O crescimento gengival induzido por ciclosporina-A (CG) é caracterizado por uma variedade de sinalizações que envolvem fatores de crescimento e proteoglicanos, porém pouco compreendidas. Investigamos a expressão gênica dos proteoheparans sulfato de superfície celular sindecam-2 (SDC-2), -4 (SDC-4) e betaglicam nesse CG. A quantidade total e relativa de glicosaminoglicanos sulfatados (GAGs) e a distribuição de SDC-2 e SDC-4 no tecido gengival também foram analisadas. Métodos: A expressão de mRNA dos proteoglicanos SDC-2, SDC-4 e betaglicam foi analisada pela reação de polimerase em cadeia e transcrição reversa (RT-PCR) em amostras gengivais de 9 indivíduos com CG (grupo CsA) e 6 com gengiva normal (grupo controle). Os GAGs foram extraídos e purificados dos tecidos gengivais e analisados tanto por eletroforese em gel de agarose quanto por espectrofotometria. Foi realizada uma avaliação imunohistoquímica dos tecidos com anticorpos para SDC-2 e SDC-4, para sua localização nos tecidos. Os grupos foram comparados pelo teste t de Student. Resultados: Todos os proteoglicanos estudados mostraramse aumentados no grupo CsA (165% para SDC-2, 308% para SDC-4 e 42% para betaglicam), comparativamente ao grupo controle (P < 0.0001). Não foram observadas diferenças significativas na quantidade total e relativa de GAGs. A imunohistoquímica mostrou uma distribuição marcante de SDC-2 e SDC-4 no componentes epitelial, conjuntivo, vascular, nervoso e inflamatório, incluindo os compartimentos celulares e matriciais de toda a casuística. Nossos resultados revelam expressão aumentada de mRNA de SDC-2, SDC-4 e betaglicam no crescimento gengival induzido pela ciclosporina-A, porém não se observaram diferenças na quantidade de glicosaminoglicanos sulfatados em relação ao tecido gengival não exposto ao fármaco. / Cyclosporin-induced gingival overgrowth (CIGO) comprises a variety of signaling pathways including growth factors and proteoglycans that remains not fully understood. We investigated the gene expression of the cell-surface heparan sulfate proteoglycans syndecan-2 (SDC-2), -4 (SDC-4) and betaglycan in CIGO. Total and relative amounts of sulfated glycosaminoglycans (GAGs) and the distribution of SDC-2 and SDC-4 in the gingival tissue were also analyzed. Methods: mRNA expression of the proteoglycans SDC-2, -4 and betaglycan was analyzed by reverse transcription polymerase chain reaction (RT-PCR) in gingival samples obtained from 9 individuals with CIGO and 6 with a normal gingiva (control group). GAGs were extracted and purified from gingival tissues and analyzed by agarose gel electrophoresis and spectrophotometry. An immunohistochemical evaluation using panels of antibodies for SDC-2 and -4 was performed for localization in the tissues. Groups were compared by the Student's t test. Results: All proteoglycans expressions revealed increase in the CIGO group (165% for SDC-2, 308% for SDC-4 and 42% for betaglycan) compared to the control group (P < 0.0001). No significant differences were observed for the total and relative amounts of GAGs. Immunohistochemistry showed a marked distribution of SDC-2 and SDC-4 in gingival epithelial, connective, vascular, neural and inflammatory components comprising cellular and matrix environments in both groups. Our results reveal increased mRNA expression of SDC-2, SDC-4 and betaglycan in CIGO, but no significant differences in the sulfated glycosaminoglycans component in situ.

O crescimento gengival induzido por ciclosporina-A (CG) é caracterizado por uma variedade de sinalizações que envolvem fatores de crescimento e proteoglicanos, porém pouco compreendidas. Investigamos a expressão gênica dos proteoheparans sulfato de superfície celular sindecam-2 (SDC-2), -4 (SDC-4) e betaglicam nesse CG. A quantidade total e relativa de glicosaminoglicanos sulfatados (GAGs) e a distribuição de SDC-2 e SDC-4 no tecido gengival também foram analisadas. Métodos: A expressão de mRNA dos proteoglicanos SDC-2, SDC-4 e betaglicam foi analisada pela reação de polimerase em cadeia e transcrição reversa (RT-PCR) em amostras gengivais de 9 indivíduos com CG (grupo CsA) e 6 com gengiva normal (grupo controle). Os GAGs foram extraídos e purificados dos tecidos gengivais e analisados tanto por eletroforese em gel de agarose quanto por espectrofotometria. Foi realizada uma avaliação imunohistoquímica dos tecidos com anticorpos para SDC-2 e SDC-4, para sua localização nos tecidos. Os grupos foram comparados pelo teste t de Student. Resultados: Todos os proteoglicanos estudados mostraramse aumentados no grupo CsA (165% para SDC-2, 308% para SDC-4 e 42% para betaglicam), comparativamente ao grupo controle (P < 0.0001). Não foram observadas diferenças significativas na quantidade total e relativa de GAGs. A imunohistoquímica mostrou uma distribuição marcante de SDC-2 e SDC-4 no componentes epitelial, conjuntivo, vascular, nervoso e inflamatório, incluindo os compartimentos celulares e matriciais de toda a casuística. Nossos resultados revelam expressão aumentada de mRNA de SDC-2, SDC-4 e betaglicam no crescimento gengival induzido pela ciclosporina-A, porém não se observaram diferenças na quantidade de glicosaminoglicanos sulfatados em relação ao tecido gengival não exposto ao fármaco. / Cyclosporin-induced gingival overgrowth (CIGO) comprises a variety of signaling pathways including growth factors and proteoglycans that remains not fully understood. We investigated the gene expression of the cell-surface heparan sulfate proteoglycans syndecan-2 (SDC-2), -4 (SDC-4) and betaglycan in CIGO. Total and relative amounts of sulfated glycosaminoglycans (GAGs) and the distribution of SDC-2 and SDC-4 in the gingival tissue were also analyzed. Methods: mRNA expression of the proteoglycans SDC-2, -4 and betaglycan was analyzed by reverse transcription polymerase chain reaction (RT-PCR) in gingival samples obtained from 9 individuals with CIGO and 6 with a normal gingiva (control group). GAGs were extracted and purified from gingival tissues and analyzed by agarose gel electrophoresis and spectrophotometry. An immunohistochemical evaluation using panels of antibodies for SDC-2 and -4 was performed for localization in the tissues. Groups were compared by the Student's t test. Results: All proteoglycans expressions revealed increase in the CIGO group (165% for SDC-2, 308% for SDC-4 and 42% for betaglycan) compared to the control group (P < 0.0001). No significant differences were observed for the total and relative amounts of GAGs. Immunohistochemistry showed a marked distribution of SDC-2 and SDC-4 in gingival epithelial, connective, vascular, neural and inflammatory components comprising cellular and matrix environments in both groups. Our results reveal increased mRNA expression of SDC-2, SDC-4 and betaglycan in CIGO, but no significant differences in the sulfated glycosaminoglycans component in situ.

