ABSTRACT
BACKGROUND: Administration of a chemically defined, liquid, elemental diet (CDD) results in intestinal microbial translocation, but the immunologic consequences of this process are unclear. This study evaluated the effects of CDD feeding and interleukin-4 (IL-4) administration on mesenteric lymphocyte, peritoneal macrophage (PMO), and hepatic Kupffer cell (KC) functions. METHODS: BALB/C mice (n = 60) were randomized to receive a paired feeding of regular diet (RD) or a CDD for 14 days. Mesenteric lymph nodes (MLN) and cecum were cultured for bacteria. Mixed lymphocyte response and cytotoxic T-lymphocyte function of MLN lymphocytes were assayed. PMO and KC were harvested to measure tumor necrosis factor production, macrophage binding of fluorescent-labeled lipopolysaccharide, and Candida albicans phagocytosis (CAP) and killing (CAK); KC-hepatocyte interaction was assessed by hepatocyte protein synthesis. In a second study 75 BALB/c mice received RD, CDD, and CDD+IL-4 (30,000 units/mouse intraperitoneally). MLN lymphocyte and PMO functions were measured and intestinal immunoglobulin A levels were determined. RESULTS: Oral feeding of a CDD resulted in significant impairment of mesenteric lymphocyte mixed lymphocyte response and cytotoxic T-lymphocyte functions and decreased PMO tumor necrosis factor production, fluorescent-labeled lipopolysaccharide binding, CAP, and CAK. KC function was preserved in CDD-fed mice. Administration of IL-4 significantly reduced the incidence of bacteria positive MLN and increased PMO superoxide production, CAP, CAK, and MLN lymphocyte mitogenesis. CONCLUSIONS: Use of IL-4 may be beneficial in situations where intestinal microbial translocation contributes to sepsis.
Subject(s)
Bacterial Physiological Phenomena , Food, Formulated , Interleukin-4/immunology , Intestines/microbiology , Animals , Cells, Cultured , Interleukin-4/pharmacology , Kupffer Cells/drug effects , Kupffer Cells/physiology , Lymphocyte Culture Test, Mixed , Lymphocytes/drug effects , Lymphocytes/physiology , Macrophages, Peritoneal/drug effects , Macrophages, Peritoneal/physiology , Mesentery/cytology , Mice , Mice, Inbred BALB C , Random Allocation , T-Lymphocytes, Cytotoxic/physiologyABSTRACT
CHO-K1D cells electroporated in buffers containing [32P]NAD incorporated the label in a voltage-dependent manner. Electroporation with 650 V/cm at 1460 microF in Ham's F12 medium supplemented with 10 mM Hepes, pH 7.1, resulted in a greater than 20-fold increase in [32P]NAD uptake, while decreasing relative cellular survival by only 6%. Exposure of cells to gamma irradiation (20 Gy) prior to electroporation increased the steady-state level of poly(ADP-ribosylated) nuclear proteins two- to four-fold over that of unirradiated control cells. These data indicate that electrotransfer of [32P]NAD is a simple and rapid means of labeling the cellular NAD pool and should prove useful in the analysis of the relationship between poly(ADP-ribosylation) of nuclear proteins and DNA repair.
Subject(s)
Adenosine Diphosphate/analysis , NAD , Nuclear Proteins/analysis , Ribose/analysis , Adenosine Diphosphate/metabolism , Adenosine Diphosphate/radiation effects , Animals , Buffers , Cells, Cultured , Cricetinae , Electricity , Electrophoresis, Polyacrylamide Gel , Gamma Rays , NAD/metabolism , Nuclear Proteins/metabolism , Nuclear Proteins/radiation effects , Ribose/metabolism , Ribose/radiation effectsABSTRACT
A method to detect single-stranded DNA damage from individual cells has been developed using a monoclonal anti-thymidine antibody (MoAb20B7). Initially, HL-60 cells were incubated with daunomycin at different concentrations, and processed by MoAb20B7. While 73.5% of the cells incubated with 5 micrograms/ml of daunomycin for 24 h reacted positively with MoAb20B7, 83.5% cells at 10 micrograms/ml daunomycin dose were positive. Next, this method was combined with unscheduled DNA synthesis to simultaneously measure repair and damage from individual cells. Finally, patients with acute myeloid leukemias were studied before and 24 h after therapy with a daunomycin containing regimen. In vivo damage could be determined in a prompt fashion.
