Abstract: Sir, Antimicrobial photodynamic therapy (aPDT) is emerging as a new promising tool to treat local infections in Veterinary Medicine [1]. In 2016, our group was the pioneer to investigate its use to inactivate pathogens related to bovine mastitis [2]. In this regard, in a recent report published in this journal, Moreira et al. [3] explored the use of toluidine blue (TBO)-mediated aPDT to treat subclinical bovine mastitis. Remarkably, bovine mastitis still remains as the most economically relevant disease for dairy industry worldwide, resulting in substantial economic losses for milk producers due to extensive antibiotic use and product losses. In this sense, antibiotic misuse or overuse to treat bovine mastitis is worrisome because it can simultaneously lead to antibiotic residues in dairy products and increase multidrug-resistant foodborne pathogens. Unfavorably, there is still no consensus on the ideal approach for bovine mastitis treatment; therefore, aPDT represents a viable option to reduce the use of antibiotics in dairy farms. The preliminary results of Moreira et al. [3] are exciting and deserve discussion on some points. In this letter, we intend to highlight other relevant aspects to be addressed in order to consolidate the use of aPDT for management of bovine mastitis. The concentration of the photosensitizer (PS) diluted in the milk plays a pivotal role. In the study by Moreira et al. [3], the authors designed a clinical assay using a 2.5% TBO solution, which promoted a significant reduction of bacterial load. It is noteworthy that clinical mastitis represents a more complex challenge for aPDT than subclinical mastitis since the alterations in the milk composition could influence the PS absorption properties. Additionally, we believe that intermolecular interactions between PS and milk components should be considered to optimize clinical protocols because it could induce aggregation of phenothiazine dyes and leads to lower photodynamic inactivation efficiency [[4], [5]]. Another important point is in respect to the light diffusion within mammary parenchyma. Appropriated light delivery systems need to reach deeper mammary tissue since pathogens normally colonize the entire mammary gland [6]. Therefore, the use of diffuser fibers should be considered to deliver homogeneous light intensities inside the gland parenchyma. Hence, investigations regarding different light delivery systems could optimize clinical protocols and facilitate the establishment of aPDT into the dairy industry. Finally, we encourage the development of innovative strategies to improve selective PS delivery to pathogens using targeted molecular and nanoparticle systems. In summary, we hope that this letter motivates further studies to develop safe and effective protocols for bovine mastitis and promote aPDT applications in Veterinary Medicine.

Palavras-Chave: veterinary medicine; cattle; mammary glands; infectious diseases; bacterial diseases; therapy; photosensitivity

RIBEIRO, MARTHA S.; GARGANO, RONALDO G.; SABINO, CAETANO P.; ANJOS, CAROLINA dos; POGLIANI, FABIO C.; SELLERA, FABIO P. Clinical challenges of antimicrobial photodynamic therapy for bovine mastitis. Photodiagnosis and Photodynamic Therapy, v. 21, p. 327-327, 2018. DOI: 10.1016/j.pdpdt.2018.01.007. Disponível em: http://repositorio.ipen.br/handle/123456789/29010. Acesso em: $DATA.

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Abstract: INTRODUCTION Photobiomodulation (PBM) has been gaining importance in a wide range of medical fields in the past few years, particularly in stem cell-based regenerative medicine. Improving in vitro cell proliferation, differentiation and viability are ways where PBM could play a pivotal role optimizing biotechnological and bioengineering applications. OBJECTIVES Here we investigated whether different wavelengths (blue, green and red) would promote distinct outcomes in human adipose-derived stem cells (hADSCs) cultured in regular and supplemented media for tenocyte differentiation. MATERIALS AND METHODS Freshly isolated hADSCs were cultured in a specific stem cell medium (MSCGM, Lonza), DMEM or a tenogenic medium (TEN-M: DMEM supplemented with growth factors and ascorbic acid). Cells were irradiated every 48 h (23.28 mW/cm 2 , 17 min 10 s delivering 24 J/cm 2 per session) using a LED irradiator (LEDbox, BioLambda). MTT and crystal violet assays were used to evaluate cell metabolic activity and proliferation. DISCUSSION AND RESULTS Red wavelength (660 nm) significantly increased metabolic activity after five irradiations, but only for cells cultured in TEN-M. Oppositely, blue (450 nm) and green (520 nm) light decreased both cell proliferation and metabolic rate, with more pronounced effects for blue light in TEN-M. Considering these findings, we examined whether irradiating only the media would generate toxic compounds that could impair cell viability. We therefore assessed reactive oxygen species (ROS) production by p-nitrosodimethylaniline/histidine assay while irradiating the three different media under the same conditions as mentioned above. Immediately after blue and green light exposure, an increment in ROS production was observed for DMEM and TEN-M, that continuously increased until reaching between 4.5 and 7.1 μM one-hour after irradiation – with higher values for TEN-M exposed to blue light. CONCLUSION Since no significant ROS formation was observed following red light exposure, we concluded that medium composition was responsible for the different effects on metabolic activity and proliferation observed after irradiation with different wavelengths.

YOSHIMURA, TANIA M.; SAUTER, ISMAEL P.; RIBEIRO, MARTHA S. Low power light triggers opposite effects on stem cells: influence of the wavelength and culture conditions. Biophysical Reviews, v. 13, n. 6, p. 1492-1493, 2021. DOI: 10.1007/s12551-021-00844-3. Disponível em: http://repositorio.ipen.br/handle/123456789/32895. Acesso em: $DATA.

