Diversity, Complexity, and Specificity of Bacterial Lipopolysaccharide (LPS) Structures Impacting Their Detection and Quantification.

Endotoxins are toxic lipopolysaccharides (LPSs), extending from the outer membrane of Gram-negative bacteria and notorious for their toxicity and deleterious effects. The comparison of different LPSs, isolated from various Gram-negative bacteria, shows a global similar architecture corresponding to a glycolipid lipid A moiety, a core oligosaccharide, and outermost long O-chain polysaccharides with molecular weights from 2 to 20 kDa. LPSs display high diversity and specificity among genera and species, and each bacterium contains a unique set of LPS structures, constituting its protective external barrier. Some LPSs are not toxic due to their particular structures. Different, well-characterized, and highly purified LPSs were used in this work to determine endotoxin detection rules and identify their impact on the host. Endotoxin detection is a major task to ensure the safety of human health, especially in the pharma and food sectors. Here, we describe the impact of different LPS structures obtained under different bacterial growth conditions on selective LPS detection methods such as LAL, HEK-blue TLR-4, LC-MS2, and MALDI-MS. In these various assays, LPSs were shown to respond differently, mainly attributable to their lipid A structures, their fatty acid numbers and chain lengths, the presence of phosphate groups, and their possible substitutions.

Early detection of oral bacteria causing gum infections and dental caries in children.

Background and Objectives: Periodontal diseases are resulted from gum infections and dental plaques, which are mainly caused by the bacterial agents. Since dental monitoring includes important prognostic roles, the aim of this study was to detect the most common periodontal pathogenic bacteria in children. Materials and Methods: A total of 200 clinical samples were collected from dental plaques and gingival grooves. Target-specific primers were designed for hbpA in Aggregatibacter actinomycetemcomitans, fimA in Porphyromonas gingivalis and 16S rRNA in Prevotella intermedia, Tannerella forsythia and Treponema denticola. Then, a multiplex polymerase chain reaction method was optimized for the highlighted bacterial agents. Results: In general, the highest and the lowest bacterial prevalence rates belonged to Tannerella forsythia (88%) and Porphyromonas gingivalis (13%), respectively. Furthermore, prevalence rates of Aggregatibacter actinomycetemcomitans, Prevotella intermedia and Treponema denticola were 25, 21 and 45% in samples, respectively. Conclusion: There were significant associations between dental/oral health and microbial community. Metabolism of the oral bacteria, including biofilm formation, can affect gums and develop dental plaques and hence dental caries, especially in children. Early diagnosis of dental caries in children via rapid, accurate molecular methods can increase the diagnostic capacity in clinical cases and therefore prevent periodontal infections in adulthood.

Integrated omics approach for the identification of HDL structure-function relationships in PCSK9-related familial hypercholesterolemia.

BACKGROUND: The role of proprotein convertase subtilisin/kexin type 9 (PCSK9) in dyslipidemia may go beyond its immediate effects on low-density lipoprotein receptor (LDL-R) activity. OBJECTIVE: This study aimed to assess PCSK9-derived alterations of high-density lipoprotein (HDL) physiology, which bear a potential to contribute to cardiovascular risk profile. METHODS: HDL was isolated from 33 patients with familial autosomal dominant hypercholesterolemia (FH), including those carrying PCSK9 gain-of-function (GOF) genetic variants (FH-PCSK9, n = 11), together with two groups of dyslipidemic patients employed as controls and carrying genetic variants in the LDL-R not treated (ntFH-LDLR, n = 11) and treated (tFH-LDLR, n = 11) with statins, and 11 normolipidemic controls. Biological evaluations paralleled by proteomic, lipidomic and glycomic analyses were applied to characterize functional and compositional properties of HDL. RESULTS: Multiple deficiencies in the HDL function were identified in the FH-PCSK9 group relative to dyslipidemic FH-LDLR patients and normolipidemic controls, which involved reduced antioxidative, antiapoptotic, anti-thrombotic and anti-inflammatory activities. By contrast, cellular cholesterol efflux capacity of HDL was unchanged. In addition, multiple alterations of the proteomic, lipidomic and glycomic composition of HDL were found in the FH-PCSK9 group. Remarkably, HDLs from FH-PCSK9 patients were systematically enriched in several lysophospholipids as well as in A2G2S2 (GP13) glycan and apolipoprotein A-IV. Based on network analysis of functional and compositional data, a novel mosaic structure-function model of HDL biology involving FH was developed. CONCLUSION: Several metrics of anti-atherogenic HDL functionality are altered in FH-PCSK9 patients paralleled by distinct compositional alterations. These data provide a first-ever overview of the impact of GOF PCSK9 genetic variants on structure-function relationships in HDL.

