Ina Fabian

Activation of human eosinophil and neutrophil functions by haematopoietic growth factors: comparisons of IL‐1, IL‐3, IL‐5 and GM‐CSF

Summary. We compared the effect of haematopoietic growth factors granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), interleukin (IL)‐1, IL‐3, and IL‐5 on the functional activation of human eosinophils and neutrophils from the same donor. All four colony‐stimulating factors (CSF) enhanced the phagocytosis of Candida albicans by eosinophils and increased staphylococcal, but not Candida, killing. GM‐CSF and IL‐5 had a profound stimulating effect on eosinophil staphylocidal activity. GM‐CSF and IL‐3 enhanced the generation of leukotriene C4 (LTC4) induced by calcium ionophore A23187 and the release of arylsulphatase and β‐glucuronidase from specific and small granules of eosinophils. In contrast, IL‐1 and IL‐5 had no effect on degranulation. GM‐CSF and IL‐1 enhanced phagocytosis of C. albicans by neutrophils, and GM‐CSF stimulated degranulation and the release of the enzymes β‐glucuronidase and arylsulphatase from neutrophils while IL‐1 stimulated the release of arylsulphatase only. This study indicates that the eosinophilactive colony‐stimulating factors can markedly enhance the host defence function of the eosinophil and even make it the equal of the neutrophil in staphylocidal activity. The CSF‐activated eosinophil, however, may cause inappropriate inflammation and normal tissue damage.

Activation of c-K-ras Mutations in Human Gastrointestinal Tumors

BACKGROUND & AIMS Ras genes are the most frequently detected oncogenes in human malignancies. Data regarding the frequency of c-K-ras mutations in esophageal, gastric, and small bowel tumors are limited and controversial. METHODS DNA was extracted from 262 formalin-fixed, paraffin-embedded sections of gastrointestinal samples and tumors, including Barretts esophagus, esophageal squamous cell carcinomas and adenocarcinomas, and small and large bowel adenomas and adenocarcinomas. The presence of c-K-ras codon 12 mutations was determined using a nonradioactive polymerase chain reaction-based restriction fragment length polymorphism assay. RESULTS c-K-ras mutations were detected in 1 of 39 (2%) patients with Barretts esophagus, 1 of 21 (5%) adenocarcinomas, 0 of 27 squamous cell carcinomas of the esophagus, and 1 of 32 (3%) gastric adenocarcinomas. It was also present in 8 of 20 (40%) and 10 of 28 (36%) small bowel adenomas and adenocarcinomas, respectively. Similar numbers were observed in 10 of 25 (40%) large bowel adenomas and 11 of 30 adenocarcinomas (37%). Mutations were not associated with age, gender, histology, grade, stage, location, or mortality. CONCLUSIONS The frequency of codon 12 c-K-ras mutations in small and large bowel tumors is approximately 10-fold higher than that of tumors in the upper gastrointestinal tract.

Anti-Inflammatory Effects of Moxifloxacin on Activated Human Monocytic Cells: Inhibition of NF-kappaB and Mitogen-Activated Protein Kinase Activation and of Synthesis of Proinflammatory Cytokines

