. There was no difference in oxygen saturation and systolic blood pressure or episodes of cranial hypertension or hypoperfusion. There was no difference in outcomes as measured by in-hospital complications, stay in the intensive care unit or hospital, mortality rates, hospital discharge or follow-up Glasgow Coma Scores, or long-term orthopedic or neurologic results. CONCLUSIONS: Patients undergoing fracture fixation with severe head injury mandate monitoring of intracranial pressure and perfusion and tailored fluid resuscitation to meet specific organ end points. Integrating end organ perfusion and pressure with meticulous fluid status during the definitive repair phase may reduce the exposure to secondary brain injury in patients undergoing early fracture fixation.

Adult↗
PubMed1998

Lipopolysaccharide pretreatment produces macrophage endotoxin tolerance via a serum-independent pathway.

BACKGROUND: Lipopolysaccharide activation (LPSa) of macrophages is thought to occur via a CD14-dependent mechanism with a requirement for the serum factor, lipopolysaccharide binding protein. LPS-stimulated, CD14-dependent signal transduction is associated with phosphorylation of mitogen-activated protein kinase (MAPK), nuclear factor-kappaB (NF-kappaB) translocation, and secretion of tumor necrosis factor (TNF) and interleukin-1 (IL-1). Macrophage endotoxin tolerance after low-dose LPS pretreatment (LPSp) is characterized by inhibition of LPSa-stimulated TNF and augmentation of IL-1 secretion. We sought to determine the role of CD14-dependent pathways in the induction of endotoxin tolerance by comparing the effects of LPSp in the presence or absence of serum. METHODS: Murine peritoneal macrophages were exposed to a range of LPSp concentrations in the presence or absence of serum. MAPK activation and NF-kappaB were assayed 30 minutes after LPSp stimulation. TNF production and IL-1 were measured 6 hours after stimulation with 100 ng/mL LPSa, with or without 24-hour 10 ng/mL LPSp. RESULTS: In the presence of serum, 100 ng/mL LPSp activated MAPK and NF-kappaB, whereas no activation of MAPK or NF-kappaB was seen at this LPSp concentration in the absence of serum. The absence of serum during 10 ng/mL LPSp did not prevent LPSp-mediated inhibition of TNF secretion, and it significantly augmented IL-1 secretion after stimulation with 100 ng/mL LPSa in the presence of serum. CONCLUSION: Induction of the alterations in subsequent LPSa-stimulated cytokine secretion characteristic of endotoxin tolerance by LPSp occurs via a serum-independent signal transduction pathway.

Acute-Phase Proteins↗
PubMed1998

Acetazolamide treatment prevents in vitro endotoxin-stimulated tumor necrosis factor release in mouse macrophages.

We previously showed that incubation in carbon dioxide (CO2), but not air or helium (He), markedly decreased macrophage intracellular pH (pHi) and resulted in reversible inhibition of lipopolysaccharide- (LPS) stimulated tumor necrosis factor (TNF) and interleukin-1 release. We sought to determine whether carbonic anhydrase inhibition with acetazolamide would prevent CO2-mediated inhibition of LPS-stimulated TNF release. Murine peritoneal macrophages were treated with acetazolamide for 1 h under control atmosphere (95% air/5% CO2) and then switched to incubator modules containing: 1) 80% CO2/20% O2, 2) 80% He/20% O2, or 3) 100% air. Before transfer to experimental atmospheric conditions the macrophages were stimulated with 0 or 1 microg/mL of LPS (Escherichia coli 0111 B4). Supernatant TNF was measured 4 h later by bioassay. In parallel experiments LPS-stimulated cytokine mRNA was estimated using reverse transcriptase polymerase chain reaction (RT-PCR) 2 h after LPS stimulation. Viability was determined using dye uptake. Incubation in CO2 or helium had no effect on TNF production in the absence of LPS. In the absence of acetazolamide CO2 produced marked inhibition of LPS-stimulated TNF release, but this was not blocked by the presence of acetazolamide. This CO2-mediated inhibition of TNF was associated with normal levels of TNF mRNA. In acetazolamide-treated macrophages, LPS resulted in a dose-dependent inhibition of TNF release when the cells were incubated in air or helium. Maintenance of normal intracellular pH is required for TNF release, but not TNF mRNA induction by LPS. Factors that alter intracellular pH regulation may modulate LPS-stimulated cytokine production.

