<A<em>b</em>stractText>The role of tumor-associated macrophages (TAMs) in the cancer immune landscape and their potential as treatment targets or modulators of response to treatment are gaining increasing interest. TAMs display high molecular and functional complexity. Therefore their o<em>b</em>jective assessment as <em>b</em>reast cancer <em>b</em>iomarkers is critical. The aims of this study were to o<em>b</em>jectively determine the in situ expression and significance of TAM <em>b</em>iomarkers (CD68, CD163, and MMP-9) in <em>b</em>reast cancer and to identify su<em>b</em>classes of patients who could <em>b</em>enefit from TAM-targeting therapies.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>We measured CD68, CD163, and MMP-9 protein expression in formalin-fixed paraffin-em<em>b</em>edded tissues of <em>b</em>reast carcinomas represented in tissue microarray format using mu<em>lt</em>iplexed quantitative immunofluorescence (QIF) in two independent Yale cohorts: cohort A-n = 398, estrogen receptor-positive (ER⁺) and ER^- cases-and the triple-negative <em>b</em>reast cancer (TN<em>B</em>C)-only cohort <em>B</em> (n = 160). Associations <em>b</em>etween macrophage markers, ER status, and survival were assessed. Protein expression measured <em>b</em>y QIF was compared with mRNA expression data from the META<em>B</em>RIC study.</p><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>All three macrophage markers were co-expressed, displaying higher expression in ER^- cancers. High pan-macrophage marker CD68 correlated with poorer overall survival (OS) only in ER^- cases of cohort A (P = 0.02). High expression of CD163 protein in TAMs was associated with improved OS in ER^- cases (cohort A, P = 0.03 and TN<em>B</em>C cohort <em>B</em>, P = 0.04, respectively) <em>b</em>ut not in ER⁺ cancers. MMP-9 protein was not individually associated with OS. High expression of MMP-9 in the CD68⁺/CD163⁺ TAMs was associated with worse OS in ER⁺ tumors (P &<em>lt</em>;0.001) <em>b</em>ut not in ER^- cancers. In the META<em>B</em>RIC dataset, mRNA levels followed the co-expression pattern o<em>b</em>served in QIF <em>b</em>ut did not always show the same trend regarding OS.</p><p><div>(<em>b</em>)CONCLUSIONS</<em>b</em>)</div>Macrophage activity markers correlate with survival differently in ER⁺ and ER^- cancers. The association <em>b</em>etween high co-expression and co-localization of MMP-9/CD163/CD68 and poor survival in ER⁺ cancers suggests that these cancers may <em>b</em>e candidates for macrophage-targeted therapies.</p>

BACKGROUND

Resection and liver transplantation (LT) are the only curative options for hepatocellular carcinoma in cirrhotic patients (HCC-cirr).

OBJECTIVE

We tried to define the best primary intention-to-treat strategy in patients undergoing either resection or LT for early single HCC-cirr (≤5 cm).

METHODS

From 1990 to 2010, 198 patients with early HCC-cirr underwent either resection (group R, n = 97) or LT (group T, n = 101) as the primary procedure. Our policy was to prioritize Childs A patients with peripheral lesions for resection rather than LT. Patient and tumor characteristics, and outcomes (recurrence-free survival [RFS] and overall survival [OS]), were studied.

RESULTS

A longer diagnosis-to-surgery interval, more Child Pugh B/C patients, and more tumor nodules (on histopathological examination) were found in group T patients. The postoperative mortality (4.1% vs 3.0%, P = 0.72) and rate of major complications (19.1% vs 24.7%, P = 0.35) were similar in groups R and T, respectively, whereas tumor recurrence was higher in group R (62% vs 10% in group T, P < 0.0001). The 5-year OS (75% vs 52%, P = 0.0008) and RFS (72% vs 20%, P < 0.0001) were better in group T; similarly, more patients were disease free at last follow-up (27% vs 62%, P < 0.0001). Resection as the surgical procedure, tumor diameter 3 cm or more on histology, and microvascular tumor invasion were poor prognostic factors for OS and RFS. Including dropout patients from LT list in the analysis, the outcomes in group T were still better (70% and 61% vs 51% and 36% at 5 and 10 years, P = 0.01).

CONCLUSIONS

On an intention-to-treat basis, LT is associated with the best survival outcomes in patients with early HCC-cirr. Resection may achieve comparable OS in patients with single HCC-cirr of size smaller than 3 cm; however, the RFS still remains lower than that in patients of group T. This study could serve as a guide for HCC-cirr patients who are candidates for either resection or LT.

