Comparative study of doxycycline, sancycline, and 4-dedimethylamino sancycline (CMT-3) on epidermal melanogenesis.

Melanogenesis is regulated by melanocytes, which synthesize the pigment melanin inside melanosomes; these melanosomes are exported through dendritic extensions to adjacent keratinocytes and result in skin coloration. Chemically modified tetracyclines (CMTs) are nonantimicrobial tetracyclines that retain the capacity to inhibit matrix metalloproteinases (MMPs) and have shown several biological benefits; in particular, CMT-3 [(4-dedimethylamino sancycline (SAN)] has emerged as a candidate for therapeutic benefits in our previous studies. However, to date, studies of the effects of CMT-3 or SAN on melanogenesis are lacking. We have previously reported the anti-melanogenic activity of CMT-308 (the 9-amino derivative of CMT-3). Herein, we have compared the three tetracycline analogs, doxycycline (DOX), SAN, and CMT-3, for their effects on melanogenesis using B16F10 mouse melanoma cells and have validated results in primary human melanocytes (HEMn-DP). DOX did not show any significant effects on intracellular melanin or melanosome export in DP cells while SAN was cytotoxic at high doses but without effects on melanogenesis at lower doses. However, CMT-3 showed a robust suppression of dendricity parameters (dendrite number, dendrite length, and proportion of dendritic cells) in DP cells which was associated, at least in part, with a significant reduction of intracellular tyrosinase activity. In spite of its inhibition of tyrosinase activity, CMT-3 had no significant effects on intracellular melanin levels, suggesting that it selectively targets melanosome export. Our results demonstrate a unique structure-activity relationship (SAR) for the effects of these compounds on melanogenesis and support the conclusion that removal of the 4-dimethylamino moiety confers the selective capacity to suppress melanosome export. Collectively, these results indicate that CMT-3 might be a candidate for diminishing hyperpigmentation skin disorders.

Effects of E-Cigarette Refill Liquid Flavorings with and without Nicotine on Human Retinal Pigment Epithelial Cells: A Preliminary Study.

Smoking is an etiologic factor for age-related macular degeneration (AMD). Although cigarette smoke has been extensively researched for retinal pigment epithelial (RPE) cell degeneration, the potential for adverse effects on the retinal epithelium following exposure to flavored e-cigarette refill liquid has never been explored. In this preliminary study, we have examined the effects of 20 e-liquids (10 different flavored nicotine-free and 10 nicotine-rich e-liquids) used in e-cigarettes on the metabolic activity, membrane integrity, and mitochondrial membrane potential of RPE cells. Our results showed that of the flavors studied over the concentration range: 0.5, 1, and 2% v/v for a duration of 48 h, cinnamon was the most toxic and menthol was the second most toxic, while other flavors showed lesser or no cytotoxicity. The presence of nicotine augmented cytotoxicity for cinnamon, menthol, strawberry, vanilla, and banana while for other flavors there was no synergism. Together, our results demonstrate that exposure of RPE to flavored e-cigarette refill liquids caused significant cytotoxicity and may be a risk factor for the development of retinal pathogenesis, although further in-depth studies are necessary.

Novel Chemically Modified Curcumin (CMC) Analogs Exhibit Anti-Melanogenic Activity in Primary Human Melanocytes.

