Pravinkumar B. Sehgal

The diagnostic and prognostic value of amniotic fluid white blood cell count, glucose, interleukin-6, and Gram stain in patients with preterm labor and intact membranes

OBJECTIVE Our goal was to compare the value of amniotic fluid tests in the detection of microbial invasion of the amniotic cavity and in the relationship with the amniocentesis-to-delivery interval and neonatal complications in patients with preterm labor and intact membranes. STUDY DESIGN Amniotic fluid was retrieved by transabdominal amniocentesis from 120 patients with preterm labor and intact membranes. Fluid was cultured for aerobic and anaerobic bacteria and for mycoplasmas. Amniotic fluid analysis included a Gram stain, white blood cell count, glucose and interleukin-6 determinations. Logistic regression and Coxs proportional hazards model were used for analysis. RESULTS (1) The prevalence of positive amniotic fluid cultures was 9.2% (11/120); (2) patients with microbial invasion had a shorter amniocentesis-to-delivery interval and a higher neonatal complications rate than patients with a negative culture; (3) the most sensitive test for the detection of microbial invasion of the amniotic cavity was amniotic fluid interleukin-6 determinations (cutoff 11.3 ng/ml) (sensitivity; for interleukin-6 100%, for glucose 81.8%, for white blood cell count 63.6%, and for Gram stain 63.6%; p < 0.05 for all comparisons); (4) the most specific test was the Gram stain of amniotic fluid (specificity: for Gram stain 99.1%, for white blood cell count 94.5%, for interleukin-6 82.6%, and for glucose 81.6%; p < 0.01 for all); (5) of all amniotic fluid tests, interleukin-6 determinations were the only ones that had significant relationship with the amniocentesis-to-delivery interval and neonatal complications. CONCLUSION Interleukin-6 concentrations in amniotic fluid are better indicators of microbial invasion of the amniotic cavity, amniocentesis-to-delivery interval, and neonatal complications than the amniotic fluid Gram stain, glucose concentration, or white blood cell count.

A cytokine network in human diploid fibroblasts: interactions of beta-interferons, tumor necrosis factor, platelet-derived growth factor, and interleukin-1.

Earlier studies demonstrated the induction of beta 2-interferon (IFN-beta 2) in human diploid fibroblasts (FS-4 strain) exposed to tumor necrosis factor (TNF). These studies suggested that IFN-beta 2 mediates an antiviral effect in TNF-treated cells and exerts a feedback inhibition of the mitogenic effect of TNF. Here we demonstrate that the expression of the antiviral action of TNF can be enhanced by prior exposure of FS-4 cells to trace amounts of IFN-beta 1. IFN-beta 1, at a higher concentration, can directly increase the expression of IFN-beta 2. Exposure of cells to TNF enhanced IFN-beta 2 (but not IFN-beta 1) mRNA expression in response to poly(I).poly(C), an IFN inducer which is also known to stimulate FS-4 cell growth. Platelet-derived growth factor and interleukin-1 also led to the increased expression of IFN-beta 2. However, platelet-derived growth factor and interleukin-1 could override the antiviral effect of TNF and also that of exogenously added IFN-beta 1. Our data suggest that a complex network of interactions that involves the endogenous production of IFN-beta 2 is triggered by several growth-modulatory cytokines. Cellular homeostasis is likely to represent a balance between the induction of IFN-beta 2 by these cytokines and their ability to override the inhibitory actions of IFN-beta 2.

The inhibition of DRB (5,6-dichloro-1-β-d-ribofuranosylbenzimidazole) of hnRNA and mRNA production in HeLa cells

Abstract 5, 6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB; 75 μM) inhibited only 60–75% of nuclear heterogeneous RNA (hnRNA) synthesis in HeLa cells, while the appearance of labeled cytoplasmic poly(A)-containing messenger RNA (mRNA) was reduced by approximately 95%. DRB-sensitive and resistant transcripts spanned the entire size range of hnRNA molecules. The relative poly(A) content of DRB-resistant hnRNA molecules was the same as that of normal hnRNA. In contrast to the effects of 3′deoxyadenosine, DRB appeared to inhibit the initiation of hnRNA chains, but did not directly interfere with labeling of poly(A). We present an interpretation of these results and propose how the results might be used to gain more information about nuclear RNA metabolism.

Halobenzimidazole Ribosides and RNA Synthesis of Cells and Viruses

Publisher Summary This chapter brings together in a systematic way the recent work as well as some of the earlier studies on the mode of action of halogenated ribofuranosylbenzimidazoles on cellular and viral biosynthesis. Investigations of the cellular and viral biosynthesis, in which these compounds have been used as exploratory tools are also reviewed in this chapter. Compounds in this class of benzimidazoles possess the unique biological activity of selective inhibition of the synthesis of nuclear heterogeneous RNA (hnRNA) in cells of cold-blooded animals, including insects and in cells of avian and mammalian species. Because of the universality of the synthesis of hnRNA in animal species and because a portion of hnRNA sequences becomes mRNA, the action of halobenzimidazole ribosides on cellular biosynthesis is of broad interest. The action of these derivatives is also relevant to the process of transcription of the viral genomes of certain RNA and DNA viruses. In addition, this chapter briefly discusses three groups of structurally distinct bcnzimidazolcs that possess biological actions entirely different from those of halobenzimidazole ribosides: 2-(α-hydroxybenzyl) benzimidazole and certain related compounds are selective inhibitors of picornavirus multiplication; 5-methyl-2-D-ribobenzimidazole and congeners and 5-hydroxy-l-methylbenzimidazole and certain related compounds restore the capacity of chorioallantoic membranes from older chicken embryos to produce influenza virus in high yield.