Identification of the specific molecular and functional signatures of pre-beta-HDL: relevance to cardiovascular disease.

While low concentrations of high-density lipoprotein-cholesterol (HDL-C) are widely accepted as an independent cardiovascular risk factor, HDL-C-rising therapies largely failed, suggesting the importance of both HDL functions and individual subspecies. Indeed HDL particles are highly heterogeneous, with small, dense pre-beta-HDLs being considered highly biologically active but remaining poorly studied, largely reflecting difficulties for their purification. We developed an original experimental approach allowing the isolation of sufficient amounts of human pre-beta-HDLs and revealing the specificity of their proteomic and lipidomic profiles and biological activities. Pre-beta-HDLs were enriched in highly poly-unsaturated species of phosphatidic acid and phosphatidylserine, and in an unexpectedly high number of proteins implicated in the inflammatory response, including serum paraoxonase/arylesterase-1, vitronectin and clusterin, as well as in complement regulation and immunity, including haptoglobin-related protein, complement proteins and those of the immunoglobulin class. Interestingly, amongst proteins associated with lipid metabolism, phospholipid transfer protein, cholesteryl ester transfer protein and lecithin:cholesterol acyltransferase were strongly enriched in, or restricted to, pre-beta-HDL. Furthermore, pre-beta-HDL potently mediated cellular cholesterol efflux and displayed strong anti-inflammatory activities. A correlational network analysis between lipidome, proteome and biological activities highlighted 15 individual lipid and protein components of pre-beta-HDL relevant to cardiovascular disease, which may constitute novel diagnostic targets in a pathological context of altered lipoprotein metabolism.

Phosphatidylserine enhances anti-inflammatory effects of reconstituted HDL in macrophages via distinct intracellular pathways.

Phosphatidylserine (PS) is a minor phospholipid constituent of high-density lipoprotein (HDL) that exhibits potent anti-inflammatory activity. It remains indeterminate whether PS incorporation can enhance anti-inflammatory effects of reconstituted HDL (rHDL). Human macrophages were treated with rHDL containing phosphatidylcholine alone (PC-rHDL) or PC and PS (PC/PS-rHDL). Interleukin (IL)-6 secretion and expression was more strongly inhibited by PC/PS-rHDL than PC-rHDL in both tumor necrosis factor (TNF)-α- and lipopolysaccharide (LPS)-stimulated macrophages. siRNA experiments revealed that the enhanced anti-inflammatory effects of PC/PS-rHDL required scavenger receptor class B type I (SR-BI). Furthermore, PC/PS-rHDL induced a greater increase in Akt1/2/3 phosphorylation than PC-rHDL. In addition, PC/PS but not PC-rHDL decreased the abundance of plasma membrane lipid rafts and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation. Finally, when these rHDL formulations were administered to dyslipidemic low-density lipoprotein (LDL)-receptor knockout mice fed a high-cholesterol diet, circulating IL-6 levels were significantly reduced only in PC/PS-rHDL-treated mice. In parallel, enhanced Akt1/2/3 phosphorylation by PC/PS-rHDL was observed in the mouse aortic tissue using immunohistochemistry. We concluded that the incorporation of PS into rHDLs enhanced their anti-inflammatory activity by modulating Akt1/2/3- and p38 MAPK-mediated signaling through SR-BI in stimulated macrophages. These data identify PS as a potent anti-inflammatory component capable of enhancing therapeutic potential of rHDL-based therapy.

