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The objective of this study was to determine whether the late failure of beta-cells in islets transplanted via the portal vein is caused by excess insulin-stimulated lipogenesis and lipotoxicity and, if so, whether the damage can be prevented by reducing lipogenesis surrounding the islets. Based on the premise that high portal vein levels of nutrients and incretins would stimulate hyperinsulinemia, thereby inducing intense lipogenesis in nearby hepatocytes, normal islets were transplanted into livers of syngeneic streptozotocin-induced diabetic recipients. Hydrolysis of the surrounding fat would flood the islet grafts with fatty acids that could damage and destroy the beta-cells. Reducing lipogenesis by leptin or caloric restriction should prevent or reduce the destruction. After a rise after transplantation, insulin levels gradually declined and hyperglycemia increased. Four weeks after transplantation mRNA of the lipogenic transcription factor, sterol regulatory element-binding protein-1c (SREBP-1c) and its lipogenic target enzymes were elevated in livers of these recipients, as was triacylglycerol content. Positive oil red O staining for lipids and immunostaining for SREBP-1 were observed in hepatocytes surrounding islets with damaged beta-cells. Leptin-induced lipopenia prevented and caloric restriction reduced steatosis, hyperglycemia, and apoptotic beta-cell destruction. Excessive SREBP-1c-mediated lipogenesis, induced in hepatocytes by insulin hypersecretion, is followed by beta-cell destruction in the grafts and reappearance of diabetes. Graft failure is prevented by blocking lipogenesis. The results suggest that strict antilipogenic intervention might improve outcomes after human islet transplantation. [ABSTRACT FROM AUTHOR]

Lycopene is a dietary antioxidant known to prevent skin photodamage. This study aimed to examine age-dependent presence of this carotenoid on the surface of the facial skin and in the serum as well as to measure the same parameters during supplementation with lycopene. Serum samples and samples from facial skin surface were obtained from 60 young (under 25 years old) and 60 middle-aged (over 50 years old) volunteers. Similar samples were taken from 15 middle-aged subjects during 4-week supplementation with lycopene (7 mg/day). Serum lycopene levels and isomer profiles were analyzed by HPLC. Lycopene in desquamated corneocytes and the sebum from facial skin surface was determined using lycopene-specific fluorescent monoclonal antibodies. The results demonstrated that there was no age-related difference in serum lycopene levels, but a higher proportion of (all-E)-lycopene was detected in the "young" group (37.5% vs 26.2% in the "middle-aged" group; p  <   0.0001). "Young" volunteers also had a higher lycopene level in both corneocytes ( p  =   0.0071) and the sebum ( p  =   0.0139) from the skin surface. Supplementation with lycopene resulted in a sharp increase of lycopene concentrations in both serum and skin surface samples. There was also a clear change in the pattern of lycopene isomers in the serum manifested by a significant increase in the proportion of (all-E)-lycopene (from 22.1% to 44.0% after supplementation, p  <   0.0001). It can be concluded that dietary supplementation with lycopene results in its accumulation in the serum and skin. This process is accompanied by significant changes in the circulating lycopene isomer profile which becomes similar to that typical for young individuals., Competing Interests: Lycotec Ltd developed GA Lycopene, which is evaluated in this study. Ivan M. Petyaev and Nigel H. Kyle are employees of Lycotec; Dmitry V. Pristensky, Marina P. Chernyshova, Marina V. Lozbiakova, and Yuriy K. Bashmakov are independent scientists who collaborate with, but are not, and have never been employees of Lycotec; Natalya E. Chalyk, Victor A. Klochkov, Victoria V. Blinova, Tatiana M. Bogdanova, Alexei A. Iljin, Larisa S. Sulkovskaya and are employees of the Institute of Cardiology in Saratov, Russian Federation; Elena Y. Morgunova, Nailya A. Zigangirova and Valeriy V. Tsibezov are employees of the Gamaleya Research Institute of Epidemiology and Microbiology in Moscow, Russian Federation. There have never been any financial relationships between Lycotec and these collaborating organisations.

In the present paper, we report that C. trachomatis can be efficiently propagated and affect mRNA expression for two major cytokines, relevant to tumor progression, in CWR-R1 cells, a malignant prostate cell line. CWR-R1 and McCoy cells, a classic cell line for chlamydial research, were grown and infected with C. trachomatis under similar conditions. Cell monolayers were harvested for RNA analysis and immunostaining with major outer membrane protein (MOMP) antibody at 24, 48, and 72 hours of the postinfection (hpi) period. It was shown that the infectious cycle of chlamydial pathogen in CWR-R1 cells resembles the progression of C. trachomatis infection in McCoy cells but with a few important differences. First of all, the initial stage of C. trachomatis propagation in CWR-R1 cells (24 hpi) was characterized by larger inclusion bodies and more intense, specific immunofluorescent staining of infected cells as compared with McCoy cells. Moreover, there was a corresponding increase in infective progeny formation in CWR-R1 cells along with mRNA for EUO, a crucial gene controlling the early phase of the chlamydial development cycle (24 hpi). These changes were more minimal and became statistically insignificant at a later time point in the infectious cycle (48 hpi). Altogether, these data suggest that the early phase of C. trachomatis infection in CWR-R1 cells is accompanied by more efficient propagation of the pathogen as compared with the growth of C. trachomatis in McCoy cells. Furthermore, propagation of C. trachomatis in CWR-R1 cells leads to enhanced transcription of interleukin-6 and fibroblast growth factor-2, genes encoding two important proinflammatory cytokines implicated in the molecular mechanisms of chemoresistance of prostate cancer and its ability to metastasize. The possible roles of reactive oxygen species and impaired mitochondrial oxidation in the prostate cancer cell line are discussed as factors promoting the early stages of C. trachomatis growth in CWR-R1 cells.

