In vitro studies have shown that the major membrane phospholipid phosphatidylcholine (PC) can positively influence the incorporation of cholesterol in lipid membranes. The influence of PC on the cellular trafficking of LDL-derived free cholesterol was investigated. Sterol regulatory-defective (SRD)-4 cells are Chinese hamster ovary (CHO)-derived fibroblasts that display vastly elevated rates for the synthesis and catabolism of PC. SRD-4 cells harbor two known gene mutations: a mutation in the functional allele for SCAP, resulting in defective feedback suppression of cholesterol biosynthesis; and a loss-of-function mutation in the functional allele for acyl-CoA:cholesterol acyl transferase (ACAT), an endoplasmic reticulum (ER)-localized enzyme that esterifies free cholesterol. Incubation of SRD-4 cells with 50 µg/ml low density lipoprotein (LDL) for 18 h resulted in lysosomal accumulation of free cholesterol as revealed by filipin staining. This accumulation was not evident following LDL treatment of parental CHO7 cells, and was blunted in SRD-2 cells that express a constitutively-active form of SREBP-2 and overproduce cholesterol but have functional ACAT activity. Treatment of SRD-2 cells with LDL in the presence of an ACAT inhibitor 58-035 resulted in robust lysosomal cholesterol accumulation that was reversible upon drug washout, supporting that cholesterol trafficking in cholesterol-loaded cells is dependent on ACAT activity and, more specifically, ER free cholesterol levels. Lysosomal accumulation of LDL-derived cholesterol was prevented in SRD-4 cells supplemented with lyso-PC (50 µM), a substrate for PC synthesis through the reacylation pathway, and also in cells treated with bromoenol lactone (BEL), an inhibitor of phospholipase A2 implicated in bulk PC turnover. In a counter study, lysosomal LDL-derived cholesterol accumulation was induced in parental CHO-7 cells using R-propranolol, which inhibits the conversion of phosphatidic acid to diacylglycerol (DAG), a substrate in the CDP-choline pathway. This blockage was also relieved through co-treatment with lyso-PC. These studies support that PC to free cholesterol ratios in downstream organellar membranes can influence cholesterol trafficking out of lysosomal compartments in cholesterol-loaded cells.

In vitro studies have shown that the major membrane phospholipid phosphatidylcholine (PC) can positively influence the incorporation of cholesterol in lipid membranes. The influence of PC on the cellular trafficking of LDL-derived free cholesterol was investigated. Sterol regulatory-defective (SRD)-4 cells are Chinese hamster ovary (CHO)-derived fibroblasts that display vastly elevated rates for the synthesis and catabolism of PC. SRD-4 cells harbor two known gene mutations: a mutation in the functional allele for SCAP, resulting in defective feedback suppression of cholesterol biosynthesis; and a loss-of-function mutation in the functional allele for acyl-CoA:cholesterol acyl transferase (ACAT), an endoplasmic reticulum (ER)-localized enzyme that esterifies free cholesterol. Incubation of SRD-4 cells with 50 µg/ml low density lipoprotein (LDL) for 18 h resulted in lysosomal accumulation of free cholesterol as revealed by filipin staining. This accumulation was not evident following LDL treatment of parental CHO7 cells, and was blunted in SRD-2 cells that express a constitutively-active form of SREBP-2 and overproduce cholesterol but have functional ACAT activity. Treatment of SRD-2 cells with LDL in the presence of an ACAT inhibitor 58-035 resulted in robust lysosomal cholesterol accumulation that was reversible upon drug washout, supporting that cholesterol trafficking in cholesterol-loaded cells is dependent on ACAT activity and, more specifically, ER free cholesterol levels. Lysosomal accumulation of LDL-derived cholesterol was prevented in SRD-4 cells supplemented with lyso-PC (50 µM), a substrate for PC synthesis through the reacylation pathway, and also in cells treated with bromoenol lactone (BEL), an inhibitor of phospholipase A2 implicated in bulk PC turnover. In a counter study, lysosomal LDL-derived cholesterol accumulation was induced in parental CHO-7 cells using R-propranolol, which inhibits the conversion of phosphatidic acid to diacylglycerol (DAG), a substrate in the CDP-choline pathway. This blockage was also relieved through co-treatment with lyso-PC. These studies support that PC to free cholesterol ratios in downstream organellar membranes can influence cholesterol trafficking out of lysosomal compartments in cholesterol-loaded cells.

