Sustained interleukin-1ß exposure modulates multiple steps in glucocorticoid receptor signaling, promoting split-resistance to the transactivation of prominent anti-inflammatory genes by glucocorticoids.

Clinical treatment with glucocorticoids (GC) can be complicated by cytokine-induced glucocorticoid low-responsiveness (GC-resistance, GCR), a condition associated with a homogeneous reduction in the expression of GC-receptor- (GR-) driven anti-inflammatory genes. However, GR level and phosphorylation changes modify the expression of individual GR-responsive genes differently. As sustained IL-1ß exposure is key in the pathogenesis of several major diseases with prevalent GCR, we examined GR signaling and the mRNA expression of six GR-driven genes in cells cultured in IL-1ß and afterwards challenged with GC. After a GC challenge, sustained IL-1ß exposure reduced the cytoplasmic GR level, GR(Ser203) and GR(Ser211) phosphorylation, and GR nuclear translocation and led to selective GCR in the expression of the studied genes. Compared to GC alone, in a broad range of GC doses plus sustained IL-1ß, FKBP51 mRNA expression was reduced by 1/3, TTP by 2/3, and IRF8 was completely knocked down. In contrast, high GC doses did not change the expression of GILZ and DUSP1, while IGFBP1 was increased by 5-fold. These effects were cytokine-selective, IL-1ß dose- and IL-1R1-dependent. The integrated gain and loss of gene functions in the "split GCR" model may provide target cells with a survival advantage by conferring resistance to apoptosis, chemotherapy, and GC.

The number of circulating monocytes as biomarkers of the clinical response to methotrexate in untreated patients with rheumatoid arthritis.

BACKGROUND: The aim of this work was to analyze the number and distribution of circulating monocytes, and of their CD14(+high)CD16(-), CD14(+high)CD16(+) and CD14(+low)CD16(+) subset cells, in treatment-naive patients with rheumatoid arthritis (RA), and to determine their value in predicting the clinical response to methotrexate (MTX) treatment. METHODS: This prospective work investigated the number of circulating monocytes, and the numbers of CD14(+high)CD16(-), CD14(+high)CD16(+) and CD14(+low)CD16(+) subset cells, in 52 untreated patients with RA before MTX treatment, and at 3 and 6 months into treatment, using flow cytometry. RESULTS: The absolute number of circulating monocytes, and the numbers of CD14(+high)CD16(-), CD14(+high)CD16(+) and CD14(+low)CD16(+) subset cells, were significantly higher in MTX non-responders than in responders and healthy controls before starting and throughout treatment. Responders showed normal numbers of monocytes, and of their subset cells, over the study period. The pre-treatment absolute number of circulating monocytes, and the numbers of CD14(+high)CD16(-) and CD14(+high)CD16(+) subset cells, were found to be predictive of the clinical response to MTX, with a sensitivity and specificity of >70% and >88%, respectively. CONCLUSIONS: Treatment-naive patients with RA showed an anomalous distribution of circulating monocyte subsets, and an anomalous number of cells in each subset. A higher pre-treatment number of circulating monocytes, and higher numbers of CD14(+high)CD16(-) and CD14(+high)CD16(+) subset cells, predict a reduced clinical response to MTX in untreated patients with RA.

Neuronal injury external to the retina rapidly activates retinal glia, followed by elevation of markers for cell cycle re-entry and death in retinal ganglion cells.

Retinal ganglion cells (RGCs) are neurons that relay visual signals from the retina to the brain. The RGC cell bodies reside in the retina and their fibers form the optic nerve. Full transection (axotomy) of the optic nerve is an extra-retinal injury model of RGC degeneration. Optic nerve transection permits time-kinetic studies of neurodegenerative mechanisms in neurons and resident glia of the retina, the early events of which are reported here. One day after injury, and before atrophy of RGC cell bodies was apparent, glia had increased levels of phospho-Akt, phospho-S6, and phospho-ERK1/2; however, these signals were not detected in injured RGCs. Three days after injury there were increased levels of phospho-Rb and cyclin A proteins detected in RGCs, whereas these signals were not detected in glia. DNA hyperploidy was also detected in RGCs, indicative of cell cycle re-entry by these post-mitotic neurons. These events culminated in RGC death, which is delayed by pharmacological inhibition of the MAPK/ERK pathway. Our data show that a remote injury to RGC axons rapidly conveys a signal that activates retinal glia, followed by RGC cell cycle re-entry, DNA hyperploidy, and neuronal death that is delayed by preventing glial MAPK/ERK activation. These results demonstrate that complex and variable neuro-glia interactions regulate healthy and injured states in the adult mammalian retina.

