Enhancement of fibroblast growth promoting activity of human blood after its irradiation in vivo (transcutaneously) and in vitro with visible and infrared polarized light

Visible and infrared (IR) irradiation of laser and non-laser sources has a pronounced wound-healing effect promoting tissue repair without hyperproduction of connective tissue elements. This effect develops as a consequence of local and systemic light effects, but many aspects of their mechanism have been yet unclear. In the present work, we have shown that in 0.5 h after irradiation of a small area of the volunteers' body surface with polychromatic visible + IR light (400-3400 nm, 95% polarization, 12 J/cm2) the amounts of PDGF and TGF-beta 1 in the blood serum increase, on average, by 20 and 43%, respectively. This effect is preserved for at least 24 h to be recorded only in volunteers with the initially normal and decreased levels of the growth factors; the initially elevated content of PDGF-AB decreases. Addition of such a plasma (2.5%) to the nutrient medium of primary cultures of human embryonal fibroblasts stimulates cell proliferation, on average, by 10 and 17%, but only in the case if the initial growth-promoting (GP) blood activity was low. Similar changes occur in parallel experiments following irradiation of blood samples of the same volunteers in vitro, as well as at mixing irradiated and non-irradiated autologous blood at the ratio 1:10 (v/v), i.e. at modeling a situation in the vascular bed, when the transcutaneously photomodified blood contacts with the rest of its volume. Similar changes in the blood GP activity under conditions in vitro were recorded as well after 4-9 daily phototherapy sessions. This allows us to suggest that changes in GP activity of circulating blood of the irradiated volunteers may be, to a large extent, the consequence of effect exerted on the blood by small amounts of transcutaneously photomodified blood. The obtained results are discussed in terms of light effect on wound healing and scar tissue formation, with regard to the authors' previous data on much higher GP of the irradiated blood in respect to keratinocytes, the fast decrease in proinflammatory cytokine levels, and the increase in IFN-gamma content.     

Stimulation of DNA repair in human cells damaged by UV and ionizing radiation with soluble growth factors of photomodified blood

Exposure of a small skin area of volunteers to UV light in 1 minimal erythemal dose is accompanied by rapid appearance in the circulating blood of soluble factors able to restore proliferation of X-ray-damaged autologous lymphocytes, to decrease frequency of chromosome breaks, and to stimulate unscheduled DNA synthesis. The appearance of such an activity in the blood can be also induced without skin irradiation. For this, one volume of a directly UV-irradiated blood is to be mixed in vitro with 10-fold volumes of intact blood, thus modeling the in vivo situation, when a small amount of transcutaneously UV-irradiated blood mixes with intact blood in the circulation. It has been found that the platelet-derived growth factor and epidermal growth factor, added at physiological concentrations to the culture medium, decrease chromosome break frequency in X-damaged cells.     

Changes in cytokine content in the peripheral blood of volunteers after their exposure to polychromatic visible and infrared light

Anti-inflammatory, immunomodulating and wound-healing effects of visible and infrared (IR) radiation from laser and non-laser sources are widely used in current medicine. However, the role of pro- and anti-inflammatory cytokines in development of these effects has been poorly studied. A randomized, placebo-controlled, double blind study was made. Using ELISA, the content of 10 cytokines was studied in the peripheral blood of volunteers after a single and four daily irradiations of the sacral area (D = 15 cm) with polychromatic visible + IR polarized light (480-3400 nm, 12 J/cm2). The phototherapeutic sessions were accompanied by four blood exfusions for the study (to a total volume of 80 ml). In the control (placebo) group, irradiation was imitated, and blood samples of the same volume were drawn at the same time intervals as in volunteers of the main group. A fast decrease in the level of pro-inflammatory cytokines was revealed as soon as in 0.5 h after the irradiation. This level was retained until the end of the phototherapeutic course. At the parameters exceeding the norm, the contents of TNF-alpha, IL-6 and IFN-gamma fell, on average, by 34, 12 and 1.5 times, respectively. By the end of the course, the levels of IFN-gamma and of IL-12 decreased by 5 and 15 times, respectively. A fast decrease (by two-fold) was also characteristic of normal values of IL-6. Neither IL-1beta, nor IL-2 were detected in blood plasma of the examined people both before and after the irradiation. In parallel with a decrease in the proinflammatory factor levels the amount of anti-inflammatory cytokines was found to rise: that of IL-10--by 2.7-3.5 times in 0.5 h and at later terms at the initially normal parameters, and that of TGF-beta1--by 1.4-1.5 times at the initially decreased level. The IL-4 content did not change. A characteristic feature of the light effect was a fast rise of IFN-gamma amount--by 3.3-4.0 times in individuals with its initially normal level, with no changes in IFN-alpha content. The above-reported regularities of the light effects were also recorded at a direct (in vitro) irradiation of the examined volunteers' blood, as well as on addition of irradiated blood to a 10-fold volume of non-irradiated autologous blood, i.e. at a modeling of mixing, of a small amount of transcutaneously photomodified blood with its main circulating volume in the vascular bed of an irradiated person. Such a similarity of effects in blood following its irradiation in vivo and in vitro enables us to associate the fast changes of the cytokine content in the entire volume of peripheral blood with the transcutaneous photomodification of its small amounts, and with a "transfer" of the light effects by photomodified blood to the whole pool of circulating blood.     