In vitro studies have shown that the major membrane phospholipid phosphatidylcholine (PC) can positively influence the incorporation of cholesterol in lipid membranes. The influence of PC on the cellular trafficking of LDL-derived free cholesterol was investigated. Sterol regulatory-defective (SRD)-4 cells are Chinese hamster ovary (CHO)-derived fibroblasts that display vastly elevated rates for the synthesis and catabolism of PC. SRD-4 cells harbor two known gene mutations: a mutation in the functional allele for SCAP, resulting in defective feedback suppression of cholesterol biosynthesis; and a loss-of-function mutation in the functional allele for acyl-CoA:cholesterol acyl transferase (ACAT), an endoplasmic reticulum (ER)-localized enzyme that esterifies free cholesterol. Incubation of SRD-4 cells with 50 µg/ml low density lipoprotein (LDL) for 18 h resulted in lysosomal accumulation of free cholesterol as revealed by filipin staining. This accumulation was not evident following LDL treatment of parental CHO7 cells, and was blunted in SRD-2 cells that express a constitutively-active form of SREBP-2 and overproduce cholesterol but have functional ACAT activity. Treatment of SRD-2 cells with LDL in the presence of an ACAT inhibitor 58-035 resulted in robust lysosomal cholesterol accumulation that was reversible upon drug washout, supporting that cholesterol trafficking in cholesterol-loaded cells is dependent on ACAT activity and, more specifically, ER free cholesterol levels. Lysosomal accumulation of LDL-derived cholesterol was prevented in SRD-4 cells supplemented with lyso-PC (50 µM), a substrate for PC synthesis through the reacylation pathway, and also in cells treated with bromoenol lactone (BEL), an inhibitor of phospholipase A2 implicated in bulk PC turnover. In a counter study, lysosomal LDL-derived cholesterol accumulation was induced in parental CHO-7 cells using R-propranolol, which inhibits the conversion of phosphatidic acid to diacylglycerol (DAG), a substrate in the CDP-choline pathway. This blockage was also relieved through co-treatment with lyso-PC. These studies support that PC to free cholesterol ratios in downstream organellar membranes can influence cholesterol trafficking out of lysosomal compartments in cholesterol-loaded cells.

M. Niemann-Pick Typ C (NPC) ist eine juvenile Demenz mit intrazellulärer Anreicherung von freiem Cholesterin, M. Alzheimer (AD) eine senile Demenz, die mit einem Polymorphismus im Gen des Cholesterintransportproteins Apolipoprotein E (ApoE) assoziiert ist. Bei beiden Erkrankungen treten im Gehirn zahlreiche neurofibrilläre Tangles (NFT), bestehend aus Protein Tau, auf. Es sollte mit einer für freies Cholesterin spezifischen Filipin-Färbung herausgefunden werden, ob sich bei beiden Erkrankungen der Cholesteringehalt Tangle-tragender Neurone von dem Tangle-freier unterscheidet. Zur Verfügung standen diverse Teile aus dem Zentralen Nervensystem von 5 NPC- und Gyri temporales superficiales von 9 AD-Fällen. In Material von 3 NPC-Fällen und 1 AD-Fall fanden sich intraneuronale, mit Tangles assoziierte Cholesterinakkumulationen. In 3 AD-Fällen fanden sich außerdem Cholesterinakkumulationen in Assoziation mit Senilen Plaques. Der Cholesteringehalt Tangle-tragender Neurone war in 6 Regionen der NPC-Fälle und in 3 AD-Fällen erhöht. Für alle Neuronenpaare der AD-Fälle insgesamt ergab sich ein erhöhter Cholesteringehalt des Tangle-tragenden Neurons. Cholesterinspeicherung im NPC-Hirn könnte über oxidativen Stress oder eine Veränderung der cholesterinabhängigen Signaltransduktion zur Tangle-Bildung führen. Argumente für die Unterstützung der Tangle-Bildung durch Cholesterin über oxidativen Stress und veränderte Signaltransduktion in AD werden angeführt. Diese Untersuchung erlaubt, neue Hypothesen zu gemeinsamen Mechanismen der Tangle-Entstehung in AD und NPC zu formulieren. / Niemann-Pick type C disease (NPC) is a juvenile dementia with intracellular accumulation of free