Scope: Previous studies show that Lab4 probiotic consortium plus Lactobacillus plantarum CUL66 (L... more Scope: Previous studies show that Lab4 probiotic consortium plus Lactobacillus plantarum CUL66 (Lab4P) reduces diet-induced weight gain and plasma cholesterol levels in C57BL/6J mice fed a high fat diet (HFD). The effect of Lab4P on atherosclerosis is not known and is therefore investigated. Methods and Results: Atherosclerosis-associated parameters are analyzed in LDL receptor deficient mice fed HFD for 12 weeks alone or supplemented with Lab4P. Lab4P increases plasma HDL and triglyceride levels and decreases LDL/VLDL levels. Lab4P also reduces plaque burden and content of lipids and macrophages, indicative of dampened inflammation, and increases smooth muscle cell content, a marker of plaque stabilization. Atherosclerosis arrays show that Lab4P alters the liver expression of 19 key disease-associated genes. Lab4P also decreases the frequency of macrophages and T-cells in the bone marrow. In vitro assays using conditioned media from probiotic bacteria demonstrates attenuation of several atherosclerosis-associated processes in vitro such as chemokine-driven monocytic migration, proliferation of monocytes and macrophages, foam cell formation and associated changes in expression of key genes, and proliferation and migration of vascular smooth muscle cells. Conclusion: This study provides new insights into the anti-atherogenic actions of Lab4P together with the underlying mechanisms and supports further assessments in human trials.
Atherosclerotic plaques are highly diverse and heterogeneous structures, even within the same ind... more Atherosclerotic plaques are highly diverse and heterogeneous structures, even within the same individual, and can vary depending on its anatomical location within the vascular bed. Early in the disease and throughout its progression, immune cells infiltrate the lesion, contributing to the plaque phenotype via different mechanisms. Detailed characterization of constituent cell populations within plaques is hence required for more accurate assessment of disease severity and inflammatory burden. A wide range of fluorophore-conjugated antibodies targeted to key cell types implicated in all stages of the disease are commercially available, enabling visualization of the dynamic cellular landscape present within lesions. This chapter describes the use of immunofluorescence staining of atherosclerotic plaque sections to study plaque cellularity and expression of key markers.
Atherosclerosis is a chronic inflammatory disease characterized by the formation of lipid-rich, f... more Atherosclerosis is a chronic inflammatory disease characterized by the formation of lipid-rich, fibrous plaques within the arterial wall of medium and large arteries. Plaques prone to rupture are typically rich in lipids and pro-inflammatory markers. Cells within the plaque can take up lipids via different mechanisms leading to the formation and accumulation of lipid-rich foam cells, a key hallmark of the disease. Evaluation of plaque burden and lipid content is hence important to determine disease progression and severity. This chapter describes the most commonly used staining methods that enable visualization and analysis of mouse atherosclerotic plaques. These methods include en face preparation of mouse aorta, and staining sections of arteries using hematoxylin and eosin, Oil Red O, and Masson's Trichrome.
Macrophage foam cell formation plays a crucial role in the initiation and progression of atherosc... more Macrophage foam cell formation plays a crucial role in the initiation and progression of atherosclerosis. Macrophages uptake native and modified low density lipoprotein (LDL) through either receptor-dependent or receptor-independent mechanisms to transform into lipid laden foam cells. Foam cells are involved in the formation of fatty streak that is seen during the early stages of atherosclerosis development and therefore represents a promising therapeutic target. Normal or modified lipoproteins labeled with fluorescent dyes such as 1,1'-dioctadecyl-3-3-3',3'-tetramethylindocarbocyanine perchlorate (Dil) are often used to monitor their internalization during foam cell formation. In addition, the fluorescent dye Lucifer Yellow (LY) is widely used as a marker for macropinocytosis activity. In this chapter, we describe established methods for monitoring modified lipoprotein uptake and macropinocytosis during macrophage foam cell formation.