Distinctive patterns of naïve/memory subset distribution and cytokine expression in CD4 T lymphocytes in ZAP-70 B-chronic lymphocytic patients.

BACKGROUND: ZAP-70 upregulation in B chronic lymphocytic leukemia (B-CLL) cells is a recognized marker of poor prognosis in these patients; the biological basis of this differential clinical outcome nonetheless remains unknown. ZAP-70 overexpression is considered a surrogate marker of a B-CLL cell subset. To test whether the differential biological characteristics of these patients also include the T helper population, we studied naïve, non-terminated memory (NTEM), terminated memory (TEM) and central memory (CM) cells, and cytokine expression by CD4 T lymphocytes from ZAP-70(+) and ZAP-70(-) B-CLL patients. METHODS: Expression of CD3, CD8, CD45RA, CD27, and CD28 antigens and intracytoplasmic cytokine production (IFNγ, IL-2, IL-4, IL-10, and TNFα) were assessed simultaneously by nine-color flow-cytometry in peripheral blood lymphocytes from B-CLL patients. B cell ZAP-70 expression in B-CLL cells was also analyzed by flow cytometry. RESULTS: Compared to ZAP-70(-) B-CLL patients, ZAP-70(+) B-CLL patients showed 1) significant reduction in the naïve T helper subset and expansion of NTEM and TEM subsets, 2) a decrease in the percentage of activated CD4 T lymphocytes expressing IFNγ, TNFα, and IL-2, and 3) an increase in the percentage of CD4 T lymphocytes expressing IL-4 or IL-10. CONCLUSIONS: In conclusion, in early stage B-CLL patients, ZAP-70 upregulation is associated with distinct patterns of activation/differentiation stage subset distribution and of cytokine expression in CD4 T lymphocytes.

Distinctive patterns of naïve/memory subset distribution and cytokine expression in CD4 T lymphocytes in ZAP-70 B-chronic lymphocytic patients.

BACKGROUND: ZAP-70 upregulation in B chronic lymphocytic leukemia (B-CLL) cells is a recognized marker of poor prognosis in these patients; the biological basis of this differential clinical outcome nonetheless remains unknown. ZAP-70 overexpression is considered a surrogate marker of a B-CLL cell subset. To test whether the differential biological characteristics of these patients also include the T helper population, we studied naïve, non-terminated memory (NTEM), terminated memory (TEM) and central memory (CM) cells and cytokine expression by CD4 T lymphocytes from ZAP-70+ and ZAP-70- B-CLL patients. METHODS: Expression of CD3, CD8, CD45RA, CD27, and CD28 antigens and intracytoplasmic cytokine production (IFNγ, IL-2, IL-4, IL-10 and TNFα) were assessed simultaneously by nine-color flow-cytometry in peripheral blood lymphocytes from B-CLL patients. B cell ZAP-70 expression in B-CLL cells was also analyzed by flow cytometry. RESULTS: Compared to ZAP-70- B-CLL patients, ZAP-70+ B-CLL patients showed 1) significant reduction in the naïve T helper subset and expansion of NTEM and TEM subsets, 2) a decrease in the percentage of activated CD4 T lymphocytes expressing IFNγ, TNFα and IL-2, and 3) an increase in the percentage of CD4 T lymphocytes expressing IL-4 or IL-10. CONCLUSIONS: In conclusion, in early stage B-CLL patients, ZAP-70 upregulation is associated with distinct patterns of activation/differentiation stage subset distribution and of cytokine expression in CD4 T lymphocytes. © 2013 Clinical Cytometry Society.

Early alterations of B cells in patients with septic shock.