<A<em>b</em>stractText>There is a need for early detection of colorectal cancer (CRC) at precancerous-stage adenoma. Here, we identified novel faecal <em>b</em>acterial markers for diagnosing adenoma.</A<em>b</em>stractText><A<em>b</em>stractText>This study included 1012 su<em>b</em>jects (274 CRC, 353 adenoma and 385 controls) from two independent Asian groups. Candidate markers were identified <em>b</em>y metagenomics and validated <em>b</em>y targeted quantitative PCR.</A<em>b</em>stractText><p><div>(<em>b</em>)RESULTS</<em>b</em>)</div>Metagenomic analysis identified '<i>m3</i>' from a <i>Lachnoclostridium</i> sp., <i>Fuso<em>b</em>acterium nucleatum</i> (<i>Fn</i>) and <i>Clostridium hathewayi</i> (<i>Ch</i>) to <em>b</em>e significantly enriched in adenoma. Faecal <i>m3</i> and <i>Fn</i> were significantly increased from normal to adenoma to CRC (p&<em>lt</em>;0.0001, linear trend <em>b</em>y one-way ANOVA) in group I (n=698), which was further confirmed in group II (n=313; p&<em>lt</em>;0.0001). Faecal <i>m3</i> may perform <em>b</em>etter than <i>Fn</i> in distinguishing adenoma from controls (areas under the receiver operating characteristic curve (AUROCs) <i>m3</i>=0.675 vs <i>Fn</i>=0.620, p=0.09), while <i>Fn</i> performed <em>b</em>etter in diagnosing CRC (AUROCs <i>Fn</i>=0.862 vs <i>m3</i>=0.741, p&<em>lt</em>;0.0001). At 78.5% specificity, <i>m3</i> and <i>Fn</i> showed sensitivities of 48.3% and 33.8% for adenoma, and 62.1% and 77.8% for CRC, respectively. In a su<em>b</em>group tested with faecal immunochemical test (FIT; n=642), <i>m3</i> performed <em>b</em>etter than FIT in detecting adenoma (sensitivities for non-advanced and advanced adenomas of 44.2% and 50.8% <em>b</em>y <i>m3</i> (specificity=79.6%) vs 0% and 16.1% <em>b</em>y FIT (specificity=98.5%)). Com<em>b</em>ining with FIT improved sensitivity of <i>m3</i> for advanced adenoma to 56.8%. The com<em>b</em>ination of <i>m3</i> with <i>Fn</i>, <i>Ch</i>, <i>Bacteroides clarus</i> and FIT performed <em>b</em>est for diagnosing CRC (specificity=81.2% and sensitivity=93.8%).</p><p><div>(<em>b</em>)CONCLUSION</<em>b</em>)</div>This study identifies a novel <em>b</em>acterial marker <i>m3</i> for the non-invasive diagnosis of colorectal adenoma.</p>

<A<em>b</em>stractText>To examine the associations among preschoolers fundamental motor skills, screen-time, physical activity (PA), and sedentary <em>b</em>ehavior (SB).</A<em>b</em>stractText><A<em>b</em>stractText>Children ages 3-4years were enrolled in a prospective o<em>b</em>servational trial of PA. Trained assessors conducted the Test of Gross Motor Development-3rdedition (TGMD-3), and the Movement Assessment Battery for Children-2nd edition, and parent-reported child screen-time and sociodemographic information. Children wore an accelerometer for 7days to examine SB and total PA (TPA). TPA was further characterized as moderate-to-vigorous PA (MVPA) or vigorous PA (VPA). Mixed linear models were calculated, controlling for age (for TGMD-3), sex, household income, and accelerometer wear time (for accelerometry models), with childcare center as a random effect. The primary analysis reported on the cross-sectional <em>b</em>aseline data of 126 children with complete fundamental motor skill and screen-time data; a su<em>b</em>analysis included 88 children with complete accelerometry data.</A<em>b</em>stractText><p><div>(<em>b</em>)Resu<em>lt</em>s</<em>b</em>)</div>Children were 3.4 ± 0.5years of age (54% girls; 46% white, 42% African American, 12% other). A total of 48% lived in households at or <em>b</em>elow the federal poverty level. Children engaged in 5.1 ± 3.6h/day of screen-time. Children's screen-time was inversely related to the Movement Assessment Battery for Children-2nd edition, manual dexterity skills percentile (<em>β</em> (SE) = -1.7 (0.8), <i>p</i> = 0.049). In the accelerometry su<em>b</em>sample, children engaged in 5.9 ± 0.9h/day of TPA of which 1.7 ± 0.6h/day was MVPA. Boys engaged in more MVPA and VPA and less SB compared with girls (all <i>p</i> &<em>lt</em>; 0.05). A higher TGMD-3, total score (<em>β</em> (SE) = 0.4 (0.2), <i>p</i> = 0.017) and locomotor score (<em>β</em> (SE) = 0.7 (0.3), <i>p</i> = 0.018) were associated with more VPA <em>b</em>ut not with TPA or MVPA. Screen-time and television in the <em>b</em>edroom were not related to SB, TPA, MVPA, or VPA.</p><A<em>b</em>stractText>Children's motor skills were positively related to VPA <em>b</em>ut inversely related to screen-time. Further inquiry into the implications of high exposure to screen-time in young children is needed.</A<em>b</em>stractText>

