Chronic Obstructive Pulmonary Disease (COPD)

Ter Arkh. 2016;88(3):32-5.

Pathogenetic effects of low-intensity laser therapy for chronic obstructive pulmonary disease.

[Article in Russian]
Aksenova IZ1, Burduli NM1.

Author information

  • 1North Ossetian State Medical Academy, Ministry of Health of Russia, Vladikavkaz, Republic of North Ossetia-Alania, Russia.

Abstract

AIM:

To study the impact of low-intensity laser therapy (LILT) on typical pathological processes of impairments in platelet aggregation and microcirculation (MC) in patients with chronic obstructive pulmonary disease (COPD).

MATERIALS AND METHODS:

A photo optical aggregatometric method was used to investigate platelet aggregation function in patients with COPD and MC was estimated by laser Doppler flowmetry over time.

RESULTS:

There were muitidirectional changes in platelet aggregation function with a predominance of hyperaggregation, as well as a significant improvement in aggregation indicators during LILT; the latter was shown to have a correcting impact on MC disorders in patients with COPD in the presence of the spastic and stagnant-stasic types of MC, which were prevalent in the majority of patients.

CONCLUSION:

Laser therapy used in the combination treatment of patients with COPD promotes correction of the typical pathological processes.

PLoS One. 2015; 10(8): e0136942.
Published online 2015 Aug 31. doi: 10.1371/journal.pone.0136942
Human Tubal-Derived Mesenchymal Stromal Cells Associated with Low Level Laser Therapy Significantly Reduces Cigarette Smoke-Induced COPD

Jean Pierre Schatzmann Peron,1 Auriléia Aparecida de Brito,2 Mayra Pelatti,3 Wesley Nogueira Brandão,1 Luana Beatriz Vitoretti,2 Flávia Regina Greiffo,2 Elaine Cristina da Silveira,2 Manuel Carneiro Oliveira-Junior,3 Mariangela Maluf,4 Lucila Evangelista,5 Silvio Halpern,5 Marcelo Gil Nisenbaum,5 Paulo Perin,4 Carlos Eduardo Czeresnia,5 Niels Olsen Saraiva Câmara,6 Flávio Aimbire,7 Rodolfo de Paula Vieira,3 Mayana Zatz,2 and Ana Paula Ligeiro de Oliveira3,*
Yuanpu Peter Di, Editor
1Neuroimmune Interactions Laboratory, Department of Immunology, Institute of Biomedical Sciences, University of Sao Paulo, São Paulo, SP, Brazil
2Laboratory of Pulmonary and Exercise Immunology–LABPEI, Nove de Julho University (UNINOVE), São Paulo, SP, Brazil
3Division of Human Genome Research Center, Biosciences Institute, University of São Paulo, São Paulo, SP, Brazil
4CEERH—Specialized Center for Human Reproduction, Division of Reproductive Medicine, São Paulo, SP, Brazil
5Division of Reproductive Medicine—Célula Mater, São Paulo, SP, Brazil
6Laboratory of Transplantation Immunobiology, Department of Immunology, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP, Brazil
7Department of Science and Technology, Federal University of São Paulo, São José dos Campos, SP, Brazil
University of Pittsburgh, UNITED STATES
Competing Interests: The authors have declared that no competing interests exist.
Conceived and designed the experiments: JPSP PP CEC APLdO. Performed the experiments: MM LE SH MGN PP CEC JPSP AAdB MP ECdS WNB LBV FRG MCO-J. Analyzed the data: JPSP NOSC FA RdPV MZ APLdO. Contributed reagents/materials/analysis tools: JPSP NOSC FA RdPV MZ APLdO. Wrote the paper: JPSP MZ APLdO.* E-mail: moc.liamg@oriegilpa
Author information ? Article notes ? Copyright and License information ?
Received 2015 Feb 2; Accepted 2015 Aug 11.
Copyright notice
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
This article has been corrected. See PLoS One. 2015 September 25; 10(9): e0139294.Abstract
Cigarette smoke-induced chronic obstructive pulmonary disease is a very debilitating disease, with a very high prevalence worldwide, which results in a expressive economic and social burden. Therefore, new therapeutic approaches to treat these patients are of unquestionable relevance. The use of mesenchymal stromal cells (MSCs) is an innovative and yet accessible approach for pulmonary acute and chronic diseases, mainly due to its important immunoregulatory, anti-fibrogenic, anti-apoptotic and pro-angiogenic. Besides, the use of adjuvant therapies, whose aim is to boost or synergize with their function should be tested. Low level laser (LLL) therapy is a relatively new and promising approach, with very low cost, no invasiveness and no side effects. Here, we aimed to study the effectiveness of human tube derived MSCs (htMSCs) cell therapy associated with a 30mW/3J—660 nm LLL irradiation in experimental cigarette smoke-induced chronic obstructive pulmonary disease. Thus, C57BL/6 mice were exposed to cigarette smoke for 75 days (twice a day) and all experiments were performed on day 76. Experimental groups receive htMSCS either intraperitoneally or intranasally and/or LLL irradiation either alone or in association. We show that co-therapy greatly reduces lung inflammation, lowering the cellular infiltrate and pro-inflammatory cytokine secretion (IL-1?, IL-6, TNF-? and KC), which were followed by decreased mucus production, collagen accumulation and tissue damage. These findings seemed to be secondary to the reduction of both NF-?B and NF-AT activation in lung tissues with a concomitant increase in IL-10. In summary, our data suggests that the concomitant use of MSCs + LLLT may be a promising therapeutic approach for lung inflammatory diseases as COPD.Introduction
According to the World Health Organization (WHO), more than five million deaths per year are the result of direct use of tobacco, whereas more than 600 000 second hand smokers also perish from cigarette exposure (World Health Organization, 2014). According to the US Centers for Disease Control and Prevention, cigarette smoking is the dominant risk factor for the development of chronic obstructive pulmonary disease (COPD) and emphysema [1,2]. A wide range of dangerous agents are found in cigarette smoke and, aside from solid particles, it includes more than 4000 chemicals, of which at least 250 are known to be highly harmful and more than 50 are tumorigenic [2]. Altogether, these factors may induce airway inflammation, cellular recruitment, lung fibrosis, mucus hypersecretion and also cancer [2,3]. Among the features of cigarette smoke-induced pathology, parenchymal fibrosis and emphysema may be considered the most characteristic and also the most deleterious, resulting in a significant organic impairment and restricted life quality of the patients [3,4].Lung inflammation induced by cigarette smoke is characterized by an initial phase of inflammatory cells recruitment to the parenchymal space, matrix metalloproteinase (MMPs) activation, extracellular matrix degradation and tissue damage [3,4]. This is followed by the intense release of pro-inflammatory cytokines, such as IL-1 [5] and IL-6 [3], chemokines as CCL2 and CXCL1 and also lipid mediators, as PGI and LTB4, as reviewed [2]. These molecules are able to recruit more inflammatory cells, as neutrophils, macrophages and also CD4 and CD8 T lymphocytes of the Th1 and Tc1 IFN-?-secreting subtypes, respectively, perpetuating lung tissue damage [6]. Chronic exposure to cigarette noxious agents leads to the perpetuation of the inflammatory and fibrotic processes, with more and more infiltrating cells recruited, progressive elastin and collagen degradation, followed by parenchymal destruction and finally the establishment of lung emphysema [3,7]. Thus, campaigns to prevent cigarette smoking and to alert the population about its morbidity are extremely important. However the need for effective and also less expensive therapeutic approaches for the treatment of cigarette smoke-induced emphysematous patients is unquestionable. In this context, mesenchymal stromal cells (MSCs) therapy seem very promising, not only due to its regenerative capacity and immunomodulatory features, but also due to its low cost and relatively easy manipulation, as reviewed [4].

Adult MSCs are typically defined as undifferentiated multipotent cells endowed with the capacity for self-renewal and the potential to differentiate into several distinct cell lineages, as recently reviewed [8]. These cells may be obtained from different organs and tissues, as bone marrow [9,10], skeletal muscle [11], adipose tissue [12,13], dental pulp [14], umbilical cord [15,16], fallopian tubes [17] and other tissues. It is noteworthy that MSCs are well tolerated in vivo, either under syngeneic [18–19] or xenogenic conditions [20, 21, 22]. Concerning its immunomodulatory function, MSCs are known to: i) secrete anti-inflammatory cytokines; ii) express reduced levels of MHC and costimulatory molecules; iii) express the tryptophan-depleting enzyme indoleamine-2,3-dioxigenase [23] iv) induce T regulatory cells [18] and many others. Altogether, these mechanisms may be useful tools for the treatment of several chronic inflammatory diseases such as multiple sclerosis, arthritis, diabetes, lupus, and also COPD and emphysema [24–26]. Moreover, the possibility to use associated approaches able to boost the overall response must be investigated.

In this sense, Low Level Laser Therapy (LLLT) seem to be an interesting approach, specially due to its non-invasiveness, lack of secondary effects, low cost [27] and ability to promote stem-cell proliferation in vitro [28]. LLLT has already shown interesting results for different human diseases, as oral mucositis [29], coronary disease [30] and also in experimental models, as for muscle dystrophy [31], asthma [32] and articular inflammation in mice [33]. Actually, our group has recently demonstrated that LLLT is able to reduce lung inflammation both during the asthma model [32] as well as secondary to gut ischemia Among the possible mechanisms proposed, we have recently demonstrated that alveolar macrophages irradiated with LLL had augmented AMPc synthesis, and this is able to reduce NF-?B activation and thus IL-6 and IL-1 secretion [21]. In this context, we decided to evaluate the effectiveness of associating human tubal mesenchymal stromal cells (htMSCs) and LLLT using the murine model of cigarette smoke-induced COPD. It is worthy to mention that we have previously reported that htMSCs display stemness properties and also miogenic and adipogenic capacity to differentiate [17].

Here, our results demonstrate that the co-therapy with htMSCs and LLLT are very effective in lowering important pulmonary inflammatory parameters, as cytokine secretion, cellular infiltrate of leukocytes, mucus secretion, collagen deposition and the activation of important transcription factors, as NF-?B and NF-AT. Altogether, we show that the association of stem cells therapy with LLL irradiation are clinically beneficial, and thus, may be considered as further interesting therapeutic approach.