INTRODUCTION: It has recently been proposed that B lymphocytes are involved in sepsis pathogenesis. The goal of this study is to investigate potential abnormalities in a subset distribution and activation of circulating B lymphocytes in patients with septic shock. METHODS: This observational prospective study was conducted in a medical-surgical ICU. All patients with septic shock were eligible for inclusion. B-cell phenotypes (CD19+CD69+, CD19+CD23+, CD19+CD5+, CD19+CD80, CD19+CD86+, CD19+CD40 and CD19+CD95+) were assessed by quantitative flow cytometry upon admission to the ICU and 3, 7, 14 and 28 d later. RESULTS: Fifty-two patients were included. Thirty-six healthy volunteers matched for age and sex were used as controls. The patients had lymphopenia that was maintained during 28 d of follow-up. In patients with septic shock who died, the percentage of CD19+CD23+ was lower during the 7 d of follow-up than it was in survival patients. Moreover, the percentage of CD80+ and CD95+ expression on B cells was higher in patients who died than in survivors. Receiver operating characteristic curve analysis showed that a CD19+CD23+ value of 64.6% at ICU admission enabled discrimination between survivors and nonsurvivors with a sensitivity of 90.9% and a specificity of 80.0% (P=0.0001). CONCLUSIONS: Patients with septic shock who survive and those who don't have different patterns of abnormalities in circulating B lymphocytes. At ICU admission, a low percentage of CD23+ and a high of CD80+ and CD95+ on B cells were associated with increased mortality of patients with septic shock. Moreover, a drop in circulating B cells persisted during 28 d of ICU follow-up.

Circulating sICAM-1 and sE-Selectin as biomarker of infection and prognosis in patients with systemic inflammatory response syndrome.

BACKGROUND: Vascular endothelium activation is a key pathogenic step in systemic inflammatory response syndrome (SIRS) that can be triggered by both microbial and sterile proinflammatory stimuli. The relevance of soluble adhesion molecules as clinical biomarkers to discriminate between infectious and non-infectious SIRS, and the individual patient prognosis, has not been established. METHODS: We prospectively measured by sandwich ELISA, serum levels of soluble E-Selectin (sE-Selectin), soluble vascular cell adhesion molecule-1 (sVCAM-1), soluble intercellular adhesion molecule-1 (sICAM-1) and soluble intercellular adhesion molecule-2 (sICAM-2) at ICU admission and at days 3, 7, 14 and 28 in patients with sepsis and at days 3 and 7 in patients with non-infectious SIRS. RESULTS: At ICU admission, sE-Selectin, sVCAM-1 and sICAM-1 in patients with infectious SIRS were significantly higher than those found in patients with non-infectious SIRS. ROC analysis revealed that the AUC for infection identification was best for sICAM-1 (0.900±0.041; 95% CI 0.819-0.981; p<0.0001). Moreover, multivariate analysis showed that 4 variables were significantly and independently associated with mortality at 28 days: male gender (OR 15.90; 95% CI, 2.54-99.32), MODS score (OR 5.60; 95% CI, 1.67-18.74), circulating sE-Selectin levels (OR 4.81; 95% CI, 1.34-17.19) and sVCAM-1 concentrations (OR 4.80; 95% CI, 1.34-17.14). CONCLUSIONS: Patients with SIRS secondary to infectious or non-infectious etiology show distinctive patterns of disturbance in serum soluble adhesion molecules. Serum ICAM-1 is a reliable biomarker for classifying patients with infectious SIRS from those with non-infectious SIRS. In addition, soluble E-Selectin is a prognostic biomarker with higher levels in patients with SIRS and fatal outcome.

Monocyte populations as markers of response to adalimumab plus MTX in rheumatoid arthritis.

INTRODUCTION: The treatment of rheumatoid arthritis (RA) patients with anti-tumor necrosis factor alpha (TNFα) biological drugs has dramatically improved the prognosis of these patients. However, a third of the treated patients do not respond to this therapy. Thus, the search for biomarkers of clinical response to these agents is currently highly active. Our aim is to analyze the number and distribution of circulating monocytes, and of their CD14⁺highCD16⁻, CD14⁺highCD16⁺ and CD14⁺lowCD16+ subsets in methotrexate (MTX) non-responder patients with RA, and to determine their value in predicting the clinical response to adalimumab plus MTX treatment. METHODS: This prospective work investigated the number of circulating monocytes, and of their CD14⁺highCD16⁻, CD14⁺highCD16⁺ and CD14⁺lowCD16⁺ subsets, in 35 MTX non-responder patients with RA before and after three and six months of anti-TNFα treatment using multiparametric flow cytometry. The number of circulating monocytes in an age- and sex-matched healthy population was monitored as a control. RESULTS: Non-responder patients with RA show an increased number of monocytes and of their CD14⁺highCD16⁻, CD14⁺highCD16⁺ and CD14⁺lowCD16⁺ subsets after three months of adalimumab plus MTX treatment that remained significantly increased at six months. In contrast, significant normalization of the numbers of circulating monocytes was found in responders at three months of adalimumab plus MTX treatment that lasts up to six months. CX3CR1 expression is increased in monocytes in non-responders. At three months of anti-TNFα treatment the number of circulating monocytes and their subsets was associated with at least 80% sensitivity, 84% specificity and an 86% positive predictive value (PPV) in terms of discriminating between eventual early responders and non-responders. CONCLUSIONS: The absolute number of circulating monocytes and of their CD14⁺highCD16⁻, CD14⁺highCD16⁺ and CD14⁺lowCD16⁺ subsets at three months of adalimumab plus MTX treatment, have a predictive value (with high specificity and sensitivity) in terms of the clinical response after six months of anti-TNFα treatment in patients with RA.