Acetazolamide↗
PubMed1997

Isolation and ultrastructural study of the flagellar basal body complex from Rhodobacter sphaeroides WS8 (wild type) and a polyhook mutant PG.

Filament-Hook-Basal Body (FHBB) complexes were isolated from the purple non-sulphur facultative anaerobic bacterium Rhodobacter sphaeroides (WS8) by lysozyme digestion of the cells followed by an alkaline treatment and ultracentrifugation, and they were analysed by electron microscopy. The structure is composed of a filament linked through an enlarged junction to the hook and a basal body composed of L and P rings, a rod, and a less well-defined cytoplasmic ring that has evidence of additional attached structures. Hook-basal body complexes isolated from a mutant (PG) which produces an extended hook but no filament shows basal body structures identical to those of wild-type FHBBs.

Bacterial Proteins↗
PubMed1997

The role of ADP-ribosylation factor and phospholipase D in adaptor recruitment.

AP-1 and AP-2 adaptors are recruited onto the TGN and plasma membrane, respectively. GTPgammaS stimulates the recruitment of AP-1 onto the TGN but causes AP-2 to bind to an endosomal compartment (Seaman, M.N.J., C.L. Ball, and M.S. Robinson. 1993. J. Cell Biol. 123:1093-1105). We have used subcellular fractionation followed by Western blotting, as well as immunofluorescence and immunogold electron microscopy, to investigate both the recruitment of AP-2 adaptors onto the plasma membrane and their targeting to endosomes, and we have also examined the recruitment of AP-1 under the same conditions. Two lines of evidence indicate that the GTPgammaS-induced targeting of AP-2 to endosomes is mediated by ADP-ribosylation factor-1 (ARF1). First, GTPgammaS loses its effect when added to ARF-depleted cytosol, but this effect is restored by the addition of recombinant myristoylated ARF1. Second, adding constitutively active Q71L ARF1 to the cytosol has the same effect as adding GTPgammaS. The endosomal membranes that recruit AP-2 adaptors have little ARF1 or any of the other ARFs associated with them, suggesting that ARF may be acting catalytically. The ARFs have been shown to activate phospholipase D (PLD), and we find that addition of exogenous PLD has the same effect as GTPgammaS or Q71L ARF1. Neomycin, which inhibits endogenous PLD by binding to its cofactor phosphatidylinositol 4,5-bisphosphate, prevents the recruitment of AP-2 not only onto endosomes but also onto the plasma membrane, suggesting that both events are mediated by PLD. Surprisingly, however, neither PLD nor neomycin has any effect on the recruitment of AP-1 adaptors onto the TGN, even though AP-1 recruitment is ARF mediated. These results indicate that different mechanisms are used for the recruitment of AP-1 and AP-2.

ADP-Ribosylation Factor 1↗
PubMed1997

LPS pretreatment reprograms macrophage LPS-stimulated TNF and IL-1 release without protein tyrosine kinase activation.

Pretreatment of macrophages with low-dose endotoxin (LPSp) profoundly alters cytokine release in response to subsequent LPSa activation. These qualitative and quantitative alterations in cytokine release have been termed macrophage reprogramming. Macrophage activation by LPS is thought to occur via a mechanism involving an early protein tyrosine kinase (PTK) phosphorylation step. PTK inhibition with genistein or herbimycin A blocks LPSa-stimulated secretion of tumor necrosis factor (TNF) and interleukin-1 (IL-1). In this study we investigated whether a PTK pathway participates in LPSp pretreatment reprogramming. We show that LPSp pretreatment inhibited TNF and augmented IL-1 release in response to subsequent LPSa stimulation. Blockade of PTK activation pathways during the interval when macrophages were exposed to LPSp prevented mitogen-activated protein kinase phosphorylation, as well as LPSp-stimulated release of TNF and IL-1, but did not block LPSp reprogramming effects. We conclude that LPSp pretreatment reprogramming of macrophage cytokine production does not require PTK activation.

Animals↗
PubMed1997

In vivo endotoxin tolerance: impaired LPS-stimulated TNF release of monocytes from patients with sepsis, but not SIRS.