Materials and Methods
Cigarette Smoke–Induced Emphysema and Treatment

Female C57BL/6 mice (6–8 weeks) were exposed to cigarette smoke during 75 days by whole body exposure using an adapted protocol from Biselli et all [34]. All experiments were performed on day 76. Briefly, animals were exposed to 14 commercially available cigarettes (containing Alcatrão 13mg, nicotine 1,10 mg and carbon monoxide 10 mg each) divided on 7 cigarettes in the morning and 7 cigarettes in the afternoon and each session lasted for 30 minutes. LLL irradiation with diode laser (30mW/3J at 660 nm) was performed twice a day from day 60 to day 75 (one hour after each cigarette exposure) during 180 seconds on the skin over the right upper bronchus with a spot size of 0.785 cm2. htMSCs (1×106 cells) were infused on days 60 and 67. Experimental groups were divided in 7 groups, as follow: 1) Basal 2) COPD 3) COPD + LLL 4) COPD + MSCs (i.p) 5) COPD + MSCs (i.n) 6) COPD + MSCs (i.p.) + LLL 7) COPD + MSCs (i.n.) + LLL. We declare that all experiments were approved by the UNINOVE animal research committee (Comitê de Ètica no uso de Animais—CEUA—#AN005/2013) and human research committee (Comitê de Ética em Pesquisa com Seres Humanos (CEPSH) of University of Sao Paulo (# CEP-IBUSP-106/2010).

htMSCs Isolation and Culture

Human tubal tissue was obtained from women (age 35–53 years) submitted to hysterectomy or tubal ligation/resection surgery. Samples were collected during the proliferative phase from fertile women who had not undergone exogenous hormonal treatment within the last three months. htMSCs experiments were approved by both the ethics committee of the Biosciences Institute of the University of São Paulo and the UNINOVE–Universidade Nove de Julho. Each sample was collected in HEPES-buffered Dulbecco Modified Eagle Medium / Hams F-12 (DMEM/F-12; Invitrogen, Carlsbad, CA) with 100 IU/mL penicillin (Invitrogen) and 100 IU/mL streptomycin (Invitrogen, Carlsbad, CA), maintained at 4°C and processed within 24 hours period. All samples were washed twice in phosphate saline buffer (PBS, Gibco, Invitrogen, Carlsbad, CA), finely minced with a scalpel and transferred to a 50 mL tube, and dissolved with collagenase IV (Sigma-Aldrich) at 0,1% diluted in PBS (Invitrogen) for 15 minutes, at 37°C, in a water bath. Further, samples were washed with 10 mL of DMEM/F-12 and added with 15 ml of pure TripLE Express, (Invitrogen, Carlsbad, CA) for 15 minutes at 37°C in a water bath. Subsequently, TripLe was inactivated with DMEM/F-12 supplemented with 20% FBS, 100 IU/mL penicillin and 100 IU/mL streptomycin, and pelleted by centrifugation at 400g for five minutes at room temperature. Supernatant was removed with a sterile Pasteur pipette. Cells were then plated in DMEM/F-12 (5mL) supplemented with 20% FBS, 100 IU/mL penicillin and 100 IU/mL streptomycin in plastic flasks (25cm2), and maintained in incubator with controlled humidified atmosphere of 5% CO2 at 37°C. The medium used for expansion was initially changed every 72 hours and routinely replaced twice a week thereafter. Data concerning osteogenic, chondrogenic and adipogenic differentiation, as well as cellular phenotype were previously published by our group [17] demonstrating the stem cell potential of this population. Human tubal tissue was obtained after written and signed informed consent approved by Comitê de Ética em Pesquisa com Seres Humanos (CEPSH) of University of Sao Paulo (# CEP-IBUSP-106/2010) and maintained at the Division of Human Genome Research Center, Biosciences Institute, University of São Paulo, São Paulo–SP—Brazil.

Bronchoalveolar Lavage Fluid Cells and Cytospin

After euthanasia, bronchoalveolar lavage fluid was obtained as previously described [35]. Briefly, 1.5 mLs of PBS as intra-tracheally injected in the lungs and 1 mL was re-collected and centrifuged at 450 g 4°C during 5 minutes. Supernatants were discarged and the pellet suspended in the desired amount of PBS 2% FBS. Total cell count was performed in Newbauer chambers and differential cell count after cytospin protocol. For cytospin, aliquots of 100 ?L were centrifuged at 300 g for 5 minutes. Samples were stained by May-Grunwald-Giemsa method and 300 cells per sample were counted on a blind fashion.

Lung Mononuclear Cells and Flow Cytometry

Lungs were obtained after right heart perfusion of 10 mLs of cold PBS. The tissue was minced with scissors and incubated for 45 minutes at 37°C with 2,5 mg/mL of Colagenase D (Roche) in HBSS. To stop collagenase, cells were suspended in Ca2+/Mg2+ free HBSS and centrifuged at 450 g and 4°C for 5 minutes. Cellular pellet was suspended in 5–6 mLs of 37% Percoll (GE) in HBSS and gently laid over 5–6mLs of 70% Percoll in HBSS using 15 mLs conical falcon tubes. Samples were centrifuged at 950 g and 4°C for 30 minutes without breaks. Cell-containing ring was collected from the percoll gradient interface and suspended in PBS 2% FBS and centrifuged again at 450 g and 4°C for 5 minutes. Further, cells were suspended in PBS 2% FBS, counted and used as desired. For flow cytometry analysis, 5×105 cells were first incubated with 0,25 ?g of anti-CD16/32 at 4°C for 20 minutes to avoid inespecific binding. Cells were then stained with 0,25 ?g of anti-CD4 APC and 0,25 ?g of anti-CD8 PE in 25 ?L of PBS 2% FBS for 20 minutes at 4°C. In the next step, cells were washed twice with 200 ?L of PBS 2% and finally suspended in paraformaldehyde 1%. Cells were acquired in the flow cytometer Accuri C6 (BD Biosciences). 5×103 T CD4+ events were collected after elimination of cell doublets by FSH-height x FSC-area plot analysis.

Cytokine Secretion

BAL lavage fluid was centrifuged at 450 g and 4°C during 5 minutes. Supernatants were used to evaluate the presence of IL-1?, IL-6, IL-10, TNF-?, IFN-? and KC by the ELISA method according to manufacturer instruction (R&D System).

Histomorphometric Analysis of Inflammation, Collagen and Mucus

After perfusion, lung samples were maintained in formaldehyde 4% for inclusion in paraffin. Slices were stained with Siriud Red for collagen detection or Periodic Acid Schiff (PAS) for the detection of mucus. Were analyzed 15 airways per each animal under a 400X magnification. H&E staining was used for the analysis of peribronchial infiltrate and mean linear intercept measurement [36]. Morphological features were analyzed using the Image Pro Plus 4.5 (Media Cybernetics, Rockville, MD, USA).

Peribronchial Inflammation

The area between the airway basal membrane and the airway adventitia was quantified using the software Image Pro-Plus and the number of mononuclear and polymorphonuclear cells was quantified in this area according to the morphological criteria. The results were expressed as the number of mononuclear and polymorphonuclear cells per square millimeter.

Mucus Production and Collagen Fibers Deposition

For the analysis of mucus production, the epithelium area of the whole airway, 15 airways per mouse were quantified. The positive stained area in the epithelium area was quantified and the results were expressed as the percentage of positive epithelial area. For the analysis of collagen fibers deposition in the airways wall, the area between the outlayer of epithelium until airway adventitia were measured and the amount of positive stained area was calculated. The results were expressed as percentage aof collagen fibers in the airway wall [37–39].

Immunohistochemistry

For immunohistochemistry analysis, all samples were previously submitted to deparaffinization. Endogenous peroxidase activity was blocked with H2O2 3% three times during 10 minutes. Samples were then washed and blocked with bovine serum albumin 10% during 1 hour and then incubated with primary rabbit anti-mouse IgG antibodies: i) anti-NF?B (Santa Cruz, CA) at 1:500; ii) anti-NF-AT 1:500 (Santa Cruz, CA) and iii) anti-IL-10 (Santa Cruz, CA) at 1:500 during 2 hours at room temperature. Samples were washed twice with TBS- BSA 10% and then incubated with secondary antibody goat anti-Rabbit IgG at 1:1000 for 1 hour. After washing samples twice with BSA 2% diaminobenzidine (DAB) was added. Finally samples were washed again and counter stained with H&E. Slides were analyzed under light microscope on a blind fashion.

Statistical Analysis

All analysis were performed using the GraphPad Prism software (GraphPad Software, Inc). For parametric analysis we used one-way ANOVA followed by Tukey post-test whereas for non-parametric analysis, we used Kruskal-Wallis followed by Dunn´s post-test. All groups were compared to COPD control group. Differences were considered significant when p<0,05.

Results
htMSCs and LLLT Reduce Bronchoalveolar Lavage Fluid Cellularity in the Lungs of COPD Animals

To evaluate whether htMSCs and/or LLLT were effective in reducing lung inflammation after cigarette smoke-induced COPD, we first analyzed BAL fluid cellularity from all experimental groups for total and differential cell counts. Interestingly, a significant reduction in total cell counts, macrophages and neutrophils was observed only when htMSCs were associated with LLL therapy, although a trend is observed in all other groups, even for LLL alone (Fig 1A, 1B and 1C). However, we had a discrepant result for lymphocyte counts, as we observed that only htMSCs by i.p. delivery was able to reduce its amount. Association with LLLT did not changed the result. It is worthy to mention that the lowest amount of total cells (Fig 1A), macrophages (Fig 1B) and neutrophils (Fig 1C) was observed in the group of htMSCs associated with LLLT, either i.p or i.n, demonstrating that association with LLLT is more important for these features than the route of htMSCs delivery. It is also noteworthy that such reduction in BAL cellularity reached levels no different from naïve controls. Moreover, intraperitoneal delivery of htMSCs seemed more effective than intranasal delivery in reducing neutrophil and lymphocyte infiltration.

Fig 1
Fig 1
Reduced cellular infiltration in the lungs of htMSC and LLLT treated COPD animals.
Next, we performed flow cytometry analysis in the BAL fluid from all experimental groups to evaluate changes in the frequency of CD4+ and CD8+ T cells in the lungs. This could be indicative, either of a reduced T cell activation in the lymph nodes, or an impairment in cell migration to the target organ. In fact, corroborating findings from Fig 1 for polymorphonuclear and total cells, the frequency of both T CD4+ (Fig 2A and 2B) and T CD8+ (Fig 2C and 2D) cells were significantly reduced irrespective of the therapeutic approach used, i.e, htMSCs alone or in association with LLLT. However, no differences were observed, when lung tissue was analyzed for perivascular infiltrate by immunohistochemistry (S1 Fig).