High-density lipoproteins (HDL): Novel function and therapeutic applications.

The failure of high-density lipoprotein (HDL)-raising agents to reduce cardiovascular disease (CVD) together with recent findings of increased cardiovascular mortality in subjects with extremely high HDL-cholesterol levels provide new opportunities to revisit our view of HDL. The concept of HDL function developed to explain these contradictory findings has recently been expanded by a role played by HDL in the lipolysis of triglyceride-rich lipoproteins (TGRLs) by lipoprotein lipase. According to the reverse remnant-cholesterol transport (RRT) hypothesis, HDL critically contributes to TGRL lipolysis via acquirement of surface lipids, including free cholesterol, released from TGRL. Ensuing cholesterol transport to the liver with excretion into the bile may reduce cholesterol influx in the arterial wall by accelerating removal from circulation of atherogenic, cholesterol-rich TGRL remnants. Such novel function of HDL opens wide therapeutic applications to reduce CVD in statin-treated patients, which primarily involve activation of cholesterol flux upon lipolysis.

Phospholipid transfer to high-density lipoprotein (HDL) upon triglyceride lipolysis is directly correlated with HDL-cholesterol levels and is not associated with cardiovascular risk.

BACKGROUND AND AIMS: While low concentrations of high-density lipoprotein-cholesterol (HDL-C) represent a well-established cardiovascular risk factor, extremely high HDL-C is paradoxically associated with elevated cardiovascular risk, resulting in the U-shape relationship with cardiovascular disease. Free cholesterol transfer to HDL upon lipolysis of triglyceride-rich lipoproteins (TGRL) was recently reported to underlie this relationship, linking HDL-C to triglyceride metabolism and atherosclerosis. In addition to free cholesterol, other surface components of TGRL, primarily phospholipids, are transferred to HDL during lipolysis. It remains indeterminate as to whether such transfer is linked to HDL-C and cardiovascular disease. METHODS AND RESULTS: When TGRL was labelled with fluorescent phospholipid 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI), time- and dose-dependent transfer of DiI to HDL was observed upon incubations with lipoprotein lipase (LPL). The capacity of HDL to acquire DiI was decreased by -36% (p<0.001) in low HDL-C patients with acute myocardial infarction (n = 22) and by -95% (p<0.001) in low HDL-C subjects with Tangier disease (n = 7), unchanged in low HDL-C patients with Type 2 diabetes (n = 17) and in subjects with high HDL-C (n = 20), and elevated in subjects with extremely high HDL-C (+11%, p<0.05) relative to healthy normolipidemic controls. Across all the populations combined, HDL capacity to acquire DiI was directly correlated with HDL-C (r = 0.58, p<0.001). No relationship of HDL capacity to acquire DiI with both overall and cardiovascular mortality obtained from epidemiological studies for the mean HDL-C levels observed in the studied populations was obtained. CONCLUSIONS: These data indicate that the capacity of HDL to acquire phospholipid from TGRL upon LPL-mediated lipolysis is proportional to HDL-C and does not reflect cardiovascular risk in subjects widely differing in HDL-C levels.

Free cholesterol transfer to high-density lipoprotein (HDL) upon triglyceride lipolysis underlies the U-shape relationship between HDL-cholesterol and cardiovascular disease.