Hyperpigmentation is a dermatological condition characterized by the overaccumulation and/or oversecretion of melanin pigment. The efficacy of curcumin as an anti-melanogenic therapeutic has been recognized, but the poor stability and solubility that have limited its use have inspired the synthesis of novel curcumin analogs. We have previously reported on comparisons of the anti-melanogenic activity of four novel chemically modified curcumin (CMC) analogs, CMC2.14, CMC2.5, CMC2.23 and CMC2.24, with that of parent curcumin (PC), using a B16F10 mouse melanoma cell model, and we have investigated mechanisms of inhibition. In the current study, we have extended our findings using normal human melanocytes from a darkly pigmented donor (HEMn-DP) and we have begun to study aspects of melanosome export to human keratinocytes. Our results showed that all the CMCs downregulated the protein levels of melanogenic paracrine mediators, endothelin-1 (ET-1) and adrenomedullin (ADM) in HaCaT cells and suppressed the phagocytosis of FluoSphere beads that are considered to be melanosome mimics. All the three CMCs were similarly potent (except CMC2.14, which was highly cytotoxic) in inhibiting melanin production; furthermore, they suppressed dendricity in HEMn-DP cells. CMC2.24 and CMC2.23 robustly suppressed cellular tyrosinase activity but did not alter tyrosinase protein levels, while CMC2.5 did not suppress tyrosinase activity but significantly downregulated tyrosinase protein levels, indicative of a distinctive mode of action for the two structurally related CMCs. Moreover, HEMn-DP cells treated with CMC2.24 or CMC2.23 partially recovered their suppressed tyrosinase activity after cessation of the treatment. All the three CMCs were nontoxic to human dermal fibroblasts while PC was highly cytotoxic. Our results provide a proof-of-principle for the novel use of the CMCs for skin depigmentation, since at low concentrations, ranging from 5 to 25 µM, the CMCs (CMC2.24, CMC2.23 and CMC2.5) were more potent anti-melanogenic agents than PC and tetrahydrocurcumin (THC), both of which were ineffective at melanogenesis at similar doses, as tested in HEMn-DP cells (with PC being highly toxic in dermal fibroblasts and keratinocytes). Further studies to evaluate the efficacy of CMCs in human skin tissue and in vivo studies are warranted.

Novel Chemically Modified Curcumin (CMC) Derivatives Inhibit Tyrosinase Activity and Melanin Synthesis in B16F10 Mouse Melanoma Cells.

Skin hyperpigmentation disorders arise due to excessive production of the macromolecular pigment melanin catalyzed by the enzyme tyrosinase. Recently, the therapeutic use of curcumin for inhibiting tyrosinase activity and production of melanin have been recognized, but poor stability and solubility have limited its use, which has inspired synthesis of curcumin analogs. Here, we investigated four novel chemically modified curcumin (CMC) derivatives (CMC2.14, CMC2.5, CMC2.23 and CMC2.24) and compared them to the parent compound curcumin (PC) for inhibition of in vitro tyrosinase activity using two substrates for monophenolase and diphenolase activities of the enzyme and for diminution of cellular melanogenesis. Enzyme kinetics were analyzed using Lineweaver-Burk and Dixon plots and nonlinear curve-fitting to determine the mechanism for tyrosinase inhibition. Copper chelating activity, using pyrocatechol violet dye indicator assay, and antioxidant activity, using a DPPH radical scavenging assay, were also conducted. Next, the capacity of these derivatives to inhibit tyrosinase-catalyzed melanogenesis was studied in B16F10 mouse melanoma cells and the mechanisms of inhibition were elucidated. Inhibition mechanisms were studied by measuring intracellular tyrosinase activity, cell-free and intracellular α-glucosidase enzyme activity, and effects on MITF protein level and cAMP maturation factor. Our results showed that CMC2.24 showed the greatest efficacy as a tyrosinase inhibitor of all the CMCs and was better than PC as well as a popular tyrosinase inhibitor-kojic acid. Both CMC2.24 and CMC2.23 inhibited tyrosinase enzyme activity by a mixed mode of inhibition with a predominant competitive mode. In addition, CMC2.24 as well as CMC2.23 showed a comparable robust efficacy in inhibiting melanogenesis in cultured melanocytes. Furthermore, after removal of CMC2.24 or CMC2.23 from the medium, we could demonstrate a partial recovery of the suppressed intracellular tyrosinase activity in the melanocytes. Our results provide a proof-of-principle for the novel use of the CMCs that shows them to be far superior to the parent compound, curcumin, for skin depigmentation.

A Novel Pro-Melanogenic Effect of Standardized Dry Olive Leaf Extract on Primary Human Melanocytes from Lightly Pigmented and Moderately Pigmented Skin.