Fig 2
Fig 2
Reduction of T CD4+ and T CD8+ T cells infiltration in the lungs of htMSC and/or LLLT treated animals.
htMSCs and/or LLLT Reduce Inflammatory Cytokines in the BAL Fluid of COPD Animals

We next analyzed the amount of cytokines as IL-1?, IL-6, TNF-?, IFN-? and the chemockine KC, to evaluate whether reduced cellularity in the lungs of our experimental culminated also in reduction of inflammatory mediators. Our results show that IL-1? was reduced when htMSCs either ip. or i.n. were associated with LLLT, although htMSCs i.n alone also reached significance (Fig 3C). The same pattern was observed for TNF-? (Fig 3A). KC, an important chemokine for the recruitment of neurotrophils, was reduced in all groups irrespective of the treatment (Fig 3F). Interestingly, IL-6 was reduced only when htMSCs were associated with LLL (Fig 3B) whereas no difference was observed for IFN-? (Fig 3D) Very interestingly, the anti-inflammatory cytokine IL-10 was up-regulated only with LLL irradiation alone (Fig 3E).

Fig 3
Fig 3
Reduced cytokine secretion in the lungs of htMSC and/or LLLT treated animals.
htMSCs and/or LLLT Reduced Mucus Secretion and Alveolar Enlargement in the Lungs of COPD Animals

Another important feature in the pathophysiology of COPD is the intense secretion of mucus, resulting in reduced airway lumen, airflow and breathlessness. Thus, we next evaluated whether either htMSCs or LLL therapies, associated or not, were able to reduce the amount of mucus observed in the airways. In fact, our data demonstrated significantly decreased amount of mucus in the bronchi of all experimental animals under therapeutic protocols, with the exception of the LLLT alone. Significant differences reached from 10 to 15-fold reduction when experimental groups were compared to COPD control. On the other hand, when associated with htMSCs, either nasally or intraperitoneally, LLL therapy restored mucus secretion to basal levels, with no difference from naïve animals (Fig 4A and 4B). We next decided to verify some possible effects of htMSCs and LLLT in the enlargement of lung parenchyma, which reflects the destruction of the alveolar septa. As indicated by S2 Fig, mean linear intercept measures were significantly reduced in the COPD + MSC(ip)+LLLT group when compared to control animals, indicating reduced lung damage.

Fig 4
Fig 4
Reduced mucus secretion in the lungs of htMSC and/or LLLT treated animals.
Collagen Deposition Was Reduced Only When htMSCs Were Associated with LLL

Aside from mucus secretion, collagen deposition is also considered an important marker for COPD. Therefore, we also evaluated the amount of peribronchial collagen deposition through Sirius Red methodology (Fig 5A and 5B). Very surprisingly, a significant collagen decrease of around 4-fold was observed only when LLLT was associated with htMSCs intranasally. Although there was a trend for the htMSCs (i.p) + LLLT, none of the other groups reached statistical significance (Fig 5B).

Fig 5
Fig 5
Reduced collagen deposition only in the lungs of htMSC i.n and LLL treated animals.
htMSCs and/or LLLT Reduces NF-?B and NF-AT Transcription Factors Expression in the Lungs of COPD Animals

Most pro-inflammatory cytokines evaluated, such as IL-1, IL-6 and TNF-?, have their transcription under the control of the transcription factor NF-?B. Moreover, these cytokines may also signal through NF-?B after engagement with its receptors cognate recptors. NF-?B is found in a great variety of cell types, as macrophages and neutrophils, but also T lymphocytes. Thus, with the aim to correlate the overall reduction in BAL cellularity and pro-inflammatory cytokine secretion with a reduction in NF-?B expression, we sought to evaluate its expression by immunohistochemistry of the perialveolar space of COPD animals (Fig 6). Corroborating the reduction in pro-inflammatory cytokine secretion (Fig 3C), all groups displayed significantly reduced NF-?B staining in the target tissue when compared to the COPD control group (Fig 6B). Differences vary from 2 to 3-fold depending on the treatment.

Fig 6
Fig 6
Reduced total NF-B staining in the lungs of htMSC i.n and/or LLLT treated animals.
Another transcription factor also important for the activation of the immune cells is the NF-AT, which is mostly expressed on T cells, both CD4+, CD8+ and NK cells, and also in the lung tissue, as shown by the literature [40]. NF-AT is the main transcription factor involved in the synthesis of IL-2 and its active form is the non-phosphorilated state, as reviewed [41]. It was very surprising to notice that LLLT by itself was already able to significantly reduce NF-AT activation (Fig 7A). This reduction was no different from that observed when LLL was associated with htMSCs i.n. (Fig 7A).

Fig 7
Fig 7
Reduced total NF-AT staining in the lungs of htMSC i.n and/or LLLT treated animals.
LLL Therapy Boosts IL-10 Secretion in Lung Tissue

In order to confirm the data obtained by the ELISA method, and concerning its importance as an anti-inflammatory factor, we also evaluated by immunohistochemistry the amount of IL-10 in lung tissue of the experimental groups. Interestingly, LLL irradiation per se was able to increase the amount of epithelial IL-10 (S3 Fig) whereas htMSCs i.p. was not able to so. Consistently, this was also observed only when htMSCs were associated with LLL therapy, although htMSCs i.n. alone could also up-regulate this cytokine (S3 Fig).

Discussion
In the present research we show that the treatment with htMSCs associated with LLL irradiation significantly reduced lung inflammation in mice with cigarette smoke-induced COPD. Many of the evaluated parameters, such as BAL cellularity, pro-inflammatory cytokine secretion, perivascular infiltrate and the presence of inflammatory transcription factors, as NF-?B and NF-AT were significantly reduced. This was associated with a better maintenance of tissue integrity when compared to COPD untreated controls, evidenced by reduced mucus secretion, collagen deposition and tissue damage. It is worthy to mention that these features are greatly responsible for tecidual destruction and further decline in patient´s life quality, as it greatly reduces airflow, lung complacence and lowering pO2 [2,42]. Moreover, our findings also demonstrate that the route of administration of htMSCs, i.e. intraperitoneal vs. intranasal, were both effective in suppressing the disease, although with some peculiarities. For instance, intranasal delivery was more effective in reducing the presence of NF-AT as well as collagen deposition when associated with LLL therapy, which was not observed after intra-peritoneal injection. This might be explained by a local regenerative/suppressive mechanism, as the cells were directly delivered to the lungs. For intra-peritoneal route, however, immune modulation on lymphoid organs, specially mediastinal lymph nodes is more likely.

Cigarette smoking-associated diseases, which may result in severe decrease of life quality represent an important social and economical burden, as billions of dollars are spent each year in the treatment of emphysematous patients worldwide. Despite the constant campaigns against tobacco, it is still the fourth leading cause of death in the United States [43]. Therefore, the need for a more efficient and yet cheaper therapeutic approach in the management of COPD patients is unquestionable. Cell therapy with MSCs has shown clinical benefits by us and other groups in conditions such as experimental autoimmune encephalomyelitis (EAE)[22], multiple sclerosis [44], chronic renal failure [13], ischemic cerebral stroke [9], myocardial stress [45] and even for COPD. In fact, several of our findings were corroborated by previous research, in which lung inflammation, weight loss and lung integrity were ameliorated after adipose-tissue MSCs treatment [26]. On the other hand, a recent clinical trial on MSCs have not shown promising results, as COPD patients treated with MSCs (Prochymal) had not shown improvement of lung function, as shown for forced expiratory volume (FEV1) and forced volume capacity (FVC). Interestingly however, it seemed that, corroborating our findings, lung inflammation was decreased, inferred by the lower level of C-reactive protein [46]. Our report however, although using an experimental model, is the first to associate htMSCs with LLL irradiation, considered a promising approach [27].

COPD is a chronic and obstructive disease of the lungs, result of the chronic exposure of the airways to the noxious agents found in cigarette smoke and droplets. It has been established that lung destruction directly correlates with the amount of total particulate matter found in each cigar [7]. Initial exposure leads to cellular infiltrate of neutrophils and blood-derived monocytes secreting pro-inflammatory and pro-fibrotic cytokines, as IL-1, IL-6, IL-12, TNF-? and TGF-?, along with chemokines, lipid mediators and several other molecules [2,3]. This is greatly accompanied by extracellular matrix degradation by MMP-1 (matrixmetalloproteinase-1) secreted by alveolar macrophages causing tissue destruction and emphysema [3]. Chronic exposure leads to intense T CD4 and T CD8 lymphocyte infiltration, and IFN-? is probably the most important T cell-derived cytokine. It is relevant that IFN-?-secreting T CD8 cells are also very important in the late phase of the disease [47]. Due to its pro-inflammatory function, and in association with other cytokines, as TNF-?, TGF-?, IFN-? induces important activation of both immune and parenchymal cells of the lung and finally leading to fibrosis and parenchymal destruction [2,3]. In this context, therapeutic approaches with the capacity to dampen such activation could greatly contribute to the maintenance of lung integrity and both MSCs and LLL therapy independently had already proven it [10,32]. However, we did not observe differences for IFN-?.

A growing body of evidence have shown several different immunosuppressive mechanisms of MSCS, such as: lowering the expression of MHC and costimulatory molecules; up-regulation of indoleamine-2,3-dioxigenase [19,48] and FAS-L expression [49]; expansion of Tregs [48]; block of IFN-? and IL-17 secretion [22], reduces tecidual caspase-3 activation [10] and many others, as reviewed [4]. Altogether, these mechanisms greatly impair immune cells activation, further avoiding or reducing tissue inflammation and destruction. However, although we may only speculate the mechanisms used by the htMSCs + LLL irradiation to suppress COPD, it is suitable to think that many of the aforementioned mechanisms may be taking place in our system.

Corroborating our findings, several other groups have already addressed the capacity of MSCs in modulating chronic diseases, including COPD. Bone marrow-derived MSCs were shown to significantly reduce tissue destruction in COPD mice by a mechanisms dependent on VEGF. Consistently, the group observed among many other cytokines, reduction in IL-1? and IL-6, in accordance with our data. More relevant was the fact that lung function, including inspiratory capacity and vital capacity were significantly higher when compared to control animals, which is again discrepant from data obtained in humans [10].

Adipose tissue-derived MSCs (ASCs) transplantation was also shown to be effective in improving COPD in mice reducing lung infiltration of inflammatory cells, as neutrophils and macrophages reduced associated with parenchymal destruction, as evaluated by active caspase-3 [12]. In line with our findings, the group also demonstrated reduced lung infiltration of inflammatory cells, as total cells, neutrophils and macrophages.