BACKGROUND: Low concentrations of high-density lipoprotein cholesterol (HDL-C) represent a well-established cardiovascular risk factor. Paradoxically, extremely high HDL-C levels are equally associated with elevated cardiovascular risk, resulting in the U-shape relationship of HDL-C with cardiovascular disease. Mechanisms underlying this association are presently unknown. We hypothesised that the capacity of high-density lipoprotein (HDL) to acquire free cholesterol upon triglyceride-rich lipoprotein (TGRL) lipolysis by lipoprotein lipase underlies the non-linear relationship between HDL-C and cardiovascular risk. METHODS: To assess our hypothesis, we developed a novel assay to evaluate the capacity of HDL to acquire free cholesterol (as fluorescent TopFluor® cholesterol) from TGRL upon in vitro lipolysis by lipoprotein lipase. RESULTS: When the assay was applied to several populations markedly differing in plasma HDL-C levels, transfer of free cholesterol was significantly decreased in low HDL-C patients with acute myocardial infarction (-45%) and type 2 diabetes (-25%), and in subjects with extremely high HDL-C of >2.59 mmol/L (>100 mg/dL) (-20%) versus healthy normolipidaemic controls. When these data were combined and plotted against HDL-C concentrations, an inverse U-shape relationship was observed. Consistent with these findings, animal studies revealed that the capacity of HDL to acquire cholesterol upon lipolysis was reduced in low HDL-C apolipoprotein A-I knock-out mice and was negatively correlated with aortic accumulation of [3H]-cholesterol after oral gavage, attesting this functional characteristic as a negative metric of postprandial atherosclerosis. CONCLUSIONS: Free cholesterol transfer to HDL upon TGRL lipolysis may underlie the U-shape relationship between HDL-C and cardiovascular disease, linking HDL-C to triglyceride metabolism and atherosclerosis.

Association of Serum Cholesterol Efflux Capacity With Mortality in Patients With ST-Segment Elevation Myocardial Infarction.

BACKGROUND: Serum cholesterol efflux capacity, a biomarker that integrates contributors and modulators of the initial step of the reverse cholesterol transport, has been associated with atherosclerosis independently of high-density lipoprotein (HDL) cholesterol level. OBJECTIVES: The authors evaluated the prognostic impact of serum cholesterol efflux capacity on mortality in a large cohort of patients hospitalized for an acute myocardial infarction (MI). METHODS: Serum cholesterol efflux capacity, cholesteryl ester transfer protein (CETP) activity, total cholesterol, low-density lipoprotein cholesterol, HDL cholesterol, and triglyceride levels were measured in 1,609 consecutive patients admitted with an acute MI. The primary endpoint was all-cause mortality evaluated at 6 years with a median follow-up of 1.9 years (interquartile range: 1.5 to 4.2 years). An analysis by quartile of serum cholesterol efflux capacity was also performed. RESULTS: In a fully adjusted model that included age, sex, traditional cardiovascular risk factors including lipid levels, and prognostic factors of MI, serum cholesterol efflux capacity was a strong predictor of survival (adjusted hazard ratio for mortality per 1-SD increase in serum cholesterol efflux capacity, 0.79; 95% confidence interval: 0.66 to 0.95; p = 0.0132). Patients displaying an elevated serum cholesterol efflux capacity had a marked lower rate of mortality at 6 years (adjusted hazard ratio: 0.54 [0.32 to 0.89]; p = 0.0165) as compared with patients with reduced serum cholesterol efflux capacity. CONCLUSIONS: Serum cholesterol efflux capacity, an integrative marker of reverse cholesterol transport pathway and efficacy, was inversely associated with all-cause mortality in MI patients independently of HDL cholesterol level and other risk factors.

Functional, structural, and phylogenetic analysis of mitochondrial cytochrome b (cytb) in insects.

Cytochrome b (Cytb, EC 1.10. 2.2) is the only cytochrome coded by mitochondrial DNA and involved in electron transport in the mitochondrial respiratory chain. In this study, characterization of cytb protein was identified on fifty-four insect protein sequences, from different orders. According to the conserved motifs obtained with MEME and MAST tools, eight motifs were common to all insects. The structural and functional analyses of 16 selected insects from different orders were performed with ProtParam, Compute PI, SOPMA, SignalP 4.1, TMHMM 2.0, ProtScale and ProDom tools in the ExPASy database and DNASTAR 12.1 software. The tertiary structure of Drosophila melanogaster as a sample of insects was predicted by the Phyre2 and TM-score servers and their similarities were verified by SuperPose servers. The tertiary structures were predicted using the 'd1ppjc2' model (PDB accession code: 1ppj C). A phylogenetic tree was constructed with MEGA 6.06 software using the neighbour-joining method. According to the results, there is a high identity of cytb in different insects so that they should be derived from a common ancestor. In protein-protein interaction analysis with STRING 10.0, twenty-three enriched pathways of KEGG were identified in D. melanogaster and other species. The obtained data provided a background for bioinformatic studies of the function and evolution of other insects and organisms.

