Effects of serotype and species dependency of bacterial lipopolysaccharides in human melanocytes from lightly and darkly-pigmented skin
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PROVIDER: S-EPMC10074968 | biostudies-literature | 2022 Jan
REPOSITORIES: biostudies-literature
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Project description:Benolea® (EFLA®943) is a standardized dry olive leaf extract (DOLE) considered safe for food consumption and has demonstrated superior pharmaceutical benefits such as antioxidant, anti-obesity, and anti-hypertensive activities. However, there is no study on its effects on melanogenesis yet. Disruption in the sequence of steps in melanogenesis can lead to hypopigmentary disorders which occur due to reduced production or export of pigment melanin in the skin. There is a need for safe and nontoxic therapeutics for the treatment of hypopigmentation disorders. Herein, we studied the effects of DOLE over a concentration range of 10-200 µg/mL on melanin synthesis and melanin secretion in B16F10 mouse melanoma cells and MNT-1 human melanoma cells and validated our results in primary human melanocytes (obtained from lightly pigmented (LP) and moderately pigmented (MP) cells) as well as their cocultures with keratinocytes. The capacity of melanocytes to export melanosomes was also estimated indirectly by the quantitation of melanocyte dendrite lengths and numbers. Our results show that DOLE significantly enhanced levels of extracellular melanin in the absence of effects on intracellular melanin, demonstrating that this plant extract's pro-melanogenic activity is primarily based on its capacity to augment melanin secretion and stimulate melanocyte dendricity. In summary, our preliminary results demonstrate that DOLE may hold promise as a pro-pigmenting agent for vitiligo therapy and gray hair treatment by its exclusive and novel mechanism of functioning as a dendrite elongator. Further studies to elucidate the mechanisms of action of the pro-melanogenic activity and effects of DOLE on melanosome export as well as the last steps of melanogenesis are warranted.
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Project description:Immortalized, amelanotic melanocytes isolted from skin of Balb/c express enzymatically-inactive tyrosinase due to a homozygous point mutation (TGT->TCT) in tyrosinase gene, resulting in a lack of melanin . To serve as a control cell line, pigmentation was restored in these cells by correcting the point mutation using an RNA-DNA oligonucleotide (kingly gift from Dr. Alexeev Y. Vitali). We used microarray to detail the effect of tyrosinase mutation on gene expression in normal versus mutant melanocytes.
2013-09-21 | GSE51073 | GEO
Project description:Immortalized, amelanotic melanocytes isolted from skin of Balb/c express enzymatically-inactive tyrosinase due to a homozygous point mutation (TGT->TCT) in tyrosinase gene, resulting in a lack of melanin . To serve as a control cell line, pigmentation was restored in these cells by correcting the point mutation using an RNA-DNA oligonucleotide (kingly gift from Dr. Alexeev Y. Vitali). We used microarray to detail the effect of tyrosinase mutation on gene expression in normal versus mutant melanocytes. Pigmented and non-pigmented melanocytes were grown for RNA extraction and hybridization on Affymetrix microarrays. We used both normal and mutant melanocyte in order to obtain expression profiles. We then examined variances in gene expression between the two cell lines.
2013-09-21 | E-GEOD-51073 | biostudies-arrayexpress
Project description:Platelets are anucleate blood cells with reported roles in hemostasis and immune responses, which possess a functional receptor for bacterial lipopolysaccharides (LPSs), the well-known inducers of inflammation. However, LPSs effects on platelets are contradictory. Here we aim to investigate mechanisms of platelet functioning in the presence of LPS and to find the cause of the discrepancy in the previously published data. Cell activity was analyzed by flow cytometry, western blotting, and aggregometry. Thrombus growth was assessed by fluorescent microscopy. LPS' activity was checked by their capability to induce PMN activation. However, LPSs did not substantially affect either thrombus growth in flow chambers, irreversible platelet aggregation, or platelet responses to strong activation. Platelet aggregation in response to 1 μM of ADP was significantly inhibited by LPSs. Flow cytometry analysis revealed that platelet activation responses to weak stimulation were also diminished by LPSs, while VASP phosphorylation was weakly increased. Additionally, LPSs were capable of inhibition of ADP-induced P2-receptor desensitization. Incubation of platelets with a pan-PDE inhibitor IBMX significantly enhanced the LPSs-induced platelet inhibition, implying cAMP/cGMP dependent mechanism. The discrepancy in the previously published data could be explained by LPS-induced weak inhibition of platelet activation and the prevention of platelet desensitization.