<A<em>b</em>stractText>Glo<em>b</em>ally, access to evidence-<em>b</em>ased psychological treatment is limited. Innovative self-help methods using smartphone applications and low-cost virtual reality have the potential to significantly improve the accessi<em>b</em>ility and scala<em>b</em>ility of psychological treatments.</A<em>b</em>stractText><A<em>b</em>stractText>To examine the effectiveness of ZeroPho<em>b</em>ia, a fully self-guided app-<em>b</em>ased virtual reality cognitive <em>b</em>ehavior therapy (VR CBT) using low-cost (card<em>b</em>oard) virtual reality goggles compared with a wait-list control group and to determine its user friendliness.</A<em>b</em>stractText><A<em>b</em>stractText>In a single-<em>b</em>lind randomized clinical trial, participants were enrolled <em>b</em>etween March 24 and Septem<em>b</em>er 28, 2017, and randomly assigned (1:1) <em>b</em>y an independent researcher to either VR CBT app or a wait-list control group. A total of 193 individuals aged 18 to 65 years from the Dutch general population with acropho<em>b</em>ia symptoms and access to an Android smartphone participated. The 6 animated modules of the VR-CBT app and gamified virtual reality environments were delivered over a 3-week period in participants' natural environment. Assessments were completed at <em>b</em>aseline, immediately after treatment, and at 3-month follow-up. Analysis <em>b</em>egan April 6, 2018, and was intention to treat.</A<em>b</em>stractText><A<em>b</em>stractText>Self-guided app-<em>b</em>ased VR CBT.</A<em>b</em>stractText><A<em>b</em>stractText>The primary outcome measure was the Acropho<em>b</em>ia Questionnaire. The hypothesis was formulated prior to data collection.</A<em>b</em>stractText><A<em>b</em>stractText>In total, 193 participants (129 women [66.84%]; mean [SD] age, 41.33 [13.64] years) were randomly assigned to intervention (n = 96) or a wait-list control group (n = 97). An intent-to-treat analysis showed a significant reduction of acropho<em>b</em>ia symptoms at posttest at 3 months for the VR-CBT app compared with the controls (<em>b</em> = -26.73 [95% CI, -32.12 to -21.34]; P &<em>lt</em>; .001; d = 1.14 [95% CI, 0.84 to 1.44]). The num<em>b</em>er needed to treat was 1.7. Sensitivity and ro<em>b</em>ustness analysis confirmed these findings. Pretreatment attrition was 22 of 96 (23%) <em>b</em>ecause of smartphone incompati<em>b</em>ility. Of the 74 participants who started using the VR-CBT app, 57 (77%) completed the intervention fully.</A<em>b</em>stractText><A<em>b</em>stractText>A low-cost fully self-guided app-<em>b</em>ased virtual reality cognitive <em>b</em>ehavioral therapy with rudimentary virtual reality goggles can produce large acropho<em>b</em>ia symptom reductions. To our knowledge, this study is the first to show that virtual reality acropho<em>b</em>ia treatment can <em>b</em>e done at home without the intervention of a therapist.</A<em>b</em>stractText><A<em>b</em>stractText>Trialregister.nl identifier: NTR6442.</A<em>b</em>stractText>

BACKGROUND

High score of model for end-stage liver diseases (MELD) before liver transplantation (LT) indicates poor prognosis. Artificial liver support system (ALSS) has been proved to effectively improve liver and kidney functions, and thus reduce the MELD score. We aim to evaluate whether downgrading MELD score could improve patient survival after LT.

RESULTS

One hundred and twenty-six LT candidates with acute-on-chronic hepatitis B liver failure and MELD score ≥30 were included in this prospective study. Of the 126 patients, 42 received emergency LT within 72 h (ELT group) and the other 84 were given ALSS as salvage treatment. Of the 84 patients, 33 were found to have reduced MELD score (<30) on the day of LT (DGM group), 51 underwent LT with persistent high MELD score (N-DGM group). The median waiting time for a donor was 10 for DGM group and 9.5 days for N-DGM group. In N-DGM group there is a significantly higher overall mortality (43.1%) than that in ELT group (16.7%) and DGM group (15.2%). N-DGM (vs. ECT and DGM) was the only independent risk factor of overall mortality (P = 0.003). Age >40 years and the interval from last ALSS to LT >48 h were independent negative influence factors of downgrading MELD.

CONCLUSIONS

Downgrading MELD for liver transplant candidates with MELD score ≥30 was effective in improving patient prognosis. An appropriate ALSS treatment within 48 h prior to LT is potentially beneficial.

BACKGROUND

Hyperlactatemia and its reduction after admission in the intensive care unit (ICU) have been related to survival. Because it is unknown whether this equally applies to different groups of critically ill patients, we compared the prognostic value of repeated lactate levels (a) in septic patients versus patients with hemorrhage or other conditions generally associated with low-oxygen transport (LT) (b) in hemodynamically stable versus unstable patients.