Associated with its immunosuppressive activity, it is also possible that MSCs exert some regenerative/reparative function in situ. For instance, intra-tracheal delivery of bone marrow-derived stem cells after hyperventilation-induced injury in rats greatly improved lung recovery, reducing cellular infiltrate of neutrophils and lymphocytes, pro-inflammatory cytokine secretion, associated with normal alveolar space [50]. In accordance, we have observed that intra-nasally treated animals have also displayed a significant reduction in several inflammatory markers, as cellular infiltrate and cytokine secretion. Moreover, we also observed amelioration in alveoli enlargement in animals treated with htMSCs(ip) + LLL, evidenced by mean linear intercept, but unfortunately lung function was not assessed by us. However, it is noteworthy that collagen deposition, a very significant factor for tissue fibrosis and lung emphysema, was significantly reduced when htMSCs were associated with LLLT. This may indicate that LLLT boosts htMSCs regerative capacity, however we may not describe the mechanisms so far.

LLLT started to be studied in the late 60`s, when reports had shown its use in improving hair growing in rats. Several other reports soon followed using different models, as wound healing and muscle regeneration. On the other hand, the effect of laser therapy is poorly understood. We have previously observed that the in vitro proliferative capacity of dental pulp derived mesenchymal stem-cells increased with low intensity laser application [28]. Interestingly, recent reports have shown that LLL therapy exert its function by increasing intracellular AMPc and thus suppressing important inflammatory transcription factors as NF-?B [51], which is consistent with our findings. In fact, the intriguing capacity of LLL therapy in suppressing the immune response has been previously shown by us and others, as mentioned. LLLT irradiation had the capacity to reduce joint inflammation in rats treated locally with papain. Consistently, irradiated groups demonstrated reduced cellular infiltration and also reduction in IL-1? and IL-6, corroborating our findings [33]. In the context of lung disease, we have previously published that LLLT reduces lung inflammation using OVA-induced lung inflammatory disease [32]. In this case, several parameters, as cellular infiltrate of the lungs, cytokine secretion, mucus secretion and collagen deposition were significantly reduced after irradiation and antigen challenge. As expected, many of these results were reproduced in the present research, and also it indicated that htMSCs and LLLT may act by an additive manner.

Aside from overall reduction in lung inflammation, another interesting observation was the reduction of the transcription factors NF-?B and NF-AT which could be secondary to the reduced infiltration of macrophages / neutrophils and lymphocytes in lung tissue, respectively. Moreover, we have also detected lower levels of IL-1? in BAL fluid, which is known to signal through IL-1r and thus induce NF-?B activation. However, we may not exclude the possibility that LLLT could also directly abrogate NF-AT activation, as observed when it was used alone. It is plausible to think that LLLT somehow changes intracellular Ca2+ levels, consequently modulating NF-AT activation by calcineurin / calmodulin. It is relevant that NF-AT, which may also be found in lung arteries, is responsible for up-regulating the expression of smooth muscle ?-actin and myosin heavy chain after hypoxia, a very important mechanism for chronic hypoxia-induced pulmonary vascular remodeling [52].

In summary, our results clearly indicate that, aside from lack of toxicity and other complications, both htMSCs and LLLT were shown to be safe for the treatment of COPD in an experimental model. More important, however, is the fact that although htMSCs and LLLT could act independently, some pathological parameters were effectively reduced when both therapies were associated. It may indicate an important additive effect that may be responsible for the overall reduction in lung inflammation and tissue destruction in cigarette smokers. Our results of the combined use of htMSCs and LLLT were effective in reducing inflammatory immune response and further overall destruction of lung parenchyma in experimental COPD. Thus, we encourage other groups to keep focus on the potential of MSCs in COPD, and also to consider the relevance of associating it with LLLT. This is not only to reinforce our data, but mainly to refine the therapeutic scheme with the aim to reach a more translational approach.

Conclusions
Our research highlights the important suppressive capacity of htMSCs in reducing overall lung inflammation during COPD in mice. Besides, we also observed a beneficial additive effect when htMSCs and LLLT are associated. Reduced lung cellularity and cytokine secretion, mucus production, collagen deposition and transcription factors activation are among the downregulated parameters. Interestingly, our data also show that this phenomenom is very consistent, irrespective of the MSCs administration via, i.e. intraperitoneal or intranasal. In summary, our data highlights the possibility of using this approach for the treatment of chronic inflammatory lung diseases.

Supporting Information
S1 Fig

Analysis of polymorphonuclear and mononuclear perivascular cells in the lungs of htMSC and/or LLLT treated animals.
COPD animals were submitted to therapeutic protocols as described in materials and methods. Further, all animals were euthanized and lungs were obtained for histomorphometric analysis. In (A) mononuclear and (B) polymorphonuclear cells per mm2 of tissue. Data representative from two experiments. n = 5–8 animals per group. One-way ANOVA.

(TIF)

Click here for additional data file.(125K, tif)
S2 Fig

Reduction in alveolar destruction in the lungs of htMSC and/or LLLT treated animals.
COPD animals were submitted to therapeutic protocols as described in materials and methods. Further, all animals were euthanized and lungs were obtained for mean linear intercept in central and peripheral measures of both lungs. In A) representative graphs and B) photomicrographs of haematoxylin- and eosin-stained pulmonary parenchyma. Data representative of two experiments. n = 5–8 animals per group. One-way ANOVA.

(TIF)

Click here for additional data file.(348K, tif)
S3 Fig

Increased IL-10 staining in the lungs of htMSC i.n and/or LLLT treated animals.
COPD animals were submitted to therapeutic protocols as described in materials and methods. Further, all animals were euthanized, lungs obtained and sections were stained with anti-IL-10. In A) representative graphs and B) photomicrographs of immunohistochemistry stained sections. Data representative of two experiments. n = 5–8 animals per group. One-way ANOVA.

(TIF)

Click here for additional data file.(366K, tif)
Go to:
Acknowledgments
We would like to thank Vanessa Roza da Silva for her great support with maintaining the experimental animals and Prof. Regiane Albertini for kindly sharing the LLL irradiator. We would like to thank also Eliane Gomes for technical support.
:
Funding Statement
Financial support from FAPESP (Fundação de Apoio à Pesquisa do Estado de São Paulo) – grants # 2011/18703-2 for Peron JPS and 2012/16498-5 for Ligeiro de Oliveira AP.

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Lasers Med Sci. 2014 Nov 21. [Epub ahead of print]

Phototherapy with combination of super-pulsed laser and light-emitting diodes is beneficial in improvement of muscular performance (strength and muscular endurance), dyspnea, and fatigue sensation in patients with chronic obstructive pulmonary disease.

Miranda EF1, de Oliveira LV, Antonialli FC, Vanin AA, de Carvalho PD, Leal-Junior EC.

Author information

  • 1Post-Graduate Program in Biophotonics Applied to Health Sciences, Nove de Julho University, Rua Vergueiro, 235, 01504-001, São Paulo, SP, Brazil.

Abstract

Phototherapy is an electrophysical intervention being considered for the retardation of peripheral muscular fatigue usually observed in chronic obstructive pulmonary disease (COPD). The objective of this study was to evaluate the acute effects of combination of super-pulsed laser and light-emitting diodes phototherapy on isokinetic performance in patients with COPD. Thirteen patients performed muscular endurance tests in an isokinetic dynamometer. The maximum voluntary isometric contraction (MVIC), peak torque (PT), and total work (TW) of the non-dominant lower limb were measured in two visits. The application of phototherapy or placebo (PL) was conducted randomly in six locations of femoral quadriceps muscle by using a cluster of 12 diodes (4 of 905 nm super-pulsed lasers, 0.3125 mW each; 4 of 875 nm LEDs, 17.5 mW each; and 4 of 640 nm LEDs, 15 mW each, manufactured by Multi Radiance Medical™). We found statistically significant increases for PT (174.7?±?35.7 N?·?m vs. 155.8?±?23.3 N?·?m, p?=?0.003) and TW after application of phototherapy when compared to placebo (778.0?±?221.1 J vs. 696.3?±?146.8 J, p?=?0.005). Significant differences were also found for MVIC (104.8?±?26.0 N?·?m vs. 87.2?±?24.0 N?·?m, p?=?0.000), sensation of dyspnea (1 [0-4] vs. 3 [0-6], p?=?0.003), and fatigue in the lower limbs (2 [0-5] vs. 5 [0.5-9], p?=?0.002) in favor of phototherapy. We conclude that the combination of super-pulsed lasers and LEDs administered to the femoral quadriceps muscle of patients with COPD increased the PT by 20.2 % and the TW by 12 %. Phototherapy with a combination of super-pulsed lasers and LEDs prior to exercise also led to decreased sensation of dyspnea and fatigue in the lower limbs in patients with COPD.

Vopr Kurortol Fizioter Lech Fiz Kult. 2014 Jul-Aug;(4):3-6.

The application of chromo- and laserotherapy for the treatment of the patients presenting with chronic obstructive pulmonary disease and concomitant arterial hypertension.

[Article in Russian]
Nikitin AV, Marks SI.

Abstract

This article gives evidence of the effectiveness of chromo- and laser therapy (using infrared and green wavelenth radiation) in combination with basal pharmacotherapy in the patients presenting with combined pathology. The analysis of the data obtained indicates that the proposed approach makes it possible to accelerate normalization of the clinical characteristics, reduce arterial pressure, improve the parameters of external respiration, and increase the duration of remission period.