cholesterol, whereas Alzheimer s disease (AD) is a senile dementia associated with a gene polymorphism of a cholesterol transport protein, apolipoprotein E (apoE). In both conditions, there are abundant neurofibrillary tangles (NFT) in brain, consisting of protein tau. This study addresses the issue whether cholesterol content of tangle-bearing neurons is the same compared to tangle-free neurons, in both diseases. For this purpose, staining with the free cholesterol-specific fluorochrome filipin was used. Several CNS tissue specimen of 5 NPC cases and superior temporal gyri of 9 AD cases were available. In 3 NPC cases and 1 AD case, intraneuronal cholesterol accumulation were found associated with neurofibrillary tangles. Furthermore, cholesterol accumulations were found associated with senile plaques in 3 AD cases. Cholesterol content of tangle-bearing neurons was increased in 6 regions of the NPC cases and in 3 AD cases. For all neurons analysed in AD, the tangle-bearing neuron showed more cholesterol than the adjacent tangle-free neuron. Cholesterol storage in NPC brain could lead to neurofibrillary degeneration by enhancing oxidative stress or by alterating cholesterol-dependent signal transduction. Cholesterol accumulation in AD neurons could lead to neurofibrillary degeneration by the same mechanisms. This study allows the creation of new hypotheses concerning common mechanisms of neurofibrillary degeneration in both Niemann-Pick type C and Alzheimer s diseases.

A doença de Niemann-Pick tipo C (NPC) é uma esfingolipidose autossômica recessiva que se caracteriza pelo acúmulo lisossômico de colesterol não-esterificado em vários tecidos, resultando em neurodegeneração progressiva, hepatoesplenomegalia e paralisia ocular vertical, entre outros sintomas. Sua manifestação ocorre geralmente entre a metade da infância e a adolescência. A morte ocorre geralmente até a terceira década de vida. Associado à sintomatologia clínica, o diagnóstico de NPC é realizado através do teste de Filipin em fibroblastos cultivados, que demonstra um intenso padrão de fluorescência perinuclear, consistente com o acúmulo de colesterol nãoesterificado. Alguns pacientes, referidos como NPC variantes, apresentam quadro clínico compatível com NPC, mas resultados inconclusivos nos testes bioquímicos, com fluorescência não característica no teste de Filipin, tornando problemático o diagnóstico. O objetivo deste trabalho foi desenvolver técnicas objetivas capazes de auxiliar na confirmação do diagnóstico de NPC, quando um fenótipo apresentar característica indefinida/variante ao teste de Filipin, tais como: quantificação do padrão de fluorescência perinuclear quanto ao número de pixels em pacientes com fenótipo clássico (NPC cl) e variante (NPC var) de NPC, comparando-os entre si e com controles saudáveis (CS); medida da quantidade de colesterol citoplasmático em fibroblastos dos dois grupos de pacientes; medida da atividade das enzimas esfingomielinase ácida (ASM), quitotriosidase (QT), beta-glicosidase ácida (GBA) e beta-galactosidase (GLB). Todos estes parâmetros foram comparados com aqueles de CS. Os resultados mostraram que a quantificação da fluorescência do colesterol no teste de Filipin nos três grupos estudados através do número de pixels da imagem, é um método prático e não subjetivo que demonstrou uma diferença significativa entre o acúmulo de colesterol intracelular em CS, NPC cl e NPC var, confirmando a eficácia do método para o esclarecimento de padrões duvidosos. A dosagem de colesterol intracelular em fibroblastos de NPC apresentou-se sete vezes maior no padrão clássico e quatro vezes maior no variante, do que aquela encontrada nos CS. Com esses dados, a dosagem intracelular de colesterol mostra-se um bom parâmetro quantitativo para auxiliar no teste de Filipin quando este apresentar padrão de fluorescência apenas moderado. A medida da atividade da ASM tanto em leucócitos quanto em SPF não apresentou diferenças estatisticamente