Mortality in patients with septic shock correlates with anti-inflammatory but not proinflammatory immunomodulatory molecules.

BACKGROUND: Mortality in patients with septic shock remains unacceptably high and the attempts to antagonize certain proinflammatory cytokines based on the results of animal model studies have failed to improve survival rates. The objective of this article is to examine the pro-/anti-inflammatory cytokine balance in patients with septic shock and its connection with mortality. METHODS: Serum levels of proinflammatory cytokines (tumor necrosis factor-α [TNF-α], interleukin 1ß [IL-1ß], interferonγ [IFN-γ], and IL-6) and soluble cytokine antagonists (soluble TNF receptor I [sTNF-RI], sTNF-RII, and IL-1Ra) were determined on admission to the intensive care unit (ICU) and 3, 7, 14, and 28 days later in 52 patients with septic shock and in 36 healthy controls. Specific sandwich enzyme-linked immunosorbent assay (ELISA) was used for all determinations. RESULTS: Serum levels of most of the pro- and anti-inflammatory molecules examined (TNF-α, IL-6, sTNF-RI, sTNF-RII, and IL-1 receptor agonist [IL-1Ra]) were significantly elevated on admission and during the 28-day observation period in patients when compared to controls. Notably, the anti-inflammatory mediators sTNF-RI, sTNF-RII, and IL-1Ra were better predictors of mortality. Receiver-operating characteristic (ROC) analysis revealed that sTNF-RI or sTNF-RII concentrations over 2767 or 4619 pg/mL, respectively, determined a high risk of death (sensitivity: 100%-100%, specificity: 57.1%-71.4%, area under the curve [AUC] 0.759-0.841, respectively), whereas IL-1Ra concentrations below 7033 pg/mL determined a high probability of survival (sensitivity: 60%, specificity: 100%, AUC 0.724). In addition, IFN-γ levels were significantly higher in survivors than in controls during the initial 2 weeks of observation. CONCLUSIONS: Our data show that serum cytokine disturbance patterns have prognostic significance in patients with septic shock admitted to the ICU. The pattern, defined by an early response to continuously elevated anti-inflammatory cytokine serum levels, is associated with an enhanced risk of a fatal outcome for patients.

Human brucellosis is characterized by an intense Th1 profile associated with a defective monocyte function.

In animal models, a defective Th1 response appears to be critical in the pathogenesis of brucellosis, but the Th1 response in human brucellosis patients remains partially undefined. Peripheral blood from 24 brucellosis patients was studied before and 45 days after antibiotherapy. Twenty-four sex- and age-matched healthy donors were analyzed in parallel. Significantly increased levels of interleukin 1beta (IL-1beta), IL-2, IL-4, IL-6, IL-12p40, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha), but not of IL-10, in serum and/or significantly increased percentages of samples with detectable levels of these cytokines, measured by enzyme-linked immunosorbent assays (ELISA), were found for untreated brucellosis patients, but these levels were reduced and/or normalized after treatment. Flow cytometry studies showed that the intracytoplasmic expression of IFN-gamma, IL-2, and TNF-alpha, but not that of IL-4, by phorbol myristate-activated CD4(+) CD3(+) and CD8(+) CD3(+) T lymphocytes was significantly increased in untreated brucellosis patients and was also partially normalized after antibiotherapy. The percentage of phagocytic cells, the mean phagocytic activity per cell, and the phagocytic indices for monocytes at baseline were defective and had only partially reverted at follow-up. T lymphocytes from untreated brucellosis patients are activated in vivo and show Th1 cytokine production polarization, with strikingly high serum IFN-gamma levels. In spite of this Th1 environment, we found deficient effector phagocytic activity in peripheral blood monocytes.

