Core-binding factor leukemia hijacks the T-cell-prone PU.1 antisense promoter.

The blood system serves as a key model for cell differentiation and cancer. It is orchestrated by precise spatiotemporal expression of crucial transcription factors. One of the key master regulators in the hematopoietic systems is PU.1. Reduced levels of PU.1 are characteristic for human acute myeloid leukemia (AML) and are known to induce AML in mouse models. Here, we show that transcriptional downregulation of PU.1 is an active process involving an alternative promoter in intron 3 that is induced by RUNX transcription factors driving noncoding antisense transcription. Core-binding factor (CBF) fusions RUNX1-ETO and CBFß-MYH11 in t(8;21) and inv(16) AML, respectively, activate the PU.1 antisense promoter that results in a shift from sense toward antisense transcription and myeloid differentiation blockade. In patients with CBF-AML, we found that an elevated antisense/sense transcript and promoter accessibility ratio represents a hallmark compared with normal karyotype AML or healthy CD34+ cells. Competitive interaction of an enhancer with the proximal or the antisense promoter forms a binary on/off switch for either myeloid or T-cell development. Leukemic CBF fusions thus use a physiological mechanism used by T cells to decrease sense transcription. Our study is the first example of a sense/antisense promoter competition as a crucial functional switch for gene expression perturbation by oncogenes. Hence, this disease mechanism reveals a previously unknown Achilles heel for future precise therapeutic targeting of oncogene-induced chromatin remodeling.

Human lactoferrin attenuates the proinflammatory response of neonatal monocyte-derived macrophages.

Bioactive components of human milk, such as human lactoferrin (hLF), play an essential role in gut microbiome homeostasis and protection against neonatal inflammatory diseases. Neonatal intestinal macrophages display a proinflammatory profile that might contribute to inflammatory mucosal injury. Therefore, the aim of the study was to investigate the immunomodulatory effects of hLF on differentiation and activation of monocyte-derived macrophages (moMϕ). Monocytes isolated from umbilical cord blood of term neonates and peripheral blood of healthy adults were differentiated in the absence or presence of hLF, and differentiation, apoptosis and phagocytosis were evaluated. Cytokine production, Toll-like receptor (TLR) signalling and activation marker expression were investigated upon activation with lipopolysaccharide (LPS) and lipoteichoic acid (LTA) challenge. We demonstrate that hLF-differentiated moMϕ exhibit decreased TLR-4 expression, TLR signalling, proinflammatory cytokine secretion and intracellular tumour necrosis factor (TNF)-α production. Investigation of differentiation markers, morphology and induction of apoptosis showed no alteration in lactoferrin-differentiated moMϕ. Taken together, hLF promote anergic/anti-inflammatory effects by TLR expression and pathway interference, resulting in a diminished proinflammatory moMϕ phenotype. The anergic/anti-inflammatory properties of hLF might contribute to the prevention of harmful TLR-mediated inflammatory disorders in the developing gut of premature infants.

Phase I Study of Lenalidomide and Sorafenib in Patients With Advanced Hepatocellular Carcinoma.

