A hanseníase é geralmente agravada pelo aparecimento de reações, que são quadros inflamatórios de difícil tratamento e a principal causa de seqüelas. Nossa hipótese é de que deficiência e/ou perda da função das células T reguladoras (Tregs) podem estar envolvidas no desenvolvimento das reações. Além da avaliação da frequência das Tregs circulantes em pacientes com reação tipo 1 (R1) e reação tipo 2 (R2), também foi avaliada a frequência in situ de FoxP3, IL-17, IL-6 e TGFbeta. Pacientes com R2 apresentaram expressiva diminuição na frequência das Tregs circulantes e in situ em comparação com pacientes com R1 e com os controles. Paralelamente a diminuição das Tregs nas R2 foi observado aumento da expressão de IL-17 in situ e diminuição da expressão de TGFbeta. Biópsias obtidas de pacientes com R1 e R2 antes do episódio reacional mostraram números de células FoxP3+ e IL-17+ similares entre os dois grupos. Entretanto, nas biópsias obtidas durante a reação foi observado diminuição de Tregs e aumento de células IL-17+ em pacientes com R2, enquanto que pacientes com R1 apresentaram o oposto: aumento de Tregs e diminuição de células IL-17+. Além disso, foi observada diminuição da expansão das Tregs frente ao estímulo in vitro com Mycobacterium leprae e uma tendência a baixa expressão de FoxP3 e da molécula imunossupressora CTLA-4 em Tregs de pacientes com R2. Nossos resultados sugerem que nas R2, a diminuição na frequência de Tregs possa estar favorecendo o desenvolvimento de uma resposta Th17, a qual é característica deste tipo de reação. Adicionalmente, com a finalidade de obter um número suficiente de Tregs para realização de ensaios funcionais com estas células, uma vez que se trata de uma subpopulação com baixa freqüência no sangue periférico ( < 10%), foram estabelecidos e avaliados três protocolos distintos para expansão in vitro de Tregs: protocolo Rapamicina, protocolo TGFbeta e protocolo Vitamina D3. Todos os protocolos foram capazes de induzir expansão de Tregs viáveis nos grupos estudados (paucibacilares e multibacilares sem reação, R1 e R2). Em todos os grupos estudados as Tregs expandidas apresentaram capacidade de suprimir a proliferação de linfócitos TCD4+ e TCD8+. Apesar dos três protocolos testados apresentarem capacidade de expandir Tregs in vitro, selecionamos para ensaios futuros os protocolos Rapamicina e TGFbeta por apresentarem melhor custo-benefício. A expansão in vitro será utilizada para estudos funcionais das Tregs buscando melhor entendimento do envolvimento desta subpopulação na patogenia das reações hansênicas / Leprosy is frequently complicated by the appearance of reactions that are difficult to treat and are the main cause of sequelae. We speculated that disturbances in regulatory T-cells (Tregs) could play a role in leprosy reactions. We determined the frequency of circulating Tregs in patients with type 1 reaction (T1R) and type 2 reaction (T2R). The in situ frequency of FoxP3 and interleukin (IL)-17, IL-6, and transforming growth factor beta (TGF)-beta expressing cells was also determined. T2R patients showed markedly lower number of circulating and in situ Tregs than T1R patients and controls. This decrease was paralleled by increased in situ IL-17 expression but decreased TGF-beta expression. Biopsies from T1R and T2R patients before the reaction episodes showed similar number of forkhead box protein P3 + (FoxP3+) and IL-17+ cells. However, in biopsies taken during the reaction, T2R patients showed a decrease in Tregs and increase in IL-17+ cells, whereas T1R patients showed the opposite: Tregs increased but IL17+ cells decreased. We also found decreased expansion of Tregs upon in vitro stimulation with Mycobacterium leprae and a trend for lower expression of FoxP3 and the immunosuppressive molecule CTLA-4 in T2R Tregs. Our results provide some evidence to the hypothesis that, in T2R, downmodulation of Tregs may favor the development of T-helper-17 responses that characterize this reaction. In addition, aiming to provide sufficient number of Tregs to perform functional assays with these cells, as they correspond to a subtle subpopulation among the peripheral blood mononuclear cells ( < 10%), we established and analyzed three different protocols for in vitro Tregs: a Rapamycin protocol, a TGFbeta protocol and a Vitamina D3 protocol. All three protocols were able to induce the expansion of viable in the four types of patients of the study (paucibacillary and multibacillary patients without reaction, and R1 and R2 patients). In these four groups the tregs were able to suppress the proliferative response of TCD4+ e TCD8+ lymphocytes. Although the three protocols resulted in expansion of Tregs, we selected two of them, Rapamycin and TGFbeta for further assays since they showed better cost-benefit. The in vitro expansion will be used to perform functional assays of the Tregs aiming at a better understanding of the involvement of this subpopulation in the pathogenesis of the leprosy reaction episodes

