A Quantitative Study of In Vivo Protoporphyrin IX Fluorescence Build Up During Occlusive Treatment Phases

C L Campbell acknowledges financial support from an UK EPSRC PhD studentship (EP/K503162/1), the Alfred Stewart Trust, the Russell trust award, the Santander mobility award and the FAPESP CEPOF grant 2013/07276.Background: Topical photodynamic therapy (PDT) is a non-invasive light based therapy used to treat non-melanoma skin cancer (NMSC) and dysplasia. During PDT, the light sensitive molecule protoporphyrin IX (PpIX) is activated, resulting in the production of singlet oxygen, which subsequently leads to cell death. PpIX is metabolised from a topically applied pro-drug and the strong fluorescence signal associated with PpIX can be utilised as an indicator of the amount of PpIX present within the tumour tissue. In this work we measure the build up PpIX during the occlusive treatment phase and investigate how the PpIX production rate is affected by different lesion and patient characteristics. Methods : Fluorescence measurements were used to investigate the build up of PpIX within the tumour tissue during the 3 hour long occlusive treatment prior to irradiation. The study included in vivo measurements of 38 lesions from 38 individual patients. Actinic keratosis (AK) and basal cell carcinoma (BCC) were the lesion types included in this study. The resulting data from the study was analysed using generalised linear mixed effects models. Results : It was found that the surface fluorescence signal linearly increased with occlusive treatment time. The predictive models suggest that there is a significant difference in PpIX production between lesion location, however no significant difference is demonstrated between different lesion types, gender and skin type. Conclusions : The study extends and supports previous knowledge of PpIX production during the occlusive treatment phase.PostprintPeer reviewe

Terapia fotodinâmica (PDT) no tratamento de queratose actínica disseminada em antebraço: estudo piloto

A terapia fotodinâmica (TFD) é um tipo de tratamento local baseado na produção de espécies reativas de oxigênio, principalmente o oxigênio singleto, a partir da fotoativação de um agente sensibilizador seletivamente presente no tecido alterado. Como consequência\ud da presença do oxigênio singleto, ocorre a oxidação de biomoléculas e a morte celular. Nesse estudo, avaliamos a resposta da ALA-TFD no tratamento de queratose disseminada em antebraço em 10 pacientes. Um dispositivo a base de LEDs foi especialmente desenvolvido. Os resultados com avaliação até 90 dias demonstraram a efetividade do tratamento com qualidade estética e boa tolerância dos pacientes. A TFD pode se constituir uma opção terapêutica para a queratose actínica disseminada.Photodynamic therapy (PDT) is a local treatment based on the production of oxygen reactive species, mainly singlet oxygen, afer the photoactivation of a sensitizer agent selectively present in the abnormal tissue. As a consequence of thepresence of singlet oxygen, oxidation of several biomolecules occurs and then the cell death. In this study we evaluate the ALA-PDT response in the treatment of disseminated keratosis ar the foream of 10 patients. A LED-based illwnination device was especially designed for this application The clinical results presented until 90 days afer treatment, showed the PDT eficacy with satisfactory aesthetic result and patient tolerante to the treatment. PDT may constitute a therapeutic option for the disseminated actinic keratosis

Immunohistochemistry for identification of neoplasic cells within brisk infiltrate of thin melanomas

Melanomas finos freqüentemente apresentam infiltrado linfocitário ativo. Melonócitos pigmentados e melanófagos dispersos no infiltrado linfocitário ativo são difíceis de se distinguir nas colorações de rotina, em lâminas coradas pela hematoxilina eosina (HE). A presença de melanócitos na derme papilar caracteriza a lesão como Clark II exigindo medida de Breslow, o que justifica a importância de vencer essas limitações técnicas. Mesmo usando técnica de imuno-histoquímica com Melan-A e diamino benzidina (DAB) como cromógenos, essa distinção é ainda difícil. O pigmento marrom formado pelo cromógeno DAB não pode ser facilmente diferenciado dos grânulos marrons do pigmento de melanina. Nós introduzimos uma simples modificação na técnica, substituindo a contracoloração de hematoxilina pelo Giemsa. Com essa modificação, o pigmento de melanina foi corado em azul-esverdeado, contrastando com a coloração positiva pelo Melan-A dos melanócitos, que permaneceu marrom. Macrófagos negativos para Melan-A continham apenas grânulos grosseiros azul-esverdeados no citoplasma. Assim, fomos capazes de identificar com precisão células Melan-A positivas na derme papilar, determinando microinvasão (Clark II) em 31 (75,5%) dos 40 casos de melanomas in situ (Clark I) associados com infiltrado linfocitário ativo. A técnica apresentada permite, portanto, diferenciar macrófagos e melanócitos dispersos no infiltrado linfocitário associado a melanomas finos, permitindo detectar invasão inicial, evitando interpretação errônea do nível de Clark e da medida de Breslow.Thin melanomas are frequently associated with brisk lymphocytic infiltrate. Pigmented melanocytes are difficult to distinguish from melanophages, which are usually seen interspersed among lymphocytes on routine hematoxylin and eosin (HE) stained slides. As the presence of melanocytes in the papillary dermis characterizes the lesion as Clark II requiring the Breslow index, it is important to identify these cells properly and overcome such technical limitations. Even using immunohistochemistry staining for Melan-A and DAB as chromogens, this distinction is still difficult because the brown pigment formed by the chromogen DBA can not be easily differentiated from the brown melanin granules. We have introduced a simple modification on the technique, by replacing hematoxylin with Giemsa as counterstain. In this regard, the melanin pigment was decorated in green-blue while the Melan-A positive melanocytes were colored brown. Negatively stained melanophages contain only course green-blue granules of melanin in their cytoplasm. Thus, we were able to identify Melan-A positive cells in the papillary dermis accurately, determining microinvasion (Clark II) in 31 (77,5%) out of 40 in situ (Clark I) melanomas associated with brisk infiltrate. This technique is useful to distinguish melanophages and melanocytes interspersed among the lymphocytic infiltrate associated to thin melanomas, allowing detection of early invasion and avoiding Clark levels and Breslow index misinterpretation