No infarto, o remodelamento da região ventricular remota tem sido pouco examinado. Previamente, observamos que a redução da pressão de perfusão coronariana (PPC) associa-se ao aparecimento de fibrose subendocárdica. O objetivo do presente estudo foi investigar o papel da PPC no remodelamento e na função ventricular esquerda após o infarto. Medidas hemodinâmicas foram determinadas em ratos Wistar imediatamente após o infarto (IM) ou cirurgia fictícia (SH) e ao final dos seguimentos de 1, 3, 7, e 28 dias. Cortes teciduais do coração foram submetidos a coloração com HE, Sirius red, e a imunohistoquímica para a-actina. Duas regiões distintas do ventrículo esquerdo remotas ao infarto foram examinadas: subendocárdio (SE) e interstício (INT). A necrose de miócitos, a infiltração leucocitária e de miofibroblastos, e a fração de volume do colágeno foram determinadas. Comparados com SH, os grupos IM apresentaram menor PPC e disfunção sistólica e diastólica do ventrículo esquerdo. A necrose foi evidente em SE no dia 1. A inflamação e a fibroplasia ocorreram predominantemente em SE até o dia 7. A fibrose foi restrita a SE e evidente desde o dia 3. Em INT, a inflamação ocorreu predominantemente nos dias 1 e 3, mas em menor grau comparada a SE. A queda da PPC associou-se à dilatação progressiva do ventrículo esquerdo. Em SE, a PPC relacionou-se inversamente com a necrose (r =- 0,65, p = 0,003, no dia 1), com a inflamação (r = -0,76, p < 0,001, no dia 1), com a fibroplasia (r = -0,47, p = 0,04, no dia 7) e com a fibrose (r = -0,83, p < 0,001, no dia 28). A necrose no dia 1, a inflamação nos dias 3 e 7 e a fibroplasia no dia 7 relacionaram-se inversamente com a função do ventrículo esquerdo. A PPC é um fator determinante da integridade de SE e interfere com o remodelamento e a função do ventrículo esquerdo. / Left ventricular (LV) subendocardial remodeling has been poorly investigated after myocardial infarction. Previously, we have demonstrated that low coronary driving pressure (CDP) early in the course of infarction was associated with the subsequent development of subendocardial fibrosis. The present study aimed at the investigation of the role of CDP in LV remodeling and function following infarction. Hemodynamics were determined in Wistar rats immediately after infarct surgery (MI group) or sham (SH group) and at days 1, 3, 7, and 28. Heart tissue sections were stained with HE, Sirius red and immunostained for a- actin. Two distinct LV regions remote to MI were examined: subendocardium (SE) and interstitium (INT). Myocyte necrosis, leukocyte and myofibroblast infiltration, and collagen volume fraction were determined. Compared with SH, MI groups showed lower CDP and LV systolic and diastolic dysfunction. Necrosis was evident in SE at day 1. Inflammation and fibroplasia occurred predominantly in SE as far as day 7. Fibrosis was restricted to SE and was evident beginning from day 3. Inflammation occurred predominantly at days 1 and 3 in INT, but at a lower extent than in SE. CDP fall was associated with progressive LV dilatation. In SE, CDP correlated inversely with necrosis (r = -0.65, p=0.003, at day 1), inflammation (r = -0.76, p < 0.001, at day 1), fibroplasia (r = -0.47, p = 0.04, at day 7) and fibrosis (r = -0.83, p < 0.001, at day 28). Necrosis at day 1, inflammation at days 3 and 7, and fibroplasia at day 7 correlated inversely with LV function. CDP is a key factor to SE integrity following infarction and interferes with LV remodeling and function.