LESSONS LEARNED: Combination therapies in patients with hepatocellular carcinoma can be associated with overlapping toxicity and are therefore poorly tolerated.Using sorafenib at the maximum tolerated dose can lead to a higher incidence of toxicities. Consequently, combination studies might evaluate sorafenib at alternative schedules or doses to improve tolerance, recognizing this could affect sorafenib efficacy.Although this combination was poorly tolerated, it does not exclude further evaluation of new-generation immunomodulator drugs or immune checkpoint inhibitors in the hope of optimizing tolerance and safety. BACKGROUND: Sorafenib is the standard treatment for advanced hepatocellular carcinoma (HCC), and to date, no combination therapy has demonstrated superior survival compared with sorafenib alone. The immunosuppressive microenvironment in HCC is a negative predictor for survival. Lenalidomide is an immunomodulator and antiangiogenic agent, with limited single-agent efficacy in HCC. Based on these data, we designed a phase I study of sorafenib plus lenalidomide to determine the safety and preliminary antitumor activity of this combination. METHODS: This was an open-label, phase I study with a 3+3 dose escalation/de-escalation design. The starting dose of sorafenib was 400 mg p.o. b.i.d. and of lenalidomide was 15 mg p.o. daily with a planned dose escalation by 5 mg per cohort up to 25 mg daily. Dose de-escalation was planned to a sorafenib dose of 400 mg p.o. daily combined with two doses of lenalidomide: 10 mg p.o. daily for a 28-day cycle (cohort 1) and 10 mg p.o. daily for a 21- or 28-day cycle (cohort 2). Patients with cirrhosis, a Child-Pugh score of A-B7, and no previous systemic therapy were eligible. RESULTS: Five patients were enrolled. Their median age was 56 years (range 39-61), and the ECOG status was 0-2. Four patients were treated at dose level (DL) 1. Because of the poor tolerance to the combination associated with grade 2 toxicities, one more patient was treated at DL -1. No dose-limiting toxicity was observed as specified per protocol. The most common toxicities were nausea, anorexia, pruritus, elevated liver enzymes, and elevated bilirubin. Three patients experienced one or more of the following grade 3 toxicities: fatigue (DL 1), increased bilirubin (DL 1), skin desquamation (DL -1), and elevated transaminase levels (DL 1). The median duration of therapy was 1 cycle (range 1-3). All patients discontinued the study, 4 because of progressive disease and 1 by patient preference. The best confirmed response was progressive disease. The median progression-free survival was 1.0 month (95% confidence interval 0.9-2.8), and the median overall survival was 5.9 months (95% confidence interval 3.68-23.4). CONCLUSION: In our small study, the combination of lenalidomide and sorafenib was poorly tolerated and showed no clinical activity. Although the study was closed early because of toxicity concerns, future studies assessing combinations of sorafenib with new-generation immunomodulator drugs or other immunomodulatory agents, should consider lower starting doses of sorafenib to avoid excessive toxicity.

Effect of preoperative fever-range whole-body hyperthermia on immunological markers in patients undergoing colorectal cancer surgery.

BACKGROUND: Previous studies have demonstrated beneficial immunological effects of fever-range whole-body hyperthermia (FR-WBH) as an adjunct to non-surgical cancer therapy. We conducted a study of preoperative FR-WBH in patients undergoing colorectal cancer surgery to evaluate perioperative, hyperthermia-induced immunomodulation. METHODS: The trial was conducted as a subject-blinded, controlled, randomized study. Subjects in the FR-WBH group (n=9) were treated with FR-WBH before operation under propofol sedation; the target core temperature was 39 (0.5)°C with 1 h warming and 2 h plateau phase. Subjects in the control group (n=9) were treated with propofol sedation only. Blood samples were acquired before and after treatment, after operation, and 24, 48 h, and 5 days after the end of surgery. The following parameters were measured: lipopolysaccharide (LPS)-induced tumour necrosis factor (TNF)-α, procalcitonin (PCT), interleukin (IL)-6/10, heat shock proteins (HSPs) 60, 70, and 90, human leucocyte antigen-DR (HLA-DR), and LPS-binding protein (LBP). RESULTS: HSPs were increased in the FR-WBH group after treatment [HSP60, 48 h postop: 143 (41)% vs 89 (42)%, P=0.04; HSP90, postop: 111 (33)% vs 64 (31)%, P=0.04; HSP70: P=0.40; FR-WBH vs control, P-values for area under the level/time curve]. TNF-α levels were elevated after surgery in the control group and remained near baseline in the FR-WBH group [24 h postop: 73 (68)% vs 151 (72)%, P=0.04]. PCT increased in both groups 24 h after surgery; in the control group, this increase was significantly higher (P=0.02). There were no significant differences for IL, HLA-DR, or LBP. CONCLUSIONS: The immune system to react to surgical stress, as measured by a panel of laboratory indicators, might be improved by preoperative FR-WBH.

Phase II trial of single agent amrubicin in patients with previously treated advanced thymic malignancies.

