Number Of Monocytes In Peripheral Blood Research Articles

Bioenergetic Failure Drives Functional Exhaustion of Monocytes in Acute-on-Chronic Liver Failure.

ObjectiveThe monocyte–macrophage system is central to the host’s innate immune defense and in resolving injury. It is reported to be dysfunctional in acute-on-chronic liver failure (ACLF). The disease-associated alterations in ACLF monocytes are not fully understood. We investigated the mechanism of monocytes’ functional exhaustion and the role of umbilical cord mesenchymal stem cells (ucMSCs) in re-energizing monocytes in ACLF.DesignMonocytes were isolated from the peripheral blood of ACLF patients (n = 34) and matched healthy controls (n = 7) and patients with compensated cirrhosis (n = 7); phagocytic function, oxidative burst, and bioenergetics were analyzed. In the ACLF mouse model, ucMSCs were infused intravenously, and animals were sacrificed at 24 h and day 11 to assess changes in monocyte function, liver injury, and regeneration.ResultsPatients with ACLF (alcohol 64%) compared with healthy controls and those with compensated cirrhosis had an increased number of peripheral blood monocytes (p < 0.0001) which displayed significant defects in phagocytic (p < 0.0001) and oxidative burst capacity (p < 0.0001). ACLF patients also showed a significant increase in the number of liver macrophages as compared with healthy controls (p < 0.001). Bioenergetic analysis showed markedly reduced oxidative phosphorylation (p < 0.0001) and glycolysis (p < 0.001) in ACLF monocytes. Patients with monocytes having maximum mitochondrial respiration of <37.9 pmol/min [AUC = 0.822, hazard ratio (HR) = 4.5] and baseline glycolysis of ≤42.7 mpH/min (AUC = 0.901, HR = 9.1) showed increased 28-day mortality (p < 0.001). Co-culturing ACLF monocytes with ucMSC showed improved mitochondrial respiration (p < 0.01) and phagocytosis (p < 0.0001). Furthermore, ucMSC therapy increased monocyte energy (p < 0.01) and phagocytosis (p < 0.001), reduced hepatic injury, and enhanced hepatocyte regeneration in ACLF animals.ConclusionBioenergetic failure drives the functional exhaustion of monocytes in ACLF. ucMSCs resuscitate monocyte energy and prevent its exhaustion. Restoring monocyte function can ameliorate hepatic injury and promote liver regeneration in the animal model of ACLF.

Protective effects of citrulline supplementation in ulcerative colitis rats.

It has been reported that supplementing certain amino acids has therapeutic effects on ulcerative colitis (UC). We intend to explore whether citrulline (Cit) supplementation has protective effects on UC. Fifteen male Wistar rats were divided into normal control group (NC group), UC group and UC+Cit group, with five rats in each group. The UC model was established by TNBS/ethanol method. Rats in UC+Cit group were intragastrically administered with Cit for 7 consecutive days after modeling. All rats were sacrificed after 7 days. Blood samples were collected to detect the number of monocytes. Colon tissues were taken for HE staining. Immunohistochemistry staining for CD68 and p-STAT3 were performed to detect the infiltration of monocytes and the phosphorylation of STAT3 in colon tissues. The concentrations of MCP-1, IL-6 and IL-17A and the protein expression of p-STAT3 in colon tissues were measured by ELISA and western blot methods, respectively. The body weight of UC group rats decreased significantly after 7 days (p<0.05). However, the weight loss of UC+Cit group rats was not statistically significant (p>0.05). The number of peripheral blood monocytes in UC+Cit group was significantly lower than that in UC group (p<0.05), and the infiltration of CD68-positive monocytes in the colon tissue of UC+Cit group was significantly reduced than that in UC group. The concentrations of MCP-1, IL-6 and IL-17A and the expression of p-STAT3 in colon tissues of UC+Cit group rats were significantly lower than those in UC group (both p<0.05). Our study suggests that Cit supplementation may be a potential therapy for UC.