Introdução: As mucopolissacaridoses (MPSs) são doenças de depósito lisossômico causadas por deficiências enzimáticas envolvidas na degradação dos glicosaminoglicanos (GAGs). As MPSs dos tipos I, II e VI decorrem da deficiência das enzimas alfa-L-iduronidase, iduronato-2-sulfatase e Nacetilgalactosamina 4-sulfatase, respectivamente. As manifestações clínicas são multissistêmicas e progressivas. Objetivo: O objetivo deste estudo foi descrever a evolução clínica e laboratorial de pacientes com Mucopolissacaridose I, II e VI, em terapia de reposição enzimática (TRE). Métodos: Foram avaliados 27 pacientes com MPS (13 MPS I, 8 MPS II e 6 MPS VI), com diagnóstico confirmado pela dosagem enzimática e GAGs urinários, acompanhados em três centros de referência para TRE. Resultados: Cinco pacientes eram casos familiares (3 MPS I e 2 MPS VI). Todos os indivíduos estudados nasceram sem alterações clínicas e as mães relataram o aparecimento dos sintomas (alteração da face, aumento do volume abdominal, rigidez articular e déficit de crescimento) a partir dos 6 meses a 8 anos de idade, MPS I-Hurler (média 7m), MPS I Hurler-Scheie (média 2a), MPS I- Scheie (6a10m), MPS II (média 3a6m) e MPS VI (média 1a). A idade ao diagnóstico foi: MPS I-H (média 1a6m), I-HS (média 4a8m) e I-S (média 13a7m), MPS II e VI (média 5a). Entre os pacientes avaliados, observaram-se cinco casos familiais, sendo uma família com duas irmãs com MPS VI e outra família com dois irmãos e um primo com MPS I-S. Todos os pacientes apresentaram dismorfismos faciais típicos, associados a outros achados frequentes: restrição articular, mãos em garra, macrocefalia, baixa estatura, déficit ponderal. Pacientes com atraso no DNPM e/ou deficiência intelectual foram: Hurler (3/3), Hurler-Scheie (3/5) e MPS II (4/8). A idade do início da TRE foi de 1a2m a 31a9m. O tempo de TRE variou de 40 semanas a 556 semanas (média 259 semanas). Ao diagnóstico, todos os pacientes I, II e VI apresentaram o nível de GAGs urinários aumentado de 2 a 13 vezes o valor de referência para a idade. Aproximadamente 26 semanas após TRE, houve redução de GAGs urinários com a normalização do nível em 12/27 (44%) e o valor ainda um pouco acima do normal em 15/27(56%). Antes da TRE, 24/26 (92%) pacientes apresentaram alterações ecocardiográficas, e a despeito da TRE, houve persistência ou progressão das anormalidades. A polissonografia foi realizada em 10 pacientes antes da TRE sendo constatada a Síndrome da Apnéia Obstrutiva do Sono (SAOS) em nove pacientes (2 Hurler, 3 HurlerScheie, 3 MPS II e 1 MPS VI). Mesmo com o uso da TRE observou-se aumento do índice de apneias. As reações às infusões foram observadas em 55% dos pacientes (15/27), a maioria, presente nas primeiras semanas de infusão. Elas consistiram de: erupção cutânea, HAS, febre e broncoespasmo, revertidas após o uso de anti-histamínicos, antitérmicos e/ou redução da velocidade da infusão. As reações mais graves foram encontradas em dois pacientes. As principais complicações clínicas diagnosticadas, antes da TRE, foram: HAS (25%), perda auditiva (37%) e hidrocefalia (15%). Durante a TRE, houve aumento das frequências das complicações: HAS (37%), perda auditiva (59%) e hidrocefalia (22%). Entre os pacientes submetidos a intervenções cirúrgicas, cinco apresentaram complicações anestésicas e dois faleceram durante o procedimento. Conclusões: Este estudo mostrou heterogeneidade clínica pela variabilidade inter e intrafamilial. A utilização da TRE é capaz de atenuar, mas não de impedir a progressão da doença, cuja mortalidade permanece elevada. O diagnóstico precoce e a instituição da TRE pode modificar substancialmente a história natural da doença e melhorar a qualidade de vida / Introduction: Mucopolysaccharidosis (MPS) are lisosomal storage disorders caused by glycosaminoglycans (GAGs) enzymatic catabolism deficiencies, leading to mucopolysaccharides organ and tissues deposition. MPS types I, II and VI are due to deficiency of respectively, alpha-L-iduronidase, iduronate-2sulfatase and N-acetylgalactosamine-4-sulfatase. Clinical manifestations are progressive and multisystemic. Objective: The aim of this study was to describe the clinical and laboratory data of patients with MPS types I, II and VI receiving Enzyme Replacement Therapy (ERT). Methods: This study involved 27 patients with MPS (13 MPS I; 8 MPS II and 6 MPS VI) diagnosed by enzymatic and urinary GAGs measurement, followed in three reference centers for ERT. Results: Five patients were familial cases (3 MPS I and 2 MPS VI). All patients. All patients were born without clinical complications and their mothers referred the first signs and symptoms (coarse face, enlarged abdomen, stiff joints, short stature) had started at 6mo to 8y: MPS I Hurler (mean 7mo), MPS I Hurler-Scheie (mean 2y), MPS I Scheie (mean 6y10mo), MPS II (mean 3y6mo) and MPS VI (mean 1y). The mean age of diagnosis was: MPS I Hurler (1y6mo), MPS I Hurler-Scheie (4y8mo), MPS I Scheie (13y7mo), MPS II and VI (5y). There were five familial cases, including a MPS VI family (two sisters) and a MPS I Scheie family (two brothers and one cousin). All patients presented progressive typical facial coarsening. Other frequent findings were: stiff joints, clowded hands, macrocrania, failure to thrive. The prevalence of developmental milestones delay and/or mental intellectual disability was: MPS I Hurler (3/3), MPS I Hurler-Scheie (3/5) and MPS II (4/8). The age of onset of ERT ranged from 1y 2mo to 31y 9mo. The follow-up time after ERT initiation ranged from 40w to 556w (mean 259w). The baseline levels of urinary GAGs were increased two to 13 folds compared to reference values according to age. Approximately 26 weeks after ERT, urinary GAGs levels decreased. Normal levels of urinary GAGs in 12/27 (44%) and slightly increased in 15/27(56%) patients were observed. Before ERT, 24/26 (92%) patients presented echocardiographic abnormalities, which persisted or worsened in spite of ERT. Polysomnography was performed in 10 patients before ERT and revealed obstructive sleep apnea in nine patients (2 MPS I Hurler, 3 MPS I Hurler-Scheie, 3 MPS II and 1 MPS VI); the apnea/hypopnea index increased after ERT. Adverse infusion reactions were reported in 55% (15/27) of patients. Most of them was observed during the first weeks of treatment and included: skin rash, arterial hypertension, fever and bronchospasm, and were solved with antihistamines, antipyretics and/or reduction of infusion rate. Severe reactions were noted in two patients. Regarding clinical complications, arterial hypertension (25%), hypoacusia (37%) and hydrocephalus (15%) were diagnosed before ERT. After ERT arterial hypertension (37%), hypoacusia (59%) and hydrocephalus (22%) were reported. Among the patients that undergone surgical procedures, five presented anesthestical complications and two patients deceased during the procedure. Conclusions: This study showed both inter and intrafamilial clinical heterogeneity. ERT is able to ameliorate but not to stop the progression of the disease that remains with high mortality rate. This study emphasizes that the early diagnosis and ERT are critical for a better outcome and for enhancing the quality of life of these patients

Introdução: As mucopolissacaridoses (MPSs) são doenças de depósito lisossômico causadas por deficiências enzimáticas envolvidas na degradação dos glicosaminoglicanos (GAGs). As MPSs dos tipos I, II e VI decorrem da deficiência das enzimas alfa-L-iduronidase, iduronato-2-sulfatase e Nacetilgalactosamina 4-sulfatase, respectivamente. As manifestações clínicas são multissistêmicas e progressivas. Objetivo: O objetivo deste estudo foi descrever a evolução clínica e laboratorial de pacientes com Mucopolissacaridose I, II e VI, em terapia de reposição enzimática (TRE). Métodos: Foram avaliados 27 pacientes com MPS (13 MPS I, 8 MPS II e 6 MPS VI), com diagnóstico confirmado pela dosagem enzimática e GAGs urinários, acompanhados em três centros de referência para TRE. Resultados: Cinco pacientes eram casos familiares (3 MPS I e 2 MPS VI). Todos os indivíduos estudados nasceram sem alterações clínicas e as mães relataram o aparecimento dos sintomas (alteração da face, aumento do volume abdominal, rigidez articular e déficit de crescimento) a partir dos 6 meses a 8 anos de idade, MPS I-Hurler (média 7m), MPS I Hurler-Scheie (média 2a), MPS I- Scheie (6a10m), MPS II (média 3a6m) e MPS VI (média 1a). A idade ao diagnóstico foi: MPS I-H (média 1a6m), I-HS (média 4a8m) e I-S (média 13a7m), MPS II e VI (média 5a). Entre os pacientes avaliados, observaram-se cinco casos familiais, sendo uma família com duas irmãs com MPS VI e outra família com dois irmãos e um primo com MPS I-S. Todos os pacientes apresentaram dismorfismos faciais típicos, associados a outros achados frequentes: restrição articular, mãos em garra, macrocefalia, baixa estatura, déficit ponderal. Pacientes com atraso no DNPM e/ou deficiência intelectual foram: Hurler (3/3), Hurler-Scheie (3/5) e MPS II (4/8). A idade do início da TRE foi de 1a2m a 31a9m. O tempo de TRE variou de 40 semanas a 556 semanas (média 259 semanas). Ao diagnóstico, todos os pacientes I, II e VI apresentaram o nível de GAGs urinários aumentado de 2 a 13 vezes o valor de referência para a idade. Aproximadamente 26 semanas após TRE, houve redução de GAGs urinários com a normalização do nível em 12/27 (44%) e o valor ainda um pouco acima do normal em 15/27(56%). Antes da TRE, 24/26 (92%) pacientes apresentaram alterações ecocardiográficas, e a despeito da TRE, houve persistência ou progressão das anormalidades. A polissonografia foi realizada em 10 pacientes antes da TRE sendo constatada a Síndrome da Apnéia Obstrutiva do Sono (SAOS) em nove pacientes (2 Hurler, 3 HurlerScheie, 3 MPS II e 1 MPS VI). Mesmo com o uso da TRE observou-se aumento do índice de apneias. As reações às infusões foram observadas em 55% dos pacientes (15/27), a maioria, presente nas primeiras semanas de infusão. Elas consistiram de: erupção cutânea, HAS, febre e broncoespasmo, revertidas após o uso de anti-histamínicos, antitérmicos e/ou redução da velocidade da infusão. As reações mais graves foram encontradas em dois pacientes. As principais complicações clínicas diagnosticadas, antes da TRE, foram: HAS (25%), perda auditiva (37%) e hidrocefalia (15%). Durante a TRE, houve aumento das frequências das complicações: HAS (37%), perda auditiva (59%) e hidrocefalia (22%). Entre os pacientes submetidos a intervenções cirúrgicas, cinco apresentaram complicações anestésicas e dois faleceram durante o procedimento. Conclusões: Este estudo mostrou heterogeneidade clínica pela variabilidade inter e intrafamilial. A utilização da TRE é capaz de atenuar, mas não de impedir a progressão da doença, cuja mortalidade permanece elevada. O diagnóstico precoce e a instituição da TRE