Extensive research has already been performed on Aloe vera therefore it is important that researchers include other aloe species, such as Aloe marlothii and Aloe ferox, in studies involving aloe plant materials (Loots et al., 2007:6891). The use of natural products has regained popularity and in recent years the demand for alternative medication has risen considerably (Walji & Wiktorowicz, 2013:86). The hydration state of the human skin is fundamental for its normal functioning (Verdier-Sévrain & Bonté, 2007:75), with healthy skin possessing a water content higher than 10% (w/v) (Blank, 1952:439). This demonstrates the importance of the topical application of skin moisturisers as part of basic skin care regime (Verdier-Sévrain & Bonté, 2007:75). The first part of this project focused on the in vivo skin hydration effects of the precipitated polysaccharide components of A. vera, A. ferox and A. marlothii leaf gel materials (3% (w/v)) after single (30, 90 and 150 min after application) and multiple applications (twice daily application over a period of four weeks) on healthy volunteers, respectively. The anti-erythema effects of these aloe materials on sodium lauryl sulphate irritated skin were also examined. The skin hydration effects of the aloe materials were determined with the Corneometer® CM 825 and Visioscan® VC 98 during the short term study (single application) and longer term study (multiple applications). In addition, as an indirect measurement of skin hydration, the Cutometer® dual MPA 580 was used to measure skin elasticity during the longer term study. To determine the anti-erythema effects of the aloe materials when applied to irritated skin areas, the haemoglobin content of the skin was measured with a Mexameter® MX 18. The results from the in vivo study indicated that A. ferox gel material dehydrated the skin, whereas A. vera and A. marlothii gel materials hydrated the skin during the short term study. Results from the longer term study showed that all the aloe leaf materials have skin dehydration effects, probably due to the aloe absorbing moisture from the skin into the applied gel layer upon drying. From the anti-erythema study, it was seen that A. vera and A. ferox materials had the potential to reduce erythema on the skin similar to that of the positive control group (i.e. hydrocortisone gel) after six days of treatment. The skin possesses exceptional barrier properties which can mostly be ascribed to the outermost layer of the skin, the stratum corneum (SC). Due to the physical barrier the skin has against drug permeation, the delivery of drug molecules into and across the skin continues to be challenging (Lane, 2013:13) and to overcome this barrier, penetration enhancers can be used to efficiently deliver drugs across the skin (Barry, 2002:522). The aim of the second part of this project was to determine the skin penetration enhancing effects of the gel and whole leaf materials of A. vera, A. marlothii and A. ferox. Ketoprofen was used as the marker compound and a high performance liquid chromatography (HPLC) method was developed and validated to determine the amount of ketoprofen present in the samples. Prior to the skin diffusion studies, membrane release studies were performed to test whether the solutions containing different concentrations of the aloe leaf materials (i.e. 3.00%, 1.50% and 0.75% (w/v)) released ketoprofen from their gel-like structures. From these studies, it was evident the 0.75% (w/v) concentration had the highest average percentage ketoprofen release, which was subsequently chosen as the concentration for the aloe leaf materials tested in the transdermal skin diffusion studies. The in vitro permeation study was conducted across dermatomed (400 μm thick) skin in Franz diffusion cells. Tape stripping was performed after completion of the diffusion studies to determine the concentration ketoprofen present in the SC-epidermis and epidermis-dermis layers of the skin. Results from the in vitro permeation study showed that A. vera gel enhanced the flux of ketoprofen to the highest extent (20.464 μg/cm2.h) when compared to the control group (8.020 μg/cm2.h). Aloe marlothii gel (12.756 μg/cm2.h) and A. ferox whole leaf material (12.187 μg/cm2.h) also enhanced the permeation of ketoprofen across the skin compared to the control group. A. vera gel material was the most efficient transdermal drug penetration enhancer of the selected aloe species investigated. In order to determine by which mechanism the aloe leaf materials enhanced the skin permeation of ketoprofen (Hadgraft et al., 2003:141), the permeation profiles were analysed using a non-linear curve-fitting procedure (Díez-Sales et al., 1991:3) to obtain α, β and kp values. A change in the α-value indicated the aloe leaf material influenced the partition coefficient (K), whereas a change in β indicated the aloe leaf material influenced the diffusivity (D) (with the assumption that h, the diffusional path length is constant) (Otto et al., 2010:278). The calculated α-values indicated the drug permeation enhancing effect of A. vera gel can be ascribed to an increased partitioning of the drug into the skin. The calculated β-values showed A. ferox whole leaf altered the diffusion characteristics of the skin for ketoprofen. The tape stripping results showed A. marlothii whole leaf delivered the highest concentration of the ketoprofen into the SC-epidermis and epidermis-dermis layers of the skin. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Extensive research has already been performed on Aloe vera therefore it is important that researchers include other aloe species, such as Aloe marlothii and Aloe ferox, in studies involving aloe plant materials (Loots et al., 2007:6891). The use of natural products has regained popularity and in recent years the demand for alternative medication has risen considerably (Walji & Wiktorowicz, 2013:86). The hydration state of the human skin is fundamental for its normal functioning (Verdier-Sévrain & Bonté, 2007:75), with healthy skin possessing a water content higher than 10% (w/v) (Blank, 1952:439). This demonstrates the importance of the topical application of skin moisturisers as part of basic skin care regime (Verdier-Sévrain & Bonté, 2007:75). The first part of this project focused on the in vivo skin hydration effects of the precipitated polysaccharide components of A. vera, A. ferox and A. marlothii leaf gel materials (3% (w/v)) after single (30, 90 and 150 min after application) and multiple applications (twice daily application over a period of four weeks) on healthy volunteers, respectively. The anti-erythema effects of these aloe materials on sodium lauryl sulphate irritated skin were also examined. The skin hydration effects of the aloe materials were determined with the Corneometer® CM 825 and Visioscan® VC 98 during the short term study (single application) and longer term study (multiple applications). In addition, as an indirect measurement of skin hydration, the Cutometer® dual MPA 580 was used to measure skin elasticity during the longer term study. To determine the anti-erythema effects of the aloe materials when applied to irritated skin areas, the haemoglobin content of the skin was measured with a Mexameter® MX 18. The results from the in vivo study indicated that A. ferox gel material dehydrated the skin, whereas A. vera and A. marlothii gel materials hydrated the skin during the short term study. Results from the longer term study showed that all the aloe leaf materials have skin dehydration effects, probably due to the aloe absorbing moisture from the skin into the applied gel layer upon drying. From the anti-erythema study, it was seen that A. vera and A. ferox materials had the potential to reduce erythema on the skin similar to that of the positive control group (i.e. hydrocortisone gel) after six days of treatment. The skin possesses exceptional barrier properties which can mostly be ascribed to the outermost layer of the skin, the stratum corneum (SC). Due to the physical barrier the skin has against drug permeation, the delivery of drug molecules into and across the skin continues to be challenging (Lane, 2013:13) and to overcome this barrier, penetration enhancers can be used to efficiently deliver drugs across the skin (Barry, 2002:522). The aim of the second part of this project was to determine the skin penetration enhancing effects of the gel and whole leaf materials of A. vera, A. marlothii and A. ferox. Ketoprofen was used as the marker compound and a high performance liquid chromatography (HPLC) method was developed and validated to determine the amount of ketoprofen present in the samples. Prior to the skin diffusion studies, membrane release studies were performed to test whether the solutions containing different concentrations of the aloe leaf materials (i.e. 3.00%, 1.50% and 0.75% (w/v)) released ketoprofen from their gel-like structures. From these studies, it was evident the 0.75% (w/v) concentration had the highest average percentage ketoprofen release, which was subsequently chosen as the concentration for the aloe leaf materials tested in the transdermal skin diffusion studies. The in vitro permeation study was conducted across dermatomed (400 μm thick) skin in Franz diffusion cells. Tape stripping was performed after completion of the diffusion studies to determine the concentration ketoprofen present in the SC-epidermis and epidermis-dermis layers of the skin. Results from the in vitro permeation study showed that A. vera gel enhanced the flux of ketoprofen to the highest extent (20.464 μg/cm2.h) when compared to the control group (8.020 μg/cm2.h). Aloe marlothii gel (12.756 μg/cm2.h) and A. ferox whole leaf material (12.187 μg/cm2.h) also enhanced