In vitro pretreatment of human monocytes (MO) with low-dose lipopolysaccharide (LPSp) inhibits TNF release in response to subsequent LPSa activation. Septic patients are often indistinguishable from patients with systemic inflammatory response syndrome (SIRS). We hypothesized that in vivo exposure to "septic" stimuli impairs subsequent LPSa-stimulated MO TNF production in vitro. Human peripheral MO were obtained after informed consent from controls or patients with sepsis, SIRS, or posttrauma [ACCP/SCCM definitions]. Cells were plated in vitro, incubated 24 hr, and then stimulated with 0-1000 ng/ml LPSa for 4 hr. Parallel control MO were incubated in vitro with 100 ng/ml LPSp for 24 hr and then stimulated with 1000 ng/ml LPSa for 4 hr. Supernatant TNF (mean U/ml +/- SEM) was measured by bioassay. ANOVA was used to determine statistical significance. In vitro LPSp pretreatment markedly inhibited subsequent LPSa-stimulated TNF release. In vitro LPSa-stimulated TNF release was likewise significantly inhibited with MO from septic patients compared to controls. Inhibition was more profound in septic patients with shock (not shown). No impaired TNF release was seen with MO from SIRS or trauma patients. In conclusion, in vivo preexposure to inflammatory stimuli in septic patients alters monocyte regulation in a manner similar to in vitro endotoxin tolerance. Provocative in vitro monocyte LPS stimulation may distinguish patients with sepsis and SIRS.

Adult↗
PubMed1997

Protein kinase C regulates macrophage tumor necrosis factor secretion: direct protein kinase C activation restores tumor necrosis factor production in endotoxin tolerance.

BACKGROUND: Macrophages pretreated in vitro with endotoxin (LPSp) secrete less tumor necrosis factor (TNF) in response to a second LPS activating (LPSa) stimulus. Protein kinase C (PKC) is required for TNF secretion in a macrophage stimulated with LPSa. In these experiments we examined the role of PKC in TNF signal transduction in naive and tolerant macrophages. METHODS: Murine macrophages were cultured +/- LPSp for 24 hours. Cultures were washed and treated for 1 hour with PKC inhibitors or phorbol myristate acetate (PMA), a direct PKC activator. Cells were then stimulated with a range of LPSa for 6 hours, and TNF was determined by bioassay. RESULTS: LPSa-stimulated TNF secretion by nontolerant macrophages was inhibited by LPSp in the absence of PMA. PKC inhibitors decreased TNF by naive macrophages and exaggerated inhibition in tolerant cells. Depletion of PKC by 24 hours of PMA decreased TNF production by both naive and tolerant macrophages. PKC activation with PMA 1 hour before LPSa augmented TNF secretion in naive cells and reversed TNF inhibition of tolerant cells. CONCLUSIONS: Direct PKC activation with PMA restored TNF secretion in LPS-tolerant macrophages. Endotoxin tolerance may alter the LPSa signal transduction pathway between the LPS receptor and PKC activation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Opposite effects on two different measures of retention following unilateral inactivation of the amygdala.

We previously reported finding differential effects of unilateral infusion of lidocaine into the right and left amygdalae prior to a retention test 24 h after one-trial inhibitory avoidance training. The present study was undertaken to determine whether the degree of training in an escape task influences the effects of unilateral inactivation of the amygdala with lidocaine prior to the retention test. Male Sprague-Dawley rats, implanted with bilateral cannulae aimed at the amygdaloid complex received 0 (no shock), 2, or 10 training trials in a footshock-motivated escape task (0.4 mA). Forty-eight h later, the rats received bilateral microinfusions into the amygdalae of either buffer, or lidocaine into one amygdala and buffer in the other, 5 min before they were tested for retention of the escape training using a continuous multiple trial inhibitory avoidance (CMIA) procedure (0.4 mA). Forty-eight h after CMIA training the animals were retested, this time without influence of drugs. As indicated by initial response latencies on the first retention test, unilateral inactivation of either the right or the left amygdala severely impaired retention performance for the escape training regardless of the number of prior escape training trials received. However, unilateral inactivation of either amygdala had no effect on acquisition of CMIA. Furthermore, unilateral inactivation of either amygdala during CMIA training did not affect retention tested 48 h later. These results suggest that the differential involvement of the right and left amygdalae may be limited to certain training conditions. Furthermore, these findings demonstrate opposite effects on 2 different measures of retention indicating that the method of testing retention affects the interpretation of the memory impairment induced by infusion of lidocaine into the amygdala.