Oxidative stress and antioxidant deficiency play a pivotal role in initiation, development, and outcomes of cardiovascular disease. Pharmacokinetic parameters as well as the impact of highly bioavailable lycopene on cardiovascular variables, markers of inflammation and oxidation were investigated during a 30-day clinical trial in patients with coronary vascular disease. The patients were randomized into two major groups and were supplemented with a single 7 mg daily dose of lycopene ingested either in the form of lactolycopene (68 patients) or in the form of lycosome-formulated GA lycopene (74 patients). The endpoints included cardiovascular function parameters, serum lipids, and four markers of oxidative stress and inflammation. Ingestion of lycosome-formulated lycopene increased serum lycopene levels by 2.9- and 4.3-fold, respectively, after 2 and 4 weeks of the trial, whereas supplementation with lactolycopene upregulated serum lycopene by half-fold only after 4 weeks of ingestion. Lycosome formulation of lycopene resulted by the end of the trial in a threefold reduction in Chlamydia pneumoniae IgG and reduction to the same degree of the inflammatory oxidative damage marker. The decrease in oxidized LDL caused by lycosome-formulated lycopene was fivefold. Moreover, supplementation with lycosome-formulated lycopene was accompanied by a significant increase in tissue oxygenation and flow-mediated dilation by the end of the observational period. In contrast, lactolycopene did not cause any significant changes in the parameters studied. Therefore, enhanced bioavailability of lycopene promotes its antioxidant and anti-inflammatory functions and endorses a positive effect of lycopene on cardiovascular system.

Ingestion of a single dose of alcohol, ranging from the intake of a moderate amount alcohol to binge drinking, is the most frequent form of alcohol consumption with poorly understood medical consequences and obscure prophylactics. The study was aimed to determine whether lycosome formulated phosphatidylcholine (PC-Lyc) containing two highly bioavailable antioxidants (PC and lycopene) ingested shortly before the alcohol-containing beverage may alleviate the biochemical markers of liver damage and parameters of biological oxidation associated with the intake of a moderate amount of alcohol. Healthy middle-aged volunteers were requested to consume a moderate amount of alcohol - 0.5 ml/kg or 1.0 ml/kg shortly after ingestion of a capsule containing 450 mg of regular phosphatidylcholine (PC, n=10), PC-Lyc (n=10), or placebo pill (PP, n=10). Serum levels of ethanol (EtOH), acetaldehyde (AA), liver-specific enzymes, total antioxidant capacity of serum (TAC), oxidized LDL (LDL-Px), and malonic dialdehyde (MDA) were measured at 1, 2.5, and 5 hours after dosing with alcohol. Ingestion of PC regardless of the formulation used had no effect on serum EtOH concentration dynamics. However, volunteers supplemented with PC-Lyc showed a better clearance of AA in serum as compared to other groups. There was a reduction in serum TAC values by 18.5% and 16.1% in both placebo groups ingesting 0.5 and 1.0 ml/kg of alcohol, respectively, at the end of observational period. This decline was preventable by supplementation of volunteers with PC and especially with PC-Lyc. Moreover, PC-Lyc promoted a reduction of serum MDA and reversed an increase in serum LDL-Px. In addition, ingestion of alcohol at 1.0 ml/kg dose caused a transient increase in serum alanine-aminotransferase activity which was abolished by both formulations of PC. Therefore, combinatory lycosomal formulation of PC and lycopene may prevent some metabolic abnormalities associated with single intake of moderate amount of alcohol. This trial is registered with ACTRN12617001335381.

Context: Docosahexaenoic acid (DHA) is an omega-3 fatty acid essential for cardiovascular health, brain development, and reproductive function. Due to hydrophobicity and low DHA bioavailability, new microencapsulated DHA formulations are under development., Aim: This study aims to evaluate DHA pharmacokinetics (PKs) and biological oxidation parameters in volunteers ingesting a newly developed lutein-containing lycosomal formulation of DHA (LF-DHA)., Materials and Methods: A total of 32 healthy volunteers (40-65 years old) with signs of oxidative stress (OS) and subclinical hypoxia were orally supplemented for a month with 250 mg of regular DHA (1 st group) or a combination of lutein (7.0 mg) and zeaxanthin (1.4 mg) (2 nd group). The third group received regular DHA (250 mg) co-ingested with lutein/zeaxanthin (7.0/1.4 mg), whereas the 4 th group was given LF-DHA containing lutein/zeaxanthin (7.0/1.4 mg). PK, OS, and oxygenation parameters were analyzed., Results: LF-DHA improved the PKs of DHA enhancing its serum concentrations time dependently by 34.6% and 94.1% after 2 nd and 4 th weeks, respectively. DHA and lutein ingested either alone or simultaneously as two separate formulations reduced the levels of OS markers. However, LF-DHA inhibited the malonicdialdehyde (MDA) and oxidized low-density lipoprotein values were better than other formulations. LF-DHA also enhanced the plasma oxygen and tissue oxygen saturation. This effect was significantly higher than in other groups., Conclusion: LF-DHA eliminates the need in high-dose DHA supplementation protocols and confers a higher DHA bioavailability, thereby improving the parameters of biological oxidation and tissue respiration in affected individuals., Competing Interests: There are no conflicts of interest.