significativas entre os grupos estudados, embora seja aparente a diminuição na atividade desta enzima no fenótipo NPC cl. Com os valores obtidos para a medida da atividade da QT em plasma e SPF, verificamos que não houve diferença significativa entre os dois grupos NPC mas sim entre estes e os CS, demostrando que tanto os indivíduos com o fenótipo clássico quanto com o variante possuem uma atividade aumentada desta enzima. A análise da atividade da GBA em leucócitos não apresentou diferenças significativas entre os três grupos, embora sua atividade em NPC cl pareça maior do em CS. Já na análise em SPF, esta diferença foi estatisticamente confirmada. A atividade da GLB em leucócitos não apresentou diferenças entre os grupos estudados, embora esta pareça maior nos grupos NPC do que em CS. / Niemann-Pick disease Type C (NPC) is an autosomal recessive sphingolipidosis which is characterized by a lysosomal accumulation of unesterified cholesterol in various tissues, resulting in progressive neurodegeneration, hepatosplenomegaly and vertical eye paralysis, among other symptoms. Its onset occurs commonly between middle childhood and adolescence. Death occurs usually until the third decade of life. Along with the clinical symptoms, the diagnosis of NPC is accomplished by Filipin test in cultured fibroblasts, showing an intense perinuclear staining pattern, which is consistent with the accumulation of unesterified cholesterol. Some patients, referred to as NPC variants, present a clinical picture of NPC, but inconclusive results in biochemical tests, with no characteristic fluorescence in Filipin test, which makes the diagnosis problematic. The objective of this study was to develop objective techniques that can assist in confirming the diagnosis of NPC, when the phenotype shows undefined/variant characteristics to Filipin test, such as: quantification of perinuclear fluorescence pattern based on pixels' luminosity pattern in patients with classical (NPC cl) and variant (NPC var) NPC phenotype, comparing them among themselves and with healthy controls (CS); the amount of cytoplasmic cholesterol in fibroblasts of both groups of patients; measurement of the activity of the enzymes acid sphingomyelinase (ASM), chitotriosidase (CT), beta-glucosidase acid (GBA), and beta-galactosidase (GLB). All of these parameters were compared with those of CS. The results showed that the fluorescence quantitation of cholesterol in the Filipin test, for the three studied groups, by counting the number of image pixels, is a practical and non-subjective method, which showed a significant difference between the accumulation of intracellular cholesterol in CS, NPC cl and NPC var, confirming its effectiveness to clarify dubious patterns. Measures of intracellular cholesterol in NPC fibroblasts showed seven times higher in classic and four times higher in variant pattern, than that found in CS. With these data, the dosage of intracellular cholesterol appears to be a good parameter to aid in the quantitative Filipin test when it presents only a moderate fluorescence pattern. The measurement of ASM activity in both leukocytes and in SPF did not show statistically significant differences between the groups, despite the remarkable decrease in the enzyme's activity in NPC cl phenotype. With the data obtained for QT's activity in plasma and SPF, we found that there was no significant difference between the two NPC groups, but a significant one between the NPC and the CS, demonstrating that both individuals with the classical phenotype and with the variant one have an increased activity of this enzyme. The analysis of GBA activity in leukocytes showed no significant differences among the three groups, although its activity in NPC cl seemed to be greater than in CS. In the SPF analysis, this difference was statistically confirmed. The activity of GLB in leukocytes did not differ between the groups, although it seems greater in the NPC group than in CS.