the permeation of ketoprofen across the skin compared to the control group. A. vera gel material was the most efficient transdermal drug penetration enhancer of the selected aloe species investigated. In order to determine by which mechanism the aloe leaf materials enhanced the skin permeation of ketoprofen (Hadgraft et al., 2003:141), the permeation profiles were analysed using a non-linear curve-fitting procedure (Díez-Sales et al., 1991:3) to obtain α, β and kp values. A change in the α-value indicated the aloe leaf material influenced the partition coefficient (K), whereas a change in β indicated the aloe leaf material influenced the diffusivity (D) (with the assumption that h, the diffusional path length is constant) (Otto et al., 2010:278). The calculated α-values indicated the drug permeation enhancing effect of A. vera gel can be ascribed to an increased partitioning of the drug into the skin. The calculated β-values showed A. ferox whole leaf altered the diffusion characteristics of the skin for ketoprofen. The tape stripping results showed A. marlothii whole leaf delivered the highest concentration of the ketoprofen into the SC-epidermis and epidermis-dermis layers of the skin. / PhD (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Objetivos: Determinar a dose eritematosa mínima (DEM) medida por exposição controlada à radiação ultravioleta-B (RUV-B), como limiar para dano solar agudo nos diversos fototipos, e medir a cor da pele constitucional pelo sistema colorimétrico CIELAB. Pacientes e Métodos: Um total de 194 voluntários, sadios, com idades acima de 18 anos, distribuídos em um mínimo de 30 participantes por fototipo. Todos foram classificados por fototipos segundo os critérios de Fitzpatrick. As regiões infra-axilar torácica e nádega foram irradiadas em 4 áreas de 1 cm2, assim como foi registrada a cor da pele desses locais pelo sistema CIELAB. Delineamento: Estudo transversal. Resultados: A média de idade dos participantes foi de 38 anos, sendo 68% do sexo feminino. A avaliação da associação entre as medidas das DEMs e dos valores colorimétricos da coordenada L*, mostrou uma correlação de Pearson negativa com r = -0,91 para um valor p<0,05. Para os valores das DEMs e os escores da classificação dos voluntários por fototipos, obteve-se correlação de Spearman (rs) de +0,95 para p<0,05 e, correlacionando os valores colorimétricos com os escores dos fototipos, encontrou-se em tórax um rs de -0,93 e em nádega -0,92 para um p < 0,05. Conclusões – Concluiu-se que: 1)- a mensuração dos valores colorimétricos da coordenada L* nas regiões infra-axilar torácica e nádega mostraram uma forte correlação com os valores das DEMs, sendo de menor poder invasivo e de maior praticidade para mensuração de sensibilidade à radiação ultravioleta; 2)- apesar de os escores de Fitzpatrick terem alta correlação com os valores das DEMs, mostraram superposição de valores nos fototipos adjacentes; 3)- o grau de associação das classes dos fototipos com a cor da pele permite dizer que a categoria numérica do fototipo aumenta à medida que a pele fica mais escura. / Objectives: Determine the minimal erythema dose (MED) measured by controlled exposure to UVB radiation (UVBR) as an acute photodamage threshold for different skin phototypes, and measure the constitutional skin color by means of the colorimetric system CIELAB. Patients and Methods: A total of 194 healthy individuals, over 18 years of age, were divided into groups with a minimum of 30 participants per phototype (Fitzpatrick critera). Four 1-cm² areas of the lateral trunk and buttocks were exposed to radiation and the color of the skin at these sites was registered by the CIELAB system. Design: Transversal study. Results: The average age of the participants out of whom 68% were females was 38 years. The assessment of the association of the MED values with the colorimetric values of the coordinate L* both on the thorax and on the buttocks showed a negative Pearson correlation coefficient (r) of - 0.91 for p<0.05. A Spearman’s rank correlation coefficient (r²) of +0.95 for p<0.05 was obtained for the MED values and the scores of the classification of the volunteers by phototypes, and rs of –0.93 on the thorax and rs of –0.92 on the buttocks for p<0.05 were found by correlating the colorimetric values with the scores of the phototypes. Conclusions – It was concluded that: 1) the colorimetric values of the coordinate L* showed a strong correlation with the DEM values, which could be a less invasive and more practical method for measuring the sensitivity to ultraviolet radiation; 2) although the Fitzpatrick’s scores have a high correlation with the MED values, they demonstrated the overlap of values in the adjoining phototypes; 3) the extent of association of the pthototype classes with the color of the skin allows us to say that the numerical category of the phototype increases as the skin gets darker.