 

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BioMed Central Biomed Central Web Site search submit a manuscript register this article Journal of Translational Medicine Journal Front Page
J Transl Med. 2010; 8: 16.
Published online 2010 Feb 16. doi:  10.1186/1479-5876-8-16
PMCID: PMC2830167

Lasers, stem cells, and COPD

Feng Lin,#1 Steven F Josephs,#1 Doru T Alexandrescu,#2 Famela Ramos,1 Vladimir Bogin,3Vincent Gammill,4 Constantin A Dasanu,5 Rosalia De Necochea-Campion,6 Amit N Patel,7 Ewa Carrier,6 and David R Kooscorresponding author1
1Entest BioMedical, San Diego, CA, USA
2Georgetown Dermatology, Washington DC, USA
3Cromos Pharma Services, Longview, WA, USA
4Center for the Study of Natural Oncology, Del Mar, CA, USA
5Department of Hematology and Medical Oncology, St Francis Hospital and Medical Center, Hartford, CT, USA
6Moores Cancer Center, University of California San Diego, CA, USA
7Department of Cardiothoracic Surgery, University of Utah, Salt Lake City, UT, USA
corresponding authorCorresponding author.
#Contributed equally.
Feng Lin: moc.liamg@dsnilfSteven F Josephs: moc.liamtoh@shpesojnevetsDoru T Alexandrescu: moc.liamtoh@uroddmFamela Ramos: moc.oohay@somaralemafVladimir Bogin: moc.amrahpsomorc@nigobvVincent Gammill: ten.aihpleda@llimmagvConstantin A Dasanu: moc.liamtoh@uroddmRosalia De Necochea-Campion: ude.dscu@oipmacaehcocenedrAmit N Patel: ude.hatu.csh@letaP.timAEwa Carrier: ude.dscu@reirraceDavid R Koos: moc.oibtsetne@ofni
Author information ? Article notes ? Copyright and License information ?
Received 2010 Jan 7; Accepted 2010 Feb 16.

Abstract

The medical use of low level laser (LLL) irradiation has been occurring for decades, primarily in the area of tissue healing and inflammatory conditions. Despite little mechanistic knowledge, the concept of a non-invasive, non-thermal intervention that has the potential to modulate regenerative processes is worthy of attention when searching for novel methods of augmenting stem cell-based therapies. Here we discuss the use of LLL irradiation as a “photoceutical” for enhancing production of stem cell growth/chemoattractant factors, stimulation of angiogenesis, and directly augmenting proliferation of stem cells. The combination of LLL together with allogeneic and autologous stem cells, as well as post-mobilization directing of stem cells will be discussed.

Introduction (Personal Perspective)

We came upon the field of low level laser (LLL) therapy by accident. One of our advisors read a press release about a company using this novel technology of specific light wavelengths to treat stroke. Given the possible role of stem cells in post-stroke regeneration, we decided to cautiously investigate. As a background, it should be said that our scientific team has been focusing on the area of cord blood banking and manufacturing of disposables for processing of adipose stem cells for the past 3 years. Our board has been interested in strategically refocusing the company from services-oriented into a more research-focused model. An unbiased exploration into the various degenerative conditions that may be addressed by our existing know-how led us to explore the condition of chronic obstructive pulmonary disease (COPD), an umbrella term covering chronic bronchitis and emphysema, which is the 4th largest cause of death in the United States. As a means of increasing our probability of success in treatment of this condition, the decision was made to develop an adjuvant therapy that would augment stem cell activity. The field of LLL therapy attracted us because it appeared to be relatively unexplored scientific territory for which large amounts of clinical experience exist. Unfortunately, it was difficult to obtain the cohesive “state-of-the-art” description of the molecular/cellular mechanisms of this therapy in reviews that we have searched. Therefore we sought in this mini-review to discuss what we believe to be relevant to investigators attracted by the concept of “regenerative photoceuticals”. Before presenting our synthesis of the field, we will begin by describing our rationale for approaching COPD with the autologous stem cell based approaches we are developing.

COPD as an Indication for Stem Cell Therapy

COPD possesses several features making it ideal for stem cell based interventions: a) the quality of life and lack of progress demands the ethical exploration of novel approaches. For example, bone marrow stem cells have been used in over a thousand cardiac patients with some indication of efficacy [,]. Adipose-based stem cell therapies have been successfully used in thousands of race-horses and companion animals without adverse effects [], as well as numerous clinical trials are ongoing and published human data reports no adverse effects (reviewed in ref []). Unfortunately, evaluation of stem cell therapy in COPD has lagged behind other areas of regenerative investigation; b) the underlying cause of COPD appears to be inflammatory and/or immunologically mediated. The destruction of alveolar tissue is associated with T cell reactivity [,], pathological pulmonary macrophage activation [], and auto-antibody production []. Mesenchymal stem cells have been demonstrated to potently suppress autoreactive T cells [,], inhibit macrophage activation [], and autoantibody responses []. Additionally, mesenchymal stem cells can be purified in high concentrations from adipose stromal vascular tissue together with high concentrations of T regulatory cells [], which in animal models are approximately 100 more potent than peripheral T cells at secreting cytokines therapeutic for COPD such as IL-10 [,]. Additionally, use of adipose derived cells has yielded promising clinical results in autoimmune conditions such as multiple sclerosis []; and c) Pulmonary stem cells capable of regenerating damaged parenchymal tissue have been reported []. Administration of mesenchymal stem cells into neonatal oxygen-damaged lungs, which results in COPD-like alveoli dysplasia, has been demonstrated to yield improvements in two recent publications [,].

Based on the above rationale for stem cell-based COPD treatments, we began our exploration into this area by performing several preliminary experiments and filing patents covering combination uses of stem cells with various pharmacologically available antiinflammatories, as well as methods of immune modulation. These have served as the basis for two of our pipeline candidates, ENT-111, and ENT-894. As a commercially-oriented organization, we needed to develop a therapeutic candidate that not only has a great potential for efficacy, but also can be easily implemented as part of the standard of care. Our search led us to the area of low level laser (LLL) therapy. From our initial perception as neophytes to this field, the area of LLL therapy has been somewhat of a medical mystery. A pubmed search for “low level laser therapy” yields more than 1700 results, yet before stumbling across this concept, none of us, or our advisors, have ever heard of this area of medicine.

On face value, this field appeared to be somewhat of a panacea: clinical trials claiming efficacy for conditions ranging from alcoholism [], to sinusitis [], to ischemic heart disease []. Further confusing was that many of the studies used different types of LLL-generating devices, with different parameters, in different model systems, making comparison of data almost impossible. Despite this initial impression, the possibility that a simple, non-invasive methodology could exist that augments regenerative potential in a tissue-focused manner became very enticing to us. Specific uses envisioned, for which intellectual property was filed included using light to concentrate stem cells to an area of need, to modulate effects of stem cells once they are in that specific area, or even to use light together with other agents to modulate endogenous stem cells.

The purpose of the current manuscript is to overview some of the previous work performed in this area that was of great interest to our ongoing work in regenerative medicine. We believe that greater integration of the area of LLL with current advancements in molecular and cellular biology will accelerate medical progress. Unfortunately, in our impression to date, this has been a very slow process.

What is Low Level Laser Irradiation?

Lasers (Light amplification by stimulated emission of radiation) are devices that typically generate electromagnetic radiation which is relatively uniform in wavelength, phase, and polarization, originally described by Theodore Maiman in 1960 in the form of a ruby laser []. These properties have allowed for numerous medical applications including uses in surgery, activation of photodynamic agents, and various ablative therapies in cosmetics that are based on heat/tissue destruction generated by the laser beam []. These applications of lasers are considered “high energy” because of their intensity, which ranges from about 10-100 Watts. The subject of the current paper will be another type of laser approach called low level lasers (LLL) that elicits effects through non-thermal means. This area of investigation started with the work of Mester et al who in 1967 reported non-thermal effects of lasers on mouse hair growth []. In a subsequent study [], the same group reported acceleration of wound healing and improvement in regenerative ability of muscle fibers post wounding using a 1 J/cm2ruby laser. Since those early days, numerous in vitro and in vivo studies have been reported demonstrating a wide variety of therapeutic effects involving LLL, a selected sample of which will be discussed below. In order to narrow our focus of discussion, it is important to first begin by establishing the current definition of LLL therapy. According to Posten et al [], there are several parameters of importance: a) Power output of laser being 10-3 to 10-1 Watts; b) Wavelength in the range of 300-10,600 nm; c) Pulse rate from 0, meaning continuous to 5000 Hertz (cycles per second); d) intensity of 10-2-10 W/cm(2) and dose of 0.01 to 100 J/cm2. Most common methods of administering LLL radiation include lasers such as ruby (694 nm), Ar (488 and 514 nm), He-Ne (632.8 nm), Krypton (521, 530, 568, and 647 nm), Ga-Al-As (805 or 650 nm), and Ga-As (904 nm). Perhaps one of the most distinguishing features of LLL therapy as compared to other photoceutical modalities is that effects are mediated not through induction of thermal effects but rather through a process that is still not clearly defined called “photobiostimulation”. It appears that this effect of LLL is not depend on coherence, and therefore allows for use of non-laser light generating devices such as inexpensive Light Emitting Diode (LED) technology [].

To date several mechanisms of biological action have been proposed, although none are clearly established. These include augmentation of cellular ATP levels [], manipulation of inducible nitric oxide synthase (iNOS) activity [,], suppression of inflammatory cytokines such as TNF-alpha, IL-1beta, IL-6 and IL-8 [], upregulation of growth factor production such as PDGF, IGF-1, NGF and FGF-2 [], alteration of mitochondrial membrane potential [,] due to chromophores found in the mitochondrial respiratory chain [,] as reviewed in [], stimulation of protein kinase C (PKC) activation [], manipulation of NF-?B activation [], direct bacteriotoxic effect mediated by induction of reactive oxygen species (ROS) [], modification of extracellular matrix components [], inhibition of apoptosis [], stimulation of mast cell degranulation [], and upregulation of heat shock proteins []. Unfortunately these effects have been demonstrated using a variety of LLL devices in non-comparable models. To add to confusion, dose-dependency seems to be confined to such a narrow range or does not seem to exist in that numerous systems therapeutic effects disappear with increased dose.

