Histological assessment of non-ablative laser stimulation of tissue repair in acellular dermal grafts.

AIM: The objective of this study was to compare integration of AlloDerm® acellular dermal grafts in animals subjected to non-ablative laser irradiation and animals not exposed to this therapy. METHODS: Standardized AlloDerm® fragments measuring 5 mm² were grafted into the subcutaneous tissue overlying the calvaria in 32 Wistar rats. Laser therapy (685 ηm), at a dose of 4 J/cm2 per session, was applied immediately after surgical intervention and every 48 hours thereafter for a total of four applications. RESULTS: Analysis of histology slides revealed significantly greater edema in the control group. There was no neutrophil infiltration in the laser-irradiated group at any point during the study period, whereas such infiltration was present in control animals at three of the four points of observation. In the laser therapy group, lymphocyte infiltration was observed from day 1, whereas in the control group, it was only apparent from day 3. Vascularization was substantially greater in the control group. In the experimental group, the AlloDerm® graft was completely replaced by fibrous tissue. CONCLUSION: These findings suggest that add-on non-ablative laser therapy is an effective stimulator of healing and graft integration after placement of AlloDerm® acellular dermal grafts.

Anti-inflammatory effect of low intensity ultrasound (LIUS) on complete Freund's adjuvant-induced arthritis synovium.

OBJECTIVES: Arthritis with intra-articular inflammation was accompanied by joint pain, swelling, and stiffness leading to significant functional impairment. Thus, regulation of joint inflammation is a good therapeutic approach for patients with arthritis. In this study, the effect of low intensity ultrasound (LIUS) applied to an adjuvant-induced arthritic rat model on the synovium was investigated. DESIGN: Synovial inflammation was induced by complete Freund's adjuvant (CFA)-injection into the rat knee joint. LIUS (200 mW/cm(2)) was applied on the ipsilateral knee everyday for 10 min beginning 1 day after inflammation induction. The expression of proinflammatory factors and immunohistochemical staining pattern of the synovium were assessed. RESULTS: CFA induced an increase of the knee circumference that was significantly diminished by LIUS. Synovial membrane hyperplasia in the ipsilateral joint was also affected by LIUS. The inflammatory mediators, COX-1/2, IL-1ß, and iNOS, but not TNF-α, in the synovial membrane were induced after 3 days, and they closely correlated with the degree of edema. In the synovial membrane, the expression of inflammatory mediators was reduced by LIUS. The chemoattractant chemokine receptor CCR5 also was involved. On immunohistochemical analysis, CFA caused increased infiltration of CD11b-positive cells in the synovium. After 3 days, neutrophils, myeloperoxidase (MPO)-positive cells filled the inflammatory core; later, monocytes and macrophages, ionized calcium binding adaptor molecule 1 (Iba1)-positive cells in the periphery infiltrated the core by day 5. LIUS markedly reduced CFA-induced inflammatory cells infiltration. CONCLUSION: LIUS showed a potent anti-inflammatory effect in this animal arthritis model with reduced infiltration of inflammatory cells into the synovium.

Langerhans cells serve as immunoregulatory cells by activating NKT cells.

Ultraviolet exposure alters the morphology and function of epidermal Langerhans cells (LCs), which play a role in UV-induced immune suppression. It is generally believed that UV exposure triggers the migration of immature LCs from the skin to the draining lymph nodes (LNs), where they induce tolerance. However, because most of the previous studies employed in vitro UV-irradiated LCs, the data generated may not adequately reflect what is happening in vivo. In this study, we isolated migrating LCs from the LNs of UV-irradiated mice and studied their function. We found prolonged LC survival in the LNs of UV-irradiated mice. LCs were necessary for UV-induced immune suppression because no immune suppression was observed in LC-deficient mice. Transferring LCs from UV-irradiated mice into normal recipient animals transferred immune suppression and induced tolerance. We found that LCs colocalized with LN NKT cells. No immune suppression was observed when LCs were transferred from UV-irradiated mice into NKT cell-deficient mice. NKT cells isolated from the LNs of UV-irradiated mice secreted significantly more IL-4 than NKT cells isolated from nonirradiated controls. Injecting the wild-type mice with anti-IL-4 blocked the induction of immune suppression. Our findings indicate that UV exposure activates the migration of mature LC to the skin draining LNs, where they induce immune regulation in vivo by activating NKT cells.