Objetivos: Determinar a dose eritematosa mínima (DEM) medida por exposição controlada à radiação ultravioleta-B (RUV-B), como limiar para dano solar agudo nos diversos fototipos, e medir a cor da pele constitucional pelo sistema colorimétrico CIELAB. Pacientes e Métodos: Um total de 194 voluntários, sadios, com idades acima de 18 anos, distribuídos em um mínimo de 30 participantes por fototipo. Todos foram classificados por fototipos segundo os critérios de Fitzpatrick. As regiões infra-axilar torácica e nádega foram irradiadas em 4 áreas de 1 cm2, assim como foi registrada a cor da pele desses locais pelo sistema CIELAB. Delineamento: Estudo transversal. Resultados: A média de idade dos participantes foi de 38 anos, sendo 68% do sexo feminino. A avaliação da associação entre as medidas das DEMs e dos valores colorimétricos da coordenada L*, mostrou uma correlação de Pearson negativa com r = -0,91 para um valor p<0,05. Para os valores das DEMs e os escores da classificação dos voluntários por fototipos, obteve-se correlação de Spearman (rs) de +0,95 para p<0,05 e, correlacionando os valores colorimétricos com os escores dos fototipos, encontrou-se em tórax um rs de -0,93 e em nádega -0,92 para um p < 0,05. Conclusões – Concluiu-se que: 1)- a mensuração dos valores colorimétricos da coordenada L* nas regiões infra-axilar torácica e nádega mostraram uma forte correlação com os valores das DEMs, sendo de menor poder invasivo e de maior praticidade para mensuração de sensibilidade à radiação ultravioleta; 2)- apesar de os escores de Fitzpatrick terem alta correlação com os valores das DEMs, mostraram superposição de valores nos fototipos adjacentes; 3)- o grau de associação das classes dos fototipos com a cor da pele permite dizer que a categoria numérica do fototipo aumenta à medida que a pele fica mais escura. / Objectives: Determine the minimal erythema dose (MED) measured by controlled exposure to UVB radiation (UVBR) as an acute photodamage threshold for different skin phototypes, and measure the constitutional skin color by means of the colorimetric system CIELAB. Patients and Methods: A total of 194 healthy individuals, over 18 years of age, were divided into groups with a minimum of 30 participants per phototype (Fitzpatrick critera). Four 1-cm² areas of the lateral trunk and buttocks were exposed to radiation and the color of the skin at these sites was registered by the CIELAB system. Design: Transversal study. Results: The average age of the participants out of whom 68% were females was 38 years. The assessment of the association of the MED values with the colorimetric values of the coordinate L* both on the thorax and on the buttocks showed a negative Pearson correlation coefficient (r) of - 0.91 for p<0.05. A Spearman’s rank correlation coefficient (r²) of +0.95 for p<0.05 was obtained for the MED values and the scores of the classification of the volunteers by phototypes, and rs of –0.93 on the thorax and rs of –0.92 on the buttocks for p<0.05 were found by correlating the colorimetric values with the scores of the phototypes. Conclusions – It was concluded that: 1) the colorimetric values of the coordinate L* showed a strong correlation with the DEM values, which could be a less invasive and more practical method for measuring the sensitivity to ultraviolet radiation; 2) although the Fitzpatrick’s scores have a high correlation with the MED values, they demonstrated the overlap of values in the adjoining phototypes; 3) the extent of association of the pthototype classes with the color of the skin allows us to say that the numerical category of the phototype increases as the skin gets darker.

Objetivos: Determinar a dose eritematosa mínima (DEM) medida por exposição controlada à radiação ultravioleta-B (RUV-B), como limiar para dano solar agudo nos diversos fototipos, e medir a cor da pele constitucional pelo sistema colorimétrico CIELAB. Pacientes e Métodos: Um total de 194 voluntários, sadios, com idades acima de 18 anos, distribuídos em um mínimo de 30 participantes por fototipo. Todos foram classificados por fototipos segundo os critérios de Fitzpatrick. As regiões infra-axilar torácica e nádega foram irradiadas em 4 áreas de 1 cm2, assim como foi registrada a cor da pele desses locais pelo sistema CIELAB. Delineamento: Estudo transversal. Resultados: A média de idade dos participantes foi de 38 anos, sendo 68% do sexo feminino. A avaliação da associação entre as medidas das DEMs e dos valores colorimétricos da coordenada L*, mostrou uma correlação de Pearson negativa com r = -0,91 para um valor p<0,05. Para os valores das DEMs e os escores da classificação dos voluntários por fototipos, obteve-se correlação de Spearman (rs) de +0,95 para p<0,05 e, correlacionando os valores colorimétricos com os escores dos fototipos, encontrou-se em tórax um rs de -0,93 e em nádega -0,92 para um p < 0,05. Conclusões – Concluiu-se que: 1)- a mensuração dos valores colorimétricos da coordenada L* nas regiões infra-axilar torácica e nádega mostraram uma forte correlação com os valores das DEMs, sendo de menor poder invasivo e de maior praticidade para mensuração de sensibilidade à radiação