METHODS

In this prospective observational two-center study (n = 394 patients), blood lactate levels at admission to the ICU (Lac(T0)) and the reduction of lactate levels from T = 0 to T = 12 hours (DeltaLac(T0-12)) and from T = 12 to T = 24 hours (DeltaLac(T12-24)), were related to in-hospital mortality.

RESULTS

Reduction of lactate was associated with a lower mortality only in the sepsis group (DeltaLac(T0-12): hazard ratio [HR] 0.34, p = 0.004 and DeltaLac(T12-24): HR 0.24, p = 0.003), but not in the LT group (DeltaLac(T0-12); HR 0.78, p = 0.52 and DeltaLac(T12-24); HR 1.30, p = 0.61). The prognostic values of Lac(T0), DeltaLac(T0-12), and DeltaLac(T12-24) were similar in hemodynamically stable and unstable patients (p = 0.43).

CONCLUSIONS

Regardless of the hemodynamic status, lactate reduction during the first 24 hours of ICU stay is associated with improved outcome only in septic patients, but not in patients with hemorrhage or other conditions generally associated with LT. We hypothesize that in this particular group a reduction in lactate is not associated with improved outcome due to irreversible damage at ICU admission.

Fully grown mouse oocytes are normally competent to progress from prophase I to metaphase II without interruption. However, growing mouse oocytes initially become only partially competent to undergo meiotic maturation. Meiotic maturation in these oocytes does not progress beyond metaphase I. In contrast to the oocytes of most strains of mice, most oocytes of strain LT/Sv mice become arrested at metaphase I even when they are fully grown. The initiation of oocyte maturation is correlated with an increase in p34cdc2 kinase activity that continues to rise until metaphase I. The transition into anaphase I is normally correlated with a decrease in p34cdc2 kinase activity. This study demonstrated that metaphase I arrest in both partially competent growing oocytes and fully grown LT/Sv oocytes is correlated with a sustained elevation of p34cdc2 kinase activity. In fact, p34cdc2 activity continued to increase during the time when activity normally decreased. In normally maturing oocytes, some, but not all, of the cyclin B, the regulatory protein associated with p34cdc2, became degraded in oocytes that entered anaphase I. In contrast, the amount of cyclin B present in the metaphase I-arrested oocytes continued to increase at the time when it was being degraded in normal oocytes progressing to metaphase II. These results suggest that the progression of meiosis is arrested at metaphase I in both groups of oocytes because of continued p34cdc2 kinase activity sustained, at least in part, by restricted degradation of cyclin B.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of the kallikrein-kinin system in lung injury has long been recognized. However, the effects of bradykinin (BK) on human lung fibroblasts (HLF) remain to be elucidated. We determined whether BK stimulates HLF to release chemotactic activity for neutrophils and monocytes (NCA and MCA, respectively). We evaluated HLF supernatant fluids for chemotactic activity through a blind-well chamber technique. HLF released NCA and MCA in a dose- and time-dependent manner in response to BK. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular sieve column chromatography revealed that both NCA and MCA had multiple chemotactic peaks. NCA was inhibited by a leukotriene (LT) B(4) receptor antagonist and by antibodies to interleukin (IL)-8 and granulocyte colony-stimulating factor (G-CSF). MCA was attenuated by the LTB(4) receptor antagonist and by antibodies to monocyte chemoattractant protein-1 (MCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and transforming growth factor (TGF)-beta. Both the LTB(4) receptor antagonist and these antibodies inhibited chemotactic activity of the molecular weights corresponding to MCP-1, GM-CSF, and TGF-beta, separated by column chromatography. The concentrations of IL-8, G-CSF, MCP-1, GM-CSF, and TGF-beta in supernatant fluids increased significantly in a time-dependent manner in response to BK. The receptors responsible for the release of NCA, MCA, and individual chemokines included both BKB(1) and BKB(2) receptors. These data suggest that BK may stimulate lung fibroblasts to release inflammatory cytokines, which may modulate lung inflammation.

Persistent neutrophilia is a feature of chronic obstructive pulmonary disease (COPD). Leukotriene synthesis inhibitors and cysteinyl leukotriene receptor antagonists have shown efficacy in the treatment of asthma. Antagonism of leukotriene (LT)B(4) receptors is being considered as a mode of treating COPD. We examined the capacity for inhibition of leukotriene synthesis and LTB(4) receptor antagonism to reduce survival of neutrophils from patients with COPD and those from normal subjects. The basal apoptosis level of these cells was 55.4 +/- 2.4% (mean +/- SEM) of total cells. Separate exposure to lipopolysaccharide (LPS), granulocyte-macrophage colony-stimulating factor (GM-CSF), dexamethasone (DEX), and LTB(4) increased neutrophil survival (p < 0. 001). The LTB(4) receptor antagonist SBBBBWA4C and of 5-LO-activating protein (FLAP) with MK886 abolished GM-CSF- and DEX-induced neutrophil survival. BWA4C and MK886 abolished GM-CSF- induced neotrophil survival in a concentration-dependent manner (1 nM to 10 microM), with IC(50) values of 182.0 nM and 63.1 nM, respectively. These findings demonstrate reversal of LPS-, GM-CSF-, and DEX-induced neutrophil survival by LTB(4) receptor antagonism and inhibitors of 5-LO and FLAP. They also suggest a potential additional antiinflammatory mode of action of these compounds through reduction of cell survival.