SBRT combined with PD-1/PD-L1 inhibitors in NSCLC treatment: a focus on the mechanisms, advances, and future challenges.

Immune checkpoint inhibitors targeting programmed cell death 1 (PD-1), programmed cell death ligand-1 (PD-L1), and others have shown potent clinical efficacy and have revolutionized the treatment protocols of a broad spectrum of tumor types, especially non-small-cell lung cancer (NSCLC). Despite the substantial optimism of treatment with PD-1/PD-L1 inhibitors, there is still a large proportion of patients with advanced NSCLC who are resistant to the inhibitors. Preclinical and clinical trials have demonstrated that radiotherapy can induce a systemic antitumor immune response and have a great potential to sensitize refractory "cold" tumors to immunotherapy. Stereotactic body radiation therapy (SBRT), as a novel radiotherapy modality that delivers higher doses to smaller target lesions, has shown favorable antitumor effects with significantly improved local and distant control as well as better survival benefits in various solid tumors. Notably, research has revealed that SBRT is superior to conventional radiotherapy, possibly because of its more powerful immune activation effects. Thus, PD-1/PD-L1 inhibitors combined with SBRT instead of conventional radiotherapy might be more promising to fight against NSCLC, further achieving more favorable survival outcomes. In this review, we focus on the underlying mechanisms and recent advances of SBRT combined with PD-1/PD-L1 inhibitors with an emphasis on some future challenges and directions that warrant further investigation.

Differential numbers of foci of lymphocytes within the brains of Lewis rats exposed to weak complex nocturnal magnetic fields during development of experimental allergic encephalomyelitis.

To discern if specific structures of the rat brain contained more foci of lymphocytes following induction of experimental allergic encephalomyelitis and exposures to weak, amplitude-modulated magnetic fields for 6 min once per hour during the scotophase, the residuals between the observed and predicted values for the numbers of foci for 320 structures were obtained. Compared to the brains of sham-field exposed rats, the brains of rats exposed to 7-Hz 50 nT (0.5 mG) amplitude-modulated fields showed more foci within hippocampal structures and the dorsal central grey of the midbrain while those exposed to 7-Hz 500 nT (5 mG) fields showed greater densities within the hypothalamus and optic chiasm. The brains of rats exposed to either the 50 nT or 500 nT amplitude-modulated 40-Hz fields displayed greater densities of foci within the midbrain structures related to rapid eye movement. Most of the enhancements of infiltrations within the magnetic field-exposed rats occurred in structures within periventricular or periaqueductal regions and were both frequency- and intensity-dependent. The specificity and complexity of the configurations of the residuals of the numbers of infiltrated foci following exposures to the different fields suggest that the brain itself may be a "sensory organ" for the detection of these stimuli.

Effect of cell phone radiation on neutrophil of mice.

Purpose: The present study aims to evaluate the effect of cell phone radiation on neutrophil of mice. Materials and methods: 40 male BALB/C mice were randomly divided into four groups as control, blank control, TD-CDMA, and LTE-advanced groups, respectively. Mice were exposed to cell phone radiation for a period of 6 weeks. Then numbers of neutrophil were detected by fully automatic hematology analyzer. Soft agar diffusion method was performed to assess the chemotaxis of neutrophils while the phagocytosis of neutrophils was determined by measuring the staphylococcus albus phagocytosis percentage. Apoptosis was analyzed by flow cytometry. Results: No significant differences were observed among the control and exposure groups regarding the numbers of neutrophils after 2 weeks' exposure to cell phone radiation, while the numbers of neutrophils in TD-SCDMA and LTE-advanced groups were seen to rise after an exposure of 4 or 6 weeks. No effect was observed on chemotaxis of neutrophils due to phone radiation. The phagocytosis of neutrophils was decreased while the apoptosis were increased both in TD-SCDMA and LTE-advanced groups after 6 weeks exposure. Conclusions: Mobile phone radiation could give rise to increase of neutrophil numbers yet with no effect whatever on neutrophils chemotaxis, and the radiation was likely to cause decrease of phagocytosis and induced apoptosis of neutrophils.

