Lycopene presence in facial skin corneocytes and sebum and its association with circulating lycopene isomer profile: Effects of age and dietary supplementation.

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.

Non-Invasive Immunofluorescence Assessment of Lycopene Supplementation Status in Skin Smears.

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.

Association with Monoclonal Antibody Promotes Intracellular Delivery of Lycopene.

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.

Structural Organization of 6B9 Molecule, a Monoclonal Antibody Against Lycopene.

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.

Generation and Application of Monoclonal Antibody Against Lycopene.

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.

Generation of monoclonal antibody against trans-resveratrol.

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.

Monoclonal antibodies against lipopolysaccharide of Chlamydia trachomatis with cross reactivity to human ApoB.

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.