Depletion of Neoplastic CD11b Positive Cells in Jak2V617F Mutant Mice Reduced Fibrocytes in Bone Marrow and Improved Myelofibrosis

Myelofibrosis (MF) associated with myeloproliferative neoplasms (MPN) has been considered to be a reactive phenomenon caused by mesenchymal stromal cells (MSCs) stimulated by cytokines such as TGFb-1 overproduced by neoplastic megakaryocytes (MKs) and platelets. TGFb-1 stimulates non-neoplastic mesenchymal cells to produce collagen and fibronectin and to induces bone marrow (BM) fibrosis. However, the involvement of neoplastic fibrocyte in MF has recently been reported (Verstovsek et al. JEM 2016), and among blood cells, monocytes in particular are considered to be the main source of neoplastic fibrocytes. In this study, we assesed the role of neoplastic fibrocytes using a mouse model of MPN induced by Jak2V617F (Shide et al. Leukemia 2008). First, the distribution of neoplastic fibrocyte in the BM of Jak2V617F transgenic (TG) mice was examined. We transplanted wild-type (WT) or Jak2V617F TG cells (B6-CD45.2), together with WT BM cells (B6-CD45.1) into irradiated WT recipient mice (B6-CD45.1). Only recipient mice transplanted with a mixture of Jak2V617F cells and WT cells developed BM fibrosis. In immunofluorescent staining of fibrotic BM, cells expressing the fibrocyte marker CD45/Collagen-1(Col-1) were observed much more than cells expressing the fibroblast marker CD90(usually positive for MSCs)/Col-1. As for CD45/Col-1 positive cells, cells expressing CD45.2/Col-1 were much more than cells expressing CD45.1/Col-1, clearly indicating that these cells were derived from Jak2V617F mutant blood cells. On the other hand, in the BM of recipient mice transplanted with control WT cells, few cells expressing CD45/Col-1 or CD90/Col-1 were present. To examine the differentiation ability of Jak2V617F blood cells to fibrocytes directly, peripheral blood (PB) mononuclear cells (MNC) of Jak2V617F mice or WT mice were cultured in vitro. After 5 days of culture, PB MNCs from Jak2V617F mice differentiated into mature fibrocytes exhibiting a long spindle shape with Col-1 expression. On the other hand, there were very few fibrocytes differentiated from PB MNC from WT mice. Next, we depleted monocytes, the main source of fibrocytes, and observed its effects on BM fibrosis in vivo. Jak2V617F TG mice were mated with CD11b-diphtheria toxin receptor (DTR) TG mice (Duffield et al. JCI 2005) to obtain Jak2V617F/CD11b-DTR double TG mice. Mice transplanted with BM cells from Jak2V617F/CD11b-DTR double TG mice (hereinafter called Jak2V617F/CD11b-DTR mice) exhibit leukocytosis, thrombocytosis, anemia, splenomegaly, and BM fibrosis with increased megakaryocytes. Jak2V617F/CD11b-DTR mice was administered diphtheria toxin (DT) intraperitoneally to deplete monocytes. One day after DT administration, the number of PB monocytes (CD11b+/F4/80+) drastically decreased in Jak2V617F/CD11b-DTR mice, and the reduction of monocyte was maintained by every-other-day DT administration. After 8 weeks DT treatment, mice were sacrificed and analyzed. As a control group, Jak2V617F/CD11b-DTR mice treated with PBS were examined. DT treatment drastically decreased the number of neoplastic fibrocytes expressing CD45.2/Col-1 in BM and spleen of Jak2V617F/CD11b-DTR mice compared with control mice treated with PBS. Consistently, reticulin fibers were eliminated almost completely and collagen fibers almost fully disappeared in BM, which led to a reversal of the decrease in BM cellularity, although the number of MKs was not affected. Similar findings were observed in the spleen, although not completely. Plasma TGF-b1 level were about 2-fold higher in Jak2V617F/CD11b-DTR mice than in WT mice. Neoplastic monocyte depletion significantly decreased TGF-b1 level. Since MK numbers did not change, this indicates that fibrocytes are one of the main sources of TGF-b1. In other features of MF in Jak2V617F/CD11b-DTR mice, splenomegaly was ameliorated by DT treatment. Microscopic analysis revealed an improvement in the damaged spleen architecture and the disappearance of splenic fibrosis. In summary, most collagen-producing cells in BM were neoplastic fibrocytes in Jak2V617F-induced MPN, indicating that neoplastic fibrocytes played an essential role and mesenchymal fibroblasts had a minor contribution in fibrosis in MPN. Depletion of neoplastic monocytes also improved splenomegaly as well as BM fibrosis in mice, and this cell fraction could be a promising therapeutic target. Disclosures No relevant conflicts of interest to declare.

