Resilience to bacterial infection: difference between species could be due to proteins in serum.

Vertebrates vary in resistance and resilience to infectious diseases, and the mechanisms that regulate the trade-off between these often opposing protective processes are not well understood. Variability in the sensitivity of species to the induction of damaging inflammation in response to equivalent pathogen loads (resilience) complicates the use of animal models that reflect human disease. We found that induction of proinflammatory cytokines from macrophages in response to inflammatory stimuli in vitro is regulated by proteins in the sera of species in inverse proportion to their in vivo resilience to lethal doses of bacterial lipopolysaccharide over a range of 10,000-fold. This finding suggests that proteins in serum rather than intrinsic cellular differences may play a role in regulating variations in resilience to microbe-associated molecular patterns between species. The involvement of circulating proteins as key molecules raises hope that the process might be manipulated to create better animal models and potentially new drug targets.

Chemical biology of inflammatory cytokine signaling.

Pro-inflammatory cytokines, such as tumor necrosis factor-alpha and interleukin-1, trigger the signal transduction pathway leading to the activation of the transcription factor, nuclear factor-kappaB (NF-kappaB). NF-kappaB induces a large number of target genes involved in many biological processes, such as inflammation, immunity, cell survival, cell death and carcinogenesis. As therapeutic agents for inflammatory diseases and cancer, as well as bioprobes for the characterization of intracellular biological response and cell function, a large number of natural and synthetic small molecules have been identified to inhibit the activation of the NF-kappaB signaling pathway. This review focuses on recent progress in the identification and biological properties of small molecules targeting the NF-kappaB signaling pathway induced by pro-inflammatory cytokines.

Physiological roles of endogenous ouabain in normal rats.

Endogenous ouabain (EO)-like compounds are synthesized in and released from the adrenal gland. Although EO has been implicated in several pathological states such as hypertension and heart and kidney failure, its physiological roles in normal animal have not been elucidated. To address this issue, we studied the effects of reduction in plasma EO resulting from antiouabain antibody administration. Normal rats were treated for 28 days with antiouabain antibodies or rabbit IgG as control. Infusions were delivered through a jugular vein cannula by osmotic pumps, and blood pressure was monitored by tail-cuff plethysmography. The animals were housed in metabolic cages to measure water and food consumption and urine excretion. After 28 days, the thoracic aorta was isolated and used to study phenylephrine-induced contraction and atrial natriuretic peptide (ANP)-induced vasorelaxation. The adrenal gland cortex was enlarged in the antiouabain antibody-treated rats. Moreover, on the second day of treatment, there was a significant transient reduction in natriuresis in the antiouabain antibody-treated rats, suggesting that EO is a natriuretic hormone. Reduction in natriuresis was also observed when EO levels were reduced by active immunization resulting from sequential injection of ouabain-albumin. Furthermore, following 28 days of treatment, the response to phenylephrine was significantly lowered and that to ANP was significantly increased in aortic rings from antiouabain antibody-treated rats. These findings show for the first time that circulatory ouabain plausibly originating in the adrenal has physiological roles controlling vasculature tone and sodium homeostasis in normal rats.

Therapeutic drug monitoring of digoxin: impact of endogenous and exogenous digoxin-like immunoreactive substances.

Digoxin is a cardioactive drug with a narrow therapeutic range. Therapeutic drug monitoring is essential in clinical practice for efficacy as well as to avoid digoxin toxicity. Immunoassays are commonly used in clinical laboratories for determination of serum or plasma digoxin concentrations. Unfortunately, digoxin immunoassays are affected by both endogenous and exogenous compounds. Endogenous compounds are termed 'digoxin-like immunoreactive substances' (DLIS), which are found in elevated concentrations in volume-expanded patients. Exogenous compounds that interfere with digoxin assays are various drugs such as spironolactone, potassium canrenoate as well as Digibind (Fab fragment of antidigoxin antibody), which is used in treating life-threatening digoxin overdose. Moreover, various Chinese medicines such as Chan Su, Lu-Shen Wan and oleander-containing herbal preparations also interfere with serum digoxin measurements by immunoassays. Monitoring unbound (free) digoxin concentration may under certain circumstances eliminate such interferences. Clinicians should be aware of limitations of therapeutic drug monitoring of digoxin using immunoassays.