Complement-derived chemotactic activity is generated in human serum containing uroporphyrin after irradiation with 405 nm light.

Patients with porphyrias have varying degrees of photosensitivity, associated with elevated levels of porphyrins in plasma, erythrocyte, urine and/or feces. To investigate the role of complement in the pathogenesis of cutaneous lesions, varying amounts of uroporphyrin were added to normal human serum (0.1-10 microgram/ml), and the mixtures were then exposed to 405 nm irradiation. Such treatments result in the diminution of total hemolytic complement activity and hemolytic titers of C1, C4, C2, C3, and C5; furthermore, cleavage products of C3 and C5 were detected. Chemotactic activity for human polymorphonuclear leukocytes was generated that was inhibitable by incubation with anti-C5, but not with anti-C3 antisera. No chemotactic activity was generated in Mg++-EGTA treated serum nor in C4-deficient guinea pig serum. These data indicate that irradiation with 405 nm light of normal human serum containing uroporphyrin results in activation of the complement system via the classical pathway, and the generation of complement (C5)-derived chemotactic activity for human polymorphonuclear leukocytes.

Activation of the complement system in patients with porphyrias after irradiation in vivo.

Irradiation of the forearms of two patients with erythropoietic protoporphyria and one patient with porphyria cutanea tarda resulted in an in vivo activation of the complement system, as assessed by diminution of the hemolytic titers of the third component of complement by 23-57%, and of the fifth component of complement (C5) by 19-47%. Such treatment also generated chemotactic activity for human polymorphonuclear cells; the chemotactic activity was stable at 56 degrees C and antigenically related to human C5. On Sephadex G-75 chromatography the chemotactic activity eluted with an apparent molecular weight of 15,000. These in vivo results extend our previous in vitro observation of photoactivation of complement in sera from patients with erythropoietic protoporphyria and porphyria cutanea tarda, and suggest that the complement system may participate in the pathogenesis of cutaneous phototoxicity in these patients.

Inflammatory-type responses after exposure to ionizing radiation in vivo: a mechanism for radiation-induced bystander effects?

Haemopoietic tissues exposed to ionizing radiation are shown to exhibit increased macrophage activation, defined by ultrastructural characteristics and increased lysosomal and nitric oxide synthase enzyme activities. Macrophage activation post-irradiation was also associated with enhanced respiratory burst activities and an unexpected neutrophil infiltration. Examination of p53-null mice demonstrated that macrophage activation and neutrophil infiltration were not direct effects of irradiation, but were a consequence of the recognition and clearance of radiation-induced apoptotic cells. Increased phagocytic cell activity was maintained after apoptotic bodies had been removed. These findings demonstrate that, contrary to expectation, recognition and clearance of apoptotic cells after exposure to radiation produces both a persistent macrophage activation and an inflammatory-type response. We also demonstrate a complexity of macrophage activation following radiation that is genotype dependent, indicating that the in vivo macrophage responses to radiation damage are genetically modified processes. These short-term responses of macrophages to radiation-induced apoptosis and their genetic modification are likely to be important determinants of the longer-term consequences of radiation exposure. Furthermore, in addition to any effects attributable to immediate radiation-induced damage, our findings provide a mechanism for the production of damage via a 'bystander' effect which may contribute to radiation-induced genomic instability and leukaemogenesis.

Solar-simulated ultraviolet radiation induces abnormal maturation and defective chemotaxis of dendritic cells.