In vitro studies of LLL

In areas of potential phenomenology, it is important to begin by assessing in vitro studies reported in the literature in which reproducibility can be attained with some degree of confidence, and mechanistic dissection is simpler as compared with in vivo systems. In 1983, one of the first studies to demonstrate in vitro effects of LLL was published. The investigators used a helium neon (He-Ne) laser to generate a visible red light at 632.8 nm for treatment of porcine granulosa cells. The paper described upregulation of metabolic and hormone-producing activity of the cells when exposed for 60 seconds to pulsating low power (2.8 mW) irradiation []. The possibility of modulating biologically-relevant signaling proteins by LLL was further assessed in a study using an energy dose of 1.5 J/cmin cultured keratinocytes. Administration of He-Ne laser emitted light resulted in upregulated gene expression of IL-1 and IL-8 []. Production of various growth factors in vitro suggests the possibility of enhanced cellular mitogenesis and mobility as a result of LLL treatment. Using a diode-based method to generate a similar wavelength to the He-Ne laser (363 nm), Mvula et al reported in two papers that irradiation at 5 J/cmof adipose derived mesenchymal stem cells resulted in enhanced proliferation, viability and expression of the adhesion molecule beta-1 integrin as compared to control [,]. In agreement with possible regenerative activity based on activation of stem cells, other studies have used an in vitro injury model to examine possible therapeutic effects. Migration of fibroblasts was demonstrated to be enhanced in a “wound assay” in which cell monolayers are scraped with a pipette tip and amount of time needed to restore the monolayer is used as an indicator of “healing”. The cells exposed to 5 J/cmgenerated by an He-Ne laser migrated rapidly across the wound margin indicating a stimulatory or positive influence of phototherapy. Higher doses (10 and 16 J/cm2) caused a decrease in cell viability and proliferation with a significant amount of damage to the cell membrane and DNA []. In order to examine whether LLL may positively affect healing under non-optimal conditions that mimic clinical situations treatment of fibroblasts from diabetic animals was performed. It was demonstrated that with the He-Ne laser dosage of 5 J/cmfibroblasts exhibited an enhanced migration activity, however at 16 J/cmactivity was negated and cellular damage observed []. Thus from these studies it appears that energy doses from 1.5 J/cmto 5 J/cmare capable of eliciting “biostimulatory effects” in vitro in the He-Ne-based laser for adherent cells that may be useful in regeneration such as fibroblasts and mesenchymal stem cells.

Studies have also been performed in vitro on immunological cells. High intensity He-Ne irradiation at 28 and 112 J/cmof human peripheral blood mononuclear cells, a heterogeneous population of T cells, B cells, NK cells, and monocytes has been described to induce chromatin relaxation and to augment proliferative response to the T cell mitogen phytohemaglutin []. In human peripheral blood mononuclear cells (PBMC), another group reported in two papers that interleukin-1 alpha (IL-1 alpha), tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), and interferon-gamma (IFN-gamma) at a protein and gene level in PBMC was increased after He-Ne irradiation at 18.9 J/cmand decreased with 37.8 J/cm[,]. Stimulation of human PBMC proliferation and murine splenic lymphocytes was also reported with He-Ne LLL [,]. In terms of innate immune cells, enhanced phagocytic activity of murine macrophages have been reported with energy densities ranging from 100 to 600 J/cm2, with an optimal dose of 200 J/cm[]. Furthermore, LLL has been demonstrated to augment human monocyte killing mycobacterial cells at similar densities, providing a functional correlation [].

Thus from the selected in vitro studies discussed, it appears that modulation of proliferation and soluble factor production by LLL can be reliably reproduced. However the data may be to some extent contradictory. For example, the over-arching clinical rationale for use of LLL in conditions such as sinusitis [], arthritis [,], or wound healing [] is that treatment is associated with anti-inflammatory effects. However the in vitro studies described above suggested LLL stimulates proinflammatory agents such as TNF-alpha or IL-1 [,]. This suggests the in vivo effects of LLL may be very complex, which to some extent should not be surprising. Factors affecting LLL in vivo actions would include degree of energy penetration through the tissue, the various absorption ability of cells in the various tissues, and complex chemical changes that maybe occurring in paracrine/autocrine manner. Perhaps an analogy to the possible discrepancy between LLL effects in vitro versus in vivo may be made with the medical practice of extracorporeal ozonation of blood. This practice is similar to LLL therapy given that it is used in treatment of conditions such as atherosclerosis, non-healing ulcers, and various degenerative conditions, despite no clear mechanistic understanding []. In vitro studies have demonstrated that ozone is a potent oxidant and inducer of cell apoptosis and inflammatory signaling []. In contrast, in vivo systemic changes subsequent to administration of ozone or ozonized blood in animal models and patients are quite the opposite. Numerous investigators have published enhanced anti-oxidant enzyme activity such as elevations in Mg-SOD and glutathione-peroxidase levels, as well as diminishment of inflammation-associated pathology []. Regardless of the complexity of in vivo situations, the fact that reproducible, in vitro experiments, demonstrate a biological effect provided support for us that there is some basis for LLL and it is not strictly an area of phenomenology.

Animal Studies with LLL

As early as 1983, Surinchak et al reported in a rat skin incision healing model that wounds exposed He-Ne radiation of fluency 2.2 J/cmfor 3 min twice daily for 14 days demonstrated a 55% increase in breaking strength over control rats. Interestingly, higher doses yielded poorer healing []. This application of laser light was performed directly on shaved skin. In a contradictory experiment, it was reported that rats irradiated for 12 days with four levels of laser light (0.0, 0.47, 0.93, and 1.73 J/cm2) a possible strengthening of wounds tension was observed at the highest levels of irradiation (1.73 J/cm2), however it did not reach significance when analyzed by resampling statistics []. In another wound-healing study Ghamsari et al reported accelerated healing in the cranial surface of teats in dairy cows by administration of He-Ne irradiation at 3.64 J/cmdose of low-level laser, using a helium-neon system with an output of 8.5 mW, continuous wave []. Collagen fibers in LLL groups were denser, thicker, better arranged and more continuous with existing collagen fibers than those in non-LLL groups. The mean tensile strength was significantly greater in LLL groups than in non-LLL groups []. In the random skin flap model, the use of He-Ne laser irradiation with 3 J/cmenergy density immediately after the surgery and for the four subsequent days was evaluated in 4 experimental groups: Group 1 (control) sham irradiation with He-Ne laser; Group 2 irradiation by punctual contact technique on the skin flap surface; Group 3 laser irradiation surrounding the skin flap; and Group 4 laser irradiation both on the skin flap surface and around it. The percentage of necrotic area of the four groups was determined on day 7-post injury. The control group had an average necrotic area of 48.86%; the group irradiated on the skin flap surface alone had 38.67%; the group irradiated around the skin flap had 35.34%; and the group irradiated one the skin flap surface and around it had 22.61%. All experimental groups reached statistically significant values when compared to control []. Quite striking results were obtained in an alloxan-induced diabetes wound healing model in which a circular 4 cmexcisional wound was created on the dorsum of the diabetic rats. Treatment with He-Ne irradiation at 4.8 J/cmwas performed 5 days a week until the wound healed completely and compared to sham irradiated animals. The laser-treated group healed on average by the 18th day whereas, the control group healed on average by the 59th day [].

In addition to mechanically-induced wounds, beneficial effects of LLL have been obtained in burn-wounds in which deep second-degree burn wounds were induced in rats and the effects of daily He-Ne irradiation at 1.2 and 2.4 J/cmwere assessed in comparison to 0.2% nitrofurazone cream. The number of macrophages at day 16, and the depth of new epidermis at day 30, was significantly less in the laser treated groups in comparison with control and nitrofurazone treated groups. Additionally, infections with S. epidermidis and S. aureus were significantly reduced [].

While numerous studies have examined dermatological applications of LLL, which may conceptually be easier to perform due to ability to topically apply light, extensive investigation has also been made in the area of orthopedic applications. Healing acceleration has been observed in regeneration of the rat mid-cortical diaphysis of the tibiae, which is a model of post-injury bone healing. A small hole was surgically made with a dentistry burr in the tibia and the injured area and LLL was administered over a 7 or 14 day course transcutaneously starting 24 h from surgery. Incident energy density dosages of 31.5 and 94.5 J/cmwere applied during the period of the tibia wound healing. Increased angiogenesis was observed after 7 days irradiation at an energy density of 94.5 J/cm2, but significantly decreased the number of vessels in the 14-day irradiated tibiae, independent of the dosage []. In an osteoarthritis model treatment with He-Ne resulted in augmentation of heat shock proteins and pathohistological improvement of arthritic cartilage []. The possibility that a type of preconditioning response is occurring, which would involve induction of genes such as hemoxygenase-1 [], remains to be investigated. Effects of LLL therapy on articular cartilage were confirmed by another group. The experiment consisted of 42 young Wistar rats whose hind limbs were operated on in order to immobilize the knee joint. One week after operation they were assigned to three groups; irradiance 3.9 W/cm2, 5.8 W/cm2, and sham treatment. After 6 times of treatment for another 2 weeks significantpreservation of articular cartilage stiffness with 3.9 and 5.8 W/cmtherapy was observed [].

Muscle regeneration by LLL was demonstrated in a rat model of disuse atrophy in which eight-week-old rats were subjected to hindlimb suspension for 2 weeks, after which they were released and recovered. During the recovery period, rats underwent daily LLL irradiation (Ga-Al-As laser; 830 nm; 60 mW; total, 180 s) to the right gastrocnemius muscle through the skin. After 2-weeks the number of capillaries and fibroblast growth factor levels exhibited significant elevation relative to those of the LLL-untreated muscles. LLL treatment induced proliferation in satellite cells as detected by BRdU [].

Other animal studies of LLL have demonstrated effects in areas that appear unrelated such as suppression of snake venom induced muscle death [], decreasing histamine-induced vasospasms [], inhibition of post-injury restenosis [], and immune stimulation by thymic irradiation [].

Clinical Studies Using LLL

Growth factor secretion by LLL and its apparent regenerative activities have stimulated studies in radiation-induced mucositis. A 30 patient randomized trial of carcinoma patients treated by radiotherapy alone (65 Gy at a rate of 2 Gy/fraction, 5 fractions per week) without prior surgery or concomitant chemotherapy suffering from radiation-induced mucositis was performed using a He-Ne 60 mW laser. Grade 3 mucositis occured with a frequency of 35.2% in controls and at 7.6% of treated patients. Furthermore, a decrease in “severe pain” (grade 3) was observed in that 23.8% in the control group experienced this level of pain, as compared to 1.9% in the treatment group []. A subsequent study reported similar effects [].

Healing ability of lasers was also observed in a study of patients with gingival flap incisions. Fifty-eight extraction patients had one of two gingival flap incisions lased with a 1.4 mW He-Ne (670 nm) at 0.34 J/cm2. Healing rates were evaluated clinically and photographically. Sixty-nine percent of the irradiated incisions healed faster than the control incisions. No significant difference in healing was noted when patients were compared by age, gender, race, and anatomic location of the incision []. Another study evaluating healing effects of LLL in dental practice examined 48 patients subjected to surgical removal of their lower third molars. Treated patients were administered Ga-Al-As diode generated 808 nm at a dose of 12 J. The study demonstrated that extraoral LLL is more effective than intraoral LLL, which was more effective than control for the reduction of postoperative trismus and swelling after extraction of the lower third molar [].