Cytokines in normal and abnormal parturition: Elevated amniotic fluid interleukin-6 levels in women with premature rupture of membranes associated with intrauterine infection

The participation of interleukin-6 (IL-6) in the pathophysiology of normal and abnormal human parturition was evaluated by determining IL-6 concentrations in amniotic fluid (AF). Biologically active IL-6 was determined (in U/ml) using the B9 hybridoma growth factor assay, while the concentrations of immunoreactive IL-6 species (in pg/ml) were assessed using a monoclonal antibody (moAb)-based ELISA. Two hundred and twenty-seven AF samples from women in normal labor and from those presenting with a clinical diagnosis of premature rupture of membranes (PROM) were assayed. In selected instances, IL-6 levels were evaluated simultaneously in AF and in maternal and fetal plasma. Women with a normal pregnancy had low titers of biologically active IL-6 in AF both at midtrimester (group 1, n = 27; median IL-6 concentration = 16 U/ml) and at term (group 2, n = 33; median = 15 U/ml). There was an increase in the IL-6 bioactivity in AF from women in normal labor at term (group 3, n = 40; median = 74 U/ml; p less than 0.001). In order to distinguish between the relative contributions of parturition per se and of intrauterine infection to the elevation of biologically active IL-6 levels in AF, IL-6 titers were compared in four different groups of women with PROM.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of interleukin-6 in regulating synthesis of C-reactive protein and serum amyloid A in human hepatoma cell lines.

Previous studies have demonstrated that synthesis of the two major human acute phase proteins, C-reactive protein (CRP) and serum amyloid A (SAA), by human hepatoma cell lines was not affected by preparations of interleukin-1 (IL-1) or tumor necrosis factor α but was increased after exposure to conditioned medium from lipopolysaccharide-activated monocytes. We report that neutralizing antibodies raised against E. coli-derived recombinant human interleukin-6 (IL-6) were capable of inhibiting induction of CRP and SAA by monocyte conditioned medium in both NPLC/PRF/5 and Hep 3B cell lines. Partially purified IL-6 from lipopolysaccharide-stimulated human fibroblasts and recombinant derived IL-6 induced both CRP and SAA synthesis in a concentration dependent manner in the NPLC/PRF/5 cell line. In contrast, in Hep 3B cells, IL-6 alone had no discernable effect on the induction of either CRP or SAA, but was capable of causing increased synthesis of α 1 -protease inhibitor and fibrinogen and reduced synthesis of albumin. The addition of IL-1 to IL-6 led to induction of both CRP and SAA in Hep 3B cells, but did not augment the response of either CRP or SAA in NPLC/PRF/5 cells. These studies indicate that IL-6 plays a central role in induction of both CRP and SAA synthesis in the two human hepatoma cell lines. The different observations in the two hepatoma lines we studied likely reflect differences between these transformed cell lines in genetic regulatory mechanisms or other intracellular mechanisms by which extracellular signals affect expression of the CRP and SAA genes. Furthermore, our findings suggest that the mechanisms by which IL-6 regulates synthesis of CRP and SAA in Hep 3B cells differ from those involved in induction of α 1 -protease inhibitor and fibrinogen and in inhibition of albumin synthesis.

Interleukin-6 gene expression in human endothelial cells: RNA start sites, multiple IL-6 proteins and inhibition of proliferation

Interleukin-6 (IL-6) is a cytokine which is not only produced by a wide variety of different cells but one which also affects the function of diverse tissues. We have studied the expression of the IL-6 gene in freshly explanted human umbilical vein endothelial cells (HUVEC) and have also evaluated the effect of IL-6 on HUVEC proliferation. Cytokines like interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor (TNF) as well as bacterial products such as the lipopolysaccharide (LPS) rapidly enhance production of biologically active IL-6 by HUVEC (IL-6 bioassay: increase in alpha 1-antichymotrypsin secretion by Hep3B2 cells and its neutralization by antiserum to E. coli-derived human IL-6). The two inducible RNA start sites in the IL-6 gene that are used in cytokine-induced fibroblasts (at +1 and -21) are also used in the same relative proportion (+1 greater than -21) in cytokine or LPS-induced HUVEC as determined by S1-nuclease protection assays for IL-6 transcripts. Immunoaffinity chromatography followed by Western blotting shows that IL-6 species secreted by IL-1 alpha-induced HUVEC are of molecular mass 23-25, 27-30 and 45 kDa as judged by SDS-PAGE under reducing conditions. Finally, rIL-6 inhibits [3H]-thymidine incorporation by HUVEC in a dose-dependent manner. Thus IL-6 is not only produced by HUVEC but may also affect its proliferation. The ability of the vascular endothelium to rapidly secrete IL-6 in response to inflammation-associated cytokines is of strategic value since it generates a circulatory signal which helps mobilize the acute phase plasma protein response and enlists the immune system in host defence.