P6390Macrophages as contributors to chronic inflammation and cardiac fibrosis in patients with myocardial infarction

Abstract Introduction The functional characteristics of tissue macrophages associated with the progression of cardiac fibrosis in clinic are still unknown. Purpose The purpose was to study cardiac macrophage phenotypes contributing to the development of chronic inflammation and fibrosis in patients with myocardial infarction (MI). Methods The study included 41 patients with fatal MI type 1. Group 1 (n=24) comprised patients who died within 72 hours of MI (the inflammatory phase) and group 2 (n=17) comprised patients who died 4–28 days after MI (the regenerative phase). Macrophage infiltration (number of cells) in the heart was assessed by double immunofluorescence in the non-infarct area. Each area was evaluated in 20 random fields. We used CD163, CD206, stabilin-1, α-smooth muscle actin (α-SMA), interleukin-10 (IL-10) as markers of M2-like macrophages. Results The number of CD163+/CD206− (p=0.087) and CD163+/206+ (p=0.072) macrophages was higher during the regenerative phase of MI. The number of CD163-/CD206+, stabilin-1+/α-SMA-, stabilin-1+/α-SMA+, stabilin-1+/IL-10−, stabilin-1+/IL-10+, stabilin-1-/IL-10+ did not significantly change throughout the entire period of MI. The comparison of various M2 macrophage subpopulations into groups revealed following. In group 1 the number of CD163+/CD206− and CD163+/CD206+ cells prevailed over CD163−/CD206+ (p=0.033 and p=0.003, respectively), stabilin1+/α-SMA− cells over stabilin-1+/α-SMA+ (p&lt;0.001), and stabilin-1+/IL-10+ cells over stabilin-1+/IL-10− (p=0.018). In group 2 the quantity of stabilin-1+/α-SMA− macrophages prevailed over stabilin-1+/α-SMA+ (p=0.005) and stabilin-1+/IL-10+ over stabilin-1−/IL-10+ (p=0.028). In group 1 the number of CD163+/CD206+ cells correlated with the absolute and the relative number of peripheral blood monocytes prior to the onset of death (R=0.97), while the quantity of stablin-1+/α-SMA+ cells correlated with the absolute number of peripheral blood monocytes at admission (R=-0.53). In group 2 the quantity of CD206+/CD163− cells correlated with the absolute and the relative number of monocytes in the peripheral blood at admission (R=0.73 and R=0.59, respectively), the quantity of CD163+/CD206− macrophages with the incidence of recurrent MI (R= 0.54), and the number of stabilin-1+/α-SMA+ macrophages with the age of patients (R=-0.58). Conclusions We have suggested that the key cardiac macrophage phenotypes contributing to the development of chronic inflammation and cardiac fibrosis in the regenerative phase of MI were stabilin-1+/α-SMA− and stabilin-1+/IL-10+. We revealed subpopulation of stabilin-1+/α-SMA+ macrophages, that indicated the possibility of cellular transdifferentiation and macrophage plasticity. Thus, our results supports that understanding the role of macrophages in initiation, progression, and resolution of cardiac fibrosis is one of the most promising goal in the design of anti-fibrotic treatment strategies for patients with MI.

Interaction of PRRS virus with bone marrow monocyte subsets

PRRSV can replicate for months in lymphoid organs leading to persistent host infections. Porcine bone marrow comprises two major monocyte subsets, one of which expresses CD163 and CD169, two receptors involved in the entry of PRRSV in macrophages. In this study, we investigate the permissiveness of these subsets to PRRSV infection. PRRSV replicates efficiently in BM CD163+ monocytes reaching titers similar to those obtained in alveolar macrophages, but with a delayed kinetics. Infection of BM CD163− monocytes was variable and yielded lower titers. This may be related with the capacity of BM CD163− monocytes to differentiate into CD163+ CD169+ cells after culture in presence of M-CSF. Both subsets secreted IL-8 in response to virus but CD163+ cells tended to produce higher amounts. The infection of BM monocytes by PRRSV may contribute to persistence of the virus in this compartment and to hematological disorders found in infected animals such as the reduction in the number of peripheral blood monocytes.