Primary Culture of Human Cumulus Cells Requires Stearoyl-Coenzyme A Desaturase 1 Activity for Steroidogenesis and Enhancing Oocyte In Vitro Maturation.

Stearoyl-coenzyme A desaturase 1 (SCD1) is a key enzyme in lipid metabolism and is expressed in cumulus cells. The objective of the present study was to evaluate the effect of SCD1 inhibition in human cumulus cells on triglyceride content, steroidogenesis, and oocyte in vitro maturation. Human cumulus cells were exposed to SCD1 inhibitor CAY10566 (SCDinhib) alone or in combination with oleic acid in primary culture. The SCDinhib markedly suppressed triglyceride accumulation (-47%, P = .01), aromatase gene expression (-36%, P = .02), and estradiol production (-49%, P = .01) even at a dose not affecting cell viability and apoptosis. Human immature oocytes at the germinal vesicle (GV) stage were cocultured with pretreated cumulus cells. The rate of oocytes reaching the metaphase II stage was significantly lower in coculture with SCDinhib-treated cumulus cells than with control cumulus cells (-18%, P < .01), which recovered by oleic acid supplementation. This finding on in vitro maturation rate was also reproducible with mouse GV oocytes. The results suggest that SCD1 activity is required for cumulus cell lipid storage and steroidogenesis. In addition, oocyte maturation is negatively affected by SCD1 inhibition in cumulus cells, possibly due to a deficient lipid-mediated paracrine support.

Small dense HDLs display potent vasorelaxing activity, reflecting their elevated content of sphingosine-1-phosphate.

The functional heterogeneity of HDL is attributed to its diverse bioactive components. We evaluated whether the vasodilatory effects of HDL differed across HDL subpopulations, reflecting their distinct molecular composition. The capacity of five major HDL subfractions to counteract the inhibitory effects of oxidized LDL on acetylcholine-induced vasodilation was tested in a rabbit aortic rings model. NO production, an essential pathway in endothelium-dependent vasorelaxation, was studied in simian vacuolating virus 40-transformed murine endothelial cells (SVECs). Small dense HDL3 subfractions displayed potent vasorelaxing activity (up to +31% vs. baseline, P < 0.05); in contrast, large light HDL2 did not induce aortic-ring relaxation when compared on a total protein basis. HDL3 particles were enriched with sphingosine-1-phosphate (S1P) (up to 3-fold vs. HDL2), with the highest content in HDL3b and -3c that concomitantly revealed the strongest vasorelaxing properties. NO generation was enhanced by HDL3c in SVECs (1.5-fold, P < 0.01), a phenomenon that was blocked by the S1P receptor antagonist, VPC 23019. S1P-enriched reconstituted HDL (rHDL) was a 1.8-fold (P < 0.01) more potent vasorelaxant than control rHDL in aortic rings. Small dense HDL3 particles displayed potent protective effects against oxidative stress-associated endothelium dysfunction, potentially reflecting their elevated content of S1P that might facilitate interaction with S1P receptors and ensuing NO generation.

Phosphatidylserine in atherosclerosis.