ABSTRACT We previously showed that moxifloxacin (MXF) exerts protective anti-inflammatory effects in immunosuppressed mice infected with Candida albicans by inhibiting interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α) production in the lung. Immunohistochemistry demonstrated inhibition of nuclear factor (NF)-κB translocation in lung epithelium and macrophages in MXF-treated mice. In the present study we investigated the effects of MXF on the production of proinflammatory cytokines (i.e., IL-8, TNF-α, and IL-1β) by activated human peripheral blood monocytes and THP-1 cells and analyzed the effects of the drug on the major signal transduction pathways associated with inflammation: NF-κB and the mitogen-activated protein kinases ERK and c-Jun N-terminal kinase (JNK). The levels of IL-8, TNF-α, and IL-1β secretion rose 20- and 6.7-fold in lipopolysaccharide (LPS)-activated monocytes and THP-1 cells, respectively. MXF (5 to 20 μg/ml) significantly inhibited cytokine production by 14 to 80% and 15 to 73% in monocytes and THP-1 cells, respectively. In THP-1 cells, the level of NF-κB nuclear translocation increased fourfold following stimulation with LPS-phorbol myristate acetate (PMA), and this was inhibited (38%) by 10 μg of MXF per ml. We then assayed the degradation of inhibitor (I)-κB by Western blotting. LPS-PMA induced degradation of I-κB by 73%, while addition of MXF (5 μg/ml) inhibited I-κB degradation by 49%. Activation of ERK1/2 and the 46-kDa p-JNK protein was enhanced by LPS and LPS-PMA and was significantly inhibited by MXF (54 and 42%, respectively, with MXF at 10 μg/ml). We conclude that MXF suppresses the secretion of proinflammatory cytokines in human monocytes and THP-1 cells and that it exerts its anti-inflammatory effects in THP-1 cells by inhibiting NF-κB, ERK, and JNK activation. Its anti-inflammatory properties should be further assessed in clinical settings.

Overexpression of cyclin D1 occurs in both squamous carcinomas and adenocarcinomas of the esophagus and in adenocarcinomas of the stomach

Increased expression of the cyclin D1 gene frequently occurs in human squamous carcinomas of the esophagus. However, the expression of cyclin D1 has not been previously examined in detail in adenocarcinomas of the esophagus or stomach. Therefore, we examined, in parallel, the expression of cyclin D1 in both squamous and adenocarcinomas of the esophagus and in adenocarcinomas of the stomach. The level of expression of the cyclin D1 protein was assessed by immunohistochemistry in 39 esophageal and 34 gastric carcinomas and correlated with clinical and pathology parameters. Within the esophagus, 71% of the squamous carcinomas and 64% of the adenocarcinomas were positive for increased cyclin D1 nuclear staining. For adenocarcinomas of the stomach, the overall positive rate was 47%; in the gastric cardia, the rate was 44%, and in other regions of the stomach, it was 50%. In esophageal and gastric adenocarcinomas of the intestinal type, increased expression of cyclin D1 was seen in 70% of the samples, whereas with the diffuse type only 13% were positive (P < .01). Tumors from patients older than the median age of 67 years were more frequently positive than tumors from patients younger than 67 years (74% v 42%, respectively) (P < .01). Positive staining was also seen more frequently in well and moderately differentiated tumors than in poorly differentiated tumors (74% v 49%, respectively) (P < .05). Cytoplasmic staining for cyclin D1 was noted in 22% of the tumors, of various types. Therefore, increased expression of cyclin D1 frequently occurs in both adenocarcinomas and squamous carcinomas of the esophagus, and in adenocarcinomas of the stomach. The increased expression in adenocarcinomas is especially frequent in the intestinal-type lesions.

Immunomodulatory and Protective Effects of Moxifloxacin against Candida albicans-Induced Bronchopneumonia in Mice Injected with Cyclophosphamide

ABSTRACT In a previous study, moxifloxacin was shown to ameliorate immunosuppression and enhance cytokine production in several tissues, including the lungs of cyclophosphamide-injected mice. We examined here the effects of moxifloxacin on Candida albicans lung infection in cyclophosphamide-injected mice. Mice were injected on day 0 with 250 mg of cyclophosphamide/kg, and on days 1 to 4 they were given moxifloxacin at 22.5 mg/kg/day compared to controls given ceftazidime at 75 mg/kg/day or saline. On day 6, C. albicans (10 7 CFU/mouse) was inoculated intratracheally, and animals were observed for the development of bronchopneumonia, weight loss, mortality, the presence of C. albicans, and lung cytokine production. Histopathology on day 10 postinoculation revealed bronchopneumonia in 50, 67, and 0% of saline-, ceftazidime-, and moxifloxacin-treated mice, respectively (P < 0.05). The mortality rates were 28, 17, and 5%, respectively (P < 0.05), and weight loss occurred at 20, 32, and 0%, respectively (P < 0.05). By day 15, C. albicans was eliminated from all moxifloxacin-treated mice but was still isolated from lung homogenates of 50 to 60% of the saline- and ceftazidime-treated groups. Among the cytokines tested on days 0 to 15, we found an increased production of tumor necrosis factor alpha, KC (functional interleukin-8), and gamma interferon in the lungs of ceftazidime- and saline-treated controls compared to the moxifloxacin pretreatment that abolished their secretion. In conclusion, moxifloxacin protected cyclophosphamide-injected mice from C. albicans-induced lung infection and significantly reduced pneumonia, weight loss, and mortality despite the lack of direct antifungal activity. This is most likely due to an immunomodulating activity conferred by moxifloxacin, as shown in this model and in our previous studies. Its potential protective role should be studied in patients undergoing chemotherapy and immune suppression.