Modulation of proliferation of peripheral blood lymphocytes after irradiation of volunteers with polychromatic visible and infrared light

Effects on human immune system of visible and infrared (IR) radiation, the dominating types of solar light on Earth, still remain poorly studied. In the present work, a small area of the volunteers' body surface was irradiated with polychromatic visible + IR polarized (VIP) light, whose spectral range is close to the natural one (400-3400 nm, 12 J/cm2), and spontaneous and phytohemagglutinin (PHA)-induced DNA syntheses were studied by radiometric method in lymphocytes (Lym) of peripheral blood. This Irradiation stimulated both spontaneous and PHA-induced DNA synthesis in Lym but only in volunteers with initially decreased parameters of synthesis (on average, by 2.5 and 2.7 times, respectively), which was recorded 24 h after irradiation of volunteers, and after a 72 h cultivation of separated mononuclears. In the parallel experiments, blood of each volunteer was irradiated in vitro. Besides, by modeling situation in vivo, when a small amount of transcutaneously photomodified blood contacts its much larger circulating volume, the irradiated and non-irradiated samples of autologous blood were mixed at a 1:10 volume ratio. In Lym with the initially decreased synthesis level, the spontaneous synthesis elevated by 2 and 3 times, respectively, whereas stimulation of PHA-synthesis was observed only after addition of the irradiated blood to the intact one (by 2.2 and 1.6 times, respectively). A high degree of positive correlation in changing the studied parameters is revealed in irradiation of blood in vivo and in vitro. This makes it possible to associate the light-stimulating effect on Lym of the entire circulating blood with transcutaneous photomodification of its small amounts, and with action of such blood on the rest of blood. A similarity in the direction and additivity of mitogenic effects of VIP light and PHA was revealed. The obtained data enable us to suggest that therapy employing polychromatic visible and IR light would promote presumably an increase in the number of Lym in peripheral blood and an enhancement of their response to antigenic stimulus.     

Hepatocyte growth factor and keratinocyte growth factor enhance IL-1-induced IL-8 secretion through different mechanisms in Caco-2 epithelial cells

A variety of cytokines have been detected in inflamed intestinal mucosal tissues, including the pro-inflammatory cytokine, interleukin-1 (IL-1), along with growth factors involved in wound healing processes such as proliferation and cell migration. However, little is known about how IL-1 and growth factors interact with intestinal epithelial cells to regulate the production of inflammatory cytokines such as interleukin-8 (IL-8). Previously, we have shown that hepatocyte growth factor (HGF) could significantly enhance IL-1-stimulated IL-8 secretion by the Caco-2 colonic epithelial cell line, yet HGF, by itself, did not stimulate IL-8 secretion. In this report, a second growth factor, keratinocyte growth factor (KGF), was also found to significantly enhance IL-1-induced IL-8 secretion by Caco-2 cells, yet KGF, by itself, also had no effect. Simultaneous addition of both IL-1 and KGF was also required for the enhancing effect. Treatment of the Caco-2 cells with wortmannin or triciribine suppressed the enhancing effect of HGF, suggesting that the effect was mediated by signaling through phosphatidylinositol-3-kinase (PI3K) and the kinase AKT. The enhancing effect of KGF was not affected by wortmannin, but was suppressed by triciribine, suggesting that the effect of KGF was through a PI3K-independent activation of AKT. These results suggest that the growth factors HGF and KGF may play a role in enhancing IL-1-stimulated production of IL-8 by epithelial cells during mucosal inflammations. However, the mechanism by which the growth factors enhance the IL-1 response may be through different initial signaling pathways.     