Circulating lycopene level is negatively associated with the prevalence of cardiovascular disease, cancers (prostate and breast), type 2 diabetes mellitus, and aging. Traditionally, lycopene is measured in biological specimens by a combination of high-performance liquid chromatography (HPLC) and mass spectrometry methods. Moreover, as we recently reported, tissue/cell lycopene depositions can be observed by the immunohistochemistry method with a newly developed monoclonal antibody (mAb) against lycopene. A main objective of this study is to evaluate the performance of a new noninvasive immunofluorescence (IF) lycopene quantification skin test with mAbs against lycopene versus HPLC lycopene assay of serum lycopene in volunteers subjected to lycopene supplementation which represents a novel approach to lycopene measurement methodology. For this purpose, 32 healthy volunteers, 30-40 years old, were supplemented with lycopene (n = 15) or placebo (n = 17) for a period of 4 weeks. It was found that lycopene supplementation leads to a significant increase in serum lycopene concentration after 2 and 4 weeks by 2.6- and 3.4-fold over control, respectively. This was accompanied by a concordant step-wise rise in IF staining of skin corneocytes and sebum, quantifiable by arbitrary IF scores. Placebo supplementation did not affect serum lycopene values or intensity of IF staining of the skin samples. There was 86.6% agreement in paired HPLC/IF variants for the intermediate time point and 80.0% agreement at the end of the study in the lycopene group. Intraclass correlation between paired values in this group was +0.49 for the 2-week time point and +0.63 for the end point. These results indicate that the new antibody-based skin assay can be used for rapid detection of lycopene deficiencies. Moreover, the noninvasive nature of the skin swab test would allow using it to monitor, optimize, and personalize lycopene supplementation protocol of risk groups in the general population.

Incubation of B10.MLM cells, a cell line of alveolar macrophages, with lycopene, a carotenoid, leads to an increase of lycopene content in their microsomal fraction. That increase was higher and developed faster when the cells were incubated with immune complexes formed by lycopene and mAb 6B9 (L-6B9 mAb), a monoclonal hapten-specific antibody raised against lycopene, as compared with dimethyl sulfoxide (DMSO)-dissolved lycopene (DMSO-L). Moreover, incubation of B10.MLM cells with L-6B9 mAb complexes was accompanied by more efficient accumulation of lipid droplets in the cultured cells and more significant inhibition of mRNA for 3-hydroxy-3-methylglutaryl-coenzyme (HMG-CoA) reductase, a rate-limiting enzyme of cholesterol biosynthesis known to be targeted by lycopene. Additionally, there was a better inhibition of Chlamydia trachomatis infection in B10.MLM cells infected with the pathogen and incubated thereafter with L-6B9 mAb complexes as compared with DMSO-L. Altogether, the results suggest that association with monoclonal antibody promotes intracellular delivery of lycopene in cultured cells possibly through Fc-receptor mediated uptake.

Twenty-nine newly diagnosed individuals with Nonalcoholic Fatty Liver Disease (NAFLD) remaining on habitual dietary regimen were supplemented with regular or lycosome formulations of phosphatidylcholine (PC) during a pilot, randomized, double-blinded clinical study. After two months of oral PC intake (450 mg daily) the liver size as well as serum levels of hepatic enzymes and markers of inflammation were evaluated by ultrasonography and biochemical analysis. It was shown that there was a statistically significant reduction of medians for the Mid-Clavicular liver size from 16.0 cm (95/5% CI: 17.1/15.5) to 15.1 cm (95/5% CI: 17.2/14.4, P = 0.021) in participants ingesting the lycosome-formulated PC (L-PC) whereas regular formulation of PC (R-PC) had only a marginal effect on this parameter ( P = 0.044). A similar tendency was observed in the Mid-Sternal liver size. Moreover, there was a reduction of medians for ALT values at the end point of the study ( P = 0.026) after ingestion of L-PC, while R-PC had no statistically significant effect. On the other hand, ingestion of both formulations was accompanied by reductions in values for Inflammatory Oxidative Damage (IOD) and oxidized LDL in serum. However, L-PC had superior activity in these terms, presumably due to the presence of lycopene, a powerful antioxidant, in the L-PC-Lycosome structure. C-reactive protein level was moderately decreased (reduction of medians from 6.5 [95/5% CI: 7.7/5.8] mg/L to 5.1 [95/5% CI: 5.6/4.3] mg/L) only after ingestion of L-PC. The greater efficacy of L-PC seen in NAFLD volunteers may reflect improved bioavailability of PC owing to better protection of the microencapsulated PC from gastrointestinal enzymes and possibly enhanced hepatic delivery of L-PC particles.