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Project description:Platelets are small circulating blood cells that play essential roles in the maintenance of haemostasis via blood clotting. However, they also play critical roles in the regulation of innate immune responses. Inflammatory receptors, specifically Toll-like receptor (TLR)-4, have been reported to modify platelet reactivity. A plethora of studies have reported controversial functions of TLR4 in the modulation of platelet function using various chemotypes and preparations of its ligand, lipopolysaccharide (LPS). The method of preparation of LPS may explain these discrepancies however this is not fully understood. Hence, to determine the impact of LPS on platelet activation, we used ultrapure preparations of LPS from Escherichia coli (LPSEC), Salmonella minnesota (LPSSM), and Rhodobacter sphaeroides (LPSRS) and examined their actions under diverse experimental conditions in human platelets. LPSEC did not affect platelet activation markers such as inside-out signalling to integrin αIIbβ3 or P-selectin exposure upon agonist-induced activation in platelet-rich plasma or whole blood whereas LPSSM and LPSRS inhibited platelet activation under specific conditions at supraphysiological concentrations. Overall, our data demonstrate that platelet activation is not largely influenced by any of the ultrapure LPS chemotypes used in this study on their own except under certain conditions.
| S-EPMC6890654 | biostudies-literature
Project description:The study of melanocyte biology is important to understand their role in health and disease. However, current methods of gene transfer into melanocytes are limited by safety or efficacy. Recombinant adeno-associated virus (rAAV) has been extensively investigated as a gene therapy vector, is safe and is associated with persistent transgene expression without genome integration. There are twelve serotypes and many capsid variants of rAAV. However, a comparative study to determine which rAAV is most efficient at transducing primary human melanocytes has not been conducted. We therefore sought to determine the optimum rAAV variant for use in the in vitro transduction of primary human melanocytes, which could also be informative to future in vivo studies. We have screened eight variants of rAAV for their ability to transduce primary human melanocytes and identified rAAV6 as the optimal serotype, transducing 7-78% of cells. No increase in transduction was seen with rAAV6 tyrosine capsid mutants. The number of cells expressing the transgene peaked at 6-12 days post-infection, and transduced cells were still detectable at day 28. Therefore rAAV6 should be considered as a non-integrating vector for the transduction of primary human melanocytes.
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Project description:Currently, Klebsiella pneumoniae is a pathogen of clinical relevance due to its plastic ability of acquiring resistance genes to multiple antibiotics. During K. pneumoniae infections, lipopolysaccharides (LPS) play an ambiguous role as they both activate immune responses but can also play a role in immune evasion. The LPS O2a and LPS O2afg serotypes are prevalent in most multidrug resistant K. pneumoniae strains. Thus, we sought to understand if those two particular LPS serotypes were involved in a mechanism of immune evasion. We have extracted LPS (serotypes O1, O2a and O2afg) from K. pneumoniae strains and, using human monocytes ex vivo, we assessed the ability of those LPS antigens to induce the production of pro-inflammatory cytokines and chemokines. We observed that, when human monocytes are incubated with LPS serotypes O1, O2a or O2afg strains, O2afg and, to a lesser extent, O2a but not O1 failed to elicit the production of pro-inflammatory cytokines and chemokines, which suggests a role in immune evasion. Our preliminary data also shows that nuclear translocation of NF-κB, a process which regulates an immune response against infections, occurs in monocytes incubated with LPS O1 and, to a smaller extent, with LPS O2a, but not with the LPS serotype O2afg. Our results indicate that multidrug resistant K. pneumoniae expressing LPS O2afg serotypes avoid an initial inflammatory immune response and, consequently, are able to systematically spread inside the host unharmed, which results in the several pathologies associated with this bacterium.