OBJECTIVES: There are limited treatment options for patients with thymic malignancies. Here we present data supporting treatment with single agent amrubicin, a third generation anthracycline and topoisomerase II inhibitor. MATERIALS AND METHODS: This was a phase 2 open-label, single arm trial of amrubicin in patients with thymoma (T) or thymic carcinoma (TC), conducted at two academic institutions. Patients were included if they had received at least one prior chemotherapy regimen. The first 18 patients received amrubicin at 40 mg/m2 IV days 1-3 repeated every 3-weeks. Due to the high incidence of febrile neutropenia, dosing was subsequently amended to 35 mg/m2 for the final 15 patients. RESULTS: A total of 33 patients (14 T/19 TC) were enrolled from 2011 to 2014. Median number of prior therapies was 2. Best response included 6 partial responses, 21 stable disease, and 6 progressive disease (all TC). Objective response rate was 18% (90% exact binomial CI 8.2%-32.8%; T = 4/14 (29%), TC = 2/19 (11%)). Median progression-free survival was 7.7 months (T: 8.3 months; TC: 7.3) and median overall survival was 29.7 months (T: 54.1 months; TC: 18 months). There was a high rate of febrile neutropenia (7 patients) that occurred despite a reduction in amrubicin dose and one related death. Five patients had reduction in LVEF below 50% during the course of treatment resulting in treatment discontinuation in one patient. CONCLUSION: Amrubicin shows promise as a single agent in heavily pre-treated patients with thymic malignancies. Notable side effects include febrile neutropenia and the use of growth factor support is essential. Further investigation of this agent is warranted.

Serum phenylalanine in patients post trauma and with sepsis correlate to neopterin concentrations.

Increased blood concentrations of phenylalanine in patients with trauma and sepsis are common but unexplained. We examined the potential relationship between serum concentrations of phenylalanine and the immune activation marker neopterin in 84 specimens of 18 patients (14 males and 4 females) post-trauma during 12-14 days of follow up. Compared to healthy controls, average phenylalanine and neopterin concentrations were elevated in patients, and there existed a positive correlation between concentrations of the two analytes (r (s) = 0.375, p < 0.001). No such association existed between neopterin and tyrosine concentrations (r (s) = -0.018), but neopterin concentrations correlated to the phenylalanine to tyrosine ratio (r (s) = 0.328, p = 0.001). Increased phenylalanine implies insufficient conversion by phenylalanine (4)-hydroxylase (PAH). Oxidative stress due to immune activation and inflammation may destroy cofactor 5,6,7,8-tetrahydrobiopterin and impair PAH activity. This assumption is further supported by the correlation found between higher neopterin concentrations and higher phenylalanine to tyrosine ratio, which estimates efficacy of PAH.

Inadequate production of hematopoietic growth factors in hairy cell leukemia: up-regulation of interleukin 6 by recombinant IFN-alpha in vitro.

The course of hairy cell leukemia (HCL) is characterized by progressive pancytopenia. The pathogenesis of this phenomenon is still not fully understood. To study if the decrease in hematopoiesis in HCL is accompanied by abnormal concentrations of growth factors, we investigated the production of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, interleukin 3 (IL-3), interleukin 6 (IL-6), and tumor necrosis factor alpha by peripheral blood mononuclear cells (PBMCs) of eight patients with HCL. The results point to a severe deficiency of production of all cytokines tested as compared to healthy donors. However, enrichment of autologous monocytes by counterflow centrifugation resulted in a marked increase of the levels of granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, IL-6, and tumor necrosis factor alpha. The most pronounced effects were seen with IL-6. Reverse transcription-PCR analysis indicated that pokeweed mitogen, IFN-alpha, and poly(I:C) are capable of inducing the expression of IL-6-specific mRNA in HCL cells. These findings are substantiated on the protein level by immunofluorescence analysis. Incubation of PBMCs with IFN-alpha resulted in a significant increase of intracellular IL-6 in HCL but not in healthy donors. This increase was also seen in hairy cells positive for CD19 and CDllc. Furthermore, IFN-alpha induced the secretion of IL-6 from PBMCs of HCL patients but not healthy donors. In conclusion, our studies with PBMCs from patients with HCL revealed an inadequate supply of hematopoietic growth factors that might, in part, be due to the monocytopenia characteristic for this disease. The findings also indicate that IFN-alpha is capable of inducing the production of IL-6 in the patients' PBMCs as well as in their hairy cells. These data from our in vitro studies support the clinical observation that treatment with IFN-alpha leads to reconstitution of hematopoiesis.