Objective: To determine if ingestion of lycosome-formulated dark chocolate (DC) containing astaxanthin (ASTX) improves bioavailability of ASTX and affects markers of hypoxia and oxidative stress in aging individuals., Design: Randomized, blinded, four-arm, prospective study., Settings: Lycotec Ltd, Cambridge, United Kingdom and Institute of Cardiology, Saratov, Russian Federation., Participants: 32 healthy individuals aged 60-70 years with confirmed signs of oxidative stress (increased serum levels of oxidized LDL and malonic dialdehyde) randomized into four study groups (8 volunteers each)., Intervention: Volunteers of first group were given orally 10 gr of dark chocolate (DC). Individuals from the second group received 7 mg of astaxanthin (ASTX). Third group of volunteers was supplemented with 10 gr of DC and 7 mg of ASTX ingested simultaneously as two separate formulations. Last group of the individuals was given 10 gr of a lycosomal formulation of DC containing 7 mg of co-crystalized ASTX (L-DC-ASTX), a newly developed highly bioavailable nutraceutical composition of DC containing 2 groups of antioxidants (cocoa flavanols and ASTX). All formulations were given orally, once daily for a month., Measurements: Serum ASTX was measured by high-performance liquid chromatography. Nitric oxide, malonic dialdehyde and oxidized LDL were quantified spectrophotometrically. Oxygenation parameters were evaluated by near-infrared spectroscopy., Results: One month ingestion of singular formulation of ASTX lead to a 20 fold buildup in serum ASTX level whereas the 4 week ingestion of L-DC-ASTX formulation was accompanied by more prominent accumulation of ASTX in serum (a 40 fold increase over the basal values) at the same daily dose of ASTX. Both antioxidants taken separately decreased serum levels of oxidized LDL and malonic dialdehyde. However effect of L-DC-ASTX formulation was more prominent. ASTX ingested alone caused a borderline increase (p=0.054) in serum nitric oxide (NO) levels, whereas DC ingestion lead to small but statistically significant increase in serum NO concentration. Higher values of NO level were seen after co-ingestion of DC and ASTX, especially in case of L-DC-ASTX formulation suggesting additive/synergistic effects of DC and ASTX on nitric oxide production. These changes were in agreement with the increase in plasma oxygen transport and tissue oxygen saturation seen in the volunteers supplemented with L-DC-ASTX formulation., Conclusion: The nutraceutical formulation of DC and ASTX with an enhanced bioavailability of ASTX can be efficiently used for the correction of oxidative status in aging individuals., Competing Interests: All authors declare no conflict of interest involved

Full cDNA and corresponding amino acid (AA) sequences of 6B9 monoclonal antibody (mAb) against lycopene was obtained using Step-Out RACE technology. Variable (V) and constant (C) regions were identified. The light chain of 6B9 contained 238 AA IgM with the highest level of identity (0.93) to both the anti-VEGF receptor antibody and anti-collagen type II FAb CIIC1. The heavy chain was composed of 634 AA with a high identity (0.9) to the Ig mu chain C region. Potential posttranslational modification regions in both chains were identified alongside with disulfide bond sites. The obtained information can be used for making chimeric constructs containing 6B9 mAb (or its fragments) and lycopene, a powerful carotenoid with antioxidant as well as antiproliferating properties, which can be implemented in the treatment of an aggressive form of prostate cancer and possibly other malignancies.

Dendrimers are unimolecular architectural nano- or microparticle entities that can accommodate various nutraceuticals and pharmaceuticals between their branches (dendrons) and provide targeted delivery of biomimetics into different tissues upon addition of functionalized groups to the dendrimer's surface. Covalent binding, hydrogen bonds, and electrostatic interactions between dendrimer-composing molecules are known to form and stabilize dendrimer structure. Carotenoids have recently been shown to form dendrimer-like structures and promote targeted delivery of "cargo" molecules into organs characterized by high-carotenoid uptake (adrenal glands, prostate, liver, and brain). The use of carotenoid dendrimers, in particular lycosome particles loaded with various xenobiotics (resveratrol, cocoa flavanols, and HMG-CoA reductase inhibitors), reportedly has a beneficial effect in diabetic foot syndrome, prehypertension, and cardiovascular disease. New applications for carotenoid dendrimers may arise from the use of complexes formed by carotenoid dendrimers and monoclonal antibodies (mAbs). The internalization of carotenoid dendrimer-mAb complexes through receptor-mediated mechanisms may prevent interactions of dendrimer-incorporated xenobiotics with membrane-associated P-glycoprotein, a major factor of drug resistance in tumor cells. The incorporation of mAb fragments with higher binding capacity to the membrane receptors and higher affinity to the target molecule may further increase the bioavailability of "cargo" molecules transported by the carotenoid dendrimer-mAb complexes and open new doors in nanodelivery technologies.