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Project description:Vertebrates, including amphibians, host diverse symbiotic microbes that contribute to host disease resistance. Globally, and especially in montane tropical systems, many amphibian species are threatened by a chytrid fungus, Batrachochytrium dendrobatidis (Bd), that causes a lethal skin disease. Bd therefore may be a strong selective agent on the diversity and function of the microbial communities inhabiting amphibian skin. In Panamá, amphibian population declines and the spread of Bd have been tracked. In 2012, we completed a field survey in Panamá to examine frog skin microbiota in the context of Bd infection. We focused on three frog species and collected two skin swabs per frog from a total of 136 frogs across four sites that varied from west to east in the time since Bd arrival. One swab was used to assess bacterial community structure using 16S rRNA amplicon sequencing and to determine Bd infection status, and one was used to assess metabolite diversity, as the bacterial production of anti-fungal metabolites is an important disease resistance function. The skin microbiota of the three Panamanian frog species differed in OTU (operational taxonomic unit, ~bacterial species) community composition and metabolite profiles, although the pattern was less strong for the metabolites. Comparisons between frog skin bacterial communities from Panamá and the US suggest broad similarities at the phylum level, but key differences at lower taxonomic levels. In our field survey in Panamá, across all four sites, only 35 individuals (~26%) were Bd infected. There was no clustering of OTUs or metabolite profiles based on Bd infection status and no clear pattern of west-east changes in OTUs or metabolite profiles across the four sites. Overall, our field survey data suggest that different bacterial communities might be producing broadly similar sets of metabolites across frog hosts and sites. Community structure and function may not be as tightly coupled in these skin symbiont microbial systems as it is in many macro-systems.
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Project description:Humans with darkly pigmented skin display superior permeability barrier function in comparison with humans with lightly pigmented skin. The reduced pH of the stratum corneum (SC) of darkly pigmented skin could account for enhanced function, because acidifying lightly pigmented human SC resets barrier function to darkly pigmented levels. In SKH1 (nonpigmented) versus SKH2/J (pigmented) hairless mice, we evaluated how a pigment-dependent reduction in pH could influence epidermal barrier function. Permeability barrier homeostasis is enhanced in SKH2/J versus SKH1 mice, correlating with a reduced pH in the lower SC that colocalizes with the extrusion of melanin granules. Darkly pigmented human epidermis also shows substantial melanin extrusion in the outer epidermis. Both acute barrier disruption and topical basic pH challenges accelerate reacidification of SKH2/J (but not SKH1) SC, while inducing melanin extrusion. SKH2/J mice also display enhanced expression of the SC acidifying enzyme, secretory phospholipase A2f (sPLA2f). Enhanced barrier function of SKH2/J mice could be attributed to enhanced activity of two acidic pH-dependent, ceramide-generating enzymes, β-glucocerebrosidase and acidic sphingomyelinase, leading to accelerated maturation of SC lamellar bilayers. Finally, organotypic cultures of darkly pigmented human keratinocytes display enhanced barrier function in comparison with lightly pigmented cultures. Together, these results suggest that the superior barrier function of pigmented epidermis can be largely attributed to the pH-lowering impact of melanin persistence/extrusion and enhanced sPLA2f expression.
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Project description:Urinary tract infections (UTI) affect a large proportion of the population, causing among other symptoms, more frequent and urgent micturition. Previous studies reported that the gram-negative bacterial wall component lipopolysaccharides (LPS) trigger acute epithelial and bladder voiding responses, but the underlying mechanisms remain unknown. The cation channel TRPV4 is implicated in the regulation of the bladder voiding. Since TRPV4 is activated by LPS in airway epithelial cells, we sought to determine whether this channel plays a role in LPS-induced responses in urothelial cells (UCs). We found that human-derived UCs display a fast increase in intracellular Ca2+ concentration upon acute application of Escherichia coli LPS. Such responses were detected also in freshly isolated mouse UCs, and found to be dependent on TRPV4, but not to require the canonical TLR4 signaling pathway of LPS detection. Confocal microscopy experiments revealed that TRPV4 is dispensable for LPS-induced nuclear translocation of NF-κB in mouse UCs. On the other hand, quantitative RT PCR determinations showed an enhanced LPS-induced production of proinflammatory cytokines in TRPV4-deficient UCs. Cystometry experiments in anesthetized wild type mice revealed that acute intravesical instillation of LPS rapidly increases voiding frequency. This effect was not observed in TRPV4-deficient animals, but was largely preserved in Tlr4 KO and Trpa1 KO mice. Our results suggest that activation of TRPV4 by LPS in UCs regulates the proinflammatory response and contributes to LPS-induced increase in voiding frequency. These findings further support the concept that TRP channels are sensors of LPS, mediating fast innate immunity mechanisms against gram-negative bacteria.
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