PURPOSE OF REVIEW: It is now widely acknowledged that phosphatidylserine is a multifunctional bioactive lipid. In this review, we focus on the function of phosphatidylserine in modulating cholesterol metabolism, influencing inflammatory response and regulating coagulation system, and discuss promising phosphatidylserine-based therapeutic approaches and detection techniques in atherosclerosis. RECENT FINDINGS: Phosphatidylserine has been suggested to play important roles in physiological processes, such as apoptosis, inflammation, and coagulation. Recent data demonstrate atheroprotective potential of phosphatidylserine, reflecting its capacity to inhibit inflammation, modulate coagulation, and enhance HDL functionality. Furthermore, modern lipidomic approaches have enabled the investigation of phosphatidylserine properties relevant to the lipid-based drug delivery and development of reconstituted HDL. SUMMARY: Studies of phosphatidylserine in relation to atherosclerosis represent an area of opportunity. Additional research elucidating mechanisms underlying experimentally observed atheroprotective effects of phosphatidylserine is required to fully explore therapeutic potential of this naturally occurring phospholipid in cardiovascular disease.

Can phosphatidylserine enhance atheroprotective activities of high-density lipoprotein?

Although high-density lipoprotein (HDL) is well known to be protective against atherosclerotic cardiovascular disease, therapeutic interventions to raise HDL-cholesterol levels do not translate into reduction in cardiovascular risk. Due to the compositional complexity of HDL particles, molecular determinants of their atheroprotective function still remain to be clarified. Recent structural and functional data identify phospholipid as a major bioactive component of HDL. Such a role has recently been specifically evidenced for phosphatidylserine (PS); indeed, HDL content of PS displayed positive correlations with all metrics of HDL functionality assessed. This review summarizes current knowledge about HDL-associated PS; possible mechanisms for its atheroprotective role are discussed and potential applications of PS to HDL-based therapies are highlighted.

Therapeutic applications of reconstituted HDL: When structure meets function.

Reconstituted forms of HDL (rHDL) are under development for infusion as a therapeutic approach to attenuate atherosclerotic vascular disease and to reduce cardiovascular risk following acute coronary syndrome and ischemic stroke. Currently available rHDL formulations developed for clinical use contain apolipoprotein A-I (apoA-I) and one of the major lipid components of HDL, either phosphatidylcholine or sphingomyelin. Recent data have established that quantitatively minor molecular constituents of HDL particles can strongly influence their anti-atherogenic functionality. Novel rHDL formulations displaying enhanced biological activities, including cellular cholesterol efflux, may therefore offer promising prospects for the development of HDL-based, anti-atherosclerotic therapies. Indeed, recent structural and functional data identify phosphatidylserine as a bioactive component of HDL; the content of phosphatidylserine in HDL particles displays positive correlations with all metrics of their functionality. This review summarizes current knowledge of structure-function relationships in rHDL formulations, with a focus on phosphatidylserine and other negatively-charged phospholipids. Mechanisms potentially underlying the atheroprotective role of these lipids are discussed and their potential for the development of HDL-based therapies highlighted.

Expression Levels of PPARγ and CYP-19 in Polycystic Ovarian Syndrome Primary Granulosa Cells: Influence of ω-3 Fatty Acid.

BACKGROUND: The omega-3 fatty acid (ω-3 fatty acid) such as eicosapentaenoic acid (EPA) is currently used in the clinic as a nutritional supplement in the treatment of poly- cystic ovarian syndrome (PCOS). The present study was designed to investigate the ef- fect of EPA on the expression levels of peroxisome proliferator-activated receptor gamma (PPARγ) and cytochrome P450 aromatase (encoded by the CYP-19) in primary cultured granulosa cells (GC) from patients undergoing in vitro fertilization (IVF), and also to compare these effects with those in GC of PCOS patients. MATERIALS AND METHODS: In this experimental study, human GC were isolated, pri- mary cultured in vitro, exposed to a range of concentrations of the EPA and in- vestigated with respect to gene expression levels of PPARγ and CYP-19 using real time-polymerase chain reaction (PCR). The participants (n=30) were the patients admitted to the IVF Center in February-March 2013 at Alzahra Hospital, Tabriz, Iran, who were divided into two groups as PCOS (n=15) and non-PCOS (n=15) women (controls). RESULTS: All doses of the EPA significantly induced PPARγ mRNA gene expression level as compared to the control recombinant follicle stimulating hormone (rFSH) alone condi- tion. High doses of EPA in the presence of rFSH produced a stimulatory effect on expres- sion level of PPARγ (2.15-fold, P=0.001) and a suppressive effect (0.56-fold, P=0.01) on the expression level of CYP-19, only in the PCOS GC. CONCLUSION: EPA and FSH signaling pathway affect differentially on the gene ex- pression levels of PPARγ and CYP-19 in PCOS GC. Altered FSH-induced PPARγ activity in PCOS GC may modulate the CYP-19 gene expression in response to EPA, and possibly modulates the subsequent steroidogenesis of these cells.

