Biomedical subjects

R D Wagner

Publications and source records attributed to R D Wagner.

23 records · Page 2Linked to original sources

PubMed1993

Analysis of cytokine mRNA expression in Listeria-resistant C57BL/6 and Listeria-susceptible A/J mice during Listeria monocytogenes infection.

This laboratory previously reported that mRNA expression for many cytokines, as determined by reverse transcription-polymerase chain reaction analysis, is induced rapidly in the spleen during murine listeriosis. In the present study, the patterns of cytokine mRNA expression in spleens and livers of Listeria-resistant C57BL/6 and Listeria-susceptible A/J mice were compared. In addition, in situ hybridization was performed to evaluate the distributions of cytokine mRNA-expressing cells in these tissues. Listeria-resistant C57BL/6 mice demonstrated greater expression of gamma interferon (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) mRNAs in the spleen than Listeria-susceptible A/J mice. Greater numbers of cells expressing IFN-gamma and GM-CSF mRNAs were observed by in situ hybridization in the spleens of C57BL/6 mice than in those of A/J mice. C57BL/6 and A/J mice did not differ in their expression of IFN-gamma mRNA in the liver. Nor did C57BL/6 and A/J mice differ in their expression of tumor necrosis factor alpha, interleukin-1 alpha (IL-1 alpha), IL-2, IL-4, or IL-6 mRNA in the liver or spleen, as determined by reverse transcription-polymerase chain reaction and in situ hybridization. These results indicate that the greater resistance of C57BL/6 mice to Listeria monocytogenes infection is associated with greater expression of IFN-gamma and GM-CSF mRNAs in the spleen and GM-CSF mRNA in the liver.

Animals↗

PubMed1992

Administration of anti-IL-4 monoclonal antibody 11B11 increases the resistance of mice to Listeria monocytogenes infection.

The role of endogenous IL-4 in resistance to Listeria monocytogenes infection was investigated by in vivo administration of an anti-IL-4 mAb (11B11). Mice treated with 0.01 to 0.4 mg of anti-IL-4 mAb before L. monocytogenes challenge demonstrated a significantly reduced peak bacterial burden in their livers and spleens and accelerated bacterial clearance from these organs. In addition, histopathologic damage to the liver was reduced. Maximal protection was achieved by i.p. injection of 0.1 mg of anti-IL-4 mAb 2 or 24 h before L. monocytogenes challenge; treatment with anti-IL-4 mAb after injection of L. monocytogenes had no effect on antilisterial resistance. Anti-IL-4 mAb-treated and control Listeria-infected mice exhibited similar patterns of IFN-gamma, IL-2, and IL-4 mRNA, as determined by polymerase chain reaction amplification of RNA extracted from spleen cells. In both anti-IL-4 mAb-treated and control mice, IFN-gamma, IL-2, and IL-4 mRNA were produced within 4 h after challenge. Cytokine mRNA levels were similar for anti-IL-4 mAb-treated and control mice, except for the greater amount of IFN-gamma mRNA in the anti-IL-4 mAb-treated mice at 4 h after L. monocytogenes challenge. IFN-gamma and IL-2 mRNA levels were sustained for at least 5 days, whereas IL-4 mRNA was undetectable by 3 days after challenge. There were no significant differences in the amounts of IL-4 and IFN-gamma detected in culture supernatants of spleen cells from anti-IL-4 mAb-treated and control Listeria-infected mice. These results suggest that endogenous IL-4, a cytokine thought to be produced principally by Th2 cells, has a deleterious effect on host defense against the facultative intracellular pathogen L. monocytogenes. Administration of an anti-IL-4 mAb increases antilisterial resistance without causing a detectable shift to a Th1 type of cytokine response.

Animals↗

PubMed1992

Apolipoprotein expression and cellular differentiation in Caco-2 intestinal cells.

Caco-2 cells, cultured for 18 days on porous filter supports and conventional plastic culture dishes, were used to study the effects of cellular differentiation on the expression of apolipoprotein (apo) genes. Media of filter-grown cells accumulated more apo B as apo B-48 and contained three times the amount of edited apo B mRNA compared with plastic-grown cells. The accumulation of apo A-I by media of plastic-grown cells was higher than accumulation by filter-grown cells, despite similar concentrations of apo A-I mRNA. The apo A-IV was detectable in the culture media earlier with filter-grown cells compared with plastic-grown cells, despite similar apo A-IV mRNA concentrations. Plastic-grown cells contained more apo E mRNA, and their media accumulated more apo E than filter-grown cells. With the exception of apo A-I, apo gene expression changed with Caco-2 cell differentiation to resemble more closely the patterns seen in adult enterocytes. There were no effects or minimal effects of added retinoic acid, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], or thyroid hormone on apo accumulation in media of filter-grown cultures of Caco-2 cells. However, 1,25(OH)2D3 and thyroid hormone increased apo B, apo A-IV, and apo A-I mRNA concentrations, retinoic acid increased apo B mRNA concentrations alone, and all three reduced apo E mRNA concentrations. Ratios of edited to unedited apo B mRNA were unaffected. In conclusion, culture substratum importantly influences Caco-2 cell differentiation. Soluble factors that influence cellular differentiation may affect apo gene expression over and above effects mediated by the culture substratum.(ABSTRACT TRUNCATED AT 250 WORDS)

Apolipoprotein A-I↗

PubMed1992

ApoB-75, a truncation of apolipoprotein B associated with familial hypobetalipoproteinemia: genetic and kinetic studies.