Modulation of IgA, IgE, and IgG Fc receptor expression on human mononuclear phagocytes by 1 alpha,25-dihydroxyvitamin D3 and cytokines.

The effects of 1 alpha,25-dihydroxyvitamin D3/calcitriol on the expression of Fc receptors (FcR) for IgA (Fc alpha R), IgE (Fc epsilon RII), and IgG (Fc gamma R) on human peripheral blood monocytes and the cell lines U937, THP-1, and Mono Mac-6, in combination with various cytokines, was examined by flow cytometry. On both monocyte-derived macrophages and the myelomonocytic cell lines, Fc alpha R/CD89 expression was induced by calcitriol alone and additively in combination with tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), and granulocyte-macrophage colony-stimulating factor. Constitutive and interleukin-4 (IL-4)-driven Fc epsilon RII/CD23 expression was markedly diminished on calcitriol-treated U937 cells and monocytes. Fc epsilon RII was also triggered by IFN-gamma, TNF-alpha, and IL-6 on all the cell lines, an effect blocked by calcitriol. On monocytes, the basal level and IFN-gamma-induced Fc gamma RI/CD64 expression was down-regulated by calcitriol and IL-4. The expression of Fc gamma RII/CD32 on monocytes was strongly suppressed by calcitriol. Transforming growth factor-beta induced Fc gamma RIII/CD16 on monocytes, an effect opposed by calcitriol. The ability of calcitriol-treated monocytes to phagocytose IgG-sensitized ox erythrocytes was diminished. Our results demonstrate that calcitriol, alone or in combination with cytokines, modulates Fc alpha R, Fc epsilon RII, Fc gamma RI, and Fc gamma RII expression on human mononuclear phagocytes, as well as Fc gamma R-mediated phagocytosis.

Ocular metastasis of colorectal cancer: An uncommon presentation of a common malignancy.

Approximately 20% of patients with colorectal cancer have metastatic disease at time of diagnosis, and another 25-35% develop metastases during the course of their disease. Liver, peritoneum, and lungs are the most common sites of metastases. We report the case of a 60-year-old female who presented with ocular metastasis 4 years after her initial curative-intent treatment for T3N1M0 rectal adenocarcinoma. After local eye radiation therapy, she received palliative systemic chemotherapy and enjoyed a good quality of life for 32 months before succumbing to progressive disease. Ocular metastasis of colorectal cancer is rare. When present, it usually occurs in the setting of diffuse hematogenous spread. In addition to local therapy, systemic chemotherapy should also be considered.

Purification of human basophils and mast cells by multistep separation technique and mAb to CDw17 and CD117/c-kit.

Basophils and mast cells represent distinct cell lineages within the hemopoietic system. Based on the unique cell surface antigen profile of both cells, we have established methods which allow the reproducible purification to homogeneity (> 99%) of normal human basophil granulocytes from the peripheral blood and of mast cells from human dispersed tissues. Basophils (n = 9) were purified by current counterflow elutriation followed by depletion of monocytes with CD14 mAb conjugated to magnetic beads, and subsequent cell sorting for CD217+ cells. Basophil purity was 99.5 +/- 0.4% (range 98.7-99.9%). Mast cells were obtained from lung (n = 6), uterus (n = 1), mastocytosis bone marrow (n = 2), and human foreskin (n = 2). Mast cells were purified by collagenase digestion followed by current counterflow elutriation and sorting with CD117/c-kit mAb. Mast cell purity was 99.4 +/- 0.7% (range: 97.5-99.9%). Purified cells were more than 90% viable and were able to release histamine on induction with IgE plus anti-IgE. Furthermore, the PCR technique could be applied on pure cells and confirmed expression of high affinity IgE receptor (Fc epsilon R1) alpha chain mRNA. Thus, by combining isolation techniques including elutriation, magnetic cell depletion and cell sorting with mAb, functionally intact normal human basophils and mast cells can be enriched to homogeneity.

Expression of MHC class II antigens on rat bone marrow cells and macrophages, and their modulation during culture with murine GM-CSF or M-CSF.