Exposure to ultraviolet (UV) light induces immunosuppression. Different evidences indicate that this phenomenon is mainly a consequence of the effect of UV light on skin dendritic cells (DC). To investigate the cellular and molecular basis of this type of immunosuppression, we assessed in vitro the effect of solar-simulated UV radiation on the phenotypic and functional characteristics of human monocyte-derived DC and Langerhans-like DC. UV radiation induced a decreased expression of molecules involved in antigen capture as DC-SIGN and the mannose receptor. This effect was accompanied by a diminished endocytic capacity, an enhanced expression of molecules involved in antigen presentation such as major histocompatibility complex-II and CD86, and a significant increase in their capability to stimulate T cells. Furthermore, irradiated DC failed to acquire a full mature phenotype upon treatment with lipopolysaccharide. On the other hand, solar-simulated radiation induced the secretion of tumor necrosis factor-alpha and interleukin (IL)-10 by DC, but no IL-12. Interestingly, solar-simulated UV radiation also caused an altered migratory phenotype, with an increased expression of CXCR4, and a lack of induction of CCR7, thus correlating with a high chemotactic response to stromal cell-derived factor 1(SDF-1) (CXCL12), but not to secondary lymphoid tissue chemokine (SLC) (CCL21). These data indicate that solar-simulated UV radiation induces a defective maturation and an anomalous migratory phenotype of DC.

Spontaneous effects of low-level laser therapy (650 nm) in acute inflammatory mouse pleurisy induced by carrageenan.

OBJECTIVE: Our aim was to investigate the effect of low-level laser therapy (LLLT), 650-nm wavelength, on acute inflammatory pleurisy. BACKGROUND DATA: There is only scattered evidence of anti-inflammatory effects from LLLT and dosage characteristics, and the effect on pleurisy inflammation has yet to be investigated. METHODS: A classical experimental model of pleurisy was used in a sample of 40 Balb male mice, randomly divided into five groups. Inflammation was induced by carrageenan (0.5 mg/cavity) administered by intrathoracic injections. Four groups received the inflammatory agent, and one received injections of sterile saline solution. At 1, 2, and 3 h after injections, LLLT irradiation was performed, with the same power (2.5 mW), but different irradiation times. The energy densities at each of the three treatment sessions were 0 J/cm(2) (placebo), 3 J/cm(2), 7.5 J/cm(2), and 15 J/cm(2), respectively. RESULTS: Total and differential cell analysis at 4 h after induction of pleurisy showed a significant reduction of inflammatory cell migration for all groups treated with active laser. However, at 4 h after injection, the most significant (p < 0.001) reduction of leukocyte cell migration was seen in the 7.5 J/cm(2) group, at 2.7 (95% CI: 2.5-2.9) x 10(6), versus 7.9 (95% CI: 6.7-9.1) x 10(6) in the placebo control group. The greatest reduction of inflammatory cells was registered for neutrophils. CONCLUSIONS: LLLT administered at 1-3 h after the induction of inflammatory pleurisy significantly reduces the inflammatory cell migration measured. Under these conditions and at 2.5 mW, 7.5 J/cm(2) was more effective than 3 J/cm(2) and 15 J/cm(2).

Effect of 8-methoxypsoralen plus UVA on psoriasis leukotactic factor.

The effect of psoralen phototherapy on the chemotactic activity of psoriasis leukotactic factor (PLF) was studied. The chemotactic activity of PLF extracted from psoriasis scales was evaluated using modified Boyden chambers. Treatment of (1) psoriasis lesions, (2) psoriasis scale and (3) extracted PLF with 8-methoxypsoralen plus UVA irradiation reduced the chemotactic activity of PLF. These results may help define the mechanism of psoralen phototherapy in psoriasis.

Alteration of lymphocyte functions by 8-methoxypsoralen and longwave ultraviolet radiation. I. Suppressive effects of PUVA on T-lymphocyte migration in vitro.

We investigated the influence of 8-methoxypsoralen (8-MOP) plus long-wave ultraviolet radiation (PUVA) on lymphocyte migration in vitro. Nylon wool-purified, mouse splenic T lymphocytes showed locomotive responses to casein, normal mouse serum (NMS), and zymosan-activated mouse serum (ZAS). Migratory responses to casein and NMS, and to ZAS were remarkably suppressed in lymphocytes exposed to 0.5 J/cm2 UVA plus 0.1 micrograms/ml 8-MOP and to 0.8 J/cm2 UVA plus 8-MOP, respectively. The PUVA treatment used in the present study had no effect on random movement and lymphocyte viability. T lymphocytes cultured in the absence of mitogenic agent for 24 h demonstrated a greater increase in their migration activity than noncultured cells, while lymphocytes cultured after 1.0 J/cm2 PUVA pretreatment remained low. These findings suggest that the therapeutic effect of PUVA on inflammatory skin disorders may be due in part to the suppression of lymphocyte migration.