Association between the baseline peripheral blood monocyte counts, the size of spleen, and the response to cardiac resynchronization therapy

BackgroundSpleen reserves monocytes, which deploy to inflammatory sites. Monocytosis is known to be observed in chronic low-grade inflammatory state, including chronic heart failure (CHF). CHF also induces splenomegaly. We tested the hypotheses that the number of peripheral blood monocytes and size of spleen at baseline could be related to the response to cardiac resynchronization therapy (CRT). MethodsFrom 2010, a total of 49 consecutive patients implanted with CRT device were evaluated at baseline and 6–8 months later. The size of spleen was evaluated at baseline by computed tomography. Blood monocyte counts (BMCs) were examined by blood test apparatus. ResultsPatients were categorized as responders (13 female, mean age 69.0±7.9 years, n=34) and nonresponders (2 female, mean age 72.0±8.8 years, n=15) according to echocardiographic findings. In non-responders, spleen index was also greater in non-responders than in responders (4504±1338mm2 vs. 3240±1115mm2; mean±SE, p<0.01). Median baseline BMCs were significantly smaller in responders than non-responders (537±211/μl vs. 336±107/μl, p<0.01). In addition, BMC is positively correlated with the spleen index (R2=0.179, p=0.02). Based on the receiver-operating characteristic curve, low BMC was set at <400/μl. Kaplan–Meier survival analysis demonstrated that the low BMC patients had lower prevalence of new hospitalization due to HF progression (log rank 5.35, p=0.02). ConclusionsOur results demonstrated that BMC and the size of spleen might be important factors for response to CRT.

Human cytomegalovirus reactivation from latency: validation of a "switch" model in vitro.

BackgroundHuman cytomegalovirus (HCMV) is an opportunistic pathogen leading to severe and even fatal diseases in ‘at-risk’ categories of individuals upon primary infection or the symptomatic reactivation of the endogenous virus. The mechanisms which make the virus able to reactivate from latency are still matter of intense study. However, the very low number of peripheral blood monocytes (an important latent virus reservoir) harbouring HCMV DNA makes it very difficult to obtain adequate viral quantities to use in such studies.Thus, the aim of the present study was to demonstrate the usefulness of human THP-1 monocytes, mostly employed as HCMV latent or lytic infection system, as a reactivation model.MethodsTHP-1 monocytes were infected with HCMV TB40E strain (latency model) at multiplicities of infection (MOI) of 0.5, 0.25 or 0.125. After infection, THP-1 aliquots were differentiated into macrophages (reactivation model). Infections were carried out for 30 h, 4, 6 and 7 days. Viral DNA evaluation was performed with viable and UV-inactivated virus by q-Real-Time PCR. RNA extracted from latency and reactivation models at 7 days post-infection (p.i.) was subjected to RT-PCR to analyse viral latency and lytic transcripts. To perform viral progeny analysis and titration, the culture medium from infected THP-1 latency and reactivation models (7 days p.i.) was used to infect human fibroblasts; it was also checked for the presence of exosomes.For viral progeny analysis experiments, the Towne strain was also used.ResultsOur results showed that, while comparable TB40E DNA amounts were present in both latent and reactivation models at 30 h p.i., gradually increased quantities of viral DNA were only evident in the latter model at 4, 6, 7 days p.i.. The completion of the lytic cycle upon reactivation was also proved by the presence of HCMV lytic transcripts and an infectious viral yield at 7 days p.i.ConclusionsOur data demonstrate the effectiveness of THP-1 cells as a “switch” model for studying the mechanisms that regulate HCMV reactivation from latency. This system is able to provide adequate quantities of cells harbouring latent/reactivated virus, thereby overcoming the intrinsic difficulties connected to the ex vivo system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0634-z) contains supplementary material, which is available to authorized users.

Accelerated apoptosis of peripheral blood monocytes in Cebpb-deficient mice

The CCAAT/enhancer-binding protein β (C/EBPβ) transcription factor is required for granulopoiesis under stress conditions. However, little is known about its roles in steady state hematopoiesis. Here, we analyzed the peripheral blood and bone marrow of Cebpb−/− mice at steady state by flow cytometry and unexpectedly found that the number of peripheral blood monocytes was severely reduced, while the number of bone marrow monocytes was maintained. The ability of Cebpb−/− bone marrow cells to give rise to macrophages/monocytes in vitro was comparable to that of wild-type bone marrow cells. Apoptosis of monocytes was enhanced in the peripheral blood, but not in the bone marrow of Cebpb−/− mice. These results indicate that C/EBPβ is required for the survival of monocytes in peripheral blood.