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.

Depigmenting effect of Xanthohumol from hop extract in MNT-1 human melanoma cells and normal human melanocytes.

Xanthohumol (XH) is the most abundant prenylated flavonoid found in the hop plant (Humulus lupulus L.) and has previously been shown to have depigmenting effects in B16F10 mouse melanoma cells; however, studies of its depigmenting efficacy in human melanocytes are still lacking. In this work, we explored the effects of XH on melanogenesis in MNT-1 human melanoma cells and normal human melanocytes from darkly-pigmented skin (HEM-DP). XH was screened for cytotoxicity over 48 h, and subsequently tested on melanogenesis in MNT-1 cells. XH was further tested in HEM-DP cells for melanin synthesis and melanosome export; dendricity was quantitated to assess effects on melanosome export. Melanosome degradation was studied in human keratinocytes (HaCaT). Our results showed that XH inhibited melanin synthesis in MNT-1 cells at 30 µM but increased intracellular tyrosinase activity without affecting ROS levels. In HEM-DP cells, XH robustly suppressed cellular tyrosinase activity at nontoxic concentrations (2.5-5 µM) without any effect on melanin synthesis. However, XH inhibited melanosome export by reducing dendrite number and total dendrite length. Further testing in HaCaT cells demonstrated that XH induced melanosome degradation at low micromolar concentrations without any cytotoxicity. In summary, our results demonstrate that XH at low micromolar concentrations might hold promise as a potent inhibitor of human pigmentation by primarily targeting melanin export and melanin degradation. Further studies to elucidate the signaling mechanisms of action of melanosome export inhibition by XH and in vivo efficacy are warranted.

Anti-melanogenic activity of ellagitannin casuarictin in B16F10 mouse melanoma cells.

Ellagitannins such as casuarictin (CAS), isolated from clove extracts, have been shown to have superior benefits such as antioxidant and anti-inflammatory activity, but there have been no reports on their capacity to inhibit melanogenesis. Inhibition of melanogenesis by novel natural products has gained attention for cosmetic applications such as skin lightening. Here, we report the effects of CAS on melanogenesis in B16F10 mouse melanoma cells. Our results showed that CAS (30 µM) significantly inhibited intracellular melanogenesis while being nontoxic to B16F10 cells or to HaCaT cells at that concentration. CAS (30 µM) also inhibited intracellular tyrosinase activity as well as mushroom tyrosinase activity; possessed robust copper chelating ability comparable to that of 500 µM kojic acid; and downregulated MITF protein levels, all of which contribute to the inhibitory mechanisms underlying its anti-melanogenic activity. In summary, our results demonstrate that CAS might hold promise as a depigmenting agent for hyperpigmentation disorders.

Effects of Fluoride Exposure on Primary Human Melanocytes from Dark and Light Skin.

Fluoride exposure has adverse effects on human health that have been studied in vitro in cell culture systems. Melanocytes are the melanin pigment-producing cells that have a significant role in the regulation of the process of melanogenesis, which provides several health benefits. Melanocytes are present in the oral cavity, skin, brain, lungs, hair, and eyes. However, to date, there has been no study on the effects of fluoride exposure on melanocytes. Hence, in the current study, we have studied the effects of sodium fluoride (NaF) exposure on neonatal human epidermal melanocytes (HEMn) derived from two different skin phototypes, lightly pigmented (LP) and darkly pigmented (DP). We have assessed the impact of a 24 h and 72 h NaF exposure on metabolic activity and membrane integrity of these cells. In addition, we have evaluated whether NaF exposure might have any impact on the physiological functions of melanocytes associated with the production of melanin, which is regulated by activity of the enzyme tyrosinase. We have also assessed if NaF exposure might induce any oxidative stress in LP and DP melanocytes, by evaluation of production of reactive oxygen species (ROS) and measurement of mitochondrial membrane potential (MMP) levels. Our results showed that HEMn-LP cells showed a higher sensitivity to NaF cytotoxicity than HEMn-DP cells, with significant cytotoxicity at concentrations >1 mM, while concentration range 0.25-1 mM were nontoxic and did not lead to oxidative stress, and also did not alter the levels of intracellular melanin or cellular tyrosinase activity, indicating that treatment up to 1 mM NaF is generally safe to melanocytes from both pigmentation phototypes.