Ascorbic Acid Inhibits Apoptosis Induced by X Irradiation in HL60 Myeloid Leukemia Cells

Exposure of cells to ionizing radiation can cause apoptosis. Since antioxidants have been shown to protect against radiation-induced apoptosis, in this study we have evaluated the putative protective effect of ascorbate against radiation-induced apoptosis as well as the production of peroxides in the cells. HL60 cells transport the oxidized form of ascorbic acid, dehydroascorbic acid (DHA), and accumulate reduced ascorbate. Exposure of the cells to 5-40 Gy X radiation resulted in induction of apoptosis. Preincubation of the cells with DHA reduced the level of apoptosis after exposure to 5-20 Gy. Exposure of the cells to 5 or 20 Gy X radiation did not affect the intracellular concentration of peroxides, while phorbol myristate acetate (PMA), which is known to induce production of H(2)O(2) in cells (and served as a control), resulted in an increase in peroxides and a decrease in intracellular ascorbate. Irradiation of the cells with 1-3 Gy resulted in up-regulation of expression of BCL2 without affecting the level of apoptosis. At higher doses of radiation, enhanced BCL2 expression did not prevent radiation-induced apoptosis. Loading of the cells with ascorbate prior to their exposure to 1-3 Gy X radiation did not affect the enhanced BCL2 expression observed in the irradiated cells. At higher doses of radiation, ascorbate decreased apoptosis and restored the level of BCL2 in the cells. Exposure of the cells to 3-20 Gy X radiation enhanced the cell surface expression of TNFRSF6 (formerly known as Fas/APO-1) antigen and enhanced anti-TNFRSF6 antibody-induced apoptosis of the cells. Ascorbate loading did not affect expression of TNFRSF6 and did not overcome the anti-TNFRSF6 antibody-induced apoptosis. In conclusion, our data demonstrate that exposure of HL60 cells to radiation enhanced BCL2 and TNFRSF6 expression. Ascorbate did not affect BCL2 or TNFRSF6 expression. We therefore conclude that it protects HL60 cells against radiation-induced apoptosis, although the mechanisms of protection must still be elucidated.

Immunomodulatory effects of moxifloxacin in comparison to ciprofloxacin and G‐CSF in a murine model of cyclophosphamide‐induced leukopenia

Abstract: We analyzed the effect of the two quinolones moxifloxacin and ciprofloxacin on the repopulation of hematopoietic organs and on the production of cytokines by various organs of cyclophosphamide (CP)‐induced leukopenic mice. The effect was compared to that of G‐CSF. Cyclophosphamide injection induced a severe leukopenia, with nadir at day 4 post‐injection. All the quinolone and G‐CSF‐treated animals showed WBC>500/μL at the nadir, compared to 50% of saline‐treated mice. Cyclophosphamide induced a marked decrease in the number of myeloid progenitors (CFU‐C) in bone marrow (BM) and spleen. Quinolone or G‐CSF treatment resulted in a 1.4–4.3‐fold increase in CFU‐C numbers in the BM; no enhancement was observed in the spleen. Treatment with CP resulted in enhanced colony‐stimulating activity (CSA) in bone shaft and spleen and decreased activity in bladder and lung. Treatment of CP‐injected mice with quinolones significantly enhanced CSA in the bone shaft, spleen, lung and bladder on different days.