Abstract 96: Changes In HDL Cholesterol During The Acute Phase of Kawasaki Disease

To determine the correlations between changes in high-density lipoprotein cholesterol (HDL-C) and other blood variables during the acute phase of Kawasaki disease (KD), we investigated changes in the plasma level of HDL-C, serum apolipoproteins and number of peripheral blood monocytes. 26 Japanese patients with KD were subjects of this study. Plasma HDL-C decreased during the 1st week of illness, and increased during the 2nd week. The mean levels of minimum HDL-C in KD patients treated with total intravenous immunoglobulin (IVIG) dose ≧ 4g/kg, was significantly lower than those treated with total IVIG dose &lt; 4g/kg (p=0.0005). There was a strong direct correlation between minimum HDL-C in KD patients and HDL-C on 5th day of KD onset (r=0.71, p &lt; 0.0001). Assessing the performance of plasma HDL-C on 5th day of KD onset in predicting the necessity for high-dose IVIG treatment to KD, ROCs were plotted and the AUC was 0.82. A cutoff value of 16mg/dl had a specificity of 0.82 and sensitivity of 0.80 for predicting those. The apo A-I and A-II levels were correlated with plasma HDL-C, respectively. And the peripheral blood monocytes counts which correlated with serum levels of macrophage-colony stimulating factor, were not correlated with plasma HDL-C. Plasma HDL-C levels during the 1st week of illness might serve as a predictor for non-responder to low-dose IVIG treatment in patients with acute KD.

Immune function in female B6C3F1 mice is modulated by DE-71, a commercial polybrominated diphenyl ether mixture

Polybrominated diphenyl ethers (PBDEs) are an important class of flame-retardants that are environmentally persistent and bioaccumulative. Toxicity of these compounds has become a concern because detectable levels of PBDEs are present in humans and wildlife and they are structurally similar to polychlorinated biphenyls (PCBs). This study examined the effects of the commercial penta-BDE mixture, DE-71, in adult female B6C3F1 mice on hematology, serum clinical chemistry, thyroid hormones, tissue histology, and several immunotoxicity end-points (lymphocyte proliferation, NK cell activity, splenic immunophenotypes, and SRBC-specific-IgM production). Mice were exposed via oral gavage for 28 days to achieve total administered doses (TAD) of 0, 0.5, 5, 50, or 100 mg/kg. No changes in histology, clinical chemistry, body or organ weights were observed. Serum total T3 and T4 levels were not altered by any of the DE-71 treatments. Peripheral blood monocyte numbers were decreased by the 0.5, 5, and 50 mg/kg treatments, but not by the 100 mg/kg TAD concentration. Compared to controls, mitogen-stimulated T- and B-cell proliferation was increased by the 100 mg/kg TAD concentration (ED50 = 60 mg/kg TAD [2.14 mg/kg/day] and 58 mg/kg TAD [2.57 mg/kg/day], respectively). NK cell activity was decreased compared to controls by the 100 mg/kg TAD concentration (ED50 = 20 mg/kg TAD [0.7 mg/kg/day]). No alterations were noted in thymic T-cell populations or in SRBC-specific-IgM production. Numbers of CD19+CD21−, CD19+CD21+, CD4+CD8−, CD4−CD8+, CD4−CD8−, and MHC-II+ cells in the spleen were not affected. However, the numbers of splenic CD4+CD8+ cells were decreased compared to the controls by 0.5, 5, and 100 mg/kg TAD. This study provides an assessment of the systemic toxicity and immunotoxicity of DE-71, and indicates that immune parameters are modulated at exposure concentrations lower than previously reported.

Dendritic Cell Depletion in Burn Patients Is Regulated by MafB Expression

Studies have shown that monocytes are hyporesponsive and that dendritic cells (DCs) are depleted in burn patients. We have recently shown in a mouse model that burn injury alters the transcriptional regulation in bone marrow progenitors and inhibits myeloid-derived DC (mDC) production. In the present study, using human burn patient peripheral blood mononuclear cells, we have shown an overexpression of MafB with a corresponding reduction in peripheral blood mononuclear cell-derived mDCs. We isolated mononuclear cells from burn patient (23–68% TBSA) and control volunteer peripheral blood samples by Ficoll gradient centrifugation and cultured mDCs by using a standard ex vivo culture system. Fluorescence-activated cell sorter analysis was used to select myeloid cells based on the cell surface expression of CD45+. The mDC fraction was identified by the expression of human leukocyte antigen (HLA)-DR+CD11c+, and we found a significant reduction in HLA-DR+ leukocytes for up to 4 weeks postburn. MafB expression was then examined in HLA-DR+CD14+ monocytes. Burn injury alters the phenotype of CD14+ monocytes augmenting MafB expression and reducing their differentiation into mDCs. MafB was then silenced in ex vivo culture prior to DC differentiation by using small interfering RNA technique. MafB gene silencing improved the differentiation potential of CD14+ cells into mDCs, increasing the percentage of mDCs by >75%. Furthermore, GATA-1+ and HLA-DR+ mDCs were increased following MafB silencing. Although burn injury augments the number of peripheral blood monocytes, the frequency of mDC is reduced. This impairment is likely secondary to the down-regulation of mDC differentiation by high MafB-expressing monocytes following burn injury.