Dark chocolate (DC) was originally introduced in human nutrition as a medicinal product consumable in a liquid form. Century-long efforts of food industry transformed this hardly appealing product into a valuable modern culinary delight with clear predominance of confectionery brands of DC on the market. However, current epidemiological data as well as multiple experimental and clinical observations reveal that DC consumption may have a profound effect on cardiovascular, central nervous systems, hemostasis, and lipid metabolism. However, despite of growing body of modern scientific evidence revealing medicinal properties of cocoa-based products, DC remains more gourmet culinary item than medicinal food product. Even today there are no clear dietary recommendations on consumption of cocoa flavonoids (flavanols) for health purpose. Clinical trials with DC rarely include monitoring of plasma flavanol concentration in volunteers. Moreover, there is no standardized assay or any quantitative requirements for flavanol content in the commercial brands of DC. High flavanol content is often sacrificed during manufacturing for a better taste of DC due to bitterness of cocoa flavonoids. All these problems including subsequently arising ethical issues need to be addressed by joint efforts of food industry and medical science. Moreover, application of microencapsulation technology in DC manufacturing, as well as molecular selection of best flavanol producers may drastically change bioavailability of DC bioactive ingredients and DC production technology. Nevertheless, only strict causative approach, linking possible health effect of DC to its bioactive ingredients considered as nutraceuticals, may change the current landscape in nutritional research related to cocoa-based products and create a trustworthy path for their medicinal use.

A monoclonal antibody (Mab) against lycopene was developed from hybridoma clones obtained from BALB/c mice immunized with trans-isomer of lycopene (t-lycopene, t-LC) conjugated with colloidal gold particles. An alternating immunization schedule which included injection of both formulations of immunogen (without and with Freund's adjuvant) was most effective in the elucidation of a measurable immune response to the t-Lycopene conjugate. Selected hybridoma clones were able to produce an Mab positive in competition assay. In particular, preincubation of 6B9 Mabs with t-LC abolished the ability of 6B9 Mabs to bind LC in the competition assay. Mabs produced by other clones (4F10, 4A3, and 3B12) worked similarly. Analysis of antigen specificity showed that 6B9 Mab raised against t-LC did not recognize other carotenoids such as lutein and carotene. Mab 6B9 was shown to recognize lycopene on a glass surface and in the settings of indirect immunofluorescence experiments performed in cultured hepatocytes and alveolar macrophages incubated with and without lycopene, as well as in sebum and corneocyte specimens from the skin of volunteers supplemented with nutraceutical formulation of lycopene. Newly generated Mabs against lycopene may provide a valuable tool for different analytical assays of lycopene content in various biological, agricultural, and food products.

Resveratrol (RESV), an antifungal compound from grapes and other plants, has a distinct ability to inhibit the Chlamydia (C.) trachomatis developmental cycle in McCoy cells, a classic cell line used for chlamydial research. Inoculation of C. trachomatis with increasing amounts of RESV (from 12.5 to 100  μ M) gave a dose-dependent reduction in the number of infected McCoy cells visualized by using monoclonal antibodies against chlamydial lipopolysaccharide. A similar trend has been observed with immunoassay for major outer membrane protein (MOMP). Furthermore, there was a step-wise reduction in the number of C. trachomatis infective progenies caused by the increasing concentrations of RESV. The ability of RESV to arrest C. trachomatis growth in McCoy cells was confirmed by a nucleic acid amplification protocol which revealed dose-dependent changes in mRNAs for different genes of chlamydial developmental cycle ( euo , incA , and omcB ). Although the precise nature of the antichlamydial activity of RESV is yet to be determined and evaluated in future studies, the observed effect of RESV on C. trachomatis infection was not related to its potential effect on attachment/entry of the pathogen into eukaryotic cells or RESV toxicity to McCoy cells. Similar inhibitory effect was shown for C. pneumoniae and C. muridarum .

Manufacturing of dark chocolate and other cocoa-based products is a complex multistage process beginning with spontaneous cocoa bean fermentation driven in the postharvest period by different microorganisms derived from the environment. Cocobiota defined as the association of microbial species involved in cocoa bean fermentation may have considerable impact on the medicinal properties of cocoa products via various primary and secondary metabolites, whose presence in dark chocolate and other cocoa-derived products has to be taken into consideration when analyzing medicinal effects of cocoa. Metabolites of acetic acid and lactic acid bacteria, two major cocobiota members, are recently shown to have considerable antifungal and cholesterol-lowering activities and promote the formation of short chain fatty acids and mannitol, an important prebiotic capable of modifying gut microbiota. Penicillium citrinum, a major type of fungi identifiable in fermented cocoa beans, produces a thermostable alkaloid, Penicitrinine A, as well as lovastatin, compounds with antineoplastic and cholesterol-lowering abilities, respectively. Moreover, recent results suggest that bacterial and fungal metabolites produced by cocobiota have a significant anti-infective potential. Therefore, various metabolites produced by cocobiota can mimic some medicinal effects of dark chocolate and other cocoa-derived products previously attributed to cocoa flavonoids and methylxanthines and need to be thoroughly investigated in in vitro and in vivo systems.