Ordering human CD34+CD10-CD19+ pre/pro-B-cell and CD19- common lymphoid progenitor stages in two pro-B-cell development pathways.

Studies here respond to two long-standing questions: Are human "pre/pro-B" CD34(+)CD10(-)CD19(+) and "common lymphoid progenitor (CLP)/early-B" CD34(+)CD10(+)CD19(-) alternate precursors to "pro-B" CD34(+)CD19(+)CD10(+) cells, and do the pro-B cells that arise from these progenitors belong to the same or distinct B-cell development pathways? Using flow cytometry, gene expression profiling, and Ig V(H)-D-J(H) sequencing, we monitor the initial 10 generations of development of sorted cord blood CD34(high)Lineage(-) pluripotential progenitors growing in bone marrow S17 stroma cocultures. We show that (i) multipotent progenitors (CD34(+)CD45RA(+)CD10(-)CD19(-)) directly generate an initial wave of Pax5(+)TdT(-) "unilineage" pre/pro-B cells and a later wave of "multilineage" CLP/early-B cells and (ii) the cells generated in these successive stages act as precursors for distinct pro-B cells through two independent layered pathways. Studies by others have tracked the origin of B-lineage leukemias in elderly mice to the mouse B-1a pre/pro-B lineage, which lacks the TdT activity that diversifies the V(H)-D-J(H) Ig heavy chain joints found in the early-B or B-2 lineage. Here, we show a similar divergence in human B-cell development pathways between the Pax5(+)TdT(-) pre/pro-B differentiation pathway that gives rise to infant B-lineage leukemias and the early-B pathway.

Somatic tetraploidy in specific chick retinal ganglion cells induced by nerve growth factor.

A subset of neurons in the normal vertebrate nervous system contains double the normal amount of DNA in their nuclei. These neurons are all thought to derive from aberrant mitoses in neuronal precursor cells. Here we show that endogenous NGF induces DNA replication in a subpopulation of differentiating chick retinal ganglion cells that express both the neurotrophin receptor p75 and the E2F1 transcription factor, but that lack the retinoblastoma protein. Many of these neurons avoid G2/M transition and remain alive in the retina as tetraploid cells with large cell somas and extensive dendritic trees, and most of them express beta2 nicotinic acetylcholine receptor subunits, a specific marker of retinal ganglion cells innervating lamina F in the stratum-griseum-et-fibrosum-superficiale of the tectal cortex. Tetraploid neurons were also observed in the adult mouse retina. Thus, a developmental program leading to somatic tetraploidy in specific retinal neurons exists in vertebrates. This program might occur in other vertebrate neurons during normal or pathological situations.

Clinical relevance of the severe abnormalities of the T cell compartment in septic shock patients.

INTRODUCTION: Given the pivotal role of T lymphocytes in the immune system, patients with septic shock may show T cell abnormalities. We have characterised the T cell compartment in septic shock and assess its clinical implications. METHODS: T lymphocytes from the peripheral blood of 52 patients with septic shock and 36 healthy control subjects were analysed on admission to the intensive care unit, baseline, and 3, 7, 14 and 28 days later. T cell phenotypes (CD3+CD4+/CD3+CD8+, CD45RA+/CD45RO+, CD62L+/CD28+) were assessed by quantitative flow cytometry. RESULTS: CD3+, CD3+CD4+ and CD3+CD8+ lymphocyte counts were significantly lower in patients with septic shock than control subjects. In surviving patients, CD3+CD4+ lymphocytes had normalised after 14 days, yet CD3+CD8+ numbers were still low. Non effector CD45RA+CD45RO- subsets of CD3+CD4+ and CD3+CD8+ were persistently low during patient follow up. CD3+CD8+CD28+ and CD3+CD8+CD62L+ were reduced in patients versus controls and survivors versus nonsurvivors in the first three days. A prediction receptor operative curve revealed that for the CD3+CD8+CD28+ subset, a cutoff of 136 cells/ml showed 70% sensitivity and 100% specificity for predicting death and the area under the curve was 0.84 at admission. Corresponding values for CD3+CD8+CD62L+ were 141 cells/ml, 60% sensitivity, 100% specificity and an area under the curve of 0.75. CONCLUSIONS: A severe redistribution of T lymphocyte subsets is found in septic shock patients. A different kinetic pattern of T cell subset involvement is observed in surviving and nonsurviving patients, with lower numbers of circulating CD3+CD8+CD28+ and CD3+CD8+CD62L+ associated with a better disease outcome.