A doença de Niemann-Pick tipo C (NPC) é uma esfingolipidose autossômica recessiva que se caracteriza pelo acúmulo lisossômico de colesterol não-esterificado em vários tecidos, resultando em neurodegeneração progressiva, hepatoesplenomegalia e paralisia ocular vertical, entre outros sintomas. Sua manifestação ocorre geralmente entre a metade da infância e a adolescência. A morte ocorre geralmente até a terceira década de vida. Associado à sintomatologia clínica, o diagnóstico de NPC é realizado através do teste de Filipin em fibroblastos cultivados, que demonstra um intenso padrão de fluorescência perinuclear, consistente com o acúmulo de colesterol nãoesterificado. Alguns pacientes, referidos como NPC variantes, apresentam quadro clínico compatível com NPC, mas resultados inconclusivos nos testes bioquímicos, com fluorescência não característica no teste de Filipin, tornando problemático o diagnóstico. O objetivo deste trabalho foi desenvolver técnicas objetivas capazes de auxiliar na confirmação do diagnóstico de NPC, quando um fenótipo apresentar característica indefinida/variante ao teste de Filipin, tais como: quantificação do padrão de fluorescência perinuclear quanto ao número de pixels em pacientes com fenótipo clássico (NPC cl) e variante (NPC var) de NPC, comparando-os entre si e com controles saudáveis (CS); medida da quantidade de colesterol citoplasmático em fibroblastos dos dois grupos de pacientes; medida da atividade das enzimas esfingomielinase ácida (ASM), quitotriosidase (QT), beta-glicosidase ácida (GBA) e beta-galactosidase (GLB). Todos estes parâmetros foram comparados com aqueles de CS. Os resultados mostraram que a quantificação da fluorescência do colesterol no teste de Filipin nos três grupos estudados através do número de pixels da imagem, é um método prático e não subjetivo que demonstrou uma diferença significativa entre o acúmulo de colesterol intracelular em CS, NPC cl e NPC var, confirmando a eficácia do método para o esclarecimento de padrões duvidosos. A dosagem de colesterol intracelular em fibroblastos de NPC apresentou-se sete vezes maior no padrão clássico e quatro vezes maior no variante, do que aquela encontrada nos CS. Com esses dados, a dosagem intracelular de colesterol mostra-se um bom parâmetro quantitativo para auxiliar no teste de Filipin quando este apresentar padrão de fluorescência apenas moderado. A medida da atividade da ASM tanto em leucócitos quanto em SPF não apresentou diferenças estatisticamente significativas entre os grupos estudados, embora seja aparente a diminuição na atividade desta enzima no fenótipo NPC cl. Com os valores obtidos para a medida da atividade da QT em plasma e SPF, verificamos que não houve diferença significativa entre os dois grupos NPC mas sim entre estes e os CS, demostrando que tanto os indivíduos com o fenótipo clássico quanto com o variante possuem uma atividade aumentada desta enzima. A análise da atividade da GBA em leucócitos não apresentou diferenças significativas entre os três grupos, embora sua atividade em NPC cl pareça maior do em CS. Já na análise em SPF, esta diferença foi estatisticamente confirmada. A atividade da GLB em leucócitos não apresentou diferenças entre os grupos estudados, embora esta pareça maior nos grupos NPC do que em CS. / Niemann-Pick disease Type C (NPC) is an autosomal recessive sphingolipidosis which is characterized by a lysosomal accumulation of unesterified cholesterol in various tissues, resulting in progressive neurodegeneration, hepatosplenomegaly and vertical eye paralysis, among other symptoms. Its onset occurs commonly between middle childhood and adolescence. Death occurs usually until the third decade of life. Along with the clinical symptoms, the diagnosis of NPC is accomplished by Filipin test in cultured fibroblasts, showing an intense perinuclear staining pattern, which is consistent with the accumulation of unesterified cholesterol. Some patients, referred to as NPC variants, present a clinical picture of NPC, but inconclusive results in biochemical tests, with no characteristic fluorescence in Filipin test, which makes the diagnosis problematic. The objective of this study was to develop objective techniques that can assist in confirming the diagnosis of NPC, when the phenotype shows undefined/variant characteristics to Filipin test, such as: quantification of perinuclear fluorescence pattern based on pixels' luminosity pattern in patients with classical (NPC cl) and variant (NPC var) NPC phenotype, comparing them among themselves and with healthy controls (CS); the amount of cytoplasmic cholesterol in fibroblasts of both groups of patients; measurement of the activity of the enzymes acid sphingomyelinase (ASM), chitotriosidase (CT), beta-glucosidase acid (GBA), and beta-galactosidase (GLB). All of these parameters were compared with those of CS. The results showed that the fluorescence quantitation of cholesterol in the Filipin test, for the three studied groups, by counting the number of image pixels, is a practical and non-subjective method, which showed a significant difference between the accumulation of intracellular cholesterol in CS, NPC cl and NPC var, confirming its effectiveness to clarify dubious patterns. Measures of intracellular cholesterol in NPC fibroblasts showed seven times higher in classic and four times higher in variant pattern, than that found in CS. With these data, the dosage of intracellular cholesterol appears to be a good parameter to aid in the quantitative Filipin test when it presents only a moderate fluorescence pattern. The measurement of ASM activity in both leukocytes and in SPF did not show statistically significant differences between the groups, despite the remarkable decrease in the enzyme's activity in NPC cl phenotype. With the data obtained for QT's activity in plasma and SPF, we found that there was no significant difference between the two NPC groups, but a significant one between the NPC and the CS, demonstrating that both individuals with the classical phenotype and with the variant one have an increased activity of this enzyme. The analysis of GBA activity in leukocytes showed no significant differences among the three groups, although its activity in NPC cl seemed to be greater than in CS. In the SPF analysis, this difference was statistically confirmed. The activity of GLB in leukocytes did not differ between the groups, although it seems greater in the NPC group than in CS.