New enzyme-linked immunosorbent digoxin assay on the ADVIA IMS 800i system is virtually free from interference of endogenous digoxin-like immunoreactive factors.

Endogenous digoxin-like immunoreactive factors (DLIF) may crossreact with antidigoxin antibody and falsely elevate immunoassay results. Recently, a new enzyme-linked immunosorbent chemiluminescent assay for digoxin has been available for use on the ADVIA IMS (Integrated Modular System) 800i analyzer (Bayer Diagnostics). We studied potential interference of DLIF with this new digoxin assay. We analyzed 30 serum specimens from patients who have pathologic conditions that may increase serum DLIF concentrations. These patients were never exposed to digoxin or other agents that may lead to a measurable digoxin concentration. We also analyzed 10 specimens from neonates, 10 cord blood specimens, and 10 amniotic fluid specimens. Apparent digoxin concentrations were measured using the new enzyme-linked immunosorbent digoxin assay (IMS-Digoxin), a fluorescence polarization immunoassay (FPIA), and also a chemiluminescent immunoassay (CLIA, run on ACS:180(R) system from Bayer Diagnostics). We observed measurable apparent digoxin levels with the FPIA in 4 uremic patients (range 0.21-0.36 ng/mL, digoxin equivalent), 7 patients with liver disease (range 0.21-0.72 ng/mL), and 3 patients in the third trimester of pregnancy (0.22-0.66 ng/mL). We also observed measurable DLIF concentrations with the FPIA in 2 neonates (0.22 and 0.36 ng/mL), 5 cord blood specimens (range 0.21-1.18 ng/mL), and 5 amniotic fluid specimens (0.21-0.50 ng/mL). None of these DLIF-positive specimens showed any measurable digoxin concentration using the IMS-Digoxin or the CLIA assay. When serum specimens containing elevated concentrations of DLIF but no digoxin (as measured by FPIA) were supplemented with known concentrations of digoxin, we observed falsely elevated digoxin concentrations, as expected, only by the FPIA. In contrast, we observed a good agreement between the target and observed concentrations when the new IMS-Digoxin or the CLIA assay was used. We conclude that the IMS-Digoxin assay is free from interference of DLIF.

Circulating bufodienolide and cardenolide sodium pump inhibitors in preeclampsia.

OBJECTIVE: To determine plasma levels of the endogenous bufodienolide Na+/K+ ATPase inhibitor, marinobufagenin-like factor (MBG), in normotensive pregnancy and in preeclampsia, to compare changes of MBG with that of ouabain-like compound (OLC), and to characterize the purified MBG immunoreactive factor from preeclamptic plasma. DESIGN AND METHODS: Consecutive sample study. The levels of MBG and OLC compounds were measured in extracted plasma by solid phase fluoroimmunoassays. MBG and ouabain immunoreactive materials were partially purified from preeclamptic plasma via reverse-phase high-performance liquid chromatography (HPLC) and studied for their ability to cross react with MBG and ouabain antibodies, and to inhibit the Na+/K+ ATPase from human mesenteric arteries. Vasoconstrictor effect of authentic MBG was studied in isolated rings of human umbilical arteries. RESULTS: In 11 nonpregnant control individuals, plasma concentrations of MBG and OLC were 0.190+/-0.04 nmol/l and 0.297+/-0.037 nmol/l, respectively. In the third trimester of noncomplicated pregnancy (n = 6), plasma MBG increased (0.625+/-0.067 nmol/l, P<0.05), and OLC did not (0.32+/-0.07 nmol/l). In 15 patients with preeclampsia, plasma levels of both MBG and OLC increased dramatically (2.63+/-0.10 nmol/l and 0.697+/-0.16 nmol/l, respectively, P<0.01 versus both control groups). When fractionated by reverse phase HPLC, OLC was eluted by 18% acetonitrile, and MBG by 48% acetonitrile. Serially diluted samples of MBG and OLC immunoreactive materials from HPLC fractions reacted with MBG and ouabain antibody in solid phase immunoassay in a concentration dependent fashion. Authentic MBG caused contractile responses of isolated rings of human mesenteric arteries in a concentration-dependent manner. Similarly to the authentic MBG, HPLC purified MBG immunoreactive material from preeclamptic plasma inhibited Na+/K+ ATPase purified from human mesenteric artery. CONCLUSIONS: Our observations demonstrate the coexistence of two endogenous cardiotonic steroids in preeclamptic plasma, a more polar OLC and a less polar MBG-like compound. Substantial increases in plasma OLC and MBG immunoreactivity in preeclampsia, along with the vasoconstrictor properties of authentic MBG and Na+,K+ ATPase inhibitory activity of human MBG immunoreactive factor, suggest, that in preeclampsia, plasma concentrations of MBG are enough to substantially inhibit the sodium pump in cardiovascular tissues, and are in accordance with the views attributing endogenous digitalis-like factors a pathogenic role in the preeclamptic hypertension.