Objective. The effect of a proprietary formulation of trans-resveratrol (t-RSV) on manifestations of diabetic foot syndrome (DFS) was studied in type 2 diabetic patients with newly diagnosed diabetic foot ulcers. Method. Placebo-controlled, examiner-blinded, parallel-group randomized controlled pilot clinical trial (ACTRN Clinical Trial Registry number 12610000629033) involving 24 patients with DFS (15 males and 9 females, average age of 56.4 ± 9.1 years) divided into the placebo and RSV-treatment groups was performed. 50 mg of t-RSV or placebo capsules was given to each patient twice a day over a 60-day time period. Results. Reduction in the parameters reflecting diabetic ulcer size was more profound in the RSV group as compared to placebo. RSV-treated patients also had a marginally improved performance in the foot pressure test. A statistically significant decline in the plasma fibrinogen level, but not CRP, was also found in the RSV-treated patients. Some improvement in the plasma lipid profile and fasting glucose levels were not related to RSV-treatment, since they have been seen on both the RSV and placebo groups, revealing the effectiveness of medical supervision and education in the newly diagnosed patients with DFS. Conclusion. t-RSV supplementation promotes reduction of the foot ulcer size and reduces plasma fibrinogen level in type 2 diabetic patients.

Inflammation in atherosclerosis, which could be associated with some subclinical infections such as C. pneumoniae, is one of the key factors responsible for the development of clinical complications of this disease. We report that a proprietary protein extract isolated from Roquefort cheese inhibits the propagation of C. pneumoniae in a human HL cell line in a dose-dependent manner, as revealed by the immunofluorescence analysis. These changes were accompanied by a significant reduction in the infective progeny formation over the protein extract range of 0.12-0.5 μg/mL. Moreover, short term feeding of mice with Roquefort cheese (twice, 10 mg per mouse with an interval of 24 hours) led to the inhibition of the migration of peritoneal leukocytes caused by intraperitoneal injection of E. coli lipopolysaccharide. These changes were complemented by a reduction in neutrophil count and a relative increase in peritoneal macrophages, suggesting that ingestion of Roquefort could promote regenerative processes at the site of inflammation. The ability of this protein to inhibit propagation of Chlamydia infection, as well as the anti-inflammatory and proregenerative effects of Roquefort itself, may contribute to the low prevalence of cardiovascular mortality in France where consumption of fungal fermented cheeses is the highest in the world.

Extragenital chlamydial complications may be associated with systemic spread of infection, but haematogenous route for C. trachomatis dissemination has not been clearly demonstrated. Here we report that serum specimens obtained from patients with chlamydiosis contain elementary bodies of C. trachomatis shown by culture and immunogold electron microscopy. We have found that 31 of the 52 patients had serum precipitates which were infective to McCoy cells. Immunostaining revealed very small inclusions resembling those reported during persistent C. trachomatis infection in vitro. DNA specimens from 49 (out of 52) patients with chlamydiosis gave positive PCR readings. The viability of the pathogen present in the sera was confirmed by chlamydial RNA detection in the cell monolayer inoculated by the serum precipitates. By using DNA isolation protocol from 1 mL of serum and quantitative TaqMan PCR, it was estimated that bacterial load in patients' sera was 2 × 10(2)-10(3) GE/mL. These findings for the first time demonstrated that C. trachomatis can be disseminated directly by the plasma, independently from blood cell, which may represent a new possible pathway of the chronic infection development. Therefore, new methodological approaches for detection of C. trachomatis in the serum of patients with complicated and chronic chlamydiosis could be important in the diagnosis of the infection regardless of its anatomical localization.

Monoclonal antibody (MAb) against trans-resveratrol (t-RSV) was obtained from hybridoma clones constructed from splenocytes of BALB/c mice immunized with carrier proteins (bovine serum albumin [BSA] and ovalbumin [OVA]) coupled with synthetic hapten mimicking t-RSV structure. The t-RSV-BSA derivate was more efficient at induction of the immune response than t-RSV-OVA. However, the use of t-RSV-OVA was advantageous during selection of hybridoma clones constructed from splenocytes of t-RSV-BSA-immunized mice. Pre-incubation of immune serum with free t-RSV inhibited the binding of antibody to t-RSV-BSA conjugate, suggesting the specific nature of antibody binding to t-RSV. Splenocytes obtained from the mouse immunized with t-RSV-BSA were used for the hybridoma construction. Expansion of the primary clones, their subsequent screening, and subcloning narrowed our search to allowed isolation of two IgG1a-producing hybridomas designated as 2H9 and 1B1. According to an indirect ELISA assay, the resulting MAb 2H9 had little or no cross-reactivity to cis-RSV. No recognition of trans-RSV-3-O-glucuronide and trans-RSV-3-sulfate was detected. The newly generated MAb against t-RSV may provide a highly valuable and cost-effective tool for the analytical assay for t-RSV in different biological and agricultural specimens.