Elevated levels of soluble CD14 in serum of patients with systemic lupus erythematosus.

A soluble form of CD14 (sCD14) was assessed with an ELISA assay in the serum of the following three clinical groups: 35 patients with an inactive phase of systemic lupus erythematosus (SLE), 17 patients with SLE relapses, and 65 normal healthy volunteers. Increased levels of sCD14 were observed in all patients suffering from SLE compared with normal controls. In addition, patients with active SLE revealed higher serum concentrations of sCD14 (median 6.9 mg/l) than patients under remission (4.1 mg/l; P < 0.0001). Serum values of sCD14 correlated neither with the number of peripheral blood monocytes bearing the CD14 membrane antigen, nor with serum concentrations of IL-1 beta. Serum sCD14 was compared with other clinical parameters used to monitor the clinical course of patients with SLE, among them complement C3, anti-dsDNA antibodies and soluble IL-2 receptor (sIL-2R). A good correlation emerged between sCD14 and C3 as well as sIL-2R concentrations, but sCD14 and anti-dsDNA titres disclosed no significant correlation in both groups of patients with SLE. Serial studies in patients with severe SLE showed that serum sCD14 closely parallels the clinical course as defined by an activity score. Our data suggest that serum sCD14 represents a promising parameter to monitor disease activity in patients with SLE.

Neuroinvasion of Fluorescein-Positive Monocytes in Acute Simian Immunodeficiency Virus Infection

Monocytes and macrophages play a central role in the pathogenesis of human immunodeficiency virus (HIV)-associated dementia. They represent prominent targets for HIV infection and are thought to facilitate viral neuroinvasion and neuroinflammatory processes. However, many aspects regarding monocyte brain recruitment in HIV infection remain undefined. The nonhuman primate model of AIDS is uniquely suited for examination of the role of monocytes in the pathogenesis of AIDS-associated encephalitis. Nevertheless, an approach to monitor cell migration from peripheral blood into the central nervous system (CNS) in primates had been lacking. Here, upon autologous transfer of fluorescein dye-labeled leukocytes, we demonstrate the trafficking of dye-positive monocytes into the choroid plexus stromata and perivascular spaces in the cerebra of rhesus macaques acutely infected with simian immunodeficiency virus between days 12 and 14 postinfection (p.i.). Dye-positive cells that had migrated expressed the monocyte activation marker CD16 and the macrophage marker CD68. Monocyte neuroinvasion coincided with the presence of the virus in brain tissue and cerebrospinal fluid and with the induction of the proinflammatory mediators CXCL9/MIG and CCL2/MCP-1 in the CNS. Prior to neuroinfiltration, plasma viral load levels peaked on day 11 p.i. Furthermore, the numbers of peripheral blood monocytes rapidly increased between days 4 and 8 p.i., and circulating monocytes exhibited increased functional capacity to produce CCL2/MCP-1. Our findings demonstrate acute monocyte brain infiltration in an animal model of AIDS. Such studies facilitate future examinations of the migratory profile of CNS-homing monocytes, the role of monocytes in virus import into the brain, and the disruption of blood-cerebrospinal fluid and blood-brain barrier functions in primates.

Clinical grade of gerneration of dendritic cells for immunotherapy

In order to develop a protocol for clinical grade generation of dendritic cells (DCs) for cancer immunotherapy, aphereses were performed with the continuous flow cell separator and materials were derived from 10 leukemia patients that had achieved complete remission. Peripheral blood monocytes were cultured in vitro with GM-CSF, IL-4 for 6 days, then TNF-(the TNF-group) or TNF-, IL-1, IL-6, PGE2 (the cytokine mixture group) were added to promote maturation. Cell number was counted by hematology analyzer, and phenotype study (CD1a, CD14, CD83) was carried out by flow cytometry, and the function of DCs was examined by mixed lymphocyte reaction. The results showed that (0.70+/-0.13)x10(7)/mL (the TNF-alpha group) and (0.79+/-0.04)x10(7)/mL (the cytokine mixture group) DCs were generated respectively in peripheral blood obtained by leucapheresis. The phenotypes were as follows: CD1a+ (74.65+/-4.45)%, CD83+ (39.50+/-4.16)%, CD14+ (2.90+/-1.76)% in TNF-alpha group, and CD1a+ (81.86+/-5.87)%, CD83+ (81.65+/-6.36)%, CD14+ (2.46+/-1.68)% in the cytokine mixture group. It was concluded that leucapheresis may be a feasible way to provide large number of peripheral blood monocytes for DC generation, and combined administration of TNF-, IL-1, IL-6, and PGE2 may greatly promote maturity.