Cardiovascular responses during a graded lower body negative pressure (LBNP) protocol were compared before and after atropine and propranolol administration to test the hypothesis that both sympathetic and parasympathetic control of cardio-acceleration are associated with syncopal predisposition to orthostatic stress in healthy subjects. Eleven men were categorized into two groups having high (HT, N = 6) or low (LT, N = 5) tolerance based on their total time before the onset of presyncopal symptoms. HT and LT groups were similar in physical characteristics, fitness, and baseline cardiovascular measurements. Atropine treatment had no effect on LBNP tolerance or mean arterial pressure at presyncope, despite an atropine-induced increase in heart rate. Propranolol treatment reduced (p<0.05) LBNP tolerance in both groups. Diminished LBNP tolerance after propranolol administration was associated with reductions in cardiac output, whereas increase in systemic peripheral resistance from baseline to presyncope was unaffected by propranolol. Reduction in cardiac output and LBNP tolerance after beta blockade reflected a chronotropic effect because lower LBNP tolerance for the HT (-50%) and LT (-39%) groups was associated with dramatic reductions (p <0.05) in the magnitude of LBNP-induced tachycardia without significant effects on stroke volume at presyncope. Absence of an atropine-induced difference in cardiac output and systemic peripheral resistance between HT and LT groups failed to support the notion that cardiac vagal withdrawal represents a predominant mechanism that could account for differences in orthostatic tolerance. Because a reduction in LBNP tolerance in both HT and LT groups after propranolol treatment was most closely associated with reduced tachycardia, the data suggest that a primary autonomically mediated mechanism for maintenance of mean arterial pressure and orthostatic tolerance in healthy subjects is beta adrenergic-induced tachycardia.

The E. coli type I heat-labile enterotoxin (LT-I) shares considerable functional, structural, and immunological homology with cholera toxin (CT). Although the ganglioside GM1 is the sole receptor for CT, LT-I also appears to utilize additional, unique receptors on intestinal cells not recognized by CT. We characterized this second class of LT-I receptors using the human intestinal epithelial cell line, CaCo-2. CaCo-2 cells bound 8-fold more LT-I than CT, and some of these additional LT-I receptors appeared to be functional, as CT-B only partially inhibited LT-I activity at concentrations that completely inhibited CT activity. Membranes from unlabeled or [3H]galactose-labeled cells were incubated with toxin B subunits and extracted with Triton X-100, and the solubilized toxin B-receptor complexes were immunoabsorbed with anti-B bound to protein A-Sepharose. When organic extracts of the complexes were separated by thin-layer chromatography and overlayed with [125I]toxin, both toxins were found to bind only GM1. Separation of the complexes from [3H]galactose-labeled membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a series of galactoproteins specifically recognized by LT-I but not by CT. Similar proteins were detected on Western blots probed with [125I]toxin. LT-I activity on intact cells and binding to membranes and the above galactoproteins were enhanced by neuraminidase treatment even in the presence of CT-B. beta-1,4-Galactosidase and endo-beta-1,4-galactosidase, but not beta-1,3-galactosidase, significantly reduced LT-I binding. LT-I binding to fetuin and transferrin exhibited a similar glycosidase sensitivity.(ABSTRACT TRUNCATED AT 250 WORDS)

Colonies of Escherichia coli from blood agar plates were suspended and lysed in saline containing polymyxin B and a detergent (Triton X-100). The lysates were assayed for heat-labile enterotoxin (LT) by a coagglutination test (coa-test). The coa-test reagent consisted of Formalin-treated and heat-killed cells of Staphylococcus aureus, strain Cowan 1, sensitized with a high-titer rabbit anti-LT serum. Purified LT was detected in the coa-test at the nanogram level (2 to 5 ng), whereas larger amounts of cholera toxin (50 ng) were required to give a positive test. The coa-test was compared with the CHO cell test for detection of LT among E. coli strains isolated from human and animal stool cultures. The results of the coa-test and the CHO cell test correlated with 63 of 67 strains of human origin. Six of nine animal strains, defined as LT positive by the CHO cell test, gave positive results in the coa-test. We conclude that the coa-test is probably accurate and sensitive enough to be used in routine diagnosis of LT-producing E. coli strains isolated from human stool cultures. No special laboratory equipment is required, which makes the coa-test suited for diagnosis of enterotoxigenic E. coli in small hospital laboratories and in developing countries.