CMT-308, a Nonantimicrobial Chemically-Modified Tetracycline, Exhibits Anti-Melanogenic Activity by Suppression of Melanosome Export.

CMT-308 is a nonantimicrobial chemically-modified tetracycline (CMT), which we have previously shown exhibits antifungal activity and pleiotropic anti-inflammatory activities, including inhibition of the enzymatic activity of matrix metalloproteinases (MMPs). Based on its chemical structure, we hypothesized that CMT-308 could inhibit melanogenesis and might be a candidate for the treatment of skin hyperpigmentation disorders which occur due to unregulated melanin biosynthesis and/or transport. CMT-308 was first studied for any effects on activity of the enzyme tyrosinase in vitro using a purified preparation of mushroom tyrosinase; the mode of inhibition of the soluble fungal enzyme was evaluated by Lineweaver-Burk and Dixon plots as well as by non-linear least squares fitting. Next, the effects of CMT-308 were tested in mammalian cell cultures using B16F10 mouse melanoma cells and further validated in darkly-pigmented human melanocytes (HEMn-DP). Our results showed that micromolar concentrations of CMT-308 inhibited mushroom tyrosinase enzyme activity, using the first two substrates in the melanogenesis pathway (l-tyrosine and l-3,4-dihydroxyphenylalanine (l-DOPA)); CMT-308 inhibited mushroom tyrosinase primarily via a mixed mode of inhibition, with the major contribution from a competitive mode. In B16F10 cell cultures, CMT-308 (10 µM) significantly diminished total melanin levels with a selective reduction of extracellular melanin levels, under both basal and hormone-stimulated conditions without any cytotoxicity over a duration of 72 h. Studies of potential mechanisms of inhibition of melanogenesis in B16F10 cells showed that, in mammalian cells, CMT-308 did not inhibit intracellular tyrosinase activity or the activity of α-glucosidase, an enzyme that regulates maturation of tyrosinase. However, CMT-308 suppressed MITF protein expression in B16F10 cells and showed copper chelating activity and antioxidant activity in a cell-free system. The significantly lower extracellular melanin levels obtained at 10 µM indicate that CMT-308's anti-melanogenic action may be attributed to a selective inhibition of melanosome export with the perinuclear aggregation of melanosomes, rather than a direct effect on the tyrosinase-catalyzed steps in melanin biosynthesis. These results were validated in HEMn-DP cells where CMT-308 suppressed dendricity in a fully reversible manner without affecting intracellular melanin synthesis. Furthermore, the capacity of CMT-308 to inhibit melanosome export was retained in cocultures of HEMn-DP and HaCaT. In summary, our results offer promise for therapeutic strategies to combat the effects of hyperpigmentation by use of CMT-308 at low micromolar concentrations.

Organogold drug Auranofin exhibits anti-melanogenic activity in B16F10 and MNT-1 melanoma cells.

