Chemopreventive and renal protective effects for docosahexaenoic acid (DHA): implications of CRP and lipid peroxides

Background

The fish oil-derived ω-3 fatty acids, like docosahexanoic (DHA), claim a plethora of health benefits. We currently evaluated the antitumor effects of DHA, alone or in combination with cisplatin (CP) in the EAC solid tumor mice model, and monitored concomitant changes in serum levels of C-reactive protein (CRP), lipid peroxidation (measured as malondialdehyde; MDA) and leukocytic count (LC). Further, we verified the capacity of DHA to ameliorate the lethal, CP-induced nephrotoxicity in rats and the molecular mechanisms involved therein.

Results

EAC-bearing mice exhibited markedly elevated LC (2-fold), CRP (11-fold) and MDA levels (2.7-fold). DHA (125, 250 mg/kg) elicited significant, dose-dependent reductions in tumor size (38%, 79%; respectively), as well as in LC, CRP and MDA levels. These effects for CP were appreciably lower than those of DHA (250 mg/kg). Interestingly, DHA (125 mg/kg) markedly enhanced the chemopreventive effects of CP and boosted its ability to reduce serum CRP and MDA levels. Correlation studies revealed a high degree of positive association between tumor growth and each of CRP (r = 0.85) and leukocytosis (r = 0.89), thus attesting to a diagnostic/prognostic role for CRP. On the other hand, a single CP dose (10 mg/kg) induced nephrotoxicity in rats that was evidenced by proteinuria, deterioration of glomerular filtration rate (GFR, -4-fold), a rise in serum creatinine/urea levels (2–5-fold) after 4 days, and globally-induced animal fatalities after 7 days. Kidney-homogenates from CP-treated rats displayed significantly elevated MDA- and TNF-α-, but reduced GSH-, levels. Rats treated with DHA (250 mg/kg, but not 125 mg/kg) survived the lethal effects of CP, and showed a significant recovery of GFR; while their homogenates had markedly-reduced MDA- and TNF-α-, but -increased GSH-levels. Significant association was detected between creatinine level and those of MDA (r = 0.81), TNF-α ) r = 0.92) and GSH (r = -0.82); implying causal relationships.

Conclusion

DHA elicited prominent chemopreventive effects on its own, and appreciably augmented those of CP as well. The extent of tumor progression in various mouse groups was highly reflected by CRP levels (thus implying a diagnostic/prognostic role for CRP). Further, this study is the first to reveal that DHA can obliterate the lethal CP-induced nephrotoxicity and renal tissue injury. At the molecular level, DHA appears to act by reducing leukocytosis, systemic inflammation, and oxidative stress.     

Increased levels of (class switched) memory B cells in peripheral blood of current smokers

There is increasing evidence that a specific immune response contributes to the pathogenesis of COPD. B-cell follicles are present in lung tissue and increased anti-elastin titers have been found in plasma of COPD patients. Additionally, regulatory T cells (Tregs) have been implicated in its pathogenesis as they control immunological reactions. We hypothesize that the specific immune response in COPD is smoke induced, either by a direct effect of smoking or as a result of smoke-induced lung tissue destruction (i.e. formation of neo-epitopes or auto antigens). Furthermore, we propose that Tregs are involved in the suppression of this smoke-induced specific immune response.The presence of B cells, memory B cells and Tregs was assessed by flow cytometry in peripheral blood of 20 COPD patients and 29 healthy individuals and related to their current smoking status.COPD patients had lower (memory) B-cell percentages and higher Treg percentages in peripheral blood than healthy individuals, with a significant negative correlation between these cells. Interestingly, current smokers had higher percentages of (class-switched) memory B cells than ex-smokers and never smokers, irrespective of COPD.This increase in (class-switched) memory B cells in current smokers is intriguing and suggests that smoke-induced neo-antigens may be constantly induced in the lung. The negative correlation between B cells and Tregs in blood is in line with previously published observations that Tregs can suppress B cells. Future studies focusing on the presence of these (class switched) memory B cells in the lung, their antigen specificity and their interaction with Tregs are necessary to further elucidate the specific B-cell response in COPD.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> effect of hyaluronic acid on erythrocyte flow properties

Background  

Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.     