Inhaled IL-2 induces systemic immunomodulation in patients with renal cell carcinoma and lung metastasis.

The peripheral blood lymphocytes of eight patients with metastatic renal cell carcinoma, and of eight healthy volunteers were analyzed by four-color flow cytometry to characterize the immunophenotypic alterations manifested, determine the prevalence of lymphocyte apoptosis, and detect evidence of the systemic effect of inhaled IL-2. The T, B and NK lymphocytes of untreated patients were found to have undergone profound changes characterized by an increase in susceptibility to both spontaneous and mitogen-induced ex vivo apoptosis, a modified distribution of the main lymphocyte populations in the peripheral blood, and alterations in activation status. An increase in the proportion of regulatory T cells was also seen in these patients. Treatment with inhaled IL-2, however, normalized the rate of apoptosis in all the lymphocyte subpopulations studied, as well as their distribution and activation status. These findings demonstrate that inhaled IL-2 has systemic immunomodulatory effects.

Treatment with AM3 restores defective T-cell function in COPD patients.

BACKGROUND: Lymphocyte alterations have been associated with an increased prevalence of acute respiratory infections in COPD patients. AM3 is an oral immunomodulator that normalizes the defective functions of peripheral blood natural killer and phagocytic cells in COPD patients and improves their health-related quality of life. OBJECTIVES: To characterize putative systemic abnormalities of the T-cell compartment in COPD patients, and to investigate whether AM3 can restore such abnormalities. DESIGN: The study was a randomized, prospective, double-blind, placebo-controlled trial in a cohort of COPD patients. The results were also compared to those of nonsmoker and ex-smoker healthy control subjects. SETTING: Outpatient departments of four hospitals. PATIENTS: Seventy COPD patients were randomized to receive either AM3 or a placebo orally for 90 consecutive days. Populations of 36 healthy nonsmokers and 36 healthy ex-smokers were used as control subjects. MEASUREMENTS: Peripheral blood mononuclear cell (PBMC) proliferation and production of interleukin (IL)-2, IL-4, IL-12p40, tumor necrosis factor-alpha, and interferon (IFN)-gamma proteins in response to the T-cell polyclonal mitogens were assessed at baseline and at the end of treatment. RESULTS: The proliferative response was significantly decreased in COPD patients. Decreased production of IFN-gamma was the only defect in the profiles of the cytokine measures, and was selectively observed in COPD patients, but not in nonsmoker and ex-smoker healthy control subjects. Treatment with AM3 significantly restored the PBMC proliferative response to polyclonal mitogens and significantly promoted stimulated IFN-gamma production in these patients. The normalization of these proliferative responses was not related to significant variations in the numbers of peripheral blood monocytes, CD3+, CD4+, CD8+ cells or of any major naïve/memory/activated T-cell subset. The increased IFN-gamma production in the AM3 study arm was associated with an increase in the mean of number of IFN-gamma molecules produced per CD8+ T cells. CONCLUSIONS: PBMCs of COPD patients showed clear functional T-lymphocyte abnormalities that are rescued by AM3 treatment.

Accurate apoptosis measurement requires quantification of loss of expression of surface antigens and cell fragmentation.

BACKGROUND: The use of ratiometric cell enumeration methods emerges as a more accurate method of measurement of the occurrence of apoptosis in cell cultures. These new flow cytometry methods were used to quantify the impact of cell fragmentation and loss of lineage antigen (LAg) expression on measurement of apoptosis. METHODS: Highly purified human lymphocyte populations were negatively sorted and cultured for 24 h. Apoptotic cells were identified using annexin V, 7-amino-actinomycin D and their LAgs were stained with antibodies. A new indicator, the apoptotic rate, was used to determine apoptosis occurrence and its validity compared with the widely accepted percentage of apoptotic cells (apoptotic index, AI). RESULTS: Loss of LAg expression and cell fragmentation were observed under all conditions assayed and for all cell populations studied. CONCLUSIONS: Current methods for quantifying of apoptosis involving AI systematically underestimate apoptosis occurrence in all populations and conditions, especially among cells undergoing spontaneous apoptosis.

Mesenteric Th1 polarization and monocyte TNF-alpha production: first steps to systemic inflammation in rats with cirrhosis.