The relationship of serum IL-6 levels to acute graft-versus-host disease and hepatorenal disease after human bone marrow transplantation

The potential involvement of cytokines in acute graft-versus-host disease led us to analyze interleukin-6 in serial serum sets from 22 allogeneic marrow recipients who developed either grade 3 or 4 GVHD (n = 10), grade 2 GVHD (n = 6), or grade 1 or no diagnosed GVHD (n = 6). A total of 279 serial serum samples taken three times weekly before day 35 were analyzed. Maximum IL-6 levels were >40 U/ml (range, 40–1536 U/ml), 11–40 U/ml, and ≤10 U/ml for six, eleven, and five patients, respectively. Serum IL-6 peaks were temporally related to onset of GVHD, onset of a syndrome of hepatorenal dysfunction (HRD), or bilateral lung infiltration. Eight of ten patients who developed grade 3 or 4 GVHD overall had IL-6 maxima of >10 U/ml an average of 1.5 ± 1.8 days before the clinical onset. Fifteen of 17 patients with peak IL-6 levels >10 U/ml developed symptoms of hepatic and renal dysfunction within three days of the peak, while none of five patients with ≤10 U/ml of IL-6 developed HRD. Regression analysis demonstrated a linkage between the log magnitudes of the serum IL-6 peaks and onset of either GVHD or HRD within three days (P = 0.001). Furthermore, IL-6 peaks tended to precede GVHD onset for the 10 patients whose GVHD onset and IL-6 peak were within three days of each other (P = 0.02). These results, confirmed by both specific bioassay and by IL-6 ELISA, support the idea that acute GVHD in humans involves a cytokine cascade that includes production of IL-6 in addition to the previously reported involvement of tumor necrosis factor alpha and interferon-gamma.

Regulation of Expression of Interleukin‐6

The human genome contains a single interleukin-6 (IL-6) gene located at 7p21.’-’ To a first approximation, the expression of 1L-6 is enhanced in almost every human tissue in response to “damage” of almost every kind (TABLES 1 and 2). Furthermore, all of the numerous effects of IL-6 on different tissue and organ systems are such that they appear to help restrict tissue damage and to allow the host to recover from injury (TABLE 3) . The regulation of expression of the IL-6 gene appears to be particularly adapted to the key function of this cytokine, namely, that of an alarm signal which recruits diverse nonspecific and specific host defense mechanisms in an attempt to limit tissue damage. Thus, every inflammation-associated cytokine, bacterial products such as endotoxin, and acute virus infections enhance IL-6 gene expression. Activation of all three of the major signal transduction pathways (protein kinase C-, CAMP-, and Ca’+-activated pathways) singly or in combination turns on IL-6 gene transcription. Perhaps the most remarkable adaptation is the paradoxical enhancement (superinduction) of IL-6 secretion by tissues as macromolecular synthesis (e.g., protein synthesis) is compromised. Therefore, even in the face of imminent cell death, this ensures that there is a net increase in the secretion of IL-6 by the damaged cell. The cell types that produce high levels of IL-6 in response to noxious stimuli are distributed all over the body. These include fibroblasts (fibrocytes), endothelial cells, keratinocytes, other epithelial cells, all cells of the monocytic series, and endometrial stromal cells to name only a few. A further principle to emerge is that not all stimuli work equally well in inducing IL-6 gene expression in every tissue. For example, interleukin-la (IL-la) and tumor necrosis factor (TNF) do not stimulate IL-6 gene expression in human peripheral blood monocytes; bacterial lipopolysaccharide (LPS) is the strongest stimulus in monocytes. In contrast, IL-la is the strongest stimulus in fibroblasts and endothelial cells. Anti-CD3 antibody thus far has been shown to induce IL-6 only in human mononuclear cell preparations. Elucidation of the biochemical basis for these tissue-specific differences in IL-6 gene expression and the

Regional localization of the interferon-β2B-cell stimulatory factor 2/hepatocyte stimulating factor gene to human chromosome 7p15-p21

The human interferon-beta 2 gene (IFNB2) is identical to the genes encoding the B-cell stimulatory factor (BSF-2), the hybridoma growth factor (HGF), and the hepatocyte stimulating factor (HSF). This protein mediates major alterations in the secretion of a wide spectrum of plasma proteins by the liver in response to tissue injury (the acute-phase response). We have used a cDNA probe specific to the human IFNB2 gene in DNA hybridization experiments and report the regional localization of this gene to human chromosome 7p15-p21. Southern blot analyses of DNA extracted from a panel of mouse X human somatic cell hybrids localized this gene to human chromosome 7p. In situ hybridization of the IFNB2 cDNA probe to prebanded human metaphase chromosome spreads allowed the further localization of this gene to 7p15-p21.