Growth-stimulating effect of UF-irradiated blood. I. The radiation dose dependence of the initial growth potencies of blood and of the functional state of the target cells

UV irradiation (UVI) of donor blood in the apparatus used in hospitals of the USSR with the therapeutic aim of autotransfusion of UV-irradiated blood (AUVIB), results in an increase of connective tissue cell growth potency: being added into culture media the supernatants of irradiated blood stimulate DNA-synthetic and proliferative activity of cultured human embryonic cells. The high activity of cells persists for about 2 days. The effect is great with low initial levels of cell proliferative activity. In this case the effect is maximum (about 125% of the control). It is suggested that the above effect may be involved in the mechanism of stimulation of regeneration processes in the organism after AUVIB.     

A comparative study of the effects of laser and LED radiation on lipid peroxidation in rat wound fluid

The effect of laser (632 nm) and LED (630 nm) on lipid peroxidation in rat wound fluid (exudate) was studied with the aim of comparing the efficiency of coherent and incoherent light on the processes that take place during wound healing. The study was performed using the model of cut aseptic wounds proposed by L.I. Slutskii. It was shown that irradiation of wounds with light of both laser and LED caused a decrease in the concentration of lipid peroxidation products in wound fluid as compared with the control group. An increase in the antioxidative activity of wound fluid was observed. It was concluded that irradiation with light of both laser and LED decreases the level of oxidative stress in wound fluid and that radiation coherence does not play a significant role.     

Changes in the expression of membrane markers and in the number of human blood monocytes after single and repeated courses of visible and infrared light at therapeutic doses

An attempt has been made to prove that the immunomodulating effect of therapeutic doses of polychromatic visible + infrared polarized (VIP) light at its application to a small body surface area is connected with a transcutaneous photomodification of a small amount of blood in superficial skin microvessels. For this purpose, in parallel experiments, using monoclonal antibodies, the membrane phenotype of circulating blood mononuclears was studied after irradiation of volunteers, of samples of their blood in vitvo, and of a mixture of the irradiated and non-irradiated autologous blood in a 1:10 volume ratio, thereby modeling events in vivo, when a small amount of the transcutaneously photomodified blood in the vascular bed contacts its main circulating volume. In this variant of experiment, a great similarity has been established of changes in expression of mononuclear membrane markers (CD3, CD4, CD8, CD20, CD16, HLA-DR and to a lesser degree of CD25); the ability has been proven of the photomodified blood to "translate" the light-induced changes to a much higher volume of non-irradiated blood, which might represent a mechanism of the systemic immunomodulating effect of phototherapy. Under conditions in vivo and in vitro, the most "reactive" were HLA-DR+, CD20+, CD16+, CD4+, and 0-cells. An increase of the total number of lymphocytes and monocytes has been shown by the end of the 10-day-long phototherapeutic course. The regulatory character of the single and course sessions of the VIP light on the blood immunocompetent cells is substantiated: depending on the initial state of the immune system, the VIP light can produce both stimulating and inhibitory effect on lymphoid cell subpopulations, which opens large possibilities of using this method for correction of immunological disturbances in diseases of different etiopathogenesis.     

Wound healing and expression of antimicrobial peptides/polypeptides in human keratinocytes,a consequence of common growth factors

In addition to acting as a physical barrier against microorganisms, the skin produces antimicrobial peptides and proteins. After wounding, growth factors are produced to stimulate the regeneration of tissue. The growth factor response ceases after regeneration of the tissue, when the physical barrier protecting against microbial infections is re-established. We found that the growth factors important in wound healing, insulin-like growth factor I and TGF-alpha, induce the expression of the antimicrobial peptides/polypeptides human cationic antimicrobial protein hCAP-18/LL-37, human beta-defensin 3, neutrophil gelatinase-associated lipocalin, and secretory leukocyte protease inhibitor in human keratinocytes. Both an individual and a synergistic effect of these growth factors were observed. These findings offer an explanation for the expression of these peptides/polypeptides in the skin disease psoriasis and in wound healing and define a host defense role for growth factors in wound healing.     