Amygdala↗
PubMed1997

Mechanism of decreased in vitro murine macrophage cytokine release after exposure to carbon dioxide: relevance to laparoscopic surgery.

OBJECTIVE: The objective of this study was to determine the effect of carbon dioxide (CO2) on the function of peritoneal macrophages. SUMMARY BACKGROUND DATA: Laparoscopic surgery is associated with minimal pain, fever, and low levels of inflammatory cytokines. To understand the mechanisms involved, the authors investigated the effect of different gases on murine peritoneal macrophage intracellular pH and correlated these alterations with alterations in LPS-stimulated inflammatory cytokine release. METHODS: Peritoneal macrophages were incubated for 2 hours in air, helium, or CO2, and the effect of the test gas on immediate or next day lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) and interleukin-1 release compared. Cytosolic pH of macrophages exposed to test gases was measured using single-cell fluorescent imaging. The in vivo effects of test gases were determined in anesthetized rats during abdominal insufflation. RESULTS: Macrophages incubated in CO2 produced significantly less TNF and interleukin-1 in response to LPS compared to incubation in air or helium. Cytokine production returned to normal 24 hours later. Exposure to CO2, but not air or helium, caused a marked cytosolic acidification. Pharmacologic induction of intracellular acidification to similar levels reproduced the inhibitory effect. In vitro studies showed that CO2 insufflation lowered tissue pH and peritoneal macrophage LPS-stimulated TNF production. CONCLUSIONS: The authors propose that cellular acidification induced by peritoneal CO2 insufflation contributes to blunting of the local inflammatory response during laparoscopic surgery.

Animals↗
PubMed1997

Minor psychiatric disorder in NHS trust staff: occupational and gender differences.

BACKGROUND: It is widely suggested that many National Health Service (NHS) workers experience high levels of minor psychiatric disorder. However, inadequacies of sampling and measurement in studies to date have not allowed this suggestion to be properly evaluated. METHOD: The present study was designed to overcome these methodological weaknesses by using a sample of over 11,000 employees from 19 NHS trusts and a well-established measure of minor psychiatric disorder for which there are comparative data. RESULTS: The findings show that 26.8% of the health service workers reported significant levels of minor psychiatric disorder, compared with 17.8% of people in the general population. Psychiatric morbidity was highest among managers, doctors, nurses and professions allied to medicine, with each of these groups recording higher rates than their professional counterparts outside the health service. It was lower among those in support occupations, such as administrative and ancillary staff. A feature of the findings was that female doctors and managers showed a much higher prevalence of minor psychiatric disorder than their male colleagues. CONCLUSION: Studies are required to establish the organisational, occupational and individual determinants of minor psychiatric disorder among NHS employees.

Administrative Personnel↗
PubMed1996

Discordant reprogramming of LPS-stimulated cytokine gene transcription and secretion by macrophages after LPS pretreatment.

Dysregulated macrophage (Mphi) cytokine release occurs during systemic inflammation and may predispose to organ failure. We showed that Mphis pretreated (PreRx) in vitro with low-dose LPSp are "reprogrammed" to release less TNF and more IL-1 in response to subsequent LPS activation (LPSa). The effects of this LPSp "reprogramming" on Mphi cytokine gene transcription were investigated in the present study. Murine peritoneal exudate Mphis were cultured in vitro 48 hr, then PreRx 24 hr +/- 100 ng/ml of LPSp. Cultures were stimulated with 0-1000 ng/ml LPSa and 6-hr supernatant TNF and IL-1 were measured using specific bioassays. Cytokine gene transcription was estimated 6 hr after LPSa using RT-PCR. PreRx with LPSp inhibited TNF and augmented IL-1 release by LPSa. PreRx with LPSp significantly inhibited cytokine gene transcription; however, messages for both TNF and IL-1 were detectable after high-dose LPSa. Despite LPSp inhibition of IL-1 transcription by most LPSa concentrations, IL-1 protein was augmented by PreRx. High-dose LPSa can override LPSp reprogrammed inhibition of cytokine gene transcription, but altered TNF and IL-1 protein release after LPSp may be regulated posttranscriptionally.

Animals↗