This is an open access article under the terms of the Creat ive Commo ns Attri bution License, wh... more This is an open access article under the terms of the Creat ive Commo ns Attri bution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Whereas 40 % to 70 % of papillary thyroid carcinomas (PTCs) are characterized by a BRAF mutation ... more Whereas 40 % to 70 % of papillary thyroid carcinomas (PTCs) are characterized by a BRAF mutation (BRAFmut), unified biomarkers for the genetically heterogeneous group of BRAF wild type (BRAFwt) PTCs are not established yet. Using state-of-the-art technology we compared RNA expression profiles between conventional BRAFwt and BRAFmut PTCs. Microarrays covering 36,079 reference sequences were used to generate whole transcript expression profiles in 11 BRAFwt PTCs including five micro PTCs, 14 BRAFmut PTCs, and 7 normal thyroid specimens. A p-value with a false discovery rate (FDR) < 0.05 and a fold change > 2 were used as a threshold of significance for differential expression. Network and pathway utilities were employed to interpret significance of expression data. BRAF mutational status was established by direct sequencing the hotspot region of exon 15. We identified 237 annotated genes that were significantly differentially expressed between BRAFwt and BRAFmut PTCs. Of these, ...
The distribution and kind of rat sarcoma viral oncogenes homolog (RAS) mutations, as well as thei... more The distribution and kind of rat sarcoma viral oncogenes homolog (RAS) mutations, as well as their clinical impact on different types of thyroid lesions, vary widely among the different populations studied. We performed a comprehensive mutational survey in the highly related RAS genes HRAS, KRAS, and NRAS in a case series of proliferative thyroid lesions with known BRAF mutational status, originating from an ethnically diverse group. Mutational hotspot regions encompassing codons 12, 13, and 61 of the RAS genes were directly sequenced in 381 cases of thyroid lesions. In addition, the putative NRAS hotspot region encompassing codon 97 was sequenced in 36 thyroid lesions. The case series included lesions of Hashimoto's thyroiditis (HT), nodular goiters, hyperplastic nodules, follicular adenomas (FAs), Hurthle cell variants of FA, papillary thyroid carcinomas (PTCs), follicular variants of PTC (FVPTCs), microcarcinomas of PTC (micro PTCs; tumor size ≤1 cm), follicular TCs (FTCs), H...
Scope: Previous studies show that Lab4 probiotic consortium plus Lactobacillus plantarum CUL66 (L... more Scope: Previous studies show that Lab4 probiotic consortium plus Lactobacillus plantarum CUL66 (Lab4P) reduces diet-induced weight gain and plasma cholesterol levels in C57BL/6J mice fed a high fat diet (HFD). The effect of Lab4P on atherosclerosis is not known and is therefore investigated. Methods and Results: Atherosclerosis-associated parameters are analyzed in LDL receptor deficient mice fed HFD for 12 weeks alone or supplemented with Lab4P. Lab4P increases plasma HDL and triglyceride levels and decreases LDL/VLDL levels. Lab4P also reduces plaque burden and content of lipids and macrophages, indicative of dampened inflammation, and increases smooth muscle cell content, a marker of plaque stabilization. Atherosclerosis arrays show that Lab4P alters the liver expression of 19 key disease-associated genes. Lab4P also decreases the frequency of macrophages and T-cells in the bone marrow. In vitro assays using conditioned media from probiotic bacteria demonstrates attenuation of several atherosclerosis-associated processes in vitro such as chemokine-driven monocytic migration, proliferation of monocytes and macrophages, foam cell formation and associated changes in expression of key genes, and proliferation and migration of vascular smooth muscle cells. Conclusion: This study provides new insights into the anti-atherogenic actions of Lab4P together with the underlying mechanisms and supports further assessments in human trials.
Atherosclerotic plaques are highly diverse and heterogeneous structures, even within the same ind... more Atherosclerotic plaques are highly diverse and heterogeneous structures, even within the same individual, and can vary depending on its anatomical location within the vascular bed. Early in the disease and throughout its progression, immune cells infiltrate the lesion, contributing to the plaque phenotype via different mechanisms. Detailed characterization of constituent cell populations within plaques is hence required for more accurate assessment of disease severity and inflammatory burden. A wide range of fluorophore-conjugated antibodies targeted to key cell types implicated in all stages of the disease are commercially available, enabling visualization of the dynamic cellular landscape present within lesions. This chapter describes the use of immunofluorescence staining of atherosclerotic plaque sections to study plaque cellularity and expression of key markers.