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Abstract: The anti-inflammatory action of Photobiomodulation (PBM) is one of its most reproducible effects in vivo. New models to study PBMeffects and to investigate light dosimetry may bring different perspectives on the field. Based on the importance of establishing new experimental models and the advantages of using zebrafish for screening new drugs with potential anti-inflammatory effects the present investigation evaluated the effect of PBM on carragenan-induced abdominal edema in zebrafish. Thirty Danio rerio (± 1 g) zebrafish were randomly divided into five aquariums with 0.25L of water to establish three treatments: T1 - Naive non-treated; T2 – Saline 0.9%-injected; T3 - carrageenan-injected (CG) 3.5% in the coelomic cavity. T4 –injected (CG) 3.5% and treated with a diode laser λ=808nm, 50mWand 0.5J T5 –same parameters with 1J and T6 -1.5J. Abdominal volumes were measured by a caliper and the area was calculated 4 hours post-indu ction. Four hours after edema induction the results revealed an increase in the abdominal area of fish injected with CG 3.5% compared to negative control. The animals injected with CG and treated with PBM exhibited a reduction in abdominal edema in a dose-dependent manner. The 0.5J energy did not present a significant reduction compare do control (p=0.18), the 1J and 1.5J groups presented a significant difference (p=0.027 and p=0.005 respectively). The 1.5J group was not different from negative control (T2) p=0.37. The present study identified that zebrafish may represent a good model to investigate PBM effects and the response was dose dependent.

Palavras-Chave: inflammation; fishes; antipyretics; dosimetry; biological effects

NUNEZ, SILVIA C.; SILVA, IVES C. da; NASCIMENTO, JONATHAN S.; RIBEIRO, MARTHA S. Methodological study of photobiomodulation anti-edematous effects on carrageenan-induced inflammation on zebrafish. Lasers in Medical Science, v. 35, n. 1, p. 288-289, 2020. DOI: 10.1007/s10103-019-02900-7. Disponível em: http://repositorio.ipen.br/handle/123456789/31112. Acesso em: $DATA.

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Abstract: Mammal fat consists ofwhite and brown adipose tissues (WATand BAT). Although most fat is energy-storing WAT, brown adipocytes dissipate energy as heat. Obese subjects exhibit BAT hyporesponsiveness to classical stimuli as cold, hence increasing BAT thermogenic capacity could be an i n t er e s t i n g t a rget f o r t r eating obesity. Photobiomodulation (PBM) seems to be a feasible alternative to activate BAT, although it has not been applied for this purpose until now. In this study, adult C57Bl/6 mice were fed with a hyperlipidic diet (HF) during 15 weeks to induce obesity. Then, they were divided into two groups: HF and HF660. The interscapular BAT of HF660 mice was exposed to a single irradiation using a 662 ± 20 nm LED (5.7 Jcm-2 at 19 mWcm-2, delivered during 300 s). The HF group was sham-irradiated. Eutrophic animals were also used. Animals received 200 mCi of 18F-FDG and were exposed to cold for basal register of PET-CT images. After PBM or sh am treat ment, mice were once again exposed to cold before PET-CT image acquisition. Our results showed that PBM was able to activate BAT in obese mice through higher glucose uptake. In contrast, PBM and shamirradiated normal fed mice presented similar levels of glucose uptake. Taking together, our findings suggest that PBM applied to the BAT is able to promote glucose uptake by obese mice and could be an interesting approach to manage obesity and its correlated morbidities.

Palavras-Chave: adipose tissue; glucose; metabolic diseases; mice; irradiation

YOSHIMURA, TANIA; RIBEIRO, MARTHA S. Photobiomodulation activates glucose uptake in brown adipose tissue of obese mice. Lasers in Medical Science, v. 35, n. 1, p. 280-280, 2020. DOI: 10.1007/s10103-019-02900-7. Disponível em: http://repositorio.ipen.br/handle/123456789/31113. Acesso em: $DATA.

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Abstract: INTRODUCTION Leishmaniasis is an important neglected disease. Photodynamic therapy (PDT) has been used to fight cutaneous leishmaniasis showing good results. However, PDT mechanisms in Leishmania parasites are not yet completely clarified. OBJECTIVES In this work, our objective was to develop a protocol to produce giant plasma membrane vesicles (GPMVs) from Leishmania amazonensis promastigotes to understand the mechanisms of action of methylene blue (MB)-mediated PDT on the cell membrane of parasites. MATERIALS AND METHODS For membrane extraction, several techniques were tested. The osmotic shock was the technique that presented the best yield and effectiveness. Phosphate and protein measurements were performed to confirm membrane extraction. For the growth of GPMVs, the best technique was electroforming using different frequencies and voltages in 4 cycles. Reconstituted GPMVs were observed by phase-contrast light microscopy. Subsequently, PDT was applied to GPMVs dispersed in an aqueous solution containing 50 μM MB and we verified the changes in permeability before and after exposure to light. The same processwas applied to giant unilamellar vesicles (GUVs) with lipid compositions similar to the parasite membrane. DISCUSSION AND RESULTS The electroforming technique with the protocol developed in this work made it possible to obtain GPMVs froma promastigote membrane isolate of L. amazonensis. The membrane isolation technique was effective to extract the parasite's membrane while preserving lipids and proteins. In GUVs we observe an increase in the area during PDT in different compositions and loss of contrast. The GPMVs showed a loss of contrast as well as the GUVs but did not show an increase in area. CONCLUSION This factor could be explained by the high degree of complexity of the membrane, which contains membrane proteins in addition to containing lipids.

SOUZA, MARESSA D.F. de; ITRI, ROSANGELA; RIBEIRO, MARTHA S. Reconstitution of Leishmania plasma membrane to understand the photodynamic effect. Biophysical Reviews, v. 13, n. 6, p. 1384-1384, 2021. DOI: 10.1007/s12551-021-00845-2. Disponível em: http://repositorio.ipen.br/handle/123456789/32887. Acesso em: $DATA.

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