The embracing end-to-side neurorrhaphy in rats A neurorrafia término-lateral abraçante em ratos

PURPOSE: Compare two new methods with the traditional end-to-side neurorrhaphy. METHODS: Rats were divided into four groups. In A-L group the peroneal nerve was sectioned and the distal stump was connected to the lateral of the tibial nerve (donor) with two 10-0 nylon points. In A-R group two perineurium flaps embraced the donor nerve. In the B-R group a suture embraced the donor nerve. Group B-L was the control. After six months tibial cranial muscle mass and morphometry of the distal stump of the peroneal nerve were evaluated. RESULTS: Muscle mass in groups A-R, A-L and B-R were lower than B-L group (p<0.0001) an equal between themselves (p>0.05). Groups A-R, B-R and A-L had a lower number of nerve fibers when compared with B-L (p=0.0155, p=0.016, p=0.0021). CONCLUSION: The three types of neurorrhaphy showed no differences related to muscle mass and number of nerve fibers suggesting that the embracing with a single suture has great potential due its simplicity and usefulness in deep areas.<br>OBJETIVO: Comparar dois novos métodos com o método tradicional da neurorrafia término-lateral. MÉTODOS: Os ratos foram separados em quatro grupos. No grupo A-E o nervo peroneal foi seccionado e o coto distal foi suturado à lateral do nervo tibial com dois pontos de nylon 10-0. No grupo A-D duas abas de epi-perineuro abraçaram o nervo doador. No grupo B-D foi realizada sutura com um único ponto abraçando o nervo doador. O grupo B-E foi o controle. Após seis meses foram observados massa do músculo tibial cranial e morfometria do coto distal do nervo peroneal. RESULTADOS: Foi encontrada menor massa muscular nos grupos A-D, A-E e B-D quando comparados com o grupo B-E (p<0.0001) e mesma massa quando comparados entre si (p>0,05). Os grupos A-D, A-E e B-D apresentaram menor número de fibras nervosas quando comparados ao grupo B-E (p=0,0155; p=0,016; p=0,0021) e mesmo número quando comparados entre si. CONCLUSÃO: Os três tipos de neurorrafia não apresentaram diferenças relacionadas à massa muscular e número de fibras nervosas sugerindo que a sutura abraçante com apenas um ponto apresente grande potencial em áreas cirúrgicas mais profundas

Dual-Agent Photodynamic Therapy with Optical Clearing Eradicates Pigmented Melanoma in Preclinical Tumor Models

Treatment using light-activated photosensitizers (photodynamic therapy, PDT) has shown limited efficacy in pigmented melanoma, mainly due to the poor penetration of light in this tissue. Here, an optical clearing agent (OCA) was applied topically to a cutaneous melanoma model in mice shortly before PDT to increase the effective treatment depth by reducing the light scattering. This was used together with cellular and vascular-PDT, or a combination of both. The effect on tumor growth was measured by longitudinal ultrasound/photoacoustic imaging in vivo and by immunohistology after sacrifice. In a separate dorsal window chamber tumor model, angiographic optical coherence tomography (OCT) generated 3D tissue microvascular images, enabling direct in vivo assessment of treatment response. The optical clearing had minimal therapeutic effect on the in control, non-pigmented cutaneous melanomas but a statistically significant effect (p &lt; 0.05) in pigmented lesions for both single- and dual-photosensitizer treatment regimes. The latter enabled full-depth eradication of tumor tissue, demonstrated by the absence of S100 and Ki67 immunostaining. These studies are the first to demonstrate complete melanoma response to PDT in an immunocompromised model in vivo, with quantitative assessment of tumor volume and thickness, confirmed by (immuno) histological analyses, and with non-pigmented melanomas used as controls to clarify the critical role of melanin in the PDT response. The results indicate the potential of OCA-enhanced PDT for the treatment of pigmented lesions, including melanoma

Oral chronic graft-versus-host disease: analysis of dendritic cells subpopulations

The graft-versus-host disease is the major cause of morbidity and mortality in patients who have undergone hematopoietic stem cell transplantation. Aiming at contributing to the understanding of the role of myeloid and plasmacytoid dendritic cells, and natural killer cells in chronic graft-versus-host disease, we examined biopsies of jugal mucosa of 26 patients with acute myeloid leukemia who had undergone allogenic hematopoietic stem cell transplantation. Half of these patients developed oral chronic graft-versus-host disease. Microscopic sections were immunohistochemically stained for anti-CD1a, anti-CD123 and anti-CD56. We calculated the number of immunostained cells in the corium per square millimeter and applied the Mann-Whitney test. Results showed a statistically significant increase of myeloid dendritic cells (CD1a+; p=0,02) and natural killer cells (CD56; p=0,04) in patients with oral chronic graft-versus-host disease. CD123 immunostaining showed no statistical difference between groups. It was concluded that myeloid dendritic cells and natural killer cells participate in the development of oral chronic graft-versus-host disease