Flow cytometric analysis employing MRC OX 6 and MRC OX17 monoclonal antibodies recognizing determinants on RT1.B or RT1.D molecules, equivalent to murine I-A and I-E, respectively, was used to detect rat MHC class II antigen (Ag) expression. Approximately 5% of freshly isolated rat bone marrow cells (BMC) expressed RT1.B and over 30% displayed RT1.D molecules. The RT1.D+ cells were W3/13+, OX 7+, OX 19- and OX 22-. After one week culture of BMC with murine recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF), regardless of concentrations, 90 to 95% of the cells were scored as bone marrow-derived macrophages (BMDM phi), and over 30% expressed both RT1.B and RT1.D Ag. GM-CSF increases the percentage of BMDM phi bearing MHC class II Ag in a concentration-dependent manner. This effect seems to be specific because antibodies to interferon-gamma, tumor necrosis factor-alpha or interleukin-4 did not reduce the number of cells expressing RT1.B and RT1.D Ag. Furthermore, GM-CSF was able to trigger expression of class II molecules on rat peritoneal macrophages (M phi) and BMDM phi resulted from cultures of BMC with mouse M phi-CSF (M-CSF), and the RT1.B and RT1.D inducing effect of GM-CSF was opposed by M-CSF, and by anti-GM-CSF antibodies. The induction of MHC class II Ag synthesis by GM-CSF on rat BMDM phi was confirmed at the mRNA level by Northern blot analysis employing cDNA probes encoding the RT1.B alpha.

Lipopolysaccharide causes atrophy of Peyer's patches and an increased expression of CD28 and B7 costimulatory ligands.

Intestinal mucosal dysfunction appears to contribute to infectious complications in critically ill patients. The current study was undertaken to investigate whether endotoxin affects lymphocyte subpopulations and the expression of costimulatory signals in Peyer's patches (PP). Female Balb/c mice were given an intraperitoneal injection of 25 microg LPS and sacrified 24 h or 72 h later to determine total cell yield, lymphocyte subpopulations (B-cells, total T-cells, CD4+- and CD8+-cells), the costimulatory molecules CD28, B7.1 (CD80) and B7.2 (CD86) and the percentage of apoptotic cells in PP and in the spleen as well as small intestinal IgA concentration. Lipopolysaccharide (LPS) challenge caused a significant decrease of total cell yield in PP at both time-points (-50+/-28% and -43+/-25%, respectively; P < 0.001). This decrease was significant for all measured lymphocyte subpopulations. In contrast, total cell yield was increased (P < 0.001) in the spleen 24 h (+52+/-13%) and 72 h (+130+/-22%) after LPS. The decrease of lymphocyte numbers in the PP was accompanied by an increased percentage of lymphocytes expressing costimulatory molecules. In this respect, an increased percentage of CD40+CD80+, CD40+CD86+, and of CD4+CD28+ could be demonstrated after LPS administration. In the spleen, the percentage of CD4+CD28+ was also elevated after LPS bolus, however, the percentage of CD40+CD80+ was reduced, and that of CD40+CD86+ was unaltered. The influence of LPS on apoptosis of lymphocytes was time-dependent. The percentage of apoptotic cells 24 h after LPS was increased in PP (P < 0.01), but was unchanged in the spleen. Seventy-two hours after LPS injection, the percentage of apoptotic cells returned to normal in PP. Luminal IgA levels remained unchanged after LPS challenge. In conclusion, our data show that LPS causes atrophy of PP which seems to be counterregulated by an enhanced expression of costimulatory molecules.

Effect of single and combined supply of glutamine, glycine, N-acetylcysteine, and R,S-alpha-lipoic acid on glutathione content of myelomonocytic cells.