Effect of antiarrhythmic drugs on In-111-labeled leukocytes: chemotaxis and adherence to nylon wool.

The influence of lidocaine (L) and procainamide (P) on the chemotactic ability and adherence to nylon wool of In-111-labeled human polymorphonuclear leukocytes (PMNs) was investigated. At the normal therapeutic levels of L (0.022 mM whole blood) or P (0.03 mM whole blood) no change in PMN function was observed. However, at and above five times the aforementioned blood levels of L, significant reduction in the chemotactic ability of PMNs was noted (p less than 0.005). The adverse effects of In-111 radiation appeared insignificant at all L or P concentrations during the 3-hr observation period. The labeled PMNs were resistant to the toxic effects of a higher concentration of P than that of L, and the reduction in PMN chemotaxis and adherence to nylon wool was not apparent until the P concentration reached 1.5 mM.

Possible application of the laser in immunobiology.

The human immune system acts a defence mechanism against exogenous or indigenous potentially harmful bodies, such as bacteria and viruses. The major histocompatibility complex (MHC class I and class II antigens) form key elements of legitimate body components, and the organization of MHC molecules allows T-lymphocytes to distinguish between legitimate and foreign bodies. On detection of a foreign component, T-cells activate the necessary pathways for destruction of the foreign body. Occasionally however the system breaks down and the result is a disease of an autoimmune nature. Both visible light and infrared low reactive-level laser therapy (LLLT) has been shown to act on immune system cells in a number of ways, activating the irradiated cells to a higher level of activity. Infrared LLLT has been shown to increase both the phagocytic and chemotactic activity of human leukocytes in vitro, for example. This is an example of photobiological activation. Photobiological cell-specific destruction is also possible using doses of low incident laser energy on cells which have been photosensitized for the specific wavelength of the laser, such as in photodynamic therapy (PDT) for superficial cancers. LLLT has also been shown to act directly and selectively on the autoimmune system, restoring immunocompetence to immunocompetence cells. Although much more research needs to be done, there are enough experimental and clinical data to show that the laser, and LLLT in particular, has a possibly exciting role both in immunobiological therapy for diseases of the immune system, and to activate and boost the normal reaction of the immune system components against harmful foreign bodies.

In vitro and in vivo expression of endothelial von Willebrand factor and leukocyte accumulation after fractionated irradiation.

Previous investigations have demonstrated an increased release of von Willebrand factor (VWF; also known as vWF) in endothelial cells after high single-dose irradiation in vitro. We have also found increased levels of Vwf protein in mouse glomeruli after a high single dose of renal irradiation in vivo. In addition, increased numbers of leukocytes were observed in the renal cortex after irradiation in vivo. The aim of the present study was to investigate and quantify these biological processes after clinically relevant fractionated irradiation and to relate them to changes in renal function. A significantly greater increase in release of VWF was observed in cultured human umbilical vein endothelial cells (HUVECs) after fractionated irradiation (20 x 1.0 Gy) than after a single dose of 20 Gy (147% compared to 115% of control, respectively, P < 0.0005). In contrast with the in vitro observations, glomerular Vwf staining was lower after fractionated irradiation in vivo (20 x 2.0 Gy or 10 x 1.6 Gy +/- re-irradiation) than after a single dose of 16 Gy. The number of leukocytes accumulating in the renal cortex was also lower after fractionated in vivo irradiation than after a single radiation dose. The onset of these events preceded renal functional and histopathological changes by approximately 10 weeks. These data indicate that radiation-induced changes in endothelial VWF expression after in vivo irradiation may be distinct from the in vitro observations. Increased VWF expression may reflect pivotal processes in the pathogenesis of late radiation nephropathy and provide a clue to appropriate timing of pharmacological intervention.

Role of CD13/aminopeptidase N in rat lymphocytic alveolitis caused by thoracic irradiation.