Influence of a natural and a synthetic inhibitor of factor XIIIa on fibrin clot rheology

We investigated the origins of greater clot rigidity associated with FXIIIa-dependent cross-linking. Fibrin clots were examined in which cross-linking was controlled through the use of two inhibitors: a highly specific active-center-directed synthetic inhibitor of FXIIIa, 1,3-dimethyl-4,5-diphenyl-2[2(oxopropyl)thio]imidazolium trifluoromethylsulfonate, and a patient-derived immunoglobulin directed mainly against the thrombin-activated catalytic A subunits of thrombin-activated FXIII. Cross-linked fibrin chains were identified and quantified by one- and two-dimensional gel electrophoresis and immunostaining with antibodies specific for the alpha- and gamma-chains of fibrin. Gamma-dimers, gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrids were detected. The synthetic inhibitor was highly effective in preventing the production of all cross-linked species. In contrast, the autoimmune antibody of the patient caused primarily an inhibition of alpha-chain cross-linking. Clot rigidities (storage moduli, G') were measured with a cone and plate rheometer and correlated with the distributions of the various cross-linked species found in the clots. Our findings indicate that the FXIIIa-induced dimeric cross-linking of gamma-chains by itself is not sufficient to stiffen the fibrin networks. Instead, the augmentation of clot rigidity was more strongly correlated with the formation of gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrid cross-links. A mechanism is proposed to explain how these cross-linked species may enhance clot rigidity.     

A DEFICIENS homologue is down-regulated during apomictic initiation in ovules of Hieracium

 Hieracium is a member of the Asteraceae family, and contains sexual species in addition to apomictic species that reproduce by apospory and produce seed without fertilization. A homologue of the floral organ-identity gene DEFICIENS (DEF) was isolated from an apomictic line of Hieracium piloselloides (Vill.) following differential display between mature ovules and those initiating autonomous embryogenesis. The gene termed HPDEF has 93% amino acid identity with GDEF2, a DEF homologue isolated from Gerbera hybrida (D. Yu et al., 1999, Plant J. 17: 51–62), another member of the Asteraceae. In-situ analysis showed that early in floral development HPDEF is expressed in stamen and petal primordia, indicating expected B-function activity, according to the ABC model of floral organ identity (J. L. Bowman et al., 1991, Development 112: 1–20; E. S. Coen and E. M. Meyerowitz, 1991, Nature 353: 31–37). However, HPDEF expression was also observed in ovule primordia and expression continued in developing ovules until anthesis, indicating that this gene may have a role in ovule development. Expression of HPDEF was not detected in megaspore mother cells, or in sexual or aposporous embryo sacs. In sexual Hieracium, HPDEF was uniformly expressed throughout the ovule integument until anthesis. In most ovules of the apomict, however, HPDEF expression was transiently down-regulated in a specific zone in the chalazal region where cells initiating aposporous embryo sac formation differentiate. Uniform low-level HPDEF expression was subsequently observed prior to anthesis in ovules from sexual and apomictic plants. HPDEF may be down-regulated as a consequence of apomictic initiation and/or its down-regulation may facilitate progression of apomictic events. Received: 15 September 1999 / Accepted: 12 October 1999     

Genetic separation of autonomous endosperm formation (AutE) from the two other components of apomixis in Hieracium

In apomictic Hieracium subgenus Pilosella species, embryo sacs develop in ovules without meiosis. Embryo and endosperm formation then occur without fertilization, producing seeds with a maternal genotype encased in a fruit (achene). Genetic analyses in H. praealtum indicate a dominant locus (LOA) controls meiotic avoidance, and another dominant locus (LOP) controls both fertilization-independent embryogenesis and endosperm formation. While cytologically examining developmental events in ovules of progeny from crosses between different wild-type and mutant Hieracium apomicts, and a sexual Hieracium species, we identified two plants, AutE196 and AutE24, which have lost the capacity for meiotic avoidance and fertilization-independent embryo formation. AutE196 and AutE24 exhibit autonomous endosperm formation and set parthenocarpic, seedless achenes at a penetrance of 18 %. Viable seed form after pollination. Cytological examination of 102 progeny from a backcross of AutE196 with sexual H. pilosella showed that autonomous endosperm formation is a heritable, dominant, qualitative trait, detected in 51 % of progeny. Variation in quantitative trait penetrance indicates other factors influence its expression. The correlation between autonomous endosperm development and mature parthenocarpic achene formation suggests the former is sufficient to trigger fruit maturation in Hieracium. The developmental component of autonomous endosperm formation is therefore genetically separable from those controlling meiotic avoidance and autonomous embryogenesis in Hieracium and has been denoted as AutE. We postulate that tight linkage of AutE and genes controlling autonomous embryogenesis at the LOP locus in H. praealtum may explain why inheritance of autonomous seed formation is typically observed as a single component.