BACKGROUND & AIMS: Several diseases are characterised by decreased glutathione (GSH) levels due to an enhanced formation of oxygen radicals. To increase GSH levels, the additional supply of GSH precursors was suggested. In this study we evaluated the potency of a single and combined administration of the GSH modulating substances glutamine (GLN), N-acetylcysteine (NAC), and glycine (GLY) as well as R,S-alpha-lipoic acid (LA) to enhance intracellular GSH content in a well-defined model system. RESULTS: Exposure of myelomonocytic U937 cells for 24 h to GLN revealed a 1.5-fold enhancement of GSH levels with a concomitant decrease in the formation of reactive oxygen species and lipid peroxidation. Addition of NAC stimulated GSH formation only at subphysiological GLN levels. GLY enhanced GSH levels under GLN starvation, but caused a diminution of GSH content under optimal GLN supply. LA in combination with 2 mmol/l GLN evoked a 3.6-fold enhancement of GSH content compared to GLN starved cells. CONCLUSION: These results demonstrate that the GSH content of U937 cells is dependent on the supply of GLN, NAC, LA, and GLY. Combinations of the single substances can enhance but also decrease the intracellular GSH content, which is of clinical importance when supplying GSH-modulating substances to patients.

Postoperative glycyl-glutamine infusion reduces immunosuppression: partial prevention of the surgery induced decrease in HLA-DR expression on monocytes.

BACKGROUND AND AIMS: Surgery, trauma and inflammation reduce HLA-DR expression on monocytes, which is associated with an increased susceptibility to infection and sepsis. Furthermore, surgery decreases plasma glutamine (GLN) levels. The expression of HLA-DR on human monocytes in vitro is dependent on the concentration of GLN in the culture medium. We therefore hypothesized that postoperative infusions of glutamine-dipeptides would prevent the decreased HLA-DR expression on monocytes. METHODS: Thirty patients undergoing major abdominal surgery were randomly allocated to receive either 1500 ml Vamin (control) or an isonitrogenic formulation containing Vamin and 500 ml glycyl-glutamine (35 g GLN; 0.5g/kg BW) (GLY-GLN), or Vamin and 500 ml alanyl-glutamine (35 g GLN; 0.5 g/kg BW) (ALA-GLN) as a continuous infusion over 48 h post-operatively. Immediately and 48 h after surgery blood samples were collected to determine HLA-DR expression on monocytes by flow cytometry. RESULTS: The groups were comparable with respect to age, gender distribution and operation time. In patients receiving GLY-GLN mean HLA-DR expression on monocytes at 48 h was significantly better preserved than in controls (65.0 %+/-7 % vs 42.5 %+/-4 %;P<0.05), whereas HLA-DR expression on monocytes in patients receiving ALA-GLN was not significantly different. CONCLUSION: This is the first study comparing the dipeptides GLY-GLN and ALA-GLN in the postoperative setting. The GLY-GLN induced preservation of HLA-DR on monocytes following surgery may prevent infectious complications in these patients.

Influence of enteral diets supplemented with key nutrients on lymphocyte subpopulations in Peyer's patches of endotoxin-boostered mice.

BACKGROUND AND AIMS: This study was undertaken to compare the effect of different key nutrients on lymphocyte subsets of Peyer's patches (PP) and spleen in endotoxemic mice. METHODS: Female Balb/c mice were fed over a period of 10 days either with an isocaloric and isonitrogenous control diet (Control), a glutamine enriched diet (Diet I) or a diet containing glutamine, arginine, glycine, and n-3 fatty acids (Diet II). On day 7 the mice were challenged intraperitoneally with 25 microg LPS. The lymphocyte subpopulations (B cells, T cells, CD4+ and CD8+) of PP and spleen were analysed by flow cytometry. Glutathione content of small intestinal mucosa and spleen was determined by HPLC and luminal small intestinal IgA by ELISA. RESULTS: Both experimental diets increased the number of B and T cells in the PP and that of T cells in the spleen (P<0.01). Glutathione content in PP and spleen was higher under administration of key nutrients (P<0.05). Diet II reduced luminal small intestinal IgA content in comparison to the two other groups. CONCLUSION: The addition of arginine, glycine and n-3 fatty acids to a glutamine supplemented diet does not enhance lymphocyte numbers in PP and spleen, but reduces intestinal IgA content.

Different transendothelial migration behaviour pattern of blood monocytes derived from patients with benign and malignant diseases of the breast.