Treatment With AM3 Restores Defective T-Cell Function in COPD Patients

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. To characterize putative systemic abnormalities of the T-cell compartment in COPD patients, and to investigate whether AM3 can restore such abnormalities. 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. Outpatient departments of four hospitals. 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. 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. 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. PBMCs of COPD patients showed clear functional T-lymphocyte abnormalities that are rescued by AM3 treatment.

Leukapheresis for the extraction of monocytes and various lymphocyte subpopulations from peripheral blood: product quality and prediction of the yield using different harvest procedures

Leukapheresis of non-mobilized healthy donors is performed to harvest monocytes and lymphocyte subpopulations for use in various therapeutic regimens. In this methodological study, we compared two different leukapheresis programs, using equivalent volumes of processed blood over similar processing periods, to determine the influence of the procedures on the donor peripheral blood count and to establish the procedure that yields the highest quality product. The target variables obtained in 41 healthy blood donors who underwent short-term leukapheresis (80-105 min) were retrospectively compared. Twenty-one volunteers were processed on a COBE Spectra machine at the MNC setting and 20 volunteers were processed at the AutoPBSC setting. Data were collected on pre- and postleukapheresis samples and on the product. AutoPBSC and MNC procedures resulted in a decrease of haemoglobin (5-7%), platelets (17-20%), monocytes (22%) and lymphocytes (23-27%), but not of granulocytes in peripheral blood. Both procedures produced nearly identical leucocyte and lymphocyte yields. AutoPBSC products contained a greater number of granulocytes, monocytes and red cells, but fewer platelets. The preleukapheresis values correlated with the yields for monocytes, T-helper and T-suppressor cells, B-lymphocytes and natural killer cells, but not for granulocytes or platelets. Leukapheresis is a safe and efficient procedure for collecting large numbers of peripheral blood monocytes and different lymphocyte populations from non-mobilized donors. The two programs yield comparable leucocyte harvests. Based on our results, yields can be predicted from the peripheral cell counts.

Concomitant Endocrine and Immune Alterations during Alcohol Intoxication and Acute Withdrawal in Alcohol-Dependent Subjects

Although both alcohol intoxication and withdrawal have been demonstrated to produce significant endocrine alterations, no data exist on the effects of acute withdrawal on immune functions. Therefore, the current study investigated the effect of alcohol intoxication and acute withdrawal on plasma cortisol, prolactin and catecholamines, and blood leukocyte subset distribution in alcohol-dependent subjects. Nine male alcoholics admitted to the university clinic for alcohol dependence and 9 age-matched controls participated in the study. Blood was drawn from the alcohol-dependent subjects at 10:30 a.m. on day 0 (chronic alcohol intoxication), at the same time during acute alcohol withdrawal (day 1) and following the resolution of acute withdrawal (day 7). Blood was drawn from age- and gender-matched healthy control subjects at the corresponding time points. Plasma was then analyzed for hormone concentrations and blood examined for leukocyte subsets by flow cytometry. Alcohol-dependent patients displayed significantly elevated plasma cortisol during intoxication and withdrawal, which decreased to control levels following resolution of acute withdrawal. Small elevations of plasma prolactin and catecholamines were also observed during intoxication. Furthermore, alcohol-dependent subjects showed reduced absolute numbers of CD4⁺ and CD8⁺ T cells and natural killer cells compared with healthy controls across all time points. In contrast, although monocyte numbers were lower in alcohol-dependent patients during intoxication, acute alcohol withdrawal increased the number of monocytes in patients. Thus, alcohol dependence produces a general suppression of leukocyte subset populations in blood. However, resolution of acute alcohol withdrawal is associated with a return of plasma cortisol to control levels, and a concomitant increase in peripheral blood monocyte numbers.