We have identified a mutation of apolipoprotein B (apoB) in a kindred with hypobetalipoproteinemia. Four affected members had plasma concentrations of total cholesterol of 115 +/- 14, low density lipoprotein (LDL)-C of 48 +/- 11, and apoB of 28 +/- 9 (mg/dl mean +/- SD). The values correspond to approximately 30% the values for unaffected relatives. Triglyceride and high density lipoprotein (HDL)-C concentrations were 92 +/- 50 and 49 +/- 4, respectively, neither significantly different from unaffected relatives. Western blots of plasma apoB of affected subjects showed two major bands: apoB-100 and an apoB-75 (mol wt of approximately 418,000). DNA sequencing of the appropriate polymerase chain reaction (PCR)-amplified genomic DNA segment revealed a deletion of the cytidine at nucleotide position 10366, resulting in a premature stop codon at amino acid residue 3387. In apoB-75/apoB-100 heterozygotes, two LDL populations containing either apoB-75 or apoB-100 could be distinguished from each other by gel permeation chromatography and by immunoblotting of nondenaturing gels using monoclonal antibodies B1B3 (epitope between apoB amino acid residues 3506-3635) and C1.4 (epitope between residues 97-526). ApoB-75 LDL were smaller and more dense than apoB-100 LDL. To determine whether the low concentration of apoB-75 was due to its enhanced LDL-receptor-mediated removal, apoB-75 LDL were isolated from the proband's d 1.063-1.090 g/ml fraction (which contained most of the apoB-75 in his plasma) by chromatography on anti-apoB and anti-apoA-I immunoaffinity columns. The resulting pure apoB-75 LDL fraction interacted with the cells 1.5-fold more effectively than apoB-100 LDL (d 1.019-1.063 g/ml). To determine the physiologic mechanism responsible for the hypobetalipoproteinemia, in vivo kinetic studies were performed in two affected subjects, using endogenous labeling of apoB-75 and apoB-100 with [13C]leucine followed by multicompartmental kinetic analyses. Fractional catabolic rates of apoB-75 VLDL and LDL were 2- and 1.3-fold those of apoB-100 very low density lipoprotein (VLDL) and LDL, respectively. Production rates of apoB-75 were approximately 30% of those for apoB-100. This differs from the behavior of apoB-89, a previously described variant, whose FCRs were also increased approximately 1.5-fold relative to apoB-100, but whose production rates were nearly identical to those of apoB-100. Thus, in contrast to the apoB-89 mutation, the apoB-75 mutation imparts two physiologic defects to apoB-75 lipoproteins that account for the hypobetalipoproteinemia, diminished production and increased catabolism.

Adult↗

PubMed1991

ApoB-54.8, a truncated apolipoprotein found primarily in VLDL, is associated with a nonsense mutation in the apoB gene and hypobetalipoproteinemia.

A new, large kindred with hypobetalipoproteinemia and a previously undescribed truncated form of apolipoprotein B (apoB) has been identified. The asymptomatic, Caucasian male proband (CK, aged 37 years) has total plasma cholesterol, triglyceride, low density lipoprotein-(LDL) cholesterol, high density lipoprotein- (HDL) cholesterol, and apoB concentrations of 108, 131, 32, 50, and 16 mg/dl, respectively. Plasma samples of 11 family members spanning three generations, which had less than 5th percentile concentrations of LDL-cholesterol, contained three apoB bands detected on immunoblots: the normal apoB-100 and apoB-48 and an unusual band of apparent molecular mass of 299,356 +/- 9580 daltons (approximately 54% the molecular weight of apoB-100). Additional immunoblotting experiments using several different anti-apoB monoclonal antibodies showed that the carboxyl terminal of apoB-100 had been deleted somewhere between amino acid residues 2148-2488. A segment of genomic DNA from the proband was amplified by polymerase chain reaction (PCR) between nucleotides 7491-7791 of Exon 26 of the apoB gene. The DNA segment was cloned into pGEM3Zf(-) and sequenced. A C----T transition was found at nucleotide 7665, resulting in a premature stop codon at amino acid residue 2486 corresponding to apoB-54.8. These results were confirmed by direct sequencing of PCR products from three apoB-54.8 positive and three apoB-54.8 negative kindred members. Allele-specific oligonucleotides were used to identify other affected family members. Cosegregation of apoB-54.8 with the C----T transition occurred in all cases. Based on haplotypes constructed from restriction fragment length polymorphism, variable number of tandem repeats, and 5' insertion/deletion analyses and from the presence or absence of apoB-54.8, it was possible to assign a single allele of apoB to the mutation throughout the family. In contrast with other shorter truncations such as apoB-31, apoB-40, and apoB-46, which are found with particles in the HDL density range, and apoB-89 that is found primarily with LDL, apoB-54.8 was found primarily in very low density lipoproteins, much less in LDL, and was virtually absent in HDL. This suggests that the length of the truncation may significantly affect the metabolism of the associated lipoprotein particles.

Adolescent↗