ultravioleta; 2)- apesar de os escores de Fitzpatrick terem alta correlação com os valores das DEMs, mostraram superposição de valores nos fototipos adjacentes; 3)- o grau de associação das classes dos fototipos com a cor da pele permite dizer que a categoria numérica do fototipo aumenta à medida que a pele fica mais escura. / Objectives: Determine the minimal erythema dose (MED) measured by controlled exposure to UVB radiation (UVBR) as an acute photodamage threshold for different skin phototypes, and measure the constitutional skin color by means of the colorimetric system CIELAB. Patients and Methods: A total of 194 healthy individuals, over 18 years of age, were divided into groups with a minimum of 30 participants per phototype (Fitzpatrick critera). Four 1-cm² areas of the lateral trunk and buttocks were exposed to radiation and the color of the skin at these sites was registered by the CIELAB system. Design: Transversal study. Results: The average age of the participants out of whom 68% were females was 38 years. The assessment of the association of the MED values with the colorimetric values of the coordinate L* both on the thorax and on the buttocks showed a negative Pearson correlation coefficient (r) of - 0.91 for p<0.05. A Spearman’s rank correlation coefficient (r²) of +0.95 for p<0.05 was obtained for the MED values and the scores of the classification of the volunteers by phototypes, and rs of –0.93 on the thorax and rs of –0.92 on the buttocks for p<0.05 were found by correlating the colorimetric values with the scores of the phototypes. Conclusions – It was concluded that: 1) the colorimetric values of the coordinate L* showed a strong correlation with the DEM values, which could be a less invasive and more practical method for measuring the sensitivity to ultraviolet radiation; 2) although the Fitzpatrick’s scores have a high correlation with the MED values, they demonstrated the overlap of values in the adjoining phototypes; 3) the extent of association of the pthototype classes with the color of the skin allows us to say that the numerical category of the phototype increases as the skin gets darker.

Erythema is a hallmark skin response to excessive ultraviolet radiation (UVR) and is associated with cutaneous inflammation. Both are mediated by inflammatory mediators including nitric oxide (NO), prostaglandin E2 (PGE2) and chemoattractants such as 12-hydroxyeicosatetraenoic acid (12-HETE) leading to vasodilation and increased leukocyte infiltration. The erythematous response is more pronounced in individuals with low basal melanin levels or who fail to respond to UVR with a robust up-regulation of melanogenesis. While melanin production is a key function of melanocytes, these cells can also produce NO and PGE2, and are located in close proximity to the dermal vasculature. It has been hypothesized that melanocytes with poor melanogenic capacity may participate in the inflammatory response to UVR. The aim of this project was to investigate the inflammatory response in the skin of individuals with either skin phototype (SPT) 1 or 4 to UVR. Sixteen normal healthy individuals were selected for study (8 SPT-1 & 8 SPT-4). Buttock skin was investigated by immunohistochemistry for leukocyte subtypes, eicosanoid producing enzymes and NO synthases under basal and UVR-stimulated conditions. In addition primary cultures of epidermal melanocytes (EM) were established from 16 individuals (8 SPT-1 & 8 SPT-4) and assessed for the presence of eicosanoid-producing enzymes, melanogenic enzymes and NO synthases, by immunocytochemistry, Polymerase Chain Reaction and Western Blotting and for the production of the main pro-inflammatory eicosanoid PGE2 by ELISA and Mass Spectrometry. Moreover, the fatty acid composition of cultured melanocytes was assessed by Gas Chromatography. Results showed that individuals with SPT-1 had significantly greater neutrophil infiltration into the epidermis than those with SPT-4 at 24 hrs post-UVR. Moreover, CD3+ lymphocyte infiltration into the dermis was significantly greater in individuals with SPT-4 than those with SPT-1 at 24 and 72 hrs post-UVR. NOS-1, NOS-3, 12-LOX and COX-2 expression were significantly increased in SPT-1 skin, while NOS-2 and 15-LOX were significantly increased in SPT-4 skin. As 12-LOX and COX-2 products are chemoattractive (for neutrophils) and pro-inflammatory respectively these data could explain the greater observed neutrophil infiltration in SPT-1. The 15-LOX product (15-HETE) is anti-inflammatory and may suggest that 15-LOX up-regulation in SPT-4 skin may aid resolution of the sunburn response, which in part may be mediated by CD3+ lymphocytes and a class-switch in eicosanoid production from COX to LOX products. Melanocyte primary cultures surprisingly showed that SPT was not correlated with melanin content or melanogenic enzyme expression/activity suggesting that all melanocytes in vitro contained the necessary cellular machinery to produce melanin. This finding may reflect also their equal treatment under these enriched culture conditions, which may or may not be available to these cells in situ. Moreover, all melanocytes expressed