Given the predominance of data supporting fibroblast proliferative ability and animal wound healing effects of LLL therapy, a clinical trial was performed on healing of ulcers. In a double-blinded fashion 23 diabetic leg ulcers from 14 patients were divided into two groups. Phototherapy was applied (<1.0 J/cm2) twice per week, using a Dynatron Solaris 705(R) LED device that concurrently emits 660 and 890 nm energies. At days 15, 30, 45, 60, 75, and 90 mean ulcer granulation and healing rates were significantly higher for the treatment group as compared to control. By day 90, 58.3% of the ulcers in the LLL treated group were fully healed and 75% achieved 90-100% healing. In the placebo group only one ulcer healed fully [].

As previously mentioned, LLL appears to have some angiogenic activity. One of the major problems in coronary artery disease is lack of collateralization. In a 39 patient study advanced CAD, two sessions of irradiation of low-energy laser light on skin in the chest area from helium-neon B1 lasers. The time of irradiation was 15 minutes while operations were performed 6 days a week for one month. Reduction in Canadian Cardiology Society (CCS) score, increased exercise capacity and time, less frequent angina symptoms during the treadmill test, longer distance of 6-minute walk test and a trend towards less frequent 1 mm ST depression lasting 1 min during Holter recordings was noted after therapy [].

Perhaps one of the largest clinical trials with LLL was the NEST trial performed by Photothera. In this double blind trial 660 stroke patients were recruited and randomized: 331 received LLL and 327 received sham. No prespecified test achieved significance, but a post hoc analysis of patients with a baseline National Institutes of Health Stroke Scale score of <16 showed a favorable outcome at 90 days on the primary end point (P < 0.044) []. Currently Photothera is in the process of repeating this trial with modified parameters.

Relevance of LLL to COPD

A therapeutic intervention in COPD would require addressing the issues of inflammation and regeneration. Although approaches such as administration of bone marrow stem cells, or fat derived cellular components have both regenerative and anti-inflammatory activity in animal models, the need to enhance their potency for clinical applications can be seen in the recent Osiris’s COPD trial interim data which reported no significant improvement in pulmonary function []. Accordingly, we sought to develop a possible rationale for how LLL may be useful as an adjunct to autologous stem cell therapy.

Table ?Table11 depicts some of the properties of LLL that provide a rationale for the combined use with stem cells. One of the basic properties of LLL seems to be ability to inhibit inflammation at the level of innate immune activation. Representative studies showed that LLL was capable of suppressing inflammatory genes and/or pathology after administration of lipopolysaccharide (LPS) as a stimulator of monocytes [] and bronchial cells [], in vitro, and leukocyte infiltration in vivo [,]. Inflammation induced by other stimulators such as zymosan, carrageenan, and TNF-alpha was also inhibited by LLL [,,]. Growth factor stimulating activity of LLL was demonstrated in both in vitro and in vivo experiments in which augmentation of FGF-2, PDGF and IGF-1 was observed [,,]. Endogenous production of these growth factors may be useful in regeneration based on activation of endogenous pulmonary stem cells [,]. Another aspect of LLL activities of relevance is ability to stimulate angiogenesis. In COPD, the constriction of blood vessels as a result of poor oxygen uptake is results in a feedback loop culminating in pulmonary hypertension. Administration of angiogenic factors has been demonstrated to be beneficial in several animal models of pulmonary pathology [,]. The ability of LLL to directly induce proliferation of HUVEC cells [], as well as to augment production of angiogenic factors such as VEGF [], supports the possibility of creation of an environment hospitable to neoangiogenesis which is optimal for stem cell growth. In fact, a study demonstrated in vivo induction of neocapillary formation subsequent to LLL administration in a hindlimb ischemia model []. The critical importance of angiogenesis in stem cell mediated regeneration has previously been demonstrated in the stroke model, where the major therapeutic activity of exogenous stem cells has been attributed to angiogenic as opposed to transdifferentiation effects [].

Table 1

Examples of LLL Properties Relevant to COPD

Direct evidence of LLL stimulating stem cells has been obtained using mesenchymal stem cells derived both from the bone marrow and from the adipose tissue [,]. Interestingly in vivo administration of LLL stimulated MSC has resulted in 50% decrease in cardiac infarct size []. Clinical translation of LLL has been performed in the area of stroke, in which a 660 patient trial demonstrated statistically significant effects in post trial subset analysis [].

Conclusions

Despite clinical use of LLL for decades, the field is still in its infancy. As is obvious from the wide variety of LLL sources, frequencies, and intensities used, no standard protocols exist. The ability of LLL to induce growth factor production, inhibition of inflammation, stimulation of angiogenesis, and direct effects on stem cells suggests the urgent need for combining this modality with regenerative medicine, giving birth to the new field of “regenerative photoceuticals”. Development of a regenerative treatment for COPD as well as for other degenerative diseases would be of considerable benefit. Regarding COPD, such treatment would be life-saving/life extending for thousands of affected individuals. Ceasing smoking or not starting to smoke would considerably impact this dise

Competing interests

David R Koos is a shareholder, as well as Chairman and CEO of Entest Bio. Feng Lin is research director of Entest Bio. All other authors declare no competing interest.

Authors’ contributions

FL, SFJ, DTA, FR, VB, VG, CAD, RDNC, ANP, EC, DRK contributed to literature review, analysis and discussion, synthesis of concepts, writing of the manuscript and proof-reading of the final draft.

Acknowledgements

The authors thank Victoria Dardov and Matthew Gandjian for critical discussions and input.

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Vopr Kurortol Fizioter Lech Fiz Kult. 2009 Mar-Apr;(2):19-22.

Low-intensity laser radiation in the combined treatment of patients with chronic obstructive bronchitis

[Article in Russian]

Kashanskaia EP, Fedorov AA.

This study included 89 patients with chronic obstructive bronchitis (COB) that were exposed to harmful occupational factors. The control group consisted of 30 healthy subjects. Chronic obstructive bronchitis is characterized by serious disturbances in the cardiorespiratory system that manifest themselves in the form of bronchogenic pneumosclerosis with moderate lung emphysema and pulmonary hypertension associated with impaired general resistance. Simultaneously, activity of lipid peroxidation reactions decreases. Application of low-intensity laser radiation in combination with other therapeutic modalities for the treatment of patients with chronic obstructive bronchitis accelerates elimination of clinical symptoms, increases its efficiency, promotes drainage function of the bronchi, facilitates normalization of the patient’s immune status, and contributes to the optimization of lipid peroxidation processes.

Klin Med (Mosk). 2007;85(9):58-61.

[The effect of intravenous laser irradiation of blood on the system hemodynamics of patients with chronic obstructive bronchitis exacerbation]

[Article in Russian]

Burduli NM, Aksenova IZ.

The aim of the study was to evaluate the effects of intravenous laser irradiation of blood (ILIB) on system hemodynamics in patients with chronic obstructive bronchitis (COB) exacerbation. Ninety-seven patients with COB exacerbation were divided into two comparable groups. The 47 patients of the control group received conventional medication, while the 50 patients of the main group received the same therapy plus ILIB, which consisted of 10 everyday 20-min procedures. Central hemodynamic variables were measured prior to and after the treatment. The results demonstrated higher effectiveness of COB treatment when ILIB was applied and that ILIB itself had a positive effect on system circulation in COB exacerbation by changing hyperkinetic hemodynamics into normokinetic one. The study shows that it is appropriate to include ILIB into treatment of COB.

Bull Exp Biol Med. 2007 Aug;144(2):238-40.

Effect of laser radiation on production of reactive oxygen species in the blood of patients with chronic obstructive pulmonary disease

 

Farkhutdinov UR, Farkhutdinov ShU.

Department of Pulmonology, Clinical Hospital No. 21, Ufa.

The effect of laser radiation on generation of reactive oxygen species in the whole blood from patients with chronic obstructive pulmonary disease was studied by in vitro recording of luminol-dependent chemiluminescence. Laser irradiation of the blood from patients with increased production of reactive oxygen species decreased the microbicidal potential of cells. In patients with low generation of reactive oxygen species and normal potential of cells, laser exposure increased production of O2 metabolites. Laser radiation had little effect on chemiluminescence of the blood in patients with low generation of reactive oxygen species and decreased functional activity of cells.

Klin Med (Mosk). 2004;82(8):34-7.

Platelet aggregatory impairments in chronic obstructive bronchitis and a role of laser therapy in their correction

[Article in Russian]

[No authors listed]

A comparative follow-up was made to study platelet aggregatory function in patients with chronic obstructive bronchitis (COB) prior to and following treatment. The patients were divided into study and control groups. In addition to conventional treatment, the patients of the study group received laser therapy as intravenous blood irradiation. According to the type of baseline platelet aggregatory changes, all the patients were divided into 3 subgroups: 1) patients with hyperaggregation; 2) those with normal aggregation; and 3) those with hypoaggregation. In the patients from the study group, the performed treatment corrected platelet aggregatory disorders–the degree of aggregation decreased from 78 +/- 8.6% to 56.8 +/- 6.9% in Subgroup 1, increased from 23 +/- 4.8% to 54.6 +/- 6.21% in Subgroup 3. The similar positive changes in aggregation rates and the cumulative aggregation index was observed in the study group. In the control group, conventional drug therapy caused no substantial changes in platelet aggregatory function. Thus, intravenous blood laser irradiation is an effective technique in correcting thrombocytic dysfunction in COB.

Klin Med (Mosk). 2001;79(8):40-3.

Efficiency of laser therapy in patients with nonspecific pulmonary disease

 

[Article in Russian]

Farkhutdinov UR, Farkhutdinov RR, Farkhutdinov ShU.

Generation of active oxygen forms (AOF) in whole blood was studied in 63 patients with acute pneumonia and 72 asthmatics by chemiluminescence (CL) registration. CL intensity depended on the intensity of inflammatory process. Groups of patients with high and low blood CL were distinguished. In 35 patients intravascular laser exposure of the blood (ILEB) was added to therapeutic complexes. Disorders of free radical oxidation persisted for a long time in the majority of patients with high CL of the blood, treated by ILEB; in many cases the inflammatory process acquired a protracted pattern. By contrast, in patients with low intensity of blood CL, ILEB stimulated the generation of AOF and increased the treatment efficiency. Hence, whole blood CL can serve as a criterion of ILEB prescription and can be used for monitoring the patient’s status during laser therapy.

Klin Med (Mosk). 2000;78(12):25-8.

Efficiency of endobronchial laser therapy in patients with chronic bronchitis

[Article in Russian]

Artem’eva EG, Latfullin IA.