Epidermal growth factor suppresses nitric oxide and hydrogen peroxide production by keratinocytes. Potential role for nitric oxide in the regulation of wound healing.

In the skin, wounding initiates a complex array of physiological processes mediated by growth factors and inflammatory mediators which stimulate tissue repair and protect against infection. We report that primary cultures of human keratinocytes and a mouse keratinocyte cell line respond to the inflammatory stimuli gamma-interferon and lipopolysaccharide or tumor necrosis factor-alpha by producing nitric oxide and hydrogen peroxide, two reactive mediators that are important in nonspecific host defense. Nitric oxide is produced by the l-arginine- and NADPH-dependent enzyme, nitric oxide synthase. In murine keratinocytes, optimal enzymatic activity was found to be dependent on Ca2+ and calmodulin as well as on glutathione. Inflammatory mediators were also found to inhibit the growth of keratinocytes, an effect that could be reversed by a nitric oxide synthase inhibitor. Epidermal growth factor (EGF), which promotes wound healing by stimulating cellular proliferation, was found to be a potent antagonist of reactive nitrogen and reactive oxygen intermediate production by keratinocytes. EGF also reversed the growth inhibitory actions of the inflammatory mediators. These data suggest that nitric oxide produced by keratinocytes is important in the control of cellular proliferation during wound healing. Our findings that EGF effectively regulates the production of free radicals by keratinocytes may represent an important pathway by which this growth factor not only stimulates epidermal cell proliferation but also facilitates the resolution of inflammation following wounding.     

Promotive effects of far-infrared ray on full-thickness skin wound healing in rats

The biological effects of far-infrared ray (FIR) on whole organisms remain poorly understood. The aim of our study was to investigate not only the hyperthermic effect of the FIR irradiation, but also the biological effects of FIR on wound healing. To evaluate the effect of FIR on a skin wound site, the speed of full-thickness skin wound healing was compared among groups with and without FIR using a rat model. We measured the skin wound area, skin blood flow, and skin temperature before and during FIR irradiation, and we performed histological inspection. Wound healing was significantly more rapid with than without FIR. Skin blood flow and skin temperature did not change significantly before or during FIR irradiation. Histological findings revealed greater collagen regeneration and infiltration of fibroblasts that expressed transforming growth factor-beta1 (TGF-beta1) in wounds in the FIR group than in the group without FIR. Stimulation of the secretion of TGF-beta1 or the activation of fibroblasts may be considered as a possible mechanisms for the promotive effect of FIR on wound healing independent of skin blood flow and skin temperature.     

Healing of burns after treatment with 670-nanometer low-power laser light

Recent investigations have reported contradictory results on the influence of low-power laser light on wound healing. Low-power laser with a power output of 250 mW and an emitted laser light of 670 nm have been insufficiently investigated to date. The effect of a 250-mW/670-nm laser light on the healing of burning wounds in rats was investigated. Thirty rats were burned on both flanks. One wound was irradiated with 670-nm laser light (2 J/cm2), whereas the other side remained untreated. Macroscopic evaluation of the wounds was performed daily; 10, 20, and 30 days after burning, 10 rats were killed and the wounds histologically evaluated. Neither macroscopic nor histologic examination of the irradiated wound showed accelerated wound healing when compared with control wounds. In the present study, irradiation of burns with a 250-mW/670-nm laser light produced no beneficial effects on wound-healing processes.     