Contribution of a single heavy chain residue to specificity of an anti-digoxin monoclonal antibody.

Two distinct spontaneous variants of the murine anti-digoxin hybridoma 26-10 were isolated by fluorescence-activated cell sorting for reduced affinity of surface antibody for antigen. Nucleotide and partial amino acid sequencing of the variant antibody variable regions revealed that 1 variant had a single amino acid substitution: Lys for Asn at heavy chain position 35. The second variant antibody had 2 heavy chain substitutions: Tyr for Asn at position 35, and Met for Arg at position 38. Mutagenesis experiments confirmed that the position 35 substitutions were solely responsible for the markedly reduced affinity of both variant antibodies. Several mutants with more conservative position 35 substitutions were engineered to ascertain the contribution of Asn 35 to the binding of digoxin to antibody 26-10. Replacement of Asn with Gln reduced affinity for digoxin 10-fold relative to the wild-type antibody, but maintained wild-type fine specificity for cardiac glycoside analogues. All other substitutions (Val, Thr, Leu, Ala, and Asp) reduced affinity by at least 90-fold and caused distinct shifts in fine specificity. The Ala mutant demonstrated greatly increased relative affinities for 16-acetylated haptens and haptens with a saturated lactone. The X-ray crystal structure of the 26-10 Fab in complex with digoxin (Jeffrey PD et al., 1993, Proc Natl Acad Sci USA 90:10310-10314) reveals that the position 35 Asn contacts hapten and forms hydrogen bonds with 2 other contact residues. The reductions in affinity of the position 35 mutants for digoxin are greater than expected based upon the small hapten contact area provided by the wild-type Asn. We therefore performed molecular modeling experiments which suggested that substitution of Gln or Asp can maintain these hydrogen bonds whereas the other substituted side chains cannot. The altered binding of the Asp mutant may be due to the introduction of a negative charge. The similarities in binding of the wild-type and Gln-mutant antibodies, however, suggest that these hydrogen bonds are important for maintaining the architecture of the binding site and therefore the affinity and specificity of this antibody. The Ala mutant eliminates the wild-type hydrogen bonding, and molecular modeling suggests that the reduced side-chain volume also provides space that can accommodate a congener with a 16-acetyl group or saturated lactone, accounting for the altered fine specificity of this antibody.

Enzyme-linked immunosorbent assay for the phytotoxin thevetin.

An enzyme-linked immunosorbent assay is reported for monitoring thevetin, an active constituent of the highly poisonous plant Thevetia nerifolia. A thevetin-BSA conjugate was employed as the immunogen and the antibodies raised in rabbits were used for the development of an ELISA. Penicillinase served as the marker enzyme and its conjugation to thevetin by the periodate method is reported for the first time. The present ELISA method could detect 2 ng/ml of thevetin. Cross-reactivity studies with structural analogues and other phytotoxins and drugs of common occurrence in clinical and forensic toxicology established the superiority of the ELISA over the existing analytical methods for determining thevetin in various biospecimens.

Oleander interference in the digoxin radioimmunoassay in a fatal ingestion.

An elderly woman allegedly ingested oleander leaves and died. Ventricular arrhythmias and asystole were unresponsive to cardiopulmonary resuscitation, pharmacologic agents, and cardioversion. The patient, who had no access to digoxin, had an initial serum digoxin concentration of 5.8 ng/mL. Cross-reactivities between oleander extract and pure oleandrin and digoxin in the digoxin radioimmunoassay were 100:1 and 29,000:1, respectively. We postulate that glycosides in oleander leaves produced the elevated serum digoxin concentration. Based on an assumed volume of distribution of the oleander glycosides of 1 L/kg, the calculated lethal dose absorbed by our patient was 200 times greater than lethal doses in several animal species and corresponded to the absorption of 4 g of oleander leaves.