Influence of ω-3 fatty acid eicosapentaenoic acid on IGF-1 and COX-2 gene expression in granulosa cells of PCOS women.

BACKGROUND: The omega-3 (ω-3) fatty acid eicosapentaenoic acid (EPA) is currently used in the clinic as a nutritional supplement to improve infertility, particularly in women with polycystic ovarian syndrome (PCOS). OBJECTIVE: The present study was designed to investigate the effect of EPA on insulin-like growth factor 1 (IGF-1) and cyclooxygenase 2 (COX-2) gene expression in primary cultured granulosa cells from patients undergoing in vitro fertilization (IVF), and also to compare this effect with those in granulosa cells of PCOS patients. MATERIALS AND METHODS: In this experimental study, human granulosa cells were isolated from follicular fluid of normal and PCOS women undergoing IVF by hyaluronidase digestions, followed by Percoll gradient centrifugation. Cells were cultured in vitro, exposed to a range of concentrations of the EPA (25-100 µM) for 24 hr, and investigated with respect to COX-2 and IGF-1 gene expression by real time-PCR. RESULTS: In both groups, all doses of the EPA significantly induced IGF-1 mRNA gene expression compared to the untreated control. High doses of EPA in the presence of recombinant (r) FSH produced a stimulatory effect on IGF-1 and a suppressive effect (p=0.01) on the COX-2 gene expression, which were more pronounced in granulosa cells from PCOS patients. CONCLUSION: EPA affect diversely the gene expression of IGF-1 and COX-2 in granulosa cells, which were more pronounced in PCOS compared to control. These findings represent the possible underlying molecular mechanisms for the positive impact of the ω-3 fatty acids on reproduction, especially in patients with PCOS.

Immunohistochemichal Assessment of the CrkII Proto-oncogene Expression in Common Malignant Salivary Gland Tumors and Pleomorphic Adenoma.

Background and aims. Various morphologies are seen in different salivary gland tumorsor within an individual tumor, and the lesions show divers biological behaviors. Experimental results support the hypothesis that increased CrkII proto-oncogene is associated with cytokine-induced tumor initiation and progression by altering cell motility signaling pathway. The aim of this study was to assess the CrkII expression in common malignant salivary gland tumors and pleomorphic ade-noma. Materials and methods. Immunohistochemical analysis of CrkII expression was performed on paraffin blocks of 64 car-cinomas of salivary glands, 10 pleomorphic adenomas, and 10 normal salivary glands. Biopsies were subjected to immu-nostaining with EnVision detection system using monoclonal anti-CrkII. Evaluation of immunoreactivity of CrkII was based on the immunoreaction intensity and percentage of stained tumor cells which were scored semi-quantitatively on a scale with four grades 0 to 3. Kruskal-wallis test and additional Mann-Whitney statistical test were used for analysis of CrkII expression levels. Results. Increased expression of CrkII was seen (P=0.005) in malignant tumors including: mucoepidermoid carcinoma, adenoid cystic carcinoma, and carcinoma ex pleomorphic adenoma, but CrkII expression in acinic cell carcinoma was weak. CrkII expression in pleomorphic adenoma was weak or negative. A weak staining was sparsely seen in normal acinar serous cell. Conclusion. Increased expression of CrkII and its higher intensity of staining in tumors with more aggressive biologic behavior in carcinomas of salivary gland is consistent with a role for this proto-oncogene in salivary gland tumorigenesis and cancer progression.