pode modificar substancialmente a história natural da doença e melhorar a qualidade de vida / Introduction: Mucopolysaccharidosis (MPS) are lisosomal storage disorders caused by glycosaminoglycans (GAGs) enzymatic catabolism deficiencies, leading to mucopolysaccharides organ and tissues deposition. MPS types I, II and VI are due to deficiency of respectively, alpha-L-iduronidase, iduronate-2sulfatase and N-acetylgalactosamine-4-sulfatase. Clinical manifestations are progressive and multisystemic. Objective: The aim of this study was to describe the clinical and laboratory data of patients with MPS types I, II and VI receiving Enzyme Replacement Therapy (ERT). Methods: This study involved 27 patients with MPS (13 MPS I; 8 MPS II and 6 MPS VI) diagnosed by enzymatic and urinary GAGs measurement, followed in three reference centers for ERT. Results: Five patients were familial cases (3 MPS I and 2 MPS VI). All patients. All patients were born without clinical complications and their mothers referred the first signs and symptoms (coarse face, enlarged abdomen, stiff joints, short stature) had started at 6mo to 8y: MPS I Hurler (mean 7mo), MPS I Hurler-Scheie (mean 2y), MPS I Scheie (mean 6y10mo), MPS II (mean 3y6mo) and MPS VI (mean 1y). The mean age of diagnosis was: MPS I Hurler (1y6mo), MPS I Hurler-Scheie (4y8mo), MPS I Scheie (13y7mo), MPS II and VI (5y). There were five familial cases, including a MPS VI family (two sisters) and a MPS I Scheie family (two brothers and one cousin). All patients presented progressive typical facial coarsening. Other frequent findings were: stiff joints, clowded hands, macrocrania, failure to thrive. The prevalence of developmental milestones delay and/or mental intellectual disability was: MPS I Hurler (3/3), MPS I Hurler-Scheie (3/5) and MPS II (4/8). The age of onset of ERT ranged from 1y 2mo to 31y 9mo. The follow-up time after ERT initiation ranged from 40w to 556w (mean 259w). The baseline levels of urinary GAGs were increased two to 13 folds compared to reference values according to age. Approximately 26 weeks after ERT, urinary GAGs levels decreased. Normal levels of urinary GAGs in 12/27 (44%) and slightly increased in 15/27(56%) patients were observed. Before ERT, 24/26 (92%) patients presented echocardiographic abnormalities, which persisted or worsened in spite of ERT. Polysomnography was performed in 10 patients before ERT and revealed obstructive sleep apnea in nine patients (2 MPS I Hurler, 3 MPS I Hurler-Scheie, 3 MPS II and 1 MPS VI); the apnea/hypopnea index increased after ERT. Adverse infusion reactions were reported in 55% (15/27) of patients. Most of them was observed during the first weeks of treatment and included: skin rash, arterial hypertension, fever and bronchospasm, and were solved with antihistamines, antipyretics and/or reduction of infusion rate. Severe reactions were noted in two patients. Regarding clinical complications, arterial hypertension (25%), hypoacusia (37%) and hydrocephalus (15%) were diagnosed before ERT. After ERT arterial hypertension (37%), hypoacusia (59%) and hydrocephalus (22%) were reported. Among the patients that undergone surgical procedures, five presented anesthestical complications and two patients deceased during the procedure. Conclusions: This study showed both inter and intrafamilial clinical heterogeneity. ERT is able to ameliorate but not to stop the progression of the disease that remains with high mortality rate. This study emphasizes that the early diagnosis and ERT are critical for a better outcome and for enhancing the quality of life of these patients

Glycosaminoglycans (GAGs) are linear polysaccharides whose disaccharide building blocks consist of an amino sugar and either uronic acid or galactose. They are expressed on virtually all mammalian cells, usually covalently attached to proteins, forming proteoglycans. GAGs are highly negatively charged due to an abundance of sulfate and carboxylic acid groups, and are structurally very diverse, with differences arising from chain length, the type of monomeric units, the linkages between each monomeric unit, the position of sulfate groups, and the degree of sulfation. GAGs are known to interact with a multitude of proteins, impacting diverse physiological and pathological processes. In addition, most of the biological interactions mediated by proteoglycans are believed to be primarily because of the GAG chains present on their surface. Considering the involvement of GAGs in multiple diseases, their use in the development of drugs has been of significant interest in the pharmaceutical field. Heparin, the first GAG-based drug developed in 1935, is still the most widely used anticoagulant in the world. The therapeutic potential of GAGs for the treatment of many other disease states, including cancer, inflammation, infection, wound healing, lung diseases, and Alzheimer’s disease, is being actively studied with many GAGs currently in clinical trials. However, challenges associated with the heterogeneous and complex structure of GAGs, limit their successful development. To combat such issues, our lab has focused on developing Non- Saccharide GAG Mimetics (NSGMs) as structural mimics of GAGs. NSGMs, being synthetic molecules, offer multiple advantages over GAGs. The studies mentioned here describe our efforts in the development of NSGMs as potential therapeutics for cancer, and cystic fibrosis.