Clinical effectiveness of endobronchial laser therapy (EBLT) was studied in 18-56-year-old patients with chronic bronchitis (CB) running for 2-18 years. A parallel luminescent and histochemical tests measured histamine, serotonin and catecholamines in bronchial mucosa. It was found that alveolar macrophages, lymphocytes and mucus of CB patients contain significantly higher amounts of histamine and serotonin though low amounts of catecholamines than those of healthy subjects. Levels of monoamines in alveolar macrophages, lymphocytes, neutrophils, mast and APUD cells, mucus in of CB patients correlated with the disease phase, duration, features of endobronchitis, bronchial obstruction and severity of respiratory insufficiency. EBLT produced a positive effect on CB course and bronchial mucosa bioamines. It was superior to conventional therapy in lowering of histamine and serotonin in alveolar macrophages, lymphocytes and mucus as well as in raising catecholamines.

EFFECTIVENESS OF DIFFERENT SCHEMES OF LOW INTENSITY LASER THERAPY IN PATIENTS WITH CHRONIC OBSTRUCTIVE BRONCHITIS (COB)

Nikitin A.V., Evstratov A.Yu., Esaulenko I.E.

Medical Academy, Voronezh, Russia

A variety of mechanisms of laser irradiation in non-specific immunity stimulation have been reported lately. One of the factors contributing to the frequent exacerba-tion’s of COB is the presence of the secondary immunological insufficiency in this category of patients. The aim of the present study is to analyze the results of different schemes of the low intensity laser therapy use and its influence on cellular immunity and the clinical course of the disease in patients with COB during the period of exac-erbation. 100 patients, 78 male and 22 female, the mean age was 51, with the diagnosis of COB and respiratory insufficiency I-II, were divided into 4 equal groups at random. Patients of the first group underwent cndovascular laser irradiation of the blood by the helium-neon laser installation “ALOK-I” with the irradiation power of 2mW;  the second group received the contact treatment on the projection of the main bronchi, intcrscapular area by the infra-red laser installation “Mustang-017” with the power of 8-10W. The third group received the combination of cndovascular laser irradiation with the contact one, The course of laser therapy in all group lasted for 15 days and was combined with conventional therapy. The control group of patients underwent only conventional therapy including antibactcrial drugs, vitamins, physiotherapy. The indices analysis of the immunity cellular link in the dynamics of laser therapy and conventional therapy in these groups has shown the evident positive dynamics of the initially decreased T-lymphocytcs (CD,J, T-suprcssors (CDg^), B-lympliocytcs (CD,^) (p<0,05). The best results were observed in patients who had received the combination of cndovascular laser irradiation with the contact one (p<0,05). In the control group the dynamic of the studied indices was positive, but not strongly marked (p>0,05).

USE OF LOW POWER LASER LIGHT IN TREATMENT OF CHRONIC PURULENT BRONCHITIS

G.G. Prosorova , A.F. Anoshkina, S.A. Afendulov

Physician in Chief, Medical Centre of the Novolipetsk, Lipetsk, Russia

Transplantation of autologic leukocytic suspension extracorporally cured with a he-lium-neon laser “TPLA” of l =o 632,8 nm and an exit light conductor power of 1,5 mW was made use of in treatment of 53 pts suffering with CPB. Curing was applied during a 7 minutes period. Transplantation of AL suspension was carried out during a sanitation fibroscopy (FN3). The treatment was conducted up to a normalisation in an cndoscoimages state and elimination of clinical and laboratory in-flainmatory signs. The results were compared to those of the conventionally treated 32 CPB pts (antibiotics , broncholytics, FBS with an infusion of mycosolvin and furagin). The following phe-nomena were revealed: 1) an improvement in the blood cells phagocytic activity under an application of HNL light cured autolcukosuspcnsion. An increase in Ph% from 71,8 up to 88,8 (p<0,05), in PhN from 5,3 up to 8,9 (p<0,05), in PhCV from 0,76 up to 1,01 (p<0,05) was evidenced, besides, these post treatment values in the experimental group didn’t differ from those of healthy persons (p>0,05 ); 2) a reduction in a number of FBS sessions from 5-7 conducted during the con vcntional treatment down to 3-4 ones carried out with LL- cured AL suspension. 3) a reduction in the quantity of anti-biotics administered: while carrying out transplantation of autoleukosuspension cured with HN-lascr light we could completely reject antibiotics administration and only 1 pt had to be subjected to a second course of administering antibactcria] preparations, whereas during .the conventional treatment antibiotics were administered to all the pts, and 47,1% of them had to be subjected to a double course of antibiotics therapy; 4) a reduction in an average hospital stay period from 30,2 down to 23,8 days. Thus, an in-troduction of transplantation of HNLL-curcd AL-suspcnsion into the CPB pts therapy causes an increase in the organism’s non-specific resistant-resistance, minimises a mcdicamcntous load upon a pt and cuts down a duration of a hosoital stay period.

Ter Arkh. 1997;69(10):34-6.

The efficacy of membrane-stabilizing therapy in patients with chronic obstructive bronchitis

[Article in Russian]

Prozorova GG, Sil’vestrov VP, Simvolokov SI, Nikitin AV.

A membrane stabilizing effect of endobronchial laser therapy and antioxidative drugs piracetam and solcoseril was studied in 83 patients with chronic bronchitis. Malonic dialdehyde was measured to evaluate effects of this treatment on cellular and humoral immunity, blood coagulation and lipid peroxidation. It was found that the addition of membrane stabilizers to standard therapy of chronic bronchitis lowered malonic dialdehyde concentrations while the addition of the stabilizers and endobronchial laser therapy relieved clinical symptoms earlier, improved parameters of immunity, hemostasis and lipid peroxidation.

Ter Arkh. 1997;69(3):17-9.

 

The treatment of patients with chronic obstructive bronchitis by using a low-power laser at a general rehabilitation center

The treatment of patients with chronic obstructive bronchitis by using a low-power laser at a general rehabilitation center]

[Article in Russian]

Vorotnev AI, Deriabin NM, Romanov AI, Sil’vestrov VP, Titov VI.

100 patients with chronic obstructive bronchitis were examined and treated in the therapeutic department of rehabilitation center. Combined treatment including low-energy laser radiation produced good results. Laser therapy has improved bronchial permeability, sensitivity of bronchial beta 2-receptors to sympathomimetics. As a result, their intake was reduced or discontinued. Laser radiation combined well with other non-pharmacological modalities.

Arkh Patol. 1995 Nov-Dec;57(6):21-5.

 

Morphologic studies of bronchial biopsies in chronic bronchitis before and after treatment

[Article in Russian]

Chumakov AA, Boikova SP, Popkova AM, Igonina NP, Boikov KA.

N. A. Semashko Moscow Medical Institute, Russia.

Bronchial biopsies in patients with chronic bronchitis were studied histochemically, light and electron microscopically before and after conventional treatment and combined therapy (standard regimen plus He-Ne laser puncture). The conclusion is made that the combined therapy is more effective especially at early stages of the disease when irreversible sclerotic changes in the submucosa and microcirculatory bed are absent. This therapy at early stages of the disease stimulates regeneration of the surface bronchial epithelium and facilitates reversibility of initial metaplasia.

Lik Sprava. 1993 Oct-Dec;(10-12):75-9.

Clinico-morphological comparisons in the laser therapy of chronic bronchitis patients

[Article in Russian]

Feshenko IuI, Dziublik AIa, Gomoliako IV, Chechel’ LV, Kulik IV.

The article presents results of endobronchial treatment with low-intensity laser in 56 patients with chronic non-obstructive bronchitis (CNOB). Irradiation was carried out in 5-7 positions. Course of the treatment consisted of 4-6 seances. 14 days after the start of the treatment patients showed improvement of general condition, normalization of body temperature, reduction of cough, disappearance of endoscopic inflammatory changes in bronchial mucosa. Morphometrical data evidenced activation of proliferative processes and normalization of bronchial secretion which indicated increase of tissue metabolism. Cytological study revealed qualitative improvement of epithelial cover. Low-intensity laser is noted to be highly effective in the treatment of patients with CNOB, especially in the early stage of pathological process.

Probl Tuberk. 1991;(6):26-9.

Effect of low-energy laser irradiation of bronchial mucosa on systemic and local immunity in patients with chronic bronchitis

[Article in Russian]

Ivaniuta OM, Chernushenko EF, Dzublik AIa, Tyshko NA, Naida IV, Kulik IV.

The effectiveness of endobronchial low-energy laser therapy was studied in 28 patients with chronic nonobstructive bronchitis concurrent with thinning of bronchial mucosa. The course of treatment made it possible to obtain positive dynamics of most parameters of immunologic reactivity in CNB patients. Systemic immunity parameters, except for the NST and concentration of circulating immune complexes, underwent essential normalization. Marked positive changes were found in the parameters characterizing functional activity of alveolar macrophages: there was a 2.5-fold increase in adhesive properties and over 1.3-fold increase in the percentage of phagocytes. The level of secretory IgA rose significantly (by 3.5 times). Hence, a manifested therapeutic effect of this therapeutic method is mainly associated with its immune-stimulating action.

Probl Tuberk. 1989;(4):50-3.

Treatment of nonspecific endobronchitis with low-intensity laser irradiation (experimental study)

[Article in Russian]

Maliev BM, Shesterina MV, Solov’eva IP, Boikov AK.

Results of endobronchial treatment of experimental purulent endobronchitis (PE) in dogs with low intensity He-Ne laser are presented. The endobronchial use of the laser energy in treatment of PE was shown expedient and valid. Comparison of the visual endoscopic picture with the findings of the histochemical and electron microscopic examination of the bronchial mucosa bioptates in the time course of the treatment provided establishment of the optimal regimen for the treatment of nonspecific PE with He-Ne laser.

Probl Tuberk. 1992;(5-6):21-4.

Optimal effectiveness of complex treatment of patients with chronic obstructive bronchitis by intravascular laser irradiation of blood

[Article in Russian]

Ivaniuta OM, Dziublik AIa, Skopichenko VN.

Results of combined treatment of 100 patients with chronic obstructive bronchitis are presented in the paper. Intravascular laser irradiation of blood had a favourable effect on the clinical disease course, normalized parameters of lipid peroxidation, produced a marked immune stimulating and anti-inflammatory action, controlled the blood coagulation system and improved rheological blood properties. Intravascular laser irradiation of blood included in the complex of therapeutic measures increased the efficiency of therapy in patients with chronic obstructive bronchitis by 12.0 +/- 2.6%.