Effects of microbeam light on growth and phototropism ofPilobolus crystallinus sporangiophores

A small blue-light beam (50 μm in diam) was used to examine light-growth response and phototropism inPilobolus crystallinus sporangiophores. Continuous irradiation by microbeam of a region 100–150 μm from the apex promoted the growth of a dark-adapted sporangiophore for about 15 min after a lag period of 1–2 min. After the promotion, the growth rate fell below that before the irradiation. Irradiation of the apex of sporangiophore slightly promoted the growth but strongly inhibited the growth after the promotion. A smaller light beam (10 μm in diam) applied continuously at grazing incidence along one side of the sporangiophore caused bending toward the shaded side, implying that the irradiated side grew more rapidly than the shaded side and that the lens effect is involved in the phototropism of young sporangiophores ofP. crystallinus. The involvement of the lens effect was confirmed by the fact that a carotenoid-less mutant was 1.5–2 times more sensitive to unilateral blue light than the wild type, probably because of a smaller intracellular light attenuation during passage through the mutant cell.     

The effect of low-intensity laser irradiation in the blue, green, and red spectral ranges on healing of experimental skin wounds in rats

The effects of low-intensity laser irradiation in the red (632.8 nm), green (532 nm), and blue (441.2 nm) spectral ranges on wound healing has been studied in rats. The effect of the traditionally used red laser irradiation has been compared with the effect caused by laser irradiation in other spectral ranges, aiming to support the provisional hypothesis that a similar healing effect could be achieved at lower doses of wound irradiation by lasers emitting in the blue and green spectral ranges. The following parameters have been used to characterize healing of the experimental wounds: the functional activity of phagocytes in the wound exudate, which was determined from luminol-dependent chemiluminescence, the phagocyte number; the wound exudates’ antioxidant activity; and the rate of healing, which was determined as the change of the wound surface area. It was found that in all cases the laser irradiation accelerated the healing of wounds. Exposure to red laser irradiation at the dose of 1.5 J/cm²), and to blue or green laser irradiation at a dose of 0.75 J/cm² shortened the time of the wound healing from 22 to 17 and 19 days, respectively. The functional activity of leukocytes in irradiated groups increased by day 5 after surgery, whereas in the control group it decreased. The superoxide dismutase activity increased in all experimental groups by day 5 after surgery. Laser irradiation in the red spectral range at a dose of 1.5 J/cm² resulted in a larger increase in superoxide dismutase activity, as compared to that found after exposure to laser irradiation in the blue and green spectral ranges at a dose of 0.75 J/cm².     

创伤渗出液对创伤成纤维细胞增殖的影响

创伤后不同时期渗出液(wound fluid,WF)的质和量的变化,在很大程度上反映伤口组织的愈合进程.研究伤口不同天数的WF对小鼠伤口组织的成纤维细胞(mouse wound fibrolast,mWFb)体外增殖能力的影响,探讨伤口微环境WF在调控mWFb的增殖规律.用两种培养基进行检测：1640培养基-10% FCS(fetal calf serum 胎牛血清)-10% WF或1640-1% FCS-10% WF.发现第1、3、7天的WF能刺激mWFb增殖.在高浓度(10%)FCS条件下,9、11、15天WF对mWFb生长产生抑制作用.而同一WF在低浓度(1%)FCS时导致mWFb死亡.结果提示,在损伤后一周期间伤口微环境能刺激mWFb增殖,但伤后更长时间的WF使细胞生长受阻止.在创伤愈合晚期的微环境中可能存在一些生长抑制因子.     

Occupational levels of radiation exposure induce surface expression of interleukin-2 receptors in stimulated human peripheral blood lymphocytes

Interleukin-2 (IL-2) is a cytokine responsible for a variety of immune and non-immune stimulatory and regulatory functions, including the activation and stimulation of cytotoxic cells able to recognize and kill human tumour cells and T-cell proliferation and differentiation. We show that low doses of radiation, in the range commonly received by atomic radiation workers or as a result of minor medical diagnostic procedures (0.25 to 10 mGy), stimulate the expression of IL-2 receptors (IL-2R) on the surface of peripheral blood lymphocytes (PBL) taken from normal human donors. This stimulated surface expression after in vitro irradiation is an indirect effect, resulting from the secretion into the medium of a soluble factor from the irradiated cells. This factor can also stimulate IL-2R surface expression in unirradiated cells. Consequently, radiation stimulation of IL-2R expression in a large population of PBL shows a triggered-type response rather than being proportional to dose. These results demonstrate that normal human cells can respond to doses of radiation in the range of common occupational or medical exposures. The data also demonstrate a possible defence mechanism against environmental stress by which a radiation-exposed cell can use an indirect signalling mechanism to communicate with and influence the biological processes in an unexposed cell.     