Auranofin (AF) is an organogold FDA-approved drug for treating rheumatism and has been repurposed for several pharmacological applications based on its anti-bacterial, anti-fungal and anti-inflammatory activities. To the best of our knowledge, there has been no study on effects of AF on melanogenesis yet. Hence, in this work, we studied the effect of AF on melanogenesis using B16F10 mouse melanoma cells and validated results in MNT-1 human melanoma cells. Melanogenesis assay was conducted with concentrations of AF determined to be nontoxic in B16F10 cells as well as HaCaT human epidermal cell line for a duration of 48 h, followed by various assays to delineate mechanisms of melanogenesis inhibition. Ultrastructural analysis was conducted to study further if AF affected melanosome maturation and protein levels of a key melanogenic protein, tyrosinase, and the maturation signaling molecule, cyclic adenosine monophosphate (cAMP), was estimated. Our results demonstrate that AF at nontoxic concentrations of 0.25-1 µM significantly inhibited melanin synthesis in a dose-dependent manner with significant inhibition of 32.85% at 1 µM. The study of mechanisms of melanogenesis inhibition revealed that AF inhibited tyrosinase activity in lysates of B16F10 cells but did not show a direct effect on purified mushroom tyrosinase activity or on copper chelation in a cell-free system, nor did it affect levels of B16F10 tyrosinase protein levels. However, AF significantly down-regulated cAMP levels, inhibited cellular ROS and increased number of melanosomes in immature stages, and also exhibited anti-melanogenic activity in B16F10-HaCaT cocultures. Furthermore, AF showed anti-melanogenic efficacy in MNT-1 cell monocultures and cocultures with an inhibition of intracellular tyrosinase activity. In summary, our results demonstrate a proof-of-principle for AF as a depigmenting agent for hyperpigmentation disorders and adjuvant for melanoma therapeutics.

Novel Isoprene Sensor for a Flu Virus Breath Monitor.

A common feature of the inflammatory response in patients who have actually contracted influenza is the generation of a number of volatile products of the alveolar and airway epithelium. These products include a number of volatile organic compounds (VOCs) and nitric oxide (NO). These may be used as biomarkers to detect the disease. A portable 3-sensor array microsystem-based tool that can potentially detect flu infection biomarkers is described here. Whether used in connection with in-vitro cell culture studies or as a single exhale breathalyzer, this device may be used to provide a rapid and non-invasive screening method for flu and other virus-based epidemics.

Inhibition of anthrax lethal factor by curcumin and chemically modified curcumin derivatives.

Curcumin (diferuloylmethane), the active ingredient in the eastern spice turmeric (Curcuma longa), has been shown to inhibit the activities of numerous enzymes and signaling molecules involved in cancer, bacterial and viral infections and inflammatory diseases. We have investigated the inhibitory activities of curcumin and chemically modified curcumin (CMC) derivatives toward lethal factor (LF), the proteolytic component of anthrax toxin produced by the bacterium Bacillus anthracis. Curcumin (Compound 1) appears to inhibit the catalytic activity of LF through a mixture of inhibitory mechanisms, without significant compromise to the binding of oligopeptide substrates, and one CMC derivative in particular, Compound 3 (4-phenylaminocarbonylbis-demethoxycurcumin), is capable of inhibiting LF with potency comparable with the parent compound, while also showing improved solubility and stability. The quantitative reduction in catalytic activity achieved by the different CMC derivatives appears to be a function of the proportion of the multiple mechanisms through which they inhibit the enzyme.

Attenuation of oxidative damage and inflammatory responses by apigenin given to mice after irradiation.

We determined the in vivo efficacy of apigenin, as an anti-oxidant and anti-inflammatory agent, given to mice after irradiation. Various concentrations of apigenin (0, 10, 20, and 40mg/kg body weight) were administered to mice by a single intraperitoneal injection 3hr after receiving 0 or 3Gy of (137)Cs gamma rays. Mice receiving vehicle only (no radiation and no apigenin) served as sham controls. We assessed the anti-oxidative activity of apigenin in vivo by measuring levels of 8-hydroxy-2-deoxy guanosine (8-OH-dG) in bone marrow (BM) cells, collected at days 3 and 10 after irradiation, from groups of mice (5 mice per treatment group) with or without apigenin treatment. Simultaneously, we evaluated the ability of apigenin to diminish radiation-induced inflammatory responses in bone-marrow-derived macrophages (BMDMs) from the same individual mice used for measuring the level of 8-OH-dG. To do this, the levels of activated nuclear factor-kappa B (NF-kappa B) and NF-kappa B-regulated pro-inflammatory cytokines [i.e. interleukin 1-beta (IL-1beta), IL-6, and tumor necrosis factor-alpha (TNF-alpha)] were measured in BMDMs. Our results indicated significant reductions (p<0.01 or <0.05) in the levels of 8-OH-dG in BM cells collected at both harvest times from irradiated mice receiving apigenin treatment, at all apigenin concentrations tested. Likewise, activation of NF-kappa B in BMDMs collected from gamma-irradiated mice that received apigenin was suppressed at both harvest times. Further, the levels of pro-inflammatory cytokines in gamma-irradiated mice treated with 20 or 40mg/kg body weight apigenin were significantly lower than those in mice receiving radiation only (p<0.01 or <0.05) even at day 10 post-irradiation. Additionally, the ratio of neutrophils to lymphocytes indicated that apigenin ameliorated radiation-induced hematological toxicity. Our study is the first to demonstrate the mitigative/therapeutic effects of apigenin given to mice after irradiation.