In contrast to our previous report (<em>B</em>iochem. <em>B</em>iophys. Res. Comm. 134:587, 1986), we now find that protein kinase C (PKC) is mobilized in human polymorphonuclear neutrophils (PMN) stimulated with platelet-activating factor (PAF) or leukotriene (<em>LT</em>)<em>B</em>4. Thus nanomolar concentrations of each compound caused PMN to lose cytosolic, PKC-specific protein phosphorylating activity, as well as receptors for phorbol myristate acetate (PMA). Smaller gains in membrane-associated PMA receptors accompanied these changes. Diacylglycerol and PMA had very similar effects on PKC. However, unlike these direct PKC activators, PAF and <em>LT</em><em>B</em>4 induced only moderate decreases in cytosolic PKC; acted only on PMN pretreated with cytochalasin <em>B</em>; did not mobilize PKC in disrupted PMN or activate PKC in a cell-free system; and with respect to PAF, induced responses that partially reversed within 30 min. Furthermore, PAF, <em>LT</em><em>B</em>4, and several of their structural analogues mobilized PKC at concentrations correlating closely with their respective affinities for cellular <em>LT</em><em>B</em>4 or PAF receptors. Thus PAF and <em>LT</em><em>B</em>4 acted by indirect and apparently receptor-mediated mechanisms. Four observations indicated that the cytochalasin <em>B</em>-dependent degranulating actions of PAF and <em>LT</em><em>B</em>4 involved PKC. First, PKC mobilization and degranulation occurred at the same stimulus concentrations. Second, 5-hydroxyicosatetraenoate dramatically enhanced both PKC mobilization and degranulation when elicited by PAF; it had relatively little influence on <em>LT</em><em>B</em>4-induced responses. Third, PAF-induced mobilization (t1/2 less than 7 sec) preceded degranulation (t1/2 approximately 20 sec). Finally, a PKC blocker, polymyxin <em>B</em>, was similarly effective in inhibiting degranulation responses to PAF, <em>LT</em><em>B</em>4, and PMA. <em>B</em>ecause stimulated PMN may produce and use PAF, <em>LT</em><em>B</em>4, and 5-hydroxyicosatetraenoate as secondary intracellular mediators, our results implicate PKC as a central and potentially critical regulator of function.

Recipients of liver transplantation (LT) may develop immunological tolerance. Factors predictive of tolerance are not clearly understood. Transplant recipients with normal liver function tests and without active viral hepatitis or autoimmune disease who presented with side effects of immunosuppression or a high risk of de novo malignancies were selected to participate in this prospective study. Twenty-four patients fulfilled the inclusion criteria and, therefore, underwent a gradual reduction of immunosuppression. Tolerance was defined as normal liver function tests after immunosuppression withdrawal. Basal clinical and immunological characteristics, including lymphocyte counts and subpopulations (T, B, natural killer, CD4(+) , CD8(+) , and regulatory T cells) and the phytohemagglutinin stimulation index (SI), were compared for tolerant and nontolerant patients. Fifteen of the 24 patients (62.5%) were tolerant at a median of 14 months (interquartile range = 8.5-22.5 months) after complete immunosuppression withdrawal. Tolerant patients had a longer median interval between transplantation and inclusion in the study (156 for tolerant patients versus 71 months for nontolerant patients, P = 0.003) and a lower median SI (7.49 for tolerant patients versus 41.73 for nontolerant patients, P = 0.01). We identified 3 groups of patients with different probabilities of tolerance: in the first group (n = 7 for an interval>> 10 years and an SI < 20), 100% reached tolerance; in the second group (n = 10 for an interval>> 10 years and an SI>> 20 or an interval < 10 years and an SI < 20), 60% reached tolerance; and in the third group (n = 7 for an interval < 10 years and an SI>> 20), 29% reached tolerance. In conclusion, a high proportion of select LT recipients can reach tolerance over the long term. Two simple basal variables-the time from transplantation and the SI-may help to identify these patients.