Proliferation of normal and tumor cells at the presence of serum from patients with breast cancer after phototherapy with visible and near infrared light

Simultaneous low-intensity visible (VIS) and near infrared (nIR) irradiation from laser and non-laser sources was used for treatment of complications developing in cancer patients after surgical tumor resection, chemo- and radiation therapy. However, the question remains about the impact of this physiotherapeutic method on proliferative activity of the patients' tumor cells and cells involved in wound healing, fibroblasts (FB) and keratinocytes (KC). In this paper, we studied the effect blood serum obtained from the patients with breast cancer after the course of irradiation with visible and NI light (480--3400 nm, 95 % polarization, 40 mW/cm2, 12 J/cm2) in postoperative period on the proliferative activity of primary cultures of human FB and KC, and of several human tumor cell lines (BT-474, HBL-100, Hs578T and A431). Seven-day course of phototherapy increase proliferation of FB (as compared to the initial level) and KC (as compared to postoperative level) by 22 and 28 %, respectively. The tumor cells BT-474, Hs578T and A431 showed statistically significant decrease in proliferative activity compared with the preoperative (initial) level by 31.5, 8.97 and 6.47%, respectively, whereas the cells BT-474, HBL-100, Hs578T and A431 also reduced their proliferative activity by 32,16, 8.65 and 6.26%, respectively, as compared with postperative level. The results obtained demonstrate the safety of the phototherapy with the visible and NI light for BC patients in the postoperative period.     

Fatty acid-induced angiogenesis in first trimester placental trophoblast cells: Possible roles of cellular fatty acid-binding proteins

Angiogenesis is involved in the growth of new blood vessels from the existing one. Consequently, angiogenesis plays an indispensable role in tissue growth and repair including early placentation processes. Besides angiogenic growth factors (vascular endothelial growth factor (VEGF), angiopoietin-like 4 (ANGPTL4), placental growth factor (PlGF), platelet derived growth factor (PDGF), fibroblast growth factors (FGF)), dietary fatty acids (c>16) also directly or indirectly modulate angiogenic processes in tumors and other cell systems. Usually n − 3 fatty acids inhibit whereas n − 6 fatty acids stimulate angiogenesis in tumors and other cells. Contrary to this, docosahexaenoic acid, 22:6n − 3 (DHA) and other fatty acids including conjugated linoleic acid stimulate angiogenesis in placental first trimester cells. In addition to the stimulation of expression of major angiogenic factors such as VEGF and ANGPTL4, fatty acids also stimulate expression of intracellular fatty acid-binding proteins (FABPs) FABP-4 and FABP-3 those are known to directly modulate angiogenesis. Emerging data indicate that FABPs may be involved in the angiogenesis process. This paper reviews the fatty acid mediated angiogenesis process and the involvement of their binding proteins in these processes.     

A comparative study of Aldactone and Diane in the treatment of hirsutism

A comparative study has been performed in order to evaluate the relative potency of Diane and Aldactone in reducing hair growth as well as the effect on blood hormone concentrations. Thirty-six women participated in the study and depending on the desire for contraception, 22 were treated with Diane and 14 with Aldactone. The results show that Aldactone (50 mg per day) had little effect on hormone concentrations, only LH was significantly reduced after 12 months of treatment. Despite the lack of effect on hormone levels, all 14 women reported reduced hair growth and after 5 months of treatment, 58% also reported a decrease in the formation in new hair growth. In contrast to the Aldactone treated group, the women on Diane medication demonstrated a marked decrease in circulating hormone levels with a subsequent effect on the hair parameters. The clinical effects were, however, not quite of the same degree as those seen with Aldactone treatment. Approximately 20% exhibited no response for any of the 3 hair-parameters (reduced hair growth, formation of new hair and a change to softer hair). The response time before any effect was observed was also longer than that seen with the Aldactone group. The data suggest that, at the dosages employed, Aldactone has a better clinical effect on the hair parameters despite a lack of effect on circulating hormone levels. One should, however, be aware that Diane contains only 2 mg cyproterone acetate (CPA) and a better effect would most probably have been obtained using a higher dosage of CPA.