Nanotechnology use with cosmeceuticals.

The skin is a complex organ and its aging is a complex process. Cutaneous aging is influenced by factors such as sun exposure, genetics, stress and the environment. While skin laxity, rhytides, and dyschromia appear on the surface, these processes originate in deeper layers including the dermis and subcutaneous tissues. Until recently, most topical skin treatments were applied to, and consequently only affected the skin surface. Skin care has evolved to be scientifically based, and as knowledge increases about the physiology of the skin, novel methods of maintaining its health and appearance are developed. New generation skin care products are targeting multiple aging mechanisms by utilizing functional active ingredients in combination with innovative delivery systems.

Chemically modified tetracycline-3 (CMT-3): a novel inhibitor of the serine proteinase, elastase.

Two classes of enzymes play an important role in connective tissue breakdown during various inflammatory diseases: serine proteinases and matrix metalloproteinases (MMPs). Tetracyclines (TCs) exhibit important anti-inflammatory and MMP-inhibitory properties that are unrelated to their antibacterial activities. Of the various TCs and their chemically modified NON-antibiotic analogs (CMTs) tested in vitro and in vivo, CMT-3 (6-demethyl-6-deoxy 4 de-dimethylamino tetracycline) has repeatedly been shown to be the most potent inhibitor of MMP activity and cytokine production. In addition to its anti-MMP function, we have shown that among all CMTs, CMT-3 is the only CMT that can also directly inhibit both the amidolytic activity of human leukocyte elastase (HLE, a serine proteinase) and the extracellular matrix degradation mediated by HLE. In addition, CMT-3 has been found to reduce leukocyte elastase activity in vivo in gingival extracts of rats with experimental periodontal disease. Thus, CMT-3 can inhibit pathologic connective tissue breakdown by (at least) two mechanisms: direct inhibition of neutral proteinases (elastase and MMPs); and protecting their endogenous inhibitors, α(1)-PI and TIMPs, from being digested and inactivated by MMPs and HLE, respectively. The pleiotropic properties of CMT-3 including (but not limited to) inhibition of serine proteinases, MMPs, and cytokines provide impressive therapeutic potential to reduce excessive connective tissue breakdown during various pathologic processes including inflammatory diseases, cancer metastasis and metabolic bone diseases.

A tetracycline analog improves acute respiratory distress syndrome survival in an ovine model.