BACKGROUND: In this study we compared the expression of selected monocyte surface antigens with the potential to transmigrate through an endothelial layer before and after surgery from breast cancer patients (CA) and patients with benign disease of the breast (BE). MATERIALS AND METHODS: Transmigration capacity of mononuclear cells was determined after isolation by Ficoll density gradient, layered over human umbilical vein endothelial cells and cultured in a two chamber plate added with fMLP as a chemotactic stimulus. We determined monocyte phenotye (HLA-DR, FcgRI/CD64, CR1/CD11b and LFA-1/CD11a) and the phagocytosis of E. coli by flow cytometry. RESULTS: Before surgery blood monocytes had an equal expression of the measured surface antigens, but were different in regard to their interaction with endothelial cells. Monocytes derived from CA had a higher transmigration potency than those of BE. Moreover, the migration through the endothelial cell layer created different populations of monocytes. Surgical stress modified transmigrated monocytes of BE into the direction of monocytes from CA. Phagocytic capacity of peripheral blood monocytes from CA was significantly diminished and was further reduced after surgery when measured in transmigrated cells. CONCLUSION: Our study shows that monocytes from CA and BE can be discriminated in regard to their interaction with endothelial cells.

Influence of short-term protein malnutrition of mice on the phenotype and costimulatory signals of lymphocytes from spleen and Peyer's patches.

The objective of this study was to investigate the impact of short-term protein malnutrition (PM) on immunoglobulin A (IgA) production and on the number and phenotype of lymphocytes in Peyer's patches (PP) and in the spleen. Balb/c mice were fed for 4, 7, or 10 d with a protein-deficient diet (0.1% protein). We determined B lymphocytes (CD40(+)), T lymphocytes (CD3(+)), T-helper (CD4(+)), and T-suppressor (CD8(+)) cells and the expression of costimulatory signals B7.1 (CD80) and B7.2 (CD86) on B cells and their counter receptors CD28 and CTLA-4 on T cells by fluorescence-activated cell-sorting analysis. Luminal IgA concentration in the small intestine was determined by an enzyme-linked immunosorbent assay. Four days of PM caused a significant reduction in the number of mononuclear cells in the spleen (5.6 x 10(7) +/- 1 x 10(7) versus 2. 4 x 10(7) +/- 0.5 x 10(7), P < 0.001) and the PP (13 x 10(6) +/- 3 x 10(6) versus 8.6 x 10(6) +/- 2 x 10(6), P < 0.01). There was a relative increase of T cells in the spleen and a relative increase of B cells in the PP. Luminal IgA content of small intestine was significantly reduced after 4 d of PM (242 +/- 55 microg versus 173 +/- 39 microg, P < 0.05) and remained at about this level until day 10 of PM. Four days after PM, the costimulatory signals B7.1 and B7. 2 on B cells were upregulated in the PP but markedly downregulated in the spleen, which was inversely related to the expression of the counter receptor CD28 on T-helper cells. We conclude that short-term PM increases the activation of B cells in the PP but reduces the relative number and activation state of splenic B cells. Only 4 d of PM caused a systemic and intestinal immunodepression, as indicated by a markedly decreased content of mononuclear cells in the PP and the spleen.

Cytotoxic effects of dental composites, adhesive substances, compomers and cements.

OBJECTIVES: Although research interest in biocompatibility of dental materials has been increasing, findings are frequently controversial and non-harmonized experimental approaches often lead to the production of contradictory results. The aim of this study was to compare the cytotoxic effects of six different light-cured dental composites, one compomer, one advanced glass-ionomer, two glass-ionomer cements, two zinc phosphate cements, one calcium hydroxide liner, one composite cement and one carboxylate cement with the same standardized cell-culture system. Two composites, one compomer and one advanced glass-ionomer were also tested in combination with the appropriate bonding substances and surface primers. METHODS: Specimens were added to the cultures immediately after production or after preincubation for 1, 2 or 7 days or 6 weeks under cell-culture conditions. Specimens were incubated with L-929 fibroblasts for 72 h and cell numbers determined by flow cytometry. RESULTS: All freshly prepared composite materials were cytotoxic. These effects diminished with increased preincubation times and were not significant after 7 days. Combinations of composites and bonding substances were still cytotoxic after preincubation for 7 days, but not after 6 weeks. Combinations of compomers and bonding substances demonstrated stronger toxicity than composites, although these effects were reduced earlier during preincubation. Glass-ionomer and phosphate cements showed similar effects to the composites with the exception of carboxylate cement, which demonstrated severe and persistent effects even after 6 weeks' preincubation. Together, our data provide evidence that all dental materials tested are cytotoxic immediately after production and that these effects are reduced after different preincubation periods in most cases. SIGNIFICANCE: Tested with a standardized cell-culture system, differences in toxicological potency between various commonly used dental materials were observed. Cytotoxicity data from standardized protocols should form the basis of screening the cytotoxic effects of new materials.