Atherosclerosis is a chronic inflammatory disease characterized by the formation of lipid-rich, f... more Atherosclerosis is a chronic inflammatory disease characterized by the formation of lipid-rich, fibrous plaques within the arterial wall of medium and large arteries. Plaques prone to rupture are typically rich in lipids and pro-inflammatory markers. Cells within the plaque can take up lipids via different mechanisms leading to the formation and accumulation of lipid-rich foam cells, a key hallmark of the disease. Evaluation of plaque burden and lipid content is hence important to determine disease progression and severity. This chapter describes the most commonly used staining methods that enable visualization and analysis of mouse atherosclerotic plaques. These methods include en face preparation of mouse aorta, and staining sections of arteries using hematoxylin and eosin, Oil Red O, and Masson's Trichrome.
Macrophage foam cell formation plays a crucial role in the initiation and progression of atherosc... more Macrophage foam cell formation plays a crucial role in the initiation and progression of atherosclerosis. Macrophages uptake native and modified low density lipoprotein (LDL) through either receptor-dependent or receptor-independent mechanisms to transform into lipid laden foam cells. Foam cells are involved in the formation of fatty streak that is seen during the early stages of atherosclerosis development and therefore represents a promising therapeutic target. Normal or modified lipoproteins labeled with fluorescent dyes such as 1,1'-dioctadecyl-3-3-3',3'-tetramethylindocarbocyanine perchlorate (Dil) are often used to monitor their internalization during foam cell formation. In addition, the fluorescent dye Lucifer Yellow (LY) is widely used as a marker for macropinocytosis activity. In this chapter, we describe established methods for monitoring modified lipoprotein uptake and macropinocytosis during macrophage foam cell formation.
This is an open access article under the terms of the Creat ive Commo ns Attri bution License, wh... more This is an open access article under the terms of the Creat ive Commo ns Attri bution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Whereas 40 % to 70 % of papillary thyroid carcinomas (PTCs) are characterized by a BRAF mutation ... more Whereas 40 % to 70 % of papillary thyroid carcinomas (PTCs) are characterized by a BRAF mutation (BRAFmut), unified biomarkers for the genetically heterogeneous group of BRAF wild type (BRAFwt) PTCs are not established yet. Using state-of-the-art technology we compared RNA expression profiles between conventional BRAFwt and BRAFmut PTCs. Microarrays covering 36,079 reference sequences were used to generate whole transcript expression profiles in 11 BRAFwt PTCs including five micro PTCs, 14 BRAFmut PTCs, and 7 normal thyroid specimens. A p-value with a false discovery rate (FDR) < 0.05 and a fold change > 2 were used as a threshold of significance for differential expression. Network and pathway utilities were employed to interpret significance of expression data. BRAF mutational status was established by direct sequencing the hotspot region of exon 15. We identified 237 annotated genes that were significantly differentially expressed between BRAFwt and BRAFmut PTCs. Of these, ...
The distribution and kind of rat sarcoma viral oncogenes homolog (RAS) mutations, as well as thei... more The distribution and kind of rat sarcoma viral oncogenes homolog (RAS) mutations, as well as their clinical impact on different types of thyroid lesions, vary widely among the different populations studied. We performed a comprehensive mutational survey in the highly related RAS genes HRAS, KRAS, and NRAS in a case series of proliferative thyroid lesions with known BRAF mutational status, originating from an ethnically diverse group. Mutational hotspot regions encompassing codons 12, 13, and 61 of the RAS genes were directly sequenced in 381 cases of thyroid lesions. In addition, the putative NRAS hotspot region encompassing codon 97 was sequenced in 36 thyroid lesions. The case series included lesions of Hashimoto's thyroiditis (HT), nodular goiters, hyperplastic nodules, follicular adenomas (FAs), Hurthle cell variants of FA, papillary thyroid carcinomas (PTCs), follicular variants of PTC (FVPTCs), microcarcinomas of PTC (micro PTCs; tumor size ≤1 cm), follicular TCs (FTCs), H...
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