BACKGROUND: Acute respiratory distress syndrome (ARDS) mortality remains high with no effective pharmacotherapy available. A chemically modified tetracycline (COL-3) is a potent inhibitor of matrix metalloproteinases. Prophylactic COL-3 administration has been shown to be effective in ARDS treatment. In the present study, the therapeutic effect of COL-3, given shortly after injury, was investigated in an ovine ARDS model. METHODS: The ovine ARDS model was induced by combined 40% body area third-degree burn, smoke inhalation, and barotrauma injuries. The sheep were randomly assigned into two groups: control (10% Solutol, n = 5) or COL-3 (200 mg/m(2), n = 5). Intravenous administration of COL-3 or vehicle was performed 1 hour after the smoke and burn injury. When ARDS criteria were met (arterial partial pressure of oxygen to fraction of inspired oxygen ratio < 200) or no later than 24 hours after injury (if criteria not met), animals underwent the ARDS Network ventilation protocol. At 96 hours after injury or at animal death, lung pathologic processes were assessed. RESULTS: Administration of COL-3 improved hemodynamics and reduced carbon dioxide levels. Administration of COL-3 also significantly delayed ARDS development and prolonged survival time compared with the control group (20.4 + or - 3.8 hours versus 12.9 + or - 3.3 hours; 94.2 + or - 4.0 hours versus 58.6 + or - 26.4 hours; p < 0.05, respectively). Survival analysis showed a higher 96-hour survival from ARDS with COL-3 administration as compared with control (80% versus 20%; p < 0.05). Lung pathologic processes were also improved by COL-3. Plasma matrix metalloproteinase-2 level increased in control but not in COL-3-treated animals. CONCLUSIONS: Our present study suggests that COL-3 may be an effective pharmacotherapy for ARDS treatment.

Dose-rate effects of protons on in vivo activation of nuclear factor-kappa B and cytokines in mouse bone marrow cells.

The objective of this study was to determine the kinetics of nuclear factor-kappa B (NF-kappaB) activation and cytokine expression in bone marrow (BM) cells of exposed mice as a function of the dose rate of protons. The cytokines included in this study are pro-inflammatory [i.e., tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6] and anti-inflammatory cytokines (i.e., IL-4 and IL-10). We gave male BALB/cJ mice a whole-body exposure to 0 (sham-controls) or 1.0 Gy of 100 MeV protons, delivered at 5 or 10 mGy min(-1), the dose and dose rates found during solar particle events in space. As a reference radiation, groups of mice were exposed to 0 (sham-controls) or 1 Gy of (137)Cs gamma rays (10 mGy min(-1)). After irradiation, BM cells were collected at 1.5, 3, 24 h, and 1 month for analyses (five mice per treatment group per harvest time). The results indicated that the in vivo time course of effects induced by a single dose of 1 Gy of 100 MeV protons or (137)Cs gamma rays, delivered at 10 mGy min(-1), was similar. Although statistically significant levels of NF-kappaB activation and pro-inflammatory cytokines in BM cells of exposed mice when compared to those in the corresponding sham controls (Student's t-test, p < 0.05 or <0.01) were induced by either dose rate, these levels varied over time for each protein. Further, only a dose rate of 5 mGy min(-1) induced significant levels of anti-inflammatory cytokines. The results indicate dose-rate effects of protons.

Doxycycline [corrected] inhibits mononuclear cell-mediated connective tissue breakdown.

Chronic periodontitis is the most common chronic inflammatory disease and has been associated with an increased risk for serious medical conditions including cardiovascular disease (CVD). Endotoxin (lipopolysaccharide), derived from periodontopathogens, can induce the local accumulation of mononuclear cells in the inflammatory lesion, increasing proinflammatory cytokines and matrix metalloproteinases (MMPs), resulting in the destruction of periodontal connective tissues including bone. In this study, we show that doxycycline, originally developed as a broad-spectrum tetracycline antibiotic (and, more recently, as a nonantimicrobial therapy for chronic inflammatory periodontal and skin diseases), can inhibit extracellular matrix degradation in cell culture mediated by human peripheral blood-derived monocytes/macrophages. The mechanisms include downregulation of cytokines and MMP-9 protein levels and the inhibition of the activities of both collagenase and MMP-9. These pleiotropic, but nonantibiotic, effects of doxycycline explain, at least in part, its therapeutic potential for various chronic inflammatory diseases including periodontitis, and may reduce the risks of systemic diseases (e.g. CVDs, less manageable diabetes) associated with this and other local diseases.

Evaluating the use of 3'-(p-Aminophenyl) fluorescein for determining the formation of highly reactive oxygen species in particle suspensions.