Protection by ascorbic acid from denaturation and release of cytochrome c, alteration of mitochondrial membrane potential and activation of multiple caspases induced by H2O2, in human leukemia cells

We investigated peroxide and superoxide accumulation, cytochrome c nature and release from mitochondria, as well as caspase activation during exposure of HL-60 cells to H(2)O(2) and the protective effect of ascorbic acid. Exposure of the cells to 100 microM H(2)O(2) resulted in intracellular accumulation of peroxides, denaturation of cytochrome c that was identified in the mitochondria and cytosol, release of native cytochrome c to the cytosol and fall in mitochondrial membrane potential (DeltaPsi(m)). Loading of cells with ascorbic acid before exposure to H(2)O(2) resulted in a dose-dependent protective effect against: intracellular accumulation of peroxides, DeltaPsi(m) alteration, cytochrome c denaturation and release. The accumulation of peroxides induced processings and activations of procaspase-8, -9 and -3. Double staining with antiserum which recognizes the p18 subunit of cleaved caspase-3 and with Hoechst had shown that a high percentage of cells exposed to 100 microM H(2)O(2) stained positively with the antibody and showed features of apoptosis. Ascorbic acid loading prevented caspase activation induced by H(2)O(2). We conclude that ascorbic acid protects against activation of apoptotic cascades in HL-60 cells exposed to H(2)O(2) and against apoptosis.

Effect of simvastatin alone and in combination with cytosine arabinoside on the proliferation of myeloid leukemia cell lines.

Background Cholesterol biosynthesis is regulated by the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Cholesterol and its derivatives are required in high concentrations by neoplastic proliferating cells for both DNA synthesis and cell growth. Thus, inhibition of HMG-CoA reductase could effect cell cycle progression and proliferation. Therefore, we examined the effect of an HMG-CoA reductase inhibitor (simvastatin) alone and in combination with cytosine arabinoside (ARA-C) on the proliferation of two AML cell lines. Methods AML blasts derived from two cell lines (HL-60 and AML-2) were incubated with increasing concentrations of either simvastatin alone or simvastatin alone for 24 hours with ARA-C added thereafter. The effect of the drugs on cell proliferation in liquid culture (3 H thymidine uptake) and on clonogenic assay was analyzed. Results We found that the number of proliferating AML blasts (suspension cultures) and colony formations (agar cultures) of both cell lines declined significantly after incubation with simvastatin. Preincubation of both cell lines with simvastatin by the addition of increasing concentrations of ARA-C produced a degree of growth inhibition that was significantly greater than that of the individual compounds. This antigrowth interaction was additive rather than synergistic. Conclusions We conclude that simvastatin has a major antiproliferative effect on AML blasts in vitro. Also, the combination of simvastatin and ARA-C significantly enhanced the antiproliferative effect of each drug. These findings may open new avenues in both the laboratory and clinical research of the treatment of leukemia.

Effects of recombinant human granulocyte and granulocyte-macrophage colony-stimulating factors on neutrophil function following autologous bone marrow transplantation

Functional activity of peripheral blood neutrophils was assessed in eight patients at 4, 6, 8, 10 and 12 weeks following autologous bone marrow transplantation (ABMT). Functions studied included superoxide generation (O2-) intracellular killing of Staphylococcus aureus, phagocytosis and killing of Candida albicans. Neutrophils were tested following in vitro preincubation with 300 pM granulocyte-macrophage colony-stimulating factor (GM-CSF), 1.2 nM granulocyte colony-stimulating factor (G-CSF) or buffered solution (diluent) as control. Our data indicate that during the early period (weeks 4-6) following ABMT most of the patients exhibited diminished neutrophil oxidative metabolism, defective phagocytosis and killing of C. albicans and reduced capacity to kill S. aureus. In some patients a gradual increase in the functional activity of neutrophils occurred with time. Both GM-CSF and G-CSF induced in vitro amplification of (a) O2- production in response to fmet-leu-phe (FMLP) (b) phagocytosis and killing of C. albicans and (c) killing of S. aureus. This study suggests that GM-CSF and G-CSF may enhance the depressed functional activity of neutrophils following ABMT.