Enterotoxigenic Escherichia coli (ETEC) strains may synthesize both thermolabile (LT-I and LT-II) and thermostable (STa and STb) enterotoxins. Whereas thermolabile enterotoxins are high molecular weight proteins (85,000 d-90,000 d) composed by a single enzymatic A subunit combined with five B subunits which enable toxin for the receptor recognition, thermostable enterotoxins are small peptide chains with molecular weight between 1,900 d and 5,000 d. In addition to the synthesis of enterotoxins, the ability of ETEC strains to cause diarrhoea is also conditioned by the possession of colonization factors which enable bacteria adhere-to and colonize the luminal surface of small bowel. Colonization factors in ETEC strains were located in rigid fimbriae and flexible fibrils constituted by protein subunits ranging in size from 14,500 d to 31,000 d and usually responsible for mannose-resistant haemagglutination with determined erythrocyte species. Both enterotoxins and colonization factors are controlled by plasmids. There exist plasmids which may code separately enterotoxins and colonization factors, and besides there also exist recombinant plasmids coding together these two virulence factors. Human ETEC strains may synthesize LT-I and/or STa enterotoxins, they may possess the colonization factors named CFA/I, CFA/II, CFA/III or CFA/IV, and they belong mainly to serogroups O6, O8, O15, O20, O25, O27, O63, O77, O78, O114, O115, O126, O128, O139, O148, O153, O159 and O167. ETEC strains from porcine origin synthesize LT-I, STa and/or STb, they possess the colonization factors K88, P987, K99 or F41, and they usually belong to serogroups O8, O9, O20, O45, O64, O101, O115, O138, O141, O147, O149 and O157. Bovine and ovine ETEC strains are usually STa producers harbouring on the bacterial surface K99 or F41 colonization factors and they belong to serogroups O8, O9 and O101. Nevertheless, some particular bovine ETEC strains synthesizing LT-II have been described. Thus, a high specificity level between ETEC strains causing diarrhoea in humans and domestic animals can be observed. This is mainly due to the specific recognition between bacterial colonization factors and the epithelial receptors during host-parasite interaction.

Lymph node (LN) stromal cells provide survival signals and adhesive substrata to lymphocytes. During an immune response, B cell follicles enlarge, questioning how LN stromal cells manage these cellular demands. Herein, we used a murine fate mapping system to describe a new stromal cell type that resides in the T cell zone of resting LNs. We demonstrated that upon inflammation, B cell follicles progressively trespassed into the adjacent T cell zone and surrounded and converted these stromal cells into CXCL13 secreting cells that in return delineated the new boundaries of the growing follicle. Acute B cell ablation in inflamed LNs abolished CXCL13 secretion in these cells, while LT-β deficiency in B cells drastically affected this conversion. Altogether, we reveal the existence of a dormant stromal cell subset that can be functionally awakened by B cells to delineate the transient boundaries of their expanding territories upon inflammation.

Type IIb heat-labile enterotoxin (LT-IIb) is produced by Escherichia coli 41. Restriction fragments of total cell DNA from strain 41 were cloned into a cosmid vector, and one cosmid clone that encoded LT-IIb was identified. The genes for LT-IIb were subcloned into a variety of plasmids, expressed in minicells, sequenced, and compared with the structural genes for other members of the Vibrio cholerae-E. coli enterotoxin family. The A subunits of these toxins all have similar ADP-ribosyltransferase activity. The A genes of LT-IIa and LT-IIb exhibited 71% DNA sequence homology with each other and 55 to 57% homology with the A genes of cholera toxin (CT) and the type I enterotoxins of E. coli (LTh-I and LTp-I). The A subunits of the heat-labile enterotoxins also have limited homology with other ADP-ribosylating toxins, including pertussis toxin, diphtheria toxin, and Pseudomonas aeruginosa exotoxin A. The B subunits of LT-IIa and LT-IIb differ from each other and from type I enterotoxins in their carbohydrate-binding specificities. The B genes of LT-IIa and LT-IIb were 66% homologous, but neither had significant homology with the B genes of CT, LTh-I, and LTp-I. The A subunit genes for the type I and type II enterotoxins represent distinct branches of an evolutionary tree, and the divergence between the A subunit genes of LT-IIa and LT-IIb is greater than that between CT and LT-I. In contrast, it has not yet been possible to demonstrate an evolutionary relationship between the B subunits of type I and type II heat-labile enterotoxins. Hybridization studies with DNA from independently isolated LT-II producing strains of E. coli also suggested that additional variants of LT-II exist.

Bacillus anthracis lethal toxin (LT) was characterized in plasma from infected African Green monkeys, rabbits, and guinea pigs. In all cases, during the terminal phase of infection only the protease-activated 63-kDa form of protective antigen (PA(63)) and the residual 20-kDa fragment (PA(20)) were detected in the plasma. No uncut PA with a molecular mass of 83 kDa was detected in plasma from toxemic animals during the terminal stage of infection. PA(63) was largely associated with lethal factor (LF), forming LT. Characterization of LT by Western blotting, capture enzyme-linked immunosorbent assay, and size exclusion chromatography revealed that the antiphagocytic poly-gamma-d-glutamic acid (gamma-DPGA) capsule released from B. anthracis bacilli was associated with LT in animal blood in variable amounts. While the nature of this in vivo association is not understood, we were able to determine that a portion of these LT/gamma-DPGA complexes retained LF protease activity. Our findings suggest that the in vivo LT complexes differ from in vitro-produced LT and that including gamma-DPGA when examining the effects of LT on specific immune cells in vitro may reveal novel and important roles for gamma-DPGA in anthrax pathogenesis.