A systemic inflammatory state with increased circulating tumor necrosis factor alpha (TNF-alpha) has been related to the bacterial infection susceptibility and hemodynamic derangement of patients with cirrhosis. We compared the activation status of immune cell subpopulations defined by 4-color cytometry in mesenteric and peripheral lymph nodes and blood of rats with CCl(4)-cirrhosis to define the immune response initiation site, the T-cell and monocyte contribution to pro-inflammatory cytokine production, as well as the pathogenic role of enteric bacteria in the cirrhosis immune response. Th1 cells and monocytes were expanded in the mesenteric nodes (P < .001) and blood (P < .001) of rats with cirrhosis, and activated to produce interferon gamma (P < .0001) and TNF-alpha (P < .0001), respectively. The greater numbers of recently activated CD134(+) Th cells in mesenteric nodes compared with blood, the correlation between their numbers in mesenteric nodes and blood (r = 0.66, P < .001), and the expansion of activated CD45RC(-) Th cells, which are unable to re-enter lymph nodes, in mesenteric nodes but not in blood or axillary nodes points to mesenteric nodes as the origin site of activated Th cells. Abrogation of bacterial translocation by bowel decontamination reduced the number of activated Th cells and monocytes, and normalized interferon gamma production by Th cells and TNF-alpha production by monocytes in mesenteric nodes and blood, respectively. In conclusion, in cirrhosis, enteric bacteria start off an orchestrated immune response cascade in mesenteric nodes involving Th1 polarization and monocyte activation to TNF-alpha production. Later, the recirculation of these activated effector immune cells into blood promotes systemic inflammation.

Serum lipopolysaccharide-binding protein prediction of severe bacterial infection in cirrhotic patients with ascites.

Serum lipopolysaccharide-binding protein is increased in a subset of non-infected ascitic cirrhotic patients, a finding previously related to bacterial passage from the gut to the circulation without overt infection. We prospectively analysed the risk factors associated with a first episode of severe bacterial infection in 84 ascitic cirrhotics, followed up for a median of 46 weeks. The cumulative probability of such infection in patients with raised and normal lipopolysaccharide-binding protein was 32.4% and 8.0% (p=0.004), respectively. Increased lipopolysaccharide-binding protein was the only factor independently associated with severe bacterial infection in a multivariate analysis (relative risk 4.49, 95% CI 1.42-14.1). Monitoring of serum lipopolysaccharide-binding protein could, therefore, help to target cirrhotic patients with ascites for antibiotic prophylaxis.

Human cord blood CD34+Pax-5+ B-cell progenitors: single-cell analyses of their gene expression profiles.

Circulating CD34(+) cells are used in reparative medicine as a stem cell source, but they contain cells already committed to different lineages. Many think that B-cell progenitors (BCPs) are confined to bone marrow (BM) niches until they differentiate into B cells and that they do not circulate in blood. The prevailing convention is that BCP transit a CD34(+)CD19(-)10(+) early-B-->CD34(+)CD19(+)CD10(+) B-cell progenitor (pro-B)-->CD34(-)CD19(+)CD10(+) B-cell precursor (pre-B) differentiation pathway within BM. However, populations of CD34(+)CD10(+) and CD34(+)CD19(+) cells circulate in adult peripheral blood and neonatal umbilical cord blood (CB) that are operationally taken as BCPs on the basis of their phenotypes, although they have not been submitted to a systematic characterization of their gene expression profiles. Here, conventional CD34(+)CD19(+)CD10(+) and novel CD34(+)CD19(+)CD10(-) BCP populations are characterized in CB by single-cell sorting and multiplex analyses of gene expression patterns. Circulating BCP are Pax-5(+) cells that span the early-B, pro-B, and pre-B developmental stages, defined by the profiles of rearranged V-D-J(H), CD79, VpreB, recombination activating gene (RAG), and terminal deoxynucleotidyl transferase (TdT) expression. Contrary to the expectation, circulating CD34(+)CD19(-)CD10(+) cells are essentially devoid of Pax-5(+) BCP. Interestingly, the novel CD34(+)CD19(+)CD10(-) BCP appears to be the normal counterpart of circulating preleukemic BCPs that undergo chromosomal translocations in utero months or years before their promotion into infant acute lymphoblastic B-cell leukemia after secondary postnatal mutations. The results underscore the power of single-cell analyses to characterize the gene expression profiles in a minor population of rare cells, which has broad implications in biomedicine.