Οι πρωτεογλυκάνες αλληλεπιδρούν μεταξύ τους και με άλλα μορφορυθμιστικά μόρια όπως γλυκοπρωτεΐνες και ιντεγκρίνες, καθώς επίσης και με αυξητικούς παράγοντες. Μεγάλο μέρος των ποικίλων λειτουργιών των πρωτεογλυκανών συνδέεται με τις γλυκοζαμινογλυκανικές αλυσίδες (GAGs) τους. Για να μελετήσουμε τη λειτουργία των πρωτεογλυκανών κατά την ανάπτυξη του πρώιμου εμβρύου όρνιθας χρησιμοποιήσαμε το β-D-xyloside, έναν εξειδικευμένο αναστολέα της βιοσύνθεσης των πρωτεογλυκανών και ειδικότερα της πρόσδεσης των αλυσίδων γλυκοζαμινογλυκανών στον πρωτεϊνικό κορμό των πρωτεογλυκανών. Χαμηλές συγκεντρώσεις β-xyloside που είναι γνωστό ότι αναστέλλουν την προσθήκη θειϊκής χονδροϊτίνης αλλά όχι της θειϊκής ηπαράνης στον πρωτεϊνικό κορμό, αποτελούν ένα χρήσιμο εργαλείο για τη μελέτη του ρόλου των πρωτεογλυκανών. Τα πρότυπα των πρωτεϊνών στα έμβρυα που μεταχειρίστηκαν με β-xyloside έδειξαν μετατόπιση των ραδιενεργών κορυφών σε μικρότερη μοριακή μάζα που φαίνεται να οφείλεται στη μείωση του μεγέθους πρωτεογλυκανών. Ήταν αξιοσημείωτο στα αποτελέσματά μας ότι το β-xyloside μετέβαλλε τη μορφή της πρωτεογλυκάνης θειϊκής χονδροϊτίνης decorin προς χαμηλότερα μοριακά βάρη ενώ δε φάνηκε να επηρεάζει το μέγεθος της πρωτεογλυκάνης θειϊκής ηπαράνης perlecan. Στα έμβρυα που μεταχειρίστηκαν με β-xyloside περισσότερη πρωτεϊνη συντέθηκε στα έμβρυα του σταδίου ΧΙΙ (μορίδιο), σε σχέση με αυτά του σταδίου ΗΗ2 (αρχή πρωτογενούς αύλακας/πρώιμο γαστρίδιο), συγκρινόμενα με τα αντίστοιχα έμβρυα μάρτυρες. Αυτό θα μπορούσε να αντανακλά μια επιταχυνόμενη κινητοποίηση και/ή μια μετάφραση των ωογενετικών μεταγράφων στα έμβρυα του σταδίου ΧΙΙ, όταν διαταράχτηκε ο μεταβολισμός των πρωτεογλυκανών. Η απορρύθμιση πρωτεογλυκανών, τροποποιώντας τη λειτουργικότητα και επηρεάζοντας το επίπεδο έκφρασής τους, είχε ως αποτέλεσμα την αδυναμία του πρώιμου εμβρύου να δομήσει την εξωκυττάρια ύλη φυσιολογικά. Το αποτέλεσμα ήταν η κατάρρευση της τυπικής αρχιτεκτονικής του πρώιμου εμβρύου στα πειράματά μας. Πρωτεογλυκάνες θειϊκής χονδροϊτίνης φαίνεται να χρειάζονται για την οργάνωση του σταδίου του βλαστιδίου της όρνιθας. Για την επαγωγή του νευρικού συστήματος φάνηκε να χρειάζονται πρωτεογλυκάνες θειϊκής χονδροϊτίνης/δερματάνης που έχουν συγκεντρωθεί από το στάδιο πριν την έναρξη των μορφογενετικών κινήσεων της γαστριδίωσης και η συνεχής βιοσύνθεση των πρωτεογλυκανών είναι απαραίτητη για τη μορφογένεση του νευρικού σωλήνα. Μελετήσαμε επίσης το πρότυπο κατανομής της συνδετικής πρωτεϊνης πρωτεογλυκανών με ανοσοφθορισμό και ανοσοκατακρήμνιση και το ρόλο αυτής της γλυκοπρωτεϊνης με τη χρήση αντισωμάτων έναντι αυτής. Η συνδετική πρωτεΐνη μεσολαβεί στην πρόσδεση στο υαλουρονικό οξύ πρωτεογλυκανών όπως οι aggrecan, neurocan, versican και brevican δημιουργώντας σταθερά σύμπλοκα στην εξωκυττάρια ύλη. Η αναγνώριση των μορφών της συνδετικής πρωτεΐνης 1 (LP1, 48kDa) και συνδετικής πρωτεΐνης 2 (LP2, 44kDa) στο πρώιμο έμβρυο ήταν επίσης ένα ενδιαφέρον εύρημα των πειραμάτων μας. Είναι γνωστό ότι συνδυασμοί των LP1 και LP2 δημιουργούν πιο σταθερά σύμπλοκα απ’ ότι μόνο του το καθένα από τα δύο μόρια αυτά. Αυτό φαίνεται και από τα πειράματά μας που δείχνουν ότι η aggrecan (180kDa) φαίνεται να συν-κατακρημνίζεται με τις συνδετικές πρωτεΐνες LP1 και LP2. Τα πειράματα ανοσοφθορισμού έδειξαν ότι η συνδετική πρωτεΐνη αρχίζει να εκφράζεται στο στάδιο του βλαστιδίου και επιδεικνύει μια διαφορική έκφραση χωρο-χρονικά κατά την ανάπτυξη του πρώιμου εμβρύου υποδεικνύοντας ότι είναι σημαντική στην κυτταρική μετανάστευση και διαφοροποίηση κυττάρων και στη μορφογένεση ιστών και οργάνων. Αυτό επιβεβαιώθηκε και από τη μελέτη του ρόλου της, στην επόμενη σειρά πειραμάτων μας με τη χρήση μονοκλωνικού αντισώματος έναντι αυτής. Η αναστολή της λειτουργίας της συνδετικής πρωτεϊνης με τη χρήση αντισωμάτων έναντι αυτής έδειξε ότι αυτή η πρωτεϊνη φαίνεται να είναι σημαντική στην οργάνωση της νευρικής πλάκας και τη μορφογένεση του νευρικού σωλήνα, στη μορφογένεση του καρδιακού σωλήνα, του εντέρου, στο σχηματισμό της ραχιαίας αορτής και στην επιθηλιοποίηση των σωμιτών. / Proteoglycans participate in cellular interactions via modulating the effects of growth factors or with other mechanisms in early embryo. The majority of the functions of proteoglycans are associated with the glycosaminoglycan (GAG) chains. We used β-D-xyloside, an inhibitor of proteoglycan synthesis