[Glutamine: effects on the immune system, protein balance and intestinal functions]. / Glutamin: Wirkungen auf das Immunsystem, auf Eiweisshaushalt und Darmfunktionen.

Glutamine is the most abundant free amino acid of the human body. In catabolic stress situations such as after operations, trauma and during sepsis the enhanced transport of glutamine to splanchnic organs and to blood cells results in an intracellular depletion of glutamine in skeletal muscle. Glutamine is an important metabolic substrate for cells cultivated under in vitro conditions and is a precursor for purines, pyrimidines and phospholipids. Increasing evidence suggests that glutamine is a crucial substrate for immunocompetent cells. Glutamine depletion in the cultivation medium decreases the mitogen-inducible proliferation of lymphocytes, possibly by arresting the cells in the G0-G1 phase of the cell cycle. Glutamine depletion in lymphocytes prevents the formation of signals necessary for late activation. In monocytes glutamine deprivation downregulates surface antigens responsible for antigen preservation and phagocytosis. Glutamine is a precursor for the synthesis of glutathionine and stimulates the formation of heat-shock proteins. Moreover, there are suggestions that glutamine plays a crucial role in osmotic regulation of cell volume and causes phosphorylation of proteins, both of which may stimulate intracellular protein synthesis. Experimental studies revealed that glutamine deficiency causes a necrotising enterocolitis and increases the mortality of animals subjected to bacterial stress. First clinical studies have demonstrated a decrease in the incidence of infections and a shortening of the hospital stay in patients after bone marrow transplantation by supplementation with glutamine. In critically ill patients parenteral glutamine reduced nitrogen loss and caused a reduction of the mortality rate. In surgical patients glutamine evoked an improvement of several immunological parameters. Moreover, glutamine exerted a trophic effect on the intestinal mucosa, decreased the intestinal permeability and thus may prevent the translocation of bacteria. In conclusion, glutamine is an important metabolic substrate of rapidly proliferating cells, influences the cellular hydration state and has multiple effects on the immune system, on intestinal function and on protein metabolism. In several disease states glutamine may consequently, become an indispensable nutrient, which should be provided exogenously during artificial nutrition.

Receptor and non-receptor mediated formation of superoxide anion and hydrogen peroxide in neutrophils of intensive care patients.

Generation of reactive oxygen intermediates (ROI) has been implicated in tissue damage in a variety of disease states including sepsis and trauma. On the other hand, generation of ROI in polymorphonuclear granulocytes (PMN) presents a crucial element in the defence of the host against invading microorganisms. In the present study we investigated the generation of superoxide anions (O2-) and hydrogen peroxide (H2O2) by neutrophils (PMN)5 of 17 critically ill patients treated at a intensive care unit (ICU) after polytrauma (n = 6), heart operation (n = 6) or during septic shock (n = 5) using flow cytometry. O2- production of PMN from ICU patients was significantly lower (p < 0.01) than that in healthy volunteers (HV) during non-receptor mediated stimulation with phorbol-myristate-acetate (PMA) but higher (p < 0.001) during receptor mediated stimulation with formylmethionine-leucine-phenylalanine (FMLP). H2O2 generation of PMN from ICU patients was increased after stimulation with FMLP (p < 0.01) and remained unchanged after stimulation with PMA. Patients in septic shock had lower O2(-)-generation of PMN than did injured patients and patients after heart operations. We conclude that receptor mediated formation of O2- and H2O2 is stimulated in ICU patients. However, in patients in septic shock O2(-)-generation decreases, which potentially might contribute to the immunoparalysis present in septic shock.