Decreased Peripheral Blood T Cell Cytokine Gene Expression in Rheumatoid Arthritis

Currently, few informations are available about spontaneous production of T cell cytokines in rheumatoid arthritis (RA) peripheral blood (PB), because these cytokines are generally under the detection threshold of ELISAs. Because the Th1/Th2 balance could help to determine the outcome of RA, we used a sensitive and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method to mesure spontaneous T cell production of IL-2, IL-4, IL-10, and IFN-gamma mRNAs using unstimulated PBMC from 25 active RA patients, not taking any DMARDs for at least 6 weeks, and 19 healthy controls. Spontaneous IL-2 and IL-4 mRNA expressions are significantly lower in RA patients compared to healthy controls. Levels of IL-10 and IFN-gamma are similar in the two groups. No correlation was found between cytokine mRNA levels and clinical parameters. Spontaneous IL-4 and IL-10 mRNA levels are respectively correlated to the number of CD4+ T cells and to the number of monocytes in PB. After in vitro stimulation, IFN-gamma mRNA production by RA PBMC is significantly decreased. Most of the patients cannot be classified as having a T cell cytokine type 1 or type 2 secretion pattern in PB. IL-2 and IL-4 mRNAs in PB of active RA are produced at a low spontaneous level and the response to in vitro activation by mitogen is weak.

Serum soluble interleukin-2 receptor level as a prognostic indicator in gastric cancer.

T lymphocytes, activated by interleukin 2 during an anti-tumour response, release soluble interleukin 2 receptors (sIL-2R) into the bloodstream. We analysed the prognostic value of the serum sIL-2R level in gastric cancer. Serum concentration of sIL-2R in 96 gastric cancer patients and 100 healthy control subjects' was measured by enzyme-linked immunosorbent assay. All survivors were followed for more than 50 months. Serum sIL-2R level was considered with respect to prognosis, clinicopathological factors, other tumour markers and peripheral blood cell count. Stage III and IV patients had significantly higher sIL-2R levels than lower stage patients and control subjects. Stage III and IV gastric cancer patients were divided into 'high' and 'low' slL-2R groups based upon the control subjects' serum sIL-2R mean value plus one standard deviation. The high group had a significantly worse prognosis than the low group, although clinicopathological features and treatments were similar. Multivariate analysis demonstrated that the serum sIL-2R level is an independent indicator. The sIL-2R level did not correlate with carbohydrate antigen 19-9, however it did correlate with carcinoembryonic antigen (r = 0.22) and with numbers of peripheral blood monocytes (r = 0.54). In conclusion, serum sIL-2R may predict the outcome of gastric cancer patients with stage III or IV disease.

Changes in CD23 expression of blood and skin in atopic eczema after Chinese herbal therapy.

Aberrant expression of CD23 (low affinity IgE receptor) on cells of the monocyte/macrophage series in peripheral blood and lesional skin of patients with atopic eczema has been demonstrated. It is not known whether this abnormality results from a fundamental systemic problem of the monocytes of these patients or reflects local changes to cell populations within the skin tissues. This study was designed to determine whether this aberrant expression was caused by local cutaneous influences on mature cells or fundamental changes in monocyte differentiation. The possible relationship between these aberrations and clinical severity was also investigated by repeating these immunopathological studies after a course of efficacious treatment with Chinese herbal therapy (CHT). Peripheral blood mononuclear cells were obtained from patients with atopic eczema before, and after 8 weeks of treatment. Efficacy of CHT was quantified on clinical grounds. Monocytes were isolated by adherence to plastic and cultured for up to 7 days. Samples were harvested at 2, 5 and 7 days of culture and cytospins prepared. Immunocytochemical staining to identify phenotypic subsets was performed on the monocytes at time 0 and on maturing cells from culture. This immunocytology was quantified using computerized image analysis equipment to determine the emergence of macrophage subsets and their level of CD23 expression. Biopsies were taken from lesional skin before and after treatment and immunohistology was performed on cryostat sections to determine the number of antigen presenting cells expressing CD23 as well as the level of expression of these molecules. The results showed that increased numbers of monocytes from patients with atopic eczema express CD23 at day 0 and that cultured monocytes from these patients differentiate faster during the 7 day culture period as compared to normal controls. Efficacious treatment did not affect the number of peripheral blood monocytes expressing CD23. However, treatment did lead to a significant decrease in the number of CD23+ mature macrophages in the skin as well as a reduction in the level of expression of this moiety. These results demonstrate that changes in clinical severity are more closely related to the expression of CD23 on mature antigen presenting cells in lesional skin rather than to differentiating peripheral blood monocyte CD23 expression. These results suggests that local factors within lesional skin govern the accumulation and the expression of CD23 on mature macrophages and that these factors may be more relevant to the pathogenesis of the disease than aberrations in CD23 expression that may occur systemically.