The low rates of cardiovascular mortality which have existed in France for decades despite high saturated fat consumption constitute an epidemiological phenomenon called the "French paradox". This phenomenon was originally attributed to consumption of red wine and its major constituent resveratrol. However, recent studies have revealed the limitations of this link outside France. These observations indicate that consumption of red wine alone cannot explain the paradox and perhaps some other constituents of the typical French diet could be responsible for reduced cardiovascular mortality. We hypothesize that cheese consumption, especially of molded varieties, may contribute to the occurrence of the "French paradox". This assumption is well supported by newly discovered facts revealing the positive effect of cheese ingestion on lipoprotein turnover and plasma lipid profile, haemorheological parameters and inflammatory status. Recent advances in cheese proteomics have allowed the identification and isolation of novel peptides capable of inhibiting the angiotensin-converting enzyme which controls systemic blood pressure. A complex time-dependent enzymatic transformation of the cheese core controlled by probiota, temperature and humidity during the ripening process has been shown to result in the formation of substances reducing major pro-inflammatory markers and cytokines (C-reactive protein, interleukin 6, tumor necrosis factor alpha). Molded cheeses, including Roquefort, may be even more favorable to cardiovascular health due to the presence of secondary metabolites produced by Penicillium roqueforti and other fungi. Among them are andrastins A-D and roquefortine, whose ability to inhibit cholesterol biosynthesis and bacterial growth may be a key mechanism in the prevention of cardiovascular disease., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Diabetic foot syndrome (DFS) is a late-stage complication of type 2 diabetes which originates from interplay among impaired tissue regeneration, vasculopathy, neuropathy and inflammation all on the background of insulin resistance. Despite astonishing mortality rate pharmacological approach in management of diabetic ulceration is almost non-existent. Foot pressure relief, wound debridement and infection control remain widely accepted options in the treatment of DFS. We hypothesize that resveratrol treatment and subsequent activation of SIRT1 pathway might be highly beneficial for patients with DFS. This prediction is based on multiple lines of evidence implicating resveratrol and sirtuins in restoration of insulin sensitivity, microcirculation, tissue regeneration, function of peripheral nerves and production of cytokines. Stabilized "nutraceutical" formulations of resveratrol with high absorption rate are essential to examine its potential medical benefits since dietary polyphenols are known to be rapidly metabolized by gut microflora and oxidized during absorption. Clinical trials with nutraceutical formulations and placebo are required to understand if resveratrol indeed holds the promise for treatment of DFS., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Splenocytes obtained from mice immunized with whole purified elementary bodies of Chlamydia trachomatis were used for hybridoma construction. The resulting clones were screened with ELISA using chlamydial lipopolysaccharide (LPS) and full-length human apolipoprotein B (ApoB). One analyzed clone producing IgG1 (MAb 7B5) showed simultaneous recognition of chlamydial LPS and human ApoB, suggesting the presence of common antigenic epitopes in their structures. MAb 7B5 exhibited agreeable activity in immunoblot analysis conducted using chlamydial extracts or full-length human ApoB as well as in immunofluorescence (IF) detecting typical inclusion bodies of C. trachomatis and C. pneumoniae in the infected eukaryotic host cells. The removal of LPS from chlamydial suspensions with lauroyl sarcosyl led to a complete disappearance of IF associated with the elementary bodies of C. trachomatis. Therefore, immunologic response to chlamydial antigen may be associated with the generation of ApoB-specific antibody. Molecular mimicry and subsequent formation of cross-reactive antibodies might be an essential mechanism explaining the appearance of circulating auto-antibodies against low density lipoproteins (LDL) in patients with atherosclerosis. Moreover, newly generated MAb 7B5 can be a useful tool in the laboratory diagnosis of chlamydial infections.

Background: Limited body of evidence suggests that lipopolysaccharide of C. pneumoniae as well as C. pneumoniae-specific immune complexes can be detected and isolated from human serum. The aim of this study was to investigate the presence of viable elementary bodies of C.pneumoniae in serum samples of patients with acute coronary syndrome and healthy volunteers., Material and Methods: Serum specimens from 26 healthy volunteers and 56 patients with acute coronary syndrome were examined subsequently by serological (C.pneumoniae-specific IgA and IgG), PCR-based and bacteriological methods. Conventional, nested and TaqMan PCR were used to detect C.pneumoniae genetic markers (ompA and 16S rRNA) in DNA from serum specimens extracted with different methods. An alternative protocol which included culturing high-speed serum sediments in HL cells and further C.pneumoniae growth evaluation with immunofluorescence analysis and TaqMan PCR was established. Pellet fraction of PCR-positive serum specimens was also examined by immunoelectron microscopy., Results: Best efficiency of final PCR product recovery from serum specimens has been shown with specific C. pneumoniae primers using phenol-chloroform DNA extraction protocol. TaqMan PCR analysis revealed that human serum of patients with acute coronary syndrome may contain genetic markers of C. pneumoniae with bacterial load range from 200 to 2000 copies/ml serum. However, reliability and reproducibility of TaqMan PCR were poor for serum specimens with low bacterial copy number (<200 /ml). Combination of bacteriological, immunofluorescence and PCR- based protocols applied for the evaluating HL cells infected with serum sediments revealed that 21.0 % of the patients with acute coronary syndrome have viable forms C.pneumoniae in serum. The detection rate of C.pneumoniae in healthy volunteers was much lower (7.7%). Immunological profile of the patients did not match accurately C.pneumoniae detection rate in serum specimens. Elementary bodies of C.pneumoniae with typical ultrastructural characteristics were also identified in serum sediments using immunoelectron microscopy., Conclusions: Viable forms C. pneumoniae with typical electron microscopic structure can be identified and isolated from serum specimens of the patients with acute coronary syndrome and some healthy volunteers. Increased detection rate of C. pneumoniae in serum among the patients with an acute coronary syndrome may contribute towards enhanced pro-inflammatory status in cardiovascular patients and development of secondary complications of atherosclerosis.