the necessary machinery (PLA2, COX-1, cPGES) to produce PGE2. However, only some cultures did so at baseline and in response to UVR, and this was not correlated with SPT. A positive correlation was found however between expression level of dopachrome tautomerase (DCT) and protection against PGE2 production in response to UVR, which may suggest a novel role for DCT unrelated to melanogenesis. In summary this research project has generated data that highlights differences between the skin of individuals with SPT-1 and those with SPT-4, and may provide evidence that the keratinocyte partner contributes significantly to the SPT-associated response. This research may also suggest DCT as a novel therapeutic target to protect EM from participation in the UVR-associated inflammatory response in skin. / Wellcome Trust

The spotted fever rickettsia, Rickettsia helvetica, is an endemic tick-borne bacteria in Sweden. It causes infections in humans, manifested as aneruptive fever, headache, arthralgia and myalgia, and sometimes an inoculation eschar or a rash. There have also been two known cases of human infections with R. felis in Sweden. The present thesis starts by investigating dispersal of ticks and Rickettsia spp. by migrating birds flying from Africa to Europe. Almost 15,000 birds were searched and 734 ticks collected, mainly of the species Hyalomma marginatum complex. Almost half (48%) of the ticks were infected with Rickettsia spp., 96% of which was R. aeschlimannii, the remaining R. africae and undefined species. The next study focused on questing ticks over a large area in Sweden and determining the prevalence of Rickettsia spp., Anaplasma spp. and Coxiella burnetii. Rickettsia spp. was found in 9.5-9.6% of the ticks and A. phagocytophilum in 0.7%; no C. burnetii was found. The last three papers in the thesis focused on the clinical presentation of rickettsiosis, the symptoms associated with the infection in general and particularly in patients with neurological complications. A tick-exposed population in Sweden was investigated to gain a better understanding of symptoms due to rickettsioses, also in relation to co-infections with other tick-borne bacteria. Based on symptoms, it was not possible to distinguish what pathogen caused the infections. Most patients had erythema migrans, some had serological reactions to Rickettsia spp., Borrelia spp. or co-infections by Rickettsia spp., Borrelia spp. and/or Anaplasma spp. In the fourth and fifth papers, we found associations between antibodies against Rickettsia spp. and sudden deafness (in 10-24% of patients) and facial nerve paralysis (in 8.3-25% of patients). In three patients R. felis was detected in the cerebrospinal fluids.    Briefly, the thesis helps to clarify our knowledge about tick dispersal, shows a narrower prevalence estimate of Rickettsia spp. in Swedish ticks, and illuminates symptoms of rickettsioses and co-infections with other tick-borne infections. It also shows that presence of erythema migrans may be explained by more than Lyme disease and indicates a possible association between rickettsiosis and sudden deafness and facial nerve paralysis.

Prior immunity to a related or unrelated pathogen greatly influences the host’s immune response to a subsequent infection and can cause a dramatic difference in disease course, a phenomenon known as heterologous immunity. Heterologous immunity can influence protective immunity, immunopathology and/or immune deviation of cytokine-producing T cell subsets. Examples of heterologous immunity have been well documented in mouse models, as well as during human infections. For example, prior immunity to lymphocytic choriomeningitis virus (LCMV) provides partial protection against vaccinia virus (VV), as LCMV-immune mice show reduced VV titers and increased survival upon lethal dose VV infection. Heterologous protection against VV challenge, as a result of LCMV immunity, is mediated by LCMV-specific CD4 and CD8 T cells, as transfer of LCMV-specific memory T cells can mediate this protective effect in naïve mice. The recognition of a single TCR with more than one MHC-peptide complex is referred to as T cell cross-reactivity. A VV Kb-restricted epitope a11r198 was identified to be able to induce cross-reactive responses from LCMV-specific CD8 T cells. During VV infection, LCMV-specific memory T cells that are cross-reactive to VV epitopes produce IFN-γ early in VV infection. IFN-γ is essential for mediating the protection against VV in LCMV-immune mice, as this heterologous protection is absent in IFN-γR-/-and IFN-γ blocking antibody-treated LCMV-immune mice. In addition to protective immunity, cross-reactive LCMV-specific memory T cells and IFN-γ also induce an altered immunopathology during heterologous VV challenge. LCMV-immune mice show moderate to severe levels of inflammation of the fat tissue, known as panniculitis, in the visceral fat pads upon VV challenge. In humans, panniculitis is a painful condition, most commonly presenting as erythema nodosum. Erythema nodosum is a disease of unknown etiology with no known treatment. It may occur following intracellular bacterial and viral