Burst firing mediated by a low-threshold spike (LTS) is the hallmark of many thalamic neurons. However, postburst afterhyperpolarizations (AHPs) are relatively uncommon in thalamus. We now report data from patch-clamp recordings in rat brain slice preparations that reveal an LTS-induced slow AHP (sAHP) in thalamic paraventricular (PVT) and other midline neurons, but not in ventrobasal or reticular thalamic neurons. The LTS-induced sAHP lasts 8.9 +/- 0.4 s and has a novel pharmacology, with resistance to tetrodotoxin and cadmium and reduction by Ni(2+) or nominally zero extracellular calcium concentration, which also attenuate both the LTS and sAHP. The sAHP is inhibited by 10 mM intracellular EGTA or by equimolar replacement of extracellular Ca(2+) with Sr(2+), consistent with select activation of LVA T-type Ca(2+) channels and subsequent Ca(2+) influx. In control media, the sAHP reverses near E(K(+)), shifting to -78 mV in 10.1 mM [K(+)](o) and is reduced by Ba(2+) or tetraethylammonium. Although these data are consistent with opening of Ca(2+)-activated K(+) channels, this sAHP lacks sensitivity to specific Ca(2+)-activated K(+) channel blockers apamin, iberiotoxin, charybdotoxin, and UCL-2077. The LTS-induced sAHP is suppressed by a beta-adrenoceptor agonist isoproterenol, a serotonin 5-HT(7) receptor agonist 5-CT, a neuropeptide orexin-A, and by stimulation of the cAMP/protein kinase A pathway with 8-Br-cAMP and forskolin. The data suggest that PVT and certain midline thalamic neurons possess an LTS-induced sAHP that is pharmacologically distinct and may be important for information transfer in thalamic-limbic circuitry during states of attentiveness and motivation.

6-Phosphogluconate dehydrogenase (6PGD), encoded by gnd, is highly polymorphic among isolates of Escherichia coli form natural populations. As a means of characterizing the growth-rate-dependent regulation of the level of 6PGD, five gnd alleles, including the E. coli B/r allele, were crossed into E. coli K-12 with bacteriophage P1. In each of the isogenic strains, the level of 6PGD was two- to threefold higher in cells grown on glucose than in cells grown on acetate. The level of enzyme activity in the acetate-grown cells varied about sixfold within the set of isogenic strains. The physiological importance of these differences in enzyme level is discussed. The gnd gene was cloned from five E. coli strains and Salmonella typhimurium LT-2 and mapped with twelve restriction endonucleases. gnd was located and oriented on the chromosomal DNAs. The restriction maps of the genes were aligned at conserved restriction sites, and the relative divergence of the genes was estimated from restriction site polymorphisms. The E. coli gnd genes differed from the S. typhimurium gene by about 11%. Most of the E. coli genes differed from one another by less than 5%, but one allele differed from the others by about 10%. Only the gnd gene from E. coli K-12 had an IS5 element located nearby.

Tumour necrosis factor (TNF) is a highly potent, proinflammatory cytokine with broad-ranging functions from the regulation of endothelial cell adhesion molecules to facilitate entry of leucocytes into tissues, to direct induction of cellular cytotoxicity. This diversity of function potentially attributable to TNF in the genesis of inflammatory disorders place TNF as a primary candidate for clinical targeting and considerable success in this regard has been achieved, particularly in rheumatoid arthritis (RA). In this article we provide a short overview of TNF and its homologue lymphotoxin (LT) alpha and beta. Particular emphasis is placed on recent discoveries regarding the cell surface expression of these cytokines and the role of TNF/LT in experimental autoimmune encephalomyelitis (EAE), an animal model of the human demyelinating disease, multiple sclerosis (MS).

Polyunsaturated fatty acids (PUFAs) and leukotriene B(4) (LTB(4)) are involved in many inflammatory and physiological conditions. The role of arachidonic acid (AA) and linoleic acid (LA) in promoting the assembly of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits is well known, but the involvement of LTB(4) and other 5-lipoxygenase (5-LO) pathway metabolites of AA in hydrogen peroxide (H(2)O(2)) production by PUFA-stimulated polymorphonuclear leukocytes (PMNs) has not been investigated. We examined this question by determining H(2)O(2) production as well as phosphorylation and membrane translocation of the p47phox subunit of NADPH oxidase. Elicited peritoneal PMNs from rats and from 5-LO-deficient or wild-type mice were pretreated with or without inhibitors of LT biosynthesis and antagonists of the receptors for LTB(4) and cysteinyl LTs for 20 min before stimulation with AA (at 5 and 20 microM) or LA (at 20 microM). PUFAs elicited H(2)O(2) production in a dose-dependent manner, and pharmacologic or genetic inhibition of LT synthesis decreased H(2)O(2) production by approximately 40% when compared with untreated controls. LTB(4) was the moiety responsible for H(2)O(2) production, as revealed by studies using receptor antagonists and its exogenous addition. LTB(4) itself also promoted p47phox phosphorylation and translocation. These results identify a heretofore unrecognized role for activation of 5-LO and subsequent production of LTB(4) in stimulation of PMN NADPH oxidase activation by PUFAs.