A doença de Niemann-Pick tipo C (NPC) é uma esfingolipidose autossômica recessiva que se caracteriza pelo acúmulo lisossômico de colesterol não-esterificado em vários tecidos, resultando em neurodegeneração progressiva, hepatoesplenomegalia e paralisia ocular vertical, entre outros sintomas. Sua manifestação ocorre geralmente entre a metade da infância e a adolescência. A morte ocorre geralmente até a terceira década de vida. Associado à sintomatologia clínica, o diagnóstico de NPC é realizado através do teste de Filipin em fibroblastos cultivados, que demonstra um intenso padrão de fluorescência perinuclear, consistente com o acúmulo de colesterol nãoesterificado. Alguns pacientes, referidos como NPC variantes, apresentam quadro clínico compatível com NPC, mas resultados inconclusivos nos testes bioquímicos, com fluorescência não característica no teste de Filipin, tornando problemático o diagnóstico. O objetivo deste trabalho foi desenvolver técnicas objetivas capazes de auxiliar na confirmação do diagnóstico de NPC, quando um fenótipo apresentar característica indefinida/variante ao teste de Filipin, tais como: quantificação do padrão de fluorescência perinuclear quanto ao número de pixels em pacientes com fenótipo clássico (NPC cl) e variante (NPC var) de NPC, comparando-os entre si e com controles saudáveis (CS); medida da quantidade de colesterol citoplasmático em fibroblastos dos dois grupos de pacientes; medida da atividade das enzimas esfingomielinase ácida (ASM), quitotriosidase (QT), beta-glicosidase ácida (GBA) e beta-galactosidase (GLB). Todos estes parâmetros foram comparados com aqueles de CS. Os resultados mostraram que a quantificação da fluorescência do colesterol no teste de Filipin nos três grupos estudados através do número de pixels da imagem, é um método prático e não subjetivo que demonstrou uma diferença significativa entre o acúmulo de colesterol intracelular em CS, NPC cl e NPC var, confirmando a eficácia do método para o esclarecimento de padrões duvidosos. A dosagem de colesterol intracelular em fibroblastos de NPC apresentou-se sete vezes maior no padrão clássico e quatro vezes maior no variante, do que aquela encontrada nos CS. Com esses dados, a dosagem intracelular de colesterol mostra-se um bom parâmetro quantitativo para auxiliar no teste de Filipin quando este apresentar padrão de fluorescência apenas moderado. A medida da atividade da ASM tanto em leucócitos quanto em SPF não apresentou diferenças estatisticamente significativas entre os grupos estudados, embora seja aparente a diminuição na atividade desta enzima no fenótipo NPC cl. Com os valores obtidos para a medida da atividade da QT em plasma e SPF, verificamos que não houve diferença significativa entre os dois grupos NPC mas sim entre estes e os CS, demostrando que tanto os indivíduos com o fenótipo clássico quanto com o variante possuem uma atividade aumentada desta enzima. A análise da atividade da GBA em leucócitos não apresentou diferenças significativas entre os três grupos, embora sua atividade em NPC cl pareça maior do em CS. Já na análise em SPF, esta diferença foi estatisticamente confirmada. A atividade da GLB em leucócitos não apresentou diferenças entre os grupos estudados, embora esta pareça maior nos grupos NPC do que em CS. / Niemann-Pick disease Type C (NPC) is an autosomal recessive sphingolipidosis which is characterized by a lysosomal accumulation of unesterified cholesterol in various tissues, resulting in progressive neurodegeneration, hepatosplenomegaly and vertical eye paralysis, among other symptoms. Its onset occurs commonly between middle childhood and adolescence. Death occurs usually until the third decade of life. Along with the clinical symptoms, the diagnosis of NPC is accomplished by Filipin test in cultured fibroblasts, showing an intense perinuclear staining pattern, which is consistent with the accumulation of unesterified cholesterol. Some patients, referred to as NPC variants, present a clinical picture of NPC, but inconclusive results in