and specifically of GAG attachment to proteoglycan core proteins, to study proteoglycan functions in early chick embryo development. Low concentrations of β-xyloside which are known to affect differentially chondroitin but not heparan sulfate proteoglycan biosynthesis have provided a convenient tool for altering proteoglycan production. The protein patterns of xyloside-treated embryos showed a shift of radioactive peaks to lower molecular mass which could be attributed to the reduction of proteoglycan size as was demonstrated by chondroitinase ABC/AC II treatments. It was notable in our data that β-xyloside altered the chondroitin sulfate proteoglycan decorin to lower molecular mass while it did not seem to affect the size of the heparin sulfate proteoglycan perlecan. More protein was synthesized from xyloside-treated embryos at stage XII (morula) than from embryos at stage HH2 (initial primitive streak/early gastrula) when compared to the controls. This could have reflected an accelerated translation and/or mobilization of oogenetic transcripts in embryos at stage XII when proteoglycan metabolism was disrupted. Misregulation of proteoglycans by modulating the functionality of the protein and by influencing their expression level resulted in an inability of the early embryo to assemble a stable extracellular matrix that would have been normally produced. These changes were associated with the collapse of the typical blastula architecture and inhibition of the induction of mesoderm in the chick embryo. Induction of neuroectoderm required proteoglycans assembled before the initiation of gastrulation movements. However, sustained proteoglycan biosynthesis was required for the morphogenetic movements to form the neural tube and the rest of the embryonic axis. We also studied the spatiotemporal distribution pattern of link protein by immunofluorescence and immunoprecipitation and the role of this glycoprotein by blocking antibodies in the early chick embryo. The recognition of the link protein 1 (LP1, 48 kDa) and link protein 2 (LP2, 44 kDa) types was an important finding of our study. Link protein links several proteoglycans, such as aggrecan to hyaluronan, creating stable aggregates in the extracellular matrix and has a general function in the organization of the extracellular matrix. It is known that combinations of LP1 and LP2 create more stable complexes than the individual link protein molecule. This was also shown in our experiments, in that aggrecan (180kDa) co-precipitated with LP1 and LP2. Our immunofluorescent experiments showed that link protein expression was first detectable at the blastula stage (st. XIII) and its presence may be fundamental as the first extracellular matrix starts to assemble before the initiation of the first major cellular migrations during the gastrula stage. Link protein influorescence was strong in the cells ingressing through the primitive streak and in the migrating cells in embryos at stage HH3 (intermediate streak/mid-gastrula). At stage HH4 (definitive streak/late gastrula), link protein fluorescence was strong at the apical surface of the neural plate. At stage HH4-5 (head process), link protein fluorescence was strong at the apical surface of the neural folds, notochord and endoderm. At stage HH13 (19 somites), link protein fluorescence was intense in the encephalic vesicles, in the extracellular matrix, in the lumen of encephalic vesicles, intense in migrating neural crest cells, neural tube and in notochord, strong in gut lower wall, hard tube and dorsal aorta wall, intense in dermomyotome and strong in sclerotome in somites. By stage HH17 (29 somites), link protein fluorescence was strong in neuroepithelium and extracellular matrix in the lumen of the diencephalon, strong in neural crest cells, in the intraretinal space in the eye, in myocardium and endocardium, in dorsal aorta, in dermomyotome, the outer surface of pharyngeal arches wall of aortic arches and intense in thyroid rudiment. Inhibition of function of link protein by blocking antibodies showed that link protein was important in neuroepithelial tissue organization and neural tube closure, in normal differentiation of the neural tube to form the brain, in the morphogenesis of the heart tube, the dorsal aorta and gut and in somite epithelialization.