infections, and occasionally happens after vaccination with VV for smallpox. During infections there can be a delicate balance between the ability of immune responses to provide protective immunity, and the tendency to induce immunopathology. By using the mouse model of heterologous immunity between LCMV and VV, we tried to understand how the immunity to LCMV biased the balance between the protective immunity and immunopathology, and what effector molecules were responsible for the pathogenesis of panniculitis in this system. TNF is a pleiotropic cytokine, which is required for normal innate and adaptive immune responses. Its functions range from inducing proliferative responses including cell survival, to destructive responses such as promoting apoptosis and programmed necrosis. In response to inflammatory stimuli, activated macrophages/ monocytes produce large amounts of TNF, and upon activation, T cells, B cells and NK cells also produce TNF. In vitro and in vivo studies have shown that TNF in synergy with IFN-γ plays an important role in mediating host defense against pathogens, such as Listeria monocytogenesand poxviruses in mice and hepatitis B virus and human immunodeficiency virus in humans. However, inappropriate expression of TNF often results in tissue damage. Considering the important role TNF plays in both host defense and mediating autoimmune diseases, we hypothesized that TNF was required for mediating both protective and pathogenic effects in the heterologous immunity between LCMV and VV. We first examined whether TNF was involved in mediating protective heterologous immunity. LCMV-immune mice, that were TNF-deficient as a consequence of genetic deletion (TNF-/-) or receptor blockade by treatment with etanercept (TNFR2: Fc fusion protein), were challenged with VV. These TNF-deficient mice showed normal recruitment and selective expansion of cross-reactive LCMV-specific memory CD8 T cells. They also exhibited efficient clearance of VV similar to LCMV-immune mice with normal TNF function. Thus, we concluded that neither TNF nor lymphotoxin (LT), which uses the same receptors as TNF, was required in mediating protective heterologous immunity against VV. Indeed, prior immunity to LCMV could completely compensate for the role of TNF in protection of naïve mice against VV infection, even under conditions of lethal dose inoculum. Thus, heterologous immunity may help explain why treatment of humans with etanercept is reasonably well tolerated with relatively few infectious complications. One of the histological characteristics of panniculitis is necrosis of adipose tissue. It is known that three members in the TNF superfamily, i.e. TNF/LT, FasL and TRAIL are able to induce necrosis of a target cell. It is also known that TNF is able to induce VV-infected cells to go through necrosis, when apoptosis is blocked in these cells by VV protein. Furthermore, TNF and FasL have already been shown to be associated with some skin and fat pathology. Thus, we hypothesized that TNF, FasL and TRAIL were involved in the pathogenesis of panniculitis in VV infected LCMV-immune mice. By using blocking antibodies or genetically deficient mice, we demonstrated that both TNF/LT and FasL were crucial for inducing panniculitis. Although TNFR1 has been reported to induce programmed necrosis, our data indicated that TNFR2, not TNFR1, was involved in mediating tissue damage in the fat pads of LCMV-immune mice infected with VV. We also found that TNF signaled through TNFR2 to up-regulate the expression of Fas on adipocytes. Thus, the engagement of Fas on the adipocytes with FasL expressed on activated VV-specific and cross-reactive LCMV-specific CD8 T cells in the fat pads could lead to panniculitis. Thus, our data may identify a potential mechanism in the pathogenesis of human panniculitis, and may suggest a possible treatment for this painful disease. Recent reports suggest that heterologous immunity may contribute to the tremendous variation in symptoms between individuals, from subclinical to death, upon viral infection. Even in genetically identical mice, variations in immunopathology from none to life-threatening levels of pathology are observed in LCMV-immune mice during VV infection. By adoptive transfer of splenocytes from a single LCMV-immune donor into two recipients, we showed that similar levels of pathology were generated in mice receiving the same splenocytes. However, the level of pathology varied among recipients receiving splenocytes from different LCMV-immune donors. The difference in levels of VV-induced pathology observed in individual LCMV-immune mice was a reflection of the private specificity of the T cell repertoire, which is a unique characteristic of each individual immune host. The goal of this doctoral thesis is to understand how heterologous immunity contributes to the pathogenesis of panniculitis. Our data demonstrate that TNF/LT and FasL directly contribute to development of panniculitis in LCMV-immune mice during VV infection, and suggest that anti-TNF treatment might be a useful treatment for diseases, such as erythema nodosum and lupus-induced acute fatty necrosis in humans.