biochemical tests, with no characteristic fluorescence in Filipin test, which makes the diagnosis problematic. The objective of this study was to develop objective techniques that can assist in confirming the diagnosis of NPC, when the phenotype shows undefined/variant characteristics to Filipin test, such as: quantification of perinuclear fluorescence pattern based on pixels' luminosity pattern in patients with classical (NPC cl) and variant (NPC var) NPC phenotype, comparing them among themselves and with healthy controls (CS); the amount of cytoplasmic cholesterol in fibroblasts of both groups of patients; measurement of the activity of the enzymes acid sphingomyelinase (ASM), chitotriosidase (CT), beta-glucosidase acid (GBA), and beta-galactosidase (GLB). All of these parameters were compared with those of CS. The results showed that the fluorescence quantitation of cholesterol in the Filipin test, for the three studied groups, by counting the number of image pixels, is a practical and non-subjective method, which showed a significant difference between the accumulation of intracellular cholesterol in CS, NPC cl and NPC var, confirming its effectiveness to clarify dubious patterns. Measures of intracellular cholesterol in NPC fibroblasts showed seven times higher in classic and four times higher in variant pattern, than that found in CS. With these data, the dosage of intracellular cholesterol appears to be a good parameter to aid in the quantitative Filipin test when it presents only a moderate fluorescence pattern. The measurement of ASM activity in both leukocytes and in SPF did not show statistically significant differences between the groups, despite the remarkable decrease in the enzyme's activity in NPC cl phenotype. With the data obtained for QT's activity in plasma and SPF, we found that there was no significant difference between the two NPC groups, but a significant one between the NPC and the CS, demonstrating that both individuals with the classical phenotype and with the variant one have an increased activity of this enzyme. The analysis of GBA activity in leukocytes showed no significant differences among the three groups, although its activity in NPC cl seemed to be greater than in CS. In the SPF analysis, this difference was statistically confirmed. The activity of GLB in leukocytes did not differ between the groups, although it seems greater in the NPC group than in CS.

No / Discoverability or visibility is a challenge that faces all researchers worldwide – with an ever increasing supply of good research entering the scholarly marketplace; this challenge is only becoming intensified as time passes. The global language of scholarly research is English and so the obstacle of getting noticed is magnified manyfold when the article is not written in the English language. Indeed, despite rapid advances in technology, the “tyranny of language” creates a segmentation inhibiting scholarly research and innovation generally. Mass translation of non-English language articles is neither feasible nor desirable. Our paper proposes a strategy for remedying this segmentation – such that, the work of non-English language scholars become more discoverable. The core piece of this strategy is a “reverse-engineering” [RE] application of Faff’s (2015, 2017) “pitching research” template. More specifically, we provide translated versions of the “cued” template across THIRTY THREE different languages: (1) Arabic; (2) Chinese; (3) Dutch; (4) French; (5) Greek; (6) Hindi; (7) Indonesian; (8) Japanese; (9) Korean; (10) Lao; (11) Norwegian; (12) Polish; (13) Portuguese; (14) Romanian; (15) Russian; (16) Sinhalese; (17) Spanish; (18) Tamil; (19) Thai; (20) Urdu; (21) Vietnamese; (22) Myanmar; (23) German; (24) Persian; (25) Bengali; (26) Filipino; (27) Italian; (28) Afrikaans; (29) Khmer (Cambodia); (30) Danish; (31) Finnish; (32) Hebrew; (33) Turkish. Further, we showcase illustrative dual language examples of the RE strategy for the Chinese, Japanese, Vietnamese and French cases.