BACKGROUND: Given the importance of highly reactive oxygen species (hROS) as reactants in a wide range of biological, photochemical, and environmental systems there is an interest in detection and quantification of these species. The extreme reactivity of the hROS, which includes hydroxyl radicals, presents an analytical challenge. 3'-(p-Aminophenyl) fluorescein (APF) is a relatively new probe used for measuring hROS. Here, we further evaluate the use of APF as a method for the detection of hydroxyl radicals in particle suspensions. RESULTS: Particle-generated hROS can be quantified with an estimated detection limit of 50 nM. Measurements of hROS in two National Institute of Standards and Technology (NIST 2709 and 2710) soil suspensions and a pyrite suspension show non-linear particle dose-response curves for hROS generation. APF can also be used in solutions containing no dissolved molecular oxygen (O2) to determine the role of O2 in the formation of hROS. Results confirm that O2 is mechanistically important in the formation of hROS by dissolved ferrous iron and in pyrite suspensions. CONCLUSION: Given the non-linear dose-response curves for particle generation of hROS, we recommend using several particle loadings in experiments aimed to compare particles for their hROS generation potential. The method presented here is specific to hROS and simple to perform. The analysis can be conducted in mobile labs as only basic laboratory equipment is required.

Protein-expression profiles in mouse blood-plasma following acute whole-body exposure to (137)Cs gamma rays.

PURPOSE: To compare the pattern of protein-expression profiles in blood-plasma after exposure of CBA/CaJ mice to 0 or 3 Gy of (137)Cs gamma rays. MATERIALS AND METHODS: Two-dimensional electrophoresis gel coupled with mass spectrometry was used to analyze blood-samples collected at days 2 and 7 post-irradiation. At each sacrifice-time, alterations in expression-level of protein spots between control- and exposed-groups were analyzed statistically by the PDQuest software using Student's t-test (at the significance level of p < 0.05). Mass spectrometry was used to identify the identity of protein-spots with significantly altered expression-level. RESULTS: At day 2, 18 proteins were significantly up-regulated in exposed-mice. These included: alpha-2-Heremans-Schmid (HS)-glycoprotein, apolipoprotein (Apo)-AII-precursor, Apo-E, beta-2-glycoprotein-I, clusterin, fibrinogen-alpha-chain, fibrinogen-gamma-polypeptide, fetuin-B, haptoglobin, high-molecular-weight (HMW)-kininogen (Kng), low-MW-Kng, Kng1-precursor, liver-carboxylesterase-I-precursor, major-urinary-protein-6-precursor, mannose-binding-protein-C-precursor, mannose-binding-lectin-C, and prothrombin-precursor. Gelsolin was detected in control-mice only. At day 7, high expression-levels of 14 proteins were detected in control-mice (i.e., alpha-1-antitrypsin-precursor, carboxylesterase-N, cholesterol-7-alpha-hydroxylase, contraspin, coagulation-factor-II, coagulation-factor-XIII, gelsolin, immunoglobulin-G-heavy-chain, neurexin, prothrombin-precursor, protein-phosphatase, putative-calcium-influx-channel, vitamin-D-binding-protein, and 1110018G07Rik); while 15 proteins were highly expressed in exposed-mice. These included: alpha-1-acid-glycoprotein, alpha-2-HS-glycoprotein, alpha-1-protease-inhibitor-2, ApoA-IV, ApoC-I, ApoH, beta-1-globin, clusterin, complement-component-3, fibrinogen-beta-chain, HMW-Kng, major-histocompatibility-complex-class-Ia-H2-K, serine-(cysteine)-proteinase-inhibitor, retinoblastoma-associated-protein-140, and vascular-cell-adhesion-molecule-1. CONCLUSION: Although different proteins (mostly involved in inflammatory responses) were detected in exposed-mice, alterations in expression-levels of clusterin, gelsolin, kininogen, and alpha-2-HS-glycoproteins were found at both times. Despite the need for validation, the results suggested that alterations in expression-levels of specific proteins may be indicative of radiation-exposure. The results also provided the important step in an eventual establishment of blood-based biomarkers of radiation-exposure in vivo.