CD13/aminopeptidase N is a cell surface glycoprotein that is widely distributed in a variety of mammalian cells. It was recently shown to have chemotactic activity for T lymphocytes. This study examined the role of CD13/aminopeptidase N in lymphocytic alveolitis in radiation-induced lung injury caused by a single-dose thoracic irradiation (15 Gy) in rats. Significantly increased aminopeptidase activity was detected in bronchoalveolar lavage fluid obtained from irradiated rats at 4 weeks after irradiation compared to the activity in unirradiated rats. Significantly higher aminopeptidase activity was detected on alveolar macrophages from irradiated rats at 2 and 4 weeks than on those from unirradiated rats. Western blot analysis showed an increased expression of CD13/aminopeptidase N protein in alveolar macrophages from irradiated rats at 4 weeks. Chemotactic activity for normal rat lymphocytes was detected in bronchoalveolar lavage fluid from irradiated rats at 4 weeks, and approximately 60% of the activity was inhibited by pretreatment of bronchoalveolar lavage fluid with bestatin, a specific aminopeptidase inhibitor. This study suggests that CD13/aminopeptidase N may play an important role as a lymphocyte chemoattractant in lymphocyte-mediated alveolitis in experimental radiation-induced lung injury.

Effect of leukaemia therapy on neutrophil chemotaxis.

The in vivo effect of various cytotoxic drugs and cranial irradiation on neutrophil chemotaxis was tested in 62 children with acute lymphoblastic leukaemia and in 10 patients with other malignant disease. Cranial radiotherapy had a transient adverse effect on neutrophil chemotaxis after completion of the course which was most marked in children. Methotrexate (MTX) and 6-mercaptopurine (6-MP) alone and in combination had a variable effect of chemotaxis, which was most marked nine days after the end of the course. The effect of 6-MP was clearly dose-related, but continuous therapy (75 mg/m2 day) had the greatest inhibitory effect of all the regimens tested. The in vitro effect was studied in 48 leukaemics and in 85 controls (adults and children); all the patients with leukaemia had been off treatment for at least six months. No difference was found between the effects of drugs tested on control or leukaemic cells. The greatest inhibitory effect was found in vinblastine, adriamycin, 6-MP, and vincristine, all of which were closely dose-dependent, MTX, prednisolone, and asparaginase had no effect on chemotaxis when tested in this way.

Structural elucidation of the 8-methoxypsoralen oxidized product that inhibits the chemotactic activity of polymorphonuclear neutrophils toward anaphylatoxin C5a.

The chemical structure of the 8-methoxypsoralen oxidized product that inhibits the chemotactic activity of anaphylatoxin C5a was determined to be 2,3-dihydro-2,9-dimethoxy-3-hydroxy-7-oxo-7H-furo[3,2-g] [1]benzopyran. Its minimal concentration required to obtain the maximum inhibition of C5a des Arg (1 x 10(-8) M) chemotactic activity is 2.5 x 10(-8) M. Bioactivity of this substance was maintained for 2 weeks, stored in a dark room at room temperature under aerobic conditions. There is a possibility that this substance may be useful in the treatment of immune complex diseases.

Effect of psoralen + ultraviolet-A on the chemotactic activity of polymorphonuclear neutrophils towards anaphylatoxin C5a des Arg.

It was shown that psoralen + UV-A inhibits the chemotactic activity of polymorphonuclear neutrophils towards anaphylatoxin C5a des Arg. This reaction required oxygen and there is a high possibility that the active oxygen species was in a singlet state. Oxygen did not act directly on C5a des Arg, but rather produced oxidized psoralen which inhibited C5a des Arg activity. The effect was dependent on the concentration and types of psoralen and on the UV-A dose. Among the psoralens, the inhibitory effect of 4,5',8-trimethylpsoralen was the strongest, followed by 8-methoxypsoralen and 3-carbethoxypsoralen and finally 4,4',6-trimethylangelicin. Psoralen + UV-A failed to inhibit the chemotactic activity towards chemotactants other than anaphylatoxin C5a such as casein, the cultured filtrate of E. coli, platelet-activating factor, leukotriene B4, 5-hydroxy-6,8,11,14-eicosatetraenoic acid and 12-L-hydroxy-5,8,10,14-eicosatetraenoic acid.