Copies of author's previously published articles inserted / Includes bibliographic references / 2 v. (various foliations) : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Summary: Studies the enzymology of some of the nine lysosomal exo-enzyme activities which act together to degrade the more highly sulphated regions of the glycosaminoglycans heparin and heparan sulphate. A deficiency of any one of these enzyme activities can result in one of the lysosomal storage disorders collectively known as the Mucopolysaccharidoses (MPS) / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 1991

Le renouvellement de la membrane basale et de la matrice extracellulaire lors de processus physiologiques ou pathologiques (réparation tissulaire, angiogenèse, inflammation, cancer) fait intervenir de nombreuses protéases dont les cathepsines à cystéine. Après avoir étudié leur localisation dans l’épiderme à proximité de la jonction dermo-épidermique et leur sécrétion par les kératinocytes, nous avons montré la capacité de la cathepsine S à dégrader efficacement les principales protéines de la membrane basale (laminine, collagène IV, perlécan) et plus particulièrement le nidogène-1, qui est essentiel à l’organisation architecturale de la membrane basale via de multiples interactions avec les autres constituants. Parmi plusieurs glycosaminoglycanes présents dans la matrice extracellulaire, le chondroïtine 4-sulfate est capable de se complexer avec la cathepsine S, via trois sites potentiels de fixation dont un au niveau de son site actif, et d’inhiber son activité enzymatique de façon dose-dépendante. L’expression et l’activité de la cathepsine S au niveau de l’épiderme diminuent au cours du vieillissement cutané, alors que l’expression du nidogène-1 reste stable. La cathepsine S jouerait donc un rôle important aux côtés d’autres protéases dans le remodelage de la membrane basale. / Basement membrane (BM) and extracellular matrix (ECM) turnover during physiological or pathological events (tissue repair, angiogenesis, inflammation, cancer) involves numerous proteases including cysteine cathepsins. Cathepsins expression in skin epidermis near dermal-epidermal junction and their secretion by keratinocytes were first analyzed. We showed that cathepsin S degrades efficiently main BM components (laminin, type IV collagen, perlecan) and particularly nidogen-1 that is essential for BM architecture. Among several glycosaminoglycans present in ECM, chondroïtin 4-sulfate is able to form a stable complex with cathepsin S. Three predicted binding sites including one closed to its active site were identified. Further, C4-S inhibits cathepsin S activity in a dose-dependent manner. The expression and activity of cathepsin S in epidermis are decreased upon skin aging while nidogen-1 expression remains unchanged. Cathepsin S besides other proteases may play an important role in BM remodeling.

Objetivos: Investigar o dano a lipídios, proteínas e ao DNA, níveis de glicosaminoglicanos (GAGs) e as concentrações de interleucina 1-β (IL1-β) em sangue de pacientes com Mucopolissacaridose (MPS) tipo II no diagnóstico e durante a terapia de reposição enzimática (TRE) e correlacioná-los com parâmetros de estresse oxidativo, bem como analisar o efeito in vitro da genisteína sob o dano ao DNA em leucócitos de pacientes com MPS tipo IVA. Métodos: Amostras de sangue e urina de doze pacientes com MPS II no diagnóstico e sob tratamento com TRE, além de controles saudáveis foram utilizados para avaliar: índice de lipoperoxidação e oxidação de proteínas (medida a partir do conteúdo de grupamentos carbonila e SH) em plasma, GAGs urinários, níveis de IL1-β plasmáticos, bem como índice de dano ao DNA em leucócitos através do ensaio cometa, também utilizada para avaliar o dano ao DNA em leucócitos de pacientes com MPS IVA, previamente incubados em diferentes concentrações de genisteína ou em tampão fosfato salino e dimetilsulfóxido. Resultados e Discussão: Foi observada a presença de dano oxidativo a biomoléculas em sangue de pacientes com MPS II, com altos níveis de lipoperoxidação, conteúdo de grupamentos carbonila, dano ao DNA e redução de grupos sulfidrila. Houve redução no dano ao DNA e na lipoperoxidação após TRE, além de aumento de grupamentos sulfidrila, embora a terapia não tenha sido capaz de reverter o dano a carbonila. Nossos resultados sugerem que o aumento dos GAGs induz o dano aos lipídios e ao DNA. A adição in vitro de genisteína (10, 30 e 50 μM) em amostras de sangue de pacientes MPS IVA acarretou em um aumento estatisticamente significativo no índice de dano ao DNA. Conclusões: Estresse oxidativo e inflamação estão envolvidos na fisiopatologia da MPS II. Além disso, a TRE mostrou ter papel protetor contra o dano ao DNA e a lipídios. A genisteína nas doses 10, 30 e 50 μM aumentou in vitro o índice de dano ao DNA em leucócitos de pacientes MPS IVA, demonstrando citotoxicidade. / Objectives: Investigate lipid, protein and DNA damage, glycosaminoglycans (GAGs) levels and the inflammatory marker interleukin 1-β (IL1-β) concentration of mucopolysaccharidosis (MPS) II patients at the moment of diagnosis and during enzyme replacement therapy (ERT), correlate these findings with oxidative stress parameters, as well as investigate the in vitro effect of genistein on DNA injury in leukocytes from MPS IVA patients. Material and Methods: Blood and urine samples from twelve MPS II patients at diagnosis and under ERT and healthy controls were evaluated regarding the parameters: lipid peroxidation index and protein oxidation (carbonyl and SH group contents) in plasma, urinary GAGs, as well as IL-1β in plasma and DNA damage index in leukocytes. Besides, blood samples from MPS IVA patients were incubated with different concentrations of genistein or phosphate buffered saline (PBS) and dimethylsulfoxide (DMSO), and the DNA damage index in leukocytes was evaluated by the comet assay. Results and Discussion: MPS II patients presented oxidative damage to biomolecules with high levels of lipid peroxidation, carbonyl content and DNA damage, as well as a reduction on SH groups. There was a decrease in DNA damage and lipid oxidative injury after ERT, and an increase in SH levels, although this therapy was not able to reverse carbonyl content. The high levels of urinary GAGs in MPS II patients were reduced after ERT and positively correlated with DNA and lipid oxidative injury, suggesting that GAGs accumulation induce lipid peroxidation and DNA damage. MPS IVA patients blood treated in vitro with genistein (10, 30 e 50 μM) had higher DNA damage index when compared to samples treated with PBS buffer and DMSO. Conclusions: Oxidative stress and inflammation process are involved in MPS II pathophysiology, and ERT protects against DNA and lipid injury, probably by reducing GAGs accumulation. Genistein increased in vitro DNA damage in leukocytes from MPS IVA patients, demonstrating cytotoxicity.