Aim: To evaluate the direct binding of two main chlamydial biovars (C. trachomatis and C. pneumoniae) to plasma lipoproteins and its effect on chlamydial infection rate in human hepatoma cell line (HepG2 cells)., Methods: Murine plasma lipoproteins were fractionated and isolated using fast-performance liquid chromatography (FPLC), spotted on nitrocellulose membrane and incubated with chlamydial suspensions. Direct binding of chlamydial particles to lipoprotein fractions has been studied using lipopolysaccharide-specific antibodies in immuno-dot blot binding assay and immunoprecipitation analysis. Immunostaining protocol as well as flow cytometry analysis have been employed to study the infectivity rate of chlamydial species in HepG2 cells., Results: Elementary bodies of both C. trachomatis and C. pneumoniae bind ApoB-containing fractions of plasma lipoproteins. That binding becomes stronger when heat-denatured FPLC fractions are used, suggesting a primary role of apolipoproteins in interaction between chlamydial particle and lipoprotein. Both chlamydial biovars efficiently propagate in human hepatoma cell line - HepG2 cells even in serum free conditions forming late-stage inclusion bodies and releasing extracellular elementary bodies. Preincubation of C. trachomatis and C. pneumoniae with native ApoB-containing lipoproteins enhances the rate of chlamydial infection in HepG2 cells., Conclusion: A productive infection caused by C. trachomatis and C. pneumoniae may take place in human-derived hepatocytes revealing hepatic cells as possible target in chlamydial infection. Obtained results may suggest the participation of lipoprotein receptors in the mechanism of attachment and/or entry of chlamydial particles into target cells.

Background: Perihepatitis is rare but consistently occurring extragenital manifestation of untreated Chlamydia trachomatis infection. Despite of possible liver involvement in generalized C. trachomatis infection, the ability of the pathogen to propagate in the hepatic cells and its impact on liver functions is not thoroughly investigated. The effect of mevastatin, an inhibitor of 3-hydroxy-3-methylglutaryl CoA reductase, on C. trachomatis growth in human hepatoma cell line HepG2 has been studied. Bacterial growth was assessed by immunostaining with FITC-labeled monoclonal antibody against chlamydial lipopolysaccharide and by RT-PCR for two chlamydial genetic markers (16S rRNA and euo)., Results: Chlamydial inclusion bodies were seen in approximately 50% of hepatocytes at 48 hours in the post infection period. Lysates obtained from infected hepatocytes were positive in the infective progeny test at 48 and especially in 72 hours after infection initiation. It has been shown that chlamydial infection in hepatocytes also leads to the decline of LDL-receptor mRNA which reflects infection multiplicity rate. Additions of mevastatin (1, 20 and 40 microM) 1 hour before inoculation restored and upregulated LDL-receptor mRNA level in a dose-dependent manner. Mevastatin treatment had no effect on internalization of chlamydial particles. However it reduced drastically the number of chlamydial 16S rRNA and euo transcripts as well as overall infection rate in HepG-2 cells. Complete eradication of infection has been seen by immunofluorescent staining at 40 microM mevastatin concentration, when expression level of chlamydial 16S rRNA and euo was undetectable. Lower concentration of mevastatin (20 microM) promoted euo expression level and the appearance of atypically small chlamydial inclusions, while there was a noticeable reduction in the number of infected cells and 16S rRNA transcripts., Conclusions: C. trachomatis can efficiently propagate in hepatocytes affecting transcription rate of some liver-specific genes. Ongoing cholesterol synthesis is essential for chlamydial growth in hepatocytes. Inhibitors of cholesterol biosynthesis can supplement conventional strategy in the management of C. trachomatis infection.

Sterol regulatory element-binding proteins (SREBPs) enhance transcription of genes encoding all of the proteins required for the cellular synthesis and uptake of cholesterol and unsaturated fatty acids. Here, we use suppression subtractive hybridization to identify a previously unrecognized SREBP-enhanced gene in mice. The gene encodes a membrane-bound enzyme that we designate SCALD, for short-chain aldehyde reductase. We expressed SCALD in bacteria, purified it extensively, and studied its catalytic properties in detergent solution. The enzyme specifically uses NADPH to reduce a variety of short-chain aldehydes, including nonanal and 4-hydroxy-2-nonenal. The enzyme also reduces retinaldehydes, showing equal activity for all-trans-retinal and 9-cis-retinal. Northern blot analysis indicates that SCALD is expressed most abundantly in mouse liver and testis. In the liver of mice, SCALD is suppressed by fasting and induced by refeeding, consistent with regulation by SREBPs. In testis, SCALD expression is restricted to pachytene spermatocytes, as revealed by visualization of mRNA and protein. SCALD is also expressed in four layers of the retina, including the outer segment of rods and cones, as revealed by immunohistochemistry. SCALD appears to be the mouse ortholog of the human protein that has been designated variously as prostate short-chain dehydrogenase/reductase 1, retinal reductase 1, and retinol dehydrogenase 11. In view of its ability to reduce short-chain aldehydes in addition to retinals, we propose that SCALD may be induced by SREBP in liver and other tissues to prevent toxicity from fatty aldehydes that are generated from oxidation of unsaturated fatty acids that are synthesized as a result of SREBP activity.